Monoclonal antibodies against human programmed cell death protein 1 (PD-1)

Abstract

The present disclosure relates to antibodies and antigen-binding fragments that specifically bind to human programmed cell death protein 1 (PD-1), and methods of using the antibodies and antigen-binding fragments in the treatment of cancer or other diseases.

Description

[0001] Cross - reference to related applications

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 378,472, filed on October 5, 2022, which is incorporated herein by reference in its entirety.

[0003] Incorporation by reference of the Sequence Listing

[0004] The Sequence Listing XML associated with this application is provided electronically in XML file format and is hereby incorporated by reference into this specification. The name of the XML file containing the Sequence Listing XML is "FMCU002_001WO_SeqList". The XML file is 311,500 bytes, created on October 4, 2023, and electronically filed via the USPTO Patent Center. Technical Field

[0005] The present disclosure relates to antibodies and antigen-binding fragments that specifically bind to human programmed cell death protein 1 (PD-1), and methods of using the antibodies and antigen-binding fragments to treat cancer or other diseases. Background Art

[0006] In the past few decades, cancer has become the leading cause of death globally, and its incidence still increases annually. Recently, cancer immunotherapy has emerged as a promising treatment strategy by harnessing the patient's immune system against cancer (Marin-Acevedo et al., J Hematol Oncol. 2018; 11(1):8; Waldman et al., Nat Rev Immunol. 2020; 20(11):651-68).

[0007] Immune checkpoint inhibitors (ICIs) are drugs that target immune checkpoint molecules and are currently the most successful cancer immunotherapy agents (Pardoll et al., Nat Rev Cancer. 2012; 12(4):252-64; Hargadon et al., Int Immunopharmacol. 2018; 62:29-39).

[0008] Approved ICIs include monoclonal antibodies against programmed cell death protein 1 (PD-1). PD-1 is an immunosuppressive co-stimulatory signal receptor that is mainly expressed on activated T cells and B cells. It controls inappropriate and extreme immune responses, such as autoimmune and excessive infectious immune responses, and curbs antigen receptor activation through programmed death ligand 1 (PD-L1) (Chamoto et al., Int J Oncol. 25:790-800 (2020)).

[0009] Despite their initial success, a large number of patients still do not respond to PD-1 targeted therapies, and side effects associated with autoimmune responses limit the clinical potential of these drugs. Thus, there remains a need for new inhibitors of PD-1 and / or PD-L1.

[0010] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. In this specification, the singular forms also include the plural forms unless the context clearly dictates otherwise. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference. The references cited herein are not admitted to be prior art to the claimed invention. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. In case of conflict between the chemical structure and the name of a compound disclosed herein, the chemical structure shall control.

[0011] Other features and advantages of the present disclosure will become apparent from the following detailed description and the claims. Summary of the Invention

[0012] In one aspect, the present disclosure provides an anti-PD-1 antibody or an antigen-binding fragment thereof, comprising

[0013] (a)(i) a heavy chain variable region (VH) comprising complementarity-determining region (CDR) 1 having the sequence shown in SEQ ID NO: 81, CDR 2 having the sequence shown in SEQ ID NO: 82, and CDR 3 having the sequence shown in SEQ ID NO: 83; and (ii) a light chain variable region (VL) comprising CDR 1 having the sequence shown in SEQ ID NO: 85, CDR 2 having the sequence shown in SEQ ID NO: 86, and CDR 3 having the sequence shown in SEQ ID NO: 87;

[0014] (b)(i) a VH comprising CDR 1 having the sequence shown in SEQ ID NO: 89, CDR 2 having the sequence shown in SEQ ID NO: 90, and CDR 3 having the sequence shown in SEQ ID NO: 91; and (ii) a VL comprising CDR 1 having the sequence shown in SEQ ID NO: 93, CDR 2 having the sequence shown in SEQ ID NO: 94, and CDR 3 having the sequence shown in SEQ ID NO: 95;

[0015] (c)(i) VH, which comprises a CDR1 containing the sequence shown in SEQ ID NO: 97, a CDR2 containing the sequence shown in SEQ ID NO: 98, and a CDR3 containing the sequence shown in SEQ ID NO: 99; and (ii) comprises VL, which comprises a CDR1 containing the sequence shown in SEQ ID NO: 101, a CDR2 containing the sequence shown in SEQ ID NO: 102, and a CDR3 containing the sequence shown in SEQ ID NO: 103;

[0016] (d)(i) VH, which comprises a CDR1 containing the sequence shown in SEQ ID NO: 105, a CDR2 containing the sequence shown in SEQ ID NO: 106, and a CDR3 containing the sequence shown in SEQ ID NO: 107; and (ii) comprises VL, which comprises a CDR1 containing the sequence shown in SEQ ID NO: 109, a CDR2 containing the sequence shown in SEQ ID NO: 110, and a CDR3 containing the sequence shown in SEQ ID NO: 111;

[0017] (e)(i) VH, which comprises a CDR1 containing the sequence shown in SEQ ID NO: 113, a CDR2 containing the sequence shown in SEQ ID NO: 114, and a CDR3 containing the sequence shown in SEQ ID NO: 115; and (ii) comprises VL, which comprises a CDR1 containing the sequence shown in SEQ ID NO: 117, a CDR2 containing the sequence shown in SEQ ID NO: 118, and a CDR3 containing the sequence shown in SEQ ID NO: 119;

[0018] (f)(i) VH, which comprises a CDR1 containing the sequence shown in SEQ ID NO: 121, a CDR2 containing the sequence shown in SEQ ID NO: 122, and a CDR3 containing the sequence shown in SEQ ID NO: 123; and (ii) comprises VL, which comprises a CDR1 containing the sequence shown in SEQ ID NO: 125, a CDR2 containing the sequence shown in SEQ ID NO: 126, and a CDR3 containing the sequence shown in SEQ ID NO: 127; or

[0019] (g)(i) VH, which comprises a CDR1 containing the sequence shown in SEQ ID NO: 129, a CDR2 containing the sequence shown in SEQ ID NO: 130, and a CDR3 containing the sequence shown in SEQ ID NO: 131; and (ii) a VL, which comprises a CDR1 containing the sequence shown in SEQ ID NO: 133, a CDR2 containing the sequence shown in SEQ ID NO: 134, and a CDR3 containing the sequence shown in SEQ ID NO: 135.

[0020] In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises a VH that comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence shown in any one of SEQ ID NOs: 80, 88, 96, 104, 112, 120, 128, 136, 137, 138, 139, 140, 144, 145, 146, 147, 148, 149, 153, 154, 155, and 156. In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises a VL that comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence shown in any one of SEQ ID NOs: 84, 92, 100, 108, 116, 124, 132, 141, 142, 143, 150, 151, 152, 157, and 158. In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises a VH that comprises the sequence shown in any one of SEQ ID NOs: 80, 88, 96, 104, 112, 120, 128, 136, 137, 138, 139, 140, 144, 145, 146, 147, 148, 149, 153, 154, 155, and 156. In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises a VL that comprises the sequence shown in any one of SEQ ID NOs: 84, 92, 100, 108, 116, 124, 132, 141, 142, 143, 150, 151, 152, 157, and 158.

[0021] In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises

[0022] (a) a VH comprising the sequence shown in SEQ ID NO: 136 and a VL comprising the sequence shown in SEQ ID NO: 143;

[0023] (b) A VH comprising the sequence shown in SEQ ID NO: 137 and a VL comprising the sequence shown in SEQ ID NO: 143;

[0024] (c) A VH comprising the sequence shown in SEQ ID NO: 138 and a VL comprising the sequence shown in SEQ ID NO: 143;

[0025] (d) A VH comprising the sequence shown in SEQ ID NO: 139 and a VL comprising the sequence shown in SEQ ID NO: 143;

[0026] (e) A VH comprising the sequence shown in SEQ ID NO: 145 and a VL comprising the sequence shown in SEQ ID NO: 150;

[0027] (f) A VH comprising the sequence shown in SEQ ID NO: 145 and a VL comprising the sequence shown in SEQ ID NO: 151;

[0028] (g) A VH comprising the sequence shown in SEQ ID NO: 145 and a VL comprising the sequence shown in SEQ ID NO: 152;

[0029] (h) A VH comprising the sequence shown in SEQ ID NO: 146 and a VL comprising the sequence shown in SEQ ID NO: 150;

[0030] (g) A VH comprising the sequence shown in SEQ ID NO: 146 and a VL comprising the sequence shown in SEQ ID NO: 151;

[0031] (h) A VH comprising the sequence shown in SEQ ID NO: 146 and a VL comprising the sequence shown in SEQ ID NO: 152;

[0032] (i) A VH comprising the sequence shown in SEQ ID NO: 153 and a VL comprising the sequence shown in SEQ ID NO: 157;

[0033] (j) A VH comprising the sequence shown in SEQ ID NO: 154 and a VL comprising the sequence shown in SEQ ID NO: 158;

[0034] (k) A VH comprising the sequence shown in SEQ ID NO: 155 and a VL comprising the sequence shown in SEQ ID NO: 158; or

[0035] (1)VH comprising the sequence shown in SEQ ID NO: 156 and VL comprising the sequence shown in SEQ ID NO: 158.

[0036] In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody or its antigen-binding fragment is a humanized antibody or its antigen-binding fragment. In some embodiments, the antibody or its antigen-binding fragment is an IgG1 antibody or its antigen-binding fragment. In some embodiments, the antigen-binding fragment is a Fab fragment, a Fab' fragment, an F(ab′)2 fragment, a scFv, a dsFv, a ds-scFv, or a nanobody. In some embodiments, the antibody has an L234A L235A mutation in the Fc region.

[0037] In some embodiments, the antibody or its antigen-binding fragment has a K of less than 0.5 nM, less than 0.3 nM, or less than 0.1 nM, less than 0.05 nM, less than 0.02 nM D that binds to PD-1. In some embodiments, the antibody or antigen-binding fragment has an IC of less than about 500 ng / mL, less than about 400 ng / mL, or less than about 300 ng / mL 50 that inhibits the interaction between PD-1 and PD-L1.

[0038] In some embodiments, the antibody or antigen-binding fragment activates T cells. In some embodiments, the antibody or antigen-binding fragment induces IL-2 secretion in T cells. In some embodiments, the antibody or its antigen-binding fragment induces IFN-γ secretion in T cells.

[0039] In another aspect, provided herein is a pharmaceutical composition comprising the anti-PD-1 antibody or its antigen-binding fragment provided herein and a pharmaceutically acceptable carrier.

[0040] In another aspect, provided herein is a method of treating cancer in a subject, which comprises administering to the subject the anti-PD-1 antibody or its antigen-binding fragment or the pharmaceutical composition provided herein. In some embodiments, the cancer is colon cancer, breast cancer, lung cancer, ovarian cancer, prostate cancer, cervical cancer, brain cancer, skin cancer, liver cancer, pancreatic cancer, or gastric cancer. In some embodiments, the cancer is leukemia or lymphoma.

[0041] In another aspect, provided herein is a method of treating an infectious disease in a subject, which comprises administering to the subject the anti-PD-1 antibody or its antigen-binding fragment or the pharmaceutical composition provided herein. In some embodiments, the infectious disease is tuberculosis, malaria, HIV infection, or HBV infection.

[0042] In another aspect, the present disclosure provides a method for treating primary immunodeficiency in a subject, which comprises administering to the subject the anti-PD-1 antibody or antigen-binding fragment thereof or pharmaceutical composition provided herein.

[0043] In another aspect, the present disclosure provides an anti-PD-1 antibody or antigen-binding fragment thereof for treating cancer. In some embodiments, the cancer is colon cancer, breast cancer, lung cancer, ovarian cancer, prostate cancer, cervical cancer, brain cancer, skin cancer, liver cancer, pancreatic cancer or gastric cancer. In some embodiments, the cancer is leukemia or lymphoma.

[0044] In another aspect, the present disclosure provides an anti-PD-1 antibody or antigen-binding fragment thereof, or a pharmaceutical composition comprising an anti-PD-1 antibody or antigen-binding fragment thereof, for treating infectious diseases. In some embodiments, the infectious disease is tuberculosis, malaria, HIV infection or HBV infection.

[0045] In another aspect, the present disclosure provides an anti-PD-1 antibody or antigen-binding fragment thereof, or a pharmaceutical composition comprising an anti-PD-1 antibody or antigen-binding fragment thereof, for treating primary immunodeficiency.

[0046] In another aspect, the present disclosure provides the use of an anti-PD-1 antibody or antigen-binding fragment thereof in the manufacture of a medicament for treating cancer. In some embodiments, the cancer is colon cancer, breast cancer, lung cancer, ovarian cancer, prostate cancer, cervical cancer, brain cancer, skin cancer, liver cancer, pancreatic cancer or gastric cancer. In some embodiments, the cancer is leukemia or lymphoma.

[0047] In another aspect, the present disclosure provides the use of an anti-PD-1 antibody or antigen-binding fragment thereof in the manufacture of a medicament for treating infectious diseases. In some embodiments, the infectious disease is tuberculosis, malaria, HIV infection or HBV infection.

[0048] In another aspect, the present disclosure provides the use of an anti-PD-1 antibody or antigen-binding fragment thereof in the manufacture of a medicament for treating primary immunodeficiency.

[0049] In another aspect, the present disclosure provides a method of inhibiting the interaction between PD-1 and PD-L1 in a cell, which comprises contacting the cell with an antibody or an antigen-binding fragment thereof provided herein. In another aspect, the present disclosure provides a method of inhibiting the interaction between PD-1 and PD-L2 in a cell, which comprises contacting the cell with an antibody or an antigen-binding fragment thereof provided herein. In another aspect, the present disclosure provides a method of stimulating IL-2 secretion in a cell, which comprises contacting the cell with an antibody or an antigen-binding fragment thereof provided herein. In another aspect, the present disclosure provides a method of stimulating IFN-γ secretion in a cell, which comprises contacting the cell with an antibody or an antigen-binding fragment thereof provided herein. In some embodiments, the cell is a human cell. In some embodiments, the cell is contacted in vitro. In some embodiments, the cell is contacted in vivo. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1A and Figure 1B show the (A) PD-1 binding curve and (B) PD-1 / PD-L1 neutralization curve of a murine anti-PD-1 antibody.

[0051] Figure 2A and Figure 2B show the (A) PD-1 binding curve and (B) PD-1 / PD-L1 neutralization curve of a chimeric anti-PD-1 antibody.

[0052] Figure 3A and Figure 3B show the PD-1 / PD-L1 neutralization curves of humanized anti-PD-1 antibodies of clone CUSB0125 and clone CUSB0136 evaluated by a PD-1 / PD-L1 blocking bioassay.

[0053] Figures 4A to 4D show the secretion of IL-2 and IFN-γ from human T cells evaluated by an MLR assay. Figure 4A and Figure 4B are the results for clone CUSB0125. Figure 4C and Figure 4D are the results for clone CUSB0136.

[0054] Figure 5A and Figure 5B show the in vivo anti-tumor efficacy of a humanized anti-PD-1 antibody at a dose of 10 mg / kg.

[0055] Figure 6A and Figure 6B show the in vivo anti-tumor efficacy of zCUSB0136.8-LALA anti-PD-1 antibody at doses of 5 mg / kg and 10 mg / kg.

[0056] Figure 7 Shows the sequence alignment of some anti-PD-1 antibodies described herein.

[0057] Figure 8A and Figure 8B Shows the sequence identity analysis of VH and VL of some anti-PD-1 antibodies described herein. Each identity consists of several stacks of symbols, with each position in the sequence corresponding to a stack of symbols. The overall height of the stack indicates the sequence conservation at that position, while the height of the symbols within the stack indicates the relative frequency of each amino acid or nucleic acid at that position.

[0058] Figure 9A and Figure 9B Shows the in vivo antitumor efficacy of the affinity matured zCUSB0136.8-2 anti-PD-1 antibody at doses of 2.5 mg / kg and 10 mg / kg, respectively.

[0059] Figure 10 Shows the PD-1 / PD-L2 neutralization curve of the affinity matured zCUSB0136.8-2 anti-PD-1 antibody evaluated by flow cytometry. Detailed Description

[0060] Definitions

[0061] It should be understood that unless otherwise specified, any description of a method of treatment or prevention includes the use of a peptide to provide such treatment or prevention as described herein. Unless otherwise specified, it should be further understood that any description of a method of treatment or prevention includes the use of a peptide to prepare a medicament for treating or preventing such a disorder. Treatment or prevention includes treating or preventing humans or non-human animals, including rodents and other disease models.

[0062] It should be understood that unless otherwise specified, any description of a method of treatment includes the use of a peptide to provide such treatment as described herein. Unless otherwise specified, it should be further understood that any description of a method of treatment includes the use of a peptide to prepare a medicament for treating such a disorder. Treatment includes treating humans or non-human animals, including rodents and other disease models.

[0063] As used herein, the terms "subject" and "subject in need" are interchangeable and both refer to a subject having a disease or an increased risk of developing the disease. "Subject" includes mammals. The mammal can be, for example, a human or a suitable non-human mammal such as a primate, mouse, rat, dog, cat, cow, horse, goat, camel, sheep or pig. The subject can also be a bird or poultry. In some embodiments, the mammal is a human. A subject in need can be a subject previously diagnosed or identified as having a disease or disorder disclosed herein. A subject in need can also be a subject suffering from a disease or disorder disclosed herein. Alternatively, a subject in need can be a subject having an increased risk of developing such a disease or disorder relative to the majority of the population (i.e., a subject more likely to develop such a disorder relative to the majority of the population). A subject in need can be refractory or resistant to a disease or disorder disclosed herein (i.e., a disease or disorder that does not respond or has not responded to treatment as disclosed herein). The subject may be resistant at the start of treatment or may become resistant during treatment. In some embodiments, a subject in need has received all known effective therapies for a disease or disorder disclosed herein and has failed. In some embodiments, a subject in need has received at least one prior therapy.

[0064] As used herein, the term "treating / treat" describes the management and care of a patient for the purpose of combating a disease, disorder or condition and includes administering a peptide of the present disclosure or a pharmaceutically acceptable salt, polymorph or solvate thereof to alleviate the symptoms or complications of the disease, disorder or condition, or to eliminate the disease, disorder or condition. The term "treatment" can also include the treatment of cells in vitro or an animal model. It should be understood that reference to "treating / treatment" includes the alleviation of established symptoms of a disorder. Thus, "treating / treatment" of a condition, disorder or disease includes: (1) preventing or delaying the appearance of clinical symptoms of a condition, disorder or disease in a human who may be predisposed to or at risk of developing the condition, disorder or disease but has not yet experienced or exhibited clinical or subclinical symptoms of the condition, disorder or disease; (2) inhibiting the condition, disorder or disease, i.e., arresting, reducing or delaying the development of the disease or its recurrence (in the case of maintenance therapy) or at least one of its clinical or subclinical symptoms; or (3) alleviating or abating the disease, i.e., causing regression of the condition, disorder or disease or at least one of its clinical or subclinical symptoms.

[0065] It should be understood that the peptide of the present disclosure or a pharmaceutically acceptable salt, polymorph or solvate thereof can or may also be used for preventing a related disease, disorder or condition, or for identifying suitable candidates for such purposes.

[0066] As used herein, the terms "preventing" or "protecting against" describe reducing or eliminating the onset of symptoms or complications of such disease, disorder or condition.

[0067] It should be understood that those skilled in the art may refer to general reference texts for a detailed description of the known techniques or equivalent techniques discussed herein. These texts include Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Inc. (2005); Sambrook et al., Molecular Cloning, A Laboratory Manual (3rd ed.), Cold Spring Harbor Press, Cold Spring Harbor, New York (2000); Coligan et al., Current Protocols in Immunology, John Wiley & Sons, N.Y.; Enna et al., Current Protocols in Pharmacology, John Wiley & Sons, N.Y.; Fingl et al., The Pharmacological Basis of Therapeutics (1975), Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA, 18th ed. (1990). Of course, these texts may also be referred to when forming or using aspects of the present disclosure.

[0068] Antibody

[0069] Antibodies that bind to PD-1 and antigen-binding fragments thereof are provided herein. Also provided herein are pharmaceutical compositions comprising the anti-PD-1 antibodies or antigen-binding fragments thereof provided herein and methods of using such pharmaceutical compositions.

[0070] In one aspect, the present disclosure provides antibodies and antigen-binding fragments thereof that bind to PD-1, also referred to as "anti-PD-1 antibodies". The term "antibody" can refer to an immunoglobulin tetrameric structure that includes two pairs of identical polypeptide chains, each pair including one light chain and one heavy chain, or to other antibody structures such as single-domain antibodies or multispecific antibodies. Thus, the term "antibody" can also refer to single-chain antibodies, bispecific antibodies, trispecific antibodies, and other multispecific antibodies such as diabodies, triabodies, and tetra-bodies. One end of each light chain has a variable domain (VL), and at its other end has a constant domain. Each heavy chain has a variable domain (VH) at the N-terminus, and has three constant domains (CH) for α and γ chains, and four CH domains for μ and ε isotypes.

[0071] The term "antigen-binding fragment" includes, but is not limited to, Fab fragments, Fab' fragments, F(ab′)2 fragments, Fd, Fv, scFv, dsFv, ds-scFv, dimers, minibodies, diabodies, and their multimers, domain antibodies, bispecific antibodies, minibodies, scap (sterol regulatory element-binding protein cleavage-activating protein), chelated recombinant antibodies, triabodies or diabodies, intracellular antibodies, nanobodies, small modular immunopharmaceuticals (SMIPs), binding domain immunoglobulin fusion proteins, camelized antibodies, and VHH-containing antibodies.

[0072] The variable region of an antibody or its antigen-binding fragment includes an antigen-binding site that is responsible for antigen-binding specificity and affinity. In particular, six hypervariable regions, three heavy-chain complementarity-determining regions (CDRs), and three light-chain CDRs form the antigen-binding site. The more highly conserved portions of the variable domain are called framework regions (FRs). Thus, the light-chain and heavy-chain variable domains include, from the N-terminus to the C-terminus, the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0073] In some embodiments, the anti-PD-1 antibody or its antigen-binding fragment includes one or more CDRs (e.g., three VH CDRs and three VL CDRs), each CDR including the CDR sequences shown in Table 1. In some embodiments, the anti-PD-1 antibody includes one or more CDRs (e.g., three VH CDRs and three VL CDRs), each CDR consisting of the CDR sequences shown in Table 1. It will be apparent to those skilled in the art that the variable heavy chains and light chains of different antibodies can be freely combined. Thus, for example, the anti-PD-1 antibody can include the VH of the antibody described as "CUSB0103" in Table 1 and the VL of the antibody described as "CUSB0123" in Table 1.

[0074] Several methods for defining the CDRs of antibodies are known in the art, including, for example, Kabat, Chothia, AbM, Contact, and IMGT. The CDRs in Table 1 are defined using the Kabat definition. However, one of ordinary skill in the art will be able to determine the CDRs of the CH and VL sequences shown in Table 1 using another definition in the art.

[0075] Table 1: Amino Acid Sequences of Anti-PD-1 Antibodies

[0076]

[0077]

[0078]

[0079]

[0080]

[0081]

[0082]

[0083]

[0084]

[0085]

[0086]

[0087]

[0088] In some embodiments, the anti-PD-1 antibody or an antigen-binding fragment thereof comprises a VH that comprises a CDR1 comprising the sequence shown in SEQ ID NO: 81, a CDR2 comprising the sequence shown in SEQ ID NO: 82, and / or a CDR3 comprising the sequence shown in SEQ ID NO: 83.

[0089] In some embodiments, the anti-PD-1 antibody or an antigen-binding fragment thereof comprises a VH that comprises a CDR1 comprising the sequence shown in SEQ ID NO: 89, a CDR2 comprising the sequence shown in SEQ ID NO: 90, and / or a CDR3 comprising the sequence shown in SEQ ID NO: 91.

[0090] In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises a VH that comprises a CDR1 comprising the sequence shown in SEQ ID NO: 97, a CDR2 comprising the sequence shown in SEQ ID NO: 98, and / or a CDR3 comprising the sequence shown in SEQ ID NO: 99.

[0091] In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises a VH that comprises a CDR1 comprising the sequence shown in SEQ ID NO: 105, a CDR2 comprising the sequence shown in SEQ ID NO: 106, and / or a CDR3 comprising the sequence shown in SEQ ID NO: 107.

[0092] In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises a VH that comprises a CDR1 comprising the sequence shown in SEQ ID NO: 113, a CDR2 comprising the sequence shown in SEQ ID NO: 114, and / or a CDR3 comprising the sequence shown in SEQ ID NO: 115.

[0093] In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises a VH that comprises a CDR1 comprising the sequence shown in SEQ ID NO: 121, a CDR2 comprising the sequence shown in SEQ ID NO: 122, and / or a CDR3 comprising the sequence shown in SEQ ID NO: 123.

[0094] In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises a VH that comprises a CDR1 comprising the sequence shown in SEQ ID NO: 129, a CDR2 comprising the sequence shown in SEQ ID NO: 130, and / or a CDR3 comprising the sequence shown in SEQ ID NO: 131.

[0095] In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises a VH that comprises a CDR1 comprising the sequence X1YDII (SEQ ID NO: 228) (wherein X1 is T or V), a CDR2 comprising the sequence VIWTGGDTX2YNSAFMS (SEQ ID NO: 229) (wherein X2 is N, W or T), and / or a CDR3 comprising the sequence DWX3Y (SEQ ID NO: 230) (wherein X3 is A, I or M).

[0096] In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises a VL that comprises a CDR1 having the sequence set forth in SEQ ID NO: 85, a CDR2 having the sequence set forth in SEQ ID NO: 86, and / or a CDR3 having the sequence set forth in SEQ ID NO: 87.

[0097] In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises a VL that comprises a CDR1 having the sequence set forth in SEQ ID NO: 93, a CDR2 having the sequence set forth in SEQ ID NO: 94, and / or a CDR3 having the sequence set forth in SEQ ID NO: 95.

[0098] In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises a VL that comprises a CDR1 having the sequence set forth in SEQ ID NO: 101, a CDR2 having the sequence set forth in SEQ ID NO: 102, and / or a CDR3 having the sequence set forth in SEQ ID NO: 103.

[0099] In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises a VL that comprises a CDR1 having the sequence set forth in SEQ ID NO: 109, a CDR2 having the sequence set forth in SEQ ID NO: 110, and / or a CDR3 having the sequence set forth in SEQ ID NO: 111.

[0100] In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises a VL that comprises a CDR1 having the sequence set forth in SEQ ID NO: 117, a CDR2 having the sequence set forth in SEQ ID NO: 118, and / or a CDR3 having the sequence set forth in SEQ ID NO: 119.

[0101] In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises a VL that comprises a CDR1 having the sequence set forth in SEQ ID NO: 125, a CDR2 having the sequence set forth in SEQ ID NO: 126, and / or a CDR3 having the sequence set forth in SEQ ID NO: 127.

[0102] In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises a VL that comprises a CDR1 having the sequence set forth in SEQ ID NO: 133, a CDR2 having the sequence set forth in SEQ ID NO: 134, and / or a CDR3 having the sequence set forth in SEQ ID NO: 135.

[0103] In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises a VL that comprises a CDR1 containing the sequence RASESVDNX4GNSFIQ (SEQ ID NO: 231) (where X4 is Y or W), a CDR2 containing the sequence FASX5LQS (SEQ ID NO: 232) (where X5 is N, Y, H or Q), and / or a CDR3 containing the sequence HQNNEDPFT (SEQ ID NO: 227).

[0104] In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises (a) a VH that comprises a CDR1 containing the sequence shown in SEQ ID NO: 81, a CDR2 containing the sequence shown in SEQ ID NO: 82, and a CDR3 containing the sequence shown in SEQ ID NO: 83; and (b) a VL that comprises a CDR1 containing the sequence shown in SEQ ID NO: 85, a CDR2 containing the sequence shown in SEQ ID NO: 86, and a CDR3 containing the sequence shown in SEQ ID NO: 87.

[0105] In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises (a) a VH that comprises a CDR1 containing the sequence shown in SEQ ID NO: 89, a CDR2 containing the sequence shown in SEQ ID NO: 90, and a CDR3 containing the sequence shown in SEQ ID NO: 91; and (b) a VL that comprises a CDR1 containing the sequence shown in SEQ ID NO: 93, a CDR2 containing the sequence shown in SEQ ID NO: 94, and a CDR3 containing the sequence shown in SEQ ID NO: 95.

[0106] In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises (a) a VH that comprises a CDR1 containing the sequence shown in SEQ ID NO: 97, a CDR2 containing the sequence shown in SEQ ID NO: 98, and a CDR3 containing the sequence shown in SEQ ID NO: 99; and (b) a VL that comprises a CDR1 containing the sequence shown in SEQ ID NO: 101, a CDR2 containing the sequence shown in SEQ ID NO: 102, and a CDR3 containing the sequence shown in SEQ ID NO: 103.

[0107] In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises (a) a VH that comprises a CDR1 having the sequence shown in SEQ ID NO: 105, a CDR2 having the sequence shown in SEQ ID NO: 106, and a CDR3 having the sequence shown in SEQ ID NO: 107; and (b) a VL that comprises a CDR1 having the sequence shown in SEQ ID NO: 109, a CDR2 having the sequence shown in SEQ ID NO: 110, and a CDR3 having the sequence shown in SEQ ID NO: 111.

[0108] In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises (a) a VH that comprises a CDR1 having the sequence shown in SEQ ID NO: 113, a CDR2 having the sequence shown in SEQ ID NO: 114, and a CDR3 having the sequence shown in SEQ ID NO: 115; and (b) a VL that comprises a CDR1 having the sequence shown in SEQ ID NO: 117, a CDR2 having the sequence shown in SEQ ID NO: 118, and a CDR3 having the sequence shown in SEQ ID NO: 119.

[0109] In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises (a) a VH that comprises a CDR1 having the sequence shown in SEQ ID NO: 121, a CDR2 having the sequence shown in SEQ ID NO: 122, and a CDR3 having the sequence shown in SEQ ID NO: 123; and (b) a VL that comprises a CDR1 having the sequence shown in SEQ ID NO: 125, a CDR2 having the sequence shown in SEQ ID NO: 126, and a CDR3 having the sequence shown in SEQ ID NO: 127.

[0110] In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises (a) a VH that comprises a CDR1 having the sequence shown in SEQ ID NO: 129, a CDR2 having the sequence shown in SEQ ID NO: 130, and a CDR3 having the sequence shown in SEQ ID NO: 131; and (b) a VL that comprises a CDR1 having the sequence shown in SEQ ID NO: 133, a CDR2 having the sequence shown in SEQ ID NO: 134, and a CDR3 having the sequence shown in SEQ ID NO: 135.

[0111] In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises (a) a VH comprising a CDR1 containing the sequence X1YDII (SEQ ID NO: 228) (where X1 is T or V), a CDR2 containing the sequence VIWTGGDTX2YNSAFMS (SEQ ID NO: 229) (where X2 is N, W or T), and a CDR3 containing the sequence DWX3Y (SEQ ID NO: 230) (where X3 is A, I or M); and (b) a VL comprising a CDR1 containing the sequence RASESVDNX4GNSFIQ (SEQ ID NO: 231) (where X4 is Y or W), a CDR2 containing the sequence FASX5LQS (SEQ ID NO: 232) (where X5 is N, Y, H or Q), and a CDR3 containing the sequence HQNNEDPFT (SEQ ID NO: 227).

[0112] In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises a VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and / or VL CDR3 having one or more conservative amino acid substitutions as shown in Table 1.

[0113] As used herein, "conservative amino acid substitution" is a substitution of one amino acid residue for another, which changes the amino acid to a different amino acid having similar biochemical properties (e.g., charge, hydrophobicity, and size). For example, lysine, arginine, and histidine have similar properties because they have basic side chains, and aspartic acid and glutamic acid have similar properties because they have acidic side chains. Additionally, glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan have similar properties because they have uncharged polar side chains, and alanine, valine, leucine, threonine, isoleucine, proline, phenylalanine, and methionine have similar properties because they have nonpolar side chains. Furthermore, tyrosine, phenylalanine, tryptophan, and histidine have similar properties because they have aromatic side chains. Thus, even when substituting an amino acid residue within a group showing similar properties as described above, it will be apparent to those skilled in the art; its properties do not show a specific change.

[0114] In some aspects, the anti-PD-1 antibodies provided herein comprise a VH and / or VL as shown in Table 1. In some embodiments, the anti-PD-1 antibodies provided herein comprise a VH and / or VL that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the VH and / or VL shown in Table 1, respectively.

[0115] To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of the first amino acid or nucleic acid sequence for optimal alignment with the second amino acid or nucleic acid sequence). The amino acid residues or nucleotides at the corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of positions shared by the sequences (i.e., percent identity = number of identical overlapping positions / total number of positions × 100%). In one embodiment, the two sequences are of the same length. The determination of the percent identity between two sequences can also be accomplished using a mathematical algorithm. A non-limiting example of a mathematical algorithm for comparing two sequences is the algorithm of Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. U.S.A. 87:2264-2268, modified according to Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. U.S.A. 90:5873-5877. This algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al., 1990, J. Mol. Biol. 215:403. The BLAST nucleotide search can be performed with the NBLAST nucleotide program parameter set (e.g., for score = 100, wordlength = 12) to obtain nucleotide sequences homologous to the nucleic acid molecules of the present application. The BLAST protein search can be performed with the XBLAST program parameter set (e.g., score - 50, wordlength = 3) to obtain amino acid sequences homologous to the protein molecules of the present invention. To obtain an alignment with gaps for comparison purposes, gapped BLAST can be utilized, as described in: Altschul et al., 1997, Nucleic Acids Res. 25:3389-3402, modified. Alternatively, PSI-BLAST can be used to perform an iterative search for detecting distant relationships between molecules (ibid.). When using the BLAST, gapped BLAST, and PSI-Blast programs, the default parameters of the corresponding programs (e.g., of XBLAST and NBLAST) can be used (see, e.g., the NCBI website). Another non-limiting example of a mathematical algorithm for sequence comparison is the algorithm of Myers and Miller, 1988, CABIOS 4:11-17. This algorithm is incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package. When using the ALIGN program to compare amino acid sequences, the PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used.Techniques similar to those described above can be used to determine the percentage identity between two sequences, with or without allowing gaps. When calculating the percentage identity, typically only exact matches are counted.

[0116] In some embodiments, the anti-PD-1 antibodies provided herein comprise a VH sequence having one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) conservative amino acid substitutions as shown in Table 1. In some embodiments, the anti-PD-1 antibodies provided herein comprise a VL sequence having one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) conservative amino acid substitutions as shown in Table 1.

[0117] In some embodiments, the anti-PD-1 antibody or an antigen-binding fragment thereof comprises a VH that comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence shown in SEQ ID NO: 80. In some embodiments, the anti-PD-1 antibody or an antigen-binding fragment thereof comprises a VH that comprises the sequence shown in SEQ ID NO: 80.

[0118] In some embodiments, the anti-PD-1 antibody or an antigen-binding fragment thereof comprises a VH that comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence shown in SEQ ID NO: 88. In some embodiments, the anti-PD-1 antibody or an antigen-binding fragment thereof comprises a VH that comprises the sequence shown in SEQ ID NO: 88.

[0119] In some embodiments, the anti-PD-1 antibody or an antigen-binding fragment thereof comprises a VH that comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence shown in SEQ ID NO: 96. In some embodiments, the anti-PD-1 antibody or an antigen-binding fragment thereof comprises a VH that comprises the sequence shown in SEQ ID NO: 96.

[0120] In some embodiments, the anti-PD-1 antibody or an antigen-binding fragment thereof comprises a VH that comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence shown in SEQ ID NO: 104. In some embodiments, the anti-PD-1 antibody or an antigen-binding fragment thereof comprises a VH that comprises the sequence shown in SEQ ID NO: 104.

[0121] In some embodiments, the anti-PD-1 antibody or an antigen-binding fragment thereof comprises a VH that comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the sequence shown in SEQ ID NO: 112. In some embodiments, the anti-PD-1 antibody or an antigen-binding fragment thereof comprises a VH that comprises the sequence shown in SEQ ID NO: 112.

[0122] In some embodiments, the anti-PD-1 antibody or an antigen-binding fragment thereof comprises a VH that comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the sequence shown in SEQ ID NO: 120. In some embodiments, the anti-PD-1 antibody or an antigen-binding fragment thereof comprises a VH that comprises the sequence shown in SEQ ID NO: 120.

[0123] In some embodiments, the anti-PD-1 antibody or an antigen-binding fragment thereof comprises a VH that comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the sequence shown in SEQ ID NO: 128. In some embodiments, the anti-PD-1 antibody or an antigen-binding fragment thereof comprises a VH that comprises the sequence shown in SEQ ID NO: 128.

[0124] In some embodiments, the anti-PD-1 antibody or an antigen-binding fragment thereof comprises a VH that comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the sequence shown in SEQ ID NO: 136. In some embodiments, the anti-PD-1 antibody or an antigen-binding fragment thereof comprises a VH that comprises the sequence shown in SEQ ID NO: 136.

[0125] In some embodiments, the anti-PD-1 antibody or an antigen-binding fragment thereof comprises a VH that comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the sequence shown in SEQ ID NO: 137. In some embodiments, the anti-PD-1 antibody or an antigen-binding fragment thereof comprises a VH that comprises the sequence shown in SEQ ID NO: 137.

[0126] In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises a VH that comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the sequence shown in SEQ ID NO: 138. In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises a VH that comprises the sequence shown in SEQ ID NO: 138.

[0127] In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises a VH that comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the sequence shown in SEQ ID NO: 139. In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises a VH that comprises the sequence shown in SEQ ID NO: 139.

[0128] In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises a VH that comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the sequence shown in SEQ ID NO: 140. In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises a VH that comprises the sequence shown in SEQ ID NO: 140.

[0129] In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises a VH that comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the sequence shown in SEQ ID NO: 144. In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises a VH that comprises the sequence shown in SEQ ID NO: 144.

[0130] In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises a VH that comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the sequence shown in SEQ ID NO: 145. In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises a VH that comprises the sequence shown in SEQ ID NO: 145.

[0131] In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises a VH that comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence shown in SEQ ID NO: 146. In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises a VH that comprises the sequence shown in SEQ ID NO: 146.

[0132] In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises a VH that comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence shown in SEQ ID NO: 147. In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises a VH that comprises the sequence shown in SEQ ID NO: 147.

[0133] In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises a VH that comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence shown in SEQ ID NO: 148. In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises a VH that comprises the sequence shown in SEQ ID NO: 148.

[0134] In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises a VH that comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence shown in SEQ ID NO: 149. In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises a VH that comprises the sequence shown in SEQ ID NO: 149.

[0135] In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises a VH that comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence shown in SEQ ID NO: 153. In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises a VH that comprises the sequence shown in SEQ ID NO: 153.

[0136] In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises a VH that comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence shown in SEQ ID NO: 154. In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises a VH that comprises the sequence shown in SEQ ID NO: 154.

[0137] In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises a VH that comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence shown in SEQ ID NO: 155. In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises a VH that comprises the sequence shown in SEQ ID NO: 155.

[0138] In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises a VH that comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence shown in SEQ ID NO: 156. In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises a VH that comprises the sequence shown in SEQ ID NO: 156.

[0139] In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises a VH that comprises the sequence X6VQLX7ESGPGLVKPSQX8LSLTCTVX9GYSITSDYAWNWIRQX 10 PGX 11 X 12 LEWX 13 GYIYYSGTTSYNPSLKSRX 14 X 15 IX 16 X17DTSKNQFX 18 LX 19 LX 20 SVTX 21 X 22 DTAX 23 YYCARNYGSAFYYFDYWGQGTTLTVSS, wherein X6 is D or Q; X7 is R or Q; X8 is S or T; X9 is T or S; X10 is F or P; X 11 is N or K; X 12 is K or G; X 13 is M or I; X 14 is I or V; X15 is S or T; X 16 is T or S; X 17 is R or V; X 18 is F or S; X 19 is Q or K; X 20 is N or S; X 21 is T or A; X 22 is E or A; and / or X 23 is T or V.

[0140] In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises a VH that comprises the sequence QVQLX 39 ESGPGLVX 40 PSX 41 X 42 LSX 43 TCTVSGFSLTX 44 YDIIWIRQX 45 PGKGLEWX 46 GVIWTGGDTX 47 YNSAFMSRX 48 X 49 ISX 50 DX 51 SKX 52 QX 53 X 54 LKX 55 X 56 SX 57 X 58 X 59 X 60 DTAX 61 YYCX 62 RDWX 63 YWGQGTLVTVSX 64 , wherein X 39 is K or Q; X 40 is A or K; X 41 is Q or E; X 42 is S or T; X 43 is I or L; X 44 is T or V; X 45 is S or P; X 46 is L or I; X 47 is N, W or T; X 48 is L or V; X 49 is T or S; X 50 is K or V; X 51 is N or T; X 52 is S or N; X 53 is I or F; X 54 is F or S; X55 is M or L; X 56 is N or S; X 57 is L or V; X 58 is Q or T; X 59 is T or A; X 60 is D or A; X 61 is I or V; X 62 is A or V; X 63 is A, I or M; and / or X 64 is T or S.

[0141] In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises a VL that comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the sequence set forth in SEQ ID NO: 84. In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises a VL that comprises the sequence set forth in SEQ ID NO: 84.

[0142] In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises a VL that comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the sequence set forth in SEQ ID NO: 92. In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises a VL that comprises the sequence set forth in SEQ ID NO: 92.

[0143] In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises a VL that comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the sequence set forth in SEQ ID NO: 100. In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises a VL that comprises the sequence set forth in SEQ ID NO: 100.

[0144] In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises a VL that comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the sequence set forth in SEQ ID NO: 108. In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises a VL that comprises the sequence set forth in SEQ ID NO: 108.

[0145] In some embodiments, the anti-PD-1 antibody or an antigen-binding fragment thereof comprises a VL that comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence shown in SEQ ID NO: 116. In some embodiments, the anti-PD-1 antibody or an antigen-binding fragment thereof comprises a VL that comprises the sequence shown in SEQ ID NO: 116.

[0146] In some embodiments, the anti-PD-1 antibody or an antigen-binding fragment thereof comprises a VL that comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence shown in SEQ ID NO: 124. In some embodiments, the anti-PD-1 antibody or an antigen-binding fragment thereof comprises a VL that comprises the sequence shown in SEQ ID NO: 124.

[0147] In some embodiments, the anti-PD-1 antibody or an antigen-binding fragment thereof comprises a VL that comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence shown in SEQ ID NO: 132. In some embodiments, the anti-PD-1 antibody or an antigen-binding fragment thereof comprises a VL that comprises the sequence shown in SEQ ID NO: 132.

[0148] In some embodiments, the anti-PD-1 antibody or an antigen-binding fragment thereof comprises a VL that comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence shown in SEQ ID NO: 141. In some embodiments, the anti-PD-1 antibody or an antigen-binding fragment thereof comprises a VL that comprises the sequence shown in SEQ ID NO: 141.

[0149] In some embodiments, the anti-PD-1 antibody or an antigen-binding fragment thereof comprises a VL that comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence shown in SEQ ID NO: 142. In some embodiments, the anti-PD-1 antibody or an antigen-binding fragment thereof comprises a VL that comprises the sequence shown in SEQ ID NO: 142.

[0150] In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises a VL that comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the sequence shown in SEQ ID NO: 143. In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises a VL that comprises the sequence shown in SEQ ID NO: 143.

[0151] In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises a VL that comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the sequence shown in SEQ ID NO: 150. In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises a VL that comprises the sequence shown in SEQ ID NO: 150.

[0152] In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises a VL that comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the sequence shown in SEQ ID NO: 151. In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises a VL that comprises the sequence shown in SEQ ID NO: 151.

[0153] In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises a VL that comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the sequence shown in SEQ ID NO: 152. In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises a VL that comprises the sequence shown in SEQ ID NO: 152.

[0154] In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises a VL that comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the sequence shown in SEQ ID NO: 157. In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises a VL that comprises the sequence shown in SEQ ID NO: 157.

[0155] In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises a VL that comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the sequence shown in SEQ ID NO: 158. In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises a VL that comprises the sequence shown in SEQ ID NO: 158.

[0156] In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises a VL that comprises the sequence DIX 24 MTQSPSSX 25 SX 26 SX 27 GDX 28 VTITCHASQGISSNIGWX 29 QQKPGKX 30 X 31 KX 32 LIYR GTNLEDGVPSRFSGSGSGX 33 DX 34 X 35 LTISSLX 36 X 37 EDFAX 38 YYCVQYAQFPPTFGGGTK LEIK, wherein X 24 is L or Q; X 25 is M or V; X 26 is V or A; X 27 is L or V; X 28 is T or R; X 29 is L or Y; X 30 is S or A; X 31 is F or P; X 32 is G or L; X 33 is A or T; X 34 is Y or F; X 35 is S or T; X 36 is E or Q; X 37 is S or P; and / or X 38 is D or T.

[0157] In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises a VL that comprises the sequence X 65 IVX 66 TQSPX 67 SLAVSLGX 68 RATIX 69 CRASESVDNX 70GNSFIQWYQQKPGQPPKLLIYF ASX 71 LQSGVPX 72 RFSGSGSX73TDFTLTIX 74 X 75 X 76 X 77 AX 78 DX 79 AX 80 YYCHQNNEDPFTF GX 81 GTKLEIK, wherein X 65 is N or D; X 66 is L or M; X 67 is A or D; X 68 is Q or E; X 69 is S or N; X 70 is Y or W; X 71 is N or Y; X 72 is A or D; X 73 is R or G; X 74 is D or S; X 75 is P or S; X 76 is V or L; X 77 is E or Q; X 78 is D or E; X 79 is A or V; X 80 is T or V; and / or X 81 is S or G.

[0158] It will be apparent to those skilled in the art that the variable heavy and light chains of different antibodies can be freely combined. Thus, for example, an anti-PD-1 antibody can include the VH of the antibody described as "CUSB0103" in Table 1 and the VL of the antibody described as "CUSB0123" in Table 1.

[0159] In some embodiments, the anti-PD-1 antibody or an antigen-binding fragment thereof comprises a VH containing the sequence shown in SEQ ID NO: 80 and a VL containing the sequence shown in SEQ ID NO: 84.

[0160] In some embodiments, the anti-PD-1 antibody or an antigen-binding fragment thereof comprises a VH containing the sequence shown in SEQ ID NO: 88 and a VL containing the sequence shown in SEQ ID NO: 92.

[0161] In some embodiments, the anti-PD-1 antibody or an antigen-binding fragment thereof comprises a VH containing the sequence shown in SEQ ID NO: 96 and a VL containing the sequence shown in SEQ ID NO: 100.

[0162] In some embodiments, the anti-PD-1 antibody or an antigen-binding fragment thereof comprises a VH containing the sequence shown in SEQ ID NO: 104 and a VL containing the sequence shown in SEQ ID NO: 108.

[0163] In some embodiments, the anti-PD-1 antibody or an antigen-binding fragment thereof comprises a VH containing the sequence shown in SEQ ID NO: 112 and a VL containing the sequence shown in SEQ ID NO: 116.

[0164] In some embodiments, the anti-PD-1 antibody or an antigen-binding fragment thereof comprises a VH containing the sequence shown in SEQ ID NO: 120 and a VL containing the sequence shown in SEQ ID NO: 124.

[0165] In some embodiments, the anti-PD-1 antibody or an antigen-binding fragment thereof comprises a VH containing the sequence shown in SEQ ID NO: 128 and a VL containing the sequence shown in SEQ ID NO: 132.

[0166] In some embodiments, the anti-PD-1 antibody or an antigen-binding fragment thereof comprises a VH containing the sequence shown in SEQ ID NO: 136 and a VL containing the sequence shown in SEQ ID NO: 143.

[0167] In some embodiments, the anti-PD-1 antibody or an antigen-binding fragment thereof comprises a VH containing the sequence shown in SEQ ID NO: 137 and a VL containing the sequence shown in SEQ ID NO: 143.

[0168] In some embodiments, the anti-PD-1 antibody or an antigen-binding fragment thereof comprises a VH containing the sequence shown in SEQ ID NO: 138 and a VL containing the sequence shown in SEQ ID NO: 143.

[0169] In some embodiments, the anti-PD-1 antibody or an antigen-binding fragment thereof comprises a VH containing the sequence shown in SEQ ID NO: 139 and a VL containing the sequence shown in SEQ ID NO: 143.

[0170] In some embodiments, the anti-PD-1 antibody or an antigen-binding fragment thereof comprises a VH containing the sequence shown in SEQ ID NO: 145 and a VL containing the sequence shown in SEQ ID NO: 150.

[0171] In some embodiments, the anti-PD-1 antibody or an antigen-binding fragment thereof comprises a VH containing the sequence shown in SEQ ID NO: 145 and a VL containing the sequence shown in SEQ ID NO: 151.

[0172] In some embodiments, the anti-PD-1 antibody or an antigen-binding fragment thereof comprises a VH containing the sequence shown in SEQ ID NO: 145 and a VL containing the sequence shown in SEQ ID NO: 152.

[0173] In some embodiments, the anti-PD-1 antibody or an antigen-binding fragment thereof comprises a VH containing the sequence shown in SEQ ID NO: 146 and a VL containing the sequence shown in SEQ ID NO: 150.

[0174] In some embodiments, the anti-PD-1 antibody or an antigen-binding fragment thereof comprises a VH containing the sequence shown in SEQ ID NO: 146 and a VL containing the sequence shown in SEQ ID NO: 151.

[0175] In some embodiments, the anti-PD-1 antibody or an antigen-binding fragment thereof comprises a VH containing the sequence shown in SEQ ID NO: 146 and a VL containing the sequence shown in SEQ ID NO: 152.

[0176] In some embodiments, the anti-PD-1 antibody or an antigen-binding fragment thereof comprises a VH containing the sequence shown in SEQ ID NO: 153 and a VL containing the sequence shown in SEQ ID NO: 157.

[0177] In some embodiments, the anti-PD-1 antibody or an antigen-binding fragment thereof comprises a VH containing the sequence shown in SEQ ID NO: 154 and a VL containing the sequence shown in SEQ ID NO: 158.

[0178] In some embodiments, the anti-PD-1 antibody or an antigen-binding fragment thereof comprises a VH containing the sequence shown in SEQ ID NO: 155 and a VL containing the sequence shown in SEQ ID NO: 158.

[0179] In some embodiments, the anti-PD-1 antibody or an antigen-binding fragment thereof comprises a VH containing the sequence shown in SEQ ID NO: 156 and a VL containing the sequence shown in SEQ ID NO: 158.

[0180] In some embodiments, the anti-PD-1 antibody or an antigen-binding fragment thereof comprises (a) a VH that comprises the sequence X6VQLX7ESGPGLVKPSQX8LSLTCTVX9GYSITSDYAWNWIRQX 10 PGX 11 X 12 LEWX 13 GYI YYSGTTSYNPSLKSRX 14 X15 IX16X 17 DTSKNQFX 18 LX 19 LX 20 SVTX 21 X 22 DTAX 23 YYCARN YGSAFYYFDYWGQGTTLTVSS, where X6 is D or Q; X7 is R or Q; X8 is S or T; X9 is T or S; X 10 is F or P; X 11 is N or K; X 12 is K or G; X 13 is M or I; X 14 is I or V; X 15 is S or T; X 16 is T or S; X 17 is R or V; X 18 is F or S; X 19 is Q or K; X 20 is N or S; X 21 is T or A; X 22 is E or A; and X 23 is T or V; and (b) VL, which contains the sequence DIX 24 MTQSPSSX 25 SX 26 SX 27 GDX 28 VTITCHASQGISSNIGWX 29 QQKPGKX30X 31 KX 32 LIYR GTNLEDGVPSRFSGSGSGX 33 DX 34 X 35 LTISSLX 36 X 37 EDFAX 38 YYCVQYAQFPPTFGGGTKLEIK, where X 24 is L or Q; X 25 is M or V; X 26 is V or A; X 27 is L or V; X 28 is T or R; X 29 is L or Y; X 30 is S or A; X 31 is F or P; X 32 is G or L; X 33 is A or T; X 34 is Y or F; X 35 is S or T; X36 is E or 0; X 37 is S or P; and X 38 is D or T.

[0181] In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises (a) a VH that comprises the sequence QVQLX 39 ESGPGLVX 40 PSX 41 X 42 LSX 43 TCTVSGFSLTX 44 YDIIWIRQX 45 PGKGLEWX 46 GVI WTGGDTX 47 YNSAFMSRX 48 X 49 ISX 50 DX 51 SKX 52 QX 53 X 54 LKX 55 X 56 SX 57 X 58 X 59 X 60 DTAX 61 YYCX 62 RDWX 63 YWGQGTLVTVSX 64 , wherein X 39 is K or Q; X 40 is A or K; X 41 is Q or E; X 42 is S or T; X 43 is I or L; X 44 is T or V; X 45 is S or P; X 46 is L or I; X 47 is N, W or T; X 48 is L or V; X 49 is T or S; X 50 is K or V; X 51 is N or T; X 52 is S or N; X 53 is I or F; X 54 is F or S; X 55 is M or L; X 56 is N or S; X 57 is L or V; X 58 is Q or T; X 59 is T or A; X 60 is D or A; X61 is I or V; X 62 is A or V; X 63 is A, I or M; and X 64 is T or S; and (b) VL, which contains the sequence

[0182] X 65 IVX 66 TQSPX 67 SLAVSLGX 68 RATIX 69 CRASESVDNX 70 GNSFIQWYQQKPGQPPKLLIYFASX 71 LQSGVPX 72 RFSGSGSX 73 TDFTLTIX 74 X 75 X 76 X 77 AX 78 DX 79 AX 80 YYCHQNNEDPFTFGX 81 GTKLEIK, where X 65 is N or D; X 66 is L or M; X 67 is A or D; X 68 is Q or E; X 69 is S or N; X 70 is Y or W; X 71 is N or Y; X 72 is A or D; X 73 is R or G; X 74 is D or S; X 75 is P or S; X 76 is V or L; X 77 is E or Q; X 78 is D or E; X 79 is A or V; X 80 is T or V; and X 81 is S or G.

[0183] The anti-PD-1 antibody or its antigen-binding fragment can belong to any class of immunoglobulins. In some embodiments, the anti-PD-1 antibody or its antigen-binding fragment is an IgG antibody or its antigen-binding fragment. In some embodiments, the anti-PD-1 antibody or its antigen-binding fragment is an IgM antibody or its antigen-binding fragment. In some embodiments, the anti-PD-1 antibody or its antigen-binding fragment is an IgA antibody or its antigen-binding fragment. In some embodiments, the anti-PD-1 antibody or its antigen-binding fragment is an IgE antibody or its antigen-binding fragment. In some embodiments, the anti-PD-1 antibody or its antigen-binding fragment is an IgD antibody or its antigen-binding fragment.

[0184] The anti-PD-1 antibody or its antigen-binding fragment can belong to any subclass of immunoglobulins. Thus, in some embodiments, the anti-PD-1 antibody or its antigen-binding fragment is an IgG1 antibody or its antigen-binding fragment, an IgG2 antibody or its antigen-binding fragment, an IgG3 antibody or its antigen-binding fragment, or an IgG4 antibody or its antigen-binding fragment. In some embodiments, the light chain of the anti-PD-1 antibody or its antigen-binding fragment is a κ chain. In some embodiments, the light chain of the anti-PD-1 antibody or its antigen-binding fragment is a λ chain.

[0185] In some embodiments, the anti-PD-1 antibody or its antigen-binding fragment is a monoclonal antibody or its antigen-binding fragment. As used herein, the term "monoclonal antibody" (MAb or mAb) refers to a population of antibody molecules that contain only one molecular species of an antibody molecule composed of a unique light chain gene product and a unique heavy chain gene product. In particular, the complementarity-determining regions (CDRs) of the monoclonal antibody are identical in all molecules of the population. Monoclonal antibodies contain antigen-binding sites that are capable of immunoreacting with a specific antigenic epitope and are characterized by a unique binding affinity for the specific antigenic epitope.

[0186] In some embodiments, the anti-PD-1 antibody or its antigen-binding fragment is a humanized antibody or its antigen-binding fragment. A humanized antibody can be produced by replacing the residues in the framework region of a non-human (e.g., murine) antibody with amino acid residues of a human framework region.

[0187] In some embodiments, the anti-PD-1 antibody or its antigen-binding fragment is a human antibody or its antigen-binding fragment. Human antibodies can be prepared by administering an antigen to a transgenic animal that has been engineered to produce such antibodies in response to antigen challenge, but whose endogenous loci have been disabled. In some embodiments, the anti-PD-1 antibody or its antigen-binding fragment is a murine antibody or its antigen-binding fragment.

[0188] In some embodiments, the anti-PD-1 antibody or its antigen-binding fragment is a chimeric antibody or its antigen-binding fragment.

[0189] In some embodiments, the anti-PD-1 antibodies provided herein bind to PD-1 with high affinity. There are non-covalent interactions between an immunoglobulin molecule and an antigen specific for the immunoglobulin. The strength or affinity of an immunobinding interaction can be represented by the dissociation constant (K D ) of the interaction, where a smaller K D represents a greater affinity. Methods well known in the art can be used to quantify the immunobinding properties of selected polypeptides. One such method requires measuring the rates of formation and dissociation of the antigen-binding site / antigen complex, where these rates depend on the concentrations of the complex partners, the affinity of the interaction, and geometric parameters that also affect the rates in both directions. Thus, the "association rate constant" (k on ) and the "dissociation rate constant" (k off ) can be determined by calculating the concentrations and the actual association and dissociation rates. (See Nature 361: 186-87 (1993)). The ratio of k off / k on cancels all parameters independent of affinity and is equal to the dissociation constant K D (see generally Davies et al., (1990) Annual Rev Biochem 59: 439-473). The antibodies disclosed herein are said to specifically bind to PD-1 when the equilibrium binding constant (K D ) ≤ 1 micromolar, ≤ 100 nM, ≤ 10 nM, or ≤ 100 pM to about 1 pM, as measured by, for example, a radioligand binding assay or a similar assay known to those of skill in the art or described herein (e.g., in the examples).

[0190] In some embodiments, the anti-PD-1 antibodies provided herein bind to PD-1 with a K D of less than 10 nM, less than 5 nM, less than 1 nM, less than 0.5 nM, less than 0.4 nM, less than 0.3 nM, less than 0.2 nM, less than 0.1 nM, less than 0.05 nM, less than 0.02 nM, or less than 0.01 nM. In some embodiments, the anti-PD-1 antibodies provided herein bind to PD-1 with a K D of 1-10 nM, 1-5 nM, 1-2 nM, 0.5-1 nM, 0.1-0.5 nM, 0.05-0.1 nM, 0.01-0.05 nM, or 0.005-0.01 nM. In some embodiments, the anti-PD-1 antibodies provided herein or antigen-binding fragments thereof inhibit the interaction between PD-1 and PD-L1. The half-maximal inhibitory concentration (IC 50) It can be determined using suitable methods known in the art or described herein (including commercially available kits). In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof provided herein has an IC of less than about 500 ng / mL, less than about 400 ng / mL, or less than about 300 ng / mL 50 Inhibit the interaction between PD-1 and PD-L1.

[0191] In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof induces the activation of T cells. The activation of T cells can be determined using any suitable method described herein or known in the art (e.g., mixed lymphocyte reaction (MLR) assay). In such assays, the antibody is added to antigen-presenting cells and effector cells, and ELISA or commercially available kits can be used to quantify the levels of cytokines such as interleukin 2 (IL-2) and / or interferons such as interferon γ (IFN-γ). In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof described herein increases the production of IL-2 by a T cell population by about 10%-25%, about 25%-50%, about 50%-75%, about 75%-100%, about 2-3 fold, about 3-4 fold, about 4-5 fold, about 5-10 fold, about 10-15 fold, or about 15-20 fold. In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof described herein increases the production of IFN-γ by a T cell population by about 10%-25%, about 25%-50%, about 50%-75%, about 75%-100%, about 2-3 fold, about 3-4 fold, about 4-5 fold, about 5-10 fold, about 10-15 fold, or about 15-20 fold.

[0192] It may be desirable to modify the antibodies disclosed herein according to effector functions to enhance, for example, the effectiveness of the antibodies in treating relevant diseases and disorders. For example, cysteine residues can be introduced into the Fc region, allowing the formation of interchain disulfide bonds in this region. The resulting homodimeric antibodies may have improved internalization ability and / or increased complement-mediated cell killing and antibody-dependent cell cytotoxicity (ADCC). Alternatively, antibodies with a dual Fc region can be engineered to have enhanced complement lysis and ADCC capabilities. Mutations that affect the effector functions of antibodies are known in the art, see, for example, Saunders et al., 20l9; Front. Immunol. 10:1296 and Wang et al., 2018, Protein Cell 9(1):63-73, each of which is incorporated herein by reference in its entirety for examples of Fc modifications that can be introduced into the antibodies described herein. Unless otherwise specified, the Fc mutations in this section are described with reference to the Kabat numbering scheme of immunoglobulins.

[0193] Compared to antibodies lacking such substitutions, such antibodies may exhibit increased or decreased binding to FcRn and thus have increased or decreased half-lives in serum, respectively. An increased affinity of an Fc variant for FcRn is expected to result in a longer serum half-life, and such molecules have useful applications in methods of treating mammals in which a longer half-life of an administered antibody is desired, e.g., to treat chronic diseases or disorders. In contrast, a decreased affinity of an Fc variant for FcRn is expected to result in a shorter half-life, and such molecules are also useful, e.g., when a shorter circulation time may be advantageous upon administration to a mammal, such as for in vivo diagnostic imaging or in cases where the starting antibody has toxic side effects when present in the circulation for an extended period of time. An Fc variant with decreased FcRn binding affinity is also less likely to cross the placenta and is thus also useful in treating diseases or disorders in pregnant mammals. Additionally, other applications in which a reduced FcRn binding affinity may be desired include those that require targeting to the brain, kidney, and / or liver. In one exemplary embodiment, the altered antibodies of the invention exhibit reduced transport across glomerular epithelium from the vasculature. In another embodiment, the altered antibodies of the invention exhibit reduced transport from the brain across the blood-brain barrier (BBB) into the vascular space. In one embodiment, an antibody with altered FcRn binding comprises an Fc domain that has one or more amino acid substitutions within the "FcRn binding loop" of the Fc domain. The FcRn binding loop comprises amino acid residues 280-299 (according to EU numbering).

[0194] Exemplary mutations and combinations of mutations that may be introduced into the Fc of the anti-FcRn antibodies provided herein include, but are not limited to, Lys326Trp / Glu333Ser, Ser267Glu / His268Phe / Ser324Thr, Lys326Trp / Glu333Ser, Lys326Ala / Glu333Ala, Lys326Met / Glu333Ser, Cys221Asp / Asp222Cys, Ser267Glu, His268Phe, Ser324Thr, Glu345Arg, S239D / I332E, S239D / I332E / A330L, Arg435His, Met252Tyr / Ser254Thr / Thr256Glu (“YTE”), Met428Leu / Asn434Ser, Thr252Leu / Thr253Ser / Thr254Phe, Leu235Glu, Leu234Ala / Leu235Ala (“LALA”), Ser228Pro / Leu235Glu, Leu234Ala / Leu235Ala / Pro329Gly, Pro331Ser / Leu234Glu / Leu235Phe, Asp265Ala, and Ala330Leu.

[0195] The present disclosure includes all antibodies and antibody fragments that bind to the same antigen or epitope as the antibodies or antibody fragments disclosed herein. Those skilled in the art will appreciate that binding assays, such as competitive binding assays, can be used to find other antibodies and antibody fragments that have the same binding specificity as the antibodies and antibody fragments disclosed herein.

[0196] Those skilled in the art will recognize that it is possible to determine whether an antibody (e.g., a monoclonal antibody) has the same specificity as an antibody or antibody fragment disclosed herein without undue experimentation by determining whether the former prevents the latter from binding to PD-1. If the tested monoclonal antibody competes with a monoclonal antibody disclosed herein, as shown by a reduction in the binding of the disclosed monoclonal antibody, then the two monoclonal antibodies bind to the same or closely related epitopes.

[0197] Methods for screening antibodies with desired specificities include, but are not limited to, enzyme-linked immunosorbent assay (ELISA) and other immunologically mediated techniques known in the art.

[0198] An illustrative method for determining whether a monoclonal antibody has the specificity of the antibodies or antibody fragments disclosed herein is to pre-incubate the monoclonal antibodies disclosed herein with soluble PD-1, and then add the monoclonal antibody being tested to determine whether the ability of the monoclonal antibody being tested to bind to PD-1 is inhibited. If the monoclonal antibody being tested is inhibited, then it is very likely that it has the same or functionally equivalent epitope specificity as the monoclonal antibodies disclosed herein.

[0199] The present disclosure also includes an immunoconjugate comprising (1) a binding protein disclosed herein, preferably an antibody or an antibody fragment, which has been attached to (2) an effector molecule.

[0200] Heteroconjugate antibodies are also within the scope of the invention. Heteroconjugate antibodies consist of two covalently linked antibodies.

[0201] In another aspect, the present disclosure provides a conjugate comprising an anti-PD-1 antibody or an antigen-binding fragment thereof provided herein and an effector molecule. In one embodiment, the effector molecule is a label that can directly or indirectly generate a detectable signal. Examples of labels include radioisotopes (i.e., radiolabeled conjugates). In another embodiment, the effector molecule is a therapeutic agent. Therapeutic agents include, but are not limited to, anti-tumor agents. In yet another embodiment, the therapeutic agent is a toxin.

[0202] As used herein, the term "cancer therapeutic agent" or "anti-tumor agent" refers to an agent having functional properties that inhibit the development or progression of a human tumor, particularly a malignant (cancerous) lesion such as carcinoma, sarcoma, lymphoma, or leukemia. Inhibiting metastasis is generally a property of anti-tumor agents.

[0203] Toxins are enzymatically active toxins of bacterial, fungal, plant, or animal origin or fragments thereof. Toxins and fragments thereof that can be used include diphtheria A chain, non-binding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii protein, dianthin protein, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), Momordica charantia inhibitor, curcin, crotin, Saponaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and tricothecenes.

[0204] Polynucleotides and vectors

[0205] In another aspect, the present disclosure relates to polynucleotide sequences encoding the anti-PD-1 antibodies and antigen-binding fragments thereof described herein. Exemplary polynucleotide sequences encoding the CDRs, as well as the heavy and light chains, of the anti-PD-1 antibodies described herein are listed in Table 2.

[0206] In some embodiments, provided herein is a polynucleotide encoding a VH that comprises a sequence shown in any of SEQ ID No: 80, 88, 96, 104, 112, 120, 128, 136, 137, 138, 139, 140, 144, 145, 146, 147, 148, 149, 153, 154, 155, and 156.

[0207] In some embodiments, the polynucleotide encoding the VH of the anti-PD-1 antibody or antigen-binding fragment thereof provided herein comprises a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the sequence shown in any one of SEQ ID NOs: 1, 9, 17, 25, 33, 41, 49, 57, 58, 59, 60, 61, 65, 66, 67, 68, 69, 70, 74, 75, 76 and 77. In some embodiments, the polynucleotide encoding the VH of the anti-PD-1 antibody or antigen-binding fragment thereof provided herein comprises the nucleic acid sequence shown in any one of SEQ ID NOs: 1, 9, 17, 25, 33, 41, 49, 57, 58, 59, 60, 61, 65, 66, 67, 68, 69, 70, 74, 75, 76 and 77.

[0208] In some embodiments, provided herein is a polynucleotide encoding a VL, which VL comprises the sequence shown in any one of SEQ ID NOs: 84, 92, 100, 108, 116, 124, 132, 141, 142, 143, 150, 151, 152, 157 and 158.

[0209] In some embodiments, the polynucleotide encoding the VL of the anti-PD-1 antibody or antigen-binding fragment thereof provided herein comprises a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the sequence shown in any one of SEQ ID NOs: 5, 13, 21, 29, 37, 45, 53, 62, 63, 64, 71, 72, 73, 78 and 79. In some embodiments, the polynucleotide encoding the VL of the anti-PD-1 antibody or antigen-binding fragment thereof provided herein comprises the nucleic acid sequence shown in any one of SEQ ID NOs: 5, 13, 21, 29, 37, 45, 53, 62, 63, 64, 71, 72, 73, 78 and 79.

[0210] Table 2: Nucleotide Sequences of Anti-PD-1 Antibodies

[0211]

[0212]

[0213]

[0214]

[0215]

[0216]

[0217]

[0218]

[0219]

[0220]

[0221]

[0222] The DNA encoding the antibodies disclosed herein can also be modified, for example, by replacing homologous murine sequences with the coding sequences of human heavy and light chain constant domains, or by covalently linking all or part of the coding sequence of a non-immunoglobulin polypeptide to the immunoglobulin coding sequence. Such non-immunoglobulin polypeptides can replace the constant domains of the antibodies disclosed herein, or can replace the variable domains of one antigen-binding site of the antibodies disclosed herein to produce chimeric bivalent antibodies.

[0223] In another aspect, the present disclosure provides a vector comprising a polynucleotide encoding VH and / or VL of an anti-PD-1 antibody or an antigen-binding fragment thereof. In some embodiments, the vector is a viral vector. In some embodiments, the vector is a retroviral vector, such as Moloney murine leukemia virus. In some embodiments, the vector is a poxvirus vector, a herpes simplex virus type I (HSV) vector, an adenovirus vector, or an adeno-associated virus vector.

[0224] In one embodiment, the present disclosure includes variants of the nucleic acid sequences encoding the anti-PD-1 antibodies or antigen-binding fragments thereof disclosed herein. For example, such variants include nucleotide sequences that hybridize to the nucleic acid sequences encoding the anti-PD-1 antibodies or antigen-binding fragments thereof disclosed herein under at least moderately stringent hybridization conditions.

[0225] "At least moderately stringent hybridization conditions" means conditions selected to promote selective hybridization between two complementary nucleic acid molecules in solution. Hybridization may occur over all or a portion of the nucleic acid sequence molecule. The length of the hybridizing portion is typically at least 15 (e.g., 20, 25, 30, 40, or 50) nucleotides. Those skilled in the art will recognize that the stability of a nucleic acid duplex or hybrid is determined by Tm, which in a sodium-containing buffer is a function of sodium ion concentration and temperature (Tm = 81.5°C - 16.6(Log10[Na+]) + 0.41(%(G+C) - 600 / l), or a similar equation). Thus, the parameters that determine hybrid stability under wash conditions are sodium ion concentration and temperature. To identify molecules that are similar but not identical to a known nucleic acid molecule, it is assumed that a 1% mismatch results in a decrease in Tm of approximately 1°C. For example, if one is seeking a nucleic acid molecule with >95% identity, the final wash temperature will be decreased by approximately 5°C. Based on these considerations, those skilled in the art will be able to readily select appropriate hybridization conditions.

[0226] Preparation method

[0227] Using known techniques, antibodies and expression libraries thereof against an anti-PD-1 antibody or antigen-binding fragment thereof can be generated, and thereafter screened for the activities described herein.

[0228] The antibodies and fragments disclosed herein can be expressed by a vector containing a DNA segment encoding VH and / or VL of an anti-PD-1 antibody or antigen-binding fragment thereof (such as those described above).

[0229] Antibodies can be purified by well-known techniques, such as affinity chromatography using Protein A or Protein G, which primarily provides the IgG portion of immune serum. Subsequently, or alternatively, a specific antigen or epitope that is the target of the sought-after immunoglobulin can be immobilized on a column to purify the immunospecific antibody by immunoaffinity chromatography. For example, D. Wilkinson (The Scientist, published by The Scientist, Inc., Philadelphia PA, Vol. 14, No. 8 (April 17, 2000), pp. 25 - 28) discusses the purification of immunoglobulins.

[0230] A variety of procedures known in the art can be used to generate monoclonal antibodies against PD-1 or against its derivatives, fragments, analogs, homologs, or orthologs. (See, e.g., Antibodies: A Laboratory Manual, Harlow E and Lane D, 1988, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, which is incorporated by reference).

[0231] Monoclonal antibodies can be produced, for example, by immunizing an animal with PD-1, such as murine, rat or human PD-1 or an immunogenic fragment, derivative or variant thereof, for example, by regular subcutaneous injection. Alternatively, the animal can be immunized with cells transfected with a vector containing a nucleic acid molecule encoding PD-1 that is expressed on the surface of the transfected cells and associated with the surface of the transfected cells. For example, mice can be immunized by regular intraperitoneal injection with CHO cells overexpressing PD-1. The serum IgG titers of the immunized animals can be monitored, and the animals with the highest titers can be used to generate hybridomas. To generate hybridomas, the spleens of the immunized animals are removed and the splenocytes are fused with myeloma cells, for example, by electrofusion.

[0232] Alternatively, antibodies can be obtained by screening a library containing sequences of antibodies or antigen-binding domains that bind to PD-1. The library is prepared, for example, as a protein or peptide fused to a phage coat protein in a phage, and the phage coat protein is expressed on the surface of the assembled phage particles and the coding DNA sequences contained within the phage particles (i.e., "phage display library"). Then, the hybridomas generated from the myeloma / B cell fusions are screened for reactivity to PD-1.

[0233] For example, monoclonal antibodies can be prepared using the hybridoma method, such as those described by Kohler and Milstein, Nature, 256:495 (1975). In the hybridoma method, a mouse, hamster or other suitable host animal is typically immunized with an immunizing agent to stimulate the production of lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, lymphocytes can be immunized in vitro.

[0234] Monoclonal antibodies can also be prepared by recombinant DNA methods. The DNA encoding a monoclonal antibody and its antigen-binding fragments can be readily isolated and sequenced using conventional methods (e.g., by using oligonucleotide probes that specifically bind to the genes encoding the heavy and light chains of a murine antibody). Hybridoma cells serve as a preferred source of such DNA. Once isolated, the DNA can be placed in an expression vector and then transfected into host cells, such as simian COS cells, Chinese hamster ovary (CHO) cells or myeloma cells, which do not otherwise produce immunoglobulins, so as to synthesize monoclonal antibodies in the recombinant host cells.

[0235] Any suitable method known in the art can be used to purify the antibodies. For example, the culture medium of the hybridoma cultures can be centrifuged and the supernatant can be filtered through a 0.45 μm PES membrane filter. Then, the antibodies can be purified using, for example, a HiTrap Protein A column. Then, the bound antibodies can be eluted and concentrated and buffer-exchanged into PBS.

[0236] The screening of monoclonal antibodies and their antigen-binding fragments can also be carried out, for example, by measuring the binding between PD-L1 and PD-1 and determining whether the test monoclonal antibody can inhibit the binding between PD-L1 and PD-1. Any suitable method known in the art can be used to screen for the binding of an antibody to PD-1. To measure PD-1 binding, the antibody to be screened can be incubated with His-tagged PD-1 and then probed with an anti-IgG Fcγ-HRP antibody. Then, a substrate solution can be used to detect HRP and determine the absorbance value, with a higher absorbance value indicating stronger PD-1 binding.

[0237] The ability of an antibody to neutralize the PD-1 / PD-L1 interaction can also be screened. To measure PD-1 / PD-L1 neutralization, the antibody to be screened can be incubated with Jurkat effector cells expressing PD-1 and recombinant PD-L1 human Fc-tagged protein. A fluorescent anti-human IgG antibody can be used to determine the binding of the PD-L1 protein to PD-1 on the cell surface, for example, using a flow cytometer. In the presence of a neutralizing antibody, the PD-L1 human Fc-tagged protein cannot bind to Jurkat cells expressing PD-1, resulting in the absence of a fluorescent signal.

[0238] Antibodies can be fragmented using conventional techniques. For example, F(ab')2 fragments can be generated by treating the antibody with pepsin. The resulting F(ab')2 fragments can be treated to reduce disulfide bonds to produce Fab' fragments. Papain digestion results in the formation of Fab fragments. Fab, Fab', and F(ab')2, scFv, dsFv, ds-scFv, dimers, minibodies, diabodies, bispecific antibody fragments, and other fragments can also be synthesized by recombinant techniques.

[0239] These techniques can be adapted to produce single-chain antibodies specific for the antigenic proteins of the present disclosure (see, for example, U.S. Patent No. 4,946,778). Additionally, these methods can be adapted to construct Fab expression libraries (see, for example, Huse et al., 1989 Science 246: 1275-1281) to allow for the rapid and efficient identification of monoclonal Fab fragments with the desired specificity for a protein or its derivatives, fragments, analogs, or homologs.

[0240] Described are various techniques for the preparation and isolation of bispecific antibody fragments directly from recombinant cell cultures. For example, bispecific antibodies have been produced using leucine zippers. Kostelny et al., J. Immunol 148(5): 1547-1553 (1992). The "diabody" described by Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993) provides another mechanism for the preparation of bispecific antibody fragments. The fragment contains a VH linked to a VL by a linker that is too short to pair between the two domains on the same chain. Thus, the VH and VL domains of one fragment are forced to pair with the complementary VL and VH domains of another fragment, thereby forming two antigen-binding sites. Another strategy for the preparation of bispecific antibody fragments by using single-chain Fv (scFv) dimers has also been reported. See Gruber et al., J. Immunol. 152: 5368 (1994).

[0241] The antibodies and antibody fragments described herein can be humanized to make them more tolerable for human use. For example, the amino acid residues in the framework region can be humanized by replacement with amino acid residues and human framework regions, provided that the replacement does not impair the ability of the antibodies and antibody fragments to bind to antigens (Vincke C, Loris R, Saerens D, Martinez-Rodriguez S, Muyldermans S, Conrath K., "General strategy to humanize a camelid single-domain antibody and identification of a universal humanized nanobody scaffold", J Biol Chem. 2008).

[0242] It should be understood that murine or antibodies from other species can be humanized or primatized using techniques well known in the art. See, for example, Winter and Harris Immunol Today 14:43-46 (1993) and Wright et al., Crit. Reviews in Immunol. 12:125-168 (1992). Antibodies of interest can be engineered by recombinant DNA techniques to replace the CH1, CH2, CH3, hinge domain, and / or framework domain with the corresponding human sequences (see WO92 / 102190 and U.S. Patent Nos. 5,530,101; 5,585,089; 5,693,761; 5,693,792; 5,714,350; and 5,777,085). In addition, it is known in the art to construct chimeric immunoglobulin genes using Ig cDNA (Liu et al., P.N.A.S. 84:3439 (1987) and J. Immunol. 139:3521 (1987)). mRNA is isolated from hybridomas or other cells producing the antibody and used to generate cDNA. cDNA of interest can be amplified by polymerase chain reaction using specific primers (U.S. Patent Nos. 4,683,195 and 4,683,202). Alternatively, a library is prepared and screened to isolate the sequence of interest. Then the DNA sequence encoding the variable region of the antibody is fused to the human constant region sequence. The sequence genes of the human constant region can be found in Kabat et al., (1991) Sequences of Proteins of immunological Interest, N.I.H. Publication No. 91-3242. The human C region genes are readily obtained from known clones. The choice of isotype will be guided by the desired effector function, such as complement fixation or antibody-dependent cell cytotoxicity activity. Optional isotypes are IgG1, IgG3, and IgG4. Either the human kappa or lambda light chain constant region can be used. Then the chimeric humanized antibody is expressed by conventional methods.

[0243] A fully human antibody is an antibody molecule in which the complete sequences of both the light and heavy chains (including the CDRs) are from human genes. Such antibodies are referred to herein as "human antibodies" or "fully human antibodies". For example, human monoclonal antibodies are prepared using the procedures described in the examples provided below. Human monoclonal antibodies can also be prepared using the following techniques: the trioma technique; human B cell hybridoma technique (see Kozbor et al., 1983 Immunol Today 4:72); and EBV hybridoma technique to produce human monoclonal antibodies (see Cole et al., 1985 in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). Human monoclonal antibodies can be used, and human monoclonal antibodies can be produced by using human hybridomas (see Cote et al., 1983. Proc Natl Acad Sci USA 80:2026-2030) or by in vitro transformation of human B cells with Epstein Barr Virus (see Cole et al., 1985 in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96).

[0244] In some methods, for example, antibody or fragments are developed using phage display methods with antibodies containing only human sequences. Such methods are well known in the art, for example in WO92 / 01047 and U.S. Patent No. 6,521,404, which are hereby incorporated by reference. In such a method, a combinatorial library of phages carrying random light and heavy chain pairs is screened using PD-1 or a fragment thereof from natural or recombinant sources. In another method, an antibody or fragment can be produced by a method in which at least one step of the method comprises immunizing a transgenic non-human animal with a PD-1 protein. In such a method, some of the endogenous heavy chain and / or kappa light chain loci of the xenogeneic / non-human animal have been disabled and are unable to undergo rearrangement required to generate genes encoding immunoglobulins responsive to an antigen. Additionally, at least one human heavy chain locus and at least one human light chain locus have been stably transfected into the animal. Thus, in response to the administered antigen, the human loci undergo rearrangement, thereby providing genes encoding human variable regions that are immunologically specific for the antigen. Thus, after immunization, the animal produces B cells that secrete fully human immunoglobulins.

[0245] In alternative methods, other approaches have employed the "minilocus" approach, which mimics the foreign Ig locus by including fragments (individual genes) from the Ig locus. Thus, one or more VH genes, one or more DH genes, one or more JH genes, the mu constant region, and a second constant region (preferably the gamma constant region) are formed into constructs for insertion into an animal.

[0246] It has also been demonstrated that large chromosomal fragments or entire chromosomes can be introduced into mice by microcell fusion, resulting in the production of human antibodies.

[0247] Those of ordinary skill in the art will recognize that a variety of possible moieties can be conjugated to the resulting antibodies and antigen-binding fragments thereof. (See, for example, "Conjugate Vaccines", Contributions to Microbiology and Immunology, J.M. Cruse and R.E. Lewis, Jr (eds.), Carger Press, Mew York, (1989), the entire contents of which are incorporated herein by reference). As long as the antibody and the other moiety retain their respective activities, the conjugation can be accomplished by any chemical reaction that binds the two molecules. The linkage can involve many chemical mechanisms, such as covalent binding, affinity binding, insertion, coordination binding, and complexation.

[0248] For example, conjugates of antibodies and effector molecules can be prepared using a variety of bifunctional protein crosslinking agents, such as N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bisazides (such as bis-(p-azidobenzoyl)hexanediamine), diazotized derivatives (such as bis-(p-diazobenzoyl)ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bifunctional fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, ricin immunotoxins can be prepared as described by Vitetta et al., Science 238:1098 (1987). Carbon-14 labeled 1-isothiocyanatobenzyl-3-methyl diethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugating radio nucleotides to antibodies (see W094 / 11026).

[0249] Pharmaceutical Compositions

[0250] In another aspect, the present invention provides pharmaceutical compositions comprising an anti-PD-1 antibody or an antigen-binding fragment thereof, and optionally a pharmaceutically acceptable carrier.

[0251] As used herein, the term "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, isotonic agents, absorption delaying agents, and the like, which are compatible with the administration of a drug. Suitable carriers are described in the latest edition of Remington's Pharmaceutical Sciences (a standard reference in the art), which is incorporated herein by reference. Optional examples of such carriers or diluents include, but are not limited to, water, saline, ringer's solution, glucose solution, and 5% human serum albumin.

[0252] The pharmaceutical composition is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, such as intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical), transmucosal, and rectal administration.

[0253] In one embodiment, the anti-PD-1 antibody or its antigen-binding fragment is prepared with a carrier that protects the compound from rapid elimination by the body, such as a sustained-release / controlled-release formulation, including implants and microencapsulation delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Methods for preparing such formulations are apparent to those skilled in the art.

[0254] In one embodiment, an oral or parenteral composition is formulated in unit dosage form for ease of administration and uniformity of dosage. As used herein, unit dosage form refers to physically discrete units suitable as unit doses for the subject to be treated; each unit contains a predetermined quantity of the active compound calculated to be associated with the required pharmaceutical carrier to produce the desired therapeutic effect. The specifications for the unit dosage form are dictated by and directly depend on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, as well as the limitations inherent in the art of compounding such active compounds for the treatment of individuals.

[0255] Depending on the needs of the particular indication being treated, the formulation may also contain more than one active compound, optionally having complementary activities and not adversely affecting each other. Alternatively, or in addition, the composition may contain agents that enhance its function, such as cytotoxic agents, cytokines, chemotherapeutic agents, or growth inhibitors. These molecules are present in a combination effective for the intended purpose in an appropriate amount.

[0256] The pharmaceutical composition of the present invention is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, such as intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical), transmucosal, and rectal administration. Solutions or suspensions for parenteral, intradermal, or subcutaneous administration may include the following components: a sterile diluent, such as water for injection, saline solution, fixed oil, polyethylene glycol, glycerol, propylene glycol, or other synthetic solvents; antibacterial agents, such as benzyl alcohol or methylparaben; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid (EDTA); buffers, such as acetate, citrate, or phosphate, and agents for adjusting tonicity, such as sodium chloride or glucose. The pH may be adjusted with an acid or base, such as hydrochloric acid or sodium hydroxide. Parenteral preparations may be sealed in ampoules, disposable syringes, or multi-dose vials made of glass or plastic.

[0257] Pharmaceutical compositions suitable for injection include sterile aqueous solutions (water-soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include saline, bacteriostatic water, Cremophor EL TM (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be a fluid that is easy to inject. It must be stable under the conditions of production and storage and must be protected against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.) and suitable mixtures thereof. Appropriate fluidity can be maintained, for example, by using a coating such as lecithin, by maintaining the required particle size in the case of a dispersion, and by using surfactants. The action of preventing microorganisms can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, it is preferred to include isotonic agents, such as sugars, polyols (e.g., mannitol), sorbitol, sodium chloride, in the composition. Prolonged absorption of injectable compositions can be achieved by including agents that delay absorption (e.g., aluminum monostearate and gelatin) in the composition.

[0258] Sterile injectable solutions can be prepared by incorporating the required amount of the active compound, as needed, into a suitable solvent with one or a combination of the ingredients listed above, followed by filtration sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and the required other ingredients from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the methods of preparation are vacuum drying and freeze-drying, which yield a powder of the active ingredient plus any additional required ingredients from a previously sterile-filtered solution.

[0259] Oral compositions generally contain an inert diluent or an edible carrier. The oral compositions can be encapsulated in gelatin capsules or compressed into tablets. For purposes of oral therapeutic administration, the active compounds can be incorporated with excipients and used in the form of tablets, lozenges, or capsules. Oral compositions can also be prepared using a fluid carrier as a mouthwash, wherein the compound in the fluid carrier is orally administered and swished and either expectorated or swallowed. Pharmaceutically compatible binding agents and / or adjuvant materials can be included as part of the composition. Tablets, pills, capsules, lozenges, etc. can contain any of the following ingredients or compounds of a similar nature: binders such as microcrystalline cellulose, acacia, or gelatin; excipients such as starch or lactose, disintegrating agents such as alginic acid, Primogel, or corn starch; lubricants such as magnesium stearate or stearates; glidants such as colloidal silicon dioxide; sweetening agents such as sucrose or saccharin; or flavoring agents such as peppermint, methyl salicylate, or orange flavoring.

[0260] For administration by inhalation, the compounds are delivered in the form of an aerosol spray from a pressurized container or dispenser containing a suitable propellant (e.g., a gas such as carbon dioxide) or nebulizer.

[0261] Systemic administration can also be effected by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art and include, for example, detergents, bile salts, and fusidic acid derivatives for transmucosal administration. Transmucosal administration can be effected using nasal sprays or suppositories. For transdermal administration, the active compound is formulated into ointments, pastes, gels, or creams as generally known in the art.

[0262] In one embodiment, the anti-PD-1 antibody or its antigen-binding fragment is prepared with a carrier that protects the compound from rapid elimination in the body, such as a controlled-release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Methods for preparing such formulations are apparent to those skilled in the art.

[0263] For ease of administration and uniformity of dosage, it is particularly advantageous to formulate oral or parenteral compositions in unit dosage form. As used herein, unit dosage form refers to physically discrete units suitable as unit doses for the subjects to be treated; each unit containing a predetermined quantity of the active compound calculated to be associated with the required pharmaceutical carrier to produce the desired therapeutic effect. The specification of the unit dosage forms of the invention depends on and is directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, as well as the inherent limitations of compounding such active compounds for the treatment of individuals in the art.

[0264] Method of Use

[0265] The present disclosure also provides a method of treating a disease in a subject, the method comprising administering to the subject an anti-PD-1 antibody or an antigen-binding fragment thereof provided herein, or a pharmaceutical composition provided herein. Further provided is the use of an anti-PD-1 antibody or an antigen-binding fragment thereof provided herein, or a pharmaceutical composition provided herein, for treating cancer in a subject. Further provided is the use of an anti-PD-1 antibody or an antigen-binding fragment thereof provided herein for manufacturing a medicament for treating cancer in a subject.

[0266] In some embodiments, the disease to be treated is cancer. In some embodiments, the disease to be treated is a solid tumor, such as colon cancer, breast cancer, lung cancer, ovarian cancer, prostate cancer, cervical cancer, brain cancer, skin cancer, liver cancer, pancreatic cancer or gastric cancer. In some embodiments, the cancer is leukemia or lymphoma. In some embodiments, the cancer has metastasized.

[0267] In some embodiments, the disease to be treated is an infectious disease, such as a chronic infectious disease, such as tuberculosis, malaria or infection with human immunodeficiency virus (HIV) or hepatitis B virus (HBV). In some embodiments, the disease to be treated is a primary immunodeficiency.

[0268] In some embodiments, a therapeutically effective amount is administered. The therapeutically effective amount of the antibody of the present disclosure generally relates to the amount required to achieve a therapeutic goal. As described above, this can be the binding interaction between the antibody and its target antigen, which in some cases interferes with the function of the target. The amount to be administered will also depend on the binding affinity of the antibody for its specific antigen, and will also depend on the rate at which the administered antibody is depleted from the free volume of the other subject to which it is administered. As a non-limiting example, a common range for the therapeutically effective dose of an antibody or antibody fragment of the present disclosure can be from about 0.1 mg / kg body weight to about 50 mg / kg body weight. Common dosing frequencies can be in the range of, for example, twice daily to once weekly.

[0269] The effectiveness of the treatment depends on any known method of diagnosing or treating the specific cancer to determine. The alleviation of one or more symptoms of the cancer indicates that the antibody confers a clinical benefit.

[0270] For example, the anti-tumor efficacy of a humanized anti-PD-1 antibody can be evaluated in a humanized mouse model, such as an MC38 allograft mouse model. To generate such an allograft model, MC38 cells can be injected into a mouse, such as the right flank of a C57BL / 6-hPD-1 humanized mouse. When the mouse develops a tumor of a suitable size (e.g., an average of about 80 mm 3After the tumor volume), the anti-PD-1 antibody can be administered by daily intraperitoneal injection. Appropriate controls can be included, such as human IgG of the same isotype as the anti-PD-1 antibody. The tumor volume can then be measured by calipers, and other clinical signs, behavior, and weight loss can be recorded throughout the study. To quantify the treatment efficacy, the relative tumor growth inhibition rate can be calculated as follows: TGI% = (1 - T / C) × 100%, where T is the tumor volume or tumor weight of the treatment group, and C is the tumor volume or tumor weight of the control group.

[0271] As used herein, "treating cancer" includes, but is not limited to, reversing, alleviating, or inhibiting the progression of cancer or cancer-related symptoms, or preventing the worsening of the severity of cancer or cancer-related symptoms or disorders. "Preventing cancer" includes preventing the occurrence of cancer and prophylactically treating a subject to prevent or reduce the incidence or recurrence of cancer.

[0272] In one embodiment, the active compound is used in combination with other therapies. Accordingly, the present application provides a method of preventing or treating cancer using a combination of the anti-PD-1 antibody or antigen-binding fragment thereof disclosed herein and at least one additional therapy. The additional therapy can be administered before, overlapping, simultaneously, and / or after the administration of the active compound. When administered simultaneously, the anti-PD-1 antibody or antigen-binding fragment thereof and the additional therapeutic agent can be administered in a single formulation or separate formulations, and if administered separately, optionally by different modes of administration. The combination of one or more anti-PD-1 antibodies or antigen-binding fragments thereof with one or more other therapies can act synergistically against cancer.

[0273] For example, the combination therapy can include using and / or co-administering the anti-PD-1 antibody or antigen-binding fragment thereof with one or more additional therapeutic agents (such as one or more cytokine and growth factor inhibitors, immunosuppressive agents, anti-inflammatory agents, metabolic inhibitors, enzyme inhibitors, anti-tumor agents, and / or cytotoxic or cytostatic agents). Such combination therapies can advantageously utilize lower doses of the administered therapeutic agents, thereby avoiding possible toxicities or complications associated with various single therapies.

[0274] In some embodiments, the subject or patient being treated according to the methods described herein has previously received chemotherapy or radiotherapy. The terms "subject" and "patient" are used interchangeably herein and can refer to, for example, a living mammalian organism such as a human, monkey, cow, horse, sheep, goat, dog, cat, mouse, rat, guinea pig, or a transgenic species thereof. In a preferred embodiment, the subject is a human.

[0275] The present invention also provides methods of inhibiting the interaction between PD-1 and one or more of its ligands using the antibodies and antigen-binding fragments described herein. For example, the antibodies or antigen-binding fragments described herein can inhibit the interaction between PD-1 and PD-L1 or between PD-1 and PD-L2. Those skilled in the art will recognize that inhibiting the interaction between PD-1 and PD-L1 and / or between PD-1 and PD-L2 can have beneficial effects on a cell or an organism containing the cell, including allowing the immune system of the organism to attack and kill tumor cells.

[0276] Accordingly, in one aspect, the present invention provides a method of inhibiting the interaction between PD-1 and PD-L1 in a cell, the method comprising contacting the cell with an antibody or an antigen-binding fragment thereof disclosed herein. In another aspect, the present invention provides a method of inhibiting the interaction between PD-1 and PD-L2 in a cell, the method comprising contacting the cell with an antibody or an antigen-binding fragment thereof disclosed herein. In some embodiments, the cell is a human cell. In some embodiments, the cell is a non-human cell. In some embodiments, the cell is not a human embryo. In some embodiments, the cell is contacted in vitro. In some embodiments, the cell is contacted in vivo.

[0277] In some embodiments, the antibody or antigen-binding fragment described herein inhibits the binding of PD-1 to PD-L1, wherein the half maximal inhibitory concentration (IC50) is from about 50 ng / mL to about 100 ng / mL, from about 100 ng / mL to about 150 ng / mL, from about 150 ng / mL to about 200 ng / mL, from about 200 ng / mL to about 250 ng / mL, from about 250 ng / mL to about 300 ng / mL, from about 300 ng / mL to about 350 ng / mL, from about 350 ng / mL to about 400 ng / mL, from about 400 ng / mL to about 450 ng / mL, from about 450 ng / mL to about 500 ng / mL, from about 500 ng / mL to about 550 ng / mL, from about 550 ng / mL to about 600 ng / mL, from about 600 ng / mL to about 650 ng / mL, from about 650 ng / mL to about 700 ng / mL, from about 700 ng / mL to about 750 ng / mL, from about 750 ng / mL to about 800 ng / mL, from about 800 ng / mL to about 850 ng / mL, from about 850 ng / mL to about 900 ng / mL, from about 900 ng / mL to about 950 ng / mL, or from about 950 ng / mL to about 1000 ng / mL.

[0278] In some embodiments, the antibodies or antigen-binding fragments described herein inhibit the binding of PD-1 to PD-L1, wherein the IC50 is about 50 ng / mL, about 60 ng / mL, about 70 ng / mL, about 80 ng / mL, about 90 ng / mL, about 100 ng / mL, about 110 ng / mL, about 120 ng / mL, about 130 ng / mL, about 140 ng / mL, about 150 ng / mL, about 160 ng / mL, about 170 ng / mL, about 180 ng / mL, about 190 ng / mL, about 200 ng / mL, about 210 ng / mL, about 220 ng / mL, about 230 ng / mL, about 240 ng / mL, about 250 ng / mL, about 260 ng / mL, about 270 ng / mL, about 280 ng / mL, about 290 ng / mL, about 300 ng / mL, about 310 ng / mL, about 320 ng / mL, about 330 ng / mL, about 340 ng / mL, about 350 ng / mL, about 360 ng / mL, about 370 ng / mL, about 380 ng / mL, about 390 ng / mL, about 400 ng / mL, about 410 ng / mL, about 420 ng / mL, about 430 ng / mL, about 440 ng / mL, about 450 ng / mL, about 460 ng / mL, about 470 ng / mL, about 480 ng / mL, about 490 ng / mL, or about 500 ng / mL.

[0279] In some embodiments, the antibodies or antigen-binding fragments described herein inhibit the binding of PD-1 to PD-L1, wherein the IC50 is about 372.2 ng / mL or less. In some embodiments, the antibodies or antigen-binding fragments described herein inhibit the binding of PD-1 to PD-L1, wherein the IC50 is about 461.0 ng / mL or less.

[0280] The IC50 for inhibiting the binding of PD-1 to PD-L1 can be determined using any suitable assay known in the art or described herein. Examples of such assays include the PD-1 / PD-L1 blocking bioassay described in Example 5 below (see also Promega Catalog No. J1250). In this assay, serial dilutions of the antibody to be tested are added to adherent PD-L1-expressing cells, which are then incubated with effector cells (e.g., Jurkat T cells stably expressing human PD-1 and NFAT-induced luciferase), and a luminescence substrate reagent is added to quantify the inhibition of PD-1 / PD-L1 blockade. PD-1 / PD-L1 interaction inhibits TCR signaling and NFAT-mediated luciferase activity. Addition of an antibody that blocks PD-1 or PD-L1 releases the inhibitory signal and results in TCR signaling and NFAT-mediated luciferase activity, both of which can be quantified.

[0281] In some embodiments, the antibodies or antigen-binding fragments described herein inhibit the binding of PD-1 to PD-L2, wherein the IC50 is from about 50 ng / mL to about 100 ng / mL, about 100 ng / mL to about 150 ng / mL, about 150 ng / mL to about 200 ng / mL, about 200 ng / mL to about 250 ng / mL, about 250 ng / mL to about 300 ng / mL, about 300 ng / mL to about 350 ng / mL, about 350 ng / mL to about 400 ng / mL, about 400 ng / mL to about 450 ng / mL, about 450 ng / mL to about 500 ng / mL, about 500 ng / mL to about 550 ng / mL, about 550 ng / mL to about 600 ng / mL, about 600 ng / mL to about 650 ng / mL, about 650 ng / mL to about 700 ng / mL, about 700 ng / mL to about 750 ng / mL, about 750 ng / mL to about 800 ng / mL, about 800 ng / mL to about 850 ng / mL, about 850 ng / mL to about 900 ng / mL, about 900 ng / mL to about 950 ng / mL or about 950 ng / mL to about 1000 ng / mL.

[0282] In some embodiments, the antibodies or antigen-binding fragments described herein inhibit the binding of PD-1 to PD-L2, wherein the IC50 is about 50 ng / mL, about 60 ng / mL, about 70 ng / mL, about 80 ng / mL, about 90 ng / mL, about 100 ng / mL, about 110 ng / mL, about 120 ng / mL, about 130 ng / mL, about 140 ng / mL, about 150 ng / mL, about 160 ng / mL, about 170 ng / mL, about 180 ng / mL, about 190 ng / mL, about 200 ng / mL, about 210 ng / mL, about 220 ng / mL, about 230 ng / mL, about 240 ng / mL, about 250 ng / mL, about 260 ng / mL, about 270 ng / mL, about 280 ng / mL, about 290 ng / mL, about 300 ng / mL, about 310 ng / mL, about 320 ng / mL, about 330 ng / mL, about 340 ng / mL, about 350 ng / mL, about 360 ng / mL, about 370 ng / mL, about 380 ng / mL, about 390 ng / mL, about 400 ng / mL, about 410 ng / mL, about 420 ng / mL, about 430 ng / mL, about 440 ng / mL, about 450 ng / mL, about 460 ng / mL, about 470 ng / mL, about 480 ng / mL, about 490 ng / mL or about 500 ng / mL.

[0283] In some embodiments, the antibodies or antigen-binding fragments described herein inhibit the binding of PD-1 to PD-L2, wherein the IC50 is about 66.76 ng / mL or less.

[0284] The IC50 for inhibiting the binding of PD-1 to PD-L2 can be determined using any suitable assay known in the art or described herein. Examples of such assays include the flow cytometry assay described in Example 15 below. In this assay, serial dilutions of the antibody to be tested are mixed with recombinant human PD-L2 protein, and the mixture is then added to Jurkat effector cells expressing PD-1. The PD-L2 protein or the test antibody bound to the PD-1-expressing Jurkat cells can be detected by flow cytometry using a fluorescently labeled secondary antibody.

[0285] Also provided herein are methods of inducing cytokine secretion in cells using the antibodies and antigen-binding fragments described herein. Inducing the secretion of cytokines in cells may enable the cells to mediate an immune response against a disease or disorder.

[0286] Thus, in one aspect, provided herein is a method of stimulating the secretion of IL-2 in cells, the method comprising contacting the cells with an antibody or an antigen-binding fragment thereof disclosed herein. In another aspect, provided herein is a method of stimulating the secretion of IFN-γ in cells, the method comprising contacting the cells with an antibody or an antigen-binding fragment thereof disclosed herein. In some embodiments, the cells are human cells. In some embodiments, the cells are non-human cells. In some embodiments, the cells are not human embryos. In some embodiments, the cells are contacted in vitro. In some embodiments, the cells are contacted in vivo.

[0287] In some embodiments, the antibodies or antigen-binding fragments described herein induce the secretion of IFN-γ from T cells, wherein the half-maximal effective concentration (EC50) is from about 1 ng / mL to about 5 ng / mL, about 5 ng / mL to about 10 ng / mL, about 10 ng / mL to about 15 ng / mL, about 15 ng / mL to about 20 ng / mL, about 20 ng / mL to about 25 ng / mL, about 25 ng / mL to about 30 ng / mL, about 30 ng / mL to about 35 ng / mL, about 35 ng / mL to about 40 ng / mL, about 40 ng / mL to about 45 ng / mL, about 45 ng / mL to about 50 ng / mL, about 50 ng / mL to about 55 ng / mL, about 55 ng / mL to about 60 ng / mL, about 60 ng / mL to about 65 ng / mL, about 65 ng / mL to about 70 ng / mL, about 70 ng / mL to about 75 ng / mL, about 75 ng / mL to about 80 ng / mL, about 80 ng / mL to about 85 ng / mL, about 85 ng / mL to about 90 ng / mL, about 90 ng / mL to about 95 ng / mL, or about 95 ng / mL to about 100 ng / mL.

[0288] In some embodiments, the antibodies or antigen-binding fragments described herein induce the secretion of IFN-γ from T cells, wherein the EC50 is about 5 ng / mL, about 6 ng / mL, about 7 ng / mL, about 8 ng / mL, about 9 ng / mL, about 10 ng / mL, about 11 ng / mL, about 12 ng / mL, about 13 ng / mL, about 14 ng / mL, about 15 ng / mL, about 20 ng / mL, about 25 ng / mL, about 30 ng / mL, about 35 ng / mL, about 40 ng / mL, about 45 ng / mL, about 50 ng / mL, about 55 ng / mL, about 60 ng / mL, about 65 ng / mL, about 70 ng / mL, about 75 ng / mL, about 75 ng / mL, about 80 ng / mL, about 85 ng / mL, about 90 ng / mL, about 95 ng / mL, or about 100 ng / mL.

[0289] In some embodiments, the antibodies or antigen-binding fragments described herein induce the secretion of IFN-γ from T cells, wherein the EC50 is about 38.4 ng / mL or less.

[0290] In some embodiments, the antibodies or antigen-binding fragments described herein induce the secretion of IL-2 from T cells, wherein the EC50 is from about 0.1 ng / mL to about 0.5 ng / mL, about 0.5 ng / mL to about 1 ng / mL, about 1 ng / mL to about 5 ng / mL, about 5 ng / mL to about 10 ng / mL, about 10 ng / mL to about 15 ng / mL, about 15 ng / mL to about 20 ng / mL, about 20 ng / mL to about 25 ng / mL, about 25 ng / mL to about 30 ng / mL, about 30 ng / mL to about 35 ng / mL, about 35 ng / mL to about 40 ng / mL, about 40 ng / mL to about 45 ng / mL, about 45 ng / mL to about 50 ng / mL, about 50 ng / mL to about 55 ng / mL, about 55 ng / mL to about 60 ng / mL, about 60 ng / mL to about 65 ng / mL, about 65 ng / mL to about 70 ng / mL, about 70 ng / mL to about 75 ng / mL, about 75 ng / mL to about 80 ng / mL, about 80 ng / mL to about 85 ng / mL, about 85 ng / mL to about 90 ng / mL, about 90 ng / mL to about 95 ng / mL, about 95 ng / mL to about 100 ng / mL, about 100 ng / mL to about 110 ng / mL, about 110 ng / mL to about 120 ng / mL, about 120 ng / mL to about 130 ng / mL, about 130 ng / mL to about 140 ng / mL, about 140 ng / mL to about 150 ng / mL, about 150 ng / mL to about 160 ng / mL, about 160 ng / mL to about 170 ng / mL, about 170 ng / mL to about 180 ng / mL, about 180 ng / mL to about 190 ng / mL, about 190 ng / mL to about 200 ng / mL, about 200 ng / mL to about 210 ng / mL, about 210 ng / mL to about 220 ng / mL, about 220 ng / mL to about 230 ng / mL, about 230 ng / mL to about 240 ng / mL, or about 240 ng / mL to about 250 ng / mL.

[0291] In some embodiments, the antibodies or antigen-binding fragments described herein induce the secretion of IL-2 from T cells, wherein the EC50 is about 0.1 ng / mL, about 0.5 ng / mL, about 1 ng / mL, about 5 ng / mL, about 10 ng / mL, about 15 ng / mL, about 20 ng / mL, about 25 ng / mL, about 30 ng / mL, about 35 ng / mL, about 40 ng / mL, about 45 ng / mL, about 25 ng / mL, about 30 ng / mL, about 35 ng / mL, about 40 ng / mL, about 50 ng / mL, about 60 ng / mL, about 70 ng / mL, about 80 ng / mL, about 90 ng / mL or about 100 ng / mL.

[0292] In some embodiments, the antibodies or antigen-binding fragments described herein induce the secretion of IL-2 from T cells, wherein the EC50 is about 0.2 ng / mL or less.

[0293] The secretion of cytokines (such as IFN-γ and / or IL-2) from immune cells can be determined using any suitable assay known in the art or described herein. Examples of such assays include the mixed lymphocyte reaction assay described in Example 8 below. In this assay, serial dilutions of the antibody to be tested are added to effector cells (e.g., human CD4 + T cells). Subsequently, antigen-presenting cells (e.g., human dendritic cells) are co-cultured with the effector cells, and commercially available kits (e.g., the human IL-2 HTRF kit and the human kit) from Cisbio can be used to quantify the secretion levels of IL-2 and IFN-γ in the culture medium.

[0294] Example

[0295] The following examples provide illustrative details of methods that can be used to generate and characterize the antibodies and antigen-binding fragments described herein. The examples are provided for illustrative purposes only and are not intended to limit the invention.

[0296] Abbreviations and Definitions

[0297] BM back mutation

[0298] CDR Complementary Determining Region

[0299] DPBS Dulbecco's Phosphate Buffered Saline

[0300] DNA Deoxyribonucleic Acid

[0301] EC 50 half maximal effective concentration

[0302] ELISA Enzyme-Linked Immunosorbent Assay

[0303] Fc crystallizable fragment

[0304] HC heavy chain

[0305] HRP horseradish peroxidase

[0306] IC 50 half maximal inhibitory concentration

[0307] IgG immunoglobulin G

[0308] LC light chain

[0309] m(prefix) murine antibody

[0310] OPD o-phenylenediamine dihydrochloride

[0311] PBS phosphate buffered saline

[0312] PBST phosphate buffered saline with tween-20

[0313] RNA ribonucleic acid

[0314] VH variable region of heavy chain

[0315] VL variable region of light chain

[0316] x(prefix) chimeric antibody

[0317] z(prefix) humanized antibody

[0318] Example 1: Generation of Murine Monoclonal Antibody Against Human PD-1 Protein

[0319] Immunization of mice with recombinant PD - 1 protein and CHO - overexpressing PD - 1 cells

[0320] To generate murine monoclonal antibodies against human PD-1, BALB / c and ICR mice were immunized with recombinant PD-1Fc chimeric protein and / or CHO cells overexpressing PD-1. The PD-1Fc chimeric protein was emulsified in AdjuLite TM Freund's complete adjuvant (primary immunization) or incomplete adjuvant (boost immunization). The emulsion mixture was injected subcutaneously into the neck (50 μL) and intramuscularly into the left and right thighs (50 μL / site) at 4-week intervals. The CHO cells overexpressing PD-1 were suspended in sterile PBS buffer (1×10 7 cells / 150 μL). The cell suspension was injected intraperitoneally at 2-week intervals. The PD-1-specific serum IgG titers were monitored regularly. Mice with the highest IgG titers were selected for final immunization, and their spleens were collected to generate hybridomas.

[0321] Generation and selection of mouse hybridomas

[0322] Anesthetize the selected mice and perform splenectomy using aseptic techniques. Wash the splenocytes 3 times with serum-free medium (360×g, for 7 minutes per round at 25 °C). Use a Countess TM 3FL Automated Cell Counter (Invitrogen TM ) to count the splenocytes and myeloma cells. Then mix the splenocytes and myeloma cells at a ratio of 2:1. Wash the cell mixture 3 times with 10 ml of sterile DPBS buffer, and then wash 3 times with 10 ml of sterile BTX buffer. Resuspend the cell mixture in ice-cold BTX buffer (5×10 7 cells / mL) and transfer it to an electrocuvette (400 μL / cuvette). Perform electrofusion using a BTX ECM 2001 machine with optimized conditions. Recover the fused cells overnight in ClonaCell TM -HY Medium C (STEMCELL TM ) in a 5% CO2 humidified incubator at 37 °C. Harvest the recovered cells (360×g for 7 minutes at 25 °C) and resuspend them in ClonaCell TM -HY Medium C (STEMCELL TM ) at a concentration of 2×10 7 cells / mL. Then, according to the ClonaCell TM -HY instruction manual, gently mix the cell suspension with ClonaCell TM -HY Medium D (STEMCELL TM ) at a ratio of 1:9 (v / v). Seed the cell mixture in a 6-well cell culture plate (2.5 mL / well) and incubate it in a 5% CO2 humidified incubator at 37 °C for 7 - 10 days. Randomly pick hybridoma colonies by pipetting and transfer them to each well (mini-pool) of a 96-well culture plate containing 200 μL of ClonaCell TM -HY Medium E (STEMCELL TM ). After culturing for 2 days, screen the mini-pool supernatants for PD-1 binding and neutralizing properties.

[0323] Example 2: Selection of Mouse Hybridoma Clones Producing Potent Anti-PD-1 Antibodies

[0324] PD - 1 specific binding screening

[0325] The ELISA technique was used to select hybridoma mini-pools that produced anti-PD-1 antibodies using the following procedure. MaxiSorp ELISA plates were coated overnight at 4 °C with PD-1 His-tag (R&D System) in PBS buffer (10 ng / 100 μL / well). The plates were washed 3 times with PBST wash buffer (0.05% Tween-20 in PBS) using a BioTek EL 406 plate washer. The culture supernatant of each mini-pool (100 μL) was added to the plate and then incubated at 37 °C for 1 hour. After washing, the plate was probed with goat anti-mouse IgG Fcγ-HRP (Jackson Immuno Research) diluted 1:8,000 in PBST (100 μL / well) and incubated at 37 °C for 1 hour. After washing 3 times, the plate was incubated with SIGMAFAST TM OPD (Sigma-Aldrich) substrate solution (100 μL / well) in the dark at room temperature for 20 minutes. 1 M H2SO4 (50 μL / well) was used to stop the reaction. Absorbance was measured at 492 nm by a Cytation TM 5 cell imaging multimode reader (BioTek). Hybridoma mini-pools with absorbance values exceeding 1.0 were selected for further screening.

[0326] PD - 1 / PD - L1 neutralization screening

[0327] Flow cytometry was used to select hybridoma mini-pools that produced PD-1 / PD-L1 neutralizing antibodies. A 96-well V-bottom plate (Nunc 4 containing Jurkat effector cells expressing PD-1 (1.5×10 TM cells / well) in FPBS buffer (10% FBS in PBS) was incubated with the culture supernatant (100 μL) from previously selected hybridoma mini-pools and recombinant PD-L1 human Fc-tag protein (GenScript) (0.5 μg) at room temperature for 30 minutes. After centrifuging the plate at 500 g for 5 minutes, the supernatant was discarded. Alexa diluted 1:100 in FPBS buffer Goat anti-human IgG (Jackson Immuno Research) was added to each well (100 μL). The plate was incubated at room temperature in the dark for 30 minutes and then washed twice with FPBS buffer. The cells were resuspended with FPBS buffer (100 μL) and analyzed by flow cytometry (IntellicytiQue screener plus). In the presence of neutralizing antibody (NAb), the PD-L1 human Fc-tagged protein could not bind to Jurkat cells expressing PD-1, resulting in the absence of a fluorescence signal. Hybridoma mini-pools with high neutralizing potency were selected for subsequent subcloning.

[0328] Hybridoma sub - cloning

[0329] Limit dilution was performed to achieve monoclonality confirmed under the microscope. Then, the PD-1 specific binding and PD-1 / PD-L1 neutralization of each single clone were evaluated using the above procedure. Finally, seven single hybridoma clones were selected based on high performance in both aspects of PD-1 specific binding and PD-1 / PD-L1 neutralization.

[0330] Example 3: Production and purification of mouse anti-PD-1 antibody

[0331] Mouse anti-PD-1 antibody was produced by culturing each hybridoma clone in HyClone SFM4MAb serum-free medium (Cytiva TM ) under static conditions at 37 °C and 5% CO2 for 5 - 7 days. The culture supernatant was harvested by centrifugation at 4000 g for 10 minutes at 25 °C. Then the supernatant was mixed with 10-fold binding buffer to reach a 1-fold concentration (100 mM HEPES, 150 mM NaCl, pH 8.0) and filtered through a 0.45 μm PES membrane filter. The antibody was purified using a HiTrap Protein A FF column (Cytiva TM ) by an AKTA purification system. Then, the bound antibody was eluted using Pierce TM IgG elution buffer (ThermoScientific TM ) and then neutralized with 3M Tris buffer. Then the eluted antibody was concentrated and the buffer was exchanged to PBS using an Ultra-15 (30K) centrifugal filter (Millipore). Antibody quantification was measured by spectrophotometry.

[0332] Example 4: Binding curve of mouse anti-PD-1 antibody

[0333] ​The purified antibody was serially diluted 2-fold starting from 2 μg / mL for eleven rounds until a final concentration of 0.977 ng / mL was reached. The PD-1 binding activity of the purified antibody was evaluated by ELISA according to the procedure described in Example 2. The half maximal effective concentration (EC ) was calculated using GraphPad 50 software.

[0334] The ELISA binding curves of the mouse anti-PD-1 antibodies are shown in Figure 1A . The EC 50 values of these antibodies are reported in Table 3.

[0335] Example 5: PD-1 / PD-L1 neutralization curves of mouse anti-PD-1 antibodies

[0336] According to the manufacturer's instructions, a PD-1 / PD-L1 blocking bioassay (Promega) was performed to evaluate the PD-1 / PD-L1 neutralization activity of the purified antibody. Briefly, adherent PD-L1a APC / CHO-K1 cells were seeded in assay plates and incubated overnight at 37 °C and 5% CO2. On the day of the assay, the medium was removed from the wells. Then, the antibody samples were serially diluted 3-fold starting from 100 μg / ml for nine rounds until a final concentration of 5.08 ng / mL was reached, and then incubated with Jurkat / PD-1 effector cells in the assay plates for 6 hours at 37 °C and 5% CO2. Bio-Glo TM substrate reagent (Promega) was added to the assay plates and incubated for 5 minutes at room temperature. The luminescence signal was measured using a Cytation TM 5 cell imaging multimode reader and reported as relative light units (RLU). The half maximal inhibitory concentration (IC ) was calculated using GraphPad 50 software. The PD-1 / PD-L1 neutralization curves are presented in Figure 1B . The IC 50 values are shown in Table 3.

[0337] Table 3: Binding characteristics of mouse anti-PD-1 antibodies

[0338]

[0339] Example 6: Sequencing of the variable regions of mouse antibodies

[0340] Total RNA was extracted from hybridoma cells using reagent, and then according to PrimeScript TMTechnical manual of the 1st strand cDNA synthesis kit, reverse transcribing it into cDNA using isotype-specific antisense primers or universal primers. Use rapid amplification of cDNA ends (RACE) technology to amplify the variable fragments of the heavy and light chains of antibodies. Clone the amplicons into a standard cloning vector and transform it into Escherichia coli Top10. Perform colony PCR to select clones with the correct inserted size. Analyze the variable fragment sequences by Sanger sequencing. The nucleotide sequences and amino acid sequences are shown in Table 2 and Table 1 respectively.

[0341] Example 7: Chimerization of anti-PD-1 antibody

[0342] Antibody chimerization

[0343] The variable regions of the mouse anti-PD-1 antibody are ligated to the human IgG4 / κ constant regions (based on the human κ constant region [UniProt accession number: P01834] and the human IgG4 constant region [UniProt accession number: P01861]). Synthesize the chimeric heavy and light chains and clone them into the pcDNA3.4 expression vector respectively.

[0344] Antibody production and purification

[0345] Use Expi293 TM expression system for small-scale transient expression of the chimeric antibody. Briefly, mix 15 μg of sterile transfection-grade expression vector (LC:HC ratio of 2:1) with the diluted ExpiFectamine TM 293 solution. Add 15 ml of Expi293F TM cell suspension (3×10 6 cells / mL) to the mixed solution, and then incubate with shaking at 37 °C and 8% CO2 for 7 days. Harvest the culture medium and purify the chimeric antibody using the method described in Example 3.

[0346] Example 8: In vitro characterization of the chimeric anti-PD-1 antibody

[0347] Binding curve of chimeric anti - PD - 1 antibody

[0348] Perform 2-fold serial dilutions of the purified chimeric antibody starting from 2 μg / ml for eleven rounds until reaching a final concentration of 0.977 ng / mL. Evaluate the PD-1 binding activity of the purified chimeric antibody by ELISA following the procedure mentioned in Example 2, while using a goat anti-human IgG Fcγ-HRP antibody (Jackson ImmunoResearch) diluted 1:10,000 in PBST to detect the chimeric antibody. The ELISA binding curve of the chimeric anti-PD-1 antibody is as Figure 2A shown. The EC of the chimeric antibody50 The values are shown in Table 4.

[0349] PD - 1 / PD - L1 neutralization curve of chimeric anti - PD - 1 antibody

[0350] To evaluate the PD-1 / PD-L1 neutralization curves, PD-1 / PD-L1 blocking bioassays were performed as described in Example 5 above. The PD-1 / PD-L1 neutralization curves of the chimeric antibodies are presented in Figure 2B The IC 50 values are shown in Table 4.

[0351] Binding kinetics of chimeric anti - PD - 1 antibody

[0352] The binding kinetics between the chimeric anti-PD-1 antibody and PD-1 protein were determined using a Biacore T200 instrument equipped with a Protein G sensor chip (GE Healthcare). Each purified chimeric anti-PD-1 antibody (1 μg / mL) was injected into the chip at a flow rate of 30 μL / min for 60 seconds. Single-cycle binding kinetics analysis was performed by sequentially injecting 1.5625, 3.125, 6.25, 12.5, and 25 nM of recombinant human PD-1 His-tag protein (R&D Systems) (with an association time of 60 seconds and a dissociation time of 120 seconds for the injection intervals). HBS-EP+ buffer (GE Healthcare) was used as the buffer blank and diluent. The association constant (k on ), dissociation constant (k off ), and equilibrium constant (K D ) values were calculated by curve fitting using the 1:1 Langmuir binding model with Biacore T200 evaluation software (version 3.1). The binding kinetics values of the chimeric anti-PD-1 antibody are shown in Table 4.

[0353] Mixed lymphocyte reaction (MLR) assay of chimeric anti - PD - 1 antibody

[0354] A two-way mixed lymphocyte reaction (MLR) assay was performed to determine the activation of human T cells by the chimeric antibodies. Human dendritic cells (DCs) were used as antigen-presenting cells (APCs) and human CD4 + T cells were used as effector cells. Serial dilutions of the antibody samples starting from 10 μg / mL were added to the assay plates containing the effector cells. Then the derived DCs were added to the assay plates, gently mixed, and co-cultured at 37 °C and 5% CO2 for 3 days. The secretion levels of human interleukin 2 (IL-2) and interferon-γ (IFN-γ) were quantified using a human IL-2 HTRF kit (Cisbio) and a human IFN-γ HTRF kit (Cisbio), respectively. Commercially available anti-PD-1 antibodies ( and ) was used as a positive control, while human IgG4 isotype control was used as a negative control. The EC of antibody-mediated IL-2 and IFN-γ secretion was reported in Table 4 50 values. The results showed that two chimeric clones xCUSB0125 and xCUSB0136 significantly induced IL-2 and IFN-γ secretion and were further selected to generate humanized anti-PD-1 antibodies.

[0355] Table 2: Characteristics of chimeric anti-PD-1 antibodies

[0356]

[0357] Note: NA; not applicable

[0358] Functional characterization of chimeric anti-PD-1 monoclonal antibodies demonstrated that these chimeric anti-PD-1 antibodies showed high PD-1 binding activity and high PD-1 / PD-L1 binding blocking activity and might activate human T cells.

[0359] Example 10: Humanization of anti-PD-1 antibodies

[0360] Antibody humanization

[0361] Antibody humanization was performed using CDR grafting technology. Briefly, the CDRs of xCUSB0125 and xCUSB0136 were grafted into the most suitable human immunoglobulin germlines. IGHV4-30-4*01 and IGKV1-12*01 were selected for xCUSB0125. IGHV4-59*01 and IGKV4-1*01 were selected for xCUSB0136. Back mutations were introduced into the antibody frameworks to restore the affinity of the humanized antibodies. Five and three versions of the variable heavy and light chains were generated from xCUSB0125. While six and three versions of the variable heavy and light chains were generated from xCUSB0136. Human IgG4 S228P and K constant regions were used as the scaffolds for all versions of the humanized antibodies. Multiple versions of each humanized antibody were generated from all combinations of their heavy and light chain versions. All nucleotide and amino acid sequences are shown in Table 1 and Table 3, respectively.

[0362] Antibody production and purification

[0363] Each version of the humanized antibody was transiently expressed using the Expi293 TM expression system as described in Example 7. The PD-1 binding kinetics of the antibodies secreted in the culture supernatants were screened using a Biacore 8K instrument to determine the K DSort the values. Select, generate, and purify humanized antibody versions with high binding kinetics against the human PD-1 protein for further characterization. The heavy and light chain pairs of the selected humanized antibody versions are shown in Table 5.

[0364] Table 3: Heavy and light chain pairs of selected humanized antibodies

[0365]

[0366] Figure 7 Sequence alignments of some of the anti-PD-1 antibodies described herein are shown. Figure 8A and Figure 8B Sequence identity analysis of the VH and VL of some of the anti-PD-1 antibodies described herein is shown. The sequence identity analysis was performed using Weblogo version 2.82 available at https: / / weblogo.berkeley.edu / logo.cgi.

[0367] In summary, four and six humanized anti-PD-1 antibodies were generated from the xCUSB0125 and xCUSB0136 antibody clones, respectively.

[0368] Example 11: In vitro characterization of humanized anti-PD-1 antibodies

[0369] PD - 1 / PD - L1 neutralization curve of humanized anti - PD - 1 antibody

[0370] To determine the PD-1 / PD-L1 neutralization curves of the humanized anti-PD-1 antibodies, a PD-1 / PD-L1 blocking bioassay was performed as described in Example 5. The PD-1 / PD-L1 neutralization curves of the top-ranked humanized anti-PD-1 antibodies are shown in Figure 3. The IC 50 values are shown in Table 6.

[0371] Binding kinetics of humanized anti - PD - 1 antibody

[0372] The Biacore T200 was used to confirm the binding kinetics of the top-ranked humanized anti-PD-1 antibodies as described in the previous Example 8. The binding kinetic values of the humanized anti-PD-1 antibodies are shown in Table 6.

[0373] Mixed lymphocyte reaction (MLR) assay of humanized anti - PD - 1 antibody

[0374] A two-way MLR assay was performed to evaluate the activation of human T cells by the humanized antibodies as described in Example 8. IL-2 and IFN-γ secretion from human T cells after stimulation with the antibodies are shown in Figure 4. The EC 50 values for antibody-mediated IL-2 and IFN-γ secretion are reported in Table 6.

[0375] Table 4: Binding and neutralizing properties of humanized anti-PD-1 antibodies

[0376]

[0377] Note: NA; not evaluated; ND; not detected

[0378] Functional characterization of selected humanized anti-PD-1 antibodies revealed that the top 3 versions each from zCUSB0125 and zCUSB0136 clones showed high PD-1 binding activity, high PD-1 / PD-L1 binding blocking activity, and could activate human T cells.

[0379] Effect of amino acid modification on binding kinetics

[0380] The effect of specific amino acid modifications on the binding kinetics of the zCUSB0136.8 antibody was evaluated and the results are shown in Table 7.

[0381] Table 7: Binding kinetics of modified zCUSB0136.8 antibodies; numbers are based on the VH and VL sequences shown in Table 1.

[0382]

[0383]

[0384] In summary, these specific amino acid modifications in the VH and VL of zCUSB0136.8 increased its binding affinity.

[0385] Example 12: In vivo antitumor efficacy of humanized anti-PD-1 antibodies

[0386] Antitumor efficacy of humanized anti - PD - 1 antibody

[0387] Based on the available functional property data of humanized anti-PD-1 antibodies, zCUSB0125.6 and zCUSB0136.8 were selected for further study. Additionally, the heavy chain Fc regions of the zCUSB0125.6 and zCUSB0136.8 anti-PD-1 antibodies were changed from IgG4(S228P) to IgG1-LALA(L234A L235A) to modulate the binding of the antibody Fc hinge region to Fcγ receptors (FcγR), which may affect antibody function and in vivo efficacy 9,10,11,12 。

[0388] The MC38 allograft / hPD-1 humanized mouse model was established by inoculating the MC38 colorectal cancer cell line (2×10 6Cells / 0.1 mL, with a cell viability > 99%) were subcutaneously inoculated into the right abdomen of C57BL / 6-hPD-1 humanized mice to establish the model. When the average tumor volume reached approximately 80 mm 3 For anti-tumor evaluation, random grouping was performed. zCUSB0125.6-LALA, zCUSB0136.8-LALA, and were used as therapeutic agents, while human IgG1 isotype control was used as a negative control. All treatments were administered intraperitoneally every 4 days for a total of 12 days.

[0389] The tumor volume was measured twice a week. The length (L) and width (W) of the tumor mass were measured using calipers. The tumor volume formula was: V = (L × W × W) / 2, where V is the tumor volume (mm 3 ), L is the tumor length (mm), and W is the tumor width (mm).

[0390] Routine monitoring included tumor growth and the effects of treatment on animal behavior, weight gain or loss, and other abnormalities. Any clinical signs observed during the experiment were fully recorded.

[0391] Efficacy evaluation criteria

[0392] The relative tumor growth inhibition rate (TGI) was calculated as follows: TGI% = (1 - T / C) × 100%, where T is the tumor volume or tumor weight of the treatment group, and C is the tumor volume or tumor weight of the control group.

[0393] To evaluate the anti-tumor efficacy of zCUSB0125.6-LALA and zCUSB0136.8-LALA anti-PD-1 antibodies, MC38 allograft / hPD-1 humanized mice (n = 5 mice / group) were treated with 10 mg / kg every 4 days (days 0, 4, 8, and 12). The tumor volume was monitored on days 0, 4, 7, 11, 14, and 18. All mice were sacrificed on day 18 to measure the tumor weight. The tumor volume and tumor weight monitoring data are shown in Figure 5. The TGI% was calculated as presented in Table 8.

[0394] Table 8: Anti-tumor efficacy of humanized anti-PD-1 monoclonal antibodies compared with commercial antibodies

[0395]

[0396] Compared with zCUSB0125.6-LALA, zCUSB0136.8-LALA exhibited excellent anti-tumor activity. Therefore, zCUSB0136.8-LALA was selected to confirm its anti-tumor activity at different doses. On days 0, 4, 8, and 12, MC38 allograft / hPD-1 humanized mice (n = 10 per group) were treated with low and high doses (5 mg / kg and 10 mg / kg) of the antibody. Tumor volume was monitored on days 0, 2, 6, 9, 13, and 16. All mice were sacrificed on day 16 to measure tumor weight. Tumor volume and tumor weight are shown in Figure 6. TGI% was calculated and presented as in Table 9.

[0397] Table 9: Anti-tumor efficacy of zCUSB0136.8-LALA compared with commercially available antibodies at the end of the study (day 16)

[0398]

[0399] In summary, in MC38 allograft / hPD-1 humanized mice, zCUSB0136.8-LALA exhibited higher anti-tumor efficacy compared with zCUSB0125.6-LALA.

[0400] Example 13: In vivo anti-tumor efficacy of affinity-matured humanized anti-PD-1 antibody

[0401] Antitumor efficacy of affinity - matured humanized anti - PD - 1 antibody

[0402] To improve the anti-tumor efficacy at low doses of the drug, affinity maturation was performed to generate multiple variants of the affinity-matured antibody. Based on previous anti-tumor efficacy data, zCUSB0136.8 was used as a template and four affinity-matured antibodies, including zCUSB0136.8-1, zCUSB0136.8-2, zCUSB0136.8-3, and zCUSB0136.8-4, were successfully generated. The binding affinity of the affinity-matured antibodies is shown in Table 6. Based on the binding kinetics data, zCUSB0136.8-2 was selected to confirm its in vivo anti-tumor efficacy at low (2.5 mg / kg) and high (10 mg / kg) doses and to compare with commercially available antibodies.

[0403] As described in Example 12, the MC38 allograft / hPD-1 humanized mouse model was used to detect the in vivo anti-tumor efficacy. zCUSB0136.8-2, and were used as therapeutic agents, while human IgG1 isotype control was used as a negative control. All treatments were administered by intraperitoneal injection once every 4 days for a total of 12 days.

[0404] As described in Example 12, tumor volume measurements, routine monitoring, and any clinical signs were observed and recorded.

[0405] Efficacy evaluation criteria

[0406] The relative tumor growth inhibition rate (TGI) was calculated as follows: TGI% = (1-T / C) x 100%, where T is the tumor volume or tumor weight of the treatment group, and C is the tumor volume or tumor weight of the control group.

[0407] To evaluate the anti-tumor efficacy of zCUSB0136.8-2 anti-PD-1 antibody, MC38 allograft / hPD-1 humanized mice (n=10 / group) were treated with 2.5 and 10 mg / kg every 4 days (i.e., on days 0, 3, 7, and 10). Tumor volume was monitored on days 0, 3, 7, 10, and 14. All mice were sacrificed on day 14 to measure tumor weight. Tumor volume and tumor weight monitoring data are shown in Figure 9. TGI% was calculated as presented in Table 10.

[0408] Table 10: Anti-tumor efficacy of affinity matured anti-PD-1 monoclonal antibodies compared with commercially available antibodies at the end of the study (Day 14)

[0409]

[0410] Data are shown as mean ± SEM

[0411] In conclusion, zCUSB0136.8-2-LALA showed similar efficacy to that of humanized hPD-1 in MC38 allograft mice. and Quite high anti-tumor efficacy.

[0412] Example 14: X-ray crystallographic analysis between zCUSB0136.8-2Fab and human PD-1 protein.

[0413] Crystallization

[0414] Crystals were obtained from a standing drop of 100 nL purified PD-1:zCUSB0136.8-2Fab complex (10 mg / mL) and 100 nL precipitant solution (composed of 0.1 M bicine pH 9.3 and 22% v / v PEG Smear Broad). The crystallization drop was set up using a mosquito crystallization robot (STP Labtech) and equilibrated with 40 μL precipitant solution at 20 °C. Crystals were cryoprotected by placing in precipitant solution supplemented with 20% v / v ethylene glycol and then rapidly cooled in liquid nitrogen.

[0415] Data collection

[0416] The best data set was collected at the Diamond Light Source beamline I03 in Didcot, UK, at 100 K and below. 3,600 images were collected, with an oscillation range of 0.10° per image. The beamline was equipped with an Eiger2 XE 16M detector. The data were processed anisotropically using autoPROC to a resolution of (see Vonrhein et al., Acta Crystallogr. D Biol. Crystallogr. 67, 293 - 302 (2011)) and STARANISO (see Tickle et al. (2018) “STARANISO”, Cambridge, United Kingdom: Global Phasing Ltd.), which includes the software XDS (see Kabsch et al., Acta Crystallogr. D Biol. Crystallogr. 66, 125 - 132 (2010)), Aimless (see Evans et al., Acta. Crystallogr. D Biol. Crystallogr. 69, 1204 - 1214 (2013)) and Pointless (see Evans et al., Acta. Crystallogr. D Biol. Crystallogr. 62, 72 - 82 (2006)). Although this data set was not used to build and refine the model, statistics for an isotropically processed data set to resolution are provided for comparison.

[0417] Structure determination and improvement

[0418] The initial phase was obtained by molecular replacement using Phaser (see McCoy et al., J. Appl. Crystallogr. 40, 658 - 674 (2007)) and the deposited structure of the PD1:zCUSB0136.8 - 2Fab complex (PDB entry: 7BXA), where the PD - 1 and Fab molecules were divided into two separate search sets. Structure refinement was carried out using Buster (see Bricogne et al., (2017) BUSTER 2.11.8, Cambridge, United Kingdom: GlobalPhasing Ltd.) and model building was performed in Coot (see Emsley et al., Acta Crystallogr. D Biol. Crystallogr. 66, 486 - 501 (2010)). The final R value (R model / R free ) was 0.204 / 0.239.

[0419] Structure of the PD - 1:zCUSB0136.8 - 2Fab complex

[0420] The structure of the PD - 1:zCUSB0136.8 - 2Fab complex was determined at resolution, and two PD - 1:zCUSB0136.8 - 2Fab complexes were used in the asymmetric unit. The first PD - 1:zCUSB0136.8 - 2Fab complex included heavy - chain residues 1 - 126 and 128 - 212 (chain A), light - chain residues 1 - 216 (chain B), and PD - 1 residues 33 - 88 and 93 - 145 (chain C). The second PD1:zCUSB0136.8 - 2Fab complex included heavy - chain residues 1 - 127 and 133 - 212 (chain H), light - chain residues 1 - 217 (chain L), and PD - 1 residues 33 - 84 and 92 - 147 (chain P). The structure was well - defined overall in the electron density map. However, some residues of PD - 1 could not be built due to weak electron density, including residues 86 - 88 in chain P, which were visible in chain C and located near the binding interface with the CDR2 loop of the light chain. Epitope interactions were calculated using CONTACT and CCP4. The epitope - paratope analysis is summarized in Table 11.

[0421] Table 11: List of epitope contacts highly likely to form hydrogen bonds or salt bridges between zCUSB0136.8 - 2Fab and human PD - 1 protein

[0422]

[0423]

[0424] In summary, the structure of the PD-1:zCUSB0136.8-2 complex was successfully evaluated, and the epitope contact residues between the complexes were identified.

[0425] Example 15: PD-1 / PD-L2 Ligand Binding Blockade of Affinity-Matured Antibodies

[0426] The PD-1 / PD-L2 ligand binding blockade of zCUSB0136.8-2 was evaluated using flow cytometry. Initially, serial 4-fold dilutions of the zCUSB0136.8-2 antibody starting at 2 μg / mL were mixed with 1.25 μg / mL of recombinant human PD-L2 protein mouse Fc tag (Sino Biological). Subsequently, this mixture was added to Jurkat effector cells expressing PD-1 in FPBS buffer (5×10 4 cells / well) and gently mixed by pipetting.

[0427] The plate was incubated at room temperature for 30 minutes and then centrifuged to harvest the cells (500 g, for 5 minutes at room temperature), and the supernatant was discarded. To detect the PD-L2 protein mouse Fc tag or the affinity-matured zCUSB0136.8-2 human IgG1-LALA antibody bound to Jurkat cells expressing hPD-1, a secondary antibody mixture (AlexaFluor488 goat anti-mouse IgG (Jackson ImmunoResearch) and Alexa Fluor 647 goat anti-human IgG (JacksonImmunoResearch) mixed at 1:250 in FPBS buffer) was added to each well (100 μL) and then gently mixed by pipetting. The plate was incubated at room temperature in the dark for 30 minutes and washed twice with FPBS buffer. The cells were resuspended in 100 μL of FPBS buffer. Fluorescence signals were detected using an IntellicytiQue Screener Plus flow cytometer, and the data were analyzed using Standard Edition 7.1 software. The half-maximal inhibitory concentration (IC ) was calculated using GraphPad 50 )

[0428] The results showed that the affinity-matured zCUSB0136.8-2 antibody could inhibit the PD-1 / PD-L2 interaction, with an IC 50 value of 66.76 ng / mL ( Figure 10 ).

[0429] In summary, zCUSB0136.8-2-LALA showed high PD-1 / PD-L2 binding blockade activity as evaluated by flow cytometry.

[0430] Equivalent

[0431] Details of one or more embodiments of the present disclosure are set forth in the foregoing description. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, the preferred methods and materials are now described. Other features, objects, and advantages of the present disclosure will become apparent from the specification and claims. In the specification and the appended claims, the singular forms include plural referents unless the context clearly dictates otherwise. All technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs, unless otherwise defined.

[0432] All publications and patent documents cited herein are incorporated herein by reference as if each such publication or document was specifically and individually indicated to be incorporated by reference. Citing publications and patent documents is not intended to admit any relevant prior art, nor does it constitute an admission of the content or date thereof. The present invention has now been described by way of written description, and those skilled in the art will recognize that the present invention can be practiced in a variety of embodiments, and the foregoing description and the following examples are for illustrative purposes only and are not limitations on the appended claims.

[0433] The foregoing description is presented for purposes of illustration only and is not intended to limit the present disclosure to the precise form disclosed, but is limited by the appended claims.

Claims

1. An anti-PD-1 antibody or antigen-binding fragment thereof, comprising (a)(i) VH, which comprises CDR1 containing the sequence X1YDII (SEQ ID NO: 228), where X1 is T or V; CDR2 containing the sequence VIWTGGDTX2YNSAFMS (SEQ ID NO: 229), where X2 is N, W or T; and CDR3 containing the sequence DWX3Y (SEQ ID NO: 230), where X3 is A, I or M; and (ii) VL, which comprises CDR1 containing the sequence RASESVDNX4GNSFIQ (SEQ ID NO: 231), where X4 is Y or W; CDR2 containing the sequence FASX5LQS (SEQ ID NO: 232), where X5 is N, Y, H or Q; and CDR3 containing the sequence HQNNEDPFT (SEQ ID NO: 227); (b)(i) VH, which comprises CDR1 containing the sequence shown in SEQ ID NO: 162, CDR2 containing the sequence shown in SEQ ID NO: 163, and CDR3 containing the sequence shown in SEQ ID NO: 164; and (ii) VL, which comprises CDR1 containing the sequence shown in SEQ ID NO: 180, CDR2 containing the sequence shown in SEQ ID NO: 181, and CDR3 containing the sequence shown in SEQ ID NO: 182; (c)(i) VH, which comprises CDR1 containing the sequence shown in SEQ ID NO: 81, CDR2 containing the sequence shown in SEQ ID NO: 82, and CDR3 containing the sequence shown in SEQ ID NO: 83; and (ii) VL, which comprises CDR1 containing the sequence shown in SEQ ID NO: 85, CDR2 containing the sequence shown in SEQ ID NO: 86, and CDR3 containing the sequence shown in SEQ ID NO: 87; (d)(i) VH, which comprises CDR1 containing the sequence shown in SEQ ID NO: 89, CDR2 containing the sequence shown in SEQ ID NO: 90, and CDR3 containing the sequence shown in SEQ ID NO: 91; and (ii) VL, which comprises CDR1 containing the sequence shown in SEQ ID NO: 93, CDR2 containing the sequence shown in SEQ ID NO: 94, and CDR3 containing the sequence shown in SEQ ID NO: 95; (e)(i) A VH that comprises a CDR1 containing the sequence shown in SEQ ID NO: 97, a CDR2 containing the sequence shown in SEQ ID NO: 98, and a CDR3 containing the sequence shown in SEQ ID NO: 99; and (ii) a VL that comprises a CDR1 containing the sequence shown in SEQ ID NO: 101, a CDR2 containing the sequence shown in SEQ ID NO: 102, and a CDR3 containing the sequence shown in SEQ ID NO: 103; (f)(i) A VH that comprises a CDR1 containing the sequence shown in SEQ ID NO: 113, a CDR2 containing the sequence shown in SEQ ID NO: 114, and a CDR3 containing the sequence shown in SEQ ID NO: 115; and (ii) a VL that comprises a CDR1 containing the sequence shown in SEQ ID NO: 117, a CDR2 containing the sequence shown in SEQ ID NO: 118, and a CDR3 containing the sequence shown in SEQ ID NO: 119; or (g)(i) A VH that comprises a CDR1 containing the sequence shown in SEQ ID NO: 121, a CDR2 containing the sequence shown in SEQ ID NO: 122, and a CDR3 containing the sequence shown in SEQ ID NO: 123; and (ii) a VL that comprises a CDR1 containing the sequence shown in SEQ ID NO: 125, a CDR2 containing the sequence shown in SEQ ID NO: 126, and a CDR3 containing the sequence shown in SEQ ID NO:

127.

2. The anti-PD-1 antibody or antigen-binding fragment thereof according to claim 1, which comprises (i) a VH that comprises a CDR1 containing the sequence shown in SEQ ID NO: 129, a CDR2 containing the sequence shown in SEQ ID NO: 130, and a CDR3 containing the sequence shown in SEQ ID NO: 131; and (ii) a VL that comprises a CDR1 containing the sequence shown in SEQ ID NO: 133, a CDR2 containing the sequence shown in SEQ ID NO: 134, and a CDR3 containing the sequence shown in SEQ ID NO:

135.

3. The anti-PD-1 antibody or antigen-binding fragment thereof according to claim 1 or 2, which comprises (a) comprising a sequence X6VQLX7ESGPGLVKPSQX8LSLTCTVX9GYSITSDYAWNWIRQX 10 PGX 11 X 12 LEWX 13 GYIYYSGTTSYNPSLKSRX 14 X 15 IX 16 X 17 DTSKNQFX 18 LX 19 LX 20 SVTX 21 X 22 DTAX 23 VH of YYC ARNYGSAFYYFDYWGQGTTLTVSS, where X6 is D or Q; X7 is R or Q; X8 is S or T; X9 is T or S; X 10 is F or P; X 11 is N or K; X 12 is K or G; X 13 is M or I; X 14 is I or V; X 15 is S or T; X 16 is T or S; X 17 is R or V; X 18 is F or S; X 19 is Q or K; X 20 is N or S; X 21 is T or A; X 22 is E or A; and / or X 23 is T or V; or (b) comprising a sequence QVQLX 39 ESGPGLVX 40 PSX 41 X 42 LSX 43 TCTVSGFSLTX 44 YDIIWIRQX 45 PGKGLEWX 46 GVIWTGGDTX 47 YNSAFMSRX 48 X 49 ISX 50 DX 51 SKX 52 QX 53 X 54 LKX 55 X 56 SX 57 X 58 X 59 X 60 DTAX 61 YYCX 62 RDWX 63 YWGQGTLVTVSX 64 of VH, where X 39 is K or Q; X 40 is A or K; X 41 is Q or E; X 42 is S or T; X 43 is I or L; X 44 is T or V; X 45 is S or P; X 46 is L or I; X 47 is N, W or T; X 48 is L or V; X 49 is T or S; X 50 is K or V; X 51 is N or T; X 52 is S or N; X 53 is I or F; X 54 is F or S; X 55 is M or L; X 56 is N or S; X 57 is L or V; X 58 is Q or T; X 59 is T or A; X 60 is D or A; X 61 is I or V; X 62 is A or V; X 63 is A, I or M; and / or X 64 is T or S.

4. The anti-PD-1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, which comprises (a) comprising a sequence DIX 24 MTQSPSSX 25 SX 26 SX 27 GDX 28 VTITCHASQGISSNIGWX 29 QQKPGKX30X 31 KX 32 LIYRGTNLEDGVPSRFSGSGSGX 33 DX 34 X 35 LTISSLX 36 X 37 EDFAX 38 The VL of YYCVQYAQFPPTFGGGTKLEIK, where X 24 is L or Q; X 25 is M or V; X 26 is V or A; X 27 is L or V; X 28 is T or R; X 29 is L or Y; X 30 is S or A; X 31 is F or P; X 32 is G or L; X 33 is A or T; X 34 is Y or F; X 35 is S or T; X 36 is E or Q; X 37 is S or P; and / or X 38 is D or T; or (b) comprising a sequence X 65 IVX 66 TQSPX 67 SLAVSLGX 68 RATIX 69 CRASESVDNX 70 GNSFIQWYQQKPGQPPKLLIYFASX 71 LQSGVPX 72 RFSGSGSX 73 TDFTLTIX 74 X 75 X 76 X 77 AX 78 DX 79 AX 80 YYCHQNNEDPFTFGX 81 VL of GTKLEIK, where X 65 is N or D; X 66 is L or M; X 67 is A or D; X 68 is Q or E; X 69 is S or N; X 70 is Y or W; X 71 is N or Y; X 72 is A or D; X 73 is R or G; X 74 is D or S; X 75 is P or S; X 76 is V or L; X 77 is E or Q; X 78 is D or E; X 79 is A or V; X 80 is T or V; and / or X 81 is S or G.

5. The anti-PD-1 antibody or antigen-binding fragment thereof according to claim 1 or 2, which comprises a VH, wherein the VH comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the sequence shown in any one of SEQ ID NOs: 80, 88, 96, 104, 112, 120, 128, 136, 137, 138, 139, 140, 144, 145, 146, 147, 148, 149, 153, 154, 155 and 156.

6. The anti-PD-1 antibody or antigen-binding fragment thereof according to claim 1 or 2, which comprises a VL, wherein the VL comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the sequence shown in any one of SEQ ID NOs: 84, 92, 100, 108, 116, 124, 132, 141, 142, 143, 150, 151, 152, 157 and 158.

7. The anti-PD-1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, which comprises a VH, wherein the VH comprises the sequence shown in any one of SEQ ID NOs: 80, 88, 96, 104, 112, 120, 128, 136, 137, 138, 139, 140, 144, 145, 146, 147, 148, 149, 153, 154, 155 and 156.

8. The anti-PD-1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, which comprises a VL, wherein the VL comprises the sequence shown in any one of SEQ ID NOs: 84, 92, 100, 108, 116, 124, 132, 141, 142, 143, 150, 151, 152, 157 and 158.

9. The anti-PD-1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, which comprises (a) a VH comprising the sequence shown in SEQ ID NO: 136 and a VL comprising the sequence shown in SEQ ID NO: 143; (b) a VH comprising the sequence shown in SEQ ID NO: 137 and a VL comprising the sequence shown in SEQ ID NO: 143; (c) a VH comprising the sequence shown in SEQ ID NO: 138 and a VL comprising the sequence shown in SEQ ID NO: 143; (d) a VH comprising the sequence shown in SEQ ID NO: 139 and a VL comprising the sequence shown in SEQ ID NO: 143; (e) a VH comprising the sequence shown in SEQ ID NO: 145 and a VL comprising the sequence shown in SEQ ID NO: 150; (f) A VH comprising the sequence shown in SEQ ID NO: 145 and a VL comprising the sequence shown in SEQ ID NO: 151; (g) A VH comprising the sequence shown in SEQ ID NO: 145 and a VL comprising the sequence shown in SEQ ID NO: 152; (h) A VH comprising the sequence shown in SEQ ID NO: 146 and a VL comprising the sequence shown in SEQ ID NO: 150; (g) A VH comprising the sequence shown in SEQ ID NO: 146 and a VL comprising the sequence shown in SEQ ID NO: 151; (h) A VH comprising the sequence shown in SEQ ID NO: 146 and a VL comprising the sequence shown in SEQ ID NO: 152; (i) A VH comprising the sequence shown in SEQ ID NO: 153 and a VL comprising the sequence shown in SEQ ID NO: 157; (j) A VH comprising the sequence shown in SEQ ID NO: 154 and a VL comprising the sequence shown in SEQ ID NO: 158; (k) A VH comprising the sequence shown in SEQ ID NO: 155 and a VL comprising the sequence shown in SEQ ID NO: 158; (1) A VH comprising the sequence shown in SEQ ID NO: 156 and a VL comprising the sequence shown in SEQ ID NO: 158; (m) Comprising the sequence X6VQLX7ESGPGLVKPSQX8LSLTCTVX9GYSITSDYAWNWIRQX 10 PGX 11 X 12 LEWX 13 GYIYYSGTTSYNPSLKSRX 14 X 15 IX 16 X 17 DTSKNQFX 18 LX 19 LX 20 SVTX 21 X 22 DTAX 23 VH of YYCARNYGSAFYYFDYWGQGTTLTVSS, where X6 is D or Q; X7 is R or Q; X8 is S or T; X9 is T or S; X 10 is F or P; X 11 is N or K; X 12 is K or G; X 13 is M or I; X 14 is I or V; X 15 is S or T; X 16 is T or S; X 17 is R or V; X 18 is F or S; X 19 is Q or K; X 20 is N or S; X 21 is T or A; X 22 is E or A; and X 23 is T or V; and contains the sequence DIX 24 MTQSPSSX 25 SX 26 SX 27 GDX 28 VTITCHASQGISSNIGWX 29 QQKPGKX30X 31 KX 32 LIYRGTNLEDGVPSRFSGSGSGX 33 DX 34 X 35 LTISSLX 36 X 37 EDFAX 38 The VL of YYCVQYAQFPPTFGGGTKLEIK, where X 24 is L or Q; X 25 is M or V; X 26 is V or A; X 27 is L or V; X 28 is T or R; X 29 is L or Y; X 30 is S or A; X 31 is F or P; X 32 is G or L; X 33 is A or T; X 34 is Y or F; X 35 is S or T; X 36 is E or Q; X 37 is S or P; and X 38 is D or T; or (k) Comprising the sequence QVQLX 39 ESGPGLVX 40 PSX 41 X 42 LSX 43 TCTVSGFSLTX 44 YDIIWIRQX 45 PGKGLEWX 46 GVIWTGGDTX 47 YNSAFMSRX 48 X 49 ISX 50 DX 51 SKX 52 QX 53 X 54 LKX 55 X 56 SX 57 X 58 X 59 X 60 DTAX 61 YYCX 62 RDWX 63 YWGQGTLVTVSX 64 of VH, wherein X 39 is K or Q; X 40 is A or K; X 41 is Q or E; X 42 is S or T; X 43 is I or L; X 44 is T or V; X 45 is S or P; X 46 is L or I; X 47 is N, W or T; X 48 is L or V; X 49 is T or S; X 50 is K or V; X 51 is N or T; X 52 is S or N; X 53 is I or F; X 54 is F or S; X 55 is M or L; X 56 is N or S; X 57 is L or V; X 58 is Q or T; X 59 is T or A; X 60 is D or A; X 61 is I or V; X 62 is A or V; X 63 is A, I or M; and X 64 is T or S; and comprising the sequence X 65 IVX 66 TQSPX 67 SLAVSLGX 68 RATIX 69 CRASESVDNX 70 GNSFIQWYQQKPGQPPKLLIYFASX 71 LQSGVPX 72 RFSGSGSX 73 TDFTLTIX 74 X 75 X 76 X 77 AX 78 DX 79 AX 80 YYCHQNNEDPFTFGX 81 The VL of GTKLEIK, where X 65 is N or D; X 66 is L or M; X 67 is A or D; X 68 is Q or E; X 69 is S or N; X 70 is Y or W; X 71 is N or Y; X 72 is A or D; X 73 is R or G; X 74 is D or S; X 75 is P or S; X 76 is V or L; X 77 is E or Q; X 78 is D or E; X 79 is A or V; X 80 is T or V; and X 81 is S or G.

10. The anti-PD-1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, wherein the antibody is a monoclonal antibody.

11. The anti-PD-1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, wherein the antibody or antigen-binding fragment thereof is a humanized antibody or antigen-binding fragment thereof.

12. The anti-PD-1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 11, wherein the antibody or antigen-binding fragment thereof is an IgG1 antibody or antigen-binding fragment thereof.

13. The anti-PD-1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, wherein the antigen-binding fragment is a Fab fragment, Fab′ fragment, F(ab′)2 fragment, scFv, dsFv, ds-scFv or nanobody.

14. The anti-PD-1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 13, wherein the antibody has an L234A L235A mutation in the Fc region.

15. The anti-PD-1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 14, wherein the antibody or antigen-binding fragment thereof has a K of less than 0.5 nM, less than 0.3 nM or less than 0.1 nM, less than 0.05 nM, less than 0.02 nM D binds to PD-1.

16. The anti-PD-1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 15, wherein the antibody or antigen-binding fragment has an IC of less than about 500 ng / mL, less than about 400 ng / mL, or less than about 300 ng / mL 50 Inhibit the interaction between PD-1 and PD-L1.

17. The anti-PD-1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 16, wherein the antibody or antigen-binding fragment activates T cells.

18. The anti-PD-1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 17, wherein the antibody or antigen-binding fragment induces IL-2 secretion in T cells.

19. The anti-PD-1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 18, wherein the antibody or antigen-binding fragment thereof induces IFN-γ secretion in T cells.

20. A pharmaceutical composition comprising the anti-PD-1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 19 and a pharmaceutically acceptable carrier.

21. A method of treating cancer in a subject, comprising administering to the subject the anti-PD-1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 19 or the pharmaceutical composition according to claim 20.

22. The method according to claim 21, wherein the cancer is colon cancer, breast cancer, lung cancer, ovarian cancer, prostate cancer, cervical cancer, brain cancer, skin cancer, liver cancer, pancreatic cancer or gastric cancer.

23. The method according to claim 21, wherein the cancer is leukemia or lymphoma.

24. A method of treating an infectious disease in a subject, comprising administering to the subject the anti-PD-1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 16 or the pharmaceutical composition according to claim 20.

25. The method according to claim 24, wherein the infectious disease is tuberculosis, malaria, HIV infection or HBV infection.

26. A method of treating primary immunodeficiency in a subject, comprising administering to the subject the anti-PD-1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 19 or the pharmaceutical composition according to claim 20.

27. The anti-PD-1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 19 or the pharmaceutical composition according to claim 20, for treating cancer.

28. The anti-PD-1 antibody or antigen-binding fragment thereof according to claim 27, wherein the cancer is colon cancer, breast cancer, lung cancer, ovarian cancer, prostate cancer, cervical cancer, brain cancer, skin cancer, liver cancer, pancreatic cancer or gastric cancer.

29. The anti-PD-1 antibody or antigen-binding fragment thereof according to claim 27, wherein the cancer is leukemia or lymphoma.

30. The anti-PD-1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 19 or the pharmaceutical composition according to claim 20, for treating infectious diseases.

31. The anti-PD-1 antibody or antigen-binding fragment thereof according to claim 30, wherein the infectious disease is tuberculosis, malaria, HIV infection or HBV infection.

32. The anti-PD-1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 19 or the pharmaceutical composition according to claim 17, for treating primary immunodeficiency.

33. Use of the anti-PD-1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 19 in the manufacture of a medicament for treating cancer.

34. The use according to claim 33, wherein the cancer is colon cancer, breast cancer, lung cancer, ovarian cancer, prostate cancer, cervical cancer, brain cancer, skin cancer, liver cancer, pancreatic cancer or gastric cancer.

35. The use according to claim 33, wherein the cancer is leukemia or lymphoma.

36. The use of the anti-PD-1 antibody or an antigen-binding fragment thereof according to any one of claims 1 to 19 in the manufacture of a medicament for the treatment of infectious diseases.

37. The use according to claim 36, wherein the infectious disease is tuberculosis, malaria, HIV infection or HBV infection.

38. The use of the anti-PD-1 antibody or an antigen-binding fragment thereof according to any one of claims 1 to 19 in the manufacture of a medicament for the treatment of primary immunodeficiency.

39. A method for inhibiting the interaction between PD-1 and PD-L1 in a cell, which comprises contacting the cell with the antibody or an antigen-binding fragment thereof according to any one of claims 1 to 19.

40. A method for inhibiting the interaction between PD-1 and PD-L2 in a cell, which comprises contacting the cell with the antibody or an antigen-binding fragment thereof according to any one of claims 1 to 19.

41. A method for stimulating the secretion of IL-2 in a cell, which comprises contacting the cell with the antibody or an antigen-binding fragment thereof according to any one of claims 1 to 19.

42. A method for stimulating the secretion of IFN-γ in a cell, which comprises contacting the cell with the antibody or an antigen-binding fragment thereof according to any one of claims 1 to 19.

43. The method according to any one of claims 39 to 42, wherein the cell is a human cell.

44. The method according to any one of claims 39 to 43, wherein the cell is contacted in vitro.

45. The method according to any one of claims 39 to 44, wherein the cell is contacted in vivo.

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