Anti-PD-1 polypeptides and their uses

Abstract

The present application provides anti-PD-1 polypeptides or fragments thereof. Further provided are methods of using the antibodies or fragments thereof to treat and diagnose diseases, such as cancer, infection, or immune diseases.

Description

Technical Field

[0001] The present application relates to anti-PD-1 polypeptides (including anti-PD-1 antibodies and immunologically active fragments thereof), isolated nucleic acids encoding anti-PD-1 antibodies or immunologically active fragments thereof, and in particular to the treatment of diseases in which diseased cells utilize the PD-1 / PD-L1 checkpoint for immune evasion. The present application particularly relates to humanized anti-PD-1 antibodies and antigen-binding fragments thereof that can enhance immune system activation against diseased tissues (including cancer cells and infected cells expressing PD-L1 and / or PD-L2). Background Art

[0002] The cDNA for programmed cell death protein 1 (PD-1) was isolated in 1992 from a mouse T cell hybridoma and an apoptotic hematopoietic progenitor cell line. Studies have shown that PD-1 deficiency leads to distinct autoimmune phenotypes in multiple mouse strains. PD-1-deficient allogeneic T cells carrying a transgenic T cell receptor (TCR) exhibit enhanced responses to allogeneic antigens, suggesting that PD-1 on T cells plays a negative regulatory role in the antigen response.

[0003] Several studies have led to the discovery of molecules that interact with PD-1. In 1999, the B7 homolog 1 of PD-1 (B7-H1, also known as programmed death ligand 1 [PD-L1]) was identified and shown to be an inhibitor of human T cell responses in vitro. B7-H1 (hereafter referred to as PD-L1) was later shown to be a binding and functional partner of PD-1. Subsequently, it was determined that mice deficient in PD-L1 (PD-L1 knockout mice) are prone to autoimmune diseases, although these mice do not spontaneously become ill. It was subsequently clarified that the PD-L1 / PD-1 interaction in vivo plays an important role in the suppression of T cell responses, especially in the tumor microenvironment.

[0004] In addition, studies have shown that tumor-associated PD-L1 promotes activated T cell apoptosis ((Dong H. et al., Tumor-associated B7-H1 promotes T-cell apoptosis: a potential mechanism of immune evasion. Nature medicine. 2002; 8(8): 793-800) and stimulates human peripheral blood T cells to produce IL-10 ((Dong H et al., B7-H1, a third member of the B7 family, co-stimulates T-cell proliferation and interleukin-10 secretion. Nature medicine. 1999; 5(12): 1365-9) to mediate immunosuppression. It is well known that the effect of PD-L1 on immunosuppression is more complex. In addition to T cell apoptosis and IL-10 induction, PD-L1 can also induce T cell dysfunction through various mechanisms. The PD pathway has also been shown to promote T cell dysfunction in vitro and in vivo.

[0005] In recent years, the FDA has approved two PD-1 monoclonal antibodies (mAbs) for the treatment of human cancers, one from Bristol-Myers Squibb (Opdivo, nivolumab, MDX-1106, BMS-936558, ONO-4538) and the other from Merck (Keytruda, pembrolizumab, lambrolizumab, MK-3475). Furthermore, multiple monoclonal antibodies targeting PD-1 or PD-L1 are being actively developed in hundreds of clinical trials involving thousands of patients. To date, anti-PD therapy has provided substantial clinical benefit by inducing regression of advanced and metastatic tumors and improving survival. More importantly, anti-PD therapy has durable effects, tolerable toxicities, and has been shown to be effective across multiple cancer types, particularly solid tumors. Due to its non-overlapping mechanisms relative to other cancer therapies, anti-PD therapy is being combined with virtually all cancer treatments in an attempt to further expand therapeutic efficacy. In addition to combining with various cancer immunotherapies (such as cancer vaccines, co-stimulatory and co-inhibitory antibodies, and adoptive cell therapy), various clinical trials have also begun to combine anti-PD-1 therapy with chemotherapy, radiotherapy, and targeted therapy.

[0006] Although antibodies against PD-1 have been developed, there is still room for improvement in PD-1 antibodies as therapeutic agents. Therefore, there is a need in the art to develop new anti-PD-1 antibodies with higher specificity and efficacy. Summary of the Invention

[0007] The present application provides antibodies and immunoreactive fragments thereof that bind to PD-1 molecules expressed on cells (e.g., cancer cells) with high affinity and promote an effective immune response against cancer cells. The antibodies and immunoreactive fragments thereof provided herein can enhance the activation of the immune system, thereby providing important therapeutic and diagnostic agents for targeting pathological conditions associated with the expression and / or activity of PD-1 molecules. In one aspect, the present application provides a separated antibody or antigen-binding fragment thereof, comprising a heavy chain (HC) variable region sequence and a light chain (LC) variable region sequence, wherein the antibody binds to the extracellular domain of PD-1 with a binding affinity better than 10nM or about 10nM, better than 8nM or about 8nM, better than 6nM or about 6nM, better than 4nM or about 4nM, better than 2nM or about 2nM, better than 1nM or about 1nM, and the affinity is determined by SPR analysis , for example, about 0.5-4 nM, about 0.8-4.0 nM, about 1.0-4.0 nM, about 2.0-4.0 nM, about 3.0-4.0 nM, about 0.6-3.5 nM, about 1.4-3.5 nM, about 2.5-3.5 nM, about 0.7-2.5 nM, about 0.8-2.0 nM, about 1.0-2.0 nM, about 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM, or better, and the affinity is determined by SPR analysis.

[0008] In certain embodiments, the present application provides an antibody or antigen-binding fragment thereof comprising at least one of the following:

[0009] (a) CDR1H sequence comprising GFTFSSYGMS (SEQ ID NO: 1).

[0010] (b) CDR2H sequence comprising IISGGGRDIYYLDSVKG (SEQ ID NO: 2).

[0011] (c) CDR3H sequence comprising PIYDAYSFAY (SEQ ID NO: 3).

[0012] (d) CDR1L sequence comprising RASQTISNNLH (SEQ ID NO: 4).

[0013] (e) comprising the CDR2L sequence of YASQSIS (SEQ ID NO: 5), and

[0014] (f) CDR3L sequence comprising QQSYSWPLT (SEQ ID NO: 6).

[0015] In certain embodiments, the present application provides an antibody or antigen-binding fragment thereof, wherein

[0016] (a) The HC comprises:

[0017] The CDR1H sequence comprises GFTFSSYGMS (SEQ ID NO: 1).

[0018] a CDR2H sequence comprising IISGGGRDIYYLDSVKG (SEQ ID NO: 2), and

[0019] The CDR3H sequence comprising PIYDAYSFAY (SEQ ID NO: 3).

[0020] (b) the LC comprises:

[0021] The CDR1L sequence comprises RASQTISNNLH (SEQ ID NO: 4).

[0022] comprising the CDR2L sequence of YASQSIS (SEQ ID NO: 5), and

[0023] The CDR3L sequence comprising QQSYSWPLT (SEQ ID NO: 6).

[0024] The CDR sequences were determined according to Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda MD (1991), vols. 1-3.

[0025] In certain embodiments, the antibody is a chimeric antibody, a humanized antibody or a human antibody. In certain embodiments, the antibody of the present invention or its antigen-binding fragment further comprises a human acceptor framework. In certain embodiments, the human acceptor framework is derived from a human immunoglobulin framework or a human consensus framework. In certain embodiments, the human acceptor framework comprises a κI subtype framework sequence for VL and a III subtype framework sequence for VH. Generally speaking, the subtype of the sequence is a subtype as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, NIH Publication 91-3242, Bethesda MD (1991), volumes 1-3. In certain embodiments, for VL, the subtype is a κI subtype as described in Kabat et al. (ibid). In certain embodiments, for VH, the subtype is a III subtype as described in Kabat et al. (ibid).

[0026] In certain embodiments, the antibodies or antigen-binding fragments thereof comprise a human consensus framework. In some embodiments, the antibodies or antigen-binding fragments thereof comprise a human consensus framework with changes in the amino acid sequence, such as, for example, changes in 1-15, 1-10, 2-9, 3-8, 4-7, or 5-6 amino acids.

[0027] In certain embodiments, the antibodies or antigen-binding fragments thereof of the present application comprise an HC variable region sequence comprising the amino acid sequence set forth in SEQ ID NO: 7 or 8, or an amino acid sequence having greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 7 or 8. In certain embodiments, the antibodies or antigen-binding fragments thereof of the present application comprise an LC variable region sequence comprising the amino acid sequence set forth in SEQ ID NO: 9 or SEQ ID NO: 10, or an amino acid sequence having greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 9 or 10. In certain embodiments, the HC variable region sequence comprises the amino acid sequence of SEQ ID NO: 7, and the LC variable region sequence comprises the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 10. In certain embodiments, the HC variable region sequence comprises the amino acid sequence of SEQ ID NO: 8, and the LC variable region sequence comprises the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 10. In certain embodiments, the antibody or antigen-binding fragment thereof comprises an HC sequence comprising the amino acid sequence of SEQ ID NO: 11 or 12, or an amino acid sequence having greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 11 or 12. In certain embodiments, the antibody or antigen-binding fragment thereof of the present application comprises an LC sequence comprising the amino acid sequence of SEQ ID NO: 13 or 14, or an amino acid sequence having greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 13 or 14. In certain embodiments, the HC sequence comprises the amino acid sequence of SEQ ID NO: 11, and the LC sequence comprises the amino acid sequence of SEQ ID NO: 13 or SEQ ID NO: 14. In certain embodiments, the HC sequence comprises the amino acid sequence of SEQ ID NO: 12, and the LC variable region sequence comprises the amino acid sequence of SEQ ID NO: 13 or SEQ ID NO: 14. In certain embodiments, the antibody is of the IgG1, IgG2, or IgG4 isotype. In certain embodiments, the antigen-binding fragment comprises any one selected from the group consisting of Fab, F(ab')2, Fab', scFv, and Fv.In certain embodiments, the antibodies or antigen-binding fragments thereof of the present application are blocking antibodies or antagonist antibodies that inhibit or reduce the biological activity of the PD-1 molecule to which they bind. Preferably, the blocking antibodies or antagonist antibodies substantially or completely inhibit the biological activity of the PD-1 molecule.

[0028] In one aspect, the present application provides a bispecific antibody comprising the antibody or antigen-binding fragment thereof of the present application and a second antibody or antigen-binding fragment thereof. In certain embodiments, the second antibody or antigen-binding fragment thereof specifically binds to a tumor antigen expressed on the surface of a tumor cell, wherein the tumor antigen comprises any one selected from the group consisting of: A33; ADAM-9; ALCAM; BAGE; β-catenin; CA125; carboxypeptidase M; CD103; CD19; CD20; CD22; CD23; CD25; CD27; CD28; CD36; CD40 / CD154; CD45; CD46; CD5; CD56; CD79a / CD79b; CDK4; CEA; CTLA4; cytokeratin 8; EGF-R; EphA 2; ErbB1; ErbB3; ErbB4; GAGE-1; GAGE-2; GD2 / GD3 / GM2; HER-2 / neu; human papillomavirus-E6; human papillomavirus-E7; JAM-3; KID3; KID31; KSA(17-1A); LUCA-2; MAGE-1; MAGE-3; MART; MUC-1; MUM-1; N-acetylglucosaminyltransferase; oncostatin M; p15; PIPA; PSA; PSMA; ROR1; TNF-β receptor; TNF-α receptor; TNF-γ receptor; transferrin receptor; and VEGF receptor. In some embodiments, the second antibody or antigen-binding fragment thereof specifically binds to a checkpoint protein expressed on the surface of an abnormal cell or immune cell, wherein the immune checkpoint protein comprises any one selected from the group consisting of: 2B4; 4-1BB; 4-1BB ligand; B7-1; B7-2; B7H2; B7H3; B7H4; B7H6; BTLA; CD155; CD160; CD19; CD200; CD27; CD27 ligand; CD28; CD40; CD40 ligand; CD47; CD48; CTLA-4; DNAM-1; Galectin-9. GITR; GITR ligand; HVEM; ICOS; ICOS ligand; IDOI; KIR; 3DL3; LAG-3; OX40; OX40 ligand; PD-L1; PD-1; PD-L2; LAG3; PGK; SIRPα; TIM-3; TIGIT; VSIG8.

[0029] In one aspect, the present application provides a polypeptide comprising the antibody or antigen-binding fragment thereof of the present application.

[0030] In one aspect, the present application provides a polypeptide comprising the HC variable region and / or LC variable region of the antibody or antigen-binding fragment thereof of the present application.

[0031] In one aspect, the present application provides a conjugate comprising an antibody or antigen-binding fragment thereof of the present application. In certain embodiments, the present application provides a conjugate consisting of an antibody or antigen-binding fragment thereof of the present application, which is connected to a therapeutic agent. In certain embodiments, the therapeutic agent is a cytotoxin or a radioisotope.

[0032] In one aspect, the present application provides a composition comprising an antibody or antigen-binding fragment thereof, a bispecific antibody, a polypeptide, a conjugate, and a pharmaceutically acceptable carrier of the present application. In certain embodiments, the composition further comprises an anticancer agent. In certain embodiments, the agent is an antibody, a chemotherapeutic agent, a radiotherapeutic agent, a hormone therapy agent, a toxin, or an immunotherapeutic agent. In certain embodiments, the composition further comprises an antibody or agent that inhibits a checkpoint.

[0033] In one aspect, the present application provides an article of manufacture or kit for treating cancer, comprising an antibody or antigen-binding fragment thereof, bispecific antibody, polypeptide, conjugate or composition of the present application, and a package insert containing necessary information about the use of the antibody or antigen-binding fragment thereof, bispecific antibody, polypeptide, conjugate or composition related to the present application.

[0034] In one aspect, the present application provides an article of manufacture or kit for diagnosing cancer or determining the presence and / or amount of PD-1, comprising the antibody or antigen-binding fragment thereof of the present application, and a package insert containing necessary information on the use of the antibody or antigen-binding fragment thereof of the present application.

[0035] In one aspect, the present application provides isolated nucleic acids encoding the antibodies or antigen-binding fragments thereof of the present application. In certain embodiments, the present application provides isolated nucleic acids encoding the HC variable region and / or LC variable region of the antibodies or antigen-binding fragments thereof of the present application. In certain embodiments, the present application provides expression vectors comprising the nucleic acids, or host cells comprising the expression vectors.

[0036] In one aspect, the present application provides a method for preparing an antibody or an antigen-binding fragment thereof, comprising expressing the antibody or the antigen-binding fragment thereof in the above-mentioned host cell, and isolating the antibody or the antigen-binding fragment thereof from the host cell.

[0037] In one aspect, the present application provides a method for treating cancer, comprising administering an effective amount of the above-mentioned antibody or antigen-binding fragment thereof, bispecific antibody, polypeptide, conjugate, composition, product, or kit of the present application to a patient suffering from a cancer disease. In certain embodiments, the cancer comprises any one selected from the group consisting of lymphoma, melanoma, colorectal adenocarcinoma, prostate cancer, breast cancer, colon cancer, lung cancer, liver cancer, gastric cancer, and clear cell renal carcinoma.

[0038] In one embodiment, an effective amount of the above-mentioned antibody or its antigen-binding fragment, bispecific antibody, polypeptide, conjugate, composition, product or kit of the present application is the only therapeutic anti-cancer agent administered to the patient. In another embodiment, they can be administered in combination with another antibody or antibody fragment or anti-cancer agent, the antibody or antibody fragment or anti-cancer agent including but not limited to, antibodies against checkpoint molecules or their receptors (e.g., anti-CTLA-4 antibodies, anti-B7S1 antibodies, anti-PD-L1 antibodies, anti-B7H3 antibodies, etc.); anti-epidermal growth factor receptor (EGFR) agents such as panitumumab, anti-EGFR antibody cetuximab (cetuximab, ), and the EGFR tyrosine kinase (TK) inhibitor gefitinib ( ) and erlotinib ( ); alkylating agents such as cisplatin, carboplatin, oxaliplatin, nedaplatin, satraplatin, triplatin tetranitrate, nitrogen mustard, cyclophosphamide, chlorambucil, and ifosfamide; paclitaxel and docetaxel; and topoisomerase inhibitors such as, for example, irinotecan, topotecan, amsacrine, etoposide, etoposide phosphate, and teniposide.

[0039] In certain embodiments, the antibodies or antigen-binding fragments thereof, bispecific antibodies, polypeptides, conjugates, compositions, preparations, or kits of the present application as described above are administered in combination with another anti-PD-1 antibody or anti-PD-L1 antibody to achieve a synergistic effect in cancer treatment.

[0040] In one aspect, the present application provides a method for treating cancer, comprising administering an effective amount of an antibody or antigen-binding fragment thereof, a bispecific antibody, a polypeptide, a conjugate, a composition, a product or a kit of the present application to a subject suffering from a cancer disease. In certain embodiments, the cancer comprises any one selected from the group consisting of prostate cancer, breast cancer, colon cancer, lung cancer, liver cancer, gastric cancer and renal clear cell carcinoma, bladder cancer, breast cancer, colorectal cancer, endometrial cancer, esophageal cancer, head and neck cancer, kidney cancer, leukemia, liver cancer, lung cancer, lymphoma, melanoma, pancreatic cancer, prostate cancer and thyroid cancer. In certain embodiments, the cancer comprises any one selected from the group consisting of colorectal cancer with high microsatellite instability, colorectal cancer with stable microsatellites, triple-negative breast cancer, Merkel cell carcinoma, endometrial cancer or esophageal cancer.

[0041] On the one hand, the application provides a method for treating cancer, including a) treating activated T cells, B cells, NK cells, dendritic cells, monocytes or their combinations, or peripheral blood mononuclear cells (PBMC) in vitro with the above-mentioned antibodies or their antigen-binding fragments, bispecific antibodies, polypeptides, conjugates, compositions, products or kits of the application; and b) administering the treated T cells, B cells, NK cells, dendritic cells, monocytes or their combinations, or peripheral blood mononuclear cells (PBMC) to the patient. In some embodiments, the method further includes, before step a), isolating T cells, B cells, NK cells, dendritic cells or monocytes from an individual. In some embodiments, the T cells and / or NK cells are from a patient to be treated. In some embodiments, the T cells are tumor infiltrating T lymphocytes, CD4+T cells, CD8+T cells, or a combination thereof.

[0042] Therefore, in one aspect, the present application also provides a lymphocyte panel comprising T cells and / or NK cells from a subject, and treated in vitro with the above-mentioned antibodies or their antigen-binding fragments, bispecific antibodies, polypeptides, conjugates, compositions, products or kits of the present application. In some embodiments, the T cells and / or NK cells are from a patient to be treated. In some embodiments, the T cells are tumor-infiltrating T lymphocytes, CD4+ T cells, CD8+ T cells, or a combination thereof.

[0043] In one aspect, the present application provides a method for treating or inhibiting an infection in a patient in need thereof, comprising administering to the patient an effective amount of the above-mentioned antibody or antigen-binding fragment thereof, bispecific antibody, polypeptide, conjugate, composition, product, or kit of the present application. In certain embodiments, the infection is a viral, bacterial, fungal, or parasitic infection. In certain specific embodiments, the infection is HIV infection.

[0044] In one aspect, the present application provides a method for detecting or quantifying the expression or activity of a PD-1 polypeptide, comprising contacting an antibody or antigen-binding fragment thereof of the present application with a sample from a subject. In certain embodiments, the antibody or antigen-binding fragment thereof is labeled with a detectable substance. In certain embodiments, the antibody or antigen-binding fragment thereof is radiolabeled, fluorescently labeled, or enzyme-labeled.

[0045] In one aspect, the present application provides a method for predicting a subject's risk of developing cancer, the method comprising detecting, quantifying or monitoring the expression or activity of a PD-1 polypeptide by using the antibody or antigen-binding fragment thereof of the present application.

[0046] In one aspect, the present application provides a method for monitoring the effectiveness of a drug in treating cancer, wherein the cancer shows elevated expression or activity of PD-1, the method comprising detecting or quantifying the expression or activity of the PD-1 polypeptide by using the antibody or antigen-binding fragment thereof of the present application.

[0047] In one embodiment, the present application provides an isolated polynucleotide encoding a human anti-PD-1 antibody or a fragment thereof, wherein the antibody comprises an amino acid sequence selected from SEQ ID NOs: 7-14, preferably selected from SEQ ID NOs: 11-14. In certain embodiments, the human PD-1 antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises SEQ ID NO: 7 and the light chain comprises SEQ ID NO: 9 or SEQ ID NO: 10. In certain embodiments, the human PD-1 antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises SEQ ID NO: 8 and the light chain comprises SEQ ID NO: 9 or SEQ ID NO: 10. In certain embodiments, the antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises SEQ ID NO: 11 and the light chain comprises SEQ ID NO: 13 or SEQ ID NO: 14. In certain embodiments, the antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises SEQ ID NO: 12 and the light chain comprises SEQ ID NO: 13 or SEQ ID NO: 14. More preferably, the antibody comprises a heavy chain and a light chain, wherein the light chain comprises SEQ ID NO: 10 or SEQ ID NO: 14, and the heavy chain comprises SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 11 or SEQ ID NO: 12. In some embodiments, the antibody or antibody fragment comprises the VH and VL domains of a single-chain antibody fragment. In some embodiments, the VH domain comprises a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3. In some embodiments, the VH domain comprises three CDRs, wherein each of the three CDRs comprises a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3. In some embodiments, the VH domain comprises SEQ ID NO: 7 or SEQ ID NO: 8. In some embodiments, the VL domain comprises a sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6. In some embodiments, the VL domain comprises three CDRs, wherein each of the three CDRs comprises a sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6. In some embodiments, the VL domain comprises SEQ ID NO: 9 or SEQ ID NO: 10.

[0048] In one embodiment, the present application provides a method for diagnosing a disease, disorder, or condition associated with the expression of PD-1 on a cell, or determining the presence and / or amount of PD-1, wherein the method comprises a) contacting the cell with a human anti-PD-1 antibody or fragment thereof, wherein the antibody or fragment thereof comprises an amino acid sequence selected from SEQ ID NOs: 7-14; and b) detecting the presence of PD-1, wherein the presence of PD-1 is diagnostic of a disease, disorder, or condition associated with the expression of PD-1. In certain embodiments, the disease, disorder, or condition associated with the expression of PD-1 is cancer.

[0049] In one embodiment, the present application provides a method for diagnosing, prognosing, or determining the risk of a PD-1-related disease in a mammal, wherein the method comprises detecting the expression of PD-1 in a sample from the mammal, comprising a) contacting the sample with a human anti-PD-1 antibody or fragment thereof, wherein the antibody or fragment thereof comprises an amino acid sequence selected from SEQ ID NOs: 7-14; and b) detecting the presence of PD-1, wherein the presence of PD-1 is diagnostic of a PD-1-related disease in the mammal. In certain embodiments, the PD-1-related disease is cancer.

[0050] In one embodiment, the present application provides a method for blocking PD-1-dependent T cell, B cell, NK cell, dendritic cell or monocyte inhibition, wherein the method comprises contacting the cell with a human anti-PD-1 antibody or a fragment thereof, wherein the antibody or fragment thereof comprises an amino acid sequence selected from SEQ ID NO: 7-14. In some embodiments, the cell is selected from B cells, T cells or NK cells. In one embodiment, the present application provides a method for blocking PD-1-dependent immunosuppression in a mammal, wherein the method comprises administering an effective amount of the above-mentioned anti-PD-1 antibody or fragment thereof to the mammal. In certain embodiments, the mammal contains abnormal cells selected from T cells, B cells, NK cells, dendritic cells or monocytes expressing PD-1 and abnormal cells expressing PD-L1 and / or PD-L2.

[0051] In one embodiment, the present application provides a method for providing anti-tumor immunity in a mammal, wherein the method comprises administering to the mammal an effective amount of genetically modified cells encoding and expressing an anti-PD-1 antibody or a fragment thereof, wherein the anti-PD-1 antibody or fragment thereof comprises an amino acid sequence selected from SEQ ID NOs: 7-14.

[0052] Exemplary embodiments of the present application include:

[0053] 1. An isolated antibody or antigen-binding fragment thereof comprising a heavy chain (HC) variable region sequence and a light chain (LC) variable region sequence, wherein the antibody binds to the extracellular domain of PD-1 with a binding affinity better than 10 nM as determined by SPR analysis, wherein

[0054] (a) The HC comprises

[0055] CDR1H, wherein the CDR1H comprises the amino acid sequence shown in GFTFSSYGMS (SEQ ID NO: 1),

[0056] CDR2H, wherein the CDR2H comprises the amino acid sequence shown in IISGGGRDIYYLDSVKG (SEQ ID NO: 2), and

[0057] CDR3H, wherein the CDR3H comprises the amino acid sequence shown by PIYDAYSFAY (SEQ ID NO: 3).

[0058] (b) the LC comprises

[0059] CDR1L, wherein the CDR1L comprises the amino acid sequence shown in RASQTISNNLH (SEQ ID NO: 4),

[0060] CDR2L, wherein the CDR2L comprises the amino acid sequence shown in YASQSIS (SEQ ID NO: 5), and

[0061] CDR3L, wherein the CDR3L comprises the amino acid sequence represented by QQSYSWPLT (SEQ ID NO: 6).

[0062] 2. The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody is a chimeric antibody, a humanized antibody or a human antibody.

[0063] 3. The antibody or antigen-binding fragment thereof according to claim 1 or 2, further comprising a human acceptor framework.

[0064] 4. An antibody or antigen-binding fragment thereof as described in any one of items 1 to 3, wherein the HC variable region sequence comprises the amino acid sequence shown in SEQ ID NO: 7 or an amino acid sequence that is greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 7.

[0065] 5. The antibody or antigen-binding fragment thereof of any one of items 1-3, wherein the HC variable region sequence comprises the amino acid sequence shown in SEQ ID NO: 8 or an amino acid sequence that is greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 8.

[0066] 6. The antibody or antigen-binding fragment thereof of claim 4 or 5, wherein the LC variable region sequence comprises the amino acid sequence shown in SEQ ID NO: 9 or an amino acid sequence that is greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 9.

[0067] 7. The antibody or antigen-binding fragment thereof of claim 4 or 5, wherein the LC variable region sequence comprises the amino acid sequence shown in SEQ ID NO: 10 or an amino acid sequence that is greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 10.

[0068] 8. An antibody or antigen-binding fragment thereof as described in any one of items 1 to 7, wherein the HC variable region sequence comprises the amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 8, or an amino acid sequence that is greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO. 7 or 8, and the LC variable region sequence comprises the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 10, or an amino acid sequence that is greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 9 or 10.

[0069] 9. The antibody or antigen-binding fragment thereof according to any one of items 1 to 8, wherein

[0070] 1) the HC variable region sequence comprises the amino acid sequence of SEQ ID NO: 7, or an amino acid sequence that is greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 7, and the LC variable region sequence comprises the amino acid sequence of SEQ ID NO: 10, or an amino acid sequence that is greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 10, or

[0071] 2) the HC variable region sequence comprises the amino acid sequence of SEQ ID NO: 8, or an amino acid sequence that is greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 8, and the LC variable region sequence comprises the amino acid sequence of SEQ ID NO: 10, or an amino acid sequence that is greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 10.

[0072] 10. The antibody or antigen-binding fragment thereof of any one of items 1-9, wherein the antibody is of the IgG isotype.

[0073] 11. The antibody or antigen-binding fragment thereof of any one of items 1 to 10, wherein the antigen-binding fragment comprises any one selected from the group consisting of Fab, F(ab')2, Fab', scFv, Fv, Fd, dAb, and diabody.

[0074] 12. A bispecific antibody comprising the antibody or antigen-binding fragment thereof according to any one of items 1 to 11 and a second antibody or antigen-binding fragment thereof.

[0075] 13. The bispecific antibody of claim 12, wherein the second antibody or antigen-binding fragment thereof specifically binds to a tumor antigen expressed on the surface of a tumor cell, wherein the tumor antigen comprises any one selected from the group consisting of: A33; ADAM-9; ALCAM; BAGE; β-catenin; CA125; carboxypeptidase M; CD103; CD19; CD20; CD22; CD23; CD25; CD27; CD28; CD36; CD40 / CD154; CD45; CD46; CD5; CD56; CD79a / CD79b; CDK4; CEA; CTLA4; cytokeratin 8; EGF-R ; EphA2; ErbB1; ErbB3; ErbB4; GAGE-1; GAGE-2; GD2 / GD3 / GM2; HER-2 / neu; human papillomavirus-E6; human papillomavirus-E7; JAM-3; KID3; KID31; KSA(17-1A); LUCA-2; MAGE-1; MAGE-3; MART; MUC-1; MUM-1; N-acetylglucosaminyltransferase; oncostatin M; p15; PIPA; PSA; PSMA; ROR1; TNF-β receptor; TNF-α receptor; TNF-γ receptor; transferrin receptor; and VEGF receptor.

[0076] 14. A conjugate comprising the antibody or antigen-binding fragment thereof according to any one of items 1 to 11, linked to a therapeutic agent.

[0077] 15. The conjugate of claim 14, wherein the therapeutic agent is a cytotoxin or a radioisotope.

[0078] 16. A composition comprising the antibody or antigen-binding fragment thereof according to any one of items 1 to 11, the bispecific antibody according to item 12 or 13, or the conjugate according to item 14 or 15, and a pharmaceutically acceptable excipient.

[0079] 17. Lymphocytes comprising T cells and / or NK cells from a subject, which are treated in vitro with the antibody or antigen-binding fragment thereof according to any one of items 1 to 11.

[0080] 18. An isolated nucleic acid encoding the antibody or antigen-binding fragment thereof according to any one of items 1 to 11.

[0081] 19. An expression vector comprising the nucleic acid of item 18.

[0082] 20. Use of the antibody or antigen-binding fragment thereof according to any one of items 1 to 11, the bispecific antibody according to item 12 or 13, the composition according to item 16, or the lymphocyte according to item 17 in the preparation of a medicament for treating cancer in a subject.

[0083] 21. The method of claim 20, wherein the cancer is selected from any one or more of the following groups: lymphoma, melanoma, colorectal adenocarcinoma, prostate cancer, breast cancer, colon cancer, lung cancer, liver cancer, gastric cancer, and renal clear cell carcinoma. BRIEF DESCRIPTION OF THE DRAWINGS

[0084] Figure 1 . SDS-PAGE analysis results of purified antibodies.

[0085] Figures 2A-2B . ELISA results to determine the binding affinity of antibodies to human (2A) and macaque (2B) PD-1.

[0086] Figure 3 .Flow cytometric analysis of antibody binding affinity to Jurkat cells expressing human PD-1.

[0087] Figure 4 . showed that humanized anti-PD-1 antibodies effectively blocked the interaction between PD-1 and PD-L1 in cell-based assays.

[0088] Figures 5A-5C . showed that in cytokine release assays, human peripheral blood mononuclear cells (PBMCs) interacting with anti-PD-1 antibodies secreted increased IL-2 (5A), IFN-γ (5B), and TNF-α (5C).

[0089] Figures 6A-6C .Shows that antibody VH7+VL6 inhibits tumor growth in vivo. Figure 6A The results are the analysis of tumor size in each mouse group. Figure 6B and 6C The changes in tumor size for each mouse are shown separately. DETAILED DESCRIPTION

[0090] The present application provides antibodies and fragments thereof that bind to PD-1 protein, especially human PD-1 protein or polypeptide. The present application also relates to the use of the antibodies and fragments thereof to enhance the activation of the immune system against, for example, cancer cells.

[0091] The present application further provides methods for producing anti-PD-1 antibodies, polynucleotides encoding anti-PD-1 antibodies, and cells comprising polynucleotides encoding anti-PD-1 antibodies.

[0092] 1. Definition

[0093] It should be understood that the present application is not limited to the aspects described herein, which can of course vary. It should also be understood that the terminology used herein is used to describe particular aspects only and is not intended to be limiting, as the scope of the present application will be limited only by the appended claims.

[0094] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those of ordinary skill in the art to which the technology belongs. All technologies and patent disclosures cited herein are incorporated herein by reference in their entirety. Unless otherwise indicated, those skilled in the art will adopt the conventional techniques of tissue culture, immunology, molecular biology, microbiology, cell biology and recombinant DNA within the scope of the art. See, for example, Sambrook and Russell (2001) Molecular Cloning: A Laboratory Manual, 3rd edition; Harlow and Lane (1999) Antibodies, A Laboratory Manual. MONOCLONAL ANTIBODIES: A PRACTICAL APROACH (Shepherd, P. et al., 2000) Oxford University Press, USA, New York NY.

[0095] As used herein, the term "PD-1" refers to a programmed cell death protein that belongs to the immunoglobulin superfamily and functions as a co-inhibitory receptor to negatively regulate the immune system. PD-1 is a member of the CD28 / CTLA-4 family and has two known ligands, including PD-L1 and PD-L2. Alternative names or synonyms for PD-1 include PDCD1, PD1, CD279, and SLEB2. The representative amino acid sequence of human PD-1 is disclosed under NCBI accession number NP 005009.2. The representative nucleic acid sequence encoding human PD-1 is shown under NCBI accession number NM 005018.2. The PD-1 protein is expressed by circulating lymphocytes (such as T cells, B cells, monocytes, natural killer T cells, NK cells, and macrophages) and is a marker of activation and exhaustion.

[0096] As used herein, the term "PD-L1" refers to programmed cell death ligand 1 (PD-L1, e.g., Freeman et al. (2000) J. Exp. Med. 192: 1027). Alternative names or synonyms for PD-L1 include PDCDIL1, PDL1, B7H1, CD274, and B7-H. A representative amino acid sequence of human PD-L1 is published under NCBI accession number NP 054862.1. A representative nucleic acid sequence encoding human PD-L1 is shown under NCBI accession number NM 014143.3. PD-L1 is expressed in the placenta, spleen, lymph nodes, thymus, heart, fetal liver, and is also found on many tumors or cancer cells. PD-L1 binds to its receptor PD-1 or B7-1, which is expressed on activated T cells, B cells, and myeloid cells. Binding of PD-L1 to its receptor induces signal transduction to inhibit TCR-mediated activation of cytokine production and T cell proliferation. Therefore, PD-L1 plays a major role in suppressing the immune system in specific events (such as pregnancy, autoimmune diseases, and tissue allotransplantation) and is believed to allow tumors or cancer cells to circumvent immune checkpoints and escape immune responses. It has been reported that PD-L1 is also highly expressed on inflammatory macrophages compared to resident peritoneal macrophages, but expression on resident macrophages can be induced by classic activation stimuli such as lipopolysaccharide, IFN-γ, and polyinosinic-polycytidylic acid.

[0097] As used herein, the term "PD-L2" refers to programmed cell death ligand 2. Alternative names or synonyms for PD-L2 include PDCDIL2, PDL2, B7-DC, Btdc, and CD273. A representative amino acid sequence of human PD-L2 is disclosed under NCBI accession number NP079515.2.

[0098] As used herein, the term "anti-PD-1 antibody" refers to an antibody that specifically binds to PD-1 (e.g., human, monkey, or simian PD-1). Advantageously, the anti-PD-1 antibody specifically binds to PD-1 with sufficient affinity for diagnosis and / or therapy. Preferably, the anti-PD-1 antibody competes for binding to PD-1 with PD-L1, PD-L2, and / or other ligands of PD-1.

[0099] As used herein, the term "antibody", also referred to as "immunoglobulin", encompasses antibodies having structural features of natural antibodies and antibody-like molecules having structural features different from natural antibodies but exhibiting binding specificity to PD-1 molecules. The term antibody is intended to encompass immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules containing antigen binding sites. Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass.

[0100] The terms "heavy chain" ("HC"), "light chain" ("LC"), "light chain variable region" ("VL"), "heavy chain variable region" ("VH"), and "framework region" ("FR") refer to domains found in naturally occurring immunoglobulins and the corresponding domains of synthetic (e.g., recombinant) binding proteins (e.g., humanized antibodies). The basic structural unit of a naturally occurring immunoglobulin (e.g., IgG) is a tetramer having two light chains and two heavy chains. The amino-terminal ("N") portion of each chain includes a variable region of about 100 to 110 or more amino acids that is primarily responsible for antigen recognition. The carboxyl terminus ("C") of each chain defines the constant region, with light chains having a single constant domain and heavy chains typically having three constant domains and a hinge region. Thus, the structure of the light chain of a naturally occurring IgG molecule is N-VL-CL-C, and the structure of the IgG heavy chain is N-VH-CH1-H-CH2-CH3-C (where H is the hinge region). The variable region of an IgG molecule is composed of the complementarity determining regions (CDRs), which contain residues that contact the antigen, and non-CDR segments, called framework segments, which maintain the structure and determine the positions of the CDR loops. Thus, the VL and VH domains have the structure N-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-C.

[0101] In natural antibodies, variability is unevenly distributed across the variable regions of the antibody. It is concentrated in three segments, called complementarity determining regions (CDRs) or hypervariable regions, in the light and heavy chain variable regions. The CDRs on the heavy chain can be referred to as CDRnH, where "n" is an integer and does not indicate the order of the CDRs on the heavy chain. Similarly, the CDRs on the light chain can be referred to as CDRnL, where "n" is an integer that labels the CDRs and does not indicate the order of the CDRs on the light chain. The more highly conserved portions of the variable domain are called frameworks (FRs). The variable regions of natural heavy and light chains each contain four FR regions connected by three CDRs. The CDRs in each chain are tightly linked by the FR regions and, together with the CDRs from the other chain, contribute to the formation of the antibody's antigen-binding site [see Kabat, EA et al., Sequences of Proteins of Immunological Interest, National Institute of Health, Bethesda, MD (1987)]. The constant regions do not directly participate in the binding of the antibody to the antigen, but exhibit various effector functions, such as the antibody's participation in antibody-dependent cellular cytotoxicity (ADCC).

[0102] As used herein, the term "antigen-binding fragment" of an antibody (or simply "antibody fragment") refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., a PD-1 molecule, such as human PD-1). The antibody fragment comprises only a portion of an intact antibody, wherein the portion preferably retains at least one, preferably most or all, functions normally associated with the portion when present in an intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

[0103] Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site, and a residual Fc fragment, whose name reflects its ability to crystallize easily. The "Fab" fragment also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. The difference between the "Fab'" fragment and the Fab fragment is that a few residues are added to the carboxyl terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. "Fab'-SH" refers to Fab' in which the cysteine ​​residues of the constant domains have free thiol groups. The "F(ab')" fragment is produced by the cleavage of the hinge cysteine ​​disulfide bonds of the pepsin digestion product "F(ab')2".

[0104] "Fd" fragments consist of the VH and CH1 domains. "dAb" fragments (Ward et al., (1989) Nature 341:544-546) consist of the VH domain. Isolated complementarity determining regions (CDRs) and combinations of two or more isolated CDRs, optionally connected by a synthetic linker.

[0105] An "Fv" fragment consists of the VL and VH domains of a single antibody arm. A single-chain Fv (scFv) consists of one heavy chain variable region and one light chain variable region covalently linked into a single polypeptide chain by a flexible peptide linker.

[0106] The term "diabody" refers to a small antibody fragment with two antigen-binding sites, which comprises a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same polypeptide chain (VH-VL). By using a linker (which is too short to allow pairing between the two domains on the same chain), the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites. Diabodies are more fully described in, for example, EP 404,097; WO 93 / 11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90: 6444-48 (1993).

[0107] These antibody fragments are obtained using conventional techniques known to those skilled in the art, for example, by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact immunoglobulins.

[0108] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., except for possible variant antibodies (e.g., containing naturally occurring mutations or mutations arising during preparation of the monoclonal antibody, such variants generally being present in minor amounts), the individual antibodies comprising the population are identical and / or bind to the same epitope.

[0109] As used herein, the term "chimeric antibody" refers to an antibody in which the Fc constant region of a monoclonal antibody from one species (e.g., a mouse Fc constant region) is replaced with the Fc constant region of an antibody from another species (e.g., a human Fc constant region) using recombinant DNA technology. See, e.g., Robinson et al., PCT / US86 / 02269; Morrison et al., European Patent Application No. 173,494.

[0110] As used herein, the term "humanized antibody" refers to an antibody comprising a human framework region and one or more CDRs from a non-human (e.g., mouse, rat, rabbit or synthetic) immunoglobulin. The non-human immunoglobulin providing CDR is referred to as a "donor," while the human immunoglobulin providing the framework is referred to as an "acceptor." On the one hand, all CDRs are from the donor immunoglobulin in the humanized immunoglobulin. Therefore, except for possible CDRs, all parts of the humanized immunoglobulin are substantially identical to the corresponding parts of natural human immunoglobulin sequences. Humanized antibodies can be constructed by means of genetic engineering (see, e.g., U.S. Patent number 5,585,089).

[0111] "Acceptor human framework" means a framework comprising the amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or a human consensus framework. An acceptor human framework "derived from" a human immunoglobulin framework or a human consensus framework may comprise the same amino acid sequence thereof, or it may contain amino acid sequence changes. In some embodiments, the number of amino acid changes is 1-10, 2-9, 3-8, 4-7, or 5-6.

[0112] "Human consensus framework" is a framework that represents the most common amino acid residues in the selection of human immunoglobulin VL or VH framework sequences. In general, the selection of human immunoglobulin VL or VH sequences is from a subtype of variable domain sequences. In general, the subtype of the sequence is a subtype such as Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda MD (1991), Volumes 1-3. In certain embodiments, for VL, the subtype is subtype κI such as Kabat et al. (supra). In certain embodiments, for VH, the subtype is subtype III such as Kabat et al. (supra).

[0113] As used herein, the term "human antibody" is intended to include antibodies with variable and constant regions derived from human germline immunoglobulin sequences. The human antibody of this technology may include amino acid residues not encoded by human germline immunoglobulin sequences (for example, by random or site-specific in vitro mutagenesis or by mutations introduced by somatic mutations in vivo). However, the term "human antibody" as used herein is not intended to include such antibodies, wherein the CDR sequences derived from the germline (such as rabbit) of another mammalian species have been transplanted into human framework sequences. Therefore, as used herein, the term "human antibody" refers to a kind of antibody, wherein substantially each part of the protein (for example, CDR, framework, CL, CH domain (for example, CH1, CH2, CH3), hinge, VL, VH) is substantially non-immunogenic in the human race, with only minor sequence variations or mutations. Therefore, human antibody is different from chimeric or humanized antibodies. It should be noted that human antibody can be produced by non-human animals or prokaryotic or eukaryotic cells that can express human immunoglobulin (for example, heavy chain and / or light chain) genes that are functionally rearranged.

[0114] As used herein, the phrases "bispecific antibodies" or "bispecific antigen-binding antibodies" or "bifunctional antibodies" are artificial hybrid antibodies with two different heavy chain / light chain pairs and two different binding sites. For the purposes of this application, "bispecific antibodies" specifically bind to PD-1 and another antigen, for example, a tumor antigen expressed on tumor cells.

[0115] A "conjugate" is an antibody conjugated to one or more heterologous molecules including, but not limited to, a cytotoxic agent.

[0116] A "blocking" antibody or "antagonist" antibody is an antibody that inhibits or reduces the biological activity of the antigen to which it binds. Preferred blocking antibodies or antagonist antibodies substantially or completely inhibit the biological activity of the antigen.

[0117] As used herein, the term "isolated" refers to a molecule or biological or cellular material that is substantially free of other materials. For example, a nucleic acid or peptide that is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or that is substantially free of chemical precursors or other chemicals when chemically synthesized. In addition, "isolated nucleic acid" is intended to include nucleic acid fragments that do not naturally exist as fragments and are not found in nature. The term "isolated" is also used herein to refer to polypeptides separated from other cellular proteins, and is intended to encompass purified and recombinant polypeptides.

[0118] As used herein, the percentage of "homology" or "identity" used in the context of two or more nucleic acid or polypeptide sequences refers to two or more sequences or subsequences that are identical or have a specified percentage of identical nucleotides or amino acid residues, for example, at least 80% identity, preferably at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity over a specified region (e.g., a nucleotide sequence encoding an antibody described herein or an amino acid sequence encoding an antibody described herein). Homology can be determined by comparing positions in each sequence, which can be aligned for comparison purposes. When a position in the compared sequences is occupied by the same base or amino acid, the molecules are homologous at that position. The degree of homology between sequences is a function of the number of matches or the number of homologous positions shared by the sequences. Software programs known in the art can be used to compare and determine percent homology or sequence identity. Preferably, default parameters are used for comparison. A preferred alignment program is BLAST using default parameters. Preferred programs are BLASTN and BLASTP. Details of these programs can be found at the following internet address: ncbi.nlm.nih.gov / cgi-bin / BLAST.

[0119] "Affinity" refers to the total strength of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). Affinity can be measured by conventional methods known in the art, including, for example, Biacore, radioimmunoassay (RIA), and ELISA.

[0120] The affinity of a molecule X for its partner Y can usually be expressed by the equilibrium dissociation constant (KD), which is expressed as the ratio k off / k on (k d / k a ) is calculated. See, for example, Chen, Y., et al., (1999) J. Mol Biol 293:865-881. Low-affinity antibodies generally bind antigen slowly and tend to dissociate easily, while high-affinity antibodies generally bind antigen faster and tend to remain bound longer. In one embodiment of the present application, the "dissociation rate (k)" is the average value of the affinity of the antigen. d )" is measured by using surface plasmon resonance. According to the present application, "association rate" or "association rate" or "association rate (k a )" or "k on” can also be determined using the same surface plasmon resonance technique and calculated by simultaneous fitting of the association and dissociation sensorgrams using a simple one-to-one Langmuir binding model (BIAcore Evaluation Software).

[0121] As used herein, the term "EC50" refers to the concentration of an antibody or antigen-binding fragment thereof that binds to PD-1 and / or induces a response in an in vitro or in vivo assay that is 50% of maximal binding or response, i.e., halfway between maximal binding or response and baseline.

[0122] The terms "cancer," "neoplasm," and "tumor" are used interchangeably herein to refer to a neoplasm or tumor that results from abnormal, uncontrolled growth of cells that renders them pathogenic to the host organism. In some embodiments, cancer refers to a benign tumor that has become localized. In other embodiments, cancer refers to a malignant tumor that has invaded and destroyed adjacent body structures and spread to distant sites. In some embodiments, the cancer is associated with a specific cancer antigen.

[0123] As used herein, "treating" or "treatment" of a disease in a subject refers to an approach for obtaining beneficial or desired results, including, but not limited to, one or more of the following: alleviation or improvement of one or more symptoms, reduction in the scope of the condition (including the disease), a stable (i.e., non-worsening) state of the condition (including the disease), delay or slowing of the condition (including the disease), progression, improvement or palliation of the condition (including the disease), state and remission (whether partial or complete), whether detectable or undetectable.

[0124] A "pharmaceutically acceptable carrier" is a carrier that constitutes a pharmaceutical preparation with an active ingredient. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0125] The term "package insert" is used to refer to instructions customarily included in commercial packages of therapeutic products. Generally, the package insert contains information about the use of the therapeutic product, such as indications, usage, dosage, administration, combination therapy, contraindications and / or warnings.

[0126] The present application will be described with respect to particular embodiments and with reference to certain drawings, but the application is not limited thereto but only by the claims. As used in the specification and claims, the term "comprising" does not exclude other elements or steps. When referring to a singular noun, an indefinite or definite article is used, for example, "a" or "an", "the", unless otherwise specifically stated, it includes the plural form of the noun.

[0127] 2. Anti-PD-1 Antibodies and Methods for Their Preparation

[0128] The present application encompasses isolated anti-PD-1 antibodies or fragments thereof, and polynucleotides comprising sequences encoding the anti-PD-1 antibodies or fragments thereof.

[0129] The isolated anti-PD-1 antibody or its fragment binds with high affinity to the PD-1 molecules expressed on cells (e.g., cancer cells), promoting an effective immune response to cancer cells. The antibodies and immunologically active fragments provided herein can enhance the activity of the immune system, thereby providing important therapeutic and diagnostic preparations for pathological conditions associated with the expression and / or activity of PD-1 molecules. In one aspect, the present application provides an isolated antibody or antigen-binding fragment thereof, comprising a heavy chain (HC) variable region sequence and a light chain (LC) variable region sequence. wherein the antibody binds to the extracellular domain of PD-1 with a binding affinity better than 10 nM or about 10 nM, better than 8 nM or about 8 nM, better than 6 nM or about 6 nM, better than 4 nM or about 4 nM, better than 2 nM or about 2 nM, better than 1 nM or about 1 nM, as determined by SPR analysis; for example, about 0.5-4 nM, about 0.8-4.0 nM, about 1.0-4.0 nM, about 2.0-4.0 nM, about 3.0-4.0 nM, about 0.6-3.5 nM, about 1.4-3.5 nM, about 2.5-3.5 nM, about 0.7-2.5 nM, about 0.8-2.0 nM, about 1.0-2.0 nM, about 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM or better, as determined by SPR analysis.

[0130] In certain embodiments, the present application provides an antibody or antigen-binding fragment thereof comprising at least one of the following:

[0131] (a) CDR1H sequence comprising GFTFSSYGMS (SEQ ID NO: 1).

[0132] (b) CDR2H sequence comprising IISGGGRDIYYLDSVKG (SEQ ID NO: 2).

[0133] (c) CDR3H sequence comprising PIYDAYSFAY (SEQ ID NO: 3).

[0134] (d) CDR1L sequence comprising RASQTISNNLH (SEQ ID NO: 4).

[0135] (e) comprising the CDR2L sequence of YASQSIS (SEQ ID NO: 5), and

[0136] (f) CDR3L sequence comprising QQSYSWPLT (SEQ ID NO: 6).

[0137] In certain embodiments, the present application provides an antibody or antigen-binding fragment thereof, wherein

[0138] (a) The HC comprises

[0139] The CDR1H sequence comprises GFTFSSYGMS (SEQ ID NO: 1).

[0140] a CDR2H sequence comprising IISGGGRDIYYLDSVKG (SEQ ID NO: 2), and

[0141] The CDR3H sequence comprising PIYDAYSFAY (SEQ ID NO: 3).

[0142] (b) the LC comprises

[0143] The CDR1L sequence comprises RASQTISNNLH (SEQ ID NO: 4).

[0144] comprising the CDR2L sequence of YASQSIS (SEQ ID NO: 5), and

[0145] The CDR3L sequence comprising QQSYSWPLT (SEQ ID NO: 6).

[0146] In certain embodiments, the antibody is a chimeric antibody, a humanized antibody, or a human antibody. In certain embodiments, the antibody or antigen-binding fragment thereof of the present application further comprises a human acceptor framework. In certain embodiments, the human acceptor framework is from a human immunoglobulin framework or a human consensus framework. In certain embodiments, the human acceptor framework comprises a subtype κI framework sequence of VL and a subtype III framework sequence of VH. Typically, the subtype sequence is a subtype as described in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda MD (1991), vols. 1-3. In certain embodiments, for VL, the subgroup is the subgroup κI described by Kabat et al. as described above. In certain embodiments, for VH, the subgroup is the subgroup III described by Kabat et al. as described above.

[0147] In certain embodiments, the antibody or its antigen-binding fragment comprises a human consensus framework. In certain embodiments, the antibody or its antigen-binding fragment comprises a human consensus framework with amino acid sequence changes, e.g., 1-15, 1-10, 2-9, 3-8, 4-7, or 5-6 amino acid changes.

[0148] In certain embodiments, the antibodies or antigen-binding fragments thereof of the present application comprise an HC variable region sequence consisting of the amino acid sequence set forth in SEQ ID NO: 7 or 8, or an amino acid sequence having greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 7 or 8. In certain embodiments, the antibodies or antigen-binding fragments thereof of the present application comprise an LC variable region sequence consisting of the amino acid sequence set forth in SEQ ID NO: 9 or SEQ ID NO: 10, or an amino acid sequence having greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 9 or 10. In certain embodiments, the HC variable region sequence comprises the amino acid sequence of SEQ ID NO: 7, and the LC variable region sequence comprises the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 10. In certain embodiments, the HC variable region sequence comprises the amino acid sequence of SEQ ID NO: 8, and the LC variable region sequence comprises the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 10. In certain embodiments, the antibodies or antigen-binding fragments thereof of the present application comprise an HC sequence consisting of the amino acid sequence of SEQ ID NO: 11 or 12, or an amino acid sequence having greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 11 or 12. In certain embodiments, the antibodies or antigen-binding fragments thereof of the present application comprise an LC sequence consisting of the amino acid sequence of SEQ ID NO: 13 or SEQ ID NO: 14, or an amino acid sequence having greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 13 or 14. In certain embodiments, the HC sequence comprises the amino acid sequence of SEQ ID NO: 11, and the LC sequence comprises the amino acid sequence of SEQ ID NO: 13 or SEQ ID NO: 14. In certain embodiments, the HC sequence comprises the amino acid sequence of SEQ ID NO: 12, and the LC variable region sequence comprises the amino acid sequence of SEQ ID NO: 13 or SEQ ID NO: 14.

[0149] In certain embodiments, the antibody is of the IgG isotype, such as IgG1, IgG2, or IgG4 isotype. In certain embodiments, the antigen-binding fragment comprises any one selected from the group consisting of Fab, F(ab')2, Fab', scFv, and Fv. In certain embodiments, the antibody or antigen-binding fragment thereof of the present application is a blocking antibody or antagonist antibody that can inhibit or reduce the biological activity of the PD-1 molecule to which it binds. Preferably, the blocking antibody or antagonist antibody substantially or completely inhibits the biological activity of the PD-1 molecule.

[0150] The anti-PD-1 antibodies of the present application are preferably monoclonal. Also encompassed within the scope of the present application are Fab, Fab', Fab'-SH and F(ab')2 fragments of the anti-PD-1 antibodies provided herein. These antibody fragments can be produced by conventional means such as enzymatic digestion, or can be generated by recombinant technology. The anti-PD-1 antibodies and their fragments can be used for diagnostic and therapeutic purposes, including the diagnosis and treatment of cancer.

[0151] A monoclonal antibody is obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in small amounts. Thus, the modifier "monoclonal" indicates that the antibody is not a mixture of different antibodies. The monoclonal anti-PD-1 antibodies of the present application can be made using hybridoma methods or recombinant DNA methods (U.S. Patent No. 4,816,567).

[0152] In the hybridoma method, mice or other appropriate host animals, such as hamsters, are immunized with the entire PD-1 molecule or a portion thereof (e.g., a polypeptide comprising the extracellular domain of PD-1), along with an adjuvant. PD-1 molecules or polypeptides comprising the extracellular domain of PD-1 molecules can be prepared using methods known in the art. In one embodiment, animals are immunized with a polypeptide comprising the extracellular domain (ECD) of PD-1 fused to the Fc portion of an immunoglobulin heavy chain. In one embodiment, animals are immunized with a PD-1-IgG1 fusion protein. Two weeks later, the animals are boosted. Seven to fourteen days later, the animals are bled and the serum anti-PD-1 titer is determined. Animals are boosted until the titer stabilizes. Alternatively, lymphocytes can be immunized in vitro. The lymphocytes are then fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, to form hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)).

[0153] The hybridoma cells thus prepared are inoculated and cultured in a suitable culture medium, which preferably contains one or more substances that inhibit the growth or survival of the unfused parental myeloma cells. Preferred myeloma cells are those that fuse efficiently, support stable, high-level production of antibodies by the selected antibody-producing cells, and are sensitive to culture medium (such as HAT culture medium). Among them, preferred myeloma cell lines are murine myeloma cell lines, such as SP-2 or X63-Ag8-653 cells. (Kozbor, J. Immunol, 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)) also describe the use of human myeloma and mouse human heteromyeloma cell lines for the production of human monoclonal antibodies.

[0154] The production of monoclonal antibodies against PD-1 is assayed in culture medium of hybridoma cells. Preferably, the binding specificity of the monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).

[0155] The binding affinity of the monoclonal antibody can then be determined by routine methods in the art. After hybridoma cells producing antibodies with the desired specificity, affinity, and / or activity are identified, they can be subcloned by limiting dilution procedures and cultured by standard methods (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)).

[0156] Suitable culture medium for the purpose includes, for example, D-MEM or RPMI-1640 culture medium. In addition, hybridoma cells can be grown in animals as ascites tumors. By conventional immunoglobulin purification procedures, the monoclonal antibodies secreted through the subclone are suitably separated from culture medium, ascites fluid or serum.

[0157] The anti-PD-1 antibodies of the present application can be made by screening synthetic antibody clones with one or more desired activities using a combinatorial library. In general, synthetic antibody clones are selected by screening a phage library containing phages displaying different fragments of antibody variable regions (Fv), wherein the Fv fragments are fused to phage coat proteins. This phage library is panned by affinity chromatography for the target antigen. The clones of the expressed Fv fragments can bind to the target antigen, which is adsorbed to the antigen and thus separated from the non-binding clones in the library. The bound clones are then eluted from the antigen and can be further enriched by additional cycles of antigen adsorption / elution. Any anti-PD-1 antibody of the present application can be obtained by the following method: designing an appropriate antigen screening program, selecting a phage clone of interest, and then constructing a full-length anti-PD-1 antibody clone using the Fv sequence from the phage clone of interest and an appropriate constant region (Fc) sequence as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, NIH Publication 91-3242, Bethesda MD (1991), Volumes 1-3.

[0158] The repertoire of VH and VL genes can be cloned separately by polymerase chain reaction (PCR) and randomly recombined in a phage library, and then the antigen binding clones therein can be searched, as described in Winter et al., Ann. Rev. Immunol, 12: 433-455 (1994). Libraries from immune sources provide high affinity antibodies to immunogens without the need to construct hybridomas. Alternatively, naive repertoires can be cloned to provide a single source of human antibodies to a wide range of non-self and self antigens without any immunization, as described in Griffiths et al., EMBO J, 12: 725-734 (1993). Finally, naive libraries can also be synthesized by cloning unrearranged V gene segments from stem cells and using PCR primers containing random sequences to encode highly variable CDR3 regions and complete rearrangement in vitro, as described in Hoogenboom and Winter, J. Mol Biol, 227: 381-388 (1992).

[0159] Antibodies generated from naive libraries (natural or synthetic) can have intermediate affinities, but affinity maturation can also be simulated in vitro by constructing a secondary library and reselecting from it. For example, mutations can be introduced randomly in vitro using an error-prone polymerase (reported in Leung et al., Technique, 1: 11-15 (1989)) in the method of Hawkins et al., J. Mol Biol., 226: 889-896 (1992) or Gram et al., Proc. Natl. Acad. Sci USA, 89: 3576-3580 (1992). Alternatively, affinity maturation can be performed in selected single Fv clones by randomly mutating one or more CDRs (e.g., using PCR and primers carrying random sequences covering the CDRs of interest) and screening for clones with higher affinity. Another efficient approach is to recombinant the selected VH or VL domains displayed by phage with a repertoire of naturally occurring V domain variants obtained from unimmunized donors and screen for higher affinity in several rounds of chain reshuffling as described in Marks et al., Biotechnol, 10:779-783 (1992).

[0160] For PD-1, it is possible to select between phage antibodies with different affinities, even if the affinities vary slightly. However, random mutagenesis of the selected antibody (e.g., as performed in some of the affinity maturation techniques described above) may produce many mutants, most of which bind to the antigen, and a few with higher affinity. To retain all higher-affinity mutants, phage can be incubated with an excess of biotinylated PD-1, but at a molar concentration lower than the target molar affinity constant for PD-1. High-affinity binding phage can then be captured by streptavidin-coated paramagnetic beads. Such "equilibrium capture" allows antibodies to be selected based on their binding affinity, and its sensitivity allows mutant clones with as little as twice as high affinity to be isolated from a large excess of phage with low affinity.

[0161] Anti-PD-1 clones can be selected based on the performance of the activity. In one embodiment, the present application provides anti-PD-1 antibodies that block the binding between the PD-1 receptor and its ligand. The anti-PD-1 antibodies of the present application having the characteristics described herein can be obtained by screening anti-PD-1 hybridoma clones for the desired characteristics by any convenient method. For example, if the desired antibody is an anti-PD-1 monoclonal antibody that blocks or does not block the binding of the PD-1 receptor to the PD-1 ligand, the candidate antibody can be tested in a binding competition assay, such as a competitive binding ELISA, in which the plate wells are coated with PD-1, a solution of antibodies with an excess of PD-1 receptors is spread on the coated plate, and the bound antibodies are detected by an enzymatic reaction, for example, the bound antibodies are contacted with an anti-Ig antibody conjugated to HRP or a biotinylated anti-Ig antibody, and an HRP color reaction is performed (for example, by developing the plate with streptavidin-HRP and / or hydrogen peroxide, and detecting the HRP color reaction by spectrophotometry at 490 nm using an ELISA plate reader).

[0162] 3. Isolated Polynucleotides, Vectors, Host Cells, and Recombinant Methods

[0163] The present application provides isolated polynucleotides, vectors or host cells, including the coding sequences of the above-mentioned anti-PD-1 antibodies or fragments thereof of the present application. In some embodiments, the anti-PD-1 antibody is a hybridoma-derived monoclonal antibody or phage-displayed Fv clone of the present application. In some embodiments, the DNA encoding the monoclonal antibody or phage-displayed Fv clone derived from the hybridoma of the present application is easy to separate and sequence using conventional procedures (for example, by using oligonucleotide primers designed to specifically amplify the heavy and light chain coding regions of interest from hybridoma or phage DNA templates). Once isolated, the DNA can be placed in an expression vector and then transfected into a host cell (such as an Escherichia coli cell, a monkey COS cell, a Chinese hamster ovary (CHO) cell, or a myeloma cell that does not otherwise produce immunoglobulins) to obtain the synthesis of the desired monoclonal antibody in the recombinant host cell.

[0164] The DNA encoding the Fv clones of the present application can be combined with known DNA sequences encoding heavy and / or light chain constant regions (e.g., suitable DNA sequences can be obtained from Kabat et al. (ibid.)) to form clones encoding full-length or partial-length heavy and / or light chains. It should be understood that any isotype of constant region can be used for the purpose, including IgG, IgM, IgA, IgD, and IgE constant regions, and such constant regions can be obtained from any human or animal species. Fv clones, which are derived from variable domain DNA of one animal (such as a human) species, are then fused with constant region DNA of another animal species to form a "hybrid," as used herein, including the coding sequences for full-length heavy and / or light chains in the definitions of "chimeric" and "hybrid" antibodies. In a preferred embodiment, Fv clones derived from human variable DNA are fused with human constant region DNA to form coding sequences for fully human, full-length, or partial-length heavy and / or light chains.

[0165] The DNA encoding the anti-PD-1 antibody derived from the hybridoma of the present application can also be modified, for example, by replacing the homologous mouse sequence derived from the hybridoma clone with the coding sequence of the human heavy and light chain constant domains (for example, as described in Morrison et al., Proc. Natl Acad. Sci. USA, 81: 6851-6855 (1984)). The DNA encoding the antibody or fragment derived from the hybridoma or Fv clone can be further modified by covalently linking all or part of the coding sequence of the non-immunoglobulin polypeptide to the immunoglobulin coding sequence. In this way, a "chimeric" or "hybrid" antibody having the binding specificity of the antibody derived from the Fv clone or hybridoma clone of the present application is prepared.

[0166] In order to recombinantly produce the antibody of the present application, the nucleic acid encoding it is separated and inserted into a reproducible vector for further cloning (amplification of DNA) or for expression. The DNA encoding the antibody is easy to separate and sequenced using conventional procedures (for example, by using oligonucleotide probes that can be specifically bound to the genes encoding the heavy and light chains of the antibody). Many vectors are available. The selection of the vector depends in part on the host cell to be used. Generally speaking, preferred host cells are prokaryotic or eukaryotic (usually mammalian) sources. It should be understood that the constant region of any isotype can be used for the purpose, including IgG, IgM, IgA, IgD and IgE constant regions, and such constant regions can be obtained from any human or animal species.

[0167] 4. Conjugates and methods for their preparation

[0168] The anti-PD-1 antibodies or fragments thereof of the present application are contemplated in the present application in combination with one or more other molecules (such as toxins, e.g., calicheamicin, maytansinoids, dolastatins, aurostatins, trichothecenes and CC1065, as well as derivatives of these toxins with toxin activity), radioactive isotopes and immunomodulators.

[0169] In some embodiments, the conjugate is used to treat T cell lymphoma, B cell lymphoma or lymphocytic leukemia, including the antibody (full length or fragment) of the present application having been put together with one or more maytansinoid molecules. Maytansinoids are mitotic inhibitors that work by suppressing tubulin polymerization. Maytansine (maytansine) was originally separated from East African shrub Maytenus serrate (Maytenus serrate) (U.S. Patent number 3,896,111). Subsequently, it was found that some microorganisms also produce maytansinoids, such as maytansinol and C-3 maytansinol esters (U.S. Patent number 4,151,042). Containing the immunoconjugate of maytansinoids, its preparation method and therapeutic use thereof are disclosed in, for example, U.S. Patent Nos. 5,208,020, 5,416,064 and European Patent EP 0 425 235B1 (its disclosure is hereby expressly incorporated by reference). Conjugates of antibodies and maytansine alkaloids can be made using a variety of bifunctional protein coupling agents, such as N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP), succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters. In some embodiments, the conjugate comprises the antibody of the present application, which is conjugated to dolastatin or dolastatin peptide analogs and derivatives, auristatin (U.S. Patent Nos. 5,635,483; 5,780,588). In order to selectively destroy tumors, the antibody may contain highly radioactive atoms. A variety of radioactive isotopes can be used to produce radioactively conjugated antibodies. Radioactive or other labels can be incorporated into the conjugate in a known manner. For example, the peptide can be biosynthesized or can be synthesized by chemical amino acid synthesis using suitable amino acid precursors, including, for example, replacing hydrogen with fluorine-9. Other methods are described in detail in "Monoclonal Antibodies in Immunoscintigraphy" (Chatal, CRC Press 1989).

[0170] In some embodiments, the conjugate is used to treat T-cell lymphoma, B-cell lymphoma or lymphocytic leukemia, which includes the antibody (full length or fragment) of the present application combined with one or more immunomodulators, wherein the immunomodulator can work synergistically with the antibody (full length or fragment) to enhance the immune response against antigens and abnormal cells (including tumor cells). In some embodiments, the immunomodulator is selected from any one of the following groups: checkpoint inhibitors (such as Atezolizumab, Avelumab, Cemiplimab, Durvalumab, Ipilimumab, Nivolumab, Pembrolizumab), cytokines (such as Aldesleukin, Granulocyte-macrophage colony-stimulating factor, IFNα-2a, IFNα-2B, Pre-IFNα-2B), agonists and adjuvants (such as Imiquimod or poly-ICLC), or molecules that act the same as them.

[0171] Typically, peptide-based drug moieties can be prepared by forming peptide bonds between two or more amino acids and / or peptide fragments. Such peptide bonds can be prepared, for example, according to liquid phase synthesis methods well known in the art of peptide chemistry. Auristatin / dolastatin drug moieties can be prepared according to the following methods: US 5635483; US 5780588. See also Doronina (2003) Nat Biotechnol 21(7):778-784.

[0172] The present application further contemplates immunoconjugates formed between an antibody and a compound having nucleolytic activity (eg, a ribonuclease or a DNA endonuclease such as a deoxyribonuclease; DNase).

[0173] 5. Antibody fragments and methods for preparing the same

[0174] This application includes antibody fragments. These antibody fragments are immunologically active fragments of the anti-PD-1 antibodies of this application. In some cases, it is advantageous to use antibody fragments rather than whole antibodies. The smaller size of fragments allows for rapid clearance and may allow them to more easily penetrate solid tumors.

[0175] A variety of techniques have been developed for producing antibody fragments. Traditionally, these fragments are obtained via proteolytic digestion of intact antibodies (see, for example, Morimoto et al., Journal of Biochemical and Biophysical Methods 24:107-117 (1992); and Brennan et al., Science, 229:81 (1985)). However, these fragments can now be produced directly by recombinant host cells. Fab, Fv, and ScFv antibody fragments can be expressed in and secreted from E. coli, thus allowing these fragments to be readily produced in large quantities. Antibody fragments can be isolated from the above-mentioned antibody phage libraries. Alternatively, Fab'-SH fragments can be directly recovered from E. coli and chemically coupled to form F(ab')2 fragments (Carter et al., Bio / Technology 10:163-167 (1992)). According to another method, F(ab')2 fragments can be isolated directly from recombinant host cell cultures. Fab and F(ab')2 fragments with increased in vivo half-life comprising salvage receptor binding epitope residues are described in US Patent No. 5,869,046. Other techniques for producing antibody fragments will be apparent to those skilled in the art.

[0176] In other embodiments, the antibody of choice is a single-chain Fv fragment (scFv). See WO 93 / 16185; U.S. Patent Nos. 5,571,894; and 5,587,458. Fv and scFv are the only known types that have complete binding sites without constant regions; therefore, they are suitable for reducing nonspecific binding during in vivo use. scFv fusion proteins can be constructed to produce fusions of effector proteins at the amino or carboxyl terminus of the scFv. See Antibody Engineering, ed. Borrebaeck, supra. The antibody fragment may also be a "linear antibody," for example, as described in U.S. Patent No. 5,641,870. Such linear antibody fragments may be monospecific or bispecific.

[0177] 6. Humanized antibodies and human antibodies

[0178] The anti-PD-1 antibodies of the present application are humanized antibodies in some embodiments. Various methods for humanizing non-human antibodies are known in the art. For example, a humanized antibody may have one or more amino acid residues introduced from a non-human source. These non-human amino acid residues are generally referred to as "introduction" residues, which are generally taken from the "introduction" variable domain. Humanization can basically be performed according to the method of Winter and colleagues (Jones et al. (1986) Nature 321: 522-525; Riechmann et al. (1988) Nature 332: 323-327; Verhoeyen et al. (1988) Science 239: 1534-1536), by replacing the corresponding sequence of a human antibody with a hypervariable region sequence. Therefore, such a "humanized" antibody is a chimeric antibody (U.S. Patent No. 4,816,567), in which a portion substantially less than the complete human variable domain is replaced by a corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some of the hypervariable region residues and possible some FR residues are replaced by residues from similar sites in rodent antibodies. The selection of human variable domains (light and heavy chains) for making humanized antibodies is very important for reducing antigenicity. According to the so-called "best-fit" method, the variable domain sequences of rodent antibodies are screened for the entire library of known human variable domain sequences. Then, the human sequence closest to the rodent is used as the human framework of the humanized antibody (Sims et al. (1993) J.Immunol.151:2296; Chothia et al. (1987) J.MoI.Biol.196:901). Another method uses a specific framework that is derived from the consensus sequence of the fully human antibodies of the specific subtype of light or heavy chain.

[0179] It is further important to humanize the antibody while retaining high affinity for the antigen and other favorable biological properties. To achieve this goal, according to one method, humanized antibodies are prepared by a process of analyzing the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are commonly available and familiar to those skilled in the art. Computer programs are available that illustrate and display the possible three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays allows analysis of the possible role of residues in the function of the candidate immunoglobulin sequence, that is, analysis of residues that affect the ability of the candidate immunoglobulin to bind its antigen. In this way, FR residues can be selected and combined from the receptor and input sequences to obtain the desired antibody properties, such as increased affinity for PD-1.

[0180] Transgenic animals (e.g., mice) that are able to produce a full repertoire of human antibodies after immunization in the absence of endogenous immunoglobulin production. For example, it has been described that homozygous deletion of the antibody heavy chain joining region (JH) gene in chimeric and germline mutant mice completely inhibits endogenous antibody production. Introduction of a human germline immunoglobulin gene array into such germline mutant mice produces human antibodies after antigen stimulation. See, for example, Jakobovits et al., Nature, 362:255 (1993); Bruggermann et al., Year in Immunol, 7:33 (1993).

[0181] Gene shuffling can also be used to obtain human antibodies from non-human (e.g., rodent) antibodies, wherein the human antibodies have similar affinity and specificity to the starting non-human antibodies. According to the method (also known as "epitope imprinting"), the heavy chain or light chain variable region of the non-human antibody fragment obtained by the above-mentioned phage display technology is replaced with a set of human V domain genes in the library to create a non-human chain / human chain scFv or Fab chimera population. Selection with antigen can separate non-human chain / human chain chimeric scFv or Fab, wherein the human chain restores the antigen binding site that was destroyed when the corresponding non-human chain in the initial phage display clone was removed, that is, the epitope controls (imprints) the selection of the human chain partner. When the process is repeated to replace the remaining non-human chain, human antibodies are obtained (see PCT WO93 / 06213 published on April 1, 1993). Unlike the humanization of traditional non-human antibodies by CDR transplantation, the technology provides completely human antibodies, which do not have FR or CDR residues of non-human origin.

[0182] 7. Bispecific antibodies and methods for preparing them

[0183] Bispecific antibodies are monoclonal antibodies, preferably human or humanized antibodies, that have binding specificities for at least two different antigens. In the present application, one binding specificity is for PD-1 and the other is for any other antigen. Exemplary bispecific antibodies can bind to two different epitopes of the PD-1 protein. Bispecific antibodies can also be used to localize cytotoxic agents to cells expressing PD-1, in which case the antibody has a PD-1 binding arm and a cytotoxic agent binding arm.

[0184] In some embodiments, the bispecific antibody has a PD-1 binding arm, which comprises an anti-PD-1 antibody or fragment thereof of the present application, and an arm that binds to a tumor antigen or immune checkpoint protein. In some embodiments, the tumor antigen comprises any one selected from the group consisting of: A33; ADAM-9; ALCAM; BAGE; β-catenin; CA125; carboxypeptidase M; CD103; CD19; CD20. CD22; CD23; CD25; CD27; CD28; CD36; CD40 / CD154; CD45; CD46; CD5; CD56; CD79a / CD79b; CDK4; CEA; CTLA4; cytokeratin 8; EGF-R; EphA2; ErbB1; ErbB3; ErbB4; GAGE-1; GAGE-2; GD2 / GD3 / GM2; HER-2 / neu; human Papillomavirus-E6; human papillomavirus-E7; JAM-3; KID3; KID31; KSA (17-1A); LUCA-2; MAGE-1; MAGE-3; MART; MUC-1; MUM-1; N-acetylglucosamine transferase; oncostatin M; p15; PIPA; PSA; PSMA; ROR1; TNF-β receptor; TNF-α receptor; TNF-γ receptor; transferrin receptor and VEGF receptor. In some embodiments, the immune checkpoint protein comprises any one selected from the group consisting of 2B4; 4-1BB; 4-1BB ligand, B7-1; B7-2; B7H2; B7H3; B7H4; B7H6; BTLA; CD155; CD160; CD19; CD200; CD27; CD27 ligand; CD28. CD40; CD40 ligand; CD47; CD48; CTLA-4; DNAM-1; galectin-9; GITR; GITR ligand; HVEM; ICOS; ICOS ligand; IDOI; KIR; 3DL3; LAG-3; OX40; OX40 ligand; PD-L1; PD-1; PD-L2; LAG3; PGK; SIRPα; TIM-3; TIGIT; VSIG8.

[0185] Bispecific antibodies can be prepared as full-length antibodies or antibody fragments (e.g., F(ab')2 bispecific antibodies). Methods for making bispecific antibodies are known in the art. Typically, the recombinant production of bispecific antibodies is based on the coexpression of two immunoglobulin heavy chain-light chain pairs, wherein the two heavy chains have different specificities. Due to the random combination of immunoglobulin heavy chains and light chains, these hybridomas (quadroma) produce a mixture of 10 different antibody molecules, only one of which has the correct bispecific structure. Purification of the correct molecule is usually accomplished by affinity chromatography steps, which is quite cumbersome and has low yields. According to different and more preferred methods, the antibody variable domains with the desired binding specificity (antibody-antigen binding site) are fused to the immunoglobulin constant domain sequence. The fusion is preferably fused to the immunoglobulin heavy chain constant domain, wherein the domain comprises at least a portion of the hinge, CH2, and CH3 regions. Preferably, in at least one fusion, there is a first heavy chain constant region (CH1), wherein the CH1 contains the site necessary for light chain binding. The DNA encoding the heavy chain immunoglobulin fusions and (if necessary) light chain immunoglobulin is inserted into a separate expression vector, and cotransfected into a suitable host organism. When three polypeptide chains with different ratios are used to provide optimal yields in a construct, the mutual ratio of the three polypeptide fragments is regulated in the embodiment and provides great flexibility. However, when at least two polypeptide chains are expressed in equal proportions and cause high yields or when ratio has no particular significance, the encoding sequence of two or all three polypeptide chains can be inserted into an expression vector.

[0186] In a preferred embodiment of the method, the bispecific antibody is composed of a hybrid immunoglobulin heavy chain with a first binding specificity in one arm and a hybrid immunoglobulin heavy chain-light chain pair (providing a second binding specificity) in another arm. It has been found that the asymmetric structure helps to separate the desired bispecific compound from unwanted immunoglobulin chain combinations because only the presence of immunoglobulin light chains in one half of the bispecific antibody provides a convenient separation method. The method is disclosed in WO 94 / 04690. For further details of generating bispecific antibodies, see, for example, Suresh et al., Methods in Enzymology, 121: 210 (1986).

[0187] 8. Pharmaceutical Compositions

[0188] The therapeutic agent comprising the anti-PD-1 antibody fragment, polynucleotide, vector, host cell, conjugate or bispecific antibody of the present application is prepared by mixing the anti-PD-1 antibody, fragment, polynucleotide, vector, host cell, conjugate or bispecific antibody of the present application having a desired purity with an optional physiologically acceptable carrier, excipient or stabilizer (Remington: The Science and Practice of Pharmacy 20th edition (2000)) in the form of an aqueous solution, lyophilized or other desiccants for storage. The acceptable carriers, excipients, or stabilizers are nontoxic to the subject at the dosages and concentrations employed, and include buffers such as phosphate, citrate, histidine, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives; low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes; and / or nonionic surfactants such as TWEEN®. TM 、PLURONICS TM or polyethylene glycol (PEG).

[0189] As required for the particular indication being treated, the formulations herein may also contain more than one active compound, preferably compounds with complementary activities that do not adversely affect each other. Such molecules are suitably present in combination in amounts that are effective for the intended purpose.

[0190] The active ingredient can also be encapsulated in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, such as hydroxymethylcellulose or gelatin-microcapsules and poly(methyl methacrylate) microcapsules, respectively, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions.

[0191] Sustained-release preparations can be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the immunoglobulins of the present application, which matrices are in the form of shaped articles, eg, films, or microcapsules.

[0192] 9. Diagnostic and therapeutic uses of anti-PD-1 antibodies

[0193] On the one hand, based on the specific binding of the antibodies disclosed herein to PD-1, the antibodies of the present application can be used to detect and quantify PD-1 polypeptides in physiological samples such as urine, plasma, cell lysates, and biopsy samples. Therefore, the anti-PD-1 antibodies disclosed herein can be used to monitor PD-1 levels in tissues for diagnosis, for example, to determine the progression of cancer and / or the efficacy of a given treatment regimen. It is known to those skilled in the art that the PD-1 antibodies disclosed herein can be coupled to a detectable material to facilitate detection. In certain embodiments, the anti-PD-1 antibodies disclosed herein or fragments thereof are attached to a solid support to facilitate detection.

[0194] On the other hand, based on the specific binding of the antibodies disclosed herein to PD-1, the antibodies of the present application can be used, for example, for separation by affinity chromatography or immunoprecipitation, for analysis or sorting of cells by flow cytometry, and for detection of PD-1 polypeptides in fixed tissue samples or cell smear samples by immunohistochemistry, cytological analysis, ELISA or immunoprecipitation.

[0195] In certain embodiments, the PD-1 molecule to be detected, quantified, or analyzed is a human PD-1 protein or a fragment thereof. In certain embodiments, the PD-1 protein or fragment thereof is placed in a solution, such as a lysis solution or a solution containing a subcellular fraction of disrupted cells, or is present on the surface of a PD-1 positive cell, or in a complex containing PD-1 and other cellular components.

[0196] The detection method of the present application can be used to detect the expression level of PD-1 polypeptide in a biological sample in vitro and in vivo. In vitro techniques for detecting PD-1 polypeptides include enzyme-linked immunosorbent assay (ELISA), western blot, flow cytometry, immunoprecipitation, radioimmunoassay, and immunofluorescence (e.g., IHC). In addition, in vivo techniques for detecting PD-1 polypeptides include introducing labeled anti-PD-1 antibodies into a subject. By way of example only, the antibody can be labeled with a radioactive marker, the presence and location of which in the subject can be detected by standard imaging techniques.

[0197] Other antibody-based methods for detecting protein gene expression include immunoassays, such as enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay (RIA). Suitable antibody assay labels are known in the art and include enzyme labels (such as glucose oxidase) and radioisotopes or other radioactive reagents, and fluorescent labels (such as fluorescein and rhodamine), and biotin.

[0198] The PD-1 antibodies or fragments thereof disclosed herein can be used as diagnostic reagents for any type of biological sample. In one aspect, the PD-1 antibodies disclosed herein can be used as diagnostic reagents for human biological samples. The PD-1 antibodies can be used to measure PD-1 polypeptides in a variety of standard assay formats. Such formats include immunoprecipitation, Western blot, ELISA, radioimmunoassay, flow cytometry, IHC, and immunohistochemical assays.

[0199] The present application also provides prognostic (or predictive) uses of anti-PD-1 antibodies and fragments thereof for determining whether a subject is at risk for a medical disease or condition associated with increased PD-1 polypeptide expression or activity (e.g., detecting precancerous cells). Therefore, the anti-PD-1 antibodies and fragments thereof disclosed herein can be used for prognostic or predictive purposes to prophylactically treat an individual before the onset of a medical disease or condition (e.g., cancer) characterized by, or associated with, increased PD-1 polypeptide expression or activity.

[0200] Another aspect of the present application provides a method for determining PD-1 expression in a subject to thereby screen for therapeutic or preventive compounds for a medical disease or condition (e.g., cancer) characterized by, or associated with, increased expression or activity of a PD-1 polypeptide.

[0201] In certain embodiments, the medical disease or condition is a precancerous condition or cancer, and the medical disease or condition is characterized by expression or activity of a PD-1 polypeptide or an increase in expression or activity of a PD-1 polypeptide, or is associated with an increase in expression or activity of a PD-1 polypeptide. In certain embodiments, a prognostic assay can be used to identify subjects suffering from cancer or at risk of developing cancer. Therefore, the present application provides a method for identifying a disease or condition (e.g., cancer) associated with an increased level of expression of a PD-1 polypeptide, wherein a test sample is obtained from a subject and the PD-1 polypeptide can be detected, wherein if there is an increase in the level of the PD-1 polypeptide compared to a control sample, the subject is predicted to have a disease or condition (e.g., cancer) associated with an increased level of expression of a PD-1 polypeptide or to be at risk of developing the disease or condition (e.g., cancer).

[0202] In another aspect, the present application provides a method for determining whether a subject can be effectively treated with a therapeutic agent for a disorder or condition (e.g., cancer) associated with increased expression of a PD-1 polypeptide, wherein a biological sample is obtained from the subject and the PD-1 polypeptide is detected using a PD-1 antibody. The expression level of the PD-1 polypeptide in the biological sample obtained from the subject is determined and compared to the PD-1 expression level found in a biological sample obtained from a subject without the disease. An increased level of the PD-1 polypeptide in a sample obtained from a subject suspected of having a disease or condition, compared to a sample obtained from a healthy subject, indicates a PD-1-related disease or condition (e.g., cancer) in the subject to be tested.

[0203] In one aspect, the present application provides methods for monitoring the therapeutic efficacy of an agent on PD-1 polypeptide expression. Such assays can be applied to drug screening and clinical trials. For example, the effectiveness of an agent in reducing PD-1 polypeptide levels can be monitored in a clinical trial of subjects who exhibit elevated PD-1 expression, such as patients diagnosed with cancer. Agents that affect PD-1 polypeptide expression can be identified by administering the agent and observing the response. In this way, the expression pattern of the PD-1 polypeptide can be used as a marker to indicate a subject's physiological response to the agent.

[0204] The foregoing is merely an exemplary assay using the anti-PD-1 antibodies and fragments thereof of the present application. Other methods for assaying PD-1 using antibodies or fragments thereof developed now or later are also within the scope of the present application.

[0205] In one aspect, the present application provides a method for treating cancer, comprising administering to a subject in need of such treatment an effective amount of an anti-PD-1 antibody or fragment thereof that specifically binds to PD-1. The antibodies of the present application can be used to treat, inhibit, delay progression, prevent / delay recurrence, improve or prevent diseases, disorders or conditions associated with the expression and / or activity of one or more antigenic molecules including PD-1 molecules, or associated with increased expression and / or activity of one or more antigenic molecules including PD-1 molecules.

[0206] For therapeutic uses of the anti-PD-1 antibodies or fragments thereof of the present application, the appropriate dose of the antibodies of the present application (when used alone or in combination with other agents) will depend on the type of disease to be treated, the type of antibody, the severity and course of the disease, whether the antibody is administered for prophylactic or therapeutic purposes, previous treatment, the patient's clinical history and response to the antibody, and the judgment of the attending physician. The antibody is suitable for one or more administrations to the patient. Depending on the type and severity of the disease, about 1 μg / kg to 15 mg / kg (e.g., 0.1 mg / kg-10 mg / kg) of the antibody is a suitable dose for administration to the patient, whether, for example, by one or more separate administrations or by continuous infusion.

[0207] The antibody of the present application can be used alone or in combination with other compositions for treatment. For example, the antibody of the present application can be co-administered with another antibody, steroid (such as inhalable, systemic or skin steroid), chemotherapeutic agent (including a mixture of chemotherapeutic agents), other cytotoxic agents, anti-angiogenic agents, cytokines and / or growth inhibitors. Such conjoint therapies described above include co-administration (wherein two or more medicaments are included in the same or separate preparations) and separate administration. In this case, before, during and / or after the administration of one or more other medicaments, the anti-PD-1 antibody of the present application or its fragment can be administered. The effective amount of the therapeutic agent administered in combination depends on factors such as the type of therapeutic agent to be used and the specific patient to be treated. And it will usually be decided by a doctor or veterinarian.

[0208] 10. Kits and Products

[0209] The present application provides a diagnostic method for determining the expression level of PD-1. In a specific aspect, the present application provides a kit for determining the expression level of PD-1. The kit comprises an anti-PD-1 antibody or fragment thereof disclosed herein and instructions on how to use the kit, for example, instructions for collecting samples and / or performing tests and / or analyzing the results. The kit can be used to detect the presence of PD-1 polypeptides in biological samples such as any body fluids, including but not limited to, for example, sputum, serum, plasma, lymph, cyst fluid, urine, feces, cerebrospinal fluid, ascites or blood, including biopsy samples of human tissue. The test sample can also be tumor cells, normal cells adjacent to the tumor, normal cells corresponding to the tumor tissue type, blood cells, peripheral blood lymphocytes, or a combination thereof.

[0210] In certain embodiments, the kit may further comprise one or more PD-1 antibodies other than the anti-PD-1 antibodies of the present application that are capable of binding to a PD-1 polypeptide in a biological sample. The one or more PD-1 antibodies may be labeled. In certain embodiments, the kit comprises, for example, a first antibody attached to a solid support that binds to a PD-1 polypeptide; and optionally: 2) a second, different antibody that binds to the PD-1 polypeptide or the first antibody and is conjugated to a detectable label.

[0211] The kit may also contain, for example, a buffer, a preservative, or a protein stabilizer. The kit may also contain components necessary for detecting the detectable label, such as an enzyme or a substrate. The kit may also contain a control sample or a series of control samples that can be assayed and compared with the test sample. Each component of the kit can be contained in a separate container, and all multiple containers can be placed in a single package, with instructions for using the kit, for example, instructions for collecting a sample and / or performing an assay and / or analyzing the results, included on the package insert.

[0212] On the other hand, the present application provides a product comprising materials for treating, preventing and / or diagnosing the above-mentioned conditions. The product comprises a container and a label or package insert on or connected to the container, the label or package insert having written instructions such as treatment indications, administration regimens and warnings. Suitable containers include, for example, bottles, vials, syringes, etc. The container can be formed from a variety of materials, such as glass or plastic. The container holds a composition comprising the anti-PD-1 antibody or fragment thereof of the present application, which composition itself or in combination with another composition is effective for treating, preventing and / or diagnosing a medical disease or condition (e.g., cancer), which is characterized by an increase in the expression and / or activity of one or more molecules comprising the PD-1 polypeptide, or is associated with an increase in the expression and / or activity of one or more molecules.

[0213] The article of manufacture may comprise: (a) a first container containing a composition, wherein the composition comprises the antibody of the present application; and (b) a second, third, or fourth container having a composition comprising another active ingredient. In addition, the article of manufacture may further comprise a container containing a pharmaceutically acceptable buffer (such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and glucose solution). It may further include other materials required from a commercial and user perspective, including other buffers, diluents, filters, needles, and syringes.

[0214] 11. Treatment Methods

[0215] The anti-PD-1 antibodies or fragments thereof of the present application can be used in specific treatment methods. The present application further includes antibody-based therapies, which involve administering an effective amount of the antibodies or antigen-binding fragments thereof, bispecific antibodies, polypeptides, conjugates, compositions, products or kits of the present application to a patient, such as a human patient or non-human primate, to treat one or more diseases or conditions described herein.

[0216] In some embodiments, the patient is a patient with a tumor. In some embodiments, the patient is a patient with an infection. In one embodiment, the patient has tumor cells or infected cells that overexpress a PD-1 ligand, such as PD-L1 and / or PD-L2.

[0217] Non-limiting examples of cancer include colorectal cancer, endometrial cancer, esophageal cancer, head and neck cancer, thyroid cancer, leukemias (including acute leukemias (e.g., acute lymphocytic leukemia, acute myelocytic (including myeloblastic, promyelocytic, myelomonocytic, monocytic and erythroleukemia) leukemia and chronic leukemias (e.g., chronic myeloid (granulocytic) leukemia and chronic lymphocytic leukemia)), polycythemia vera, lymphomas (e.g., Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, and solid tumors (including but not limited to sarcomas and malignant epithelial tumors such as fibrosarcoma, myeloma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, and malignant epithelial tumors). In some embodiments, the infection is a viral, bacterial, fungal, or parasitic infection. In some specific embodiments, the infection is an HIV infection.

[0218] The application also provides cell therapy, and chimeric antigen receptor (CAR) T cell therapy in certain embodiments. Suitable T cells can be used, and the T cells are contacted with the anti-PD-1 antibody or its fragment of the application (or optionally engineered to express the anti-PD-1 antibody or its binding fragment of the application). After such contact or engineering, the T cells can be introduced into a cancer patient in need of treatment. The cancer patient may suffer from any type of cancer disclosed herein. The T cells can be, for example, tumor infiltrating T lymphocytes, CD4+ T cells, CD8+ T cells, or a combination thereof, without limitation. In some embodiments, the T cells are isolated from a cancer patient. In some embodiments, the T cells are provided by a donor or from a cell bank. When the T cells are isolated from a cancer patient, the immune response that is not desired can be minimized. When the T cells are provided by a donor other than the patient himself or from a cell bank, one or more genes encoding T cell receptors and HLA genes are knocked out.

[0219] The specific dosage and treatment regimen for any particular patient will depend on various factors, including the anti-PD-1 antibody or fragment thereof used, the patient's age, weight, general health, sex and diet, as well as the time of administration, excretion rate, drug combination and the severity of the specific disease being treated. The judgment of these factors by medical care personnel is within the routine skills of the art. The dosage also depends on the individual patient to be treated, the route of administration, the type of drug, the characteristics of the compound used, the severity of the disease and the desired effect. The dosage can be determined by pharmacological and pharmacokinetic principles well known in the art.

[0220] In some embodiments, the antibodies or antigen-binding fragments thereof, bispecific antibodies, polypeptides, conjugates, compositions, products or kits of the present application are administered in combination with an antitumor agent, an antiviral agent, an antibacterial or antibiotic agent or an antifungal agent. Any of these agents known in the art can be administered in the presently disclosed compositions.

[0221] In another embodiment, the antibody or antigen-binding fragment thereof, bispecific antibody, polypeptide, conjugate, composition, product or kit of the present application is administered in combination with a chemotherapeutic agent. Chemotherapeutic agents that can be administered with the composition of the present application include, but are not limited to, antibiotic derivatives (such as doxorubicin, bleomycin, daunorubicin and actinomycin D); antiestrogens (e.g., tamoxifen); antimetabolites (e.g., fluorouracil, 5-FU, methotrexate, fluorouracil, interferon α-2b, glutamic acid, plikamycin, mercaptopurine and 6-thioguanine); cytotoxic agents (e.g., carmustine, BCNU, lomustine, CCNU, cytarabine, cyclophosphamide, estramustine, hydroxyurea, procarbazine, mitomycin, busulfan, cisplatin and vincristine sulfate); hormones (e.g., medroxyprogesterone, estramustine sodium phosphate, ethinyl estradiol, estradiol, megestrol acetate, methyltestosterone, diethylstilbestrol phosphate (diethylstilbestrol); diphosphate), chlorfenapyr, and testolactone); nitrogen mustard derivatives (e.g., melphalan, chlorambucil, dichloromethyl diethylamide (nitrogen mustard), and thiotepa); steroids and combinations thereof (e.g., betamethasone sodium phosphate); and others (e.g., dacarbazine, asparaginase, mitotane, vincristine sulfate, vinblastine sulfate, and etoposide).

[0222] In another embodiment, the antibody or antigen-binding fragment thereof, bispecific antibody, polypeptide, conjugate, composition, product or kit of the present application is administered in combination with a cytokine, wherein the cytokine includes but is not limited to IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12, IL-13, IL-15, anti-CD40, CD40L, and TNF-α. In another embodiment, the composition of the present application is administered in combination with other treatment or prevention regimens (e.g., radiotherapy).

[0223] The antibodies or antigen-binding fragments thereof, bispecific antibodies, polypeptides, conjugates, compositions, products or kits of the present application may be used in some embodiments with immune checkpoint inhibitors. Immune checkpoints are the following molecules in the immune system that either turn up the signal (co-stimulatory molecules) or turn down the signal. Many cancers protect themselves from the immune system by inhibiting T cell signals. Immune checkpoint inhibitors can help prevent this protective mechanism. Immune checkpoint inhibitors can be directed against any one or more of the following checkpoint molecules: 2B4; 4-1BB; 4-1BB ligands, B7-1; B7-2; B7H2; B7H3; B7H4; B7H6; BTLA; CD155; CD160; CD19; CD200; CD27; CD27 ligands. CD28; CD40; CD40 ligand; CD47; CD48; CTLA-4; DNAM-1; Galectin-9; GITR; GITR ligand; HVEM; ICOS; ICOS ligand; IDOI; KIR; 3DL3; LAG-3; OX40; OX40 ligand; PD-L1; PD-1; PD-L2; LAG3; PGK; SIRPα; TIM-3; PD-1; VSIG8.

[0224] Programmed T-cell death 1 (PD-1) is a transmembrane protein found on the surface of T cells that, when bound to programmed T-cell death ligand 1 (PD-L1) on tumor cells, leads to inhibition of T-cell activity and reduction of T-cell-mediated cytotoxicity. Therefore, PD-1 and PD-L1 are immune downregulators or immune checkpoint "off switches." Examples of PD-1 inhibitors include, but are not limited to, nivolumab (Opdivo) (BMS-936558), pembrolizumab (Keytruda, pidilizumab, AMP-224, MEDI0680 (AMP-514, PDR001, MPDL3280A, MEDI4736, BMS-936559, and MSB0010718C. Programmed death ligand 1 (PD-L1), also known as cluster of differentiation 274 (CD274) or B7 homolog 1 (B7-H1), is a protein that, in humans, is encoded by the CD274 gene. Non-limiting examples of PD-L1 inhibitors include atezolizumab (Tecentriq), durvalumab (MEDI4736), avelumab (MSB0010718C), MPDL3280A, BMS S935559 (MDX-105) and AMP-224. CTLA-4 is a protein receptor that downregulates the immune system. Non-limiting examples of CTLA-4 inhibitors include ipilimumab (Yervoy) (also known as BMS-734016, MDX-010, MDX-101) and tremelimumab (formerly ticilimumab, CP-675,206). Lymphocyte activation gene 3 (LAG-3) is an immune checkpoint receptor on the surface of cells that inhibits immune responses by acting on Tregs and directly on CD8+ T cells. LAG-3 inhibitors include but are not limited to LAG525 and BMS-986016. CD28 is expressed on almost all human CD4+ T cells and approximately half of CD8 Constitutively expressed on T cells. Promotes T cell expansion. Non-limiting examples of CD28 inhibitors include TGN1412. CD122 increases the proliferation of CD8+ effector T cells. Non-limiting examples include NKTR-214. 4-IBB (also known as CD137) is involved in T cell proliferation. It is known that CD137-mediated signaling can also protect T cells, especially CD8+ T cells from activation-induced cell death. PF-05082566, Urelumab (BMS-663513) and lipocalin are examples of CD137 inhibitors.

[0225] For any of the above combination therapies, the antibodies or antigen-binding fragments thereof, bispecific antibodies, polypeptides, conjugates, compositions, preparations or kits of the present application can be administered simultaneously or separately with other anticancer agents.

[0226] In one embodiment, a method of treating or inhibiting an infection in a patient in need thereof is provided, comprising administering to the patient an effective amount of the antibody or antigen-binding fragment thereof, bispecific antibody, polypeptide, conjugate, composition, preparation or kit of the present application.

[0227] Example

[0228] Example 1. Production of anti-PD-1 antibodies

[0229] BALB / c mice (6 weeks old, purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd.) were immunized subcutaneously with his-tagged human PD-1 recombinant protein (PD-1 / 6His, produced in-house, NCBI accession number: NP_005009.2, extracellular domain Pro21-Gln167) and complete Freund's adjuvant (Sigma Aldrich #F5881). Immunizations were repeated four times, with a 3-day interval between each. Following protein immunization, mice were immunized twice with irradiated Jurkat cells expressing human PD-1 (generated in Example 6). Three days after the final immunization, lymph nodes near the injection site were carefully dissected. Lymphocytes were fused with P3X63Ag8.653 myeloma cells (cell bank, Chinese Academy of Sciences, #TCM10) using PEG1500 (polyethylene glycol 1500, Roche #783641, 10x4 mL, dissolved in 75 mM Hepes, PEG 50% W / V) and cloned using HAT selection (Sigma #H0262) and HFCS (hybridoma fusion and cloning supplement, 50x, Roche #11-363-735-001). Hybridoma supernatants were screened by ELISA and cell matrix assays to obtain antibodies that bind to human PD-1. Selected mouse anti-PD-1 clones were humanized using CDR grafting and reverse mutation.

[0230] Humanization of antibodies is achieved by CDR transplantation: the acceptor framework is selected. The variable region sequence of the parent antibody is searched in the human germline database using NCBI Ig-Blast (http: / / www.ncbi.nlm.nih.gov / projects / igblast). Five different human acceptors (i.e., human variable regions with a high homology to the parent antibody) are selected for each heavy chain and light chain. The CDRs of the human acceptors are replaced with the CDRs of the mouse to form a humanized variable region sequence. Then, reverse mutations are performed to produce four heavy and light chains. The mouse CDR sequences of the heavy and light chains (SEQ ID NO: 1-6) are shown below, respectively. The coding genes for 9 humanized heavy chains and 9 humanized light chains were designed, synthesized, and inserted into expression vectors. The humanized antibodies are expressed and then used to test affinity rankings.

[0231] Example 2: Expression and purification of anti-PD-1 antibodies

[0232] The DNA sequence encoding the humanized IgG heavy and light chains was synthesized and inserted into the pTT5 vector (available at Genscript Biotech) to construct an expression plasmid for full-length IgG. The chimeric antibody was expressed in HEK 293 cell cultures (available at Thermo Fisher Scientific), and the supernatant was purified using a protein A affinity column (Yeasen #36410ES08). The purified antibody was buffer exchanged into PBS using a PD-10 desalting column (available at Thermo Fisher Scientific). The concentration and purity of the purified antibody were measured by OD280 and SDS-PAGE, respectively. The humanized antibody was expressed in HEK 293 cell cultures. The cells were centrifuged and precipitated. The supernatant was filtered and subjected to SDS-PAGE analysis ( Figure 1 A random mixture of human IgG (available from Genscript Biotechnology) was used as a control. The results showed that the humanized antibodies were successfully expressed and purified.

[0233] Example 3. SPR analysis of the binding affinity of anti-PD-1 antibodies to human PD-1

[0234] Anti-human Fc gamma-specific antibodies (Jackson ImmunoResearch #109-005-098) were immobilized on the sensor chip using an amine coupling method. The humanized antibodies secreted into the culture medium plus chimeric VH+VL (parent mouse VH+VL bound to human Fc) were injected separately and captured by Fc (capture phase) using anti-human Fc antibodies. After equilibrium, PD-1 was injected for 200 seconds (binding phase) and then the electrophoresis buffer was injected for 600 seconds (dissociation phase). The response value of the reference flow cell (flow cell 1) was subtracted from the response value of the humanized antibody flow cell for each cycle. The surface was regenerated before injecting other humanized antibodies. The process was repeated until all antibodies were analyzed. The shedding rate of the humanized antibodies was obtained by locally fitting the experimental data to a 1:1 interaction model using Biacore 8K evaluation software. Antibodies were sorted by their dissociation rate constant (shedding rate, kd). Binders with similar affinity to PD-1 as the parent antibody were selected (Table 1).

[0235] Table 1. Affinity measurement data

[0236]

[0237] Therefore, VH6+VL1, VH6+VL6, VH7+VL1 and VH7+VL6 were selected for further characterization. The sequences of the antibodies or fragments thereof in Table 2 are shown below.

[0238] CDR1H amino acid sequence (SEQ ID NO: 1)

[0239] GFTFSSYGMS

[0240] CDR2H amino acid sequence (SEQ ID NO: 2)

[0241] IISGGGRDIYYLDSVKG

[0242] CDR3H amino acid sequence (SEQ ID NO: 3)

[0243] PIYDAYSFAY

[0244] CDR1L amino acid sequence (SEQ ID NO: 4)

[0245] RASQTISNNLH

[0246] CDR2L amino acid sequence (SEQ ID NO: 5)

[0247] YASQSIS

[0248] CDR3L amino acid sequence (SEQ ID NO: 6)

[0249] QQSYSWPLT

[0250] Heavy chain variable region (VH6) amino acid sequence (SEQ ID NO: 7)

[0251] EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYGMSWVRQAPGKRLEWAIISGGGRDIYYLDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCSSPIYDAYSFAYWGQGTLVTVSS

[0252] Heavy chain variable region (VH7) amino acid sequence (SEQ ID NO: 8)

[0253] EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYGMSWVRQAPGKGLEWAIISGGGRDIYYLDSVKGRFTISRDNSKNNLYLQMNSLRAEDTAVYYCSSPIYDAYSFAYWGQGTLVTVSS

[0254] Light chain variable region (VL1) amino acid sequence (SEQ ID NO: 9).

[0255] EIVMTQSPATLSVSPGERATLSCRASQTISNNLHWYQQKPGQAPRLLIYYASQSISGIPARFSGSGTEFTLTISSLQSEDFAVYYCQQSYSWPLTFGGGTKLEIK

[0256] Light chain variable region (VL6) amino acid sequence (SEQ ID NO: 10)

[0257] EIVLTQSPATLSVSPGERATLSCRASQTISNNLHWYHQKPGQAPRLLIKYASQSISGIPSRFSGSGTDFTLTISSLQSEDFAVYFCQQSYSWPLTFGGGTKLEIK

[0258] The heavy chain amino acid sequence 1 (HC1) of VH6 is included (SEQ ID NO: 11, full-length sequence).

[0259] EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYGMSWVRQAPGKRLEWVAIISGGGRDIYYLDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCSSPIYDAYSFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0260] Heavy chain amino acid sequence 2 (HC2) containing VH7 (SEQ ID NO:12, full-length sequence).

[0261] EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYGMSWVRQAPGKGLEWVAIISGGGRDIYYLDSVKGRFTISRDNSKNNLYLQMNSLRAEDTAVYYCSSPIYDAYSFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0262] Contains the light chain amino acid sequence 1 (LC1) of VL1 (SEQ ID NO: 13, full-length sequence).

[0263] EIVMTQSPATLSVSPGERATLSCRASQTISNNLHWYQQKPGQAPRLLIYYASQSISGIPARFSGSGTEFTLTISSLQSEDFAVYYCQQSYSWPLTFGGGTKLE IKRTVAAPSVFPPSDEQLKSGTASVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0264] Contains the light chain amino acid sequence 2 (LC2) of VL6 (SEQ ID NO: 14, full-length sequence).

[0265] EIVLTQSPATLSVSPGERATLSCRASQTISNNLHWYHQKPGQAPRLLIKYASQSISGIPSRFSGSGTDFTLTISSLQSEDFAVYFCQQSYSWPLTFGGGTKLEI KRTVAAPSVFIFPPSDEQLKSGTASVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0266] Heavy chain amino acid sequence comprising the chimeric VH (SEQ ID NO: 15, full-length sequence)

[0267] EVKLVESGGGLVKPGGSLKLSCAASGFTFSSYGMSWVRQTPEKRLEWVAIISGGGRDIYYLDSVKGRFTISRDNAKNNLYLQMSSLRSEDTAFYYCSSPIYDAYSFAYWGQG TLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0268] The light chain amino acid sequence comprising the chimeric VL (SEQ ID NO: 16, full-length sequence).

[0269] DIVLVQSPATLSVTPGDSVSLSCRASQTISNNLHWYHQKSHESPRLLIKYASQSISGIPSRFSGSGTDFTLSINSVETEDFGMYFCQQSYSWPLTFGAGTNLELKRTVAAPSVFIFPPSDEQLKSGTASVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0270] For further explanation, the inclusion relationship between the above sequences is shown in Table 2. The sequence on the right is included in the sequence on the left in the same row.

[0271] Table 2:

[0272]

[0273]

[0274] Example 4. Measurement of binding to human and macaque PD-1 by ELISA

[0275] MaxiSorp 96-well plates (NUNC #449824) were coated with 2 μg / mL human PD-1 / His (self-produced) or macaque PD-1 / His protein (ACROBiosystems #PD1-C5223) dissolved in 1x PBS (50 μL / well). The plates were incubated overnight at 4°C. The coating solution was removed and the plates were washed once with 200 μL / well PBST (1x PBS containing 0.05% Tween-20). Then, 200 μL / well blocking buffer (1x PBS containing 0.05% Tween-20, 3% BSA) was added and incubated at room temperature for 1 hour. The blocking buffer was removed and the plates were washed three times with 200 μL / well PBST. Antibodies VH7+VL6 (generated in Example 2) and human IgG1 isotype control (hIgG1, Sigma #I5154-1MG) were diluted with 1x PBS and added to the plate (50 μL / well). The plate was incubated at room temperature for 2 hours. The antibodies in the wells were removed and the plates were washed three times with 200 μL / well of PBST. Goat anti-human IgG (H&L)-HRP secondary antibody (Jackson Immuno Research #109-035-088) was diluted 1:5000 in 1x PBS and added to each well (50 μL / well). The plate was incubated at room temperature for 1 hour. The secondary antibody was removed and the plate was washed 5 times with 200 μL / well of PBST. 50 μL / well TMB (eBioscience #85-00-4201-56) was added and incubated at room temperature for several minutes. 50 μL / well of 2N H2SO4 was then added to stop the reaction. Optical density was measured at 450 nm.

[0276] The anti-PD-1 antibody VH7+VL6 binds to human and macaque PD-1 with EC50 of 0.10 nM and 0.40 nM, respectively (Table 3). These results indicate that the anti-PD-1 antibody can bind to human and macaque PD-1 with high affinity ( Figure 2A and 2B ).

[0277] Table 3. Binding to human and macaque PD-1

[0278]

[0279] Example 5. Binding to human PD-1 on Jurkat cells

[0280] Jurkat cells expressing human PD-1 (generated in Example 6) were incubated with different concentrations of anti-PD-1 antibodies VH7+VL6 or human IgG1 isotype control (Sigma #I5154-MG) at 4°C for 30 minutes. The cells were then washed once with FACS buffer (PBS plus 2% FBS) and incubated with Alexa Fluor 594 AffiniPure goat anti-human IgG secondary antibody (Jackson ImmunoResearch #109-585-088) at 4°C for 30 minutes. After washing once with FACS buffer, the cells were resuspended in 200 μL FACS buffer. The stained cells were analyzed using a BD LSRFortessa flow cytometer.

[0281] like Figure 3 As shown, the anti-PD-1 antibody VH7+VL6 bound to human PD-1 expressed on Jurkat with an EC50 of 2.16 nM.

[0282] Example 6. Blocking effect of anti-PD-1 antibodies on the interaction between PD-1 and PD-L1 in a cell-based assay

[0283] In order to carry out the test, two stable cell lines were first generated. DNA encoding chimeric PD-1 receptors (extracellular and transmembrane domains of human PD-1 fused to the cytoplasmic domain of human CD3ζ chain (NCBI accession number: NP_932170.1)) and DNA encoding NFAT-luciferase (amplified from pGL4.30 [luc2P / NFAT-RE / Hygro], Sigma # E8481) were cloned into pcDNA3.4 vectors (Invitrogen # A14697) and transfected into Jurkat cells (cell bank, Chinese Academy of Sciences, # TCHU123) by electroporation. Stable cell lines were generated by G418 selection and limiting dilution and named Jurkat / PD-1-CD3z / NF-luc cells. Using the same method, a stable cell line expressing full-length human PD-L1 (NCBI accession number: NP_054862.1) was generated on CHO-K1 cells (cell bank, Chinese Academy of Sciences, #GNHa7) and named CHO / PD-L1 cells.

[0284] One day before co-culture, CHO / PD-L1 cells were seeded in 96-well flat-bottom plates (NUNC#167008) (5×10 4cells / well) in complete RPMI1640 medium (Thermo Fisher#C11875500BT) containing 10% FBS (Gibco#16000-044) and 1% penicillin-streptomycin (Corning#30-002-CI) and cultured overnight in a CO2 incubator. Jurkat / PD-1-CD3z / NF-luci cells were pre-cultured with anti-PD-1 antibody or human IgG1 isotype control (Sigma#I5154-1MG) for 30 minutes before co-culture with CHO / PD-L1 cells. The culture medium of CHO / PD-L1 cells was then removed, and Jurkat / PD-1-CD3z / NF-luci cells with antibodies were seeded into the wells (1×10 5 After 6 hours, the fluorescence signal was detected using the Luciferase Assay System kit (Promega #E1500).

[0285] The results are shown in Table 4 and Figure 4 The VH7+VL6 antibody completely inhibited the fluorescence signal induced by CHO / PD-L1 cells, indicating that the VH7+VL6 antibody can effectively block the interaction between PD-1 and PD-L1. Nivolumab (CAS#946414-94-4, produced by Shanghai Cabot Corporation) and pembrolizumab (CAS#1374853-91-4, produced by Shanghai Cabot Corporation) inhibited the fluorescence signal by 75% and 71% respectively.

[0286] Table 4. Inhibition of luciferase signal by anti-PD-1 antibodies

[0287] Antibody EC50 (nM) Maximum inhibition (%) VH7+VL6 0.38 100 Nivolumab 0.04 75 Pembrolizumab 0.16 71

[0288] Example 7. Enhancement of IL-2, IFN-γ and TNF-α production on human PBMCs

[0289] IL-2 release assay

[0290] Staphylococcal enterotoxin B (SEB, 0.1 μg / mL, dissolved in PBS, 100 μL / well) (provided by the Chinese Academy of Military Medical Sciences) was coated on a 96-well flat-bottom plate (NUNC #167008) overnight at 4°C. The next day, human peripheral blood mononuclear cells (PBMCs) isolated from healthy donors were suspended in complete RPMI1640 medium (Thermo Fisher #C11875500BT) containing 10% FBS (Gibco #16000-044) and 1% penicillin-streptomycin (Corning #30-002-CI). PBMCs were then seeded onto the pre-coated plates (3 × 10 5Cells were plated at 400 nmol / well and incubated with different concentrations of anti-PD-1 antibody or human IgG1 isotype control (Sigma #I5154-1MG) in a CO2 incubator for 72 hours. Culture supernatants were collected and IL-2 levels were assessed using the Human IL-2 DuoSet ELISA Kit (R&D systems #DY202) according to the manufacturer's instructions.

[0291] Figure 5A showed that antibodies VH7+VL6, nivolumab, and pembrolizumab increased IL-2 secretion by PBMCs at equivalent levels.

[0292] IFN-γ and TNF-α release assay

[0293] SEB (40 ng / ml, dissolved in 17 ml PBS) was used to coat 100 mm TC-treated cell culture dishes (BD Falcon #353003) at 4°C overnight. 7 Individual PBMC cells were suspended in 20 mL of complete RPMI1640 medium containing 10% FBS and 1% penicillin-streptomycin and seeded on pre-coated dishes. The dishes were cultured in a CO2 incubator. 96-well flat-bottom plates were coated with SEB (4 ng / ml, dissolved in PBS, 100 μL / well) overnight at 4°C. After 72 hours of culture, PBMCs were collected by centrifugation and washed once with complete RPMI1640 medium. PBMCs were then pre-incubated with different concentrations of anti-PD-1 antibodies or human IgG1 isotype control (Sigma#I5154-1MG) for 30 minutes and seeded on plates pre-coated with SEB (3×10 5 After culturing in a CO2 incubator for 24 hours, the culture supernatants were collected and the levels of IFN-γ and TNF-α were assessed using human IFN-γ DuoSet ELISA (R&D systems #DY285B) and human TNF-α DuoSet ELISA (R&D systems #DY210) kits according to the manufacturer's instructions.

[0294] Consistent with the results of the IL-2 release assay, all anti-PD-1 antibodies significantly increased the secretion of IFN-γ and TNF-α. The increase in IFN-γ and TNF-α secretion induced by antibody VH7+VL6 was greater than that induced by nivolumab and pembrolizumab ( Figure 5B and 5C ).

[0295] Example 8. In vivo study of anti-tumor activity in animals

[0296] Antibody expression and purification for animal research

[0297] DNA sequences encoding VH7 (SEQ ID NO: 8) and VL6 (SEQ ID NO: 10) were subcloned into the pcDNA3.4 vector (Invitrogen #A14697) to construct two plasmids, pcDNA3.4-VH7 and pcDNA3.4-VL6. pcDNA3.4-VH7 and pcDNA3.4-VL6 were prepared using the endotoxin-free Plasmid DNA Maxiprep Kit (TIANGEN #DP117). Antibody expression was performed in 293-F (Invitrogen #R79007). Antibodies in the culture supernatant were purified using a protein A affinity column (Yeasen #36410ES08). The purified antibodies were then dialyzed and buffer-exchanged into histidine buffer (20 mM histidine, 5% sucrose, 0.02% Tween 80, pH 5.5). The concentration and purity of the purified antibodies were determined by OD280 and SDS-PAGE, respectively.

[0298] Animal studies

[0299] In this study, a human PD-1 knockout mouse tumor model bearing CT26 was used to investigate the antitumor activity of antibody VH7+VL6.

[0300] Mouse colon cancer cells CT26 (cell bank, Chinese Academy of Sciences, #TCM37) were cultured in RPMI1640 medium supplemented with 10% FBS and 1% penicillin-streptomycin. 5×10 5 CT26 cells were injected subcutaneously into the right dorsal flank of each human PD-1 knockout mouse (BALB / c, female, 6-8 weeks old, GemPharmatech). 3 Mice were randomly divided into groups of 8 and administered with antibodies. Anti-PD-1 antibodies VH7+VL6 were injected intraperitoneally at a dose of 5 mg / kg on days 5, 8, 11, 14, and 17. Mice in the control group were injected with a human IgG1 isotype control (Bioxcell #BP0085). Tumors were measured with a caliper every two days. Tumor volume was calculated according to the following formula: width 2 × length / 2(mm 3 When the mean tumor volume of any group reached 2000 mm 3 The mice were euthanized when .

[0301] Figure 6A The results showed that antibody VH7+VL6 strongly inhibited tumor growth in vivo, with 50% of tumors completely regressed on day 20 ( Figure 6B and 6CIn the control group treated with human IgG1 isotype antibody, tumors grew significantly faster and larger. There was no significant change in body weight associated with antibody administration. Sequence Listing <110> Suzhou Xinkanghe Biopharmaceutical Technology Co., Ltd. Beijing Xinkanghe Biopharmaceutical Technology Co., Ltd. <120> Anti-PD-1 polypeptides and their uses <130> PF02114 <150> PCT / CN2021 / 094422 <151> 2021-05-18 <160> 16 <170> PatentIn version 3.5 <210> 1 <211> 10 <212> PRT <213> Artificial sequence <220> <223> CDR1H <400> 1 Gly Phe Thr Phe Ser Ser Tyr Gly Met Ser 1 5 10 <210> 2 <211> 17 <212> PRT <213> Artificial sequence <220> <223> CDR2H <400> 2 Ile Ile Ser Gly Gly Gly Arg Asp Ile Tyr Tyr Leu Asp Ser Val Lys 1 5 10 15 Gly <210> 3 <211> 10 <212> PRT <213> Artificial sequence <220> <223> CDR3H <400> 3 Pro Ile Tyr Asp Ala Tyr Ser Phe Ala Tyr 1 5 10 <210> 4 <211> 11 <212> PRT <213> Artificial sequence <220> <223> CDR1L <400> 4 Arg Ala Ser Gln Thr Ile Ser Asn Asn Leu His 1 5 10 <210> 5 <211> 7 <212> PRT <213> Artificial sequence <220> <223> CDR2L <400> 5 Tyr Ala Ser Gln Ser Ile Ser 1 5 <210> 6 <211> 9 <212> PRT <213> Artificial sequence <220> <223> CDR3L <400> 6 Gln Gln Ser Tyr Ser Trp Pro Leu Thr 1 5 <210> 7 <211> 119 <212> PRT <213> Artificial sequence <220> <223> VH6 <400> 7 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Arg Leu Glu Trp Val 35 40 45 Ala Ile Ile Ser Gly Gly Gly Arg Asp Ile Tyr Tyr Leu Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ser Ser Pro Ile Tyr Asp Ala Tyr Ser Phe Ala Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 8 <211> 119 <212> PRT <213> artificial sequence <220> <223> VH7 <400> 8 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Ile Ile Ser Gly Gly Gly Arg Asp Ile Tyr Tyr Leu Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Asn Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ser Ser Pro Ile Tyr Asp Ala Tyr Ser Phe Ala Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 9 <211> 107 <212> PRT <213> artificial sequence <220> <223> VL1 <400> 9 Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Thr Ile Ser Asn Asn 20 25 30 Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Tyr Ala Ser Gln Ser Ile Ser Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Tyr Ser Trp Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 10 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> VL6 <400> 10 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Thr Ile Ser Asn Asn 20 25 30 Leu His Trp Tyr His Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Lys Tyr Ala Ser Gln Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser 65 70 75 80 Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Ser Tyr Ser Trp Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 11 <211> 449 <212> PRT <213> artificial sequence <220> <223> HC1 <400> 11 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Arg Leu Glu Trp Val 35 40 45 Ala Ile Ile Ser Gly Gly Gly Arg Asp Ile Tyr Tyr Leu Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ser Ser Pro Ile Tyr Asp Ala Tyr Ser Phe Ala Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445 Lys <210> 12 <211> 449 <212> PRT <213> Artificial Sequence <220> <223> HC2 <400> 12 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Ile Ile Ser Gly Gly Gly Arg Asp Ile Tyr Tyr Leu Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Asn Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ser Ser Pro Ile Tyr Asp Ala Tyr Ser Phe Ala Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445 Lys <210> 13 <211> 214 <212> PRT <213> Artificial sequence <220> <223> LC1 <400> 13 Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Thr Ile Ser Asn Asn 20 25 30 Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Tyr Ala Ser Gln Ser Ile Ser Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Tyr Ser Trp Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 14 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> LC2 <400> 14 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Thr Ile Ser Asn Asn 20 25 30 Leu His Trp Tyr His Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Lys Tyr Ala Ser Gln Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser 65 70 75 80 Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Ser Tyr Ser Trp Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 15 <211> 449 <212> PRT <213> Artificial Sequence <220> <223> HC Containing Chimeric VH <400> 15 Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly<000102$7>1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Gly Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val 35 40 45 Ala Ile Ile Ser Gly Gly Gly Arg Asp Ile Tyr Tyr Leu Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Asn Leu Tyr 65 70 75 80 Leu Gln Met Ser Ser Leu Arg Ser Glu Asp Thr Ala Phe Tyr Tyr Cys 85 90 95 Ser Ser Pro Ile Tyr Asp Ala Tyr Ser Phe Ala Tyr Tr p Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ala Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445 Lys <210> 16 <211> 214 <212> PRT <213> Artificial sequence <220> <223> LC containing chimeric VL <400> 16 Asp Ile Val Leu Val Gln Ser Pro Ala Thr Leu Ser Val Thr Pro Gly 1 5 10 15 Asp Ser Val Ser Leu Ser Cys Arg Ala Ser Gln Thr Ile Ser Asn Asn 20 25 30 Leu His Trp Tyr His Gln Lys Ser His Glu Ser Pro Arg Leu Leu Ile 35 40 45 Lys Tyr Ala Ser Gln Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Thr 65 70 75 80 Glu Asp Phe Gly Met Tyr Phe Cys Gln Gln Ser Tyr Ser Trp Pro Leu 85 90 95 Thr Phe Gly Ala Gly Thr Asn Leu Glu Leu Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210

Claims

1. An isolated antibody or antigen-binding fragment thereof comprising a heavy chain (HC) variable region sequence and a light chain (LC) variable region sequence, wherein the antibody binds to the extracellular domain of PD-1 with a binding affinity greater than 10 nM as determined by SPR analysis, wherein (a) The HC comprises CDR1H, the amino acid sequence of CDR1H is GFTFSSYGMS, as shown in SEQ ID NO: 1, CDR2H, the amino acid sequence of CDR2H is IISGGGRDIYYLDSVKG, as shown in SEQ ID NO: 2, and CDR3H, wherein the amino acid sequence of CDR3H is PIYDAYSFAY, as shown in SEQ ID NO: 3; (b) the LC comprises CDR1L, the amino acid sequence of CDR1L is RASQTISNNLH, as shown in SEQ ID NO: 4, CDR2L, the amino acid sequence of CDR2L is YASQSIS, as shown in SEQ ID NO: 5, and CDR3L, the amino acid sequence of CDR3L is QQSYSWPLT, as shown in SEQ ID NO:

6.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody is a chimeric antibody, a humanized antibody or a human antibody.

3. The antibody or antigen-binding fragment thereof according to claim 1 or 2, further comprising a human acceptor framework.

4. The antibody or antigen-binding fragment thereof of claim 1 , wherein the HC variable region sequence comprises the amino acid sequence of SEQ ID NO: 7, or an amino acid sequence that is greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:

7.

5. The antibody or antigen-binding fragment thereof of claim 1 , wherein the HC variable region sequence comprises the amino acid sequence of SEQ ID NO: 8, or an amino acid sequence that is greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:

8.

6. The antibody or antigen-binding fragment thereof of claim 4 or 5, wherein the LC variable region sequence comprises the amino acid sequence shown in SEQ ID NO: 9 or an amino acid sequence having greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with SEQ ID NO:

9.

7. The antibody or antigen-binding fragment thereof of claim 4 or 5, wherein the LC variable region sequence comprises the amino acid sequence shown in SEQ ID NO: 10 or an amino acid sequence having greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with SEQ ID NO:

10.

8. The antibody or antigen-binding fragment thereof of claim 1 , wherein the HC variable region sequence comprises the amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 8, or an amino acid sequence that is greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO. 7 or 8, and the LC variable region sequence comprises the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 10, or an amino acid sequence that is greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 9 or 10.

9. The antibody or antigen-binding fragment thereof according to claim 1, wherein 1) the HC variable region sequence comprises the amino acid sequence of SEQ ID NO: 7, or an amino acid sequence that is greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 7, and the LC variable region sequence comprises the amino acid sequence of SEQ ID NO: 10, or an amino acid sequence that is greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 10, or 2) the HC variable region sequence comprises the amino acid sequence of SEQ ID NO: 8, or an amino acid sequence that is greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 8, and the LC variable region sequence comprises the amino acid sequence of SEQ ID NO: 10, or an amino acid sequence that is greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:

10.

10. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody is of the IgG isotype.

11. The antibody or antigen-binding fragment thereof according to claim 1, wherein the antigen-binding fragment comprises any one selected from the group consisting of Fab, F(ab')2, Fab', scFv, Fv, Fd, dAb and diabody. 12 . A bispecific antibody comprising the antibody or antigen-binding fragment thereof according to claim 1 and a second antibody or antigen-binding fragment thereof.

13. The bispecific antibody according to claim 12, wherein the second antibody or antigen-binding fragment thereof specifically binds to a tumor antigen expressed on the surface of a tumor cell, wherein the tumor antigen comprises any one selected from the group consisting of A33; ADAM-9; ALCAM; BAGE; β-catenin; CA125; carboxypeptidase M; CD103; CD19; CD20; CD22; CD23; CD25; CD27; CD28; CD36; CD40 / CD154; CD45; CD46; CD5; CD56; CD79a / CD79b; CDK4; CEA; CTLA4; cytokeratin 8; EGF-R; EphA2; ErbB1; ErbB3; ErbB4; GAGE-1; GAGE-2; GD2 / GD3 / GM2; HER-2 / neu; human papillomavirus-E6; human papillomavirus-E7; JAM-3; KID3; KID31; KSA (17-1A); LUCA-2; MAGE-1; MAGE-3; MART; MUC-1; MUM-1; N-acetylglucosaminyltransferase; oncostatin M; p15; PIPA; PSA; PSMA; ROR1; TNF-β receptor; TNF-α receptor; TNF-γ receptor; transferrin receptor; and VEGF receptor.

14. A conjugate comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 11, linked to a therapeutic agent.

15. The conjugate of claim 14, wherein the therapeutic agent is a cytotoxin or a radioisotope.

16. A composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 11, the bispecific antibody according to claim 12 or 13, or the conjugate according to claim 14 or 15, and a pharmaceutically acceptable excipient.

17. Lymphocytes comprising T cells and / or NK cells from a subject, which are treated in vitro with the antibody or antigen-binding fragment thereof according to any one of claims 1 to 11.

18. An isolated nucleic acid encoding the antibody or antigen-binding fragment thereof of any one of claims 1-11.

19. An expression vector comprising the nucleic acid of claim 18.

20. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 11, the bispecific antibody according to claim 12 or 13, the composition according to claim 16, or the lymphocyte according to claim 17 in the preparation of a medicament for treating cancer in a subject; in, The HC variable region sequence comprises the amino acid sequence of SEQ ID NO: 8, or an amino acid sequence having greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 8, and the LC variable region sequence comprises the amino acid sequence of SEQ ID NO: 10, or an amino acid sequence that is greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 10; The cancer is selected from any one or more of the following groups: lymphoma, melanoma, colorectal adenocarcinoma, prostate cancer, breast cancer, lung cancer, liver cancer, gastric cancer and renal clear cell carcinoma.

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