method for the expansion of tumor infiltrating lymphocytes (tils) in a therapeutic population of til

专利摘要:
The present invention provides methods for expanding populations of TILs present in fine needle aspirates (FNAs) or small biopsies, which contain reduced numbers of TILs, using the methods described here in a closed system that leads to improved phenotype and increased metabolic health of TILs in a shorter period of time.
公开号:BR112020009663A2
申请号:R112020009663-6
申请日:2018-11-19
公开日:2020-11-10
发明作者:Michelle Simpson-Abelson；Cecile Chartier-Courtaud
申请人:Iovance Biotherapeutics, Inc.；
IPC主号:

专利说明:

[001] [001] This patent application claims its priority to Provisional US Patent Application No. 62/588 044, filed on November 17, 2017, to Provisional US Patent Application No. 62/621 515, filed on January 24, 2018 and to US Provisional Patent Application No. 62/756 038, filed on November 5, 2018, the contents of which are incorporated herein, in their entirety, by reference. Fundamentals of the invention
[002] [002] The treatment of large, refractory cancers through the adoptive transfer of tumor infiltrating lymphocytes (TILs) represents a powerful approach to therapy for patients with poor prognosis. Gattinoni, et al., Nat. Rev. Immunol. 2006, 6, 383-393. The success of immunotherapy requires a large number of TILs, and a robust and reliable process is required for commercialization. This has been a challenge to be achieved due to technical, logistical and regulatory issues involved with cell expansion. The expansion of IL-2-based TILs, followed by a “rapid expansion process” (REP) has become a preferred method for expanding TILs due to their speed and efficiency. Dudley, et al., Science 2002, 298, 850-54; Dudley, et al., J. Clin. Oncol. 2005, 23, 2346-57; Dudley, et al., J. Clin. Oncol. 2008, 26, 5233-39; Riddell, et al., Science 1992, 257, 238-41; Dudley, et al., J. Immunother. 2003, 26, 332-42. REP can result in a 1,000-fold expansion of TILs over a 14-day period, although it requires a large excess (e.g., 200-fold) of peripheral blood mononuclear cells (PBMCs, also known as mononuclear cells ( Irradiated allogeneic MNCs), often of
[003] [003] Current processes for producing TILs are limited by the way in which TILs are obtained from the patient. In some cases, when the tumor is small enough or is in a place where tumor resection is not possible, there is still a need for additional methods of obtaining TILs for expansion and treatment. The present invention addresses this need by providing methods for expanding TILs from a fine needle aspirate (FNA) or a core needle biopsy fragment, which contains reduced numbers of TILs, and using these TILs expanded in treatment methods. Brief summary of the invention
[004] [004] The present invention provides improved and / or shortened methods for expanding TILs isolated from a fine needle aspirate or a thick needle biopsy fragment and producing therapeutic populations of TILs.
[005] [005] The present invention provides a method for the expansion of tumor infiltrating lymphocytes (TILs) to a therapeutic population of TILs, which comprises: (i) obtaining a first TIL population of at least one fine needle aspirate (FNA) ) or at least a small biopsy of a tumor in a patient; (ii) carry out a first expansion, cultivating the first
[006] [006] In some embodiments, the cell culture medium comprising IL-2 in step (ii) further comprises OKT-3 and is not optionally supplemented with OKT-3 on any of days 1-3, and in which step ( i) it is a priming step for the first expansion and step (ii) is a second rapid expansion.
[007] [007] Method according to claim 1 or 2, in which, after step (iii), the cells are removed from the cell culture and cryopreserved in a storage medium before performing step (iv).
[008] [008] In some modalities, the cells are thawed before performing step (iv).
[009] [009] In some embodiments, step (iv) is repeated one to four times in order to obtain sufficient TILs in the therapeutic population of TILs for a therapeutically effective dose of TILs.
[0010] [0010] In some modalities, steps (i) to (iii) or (iv) are
[0011] [0011] In some modalities, steps (i) to (iii) or (iv) are carried out within a period between approximately 18 days and 22 days.
[0012] [0012] In some modalities, steps (i) to (iii) or (iv) are carried out within a period between approximately 20 days and 22 days.
[0013] [0013] In some modalities, steps (i) to (iii) or (iv) are performed within approximately 22 days.
[0014] [0014] In some embodiments, the cells in steps (iii) or (iv) express CD4, CD8 and TCR α β at levels similar to those of recently collected cells.
[0015] [0015] In some modalities, APCs are peripheral blood mononuclear cells (PBMCs).
[0016] [0016] In some embodiments, PBMCs are added to the cell culture on any of days 3 to 12 in step (ii) and / or any of days 11 to 14 in step (iii).
[0017] [0017] In some embodiments, the third population of TILs comprises an increased subpopulation of effector T cells and / or central memory T cells compared to the second population of TILs, in which effector T cells and / or central memory T cells in the therapeutic population of TILs in step (iv) exhibit one or more characteristics selected from the group consisting of CD27 expression, CD28 expression, longer telomeres, increased CD57 expression and decreased CD56 expression, in relation to cells Effector T and / or central memory T cells in the third cell population.
[0018] [0018] In some embodiments, effector T cells and / or central memory T cells exhibit increased CD57 expression and decreased CD56 expression.
[0019] [0019] In some modalities, APCs are artificial APCs (aAPCs) or are autologous APCs.
[0020] [0020] In some modalities, the therapeutic population of TILs is infused in one patient.
[0021] [0021] In some modalities, the first expansion in step (ii) is performed by further supplementing the cell culture medium of the second TIL population with OKT-3, IL-15, OX40 agonist antibody and / or 4 agonist antibody -1BB.
[0022] [0022] In some embodiments, the second expansion in step (iii) is performed by further supplementing the cell culture medium of the second population of TILs with IL-15, OX40 agonist antibody and / or 4-1BB agonist antibody.
[0023] [0023] In some modalities, the FNA in step (i) comprises at least 400,000 TILs.
[0024] [0024] In some modalities, the small biopsy is obtained from a tumor selected from the group consisting of pancreatic tumor, melanoma, breast and ovarian.
[0025] [0025] In some modalities, FNA is obtained from a tumor selected from the group consisting of lung, melanoma, head and neck, cervical, ovarian, pancreatic, glioblastoma, colorectal and sarcoma.
[0026] [0026] In some modalities, the lung tumor is a non-small cell lung carcinoma (NSCLC) and, optionally, in which the patient has previously undergone surgical treatment.
[0027] [0027] In some modalities, the TILs in step (i) are obtained from an FNA.
[0028] [0028] In some embodiments, FNA is obtained using a 25-18 gauge needle.
[0029] [0029] In some modalities, the TILs in step (i) are obtained from a small biopsy.
[0030] [0030] In some modalities, the small biopsy is obtained using a 16-11 gauge needle.
[0031] [0031] In some embodiments, step (iii) is repeated one to four times in order to obtain sufficient TILs in the therapeutic population of TILs for a therapeutically effective dose of TILs.
[0032] [0032] In some embodiments, the number of TILs sufficient for a therapeutically effective dose is between approximately 2.3x1010 and 13.7x1010.
[0033] [0033] In some embodiments, the present invention provides a method for the expansion of tumor infiltrating lymphocytes (TILs) in a therapeutic population of TILs, which comprises: (i) performing a first expansion by cultivating a first population of TILs, from of a fine needle aspirate (ANF) or a small biopsy of a tumor in a patient, in a cell culture medium comprising IL-2 in order to obtain a second population of TILs, in which the cell culture medium is supplemented with OKT-3 on any of days 1-3, where the first expansion is performed for approximately 3 days to 12 days in order to obtain the second population of TILs, where the second population of TILs comprises at least 5 x 107 TILs for approximately 3 days to 12 days when the first population of TILs is from a small biopsy; and (ii) perform a second expansion, supplementing the cell culture medium of the second population of TILs with IL-2, OKT-3 and additional antigen presenting cells (APCs) in order to obtain a third population of TILs, in which the third population of TILs has a number at least 25 times greater than the second population of TILs, and in which the second expansion is performed for approximately 3 days to 12 days in order to obtain the third population of TILs, in which the third population of TILs is a therapeutic population of TILs.
[0034] [0034] In some embodiments, the cell culture medium comprising IL-2 in step (ii) still comprises OKT-3 and is not
[0035] [0035] In some embodiments, the cells of the cell culture medium in step (ii) are removed and cryopreserved in a storage medium before step (iii).
[0036] [0036] In some embodiments, the cells are thawed before step (iii).
[0037] [0037] In some modalities, APCs are artificial APCs (aAPCs) or are autologous APCs.
[0038] [0038] In some modalities, the therapeutic population of TILs is infused in one patient.
[0039] [0039] In some embodiments, the first expansion in step (i) is carried out by further supplementing the cell culture medium of the second TIL population with OKT-3, IL-15, OX40 agonist antibody and / or 4 agonist antibody -1BB.
[0040] [0040] In some embodiments, the second expansion in step (ii) is performed by further supplementing the cell culture medium of the second TIL population with IL-15, OX40 agonist antibody and / or 4-1BB agonist antibody.
[0041] [0041] In some embodiments, the second additional expansion in step (iii) is performed by further supplementing the cell culture medium of the third TIL population with IL-15, OX40 agonist antibody and / or 4-1BB agonist antibody.
[0042] [0042] In some modalities, the FNA in step (i) comprises at least 400,000 TILs.
[0043] [0043] In some modalities, the small biopsy is obtained from a tumor selected from the group consisting of pancreatic, melanoma, breast and ovarian.
[0044] [0044] In some modalities, FNA is obtained from a tumor selected from the group consisting of lung, melanoma, head and neck, cervical, ovarian, pancreatic, glioblastoma, colorectal and sarcoma.
[0045] [0045] In some embodiments, the lung tumor is a non-small cell lung carcinoma (NSCLC) and, optionally, in which the patient has previously undergone surgical treatment.
[0046] [0046] In some modalities, the TILs in step (i) are obtained from an FNA.
[0047] [0047] In some modalities, FNA is obtained using a 25-18 gauge needle.
[0048] [0048] In some modalities, the TILs in step (i) are obtained from a small biopsy.
[0049] [0049] In some modalities, the small biopsy is obtained using a 16-11 gauge needle.
[0050] [0050] In some embodiments, step (ii) is repeated one to four times in order to obtain sufficient TILs in the therapeutic population of TILs for a therapeutically effective dose of TILs.
[0051] [0051] In some embodiments, the number of TILs sufficient for a therapeutically effective dose is between approximately 2.3x1010 and 13.7x1010.
[0052] [0052] In some embodiments, the third population of TILs comprises an increased subpopulation of effector T cells and / or central memory T cells compared to the second population of TILs, in which effector T cells and / or central memory T cells exhibit one or more characteristics selected from the group consisting of CD27 expression, CD28 expression, longer telomeres, increased CD57 expression and decreased CD56 expression, in relation to effector T cells and / or central memory T cells in the third cell population.
[0053] [0053] In some embodiments, effector T cells and / or T cells
[0054] [0054] The present invention also provides a method for the treatment of an individual with cancer comprising administering expanded tumor infiltrating lymphocytes (TILs), which comprises: (i) obtaining a first population of TILs from a fine needle aspirate (FNA ) or a small biopsy obtained from a tumor in a patient; (ii) perform a first expansion, cultivating the first population of TILs in a cell culture medium comprising IL-2, to produce a second population of TILs, in which the cell culture medium is supplemented with OKT-3 in any of the days 1-3, where the first expansion is performed for approximately 3 days to 12 days in order to obtain the second population of TILs, where the second population of TILs comprises at least 5 x 107 TILs for approximately 3 days to 12 days when the first population of TILs is from a small biopsy; (iii) perform a second expansion, supplementing the cell culture medium of the second population of TILs with IL-2, OKT-3 and additional antigen presenting cells (APCs), to produce a third population of TILs, in which the third population of TILs has a number at least 25 times greater than the second population of TILs, and in which the second expansion is performed for approximately 3 days to 12 days in order to obtain the third population of TILs, in which the third population of TILs it is a therapeutic population of TILs; and (iv) administering a therapeutically effective dose of the third population of TILs to the patient.
[0055] [0055] In some embodiments, the cell culture medium comprising IL-2 in step (ii) further comprises OKT-3 and is not optionally supplemented with OKT-3 on any of days 1-3, and in
[0056] [0056] In some embodiments, after step (ii), the cells are removed from the cell culture medium and cryopreserved in a storage medium before step (iv).
[0057] [0057] In some embodiments, the cells are thawed before step (iv).
[0058] [0058] In some embodiments, step (iii) is repeated one to four times in order to obtain sufficient TILs in the therapeutic population of TILs for a therapeutically effective dose of TILs.
[0059] [0059] In some modalities, APCs are artificial APCs (aAPCs) or are autologous APCs.
[0060] [0060] In some embodiments, the first expansion in step (ii) is performed by further supplementing the cell culture medium of the second TIL population with OKT-3, IL-15, OX40 agonist antibody and / or 4 agonist antibody -1BB.
[0061] [0061] In some embodiments, the second expansion in step (iii) is performed by further supplementing the cell culture medium of the second TIL population with IL-15, OX40 agonist antibody and / or 4-1BB agonist antibody.
[0062] [0062] In some modalities, the FNA in step (i) comprises at least 400,000 TILs.
[0063] [0063] In some modalities, FNA is obtained from a tumor selected from the group consisting of lung, melanoma, head and neck, cervical, ovarian, pancreatic, glioblastoma, colorectal and sarcoma.
[0064] [0064] In some embodiments, the lung tumor is a non-small cell lung carcinoma (NSCLC) and, optionally, in which the individual has previously undergone surgical treatment.
[0065] [0065] In some modalities, the TILs in step (i) are obtained from
[0066] [0066] In some embodiments, FNA is obtained using a 25-18 gauge needle.
[0067] [0067] In some modalities, the TILs in step (i) are obtained from a small biopsy.
[0068] [0068] In some modalities, the small biopsy is obtained using a 16-11 gauge needle.
[0069] [0069] In some embodiments, step (iii) is repeated one to four times in order to obtain sufficient TILs in the therapeutic population of TILs for a therapeutically effective dose of TILs.
[0070] [0070] In some embodiments, the number of TILs sufficient for a therapeutically effective dose is between approximately 2.3x1010 and 13.7x1010.
[0071] [0071] The present invention also provides a method according to any one of claims 50 to 66, wherein the third population of TILs comprises an increased subpopulation of effector T cells and / or central memory T cells relative to the second population of TILs, in which effector T cells and / or central memory T cells exhibit one or more characteristics selected from the group consisting of CD27 expression, CD28 expression, longer telomeres, increased CD57 expression and decreased expression of CD56, in relation to effector T cells and / or central memory T cells in the third cell population.
[0072] [0072] In some embodiments, effector T cells and / or central memory T cells exhibit increased CD57 expression and decreased CD56 expression.
[0073] [0073] In some modalities, cancer is selected from the group consisting of melanoma, cervical cancer, head and neck cancer, glioblastoma, ovarian cancer, sarcoma, pancreatic cancer, bladder cancer, breast cancer, breast cancer triple negative breast and carcinoma of
[0074] [0074] The present invention also provides a method for the treatment of an individual with cancer, comprising administering expanded tumor infiltrating lymphocytes (TILs), which comprises: (i) performing a first expansion by cultivating a first population of TILs, from a fine needle aspirate (FNA) or a small biopsy of a tumor in a patient, in a cell culture medium comprising IL-2 in order to obtain a second population of TILs, in which the cell culture medium is supplemented with OKT -3 on any of days 1-3, where the first expansion is performed for approximately 3 days to 12 days in order to obtain the second population of TILs, where the second population of TILs comprises at least 5 x 107 TILs per approximately 3 days to 12 days when the first population of TILs is from a small biopsy; (ii) perform a second expansion, supplementing the cell culture medium of the second population of TILs with IL-2, OKT-3 and additional antigen presenting cells (APCs) in order to obtain a third population of TILs, in which the third population of TILs has a number at least 25 times greater than the second population of TILs, and in which the second expansion is carried out for approximately 3 days to 12 days in order to obtain the third population of TILs, in which the third population of TILs are a therapeutic population of TILs; and (iii) administering a therapeutically effective dose of the therapeutic population of TILs to the patient.
[0075] [0075] In some embodiments, the cell culture medium comprising IL-2 in step (ii) further comprises OKT-3 and is not optionally supplemented with OKT-3 on any of days 1-3, and in which step ( i) it is a stage of preparation of the first expansion and step (ii) is a stage of rapid second expansion.
[0076] [0076] In some embodiments, the cells of the cell culture medium in step (ii) are removed and cryopreserved in a storage medium before step (iii).
[0077] [0077] In some embodiments, the cells are thawed before step (iii).
[0078] [0078] In some modalities, APCs are artificial APCs (aAPCs) or are autologous APCs.
[0079] [0079] In some embodiments, APCs are peripheral blood mononuclear cells (PBMCs).
[0080] [0080] In some modalities, the therapeutic population of TILs is infused in one patient.
[0081] [0081] In some embodiments, the first expansion in step (i) is carried out by further supplementing the cell culture medium of the second TIL population with OKT-3, IL-15, OX40 agonist antibody and / or 4 agonist antibody -1BB.
[0082] [0082] In some embodiments, the second expansion in step (iii) is performed by further supplementing the cell culture medium of the second TIL population with IL-15, OX40 agonist antibody and / or 4-1BB agonist antibody.
[0083] [0083] In some modalities, the FNA in step (i) comprises at least 400,000 TILs.
[0084] [0084] In some modalities, the small biopsy is obtained from a tumor selected from the group consisting of pancreatic, melanoma, breast and ovarian.
[0085] [0085] In some modalities, FNA is obtained from a tumor selected from the group consisting of lung, melanoma, head and neck, cervical, ovarian, pancreatic, glioblastoma, colorectal and sarcoma.
[0086] [0086] In some embodiments, the lung tumor is a non-small cell lung carcinoma (CPNP) and, optionally, in which the
[0087] [0087] In some embodiments, the TILs in step (i) are obtained from an FNA.
[0088] [0088] In some embodiments, FNA is obtained using a 25-18 gauge needle.
[0089] [0089] In some modalities, the TILs in step (i) are obtained from a small biopsy.
[0090] [0090] In some modalities, the small biopsy is obtained using a 16-11 gauge needle.
[0091] [0091] In some embodiments, step (ii) is repeated one to four times in order to obtain sufficient TILs in the therapeutic population of TILs for a therapeutically effective dose of TILs.
[0092] [0092] In some embodiments, the number of TILs sufficient for a therapeutically effective dose is between approximately 2.3x1010 and 13.7x1010.
[0093] [0093] In some embodiments, the third population of TILs comprises an increased subpopulation of effector T cells and / or central memory T cells compared to the second population of TILs, in which effector T cells and / or central memory T cells exhibit one or more characteristics selected from the group consisting of CD27 expression, CD28 expression, longer telomeres, increased CD57 expression and decreased CD56 expression, in relation to effector T cells and / or central memory T cells in the third cell population.
[0094] [0094] In some embodiments, effector T cells and / or central memory T cells exhibit increased CD57 expression and decreased CD56 expression.
[0095] [0095] The present invention also provides a method for the expansion of tumor infiltrating lymphocytes (TILs) in a therapeutic population of TILs, which comprises:
[0096] [0096] In some embodiments, the cell culture medium comprising IL-2 in step (ii) further comprises OKT-3 and is not optionally supplemented with OKT-3 on any of days 1-3, and in which step ( i) it is a stage of preparation of the first expansion and step (ii) is a stage of rapid second expansion.
[0097] [0097] In some modalities, the method also comprises the cryopreservation step of the infusion bag comprising the population of TILs collected in step (f) using a cryopreservation process.
[0098] [0098] In some modalities, the cryopreservation process is performed in the ratio of 1: 1 of the population of TILs collected for CS10 medium.
[0099] [0099] In some modalities, APCs are peripheral blood mononuclear cells (PBMCs).
[00100] [00100] In some modalities, PBMCs are irradiated and allogeneic.
[00101] [00101] In some embodiments, PBMCs are added to the cell culture on any of days 3 to 12 in step (c) and / or any of days 3 to 12 in step (d).
[00102] [00102] In some embodiments, antigen presenting cells are artificial antigen presenting cells (aAPCs) or are autologous APCs.
[00103] [00103] In some modalities, the therapeutic population of TILs is infused in a patient.
[00104] [00104] In some embodiments, the first expansion in step (c) is carried out by further supplementing the cell culture medium of the second TIL population with OKT-3, IL-15, OX40 agonist antibody and / or
[00105] [00105] In some embodiments, the second expansion in step (d) is performed by further supplementing the cell culture medium of the second population of TILs with IL-15, OX40 agonist antibody and / or 4-1BB agonist antibody.
[00106] [00106] In some modalities, the FNA in step (a) comprises at least 400,000 TILs.
[00107] [00107] In some modalities, the small biopsy is obtained from a tumor selected from the group consisting of pancreatic, melanoma, breast and ovarian.
[00108] [00108] In some modalities, FNA is obtained from a tumor selected from the group consisting of lung, melanoma, head and neck, cervical, ovarian, pancreatic, glioblastoma, colorectal and sarcoma.
[00109] [00109] In some embodiments, the lung tumor is a non-small cell lung carcinoma (CPNP) and, optionally, in which the individual has previously undergone surgical treatment.
[00110] [00110] In some modalities, the TILs in step (a) are obtained from an FNA.
[00111] [00111] In some embodiments, FNA is obtained using a 25-18 gauge needle.
[00112] [00112] In some modalities, the TILs in step (a) are obtained from a small biopsy.
[00113] [00113] In some modalities, the small biopsy is obtained using a 16-11 gauge needle.
[00114] [00114] In some modalities, the collection in step (e) is performed using a LOVO system for processing the cells.
[00115] [00115] In some embodiments, the cell culture medium is supplied in a container selected from the group consisting of a G-container and an Xuri Cellbag.
[00116] [00116] In some embodiments, the infusion bag in step (f) is a HyperThermosol infusion bag.
[00117] [00117] In some modalities, steps (a) to (f) are carried out within a period of approximately 17 days to 24 days.
[00118] [00118] In some modalities, steps (a) to (f) are performed within a period of approximately 18 days to 22 days.
[00119] [00119] In some modalities, steps (a) to (f) are performed within a period of approximately 20 days to 22 days.
[00120] [00120] In some modalities, steps (a) to (f) are performed in 22 days or less.
[00121] [00121] In some modalities, steps (a) to (f) and cryopreservation are carried out in 22 days or less.
[00122] [00122] In some embodiments, the therapeutic population of TILs collected in step (e) comprises sufficient TILs for a therapeutically effective dose of TILs.
[00123] [00123] In some embodiments, the number of TILs sufficient for a therapeutically effective dose is between approximately 2.3x1010 and 13.7x1010.
[00124] [00124] In some modalities, steps (b) to (e) are performed in a single container, in which steps (b) to (e) performed in a single container result in an increase in the TILs yield per resected tumor compared to performing steps (b) to (e) in more than one container.
[00125] [00125] In some embodiments, the antigen presenting cells are added to the TILs during the second period in step (d) without reopening the system.
[00126] [00126] In some embodiments, the third population of TILs comprises an increased subpopulation of effector T cells and / or central memory T cells compared to the second population of TILs, in which the
[00127] [00127] In some embodiments, effector T cells and / or central memory T cells obtained from the third population of TILs exhibit increased expression of CD57 and decreased expression of CD56, in relation to effector T cells and / or T cells of central memory obtained from the second cell population.
[00128] [00128] In some modalities, the risk of microbial contamination is reduced compared to an open system.
[00129] [00129] In some modalities, the TILs of step (g) are infused in a patient.
[00130] [00130] The present invention also provides a method for the treatment of an individual with cancer, wherein the method comprises administering expanded tumor infiltrating lymphocytes (TILs), comprising: (a) obtaining a first population of TILs from a needle aspirate thin (FNA) or a small biopsy of a resected tumor from an individual; (b) adding the first population to a closed system; (c) performing a first expansion, cultivating the first population of TILs in a cell culture medium comprising IL-2, to produce a second population of TILs, in which the cell culture medium is supplemented with OKT-3 in any of the days 1-3, in which the first expansion is performed for approximately 3 days to 12 days in order to obtain the second population of TILs, in which the second population of TILs
[00131] [00131] In some modalities, the therapeutic population of TILs
[00132] [00132] In some embodiments, the cell culture medium comprising IL-2 in step (ii) further comprises OKT-3 and is not optionally supplemented with OKT-3 on any of days 1-3, and in which step ( i) it is a stage of preparation of the first expansion and step (ii) is a stage of rapid second expansion.
[00133] [00133] In some embodiments, APCs are artificial APCs (aAPCs) or are autologous APCs.
[00134] [00134] In some modalities, the therapeutic population of TILs is infused in a patient.
[00135] [00135] In some embodiments, the first expansion in step (c) is performed by further supplementing the cell culture medium of the second TIL population with OKT-3, IL-15, OX40 agonist antibody and / or 4 agonist antibody -1BB.
[00136] [00136] In some embodiments, the second expansion in step (d) is performed by further supplementing the cell culture medium of the second TIL population with OKT-3, IL-15, OX40 agonist antibody and / or 4 agonist antibody -1BB.
[00137] [00137] In some modalities, the FNA in step (a) comprises at least 400,000 TILs.
[00138] [00138] In some modalities, the small biopsy is obtained from a tumor selected from the group consisting of pancreatic, melanoma, breast and ovarian.
[00139] [00139] In some modalities, FNA is obtained from a tumor selected from the group consisting of pulmonary, melanoma, head and neck, cervical, ovarian, pancreatic, glioblastoma, colorectal and sarcoma.
[00140] [00140] In some embodiments, the lung tumor is a non-small cell lung carcinoma (CPNP) and, optionally, in which the
[00141] [00141] In some embodiments, the TILs in step (i) are obtained from an FNA.
[00142] [00142] In some embodiments, FNA is obtained using a 25-18 gauge needle.
[00143] [00143] In some modalities, the TILs in step (i) are obtained from a small biopsy.
[00144] [00144] In some modalities, the small biopsy is obtained using a 16-11 gauge needle.
[00145] [00145] In some embodiments, the number of TILs sufficient to deliver a therapeutically effective dose in step (h) is between approximately 2.3x1010 and 13.7x1010.
[00146] [00146] In some embodiments, the antigen presenting cells (APCs) are PBMCs.
[00147] [00147] In some embodiments, PBMCs are added to the cell culture on any of days 3 to 12 in step (c) and / or any of days 3 to 12 in step (d).
[00148] [00148] In some embodiments, before a therapeutically effective dose of TIL cells was administered in step (h), a non-myeloablative lymph-depletion scheme was administered to the patient.
[00149] [00149] In some embodiments, the non-myeloablative lymphodepletion schedule comprises the steps of administering cyclophosphamide at a dose of 60 mg / m2 / day for two days, followed by the administration of fludarabine at a dose of 25 mg / m2 / day for five days.
[00150] [00150] In some modalities, the method also comprises the step of treating the patient with a high dose IL-2 schedule, starting the day after the administration of TIL cells to the patient in step (h).
[00151] [00151] In some embodiments, the high-dose IL-2 schedule comprises 600,000 or 720,000 IU / kg, administered by intravenous infusion
[00152] [00152] In some embodiments, the third population of TILs comprises an increased subpopulation of effector T cells and / or central memory T cells compared to the second population of TILs, in which effector T cells and / or central memory T cells in the therapeutic population of TILs exhibit one or more characteristics selected from the group consisting of CD27 + expression, CD28 + expression, longer telomeres, increased CD57 expression and decreased CD56 expression, in relation to effector T cells, and / or central memory T cells obtained from the second cell population.
[00153] [00153] In some embodiments, effector T cells and / or central memory T cells in the therapeutic population of TILs exhibit increased CD57 expression and decreased CD56 expression, compared to effector T cells and / or memory T cells obtained from the second population of cells.
[00154] [00154] The present invention also provides a method for the expansion of tumor infiltrating lymphocytes (TILs) in a therapeutic population of TILs, which comprises: (a) obtaining a first population of TILs from a fine needle aspirate (FNA) , a small biopsy, a thick needle biopsy or a small biopsy of a tumor in a patient; (b) perform a first preparation expansion, cultivating the first population of TILs in a cell culture medium comprising IL-2, OKT-3 and antigen presenting cells (APCs), to produce a second population of TILs, in which the first preparation expansion is carried out for a first period of approximately 1 to 14 days in a container comprising a first gas-permeable surface area to obtain the second population of TILs, where the second population of TILs has a greater number than first population of TILs;
[00155] [00155] In some modalities, the first period is approximately 6 to 12 days.
[00156] [00156] In some modalities, the second period is approximately 6 to 12 days.
[00157] [00157] In some modalities, the first period is selected from the group consisting of 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days or 12 days.
[00158] [00158] In some modalities, the second period is selected from the group consisting of 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days or 12 days.
[00159] [00159] In some embodiments, APCs are peripheral blood mononuclear cells (PBMCs).
[00160] [00160] In some embodiments, the ratio of APCs used in step (b) and for the APCs used in step (c) is approximately 1.1: 1; 1.2: 1; 1.3: 1; 1.4: 1; 1.5: 1; 1.6: 1; 1.7: 1; 1.8: 1; 1.9: 1; 2: 1; 2.1: 1; 2.2: 1; 2.3: 1; 2.4: 1; 2.5: 1; 2.6: 1; 2.7: 1; 2.8: 1; 2.9: 1; 3: 1; 3.1: 1; 3.2: 1; 3.3: 1; 3.4: 1; 3.5: 1; 3.6: 1; 3.7: 1; 3.8: 1; 3.9: 1; 4: 1; 4.1: 1; 4.2: 1; 4.3: 1; 4.4: 1; 4.5: 1; 4.6: 1; 4.7: 1; 4.8: 1; 4.9: 1 or 5: 1 or, preferably, approximately 1 to 2.
[00161] [00161] In some modalities, the first population of TILs is obtained from a thick needle biopsy.
[00162] [00162] In some modalities, a thick needle biopsy is obtained from a tumor selected from the group consisting of a tumor
[00163] [00163] The present invention also provides compositions that comprise expanded TILs using the methods described herein.
[00164] [00164] In some modalities, the composition also includes a cryopreservative.
[00165] [00165] In some embodiments, the cryopreservative comprises dimethyl sulfoxide.
[00166] [00166] In some modalities, the composition also includes a cryopreservative and an isotonic agent.
[00167] [00167] In some embodiments, the composition further comprises a cryopreservative, comprising dimethylsulfoxide, and an isotonic agent comprising sodium chloride, sodium gluconate and sodium acetate.
[00168] [00168] In some embodiments, the composition further comprises a cryopreservative, comprising dimethyl sulfoxide and dextran 40, and an isotonic agent comprising sodium chloride, sodium gluconate and sodium acetate.
[00169] [00169] In some embodiments, the composition comprising the TILs is supplied in a sterile infusion pouch. Brief description of the drawings
[00170] [00170] Figure 1: Diagram of the exemplary Process 2A, providing an overview of Steps A to F.
[00171] [00171] Figure 2: Flowchart of Process 2A.
[00172] [00172] Figure 3: Diagram of a modality of a process of
[00173] [00173] Figure 4: Diagram of a modality of process 2A, a 22-day process for producing TILs.
[00174] [00174] Figure 5: Comparative table of Steps A to F of exemplary modalities of process 1C and process 2A.
[00175] [00175] Figure 6: Detailed comparison of a 1C process modality and a 2A process modality.
[00176] [00176] Figure 7: Diagram of an exemplary Small Biopsy providing an overview of Steps A to F of the Small Biopsy Expansion process.
[00177] [00177] Figure 8: Image of viable cells expanded from thick needle biopsy samples from a patient with pancreatic cancer (P7015). A) Thick needle biopsies treated with IL-2, and B) Thick needle biopsies treated with a combination of IL-2, IL-15 and IL-21.
[00178] [00178] Figure 9: FACS analysis of expanded TILs from pancreatic biopsy samples. A) T cells expanded from thick needle biopsies treated with IL-2 are CD4 + and CD8 +. B) CD8 + cells expanded from thick needle biopsies treated with IL-2 are CD107a +. C) CD8 + cells expanded from thick needle biopsies treated with a combination of IL-2, IL-15 and IL-21 are CD4 + and CD8 +. D) CD8 + T cells expanded from thick needle biopsies treated with a combination of IL-2, IL-15 and IL-21 are CD107a +.
[00179] [00179] Figure 10: FACS analysis of expanded TILs from fine needle aspiration of a patient with cervical carcinoma (C2005). A) Fine needle aspirated treated with IL-2, and B) Fine needle aspirated treated with a combination of IL-2, IL-15 and IL-21.
[00180] [00180] Figure 11: FACS analysis of expanded TILs from fine needle aspiration of a patient with lung carcinoma (L4032). A) Fine needle aspirated treated with IL-2, and B) needle aspirated
[00181] [00181] Figure 12: TILs from the FNA sample from a patient with lung carcinoma (L4032) were further expanded using a Rapid Expansion Protocol. A) Total cell counts after rapid expansion of expanded TILs from aspirates that were initially treated with IL-2 alone or with a combination of IL-2, IL-15 and IL-21, or initially a combination of IL-2 , IL-15 and IL-21 and then IL-2 alone. B) CD4 + and CD8 + T cells were present in the population of expanded IL-2 treated TILs.
[00182] [00182] Figure 13: FACS analysis of expanded TILs from fine needle aspiration of a patient with lung carcinoma (L4033). A) T cells from fine needle aspirates treated with IL-2, and B) as a control, T cells from lung tumor fragments cultured with IL-2.
[00183] [00183] Figure 14: TILs expanded from fine needle aspiration of a patient with lung carcinoma (L4033) were further expanded using a Rapid Expansion Protocol. A) Total cell counts after rapid expansion of TILs obtained from fine needle aspirates treated with IL-2.
[00184] [00184] Figure 15: TILs from fine needle aspirated ovarian tumors taken from patients with ovarian carcinoma (OV8011, OV8012, and OV8013). Total cell counts after culture of fine needle aspirates with IL-2 or a combination of IL-2, IL-15 and IL-21.
[00185] [00185] Figure 16: FACS analysis of expanded TILs from fine needle aspirations of a melanoma patient (M1101). A) T cells from fine needle aspirates treated with IL-2, and B) as a control, T cells from lung tumor fragments cultured with IL-2.
[00186] [00186] Figure 17: Phenotypic and functional status of TILs derived from
[00187] [00187] Figure 18: Summary table of biopsy results for thick needle and fine needle aspiration. A) Results of pancreatic cancer and lung cancer samples. B) Results of samples of colorectal cancer, ovarian cancer, cervical cancer and melanoma.
[00188] [00188] Figure 19: Phenotypic and functional status of TILs derived from melanoma samples obtained by biopsies with fine needle aspiration. A) Total cell counts of cell cultures after 0 days, 11 days, 18 days and 21 days. B) Phenotypic analysis of cells derived from fragments and aspirates. B) Functional analysis (expression of CD107a) of cells derived from fragments and aspirated after stimulation with PMA.
[00189] [00189] Figure 20: Summary of data showing that TILs can be expanded from the Pancreatic Cancer Biopsy Samples obtained from the UPMC (University of Pittsburgh Medical Center).
[00190] [00190] Figure 21: Data showing the general phenotyping of TIL derived from pancreatic tumors.
[00191] [00191] Figure 22: TILs isolated from the pre-REP and REP of pancreatic biopsy samples are Functional, as determined by IFNγ and CD107a.
[00192] [00192] Figure 23: An increase in the population of CD3 + / NK cells was observed in poorly expanded Pre-REP pulmonary TILs.
[00193] [00193] Figure 24: CD4 / 8 ratio is greater than 1 in most pre-REP pulmonary TILs.
[00194] [00194] Figure 25: During REP, comparable relative expansion was observed in both high and low yields.
[00195] [00195] Figure 26: Sarcoma data (UPMC 58).
[00196] [00196] Figure 27: Cervical cancer data (UPMC45). Pre-REP TILs were expanded from 4 fragments in G-REX 10 for 21 days.
[00197] [00197] Figure 28: Data referring to ES19001 (UPMC46). Pre-REP TILs were expanded from 4 fragments in G-REX 10 for 21 days.
[00198] [00198] Figure 29: Data referring to Cancer of the Hard Palate (UPMC 67) and Oral (UPMC 68). Pre-REP TILs were expanded from 4 fragments in G-REX 10 for 21 days.
[00199] [00199] Figure 30: Summary of data showing that TILs can be expanded from the Pancreatic Cancer Biopsy Samples provided by the UPMC.
[00200] [00200] Figure 31: Pancreatic biopsy samples: TILs can be expanded from Pancreatic Cancer Biopsy Samples. Summary and experimental results of P7015: A sample was placed in a well of a plate with 24 wells in total of 5 wells. Two samples were placed in two wells, however, there was little to no growth in those wells. Three wells were treated with 1) IL-2 and two wells with 2) IL-2 / IL-15 / IL-21.
[00201] [00201] Figure 32: Data showing that most cells expanded from pancreatic biopsy samples were CD4 and CD8 + T cells and were functional as determined by the expression of CD107a (P7015). Experimental results for P7015: Most cells expanded from pancreatic biopsy samples are T cells (approximately 80% were CD4 + and CD8 +). PMA / I stimulation demonstrated the CD8 + cell's ability to degranulate as
[00202] [00202] Figure 33: Pancreatic biopsy samples: TILs can be expanded from pancreatic cancer biopsy samples. Summary and Experimental Results of pre-REP of P7028 and P7031: 11 Biopsy samples were placed in G-Rex 10 with the triple cocktail (IL-2 / IL-15 / IL-21). P7028 TILs were evaluated on Day 12 and maintained in culture until Day
[00203] [00203] Figure 34: Pancreatic biopsy samples: TILs can be expanded from thick needle biopsy samples and fragments of pancreatic cancer treated with IL-2 / IL-15 / IL-21. The collection day for TILs derived from fragments ranged from Day 11-21. The collection day for TILs derived from the samples ranged from Day 21-28.
[00204] [00204] Figure 35: Phenotypic characterization of TILs derived from tumor fragments and biopsies. The collection day for TILs derived from fragments ranged from Day 11-21. The collection day for TILs derived from samples ranged from Day 21-28.
[00205] [00205] Figure 36: Phenotypic characterization of TILs derived from fragments and tumor biopsy samples.
[00206] [00206] Figure 37: TILs from biopsy samples can express m CD107a and secrete IFNγ in the same way as TILs isolated from fragments.
[00207] [00207] Figure 38: A) Exemplary process for expansion procedure with thick needle biopsy. 1-2 coarse needle biopsy samples (3 from the pancreas, 4 from melanoma, 1 from the breast and 1 ovarian) were subjected to an exemplary procedure that included the addition of OKT3 +
[00208] [00208] Figure 39: Summary table of the procedure for expanding the pancreatic biopsy sample with a thick needle.
[00209] [00209] Figure 40: Data referring to the number of cells for expansions with pancreatic biopsy by thick needle.
[00210] [00210] Figure 41: Summary table of the expansion procedure with thick needle biopsy of melanoma.
[00211] [00211] Figure 42: Data referring to the number of cells for large needle biopsy melanoma sample expansions.
[00212] [00212] Figure 43: The phenotypic expression of exhaustion and activation markers are similar between REP1 and REP2 (ie, first expansion and second expansion).
[00213] [00213] Figure 44: The addition of OKT3 + feeders to biopsy samples on Day 3 does not significantly alter the TIL phenotype compared to TIL derived from Gen2 fragments (ie process 2A).
[00214] [00214] Figure 45: Summary table of the expansion procedure with thick needle biopsy of the breast and ovary.
[00215] [00215] Figure 46: Data referring to the number of cells for expansions of breast (left panel) and ovary (right panel) samples of thick needle biopsy.
[00216] [00216] Figure 47: Experiment showing that TILs can be expanded from pancreatic cancer biopsy samples. Summary and pre-REP experimental results for P7028 and P7031. Biopsy samples
[00217] [00217] Figure 48: TILs can be expanded from biopsy samples and fragments treated with pancreatic cancer IL-2 / IL-15 / IL-21. Graphs showing cell expansion numbers for fragments and biopsy samples. The collection day for TIL derived from fragments ranged from Day 11-21. The collection day for TIL derived from samples ranged from Day 21-28.
[00218] [00218] Figure 49: TIL derived from biopsy samples and fragments are phenotypically similar. The collection day for TIL derived from fragments ranged from Day 11-21. The collection day for TIL derived from samples ranged from Day 21-28.
[00219] [00219] Figure 50: The "exhaustion markers" are differentially expressed when comparing TILs from biopsy samples and fragments.
[00220] [00220] Figure 51: TIL of thick needle biopsies can express CD107a and secrete IFNγ in a similar way to TIL isolated from fragments.
[00221] [00221] Figure 52: The TC (triple cocktail; IL-2 / IL-15 / IL-21) does not significantly change the total cell count in pancreatic samples in REP2 (second expansion), regardless of the initial culture conditions ( REP1; first expansion).
[00222] [00222] Figure 53: CT (triple cocktail; IL-2 / IL-15 / IL-21) does not significantly alter the CD4 / CD8 ratio or the REP2 (second expansion) Memory subsets in pancreatic biopsy samples.
[00223] [00223] Figure 54: CD107a is similar in CD4 populations and
[00224] [00224] Figure 55: IFNγ secretion is similar regardless of treatment conditions in REP2 (second expansion) in pancreatic biopsy samples.
[00225] [00225] Figure 56: The addition of OKT3 and feeders on Day 3 (REP1; first expansion) does not significantly change phenotypic expression, compared to pre-REP conditions in pancreatic biopsy samples.
[00226] [00226] Figure 57: The addition of OKT3 and feeders on Day 3 (REP1; first expansion) does not significantly alter the phenotypic expression of "exhaustion markers" in pancreatic biopsy samples.
[00227] [00227] Figure 58: The addition of OKT3 from feeders on Day 3 (REP1; first expansion) does not significantly alter the phenotypic expression of activation markers in pancreatic biopsy samples.
[00228] [00228] Figure 59: CD107a is similar in populations of CD4 and CD8 in pre-REP, REP1 (first expansion) and REP2 (second expansion) in pancreatic biopsy samples.
[00229] [00229] Figure 60: IFNγ secretion is reduced in REP2 (second expansion), when compared to REP1 (first expansion) in pancreatic biopsy samples.
[00230] [00230] Figure 61: The TCM population (central memory T cells) is increased and TEM (effector memory T cells) decreased in REP2 (second expansion), but CD27 and CD28 do not change in melanoma. TEMRA: effector memory cells terminally differentiated and expressing CD45RA again. TSCM: Stem cell-like memory T cells.
[00231] [00231] Figure 62: IFNγ and CD107a are similar in REP1 (first expansion) and REP2 (second expansion) of biopsy samples from
[00232] [00232] Figure 63: Data showing phenotypic CD4 and CD8 markers in melanoma fragments versus melanoma biopsy samples.
[00233] [00233] Figure 64: Data showing phenotypic CD4 and CD8 markers in melanoma fragments versus melanoma biopsy samples.
[00234] [00234] Figure 65: Data showing the level of CD107a in CD4 and CD8 in melanoma fragments versus melanoma biopsy samples.
[00235] [00235] Figure 66: Treatment with OX40 improved the percentage of CD8 + cells in ovarian biopsy samples.
[00236] [00236] Figure 67: Expression of CD107a and similar IFNγ secretion in ovarian biopsy samples treated with OX40.
[00237] [00237] Figure 68: Improved TCM expression and differential of exhaustion markers and activation of breast biopsy samples in REP1 (first expansion) and REP2 (second expansion).
[00238] [00238] Figure 69: CD107a and IFNγ are similar in REP1 (first expansion) and REP2 (second expansion) of breast biopsy samples.
[00239] [00239] Figure 70: Schematic drawing of an exemplary TIL expansion procedure with thick needle biopsy of tumor.
[00240] [00240] Figure 71: TIL derived from pancreatic biopsy samples treated with OKT3 + feeders secrete IFNγ with restimulation.
[00241] [00241] Figure 72: TIL derived from melanoma biopsy samples express CD107a and secrete IFNγ upon restimulation.
[00242] [00242] Figure 73: Expansion of TIL from biopsies by thick needle of breast and ovary.
[00243] [00243] Figure 74: The phenotype of the final TIL product derived from breast biopsy samples treated with OKT3 + feeders during REP1 was similar to that of biopsy samples treated with IL-2 only during REP1.
[00244] [00244] Figure 75: The phenotype of the final TIL product derived from breast biopsy samples treated with OKT3 + feeders during REP1 was similar to that of biopsy samples treated with IL-2 only during REP1.
[00245] [00245] Figure 76: Increased IFNγ expression and secretion in breast biopsy samples treated with OKT3 + feeders during REP1 when compared to biopsy samples treated with IL-2 only during REP1.
[00246] [00246] Figure 77: Treatment with OX40 improved the percentage of CD8 + cells in ovarian biopsy samples in REP2.
[00247] [00247] Figure 78: Treatment with OX40 during the two REPs changed the expression of PD-1, KLRG1 and CD25 in REP2.
[00248] [00248] Figure 79: IFNγ secretion in ovarian biopsy samples treated with OX40 is slightly higher in the final product (REP2).
[00249] [00249] Figure 80: Expression of CD107a in ovarian biopsy samples treated with OX40.
[00250] [00250] Figure 81: Treatment with OX40 through 2 REPs altered the expression of markers associated with T-cell “youth”
[00251] [00251] Figure 82: The treatment with OX40 during the two REP slightly altered the expression of PD-1, KLRG1 and CD25 in REP2.
[00252] [00252] Figure 83: Differential expression of exhaustion and activation markers in breast biopsy samples from REP1 and REP 2.
[00253] [00253] Figure 84: CD107a and IFNγ are similar in REP1 and REP2 of breast biopsy samples.
[00254] [00254] Figure 85A-85B: A) Shows a comparison between process 2A (process with approximately 22 days) and a modality of the Gen 3 process with a small biopsy for the production of TIL (process with approximately 17 days to 24 days). B) Diagram of the exemplary Gen3 process providing an overview of Steps A to F (process with
[00255] [00255] Figure 86: Provides an experimental flow chart for comparability between GEN 2 (process 2A) versus GEN 3.
[00256] [00256] Figure 84A-87C: A) L4054 - Phenotypic characterization of TIL produced in the Gen 2 and Gen 3 process. B) L4055-Phenotypic characterization of TIL produced in the Gen 2 and Gen 3 process. C) M1085T- Phenotypic characterization of TIL produced in the Gen 2 and Gen 3 process.
[00257] [00257] Figure 88A-88C: A) L4054 - analysis of TIL memory markers produced in Gen 2 and Gen 3 processes. B) L4055 - analysis of TIL memory markers produced in Gen 2 and Gen 3 processes. C) M1085T- analysis of TIL memory markers produced in the Gen 2 and Gen 3 processes.
[00258] [00258] Figure 89: L4054 activation and exhaustion markers (A) with CD4 + gate (window), (B) with CD8 + gate.
[00259] [00259] Figure 90: L4055 activation and exhaustion markers (A) with CD4 + gate, (B) with CD8 + gate.
[00260] [00260] Figure 91: IFNγ production (pg / mL): (A) L4054, (B) L4055, and (C) M1085T for the Gen 2 and Gen 3 processes: Each bar represented is the mean + SEM for the levels of IFNγ of stimulated, unstimulated and control medium. Optical density measured at 450 nm.
[00261] [00261] Figure 92: ELISA analysis of IL-2 concentration in cell culture supernatant: (A) L4054 and (B) L4055. Each bar represented is the mean + SEM for IL-2 levels in spent media. Optical density measured at 450 nm.
[00262] [00262] Figure 93: Quantification of glucose and lactate (g / L) in depleted medium: (A) Glucose and (B) Lactate: In both tumor lines, and in both processes, a decrease in glucose was observed throughout the expansion CZECH REP. On the other hand, as predicted, an increase in lactate was observed. Both the decrease in glucose and the increase in lactate were
[00263] [00263] Figure 94: A) Quantification of L-glutamine in depleted medium for L4054 and L4055. B) Quantification of depleted Glutamax for L4054 and L4055. C) Quantification of depleted ammonia for L4054 and L4055.
[00264] [00264] Figure 95: Telomere length analysis: The relative telomere length (RTL) value indicates the average fluorescence of the telomer by chromosome / genome in the Gen 2 and Gen 3 process of the telomere fluorescence by chromosome / genome in the lineage control cells (leukemia cell line 1301) using the DAKO kit.
[00265] [00265] Figure 96: Analysis of the unique CDR3 sequence for TIL as final product in L4054 and L4055 under the Gen 2 and Gen 3 process. The columns show the number of unique TCR B clonotypes identified in 1 x 106 cells on Collection Day of the Gen 2 process (eg, day 22) and Gen 3 (eg, day 14-16). Gen 3 showed greater clonal diversity when compared to Gen 2 based on the number of unique peptide CDRs within the sample.
[00266] [00266] Figure 97: Frequency of single CDR3 sequences in the final cell product collected from L4054 IL (Gen 2 process (eg, day 22) and Gen 3 (eg, day 14-16)).
[00267] [00267] Figure 98: Frequency of unique CDR3 sequences in the final cell product collected from L4055 TIL (Gen 2 process (eg, day 22) and Gen 3 (eg, day 14-16)).
[00268] [00268] Figure 99: Diversity Index for the final TIL product in L4054 and L4055 under the Gen 2 and Gen 3 process. The Shannon diversity index for entropy is a more reliable and common metric for comparison. Gen40's L4054 and L4055 show slightly greater diversity than Gen 2.
[00269] [00269] Figure 100: Raw cell count data Day 7-start
[00270] [00270] Figure 101: Raw data for cell counts Day 11 - start of REP Gen 2 and Scale Up (vertical scaling) of Gen 3, shown in Table 37 (see Example 14 below).
[00271] [00271] Figure 102: Raw cell count data for Day 16 - Gen 2 Scale Up and Gen 3 Collection (eg, day 16), shown in Table 38 (see Example 14 below).
[00272] [00272] Figure 103: Raw cell count data Day 22- Gen 2 collection (eg, day 22), shown in Table 38 (see Example 14 below). For L4054 Gen 2, the post-LOVO count was extrapolated to 4 vials, because it was the total study number. 1 bottle was contaminated, and the scale was expanded to a total of 6.67 x 1010.
[00273] [00273] Figure 104: Raw data of the results of flow cytometry presented in Figures 87A, 87th and 87B.
[00274] [00274] Figure 105: Raw data from the results of flow cytometry presented in Figures 87C and 87C.
[00275] [00275] Figure 106: Raw data of the results of flow cytometry presented in Figures 89 and 90.
[00276] [00276] Figure 107: Raw data of the results of the IFNγ production assay for L4054 samples, shown in Figure 91.
[00277] [00277] Figure 108: Raw data from the results of the IFNγ production assay for L4055 samples, shown in Figure 91.
[00278] [00278] Figure 109: Raw data of the results of the IFNγ production assay for M1085T samples, shown in Figure 91.
[00279] [00279] Figure 110: Raw data from IL-2 ELISA results shown in Figure 92.
[00280] [00280] Figure 111: Raw data of the results of the substrate and metabolic analysis presented in Figures 93 and 94.
[00281] [00281] Figure 112: Raw data from the results of the analysis of
[00282] [00282] Figure 113: Raw data from the results of the CD3 single sequence and clonal diversity analyzes presented in Figures 96 and
[00283] [00283] Figure 114: Shows a comparison between several modalities of the Gen 2 process (process 2A) and Gen 3.1.
[00284] [00284] Figure 115: Table describing several characteristics of the Gen 2, Gen 2.1 and Gen 3.0 process modalities.
[00285] [00285] Figure 116: Overview of the conditions of the medium for a modality of the Gen 3 process, referred to as Gen 3.1
[00286] [00286] Figure 117: Table describing various characteristics of the Gen 2, Gen 2.1 and Gen 3.0 process modalities.
[00287] [00287] Figure 118: Table comparing several characteristics of modalities of the Gen 2 and Gen 3.0 processes.
[00288] [00288] Figure 119: Table providing uses of medium in the various modalities of the described expansion processes.
[00289] [00289] Figure 120: Phenotype comparison: Gen 3.0 and Gen 3.1 process modalities showed comparable expression of CD28, CD27 and CD57.
[00290] [00290] Figure 121: Higher IFNγ production in the final Gen product
[00291] [00291] Figure 122: Top: Single sequence analysis of CDR3 for the final TIL product: The columns show the number of unique TCR B clonotypes identified in 1 x 106 cells collected in the Gen 2 process
[00292] [00292] Figure 123: 199 sequences are shared between the final product of Gen 3 and Gen 2, corresponding to 97.07% of 80% of the best single CDR3 of Gen 2 shared with the final product of Gen 3.
[00293] [00293] Figure 124: 1833 sequences are shared between the final product of Gen 3 and Gen 2, corresponding to 99.45% of 80% of the best single CDR3 of Gen 2 shared with the final product of Gen 3.
[00294] [00294] Figure 125: Schematic drawing of an exemplary modality of the Gen 3 process (16-day process).
[00295] [00295] Figure 126: Schematic drawing of an exemplary modality of a method for the expansion of TILS of hematopoietic malignancies using the Gen 3 expansion platform.
[00296] [00296] Figure 127: Provides data showing that adding OKT3 + feeders to biopsy samples on Day 3 did not significantly alter the TIL phenotype compared to Gen2, indicating that the TILs were healthy TILs.
[00297] [00297] Figure 128: Provides data showing that the expression of exhaustion and TIL activation markers derived from melanoma biopsy samples followed trends similar to that of TIL derived from pancreatic biopsy sample. There were no differences observed in phenotypic expression between IL-2 and the triple cocktail, in REP1 and REP2.
[00298] [00298] Figure 129: Provides data regarding the expansion of TIL
[00299] [00299] Figure 130: Provides data showing that similar phenotypic profiles are seen on Day 0 versus Day 3 (OKT3 + feeders) in treated lung biopsy samples.
[00300] [00300] Figure 131: Provides data showing improved phenotypic expression of activation markers and T cells residing in biopsy samples treated with OKT3 and feeders on Day 3.
[00301] [00301] Figure 132: TIL of lung biopsy samples are functional as determined by IFNγ secretion and CD107a mobilization in response to non-specific stimulation.
[00302] [00302] Figure 133: CD8 + TILs from lung biopsy samples treated on Day 3 are oligoclonal when compared to TILs treated on Day 0. Brief description of the sequence listing
[00303] [00303] SEQ ID NO: 1 is the amino acid sequence of the muromonab heavy chain.
[00304] [00304] SEQ ID NO: 2 is the amino acid sequence of the muromonab light chain.
[00305] [00305] SEQ ID NO: 3 is the amino acid sequence of a recombinant human IL-2 protein.
[00306] [00306] SEQ ID NO: 4 is the amino acid sequence of aldesleukin.
[00307] [00307] SEQ ID NO: 5 is the amino acid sequence of a recombinant human IL-4 protein.
[00308] [00308] SEQ ID NO: 6 is the amino acid sequence of a recombinant human IL-7 protein.
[00309] [00309] SEQ ID NO: 7 is the amino acid sequence of a recombinant human IL-15 protein.
[00310] [00310] SEQ ID NO: 8 is the amino acid sequence of a recombinant human IL-21 protein.
[00311] [00311] SEQ ID NO: 9 is the human 4-1BB amino acid sequence.
[00312] [00312] SEQ ID NO: 10 is the amino acid sequence of murine 4-1BB.
[00313] [00313] SEQ ID NO: 11 is the heavy chain of the 4-1BB agonist monoclonal antibody, utomilumab (PF-05082566).
[00314] [00314] SEQ ID NO: 12 is the 4-1BB agonist monoclonal antibody light chain, utomilumab (PF-05082566).
[00315] [00315] SEQ ID NO: 13 is the variable region of the heavy chain (VH) of the 4-1BB agonist monoclonal antibody, utomilumab (PF-05082566).
[00316] [00316] SEQ ID NO: 14 is the variable region of the light chain (VL) of the 4-1BB agonist monoclonal antibody, utomilumab (PF-05082566).
[00317] [00317] SEQ ID NO: 15 is the CDR1 of the 4-1BB agonist monoclonal antibody heavy chain, utomilumab (PF-05082566).
[00318] [00318] SEQ ID NO: 16 is the CDR2 of the 4-1BB agonist monoclonal antibody heavy chain, utomilumab (PF-05082566).
[00319] [00319] SEQ ID NO: 17 is the CDR3 of the 4-1BB agonist monoclonal antibody heavy chain, utomilumab (PF-05082566).
[00320] [00320] SEQ ID NO: 18 is the CDR1 of the 4-1BB agonist monoclonal antibody, utomilumab (PF-05082566).
[00321] [00321] SEQ ID NO: 19 is the CDR2 of the 4-1BB agonist monoclonal antibody, utomilumab (PF-05082566).
[00322] [00322] SEQ ID NO: 20 is the CDR3 of the 4-1BB agonist monoclonal antibody, utomilumab (PF-05082566).
[00323] [00323] SEQ ID NO: 21 is the heavy chain of the 4-1BB agonist monoclonal antibody, urelumab (BMS-663513).
[00324] [00324] SEQ ID NO: 22 is the light chain of the 4-1BB agonist monoclonal antibody, urelumab (BMS-663513).
[00325] [00325] SEQ ID NO: 23 is the variable region of the heavy chain (VH) of
[00326] [00326] SEQ ID NO: 24 is the variable region of the light chain (VL) of the 4-1BB agonist monoclonal antibody, urelumab (BMS-663513).
[00327] [00327] SEQ ID NO: 25 is the CDR1 heavy chain of the 4-1BB agonist monoclonal antibody, urelumab (BMS-663513).
[00328] [00328] SEQ ID NO: 26 is the CDR2 of the 4-1BB agonist monoclonal antibody heavy chain, urelumab (BMS-663513).
[00329] [00329] SEQ ID NO: 27 is the CDR3 of the 4-1BB agonist monoclonal antibody heavy chain, urelumab (BMS-663513).
[00330] [00330] SEQ ID NO: 28 is the CDR1 of the 4-1BB agonist monoclonal antibody, urelumab (BMS-663513).
[00331] [00331] SEQ ID NO: 29 is the CDR2 of the 4-1BB agonist monoclonal antibody, urelumab (BMS-663513).
[00332] [00332] SEQ ID NO: 30 is the CDR3 of the 4-1BB agonist monoclonal antibody, urelumab (BMS-663513).
[00333] [00333] SEQ ID NO: 31 is an Fc domain of a TNFRSF agonist fusion protein.
[00334] [00334] SEQ ID NO: 32 is a linker for a TNFRSF agonist fusion protein.
[00335] [00335] SEQ ID NO: 33 is a linker for a TNFRSF agonist fusion protein.
[00336] [00336] SEQ ID NO: 34 is a linker of a TNFRSF agonist fusion protein.
[00337] [00337] SEQ ID NO: 35 is a linker for a TNFRSF agonist fusion protein.
[00338] [00338] SEQ ID NO: 36 is a linker for a TNFRSF agonist fusion protein.
[00339] [00339] SEQ ID NO: 37 is a linker for a TNFRSF agonist fusion protein.
[00340] [00340] SEQ ID NO: 38 is a linker for a TNFRSF agonist fusion protein.
[00341] [00341] SEQ ID NO: 39 is a linker for a TNFRSF agonist fusion protein.
[00342] [00342] SEQ ID NO: 40 is a linker for a TNFRSF agonist fusion protein.
[00343] [00343] SEQ ID NO: 41 is a linker for a TNFRSF agonist fusion protein.
[00344] [00344] SEQ ID NO: 42 is an Fc domain of a TNFRSF agonist fusion protein.
[00345] [00345] SEQ ID NO: 43 is a linker for a TNFRSF agonist fusion protein.
[00346] [00346] SEQ ID NO: 44 is a linker for a TNFRSF agonist fusion protein.
[00347] [00347] SEQ ID NO: 45 is a linker for a TNFRSF agonist fusion protein.
[00348] [00348] SEQ ID NO: 46 is an amino acid sequence of a 4-1BB (4-1BBL) linker.
[00349] [00349] SEQ ID NO: 47 is a soluble portion of a 4- 1BBL polypeptide.
[00350] [00350] SEQ ID NO: 48 is a heavy chain variable (VH) region of the 4-1BB, 4B4-1-1 version 1 agonist antibody.
[00351] [00351] SEQ ID NO: 49 is a light chain variable (VL) region of the 4-1BB, 4B4-1-1 version 1 agonist antibody.
[00352] [00352] SEQ ID NO: 50 is a heavy chain variable (VH) region of the 4-1BB, 4B4-1-1 version 2 agonist antibody.
[00353] [00353] SEQ ID NO: 51 is a light chain variable (VL) region of the 4-1BB, 4B4-1-1 version 2 agonist antibody.
[00354] [00354] SEQ ID NO: 52 is a variable region of the heavy chain (VH)
[00355] [00355] SEQ ID NO: 53 is a light chain variable (VL) region of the 4-1BB agonist antibody, H39E3-2.
[00356] [00356] SEQ ID NO: 54 is the amino acid sequence of human OX40.
[00357] [00357] SEQ ID NO: 55 is the amino acid sequence of murine OX40.
[00358] [00358] SEQ ID NO: 56 is the heavy chain of the OX40 agonist monoclonal antibody, tavolixizumab (MEDI-0562).
[00359] [00359] SEQ ID NO: 57 is the light chain of the OX40 agonist monoclonal antibody, tavolixizumab (MEDI-0562).
[00360] [00360] SEQ ID NO: 58 is the variable region of the heavy chain (VH) of the OX40 agonist monoclonal antibody, tavolixizumab (MEDI-0562).
[00361] [00361] SEQ ID NO: 59 is the variable region of the light chain (VL) of the OX40 agonist monoclonal antibody, tavolixizumab (MEDI-0562).
[00362] [00362] SEQ ID NO: 60 is the CDR1 of the OX40 agonist monoclonal antibody heavy chain, tavolixizumab (MEDI-0562).
[00363] [00363] SEQ ID NO: 61 is the CDR2 of the heavy chain of the OX40 agonist monoclonal antibody, tavolixizumab (MEDI-0562).
[00364] [00364] SEQ ID NO: 62 is the CDR3 of the heavy chain of the OX40 agonist monoclonal antibody, tavolixizumab (MEDI-0562).
[00365] [00365] SEQ ID NO: 63 is the CDR1 of the OX40 agonist monoclonal antibody tavolixizumab (MEDI-0562).
[00366] [00366] SEQ ID NO: 64 is the CDR2 of the OX40 agonist monoclonal antibody tavolixizumab (MEDI-0562).
[00367] [00367] SEQ ID NO: 65 is the CDR3 of the OX40 agonist monoclonal antibody tavolixizumab (MEDI-0562).
[00368] [00368] SEQ ID NO: 66 is the heavy chain of the OX40 agonist monoclonal antibody, 11D4.
[00369] [00369] SEQ ID NO: 67 is the light chain of the OX40 agonist monoclonal antibody, 11D4.
[00370] [00370] SEQ ID NO: 68 is the heavy chain (VH) variable region of the OX40, 11D4 agonist monoclonal antibody.
[00371] [00371] SEQ ID NO: 69 is the variable region of the light chain (VL) of the OX40, 11D4 agonist monoclonal antibody.
[00372] [00372] SEQ ID NO: 70 is the CDR1 of the OX40 agonist monoclonal antibody, 11D4.
[00373] [00373] SEQ ID NO: 71 is the CDR2 of the OX40 agonist monoclonal antibody, 11D4.
[00374] [00374] SEQ ID NO: 72 is the CDR3 of the heavy chain of the OX40 agonist monoclonal antibody, 11D4.
[00375] [00375] SEQ ID NO: 73 is the CDR1 of the OX40 agonist monoclonal antibody, 11D4.
[00376] [00376] SEQ ID NO: 74 is the CDR2 of the OX40 agonist monoclonal antibody, 11D4.
[00377] [00377] SEQ ID NO: 75 is the CDR3 of the OX40 agonist monoclonal antibody, 11D4.
[00378] [00378] SEQ ID NO: 76 is the heavy chain of the OX40 agonist monoclonal antibody, 18D8.
[00379] [00379] SEQ ID NO: 77 is the OX40 agonist monoclonal antibody light chain, 18D8.
[00380] [00380] SEQ ID NO: 78 is the heavy chain (VH) variable region of the OX40, 18D8 agonist monoclonal antibody.
[00381] [00381] SEQ ID NO: 79 is the variable region of the light chain (VL) of the OX40, 18D8 agonist monoclonal antibody.
[00382] [00382] SEQ ID NO: 80 is the CDR1 of the OX40 agonist monoclonal antibody, 18D8.
[00383] [00383] SEQ ID NO: 81 is the antibody heavy chain CDR2
[00384] [00384] SEQ ID NO: 82 is the CDR3 of the OX40 agonist monoclonal antibody, 18D8.
[00385] [00385] SEQ ID NO: 83 is the CDR1 of the OX40 agonist monoclonal antibody, 18D8.
[00386] [00386] SEQ ID NO: 84 is the CDR2 of the OX40 agonist monoclonal antibody, 18D8.
[00387] [00387] SEQ ID NO: 85 is the CDR3 of the OX40 agonist monoclonal antibody, 18D8.
[00388] [00388] SEQ ID NO: 86 is the heavy chain variable (VH) region of the OX40 agonist monoclonal antibody, Hu119-122.
[00389] [00389] SEQ ID NO: 87 is the variable region of the light chain (VL) of the OX40 agonist monoclonal antibody, Hu119-122.
[00390] [00390] SEQ ID NO: 88 is CDR1 of the OX40 agonist monoclonal antibody heavy chain, Hu119-122.
[00391] [00391] SEQ ID NO: 89 is the CDR2 of the OX40 agonist monoclonal antibody heavy chain, Hu119-122.
[00392] [00392] SEQ ID NO: 90 is the CDR3 of the heavy chain of the OX40 agonist monoclonal antibody, Hu119-122.
[00393] [00393] SEQ ID NO: 91 is the CDR1 of the OX40 agonist monoclonal antibody, Hu119-122.
[00394] [00394] SEQ ID NO: 92 is the CDR2 of the OX40 agonist monoclonal antibody, Hu119-122.
[00395] [00395] SEQ ID NO: 93 is the CDR3 of the OX40 agonist monoclonal antibody, Hu119-122.
[00396] [00396] SEQ ID NO: 94 is the variable region of the heavy chain (VH) of the OX40 agonist monoclonal antibody, Hu106-222.
[00397] [00397] SEQ ID NO: 95 is the variable region of the light chain (VL) of the OX40 agonist monoclonal antibody, Hu106-222.
[00398] [00398] SEQ ID NO: 96 is the CDR1 of the heavy chain of the OX40 agonist monoclonal antibody, Hu106-222.
[00399] [00399] SEQ ID NO: 97 is the CDR2 of the heavy chain of the OX40 agonist monoclonal antibody, Hu106-222.
[00400] [00400] SEQ ID NO: 98 is the CDR3 of the heavy chain of the OX40 agonist monoclonal antibody, Hu106-222.
[00401] [00401] SEQ ID NO: 99 is the CDR1 of the OX40 agonist monoclonal antibody, Hu106-222.
[00402] [00402] SEQ ID NO: 100 is the CDR2 of the OX40 agonist monoclonal antibody, Hu106-222.
[00403] [00403] SEQ ID NO: 101 is the CDR3 of the OX40 agonist monoclonal antibody, Hu106-222.
[00404] [00404] SEQ ID NO: 102 is an amino acid sequence of the OX40 linker (OX40L).
[00405] [00405] SEQ ID NO: 103 is a soluble portion of the OX40L polypeptide.
[00406] [00406] SEQ ID NO: 104 is an alternative soluble portion of the OX40L polypeptide.
[00407] [00407] SEQ ID NO: 105 is the variable region of the heavy chain (VH) of the OX40, 008 agonist monoclonal antibody.
[00408] [00408] SEQ ID NO: 106 is the variable region of the light chain (VL) of the OX40, 008 agonist monoclonal antibody.
[00409] [00409] SEQ ID NO: 107 is the heavy chain variable (VH) region of the OX40, 011 agonist monoclonal antibody.
[00410] [00410] SEQ ID NO: 108 is the variable region of the light chain (VL) of the OX40, 011 agonist monoclonal antibody.
[00411] [00411] SEQ ID NO: 109 is the variable region of the heavy chain (VH) of the OX40, 021 agonist monoclonal antibody.
[00412] [00412] SEQ ID NO: 110 is the variable region of the light chain (VL) of
[00413] [00413] SEQ ID NO: 111 is the heavy chain variable (VH) region of the OX40, 023 agonist monoclonal antibody.
[00414] [00414] SEQ ID NO: 112 is the variable region of the light chain (VL) of the OX40, 023 agonist monoclonal antibody.
[00415] [00415] SEQ ID NO: 113 is the heavy chain variable (VH) region of an OX40 agonist monoclonal antibody.
[00416] [00416] SEQ ID NO: 114 is the light chain variable region (VL) of an OX40 agonist monoclonal antibody.
[00417] [00417] SEQ ID NO: 115 is the heavy chain (VH) variable region of an OX40 agonist monoclonal antibody.
[00418] [00418] SEQ ID NO: 116 is the variable region of the light chain (VL) of an OX40 agonist monoclonal antibody.
[00419] [00419] SEQ ID NO: 117 is the heavy chain variable region (VH) of a humanized OX40 agonist monoclonal antibody.
[00420] [00420] SEQ ID NO: 118 is the heavy chain variable region (VH) of a humanized OX40 agonist monoclonal antibody.
[00421] [00421] SEQ ID NO: 119 is the light chain variable region (VL) of a humanized OX40 agonist monoclonal antibody.
[00422] [00422] SEQ ID NO: 120 is the light chain variable region (VL) of a humanized OX40 agonist monoclonal antibody.
[00423] [00423] SEQ ID NO: 121 is the heavy chain variable region (VH) of a humanized OX40 agonist monoclonal antibody.
[00424] [00424] SEQ ID NO: 122 is the heavy chain variable region (VH) of a humanized OX40 agonist monoclonal antibody.
[00425] [00425] SEQ ID NO: 123 is the light chain variable region (VL) of a humanized OX40 agonist monoclonal antibody.
[00426] [00426] SEQ ID NO: 124 is the light chain variable region (VL) of a humanized OX40 agonist monoclonal antibody.
[00427] [00427] SEQ ID NO: 125 is the heavy chain (VH) variable region of an OX40 agonist monoclonal antibody.
[00428] [00428] SEQ ID NO: 126 is the light chain variable region (VL) of an OX40 agonist monoclonal antibody. Detailed description of the invention I. Introduction
[00429] [00429] Adoptive cell therapy using TILs cultured ex vivo by the Rapid Expansion Protocol (REP) was successful after host immunosuppression in patients with melanoma. The current parameters for infusion acceptance are based on readings of the TILs composition (eg, positivity for CD28, CD8 or CD4) and the numerical times of expansion and viability of the REP product.
[00430] [00430] Current REP protocols do not reveal much about the health of the TIL that will be infused into the patient. T cells over a profound metabolic deviation during their maturation of virgin to effector T cells (see Chang, et al., Nat. Immunol. 2016, 17, 364, here expressly incorporated, in their entirety and, in particular, for discussion and markers of anaerobic and aerobic metabolism). For example, virgin T cells rely on mitochondrial respiration to produce ATP, while mature, healthy, effector T cells, such as TIL, are highly glycolytic, relying on aerobic glycolysis to provide bioenergetic substrates they need for proliferation, migration, activation and antitumor efficacy.
[00431] [00431] Previous articles report that limiting glycolysis and promoting mitochondrial metabolism in TILs, prior to transfer, is desirable, as cells that are heavily dependent on glycolysis will be deprived of nutrients upon adoptive transfer, resulting in the death of most cells transferred. Thus, the technique teaches that promoting mitochondrial metabolism could promote longevity in vivo and, in fact, suggests the use of glycolysis inhibitors before inducing the immune response. See Chang et
[00432] [00432] The present invention is also directed, in some modalities, to methods to evaluate and quantify this increase in metabolic health. Thus, the present invention provides methods of assessing the relative health of a TIL population through one or more general assessments of metabolism, including, but not limited to, rates and levels of glycolysis, oxidative phosphorylation, respiratory reserve capacity (CRS) and glycolytic reserve.
[00433] [00433] In addition, the present invention is also directed, in some modalities, to methods to evaluate and quantify this increase in metabolic health. Thus, the present invention provides methods of assessing the relative health of a TIL population through one or more general assessments of metabolism, including, but not limited to, rates and levels of glycolysis, oxidative phosphorylation, respiratory reserve capacity (CRS) and glycolytic reserve.
[00434] [00434] In addition, additional optional assessments include, among others, ATP production, mitochondrial mass and glucose absorption. II. Definitions
[00435] [00435] The term "in vivo" refers to an event that happens in an individual's body.
[00436] [00436] The term "in vitro" refers to an event that happens outside an individual's body. In vitro assays include cell assays, in which living or dead cells are used, and can also cover a cell-free assay in which intact cells are not used.
[00437] [00437] The term "rapid expansion" means an increase in the number of antigen-specific TILs of at least approximately 3 times (or 4, 5, 6, 7, 8 or 9 times) over a period of one week, more preferably at least 10 times (or 20, 30, 40, 50, 60, 70, 80 or 90 times) over a period of one week or, most preferably, at least
[00438] [00438] By "tumor infiltrating lymphocytes" or "TILs", in this specification, we understand a population of cells originally obtained as leukocytes that left an individual's bloodstream and migrated to a tumor. TILs include, among others, cytotoxic CD8 + T cells (lymphocytes), CD4 + Th1 and Th17 T cells, natural killer cells, dendritic cells and M1 macrophages. TILs include primary and secondary TILs. “Primary TILs” are those that are obtained from tissue samples from the patient, as outlined here (sometimes referred to as “recently collected”), and “Secondary TILs” are any cell populations of TIL that have been expanded or proliferated as discussed here, including, but not limited to, bulk TILs and expanded TILs (“REP TILs” or “post-REP TILs”).
[00439] [00439] By "cell population" (including TILs), this specification is understood to mean some cells that share common features. In general, the number in populations ranges from 1 x 106 to 1 x 1010, with different TIL populations comprising different numbers. For example, the initial growth of primary TILs in the presence of IL-2 results in a population of bulk TILs of approximately 1 x 108 cells. REP expansion is generally performed to provide populations from 1.5 x 109 to 1.5 x 1010 cells for infusion.
[00440] [00440] By "small biopsy" in this specification, it is understood that a biopsy removes a volume of tissue from a tumor that is typically lower and, in some modalities, substantially lower than the tumor volume. Small biopsy includes thick needle biopsies, puncture biopsies and the like. Any small biopsy is included, for example, any biopsy with a volume of less than approximately 1 mm3, 2 mm3, approximately 5 mm3, approximately 10 mm3, approximately 25 mm3, approximately 50 mm3 or approximately 100 mm3 or with area
[00441] [00441] By "cryopreserved TILs", it is understood in this specification that TILs, whether primary, bulk or expanded (TILs REP), are treated and stored in the range between about -150 ºC and -60 ºC. General methods for cryopreservation are also described elsewhere in the present, including in the Examples. For the sake of clarity, “cryopreserved TILs” are distinguished from frozen tissue samples that can be used as a source of primary TILs.
[00442] [00442] “Thawed cryopreserved TILs” means, in this specification, a population of TILs that were previously cryopreserved and then treated to return to room temperature or above, including, but not limited to, cell culture temperatures or temperatures at which TILs can be administered to a patient.
[00443] [00443] TILs can generally be defined either biochemically, using cell surface markers, or functionally, for their ability to infiltrate tumors and perform treatment. TILs can generally be classified by the expression of one or more of the following biomarkers: CD4, CD8, TCR αβ, CD27, CD28, CD56, CCR7, CD45Ra, CD95, PD-1 and CD25. In addition to and alternatively, TILs can be functionally defined by their ability to infiltrate solid tumors upon reintroduction into a patient.
[00444] [00444] The term "cryopreservation medium" or "cryopreservation medium" refers to any medium that can be used for cryopreservation of cells. Such means may include means that
[00445] [00445] By "cell population" (including TILs), in this specification, we mean some cells that share common features. In general, the number of populations ranges from 1 x 106 to 1 x 1010, with different TIL populations comprising different numbers. For example, the initial growth of primary TILs in the presence of IL-2 results in a population of bulk TILs of approximately 1 x 108 cells. REP expansion is generally performed to provide populations from 1.5 x 109 to 1.5 x 1010 cells for infusion.
[00446] [00446] The term "central memory T cell" refers to a subset of T cells that, in man, are CD45R0 + and constitutively express CCR7 (CCR7hi) and CD62L (CD62hi). The surface phenotype of central memory T cells also includes TCR, CD3, CD127 (IL-7R) and IL-15R. Transcription factors for central memory T cells include BCL-6, BCL-6B, MBD2 and BMI1. Central memory T cells primarily secrete IL-2 and CD40L as effector molecules after being triggered by TCR. Central memory T cells are predominant in the CD4 compartment in the blood and, in humans, are proportionally enriched in lymph nodes and tonsils.
[00447] [00447] The term "effector memory T cell" refers to a subset of human or mammalian T cells that, like central memory T cells, are CD45R0 +, but which have lost the constitutive expression of CCR7 (CCR7lo) and with heterogeneous or low expression of CD62L (CD62Llo). The surface phenotype of central memory T cells also
[00448] [00448] The terms "fragment", "fragment" and "fragmented", used here in describing processes to rupture a tumor, include methods of mechanical fragmentation such as crushing, slicing, dividing and morcreasing the tumor tissue, as well as any other method to disrupt the physical structure of tumor tissue.
[00449] [00449] The terms "peripheral blood mononuclear cells" and "PBMCs" refer to a peripheral blood cell with a round nucleus, including lymphocytes (T cells, B cells, NK cells) and monocytes. When used as antigen presenting cells (PBMCs are a type of antigen presenting cell), peripheral blood mononuclear cells are peripheral blood mononuclear cells that are irradiated allogeneic.
[00450] [00450] The terms "peripheral blood lymphocytes" and "PBLs" refer to expanded T cells in the peripheral blood. In some modalities, PBLs are separated from whole blood or are the product of a donor's apheresis. In
[00451] [00451] The term "anti-CD3 antibody" refers to an antibody or variant thereof, e.g. , a monoclonal antibody and including human, humanized, chimeric or murine antibodies that are directed against the CD3 receptor on the mature T cell T cell antigen receptor. Anti-CD3 antibodies include OKT-3, also known as muromonab. Other anti-CD3 antibodies include, for example, otelixizumab, teplizumab and visilizumab.
[00452] [00452] The term "OKT-3" (also referred to herein as "OKT3") refers to a monoclonal or biosimilar antibody or variant thereof, including human, humanized, chimeric or murine antibodies that are directed against the CD3 receptor at the mature T cell T cell antigen receptor, and includes commercially available forms such as OKT-3 (30 ng / mL, MACS GMP CD3 pure, Miltenyi Biotech, Inc., San Diego, CA, USA) and muromonabe or variants, conservative substitutions of amino acids, glycoforms or biosimilars thereof. The amino acid sequences of muromonab heavy and light chains are given in Table 1 (SEQ ID NO: 1 and SEQ ID NO: 2). A hybridoma capable of producing OKT-3 is deposited in the American Type Culture Collection and has received the accession number ATCC CRL 8001. A hybridoma capable of producing OKT-3 is also deposited in the European Collection of Authenticated Cell Cultures (ECACC) and received No de Catalog 86022706. Table 1. Amino acid sequences of muromonabe
[00453] [00453] The term "IL-2" (also referred to herein as "IL2") refers to the T cell growth factor known as interleukin-2, and includes all forms of IL-2, including human and human forms. mammals, conservative amino acid substitutions, glycoforms, biosimilars and variants thereof. IL-2 is described, e.g. in Nelson, J. Immunol. 2004, 172, 3983-88 and Malek, Annu. Rev. Immunol. 2008, 26, 453-79, whose contents are incorporated by reference. The amino acid sequence of recombinant human IL-2 suitable for use in the invention is provided in Table 2 (SEQ ID NO: 3). For example, the term IL-2 encompasses the human form, recombinants of IL-2 such as aldesleukin (PROLEUKIN, commercially available from multiple suppliers in a 22 million IU vial for single use), as well as the form of IL- 2 recombinant commercially supplied by CellGenix, Inc., Portsmouth, NH, USA (CELLGRO GMP) or ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (Cat. No CYT-209-b) and other commercial equivalents from others Providers. Aldesleukin (des-alanyl-1, serine-125 human IL-2) is a recombinant non-glycosylated human form of IL-2 with a molecular weight close to 15 kDa. The appropriate aldesleukin amino acid sequence
[00454] [00454] The term "IL-4" (also referred to here as "IL4") refers to the cytokine known as interleukin 4, which is produced by Th2 T cells
[00455] [00455] The term "IL-7" (also referred to herein as "IL7") refers to a glycosylated tissue-derived cytokine known as interleukin 7, which can be obtained from stromal and epithelial cells, as well as from dendritic cells. Fry and Mackall, Blood 2002, 99, 3892-904. IL-7 can stimulate the development of T cells. IL-7 binds to the IL-7 receptor, a heterodimer consisting of an IL-7 alpha receptor and a common gamma receptor, which emits a series of important signals for the development of T cells within the thymus and survival in the periphery. Recombinant human IL-4 suitable for use in the invention is commercially available from multiple suppliers, including ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (Cat. No CYT-254) and ThermoFisher Scientific, Inc., Waltham, MA , USA (recombinant human IL-15 protein, Cat. No Gibco PHC0071). The amino acid sequence of recombinant human IL-7 suitable for use in the invention is provided in Table 2 (SEQ ID NO: 6).
[00456] [00456] The term “IL-15” (also referred to here as “IL15”) refers to
[00457] [00457] The term "IL-21" (also referred to herein as "IL21") refers to the pleiotropic cytokine protein known as interleukin-21, and includes all forms of IL-21 including human and mammalian forms, conservative substitutions amino acids, glycoforms, biosimilars and variants thereof. IL-21 is described, e.g. in Spolski and Leonard, Nat. Rev. Drug. Disc. 2014, 13, 379-95, the content of which is incorporated herein by reference. IL-21 is produced primarily by natural killer T cells and activated human CD4 + T cells. Recombinant human IL-21 is a single, non-glycosylated polypeptide chain containing 132 amino acids with a molecular mass of 15.4 kDa. Recombinant human IL-21 is commercially available from multiple suppliers, including ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (Cat. No CYT-408-b) and ThermoFisher Scientific, Inc., Waltham, MA, USA (protein Recombinant human IL-21, Cat. No 14-8219-80). The amino acid sequence of IL-21
[00458] [00458] When "an effective antitumor amount", "an effective tumor inhibiting amount" or "therapeutic amount" is indicated, the precise amount of the compositions of the present invention to be administered can be determined by a physician taking into account individual differences in age, weight, tumor size, extent of infection or metastasis and condition of the patient (individual). It can generally be said that a pharmaceutical composition comprising the genetically modified cytotoxic lymphocytes described herein can be administered at a dose of 104 to 1011 cells / kg of body weight (eg, 105 to 106, 105 to 1010, 105 to 1011, 106 to 1010, 106 to 1011,107 to 1011, 107 to 1010, 108 to 1011, 108 to 1010, 109 to 1011 or 109 to 1010 cells / kg body weight), including all integer values within those ranges. Compositions with genetically modified cytotoxic lymphocytes can also be administered multiple times at these doses. Genetically modified cytotoxic lymphocytes can be administered using infusion techniques that are commonly known in immunotherapy (see, eg, Rosenberg et al., New Eng. J. of Med. 319: 1676, 1988). The optimum dose and treatment schedule for a particular patient can be easily determined by the medically skilled technician, monitoring the patient for signs of illness and adjusting treatment accordingly.
[00459] [00459] The term "hematological malignancy", "hematological malignancy" or related terms of meaning refer to cancers and tumors in mammals of hematopoietic and lymphoid tissue, including, among others, blood tissues, bone marrow, lymph nodes and system lymphatic. Hematological malignancies are also referred to as "liquid tumors". Haematological malignancies include, but are not limited to, acute lymphoblastic leukemia (ALL), chronic lymphocytic lymphoma (CLL), small lymphocytic lymphoma
[00460] [00460] The term "solid tumor" refers to an abnormal mass of tissue that normally does not contain cysts or liquid areas. Solid tumors can be benign or malignant. The term "solid tumor cancer" refers to solid malignant, neoplastic or cancerous tumors. Solid tumor cancers include, but are not limited to, sarcomas, carcinomas and lymphomas, such as cancers of the lung, breast, prostate, colon, rectum and bladder. The tissue structure of solid tumors includes interdependent tissue compartments including the parenchyma (cancer cells) and stromal support cells in which cancer cells are dispersed and which can provide a supporting microenvironment.
[00461] [00461] The term “fine needle aspiration” or FNA refers to a type of biopsy procedure that can be used for sampling or diagnostic procedures, including sampling the tumor, in which a sample is taken, but the tumor is not is removed or resected. In fine needle aspiration, a hollow needle, for example, 25-18 gauge, is inserted into the tumor or an area containing the tumor, and liquid and cells (including tissue) are obtained for analysis or future expansion, as described here . With an FNA, cells are removed without preserving the histological architecture of the tissue cells. An FNA can comprise TILs. In some cases, a fine needle aspirate biopsy is performed using an ultrasound-guided biopsy needle aspiration. FNA needles are commercially available from Becton Dickinson, Covidien, and the like.
[00462] [00462] The term “thick needle biopsy” refers to a type of biopsy procedure that can be used for biopsy procedures
[00463] [00463] The term "liquid tumor" refers to an abnormal mass of cells that is liquid in nature. Liquid tumor cancers include, among others, leukemias, myelomas and lymphomas, as well as other hematological malignancies. TILs obtained from liquid tumors can also be referred to here as marrow infiltrating lymphocytes (MILs). TILs obtained from liquid tumors, including liquid tumors circulating in the peripheral blood, can also be referred to herein as PBLs. The terms MIL, TIL and PBL are used alternatively at present and differ only based on the type of tissue from which the cells are derived.
[00464] [00464] The term "microenvironment", in this specification, may refer to a solid or hematological tumor microenvironment, in whole or as a subset of individual cells within the microenvironment. O
[00465] [00465] In one embodiment, the invention includes a method of treating cancer with a population of remaining TILs (rTILs), in which a patient is pretreated with non-myeloablative chemotherapy prior to the infusion of rTILs according to the invention. In some embodiments, the rTIL population may be provided with a population of normal emigrant TILs (eTILs), in which a patient is pre-treated with non-myeloablative chemotherapy prior to an infusion of rTILs and eTILs according to the invention. In one embodiment, non-myeloablative chemotherapy is cyclophosphamide 60 mg / kg / d for 2 days (days 27 and 26 before rTIL infusion) and fludarabine 25 mg / m2 / d for 5 days (days 27 to 23 before infusion of rTIL). In one embodiment, after non-myeloablative chemotherapy and infusion of rTIL (on day 0) according to the invention, the patient receives an intravenous infusion of IL-2 intravenously at 720,000 IU / kg every 8 hours according to tolerance physiological.
[00466] [00466] Experimental findings indicate that lymphodepletion prior to the adoptive transfer of tumor-specific T lymphocytes plays a key role in improving treatment effectiveness by eliminating regulatory T cells and competitive elements of the immune system ("cytokine dissipators"). Thus, some embodiments of the invention use a
[00467] [00467] The terms “coadministration”, “coadministrar”, “administered in combination with”, “administer in combination with”, “simultaneous” and “concomitant”, in this specification, cover the administration of two or more active pharmaceutical ingredients ( in a preferred embodiment of the present invention, for example, at least one potassium channel agonist in combination with a plurality of TILs) to an individual so that both active pharmaceutical ingredients and / or their metabolites are present in the individual at the same time . Co-administration includes simultaneous administration in separate compositions, administration at different times in separate compositions or administration of a composition in which two or more active pharmaceutical ingredients are present. Simultaneous administration in separate compositions and administration of a composition in which both agents are present are preferred.
[00468] [00468] The term "effective amount" or "therapeutically effective amount" refers to that amount of a compound or combination of compounds as described herein that is sufficient for the purpose of the intended application, including, among others, treatment of the disease. The therapeutically effective amount may vary depending on the intended application (in vitro or in vivo) or the individual and condition of the disease being treated (eg, the individual's weight, age and sex), the severity of the disease condition or the mode of administration. The term also applies to a dose that will induce a particular response in target cells (eg, reduced platelet adhesion and / or cell migration). The specific dose will vary depending on the specially chosen compounds, the administration schedule to be followed, whether the compound is administered in combination with other compounds, the time of administration, the tissue to which it is administered
[00469] [00469] The terms "treatment", "treating", "treating" and the like refer to obtaining a desired pharmacological and / or physiological effect. The effect can be prophylactic in terms of preventing a disease or symptom of it completely or partially and / or it can be therapeutic in terms of partial or complete cure for a disease and / or adverse effect attributable to the disease. “Treatment” in this specification covers any treatment of a disease in a mammal, particularly a human, and includes: (a) preventing the occurrence of the disease in an individual who may be predisposed to the disease, but who has not yet been diagnosed as carrier; (b) inhibit the disease, that is, stop its development or progression; and (c) alleviate the disease, that is, cause the disease to regress and / or relieve or more symptoms of the disease. "Treatment" is also intended to encompass the release of an agent for a pharmacological effect, even in the absence of a disease or condition. For example, "treatment" encompasses the release of a composition that can elicit an immune response or confer immunity in the absence of a disease condition, e.g. in the case of a vaccine.
[00470] [00470] The term "heterologous" when used with reference to parts of a nucleic acid or protein indicates that the nucleic acid or protein comprises two or more subsequences that are not found in the same relationship with each other in nature. For example, nucleic acid is typically produced by recombination, having two or more sequences of unrelated genes arranged to produce a new functional nucleic acid, e.g. e.g., a promoter from one source and a coding region from another source, or coding regions from different sources. Likewise, a heterologous protein indicates that the protein comprises two or more subsequences that are not found in the same relationship with each other in nature (eg, a fusion protein).
[00471] [00471] The terms “sequence identity”, “percentage of
[00472] [00472] In this specification, the term "variant" encompasses, among others, antibodies or fusion proteins that comprise an amino acid sequence that differs from the amino acid sequence of a reference antibody by means of one or more substitutions, deletions and / or additions at certain positions within or adjacent to the antibody's amino acid sequence
[00473] [00473] The term "in vivo" refers to an event that happens in an individual's body.
[00474] [00474] The term "in vitro" refers to an event that happens outside an individual's body. In vitro assays include cell assays, in which living or dead cells are used, and can also cover a cell-free assay in which intact cells are not used.
[00475] [00475] The term "rapid expansion" means an increase in the number of antigen-specific TILs of at least approximately 3 times (or 4, 5, 6, 7, 8 or 9 times) over a period of one week, more preferably at least 10 times (or 20, 30, 40, 50, 60, 70, 80 or 90 times) over a period of one week or, more preferably, at least about 100 times over a period one week period. Some rapid expansion protocols are outlined below.
[00476] [00476] By "tumor infiltrating lymphocytes" or "TILs" we mean, in this specification, a population of cells originally obtained as leukocytes that left an individual's bloodstream and migrated to a tumor. TILs include, among others, cytotoxic CD8 + T cells (lymphocytes), CD4 + Th1 and Th17 T cells, natural killer cells, dendritic cells and M1 macrophages. TILs include primary and secondary TILs. “Primary TILs” are those obtained from tissue samples from the patient as outlined here (sometimes referred to as “recently collected”), and “Secondary TILs” are any cell populations of TIL
[00477] [00477] TILs can generally be defined either biochemically, using cell surface markers, or functionally, for their ability to infiltrate tumors and effect treatment. TILs can generally be classified by the expression of one or more of the following biomarkers: CD4, CD8, TCR αβ, CD27, CD28, CD56, CCR7, CD45Ra, CD95, PD-1 and CD25. In addition and alternatively, TILs can be functionally defined by their ability to infiltrate solid tumors upon reintroduction into a patient. TILs can also be characterized by potency - for example, TILs can be considered potent if, for example, the release of interferon (IFN) is greater than approximately 50 pg / mL, greater than approximately 100 pg / mL, greater than approximately 150 pg / ml or greater than approximately 200 pg / ml.
[00478] [00478] By "cell population" (including TILs), in this specification, we mean some cells that share common features. In general, the number in populations ranges from 1 x 106 to 1 x 1010, with different TIL populations comprising different numbers. For example, the initial growth of primary TILs in the presence of IL-2 results in a population of bulk TILs of approximately 1 x 108 cells. REP expansion is generally performed to provide populations from 1.5 x 109 to 1.5 x 1010 cells for infusion.
[00479] [00479] The term "pharmaceutically acceptable salt" refers to salts derived from a variety of organic and inorganic counterions known in the art. Pharmaceutically acceptable acid addition salts can be
[00480] [00480] The terms "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" are intended to include any and all solvents, dispersion media, coatings, agents
[00481] [00481] The terms "about" and "approximately" mean within a statistically significant range of a value. Such a range may be within an order of magnitude, preferably within 50%, more preferably within 20%, even more preferably within 10%, and even more preferably within 5% of a given value or range. The allowed variation covered by the terms "about" or "approximately" depends on the specific system under study, and can be easily recognized by any technician in the subject. In addition, in this specification, the terms “about” and “approximately” mean that dimensions, sizes, formulations, parameters, shapes and other quantities and characteristics are not and need not be exact, but can be approximated and / or greater or smaller, as desired, reflecting tolerances, conversion factors, rounding, measurement error and the like, in addition to other factors known to those skilled in the art. In general, a dimension, size, formulation, parameter, shape or other quantity or characteristic is “about” or “approximate” whether expressly stated or not as such. Note that very different sizes, shapes and dimensions can use the arrangements described.
[00482] [00482] The transition terms “comprising”, “consisting of
[00483] [00483] In some embodiments, the invention provides the method of any of the GEN 2 processes (e.g., process 2A) modified to use a small biopsy, thick needle biopsy or fine needle aspiration as the source of T cells for expansion in the first expansion of any of such GEN 2 processes, where (1) the duration of the first expansion is extended to achieve the desired cell count of TILs in the population of TILs collected after the second expansion of such a GEN 2 process or ( 2) the duration of the second expansion of such a GEN 2 process is extended to achieve the desired cell count of TILs in the population of TILs collected after the second expansion or (3) the duration of the first expansion is prolonged and the duration of the second expansion of such GEN 2 process is prolonged to achieve the desired TIL cell count in the collected TIL population
[00484] [00484] In some embodiments, the invention provides a population of TILs produced by the method of any of the GEN 2 processes (eg, process 2A) modified to use a small biopsy, thick needle biopsy or fine needle aspiration as the source of T cells for expansion in the first expansion of any of such GEN 2 processes, where (1) the duration of the first expansion is extended to achieve the desired cell count of TILs in the population of TILs collected after the second expansion of such GEN 2 process or (2) the duration of the second expansion of such a GEN 2 process is extended to achieve the desired cell count of TILs in the population of TILs collected after the second expansion or (3) the duration of the first expansion is prolonged and the duration the second expansion of such a GEN 2 process is prolonged to achieve the desired cell count of TILs in the population of TILs collected after the second expansion.
[00485] [00485] An exemplary process for TIL, known as process 2A and the small biopsy process containing some of these characteristics is shown in Figure 7, and some of the advantages of this modality of the present invention in relation to process 1C are described in Figures 5 and 6 A modality of process 2A is shown in Figure 1. In addition, an overview as well as a comparison with process 2A in relation to the exemplary process with thick needle biopsy process is provided in Figure 7. Process 2A (Gen 2) , treatment methods using TILs from the 2A process and TIL compositions prepared by the 2A process can be used in conjunction with a small needle biopsy / biopsy with lengths of expansion periods adjusted as necessary to achieve the required cell counts, as arranged below and throughout this patent application.
[00486] [00486] As discussed herein, the present invention may include a step related to the restimulation of cryopreserved TILs to increase the
[00487] [00487] In some modalities, TILs can be cryopreserved. Once thawed, they can also be stimulated to increase their metabolism before infusion into a patient.
[00488] [00488] In some embodiments, the first expansion (including processes referred to as the pre-REP as well as the processes shown in Figure 7 as Step A) is 1-19 days and the second expansion (including processes referred to as the REP as well as processes shown in Figure 7 as Step B) is shortened to 11-14 days, as discussed in detail below, as well as in the examples and figures. In some embodiments, the first expansion (for example, an expansion described as Step B in Figure 7) is divided into two periods of 1-2 days and 3-19 or, in some cases, 1-2 days and 3-10 days , and the second expansion (for example, an expansion described as step D in Figure 7) is 11-14 days, as discussed in the Examples and shown in Figures 4, 5, and 7. In some embodiments, the combination of the first expansion and second expansion (for example, expansions described as Step B and Step D in Figure 7) is shortened to 22 days, as discussed in detail below and in the examples and figures.
[00489] [00489] The “Step” Designations A, B, C, etc., below are in reference to Figure 7. The ordering of the Steps below and in Figure 7 is exemplary and any combination or order of steps, as well as additional steps, repetition of steps and / or omission of steps is covered by the present patent application and the methods described here. A. Step A: Obtain a tumor sample from the patient
[00490] [00490] In general, TILs are initially obtained from a sample of the patient's tumor ("primary TILs"), obtained by a thick needle biopsy or similar procedure, and then expanded into a larger population for future manipulation, as here described, optionally cryopreserved and optionally evaluated for phenotype and metabolic parameters.
[00491] [00491] The patient's tumor sample can be obtained by methods known in the art, usually through small biopsy, thick needle biopsy, needle biopsy or other means to obtain a sample containing a mixture of tumor and TIL cells. In general, the tumor sample can be any solid tumor, including primary tumors, invasive tumors or metastatic tumors. The tumor sample can also be a liquid tumor, such as a tumor obtained from a hematological malignancy. In some embodiments, the sample may be multiple small samples of the tumor or biopsies. In some embodiments, the sample may comprise multiple tumor samples from a single tumor from the same patient. In some embodiments, the sample may comprise multiple tumor samples from one, two, three or four tumors from the same patient. In some embodiments, the sample may comprise multiple tumor samples from multiple tumors from the same patient. The solid tumor can be of any type of cancer, including, but not limited to, breast, pancreatic, prostate, colorectal, lung, brain, kidney, stomach and skin (including, but not limited to, squamous cell carcinoma, basal cell carcinoma and melanoma ). In some modalities, cancer is selected from cervical cancer, head and neck cancer (including, for example, squamous cell carcinoma of the head and neck (CCECP)), glioblastoma (GBM), gastrointestinal cancer, ovarian cancer, sarcoma, pancreatic cancer, bladder cancer, breast cancer, triple negative breast cancer and non-small cell lung cancer (NSCLC). In some modalities, TILs
[00492] [00492] In general, the cell suspension obtained from the tumor sample or fragment is called the "primary cell population" or "recently obtained" or "recently isolated" cell population. In certain embodiments, the cell population recently obtained from TILs is exposed to a cell culture medium that comprises antigen presenting cells, IL-2 and OKT-3.
[00493] [00493] In some modalities, if the tumor is metastatic and the primary lesion has been treated / removed efficiently in the past, removal of one of the metastatic lesions may be necessary. In some modalities, the less invasive approach is to remove a skin lesion, or a lymph node in the neck or axillary area when available. In some modalities, a skin lesion or a small biopsy is removed. In some modalities, a lymph node or small biopsy is removed. In some modalities, a metastatic lung or liver injury, or an intra-abdominal or thoracic lymph node or a small biopsy of the same may be employed.
[00494] [00494] In some modalities, the tumor is a melanoma. In some embodiments, the small biopsy for a melanoma comprises a nevus (mole) or portion thereof.
[00495] [00495] In some modalities, the small biopsy is a biopsy by puncture. In some modalities, the puncture biopsy is obtained with a circular blade pressed into the skin. In some modalities, puncture biopsy is obtained with a circular blade pressed to the skin around a suspected nevus. In some modalities, the puncture biopsy is obtained with a circular blade pressed into the skin, and a round piece of skin is removed. In some modalities, the small biopsy is a puncture biopsy and the round portion of the tumor is removed.
[00496] [00496] In some modalities, the small biopsy is an excisional biopsy. In some embodiments, the small biopsy is an excisional biopsy and the entire nevus or growth is removed. In some embodiments, the small biopsy is an excisional biopsy and the entire nevus or growth is removed together with a small, normal looking skin border.
[00497] [00497] In some modalities, the small biopsy is an incisional biopsy. In some modalities, the small biopsy is an incisional biopsy and only the most irregular part of a nevus or growth is removed. In some modalities, the small biopsy is an incisional biopsy and the incisional biopsy is used when other techniques cannot be completed, such as if a suspected nevus is very large.
[00498] [00498] In some modalities, the small biopsy is a lung biopsy. In some modalities, a small biopsy is obtained by bronchoscopy. Generally, in bronchoscopy, the patient is placed under anesthesia and a small tool enters through the nose or mouth, down the throat and enters the bronchial passages, where small tools are used for some tissue removal. In some modalities, when the tumor or growth cannot be reached through bronchoscopy, a transthoracic needle biopsy can be used. Generally, for a transthoracic needle biopsy, the patient is also under anesthesia and a needle is inserted through the skin directly into the suspect site to remove a small sample of tissue. In some modalities, a transthoracic needle biopsy may require interventional radiology (for example, the use of radiographs or CT examination to orient the needle). In some modalities, the small biopsy is obtained by needle biopsy. In some modalities, the small biopsy is obtained by endoscopic ultrasound (for example, an endoscope with a light is placed through the mouth into the esophagus). In some modalities, the small biopsy is obtained
[00499] [00499] In some modalities, the small biopsy is a biopsy on the head and neck. In some modalities, the small biopsy is an incisional biopsy. In some modalities, the small biopsy is an incisional biopsy, in which a small piece of tissue is cut from an area that looks abnormal. In some modalities, if the abnormal region has easy access, the sample can be collected without hospitalization. In some modalities, if the tumor is deeper inside the mouth or throat, the biopsy may need to be performed in an operating room, under general anesthesia. In some modalities, the small biopsy is an excisional biopsy. In some modalities, the small biopsy is an excisional biopsy, in which the entire area is removed. In some modalities, the small biopsy is a fine needle aspiration (FNA). In some modalities, the small biopsy is a fine needle aspiration (FNA), in which a very fine needle attached to a syringe is used to extract (aspirate) cells from a tumor or nodule. In some modalities, the small biopsy is a puncture biopsy. In some modalities, the small biopsy is a puncture biopsy, in which puncture forceps are used to remove a piece of the suspect area.
[00500] [00500] In some modalities, the small biopsy is a cervical biopsy. In some modalities, the small biopsy is obtained through colposcopy. In general, colposcopy methods employ the use of a light magnification instrument attached to magnification binoculars (a colposcope) which is then used to biopsy a small section of the cervical surface. In some embodiments, the small biopsy is a conization / cone biopsy. In some modalities, the small biopsy is a conization / cone biopsy, in which outpatient surgery may be necessary to remove a larger piece of tissue from the cervix. In some modalities, cone biopsy, in addition to helping to
[00501] [00501] The term "solid tumor" refers to an abnormal mass of tissue that normally does not contain cysts or areas of fluid. Solid tumors can be benign or malignant. The term "solid tumor" cancer refers to solid malignant, neoplastic or cancerous tumors. Solid tumor cancers include, but are not limited to, sarcomas, carcinomas and lymphomas, such as cancers of the lung, breast, triple negative breast cancer, prostate, colon, rectum and bladder. In some modalities, cancer is selected from cervical cancer, head and neck cancer, glioblastoma, ovarian cancer, sarcoma, pancreatic cancer, bladder cancer, breast cancer, triple negative breast cancer and non-small lung cancer cells. The tissue structure of solid tumors includes interdependent tissue compartments including the parenchyma (cancer cells) and the stromal supporting cells in which the cancer cells are dispersed and which can provide a supporting microenvironment.
[00502] [00502] In some embodiments, the tumor sample is obtained as a fine needle aspirate (FNA), a thick needle biopsy, a small biopsy (including, for example, a puncture biopsy). In some modalities, the sample is placed first in G-Rex 10. In some modalities, the sample is placed first in G-Rex 10 where there are 1 or 2 samples by thick needle biopsy and / or small biopsy. In some embodiments, the sample is placed first in G-Rex 100 where 3, 4, 5, 6, 8, 9 or 10 or more biopsy samples are used with a thick needle and / or small biopsy. In some embodiments, the sample is placed first on G-Rex 500 where 3, 4, 5, 6, 8, 9 or 10 or more biopsy samples are used with a thick needle and / or small biopsy.
[00503] [00503] FNA can be obtained from a tumor selected from the group consisting of lung, melanoma, head and neck, cervical, ovarian, pancreatic, glioblastoma, colorectal and sarcoma. In some
[00504] [00504] TILs described herein can be obtained from an FNA sample. In some cases, the ANF sample is obtained from or isolated from the patient using a fine gauge needle ranging from an 18 gauge needle to a 25 gauge needle. The fine gauge needle can be 18 gauge, 19 gauge, 20 gauge, caliber 21, caliber 22, caliber 23, caliber 24 or caliber 25. In some embodiments, the patient's ANF sample may contain at least
[00505] [00505] In some cases, the TILs described here are obtained from a thick needle biopsy sample. In some cases, the thick needle biopsy sample is obtained or isolated from the patient using a surgical or clinical needle ranging from 11 gauge needle to 16 gauge needle. The needle can be 11 gauge, 12 gauge, 13 gauge, gauge 14, gauge 15 or gauge 16. In some embodiments, the patient's thick needle biopsy sample may contain at least 400,000 TILs, p. e.g. 400,000 TILs,
[00506] [00506] In general, the collected cell suspension is called the "primary cell population" or "recently collected" cell population.
[00507] [00507] In some embodiments, TILs are not obtained from tumor-digested material. In some embodiments, samples of the solid tumor are not fragmented.
[00508] [00508] In some embodiments, TILs are obtained from material digested from the tumor. In some embodiments, tumor digested material was generated by incubation in enzyme media, for example, among others, RPMI 1640, 2 mM GlutaMAX, 10 mg / mL gentamicin, 30 U / mL DNase and 1.0 mg / mL collagenase , followed by mechanical dissociation (GentleMACS, Miltenyi Biotec, Auburn, CA). After being placed in an enzyme medium, the tumor can be mechanically dissociated for approximately 1 minute. The solution can then be incubated for 30 minutes at 37 ºC in 5% CO2 and then mechanically broken again for approximately 1 minute. After being incubated again for 30 minutes at 37 ºC in 5% CO2, the tumor can be mechanically broken a third time for approximately 1 minute. In some modalities, after the third mechanical rupture, if large pieces of tissue were present, 1 or 2 additional mechanical dissociations were applied to the sample, with or without an additional 30 minutes of incubation at 37 ºC in 5% CO2. In some embodiments, at the end of the final incubation if the cell suspension contained a large number of erythrocytes or dead cells, a density gradient separation, using Ficoll, could be performed to remove those cells.
[00509] [00509] In some modalities, the cell suspension collected, before the first expansion stage, is called "primary cell population" or "recently collected" cell population.
[00510] [00510] In some embodiments, the cells can be optionally frozen, after collection in the sample, and frozen before being admitted to the expansion described in step B, which is described in more detail below, as well as exemplified in Figure 7. B. Step B : First expansion
[00511] [00511] In some modalities, the present methods allow to obtain young TILs, which are capable of increased cycles of replication when administered to an individual / patient and, therefore, can
[00512] [00512] The various T and B lymphocyte antigen receptors are produced by somatic recombination of a limited but large number of gene segments. These gene segments: V (variable), D (diversity), J (junction) and C (constant), determine the specificity of binding and applications downstream of immunoglobulins and T cell receptors (TCRs). The present invention provides a method for generating TILs that exhibit and increase the diversity of the T cell repertoire. In some embodiments, the TILs obtained by the present method exhibit an increase in the diversity of the T cell repertoire. In some embodiments, the TILs obtained by the present method show an increase in the diversity of the T cell repertoire when compared to recently collected TILs and / or TILs prepared using methods other than those provided here, for example, methods other than those incorporated in Figure 1. In some modalities, the TILs obtained by the present method exhibit an increase in the diversity of the T cell repertoire when compared to recently collected TILs and / or TILs prepared using methods referred to as process 1C, as exemplified in Figure 3 and / or Figure 4. In some modalities, TILs
[00513] [00513] After obtaining the tumor fragment or fragments by small biopsy, thick needle biopsy or fine needle aspiration (which can be referred to as "samples" or "fragments"), for example, as described in step A of Figure 7, the resulting cells are cultured in serum containing IL-2 under conditions that favor the growth of TILs more than tumor and other cells. In some embodiments, the digested tumor material is incubated in 2 ml wells in medium comprising inactivated human AB serum with 6000 IU / ml IL-2. This primary cell population is cultured for a period of days, usually 3 to 14 days, resulting in a population of bulk TILs, usually close to 1 x 108 bulk TIL cells. In some embodiments, this primary cell population is cultured for a period of 7 to 14 days, resulting in a population of bulk TILs, usually close to 1 x 108 bulk TIL cells. In some modalities, this population of primary cells is cultivated for a period of 1 to 149 days, resulting in a
[00514] [00514] In modalities in which TIL cultures are started in 24-well plates, for example, using a Costar flat-bottom cell culture plate with 24 wells (Corning Incorporated, Corning, NY, each well can be seeded with 1 x 106 small biopsy tumor cells, thick needle biopsy or fine needle aspiration or a tumor fragment by small biopsy, thick needle biopsy or fine needle aspiration in 2 mL of complete medium (CM) with IL-2 (6000 IU / mL; Chiron Corp., Emeryville, CA).
[00515] [00515] In some modalities, the culture medium of the first expansion is referred to as "CM", an abbreviation for culture medium. In some modalities, CM for Step B consists of RPMI 1640 with GlutaMAX, supplemented with 10% AB human serum, 25 mM Hepes and 10 mg / mL gentamicin. In modalities in which cultures are started in gas-permeable flasks with 40 mL capacity and 10 cm2 gas-permeable silicone bottom (eg, G-Rex10; Wilson Wolf Manufacturing, New Brighton, MN) (Figure 1), each flask was loaded with 10–40 x 106 viable small biopsy tumor cells, thick needle biopsy or fine needle aspiration or 5-30 small biopsy tumor fragments, thick needle biopsy or fine needle aspiration in 10-40 ml of CM with IL-2. Both G-Rex10 and 24-well plates were incubated in a humidified incubator at 37 ºC in 5% CO2 and, 5 days after the start of culture, half of the medium was removed and replaced with fresh CM and IL-2 and , after 5 days, half of the medium was changed every 2–3 days.
[00516] [00516] After collection in tumor fragments from the small biopsy, thick needle biopsy or fine needle aspiration, the resulting cells (ie fragments) are cultured in serum containing IL-2 under conditions that favor the growth of TILs more than than tumor cells and other cells. In some embodiments, the tumor fragments are incubated in cavities
[00517] [00517] In some embodiments, the first expansion is performed by further supplementing the cell culture medium of the second population of TILs with OKT-3, IL-15, OX40 agonist antibody and / or 4-1BB agonist antibody. For example, the cell culture medium of the second population of TILs is supplemented with OKT3, IL-15, an OX40 agonist antibody, a 4-1BB agonist antibody, a combination of OKT3 and IL-15, a combination of OKT3 and an OX40 agonist antibody, a combination of OKT3 and a 4-1BB agonist antibody, a combination of IL-15 and an OX40 agonist antibody, a combination of IL-15 and a 4-1BB agonist antibody, a combination of an OX40 agonist antibody and a 4-1BB agonist antibody, a combination of OKT3, IL-15 and an OX40 agonist antibody, a combination of OKT3, IL-15 and a 4-1BB agonist antibody, a combination of OKT3 , an antibody
[00518] [00518] After obtaining the tumor fragments by small biopsy, thick needle biopsy or fine needle aspiration, for example, as described in step A of Figure 7A, the resulting cells are cultured in serum containing IL-2 under conditions that favor more growth of TILs than tumor and other cells. In some embodiments, tumor fragments are incubated in 2 ml wells in medium comprising inactivated human AB serum with 6000 IU / ml IL-2. This primary cell population is cultured for a period of days, usually from 1 to 19 days, resulting in a TIL population of at least 5 x 107 (that is, 50 million) TIL cells in at least 11 to 19 days. In some embodiments, this primary cell population is cultured for a period of 1 to 18 days, resulting in a population of bulk TILs of at least 5 x 10 7 (that is, 50 million) TIL cells in at least 18 days. In some embodiments, this primary cell population is cultured for a period of 1 to 17 days, resulting in a bulk TIL population of at least 5 x 10 7 (that is, 50 million) TIL cells in at least 17 days. In some embodiments, this primary cell population is cultured for a period of 1 to 16 days, resulting in a population of bulk TILs of at least 5 x 10 7 (that is, 50 million) TIL cells in at least 16 days. In some embodiments, this primary cell population is cultured for a period of 1 to 15 days, resulting in a bulk TIL population of at least 5 x 10 7 (that is, 50 million) TIL cells in at least 15 days. In some embodiments, this primary cell population is grown for a period of 1 to 14 days, resulting in a population of bulk TILs of at least
[00519] [00519] In a preferred embodiment, the expansion of TILs can be performed using an initial step of expanding TILs in bulk (for example, such as those described in step B of Figure 7, which may include processes referred to as pre-REP) as described below and here, followed by a second expansion (Step D, including processes referred to as Rapid Expansion Protocol (REP) steps), as described below under Step D and here, followed by optional cryopreservation and followed by a second Step D (including processes referred to as REP steps with restimulation) as described below and here. The TILs obtained from this process can be optionally classified according to phenotypic characteristics and metabolic parameters as described here.
[00520] [00520] In some modalities, the culture medium of the first expansion is referred to as "CM", an abbreviation for culture medium. In some modalities, CM for Stage B consists of RPMI 1640 with GlutaMAX, supplemented with 10% human AB serum, 25 mM Hepes, and
[00521] [00521] The fragments or small biopsy samples are cultured in serum containing IL-2 under conditions that favor the growth of TILs more than the tumor and other cells. In some embodiments, fragments or small biopsy samples are incubated in 2 mL wells in medium comprising inactivated human AB serum (or, in some cases, as outlined here, in the presence of aAPC cell population) with 6000 IU / mL of IL-2. This primary cell population is cultivated over a period of days, usually from 1 to 19 days, resulting in a population of bulk TILs, of at least approximately 5x107 TIL cells on day 11 and, in some modalities, 1x108 TILs on day 19 to 28. In some embodiments, the growth medium during the first expansion comprises IL-2 or a variant thereof. In some modalities, IL is IL-2
[00522] [00522] In some embodiments, the culture medium of the first expansion comprises about 500 IU / ml IL-15, about 400 IU / ml IL-15, about 300 IU / ml IL-15, about 200 IU / ml IL-15, about 180 IU / ml IL-15, about 160 IU / ml IL-15, about 140 IU / ml IL-15, about 120 IU / ml IL -15 or about 100 IU / ml IL-15. In some embodiments, the first expansion culture medium comprises about 500 IU / ml IL-15 to 100 IU / ml IL-15. In some embodiments, the first expansion culture medium comprises about 400 IU / ml IL-15 to 100 IU / ml IL-15. In some embodiments, the first expansion culture medium comprises about 300 IU / ml IL-15 to 100 IU / ml IL-15. In some embodiments, the first expansion culture medium comprises about 200 IU / ml of IL-15. In some embodiments, the cell culture medium comprises about 180 IU / ml IL-15. In one embodiment, the cell culture medium also comprises
[00523] [00523] In some embodiments, the culture medium of the first expansion comprises approximately 20 IU / mL of IL-21,
[00524] [00524] In one embodiment, the cell culture medium comprises OKT-3 antibody. In some embodiments, the cell culture medium comprises approximately 30 ng / ml of OKT-3 antibody. In one embodiment, the cell culture medium comprises approximately 0.1 ng / mL, approximately 0.5 ng / mL, approximately 1 ng / mL, approximately 2.5 ng / mL, approximately 5 ng / mL, approximately
[00525] [00525] In some embodiments, the cell culture medium comprises one or more TNFRSF agonists in a culture medium
[00526] [00526] In some embodiments, in addition to one or more TNFRSF agonists, the cell culture medium further comprises IL-2 at an initial concentration of approximately 3000 IU / mL and OKT-3 antibody at an initial concentration of approximately 30 ng / ml, and wherein one or more of the TNFRSF agonists comprises a 4-1BB agonist.
[00527] [00527] In some modalities, the culture medium of the first expansion is referred to as "CM", an abbreviation for culture medium. In some embodiments, it is referred to as CM1 (culture medium 1). In some modalities, CM consists of RPMI 1640 with GlutaMAX, supplemented with 10% human AB serum, 25 mM Hepes and 10 mg / mL gentamicin. In modalities in which cultures are started in gas-permeable flasks with 40 mL capacity and 10 cm2 gas-permeable silicone bottom (eg, G-Rex10; Wilson Wolf Manufacturing, New Brighton, MN). In some embodiments, the CM is the CM1 described in the Examples, see, Example 1 In some embodiments, the first expansion occurs in an initial cell culture medium or a first cell culture medium. In some embodiments, the initial cell culture medium or the first cell culture medium comprises IL-2.
[00528] [00528] In some modalities, the process of the first expansion
[00529] [00529] In some modalities, the first expansion of TILs can proceed for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days , 13 days, 14 days, 14 days, 14 days, 14 days, 14 days, 14 days, in order to obtain at least 5 x 107 (ie 50 million) TILs. In some modalities, the first expansion of TILs can proceed for 1 days to 19 days, in order to obtain at least 5 x 107 (ie 50 million) TILs. In some modalities, the first expansion of TILs can proceed for 2 days to 19 days, in order to obtain at least 5 x 107 (that is, 50 million) TILs. In some modalities, the first expansion of TILs can proceed for 3 days to 19 days, in order to obtain at least 5 x 107 (ie 50 million) TILs. In some modalities, the first expansion of TILs can proceed for 6 days to 19 days, in order to obtain at least 5 x 107 (that is, 50 million) TILs. In some modalities, the first expansion of TILs can proceed for 7 days to 19 days, in order to obtain at least 5 x 107 (that is, 50 million) TILs. In some modalities, the first expansion of TILs can proceed for 8 days to 19 days, in order to obtain at least 5 x 107 (that is, 50 million) TILs. In some modalities, the first expansion of TILs can proceed from 9 days to 19 days, in order to obtain at least 5 x 107 (ie 50 million) TILs. In
[00530] [00530] In some embodiments, a combination of IL-2, IL-7, IL-15 and / or IL-21 is employed as a combination during the first expansion. In some embodiments, IL-2, IL-7, IL-15 and / or IL-21 as well as any combinations thereof can be included during the first expansion, including, for example, during Step B processes according to the Figure 7, as well as described herein. In some embodiments, a combination of IL-2, IL-15 and IL-21 is used as a combination during the first expansion. In some modalities, IL-2, IL-15 and IL-21 as well as any combinations of them may be included during
[00531] [00531] In some modalities, the first expansion, for example, Stage B according to Figure 7, is carried out in a closed system bioreactor. In some embodiments, a closed system is employed for the expansion of TIL, as described here. In some modalities, a single bioreactor is employed. In some modalities, the only bioreactor employed is, for example, a GREX-10 or a GREX-100. In some embodiments, the closed system bioreactor is a unique bioreactor.
[00532] [00532] In some modalities, the first TIL expansion generates at least 50 x 106 TILs, p. 50 x 106 (i.e. 5 x 107), 55 x 106, 60 x 106, 65 x 106, 70 x 106, 75 x 106, 80 x 106, 85 x 106, 90 x 106, 95 x 106 , 100 x 106, 105 x 106, 110 x 106, 115 x 106, 120 x 106, 125 x 106, 150 x 106, 200 x 106, 300 x 106, 400 x 106 or more. C. Step C: Transition from the first expansion to the second expansion
[00533] [00533] In some cases, the population of bulk TILs obtained in the first expansion, including for example, the population of TILs obtained, for example, in Step B as indicated in Figure 7, can be cryopreserved immediately, using the protocols discussed below . Alternatively, the population of TILs obtained in the first expansion, referred to as the second population of TILs, can be subjected to a second expansion (which may include expansions sometimes referred to as REP) and then cryopreserved as discussed below. Likewise, if genetically modified TILs are to be used in therapy, the first population of TILs (sometimes
[00534] [00534] In some modalities, the TILs obtained in the first expansion (for example, in Step B as indicated in Figure 7) are stored until phenotyped for selection. In some embodiments, the TILs obtained in the first expansion (for example, in Step B as shown in Figure 7) are not stored and proceed directly to the second expansion. In some modalities, TILs obtained in the first expansion are not cryopreserved after the first expansion and before the second expansion. In some modalities, the transition from the first expansion to the second expansion occurs in approximately 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, or 19 days since the fragments are added to the culture medium for first expansion. In some modalities, the transition from the first expansion to the second expansion occurs in approximately 3 days to 19 days from when the fragments are added to the culture medium for the first expansion. In some embodiments, the transition from the first expansion to the second expansion occurs in approximately 4 days to 19 days from when the fragments are added to the culture medium for the first expansion. In some embodiments, the transition from the first expansion to the second expansion occurs in approximately 4 days to 19 days from when the fragments are added to the culture medium for the first expansion. In some embodiments, the transition from the first expansion to the second expansion occurs in approximately 7 days to 19 days since the fragments are added to the culture medium for the first expansion. In some modalities, the transition from the first
[00535] [00535] In some modalities, TILs are not stored after the first expansion and before the second expansion, and the TILs continue
[00536] [00536] In some modalities, the transition from the first expansion to the second expansion, for example, Step C according to Figure 7, is carried out in a closed system bioreactor. In some embodiments, a closed system is employed for the expansion of TIL, as described here. In some modalities, a single bioreactor is employed. In some modalities, the only bioreactor employed is, for example, a GREX-10 or a GREX-100. In some embodiments, the closed system bioreactor is a unique bioreactor. D. Step D: Second expansion
[00537] [00537] In some modalities, the cell population of TILs has the number expanded after the collection and initial bulk processing, for example, after Step A and Step B, and the transition referred to as Step C, as shown in Figure 7 ) [sic]. This additional expansion is referred to as the second expansion, which may include expansion processes generally referred to in the art as a rapid expansion process (REP; as well as processes indicated in step D of Figure 7). The second expansion is usually performed using a culture medium that comprises some components, including feeder cells, a source of cytokines and an anti-CD3 antibody, in a gas-permeable container.
[00538] [00538] In some embodiments, the second expansion or second expansion of TILs (which may include expansions sometimes referred to as REP; as well as processes as indicated in step D of Figure 7) can be performed using any known TIL flasks or containers hair
[00539] [00539] In one embodiment, the second expansion can be performed in a gas-permeable container by the methods of the present invention (including, for example, expansions referred to as REP; as well as processes as indicated in step D of Figure 7). For example, TILs can be expanded rapidly using non-specific stimulation of T cell receptors in the presence of interleukin-2 (IL-2) or interleukin-15 (IL-15). The non-specific stimulation of T cell receptors may include, for example, about 30 ng / mL of OKT3, an anti-CD3 mouse monoclonal antibody (commercially available from Ortho-McNeil, Raritan, NJ or Miltenyi Biotech, Auburn, CA ). TILs can be expanded to induce additional stimulation of TILs in vitro including one or more antigens during the second expansion, including antigenic portions thereof, such as cancer epitope (s), which can optionally be expressed from a vector, such as a human leukocyte antigen-binding peptide A2 (HLA-A2), e.g. eg MART-1 0.3 μΜ: 26-35 (27 L) or gpl 00: 209-217 (210M), optionally in the presence of a T cell growth factor, such as IL-2 or IL-15 300 IU / mL. Other suitable antigens may include, e.g. e.g., NY-ESO-1, TRP-1, TRP-2, tyrosinase cancer antigen, MAGE-A3, SSX-2 and VEGFR2, or portions
[00540] [00540] In one embodiment, the cell culture medium further comprises IL-2. In some embodiments, the cell culture medium comprises about 3000 IU / ml of IL-2. In one embodiment, the cell culture medium comprises about 1000 IU / mL, about 1500 IU / mL, about 2000 IU / mL, about 2500 IU / mL, about 3000 IU / mL, about 3500 IU / mL. ml, about 4000 IU / ml, about 4500 IU / ml, about 5000 IU / ml, about 5500 IU / ml, about 6000 IU / ml, about 6500 IU / ml, about 7000 IU / ml , about 7500 IU / ml or about 8000 IU / ml IL-2. In one embodiment, the cell culture medium comprises between 1000 and 2000 IU / mL, between 2000 and 3000 IU / mL, between 3000 and 4000 IU / mL, between 4000 and 5000 IU / mL, between 5000 and 6000 IU / mL, between 6000 and 7000 IU / mL, between 7000 and 8000 IU / mL or between 8000 IU / mL of IL-2.
[00541] [00541] In one embodiment, the cell culture medium comprises OKT3 antibody. In some embodiments, the cell culture medium comprises about 30 ng / ml of OKT3 antibody. In one embodiment, the cell culture medium comprises approximately 0.1 ng / mL, approximately 0.5 ng / mL, approximately 1 ng / mL, approximately
[00542] [00542] In some embodiments, a combination of IL-2, IL-7, IL-15 and / or IL-21 is employed as a combination during the second expansion. In some embodiments, IL-2, IL-7, IL-15 and / or IL-21 as well as any combinations thereof can be included during the second expansion, including, for example, during Step D processes according to the Figure 7, as well as described herein. In some embodiments, a combination of IL-2, IL-15 and IL-21 is employed as a combination during the second expansion. In some embodiments, IL-2, IL-15 and IL-21 as well as any combinations thereof can be included during Step D processes according to Figure 7 and as described herein.
[00543] [00543] In some embodiments, the second expansion can be conducted in a supplemented cell culture medium comprising IL-2, OKT-3 and antigen presenting feeder cells. In some embodiments, the second expansion occurs in a supplemented cell culture medium. In some embodiments, the supplemented cell culture medium comprises IL-2, OKT-3 and antigen-presenting feeder cells. In some modalities, the second means of
[00544] [00544] In some embodiments, the second expansion is performed by further supplementing the cell culture medium of the TIL population in the second expansion with IL-15, OX40 agonist antibody and / or 4-1BB agonist antibody (sometimes referred to as the second population of TILs). For example, the cell culture medium of the TIL population in the second expansion is supplemented with IL-15, an OX40 agonist antibody, a 4-1BB agonist antibody, a combination of IL-15 and an OX40 agonist antibody, an combination of IL-15 and a 4- 1BB agonist antibody, a combination of an OX40 agonist antibody and a 4-1BB agonist antibody, a combination of IL-15, an OX40 agonist antibody and a 4- agonist antibody 1BB, and any combinations thereof. In some embodiments, OX40 is included when the fragments are from an ovarian tumor (eg, of ovarian origin). In some embodiments, OKT-3 is added during the first and / or second expansion. In some modalities, OKT-3 is added on day-3, along with antigen presenting feeder cells (APCs). In some modalities, when the sample is from an FNA, OKT-3 is added on day-1, together with antigen presenting feeder cells (APCs).
[00545] [00545] In some embodiments, the antigen presenting feeder cells (APCs) are PBMCs. In one embodiment, the ratio of TILs to PBMCs and / or antigen presenting cells in the rapid expansion and / or the second expansion is approximately 1 to 25, approximately 1 to 50, approximately 1 to 100, approximately
[00546] [00546] In one embodiment, REP and / or the second expansion is performed in vials, with the bulk TILs being mixed with 100 or 200 times excess of inactivated feeder cells, anti-CD3 antibody OKT3 30 mg / mL and IL-2 3000 IU / mL in 150 mL of medium. The medium is replaced (usually 2/3 of the medium is replaced by breathing with fresh medium) until the cells are transferred to an alternative growth chamber. Alternative growth chambers include GREX flasks and gas-permeable containers as discussed more fully below.
[00547] [00547] In some modalities, the second expansion (which may include processes referred to as the REP process) is 11-14 days, as discussed in the examples and figures. In some modalities, the second expansion is 11 days. In some modalities, the second expansion is 12 days. In some modalities, the second expansion is 13 days. In some modalities, the second expansion is e 14 days.
[00548] [00548] In one embodiment, the REP and / or the second expansion can be performed using T-175 flasks and gas-permeable bags as described previously (Tran, et al., J. Immunother. 2008, 31, 742-51; Dudley, et al., J. Immunother. 2003, 26, 332-42) or gas-permeable culture tools
[00549] [00549] In one embodiment, the second expansion (which may include expansions referred to as REP, as well as those referred to in step D of Figure 7) can be performed in gas-permeable flasks with a capacity of 500 mL and a gas-permeable silicone bottom 100 cm (G-Rex 100, commercially available from Wilson Wolf Manufacturing Corporation, New Brighton, MN, USA), 5 x 106 or 10 x 106 TILs can be grown with PBMCs in 400 mL of 50/50 medium, supplemented with serum 5% human AB, 3000 IU per ml of IL-2 and 30 ng per ml of anti-CD3 (OKT3). G-Rex 100 flasks can be incubated at 37 ºC in 5% CO2. On day 5, 250 mL of supernatant can be removed and placed in centrifuge bottles and centrifuged at 1500 rpm (491 x g) for 10 minutes. TIL pellets can be resuspended with 150 ml of fresh medium with 5% human AB serum, 3000 IU per ml of IL-2, and added back to the original 10 G-Rex vials. When TILs are expanded serially in G-Rex 100 flasks on day 7, the TILs in each G-Rex 100 can be suspended in the 300 mL of medium present in each flask, and the cell suspension can be divided into
[00550] [00550] In one embodiment, the second expansion (including expansions referred to as REP) is performed in flasks, with the bulk TILs being mixed with 100 or 200 times excess inactivated feeder cells, anti-CD3 antibody OKT3 30 mg / mL and IL-2 3000 IU / ml in 150 ml of medium. In some embodiments, the medium is replaced until the cells are transferred to an alternative growth chamber. In some modalities, 2/3 of the medium is replaced by aspiration of the exhausted medium and replacement with an equivalent volume of fresh medium. In some embodiments, alternative growth chambers include GREX flasks and gas-permeable containers as discussed more fully below.
[00551] [00551] In one embodiment, the second expansion (including expansions referred to as REP) is performed and also comprises a stage in which TILs are selected for superior reactivity with the tumor. Any selection method known in the art can be used. For example, the methods described in U.S. Patent Application Publication No. 2016/0010058 A1, the content of which is incorporated herein by reference, can be used for the selection of TILs by superior reactivity with the tumor.
[00552] [00552] Optionally, a cell viability assay can be performed after the second expansion (including expansions referred to as
[00553] [00553] In some embodiments, the second expansion (including expansions referred to as REP) of TILs can be performed using T-175 flasks and gas-permeable bags as previously described (Tran KQ, Zhou J, Durflinger KH, et al., 2008 , J Immunother., 31: 742–751, and Dudley ME, Wunderlich JR, Shelton TE, et al. 2003, J Immunother., 26: 332-342) or gas-permeable G-Rex flasks. In some embodiments, the second expansion is performed using bottles. In some embodiments, the second expansion is performed using gas-permeable G-Rex flasks. In some embodiments, the second expansion is performed in T-175 flasks, and about 1 x 106 TIL are suspended in approximately 150 ml of medium that are added to each T-175 flask. TILs are grown with irradiated allogeneic PBMC (50 Gy) as "feeder" cells in a 1 to 100 ratio and the cells were grown in a 1 to 1 mixture of CM and AIM-V medium (50/50 medium), supplemented with 3000 IU / ml IL-2 and 30 ng / ml anti-CD3. The T-175 flasks are incubated at 37 ºC in 5% CO2. In some modalities, half of the medium is changed in 5 days, using the 50/50 medium with 3000 IU / mL of IL-2. In some embodiments, on day 7, cells from 2 T-175 vials are combined in a 3 L pouch, and 300 mL of AIM-V with 5% human AB serum and 3000 IU / mL of IL-2 are added to the 300 mL of the TILs suspension. The number of cells in each bag can be counted every day or two days and fresh medium can be added to maintain
[00554] [00554] In some modalities, the second expansion (including expansions referred to as REP) are carried out in flasks with a capacity of 500 mL and a 100 cm2 gas-permeable silicone bottom (G-Rex100, Wilson Wolf) (Figure 1), approximately 5 x 106 or 10 x 106 TILs are grown with allogeneic PBMC irradiated in a 1 to 100 ratio in 400 ml of 50/50 medium, supplemented with 3000 IU / ml IL-2 and 30 ng / ml anti-CD3. The G-Rex100 flasks are incubated at 37 ºC in 5% CO2. In some embodiments, on day 5, 250 mL of supernatant is removed and placed in centrifuge bottles and centrifuged at 1500 rpm (491 x g) for 10 minutes. The TIL pellets can then be resuspended with 150 ml of fresh 50/50 medium with 3000 IU / ml IL-2 and added back to the original G-Rex100 vials. In modalities in which TILs are expanded serially in G-Rex100 flasks, on day 7, the TILs in each G-Rex100 are suspended in the 300 ml of medium present in each flask, and the cell suspension was divided into three 100 ml aliquots that are used to seed 3 G-Rex100 flasks. Then, 150 ml of AIM-V with 5% human AB serum and 3000 IU / ml IL-2 are added to each vial. The G-Rex100 flasks are incubated at 37 ºC in 5% CO2 and, after 4 days, 150 ml of AIM-V with 3000 IU / ml IL-2 are added to each G-Rex100 flask. The cells are collected in 14 days of culture. In some embodiments, cultures are expanded on a G-Rex 500.
[00555] [00555] In some embodiments, the second expansion culture medium (eg, sometimes referred to as CM2 or the second cell culture medium) comprises IL-2, OKT-3, as well as antigen presenting feeder cells (APCs), as discussed in more detail below.
[00556] [00556] In some modalities, the second expansion, for example, the
[00557] [00557] In one embodiment, the second expansion procedures described here (for example, including expansion such as those described in step D of Figure 7, as well as those referred to as REP) require an excess of feeder cells during REP expansion of TILs and / or during the second expansion. In many embodiments, feeder cells are peripheral blood mononuclear cells (PBMCs) obtained from standard whole blood units from healthy blood donors. PBMCs are obtained using standard methods such as Ficoll-Paque gradient separation.
[00558] [00558] In general, allogeneic PBMCs are inactivated, either by irradiation or heat treatment, and used in REP procedures, as described in the examples, especially in Example 4, which provides an exemplary protocol for assessing incompetence in replicating PBMCs allogeneic rays.
[00559] [00559] In some embodiments, PBMCs are considered incompetent for replication and accepted for use in the TIL expansion procedures described here if the total number of viable cells on day 14 is less than the initial number of viable cells placed in culture on day 0 of the REP and / or day 0 of the second expansion (ie, the day of the start of the second expansion). See, for example, Examples 3 and / or 4.
[00560] [00560] In some modalities, PBMCs are considered incompetent for replication and accepted for use in
[00561] [00561] In some modalities, PBMCs are considered incompetent for replication and accepted for use in the TILs expansion procedures described here if the total number of viable cells, cultured in the presence of OKT3 and IL-2, on day 7 and day 14, has not increased since the initial number of viable cells cultured on day 0 of the REP and / or day 0 of the second expansion (ie, the day of the start of the second expansion). In some embodiments, PBMCs are cultured in the presence of OKT3 antibody 5-60 ng / mL and IL-2 1000-6000 IU / mL. In some embodiments, PBMCs are cultured in the presence of OKT3 antibody 10-50 ng / mL and IL-2 2000-5000 IU / mL. In some embodiments, PBMCs are cultured in the presence of OKT3 antibody 20-40 ng / mL and IL-2 2000-4000 IU / mL. In some embodiments, PBMCs are cultured in the presence of OKT3 antibody 25-35 ng / mL and IL-2 2500-3500 IU / mL.
[00562] [00562] In some embodiments, the antigen presenting feeder cells are PBMCs. In some embodiments, antigen-presenting feeder cells are artificial antigen-presenting feeder cells. In one embodiment, the ratio of TILs to antigen presenting feeder cells in the second expansion is approximately 1 to 25, approximately 1 to 50, approximately 1 to 100, approximately 1 to 125, approximately 1 to 150, approximately 1 to 175, approximately 1 for 200, approximately 1 to 225, approximately 1 to 250,
[00563] [00563] In one embodiment, the second expansion procedures described here require an excess of feeder cells during the second expansion. In many embodiments, feeder cells are peripheral blood mononuclear cells (PBMCs) obtained from standard whole blood units from healthy blood donors. PBMCs are obtained using standard methods such as Ficoll-Paque gradient separation. In one embodiment, artificial antigen presenting cells (aAPC) are used in place of PBMCs.
[00564] [00564] In general, allogeneic PBMCs are inactivated, either by irradiation or heat treatment, and used in the TIL expansion procedures described here, including the exemplary procedures described in, for example, Figure 7.
[00565] [00565] In one embodiment, artificial antigen presenting cells are used in the second expansion in replacement or in combination with PBMCs.
[00566] [00566] The expansion methods described herein generally use culture media with high doses of a cytokine, in particular IL-2, as is known in the art.
[00567] [00567] Alternatively, the use of combinations of cytokines for rapid expansion and / or the second expansion of TILs is also possible, with combinations of two or more among IL-2, IL-15 and IL-21 as is in general
[00568] [00568] After the second expansion step, the cells can be collected. In some modalities, TILs are collected after one, two, three, four or more expansion steps, for example, as shown in Figure 7. In some modalities, TILs are collected after two expansion steps, for example, as shown in Figure 7.
[00569] [00569] TILs can be collected in any suitable and sterile manner, including for example, by centrifugation. Methods for collecting TILs are well known in the art and any of such known methods can be employed with the present process. In some modalities, TILS are collected using an automatic system.
[00570] [00570] In some modalities, the collection, for example, Step E according to Figure 7, is carried out from a closed system bioreactor. In some embodiments, a closed system is employed for the expansion of TIL, as described here. In some modalities, a single bioreactor is employed. In some modalities, the only bioreactor employed is, for example, a GREX-10 or a GREX-100. In some embodiments, the closed system bioreactor is a unique bioreactor. F. Step F: Final formulation / Transfer to infusion bag
[00571] [00571] After completing Steps A through E, as arranged in an exemplary order in Figure 7 and as outlined in detail above and here, the cells are transferred to a container for use in administration
[00572] [00572] In one embodiment, TILs expanded by the processes of the present invention are administered to a patient as a pharmaceutical composition. In one embodiment, the pharmaceutical composition is a suspension of TILs in a sterile buffer. TILs expanded by the processes of the present invention can be administered by any suitable route known in the art. In some embodiments, T cells are administered as a single intra-arterial or intravenous infusion, which preferably lasts approximately 30 to 60 minutes. Other suitable routes of administration include intraperitoneal, intrathecal and intralymphatic.
[00573] [00573] In one embodiment, TILs expanded by the processes described above can be administered as compositions that further comprise a cryopreservative. In one embodiment, TILs expanded by the processes described above can be administered as compositions that further comprise a cryopreservative and an isotonic agent. In one embodiment, TILs expanded by the processes described above can be administered as compositions which further comprise a cryopreservative, comprising dimethyl sulfoxide, and an isotonic agent, comprising sodium chloride, sodium gluconate and sodium acetate. In one embodiment, TILs expanded by the processes described above can be administered as compositions which further comprise a cryopreservative, comprising dimethyl sulfoxide and dextran 40, and an isotonic agent comprising sodium chloride, sodium gluconate and sodium acetate. In one embodiment, TILs expanded by the processes described above can be administered as compositions delivered in a sterile infusion pouch, such as compositions that further comprise a cryopreservative,
[00574] [00574] In one embodiment, TILs expanded by the methods of the present invention are administered to a patient as a pharmaceutical composition. In one embodiment, the pharmaceutical composition is a suspension of TILs in a sterile buffer. TILs expanded by the processes of the present invention can be administered by any suitable route known in the art. In some embodiments, the T cells are administered in a single intra-arterial or intravenous infusion, which preferably lasts approximately 30 to 60 minutes. Other suitable routes of administration include intraperitoneal, intrathecal and intralymphatic.
[00575] [00575] Any suitable dose of TILs can be administered. In some embodiments, a therapeutically sufficient number of TILs is required for an adequate dose. In some modalities, approximately 2.3x1010 to 13.7x1010 TILs are administered, on average, around 7.8x1010 TILs, especially if the cancer is melanoma. In one embodiment, approximately 1.2x1010 to 4.3x1010 TILs are administered. In some embodiments, approximately 3x1010 to approximately 12x1010 TILs are administered. In some embodiments, approximately 4x1010 to approximately 10x1010 TILs are administered. In some embodiments, approximately 5x1010 to approximately 8x1010 TILs are administered. In some embodiments, approximately 6x1010 to approximately 8x1010 TILs are administered. In some embodiments, approximately 7x1010 to approximately 8x1010 TILs are administered. In some embodiments, the therapeutically effective dose is approximately 2.3x1010 to 13.7x1010. In some embodiments, the therapeutically effective dose is approximately 7.8x1010 TILs, especially if the cancer is melanoma. In some embodiments, the therapeutically effective dose is
[00576] [00576] In some embodiments, the number of TILs provided in the pharmaceutical compositions of the invention is approximately 1x106, 2x106, 3x106, 4x106, 5x106, 6x106, 7x106, 8x106, 9x106, 1x107, 2x107, 3x107, 4x107, 5x107, 6x107, 7x107 , 8x107, 9x107, 1x108, 2x108, 3x108, 4x108, 5x108, 6x108, 7x108, 8x108, 9x108, 1x109, 2x109, 3x109, 4x109, 5x109, 6x109, 7x109, 8x109, 9x109, 1x1010, 2x1010, 3x1010, 4x10, 3x1010, 4x10, 3x1010, 4x10, , 6x1010, 7x1010, 8x1010, 9x1010, 1x1011, 2x1011, 3x1011, 4x1011, 5x1011, 6x1011, 7x1011, 8x1011, 9x1011, 1x1012, 2x1012, 3x1012, 4x1012, 5x1012, 6x1012, 7x1012, 8x1012, 9x1012, 1x1013, 2x10, 1x1013, 2x10, , 4x1013, 5x1013, 6x1013, 7x1013, 8x1013 and 9x1013. In one embodiment, the number of TILs provided in the pharmaceutical compositions of the invention is in the range of 1x106 to 5x106, 5x106 to 1x107, 1x107 to 5x107, 5x107 to 1x108, 1x108 to 5x108, 5x108 to 1x109, 1x109 to 5x109, 5x109 to 1x1010, 1x1010 to 5x1010, 5x1010 to 1x1011, 5x1011 to 1x1012, 1x1012 to 5x1012 and 5x1012 to 1x1013. In some embodiments, the therapeutically effective dose is approximately 1x106, 2x106, 3x106, 4x106, 5x106, 6x106, 7x106, 8x106, 9x106, 1x107, 2x107, 3x107, 4x107, 5x107, 6x107, 7x107, 8x107, 9x107, 1x108, 2x108, 2x108, 3x108, 4x108, 5x108, 6x108, 7x108, 8x108, 9x108, 1x109, 2x109, 3x109, 4x109, 5x109, 6x109, 7x109, 8x109, 9x109, 1x1010, 2x1010, 3x1010, 4x1010, 5x1010, 6x1010, 7x1010, 8x1010, 7x1010, 8x1010, 1x1011, 2x1011, 3x1011, 4x1011, 5x1011, 6x1011, 7x1011, 8x1011, 9x1011, 1x1012, 2x1012, 3x1012, 4x1012, 5x1012, 6x1012, 7x1012, 8x1012, 9x1012, 1x1013, 2x1013, 3x1013, 4x1013, 5x1013, 6x1013, 7x10 8x1013 and 9x1013.
[00577] [00577] In some embodiments, the concentration of TILs provided in the pharmaceutical compositions of the invention is less than, for example, 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20% , 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3 %, 2%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07%, 0, 06%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002 %, 0.001%, 0.0009%, 0.0008%, 0.0007%, 0.0006%, 0.0005%, 0.0004%, 0.0003%, 0.0002% or 0.0001% p / w, w / v or v / v of the pharmaceutical composition.
[00578] [00578] In some embodiments, the concentration of TILs provided in the pharmaceutical compositions of the invention is greater than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 19.75%, 19 , 50%, 19.25% 19%, 18.75%, 18.50%, 18.25% 18%, 17.75%, 17.50%, 17.25% 17%, 16.75%, 16.50%, 16.25% 16%, 15.75%, 15.50%, 15.25% 15%, 14.75%, 14.50%, 14.25% 14%, 13.75% , 13.50%, 13.25% 13%, 12.75%, 12.50%, 12.25% 12%, 11.75%, 11.50%, 11.25% 11%, 10.75 %, 10.50%, 10.25% 10%, 9.75%, 9.50%, 9.25% 9%, 8.75%, 8.50%, 8.25% 8%, 7, 75%, 7.50%, 7.25% 7%, 6.75%, 6.50%, 6.25% 6%, 5.75%, 5.50%, 5.25% 5%, 4 , 75%, 4.50%, 4.25%, 4%, 3.75%, 3.50%, 3.25%, 3%, 2.75%, 2.50%, 2.25%, 2%, 1.75%, 1.50%, 125%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0 , 08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01%, 0.009%, 0.008%, 0.007%, 0.006% , 0.005%, 0.004%, 0.003%, 0.002%, 0.001%, 0.0009%, 0.0008%, 0.0007%, 0.0006%, 0.0005%, 0.0004%, 0.0003% , 0.0002% or 0.0001% w / w, w / v, or v / v of the pharmaceutical composition.
[00579] [00579] In some embodiments, the concentration of TILs provided in the pharmaceutical compositions of the invention is in the range between approximately 0.0001% and 50%, approximately 0.001% and 40%, approximately 0.01% and 30%, approximately 0.02 % and 29%, approximately 0.03% and 28%, approximately 0.04% and 27%,
[00580] [00580] In some embodiments, the concentration of TILs provided in the pharmaceutical compositions of the invention is in the range between approximately 0.001% and 10%, approximately 0.01% and 5%, approximately 0.02% and 4.5%, approximately 0 , 03% and 4%, approximately 0.04% and 3.5%, approximately 0.05% and 3%, approximately 0.06% and 2.5%, approximately 0.07% and 2%, approximately 0, 08% and 1.5%, approximately 0.09% and 1%, approximately 0.1% and 0.9% w / w, w / v or v / v of the pharmaceutical composition.
[00581] [00581] In some embodiments, the amount of TILs provided in the pharmaceutical compositions of the invention is equal to or less than 10 g, 9.5 g, 9.0 g, 8.5 g, 8.0 g, 7.5 g, 7.0 g, 6.5 g, 6.0 g, 5.5 g, 5.0 g, 4.5 g, 4.0 g, 3.5 g, 3.0 g, 2.5 g, 2.0 g, 1.5 g, 1.0 g, 0.95 g, 0.9 g, 0.85 g, 0.8 g, 0.75 g, 0.7 g, 0.65 g, 0.6 g, 0.55 g, 0.5 g, 0.45 g, 0.4 g, 0.35 g, 0.3 g, 0.25 g, 0.2 g, 0.15 g, 0.1 g, 0.09 g, 0.08 g, 0.07 g, 0.06 g, 0.05 g, 0.04 g, 0.03 g, 0.02 g, 0.01 g, 0.009 g, 0.008 g, 0.007 g, 0.006 g, 0.005 g, 0.004 g, 0.003 g, 0.002 g, 0.001 g, 0.0009 g, 0.0008 g, 0.0007 g, 0.0006 g, 0.0005 g, 0.0004 g, 0.0003 g, 0.0002 g or 0.0001 g.
[00582] [00582] In some embodiments, the amount of TILs provided in the pharmaceutical compositions of the invention is greater than 0.0001 g, 0.0002 g, 0.0003 g, 0.0004 g, 0.0005 g, 0.0006 g, 0.0007 g, 0.0008 g, 0.0009 g, 0.001 g, 0.0015 g, 0.002 g, 0.0025 g, 0.003 g, 0.0035 g, 0.004 g, 0.0045 g, 0.005 g,
[00583] [00583] The TILs provided in the pharmaceutical compositions of the invention are effective over a wide dose range. The exact dose will depend on the route of administration, the form in which the compound is administered, the sex and age of the individual to be treated, the body weight of the individual to be treated and the preference and experience of the attending physician. Clinically established doses of TILs can also be used, if appropriate. The amounts of pharmaceutical compositions administered by the present methods, such as doses of TILs, will depend on the human or mammal being treated, the severity of the disorder or condition, the rate of administration, the disposition of the active pharmaceutical ingredients and at the discretion of the prescribing physician.
[00584] [00584] In some modalities, TILs can be administered in a single dose. Such administration can be by injection, e.g. eg, intravenous injection. In some modalities, TILs can be administered in multiple doses. Administration can be once, twice, three times, four times, five times, six times or more than six times a year. Administration can be once a month, once every two weeks, once a week or once every two days. The administration of TILs can continue as long as necessary.
[00585] [00585] In some embodiments, an effective dose of TILs is approximately 1x106, 2x106, 3x106, 4x106, 5x106, 6x106, 7x106, 8x106, 9x106, 1x107, 2x107, 3x107, 4x107, 5x107, 6x107, 7x107, 8x107, 9x107, 1x108, 2x108, 3x108, 4x108, 5x108, 6x108, 7x108, 8x108, 9x108, 1x109, 2x109, 3x109,
[00586] [00586] In some embodiments, an effective dose of TILs is in the range between approximately 0.01 mg / kg and 4.3 mg / kg, approximately 0.15 mg / kg and 3.6 mg / kg, approximately 0.3 mg / kg and 3.2 mg / kg, approximately 0.35 mg / kg and 2.85 mg / kg, approximately 0.15 mg / kg and 2.85 mg / kg, approximately 0.3 mg and 2.15 mg / kg, approximately 0.45 mg / kg and 1.7 mg / kg, approximately 0.15 mg / kg and 1.3 mg / kg, approximately 0.3 mg / kg and 1.15 mg / kg, approximately 0.45 mg / kg and 1 mg / kg, approximately 0.55 mg / kg and 0.85 mg / kg, approximately 0.65 mg / kg and 0.8 mg / kg, approximately 0.7 mg / kg and 0.75 mg / kg, approximately 0.7 mg / kg and 2.15 mg / kg, approximately 0.85 mg / kg and 2 mg / kg, approximately 1 mg / kg and 1.85 mg / kg, approximately 1 , 15 mg / kg and 1.7 mg / kg, approximately 1.3 mg / kg mg and 1.6 mg / kg, approximately 1.35 mg / kg and 1.5 mg / kg, approximately 2.15 mg / kg kg and 3.6 mg / kg, approximately 2.3 mg / kg and 3.4 mg / kg, approximately 2.4 mg / kg and 3.3 mg / kg, approximately 2.6 mg / kg and 3.15 mg / kg, approx approximately 2.7 mg / kg and 3 mg / kg, approximately 2.8 mg / kg and 3 mg / kg or approximately 2.85 mg / kg and 2.95 mg / kg.
[00587] [00587] In some embodiments, an effective dose of TILs is in the range between approximately 1 mg and 500 mg, approximately 10 mg and 300 mg, approximately 20 mg and 250 mg, approximately 25 mg and 200 mg, approximately 1 mg and 50 mg , approximately 5 mg and 45 mg,
[00588] [00588] An effective amount of TILs can be administered in a single or multiple dose by any of the accepted modes for administration of agents of similar utility, including intranasal and transdermal, by intra-arterial injection, intravenous, intraperitoneal, parenteral, intramuscular, subcutaneous, topical, by transplant or by inhalation. IV. Production processes of TILs - Gen 3
[00589] [00589] In some embodiments, the invention provides a population of TILs produced by the method any of the GEN 3 processes, modified to use a small biopsy, thick needle biopsy or fine needle aspiration. In some embodiments, the invention provides the method any of the GEN 3 processes, modified to use a small biopsy, thick needle biopsy or fine needle aspiration as the source of T cells for expansion in the first expansion of preparation of any such GEN 3 processes, in which (1) the duration of the first preparation expansion is extended to achieve the desired TIL cell count in the population of TILs collected after the second rapid expansion of such a GEN 3 process or (2) the duration of the second expansion such a process GEN 3 is prolonged to achieve the desired cell count of TILs in the population of TILs collected after the second rapid expansion or (3) the duration of the first preparation expansion is prolonged and the duration of the second
[00590] [00590] In some embodiments, the invention provides a population of TILs produced by any of the GEN 3 methods, modified to use a small biopsy, thick needle biopsy or fine needle aspiration as the source of T cells for expansion in the first preparation expansion of any of such GEN 3 processes, wherein (1) the duration of the first preparation expansion is extended to achieve the desired cell count of TILs in the population of TILs collected after the second rapid expansion of such GEN 3 process or (2) the duration of the second rapid expansion of such a GEN 3 process is extended to achieve the desired cell count of TILs in the population of TILs collected after the second rapid expansion or (3) the duration of the first preparation expansion is prolonged and the duration the second rapid expansion of such a GEN 3 process is prolonged to achieve the desired cell count of TILs in the population of TILs collected after the second rapid expansion.
[00591] [00591] Without being limited to any particular theory, it is believed that the first expansion of preparation, which prepares an activation of T cells, obtained from a tumor fragment or fragment obtained from a donor, followed by the second rapid expansion that reinforces activation of T cells, as described in some methods of the invention, allows the preparation of expanded T cells that retain a "younger" phenotype, and thus, it is predicted that the expanded T cells of the invention will exhibit greater cytotoxicity against cancer cells than T cells expanded by other methods. Specifically, it is believed that an activation of T cells that is prepared by exposure to an anti-CD3 antibody (eg, OKT-3), IL-2 and, optionally, antigen presenting cells (APCs) and then enhanced subsequent exposure to an anti-CD-3 antibody (eg, OKT-3), IL-2 and
[00592] [00592] In some embodiments, the second rapid expansion step is divided into a plurality of steps to achieve the vertical scaling of the culture: (a) performing a second rapid expansion in which T cells are grown in a small-scale culture in one first container, p. eg, a G-REX 100MCS container, for a period of approximately 3 to 4 days and then (b) transferring the T cells in the small-scale culture to a second container larger than the first container, e.g. eg, a G-REX 500MCS container, and growing the
[00593] [00593] In some embodiments, the second rapid expansion is performed after the activation of T cells made by the first preparation expansion begins to decrease, reduce, decay or lower.
[00594] [00594] In some embodiments, the second rapid expansion is performed after the activation of T cells carried out by the first preparation expansion has decreased by or near 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%.
[00595] [00595] In some embodiments, the second rapid expansion is carried out after the activation of T cells made by the first preparation expansion has decreased by a percentage in the range between or close to 1% and 100%.
[00596] [00596] In some embodiments, the second rapid expansion is performed after the activation of T cells carried out by the first preparation expansion has decreased by a percentage in the range between or close to 1% and 10%, 10% and 20%, 20 % and 30%, 30% and 40%, 40% and 50%, 50% and 60%, 60% and 70%, 70% and 80%, 80% and 90% or 90% and 100%.
[00597] [00597] In some modalities, the second rapid expansion is
[00598] [00598] In some embodiments, the second rapid expansion is performed after the activation of T cells made by the first preparation expansion has decreased by up to or close to 1, 2, 3, 4, 5, 6, 7, 8, 9 , 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 , 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 , 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84 , 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%.
[00599] [00599] In some embodiments, the decrease in T cell activation effected by the first preparation expansion is determined by a reduction in the amount of gamma interferon released by T cells in response to antigen stimulation.
[00600] [00600] In some modalities, the first expansion of T cell preparation is performed during a period of up to or close to 7 days.
[00601] [00601] In some modalities, the first expansion of T cell preparation is performed during a period of up to or near 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days.
[00602] [00602] In some embodiments, the first expansion of T cell preparation is performed over a period of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days or 7 days.
[00603] [00603] In some modalities, the second rapid expansion of T cells is performed during a period of up to or close to 11 days.
[00604] [00604] In some embodiments, the second rapid expansion of T cells is performed over a period of up to or near 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days , 10 days or 11 days.
[00605] [00605] In some embodiments, the second rapid expansion of T cells is performed over a period of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days or 11 days.
[00606] [00606] In some embodiments, the first expansion of T cell preparation is carried out for a period between approximately 1 day and 7 days and the second rapid expansion of T cells is carried out for a period of between approximately 1 day and 11 days.
[00607] [00607] In some embodiments, the first expansion of T cell preparation is carried out over a period of up to or close to 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days and the second expansion T-cell rapid is performed over a period of up to or around 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days or 11 days.
[00608] [00608] In some embodiments, the first expansion of T cell preparation is performed over a period of between approximately 1 day and 7 days and the second rapid expansion of T cells is performed over a period between approximately 1 day and 9 days.
[00609] [00609] In some embodiments, the first expansion of T cell preparation is performed over a period of 7 days and the second rapid expansion of T cells is performed over a period of 9 days.
[00610] [00610] In some embodiments, T cells are tumor infiltrating lymphocytes (TILs).
[00611] [00611] In some embodiments, T cells are marrow infiltrating lymphocytes (MILs).
[00612] [00612] In some embodiments, T cells are obtained from a donor who suffers from cancer.
[00613] [00613] In some embodiments, T cells are TILs obtained from
[00614] [00614] In some modalities, T cells are MILs obtained from the bone marrow of a patient suffering from a hematological malignancy.
[00615] [00615] In some modalities, the donor is suffering from cancer. In some modalities, cancer is selected from the group consisting of melanoma, ovarian cancer, cervical cancer, non-small cell lung cancer (NSCLC), lung cancer, bladder cancer, breast cancer, cancer caused by papillomavirus human, head and neck cancer (including squamous cell carcinoma of the head and neck (CCECP)), glioblastoma (including GBM), gastrointestinal cancer, kidney cancer and renal cell carcinoma. In some modalities, cancer is selected from the group consisting of melanoma, ovarian cancer, cervical cancer, non-small cell lung cancer (NSCLC), lung cancer, bladder cancer, breast cancer, cancer caused by papillomavirus human, head and neck cancer (including head and neck squamous cell carcinoma (CCECP)), glioblastoma (including GBM), gastrointestinal cancer, kidney cancer, and renal cell carcinoma. In some modalities, the donor is suffering from a tumor. In some embodiments, the tumor is a liquid tumor. In some embodiments, the tumor is a solid tumor. In some modalities, the donor is suffering from a hematological malignancy.
[00616] [00616] An exemplary TIL process, known as process 3 (also referred to here as GEN3), containing some of these characteristics is represented in Figure 85 (in particular, eg, Figure 85B), and some of the advantages of this modality of the present invention in relation to process 2A are described in Figures 1, 2, 30 and 31 (in particular, e.g., Figure 85B). Two modalities of process 3 are shown in Figures 1 and 30 (in particular, eg, Figure 85B). Process 2A or Gen 2 is also described in
[00617] [00617] As discussed and outlined in general terms at present, TILs are taken from a patient's sample and manipulated to expand their number before transplantation into a patient, using the TIL expansion process described here and referred to as Gen 3. In some embodiments, TILs can optionally be genetically engineered as discussed below. In some modalities, TILs can be cryopreserved before or after expansion. Once thawed, they can also be stimulated to increase their metabolism before infusion into a patient.
[00618] [00618] In some embodiments, the first preparation expansion (including processes referred to here as the Rapid pre-Expansion (Pre-REP), as well as processes shown in Figure 85 (in particular, eg, Figure 85B) as Step B) is shortened to 1 to 7 days and the second rapid expansion (including processes referred to herein as the Rapid Expansion Protocol (REP) as well as processes shown in Figure 85 (in particular, eg, Figure 85B) as Step D) it is shortened to 1 to 9 days, as discussed in detail below, as well as in the examples and figures. In some embodiments, the first preparation expansion (for example, an expansion described as Step B in Figure 85 (in particular, eg, Figure 85B)) is shortened to 7 days and the second rapid expansion (for example, a expansion as described in step D in Figure 85 (in particular, eg, Figure 85B)) is 7 to 9 days. In some embodiments, the combination of the first preparation expansion with the second rapid expansion (for example, expansions described as Step B and Step D in Figure 85 (in particular, eg, Figure 85B)) is 14-16 days , as discussed in detail below and in the examples and figures. Specifically, it is considered that, certain
[00619] [00619] The "Stage" Designations A, B, C etc., below are in reference to the non-limiting example in Figure 85 (in particular, eg, Figure 85B) and in reference to certain non-limiting modalities described herein . The ordering of the Steps below and in Figure 85 (in particular, eg, Figure 85B) is exemplary and any combination or order of steps, as well as additional steps, repetition of steps and / or omission of steps is contemplated by this application patent and the methods described herein.
[00620] [00620] In some modalities, if the tumor is metastatic and the primary lesion has been effectively treated / removed in the past, removal of one of the metastatic lesions may be necessary. In some modalities, the least invasive approach is to remove a skin lesion or lymph node in the neck or axillary area when available. In some modalities, a skin lesion is removed or a small biopsy is removed. In some modalities, a lymph node or small biopsy is removed. In some modalities, a metastatic pulmonary or hepatic lesion or an intra-abdominal or thoracic lymph node or a small biopsy of the same may be used.
[00621] [00621] In some modalities, the tumor is a melanoma. In some modalities, the small biopsy of a melanoma comprises a nevus or part of it.
[00622] [00622] In some modalities, the small biopsy is a biopsy by puncture. In some modalities, puncture biopsy is obtained with
[00623] [00623] In some modalities, the small biopsy is an excisional biopsy. In some embodiments, the small biopsy is an excisional biopsy and the entire nevus or growth is removed. In some embodiments, the small biopsy is an excisional biopsy and the entire nevus or growth is removed along with a small border of normal looking tissue.
[00624] [00624] In some modalities, the small biopsy is an incisional biopsy. In some modalities, the small biopsy is an incisional biopsy and only the most irregular part of a nevus or growth is removed. In some modalities, the small biopsy is an incisional biopsy and the incisional biopsy is used when other techniques cannot be completed, such as if a suspected nevus is very large.
[00625] [00625] In some modalities, the small biopsy is a lung biopsy. In some modalities, a small biopsy is obtained by bronchoscopy. In general, in bronchoscopy, the patient is placed under anesthesia, and a small [sic] passes through the nose or mouth, goes down the throat and enters the bronchial passages, where small tools are used to remove some tissue. In some modalities, when the tumor or growth cannot be reached by bronchoscopy, a transthoracic needle biopsy can be used. In general, for a transthoracic needle biopsy, the patient is also under anesthesia and a needle is inserted through the skin directly into the suspect site to remove a small sample of tissue. In some modalities, transthoracic needle biopsy may
[00626] [00626] In some modalities, the small biopsy is a biopsy on the head and neck. In some modalities, the small biopsy is an incisional biopsy. In some modalities, the small biopsy is an incisional biopsy, in which a small piece of tissue is cut from an area that looks abnormal. In some modalities, if the abnormal region has easy access, the sample can be collected without hospitalization. In some modalities, if the tumor is deeper inside the mouth or throat, the biopsy may need to be performed in an operating room, under general anesthesia. In some modalities, the small biopsy is an excisional biopsy. In some modalities, the small biopsy is an excisional biopsy, in which the entire area is removed. In some modalities, the small biopsy is a fine needle aspiration (FNA). In some modalities, the small biopsy is a fine needle aspiration (FNA), in which a very fine needle attached to a syringe is used to extract (aspirate) cells from a tumor or nodule. In some modalities, the small biopsy is a puncture biopsy. In some modalities, the small biopsy is a puncture biopsy, in which puncture forceps are used to remove a piece of the suspect area.
[00627] [00627] In some modalities, the small biopsy is a cervical biopsy. In some modalities, the small biopsy is obtained through colposcopy. In general, colposcopy methods employ the use of a light magnification instrument attached to magnification binoculars (a colposcope) which is then used to biopsy a small section of the
[00628] [00628] The term "solid tumor" refers to an abnormal mass of tissue that normally does not contain cysts or areas of fluid. Solid tumors can be benign or malignant. The term "solid tumor" cancer refers to solid malignant, neoplastic or cancerous tumors. Solid tumor cancers include, but are not limited to, sarcomas, carcinomas, and lymphomas, such as cancers of the lung, breast, triple negative breast cancer, prostate, colon, rectum, and bladder. In some modalities, cancer is selected from cervical cancer, head and neck cancer, glioblastoma, ovarian cancer, sarcoma, pancreatic cancer, bladder cancer, breast cancer, triple negative breast cancer and non-small lung cancer cells. The tissue structure of solid tumors includes interdependent tissue compartments including the parenchyma (cancer cells) and the stromal supporting cells in which the cancer cells are dispersed and which can provide a supporting microenvironment.
[00629] [00629] In some embodiments, the tumor sample is obtained as a fine needle aspirate (FNA), a thick needle biopsy, or a small biopsy (including, for example, puncture biopsy). In some modalities, the sample is placed first in G-Rex 10. In some modalities, the sample is placed first in G-Rex 10 where there are 1 or 2 biopsy samples by thick needle and / or small biopsy. In some embodiments, the sample is placed first on G-Rex 100 where 3, 4, 5, 6, 8, 9 or 10 or more biopsy samples are used with a thick needle and / or a small biopsy. In some modalities, the sample is placed
[00630] [00630] FNA can be obtained from a tumor selected from the group consisting of lung, melanoma, head and neck, cervical, ovarian, pancreatic, glioblastoma, colorectal and sarcoma. In some embodiments, FNA is obtained from a lung tumor, such as a lung tumor from a patient with non-small cell lung cancer (NSCLC). In some cases, the patient with NSCLC has previously undergone surgical treatment.
[00631] [00631] TILs described herein can be obtained from an FNA sample. In some cases, the ANF sample is obtained or isolated from the patient using a fine gauge needle ranging from an 18 gauge needle to a 25 gauge needle. The fine gauge needle can be 18 gauge, 19 gauge, 20 gauge , caliber 21, caliber 22, caliber 23, caliber 24 or caliber
[00632] [00632] In some cases, the TILs described here are obtained from a thick needle biopsy sample. In some cases, the small biopsy sample or thick needle biopsy is obtained or isolated from the patient by a surgical or clinical needle ranging from an 11 gauge needle to a 16 gauge needle. The needle can be 11 gauge, 12 gauge , gauge 13, gauge 14, gauge 15, or gauge 16. In some embodiments, the patient's thick needle biopsy sample may contain at least 400,000 TILs, p. e.g., 400,000 TILs, 450,000 TILs, 500,000 TILs, 550,000 TILs, 600,000 TILs, 650,000 TILs, 750,000 TILs, 800,000 TILs, 850,000 TILs, 900,000 TILs, 950,000 TILs or more.
[00633] [00633] In some modalities, TILs are obtained from material
[00634] [00634] In some modalities, the cell suspension collected before the first expansion stage is called a "primary cell population" or a "recently collected" cell population.
[00635] [00635] In some embodiments, the cells can be optionally frozen, after collection in the sample, and frozen before being admitted to the expansion described in step B, which is described in more detail below, as well as exemplified in Figure 85. A. Step A : Obtaining a tumor sample from the patient
[00636] [00636] In general, TILs are initially obtained from a patient's tumor sample ("primary TILs"), obtained by a thick needle biopsy or similar procedure, and are then expanded into a larger population for further manipulation as described here,
[00637] [00637] A tumor sample from the patient can be obtained by methods known in the art, usually via surgical resection, thick needle biopsy, needle biopsy or other means to obtain a sample containing a mixture of tumor cells and TILs. In general, the tumor sample can be any solid tumor, including primary tumors, invasive tumors or metastatic tumors. In some embodiments, the sample may be multiple small samples of the tumor or biopsies. In some embodiments, the sample may comprise multiple tumor samples (such as multiple fragments) from a single tumor from the same patient. In some embodiments, the sample may comprise multiple tumor samples from one, two, three or four tumors from the same patient (such as thick needle biopsies obtained from multiple lesions in metastatic disease). In some embodiments, the sample may comprise multiple tumor samples from multiple tumors from the same patient. The tumor sample can also be a liquid tumor, such as a tumor resulting from a hematological malignancy. The solid tumor can be of any type of cancer, including, but not limited to, breast, pancreatic, prostate, colorectal, lung, brain, kidney, stomach and skin (including, but not limited to, squamous cell carcinoma, basal cell carcinoma and melanoma ). In some modalities, cancer is selected from cervical cancer, head and neck cancer (including, for example, squamous cell carcinoma of the head and neck (CCECP)), glioblastoma (GBM), gastrointestinal cancer, ovarian cancer, sarcoma, pancreatic cancer, bladder cancer, breast cancer, triple negative breast cancer and non-small cell lung carcinoma. In some modalities, useful TILs are obtained from malignant melanoma tumors, as these, according to reports, have particularly high levels of TILs.
[00638] [00638] As indicated above, in some embodiments, TILs are derived from solid tumor colors or fragments. In some embodiments, the samples or fragments of the solid tumor are subjected to enzymatic digestion. In some embodiments, the tumor samples or fragments are digested in an enzyme mixture comprising collagenase, DNase and hyaluronidase. In some embodiments, the tumor samples or fragments are digested in an enzyme mixture comprising collagenase, DNase and hyaluronidase for 1-2 hours. In some embodiments, tumors are digested in a mixture of enzymes comprising collagenase, DNase and hyaluronidase for 1-2 hours at 37 ºC, 5% CO2. In some embodiments, the tumor samples or fragments are digested in an enzyme mixture comprising collagenase, DNase and hyaluronidase for 1-2 hours at 37 ºC, 5% CO2 with rotation. In some embodiments, tumor samples or fragments are digested overnight with constant rotation. In some embodiments, the tumor samples or fragments are digested overnight at 37 ºC, CO2 5% with constant rotation. In some embodiments, the tumor samples or fragments are combined with the enzymes to form a digested reaction mixture of the tumor.
[00639] [00639] In some embodiments, TILs are not obtained from material digested from the tumor. In some embodiments, samples of the solid tumor are not fragmented.
[00640] [00640] In some embodiments, the tumor samples or fragments are reconstituted with lyophilized enzymes in a sterile buffer. In some embodiments, the buffer is sterile HBSS.
[00641] [00641] In some embodiments, the enzyme mixture comprises collagenase. In some embodiments, collagenase is collagenase IV. In some embodiments, the working stock for collagenase is 10X a working stock at 100 mg / mL.
[00642] [00642] In some embodiments, the enzyme mixture comprises
[00643] [00643] In some embodiments, the enzyme mixture comprises hyaluronidase. In some embodiments, the working stock for hyaluronidase is 10X a working stock at 10 mg / mL.
[00644] [00644] In some embodiments, the enzyme mixture comprises collagenase 10 mg / mL, DNAse 1000 IU / mL and hyaluronidase 1 mg / mL hyaluronidase.
[00645] [00645] In some embodiments, the enzyme mixture comprises collagenase 10 mg / mL, DNAse 500 IU / mL and hyaluronidase 1 mg / mL.
[00646] [00646] In general, the cell suspension obtained from the tumor sample or fragment is called a "primary cell population" or a "recently obtained" or "recently isolated" cell population. In certain embodiments, the cell population recently obtained from TILs is exposed to a cell culture medium comprising antigen presenting cells, IL-12 and OKT-3.
[00647] [00647] In some embodiments, TILs can be initially cultured from enzymatically digested material from tumor sample or fragment and tumor samples or fragments obtained from patients. In one embodiment, TILs can be initially cultured from enzymatically digested material from the tumor sample or fragment and tumor samples or fragments obtained from patients.
[00648] [00648] In some embodiments, TILs are obtained from material digested from the tumor fragment or sample. In some embodiments, the digested material of the tumor fragment or sample is generated by incubation in a medium with enzymes, for example, among others, RPMI 1640, GlutaMAX 2 mM, gentamicin 10 mg / mL, DNase 30 U / mL and collagenase 1, 0 mg / mL, followed by mechanical dissociation (GentleMACS, Miltenyi Biotec, Auburn, CA). After being placed in an enzyme medium, the fragment or sample of the
[00649] [00649] In some modalities, if the tumor is metastatic and the primary lesion has been effectively treated / removed in the past, removal of one of the metastatic lesions may be necessary. In some modalities, the less invasive approach is to remove a skin lesion, or a lymph node in the neck or axillary area when available. In some modalities, a skin lesion or a small biopsy is removed. In some modalities, a lymph node or small biopsy is removed. In some modalities, a metastatic lung or liver injury, or an intra-abdominal or thoracic lymph node or a small biopsy of the same may be employed.
[00650] [00650] In some modalities, the tumor is a melanoma. In some modalities, the small biopsy for melanoma comprises a nevus or part of it.
[00651] [00651] In some modalities, the small biopsy is a biopsy by puncture. In some modalities, the puncture biopsy is obtained with a circular blade pressed into the skin. In some modalities, biopsy
[00652] [00652] In some modalities, the small biopsy is an excisional biopsy. In some embodiments, the small biopsy is an excisional biopsy and the entire nevus or growth is removed. In some modalities, the small biopsy is an excisional biopsy and the entire nevus or growth is removed along with a small margin of normal looking skin.
[00653] [00653] In some modalities, the small biopsy is an incisional biopsy. In some modalities, the small biopsy is an incisional biopsy and only the most irregular part of a nevus or growth is removed. In some modalities, the small biopsy is an incisional biopsy and the incisional biopsy is used when other techniques cannot be completed, such as if a suspected nevus is very large.
[00654] [00654] In some modalities, the small biopsy is a lung biopsy. In some modalities, a small biopsy is obtained by bronchoscopy. In general, in bronchoscopy, the patient is placed under anesthesia, and a small [sic] passes through the nose or mouth, goes down the throat and enters the bronchial passages, where small tools are used to remove some tissue. In some modalities, when the tumor or growth cannot be reached by bronchoscopy, a transthoracic needle biopsy can be used. In general, for a transthoracic needle biopsy, the patient is also under anesthesia and a needle is inserted through the skin directly into the suspect site to remove a small sample of tissue. In some modalities, transthoracic needle biopsy may require interventional radiology (for example, the use of radiographs or
[00655] [00655] In some modalities, the small biopsy is a biopsy on the head and neck. In some modalities, the small biopsy is an incisional biopsy. In some modalities, the small biopsy is an incisional biopsy, in which a small piece of tissue is cut from an area that looks abnormal. In some modalities, if the abnormal region has easy access, the sample can be collected without hospitalization. In some modalities, if the tumor is deeper inside the mouth or throat, the biopsy may need to be performed in an operating room, under general anesthesia. In some modalities, the small biopsy is an excisional biopsy. In some modalities, the small biopsy is an excisional biopsy, in which the entire area is removed. In some modalities, the small biopsy is a fine needle aspiration (FNA). In some modalities, the small biopsy is a fine needle aspiration (FNA), in which a very fine needle attached to a syringe is used to extract (aspirate) cells from a tumor or nodule. In some modalities, the small biopsy is a puncture biopsy. In some modalities, the small biopsy is a puncture biopsy, in which puncture forceps are used to remove a piece of the suspect area.
[00656] [00656] In some modalities, the small biopsy is a cervical biopsy. In some modalities, the small biopsy is obtained through colposcopy. In general, colposcopy methods employ the use of a light magnification instrument attached to magnification binoculars (a colposcope) which is then used to biopsy a small section of the cervical surface. In some modalities, the small biopsy is
[00657] [00657] The term "solid tumor" refers to an abnormal mass of tissue that normally does not contain cysts or areas of fluid. Solid tumors can be benign or malignant. The term "solid tumor" cancer refers to solid malignant, neoplastic or cancerous tumors. Solid tumor cancers include, but are not limited to, sarcomas, carcinomas, and lymphomas, such as cancers of the lung, breast, triple negative breast cancer, prostate, colon, rectum, and bladder. In some modalities, cancer is selected from cervical cancer, head and neck cancer, glioblastoma, ovarian cancer, sarcoma, pancreatic cancer, bladder cancer, breast cancer, triple negative breast cancer and non-small lung cancer cells. The tissue structure of solid tumors includes interdependent tissue compartments including the parenchyma (cancer cells) and the stromal supporting cells in which the cancer cells are dispersed and which can provide a supporting microenvironment.
[00658] [00658] In some embodiments, the tumor sample is obtained as a fine needle aspirate (FNA), a thick needle biopsy, a small biopsy (including, for example, puncture biopsy). In some modalities, the sample is placed first in G-Rex 10. In some modalities, the sample is placed first in G-Rex 10 where there are 1 or 2 biopsy samples by thick needle and / or small biopsy. In some embodiments, the sample is placed first on G-Rex 100 where 3, 4, 5, 6, 8, 9 or 10 or more biopsy samples are used with a thick needle and / or small biopsy. In some modalities, the sample is placed first in G-Rex 500 where 3, 4, 5, 6, 8, 9 or 10 or more are
[00659] [00659] FNA can be obtained from a tumor selected from the group consisting of pulmonary, melanoma, head and neck, cervical, ovarian, pancreatic, glioblastoma, colorectal and sarcoma. In some embodiments, FNA is obtained from a lung tumor, such as a lung tumor from a patient with non-small cell lung cancer (NSCLC). In some cases, the patient with NSCLC has previously undergone surgical treatment.
[00660] [00660] TILs described herein can be obtained from an FNA sample. In some cases, the ANF sample is obtained from or isolated from the patient using a fine gauge needle ranging from an 18 gauge needle to a 25 gauge needle. The fine gauge needle can be 18 gauge, 19 gauge, 20 gauge, caliber 21, caliber 22, caliber 23, caliber 24 or caliber 25. In some embodiments, the patient's ANF sample may contain at least
[00661] [00661] In some cases, the TILs described here are obtained from a thick needle biopsy sample. In some cases, the coarse needle or small biopsy sample is obtained or isolated from the patient using a surgical or clinical needle ranging from 11 gauge needle to 16 gauge needle. The needle can be 11 gauge, 12 gauge, gauge 13, 14 gauge, 15 gauge or 16 gauge. In some embodiments, the patient's thick needle biopsy sample may contain at least 400,000 TILs, p. ex.,
[00662] [00662] In some embodiments, TILs are obtained from material digested from the tumor fragment or sample. In some modalities, the
[00663] [00663] In some embodiments, the cell suspension, before the first expansion stage of preparation is called a "primary cell population" or a "recently obtained" or "recently isolated" cell population.
[00664] [00664] In some embodiments, cells may optionally be frozen after sample isolation (eg, after obtaining the tumor sample and / or after obtaining the cell suspension of the tumor sample) and frozen before being admitted in the expansion described in step B, which is described in more detail below, as well as exemplified in Figure 85 (in particular, eg, Figure 85B). B. Step B: First preparation expansion
[00665] [00665] In some embodiments, the present methods provide
[00666] [00666] After biopsy or digestion of tumor fragments tumor fragments and / or samples, for example, as described in step A of Figure 85 (in particular, e.g., Figure 85B), the resulting cells are cultured in serum containing IL-2, OKT-3 and feeder cells (eg, antigen presenting feeder cells), under conditions that favor the growth of TILs more than tumor cells and other cells. In some embodiments, IL-2, OKT-3 and feeder cells are added at the beginning of the culture along with the digested material from the tumor fragment or sample and / or tumor fragments or samples (eg, on Day 0) . In some embodiments, the digested material from the tumor fragment or sample and / or tumor fragments or samples is incubated in a container with up to 60 fragments or samples per container and with 6000 IU / mL of IL-2. This primary cell population is cultivated over a period of days, usually 1 to 7 days, resulting in a population of bulk TILs, usually close to 1 x 108 bulk TIL cells. In some modalities, the first expansion of preparation takes place over a period of 1 to 7 days, resulting in a population of bulk TILs, usually close to 1 x 108
[00667] [00667] In a preferred embodiment, the expansion of TILs can be performed using a first preparation expansion step (for example, such as those described in step B of Figure 85 (in particular, e.g., Figure 85B), which may include processes referred to as pre-REP or preparation REP and containing feeder cells since Day 0 and / or since the start of culture), as described below and here, followed by a second rapid expansion (Step D, including processes referred to as Rapid Expansion Protocol (REP) steps as described below below Step D, followed by optional cryopreservation and then by a second Step D (including processes referred to as REP steps with restimulation) as described below. The TILs obtained from this process can be optionally classified according to phenotypic characteristics and metabolic parameters as described here. In some embodiments, the tumor fragment is between approximately 1 mm3 and 10 mm3.
[00668] [00668] In some modalities, the culture medium of the first expansion is referred to as "CM", an abbreviation for culture medium. In some modalities, the CM for Step B consists of RPMI 1640 with GlutaMAX, supplemented with 10% human AB serum, 25 mM Hepes and 10 µg / mL gentamicin.
[00669] [00669] In some modalities, there are 240 or less fragments or
[00670] [00670] After the preparation of the tumor fragments or samples, the resulting cells (that is, those that come from the fragments or samples and that constitute a population of primary cells) are cultured in medium containing IL-2, antigen presenting feeder cells and OKT-3 under conditions that favor the growth of TILs more than that of tumor cells and other cells and which allow the preparation of TILs and the accelerated growth since the beginning of culture on Day 0. In some embodiments, the digested material of the tumor fragments or samples and / or tumor fragments or samples are incubated with 6000 IU / mL of IL-2, as well as antigen presenting OKT-3 feeder cells. This primary cell population is grown for a period of days, usually
[00671] [00671] In some embodiments, the culture medium of the first preparation expansion comprises approximately 500 IU / ml IL-15, approximately 400 IU / ml IL-15, approximately 300 IU / ml IL-15, approximately 200 IU / ml IL-15, approximately 180 IU / ml IL-15, approximately 160 IU / ml IL-15, approximately 140 IU / ml IL-15, approximately 120 IU / ml IL-15 or approximately 100 IU / ml IL-15. In some modalities, the culture medium of the first
[00672] [00672] In some embodiments, the culture medium of the first preparation expansion comprises approximately 20 IU / ml IL-21, approximately 15 IU / ml IL-21, approximately 12 IU / ml IL-21, approximately 10 IU / ml IL-21, approximately 5 IU / ml IL-21, approximately 4 IU / ml IL-21, approximately 3 IU / ml IL-21, approximately 2 IU / ml IL-21, approximately 1 IU / ml IL-21 or approximately 0.5 IU / ml IL-21. In some embodiments, the culture medium of the first preparation expansion comprises approximately 20 IU / ml IL-21 to approximately 0.5 IU / ml IL-21. In some embodiments, the culture medium of the first preparation expansion comprises approximately 15 IU / ml IL-21 to approximately 0.5 IU / ml IL-21. In some embodiments, the culture medium of the first expansion of preparation comprises approximately 12 IU / ml IL-21 to approximately 0.5 IU / ml IL-21. In some embodiments, the culture medium of the first preparation expansion comprises approximately 10
[00673] [00673] In one embodiment, the cell culture medium of the first expansion of preparation comprises OKT-3 antibody. In some embodiments, the cell culture medium of the first preparation expansion comprises approximately 30 ng / ml of OKT-3 antibody. In one embodiment, the cell culture medium of the first preparation expansion comprises approximately 0.1 ng / mL, approximately 0.5 ng / mL, approximately 1 ng / mL, approximately 2.5 ng / mL, approximately 5 ng / mL , approximately 7.5 ng / mL, approximately 10 ng / mL, approximately 15 ng / mL, approximately 20 ng / mL, approximately 25 ng / mL, approximately 30 ng / mL, approximately 35 ng / mL, approximately 40 ng / mL , approximately 50 ng / mL, approximately 60 ng / mL, approximately 70 ng / mL, approximately 80 ng / mL, approximately 90 ng / mL, approximately 100 ng / mL, approximately 200 ng / mL, approximately 500 ng / mL, and approximately 1 µg / mL of OKT-3 antibody. In one embodiment, the cell culture medium comprises between 0.1 ng / ml and 1 ng / ml, between 1 ng / ml and 5 ng / ml, between 5 ng / ml and 10 ng / ml, between 10 ng / ml and 20 ng / mL, between 20 ng / mL and 30 ng / mL, between 30
[00674] [00674] In some embodiments, the cell culture medium of the first preparation expansion comprises one or more TNFRSF agonists in a cell culture medium. In some embodiments, the TNFRSF agonist comprises a 4-1BB agonist. In some embodiments, the TNFRSF agonist is a 4-1BB agonist, and the 4-1BB agonist is selected from the group consisting of urelumab, utomilumab, EU-101, a fusion protein and fragments, derivatives, variants, biosimilars and combinations thereof. In some embodiments, the TNFRSF agonist is added to a concentration sufficient to achieve a concentration in the cell culture medium between 0.1 µg / ml and 100 µg / ml. In some modalities, the TNFRSF agonist is
[00675] [00675] In some embodiments, in addition to one or more TNFRSF agonists, the cell culture medium of the first preparation expansion further comprises IL-2 at an initial concentration of approximately 3000 IU / mL and OKT-3 antibody at an initial concentration approximately 30 ng / ml, and wherein one or more of the TNFRSF agonists comprise a 4-1BB agonist. In some embodiments, in addition to one or more TNFRSF agonists, the cell culture medium of the first expansion of preparation further comprises IL-2 at an initial concentration of approximately 6000 IU / mL and OKT-3 antibody at an initial concentration of approximately 30 ng / ml, and wherein one or more of the TNFRSF agonists comprises a 4-1BB agonist.
[00676] [00676] In some modalities, the culture medium of the first preparation expansion is referred to as "CM", an abbreviation for culture medium. In some embodiments, it is referred to as CM1 (culture medium 1). In some modalities, the CM consists of RPMI 1640 with GlutaMAX, supplemented with 10% human AB serum, 25 mM Hepes and 10 µg / mL gentamicin. In some embodiments, the CM is the CM1 described in the Examples, see, Examples 1 and 14. In some embodiments, the first preparation expansion occurs in an initial cell culture medium or a first cell culture medium. In some embodiments, the culture medium of the first preparation expansion or the initial cell culture medium or the first cell culture medium comprises IL-2, OKT-3 and antigen presenting feeder cells (also referred to herein as feeder cells).
[00677] [00677] In some embodiments, the process of the first preparation expansion (including processes such as, for example, those described in step B of Figure 85 (in particular, eg, Figure 85B), which may include
[00678] [00678] In some embodiments, the first expansion of TIL preparation can proceed for 1 days to 7 days since when samples or tumor fragments are added to the cell culture medium and / or
[00679] [00679] In some embodiments, the first expansion of TIL preparation can proceed for 8 days to 17 days since when samples or tumor fragments are added to the cell culture and / or when the first expansion expansion step is initiated. In some embodiments, the first expansion of TIL preparation can proceed for 9 days to 17 days from when samples or tumor fragments are added to the cell culture and / or when the first expansion preparation step is initiated. In some embodiments, the first expansion of TIL preparation can proceed for 10 days to 17 days from when samples or tumor fragments are added to the cell culture and / or when the first preparation expansion step is initiated. In some embodiments, the first expansion of TIL preparation can proceed for 11 days to 17 days from when samples or tumor fragments are added to the cell culture and / or when the first expansion preparation step is initiated. In some embodiments, the first expansion of TIL preparation can proceed for 12 days to 17 days from when samples or tumor fragments are added to the cell culture and / or when the first expansion expansion step is initiated. In some embodiments, the first expansion of TIL preparation can proceed for 13 days to 17 days from when samples or tumor fragments are added to the cell culture and / or when the first expansion expansion step is initiated. In some embodiments, the first expansion of TIL preparation can proceed for 14 days to 17 days from when samples or tumor fragments are added to the cell culture and / or when the first expansion expansion step is initiated. In some embodiments, the first expansion of TIL preparation can proceed for 15 days to 17 days from when samples or tumor fragments are added to the cell culture and / or when the first preparation expansion step is initiated. In some modalities, the first expansion of TIL preparation can
[00680] [00680] In some modalities, the first expansion of TIL preparation can proceed for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9, days or 10 days. In some modalities, the first expansion of TILs can proceed for 1 day to 7 days. In some modalities, the first expansion of TILs can continue for 2 days to 7 days. In some modalities, the first expansion of TILs can continue for 3 days to 7 days. In some modalities, the first expansion of TILs can continue for 4 days to 7 days. In some modalities, the first expansion of TILs can proceed for 5 days to 7 days. In some modalities, the first expansion of TILs can continue for 6 days to 7 days. In some modalities, the first expansion of TILs can proceed for 1 day to 10 days. In some modalities, the first expansion of TILs can continue for 2 days to 10 days. In some modalities, the first expansion of TILs can proceed for 3 days to 10 days. In some modalities, the first expansion of TILs can proceed for 4 days to 10 days. In some modalities, the first expansion of TILs can proceed for 5 days to 10 days. In some modalities, the first expansion of TILs can continue for 6 days to 10 days. In some modalities, the first expansion of TILs can continue for 7 days to 10 days. In some modalities, the first expansion of TILs can continue for 8 days to 10 days. In some modalities, the first expansion of TILs can continue for 9 days to 10 days. In some modalities, the first expansion of TILs can continue for 7 days. In some modalities, the first expansion of TILs can continue for 8 days. In some modalities, the first expansion of TILs can continue for 9 days. In some modalities, the first expansion of TILs can continue for 10 days.
[00681] [00681] In some modalities, the first expansion of TIL preparation can continue for 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days or 17 days. In some modalities, the first expansion of TILs can continue for 8 days to 17 days. In some modalities, the first expansion of TILs can proceed from 9 days to 17 days. In some modalities, the first expansion of TILs can continue for 10 days to 17 days. In some modalities, the first expansion of TILs can continue for 11 days to 17 days. In some modalities, the first expansion of TILs can proceed for 12 days to 17 days. In some modalities, the first expansion of TILs can continue for 13 days to 17 days. In some modalities, the first expansion of TILs can continue for 14 days to 17 days. In some modalities, the first expansion of TILs can continue for 15 days to 17 days. In some modalities, the first expansion of TILs can continue for 16 days to 17 days. In some modalities, the first expansion of TILs can continue for 17 days.
[00682] [00682] In some embodiments, a combination of IL-2, IL-7, IL-15 and / or IL-21 is employed as a combination during the first preparation expansion. In some embodiments, IL-2, IL-7, IL-15 and / or IL-21 as well as any combinations thereof can be included during the first expansion of preparation, including, for example, during Step B processes according to Figure 85 (in particular, e.g., Figure 85B), as well as described herein. In some embodiments, a combination of IL-2, IL-15 and IL-21 is employed as a combination during the first expansion of preparation. In some embodiments, IL-2, IL-15 and IL-21 as well as any combinations thereof can be included during Step B processes according to Figure 85 (in particular, eg, Figure 85B) and as described here.
[00683] [00683] In some embodiments, the first expansion of preparation, for example, Step B according to Figure 85 (in particular, eg, Figure
[00684] [00684] In one embodiment, the first preparation expansion procedures described herein (for example, including expansions such as those described in step B of Figure 85 (in particular, eg, Figure 85B), as well as those referred to as pre-REP or preparation REP) require feeder cells (also referred to herein as “antigen presenting cells”) at the beginning of the TIL expansion and during the first preparation expansion. In many modalities, feeder cells are peripheral blood mononuclear cells (PBMCs) obtained from standard whole blood units from healthy allogeneic blood donors. PBMCs are obtained using standard methods such as Ficoll-Paque gradient separation. In some embodiments, 2.5 x 108 feeder cells are used during the first preparation expansion. In some embodiments, 2.5 x 108 feeder cells per container are used during the first preparation expansion. In some embodiments, 2.5 x 108 feed cells per GREX-10 are used during the first preparation expansion. In some embodiments, 2.5 x 108 feed cells per GREX-100 are used during the first preparation expansion.
[00685] [00685] In general, allogeneic PBMCs are inactivated, either by irradiation or heat treatment, and used in REP procedures, as described in the examples, which provides an exemplary protocol for evaluating the
[00686] [00686] In some embodiments, PBMCs are considered incompetent for replication and acceptable for use in the TIL expansion procedures described here if the total number of viable cells on day 14 is less than the initial number of viable cells placed in culture on day 0 of the first preparation expansion.
[00687] [00687] In some embodiments, PBMCs are considered incompetent for replication and acceptable for use in the TILs expansion procedures described here if the total number of viable cells, cultured in the presence of OKT3 and IL-2, on day 7 has not increased from the initial number of viable cells cultured on day 0 of the first preparation expansion. In some embodiments, PBMCs are cultured in the presence of OKT3 antibody 30 ng / mL and IL-2 3000 IU / mL. In some embodiments, PBMCs are cultured in the presence of OKT3 antibody 30 ng / mL and 6000 IU / mL IL-2.
[00688] [00688] In some embodiments, PBMCs are considered incompetent for replication and acceptable for use in the TILs expansion procedures described here if the total number of viable cells, cultured in the presence of OKT3 and IL-2, on day 7 has not increased from the initial number of viable cells cultured on day 0 of the first preparation expansion. In some embodiments, PBMCs are cultured in the presence of OKT3 antibody 5-60 ng / mL and IL-2 1000-6000 IU / mL. In some embodiments, PBMCs are cultured in the presence of OKT3 antibody 10-50 ng / mL and IL-2 2000-5000 IU / mL. In some embodiments, PBMCs are cultured in the presence of OKT3 antibody 20-40 ng / mL and IL-2 2000-4000 IU / mL. In some embodiments, PBMCs are cultured in the presence of OKT3 antibody 25-35 ng / mL and IL-2 2500-3500 IU / mL. In some embodiments, PBMCs are cultured in the presence of OKT3 antibody 30 ng / mL and 6000 IU / mL IL-2. In some modalities, PBMCs
[00689] [00689] In some embodiments, the antigen presenting feeder cells are PBMCs. In some embodiments, antigen-presenting feeder cells are artificial antigen-presenting feeder cells. In one embodiment, the ratio of TILs to antigen presenting feeder cells in the second expansion is approximately 1 to 25, approximately 1 to 50, approximately 1 to 100, approximately 1 to 125, approximately 1 to 150, approximately 1 to 175, approximately 1 for 200, approximately 1 to 225, approximately 1 to 250, approximately 1 to 275, approximately 1 to 300, approximately 1 to 325, approximately 1 to 350, approximately 1 to 375, approximately 1 to 400 or approximately 1 to 500. In one modality, the ratio of TILs to antigen presenting cells in the second expansion is between 1 to 50 and 1 to 300. In one embodiment, the ratio of TILs to antigen presenting cells in the second expansion is between 1 to 100 and 1 to 200.
[00690] [00690] In one embodiment, the first preparation expansion procedures described here require a ratio of approximately 2.5 x 108 feeder cells to approximately 100 x 106 TILs. In another embodiment, the first preparation expansion procedures described herein require a ratio of approximately 2.5 x 10 8 feeder cells to approximately 50 x 10 6 TILs. In yet another embodiment, the first preparation expansion described herein requires approximately 2.5 x 10 8 feeder cells for approximately 25 x 10 6 TILs. In yet another modality, the first expansion of preparation
[00691] [00691] In some embodiments, the medium in the first preparation expansion comprises IL-2. In some embodiments, the medium in the first preparation expansion comprises 6000 IU / ml IL-2. In some embodiments, the medium in the first expansion of preparation comprises antigen presenting feeder cells. In some embodiments, the medium in the first expansion of preparation comprises 2.5 x 108 antigen presenting feeder cells per container. In some modalities, the medium in the first preparation expansion comprises OKT-
[00692] [00692] In one embodiment, the first preparation expansion procedures described here require an excess of feeder cells over TILs during the second expansion. In many modalities, feeder cells are peripheral blood mononuclear cells (PBMCs) obtained from standard whole blood units from healthy allogeneic blood donors. PBMCs are obtained using standard methods such as Ficoll-Paque gradient separation. In one embodiment, artificial antigen presenting cells (aAPC) are used in place of PBMCs.
[00693] [00693] In general, allogeneic PBMCs are inactivated, either by irradiation or heat treatment, and used in the TIL expansion procedures described here, including the exemplary procedures described in the figures and examples.
[00694] [00694] In one embodiment, artificial antigen presenting cells are used in the first expansion of preparation in substitution or in combination with PBMCs.
[00695] [00695] The expansion methods described herein generally use culture media with high doses of a cytokine, in particular IL-2, as is known in the art.
[00696] [00696] Alternatively, the use of combinations of cytokines for the first expansion of TIL preparation is also possible, with combinations of two or more among IL-2, IL-15 and IL-21 as is generally outlined in International Publication No. WO 2015/189356 and WO 2015/189357, herein expressly incorporated, in their entirety, by reference. Thus, possible combinations include IL-2 and IL-15, IL-2 and IL-21, IL-15 and IL-21 and IL-2, IL-15 and IL-21, the latter finding utility especially in many modalities. The use of combinations of cytokines
[00697] [00697] In some cases, the bulk TIL population obtained in the first preparation expansion (which may include expansions sometimes referred to as pre-REP), including, for example, the TIL population obtained, for example, in Step B as indicated in Figure 85 (in particular, eg, Figure 85B), it can undergo a second rapid expansion (which may include expansions sometimes referred to as the Rapid Expansion Protocol (REP)) and then cryopreserved as discussed below. Likewise, if genetically modified TILs are to be used in therapy, the population of expanded TILs from the first preparation expansion or the population of expanded TILs from the second rapid expansion can be
[00698] [00698] In some embodiments, the TILs obtained in the first preparation expansion (for example, from Step B as indicated in Figure 85 (in particular, eg, Figure 85B)) are stored until phenotyped for selection. In some embodiments, the TILs obtained in the first preparation expansion (for example, from Step B as indicated in Figure 85 (in particular, eg, Figure 85B)) are not stored and proceed directly to the second rapid expansion. In some embodiments, the TILs obtained in the first preparation expansion are not cryopreserved after the first preparation expansion and before the second rapid expansion. In some embodiments, the transition from the first preparation expansion to the second expansion occurs in approximately 2 days, 3 days, 4 days, 5 days, 6 days or 7 days, since when samples or tumor fragments are added to the culture medium cell phone and / or when the first preparation expansion step starts. In some embodiments, the transition from the first preparation expansion to the second rapid expansion occurs in approximately 3 days to 7 days from when samples or fragments are added to the cell culture medium and / or when the first preparation expansion step is initiated . In some embodiments, the transition from the first expansion of preparation to the second expansion occurs in approximately 4 days to 7 days from when samples or fragments are added to the cell culture medium and / or when the first expansion expansion step is initiated. In some embodiments, the transition from the first preparation expansion to the second expansion occurs in approximately 5 days to 7 days from when samples or fragments are added to the cell culture medium and / or when the first preparation expansion step is initiated. In some modalities, the transition from
[00699] [00699] In some modalities, the transition from the first preparation expansion to the second rapid expansion occurs in 1 day, 2 days, 3 days, 4 days, 5 days, 6 days or 7 days, up to 10 days from when samples or fragments they are added to the cell culture medium and / or when the first expansion expansion step is started. In some embodiments, the transition from the first preparation expansion to the second rapid expansion occurs in 1 day to 7 days from when samples or fragments are added to the cell culture medium and / or when the first preparation expansion step is initiated. In some embodiments, the transition from the first preparation expansion to the second expansion occurs in 2 days to 7 days from when samples or fragments are added to the cell culture medium and / or when the first preparation expansion step is initiated. In some embodiments, the transition from the first preparation expansion to the second expansion occurs in 3 days to 7 days from when samples or fragments are added to the cell culture medium and / or when the first preparation expansion step is initiated. In some embodiments, the transition from the first preparation expansion to the second rapid expansion occurs in 4 days to 7 days from when samples or fragments are added to the cell culture medium and / or when the first preparation expansion step is initiated. In some embodiments, the transition from the first expansion to the second rapid expansion
[00700] [00700] In some modalities, TILs are not stored after the first primary expansion and before the second rapid expansion, and TILs proceed directly to the second rapid expansion (for example, in some modalities, there is no storage during the transition from Step B for Step D as shown in Figure 85 (in particular, eg, Figure 85B)). In some modalities, the transition occurs in a closed system, as described here. In some modalities, the TILs from the first preparation expansion, the second population of TILs, proceed directly to the second rapid expansion without a transition period.
[00701] [00701] In some embodiments, the transition from the first preparation expansion to the second rapid expansion, for example, Step C according to Figure 85 (in particular, eg, Figure 85B), is carried out in a bioreactor of closed system. In some modalities, a closed system is employed for the expansion of TIL, as described here. In some modalities, a single bioreactor is employed. In some modalities, the only bioreactor employed is, for example, a GREX-10 or a GREX-100. In some embodiments, the closed system bioreactor is a unique bioreactor. In some embodiments, the transition from the first expansion to the second rapid expansion involves scaling
[00702] [00702] In some modalities, the TILs cell population is expanded more in number after collection and the first preparation expansion, after Step A and Step B, and the transition referred to as Step C, as shown in Figure 85 (in particular, eg, Figure 85B)). This additional expansion is referred to here as the second rapid expansion, which may include expansion processes generally referred to in the art as a rapid expansion process (Rapid Expansion Protocol or REP; as well as processes as indicated in step D of Figure 85 (in particular, eg, Figure 85B)). The second rapid expansion is generally carried out using a culture medium comprising some components, including feeder cells, a source of cytokines and an anti-CD3 antibody, in a gas-permeable container. In some modalities, 1 day, 2 days, 3 days or 4 days after the start of the second rapid expansion (that is, in 8, 9, 10 or 11 days of the global Gen 3 process), the TILs are transferred to a container of higher volume.
[00703] [00703] In some embodiments, the second rapid expansion (which may include expansions sometimes referred to as REP; as well as processes as indicated in step D of Figure 85 (in particular, eg, Figure 85B)) of TILs can be performed using any TIL bottles or containers known to those skilled in the art. In some embodiments, the second expansion of TILs may continue for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days or 9 days after the start of the second rapid expansion. In some modalities, the second expansion of TILs may proceed for
[00704] [00704] In one embodiment, the second rapid expansion can be carried out in a gas-permeable container by the methods of the present invention (including, for example, expansions referred to as REP; as well as processes as indicated in step D of Figure 85 (in particular , eg, Figure 85B)). In some embodiments, TILs are expanded in the second rapid expansion in the presence of IL-2, OKT-3 and feeder cells (also referred to herein as “antigen presenting cells”). In some embodiments, TILs are expanded in the second rapid expansion in the presence of IL-2, OKT-3 and feeder cells, in which feeder cells are added to a final concentration that is twice, 2.4 times, 2.5 times, 3 times, 3.5 times or 4 times the concentration of feeder cells present in the first preparation expansion. For example, TILs can be expanded rapidly using non-specific stimulation of T cell receptors in the presence of interleukin-2 (IL -2) or interleukin-15 (IL-15). Non-specific stimulation of T cell receptors may include, for example, an anti-CD3 antibody, such as about 30 ng / mL OKT3, a mouse anti-CD3 monoclonal antibody (commercially available from Ortho-McNeil, Raritan, NJ or Miltenyi Biotech, Auburn, CA) or UHCT-1 (commercially available from BioLegend, San Diego, CA, USA). TILs can be expanded to induce
[00705] [00705] In one embodiment, the cell culture medium further comprises IL-2. In some embodiments, the cell culture medium comprises approximately 3000 IU / ml of IL-2. In one embodiment, the cell culture medium comprises approximately 1000 IU / mL, approximately 1500 IU / mL, approximately 2000 IU / mL, approximately 2500 IU / mL, approximately 3000 IU / mL, approximately 3500 IU / mL, approximately 4000 IU / mL. mL, approximately 4500 IU / mL, approximately 5000 IU / mL, approximately 5500 IU / mL, approximately 6000 IU / mL, approximately 6500 IU / mL, approximately 7000 IU / mL, approximately 7500 IU / mL or approximately 8000 IU / mL IL-2. In one embodiment, the means of
[00706] [00706] In one embodiment, the cell culture medium comprises OKT-3 antibody. In some embodiments, the cell culture medium comprises approximately 30 ng / ml of OKT-3 antibody. In one embodiment, the cell culture medium comprises approximately 0.1 ng / mL, approximately 0.5 ng / mL, approximately 1 ng / mL, approximately 2.5 ng / mL, approximately 5 ng / mL, approximately 7.5 ng / mL, approximately 10 ng / mL, approximately 15 ng / mL, approximately 20 ng / mL, approximately 25 ng / mL, approximately 30 ng / mL, approximately 35 ng / mL, approximately 40 ng / mL, approximately 50 ng / mL mL, approximately 60 ng / mL, approximately 70 ng / mL, approximately 80 ng / mL, approximately 90 ng / mL, approximately 100 ng / mL, approximately 200 ng / mL, approximately 500 ng / mL and approximately 1 µg / mL OKT-3 antibody. In one embodiment, the cell culture medium comprises between 0.1 ng / ml and 1 ng / ml, between 1 ng / ml and 5 ng / ml, between 5 ng / ml and 10 ng / ml, between 10 ng / ml and 20 ng / mL, between 20 ng / mL and 30 ng / mL, between 30 ng / mL and 40 ng / mL, between 40 ng / mL and 50 ng / mL and between 50 ng / mL and 100 ng / mL of OKT-3 antibody. In one embodiment, the cell culture medium comprises between 30 ng / ml and 60 ng / ml of OKT-3 antibody. In one embodiment, the cell culture medium comprises approximately 30 ng / ml OKT-3. In one embodiment, the cell culture medium comprises approximately 60 ng / ml OKT-3. In some embodiments, the OKT-3 antibody is muromonab.
[00707] [00707] In some embodiments, the medium in the second rapid expansion comprises IL-2. In some embodiments, the medium comprises 6000 IU / ml IL-2. In some modalities, the medium in the second rapid expansion
[00708] [00708] In some embodiments, the medium in the second rapid expansion comprises IL-2. In some embodiments, the medium comprises 6000 IU / ml IL-2. In some embodiments, the medium in the second rapid expansion comprises antigen presenting feeder cells. In some embodiments, the medium comprises between 5 x 108 and 7.5 x 108 antigen presenting feeder cells per container. In some embodiments, the medium in the second rapid expansion comprises OKT-3. In some embodiments, the medium in the second rapid expansion comprises 500 ml of culture medium and 30 µg of OKT-3 per container. In some embodiments, the container is a GREX100 MCS bottle. In some embodiments, the medium in the second rapid expansion comprises 6000 IU / ml IL-2, 60 ng / ml OKT-3 and between 5 x 108 and 7.5 x 108 antigen presenting cells. In some embodiments, the medium in the second rapid expansion comprises 500 ml of culture medium and 6000 IU / ml of IL-2, 30 µg of OKT-3 and between 5 x 108 and 7.5 x 108 antigen-presenting feeder cells per container.
[00709] [00709] In some modalities, the cell culture medium
[00710] [00710] In some embodiments, in addition to one or more TNFRSF agonists, the cell culture medium further comprises IL-2 at an initial concentration of approximately 3000 IU / mL and OKT-3 antibody at an initial concentration of approximately 30 ng / ml, and wherein one or more of the TNFRSF agonists comprises a 4-1BB agonist.
[00711] [00711] In some embodiments, a combination of IL-2, IL-7, IL-15 and / or IL-21 is employed as a combination during the second expansion. In some embodiments, IL-2, IL-7, IL-15 and / or IL-21, as well as any combinations thereof, may be included during the second expansion, including, for example, during Step D processes according to Figure 85 (in particular, e.g., Figure 85B), as well as described herein. In some embodiments, a combination of IL-2, IL-15 and IL-21 is employed as a combination during the second expansion. In some embodiments, IL-2, IL-15 and IL-21 as well as any combinations thereof can be included during Step D processes according to Figure 85 (in particular, eg, Figure 85B) and as here described.
[00712] [00712] In some embodiments, the second expansion can be conducted in a supplemented cell culture medium comprising IL-2,
[00713] [00713] In some embodiments, the second expansion culture medium comprises approximately 500 IU / ml IL-15, approximately 400 IU / ml IL-15, approximately 300 IU / ml IL-15, approximately 200 IU / ml IL-15, approximately 180 IU / mL IL-15, approximately 160 IU / mL IL-15, approximately 140 IU / mL IL-15, approximately 120 IU / mL IL-15 or approximately 100 IU / mL of IL-15. In some embodiments, the second expansion culture medium comprises approximately 500 IU / ml IL-15 to approximately 100 IU / ml IL-15. In some embodiments, the second expansion culture medium comprises approximately 400 IU / ml IL-15 to approximately 100 IU / ml IL-15. In some embodiments, the second expansion culture medium comprises approximately 300 IU / ml IL-15 to approximately 100 IU / ml IL-15. In some embodiments, the second expansion culture medium comprises approximately 200 IU / ml of IL-15. In some embodiments, the cell culture medium comprises approximately 180 IU / ml of IL-15. In one embodiment, the cell culture medium further comprises IL-15. In a preferred embodiment, the cell culture medium comprises
[00714] [00714] In some embodiments, the second expansion culture medium comprises approximately 20 IU / mL IL-21, approximately 15 IU / mL IL-21, approximately 12 IU / mL IL-21, approximately 10 IU / mL of IL-21, approximately 5 IU / mL of IL-21, approximately 4 IU / mL of IL-21, approximately 3 IU / mL of IL-21, approximately 2 IU / mL of IL-21, approximately 1 IU / mL IL-21 or approximately 0.5 IU / mL IL-21. In some embodiments, the second expansion culture medium comprises approximately 20 IU / ml IL-21 to approximately 0.5 IU / ml IL-21. In some embodiments, the second expansion culture medium comprises approximately 15 IU / ml IL-21 to approximately 0.5 IU / ml IL-21. In some embodiments, the second expansion culture medium comprises approximately 12 IU / ml IL-21 to approximately 0.5 IU / ml IL-21. In some embodiments, the second expansion culture medium comprises approximately 10 IU / ml IL-21 to approximately 0.5 IU / ml IL-21
[00715] [00715] In some embodiments, the antigen presenting feeder cells (APCs) are PBMCs. In one embodiment, the ratio of TILs to PBMCs and / or antigen presenting cells in the expansion
[00716] [00716] In one embodiment, the REP and / or the second rapid expansion is performed in flasks with the bulk TILs mixed with 100 or 200 times excess of inactivated feeder cells, where the concentration of feeder cells is at least 1, 1 times (1.1X), 1.2x, 1.3x, 1.4X, 1.5X, 1.6X, 1.7X, 1.8x, 1.8x, 2X, 2.1X2.2x, 2, 3x, 2.4X, 2.5X, 2.6X, 2.7X, 2.8x, 2.9X, 3.0X, 3.1X, 3.2x, 3.3x, 3.4X, 3.5X, 3.6X, 3.7X, 3.8x, 3.9X or 4.0X the concentration of feeder cells in the first preparation expansion, anti-CD3 antibody, OKT3, 30 ng / mL and IL-2 6000 IU / mL in 150 mL of medium. The medium is replaced (usually 2/3 of the medium is replaced by aspiration of 2/3 of the depleted medium and replacement with an equal volume of fresh medium) until the cells are transferred to an alternative growth chamber. Alternative growth chambers include G-REX flasks and gas-permeable containers as discussed more fully below.
[00717] [00717] In some modalities, the second rapid expansion (which may include processes referred to as the REP process) is 7 to 10 days, as discussed in the examples and figures. In some embodiments, the second rapid expansion (which may include processes referred to as the REP process) is 7 to 9 days, as discussed in the examples and figures. In some modalities, the second expansion is 7 days. In some modalities, the second expansion is 8 days. In some modalities, the second expansion is 9 days. In some modalities, the second expansion is 10 days.
[00718] [00718] In one embodiment, the second expansion (which may include expansions referred to as REP, as well as those referred to in step D of Figure 85 (in particular, eg, Figure 85B)) can be performed in gas-permeable flasks with 500 mL capacity and 100 cm gas permeable silicone bottom (G-Rex 100, commercially available from Wilson Wolf Manufacturing Corporation, New Brighton, MN, USA), 5 x 106 or 10 x 106 TILs can be grown with PBMCs in 400 ml of 50/50 medium, supplemented with 5% human AB serum, 3000 IU per ml of IL-2 and 30 ng per ml of anti-CD3 (OKT3). G-Rex 100 flasks can be incubated at 37 ºC in 5% CO2. On day 5, 250 mL of supernatant can be removed and placed in centrifuge bottles and centrifuged at 1500 rpm (491 x g) for 10 minutes. TIL pellets can be resuspended with 150 ml of fresh medium with 5% human AB serum, 6000 IU per ml of IL-2, and added back to the original GREX-100 vials. When TILs are expanded in series in GREX-100 vials on the 10th or 11th, TILs can be transferred to a larger vial, such as a GREX-500. The cells can be collected in 14 days of culture. The cells can be collected in 15 days of culture. The cells can be collected in 16 days of culture. In some embodiments, the medium is replaced until the cells are transferred to an alternative growth chamber. In some modalities, 2/3 of the medium is replaced by aspiration of the exhausted medium with an equal volume of
[00719] [00719] In one embodiment, the second rapid expansion (including expansions referred to as REP) is performed and further comprises a stage in which TILs are selected for superior reactivity with the tumor. Any selection method known in the art can be used. For example, the methods described in U.S. Patent Application Publication No. 2016/0010058 A1, the content of which is hereby incorporated by reference, can be used for the selection of TILs for superior reactivity with the tumor.
[00720] [00720] Optionally, a cell viability assay can be performed after the second rapid expansion (including expansions referred to as the REP expansion), using standard assays known in the art. For example, a Trypan blue exclusion assay can be performed on a sample of the bulk TILs, which selectively marks dead cells and allows to assess viability. In some embodiments, samples of TILs can be counted and viability determined using an automatic Cellometer K2 cell counter (Nexcelom Bioscience, Lawrence, MA). In some modalities, viability is determined according to the standard protocol of the Cellometer K2 Image Cytometer Automatic Cell Counter.
[00721] [00721] The various T and B lymphocyte antigen receptors are produced by somatic recombination of a limited but large number of gene segments. These gene segments: V (variable), D (diversity), J (junction) and C (constant), determine the specificity of binding and applications downstream of immunoglobulins and T cell receptors (TCRs). The present invention provides a method for generating TILs that exhibit and increase the diversity of the T cell repertoire. In some embodiments, the TILs obtained by the present method exhibit an increase in the diversity of the T cell repertoire. In some embodiments, the TILs obtained in the Monday
[00722] [00722] In some embodiments, the second rapid expansion culture medium (eg, sometimes referred to as CM2 or the second cell culture medium), comprises IL-2, OKT-3, as well as the presenting feeder cells antigen (APCs), as discussed in more detail below. In some embodiments, the second rapid expansion culture medium (eg, sometimes referred to as CM2 or the second cell culture medium), comprises IL-2 6000 IU / mL, OKT-3 30 µg / vial, as well such as 7.5 x 108 antigen presenting feeder cells (APCs), as discussed in more detail below. In some embodiments, the second rapid expansion culture medium (eg, sometimes referred to as CM2 or the second cell culture medium), comprises IL-2, OKT-3, as well as antigen presenting feeder cells ( APCs), as discussed in more detail below. In some embodiments, the second rapid expansion culture medium (eg, sometimes referred to as CM2 or the second cell culture medium), comprises IL-2
[00723] [00723] In some embodiments, the second rapid expansion, for example, Step D according to Figure 85 (in particular, eg, Figure 85B), is performed in a closed system bioreactor. In some modalities, a closed system is employed for the expansion of TIL, as described here. In some modalities, a bioreactor is employed. In some embodiments, a bioreactor is employed as the container. In some embodiments, the bioreactor employed is, for example, the G-REX-100 or the G-REX-500. In some embodiments, the bioreactor used is the G-REX-100. In some modalities, the bioreactor employed is the G-REX-
[00724] [00724] In one embodiment, the second rapid expansion procedures described here (for example, including the expansion such as those described in step D of Figure 85 (in particular, eg, Figure 85B), as well as those referred to as REP) require an excess of feeder cells during the REP expansion of TILs and / or during the second rapid expansion. In many embodiments, feeder cells are peripheral blood mononuclear cells (PBMCs) obtained from standard whole blood units from healthy blood donors. PBMCs are obtained using standard methods such as Ficoll-Paque gradient separation.
[00725] [00725] In general, allogeneic PBMCs are inactivated, either by irradiation or heat treatment, and used in REP procedures, as described in the examples, which provides an exemplary protocol for assessing incompetence in the replication of irradiated allogeneic PBMCs.
[00726] [00726] In some modalities, PBMCs are considered incompetent for replication and acceptable for use in
[00727] [00727] In some embodiments, PBMCs are considered incompetent for replication and acceptable for use in the TILs expansion procedures described here if the total number of viable cells, cultured in the presence of OKT3 and IL-2, on day 7 and day 14, has not increased since the initial number of viable cells cultured on day 0 of the REP and / or day 0 of the second expansion (ie, the day of the start of the second expansion). In some embodiments, PBMCs are cultured in the presence of OKT3 antibody 30 ng / mL and IL-2 3000 IU / mL. In some embodiments, PBMCs are cultured in the presence of OKT3 antibody 30 ng / mL and IL-2 6000 IU / mL. In some embodiments, PBMCs are cultured in the presence of OKT3 antibody 15 ng / mL and IL-2 3000 IU / mL. In some embodiments, PBMCs are cultured in the presence of OKT3 antibody 15 ng / mL and IL-2 6000 IU / mL. In some embodiments, PBMCs are cultured in the presence of OKT3 60 ng / mL and IL-2 3000 IU / mL. In some embodiments, PBMCs are cultured in the presence of OKT3 60 ng / mL and IL-2 6000 IU / mL.
[00728] [00728] In some embodiments, PBMCs are considered incompetent for replication and acceptable for use in the TILs expansion procedures described here if the total number of viable cells, cultured in the presence of OKT3 and IL-2, on day 7 and day 14, has not increased since the initial number of viable cells cultured on day 0 of the REP and / or day 0 of the second expansion (ie, the day of the start of the second expansion). In some embodiments, PBMCs are cultured in the presence of 30-OKT3 antibody 60 ng / mL and IL-2 1000-6000 IU / mL. In some embodiments, PBMCs are grown in the presence of 30-antibody
[00729] [00729] In some embodiments, the antigen-presenting feeder cells are PBMCs. In some embodiments, antigen-presenting feeder cells are artificial antigen-presenting feeder cells. In one embodiment, the ratio of TILs to antigen presenting feeder cells in the second expansion is approximately 1 to 10, approximately 1 to 25, approximately 1 to 50, approximately 1 to 100, approximately 1 to 125, approximately 1 to 150, approximately 1 for 175, approximately 1 for 200, approximately 1 for 225, approximately 1 for 250, approximately 1 for 275, approximately 1 for 300, approximately 1 for 325, approximately 1 for 350, approximately 1 for 375, approximately 1 for 400 or approximately 1 to 500. In one embodiment, the ratio of TILs to antigen presenting cells in the second expansion is between 1 to 50 and 1 to 300. In one embodiment, the ratio of TILs to antigen presenting cells in the second expansion is between 1 for 100 and 1 for 200.
[00730] [00730] In one embodiment, the second expansion procedures described here require a ratio of approximately 5 x 108 feeder cells to approximately 100 x 106 TILs. In one embodiment, the second expansion procedures described herein require a ratio of approximately 7.5 x 10 8 feeder cells to approximately 100 x 10 6 TILs. In another modality, the procedures of the second expansion
[00731] [00731] In one embodiment, the second rapid expansion procedures described here require an excess of feeder cells during the second rapid expansion. In many modalities, feeder cells are peripheral blood mononuclear cells (PBMCs) obtained from standard whole blood units from healthy allogeneic blood donors. PBMCs are obtained using standard methods such as Ficoll-Paque gradient separation. In one embodiment, artificial antigen presenting cells (aAPC) are used in place of PBMCs. In some embodiments, PBMCs are added to the second rapid expansion at twice the rate
[00732] [00732] In general, allogeneic PBMCs are inactivated, either by irradiation or heat treatment, and used in the TIL expansion procedures described here, including the exemplary procedures described in the figures and examples.
[00733] [00733] In one embodiment, artificial antigen presenting cells are used in the second rapid expansion in replacement or in combination with PBMCs.
[00734] [00734] The second rapid expansion methods described herein generally use culture media with high doses of a cytokine, in particular IL-2, as is known in the art.
[00735] [00735] Alternatively, the use of cytokine combinations for the second rapid expansion of TILs is also possible, with combinations of two or more among IL-2, IL-15 and IL-21 as is generally outlined in WO 2015/189356 and WO 2015/189357, herein expressly incorporated, in their entirety, by reference. Thus, possible combinations include IL-2 and IL-15, IL-2 and IL-21, IL-15 and IL-21 and IL-2, IL-15 and IL-21, the latter finding utility especially in many modalities. The use of combinations of cytokines specifically favors the generation of lymphocytes and, in particular, T cells as described there. E. Step E: Collection of TILs
[00736] [00736] After the second rapid expansion step, cells can be harvested. In some embodiments, TILs are collected after one, two, three, four or more expansion steps, for example, as shown in Figure 85 (in particular, eg, Figure 85B). In some modalities, TILs are collected after two expansion steps, for example, as shown in Figure 85 (in particular, eg, Figure 85B). In some
[00737] [00737] TILs can be collected in any suitable and sterile manner, including, for example, by centrifugation. Methods for collecting TILs are well known in the art and any of such known methods can be employed with the present process. In some modalities, TILS are collected using an automatic system.
[00738] [00738] Cell collectors and / or cell processing systems are commercially available from a variety of sources, including, for example, Fresenius Kabi, Tomtec Life Science, Perkin Elmer and Inotech Biosystems International, Inc. Any cell collector can be employed with the present methods. In some embodiments, the cell collector and / or cell processing system is a membrane-based cell collector. In some modalities, the collection of cells is by a cell processing system, such as the LOVO system (manufactured by Fresenius Kabi). The term "LOVO cell processing system" also refers to any instrument or device manufactured by any supplier that can pump a solution comprising cells through a membrane or filter, such as a spinning membrane or spinning filter in an environment sterile and / or closed system, allowing continuous flow and processing of cells to remove the supernatant or cell culture medium without pelletization. In some embodiments, the cell collector and / or cell processing system can perform cell separation, washing, fluid exchange, concentration, and / or other stages of cell processing in a closed, sterile system.
[00739] [00739] In some embodiments, the second rapid expansion, for example, Step D according to Figure 85 (in particular, eg, Figure
[00740] [00740] In some embodiments, Step E according to Figure 85 (in particular, eg, Figure 85B), is performed according to the processes described here. In some embodiments, access to the closed system is through syringes under sterile conditions in order to maintain the sterility and closed nature of the system. In some embodiments, a closed system as described herein is employed.
[00741] [00741] In some modalities, TILs are collected according to the methods described here. In some modalities, TILs are collected between 14 and 16 days using the methods described here. In some modalities, TILs are collected in 14 days using the methods described here. In some modalities, TILs are collected in 15 days using the methods described here. In some modalities, TILs are collected in 16 days using the methods described here. F. Step F: Final formulation / Transfer to infusion bag
[00742] [00742] After Steps A through E, as arranged in an exemplary order in Figure 85 (in particular, eg, Figure 85B) and as outlined in detail above are completed, the cells are transferred to a container for use in administration to a patient. In some embodiments, once sufficient therapeutically obtained by the expansion methods described above, the TILs are transferred to a container for use in administering to a patient.
[00743] [00743] In one modality, TILs expanded by the methods of
[00744] [00744] In one embodiment, TILs expanded by the processes described above can be administered as compositions that further comprise a cryopreservative. In one embodiment, TILs expanded by the processes described above can be administered as compositions that further comprise a cryopreservative and an isotonic agent. In one embodiment, TILs expanded by the processes described above can be administered as compositions which further comprise a cryopreservative, comprising dimethyl sulfoxide, and an isotonic agent comprising sodium chloride, sodium gluconate and sodium acetate. In one embodiment, TILs expanded by the processes described above can be administered as compositions that further comprise a cryopreservative, comprising dimethyl sulfoxide and dextran 40, and an isotonic agent comprising sodium chloride, sodium gluconate and sodium acetate. In one embodiment, TILs expanded by the processes described above can be administered as compositions delivered in a sterile infusion pouch, wherein such compositions further comprise a cryopreservative, comprising dimethylsulfoxide and dextran 40, and an isotonic agent comprising sodium chloride, sodium gluconate and sodium acetate. G. Reasons for PBMP-feeder cells
[00745] [00745] In some embodiments, the culture medium used in the expansion methods described here (see, for example, Figure 85 (in
[00746] [00746] In one embodiment, the number of feeder PBMC layers is calculated as follows: A. T cell volume (10 µm in diameter): V = (4/3) πr3 = 523.6 µm3 B. G column -Rex 100 (M) 40 µm (4 cells) high: V = (4/3) πr3 = 4x1012 µm3 C. Number of cells required to fill column B: 4x1012 µm3 / 523.6 µm3 = 7.6x108 µm3 * 0.64 = 4.86x108 D. Number of cells that can be optimally activated in 4D space: 4.86x108 / 24 = 20.25x106 E. Number of feeders and TIL extrapolated to G-Rex 500: TIL: 100x106 and Feeder: 2.5x109
[00747] [00747] In this calculation, an approximation of the number of mononuclear cells required to provide an icosahedron geometry for TIL activation in a cylinder with a base of 100 cm2 is used. The calculation derives from the experimental result of ~ 5x108 for the T cell activation limit that closely reflects experimental NCI data. (1) (C) The multiplier (0.64) is the random packing density for equivalent spheres (2) as calculated by Jaeger and Nagel in 1992. (D) The divisor 24 is the number of equivalent spheres that could come in contact with a similar object in four-dimensional space "Newton's number" (3). (1)
[00748] [00748] Jin, Jianjian, et. al., Simplified Method of the Growth of Human Tumor Infiltrating Lymphocytes (TIL) in Gas-Permeable Flasks to
[00749] [00749] Jaeger HM, Nagel SR. Physics of the granular state. Science. 1992 Mar 20; 255 (5051): 1523-31. (3)
[00750] [00750] O. R. Musin (2003). "The problem of the twenty-five spheres". Russ. Math. Surv. 58 (4): 794-795.
[00751] [00751] In one embodiment, the number of antigen presenting feeder cells provided exogenously during the first preparation expansion is approximately half the number of antigen presenting feeder cells provided exogenously during the second rapid expansion. In certain embodiments, the method comprises performing the first expansion of preparation in a cell culture medium which comprises approximately 50% less antigen presenting cells compared to the cell culture medium of the second rapid expansion.
[00752] [00752] In another embodiment, the number of antigen presenting feeder cells (APCs) supplied exogenously during the second rapid expansion is greater than the number of APCs provided exogenously during the first preparation expansion.
[00753] [00753] In another embodiment, the ratio of the number of APCs supplied exogenously during the second rapid expansion to the number of APCs provided exogenously during the first preparation expansion is selected from a range of equal to or close to 1.1: 1 to equal or close to 20: 1.
[00754] [00754] In another embodiment, the ratio of the number of APCs provided exogenously during the second rapid expansion to the number of APCs provided exogenously during the first expansion of preparation is selected from a range of equal to or close to 1.1: 1 to equal or close to 10: 1.
[00755] [00755] In another modality, the reason for the number of APCs provided
[00756] [00756] In another embodiment, the ratio of the number of APCs supplied exogenously during the second rapid expansion to the number of APCs supplied exogenously during the first preparation expansion is selected from the range of equal to or close to 1.1: 1 to equal or close to 8: 1.
[00757] [00757] In another embodiment, the ratio of the number of APCs supplied exogenously during the second rapid expansion to the number of APCs provided exogenously during the first preparation expansion is selected from the range of equal to or close to 1.1: 1 to equal or close to 7: 1.
[00758] [00758] In another embodiment, the ratio of the number of APCs provided exogenously during the second rapid expansion to the number of APCs provided exogenously during the first expansion of preparation is selected from a range of equal to or close to 1.1: 1 to equal or close to 6: 1.
[00759] [00759] In another embodiment, the ratio of the number of APCs supplied exogenously during the second rapid expansion to the number of APCs supplied exogenously during the first preparation expansion is selected from the range of equal to or close to 1.1: 1 to equal or close to 5: 1.
[00760] [00760] In another embodiment, the ratio of the number of APCs supplied exogenously during the second rapid expansion to the number of APCs provided exogenously during the first expansion of preparation is selected from a range of equal or close to 1.1: 1 to equal or close to 4: 1.
[00761] [00761] In another embodiment, the ratio of the number of APCs supplied exogenously during the second rapid expansion to the number of APCs supplied exogenously during the first preparation expansion) is selected from a range of equal to or close to 1.1: 1 to equal to or close to 3: 1.
[00762] [00762] In another embodiment, the ratio of the number of APCs provided exogenously during the second rapid expansion to the number of APCs provided exogenously during the first preparation expansion is selected from a range of equal to or close to 1.1: 1 to equal or close to 2.9: 1.
[00763] [00763] In another embodiment, the ratio of the number of APCs supplied exogenously during the second rapid expansion to the number of APCs provided exogenously during the first preparation expansion is selected from a range of equal or close to 1.1: 1 to equal or close to 2.8: 1.
[00764] [00764] In another embodiment, the ratio of the number of APCs provided exogenously during the second rapid expansion to the number of APCs provided exogenously during the first expansion of preparation is selected from a range of equal to or close to 1.1: 1 to equal or close to 2.7: 1.
[00765] [00765] In another embodiment, the ratio of the number of APCs provided exogenously during the second rapid expansion to the number of APCs provided exogenously during the first expansion of preparation is selected from a range of equal or close to 1.1: 1 to equal or close to 2.6: 1.
[00766] [00766] In another embodiment, the ratio of the number of APCs supplied exogenously during the second rapid expansion to the number of APCs provided exogenously during the first preparation expansion is selected from a range of equal or close to 1.1: 1 to equal or
[00767] [00767] In another embodiment, the ratio of the number of APCs supplied exogenously during the second rapid expansion to the number of APCs provided exogenously during the first preparation expansion is selected from a range of equal to or close to 1.1: 1 to equal or close to 2.4: 1.
[00768] [00768] In another embodiment, the ratio of the number of APCs provided exogenously during the second rapid expansion to the number of APCs provided exogenously during the first expansion of preparation is selected from a range of equal to or close to 1.1: 1 to equal or close to 2.3: 1.
[00769] [00769] In another embodiment, the ratio of the number of APCs provided exogenously during the second rapid expansion to the number of APCs provided exogenously during the first preparation expansion is selected from a range of equal to or close to 1.1: 1 to equal or close to 2.2: 1.
[00770] [00770] In another embodiment, the ratio of the number of APCs provided exogenously during the second rapid expansion to the number of APCs provided exogenously during the first preparation expansion is selected from a range of equal to or close to 1.1: 1 to equal or close to 2.1: 1.
[00771] [00771] In another embodiment, the ratio of the number of APCs supplied exogenously during the second rapid expansion to the number of APCs supplied exogenously during the first preparation expansion is selected from the range of equal to or close to 1.1: 1 to equal or close to 2: 1.
[00772] [00772] In another embodiment, the ratio of the number of APCs supplied exogenously during the second rapid expansion to the number of APCs supplied exogenously during the first preparation expansion is
[00773] [00773] In another embodiment, the ratio of the number of APCs provided exogenously during the second rapid expansion to the number of APCs provided exogenously during the first preparation expansion is selected from a range of equal to or close to 2: 1 to equal to or close to to 5: 1.
[00774] [00774] In another embodiment, the ratio of the number of APCs provided exogenously during the second rapid expansion to the number of APCs provided exogenously during the first expansion of preparation is selected from a range of equal or close to 2: 1 to equal or close to 4: 1.
[00775] [00775] In another embodiment, the ratio of the number of APCs provided exogenously during the second rapid expansion to the number of APCs provided exogenously during the first expansion of preparation is selected from a range of equal or close to 2: 1 to equal or close to 3: 1.
[00776] [00776] In another embodiment, the ratio of the number of APCs supplied exogenously during the second rapid expansion to the number of APCs provided exogenously during the first preparation expansion is selected from a range of equal or close to 2: 1 to equal or close to 2.9: 1.
[00777] [00777] In another embodiment, the ratio of the number of APCs supplied exogenously during the second rapid expansion to the number of APCs supplied exogenously during the first preparation expansion is selected from a range of equal to or close to 2: 1 to equal or close 2.8: 1.
[00778] [00778] In another embodiment, the ratio of the number of APCs provided exogenously during the second rapid expansion to the number of APCs
[00779] [00779] In another embodiment, the ratio of the number of APCs provided exogenously during the second rapid expansion to the number of APCs provided exogenously during the first preparation expansion is selected from a range of equal or close to 2: 1 to equal or close to 2.6: 1.
[00780] [00780] In another embodiment, the ratio of the number of APCs supplied exogenously during the second rapid expansion to the number of APCs provided exogenously during the first preparation expansion is selected from a range of equal to or close to 2: 1 to equal to or close to to 2.5: 1.
[00781] [00781] In another embodiment, the ratio of the number of APCs provided exogenously during the second rapid expansion to the number of APCs provided exogenously during the first preparation expansion is selected from a range of equal or close to 2: 1 to equal or close to 2.4: 1.
[00782] [00782] In another embodiment, the ratio of the number of APCs supplied exogenously during the second rapid expansion to the number of APCs provided exogenously during the first preparation expansion is selected from a range of equal to or close to 2: 1 to equal or close to 2.3: 1.
[00783] [00783] In another embodiment, the ratio of the number of APCs supplied exogenously during the second rapid expansion to the number of APCs provided exogenously during the first preparation expansion is selected from a range of equal or close to 2: 1 to equal or close to 2.2: 1.
[00784] [00784] In another modality, the ratio of the number of APCs provided
[00785] [00785] In another embodiment, the ratio of the number of APCs supplied exogenously during the second rapid expansion to the number of APCs provided exogenously during the first expansion of preparation is equal to or close to 2: 1.
[00786] [00786] In another embodiment, the ratio of the number of APCs supplied exogenously during the second rapid expansion to the number of APCs provided exogenously during the first preparation expansion is equal to or close to 1.1: 1, 1.2: 1, 1.3: 1, 1.4: 1, 1.5: 1, 1.6: 1, 1.7: 1, 1.8: 1, 1.9: 1, 2: 1, 2.1: 1, 2.2: 1, 2.3: 1, 2, 4: 1, 2.5: 1, 2.6: 1, 2.7: 1, 2.8: 1, 2.9: 1, 3: 1, 3.1: 1, 3.2: 1,
[00787] [00787] In another embodiment, the number of APCs supplied exogenously during the first expansion of preparation is equal to or close to 1 x 108, 1.1 x 108, 1.2 x 108, 1.3 x 108, 1.4 x 108, 1.5 x 108, 1.6 x 108, 1.7 x 108, 1.8 x 108, 1.9 x 108, 2 x 108, 2.1 x 108, 2.2 x 108, 2, 3 x 108, 2.4 x 108, 2.5 x 108, 2.6 x 108, 2.7 x 108, 2.8 x 108, 2.9 x 108, 3 x 108, 3.1 x 108, 3.2 x 108, 3.3 x 108, 3.4 x 108 or 3.5 x 108 APCs, and the number of APCs provided exogenously during the second rapid expansion is equal to or close to 3.5 x 108, 3, 6 x 108, 3.7 x 108, 3.8 x 108, 3.9 x 108, 4 x 108, 4.1 x 108, 4.2 x 108, 4.3 x 108, 4.4 x 108, 4.5 x 108, 4.6 x 108, 4.7 x 108, 4.8 x 108, 4.9 x 108, 5 x 108, 5.1 x 108, 5.2 x 108, 5.3 x 108, 5.4 x 108, 5.5 x 108, 5.6 x 108, 5.7 x 108, 5.8 x 108, 5.9 x 108, 6 x 108, 6.1 x 108, 6, 2 x 108, 6.3 x 108, 6.4 x 108, 6.5 x 108, 6.6 x 108, 6.7 x 108, 6.8 x 108, 6.9 x 108, 7 x 108, 7.1 x 108, 7.2 x 108, 7.3 x 108, 7.4 x 108, 7.5 x 108, 7.6 x 108, 7.7 x 108, 7.8 x 108, 7, 9 x 108, 8 x 108, 8.1 x 108, 8.2 x 108, 8.3 x 108, 8.4 x 108, 8.5 x 108, 8.6 x 108, 8.7 x 108, 8, 8 x 108,
[00788] [00788] In another embodiment, the number of APCs supplied exogenously during the first preparation expansion is selected from the range of equal to or close to 1.5 x 108 APCs to equal to or close to 3 x 108 APCs, and the number of APCs exogenously supplied during the second rapid expansion is selected from the range of 4 x 108 APCs or close to or close to 7.5 x 108 APCs.
[00789] [00789] In another embodiment, the number of APCs supplied exogenously during the first preparation expansion is selected from the range of equal to or close to 2 x 108 APCs to equal to or close to 2.5 x 108 APCs, and the number of APCs exogenously supplied during the second rapid expansion is selected from the range of equal to or close to 4.5 x 108 APCs to or equal to 5.5 x 108 APCs.
[00790] [00790] In another embodiment, the number of APCs supplied exogenously during the first preparation expansion is equal to or close to 2.5 x 108 APCs, and the number of APCs provided exogenously during the second rapid expansion is equal to or close to 5 x 108 APCs.
[00791] [00791] In one embodiment, the number of APCs (including, for example, PBMCs) added on day 0 of the first preparation expansion is approximately half the number of PBMCs added on day 7 of the first preparation expansion (e.g. , day 7 of the method). In certain embodiments, the method comprises adding antigen presenting cells on day 0 of the first preparation expansion to the first TIL population and adding antigen presenting cells on day 7 to the second TIL population, where the number of antigen presenting cells added on day 0 is approximately 50% of the number of antigen presenting cells added on day 7 of the first preparation expansion (eg, day 7 of the method).
[00792] [00792] In another embodiment, the number of APCs (including, for example, PBMCs) provided exogenously on day 7 of the second rapid expansion is greater than the number of PBMCs provided exogenously on day 0 of the first preparation expansion.
[00793] [00793] In another embodiment, the APCs supplied exogenously in the first preparation expansion are sown in the culture flask at a density selected from a range of 1.0 x 106 APCs / cm2 or equal to or close to 4.5 x 106 APCs / cm2.
[00794] [00794] In another modality, the APCs supplied exogenously in the first preparation expansion are seeded in the culture flask at a selected density within a range of 1.5 x 106 APCs / cm2 or equal to or close to 3.5 x 106 APCs / cm2.
[00795] [00795] In another embodiment, the APCs provided exogenously in the first preparation expansion are seeded in the culture flask at a density selected from an interval equal to or close to 2 x 106 APCs / cm2 at or close to 3 x 106 APCs / cm2.
[00796] [00796] In another embodiment, the APCs supplied exogenously in the first preparation expansion are seeded in the culture flask at a density equal to or close to 2 x 106 APCs / cm2.
[00797] [00797] In another embodiment, the APCs provided exogenously in the first preparation expansion are seeded in the culture flask at a density equal to or close to 1.0 x 106, 1.1 x 106, 1.2 x 106, 1.3 x 106, 1.4 x 106, 1.5 x 106, 1.6 x 106, 1.7 x 106, 1.8 x 106, 1.9 x 106, 2 x 106, 2.1 x 106, 2 , 2 x 106, 2.3 x 106, 2.4 x 106, 2.5 x 106, 2.6 x 106, 2.7 x 106, 2.8 x 106, 2.9 x 106, 3 x 106 , 3.1 x 106, 3.2 x 106, 3.3 x 106, 3.4 x 106, 3.5 x 106, 3.6 x 106, 3.7 x 106, 3.8 x 106, 3 , 9 x 106, 4 x 106, 4.1 x 106, 4.2 x 106, 4.3 x 106, 4.4 x 106 or 4.5 x 106 APCs / cm2.
[00798] [00798] In another embodiment, the APCs supplied exogenously in the second rapid expansion are sown in the culture flask at a density
[00799] [00799] In another modality, the APCs supplied exogenously in the second rapid expansion are sown in the culture flask at a selected density within a range of equal to or close to 3.5 x 106 APCs / cm2 at approximately 6.0 x 106 APCs / cm2.
[00800] [00800] In another embodiment, the APCs supplied exogenously in the second rapid expansion are seeded in the culture flask at a selected density from a range of 4.0 x 106 APCs / cm2 or approximately 5.5 x 106 APCs / cm2.
[00801] [00801] In another modality, the APCs supplied exogenously in the second rapid expansion are sown in the culture flask at a selected density from a range of equal to or close to 4.0 x 106 APCs / cm2.
[00802] [00802] In another embodiment, the APCs supplied exogenously in the second rapid expansion are sown in the culture flask at a density equal to or close to 2.5 x 106 APCs / cm2, 2.6 x 106 APCs / cm2, 2.7 x 106 APCs / cm2, 2.8 x 106, 2.9 x 106, 3 x 106, 3.1 x 106, 3.2 x 106, 3.3 x 106, 3.4 x 106, 3.5 x 106 , 3.6 x 106, 3.7 x 106, 3.8 x 106, 3.9 x 106, 4 x 106, 4.1 x 106, 4.2 x 106, 4.3 x 106, 4.4 x 106, 4.5 x 106, 4.6 x 106, 4.7 x 106, 4.8 x 106, 4.9 x 106, 5 x 106, 5.1 x 106, 5.2 x 106, 5 , 3 x 106, 5.4 x 106, 5.5 x 106, 5.6 x 106, 5.7 x 106, 5.8 x 106, 5.9 x 106, 6 x 106, 6.1 x 106 , 6.2 x 106, 6.3 x 106, 6.4 x 106, 6.5 x 106, 6.6 x 106, 6.7 x 106, 6.8 x 106, 6.9 x 106, 7 x 106, 7.1 x 106, 7.2 x 106, 7.3 x 106, 7.4 x 106 or 7.5 x 106 APCs / cm2.
[00803] [00803] In another embodiment, the APCs provided exogenously in the first preparation expansion are seeded in the culture flask at a density equal to or close to 1.0 x 106, 1.1 x 106, 1.2 x 106, 1.3 x 106, 1.4 x 106, 1.5 x 106, 1.6 x 106, 1.7 x 106, 1.8 x 106, 1.9 x 106, 2 x 106, 2.1 x 106, 2 , 2 x 106, 2.3 x 106, 2.4 x 106, 2.5 x 106, 2.6 x 106, 2.7 x 106, 2.8 x 106, 2.9 x 106, 3 x 106 , 3.1 x 106, 3.2 x 106, 3.3 x 106, 3.4 x 106, 3.5 x 106, 3.6 x 106, 3.7 x
[00804] [00804] In another embodiment, the APCs supplied exogenously in the first expansion of preparation are seeded in the culture flask at a selected density within a range of 1.0 x 106 APCs / cm2 or equal to or close to 4.5 x 106 APCs / cm2, and the APCs provided exogenously in the second rapid expansion are seeded in the culture flask at a selected density within a range of 2.5 x 106 APCs / cm2 at or near 7.5 x 106 APCs / cm2.
[00805] [00805] In another embodiment, the APCs supplied exogenously in the first preparation expansion are sown in the culture flask at a selected density within a range of 1.5 x 106 APCs / cm2 at or near 3.5 x 106 APCs / cm2, and the APCs supplied exogenously in the second rapid expansion are seeded in the culture flask at a selected density within a range of equal to or close to 3.5 x 106 APCs / cm2 at or near 6 x 106 APCs / cm2.
[00806] [00806] In another modality, the APCs supplied exogenously in the first preparation expansion are sown in the culture flask at a selected density within a range of equal to or close to 2 x 106 APCs / cm2 at or close to 3 x 106 APCs / cm2, and the APCs provided
[00807] [00807] In another embodiment, the APCs provided exogenously in the first preparation expansion are seeded in the culture flask at a density equal to or close to 2 x 106 APCs / cm2 and the APCs provided exogenously in the second rapid expansion are seeded in the culture flask at a density equal to or close to 4 x 106 APCs / cm2.
[00808] [00808] In another embodiment, the ratio of the number of APCs (including, for example, PBMCs) provided exogenously on day 7 of the second rapid expansion to the number of PBMCs provided exogenously on day 0 of the first preparation expansion is selected from an interval from equal to or close to 1.1: 1 to equal to or close to 20: 1.
[00809] [00809] In another embodiment, the ratio of the number of APCs (including, for example, PBMCs) provided exogenously on day 7 of the second rapid expansion to the number of PBMCs provided exogenously on day 0 of the first preparation expansion is selected from an interval from equal to or close to 1.1: 1 to equal to or close to 10: 1.
[00810] [00810] In another embodiment, the ratio of the number of APCs (including, for example, PBMCs) provided exogenously on day 7 of the second rapid expansion to the number of PBMCs provided exogenously on day 0 of the first preparation expansion is selected from an interval from equal to or close to 1.1: 1 to equal to or close to 9: 1.
[00811] [00811] In another embodiment, the ratio of the number of APCs (including, for example, PBMCs) provided exogenously on day 7 of the second rapid expansion to the number of APCs (including, for example, PBMCs) provided exogenously on day 0 of the first preparation expansion is selected from a range of equal to or close to 1.1: 1 to equal to or close to 8: 1.
[00812] [00812] In another embodiment, the ratio of the number of APCs (including, for example, PBMCs) provided exogenously on day 7 of the second rapid expansion to the number of APCs (including, for example, PBMCs) provided exogenously on day 0 of the first preparation expansion is selected from a range of equal to or close to 1.1: 1 to equal to or close to 7: 1.
[00813] [00813] In another embodiment, the ratio of the number of APCs (including, for example, PBMCs) provided exogenously on day 7 of the second rapid expansion to the number of APCs (including, for example, PBMCs) provided exogenously on day 0 of the first preparation expansion is selected from a range of equal to or close to 1.1: 1 to equal to or close to 6: 1.
[00814] [00814] In another embodiment, the ratio of the number of APCs (including, for example, PBMCs) provided exogenously on day 7 of the second rapid expansion to the number of APCs (including, for example, PBMCs) provided exogenously on day 0 of the first preparation expansion is selected from a range of equal to or close to 1.1: 1 to equal to or close to 5: 1.
[00815] [00815] In another embodiment, the ratio of the number of APCs (including, for example, PBMCs) provided exogenously on day 7 of the second rapid expansion to the number of APCs (including, for example, PBMCs) provided exogenously on day 0 of the first preparation expansion is selected from a range of equal to or close to 1.1: 1 to equal to or close to 4: 1.
[00816] [00816] In another embodiment, the ratio of the number of APCs (including, for example, PBMCs) provided exogenously on day 7 of the second rapid expansion to the number of APCs (including, for example, PBMCs) provided exogenously on day 0 of the first preparation expansion is selected from a range of equal to or close to 1.1: 1 to equal to or
[00817] [00817] In another embodiment, the ratio of the number of APCs (including, for example, PBMCs) provided exogenously on day 7 of the second rapid expansion to the number of APCs (including, for example, PBMCs) provided exogenously on day 0 of the first preparation expansion is selected from a range of equal to or close to 1.1: 1 to equal to or close to 2.9: 1.
[00818] [00818] In another embodiment, the ratio of the number of APCs (including, for example, PBMCs) provided exogenously on day 7 of the second rapid expansion to the number of APCs (including, for example, PBMCs) provided exogenously on day 0 of the first preparation expansion is selected from a range of equal to or close to 1.1: 1 to equal to or close to 2.8: 1.
[00819] [00819] In another embodiment, the ratio of the number of APCs (including, for example, PBMCs) provided exogenously on day 7 of the second rapid expansion to the number of APCs (including, for example, PBMCs) provided exogenously on day 0 of the first preparation expansion is selected from a range of equal to or close to 1.1: 1 to equal to or close to 2.7: 1.
[00820] [00820] In another embodiment, the ratio of the number of APCs (including, for example, PBMCs) provided exogenously on day 7 of the second rapid expansion to the number of APCs (including, for example, PBMCs) provided exogenously on day 0 of the first preparation expansion is selected from a range of equal to or close to 1.1: 1 to equal to or close to 2.6: 1.
[00821] [00821] In another embodiment, the ratio of the number of APCs (including, for example, PBMCs) provided exogenously on day 7 of the second rapid expansion to the number of APCs (including, for example, PBMCs) provided exogenously on day 0 of the first preparation expansion is
[00822] [00822] In another embodiment, the ratio of the number of APCs (including, for example, PBMCs) provided exogenously on day 7 of the second rapid expansion to the number of APCs (including, for example, PBMCs) provided exogenously on day 0 of the first preparation expansion is selected from a range of equal to or close to 1.1: 1 to equal to or close to 2.4: 1.
[00823] [00823] In another embodiment, the ratio of the number of APCs (including, for example, PBMCs) provided exogenously on day 7 of the second rapid expansion to the number of APCs (including, for example, PBMCs) provided exogenously on day 0 of the first preparation expansion is selected from a range of equal to or close to 1.1: 1 to equal to or close to 2.3: 1.
[00824] [00824] In another embodiment, the ratio of the number of APCs (including, for example, PBMCs) provided exogenously on day 7 of the second rapid expansion to the number of APCs (including, for example, PBMCs) provided exogenously on day 0 of the first preparation expansion is selected from a range of equal to or close to 1.1: 1 to equal to or close to 2.2: 1.
[00825] [00825] In another embodiment, the ratio of the number of APCs (including, for example, PBMCs) provided exogenously on day 7 of the second rapid expansion to the number of APCs (including, for example, PBMCs) provided exogenously on day 0 of the first preparation expansion is selected from a range of equal to or close to 1.1: 1 to equal to or close to 2.1: 1.
[00826] [00826] In another embodiment, the ratio of the number of APCs (including, for example, PBMCs) provided exogenously on day 7 of the second rapid expansion to the number of APCs (including, for example, PBMCs)
[00827] [00827] In another embodiment, the ratio of the number of APCs (including, for example, PBMCs) provided exogenously on day 7 of the second rapid expansion to the number of APCs (including, for example, PBMCs) provided exogenously on day 0 of the first preparation expansion is selected from a range of equal to or close to 2: 1 to equal to or close to 10: 1.
[00828] [00828] In another embodiment, the ratio of the number of APCs (including, for example, PBMCs) provided exogenously on day 7 of the second rapid expansion to the number of APCs (including, for example, PBMCs) provided exogenously on day 0 of the first preparation expansion is selected from a range of equal to or close to 2: 1 to equal to or close to 5: 1.
[00829] [00829] In another embodiment, the ratio of the number of APCs (including, for example, PBMCs) provided exogenously on day 7 of the second rapid expansion to the number of APCs (including, for example, PBMCs) provided exogenously on day 0 of the first preparation expansion is selected from a range of equal to or close to 2: 1 to equal to or close to 4: 1.
[00830] [00830] In another embodiment, the ratio of the number of APCs (including, for example, PBMCs) provided exogenously on day 7 of the second rapid expansion to the number of APCs (including, for example, PBMCs) provided exogenously on day 0 of the first preparation expansion is selected from a range of equal to or close to 2: 1 to equal to or close to 3: 1.
[00831] [00831] In another embodiment, the ratio of the number of APCs (including, for example, PBMCs) provided exogenously on day 7 of the second
[00832] [00832] In another embodiment, the ratio of the number of APCs (including, for example, PBMCs) provided exogenously on day 7 of the second rapid expansion to the number of APCs (including, for example, PBMCs) provided exogenously on day 0 of the first preparation expansion is selected from a range of equal to or close to 2: 1 to equal to or close to 2.8: 1.
[00833] [00833] In another embodiment, the ratio of the number of APCs (including, for example, PBMCs) provided exogenously on day 7 of the second rapid expansion to the number of APCs (including, for example, PBMCs) provided exogenously on day 0 of the first preparation expansion is selected from a range of equal to or close to 2: 1 to equal to or close to 2.7: 1.
[00834] [00834] In another embodiment, the ratio of the number of APCs (including, for example, PBMCs) provided exogenously on day 7 of the second rapid expansion to the number of APCs (including, for example, PBMCs) provided exogenously on day 0 of the first preparation expansion is selected from a range of equal to or close to 2: 1 to equal to or close to 2.6: 1.
[00835] [00835] In another embodiment, the ratio of the number of APCs (including, for example, PBMCs) provided exogenously on day 7 of the second rapid expansion to the number of APCs (including, for example, PBMCs) provided exogenously on day 0 of the first preparation expansion is selected from a range of equal to or close to 2: 1 to equal to or close to 2.5: 1.
[00836] [00836] In another modality, the ratio of the number of APCs (including,
[00837] [00837] In another embodiment, the ratio of the number of APCs (including, for example, PBMCs) provided exogenously on day 7 of the second rapid expansion to the number of APCs (including, for example, PBMCs) provided exogenously on day 0 of the first preparation expansion is selected from a range of equal to or close to 2: 1 to equal to or near 2.3: 1.
[00838] [00838] In another embodiment, the ratio of the number of APCs (including, for example, PBMCs) provided exogenously on day 7 of the second rapid expansion to the number of APCs (including, for example, PBMCs) provided exogenously on day 0 of the first preparation expansion is selected from a range of equal to or close to 2: 1 to equal to or close to 2.2: 1.
[00839] [00839] In another embodiment, the ratio of the number of APCs (including, for example, PBMCs) provided exogenously on day 7 of the second rapid expansion to the number of APCs (including, for example, PBMCs) provided exogenously on day 0 of the first Preparation expansion is selected from a range of 2: 1 or close to 2.1: 1.
[00840] [00840] In another embodiment, the ratio of the number of APCs (including, for example, PBMCs) provided exogenously on day 7 of the second rapid expansion to the number of APCs (including, for example, PBMCs) provided exogenously on day 0 of the first preparation expansion is equal to or close to 2: 1.
[00841] [00841] In another modality, the ratio of the number of APCs (including,
[00842] [00842] In another embodiment, the number of APCs (including, for example, PBMCs) supplied exogenously on day 0 of the first preparation expansion is equal to or close to 1 x 108, 1.1 x 108, 1.2 x 108, 1.3 x 108, 1.4 x 108, 1.5 x 108, 1.6 x 108, 1.7 x 108, 1.8 x 108, 1.9 x 108, 2 x 108, 2.1 x 108, 2.2 x 108, 2.3 x 108, 2.4 x 108, 2.5 x 108, 2.6 x 108, 2.7 x 108, 2.8 x 108, 2.9 x 108, 3 x 108, 3.1 x 108, 3.2 x 108, 3.3 x 108, 3.4 x 108 or 3.5 x 108 APCs (including, for example, PBMCs), and the number of APCs (including , for example, PBMCs) delivered exogenously on day 7 of the second rapid expansion is equal to or close to 3.5 x 108, 3.6 x 108, 3.7 x 108, 3.8 x 108, 3.9 x 108, 4 x 108, 4.1 x 108, 4.2 x 108, 4.3 x 108, 4.4 x 108, 4.5 x 108, 4.6 x 108, 4.7 x 108, 4.8 x 108, 4.9 x 108, 5 x 108, 5.1 x 108, 5.2 x 108, 5.3 x 108, 5.4 x 108, 5.5 x 108, 5.6 x 108, 5, 7 x 108, 5.8 x 108, 5.9 x 108, 6 x 108, 6.1 x 108, 6.2 x 108, 6.3 x 108, 6.4 x 108, 6.5 x 108, 6.6 x 108, 6.7 x 108, 6.8 x 108, 6.9 x 108, 7 x 108, 7.1 x 108, 7.2 x 108, 7.3 x 108, 7.4 x 108, 7.5 x 108, 7.6 x 108, 7.7 x 108, 7.8 x 108, 7.9 x 108, 8 x 108, 8.1 x 108, 8.2 x 108, 8.3 x 108, 8.4 x 108, 8.5 x 108, 8.6 x 108, 8.7 x 108, 8.8 x 108, 8.9 x 108, 9 x 108, 9.1 x 108, 9.2 x 108, 9.3 x 108, 9.4 x 108, 9.5 x 108, 9, 6 x 108, 9.7 x 108, 9.8 x 108, 9.9 x 108 or 1 x 109 APCs (including, for example, PBMCs).
[00843] [00843] In another embodiment, the number of APCs (including, for example, PBMCs) provided exogenously on day 0 of the first preparation expansion is selected from the range of equal to or close to 1 x 108 APCs (including, for example, PBMCs ) equal to or close to 3.5 x 108 APCs
[00844] [00844] In another embodiment, the number of APCs (including, for example, PBMCs) provided exogenously on day 0 of the first preparation expansion is selected from the range of 1.5 x 108 APCs at or near 3 x 108 APCs (including, for example, PBMCs), and the number of APCs (including, for example, PBMCs) provided exogenously on day 7 of the second rapid expansion is selected from the range of equal to or close to 4 x 108 APCs ( including, for example, PBMCs) at or near 7.5 x 108 APCs (including, for example, PBMCs).
[00845] [00845] In another embodiment, the number of APCs (including, for example, PBMCs) provided exogenously on day 0 of the first preparation expansion is selected from the range of equal to or close to 2 x 108 APCs (including, for example, PBMCs ) at or near 2.5 x 108 APCs (including, for example, PBMCs), and the number of APCs (including, for example, PBMCs) provided exogenously on day 7 of the second rapid expansion is selected from the equal range or close to 4.5 x 108 APCs (including, for example, PBMCs) at or close to 5.5 x 108 APCs (including, for example, PBMCs).
[00846] [00846] In another embodiment, the number of APCs (including, for example, PBMCs) provided exogenously on day 0 of the first preparation expansion is equal to or close to 2.5 x 108 APCs (including, for example, PBMCs) and the number of APCs (including, for example, PBMCs) provided exogenously on day 7 of the second rapid expansion is equal to or close to 5 x 108 APCs (including, for example, PBMCs).
[00847] [00847] In one embodiment, the number of APC layers (including, for example, PBMCs) added on day 0 of the first preparation expansion is approximately half the number of APC layers (including, for example, PBMCs) added in day 7 of the second rapid expansion. In certain embodiments, the method comprises adding layers of antigen presenting cells on day 0 of the first preparation expansion to the first population of TILs and adding layers of antigen presenting cells on day 7 to the second population of TILs, where the number of layers of antigen presenting cells added on day 0 is approximately 50% of the number of layers of antigen presenting cells added on day 7.
[00848] [00848] In another embodiment, the number of layers of APCs (including, for example, PBMCs) provided exogenously on day 7 of the second rapid expansion is greater than the number of layers of APCs (including, for example, PBMCs) provided exogenously on day 0 of the first preparation expansion.
[00849] [00849] In another embodiment, day 0 of the first preparation expansion occurs in the presence of APCs arranged in layers (including, for example, PBMCs) with an average thickness equal to that of 2 layers of cells and day 7 of the second expansion rapid occurs in the presence of APCs arranged in layers (including, for example, PBMCs) with an average thickness equal to or close to that of 4 cell layers.
[00850] [00850] In another embodiment, day 0 of the first preparation expansion occurs in the presence of APCs arranged in layers (including, for example, PBMCs) with an average thickness equal to or close to that of a cell layer and day 7 of the second rapid expansion occurs in the presence of APCs arranged in layers (including, for example, PBMCs) with an average thickness equal to or close to that of 3 layers of cells.
[00851] [00851] In another modality, day 0 of the first expansion of
[00852] [00852] In another embodiment, day 0 of the first preparation expansion occurs in the presence of APCs arranged in layers (including, for example, PBMCs) with an average thickness equal to or close to that of a cell layer and day 7 of the second rapid expansion occurs in the presence of APCs arranged in layers (including, for example, PBMCs) with an average thickness equal to that of 2 layers of cells.
[00853] [00853] In another embodiment, day 0 of the first preparation expansion occurs in the presence of APCs arranged in layers (including, for example, PBMCs) with an average thickness equal to or close to 1; 1.1; 1.2, 1.3; 1.4; 1.5; 1.6; 1.7; 1.8; 1.9; two; 2.1; 2.2, 2.3; 2.4; 2.5; 2.6; 2.7; 2.8; 2.9 or 3 layers of cells and day 7 of the second rapid expansion occurs in the presence of layered APCs (including, for example, PBMCs) with an average thickness equal to or close to 3.1; 3.2; 3.3; 3.4; 3.5; 3.6; 3.7; 3.8; 3.9; 4; 4.1; 4.2; 4.3; 4.4; 4.5; 4.6; 4.7; 4.8; 4.9; 5; 5.1; 5.2; 5.3; 5.4; 5.5; 5.6; 5.7; 5.8; 5.9; 6; 6.1; 6.2; 6.3; 6.4; 6.5; 6.6; 6.7; 6.8; 6.9; 7; 7.1; 7.2; 7.3; 7.4; 7.5; 7.6; 7.7; 7.8; 7,9 or 8 layers of cells.
[00854] [00854] In another embodiment, day 0 of the first preparation expansion occurs in the presence of APCs arranged in layers (including, for example, PBMCs) with an average thickness equal to or close to that of 1 layer of cells equal to or close to that of 2 layers of cells and day 7 of the second rapid expansion occurs in the presence of APCs arranged in layers (including, for example, PBMCs) with an average thickness equal to or close to 3 layers of cells equal to or close to 10 layers of
[00855] [00855] In another embodiment, day 0 of the first preparation expansion takes place in the presence of APCs arranged in layers (including, for example, PBMCs) with an average thickness equal to that of 2 layers of cells equal to or close to that of 3 layers of cells and day 7 of the second rapid expansion occurs in the presence of APCs arranged in layers (including, for example, PBMCs) with an average thickness equal to or close to 4 layers of cells equal to or close to 8 layers of cells .
[00856] [00856] In another embodiment, day 0 of the first preparation expansion occurs in the presence of APCs arranged in layers (including, for example, PBMCs) with an average thickness equal to that of 2 layers of cells and day 7 of the second expansion rapid occurs in the presence of APCs arranged in layers (including, for example, PBMCs) with an average thickness equal to or close to 4 layers of cells at or close to 8 layers of cells.
[00857] [00857] In another embodiment, day 0 of the first preparation expansion occurs in the presence of APCs arranged in layers (including, for example, PBMCs) with an average thickness equal to or close to 1, 2 or 3 layers of cells and day 7 of the second rapid expansion occurs in the presence of APCs arranged in layers (including, for example, PBMCs) with an average thickness equal to or close to 3, 4, 5, 6, 7, 8, 9 or 10 layers of cells.
[00858] [00858] In another embodiment, day 0 of the first preparation expansion occurs in the presence of APCs arranged in layers (including, for example, PBMCs) with a first average thickness equal to a first number of APC layers (including, for example) , PBMCs) and day 7 of the second rapid expansion occurs in the presence of layered APCs (including, for example, PBMCs) with a second average thickness equal to
[00859] [00859] In another embodiment, day 0 of the first preparation expansion takes place in the presence of layered APCs (including, for example, PBMCs) with a first average thickness equal to a first number of APC layers (including, for example) , PBMCs) and day 7 of the second rapid expansion occurs in the presence of layered APCs (including, for example, PBMCs) with a second average thickness equal to a second number of APC layers (including, for example, PBMCs), wherein the ratio of the first number of APC layers (including, for example, PBMCs) to the second number of APC layers (including, for example, PBMCs) is selected from the range of equal to or close to 1: 1.1 equal to or close to 1: 8.
[00860] [00860] In another embodiment, day 0 of the first preparation expansion occurs in the presence of APCs arranged in layers (including, for example, PBMCs) with a first average thickness equal to a first number of layers of APCs (including, for example , PBMCs) and day 7 of the second rapid expansion occurs in the presence of layered APCs (including, for example, PBMCs) with a second average thickness equal to a second number of APC layers (including, for example, PBMCs), wherein the ratio of the first number of APC layers (including, for example, PBMCs) to the second number of APC layers (including, for example, PBMCs) is selected from the range of equal to or close to 1: 1.1 equal to or close to 1: 7.
[00861] [00861] In another modality, day 0 of the first preparation expansion occurs in the presence of APCs arranged in layers (including, for example,
[00862] [00862] In another embodiment, day 0 of the first preparation expansion occurs in the presence of APCs arranged in layers (including, for example, PBMCs) with a first average thickness equal to a first number of layers of APCs (including, for example , PBMCs) and day 7 of the second rapid expansion occurs in the presence of layered APCs (including, for example, PBMCs) with a second average thickness equal to a second number of APC layers (including, for example, PBMCs), wherein the ratio of the first number of APC layers (including, for example, PBMCs) to the second number of APC layers (including, for example, PBMCs) is selected from the range of equal to or close to 1: 1.1 equal to or close to 1: 5.
[00863] [00863] In another embodiment, day 0 of the first preparation expansion occurs in the presence of APCs arranged in layers (including, for example, PBMCs) with a first average thickness equal to a first number of layers of APCs (including, for example , PBMCs) and day 7 of the second rapid expansion occurs in the presence of layered APCs (including, for example, PBMCs) with a second average thickness equal to a second number of APC layers (including, for example, PBMCs), wherein the ratio of the first number of APC layers (including, for example, PBMCs) to the second number of APC layers (including,
[00864] [00864] In another embodiment, day 0 of the first preparation expansion occurs in the presence of APCs arranged in layers (including, for example, PBMCs) with a first average thickness equal to a first number of layers of APCs (including, for example , PBMCs) and day 7 of the second rapid expansion occurs in the presence of layered APCs (including, for example, PBMCs) with a second average thickness equal to a second number of APC layers (including, for example, PBMCs), wherein the ratio of the first number of APC layers (including, for example, PBMCs) to the second number of APC layers (including, for example, PBMCs) is selected from the range of equal to or close to 1: 1.1 equal to or close to 1: 3.
[00865] [00865] In another embodiment, day 0 of the first preparation expansion occurs in the presence of APCs arranged in layers (including, for example, PBMCs) with a first average thickness equal to a first number of layers of APCs (including, for example , PBMCs) and day 7 of the second rapid expansion occurs in the presence of layered APCs (including, for example, PBMCs) with a second average thickness equal to a second number of APC layers (including, for example, PBMCs), wherein the ratio of the first number of APC layers (including, for example, PBMCs) to the second number of APC layers (including, for example, PBMCs) is selected from the range of equal to or close to 1: 1.1 equal to or close to 1: 2.
[00866] [00866] In another embodiment, day 0 of the first preparation expansion takes place in the presence of layered APCs (including, for example, PBMCs) with a first average thickness equal to a first number of APC layers (including, for example) , PBMCs) and day 7 of the second rapid expansion occurs in the presence of layered APCs
[00867] [00867] In another embodiment, day 0 of the first preparation expansion occurs in the presence of APCs arranged in layers (including, for example, PBMCs) with a first average thickness equal to a first number of layers of APCs (including, for example , PBMCs) and day 7 of the second rapid expansion occurs in the presence of layered APCs (including, for example, PBMCs) with a second average thickness equal to a second number of APC layers (including, for example, PBMCs), where the ratio of the first number of APC layers (including, for example, PBMCs) to the second number of APC layers (including, for example, PBMCs) is selected from the range of equal to or close to 1: 1.3 equal to or close to 1: 7.
[00868] [00868] In another embodiment, day 0 of the first preparation expansion occurs in the presence of APCs arranged in layers (including, for example, PBMCs) with a first average thickness equal to a first number of APC layers (including, for example , PBMCs) and day 7 of the second rapid expansion occurs in the presence of layered APCs (including, for example, PBMCs) with a second average thickness equal to a second number of APC layers (including, for example, PBMCs), wherein the ratio of the first number of APC layers (including, for example, PBMCs) to the second number of APC layers (including, for example, PBMCs) is selected from the range of 1: 1.4 equal to or close to 1: 6.
[00869] [00869] In another modality, day 0 of the first expansion of
[00870] [00870] In another embodiment, day 0 of the first preparation expansion occurs in the presence of APCs arranged in layers (including, for example, PBMCs) with a first average thickness equal to a first number of layers of APCs (including, for example , PBMCs) and day 7 of the second rapid expansion occurs in the presence of layered APCs (including, for example, PBMCs) with a second average thickness equal to a second number of APC layers (including, for example, PBMCs), wherein the ratio of the first number of APC layers (including, for example, PBMCs) to the second number of APC layers (including, for example, PBMCs) is selected from the range of 1: 1.6 equal to or close to 1: 4.
[00871] [00871] In another embodiment, day 0 of the first preparation expansion occurs in the presence of APCs arranged in layers (including, for example, PBMCs) with a first average thickness equal to a first number of APC layers (including, for example , PBMCs) and day 7 of the second rapid expansion occurs in the presence of layered APCs (including, for example, PBMCs) with a second average thickness equal to a second number of APC layers (including, for example, PBMCs), where the reason for the first number of APC layers (including, for example,
[00872] [00872] In another embodiment, day 0 of the first preparation expansion occurs in the presence of layered APCs (including, for example, PBMCs) with a first average thickness equal to a first number of APC layers (including, for example) , PBMCs) and day 7 of the second rapid expansion occurs in the presence of layered APCs (including, for example, PBMCs) with a second average thickness equal to a second number of APC layers (including, for example, PBMCs), wherein the ratio of the first number of APC layers (including, for example, PBMCs) to the second number of APC layers (including, for example, PBMCs) is selected from the range of 1: 1.8 equal to or close to 1: 3.
[00873] [00873] In another embodiment, day 0 of the first preparation expansion occurs in the presence of APCs arranged in layers (including, for example, PBMCs) with a first average thickness equal to a first number of layers of APCs (including, for example , PBMCs) and day 7 of the second rapid expansion occurs in the presence of layered APCs (including, for example, PBMCs) with a second average thickness equal to a second number of APC layers (including, for example, PBMCs), where the ratio of the first number of APC layers (including, for example, PBMCs) to the second number of APC layers (including, for example, PBMCs) is selected from the range of 1: 1.9 equal to or close to 1: 2.5.
[00874] [00874] In another embodiment, day 0 of the first preparation expansion occurs in the presence of APCs arranged in layers (including, for example, PBMCs) with a first average thickness equal to a first number of layers of APCs (including, for example , PBMCs) and day 7 of
[00875] [00875] In another embodiment, day 0 of the first preparation expansion occurs in the presence of APCs arranged in layers (including, for example, PBMCs) with a first average thickness equal to a first number of APC layers (including, for example , PBMCs) and day 7 of the second rapid expansion occurs in the presence of layered APCs (including, for example, PBMCs) with a second average thickness equal to a second number of APC layers (including, for example, PBMCs), wherein the ratio of the first number of APC layers (including, for example, PBMCs) to the second number of APC layers (including, for example, PBMCs) is selected at or close to 1: 1.1; 1: 1.2; 1: 1.3; 1: 1.4; 1: 1.5; 1: 1.6; 1: 1.7; 1: 1.8; 1: 1.9; 1: 2; 1: 2.1; 1: 2.2; 1: 2.3; 1: 2.4; 1: 2.5; 1: 2.6; 1: 2.7; 1: 2.8; 1: 2.9; 1: 3; 1: 3.1; 1: 3.2; 1: 3.3; 1: 3.4; 1: 3.5; 1: 3.6; 1: 3.7; 1: 3.8; 1: 3.9; 1: 4; 1: 4.1; 1: 4.2; 1: 4.3; 1: 4.4; 1: 4.5; 1: 4.6; 1: 4.7; 1: 4.8; 1: 4.9; 1: 5; 1: 5.1; 1: 5.2; 1: 5.3; 1: 5.4; 1: 5.5; 1: 5.6; 1: 5.7; 1: 5.8; 1: 5.9; 1: 6; 1: 6.1; 1: 6.2; 1: 6.3; 1: 6.4; 1: 6.5; 1: 6.6; 1: 6.7; 1: 6.8; 1: 6.9; 1: 7; 1: 7.1; 1: 7.2; 1: 7.3; 1: 7.4; 1: 7.5; 1: 7.6; 1: 7.7; 1: 7.8; 1: 7.9; 1: 8; 1: 8.1; 1: 8.2; 1: 8.3; 1: 8.4; 1: 8.5; 1: 8.6; 1: 8.7; 1: 8.8; 1: 8.9; 1: 9; 1: 9.1; 1: 9.2; 1: 9.3; 1: 9.4; 1: 9.5; 1: 9.6; 1: 9.7; 1: 9.8; 1: 9,9 or 1:10. V. Optional components of the cellular environment
[00876] [00876] In some embodiments of Process 2A, the culture medium used in the expansion methods described herein (including those referred to as REP, see, for example, Figure 1) also includes an anti-
[00877] [00877] As will be recognized by those skilled in the art, there are a number of suitable anti-human CD3 antibodies which find use in the invention, including polyclonal and monoclonal anti-human CD3 antibodies to various mammals, including, but not limited to, murine, human, antibodies primates, rats and canines. In particular embodiments, the OKT3 anti-CD3 antibody is used (commercially available from Ortho-McNeil, Raritan, NJ or Miltenyi Biotech, Auburn, CA). Table 4: Muromonabe amino acid sequences (exemplary OKT-3 antibody) Identifier Sequence (One-letter amino acid symbols) SEQ ID NO: 1 QVQLQQSGAE LARPGASVKM SCKASGYTFT RYTMHWVKQR Heavy chain
[00878] [00878] In one embodiment, the TNFRSF agonist is a 4-1BB agonist (CD137). The 4-1BB agonist can be any 4-1BB-binding molecule known in the art. The 4-1BB binding molecule can be a
[00879] [00879] In a preferred embodiment, the 4-1BB agonist or 4-1BB binding molecule can also be a fusion protein. In a preferred embodiment, a 4-1BB multimeric agonist, such as a 4-1BB trimeric or hexameric agonist (with three or six ligand-binding domains), can induce superior clustering (aggregation) with the receptor (4- 1BBL) and formation of the internal cell signaling complex when compared to an agonist monoclonal antibody, which typically has two ligand binding domains. Trimeric (trivalent) or hexameric (hexavalent) or larger fusion proteins comprising three TNFRSF and IgG1-Fc binding domains and optionally linking two or more of these fusion proteins are described, e.g. e.g., in Gieffers, et al., Mol. Cancer Therapeutics 2013, 12, 2735-47.
[00880] [00880] 4-1BB agonist fusion antibodies and proteins are known to induce strong immune responses. In a preferred embodiment, the 4-1BB agonist is a monoclonal antibody or fusion protein that specifically binds to a 4-1BB antigen sufficiently to reduce toxicity. In some embodiments, the 4-1BB agonist is a monoclonal antibody or 4-1BB agonist fusion protein that cancels antibody dependent cell toxicity (ADCC), for example, NK cell cytotoxicity. In some embodiments, the 4-1BB agonist is a monoclonal antibody or 4-1BB agonist fusion protein that nullifies antibody-dependent cell phagocytosis (ADCP). In some
[00881] [00881] In some modalities, 4-1BB agonists are characterized by binding to human 4-1BB (SEQ ID NO: 9) with high affinity and agonist activity. In one embodiment, the 4-1BB agonist is a binding molecule that binds to human 4-1BB (SEQ ID NO: 9). In one embodiment, the 4-1BB agonist is a binding molecule that binds to the murine 4-1BB (SEQ ID NO: 10). The amino acid sequences of the 4-1BB antigen to which a 4-1BB agonist or binding molecule is attached are summarized in Table DD. Table 5. 4-1BB Antigen Amino Acid Sequences Identifier Sequence (One-letter symbols of amino acids) SEQ ID NO: 9 4- MGNSCYNIVA TLLLVLNFER TRSLQDPCSN CPAGTFCDNN 1BB human, RNQICSPCPP NSFSSAGGQR 60 120 tumor necrosis CFGTFNDQKR GICRPWTNCS LDGKSVLVNG TKERDVVCGP member 9 (Homo sapiens SPADLSPGAS SVTPPAPARE 180) PGHSPQIISF FLALTSTALL FLLFFLTLRF SVVKRGRKKL LYIFKQPFMR PVQTTQEEDG CSCRFPEEEE GGCEL 255 240 SEQ ID NO: 10 MGNNCYNVVV 4- 1BB IVLLLVGCEK VGAVQNSCDN CQPGTFCRKY murine NPVCKSCPPS TFSSIGGQPN 60 Superfamily CNICRVCAGY FRFKKFCSST HNAECECIEG FHCLGPQCTR tumor necrosis factor CEKDCRPGQE LTKQGCKTCS 120 receptors, LGTFNDQNGT GVCRPWTNCS LDGRSVLKTG TTEKDVVCGP member 9 (Mus PVVSFSPSTT ISVTPEGGTKLGKTKKFKKLKKFKKFKKKKKKKKKKKKKKKKLKKKKKKKKKKKKKKKKKKKKKKKKKLKLQQQ ...
[00882] [00882] In some embodiments, the described compositions, processes and methods include a 4-1BB agonist that binds to human or murine 4-1BB with KD close to or less than 100 pM, binds to human or murine 4-1BB with KD close to or below 90 pM, binds to human or murine 4-1BB with KD close to or below 80 pM, binds to human or murine 4-1BB with KD close to or less than 70 pM, binds to 4-1BB
[00883] [00883] In some embodiments, the compositions, processes and methods described include a 4-1BB agonist that binds to human or murine 4-1BB with kassoc close to 7.5 x 105 1 / M · s or faster, binds - if human or murine 4-1BB with kassoc close to 7.5 x 105 1 / M · s or faster, binds to human or murine 4-1BB with kassoc close to 8 x 105 l / M · s or faster, binds to human or murine 4-1BB with kassoc close to 8.5 x 105 1 / M · s or faster, binds to human or murine 4-1BB with kassoc close to 9 x 105 1 / M · s or faster, binds to human or murine 4-1BB with kassoc close to 9.5 x 105 1 / M · s or faster or binds to human or murine 4-1BB with kassoc close to 1 x 106 1 / M · s or faster.
[00884] [00884] In some embodiments, the described compositions, processes and methods include a 4-1BB agonist that binds to human or murine 4-1BB with kdissoc close to 2 x 10-5 1 / s or slower, binds human or murine 4-1BB with kdissoc close to 2.1 x 10-5 1 / s or slower, binds to human or murine 4-1BB with kdissoc close to 2.2 x 10-5 1 / s or slower, binds to human or murine 4-1BB with kdissoc close to 2.3 x 10-5 1 / s or slower, binds to human or murine 4-1BB with kdissoc close to 2.4 x 10 -5 1 / s or slower, binds to human or murine 4-1BB with kdissoc close to 2.5 x 10-5 1 / s or slower, binds to human or murine 4-1BB with close kdissoc at 2.6 x 10-5 1 / s or slower or binds to human or murine 4- 1BB with kdissoc close to 2.7 x 10-5 1 / s or slower, binds to 4-1BB human or murine with kdissoc close to 2.8 x 10-5 1 / s or slower, binds to human or murine with kdissoc close to 2.9 x 10-5 1 / s or slower, or binds to human 4-1BB or murine with kdissoc close to 3 x 10-5 1 / s or slower.
[00885] [00885] In some embodiments, the described compositions, processes and methods include a 4-1BB agonist that binds to human or murine 4-1BB with an IC50 close to or less than 10 nM, binds to human or murine 4-1BB with IC50 close to or below 9 nM, binds to human or murine 4-1BB with IC50 close to or below 8 nM, binds to human or murine 4-1BB with close to or below 7 nM IC50, binds to human or murine 4-1BB with IC50 close to or below 6 nM, binds to human or murine 4-1BB with IC50 close to or below 5 nM, binds to human or murine 4-1BB with close to or below IC50 at 4 nM, binds to human or murine 4-1BB with IC50 close to or less than 3 nM, binds to human or murine 4-1BB with IC50 close to or less than 2 nM, or binds to 4-1BB human or murine with IC50 close to or less than 1 nM.
[00886] [00886] In a preferred embodiment, the 4-1BB agonist is utomilumab, also known as PF-05082566 or MOR-7480, or a fragment, derivative, variant or biosimilar thereof. Utomilumab is available from Pfizer, Inc. Utomilumab is a G2-lambda immunoglobulin, anti- [TNFRSF9 of Homo sapiens (member of the tumor necrosis factor (TNFR) receptor superfamily, 4-1BB, T cell ILA antigen, CD137) ], monoclonal (entirely human) antibody to Homo sapiens. The amino acid sequences of utomilumab are shown in Table EE. Utomilumab comprises glycosylation sites on Asn59 and Asn292; intrachain disulfide bridges - heavy chain at positions 22-96 (VH-VL), 143-199 (CH1-CL), 256-316 (CH2) and 362-420 (CH3); intrachain disulfide bridges - light chain at positions 22'-87 '(VH-VL) and 136'-195' (CH1-CL); disulfide bridges interchanges heavy-heavy chain at positions 218- 218, 219-219, 222-222 and 225-225 of the IgG2A isoform, at positions 218-130, 219-219, 222-222, and 225-225 of the IgG2A isoform / B and positions 219-130 (2), 222-222 and 225-225 of the IgG2B isoform; and disulfide bridges interchain heavy-light chain at positions 130-213 'of the IgG2A isoform (2), at
[00887] [00887] In one embodiment, a 4-1BB agonist comprises a heavy chain provided by SEQ ID NO: 11 and a light chain provided by SEQ ID NO: 12. In one embodiment, a 4-1BB agonist comprises heavy and light chains with the sequences shown in SEQ ID NO: 11 and SEQ ID NO: 12, respectively, or antigen-binding fragments, Fab fragments, unique fragments of the variable chain ( scFv), variants or conjugates thereof. In one embodiment, a 4-1BB agonist comprises heavy and light chains that are each at least 99% identical to the sequences shown in SEQ ID NO: 11 and SEQ ID NO: 12, respectively. In one embodiment, an agonist of 4-1BB heavy and light chains that are each at least 98% identical to the sequences shown in SEQ ID NO: 11 and SEQ ID NO: 12, respectively. In one embodiment, a 4-1BB agonist comprises heavy and light chains that are each at least 97% identical to the sequences shown in SEQ ID NO: 11 and SEQ ID NO: 12, respectively. In one embodiment, a 4- 1BB agonist comprises heavy and light chains that are each at least 96% identical to the sequences shown in SEQ ID NO: 11 and SEQ ID NO: 12, respectively. In one embodiment, a 4-1BB agonist comprises
[00888] [00888] In one embodiment, the 4-1BB agonist comprises the heavy and light chain CDRs or the variable regions (VRs) of utomilumab. In one embodiment, the heavy chain variable (VH) region of the 4-1BB agonist comprises the sequence shown in SEQ ID NO: 13, and the light chain variable region (VL) of the 4-1BB agonist comprises the sequence shown in SEQ ID NO: 14, and conservative amino acid substitutions thereof. In one embodiment, a 4-1BB agonist comprises VH and VL regions that are each at least 99% identical to the sequences shown in SEQ ID NO: 13 and SEQ ID NO: 14, respectively. In one embodiment, a 4-1BB agonist comprises VH and VL regions that are each at least 98% identical to the sequences shown in SEQ ID NO: 13 and SEQ ID NO: 14, respectively. In one embodiment, a 4- 1BB agonist comprises VH and VL regions that are each at least 97% identical to the sequences shown in SEQ ID NO: 13 and SEQ ID NO: 14, respectively. In one embodiment, a 4-1BB agonist comprises VH and VL regions that are each at least 96% identical to the sequences shown in SEQ ID NO: 13 and SEQ ID NO: 14, respectively. In one embodiment, a 4-1BB agonist comprises VH and VL regions that are each at least 95% identical to the sequences shown in SEQ ID NO: 13 and SEQ ID NO: 14, respectively. In one embodiment, a 4- 1BB agonist comprises a scFV antibody comprising VH and VL regions that are each at least 99% identical to the sequences shown in SEQ ID NO: 13 and SEQ ID NO: 14.
[00889] [00889] In one embodiment, a 4-1BB agonist comprises the heavy chain CDR1, CDR2 and CDR3 domains having the sequences shown in SEQ ID NO: 15, SEQ ID NO: 16 and SEQ ID NO: 17, respectively, and conservative amino acid substitutions thereof, and
[00890] [00890] In one embodiment, the 4-1BB agonist is a 4-1BB agonist biosimilar monoclonal antibody, approved by the drug regulatory authorities with reference to utomilumab. In one embodiment, the biosimilar monoclonal antibody comprises an antibody against 4-1BB comprising an amino acid sequence with at least 97% sequence identity, e.g. 97%, 98%, 99% or 100% sequence identity with the amino acid sequence of a reference medicine or biological reference product and which comprises one or more post-translational modifications when compared to the reference medicine or reference biological product, where the reference medicine or reference biological product is utomilumab. In some modalities, one or more of the post-translational modifications are selected from one or more of: glycosylation, oxidation, deamidation and truncation. In some embodiments, the biosimilar is a 4-1BB agonist antibody authorized or submitted for authorization, in which the 4-1BB agonist antibody is supplied in a formulation that differs from the formulations of a reference drug or reference biological product, in that the reference medicine or reference biological product is utomilumab. The 4-1BB agonist antibody may be authorized by a drug regulatory authority, such as the United States FDA agency and / or the European Union EMA. In some embodiments, the biosimilar is provided as a composition that further comprises one or more excipients, in which one or more of the excipients are the same or different from the excipients included in a reference medicine or reference biological product, in which the reference medicine or biological reference product is utomilumab. In some modalities, the
[00891] [00891] In a preferred embodiment, the 4-1BB agonist is the monoclonal antibody urelumab, also known as BMS-663513 and 20H4.9.h4a, or a fragment, derivative, variant or biosimilar thereof. Urelumab is available from Bristol-Myers Squibb, Inc., and Creative Biolabs, Inc. Urelumab is a G4-kappa immunoglobulin, anti- [TNFRSF9 of Homo sapiens (member of the tumor necrosis factor (TNFR) receptor superfamily, 4- 1BB, T cell ILA antigen, CD137)], monoclonal (entirely human) antibody to Homo sapiens. The amino acid sequences of urelumab are shown in Table EE. Urelumab comprises N-glycosylation sites at positions 298 (and 298 ’’); intrachain disulfide bridges - heavy chain at positions 22-95 (VH-VL), 148-204 (CH1-CL), 262-322 (CH2) and 368-426 (CH3) (and at positions 22 '' - 95 '' , 148 '' - 204 '', 262 '' - 322 '' and 368 '' - 426 ''); intrachain disulfide bridges - light chain at positions 23'-88 '(VH-VL) and 136'-196' (CH1-CL) (and at positions 23 '' '- 88' '' and 136 '' '- 196' ''); disulfide bridges interchain heavy chain-heavy chain at positions 227-227 ’’ and 230-230 ’’; and disulfide bridges interchanging heavy chain-light chain at 135-216 ’and 135’ ’- 216’ ’’. The preparation and properties of urelumab and its variants and fragments are described in U.S. Patents Nos. 7,288,638 and 8,962,804, the contents of which are hereby incorporated by reference. The pre-clinical and clinical characteristics of urelumab are described in Segal, et al., Clin. Cancer Res. 2016, available at http: /dx.doi.org/10.1158/1078-0432.CCR-16-1272. Current clinical studies of urelumab in a variety of hematological indications and solid tumors include the identifiers NCT01775631, NCT02110082, NCT02253992 and NCT01471210 of the U.S. National Institutes of Health
[00892] [00892] In one embodiment, a 4-1BB agonist comprises a heavy chain provided by SEQ ID NO: 21 and a light chain provided by SEQ ID NO: 22. In one embodiment, a 4-1BB agonist comprises heavy and light chains with the sequences shown in SEQ ID NO: 21 and SEQ ID NO: 22, respectively, or antigen-binding fragments, Fab fragments, unique fragments of the variable chain ( scFv), variants or conjugates thereof. In one embodiment, a 4-1BB agonist comprises heavy and light chains that are each at least 99% identical to the sequences shown in SEQ ID NO: 21 and SEQ ID NO: 22, respectively. In one embodiment, an agonist of 4-1BB heavy and light chains that are each at least 98% identical to the sequences shown in SEQ ID NO: 21 and SEQ ID NO: 22, respectively. In one embodiment, a 4-1BB agonist comprises heavy and light chains that are each at least 97% identical to the sequences shown in SEQ ID NO: 21 and SEQ ID NO: 22, respectively. In one embodiment, a 4- 1BB agonist comprises heavy and light chains that are each at least 96% identical to the sequences shown in SEQ ID NO: 21 and SEQ ID NO: 22, respectively. In one embodiment, a 4-1BB agonist comprises heavy and light chains that are each at least 95% identical to the sequences shown in SEQ ID NO: 21 and SEQ ID NO: 22, respectively.
[00893] [00893] In one embodiment, the 4-1BB agonist comprises the heavy and light chain CDRs or the urelumab variable regions (VRs). In one embodiment, the heavy chain variable (VH) region of the 4-1BB agonist comprises the sequence shown in SEQ ID NO: 23, and the light chain variable region (VL) of the 4-1BB agonist comprises the sequence shown in SEQ ID NO: 24, and conservative amino acid substitutions thereof. In one embodiment, a 4-1BB agonist comprises VH and VL regions that are each at least 99% identical to the sequences
[00894] [00894] In one embodiment, a 4-1BB agonist comprises the heavy chain CDR1, CDR2 and CDR3 domains having the sequences shown in SEQ ID NO: 25, SEQ ID NO: 26 and SEQ ID NO: 27, respectively, and substitutions conservative amino acid domains thereof, and the light chain CDR1, CDR2 and CDR3 domains having the sequences shown in SEQ ID NO: 28, SEQ ID NO: 29 and SEQ ID NO: 30, respectively, and conservative amino acid substitutions thereof.
[00895] [00895] In one embodiment, the 4-1BB agonist is a 4-1BB agonist biosimilar monoclonal antibody, approved by drug regulatory authorities with reference to urelumab. In one embodiment, the biosimilar monoclonal antibody comprises an antibody against 4-1BB comprising an amino acid sequence with at least 97% sequence identity, e.g. 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of a drug
[00896] [00896] In one embodiment, the 4-1BB agonist is selected from the group consisting of 1D8, 3Elor, 4B4 (BioLegend 309809), H4- 1BB-M127 (BD Pharmingen 552532), BBK2 (Thermo Fisher MS621PABX), 145501 (Leinco Technologies B591), the antibody produced by the
[00897] [00897] In one embodiment, the 4-1BB agonist is a 4-1BB agonist fusion protein described in International Patent Application Publication WO 2008/025516 A1, WO 2009/007120 A1, WO 2010/003766 A1, WO 2010/010051 A1 and WO 2010/078966 A1; U.S. Patent Application Publication Nos. US 2011/0027218 A1, US 2015/0126709 A1, US 2011/0111494 A1, US 2015/0110734 A1 and US 2015/0126710 A1; and U.S. Patents Nos. 9,359,420, 9,340,599, 8,921,519 and 8,450,460, the contents of which are hereby incorporated by reference.
[00898] [00898] In one embodiment, the 4-1BB agonist is a 4-1BB agonist fusion protein as represented in Structure IA (C-terminal antibody Fc fragment fusion protein) or Structure IB (Fragment fusion protein) N-terminal antibody Fc), or a fragment, derivative, conjugate, variant or biosimilar thereof: (IA) (IB)
[00899] [00899] In structures I-A and I-B, the cylinders refer to the binding domains of individual polypeptides. Structures I-A and I-B comprise three linearly linked TNFRSF binding domains, derived from, e.g. 4-1BBL or an antibody that binds to 4-1BB, which fold to form a trivalent protein, which is then linked to a second trivalent protein via IgG1-Fc (including the CH3 and CH2 domains) is used to join two of the trivalent proteins through disulfide bonds (small elongated ovals), stabilizing the structure and providing an agonist capable of joining the intracellular signaling domains of the six receptors and signaling proteins to form a signaling complex. TNFRSF binding domains indicated as cylinders can be scFv domains comprising, e.g. eg a VH chain and a VL connected by a linker that can comprise hydrophilic residues and Gly and Ser sequences for flexibility, as well as Glu and Lys for
[00900] [00900] Domain amino acid sequences for other polypeptides of structure I-A are provided in Table GG. Preferably, the Fc domain comprises a complete constant domain (amino acids 17- 230 of SEQ ID NO: 31), the complete hinge domain (amino acids 1-16 of SEQ ID NO: 31) or a part of the hinge domain (e.g. ., amino acids 4-16 of SEQ ID NO: 31). Preferred linkers for connecting an Fc C-terminal Fc antibody can be selected from the modalities provided in SEQ ID NO: 32 to SEQ ID NO: 41, including linkers suitable for fusion of additional polypeptides. Table 8: Amino acid sequences for TNFRSF fusion proteins, including 4-1BB fusion proteins with fusion protein design with C-terminal antibody Fc fragment (structure I-A). String Identifier (One-letter symbols for amino acids) SEQ ID NO: 31 KSCDKTHTCP PCPAPELLGG PSVFLFPPKP KDTLMISRTP EVTCVVVDVS Fc Domain HEDPEVKFNW 60
[00901] [00901] Domain amino acid sequences for other polypeptides of structure IB are provided in Table 9. If an antibody Fc fragment is fused to the N-terminus of a TNRFSF fusion protein as in structure IB, the sequence of the Fc module is preferably, that shown in SEQ ID NO: 42, and the linker sequences are preferably selected from those modalities presented in SED ID NO: 43 to SEQ ID NO: 45. Table 9: Amino acid sequences for TNFRSF fusion proteins, including 4-1BB fusion proteins with fusion protein design with N-terminal antibody Fc fragment (structure I-B). String Identifier (One-letter symbols for amino acids) SEQ ID NO: 42 METDTLLLWV LLLWVPAGNG DKTHTCPPCP APELLGGPSV FLFPPKPKDT Fc Domain LMISRTPEVT 60
[00902] [00902] In one embodiment, a 4- 1BB agonist fusion protein according to structures IA or IB comprises one or more 4-1BB binding domains selected from the group consisting of a variable heavy chain and a variable light chain utomilumab, a variable heavy chain and variable light chain of urelumab, a variable heavy chain and variable light chain of utomilumab, a variable heavy chain and variable light chain selected from heavy chains
[00903] [00903] In one embodiment, a 4- 1BB agonist fusion protein according to structures I-A or I-B comprises one or more 4-1BB binding domains comprising a 4-1BBL sequence. In one embodiment, a 4-1BB agonist fusion protein according to structures I-A or I-B comprises one or more 4-1BB binding domains comprising a sequence according to SEQ ID NO: 46. In one embodiment, a 4-1BB agonist fusion protein according to structures I-A or I-B comprises one or more 4-1BB binding domains comprising a soluble 4-1BBL sequence. In one embodiment, a 4-1BB agonist fusion protein according to structures I-A or I-B comprises one or more 4-1BB binding domains comprising a sequence according to SEQ ID NO: 47.
[00904] [00904] In one embodiment, a 4- 1BB agonist fusion protein according to structures IA or IB comprises one or more 4-1BB binding domains which is a scFv domain comprising VH and VL regions which are each , at least 95% identical to the sequences shown in SEQ ID NO: 13 and SEQ ID NO: 14, respectively, in which the VH and VL domains are connected by a linker. In one embodiment, a 4-1BB agonist fusion protein according to structures IA or IB comprises one or more 4-1BB binding domains which is an scFv domain comprising VH and VL regions that are each at least 95% identical to the sequences shown in SEQ ID NO: 23 and SEQ ID NO: 24, respectively, in which the VH and VL domains are connected by a linker. In one embodiment, a 4-1BB agonist fusion protein according to structures I-A or I-B comprises one or more 4-1BB binding domains which is an scFv domain comprising VH and VL regions which are each
[00905] [00905] In one embodiment, the 4-1BB agonist is a 4-1BB agonist single-chain fusion polypeptide comprising (i) a first soluble 4-1BB binding domain, (ii) a first peptide linker, ( iii) a second soluble 4-1BB binding domain, (iv) a second peptide linker and (v) a third soluble 4-1BB binding domain,
[00906] [00906] In one embodiment, the 4-1BB agonist is a 4-1BB agonist single-chain fusion polypeptide comprising (i) a first soluble cytokine domain of the tumor necrosis factor (TNF) superfamily, (ii) a first peptide linker, (iii) a second soluble cytokine domain of the TNF superfamily, (iv) a second peptide linker and (v) a third soluble cytokine domain of the TNF superfamily, where each of the soluble cytokines of the TNF superfamily does not it contains a stem region and the first and second peptide linkers are independently 3-8 amino acids long, and each cytokine domain of the TNF superfamily is a 4-1BB binding domain.
[00907] [00907] In one embodiment, the 4-1BB agonist is a 4-1BB scFv agonist antibody comprising any of the previous VH domains linked to any of the previous VL domains.
[00908] [00908] In one embodiment, the 4-1BB agonist is the 4-1BB agonist antibody of BPS Bioscience, catalog number 79097-2,
[00909] [00909] In one embodiment, the TNFRSF agonist is an OX40 agonist (CD134). The OX40 agonist can be any OX40-binding molecule known in the art. The OX40 binding molecule can be a monoclonal antibody or fusion protein capable of binding to human or mammalian OX40. OX40 agonists or OX40 binding molecules can comprise an immunoglobulin heavy chain of any isotype (eg, IgG, IgE, IgM, IgD, IgA, and IgY), class (eg, IgG1, IgG2 , IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule. The OX40 agonist or OX40 binding molecule can have both a heavy and a light chain. In this specification, the term binding molecule also includes antibodies (including complete antibodies), monoclonal antibodies (including complete monoclonal antibodies), polyclonal antibodies, multispecific antibodies (eg, bispecific antibodies), human, humanized or chimeric antibodies and fragments of antibodies, e.g. , Fab fragments, fragments, F (ab ′), fragments produced by a Fab expression library, epitope-binding fragments of any of the above and modified forms of antibodies, e.g. , scFv molecules that bind to OX40. In one embodiment, the OX40 agonist is an antigen-binding protein that is an entirely human antibody. In one embodiment, the OX40 agonist is an antigen-binding protein that is a humanized antibody. In some embodiments, OX40 agonists for use in the methods and compositions described herein include anti-OX40 antibodies, human anti-OX40 antibodies, mouse anti-OX40 antibodies, mammalian anti-OX40 antibodies,
[00910] [00910] In a preferred embodiment, the OX40 agonist or OX40 binding molecule can also be a fusion protein. OX40 fusion proteins comprising an Fc domain fused to OX40L are described, for example, in Sadun, et al., J. Immunother. 2009, 182, 1481-89. In a preferred embodiment, an OX40 multimeric agonist, such as an OX40 trimeric or hexameric agonist (with three or six ligand-binding domains), can induce superior receptor clustering (OX40L) and internal cell signaling complex formation when compared to an agonist monoclonal antibody, which typically has two ligand-binding domains. Trimeric (trivalent) or hexameric (hexavalent) or larger fusion proteins comprising three TNFRSF and IgG1-Fc binding domains and optionally linking two or more of these fusion proteins are described, e.g. e.g., in Gieffers, et al., Mol. Cancer Therapeutics 2013, 12, 2735-47.
[00911] [00911] OX40 agonist antibodies and fusion proteins are known to induce strong immune responses. Curti, et al., Cancer Res. 2013, 73, 7189-98. In a preferred embodiment, the OX40 agonist is a monoclonal antibody or fusion protein that specifically binds to an OX40 antigen in a manner sufficient to reduce toxicity. In some embodiments, the OX40 agonist is a monoclonal antibody or
[00912] [00912] In some embodiments, OX40 agonists are characterized by binding to human OX40 (SEQ ID NO: 54) with high affinity and agonist activity. In one embodiment, the OX40 agonist is a binding molecule that binds to human OX40 (SEQ ID NO: 54). In one embodiment, the OX40 agonist is a binding molecule that binds to the murine OX40 (SEQ ID NO: 55). The amino acid sequences of the OX40 antigen to which the OX40 binding agonist or molecule binds are summarized in Table 11. Table 11: Amino acid sequences of OX40 antigens Identifier Sequence (Symbols of a letter of the amino acids) SEQ ID NO: 54 MCVGARRLGR GPCAALLLLG LGLSTVTGLH CVGDTYPSND RCCHECRPGN human OX40 GMVSRCSRSQ 60 (Homo sapiens) NTVCRPCGPG FYNDVVSSKP CKPCTWCNLR SGSERKQLCT ATQDTVCRCR AGTQPLDSYK 120
[00913] [00913] In some embodiments, the described compositions, processes and methods include an OX40 agonist that binds to OX40
[00914] [00914] In some embodiments, the described compositions, processes and methods include an OX40 agonist that binds to human or murine OX40 with kassoc close to 7.5 x 105 1 / M · s or faster, binds to OX40 human or murine with kassoc close to 7.5 x 105 1 / M · s or faster, binds to OX40 human or murine with kassoc close to 8 x 105 1 / M · s or faster, binds to OX40 human or murine with kassoc close to 8.5 x 105 1 / M · s or faster, binds to OX40 human or murine with kassoc close to 9 x 105 1 / M · s or faster, binds to OX40 human or murine with kassoc close to 9.5 x 105 1 / M · s or faster or binds to human or murine OX40 with kassoc close to 1 x 106 1 / M · s or faster.
[00915] [00915] In some embodiments, the described compositions, processes and methods include an OX40 agonist that binds to human or murine OX40 with kdissoc close to 2 x 10-5 1 / s or slower, binds to human OX40 or murine with kdissoc close to 2.1 x 10-5 1 / s or slower, binds to human OX40 or murine with kdissoc close to 2.2 x 10-5 1 / s or slower, binds to OX40 human or murine with kdissoc close to 2.3 x 10-5 1 / s or slower, binds to human OX40 or murine with kdissoc close to 2.4 x 10-5 1 / s or slower, binds human or murine OX40 with kdissoc close to 2.5 x 10-5 1 / s or slower, binds to human or murine OX40 with kdissoc close to 2.6 x 10-5 1 / s or slower or alloy to human or murine OX40 with kdissoc close to 2.7 x 10-5 1 / s or slower, binds to OX40
[00916] [00916] In some embodiments, the described compositions, processes and methods include an OX40 agonist that binds to human or murine OX40 with an IC50 close to or below 10 nM, binds to human or murine OX40 with an IC50 close to or below 9 nM, binds to human or murine OX40 with an IC50 close to or less than 8 nM, binds to human or murine OX40 with an IC50 close to or less than 7 nM, binds to human or murine OX40 with close or less IC50 at 6 nM, binds to human or murine OX40 with an IC50 close to or below 5 nM, binds to human or murine OX40 with an IC50 close to or less than 4 nM, binds to human or murine OX40 with close to or less than 3 nM, binds to human or murine OX40 with an IC50 close to or less than 2 nM or binds to human or murine OX40 with an IC50 close to or less than 1 nM.
[00917] [00917] In some modalities, the OX40 agonist is tavolixizumab, also known as MEDI0562 or MEDI-0562. Tavolixizumab provided by AstraZeneca, Inc.'s MedImmune subsidiary Tavolixizumab is a G1-kappa immunoglobulin, humanized and chimeric monoclonal antibody against Homo sapiens [TNFRSF4 (member 4 of the tumor necrosis factor receptor (TNFR) superfamily, OX40, CD134) ]. The amino acid sequences of tavolixizumab are shown in Table KK. Tavolixizumab comprises N-glycosylation sites at positions 301 and 301 ’’, with fucosylated glycan complexes of the CHO type; intrachain disulfide bridges - heavy chain at positions 22-95 (VH-VL), 148-204 (CH1-CL), 265-325 (CH2) and 371-429 (CH3) (and at positions 22 '' - 95 '' , 148 '' - 204 '', 265 '' - 325 '', and 371 '' - 429 ''); intrachain disulfide bridges - light chain at positions 23'-88 '(VH-VL) and 134'-194'
[00918] [00918] In one embodiment, an OX40 agonist comprises a heavy chain provided by SEQ ID NO: 56 and a light chain provided by SEQ ID NO: 57. In one embodiment, an OX40 agonist comprises heavy and light chains with the sequences shown in SEQ ID NO: 56 and SEQ ID NO: 57, respectively, or antigen-binding fragments, Fab fragments, unique fragments of the variable chain (scFv) , variants or conjugates thereof. In one embodiment, an OX40 agonist comprises heavy and light chains that are each at least 99% identical to the sequences shown in SEQ ID NO: 56 and SEQ ID NO: 57, respectively. In one embodiment, heavy and light chains of an OX40 agonist that are each at least 98% identical to the sequences shown in SEQ ID NO: 56 and SEQ ID NO: 57, respectively. In one embodiment, an OX40 agonist comprises heavy and light chains that are each at least 97% identical to the sequences shown in SEQ ID NO: 56 and SEQ ID NO: 57, respectively. In one embodiment, an OX40 agonist comprises heavy and light chains that are each at least 96% identical to the sequences shown in SEQ ID NO: 56 and SEQ ID NO: 57, respectively. In one embodiment, an OX40 agonist comprises heavy and light chains that are each at least 95% identical to the sequences shown in SEQ ID NO: 56 and SEQ ID NO: 57, respectively.
[00919] [00919] In one embodiment, the OX40 agonist comprises the heavy and light chain CDRs or the variable regions (VRs) of
[00920] [00920] In one embodiment, an OX40 agonist comprises the heavy chain CDR1, CDR2 and CDR3 domains having the sequences shown in SEQ ID NO: 60, SEQ ID NO: 61 and SEQ ID NO: 62, respectively, and conservative substitutions amino acid domains, and the light chain CDR1, CDR2 and CDR3 domains having the sequences shown in SEQ ID NO: 63, SEQ ID NO: 64 and SEQ ID NO: 65, respectively, and conservative amino acid substitutions thereof.
[00921] [00921] In one embodiment, the OX40 agonist is an antibody
[00922] [00922] In some embodiments, the OX40 agonist is 11D4, which is an entirely human antibody available from Pfizer, Inc. The preparation and properties of 11D4 are described in U.S. Patent Nos. 7,960,515; 8,236,930; and 9 028 824, the contents of which are hereby incorporated by reference. The 11D4 amino acid sequences are shown in Table 13.
[00923] [00923] In one embodiment, an OX40 agonist comprises a heavy chain provided by SEQ ID NO: 66 and a light chain provided by SEQ ID NO: 67. In one embodiment, an OX40 agonist comprises heavy and light chains with the sequences shown in SEQ ID NO: 66 and SEQ ID NO: 67, respectively, or antigen binding fragments, Fab fragments, unique fragments of the variable chain (scFv) , variants or conjugates thereof. In one embodiment, an OX40 agonist comprises heavy and light chains that are each at least 99% identical to the sequences shown in SEQ ID NO: 66 and SEQ ID NO: 67, respectively. In one embodiment, heavy and light chains of an OX40 agonist that are each at least 98% identical to the sequences shown in SEQ ID NO: 66 and SEQ ID NO: 67, respectively. In one embodiment, an OX40 agonist comprises heavy and light chains that are each at least 97% identical to the sequences shown in SEQ ID NO: 66 and SEQ ID NO: 67, respectively. In one embodiment, an OX40 agonist comprises heavy and light chains that are each at least 96% identical to the sequences shown in SEQ ID NO: 66 and SEQ ID NO: 67, respectively. In one embodiment, an OX40 agonist comprises heavy and light chains that are each at least 95% identical to the sequences shown in SEQ ID NO: 66 and SEQ ID NO: 67, respectively.
[00924] [00924] In one embodiment, the OX40 agonist comprises the heavy and light chain CDRs or the variable regions (VRs) of 11D4. In one embodiment, the OX40 agonist variable region of the heavy chain (VH) comprises the sequence shown in SEQ ID NO: 68, and the OX40 agonist variable region of the light chain (VL) comprises the sequence shown in SEQ ID NO: 69 , and conservative amino acid substitutions thereof. In one embodiment, an OX40 agonist comprises VH and VL regions that are each at least 99% identical to the sequences shown in SEQ ID NO: 68 and SEQ ID NO: 69, respectively. In one embodiment, an OX40 agonist comprises VH and VL regions that are each at least 98% identical to the sequences shown in SEQ ID NO: 68 and SEQ ID NO: 69, respectively. In one embodiment, an OX40 agonist comprises VH and VL regions that are each at least 97% identical to the sequences shown in SEQ ID NO: 68 and SEQ ID NO: 69, respectively. In one embodiment, an OX40 agonist comprises VH and VL regions that are each at least 96% identical to the sequences shown in SEQ ID NO: 68 and SEQ ID NO: 69, respectively. In one embodiment, an OX40 agonist comprises VH and VL regions that are each at least 95% identical to the sequences shown in SEQ ID NO: 68 and SEQ ID NO: 69, respectively.
[00925] [00925] In one embodiment, an OX40 agonist comprises the heavy chain CDR1, CDR2 and CDR3 domains having the sequences shown in SEQ ID NO: 70, SEQ ID NO: 71 and SEQ ID NO: 72, respectively, and conservative substitutions of amino acids thereof, and the light chain CDR1, CDR2 and CDR3 domains having the sequences shown in SEQ ID NO: 73, SEQ ID NO: 74 and SEQ ID NO: 75, respectively, and conservative amino acid substitutions thereof.
[00926] [00926] In one embodiment, the OX40 agonist is a biosimilar monoclonal antibody to the OX40 agonist, approved by drug regulatory authorities with reference to 11D4. In one mode, the
[00927] [00927] In some embodiments, the OX40 agonist is 18D8, which is an entirely human antibody available from Pfizer, Inc. The preparation and properties of 18D8 are described in U.S. Patent Nos. 7,960,515; 8,236,930; and 9 028 824, the contents of which are hereby incorporated by reference. The 18D8 amino acid sequences are shown in
[00928] [00928] In one embodiment, an OX40 agonist comprises a heavy chain provided by SEQ ID NO: 76 and a light chain provided by SEQ ID NO: 77. In one embodiment, an OX40 agonist comprises heavy and light chains with the sequences shown in SEQ ID NO: 76 and SEQ ID NO: 77, respectively, or antigen binding fragments, Fab fragments, unique fragments of the variable chain (scFv) , variants or conjugates thereof. In one embodiment, an OX40 agonist comprises heavy and light chains that are each at least 99% identical to the sequences shown in SEQ ID NO: 76 and SEQ ID NO: 77, respectively. In one embodiment, heavy and light chains of an OX40 agonist that are each at least 98% identical to the sequences shown in SEQ ID NO: 76 and SEQ ID NO: 77, respectively. In one embodiment, an OX40 agonist comprises heavy and light chains that are each at least 97% identical to the sequences shown in SEQ ID NO: 76 and SEQ ID NO: 77, respectively. In one embodiment, an OX40 agonist comprises heavy and light chains that are each at least 96% identical to the sequences shown in SEQ ID NO: 76 and SEQ ID NO: 77, respectively. In one embodiment, an OX40 agonist comprises heavy and light chains that are each at least 95% identical to the sequences shown in SEQ ID NO: 76 and SEQ ID NO: 77, respectively.
[00929] [00929] In one embodiment, the OX40 agonist comprises the heavy and light chain CDRs or the 18D8 variable regions (VRs). In one embodiment, the OX40 agonist variable region of the heavy chain (VH) comprises the sequence shown in SEQ ID NO: 78, and the OX40 agonist variable region of the light chain (VL) comprises the sequence shown in SEQ ID NO: 79 , and conservative amino acid substitutions thereof. In one embodiment, an OX40 agonist comprises VH and VL regions that
[00930] [00930] In one embodiment, an OX40 agonist comprises the heavy chain CDR1, CDR2 and CDR3 domains having the sequences shown in SEQ ID NO: 80, SEQ ID NO: 81 and SEQ ID NO: 82, respectively, and conservative substitutions of amino acids thereof, and the light chain CDR1, CDR2 and CDR3 domains having the sequences shown in SEQ ID NO: 83, SEQ ID NO: 84 and SEQ ID NO: 85, respectively, and conservative amino acid substitutions thereof.
[00931] [00931] In one embodiment, the OX40 agonist is a biosimilar monoclonal antibody to the OX40 agonist, approved by drug regulatory authorities with reference 18D8. In one embodiment, the biosimilar monoclonal antibody comprises an antibody against OX40 comprising an amino acid sequence with at least 97% sequence identity, e.g. 97%, 98%, 99% or 100% sequence identity with the amino acid sequence of a reference drug or biological reference product and which comprises one or more post-translational modifications when compared to the reference drug
[00932] [00932] In some embodiments, the OX40 agonist is Hu119-122, which is a humanized antibody made available by GlaxoSmithKline plc. The preparation and properties of Hu119-122 are described in U.S. Patents Nos. 9,006,399 and 9,163,085 and in International Patent Publication No. WO 2012/027328, the contents of which are incorporated herein by reference. At
[00933] [00933] In one embodiment, the OX40 agonist comprises the CDRs of the heavy and light chain or the variable regions (VRs) of Hu119-122. In one embodiment, the heavy chain variable (VH) region of the OX40 agonist comprises the sequence shown in SEQ ID NO: 86, and the light chain variable region (VL) of the OX40 agonist comprises the sequence shown in SEQ ID NO. : 87, and conservative amino acid substitutions thereof. In one embodiment, an OX40 agonist comprises VH and VL regions that are each at least 99% identical to the sequences shown in SEQ ID NO: 86 and SEQ ID NO: 87, respectively. In one embodiment, an OX40 agonist comprises VH and VL regions that are each at least 98% identical to the sequences shown in SEQ ID NO: 86 and SEQ ID NO: 87, respectively. In one embodiment, an OX40 agonist comprises VH and VL regions that are each at least 97% identical to the sequences shown in SEQ ID NO: 86 and SEQ ID NO: 87, respectively. In one embodiment, an OX40 agonist comprises VH and VL regions that are each at least 96% identical to the sequences shown in SEQ ID NO: 86 and SEQ ID NO: 87, respectively. In one embodiment, an OX40 agonist comprises VH and VL regions that are each at least 95% identical to the sequences shown in SEQ ID NO: 86 and SEQ ID NO: 87, respectively.
[00934] [00934] In one embodiment, an OX40 agonist comprises the heavy chain CDR1, CDR2 and CDR3 domains having the sequences shown in SEQ ID NO: 88, SEQ ID NO: 89 and SEQ ID NO: 90, respectively, and conservative substitutions amino acid domains, and the light chain CDR1, CDR2 and CDR3 domains having the sequences shown in SEQ ID NO: 91, SEQ ID NO: 92 and SEQ ID NO: 93, respectively, and conservative amino acid substitutions thereof.
[00935] [00935] In one embodiment, the OX40 agonist is an antibody
[00936] [00936] In some embodiments, the OX40 agonist is Hu106-222, which is a humanized antibody made available by GlaxoSmithKline plc. The preparation and properties of Hu106-222 are described in U.S. Patent Nos. 9 006 399 and 9 163 085 and in International Patent Publication No. WO 2012/027328, the contents of which are incorporated herein by reference. The amino acid sequences of Hu106-222 are shown in Table 16.
[00937] [00937] In one embodiment, the OX40 agonist comprises the CDRs of the heavy and light chain or the variable regions (VRs) of Hu106-222. In one embodiment, the heavy chain variable (VH) region of the OX40 agonist comprises the sequence shown in SEQ ID NO: 94, and the light chain variable region (VL) of the OX40 agonist comprises the sequence shown in SEQ ID NO. : 95, and conservative amino acid substitutions
[00938] [00938] In one embodiment, an OX40 agonist comprises the heavy chain CDR1, CDR2 and CDR3 domains having the sequences shown in SEQ ID NO: 96, SEQ ID NO: 97 and SEQ ID NO: 98, respectively, and conservative substitutions of amino acids thereof, and the light chain CDR1, CDR2 and CDR3 domains having the sequences shown in SEQ ID NO: 99, SEQ ID NO: 100 and SEQ ID NO: 101, respectively, and conservative amino acid substitutions thereof.
[00939] [00939] In one embodiment, the OX40 agonist is a biosimilar monoclonal antibody to the OX40 agonist, approved by drug regulatory authorities with reference to Hu106-222. In one embodiment, the biosimilar monoclonal antibody comprises an antibody against OX40 comprising an amino acid sequence with at least 97% sequence identity, e.g. 97%, 98%, 99% or 100% sequence identity with the amino acid sequence of a reference medicine or reference biological product and comprising one or more
[00940] [00940] In some embodiments, the OX40 agonist antibody is MEDI6469 (also referred to as 9B12). MEDI6469 is a murine monoclonal antibody. Weinberg, et al., J. Immunother. 2006, 29, 575-585. In some embodiments, the OX40 agonist is an antibody produced by the hybridoma 9B12, deposited with Biovest Inc. (Malvern, MA, USA), as described in Weinberg, et al., J. Immunother. 2006, 29, 575-585, whose content is incorporated herein, in its entirety, by reference. In some embodiments, the antibody comprises the CDI sequences of MEDI6469. In some embodiments, the antibody comprises a heavy chain variable region sequence and / or a MEDI6469 light chain variable region sequence.
[00941] [00941] In one embodiment, the OX40 agonist is L106 BD (Pharmingen, Product no 340420). In some embodiments, the OX40 agonist comprises the CDRs of the L106 antibody (BD Pharmingen, Product No. 340420). In some modalities, the OX40 agonist comprises a
[00942] [00942] In one embodiment, the OX40 agonist is selected from the OX40 agonists described in International Patent Application Publication Nos WO 95/12673, WO 95/21925, WO 2006/121810, WO 2012/027328, WO 2013 / 028231, WO 2013/038191 and WO 2014/148895; European Patent Application EP 0672141; U.S. Patent Application Publication Nos. US 2010/136030, US 2014/377284, US 2015/190506 and US 2015/132288 (including clones 20E5 and 12H3); and US Patents Nos. 7,504 101, 7 550 140, 7 622 444, 7 696 175, 7 960 515, 7 961 515, 8 133 983, 9 006 399, and 9 163 085, the contents of which are incorporated herein in their entirety , by reference.
[00943] [00943] In one embodiment, the OX40 agonist is an OX40 agonist fusion protein as represented in Structure IA (C-terminal antibody Fc fragment fusion protein) or Structure IB (N-antibody Fc fragment fusion protein) -terminal), or a fragment, derived, conjugated, variant or biosimilar thereof. The properties of structures I-A and I-B are described above and in U.S. Patent Nos. 9,359,420, 9 340 599, 8 921 519 and 8 450 460, the contents of which are incorporated herein by
[00944] [00944] In one embodiment, an OX40 agonist fusion protein according to structures IA or IB comprises one or more OX40 binding domains selected from the group consisting of a variable heavy chain and a variable light chain of tavolixizumab, a variable heavy chain and variable light chain of 11D4, a variable heavy chain and variable light chain of 18D8, the variable heavy chain and variable light chain of Hu119-122, a variable heavy chain and variable light chain of Hu106-222, a heavy chain variable and variable light chain selected from the variable heavy chains and variable light chains described in Table OO, any combination of variable heavy chain and variable light chain from the previous ones, and fragments, derivatives, conjugates, variants and biosimilars thereof.
[00945] [00945] In one embodiment, an OX40 agonist fusion protein according to structures I-A or I-B comprises one or more domains of
[00946] [00946] In one embodiment, an OX40 agonist fusion protein according to structures IA or IB comprises one or more OX40 binding domains which is a scFv domain comprising VH and VL regions which are each at least 95 % identical to the sequences shown in SEQ ID NO: 58 and SEQ ID NO: 59, respectively, in which the VH and VL domains are connected by a linker. In one embodiment, an OX40 agonist fusion protein according to structures IA or IB comprises one or more OX40 binding domains which is an scFv domain comprising VH and VL regions which are each at least 95% identical to sequences shown in SEQ ID NO: 68 and SEQ ID NO: 69, respectively, in which the VH and VL domains are connected by a linker. In one embodiment, an OX40 agonist fusion protein according to structures IA or IB comprises one or more OX40 binding domains which is an scFv domain comprising VH and VL regions which are each at least 95% identical to sequences shown in SEQ ID NO: 78 and SEQ ID NO: 79, respectively, in which the VH and VL domains are connected by a linker.
[00947] [00947] In one embodiment, the OX40 agonist is a single-chain fusion polypeptide OX40 agonist comprising (i) a first soluble OX40 binding domain, (ii) a first peptide linker, (iii) a second peptide domain binding to soluble OX40, (iv) a second peptide linker and (v) a third soluble OX40 binding domain, further comprising an additional domain at the N-terminal and / or C-terminal end, and where the additional domain is a Fab or Fc fragment domain. In one embodiment, the OX40 agonist is an OX40 agonist single-chain fusion polypeptide comprising (i) a first soluble OX40 binding domain, (ii) a first peptide linker, (iii) a second OX40 binding domain soluble, (iv) a second peptide linker and (v) a third soluble OX40 binding domain, further comprising a domain
[00948] [00948] In one embodiment, the OX40 agonist is a single chain fusion polypeptide OX40 agonist comprising (i) a first soluble cytokine domain of the tumor necrosis factor (TNF) superfamily, (ii) a first peptide linker , (iii) a second soluble cytokine domain of the TNF superfamily, (iv) a second peptide linker and (v) a third soluble cytokine domain of the TNF superfamily, where each of the soluble cytokines of the TNF superfamily does not contain a region of rod and the first and the second peptide linker are independently 3-8 amino acids long, and where the cytokine domain of the TNF superfamily is an OX40 binding domain.
[00949] [00949] In some modalities, the OX40 agonist is MEDI6383. MEDI6383 is an OX40 agonist fusion protein and can be prepared as described in U.S. Patent No. 6,312,700, the content of which is incorporated herein by reference.
[00950] [00950] In one embodiment, the OX40 agonist is an OX40 agonist scFv antibody comprising any of the previous VH domains linked to any of the previous VL domains.
[00951] [00951] In one embodiment, the OX40 agonist is the OX40 agonist monoclonal antibody from Creative Biolabs MOM-18455, commercially available from Creative Biolabs, Inc., Shirley, NY, USA.
[00952] [00952] In one embodiment, the OX40 agonist is the Ber-ACT35 clone of OX40 agonist antibody, commercially available from
[00953] [00953] Optionally, a cell viability assay can be performed after Step B first expansion of Process 2A (Gen 2) or Gen 3 processes, using standard assays known in the art. For example, a Trypan blue exclusion assay can be performed on a sample of the bulk TILs, which selectively marks dead cells and allows to assess viability. Other tests for use in viability tests may include, among others, the Alamar test; and the MTT assay.
[00954] [00954] In some modalities of Process 2A (Gen 2) or Gen 3, cell counts and / or viability are measured. The expression of markers such as, among others, CD3, CD4, CD8 and CD56, as well as any other described or described herein, can be measured by flow cytometry with antibodies, for example, among others, those commercially available from BD Bio- sciences (BD Biosciences, San Jose, CA) using a FACSCantoTM flow cytometer (BD Biosciences). Cells can be counted manually using a disposable c-chip hemocytometer (VWR, Batavia, IL), and viability can be assessed by any method known in the art, including, but not limited to, Trypan blue staining. In some modalities of Process 2A (Gen 2) and / or Gen 3, TILs exhibit an increase in CD8 + cells after the first expansion, the second expansion, the first preparation expansion or the second rapid expansion. In some modalities of Process 2A (Gen 2), TILs exhibit an increase in CD8 + cells after the first expansion and / or the second expansion. In some Gen 3 modalities, TILs exhibit an increase in CD8 + cells after the first preparation expansion or the second rapid expansion.
[00955] [00955] In some cases, the bulk TIL population can be cryopreserved immediately, using the protocols discussed below. Alternatively, the bulk TIL population can be subjected to REP and then cryopreserved as discussed below. Likewise, if genetically modified TILs are to be used in therapy, populations of bulk TIL or REP can be subjected to genetic modification by appropriate treatments.
[00956] [00956] In one embodiment, a method for expanding TILs may include using between approximately 5,000 mL and 25,000 mL of cellular medium, between approximately 5,000 mL and 10,000 mL of cellular medium or between approximately 5,800 mL and 8,700 mL of cellular medium. In one embodiment, expanding the number of TILs does not use more than one type of cell culture medium. Any suitable cell culture medium can be used, e.g. eg AIM-V cell culture medium (L-glutamine, 50 μM streptomycin sulfate and 10 μM gentamicin sulfate) (Invitrogen, Carlsbad CA). In this regard, the methods of the invention advantageously reduce the amount of medium and the number of types of medium required to expand the number of TIL. In one embodiment, expanding the number of TIL may comprise adding fresh cell culture medium to the cells (also referred to as feeding the cells) no more often than every three or four days. Expanding the number of cells in a gas-permeable container simplifies the procedures required to expand the number of cells by reducing the feed frequency required to expand the cells.
[00957] [00957] In one embodiment, the cellular medium in the first and / or second gas-permeable container is not filtered. The use of unfiltered cellular media can simplify the procedures necessary to expand the number of cells. In one embodiment, the cellular environment in the first and / or
[00958] [00958] In one embodiment, TILs are expanded into gas-permeable containers. Gas permeable containers have been used to expand TILs using PBMCs by methods, compositions and devices known in the art, including those described in U.S. Patent Application Publication No. 2005/0106717 A1, the content of which is incorporated herein by reference. In one embodiment, TILs are expanded into gas-permeable bags. In one embodiment, TILs are expanded using a cell expansion system that expands TILs in gas-permeable bags, such as the Xuri Cell Expansion System W25 (GE Healthcare). In one embodiment, TILs are expanded using a cell expansion system that expands TILs in gas-permeable bags, such as the WAVE Bioreactor System, also known as the Xuri Cell Expansion System W5 (GE Healthcare). In one embodiment, the cell expansion system includes a gas-permeable cell bag with a volume selected from the group consisting of approximately 100 mL, approximately 200 mL, approximately 300 mL, approximately 400 mL, approximately 500 mL, approximately 600 mL , approximately 700 mL, approximately 800 mL, approximately 900 mL, approximately 1 L, approximately 2 L, approximately 3 L, approximately 4 L, approximately 5 L, approximately 6 L, approximately 7 L, approximately 8 L, approximately 9 L and approximately 10 L. In one embodiment, TILs can be expanded into G-Rex flasks (commercially available from Wilson Wolf Manufacturing). Such modalities allow the expansion of cell populations from approximately 5 x 105 cells / cm2 to between 10 x 106 and 30 x 106 cells / cm2. In one embodiment, this expansion is conducted without adding fresh cell culture medium to the cells (also referred to as feeding the cells).
[00959] [00959] Respiratory reserve capacity (CRS) and glycolytic reserve can be assessed from expanded TILs with the methods described here. The Seahorse XF Cell Mito Stress test measures mitochondrial function by the oxygen consumption rate (OCR) of cells, measured directly with respiration modulators that target components of the electron transport chain in the mitochondria. The test compounds (oligomycin, FCCP and a mixture of rotenone and antimycin A, described below) are injected in series to measure ATP production, maximum breath and non-mitochondrial breath, respectively. Proton leakage and
[00960] [00960] In some modalities, the metabolic test is baseline breathing.
[00961] [00961] In general, TILs have a basal breathing rate that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70% ¸ at least 75%, at least 80% ¸ at least 85% ¸ at least 90% ¸ at least 95%, at least 97%, at least 98% or at least 99% of the baseline respiration rate of recently collected TILs. In some modalities, the baseline respiration rate is between approximately 50% and 99% of the baseline respiration rate of recently collected TILs. In some modalities, the baseline respiration rate is between approximately 60% and 99% of the baseline respiration rate of recently collected TILs. In
[00962] [00962] In some modalities, the metabolic test is respiratory reserve capacity.
[00963] [00963] In some modalities, the metabolic test is glycolytic reserve. In some modalities, the metabolic test is a reserve respiratory capacity. To measure cellular (respiratory) metabolism, cells were treated with mitochondrial respiration and glycolysis inhibitors to determine a TIL metabolic profile that consisted of the following measures: basal oxidative phosphorylation (as measured by OCR), respiratory reserve capacity, glycolytic activity baseline (as measured by ECAR) and glycolytic reserve. The metabolic profiles were performed using the Seahorse Combination Mitochondrial / Glycolysis Stress Test Assay assay (including the kit commercially available from Agilent®), which allows determining the capacity with which cells perform glycolysis when blocking the production of mitochondrial ATP. In some modalities, the cells are deprived of glucose, so glucose is injected, followed by a stressor. In some modalities, the stress agent is selected from the group consisting of oligomycin, FCCP, rotenone, antimycin A and / or 2-deoxyglucose (2-DG),
[00964] [00964] In some modalities, the metabolic assay is basal glycolysis. In some, second expansion TILs or additional second expansion TILs (such as, for example, those described in step D of Figure 7, including TILs referred to as reREP TILs) show an increase in basal glycolysis of at least twice, at least at least three times, at least four times, at least five times, at least six times, at least 7
[00965] [00965] In general, second expansion TILs or additional second expansion TILs (such as, for example, those described in step D of Figure 7, including TILs referred to as reREP TILs) have a reserve respiratory capacity that is at least is at least 50%, at least 55%, at least 60%, at least 65%, at least 70% ¸ at least 75%, at least 80% ¸ at least 85% ¸ at least 90% ¸ at least 95% at least 97%, at least 98% or at least 99% of the baseline respiration rate of recently collected TILs. In some modalities, the capacity
[00966] [00966] Granzyme B Production: Granzyme B is another measure of TIL's ability to eliminate target cells. Supernatants from the medium, restimulated as described above with antibodies against CD3, CD28 and CD137 / 4-1BB, also had their Granzyme B levels evaluated using the Human Granzyme B DuoSet ELISA kit (R & D Systems, Minneapolis, MN) according to the manufacturer's instructions. In some embodiments, the second expansion TILs or additional second expansion TILs (such as, for example, those described in step D of Figure 7,
[00967] [00967] In some embodiments, the length of the telomeres can be used as a measure of cell viability and / or cell function. Telomere length measurement: Several methods have been used to measure telomeres length in genomic and cytological DNA preparations. Terminal restriction fragment analysis (TRF) is the gold standard for measuring telomere length (de Lange et al., 1990). However, the main limitation of the TRF analysis is the need for a large amount of DNA (1.5 µg). Two techniques widely used to measure telomere length, namely, fluorescent in situ hybridization (FISH; Agilent Technologies, Santa Clara, CA) and quantitative PCR can be employed with the present invention. In some modalities, there is no change in telomere length between the TILs collected initially in step A and the expanded TILs of, for example, Step D as shown in Figure 7.
[00968] [00968] In some modalities, the health of TILs is measured by the secretion of IFN-gamma. In some modalities, a power test is used for the production of IFN-γ. IFN-γ production is another measure of cytotoxic potential. IFN-γ production can be measured by determining the levels of the IFN-γ cytokine in the middle of TILs stimulated with antibodies against CD3, CD28 and CD137 / 4-1BB. The levels of IFN-γ in the middle of these stimulated TILs can be determined by measuring the release of IFN-γ. In some modalities, an increase in IFN-γ production in, for example, TILS from Step D as shown in Figure 7, when compared to the TILs collected initially in, for example, Step A as shown in Figure 7
[00969] [00969] In some embodiments, the redirected lysis bioluminescence assay: The cytotoxic potential of TILs to lyse target cells was assessed with a TIL co-culture assay with the bioluminescent cell line, P815 (Clone G6), according to a bioluminescence assay of redirected lysis (potency assay) for TILs analysis that measures the cytotoxicity of TILs in a highly sensitive dose-dependent manner.
[00970] [00970] In some embodiments, the present methods include an assay to assess the viability of TILs, by the methods described above and here, including, for example, as described in the Examples and Figures. In some modalities, TILs are expanded as discussed above, including for example, as shown in Figure 7. In some modalities, TILs are cryopreserved before being assessed for viability. In some modalities, the feasibility assessment includes thawing TILs before carrying out a first expansion, a second expansion and an additional second expansion. In some embodiments, the present methods provide an assay to assess cell proliferation, cell toxicity, cell death and / or other terms related to the viability of the TIL population. Viability can be measured by any of the TIL metabolic assays described above as well as any known methods for assessing the viability of cells that are known in the art. In some embodiments, the present methods provide an assay to assess cell proliferation, cell toxicity, cell death and / or other terms related to the viability of TILs expanded by the methods described here, including those exemplified in Figure 7.
[00971] [00971] In some modalities, the metabolic test is baseline breathing. In general, second expansion TILs have a baseline respiration rate that is at least 50%, at least 55%, at least 60%,
[00972] [00972] In general, second expansion TILs or additional second expansion TILs (such as, for example, those described in step D of Figure 7, including TILs referred to as reREP TILs) have a reserve respiratory capacity that is at least is at least 50%, at least 55%, at least 60%, at least 65%, at least 70% ¸ at least 75%, at least 80% ¸ at least 85% ¸ at least 90% ¸ at least 95% , fur
[00973] [00973] In general, second expansion TILs or additional second expansion TILs (such as, for example, those described in step D of Figure 7, including TILs referred to as reREP TILs) have a reserve respiratory capacity that is at least is at least 50%, at least 55%,
[00974] [00974] In some modalities, the metabolic assay is basal glycolysis. In some embodiments, second expansion TILs or additional second expansion TILs (such as, for example, those described in step D of Figure 7, including TILs referred to as reREP TILs) show an increase in basal glycolysis of at least twice, at least at least three times, at least four times, at least five times, at least six times, at least 7 times, at least eight times, at least nine times or at least ten times. In some embodiments, second expansion TILs or additional second expansion TILs (such as, for example, those described in step D of Figure 7, including TILs referred to as reREP TILs) show an increase in basal glycolysis between approximately twice and ten times. In some embodiments, the second expansion TILs or additional second expansion TILs (such as, for example, those described in step D of Figure 7, including TILs referred to as TILs
[00975] [00975] In general, second expansion TILs or additional second expansion TILs (such as, for example, those described in step D of Figure 7, including TILs referred to as reREP TILs) have a glycolytic reserve that is at least 50% , at least 55%, at least 60%, at least 65%, at least 70% ¸ at least 75%, at least 80% ¸ at least 85% ¸ at least 90% ¸ at least 95%, at least 97% , at least 98% or at least 99% of the baseline TIL breath rate of process 2A (see, for example, Figure 7). In some embodiments, the glycolytic reserve is between approximately 50% and 99% of the baseline respiration rate of TILs in process 2A (see, for example, Figure 7). In some embodiments, the glycolytic reserve is between approximately 60% and 99% of the baseline respiration rate of TILs in process 2A (see, for example, Figure 7). In some embodiments, the glycolytic reserve is between approximately 70% and 99% of the baseline respiration rate of TILs in process 2A (see, for example, Figure 7). In some modalities, the glycolytic reserve is among
[00976] [00976] Granzyme B Production: Granzyme B is another measure of the ability of TILs to eliminate target cells. Supernatants from the medium, restimulated as described above with antibodies against CD3, CD28 and CD137 / 4-1BB, also had their Granzyme B levels evaluated using the Human Granzyme B DuoSet ELISA kit (R & D Systems, Minneapolis, MN) according to the manufacturer's instructions.
[00977] [00977] In some embodiments, the present methods provide an assay to assess the viability of TILs by the methods described above. In some modalities, TILs are expanded as discussed above, including for example, as shown in Figure 7. In some modalities, TILs are cryopreserved before being assessed for viability. In some modalities, the feasibility assessment includes thawing TILs before carrying out a first expansion, a second expansion and an additional second expansion. In some embodiments, the present methods provide an assay to assess cell proliferation, cell toxicity, cell death and / or other terms related to the viability of the TIL population. Viability can be measured by any of the TIL metabolic assays described above as well as any known methods for assessing the viability of cells that are known in the art. In some embodiments, the present methods provide an assay to assess cell proliferation, cell toxicity, cell death and / or other terms related to the viability of TILs expanded by the methods
[00978] [00978] The present invention also provides test methods to determine the viability of TILs. The present invention provides methods for assessing TILs for viability by expanding tumor infiltrating lymphocytes (TILs) into a larger population of TILs, comprising: (i) obtaining a first population of TILs that has been previously expanded; (ii) perform a first expansion, cultivating the first TIL population in a cell culture medium comprising IL-2, to produce a second TIL population, in which the cell culture medium is supplemented with OKT-3 on day 3, where the first expansion is performed for approximately 3 days to 19 days in order to obtain the second population of TILs, where the second population of TILs comprises at least 5 x 107 TILs in approximately 10 days to 19 days when the first population of TILs are from a thick needle biopsy; and perform a second expansion, supplementing the cell culture medium of the second population of TILs with IL-2, OKT-3 and additional antigen presenting cells (APCs), to produce a third population of TILs, in which the number of the third population of TILs is at least 100 times greater than that of the second population of TILs, and in which the second expansion is performed for approximately 11 days to approximately 14 days in order to obtain the third population of TILs, in which the third population of TILs it comprises an increased subpopulation of effector T cells and / or central memory T cells in relation to the second population of TILs, and in which the third population is further analyzed for viability. In some embodiments, the method further comprises: (iv) performing an additional second expansion, supplementing the cell culture medium of the third TIL population with additional IL-2, additional OKT-3 and additional APCs, where the second additional expansion is
[00979] [00979] In some modalities, before step (i), the cells are cryopreserved.
[00980] [00980] In some embodiments, the cells are thawed before performing step (i).
[00981] [00981] In some modalities, step (iv) is repeated one to four times in order to obtain sufficient TILs for analysis.
[00982] [00982] In some modalities, steps (i) to (iii) or (iv) are performed within a period between approximately 21 days and 33 days.
[00983] [00983] In some modalities, steps (i) to (iii) or (iv) are performed within a period between approximately 21 days and 30 days.
[00984] [00984] In some modalities, steps (i) to (iii) or (iv) are performed within a period between approximately 21 days and 28 days.
[00985] [00985] In some modalities, steps (i) to (iii) or (iv) are performed within approximately 24 days.
[00986] [00986] In some embodiments, the cells in steps (iii) or (iv) express CD4, CD8 and TCR α β at levels similar to those of recently collected cells.
[00987] [00987] In some embodiments, the antigen presenting cells are peripheral blood mononuclear cells (PBMCs).
[00988] [00988] In some embodiments, PBMCs are added to the cell culture on any of the days 9 to 17 in step (iii).
[00989] [00989] In some embodiments, effector T cells and / or central memory T cells in the larger population of TILs in step (iv) exhibit one or more characteristics selected from the group consisting of expression of
[00990] [00990] In some embodiments, effector T cells and / or central memory T cells exhibit increased CD57 expression and decreased CD56 expression.
[00991] [00991] In some modalities, APCs are artificial APCs (aAPCs).
[00992] [00992] In some embodiments, the method further comprises the step of transducing the first population of TILs with an expression vector comprising a nucleic acid that encodes a high affinity T cell receptor.
[00993] [00993] In some modalities, the transduction step occurs before step (i).
[00994] [00994] In some embodiments, the method further comprises the step of transducing the first population of TILs with an expression vector comprising a nucleic acid encoding a chimeric antigen receptor (CAR) comprising a fused single chain variable fragment antibody with at least one endodomain of a T cell signaling molecule.
[00995] [00995] In some modalities, the transduction step occurs before step (i).
[00996] [00996] In some modalities, TILs are analyzed for viability.
[00997] [00997] In some modalities, TILs are analyzed for viability after cryopreservation.
[00998] [00998] In some modalities, TILs are analyzed for viability after cryopreservation and after step (iv).
[00999] [00999] In some embodiments, the present invention provides a
[001000] [001000] In accordance with the present invention, a method is provided for analyzing TILs for viability and / or additional use in administration to an individual. In some embodiments, the method for analyzing tumor infiltrating lymphocytes (TILs) comprises: (i) obtaining a first population of TILs; (ii) carrying out a first expansion, cultivating the first population of TILs in a cell culture medium comprising IL-2, to produce a second population of TILs, in which the cell culture medium is
[001001] [001001] In some modalities, the reREP period is performed until the ratio of the number of TILs in the fourth population of TILs to the number of TILs in the third population of TILs is greater than 50: 1.
[001002] [001002] In some embodiments, the number of TILs sufficient for a therapeutically effective dose is between approximately 2.3x1010 and 13.7x1010.
[001003] [001003] In some modalities, steps (i) to (vii) are performed within a period between approximately 21 days and 33 days. In some embodiments, steps (i) to (vii) are carried out within a period of approximately 21 days to 30 days. In some embodiments, steps (i) to (vii) are carried out within a period of approximately 21 days to approximately 28 days. In some embodiments, steps (i) to (vii) are performed within approximately 24 days. In some embodiments, the cells in steps (iii) or (vii) express CD4, CD8 and TCR α β at levels similar to those of recently collected cells. In some embodiments, the cells are TILs.
[001004] [001004] In some embodiments, the antigen presenting cells are peripheral blood mononuclear cells (PBMCs). In some embodiments, PBMCs are added to the cell culture on any of days 3 to 12 in step (ii) and / or any of days 11 to 14 in step (iii).
[001005] [001005] In some embodiments, effector T cells and / or central memory T cells in the larger population of TILs in steps (iii) or (vii) exhibit one or more characteristics selected from the group consisting of CD27 expression, expression CD28, longer telomeres, increased CD57 expression and decreased CD56 expression, in relation to effector T cells and / or central memory T cells in the third population of
[001006] [001006] In some embodiments, effector T cells and / or central memory T cells exhibit increased CD57 expression and decreased CD56 expression.
[001007] [001007] In some modalities, APCs are artificial APCs (aAPCs).
[001008] [001008] In some embodiments, the stage of transduction of the first population of TILs with an expression vector comprising a nucleic acid encoding a high affinity T cell receptor.
[001009] [001009] In some modalities, the transduction step occurs before step (i).
[001010] [001010] In some embodiments, the step of transducing the first population of TILs with an expression vector comprising a nucleic acid encoding a chimeric antigen (CAR) receptor comprising a single chain variable fragment antibody fused to at least one endodomain of a T-cell signaling molecule
[001011] [001011] In some modalities, the transduction step occurs before step (i).
[001012] [001012] In some modalities, TILs are analyzed for viability after step (vii).
[001013] [001013] The present invention also provides for methods for analyzing TILs. In some embodiments, the invention provides a method for analyzing TILs that comprises: (i) obtaining a portion of a first population of cryopreserved TILs obtained from a coarse needle biopsy sample; (ii) thawing part of the first population of cryopreserved TILs; (iii) perform a first expansion, cultivating part of the first population of TILs in a cell culture medium comprising
[001014] [001014] In some embodiments, the ratio of the number of TILs in the second population of TILs to the number of TILs in the part of the first population of TILs is greater than 50: 1.
[001015] [001015] In some modalities, the method also comprises expanding the first entire population of cryopreserved TILs from step (i) according to the methods described in any of the modalities provided here.
[001016] [001016] In some modalities, the method also comprises administering the first entire population of cryopreserved TILs from step (i) to the patient. IX. Closed systems for TIL production
[001017] [001017] The present invention provides the use of closed systems during the process of cultivating TIL by the Gen 2 processes or Gen 3 processes. Such closed systems allow to prevent and / or reduce contamination
[001018] [001018] Such closed systems are well known in the art and can be found, for example, at http://www.fda.gov/cber/guidelines.htm and https://www.fda.gov/BiologicsBloodVaccines/GuidanceComplianceRegulator yInformation /Guidances/Blood/ucm076779.htm.
[001019] [001019] Sterile connection devices (STCDs) produce sterile welds between two pieces of compatible tubes. This procedure allows the sterile connection of a variety of containers and tube diameters. In some embodiments, closed systems include luer lock and thermally sealed systems. In some embodiments, access to the closed system is through syringes under sterile conditions in order to maintain the sterility and closed nature of the system. In some embodiments, a closed system is employed. In some embodiments, TILs are formulated in a container for formulating the final product.
[001020] [001020] In some embodiments, the closed system uses a container from the moment the tumor fragments are obtained until the TILs are ready for administration to the patient or for cryopreservation. In some embodiments, when two containers are used, the first container is a closed G container and the population of TILs is centrifuged and transferred to an infusion bag without opening the first closed G container. In some embodiments, when two containers are used, the infusion bag is an infusion bag containing HyperThermosol. A closed system or closed system for culture of TILs is characterized by the fact that, once the tumor sample or tumor fragments have been added, the system is hermetically closed from the outside to form a closed environment free from bacterial invasion, fungi and / or any other microbial contamination.
[001021] [001021] In some modalities, the reduction in microbial contamination is between approximately 5% and 100%. In some modalities, the reduction in microbial contamination is between approximately 5% and 95%. In some modalities, the reduction in microbial contamination is between approximately 5% and 90%. In some modalities, the reduction in microbial contamination is between approximately 10% and 90%. In some modalities, the reduction in microbial contamination is between approximately 15% and 85%. In some embodiments, the reduction in microbial contamination is approximately 5%, approximately 10%, approximately 15%, approximately 20%, approximately 25%, approximately 30%, approximately 35%, approximately 40%, approximately 45%, approximately 50%, approximately 55%, approximately 60%, approximately 65%, approximately 70%, approximately 75%, approximately 80%, approximately 85%, approximately 90%, approximately 95%, approximately 97%, approximately 98%, approximately 99% or approximately 100 %.
[001022] [001022] The closed system allows the growth of TILs in the absence and / or with a significant reduction in microbial contamination.
[001023] [001023] In addition, the pH, the partial pressure of carbon dioxide and the partial pressure of oxygen in the TIL cell culture environment vary each as the cells are cultured. Consequently, even if an appropriate medium for cell culture is circulated, the closed environment still needs to be constantly maintained as an optimal environment for the proliferation of TILs. For this, it is desirable that the physical factors of pH, partial pressure of carbon dioxide and partial pressure of oxygen within the culture liquid of the closed environment be monitored by a sensor, whose signal is used to control a gas exchanger installed at the entrance of the culture environment, and that the partial pressure of that gas in the closed environment is adjusted in real time according to changes in the culture liquid to
[001024] [001024] In some modalities, the pressure inside the closed environment is controlled continuously or intermittently. That is, the pressure in the closed environment can be varied by means of a pressure maintenance device, for example, thus ensuring that the space is suitable for the growth of TILs in a state of positive pressure, or by promoting the exudation of liquid in a state of negative pressure and thus promoting cell proliferation. In addition, with the intermittent application of negative pressure, it is possible to uniformly and efficiently replace the circulating liquid in the closed environment.
[001025] [001025] In some modalities, components of the culture, excellent for the proliferation of TILs, can be replaced or added, including factors such as IL-2 and / or OKT3, in addition to combinations, which can be added. X. Optional genetic engineering of TILs
[001026] [001026] In some modalities of Gen 2 or Gen 3 processes, the expanded TILs of the present invention are manipulated more before, during or after an expansion step, including during closed, sterile processes of production, each as provided herein, aiming alter protein expression in a transient manner. In some modalities, protein expression transiently altered is due to transient gene editing. In some embodiments, the expanded TILs of the present invention are treated with transcription factors (TFs) and / or other molecules capable of altering
[001027] [001027] In certain modalities, the method involves genetically editing a population of TILs. In certain embodiments, the method comprises genetically editing the first population of TILs, the second population of TILs and / or the third population of TILs.
[001028] [001028] In some embodiments, the present invention includes genetic editing through the insertion of nucleotides, such as through the insertion of ribonucleic acid (RNA), including the insertion of messenger RNA (mRNA) or small (or short) interfering RNA (siRNA), in a population of TILs to promote the expression of one or more proteins or inhibit the expression of one or more proteins, as well as simultaneous combinations of promotion of a set of proteins with inhibition of another set of proteins.
[001029] [001029] In some embodiments, the expanded TILs of the present invention undergo transient change in protein expression. In some embodiments, the transient change in protein expression occurs in a population of TIL in bulk prior to the first expansion, including, for example, in a population of TIL obtained, for example, in Step A as indicated in Figure 85 (especially in Figure 85B). In some embodiments, the transient change in protein expression occurs during the first expansion, including, for example, in the population of TILs expanded in, for example, Step B as indicated in Figure 85 (for example, Figure 85B). In some embodiments, the transient change in protein expression occurs after the first expansion, including, for example, in the population of TILs in transition between the first and the second expansion (eg, the second
[001030] [001030] In one embodiment, the method for transiently changing protein expression in a population of TILs includes the electroporation step. Electroporation methods are known in the art and are described, e.g. in Tsong, Biophys. J. 1991, 60, 297-306, and in U.S. Patent Application Publication No. 2014/0227237 A1, the contents of which are hereby incorporated by reference. In one embodiment, a method for transiently altering protein expression in the TIL population includes the step of transfection with calcium phosphate. Calcium phosphate transfection methods (precipitation of calcium phosphate DNA, cell surface coating and endocytosis) are known in the art and are described in Graham and van der Eb, Virology 1973, 52, 456-467; Wigler, et al., Proc. Natl. Acad. Sci. 1979, 76, 1373-1376; and Chen and Okayarea, Mol. Cell. Biol. 1987, 7, 2745-2752; and in U.S. Patent No. 5,593,875, the contents of which are hereby incorporated by reference. In one embodiment, a method for transiently altering protein expression in a population of TILs includes the liposome transfection step. Transfection methods with liposomes, such as methods
[001031] [001031] In some modalities, the transient alteration of protein expression results in an increase in memory T cells similar to stem cells (TSCMs). TSCMs are early progenitors of central memory T cells with antigen experience. TSCMs generally exhibit the long-term survival, self-renewal and multipotency skills that define stem cells and, in general, are desirable for generating effective TIL products. TSCM demonstrated enhanced antitumor activity compared to other subsets of T cells in models of adoptive cell transfer in mice (Gattinoni et al. Nat Med 2009, 2011; Gattinoni, Nature Rev. Cancer, 2012; Cieri et al. Blood 2013). In some embodiments, the transient change in protein expression results in a population of TILs with a composition that comprises a high proportion of TSCM. In some embodiments, the transient change in protein expression results in an increase of at least 5%, at least 10%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35% at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85% , at least 90% or at least
[001032] [001032] In some modalities, the transient alteration of protein expression results in the rejuvenation of T cells with experience in antigens. In some modalities, rejuvenation includes, for example, increased proliferation, increased activation of T cells and / or increased recognition of antigens.
[001033] [001033] In some modalities, the transient alteration of protein expression alters expression in a large fraction of T cells in order to preserve the TCR repertoire derived from the tumor. In some modalities, the transient alteration of protein expression does not alter the repertoire of TCR derived from the tumor. In some modalities, the transient alteration of protein expression maintains the repertoire of TCR derived from the tumor.
[001034] [001034] In some modalities, the transient alteration of the protein
[001035] [001035] In some embodiments, the transient change in protein expression results in increased expression and / or overexpression of a chemokine receptor. In some embodiments, the chemokine receptor that is overexpressed by transient protein expression includes a receptor with a linker that includes, among others, CCL2 (MCP-1), CCL3 (MIP-1α), CCL4 (MIP1-β), CCL5 ( RANTES), CXCL1, CXCL8, CCL22 and / or CCL17.
[001036] [001036] In some modalities, the transient alteration of protein expression results in a decrease and / or reduced expression of PD-1, CTLA-4, TIM-3, LAG-3, TIGIT, TGFβR2 and / or TGFβ (including resulting in blocking, for example, the TGFβ pathway). In some modalities, the transient alteration of protein expression results in a decrease and / or reduced expression CBLB (CBL-B). In some embodiments, the transient change in protein expression results in a decrease and / or reduced expression of CISH.
[001037] [001037] In some embodiments, the transient change in protein expression results in increased expression and / or overexpression of chemokine receptors in order, for example, to improve TIL traffic or movement to the tumor site. In some modalities, the transient alteration of protein expression results in increased expression and / or overexpression of a CCR (chimeric co-stimulatory receptor). In some modalities, the transient change in protein expression results in increased expression and / or overexpression of a chemokine receptor selected from the group consisting of CCR1, CCR2, CCR4, CCR5, CXCR1, CXCR2 and / or CSCR3.
[001038] [001038] In some embodiments, the transient change in protein expression results in increased expression and / or overexpression of an interleukin. In some embodiments, the transient change in protein expression results in increased expression and / or overexpression of a protein.
[001039] [001039] In some embodiments, the transient change in protein expression from protein expression results in increased expression and / or overexpression of NOTCH 1/2 ICD. In some modalities, the transient change in protein expression results in increased expression and / or overexpression of VHL. In some modalities, the transient alteration of protein expression results in increased expression and / or overexpression of CD44. In some embodiments, the transient change in protein expression results in increased expression and / or overexpression of PIK3CD. In some modalities, the transient change in protein expression results in increased expression and / or overexpression of SOCS1.
[001040] [001040] In some embodiments, the transient change in protein expression results in decreased and / or reduced expression of cAMP-dependent protein kinase A (PKA).
[001041] [001041] In some embodiments, the transient change in protein expression results in decreased and / or reduced expression of a molecule selected from the group consisting of PD-1, LAG3, TIM3, CTLA-4, TIGIT, CISH, TGFβR2, PKA , CBLB, BAFF (BR3) and combinations thereof. In some modalities, the transient alteration of protein expression results in decreased and / or reduced expression of two molecules selected from the group consisting of PD-1, LAG3, TIM3, CTLA-4, TIGIT, CISH, TGFβR2, PKA, CBLB, BAFF (BR3) and combinations thereof. In some embodiments, the transient change in protein expression results in decreased and / or reduced expression of PD-1 of a molecule selected from the group consisting of LAG3, TIM3, CTLA-4, TIGIT, CISH, TGFβR2, PKA, CBLB, BAFF (BR3) and combinations thereof. In some embodiments, the transient change in protein expression results in decreased and / or reduced expression of PD-1, LAG-3,
[001042] [001042] In some modalities, an adhesion molecule, selected from the group consisting of CCR2, CCR4, CCR5, CXCR2, CXCR3, CX3CR1 and combinations thereof, is inserted by a method with gamma retrovirus or lentivirus in the first population of TILs , in the second population of TILs or in the population of collected TILs (eg, the expression of the adhesion molecule is increased).
[001043] [001043] In some embodiments, the transient change in protein expression results in decreased and / or reduced expression of a molecule selected from the group consisting of PD-1, LAG3, TIM3, CTLA-4, TIGIT, CISH, TGFβR2, PKA , CBLB, BAFF (BR3) and combinations thereof, and in increased and / or improved expression of CCR2, CCR4, CCR5, CXCR2, CXCR3, CX3CR1 and combinations thereof. In some modalities, the transient alteration of protein expression results in decreased and / or reduced expression of a molecule selected from the group consisting of PD-1, LAG3, TIM3, CISH, CBLB and combinations thereof, and in increased expression and / or improved by CCR2, CCR4, CCR5, CXCR2, CXCR3, CX3CR1 and combinations thereof.
[001044] [001044] In some modalities, there is a reduction in expression of approximately 5%, approximately 10%, approximately 10%, approximately 20%, approximately 25%, approximately 30%, approximately 35%, approximately 40%, approximately 45%, approximately 50%, approximately 55%, approximately 60%, approximately 65%, approximately 70%, approximately 75%, approximately 80%, approximately 85%, approximately 90% or approximately 95%. In some embodiments, there is a reduction in expression of at least approximately 65%, approximately 70%, approximately 75%, approximately 80%, approximately 85%, approximately 90% or approximately 95%. In some embodiments, there is a reduction in expression of at least approximately 75%, approximately 80%, approximately 85%, approximately 90% or approximately 95%. In some embodiments, there is a reduction in expression of at least approximately 80%, approximately 85%, approximately 90% or approximately 95%. In some embodiments, there is a reduction in expression of at least approximately 85%, approximately 90% or approximately 95%. In some modalities,
[001045] [001045] In some modalities, there is an increase in expression of approximately 5%, approximately 10%, approximately 10%, approximately 20%, approximately 25%, approximately 30%, approximately 35%, approximately 40%, approximately 45%, approximately 50%, approximately 55%, approximately 60%, approximately 65%, approximately 70%, approximately 75%, approximately 80%, approximately 85%, approximately 90% or approximately 95%. In some embodiments, there is an increase in expression of at least approximately 65%, approximately 70%, approximately 75%, approximately 80%, approximately 85%, approximately 90% or approximately 95%. In some embodiments, there is an increase in expression of at least approximately 75%, approximately 80%, approximately 85%, approximately 90% or approximately 95%. In some embodiments, there is an increase in expression of at least approximately 80%, approximately 85%, approximately 90% or approximately 95%. In some modalities, there is an increase in expression of at least approximately 85%, approximately 90%, or approximately 95%. In some modalities, there is an increase in expression of at least approximately 80%. In some modalities, there is an increase in expression of at least approximately 85%. In some modalities, there is an increase in expression of at least approximately 90%. In some modalities,
[001046] [001046] In some modalities, the transient alteration of protein expression is induced by the treatment of TILs with transcription factors (TFs) and / or other molecules capable of transiently altering protein expression in TILs. In some modalities, the microfluidic platform without SQZ vector is used for intracellular delivery of transcription factors (TFs) and / or other molecules capable of transiently altering protein expression. Such methods, which demonstrate the ability to deliver proteins, including transcription factors, to a variety of primary human cells, including T cells (Sharei et al. PNAS 2013, in addition to Sharei et al., PLOS ONE 2015 and Greisbeck et al. , J. Immunology, vol. 195, 2015) have already been described; see, for example, International Patent Publications WO 2013 / 059343A1, WO 2017 / 008063A1 and WO 2017 / 123663A1, all of which are incorporated herein in their entirety by reference. Such methods, as described in International Patent Publications WO 2013 / 059343A1, WO 2017 / 008063A1 and WO 2017 / 123663A1 can be employed with the present invention in order to expose a population of TILs to transcription factors (TFs) and / or other molecules capable of inducing transient protein expression, wherein said TFs and / or other molecules capable of inducing transient protein expression increase the expression of tumor antigens and / or the number of T cells specific for tumor antigens in the TIL population, resulting, thus, in the reprogramming of the TIL population and an increase in the therapeutic efficacy of the reprogrammed TIL population compared to an unreprogrammed TIL population. In some embodiments, reprogramming results in an increased subpopulation of effector T cells and / or central memory T cells in relation to the initial or previous population (ie, before reprogramming) of TILs, as here
[001047] [001047] In some modalities, the transcription factor (TF) includes, among others, TCF-1, NOTCH 1/2 ICD and / or MYB. In some modalities, the transcription factor (TF) is TCF-1. In some embodiments, the transcription factor (TF) is NOTCH 1/2 ICD. In some embodiments, the transcription factor (TF) is MYB. In some embodiments, the transcription factor (TF) is administered with a culture of induced pluripotent stem cells (iPSC), such as the commercially available KNOCKOUT Serum Replacement (Gibco / ThermoFisher), to induce additional reprogramming of TILs. In some modalities, the transcription factor (TF) is administered with an iPSC cocktail to induce additional reprogramming of TILs. In some modalities, the transcription factor (TF) is administered without an iPSC cocktail. In some modalities, reprogramming results in an increase in the percentage of TSCMs. In some modalities, reprogramming results in an increase in the percentage of TSCMs of approximately 5%, approximately 10%, approximately 10%, approximately 20%, approximately 25%, approximately 30%, approximately 35%, approximately 40%, approximately 45%, approximately 50%, approximately 55%, approximately 60%, approximately 65%, approximately 70%, approximately 75%, approximately 80%, approximately 85%, approximately 90% or approximately 95% of TSCMs.
[001048] [001048] In some embodiments, a method of transient alteration of protein expression, as described above, can be combined with a method to genetically modify a population of TILs and includes the stage of stable gene incorporation for the production of one or more proteins. In certain modalities, the method comprises a stage of genetic modification of a population of TILs. In certain modalities, the method involves genetically modifying the first population of TILs, the
[001049] [001049] In some embodiments, the transient change in protein expression is a reduction in RNA-induced expression of interference from
[001050] [001050] In some modalities, sdRNA is inserted into a population of TILs during production. In some embodiments, sdRNA encodes the RNA that interferes with NOTCH 1/2 ICD, PD-1, CTLA-4 TIM-3, LAG-3, TIGIT, TGFβ, TGFBR2, cAMP-dependent protein kinase A (PKA), BAFF BR3, CISH and / or CBLB. In some embodiments, the reduction in expression is determined based on a percentage of gene silencing, for example, as assessed by flow cytometry and / or qPCR. In some modalities, there is a reduction in expression of approximately 5%, approximately 10%, approximately 10%, approximately 20%, approximately 25%, approximately 30%, approximately 35%, approximately 40%, approximately 45%, approximately 50%, approximately 55%, approximately 60%, approximately 65%, approximately 70%, approximately 75%, approximately 80%, approximately 85%, approximately 90% or approximately 95%. In some embodiments, there is a reduction in expression of at least approximately 65%, approximately 70%, approximately 75%, approximately 80%, approximately 85%, approximately 90%, or
[001051] [001051] The self-deliverable RNAi (self-delivery) technology, based on the chemical modification of siRNAs, can be employed with the methods of the present invention for successful delivery of sdRNAs to TILs as described herein. The combination of modifications in the main chain with asymmetric siRNA structure and a hydrophobic ligand (see, for example, Ligtenberg, et al., Mol. Therapy, 2018 and US20160304873) allows sdRNAs to penetrate cultured mammalian cells, without formulations and methods by simply adding it to the culture medium, capitalizing on the stability of the sdRNAs nuclease. This stability allows the support of constant levels of RNAi-mediated reduction in the activity of target genes, simply maintaining the active concentration of sdRNA in the medium. While not bound by theory, stabilization of the sdRNA backbone prolongs the reduction in the effects of gene expression that can last for months in cells that are not dividing.
[001052] [001052] In some modalities, the efficiency in transfection of TILs
[001053] [001053] Small interference RNA (siRNA), sometimes known as short interfering RNA or silencing RNA, is a double-stranded RNA molecule, generally 19-25 base pairs in length. siRNA is used in interference RNA (RNAi), which interferes with the expression of specific genes with complementary nucleotide sequences.
[001054] [001054] Double-stranded DNA (dsRNA) can generally be used to define any molecule that comprises a pair of complementary RNA strands, usually a sense strand (passenger) and antisense strips (guide), and can include protruding regions (overhang) simple ribbon. The term dsRNA, in contrast to siRNA, generally refers to a precursor molecule that includes the sequence of a siRNA molecule that is released from the larger molecule of dsRNA by the action of enzymatic cleavage systems, including Dicer.
[001055] [001055] sdRNA (self-delivery RNA) is a new class of covalently modified RNAi compounds that do not require a delivery vehicle to enter cells and with improved pharmacology compared to traditional siRNAs. "Self-delivered RNA" or "sdRNA" is a hydrophobically modified interfering RNA antisense hybrid, shown to be highly effective in vitro in primary cells and in vivo by local administration. Robust absorption and / or silencing without toxicity has been demonstrated. sdRNAs are usually chemically modified asymmetric nucleic acid molecules with minimal double-stranded regions. SdRNA molecules typically contain single-stranded and double-stranded regions, and can contain a variety of chemical modifications in both single- and double-stranded regions of the molecule. In addition, sdRNA molecules can be attached to a hydrophobic conjugate, such as a conventional and advanced sterol-like molecule, as described herein. sdRNAs and associated methods for producing such sdRNAs have also been extensively described in, for example, US20160304873, WO2010033246, WO2017070151, WO2009102427, WO2011119887, WO2010033247A2, WO2009045457, WO2011119852, all of which are incorporated herein by reference, in their entirety, by reference, in their entirety, by reference to all . To optimize structure, chemistry, targeting position, preferences in relation to the sdRNA sequence, a proprietary algorithm was developed and used to predict sdRNA potency (see, for example, US 20160304873). Based on these analyzes, functional sdRNA sequences were generally defined as presenting a reduction of over 70% in expression at the concentration of 1 µM, with more than 40% probability.
[001056] [001056] In some embodiments, the sdRNA sequences used in the invention exhibit a 70% reduction in expression of the target gene. In some embodiments, the sdRNA sequences used in the invention exhibit reduced
[001057] [001057] In some embodiments, the oligonucleotide agents comprise one or more modifications to increase the stability and / or effectiveness of the therapeutic agent, and to effect an efficient delivery of the oligonucleotide to the cells or tissue to be treated. Such modifications may include a 2'-O-methyl modification, a 2'-O-fluoro modification, a diphosphorothioate modification, 2 'F-modified nucleotide, 2'-O- nucleotide
[001058] [001058] In some embodiments, the double-stranded oligonucleotide of the invention is double-stranded in its entire length, that is, with no single stranded sequence protruding at either end of the molecule, that is, it is blunt-ended (with blunt end) ). In some embodiments, the individual nucleic acid molecules can be of different lengths. In other words, the double-stranded oligonucleotide of the invention is not double-stranded in its entirety. For example, when two separate nucleic acid molecules are used, one of the molecules, e.g. e.g., the first molecule comprising an antisense sequence, may be longer than the second molecule that hybridizes to it (leaving a portion of the molecule with simple tape). In some embodiments, when a single nucleic acid molecule is used, a portion of the molecule at either end may remain single-stranded.
[001059] [001059] In some embodiments, a double-stranded oligonucleotide of the invention contains mismatches and / or handles or
[001060] [001060] In some embodiments, the oligonucleotide can be substantially protected from nucleases, e.g. modifying the 3 'or 5' bonds (e.g., U.S. Patent No. 5,849,902 and WO 98/13526). For example, it is possible to make oligonucleotides resistant by the inclusion of a "blocking group". The term “blocking group” in this specification refers to substituents (eg, in addition to OH groups) that can be attached to oligonucleotides or nucleomonomers, either as protecting groups or coupling groups for synthesis (eg ., FITC, propyl (CH2-CH2-CH3), glycol (-0-CH2-CH2-O-), phosphate (PO32 +), hydrogenphosphonate or phosphoramidite). "Blocking groups" can also include "end blocking groups" or "exonuclease blocking groups" that protect the 5 'and 3' termini of the oligonucleotide, including modified nucleotides and non-nucleotide structures resistant to exonucleases.
[001061] [001061] In some embodiments, at least part of the contiguous polynucleotides within the sdRNA is joined by a substitute bond, e.g. a phosphorothioate bond.
[001062] [001062] In some modalities, chemical modification can lead to at least 1.5; two; 3; 4; 5; 6; 7; 8; 9; 10; 15; 20; 25; 30; 35; 40; 45; 50; 55;
[001063] [001063] In some embodiments, sdRNA or sd-rxRNAs exhibit improved endosomal release of sd-rxRNA molecules through protonable amines. In some embodiments, protonable amines are incorporated into the sense tape (the part of the molecule that is discarded after loading the RISC). In some embodiments, the sdRNA compounds of the invention comprise an asymmetric compound comprising a duplex region (necessary for efficient RISC entry 10-15 bases in length) and a single strand region 4-12 nucleotides in length; with a 13 nucleotide duplex. In some embodiments, a single strand region with 6 nucleotides is used. In some embodiments, the single strand region of the sdRNA comprises 2-12 internucleotide phosphorothioate bonds (referred to as phosphorothioate modifications). In some embodiments, 6-8 internucleotide phosphorothioate bonds are employed. In some embodiments, the sdRNA compounds of the invention also include a unique pattern of chemical modifications, which provides stability and is compatible with RISC input.
[001064] [001064] The guide tape, for example, can also be modified by any chemical modification that confirms the stability without interfering with the input of the RISC. In some modalities, the pattern of changes
[001065] [001065] In some embodiments, at least 30% of the nucleotides in the sdRNA or sd-rxRNA are modified. In some modalities, at least 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44% , 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61 %, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% , 95%, 96%, 97%, 98% or 99% of the nucleotides in the sdRNA or sd-rxRNA are modified. In some embodiments, 100% of the nucleotides in the sdRNA or sd-rxRNA are modified.
[001066] [001066] In some embodiments, sdRNA molecules have minimal double-stranded regions. In some embodiments, the region of the double-stranded molecule ranges from 8-15 nucleotides in length. In some embodiments, the region of the double-stranded molecule is 8, 9, 10, 11, 12, 13, 14 or 15 nucleotides in length. In some embodiments, the double-stranded region is 13 nucleotides in length. There can be 100% complementarity between the guide and passenger tapes, or there may be one or more pairing errors between the guide and passenger tapes. In some embodiments, at one end of the double-stranded molecule, the molecule is blunt-ended or has a protrusion of a nucleotide. The single strand region of the molecule is, in some embodiments, between 4-12 nucleotides in length. In some embodiments, the single strand region may be 4, 5, 6, 7, 8, 9, 10, 11 or 12 nucleotides in length. In some embodiments, the single strand region may be less than 4 or more than 12 nucleotides in length. In certain embodiments, the single strand region is 6 or 7 nucleotides in length.
[001067] [001067] In some embodiments, sdRNA molecules have
[001068] [001068] In some modalities, sdRNA is optimized to increase potency and / or reduce toxicity. In some embodiments, the nucleotide length of the guide and / or passenger strand and / or the number of phosphorothioate modifications in the guide and / or passenger strand may, in some respects, influence the potency of the RNA molecule, while replacing 2'- modifications. fluoro (2'F) by modifications 2'-0-methyl (2'OMe) can, in some aspects, influence the toxicity of the molecule. In some embodiments, a reduction in the 2'F content of a molecule is expected to reduce the toxicity of the molecule. In some embodiments, the number of phosphorothioate modifications in an RNA molecule can influence the absorption of the molecule in a cell, for example, the efficiency of passive absorption of the molecule in a cell. In some embodiments, sdRNA has no 2'F modification and is still characterized by equal effectiveness in cell absorption and tissue penetration.
[001069] [001069] In some embodiments, a guide strip approximately 18-19 nucleotides in length and approximately 2-14 phosphate modifications. For example, a guide strip can contain 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or more than 14 nucleotides that are modified in the phosphate. The guide tape may contain one or more modifications that provide increased stability without interfering with RISC input. The phosphate-modified nucleotides, such as phosphorothioate-modified nucleotides, can be at the 3 ', 5' end or spread across the guide strip. In some embodiments, the 10 terminal 3 'nucleotides
[001070] [001070] The self-deliverable RNAi technology provides a method to directly transfect cells with the RNAi agent, without the need for additional formulations or techniques. The ability to transfect difficult to transfect cell lines, high in vivo activity and simplicity of use are characteristics of the compositions and methods that have significant functional advantages over traditional siRNA-based techniques and, thus, sdRNA methods are used in several modalities related to methods of reducing target gene expression in the TILs of the present invention. Methods with sdRNAi allow
[001071] [001071] sdRNA are formed as hydrophobically modified antisense oligonucleotide siRNA hybrid structures, and are described, for example, in Byrne et al., December 2013, J. Ocular Pharmacology and Therapeutics, 29 (10): 855-864, here incorporated in its entirety by reference.
[001072] [001072] In some embodiments, sdRNA oligonucleotides can be delivered to the TILs described here by sterile electroporation. In certain modalities, the method comprises the sterile electroporation of a population of TILs to between the oligonucleotides-sdRNA.
[001073] [001073] In some embodiments, oligonucleotides can be delivered to cells in combination with a system for transmembrane delivery. In some embodiments, this system for transmembrane delivery comprises lipids, viral vectors and the like. In some embodiments, the oligonucleotide agent is a self-delivering RNAi agent, which requires no agents for delivery. In certain embodiments, the method comprises the use of a system for transmembrane delivery to deliver oligonucleotides-sdRNA to a population of TILs.
[001074] [001074] Oligonucleotides and oligonucleotide compositions are brought into contact with (e.g., brought into contact with, also referred to herein as administered or released to) and absorbed by the described TILs, including through passive absorption by the TILs. SdRNA can be added to the TILs described here during the first expansion, for example, Figure 7 and / or Figure 85, Step B, after the first expansion, for example, during Step C, before or during the second expansion, for example , before or during Step D, after Step D and before collection at
[001075] [001075] The oligonucleotide compositions of the invention, including sdRNA, can be brought into contact with TILs as described here during the expansion process, for example, by dissolving sdRNA in high concentrations in the cell culture medium and allowing sufficient time for absorption to occur passive. In certain embodiments, the method
[001076] [001076] In some embodiments, the delivery of oligonucleotides to cells can be increased by suitable methods recognized in the art, including with calcium phosphate, DMSO, glycerol or dextran, electroporation, or by transfection, e.g. using cationic, anionic or neutral lipid compositions or liposomes by methods known in the art (see, e.g., WO 90/14074; WO 91/16024; WO 91/17424; US Patent No. 4 897 355; Bergan et al, 1993. Nucleic Acids Research. 21: 3567).
[001077] [001077] In some embodiments, more than one sdRNA is used to reduce the expression of a target gene. In some modalities, sdRNA targeting one or more of PD-1, TIM-3, CBLB, LAG3 and / or CISH are used together. In some embodiments, a PD-1 sdRNA is used with one or more of TIM-3, CBLB, LAG3 and / or CISH to reduce the expression of more than one target gene. In some embodiments, a LAG3 sdRNA is used in combination with a CISH-directed sdRNA to reduce the gene expression of both targets. In some embodiments, sdRNAs targeting one or more of PD-1, TIM-3, CBLB, LAG3 and / or CISH are commercially available from Advirna LLC, Worcester, MA, USA.
[001078] [001078] In some modalities, sdRNA is directed to a gene selected from the group consisting of PD-1, LAG3, TIM3, CTLA-4, TIGIT, CISH, TGFβR2, PKA, CBLB, BAFF (BR3) and combinations of themselves. In some modalities, sdRNA is directed to a gene selected from the group consisting of PD-1, LAG3, TIM3, CTLA-4,
[001079] [001079] As discussed above, modalities of the present invention provide tumor infiltrating lymphocytes (TILs) that have been genetically modified, through gene editing, to improve their therapeutic effect. Modalities of the present invention adopt genetic editing through the insertion of nucleotides (RNA or DNA) in a population of TILs to promote the expression of one or more proteins and to inhibit the expression of one or more proteins, as well as combinations of them.
[001080] [001080] In some modalities, the method comprises a method to genetically modify a population of TILs, which includes the stage of stable incorporation of genes to produce one or more proteins. In one embodiment, a method for genetically modifying a population of TILs includes the retroviral transduction step. In one embodiment, a method for genetically modifying a population of TILs includes the step of lentiviral transduction. Lentiviral transduction systems are known in the art and are described, e.g. in Levine, et al., Proc. Nat’l Acad. Sci. 2006, 103, 17372-77; Zufferey, et al., Nat. Biotechnol. 1997, 15, 871-75; Dull, et al., J. Virology 1998, 72, 8463-71, and U.S. Patent No. 6,627,442, the contents of which are hereby incorporated by reference. In one embodiment, a method for genetically modifying a population of TILs includes the gamma retroviral transduction step. Gamma retroviral transduction systems are known in the art and are described, e.g. e.g., Cepko and Pear, Cur. Prot. Mol. Biol. 1996, 9.9.1-9.9.16, the content of which is incorporated herein by reference. In one embodiment, a method for genetically modifying a population of TILs includes the transposon-mediated gene transfer step. Transposon-mediated gene transfer systems are known in the art and include systems in which transposase is provided as a DNA expression vector or as an expressible RNA or protein, such that long-term expression of transposase does not occur in transgenic cells, for example, a transposase provided as mRNA (eg, an mRNA comprising a cap and a poly-A tail). Suitable systems for transposon-mediated gene transfer,
[001081] [001081] In one embodiment, the method comprises a method for genetically modifying a population of TILs, p. a first population, a second population and / or a third population as described herein. In one embodiment, a method for genetically modifying a population of TILs includes the step of stable gene incorporation for the production or inhibition (eg, silencing) of one or more proteins. In one embodiment, a method for genetically modifying a population of TILs includes the electroporation step. Electroporation methods are known in the art and are described, e.g. in Tsong, Biophys. J. 1991, 60, 297-306, and U.S. Patent Application Publication No. 2014/0227237 A1, the contents of which are hereby incorporated by reference. Other electroporation methods known in the art, such as those described in U.S. Patent Nos. 5,019,034; 5,128,257; 5,137,817; 5,173,158; 5,232,856; 5,273,525; 5,304,120; 5,318,514; 6,010,613 and 6,078,490, the contents of which are hereby incorporated by reference, may be used. In one embodiment, the electroporation method is a sterile electroporation method. In one embodiment, the electroporation method is a pulsed electroporation method. In one embodiment, the electroporation method is a pulsed electroporation method, which comprises the steps of treating TILs with pulses of electric fields to alter, manipulate or cause definite and controlled, permanent or temporary changes to the TILs, comprising the stage of applying a sequence of at least three unique DC electrical pulses, controlled by the operator, independently programmed, with field strengths equal to or greater than 100 V / cm, to the TILs, in which the
[001082] [001082] According to one modality, the gene editing process may include the use of a programmable nuclease that mediates the generation of a double-stranded or single-stranded break in one or more immunological checkpoint (control) genes. Such programmable nucleases allow the genome to be accurately edited by introducing breaks at specific genomic loci, that is, they are based on the recognition of a specific DNA sequence within the genome to reach a nuclease domain at that location and mediate the generation of a break double tape in the target sequence. A double strand break in DNA subsequently recruits repair machinery endogenous to the site of the break to mediate genome editing by joining non-homologous ends (NHEJ) or homology-directed repair (HDR). Thus, repair of the break may result in the introduction of insertion / deletion mutations that target (eg, silence, repress or increase) the target gene product.
[001083] [001083] The main classes of nucleases that were developed
[001084] [001084] Non-limiting examples of methods for editing genes that can be used according to the TIL expansion methods of the present invention include methods with CRISPR, methods with TALE and methods with ZFN, which are described in more detail below. According to one modality, a method for the expansion of TILs in a therapeutic population can be carried out according to any modality of the methods described here (eg, GEN 3 processes) or as described in PCT / US2017 / 058610, PCT / US2018 / 012605, or PCT / US2018 / 012633, in which the method further comprises the gene editing of at least part of the TILs by one or more of a method with CRISPR, a method with TALE or a method with ZFN, a in order to generate TILs that can provide an increased therapeutic effect. According to one embodiment, TILs from edited genes can be evaluated for an improved therapeutic effect by comparing them with unmodified TILs in vitro, e.g. eg, evaluating effector function in vitro, cytokine profiles etc. compared to unmodified TILs. In certain embodiments, the method comprises editing genes from a population of TILs using CRISPR, TALE and / or ZFN methods.
[001085] [001085] In some embodiments of the present invention, electroporation is used to deliver a gene editing system, such as CRISPR, TALEN and ZFN system. In some embodiments of the present invention, the electroporation system is an electroporation flow system. An example of an electroporation flow system suitable for use with some embodiments of the present invention is the commercially available MaxCyte STX system. There are several alternative electroporation instruments available commercially that may be suitable for use with the present invention, such as the AgilePulse or ECM 830 system available from BTX-Harvard Apparatus, Cellaxess Elektra (Cellectricon), Nucleofector (Lonza / Amaxa), GenePulser MXcell (BIORAD), iPorator-96 (Primax) or siPORTer96 (Ambion). In some embodiments of the present invention, the electroporation system forms a closed, sterile system with the rest of the TIL expansion method. In some embodiments of the present invention, the electroporation system is a pulsed electroporation system as described herein, and forms a closed, sterile system with the rest of the TIL expansion method.
[001086] [001086] A method for expanding TILs in a therapeutic population can be performed according to any modality of the methods described here (eg, GEN 3 process), or as described in PCT / US2017 / 058610, PCT / US2018 / 012605 or PCT / US2018 / 012633, where the method also comprises the gene editing of at least part of the TILs by a CRISPR method (eg, CRISPR / Cas9 or CRISPR / Cpf1). According to particular modalities, the use of a CRISPR method during the TIL expansion process causes the expression of one or more immunological checkpoint genes to be silenced or reduced in at least part of the therapeutic population of TILs. Alternatively, the use of a CRISPR method during the expansion process causes the expression of one or more immunological checkpoint genes to be enhanced by at least
[001087] [001087] CRISPR stands for “Clustered Regularly Interspaced Short Palindromic Repeats” [Short palindromic repetitions, interspersed regularly grouped]. A method of using a CRISPR system for gene editing is also referred to here as the CRISPR method. There are three types of CRISPR systems that incorporate RNAs and Cas proteins, and that can be used in accordance with the present invention: Types I, II, and III. CRISPR Type II (exemplified by Cas9) is one of the best characterized systems.
[001088] [001088] CRISPR technology was adapted from the natural defense mechanisms of bacteria and archaea (the domain of single-celled microorganisms). These organisms use CRISPR-derived RNA and various Cas proteins, including Cas9, to thwart attacks by viruses and other foreign bodies by cutting into pieces and destroying the DNA of a foreign invader. CRISPR is a specialized region of DNA with two distinct characteristics: the presence of repeats of nucleotides and spacers. Repeated nucleotide sequences are distributed throughout a CRISPR region with short segments of foreign DNA (spacers) interspersed between the repeated sequences. In the CRISPR / Cas type II system, the spacers are integrated into the CRISPR genomic loci and transcribed and processed into CRISPR short RNA (crRNA). These crRNAs anneal to trans transactivation crRNAs (tracrRNAs) and direct direct sequence-specific cleavage and silencing of pathogenic DNA by Cas proteins. Recognition of the target by the Cas9 protein requires a "seed" sequence within the crRNA and a motif sequence adjacent to the protospace (PAM) containing conserved dinucleotide upstream of the crRNA binding region. The CRISPR / Cas system can thus be redirected to cleave virtually any DNA sequence when redesigning the crRNA. The crRNA and tracrRNA in the native system can be
[001089] [001089] Non-limiting examples of genes that can be silenced or inhibited by permanent gene editing of TILs using a CRISPR method include PD-1, CTLA-4, LAG-3, HAVCR2 (TIM-3), Cish, TGFβ, PKA , CBL-B, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, BTLA, CD160, TIGIT, CD96, CRTAM, LAIR1, SIGLEC7, SIGLEC9, CD244, TNFRSF10B, TNFRSF10A, CASP8, CASP10, CASP3, CASP3, CASD , SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1, IL10RA, IL10RB, HMOX2, IL6R, IL6ST, EIF2AK4, CSK, PAG1, SIT1, FOXP3, PRDM1, BATF, GUCY1A2, GUCY1A3, GUCY1A3, GUCY1A3, GUCY1A3, GUCY1A3, GUCY1A3, GUCY1A3
[001090] [001090] Non-limiting examples of genes that can be enhanced by permanent gene editing of TILs using a CRISPR method include CCR2, CCR4, CCR5, CXCR2, CXCR3, CX3CR1, IL-2, IL12, IL-15 and IL-21.
[001091] [001091] Examples of systems, methods and compositions for altering the expression of a target gene sequence by a CRISPR method, and which can be used in accordance with embodiments of the present invention, are described in U.S. Patent Nos. 8,697,359; 8,993,233; 8,795,965; 8,771,945; 8 889 356; 8 865 406; 8 999 641; 8 945 839; 8 932 814; 8 871 445; 8,906,616; and 8 895 308, the contents of which are hereby incorporated by reference. Resources for carrying out the CRISPR methods, such as plasmids for the expression of CRISPR / Cas9 and CRISPR / Cpf1, are commercially available from companies such as GenScript.
[001092] [001092] In one embodiment, genetic modifications of TIL populations, as described herein, can be performed by the CRISPR / Cpf1 system as described in U.S. Patent No. US 9790490, the content of which is incorporated herein by reference.
[001093] [001093] A method for expanding TILs in a therapeutic population can be carried out according to any modality of the methods described here (eg, process 2A) or as described in PCT / US2017 / 058610, PCT / US2018 / 012605 or PCT / US2018 / 012633, in which the method also includes the gene editing of at least part of the TILs by a TALE method. According to particular modalities, the use of a TALE method during the expansion process of TILs causes the expression of one or more immunological checkpoint genes to be silenced or reduced in at least a part of the therapeutic population of TILs. Alternatively, the use of a TALE method during the TIL expansion process causes the expression of one or more immunological checkpoint genes to be enhanced in at least part of the therapeutic population of TILs.
[001094] [001094] TALE stands for "Transcription Activator-Like Effector" proteins, which include TALENs ("Transcription Activator-Like Effector Nucleases"). A method of using a TALE system for gene editing can also be referred to here as the TALE method. TALEs are natural proteins of plant pathogenic bacteria, belonging to the genus Xanthomonas, and contain DNA-binding domains composed of a series of 33-35 amino acid repeat domains that each recognize a single base pair. The specificity of TALE is determined by two hypervariable amino acids that are known as the repeated variable di-residues (RVDs). Modular TALE repetitions are joined to recognize sequences
[001095] [001095] Several large systematic studies, using various assembly methods, have indicated that TALE repetitions can be combined to recognize virtually any user-defined sequence. Custom design TALE arrangements are also commercially available from Cellectis Bioresearch (Paris, France), Transposagen Biopharmaceuticals (Lexington, KY, USA) and Life Technologies (Grand Island, NY, USA). TALE and TALEN methods suitable for use in the present invention are described in U.S. Patent Application Publication No. US 2011/0201118 A1; US 2013/0117869 A1; US 2013/0315884 A1; US 2015/0203871 A1 and US 2016/0120906 A1, the contents of which are hereby incorporated by reference.
[001096] [001096] Non-limiting examples of genes that can be silenced or inhibited by permanent gene editing of TILs using a TALE method include PD-1, CTLA-4, LAG-3, HAVCR2 (TIM-3), Cish, TGFβ, PKA , CBL-B, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, BTLA, CD160, TIGIT, CD96, CRTAM, LAIR1, SIGLEC7, SIGLEC9, CD244, TNFRSF10B, TNFRSF10A, CASP8, CASP10, CASP3, CASP3, CASD , SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1, IL10RA, IL10RB, HMOX2, IL6R, IL6ST, EIF2AK4, CSK, PAG1, SIT1, FOXP3, PRDM1, BATF, GUCY1A2, GUCY1A3, GUCY1A3, GUCY1A3, GUCY1A3, GUCY1A3, GUCY1A3, GUCY1A3
[001097] [001097] Non-limiting examples of genes that can be enhanced by permanent gene editing of TILs using a TALE method include CCR2, CCR4, CCR5, CXCR2, CXCR3, CX3CR1, IL-2, IL12, IL-15 and IL-21.
[001098] [001098] Examples of systems, methods and compositions for altering the expression of a target gene sequence by a TALE method, and which can be used according to the modalities of the present invention, are described in US Patent No. 8,586,526, the content of which is incorporated herein by reference.
[001099] [001099] A method for expanding TILs in a therapeutic population can be carried out according to any modality of the methods described here (eg, GEN 3 process) or as described in PCT / US2017 / 058610, PCT / US2018 / 012605 or PCT / US2018 / 012633, in which the method also comprises the gene editing of at least part of the TILs by a zinc finger method or with a zinc finger nuclease. According to particular modalities, the use of a zinc finger method during the expansion process of TILs causes the expression of one or more immunological checkpoint genes to be silenced or reduced in at least a part of the therapeutic population of TILs. Alternatively, the use of a zinc finger method during the TIL expansion process causes the expression of one or more immunological checkpoint genes to be enhanced in at least part of the therapeutic population of TILs.
[001100] [001100] An individual zinc finger contains approximately 30 amino acids in a conserved ββα configuration. Several amino acids on the α helix surface typically come in contact with 3 bp in the larger DNA groove, with varying levels of selectively. Zinc fingers have two protein domains. The first domain is the DNA binding domain, which includes eukaryotic transcription factors and contains the zinc finger. The second domain is the nuclease domain, which includes the restriction enzyme FokI and is responsible for the catalytic cleavage of DNA.
[001101] [001101] The DNA binding domains of individual ZFNs contain
[001102] [001102] Non-limiting examples of genes that can be silenced or inhibited by permanent gene editing of TILs using a zinc finger method include PD-1, CTLA-4, LAG-3, HAVCR2 (TIM-3), Cish, TGFβ, PKA, CBL-B, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, BTLA, CD160, TIGIT, CD96, CRTAM, LAIR1, SIGLEC7, SIGLEC9, CD244, TNFRSF10B, TNFRSF10A, CASP8, CASP10, CASD3 FAS, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1, IL10RA, IL10RB, HMOX2, IL6R, IL6ST, EIF2AK4, CSK, PAG1, SIT1, FOXP3, PRDM1, BATF, GUCY1A2, GUCY1B3, GUCY1B3 and GUCY1B3
[001103] [001103] Non-limiting examples of genes that can be enhanced by permanent gene editing of TILs using a zinc finger method
[001104] [001104] Examples of systems, methods and compositions for altering the expression of a target gene sequence by a zinc finger method, which can be used in accordance with the modalities of the present invention, are described in US Patent Nos. 6,534,261, 6,607,882 , 6 746 838, 6 794 136, 6 824 978, 6 866 997, 6 933 113, 6 979 539, 7 013 219, 7 030 215, 7 220 719, 7 241 573, 7 241 574, 7 585 849, 7 595 376, 6 903 185 and 6 479 626, the contents of which are hereby incorporated by reference.
[001105] [001105] Other examples of systems, methods and compositions for altering the expression of a target gene sequence by a zinc finger method, which can be used according to modalities of the present invention, are described in Beane, et al., Mol. Therapy , 2015, 23 1380-1390, the content of which is incorporated herein by reference.
[001106] [001106] In some embodiments, TILs are optionally genetically modified to include additional functionality, including, among others, a high affinity T cell receptor (TCR), p. a TCR targeting a tumor-associated antigen, such as MAGE-1, HER2 or NY-ESO-1, or a chimeric antigen (CAR) receptor that binds to a molecule on the tumor-associated cell surface (p eg, mesothelin) or a molecule on the cell surface of restricted lineage (eg, CD19). In certain embodiments, the method comprises genetically modifying a population of TILs to include a high affinity T cell receptor (TCR), e.g. , a TCR targeting a tumor-associated antigen, such as MAGE-1 HER2, or NY-ESO-1, or a chimeric antigen (CAR) receptor that binds to a molecule on the tumor-associated cell surface (p eg, mesothelin) or a molecule on the cell surface of restricted lineage (eg, CD19). Suitably, the population of TILs can be a first population, a second population
[001107] [001107] The present invention provides the use of closed systems during the process of TIL cultivation, for example, in conjunction with the Gen 2 or Gen 3 processes. Such closed systems allow to prevent and / or reduce microbial contamination, allow the use fewer bottles and allow cost savings. In some embodiments, the closed system uses two containers.
[001108] [001108] Such closed systems are well known in the art and can be found, for example, at http://www.fda.gov/cber/guidelines.htm and https://www.fda.gov/BiologicsBloodVaccines/GuidanceComplianceRegulator yInformation /Guidances/Blood/ucm076779.htm.
[001109] [001109] Sterile connection devices (STCDs) produce sterile welds between two pieces of compatible tubes. This procedure allows the sterile connection of a variety of containers and tube diameters. In some embodiments, closed systems include luer lock and thermally sealed systems as described. In some embodiments, access to the closed system is through syringes under sterile conditions in order to maintain the sterility and closed nature of the system. In some embodiments, a closed system as described herein is employed. In some embodiments, TILs are formulated in a container for formulating the final product according to the method described here.
[001110] [001110] In some embodiments, the closed system uses a container from the moment the tumor fragments are obtained until the TILs are ready for administration to the patient or for cryopreservation. In some embodiments when two containers are used, the first container is a closed G container and the population of TILs is centrifuged and transferred to an infusion bag without opening the first G container
[001111] [001111] In some modalities, the reduction in microbial contamination is between approximately 5% and 100%. In some modalities, the reduction in microbial contamination is between approximately 5% and 95%. In some modalities, the reduction in microbial contamination is between approximately 5% and 90%. In some modalities, the reduction in microbial contamination is between approximately 10% and 90%. In some modalities, the reduction in microbial contamination is between approximately 15% and 85%. In some embodiments, the reduction in microbial contamination is approximately 5%, approximately 10%, approximately 15%, approximately 20%, approximately 25%, approximately 30%, approximately 35%, approximately 40%, approximately 45%, approximately 50%, approximately 55%, approximately 60%, approximately 65%, approximately 70%, approximately 75%, approximately 80%, approximately 85%, approximately 90%, approximately 95%, approximately 97%, approximately 98%, approximately 99% or approximately 100 %.
[001112] [001112] The closed system allows the growth of TILs in the absence and / or with a significant reduction in microbial contamination.
[001113] [001113] Furthermore, the pH, the partial pressure of carbon dioxide and the partial pressure of oxygen in the TIL cell culture environment vary each as the cells are cultured. Consequently, even if an appropriate medium for cell culture is circulated, the environment
[001114] [001114] In some modalities, the pressure inside the closed environment is controlled continuously or intermittently. That is, the pressure in the closed environment can be varied by means of a pressure maintenance device, for example, thus ensuring that the space is suitable for the growth of TILs in a state of positive pressure, or by promoting the exudation of liquid in a state of negative pressure and thus promoting cell proliferation. In addition, with the intermittent application of negative pressure, it is possible to uniformly and efficiently replace the circulating liquid in the closed environment.
[001115] [001115] In some modalities, components of the culture, excellent for the proliferation of TILs, can be replaced or added, including factors such as IL-2 and / or OKT3, in addition to combinations, which can be added. XII. Cryopreservation of TILs
[001116] [001116] A bulk TIL population (for example, the second
[001117] [001117] When appropriate, the cells are removed from the freezer and thawed in a water bath at 37 ºC until approximately 4/5 of the solution has thawed. The cells are generally suspended in complete medium and, optionally, washed one or more times. In some embodiments, thawed TILs can be counted and assessed for viability as is known in the art.
[001118] [001118] In a preferred embodiment, a population of TILs is cryopreserved with CS10 cryopreservation medium (CryoStor 10, BioLife Solutions). In a preferred embodiment, a population of TILs is cryopreserved using a cryopreservation medium containing
[001119] [001119] As discussed above, and exemplified in steps A to E as shown in Figure 85 (in particular, eg, Figure 85B), cryopreservation can occur at numerous points throughout the TIL expansion process. In some embodiments, the population of expanded TILs after the second expansion (as arranged, for example, according to Step D of Figure 85 (in particular, eg, Figure 85B)) can be cryopreserved. Cryoconservation can be performed, in general, by placing the TIL population in a freezing solution, e.g. eg, inactivated AB serum with 85% complement and 15% dimethyl sulfoxide (DMSO). The cells in solution are placed in cryogenic tubes and stored for 24 hours at -80 ° C, with optional transfer to freezers containing nitrogen gas for cryopreservation. See Sadeghi, et al., Acta Oncologica 2013, 52, 978-986. In some modalities, TILs are cryopreserved in DMSO 5%. In some modalities, TILs are cryopreserved in cell culture medium plus 5% DMSO. In some modalities, TILs are cryopreserved according to the methods provided here.
[001120] [001120] When appropriate, the cells are removed from the freezer and thawed in a water bath at 37 ºC until approximately 4/5 of the solution has thawed. The cells are generally suspended in complete medium and, optionally, washed one or more times. In some embodiments, thawed TILs can be counted and assessed for viability as is known in the art.
[001121] [001121] In some cases, the Stage B TIL population can be cryopreserved immediately, using the protocols discussed below.
[001122] [001122] In some modalities, TILs are analyzed for the expression of numerous phenotypic markers after expansion, including those described here and in the Examples. In one embodiment, the expression of one or more phenotypic markers is examined. In some modalities, the phenotypic characteristics of TILs are analyzed after the first expansion in step B. In some modalities, the phenotypic characteristics of TILs are analyzed during the transition in step C. In some modalities, the phenotypic characteristics of TILs are analyzed during transition according to Step C and then cryopreservation. In some modalities, the phenotypic characteristics of TILs are analyzed after the second expansion according to Step D. In some modalities, the phenotypic characteristics of TILs are analyzed after two or more expansions according to Step D.
[001123] [001123] In some modalities, the marker is selected from the group consisting of CD8 and CD28. In some embodiments, the expression of CD8 is examined. In some embodiments, CD28 expression is examined. In some embodiments, the expression of CD8 and / or CD28 is greater in TILs produced according to the process of the present invention, as compared to other processes (eg, the Gen 3 process as arranged, for example, in Figure 85 (in particular, e.g., Figure 85B), compared to process 2A as arranged, for example, in Figure 85 (in particular, e.g., Figure 85B)). In some modalities, CD8 expression and CD8 and / or CD28 expression is higher in TILs produced according to
[001124] [001124] In one embodiment, no selection of the first population of TILs, the second population of TILs, the third population of TILs or the population of TILs collected based on the expression of CD8 and / or CD28 is performed during any of the steps of the method for the expansion of tumor infiltrating lymphocytes (TILs) described here.
[001125] [001125] In some embodiments, the percentage of central memory cells is higher in TILs produced according to the process of the present invention, compared to other processes (eg, the Gen 3 process as arranged, for example, in Figure 85 (in particular, e.g., Figure 85B), compared to process 2A as arranged, for example, in Figure 85 (in particular, e.g., Figure 85A)). In some modalities, the memory marker for central memory cells is selected from the group consisting of CCR7 and CD62L.
[001126] [001126] In one embodiment, restimulated TILs can also be evaluated for cytokine release, through cytokine release assays. In some modalities, TILs can be evaluated for the secretion of interferon-γ (IFN-γ). In some embodiments, IFN-γ secretion is measured by an ELISA assay. In some modalities, secretion
[001127] [001127] The various T and B lymphocyte antigen receptors are produced by somatic recombination of a limited but large number
[001128] [001128] In some modalities, phenotypic characterization is examined after cryopreservation. XIV. Additional modalities of the process
[001129] [001129] In some embodiments, the invention provides a method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic population of TILs comprising: (a) obtaining a first population of TILs from one or more fragments or samples of the tumor of an individual; (b) performing a first preparation expansion by cultivating the first population of TILs in a cell culture medium comprising IL-2 and OKT-3, where the first preparation expansion is performed for approximately 1 to 17 days to obtain the second population TILs, where the second population of TILs has a greater number than the first population of TILs; (c) perform a second rapid expansion by placing the second population of TILs in contact with a cell culture medium comprising IL-2, OKT-3 and exogenous antigen presenting cells (APCs) to produce a third population of TILs, in which the second rapid expansion is performed for approximately 1 to 11 days to obtain the third population of TILs, where the third population of TILs is a therapeutic population of TILs; and (d) collect the therapeutic population of TILs obtained in step (c). In some embodiments, the second rapid expansion step is divided into a plurality of steps to achieve a vertical crop scaling: (1) performing the second rapid expansion by cultivating the second population of TILs in a small crop
[001130] [001130] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that, in step (b), the first primary expansion is carried out by placing the first population of TILs in contact with a culture medium that further comprises exogenous antigen presenting cells (APCs), in which the number of APCs in the culture medium in step (c) is greater than the number of APCs in the culture medium in step (b).
[001131] [001131] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that, in step (c), the culture medium is supplemented with additional exogenous APCs.
[001132] [001132] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that the ratio of the number of APCs added in the second
[001133] [001133] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that the ratio of the number of APC added in the second rapid expansion to the number of APC added in step (b ) is selected from a range of equal to or close to 1.1: 1 to equal to or close to 10: 1.
[001134] [001134] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that the ratio of the number of APC added in the second rapid expansion to the number of APC added in step (b ) is selected from a range of equal to or close to 1.1: 1 to equal to or close to 9: 1.
[001135] [001135] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that the ratio of the number of APC added in the second rapid expansion to the number of APC added in step (b ) is selected from a range of equal to or close to 1.1: 1 to equal to or close to 8: 1.
[001136] [001136] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that the ratio of the number of APC added in the second rapid expansion to the number of APC added in step (b ) is selected from a range of equal to or close to 1.1: 1 to equal to or close to 7: 1.
[001137] [001137] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that the ratio of the number of APC added in the second rapid expansion to the number of APC added in step (b ) is selected from a range of equal to or close to 1.1: 1 to equal to or close to 6: 1.
[001138] [001138] In another embodiment, the invention provides the method described in
[001139] [001139] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable, modified in such a way that the ratio of the number of APC added in the second rapid expansion to the number of APC added in step (b) is selected from a range of 1.1: 1 or close to or close to 4: 1.
[001140] [001140] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that the ratio of the number of APC added in the second rapid expansion to the number of APC added in step (b ) is selected from a range of equal to or close to 1.1: 1 to equal to or close to 3: 1.
[001141] [001141] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that the ratio of the number of APC added in the second rapid expansion to the number of APC added in step (b ) is selected from a range of equal to or close to 1.1: 1 to equal to or close to 2.9: 1.
[001142] [001142] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that the ratio of the number of APC added in the second rapid expansion to the number of APC added in step (b ) is selected from a range of equal to or close to 1.1: 1 to equal to or close to 2.8: 1.
[001143] [001143] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that the ratio of the number of APC added in the second rapid expansion to the number of APC added in step (b ) is selected from a range of equal to or close to 1.1: 1 to equal to or close to 2.7: 1.
[001144] [001144] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that the ratio of the number of APC added in the second rapid expansion to the number of APC added in step (b ) is selected from a range of equal to or close to 1.1: 1 to equal to or close to 2.6: 1.
[001145] [001145] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that the ratio of the number of APC added in the second rapid expansion to the number of APC added in step (b ) is selected from a range of equal to or close to 1.1: 1 to equal to or close to 2.5: 1.
[001146] [001146] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that the ratio of the number of APC added in the second rapid expansion to the number of APC added in step (b ) is selected from a range of equal to or close to 1.1: 1 to equal to or close to 2.4: 1.
[001147] [001147] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that the ratio of the number of APC added in the second rapid expansion to the number of APC added in step (b ) is selected from a range of equal to or close to 1.1: 1 to equal to or close to 2.3: 1.
[001148] [001148] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that the ratio of the number of APC added in the second rapid expansion to the number of APC added in step (b ) is selected from a range of equal to or close to 1.1: 1 to equal to or close to 2.2: 1.
[001149] [001149] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that the ratio of the number of APC added in the second rapid expansion to the number of APC added in step (b ) is selected from among
[001150] [001150] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that the ratio of the number of APC added in the second rapid expansion to the number of APC added in step (b ) is selected from a range of equal to or close to 1.1: 1 to equal to or close to 2: 1.
[001151] [001151] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that the ratio of the number of APC added in the second rapid expansion to the number of APC added in step (b ) is selected from a range of equal to or close to 2: 1 to equal to or close to 10: 1.
[001152] [001152] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that the ratio of the number of APC added in the second rapid expansion to the number of APC added in step (b ) is selected from a range of equal to or close to 2: 1 to equal to or close to 5: 1.
[001153] [001153] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that the ratio of the number of APC added in the second rapid expansion to the number of APC added in step (b ) is selected from a range of equal to or close to 2: 1 to equal to or close to 4: 1.
[001154] [001154] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that the ratio of the number of APC added in the second rapid expansion to the number of APC added in step (b ) is selected from a range of equal to or close to 2: 1 to equal to or close to 3: 1.
[001155] [001155] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that the ratio of the number of APC added in the second expansion
[001156] [001156] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that the ratio of the number of APC added in the second rapid expansion to the number of APC added in step (b ) is selected from a range of equal to or close to 2: 1 to equal to or close to 2.8: 1.
[001157] [001157] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that the ratio of the number of APC added in the second rapid expansion to the number of APC added in step (b ) is selected from a range of equal to or close to 2: 1 to equal to or close to 2.7: 1.
[001158] [001158] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that the ratio of the number of APC added in the second rapid expansion to the number of APC added in step (b ) is selected from a range of equal to or close to 2: 1 to equal to or close to 2.6: 1.
[001159] [001159] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that the ratio of the number of APC added in the second rapid expansion to the number of APC added in step (b ) is selected from a range of equal to or close to 2: 1 to equal to or close to 2.5: 1.
[001160] [001160] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that the ratio of the number of APC added in the second rapid expansion to the number of APC added in step (b ) is selected from a range of equal to or close to 2: 1 to equal to or close to 2.4: 1.
[001161] [001161] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified
[001162] [001162] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that the ratio of the number of APC added in the second rapid expansion to the number of APC added in step (b ) is selected from a range of equal to or close to 2: 1 to equal to or close to 2.2: 1.
[001163] [001163] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that the ratio of the number of APC added in the second rapid expansion to the number of APC added in step (b ) is selected from a range of equal to or close to 2: 1 to equal to or close to 2.1: 1.
[001164] [001164] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that the ratio of the number of APC added in the second rapid expansion to the number of APC added in step (b ) is equal to or close to 2: 1.
[001165] [001165] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that the ratio of the number of APC added in the second rapid expansion to the number of APC added in step (b ) is equal to or close to 1.1: 1; 1.2: 1; 1.3: 1; 1.4: 1; 1.5: 1; 1.6: 1; 1.7: 1; 1.8: 1; 1.9: 1; 2: 1; 2.1: 1; 2.2: 1; 2.3: 1; 2.4: 1; 2.5: 1; 2.6: 1; 2.7: 1; 2.8: 1; 2.9: 1; 3: 1; 3.1: 1; 3.2: 1; 3.3: 1; 3.4: 1; 3.5: 1; 3.6: 1; 3.7: 1; 3.8: 1; 3.9: 1; 4: 1; 4.1: 1; 4.2: 1; 4.3: 1; 4.4: 1; 4.5: 1; 4.6: 1; 4.7: 1; 4.8: 1; 4.9: 1 or 5: 1.
[001166] [001166] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that the number of APCs added in the first primary expansion
[001167] [001167] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that the number of APCs added in the first primary expansion is selected from the range of equal to or close to 1 x 108 APCs equal to or close to 3.5 x 108 APCs, and where the number of APCs added in the second rapid expansion is selected from the range of equal to or close to 3.5 x 108 APCs equal to or close to 1 x 109 APCs.
[001168] [001168] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that the number of APC added in the first primary expansion is selected from the range of equal to or close to 1, 5 x 108 APCs at or near 3 x 108 APCs, and where the number of APCs added in the second rapid expansion is selected from the range of 4 x 108 APCs at or near 7.5 x 108 APCs.
[001169] [001169] In another embodiment, the invention provides the method described in
[001170] [001170] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that 2.5 x 108 APCs, or next, are added to the first primary expansion and 5 x 108 APCs , or next, are added to the second rapid expansion.
[001171] [001171] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that the antigen presenting cells are peripheral blood mononuclear cells (PBMCs).
[001172] [001172] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that the multiple fragments of the tumor are distributed in a plurality of separate containers, in which, each of these containers separate, the first population of TILs is obtained in step (a), the second population of TILs is obtained in step (b) and the third population of TILs is obtained in step (c), and the therapeutic populations of TILs of the plurality of containers in step (c) are combined to produce the population of TILs collected from step (d).
[001173] [001173] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that the first population of TILs is obtained from multiple fragments or samples of the individual's tumor in step ( The).
[001174] [001174] In another embodiment, the invention provides the method described in
[001175] [001175] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that the plurality of separate containers comprises at least two separate containers.
[001176] [001176] In another embodiment, the invention provides the method described in any of the preceding paragraphs, as applicable above, modified in such a way that the plurality of separate containers comprises from two to twenty separate containers.
[001177] [001177] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that the plurality of separate containers comprises from two to fifteen separate containers.
[001178] [001178] In another embodiment, the invention provides the method described in any of the preceding paragraphs, as applicable above, modified in such a way that the plurality of separate containers comprises from two to ten separate containers.
[001179] [001179] In another embodiment, the invention provides the method described in any of the preceding paragraphs, as applicable above, modified in such a way that the plurality of separate containers comprises from two to five separate containers.
[001180] [001180] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that the plurality of separate containers comprises 2, 3, 4, 5, 6, 7, 8, 9 , 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 separate containers.
[001181] [001181] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified
[001182] [001182] In another embodiment, the invention provides the method described in any of the preceding paragraphs, as applicable above, modified in such a way that each of the separate containers comprises a first gas-permeable surface area.
[001183] [001183] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that the multiple fragments of the tumor are distributed in a single container.
[001184] [001184] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that the single container comprises a first gas-permeable surface area.
[001185] [001185] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that, in step (b), the first primary expansion is performed by supplementing the cell culture medium of the first population of TILs with additional antigen presenting cells (APCs), where the number of APCs added in step (c) is greater than the number of APCs added in step (b), and where, in step (b), APCs they are arranged in layers on the first gas-permeable surface area with an average thickness between or close to that of a layer of cells and equal to or close to three layers of cells.
[001186] [001186] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified
[001187] [001187] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that, in step (b), the APCs are layered on the first permeable surface area to gases with an average thickness between or equal to that of 2 layers of cells.
[001188] [001188] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that, in step (b), the APCs are layered on the first permeable surface area to gases with an average thickness equal to or close to 1; 1.1; 1.2; 1.3; 1.4; 1.5; 1.6; 1.7; 1.8; 1.9; two; 2.1; 2.2; 2.3; 2.4; 2.5; 2.6; 2.7; 2.8; 2.9 or 3 layers of cells.
[001189] [001189] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that, in step (c), the APCs are layered on the first permeable surface area to gases with an average thickness between or close to that of 3 layers of cells and equal to or close to that of 10 layers of cells.
[001190] [001190] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that, in step (c), the APCs are layered on the first permeable surface area to gases with an average thickness between or close to that of 4 layers of cells and equal to or close to that of 8 layers of cells.
[001191] [001191] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified
[001192] [001192] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that, in step (c), the APCs are layered on the first permeable surface area to gases with an average thickness equal to or close to 4; 4.1; 4.2; 4.3; 4.4; 4.5; 4.6; 4.7; 4.8; 4.9; 5; 5.1; 5.2; 5.3; 5.4; 5.5; 5.6; 5.7; 5.8; 5.9; 6; 6.1; 6.2; 6.3; 6.4; 6.5; 6.6; 6.7; 6.8; 6.9; 7; 7.1; 7.2; 7.3; 7.4; 7.5; 7.6; 7.7; 7.8; 7,9 or 8 layers of cells.
[001193] [001193] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that, in step (b), the first preparation expansion is carried out in a first container comprising a first gas permeable surface area and, in step (c), the second rapid expansion is carried out in a second container comprising a second gas permeable surface area.
[001194] [001194] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that the second container is larger than the first container.
[001195] [001195] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that, in step (b), the first expansion of preparation is performed by supplementing the cell culture medium of first population of TILs with additional antigen presenting cells (APCs), where the number of APCs added in step (c) is greater than the number of APCs added in step (b), and where, in step (b), the APCs are layered on the first gas-permeable surface area with an average thickness between or close to that of a cell layer and equal to or close to three
[001196] [001196] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that, in step (b), the APCs are layered on the first permeable surface area to gases with an average thickness between or equal to or equal to 1.5 layers of cells and equal to or close to 2.5 layers of cells.
[001197] [001197] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that, in step (b), the APCs are layered on the first permeable surface area to gases with an average thickness equal to or close to that of 2 layers of cells.
[001198] [001198] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable, modified in such a way that, in step (b), the APCs are layered on the first gas-permeable surface area with an average thickness equal to or close to 1; 1.1; 1.2; 1.3; 1.4; 1.5; 1.6; 1.7; 1.8; 1.9; two; 2.1; 2.2; 2.3; 2.4; 2.5; 2.6; 2.7; 2.8, 2.9 or 3 layers of cells.
[001199] [001199] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that, in step (c), the APCs are layered on the second permeable surface area to gases with an average thickness between or close to that of 3 layers of cells and equal to or close to that of 10 layers of cells.
[001200] [001200] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that, in step (c), the APCs are layered on the second permeable surface area to gases with an average thickness between or close to that of 4 layers of cells and equal to or close to that of 8
[001201] [001201] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that, in step (c), the APCs are layered on the second permeable surface area to gases with an average thickness equal to or close to 3, 4, 5, 6, 7, 8, 9 or 10 layers of cells.
[001202] [001202] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable, modified in such a way that, in step (c), the APCs are layered on the second gas-permeable surface area with an average thickness between or close to 4; 4.1; 4.2; 4.3; 4.4; 4.5; 4.6; 4.7; 4.8; 4.9; 5; 5.1; 5.2; 5.3; 5.4; 5.5; 5.6; 5.7; 5.8; 5.9; 6; 6.1; 6.2; 6.3; 6.4; 6.5; 6.6; 6.7; 6.8; 6.9; 7; 7.1; 7.2; 7.3; 7.4; 7.5; 7.6; 7.7; 7.8; 7,9 or 8 layers of cells.
[001203] [001203] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that, in step (b), the first preparation expansion is carried out in a first container comprising a first surface area permeable to gases and, in step (c), the second rapid expansion is carried out in the first container.
[001204] [001204] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that, in step (b), the first primary expansion is performed by supplementing the cell culture medium of the first population of TILs with additional antigen presenting cells (APCs), where the number of APCs added in step (c) is greater than the number of APCs added in step (b), and where, in step (b), APCs they are arranged in layers on the first gas-permeable surface area with an average thickness between or close to that of a layer of cells and equal to or close to three layers of cells.
[001205] [001205] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that, in step (b), the APCs are layered on the first permeable surface area to gases with an average thickness between or equal to or equal to 1.5 layers of cells and equal to or close to 2.5 layers of cells.
[001206] [001206] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that, in step (b), the APCs are layered on the first permeable surface area to gases with an average thickness equal to or close to that of 2 layers of cells.
[001207] [001207] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that, in step (b), the APCs are layered on the first permeable surface area to gases with an average thickness equal to or close to 1; 1.1; 1.2; 1.3; 1.4; 1.5; 1.6; 1.7; 1.8; 1.9; two; 2.1; 2.2; 2.3; 2.4; 2.5; 2.6; 2.7; 2.8; 2.9 or 3 layers of cells.
[001208] [001208] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that, in step (c), the APCs are layered on the first permeable surface area to gases with an average thickness between or close to that of 3 layers of cells and equal to or close to that of 10 layers of cells.
[001209] [001209] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that, in step (c), the APCs are layered on the first permeable surface area to gases with an average thickness between or close to that of 4 layers of cells and equal to or close to that of 8 layers of cells.
[001210] [001210] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that, in step (c), the APCs are layered on the first permeable surface area to gases with an average thickness equal to or close to 3, 4, 5, 6, 7, 8, 9 or 10 layers of cells.
[001211] [001211] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that, in step (c), the APCs are layered on the first permeable surface area to gases with an average thickness equal to or close to 4; 4.1; 4.2; 4.3; 4.4; 4.5; 4.6; 4.7; 4.8; 4.9; 5; 5.1; 5.2; 5.3; 5.4; 5.5; 5.6; 5.7; 5.8; 5.9; 6; 6.1; 6.2; 6.3; 6.4; 6.5; 6.6; 6.7; 6.8; 6.9; 7; 7.1; 7.2; 7.3; 7.4; 7.5; 7.6; 7.7; 7.8; 7,9 or 8 layers of cells.
[001212] [001212] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that, in step (b), the first primary expansion is performed by supplementing the cell culture medium of the first population of TILs with additional antigen presenting cells (APCs), where the number of APCs added in step (c) is greater than the number of APCs added in step (b), and where the average number of APC layers layered in step (b) for the average number of layers of APCs layered in step (c) is selected from the range of equal to or close to 1: 1.1 to equal to or close to 1:10.
[001213] [001213] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that, in step (b), the first primary expansion is performed by supplementing the cell culture medium of the first population of TILs with additional antigen presenting cells (APCs), where the number of APCs added in step (c) is greater than the number of APCs added in step (b), and where the average number of APC layers willing
[001214] [001214] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that, in step (b), the first primary expansion is performed by supplementing the cell culture medium of the first population of TILs with additional antigen presenting cells (APCs), where the number of APCs added in step (c) is greater than the number of APCs added in step (b), and where the average number of APC layers layered in step (b) for the average number of layers of APCs layered in step (c) is selected from the range of equal to or close to 1: 1.1 to equal to or close to 1: 8.
[001215] [001215] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that, in step (b), the first primary expansion is performed by supplementing the cell culture medium of the first population of TILs with additional antigen presenting cells (APCs), where the number of APCs added in step (c) is greater than the number of APCs added in step (b), and where the average number of APC layers layered in step (b) for the average number of layers of APCs layered in step (c) is selected from the range of equal to or close to 1: 1.1 to equal to or close to 1: 7.
[001216] [001216] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that, in step (b), the first primary expansion is performed by supplementing the cell culture medium of the first population of TILs with additional antigen presenting cells (APCs), where the number of APCs added in step (c) is greater than the number of APCs added in
[001217] [001217] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that, in step (b), the first primary expansion is performed by supplementing the cell culture medium of the first population of TILs with additional antigen presenting cells (APCs), where the number of APCs added in step (c) is greater than the number of APCs added in step (b), and where the average number of APC layers layered in step (b) for the average number of layers of APCs layered in step (c) is selected from the range of equal to or close to 1: 1.1 to equal to or close to 1: 5.
[001218] [001218] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that, in step (b), the first primary expansion is performed by supplementing the cell culture medium of the first population of TILs with additional antigen presenting cells (APCs), where the number of APCs added in step (c) is greater than the number of APCs added in step (b), and where the average number of APC layers layered in step (b) for the average number of layers of APCs layered in step (c) is selected from the range of equal to or close to 1: 1.1 to equal to or close to 1: 4.
[001219] [001219] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that, in step (b), the first primary expansion is performed by supplementing the cell culture medium of the first population of TILs with additional antigen presenting cells (APCs), in which the number of
[001220] [001220] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that, in step (b), the first primary expansion is performed by supplementing the cell culture medium of the first population of TILs with additional antigen presenting cells (APCs), where the number of APCs added in step (c) is greater than the number of APCs added in step (b), and where the average number of APC layers layered in step (b) for the average number of layers of APCs layered in step (c) is selected from the range of equal to or close to 1: 1.1 to equal to or close to 1: 2.
[001221] [001221] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that, in step (b), the first primary expansion is performed by supplementing the cell culture medium of the first population of TILs with additional antigen presenting cells (APCs), where the number of APCs added in step (c) is greater than the number of APCs added in step (b), and where the average number of APC layers layered in step (b) for the average number of layers of APCs layered in step (c) is selected from the range of equal to or close to 1: 1.2 to equal to or close to 1: 8.
[001222] [001222] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that, in step (b), the first primary expansion is performed by supplementing the cell culture medium of the first population of TILs with
[001223] [001223] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that, in step (b), the first primary expansion is performed by supplementing the cell culture medium of the first population of TILs with additional antigen presenting cells (APCs), where the number of APCs added in step (c) is greater than the number of APCs added in step (b), and where the average number of APC layers layered in step (b) for the average number of layers of APCs layered in step (c) is selected from the range of equal to or close to 1: 1.4 to equal to or close to 1: 6.
[001224] [001224] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that, in step (b), the first primary expansion is performed by supplementing the cell culture medium of the first population of TILs with additional antigen presenting cells (APCs), where the number of APCs added in step (c) is greater than the number of APCs added in step (b), and where the average number of APC layers layered in step (b) for the average number of layers of APCs layered in step (c) is selected from the range of equal to or close to 1: 1.5 to equal to or close to 1: 5.
[001225] [001225] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that, in step (b), the first primary expansion is performed
[001226] [001226] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that, in step (b), the first primary expansion is performed by supplementing the cell culture medium of the first population of TILs with additional antigen presenting cells (APCs), where the number of APCs added in step (c) is greater than the number of APCs added in step (b), and where the average number of APC layers layered in step (b) for the average number of layers of APCs layered in step (c) is selected from the range of equal to or close to 1: 1.7 to equal to or close to 1: 3.5.
[001227] [001227] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that, in step (b), the first primary expansion is performed by supplementing the cell culture medium of the first population of TILs with additional antigen presenting cells (APCs), where the number of APCs added in step (c) is greater than the number of APCs added in step (b), and where the average number of APC layers layered in step (b) for the average number of layers of APCs layered in step (c) is selected from the range of equal to or close to 1: 1.8 to equal to or close to 1: 3.
[001228] [001228] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified
[001229] [001229] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that, in step (b), the first primary expansion is performed by supplementing the cell culture medium of the first population of TILs with additional antigen presenting cells (APCs), where the number of APCs added in step (c) is greater than the number of APCs added in step (b), and where the average number of APC layers layered in step (b) for the average number of layers of APCs layered in step (c) is selected from the range of equal to or close to 1: 2.
[001230] [001230] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that, in step (b), the first primary expansion is performed by supplementing the cell culture medium of the first population of TILs with additional antigen presenting cells (APCs), where the number of APCs added in step (c) is greater than the number of APCs added in step (b), and where the average number of APC layers layered in step (b) for the average number of layers of APCs layered in step (c) is selected from or equal to 1: 1.1; 1: 1.2; 1: 1.3; 1: 1.4; 1: 1.5; 1: 1.6; 1: 1.7; 1: 1.8; 1: 1.9; 1: 2; 1: 2.1; 1: 2.2; 1: 2.3; 1: 2.4; 1: 2.5; 1: 2.6; 1: 2.7; 1: 2.8; 1: 2.9; 1: 3; 1: 3.1; 1: 3.2; 1: 3.3; 1: 3.4; 1: 3.5; 1: 3.6;
[001231] [001231] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that the ratio of the number of TILs in the second population of TILs to the number of TILs in the first population of TILs is equal to or close to
[001232] [001232] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that the ratio of the number of TILs in the second population of TILs to the number of TILs in the first population of TILs is equal to or close to 50: 1.
[001233] [001233] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that the ratio of the number of TILs in the second population of TILs to the number of TILs in the first population of TILs is equal to or close to 25: 1.
[001234] [001234] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that the ratio of the number of TILs in the second population of TILs to the number of TILs in the first population of TILs is equal to or close to 20: 1.
[001235] [001235] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that the ratio of the number of TILs in the second population of TILs
[001236] [001236] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that the second population of TILs is at least equal to or close to 50 times greater in number than the first population of TILs.
[001237] [001237] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that the second population of TILs is at least equal to or close to 1, 2, 3, 4, 5 , 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 , 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 times greater in number than the first population of TILs.
[001238] [001238] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that in or close to 2 days or in or close to 3 days after the start of the second period in the step (c), the cell culture medium is supplemented with additional IL-2.
[001239] [001239] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified to further understand the cryopreservation stage of the population of TILs collected in step (d) using a cryopreservation process.
[001240] [001240] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified to understand, after step (d), the additional step of (e) transferring the population of TILs collected in the step (d) for an infusion bag that optionally contains HyperThermosol.
[001241] [001241] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified
[001242] [001242] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that the cryopreservation process is carried out using a 1: 1 ratio of the population of TILs collected to the cryopreservation medium. .
[001243] [001243] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that the antigen presenting cells are peripheral blood mononuclear cells (PBMCs).
[001244] [001244] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that the PBMCs are irradiated and allogeneic.
[001245] [001245] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that the total number of APC added to the cell culture in step (b) is 2.5 x 108.
[001246] [001246] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that the total number of APC added to the cell culture in step (c) is 5 x 108.
[001247] [001247] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that the APCs are PBMCs.
[001248] [001248] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that PBMCs are irradiated and allogeneic.
[001249] [001249] In another embodiment, the invention provides the method described in
[001250] [001250] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that the collection in step (d) is performed using a membrane-based cell processing system.
[001251] [001251] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that the collection in step (d) is performed using a LOVO system for processing the cells.
[001252] [001252] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that the multiple fragments comprise between equal to or close to 5 and equal to or close to 60 samples or fragments per container in step (b).
[001253] [001253] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that the multiple fragments comprise between equal or close to 10 and between equal or close to 60 samples or fragments per container in step (b).
[001254] [001254] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that the multiple fragments comprise between equal or close to 15 and between equal or close to 60 samples or fragments per container in step (b).
[001255] [001255] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that the multiple fragments comprise between equal or
[001256] [001256] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that the multiple fragments comprise between equal or close to 25 and between equal or close to 60 samples or fragments per container in step (b).
[001257] [001257] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that the multiple fragments comprise between equal or close to 30 and between equal or close to 60 samples or fragments per container in step (b).
[001258] [001258] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that the multiple fragments comprise between equal or close to 35 and between equal or close to 60 samples or fragments per container in step (b).
[001259] [001259] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that the multiple fragments comprise between equal or close to 40 and between equal or close to 60 samples or fragments per container in step (b).
[001260] [001260] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that the multiple fragments comprise between equal or close to 45 and between equal or close to 60 samples or fragments per container in step (b).
[001261] [001261] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified
[001262] [001262] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that the multiple samples or fragments comprise equal to or close to 2, 3, 4, 5, 6, 7 , 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 , 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57 , 58, 59 or 60 samples or fragment (s) per container in step (b).
[001263] [001263] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that each sample or fragment has a volume equal to or close to 1 mm3.
[001264] [001264] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that each sample or fragment has a volume between or equal to 1 mm3 and equal to or close to 10 mm3 .
[001265] [001265] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that each sample or fragment has a volume between or equal to 1 mm3 and equal to or close to 9 mm3 .
[001266] [001266] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that each fragment has a volume between or equal to 1 mm3 and equal to or close to 8 mm3.
[001267] [001267] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that each fragment has a volume between or equal to 1
[001268] [001268] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that each fragment has a volume between or equal to 1 mm3 and equal to or close to 6 mm3.
[001269] [001269] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that each fragment has a volume between or equal to 1 mm3 and equal to or close to 5 mm3.
[001270] [001270] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that each fragment has a volume between or equal to 1 mm3 and equal to or close to 4 mm3.
[001271] [001271] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that each fragment has a volume between equal to or close to 1 mm3 and equal to or close to 3 mm3.
[001272] [001272] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that each fragment has a volume between equal to or close to 1 mm3 and equal to or close to 2 mm3.
[001273] [001273] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that each fragment has a volume equal to or close to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mm3.
[001274] [001274] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that the cell culture medium is supplied in a container that is a G container or a Xuri Cellbag.
[001275] [001275] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that the concentration of IL-2 in the cell culture medium is between approximately 10,000 IU / mL and 5,000 IU / ml.
[001276] [001276] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that the concentration of IL-2 in the cell culture medium is approximately 6,000 IU / ml.
[001277] [001277] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that the cryopreservation medium comprises dimethyl sulfoxide (DMSO).
[001278] [001278] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that the cryopreservation medium comprises DMSO 7% to 10%.
[001279] [001279] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that the first period in step (b) is carried out within a period of equal or close to 1 day , 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days or 17 days.
[001280] [001280] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that the second period in step (c) is carried out within a period of equal or close to 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days or 11 days.
[001281] [001281] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that the first period in step (b) and the second period in step
[001282] [001282] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that the first period in step (b) and the second period in step (c) are performed, each one individually, within a period of 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, or 12 days.
[001283] [001283] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that the first period in step (b) and the second period in step (c) are performed, each one individually, within a period of equal or close to 7 days.
[001284] [001284] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that steps (a) to (d) are carried out, in total, between 14 days or almost e 28 days or so.
[001285] [001285] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that steps (a) to (d) are carried out, in total, between 15 days or almost e 28 days or so.
[001286] [001286] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that steps (a) to (d) are carried out, in total, between 16 days or almost e 28 days or so.
[001287] [001287] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that steps (a) to (d) are carried out, in total, between 17 days or almost e 28 days or so.
[001288] [001288] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that steps (a) to (d) are carried out, in total, between 18 days or almost e 28 days or so.
[001289] [001289] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that steps (a) to (d) are carried out, in total, between 19 days or almost e 28 days or so.
[001290] [001290] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that steps (a) to (d) are carried out, in total, between 20 days or almost e 28 days or so.
[001291] [001291] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that steps (a) to (d) are carried out, in total, between 21 days or almost e 28 days or so.
[001292] [001292] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that steps (a) to (d) are carried out, in total, between 22 days or almost e 28 days or so.
[001293] [001293] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that steps (a) to (d) are carried out, in total, between 23 days or almost e 28 days or so.
[001294] [001294] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that steps (a) to (d) are performed, in total, between 24 days or almost e 28 days or so.
[001295] [001295] In another embodiment, the invention provides the method described in
[001296] [001296] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that steps (a) to (d) are carried out, in total, between 26 days or almost e 28 days or so.
[001297] [001297] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that steps (a) to (d) are carried out, in total, between 27 days or almost e 28 days or so.
[001298] [001298] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that steps (a) to (d) are carried out, in total, in or almost 14 days.
[001299] [001299] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that steps (a) to (d) are carried out, in total, in or almost 15 days.
[001300] [001300] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that steps (a) to (d) are carried out, in total, in or almost 16 days.
[001301] [001301] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that steps (a) to (d) are carried out, in total, in or almost 17 days.
[001302] [001302] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified
[001303] [001303] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that steps (a) to (d) are carried out, in total, in or almost 19 days.
[001304] [001304] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that steps (a) to (d) are carried out, in total, in or almost 20 days.
[001305] [001305] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that steps (a) to (d) are carried out, in total, in or almost 21 days.
[001306] [001306] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that steps (a) to (d) are carried out, in total, in or almost 22 days.
[001307] [001307] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that steps (a) to (d) are carried out, in total, in or almost 23 days.
[001308] [001308] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that steps (a) to (d) are performed, in total, in or almost 24 days.
[001309] [001309] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that steps (a) to (d) are performed, in total, in or nearly 25
[001310] [001310] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that steps (a) to (d) are carried out, in total, in or almost 26 days.
[001311] [001311] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that steps (a) to (d) are carried out, in total, in or almost 27 days.
[001312] [001312] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that steps (a) to (d) are carried out, in total, in or almost 28 days.
[001313] [001313] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that steps (a) to (d) are performed, in total, in or less than 16 days .
[001314] [001314] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that steps (a) to (d) are performed, in total, in or less than 20 days .
[001315] [001315] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that steps (a) to (d) are performed, in total, in or less than 24 days .
[001316] [001316] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that steps (a) to (d) are performed, in total, in or less than 28 days .
[001317] [001317] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that the therapeutic population of TILs harvested in step (d) comprises sufficient TILs for a therapeutically effective dose of TILs .
[001318] [001318] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that the number of TILs sufficient for a therapeutically effective dose is between or equal to 2.3x1010 and equal or close to 13.7x1010.
[001319] [001319] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that the third population of TILs in step (c) provides increased efficacy, increased production of interferon-gamma and / or increased polyclonality.
[001320] [001320] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that the third population of TILs in step (c) provides at least one to five times or more production of interferon-gamma compared to TILs prepared by a process of more than 16 days.
[001321] [001321] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that the effector T cells and / or central memory T cells obtained from the third population of TILs in step ( c) exhibit increased expression of CD8 and CD28 in relation to effector T cells and / or central memory T cells obtained from the second cell population in step (b).
[001322] [001322] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that each container mentioned in the method is a closed container.
[001323] [001323] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that each container mentioned in the method is a G container.
[001324] [001324] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that each container mentioned in the method is a GREX-10.
[001325] [001325] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that each container mentioned in the method is a GREX-100.
[001326] [001326] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that each container mentioned in the method is a GREX-500.
[001327] [001327] In another embodiment, the invention provides the therapeutic population of tumor infiltrating lymphocytes (TILs) produced by the method described in any of the previous paragraphs, as applicable above.
[001328] [001328] In another embodiment, the invention provides a therapeutic population of tumor infiltrating lymphocytes (TILs) prepared from a patient's tumor tissue, wherein the therapeutic population of TILs provides increased efficacy, increased production of interferon-gamma and / or increased polyclonality when compared to TILs prepared by a process in which the first expansion of TILs is performed without any added antigen presenting cells (APCs) or OKT3.
[001329] [001329] In another embodiment, the invention provides a therapeutic population of tumor infiltrating lymphocytes (TILs) prepared from a patient's tumor tissue, wherein the therapeutic population of TILs provides increased efficacy, increased production of interferon-gamma and / or increased polyclonality when compared to TILs prepared by a process in which the first expansion of TILs is performed without any added antigen presenting cells (APCs).
[001330] [001330] In another embodiment, the invention provides a therapeutic population of tumor infiltrating lymphocytes (TILs) prepared from a patient's tumor tissue, wherein the therapeutic population of TILs provides increased efficacy, increased production of interferon-gamma and / or increased polyclonality when compared to TILs prepared by a process in which the first expansion of TILs is performed without any added OKT3.
[001331] [001331] In another embodiment, the invention provides a therapeutic population of tumor infiltrating lymphocytes (TILs) prepared from a patient's tumor tissue, wherein the therapeutic population of TILs provides increased efficacy, increased production of interferon-gamma and / or increased polyclonality when compared to TILs prepared by a process in which the first expansion of TILs is performed without added antigen presenting cells (APCs) and without added OKT3.
[001332] [001332] In another embodiment, the invention provides a therapeutic population of tumor infiltrating lymphocytes (TILs) prepared from a patient's tumor tissue, wherein the therapeutic population of TILs provides increased efficacy, increased production of interferon-gamma and / or increased polyclonality when compared to TILs prepared by a process of more than 16 days.
[001333] [001333] In another embodiment, the invention provides the therapeutic population of TILs described in any of the previous paragraphs, as applicable above, which provides increased production of interferon-gamma.
[001334] [001334] In another embodiment, the invention provides the therapeutic population of TILs described in any of the previous paragraphs, as applicable above, which provides increased polyclonality.
[001335] [001335] In another embodiment, the invention provides the therapeutic population of TILs described in any of the previous paragraphs,
[001336] [001336] In another embodiment, the invention provides the therapeutic population of TILs described in any of the previous paragraphs, as applicable above, modified in such a way that the therapeutic population of TILs is capable of producing interferon-gamma at least once greater when compared to TILs prepared by a process of more than 16 days.
[001337] [001337] In another embodiment, the invention provides the therapeutic population of TILs described in any of the previous paragraphs, as applicable above, modified in such a way that the therapeutic population of TILs is capable of producing interferon-gamma at least twice greater when compared to TILs prepared by a process of more than 16 days.
[001338] [001338] In another embodiment, the invention provides for the therapeutic population of TILs described in any of the previous paragraphs, as applicable above modified in such a way that the therapeutic population of TILs is capable of producing interferon-gamma at least three times greater when compared to TILs prepared by a process of more than 16 days.
[001339] [001339] In another embodiment, the invention provides a therapeutic population of tumor-infiltrating lymphocytes (TILs) that are capable of at least an increased production of interferon-gamma when compared to TILs prepared by a process in which the first expansion of TILs is performed without any added antigen presenting cells (APCs).
[001340] [001340] In another embodiment, the invention provides a therapeutic population of tumor-infiltrating lymphocytes (TILs) that are capable of at least an increased production of interferon-gamma when compared to TILs prepared by a process in which the first expansion of TILs is
[001341] [001341] In another embodiment, the invention provides a therapeutic population of TILs that is capable of at least twice the production of interferon-gamma when compared to TILs prepared by a process in which the first expansion of TILs is performed without any APCs added.
[001342] [001342] In another embodiment, the invention provides a therapeutic population of TILs that is capable of at least twice the production of interferon-gamma when compared to TILs prepared by a process in which the first expansion of TILs is performed without any OKT3 added.
[001343] [001343] In another embodiment, the invention provides a therapeutic population of TILs that is capable of at least three times greater production of interferon-gamma when compared to TILs prepared by a process in which the first expansion of TILs is performed without any APCs added.
[001344] [001344] In another embodiment, the invention provides a therapeutic population of TILs that is capable of at least three times greater production of interferon-gamma when compared to TILs prepared by a process in which the first expansion of TILs is performed without any OKT3 added.
[001345] [001345] In another embodiment, the invention provides a method for expansion of T cells comprising: (a) performing a first expansion of preparation of a first population of T cells, obtained from a fragment or sample of tumor obtained from a donor, cultivating the first population of T cells to effect growth and prepare for activation of the first population of T cells; (b) after the activation of the first population of T cells prepared in step (a) begins to decay, perform a second rapid expansion of the first population of T cells, cultivating the first
[001346] [001346] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that the first expansion of preparation of step (a) is carried out over a period of up to 17 days.
[001347] [001347] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that the second rapid expansion of step (b) is carried out over a period of up to 11 days.
[001348] [001348] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that the first preparation expansion in step (a) is carried out over a period of 17 days and the second rapid expansion of step (b) is carried out over a period of up to 9 days.
[001349] [001349] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that, in step (a), the first T cell population is cultured in a first culture medium comprising OKT-3 and IL-2.
[001350] [001350] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that the first culture medium comprises OKT-3, IL-2 and antigen presenting cells (APCs) .
[001351] [001351] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that, in step (b), the first T cell population is cultured in a second culture medium comprising OKT-3, IL-2 and antigen presenting cells (APCs).
[001352] [001352] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that, in step (a), the first population of T cells is cultured in a first culture medium in a container comprising a first gas-permeable surface, in which the first culture medium comprises OKT-3, IL-2 and the first population of antigen presenting cells (APCs), in which the first population of APCs is exogenous to the donor of the first population of T cells and the first population of APCs is layered on the first gas-permeable surface, in which, in step (b), the first population of T cells is grown in a second culture medium in the container , in which the second culture medium comprises
[001353] [001353] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that the ratio of the number of APCs in the second population of APCs to the number of APCs in the first population of APCs is approximately 2: 1.
[001354] [001354] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that the number of APCs in the first population of APCs is approximately 2.5 x 108 and the number of APCs in the second population of APCs it is approximately 5 x 108.
[001355] [001355] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that, in step (a), the first population of APCs is layered on the first permeable surface to gases with an average thickness of 2 layers of APCs.
[001356] [001356] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that, in step (b), the second population of APCs is layered on the first permeable surface to gases with an average thickness selected from the range of 4 to 8 layers of APCs.
[001357] [001357] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that the ratio of the average number of layers of APCs layered on the first gas-permeable surface in the step (b) for the
[001358] [001358] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that, in step (a), the first population of APCs is sown on the first gas-permeable surface at a density selected from the range of equal to or close to 1.0 x 106 APCs / cm2 to or equal to 4.5 x 106 APCs / cm2.
[001359] [001359] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that, in step (a), the first APC population is sown on the first gas-permeable surface at a density selected from the range of 1.5 x 106 APCs / cm2 or close to 3.5 x 106 APCs / cm2.
[001360] [001360] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that, in step (a), the first population of APCs is sown on the first gas-permeable surface at a density selected from the range of 2.0 x 106 APCs / cm2 or close to 3.0 x 106 APCs / cm2.
[001361] [001361] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that, in step (a), the first population of APCs is sown on the first gas-permeable surface at a density equal to or close to 2.0 x 106 APCs / cm2.
[001362] [001362] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that, in step (b), the second population of APCs is sown on the first gas-permeable surface at a density selected from the
[001363] [001363] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that, in step (b), the second population of APCs is sown on the first gas-permeable surface at a density selected from the range of 3.5 x 106 APCs / cm2 or close to 6.0 x 106 APCs / cm2.
[001364] [001364] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that, in step (b), the second population of APCs is sown on the first gas-permeable surface at a density selected from the range of 4.0 x 106 APCs / cm2 or equal to 5.5 x 106 APCs / cm2.
[001365] [001365] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that, in step (b), the second population of APCs is sown on the first gas-permeable surface at a density equal to or close to 4.0 x 106 APCs / cm2.
[001366] [001366] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that, in step (a), the first population of APCs is sown on the first gas-permeable surface at a density selected from the range equal to or close to 1.0 x 106 APCs / cm2 to or equal to 4.5 x 106 APCs / cm2 and, in step (b), the second APC population is sown on the first gas-permeable surface at a density selected from the range of 2.5 x 106 APCs / cm2 or close to 7.5 x 106 APCs / cm2.
[001367] [001367] In another embodiment, the invention provides the method described in
[001368] [001368] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that, in step (a), the first population of APCs is sown on the first gas-permeable surface at a density selected from the range of equal to or close to 2.0 x 106 APCs / cm2 to or equal to 3.0 x 106 APCs / cm2 and, in step (b), the second APC population is sown on the first gas-permeable surface at a density selected from the range of 4.0 x 106 APCs / cm2 or less than or equal to 5.5 x 106 APCs / cm2.
[001369] [001369] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that, in step (a), the first population of APCs is sown on the first gas-permeable surface at a density equal to or near 2.0 x 106 APCs / cm2 and, in step (b), the second population of APCs is sown on the first gas-permeable surface at a density equal to or near 4.0 x 106 APCs / cm2.
[001370] [001370] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that APCs are peripheral blood mononuclear cells (PBMCs).
[001371] [001371] In another embodiment, the invention provides the method described in
[001372] [001372] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that T cells are tumor-infiltrating lymphocytes (TILs), the first population of T cells is a first population of TILs in step (a), the second population of T cells is a second population of TILs in step (b), the second population of TILs is a therapeutic population of TILs and the therapeutic population of TILs is harvested in step (c ).
[001373] [001373] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that T cells are marrow infiltrating lymphocytes (MILs).
[001374] [001374] In another embodiment, the invention provides the method described in any of the previous paragraphs, as applicable above, modified in such a way that the T cell phenotype is CD3 + and CD45 +. XV. Pharmaceutical compositions, doses and administration schedules
[001375] [001375] In one embodiment, TILs expanded by the methods of the present invention are administered to a patient as a pharmaceutical composition. In one embodiment, the pharmaceutical composition is a suspension of TILs in a sterile buffer. Expanded TILs using PBMCs of the present invention can be administered by any suitable route known in the art. In some embodiments, T cells are administered as a single intra-arterial or intravenous infusion, which preferably lasts approximately 30 to 60 minutes. Other suitable routes of administration include intraperitoneal, intrathecal and intralymphatic administration.
[001376] [001376] Any suitable dose of TILs can be administered. In some modalities, between approximately 2.3x1010 and 13.7x1010 TILs are
[001377] [001377] In some embodiments, the number of TILs provided in the pharmaceutical compositions of the invention is approximately 1x106, 2x106, 3x106, 4x106, 5x106, 6x106, 7x106, 8x106, 9x106, 1x107, 2x107, 3x107, 4x107, 5x107, 6x107, 7x107 , 8x107, 9x107, 1x108, 2x108, 3x108, 4x108, 5x108, 6x108, 7x108, 8x108, 9x108, 1x109, 2x109, 3x109, 4x109, 5x109, 6x109, 7x109, 8x109, 9x109, 1x1010, 2x1010, 3x1010, 4x10, 3x1010, 4x10, 3x1010, 4x10, , 6x1010, 7x1010, 8x1010, 9x1010, 1x1011, 2x1011, 3x1011, 4x1011, 5x1011, 6x1011, 7x1011, 8x1011, 9x1011, 1x1012, 2x1012, 3x1012, 4x1012, 5x1012, 6x1012, 7x1012, 8x1012, 9x1012, 1x1013, 2x10, 1x1013
[001378] [001378] In some embodiments, the concentration of TILs provided in the pharmaceutical compositions of the invention is less than, for example, 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20% , 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3 %, 2%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07%, 0, 06%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002 %, 0.001%, 0.0009%, 0.0008%, 0.0007%, 0.0006%, 0.0005%, 0.0004%, 0.0003%, 0.0002% or 0.0001% p / w, w / v or v / v of the pharmaceutical composition.
[001379] [001379] In some embodiments, the concentration of TILs provided in the pharmaceutical compositions of the invention is greater than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 19.75%, 19 , 50%, 19.25% 19%, 18.75%, 18.50%, 18.25% 18%, 17.75%, 17.50%, 17.25% 17%, 16.75%, 16.50%, 16.25% 16%, 15.75%, 15.50%, 15.25% 15%, 14.75%, 14.50%, 14.25% 14%, 13.75% , 13.50%, 13.25% 13%, 12.75%, 12.50%, 12.25% 12%, 11.75%, 11.50%, 11.25% 11%, 10.75 %, 10.50%, 10.25% 10%, 9.75%, 9.50%, 9.25% 9%, 8.75%, 8.50%, 8.25% 8%, 7, 75%, 7.50%, 7.25% 7%, 6.75%, 6.50%, 6.25% 6%, 5.75%, 5.50%, 5.25% 5%, 4 , 75%, 4.50%, 4.25%, 4%, 3.75%, 3.50%, 3.25%, 3%, 2.75%, 2.50%, 2.25%, 2%, 1.75%, 1.50%, 125%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0 , 08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01%, 0.009%, 0.008%, 0.007%, 0.006% , 0.005%, 0.004%, 0.003%, 0.002%, 0.001%, 0.0009%, 0.0008%, 0.0007%, 0.0006%, 0.0005%, 0.0004%, 0.0003% , 0.0002% or 0.0001% w / w, w / v, or v / v of the pharmaceutical composition,
[001380] [001380] In some embodiments, the concentration of TILs provided in the pharmaceutical compositions of the invention is in the range between approximately 0.001% and 10%, between approximately 0.01% and 5%, between approximately 0.02% and 4.5%, between approximately 0.03% and 4%, between approximately 0.04% and 3.5%, between approximately 0.05% and 3%, between approximately 0.06% and 2.5%, between approximately 0.07% and 2%, between approximately 0.08% and 1.5%, between approximately 0.09% and 1%, between approximately 0.1% and 0.9% w / w, w / v or v / v of the composition pharmaceutical company.
[001381] [001381] In some embodiments, the amount of TILs provided in the pharmaceutical compositions of the invention is equal to or less than 10 g, 9.5 g, 9.0 g, 8.5 g, 8.0 g, 7.5 g, 7.0 g, 6.5 g, 6.0 g, 5.5 g, 5.0 g, 4.5 g, 4.0 g, 3.5 g, 3.0 g, 2.5 g, 2.0 g, 1.5 g, 1.0 g, 0.95 g, 0.9 g, 0.85 g, 0.8 g, 0.75 g, 0.7 g, 0.65 g, 0.6 g, 0.55 g, 0.5 g, 0.45 g, 0.4 g, 0.35 g, 0.3 g, 0.25 g, 0.2 g, 0.15 g, 0.1 g, 0.09 g, 0.08 g, 0.07 g, 0.06 g, 0.05 g, 0.04 g, 0.03 g, 0.02 g, 0.01 g, 0.009 g, 0.008 g, 0.007 g, 0.006 g, 0.005 g, 0.004 g, 0.003 g, 0.002 g, 0.001 g, 0.0009
[001382] [001382] In some embodiments, the amount of TILs provided in the pharmaceutical compositions of the invention is greater than 0.0001 g, 0.0002 g, 0.0003 g, 0.0004 g, 0.0005 g, 0.0006 g, 0.0007 g, 0.0008 g, 0.0009 g, 0.001 g, 0.0015 g, 0.002 g, 0.0025 g, 0.003 g, 0.0035 g, 0.004 g, 0.0045 g, 0.005 g, 0.0055 g, 0.006 g, 0.0065 g, 0.007 g, 0.0075 g, 0.008 g, 0.0085 g, 0.009 g, 0.0095 g, 0.01 g, 0.015 g, 0.02 g, 0.025 g, 0.03 g, 0.035 g, 0.04 g, 0.045 g, 0.05 g, 0.055 g, 0.06 g, 0.065 g, 0.07 g, 0.075 g, 0.08 g, 0.085 g , 0.09 g, 0.095 g, 0.1 g, 0.15 g, 0.2 g, 0.25 g, 0.3 g, 0.35 g, 0.4 g, 0.45 g, 0 , 5 g, 0.55 g, 0.6 g, 0.65 g, 0.7 g, 0.75 g, 0.8 g, 0.85 g, 0.9 g, 0.95 g, 1 g, 1.5 g, 2 g, 2.5, 3 g, 3.5, 4 g, 4.5 g, 5 g, 5.5 g, 6 g, 6.5 g, 7 g, 7, 5 g, 8 g, 8.5 g, 9 g, 9.5 g or 10 g.
[001383] [001383] The TILs provided in the pharmaceutical compositions of the invention are effective over a wide dose range. The exact dose will depend on the route of administration, the form in which the compound is administered, the sex and age of the individual to be treated, the body weight of the individual to be treated and the preference and experience of the attending physician. Clinically established doses of TILs can also be used, if appropriate. The amounts of pharmaceutical compositions administered by the present methods, such as doses of TILs, will depend on the human or mammal being treated, the severity of the disorder or condition, the rate of administration, the disposition of the active pharmaceutical ingredients and at the discretion of the prescribing physician.
[001384] [001384] In some modalities, TILs can be administered in a single dose. Such administration can be by injection, e.g. eg, intravenous injection. In some modalities, TILs can be administered in multiple doses. Administration can be once, twice, three times, four times, five times, six times or more than six times a year. Administration can be once a month, once every two weeks, once a month.
[001385] [001385] In some embodiments, an effective dose of TILs is approximately 1x106, 2x106, 3x106, 4x106, 5x106, 6x106, 7x106, 8x106, 9x106, 1x107, 2x107, 3x107, 4x107, 5x107, 6x107, 7x107, 8x107, 9x107, 1x108, 2x108, 3x108, 4x108, 5x108, 6x108, 7x108, 8x108, 9x108, 1x109, 2x109, 3x109, 4x109, 5x109, 6x109, 7x109, 8x109, 9x109, 1x1010, 2x1010, 3x1010, 4x1010, 5x10, 6x10, 5x10, 6x10, 7x10 8x1010, 9x1010, 1x1011, 2x1011, 3x1011, 4x1011, 5x1011, 6x1011, 7x1011, 8x1011, 9x1011, 1x1012, 2x1012, 3x1012, 4x1012, 5x1012, 6x1012, 7x1012, 8x1012, 9x1012, 1x1013, 2x1013, 3x1013, 4x1013, 5x1013, 4x1013 6x1013, 7x1013, 8x1013 and 9x1013. In some modalities, an effective dose of TILs is in the range of 1x106 to 5x106, 5x106 to 1x107, 1x107 to 5x107, 5x107 to 1x108, 1x108 to 5x108, 5x108 to 1x109, 1x109 to 5x109, 5x109 to 1x1010, 1x1010 to 5x1010, 5x1010 to 1x1011, 5x1011 to 1x1012, 1x1012 to 5x1012 and 5x1012 to 1x1013.
[001386] [001386] In some embodiments, an effective dose of TILs is in the range between approximately 0.01 mg / kg and 4.3 mg / kg, between approximately 0.15 mg / kg and 3.6 mg / kg, between approximately 0 , 3 mg / kg and 3.2 mg / kg, between approximately 0.35 mg / kg and 2.85 mg / kg, between approximately 0.15 mg / kg and 2.85 mg / kg, between approximately 0.3 mg and 2.15 mg / kg, between approximately 0.45 mg / kg and 1.7 mg / kg, between approximately 0.15 mg / kg and 1.3 mg / kg, between approximately 0.3 mg / kg and 1.15 mg / kg, between approximately 0.45 mg / kg and 1 mg / kg, between approximately 0.55 mg / kg and 0.85 mg / kg, between approximately 0.65 mg / kg and 0.8 mg / kg, between approximately 0.7 mg / kg and 0.75 mg / kg, between approximately 0.7 mg / kg and 2.15 mg / kg, between approximately 0.85 mg / kg and 2 mg / kg, between approximately 1 mg / kg and 1.85 mg / kg, between approximately 1.15 mg / kg and 1.7 mg / kg, between approximately 1.3 mg / kg mg and 1.6 mg / kg, between approximately 1, 35 mg / kg and 1.5 mg / kg, between approximately 2.15 mg / kg and 3.6 mg / kg, between approx. approximately 2.3 mg / kg
[001387] [001387] An effective amount of TILs can be administered in a single or multiple dose by any of the accepted modes for administration of agents of similar utility, including intranasal and transdermal, by intra-arterial injection, intravenous, intraperitoneal, parenteral , intramuscular, subcutaneous, topical, by transplant or by inhalation.
[001388] [001388] In another embodiment, the invention provides an infusion bag comprising the therapeutic population of TILs described in any of the previous paragraphs, as applicable above.
[001389] [001389] In another embodiment, the invention provides a tumor infiltrating lymphocyte (TIL) composition comprising the therapeutic population
[001390] [001390] In another embodiment, the invention provides an infusion pouch that comprises the composition of TIL described in any of the previous paragraphs, as applicable above.
[001391] [001391] In another embodiment, the invention provides a preparation of the therapeutic population of cryopreserved TILs described in any of the previous paragraphs, as applicable above.
[001392] [001392] In another embodiment, the invention provides a tumor infiltrating lymphocyte (TIL) composition that comprises the therapeutic population of TILs described in any of the previous paragraphs, as applicable above and a cryopreservation medium.
[001393] [001393] In another embodiment, the invention provides the composition of TIL described in any of the previous paragraphs, as applicable above, modified in such a way that the cryopreservation medium contains DMSO.
[001394] [001394] In another embodiment, the invention provides the TIL composition described in any of the previous paragraphs, as applicable above, modified in such a way that the cryopreservation medium contains 7-10% DMSO.
[001395] [001395] In another embodiment, the invention provides a preparation of the composition of cryopreserved TILs described in any of the previous paragraphs, as applicable above. XVI. Methods of treating patients
[001396] [001396] Treatment methods begin with the initial collection of TIL and culture of TILs. Such methods have both been described in the art, for example, by Jin et al., J. Immunotherapy, 2012, 35 (3): 283-292, hereby incorporated in their entirety by reference. Modalities of methods of treatment are described in all sections below, including the Examples.
[001397] [001397] Expanded TILs produced according to the methods described here, including, for example, as described in steps A to F above or according to Steps A to F above (also shown, for example, in Figure 85 (in in particular, eg, Figure 85B)) find particular utility in the treatment of cancer patients (for example, as described in Goff, et al., J. Clinical Oncology, 2016, 34 (20): 2389-239, as well as in the supplementary content; here incorporated in their entirety by reference. In some embodiments, TIL were cultured from resected metastatic melanoma deposits as previously described (see, Dudley, et al., J Immunother., 2003, 26 : 332-342; incorporated here, in its entirety, by reference). Fresh tumor can be dissected under sterile conditions. A representative sample can be collected for formal pathological analysis. Isolated fragments of 2 mm3 to 3 mm3 can be used. modalities, 5, 10, 15, 20, 25 or 30 are obtained from samples per patient. In some modalities, 20, 25 or 30 samples are obtained per patient. In some modalities, 20, 22, 24, 26, or 28 samples are obtained per patient. In some modalities, 24 samples are obtained per patient. The samples can be placed in individual wells of a 24-well plate, kept in growth medium with a high dose of IL-2 (6,000 IU / mL) and monitored for tumor destruction and / or TIL proliferation. Any tumor with viable cells that remains after processing can be digested enzymatically in a single cell suspension and cryopreserved, as described herein.
[001398] [001398] In some modalities, samples of successfully cultured TILs can be collected for phenotype analysis (CD3, CD4, CD8 and CD56) and tested against autologous tumor when available. TIL can be considered reactive if overnight coculture produces levels of gamma interferon (IFN-γ) ˃ 200 pg / mL and twice the background level (Goff, et al., J Immunother., 2010, 33: 840 -847; here incorporated, in its entirety,
[001399] [001399] In some modalities, the patient is not referred directly to ACT (adoptive cell transfer), for example, in some modalities, after collection in the tumor and / or a first expansion, as the cells are not used immediately. In some modalities, TILs can be cryopreserved and thawed 2 days before administration to a patient. In some modalities, TILs can be cryopreserved and thawed 1 day before administration to a patient. In some modalities, TILs can be cryopreserved and thawed immediately before administration to a patient.
[001400] [001400] The cell phenotype of conserved TILs samples in the infusion bag can be analyzed by flow cytometry (eg FlowJo ™) for surface markers CD3, CD4, CD8, CCR7 and CD45RA (BD BioSciences), as well as by any of the methods described here. Serum cytokines were measured using standard immunoenzymatic absorption assay techniques. Elevation in serum IFN-g was defined as ˃100 pg / mL and greater than 43 3 at baseline levels.
[001401] [001401] In some embodiments, the TILs produced by the methods provided herein, for example, those exemplified in Figure 85 (in particular, e.g., Figure 85B), provide a surprising improvement in the clinical effectiveness of TILs. In some embodiments, the TILs produced by the methods provided herein, for example, those exemplified in Figure 85 (in particular, eg, Figure 85B), exhibited increased clinical efficacy compared to TILs produced by methods other than those described herein. , including, for example, methods other than those exemplified in Figure 85 (in particular, e.g., Figure 85B). In some embodiments, methods other than those described herein include methods referred to as process 1C and / or Generation 1 (Gen 1). In some modalities, the increased effectiveness is measured by the disease control rate (TCD), global response rate (TRG) and / or other clinical responses. In some embodiments, TILs produced by the methods provided herein, for example, those exemplified in Figure 85 (in particular, eg, Figure 85B) exhibit similar security profile response times compared to TILs produced by other methods than those described herein, including, for example, methods other than those exemplified in Figure 85 (in particular, e.g., Figure 85B), for example, the Gen 1 process.
[001402] [001402] In some embodiments, IFN-gamma (IFN-γ) is indicative of treatment efficacy and / or increased clinical efficacy. In some embodiments, IFN-γ in the blood of individuals treated with TILs is indicative of active TILs. In some modalities, a power test is used to produce IFN-γ. IFN-γ production is another measure of cytotoxic potential. IFN-γ production can be measured by determining the levels of the IFN-γ cytokine in the blood, serum or in ex vivo TILs of an individual treated with TILs prepared by the methods of the present invention, including those described, for example, in Figure 85 ( in particular, e.g., Figure 85B). In
[001403] [001403] In some embodiments, TILs prepared by the methods of the present invention, including those described, for example, in Figure 85 (in particular, e.g., Figure 85B) exhibit increased polyclonality compared to TILs produced by other methods, including those not exemplified in Figure 85 (in particular, e.g., Figure 85B), such as, for example, methods referred to as process methods 1C. In some modalities, significantly improved polyclonality and / or increased polyclonality is indicative of treatment efficacy and / or increased clinical efficacy. In some embodiments, polyclonality refers to the diversity of the T cell repertoire. In some embodiments, an increase in polyclonality may be indicative of treatment effectiveness in relation to the administration of the TILs produced by the methods of the present invention. In some embodiments, polyclonality is increased once, twice, ten times, 100 times, 500 times or 1000 times compared to TILs prepared by methods other than those provided here, for example, methods different from those incorporated in Figure 85 (in particular , e.g., Figure 85B). In some modalities, polyclonality is increased a
[001404] [001404] Efficacy measures may include the disease control rate (DCR) as well as the overall response rate (TRG), as known in
[001405] [001405] The compositions and methods described herein can be used in a method to treat diseases. In one embodiment, they are used to treat hyperproliferative disorders. They can also be used to treat other disorders as described here and in the following paragraphs.
[001406] [001406] In some modalities, the hyperproliferative disorder is cancer. In some modalities, hyperproliferative disorder is a solid tumor cancer. In some modalities, solid tumor cancer is selected from the group consisting of glioblastoma (GBM), gastrointestinal cancer, melanoma, ovarian cancer, cervical cancer, non-small cell lung cancer (NSCLC), lung cancer, cancer bladder cancer, breast cancer, cancer caused by human papillomavirus, cancer of the head and neck (including squamous cell carcinoma of the head and neck (CCECP)), kidney cancer and renal cell carcinoma. In some modalities, hyperproliferative disorder is a hematological malignancy. In some modalities, solid tumor cancer is selected from the group consisting of chronic lymphocytic leukemia, acute lymphoblastic leukemia, diffuse large B cell lymphoma, non-Hodgkin's lymphoma, Hodgkin's lymphoma, follicular lymphoma and mantle cell lymphoma.
[001407] [001407] In some modalities, cancer is a hypermutated cancer phenotype. Hypermutated cancers are extensively described in Campbell, et al. (Cell, 171: 1042-1056 (2017); incorporated here, in its entirety, by reference for all purposes). In some modalities, hypermutated tumors comprise between 9 and 10 mutations per megabase (Mb). In some modalities, hypermutated pediatric tumors comprise 9.91 mutations per megabase (Mb). In some modalities, adult hypermutated tumors comprise 9 mutations per megabase (Mb). In
[001408] [001408] In some embodiments, hypermutated tumors exhibit mutations in the path of repair in replication. In some modalities, hypermutated tumors exhibit mutations in DNA polymerases associated with repair in replication. In some modalities, hypermutated tumors exhibit microsatellite instability. In some modalities, ultra-hypermutated tumors exhibit mutations in DNA polymerases associated with repair in the replication and instability of microsatellites. In some modalities, hypermutation in the tumor is correlated with the response to immune checkpoint inhibitors. In some modalities, hypermutated tumors are resistant to treatment with immunological checkpoint inhibitors. In some embodiments, hypermutated tumors can be treated with the TILs of the present invention. In some modalities, hypermutation in the tumor is caused by environmental factors (extrinsic exposures). For example, UV light may be the primary cause of the high numbers of mutations in malignant melanoma (see, for example, Pfeifer, GP, You, YH, and Besaratinia, A. (2005). Mutat. Res. 571, 19-31 .; Sage, E. (1993). Photochem. Photobiol. 57, 163-174.). In some embodiments, tumor hypermutation can be caused by more than 60 carcinogens in tobacco smoke from lung and laryngeal tumors, as well as other tumors,
[001409] [001409] In one embodiment, the invention includes a method of treating cancer with a population of TILs, in which the cancer is a hypermutated cancer. In one embodiment, the invention includes a method of
[001410] [001410] In one embodiment, the invention includes a method of treating cancer with a population of TILs, in which a patient is pre-treated with non-myeloablative chemotherapy before an infusion of TILs in accordance with the present invention. In one embodiment, non-myeloablative chemotherapy is cyclophosphamide 60 mg / kg / d for 2 days (27 and 26 days before the infusion of TILs) and fludarabine 25 mg / m2 / d for 5 days (27 to 23 days before the infusion of TILs). In one embodiment, after non-myeloablative chemotherapy and infusion of TILs (on day 0) according to the present invention, the patient receives an intravenous infusion of IL-2 intravenously at 720,000 IU / kg every 8 hours according to physiological tolerance.
[001411] [001411] The effectiveness of the compounds and combinations of compounds described herein in treating, preventing and / or controlling the indicated diseases or disorders can be tested using various models known in the art, which provide guidance for the treatment of human diseases. For example, models for determining the effectiveness of treatments for ovarian cancer are described, e.g. in Mullany, et al., Endocrinology 2012, 153, 1585-92; and Fong, et al., J. Ovariano Res. 2009, 2, 12. Models for determining the effectiveness of treatments for pancreatic cancer are described in Herreros-Villanueva, et al., World J. Gastroenterol. 2012, 18, 1286-1294. Models for determining the effectiveness of breast cancer treatments are described, p. in Fantozzi, Breast Cancer Res. 2006, 8, 212. Models for determining the effectiveness of treatments for melanoma are described, p. e.g., in Damsky, et al., Pigment Cell & Melanoma Res. 2010, 23, 853-859. Models for determining the effectiveness of treatments for lung cancer are described,
[001412] [001412] In some modalities, IFN-gamma (IFN-γ) is indicative of the effectiveness of the treatment for hyperproliferative disorder. In some embodiments, IFN-γ in the blood of individuals treated with TILs is indicative of active TILs. In some modalities, a power test is used to produce IFN-γ. IFN-γ production is another measure of cytotoxic potential. IFN-γ production can be measured by determining the levels of the IFN-γ cytokine in the blood of an individual treated with TILs prepared by the methods of the present invention, including those described, for example, in Figure 85 (in particular, e.g. , Figure 85B). In some embodiments, the TILs obtained by the present method increase IFN-γ in the blood of individuals treated with the TILs of the present method when compared to individuals treated with TILs prepared by methods referred to as the Gen 3 process, as exemplified in Figure 85 (in particular , eg, Figure 85B) and throughout this patent application. In some embodiments, an increase in IFN-γ is indicative of treatment efficacy in a patient treated with the TILs produced by the methods of the present invention. In some embodiments, IFN-γ is increased once, twice, three times, four times, five times or more compared to an untreated patient and / or when compared to a patient treated with TILs prepared by methods other than those here described, for example, methods other than those incorporated in Figure 85 (in particular, e.g., Figure 85B). In some embodiments, IFN-γ secretion is increased once compared to an untreated patient and / or when compared to a patient treated with TILs prepared by methods other than those described here, for example, methods other than those incorporated in the Figure 85 (in
[001413] [001413] In some embodiments, TILs prepared by the methods of the present invention, including those described, for example, in Figure 85
[001414] [001414] In some embodiments, TILs produced as described herein, including, for example, TILs derived from a method described in steps A to F of Figure 85 (in particular, e.g., Figure 85B), can be administered in combination with one or more immune checkpoint regulators, such as the antibodies described below. For example, antibodies directed to PD-1 and which can be co-administered with the TILs of the present invention include, e.g. e.g., among others, nivolumab (BMS-936558, Bristol-Myers Squibb; Opdivo®), pembrolizumab (lambrolizumab, MK03475 or MK-3475, Merck; Keytruda®), the humanized anti-PD-1 JS001 antibody (ShangHai JunShi) , the anti-PD-1 monoclonal antibody TSR-042 (Tesaro, Inc.), Pidilizumab (anti-PD-1 mAb CT-011, Medivation), the anti-PD-1 monoclonal antibody BGB-A317 (BeiGene) and / or the SHR-1210 anti-PD-1 antibody (ShangHai HengRui), the human monoclonal antibody REGN2810 (Regeneron), the human monoclonal antibody MDX-1106 (Bristol-Myers Squibb) and / or the humanized anti-PD-1 IgG4 antibody
[001415] [001415] In one embodiment, the invention includes a method of treating cancer with a population of TILs, in which a patient is pre-treated with non-myeloablative chemotherapy before an infusion of TILs in accordance with the present invention. In one embodiment, the invention includes a population of TILs for use in the treatment of cancer in a patient who has been pretreated with non-myeloablative chemotherapy. In one embodiment, the TIL population is for infusion administration. In one embodiment, non-myeloablative chemotherapy is cyclophosphamide 60 mg / kg / d for 2 days (27 and 26 days before the infusion of TILs) and fludarabine 25 mg / m2 / d for 5 days (27 to 23 days before the infusion of TILs). In one embodiment, after non-myeloablative chemotherapy and infusion of TILs (on day 0) according to the present invention, the patient receives an intravenous infusion of IL-2 (aldesleukin, commercially available as PROLEUKIN) intravenously at 720,000 IU / kg every 8 hours according to physiological tolerance. In certain embodiments, the TIL population is for use in the treatment of cancer in combination with IL-2, where IL-2 is administered after the TIL population.
[001416] [001416] Experimental findings indicate that lymphodepletion before the adoptive transfer of tumor-specific T lymphocytes plays a key role in increasing the effectiveness of treatment by eliminating regulatory T cells and competing elements of the immune system ("cytokine dissipators"). Thus, some embodiments of the invention use a lymphodepletion step (sometimes also referred to as "immunosuppressive conditioning") in the patient prior to the introduction of the rTILs of the invention.
[001417] [001417] In general, lymphodepletion is achieved by administering fludarabine or cyclophosphamide (the active form referred to as mafosfamide) and combinations thereof. Such methods are described in Gassner, et al., Cancer Immunol. Immunother. 2011, 60, 75-85, Muranski, et al., Nat. Clin. Pract. Oncol., 2006, 3, 668-681, Dudley, et al., J. Clin. Oncol. 2008, 26,
[001418] [001418] In some embodiments, fludarabine is administered at a concentration of fludarabine 0.5 μg / mL-10 μg / mL. In some embodiments, fludarabine is administered at a concentration of fludarabine 1 μg / mL. In some modalities, treatment with fludarabine is administered for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days or more. In some embodiments, fludarabine is administered at a dose of 10 mg / kg / day, 15 mg / kg / day, 20 mg / kg / day¸ 25 mg / kg / day, 30 mg / kg / day, 35 mg / day kg / day, 40 mg / kg / day or 45 mg / kg / day. In some modalities, treatment with fludarabine is administered for 2-7 to 35 mg / kg / day. In some modalities, treatment with fludarabine is administered for 4-5 days at 35 mg / kg / day. In some modalities, treatment with fludarabine is administered for 4-5 days at 25 mg / kg / day.
[001419] [001419] In some embodiments, mafosfamide, the active form of cyclophosphamide, is obtained at a concentration of 0.5 μg / mL-10 μg / mL by administering cyclophosphamide. In some embodiments, mafosfamide, the active form of cyclophosphamide, is obtained at a concentration of 1 μg / mL by administering cyclophosphamide. In some modalities, cyclophosphamide treatment is administered for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days or more. In some embodiments, cyclophosphamide is administered at a dose of 100 mg / m2 / day, 150 mg / m2 / day, 175 mg / m2 / day¸ 200 mg / m2 / day, 225 mg / m2 / day, 250 mg / m2 / day m2 / day, 275 mg / m2 / day or 300 mg / m2 / day. In some embodiments, cyclophosphamide is administered intravenously (ie i.v.) In some embodiments, treatment with cyclophosphamide is administered for 2-7 days at 35 mg / kg / day. In some embodiments, treatment with cyclophosphamide is administered for 4-5 days at 250 mg / m2 / day i.v. In some modalities, treatment with cyclophosphamide is administered for 4 days at 250 mg / m2 / day i.v.
[001420] [001420] In some modalities, lymphodepletion is performed by administering fludarabine and cyclophosphamide together to a patient. In some embodiments, fludarabine is administered at 25 mg / m2 / day i.v. and cyclophosphamide is administered at 250 mg / m2 / day i.v. for 4 days.
[001421] [001421] In one embodiment, lymphodepletion is performed by administering cyclophosphamide at a dose of 60 mg / m2 / day for two days, followed by administration of fludarabine at a dose of 25 mg / m2 / day for five days.
[001422] [001422] In one embodiment, the IL-2 schedule comprises a high-dose IL-2 schedule, in which the high-dose IL-2 schedule comprises aldesleukin, or a biosimilar or variant thereof, administers intravenously starting the day after the administration of a therapeutically effective part of the therapeutic population of TILs, in which aldesleukin or a biosimilar or variant thereof is administered at a dose of 0.037 mg / kg or 0.044 mg / kg IU / kg (patient's body mass ) by intravenous bolus infusions of 15 minutes every eight hours according to tolerance, for a maximum of 14 doses. After 9 days of rest, this scheme can be repeated for another 14 doses, totaling a maximum of 28 doses.
[001423] [001423] In one embodiment, the IL-2 scheme comprises a decreasing IL-2 scheme. Descending schemes of IL-2 have been described in O'Day, et al., J. Clin. Oncol. 1999, 17, 2752-61 and Eton, et al., Cancer 2000, 88, 1703-9, the content of which is incorporated herein by reference. In one embodiment, a decreasing schedule of IL-2 comprises 18 x 106 IU / m2 administered intravenously over 6 hours, followed by 18 x 106 IU / m2 administered intravenously over 12 hours, followed by 18 x 106 IU / m2 administered intravenously. intravenous for 24 hours, followed by 4.5 x 106 IU / m2 administered intravenously for 72 hours. This treatment cycle can
[001424] [001424] In one embodiment, the IL-2 schedule comprises the administration of pegylated IL-2 every 1, 2, 4, 6, 7, 14 or 21 days at a dose between 0.10 mg / day and 50 mg /day.
[001425] [001425] Adoptive cell transfer (ACT) is an effective form of immunotherapy and involves the transfer of immune cells with antitumor activity to cancer patients. ACT is a therapeutic approach that involves the identification, in vitro, of lymphocytes with antitumor activity, the in vitro expansion of these cells to large numbers and their infusion in the host with cancer. The lymphocytes used for adoptive transfer can be derived from the stroma of resected tumors (tumor infiltrating lymphocytes or TILs). ACT TILs can be prepared as described herein. In some embodiments, TILs are prepared, for example, according to a method described in Figure 85 (in particular, e.g., Figure 85B). They can also be derived from blood if they are genetically modified to express antitumor T cell receptors (TCRs) or chimeric antigen receptors (CARs), enriched with tumor cell-mixed lymphocyte cultures (MLTCs), or cloned using autologous and antigen presenting cells. tumor-derived peptides. The ACT in which the lymphocytes originate from the cancer-bearing host to be infused is called an autologous ACT. US Publication No. 2011/0052530 refers to a method for adoptive cell therapy aimed at promoting cancer regression, primarily for the treatment of patients suffering from metastatic melanoma, the content of which is incorporated herein in its entirety by reference to what regards these methods. In some modalities, TILs can be administered as
[001426] [001426] In another embodiment, the invention provides a method for treating an individual with cancer, which comprises administering to the individual a therapeutically effective dose of the therapeutic population of TILs described in any of the preceding paragraphs, as applicable above.
[001427] [001427] In another embodiment, the invention provides a method for treating an individual with cancer, which comprises administering to the individual a therapeutically effective dose of the TIL composition described in any of the previous paragraphs, as applicable above
[001428] [001428] In another embodiment, the invention provides the method for the treatment of an individual with cancer, described in any of the previous paragraphs, as applicable above, modified in such a way that, before administering the therapeutically effective dose to the therapeutic population of TILs and the composition of TILs described in any of the previous paragraphs, as applicable above, respectively, a non-myeloablative lymphodepletion scheme was administered to the individual.
[001429] [001429] In another embodiment, the invention provides the method for treating an individual with cancer, described in any of the previous paragraphs, as applicable above, modified in such a way that
[001430] [001430] In another embodiment, the invention provides the method for treating an individual with cancer, described in any of the previous paragraphs, as applicable above, modified to further understand the step of treating the individual with a high dose regimen of IL-2, starting on the day following the administration of TIL cells to the individual.
[001431] [001431] In another embodiment, the invention provides the method for the treatment of an individual with cancer, described in any of the previous paragraphs, as applicable above modified, in such a way that the high-dose schedule of IL-2 comprises 600,000 or 720,000 IU / kg, administered by intravenous bolus infusion over 15 minutes every eight hours while tolerated.
[001432] [001432] In another embodiment, the invention provides the method for treating an individual with cancer, described in any of the previous paragraphs, as applicable above, modified in such a way that the cancer is a solid tumor.
[001433] [001433] In another embodiment, the invention provides the method for treating an individual with cancer, described in any of the previous paragraphs, as applicable above, modified in such a way that the cancer is melanoma, ovarian cancer, cervical cancer, non-small cell lung cancer (NSCLC), lung cancer, bladder cancer, breast cancer, cancer caused by human papillomavirus, head and neck cancer (including head and neck squamous cell carcinoma (CCECP)), glioblastoma (including GBM), gastrointestinal cancer, kidney cancer or kidney cell carcinoma.
[001434] [001434] In another embodiment, the invention provides the method for treating an individual with cancer, described in any of the previous paragraphs, as applicable above, modified in such a way that the cancer is melanoma, CCECP, cervical cancer, CPNPC , glioblastoma (including GBM) and gastrointestinal cancer.
[001435] [001435] In another embodiment, the invention provides the method for treating an individual with cancer, described in any of the previous paragraphs, as applicable above, modified in such a way that the cancer is melanoma.
[001436] [001436] In another embodiment, the invention provides the method for treating an individual with cancer, described in any of the previous paragraphs, as applicable above modified in such a way that the cancer is CCECP.
[001437] [001437] In another embodiment, the invention provides the method for treating an individual with cancer, described in any of the previous paragraphs, as applicable above, modified in such a way that the cancer is cervical cancer.
[001438] [001438] In another embodiment, the invention provides the method for treating an individual with cancer, described in any of the previous paragraphs, as applicable above, modified in such a way that the cancer is NSCLC.
[001439] [001439] In another embodiment, the invention provides the method for treating an individual with cancer, described in any of the preceding paragraphs, as applicable above, modified in such a way that the cancer is glioblastoma (including GBM).
[001440] [001440] In another embodiment, the invention provides the method for the treatment of an individual with cancer, described in any of the previous paragraphs, as applicable above modified in such a way that the cancer is gastrointestinal cancer.
[001441] [001441] In another embodiment, the invention provides the method for treating an individual with cancer described in any of the previous paragraphs, as applicable above, modified in such a way that the cancer is a hypermutated cancer.
[001442] [001442] In another embodiment, the invention provides the method for treating an individual with cancer, described in any of the previous paragraphs, as applicable above modified in such a way that the cancer is a pediatric hypermutated cancer.
[001443] [001443] In another embodiment, the invention provides the therapeutic population of TILs described in any of the preceding paragraphs, as applicable above, for use in a method for treating an individual with cancer, which comprises administering to the individual a therapeutically dose therapeutic population of TILs.
[001444] [001444] In another embodiment, the invention provides the composition of TILs described in any of the preceding paragraphs, as applicable above, for use in a method for the treatment of an individual with cancer, which comprises administering to the individual a therapeutically effective dose composition of TILs.
[001445] [001445] In another embodiment, the invention provides the therapeutic population of TILs described in any of the previous paragraphs, as applicable above, or the composition of TILs described in any of the previous paragraphs, as applicable above, modified in such a way that, prior to administering to the individual, the therapeutically effective dose of the therapeutic population of TILs described in any of the preceding paragraphs, as applicable above, or of the composition of TILs described in any of the previous paragraphs, as applicable above, a non-lymph-depletion scheme myeloablativa was administered to the individual.
[001446] [001446] In another embodiment, the invention provides the therapeutic population of TILs or the composition of TILs described in any of the
[001447] [001447] In another embodiment, the invention provides the therapeutic population of TILs or the composition of TILs described in any of the previous paragraphs, as applicable above, modified to further understand the step of treating a patient with a high-dose IL-regimen 2, starting the day after TIL cell administration to the patient.
[001448] [001448] In another embodiment, the invention provides the therapeutic population of TILs or the composition of TILs described in any of the previous paragraphs, as applicable above modified in such a way that the high dose IL-2 schedule comprises 600,000 or 720,000 IU / kg, administered by intravenous bolus infusion over 15 minutes every eight hours while tolerated.
[001449] [001449] In another embodiment, the invention provides the therapeutic population of TILs or the composition of TILs described in any of the previous paragraphs, as applicable above, modified in such a way that the cancer is a solid tumor.
[001450] [001450] In another embodiment, the invention provides the therapeutic population of TILs or the composition of TILs described in any of the previous paragraphs, as applicable above, modified in such a way that the cancer is melanoma, ovarian cancer, cervical cancer, cancer non-small cell lung cancer (NSCLC), lung cancer, bladder cancer, breast cancer, cancer caused by human papillomavirus, head and neck cancer (including head and neck squamous cell carcinoma (CCECP)), glioblastoma ( including GBM), gastrointestinal cancer, kidney cancer, or renal cell carcinoma.
[001451] [001451] In another embodiment, the invention provides the therapeutic population of TILs or the composition of TILs described in any of the previous paragraphs, as applicable above, modified in such a way that the cancer is melanoma, CCECP, cervical cancer, CPNPC, glioblastoma (including GBM) and gastrointestinal cancer.
[001452] [001452] In another embodiment, the invention provides the therapeutic population of TILs or the composition of TILs described in any of the previous paragraphs, as applicable above, modified in such a way that the cancer is melanoma.
[001453] [001453] In another embodiment, the invention provides the therapeutic population of TILs or the composition of TILs described in any of the previous paragraphs, as applicable above, modified in such a way that the cancer is CCECP.
[001454] [001454] In another embodiment, the invention provides the therapeutic population of TILs or the composition of TILs described in any of the previous paragraphs, as applicable above, modified in such a way that the cancer is cervical cancer.
[001455] [001455] In another embodiment, the invention provides the therapeutic population of TILs or the composition of TILs described in any of the previous paragraphs, as applicable above, modified in such a way that the cancer is NSCLC.
[001456] [001456] In another embodiment, the invention provides the therapeutic population of TILs or the composition of TILs described in any of the previous paragraphs, as applicable above, modified in such a way that the cancer is glioblastoma (including GBM).
[001457] [001457] In another embodiment, the invention provides the therapeutic population of TILs or the composition of TILs described in any of the previous paragraphs, as applicable above, modified in such a way that the cancer is gastrointestinal cancer.
[001458] [001458] In another embodiment, the invention provides the therapeutic population of TILs or the composition of TILs described in any of the previous paragraphs, as applicable above, modified in such a way that the cancer is a hypermutated cancer.
[001459] [001459] In another embodiment, the invention provides the therapeutic population of TILs or the composition of TILs described in any of the previous paragraphs, as applicable above, modified in such a way that the cancer is a pediatric hypermutated cancer.
[001460] [001460] In another embodiment, the invention provides the use of the therapeutic population of TILs described in any of the preceding paragraphs, as applicable above, in a method of treating cancer in an individual, which comprises administering to the individual a therapeutically effective dose therapeutic population of TILs.
[001461] [001461] In another embodiment, the invention provides the use of the composition of TILs described in any of the preceding paragraphs, as applicable above, in a method of treating cancer in an individual, which comprises administering to the individual a therapeutically effective dose of composition of TILs.
[001462] [001462] In another embodiment, the invention provides the use of the therapeutic population of TILs described in any of the previous paragraphs, as applicable above, or of the composition of TILs described in any of the previous paragraphs, as applicable above, in a method of treatment of cancer in an individual, which comprises administering to the individual a non-myeloablative lymphodepletion regimen and then administering to the individual a therapeutically effective dose of the therapeutic population of TILs described in any of the previous paragraphs, as applicable above, or to therapeutically effective dose of the TILs composition described in any of the preceding paragraphs, as applicable above.
[001463] [001463] In another embodiment, the invention provides the use of the therapeutic population of TILs or the composition of TILs described in any of the previous paragraphs, as applicable above, modified in such a way that the scheme of non-myeloablative lymph node depletion comprises the stages of administration cyclophosphamide at a dose of 60 mg / m2 / day for two days, followed by the administration of fludarabine at a dose of 25 mg / m2 / day for five days.
[001464] [001464] In another embodiment, the invention provides for the use of the therapeutic population of TILs described in any of the preceding paragraphs, as applicable above, or the use of the composition of TILs described in any of the preceding paragraphs, as applicable above, modified to further understand the step of treating a patient with a high dose IL-2 regimen, starting the day after the administration of TIL cells to the patient.
[001465] [001465] In another embodiment, the invention provides for the use of the therapeutic population of TILs or the composition of TILs described in any of the previous paragraphs, as applicable above, modified in such a way that the high-dose schedule of IL-2 comprises 600,000 or 720,000 IU / kg, administered by intravenous bolus infusion over 15 minutes every eight hours while tolerated.
[001466] [001466] In another embodiment, the invention provides for the use of the therapeutic population of TILs or the composition of TILs described in any of the previous paragraphs, as applicable above, modified in such a way that the cancer is a solid tumor.
[001467] [001467] In another embodiment, the invention provides for the use of the therapeutic population of TILs or the composition of TILs described in any of the previous paragraphs, as applicable above, modified in such a way that the cancer is melanoma, ovarian cancer, cervical cancer , non-small cell lung cancer (NSCLC), lung cancer, bladder cancer, cancer
[001468] [001468] In another embodiment, the invention provides for the use of the therapeutic population of TILs or the composition of TILs described in any of the previous paragraphs, as applicable above, modified in such a way that the cancer is melanoma, CCECP, cervical cancer, CPNPC , glioblastoma (including GBM) and gastrointestinal cancer.
[001469] [001469] In another embodiment, the invention provides for the use of the therapeutic population of TILs or the composition of TILs described in any of the previous paragraphs, as applicable above, modified in such a way that the cancer is melanoma.
[001470] [001470] In another embodiment, the invention provides for the use of the therapeutic population of TILs or the composition of TILs described in any of the previous paragraphs, as applicable above, modified in such a way that the cancer is CCECP.
[001471] [001471] In another embodiment, the invention provides for the use of the therapeutic population of TILs or the composition of TILs described in any of the previous paragraphs, as applicable above, modified in such a way that the cancer is cervical cancer.
[001472] [001472] In another embodiment, the invention provides for the use of the therapeutic population of TILs or the composition of TILs described in any of the previous paragraphs, as applicable above, modified in such a way that the cancer is NSCLC.
[001473] [001473] In another embodiment, the invention provides for the use of the therapeutic population of TILs or the composition of TILs described in any of the previous paragraphs, as applicable above, modified in such a way that the cancer is glioblastoma (including GBM).
[001474] [001474] In another embodiment, the invention provides for the use of the therapeutic population of TILs or the composition of TILs described in any of the previous paragraphs, as applicable above, modified in such a way that the cancer is gastrointestinal cancer.
[001475] [001475] In another embodiment, the invention provides the use of the therapeutic population of TILs or the composition of TILs described in any of the previous paragraphs, as applicable above, modified in such a way that the cancer is a hypermutated cancer.
[001476] [001476] In another embodiment, the invention provides for the use of the therapeutic population of TILs or the composition of TILs described in any of the previous paragraphs, as applicable above, modified in such a way that the cancer is a pediatric hypermutated cancer.
[001477] [001477] In some embodiments, the present invention provides a method of treating cancer with a population of tumor infiltrating lymphocytes (TILs), which comprises the steps of (a) obtaining a first population of TILs from a fragment or sample a patient's tumor; (b) perform an initial expansion of the first population of TILs in a first cell culture medium in order to obtain a second population of TILs, in which the second population of TILs has a number at least 5 times greater than the first population of TILs, and wherein the first cell culture medium comprises IL-2; (c) perform a rapid expansion of the second population of TILs using a population of artificial myeloid antigen presenting cells (myeloid aAPCs) in a second cell culture medium in order to obtain a third population of TILs, in which the third population of TILs has a number at least 50 times greater than the second population of TILs after 7 days from the start of rapid expansion; and wherein the second cell culture medium comprises IL-2 and OKT-3; (d) administering a therapeutically effective part of the third
[001478] [001478] In some embodiments, the present invention provides a method of treating an individual with cancer, which comprises administering expanded tumor infiltrating lymphocytes (TILs) comprising: (i) obtaining a first population of TILs from a fine needle aspirate (FNA) or a small biopsy obtained from a tumor in a patient; (ii) perform a first expansion, cultivating the first TIL population in a cell culture medium comprising IL-2, to produce a second TIL population, in which the cell culture medium is supplemented with OKT-3 on day 3, where the first expansion is performed between approximately 3 days and 19 days in order to obtain the second population of TILs, where the second population of TILs comprises at least 5 x 107 TILs between approximately 10 days and 19 days when the first population of TILs are from a small biopsy; (iii) perform a second expansion, supplementing the cell culture medium of the second population of TILs with IL-2, OKT-3 and additional antigen presenting cells (APCs), to produce a third population of TILs, in which the third population of TILs has a number at least 25 times greater than the second population of TILs, and in which the second expansion is carried out between approximately 11 days and 14 days in order to obtain the third population of TILs, in which the third population of TILs it is a therapeutic population of TILs; and (iv) administering a therapeutically effective dose of the third population of TILs to the patient. In some embodiments, IL-2 is present at an initial concentration of approximately 3000 IU / ml and OKT-3 antibody is present at an initial concentration of approximately 30 ng / ml in the second cell culture medium. In some embodiments, the first expansion is performed using a permeable container
[001479] [001479] 1. A method for the expansion of tumor infiltrating lymphocytes (TILs) in a therapeutic population of TILs comprising:
[001480] [001480] 2. Method according to claim 1, wherein the method further comprises: (iv) performing an additional second expansion by supplementing the cell culture medium of the third TIL population with additional IL-2, additional OKT-3 and Additional APCs, where the second additional expansion is performed for at least 14 days in order to obtain a larger therapeutic population of TILs than that obtained in step (iii), where the larger therapeutic population of TILs comprises an increased subpopulation of cells Effector T and / or central memory T cells in relation to the third population of TILs.
[001481] [001481] 3. Method according to claim 2, wherein after step (iii), the cells are removed from the cell culture and cryopreserved in a storage medium before performing step (iv).
[001482] [001482] 4. Method according to claim 3, wherein the cells are thawed before performing step (iv).
[001483] [001483] 5. Method according to any of the preceding claims, wherein step (iv) is repeated one to four times in order to obtain sufficient TILs in the therapeutic population of TILs for a therapeutically effective dose of TILs.
[001484] [001484] 6. Method according to any of the preceding claims, wherein steps (i) to (iii) or (iv) are carried out within a period between approximately 21 days and 33 days.
[001485] [001485] 7. Method according to any one of the preceding claims, wherein steps (i) to (iii) or (iv) are performed within a period between approximately 21 days and 30 days.
[001486] [001486] 8. Method according to any of the preceding claims, wherein steps (i) to (iii) or (iv) are carried out within a period between approximately 21 days and 28 days.
[001487] [001487] 9. Method according to any of the preceding claims, wherein steps (i) to (iii) or (iv) are performed within approximately 28 days.
[001488] [001488] 10. Method according to any one of the preceding claims, wherein the cells of steps (iii) or (iv) express CD4, CD8 and TCR α β at levels similar to those of recently collected cells.
[001489] [001489] 11. Method according to claim 1, wherein the APCs are peripheral blood mononuclear cells (PBMCs).
[001490] [001490] 12. Method according to claim 11, wherein the PBMCs are added to the cell culture on any of days 3 to 19 in step (ii) and / or any of days 11 to 14 in step (iii) .
[001491] [001491] 13. Method according to claims 2 to 12, wherein the effector T cells and / or central memory T cells in the therapeutic population of TILs in step (iv) exhibit one or more characteristics selected from the group consisting of in CD27 expression, CD28 expression, longer telomeres, increased CD57 expression and decreased CD56 expression, in relation to effector T cells and / or central memory T cells in the third cell population.
[001492] [001492] 14. The method of claim 13, wherein effector T cells and / or central memory T cells exhibit increased CD57 expression and decreased CD56 expression.
[001493] [001493] 15. Method according to any of the preceding claims, wherein the APCs are artificial APCs (aAPCs) or are autologous APCs.
[001494] [001494] 16. Method according to any of the preceding claims, wherein the therapeutic population of TILs is infused into a patient.
[001495] [001495] 17. Method according to claim 1, wherein the first expansion in step (ii) is carried out by further supplementing the cell culture medium of the second population of TILs with OKT-3, IL-15, agonist antibody of OX40 and / or 4-1BB agonist antibody.
[001496] [001496] 18. Method according to claim 1, wherein the second expansion in step (iii) is performed by further supplementing the cell culture medium of the second TIL population with IL-15, OX40 agonist antibody and / or 4-1BB agonist antibody.
[001497] [001497] 19. Method according to claim 2, wherein the second additional expansion in step (iv) is carried out by further supplementing the cell culture medium of the third TIL population with IL-15, OX40 agonist antibody and / or 4-1BB agonist antibody.
[001498] [001498] 20. Method according to claim 1, wherein the FNA
[001499] [001499] 21. Method according to claim 1, in which the thick needle biopsy is obtained from a tumor selected from the group consisting of pancreatic, melanoma, mammary and ovarian.
[001500] [001500] 22. Method according to claim 1, in which the FNA is obtained from a tumor selected from the group consisting of lung, melanoma, head and neck, cervical, ovarian, pancreatic, glioblastoma, colorectal and sarcoma.
[001501] [001501] 23. The method of claim 22, wherein the lung tumor is a non-small cell lung carcinoma (CPNP) and, optionally, in which the patient has previously undergone surgical treatment.
[001502] [001502] 24. The method of claim 1, wherein the TILs in step (i) are obtained from an FNA.
[001503] [001503] 25. The method of claim 1, wherein the FNA is obtained using a 25-18 gauge needle.
[001504] [001504] 26. The method of claim 1, wherein the TILs in step (i) are obtained from a thick needle biopsy.
[001505] [001505] 27. The method of claim 1, wherein the thick needle biopsy is obtained using a 16-11 gauge needle.
[001506] [001506] 28. The method of claim 1, wherein step (iii) is repeated one to four times in order to obtain sufficient TILs in the therapeutic population of TILs for a therapeutically effective dose of TILs.
[001507] [001507] 29. The method of claim 28, wherein the number of TILs sufficient for a therapeutically effective dose is between approximately 2.3x1010 and 13.7x1010.
[001508] [001508] 30. A method for the expansion of tumor infiltrating lymphocytes (TILs) in a therapeutic population of TILs comprising:
[001509] [001509] 31. The method of claim 30, wherein the method further comprises: (iii) performing an additional second expansion of the third population of TILs by supplementing the cell culture medium of the third population of TILs with additional IL-2, OKT- Additional 3 and additional APCs, where the second additional expansion is performed for at least 14 days to obtain a larger therapeutic population of TILs than that obtained in step (ii), where the larger therapeutic population of TILs exhibits an increased subpopulation of effector T cells and / or central memory T cells in relation to the third population of TILs.
[001510] [001510] 32. The method of claim 31, wherein the
[001511] [001511] 33. The method of claim 32, wherein the cells are thawed before step (iii).
[001512] [001512] 34. The method of any one of claims 30 to 33, wherein the APCs are artificial APCs (aAPCs) or are autologous APCs.
[001513] [001513] 35. The method of any one of claims 30 to 34, wherein the therapeutic population of TILs is infused into a patient.
[001514] [001514] 36. The method of claim 30, wherein the first expansion in step (i) is carried out by further supplementing the cell culture medium of the second TIL population with OKT-3, IL-15, agonist antibody from OX40 and / or 4-1BB agonist antibody.
[001515] [001515] 37. The method of claim 30, wherein the second expansion in step (ii) is carried out by further supplementing the cell culture medium of the second TIL population with IL-15, OX40 agonist antibody and / or 4-1BB agonist antibody.
[001516] [001516] 38. The method of claim 30, wherein the second further expansion in step (iii) is performed by further supplementing the cell culture medium of the third TIL population with IL-15, OX40 agonist antibody and / or 4-1BB agonist antibody.
[001517] [001517] 39. The method of claim 30, wherein the FNA in step (i) comprises at least 400,000 TILs.
[001518] [001518] 40. Method according to claim 30, in which the thick needle biopsy is obtained from a tumor selected from the group consisting of pancreatic, melanoma, mammary and ovarian.
[001519] [001519] 41. The method of claim 30, wherein the FNA is obtained from a tumor selected from the group consisting of lung, melanoma, head and neck, cervical, ovarian, pancreatic,
[001520] [001520] 42. The method of claim 41, wherein the lung tumor is a non-small cell lung carcinoma (CPNP) and, optionally, in which the patient has previously undergone surgical treatment.
[001521] [001521] 43. The method of claim 30, wherein the TILs in step (i) are obtained from an FNA.
[001522] [001522] 44. The method of claim 30, wherein the FNA is obtained using a 25-18 gauge needle.
[001523] [001523] 45. The method of claim 30, wherein the TILs in step (i) are obtained from a thick needle biopsy.
[001524] [001524] 46. The method of claim 30, wherein the thick needle biopsy is obtained using a 16-11 gauge needle.
[001525] [001525] 47. The method of claim 30, wherein step (ii) is repeated one to four times in order to obtain sufficient TILs in the therapeutic population of TILs for a therapeutically effective dose of TILs.
[001526] [001526] 48. The method of claim 47, and that the number of TILs sufficient for a therapeutically effective dose is between approximately 2.3x1010 and 13.7x1010.
[001527] [001527] 49. The method of any one of claims 30 to 48, wherein the effector T cells and / or central memory T cells exhibit one or more characteristics selected from the group consisting of CD27 expression, CD28 expression , longer telomeres, increased CD57 expression and decreased CD56 expression, in relation to effector T cells and / or central memory T cells in the third cell population.
[001528] [001528] 50. The method of claim 49, wherein the effector T cells and / or central memory T cells exhibit increased
[001529] [001529] 51. A method for the treatment of an individual with cancer, which comprises administering expanded tumor infiltrating lymphocytes (TILs) comprising: (i) obtaining a first population of TILs from a fine needle aspirate (FNA) or a thick needle biopsy obtained from a tumor in a patient; (ii) perform a first expansion, cultivating the first TIL population in a cell culture medium comprising IL-2, to produce a second TIL population, in which the cell culture medium is supplemented with OKT-3 on day 3, where the first expansion is performed between approximately 3 days and 19 days in order to obtain the second population of TILs, where the second population of TILs comprises at least 5 x 107 TILs between approximately 10 days and 19 days when the first population of TILs are from a thick needle biopsy; (iii) perform a second expansion, supplementing the cell culture medium of the second population of TILs with IL-2, OKT-3 and additional antigen presenting cells (APCs), to produce a third population of TILs, in which the third population of TILs has a number at least 25 times greater than the second population of TILs, and in which the second expansion is carried out between approximately 11 days and 14 days in order to obtain the third population of TILs, in which the third population of TILs it is a therapeutic population of TILs; and (iv) administering a therapeutically effective dose of the third population of TILs to the patient.
[001530] [001530] 52. The method of claim 51, wherein the method further comprises, prior to step (iv), the step in which a second additional expansion is performed by supplementing the cell culture medium of the third population of TILs with IL-2 additional, additional OKT-3 and additional APCs,
[001531] [001531] 53. The method of claim 51, wherein after step (ii) the cells are removed from the cell culture medium and cryopreserved in a storage medium prior to the second further expansion of claim 52.
[001532] [001532] 54. The method of claim 51, wherein the cells are thawed prior to the second further expansion of claim 52.
[001533] [001533] 55. The method of claim 51, wherein step (iii) is repeated one to four times in order to obtain sufficient TILs in the therapeutic population of TILs for a therapeutically effective dose of TILs.
[001534] [001534] 56. The method of claims 51 to 55, wherein the APCs are artificial APCs (aAPCs) or are autologous APCs.
[001535] [001535] 57. The method of claim 51, wherein the first expansion in step (ii) is carried out by further supplementing the cell culture medium of the second population of TILs with OKT-3, IL-15, agonist antibody from OX40 and / or 4-1BB agonist antibody.
[001536] [001536] 58. The method of claim 51, wherein the second expansion in step (iii) is performed by further supplementing the cell culture medium of the second TIL population with IL-15, OX40 agonist antibody and / or 4-1BB agonist antibody.
[001537] [001537] 59. Method according to claim 52, wherein the second additional expansion is performed by further supplementing the cell culture medium of the third TIL population with IL-15, agonist antibody
[001538] [001538] 60. The method of claim 51, wherein the FNA in step (i) comprises at least 400,000 TILs.
[001539] [001539] 61. The method of claim 51, wherein the FNA is obtained from a tumor selected from the group consisting of lung, melanoma, head and neck, cervical, ovarian, pancreatic, glioblastoma, colorectal and sarcoma.
[001540] [001540] 62. The method of claim 61, wherein the lung tumor is a non-small cell lung carcinoma (CPNP) and, optionally, in which the individual has previously undergone surgical treatment.
[001541] [001541] 63. The method of claim 51, wherein the TILs in step (i) are obtained from an FNA.
[001542] [001542] 64. The method of claim 51, wherein the FNA is obtained using a 25-18 gauge needle.
[001543] [001543] 65. The method of claim 51, wherein the TILs in step (i) are obtained from a thick needle biopsy.
[001544] [001544] 66. The method of claim 51, wherein the thick needle biopsy is obtained using a 16-11 gauge needle.
[001545] [001545] 67. The method of claim 51, wherein step (iii) is repeated one to four times in order to obtain sufficient TILs in the therapeutic population of TILs for a therapeutically effective dose of TILs.
[001546] [001546] 68. The method of claim 67, wherein the number of TILs sufficient for a therapeutically effective dose is between approximately 2.3x1010 and 13.7x1010.
[001547] [001547] 69. The method of any one of claims 51 to 68, wherein the effector T cells and / or central memory T cells exhibit one or more characteristics selected from the group consisting of
[001548] [001548] 70. The method of claim 69, wherein the effector T cells and / or central memory T cells exhibit increased CD57 expression and decreased CD56 expression.
[001549] [001549] 71. Method according to any one of claims 51 to 70, in which the cancer is selected from the group consisting of melanoma, cervical cancer, head and neck cancer, glioblastoma, ovarian cancer, sarcoma, cancer of pancreas, bladder cancer, breast cancer, triple negative breast cancer and non-small cell lung carcinoma.
[001550] [001550] 72. A method for the treatment of an individual with cancer, which comprises administering expanded tumor infiltrating lymphocytes (TILs) comprising: (i) performing a first expansion, cultivating the first population of TILs from an aspirate by fine needle (FNA) or a thick needle biopsy of a tumor in a patient in a cell culture medium comprising IL-2 in order to obtain a second population of TILs, in which the cell culture medium is supplemented with OKT-3 on day 3, when the first expansion is performed between approximately 3 days and 19 days in order to obtain the second population of TILs, where the second population of TILs comprises at least 5 x 107 TILs between approximately 10 days and 19 days when first population of TILs is from a thick needle biopsy; (ii) perform a second expansion, supplementing the cell culture medium of the second population of TILs with IL-2, OKT-3 and additional antigen presenting cells (APCs) to obtain a third
[001551] [001551] 73. The method of claim 72, wherein the method further comprises, prior to step (iii), a step in which an additional second expansion is performed by supplementing the cell culture medium of the third population of TILs with IL-2 additional, additional OKT-3 and additional APCs, where the second additional expansion is performed for at least 14 days to obtain a larger therapeutic population of TILs than that obtained in step (ii), where the larger therapeutic population of TILs comprises an increased subpopulation of effector T cells and / or central memory T cells compared to the third population of TILs.
[001552] [001552] 74. The method of claim 72, wherein the cells from the cell culture medium in step (ii) are removed and cryopreserved in a storage medium prior to the second further expansion in claim 73.
[001553] [001553] 75. The method of claim 74, wherein the cells are thawed before the second additional expansion in claim 73.
[001554] [001554] 76. The method of claim 74, wherein the cells are thawed before step (iii).
[001555] [001555] 77. The method of any one of claims 72 to 76, wherein the APCs are artificial APCs (aAPCs) or are autologous APCs.
[001556] [001556] 78. Method according to any of the claims
[001557] [001557] 79. The method of any one of claims 72 to 78, wherein the therapeutic population of TILs is infused into a patient.
[001558] [001558] 80. The method of claim 72, wherein the first expansion in step (i) is carried out by further supplementing the cell culture medium of the second TIL population with OKT-3, IL-15, agonist antibody from OX40 and / or 4-1BB agonist antibody.
[001559] [001559] 81. The method of claim 72, wherein the second expansion in step (iii) is performed by further supplementing the cell culture medium of the second TIL population with IL-15, OX40 agonist antibody and / or 4-1BB agonist antibody.
[001560] [001560] 82. The method of claim 81, wherein the second additional expansion is performed by further supplementing the cell culture medium of the third TIL population with IL-15, OX40 agonist antibody and / or 4 agonist antibody. -1BB.
[001561] [001561] 83. The method of claim 72, wherein the FNA in step (i) comprises at least 400,000 TILs.
[001562] [001562] 84. The method of claim 72, wherein the thick needle biopsy is obtained from a tumor selected from the group consisting of pancreatic, melanoma, mammary and ovarian.
[001563] [001563] 85. The method of claim 72, wherein the FNA is obtained from a tumor selected from the group consisting of lung, melanoma, head and neck, cervical, ovarian, pancreatic, glioblastoma, colorectal and sarcoma.
[001564] [001564] 86. The method of claim 85, wherein the lung tumor is a non-small cell lung carcinoma (CPNP) and, optionally, in which the individual has previously undergone surgical treatment.
[001565] [001565] 87. The method of claim 72, wherein the TILs in step (i) are obtained from an FNA.
[001566] [001566] 88. The method of claim 72, wherein the FNA is obtained using a 25-18 gauge needle.
[001567] [001567] 89. The method of claim 72, wherein the TILs in step (i) are obtained from a thick needle biopsy.
[001568] [001568] 90. The method of claim 72, wherein the thick needle biopsy is obtained using a 16-11 gauge needle.
[001569] [001569] 91. The method of claim 72, wherein step (ii) is repeated one to four times in order to obtain sufficient TILs in the therapeutic population of TILs for a therapeutically effective dose of TILs.
[001570] [001570] 92. The method of claim 91, wherein the number of TILs sufficient for a therapeutically effective dose is between approximately 2.3x1010 and 13.7x1010.
[001571] [001571] 93. The method of any one of claims 72 to 92, wherein the effector T cells and / or central memory T cells exhibit one or more characteristics selected from the group consisting of CD27 expression, CD28 expression , longer telomeres, increased CD57 expression and decreased CD56 expression, in relation to effector T cells and / or central memory T cells in the third cell population.
[001572] [001572] 94. The method of claim 93, wherein the effector T cells and / or central memory T cells exhibit increased CD57 expression and decreased CD56 expression.
[001573] [001573] 95. A method for the expansion of tumor infiltrating lymphocytes (TILs) in a therapeutic population of TILs comprising: (a) obtaining a first population of TILs from a fine needle aspirate (FNA) or a biopsy by thick needle obtained from
[001574] [001574] 96. The method of claim 95, further comprising the cryopreservation step of the infusion bag comprising the population of TILs collected in step (f) using a cryopreservation process.
[001575] [001575] 97. The method of claim 96, wherein the cryopreservation process is performed using a 1: 1 ratio of collected TILs / CS10 medium.
[001576] [001576] 98. The method of claim 96, wherein the antigen presenting cells are peripheral blood mononuclear cells (PBMCs).
[001577] [001577] 99. The method of claim 98, wherein the PBMCs are irradiated and allogeneic.
[001578] [001578] 100. The method of claim 98, wherein the PBMCs are added to the cell culture on any of days 3 to 19 in step (c) and / or any of days 11 to 14 in step (d) .
[001579] [001579] 101. The method of claim 95, wherein the antigen presenting cells are artificial antigen presenting cells (aAPCs) or are autologous APCs.
[001580] [001580] 102. The method of any one of claims 95 to 101, wherein the therapeutic population of TILs is infused into a patient.
[001581] [001581] 103. The method of claim 95, wherein the first expansion in step (c) is performed by further supplementing the cell culture medium of the second TIL population with OKT-3, IL-15, agonist antibody from OX40 and / or 4-1BB agonist antibody.
[001582] [001582] 104. The method of claim 95, wherein the second expansion in step (d) is performed by further supplementing the cell culture medium of the second TIL population with IL-15, OX40 agonist antibody and / or 4-1BB agonist antibody.
[001583] [001583] 105. The method of claim 95, wherein the FNA in step (a) comprises at least 400,000 TILs.
[001584] [001584] 106. The method of claim 95, wherein the thick needle biopsy is obtained from a tumor selected from the group consisting of pancreatic, melanoma, breast and ovarian.
[001585] [001585] 107. The method of claim 95, wherein the FNA is obtained from a tumor selected from the group consisting of lung, melanoma, head and neck, cervical, ovarian, pancreatic, glioblastoma, colorectal and sarcoma.
[001586] [001586] 108. The method of claim 107, wherein the lung tumor is a non-small cell lung carcinoma (CPNP) and, optionally, in which the individual has previously undergone surgical treatment.
[001587] [001587] 109. The method of claim 95, wherein the TILs in step (a) are obtained from an FNA.
[001588] [001588] 110. The method of claim 95, wherein the FNA is obtained using a 25-18 gauge needle.
[001589] [001589] 111. The method of claim 95, wherein the TILs in step (a) are obtained from a thick needle biopsy.
[001590] [001590] 112. The method of claim 95, wherein the thick needle biopsy is obtained using a 16-11 gauge needle.
[001591] [001591] 113. Method according to claim 95, in which the collection in step (e) is performed using a LOVO system for processing the cells.
[001592] [001592] 114. The method of claim 95, wherein the medium
[001593] [001593] 115. The method of claim 95, wherein the infusion bag in step (f) is a HyperThermosol infusion bag.
[001594] [001594] 116. The method of claim 95, wherein steps (a) to (f) are carried out within a period between approximately 21 days and 33 days.
[001595] [001595] 117. The method of claim 95, wherein steps (a) to (f) are carried out within a period between approximately 21 days and 30 days.
[001596] [001596] 118. The method of claim 95, wherein steps (a) to (f) are carried out within a period between approximately 21 days and 28 days.
[001597] [001597] 119. The method of claim 95, wherein steps (a) to (f) are carried out in 22 days or less.
[001598] [001598] 120. The method of claim 96, wherein steps (a) to (f) and cryopreservation are carried out in 22 days or less.
[001599] [001599] 121. The method of any one of claims 95 to 120, wherein the therapeutic population of TILs harvested in step (e) comprises sufficient TILs for a therapeutically effective dose of the TILs.
[001600] [001600] 122. The method of claim 121, wherein the number of TILs sufficient for a therapeutically effective dose is between approximately 2.3x1010 and 13.7x1010.
[001601] [001601] 123. Method according to any one of claims 95 to 122, wherein steps (b) to (e) are carried out in a single container, wherein carrying out steps (b) to (e) in a single container results in an increase in TIL yield per resected tumor, compared to performing steps (b) to (e) in more than one container.
[001602] [001602] 124. The method of any one of claims 95 to 123, wherein the antigen presenting cells are added to the TILs during the second period in step (d) without reopening the system.
[001603] [001603] 125. The method of any one of claims 95 to 124, wherein the effector T cells and / or central memory T cells in the therapeutic population of TILs exhibit one or more characteristics selected from the group consisting of expression of CD27 +, CD28 + expression, longer telomeres, increased CD57 expression and decreased CD56 expression in relation to effector T cells, and / or central memory T cells obtained from the second cell population.
[001604] [001604] 126. The method of any one of claims 95 to 125, wherein effector T cells and / or central memory T cells obtained from the third population of TILs exhibit increased expression of CD57 and decreased expression of CD56 in relation to effector T cells and / or central memory T cells obtained from the second cell population.
[001605] [001605] 127. Method according to any of claims 95 to 126, wherein the risk of microbial contamination is reduced compared to an open system.
[001606] [001606] 128. The method of any one of claims 95 to 128, wherein the TILs of step (g) are infused into a patient.
[001607] [001607] 129. A method for the treatment of an individual with cancer, wherein the method comprises administering expanded tumor infiltrating lymphocytes (TILs) comprising: (a) obtaining a first population of TILs from a fine needle aspirate ( FNA) or a thick needle biopsy of a resected tumor from an individual; (b) adding the first population to a closed system; (c) perform a first expansion, cultivating the first population of TILs in a cell culture medium comprising IL-2, to
[001608] [001608] 130. The method of claim 129, wherein the therapeutic population of TILs collected in step (e) comprises sufficient TILs to deliver a therapeutically effective dose of TILs in step (h).
[001609] [001609] 131. The method of claim 129 or 130, wherein the APCs are artificial APCs (aAPCs) or are autologous APCs.
[001610] [001610] 132. The method of any of claims 129 to 131, wherein the therapeutic population of TILs is infused into a patient.
[001611] [001611] 133. The method of claim 129, wherein the first expansion in step (c) is carried out by further supplementing the cell culture medium of the second TIL population with OKT-3, IL-15, agonist antibody from OX40 and / or 4-1BB agonist antibody.
[001612] [001612] 134. The method of claim 129, wherein the second expansion in step (d) is performed by further supplementing the cell culture medium of the second TIL population with OKT-3, IL-15, agonist antibody from OX40 and / or 4-1BB agonist antibody.
[001613] [001613] 135. The method of claim 129, wherein the FNA in step (a) comprises at least 400,000 TILs.
[001614] [001614] 136. The method of claim 129, wherein the thick needle biopsy is obtained from a tumor selected from the group consisting of pancreatic, melanoma, breast and ovarian.
[001615] [001615] 137. The method of claim 129, wherein the FNA is obtained from a tumor selected from the group consisting of
[001616] [001616] 138. The method of claim 137, wherein the lung tumor is a non-small cell lung carcinoma (CPNP) and, optionally, in which the individual has previously undergone surgical treatment.
[001617] [001617] 139. The method of claim 129, wherein the TILs in step (i) are obtained from an FNA.
[001618] [001618] 140. The method of claim 139, wherein the FNA is obtained using a 25-18 gauge needle.
[001619] [001619] 141. The method of claim 129, wherein the TILs in step (i) are obtained from a thick needle biopsy.
[001620] [001620] 142. The method of claim 141, wherein the thick needle biopsy is obtained using a 16-11 gauge needle.
[001621] [001621] 143. The method of claim 129, wherein the number of TILs sufficient to deliver a therapeutically effective dose in step (h) is between approximately 2.3x1010 and 13.7x1010.
[001622] [001622] 144. The method of claim 129, wherein the antigen presenting cells (APCs) are PBMCs.
[001623] [001623] 145. The method of claim 144, wherein the PBMCs are added to the cell culture on any of days 3 to 19 in step (c) and / or any of days 9 to 14 in step (d) .
[001624] [001624] 146. The method of any one of claims 129 to 145, wherein before a therapeutically effective dose of TIL cells in step (h) is administered, a non-myeloablative lymph-depletion scheme was administered to the patient.
[001625] [001625] 147. The method of claim 146, wherein the non-myeloablative lymphodepletion scheme comprises the steps of administering cyclophosphamide at a dose of 60 mg / m2 / day for two days,
[001626] [001626] 148. The method of any one of claims 129 to 147, further comprising the step of treating the patient with a high dose IL-2 schedule, starting the day after the administration of TIL cells to the patient in step ( H).
[001627] [001627] 149. The method of claim 148, wherein the high-dose IL-2 schedule comprises 600,000 or 720,000 IU / kg, administered by intravenous bolus infusion over 15 minutes every eight hours while tolerated.
[001628] [001628] 150. The method of any one of claims 129 to 149, wherein the effector T cells and / or central memory T cells in the therapeutic population of TILs exhibit one or more characteristics selected from the group consisting of expression of CD27 +, CD28 + expression, longer telomeres, increased CD57 expression and decreased CD56 expression in relation to effector T cells, and / or central memory T cells obtained from the second cell population.
[001629] [001629] 151. The method of any one of claims 129 to 150, wherein effector T cells and / or central memory T cells in the therapeutic population of TILs exhibit increased CD57 expression and decreased CD56 expression relative to effector T cells and / or central memory T cells obtained from the second cell population.
[001630] [001630] 152. A method for the expansion of tumor infiltrating lymphocytes (TILs) in a therapeutic population of TILs comprising: (a) obtaining a first population of TILs from a fine needle aspirate (FNA), the small biopsy , or a thick needle biopsy of a tumor in a patient; (b) perform a first preparation expansion, cultivating the first population of TILs in a cell culture medium comprising
[001631] [001631] The modalities covered here are now described with reference to the following examples. These examples are provided for purposes of illustration only, and the description contained therein should not be construed in any way as a limitation to these examples, but should be interpreted to cover any and all variations that become evident as a result of the teachings now provided. Example 1: Preparation of means for the pre-REP and REP processes
[001632] [001632] This example describes the procedure for preparing tissue culture media for use in protocols involving the culture of tumor infiltrating lymphocytes (TIL) derived from various types of tumor including, but not limited to, metastatic melanoma, squamous cell carcinoma of head and neck (CCECP), ovarian carcinoma, triple negative breast carcinoma and pulmonary adenocarcinoma. These means can be used to prepare any of the TILs described in the present application and in the Examples.
[001633] [001633] The following reagents were removed from cold storage and heated in a water bath at 37 ºC: (RPMI1640, human AB serum, L-glutamine 200mM L-). Prepared CM1 medium according to Table 32 below, adding each of the ingredients to the upper section of a 0.2 µm unit filter appropriate to the volume to be filtered. Store at 4 ºC. Table 32: Preparation of CM1 Ingredient Final concentration Final volume 500 ml Final volume IL RPMI1640 NA 450 mL 900 mL Human AB serum, 50 mL 100 mL heat inactivated 10% L-glutamine 200 mM 2 mM 5 mL 10 mL BME 55 mM 55 µM 0.5 mL 1 mL Gentamicin sulfate 50 50 µg / mL 0.5 mL 1 mL mg / mL
[001634] [001634] On the day of use, required amount of CM1 preheated in a water bath at 37 ºC and added IL-2 6000 IU / mL.
[001635] [001635] Additional supplementation - when necessary according to Table 33. Table 33: Additional supplementation of CM1, if necessary Supplement Stock concentration Dilution Final concentration GlutaMAXTM 200mM 1: 100 2mM Penicillin 10,000 U / mL Penicillin 100 U / mL Penicillin / streptomycin Streptomycin 1: 100 Streptomycin
[001636] [001636] Removed prepared CM1 from the refrigerator or prepare fresh CM1 according to Section 7.3 above. AIM-V® was removed from the refrigerator and the required amount of CM2 was prepared, mixing CM1 prepared with an equal volume of AIM-V® in a sterile medium bottle. IL-2 3000 IU / mL was added to the CM2 medium on the day of use. Sufficient amount of CM2 was prepared with IL-2 3000 IU / mL on the day of use. Once the CM2 medium bottle was identified with your name, the initials of the preparer, the date it was filtered / prepared, the expiration date of two weeks and store at 4 ºC until necessary for tissue culture.
[001637] [001637] Prepared CM3 on the day that required for use. CM3 was equal to the AIM-V® medium, supplemented with IL-2 3000 IU / mL on the day of use. A sufficient amount of CM3 was prepared for the experimental needs, adding IL-2 stock solution directly to the AIM-V vial or bag. Mixed well by gentle stirring. Vial identification with “IL-2 3000 IU / mL” immediately after adding to AIM-V. In case of excess, CM3 was stored in bottles at 4 ºC identified with the name of the medium, the initials of the preparer, the date on which the medium was prepared and its expiration date (7 days after preparation). Medium supplemented with IL-2 discarded after 7 days storage at 4 ºC. Preparation of CM4
[001638] [001638] CM4 was equal to CM3, with the additional supplement of GlutaMAX 2 mMTM (final concentration). For each 1 L of CM3, 10 mL of 200 mM GlutaMAXTM is added. A sufficient amount of CM4 was prepared for the experimental needs, adding IL-2 stock solution and GlutaMAXTM stock solution directly to the AIM-V flask or bag. Mixed well by gentle agitation. Weak identification made with “3000 IL / ml IL-2 and GlutaMAX” immediately after adding to AIM-V. In case of excess, CM4 was stored in bottles at 4 ºC, identified with the middle name, “GlutaMAX”, and its expiration date (7 days after preparation). Medium supplemented with IL-2 discarded after 7 days storage at 4 ºC. Example 2: Use of the IL-2, IL-15 and IL-21 cytokine cocktail
[001639] [001639] This example describes the use of the cytokines IL-2, IL-15 and IL-21, which serve as additional T cell growth factors, in combination with the TILs process of the Examples.
[001640] [001640] Using the processes described here, TILs of colorectal, melanoma, cervical, triple negative breast, lung and kidney tumors
[001641] [001641] The results showed that the enhanced expansion of TILs (> 20%), in CD4 + and CD8 + cells under the conditions treated with IL-2, IL-15 and IL-21, was observed in multiple histologies in relation to the conditions with IL-2 only. There was a skew towards a predominantly CD8 + population with a skewed repertoire for TCR Vβ in the TILs obtained from cultures treated with IL-2, IL-15 and IL-21 in relation to cultures with IL-2 only. IFNγ and CD107a were elevated in TILs treated with IL-2, IL-15 and IL-21, compared to TILs treated with IL-2 only. Example 3: Qualification of individual lots of peripheral blood mononuclear cells subjected to gamma radiation
[001642] [001642] This example describes an innovative abbreviated procedure for qualifying individual batches of peripheral blood mononuclear cells (PBMCs, also known as MNC) subjected to gamma irradiation for use as allogeneic feeder cells in the exemplary methods described here.
[001643] [001643] Each batch of irradiated feeder MNC was prepared from an individual donor. Each lot or donor had its capacity to expand TIL in the REP in the presence of purified anti-CD3 antibody (clone OKT3) and interleukin-2 (IL-2). In addition, each batch of feeder cells was tested without the addition of TIL to verify that the dose
[001644] [001644] Gamma-irradiated MNC feeder cells, with sustained growth, were required for TIL REP. Membrane receptors on feeder MNCs bind to the anti-CD3 antibody (OKT3 clone) and cross-link with TIL in the REP flask, stimulating the TIL to expand. Lots of feeders were prepared from leukopheresis of whole blood collected from individual donors. The leukopheresis product was subjected to centrifugation with Ficoll-Hypaque, washed, irradiated and cryopreserved under GMP conditions.
[001645] [001645] It is important that patients who received TIL therapy are not infused with viable feeder cells, as this can result in graft versus host disease (GVHD). The growth of the feeder cells is therefore stopped by subjecting the cells to gamma irradiation, resulting in double stranded DNA breaks and loss of cell viability of the MNC cells when cultured. Evaluation criteria and experimental configuration
[001646] [001646] Lots of feeders were evaluated based on two criteria: 1) the ability to expand TIL in coculture> 100 times and 2) the incompetence for replication.
[001647] [001647] Feeder batches were tested in mini-REP format using two strains of primary pre-REP TILs grown in T25 tissue culture flasks in an upright position. The feeder batches were tested against two distinct TIL strains, since each TIL strain is unique in its ability to proliferate in response to activation in a REP. As a control, a batch of MNC irradiated MNC feeder cells that, historically, have demonstrated to meet the above criteria, tested side by side with the batches under test.
[001648] [001648] To ensure that all batches tested in a single experiment received equivalent testing, sufficient stocks of the same pre-REP TIL strains were available to test all conditions and all feeder batches.
[001649] [001649] For each batch of feeder cells tested, there were, in total, six flasks T25: Line 1 of TIL Pre-REP (2 flasks); Lineage 2 of TIL Pre-REP (2 vials); and Control feeder (2 bottles). Flasks containing lines 1 and 2 of TIL evaluated the capacity of the batch of feeders to expand TIL. The control feeder bottles evaluated the incompetence to replicate the batch of feeders. Experimental protocol Day -2/3, Thawing of TIL strains
[001650] [001650] CM2 medium prepared. CM2 heated in a water bath at 37 ºC. 40 ml of CM2 supplemented with IL-2 3000 IU / ml was prepared. Kept warm until use. 20 mL of pre-heated CM2 without IL-2 were placed in each of two 50 mL conical tubes, identified with the names of the TIL strains used. The two designated strains of TIL pre-REP were removed from storage with LN2 and the flasks were transferred to the tissue culture room. Thawed vials by placing them in a closed storage bag with a zipper in a water bath at 37 ºC until a small amount of ice remained.
[001651] [001651] Using a sterile transfer pipette, immediately transfer the contents of the vial in the 20 mL of CM2 into the prepared, marked 50 mL conical tube. QS to 40 mL with CM2 without IL-2 to wash the cells. Centrifuged at 400 x CF for 5 minutes. The supernatant was aspirated and resuspended in 5 ml of warm CM2, supplemented with IL-2 3000 IU / ml.
[001652] [001652] Removed a small aliquot (20 µL) in duplicate for counting cells, using an automatic cell counter. Record the counts. During counting, the 50 mL conical tube was placed with
[001653] [001653] Cultivated in 2 ml / well of a tissue culture plate with 24 wells in as many wells as necessary in a humidified incubator at 37 ºC until Day 0 of the mini-REP. The different strains of TILs grown in tissue culture plates with 24 wells, separated to avoid confusion and potential cross-contamination. Day 0, start of Mini-REP
[001654] [001654] Prepared enough CM2 medium for the number of feeder batches to be tested (eg, to test 4 feeder batches at the same time, prepared 800 mL of CM2 medium). An aliquot of the CM2 prepared above was obtained and supplemented with IL-2 3000 IU / mL for cell culture. (eg, to test 4 batches of feeders at the same time, prepare 500 mL of CM2 medium with IL-2 3000 IU / mL).
[001655] [001655] Work each TIL strain separately to prevent cross contamination, remove the 24 well plate with TIL culture from the incubator and transfer to the BSC.
[001656] [001656] With a sterile transfer pipette or 100-1000 µL Pipettor and tip, remove approximately 1 mL of medium from each TIL well to be used and placed in an unused well of the 24 well tissue culture plate.
[001657] [001657] With a fresh sterile transfer pipette or Pipettor 100-1000 µL and tip, mixed the remaining medium with TIL in the wells to resuspend the cells and then transfer the cell suspension to a 50 mL conical tube, identified with the TIL name and the volume registered.
[001658] [001658] Wash the wells with the reserved medium and transfer that volume to the same 50 mL conical tube. Centrifuged the cells
[001659] [001659] With a serological pipette, mixed the cell suspension a lot and recorded the volume. 200 µL removed for a cell count, using an automatic cell counter. During counting, the 50 ml conical tube with TIL cells was placed in a humidified incubator at 37 ºC, 5% CO2, with the cap loosened to allow gas exchange. Counts recorded.
[001660] [001660] The 50 ml conical tube containing the TIL cells from the incubator was removed and the cells were resuspended at a concentration of 1.3 x 106 cells / ml in warm CM2 supplemented with IL-2 3000 IU / ml. The 50 mL conical tube was returned to the incubator with a loose cap.
[001661] [001661] Repeated the steps above for the second strain of TIL.
[001662] [001662] Just before plating the TILs in the T25 flasks for the experiment, the TILs were diluted 1:10 to a final concentration of 1.3 x 105 cells / mL as shown below. Preparation of the pure MACS GMP CD3 working solution (OKT3)
[001663] [001663] OKT3 stock solution (1 mg / mL) was removed from the refrigerator at 4 ºC and placed in BSC. The final concentration of 30 ng / mL OKT3 was used in the mini-REP medium.
[001664] [001664] 600 ng of OKT3 was needed for 20 mL in each T25 flask of the experiment; this was the equivalent of 60 µL of a 10 µg / mL solution for every 20 mL, or 360 µL for all 6 vials tested for each batch of feeders.
[001665] [001665] For each batch of feeders tested, 400 µL of a 1: 100 dilution of OKT-3 were prepared for a working concentration of 10
[001666] [001666] Identify each vial and fill each vial with CM2 medium before preparing the feeder cells. The bottles were placed in a humidified incubator at 37 ºC, 5% CO2 to keep the medium warm while waiting for the addition of the other components. Once the feeder cells have been prepared, the components will be added to CM2 in each vial. Table 34: Solutions Component Volume in vials Volume in control coculture vials (feeders only) CM2 + IL-2 3000 IU / mL 18 mL 19 mL MNC: 1.3 x 107 / mL in CM2 + IL-2 3000 UI 1 mL 1 mL (final concentration 1.3 x 107 / vial) OKT3: 10 µg / mL in CM2 + IL-2 3000 IU 60 µL 60 µL TIL: 1.3 x 105 / mL in CM2 + IL-2 3000 IU 1 mL 0 (final concentration 1.3 x 105 / vial) Prepare feeder cells
[001667] [001667] At least 78 x 106 feeder cells were needed per batch tested for this protocol. Each 1 ml flask frozen by SDBB had 100 x 10 6 viable cells upon freezing. Assuming 50% recovery when defrosting, it was recommended to defrost at least two 1 ml vials of feeder cells per batch, providing an estimate of 100 x 106 viable cells for each REP. Alternatively, if supplied in 1.8 mL bottles, only one bottle provided sufficient feeder cells.
[001668] [001668] Before defrosting the feeder cells, preheated approximately 50 mL of CM2 without IL-2 for each batch of feeders to be tested. Removed designated flasks from feeder batches from LN2 storage, placed in a zippered storage bag and placed on ice. Thawed the vials in the pouch
[001669] [001669] With a transfer pipette, immediately transfer the contents of the feeder bottles in 30 mL of warm CM2 in a 50 mL conical tube. Wash the flask with a small volume of CM2 to remove any residual cells in the flask. Centrifuged at 400 x CF for 5 minutes. The supernatant was aspirated and resuspended in 4 ml of warm CM2 plus IL-2 3000 IU / ml. 200 µL removed for cell counting using the Automatic Cell Counter. Counts recorded.
[001670] [001670] Resuspend cells at 1.3 x 107 cells / ml in warm CM2 plus IL-2 3000 IU / ml. Diluted TIL cells from 1.3 x 10 6 cells / ml to 1.3 x 10 5 cells / ml. Co-culture preparation
[001671] [001671] Diluted TIL cells from 1.3 x 10 6 cells / ml to 1.3 x 10 5 cells / ml. 4.5 ml of CM2 medium was added to a 15 ml conical tube. TIL cells were removed from the incubator and resuspended well with a 10 mL serological pipette. 0.5 ml of cells removed from 1.3 x 106 cells / ml TIL suspension and added to 4.5 ml of medium in the 15 ml conical tube. The TIL stock bottle returned to the incubator. Mixed well. Repeated for the second strain of TIL. Bottles with preheated medium were transferred to a single batch of feeders from the incubator to the BSC. Mixed feeder cells, stirring the tip of a 1 mL pipette up and down several times and transferring 1 mL (1.3 x 107 cells) to each flask in that batch of feeders. 60 µL of OKT3 working stock (10 µg / mL) was added to each vial. The two control bottles were returned to the incubator.
[001672] [001672] 1 ml (1.3 x 105) of each batch of TIL was transferred to the correspondingly identified T25 flask. Bottles returned to the
[001673] [001673] Repeated for all tested feeder batches. Day 5, Change of medium
[001674] [001674] Prepared CM2 with IL-2 3000 IU / mL. 10 mL is required for each vial. With a 10 mL pipette, 10 mL of warm CM2 with IL-2 3000 IU / mL was transferred to each vial. Return the bottles to the incubator and incubate in an upright position until Day 7. Repeated for all batches of feeders tested. Day 7, Collection
[001675] [001675] Removed the flasks from the incubator and transfer to the BSC, taking care not to disturb the cell layer at the bottom of the flask. Without disturbing the cells growing at the bottom of the flasks, 10 ml of medium were removed from each test flask and 15 ml of medium from each of the control flasks.
[001676] [001676] With a 10 mL serological pipette, resuspend the cells in the remaining medium and mix well to break up any cell aggregates. After thoroughly mixing the cell suspension with the pipette, 200 µL was removed for cell counting. TILs counted using the appropriate standard operating procedure in conjunction with automatic cell counter equipment. Day 7 counts recorded.
[001677] [001677] Repeated for all tested feeder batches.
[001678] [001678] Feeder control flasks were assessed for incompetence for replication and flasks containing TIL were assessed for expansion times since Day 0 according to Table TT below. Day 7, Continuation of feeder control bottles until Day 14
[001679] [001679] After Day 7 counts of feeder control vials were completed, 15 mL of fresh CM2 medium containing IL-2 3000 IU / mL was added to each of the control vials. Control flasks returned to the incubator and incubated in an upright position until Day 14.
[001680] [001680] Removed flasks from the incubator and transfer to the BSC, taking care not to disturb the cell layer at the bottom of the flask. Without disturbing the cells growing at the bottom of the flasks, approximately 17 mL of medium was removed from each of the control flasks. Using a 5 mL serological pipette, resuspend the cells in the remaining medium and mix well to break up any cell aggregates. The volumes for each bottle were recorded.
[001681] [001681] After thoroughly mixing the cell suspension with the pipette, 200 µl was removed for cell counting. TILs counted using the appropriate standard operating procedure in conjunction with automatic cell counter equipment. Registered.
[001682] [001682] Repeated for all tested feeder batches. Results and acceptance criteria Results
[001683] [001683] The dose of gamma irradiation was sufficient to render the feeder cells incompetent for replication. All batches, it was expected, would meet the evaluation criteria and also demonstrated a reduction in the total number of viable feeder cells that remained on day 7 of the REP culture compared to Day 0.
[001684] [001684] All feeder batches, it was expected, would meet the criteria of 100-fold expansion of TIL growth on Day 7 of the REP culture.
[001685] [001685] It was expected that the 14th counts of control feeder bottles would continue the non-proliferative trend seen on the 7th. Acceptance criteria
[001686] [001686] The following acceptance criteria were met for each replica of TIL lineage tested for each batch of feeder cells.
[001687] [001687] The acceptance was twice, as follows (shown in Table 35 below). Table 35: Acceptance criteria Test Acceptance criteria MNC irradiation / Incompetence for No growth observed in 7 and 14 days replication At least 100 expansion times for each TIL TIL expansion (minimum 1.3 x 107 viable cells)
[001688] [001688] Evaluated if the radiation dose was sufficient to make MNC feeder cells incompetent for replication when cultured in the presence of OKT3 30 ng / mL and IL-2 3000 IU / mL. Incompetence for replication was assessed by total viable cell count (TVC) as determined by automatic cell count on day 7 and day 14 of the REP.
[001689] [001689] The acceptance criteria was "No growth", meaning that the number of total viable cells did not increase on day 7 and on day 14 of the initial number of viable cells placed in culture on day 0 of the REP.
[001690] [001690] The capacity of the feeder cells to support the expansion of TIL was evaluated. TIL growth was measured in terms of times of expansion of viable cells from the start of culture on Day 0 of the REP to Day 7 of the REP. On day 7, the TIL cultures achieved an expansion of at least 100 times (that is, 100 times greater number of total viable TILs placed in culture on REP Day 0), as assessed by automatic cell counting. Contingency testing of MNC feeder batches that do not meet the acceptance criteria
[001691] [001691] If one of the batches of MNC feeders had not met any of the acceptance criteria presented above, the following steps would be re-tested in the batch to rule out the evaluator's simple error as its cause.
[001692] [001692] If there were two or more satellite flasks remaining for
[001693] [001693] To be qualified, the lot in question and the control lot had to reach the acceptance criteria above. Upon meeting these criteria, the batch was then released for use. Example 4: Qualification of individual lots of peripheral blood mononuclear cells subjected to gamma radiation
[001694] [001694] This example describes an innovative abbreviated procedure for qualifying individual batches of gamma-irradiated peripheral blood mononuclear cells (PBMC) for use as allogeneic feeder cells in the exemplary methods described here. This example provides a protocol for evaluating irradiated PBMC cell batches for use in producing clinical TIL batches. Each batch of irradiated PBMC was prepared from an individual donor. Over more than 100 qualification protocols, it has been shown that, in all cases, the PBMC lots irradiated from the SDBB (San Diego Blood Bank) expand TIL> 100 times on day 7 of a REP. This modified qualification protocol was intended to be applied to batches of PBMC irradiated from the SDBB donor who were then further tested to see if the received dose of gamma radiation was sufficient to render them incompetent for replication. Once it was demonstrated that they remained incompetent for replication during the 14-day cycle, the donor's PBMC batches were considered “qualified” for use in the production of clinical TIL batches. Foundations
[001695] [001695] Gamma-irradiated PBMC, with sustained growth, was required for current standard TIL REP. Membrane receptors in PBMCs bind to the anti-CD3 antibody (clone OKT3) and cross-link with TIL in culture, stimulating TIL to expand. Lots of PBMC were
[001696] [001696] 7.2.1. The evaluation criterion for irradiated PBMC lots was their incompetence for replication. Experimental setup
[001697] [001697] Lots of feeders were tested in the mini-REP format as if they were to be co-cultivated with TIL, using T25 tissue culture flasks in the upright position. Control batch: A batch of irradiated PBMCs, which had historically demonstrated to meet the criterion of 7.2.1, was tested side by side with the experimental batches as a control. For each tested batch of PBMC irradiated from the donor, duplicate flasks were tested. Day 0 experimental protocol
[001698] [001698] Prepared ~ 90 mL of CM2 medium for each batch of PBMC from the donor to be tested. CM2 kept warm in a water bath at 37 ºC. An aliquot of IL-2 6 x 106 IU / mL was thawed. The CM2 medium was returned to the BSC, cleaning with 70% EtOH before placing it in the chapel. For each batch of PBMC tested, about 60 mL of CM2 was removed into a separate sterile flask. IL-2 of the thawed stock solution 6 x 106 IU / mL was added to this medium to a final concentration of 3000 IU / mL. This bottle was identified as “CM2 / IL2” (or similar) to distinguish it from the
[001699] [001699] The anti-CD3 stock solution (OKT3) was removed from the refrigerator at 4 ºC and placed in the BSC. A final concentration of OKT3 30 ng / ml was used in the mini-REP medium. A 10 µg / mL anti-CD3 working solution (OKT3) was prepared from the 1 mg / mL stock solution. Put in the refrigerator until needed.
[001700] [001700] For each batch of PBMC tested, prepare 150 µL of a 1: 100 dilution of the anti-CD3 stock (OKT3). For example, to test 4 batches of PBMC at the same time, prepare 600 µL of anti-CD3 10 µg / mL (OKT3) by adding 6 µL of the 1 mg / mL stock solution to 594 µL of CM2 supplemented with IL-2 3000 IU / ml. Preparation of Bottles
[001701] [001701] 19 mL per bottle of CM2 / IL-2 were added to the identified T25 flasks and the flasks were placed in a humidified incubator at 37 ºC in 5% CO2 during cell preparation. Preparation of irradiated PBMC
[001702] [001702] Vials of PBMC batches to be tested from storage with LN2 were recovered. These were placed at -80 ºC or kept on dry ice before thawing. 30 ml of CM2 (without IL-2 supplement) were placed in 50 ml conical tubes for each batch to be defrosted. Identification of each tube with the different batch numbers of PBMCs to be defrosted. The tubes are hermetically closed and placed in a water bath at 37 ºC before use. If necessary, return the 50 mL conical tubes to the BSC, cleaning with 70% EtOH before placing in the chapel.
[001703] [001703] A PBMC bottle was removed from cold storage and placed in an oscillating rack of tubes in a water bath at 37 ºC to thaw. Allow the defrost to continue until a
[001704] [001704] Centrifuged at 400 x g for 5 minutes at room temperature. Aspirate the supernatant and resuspend the cell pellet in 1 mL of warm CM2 / IL-2 using a 1000 µL pipette tip. Alternatively, before adding the medium, I resuspend the cell pellet by dragging the closed tube along the rack without tubes. After resuspension of the cell pellet, make up to 4 mL with CM2 / IL-2 medium. Recorded the volume.
[001705] [001705] A small aliquot (eg, 100 µL) was removed for cell counting using an automatic cell counter. Duplicate counts were performed according to the specific POP for the automatic cell counter. Most likely, it was necessary to dilute PBMCs before performing cell counts. A recommended initial dilution was 1:10, but this varied depending on the type of cell counter used. Counts recorded.
[001706] [001706] PBMC concentration adjusted to 1.3 x 107 cells / mL with CM2 / IL-2 medium. Mix well, swirling gently or gently aspirating up and down with a serological pipette. Preparation of culture flasks
[001707] [001707] Two identified T25 flasks returned to the BSC from the tissue culture incubator. The 10 µg / mL anti-CD3 / OKT3 vial was returned to the BSC. Added 1 mL of the cell suspension with 1.3 x 107 PBMC to each vial. 60 µL of anti-CD3 / OKT3 10 µg / mL was added to each vial. The capped vials were returned to tissue culture incubators for 14 days of growth without disturbance. Placed the bottle with
[001708] [001708] T25 bottles returned in duplicate to the BSC. For each vial, with a fresh 10 mL serological pipette, remove ~ 17 mL from each vial, then carefully pulling the rest of the medium to measure the remaining volume in the vials. Recorded the volume.
[001709] [001709] Mix the sample well, stirring it up and down with the same serological pipette.
[001710] [001710] A 200 µL sample was removed from each vial for counting. Counted the cells using an automatic cell counter. The steps were repeated for each batch of PBMC under evaluation. Results and acceptance criteria Results
[001711] [001711] The dose of gamma radiation, it was hoped, was sufficient to render the feeder cells rendering incompetent for replication. All batches, it was expected, would meet the evaluation criteria, demonstrating a reduction in the number of total viable feeder cells remaining on Day 14 of the REP culture compared to Day 0. Evaluation criteria
[001712] [001712] The following acceptance criteria were met for each batch of irradiated PBMC from the donor tested: “No growth” - meaning that the total number of viable cells on Day 14 was less than the total number of initial viable cells cultured in the day 0 of the REP. Contingency testing of PBMC batches that do not meet the acceptance criteria
[001713] [001713] If a batch of PBMC irradiated from the donor did not meet the acceptance criteria above, the following steps would be re-tested in the batch to rule out the evaluator's simple error as the cause of its failure. Case
[001714] [001714] To be qualified, the batch of PBMC submitted to the contingency tests had, both the control batch and both replicas of the batch in question, that reach the acceptance criterion. Upon satisfying this criterion, the batch was then released for use. Example 5: Preparation of IL-2 stock solution (CELLGENIX)
[001715] [001715] This example describes the process of dissolving purified recombinant human interleukin-2, lyophilized into stock samples suitable for use in future tissue culture protocols, including all those described in the present patent application and the Examples, including those that involve the use of rhIL-2. Procedure
[001716] [001716] 0.2% acetic acid (HAc) solution prepared. 29 mL of sterile water was transferred to a 50 mL conical tube. Added 1 mL of 1 N acetic acid to the 50 mL conical tube. Mixed well by inverting the tube 2-3 times. Sterilized the HAc solution by filtration using a Steriflip6 filter
[001717] [001717] Prepare 1% HSA in PBS. 4 mL of 25% HSA stock solution is added to 96 mL of PBS in a sterile filter unit.
[001718] [001718] RhIL-2 stock solution prepared (final concentration: 6 x 106 IU / mL). Each batch of rhIL-2 was different and required information found in the manufacturer's Certificate of Analysis (COA), such as: 1) Mass of rhIL-2 per bottle (mg), 2) Specific activity of rhIL-2 (UI / mg ) and 3) Recommended volume for reconstitution of 0.2% HAc (mL).
[001719] [001719] Calculated the volume of HSA 1%, necessary for the batch of
[001720] [001720] For example, according to the COA of CellGenix lot10200121 rhIL-2, the specific activity for the 1 mg vial is 25 x 106 IU / mg. It recommends reconstituting rhIL-2 in 2 mL of 0.2% HAc.
[001721] [001721] Cleaned the IL-2 rubber stopper with an alcohol wipe. With a 16G needle attached to a 3 mL syringe, the recommended volume of 0.2% HAc is injected into the vial. Care is taken not to dislodge the stopper when the needle is removed. Invert the bottle 3 times and swirl until all the powder is dissolved. Carefully removed the cap and placed on its side over an alcohol wipe. Added the calculated volume of 1% HSA to the vial.
[001722] [001722] Storage of the rhIL-2 solution. For short-term storage (<72 h), the bottle is stored at 4 ºC. For prolonged storage (> 72 h), Volume of the bottle divided into smaller aliquots and stored in cryovials at -20 ºC until ready for use. Freeze / thaw cycles are avoided. Due 6 months after the date of preparation. Rh-IL-2 labels included supplier and catalog number, lot number, operator initials, concentration and aliquot volume. Example 6: Cryo-preservation process
[001723] [001723] This example describes the method of the cryopreservation process for TILs prepared with the abbreviated, closed procedure described in Example G using the CryoMed Controlled Rate Freezer,
[001724] [001724] The equipment used was as follows: rack with support for aluminum cassettes (compatible with CS750 freezer pouches), cryo-storage cassettes for 750 mL pouches, low pressure liquid nitrogen tank (22 psi), refrigerator, sensor thermocouple (strap type for bags) and CryoStore CS750 Freezing bags (oriGen Scientific).
[001725] [001725] The freezing process allows a rate of 0.5 ºC from the nucleation to -20 ºC and a cooling rate of 1 ºC per minute to a final temperature of -80 ºC. The program parameters are as follows: Step 1 - waiting time at 4 ºC; Step 2: 1.0 ºC / min. (sample temperature) up to -4 ºC; Step 3: 20.0 ºC / min. (chamber temperature) up to -45 ºC; Step 4: 10.0 ºC / min (chamber temperature) to -10.0 ºC; Stage 5: 0.5 ºC / min. (chamber temperature) to -20 ° C; and Step 6: 1.0 ºC / min (sample temperature) to -80 ºC. Example 7: Use of the cytokine cocktail with IL-2, IL-15 and IL-21
[001726] [001726] This example describes the use of cytokines IL-2, IL-15 and IL-21, which serve as additional T cell growth factors, in combination with the TIL process of Examples 1 to 6.
[001727] [001727] Using the procedures of Examples 1 to 9, TILs of colorectal, melanoma, cervical, triple negative breast, lung and kidney tumors were cultured in the presence of IL-2, in one arm of the experiment and, in place of IL-2, a combination of IL-2, IL-15 and IL-21 in another arm at the beginning of the culture. At the conclusion of the pre-REP, cultures were evaluated for expansion, phenotype, function (CD107a + and IFNγ) and TCR Vβ repertoire. IL-15 and IL-21 are described elsewhere in the present and in Gruijl, et al., IL-21 promotes the expansion of tumor infiltrating lymphocytes CD27 + CD28 + with high cytotoxic potential and low collateral expansion of regulatory T cells, Santegoets, SJ, J Transl Med., 2013, 11:37 (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3626797/).
[001728] [001728] The results showed that the enhanced expansion of TILs (> 20%), in CD4 + and CD8 + cells under the conditions treated with IL-2, IL-15 and IL-21, was observed in multiple histologies in relation to the conditions with IL-2 only. There was a skew towards a predominantly CD8 + population with a skewed repertoire for TCR Vβ in the TILs obtained from cultures treated with IL-2, IL-15 and IL-21 in relation to cultures with IL-2 only. Example 8: Expansion of tumor infiltrating lymphocytes (TIL) from fine needle aspirates (FNAs) and thick needle biopsies I. Background
[001729] [001729] This example provides data related to the expansion of tumor infiltrating lymphocytes (TILs) from tumor samples obtained by fine needle aspirations or thick needle biopsies.
[001730] [001730] The objective of the study was to develop a method for expanding TILs from biopsy samples with a thick needle or aspirated with a fine needle. In some cases, 3 to 5 tumor samples were obtained from cancer patients, such as lung cancer (n = 3), melanoma (n = 1), head and neck cancer (n = 1), cervical cancer (n = 1), ovarian cancer (n = 3), pancreatic cancer (n = 2), glioblastoma (GBM) and colorectal cancer (n = 1). II. Expanded TILs from thick needle biopsies of a patient with pancreatic cancer (P7015).
[001731] [001731] Pancreatic biopsy samples were received for the study. A fragment was placed in a well of a 24-well plate, totaling 5 wells. When two fragments were placed in two wells, little to no growth was observed in these wells. Three wells were treated with culture medium supplemented with IL-2 and two wells were treated with culture medium supplemented with a cytokine cocktail containing IL-2, IL-15 and IL-21.
[001732] [001732] Figures 17A, 17B and 17C show the phenotyping and functional status of TILs derived from a thick needle biopsy of pancreatic tumors. TILs were expanded from pancreatic tumors and culture in medium containing IL-2 or medium supplemented with cell culture additives. TILs were expanded from pancreatic tumors in culture medium supplemented with IL-2 and in culture medium containing cell culture additives (Figure 17D). Figure 17A shows the percentage of cells that were CD3 +, CD4 +, CD8 +, CD19 +, CD3 + / CD56 + or CD3- / CD56 +. Figure 17B shows the percentage of CD107a cells in the CD4 + subpopulation after stimulation with phorbol 12-myristate 13-acetate (PMA) compared to an unstimulated condition. Figure 17C shows functional analysis of expanded TILs from the pancreatic thick needle biopsy. The graph shows the percentage of CD107a cells in the CD8 + subpopulation after stimulation with phorbol 12-myristate 13-acetate (PMA) compared to an unstimulated condition.
[001733] [001733] Most cells expanded from pancreatic biopsy samples are T cells (CD4 + cells and CD8 + cells). PMA stimulation demonstrated the ability of CD8 + to degranulate, as indicated by the expression of CD107a +, thereby suggesting that these cells are functional. The% of CD8 + expressing CD107a + was higher in the wells treated with the culture additives, when compared to IL-2 only (Figure 17C).
[001734] [001734] A cervical tumor was received for the study. Five fine needle aspirates were isolated from the tumor using a 22 gauge needle and syringe. Each FNA sample was placed in at least three wells on a 24-well plate. Two wells from each sample were treated with IL-2 and one well from each sample was treated with the cytokine cocktail (IL-2, IL-15 and IL-21).
[001735] [001735] Figure 10A shows that CD4 + and CD8 + T cells were present in IL-2 treated FNAs. On average, 3.45 x 105 viable cells / well were present in the IL-2 treated wells. Figure 10B shows that CD4 + and CD8 + T cells were present in FNAs treated with the cytokine cocktail. On average, 8.69 x 105 viable cells / well were present in these wells. Aspirates treated with IL-2. IL-15 and IL-21 demonstrated more cells / cavity when compared to aspirates treated with IL-2. The percentage of CD8 + cells was higher in the cavities treated with the cocktail (20.1%) when compared to the cavities treated with IL-2 (8.58%). IV. Expanded TILs from fine needle aspirations of a patient with lung carcinoma (L4032)
[001736] [001736] Three lung tumors were received for the study. Fine needle aspirations were isolated from the tumors using a 22 gauge needle and syringe. An FNA sample included 1.6 x 10 6 cells with 28% viable cells. Two wells (each condition) were treated with culture medium supplemented with (1) IL-2 only or (2) the cytokine cocktail. In addition, lung tumor fragments were cultured in a 24-well plate (two wells each) as a control. Cells were collected on day 13 and evaluated by FACS analysis.
[001737] [001737] Figure 11A shows that T cells expanded from
[001738] [001738] Then, the TILs grown from the FNA samples were expanded with a Rapid Expansion Protocol (eg, protocol with second expansion or step D). The cells were collected on day 14 and evaluated by FACS analysis. Figure 12A shows the total cell count of the expanded TILs from the FNA samples treated with (i) IL-2, (ii) IL-2, IL-15 and IL-21 and (iii) IL-2, IL -15 and IL-21 in IL-2. Figure 12A shows that, after the second expansion, TILs included CD4 + and CD8 + T cells (84.1% and 10.3%, respectively). V. Expanded TILs from fine needle aspirations of a patient with lung carcinoma (L4033)
[001739] [001739] A fine needle aspirate was isolated from a lung tumor using a 22 gauge needle and syringe. The FNA sample included 5.5 x 10 6 cells with 76% viable cells. A G-Rex 10 vial (each condition) was treated with culture medium containing either (1) IL-2 only or (2) the cytokine cocktail. In addition, four lung tumor fragments were cultured in a G-Rex 10 flask as a control. Cells were collected on day 12 and evaluated by FACS analysis. Figure 13A shows that CD4 + T cells (4.25%) and CD8 + T cells (55.1%) were obtained from the IL-2 treated FNA sample. Figure 13B that CD4 + T cells (7.76%) and CD8 + T cells (54.5%) were obtained from the tumor fragments treated with IL-2.
[001740] [001740] Next, the TILs grown from the FNA samples were
[001741] [001741] In a similar experiment, FNA samples were isolated and three lung tumor samples from a patient with lung carcinoma (L4034). Approximately 1.0 x 10 6 cells from an FNA sample were cultured in a G-Rex 10 flask for 15 days in culture medium supplemented with IL-2 only. As a control, four tumor fragments were cultured in a G-Rex 10 flask in culture medium supplemented with IL-2 only. Few or no detectable cells were collected in the culture from FNA. SAW. Expanded TILs from fine needle aspirations of patients with ovarian carcinoma (OV8011, OV8012, and OV8013)
[001742] [001742] Three ovarian tumors (OV8011, OV8012, and OV8013) were received for the study. Fine needle aspirations were isolated from the tumors using a 22 gauge needle and syringe.
[001743] [001743] The FNA sample from OV8011 included 3.19 x 105 cells with 28% viable cells. This FNA sample was divided into four wells; two wells were grown in culture medium containing IL-2 and two wells were grown in culture medium containing IL-2, IL-15 and IL-21. The OV8012 FNA sample included 8.3 x 105 cells with 11% viable cells. This FNA sample was divided into two wells; one well was grown in culture medium containing IL-2 and the other well was grown in culture medium containing IL-2, IL-15 and IL-21. The OV8013 FNA sample included 1.5 x 105 cells with 7% cells
[001744] [001744] As a control, four tumor fragments from each tumor were grown in a G-Rex 10 flask. Some flasks received culture medium containing only IL-2 and other flasks received culture medium containing IL-2, IL-15 and IL -21. Figure 15 shows the total cell counts of the cells derived from the ovarian tumor fragments grown in culture medium containing (1) IL-2 only or (2) the cytokine cocktail. The results show that ovarian tumors expanded little in the first expansion. VII. Expanded TILs from fine needle aspirations of a melanoma patient (M1101)
[001745] [001745] A melanoma tumor (M1101) was received for the study. An FNA sample was obtained from the tumor. The sample included 1.96 x 10 6 cells with 65% viable cells. The sample was placed in a G-Rex 10 flask and treated with culture medium supplemented with IL-2. As a control, four melanoma tumor fragments were placed in a G-Rex 10 flask and treated with culture medium supplemented with IL-2. The cells were collected on Day 12. Figure 16A shows that CD4 + T cells (21.4%) and CD8 + T cells (52.8%) expanded from the IL-2 treated FNA sample. Figure 16B shows that CD4 + T cells (18.0%) and CD8 + T cells (71.5%) expanded from the tumor fragments treated with IL-2.
[001746] [001746] Figures 18A and 18B provide a summary of the results obtained from thick needle biopsies and fine needle aspirations. VIII. Expanded TILs from fine needle aspirations of a melanoma patient (M1101)
[001747] [001747] On Day 11 of the pre-REP, cell counts were performed in the vials containing the aspirate and fragments (n = 2). Pre-REP cultures containing the fragments were collected on Day 11 (Gen2). Pre-REP culture containing the aspirate was continued due to low cell count on Day 11. The aspirate was recounted on Day 18 and then again on Day 21 (Figure 19A). On Day 21, the culture had reached almost 60 x 106 cells, the cells were collected at that time and evaluated by flow cytometry. The phenotype (Figure 19B) and the function (CD107a expression, Figure 19C) of the pre-REP fragments and aspirator TILs proved to be comparable.
[001748] [001748] Figures 19A, 19B and 19C show the expansion of TILs from an FNA sample from another melanoma patient (M1107). Figure 19A shows the total cell count at a different time during the expansion. Figure 19B shows the phenotype of expanded TILs. Figure 19C shows the functional analysis of the TILs expanded from the fragments and aspirated. The graph shows the percentage of CD107a cells in the CD4 + and CD8 + subpopulations after PMA stimulation compared to an unstimulated condition. Example 9: Expansion of tumor infiltrating lymphocytes (TIL) from fine needle aspirates (FNAs) and thick needle biopsies I. Pancreatic biopsy samples: Data shown in Figures 20-29
[001749] [001749] Fifteen tumors (Pancreatic, Pulmonary, Cervical, Mesothelioma, Colorectal) have been reported. To date, 37 tumor samples of different histology, including pancreatic, cervical, lung, oral and esophageal cancer, sarcoma, have been received.
[001750] [001750] Upon arrival, the tumors were fragmented and placed in vials / plate with IL-2 for a Rapid Pre-Expansion (REP) protocol. Pre-REP TILs were further propagated in a REP protocol in the presence of irradiated PBMCs, anti-CD3 antibody (30 ng / mL) and IL-2 (3000 IU / mL). Pre-REP lung and sarcoma TILs were expanded from 4
[001751] [001751] Growth kinetics demonstrated very few cells on Day 11-12 in both P7028 and P7031, however significant expansion was demonstrated between Day 21-28.
[001752] [001752] Most cells expanded from pancreatic biopsy samples are T cells and with a CD4 + phenotype. TILs isolated from pancreatic tumors are typically CD4 +.
[001753] [001753] PMA stimulation demonstrated the ability of CD4 + to degranulate, as indicated by the expression of CD107a +, thereby suggesting that these cells are functional, as shown in previous studies with P7015. CD107a expression was similar in TIL derived from fragments.
[001754] [001754] TILs derived from thick needle fragments secreted significant levels of IFNγ in a plate-bound anti-CD3 restimulation assay, similarly to TIL derived from fragments.
[001755] [001755] Extensive phenotyping demonstrated similar expression of most of the markers evaluated in TIL derived from tumor fragments, suggesting that the result of cultivating TILs from thick needle fragments is similar to that of fragments.
[001756] [001756] The exhaustion / activation panel demonstrated significant differences in PD-1, Tim3 and KLRG1 in CD4 + cells and LAG3, Tim3 and KLRG1 in CD8 + cells. Additional studies with pancreatic fragments of thick needle biopsies are needed to validate these findings. General propositions:
[001757] [001757] These results suggest that the process for successfully culturing TIL from pancreatic biopsy samples (and other histologies) will likely require additional time in culture (in addition to the requirements for Gen2 pre-REP), unless used Additional “tuning strategies” to reinforce growth (see below).
[001758] [001758] Based on preliminary growth data in pancreatic biopsy samples, it is recommended that the pre-REP process take place between Day 21-28, in order to obtain sufficient cells.
[001759] [001759] For this process, the pre-REP must be started in bottles G-Rex 10 or G-Rex 10M with CM1 medium and IL-2 / IL-15 / IL-21.
[001760] [001760] The REP must be started on a G-Rex 100M or G-Rex 500M, depending on the pre-REP cell count.
[001761] [001761] G-Rex 500M: if the cell count is 5 x 107.
[001762] [001762] G-Rex 100M: if the cell count is between 1-5 x 107.
[001763] [001763] Future experiments will be used to determine the minimum number of cells required for sufficient cell growth in each vessel.
[001764] [001764] Future experiments will compare the growth, phenotype and function of expanded pancreas biopsy samples in IL-2 / IL-15 / IL-21 alone or with IL-2 / IL-15 / IL-21 with OKT3 and feeders. Example 10: TIL expansion with thick needle biopsies Justification:
[001765] [001765] The removal / resection of the tumor is neither appropriate nor feasible for all patients. In this cohort of patients, thick needle biopsies can be a source of TIL for ACT (adoptive cell therapy). Such thick needle biopsies allow samples to be collected from multiple lesions and potentially obtain a wider repertoire of TIL. Strategy:
[001766] [001766] The present example provides a method to expand TIL to
[001767] [001767] The ability to expand TIL from biopsies has been demonstrated for thick needle biopsy samples from pancreas using Gen2, which also showed that TIL obtained from biopsies are phenotypically and functionally comparable to TIL obtained from fragments.
[001768] [001768] Seven sets of samples per pancreas thick needle (between 7-10 samples) were treated with IL-2 / IL-15 / IL-21 and subjected to pre-REP and evaluated for expansion. Three managed to expand, however, the growth was extremely variable. Pre-REP cultures required at least 21-28 days to acquire sufficient numbers of cells (eg, 21-28 days for Steps A to E in Figure 7). Extensive phenotyping demonstrated similar expression of most of the markers evaluated in TIL derived from a pancreatic tumor fragment. The expression of CD107a and IFNγ was similar in TIL derived from fragments Current protocol:
[001769] [001769] The addition of OKT3 and feeders on Day 3 of culture reinforced the production of TILs. Fewer samples were grown per coarse needle / vial and growth was evaluated (for example, 1-2 cultured samples).
[001770] [001770] 1-2 thick needle biopsies (3 from pancreas, 4 from melanoma, 1 from breast and 1 ovarian) were subjected to a modified research scale process that included the addition of OKT3 + feeders on Day 3 (See, Figures 39-40).
[001771] [001771] • TIL underwent a second REP (eg, a second expansion).
[001772] [001772] • Pancreatic biopsy samples were expanded +/- IL15 and 21 (the triple cocktail; REP1 and REP2; eg, a first expansion and a second expansion)
[001773] [001773] • Ovarian biopsy samples were treated +/- OX40 (BMS) (n = 1, preliminary data).
[001774] [001774] • All TILs were submitted to a second REP. Pancreatic
[001775] [001775] TIL of pancreatic biopsy samples were successfully expanded in the presence of OKT3 + feeders. > 506 cells were purchased between Day 11-17 of the first expansion.
[001776] [001776] Two samples per coarse needle were generally needed to acquire the appropriate cell numbers.
[001777] [001777] The addition of the triple cocktail (TC: IL-2, IL-15 / IL-21) to the 2nd REP (either cultivated before with IL-2 or with the TC) did not significantly change the expansion of TIL compared to IL -2 alone.
[001778] [001778] The addition of OKT3 + feeders on Day 3 to thick needle biopsies does not significantly alter the phenotype (memory, CD27 / CD28, exhaustion, activation) of TIL compared to thick needle samples using conditions similar to those of Gen2. The phenotype (memory, CD27 / CD28, exhaustion, activation) of TIL expanded through 2 REPs did not differ significantly from that of TIL grown using Gen2 conditions.
[001779] [001779] CD107a expression did not vary significantly with the addition of OKT3 and feeders. TIL expanded through 2 REPs maintained the ability to respond to non-specific stimuli, as assessed by CD107a induction.
[001780] [001780] TILs expanded through 2 REPs maintained the ability to respond to non-specific stimuli, as assessed by CD107a induction.
[001781] [001781] IFNγ secretion was assessed. TIL produce IFNγ upon stimulation with OKT3.
[001782] [001782] Pancreatic TILs can be expanded from thick needle biopsies of pancreatic cancer. Summary and pre-REP experimental results for P7028 and P7031
[001783] [001783] Thick needle biopsies were placed on a G-Rex 10 with the triple cocktail (IL-2 / IL-15 / IL-21)
[001784] [001784] TILS P7028 were evaluated on Day 12 and maintained in culture until Day 19
[001785] [001785] Cell counts: Day 12 = 1.61 x 106 viable cells; Day 213.49 x 107 viable cells
[001786] [001786] P7030 TIL were evaluated on Day 12 and maintained in culture until Day 27
[001787] [001787] Cell counts: Day 12 = 1.52 x 105 viable cells; Day 27: 1.14 x 107 viable Melanoma cells
[001788] [001788] TIL derived from melanoma biopsy samples were successfully expanded in the presence of OKT3 + feeders (n = 4). > 50 x 106 cells were purchased on Day 12 of the first expansion.
[001789] [001789] One to two fragments per thick needle were generally required to acquire the appropriate numbers of cells. The addition of OKT3 + feeders on Day 3 to thick needle biopsies does not significantly alter the phenotype (memory, CD27 / CD28, exhaustion, activation) of TIL compared to fragments using conditions similar to Gen2.
[001790] [001790] TILs expanded through 2 REPs responded to non-specific stimuli, as assessed by CD107a mobilization and IFNγ secretion. Pulmonary
[001791] [001791] TILs of 1 fragment per thick needle of the lung were
[001792] [001792] The phenotype (memory, CD27 / CD28, exhaustion, activation) of TIL expanded through 2-REPs varied slightly from TIL expanded from coarse needle samples treated with OKT3 + feeders on Day 3 of the start of culture. There was an increase in the CD8 + / CD4 + ratio in the thick needle samples treated on Day 3.
[001793] [001793] There were slight differences in the expression of CD28, CD69, CD39, CD49a, Tim3 and CD101 that were observed between Day 0 and Day 3 of conditions in culture.
[001794] [001794] TILs produced IFNγ upon stimulation with anti-CD3. Day 0 lung samples treated with OKT3 + feeders had higher levels of IFNγ compared to Day 3 lung samples treated with OKT3 + feeders.
[001795] [001795] TILs expanded through 2 REPs maintained the ability to respond to non-specific stimuli, as assessed by CD107a induction.
[001796] [001796] CD8 + TILs from Day 3 thick needle lung samples treated with OKT3 + feeders were oligoclonal when compared to Day 0 lung samples TILs treated with OKT3 + feeders, indicating a different repertoire of TCR depending on the moment the addition of OKT3 + feeders. Conclusions
[001797] [001797] The addition of OKT3 + feeders to coarse needle samples on Day 3 of culture significantly improved TIL production. > 50 x 106 cells (limit for beginning of REP2) were acquired within 11-15 days of the first expansion in all evaluated histologies (pancreatic, melanoma, mammary and ovarian). Two samples (in the evaluated histologies) were sufficient to acquire the required number of cells.
[001798] [001798] TILs from at least 2 breast samples were successfully expanded in the presence of IL-2 + OKT3 + feeders, but not with IL-2 alone. > 50 x 106 cells were purchased on Day 11. One to two samples are sufficient to acquire the required number of cells.
[001799] [001799] The data suggest that the expanded TIL phenotype and function from coarse needle samples (with OKT3 + feeders early) are similar to those from expanded samples / fragments under Gen2 conditions (IL-2 only). The addition of OX40 to ovarian biopsy samples improved the expansion of TILs compared to IL-2 alone.
[001800] [001800] 1-2 pancreatic biopsy samples failed to expand on IL-2 only.
[001801] [001801] Experiments side by side will be performed with fragments +/- OKT3 + feeders on Day 3. TILs produced IFNγ when stimulating with OKT3 and additional experiments are underway. Differences in KLRG1, CD25 and PD1 were observed in ovarian biopsy samples treated +/- OX40 (therapeutic population of TILs).
[001802] [001802] The TCR phenotype, function (power) and repertoire have been and continue to be evaluated (currently underway). The addition of OKT3 + feeders on Day 3 to thick needle biopsies did not significantly alter the phenotype (memory, CD27 / CD28, exhaustion, activation) compared to fragments using conditions similar to Gen2. TILs expanded through 2 REPs responded to non-specific stimuli, as assessed by CD107a mobilization and IFNγ secretion. The phenotype (memory, CD27 / CD28, exhaustion, activation) of TILs expanded through 2-REPs did not differ significantly from that of TILs expanded using the 1-REP process (ie, without OKT3 + feeders during the 1st expansion) in samples of breast biopsy.
[001803] [001803] Discreet differences were observed in the expression of
[001804] [001804] TILs expanded through 2 REPs maintained the ability to respond to non-specific stimuli, as assessed by CD107a induction.
[001805] [001805] The future evaluation will include +/- the removal of samples with a thick needle on Day 3, as well as additional evaluation of the effect of anti-OX40 on expanded TILs. Example 11: Expansion of TIL from thick needle and breast biopsy using OKT3 + Feeders: Summary of Examples 9-10
[001806] [001806] TIL from at least 2 breast samples were successfully expanded in the presence of IL-2 + OKT3 + feeders, but not IL-2 alone. > 50 x 106 cells were purchased on Day 11.
[001807] [001807] The addition of OX40 to ovarian biopsy samples improved the expansion of TIL compared to IL-2 alone.
[001808] [001808] The phenotype (memory, CD27 / CD28, exhaustion, activation) of TIL expanded through 2-REPs did not differ significantly from that of TIL expanded using 1 REP process (ie, without OKT3 + feeders during the 1st expansion) in breast biopsy samples.
[001809] [001809] Slight differences were observed in the expression of KLRG1 and PD1.
[001810] [001810] Differences in KLRG1, CD25 and PD1 were observed in ovarian biopsy samples treated +/- OX40 (final product).
[001811] [001811] TIL produce IFNγ upon stimulation with OKT3.
[001812] [001812] TILs expanded through 2 REPs maintained the ability to respond to non-specific stimuli, as assessed by CD107a induction.
[001813] [001813] The addition of OKT3 + feeders to coarse needle samples on Day 3 significantly improved TIL production. > 50 x 106
[001814] [001814] Expand TIL from needle aspirates of multiple histologies and develop a defined process that is viable for TIL production. Preliminary studies have shown that TIL could be expanded from “some” aspirates performed under optimal conditions (ie, taken directly from tumor biopsies).
[001815] [001815] 22 aspirates were isolated from tumor biopsies of various histologies using Gen Iovance conditions (ie, without OKT3 and feeders on Day 3). Growth was observed in 6 tumors (1 cervical, 2 lung and 3 melanomas). The number of cells isolated during pre-REP was well below the 50 x 106 cell limit.
[001816] [001816] In addition, 10 needle aspirates were received, including three for melanoma (n-3) and seven for breast (n-7). The aspirates were treated with OKT3 and feeders.
[001817] [001817] Due to the small initial numbers of cells, most experiments were started in a 96-well plate. <50 x 106 cells were obtained on Day 11 in all aspirates. Several subcultures would be required to acquire the necessary number of cells. Example 13: TIL expansion from thick needle biopsies of tumors Introduction
[001818] [001818] T-cell adoptive therapy (ACT) with autologous tumor infiltrating lymphocytes (TIL) has demonstrated clinical efficacy in patients with metastatic melanoma and cervical carcinoma. TILs are currently expanded from a fragmented tumor that is surgically removed. Unfortunately, patients with unresectable tumors do not currently qualify for TIL therapy. If TIL expansion were feasible from the type of small tumor samples used by doctors for diagnostic and staging purposes, correlative studies and biomarker analyzes, TIL therapy would become available to a greater number of cancer patients.
[001819] [001819] A thick needle biopsy is an example of such small tumor samples. This procedure is inexpensive, safe, simple and less invasive than surgical resection. A thick needle biopsy removes a small cylinder of tumor tissue using a small incision and needle. Typically, a needle can be inserted into the incision up to 5 times to excise an adequate amount of tissue. Thick needle biopsies are small samples of the intact tumor microenvironment and therefore contain tumor cells, stroma and immune cells, including TIL. To date, there is a clinical study in melanoma that used tissue derived from thick needle biopsies to expand TIL (Ullenhag, G.J., et al., Cancer Immunol Immunother, 61 (5): 725-732 (2014)). The absolute number of cells and the expansion times were similar between TIL derived from surgical resection versus TIL derived from thick needle biopsies. Interestingly, most of the objective responses (4/5) in this study were treated with cells derived from thick needle biopsies. No study further corroborated these findings.
[001820] [001820] In some embodiments, a 2-REP process is employed to expand TIL derived from thick needle biopsies, as described above and in the present example.
[001821] [001821] The present example provides a protocol to expand TIL to clinically adequate numbers from small tumor samples to ACT (adoptive cell transfer). This protocol was developed using thick needle biopsies of pancreatic tumors, melanoma, breast and ovarian tumors. Expanded TILs were evaluated for growth, viability, phenotype, function (IFNγ secretion, CD107a mobilization) and TCR Vβ repertoire (RNA sequencing). Materials
[001822] [001822] Tumor sample tissue with a thick needle. Samples by thick needle of the pancreas, melanoma, breast and ovary are received from UPMC, Biotheme and MT Group. Procedure Sample processing and start of culture
[001823] [001823] Thick needle biopsies of recently resected tumors are received from research alliances (UPMC) and third party tissue suppliers (Biotheme and MTG Group). Thick needle biopsies of the tumor are sent overnight in HyperThermosol (Biolife Solutions, Washington, Cat. No 101104) (with antibiotic) or RPMI 1640 (Fisher Scientific, Pennsylvania, Cat. No 11875-085) + human male AB serum (Access Biologicals, California, A13012).
[001824] [001824] Using a 0.22 µM filter on the top of a 50 mL conical tube, carefully and slowly pour the contents of the shipping container including the thick needle biopsies of the tumor through the filter. The samples are retained in the filter. Wash the samples by gently adding 10-20 mL of HBSS over the samples.
[001825] [001825] Gently remove each sample from the filter membrane, while trying to keep the sample intact. With sterile plastic forceps or a pipette gently (1 mL pipette containing approximately 200
[001826] [001826] On Day 3 post-culture, OKT3 (30 ng / mL) (Miltenyi Biotec, Germany, Cat. No. 170-076-116) and Feeders (1: 100) are added to G-Rex 10 without disturbing the culture when adding.
[001827] [001827] Evaluate the growth in the culture daily, by microscopy. REP1 is conducted for 8 days after adding OKT3 + Feeders and ends on Day 11. The cells remain untouched throughout the culture period.
[001828] [001828] To count, remove about 30 ml of medium and resuspend the cells in the remaining 10 ml of medium, with a pipette up and down. Place the cells in a 50 mL conical tube and centrifuge at 1500 rpm and room temperature for 5 minutes in a Sorvell Legend XFR centrifuge and bucket-type mobile rotor (ThermoScientific, CA, Cat. No 75003180).
[001829] [001829] Determine cell counts and viability using the Nexcelom Cellometer K2 (Nexcelom, MA).
[001830] [001830] If, after centrifugation, there is no pellet, or if the pellet is <200 µL, resuspend the pellet in 2 mL of counting medium. If the pellet is> 200 µL, resuspend in 5 mL of CM2 medium for counting.
[001831] [001831] Experiments are collected on Day 11 post-start of culture (8 days after adding OKT3 + Feeders). The REP1 product is evaluated for cell count, viability, phenotype (TIL1, TIL2 and TIL3 (TIL2 and TIL3 panel for TIL Surface Antigen Staining), TIL3) and function (CD107a (TIL function evaluation by mobilization of CD107a), IFNγ (WRK LAB-059)). The remaining cells are frozen and stored in liquid nitrogen. Start of REP2 (eg, second rapid expansion)
[001832] [001832] To start mini-REP2, 1 x 105 cells are placed in a G-Rex 10 with 40 ml of CM2 medium and 3000 IU / ml of IL-2. Anti-CD3 (clone: OKT3, 30 ng / mL) and Feeders (ratio 1: 100, TIL: Feeders) are added at the beginning of the culture. All vials are treated with OKT3 + Feeders in REP2, even if OKT3 + feeders were not added during REP1.
[001833] [001833] A change of medium is performed on Day 5-7 of REP2 (Day 16-18 of the process). The medium in the G-Rex 10 vial is reduced in volume to about 10 mL and supplemented to 40 mL with CM2 or AimV + IL-2 3000 IU / mL.
[001834] [001834] On Day 11 of REP2, the volume is reduced as indicated above and the cells are centrifuged at 1500 rpm and room temperature for 5 minutes.
[001835] [001835] The final product is evaluated for cell count, viability, phenotype (TIL1, TIL2 and TIL3 (TIL2 and TIL3 panel for TIL Surface Antigen Staining, v1 and v2), TIL3 and function (CD107a (Evaluation of TIL function by mobilization of CD107a, IFNγ). Additional cells (1 x 106 - 5 x 106 cells) are pelleted and frozen for RNA sequencing and analysis. The remaining cells are frozen and stored in liquid nitrogen. acceptance
[001836] [001836] The expected yield for REP1 is> 50 x 106 in D11 in bottles that have been treated with OKT3 + Feeders. Expansion times during REP2 are not expected to be affected by the addition of OKT3 and feeders during REP1 and are consistent with the Gen2 REP expansion times. The TIL phenotype derived from thick needle biopsies and exposed to OKT3 + Feeders early is expected to be similar to that of TIL derived from surgical resections (or small biopsies) and expanded according to the Gen 2 process.
[001837] [001837] The example provides a comparison between the Gen 2 and Gen 3 processes. This example describes the development of a robust platform for expanding TIL. Modifications to the Gen 2 process reduce risk and streamline the manufacturing process by reducing the number of interventions by the operator, reducing overall manufacturing time, optimizing the use of reagents and facilitating a flexible, semi-closed and semi-automatic cell production process, conducive to high-performance manufacturing on a commercial scale.
[001838] [001838] Gen 2 and Gen 3 process TILs are composed of autologous TILs derived from an individual patient through surgical resection of a tumor and then expanded ex vivo. The First Expansion Preparation Stage of the Gen 3 process was a cell culture in the presence of interleukin-2 (IL-2) and the monoclonal antibody OKT3, directed to the CD3 T cell co-receptor in a peripheral blood mononuclear cell framework (PBMCs) irradiated.
[001839] [001839] The manufacture of Gen 2 TIL products consisted of two phases: 1) Rapid Pre-Expansion (Pre-REP) and 2) Rapid Expansion Protocol (REP). During Pre-REP, resected tumors were cut into ≤ 50 2-3 mm fragments in each dimension that were cultured with serum-containing culture medium (RPMI 1640 medium containing 10% supplemented HuSAB) and 6,000 IU / mL of Interleukin-2 (IL-2) for a period of 11 days. On day 11, TILs were collected and introduced in the secondary large scale REP expansion. The REP consists of activation of ≤200 x 106 viable cells from pre-REP in a coculture of 5 x 109 irradiated allogeneic PBMC feeder cells, loaded with 150 µg of anti-CD3 monoclonal antibody (OKT3) in a volume of 5L CM2 supplemented with 3000 IU / mL rhIL-2 for 5 days. On day 16, the culture has a reduced volume to 90% and the cell fraction is divided into
[001840] [001840] The manufacture of Gen 3 TIL products consisted of three phases: 1) First Preparation Expansion Protocol 2) Second Rapid Expansion Protocol (also referred to as rapid expansion phase or REP) and 3) Subcultures Division. To propagate TILs in the First Preparation Expansion, the resected tumor was cut into ≤ 120 fragments of 2-3 mm in each dimension. On day 0 of the First Preparation Expansion, a layer of feeders of approximately 2.5 x 108 irradiated allogeneic PBMCs feeder cells, loaded with OKT-3, was established over a surface area of approximately 100 cm2 in each of 3 containers 100 MCS. The tumor fragments were distributed in 3 100 MCS containers, each with 500 ml of CM1 culture medium containing serum and 6,000 IU / ml of Interleukin-2 (IL-2) and 15 µg of OKT-3 for a period of 7 days . On day 7, REP was initiated, incorporating a layer of additional feeder cells with approximately 5 x 108 irradiated allogeneic PBMC feeder cells, loaded with OKT-3 in the fragmented tumor culture phase in each of the 3 100 MCS containers and cultured with 500 mL of culture medium CM2 and IL-2 6,000 IU / mL and 30 µg of OKT-3. The start of REP was reinforced by activating the entire First Preparation Expansion culture in the same container, using a closed system for transferring liquids from OKT3-loaded feeder cells to the 100MCS container. For Gen 3, vertical scaling of TIL or division involved process steps in which the entire cell culture was scaled to a larger container by transferring liquid in a closed system, and was transferred (from a vial of
[001841] [001841] In general, the Gen 3 process is a shorter platform, with more flexible and easily modifiable scaling for expansion that will accommodate with adjustment for robust manufacturing and comparability between processes. Table 36: Comparison between the exemplary Gen 2 and Gen 3 exemplary manufacturing processes Step Process (Gen 2) Process (Gen 3) Whole tumor up to 120 fragments evenly divided between up to 3 vials. 1 vial: 1-60 fragments Up to 50 fragments / 1-G-Rex 2 vials: 61-89 fragments 100MCS - 11 days Pre-REP Day 0 3 vials 90-120 fragments In 1 L of CM1 medium 7 days in 500 mL of medium CM1 + IL-2 (6000 IU / mL) + IL-2 (6000 IU / mL) 2.5 x 108 feeder cells / flask 15 µg OKT-3 / flask Direct to REP- Day 11- Direct to REP- Day 7- all cells <200 x 106 TIL TIL- same G-Rex 100MCS (1) G-Rex 500MCS in 5L of Add 500 of M2 medium Start of REP CM2 medium IL-2 (6000 IU / mL) IL-2 ( 3000 IU / mL) 5 x 108 feeder cells / vial 5 x 109 feeder cells 30 µg OKT-3/150 µg bottle OKT-3 Reduce volume, divide the cell fraction up to 5 G-REX Each G-REX 100MCS (1L) transferred to Propagation of TIL 500MCS 1 G-REX 500MCS or Scaling 4.5 L of CM4 + IL-2 medium Add 4 L of CM4 + IL-2 medium (3000 vertical (3000 IU / mL) IU / mL) ≥ 1 x 109 TVC / bottle Vertical staging on days 9 to 11 Division on day 16 Collection day 22, Collection day 16 Collection Automatic cell washer Washer automatic LOVO cells
[001842] [001842] On day 0, for both processes, the tumor was washed 3 times and the fragments were randomized and divided into two pools; one pool per
[001843] [001843] Three different tumors were included in the comparison, two lung tumors (L4054 and L4055) and one melanoma tumor (M1085T).
[001844] [001844] CM1 (culture medium 1), CM2 (culture medium 2) and CM4 (culture medium 4) media were prepared beforehand and kept at 4 ºC for L4054 and L4055. CM1 and CM2 media were prepared without filtration to compare cell growth with and without filtration of the medium.
[001845] [001845] The medium was heated to 37 ºC up to 24 hours in advance for the L4055 tumor at the beginning of REP and vertical scaling.
[001846] [001846] Gen 3 was 30% below Gen 2 in relation to the total viable cells obtained. The final product of Gen 3 exhibited higher production of INF-γ after restimulation. The final Gen 3 product exhibited increased clonal diversity, as measured by the total unique CDR3 sequences present. The final product of Gen 3 exhibited longer average telomere length. Results achieved Cell count and% viability:
[001847] [001847] Pre-REP and REP expansion in the Gen 2 and Gen 3 processes followed the details described above.
[001848] [001848] Table 37: Pre-REP cell counts. For each tumor, the two pools contained an equal number of fragments. Due to the small size of the tumors, the maximum number of fragments per vial has not been reached. The total pre-REP cells (TVC) were collected and counted on day 11, for the Gen 2 process, and on day 7 for the Gen 3 process. To compare the two pre-REP arms, the cell count was divided by the number of fragments supplied in the culture in order to calculate an average of viable cells per fragment. As indicated in the table below, the Gen 2 process consistently cultivated more cells per fragment when compared to the Gen 3 process. An extrapolated calculation of the number of TVC predicted for the Gen 3 process on day 11 was used, which was calculated by dividing pre- REP by 7 and then multiplying by 11. Table 37: Pre-REP cell counts Tumor ID L4054 L4055 * M1085T Process Gen 2 Gen 3 Gen 2 Gen 3 Gen 2 Gen 3 TVC pre-REP 1.42E + 08 4.32E +07 2.68E + 07 1.38E + 07 1.23E + 07 3.50E + 06 Number of fragments 21 21 24 24 16 16 Average CVT per fragment in pre-REP 6.65E + 06 2.06E + 06 1 , 12E + 06 5.75E + 05 7.66E + 05 2.18E + 05
[001849] [001849] Table 38: Total viable cell count and expansion times in the final product TIL: For the Gen 2 and Gen 3 processes, TVC was counted by process condition, and the percentage of viable cells was generated for each day of the process . At collection, day 22 (Gen 2) and day 16 (Gen 3), cells were collected and the CVT count was established. TVC was then divided by the number of fragments provided on day 0, to calculate an average of viable cells per fragment. The expansion times were calculated by dividing the TVC collected by TVC at the beginning of REP. As shown in the table, comparing Gen 2 and Gen 3, the number of times for expansions was similar for L4054; in the case of L4055, the number of expansion times was greater for the Gen 2 process. Specifically, in this case, the medium was heated up to 24 in advance of the REP start day. A greater number of expansion times were also observed in Gen 3 for M1085T. An extrapolated calculation of the number of TVC predicted for the Gen 3 process on day 22 was used, which was calculated by dividing pre-REP TVC by 16 and then multiplying by 22. Table 38: Total viable cell count and product expansion times final TIL tumor ID L4054 L4055 M1085T Process Gen 2 Gen 3 Gen 2 Gen 3 Gen 2 Gen 3 No. 21 21 24 24 16 16 Fragments
[001850] [001850] Table 39:% viability of the final product TIL: At collection, the final products TIL of REP were compared against release criteria for% viability. All conditions for the Gen 2 and Gen 3 processes exceeded the 70% viability criterion and were comparable between processes and tumors. Table 39:% viability of REP tumor ID L4054 L4055 M1085T Process Gen 2 Gen 3 Gen 2 Gen 3 Gen 2 Gen 3 Start of REP 98.23% 97.97% 97.43% 92.03% 81.85% 68.27% Vertical scheduling 94.00% 93.57% 90.50% 95.93% 78.55% 71.15% Collection 87.95% 89.85% 87.50% 86.70% 86.10 % 87.45%
[001851] [001851] Table 40: Estimation of cell count per additional bottle for the Gen 3 process. Because the number of fragments per bottle is below the maximum number required, an estimate for cell count on the day of collection was calculated for each tumor. The estimate was based on the expectation that the clinical tumors would be large enough to seed 2 or 3 vials on day 0. Table 40: Extrapolated estimate for calculating cell counts for a full scale of 2 and 3 vials in the Gen 3 ID Process tumor L4054 L4055 M1085T 3 Frasc Gen 3 process 2 vials 3 Vials 2 vials 3 Vials 2 vials 2.30E Collection estimate 3.68E + 10 5.52E + 10 1.75E + 10 2.63E + 10 1.54E + 10 +10 Immunophenotyping: Comparison of phenotypic markers in the final TIL product:
[001852] [001852] Three tumors L4054, L4055 and M1085T underwent expansion of TIL in both processes, Gen 2 and Gen 3. Upon collection, the final products TIL REP TIL were subjected to analysis by flow cytometry to test for purity, markers of differentiation and memory. For all conditions, the percentage of TCR a / b + cells was above 90%.
[001853] [001853] TILs collected from the Gen 3 process showed a greater expression of CD8 and CD28 when compared to the TILs collected from the Gen 2 process. The Gen 2 process showed a higher percentage of CD4 +. See Figure 3 (A, B, C). Comparison of memory marker markers in the final TIL product:
[001854] [001854] TILs collected from the Gen 3 process showed greater expression in central memory compartments when compared to the TILs collected from the Gen 2 process. See Figure 4 (A, B, C). Comparison of activation and exhaustion markers in the final TIL product:
[001855] [001855] Activation and exhaustion markers were analyzed in TIL of two L4054 and L4055 tumors to compare the final TIL product by the TILs expansion processes, Gen 2 and Gen 3. The activation and exhaustion markers were comparable between the Gen processes 2 and Gen 3. See Figure 5 (A, B); Figure 6 (A, B). Gamma interferon secretion upon restimulation:
[001856] [001856] On the day of collection, day 22 for Gen 2 and day 16 for Gen 3, TIL were submitted to restimulation during the night with plates coated with anti-CD3 for L4054 and L4055. M1085T restimulation was performed using anti-CD3, CD28 and CD137 beads. The supernatant was collected after 24 hours of restimulation under all conditions and the supernatant was frozen. IFNγ analysis by ELISA was verified in the supernatant of both processes at the same time, using the same ELISA plate. Higher IFNγ production was observed in the Gen 3 process in the three analyzed tumors. See Figure 7 (A, B, C). Measurement of IL-2 levels in the culture medium:
[001857] [001857] To compare the consumption of IL-2 between the Gen 2 and Gen 3 processes, the cell supernatant was collected at the beginning of REP, in the vertical scale and on the day of collection, in tumor L4054 and L4055. The amount of IL-2 in the cell culture supernatant was measured by the kit
[001858] [001858] The levels of metabolic substrates, such as D-glucose and L-glutamine, were measured as substitutes for the overall consumption of the medium. Their respective metabolites, such as lactic acid and ammonia, were measured. Glucose is a simple sugar in the medium that is used by the mitochondria to produce energy in the form of ATP. When glucose is oxidized, lactic acid is produced (lactate is an ester of lactic acid). Lactate is strongly produced during the exponential cell growth phase. Elevated lactate levels have a negative impact on cell culture processes. See Figure 9 (A, B).
[001859] [001859] The medium exhausted by L4054 and L4055 was collected on the days of beginning of REP, vertical scaling and collection for both processes, Gen 2 and Gen 3. The collection of the exhausted medium was on Day 11, day 16 and day 22 for Gen 2; for Gen 3 it was on day 7, day 11 and day 16. The supernatant was analyzed in a CEDEX Bio-analyzer for concentrations of glucose, lactic acid, glutamine, glutamax and ammonia.
[001860] [001860] L-glutamine is an unstable essential amino acid needed in cell culture medium formulations. Glutamine contains an amine, and this structural amide group can transport and deliver nitrogen to cells. When L-glutamine is oxidized, a toxic by-product, ammonia, is generated by the cell. To neutralize the degradation of L-glutamine, the medium for the Gen 2 and Gen 3 processes has been supplemented with Glutamax, which is more stable in aqueous solutions and does not spontaneously degrade. In the two tumor lines, the Gen 3 arm showed a decrease in L-glutamine and
[001861] [001861] Flow-FISH technology was used to measure the average length of the telomere repetition in L4054 and L4055 in the Gen 2 and Gen 3 process. The determination of a relative telomere length (RTL) was calculated with the Telomere PNA kit / FITC for flow cytometry analysis by DAKO. Gen 3 showed telomere length comparable to that of Gen 2. CD3 analysis
[001862] [001862] To determine the clonal diversity of the cellular products generated in each process, the final product TIL, collected for L4054 and L4055, was sampled and analyzed for clonal diversity by sequencing the CDR3 portion of T cell receptors.
[001863] [001863] Table 41: Comparison between Gen 2 and Gen3 of the percentage of shared single sequences of CDR3 in L4054 in the collected TIL cell product. 199 sequences are shared between the final Gen 3 and Gen 2 product, corresponding to 97.07% of 80% of the best unique Gen 2 CDR3 sequences shared with the final Gen 3 product. Table 41: Comparison of shared sequences from uCDR3 between the Gen 2 and Gen 3 processes in L4054. No. of uCDR3 All uCDR3 80% main of uCDR3 (% overlap) Gen 2 Gen 3 Gen 2 Gen 3 Gen 2-L4054 8915 4355 (48.85%) 205 199 (97.07%) Gen 3-L4054 - 18130 - 223
[001864] [001864] Table 42: Comparison between Gen 2 and Gen3 of the percentage of unique shared CDR3 sequences in L4055 in the collected TIL cell product. 1833 strings are shared between the final product of
[001865] [001865] CM1 and CM2 media were previously prepared without filtration and kept at 4 ºC until use for tumor L4055 in the Gen 2 and Gen 3 process.
[001866] [001866] The medium was heated to 37 ºC for 24 hours in advance for tumor L4055 on the day of beginning of REP for the Gen 2 and Gen 3 process.
[001867] [001867] LDH was not measured in the supernatants collected in the processes.
[001868] [001868] The T cell count in M1085T was performed with the cell counter K2 cellometer.
[001869] [001869] In the M1085T tumor, there were no samples available, such as supernatant for metabolic analysis, TIL product for analysis of activation and exhaustion markers, telomere length and CD3 - TCR vb analysis. Conclusions
[001870] [001870] This example compares 3 tumor tissues from independent donors in terms of functional quality attributes, broader phenotypic characterization and medium consumption between the Gen 2 and Gen processes
[001871] [001871] The comparison of pre-REP and REP expansion between Gen 2 and Gen was evaluated in terms of total viable cells generated and viability of the total nucleated cell population. Cell doses on the day of collection were not comparable between Gen 2 (22 days) and Gen 3 (16 days). The doses of Gen 3 cells were lower than those of Gen 2 by around 40% of the total viable cells collected in the collection.
[001872] [001872] An extrapolated number of cells was calculated for the Gen 3 process, assuming that pre-REP collection occurred on day 11 instead of day 7 and REP collection on Day 22 instead of day 16. Both cases show a closer number in TVC, compared to the Gen 2 process, indicating that early activation would allow a better overall performance in the growth of TIL. Bottom row of Tables 4 and 5.
[001873] [001873] In the case of extrapolated value for extra flasks (2 or 3) in the Gen 3 process, assuming a larger size of processed tumor, and reaching the number of fragments required according to the process as described. It was observed that a similar dose may be achievable in TVC in the Collection on Day 16 for the Gen 3 process when compared to the Gen 2 process on Day 22. This observation is important and indicates that an early activation of the culture can provide better performance of TIL in less processing time.
[001874] [001874] The pre-REP comparison and REP expansion, between Gen 2 and Gen 3, was evaluated in terms of total viable cells generated and viability of the total nucleated cell population. total nucleated cells. Cell doses on the day of collection were not comparable between Gen 2 (22 days) and Gen 3 (16 days). The doses of Gen 3 cells were lower than those of Gen 2 by around 40% of the total viable cells collected in the collection.
[001875] [001875] In terms of phenotypic characterization, a greater expression of CD8 + and CD28 + was observed in three tumors in the Gen 3 process when compared to the Gen 2 process. These data indicate that the Gen 3 process further improves the attributes of the final TIL product when compared to Gen 2.
[001876] [001876] The Gen 3 process revealed slightly larger central memory compartments compared to the Gen 2 process.
[001877] [001877] The Gen 2 and Gen 3 process showed comparable activation and exhaustion markers, despite the shorter duration of the Gen 3 process.
[001878] [001878] IFN gamma (IFNγ) production was 3 times higher in the product
[001879] [001879] The length of the telomeres in the final product TIL was comparable between Gen 2 and Gen 3.
[001880] [001880] Glucose and Lactate levels were comparable between the final products of Gen 2 and Gen 3, suggesting that the levels of nutrients in the middle of the Gen 3 process were not affected by the volume having ceased to be removed on each day of the process and less overall volume of the medium in the process when compared to Gen 2.
[001881] [001881] The global Gen 3 process showed a reduction of almost times in the process time when compared to Gen 2, which would result in a substantial reduction in the cost of goods (COGs) for the TIL product expanded by the Gen 3 process.
[001882] [001882] IL-2 consumption indicates a general trend of IL-2 consumption in the Gen 2 process and, in the Gen 3 process, IL-2 became higher due to the non-removal of the old medium.
[001883] [001883] The Gen 3 process revealed higher clonal diversity, as measured by CDR3 TCRab sequence analysis.
[001884] [001884] The addition of feeders and OKT-3 on day 0 of the pre-REP allowed an early activation of TIL and better overall performance in TIL growth by the Gen 3 process.
[001885] [001885] Table 43 describes various modalities and results for the Gen 3 process, compared to the current Gen 2 process. Table 43: Exemplary Gen 3 process
[001886] [001886] The examples presented above are offered to provide those skilled in the art with a full exposition and description of how to prepare and use the modalities of the compositions, systems and methods of the invention, and are not intended to limit the scope of what the inventors regard as your invention. Modifications to the modes described above for carrying out the invention that are obvious to those skilled in the art are intended to fall within the scope of the following claims. All patents and publications mentioned in the specification are indicative of the skill levels of the technicians in the subject to which the invention relates. All references cited in this exhibition are incorporated by reference to the same extent as they would be if each reference had been incorporated, in its entirety, by reference individually.
[001887] [001887] TILs have been successfully expanded from thick needle biopsies isolated from pancreatic tumors. TILs have also been expanded from the FNA of certain types of tumor. In the study, no growth of TIL was observed from ovarian tumors (n = 3) and a colorectal tumor (n = 1). The growth of TIL was observed from the FNA obtained from lung tumor, melanoma and cervical tumor. Phenotyping and functional testing of the first and second expansion TIL populations is currently underway. The study data reveal that a significant population of CD4 + and CD8 + T cells are present in the pre-REP aspirate.
[001888] [001888] All section titles and designations are used for clarity and reference purposes only and should not be considered limiting in any way. For example, those skilled in the art will appreciate the usefulness of combining various aspects of different titles and sections, as appropriate, in accordance with the spirit and scope of the invention described herein.
[001889] [001889] All references cited herein are now incorporated, in their entirety, by reference in this patent application and for all purposes to the same extent that each individual publication or patent or patent application would have been specifically and individually indicated for. be incorporated, in its entirety, by reference and for all purposes.
[001890] [001890] Many modifications and variations of this patent application can be made without departing from its spirit and scope, as will be obvious to those skilled in the art. The specific modalities and examples described herein are offered by way of example only, and the patent application should be limited only by the terms of the attached claims, together with the full range of equivalents to which the claims are entitled.

权利要求:
Claims (165)
[1]
1. Method for the expansion of tumor infiltrating lymphocytes (TILs) in a therapeutic population of TILs, characterized by the fact that it comprises: (i) obtaining a first population of TILs from at least one fine needle aspirate (FNA) or at least a small biopsy of a tumor in a patient; (ii) performing a first expansion, cultivating the first population of TILs in a cell culture medium comprising IL-2, to produce a second population of TILs, in which the cell culture medium is optionally supplemented with OKT-3 in either days 1-3, where the first expansion is performed for approximately 3 days to 10 days in order to obtain the second population of TILs, where the second population of TILs comprises at least 5 × 107 TILs between approximately 3 days and 12 days when the first population of TILs is from a small biopsy; and (iii) perform a second expansion, supplementing the cell culture medium of the second population of TILs with additional IL-2, OKT-3 and antigen presenting cells (APCs), to produce a third population of TILs, in which the third population of TILs has a number at least 25 times greater than the second population of TILs, and in which the second expansion is carried out for approximately 3 days to 12 days in order to obtain the third population of TILs, in which the third population of TILs are a therapeutic population of TILs.
[2]
2. Method according to claim 1, characterized by the fact that the cell culture medium comprising IL-2 in step (ii) further comprises OKT-3 and is not optionally supplemented with OKT-3 on any of the days 1- 3, and where step (i) is a step of preparing the first expansion and step (ii) is a second rapid expansion.
[3]
Method according to claim 1 or 2, characterized
2/26 due to the fact that, after step (iii), the cells are removed from the cell culture and cryopreserved in a storage medium before performing step (iv).
[4]
4. Method according to claim 3, characterized by the fact that the cells are thawed before performing step (iv).
[5]
5. Method according to any of the preceding claims, characterized in that step (iv) is repeated one to four times in order to obtain sufficient TILs in the therapeutic population of TILs for a therapeutically effective dose of TILs.
[6]
6. Method according to any of the preceding claims, characterized by the fact that steps (i) to (iii) or (iv) are carried out within a period of approximately 17 days and 24 days.
[7]
7. Method according to any of the preceding claims, characterized by the fact that steps (i) to (iii) or (iv) are carried out within a period of approximately 18 days and 22 days.
[8]
8. Method according to any of the preceding claims, characterized by the fact that steps (i) to (iii) or (iv) are carried out within a period of approximately 20 days and 22 days.
[9]
9. Method according to any of the preceding claims, characterized by the fact that steps (i) to (iii) or (iv) are carried out within approximately 22 days.
[10]
10. Method according to any one of the preceding claims, characterized by the fact that the cells of steps (iii) or (iv) express CD4, CD8 and TCR α β at levels similar to those of recently harvested cells.
[11]
11. Method according to claim 1, characterized by the fact that APCs are peripheral blood mononuclear cells (PBMCs).
[12]
12. Method according to claim 11, characterized
3/26 due to the fact that PBMCs are added to the cell culture on any of days 3 to 12 in step (ii) and / or any of days 11 to 14 in step (iii).
[13]
Method according to claims 2 to 12, characterized in that the third population of TILs comprises an increased subpopulation of effector T cells and / or central memory T cells in relation to the second population of TILs, in which the cells Effector T and / or central memory T cells in the therapeutic population of TILs in step (iv) exhibit one or more characteristics selected from the group consisting of CD27 expression, CD28 expression, longer telomeres, increased CD57 expression and decreased expression of CD56, in relation to effector T cells and / or central memory T cells in the third cell population.
[14]
Method according to claim 13, characterized in that effector T cells and / or central memory T cells exhibit increased CD57 expression and decreased CD56 expression.
[15]
15. Method according to any of the preceding claims, characterized by the fact that APCs are artificial APCs (aAPCs) or are autologous APCs.
[16]
16. Method according to any of the preceding claims, characterized by the fact that the therapeutic population of TILs is infused into a patient.
[17]
17. Method according to claim 1, characterized by the fact that the first expansion in step (ii) is carried out by further supplementing the cell culture medium of the second population of TILs with OKT-3, IL-15, an agonist antibody of OX40 and / or 4-1BB agonist antibody.
[18]
18. Method according to claim 1, characterized by the fact that the second expansion in step (iii) is performed by further supplementing the cell culture medium of the second population of TILs with IL-
4/26 15, OX40 agonist antibody and / or 4-1BB agonist antibody.
[19]
19. Method according to claim 1, characterized by the fact that the FNA in step (i) comprises at least 400,000 TILs.
[20]
20. Method according to claim 1, characterized by the fact that the small biopsy is obtained from a tumor selected from the group consisting of pancreatic, melanoma, breast and ovarian.
[21]
21. Method according to claim 1, characterized by the fact that the ANF is obtained from a tumor selected from the group consisting of lung, melanoma, head and neck, cervical, ovarian, pancreatic, glioblastoma, colorectal and sarcoma.
[22]
22. Method according to claim 21, characterized by the fact that the lung tumor is a non-small cell lung carcinoma (NSCLC) and, optionally, in which the patient has previously undergone surgical treatment.
[23]
23. Method according to claim 1, characterized by the fact that the TILs in step (i) are obtained from an FNA.
[24]
24. Method according to claim 1, characterized in that the FNA is obtained using a 25-18 gauge needle.
[25]
25. Method according to claim 1, characterized by the fact that the TILs in step (i) are obtained from a small biopsy.
[26]
26. Method according to claim 1, characterized by the fact that the small biopsy is obtained using a 16-11 gauge needle.
[27]
27. Method according to claim 1, characterized by the fact that step (iii) is repeated one to four times in order to obtain sufficient TILs in the therapeutic population of TILs for a therapeutically effective dose of TILs.
[28]
28. Method according to claim 27, characterized in that the number of TILs sufficient for a therapeutically dose
Effective 5/26 is between approximately 2.3 × 1010 and 13.7 × 1010.
[29]
29. Method for the expansion of tumor infiltrating lymphocytes (TILs) in a therapeutic population of TILs, characterized by the fact that it comprises: (i) performing a first expansion by cultivating a first population of TILs from a fine needle aspirate ( FNA) or a small biopsy of a tumor in a patient in a cell culture medium, comprising IL-2, to obtain a second population of TILs, in which the cell culture medium is supplemented with OKT-3 on any of the days 1-3, where the first expansion is performed for approximately 3 days to 12 days in order to obtain the second population of TILs, where the second population of TILs comprises at least 5 × 107 TILs between approximately 3 days and 12 days, when the first population of TILs is from a small biopsy; and (ii) perform a second expansion, supplementing the cell culture medium of the second population of TILs with additional IL-2, OKT-3 and antigen presenting cells (APCs) to obtain a third population of TILs, in which the third population of TILs has a number at least 25 times greater than the second population of TILs, and in which the second expansion is performed for approximately 3 days to 12 days in order to obtain the third population of TILs, in which the third population of TILs is a therapeutic population of TILs.
[30]
30. The method of claim 29, characterized in that the cell culture medium comprising IL-2 in step (ii) further comprises OKT-3 and is not optionally supplemented with OKT-3 on any of the days 1- 3, and where step (i) is a first expansion preparation step and step (ii) is a rapid second expansion step.
[31]
31. Method according to claim 29 or 30, characterized in that the cells of the cell culture medium in the step
6/26 (ii) are removed and cryopreserved in a storage medium before step (iii).
[32]
32. Method according to claim 30, characterized in that the cells are thawed before step (iii).
[33]
33. Method according to any of claims 29 to 31, characterized in that the APCs are artificial APCs (aAPCs) or are autologous APCs.
[34]
34. Method according to any one of claims 29 to 32, characterized in that the therapeutic population of TILs is infused into a patient.
[35]
35. Method according to claim 29, characterized by the fact that the first expansion in step (i) is carried out by further supplementing the cell culture medium of the second population of TILs with OKT-3, IL-15, agonist antibody of OX40 and / or 4-1BB agonist antibody.
[36]
36. Method according to claim 29, characterized by the fact that the second expansion in step (ii) is performed by further supplementing the cell culture medium of the second population of TILs with IL-15, OX40 agonist antibody and / or 4-1BB agonist antibody.
[37]
37. Method according to claim 29, characterized in that the second additional expansion in step (iii) is performed by further supplementing the cell culture medium of the third TIL population with IL-15, OX40 agonist antibody and / or 4- 1BB agonist antibody.
[38]
38. Method according to claim 29, characterized by the fact that the FNA in step (i) comprises at least 400,000 TILs.
[39]
39. Method according to claim 29, characterized by the fact that the small biopsy is obtained from a tumor selected from the group consisting of pancreatic, melanoma, breast and ovarian.
[40]
40. Method according to claim 29, characterized
7/26 due to the fact that the ANF is obtained from a tumor selected from the group consisting of lung, melanoma, head and neck, cervical, ovarian, pancreatic, glioblastoma, colorectal and sarcoma.
[41]
41. Method according to claim 39, characterized in that the lung tumor is a non-small cell lung carcinoma (NSCLC) and, optionally, in which the patient has previously undergone surgical treatment.
[42]
42. Method according to claim 29, characterized in that the TILs in step (i) are obtained from an FNA.
[43]
43. Method according to claim 29, characterized in that the FNA is obtained using a 25-18 gauge needle.
[44]
44. Method according to claim 29, characterized in that the TILs in step (i) are obtained from a small biopsy.
[45]
45. Method according to claim 29, characterized in that the small biopsy is obtained using a 16-11 gauge needle.
[46]
46. Method according to claim 29, characterized in that step (ii) is repeated one to four times in order to obtain sufficient TILs in the therapeutic population of TILs for a therapeutically effective dose of TILs.
[47]
47. Method according to claim 45, characterized in that the number of TILs sufficient for a therapeutically effective dose is between approximately 2.3 × 1010 and 13.7 × 1010.
[48]
48. Method according to any one of claims 29 to 47, characterized in that the third population of TILs comprises an increased subpopulation of effector T cells and / or central memory T cells in relation to the second population of TILs, wherein effector T cells and / or central memory T cells exhibit one or more characteristics selected from the group consisting of expression of
8/26 CD27, CD28 expression, longer telomeres, increased CD57 expression and decreased CD56 expression, in relation to effector T cells and / or central memory T cells in the third cell population.
[49]
49. Method according to claim 48, characterized by the fact that effector T cells and / or central memory T cells exhibit increased expression of CD57 and decreased expression of CD56.
[50]
50. Method for the treatment of an individual with cancer, characterized by the fact that it comprises administering expanded tumor infiltrating lymphocytes (TILs) and comprising: (i) obtaining a first population of TILs from a fine needle aspirate (FNA) or a small biopsy obtained from a tumor in a patient; (ii) perform a first expansion, cultivating the first population of TILs in a cell culture medium comprising IL-2, to produce a second population of TILs, in which the cell culture medium is supplemented with OKT-3 in any of the days 1-3, where the first expansion is performed for approximately 3 days to 12 days in order to obtain the second population of TILs, where the second population of TILs comprises at least 5 × 107 TILs between approximately 3 days and 12 days when the first population of TILs is from a small biopsy; (iii) perform a second expansion, supplementing the cell culture medium of the second population of TILs with additional IL-2, OKT-3 and antigen presenting cells (APCs), to produce a third population of TILs, in which the third population of TILs has a number at least 25 times greater than the second population of TILs, and in which the second expansion is performed for approximately 3 days to 12 days in order to obtain the third population of TILs, in which the third population of TILs it is a therapeutic population of TILs; and (iv) administering a therapeutically effective dose of the third
9/26 population of TILs to the patient.
[51]
51. Method according to claim 50, characterized in that the cell culture medium comprising IL-2 in step (ii) further comprises OKT-3 and is not optionally supplemented with OKT-3 on any of the days 1- 3, and where step (i) is a first expansion preparation step and step (ii) is a rapid second expansion step.
[52]
52. Method according to claim 50, characterized in that, after step (ii), the cells are removed from the cell culture medium and cryopreserved in a storage medium before step (iv).
[53]
53. Method according to claim 50, characterized in that the cells are thawed before step (iv).
[54]
54. Method according to claim 50, characterized in that step (iii) is repeated one to four times in order to obtain sufficient TILs in the therapeutic population of TILs for a therapeutically effective dose of TILs.
[55]
55. Method according to claims 50 to 54, characterized by the fact that APCs are artificial APCs (aAPCs) or are autologous APCs.
[56]
56. Method according to claim 50, characterized by the fact that the first expansion in step (ii) is carried out by further supplementing the cell culture medium of the second population of TILs with OKT-3, IL-15, an agonist antibody of OX40 and / or 4-1BB agonist antibody.
[57]
57. Method according to claim 50, characterized by the fact that the second expansion in step (iii) is performed by further supplementing the cell culture medium of the second population of TILs with IL-15, OX40 agonist antibody and / or 4-1BB agonist antibody.
[58]
58. Method according to claim 50, characterized by the fact that the FNA in step (i) comprises at least 400,000 TILs.
10/26
[59]
59. Method according to claim 50, characterized by the fact that FNA is obtained from a tumor selected from the group consisting of lung, melanoma, head and neck, cervical, ovarian, pancreatic, glioblastoma, colorectal and sarcoma.
[60]
60. The method of claim 59, characterized in that the lung tumor is a non-small cell lung carcinoma (NSCLC) and, optionally. in which the individual has previously undergone surgical treatment.
[61]
61. Method according to claim 50, characterized in that the TILs in step (i) are obtained from an FNA.
[62]
62. Method according to claim 50, characterized in that the FNA is obtained using a 25-18 gauge needle.
[63]
63. Method according to claim 50, characterized by the fact that the TILs in step (i) are obtained from a small biopsy.
[64]
64. Method according to claim 50, characterized in that the small biopsy is obtained using a 16-11 gauge needle.
[65]
65. The method of claim 50, characterized in that step (iii) is repeated one to four times in order to obtain sufficient TILs in the therapeutic population of TILs for a therapeutically effective dose of TILs.
[66]
66. The method of claim 65, characterized in that the number of TILs sufficient for a therapeutically effective dose is between approximately 2.3 × 1010 and 13.7 × 1010.
[67]
67. Method according to any one of claims 50 to 66, characterized in that the third population of TILs comprises an increased subpopulation of effector T cells and / or central memory T cells in relation to the second population of TILs, wherein effector T cells and / or central memory T cells exhibit one or more
11/26 characteristics selected from the group consisting of CD27 expression, CD28 expression, longer telomeres, increased CD57 expression, and decreased CD56 expression, in relation to effector T cells and / or central memory T cells in the third cell population.
[68]
68. The method of claim 67, characterized by the fact that effector T cells and / or central memory T cells exhibit increased CD57 expression and decreased CD56 expression.
[69]
69. Method according to any of claims 50 to 68, characterized by the fact that the cancer is selected from the group consisting of melanoma, cervical cancer, head and neck cancer, glioblastoma, ovarian cancer, sarcoma, cancer of pancreas, bladder cancer, breast cancer, triple negative breast cancer and non-small cell lung carcinoma.
[70]
70. Method for treating an individual with cancer, characterized by the fact that it comprises administering expanded tumor infiltrating lymphocytes (TILs) and comprising: (i) performing a first expansion by cultivating a first population of TILs from a fine needle aspirate (FNA) or a small biopsy of a tumor in a patient in a cell culture medium comprising IL-2 to obtain a second population of TILs, in which the cell culture medium is supplemented with OKT-3 on any of the days 1 -3, where the first expansion is performed for approximately 3 days to 12 days in order to obtain the second population of TILs, where the second population of TILs comprises at least 5 × 107 TILs between approximately 3 days and 12 days when the first population of TILs is from a small biopsy; (ii) perform a second expansion, supplementing the cell culture medium of the second population of TILs with additional IL-2, OKT-3 and antigen presenting cells (APCs) to obtain a third population
12/26 TILs, where the third population of TILs has a number at least 25 times that of the second population of TILs, and where the second expansion is performed for approximately 3 days to 12 days in order to obtain the third population of TILs, where the third population of TILs is a therapeutic population of TILs; and (iii) administering a therapeutically effective dose of the therapeutic population of TILs to the patient.
[71]
71. Method according to claim 70, characterized in that the cell culture medium comprising IL-2 in step (ii) further comprises OKT-3 and is not optionally supplemented with OKT-3 on any of the days 1- 3, and where step (i) is a first expansion preparation step and step (ii) is a rapid second expansion step.
[72]
72. Method according to claim 70 or 71, characterized in that the cells of the cell culture medium in step (ii) are removed and cryopreserved in a storage medium before step (iii).
[73]
73. Method according to claim 72, characterized in that the cells are thawed before step (iii).
[74]
74. Method according to any of claims 70 to 73, characterized in that the APCs are artificial APCs (aAPCs) or are autologous APCs.
[75]
75. Method according to any one of claims 70 to 74, characterized in that the APCs are peripheral blood mononuclear cells (PBMCs).
[76]
76. Method according to any of claims 69 to 75, characterized in that the therapeutic population of TILs is infused into a patient.
[77]
77. Method according to claim 70, characterized by the fact that the first expansion in step (i) is carried out by supplementing
13/26 further the cell culture medium of the second population of TILs with OKT-3, IL-15, OX40 agonist antibody and / or 4-1BB agonist antibody.
[78]
78. Method according to claim 70, characterized by the fact that the second expansion in step (iii) is carried out by further supplementing the cell culture medium of the second population of TILs with IL-15, OX40 agonist antibody and / or 4-1BB agonist antibody.
[79]
79. The method of claim 70, characterized by the fact that the FNA in step (i) comprises at least 400,000 TILs.
[80]
80. Method according to claim 70, characterized by the fact that the small biopsy is obtained from a tumor selected from the group consisting of pancreatic, melanoma, breast and ovarian.
[81]
81. Method according to claim 70, characterized by the fact that FNA is obtained from a tumor selected from the group consisting of lung, melanoma, head and neck, cervical, ovarian, pancreatic, glioblastoma, colorectal and sarcoma.
[82]
82. Method according to claim 81, characterized in that the lung tumor is a non-small cell lung carcinoma (NSCLC) and, optionally, in which the individual has previously undergone surgical treatment.
[83]
83. The method of claim 70, characterized in that the TILs in step (i) are obtained from an FNA.
[84]
84. Method according to claim 70, characterized in that the FNA is obtained using a 25-18 gauge needle.
[85]
85. Method according to claim 70, characterized in that the TILs in step (i) are obtained from a small biopsy.
[86]
86. Method according to claim 70, characterized in that the small biopsy is obtained using a 16-11 gauge needle.
[87]
87. The method of claim 70, characterized
14/26 by the fact that step (ii) is repeated one to four times in order to obtain sufficient TILs in the therapeutic population of TILs for a therapeutically effective dose of TILs.
[88]
88. Method according to claim 87, characterized in that the number of TILs sufficient for a therapeutically effective dose is between approximately 2.3 × 1010 and 13.7 × 1010.
[89]
89. Method according to any one of claims 70 to 88, characterized in that the third population of TILs comprises an increased subpopulation of effector T cells and / or central memory T cells in relation to the second population of TILs, wherein effector T cells and / or central memory T cells exhibit one or more characteristics selected from the group consisting of CD27 expression, CD28 expression, longer telomeres, increased CD57 expression and decreased CD56 expression, in effector T cells and / or central memory T cells in the third cell population.
[90]
90. Method according to claim 89, characterized by the fact that effector T cells and / or central memory T cells exhibit increased expression of CD57 and decreased expression of CD56.
[91]
91. Method for the expansion of tumor infiltrating lymphocytes (TILs) in a therapeutic population of TILs, characterized by the fact that it comprises: (a) obtaining a first population of TILs from a fine needle aspirate (FNA) or a small biopsy obtained from a tumor in a patient; (b) adding the first population to a closed system; (c) performing a first expansion, cultivating the first population of TILs in a cell culture medium comprising IL-2, to produce a second population of TILs, in which the cell culture medium is supplemented with OKT-3 in any of the days 1-3, when the first
15/26 expansion is performed for approximately 3 days to 12 days in order to obtain that second population of TILs, in which the second population of TILs comprises at least 5 × 107 TILs between approximately 3 days and 12 days when the first population of TILs is from a small biopsy, in which the first expansion is carried out in a closed container providing a first gas permeable surface area, in which the first expansion is carried out for approximately 3 days to 12 days to obtain the second population of TILs, in whereas the second population of TILs has a number at least 25 times greater than the first population of TILs, and in which the transition from step (b) to step (c) occurs without opening the system; (d) perform a second expansion, supplementing the cell culture medium of the second population of TILs with additional IL-2, OKT-3 and antigen presenting cells (APCs), to produce a third population of TILs, in which the second expansion is performed for approximately 3 days to 12 days to obtain the third population of TILs, where the third population of TILs is a therapeutic population of TILs, where the second expansion is performed in a closed container, providing a second permeable surface area gas, and where the transition from step (c) to step (d) occurs without opening the system; (e) harvesting the therapeutic population of TILs obtained in step (d), in which the transition from step (d) to step (e) occurs without opening the system; and (f) transfer the population of TILs harvested in step (e) to an infusion bag, in which the transfer from step (e) to (f) occurs without opening the system.
[92]
92. Method according to claim 91, characterized in that the cell culture medium comprising IL-2 in step (ii) further comprises OKT-3 and is not optionally supplemented with OKT-3 on any of the days 1- 3, and where step (i) is a preparation step
16/26 of the first expansion and step (ii) is a second phase of rapid expansion.
[93]
93. Method according to claim 91 or 92, characterized by the fact that it further comprises the cryopreservation step of the infusion bag comprising the population of TILs harvested in step (f) using a cryopreservation process.
[94]
94. Method according to claim 93, characterized by the fact that the cryopreservation process is performed using a 1: 1 ratio of harvested TILs / CS10 medium.
[95]
95. Method according to any one of claims 91 to 94, characterized in that the APCs are peripheral blood mononuclear cells (PBMCs).
[96]
96. Method according to any of claims 91 to 95, characterized by the fact that PBMCs are irradiated and allogeneic.
[97]
97. Method according to claim 96, characterized by the fact that PBMCs are added to the cell culture on any of days 3 to 12 in step (c) and / or any of days 3 to 12 in step (d) .
[98]
98. Method according to any of claims 91 to 95, characterized in that the antigen presenting cells are artificial antigen presenting cells (aAPCs) or are autologous APCs.
[99]
99. Method according to any one of claims 91 to 98, characterized in that the therapeutic population of TILs is infused into a patient.
[100]
100. Method according to claim 91, characterized by the fact that the first expansion in step (c) is carried out by further supplementing the cell culture medium of the second population of TILs with OKT-3, IL-15, an agonist antibody of OX40 and / or 4-1BB agonist antibody.
[101]
101. Method according to claim 91, characterized by the fact that the second expansion in step (d) is performed by supplementing
17/26 further the cell culture medium of the second population of TILs with IL-15, OX40 agonist antibody and / or 4-1BB agonist antibody.
[102]
102. Method according to claim 91, characterized by the fact that the FNA in step (a) comprises at least 400,000 TILs.
[103]
103. Method according to claim 91, characterized by the fact that the small biopsy is obtained from a tumor selected from the group consisting of pancreatic, melanoma, breast and ovarian.
[104]
104. Method according to claim 91, characterized by the fact that FNA is obtained from a tumor selected from the group consisting of lung, melanoma, head and neck, cervical, ovarian, pancreatic, glioblastoma, colorectal and sarcoma.
[105]
105. Method according to claim 104, characterized by the fact that the lung tumor is a non-small cell lung carcinoma (NSCLC) and, optionally, in which the individual has previously undergone surgical treatment.
[106]
106. Method according to claim 91, characterized in that the TILs in step (a) are obtained from an FNA.
[107]
107. Method according to claim 91, characterized in that the FNA is obtained using a 25-18 gauge needle.
[108]
108. Method according to claim 91, characterized by the fact that the TILs in step (a) are obtained from a small biopsy.
[109]
109. Method according to claim 91, characterized in that the small biopsy is obtained using a 16-11 gauge needle.
[110]
110. Method according to claim 91, characterized by the fact that the harvest in step (e) is carried out using a LOVO system for processing the cells.
[111]
111. Method according to claim 91, characterized in that the cell culture medium is supplied in a container
18/26 selected from the group consisting of a G-container and a Xuri Cellbag.
[112]
112. Method according to claim 91, characterized in that the infusion bag in step (f) is a Hypothermosol infusion bag.
[113]
113. Method according to claim 91, characterized by the fact that steps (a) to (f) are carried out within a period of approximately 17 days to 24 days.
[114]
114. Method according to claim 91, characterized by the fact that steps (a) to (f) are carried out within a period of approximately 18 days to 22 days.
[115]
115. Method according to claim 91, characterized by the fact that steps (a) to (f) are carried out within a period of approximately 20 days to 22 days.
[116]
116. Method according to claim 91, characterized by the fact that steps (a) to (f) are carried out in 22 days or less.
[117]
117. The method of claim 91, wherein steps (a) to (f) and cryopreservation are carried out in 22 days or less.
[118]
118. Method according to any of claims 91 to 117, characterized in that the therapeutic population of TILs harvested in step (e) comprises sufficient TILs for a therapeutically effective dose of TILs.
[119]
119. Method according to claim 118, characterized in that the number of TILs sufficient for a therapeutically effective dose is between approximately 2.3 × 1010 and 13.7 × 1010.
[120]
120. Method according to any of claims 91 to 119, characterized by the fact that steps (b) to (e) are carried out in a single container, in which steps (b) to (e) are carried out in one single container results in an increase in TILs yield per resected tumor
19/26 compared to performing steps (b) to (e) in more than one container.
[121]
121. Method according to any of claims 91 to 120, characterized in that the antigen presenting cells are added to the TILs during the second period in step (d) without reopening the system.
[122]
122. Method according to any one of claims 91 to 121, characterized in that the third population of TILs comprises an increased subpopulation of effector T cells and / or central memory T cells relative to the second population of TILs, wherein effector T cells and / or central memory T cells in the therapeutic population of TILs exhibit one or more characteristics selected from the group consisting of CD27 + expression, CD28 + expression, longer telomeres, increased CD57 expression and decreased expression of CD56 in relation to effector T cells, and / or central memory T cells obtained from the second cell population.
[123]
123. Method according to any one of claims 91 to 122, characterized in that the effector T cells and / or central memory T cells obtained from the third population of TILs exhibit increased expression of CD57 and decreased expression of CD56 in relation to effector T cells and / or central memory T cells obtained from the second cell population.
[124]
124. Method according to any one of claims 91 to 123, characterized in that the risk of microbial contamination is reduced compared to an open system.
[125]
125. Method according to any of claims 91 to 124, characterized in that the TILs of step (g) are infused into a patient.
[126]
126. Method for the treatment of an individual with cancer, characterized by the fact that the method comprises administering lymphocytes
20/26 expanded tumor infiltrators (TILs) and comprising: (a) obtaining a first population of TILs from a fine needle aspirate (FNA) or a small biopsy of a resected tumor from an individual; (b) adding the first population to a closed system; (c) performing a first expansion, cultivating the first population of TILs in a cell culture medium comprising IL-2, to produce a second population of TILs, in which the cell culture medium is supplemented with OKT-3 in any of the days 1-3, where the first expansion is performed for approximately 3 days to 12 days in order to obtain the second population of TILs, where the second population of TILs comprises at least 5 × 107 TILs between approximately 3 days and 12 days when the first population of TILs is from a small biopsy, in which the first expansion is carried out in a closed container providing a first gas-permeable surface area, in which the first expansion is carried out for approximately 3-14 days to obtain the second population of TILs, where the second population of TILs has a number at least 25 times greater than the first population of TILs and where the transition from step (b) to step (c) occurs without opening the system; (d) perform a second expansion, supplementing the cell culture medium of the second population of TILs with additional IL-2, OKT-3 and antigen presenting cells (APCs), to produce a third population of TILs, in which the second expansion is performed for approximately 3 days to 12 days to obtain the third population of TILs, where the third population of TILs is a therapeutic population of TILs, where the second expansion is performed in a closed container providing a second area of permeable surface to gas and in which the transition from step (c) to step (d) occurs without opening the system; (e) harvesting the therapeutic population of TILs obtained in step (d),
21/26 in which the transition from step (d) to step (e) occurs without opening the system; and (f) transfer the population of TILs harvested in step (e) to an infusion bag, in which the transfer from step (e) to (f) occurs without opening the system; (g) optionally cryopreserving the infusion bag comprising the population of TILs harvested in step (f) using a cryopreservation process; and (h) administering a therapeutically effective dose of the third population of TILs from the infusion bag in step (g) to the patient.
[127]
127. The method of claim 126, characterized in that the therapeutic population of TILs harvested in step (e) comprises sufficient TILs to deliver a therapeutically effective dose of TILs in step (h).
[128]
128. Method according to claim 126 or 127, characterized in that the cell culture medium comprising IL-2 in step (ii) further comprises OKT-3 and is not optionally supplemented with OKT-3 on any of the days 1-3, and where step (i) is a first expansion preparation step and step (ii) is a rapid second expansion step.
[129]
129. Method according to any of claims 126 to 128, characterized in that the APCs are artificial APCs (aAPCs) or are autologous APCs.
[130]
130. The method of any one of claims 126 to 129, characterized in that the therapeutic population of TILs is infused into a patient.
[131]
131. Method according to claim 126, characterized by the fact that the first expansion in step (c) is carried out by further supplementing the cell culture medium of the second population of TILs with
22/26 OKT-3, IL-15, OX40 agonist antibody and / or 4-1BB agonist antibody.
[132]
132. Method according to claim 126, characterized by the fact that the second expansion in step (d) is carried out by further supplementing the cell culture medium of the second population of TILs with OKT-3, IL-15, an agonist antibody of OX40 and / or 4-1BB agonist antibody.
[133]
133. Method according to claim 126, characterized by the fact that the FNA in step (a) comprises at least 400,000 TILs.
[134]
134. Method according to claim 126, characterized by the fact that the small biopsy is obtained from a tumor selected from the group consisting of pancreatic, melanoma, breast and ovarian.
[135]
135. Method according to claim 126, characterized by the fact that FNA is obtained from a tumor selected from the group consisting of lung, melanoma, head and neck, cervical, ovarian, pancreatic, glioblastoma, colorectal and sarcoma.
[136]
136. Method according to claim 135, characterized in that the lung tumor is a non-small cell lung carcinoma (NSCLC) and, optionally, in which the individual has previously undergone surgical treatment.
[137]
137. Method according to claim 126, characterized in that the TILs in step (i) are obtained from an FNA.
[138]
138. Method according to claim 137, characterized in that the FNA is obtained using a 25-18 gauge needle.
[139]
139. Method according to claim 126, characterized by the fact that the TILs in step (i) are obtained from a small biopsy.
[140]
140. Method according to claim 139, characterized by the fact that the small biopsy is obtained using a 16-11 gauge needle.
[141]
141. Method according to claim 126, characterized by the fact that the number of TILs sufficient to deliver a dose
23/26 therapeutically effective in step (h) is between approximately 2.3 × 1010 and 13.7 × 1010.
[142]
142. Method according to claim 126, characterized by the fact that the antigen presenting cells (APCs) are PBMCs.
[143]
143. Method according to claim 142, characterized by the fact that PBMCs are added to the cell culture on any of days 3 to 12 in step (c) and / or any of days 3 to 12 in step (d) .
[144]
144. Method according to any one of claims 126 to 143, characterized in that, prior to administering a therapeutically effective dose of TIL cells in step (h), a non-myeloablative lymph-depletion scheme was administered to the patient.
[145]
145. Method according to claim 144, characterized in that the non-myeloablative lymphodepletion scheme comprises the steps of administering cyclophosphamide at a dose of 60 mg / m2 / day for two days, followed by the administration of fludarabine at a dose 25 mg / m2 / day for five days.
[146]
146. Method according to any one of claims 126 to 145, characterized in that it further comprises the step of treating the patient with a high dose IL-2 schedule, starting the day after the administration of TIL cells to the patient in the step (h).
[147]
147. Method according to claim 146, characterized in that the high-dose IL-2 schedule comprises 600,000 or
720,000 IU / kg, administered by intravenous bolus infusion over 15 minutes every eight hours until tolerance.
[148]
148. Method according to any one of claims 126 to 147, characterized in that the third population of TILs comprises an increased subpopulation of effector T cells and / or central memory T cells in relation to the second population of TILs, wherein effector T cells and / or central memory T cells in the therapeutic population
24/26 of TILs exhibit one or more characteristics selected from the group consisting of CD27 + expression, CD28 + expression, longer telomeres, increased CD57 expression and decreased CD56 expression in relation to effector T cells and / or cells Central memory T obtained from the second cell population.
[149]
149. Method according to any one of claims 126 to 148, characterized by the fact that effector T cells and / or central memory T cells in the therapeutic population of TILs exhibit increased expression of CD57 and decreased expression of CD56 in effector T cells and / or central memory T cells obtained from the second cell population.
[150]
150. Method for the expansion of tumor infiltrating lymphocytes (TILs) in a therapeutic population of TILs, characterized by the fact that it comprises: (a) obtaining a first population of TILs from a fine needle aspirate (FNA), a small biopsy , a thick needle biopsy or a small biopsy of a tumor in a patient; (b) perform a first preparation expansion, cultivating the first population of TILs in a cell culture medium comprising IL-2, OKT-3 and antigen presenting cells (APCs), to produce a second population of TILs, in which the first preparation expansion is carried out for a first period of approximately 1 to 14 days in a container comprising a first gas permeable surface area to obtain the second population of TILs, where the second population of TILs has a greater number than the first population of TILs; (c) perform a second rapid expansion, supplementing the cell culture medium of the second population of TILs with additional IL-2, OKT-3 and APCs, to produce a third population of TILs, in which the second rapid expansion is performed by a second period
25/26 approximately 1 to 14 days to obtain the third population of TILs, where the third population of TILs is a therapeutic population of TILs; and (d) harvesting the therapeutic population of TILs from step (c).
[151]
151. Method according to claim 150, characterized by the fact that the first period is approximately 6 to 12 days.
[152]
152. Method according to claim 150 or claim 151, characterized in that the second period is approximately 6 to 12 days.
[153]
153. Method according to claim 150, characterized by the fact that the first period is selected from the group consisting of 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days or 12 days.
[154]
154. Method according to claim 150 or claim 151, characterized by the fact that the second period is selected from the group consisting of 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days or 12 days.
[155]
155. Method according to any of claims 150 to 154, characterized by the fact that APCs are peripheral blood mononuclear cells (PBMCs).
[156]
156. Method according to claim 155, characterized in that the ratio between APCs used in step (b) and the APCs used in step (c) is approximately 1.1: 1; 1.2: 1; 1.3: 1; 1.4: 1; 1.5: 1; 1.6: 1; 1.7: 1; 1.8: 1; 1.9: 1; 2: 1; 2.1: 1; 2.2: 1; 2.3: 1; 2.4: 1; 2.5: 1; 2.6: 1; 2.7: 1; 2.8: 1; 2.9: 1; 3: 1; 3.1: 1; 3.2: 1; 3.3: 1; 3.4: 1; 3.5: 1; 3.6: 1; 3.7: 1; 3.8: 1; 3.9: 1; 4: 1; 4.1: 1; 4.2: 1; 4.3: 1; 4.4: 1; 4.5: 1; 4.6: 1; 4.7: 1; 4.8: 1; 4.9: 1; 5: 1 or, preferably, approximately 1 to 2.
[157]
157. Method according to claim 156, characterized by the fact that the first population of TILs is obtained from a thick needle biopsy.
[158]
158. Method according to claim 157, characterized
26/26 by the fact that the thick needle biopsy is obtained from a tumor selected from the group consisting of a melanoma tumor, an ovarian cancer tumor, a cervical cancer tumor, a non-small lung cancer tumor cells (CPNPC), a lung cancer tumor, a bladder cancer tumor, a breast cancer tumor, a cancer tumor caused by human papillomavirus, a head and neck cancer tumor (including squamous cell carcinoma head and neck (CCECP)), a glioblastoma tumor, a gastrointestinal cancer tumor and a kidney cancer tumor.
[159]
159. Composition, characterized by the fact that it comprises expanded TILs using the methods as defined in any of claims 150 to 158.
[160]
160. Composition according to claim 159, characterized by the fact that it also comprises a cryopreservative.
[161]
161. Composition according to claim 160, characterized in that the cryopreservative comprises dimethylsulfoxide.
[162]
162. Composition according to claim 159, characterized by the fact that it further comprises a cryopreservative and an isotonic agent.
[163]
163. Composition according to Claim 162, characterized in that it further comprises a cryopreservative comprising dimethylsulfoxide and an isotonic agent comprising sodium chloride, sodium gluconate and sodium acetate.
[164]
164. Composition according to claim 162, characterized in that it further comprises a cryopreservative comprising dimethyl sulfoxide and dextran 40 and an isotonic agent comprising sodium chloride, sodium gluconate and sodium acetate.
[165]
165. Composition according to any one of claims 159 to 164, characterized in that the composition is supplied in a sterile infusion pouch.

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同族专利:

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公开号 | 公开日

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CN111601883A|2020-08-28|

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IL274584D0|2020-06-30|

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CR20200251A|2020-07-17|

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US20210309968A1|2021-10-07|

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KR20200100060A|2020-08-25|

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TW201932594A|2019-08-16|

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AU2018368786A1|2020-06-18|

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JP2021503281A|2021-02-12|

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SG11202004457XA|2020-06-29|

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CA3082484A1|2019-05-23|

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PH12020550642A1|2021-03-22|

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WO2019100023A1|2019-05-23|

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EP3710576A1|2020-09-23|

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US20200277573A1|2020-09-03|

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WO2021081378A1|2019-10-25|2021-04-29|Iovance Biotherapeutics, Inc.|Gene editing of tumor infiltrating lymphocytes and uses of same in immunotherapy|

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TW202131946A|2020-01-10|2021-09-01|加拿大商融合製藥公司|Sustained immunotherapy|

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WO2021219990A1|2020-04-28|2021-11-04|Achilles Therapeutics Uk Limited|T cell therapy|

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WO2021226061A1|2020-05-04|2021-11-11|Iovance Biotherapeutics, Inc.|Processes for production of tumor infiltrating lymphocytes and uses of the same in immunotherapy|

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GB202006989D0|2020-05-12|2020-06-24|Gammadelta Therapeutics Ltd|Methods for isolating gamma delta t cells|

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GB202109886D0|2021-07-08|2021-08-25|Achilles Therapeutics Uk Ltd|Assay|

法律状态:
2021-12-07| B350| Update of information on the portal [chapter 15.35 patent gazette]|

优先权:

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申请号 | 申请日 | 专利标题

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US201762588044P| true| 2017-11-17|2017-11-17|

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US62/588044|2017-11-17|

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US201862621515P| true| 2018-01-24|2018-01-24|

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US62/621515|2018-01-24|

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US201862756038P| true| 2018-11-05|2018-11-05|

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US62/756038|2018-11-05|

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PCT/US2018/061865|WO2019100023A1|2017-11-17|2018-11-19|Til expansion from fine needle aspirates and small biopsies|

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