ANTI-4-1BB ANTIBODIES, USE THEREOF, PHARMACEUTICAL COMPOSITION, METHODS FOR DETERMINING A DOSE, FOR

专利摘要:
Anti-human 4-1bb antibodies and fragments thereof with one or more structural features not found in a reference anti-human 4-1bb antibody are provided, wherein said features may improve certain features of the antibody with respect to the reference antibody. various in vitro and in vivo methods and reagents related to the anti-human 4-1bb antibodies described herein are also provided. methods include, for example, inducing t-cell proliferation, inducing t-cell secretion of ifn?, as well as detecting, preventing, and/or therapeutically treating cancer using an anti-human 4-1bb antibody or fragment thereof. .
公开号:BR112019007714B1
申请号:R112019007714-6
申请日:2018-01-05
公开日:2022-01-18
发明作者:Byoung S. Kwon；Seoung-Joo Lee；Young Ho Kim；Ho-Sik Oh；Joong Won Lee
申请人:Eutilex Co., Ltd；
IPC主号:

专利说明:

CROSS REFERENCE TO RELATED ORDERS
[001] This application claims priority to and benefit from United States Patent Application No. 62/443,281, filed January 6, 2017, the disclosure of which is incorporated herein by reference in its entirety. BACKGROUND
[002] Cancer remains one of the leading causes of death worldwide. Recent statistics report that 13% of the world's population die from cancer. According to estimates by the International Agency for Research on Cancer (IARC), in 2012, there were 14.1 million new cancer cases and 8.2 million cancer deaths worldwide. By 2030, the global burden is expected to grow to 21.7 million new cancer cases and 13 million cancer deaths due to population growth and aging and exposure to risk factors such as smoking, poor diet and physical inactivity. In addition, the pain and medical expenses for cancer treatment cause reduced quality of life for both cancer patients and their families. It is evident that, above all, cancer is a disease for which improved treatment methods are urgently needed. SUMMARY
[003] The present invention provides, among other things, antibodies and fragments thereof that bind to a human 4-1BB polypeptide. In some aspects, provided anti-human 4-1BB antibodies and fragments thereof are variants of a reference human anti-4-1BB antibody in that they contain one or more particular structural features that are not found in the anti-human 4-1BB antibody. reference human anti-4-1BB. The present invention encompasses a recognition that provided variant anti-human 4-1BB antibodies have improved properties over a reference human anti-4-1BB antibody lacking one or more of the structural features described herein. In some embodiments, the provided human anti-4-1BB antibodies and fragments thereof have one or more improved properties, such as, for example, improved binding affinity, improved induction of T cell proliferation (e.g., T cell proliferation). CD8+ T cells), increased ability to induce IFNY production by T cells (eg, proliferation of CD8+ T cells), improved ability to reduce and/or eliminate cancer proliferation in vivo (eg, at a lower dose).
[004] In some embodiments, an anti-4-1BB antibody or antigen-binding antibody fragment includes 1, 2, or 3 heavy chain CDR sequences that are or include a sequence of SEQ ID NOs: 5 to 8. In some embodiments, an anti-4-1BB antibody or antigen-binding antibody fragment includes one or more of: a heavy chain CDR1 that is or includes a sequence of SEQ ID NO: 5, a heavy chain CDR2 that is or includes a sequence of SEQ ID NO: 6 and a heavy chain CDR3 that is or includes a sequence of SEQ ID NO: 7 or 8. In some embodiments, an anti-4-1BB antibody or antigen-binding antibody fragment includes each one of: a heavy chain CDR1 that is or includes a sequence of SEQ ID NO: 5, a heavy chain CDR2 that is or includes a sequence of SEQ ID NO: 6, and a heavy chain CDR3 that is or includes a sequence of SEQ ID NO: 7 or 8.
[005] In some embodiments, an anti-4-1BB antibody or antigen-binding antibody fragment includes 1, 2, or 3 light chain CDR sequences that are or include a sequence of SEQ ID NOs: 1-4. In some embodiments, an anti-4-1BB antibody or antigen-binding antibody fragment includes one or more of: a light chain CDR1 that is or includes a sequence of SEQ ID NO: 1, a light chain CDR2 that is or includes a sequence of SEQ ID NO: 2 and a light chain CDR1 that is or includes a sequence of SEQ ID NO: 3 or 4. In some embodiments, an anti-4-1BB antibody or antigen-binding antibody fragment includes each of: a light chain CDR1 that is or includes a sequence of SEQ ID NO: 1, a light chain CDR2 that is or includes a sequence of SEQ ID NO: 2, and a light chain CDR1 that is or includes a sequence of SEQ ID NO: 3 or 4.
[006] In some embodiments, an anti-4-1BB antibody or antigen-binding antibody fragment includes a heavy chain variable domain that includes a heavy chain CDR1 that is or includes a sequence of SEQ ID NO: 5, a CDR2 chain that is or includes a sequence of SEQ ID NO: 6 and a heavy chain CDR3 that is or includes a sequence of SEQ ID NO: 7 or 8, and/or a light chain variable domain that includes a CDR1 of light chain that is or includes a sequence of SEQ ID NO: 1, a light chain CDR2 that is or includes a sequence of SEQ ID NO: 2 and a light chain CDR1 that is or includes a sequence of SEQ ID NO: 4 .
[007] In some embodiments, an anti-4-1BB antibody or antigen-binding antibody fragment includes a heavy chain variable domain that includes a light chain framework region 1 (FR1) comprising a sequence of SEQ ID NO: 16 or 17. In some embodiments, an anti-4-1BB antibody or antigen-binding antibody fragment includes a heavy chain variable domain that includes a heavy chain framework 3 (FR3) region comprising a sequence of any one of SEQ ID NOs : 1820. In some embodiments, an anti-4-1BB antibody or antigen-binding antibody fragment includes a heavy chain variable domain that includes a light chain framework region 1 (FR1) comprising a sequence of SEQ ID NO: 16 or 17 and a heavy chain framework region 3 (FR3) comprising a sequence of any one of SEQ ID NOs: 18-20.
[008] In some embodiments, an anti-4-1BB antibody or antigen-binding antibody fragment includes substantial homology to an antibody or antibody fragment that includes a heavy chain variable domain that is or includes a sequence selected from SEQ ID NOs : 11-14. In some embodiments, an anti-4-1BB antibody or antigen-binding antibody fragment includes a heavy chain variable domain that is or includes a sequence at least 90%, 91%, 92%, 93%, 94%, 95 %, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4% or 99.5% identical to a selected sequence of SEQ ID NOs: 11- 14. In some embodiments, an anti-4-1BB antibody or antigen-binding antibody fragment includes a heavy chain variable domain that is or includes a sequence selected from SEQ ID NOs: 11-14.
[009] In some embodiments, an anti-4-1BB antibody or antigen-binding antibody fragment includes substantial homology to an antibody or antibody fragment that includes a light chain variable domain that is or includes a sequence of SEQ ID NO: 9 or 10. In some embodiments, an anti-4-1BB antibody or antigen-binding antibody fragment includes a light chain variable domain that is or includes a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4% or 99.5% identical to a sequence of SEQ ID NO: 9 or 10. In In some embodiments, an anti-4-1BB antibody or antigen-binding antibody fragment includes a light chain variable domain that is or includes a sequence of SEQ ID NO: 9 or 10.
[0010] In some embodiments, an anti-4-1BB antibody or antigen-binding antibody fragment includes substantial homology to an antibody or antibody fragment that includes a heavy chain variable domain that is or includes a sequence selected from SEQ ID NOs : 11-14 and a light chain variable domain that is or includes a sequence of SEQ ID NO: 10. In some embodiments, an anti-4-1BB antibody or antigen-binding antibody fragment includes a heavy chain variable domain. which is or includes a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99, 3%, 99.4%, or 99.5% identical to a selected sequence of SEQ ID NOs: 11-14 and a light chain variable domain that is or includes a sequence at least 90%, 91%, 92%, 93 %, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4% or 99.5% identical to a SEQ sequence ID NO: 10. In some embodiments, an anti-4-1BB antibody or antigen-binding antibody fragment is includes a heavy chain variable domain that is or includes a sequence selected from SEQ ID NOs: 1114 and a light chain variable domain that is or includes a sequence of SEQ ID NOs: 10.
[0011] In some embodiments, a provided anti-human 4-1BB antibody or fragment thereof is an agonistic antibody. In some embodiments, a provided anti-human 4-1BB antibody or fragment thereof is characterized as having greater agonistic activity than a 94G1 humanized anti-human 4-1BB antibody (i.e., an antibody including light chain variable domains). and heavy chain of SEQ ID NOs: 9 and 11, respectively). In some embodiments, a provided human anti-4-1BB antibody or fragment thereof is characterized as having better binding affinity than a 94G1 humanized anti-human 4-1BB antibody (i.e., an antibody including light chain and variable domains). heavy chain of SEQ ID NOs: 9 and 11, respectively).
[0012] In some embodiments, a provided anti-human 4-1BB antibody or fragment thereof is or comprises a humanized antibody. In some embodiments, a provided human anti-4-1BB antibody or fragment thereof includes a human immunoglobulin constant domain, wherein the constant domain is selected from an IgG1 or a variant thereof, an IgG2 or a variant thereof, an IgG4 or a variant thereof, an IgA or a variant thereof, an IgE or a variant thereof, an IgM or a variant thereof, and an IgD or a variant thereof. In some embodiments, a provided human anti-4-1BB antibody or fragment thereof is or comprises a human IgG1. In some embodiments, an IgG1 is or comprises a sequence that is at least 95% identical to SEQ ID NO: 22 or 23.
[0013] In some embodiments, a provided anti-human 4-1BB antibody or fragment thereof is a monoclonal antibody.
[0014] In some embodiments, a provided anti-human 4-1BB antibody or fragment thereof is a full-size antibody. In some embodiments, a provided human anti-4-1BB antibody or fragment thereof is a Fab fragment, a Fab' fragment, an F(ab')2 fragment, an Fv fragment, a linked Fv fragment by disulfide, an scFv fragment, a single domain antibody, humabody, nanobody, or a diabody.
[0015] In some embodiments, a provided anti-human 4-1BB antibody or fragment thereof has a binding affinity (KD) for a human 4-1BB molecule from 1xio-7 to ixio-12 M. In some embodiments, a provided human anti-4-1BB antibody or fragment thereof has a binding affinity (KD) for a human 4-1BB molecule from 1xio-8 to ixio-12 M. In some embodiments, an anti-4 Provided human -1BB or fragment thereof has a binding affinity (KD) for a human 4-1BB molecule of 1x10 -9 to 1x10 -12 M. In some embodiments, a provided human anti-4-1BB antibody or fragment of the same has a binding affinity (KD) for a human 4-1BB molecule of 1x10-10 to 1x10-12 M.
[0016] In some embodiments, a provided anti-human 4-1BB antibody or fragment thereof binds to an epitope within the extracellular domain of a human 4-1BB polypeptide. In some embodiments, a provided anti-human 4-1BB antibody or fragment thereof binds to an epitope within the extracellular domain of human 4-1BB. In some embodiments, binding of a provided human anti-4-1BB antibody or fragment thereof is abrogated by one or more mutations at positions N30, D38, N39, and R41 of SEQ ID NO:44.
[0017] In some embodiments, a provided anti-human 4-1BB antibody or fragment thereof does not or weakly binds to a non-primate 4-1BB polypeptide. In some embodiments, a provided human anti-4-1BB antibody or fragment thereof does not or weakly binds to a canine 4-1BB polypeptide.
[0018] In some embodiments, the present invention provides nucleic acid molecules encoding an anti-4-1BB antibody or antigen-binding fragment. In some embodiments, the present invention provides vectors that include a nucleic acid molecule encoding an anti-4-1BB antibody or antigen-binding fragment. In some embodiments, the present invention provides host cells that include a vector and/or nucleic acid molecule encoding an anti-4-1BB antibody or antigen-binding fragment. In some embodiments, a host cell is selected from a bacterial, yeast, insect, or mammalian cell. In some embodiments, a is selected from the group consisting of E.coli, P.pastoris, Sf9, COS, HEK293, CHO, and a mammalian lymphocyte.
[0019] In some embodiments, the present invention provides pharmaceutical compositions that include an anti-4-1BB antibody or antigen-binding fragment and a pharmaceutically acceptable carrier. In some embodiments, the present invention provides pharmaceutical compositions that include a nucleic acid and/or vector encoding an anti-4-1BB antibody or antigen-binding fragment and a pharmaceutically acceptable carrier.
[0020] In some embodiments, the present invention provides methods of treating a subject in need thereof, the method comprising administering to the subject a composition that comprises or releases an anti-4-1BB antibody or antigen-binding fragment. In some embodiments, the present invention provides methods of treating a subject in need thereof, the method comprising administering to the subject a composition that comprises or delivers a nucleic acid and/or vector encoding an anti-4-1BB antibody or binding fragment. to the antigen. In some modalities, an individual has or is at risk of developing cancer.
[0021] In some embodiments, the present invention provides methods of inducing an immune response in a subject in need thereof, the method comprising administering to the subject a composition that comprises or releases an anti-4-1BB antibody or antigen-binding fragment . In some embodiments, the present invention provides methods of inducing an immune response in a subject in need thereof, the method comprising administering to the subject a composition that comprises or delivers a nucleic acid and/or vector encoding an anti-4-1BB antibody. or antigen-binding fragment. In some modalities, an individual has or is at risk of developing cancer.
[0022] In some embodiments, the present invention provides methods of enhancing an immune response in a subject in need thereof, the method comprising administering to the subject a composition that comprises or releases an anti-4-1BB antibody or antigen-binding fragment . In some embodiments, the present invention provides methods of enhancing an immune response in a subject in need thereof, the method comprising administering to the subject a composition that comprises or delivers a nucleic acid and/or vector encoding an anti-4-1BB antibody. or antigen-binding fragment. In some modalities, an individual has or is at risk of developing cancer.
[0023] In some embodiments, a cancer to be treated by a method of the present invention in a subject is selected from bladder cancer, breast cancer, cervical cancer, colon cancer, endometrial cancer, esophageal cancer, fallopian tube cancer, fallopian tube, gallbladder cancer, gastrointestinal cancer, head and neck cancer, hematologic cancer, laryngeal cancer, liver cancer, lung cancer, lymphoma, melanoma, mesothelioma, ovarian cancer, primary peritoneal cancer, salivary gland cancer, sarcoma , stomach cancer, thyroid cancer, pancreatic cancer, and prostate cancer.
[0024] In some embodiments, a composition comprises or delivers an anti-human 4-1BB antibody of the present invention or an antigen-binding fragment thereof at a dose of 0.01 mg/kg to 100 mg/kg. In some embodiments, a composition comprises or delivers a human anti-4-1BB antibody or an antigen-binding fragment thereof at a dose of about 0.01 mg/kg, 0.025 mg/kg, 0.05 mg/kg. kg, 0.075 mg/kg, 0.1 mg/kg, 0.25 mg/kg, 0.5 mg/kg, 0.75 mg/kg, 1 mg/kg, 2 mg/kg, 3 mg/kg, 4mg/kg, 5mg/kg, 8mg/kg, 10mg/kg, 20mg/kg, 25mg/kg, 30mg/kg, 40mg/kg, 50mg/kg, 50mg/kg, 70 mg/kg, 80 mg/kg, 90 mg/kg, or 100 mg/kg.
[0025] In some embodiments, human anti-4-1BB antibodies and/or fragments thereof and/or compositions comprising the same are characterized by inducing increased T cell proliferation (e.g., CD8+ T cell proliferation) and/or increased IFNY secretion by T cells (eg, CD8+ T cells) in an individual.
[0026] In some embodiments, the present invention provides methods that include administering to the subject a composition that comprises or releases an anti-4-1BB antibody or antigen-binding fragment to a subject who has been or will be administered one or more anti-cancer therapies additional. In some embodiments, the present invention provides methods that include administering to the subject a composition that comprises or releases an anti-4-1BB antibody or antigen-binding fragment to a subject who has been or will be administered with one or more of ionizing radiation, a chemotherapeutic agent, an antibody agent, and a cell-based therapy, so that the subject receives treatment with both.
[0027] In some embodiments, the present invention provides methods that include administering to the subject a composition that comprises or releases an anti-4-1BB antibody or antigen-binding fragment to a subject who has been or will be administered with one or more of a immunological checkpoint inhibitor, IL-12, GM-CSF, an anti-CD4 agent, fluorouracil, doxorubicin, irinotecan, paclitaxel, cisplatin, or cyclophosphamide.
[0028] In some embodiments, the present invention provides methods that include administering to the subject a composition that comprises or releases an anti-4-1BB antibody or antigen-binding fragment to a subject who has been or will be administered a composition comprising an inhibitor checkpoint, so that the individual receives treatment with both. In some embodiments, an immunological checkpoint inhibitor is an agent that inhibits PD-1 signaling. In some embodiments, an agent that inhibits PD-1 signaling is an anti-PD-1 antibody. In some embodiments, an anti-PD-1 antibody is nivolumab, pembrolizumab, atezolizumab, durvalumab, or avelumab.
[0029] In some embodiments, the present invention provides methods of determining a dose of an anti-4-1BB antibody or antigen-binding fragment thereof for the therapeutic treatment of an individual in need thereof. In some embodiments, such a method includes (i) providing or obtaining a measurement of secreted IFN-gamma in a biological sample from the subject, wherein the subject has been administered a composition that comprises or releases an amount of an anti-4-1BB antibody or antigen-binding fragment described herein; and (ii) comparing the measurement of secreted IFN-gamma to a reference value, where whether the measurement of secreted IFN-gamma is greater or less than the reference value, the adjustment of an anti-4-1BB antibody or fragment of antigen binding thereof will be administered, thereby determining a dose for the therapeutic treatment of an individual. In some embodiments, a reference value comprises an index value that includes a value derived from one or more healthy individuals, a value derived from one or more individuals diagnosed with cancer, or a value derived from a cancer risk prediction algorithm. In some embodiments, a biological sample is a sample of whole blood, plasma, or serum. In some modalities, an individual has or is at risk of developing cancer. In some embodiments, the cancer is selected from bladder cancer, breast cancer, cervical cancer, colon cancer, endometrial cancer, esophageal cancer, fallopian tube cancer, gallbladder cancer, gastrointestinal cancer, head and neck cancer. neck, hematologic cancer, laryngeal cancer, liver cancer, lung cancer, lymphoma, melanoma, mesothelioma, ovarian cancer, primary peritoneal cancer, salivary gland cancer, sarcoma, stomach cancer, thyroid cancer, pancreatic cancer, and cancer of prostate.
[0030] In some embodiments, the present invention provides methods for increasing IFN-Y secretion by a cell in vivo or in vitro that include: contacting the cell with an anti-4-1BB antibody or antigen-binding fragment described herein.
[0031] In some embodiments, the present invention provides methods of ex vivo proliferation, proliferation, or isolation of activated T cells that include: contacting a population of T cells with an anti-4-1BB antibody or antigen-binding fragment described herein, thereby increasing the proliferation of activated T cells.
[0032] In some embodiments, the present invention provides methods for isolating non-antigen-specific activated T cells that include one or more steps of: (a) culturing peripheral blood mononuclear cells (PBMCs) in a medium together with a peptide from an epitope of interest is IL-2; (b) inducing 4-1BB expression in the cultured cells by adding the epitope peptide of interest; (c) contacting the cultured cells with a surface coated with an anti-4-1BB antibody or antigen-binding fragment described herein, wherein the cultured cells expressing 4-1BB adhere to the coated surface; and (d) removing the unbound cells, thereby isolating the antigen-specific activated T cells. In some embodiments, activated T cells are CD8+ T cells.
[0033] In some embodiments, the present invention provides methods for treating or preventing cancer in a subject in need thereof which include administering to the subject a composition that includes a therapeutically effective amount of activated T cells produced by any of the methods described herein. In some embodiments, the cancer is selected from bladder cancer, breast cancer, cervical cancer, colon cancer, endometrial cancer, esophageal cancer, fallopian tube cancer, gallbladder cancer, gastrointestinal cancer, head and neck cancer. neck, hematologic cancer, laryngeal cancer, liver cancer, lung cancer, lymphoma, melanoma, mesothelioma, ovarian cancer, primary peritoneal cancer, salivary gland cancer, sarcoma, stomach cancer, thyroid cancer, pancreatic cancer, and cancer of prostate. In some embodiments, a composition includes less than 109, at least 1010 cells, or more than 1010 activated T cells. In some embodiments, activated T cells are CD8+ T cells.
[0034] Also provided, among other things, are technologies for characterizing human anti-4-1BB antibodies and/or fragments thereof as described herein and/or compositions comprising the same. In some embodiments, methods are provided for characterizing human anti-4-1BB antibodies and/or fragments thereof and/or compositions comprising the same that bind to AML cells (e.g., HL60). In some embodiments, methods for characterizing human anti-4-1BB antibodies and/or fragments thereof and/or compositions comprising the same are provided by ELISA, immunohistochemistry, Biacore binding assays, mass spectrometry, isoelectric focusing (IEF), and/or western blot.
[0035] The present invention provides various technologies related to preparing or manufacturing the human anti-4-1BB antibodies and/or fragments thereof as described herein and/or compositions containing said antibodies or fragments thereof.
[0036] As used in this application, the terms "about" and "approximately" are used interchangeably. Any citations to publications, patents or patent applications herein are incorporated by reference in their entirety. Any numerals used in this application with or without about/approximately are not intended to encompass any normal fluctuations appreciated by one skilled in the relevant art.
[0037] Other features, objects and advantages of the present invention are evident from the detailed description that follows. It is to be understood, however, that the detailed description, while indicating embodiments of the present invention, is given in an illustrative, non-limiting manner only. Various changes and modifications in the scope of the invention will become apparent to those skilled in the art from the detailed description. BRIEF DESCRIPTION OF THE DRAWINGS
[0038] The drawing included here, which is comprised of the following figures, is for illustration purposes only and not for limitation. The foregoing and other objects, aspects, features and advantages of the present invention will become more apparent and better understood by referring to the following description taken in conjunction with the accompanying figures in which:
[0039] FIG. 1A describes human 4-1BB extracellular domain (ECD) constructs. At the top is a schematic of a full-size 4-1BB ECD (167 amino acids), and below are several fragments of a 4-1BB ECD: R1 (1-55 aa), R2 (56-110 aa), R3 (110-167 aa), R1.1 (1-45 aa), R1.2 (1-35 aa), R1.3 (11-55 aa), R1.4 (21-55 aa) R1.5 (1-25 aa) and R1.6 (1-30 aa). Each of these 4-1BB ECD constructs were fused with GST. FIG. 1B depicts a western blot showing the binding of an exemplary humanized anti-4-1BB antibody to the 4-1BB ECD fusion constructs as described in FIG. 1A. As shown, an exemplary humanized anti-4-1BB antibody binds a full-size 4-1BB ECD fusion polypeptide and the R1 fusion polypeptide, but not the R2 or R3 fusion polypeptides. Molecular size markers are indicated in kDa on the left.
[0040] FIG. 2A depicts an SDS-PAGE gel of whole cell extracts of cells expressing 4-1BB ECD fusion constructs. Fusion constructs as described above in FIG. 2A were expressed in E. coli BL21 cells induced with 1 mM IPTG, and whole cell extracts resolved by 12% SDS-PAGE. As shown, all of the fusion constructs (ECD, R1, R1.1, R1.2, R1.3, R1.4, R1.5, and R1.6) have robust protein expression. FIG. 2B depicts a western blot showing the binding of an exemplary humanized anti-4-1BB antibody to the full-length 4-1BB ECD fusion peptide, and fusion polypeptides of R1.1, R1.2, R1.3, and R1.6, but not to R1.4 or R1.5 fusion polypeptides. Immunoblots have been reported with exemplary human anti-4-1BB antibody. Molecular size markers are indicated in kDa on the left.
[0041] FIG. 3 describes the binding affinity of anti-4-1BB monoclonal antibodies to monoclonal antibodies to recombinant human 4-1BB antigen, as measured by ELISA. OD450nm values are plotted on the y-axis, and increasing concentrations of anti-4-1BB antibodies (in μg/ml) along the x-axis. BBK-4 (circles) is a murine human anti-4-1BB antibody, 94G1 (squares), 94K (upward-pointing triangles), 94KV (diamonds), 94KVT (stars) and EU101 (downward-pointing triangles) are antibodies exemplary humanized variant anti-4-1BB.
[0042] FIG. 4 describes the binding of anti-4-1BB monoclonal antibodies to 4-1BB expressing Jurkat T cells (Jurkat 8-1). Mean Fluorescence Intensity (MFI) values are plotted on the y-axis and Log10 antibody concentration (in μg/ml) along the x-axis. BBK-4 (circles) is a murine human anti-4-1BB antibody, 94G1 (squares), 94K (upward-pointing triangles), 94KV (diamonds), 94KVT (stars) and EU101 (downward-pointing triangles) are antibodies exemplary humanized variant anti-4-1BB.
[0043] FIG. 5 provides a table listing in vitro binding affinities of variant anti-4-1BB antibodies to 4-1BB. Binding affinity was measured using surface plasmon resonance (SPR, Biacore 3000). 94G1 and EU101 are exemplary humanized variant anti-4-1BB antibodies.
[0044] FIG. 6 describes the binding of anti-4-1BB monoclonal antibodies to 4-1BB expressing CD8+ T cells. CD8+ T cells were isolated from human PBMCs and activated by anti-CD3 antibody for 2 days. 4-1BB-PE is a commercially available anti-4-1BB antibody, BBK-4 is a murine human anti-4-1BB antibody, 94G1, 94K, 94KV, 94KVT and EU101 are exemplary humanized variant anti-4-1BB antibodies . The graph in the lower panel reflects the values shown for each antibody in the FACS data in the upper panels.
[0045] FIG. 7 depicts a graph quantifying the in vitro proliferation of CD8+ T cells treated with anti-4-1BB antibodies. Proliferating CD8+ T cells were treated without any antibody, human IgG alone, BBK-4, or exemplary humanized variant anti-4-1BB antibody: 94G1, 94K, 94KV, 94KVT, and EU101 and treated with WST-1 (water-soluble tetrazolium salt). water) to stain proliferating (i.e. metabolically active) cells.
[0046] FIG. 8 depicts a graph quantifying in vitro IFNY secretion by CD8+ T cells treated with anti-4-1BB antibodies. CD8+ T cells were isolated from human PBMCs and treated without any antibody, human IgG alone, or 1 µg/ml of an anti-4-1BB antibody: BBK-4, 94G1, 94K, 94KV, 94KVT and EU101. IFNy secretion was assessed on days 1, 3, and 5.
[0047] FIG. 9 shows graphs depicting IFN-γ secretion on (A) CD4+ and (B) CD8+. After being isolated from PBMCs from a healthy donor, activated T cells present in the PBMCs were rested in RPMI-1640+2% FBS medium for 24 hours, and the rested PBMCs were treated with anti-CD4 antibody or anti-CD8 antibody. bound to iron beads, and CD4+ cells or CD8+ cells were isolated using a MACS magnetic separator. Isolated CD4+ T cells or CD8+ T cells were treated with an anti-CD3 T cell activator to induce 4-1BB expression, and treated with EU101 at different concentrations (0.5, 1.0, 2.5 , and 5.0 µg/ml) or a control human IgG (5.0 µg/ml) for 3 days. After 3 days, a culture medium excluding the cells was obtained, and the fluorescence of human IFN-γ in the culture medium was evaluated by ELISA (ebioscience). Results were compared to a standard curve as provided in an IFN-γ ELISA kit.
[0048] FIG. 10A shows a graph depicting the antibody-dependent cytotoxicity (ADCC) of exemplary human anti-4-1BB antibodies BBK4, 94G1, 94KVT, and EU101. FIG. 10B shows a graph depicting the complement cytotoxicity (CDC) of exemplary human anti-4-1BB antibodies BBK4, 94G1, and EU101.
[0049] FIG. 11 shows in vivo anti-cancer effects of an exemplary human anti-4-1BB antibody (EU101) by concentration, which are measured as tumor sizes after colon cancer tumor cells (HT29) are subcutaneously injected into humanized mice, and when tumor sizes reach 100 to 200 mm3, an exemplary human anti-4-1BB antibody (EU101) was intravenously administered to the mice at doses of 1.0 mg, 5.0 mg, and 10.0 mg per 1 kg of body weight. body once every 5 days 3 times (representative data).
[0050] FIG. 12 shows the anti-cancer effects of an exemplary human anti-4-1BB antibody (EU101) and an exemplary anti-PD-1 antibody (Keytruda, "KD") by concentration. Anticancer effects were measured as tumor sizes after subcutaneous injection of colon cancer tumor cells (HT29) into humanized mice and antibody treatment. When tumor sizes reached 100 to 150 mm3, mice were treated with an exemplary human anti-4-1BB antibody (EU101) or an exemplary anti-PD-1 antibody (Keytruda) by intraperitoneal injection at a dose of 5, 0 mg and 10.0 mg per 1 kg of body weight once every 5 days three times.
[0051] FIG. 13 shows comparative anticancer effects of single treatment and combination therapy of an exemplary human anti-4-1BB antibody (EU101) and an exemplary anti-PD-1 antibody (Keytruda). Anticancer effects were measured as tumor sizes after colon cancer tumor cells (HT29) were subcutaneously injected into humanized mice and treated with antibody. When tumor sizes reached 300 to 450 mm3, an exemplary human anti-4-1BB antibody (EU101) was administered at 2.5 mpk for individual treatment, an exemplary anti-PD-1 antibody (Keytruda) was administered at 2 .5 mpk for individual treatment, and EU101, 2.5 mpk + Keytruda, 2.5 mpk were given for combination therapy. Administration was by intraperitoneal injection of mice once every three days for a total of three times.
[0052] FIG. 14A shows the numbers of CD4+ T cells and CD8+ T cells circulated in mouse blood or 1 gram of tumor tissue at 34 days after treatment with an exemplary human anti-4-1BB antibody (EU101) and an anti-PD-1 antibody. (Keytruda), individually and in combination, in tumor-implanted humanized mice, as described in FIG. 13. The number of T cell infiltrating lymphocytes (TILs) in tumor was measured by calculating the proportional ratios of the total cell numbers by measuring the ratios (%) of CD4+ T cells and CD8+ T cells using a flow cytometer. Flow cytometry was performed to measure the ratios (%) of CD4+ T cells to CD8+ T cells after cells were stained with FITC-labeled CD4 antibody, a fluorescent BV510-labeled CD8 antibody, and a fluorescent APC-cy7-labeled CD45 antibody, and a human blood cell marker, CD45 positive cells were separated by a flow cytometry (gating) program. FIG. 14B shows a ratio of Treg (CD4+Foxp3high T cells) to CD8+IFN-Y+ T cell ratio measured by calculating a proportional ratio between CD8+IFN-Y+ T cell ratio and Treg ratio (CD4+Foxp3high T cells ) using a flow cytometer after cells were stained with a fluorescent APC-cy7-labeled CD45 antibody, a fluorescent BV510-labeled CD8 antibody, a fluorescent FITC-labeled CD4 antibody, a fluorescent PE-labeled INFY antibody, and a fluorescent APC-labeled Foxp3 antibody. .
[0053] FIG. 15A and FIG. 15B show the results of analysis of IFN-Y through serum and tumor fluid after individual and combination treatment of an exemplary human anti-4-1BB antibody (EU101) and an exemplary anti-PD-1 antibody (Keytruda). After dissection performed on all treated groups shown in FIGS. 15A and 15B, 10 µl of serum and 100 µl of tumor fluid were analyzed with human IFN-Y and human TGF-β ELISA kits.
[0054] FIG. 16A shows antigen-specific CD8+ T cell ratios (4-1BB+CD8+ T cell ratio: 43.2%, CD8+ T cell ratio: 58.6%) measured before paniculation with a human anti-4-1BB antibody. copy (EU101). FIG. 16B shows antigen-specific CD8+ T cell ratios (pCMV+CD8+ T cell ratio: 60.0%, CD8+ T cell ratio: 79.3%) measured after paniculation with an exemplary human anti-4-1BB antibody ( EU101). CERTAIN DEFINITIONS
[0055] In the description that follows, several terms used in recombinant DNA and immunology are used extensively. In order to provide a clearer and more consistent understanding of the specification and claims, including the scope to be given to such terms, the following definitions are provided.
[0056] About: The term "about", when used here in reference to a value, refers to a value that is similar, in context, to the referenced value. In general, those skilled in the art, familiar with the context, will appreciate the relevant degree of variance encompassed by "about" in that context. For example, in some embodiments, the term "about" can encompass a range of values that include 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12% , 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of the referenced value.
[0057] Administration: As used herein, the term "administration" typically refers to the administration of a composition to an individual or system to obtain release of an agent that is, or is included in the composition. Those skilled in the art will be aware of a variety of routines which may, in appropriate circumstances, be used for administration to an individual, for example, a human. For example, in some embodiments, administration may be ocular, oral, parenteral, topical, etc. In some particular embodiments, administration may be bronchial (e.g., by bronchial instillation), buccal, dermal (which may be or comprise, for example, one or more of topical dermal, intradermal, interdermal, transdermal, etc.), enteral, intraarterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, within a specific organ (eg, intrahepatic), mucosal, nasal, oral, rectal, subcutaneous, sublingual, topical, tracheal (e.g. by intratracheal placement), vaginal, vitreous, etc. In some embodiments, administration may involve only a single dose. In some embodiments, administration may involve the application of a fixed number of doses. In some embodiments, administration may involve dosing that is intermittent (e.g., a plurality of doses separated in time) and/or periodic (e.g., individual doses separated by a common period of time) dosing. In some embodiments, administration may involve continuous dosing (e.g., infusion) for at least a selected period of time.
[0058] Affinity: As is known in the art, "affinity" is a measure of the tension with which a particular ligand binds to its partner. Affinities can be measured in different ways. In some embodiments, affinity is measured by a quantitative assay. In some such embodiments, the binding partner concentration may be set to be in excess of the ligand concentration in order to mimic physiological conditions. Alternatively, or additionally, in some embodiments, the binding partner concentration and/or linker concentration may be varied. In some such embodiments, the affinity can be compared to a reference under comparable conditions (eg, concentrations).
[0059] Agonist: Those skilled in the art will appreciate that the term "agonist" can be used to refer to an agent condition, or event whose presence, level, degree, type, or form correlates with an increased level of activity of another agent. . (i.e., the agonized agent). In general, an agonist can be or include an agent of any chemical class including, for example, small molecules, polypeptides, nucleic acids, carbohydrates, lipids, metals, and/or any other entity that shows relevant activating activity. In some embodiments, an agonist may be direct (in which case it exerts its influence directly on its target); in some embodiments, an agonist may be indirect (in which case it exerts its influence by something other than binding to its target; for example, interacting with a regulator of the target such that the target's level or activity is altered).
[0060] Animal: as used here refers to any member of the animal kingdom. In some embodiments, "animal" refers to humans, of either sex and at any stage of development. In some embodiments, "animal" refers to non-human animals at any stage of development. In certain embodiments, the non-human animal is a mammal (e.g., a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a sheep, cattle, a primate, and/or a pig ). In some embodiments, animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, insects, and/or worms. In some embodiments, an animal may be a transgenic animal, genetically modified animal, and/or a clone.
[0061] Antagonist: Those skilled in the art will appreciate that the term "antagonist", as used herein, can be used to refer to an agent condition, or event whose presence, level, degree, type, or form correlates with a decreased degree or activity of another agent (ie, the inhibited agent, or target). In general, an antagonist can be or include an agent of any chemical class including, for example, small molecules, polypeptides, nucleic acids, carbohydrates, lipids, metals, and/or any other entity that shows the relevant inhibitory activity. In some embodiments, an antagonist may be direct (in which case he exerts his influence directly on his target); in some embodiments, an antagonist may be indirect (in which case it exerts its influence by something other than binding to its target; for example, interacting with a regulator of the target such that the target's level or activity is altered).
[0062] Antibody: As used herein, the term "antibody" refers to a polypeptide that includes sufficient canonical immunoglobulin sequence elements to confer specific binding to a particular target antigen. As is known in the art, intact antibodies as produced in nature are tetrameric agents of approximately 150 kD comprised of two identical heavy chain polypeptides (about 50 kD each) and two identical light chain polypeptides (about 25 kD each) that bind to each other. associate with each other in what is commonly referred to as a "Y-shaped" structure. Each heavy chain is comprised of at least four domains (each about 110 amino acids long) - an amino-terminal variable (VH) domain (located at the ends of the Y structure), followed by three constant domains: CH1, CH2, and the CH3 carboxy terminal (located at the base of the Y stem). A short region, known as an "exchanger", connects the heavy chain constant and variable regions. The "hinge" connects the CH2 and CH3 domains to the rest of the antibody. Two disulfide bonds in this hinge region link the two heavy chain polypeptides together in an intact antibody. Each light chain is comprised of two domains - an amino-terminal variable (VL) domain, followed by a carboxy-terminal constant (CL) domain, separated from one another by another "exchanger". Intact antibody tetramers are comprised of two heavy chain-light chain dimers in which the heavy and light chains are linked together by a disulfide bond; two other disulfide bonds connect the heavy chain hinge regions to one another so that the dimers are connected together and the tetramer is formed. Naturally produced antibodies are also glycosylated, typically in the CH2 domain. Each domain in a natural antibody has a structure characterized by an "immunoglobulin fold" formed from two beta sheets (e.g., 3-, 4-, or 5-strand sheets) packed against each other in an antiparallel beta drum. pill. Each variable domain contains three hypervariable loops known as "complement determining regions" (CDR1, CDR2, and CDR3) and four somewhat invariant "framework" regions. (FR1, FR2, FR3 and FR4). When natural antibodies fold, the FR regions form the beta sheets that provide the structural framework for the domains, and the CDR loop regions of both heavy and light chains are brought together in three-dimensional space to create a single hypervariable antibody-binding site. antigen located at the tip of the Y structure. The Fc region of naturally occurring antibodies binds to elements of the complement system, as well as to receptors on effector cells, including, for example, effector cells that mediate cytotoxicity. As is known in the art, affinity and/or other binding attributes of Fc regions to Fc receptors can be modulated through glycosylation or other modification. In some embodiments, antibodies produced and/or used in accordance with the present invention include glycosylated Fc domains, including Fc domains modified or engineered with such glycosylation. For purposes of the present invention, in certain embodiments, any polypeptide or complex of polypeptides that includes sufficient immunoglobulin domain sequences as found in natural antibodies may be referred to and/or used as an "antibody", if such a polypeptide is naturally produced (eg. (eg, generated by an organism reacting to an antigen), or produced by recombinant engineering, chemical synthesis, or other artificial system or methodology. In some embodiments, an antibody is polyclonal, in some embodiments, an antibody is monoclonal. In some embodiments, an antibody is polyclonal; in some embodiments, an antibody is monoclonal. In some embodiments, an antibody has constant region sequences that are characteristic of mouse, rabbit, primate, or human antibodies. In some embodiments, antibody sequence elements are humanized, primatized, chimeric, etc., as is known in the art. Furthermore, the term "antibody" as used herein may refer in appropriate embodiments (unless otherwise stated or clear from context) to any of the formats or constructs known in the art or developed to utilize the features structural and functional of the antibody in alternative presentation. For example, in embodiments, an antibody used in accordance with the present invention is in a format selected from, but not limited to, intact IgA, IgG, IgE, or IgM antibodies; bi- or multi-specific antibodies (e.g., Zybodies®, etc.); antibody fragments such as Fab fragments, Fab' fragments, F(ab')2 fragments, Fd' fragments, Fd fragments, and isolated CDRs or groups thereof; Single-chain Fvs; polypeptide-Fc fusions; single domain antibodies (e.g., single domain shark antibodies such as IgNAR or fragments thereof); cameloid antibodies; masked antibodies (e.g., Probodies®); Small Modular ImmunoPharmaceuticals ("SMIPsTM"); single-chain or Tandem diabodies (TandAb®); humabodies, VHHs; Anticalins®; Nanobodies® minibodies; BiTE®s; ancyrin repeat proteins or DARPINs®; Avimers®; DARTs; TCR-like antibodies; Adnectins®; Affilins®; Trans-bodies®; Affibodies®; TrimerX®; Microproteins; Fynomers®, Centyrins®; and KALBITOR®s. In some embodiments, an antibody may lack a covalent modification (eg, attachment of a glycan) that it would have if it were produced naturally. In some embodiments, an antibody may contain a covalent modification (e.g., linkage of a glycan, a payload [e.g., a detectable moiety, a therapeutic moiety, a photocatalytic moiety, etc.], or another pendant group [e.g. , polyethylene glycol, etc.]
[0063] Antibody Fragment: As used herein, an "antibody fragment" refers to a portion of an antibody or antibody agent as described herein, and typically refers to a portion that includes an antigen-binding portion or variable region of the same. An antibody fragment can be produced by any means. For example, in some embodiments, an antibody fragment can be enzymatically or chemically produced by fragmenting an intact antibody or antibody agent. Alternatively, in some embodiments, an antibody fragment can be produced recombinantly (i.e., by expression of an engineered nucleic acid sequence). In some embodiments, an antibody fragment may be wholly or partially produced synthetically. In some embodiments, an antibody fragment (particularly, an antigen-binding antibody fragment) can be at least about 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150 in length. , 160, 170, 180, 190 amino acids or more, in some embodiments at least about 200 amino acids.
[0064] Binding: It will be understood that the term "binding", as used herein, typically refers to a non-covalent association between or within two or more entities. "Direct" bonding involves physical contact between entities or portions; indirect linkage involves physical interaction through physical contact with one or more intermediary entities. The link between two or more entities can typically be assessed in any of a variety of contexts - including where the interactions of entities or portions are studied in isolation or in the context of more complex systems (e.g., while covalently or otherwise associated with a carrier entity and/or in a biological system or cell).
[0065] Cancer: The terms "cancer", "malignancy", "neoplasm", "tumor", and "carcinoma", are used here to refer to cells that exhibit relatively abnormal, uncontrolled, and/or autonomous growth. , such that they exhibit an aberrant growth phenotype characterized by a significant loss of control over cell proliferation. In some embodiments, a tumor can be or comprise cells that are precancerous (e.g., benign), malignant, pre-metastatic, metastatic, and/or non-metastatic. The present invention specifically identifies certain cancers to which its teachings may be particularly relevant. In some embodiments, a relevant cancer may be characterized by a solid tumor. In some embodiments, a relevant cancer may be characterized by a hematologic tumor. In general, examples of different types of cancers known in the art include, for example, hematopoietic cancers including leukemias, lymphomas (Hodgkin's and non-Hodgkin's), myelomas and myeloproliferative disorders; sarcomas, melanomas, adenomas, solid tissue carcinomas, squamous cell carcinomas of the mouth, throat, larynx and lung, liver cancer, genitourinary cancers such as prostate, cervical, bladder, uterine, endometrial and renal cell carcinomas, cancer bone, pancreatic cancer, skin cancer, cutaneous or intraocular melanoma, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancers of the head and neck, breast cancer, gastrointestinal cancers and cancers of the nervous system, benign lesions such as papillomas, and the like.
[0066] CDR: as used herein, refers to a complementarity determining region within an antibody variable region. There are three CDRs in each of the heavy chain and light chain variable regions, which are designated CDR1, CDR2 and CDR3, for each of the variable regions. A "group of CDRs" or "group of CDRs" refers to a group of three or six CDRs that occur in a single variable region capable of binding antigen or in the CDRs of cognate heavy and light chain variable regions. Certain systems have been established in the art for defining CDR limits (eg Kabat, Chothia, etc.); those skilled in the art appreciate the differences between and among these systems and are capable of understanding the limits of CDR to the level required to understand and practice the claimed invention.
[0067] Chemotherapeutic agent: The term "chemotherapeutic agent" as used herein has its art-understood meaning referring to one or more pro-apoptotic, cytostatic and/or cytotoxic agents, e.g. specifically including agents used and/or recommended for use in the treatment of one or more diseases, disorders, or conditions associated with undesirable cell proliferation. In some embodiments, chemotherapeutic agents are useful in treating cancer. In some embodiments, a chemotherapeutic agent can be or comprise one or more alkylating agents, one or more anthracyclines, one or more cytoskeletal disruptors (e.g., microtubule targeting agents such as taxanes, maytansine, and analogs thereof, of), one or more epothilones, one or more histone deacetylase inhibitors, HDACs), one or more topoisomerase inhibitors (e.g. topoisomerase I and/or topoisomerase II inhibitors), one or more kinase inhibitors, one or more nucleotide analogues or nucleotide precursor analogues, one or more peptide antibiotics, one or more platinum-based agents, one or more retinoids, one or more vinca alkaloids, and/or one or more analogues of one or more of the following (i.e. that is, they share relevant antiproliferative activity). In some particular embodiments, a chemotherapeutic agent may be or comprise one or more of Actinomycin, an all-trans retinoic acid, an Auristatin, Azacitidine, Azathioprine, Bleomycin, Bortezomib, Carboplatin, Capecitabine, Cisplatin, Chlorambucil, Cyclophosphamide, Curcumin, Cytarabine, Daunorubicin , Docetaxel, Doxifluridine, Doxorubicin, Epirubicin, Epothilone, Etoposide, Fluorouracil, Gemcitabine, Hydroxyurea, Idarubicin, Imatinib, Irinotecan, Maytansine and/or analogues thereof (e.g. DM1) Mechlorethamine, Mercaptopurine, Methotrexate, Mitoxantrone, a Maytansinoid, Oxaliplatin, Paclitaxel, Pemetrexed, Teniposide, Thioguanine, Topotecan, Valrubicin, Vinblastine, Vincristine, Vindesine, Vinorelbine, and combinations thereof. In some embodiments, a chemotherapeutic agent may be used in the context of an antibody-drug conjugate. In some embodiments, a chemotherapeutic agent is one found in an antibody-drug conjugate selected from the group consisting of: hLL1-doxorubicin, hRS7-SN-38, hMN-14-SN-38, hLL2-SN-38, hA20- SN-38, hPAM4-SN-38, hLL1-SN-38, hRS7-Pro-2-P-Dox, hMN-14-Pro-2-P-Dox, hLL2-Pro-2-P-Dox, hA20- Pro-2-P-Dox, hPAM4-Pro-2-P-Dox, hLL1-Pro-2-P-Dox, P4/D10-doxorubicin, gentuzumab ozogamycin, brentuximab vedotin, trastuzumab emtansine, inotuzumab ozogamicin, glembatumomab vedotin, SAR3419 , SAR566658, BIIB015, BT062, SGN-75, SGN-CD19A, AMG-172, AMG-595, BAY-94-9343, ASG-5ME, ASG-22ME, ASG-16M8F, MDX-1203, MLN-0264, anti -PSMA ADC, RG-7450, RG-7458, RG-7593, RG-7596, RG-7598, RG-7599, RG-7600, RG-7636, ABT-414, IMGN-853, IMGN-529, vorsetuzumab mafodotin , and lorvotuzumab mertansine.
[0068] Combination Therapy: As used herein, the term "combination therapy" refers to those situations where an individual is simultaneously exposed to one or more therapeutic regimens (eg, two or more therapeutic agents). In some embodiments, the two or more therapeutic regimens may be administered simultaneously. In some embodiments, the two or more therapeutic regimens may be administered sequentially (e.g., a first regimen administered prior to administration of any of the doses of a second regimen). In some embodiments, the two or more therapeutic regimens are administered in overlapping dosage regimens. In some embodiments, administration of combination therapy may involve administration of one or more therapeutic agents or modalities to an individual receiving the other agent(s) or modality.
[0069] Corresponding to: As used herein, the term "corresponding to" may be used to denote the position/identity of a structural element in a compound or composition by comparison with an appropriate reference compound or composition. For example, in some embodiments, a monomeric residue in a polymer (e.g., an amino acid residue in a polypeptide or a nucleic acid residue in a polynucleotide) may be identified as "corresponding to" a residue in an appropriate reference polymer. . For example, those skilled in the art will appreciate that, for purposes of simplicity, residues in a polypeptide are often designated using a canonical numbering system based on a related polypeptide reference, such that an amino acid "corresponding to" a residue at position 190, for example, need not actually be the 190th amino acid in a given amino acid chain, but rather correspond to the residue found at 190 in the reference polypeptide; those skilled in the art will readily appreciate how to identify the "corresponding" amino acids. For example, those skilled in the art will be aware of various sequence alignment strategies, including software programs such as, for example, BLAST, CS-BLAST, CUSASW++, DIAMOND, FASTA, GGSEARCH/GLSEARCH, Genoogle, HMMER, HHpred/HHsearch , IDF, Infernal, KLAST, USEARCH, parasail, PSI-BLAST, PSI-Search, ScalaBLAST, Sequilab, SAM, SSEARCH, SWAPHI, SWAPHI-LS, SWIMM, or SWIPE that can be used, for example, to identify residues " corresponding" in polypeptides and/or nucleic acids according to the present invention.
[0070] Engineered: In general, the term "engineered" refers to the appearance of having been manipulated by the hand of man. For example, a polypeptide is considered to be "engineered" when the polypeptide sequence is manipulated by human hand. For example, in some embodiments of the present invention, an engineered polypeptide comprises a sequence that includes one or more amino acid mutations, deletions and/or insertions that have been introduced by human hand into a reference polypeptide sequence. Comparably, a cell or organism is considered to be "engineered" if it has been manipulated so that its genetic information is altered (e.g., new genetic material not previously present has been introduced, e.g. by transformation, mating, somatic hybridization, transfection, transduction, or other mechanism, or genetic material previously present is altered or removed, for example, by substitution or deletion mutation, or by mating protocols). As is common practice and is understood by those in the art, derivatives and/or progeny of an engineered polypeptide or cell are still termed "engineered" even though the actual manipulation has been performed on a prior entity.
[0071] Epitope: as used herein, includes any portion that is specifically recognized by an immunoglobulin binding component (e.g., antibody or receptor). In some embodiments, an epitope is comprised of a plurality of atoms or chemical groups on an antigen. In some embodiments, such chemical groups or atoms are exposed to the surface when the antigen adopts a relevant three-dimensional conformation. In some embodiments, such chemical groups or atoms are physically close together in space when the antigen adopts such a conformation. In some embodiments, at least some such atoms are groups that are physically separated from one another when the antigen adopts an alternative conformation (eg, is linearized).
[0072] Ex vivo: as used herein refers to biological events that occur outside the context of a multicellular organism. For example, in the context of cell-based systems, the term can be used to refer to events that occur among a population of cells (eg, cell proliferation, cytokine secretion, etc.) in an artificial environment.
[0073] Framework or framework region: as used herein, refers to the variable region sequences minus the CDRs. Because a CDR sequence can be determined by different systems, a structural sequence is also subjected to different corresponding interpretations. The six CDRs divide the framework regions in the heavy and light chains into four sib-regions (FR1, FR2, FR3, and FR4) on each chain, where CDR1 is positioned between FR1 and FR2, CDR2 between FR2 and FR3, and CDR3 between FR3 and FR4. Without specifying the particular subregions such as FR1, FR2, FR3 or FR4, a framework region, as noted by others, represents the FRs combined with the variable region of a naturally occurring single chain immunoglobulin. As used herein, a FR represents one of the four FR1 subregions, for example, it represents the first framework region near the amino-terminal end of the variable region and 5' with respect to CDR1, and FRs represent two or more subregions constituting a structural region.
[0074] Humanized: As is known in the art, the term "humanized" is commonly used to refer to antibodies (or antibody components) whose amino acid sequence includes VH and VL region sequences of a reference antibody engineered in a human species (eg, a mouse), but also includes modifications to those sequences relative to the reference antibody intended to make them more "human-like", i.e., more similar to human germline variable sequences. In some embodiments, a "humanized" antibody (or antibody component) is one that immunospecifically binds to an antigen of interest and which has a framework region (FR) having the amino acid sequence substantially like that of a human antibody, and a complementary determining region (CDR) having the amino acid sequence substantially like that of a non-human antibody. A humanized antibody comprises substantially all of at least one, and typically two, variable domains (Fab, Fab', F(ab')2, FabC, Fv) wherein all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin. (i.e., donor immunoglobulin) and all or substantially all of the framework regions are those of a human immunoglobulin consensus sequence. In some embodiments, a humanized antibody also comprises at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin constant region. In some embodiments, the humanized antibody contains both the light chain as well as at least the variable domain of a heavy chain. The antibody may also include a CH1, hinge, CH2, CH3, and, optionally, a CH4 region of a heavy chain constant region.
[0075] In vitro: The term "in vitro" as used herein refers to events that occur in an artificial environment, for example, in a test tube or reaction vessel, in a cell culture, etc., rather than within a multicellular organism.
[0076] In vivo: as used herein refers to events that occur within a multicellular organism, such as a human and a non-human animal. In the context of cell-based systems, the term can be used to refer to events that occur within a living cell (as opposed to, for example, in vitro systems).
[0077] Isolated: as used herein, refers to a substance and/or entity that has been (1) separated from at least some of the components with which it was associated when initially produced (either in nature and/or in an experimental setting) , and/or (2) designed, produced, prepared, and/or manufactured by the hand of a man. Isolated substances and/or entities can be separated by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% of the other components with which they were initially associated. In some embodiments, single agents are about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96 %, about 97%, about 98%, about 99%, or more than about 99% pure. As used herein, a substance is "pure" if it is substantially free of other components. In some embodiments, as will be understood by those skilled in the art, a substance may still be considered "isolated" or even "pure", after it has been combined with other components, such as, for example, one or more carriers or excipients (e.g. e.g. buffer, solvent, water, etc.); in such embodiments, the isolation percentage or purity of the substance is calculated without including such carriers or excipients. To provide, however, an example, in some embodiments, a biological polymer such as a naturally occurring polypeptide or polynucleotide is considered to be "isolated" when, a) by virtue of its origin or source of derivation it is not associated with some or all the components that accompany it in its native state in nature; b) it is substantially free of other polypeptides or nucleic acids of the same species as the species that produces it in nature; c) is expressed by or otherwise in association with components of a cell or other expression system that is not of the species that produces it in nature. Thus, for example, in some embodiments, a polypeptide that is chemically synthesized or synthesized in a cellular system other than the one that produces it in nature is considered an "isolated" polypeptide. Alternatively or additionally, in some embodiments, a polypeptide that has been subjected to one or more purification techniques may be considered an "isolated" polypeptide insofar as it has been separated from other components a) with which it is associated in nature; and/or b) with which it was associated when initially produced.
[0078] KD: as used herein, refers to the dissociation constant of a binding agent (e.g., a binding antibody or binding component thereof) from a complex with its partner (e.g., the epitope to which the antibody or binding component thereof binds).
[0079] Operatively linked: as used here, refers to a juxtaposition, in which the described components are in a relationship that allows them to function in their intended manner. A control element "operably linked" to a functional element is associated in such a way that the expression and/or activity of the functional element is obtained under conditions compatible with the control element. In some embodiments, "operably linked" control elements are contiguous (eg, covalently linked) with the encoding elements of interest; in some embodiments, control elements act in trans or otherwise from the functional element of interest.
[0080] Pharmaceutical Composition: As used herein, the term "pharmaceutical composition" refers to a composition wherein an active agent is formulated together with one or more pharmaceutically acceptable carriers. In some embodiments, the composition is suitable for administration to a human or animal subject. In some embodiments, the active agent is present in a unit dose amount appropriate for administration in a therapeutic regimen that shows a statistically significant probability of achieving a predetermined therapeutic effect when administered to a relevant population.
[0081] Polypeptide: The term "polypeptide" as used herein generally has its art-recognized meaning of a polymer of at least three amino acids. Those skilled in the art will appreciate that the term "polypeptide" is intended to be sufficiently general to encompass not only polypeptides having a complete sequence recited herein, but also encompass polypeptides that represent functional fragments (i.e., fragments retaining at least one activity) of such polypeptides. complete. Furthermore, those skilled in the art understand that protein sequences generally tolerate some substitution without destroying activity. Thus, any polypeptide that maintains activity and shares at least about 30-40% overall sequence identity, often greater than about 50%, 60%, 70%, or 80%, and also generally including at least a much larger region of identity, often greater than 90% or even 95%, 96%, 97%, 98%, or 99% in one or more highly conserved regions, usually spanning at least 3-4 and often up to 20 or more amino acids, with another polypeptide of the same class, is encompassed within the relevant term "polypeptide" as used herein. Polypeptides can contain L-amino acids, D-amino acids, or both and can contain any of a variety of amino acid modifications or analogs known in the art. Useful modifications include, for example, terminal acetylation, amidation, methylation, etc. In some embodiments, proteins may comprise natural amino acids, unnatural amino acids, synthetic amino acids, and combinations thereof. The term "peptide" is generally used to refer to a polypeptide having a length of less than about 100 amino acids, less than about 50 amino acids, less than 20 amino acids, or less than 10 amino acids. In some embodiments, proteins are antibodies, antibody fragments, biologically active portions thereof, and/or characteristic portions thereof.
[0082] To prevent or prevent: as used herein when used in connection with the occurrence of a disease, disorder, and/or condition, refers to reducing the risk of developing the disease, disorder and/or condition and/or delaying the onset and/or severity of one or more features or symptoms of the disease, disorder or condition. In some embodiments, prevention is assessed on a population basis so that an agent is considered to "prevent" a particular disease, disorder, or condition if a statistically significant decrease in the development, frequency, and/or intensity of one or more symptoms of the disease , disorder condition is observed in a population susceptible to the disease, disorder or condition.
[0083] Recombinant: As used herein, it is intended to refer to polypeptides that are designated, engineered, prepared, expressed, engineered, manufactured, and/or isolated by recombinant methods, such as polypeptides expressed using a recombinant expression vector transfected into a host cell; polypeptides isolated from a recombinant human polypeptide combinatorial library; polypeptides isolated from an animal (e.g., a mouse, rabbit, sheep, fish, etc.) that is transgenic for or has otherwise been manipulated to express a gene or genes, or gene components that encode and/or direct expression of the polypeptide or one or more components, portions, elements, or domains thereof; and/or polypeptides prepared, expressed, engineered, or isolated by any other means that involves joining or linking selected nucleic acid sequence elements together, chemically synthesizing the selected sequence elements, and/or otherwise generating a nucleic acid that encodes and/or directs the expression of the polypeptide or one or more components, portions, elements, or domains thereof. In some embodiments, one or more such selected sequence elements are found in nature. In some embodiments, one or more of such selected sequence elements are designated in silico. In some embodiments, one or more such selected sequence elements result from mutagenesis (e.g., in vivo or in vitro) of a known sequence element, e.g., from a natural or synthetic source such as, e.g., in the germ line. from a source organism of interest (e.g. a human, a mouse, etc).
[0084] Specific Binding: As used herein, the term "specific binding" refers to any ability to discriminate between possible binding partners in the environment in which binding is to occur. A binding agent that interacts with a particular target when other potential targets are present is said to "bind specifically to the target with which it interacts. In some embodiments, specific binding is assessed by detecting or determining the degree of association between the agent binding agent and its partner; in some embodiments, specific binding is assessed by detecting or determining the degree of dissociation of a binding partner-binding agent complex; in some embodiments, specific binding is assessed by detecting or determining the binding agent's ability to compete by an alternative interaction between its partner and another entity In some embodiments, specific binding is assessed by performing such detections or determinations across a range of concentrations.
[0085] Subject: As used herein, the term "subject" refers to an organism, typically a mammal (e.g., a human, in some embodiments including prenatal human forms). In some embodiments, an individual is suffering from a relevant disease, disorder or condition. In some embodiments, an individual is susceptible to a disease, disorder, or condition. In some embodiments, an individual exhibits one or more symptoms or features of a disease, disorder, or condition. In some embodiments, an individual does not exhibit any symptoms or features of a disease, disorder, or condition. In some embodiments, an individual is someone with one or more characteristic features of risk susceptibility to a disease, disorder, or condition. In some embodiments, an individual is a patient. In some embodiments, an individual is an individual to whom the diagnosis and/or therapy is and/or has been administered.
[0086] Therapeutic agent: As used herein, the phrase "therapeutic agent" in general refers to any agent that evokes a desired pharmacological effect when administered to an organism. In some embodiments, an agent is considered to be a therapeutic agent if it demonstrates a statistically significant effect across an appropriate population. In some embodiments, the appropriate population may be a population of model organisms. In some modalities, an appropriate population may be defined by various criteria, such as a certain age group, gender, genetic background, pre-existing clinical conditions, etc. In some embodiments, a therapeutic agent is a substance that can be used to alleviate, ameliorate, mitigate, inhibit, prevent, delay the onset of, reduce the severity of, and/or reduce the incidence of one or more symptoms or aspects of a disease. , disorder, and/or condition. In some embodiments, a "therapeutic agent" is an agent that has been or is required to be approved by a government agency before it can be marketed for administration to humans. In some embodiments, a "therapeutic agent" is an agent for which a prescription is required for administration to humans.
[0087] Therapeutically Effective Amount: As used herein, the term "therapeutically effective amount" means an amount that is sufficient when administered to a population suffering from or susceptible to a disease, disorder, and/or condition in accordance with a dosage regimen therapy, to treat the disease, disorder, and/or condition. In some embodiments, a therapeutically effective amount is one that reduces the incidence and/or severity of, stabilizes one or more features of, and/or delays the onset of one or more symptoms of the disorder, and/or condition. Those skilled in the art will appreciate that the term "therapeutically effective amount" does not actually require successful treatment in a given individual. Preferably, a therapeutically effective amount can be that amount which provides a particular desired pharmacological response in a significant number of individuals when administered to patients in need of such treatment. For example, in some embodiments, the term "therapeutically effective amount" refers to an amount that, when administered to an individual in need thereof in the context of inventive therapy, will block, stabilize, attenuate, or reverse a supportive process. to cancer occurring in said individual, or enhance or enhance a cancer suppressive process in said individual. In the context of cancer treatment, a "therapeutically effective amount" is an amount that, when administered to an individual diagnosed with cancer, will prevent, stabilize, inhibit, or reduce the further development of cancer in the individual. A particularly preferred "therapeutically effective amount" of a composition described herein reverses (in a therapeutic treatment) the treatment of a malignancy such as a pancreatic carcinoma or assists in achieving or prolonging remission of a malignancy. A therapeutically effective amount administered to a subject to treat cancer in that subject can be the same as or different from a therapeutically effective amount administered to promote remission or inhibit metastasis. As with most cancer therapies, the therapeutic methods described here should not be interpreted as, restricted to, or otherwise limited to a "cure" for cancer; preferably, the methods of treatment are directed to using the described compositions to "treat" a cancer, i.e., to effect a beneficial or desirable change in the health of an individual having cancer. Such benefits are recognized by qualified healthcare professionals in the field of oncology and include, but are not limited to, a stabilization of the patient's condition, a decrease in tumor size (tumor regression), an improvement in vital functions (eg, improved function of cancerous tissues or organs, decreased or inhibited new metastases, decreased opportunistic infections, increased survivability, decreased pain, improved motor function, improved cognitive function, improved feeling of energy (vitality, decreased malaise) improved sense of well-being, restoration of normal appetite, restoration of healthy weight gain, and combinations thereof. Also, regression of a particular tumor in an individual (e.g., as a result of treatments described here) can also be evaluated by taking samples of cancer cells from the site of a tumor, such as a pancreatic adenocarcinoma (for example, during treatment ) and testing cancer cells for the level of metabolic and signaling markers to monitor the state of cancer cells to verify, at a molecular level, the regression of cancer cells to a less malignant phenotype. For example, tumor regression induced by employing the methods of this invention would be indicated by finding a decrease in any of the pro-angiogenic markers described above, an increase in the anti-angiogenic markers described herein, normalization (i.e., change toward a state found in normal individuals who do not suffer from cancer) of metabolic pathways, intercellular signaling pathways, or intracellular signaling pathways that exhibit abnormal activity in individuals with diagnosed cancer. Those skilled in the art will appreciate that, in some embodiments, a therapeutically effective amount may be formulated and/or administered in a single dose. In some embodiments, a therapeutically effective amount may be formulated and/or administered in a plurality of doses, for example, as part of a dosing regimen.
[0088] Variant: As used here in the context of molecules, e.g., nucleic acids, proteins, or small molecules, the term "variant" refers to a molecule that shows significant structural identity with a reference molecule, but differs structurally from the reference molecule, for example, in the presence or absence at the level of one or more chemical moieties in relation to the reference entity. In some embodiments, a variant also functionally differs from its reference molecule. In general, whether a given molecule is properly considered a "variant" of a reference molecule is based on its degree of structural identity with the reference molecule. As will be appreciated by those skilled in the art, any biological or chemical reference molecule has certain characteristic structural elements. A variant, by definition, is a distinct molecule that shares one or more of such characteristic structural elements, but differs in at least one respect from the reference molecule. To provide just a few examples, a polypeptide may have a characteristic sequence element comprised of a plurality of amino acids having designated positions from one another in linear or three-dimensional space and/or contributing to a particular structural motif and/or biological function; a nucleic acid may have a characteristic sequence element comprised of a plurality of nucleotide residues having designated positions relative to one another in linear or three-dimensional space. In some embodiments, a variant polypeptide or nucleic acid differs from a reference polypeptide or nucleic acid as a result of one or more differences in nucleic acid or amino acid sequence. In some embodiments, a variant polypeptide or nucleic acid shows a full sequence identity to a reference polypeptide or nucleic acid that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92 %, 93%, 94%, 95%, 96%, 97%, or 99%. In some embodiments, a variant polypeptide or nucleic acid does not share at least one characteristic sequence element with a reference polypeptide or nucleic acid. In some embodiments, a reference polypeptide or nucleic acid has one or more biological activities. In some embodiments, a variant polypeptide or nucleic acid shares one or more of the biological activities of the reference polypeptide or nucleic acid.
[0089] Vector: as used herein, refers to a nucleic acid molecule capable of carrying another nucleic acid to which it has been linked. One type of vector is a "plasmid", which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, in which additional segments of DNA can be linked to the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (eg, bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (eg, non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thus are replicated along with the host genome. Furthermore, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "expression vectors". Standard techniques can be used for recombinant DNA, oligonucleotide synthesis, and tissue culture transformation (eg, electroporation, lipofection). Enzymatic reactions and purification techniques may be performed according to the manufacturer's specifications or as commonly performed in the art or as described herein. The foregoing techniques and procedures may generally be performed according to conventional methods well known in the art, as described in various general and more specific references that are cited and described throughout the present specification. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)), which is incorporated herein by reference for any purpose. DETAILED DESCRIPTION OF EXEMPLARY MODALITIES
[0090] The present invention relates, inter alia, to 4-1BB, which is an inducible costimulatory molecule, and therapeutic antibodies that bind thereto that has been modified to have improved characteristics over a reference anti-4-1BB antibody. For example, the modified antibodies provided herein have been modified to enhance antigen affinity over that of a reference agonist antibody that specifically recognizes an epitope within the extracellular domain of human 4-1BB (Korean Patent No. 100500286, Accession No. : KCTC 0952BP). Specifically, as described herein, the inventors designed a reference humanized anti-human 41BB antibody, 94G1 (U.S. Patent No. 7,932,045). As examples described herein, the light chain and heavy chain CDR sequences of a reference 94G1 antibody were separately engineered to remove the affinity of each chain. Furthermore, as described herein, exemplary engineered anti-4-1BB antibodies can effectively induce the proliferation of activated T cells. Notably, the exemplary engineered anti-4-1BB antibodies are capable of inducing surprisingly improved activities of CD8+ T cells due to the stimulation caused by the binding of the humanized antibody 4-1BB to a 4-1BB molecule and inhibition of activation-induced cell death ( IACD). Thus, the present invention provides engineered human anti-4-1BB antibodies with improved properties over a reference antibody, and further demonstrates that these antibodies have surprisingly beneficial activity in vitro and in vivo.4-1BB
[0091] 4-1BB (also referred to as CD137, TNFRSF9, etc.) is a receptor belonging to the tumor necrosis factor receptor (TNFR) superfamily. 4-1BB is a costimulatory molecule generally expressed on activated T lymphocytes and involved in immunity and autoimmune diseases (Kwon et al. PNAS 84:2896,1987; Kwon et al. PNAS (1989) 86:1963; Son et al. Journal of Immunological Methods (2004) 286(1-2):187-201, each of which is incorporated herein by reference in its entirety). Human 4-1BB is a 255 amino acid protein (Accession No. NM_001561; NP_001552). The complete human amino acid sequence of 4-1BB is provided in SEQ ID NO: 44. 4-1BB is expressed on the cell surface in monomer (30 kDa) and dimer (55 kDa) forms and likely trimerizes with 4-1BB linker the signal.
[0092] Current understanding of 4-1BB suggests that it is constitutively expressed in several cells, albeit at low levels, including Foxp3+ Tregs and dendritic cells (DC). (See, Vinay and Kwon (2014) BMB Rep. 47(3): 122-129, which is incorporated by reference herein). Activation with various agonists, such as cytokines (eg, IL-2, IL-4), polyclonal activators (eg, Con A and PHA), cell surface molecules (eg, anti-CD3 and anti-CD28) and promoters of Ca2+ induction and PKC activity (eg, ionomycin myristate acetate and photobol) also enhance 4-1BB expression. ID
[0093] Several studies of murine and human T cells indicate that 4-1BB promotes enhanced cell proliferation, survival, and cytokine production (Croft, 2009, Nat. Rev. Immunol. 9:271-285). Studies have indicated that some 4-1BB agonist monoclonal antibodies can increase costimulatory molecule expression and markedly enhance cytolytic T lymphocyte responses, resulting in antitumor efficacy in various models. 4-1BB agonist monoclonal antibodies have demonstrated efficacy in both prophylactic and therapeutic settings. In addition, 4-1BB monotherapy and combination therapy tumor models have established durable antitumor protective T cell memory responses (Lynch (2008) Immunol. Rev. 22: 277-286). 4-1BB agonists have also been shown to inhibit autoimmune reactions in several art-recognized models of autoimmunity ( Vinay (2006) J. Mol. Med. 84:726-736 ). This dual activity of 4-1BB provides the potential to provide antitumor activity while buffering autoimmune side effects that may be associated with immunotherapy methods. 4-1BB antibodies and fragments thereof
[0094] The present invention provides, at least in part, engineered human anti-4-1BB antibodies and fragments thereof that exhibit markedly, and unexpectedly, superior characteristics in vitro and/or in vivo. For example, certain antibodies provided have increased affinity over a reference humanized anti-human 41BB antibody.
[0095] In some embodiments, an anti-4-1BB antibody or antigen-binding antibody fragment includes 1, 2, or 3 heavy chain CDR sequences that are or include a sequence of SEQ ID NOs: 5 to 8. In some embodiments, an anti-4-1BB antibody or antigen-binding antibody fragment includes one or more of: a heavy chain CDR1 that is or includes a sequence of SEQ ID NO: 5, a heavy chain CDR2 that is or includes a sequence of SEQ ID NO: 6 and a heavy chain CDR3 that is or includes a sequence of SEQ ID NO: 7 or 8. In some embodiments, an anti-4-1BB antibody or antigen-binding antibody fragment includes each one of: a heavy chain CDR1 that is or includes a sequence of SEQ ID NO: 5, a heavy chain CDR2 that is or includes a sequence of SEQ ID NO: 6, and a heavy chain CDR3 that is or includes a sequence of SEQ ID NO: 7 or 8.
[0096] In some embodiments, an anti-4-1BB antibody or antigen-binding antibody fragment includes 1, 2, or 3 light chain CDR sequences that are or include a sequence of SEQ ID NOs: 1-4. In some embodiments, an anti-4-1BB antibody or antigen-binding antibody fragment includes one or more of: a light chain CDR1 that is or includes a sequence of SEQ ID NO: 1, a light chain CDR2 that is or includes a sequence of SEQ ID NO: 2 and a light chain CDR1 that is or includes a sequence of SEQ ID NO: 3 or 4. In some embodiments, an anti-4-1BB antibody or antigen-binding antibody fragment includes each of: a light chain CDR1 that is or includes a sequence of SEQ ID NO: 1, a light chain CDR2 that is or includes a sequence of SEQ ID NO: 2, and a light chain CDR1 that is or includes a sequence of SEQ ID NO: 3 or 4.
[0097] In some embodiments, an anti-4-1BB antibody or antigen-binding antibody fragment includes a heavy chain variable domain that includes a heavy chain CDR1 that is or includes a sequence of SEQ ID NO: 5, a CDR2 chain that is or includes a sequence of SEQ ID NO: 6 and a heavy chain CDR3 that is or includes a sequence of SEQ ID NO: 7 or 8 and/or a light chain variable domain that includes a chain CDR1 light chain that is or includes a sequence of SEQ ID NO: 1, a light chain CDR2 that is or includes a sequence of SEQ ID NO: 2 and a light chain CDR1 that is or includes a sequence of SEQ ID NO: 4.
[0098] In some embodiments, an anti-4-1BB antibody or antigen-binding antibody fragment includes a heavy chain variable domain that includes a heavy chain CDR2 that is or includes a sequence of SEQ ID NO: 6 wherein the The 5th amino acid, asparagine (N), was substituted with glutamine (Q), glutamic acid (E) or serine (S). In some embodiments, an anti-4-1BB antibody or antigen-binding antibody fragment includes a heavy chain variable domain that includes a heavy chain CDR2 that is or includes a sequence of SEQ ID NO: 6 wherein the 5th amino acid, asparagine (N), has been replaced with valine (V), glycine (G), or proline (P).
[0099] In some embodiments, an anti-4-1BB antibody or antigen-binding antibody fragment includes a light chain variable domain that includes a light chain CDR1 that is or includes a sequence of SEQ ID NO: 3 or 4 in that the 6th amino acid position of LCDR3 is changed.
[00100] In some embodiments, an anti-4-1BB antibody or antigen-binding antibody fragment includes a heavy chain variable domain that includes a light chain framework region 1 (FR1) comprising a sequence of SEQ ID NO: 16 or 17. In some embodiments, an anti-4-1BB antibody or antigen-binding antibody fragment includes a heavy chain variable domain that includes a heavy chain framework 3 (FR3) region comprising a sequence of any of SEQ ID's NOs: 1820. In some embodiments, an anti-4-1BB antibody or antigen-binding antibody fragment includes a heavy chain variable domain that includes a light chain framework region 1 (FR1) comprising a sequence of SEQ ID NO: 16 or 17 and a heavy chain framework region 3 (FR3) comprising a sequence of any one of SEQ ID NOs: 18-20.
[00101] In some embodiments, an anti-4-1BB antibody or antigen-binding antibody fragment includes substantial homology to an antibody or antibody fragment that includes a heavy chain variable domain that is or includes a sequence selected from SEQ ID NOS: 11-14. In some embodiments, an anti-4-1BB antibody or antigen-binding antibody fragment includes a heavy chain variable domain that is or includes a sequence at least 90%, 91%, 92%, 93%, 94%, 95 %, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4% or 99.5% identical to a selected sequence of SEQ ID NOs: 11- 14. In some embodiments, an anti-4-1BB antibody or antigen-binding antibody fragment includes a heavy chain variable domain that is or includes a sequence selected from SEQ ID NOs: 11-14.
[00102] In some embodiments, an anti-4-1BB antibody or antigen-binding antibody fragment includes substantial homology to an antibody or antibody fragment that includes a light chain variable domain that has or includes a sequence of SEQ ID NO : 9 or 10. In some embodiments, an anti-4-1BB antibody or antigen-binding antibody fragment includes a light chain variable domain that is or includes a sequence at least 90%, 91%, 92%, 93% , 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4% or 99.5% identical to a SEQ ID sequence NO: 9 or 10. In some embodiments, an anti-4-1BB antibody or antigen-binding antibody fragment includes a light chain variable domain that is or includes a sequence of SEQ ID NO: 9 or 10.
[00103] In some embodiments, an anti-4-1BB antibody or antigen-binding antibody fragment includes substantial homology to an antibody or antibody fragment that includes a heavy chain variable domain that is or includes a sequence selected from SEQ ID NOs: 11-14 and a light chain variable domain that is or includes a sequence of SEQ ID NO: 10. In some embodiments, an anti-4-1BB antibody or antigen-binding antibody fragment includes a chain variable domain heavy that is or includes a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99 .3%, 99.4% or 99.5% identical to a selected sequence of SEQ ID NOs: 11-14 and a light chain variable domain that is or includes a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4% or 99.5% identical to a sequence of SEQ ID NO: 10. In some embodiments, an anti-4-1BB antibody or antigen-binding antibody fragment includes a heavy chain variable domain that is or includes a sequence selected from SEQ ID NOs: 1114 and a light chain variable domain that is or includes a sequence of SEQ ID NOs: 10.
[00104] Anti-human 4-1BB binding antigen binding fragment or antibody amino acid sequences of the present invention may be substituted through conservative substitution. The term "conservative substitution" used herein refers to the modification of a polypeptide wherein one or more amino acids are replaced with an amino acid having a similar biochemical property so as not to cause the corresponding polypeptide to lose a biological or biochemical function. The term "conservative sequence variant" or "conservative amino acid substitution" used herein is the substitution of an amino acid residue with an amino acid residue having a similar side chain. Amino acid residues having a similar side chain are defined in the art. Those residues encompass amino acids with a basic side chain (e.g., lysine, arginine, and histidine), amino acids with an acidic side chain (e.g., aspartic acid and glutamate), amino acids with an uncharged polar side chain (e.g., glycine , asparagine, glutamine, serine, threonine, tyrosine, and cysteine), amino acids with a nonpolar side chain (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan), amino acids with a beta side chain -branched (eg, threonine, valine, and isoleucine) and amino acids with an aromatic side chain (eg, tyrosine, phenylalanine, tryptophan, and histidine). Consequently, it is expected that the antibody of the present invention may have conservative amino acid substitution, and still ensure activity.
[00105] In some embodiments, a human anti-4-1BB antibody or antigen-binding fragment of the present invention may include a constant region selected from an IgG1 constant domain, an IgG2 constant domain, an IgG1 hybrid constant domain /IgG2, a human IgG4 constant domain, an IgA constant domain, an IgE constant domain, an IgM constant domain, and an IgD constant domain.
[00106] In some embodiments, an anti-human 4-1BB antibody or antigen-binding fragment of the present invention is or includes an IgA, IgD, IgE, IgM, IgG, or variants thereof.
[00107] In some embodiments, an anti-human 4-1BB antibody of the present invention includes a variant Fc region that has mutations and/or amino acid substitutions at one or more positions at 234, 235, 236, 237, 238, 239 , 253, 254, 265, 266, 267, 268, 269, 270, 288, 297, 298, 299, 307, 311, 322, 327, 328, 329, 330, 331, 332, 434 and 435.
[00108] In some embodiments, an anti-human 4-1BB antibody or antigen-binding fragment of the present invention is isotope of human IgG1. In some embodiments, a human anti-4-1BB antibody or antigen-binding fragment of the present invention includes a variant IgG1. In some embodiments, a human anti-4-1BB antibody or antigen-binding fragment includes an IgG1 polypeptide that has an amino acid mutation at one or more positions at 233, 234, 235, 236, 265, 297, 329, 331 and 322.
[00109] In some embodiments, a human anti-4-1BB antibody or antigen-binding fragment includes an IgG1 polypeptide containing one or more mutations in L234, L235, D270, N297, E318, K320, K322, P331, and P329 . In some embodiments, a human anti-4-1BB antibody or antigen-binding fragment includes an IgG1 polypeptide containing two, three, four, or more mutations in L234, L235, D270, N297, E318, K320, K322, P331, and P329. In some embodiments, a human anti-4-1BB antibody or antigen-binding fragment includes an IgG1 polypeptide with mutations in L234A and L235A.
[00110] In some embodiments, a human anti-4-1BB antibody or antigen-binding fragment includes a light chain constant region. In some embodiments, a human anti-4-1BB antibody or antigen-binding fragment includes a kappa (K) and/or lambda (α) chain and/or a variant thereof.
[00111] In some embodiments, a human anti-4-1BB antibody or antigen-binding fragment is a monoclonal antibody. In some embodiments, a human anti-4-1BB antibody or antigen-binding fragment is a Fab fragment, a Fab' fragment, an F(ab')2 fragment, an Fv fragment, an Fv fragment disulfide-linked, an scFv fragment, a single domain antibody, humabody, nanobody, and/or a diabody. In some embodiments, a human anti-4-1BB antibody or antigen-binding fragment is a monovalent antibody. In some embodiments, a human anti-4-1BB antibody or antigen-binding fragment is a multivalent antibody. In some embodiments, a human anti-4-1BB antibody or antigen-binding fragment is a multispecific antibody (eg, a bispecific antibody).
[00112] In some embodiments, the present invention encompasses methods of modifying the carbohydrate content of an antibody of the disclosure by adding or deleting a glycosylation site. Methods for modifying the carbohydrate content of antibodies are well known in the art and embraced by the description, see, for example, United States Patent No. 6,218,149; EP 0 359 096 B1; United States Publication No. US 2002/0028486 ; WO 03/035835; United States Publication No. 2003/0115614; United States Patent No. 6,218,149; United States Patent No. 6,472,511; all of which are incorporated herein by reference in their entirety. In other embodiments, the present invention encompasses methods of modifying the carbohydrate content of an antibody of the present invention by excluding one or more endogenous carbohydrate portions of the antibody. In a specific embodiment, the present invention encompasses deleting the glycosylation site from the Fc region of an antibody by changing position 297 from asparagine to alanine. In some embodiments, a human anti-4-1BB antibody or antigen-binding fragment comprises a mutation of N297A in the CH2 domain. In some embodiments, the N297A mutation results in glycosylation, which reduces FcR or C1q binding. In some embodiments, an anti-human 4-1BB antibody or antigen-binding fragment comprises a heavy chain comprising an Fc region comprising an N297A mutation and a K322A mutation. In some embodiments, a human anti-4-1BB antibody or antigen-binding fragment comprises a heavy chain comprising an Fc region comprising an N297A mutation and a D265A mutation. In some embodiments, a human anti-4-1BB antibody or antigen-binding fragment comprises a heavy chain comprising an Fc region comprising an N297A mutation, a D265A mutation, and a K322A mutation. In some embodiments, a human anti-4-1BB antibody or antigen-binding fragment comprises an Fc region with an L234A mutation and/or an L235A mutation. In some embodiments, a human anti-4-1BB antibody or antigen-binding fragment comprises an Fc region with one or more mutations selected from L234A, L235A, N297A, D265A, and K322A. In some embodiments, a human anti-4-1BB antibody or antigen-binding fragment comprises the Fc region with two or more mutations selected from L234A, L235A, N297A, D265A, and K322A. In some embodiments, a human anti-4-1BB antibody or antigen-binding fragment comprises the Fc region with three, four, or five selected mutations from L234A, L235A, N297A, D265A, and K322A.
[00113] Engineered glycoforms may be useful for a variety of purposes, including, but not limited to, enhancing or reducing effector function. Engineered glycoforms can be generated by any method known to one skilled in the art, e.g. using engineered expression lines or variants, by co-expression with one or more enzymes, e.g. DI N-acetylglucosaminyltransferase III (GnTI11), expressing a molecule comprising an Fc region is made in various organisms or cell lines of various organisms, or by modifying carbohydrate(s) after the molecule comprising the Fc region has been expressed. Methods for generating engineered glycoforms are known in the art, and include, but are not limited to, those described in Umana et al, 1999, Nat. Biotechnol 17:176-180; Davies et al., 20017 Biotechnol Bioeng 74:288-294 ; Shields et al, 2002, J Biol Chem 277:26733-26740; Shinkawa et al., 2003, J Biol Chem 278:34663473) US Patent No. 6,602,684; United States Serial Number 10/277,370; United States Serial Number 10/113,929; PCT WO 00/61739A1; PCT WO 01/292246A1; PCT WO 02/311140A1; PCT WO 02/30954A1; POTILLEGENT™ technology (Biowa, Inc. Princeton, N.J.); GLYCOMAB™glycosylation engineering technology (GLYCART biotechnology AG, Zurich, Switzerland); each of which is incorporated herein by reference in their entirety. See, for example, WO 00061739; EA01229125; US 20030115614 ; Okazaki et al., 2004, JMB, 336: 1239-49 each of which is incorporated herein by reference in their entirety.
[00114] In some embodiments, an anti-human 4-1BB antibody or antigen-binding fragment of the present invention is as an agonist for human 4-1BB.
[00115] In some embodiments, an anti-human 4-1BB antibody or antigen-binding fragment of the present invention binds to a human 4-1BB molecule. In some embodiments, an anti-human 4-1BB antibody or antigen-binding fragment of the present invention specifically binds to a human 4-1BB molecule.
[00116] In some embodiments, a human anti-4-1BB antibody or antigen-binding fragment binds to a binding that is or includes that of SEQ ID NO: 15. In some embodiments, an anti-4- Human 1BB or antigen binding fragment binds to an epitope extracellular domain of 4-1BB that is or includes a sequence of SEQ ID NO: 15.
[00117] In some embodiments, binding of an anti-human 4-1BB antibody or antigen-binding fragment of the present invention with the extracellular domain of human 4-1BB is abrogated by one or more mutations of selected SEQ ID NO: 44 of N30, D38, N39, R41, A56, G57, R60 or T61.
[00118] In some embodiments, an anti-human 4-1BB antibody or antigen-binding fragment of the present invention binds to a human 4-1BB molecule with a binding affinity (KD) of 1X10-7 to 1x10-12 M. In some embodiments, an anti-human 4-1BB antibody or antigen-binding fragment of the present invention binds to a human 4-1BB molecule with a binding affinity (KD) of 1X10-8 to 1x10- 12 M. Binding affinity (KD) can be measured, for example, by surface plasmon resonance, for example using a BIACORE system.
[00119] In some embodiments, an anti-human 4-1BB antibody or antigen-binding fragment of the present invention binds to a human 4-1BB molecule or a fragment thereof at a binding affinity (KD) of less than 1.0x10 -8 M. In some embodiments, an anti-humanized 4-1BB antibody or antigen-binding fragment of the present invention binds to a human 4-1BB molecule or a fragment thereof in a binding affinity (KD) less than 1.0x10 -9 M. In some embodiments, an anti-humanized 4-1BB antibody or antigen-binding fragment of the present invention binds to a human 4-1BB molecule or a fragment of the present invention. same at a binding affinity (KD) of less than 1.0x10-10 M.
[00120] In some embodiments, an anti-4-1BB antibody or antigen-binding fragment of the present invention does not bind or weakly binds to a non-primate 4-1BB polypeptide (e.g., a 4-1BB polypeptide from canine, mouse and rat). In some embodiments, an anti-4-1BB antibody or antigen-binding fragment of the present invention effectively binds to human or monkey 4-1BB. This binding affinity suggests that the structure and/or epitope sequence for a primate 4-1BB antibody may be quite different from canine, mouse and rat.
[00121] In some embodiments, an anti-human 4-1BB antibody or antigen-binding fragment of the present invention is an agonistic antibody. In some embodiments, an anti-human 4-1BB antibody or antigen-binding fragment of the present invention mediates T cell activation. In some embodiments, an anti-human 4-1BB antibody or antigen-binding fragment of the present invention invention binds to CD8+ and/or CD4+ T cells that express human 4-1BB.
[00122] In some embodiments, a human anti-4-1BB antibody or antigen-binding fragment of the present invention has no or low ADCC activity. In some embodiments, a human anti-4-1BB antibody or antigen-binding fragment of the present invention has little or no CDC activity. In some embodiments, a human anti-4-1BB antibody or antigen-binding fragment of the present invention has no or low ADCC activity and CDC activity. In some embodiments, a human anti-4-1BB antibody or antigen-binding fragment of the present invention has an ADCC cell killing activity of less than about 20%, less than about 10%, less than about 8%, or less than about 5%. In some embodiments, a human anti-4-1BB antibody or antigen-binding fragment of the present invention has an ADCC cell killing activity of less than about 10%. In some embodiments, a human anti-4-1BB antibody or antigen-binding fragment of the present invention has a CDC cell killing activity of less than about 30%, less than about 20%, less than about 10%, less than about 8%, or less than about 5%. In some embodiments, a human anti-4-1BB antibody or antigen-binding fragment of the present invention has a CDC cell killing activity of less than about 20%.
[00123] In some embodiments, an anti-human 4-1BB antibody or antigen-binding fragment of the present invention is characterized by low toxicity (eg, a low degree of post-administration cell death). In some embodiments, a human anti-4-1BB antibody or antigen-binding fragment of the present invention is characterized by low hepatotoxicity. In some embodiments, a subject administered an anti-human 4-1BB antibody or antigen-binding fragment of the present invention at a therapeutic dose has levels of one or more of ALT, AST, and total bilirubin in a normal range. In some embodiments, an anti-human 4-1BB antibody or antigen-binding fragment of the present invention is characterized by an ability to treat patients for prolonged periods with measurable relief of symptoms and low and/or acceptable toxicity. Low or acceptable immunogenicity and/or high affinity, as well as other suitable properties, may contribute to the therapeutic results obtained. "Low immunogenicity" is defined herein as elevation of significant HAHA, HACA or HAMA responses of less than about 75%, or preferably less than about 50% of treated patients and/or elevation of low titers in the treated patient (Elliott et al., Lancet 344:1125-1127 (1994), incorporated herein by reference in its entirety). Nucleic acids
[00124] The description provides polynucleotides comprising a nucleotide sequence encoding human anti-4-1BB antibodies of the present invention and fragments thereof. Human anti-4-1BB antibodies and fragments thereof as described herein can be produced from nucleic acid molecules using molecular biological methods known in the art. Nucleic acids of the present invention include, for example, DNA and/or RNA.
[00125] In some embodiments, the nucleic acid constructs include regions encoding a human anti-4-1BB antibody or fragment thereof (e.g., 94K, 94KV, 94KVT, EU101). In some embodiments, such antibodies or fragments thereof will include VH and/or VL regions. A human anti-4-1BB antibody or fragment thereof can be identified and/or selected by functional properties and/or desired binding, and variable regions of said isolated, amplified, cloned and/or sequenced antibody. Modifications may be made to the VH and VL nucleotide sequences, including additions of nucleotide sequences that encode amino acids and/or carry restriction sites, and/or substitution of nucleotide sequences that encode amino acids. In some embodiments, a nucleic acid sequence may or may not include an intron sequence.
[00126] Where appropriate, nucleic acid sequences encoding human anti-4-1BB antibodies and fragments thereof (e.g., 94K, 94KV, 94KVT, EU101) can be modified to include codons that are optimized for expression in a particular cell type or organism (for example, see United States Patent No. 5,670,356 and United States Patent No. 5,874,304). Codon-optimized sequences are synthetic sequences, and preferably encode the identical polypeptide (or a biologically active fragment of a full-length polypeptide that has substantially the same activity as that of the full-length polypeptide) encoded by the non-codon-optimized source polynucleotide. In some embodiments, the coding region of the genetic material that encodes the antibody components, in whole or in part, may include an altered sequence to optimize codon usage for a particular cell type (e.g., a eukaryotic or prokaryotic cell). For example, a coding sequence for a humanized heavy (or light) chain variable region as described here can be optimized for expression in a bacterial cell. Alternatively, the coding sequence can be optimized for expression in a mammalian cell (e.g., a CHO cell). Such a sequence can be described as a codon-optimized sequence.
[00127] The nucleic acid constructs of the present invention can be inserted into an expression vector or viral vector by methods known in the art, and the nucleic acid molecules can be operably linked to an expression control sequence. A vector comprising any of the above-described nucleic acid molecules, or fragments thereof, is also provided by the present invention. Any of the above nucleic acid molecules, or fragments thereof, can be cloned into any suitable vector and can be used to transform or transfect any suitable host. The selection of vectors and the methods for constructing them are commonly known to persons skilled in the art and are described in the general technical references (See, in general, "Recombinant DNA Part D," Methods in Enzymology, Volume 153, Wu and Grossman, editions , Academic Press (1987)).
[00128] In some embodiments, conventionally used techniques such as, for example, electrophoresis, calcium phosphate precipitation, DEA-dextran transfection, lipofection, etc. can be used to introduce a foreign nucleic acid (DNA or RNA) into a prokaryotic or eukaryotic cell. Desirably, a vector may include regulatory sequences, such as transcriptional and translational initiation and termination codons, that are specific to the type of host (e.g., bacterium, fungus, plant, or animal) into which the vector is to be introduced, as appropriate. and taking into account whether the vector is DNA or RNA. In some embodiments, a vector comprises regulatory sequences that are specific to the host's gender. Preferably, a vector comprises regulatory sequences that are specific to the host species.
[00129] In addition to the replication system and the inserted nucleic acid, a nucleic acid construct may include one or more marker genes, which allow for the selection of transformed or transfected hosts. Marker genes include biocide resistance, for example, resistance to antibiotics, heavy metals, etc., complementation in an auxotrophic host can provide prototrophy, and the like.
[00130] Suitable vectors include those engineered for propagation and expansion or for expression or both. For example, a cloning vector is selected from the group consisting of the pUC series, the pBluescript series (Stratagene, LaJolla, Calif.), the pET series (Novagen, Madison, Wis.), the pGEX series (Pharmacia Biotech, Uppsala, Sweden), and in the pEX series (Clontech, Palo Alto, Calif.). Bacteriophage vectors, such as ΔGT10, ΔGT11, ΔZapII (Stratagene), ΔEMBL4, and ΔNM1149, can also be used. Examples of plant expression vectors include pBI110, pBI101.2, pBI101.3, pBI121 and pBIN19 (Clontech). Examples of animal expression vectors include pEUK-C1, pMAM and pMAMneo (Clontech). The TOPO cloning system (Invitrogen, Carlsbad, Calif.) can also be used according to the manufacturer's recommendations.
[00131] An expression vector may comprise a native or non-native promoter operably linked to an isolated or purified nucleic acid molecule as described above. Selection of promoters, eg strong, weak, inducible, tissue-specific and developmental-specific, is within the skill of the art. Similarly, combining a nucleic acid molecule, or fragment thereof, as described above with a promoter is also within the skill of the art.
[00132] Suitable viral vectors include, for example, retroviral vectors, parovirus-based vectors, for example, adeno-associated virus (AAV)-based vectors, AAV-adenoviral chimeric vectors, and adenovirus-based vectors, and vectors lentivirals, such as Herpes simplex (HSV)-based vectors. These viral vectors can be prepared using standard recombinant DNA techniques described in, for example, Sambrook et al., Molecular Cloning, a Laboratory Manual, 2nd edition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989); and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, New York, N.Y. (1994).
[00133] A retroviral vector is derived from a retrovirus. Retrovirus is an RNA virus capable of infecting a wide variety of host cells. Upon infection, the retroviral genome integrates into its host cell's genome and is replicated along with the host cell's DNA, thereby constantly producing viral RNA and any nucleic acid sequences incorporated into the retroviral genome. As such, "long term" expression of a therapeutic factor(s) is obtainable when using retroviruses. Retroviruses contemplated for use in gene therapy are relatively nonpathogenic, although pathogenic retroviruses do exist. When using pathogenic retroviruses, eg human immunodeficiency virus (HIV) or human T-cell lymphotrophic virus (HTLV), care must be taken to alter the viral genome to eliminate host toxicity. A retroviral vector can additionally be engineered to render the virus replication deficient. As such, retroviral vectors are considered particularly useful for stable gene transfer in vivo. Lentiviral vectors, such as HIV-based vectors, are exemplary retroviral vectors used for gene delivery. Unlike other retroviruses, HIV-based vectors are known to incorporate their passenger genes into undivided cells and, consequently, may be of use in the treatment of persistent forms of disease.
[00134] Additional sequences may be added to such cloning and/or expression sequences to optimize their function in cloning and/or expression, to aid in the isolation of the polynucleotide, or to enhance the introduction of the polynucleotide into a cell. The use of cloning vectors, expression vectors, adapters, and linkers is well known in the art. (See, for example, Ausubel, supra; or Sambrook, supra).
[00135] In some embodiments, the nucleic acids and vectors of the present invention can be isolated and/purified. The present invention also provides a composition comprising the isolated or purified nucleic acid molecule described above, optionally in the form of a vector. Vectors and isolated nucleic acids can be prepared using standard techniques known in the art including, for example, alkali/SDS treatment, CsCl binding, column chromatography, agarose gel electrophoresis and other techniques well known in the art. The composition may comprise other components, as also described herein.
[00136] In some embodiments, the nucleic acid molecules are inserted into a vector that is capable of expressing a human anti-4-1BB antibody or fragment thereof when introduced into an appropriate host cell. Suitable host cells include, but are not limited to, bacterial, yeast, insect, and mammalian cells. Exemplary host cells include prokaryotic (e.g., E. coli) and eukaryotic (e.g., a COS cell or a CHO cell). Mammalian host cells that would be used include human Hela 293, H9, and Jurkat cells, mouse NIH3T3 and C127 cells, Cos 1, Cos 7, and CV 1, quail QC1-3 cells, mouse L cells, and ovarian ovarian cells. Chinese hamster (CHO) (eg DG44 cells). In some embodiments, a mammalian host cell suitable for antibody expression may be a Chinese Hamster Ovary (CHO) cell (e.g., including DHFR-CHO cells used in conjunction with a DHFR selectable marker), a myeloma cell of NSO, a cell of COS or a cell of SP2.
[00137] Any method known to one skilled in the art for inserting DNA fragments into a vector can be used to construct expression vectors encoding a human anti-4-1BB antibody or fragment thereof of the present invention under the control of transcriptional/translational control signals. These methods may include in vitro recombinant DNA and synthetic techniques and in vivo recombination (See, for example, Ausubel, supra; or Sambrook, supra). Antibody production
[00138] The antibodies and antigen-binding fragments of the present invention can be prepared and/purified by any technique known in the art, which allows for the subsequent formation of a stable antibody or antibody fragment.
[00139] A nucleic acid encoding a human anti-4-1BB antibody and/or antigen-binding fragment of the present invention can be readily isolated and sequenced by conventional procedures. For example, an oligonucleotide primer designed to specifically amplify corresponding light chain and heavy chain coding regions of a hybridoma or phage template DNA can be used. Isolated nucleic acids can be inserted into an expression vector, and then monoclonal antibodies can be produced from a suitable host cell (i.e., transformant) transformed by introducing the expression vector into the host cell. In some embodiments, a method for preparing the human anti-4-1BB antibody and/or antigen-binding fragment of the present invention may include amplifying an expression vector including a nucleic acid encoding the antibody, but is not limited to this.
[00140] In some embodiments, a host cell is a eukaryotic host cell, including, for example, yeast, higher plant, insect, and mammalian cells. Depending on the host employed in a recombinant production procedure, antibodies and antibody fragments of the present invention may be glycosylated or may be non-glycosylated. In some embodiments, a recombinant expression vector encoding a human anti-4-1BB antibody and/or antigen-binding fragment of the present invention is introduced into a mammalian host cell, and an antibody can be prepared by culturing the host cell. for a time sufficient to express the antibody. In some embodiments, a mammalian host cell is cultured for a time sufficient to secrete an antibody or antibody fragment of the present invention into a culture medium.
[00141] In some embodiments, an expressed antibody of the present invention can be uniformly purified after being isolated from the host cell. Isolation and/or purification of an antibody of the present invention can be carried out by a conventional method for isolating and purifying a protein. For example, not wishing to be bound by theory, a human anti-4-1BB antibody and/or antigen-binding fragment of the present invention can be recovered and purified from recombinant cell cultures by well-known methods including, but not limited to, , protein A purification, protein G purification, ammonium sulfate or ethanol precipitation, acid extraction, anion and cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lecithin chromatography . High performance liquid chromatography ("HPLC") can also be employed for purification. See, for example, Colligan, Current Protocols in Immunology, or Current Protocols in Protein Science, John Wiley & Sons, NY, NY, (1997-2001), for example, chapters 1, 4, 6, 8, 9, and 10 , each fully incorporated herein by reference. In some embodiments, an antibody of the present invention can be isolated and/or further purified by combining filtration, superfiltration, saturation precipitation, dialysis, etc.
[00142] Purified human anti-4-1BB antibody and/or antigen-binding fragments of the present invention can be characterized by, for example, ELISA, ELISPOT, flow cytometry, immunocytology, BIACORE™ analysis, exclusion assay by SAPIDYNE KINEXA™ kinetics, SDS-PAGE and Western blot, or by HPLC analysis as well as by various other functional assays described herein. therapeutic applications
[00143] The present invention encompasses a recognition that engineered human anti-4-1BB antibodies and antigen-binding fragments may be useful for diagnosing, preventing, and/or treating certain diseases such as, for example, cancer. Any of the anti-4-1BB antibodies or antigen-binding fragments provided herein can be used in therapeutic methods. For example, an anti-4-1BB antibody or antigen-binding fragment of the present invention can be used as immunotherapeutic agents, for example, in the treatment of a malignant disease (e.g., cancer).
[00144] The present invention provides methods for treating and/or preventing a malignant disease, which methods include administering an anti-4-1BB antibody or antigen-binding fragment of the present invention to a subject. Methods for modulating or treating at least one malignancy in a cell, tissue, organ, animal or patient, include, but are not limited to, cancer and/or and the treatment of inflammatory diseases.
[00145] Anticancer treatments in connection with the present invention may be mediated through the increase in cytotoxic T cells and anticancer cytokines. Generally, antigen-specific cell-mediated immunity is caused by cytotoxic T cells, and includes two signaling events: a first signaling event is induced when a T cell recognizes an antigen from an antigen-presenting cell via a receptor , and a second signaling is induced by costimulatory molecules. Due to the first and second stimuli, T cell activity and related factors are increased, thereby forming T cells specifically functioning in cancer treatment, and the T cells formed are increased in cytotoxicity, cell division, cell viability and anticancer cytokine secretion. due to stimulation with costimulatory molecules.
[00146] Specifically, stimulation by 4-1BB has been shown to enhance CD8+ T cell activity, increase secretion of anti-cancer cytokines such as interferon gamma (IFNY), increase expression of anti-apoptotic molecules such as Bcl-2, BclXL and Bfl-1, and/or inhibit activation-induced cell death (AICD). In some embodiments, an anti-human 4-1BB antibody or antigen-binding fragment of the present invention can enhance or increase one or more of CD8+ T cell activity, secretion of anti-cancer cytokines such as interferon gamma (IFNY), expression of anti-apoptotic molecules such as Bcl-2, BclXL and Bfl-1, and inhibition of activation-induced cell death (AICD). In some embodiments, therapeutic treatment with an anti-human 4-1BB antibody or antigen-binding fragment of the present invention can reduce and/or inhibit the growth of cancer cells.
[00147] In some embodiments, the present invention provides a method of delaying or inhibiting tumor growth, comprising regulating cytokine secretion in vivo or in vitro by administering an anti-human 4-1BB antibody or binding fragment. to the antigen of the present invention. In some embodiments, the present invention provides a method for reducing tumor burden, comprising regulating cytokine secretion in vivo or in vitro by administering an anti-human 4-1BB antibody or antigen-binding fragment of the present invention.
[00148] In some embodiments, the present invention provides a method for treating cancer or tumor by monitoring a biological subject for cancer or tumor to be treated, comprising: (i) administering an anti-human 4-1BB antibody or antigen-binding fragment of the present invention to a subject, (ii) separating, then isolating a biological sample from the subject, (iii) measuring an amount of INFY or TGFβ secretion from the sample and estimating a ratio of proportion, and ( iv) determining a therapeutically effective amount of the antibody or antigen-binding fragment thereof by comparing control samples that are administered or not administered with the antigen-binding fragment or antibody of human anti-4-1BB thereof.
[00149] In some embodiments, the present invention provides a method of treating a subject in need thereof, the method comprising a step of administering to the subject a composition that comprises or releases an anti-4-1BB antibody or binding fragment to the antigen of the present invention and/or a nucleic acid thereof. In some modalities, an individual has or is at risk of developing cancer. In some embodiments, the present invention provides a method for preventing or treating a patient's cancer or tumor, which includes administering a therapeutically effective amount of the humanized 4-1BB antibody or antigen-binding fragment thereof to a cancer patient. or tumor.
[00150] In some embodiments, the present invention provides a method of inducing an immune response in a subject in need thereof, the method comprising a step of administering to the subject a composition that comprises or releases an anti-4-1BB antibody or antigen-binding fragment of the present invention and/or a nucleic acid thereof. In some modalities, an individual has or is at risk of developing cancer.
[00151] In some embodiments, the present invention provides a method of enhancing an immune response or increasing the activity of an immune cell in a subject in need thereof, the method comprising a step of administering to the subject a composition comprising or releases an anti-4-1BB antibody or antigen-binding fragment of the present invention and/or a nucleic acid thereof. In some modalities, an individual has or is at risk of developing cancer.
[00152] Cancers suitable for treatment with the method of the present invention may include, but are not limited to, bladder cancer, breast cancer, cervical cancer, colon cancer, endometrial cancer, esophageal cancer, fallopian tube cancer, gallbladder cancer, gastrointestinal cancer, head and neck cancer, hematologic cancer, larynx cancer, liver cancer, lung cancer, lymphoma, melanoma, mesothelioma, ovarian cancer, primary peritoneal cancer, salivary gland cancer, sarcoma, cancer stomach cancer, thyroid cancer, pancreatic cancer, and prostate cancer. In some embodiments, a cancer for treatment with an anti-4-1BB antibody or antigen-binding fragment of the present invention may include, but is not limited to, carcinoma, lymphoma (e.g., Hodgkin's and non-Hodgkin's lymphomas), blastoma , sarcoma and leukemia. In some embodiments, cancer may include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, squamous cell carcinoma of the lung, peritoneal cancer, hepatocellular carcinoma, gastric cancer, pancreatic cancer, glioma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatocellular carcinoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary carcinoma, kidney cancer, prostate cancer, vulvular cancer, cancer thyroid disease, liver carcinoma, leukemia and other lymphoproliferative disorders, and various types of head and neck cancer.
[00153] A composition including an anti-4-1BB antibody or antigen-binding fragment of the present invention can be administered in a pharmaceutically effective amount to treat cancer cell or metastasis thereof, or inhibit the growth of cancer. For use in therapeutic methods, an anti-4-1BB antibody or antigen-binding fragment of the present invention would be formulated, dosed, and administered in a pattern consistent with good medical practice. Factors for consideration in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the patient's age, the patient's weight, the cause of the disorder, the agent delivery site, the method administration, time of administration, and other known factors other factors known to physicians.
[00154] The present invention provides high affinity human anti-4-1BB antibodies that may have superior properties over a reference antibody. The present invention encompasses a recognition that these antibodies may have an improved ability to induce T cell activation and/or secretion of cytokines such as IFNY. Accordingly, the present invention encompasses a recognition that a human anti-4-1BB antibody or antigen-binding fragment of the present invention can be administered at a lower dose than the reference antibody.
[00155] In some embodiments, the composition that includes an anti-4-1BB antibody or antigen-binding fragment of the present invention can be administered to a patient as a bolus or by continuous injection as needed. In some embodiments, the bolus administration is an anti-4-1BB Fab of the present invention and can be administered at a dose of 0.0025 to 100 mg/kg, 0.025 to 0.25 mg/kg, 0.010 to 0. 10 mg/kg, or 0.10 to 0.50 mg/kg. In the case of continuous injection, the antibody of the present invention presented as a Fab fragment can be administered at a dose of 0.001 to 100 mg/kg/minute, 0.0125 to 1.25 mg/kg/minute, 0.010 to 0.010 to 0.0125 to 1.25 mg/kg/minute. 75 mg/kg/minute, 0.010 to 1.0 mg/kg/minute or 0.10 to 0.50 mg/kg/minute for 1 to 24 hours, 1 to 12 hours, 2 to 12 hours, 6 to 12 hours , 2 to 8 hours, or 1 to 2 hours. In some embodiment, an antibody of the present invention is a full-length antibody (having a complete constant domain). In some embodiments, a full-size antibody is administered at a dose of approximately 0.01 to 10 mg/kg, 1 to 8 mg/kg, or 2 to 6 mg/kg. In some embodiments, a full-size antibody is administered by injection over 30 to 35 minutes. The frequency of administration may vary depending on the severity of the condition. For example, the frequency could be once every 2 to 7 days, once a week, or once every 1, 2, 3, or 4 weeks.
[00156] In some embodiments, a composition may be administered to a patient by subcutaneous injection. Specifically, the antibody can be administered to a patient at a dose of 0.1 to 100 mg by subcutaneous injection once every 2 to 7 days, every week, once every two weeks, or every month. Combination Therapies
[00157] The present invention provides therapeutic methods that include administration of an anti-human 4-1BB antibody or antigen-binding fragment of the present invention in combination with one or more other therapies.
[00158] In some embodiments, an anti-human 4-1BB antibody or antigen-binding fragment is administered in combination with one or more therapies that have been approved for treating cancer. For example, combination treatment with an anti-4-1BB antibody and a conventional chemotherapeutic material, cisplatin, has been shown to have synergistic activity in killing tumors and preventing organ-specific toxicity. (Kim et al., Cancer Research (2008) 68(18):7264-9 )
[00159] In some embodiments, a human anti-4-1BB antibody or antigen-binding fragment of the present invention is administered in combination with a second therapy selected from an immune checkpoint inhibitor, Interleukin 12 (IL-12) , Granulocyte Macrophage Colony Stimulating Factor (GM-CSF), an anti-CD4 agent, and a chemotherapeutic agent, so that the individual receives treatment with both.
[00160] In some embodiments, an anti-human 4-1BB antibody or antigen-binding fragment of the present invention is administered to a subject who has been or will be administered a composition comprising a chemotherapeutic agent, such that the subject receives treatment with both. The therapeutic methods of the present invention may include administration of any chemotherapeutic agent known in the art. In some embodiments, the chemotherapeutic agent is administered to a subject who has been or will be administered a composition comprising an anti-human 4-1BB antibody or antigen-binding fragment.
[00161] In some embodiments, an anti-human 4-1BB antibody or antigen-binding fragment is administered to a subject who has been administered or will be administered a composition comprising fluorouracil. In some embodiments, fluorouracil is administered to a subject who has been or will be administered a composition comprising an anti-human 4-1BB antibody or antigen-binding fragment. In some embodiments, an anti-human 4-1BB antibody or antigen-binding fragment is administered to a subject who has been or will be administered a composition comprising doxorubicin. In some embodiments, doxorubicin is administered to a subject who has been or will be administered a composition comprising an anti-human 4-1BB antibody or antigen-binding fragment. In some embodiments, an anti-human 4-1BB antibody or antigen-binding fragment is administered to a subject who has been or will be administered a composition comprising irinotecan. In some embodiments, the irinotecan is administered to a subject who has been or will be administered a composition comprising an anti-human 4-1BB antibody or antigen-binding fragment. In some embodiments, an anti-human 4-1BB antibody or antigen-binding fragment is administered to a subject who has been or will be administered a composition comprising paclitaxel. In some embodiments, paclitaxel is administered to a subject who has been or will be administered a composition comprising an anti-human 4-1BB antibody or antigen-binding fragment.
[00162] In some embodiments, an anti-human 4-1BB antibody or antigen-binding fragment is administered to a subject who has been administered or will be administered a composition comprising cisplatin. In some embodiments, cisplatin is administered to a subject who has been or will be administered a composition comprising an anti-human 4-1BB antibody or antigen-binding fragment. In some embodiments, an anti-human 4-1BB antibody or antigen-binding fragment is administered to a subject who has been or will be administered a composition comprising cyclophosphamide. In some embodiments, cyclophosphamide is administered to a subject who has been or will be administered a composition comprising an anti-human 4-1BB antibody or antigen-binding fragment.
[00163] In some embodiments, a human anti-4-1BB antibody or antigen-binding fragment of the present invention is administered to a subject who has been or will be administered a composition comprising GM-CSF, such that the subject receives treatment with both. In some embodiments, GM-CSF is administered to a subject who has been or will be administered a composition comprising an anti-human 4-1BB antibody or antigen-binding fragment.
[00164] In some embodiments, an anti-human 4-1BB antibody or antigen-binding fragment of the present invention is administered to a subject who has been or will be administered a composition comprising IL-12, such that the subject receives treatment with both. In some embodiments, IL-12 is administered to a subject who has been or will be administered a composition comprising an anti-human 4-1BB antibody or antigen-binding fragment.
[00165] In some embodiments, an anti-human 4-1BB antibody or antigen-binding fragment of the present invention is administered to a subject who has been or will be administered a composition comprising an anti-CD4 agent, such that the subject receive treatment with both. In some embodiments, an anti-CD4 agent is administered to a subject who has been or will be administered a composition comprising an anti-human 4-1BB antibody or antigen-binding fragment.
[00166] In some embodiments, an anti-human 4-1BB antibody or antigen-binding fragment of the present invention is administered to a subject who has been administered or will be administered a composition comprising a checkpoint inhibitor (e.g., an immune checkpoint inhibitor) so that the individual receives treatment with both. In some embodiments, an immunological checkpoint inhibitor is administered to a subject who has been administered or will be administered a composition comprising a human anti-4-1BB antibody or antigen-binding fragment.
[00167] A checkpoint inhibitor used in combination with a human anti-4-1BB antibody or antigen-binding fragment of the present invention can be, for example, any immunological checkpoint inhibitor. Examples of inhibitory checkpoint molecules include A2AR, B7-H3, B7-H4, BTLA, CTLA-4, CD277, IDO, KIR, PD-1, LAG-3, TIM-3, TIGIT and VISTA. An immune checkpoint inhibitor can refer to any compound that inhibits the function of an immune checkpoint protein. Inhibition includes reduced function and total block. In some embodiments, an immune checkpoint inhibitor is an antibody that specifically recognizes an immune checkpoint protein. Several immune checkpoint inhibitors are known and in analogy to these known checkpoint inhibitors, alternative immune checkpoint inhibitors may be developed in the future. Immune checkpoint inhibitors include, but are not limited to, peptides, antibodies, nucleic acid molecules, and small molecules.
[00168] In some embodiments, an immunological checkpoint inhibitor is a CTLA-4 inhibitor. In some embodiments, a checkpoint inhibitor is an antibody that targets CTLA-4, such as, for example, ipilimumab. In some embodiments, a checkpoint inhibitor targets CD366, which is a transmembrane protein also known as a T cell immunoglobulin and mucin domain containing protein-3 (TIM-3). In some embodiments, an immunological checkpoint inhibitor is an agent that inhibits PD-1 signaling.
[00169] PD-1 (i.e. programmed cell death protein-1), is a protein that is distributed on the surface of an immune cell such as a T or B cell and is also known as CD279. In a human, PD-1 is expressed by a PDCD1 gene located at position 2p37,3 on chromosome 2. PD-1 is known to bind to two ligands, PD-L1 and PD-L2.
[00170] In some embodiments, an anti-PD-1 agent is administered to the patient who is receiving, has received, or will receive treatment with an anti-human 4-1BB antibody or antigen-binding fragment of the present invention. In some certain embodiments, a human anti-4-1BB antibody or antigen-binding fragment of the present invention is administered to a patient who is receiving, has received, or will receive treatment with an anti-PD-1 agent.
[00171] In some embodiments, an anti-PD-L1 agent is administered to the patient who is receiving, has received, or will receive treatment with an anti-human 4-1BB antibody or antigen-binding fragment of the present invention. In some certain embodiments, a human anti-4-1BB antibody or antigen-binding fragment of the present invention is administered to a patient who is receiving, has received, or will receive treatment with an anti-PD-L1 agent. In some embodiments, agents that inhibit PD-L1 include, for example, AMP-244, MEDI-4736, MPDL328 OA, MIH1.
[00172] In some embodiments, an anti-PD-1 agent is an agent that inhibits PD-1. In some embodiments, an anti-PD-1 agent is an agent that inhibits PD-L1 and/or PD-L2. In some embodiments, an antibody agent that inhibits PD-1 signaling is a monoclonal antibody or a fragment thereof. In some embodiments, an antibody agent that inhibits PD-1 signaling is an anti-PD-1 antibody or fragment thereof.
[00173] In some embodiments, an anti-PD-1 antibody is administered to the patient who is receiving, has received, or will receive treatment with an anti-human 4-1BB antibody or antigen-binding fragment of the present invention. In some certain embodiments, a human anti-4-1BB antibody or antigen-binding fragment of the present invention is administered to a patient who is receiving, has received, or will receive treatment with an anti-PD-1 antibody. Anti-PD-1 antibodies include, for example, nivolumab, pembrolizumab, atezolizumab, durvalumab, and avelumab. Pembrolizumab (Keytruda, Merck) is a therapeutic antibody that inhibits PD-1 activity.
[00174] As described in the examples of the present application, administration of an anti-human 4-1BB antibody or antigen-binding fragment of the present invention in combination with an anti-PD-1 antibody can enhance efficacy with respect to any treatment alone, and may also reduce conventionally known side effects.
[00175] In some certain embodiments, pembrolizumab is administered to the patient who is receiving, has received, or will receive treatment with an anti-human 4-1BB antibody or antigen-binding fragment of the present invention. In some certain embodiments, an anti-human 4-1BB antibody or antigen-binding fragment of the present invention is administered to the patient who is receiving, has received, or will receive treatment with pembrolizumab.
[00176] In some embodiments, an immunological checkpoint inhibitor (e.g., an anti-PD-1 agent) is administered to a patient in an amount from about 0.01 mg/kg to about 100 mg/kg . In some embodiments, an immunological checkpoint inhibitor (e.g., an anti-PD-1 agent) is administered to a patient in an amount within a range bound by a lower limit and an upper limit, the upper limit being greater. than the lower limit. In some embodiments, the lower limit may be about 0.01 mg/kg, 0.025 mg/kg, 0.05 mg/kg, 0.075 mg/kg, 0.1 mg/kg, 0.25 mg/kg, 0.5mg/kg, 0.75mg/kg, 1mg/kg, 2mg/kg, 3mg/kg, 4mg/kg, 5mg/kg, 8mg/kg, 10mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 40 mg/kg, 50 mg/kg, 50 mg/kg, 70 mg/kg, 80 mg/kg, or 90 mg/kg. In some embodiments, the upper limit may be about 0.025 mg/kg, 0.05 mg/kg, 0.075 mg/kg, 0.1 mg/kg, 0.25 mg/kg, 0.5 mg/kg, 0.75mg/kg, 1mg/kg, 2mg/kg, 3mg/kg, 4mg/kg, 5mg/kg, 8mg/kg, 10mg/kg, 20mg/kg, 25mg/ kg, 30 mg/kg, 40 mg/kg, 50 mg/kg, 50 mg/kg, 70 mg/kg, 80 mg/kg, 90 mg/kg, or 100 mg/kg. In some embodiments, an immunological checkpoint inhibitor (e.g., an anti-PD-1 agent) can be administered to a patient in an amount from about 1 mg/kg to about 20 mg/kg, from about 1 mg/kg to about 20 mg/kg. 1 mg/kg to about 10 mg/kg, from about 1 mg/kg to about 5 mg/kg, from about 2 mg/kg to about 5 mg/kg, from about 2 mg/kg to about 4 mg/kg, from about 3 mg/kg to about 5 mg/kg, or from about 3 mg/kg to about 4 mg/kg. In some embodiments, an immunological checkpoint inhibitor (e.g., an anti-PD-1 agent) can be administered to a patient in an amount of about 1 mg/kg, about 2 mg/kg, about 3 mg/kg, about 4 mg/kg, or about 5 mg/kg.
[00177] In some embodiments, treatment with a combination of an immunological checkpoint inhibitor and an anti-human 4-1BB antibody or antigen-binding fragment of the present invention can enhance proliferation, migration, persistence, and/or cytotoxic activity of CD8+ T cells in an individual. Cell based applications
[00178] Yet another object of the present invention is to provide a method for proliferating activated T cells ex vivo by administering the humanized 4-1BB antibody or antigen-binding fragment thereof.
[00179] In some embodiments, a method for ex vivo proliferation and/or isolation of activated T cells includes contacting a population of T cells with an anti-4-1BB antibody or antigen-binding fragment of the present invention, thereby increasing the proliferation of activated T cells.
[00180] In some embodiments, a method for proliferating activated T cells ex vivo includes administering an anti-4-1BB antibody or antigen-binding fragment of the present invention. In some embodiments, activated T cells are proliferated and/or isolated from a sample of peripheral blood mononuclear cells (PBMC). PBMCs can be obtained/isolated using methods known in the art.
[00181] In some embodiments, a method for ex vivo proliferation and/or isolation of activated T cells includes administering an anti-CD3 monoclonal antibody to the culture medium (e.g., at a concentration of at least about 0.5 ng /ml). In some embodiments, a method for ex vivo proliferation and/or isolation of activated T cells includes administering IL-2 and/or IL-15 to the culture medium (e.g., at a concentration that is at least about 10 units/ml ).
[00182] In some embodiments, a method for isolating non-antigen-specific activated T cells includes (a) culturing peripheral blood mononuclear cells (PBMC) in a medium together with a peptide of an epitope of interest and IL-2; (b) inducing 4-1BB expression in the cultured cells by adding the epitope peptide of interest; (c) contacting the cultured cells with a surface coated with an anti-4-1BB antibody or antigen-binding fragment, wherein the cultured cells expressing 4-1BB adhere to the coated surface; and (d) removing the unbound cells, thereby isolating the antigen-specific activated T cells.
[00183] In some embodiments, the activated T cells are CD8+ T cells.
[00184] In some embodiments, lymphocytes (e.g., T cells) are cultured at a temperature of at least about 25°C, preferably at least about 30°C, more preferably about 37°C.
[00185] The present invention encompasses the recognition that activated T cells (e.g., CD8+ T cells), generated by the methods described herein, may be therapeutically useful (e.g., for the treatment of cancer). Cell-Based Therapies
[00186] The present invention provides methods for selectively isolating and culturing CD8+ T cells that recognize an outlogous cancer antigen (autotumor antigen), e.g., autologous cancer antigen that has been overexpressed on cancer cells while present in a low ratio in normal cells. The invention shows that cells (e.g. CD8+ T) isolated by these methods may be useful for the treatment of cancer.
[00187] In some embodiments, a method of treating and/or preventing cancer in a subject in need thereof includes administering to the subject a therapeutically effective amount of activated T cells produced by an ex vivo method such as those described herein.
[00188] On proper reactivation, tumor antigen-specific T cells can recognize and eliminate autologous tumor cells. For example, tumor antigen-specific T cells can be generated ex vivo using methods such as those described herein. In adoptive transfer, specifically activated T cells from cancer patients can effectively reject autologous human tumors in vivo.
[00189] The present invention provides methods for preventing and/or treating a patient's cancer and/or tumor, which includes administering a therapeutically effective amount of activated T cells prepared ex vivo by administering an anti-4-1BB antibody or fragment binding to the antigen of the present invention.
[00190] In some embodiments, the T cells for use in a therapeutic method are allogeneic (of the same species but from a different donor) as the recipient individual. In some embodiments, T cells for use in a therapeutic method are autologous (the donor and recipient are the same). In some modalities, T cells for use in a therapeutic method are synergistic (the donor and recipients are different but identical twins).
[00191] In some embodiments, cells are formulated by first harvesting them from their culture medium, then washing and concentrating them in a suitable medium and container system for administration (a "pharmaceutically acceptable" vehicle) in an effective amount in treatment. The suitable infusion medium can be any isotonic medium formulation, typically saline formal, Normosol R (Abbott) or Plasma-Lyte A (Baxter), but also 5% dextrose in water or lactated Ringer's can be used. The infusion medium can be supplemented with human serum albumin.
[00192] A cell treatment effective amount in the composition is at least 108, typically greater than 108, at least 109 cells, and generally more than 1010. The number of cells will depend on the end use for which the composition is intended, as will be the type of cells included here. For example, if cells that are specific to a particular antigen are desired, then the population will contain more than 70%, generally more than 80%, 85%, and 90-95% of such cells. For the uses provided here, the cells are usually in a volume of one liter or less. In some embodiments, the cells for administration are in a volume less than 500 ml, less than 250 ml, or 100 ml or less. In some embodiments, a desired cell density is typically greater than 10 6 cells/ml and is generally greater than 10 7 cells/ml, generally 10 8 cells/ml or greater. A clinically relevant number of immune cells can be apportioned into multiple infusions that cumulatively equal or exceed 108 cells, 109 cells, 1010 cells, 1011 cells, or 1012 cells. compositions
[00193] Compositions comprising antibodies and antigen-binding fragments that specifically bind to an epitope of a human 4-1BB polypeptide are provided herein. Compositions of the present invention (e.g., compositions that release a human anti-4-1BB antibody or antibody fragment) can include any suitable or effective amount of a composition for use in releasing a given anti-4-1BB antibody. human or antibody fragment to a cell, tissue, organ, animal or patient in need of such modulation, treatment or therapy. In addition, compositions are also provided herein that include activated cell populations (e.g., activated T cell population) that have been generated by a method of the present invention (e.g., a method that includes a step of contacting a cell with an anti-human 4-1BB antibody or antibody fragment).
[00194] The compositions of the present invention include pharmaceutical compositions that include an anti-human 4-1BB antibody or antigen-binding fragment described herein and/or a cell population obtained by a method described herein. In some embodiments, a pharmaceutical composition may include a buffer, a diluent, an excipient, or any combination thereof. In some embodiments, a composition, if desired, may also contain one or more additional therapeutically effective substances.
[00195] In some embodiments, an anti-4-1BB antibody, antigen-binding fragment, and/or cell population of the present invention are suitable for administration to a mammal (e.g., a human). While the descriptions of pharmaceutical compositions provided herein are primarily directed to pharmaceutical compositions that are suitable for ethical administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to animals of all types. Modification of pharmaceutical compositions suitable for administration to humans to make the compositions suitable for administration to various animals is well understood, and the veterinary pharmacologist skilled in the art can plan and/or carry out such modification with merely ordinary experimentation, if any.
[00196] In some embodiments, the compositions are formulated for parenteral administration. For example, a pharmaceutical composition provided herein may be provided in a sterile injectable form (e.g., a form that is suitable for subcutaneous injection or intravenous infusion). For example, in some embodiments, a pharmaceutical composition is provided in a liquid dosage form that is suitable for injection. In some embodiments, a pharmaceutical composition is provided as a powder (e.g., lyophilized and/or sterilized), optionally under vacuum, which may be reconstituted with an aqueous diluent (e.g., water, buffer, saline, etc.) before of the injection. In some embodiments, a pharmaceutical composition is diluted and/or reconstituted in water, sodium chloride solution, sodium acetate solution, benzyl alcohol solution, phosphate buffered saline, etc. In some embodiments, a powder must be mixed gently with the aqueous diluent (eg, not shaken).
[00197] In some embodiments, an anti-4-1BB antibody, antigen-binding fragment, and/or cell population of the present invention is formulated with a pharmaceutically acceptable parenteral vehicle. Examples of such vehicles are water, saline, Ringer's solution, dextrose solution, and 1 to 10% human serum albumin. Liposomes and non-aqueous vehicles such as fixed oils can also be used. A lyophilized vehicle or powder may contain additives that maintain isotonicity (eg, sodium chloride, mannitol) and chemical stability (eg, buffers and preservatives). In some embodiments, a formulation is sterilized by known or suitable techniques.
[00198] The formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereinafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the active ingredient into association with a diluent or other excipient and/or one or more other accessory ingredients, and then, if necessary and/or desirable, shaping and/or packaging the product. in a single dose unit or multiple desired doses.
[00199] In some embodiments, a pharmaceutical composition including an anti-4-1BB antibody, antigen-binding fragment, and/or cell population of the present invention may be included in a container for storage or administration, for example, a vial, a syringe (eg, an IV syringe), or a bag (eg, an IV bag). A pharmaceutical composition according to the present invention may be prepared, packaged, and/or sold in bulk, as a single unit dose, and/or as a plurality of single unit doses. As used herein, a "unit dose" is a discrete amount of pharmaceutical composition comprising a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient that would be administered to a subject and/or a convenient fraction of such dosage such as, for example, one-half or one-third of such dosage.
[00200] The relative amounts of the active ingredient, the pharmaceutically acceptable excipient, and/or any additional ingredients in a pharmaceutical composition as described will vary, depending on the identity, size, and/or condition of the subject treated and also depending on the routine by which the composition is to be administered. The examples below describe, in part, the dosing of an exemplary human anti-4-1BB antibody to a rodent. Standard methods are known in the art for measuring dosage in animal systems. See, for example, J Basic Clin Pharm. March 2016-May 2016; 7(2): 27-31, which is incorporated herein by reference in its entirety. By way of example, the composition may comprise between 0.1% and 100% (weight/weight) of active ingredient.
[00201] In some embodiments, a composition comprises or delivers an anti-human 4-1BB antibody or antigen-binding fragment of the present invention at a dose of 0.01 mg/kg to 100 mg/kg. In some embodiments, a composition comprises or releases a human anti-4-1BB antibody or antigen-binding fragment in a dose in an amount within a range bound by a lower limit and an upper limit, the upper limit being greater than the than the lower limit. In some embodiments, the lower limit may be about 0.01 mg/kg, 0.025 mg/kg, 0.05 mg/kg, 0.075 mg/kg, 0.1 mg/kg, 0.25 mg/kg, 0.5mg/kg, 0.75mg/kg, 1mg/kg, 2mg/kg, 3mg/kg, 4mg/kg, 5mg/kg, 8mg/kg, 10mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 40 mg/kg, 50 mg/kg, 50 mg/kg, 70 mg/kg, 80 mg/kg, or 90 mg/kg. In some embodiments, the upper limit may be about 0.025 mg/kg, 0.05 mg/kg, 0.075 mg/kg, 0.1 mg/kg, 0.25 mg/kg, 0.5 mg/kg, 0.75mg/kg, 1mg/kg, 2mg/kg, 3mg/kg, 4mg/kg, 5mg/kg, 8mg/kg, 10mg/kg, 20mg/kg, 25mg/ kg, 30 mg/kg, 40 mg/kg, 50 mg/kg, 50 mg/kg, 70 mg/kg, 80 mg/kg, 90 mg/kg, or 100 mg/kg.
[00202] A pharmaceutical composition may further comprise a pharmaceutically acceptable excipient, which, as used herein, includes any and all solvents, dispersion media, diluents, or other liquid carriers, dispersion or suspension aids, surface active agents, isotonics, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as appropriate for the particular dosage form desired. Remington's The Science and Practice of Pharmacy, 21st Edition, A.R. Gennaro (Lippincott, Williams & Wilkins, Baltimore, MD, 2006) describes various excipients used in formulating pharmaceutical compositions and known techniques for preparing the same. Except to the extent that any conventional excipient medium is incompatible with a substance or its derivatives, such as by producing any undesired biological effect or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutical composition, its use is contemplated to be within the scope of this description.
[00203] In some embodiments, a pharmaceutically acceptable excipient is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% pure. In some embodiments, an excipient is approved for use in humans and for veterinary use. In some embodiments, an excipient is approved by the US Food and Drug Administration. In some embodiments, an excipient is pharmaceutical grade. In some embodiments, an excipient meets United States Pharmacopeia (USP), European Pharmacopeia (EP), British Pharmacopeia, and/or International Pharmacopeia standards.
[00204] Pharmaceutically acceptable excipients used in the manufacture of pharmaceutical compositions include, but are not limited to, inert diluents, dispersing and/or granulating agents, surface active agents and/or emulsifiers, disintegrating agents, binding agents, preservatives , buffering agents, lubricating agents, and/or oils. Such excipients may optionally be included in pharmaceutical formulations. Excipients such as cocoa butter and suppository waxes, coloring agents, coating agents, sweetening, flavoring, and/or perfuming agents may be present in the composition, at the discretion of the formulator.
[00205] In some embodiments, a given pharmaceutical composition comprises one or more pharmaceutically acceptable excipients (e.g., preservative, inert diluent, dispersing agent, surface active agent and/or emulsifier, buffering agent, etc.). In some embodiments, a pharmaceutical composition comprises one or more preservatives. In some embodiments, the pharmaceutical compositions do not comprise any preservatives.
[00206] In some embodiments, a composition including an anti-human 4-1BB antibody or antigen-binding fragment of the present invention is stably formulated. In some embodiments, a stable formulation of a human anti-4-1BB antibody or antigen-binding fragment of the present invention may comprise a phosphate buffer with saline or chosen salt, as well as solutions and preserved formulations containing a preservative as well as formulations. preserved from multiple uses suitable for pharmaceutical or veterinary use. The preserved formulations contain at least one known preservative or optionally selected from the group consisting of at least one phenol, m-cresol, p-cresol, o-cresol, chlorocresol, benzyl alcohol, phenylmercuric nitrite, phenoxyethanol, formaldehyde, chlorobutanol, magnesium (e.g. hexahydrate), alkyl paraben (methyl, ethyl, propyl, butyl and the like), benzalkonium chloride, benzaltonium chloride, sodium dehydroacetate and thimerosal, or mixtures thereof in an aqueous diluent. Any suitable mixture or concentration may be used as known in the art, such as 0.001 to 5%, or any range or value herein, such as, but not limited to, 0.001,0.003, 0.005, 0.009, 0.01, 0, 02, 0.03, 0.05, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1.2, 2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7,3.8, 3.9, 4.0, 4.3, 4.5, 4.6, 4.7, 4.8, 4.9, or any range or value here. Non-limiting examples include, in the preservative, 0.1 to 2% m-cresol (e.g., 0.2, 0.3, 0.4, 0.5, 0.9, 1.0%), 0 .1 to 3% benzyl alcohol (e.g. 0.5, 0.9, 1.1, 1.5, 1.9, 2.0, 2.5%), 0.001 to 0.5% thimerosal (e.g. 0.005, 0.01), 0.001-2.0% phenol (e.g. 0.05, 0.25, 0.28, 0.5, 0.9, 1.0%), 0 .0005-1.0%alkylparaben(s) (e.g. 0.00075, 0.0009, 0.001, 0.002, 0.005, 0.0075, 0.009, 0.01, 0.02, 0.05, 0.075, 0 .09, 0.1, 0.2, 0.3, 0.5, 0.75, 0.9, 1.0%), and the like.
[00207] In some embodiments, a pharmaceutical composition is provided in a form that can be refrigerated and/or frozen. In some embodiments, a pharmaceutical composition is provided in a form that cannot be refrigerated and/or frozen. In some embodiments, reconstituted solutions and/or liquid dosage forms may be stored for a certain period of time after reconstitution (e.g., 2 hours, 12 hours, 24 hours, 2 days, 5 days, 7 days, 10 days, 2 weeks, one month, two months, or longer). In some embodiments, storage of antibody compositions for a longer time than specified results in antibody degradation.
[00208] Liquid dosage forms and/or reconstituted solutions may comprise particulate matter and/or discoloration prior to administration. In some embodiments, a solution should not be used if it is discolored or cloudy and/or if particulate matter remains after filtration.
[00209] General considerations in the formulation and/or manufacture of pharmaceutical agents can be found, for example, in Remington: The Science and Practice of Pharmacy, 21st edition, Lippincott Williams & Wilkins, 2005.Kits
[00210] The present invention further provides a pharmaceutical package or kit comprising one or more containers loaded with at least one human anti-4-1BB antibody or antibody fragment as described herein. The kits can be used in any applicable method, including, for example, therapeutic methods, diagnostic methods, cell proliferation and/or isolation methods, etc. Optionally, a notice may be associated with such container(s) in a manner prescribed by a government agency regulating the manufacture, use or sale of pharmaceutical or biological products, which notice reflects (a) approval by the agency of manufacture, use or sale for human administration, (b) instructions for use, or both.
[00211] In some embodiments, a kit may include one or more reagents for detection (e.g., detection of an anti-human 4-1BB antibody or antibody fragment). In some embodiments, a kit may include an anti-human 4-1BB antibody or antibody fragment, in a detectable manner (e.g., covalently associated with a detectable portion or entity).
[00212] In some embodiments, an anti-human 4-1BB antibody or antibody fragment, as provided herein, may be included in a kit used to treat subjects. In some embodiments, an anti-human 4-1BB antibody or antibody fragment, as indicated herein, may be included in a kit used for the proliferation and/or isolation of T cells (e.g., CD8+ T cells).
[00213] The contents of all cited references (including bibliographic references, issued patents, published patent applications, coping patent applications) cited throughout this application are hereby expressly incorporated by reference.
[00214] Other features of the invention will become apparent in the course of the following descriptions of exemplary embodiments. However, the following examples are merely provided to illustrate the present invention, but the scope of the present invention is not limited to the following examples. EXAMPLES
[00215] The present invention provides, at least in part, humanized anti-human antibodies and fragments thereof with improved properties that contain one or more structural features not found in a reference humanized anti-4-1BB human antibody, 94G1 . 94G1 was generated by humanization of murine anti-human 4-1BB antibody BBK-4 antibody. Antigen recognition sites (CDR regions) were determined using CDR loop assignment (IMGT: Lefranc, 1997) and a 3-D model (Swiss-Pdb Viewer (www.expasy.org)). A phage display library was prepared with diversity at a total of 10 sites including 4 sites in the light chain amino acid sequence and 6 sites in the heavy chain were constructed. After paniculation, approximately 14 humanized antibody clones out of 1000 clones were selected (for a total of six humanized scFvs), and among the selected clones, 94G1 was obtained (Son et al., J. Immunol. Methods (2004) 286: 187-201). These humanized antibodies, including 94G1, had affinities for the human 4-1BB antigen that were less than 1/10° of that of BBK-4, but were active in vitro. The present invention encompassed the recognition that structural variants of 94G1 may have improved properties. Generation and characterization of variant humanized anti-4-1BB human antibodies and their fragments are described in greater detail in the following examples.Example 1 - Preparation of humanized human anti-4-1BB antibodies
[00216] This example describes the production of an exemplary human anti-4-1BB antibody with improved affinity relative to a reference 94G1 antibody. 94G1 was generated by humanization of a murine human anti-4-1BB antibody (BBK4) as described in Son et al. J. Immunol. Methods (2004) 286: 187-201, which is incorporated herein by reference in its entirety. Also used here is an H4-1BB antigen (Accession No: KCTC 0952BP) that is specifically isolated from activated T cells (eg, activated T cell lineage), and has not been identified from unstimulated T cells. For example, an H4-1BB antigen can be isolated from T cells that have been matured by phorbol myristate acetate (PMA), ionomycin, Concanavalin A, or anti-CD3i. This H4-1BB antigen has a size of 1.4 kb, and 60% homology to mouse 4-1BB ( Garni-Wagner et al., Cellular Immunology (1996) 169: 91-98 , which is incorporated herein by reference in its entirety). In this example, 94G1 has been split into a light chain and a heavy chain vector, each optimized to generate improved humanized antibodies.
[00217] The present invention encompasses a recognition that a suitable method for generating improved humanized anti-4-1BB human antibodies or fragments thereof is through simple, step-by-step amino acid substitutions and/or combinations thereof. The present invention provides various structural variants of humanized human anti-4-1BB antibodies and fragments thereof with one or more structural features (e.g., amino acid substitutions) that are not found in a 94G1 antibody. The present invention also encompasses a recognition that structural features can be combined for step-by-step improvements in one or more antibody properties (e.g., increased antigen affinity).
[00218] First, a humanized anti-4-1BB human antibody with higher affinity compared to the 94G1 reference antibody was obtained by altering a CDR region of a light chain rather than a heavy chain. This light chain structural variant was fixed, and combined with anti-human heavy chain structural variants of humanized anti-human 4-1BB antibody with, for example, mutations in the CDR region of 94G1. Other structural features were integrated to generate humanized anti-4-1BB human antibodies with high affinity and/or other improved features. Vector Construction
[00219] Vectors with a 94G1 light chain and 94G1 heavy chain, respectively, were constructed by altering pComb3H-HA to be expressed in a Fab type to improve a heavy chain and a light chain of a humanized antibody in E. coli ( J. Immunol Methods (2008) 329 (1-2): 176-83; Virology (2004) 318: 598 ). Specifically, a 94G1 light chain was inserted into a vector designated by altering an AP2 marker (SEQ ID NO: 42 - NANNPDWDFNP) with a flag marker (SEQ ID NO: 43 -DYKDDDDK), a flag marker was designated to be located downstream of the same, and having a human heavy chain sequence (Accession No. AB019438) obtained from known NCBI GenBank data, placed as a constant domain at a heavy chain position. Furthermore, after a 94G1 light chain sequence was cloned into the vector, it was transferred into E. coli (eg, TG1) (F'[traD36 proAB + lacIqlacZΔM15]supE thi-1 Δ (lac-proAB) Δ (mcrB-hsdSM)5, (rK-mK-) by transformation, followed by selection of a transformed vector called pCOM-Fab-94G1-L, which was used as a backbone to induce light chain affinity maturation (Table 1) The above-described method was similarly performed for the 94G1 heavy chain, and a selected vector was named pCOM-Fab-94G1-H. An improved light chain, 94/w, was designated as the pCOM-Fab- 94G1, which served as the backbone for the production of heavy chain variants with improved affinity.Table 1 - LCDR 94G1 and 94/w amino acid sequences
1.2 . Affinity maturation of humanized human anti-4-1BB antibody light chain
[00220] The development of a humanized anti-human 4-1BB antibody with a light chain variant having improved binding affinity is described herein. An antibody with a high affinity was obtained by altering LCDR3 (SEQ ID NO: 3) of a 94G1 light chain in the context of the vector pCOM-Fab-94G1-L described above as follows. Various DNA sequences encoding a light chain were amplified by PCR using "primers" [using NNS (N: A, T, C, G; S: C, G)] designed to insert 19 different amino acids at each amino acid position of the 9 amino acids SEQ. ID NO: 3, constituting the LCDR3 part of the 94G1 light chain. Amplified products were ligated to a light chain position of the vector and then transformed into E. coli TG1. All clones with variants of the LCDR3 light chain structure were replaced in different ways and collected to prepare nine position mixtures. To assess whether each amino acid position was replaced by a different amino acid, two clones altered at the respective positions were chosen at random and analyzed by sequencing using an ABI-3730xl sequencer, which showed that the amino acid residues at the respective positions were replaced at various positions.
[00221] To see if 94G1 Fab variants with mutations at different positions of LCDR3 had increased antibody affinity, each position mixture was expressed by adding IPTG (to a final concentration of 1 mM) to E. coli TG1, and then Fab antibody present in a supernatant was subjected to ELISA. Specifically, each position mixture was cultured with shaking in 2YT medium in a 37 °C incubator until the culture had an absorbance at 600 nm of 0.8 or greater, then cultured overnight at 30 °C with IPTG ( e.g. at a final concentration of 1 mM). ELISA was performed the next day on a supernatant obtained by centrifugation at 12,000 rpm for 10 minutes at 4 °C. Binding affinities were determined for the various variant 94G1 LCDR3 Fabs by dividing the binding activities of each clone relative to the 4-1BB Fab by the expression levels for the respective mutant clone. A 94G1 LCDR3 variant with a mutation position 6 of LCDR3 (LCDR3.6) showed the highest binding affinity.
[00222] Subsequently, to determine how various 94G1 mutations at the LCDR3.6 position impacted antibody affinity, 25 monoclonal antibodies were isolated from the mixture of pCOM-Fab94G1-LCDR3.6 position and expressed by the addition of IPTG (e.g. in a final concentration of 1 mM) to E. coli (eg, TG1), cultured, and ELISA was performed on a Fab molecule present in a supernatant. Binding affinities were determined for the various 94G1 LCDR3.6 clones by dividing the 4-1BB Fab binding activities of each clone by the expression levels for each.
[00223] A 94G1 LCDR3.6 variant with phenylalanine at the LCDR3.6 position substituted with tryptophan exhibited the highest binding affinity. A Fab antibody prepared by replacing the constant heavy chain of pCOM-Fab94Gl-L with the heavy chain of the 94G1 backbone in the improved 94G1 light chain was named 94/w. Thus, a 94/w variant includes an improved 94G1 light chain wherein the 6th amino acid of LCDR3 is substituted with tryptophan (W) (QDGHSWPPT - SEQ ID NO: 4) and a 94G1 heavy chain. IPTG-induced expression in E. coli and ELISA of a 94/w Fab was used to determine binding affinity as described above. Using this method, a 94/w Fab antibody was determined to have a 3.5-fold greater binding activity than that of 94G1 (Fab antibody) (data not shown).1.3. Affinity maturation of humanized humanized anti-4-1BB antibody heavy chain CDRs
[00224] The development of humanized human anti-4-1BB antibodies with heavy chain structural variants that have improved binding affinity is described herein. To achieve more improved human anti-4-1BB antibodies, a 94/w light chain was used as described above and the 94G1 heavy chain was affinity matured. In Table 2, the HCDR amino acid sequences for a reference 94G1 antibody heavy chain are shown.Table 2 - 94G1 and 94K HCDR Amino Acid Sequences

[00225] Upgrading a heavy chain using 94/w as a starting sequence was performed by methods similar to those described for the 94G1 light chain above. Particularly, to enhance a 94G1 heavy chain, amino acid residues were substituted with various amino acids at the respective amino acid positions of HDR2 and/or HCDR3. In the case of the third heavy chain CDR (HCDR3, SEQ ID NO: 7), clones that were produced with random replacement amino acid residues of 94/w HCDR3 by different amino acids were collected to prepare 12 pools of positions. A mutant clone that increases the length of HCDR3 was also prepared. When the 5th amino acid residue of HCDR3 was replaced with a different amino acid, an increase in affinity was observed. Subsequently, to determine how various mutations at the HCDR3.5 position impacted the affinity of the 94/w antibody, 19 monoclonal antibodies were isolated from a position mixture in which the HCDR3.5 position of the 94/w antibody was randomly substituted. HCDR3.5 variant Fabs were expressed in E. coli by addition of IPTG (eg to a concentration of 1 mM) and ELISA was performed using a Fab antibody present in a supernatant. Sequencing identified that when threonine was replaced by lysine at position HCDR3.5 (5th position) (SEQ ID NO: 8 - ARSFKTARAFAY), the highest affinity was shown, and the resulting product was called 94K/w.
[00226] In the case of the second heavy chain CDR (HCDR2), a position mixture was prepared by randomly substituting each of the 9 amino acids of a 94G1 HCDR2 (SEQ ID NO: 6) for ELISA. ELISA results showed that when amino acid residues in the 2nd, 5th. and 6a. positions were changed, affinity increased. From each of position 94/w mixtures HCDR2.2, HCDR2.5 and HCDR2.6, monoclonal antibodies 22, 19, and 36 were isolated, respectively, and the binding activity of each clone towards 4-1BB was analyzed depending on a level of Fab expression. In the case of HCDR2.5, an ELISA value was relatively higher than when asparagine was substituted for valine (V), glycine (G), or proline (P). Furthermore, according to sequencing data for antibody heavy chains, there was a risk of deamination at the 5th amino acid, asparagine (N), of HCDR2 (SEQ ID NO: 6), and variant HCDR2 sequences were also prepared with substitutions in this residue with each of glutamine (Q), glutamic acid (E), and serine (S).
[00227] DNAs from structural variants of 94G1 with mutations in HCDR3 and/or HDR2 of the heavy chain, prepared as described above, were amplified by PCR using a sequence of three bases NNS, linked to the position of the heavy chain of a vector having a domain light chain constant of 94/w, and then transformed into E. coli TG1 by the method used in light chain enhancement as described above.1.4 Optimization of humanized anti-4-1BB human antibody heavy chain framework regions
[00228] Heavy chain variants with optimized framework sequences were also produced. For example, heavy chain framework 1 (FR1) regions were produced where heavy chain FR1 (SEQ ID NO: 16) was modified so that the 5th amino acid, glutamine (Q), was replaced by valine (V) . Exemplary FR1 regions are provided in Table 3 below. Table 3 - 94G1 heavy chain FR1 and variations thereof.

[00229] In addition, framework regions 3 (FR3) were produced where the heavy chain FR3 (SEQ ID NO: 18) was modified as such: the 10th amino acid, alanine (A), and/or the 33rd amino acid, serine (S), which were murine sequences, were replaced by valine (V) and threonine (T), respectively. Exemplary FR3 regions are provided in Table 4 below. Table 4 - 94G1 heavy chain FR3 and variations thereof
1.5 Preparation of humanized human anti-4-1BB variable regions and full-size antibodies
[00230] Human anti-4-1BB antibody variable regions were produced containing various combinations of the above-described heavy chain and light chain CDRs and framework regions. For example, a Fab-like 94KVT/w antibody was produced with the 5th amino acid, threonine, in the CDR3 of a heavy chain was replaced by lysine (K), and the 10th amino acid of the heavy chain FR3, alanine, and the 33rd The heavy chain FR3 amino acid, serine, was substituted for valine (V) and threonine (T), respectively, to produce heavy chain and light chain variable region sequences that are or include SEQ ID NO: 30 and SEQ ID NO : 34, respectively. In addition, heavy chain variants of 94KVT were produced where the 5th amino acid of HCDR2 (SEQ ID NO: 6), (asparagine (N) was replaced by glutamine (Q), glutamic acid (E) or serine (S). Exemplary heavy chain and light chain variable domain sequences are provided in Table 5 below (CDR sequences underlined). Table 5 - Exemplary humanized anti-4-1BB human antibody variable domains

[00231] For conversion to a full-size human anti-4-1BB antibody (full-length Ig type), an Fc domain was linked to the respective Fab. For example, a 94K/w Fab composed of a heavy chain in which threonine is replaced by lysine in HCDR3.5 and a light chain with a 94/w variant in which the 6th amino acid of LCDDR3 is replaced by tryptophan (W), and respective extended regions of the CH2 and CH3 domains and a human IgG1 sequence were amplified by PCR to overlap and subjected to splicing PCR to produce full length IgG DNA, and then the resulting DNA was cloned into a mammalian expression vector. Full-size antibodies to other humanized anti-4-1BB human antibodies described herein were produced in a similar manner. Exemplary immunoglobulin constant region sequences are provided in Table 6 below.Table 6 - Exemplary immunoglobulin constant domains

[00232] As used herein, a full-length 94KVT/w antibody includes an IgG1 sequence, such as that of SEQ ID NO: 22. In addition, a full-length antibody, referred to herein as EU101, was produced which includes 94KVT/w variable domains. w described above (SEQ ID NOs: 10 and 14, for light chain and heavy chain variable domains, respectively), with a variant IgG1 constant domain that includes 3 mutations: L234, L235, and K322 (SEQ ID NO: 23) . Thus, the example provides several examples of humanized human anti-4-1BB antibodies and antibody fragments that have been modified to potentially enhance antigen-binding affinity. These exemplary antibodies and fragments are characterized in the following examples.Example 2 - Binding characterization of humanized human anti-4-1BB antibodies2.1 Determination of the binding epitope of human anti-4-1BB antibodies
[00233] The present invention encompasses a recognition that humanized human anti-4-1BB antibodies provided herein may be useful for costimulation of 4-1BB. Therapeutic applications of antibodies of the present invention may include promoting anticancer immunity and/or antiviral immunity. However, for clinical applications, it is important to identify which part of human 4-1BB is recognized by and/or reacts with an anti-humanized 4-1BB antibody (ie, a binding epitope). 4-1BB antibodies that recognize different epitopes of the 4-1BB molecule have been identified, and these antibodies may have been proven to have different clinical effects. (See, for example, Kwon et al., Eur. J. Immunogenetics (2002) 29: 449-452, incorporated herein by reference in its entirety). Epitope mapping encompasses methods for identifying an antibody antigen-recognition molecular determinant. This example describes epitope mapping of an exemplary human anti-4-1BB antibody as modified in Example 1 above. Specifically, this example assesses the epitope binding of a humanized anti-human 4-1BB antibody with 94KVT/w variable domains, EU101.
[00234] A human 4-1BB antigen for investigation of a humanized 4-1BB antibody epitope is derived from a cDNA library manufactured from human peripheral blood lymphocytes that was generated by at least some of the inventors of the present application (see, for for example, Kwon et al., Cellular Immunology (1996) 169: 91-98; Immunol Lett. (1995) 45: 67-73; and Korean Patent No. 10-0500286, each of which is incorporated herein by reference). The cDNA encoding an extracellular domain (ECD) of the human homolog obtained from cDNA 4-1BB (hereinafter referred to as H4-1BB) was selected, fused to GST, and then inserted into a vector (pGEX-6T) for expression. A cell line producing a GST-4-1BB fusion polypeptide as used herein has been deposited as part of the invention of Korean Patent No. 10-0500286, Accession No.: KCTC 0952BP. A full-length human 4-1BB sequence is provided as SEQ ID NO: 44, below. The extracellular domain of human 4-1BB corresponds to amino acids 1 to 167 of the sequence H4-1BB size natural.SEQ ID NO: 44 native-sequence human 4-1BB size MGNSCYNIVATLLLVLNFERTRSLQDPCSNCPAGTFCDNNRNQICSP CPPNSFSSAGGQRTCDICRQCKGVFRTRKECSSTSNAECDCTPGF HCLGAGCSMCEQDCKQGQELTKKGCKDCCFGTFNDQKRGICRPW TNCSLDGKSVLVNGTKERDVVCGPSPADLSPGASSVTPPAPAREPG HSPQIISFFLALTSTALLFLLFFLTLRFSVVKRGRKKLLYIFKQPFMRPV QTTQEEDGCSCRFPEEEEGGCEL
[00235] To determine a 4-1BB epitope recognized by humanized human anti-4-1BB antibodies of the present invention, constructs were generated with fragments of a 4-1BB extracellular domain of various sizes (e.g. R1, R2, R3) , fused to GST, and replicated. A schematic of GST-4-1BB polypeptides as used in the present example is provided in FIG. 1a, and exemplary primers used herein to generate different 4-1BB extracellular domain constructs are provided in Table 7 below. Single recombinant GST-H-4-1BB constructs were grown with 1 mM culture IPTG and produced in E. coli BL21DX5α cells, and the fusion polypeptides were purified using a glutathione-agarose column.Table 7 - Exemplary primers used to generate extracellular domain fragments of 4-1BB useful for epitope mapping

[00236] Purified protein samples were obtained from bacterial cells transformed by a lysis buffer (e.g. 10 mM Tris-HCl - pH 7.4, 50 mM NaCl, 5 mM EDTA, 30 mM NaF, 30 mM Na3VO4 0.1 mM, 1% Triton X-100, 0.5% Nonidet P-40, 1 mM PMSF and protease inhibitor mixture). Approximately 20 μg of each fusion polypeptide sample was diluted in a 4X SDS sample buffer, electrophoresed on SDS-PAGE gels, and then transferred to nitrocellulose membranes (Millipore, Bedford, MA). On cellulose membranes, human anti-4-1BB mAb was reacted with anti-mouse IgG horseradish peroxide (HRP). Binding antibodies were recognized by enhanced chemiluminescence (ECL) (Amersham Pharmacia Biotech, Little Chalfont, UK).
[00237] As described above and shown in FIG. 1B, when each of the three non-overlapping GST-H4-1BB ECD fragment fusion polypeptides, R1, R2, and R3, were treated with GST binding, respectively. An exemplary anti-4-1BB humanized antibody encompassed by the present invention (EU101) was determined to bind to an approximately 32 kDa N-terminal fragment construct (R1) fusion construct (amino acids 1 to 55 of 4- 1BB) by Western Blotting. Furthermore, this binding was specific, as no binding was observed with either the R2 or R3 fusion constructs. See FIG. 1B.
[00238] In addition, to determine the minimal binding site of the humanized anti-4-1BB antibody, an extracellular domain R1 fragment was further divided into 6 smaller fragments: R1.1 polypeptide fragments (amino acids 1 to 45 of 4- 1BB), R1. 2 (amino acids 1 to 35 of 4-1BB), R1.3 (amino acids 11 to 55 of 4-1BB), R1.4 (amino acids 21 to 55 of 4-1BB), R1.5 (amino acids 1 to 25 of 4 - 1BB), and R1.6 (amino acids 1 to 30 of 4-1BB), as depicted in FIG. 1A, and fused to GST (Glutathione S-Transferase, 27 kDa). Exemplary primer pairs used to generate these constructs are provided in Table 7 above. The fusion polypeptide constructs were produced in E. coli BL21 cells with IPTG induction (eg, 1 mM IPTG) and the whole bacterial cell extract was resolved by 12% SDS-PAGE. As shown in FIG. 2A, SDS-PAGE confirmed that individual 4-1BB fusion polypeptides are well expressed.
[00239] SDS-PAGE was transferred to a nitrocellulose membrane and immunostaining was done using an exemplary human anti-4-1BB antibody, EU101. As shown in FIG. 2B, an amino acid sequence 10 to 30 of the extracellular domain of H4-1BB was confirmed to be significant for binding an exemplary humanized anti-4-1BB antibody. This analysis indicates that an exemplary anti-human 4-1BB antibody of the present invention (EU101) binds to an epitope of human 4-1BB whose sequence is or includes CPAGTFCDNNRNQICSPCPP (SEQ ID NO: 15). It was also confirmed that a sequence including amino acids 35 to 50 of the 4-1BB extracellular domain is not significant for binding of an exemplary humanized antibody described herein (Figure 2B). 2.2 Assessment of binding affinity of humanized human anti-4-1BB antibodies specimens to 4-1BB antigen Binding capacity of human anti-4-1BB antibodies specimens
[00240] To examine the binding ability of exemplary humanized anti-4-1BB human antibodies described in Example 1 to a human 4-1BB antigen (H4-1BB), ELISA was performed. Recombinant human 4-1BB expressed by E. coli was used for the antigen.
[00241] A murine BBK-4 antibody, a humanized reference 94G1 antibody, and exemplary modified antibodies 94K, 94KV, 94KVT and EU101 as described in Example 1 were each treated in 96-well plates coated with the extracellular domain recombinant protein. Histidine-tagged 4-1BB (H4-1BB). The exemplary ELISA affinity analysis employed a total volume of 100 µl at a concentration of 1.0 µg/ml, and the reaction was allowed to proceed at room temperature for 1 hour. Horseradish peroxidase (HRP) labeled anti-human IgG and anti-mIgG-HRP, as appropriate, recognizing an antibody was treated by it, and allowed to react at room temperature for 40 minutes. After washing, treatment with ABTS solution (Sigma-Aldrich), which is a substrate for a staining reaction, and allowing the reaction to proceed at room temperature for 30 minutes, and an absorbance at 450 nm in the staining reaction was detected using an ELISA reader to analyze antibody binding activity. The results are shown in FIG. 3. As shown in FIG. 3, as the antibody concentration increases, the binding between each antibody and the 4-1BB (H4-1BB) antigen is improved. These data confirm that the antibodies encompassed by the present invention specifically bind to 4-1BB. Binding of exemplary human anti-4-1BB antibodies to the antigen expressed on the cell
[00242] The ability of exemplary humanized human anti-4-1BB antibodies to bind to a human 4-1BB antigen (H4-1BB) in a cellular context was evaluated. Jurkat 8-1 cells were genetically engineered to overexpress 4-1BB. Exemplary modified antibodies 94K, 94KV, 94KVT and EU101 as described in Example 1, along with that of a murine BBK-4 antibody, and a reference humanized 94G1 antibody were evaluated for binding to Jurkat 8-1 cells using a secondary antibody. anti-mIgG-HRP or anti-hIgG-HRP, as appropriate, and analyzed by FACS. As shown in FIG. 4, each of the antibodies was able to effectively bind 4-1BB expressed by Jurkat 8-1 cells, and the affinity of 94KVT and EU101 was greater than that of BBK-4 and 94G1.In vitro binding affinity of anti -4-1BB human exemplary to the antigen
[00243] In vitro binding affinity of the exemplary modified EU101 antibody as described in Example 1, along with that of a reference humanized 94G1 antibody, was each determined by Biacore analysis. Anti-human IgG was immobilized on a CM5 chip, and coupled to the Fab antibodies prepared above flowing over the chip, and finally reacted with a human 4-1BB antigen (H4-1BB) to measure the binding activity between the antibody and the antigen. (Biacore3000, CM5 sensor chip). The results of the affinity measurement are shown in FIG. 5. The Ka (1/Ms) and Kd (1/s) values represent how quickly an antibody associates with and dissociates from an antigen, respectively. A dissociation constant (KD) is obtained by dividing Kd by Ka (Kd / Ka = KD).
[00244] As a dissociation constant decreases, it can be interpreted that dissociation occurs at a lower concentration and that the affinity is increasing. As shown in FIG. 5, exemplary modified human anti-4-1BB antibody had improved binding affinity relative to a reference 94G1. Exemplary human anti-4-1BB antibodies recognize 4-1BB expressed by activated CD8+ T cells
[00245] CD8+ T cells were isolated from human PBMCs and activated with 1 μg/mL anti-CD3 antibody for 2 days. The ability of exemplary humanized humanized anti-4-1BB antibodies (94K, 94KV, 94KVT and EU101) described in Example 1 to detect a 4-1BB on the surface of activated CD8+ T cells was evaluated against an anti-4-1BB antibody. 1BB commercially available exemplary (4-1BB-PE). Detection with a murine anti-human 4-1BB antibody BBk-4 and a humanized reference antibody 94G1 is also shown. Treatment with 4-1BB antibodies was at a concentration of 25 ng/ml.
[00246] Exemplary antibodies were detected with an anti-mIgG-Dylight488 or anti-hIgG-Dylight488, as appropriate, and analyzed by FACS. The results are shown in FIG. 6. While a reference 94G1 antibody detected 4-1BB on 17.93% of CD8+ T cells, each of a 94KVT and EU101 antibody showed robust detection of 25.3% and 28.33%, respectively. Demonstrating that exemplary antibodies 94KVT and EU101 both had improved binding properties over BBK-4 and 94G1. Thus, humanized variant antibodies of the present invention have superior binding to activated T cells in vitro.Example 3 - In vitro efficacy analysis of humanized human anti-4-1BB antibodies
[00247] Anti-4-1BB antibodies have previously been shown to provide signal stimulation to a co-stimulation molecule expressed on activated CD8+ T cells, 4-1BB, to activate CD8+ T cells, induce proliferation and increase expression of TH1-type cytokines. In this example, the activity of exemplary humanized human anti-4-1BB antibodies described in Example 1 inducing CD8+ T cell proliferation and TH1.3.1-type cytokine expression was examined. Exemplary human anti-4-1BB antibodies induce cell proliferation of CD8+ T cells
[00248] To assess CD8+ T cell proliferation, cells were stained with WST-1 (water soluble tetrazolium salt) which is a cell proliferation reagent. WST-1 labeled CD8+ T cells were primed and activated with 0.5 µg/ml anti-CD3 antibody. Activated CD8+ T cells were treated with 1.0 μg/ml isotype control antibody, murine BBK-4 antibody, reference 94G1 antibody, and exemplary humanized anti-4-1BB human antibodies (94K, 94KV, 94KVT, and EU101 ) described in Example 1. Cells were analyzed using a MACS system and the results are shown in FIG. 7. Referring to FIG. 7 , exemplary humanized human anti-4-1BB antibodies of the present invention were confirmed to induce cell proliferation of CD8+ T cells. Furthermore, a degree of CD8+ T cell activation increases by an order of 94G1 < 94K / 94KV < 94KVT / EU101.3.2 Exemplary human anti-4-1BB antibodies stimulate cytokine secretion
[00249] IFN-Y is a representative cytokine primarily secreted from a T lymphocyte or a natural killer cell (NK cell) and exhibiting proliferation and antiviral activities. In addition, IFN-γ is an important activator for a macrophage and, particularly, an important cytokine that distinguishes TH1 cells from other cell types. IFN-Y secretion plays an important role in the activation of cytotoxic T cells, phagocytes and B cells. Consequently, the efficiency of an anticancer agent can be assessed with an increased amount of TH1-inducing IFN-Y. For this reason, measurement of IFN-Y secretion by specific stimulation may be an ideal standard that can be used as a quantitative criterion for a functional change of T cells.
[00250] CD8+ T cells were isolated from human PBMCs treated with 0.5 µg/ml of an anti-CD3 mAb antibody and then treated with no antibody, or with 1.0 µg/ml of an anti-4 antibody mAb. -1BB: BBK-4, 94G1,94K, 94KV, 94KVT and EU101. IFNY secretion was assessed on days 1, 3, and 5. Results are shown in FIG. 8. As can be seen in FIG. 8 , IFNY secretion increased in all samples treated with the anti-4-1BB antibody, and this increase correlated with the duration of antibody treatment. Treatment with the 94KVT and EU101 antibodies reached a level of secretion that was 13-fold higher than the control group on day 5. Therefore, the exemplary humanized antibodies 94KVT and EU101 could both induce IFNy secretion more efficiently than the antibody reference 94G1.3.3 Increase in IFN-Y level according to treatment of activated CD4+ T cells or CD8+ T cells with an exemplary human anti-4-1BB antibody
[00251] Blood was collected from three healthy donors, PBMCs obtained from them were isolated by Ficoll plate gradient centrifugation, active T cells present in the PBMCs were rested in a medium RPMI-1640 + 2% FBS for 24 hours. Rested PBMCs were treated with an anti-CD4 antibody or anti-CD8 antibody bound to iron beads, and CD4+ T cells or CD8+ T cells were isolated using a MACS magnetic separator. CD4+ T cells or isolated CD8+ T cells were treated with an anti-CD3 T cell activator to induce 4-1BB expression, and treated with EU101 at different concentrations (0.5, 1.0, 2.5, and 5.0 µg/ml) for 3 days. After 3 days, a culture medium excluding the cells was obtained, and IFN-y fluorescence in the culture medium was evaluated by ELISA (ebioscience), and the result was compared with the standard curve provided in an IFNy-ELISA kit (FIG 9).
[00252] As shown in FIG. 9 , IFN-γ expression levels on CD4+ T cells and CD8+ T cells increased in a dose-dependent manner. Particularly, when 5.0 µg/ml of EU101 was treated, compared to a 278% increase in CD4+ T cells, the expression level of IFN-γ increased by 612% in CD8+ T cells. In accordance with the T cell-specific expression pattern of IFN-γ involved in the conversion of T cells to TH1, an exemplary human anti-4-1BB antibody of the present invention, EU101, has sufficient in vitro activity to suggest that it may be efficacious. for the prevention and/or treatment of cancer.3.4 Measurement of ADCC and CDC activities of an exemplary human anti-4-1BB antibody
[00253] An immune system recognizes and attacks virus-infected cells or cancer cells, and antibodies can be used to induce cytotoxicity-mediated apoptosis. For such an immune system, two types of mechanisms, such as antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), can be used. In either case, apoptosis can be mediated by an antibody that binds to a target on the cell surface. That is, when an antibody has an ADCC activity, a cell recognized by the molecule results in apoptosis mediated by a natural killer (NK) cell, and when an antibody has a CDC activity, death is mediated by a complement protein. Therefore, in the case of development of an antibody antagonistic therapy, a degree of killer cells recognized by an antibody can be identified by analyzing ADCC and CDC activities. However, a target for the humanized 4-1BB antibody described in the present invention is T cells, not cancer cells. That is, considering a mechanism for inducing T cell activation by binding a 4-1BB antibody as an agonistic antibody, an antibody that lacks the activities of ADCC and CDC may be preferably for therapeutic uses.
[00254] In the present invention, for an ADCC assay, human PBMCs were isolated by Ficoll centrifugation using the same density difference. PBMCs were incubated in RPMI (Thermo Fisher Scientific) and 10% FBS with IL-2 (100 U/ml) overnight cultured. Target cells (cell lines expressing 4-1BB) were collected, resuspended in culture medium at 1 ml, and labeled with 5μM CFSE at 37°C for 5 minutes. Effector/target cells of the present invention were washed at a ratio of 10:1, counted and then dispensed. For analysis, an antibody of the present invention was prepared to a final concentration of 10 nM (1.5 μg/mL), and plated at 37°C for 4 hours. 5 μl of 7-AAD was added to each well and transferred to a FACS tube, and then the sample was analyzed by FACS manufactured by BDFACScan. The frequencies of non-viable target cells (CFSE+ 7-AAD+) and viable target cells (CFSE+ 7-AAD-) were measured. ADCC was assessed as a viable cell frequency of total cells (FIG. 10A).
[00255] A complement dependent cytotoxicity (CDC) assay was conducted similarly to the ADCC assay described above, using FACS as a readout, with the above target cells incubated with anti-4-1BB antibodies on ice for 30 minutes and then added the supplemented human serum at a final concentration of 20% at 37°C for 30 minutes. Subsequently, the resulting samples were transferred to a FACS tube, and evaluated by FACS manufactured by BDFACScan (FIG. 10B). The results in FIG. 10A and FIG. 10B confirm that an exemplary humanized 4-1BB antibody, EU101, has almost no ADCC and CDC effects. Therefore, it can be said that an exemplary EU101 antibody of the present invention has beneficial ADCC and CDC properties for an agonist antibody, and is a good candidate for anti-cancer treatment in vivo.Example 4 - Confirmation of the in vivo efficiency of an anti-4 antibody -1BB exemplary humanized human
[00256] The anti-human 4-1BB antibody, EU101, of the present invention showed a dose-dependent effect in an in vitro example, and showed an effect considerably superior to a conventional antibody. This example is to verify whether the anti-human anti-human 4-1BB, EU101, is capable of being used alone or in combination with a different composition to diagnose, prevent or treat cancer or tumor in vivo, and to effectively inhibit growth. of tumor.4.1 NOD-scid mouse IL2Rgamanull graft from human peripheral blood mononuclear cells and anti-tumor activity of human anti-4-1BB antibody.
[00257] Peripheral venous blood collected from a healthy HLA-A24 donor was treated with heparin, and subjected to concentration gradient centrifugation in Ficoll-paque (GE Healthcare, Piscataway, NJ) to collect PBMCs. PBMCs were washed with RPMI-1640 medium, and 3x10 6 cells were injected intraperitoneally into immunodeficient mice, ie, NSG mice (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ; NOD-scid IL2rynull, Jackson Laboratory).
[00258] Analysis of humanized mice was performed by flow cytometry to verify if human T cells were present in blood collected from mice by collecting mouse orbital blood after 5 weeks of engraftment of human PBMCs. 7-week-old NSG mice (Jackson Laboratory, Barharbor, ME) were engineered under a specific pathogen-free environment (SPF).
[00259] Flow cytometry was performed to verify CD4 and CD8 ratios after cells were stained with human blood cell markers such as a fluorescently labeled CD45 antibody APC-cy7 and a fluorescently labeled CD4 antibody FITC and a CD8 antibody fluorescently labeled BV510. After collecting orbital blood from each mouse, human T cells from mouse blood samples were observed to see if a human immune system is engrafted into the mouse. Human tumor cells were prepared in an HLA-like humanized mouse model and 1 x 10 7 cells were subcutaneously injected into the back of each mouse. When a tumor size reached 100 to 200 mm3, an exemplary human anti-4-1BB antibody preparation (EU101) was intravenously administered at 1.0 mg, 5.0 mg, or 10.0 mg per 1 kg of body weight once every 5 days for a total of 3 times. As a control, human IgG was used. Tumor volume (mm 3 ) of each mouse was measured every 3 days (FIG. 11). Results shown in FIG. 11 confirm that tumor size in mice treated with an exemplary human anti-4-1BB antibody (EU101) was reduced relative to mice treated with human IgG, and furthermore that this reduction was proportional to antibody concentration. Particularly, tumor regression in a group administered 5 mg/kg of antibody occurred rapidly. Within one week after administration at a dose of 5 mg/kg, tumor size was established in a humanized mouse and tumor growth was eradicated. Therefore, an exemplary antibody EU101 of the present invention shows an anti-cancer effect in vivo.
[00260] Accordingly, the above results show that an exemplary human anti-4-1BB antibody (EU101) that specifically recognizes an epitope (SEQ ID NO: 15) of H4-1BB, however due to improved characteristics of this exemplary antibody, such as , for example, improved affinity, this antibody shows superior effects in an in vivo mouse model. Thus, the example suggests that an antibody encompassed by the present invention can be used as an anti-cancer agent at a lower dosage than the reference antibody. 4.2 Effects of tumor growth inhibition with an exemplary human anti-4-1BB antibody and an agent anti-PD-1 Comparison of effects caused by individual treatment of an exemplary human anti-4-1BB antibody (EU101) and an exemplary anti-PD-1 agent after tumor injection into humanized mice.
[00261] Humanized mice were prepared by the same method described in Example 4.1 above. To perform an experiment confirming an increase in anti-cancer effect according to doses of an exemplary human anti-4-1BB antibody (EU101) and an exemplary anti-PD-1 agent (Keytruda) (purchased from MSD, GER), 1x107 cells of a human colorectal adenocarcinoma cell line combined with the HLA-A, HT29, were subcutaneously injected into the previously prepared humanized mice. When the injected tumor volume reached 100 to 150 mm3, the mice were divided into a total of 5 groups of three mice, and to compare the effect of EU101 on tumor inhibition, each group of mice was treated with each of three conditions of administration (Control: IgG, treated group 1: 5 mg/kg, and treated group 2: 10 mg/kg) at 5-day intervals 3 times, and for anti-PD-1, the same procedures were performed (FIG. 12 ). As a result of the experiment, in both cases of EU101 and keytruda (anti-PD1), tumor volumes were reduced in a dose-dependent manner. However, in FIG. 12, 5 mg/kg of EU101 did not have an influence on tumor growth, however according to treatment with 5 mg/kg and 10 mg/kg of EU101, an antitumor activity was displayed in a dose-dependent manner. Furthermore, it was confirmed that EU101 exhibited greater efficiency at a lower dose than keytruda (anti-PD-1), and tumor growth was completely blocked particularly by treatment with 5 mg/kg of EU101.Treatment of humanized mice with combination of EU101 and an anti-PD-1 agent after tumor injection
[00262] Since co-inhibitory receptor signals (PD-1 and CTLA-4) and a costimulatory T cell signal (CD137) are differentiated for the same purpose of inhibiting tumor growth, stimulation of the two receptors can expect a synergistic effect ( Chen et al., Cancer Immunol. Res. (2015) 3: 149-160 ; Bartkowiak et al., Front. Oncol. (2015) 5: 117 , both incorporated herein by reference . In addition, PD1 immunotherapy has shown the possibility of an anticancer treatment effect for some cancer patient populations, but administration of a low dose in combination therapy with a different anticancer agent may still be required in a larger patient population. To investigate the antitumor effect caused by a combination therapy of an exemplary human anti-4-1BB antibody (EU101) and an exemplary anti-PD-1 agent (Keytruda), humanized tumor-bearing mice were treated with the combination therapy of EU101 and Keytruda. The preparation of humanized mice was carried out by the same method as described in Example 4.1.
[00263] Eye bleed was performed to identify humanized mice. Among humanized mice, HT29 colon carcinoma was subcutaneously injected into HLA-A24 mice maintaining a normal condition at 1x107 cells/mice. When the tumor size was 300 to 450 mm3, an experiment was performed as follows.
[00264] As known from this example, although tumor growth was not retarded with single injection at minimum concentration or less (EU101: 2.5 mg/kg, Keytruda (manufactured by MSD, GER): 2.5 mg/kg), tumor was greatly regressed with combination therapy of EU101 and Keytruda. This is the result that shows that exemplary human anti-4-1BB antibodies provided herein (eg, EU101) are good candidates for combination therapy with different anticancer agents, including in combination with one or more immunological checkpoint inhibitors. (FIG. 13).Analysis of T-cell-infiltrating lymphocytes (TILs) in normal tissue and human colorectal adenocarcinoma tissue after treatment alone and in combination with an exemplary human anti-4-1BB antibody and an anti-PD-1 agent exemplar.
[00265] After individual administration of an exemplary human anti-4-1BB antibody (EU101) and an exemplary anti-PD-1 agent (Keytruda) (purchased from MSD, GER) and combination administration of EU101 and Keytruda to implanted humanized mice HT29, on the day the effect analysis is finished, all groups were dissected to separate tumor and blood. After the detached tumor was treated with collagenase IV at 37°C for 30 minutes, the tumor tissue cells were dissociated by a mechanical method and then washed with 1xPBS. PBMCs were separated from separated blood by Ficoll gradient centrifugation, and tumor cells separated and PBMCs were subjected to the following experiment. Red blood cells (RBCs) were removed from washed cells using RBC lysis buffer and then washed with 1xPBS. Entangled cell fragments were removed from washed cells using a 40 μm nylon cell filter to create a single cell state, and single cells were washed with 1xPBS, followed by counting separate T cells from each group using a cell counter. .
[00266] The sorted T cells were stained with human blood cell markers such as a CD45 antibody (labeled by fluorescent APC-cy7), a fluorescent FITC-labeled human CD4 antibody, and a fluorescent BV510-labeled human CD8 antibody and then subjected to the FACS test. The FACS assay was performed based on a ratio (%) of groups of CD4 and CD8 cells, which were excluded from the CD45 group (FIG. 14A).
[00267] Particularly, to identify a Treg group among the separated T cells, the cell surfaces were stained with human blood cell markers, such as a CD45 antibody (labeled with fluorescent APC-cy7), a human FITC-labeled CD4 antibody fluorescent antibody and a human fluorescent PE-cy5 labeled CD25 antibody, and intracellular and intranuclear staining with a Foxp3 cellular transcription factor (human fluorescent APC labeled Foxp3 antibody) was performed using a Foxp3/Transcription Factor Staining Buffer Set kit (ebioscience). In the FACS trial, a CD45 group was split to close the R1 channel, a CD4+ CD25high group was split to close R2, and a ratio (%) of a Foxp3high group was measured in the R1 and R2 groups. To identify IFN-γ+ CD8+ T cells in the sorted cells, cell surfaces were stained with blood cell markers such as fluorescent APC-cy7-labeled human CD45 antibody and fluorescent BV510-labeled human CD8 antibody, fixed with PFA at 2%, and reacted with a solution of 0.5% saponin and fluorescent PE-cy7 labeled human IFN-γ antibody. Subsequently, IFN-γ+ cytokine cells in the CD8 T cell group were measured by FACS assay. Cells were identified in a ratio by the same method as described above and a proportional ratio of the CD8+IFN-y+ ratio and the Treg ratio was calculated, shown in FIG. 14B.
[00268] According to the result of this modality, different from the individual administration, the combination administration of EU101 and Keytruda greatly increased the infiltration of the combination of tumor tissue and a T lymphocyte. Other more specific results of the combination treatment are as follows. When combination treatment was performed on the PBMCs in the healthy humanized mouse as a control, the number of lymphocytes increased approximately 3-fold, and the lymphocytes infiltrated by 1 g of tumor increased 76-fold in the tumor tissue. This means that most tumor-specific lymphocytes were activated and recruited into the tumor tissue to kill the target cells. Particularly, when PBMCs in the combination therapy group were measured, as shown in FIG. 14A, CD4+ T cells did not increase greatly, but CD8+ cytotoxic T cells were increased approximately 5-fold. In addition, the combination therapy group showed a 100-fold increase in CD8+ T cell counts per 1 g of tumor tissue. Furthermore, as a result, a ratio of CD8+ T cells secreting IFN-γ and regulatory T cells was also greatly increased (FIG. 14B). That is, it can be said that the treatment with a combination of EU101 and anti-PD-1 agent provides a marked increase in effector T cells and, thus, tumor inhibition is effectively performed.Analyses of IFN-Y in serum or tumor fluid obtained from human colorectal adenocarcinoma tissue after individual and combination treatment with an exemplary human anti-4-1BB antibody (EU101) and an exemplary anti-PD-1 agent (Keytruda)
[00269] After single administration and a combination administration of an exemplary human anti-4-1BB antibody (EU101) and an exemplary anti-PD-1 agent (Keytruda) to humanized mice implanted with HT29. On the day the effect analyzes were completed, all groups were dissected to separate tumor and blood. In tumor dissection to separate a tumor fluid present in the separated tumor, 300 μl of 1x PBS was injected into the upper portion of a tumor membrane using a 1 cc syringe, and a fluent solution is withdrawn from the lower portion of the tumor membrane using a insulin syringe. Furthermore, in the dissociation of tumor tissue, the taken solution was added to dissociate the tumor tissue and then stored. In addition, as serum, serum was stored when PBMCs were separated from blood by Ficoll gradient centrifugation. Stored serum and a tumor fluid were dissolved and filtered using a 0.22 μm filter unit (manufacturer: Corning). 10 μl of serum was used for each group, and 100 μl of tumor fluid was used to measure human IFN-β and human TGF-β using a Human IFN-Y ELISA Ready-SET-Go kit (eBioscience) and a kit Human TGF beta 1 Ready-SET-Go ELISA (eBioscience). Results were analyzed by comparing the standard curve provided in each ELISA kit.
[00270] As a result, compared to the individual administration of EU101 and Keytruda, in the combination administration, the interferon concentration in the serum of the tumor group was the highest. Since an EU101 mechanism can be explained with a correlation between IFN-y and an antitumor effect, expression levels of IFN-y and TGF-β in serum from a healthy donor and serum from a tumor group to which therapy combination was applied, were evaluated. According to the example material in healthy donor serum, in the combination therapy group shown in FIG. 15A, IFN-y was increased approximately 16-fold, however a cytokine secreted from Treg cells, TGF-β, was reduced approximately 65%. Furthermore, in FIG. 15B, the concentration of IFN-γ caused by administration of the combination in the tumor fluid was considerably higher (approximately 213-fold) than in the control. As a result of the examples, due to EU101 particularly, compared to the control group, the combination group showed marked increases in IFN-γ secretion. Therefore, it can be confirmed that the anti-cancer effect caused by an improved anti-humanized 4-1BB antibody of the present invention provides effective tumor infiltration of effector T cells directly related to cancer cell apoptosis, and a considerably tissue-specific effect. tumor compared to the untreated group. In other words, in the present invention, it was confirmed that EU101, as an anti-cancer agent, has the ideal conditions for apoptosis of cancer cells. Conventionally, in cancer patients, anti-cancer cytokine and anti-cancer cellular immunity were considerably reduced, however, EU101 can be expected in the present invention to induce increases in anti-cancer cytokine and anti-cancer cellular immunity, resulting in considerable therapeutic effect.
[00271] Thus, an exemplary human anti-4-1BB antibody EU101 exhibits an antitumor effect mediated by high expression of IFN-γ, and such an effect is exhibited in a dose-dependent manner, as such, a concentration of IFN-γ in a serum from a cancer patient can be used as a biomarker to diagnose and estimate the tumor. Therefore, according to the effective treatment of cancer or tumor by the combination treatment of EU101 and anti-PD-1 and prognosis by measuring an IFN-γ concentration, it is expected to carry out more effective treatment with respect to each patient. . Example 5 - Ex vivo separation and massive proliferation of 4-1BB+CD8+ T cells using an exemplary humanized humanized anti-4-1BB antibody
[00272] The inventors used expression of 4-1BB on antigen-specifically activated CD8+ T cells in isolation and purification of various antigen-specific 4-1BB+CD8+ T cells using an anti-4-1BB antibody (Korean Patent No. 10 -1503341). A subsequent experiment was performed to examine whether the EU101 antibody developed here is also used for isolation and mass proliferation of antigen-specific CD8+ T cells.
[00273] Construction of PBMCs from peripheral blood from a cancer patient was performed as described in Example 4.1. However, in this example, cancer antigen-specific undifferentiated T cells can be obtained by the method described in Korean Patent Application No. 10-2016-0165224, filed by the inventors. In this example, for efficient sorting of 4-1BB+CD8+ T cells and mass production of 4-1BB+CD8+ T cells with high purity, a paniculation method using a human anti-4-1BB antibody (EU101) was used. 10 μg/ml of the human anti-4-1BB antibody (EU101) diluted in PBS was added to a 10 ml vial and stored at 4°C for 20 to 24 hours. After storage, a supernatant containing the antibody was removed, and without washing, a 2.5% solution of BSA dissolved in PBS was added to the cell pellets in the 10 ml flask and then stored at 4 °C for 20 to 24 hours. . Subsequently, the BSA solution was removed, and each vial was washed twice with 15 ml of PBS. The previously prepared cells were suspended in X-VIVO10 medium, added to a vial coated with EU101 antibody and then incubated at 37°C in a CO2 incubator for 1 hour. After incubation, a supernatant was removed, and cell pellets were washed twice with 10 ml of RPMI1640 medium to remove non-specifically binding cells. 1% autoserum and an X-VIVO10 medium containing IL-2 at 1000 IU/ml was added to the flask, followed by culture for 14 days. In the example, some cells were harvested and then stained to measure the purity and phenotypes of the isolated cells. As shown in FIGS. 16A and 16B, it was confirmed that, prior to paniculation with the 94 kvt antibody, an antigen-specific 4-1BB+CD8+ T cell ratio increased by 43.2% (CD8+ T cell ratio: 58.6%), and in after paniculation with the EU101 antibody, an antigen-specific pCMV+CD8+ T cell ratio increased by 60.0% (CD8+ T cell ratio: 79.3%). This means that antigen-specific 4-1BB+CD8+ T cells can be isolated with high purity using an EU101. Antigen-specific 4-1BB+CD8+ T cells isolated as described above can be easily mass produced as described in Korean Patent Application No. 10-2016-0165224 filed by the inventors.
[00274] From the above description, it will be understood by those skilled in the art that the present invention can be carried out in different specific ways without altering the technical idea or essential features of the present invention. However, it is not intended to limit the present invention to specific exemplary embodiments, and it is to be understood that all modifications or modified forms deduced from the meaning and scope of the following claims and equivalents thereof are included within the scope of the present invention, in instead of the detailed description.
[00275] Human anti-4-1BB antibodies encompassed by the present invention have demonstrated several beneficial properties, such as, for example, greater affinity than a reference antibody, and/or can be used alone or in combination with another anti-cancer agent to diagnose , prevent or treat cancer or tumor, or used to inhibit the growth of cancer.
[00276] Above, the present invention has been described with reference to the examples, but it will be understood by those skilled in the art that the present invention may be changed or modified in various ways without departing from the spirit and scope of the present invention, which is described in the accompanying claims.EQUIVALENTS
[00277] Those skilled in the art will recognize, or be able to verify using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. The scope of the present invention is not intended to be limited by the above description, but rather is as set out in the claims.

权利要求:
Claims (23)
[0001]
1. Anti-4-1BB antibody, characterized by a heavy chain variable domain consisting of SEQ ID NO: 14, a light chain variable domain consisting of SEQ ID NO: 10 and a heavy chain constant domain consisting of SEQ ID NO : 23.
[0002]
2. Anti-4-1BB antibody according to claim 1, characterized in that it has a binding affinity (KD) for a human 4-1BB molecule from 1xio-7 to ixio-12 M.
[0003]
3. Anti-4-1BB antibody according to claim 1 or 2, characterized in that it binds to an epitope within the extracellular domain of human 4-1BB polypeptide.
[0004]
4. Anti-4-1BB antibody according to claim 3, characterized in that binding to an epitope within the extracellular domain of human 4-1BB is abrogated by one or more mutations at positions N30, D38, N39, and R41 of SEQ ID NO: 44.
[0005]
5. Anti-4-1BB antibody according to any one of claims 1 to 4, characterized in that the antibody does not bind or binds weakly to a canine 4-1BB polypeptide.
[0006]
6. Anti-4-1BB antibody according to any one of claims 1 to 5, characterized in that the antibody is a humanized antibody.
[0007]
7. Pharmaceutical composition, characterized in that it comprises: (a) the anti-4-1BB antibody as defined in any one of claims 1 to 6; and (b) a pharmaceutically acceptable carrier.
[0008]
8. Use of an anti-4-1BB antibody as defined in any one of claims 1 to 6, characterized in that it is in the manufacture of a drug and/or composition to treat an individual in need thereof.
[0009]
9. Use of an anti-4-1BB antibody as defined in any one of claims 1 to 6, characterized in that it is in the manufacture of a drug and/or composition to induce an immune response in an individual in need thereof.
[0010]
10. Use of an anti-4-1BB antibody as defined in any one of claims 1 to 6, characterized in that it is in the manufacture of a drug and/or composition to enhance an immune response or increase the activity of an immune cell in an individual in need of the same.
[0011]
11. Use according to any one of claims 8 to 10, characterized in that the individual has or is at risk of developing cancer.
[0012]
12. Use according to claim 11, characterized in that the cancer is selected from bladder cancer, breast cancer, cervical cancer, colon cancer, endometrial cancer, esophageal cancer, fallopian tube cancer, cancer gallbladder cancer, gastrointestinal cancer, head and neck cancer, hematologic cancer, laryngeal cancer, liver cancer, lung cancer, lymphoma, melanoma, mesothelioma, ovarian cancer, primary peritoneal cancer, salivary gland cancer, sarcoma, ovarian cancer stomach cancer, thyroid cancer, pancreatic cancer, and prostate cancer.
[0013]
13. A method of determining a dose of an anti-4-1BB antibody thereto for the therapeutic treatment of an individual in need thereof, comprising; (a) providing or obtaining a measurement of secreted IFN-gamma in a biological sample from the subject, wherein the subject has been administered a composition that comprises or releases an amount of an anti-4-1BB antibody as defined in any one of claims 1 to 6; and (b) comparing the measurement of secreted IFN-gamma to a reference value, wherein if the measurement of secreted IFN-gamma is greater or less than the reference value, adjusting the amount of an anti-4-1BB antibody thereof will be administered, to determine a dose for therapeutic treatment of a subject.
[0014]
14. Method according to claim 13, characterized in that the reference value comprises an index value that includes a value derived from one or more healthy individuals, a value derived from one or more individuals diagnosed with cancer or a value derived from a cancer risk prediction algorithm.
[0015]
15. Method according to claim 13 or 14, characterized in that the biological sample is a sample of whole blood, plasma or serum.
[0016]
16. Method according to any one of claims 13 to 15, characterized in that the individual has or is at risk of developing cancer.
[0017]
17. Method according to claim 16, characterized in that the cancer is selected from bladder cancer, breast cancer, cervical cancer, colon cancer, endometrial cancer, esophageal cancer, fallopian tube cancer, cancer gallbladder cancer, gastrointestinal cancer, head and neck cancer, hematologic cancer, laryngeal cancer, liver cancer, lung cancer, lymphoma, melanoma, mesothelioma, ovarian cancer, primary peritoneal cancer, salivary gland cancer, sarcoma, ovarian cancer stomach cancer, thyroid cancer, pancreatic cancer, and prostate cancer.
[0018]
18. Use of an anti-4-1BB antibody as defined in any one of claims 1 to 6, characterized in that it is in the manufacture of a medicament and/or composition for increasing IFN-Y secretion by an ingrown cell. in vivo, in which the cell comes into contact with the anti-4-1BB antibody.
[0019]
19. Method for increasing IFN-y secretion by a cell in vitro, characterized in that it comprises: contacting the cell with an anti-4-1BB antibody as defined in any one of claims 1 to 6.
[0020]
20. Method for ex vivo proliferation or isolation of activated T cells, comprising: contacting a population of T cells with an anti-4-1BB antibody as defined in any one of claims 1 to 6, thereby increasing proliferation of activated T cells.
[0021]
21. Method for isolating antigen-specific activated T cells, characterized in that it comprises: (a) culturing peripheral blood mononuclear cells (PBMC) in a medium together with a peptide of an epitope of interest and IL-2;( b) inducing 4-1BB expression in the cultured cells by adding the epitope peptide of interest; (c) contacting the cultured cells with a surface coated with an anti-4-1BB antibody as defined in any one of claims 1 to 6, in that cultured cells expressing 4-1BB adhere to the coated surface; and (d) removing the unbound cells, thereby isolating the antigen-specific activated T cells.
[0022]
22. Method according to claim 23 or 24, characterized in that the activated T cells are CD8+ T cells.
[0023]
23. Use of activated T cells produced by the method as defined in any one of claims 23 to 25, characterized in that it is in the manufacture of a drug and/or composition to treat or prevent cancer in an individual.

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WO2022029496A1|2020-08-07|2022-02-10|Eutilex Co., Ltd.|Anti-her2 / anti-4-1bb bispecific antibodies and uses thereof|

法律状态:
2020-02-11| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|

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2020-05-12| B07E| Notification of approval relating to section 229 industrial property law [chapter 7.5 patent gazette]|

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2020-10-27| B07A| Application suspended after technical examination (opinion) [chapter 7.1 patent gazette]|

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2021-06-08| B06A| Patent application procedure suspended [chapter 6.1 patent gazette]|

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2021-10-05| B350| Update of information on the portal [chapter 15.35 patent gazette]|

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2021-11-09| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2022-01-18| 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 05/01/2018, OBSERVADAS AS CONDICOES LEGAIS. |

优先权:

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申请号 | 申请日 | 专利标题

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US201762443281P| true| 2017-01-06|2017-01-06|

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US62/443,281|2017-01-06|

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PCT/IB2018/000043|WO2018127787A1|2017-01-06|2018-01-05|Anti-human 4-1 bb antibodies and use thereof|

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