POLYNUCLEOTIDE, CAR POLYPEPTIDE CODED BY THE SAID POLINUCLEOTIDE, VECTOR UNDERSTANDING THE SAID POLY

专利摘要:
the invention provides improved compositions for adoptive t cell therapies for b cell related conditions.
公开号:BR112017012502B1
申请号:R112017012502-1
申请日:2015-12-07
公开日:2020-09-15
发明作者:Richard Morgan；Kevin Friedman
申请人:Bluebird Bio, Inc.；
IPC主号:

专利说明:

CROSS REFERENCE TO RELATED REQUESTS
[001] This claim claims the benefit under 35 USC § 119 (e) of US Provisional Application No. 62 / 200,505, filed on August 3, 2015 and US Provisional Application No. 62 / 091,419, filed on December 12, 2014, each one of which is incorporated herein by reference in its entirety. DECLARATION REGARDING THE SEQUENCE LISTING
[002] The Sequence Listing associated with this application is provided in text format instead of a hard copy, and is hereby incorporated by reference in the specification. The name of the text file containing the String Listing is BLBD_043_02WO_ST25.txt. The text file is 27 KB, was created on December 4, 2015, and is being submitted electronically through EFS-Web, along with the presentation of the specification TECHNICAL FIELD FUNDAMENTALS
The present invention relates to improved compositions and methods for treating B cell-related conditions. More particularly, the invention relates to improved chimeric antigen receptors (CARs) comprising humanized murine anti-BCMA antibodies or binding fragment. to the antigens of these, immune effector cells genetically modified to express these CARs, and use of these compositions to effectively treat conditions related to B cells. DESCRIPTION OF RELATED TECHNIQUE
[004] Several significant diseases involve B lymphocytes, ie B cells. Abnormal B cell physiology can also lead to the development of autoimmune diseases including, but not limited to, systemic lupus erythematosus (SLE). Malignant B-cell transformation leads to cancers including, but not limited to lymphomas, for example, multiple myeloma and non-Hodgkin's lymphoma. The vast majority of patients with B-cell malignancies, including non-Hodgkin's lymphoma (NHL) and multiple myeloma (MM), are significant contributors to cancer mortality. The response of B-cell malignancies to various forms of treatment is varied. Traditional methods of treating B-cell malignancies, including chemotherapy and radiation, have limited utility due to toxic side effects. Immunotherapy with therapeutic anti-CD19, anti-CD20, anti-CD22, anti-CD23, anti-CD52, anti-CD80 and anti-HLA-DR antibodies has provided limited success, due in part to deficient pharmacokinetic profiles, rapid elimination of antibodies for serum proteases and filtration in the glomerulus, and limited penetration at the tumor site and levels of expression of the target antigen in cancer cells. Attempts to use genetically modified cells that express chimeric antigen receptors (CARs) have also met with limited success. In addition, the therapeutic efficacy of a particular antigen-binding domain used in a CAR is unpredictable: if the antigen-binding domain binds too tightly, CAR T cells induce massive cytokine release, resulting in an immune reaction potentially fatal, considered a "cytokine storm" and if the antigen-binding domain binds too weakly, CAR T cells do not have sufficient therapeutic efficacy to clear cancer cells. BRIEF SUMMARY
[005] The invention generally provides improved vectors for generating T cell therapies and methods of using them.
[006] In several embodiments, a chimeric antigen (CAR) receptor is provided comprising: an extracellular domain comprising a humanized murine anti-BCMA antibody (B cell maturation antigen) or antigen-binding fragment of the same that binds one or more epitopes of a human BCMA polypeptide; a transmembrane domain, one or more intracellular co-stimulatory signaling domains and a primary signaling domain.
[007] In particular embodiments, the murine anti-BCMA antibody or antigen-binding fragment that binds to the human BCMA polypeptide is selected from the group consisting of: a lg Camel, lg NAR, Fab fragments, Fab 'fragments, F (ab) '2 fragments, F (ab)' 3 fragments, Fv, single chain Fv antibody (“scFv”), bis-scFv, (scFv) 2, diavalent, triavalent, tetravalent minivalent, disulfide stabilized Fv protein (“DsFv”) and single domain antibody (sdAb, Nanocorpo).
[008] In additional embodiments, the murine anti-BCMA antibody or antigen-binding fragment that binds to the human BCMA polypeptide is a scFv.
[009] In some embodiments, the murine anti-BCMA antibody or antigen-binding fragment thereof comprises one or more CDRs as presented in any of SEQ ID NOs: 1 to 3.
[010] In particular embodiments, the murine anti-BCMA antibody or antigen-binding fragment thereof comprises one or more CDRs as presented in any of SEQ ID NOs: 4 to 6.
[011] In certain embodiments, the murine anti-BCMA antibody or antigen-binding fragment thereof comprises a variable light chain sequence as shown in SEQ ID NO: 7.
[012] In particular embodiments, the variable light chain sequence comprises the CDR sequences presented in SEQ ID NOs: 1 to 3.
[013] In other embodiments, the murine anti-BCMA antibody or antigen-binding fragment thereof comprises a variable heavy chain sequence as shown in SEQ ID NO: 8.
[014] In additional embodiments, the variable heavy chain sequence comprises the CDR sequences shown in SEQ ID NOs: 4 to 6.
[015] In other embodiments, the transmembrane domain is a polypeptide selected from the group consisting of: alpha, beta or zeta chain of the T cell receptor, CD3ε, CD3Ç, CD4, CD5, CD8α, CD9, CD 16, CD22 , CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD 134, CD137, CD152, CD 154 and PD1.
[016] In some embodiments, the transmembrane domain is a polypeptide selected from the group consisting of: CD8α; CD4, CD45, PD1 and CD152.
[017] In certain modalities, the transmembrane domain is CD8a.
[018] In particular modalities, one or more co-stimulatory signaling domains come from a co-stimulatory molecule selected from the group consisting of: CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM) , CD83, CD134 (0X40), CD137 (4-1 BB), CD150 (SLAMF1), CD152 (CTLA4), CD223 (LAG3), CD270 (HVEM), CD273 (PD-L2), CD274 (PD-L1), CD278 (ICOS), DAP10, LAT, NKD2C SLP76, TRIM and ZAP70.
[019] In particular modalities, one or more co-stimulatory signaling domains come from a co-stimulatory molecule selected from the group consisting of: CD28, CD134 and CD137.
[020] In additional modalities, one or more co-stimulating signaling domains are from a co-stimulatory molecule selected from the group consisting of: CD28, CD134 and CD137.
[021] In additional modalities, one or more co-stimulating signaling domains is CD28.
[022] In particular modalities, one or more co-stimulating signaling domains is CD134.
[023] In other modalities, one or more co-stimulating signaling domains is CD137.
[024] In certain embodiments, a CAR comprises a polypeptide from the hinge region.
[025] In other embodiments, the hinge region polypeptide comprises a CD8a hinge region.
[026] In some modalities, a CAR is a spacer region.
[027] In additional embodiments, the spacer region polypeptide comprises CH2 and CH3 regions of IgG1 or IgG4.
[028] In particular embodiments, a CAR comprises a signal peptide.
[029] In other embodiments, the signal peptide comprises a lgG1 heavy chain signal polypeptide, a granulocyte and macrophage colony stimulating factor receptor 2 polypeptide (GM-CSFR2) or a CD8a signal polypeptide.
[030] In an embodiment formula, a CAR comprises an amino acid sequence as presented in SEQ ID NO: 9.
[031] In several embodiments, a polynucleotide encoding a CAR provided for here is provided.
[032] In several particular embodiments, a polynucleotide encoding a CAR is provided, wherein the polynucleotide sequence is shown in SEQ ID NO: 10.
[033] In several certain embodiments, a vector comprising a polynucleotide encoding a CAR considered here or as set out in SEQ ID NO: 10 is provided.
[034] In certain modalities, the vector is an expression vector.
[035] In additional modalities, the vector is an episomal vector.
[036] In particular modalities, the vector is a viral vector.
[037] In other modalities, the vector is a retroviral vector.
[038] In other modalities, the vector is a lentiviral vector.
[039] In one embodiment, a vector encoding a BCMA CAR comprises the polynucleotide sequence shown in SEQ ID NO: 36.
[040] In additional modalities, the lentiviral vector is selected from the group consisting essentially of: human immunodeficiency virus 1 (HIV-1); human immunodeficiency virus 2 (HIV-2), visna-maedi virus (VMV); caprine arthritis-encephalitis virus (CAEV); equine infectious anemia virus (EIAV); feline immunodeficiency virus (IVF); bovine immunodeficiency virus (BIV); and simian immunodeficiency virus (SIV).
[041] In particular modalities, a vector comprises a retroviral LTR from the left (5 '), a Psi packaging signal (ψ), a polypurine tract (polypurine tract) / central DNA flap (cPPT / FLAP) , a retroviral export element; a promoter operably linked to the polynucleotide defined according to claim 26 or claim 27 and a right-hand retroviral LTR (3 ').
[042] In other embodiments, a CAR comprises a heterologous polyadenylation sequence.
[043] In some embodiments, a CAR comprises a post-transcriptional regulatory element of the hepatitis B virus (HPRE) or a post-transcriptional regulatory element of the groundhog (WPRE).
[044] In certain embodiments, the LTR 5 'promoter will be replaced by a heterologous promoter
[045] In other embodiments, the heterologous promoter is a heterologous promoter being a cytomegalovirus (CMV) enhancer, a Rous Sarcoma virus (RSV) promoter or a Simian 40 (SV40) virus promoter.
[046] In particular modalities, LTR5 'or LTR3' is a Lentivirus LTR.
[047] In particular modalities, the LTR3 'comprises one or more modifications.
[048] In some modalities, the LTR3 'comprises one or more deletions.
[049] In certain modalities, the LTR3 'is a self-inactivating LTR (SIN).
[050] In some embodiments, the polyadenylation sequence is a bovine growth hormone polyadenylation or a rabbit signal β-globin polyadenylation sequence
[051] In additional embodiments, a polynucleotide encoding a CAR provided herein comprises an optimized Kozak sequence.
[052] In other embodiments, the promoter operationally linked to the polynucleotide encoding a CAR contemplated here is selected from the group consisting of: a cytomegalovirus (CMV) immediate early gene promoter, an elongation factor alpha 1 promoter ( EF1-a), a phosphoglycosis kinase-1 promoter (PGK), a ubiquitin-C promoter (UBQ-C), a cytomegalovirus enhancer / chicken beta-actin promoter (CAG), polyoma enhancer / promoter herpes simplex thymidine kinase (MC1), a beta actin promoter (β-ACT), a simian 40 virus promoter (SV40) and a myeloproliferative sarcoma virus enhancer, deletion of the negative control region, substituted site promoter dl587rev initiator (MND) connection.
[053] In several modalities, an immune effector cell is provided, comprising a vector contemplated here. In several modalities, the immune effector cell is transduced with a vector contemplated here.
[054] In other modalities, the immune effector cell is selected from the group consisting of: a T lymphocyte and a natural killer cell (NK).
[055] In some embodiments, the immune effector cell is transduced with the vector according to any of claims 28 to 46 and is activated and stimulated in the presence of an inhibitor of the PI3K pathway, thus maintaining the proliferation of immune effector cells transduced in comparison with the proliferation of transduced immune effector cells that were activated and stimulated in the absence of the PI3K pathway inhibitor.
[056] In particular modalities, the immune effector cell activated and stimulated in the presence of the PI3K pathway inhibitor increased the expression of i) one or more markers selected from the group consisting of: CD62L, CD127, CD197 and CD38 or ii) all CD62L, CD127, CD197 and CD38 markers compared to an immune effector cell activated and stimulated in the absence of the PI3K pathway inhibitor.
[057] In one embodiment, the PI3K inhibitor is ZSTK474.
[058] In various embodiments, a composition is provided comprising an immune effector cell contemplated herein and a physiologically acceptable excipient.
[059] In several modalities, a method of generating an immune effector cell comprising a CAR contemplated herein is provided, comprising introducing into a immune effector cell a vector comprising a polynucleotide encoding the CAR.
[060] Method for generating an immune effector cell characterized in that it comprises a CAR defined according to any one of claims 1 to 25 comprising introducing into an immune effector cell the vector defined according to one of claims 28 to 46.
[061] In additional modalities, the method further comprises stimulating the immune effector cell and inducing cell proliferation by contacting the cell with antibodies that bind to CD3 and antibodies that bind to CD28; thus generating a population of immune effector cells.
[062] In particular modalities, the immune effector cell is stimulated and induced to proliferate before introducing the vector.
[063] In certain embodiments, immune effector cells comprise T lymphocytes.
[064] In particular embodiments, immune effector cells comprise NK cells.
[065] In particular embodiments, cells are activated and stimulated in the presence of an inhibitor of the PI3K pathway, thus maintaining the proliferation of transduced immune effector cells compared to the proliferation of immune effector cells that are activated and stimulated in the absence of the inhibitor of via PI3K.
[066] In some embodiments, immune effector cells activated and stimulated in the presence of the PI3K pathway inhibitor increased the expression of i) one or more markers selected from the group consisting of: CD62L, CD127, CD197 and CD38 or ii) all markers CD62L, CD127, CD197 and CD38 compared to immune effector cells activated and stimulated in the absence of the PI3K pathway inhibitor.
[067] In one embodiment, the PI3K inhibitor is ZSTK474.
[068] In various embodiments, a method of treating a condition related to B cells in an individual in need thereof is provided, comprising administering to the individual a therapeutically effective amount of a composition comprising BCMA CAR T cells considered here and optionally, an excipient pharmaceutically acceptable.
[069] In other modalities, the B-cell-related condition is multiple myeloma, non-Hodgkin's lymphoma, B-cell proliferations of uncertain malignant potential, lymphomatoid granulomatosis, post-transplantation lymphoproliferative disorder, an immunoregulatory disorder, rheumatoid arthritis, myvisthenia gravis, purple idiopathic thrombocytopenic, anti-phospholipid syndrome, Chagas disease, Grave's disease, Wegener's granulomatosis, polyarteritis nodosa, Sjogren's syndrome, pemphigus vulgaris, scleroderma, multiple sclerosis, antiphospholipid syndrome, ANCA-associated vasculitis, Goodpasture's disease de Kawasaki, autoimmune hemolytic anemia, and rapidly progressive glomerulonephritis, heavy chain disease, primary or immunocyte-associated amyloidosis or monoclonal gammopathy of undetermined significance.
[070] In other embodiments, the B cell related condition is a malignant B cell disease.
[071] In certain embodiments, B-cell malignancy is multiple myeloma (MM) or non-Hodgkin's lymphoma (NHL).
[072] In certain modalities, MM is selected from the group consisting of: open multiple myeloma, burning multiple myeloma, plasma cell leukemia, non-secretory myeloma, IgD myeloma, osteosclerotic myeloma, solitary bone plasmocytoma and extramedullary plasmacytoma.
[073] In some modalities, NHL is selected from the group consisting of: Burkitt's lymphoma, chronic lymphocytic leukemia / small lymphocytic lymphoma (CLL / SLL), diffuse large B cell lymphoma, follicular lymphoma, large cell immunoblastic lymphoma, precursor B lymphoblastic lymphoma and mantle cell lymphoma.
[074] In particular embodiments, the B cell-related condition is a plasma cell malignancy.
[075] In other embodiments, the B cell-related condition is an autoimmune disease.
[076] In additional modalities, the autoimmune disease is systemic lupus erythematosus.
[077] In certain embodiments, the B cell-related condition is rheumatoid arthritis.
[078] In particular modalities, the B cell-related condition is idiopathic thrombocytopenia purpura, or myasthenia gravis, or autoimmune hemolytic anemia. BRIEF DESCRIPTION OF VARIOUS VIEWS OF THE DRAWINGS
[079] Figure 1 shows a schematic of murine B cell maturation antigen (muBCMA) CAR constructs.
[080] Figure 2a shows the amount of IFNg released from anti-BCMA02 CAR T cells, anti-BCMA10 CAR T cells and CAR19Δ T cells after the cells were co-cultured for 24 hours with K562 cells that express BCMA.
[081] Figure 2b shows the amount of IFNg released from anti-BCMA02 CAR T cells, anti-BCMA10 CAR T cells and CAR19Δ T cells after the cells were co-cultured for 24 hours with K562 cells that lack BCMA expression compared to K562 cells that express BCMA.
[082] Figure 3 shows the amount of inflammatory cytokines in growth media from untransformed control T cells, anti-BCMA02 CAR T cells, anti-BCMA10 T cells and CAR19Δ T cells, stimulated 10 days before of the test.
[083] Figure 4 shows the amount of inflammatory cytokines produced by anti-BCMA02 CAR T cells, anti-BCMA10 T cells and CAR19Δ T cells in the absence of antigenic stimulation.
[084] Figure 5 shows the expression of phenotypic activation markers at the end of the manufacture of anti-BCMA CAR T cells. The expression of HLA-DR and CD25 was measured in anti-BCMA02 CAR T cells, anti-BCMA10 CAR T cells and CAR19Δ T cells.
[085] Figure 6 shows the levels of activated caspase-3, a necessary step in apoptosis and important for IACD in anti-BCMA10 CAR T cells and anti-BCMA02 CAR T cells in the absence of antigenic stimulation.
[086] Figure 7 shows the amount of inflammatory cytokine release in anti-BCMA02 and anti-BCMA10 CAR T cells in medium containing fetal bovine serum (FBS), human AB serum (HABS) or 100ng / ml soluble BCMA.
[087] Figures 8A show the tumor volume in NOD scid gamma (NSG) mice with subcutaneous human cutaneous multiple myeloma tumors <100mm3 (RPMI-8226). The mice were treated with vehicle, 107 T cells of anti-BCMA02 CAR, 107 T cells of anti-BCMA10 CAR or Bortezomib (velcade).
[088] Figures 8B show tumor volume in NOD scid gamma (NSG) mice with 100mm3 human subcutaneous multiple myeloma tumors (RPMI-8226). The mice were treated with vehicle, 107 T cells of anti-BCMA02 CAR, 107 T cells of anti-BCMA10 CAR or Bortezomib (velcade).
[089] Figure 9 shows the level of BCMA expression in lymphoma and leukemia cell lines (circles) and the activity of anti-BCMA CAR T cells for each cell line (IFNy release, boxes). BCMA-negative tumor cell lines (BCMA): myelogenic leukemia (K562), acute lymphoblastic leukemia (NALM-6 and NALM-16); Mantle cell lymphoma (REC-1); Or Hodgkin's lymphoma (HDLM-2) showed little or no IFNy release. BCMA-positive tumor cell lines (BCMA +): chronic B-cell lymphoblastic leukemia (MEC-1), Mantle cell lymphoma (JeKo-1), Hodgkin's lymphoma (RPMI-6666), Burkitt's lymphoma (Daudi cells and Ramos cells ) and multiple myeloma (RPMI-8226) showed substantial release of IFNy.
[090] Figure 10A shows the in vivo activity of vehicle CAR T cells, anti-CD19Δ CAR T cells, anti-CD19 CAR T cells and anti-BCMA CAR T cells for BCMA expressing lymphoma cells from Burkitt (Daudi cells) in an NSG mouse model when CAR T cells are administered to mice at 8 days after tumor induction.
[091] Figure 10B shows the vehicle's in vivo activity, anti-CD19Δ CAR T cells, anti-CD19 CAR T cells and BCMA anti-BCMA T cells expressing Burkitt's lymphoma cells (Daudi cells ) in an NSG mouse model when CAR T cells are administered to mice at 18 days after tumor induction.
[092] Figure 11 shows the potent in vitrode activity of anti-BCMA CAR T cells obtained with a 50% reduction expression of CAR BCMA reduction. (A) T cell populations were transduced with 4x108 and 5x107 transduction units from a lentivirus encoding an anti-BCMA CAR molecule (MOI 5 to 40). The resulting T cell populations were normalized to contain 26 ± 4% positive anti-BCMA + CAR T cells. (B) MFI of normalized anti-BCMA CAR T cells ranged from 885 to 1875 as tested by flow cytometry. (C) K562 cells and K562-BCMA cells were co-cultured with standard anti-BCMA CAR T cells in a 20: 1 or 10: 1 effector (E; T) to achieve (T mixture; 1: 1 of K562 and K562 BCMA cells) showed comparable cytolytic activity.
[093] Figure 12 shows the reliability of the manufacturing process for anti-BCMA CAR T cells. (A), anti-BCMA CAR T products manufactured from PBMCs from 11 individual donors show comparable levels of expansion compared to a corresponding culture of non-transposed donor T cells. (B) T products of anti-BCMA CAR manufactured from the 11 donors showed comparable lentiviral transduction efficiency (VCN). (C) The frequency of positive anti-BCMA CAR T cells was measured by flow cytometry and BCMA expression was comparable across all donors. (D) anti-BCMA CAR T cell products manufactured from 11 donors showed therapeutically relevant levels of IFNy release when exposed to BCMA-expressing K562 cells.
[094] Figure 13 shows Venn diagrams for co-expression of CD127, CD197 and CD38 in CD62L positive anti-BCMA02 T cells that were cultured in the presence of IL-2 or IL-2 and ZSTK474 for ten days. ZSTK474-treated CAR anti-BCMA02 T cells showed an increase in the percentage of cells that co-expressed CD127, CD197 and CD38 compared to anti-BCMA CAR T cells cultured with IL-2 alone.
[095] Figure 14 shows an increased percentage of anti-BCMA02 CAR T cells that express CD8 in cultures treated IL-2 and ZSTK474 (n = 7) compared to cultures treated with IL-2 alone. CD8 expression was determined using a fluorescently labeled anti-CD8 antibody and flow cytometry.
[096] Figure 15 shows the amount of IFN-y released by anti-BCMA02 CAR T cells from 14 donors after culturing with IL-2 alone or with IL-2 and ZSTK474. At the end of the culture period, an equivalent number of anti-BCMA02 CAR T cells were re-cultured for 24 hours in isolated media. The amount of IFN-y released in 24 hours was quantified by ELISA. Culture in ZSTK474 did not significantly increase the release of tonic cytokines from anti-BCMA02 CAR T cells compared to anti-BCMA02 CAR T cells cultured with IL-2 alone.
[097] Figure 16 shows the antitumor activity of anti-BCMA02 T CAR CARs treated with IL-2, or IL-2 and ZSTK474, or an anti-BCMA02 T CAR CAR cell (tBCMA02) with truncated signaling deficiency treated with IL-2 and ZSTK474 in an aggressive tumor model from Daudi. Complete tumor regression was observed in 50% of mice administered by anti-BCMA02 CAR T cells treated with IL-2 and ZSTK474.
[098] Figure 17 shows the anti-tumor activity of anti-BCMA02 CAR T cells treated with IL-2, or IL-2 and ZSTK474 in a multiple myeloma tumor model (RPMI-8226). Animals treated with IL-2 or IL-2 and ZSTK474-cultured anti-BCMA02 CAR T cells completely prevented tumor growth. BRIEF DESCRIPTION OF SEQUENCE IDENTIFIERS
[099] SEQ ID NOs: 1-3 establishes the amino acid sequences of exemplary light chain CDR sequences for BCMA CARs contemplated herein.
[0100] SEQ ID NOs: 4-6 establishes amino acid sequences of exemplary heavy chain CDR sequences for CARS BCMA contemplated herein.
[0101] SEQ ID NO: 7 establishes an amino acid sequence of an exemplary light chain sequence for BCMA CARs contemplated herein.
[0102] SEQ ID NO: 8 sets out an amino acid sequence of an exemplary heavy chain sequence for BCMA CARs contemplated herein.
[0103] SEQ ID NO: 9 establishes an amino acid sequence of an exemplary BCMA CAR contemplated herein.
[0104] SEQ ID NO: 10 establishes a polynucleotide sequence that encodes an example of CAR BCMA contemplated herein.
[0105] SEQ ID NO: 11 establishes the amino acid sequence of human BCMA.
[0106] SEQ ID NO: 12-22 establishes the amino acid sequence of various linkers.
[0107] SEQ ID NO: 23-35 establishes the amino acid sequence of protease cleavage sites and self-cleavage polypeptide cleavage sites.
[0108] SEQ ID NO: 36 establishes the polynucleotide sequence of a vector encoding a BCMA CAR. DETAILED DESCRIPTION 1. Overview
[0109] The invention generally relates to improved compositions and methods for treating B cell related conditions. As used here, the term "B cell related conditions" refers to conditions involving inappropriate B cell activity and malignancies of B cells.
[0110] In particular embodiments, the invention relates to improved adoptive cell therapy for B cell-related conditions using genetically modified immune effector cells. Genetic approaches offer a potential means of enhancing immune recognition and elimination of cancer cells. A promising strategy is for immune effector cells genetically engineered to express chimeric antigen receptors (CAR) that redirect cytotoxicity towards cancer cells. However, existing adoptive cell immunotherapies to treat B-cell disorders pose a serious risk of compromising humoral immunity because the antigens that target cells expressed in all, or most, B cells. Consequently, such therapies are not clinically desirable and so , a need remains in the art for more efficient therapies for B cell related conditions that spare humoral immunity.
[0111] The improved adoptive cell therapy compositions and methods disclosed here, provide genetically modified immune effector cells that can be easily expanded, exhibit long-term persistence in vivo, and reduce humoral immunity deficiency by targeting antigen expression B cells cell maturation (BCMA, also known as CD269 or tumor necrosis factor receptor superfamily, member 17; TNFRSF17).
[0112] BCMA is a member of the tumor necrosis factor receptor superfamily (see, for example, Thompson et al., J. Exp. Medicine, 192 (1): 129-135, 2000, and Mackay et al., Annu. Rev. Immunol, 21: 231-264, 2003. BCMA binds to cell activation factor B (BAFF) and a proliferation-inducing ligand (APRIL) (see, for example, Mackay et al., 2003 and Kalled et al., Immunological Reviews, 204: 43-54, 2005) Among non-malignant cells, BCMA has been reported to be expressed primarily in plasma cells and subsets of mature B cells (see, for example, Laabi et al., EMBO J., 77 (1): 3897-3904, 1992; Laabi et al., Nucleic Acids Res, 22 (7): 1147-1154, 1994; Kalled et al., 2005; O'Connor et al., J. Exp. Medicine, 199 (1): 91-97, 2004; and Ng et al., J. Immunol., 73 (2): 807-817, 2004. BCMA-deficient mice are healthy and have normal numbers of B cells , but the survival of long-lived plasma cells is impaired (see, for example, O'Connor et al., 2004; Xu et al., M ol. Biol, 21 (12): 4067-4074, 2001; and Schiemann et al., Science, 293 (5537): 211-21 14, 2001). BCMA RNA has been detected universally in multiple myeloma cells and other lymphomas, and BCMA protein has been detected on the surface of plasma cells in patients with multiple myeloma by several investigators (see, for example, Novak et al., Blood, 103 (2): 689- 694, 2004; Neri et al., Clinical Cancer Research, 73 (19): 5903-5909, 2007; Bellucci et al., Blood, 105 (10): 3945-3950, 2005; and Moreaux et al., Blood, 703 (8): 3148-3157, 2004.
[0113] In several embodiments, CARs comprising murine anti-BCMA antibody sequences are highly effective compared to BCMA CARs comprising human antibody sequences; undergo robust in vivo expansion; and recognize human B cells that express BCMA and show cytotoxic activity against BCMA that express B cells, and show no signs of inducing a cytokine storm, a potentially fatal condition, where the cytokines released by activated T cells create a sudden inflammatory response in the system that stimulates a non-infectious fever.
[0114] In one embodiment, a CAR comprising a murine anti-BCMA antibody or antigen binding fragment, a transmembrane domain and one or more intracellular signaling domains is provided.
[0115] In one embodiment, an immune effector cell is genetically modified to express a CAR contemplated here. T cells expressing a CAR are referred to herein as CAR T cells or modified CAR T cells.
[0116] In various embodiments, the genetically modified immune effector cells contemplated herein are administered to a patient with a condition related to B cells, for example, an autoimmune disease associated with B cells or a malignancy of B cells.
[0117] The practice of the invention will use, unless specifically stated otherwise, conventional methods of chemistry, biochemistry, organic chemistry, molecular biology, microbiology, recombinant DNA techniques, genetics, immunology, and cell biology that are within the skill of technique, many of which are described below for the purpose of illustration. Such techniques are explained completely in the literature. See, for example, Sambrook, et al., Molecular Cloning: A Labocamundongory Manual (3rd Edition, 2001); Sambrook, et al., Molecular Cloning: A Labocamundongory Manual (2nd Edition, 1989); Maniatis et al., Molecular Cloning: A Labocamundongory Manual (1982); Ausubel et al., Current Protocols in Molecular Biology (John Wiley and Sons, updated in July 2008); Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; Glover, DNA Cloning: A Practical Approach, vol. I & II (IRL Press, Oxford, 1985); Anand, Techniques for the Analysis of Complex Genomes, (Academic Press, New York, 1992); Transcription and Translation (B. Hames & S. Higgins, Eds., 1984); Perbal, A Practical Guide to Molecular Cloning (1984); Harlow and Lane, Antibodies, (Cold Spring Harbor Labocamundongory Press, Cold Spring Harbor, N.Y., 1998) Current Protocols in Immunology Q. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach and W. Strober, eds., 1991); Annual Review of Immunology; as well as monographs in newspapers such as Advances in Immunology. 8. Definitions
[0118] Unless otherwise defined, all technical and scientific terms used here have the same meaning as commonly understood by those of ordinary skill in the technique to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, preferred embodiments of compositions, methods and materials are described here. For the purposes of the present invention, the following terms are defined below.
[0119] The articles "one / a" and "the" are used here to refer to one or more than one (that is, at least one, or one or more) of the grammatical object of the article. For example, “an element” means an element or one or more elements.
[0120] The use of the alternative (for example, "or") must be understood to mean one, both or any combination of these alternatives thereof.
[0121] The term “and / or” should be understood as one or both alternatives.
[0122] As used here, the term "about" or "approximately" refers to an amount, level, value, number, frequency, percentage, dimension, size, quantity, weight or length that varies as much as 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% to a reference quantity, level, value, number, frequency, percentage, dimension, size, quantity , weight or length. In one embodiment, the term "about" or "approximately" refers to a range of quantity, level, value, number, frequency, percentage, dimension, size, quantity, weight or length ± 15%, ± 10%, ± 9%, ± 8%, ± 7%, ± 6%, ± 5%, ± 4%, ± 3%, ± 2%, or ± 1% about a reference quantity, level, value, number, frequency, percentage, dimension, size, quantity, weight or length.
[0123] Throughout this specification, unless the context otherwise requires, the words "understand", "understand" and "understanding" will be understood to imply the inclusion of a step or element or set of established steps or elements, but not the exclusion of any other step or element or group of steps or elements. By "consisting of" is meant to include, and limited to, whatever follows the phrase "consisting of". In addition, the phrase “consisting of” indicates that the elements listed are necessary or mandatory, and that no other elements may be present. By "consisting essentially of" is meant to include any element listed after the sentence, and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. In addition, the phrase “consisting essentially of” indicates that the elements listed are necessary or mandatory, but that no other elements are present that materially affect the activity or action of the listed elements.
[0124] Reference for this entire specification to "one modality", "one modality", "a particular modality", "a related modality", "a certain modality", "an additional modality", or "another modality" or combinations of these means that a particular feature, structure or feature described in relation to the embodiment is included in at least one embodiment of the present invention. Furthermore, the appearances of the previous phrases in various places throughout this specification are not necessarily all referring to the same modality. In addition, the particular characteristics, structures or characteristics can be combined in any one or more modalities. It is also understood that positive recitation of a characteristic in a modality, serves as a basis for excluding the characteristic in a particular modality. C. Chimeric antigen receptors
[0125] In various modalities, genetically engineered receptors that redirect cytotoxicity of immune effector cells towards B cells are provided. These genetically engineered receptors referred to here as chimeric antigen receptors (CARs). CARs are molecules that combine antibody-based specificity for a desired antigen (eg, BCMA) with an intracellular domain that activates the T cell receptor to generate a chimeric protein that exhibits specific anti-BCMA cellular immune activity. As used here, the term "chimeric" describes being composed of different parts of proteins or DNAs of different origins.
[0126] CARs considered here, comprise an extracellular domain (also referred to as an antigen-specific binding domain or binding domain) that binds to BCMA, a transmembrane domain and an intracellular signaling domain. Engagement of the anti-BCMA antigen-binding domain of CAR with BCMA on the surface of a target cell results in clustering of the CAR and delivers an activation stimulus to the cell containing CAR. The main characteristic of CARs is their ability to redirect specificity of immune effector cells, thereby triggering proliferation, cytokine production, phagocytosis or production of molecules that can mediate cell death of the target antigen that expresses the cell in a manner independent of major histocompatibility (MHC ), exploring the cell-specific targeting ability of monoclonal antibodies, soluble ligands or cell-specific co-receptors.
[0127] In several embodiments, a CAR comprises an extracellular binding domain that comprises a mouse specific BCMA binding domain; a transmembrane domain; one or more intracellular costimulatory signaling domains; and a primary signaling domain.
[0128] In particular embodiments, a CAR is an extracellular binding domain comprising a murine anti-BCMA antibody or an antigen binding fragment thereof; one or more hinge domains or spacer domains; a transmembrane domain including; one or more intracellular costimulatory signaling domains; and a primary signaling domain. 10. Domain Connection
[0129] In particular embodiments, CARs considered here comprise an extracellular binding domain that comprises a murine anti-BCMA antibody or antigen binding fragment thereof specifically binds to a human BCMA polypeptide expressed in a B cell. As used here , the terms “binding domain”, “extracellular domain”, “extracellular binding domain”, “antigen specific binding domain” and “extracellular antigen specific binding domain”, are used interchangeably and provide a CAR with the capacity to bind specifically to the target antigen of interest, for example, BCMA. The binding domain can be derived from a natural, synthetic, semi-synthetic or recombinant source.
[0130] The terms "specific binding affinity" or "specifically bound" or "specifically bound" or "specific binding" or "specifically targeted" as used herein, describe binding of an anti-BCMA antibody or antigen binding fragment of this (or a CAR comprising the same) the BCMA in greater bonding affinity than the previous bond. A binding domain (or a CAR comprising a binding domain or a fusion protein containing a binding domain) "specifically binds" to a BCMA if it binds or associates with BCMA with an affinity or Ka (i.e., an equilibrium association constant of a particular bonding interaction with 1 / M units), for example, greater than or equal to about 105 M'1. In certain embodiments, a binding domain (or a fusion protein thereof) binds to a target with a Ka greater than or equal to about 106 M'1, 107 M'1, 108 M'1, 109 M ' 1, 1010 M'1, 1011 M'1, 1012 M'1, or 1013 M'1. “High affinity” binding domains (or single-chain fusion proteins of these) refer to those binding domains with a Ka of at least 107 M'1, at least 108 M-1, at least 109 M'1, at least 1010 M'1, at least 1011 M'1, at least 1012 M'1, at least 1013 M-1 or greater.
[0131] Alternatively, affinity can be defined as an equilibrium dissociation constant (Kd) of a particular bonding interaction with units of M (for example, 10'5 M to 10'13 M or less). Affinities of binding domain polypeptides and CAR proteins according to the present disclosure can be easily determined using conventional techniques, for example, by competitive ELISA (enzyme linked immunosorbent assay), or by binding association, or displacement assays using labeled ligands, or using a surface plasmon resonance device such as Biacore T100, which is available from Biacore, Inc., Piscataway, NJ, or optical biosensor technology such as the EPIC or EnSpire system which are available from Corning and Perkin Elmer respectively (see also, for example, Scatchard et al. (1949) Ann. NY Acad. Sci. 51: 660; and US Patent Nos. 5,283,173; 5,468,614 or equivalent).
[0132] In one embodiment, the specific binding affinity is about 2 times greater than the antecedent bond, about 5 times greater than the antecedent bond, about 10 times greater than the antecedent bond, about 20 times greater than the leading link, about 50 times greater than the leading link, about 100 times greater than the leading link, or about 1000 times greater than the leading link or more.
[0133] In particular embodiments, the extracellular binding domain of a CAR comprises an antibody or antigen-binding fragment thereof. An "antibody" refers to a binding agent that is a polypeptide comprising at least one variable region of light chain or heavy chain immunoglobulin that specifically recognizes and binds to an epitope of an antigen, such as a peptide, lipid, polysaccharide or nucleic acid containing an antigenic determinant, such as that recognized by an immune cell.
[0134] An "antigen (Ag)" refers to a compound, composition or substance that can stimulate the production of antibodies or a T cell response in an animal, including compositions (such as one that includes a specific cancer protein ) that are injected or absorbed into an animal. An antigen reacts with specific cellular or humoral immunity products, including those induced by heterologous antigens, such as the disclosed antigens. In particular embodiments, the target antigen is an epitope of a BCMA polypeptide.
[0135] An "epitope" or "antigenic determinant" refers to the region of an antigen to which a binding agent binds. Epitopes can be formed either from contiguous amino acids or non-contiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents while epitopes formed by tertiary folding are typically lost in treatment with denaturing solvents. An epitope typically includes at least 3, and more usually, at least 5, about 9, or about 8 to 10 amino acids in a single spatial conformation.
[0136] Antibodies include antigen-binding fragments, such as lg Camel, lg NAR, Fab fragments, Fab 'fragments, F (ab)' 2 fragments, F (ab) '3 fragments, Fv, single chain Fv proteins (scFv) Bis-scFv, (scFv) 2, minibodies, divalents, tribodies, tetribodies, disulfide stabilized Fv proteins (“dsFv”) and single domain antibodies (sdAb, Nanocorp) and portions of full-length antibodies responsible for binding to the antigen. The term also includes genetically engineered forms such as chimeric antibodies (e.g., humanized murine antibodies), heteroconjugate antibodies (such as, bispecific antibodies) and antigen-binding fragment thereof. See also, Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, IL); Kuby, J., Immunology, 3rd Ed., W. H. Freeman & Co., New York, 1997.
[0137] As would be understood by the skilled person and as described elsewhere here, a complete antibody comprises two heavy chains and two light chains. Each heavy chain consists of a variable region and a first, second and third constant region, while each light chain consists of a variable region and a constant region. Heavy mammal chains are classified as α, δ, ε, Y and p. Mammalian light chains are classified as À or K. Immunoglobulins comprising the heavy chains α, δ, ε, y and p are classified as immunoglobulin (lg) A, IgD, IgE, IgG and IgM. The complete antibody forms a "Y" shape. The Y stem consists of the second and third constant region (and for IgE and IgM, the fourth constant region) of two heavy chains linked together and disulfide (inter-chain) bonds are formed on the hinge. Heavy chains y, α and δ have a constant region made up of three tandem (in a row) lg domains, and a hinge region for added flexibility; heavy chains p and ε have a constant region made up of four immunoglobulin domains. The second and third constant regions are referred to as “CH2 domain” and “CH3 domain”, respectively. Each Y stem includes the variable and first constant region of a single heavy chain linked to the variable and constant region of a single light chain. The variable regions of the light and heavy chains are responsible for binding to the antigen.
[0138] The light and heavy chain variable regions contain a "structure" region interrupted by three hypervariable regions, also called "complementarity determining regions" or "CDRs". CDRs can be defined or identified by conventional methods, such as by sequence according to Kabat et al (Wu, TT and Kabat, EA, J Exp Med. 132 (2): 211-50, (1970); Borden, P and Kabat EA, PNAS, 84: 2440-2443 (1987); (see, Kabat et al., Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, 1991, which is incorporated herein by reference), or by structure according to Chothia et al. (Chothia, C. and Lesk, AM, J Mol. Biol., 196 (4): 901-917 (1987), Chothia, C. et al., Nature, 342: 877 -883 (1989)).
[0139] The sequences of the different light or heavy chain structure regions are relatively conserved within a species, such as a human. The structure region of an antibody, which is the combined structure regions of the constituent light and heavy chains, serves to position and align the CDRs in three-dimensional spaces. CDRs are primarily responsible for binding to an antigen epitope. The CDRs of each chain are typically referred to as CDR1, CDR2 and CDR3, numbered sequentially from the N-terminus, and are also typically identified by the chain on which the particular CDR is located. Thus, CDRs located in the variable domain of the antibody heavy chain are referred to as CDRH1, CDRH2 and CDRH3, while CDRs located in the variable domain of the antibody light chain are referred to as CDRL1, CDRL2 and CDRL3. Antibodies with different specificities (that is, different combination sites for different antigens) have different CDRs. Although it is CDRs that vary from antibody to antibody, only a limited number of amino acid positions within CDRs are directly involved in binding to the antigen. These positions within the CDRs are called specificity-determining residues (SDRs). Illustrative examples of light chain CDRs that are suitable for constructing humanized anti-BCMA CARs considered here include, but are not limited to, the CDR sequences presented in SEQ ID NOs: 1 to 3. Illustrative examples of heavy chain CDRs that are suitable for building humanized anti-BCMA CARs considered here include, but are not limited to, the CDR sequences presented in SEQ ID NOs: 4 to 6
[0140] References to "VH" or "VH" refer to the variable region of a heavy chain immunoglobulin, including that of an antibody, Fv, scFv, dsFv, Fab, or other antibody fragment as disclosed herein. References to "VL" or "VL" refer to the variable region of a light chain immunoglobulin, including that of an antibody, Fv, scFv, dsFv, Fab or other antibody fragment as disclosed herein.
[0141] A "monoclonal antibody" is an antibody produced by a single B lymphocyte clone or by a cell in which the light and heavy chain genes of a single antibody have been transfected. Monoclonal antibodies are produced by methods known to those of skill in the art, for example, by making hybrid antibody-forming cells from a fusion of myeloma cells with immune spleen cells. Monoclonal antibodies include humanized monoclonal antibodies.
[0142] A "chimeric antibody" has structural residues from one species, such as human, and CDRs (which generally confer binding and antigen) from another species, such as a mouse. In particular preferred embodiments, a CAR considered herein comprises an antigen-specific binding domain that is a chimeric antibody or antigen-binding fragment thereof.
[0143] An "humanized" antibody is an immunoglobulin including a region of human structure and one or more CDRs of a non-human immunoglobulin (for example, a mouse, mouse or synthetic). The non-human immunoglobulin that provides CDRs is called "donor", and the human immunoglobulin that provides the structure is called "acceptor".
[0144] In particular embodiments, a murine anti-BCMA antibody or antigen-binding fragment thereof, includes but is not limited to a lg Camel (a camelid antibody (VHH)), lg NAR, Fab fragments, Fab 'fragments , F (ab) '2 fragments, F (ab)' 3, Fv fragments, single chain Fv antibody (“scFv”), bis-scFv, (scFv) 2, minibody, diabody, triacorp, tetribody, stabilized Fv protein disulfide (“dsFv”), and single domain antibody (sdAb, Nanovalent).
[0145] The "Camel lg" or "VHH camelid" as used here, refers to the smallest antigen binding unit known to a heavy chain antibody (KochNolte, et al, FASEB J., 21: 3490-3498 ( 2007)). A "heavy chain antibody" or a "camelid antibody" refers to an antibody that contains two VH domains and no light chain (Riechmann L. et al, J. Immunol., Methods 231: 25-38 (1999); WO94 / 04678; WO94 / 25591; US Patent No. 6,005,079).
[0146] The "new immunoglobulin antigen receptor" IgNAR refers to the class of antibodies from the shark's immune repertoire consisting of homodimers of a variable new antigen receptor (VNAR) domain and five domains of the constant new antigen receptor (CNAR). IgNARs represent some of the least known immunoglobulin-based protein scaffolds and are highly stable and have efficient binding characteristics. The inherent stability can be attributed to both (i) underlying lg scaffolds, which have a considerable number of residues exposed to the charged and hydrophilic surface compared to the conventional antibody VH and VL domains found in murine antibodies; as for (ii) stabilizing structural characteristics in the loops of the complementarity determining region (CDR) including inter-loop disulfide bridges, and intra-loop hydrogen bonding patterns.
[0147] Papain digestion of antibodies produces two identical antigen binding fragments, called "Fab" fragments, each with a unique antigen binding site, and a residual "Fc" fragment, whose name reflects its ability to crystallize easily. Pepsin treatment produces an F (ab ') 2 fragment that has two antigen combining sites and is also capable of cross-linking to the antigen.
[0148] The "Fv" is the minimum antibody fragment that contains a complete antigen binding site. In one embodiment, a double-stranded Fv species consists of a dimer from a heavy and light chain variable domain in tight, non-covalent association. In a single chain Fv (scFv) species, a heavy and light chain variable domain can be covalently linked by a flexible peptide ligand such that the light and heavy chains can associate in a "dimeric" structure analog to those in a kind of double-stranded Fv. It is in this configuration that the three hypervariable regions (HVRs) of each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. Collectively, the six HVRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three antigen-specific HVRs) has the ability to recognize and bind to the antigen, albeit at a lower affinity than the entire binding site.
[0149] The Fab fragment contains the variable domains of the heavy and light chains and also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab1 fragments differ from Fab fragments by the addition of some residues in the terminal carboxy of the heavy chain CH1 domain including one or more cysteines from the antibody hinge region. Fab1- SH is the designation here for Fab 'in which the cysteine residue (s) of the constant domains have a free thiol group. The F (ab ') 2 antibody fragments were originally produced as pairs of Fab' fragments that have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.
[0150] The term "divalents" refers to antibody fragments with two antigen-binding sites, whose fragments comprise a heavy chain variable domain (VH) linked to a light chain variable domain (VL) on the same chain polypeptide (VH-VL). Using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with complementary domains on another chain and create two antigen-binding sites. The divalents can be divalent or bispecific. Divalents are described more fully, for example, in EP 404 097; WO 1993/01161; Hudson et al., Nat. Med. 9: 129-134 (2003); And Hollinger et al., PNAS USA 90: 6444-6448 (1993). Tribodies and tetrabodies are also described in Hudson et al., Nat. Med. 9: 129-134 (2003).
[0151] The "single domain antibody" or "sdAb" or "nanovalent" refers to an antibody fragment consisting of the variable region of an antibody heavy chain (VH domain) or the variable region of a light chain of antibody (VL domain) (Holt, L., Et al., Trends in Biotechnology, 21 (11): 484-490).
[0152] The "single chain Fv" or antibody fragments "scFv" comprise the VH and VL domains of the antibody, where these domains are present in a single polypeptide chain and in any orientation (for example, VL-VH or VHVL) . Generally, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains that allow scFv to form the desired structure for antigen binding. For a review of scFv, see, for example, Pluckthün, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenborg and Moore eds., (Springer-Verlag, New York, 1994), p. 269-315.
[0153] In preferred embodiments, a CAR considered here comprises a specific antigen binding domain that is a murine scFv. Single chain antibodies can be cloned from the V region genes of a target-specific hybridoma. The production of such hybridomas has become routine. A technique that can be used for cloning the variable region heavy chain (VH) and the variable region light chain (VL) has been described, for example, in Orlandi et al., PNAS, 1989; 86: 3833-3837.
[0154] In particular embodiments, the antigen-specific binding domain that is a murine scFv that binds to a human BCMA polypeptide. Illustrative examples of variable heavy chains that are suitable for constructing anti-BCMA CARs contemplated herein include, but are not limited to, the amino acid sequences presented in SEQ ID NO: 8. Illustrative examples of variable light chains that are suitable for constructing antiBCMA CARs contemplated herein include, but are not limited to, the amino acid sequences shown in SEQ ID NO: 7.
[0155] The BCMA specific binding domains provided here also comprise one, two, three, four, five or six CDRs. Such CDRs can be non-human CDRs or altered non-human CDRs selected from light chain CDRL1, CDRL2 and CDRL3 and heavy chain CDRH1, CDRH2 and CDRH3. In certain embodiments, a BCMA-specific binding domain comprises (a) a light chain variable region comprising a light chain CDRL1, a light chain CDRL2 and a light chain CDRL3, and (b) a heavy chain variable region which comprises a heavy chain CDRH1, a heavy chain CDRH2 and a heavy chain CDRH3. 11. Binders
[0156] In certain embodiments, the CARs considered here may comprise binding residues between the various domains, for example, added for proper conformation and spacing of the molecule. In particular embodiments, the ligand is a variable region binding sequence. A "variable region binding sequence" is an amino acid sequence that connects to the VH and VL domains and provides a spacer function compatible with the interaction of the two binding subdomains so that the resulting polypeptide retains a specific binding affinity to the same target molecule as an antibody comprising the same variable regions of light and heavy chains. CARs considered here, may comprise one, two, three, four or five, or more ligands. In particular embodiments, the length of a linker is about 1 to about 25 amino acids, about 5 to about 20 amino acids, or about 10 to about 20 amino acids, or any length of amino acid intervention. In some embodiments, the ligand is 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 or more long amino acids.
[0157] Illustrative examples of binders include polymers of glycine (G) n; glycine-serine polymers (Gi-sSi-5) n, where n is an integer of at least one, two, three, four or five; glycine-alanine polymers; alaninaserine polymers; and other flexible binders known in the art. Glycine and glycine-serine polymers are relatively unstructured and therefore may be able to serve as a neutral boundary between domains of fusion proteins such as the CARs described herein. Glycine accesses significantly more phi-psi space than still alanine, and is much less restricted than residues with longer side chains (see Scheraga, Rev. Computational Chem. 11173-142 (1992)). The skilled technician will recognize that the design of a CAR in particular modalities may include binders that are all or partly flexible, such that the binder may include a flexible binder as well as one or more portions that provide a less flexible structure to provide a desired CAR structure .
[0158] Other exemplary linkers include, but are not limited to, the following amino acid sequences: GGG; DGGGS (SEQ ID NO: 46); TGEKP (SEQ ID NO: 47) (see, for example, Liu et al., PNAS 5525-5530 (1997)); GGRR (SEQ ID NO: 48) (Pomerantz et al. 1995, supra); (GGGGS) n where = 1, 2, 3, 4 or 5 (SEQ ID NO: 49) (Kim et al., PNAS 93, 1156-1160 (1996.); EGKSSGSGSESKVD (SEQ ID NO: 50) (Chaudhary et al., 1990, Proc. Natl. Acad. Sci. USA 87: 1066-1070); KESGSVSSEQLAQFRSLD (SEQ ID NO: 51) (Bird et al., 1988, Science 242: 423-426), GGRRGGGS (SEQ ID NO: 52); LRQRDGERP (SEQ ID NO: 53); LRQKDGGGSERP (SEQ ID NO: 54); LRQKD (GGGS) 2 ERP (SEQ ID NO: 55) Alternatively, flexible binders can be rationally designed using a computer program capable of modeling both DNA binding sites and peptides by themselves (Desjarlais & Berg, PNAS 90: 2256-2260 (1993), PNAS 91: 11099-11103 (1994) or by phage display methods. , the linker comprises the following amino acid sequence: GSTSGSGKPGSGEGSTKG (SEQ ID NO: 56) (Cooper et al., Blood, 101 (4): 1637-1644 (2003)).
[0159] In particular modalities, the CAR binding domain is followed by one or more "spacer domains", which refer to the region that moves the antigen binding domain away from the effector cell surface to enable cell / appropriate cell, antigen binding and activation (Patel et al., Gene Therapy, 1999; 6: 412-419). The folding domain can be derived from a natural, synthetic, semi-synthetic or recombinant source. In certain embodiments, a spacer domain is a portion of an immunoglobulin, including, but not limited to one or more heavy chain constant regions, for example, CH2 and CH3. The spacer domain can include the amino acid sequence of a naturally occurring immunoglobulin hinge region or an altered immunoglobulin hinge region.
[0160] In one embodiment, the spacer domain comprises the CH2 and CH3 domains of lgG1 or lgG4. 13. Domain Hinge
[0161] The CAR binding domain is generally followed by one or more "hinge domains", which play a role in positioning the antigen binding domain away from the effector cell surface to enable the appropriate cell / cell contact, binding and activation to the antigen. A CAR generally comprises one or more hinge domains between the binding domain and the transmembrane (TM) domain. The folding domain can be derived from a natural, synthetic, semi-synthetic or recombinant source. The folding domain can include the amino acid sequence of a naturally occurring immunoglobulin hinge region or an altered immunoglobulin hinge region.
[0162] An “altered hinge region” refers to (a) a naturally occurring hinge region with up to 30% amino acid changes (for example, up to 25%, 20%, 15%, 10% or 5 % amino acid substitutions or deletions), (b) a portion of a naturally occurring hinge region that is at least 10 amino acids (for example, at least 12, 13, 14 or 15 amino acids) in length with up to 30% amino acid changes (for example, up to 25%, 20%, 15%, 10% or 5% of amino acid substitutions or deletions), or (c) a naturally occurring portion of a hinge region that comprises the hinge region core (which can be 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15, or at least 4, 5, 6, 7, 8, 9, 10, 11, 12 , 13, 14, or 15 amino acids in length). In certain embodiments, one or more cysteine residues in a naturally occurring immunoglobulin hinge region can be replaced by one or more other amino acid residues (for example, one or more serine residues). An altered immunoglobulin hinge region may alternatively or additionally have a proline residue from a wild-type immunoglobulin hinge region replaced by another amino acid residue (e.g., a serine residue).
[0163] Other illustrative hinge domains suitable for use in the CARs described here include the hinge region derived from extracellular regions of type 1 membrane proteins such as CD8α, CD4, CD28 and CD7, which may be hinge regions of wild type from these molecules or can be altered. In another embodiment, the hinge domain comprises a CD8a hinge region. 14. Transmembrane Domain (TM)
[0164] The "transmembrane domain" is the portion of the CAR that fuses the portion of extracellular binding and intracellular signaling domain and anchors the CAR to the plasma membrane of the immune effector cell. The TM domain can be derived from a natural, synthetic, semi-synthetic or recombinant source. The TM domain can be derived from (that is, comprises at least the transmembrane regions of) alpha, beta or zeta chain of the T cell receptor, CD3ε, CD3Ç, CD4, CD5, CD8α, CD9, CD 16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD 134, CD137, CD152, CD154 and PD1. In a particular embodiment, the TM domain is synthetic and predominantly comprises hydrophobic residues such as leucine and valine.
[0165] In one embodiment, the CARs considered here comprise a TM domain derived from CD8a. In another embodiment, a CAR considered here comprises a TM domain derived from CD8a and a short oligo- or polypeptide linker, preferably between 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids in length that binds to the TM domain and the CAR intracellular signaling domain. A glycine-serine-based binder provides a particularly suitable binder. 15. Domain of Intracellular Signaling
[0166] In particular modalities, CARs considered here comprise an intracellular signaling domain. An "intracellular signaling domain" refers to the part of a CAR that participates in transducing the effective anti-BCMA CAR message that binds a human BCMA polypeptide within the immune effector cell to evoke effector cell function, for example , activation, cytokine production, proliferation and cytotoxic activity, including the release of cytotoxic factors to the target cell linked to CAR, or other cellular responses induced with antigen that binds to the extracellular CAR domain.
[0167] The term "effector function" refers to a specialized function of an immune effector cell. The effector function of the T cell, for example, can be cytolytic activity or help or activity including the secretion of a cytokine. In addition, the term "intracellular signaling domain" refers to the portion of a protein that transduces the effector function signal and directs the cell to perform a specialized function. Although usually the entire intracellular signaling domain can be used, in many cases it is not necessary to use the entire domain. Insofar as a truncated portion of an intracellular signaling domain is used, such a truncated portion can be used in place of the entire domain as long as it transduces the effector function signal. The term intracellular signaling domain means to include any truncated portion of the intracellular signaling domain sufficient to transduce effector function signal.
[0168] It is known that signals generated through TCR alone are insufficient for complete activation of the T cell and that a secondary or co-stimulatory signal is also needed. In addition, T cell activation can be said to be mediated by two distinct classes of intracellular signaling domains: primary signaling domains that initiate antigen-dependent primary activation through the TCR (for example, a TCR / CD3 complex) and domains co-stimulatory signaling agents that act in an antigen-independent manner to provide a secondary or co-stimulatory signal. In preferred embodiments, a CAR considered here comprises an intracellular signaling domain that comprises one or more "co-stimulatory signaling domain" and a "primary signaling domain".
[0169] The primary signaling domains regulate primary activation of the TCR complex in a stimulatory form, or in an inhibitory form. The primary signaling domains that act in a stimulatory manner can contain signaling motifs that are known as the tyrosine-based immunoreceptor activation motif or ITAMs.
[0170] Illustrative examples of ITAM containing primary signaling domains that are of particular use in the invention include those derived from TCRÇ, FcRy, FcRβ, CD3Y, CD3δ, CD3ε, CD3Ç, CD22, CD79a, CD79b and CD66d. In particular preferred embodiments, a CAR comprises a primary CD3Ç signaling domain and one or more co-stimulatory signaling domains. The primary intracellular signaling and co-stimulatory signaling domains can be linked in any order together with terminal carboxyl of the transmembrane domain.
[0171] The CARs considered here comprise one or more co-stimulatory signaling domains to enhance the efficacy and expansion of T cells that express CAR receptors. As used here, the term "co-stimulatory signaling domain" or "co-stimulatory domain" refers to an intracellular signaling domain of a co-stimulatory molecule. Co-stimulatory molecules are cell surface molecules except antigen receptors or Fc receptors that provide a second signal necessary for efficient activation and function of T lymphocytes after binding to the antigen. Illustrative examples of such co-stimulatory molecules include CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (0X40), CD137 (4-1 BB), CD150 (SLAMF1), CD152 (CTLA4) , CD223 (LAG3), CD270 (HVEM), CD273 (PD-L2), CD274 (PD-L1), CD278 (ICOS), DAP10, LAT, NKD2C SLP76, TRIM and ZAP70. In one embodiment, a CAR comprises one or more co-stimulatory signaling domains selected from the group consisting of CD28, CD137 and CD134 and a primary CD3Ç signaling domain.
[0172] In another embodiment, a CAR comprises co-stimulatory signaling domains CD28 and CD137 and a primary signaling domain CD3Ç.
[0173] In yet another modality, a CAR comprises co-stimulatory signaling domains CD28 and CD137 and a primary signaling domain CD3Ç.
[0174] In one embodiment, a CAR comprises co-stimulatory signaling domains CD28 and CD134 and a primary signaling domain CD3Ç
[0175] In still modality, a CAR comprises co-stimulatory signaling domains CD137 and CD134 and a primary signaling domain CD3Ç.
[0176] In particular embodiments, CARs considered herein comprise a murine anti-BCMA antibody or antigen-binding fragment thereof specifically bound to a BCMA polypeptide expressed on B cells.
[0177] In one embodiment, a CAR comprises a murine anti-BCMA scFv that binds a BCMA polypeptide; a transmembrane domain derived from a polypeptide selected from the group consisting of: alpha, beta or zeta chain of the T cell receptor, CD3ε, CD3Ç, CD4, CD5, CD8α, CD9, CD 16, CD22, CD27, CD28, CD33 , CD37, CD45, CD64, CD80, CD86, CD 134, CD137, CD152, CD154 and PD1; and one or more intracellular co-stimulatory signaling domains of a co-stimulatory molecule selected from the group consisting of: CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (0X40 ), CD137 (4-1 BB), CD150 (SLAMF1), CD152 (CTLA4), CD223 (LAG3), CD270 (HVEM), CD273 (PD-L2), CD274 (PD-L1), CD278 (ICOS), DAP10 , LAT, NKD2C SLP76, TRIM and ZAP70; and a primary signaling domain of TCRÇ, FcRy, FcRβ, CD3Y, CD3δ, CD3ε, CD3Ç, CD22, CD79a, CD79b and CD66d.
[0178] In one embodiment, a CAR comprises a murine anti-BCMA scFv that binds to a BCMA polypeptide; a hinge domain selected from the group consisting of: lgG1 hinge / CH2 / CH3, lgG4 hinge / CH2 / CH3, a CD8α hinge; a transmembrane domain derived from a polypeptide selected from the group consisting of: alpha, beta or zeta chain of the T cell receptor, CD3ε, CD3Ç, CD4, CD5, CD8α, CD9, CD 16, CD22, CD27, CD28, CD33 , CD37, CD45, CD64, CD80, CD86, CD 134, CD137, CD152, CD 154 and PD1; and one or more intracellular co-stimulatory signaling domains of a co-stimulatory molecule selected from the group consisting of: CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (0X40 ), CD137 (4-1 BB), CD150 (SLAMF1), CD152 (CTLA4), CD223 (LAG3), CD270 (HVEM), CD273 (PD-L2), CD274 (PD-L1), CD278 (ICOS), DAP10 , LAT, NKD2C SLP76, TRIM and ZAP70; and a primary signaling domain of TCRÇ, FcRy, FcRβ, CD3Y, CD3δ, CD3ε, CD3Ç, CD22, CD79a, CD79b and CD66d.
[0179] In one embodiment, a CAR comprises a mu anti-BCMA scFv that binds a BCMA polypeptide; a hinge domain selected from the group consisting of: lgG1 hinge / CH2 / CH3, lgG4 hinge / CH2 / CH3, a CD8α hinge; a transmembrane domain derived from a polypeptide selected from the group consisting of: alpha, beta or zeta chain of the T cell receptor, CD3ε, CD3Ç, CD4, CD5, CD8α, CD9, CD 16, CD22, CD27, CD28, CD33 , CD37, CD45, CD64, CD80, CD86, CD 134, CD137, CD152, CD 154e PD1; a polypeptide or short oligo linker, preferably between 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids in length that link the TM domain to the CAR intracellular signaling domain; and one or more intracellular co-stimulatory signaling domains of a co-stimulatory molecule selected from the group consisting of: CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (0X40), CD137 (4-1 BB), CD150 (SLAMF1), CD152 (CTLA4), CD223 (LAG3), CD270 (HVEM), CD273 (PD-L2), CD274 (PD-L1), CD278 (ICOS), DAP10, LAT , NKD2C SLP76, TRIM and ZAP70; and a primary signaling domain of TCRÇ, FcRy, FcRβ, CD3Y, CD3δ, CD3ε, CD3Ç, CD22, CD79a, CD79b and CD66d.
[0180] In a particular embodiment, a CAR comprises a mu anti-BCMA scFv that binds a BCMA polypeptide; a hinge domain comprising a hGG1 / CH2 / CH3 hinge polypeptide and a CD8α polypeptide; a CD8a transmembrane domain comprising a polypeptide linker of about 3 to about 10 amino acids; a CD137 intracellular co-stimulatory signaling domain; and a CD3Ç primary signaling domain
[0181] In a particular embodiment, a CAR comprises an anti-BCMA nocturnal scFv that binds a BCMA polypeptide; a hinge domain comprising a CD8α polypeptide; a CD8a transmembrane domain comprising a polypeptide linker of about 3 to about 10 amino acids; a CD134 intracellular co-stimulatory signaling domain; and a CD3Ç primary signaling domain
[0182] In a particular embodiment, a CAR comprises a murine anti-BCMA scFv that binds a BCMA polypeptide; a hinge domain comprising a CD8α polypeptide; a CD8a transmembrane domain comprising a polypeptide linker of about 3 to about 10 amino acids; a CD28 intracellular co-stimulatory signaling domain; and a CD3Ç primary signaling domain
[0183] In addition, the design of the CARs considered here allows for improved expansion, long-term persistence, and cytotoxic properties in T cells that express CARs compared to unmodified T cells or T cells modified to express other CARs. D. Polypeptides
[0184] The present invention contemplates, in part, CAR polypeptides and fragments thereof, cells and compositions comprising the same, and vectors that express polypeptides. In preferred embodiments, a polypeptide comprising one or more CARs as shown in SEQ ID NOs: 9.
[0185] "Polypeptide", "polypeptide fragment", "peptide" and "protein" are used interchangeably, unless otherwise specified, and in accordance with conventional meaning, that is, as a sequence of amino acids. Polypeptides are not limited to a specific length, for example, they may comprise a full-length protein sequence or a fragment of a full-length protein, and may include post-translational modifications of the polypeptide, for example, glycosylations, acetylations, phosphosylations and the like, as well as other known modifications in the art, both naturally occurring and naturally occurring. In various embodiments, the CAR polypeptides considered here comprise a signal sequence (or leader) at the N-terminal end of the protein, which co-translationally or post-transdially directs protein transfer. Illustrative examples of suitable signal sequences useful in CARs disclosed herein include, but are not limited to, the IgG 1 heavy chain signal polypeptide, a CD8a signal polypeptide or a human GM-CSF receptor alpha signal polypeptide. Polypeptides can be prepared using any of a variety of well-known recombinant and / or synthetic techniques. Polypeptides considered here specifically encompass the CARs of the present disclosure, or sequences that have deletions of additions and / or substitutions of one or more amino acids of a CAR as disclosed herein.
[0186] An "isolated peptide" or an "isolated polypeptide" and the like, as used herein, refers to isolation and / or in vitro purification of a peptide or polypeptide molecule from a cellular environment, and in association with other components of the that is, it is not significantly associated with substances in vivo. Similarly, an "isolated cell" refers to a cell that was obtained from a tissue or organ in vivo and is substantially free of extracellular matrix.
[0187] Polypeptides include "polypeptide variants". Polypeptide variants can differ from a naturally occurring polypeptide in one or more substitutions, deletions, additions and / or insertions. Such variants can be naturally occurring or can be synthetically generated, for example, by modifying one or more of the above polypeptide sequences. For example, in particular modalities, it may be desirable to improve the binding affinity and / or other biological properties of CARs by introducing one or more substitutions, deletions, additions and / or insertions in a link domain, hinge, TM domain, domain co-stimulatory signaling or primary signaling domain of a CAR polypeptide. Preferably, polypeptides of the invention include polypeptides having at least about 65%, 70%, 75%, 85%, 90%, 95%, 98%, or 99% identity thereof.
[0188] Polypeptides include "fragment of polypeptides". Polypeptide fragment refers to a polypeptide, which can be monomeric or multimeric, which has an amino-terminal deletion, a carboxyl-terminal deletion, and / or an internal deletion or substitution of a recombinantly produced or naturally occurring polypeptide . In certain embodiments, a polypeptide fragment may comprise an amino acid chain of at least 5 to about 500 long amino acids. It will be evaluated that in certain modalities, fragments are at least 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, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 150, 200, 250, 300, 350, 400, or 450 long amino acids. Particularly useful polypeptide fragments include functional domains, including antigen-binding domains or antibody fragments. In the case of a humanized antiBCMA antibody, useful fragments include, but are not limited to: a CDR region, a CDR3 region of the light or heavy chain; a variable region of a light or heavy chain; a portion of an antibody chain or variable region including two CDRs; and the like.
[0189] The polypeptide can also be fused in structure or conjugated to a linker or other sequence for ease of synthesis, purification or identification of the polypeptide (e.g., poly-His), or to enhance binding of the polypeptide to a solid support.
[0190] As noted above, polypeptides of the invention can be altered in several ways including amino acid substitutions, deletions, truncations and insertions. Methods for such manipulations are generally known in the art. For example, amino acid sequence variants of a reference polypeptide can be prepared by mutations in the DNA. Methods for mutagenesis and nucleotide sequence changes are well known in the art. See, for example, Kunkel (1985, Proc. Natl. Acad. Sci. USA. 82: 488-492), Kunkel et al., (1987, Methods in Enzymol, 154: 367-382), Pat. No. 4,873,192, Watson, J. D. et al., (Molecular Biology of the Gene, Fourth Edition, Benjamin / Cummings, Menlo Park, Calif., 1987) and the references cited here. Guidance on how appropriate amino acid substitutions that do not affect biological activity of the protein of interest can be found in the model by Dayhoff et al., (1978) Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found., Washington, DC) .
[0191] In certain embodiments, a variant will contain conservative substitutions. A "conservative substitution" is one in which an amino acid is replaced in place of another amino acid that has similar properties, such that one skilled in the technique of peptide chemistry would expect the secondary structure and hydropathic nature of the polypeptide to be substantially unchanged. Modifications can be made in the structure of the polynucleotides and polypeptides of the present invention and still obtain a functional molecule that encodes a variant of polypeptide or derivative with desirable characteristics. When it is desired to change the amino acid sequence of a polypeptide to create an equivalent, or even an improved, polypeptide variant of the invention, one skilled in the art, for example, can change one or more of the codons in the coding DNA sequence, for example , according to Table 1. TABLE 1- Amino Acid Codons


[0192] Guidance on determining which amino acid residues can be replaced, inserted or deleted without abolishing biological activity can be found using computer programs well known in the art, such as DNASTARTM software. Preferably, amino acid changes in the protein variants disclosed herein are conservative amino acid changes, i.e., substitutions of similarly charged or uncharged amino acids. A conservative amino acid change involves replacing one of a family of amino acids that are related in its side chains. Naturally occurring amino acids are generally divided into four families: acid (aspartate, glutamate), basic (lysine, arginine, histidine), non-polar (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), and polar amino acids not loaded (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine). Phenylalanine, tryptophan and tyrosine are sometimes classified together as aromatic amino acids. In a peptide or protein, suitable conservative amino acid substitutions are known to those skilled in the art and can generally be made without altering the biological activity of a resulting molecule. Those of skill in this technique recognized that, in general, unique amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity (see, for example, Watson et al. Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin / Cummings Pub. Co., p. 224). Exemplary conservative substitutions are described in U.S. Provisional Patent Application No. 61 / 241,647, the disclosure of which is incorporated herein by reference.
[0193] When making such changes, the hydropathic index of amino acids can be considered. The importance of the hydropathic amino acid index in conferring interactive biological function on a protein is generally understood in the art (Kyte and Doolittle, 1982, incorporated here as a reference). Each amino acid was assigned a hydropathic index based on its hydrophobicity and charge characteristics (Kyte and Doolittle, 1982). These values are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cysteine (+2.5); methionine (+1.9); alanine (+1.8); glycine (0.4); threonine (0.7); serine (0.8); tryptophan (0.9); tyrosine (1,3); proline (1.6); Histidine (3.2); glutamate (3.5); glutamine (3.5); aspartate (3.5); asparagine (3.5); Lysine (3.9); and arginine (4.5).
[0194] It is known in the art that certain amino acids can be replaced by other amino acids having a similar hydropathic index or point and still results in a protein with similar biological activity, that is, it still obtains a functionally equivalent biological protein. In making such changes, substitution of amino acids whose hydropathic indices are within ± 2 is preferred, those within ± 1 are particularly preferred, and those within ± 0.5 are even more particularly preferred. It is also understood in the art that the substitution of similar amino acids can be done effectively on the basis of hydrophilicity.
[0195] As detailed in U.S. Patent No. 4,554,101, the following hydrophilicity values have been assigned to the amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0 ± 1); glutamate (+3.0 ± 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5 ± 1); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4). It is understood that an amino acid can be substituted in place of another having a similar hydrophilicity value and still obtain a biologically equivalent protein, and in particular, an immunologically equivalent one. In such changes, substitution of amino acids whose hydrophilicity values are within ± 2 is preferred, those within ± 1 are particularly preferred, and those within ± 0.5 are even more particularly preferred.
[0196] As outlined above, amino acid substitutions can be based on the relative similarity of the amino acid side chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size and the like.
[0197] Polypeptide variants still include glycosylated forms, aggregation conjugates with other molecules and covalent conjugates with unrelated chemical moieties (eg, pegylated molecules). Covalent variants can be prepared by linking functionalities to groups that are on the amino acid chain or at the N or C-terminal residue, as is known in the art. Variants also include allelic variants, species variants and muteins. Truncations or deletions of regions that do not affect the functional activity of proteins are also variants.
[0198] In one embodiment, where expression of two or more polypeptides is desired, the polynucleotide sequences that are encoded can be separated by and the IRES sequence as discussed here elsewhere. In another embodiment, two or more polypeptides can be expressed as a fusion protein that comprises one or more self-cleaving polypeptide sequences.
[0199] The polypeptides of the present invention include fusion polypeptides. In preferred embodiments, fusion polypeptides and polynucleotides that encode fusion polypeptides are provided, for example, CARs. Fusion polypeptides and fusion proteins refer to a polypeptide having at least two, three, four, five, six, seven, eight, nine or ten or more polypeptide segments. Fusion polypeptides are typically linked from C-terminal to N-terminal, although they can also be linked from C-terminal to C-terminal, N-terminal to Nterminal, or N-terminal to C-terminal. The polypeptides of the fusion protein can be in any order or a specified order. Fusion polypeptides or fusion proteins can also include conservatively modified variants, polymorphic variants, alleles, mutants, subsequences, and interspecies homologs, as long as the desired transcriptional activity of the fusion polypeptide is preserved. Fusion polypeptides can be produced by synthetic chemical methods or by chemical bonding between the two portions or can generally be prepared using other standard techniques. Linked DNA sequences comprising the fusion polypeptide are operationally linked to the appropriate transcriptional or translational control elements as discussed elsewhere here.
[0200] In one embodiment, a fusion partner comprises a sequence that assists in expressing the protein (an expression enhancer) in higher yields than the native recombinant protein. Other fusion partners can be selected in order to increase the solubility of the protein or to allow the protein to be directed to the desired intracellular compartments or to facilitate the transport of the fusion protein across the cell membrane.
[0201] Fusion polypeptides may further comprise a polypeptide cleavage signal between each of the polypeptide domains described herein. In addition, the polypeptide site can be placed on any peptide linker sequence. Examples of polypeptide cleavage signals include polypeptide cleavage recognition sites such as protease cleavage sites, nuclease cleavage sites (for example, rare restriction enzyme recognition sites, self-cleavage ribozyme recognition sites ) and viral self-cleaving oligopeptides (see Felipe and Ryan, 2004. Traffic, 5 (8); 616-26).
[0202] The suitable protease cleavage sites and autocleaving peptides are known to the skilled person (see, for example, in Ryan et al., 1997. J. Gener. Virol. 78, 699-722; Scymczak et al. ( 2004) Nature Biotech. 5, 589-594). Exemplary protease cleavage sites include, but are not limited to, potivirus Nla protease cleavage sites (e.g., tobacco attack virus protease), HC potivirus proteases, (P35) P1 potivirus proteases, Nla proteases biovirus proteases, biovirus RNA-2 proteases, aptovirus L proteases, enterovirus 2A proteases, rhinovirus 2A proteases, picorea 3C proteases, comovirus 24K proteases, πepovirus 24K proteases, RTSV 3C-like protease (virus spherical rice tungro), protease similar to PYVF 3C (parsnip yellow spot virus), heparin, thrombin, factor Xa and enterokinase. Due to their high stringency to cleavage, TEV (tobacco attack virus) protease cleavage sites are preferred in one embodiment, for example, EXXYXQ (GZS) (SEQ ID NO: 23), for example, ENLYFQG (SEQ ID NO: 24) and ENLYFQS (SEQ ID NO: 25), where X represents any amino acid (cleavage by TEV occurs between Q and G or Q and S).
[0203] In a particular embodiment, self-cleaving peptides include those polypeptide sequences obtained from 2A potivirus and cardiovascular peptides, FMDV (foot-and-mouth disease virus), equine rhinitis virus, Thosea asigna virus and porcine teschovirus.
[0204] In certain embodiments, the self-cleaving polypeptide site comprises a similar 2A or 2A sequence or domain (Donnelly et al., 2001. J. Gen. Virol. 82: 1027-1041). TABLE 2: Exemplary 2A sites include the following sequences:

[0205] In preferred embodiments, a polypeptide considered herein comprises a CAR polypeptide. E. Polynucleotides
[0206] In preferred embodiments, a polynucleotide encoding one or more CAR polypeptides is provided, for example, SEQ ID Nos10. As used here, the terms "polynucleotide" or "nucleic acid" refer to messenger RNA (mRNA), RNA, genomic RNA (gRNA), positive strand RNA (RNA (+)), negative strand RNA (RNA ( -)), genomic DNA (gDNA), complementary DNA (cDNA) or recombinant DNA. Polynucleotides include single and double-stranded polynucleotides. Preferably, the polynucleotides of the invention include polynucleotides or variants having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97% , 98%, 99% or 100% sequence identity to any of the reference sequences described here (see, for example, Sequence Listing), typically where the variant maintains at least one biological activity of the reference sequence. In various illustrative embodiments, the present invention contemplates, in part, polynucleotides comprising expression vectors, viral vectors, and transfer plasmids, and compositions, and cells comprising them.
[0207] In particular embodiments, polynucleotides are provided by this invention that encode at least about 5, 10, 25, 50, 100, 150, 200, 250, 300, 350, 400, 500, 1000, 1250, 1500, 1750 , or 2000 or more contiguous amino acid residues of a polypeptide of the invention, as well as all intermediate lengths. It will be easily understood that "intermediate lengths", in this context, mean any length between the quoted values, such as 6, 7, 8, 9, etc., 101, 102, 103, etc .; 151, 152, 153, etc .; 201,202, 203, etc.
[0208] As used herein, the terms "polynucleotide variant" and "variant" and the like refer to polynucleotides that exhibit substantial sequence identity with a polynucleotide or polynucleotide reference sequence that hybridize to a reference sequence under stringent conditions which are defined below. These terms include polynucleotides in which one or more nucleotides have been added or deleted, or replaced with different nucleotides compared to a reference polynucleotide. In this regard, it is well understood in the art that certain changes including mutations, additions, deletions and substitutions can be made to a reference polynucleotide whereby altered polynucleotide retains the biological function or activity of the reference polynucleotide.
[0209] The recitations "sequence identity" or, for example, comprising a "sequence 50% identical to", as used here, refer to the extent that sequences are identities on a nucleotide per nucleotide basis or an amino acid base per amino acid over a comparison window. In addition, a “percentage of sequence identity” can be calculated by comparing two sequences optimally aligned over the comparison window, determining the number of positions at which the nucleic acid base of identity (for example, A, T, C , G, I) or the identical amino acid residue (for example, Ala, Pro, Ser, Thr, Gly, Vai, Leu, lie, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gin , Cys and Met) occurs in both sequences to produce the number of combined positions, dividing the number of combined positions by the total number of positions in the comparison window (that is, the window size), and multiplying the result by 100 to produced a percentage of string identity. Included are nucleotides and polypeptides having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99 % or 100% sequence identity to any of the reference sequences described here, typically where the polypeptide variant maintains at least one biological activity of the reference polypeptide.
[0210] Terms used to describe sequence relationships between two or more polynucleotides or polypeptides include "reference sequence", "comparison window", "sequence identity", "sequence identity percentage", and "substantial identity". A "reference sequence" is at least 12, but often 15 to 18 and often at least 25 units of monomer, including nucleotide and amino acid residues, in length. Because two polynucleotides can each comprise (1) a sequence (that is, only a portion of the complete polynucleotide sequence) that is similar between the two polynucleotides, and (2) a sequence that is divergent between the two polynucleotides, sequence comparisons between two (or more) polynucleotides are typically performed by comparing sequences of the two polynucleotides over a "comparison window" to identify and compare local regions of sequence similarity. A "comparison window" refers to a conceptual segment of at least 6 contiguous positions, usually about 50 to about 100, more usually about 100 to about 150 where a sequence is compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. The comparison window can comprise additions or deletions (i.e., gaps) of about 20% or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. Optimal alignment of sequences to align a comparison window can be conducted by computerizing implementations of algorithms (GAP, BESTFIT, FASTA and TFASTA Wisconsin Genetics Software Package Version 7.0, Genetics Computer Group, 575 Science Drive Madison, Wl, USA) or by inspection and the best alignment (that is, resulting in the highest percentage homology over the comparison window) generated by any of the several selected methods. Reference can also be made to the BLAST family of programs, as, for example, published by Altschul et al., 1997, Nucl. Acids Res. 25: 3389. A detailed debate on sequence analysis can be found in Unit 19.3 of Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons Inc, 1994-1998, Chapter 15.
[0211] As used here, "isolated polynucleotide" refers to a polynucleotide that has been purified from sequences that flank in a naturally occurring state, for example, a fragment of DNA that has been removed from sequences that are normally adjacent to the fragment. An "isolated polynucleotide" also refers to complementary DNA (cDNA), recombinant DNA, or another polynucleotide that does not exist in nature and was made by the hand of man.
[0212] Terms describing the orientation of polynucleotides include: 5 '(usually the end of the polynucleotide having a free phosphate group) and 3' (usually the end of the polynucleotide having a free hydroxyl (OH) group). Polynucleotide sequences can be annotated in the 5 'to 3' orientation or 3 'to 5' orientation. For DNA and mRNA, the 5 'to 3' strand is called the “sense”, “positive”, or “coding” strand because its sequence is identical to the pre-messenger (premRNA) sequence [except for uracil (U) in RNA, instead of thymine (T) in DNA], For DNA and mRNA, the complementary 3 'to 5' strand, which is the strand transcribed by RNA polymerase, is designated as “model”, “antisense”, “negative” ”, Or“ unencrypted ”. As used herein, the term "reverse orientation" refers to a sequence 5 'to 3' written in orientation 3 'to 5' or a sequence 3 'to 5' written in orientation 5 'to 3'.
[0213] The terms "complementary" and "complementarity" refer to polynucleotides (that is, a sequence of nucleotides) related by the basic pairing rules. For example, the complementary strand of the 5 'A GTC ATG 3' DNA sequence is 3 'TC AGT AC 5'. The last string is often written as the reverse complement with the 5 'end on the left and the 3' end on the right, 5 'C A T G A C T 3'. A sequence that is equal to its reverse complement is said to be a palindromic sequence. Complementarity can be “partial”, in which only some of the nucleic acid bases ’are combined according to the basic pairing rules. Or, it can be “complete” or “total” complementarity between nucleic acids.
[0214] Furthermore, it will be assessed by one of ordinary skill in the art that, as a result of the degeneracy of the genetic code, there are many nucleotide sequences that encode a polypeptide, or fragment of a variant thereof, as described here. Some of these polynucleotides have minimal homology to the nucleotide sequence of any native gene. However, polynucleotides that vary due to differences in codon usage are specifically considered by the present invention, for example, polynucleotides that are optimized by selecting human and / or primate codons. In addition, alleles of the genes comprising the polynucleotide sequences provided here can also be used. Alleles are endogenous genes that are altered as a result of one or more mutations, such as deletions, additions and / or substitutions of nucleotides.
[0215] The term "nucleic acid cassette" as used here refers to the genetic sequences within a vector that can express an RNA, and subsequently a protein. The nucleic acid cassette contains the gene of interest, for example, a CAR. The nucleic acid cassette is positioned and sequentially oriented within the vector such that the nucleic acid in the cassette can be transcribed into RNA, and when necessary, translated into a protein or polypeptide, undergoes appropriate post-translational modifications necessary for activity in the transformed cell , and be translocated to the appropriate compartment by biological activity targeting the appropriate intracellular compartments or secretion in extracellular compartments. Preferably, the cassette has its 3 'and 5' ends adapted for prompt insertion into a vector, for example, it has restriction endonuclease sites at each end. In a preferred embodiment of the invention, the nucleic acid cassette contains the sequence of a chimeric antigen receptor used to treat a B cell malignancy. The cassette can be removed and inserted into a plasmid or viral vector as a single unit.
[0216] In particular embodiments, polynucleotides include at least one polynucleotide of interest. As used herein, the term "polynucleotide of interest" refers to a polynucleotide that encodes a polypeptide (i.e., a polypeptide of interest), inserted into an expression vector that is desired to be expressed. A vector can comprise 1,2,3, 4, 5, 6, 7, 8, 9 or 10 polynucleotides of interest. In certain embodiments, the polynucleotide-of-interest that encodes a polypeptide that provides a therapeutic effect in the treatment or prevention of a disease or disorder. Polynucleotides of interest, and polypeptides encoded therefrom, both include polynucleotides that encode wild-type polypeptides, as well as functional variants and fragments thereof. In particular embodiments, a functional variant has at least 80%, at least 90%, at least 95%, or at least 99% identity to a corresponding wild-type reference polynucleotide or polypeptide sequence. In certain embodiments, a functional variant or fragment has at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of a biological activity of a corresponding wild type polypeptide.
[0217] In one embodiment, the polynucleotide of interest does not encode a polypeptide but serves as a model for transcribing miRNA, siRNA, or shRNA, ribozyme, or other inhibitory RNA. In several other embodiments, a polynucleotide comprises a polynucleotide of interest encoding a CAR and one or more additional polynucleotides of interest including, but not limited to an inhibitory nucleic acid sequence including, but not limited to: a siRNA, a miRNA, a shRNA and a ribozyme.
[0218] As used here, the terms "siRNA" or "Short interfering RNA" refer to a short polynucleotide sequence that mediates a sequence-specific post-transcriptional gene silencing process, translational inhibition, transcriptional inhibition, or epigenetic RNAi in animals (Zamore et al., 2000, Cell, 101, 25-33; Fire et al., 1998, Nature, 391, 806; Hamilton et al., 1999, Science, 286, 950-951; Lin et al. , 1999, Nature, 402, 128-129; Sharp, 1999, Genes & Dev., 13, 139-141; and Strauss, 1999, Science, 286, 886). In certain embodiments, a siRNA comprises a first strand and a second strand that have the same number of nucleosides; however, the first and second strands are displaced such that the two terminal nucleosides in the first and second strands are not paired with a residue in the complementary strand. In certain cases, the two nucleosides that are not paired are thymidine residues. The siRNA must include a region of sufficient homology to the target gene, and be of sufficient length in terms of nucleotides, such that siRNA, or a fragment thereof, can mediate down regulation of the target gene. In addition, a siRNA includes a region that is at least partially complementary to the target RNA. It is not necessary that there is perfect complementarity between the siRNA and the target, but the correspondence must be sufficient to allow siRNA, or a divaring product thereof, for direct sequence specific silencing, such as by RNAi dividing from the target RNA. Complementarity, or degree of homology with the target filament, is more critical in the antisense filament. Although perfect complementarity, particularly in the antisense strand, is often desired, some embodiments include one or more, but preferably 10, 8, 6, 5, 4, 3, 2, or less discrepancies with respect to the target RNA. Discrepancies are more tolerated in the terminal regions, and if present they are preferably in a terminal region or regions, for example, within 6, 5, 4 or 3 nucleotides of the 5 'and / or 3' terminals. The felt filament need only be sufficiently complementary with the antisense filament to maintain the overall double filament character of the molecule.
[0219] In addition, a siRNA can be modified or include nucleoside analogs. Single strand regions of a siRNA can be modified or include nucleoside analogues, for example, the unpaired region or regions of a hairping structure, for example, a region that connects two complementary regions, may have nucleoside modifications or analogs. Modification to stabilize one or more 3'- or 5'-terminals of a siRNA, for example, against exonucleases, or to favor the antisense siRNA agent to enter RISC are also useful. Modifications may include C3 amino (or C6, C7, C12) ligands, thiol ligands, carboxyl ligands, non-nucleotide spacers (C3, C6, C9, C12, abasic, triethylene glycol, hexaethylene glycol), special biotin or fluorescein reagents that come as phosphoramidites and that they have another hydroxyl group protected by DMT, allowing multiple couplings during RNA synthesis. Each strand of a siRNA can be equal to or less than 30, 25, 24, 23, 22, 21 or 20 nucleotides in length. The filament is preferably at least 19 nucleotides in length. For example, each strand can be between 21 and 25 nucleotides in length. Preferred siRNAs have a duplex region of 17, 18, 19, 29, 21,22, 23, 24 or 25 pairs of nucleotides, and one or more projections of 2 to 3 nucleotides, preferably one or two 3 'projections, of 2 to 3 nucleotides.
[0220] As used here, the terms "miRNA" or "microRNA" refer to small non-coding RNAs of 20 to 22 nucleotides, typically excised from ~ 70 nucleotides do not translate RNA precursor structures known as pre-miRNAs. MiRNAs negatively regulate their targets in one of two ways depending on the degree of complementarity between the miRNA and the target. First, miRNAs that bind with perfect or near perfect complementarity to the protein-encoding mRNA sequences induce the RNA-mediated interference pathway (RNAi). MiRNAs that exert their regulatory effects by binding to imperfect complementary sites within the 3 'untranslated regions (RTUs) of their mRNA targets, suppress post-transcriptional target gene expression, apparently at the translation level, through a RISC complex that it is similar to, or possibly identical to, one used for the RNAi pathway. Compatible with translational control, miRNAs that use these mechanisms reduce the protein levels of their target genes, but the mRNA levels of these genes are only minimally affected. MiRNAs encompass both naturally occurring miRNAs as well as artificially designed miRNAs that can specifically target any mRNA sequence. For example, in one embodiment, the skilled technician can design Short Hairping RNA constructs expressed as human miRNA (for example, miR-30 or miR-21) primary transcripts. This project adds a Drosha processing site to the hairping construction and has been shown to significantly increase knockdown efficiency (Pusch et al., 2004). Hairpin stem consisting of 22-nt of dsRNA (for example, antisense has perfect complementarity to the desired target) and a 15-19-nt loop of a human miR. Adding the miR loop and miR30 flanking sequences on both sides of the hairping results in more than a 10-fold increase in Drosha and Dicer processing of the expressed hairpins when compared to conventional shRNA designs without microRNA. Increased Drosha and Dicer processing translates into greater siRNA / miRNA production and greater potency for expressed hairpins.
[0221] As used here, the terms "shRNA" or "Short hairping RNA" refer to the double stranded structure that is formed by a single strand of self-complementary RNA. ShRNA constructs containing a nucleotide sequence identical to a portion, coding or non-coding sequence, of the target gene are preferred for inhibition. RNA sequences with insertions, deletions, and single point mutations relative to the target sequence have also been found to be effective for inhibition. Greater than 90% sequence identity, or even 100% sequence identity, between the inhibitory RNA and the target gene portion is preferred. In certain preferred embodiments, the length of the duplex forming portion of a shRNA is at least 20, 21 or 22 nucleotides in length, for example, corresponding in size to the RNA products produced by Dicer-dependent cleavage. In certain embodiments, the shRNA construct is at least 25, 50, 100, 200, 300 or 400 bases in length. In certain embodiments, the shRNA construct is 400-800 bases in length. The shRNA constructs are highly tolerant of variation in loop sequence and loop size.
[0222] As used herein, the term "ribozyme" refers to a catalytically active RNA molecule capable of specific cleavage of the target mRNA site. Several subtypes have been described, for example, hammerhead ribozymes and hairping. Catalytic activity of ribozyme and stability can be improved by replacing deoxyribonucleotides with ribonucleotides on non-catalytic bases. While ribozymes that cleave mRNA in site-specific recognition sequences can be used to destroy particular mRNAs, the use of hammerhead ribozymes is preferred. Hammerhead ribozymes cleave mRNAs at sites dictated by flanking regions that form complementary base pairs with the target mRNA. The only requirement is that the target mRNA has the following two base sequence: 5'-UG-3 '. The construction and production of hammerhead ribozymes is well known in the art.
[0223] A preferred method of releasing a polynucleotide of interest that comprises a siRNA, a miRNA, a shRNA, or a ribozyme comprises one or more regulatory sequences, such as, for example, a strong constitutive pol III, for example , human U6 snRNA promoter, mouse U6 snRNA promoter, mouse and human H1 RNA promoter, and human tRNA-val promoter, or a strong constitutive pol II promoter, as described elsewhere place here.
[0224] The polynucleotides of the present invention, regardless of the length of their coding sequence, can be combined with other DNA sequences, such as promoters and / or enhancers, untranslated regions (RTUs), signal sequences, Kozak sequences, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, internal ribosomal entry sites (IRES), recombinase recognition sites (for example, LoxP, FRT and Att sites), termination codons, transcription termination signals , and self-cleaving polypeptides encoding polynucleotides, epitope markers, as disclosed elsewhere herein or as known in the art, such that their overall length can vary considerably. It is therefore considered that a polynucleotide fragment of almost any length can be used, with the total length being preferably limited by the ease of preparation and use in the intended recombinant DNA protocol.
[0225] Polynucleotides can be prepared, manipulated and / or expressed using any of a variety of well-presented techniques known and available in the art. In order to express a desired polypeptide, a nucleotide sequence encoding the polypeptide, can be inserted into an appropriate vector. Examples of vectors are plasmid, autonomously replicating sequences, and transposable elements. Additional exemplary vectors include, without limitation, plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC), or P1-derived artificial chromosome (PAC), bacteriophages such as lambda phage or phage M13, and animal viruses. Examples of categories of animal viruses useful as vectors include, without limitation, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (for example, herpes simplex viruses), poxviruses, baculoviruses, papillomaviruses and papovaviruses (for example, SV40 ). Examples of expression vectors are pCIneo vectors (Promega) for expression in mammalian cells; pl_enti4 / V5-DEST ™, pl_enti6 / V5-DEST ™ and pl_enti6.2 / V5-GW / lacZ (Invitrogen) for lentivirus-mediated gene transfer and expression in mammalian cells. In particular embodiments, the chimeric protein coding sequences disclosed herein can be linked in such expression vectors for the expression of the chimeric protein in mammalian cells.
[0226] In one embodiment, a vector encoding a CAR provided here comprises the polynucleotide sequence shown in SEQ ID NO: 36.
[0227] In particular modalities, the vector is either an episomal vector or a vector that is maintained extrachromosomally. As used here, the term "episomal" refers to a vector that is capable of replicating without integration into host chromosomal DNA and without gradual loss of a dividing host cell also meaning that said vector replicates extracromosomally or episomally. The vector is engineered to house the coding sequence for the origin of DNA replication or “oh” of an Iinfotrophic herpes virus or a gamma herpesvirus, an adenovirus, SV40, a bovine papilloma virus, or a yeast, specifically an origin of replication of a lymphotrophic herpes virus or a herpesvirus gamma corresponding to EBV oriP. In a particular aspect, the lymphotrophic herpes virus can be Epstein Barr virus (EBV), Kaposi's sarcoma herpes virus (KSHV), Herpes saimiri virus (HS), or Marek's disease virus (MDV). Epstein Barr virus (EBV) and Kaposi herpes sarcoma virus (KSHV) are also examples of a gamma herpes virus. Typically, the host cell comprises the transactivating viral replication protein that activates replication.
[0228] The “control elements” or “regulatory sequences” present in an expression vector are those untranslated regions of the vector — origin of replication, selection cassettes, promoters, enhancers, intron translation initiation signals (Shine sequence Dalgarno or Kozak sequence), a polyadenylation sequence, 5 'and 3' untranslated regions — that interact with host cell proteins to perform transcription and translation. Such elements may vary in strength and specificity. Depending on the host and vector system used, any number of suitable transcription and translation elements, including ubiquitous promoters and inducible promoters can be used.
[0229] In particular embodiments, a vector for use in practice the invention including, but not limited to expression vectors and viral vectors, will include exogenous, endogenous or heterologous control sequences such as promoters and / or enhancers. An "endogenous" control sequence is one that is naturally linked with a given gene in the genome. An “exogenous” control sequence is one that is placed in juxtaposition to a gene by means of genetic manipulation (ie, molecular biological techniques) such that the transcription of that gene is directed by the linked enhancer / promoter. A "heterologous" control sequence is an exogenous sequence that is of a different species than the cell being manipulated genetically.
[0230] The term "promoter" as used here refers to a polynucleotide (DNA or RNA) recognition site to which an RNA polymerase binds. An RNA polymerase initiates and transcribes polynucleotides operably linked to the promoter. In particular embodiments, mammalian cell operative promoters comprise an AT-rich region located approximately 25 to 30 bases upstream from the site where transcription is initiated and / or another sequence found 70 to 80 bases upstream from the start of transcription, a CNCAAT region where N can be any nucleotide.
[0231] The term "enhancer" refers to a segment of DNA that contains sequences capable of providing improved transcription and in some cases can function regardless of its orientation relative to another control sequence. An enhancer can work cooperatively or additively with promoter elements and / or another enhancer. The term "promoter / enhancer" refers to a segment of DNA that contains sequences capable of providing both promoter and enhancer functions.
[0232] The term "operationally linked", refers to a juxtaposition in which the components described are in a relationship allowing them to function in their intended manner. In one embodiment, the term refers to a functional link between a nucleic acid expression control sequence (such as a promoter, and / or enhancer) and a second polynucleotide sequence, for example, a polynucleotide of interest, wherein the expression control sequence directs transcription of the nucleic acid corresponding to the second sequence.
[0233] As used herein, the term "constitutive expression control sequence" refers to a promoter, enhancer, or promoter / enhancer that continuously or continuously allows for transcription of an operably linked sequence. A constitutive expression control sequence can be a "ubiquitous" promoter, enhancer, or promoter / enhancer that allows expression in a wide variety of cell and tissue types or a "cell-specific" promoter, enhancer, or promoter / enhancer, " cell type specific ”,“ cell line specific ”, or“ tissue specific ”that allows expression in a restricted variety of cell and tissue types, respectively.
[0234] Illustrative ubiquitous expression control sequences suitable for use in particular embodiments of the invention include, but are not limited to, an immediate cytomegalovirus (CMV) early promoter, a simian viral virus 40 (SV40) (for example, early or late), a Moloney murine leukemia virus (MoMLV) LTR promoter, a Rous sarcoma virus (RSV) LTR, a herpes simplex virus (HSV) promoter (thymidine kinase), H5, P7 promoters, 5, and vaccinia virus P11, an elongation factor 1-alpha (EF1a) promoter, early growth response 1 (EGR1), ferritin H (FerH), ferritin L (FerL), Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) , eukaryotic translation initiation factor 4A1 (EIF4A1), heat shock protein 70KDa 5 (HSPA5), heat shock protein 90kDa beta, member 1 (HSP90B1), heat shock protein 70kDa (HSP70), β-kinesin (β- KIN), the human ROSA 26 locus (Irions et al., Nature Biotechnology 25, 1477-1482 (2007)), a promoter from Ubiqu itin C (UBC), a promoter of phosphoglycemong kinase-1 (PGK), a promoter of chicken cytomegalovirus / β-actin enhancer (CAG), a β-actin promoter and a myeloproliferative sarcoma virus enhancer, deletion of negative control region, promoter substituted at the dl587rev primer binding site (MND) (Challita et al., J Virol. 69 (2) 748-55 (1995)).
[0235] In one embodiment, a vector of the invention comprises an MND promoter.
[0236] In one embodiment, a vector of the invention comprises an EF1a promoter comprising the first intron of the human EF 1a gene.
[0237] In one embodiment, a vector of the invention comprises an EF1a promoter that lacks the first intron of the human EF 1a gene.
[0238] In a particular embodiment, it may be desirable to express a polynucleotide comprising a CAR from a specific T cell promoter.
[0239] As used here, "conditional expression" can refer to any type of conditional expression including, but not limited to, inducible expression; repressive expression !; expression in cells or tissues having a particular physiological, biological, or disease state, etc. This definition is not intended to exclude specific cell type or tissue expression. Certain embodiments of the invention provide conditional expression of a polynucleotide of interest, for example, expression is controlled by subjecting a cell, tissue, organism, etc., to a treatment or condition that causes the polynucleotide to be expressed or that causes a increase or decrease in expression of the polynucleotide encoded by the polynucleotide of interest.
[0240] Illustrative examples of inducible promoters / systems include, but are not limited to, inducible steroid promoters such as promoters for genes encoding glucocorticoid or estrogen receptors (inducible by treatment with the corresponding hormone), metallothionine promoter (inducible by treatment with various heavy metals), MX-1 promoter (interferon inducible), the mifepristone regulating system “GeneSwitch” (Sirin et al., 2003, Gene, 323: 67), the coupling inducible switch gene (WO 2002/088346) , tetracycline-dependent regulatory systems, etc.
[0241] Conditional expression can also be obtained using a site-specific DNA recombinase. According to certain embodiments of the invention the vector comprises at least one (typically two) site (s) for recombination mediated by a site specific recombinase. As used here, the terms "recombinase" or "site-specific recombinase" include excisive or integrative proteins, enzymes, cofactors or associated proteins that are involved in recombination reactions involving one or more recombination sites (for example, two, three, four, five, seven, ten, twelve, fifteen, twenty, thirty, fifty, etc.), which can be wild type proteins (see Landy, Current Opinion in Biotechnology 3: 699-707 (1993)), or mutants, derivatives (e.g., fusion proteins containing the recombination protein sequences or fragments thereof), fragments, and variants thereof. Illustrative examples of recombinases suitable for use in particular embodiments of the present invention include, but are not limited to: Cre, Int, IHF, Xis, Flp, Fis, Hin, Gin, <t> C31, Cin, Tn3 resolvase, TndX, XerC, XerD, TnpX, Hjc, Gin, SpCCEI and ParA.
[0242] The vectors can comprise one or more recombination sites for any of a wide variety of site-specific recombinase. It should be understood that the target site for a site-specific recombinase is beyond any site necessary for integration of a vector, for example, a retroviral or lentiviral vector. As used herein, the terms "recombination sequence", "recombination site", or "site-specific recombination site" refer to a particular nucleic acid sequence that a recombinase recognizes and binds to.
[0243] For example, a recombination site for Cre recombinase is loxP which is a 34 base pair sequence comprising two inverted repeated 13 base pairs (serving as the recombinase binding sites) flanking an 8 pair core sequence baseline (see FIG. 1 by Sauer, B., Current Opinion in Biotechnology 5: 521-527 (1994)). Other exemplary loxP sites include, but are not limited to: Iox511 (Hoess et al., 1996; Bethke and Sauer, 1997), Iox5171 (Lee and Saito, 1998), Iox2272 (Lee and Saito, 1998), m2 (Langer et al., 2002), Iox71 (Albert et al., 1995), and Iox66 (Albert et al., 1995).
[0244] Suitable recognition sites for FLP recombinase include, but are not limited to: FRT (McLeod, et al., 1996), F1, F2, F3 (Schlake and Bode, 1994), F4, F5 (Schlake and Bode , 1994), FRT (LE) (Senecoff et al., 1988), FRT (RE) (Senecoff et al., 1988).
[0245] Other examples of recognition sequences are the attB, attP, attL and attR sequences, which are recognized by the enzyme À integrase recombinase, for example, phi-c31. The ψC31 SSR mediates recombination only between the heterotypic sites attB (34 bp in length) and attP (39 bp in length) (Groth et al., 2000). attB and attP, named for the binding sites for integrase phage in the bacterial and phage genomes, respectively, both contain imperfect inverted repeats that are probably linked by cpC31 homodimers (Groth et al., 2000). The product sites, attL and attR, are effectively inert for another cpC31-mediated recombination (Belteki et al., 2003), making the reaction irreversible. To catalyze insertions, it was found that attB-containing DNA inserts into a genomic attP site more easily than an attP site in a genomic attB site (Thyagarajan et al., 2001; Belteki et al., 2003). In addition, typical strategy positions by recombination counterparts include a "coupling site" containing attP at a defined locus, which is then associated with an input sequence containing attB for insertion.
[0246] As used here, an “internal ribosome entry site” or “IRES” refers to an element that promotes entry of an internal ribosome directly into the initiation codon, such as ATG, of a cystron (a region that encodes protein), thereby leading to cap-independent translation of the gene. See, for example, Jackson et al., 1990. Trends Biochem Sci 15 (12): 477-83) and Jackson and Kaminski. 1995. RNA 1 (10): 985-1000. In particular embodiments, the vectors considered by the invention include one or more polynucleotides of interest that encode one or more polypeptides. In particular embodiments, to obtain efficient translation of each of the plurality of polypeptides, the polynucleotide sequences can be separated by one or more self-cleaving polypeptides that encode IRES sequences or polynucleotide sequences.
[0247] As used here, the term "Kozak sequence" refers to a short nucleotide sequence that greatly facilitates initial mRNA binding to the small subunit of the ribosome and increases translation. The Kozak consensus sequence is (GCC) RCCATGG, where R is a purine (A or G) (Kozak, 1986. Cell. 44 (2): 283-92, and Kozak, 1987. Nucleic Acids Res. 15 (20) : 8125-48). In particular embodiments, the vectors considered by the invention comprise polynucleotides that have a Kozak consensus sequence and that encode a desired polypeptide, for example, a CAR.
[0248] In some embodiments of the invention, a polynucleotide or cell that houses the polynucleotide uses a suicide gene, including an inducible suicide gene to reduce the risk of direct toxicity and / or uncontrolled proliferation. In specific aspects, the suicide gene is not immunogenic to the host that houses the polynucleotide or cell. A certain example of a suicide gene that can be used is caspase-9 or caspase-8 or cytosine deaminase. Caspase-9 can be activated using a specific chemical dimerization inducer (CID).
[0249] In certain embodiments, vectors comprise gene segments that cause the immune effector cells of the invention, for example, T cells, to be susceptible to negative selection in vivo. By "negative selection" it means that the infused cell can be eliminated as a result of a change in the individual's in vivo condition. The negative selectable phenotype can result from the insertion of a gene that confers sensitivity to an administered agent, for example, a compound. Selectable negative genes are known in the art, and include, inter alia, the Herpes simplex virus type I thymidine kinase (HSV-I TK) gene (Wigler et al., Cell. 11: 223, 1977) which confers sensitivity to ganciclovir; the hypoxanthine cell phosphoribosyltransferase (HPRT) gene, the adenine cell phosphoribosyltransferase (APRT) gene, and bacterial cytosine deaminase, (Mullen et al., Proc. Natl. Acad. Sci. USA. 89:33 (1992)).
[0250] In some embodiments, genetically modified immune effector cells, such as T cells, comprise a polynucleotide still comprising a positive marker that allows selection of cells of the negative selectable phenotype in vitro. The positive selectable marker may be a gene that, even being introduced into the host cell it expresses a dominant phenotype allowing positive selection of cells carrying the gene. Genes of this type are known in the art, and include, inter alia, the hygromycin B phosphotransferase (hph) gene that confers resistance to hygromycin B, the aminoglycoside phosphotransferase (neo or aph) gene that codes for antibiotic resistance G418, the gene dihydrofolate reductase (DHFR), the adenosine deaminase gene (ADA), and the multi-drug resistance (MDR) gene.
[0251] Preferably, the positive selectable marker and the negative selectable element are linked such that the loss of the negative selectable element is necessarily also accompanied by the loss of the positive selectable marker. Even more preferably, the positive and negative selectable markers are merged so that the loss of one necessarily leads to the loss of the other. An example of a fused polynucleotide that produces as a expression product a polypeptide that confers both the desired negative and positive selection characteristics described above is a hygromycin phosphotransferase (HyTK) thymidine kinase fusion gene. Expression of this gene produces a polypeptide that confers resistance to hygromycin B for positive selection in vitro, and sensitivity to ganciclovir for negative selection in vivo. See Lupton S. D., et al, Mol. And Cell. Biology 1 1: 3374-3378, 1991. In addition, in preferred embodiments, the polynucleotides of the invention encoding chimeric receptors are retroviral vectors containing the fused gene, particularly those that confer resistance to hygromycin B for positive selection in vitro, and sensitivity to ganciclovir for negative selection in vivo, for example, the retroviral vector HyTK described in Lupton, SD et al. (1991), supra. See also PCT publications US91 / 08442 and PCT / US94 / 05601, by S. D. Lupton, describing the use of selectable bifunctional fusion genes derived from fusion with dominant positive selectable markers with negative selectable markers.
[0252] Preferred positive selectable markers are derived from genes selected from the group consisting of hph, nco and gpt, and preferred negative selectable markers are derived from genes selected from the group consisting of cytosine deaminase, HSV-I TK, VZVTK , HPRT, APRT and gpt. Especially preferred markers are bifuπtional selectable fusion genes in which the positive selectable marker is derived from hph or neo, and the negative selectable marker is derived from cytosine deaminase or a TK gene or selectable marker. Inducible suicidal genes. F. Viral vectors
[0253] In particular modalities, a cell (for example, an immune effector cell) is transduced with a retroviral vector, for example, a lentiviral vector, which encodes a CAR. For example, an immune effector cell is transduced with a vector encoding a CAR that comprises a murine anti-BCMA antibody or antigen-binding fragment that binds to a BCMA polypeptide, with an intracellular signaling domain of CD3Ç, CD28, 4- 1BB, 0x40, or any combination of these. In addition, these transduced cells can evoke a CAR-mediated cytotoxic response.
[0254] Retroviruses are a common tool for gene release (Miller, 2000, Nature. 357: 455-460). In particular embodiments, a retrovirus is used to deliver a polynucleotide that encodes a chimeric antigen receptor (CAR) to a cell. As used here, the term "retrovirus" refers to an RNA virus that reverse transcribes its genomic RNA into a linear double-stranded DNA copy and subsequently covalently integrates its genomic DNA into a host genome. Since the virus is integrated into the host genome, it is referred to as a "provirus". The provirus serves as a model for RNA polymerase II and directs the expression of RNA molecules that encode the proteins and structural enzymes needed to produce new viral particles.
[0255] Illustrative retroviruses suitable for use in particular modalities include, but are not limited to: Moloney murine leukemia virus (M-MuLV), Moloney murine sarcoma virus (MoMSV), Harvey murine sarcoma virus ( HaMuSV), murine mammary tumor virus (MuMTV), gibbon monkey leukemia virus (GaLV), feline leukemia virus (FLV), foam virus, Friend murine leukemia virus, Murine Stem Cell Virus (MSCV) and Virus of the Rous sarcoma (RSV)) and lentivirus.
[0256] As used here, the term "lentivirus" refers to a group (or genus) of complex retroviruses. Illustrative lentiviruses include, but are not limited to: HIV (human immunodeficiency virus; including HIV type 1, and HIV type 2); visnamaedi virus (VMV); the caprine arthritis-encephalitis virus (CAEV); equine infectious anemia virus (EIAV); feline immunodeficiency virus (IVF); bovine immunodeficiency virus (BIV); and simian immunodeficiency virus (SIV). In one embodiment, HIV-based vector structures (that is, sequence elements that act with HIV cis) are preferred. In particular embodiments, a lentivirus is used to release a polynucleotide comprising a CAR to a cell.
[0257] Retroviral vectors and more particularly lentiviral vectors can be used in the practice of particular embodiments of the present invention. Consequently, the term "retrovirus" or "retroviral vector", as used here means to include "lentivirus" and "lentiviral vectors" respectively.
[0258] The term "vector" is used here to refer to a nucleic acid molecule capable of transferring or transporting another nucleic acid molecule. The transferred nucleic acid is generally bound to, for example, inserted into the vector nucleic acid molecule. A vector can include sequences that direct autonomous replication in a cell, or it can include enough sequences to allow integration into host cell DNA. Useful vectors include, for example, plasmids (for example, DNA plasmids or RNA plasmids), transposons, cosmids, bacterial artificial chromosomes, and viral vectors. Useful viral vectors include, for example, replication defective retrovirus and lentivirus.
[0259] As will be apparent to a person of skill in the art, the term "viral vector" is widely used to refer to a nucleic acid molecule (for example, a transfer plasmid) that includes nucleic acid elements derived from viruses that typically facilitate the transfer of the nucleic acid molecule or integration into the genome of a cell or a viral particle that mediates transfer of nucleic acid. Viral particles will typically include several viral components and sometimes also host cell components in addition to nucleic acid (s).
[0260] The term viral vector can refer to a virus or viral particle capable of transferring a nucleic acid in a cell or its transferred nucleic acid. Viral vectors and transfer plasmids contain structural and / or functional genetic elements that are mainly derived from a virus. The term "retroviral vector" refers to a viral vector or plasmid containing structural and functional genetic elements, or portions thereof, which are mainly derived from a retrovirus. The term "lentiviral vector" refers to a viral vector or plasmid containing structural and functional genetic elements, or portions thereof, including LTRs that are primarily derived from a lentivirus. The term "hybrid vector" refers to a vector, LTR or other nucleic acid containing both retroviral sequences, for example, lentiviral and non-lentiviral viral sequences. In one embodiment, a hybrid vector refers to a transfer vector or plasmid comprising retroviral sequences, for example, lentiviral for reverse transcription, replication, integration and / or packaging.
[0261] In particular modalities, the terms "lentiviral vector", "lentiviral expression vector" can be used to refer to lentiviral transfer plasmids and / or infectious lentiviral particles. Where reference is made here to elements such as cloning sites, promoters, regulatory elements, heterologous nucleic acids, etc., it should be understood that the sequences of these elements are present in the form of RNA in the lentiviral particles of the invention and are present in the form of DNA in the DNA plasmids of the invention.
[0262] At each end of the provirus are structures called "long terminal repetitions" or "LTRs". The term "long terminal repeat (LTR)" refers to the base pair domains located at the ends of retroviral DNAs that, in their natural sequence context, are direct repeats and contain U3, R and U5 regions. LTRs generally provide fundamental functions for the expression of retroviral genes (for example, promotion, initiation and polyadenylation of gene transcriptions) and for viral replication. The LTR contains numerous regulatory signals including transcriptional control elements, polyadenylation signals and sequences necessary for replication and integration of the viral genome. The viral LTR is divided into three regions called U3, R and U5. The U3 region contains the enhancing and promoting elements. The U5 region is the sequence between the primer binding site and the R region and contains the polyadenylation sequence. The R (repeat) region is flanked by the U3 and U5 regions. The LTR consists of U3, R and U5 regions and appears at both the 5 'and 3' ends of the viral genome. Adjacent to the 5 'LTR are the sequences necessary for reverse transcription of the genome (the tRNA primer binding site) and for efficient packaging of particulate viral RNA (the Psi site).
[0263] As used here, the term "packaging signal" or "packaging sequence" refers to the sequences located within the retroviral genome that are required for insertion of the viral RNA into the capsid or viral particle, see, for example, Clever et al., 1995. J. of Virology, Vol. 69, No 4; pages 2101-2109. Several retroviral vectors use the minimal packaging signal (also referred to as the psi [ψ] sequence) necessary for encapsidating the viral genome. In addition, as used here, the terms "packaging sequence", "packaging signal", "psi" and the symbol "ψ", are used in reference to the non-coding sequence required for encapsidation of retroviral RNA strands during formation of viral particle.
[0264] In various embodiments, vectors comprise 5 'LTR and / or modified 3' LTRs. One or both LTRs may comprise one or more modifications including, but not limited to, one or more deletions, insertions or substitutions. Modifications of LTR 3 'are often made to improve the safety of lentiviral or retroviral systems becoming the virus replication defect. As used here, the term "replication defect" refers to the virus that is not capable of complete replication, effective such that infectious virions are not produced (for example, lentiviral progeny of replication defect). The term "competent for replication" refers to the wild-type virus or mutant virus that is capable of replication, such that viral replication of the virus is capable of producing infectious virions (for example, lentiviral progeny competent for replication).
[0265] "Self-inactivating" (SIN) vectors refers to replication defect vectors, for example, retroviral or lentiviral vectors, in which the right LTR (3 ') enhancer region promoter, known as the U3 region , has been modified (for example, by deletion or substitution) to prevent viral transcription beyond the first round of viral replication. This is because the right U3 LTR (3 ') region is used as a template for the left U3 LTR (5') region during viral replication and thus viral transcription cannot be done without the U3 enhancer promoter. In another embodiment of the invention, LTR 3 'is modified such that the U5 region is replaced, for example, with an ideal poly (A) sequence. It should be noted that modifications to LTRs such as modifications to LTR 3 ', LTR 5', or both LTRs 3 'and 5', are also included in the invention.
[0266] Additional security enhancement is provided by replacing the U3 region of the LTR 5 'with a heterologous promoter to conduct transcription of the viral genome during production of viral particles. Examples of heterologous promoters that can be used include, for example, simian viral virus 40 (SV40) (for example, early or late), cytomegalovirus (CMV) (for example, early immediate), Moloney murine leukemia virus (MoMLV ), Rous sarcoma virus (RSV), and promoters of the herpes simplex virus (HSV) (thymidine kinase). Typical promoters are able to conduct high levels of transcription in a Tat-independent manner. This substitution reduces the possibility of recombination to generate viruses competent for replication because there is no complete U3 sequence in the virus production system. In certain embodiments, the heterologous promoter has additional advantages in controlling the way in which the viral genome is transcribed. For example, the heterologous promoter may be inducible, such that transcription of all or part of the viral genome will occur only when the inducing factors are present. Induction factors include, but are not limited to, one or more chemical compounds or physiological conditions such as temperature or pH, in which host cells are cultured.
[0267] In some modalities, viral vectors comprise a TAR element. The term "TAR" refers to the "transactivation response" genetic element located in the Lentiviral R region LTRs (for example, HIV). This element interacts with the trans-activating lentiviral genetic element (tat) to enhance viral replication. However, this element is not necessary in modalities in which the U3 region of the LTR 5 'is replaced by a heterologous promoter.
[0268] The “R region” refers to the region within retroviral LTRs beginning at the beginning of the leveling group (ie, the beginning of transcription) and ending immediately before the start of the poly mouse A. The R region is also defined as being flanked by the U3 and U5 regions. The R region plays a role during reverse transcription allowing the transfer of nascent DNA from one end of the genome to the other.
[0269] As used here, the term "FLAP element" refers to a nucleic acid whose sequence includes the central polipurin tract and central termination sequences (cPPT and CTS) of a retrovirus, for example, HIV-1 or HIV -2. Suitable FLAP elements are described in Pat. No. 6,682,907 and in Zennou, et al., 2000, Cell, 101: 173. During HIV-1 reverse transcription, the central initiation of positive strand DNA in the central polipurin tract (cPPT) and central termination in the central termination sequence (CTS) leads to the formation of a three-stranded DNA structure: the flap ( flap) of central HIV-1 DNA. Although not wishing to be bound by any theory, the DNA tab can act as a cisative determinant of nuclear import from the lentiviral genome and / or can increase the virus titer. In particular embodiments, retroviral or lentiviral vector structures comprise one or more FLAP elements upstream or downstream of the heterologous genes of interest in the vectors. For example, in particular embodiments, a transfer plasmid includes a FLAP element. In one embodiment, a vector of the invention comprises a FLAP element isolated from HIV-1.
[0270] In one embodiment, retroviral or lentiviral transfer vectors comprise one or more export elements. The term "export element" refers to a post-transcriptional regulatory element acting with cis that regulates the transport of an RNA Transcription from the nucleus to the cytoplasm of a cell. Examples of RNA export elements include, but are not limited to, the human immunodeficiency virus (RRE) rev (HIV) response elements (see, for example, Cullen et al., 1991. J. Virol. 65: 1053; and Cullen et al., 1991. Cell. 58: 423), and the post-transcriptional regulatory element of the hepatitis B virus (HPRE). Generally, the RNA export element is placed within the 3 'RTU of a gene, and can be inserted as one or multiple copies.
[0271] In particular modalities, expression of heterologous sequences in viral vectors is increased by incorporating post-transcriptional regulatory elements, efficient polyadenylation sites, and optionally, transcription termination signals in the vectors. A variety of post-transcriptional regulatory elements can increase expression of a heterologous nucleic acid in the protein, for example, post-transcriptional regulatory element of the hepatitis woodchuck virus (WPRE; Zufferey et al., 1999, J. Virol., 73: 2886 ); the post-transcriptional regulatory element present in the hepatitis B virus (HPRE) (Huang et al., Mol. Cell. Biol., 5: 3864); and the like (Liu et al., 1995, Genes Dev., 9: 1766). In particular embodiments, vectors of the invention comprise a post-transcriptional regulatory element such as a WPRE or HPRE.
[0272] In particular embodiments, vectors of the invention lack or do not comprise a post-transcriptional regulatory element such as a WPRE or HPRE because in some cases these elements increase the risk of cell transformation and / or do not substantially or significantly increase the amount of mRNA transcription or increase mRNA stability. Therefore, in some embodiments, vectors of the invention lack or do not comprise a WPRE or HPRE as an added security measure.
[0273] Elements that direct efficient termination and polyadenylation of heterologous gene expression increase heterologous nucleic acid transcripts. Transcription termination signals are generally found downstream of the polyadenylation signal. In particular embodiments, vectors comprise a 3 'polyadenylation sequence of a polynucleotide that encodes a polypeptide to be expressed. The term "polyA site" or "polyA sequence" as used here denotes a DNA sequence that directs both the termination and the polyadenylation of the transcription of nascent RNA by RNA polymerase II. Polyadenylation sequences can promote mRNA stability by adding a polyA tail to the 3 'end of the sequence that they encode and then contribute to increasing translational efficiency. Efficient polyadenylation of recombinant transcription is desirable as the absence of transcripts a poly A tail are unstable and are rapidly degraded. Illustrative examples of polyA signals that can be used in a vector of the invention include an ideal polyA sequence (for example, AATAAA, ATTAAA, AGTAAA), a bovine growth hormone polyA sequence (BGHpA), a β- polyA sequence rabbit globin (rβgpA), or another suitable heterologous or endogenous polyA sequence known in the art.
[0274] In certain embodiments, a retroviral or lentiviral vector further comprises one or more insulating elements. Isolating elements can contribute to protect sequences expressed by lentiviruses, for example, therapeutic polypeptides, from integration site effects, which can be mediated by elements acting as cis present in genomic DNA and lead to unregulated expression of transferred sequences (ie, effect position, see, for example, Burgess-Beusse et al., 2002, Proc. Natl. Acad. Sci., USA, 99: 16433; and Zhan et al., 2001, Hum. Genet., 109: 471). In some embodiments, transfer vectors comprise one or more isolating elements at 3 'LTR and after integration of the provirus into the host genome, the provirus comprises one or more isolators at both 5' or 3 'LTR, due to duplication of LTR 3 '. Isolators suitable for use in the invention include, but are not limited to, the chicken β-globin isolator (see Chung et al., 1993. Cell 74: 505; Chung et al., 1997. PNAS 94: 575; and Bell et al., 1999. Cell 98: 387, incorporated by reference here). Examples of insulating elements include, but are not limited to, an isolator of a β-globin locus, such as chicken HS4.
[0275] According to certain specific embodiments of the invention, more or all of the viral vector structure sequences are derived from a lentivirus, for example, HIV-1. However, it should be understood that many different sources of retroviral and / or lentiviral sequences can be used, or combined and numerous substitutions and changes in certain of the lentiviral sequences can be accommodated without impairing the ability of a transfer vector to perform the functions described here . In addition, a variety of lentiviral vectors are known in the art, see Naldini et al., (1996a, 1996b, and 1998); Zufferey et al., (1997); Dull et al., 1998, Pat. No. 6,013,516; and 5,994,136, many of which can be adapted to produce a viral vector or transfer plasmid of the present invention.
[0276] In several embodiments, the vectors of the invention comprise a promoter operably linked to a polynucleotide that encodes a CAR polypeptide. Vectors can have one or more LTRs, where LTR comprises one or more modifications, such as one or more nucleotide substitutions, additions, or deletions. The vectors may further comprise one of more accessory elements to increase transduction efficiency (for example, a cPPT / FLAP), viral packaging (for example, a Psi (ψ) packaging signal, RRE), and / or other elements that increase expression of the therapeutic gene (for example, poly (A) sequences), and can optionally comprise a WPRE or HPRE.
[0277] In a particular embodiment, the transfer vector of the invention comprises a retroviral LTR (5 ') to the left; a polipurin / DNA flap tract (cPPT / FLAP); a retroviral export element; a promoter active in a T cell, operably linked to a CAR polypeptide encoding polynucleotide considered here; and a retroviral LTR (3 ') on the right; and optionally a WPRE or HPRE.
[0278] In a particular embodiment, the transfer vector of the invention comprises a retroviral LTR (5 ') to the left; a retroviral export element; a promoter active in a T cell, operably linked to a CAR polypeptide encoding polynucleotide considered herein; a retroviral LTR (3 ') on the right; and a poly (A) sequence; and optionally a WPRE or HPRE. In another particular embodiment, the invention provides a lentiviral vector comprising: an LTR (5 ') to the left; a cPPT / FLAP; an RRE; a promoter active in a T cell, operably linked to a CAR polypeptide encoding polynucleotide considered herein; an LTR (3 ') on the right; and a polyadenylation sequence; and optionally a WPRE or HPRE.
[0279] In a certain embodiment, the invention provides a lentiviral vector comprising: an HIV-1 (5 ') LTR to the left; a packaging signal Psi (ψ); a cPPT / FLAP; an RRE; a promoter active in a T cell, operably linked to a CAR polypeptide encoding polynucleotide considered herein; a self-inactivating HIV-1 (SIN) LTR (3 ') on the right; and a rabbit β-globin polyadenylation sequence; and optionally a WPRE or HPRE.
[0280] In another embodiment, the invention provides a vector comprising: at least one LTR; a polipurin / DNA flap tract (cPPT / FLAP); a retroviral export element; and a promoter active in a T cell, operably linked to a CAR polypeptide encoding polynucleotide considered herein; and optionally a WPRE or HPRE.
[0281] In a particular embodiment, the present invention provides a vector comprising at least one LTR; a cPPT / FLAP; an RRE; a promoter active in a T cell, operably linked to a CAR polypeptide encoding polynucleotide considered herein; and a polyadenylation sequence; and optionally a WPRE or HPRE.
[0282] In a certain embodiment, the present invention provides at least one HIV-1 SIN LTR; a packaging signal Psi (ψ); a cPPT / FLAP; an RRE; a promoter active in a T cell, operably linked to a CAR polypeptide encoding polynucleotide considered herein; and a rabbit β-globin polyadenylation sequence; and optionally a WPRE or HPRE.
[0283] In several modalities, the vector is a viral integration vector.
[0284] In several other modalities, the vector is an episomal or non-integrating viral vector.
[0285] In various modalities, the vectors contemplated here, comprise defective non-integrating or integrating retrovirus. In one embodiment, a "defective integration" retrovirus or lentivirus refers to a retrovirus or lentivirus with an integrase that lacks the ability to integrate the viral genome into the host cell genome. In several embodiments, the integrase protein is mutated to specifically decrease its integrase activity. Lentiviral vectors incompatible with integration are obtained by modifying the pol gene that encodes the integrase protein, resulting in a mutated pol gene that encodes a deficient integrative integrase. Such integration incompatibility viral vectors have been described in patent application WO 2006/010834, which is incorporated herein by reference in its entirety.
[0286] Illustrative mutations in the pol HIV-1 gene suitable for reducing integrase activity include, but are not limited to: H12N, H12C, H16C, H16V, S81 R, D41A, K42A, H51A, Q53C, D55V, D64E, D64V, E69A, K71A E85A, E87A, D116N, D1161, D116A, N120G, N1201, N120E, E152G, E152A, D35E, K156E, K156A, E157A, K159E, K159A, K160A, R166, , K186T, K188T E198A, R199c, R199T, R199A, D202A, K211A, Q214L, Q216L, Q221 L, W235F, W235E, K236S, K236A, K246A, G247W, D253A, R262A, 263
[0287] Illustrative mutations in the HIV-1 pol gene suitable for reducing integrase activity include, but are not limited to: D64E, D64V, E92K, D116N, D1161, D116A, N120G, N1201, N120E, E152G, E152A, D35E, K156E, K156A, E157A, K159E, K159A, W235F and W235E.
[0288] In a particular embodiment, an integrase comprises a mutation in one or more of the amino acids, D64, D116 or E152. In one embodiment, an integrase comprises a mutation in the amino acids, D64, D116 and E152. In a particular embodiment, a defective HIV-1 integrase comprises a D64V mutation.
[0289] A "host cell" includes cells electroporated, transfected, infected, or transduced in vivo, ex vivo, or in vitro with a recombinant vector or polynucleotide of the invention. Host cells can include packaging cells, producer cells, and cells infected with viral vectors. In particular embodiments, host cells infected with the viral vector of the invention are administered to an individual in need of therapy. In certain embodiments, the term "target cell" is used interchangeably with a host cell and refers to cells transfected, infected, or transduced from a desired cell type. In preferred embodiments, the target cell is a T cell.
[0290] Large scale viral particle production is often necessary to obtain a reasonable viral titer. Viral particles are produced by transfecting a transfer vector into a packaging cell line that comprises structural genes and / or viral accessories, for example, gag, pol, env, tat, rev, vif, vpr, vpu, vpx or nef genes or other retroviral genes.
[0291] As used here, the term "packaging vector" refers to an expression vector or viral vector that lacks a packaging signal and comprises a polynucleotide that encodes one, two, three, four or more structural genes and / or viral accessories. Typically, packaging vectors are included in a packaging cell, and are introduced into the cell via transfection, transduction or infection. Methods for transfection, transduction or infection are well known to those of skill in the art. A retroviral / lentiviral transfer vector of the present invention can be introduced into a packaging cell line, via transfection, transduction or infection, to generate a producing cell or cell line. The packaging vectors of the present invention can be introduced into human cells or cell lines by standard methods including, for example, transfection, lipofection or electroporation of calcium phosphate. In some embodiments, packaging vectors are introduced into cells along with a selectable dominant marker, such as neomycin, hygromycin, puromycin, blastocidine, zeocin, thymidine kinase, DHFR, Gin synthetase or ADA, followed by selection in the presence of the appropriate drug and isolation of clones. A selectable marker gene can be physically linked to genes that encode by the packaging vector, for example, by IRES or self-cleaving viral peptides.
[0292] Viral envelope proteins (env) determine the range of host cells that can finally be infected and transformed by recombinant retroviruses generated from cell lines. In the case of lentiviruses, such as HIV-1, HIV-2, SIV, FIV and EIV, env proteins include gp41 and gp120. Preferably, the viral env proteins expressed by packaging cells of the invention are encoded in a separate vector from the gag and pol viral genes, as previously described.
[0293] Illustrative examples of retroviral-derived env genes that can be used in the invention include, but are not limited to: MLV envelopes, 10A1 envelope, BAEV, FeLV-B, RD114, SSAV, Ebola, Sendai, FPV ( plague virus), and envelopes of influenza viruses. Similarly, genes encoding RNA virus envelopes (for example, RNA virus families from Picornaviridae, Calciviridae, Astroviridae, Togaviridae, Flaviviridae, Coronaviridae, Paramixoviridae, Rhabdoviridae, Filoviridae, Orthomyxoviridae, Retromyviridae, Aria, Bunia as well as from DNA viruses (families of Hepadnaviridae, Circoviridae, Parvoviridae, Papovaviridae, Adenoviridae, Herpesviridae, Poxiiridae and Iridoviridae) can be used. Representative examples include, FeLV, VEE, HFVW, WDSV, SFV, Rabies, ALV, BIV, BLV, EBV, CAEV, SNV, ChTLV, STLV, MPMV, SMRV, RAV, FuSV, MH2, AEV, AMV, CT10 and EIAV.
[0294] In other embodiments, envelope proteins for pseudotyping the viruses of the present invention include, but are not limited to any of the following viruses: Influenza A such as H1N1, H1N2, H3N2 and H5N1 (avian influenza), Influenza B, viruses Influenza C, Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Hepatitis D virus, Hepatitis E virus, Rotavirus, any virus in the Norwalk virus group, enteric adenovirus, parvovirus, Dengue virus, Monkey pox , Mononegavirales, Lyssaviruses such as rabies virus, Lagos bat virus, Mokola virus, Duvenhage virus, European bat virus 1 & 2 and Australian bat virus, Ephemerovirus, Vesiculovirus, Vesicular Stomatitis Virus (VSV), Herpesvirus such as viruses Herpes simplex types 1 and 2, chickenpox zoster, cytomegalovirus, Epstein-Bar virus (EBV), human herpesvirus (HHV), human herpesvirus types 6 and 8, Human immunodeficiency virus (HIV), papilloma virus, murine gamma herpesvirus, Arenavirus such such as Argentine hemorrhagic fever virus, Bolivian hemorrhagic fever virus, Sabia-associated hemorrhagic fever virus, Venezuelan hemorrhagic fever virus, Lassa fever virus, Machupo virus, lymphocytic choriomeningitis virus (LCMV), Bunyaviridiae as well as fever virus hemorrhagic Crimean-Congo, Hantavirus, hemorrhagic fever with kidney syndrome causing virus, Rift Valley fever virus, Filoviridae (filovirus) including Ebola hemorrhagic fever and Marburg hemorrhagic fever, Flaviviridae including Kaysanur Forest disease virus, hemorrhagic fever virus Omsk, Tick-borne encephalitis causing viruses and Paramixoviridae such as Hendra virus and Nipah virus, major smallpox and minor smallpox (smallpox), alphaviruses such as Venezuelan equine encephalitis virus, eastern equine encephalitis virus, western equine encephalitis virus, associated coronavirus with SARS (SARS-CoV), West Nile virus, any virus causing encephalitis.
[0295] In one embodiment, the invention provides packaging cells that produce recombinant retrovirus, for example, lentivirus, pseudotyped with the VSV-G glycoprotein.
[0296] The terms "pseudotype" or "pseudotyping" as used here, refer to a virus whose viral envelope proteins have been replaced with those of another virus having preferential characteristics. For example, HIV can be pseudotyped with vesicular stomatitis virus (VSV-G) protein G envelope proteins, which allows HIV to infect a broader range of cells because HIV envelope proteins (encoded by the env gene) normally target the virus to cells that have CD4 +. In a preferred embodiment of the invention, lentiviral envelope proteins are pseudotyped with VSV-G. In one embodiment, the invention provides packaging cells that produce recombinant retroviruses, for example, lentivirus, pseudotyped with the VSV-G envelope glycoprotein.
[0297] As used here, the term “packaging cell lines” is used in reference to cell lines that do not contain a packaging signal but stably or transiently express viral structural proteins and replication enzymes (for example, gag, pol and env) that are necessary for the correct packaging of viral particles. Any suitable cell line can be used to prepare packaging cells of the invention. The cells are usually mammalian cells. In a particular embodiment, the cells used to produce the packaging cell line are human cells. Suitable cell lines that can be used include, for example, CHO cells, BHK cells, MDCK cells, C3H 10T1 / 2 cells, FLY cells, Psi-2 cells, BOSC 23 cells, PA317 cells, WEHI cells, COS cells, cells BSC 1, BSC 40 cells, BMT 10 cells, VERO cells, W138 cells, MRC5 cells, A549 cells, HT1080 cells, 293 cells, 293 T cells, B-50 cells, 3T3 cells, NIH3T3 cells, HepG2 cells, Saos cells 2, Huh7 cells, HeLa cells, W163 cells, 211 cells and 211A cells. In preferred embodiments, the packaging cells are 293 cells, T 293 cells or A549 cells. In another preferred embodiment, the cells are A549 cells.
[0298] As used herein, the term "producing cell line" refers to a cell line that is capable of producing recombinant retroviral particles, comprising a packaging cell line and a transfer vector construct comprising a packaging signal. The production of infectious viral particles and viral stock solutions can be performed using conventional techniques. Methods of preparing viral stock solutions are known in the art and are illustrated by, for example, Y. Soneoka et al. (1995) Nucl. Acids Res. 23: 628-633, and N. R. Landau et al. (1992) J. Virol. 66: 5110-5113. Infectious virus particles can be collected from the packaging cells using conventional techniques. For example, infectious particles can be collected by cell lysis, or by collecting cell culture supernatant, as is known in the art. Optionally, the collected virus particles can be purified if desired. Suitable purification techniques are well known to those skilled in the art.
[0299] The release of a gene or other polynucleotide sequence using a retroviral or lentiviral vector by means of viral infection rather than by transfection is referred to as "transduction". In one embodiment, retroviral vectors are transduced in a cell through infection and integration of the provirus. In certain embodiments, a target cell, for example, a T cell, is "transduced" if it comprises a gene or other polynucleotide sequence released to the cell by infection using a viral or retroviral vector. In particular embodiments, a transduced cell comprises one or more genes or other polynucleotide sequences released by a retroviral or lentiviral vector in its cell genome.
[0300] In particular embodiments, host cells transduced with a viral vector of the invention that express one or more polypeptides, are administered to an individual to treat and / or prevent a B cell malignancy. Other methods refer to the use of viral vectors in gene therapy, which can be used according to certain embodiments of the present invention, can be found in, for example, Kay, MA (1997) Chest 111 (6 Supp.): 138S-142S; Ferry, N. and Heard, J. M. (1998) Hum. Gene Then 9: 1975-81; Shicamundongory, Y. et al. (1999) Liver 19: 265-74; Oka, K. et al. (2000) Curr. Opin. Lipidol. 11: 179-86; Thule, P. M. and Liu, J. M. (2000) Gene Then 7: 1744-52; Yang, N. S. (1992) Crit. Rev. Biotechnol. 12: 335-56; Alt, M. (1995) J. Hepatol. 23: 746-58; Brody, S. L. and Cristal, R. G. (1994) Ann. N.Y. Acad. Sci. 716: 90-101; Strayer, D. S. (1999) Expert Opin. Investig. Drugs 8: 2159-2172; Smith-Arica, J. R. and Bartlett, J. S. (2001) Cum Cardiol. Rep. 3: 43-49; and Lee, H. C. et al. (2000) Nature 408: 483-8. G. Genetically Modified Cells
[0301] The present invention contemplates, in particular modalities, cells genetically modified to express the CARs considered here, for use in the treatment of conditions related to B cells. As used here, the term "genetically engineered" or "genetically modified" refers to the addition of extra genetic material in the form of DNA or RNA to the total genetic material in a cell. The terms, "genetically modified cells", "modified cells", and "redirected cells", are used interchangeably. As used here, the term "gene therapy" refers to the introduction of extra genetic material in the form of DNA or RNA into the total genetic material in a cell that restores, corrects or modifies expression of a gene, or for the purpose of expressing a therapeutic polypeptide, for example, a CAR.
[0302] In particular modalities, the CARs considered here are introduced and expressed in immune effector cells in order to redirect their specificity to a target antigen of interest, for example, a BCMA polypeptide. An “immune effector cell” is any cell in the immune system that has one or more effector functions (for example, cytotoxic cell death activity, cytokine secretion, ADCC and / or CDC induction).
[0303] Immune effector cells of the invention can be autologous / autogenic ("own") or non-autologous ("non-own", for example, allogeneic, syngeneic or xenogenic).
[0304] "Autologous", as used here, refers to cells from the same individual.
[0305] "Allogeneic", as used here, refers to cells of the same species that differ genetically from the cell in comparison.
[0306] "Unique", as used here, refers to cells from a different individual that are genetically identical to the cell in comparison.
[0307] "Xenogenic", as used here, refers to cells of a different species than the cell in comparison. In preferred embodiments, the cells of the invention are allogeneic.
[0308] Illustrative immune effector cells used with the CARs considered here include T lymphocytes. The terms "T cells" or "T lymphocyte" are recognized techniques and are intended to include thymocytes, immature T lymphocytes, mature T lymphocytes, T lymphocytes in resting, or activated T lymphocytes. A T cell can be an auxiliary T cell (Th), for example, an auxiliary T cell 1 (Th1) or an auxiliary T cell 2 (Th2). The T cells can be a helper T cell (HTL; CD4 + T cells) CD4 + T cells, a cytotoxic T cell (CTL; CD8 + T cells), CD4 + CD8 + T cells, CD4-CD8- T cells, or any other subset of T cells. Other illustrative populations of T cells suitable for use in particular embodiments include naive T cells and memory T cells.
[0309] As would be understood by the qualified person, other cells can also be used as immune effector cells with CARs as described here. In particular, immune effector cells also include NK cells, NKT cells, neutrophils and macrophages. Immune effector cells also include effector cell progenitors in which such progenitor cells can be induced to differentiate into immune effector cells in vivo or in vitro. In addition, in particular embodiments, immune effector cell includes progenitors of immune effector cells such as hematopoietic stem cells (HSCs) contained within the CD34 + population of cells derived from cord blood, bone marrow or mobilized peripheral blood that after administration to an individual to differentiate into mature immune effector cells, or that can be induced in vitro to differentiate into mature immune effector cells.
[0310] As used here, genetically engineered immune effector cells contain BCMA specific CAR can be referred to as, “BCMA specific redirected immune effector cells”.
[0311] The term, "CD34 + cell", as used here refers to a cell that expresses the CD34 protein on its cell surface. "CD34" as used here refers to a cell surface glycoprotein (for example, sialomucine protein) that often acts as a cell-cell adhesion factor and is involved in the entry of T cells into lymph nodes. The CD34 + cell population contains hematopoietic stem cells (HSC), which after administration to a patient differentiate and contribute to all hematopoietic lineages, including T cells, NK cells, NKT cells, neutrophils and cells from the monocyte / macrophage lineage.
[0312] The present invention provides methods for making the immune effector cells that express the CAR considered here. In one embodiment, the method comprises transfecting or transducing immune effector cells isolated from an individual such that the immune effector cells express one or more CAR as described herein. In certain embodiments, immune effector cells are isolated from an individual and genetically modified without further manipulation in vitro. These cells can then be directly re-administered to the individual. In additional embodiments, immune effector cells are activated and stimulated first to proliferate in vitro before being genetically modified to express a CAR. In this regard, immune effector cells can be cultured before and / or after being genetically modified (i.e., transduced or transfected to express a CAR considered here).
[0313] In particular modalities, prior to manipulation or genetic modification in the immune effector cells described here, the source of cells is obtained from an individual. In particular embodiments, CAR-modified immune effector cells comprise T cells. T cells can be obtained from a variety of sources including, but not limited to, peripheral blood mononuclear cells, bone marrow, lymph tissue, cord blood, thymus emission, tissue from an infection site, ascites, pleural effusion, spleen tissue and tumors. In certain embodiments, T cells can be obtained from a unit of blood collected from an individual using any number of techniques known to the skilled person, such as sedimentation, for example, FICOLLTM separation. In one embodiment, cells from an individual's circulating blood are obtained by apheresis. The apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other white blood cells, nucleated red blood cells and platelets. In one embodiment, the cells collected by apheresis can be washed to remove the plasma fraction and to place the cells in a buffer or appropriate media for further processing. The cells can be washed with PBS or another suitable solution that lacks calcium, magnesium, and most, if not all, divalent cations. As would be judged by one of ordinary skill in the art, a washing step can be performed by methods known to those in the art, such as using a centrifuge through semi-automatic flow. For example, the Cobe 2991 cell processor, the Baxter CytoMate, or the like. After washing, the cells can be resuspended in a variety of biocompatible buffers or another saline solution with or without a buffer. In certain embodiments, the undesirable components of the apheresis sample can be removed in the cell directly returned to suspended culture media.
[0314] In certain embodiments, T cells are isolated from peripheral blood mononuclear cells (PBMCs) by lysing red blood cells and depleting monocytes, for example, by centrifugation using a PERCOLLTM gradient. A specific subpopulation of T cells, which expresses one or more of the following markers: CD3, CD28, CD4, CD8, CD45RA, and CD45RO, can be further isolated by positive or negative selection techniques. In one embodiment, a specific subpopulation of T cells, which express CD3, CD28, CD4, CD8, CD45RA and CD45RO is further isolated by positive or negative selection techniques. For example, enrichment of a population of T cells by negative selection can be accomplished with a combination of antibodies directed to surface markers unique to the negatively selected cells. One method for use here is cell separation and / or selection by means of negative magnetic immunoadhesion or flow cytometry that uses a cocktail of monoclonal antibodies directed at cell surface markers present in the negatively selected cells. For example, enriching for CD4 + cells by negative selection, a monoclonal antibody cocktail typically includes antibodies to CD14, CD20, CD11b, CD16, HLA-DR and CD8. Flow cytometry and cell separation can also be used to isolate cell populations of interest for use in the present invention.
[0315] PBMC can be directly modified genetically to express CARs using methods considered here. In certain embodiments, after PBMC isolation, T lymphocytes are still isolated and in certain embodiments, both cytotoxic and helper T lymphocytes can be separated into naive, memory, and subpopulations of effector T cells before or after genetic modification and / or expansion .
[0316] CD8 + cells can be obtained using standard methods. In some embodiments, CD8 + cells are further separated into central memory cells, and effector cells identifying cell surface antigens that are associated with each of those types of CD8 + cells.
[0317] In certain embodiments, naive T CD8 + lymphocytes are characterized by the expression of naive T cell phenotypic markers including CD62L, CCR7, CD28, CD3, CD 127 and CD45RA.
[0318] In particular modalities, memory T cells are present in both CD62L + and CD62L- subsets of CD8 + peripheral blood lymphocytes. PBMCs are separated into CD62L-CD8 + and CD62L + CD8 + fractions after staining with anti-CD8 and anti-CD62L antibodies. In some embodiments, the expression of central memory T cell phenotypic markers includes CD45RO, CD62L, CCR7, CD28, CD3 and CD127 and are negative for granzyme B. In some embodiments, central memory T cells are CD45RO +, CD62L + and T cells CD8 +.
[0319] In some modalities, effector T cells are negative for CD62L, CCR7, CD28 and CD127, and positive for granzyme B and perforin.
[0320] In certain embodiments, CD4 + T cells are further separated into subpopulations. For example, CD4 + helper T cells can be separated into naive, central memory, and effector cells by identifying cell populations that have cell surface antigens. CD4 + lymphocytes can be obtained by standard methods. In some modalities, naive CD4 + T lymphocytes are CD45RO-, CD45RA + T cells, CD62L + CD4 +. In some embodiments, central memory CD4 + cells are CD62L positive and CD45RO positive. In some embodiments, CD4 + effector cells are CD62L and CD45RO negative.
[0321] Immune effector cells, such as T cells, can be genetically modified after isolation using known methods, or immune effector cells can be activated and expanded (or differentiated in the case of parents) in vitro before being genetically modified. In a particular embodiment, immune effector cells, such as T cells, are genetically modified with the chimeric antigen receptors considered here (for example, transduced with a viral vector comprising a nucleic acid encoding a CAR) and are then activated and expanded in vitro. In various embodiments, T cells can be activated and expanded before or after genetic modification to express a CAR, using methods as described, for example, in US Patents 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041; and Publication of U.S. Patent Application No. 20060121005.
[0322] T cells are usually expanded by contact with a surface with an agent that stimulates a signal associated with the CD3 TCR complex and a ligand that stimulates a co-stimulatory molecule on the surface of T cells bound to them. T can be stimulated by contact with an anti-CD3 antibody, or antigen binding fragment thereof, or an anti-CD2 antibody immobilized on a surface, or by contact with a protein kinase C activator (eg, briostatin) in conjunction with a harmless calcium. Co-stimulation of accessory molecules on the surface of T cells is also considered.
[0323] Generally, T cells are expanded by contact with a surface with an agent that stimulates a signal associated with the CD3 TCR complex and a ligand that stimulates a co-stimulatory molecule on the surface of T cells bound to them. T can be stimulated by contact with an anti-CD3 antibody, or antigen binding fragment thereof, or an anti-CD2 antibody immobilized on a surface, or by contact with a protein kinase C activator (eg, briostatin) in conjunction with a harmless calcium. Co-stimulation of accessory molecules on the surface of T cells is also considered.
[0324] In particular embodiments, PBMCs or isolated T cells are contacted with a stimulating and co-stimulating agent, such as anti-CD3 and anti-CD28 antibodies, usually attached to a pearl or other surface, in a culture medium with appropriate cytokines, such as IL-2, IL-7, and / or IL-15. To stimulate proliferation of CD4 + T cells or CD8 + T cells, an anti-CD3 antibody and an anti-CD28 antibody. Examples of an anti-CD28 antibody include 9.3, B-T3, XR-CD28 (Diacione, Besancon, France) can be used with other methods commonly known in the art (Berg et al., Transplant Proc. 30 (8): 3975 - 3977, 1998; Haanen et al., J. Exp. Med. 190 (9): 13191328, 1999; Garland et al., J. Immunol Meth. 227 (1-2): 53 - 63, 1999). Anti-CD3 and anti-CD28 antibodies attached to the same bead serve as a “substitute” antigen presenting cell (APC). In other embodiments, T cells can be activated and stimulated to proliferate with appropriate feeder cells and antibodies and cytokines using methods such as those described in US6040177; US5827642; and WO2012129514.
[0325] In other modalities, artificial APC (aAPC) engineered by K562, U937, 721,221, T2 and C1R cells to direct the stable expression and secretion of a variety of co-stimulatory molecules and cytokines. In a particular embodiment, K32 or U32 aAPCs are used to direct the display of one or more antibody-based stimulatory molecules on the AAPC cell surface. Expression of various combinations of genes in aAPC allows the precise determination of human T cell activation requirements, such that aAPCs can be adapted for optimal propagation of subsets of T cells with specific growth requirements and distinct functions. AAPCs support ex vivo growth and long-term expansion of functional human CD8 T cells without requiring the addition of exogenous cytokines, unlike the use of natural APCs. T cell populations can be expanded by aAPCs that express a variety of costimulatory molecules including, but not limited to, CD137L (4-1 BBL), CD134L (OX40L), and / or CD80 or CD86. Finally, aAPCs provide an efficient platform for expanding genetically modified T cells and for maintaining CD28 expression in CD8 T cells. The aAPCs provided in WO 03/057171 and US2003 / 0147869 are hereby incorporated by reference in their entirety.
[0326] In one embodiment, CD34 + cells are transduced with a nucleic acid construct according to the invention. In certain embodiments, the transduced CD34 + cells differentiate into mature immune effector cells in vivo after administration to an individual, generally the individual from whom the cells were originally isolated. In another embodiment, CD34 + cells can be stimulated in vitro before exposure to or after being genetically modified with a CAR as described here, with one or more of the following cytokines: Flt-3 ligand (FLT3), Stem cell factor (SCF ), megakaryocyte growth and differentiation factor (TPO), IL-3 and IL-6 according to the methods previously described (Asheuer et al., 2004; Imren, et al., 2004).
[0327] The invention provides a population of modified immune effector cells for the treatment of cancer, the modified immune effector cells comprising a CAR as disclosed herein. For example, a population of modified immune effector cells is prepared from peripheral blood mononuclear cells (PBMCs) obtained from a patient diagnosed with B cell malignancy described here (autologous donors). PBMCs form a heterogeneous population of T lymphocytes that can be CD4 +, CD8 +, or CD4 + and CD8 +.
[0328] PBMCs can also include other cytotoxic lymphocytes such as NK cells or NKT cells. An expression vector carrying the CAR coding sequence considered here can be introduced into a population of human donor T cells, NK cells or NKT cells. Successfully transduced T cells that carry the expression vector can be separated using flow cytometry to isolate CD3 positive T cells and then further propagated to increase the number of this CAR protein that expresses T cells in addition to cell activation using anti- CD3 and or anti-CD28 and IL-2 antibodies or any other methods known in the art as described elsewhere herein. Standard procedures are used for cryopreservation of T cells that express CAR protein T cells to store and / or prepare for use in a human individual. In one embodiment, in vitro transduction, culture and / or expansion of T cells are performed in the absence of products derived from non-human animals such as fetal calf serum and fetal bovine serum. Since a heterogeneous population of PBMCs is genetically modified, the resulting transduced cells are a heterogeneous population of modified cells comprising a BCMA-targeted CAR as considered here.
[0329] In an additional embodiment, a mixture of, for example, one, two, three, four, five or more, different expression vectors can be used by genetically modifying a donor population of immune effector cells in which each vector encodes a protein different chimeric antigen receptor as considered here. The resulting modified immune effector cells form a population of mixed modified cells, with a proportion of the modified cells expressing more than different CAR proteins.
[0330] In one embodiment, the invention provides a method of storing immune effector cells that express genetically modified murine, human or humanized CAR protein that targets a BCMA protein, comprising cryopreserving immune effector cells such that the cells remain viable until thawing . A fraction of the immune effector cells that express the CAR proteins can be cryopreserved by methods known in the art to provide a permanent source of such cells for the future treatment of patients afflicted with the B cell related condition. When necessary, the transformed immune effector cells cryopreserved cells can be thawed, cultured and expanded to further such cells.
[0331] As used here, "cryopreserve" refers to the preservation of cells by cooling to sub-zero temperatures, such as (typically) 77 K or -196 ° C. (the boiling point of liquid nitrogen). Cryoprotective agents are often used at sub-zero temperatures to prevent cells from being preserved from damage due to freezing at low temperatures or warming up to room temperature. Cryopreservative agents and optimal cooling rates can protect against cell damage. Cryoprotectants that can be used include, but are not limited to, dimethyl sulfoxide (DMSO) (Lovelock and Bishop, Nature, 1959; 183: 1394 - 1395; Ashwood-Smith, Nature, 1961; 190: 1204 - 1205), glycerol , polyvinylpyrrolidine (Rinfret, Ann. NY Acad. Sci., 1960; 85: 576), and polyethylene glycol (Sloviter and Ravdin, Nature, 1962; 196: 48). The preferred cooling rate is 1 to 3 ° C / minute. After at least two hours, T cells have reached a temperature of -80 ° C and can be placed directly in liquid nitrogen (-196 ° C) for permanent storage, such as in a long-term cryogenic storage vessel. H. T cell manufacturing methods
[0332] T cells manufactured by the methods contemplated herein provide improved adoptive immunotherapy compositions. Without wishing to be bound by any particular theory, it is believed that the T cell compositions manufactured by the methods contemplated herein are imbued with superior properties, including increased survival, expansion in the relative absence of differentiation and persistence in vivo. In one embodiment, a method of making T cells comprises contacting the cells with one or more agents that modulate a PI3K cell signaling pathway. In one embodiment, a method of making T cells comprises contacting the cells with one or more agents that modulate a PI3K / Akt / mTOR cell signaling pathway. In various modalities, T cells can be obtained from any source and contacted with the agent during the activation and / or expansion phases of the manufacturing process. The resulting T cell compositions are enriched in potent developer T cells that have the ability to proliferate and express one or more of the following biomarkers: CD62L, CCR7, CD28, CD27, CD122, CD127, CD197 and CD38. In one embodiment, populations of cells that make up T cells, which are treated with one or more PI3K inhibitors, are enriched for a population of CD8 + T cells that coexpress one or more, or all of the following biomarkers: CD62L, CD127, CD197 and CD38.
[0333] In one embodiment, modified T cells that comprise maintained levels of proliferation and decreased differentiation are manufactured. In a particular embodiment, T cells are manufactured by stimulating T cells to be activated and to proliferate in the presence of one or more stimulating signals and an agent that is an inhibitor of a PI3K cell signaling pathway.
[0334] T cells can then be modified to express an anti-BCMA CARs. In one embodiment, T cells are modified by transducing T cells with a viral vector comprising an anti-BCMA CAR contemplated herein. In a particular embodiment, T cells are modified prior to stimulation and activation in the presence of an inhibitor of a PI3K cell signaling pathway. In another embodiment, T cells are modified after stimulation and activation in the presence of an inhibitor of a PI3K cell signaling pathway. In a particular embodiment, T cells are modified within 12 hours, 24 hours, 36 hours or 48 hours of stimulation and activation in the presence of an inhibitor of a PI3K cell signaling pathway.
[0335] After T cells are activated, cells are cultured to proliferate. T cells can be cultured for at least 1,2,3, 4, 5, 6 or 7 days, at least 2 weeks, at least 1, 2, 3, 4, 5 or 6 months or more with 1, 2 3 , 4, 5, 6, 7, 8, 9 or 10 or more expansion rounds.
[0336] In various embodiments, T cell compositions are manufactured in the presence of one or more inhibitors of the PI3K pathway. Inhibitors can direct one or more activities on the pathway or in a single activity. Without pretending to be linked to any particular theory, it is contemplated that the treatment or contact of T cells with one or more inhibitors of the PI3K pathway during the stages of stimulation, activation and / or expansion of the manufacturing process preferably increases young T cells, thus producing superior therapeutic T cell compositions.
[0337] In a particular embodiment, a method is provided to increase the proliferation of T cells that express a manipulated T cell receptor. Such methods may comprise, for example, harvesting a source of T cells from an individual, stimulating and activating T cells in the presence of one or more inhibitors of the PI3K pathway, modification of T cells to express an anti-BCMA CAR, for example, Anti-BCMA02 CAR, and expanding T cells in culture.
[0338] In a particular embodiment, a method for producing enriched T cell populations for the expression of one or more of the following biomarkers: CD62L, CCR7, CD28, CD27, CD122, CD127, CD197 and CD38. In one embodiment, young T cells comprise one or more of, or all of the following biological markers: CD62L, CD127, CD197 and CD38. In one embodiment, young T cells lack CD57, CD244, CD160, PD-1, CTLA4, TIM3 and LAG3 expression. As discussed elsewhere here, the expression levels of young T cell biomarkers are relative to the expression levels of such markers in more differentiated T cells or populations of immune effector cells.
[0339] In one embodiment, peripheral blood mononuclear cells (PBMCs) are used as a source of T cells in the T cell manufacturing methods contemplated here. PBMCs form a heterogeneous population of T lymphocytes that can be CD4 +, CD8 + or CD4 + and CD8 + and can include other mononuclear cells such as monocytes, B cells, NK cells and NKT cells. An expression vector comprising a polynucleotide encoding an engineered TCR or CAR contemplated herein can be introduced into a population of human donor T cells, NK cells or NKT cells. Successfully transduced T cells carrying the expression vector can be classified using flow cytometry to isolate CD3 positive T cells and then propagated to increase the number of modified T cells, in addition to cell activation using anti-CD3 antibodies and or anti antibodies -CD28 AND IL-2, IL-7 and / or IL-15 or any other methods known in the art, as described elsewhere herein.
[0340] The manufacturing methods contemplated herein may further comprise cryopreservation of modified T cells for storage and / or preparation for use in a human individual. T cells are cryopreserved in such a way that the cells remain viable after thawing. When necessary, immune effector cells transformed by cryopreserved can be thawed, grown and expanded to more of these cells. As used herein, "cryopreservation" refers to the preservation of cells by cooling to temperatures below zero, such as (typically) 77 K or -196 ° C (the boiling point of liquid nitrogen). Cryoprotective agents are often used at sub-zero temperatures to prevent cells from being preserved from damage caused by freezing at low temperatures or heating to room temperature. Cryopreservative agents and optimal cooling rates can protect against cell damage. Cryoprotective agents that can be used include, but are not limited to, dimethyl sulfoxide (DMSO) (Lovelock and Bishop, Nature, 1959; 183: 1394-1395; Ashwood-Smith, Nature, 1961; 190: 1204-1205) , glycerol, polyvinylpyrrolidine (Rinfret, Ann. NY Acad. Sci., 1960; 85: 576) and polyethylene glycol (Sloviter and Ravdin, Nature, 1962; 196: 48). The preferred cooling rate is 10 to 3 ° C / minute. After at least two hours, T cells have reached a temperature of -80 ° C and can be placed directly in liquid nitrogen (- 196 0 C) for permanent storage, such as in a long-term cryogenic storage container. 1. T cells
[0341] The present invention contemplates the manufacture of improved CAR T cell compositions. The T cells used for the production of CAR T cells can be autologous / autogenic ("auto") or non-autologous ("non-auto", for example, allogeneic, synergistic or xenogenic). In preferred embodiments, T cells are obtained from a mammalian individual. In a more preferred embodiment, T cells are obtained from a primate individual. In the most preferred embodiment, T cells are obtained from a human individual.
[0342] T cells can be obtained from various sources including, but not limited to, peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus problem, tissue from an infection site , ascites, pleural effusion, spleen tissue, and tumors. In certain embodiments, T cells can be obtained from a blood unit taken from an individual using any number of techniques known to the person skilled in the art, such as sedimentation, for example, FICOLLTM separation. In one embodiment, an individual's circulating blood cells are obtained by apheresis. The apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells and platelets. In one embodiment, the cells collected by apheresis can be washed to remove the plasma fraction and place the cells in an appropriate buffer or medium for further processing. The cells can be washed with PBS or another suitable solution that lacks calcium, magnesium and most, if not all, divalent cations. As will be appreciated by those skilled in the art, a washing step can be performed by methods known to those in the art, for example, using a semi-automatic pass-through centrifuge. For example, the Cobe 2991 cell processor, CytoMate Baxter or similar. After washing, cells can be resuspended in a variety of biocompatible buffers or another saline solution with or without a buffer. In certain embodiments, the undesirable components of the apheresis sample can be removed in the culture from directly resuspended culture media.
[0343] In particular embodiments, a cell population comprising T cells, for example, PBMCs, is used in the manufacturing methods contemplated here. In other embodiments, an isolated or purified population of T cells is used in the manufacturing methods contemplated here. The cells can be isolated from peripheral blood mononuclear cells (PBMCs) by lysing red blood cells and emptying the monocytes, for example, by centrifugation using a PERCOLL ™ gradient. In some modalities, after isolation of PBMC, both cytotoxic and auxiliary T lymphocytes can be classified into subpopulations of native T cells, memory and effector before or after activation, expansion and / or genetic modification.
[0344] A specific sub-population of T cells, which expresses one or more of the following markers: CD3, CD4, CD8, CD28, CD45RA, CD45RO, CD62, CD127 and HLA-DR can be further isolated by positive or negative selection techniques. In one embodiment, a specific sub-population of T cells, which expresses one or more of the markers selected from the group consisting of i) CD62L, CCR7, CD28, CD27, CD122, CD127, CD197; or ii) CD38 or CD62L, CD127, CD197 and CD38, is further isolated by positive or negative selection techniques. In various embodiments, the T cell compositions manufactured do not express or substantially express one or more of the following markers: CD57, CD244, CD160, PD-1, CTLA4, TIM3 and LAG3.
[0345] In one embodiment, the expression of one or more of the markers selected from the group consisting of CD62L, CD127, CD197 and CD38 is increased at least 1.5 times, at least 2 times, at least 3 times, by at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 25 times, or more compared to a population of activated T cells and expanded without a PI3K inhibitor.
[0346] In one embodiment, the expression of one or more of the selected markers from the group consisting of CD57, CD244, CD160, PD-1, CTLA4, TIM3 and LAG3 is decreased at least 1.5 times, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 25 times, or more in compared to a population of T cells activated and expanded with a PI3K inhibitor.
[0347] In one embodiment, the manufacturing methods contemplated here increase the number of CAR T cells comprising one or more activated T cell markers or potent developers. Without wishing to be bound by any particular theory, the present inventors believe that treating a population of cells that make up T cells with one or more PI3K inhibitors results in an increase in the expansion of potent developer T cells and provides adoptive CAR T cells more robust and effective in cellular immunotherapy compared to existing CAR T cell therapies.
[0348] Illustrative examples of potent developer T cell markers augmented on T cells manufactured using the methods contemplated herein include, but are not limited to CD62L, CD127, CD197 and CD38. In particular embodiments, naive T cells that are not expressed do not express or do not substantially express one or more of the following markers: CD57, CD244, CD160, PD-1, BTLA, CD45RA, CTLA4, TIM3 and LAG3.
[0349] With respect to T cells, the T cell populations resulting from the various expansion methodologies contemplated here may have a variety of specific phenotypic properties, depending on the conditions employed. In various embodiments, expanded T cell populations comprise one or more of the following phenotypic markers: CD62L, CD127, CD197, CD38 and HLA-DR.
[0350] In one embodiment, such phenotypic markers include increased expression of one or more of CD62L, CD127, CD197 and CD38 or all CD62. In particular embodiments, CD8 + T lymphocytes characterized by the expression of naive T cell phenotypic markers including CD62L, CD127, CD197 and CD38 are expanded.
[0351] In particular embodiments, T cells characterized by the expression of centric memory T cell phenotypic markers, including CD45RO, CD62L, CD127, CD197 and CD38 and negative for granzyme B, are expanded. In some embodiments, the T cells in the memory center are CD45RO +, CD62L +, CD8 + T cells.
[0352] In certain embodiments, CD4 + T lymphocytes characterized by the expression of phenotypic markers of CD4 + naive cells including CD62L and negative for CD45RA and / or CD45RO expression are expanded. In some embodiments, CD4 + cells characterized by the expression of phenotypic markers of central memory CD4 + cells including CD62L and CD45RO positive. In some embodiments, CD4 + effector cells are CD62L positive and CD45RO negative.
[0353] In certain embodiments, T cells are isolated from an individual and activated and stimulated to proliferate in vitro before being genetically modified to express an anti-BCMA CAR. In this regard, T cells can be cultured before and / or after being genetically modified (i.e., transduced or transfected to express an anti-BCMA CAR contemplated herein). 2. ACTIVATION AND EXPANSION
[0354] In order to obtain sufficient therapeutic doses of T cell compositions, T cells are often subjected to one or more rounds of stimulation, activation and / or expansion. T cells can be activated and expanded generally using methods as described, for example, in U.S. Patents 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; And 6,867,041, each of which is incorporated herein by reference in its entirety. T cells modified to express an anti-BCMA CAR can be activated and expanded before and / or after T cells are modified. In addition, T cells can be contacted with one or more agents that modulate the PI3K cell signaling pathway before, during and / or after activation and / or expansion. In one embodiment, the T cells manufactured by the methods contemplated herein undergo one, two, three, four or five or more rounds of activation and expansion, each of which may include one or more agents that modulate the signaling pathway of PI3K cells .
[0355] In one embodiment, a costimulatory ligand is established in an antigen-establishing cell (for example, aAPC, dendritic cell, B cell and the like) that specifically binds a cognated costimulatory molecule to a T cell, thereby providing a signal that, in addition, to the primary signal provided, for example, by binding a TCR / CD3 complex, mediates a desired T cell response. Suitable costimulatory ligands include, but are not limited to, CD7, B7-1 (CD80), B7-2 (CD86), PD-L 1, PD-L2, 4-1 BBL, OX40L, inducible stimulator ligand ( ICOS-L), intercellular adhesion molecule (ICAM), CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB, HVEM, beta lymphotoxin receptor, ILT3, ILT4, an agonist or antibody that binds to the receptor of the Toll linker and a linker that specifically binds with B7-H3.
[0356] In a particular embodiment, a costimulatory ligand comprises an antibody or its antigen binding fragment that specifically binds to a costimulatory molecule present in a T cell, including, but not limited to, CD27, CD28, 4 - IBB, 0X40, CD30, CD40, PD-1, 1COS, antigen-1 associated with lymphocyte function (LFA-1), CD7, LIGHT, NKG2C, B7-H3 and a ligand that specifically binds with CD83.
[0357] Suitable costimulatory ligands further include target antigens, which can be provided in soluble form or expressed in APCs or aAPCs that bind to manipulated TCRs or RAC expressed in modified T cells.
[0358] In several embodiments, a method for making T cells contemplated here comprises activating a cell population comprising Tea cells and expanding the T cell population. Activation of T cells can be achieved by providing a primary stimulation signal through the TCR / CD3 complex of T cells or by stimulating the CD2 surface protein and providing a secondary costimulation signal through an accessory molecule, for example, CD28.
[0359] The TCR / CD3 complex can be stimulated by contacting the T cell with a suitable CD3 binding agent, for example, a CD3 linker or an anti-CD3 monoclonal antibody. Illustrative examples of CD3 antibodies include, but are not limited to, OKT3, G19-4, BC3 and 64.1.
[0360] In another embodiment, a CD2 binding agent can be used to provide a primary stimulation signal to T cells. Illustrative examples of CD2 binding agents include, but are not limited to, CD2 ligands and anti-CD2 antibodies, for example, the T11.3 antibody in combination with the T11.1 or T11.2 antibody (Meuer, SC et al. (1984) Cell 36: 897-906) and the 9.6 antibody (which recognizes the same epitope as T11.1) in combination with the 9-1 antibody (Yang, SY et al. (1986) J. Immunol. 137: 1097-1100). Other antibodies that bind to the same epitopes as any of the antibodies described above can also be used. Additional antibodies, or combinations of antibodies, can be prepared and identified by standard techniques, as described elsewhere here.
[0361] In addition to the primary stimulation signal provided through the TCR / CD3 complex, or through CD2, the induction of T cell responses requires a second costimulatory signal. In particular embodiments, a CD28 binding agent can be used to provide a costimulatory signal. Illustrative examples of CD28 binding agents include, but are not limited to: natural CD28 ligands, for example, a natural CD28 ligand (for example, a member of the B7 protein family, such as B7-1 (CD80) and B7- 2 (CD86) and anti-CD28 monoclonal antibody or a fragment thereof capable of crosslinking the CD28 molecule, for example, monoclonal antibodies 9.3, B-T3, XR-CD28, KOLT-2, 15E8, 248.23.2 and EX5.3D10.
[0362] In one embodiment, the molecule providing the primary stimulation signal, for example a molecule that provides stimulation through the TCR / CD3 or CD2 complex, and the co-stimulatory molecule is coupled to the same surface.
[0363] In certain embodiments, binding agents that provide stimulating and costimulatory signals are located on the surface of a cell. This can be accomplished by transfecting or transducing a cell with a nucleic acid that encodes the binding agent in a form suitable for its expression on the cell surface or, alternatively, by coupling a binding agent to the cell surface.
[0364] In another embodiment, the molecule providing the primary stimulation signal, for example, a molecule that provides stimulation through the TCR / CD3 or CD2 complex, and the co-stimulatory molecule is displayed in the antigen-establishing cells.
[0365] In one embodiment, the molecule providing the primary stimulation signal, for example a molecule that provides stimulation through the TCR / CD3 or CD2 complex, and the co-stimulatory molecule is provided on separate surfaces.
[0366] In a certain embodiment, one of the binding agents that provide stimulating and co-stimulating signals is soluble (provided in the solution) and the other agent (s) is provided on one or more surfaces.
[0367] In a particular embodiment, the binding agents that provide stimulating and co-stimulating signals are both provided in a soluble form (provided in the solution).
[0368] In various modalities, the methods for making T cells contemplated here comprise the activation of T cells with anti-CD3 and anti-CD28 antibodies.
[0369] T cell compositions manufactured by the methods contemplated herein comprise activated and / or expanded T cells in the presence of one or more agents that inhibit a PI3K cell signaling pathway. T cells modified to express an anti-BCMA CAR can be activated and expanded before and / or after T cells are modified. In particular embodiments, a population of T cells is activated, modified to express an anti-BCMA CAR, and then cultured for expansion.
[0370] In one embodiment, T cells manufactured by the methods contemplated herein comprise an increased number of T cells that express indicative markers of high proliferative potential and the ability to self-renew, but that do not express or express substantially undetectable markers of cell differentiation T. These T cells can be repeatedly activated and expanded robustly and thus provide an improved therapeutic T cell composition.
[0371] In one embodiment, a population of activated and expanded T cells in the presence of one or more agents that inhibit a PI3K cell signaling pathway is expanded at least 1.5 times, at least 2 times, at least 3 times, at least 4 times, a At least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 25 times, at least 50 times, at least 100 times at least 250 times, at least 500 times, at least 1,000 times, or more compared to a population of activated and expanded T cells without a PI3K inhibitor.
[0372] In one embodiment, a population of T cells characterized by the expression of young T cell markers are activated and expanded in the presence of one or more agents that inhibit a PI3K cell signaling pathway is expanded at least 1.5 times, by at least 2 times, at least 5 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 9 times, at least 9 times, at least 10 times, at least 25 times, at least 50 times, a At least 100 times, at least 250 times, at least 500 times, at least 1,000 times, or more, compared the population of activated and expanded T cells without a PI3K inhibitor.
[0373] In one embodiment, expanding the T cells activated by the methods contemplated herein also includes culturing a population of cells comprising T cells for several hours (about 3 hours) to about 7 days to about 28 days or whatever integer value between hours. In another embodiment, the T cell composition can be cultured for 14 days. In a particular embodiment, T cells are cultured for about 21 days. In another embodiment, the T cell compositions are grown for about 2-3 days. Various stimulation / activation / expansion cycles can also be desired in such a way that the T cell culture time can be 60 days or more.
[0374] In particular embodiments, suitable conditions for T cell culture include an appropriate medium (for example, Minimum Essential Medium or RPMI Media 1640 or X-vivo 15, (Lonza)) and one or more factors necessary for proliferation and viability including, but not limited to, serum (eg, fetal bovine or human serum), interleukin-2 (IL-2), insulin, IFN-y, IL-4, IL-7, IL-21, GM-CSF , IL-10, IL-12, IL-15, TGFβ and TNF-a or any other additives suitable for cell growth known to those skilled in the art.
[0375] Other illustrative examples of cell culture media include, but are not limited to RPMI 1640, Clicks, AIM-V, DMEM, MEM, a-MEM, F-12, X-Vivo 15 and X-Vivo 20, optimizer, with added amino acids, sodium pyruvate and vitamins, without serum or supplemented with an appropriate amount of serum (or plasma) or a defined set of hormones, and / or an amount of cytokines sufficient for the growth and expansion of T cells.
[0376] Illustrative examples of other additives for T cell expansion include, but are not limited to, surfactant, piasmanate, pH buffers such as HEPES and reducing agents such as N-acetyl-cysteine and 2-mercaptoethanol.
[0377] Antibiotics, for example, penicillin and streptomycin, are included only in experimental cultures, not in cell cultures that must be infused into an individual. The target cells are maintained in the conditions necessary to support growth, for example, at an appropriate temperature (for example, 37 ° C) and atmosphere (for example, air plus 5% CO2).
[0378] In particular embodiments, isolated PBMCs or T cells are brought into contact with a stimulating agent and a co-stimulating agent, such as anti-CD3 and anti-CD28 antibodies, usually attached to a pearl or other surface, in a culture with appropriate cytokines, such as IL -2, IL-7 and / or IL-15.
[0379] In other embodiments, artificial APC (aAPC) produced by K562, U937, 721.221, T2 and C1R cells engineered to direct the stable expression and secretion of a variety of co-stimulating molecules and cytokines. In a particular embodiment, the K32 or U32 AAPCs are used to direct the display of one or more antibody-based stimulating molecules on the AAPC cell surface. T cell populations can be expanded by aAPCs that express a variety of costimulatory molecules including, but not limited to, CD137L (4-1 BBL), CD134L (OX40L) and / or CD80 or CD86. Finally, aAPCs provide an efficient platform to expand genetically modified T cells and to maintain CD28 expression in CD8 T cells. AAPCs provided in WO 03/057171 and US2003 / 0147869 are hereby incorporated by reference in their entirety. 3. Agents
[0380] In various embodiments, a method for making T cells is provided that expands undifferentiated or developmental T cells comprising contacting T cells with an agent that modulates a PI3K pathway in the cells. In various embodiments, a method for making T cells is provided that expands undifferentiated or developmental T cells comprising contacting T cells with an agent that modulates a PI3K / AKT / mT0R pathway in the cells. The cells can be contacted before, during and / or after activation and expansion. T cell compositions retain sufficient T cell potency so that they can undergo multiple rounds of expansion without a substantial increase in differentiation.
[0381] As used herein, the terms "modular", "modulator" or "modulating agent" or comparable term refer to an agent's ability to cause a change in a cell's signaling pathway. A modulator can increase or decrease a quantity, activity of a pathway component or increase or decrease the desired effect or the output of a cell signaling pathway. In one embodiment, the modulator is an inhibitor. In another embodiment, the modulator is an activator.
[0382] An "agent" refers to a compound, small molecule, for example, small organic molecule, nucleic acid, polypeptide or a fragment, isoform, variant, analog or derivative thereof used in modulating a PI3K / AKT pathway / mTOR.
[0383] A "small molecule" refers to a composition that has a molecular weight of less than about 5 kD, less than about 4 kD, less than about 3 kD, less than about 2 kD, less than about 1 kD, or less than about. 5kD. Small molecules can comprise nucleic acids, petids, polypeptides, peptidomimetics, peptides, carbohydrates, lipids, their components or other organic or inorganic molecules. Libraries of chemical and / or biological mixtures, such as fungal, bacterial or algal extracts, are known in the art and can be screened with any of the assays of the invention. Examples of methods for the synthesis of molecular libraries can be found at: (Carell et al., 1994a; Carell et al., 1994b; Cho et al., 1993; DeWitt et al., 1993; Gallop et al., 1994; Zuckermann et al., 1994).
[0384] An "analogue" refers to a small organic compound, nucleotide, protein or polypeptide that has activity or function (s) similar or identical to that of the compound, nucleotide, protein or polypeptide or compound that has activity of the present invention, but need not necessarily comprise a sequence or structure that is similar or identical to the sequence or structure of the preferred embodiment.
[0385] A "derivative" refers to a compound, protein or polypeptide that comprises an amino acid sequence of an original protein or polypeptide that has been altered by introducing substitutions, deletions or additions of amino acid residues or a nucleic acid or nucleotide that has been modified by introducing nucleotide substitutions or deletions, additions or mutations. The derived nucleic acid, nucleotide, protein or polypeptide has a similar or identical function to the original polypeptide.
[0386] In several modalities, the agent that modulates a PI3K pathway activates a component of the pathway. An "activator" or "agonist" refers to an agent that promotes, increases or induces one or more activities of a molecule in a PI3K / AKT / mT0R pathway including, without limitation, a molecule that inhibits one or more activities of a PI3K.
[0387] In several modalities, the agent that modulates a PI3K pathway inhibits a component of the pathway. An "inhibitor" or "antagonist" refers to an agent that inhibits, decreases or reduces one or more activities of a molecule in a PI3K pathway including, without limitation, a PI3K. In one embodiment, the inhibitor is a double molecule inhibitor. In particular, the inhibitor can inhibit a class of molecules that have the same or substantially similar activities (a pan inhibitor) or can specifically inhibit the activity of a molecule (a selective or specific inhibitor). The inhibition can also be irreversible or reversible.
[0388] In one embodiment, the inhibitor has an ICsode of at least 1 nM, at least 2 nM, at least 5 nM, at least 10 nM, at least 50 nM, at least 100 nM, at least 200 nM, at least 500 nM, at least 1 pM, at least 10 pM, At least 50 pM, or at least 100 pM. ICso determinations can be performed using any conventional techniques known in the art. For example, an ICso can be determined by measuring the activity of a given enzyme in the presence of a range of concentrations of the inhibitor being studied. The experimentally obtained values of the enzyme activity are then plotted against the concentrations of inhibitors used. The concentration of the inhibitor showing 50% enzyme activity (compared to the activity in the absence of any inhibitor) is taken as the "ICso" value. Similarly, other inhibitory concentrations can be defined through appropriate activity determinations.
[0389] In various embodiments, T cells are contacted or treated or cultured with one or more modulators of a PI3K pathway at a concentration of at least 1 nM, at least 2 nM, at least 5 nM, at least 10 nM, at least at least 50 nM, at least 100 nM, at least 200 nM, at least 500 nM, at least 1 pM, at least 10 pM, at least 50 pM, at least 100 pM or at least 1 M.
[0390] In particular embodiments, T cells can be contacted or treated or cultured with one or more PI3K pathway modulators for at least 12 hours, 18 hours, at least 1, 2, 3, 4, 5, 6 or 7 days, a At least 2 weeks, at least 1,2, 3, 4, 5 or 6 months or more with 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more expansion rounds . The. PI3K / Akt / mTOR path
[0391] The phosphatidyl-inositol-3 kinase / Akt / mammalian target of the rapamycin pathway serves as a channel to integrate growth factor signaling with cell proliferation, differentiation, metabolism and survival. PI3Ks are a family of highly conserved intracellular lipid chimeras. Class IA PI3Ks are activated by the growth factor receptor tyrosine kinases (RTKs), either directly or by interaction with the insulin receptor substrate family of adapter molecules. This activity results in the production of phosphatidyl-inositol-3,4,5-trisphospate (PIP3) a seri / threonine chiπase Akt regulator. MTOR works through the PI3K canonical pathway through 2 different complexes, each characterized by different liaison partners that confer different activities. MT0RC1 (mTOR in the complex with PRAS40, raptor and ml_ST8 / Gbl_) acts as an effector downstream of PI3K / Akt signaling, linking growth factor signals to protein translation, cell growth, proliferation and survival. MT0RC2 (mTOR in the complex with rictor, mSIN1, protor and ml_ST8) acts as an activator upstream of Akt.
[0392] After PI3K growth factor receptor-mediated activation, Akt is recruited to the membrane through the interaction of its pleckstrin homology domain with PIP3, thereby exposing its activation cycle and enabling phosphorylation in threonine 308 ( Thr308) by phospho -inositide-dependent protein kinase 1 (PDK1). For maximum activation, Akt is also phosphorylated by mT0RC2, in serine 473 (Ser473) of its C-terminal hydrophobic motif. DNA-PK and HSP have also been shown to be important in regulating Akt activity. Akt activates mTORCI through inhibitory phosphorylation of TSC2, which together with TSC1, down-regulates mTORCI by inhibiting Rheb GTPase, a positive regulator of mTORCI. MTORC1 has 2 well-defined substrates, p70S6K (referred to below as S6K1) and 4E-BP1, both of which critically regulate protein synthesis. Thus, mTORCI is an important effector downstream of PI3K, which links growth factor signaling with protein translation and cell proliferation. B. PI3K inhibitors
[0393] As used herein, the term "PI3K inhibitor" refers to a nucleic acid, peptide, compound or small organic molecule that binds to and inhibits at least one PI3K activity. PI3K proteins can be divided into three classes, PI3Ks of class 1, PI3Ks of class 2 and PI3Ks of class 3. PI3Ks of Class 1 exist as heterodimers consisting of one of the four catalytic subunits p110 (p110α, p110β, p110δ and pHOy) and one of two families of regulatory subunits. A PI3K inhibitor of the present invention preferably targets PI3K class 1 inhibitors. In one embodiment, a PI3K inhibitor will exhibit selectivity for one or more isoforms of class 1 PI3K inhibitors (ie selectivity for p110a, p110β, p110β, and pUOy or one or more of p110a, p110β, p110δ and p110y). In another aspect, a PI3K inhibitor will not exhibit isoform selectivity and will be considered a "pan-PI3K inhibitor". In one embodiment, a PI3K inhibitor will compete for binding with ATP to the PI3K catalytic domain.
[0394] In certain embodiments, a PI3K inhibitor can, for example, target PI3K, as well as additional proteins in the PI3K-AKT-mTOR pathway. In particular embodiments, a PI3K inhibitor that targets both mTOR and PI3K can be referred to as an mTOR inhibitor or a PI3K inhibitor. A PI3K inhibitor that only targets PI3K can be referred to as a selective PI3K inhibitor. In one embodiment, a selective PI3K inhibitor can be understood as referring to an agent that exhibits a 50% inhibitory concentration compared to PI3K that is at least 10 times, at least 20 times, at least 30 times, at least 50 times at least 100 times, at least 1,000 times, or more, lower than the ICso of the inhibitor in relation to mTOR and / or other proteins in the pathway.
[0395] In a particular embodiment, exemplary PI3K inhibitors inhibit PI3K with an ICso (concentration that inhibits 50% of activity) of about 200 nM or less, preferably about 100 nm or less, even more preferably about 60 nM or less , about 25 nM, About 10 nM, about 5 nM, about 1 nM, 100 pM, 50 pM, 25 pM, 10 pM, 1 pM or less. In one embodiment, a PI3K inhibitor inhibits PI3K with an ICso of about 2 nM to about 100 nm, more preferably from about 2 nM to about 50 nM, even more preferably from about 2 nM to about 15 nM .
[0396] Illustrative examples of PI3K inhibitors suitable for use in the T cell manufacturing methods contemplated herein include, but are not limited to, BKM120 (class 1 PI3K inhibitor, Novartis), XL147 (class 1 PI3K inhibitor, Exelixis), (pan-PI3K inhibitor, GlaxoSmithKline) and PX-866 (class 1 PI3K inhibitor, p110a, p110β and p11Oy, Oncothyreon isoforms).
[0397] Other illustrative examples of selective PI3K inhibitors include, but are not limited to BYL719, GSK2636771, TGX-221, AS25242, CAL-101, ZSTK474 and IPI-145.
[0398] Other illustrative examples of pan-PI3K inhibitors include, but are not limited to BEZ235, LY294002, GSK1059615, TG100713 and GDC-0941. ç. AKT inhibitors
[0399] As used herein, the term "AKT inhibitor" refers to a nucleic acid, peptide, compound or small organic molecule that inhibits at least one AKT activity. AKT inhibitors can be grouped into several classes, including lipid-based inhibitors (for example, inhibitors that target the AKT pleckstrin homology domain that prevents AKT from being located in plasma membranes), competitive ATP inhibitors and allosteric inhibitors . In one embodiment, AKT inhibitors work by binding to the AKT catalytic site. In a particular embodiment, Akt inhibitors work by inhibiting phosphorylation of AKT targets downstream such as with TOR. In another embodiment, AKT activity is inhibited by inhibiting the input signals to activate Akt by inhibiting, for example, the DNA-PK activation of AKT activation, AKT PDK-1 and / or Akt mT0RC2 activation.
[0400] AKT inhibitors can target all three isoforms AKT, AKT1, AKT2, AKT3 or can be selective isoforms and target only one or two of the AKT isoforms. In one embodiment, an AKT inhibitor can target AKT, as well as additional proteins in the PI3K-AKT-mT0R path. An AKT inhibitor that targets only AKT can be referred to as a selective AKT inhibitor. In one embodiment, a selective AKT inhibitor can be understood to refer to an agent that exhibits a 50% inhibitory concentration compared to AKT that is at least 10 times, at least 20 times, at least 30 times, at least 50 times at least 100 times, at least 1,000 times, or less than the ICso of the inhibitor in relation to other proteins in the pathway.
[0401] In a particular embodiment, exemplary AKT inhibitors inhibit AKT with an ICso (concentration that inhibits 50% of activity) of about 200 nM or less, preferably about 100 nm or less, even more preferably about 60 nM or less , about 25 nM, About 10 nM, about 5 nM, about 1 nM, 100 pM, 50 pM, 25 pM, 10 pM, 1 pM or less. In one embodiment, an AKT inhibits AKT with an ICso of about 2 nM to about 100 nm, more preferably from about 2 nM to about 50 nM, even more preferably from about 2 nM to about 15 nM.
[0402] Illustrative examples of AKT inhibitors for use in combination with auristatin-based antibody-drug conjugates include, for example, perifosine (Keryx), MK2206 (Merck), VQD-002 (VioQuest), XL418 (Exelixis), GSK690693 , GDC-0068 and PX316 (PROLX Pharmaceuticals).
[0403] An illustrative, non-limiting example of a selective Akt1 inhibitor is A-674563.
[0404] An illustrative, non-limiting example of a selective Akt2 inhibitor is CCT128930.
[0405] In particular embodiments, inhibition of Akt DNA-PK activation of Akt, PDK-1 activation of Akt, mTORC2 activation of Akt, or HSP activation of Akt.
[0406] Illustrative examples of DNA-PK inhibitors include, but are not limited to, NU7441, PI-103, NU7026, PIK-75 and PP-121. d. MTOR inhibitors
[0407] The terms "mTOR inhibitor" or "agent that inhibits mTOR" refer to a nucleic acid, peptide, compound or small organic molecule that inhibits at least one activity of an mTOR protein, such as, for example, serine / threonine protein kinase activity in at least one of its substrates (for example, p70S6 kinase 1, 4E-BP1, AKT / PKB and eEF2). MTOR inhibitors are able to bind directly and inhibit mTORCI, mT0RC2 or mTORCI and mT0RC2.
[0408] Inhibition of mTORCI and / or mT0RC2 activity can be determined by a reduction in signal transduction of the PI3K / Akt / mTOR pathway. A wide variety of readings can be used to establish a reduction in the output of this signaling path. Some non-limiting exemplary readings include (1) a decrease in Akt phosphorylation in residues, including, but not limited to, 5473 and T308; (2) a decrease in Akt activation as evidenced, for example, by a reduction in phosphorylation of Akt substrates including, but not limited to, Fox01 / O3a T24 / 32, GSK3a / β; S21 / 9 and TSC2 T1462; (3) a decrease in phosphorylation of signaling molecules downstream of mTOR, including but not limited to S6 S240 / 244, 70S6K T389 ribosomal and 4EBP1 T37 / 46; And (4) inhibition of cancer cell proliferation.
[0409] In one embodiment, mTOR inhibitors are active site inhibitors. These are mTOR inhibitors that bind to the ATP binding site (also referred to as ATP binding pocket) of mTOR and inhibit the catalytic activity of both mTORCI and mT0RC2. A class of active site inhibitors suitable for use in the T cell manufacturing methods contemplated herein are dual specificity inhibitors that target and directly inhibit PI3K and mTOR. Dual specificity inhibitors bind to both the ATP binding site of mTOR and PI3K. Illustrative examples of such inhibitors include, but are not limited to: imidazoquinazolines, wortmannin, LY294002, PI-103 (Cayman Chemical), SF1126 (Semafore), BGT226 (Novartis), XL765 (Exelixis) and NVP-BEZ235 (Novartis).
[0410] Another class of mTOR active site inhibitors suitable for use in the methods contemplated herein selectively inhibit the activity of mTORCI and mTORC2 over one or more type I foptidylinositol 3-kinases, for example, Plinase kinase α, β , y, or δ. These active site inhibitors bind to the mTOR active site, but not to PI3K. Illustrative examples of such inhibitors include, but are not limited to: pyrazolopyrimidines, Torinl (Guertin and Sabatini), PP242 (2- (4-amino-1-isopropyl-1 H-pyrazolo [3,4-d] pyrimidin-3- yl) -1 H-indole-5-ol), PP30, Ku- 0063794, WAY-600 (Wyeth), WAY-687 (Wyeth), WAY-354 (Wyeth) and AZD8055 (Liu et al., Nature Review, 8,627-644, 2009).
[0411] In one embodiment, a selective mTOR inhibitor refers to an agent that exhibits a 50% inhibitory concentration (ICso) in relation to mTORCI and / or mTORC2, which is at least 10 times, at least 20 times, at least 50 times, at least 100 times, at least 1,000 times, or more, less than the ICso of the inhibitor in relation to one type I PI3-kinases, two, three or more or all type I PI3-kinases .
[0412] Another class of mTOR inhibitors for use in the present invention are referred to herein as "rapalogs". As used herein, the term "rapalogs" refers to compounds that specifically bind to the mTOR FRB domain (FKBP binding domain rapamycin), are structurally related to rapamycin and retain the inhibitory properties of mTOR. The term rapalogs excludes rapamycin. De-icers include rapamycin esters, ethers, oximes, hydrazones and hydroxylamines, as well as compounds in which functional groups in the rapamycin core structure have been modified, for example, by reduction or oxidation. Pharmaceutically acceptable salts of such compounds are also considered to be rapamycin derivatives. Illustrative examples of rapalogs suitable for use in the methods contemplated herein include, without limitation, temsirolimus (CC1779), everolimus (RAD001), deforolimus (AP23573), AZD8055 (AstraZeneca) and OSI-027 (OSI).
[0413] In one embodiment, the agent is the mTOR inhibitor rapamycin (sirolimus).
[0414] In a particular embodiment, exemplary mTOR inhibitors for use in the present invention inhibit mTORCI, mT0RC2 or both mTORCI and mT0RC2 with an IC 50 (concentration that inhibits 50% of activity) of about 200 nM or less, preferably about 100 nm or less, even more preferably about 60 nM or less, about 25 nM, about 10 nM, about 5 nM, about 1 nM, 100 pM, 50 pM, 25 pM, 10 pM, 1 pM or less . In one aspect, an mTOR inhibitor for use in the present invention inhibits mTORCI, mTORC2 or both mTORCI and mTORC2 with an ICso of about 2 nM to about 100 nm, more preferably from about 2 nM to about 50 nM, even more preferably from about 2 nM to about 15 nM.
[0415] In one embodiment, exemplary mTOR inhibitors inhibit PI3K and mTORCI or mTORC2 or both mTORCI and mTORC2 and PI3K with an ICso (concentration that inhibits 50% of activity) of about 200 nM or less, preferably about 100 nm or less, even more preferably about 60 nM or less, about 25 nM, about 10 nM, about 5 nM, about 1 nM, 100 pM, 50 pM, 25 pM, 10 pM, 1 pM or less. In one aspect, an mTOR inhibitor for use in the present invention inhibits PI3K and mTORCI or mTORC2 or both mTORCI and mTORC2 and PI3K with an ICso of about 2 nM to about 100 nm, more preferably from about 2 nM to about 50 nM , even more preferably from approximately 2 nM to about 15 nM.
[0416] Other illustrative examples of mTOR inhibitors suitable for use in particular embodiments contemplated include, but are not limited to, AZD8055, INK128, rapamycin, PF-04691502 and everolimus.
[0417] mTOR demonstrated robust and specific catalytic activity in relation to proteins of the physiological substrate, protein kinase I protein 1 (p70S6K1) and elF4E binding protein 1 (4EBP1) measured by phosphorus-specific antibodies in Western blot .
[0418] In one embodiment, the inhibitor of the PI3K / AKT / mT0R pathway is an s6 kinase inhibitor selected from the group consisting of: BI-D1870, H89, PF-4708671, FMK and AT7867. l. Compositions and Formulations
[0419] The compositions considered here may comprise one or more polypeptides, polynucleotides vectors, comprising the same, genetically modified immune effector cells, etc., as considered here. Compositions include, but are not limited to pharmaceutical compositions. A "pharmaceutical composition" refers to a composition formulated in pharmaceutically acceptable or physiologically acceptable solutions for administration to a cell or an animal, alone, or in combination with one or more other modalities of therapy. It will also be understood that, if desired, the compositions of the invention can be administered in combination with other agents as well, such as, for example, cytokines, growth factors, hormones, small molecules, chemotherapeutics, prodrugs, drugs, antibodies, or other various pharmaceutically active agents. There is practically no limit to other components that can also be included in the compositions, provided that the additional agents do not adversely affect the composition's ability to deliver the intended therapy.
[0420] The phrase "pharmaceutically acceptable" is used here to refer to those compounds, materials, compositions and / or dosage forms that are, within the scope of the relevant clinical judgment suitable for use in contact with the tissues of human beings. humans and animals without excessive toxicity, irritation, allergic response, or other problem or complication, compatible with a reasonable risk / benefit ratio.
[0421] As used herein, "pharmaceutically acceptable carrier, diluent or excipient" includes without limitation any adjuvant, carrier, excipient, glidant, sweetening agent, diluent, preservative, pigment / dye, flavor enhancer, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonic agent, solvent, surfactant, or emulsifier that has been approved by the United States Food and Drug Administration as being acceptable for use in humans or pets. Exemplary pharmaceutically acceptable carriers include, but are not limited to, sugars such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; tragacanth; malt; gelatine; baby powder; cocoa butter, waxes, animal and vegetable fats, paraffins, silicones, bentonites, silicic acid, zinc oxide; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laucamongong; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffer solution; and any other compatible substances used in pharmaceutical formulations.
[0422] In particular embodiments, compositions of the present invention comprise a number of immune effector cells that express CAR considered herein. As used herein, the term "amount" refers to "an effective amount" or "an effective amount" of a genetically modified Therapeutic cell, for example, T cells, to obtain a prophylactic or therapeutic result, beneficial or desired, including clinical results.
[0423] A "prophylactically effective amount" refers to an amount of a genetically modified Therapeutic cell effective to obtain the desired prophylactic result. Typically, but not necessarily, since a prophylactic dose is used in individuals before or at an earlier stage of the disease, the prophylactically effective amount is less than the therapeutically effective amount.
[0424] A "therapeutically effective amount" of a genetically modified Therapeutic cell can vary according to factors such as the individual's disease state, age, sex and weight, and the ability of stem and progenitor cells to evoke a desired response in the individual. A therapeutically effective amount is also one in which any toxic or harmful effects of the virus or transduced therapeutic cells are outweighed by the therapeutically beneficial effects. The term "therapeutically effective amount" includes an amount that is effective for "treating" an individual (for example, a patient). When a therapeutic amount is indicated, the precise amount of the compositions of the present invention to be administered can be determined by a physician with consideration for individual differences in age, weight, tumor size, extent of infection or metastasis, and condition of the patient (individual ). It can generally be established that a pharmaceutical composition comprising the T cells described herein can be administered in a dosage of 102 to 10 10 cells / kg of body weight, preferably 105 to 106 cells / kg of body weight, including all integer values within those ranges. The number of cells will depend after the end use so that the composition is intended as the type of cells included in it. For the use provided here, the cells are usually in a volume of one liter or less, it can be 500 ml_s or less, even 250 mls or 100 mls or less. Consequently the density of the desired cells is typically greater than 106 cells / ml and is generally greater than 107 cells / ml, generally 108 cells / ml or greater. The clinically relevant number of immune cells can be divided into multiple infusions that cumulatively equals or exceeds 105, 106, 107, 108, 109, 1010, 1011 or 1012 cells. In some aspects of the present invention, particularly since all infused cells will be redirected to a particular target antigen, lower numbers of cells, in the range of 106 / kg (106 to 1011 per patient) can be administered. Cell compositions expressing CAR can be administered multiple times at dosages within these ranges. The cells can be allogeneic, syngeneic, xenogenic or autologous to the patient undergoing therapy. If desired, treatment may also include administration of mitogens (eg, PHA) or lymphokines, cytokines and / or chemokines (eg, IFN-y, IL-2, IL-12, TNF-alpha, IL-18 and TNF -beta, GM-CSF, IL-4, IL-13, Flt3-L, RANTES, MIP1α, etc.) as described here to enhance induction of the immune response.
[0425] Generally, compositions comprising the activated and expanded cells as described here can be used in the treatment and prevention of diseases that arise in individuals who are immunocompromised. In particular, compositions comprising the CAR modified T cells considered herein are used in the treatment of B cell malignancy. The CAR modified T cells of the present invention can be administered alone, or as a pharmaceutical composition in combination with carriers, diluents, excipients , and / or with other components such as IL-2 or other cytokines or cell populations. In particular embodiments, pharmaceutical compositions considered herein comprise an amount of genetically modified T cells, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients.
[0426] Pharmaceutical compositions of the present invention comprising a population of immune cells that express CAR, such as T cells, may comprise buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (for example, aluminum hydroxide); and preservatives. Compositions of the present invention are preferably formulated for parenteral administration, for example, intravascular (intravenous or intraarterial), intraperitoneal or intramuscular administration.
[0427] Liquid pharmaceutical compositions, whether they are solutions, suspensions or other similar forms, may include one or more of the following: sterile diluents such as water for injection, saline, preferably physiological saline, Ringer's solution, sodium chloride isotonic, fixed oils such as synthetic mono or diglycerides which can serve as the solvent or suspending medium, polyethylene glycols, glycerin, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylene diaminetetraacetic acid; buffers such as acetates, cyto mice or phosphates and agents for adjusting tonicity such as sodium chloride or dextrose. The parenteral preparation can be included in ampoules, disposable syringes or multiple dose vials made of glass or plastic. An injectable pharmaceutical composition is preferably sterile.
[0428] In a particular embodiment, compositions considered here comprise an effective amount of immune effector cells that express CAR, either alone or in combination with one or more therapeutic agents. In addition, CAR-expressing immune effector cell compositions can be administered alone or in combination with other known cancer treatments, such as radiation therapy, chemotherapy, transplantation, immunotherapy, hormonal therapy, photodynamic therapy, etc. The compositions can also be administered in combination with antibiotics. Such therapeutic agents can be accepted in the art as a standard treatment for a particular disease state as described herein, such as a particular cancer. Therapeutic agents considered exemplary include cytokines, growth factors, steroids, NSAIDs, DMARDs, anti-inflammatory, chemotherapeutic, radiotherapeutic, therapeutic antibodies, or other active and auxiliary agents.
[0429] In certain embodiments, compositions comprising immune effector cells expressing CAR disclosed herein can be administered in conjunction with any number of chemotherapeutic agents. Illustrative examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide (CYTOXAN ™); alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa and uredopa; ethylenimines and methylamelamines including altretamine, triethylenomelamine, triethylenophosphoramide, triethylenethiophosphoramide and trimethylolomelamine; nitrogen mustards such as chlorambucil, chlornafazine, colophosphamide, estramustine, ifosfamide, mecloretamine, meclorethamine oxide hydrochloride, melphalan, novembicin, phenesterine, prednimustine, trophosphamide, uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics such as aclacinomycin, actinomycin, autramycin, azasserine, bleomycins, cactinomycin, calicheamicin, carabicin, carminomycin, carzinophylline, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-lororubin, idorubin, eorubrub, eorubin, , marcellomycin, mitomycins, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, chelamycin, rhodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, tiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacytidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU; androgens such as calusterone, dromostanolone propionate, epithiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutetimide, mitotane, trilostane; folic acid booster such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; ansacrine; bestrabucila; bisanthrene; edatraxate; defofamine; demecolcine; diaziquone; elformitin; ellipinium acetate; etoglucide; nitium mouse of gallium; hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; fenamet; pirarrubicin; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK®; razoxane; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2.2 ', 2 "- trichlorotriethylamine; urethane; vindesina; dacarbazine; manomustine; mitobronitol; mitolactol; pipobromano; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, for example, paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, N.J.) and doxetaxel (TAXOTERE®, Rhne-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine; 6- thioguanine; mercaptopurine; methotrexate; platinum analogues such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; new chair; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS 2000; difluoromethylomitine (DMFO); retinoic acid derivatives such as Targretin ™ (bexarotene), Panretin ™ (alitretinoin); ONTAK ™ (denileucine diftitox); speramicins; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above. Also included in this definition are anti-hormonal agents that regulate age or inhibit hormone action in cancers such as anti-estrogens including for example tamoxifen, raloxifene, 4 (5) -imidazoles, 4-hydroxy tamoxifen, trioxifene, ceoxifene aromatase inhibitors, LY117018, onapristone and toremifene (Fareston); and anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the above.
[0430] A variety of other therapeutic agents can be used in conjunction with the compositions described here. In one embodiment, the composition comprising immune effector cells that express CAR is administered with an anti-inflammatory agent. Anti-inflammatory agents or drugs include, but are not limited to, steroids and glucocorticoids (including betamethasone, budesonide, dexamethasone, hydrocortisone acetate, hydrocortisone, hydrocortisone, methylprednisolone, prednisolone, prednisone, triamcinolone), non-inflammatory drugs (anti-inflammatory drugs) including aspirin, ibuprofen, naproxen, methotrexate, sulfasalazine, leflunomide, anti-TNF medications, cyclophosphamide and mycophenolate.
[0431] Other exemplary NSAIDs are chosen from the group consisting of ibuprofen, naproxen, naproxen sodium, Cox-2 inhibitors such as VIOXX® (rofecoxib) and CELEBREX® (celecoxib) and siallylates. Exemplary painkillers are chosen from the group consisting of acetaminophen, oxycodone, tramadol of propoxyphene hydrochloride. Exemplary glucocorticoids are chosen from the group consisting of cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisolone or prednisone. Exemplary biological response modifiers include molecules directed against cell surface markers (eg, CD4, CD5, etc.), cytokine inhibitors, such as TNF antagonists (eg, etanercept (ENBREL®), adalimumab (HUMIRA®) and infliximab (REMICADE®), chemokine inhibitors and adhesion molecule inhibitors Biological response modifiers include monoclonal antibodies as well as recombinant forms of molecules. Exemplary DMARDs include azathioprine, cyclophosphamide, cyclosporine, methotrexate, penicillamine, lefluninine, hydroxalazine , gold (oral (auranofin) and intramuscular) and minocycle.
[0432] Illustrative examples of therapeutic antibodies suitable for combination with the CAR modified T cells considered here, include, but are not limited to, bavituximab, bevacizumab (avastin), bivatuzumab, blinatumomab, conatumumab, daratumumab, duligotumab, dacetuzumab, dalotuzumab, dalotuzumab, dalotuzumab (HuLuc63), gemtuzumab, ibritumomab, indatuximab, inotuzumab, lorvotuzumab, lucatumumab, milatuzumab, moxetumomab, ocaratuzumab, ofatumumab, rituximab, siltuximab, teprotumumab and ublituximab.
[0433] In certain embodiments, the compositions described here are administered in conjunction with a cytokine. By "cytokine" as used here it means a generic term for proteins released by a population of cells that act in another cell as intercellular mediators. Examples of such cytokines are lymphokines, monocytes and traditional polypeptide hormones. Included among cytokines are growth hormones such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH); liver growth factor; fibroblastic growth factor; prolactin; placental lactogen; tumor necrosis factor alpha and beta; mullerian inhibiting substance; peptide associated with mouse gonadotropin; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factors such as NGF-beta; platelet growth factors; transforming growth factors (TGFs) such as TGF-alpha and TGF-beta; insulin-like growth factor-l and -II; erythropoietin (EPO); osteoinductive factors; interferons such as interferon-alpha, beta and gamma; colony stimulating factors (CSFs) such as macrophage-CSF (M-CSF); granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF); interleukins (ILs) such as IL-1, IL-1 alpha, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10 , IL-11, IL-12; IL-15, a tumor necrosis factor such as TNF-alpha or TNF-beta; and other polypeptide factors including LIF and ligand kit (KL). As used herein, the term cytokine includes proteins from natural sources or recombinant cell culture, and biologically active equivalents of native sequence cytokines.
[0434] In particular embodiments, a composition comprises CAR T cells contemplated herein that are cultured in the presence of a PI3K inhibitor as disclosed herein and express one or more of the following markers: CD3, CD4, CD8, CD28, CD45RA, CD45RO, CD62 , CD127, AND HLA-DR can be further isolated by positive or negative selection techniques. In one embodiment, a composition comprises a specific subpopulation of T cells, which expresses one or more of the markers selected from the group consisting of CD62L, CCR7, CD28, CD27, CD122, CD127, CD197; And CD38 or CD62L, CD127, CD197 and CD38, is further isolated by positive or negative selection techniques. In various embodiments, the compositions do not express or substantially express one or more of the following markers: CD57, CD244, CD160, PD-1, CTLA4, TIM3 and LAG3.
[0435] In one embodiment, the expression of one or more of the markers selected from the group consisting of CD62L, CD127, CD197 and CD38 is increased at least 1.5 times, at least 2 times, at least 3 times, by at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 25 times, or more compared to a population of activated T cells and expanded without a PI3K inhibitor.
[0436] In one embodiment, the expression of one or more of the selected markers from the group consisting of CD57, CD244, CD160, PD-1, CTLA4, TIM3 and LAG3 is decreased at least 1.5 times, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 25 times, or more in compared to a population of T cells activated and expanded with a PI3K inhibitor. m. Therapeutic Methods
[0437] The genetically modified immune effector cells considered here provide improved methods of adoptive immunotherapy for use in the treatment of conditions related to B cells that include, but are not limited to immunoregulatory conditions and hematological malignancies.
[0438] In particular modalities, the specificity of a primary immune effector cell is redirected to B cells by genetically modifying the primary immune effector cell with a CAR considered here. In several embodiments, a viral vector is used to genetically modify an immune effector cell with a particular polynucleotide encoding a CAR comprising a murine anti-BCMA antigen binding domain that binds a BCMA polypeptide; a hinge domain; a transmembrane (TM) domain, a short oligo- or polypeptide linker, which links the TM domain to the intracellular signaling domain of CAR; and one or more intracellular co-stimulatory signaling domains; and a primary signaling domain.
[0439] In one embodiment, the present invention includes a type of cell therapy in which T cells are genetically modified to express a CAR that targets B cells that express BCMA. In another embodiment, anti-BCMA T fr CAR cells are cultured in the presence of IL-2 and a PI3K inhibitor to increase the therapeutic properties and persistence of CAR T cells. The CAR T cell is then infused to a recipient that needs it. The infused cell is capable of killing diseases that cause B cells in the recipient. Unlike antibody therapies, CAR T cells are able to replicate in vivo, resulting in long-term persistence that can lead to sustained cancer therapy.
[0440] In one embodiment, the CAR T cells of the invention can undergo a robust expansion of T cells in vivo and can persist for an extended period of time. In another embodiment, the CAR T cells of the invention evolve into specific memory T cells that can be reactivated to inhibit any further tumor formation or growth.
[0441] In particular embodiments, compositions comprising immune effector cells that comprise the CARs contemplated herein are used to treat conditions associated with abnormal B-cell activity.
[0442] Illustrative examples of conditions that can be treated, prevented or improved using immune effector cells comprising the CARs considered here include, but are not limited to: systemic lupus erythematosus, rheumatoid arthritis, myasthenia gravis, autoimmune hemolytic anemia, idiopathic thrombocytopenic purpura , anti-phospholipid syndrome, Chagas disease, Grave's disease, Wegener's granulomatosis, polyarteritis nodosa, Sjogren's syndrome, pemphigus vulgaris, scleroderma, multiple sclerosis, antiphospholipid syndrome, ANCA-associated vasculitis, Goodpasture's disease, Kawasaki disease and rapidly progressive glomerulonephritis.
[0443] Modified immune effector cells may also have applications in plasma cell disorders such as heavy chain disease, primary or immunocyte-associated amyloidosis, and monoclonal gammopathy of undetermined significance (MGUS).
[0444] As used here, “B cell malignancy” refers to a type of cancer that forms in B cells (a type of immune cell) as discussed below.
[0445] In particular embodiments, compositions comprising CAR modified T cells considered herein are used in the treatment of hematological malignancies, including, but not limited to, B cell malignancy such as, for example, multiple myeloma (MM) and non-lymphoma -Hodgkin (NHL).
[0446] Multiple myeloma is a B-cell malignancy of mature plasma cell morphology characterized by the neoplastic transformation of a single clone of these cell types. These plasma cells proliferate in the BM and can invade the adjacent bone and sometimes the blood. Variant forms of multiple myeloma include evident multiple myeloma, latent multiple myeloma, plasma cell leukemia, non-secretory myeloma, IgD myeloma, osteosclerotic myeloma, solitary bone plasmacytoma and extramedullary plasmacytoma (see, for example, Braunwald, et al. (Eds ), Harrison's Principles of Internal Medicine, 15th Edition (McGraw-Hill 2001)).
[0447] Non-Hodgkin's lymphoma covers a large group of lymphocyte (white blood cell) cancers. Non-Hodgkin's lymphomas can occur at any age and are often marked by lymph nodes that are larger than normal, fever and weight loss. There are many different types of non-Hodgkin's lymphoma. For example, non-Hodgkin's lymphoma can be divided into aggressive (rapid growth) and indolent (slow growth) types. Although non-Hodgkin's lymphomas can be derived from B cells and T cells, as used here, the terms "non-Hodgkin's lymphoma" and "non-Hodgkin's lymphoma of B cells" are used interchangeably. Non-Hodgkin's B-cell lymphomas (NHL) include Burkitt's lymphoma, chronic lymphocytic leukemia / small lymphocytic lymphoma (CLL / SLL), diffuse large B-cell lymphoma, follicular lymphoma, large cell immunoblastic lymphoma, precursor B lymphoblastic lymphoma and mantle cell lymphoma. Lymphomas that occur after bone marrow or stem cell transplantation are usually B-cell non-Hodgkin's lymphomas.
[0448] Chronic lymphocytic leukemia (CLL) is an indolent (slow-growing) cancer that causes a slow increase in immature white blood cells called B lymphocytes or B cells. Cancer cells spread through blood and bone marrow, and can also affect lymph nodes or other organs such as the liver and spleen. CLL eventually causes the bone marrow to fail. Sometimes, in more advanced stages of the disease, the disease is called small lymphocytic lymphoma.
[0449] In particular embodiments, methods comprising administering a therapeutically effective amount of immune effector cells expressing CAR considered herein or a composition comprising the same, to a patient in need thereof, alone or in combination with one or more therapeutic agents, are provided . In certain embodiments, the cells of the invention are used to treat patients at risk for developing a condition associated with abnormal B-cell activity or B-cell malignancy. In addition, the present invention provides methods for treating or preventing a condition associated with abnormal B cell activity or a B cell malignancy comprising administering to a subject in need thereof, a therapeutically effective amount of the CAR modified cells considered herein.
[0450] As used herein, the terms "individual" and "individual" are often used interchangeably and refer to any animal that exhibits a symptom of a disease, disorder, or condition that can be treated with the therapy vectors. genes, cell-based therapies, and methods disclosed elsewhere here. In preferred embodiments, an individual includes any animal that exhibits symptoms of a disease, disorder, or condition of the hematopoietic system, for example, a B-cell malignancy, which can be treated with gene therapy vectors, cell-based therapies , and methods disclosed elsewhere here. Suitable individuals (for example, patients) include laboratory animals (such as mouse, mouse, rabbit, or guinea pig), farm animals, and domestic animals or pets (such as a cat or dog). Non-human primates and, preferably, human patients, are included. Typical individuals include human patients who have B-cell malignancy, have been diagnosed with B-cell malignancy, or are at risk or have B-cell malignancy.
[0451] As used herein, the term "patient" refers to an individual who has been diagnosed with a particular disease, disorder, or condition that can be treated with gene therapy vectors, cell-based therapies, and methods released elsewhere here.
[0452] As used herein, "treatment" or "treating" includes any beneficial or desirable effect on the symptoms or pathology of a disease or pathological condition, and may even include minimal reductions in one or more measurable markers of the disease or condition being treated. Treatment may optionally involve reducing or ameliorating symptoms of the disease or condition, or delaying the progression of the disease or condition. "Treatment" does not necessarily indicate complete eradication or cure of the disease or condition, or associated symptoms thereof.
[0453] As used herein, "prevent", words and the like such as "prevented", "preventing" etc., indicate a method for preventing, inhibiting, or reducing the likelihood of the occurrence or recurrence of a disease or condition. It also refers to delaying the onset or recurrence of a disease or condition or delaying the occurrence or recurrence of symptoms of a disease or condition. As used here, "prevention" and similar words also include reducing the intensity, effect, symptoms and / or burden of a disease or condition before the onset or recurrence of the disease or condition.
[0454] By "improving" or "promoting", or "increasing" or "expanding" generally refers to the ability of a composition considered here, for example, a genetically modified T cell or vector encoding a CAR, to produce, evoke , or cause a greater physiological response (ie, adjusting effects) compared to the response caused by a vehicle or a control molecule / composition. A measurable physiological response may include an increase in expansion, activation, persistence, and / or an increase in T cells in the ability to exterminate cancer cells, among others evident from the understanding in the art and the description here. An "increased" or "enhanced" amount is typically an "statistically significant" amount, and can include an increase that is 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8 , 9, 10, 15, 20, 30 or more times (e.g. 500, 1,000 times) (including all integers and decimal points between and above 1, e.g. 1.5, 1.6, 1.7 , 1.8, etc.) the response produced by the vehicle or a control composition.
[0455] By “decreasing” or “lowering”, or “attenuating”, or “reducing”, or “slaughtering” generally refers to the capacity of composition considered here to produce, evoke, or cause an attenuated physiological response (that is, adjusting effects) compared to the response caused by a vehicle or a control molecule / composition. A "decreased" or "reduced" amount is typically a "statistically significant" amount, and may include a decrease that is 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8 , 9, 10, 15, 20, 30 or more times (e.g. 500, 1,000 times) (including all integers and decimal points between and above 1, e.g. 1.5, 1.6, 1.7 , 1.8, etc.) the response (reference response) produced by vehicle, a control composition, or the response in a particular cell line.
[0456] By "maintain", or "preserve", or "maintain", or "no change", or "no substantial change", or "no substantial decrease" generally refers to the ability of a composition considered here to produce , evoke, or cause an attenuated physiological response (i.e., adjusting effects) in a cell, as compared to the response caused by a vehicle, a control molecule / composition, or the response in a particular cell line. A comparable response is one that is not significantly different or measurably different from the reference response.
[0457] In one embodiment, a method of treating a condition related to B cells in an individual in need thereof comprises administering an effective amount, for example, the therapeutically effective amount of a composition comprising genetically modified immune effector cells considered herein. The amount and frequency of administration will be determined by such factors as the patient's condition, and the type and severity of the patient's illness, although appropriate dosages can be determined by clinical trials.
[0458] In one embodiment, the amount of T cells in the composition administered to an individual is at least 0.1 x 105 cells, at least 0.5 x 105 cells, at least 1 x 105 cells, at least 5 x 105 cells at least 1 x 106 cells, at least 0.5 x 107 cells, at least 1 x 107 cells, at least 0.5 x 108 cells, at least 1 x 108 cells, at least 0.5 x 109 cells, at least at least 1 x 109 cells, at least 2 x 109 cells, at least 3 x 109 cells, at least 4 x 109 cells, at least 5 x 109 cells, or at least 1 x 1010 cells. In particular embodiments, about 1 x 107 CAR T cells to about 1 x 109 CAR T cells, about 2 x 107 CAR T cells to about 0.9 x 109 CAR T cells, about 3 x 107 CAR T cells to about 0.8 x 109 CAR T cells, about 4 x 107 CAR T cells to about 0.7 x 109 CAR T cells, about 5 x 107 CAR T cells to about 0.6 x 109 CAR T cells, or about 5 x 107 CAR T cells to about 0.5 x 109 CAR T cells are administered to an individual.
[0459] In one embodiment, the amount of T cells in the composition administered to an individual is at least 0.1 x 104 cells / kg body weight, at least 0.5 x 104 cells / kg body weight, at least 1 x 104 cells / kg body weight, at least 5 x 104 cells / kg body weight, at least 1 x 105 cells / kg body weight, at least 0.5 x 106 cells / kg body weight, at least 1 x 106 cells / kg body weight, at least 0.5 x 107 cells / kg body weight, at least 1 x 107 cells / kg body weight, at least 0.5 x 108 cells / kg body weight, at least at least 1 x 108 cells / kg body weight, at least 2 x 108 cells / kg body weight, at least 3 x 108 cells / kg body weight, at least 4 x 108 cells / kg body weight 5 x 108 cells / kg body weight, or at least 1 x 109 cells / kg body weight. In particular embodiments, about 1 x 10 6 T cells from CAR / kg body weight to about 1 x 10 8 T cells from CAR / kg body weight, about 2 x 10 6 T cells from CAR / kg body weight about 0.9 x 108 CAR T cells / kg Body weight, about 3 x 106 CAR T cells / kg body weight to about 0.8 x 108 CAR T cells / kg body weight, about 4 x 106 CAR T cells / kg body weight to about 0.7 x 108 CAR T cells / kg body weight, about 5 x 106 CAR T cells / kg body weight to about 0.6 x 108 CAR T cells / kg body weight or about 5 x 106 CAR T cells / kg body weight to about 0.5 x 108 CAR T cells / kg body weight are administered to an individual.
[0460] One skilled in the art would recognize that multiple administrations of the compositions of the invention may be necessary to effect the desired therapy. For example, a composition can be administered 1,2,3, 4, 5, 6, 7, 8, 9 or 10 or more times over an interval of 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 Months, 4 months, 5 months, 6 months, 1 year, 2 years, 5 years, 10 years or more.
[0461] In certain embodiments, it may be desirable to administer activated immune effector cells to a subject and then subsequently collect blood (or have an apheresis performed), activate immune effector cells in accordance with the present invention, and reinfuse the patient with these immune effector cells activated and expanded. This process can be performed several times in a few weeks. In certain embodiments, immune effector cells can be activated from blood collections from 10cc to 400cc. In certain modalities, immune effector cells are activated from blood collections of 20cc, 30cc, 40cc, 50cc, 60cc, 70cc, 80cc, 90cc, 100cc, 150cc, 200cc, 250cc, 300cc, 350cc, or 400cc or more. Not being limited by theory, using this multiple reinfusion / multiple blood collection protocol can serve to select certain populations of immune effector cells.
[0462] The administration of the compositions considered here can be carried out in any convenient manner, including by aerosol inhalation, injection, ingestion, transfusion, implantation or transplantation. In a preferred embodiment, compositions are administered parenterally. The phrases “parenteral administration” and “administered parenterally” as used here refer to modes of administration except enteral and topical administration, usually by injection, and include, without limitation injection and infusion, intravascular, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intra-orbital, intratumoral, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intra-articular, subcapsular, subarachnoid, intraspinal and intrasternal. In one embodiment, the compositions considered here are administered to a subject by direct injection into a tumor, lymph node, or infection site.
[0463] In one embodiment, a subject in need of this is administered an effective amount of a composition to enhance a cellular immune response to a condition related to B cells in the subject. The immune response can include cellular immune responses mediated by cytotoxic T cells capable of exterminating infected cells, regulatory T cells, and helper T cell responses. Humoral immune responses, mediated mainly by helper T cells capable of activating B cells, thus leading to antibody production, can also be induced. A variety of techniques can be used to analyze the type of immune responses induced by the compositions of the present invention, which are well described in the art; for example, Current Protocols in Immunology, edited by: John E. Coligan, Ada M. Kruisbeek, David H. Margulies, Ethan M. Shevach, Warren Strober (2001) John Wiley & Sons, NY, N.Y.
[0464] In the case of T cell-mediated extermination, CAR ligand binding initiates CAR signaling to T cells, resulting in activation of a variety of T cell signaling pathways that induce T cells to produce or release proteins capable of to induce target cell apoptosis by various mechanisms. These T-cell-mediated mechanisms include (but are not limited to) the transfer of intracellular cytotoxic granules from the T cells in the target cell, T-cell secretion of pro-inflammatory cytokines that can induce the target cell to exterminate directly (or indirectly by through recruitment of other effector cell exterminators), and even regulation of death receptor ligands (eg, FasL) on the surface of T cells that induce target cell apoptosis after binding to their cognate death receptor (eg, Fas ) in the target cell.
[0465] In one embodiment, the invention provides a method of treating a subject diagnosed with a condition related to B cells comprising removing immune effector cells from a subject diagnosed with a condition related to B cells expressing BCMA, genetically modifying said effector cells immune with a vector comprising a nucleic acid encoding a CAR as considered herein, thereby producing a population of modified immune effector cells, and administering the population of modified immune effector cells to the same subject. In a preferred embodiment, the immune effector cells comprise T cells.
[0466] In certain embodiments, the present invention also provides methods for stimulating an immune modulator response mediated by an immune effector cell to a target cell population in a subject comprising the steps of administering to the subject an immune effector cell population that expresses a nucleic acid construction that encodes a CAR molecule.
[0467] Methods for administering the cell compositions described herein include any method that is effective to result in the reintroduction of ex vivo genetically modified immune effector cells that directly express a CAR of the invention in the subject or in reintroduction of the genetically modified progenitors of immune effector cells that on introduction into a subject to differentiate into mature immune effector cells that express CAR. One method comprises transducing peripheral blood T cells ex vivo with a nucleic acid construct according to the invention and returning the transduced cells in the subject.
[0468] All publications, patent applications, and granted patents cited in this specification are incorporated herein by reference as if each individual publication, patent application, or granted patent was specifically and individually indicated to be incorporated by reference.
[0469] Although the previous invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be easily apparent to one of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications can be made to this without departing from the spirit or scope of the attached claims. The following examples are provided by way of illustration only and not by way of limitation. Those of skill in the art will easily recognize a variety of non-critical parameters that can be altered or modified to produce essentially similar results. EXAMPLES EXAMPLE 1 BCMA CARS CONSTRUCTION
[0470] CARs containing mouse anti-BCMA scFv antibodies were designed to contain an MND promoter operatively linked to antiBCMA scFv, a hinge and transmembrane domain from co-stimulatory domains of CD8α and CD137 followed by the intracellular signaling domain of the CD3Ç chain Figure 1. BCMA CARs comprise a CD8a signal peptide (SP) sequence for surface expression in immune effector cells. The BCMA CAR polynucleotide sequences are shown in SEQ ID NOs: 10; polypeptide sequences of anti-BCMA CARs are shown in SEQ ID NOs: 9; and the vector maps are shown in Figure 1. Tables 3 shows the Identity, Genbank Reference, Source Name and Citation for the various nucleotide segments of several exemplary CAR BCMA lentiviral vectors. Table 3


EXAMPLE 2 BCMA CAR EVALUATION OF A MURINE Introduction
[0471] The adoptive transfer of genetically modified T cells with chimeric antigen receptors (CAR) has emerged as a promising approach to treat cancer. CAR is an artificial molecule composed of a variable fragment of single reactive antigen chain (scFv) fused in signaling domains of T cells through a transmembrane region. In this example, a specific CAR molecule was evaluated for the B cell maturation antigen (BCMA). BCMA is expressed in multiple myeloma, plasmacytoma and some lymphomas, but normal expression is limited to plasma cells (Avery et al., 2003; Carpenito et al., 2009; Chiu et al., 2007).
[0472] The anti-BCMA02 CAR was constructed using sequences of a murine anti-BCMA antibody (C11D5.3). The anti-BCMA10 CAR was built using modified sequences and is a "humanized" version of the anti-BCMA02 CAR. In a series of in vitro assays, anti-BCMA02 CAR T cells and anti-BCMA10 CAR T cells showed tumor specificity, high CAR expression and caused potent reactivity to targets that express the antigen. Anti-BCMA02 CAR T cells and anti-BCMA10 CAR T cells have been shown to have a reactivity comparable to BCMA expressing tumor cell lines. Although both anti-BCMA02 CAR T cells and anti-BCMA10 CAR T cells were able to cause regressions in a mouse tumor model, anti-BCMA10 CAR T cells exhibited antigen-independent and inflammatory cytokine secretion therefore have the potential to cause clinical toxicities associated with high levels of cytokines. Results Release of tonic inflammatory cytokines from anti-BCMA10 T cells associated with apoptosis
[0473] BCMA protein is detectable in the serum of patients with multiple myeloma (Sanchez et al., 2012). The mean serum BCMA in patients with multiple myeloma was 10 ng / mL, but peaked at levels up to 100 ng / mL. The impact of physiological soluble BCMA levels on anti-BCMA CAR T cell candidates was assessed.
[0474] The release of IFNy from anti-BCMA02 CAR T cells, anti-BCMA10 CAR T cells and CAR19Δ T cells was examined after a 24-hour culture with soluble BCMA (Figure 2a). The anti-BCMA02 CAR T cells responded with minimal cytokine release after 24 hours of culture with up to 1 pg / mL BCMA. In contrast, anti-BCMA10 CAR T cells responded with increasing levels of IFNy that were proportional to the concentration of soluble BCMA added to the culture. In 100 pg / mL of BCMA, the maximum levels reported in patients with multiple myeloma, anti-BCMA10 CAR T cells secreted 821ng / ml IFNy compared to 28.8ng / ml IFNy secreted by anti-CARMA T cells BCMA02. IFNy was even detected in several co-culture experiments with anti-BCMA10 CAR T cells plus control cell lines that lacked BCMA antigen (Figure 2b, K562 co-culture). These data suggest that anti-BCMA10 CAR T cells had increased sensitivity to soluble BCMA stimulation and potential for antigen-independent cytokine responses in T cells.
[0475] The potential for tonic cytokine secretion from anti-BCMA02 CAR T cells, anti-BCMA10 CAR T cells (10 days after culture initiation) and CAR19 Δ T cells was examined. After the manufacture of CAR T cells, the growth media of the anti-BCMA02 CAR cells, CAR anti-BCMA10 cells and CAR19 Δ T cells were analyzed for the presence of inflammatory cytokines. Despite the lack of antigen stimulation, anti-BCMA10 CAR T cell cultures contained greater than 10 ng / mL IFNy compared to less than 1 ng / mL IFNy in anti-BCMA02 CAR T cell cultures (Figure 3). The anti-BCMA10 CAR T cell cultures also contained significantly (p <0.001) more TNFα. To further quantify the amount of cytokine produced by anti-BCMA10 CAR T cells without antigen stimulation, cytokine release was measured from 5 x 104 CAR T cells during a 24-hour culture. CAR anti-BCMA10 T cells produced significantly greater amounts of inflammatory cytokines MIP1 α, IFNy, GMCSF, MIP1 β, IL-8 and TNFa compared to anti-BCMA02 CAR T cells (Figure 4, p <0.0001) . The concentrations of MIP1 α and IFNy were the highest among all examined cytokines. The anti-BCMA10 CAR T cells produced 4.7ng MIP1α / 5 x 104 cells / 24 hours, 3.0 ng IFN / 5 x 104 cells / 24 hours and ~ 1 ng / 5 x 104 cells / 24 hours or less of the other cytokines. No significant differences were detected in the anti-inflammatory cytokines IL-10, IL-2 and IL-4.
[0476] The expression of phenotypic T cell activation markers at the end of the manufacture of anti-BCMA10 CAR T cells was measured to examine whether the secretion of tonic inflammatory cytokines was indicative of a hyperactive state in anti-BCMA10 T T cells. HLA-DR and CD25 are surface markers that normally show maximum expression 12-24 hours after T cell activation and then decrease over time. CAR T cells prepared from three normal donors showed that an average of 40 ± 2% of anti-BCMA02 CAR T cells expressed HLA-DR. HLA-DR expression in these cells was comparable to untransformed T cells (43 ± 2.3%) and CAR19 Δ control T cells (32 ± 2.2%). In contrast, 88 ± 1.2% anti-BCMA10 CAR T cells expressed HLA-DR (Figure 5). The expression of another CD25 activation marker was also higher in anti-BCMA10 CAR T cells compared to anti-BCMA02 CAR T cells (53 ± 0.9% vs 35 ± 2.4%). Therefore, anti-BCMA10 CAR T cells exhibited phenotypic characteristics of activated T cells in the absence of added antigens.
[0477] T-cell hyperactivity is often associated with activation-induced cell death (AICD) by apoptosis. Levels of activated caspase-3 were measured to examine whether hyperactivity of anti-BCMA10 CAR T cells could result in higher apoptotic levels compared to anti-BCMA02 CAR T cells. 48% of the anti-BCMA10 CAR T cells from two donors showed active caspase-3, compared with 16% of the anti-BCMA02 CAR T cells (Figure 6). Thus, in the absence of added BCMA antigen, anti-BCMA10 CAR T cells contain a higher frequency of apoptotic cells associated with increased inflammatory cytokine activation and secretion compared to anti-BCMA02 CAR T cells.
[0478] Anti-BCMA02 CAR T cells and anti-BCMA10 CAR T cells were evaluated to see whether CAR T cells could respond selectively to low levels of BCMA or be reactive to an unrelated antigen in human serum, used for T cell growth. Anti-BCMA02 CAR T cells and anti-BCMA10 CAR T cells were maintained in media that did not contain human serum for two days and then switched to medium containing fetal bovine serum (FBS), serum (HABS) or HABS in the presence or absence of soluble BCMA 10Ong / mL (Figure 7). IFNy release was assayed 24 hours later by ELISA. Both anti-BCMA02 CAR T cells and anti-BCMA10 CAR T cells responded to soluble BCMA. However, anti-BCMA10 CAR T cells secreted 10 times more IFNy than anti-BCMA02 CAR T cells. In the absence of BCMA, only anti-BCMA10 CAR T cells released IFNy regardless of culture in fetal bovine serum (FBS) (p = 0.0002) or human AB serum (HABS) (p = 0.0007). These data suggest that inflammatory cytokine secretion was intrinsic to anti-BCMA10 CAR T cells. Lower anti-tumor function of anti-BCMA10 CAR T cells in the multiple myeloma mouse model
[0479] Hyperactivation and increased apoptosis can negatively affect the persistence of CAR T cells in patients and, ultimately, clinical efficacy. The anti-tumor function of anti-BCMA02 CAR T cells and anti-BCMA10 CAR T cells was examined in a mouse tumor model. NOD scídgamma (NSG) mice with cutaneous human subcutaneous multiple myeloma tumors (RPMI-8226) of ~ 100mm3 were treated with 107 anti-BCMA02 CAR T cells, 107 anti-BCMA10 CAR T cells or Bortezomib (velcade). The growth of RPMI-8226 was monitored with calipers. In two independent experiments (Figures 8a and 8b), Bortezomib controlled the growth of the tumor in comparison with vehicle control animals. Animals adopted with anti-BCMA02 CAR T cells exhibited rapid and durable tumor clearance (inserted graphics magnify early tumor regressions). The adoptive transfer of anti-BCMA10 CAR T cells also caused tumor regressions, but was postponed in both experiments compared to anti-BCMA02 CAR T cells. Conclusions
[0480] Anti-BCMA02 CAR T cells and anti-BCMA10 CAR T cells exhibited comparable anti-tumor function in in vitro assays, but anti-BCMA10 CAR T cells showed characteristics that could negatively affect safety and efficacy in patient treatment. The anti-BCMA10 CAR T cells responded robustly to inflammatory cytokine secretion after exposure to physiological levels of the BCMA protein. Cytokine release or cytokine release syndrome is a known clinical toxicity associated with CAR T cell therapies. Concerns about the release of cytokines for BCMA were worsened after observing the tonic activity of anti-BCMA CAR T cells10. Even in the absence of antigen stimulation, anti-BCMA10 CAR T cells released high levels of inflammatory cytokines. Persistent cytokine secretion has the potential to cause substantial clinical toxicities, as well as negatively impact antitumor function. In fact, we found greater composition of apoptotic cells and inferior anti-tumor function in anti-BCMA10 CAR T cells compared to anti-BCMA02 CAR T cell cultures in a mouse multiple bush model. References Avery et al., (2003). BAFF selectively enhances the survival of plasmablasts generated from human memory B cells. J Clin Invest, 112 (2), 286-297. Carpenito et al., (2009). Control of large, established tumor xenografts with genetically retargeted human T cells containing CD28 and CD137 domains. Proc Natl Acad Sci USA, 106 (9), 3360-3365. Chiu et al., (2007). Hodgkin lymphoma cells express TACI and BCMA receptors and generate survival and proliferation signals in response to BAFF and APRIL. Blood, 109 (2), 729-739. Sanchez et al. (2012). Serum B-cell maturation antigen is elevated in multiple myeloma and correlates with disease status and survival. Br J Haematol, 158 (6), 727- 738. EXAMPLE 3 THE MINIMUM EXPRESSION OF BCMA ON ACTIVE ANTI-BCMA LYMPHOMA CAR T CELLS
[0481] The level of BCMA expression in the lymphoma and leukemia cell lines (Daudi and Raji) was measured to determine whether the expression was sufficient to activate anti-BCMA02 CAR T cells.
[0482] BCMA expression in lymphoma, leukemia and multiple myeloma cells was quantified using flow cytometry. In this assay, the relative expression of BCMA in cells was assessed by correlating the fluorescence intensity of BCMA expression with a known number of bound antibodies (antibody binding capacity, ABC). The expression levels of BCMA in the lymphoma cell lines were compared to the expression levels of BCMA, a multiple myeloma cell line (RPMI-8226) that is known to activate anti-BCMA02 CAR T cells. 12590 ± 1275 BCMA02 molecules were expressed on the surface of RPMI-8226 cells. In contrast, Daudi cells expressed 1173 ± 234 BCMA02 molecules and JeKo-1 cells (a cell line of Mantle cell lymphoma) expressed only 222 ± 138 BCMA02 molecules (Figure 9, circles).
[0483] In another set of experiments, the activity of anti-BCMA02 CAR T cells at the minimum levels of BCMA observed in the lymphoma and leukemia cell lines was tested (Figure 9, boxes). Anti-BCMA02 CAR T cells were generated using standard methods and activity was assessed by IFNy ELISA after co-culture with BCMA-positive and BCMA-negative tumor cell lines. The reactivity of anti-BCMA02 CAR T cells correlated with the relative amount of BCMA mRNA expression (above a threshold) and / or the density of the BCMA receptor on the surface of various tumor cell lines after co-culture ( Figure 9). Little, if any, IFNy is released after co-culture of CAR BCMA T cells with BCMA-negative (BCMA-) tumor cells: myelogenic leukemia (K562), acute lymphoblastic leukemia (NALM-6 and NALM-16); Mantle cell lymphoma (REC-1); Or Hodgkin's lymphoma (HDLM-2). In contrast, substantial amounts of IFNy were released after BCMA02 CAR T cell co-culture with BCMA-positive tumor cell lines (BCMA +): chronic B-cell lymphoblastic leukemia (MEC-1), Mantle cell lymphoma (JeKo-1 ), Hodgkin Lymphoma (RPMI-6666), Burkitt's lymphoma (Daudi cells and Ramos cells) and multiple myeloma (RPMI-8226).
[0484] The reactivity of anti-BCMA02 CAR T cells to BCMA that express Burkitt's lymphoma cells (Daudi cells) extended to in vivo animal studies. Daudi cells also express CD19. The in vivo activity of anti-BCMA02 CAR T cells was compared with the in vivo activity of anti-CD19 CAR T cells. NOD scid gamma (NSG) mice were injected IV with 2 x 106 Daudi cells and a large systemic tumor load was allowed to accumulate before being treated with CAR T cells. CAR T cells were administered at 8 days and 18 days after tumor induction (Figures 10A and 10B, respectively). The vehicle and the negative control (anti-CD19Δ CAR T cells) failed to prevent tumor growth, as demonstrated by increases in the logarithmic phase in bioluminescence, resulting in weight loss and death (Figure 10A, two leftmost mouse panels ). The anti-CD19 and anti-BCMA02 CAR T cells prevented tumor growth, resulting in maintenance of body weight and survival. The anti-CD19 and anti-BCMA02 CAR T cells were equally effective when administered on Day 8 (Figure 10A, two rightmost mouse panels). The anti-BCMA02 CAR T cells were also effective in decreasing tumor burden when administered at 18 days after tumor induction. Figure 10B, rightmost panel. EXAMPLE 4 POWERFUL ACTIVITY IN VITRODE ANTI-BCMA CAR T CELLS
[0485] The in vitropotent activity of the anti-BCMA02 CAR T cells was achieved with an anti-BCMA02 RCA expression with a 50 percent reduction. T cell populations were transduced with 4x108 and 5x107 transduction units of a lentivirus encoding an anti-BCM02A CAR molecule. The resulting T cell populations showed a low frequency of anti-BCMA02 CAR T cells (tested as a positive percentage) and reduced expression of anti-BCMA02 CAR molecules (assayed as mean fluorescence intensity: MFI).
[0486] The impact of reduced expression of the CAR molecule on anti-BCMA02 activity has been determined. The frequency of anti-BCMA-positive CAR T cells was normalized with non-transposed T cells to contain 26 ± 4% BCMA reactive T cells (Figure 11 A). The MFI of normalized anti-BCMA02 CAR T cells ranged from 885 to 1875 (Figure 11B). K562 is a CML cell line that has no BCMA expression. K562 cells were designed to express BCMA and were used in an in vitro cytolytic test to assess the activity of anti-BCMA02 CAR T cells with varying CAR BCMA expression (Figure 11C). K562 cells were labeled with a violet cell line, while K562 cells that stably express BCMA (K562-BCMA) were labeled with CFSE. T cells, K562 cells and K562-BCMA cells were harvested, washed and resuspended in media that lacked exogenous cytokines. The cells were cultured in a 20: 1 or 10: 1 effector (E; T) cell to achieve the target ratio (T; 1: 1 mixture of K562 and K562 BCMA cells) for 4 h in a 37 ° C CO2 incubator ° C and 5%. The cells were then stained with Live / Dead and analyzed by FACS. Cytotoxicity was determined by the difference in the proportion of K562: K562-BCMA cells normalized to conditions that do not have T cells. EXAMPLE 5 ANTI-BCMA CAR T-CELL MANUFACTURING PROCESS
[0487] Genuine anti-BCMA02 CAR T cell products are manufactured for each patient's treatment. The reliability of the manufacturing process for anti-BCMA02 CAR T cell products was assessed by the generation of anti-BCMA02 CAR T cells from 11 individual PBMC normal donors. The expansion of anti-BCMA02 CAR T cells from each donor was comparable to a combined non-transposed culture performed in parallel (Figure 12A).
[0488] At the end of the culture period (day 10), T cell transduction efficiency was assessed by quantifying the number of lentiviruses integrated with qPCR and specific lentivirus primer sets (vector copy number, VCN). Cultures of anti-BCMA02 CAR T cells from 11 donors showed comparable lentiviral transduction efficiency (Figure 12B). The frequency of positive anti-BCMA02 CAR T cells was measured by flow cytometry and BCMA expression was comparable across all donors (Figure 12C).
[0489] The activity of each anti-BCMA02 CAR T cell product was assessed by releasing IFNy after co-culture with K562 cells, designed to express BCMA. All anti-BCMA 02 CAR T cell products showed relevant therapeutic levels of IFNy release when exposed to BCMA-expressing K562 cells (Figure 12D). EXAMPLE 6 CD62L, CD127, CD197 AND CD38 EXPRESSION ON CAR T CELLS TREATED WITH IL-2 OR IL-2 AND ZSTK474
[0490] CAR T cells cultured with IL-2 and ZSTK474 show increased expression of CD62L compared to CAR T cells cultured with IL-2 alone. Expression analysis of 29 additional cell surface markers in anti-BCMA02 CAR T cells cultured with IL-2 and ZSTK474 was performed using multi-parameter mass cytometry (CyTOF) and compared to CAR T cells cultured in IL-2 alone . Three additional markers (CD127, CD197 and CD38) showed increased expression in the CAR T cells treated with IL-2 + ZSTK474 compared to the CAR T cells treated with IL-2 alone. Thus, the coexpression of CD62L, CD127, CD197 and CD38 added the CAR T cells from ZSTK474 culture. After culturing on media containing IL-2, 7.44% of CD127, CD197 and CD38 co-expressed anti-BCMA02 CAR T compared to 24.5% anti-BCMA02 CAR T cells cultured with IL-2 and ZSTK474. The Venn diagram in Figure 13 illustrates the co-expression of CD127, CD197 and CD38 in CD62L positive anti-BCMA02 T cells. EXAMPLE 7 TREATMENT WITH ZSTK474 INCREASES CD8 T T CELL FREQUENCY
[0491] CD8 expression was quantified in anti-BCMA02 CAR T cells treated with IL-2 alone or IL-2 and ZSTK474. CD8 expression was determined using a fluorescently labeled anti-CD8 antibody and flow cytometry. CAR anti-BCMA02 T cells from seven normal donors cultured with IL-2 and ZSTK474 showed significantly higher CD8 expressions compared to anti-BCMA02 CAR T cells cultured with IL-2 alone. Figure 14. EXAMPLE 8 LACK OF INDEPENDENT ANTIGEN ACTIVITY IN ANTI-BCMA CAR T CELLS TREATED WITH ZSTK474
[0492] The tonic activity of CAR T cells in the absence of antigen has been associated with reduced biological activity. The tonic activity of anti-BCMA02 CAR T cells was assessed by quantifying the release of interferon-y (IFN-y) in the absence of antigen after culture in the presence of IL-2 and ZSTK474, compared to standard culture conditions with IL -2 alone. The anti-BCMA CAR T cell cultures were prepared using a directly scalable system for large clinical manufacturing processes. Briefly, peripheral blood mononuclear cells (PBMC) were cultured in static flasks in medium containing IL-2 (CelIGenix) and specific antibodies for CD3 and CD28 (Miltenyi Biotec). 2x108 units of BCMA anti-CARs lentivirus encoding transduction were added one day after culture initiation.
[0493] Anti-BCMA02 CAR T cells were maintained in a log phase by adding fresh medium containing IL-2 and an optimized dose of ZSTK474, for a total of ten days of culture. At the end of manufacture, an equivalent number of anti-BCMA02 CAR T cells were re-cultured for 24 hours in isolated media. The amount of IFN-y released in 24 hours was quantified by ELISA. In this assay, IFN-y levels below 200pg / mL do not represent tonic activity. Figure 15 shows the amount of IFN-y released by anti-BCMA02 CAR T cells from 14 donors is consistent with the lack of tonic activity, regardless of whether CAR T cells are cultured with ZSTK474. EXAMPLE 9 ANTI-BCMA02 CAR T CELLS TREATED WITH ZSTK474 SHOW THERAPEUTIC ACTIVITY IN A TUMOR LYMPHOMA MODEL
[0494] Daudi's tumors were used to interrogate the anti-tumor activity of anti-BCMA02 CAR T cells cultured with IL-2 or IL-2 and ZSTK474. Daudi cells express a low level of BCMA protein and provide an aggressive and difficult-to-treat tumor lymphoma model.
[0495] 2 x 106 Daudi tumor cells were tagged with a firefly luciferase gene and injected into NOD scid IL-2 receptor (NSG) gamma chain knockout mice by intravenous injection. After the tumors were allowed to form, 1x107 CAR T cells were injected into tumor-bearing mice. The mice were injected with i) CAR anti-BCMA02 T cells treated for ten days with IL-2 or IL-2 and ZSTK474; or ii) an anti-BCMA02 CAR T cell with truncated signaling deficiency treated for ten days with IL-2 and ZSTK474. Tumor growth was monitored by bioluminescence using a Xenogen-IVIS Imaging system.
[0496] Complete tumor regression was observed in 50% of mice administered by anti-BCMA02 T DE CAR cells treated with IL-2 and ZSTK474. Figure 16. EXAMPLE 10 CAR T CELLS TREATED WITH ZSTK474 SHOW THERAPEUTIC ACTIVITY IN A HUMAN MYELOMA MOUNTAIN MODEL
[0497] Animals with 100mm3 multiple myeloma subcutaneous tumors (RPMI-8226) were infused with equivalent doses of CAR T cells (1x106 anti-BCMA02 CAR-positive T cells) or unmodified T cells from a donor of corresponding T cells (not transduced). The anti-BCMA CAR T cells were treated with IL-2 or IL-2 and ZSTK474 as described in Example 8.
[0498] Animals treated with anti-BCMA02 CAR T cells with IL-2 or IL-2 and cultured ZSTK474 completely prevented tumor growth. Figure 17. In contrast, animals treated with non-transduced or vehicle were unable to control tumor growth. Figure 17.
[0499] In general, in the following claims, the terms used should not be interpreted to limit the claims to the specific modalities disclosed in the specification and in the claims, but should be interpreted to include all possible modalities along with the full scope of equivalents to which claims are entitled. Consequently, claims are not limited to disclosure.

权利要求:
Claims (16)
[0001]
1. Polynucleotide CHARACTERIZED by the fact that it encodes a chimeric antigen receptor (CAR), in which the polynucleotide sequence is presented in SEQ ID NO: 10.
[0002]
2. CAR polypeptide CHARACTERIZED by the fact that it is encoded by the polynucleotide, as defined in claim 1, in which the polypeptide sequence is shown in SEQ ID NO: 9.
[0003]
3. Vector CHARACTERIZED by the fact that it comprises the polynucleotide as defined in claim 1.
[0004]
4. Vector, according to claim 3, CHARACTERIZED by the fact that the vector is an expression vector, an episomal vector, a viral vector, a retroviral vector or a lentiviral vector.
[0005]
5. Vector, according to claim 3, CHARACTERIZED by the fact that the lentiviral vector is selected from the group consisting of: human immunodeficiency virus 1 (HIV-1); human immunodeficiency virus 2 (HIV-2), visna-maedi virus (VMV); caprine arthritis-encephalitis virus (CAEV); equine infectious anemia virus (EIAV); feline immunodeficiency virus (IVF); bovine immunodeficiency virus (BIV) and simian immunodeficiency virus (SIV).
[0006]
6. Vector, according to any one of claims 3 to 5, CHARACTERIZED by the fact that it comprises a retroviral LTR from the left (5 '); a packaging signal Psi (ψ); a central tract of polipurin / DNA flap (cPPT / FLAP); a retroviral export element; a promoter operably linked to the polynucleotide shown in SEQ ID NO: 10; a retroviral LTR from the right (3 ') and a heterologous polyadenylation sequence.
[0007]
7. Vector, according to claim 6, CHARACTERIZED by the fact that the LTR 5 'promoter is replaced by a heterologous promoter.
[0008]
8. Vector, according to claim 7, CHARACTERIZED by the fact that the heterologous promoter is a promoter of cytomegalovirus (CMV), a promoter of the Rous Sarcoma virus (RSV) or a promoter of the Simian 40 virus (SV40).
[0009]
9. Vector, according to any of claims 6 to 8, CHARACTERIZED by the fact that the 5 'or 3' LTR is a Lentivirus LTR.
[0010]
10. Vector, according to any one of claims 6 to 9, CHARACTERIZED by the fact that the 3 'LTR is a self-inactivating LTR (SIN).
[0011]
11. Vector according to any of claims 6 to 10, CHARACTERIZED by the fact that the polyadenylation sequence is a bovine growth hormone polyadenylation or a rabbit β-globin polyadenylation signal sequence.
[0012]
12. Vector according to any one of claims 6 to 11, CHARACTERIZED by the fact that the promoter is selected from the group consisting of: an immediate early cytomegalovirus (CMV) promoter, an elongation factor alpha 1 promoter (EF1-a), a phosphoglycerate kinase-1 promoter (PGK), a ubiquitin-C promoter (UBQ-C), a cytomegalovirus enhancer / chicken beta-actin promoter (CAG), polyoma enhancer / promoter of herpes simplex thymidine kinase (MC1), a beta actin promoter (β-ACT), a simian 40 virus promoter (SV40) and a myeloproliferative sarcoma virus enhancer, deletion of the negative control region, substituted promoter of dl587rev (MND) primer binding site.
[0013]
13. Composition CHARACTERIZED by the fact that it comprises an immune effector cell ex vivo or in vitro that comprises the vector, as defined in claims 3 to 12, and a physiologically acceptable cell culture medium or cryoprotection agent.
[0014]
14. Composition, according to claim 13, CHARACTERIZED by the fact that the immune effector cell is a T lymphocyte or a natural killer cell (NK).
[0015]
15. Ex vivo or in vitro method for generating an effector immune cell comprising a CAR, CHARACTERIZED by the fact that it comprises introducing the vector into an immune effector cell as defined in any of claims 3 to 12.
[0016]
16. Ex vivo or in vitro method, according to claim 15, CHARACTERIZED by the fact that the immune effector cell is selected from the group consisting of: a T lymphocyte and a natural killing cell (NK).

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2019-04-09| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|

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2019-08-20| B07E| Notification of approval relating to section 229 industrial property law [chapter 7.5 patent gazette]|Free format text: NOTIFICACAO DE ANUENCIA RELACIONADA COM O ART 229 DA LPI |

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2020-03-31| B06A| Patent application procedure suspended [chapter 6.1 patent gazette]|

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2020-09-15| B16A| Patent or certificate of addition of invention granted [chapter 16.1 patent gazette]|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 07/12/2015, OBSERVADAS AS CONDICOES LEGAIS. |

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申请号 | 申请日 | 专利标题

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US201462091419P| true| 2014-12-12|2014-12-12|

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US62/091,419|2014-12-12|

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US201562200505P| true| 2015-08-03|2015-08-03|

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US62/200,505|2015-08-03|

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PCT/US2015/064269|WO2016094304A2|2014-12-12|2015-12-07|Bcma chimeric antigen receptors|

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