A pd-1 binding protein and uses thereof

Abstract

Provided are a PD-1 binding protein and applications thereof, and uses of the antigen binding protein.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, in particular to a PD-1 binding protein and application thereof. BACKGROUND

[0002] Programmed Death-1 (PD-1) is a type I membrane protein with 288 amino acids, mainly expressed on the surface of activated T cells. PD-1 has two ligands, namely Programmed Death Ligand-1 (PD-L1) and PD-L2. The interaction of PD-1 with PD-L1 and PD-L2 down-regulates the activity of T cells and reduces the secretion of cytokines, playing an immunosuppressive role. PD-1 / PD-L1 pathway inhibitors can block the binding of PD-1 and PD-L1, block the negative regulatory signal, restore the activity of T cells, and play a role in killing tumor cells, thereby inhibiting tumor growth. Therefore, the immune regulation of PD-1 / PD-L1 as a target has important significance for tumor inhibition. At present, the blocking antibody drugs of PD-1 / PD-L1 pathway still face many challenges in clinical practice, such as low effectiveness, drug resistance and side effects, and it is still necessary to continue to develop more effective anti-PD-1 antibodies and humanized antibodies thereof. SUMMARY

[0003] The present application provides an antigen binding protein that binds to PD-1, which exhibits one or more desirable functional properties, such as high affinity binding to PD-L1, the ability to inhibit the binding of PD-1 to PD-1, the ability to enhance T cell activation including proliferation, the ability to secrete IFN-γ and / or IL-2, the ability to stimulate antibody response, and / or the ability to reverse the inhibitory function of immunosuppressive cells such as T regulatory cells. In one embodiment, the present application also provides a scFv-huIgG1 Fc type antibody, which is convenient to use in combination with cell drugs in an autocrine form. The present application also provides nucleic acid molecules encoding the isolated antigen binding protein, expression vectors, host cells, and methods for preparing the isolated antigen binding protein. The PD-1 binding antigen binding protein disclosed in the present application can be used (alone or in combination with other active agents or treatment forms) to treat, prevent and / or diagnose diseases, such as cancer diseases (for example, solid and soft tissue tumors).

[0004] In one aspect, the present application provides an isolated antigen binding protein comprising a light chain variable region and a heavy chain variable region, the heavy chain variable region comprising a HCDR1 comprising an amino acid sequence set forth in SEQ ID NO: 1; a HCDR2 comprising an amino acid sequence set forth in SEQ ID NO: 2; and a HCDR3 comprising an amino acid sequence set forth in SEQ ID NO: 3; the light chain variable region comprising a LCDR1 comprising an amino acid sequence set forth in SEQ ID NO: 4; a LCDR2 comprising an amino acid sequence set forth in SEQ ID NO: 5 (YAS); and a LCDR3 comprising an amino acid sequence set forth in SEQ ID NO: 6; and the heavy chain variable region of the antigen binding protein comprising at least one FR in an antibody heavy chain variable region VH comprising an amino acid sequence set forth in SEQ ID NO: 67.

[0005] In another preferred embodiment, the heavy chain variable region of the antigen binding protein comprises at least one FR in an antibody heavy chain variable region VH comprising an amino acid sequence set forth in any one of SEQ ID NOs: 54 to 56.

[0006] In another preferred embodiment, the light chain variable region of the antigen binding protein comprises at least one FR in an antibody light chain variable region VL comprising an amino acid sequence set forth in SEQ ID NO: 68.

[0007] In another preferred embodiment, the light chain variable region of the antigen binding protein comprises at least one FR in an antibody light chain variable region VL comprising an amino acid sequence set forth in any one of SEQ ID NOs: 57 to 59.

[0008] In another preferred embodiment, the heavy chain variable region of the antigen binding protein comprises a H-FR1, a H-FR2, a H-FR3, and a H-FR4, the H-FR4 comprising an amino acid sequence set forth in SEQ ID NO: 45.

[0009] In another preferred embodiment, the heavy chain variable region of the antigen binding protein comprises a H-FR1, a H-FR2, a H-FR3, and a H-FR4, the H-FR3 comprising an amino acid sequence set forth in SEQ ID NO: 63.

[0010] In another preferred embodiment, the heavy chain variable region of the antigen binding protein comprises a H-FR1, a H-FR2, a H-FR3, and a H-FR4, the H-FR3 comprising an amino acid sequence set forth in any one of SEQ ID NOs: 42 to 44.

[0011] In another preferred embodiment, the heavy chain variable region of the antigen binding protein comprises a H-FR1, a H-FR2, a H-FR3, and a H-FR4, the H-FR2 comprising the amino acid sequence set forth in SEQ ID NO: 41.

[0012] In another preferred embodiment, the heavy chain variable region of the antigen binding protein comprises a H-FR1, a H-FR2, a H-FR3, and a H-FR4, the H-FR1 comprising the amino acid sequence set forth in SEQ ID NO: 62.

[0013] In another preferred embodiment, the heavy chain variable region of the antigen binding protein comprises a H-FR1, a H-FR2, a H-FR3, and a H-FR4, the H-FR1 comprising the amino acid sequence set forth in any one of SEQ ID NOs: 39-40.

[0014] In another preferred embodiment, the light chain variable region of the antigen binding protein comprises a L-FR1, a L-FR2, a L-FR3, and a L-FR4, the L-FR4 comprising the amino acid sequence set forth in SEQ ID NO: 53.

[0015] In another preferred embodiment, the light chain variable region of the antigen binding protein comprises a L-FR1, a L-FR2, a L-FR3, and a L-FR4, the L-FR3 comprising the amino acid sequence set forth in SEQ ID NO: 66.

[0016] In another preferred embodiment, the light chain variable region of the antigen binding protein comprises a L-FR1, a L-FR2, a L-FR3, and a L-FR4, the L-FR3 comprising the amino acid sequence set forth in any one of SEQ ID NOs: 51-52.

[0017] In another preferred embodiment, the light chain variable region of the antigen binding protein comprises a L-FR1, a L-FR2, a L-FR3, and a L-FR4, the L-FR2 comprising the amino acid sequence set forth in SEQ ID NO: 65.

[0018] In another preferred embodiment, the light chain variable region of the antigen binding protein comprises a L-FR1, a L-FR2, a L-FR3, and a L-FR4, the L-FR2 comprising the amino acid sequence set forth in any one of SEQ ID NOs: 49-50.

[0019] In another preferred embodiment, the light chain variable region of the antigen binding protein comprises a L-FR1, a L-FR2, a L-FR3, and a L-FR4, the L-FR1 comprising the amino acid sequence set forth in SEQ ID NO: 64.

[0020] In another preferred embodiment, the heavy chain variable region of the antigen binding protein comprises the amino acid sequence set forth in SEQ ID NO: 54.

[0021] In another preferred embodiment, the heavy chain variable region of the antigen binding protein comprises the amino acid sequence set forth in SEQ ID NO: 54.

[0022] In another preferred embodiment, the heavy chain variable region of the antigen binding protein comprises the amino acid sequence set forth in SEQ ID NO: 54.

[0023] In another preferred embodiment, the heavy chain variable region of the antigen binding protein comprises the amino acid sequence set forth in SEQ ID NO: 54.

[0024] In another preferred embodiment, the heavy chain variable region of the antigen binding protein comprises the amino acid sequence set forth in SEQ ID NO: 54.

[0025] In another preferred embodiment, the heavy chain variable region of the antigen binding protein comprises the amino acid sequence set forth in SEQ ID NO: 54.

[0026] In another preferred embodiment, the heavy chain variable region of the antigen binding protein comprises the amino acid sequence set forth in SEQ ID NO: 54.

[0027] In another preferred embodiment, the heavy chain variable region of the antigen binding protein comprises the amino acid sequence set forth in SEQ ID NO: 54.

[0028] In another preferred embodiment, the heavy chain variable region of the antigen binding protein comprises the amino acid sequence set forth in SEQ ID NO: 54.

[0029] In another preferred embodiment, the antigen binding protein further comprises an antibody heavy chain constant region.

[0030] In another preferred embodiment, the antigen binding protein comprises an antibody heavy chain constant region derived from a human IgG constant region.

[0031] In another preferred embodiment, the antibody heavy chain constant region of the antigen binding protein comprises the amino acid sequence set forth in SEQ ID NO: 60.

[0032] In another preferred embodiment, the antigen binding protein comprises an antibody light chain constant region.

[0033] In another preferred embodiment, the antibody light chain constant region of the antigen binding protein comprises the amino acid sequence set forth in SEQ ID NO: 61.

[0034] In another preferred embodiment, the antigen binding protein comprises an antibody or an antigen binding fragment thereof.

[0035] In another preferred embodiment, the antibody comprises a Fab, Fab', Fv fragment, F(ab')2, scFv, di-scFv and / or dAb.

[0036] In another preferred embodiment, the antigen binding fragment is selected from the group consisting of a humanized antibody, a chimeric antibody and a fully human antibody.

[0037] In another preferred embodiment, the antigen binding protein has one or more of the properties selected from the group consisting of the ability to bind human PD-1, the ability to block PD-1 and PD-L1 binding, the ability to block PD-1 and PD-L2 binding, the ability to stimulate secretion of IL-2, TNF-alpha and / or IFN-gamma in immune cells, the ability to inhibit tumor growth and / or tumor cell proliferation, and the ability to increase the killing capacity of immune cells.

[0038] In another aspect, the present application provides a polypeptide comprising the isolated antigen binding protein, and optionally a tag sequence to facilitate expression and / or purification.

[0039] In another aspect, the present application provides one or more isolated nucleic acid molecules encoding the isolated antigen binding protein and / or the polypeptide.

[0040] In another aspect, the present application provides a vector comprising the nucleic acid molecule.

[0041] In another aspect, the present application provides a cell comprising the nucleic acid molecule or the vector.

[0042] In another aspect, the present application provides a method of producing the isolated antigen binding protein, the method comprising culturing the cell under conditions such that the isolated antigen binding protein is expressed.

[0043] In another aspect, the present application provides a pharmaceutical composition comprising the isolated antigen binding protein, the nucleic acid molecule, the vector and / or the cell, and optionally a pharmaceutically acceptable adjuvant.

[0044] In another aspect, there is provided an immunoconjugate comprising the isolated antigen binding protein described, and a conjugating moiety selected from the group consisting of a detectable label, a drug, a toxin, a cytokine, a radionuclide, or an enzyme.

[0045] In another aspect, the present application provides use of the isolated antigen binding protein described, the nucleic acid molecule described, the vector described, the cell described, the pharmaceutical composition described, and / or the immunoconjugate described, in the manufacture of a medicament for treating a PD-1 mediated disease or disorder.

[0046] In certain embodiments, the PD-1 mediated disease or disorder comprises a cancer or a tumor.

[0047] In another aspect, the present application provides use of the isolated antigen binding protein described and / or the immunoconjugate described, in the manufacture of a detection reagent, a detection plate, or a detection kit for detecting a PD-1 molecule in a sample.

[0048] In another aspect, the present application provides a method of detecting a PD-1 protein in a sample (including non-diagnostic purposes and diagnostic purposes), the method comprising the steps of:

[0049] (1) contacting the sample with the isolated antigen binding protein described and / or the immunoconjugate described;

[0050] (2) detecting whether an antigen-antibody complex is formed, wherein the formation of the complex indicates the presence of the PD-1 protein in the sample.

[0051] In another aspect, the present application provides a method for affecting the production of a cytokine by a target cell for non-therapeutic and / or diagnostic purposes, the method comprising administering the antigen binding protein described, the polypeptide described, the nucleic acid described, the vector described, and / or a cell expressing the antigen binding protein, the polypeptide, or comprising the nucleic acid and / or the vector.

[0052] In another preferred embodiment, the target cell comprises an immune cell, such as a tumor infiltrating lymphocyte (TIL), a peripheral blood mononuclear cell (PBMC).

[0053] In another preferred embodiment, the cytokine comprises IL-2, TNF-a, and / or IFN-γ.

[0054] In another aspect, the present application provides a method of inhibiting the binding of PD-1 to PD-L1, comprising administering to a subject in need thereof an effective amount of the antigen binding protein described, the nucleic acid molecule described, the vector described, the cell described, and / or the pharmaceutical composition described.

[0055] In another aspect, the present application provides a method of inhibiting the binding of PD-1 to PD-L2, comprising administering to a subject in need thereof an effective amount of the antigen binding protein, the nucleic acid molecule, the vector, the cell and / or the pharmaceutical composition.

[0056] In another aspect, the present application provides a method of preventing, alleviating or treating a PD-1 mediated disease or disorder, comprising administering to a subject in need thereof an effective amount of the antigen binding protein, the nucleic acid molecule, the vector, the cell and / or the pharmaceutical composition.

[0057] In certain embodiments, the PD-1 mediated disease or disorder comprises a cancer or a tumor.

[0058] In another preferred embodiment, the cancer or tumor comprises a solid tumor and a hematological tumor.

[0059] Other aspects and advantages of the present application will become apparent from the following detailed description, taken in conjunction with the accompanying drawings. Only illustrative embodiments of the present application are described below. As will be realized by those skilled in the art, the application is capable of modifications in various obvious aspects, all without departing from the spirit and scope of the inventive aspects of the application. Accordingly, the drawings and descriptions are to be regarded as illustrative in nature and not restrictive. BRIEF DESCRIPTION OF DRAWINGS

[0060] The specific features of the inventive aspects of the present application are shown in the appended claims. The features and advantages of the inventive aspects of the present application can be better understood from the exemplary embodiments described in detail below, in conjunction with the accompanying drawings. Brief description of the drawings is as follows:

[0061] Figure 1 The figure shows the detection curve of the affinity of the four valent human PD-L1 extracellular segment mutant of the present application to PD-1-huIgG1 Fc (based on BLI method);

[0062] Figure 2 The figure shows the inhibition curve of the huIgG1 antibody of 6H6 and pembrolizumab to the binding of PD-1 and PD-L1;

[0063] Figure 3 The figure shows the binding curve of the huIgG1 antibody of 6H6 and pembrolizumab to PD-1.

[0064] Figure 4 The figure shows the result graph of the proportion of CD107a cells measured after adding PD-1 antibody to the tumor infiltrating lymphocyte (TIL) cell culture medium derived from donor A and donor B. The figure shows the result graph of the proportion of CD107a cells measured after adding PD-1 antibody to the tumor infiltrating lymphocyte (TIL) cell culture medium derived from donor A and donor B.

[0065] Figure 5 The graph shows the cytokine secretion results measured after adding PD-1 antibody to the culture medium of tumor-infiltrating lymphocytes (TILs) derived from donors A and B. AC shows the cytokine secretion by TILs derived from donor A; DF shows the cytokine secretion by TILs derived from donor B.

[0066] Figure 6 The image shows the results of a mixed lymphocyte reaction (MLR) assay to detect the stimulation of T lymphocytes by PD-1 antibody.

[0067] Figure 7 The graph shows the enhanced killing effect of PD-1 antibody on TCR-T cells.

[0068] Figure 8 The results show the antigen-binding affinity of the 6H6 antibody.

[0069] Figure 9 The results show the antigen-binding affinity of the 6H6-5 antibody.

[0070] Figure 10 The results show the antigen-binding affinity of the 6H6-25 antibody.

[0071] Figure 11 The results show the antigen-binding affinity of the 6H6-29 antibody.

[0072] Figure 12 The figure shows the half-maximal inhibitory concentration (IC50) of 6H6 and humanized 6H6-5, 6H6-25, and 6H6-29 huIgG1 antibodies against PD-1 and PD-L1 binding. 50 . Detailed Implementation

[0073] The following specific embodiments illustrate the implementation of the invention. Those skilled in the art can easily understand other advantages and effects of the invention from the content disclosed in this specification.

[0074] Terminology Definition

[0075] In the present application, the term "PD-1" generally refers to Programmed Cell Death 1, also known as "Programmed Death 1," "CD279," "Cluster of Differentiation 279," "PD1," "PDCD1." PD-1 is generally expressed on T cells, B cells, natural killer T cells, activated monocytes, and dendritic cells (DCs) and is involved in apoptosis. PD-1 generally contains an extracellular IgV domain, a transmembrane region, and an intracellular domain. PD-1 can bind to two ligands, PD-L1 and PD-L2. The "PD-1" includes any native PD-1 of any vertebrate origin, including mammals such as primates (e.g., humans and cynomolgus monkeys) and rodents (e.g., mice and rats). The term encompasses "full-length," unprocessed PD-1 as well as any form of PD-1 that results from cellular processing. PD-1 can exist as a transmembrane protein or as a soluble protein. "PD-1" includes intact PD-1 as well as fragments thereof, and further includes functional variants, isoforms, species homologs, derivatives, analogs of PD-1, and analogs that have at least one epitope in common with PD-1. The amino acid sequence of human PD1 is shown at UniProt (www.uniprot.org) under accession number Q15116.

[0076] In the present application, the term "PD-L1" generally refers to Programmed Cell Death 1 Ligand 1, also known as B7 Homolog 1, B7-H1, Cluster of Differentiation 274, (3) 274, or CD274, which downregulates T cell activation and cytokine secretion upon binding to PD-1. The "PD-L1" includes any native PD-L1 of any vertebrate origin, including mammals such as primates (e.g., humans and cynomolgus monkeys) and rodents (e.g., mice and rats). The term encompasses "full-length," unprocessed PD-L1 as well as any form of PD-L1 that results from cellular processing. PD-L1 can exist as a transmembrane protein or as a soluble protein. "PD-L1" includes intact PD-L1 as well as fragments thereof, and further includes functional variants, isoforms, species homologs, derivatives, analogs of PD-L1, and analogs that have at least one epitope in common with PD-L1. The basic structure of PD-L1 includes four domains: an extracellular Ig-like V-type domain and Ig-like C2-type domain, a transmembrane domain, and a cytoplasmic domain. The complete hPD-L1 sequence can be found under GenBank Accession No. Q9NZQ7.

[0077] In the present application, the terms "isolated" or "purified" generally refer to a molecule (e.g., an antibody, a nucleic acid, etc.) that is at least partially separated from other molecules normally associated with it in its natural state. An "isolated or purified polypeptide" is substantially free of other biological molecules, such as nucleic acids, proteins, lipids, carbohydrates, cellular debris, and growth media. An "isolated or purified nucleic acid" is at least partially separated from nucleic acids normally flanking the polynucleotide in its natural state. Thus, a polynucleotide that, for example, fuses regulatory or coding sequences that are not normally associated is considered isolated in the present application, even when present in, for example, a host cell chromosome or in solution. Generally, the terms "isolated" and "purified" are not intended to mean the complete absence of such substances or the absence of water, buffers, or salts, unless they are present in amounts that substantially interfere with the experimental or therapeutic use of the molecule. The antigen binding proteins of the present application and nucleic acids encoding the antigen binding proteins of the present application are isolated / purified.

[0078] In the present application, the term "antigen binding protein" is used in its broadest sense and means a protein that comprises a portion that binds to an antigen or target and optionally comprises a framework or framework portion that allows the antigen binding portion to adopt a conformation that facilitates binding of the antigen binding protein to the antigen. Examples of antigen binding proteins include a human antibody, a humanized antibody; a chimeric antibody; a recombinant antibody; a single chain antibody; a single domain antibody (or nanobody); a diabody; a triabody; a tetrabody; a Fab fragment; a F(ab')2 fragment; an IgD antibody; an IgE antibody; an IgM antibody; an IgGl antibody; an IgG2 antibody; an IgG3 antibody; or an IgG4 antibody and fragments thereof. The antigen binding protein can include, for example, an alternative protein framework or an artificial framework with grafted CDRs or CDR derivatives. Such frameworks include, but are not limited to, antibody-derived frameworks comprising mutations introduced, for example, to stabilize the three-dimensional structure of the antigen binding protein; and completely synthetic frameworks comprising, for example, biocompatible polymers. See, for example, Korndorfer et al., 2003, Proteins: Structure, Function, and Bioinformatics, 53(1): 121-129 (2003); Roque et al., Biotechnol. Prog. 20:639-654 (2004). In addition, peptide antibody mimetics ("PAMs") can be used, as can antibody mimetic-based frameworks that utilize a fibronectin component as a framework.

[0079] In the present application, the term "antibody" is used in the broadest sense and specifically covers, but is not limited to, monoclonal antibodies (including full-length monoclonal antibodies comprising two heavy chains and two light chains), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), humanized antibodies, fully human antibodies, chimeric antibodies, heavy chain antibodies, and camelized single domain antibodies (e.g., heavy chain variable domain antibodies). An antibody typically has the structure of an immunoglobulin and can comprise a protein of at least two heavy (HC) chains and two light (LC) chains interconnected by disulfide bonds, or an antigen binding fragment thereof. Each heavy chain comprises a heavy chain variable region (VH) and a heavy chain constant region. The amino acid composition and arrangement of the heavy chain constant region of an immunoglobulin differs, and thus its antigenicity differs. Accordingly, immunoglobulins can be classified into five classes, or isotypes, of immunoglobulins, namely IgM, IgD, IgG, IgA, and IgE, with the corresponding heavy chains being μ, δ, γ, α, and ε, respectively. The same class of Ig can be further divided into subclasses, e.g., IgG can be divided into IgGl, IgG2, IgG3, and IgG4, based on differences in the amino acid composition of the hinge region and the number and location of disulfide bonds in the heavy chain. The light chains can be classified into κ or λ chains by the differences in the constant region. Each of the five classes of Ig can have either κ or λ chains.

[0080] In certain naturally occurring IgG, IgD, and IgA antibodies, the heavy chain constant region comprises three domains, CH1, CH2, and CH3. In certain naturally occurring antibodies, each light chain comprises a light chain variable region (VL) and a light chain constant region. The light chain constant region comprises one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), that are interspersed with regions that are more conserved, termed framework regions (FRs). Each VH and VL comprises three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable domains of the heavy and light chains each comprise four FR regions (HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3, LFR4), mostly from β-sheet forming sequences, connected by three CDRs, which form loops connecting, and in some cases forming part of, the β-sheet structure. The CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs from the other chain, contribute to the formation of the antigen binding site of antibodies. The constant regions of the antibodies can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

[0081] In the present application, the term "variable" generally refers to the fact that certain portions of the variable domain sequence of an antibody vary widely in intensity, it forms the binding and specificity of various specific antibodies to their specific antigens. However, variability is not evenly distributed throughout the variable region of an antibody. It is concentrated in three segments of the light chain and heavy chain variable regions, known as complementarity determining regions (CDRs) or hypervariable regions (HVRs). The more highly conserved portions of the variable domains are called the framework (FR). The variable domains of the naturally occurring heavy and light chains each comprise four FR regions, mostly adopting a beta-sheet configuration, connected by three CDRs, forming loops connecting, and in some cases, forming part of the beta-sheet structure. The CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs from the other chain, form the antigen binding site of the antibody, the constant region is not directly involved in the binding of the antibody to the antigen, but they exhibit different effector functions, such as participating in antibody-dependent cellular cytotoxicity. In the art, the CDRs of an antibody can be defined by various methods, such as the Kabat definition rule based on sequence variability (see, Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, Bethesda, MD (1991). In the present application, the amino acid residues in the variable domain sequence and the full-length antibody sequence are determined using the Kabat definition rule (see Table 1).

[0082] Table 1 CDR definition of the antibodies of the present application based on Kabat definition rule

[0083] Kabat Residues LCDR1 L24-L34 LCDR2 L50-L56 LCDR3 L89-L97 HCDR1 H31-H35 HCDR2 H50-H66 HCDR3 H99-H107

[0084] Wherein, Laa-Lbb can refer to the amino acid sequence from the N-terminal of the light chain of the antibody aa to bb; Haa-Hbb can refer to the amino acid sequence from the N-terminal of the heavy chain of the antibody aa to bb. For example, L24-L34 can refer to the amino acid sequence from the N-terminal of the light chain of the antibody aa to bb; H231-H35 can refer to the amino acid sequence from the N-terminal of the heavy chain of the antibody aa to bb.

[0085] In the present application, the term "Fab" refers to an antigen-binding fragment of an antibody. As described above, papain digestion of an intact antibody produces two identical antigen-binding fragments, i.e. "Fab" fragments, and a residual "Fc" fragment (i.e. Fc region, supra). The Fab fragment consists of one complete L chain and the variable region of one heavy chain and the first constant region (CH1) of that H chain (VH-CH1). H ) of the H chain. H 1).

[0086] In the present application, the term "Fab' fragment" refers to a monovalent antigen-binding fragment of a human monoclonal antibody that is slightly larger than a Fab fragment. For example, a Fab' fragment includes all light chains, all heavy chain variable regions, and all or part of the first and second constant regions of the heavy chain. For example, a Fab' fragment can also include part or all of the 220-330 amino acid residues of the heavy chain.

[0087] In the present application, the term "F(ab')2" refers to an antibody fragment produced by pepsin digestion of an intact antibody. An F(ab')2 fragment contains two Fab fragments and part of the hinge region held together by disulfide bonds. An F(ab')2 fragment has bivalent antigen-binding activity and is capable of cross-linking antigens.

[0088] In the present application, the term "Fv fragment" refers to a monovalent antigen-binding fragment of a human monoclonal antibody, including all or part of the heavy chain variable region and the light chain variable region, and lacking the heavy chain constant region and the light chain constant region. The heavy chain variable region and the light chain variable region include, for example, CDRs. For example, an Fv fragment includes all or part of the approximately 110 amino acid amino-terminal variable regions of the heavy and light chains.

[0089] In the present application, the term "scFv" generally refers to a fusion protein comprising at least one antibody fragment including a variable region of a light chain and at least one antibody fragment including a variable region of a heavy chain, wherein the light and heavy chain variable regions are contiguous (e.g., via a synthetic linker such as a short flexible polypeptide linker) and capable of being expressed as a single chain polypeptide, and wherein the scFv retains the specificity of the intact antibody from which it is derived. Unless otherwise specified, as used in the present application, the scFv can have the VL and VH variable regions in any order (e.g., with respect to the N-terminus and C-terminus of the polypeptide), the scFv can include VL-linker-VH or can include VH-linker-VL.

[0090] In the present application, the term "dAb" generally refers to an antigen-binding fragment having a VH domain, a VL domain, or having a VH domain or a VL domain, with reference to, for example, Ward et al. (Nature, 1989 Oct 12; 341(6242): 544-6), with reference to Holt et al., Trends Biotechnol., 2003, 21(11): 484-490; and with reference to, for example, WO 06 / 030220, WO 06 / 003388, and other published patent applications of Domantis Ltd.

[0091] In the present application, the term "monoclonal antibody" generally refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that can be present. Monoclonal antibodies typically display high specificity for a single antigenic site. Also, in contrast to conventional polyclonal antibody preparations, which typically include different antibodies directed against different determinants, each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they can be synthesized in large amounts through cell culture, uncontaminated by other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as being obtained from a population of substantially homogeneous antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present application can be made by the hybridoma method, or can be prepared / obtained by recombinant DNA methods.

[0092] In the present application, the term "chimeric antibody" generally refers to an antibody in which the variable region is derived from one species and the constant region is derived from another species. Typically, the variable region is derived from an antibody of an experimental animal such as a rodent ("parental antibody"), and the constant region is derived from a human antibody, such that the resulting chimeric antibody has a reduced likelihood of eliciting an adverse immune response in a human individual as compared to the parental (e.g., mouse-derived) antibody.

[0093] In the present application, the term "humanized antibody" generally refers to an antibody in which some or all of the amino acids outside the CDR regions of a non-human antibody (e.g., a mouse antibody) are replaced with corresponding amino acids from a human immunoglobulin. Small additions, deletions, insertions, substitutions or modifications of amino acids in the CDR regions can also be allowed, as long as they do not alter the ability of the antibody to bind to a particular antigen. A humanized antibody can optionally contain at least a portion of a human immunoglobulin constant region. A "humanized antibody" retains antigenic specificity similar to that of the original antibody. "Humanized" forms of non-human (e.g., murine) antibodies can minimally contain a chimeric antibody having sequences derived from non-human immunoglobulin. In some cases, CDR region residues in a human immunoglobulin (recipient antibody) are replaced by CDR region residues of a non-human species (donor antibody) such as mouse, rat, rabbit, or non-human primate that have the desired properties, affinities, and / or capabilities. In some cases, FR region residues of a human immunoglobulin are replaced by corresponding non-human residues. Additionally, a humanized antibody can contain amino acid modifications not found in the recipient antibody or in the donor antibody. These modifications can be made to further improve antibody performance, such as binding affinity.

[0094] In the present application, the term "reference antibody" generally refers to any antibody that can bind to an antigen (e.g., PD-1). In some cases, an antigen binding protein described herein can compete with a reference antibody for binding to an antigen (e.g., PD-1).

[0095] In the present application, the term "isolated nucleic acid molecule" or "isolated polynucleotide" generally refers to a genomic, mRNA, cDNA or synthetically derived DNA or RNA or a certain combination thereof, which is not associated with all or a portion of a polynucleotide as it occurs in nature, or linked to a polynucleotide to which it is not linked in nature.

[0096] In the present application, the term "vector" generally refers to a nucleic acid molecule capable of self-replication in a suitable host, which transfers an inserted nucleic acid molecule into and / or between host cells. The vector can include a vector mainly for inserting DNA or RNA into a cell, a vector mainly for replicating DNA or RNA, and a vector mainly for expression of transcription and / or translation of DNA or RNA. The vector also includes a vector having a plurality of the above-mentioned functions. The vector can be a polynucleotide that can be transcribed and translated into a polypeptide when introduced into a suitable host cell. Generally, the vector can produce a desired expression product by culturing a suitable host cell containing the vector.

[0097] In the present application, the term "cell" generally refers to an individual cell, cell line or cell culture that can or has been made to contain a plasmid or vector including a nucleic acid molecule described in the present application, or is capable of expressing an antibody or antigen-binding fragment thereof described in the present application. The cell can include progeny of a single host cell. The progeny cell can not necessarily be identical to the original parent cell in morphology or in genomic or total cellular DNA complement as a result of natural, accidental, or deliberate mutation, but is capable of expressing an antibody or antigen-binding fragment thereof described in the present application. The cell can be obtained by transfecting a cell in vitro using a vector described in the present application. The cell can be a prokaryotic cell (e.g., E. coli), or a eukaryotic cell (e.g., a yeast cell, e.g., a COS cell, a Chinese hamster ovary (CHO) cell, a HeLa cell, a HEK293 cell, a COS-1 cell, an NSO cell, or a myeloma cell). In some cases, the cell can be a mammalian cell. For example, the mammalian cell can be a 293T cell. In the present application, the term "recombinant cell" generally refers to a cell into which a recombinant expression vector has been introduced. The recombinant host cell includes not only a certain particular cell, but also progeny of the cell.

[0098] In the present application, the term "pharmaceutically acceptable adjuvant" generally includes a pharmaceutically acceptable carrier, excipient, or stabilizer, which is non-toxic to the cells or mammals exposed thereto at the dosages and concentrations employed. Typically, a physiologically acceptable carrier is an aqueous pH buffered solution. Examples of physiologically acceptable carriers can include buffers, antioxidants, low molecular weight (less than about 10 residues) polypeptides, proteins, hydrophilic polymers, amino acids, monosaccharides, disaccharides and other carbohydrates, chelating agents, sugar alcohols, salt-forming counterions, such as sodium; and / or nonionic surfactants.

[0099] In the present application, the protein, polypeptide and / or amino acid sequence referred to should also be understood to encompass at least the range of variants or homologues of the said protein or polypeptide having the same or similar function.

[0100] In the present application, the variant can be a protein or polypeptide having one or more amino acids substituted, deleted or added in the amino acid sequence of the protein and / or the polypeptide (e.g., an antibody or fragment thereof specifically binding to a PD-1 protein). For example, the functional variant can comprise a protein or polypeptide having an amino acid alteration by at least 1, for example, 1-30, 1-20 or 1-10, and for example, 1, 2, 3, 4 or 5 amino acid substitutions, deletions and / or insertions. The functional variant can substantially maintain the biological properties of the protein or the polypeptide before the alteration (e.g., substitution, deletion or addition). For example, the functional variant can maintain at least 60%, 70%, 80%, 90%, or 100% of the biological activity (e.g., antigen binding ability) of the protein or the polypeptide before the alteration. For example, the substitution can be a conservative substitution.

[0101] In the present application, the homologue can be a protein or polypeptide having at least about 85% (e.g., having at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more) sequence homology to the amino acid sequence of the protein and / or the polypeptide (e.g., an antibody or fragment thereof specifically binding to a PD-1 protein).

[0102] In the present application, the homology generally refers to similarity, analogy or correlation between two or more sequences. The "percent sequence homology" can be calculated by comparing two sequences to be aligned in a comparison window, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, I) or identical amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, lie, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gin, Cys and Met) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window (i.e., the window size), and multiplying the result by 100 to yield the percent sequence homology. The alignment for determining percent sequence homology can be achieved in various ways known in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length sequence or a desired region of the sequence under comparison. The homology can also be determined by FASTA and BLAST. The description of the FASTA algorithm can be found in W. R. Pearson and D. J. Lipman, "Improved tools for biological sequence comparison", Proc. Natl. Acad. Sci., 85:2444-2448, 1988; and D. J. Lipman and W. R. Pearson, "Fast

[0103] In the present application, the term "optional" or "optionally" means that the event or circumstance subsequently described can occur, but does not have to occur.

[0104] In the present application, the term "comprising" generally means the inclusive meaning of the term "comprising", "containing", "including" or "encompassing". In some cases, it also means the meaning of "consisting of".

[0105] In the present application, the terms "about" and "approximately" shall generally mean an acceptable degree of error for the quantity measured considering the nature of the measurement or the precision of the instrument used to make the measurement. An exemplary acceptable degree of error is within 20 percent (%), typically within 10%, and more typically within 5% of a given value or range of values.

[0106] In the present application, the term "therapeutically effective amount" refers to the amount of an antibody that when administered to a human or animal elicits a response sufficient to produce a therapeutic effect in the human or animal. The effective amount is readily determined by one of ordinary skill in the art following conventional methods. DETAILED DESCRIPTION

[0108] Antigen binding protein

[0109] In one aspect, the present application provides an antigen binding protein, which can comprise at least one CDR in an antibody heavy chain variable region VH, which comprises an amino acid sequence set forth in SEQ ID NO: 7 or 9.

[0110] The antigen binding protein described in the present application includes an antibody or an antigen binding fragment thereof. The antibody described in the present application can be a monoclonal antibody, a chimeric antibody, a humanized antibody and / or a fully human antibody. The antigen binding fragment of the antibody described in the present application can be a Fab, Fab', Fv fragment, F(ab')2, scFv, di-scFv and / or dAb.

[0111] The antigen binding protein described in the present application can compete with a reference antibody for binding to PD-1. The reference antibody can comprise a light chain variable region and a heavy chain variable region. For example, the light chain variable region of the reference antibody comprises a LCDR1 comprising an amino acid sequence set forth in SEQ ID NO: 4, a LCDR2 comprising an amino acid sequence set forth in SEQ ID NO: 5 (YAS), and a LCDR3 comprising an amino acid sequence set forth in SEQ ID NO: 6; and the heavy chain variable region of the reference antibody comprises a HCDR1 comprising an amino acid sequence set forth in SEQ ID NO: 1, a HCDR2 comprising an amino acid sequence set forth in SEQ ID NO: 2, and a HCDR3 comprising an amino acid sequence set forth in SEQ ID NO: 3.

[0112] The antigen binding protein described in the present application can comprise heavy chain complementarity determining regions HCDR1, HCDR2 and HCDR3.

[0113] In the present application, the HCDR3 of the antigen binding protein can comprise an amino acid sequence set forth in SEQ ID NO: 3.

[0114] In the present application, the HCDR2 of the antigen binding protein can comprise the amino acid sequence set forth in SEQ ID NO: 2.

[0115] In the present application, the HCDR1 of the antigen binding protein can comprise the amino acid sequence set forth in SEQ ID NO: 1.

[0116] In the present application, the HCDR1, HCDR2 and HCDR3 of the antigen binding protein can comprise, in order, the amino acid sequences set forth in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively.

[0117] The antigen binding protein described in the present application can further comprise heavy chain framework regions HFR1, HFR2, HFR3 and HFR4.

[0118] In the present application, the HFR1 of the antigen binding protein can comprise the amino acid sequence set forth in SEQ ID NO: 11 or SEQ ID NO: 19, and the C-terminus of the HFR1 is directly or indirectly connected to the N-terminus of the HCDR1.

[0119] In the present application, the HFR2 of the antigen binding protein can comprise the amino acid sequence set forth in SEQ ID NO: 12 or SEQ ID NO: 20, and the HFR2 is located between the HCDR1 and the HCDR2.

[0120] In the present application, the HFR3 of the antigen binding protein can comprise the amino acid sequence set forth in SEQ ID NO: 13 or SEQ ID NO: 21, and the HFR3 is located between the HCDR2 and the HCDR3.

[0121] In the present application, the HFR4 of the antigen binding protein can comprise the amino acid sequence set forth in SEQ ID NO: 14 or SEQ ID NO: 22, and the N-terminus of the HFR4 is connected to the C-terminus of the HCDR3.

[0122] In the present application, the antigen binding protein can comprise an antibody heavy chain variable region (VH), and the VH can comprise the amino acid sequence set forth in SEQ ID NO: 7 or 9.

[0123] In some embodiments, the HFR1, HFR2, HFR3 and HFR4 of the antigen binding protein can comprise, in order, the amino acid sequences set forth in SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14, respectively.

[0124] For example, the HCDR1, HCDR2, and HCDR3 of the antigen binding protein can comprise, in order, the amino acid sequences set forth in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively, and the HFR1, HFR2, HFR3, and HFR4 of the antigen binding protein can comprise, in order, the amino acid sequences set forth in SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14, respectively.

[0125] In some embodiments, the HFR1, HFR2, HFR3, and HFR4 of the antigen binding protein can comprise, in order, the amino acid sequences set forth in SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22, respectively.

[0126] For example, the HCDR1, HCDR2, and HCDR3 of the antigen binding protein can comprise, in order, the amino acid sequences set forth in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively, and the HFR1, HFR2, HFR3, and HFR4 of the antigen binding protein can comprise, in order, the amino acid sequences set forth in SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22, respectively.

[0127] The antigen binding proteins described herein can comprise a heavy chain constant region.

[0128] In the present application, the heavy chain constant region can comprise a constant region of human IgG. In some cases, the heavy chain constant region can include a human IgG4 constant region and / or a heavy chain constant region of human IgG1.

[0129] In some embodiments, the heavy chain constant region can comprise the amino acid sequence set forth in any one of SEQ ID NO: 27 or SEQ ID NO: 28.

[0130] For example, the HCDR1, HCDR2, and HCDR3 of the antigen binding protein can comprise, in order, the amino acid sequences set forth in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively, and the heavy chain constant region of the antigen binding protein can comprise the amino acid sequence set forth in any one of SEQ ID NO: 27 or SEQ ID NO: 28.

[0131] For example, the HCDR1, HCDR2, and HCDR3 of the antigen binding protein can comprise, in order, the amino acid sequences set forth in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively, and the HFR1, HFR2, HFR3, and HFR4 of the antigen binding protein can comprise, in order, the amino acid sequences set forth in SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14, respectively, and the heavy chain constant region of the antigen binding protein can comprise the amino acid sequence set forth in any one of SEQ ID NO: 27 or SEQ ID NO: 28.

[0132] For example, the HCDR1, HCDR2, and HCDR3 of the antigen binding protein can comprise, in order, the amino acid sequences set forth in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively, and the HFR1, HFR2, HFR3, and HFR4 of the antigen binding protein can comprise, in order, the amino acid sequences set forth in SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22, respectively, and the heavy chain constant region of the antigen binding protein can comprise the amino acid sequence set forth in any one of SEQ ID NO: 27 or SEQ ID NO: 28.

[0133] The antigen binding protein described in the present application can comprise at least one CDR in an antibody light chain variable region VL comprising the amino acid sequence set forth in SEQ ID NO: 8 or 10.

[0134] In the present application, the HCDR1, HCDR2, and HCDR3 of the antigen binding protein can comprise, in order, the amino acid sequences set forth in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively.

[0135] In some embodiments, the HCDR1, HCDR2, and HCDR3 of the antigen binding protein can comprise, in order, the amino acid sequences set forth in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively, and the antigen binding protein can comprise at least one CDR in an antibody light chain variable region VL comprising the amino acid sequence set forth in SEQ ID NO: 8.

[0136] In some embodiments, the HCDR1, HCDR2, and HCDR3 of the antigen binding protein can comprise, in order, the amino acid sequences set forth in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively, and the antigen binding protein can comprise at least one CDR in the antibody light chain variable region VL, which comprises the amino acid sequence set forth in SEQ ID NO: 16.

[0137] The antigen binding protein described herein can comprise light chain complementarity determining regions LCDR1, LCDR2, and LCDR3.

[0138] In this application, the LCDR1 of the antigen binding protein comprises the amino acid sequence set forth in SEQ ID NO: 4.

[0139] In this application, the LCDR2 of the antigen binding protein comprises the amino acid sequence set forth in SEQ ID NO: 5 (YAS).

[0140] In this application, the LCDR3 of the antigen binding protein comprises the amino acid sequence set forth in SEQ ID NO: 6.

[0141] In this application, the LCDR1, LCDR2, and LCDR3 of the antigen binding protein can comprise, in order, the amino acid sequences set forth in SEQ ID NO: 4, SEQ ID NO: 5 (YAS), and SEQ ID NO: 6, respectively.

[0142] In some embodiments, the antigen binding protein can comprise HCDR1, HCDR2, HCDR3, and LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, HCDR3, and LCDR1, LCDR2, and LCDR3 can comprise, in order, the amino acid sequences set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 (YAS), and SEQ ID NO: 6, respectively.

[0143] In some embodiments, the LCDR1, LCDR2, and LCDR3 of the antigen binding protein can comprise, in order, the amino acid sequences set forth in SEQ ID NO: 4, SEQ ID NO: 5 (YAS), and SEQ ID NO: 6, respectively, and the VH of the antigen binding protein can comprise the amino acid sequence set forth in SEQ ID NO: 7.

[0144] In some embodiments, the LCDR1, LCDR2 and LCDR3 of the antigen binding protein can comprise, in order, the amino acid sequences set forth in SEQ ID NO: 4, SEQ ID NO: 5 (YAS), and SEQ ID NO: 6, respectively, and the VH of the antigen binding protein can comprise the amino acid sequence set forth in SEQ ID NO: 9.

[0145] In some embodiments, the LCDR1, LCDR2 and LCDR3 of the antigen binding protein can comprise, in order, the amino acid sequences set forth in SEQ ID NO: 4, SEQ ID NO: 5 (YAS), and SEQ ID NO: 6, respectively, and the VH of the antigen binding protein can comprise the amino acid sequence set forth in SEQ ID NO: 7, and the antigen binding protein can comprise a heavy chain constant region, and the heavy chain constant region can comprise the amino acid sequence set forth in any one of SEQ ID NO: 27 or SEQ ID NO: 28.

[0146] In some embodiments, the LCDR1, LCDR2 and LCDR3 of the antigen binding protein can comprise, in order, the amino acid sequences set forth in SEQ ID NO: 4, SEQ ID NO: 5 (YAS), and SEQ ID NO: 6, respectively, and the VH of the antigen binding protein can comprise the amino acid sequence set forth in SEQ ID NO: 9, and the antigen binding protein can comprise a heavy chain constant region, and the heavy chain constant region can comprise the amino acid sequence set forth in any one of SEQ ID NO: 27 or SEQ ID NO: 28.

[0147] The antigen binding protein described herein can comprise light chain framework regions LFR1, LFR2, LFR3 and LFR4.

[0148] In this application, the LFR1 of the antigen binding protein can comprise the amino acid sequence set forth in SEQ ID NO: 15 or SEQ ID NO: 23, and the C-terminus of the LFR1 is directly or indirectly connected to the N-terminus of the LCDR1.

[0149] In this application, the LFR2 of the antigen binding protein can comprise the amino acid sequence set forth in SEQ ID NO: 16 or SEQ ID NO: 24, and the LFR2 is located between the LCDR1 and the LCDR2.

[0150] In this application, the LFR3 of the antigen binding protein can comprise the amino acid sequence set forth in SEQ ID NO: 17 or SEQ ID NO: 25, and the LFR3 is located between the LCDR2 and the LCDR3.

[0151] In the present application, the LFR4 of the antigen binding protein can comprise the amino acid sequence set forth in SEQ ID NO: 18 or SEQ ID NO: 26, and the N-terminus of the LFR4 is connected to the C-terminus of the LCDR3.

[0152] In the present application, the antigen binding protein can comprise a VL, and the VL can comprise the amino acid sequence set forth in SEQ ID NO: 8 or 10.

[0153] In some embodiments, the LFR1, LFR2, LFR3 and LFR4 of the antigen binding protein can comprise, in sequence, the amino acid sequences set forth in SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18.

[0154] For example, the LCDR1, LCDR2 and LCDR3 of the antigen binding protein can comprise, in sequence, the amino acid sequences set forth in SEQ ID NO: 4, SEQ ID NO: 5 (YAS) and SEQ ID NO: 6, respectively, and the LFR1, LFR2, LFR3 and LFR4 of the antigen binding protein can comprise, in sequence, the amino acid sequences set forth in SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18.

[0155] For example, the HCDR1, HCDR2 and HCDR3 of the antigen binding protein can comprise, in sequence, the amino acid sequences set forth in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively, the LCDR1, LCDR2 and LCDR3 of the antigen binding protein can comprise, in sequence, the amino acid sequences set forth in SEQ ID NO: 4, SEQ ID NO: 5 (YAS) and SEQ ID NO: 6, respectively, and the LFR1, LFR2, LFR3 and LFR4 of the antigen binding protein can comprise, in sequence, the amino acid sequences set forth in SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18.

[0156] In some embodiments, the LFR1, LFR2, LFR3 and LFR4 of the antigen binding protein can comprise, in sequence, the amino acid sequences set forth in SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25 and SEQ ID NO: 26.

[0157] For example, the LCDR1, LCDR2, and LCDR3 of the antigen binding protein can comprise, in order, the amino acid sequences set forth in SEQ ID NO:4, SEQ ID NO:5 (YAS), and SEQ ID NO:6, respectively, and the LFR1, LFR2, LFR3, and LFR4 of the antigen binding protein can comprise, in order, the amino acid sequences set forth in SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, and SEQ ID NO:26, respectively.

[0158] For example, the HCDR1, HCDR2, and HCDR3 of the antigen binding protein can comprise, in order, the amino acid sequences set forth in SEQ ID NO: 1, SEQ ID NO:2, and SEQ ID NO:3, respectively, and the LCDR1, LCDR2, and LCDR3 of the antigen binding protein can comprise, in order, the amino acid sequences set forth in SEQ ID NO:4, SEQ ID NO:5 (YAS), and SEQ ID NO:6, respectively, and the LFR1, LFR2, LFR3, and LFR4 of the antigen binding protein can comprise, in order, the amino acid sequences set forth in SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, and SEQ ID NO:26, respectively.

[0159] In the present application, the antigen binding protein can comprise a VH and a VL, the VH can comprise the amino acid sequence set forth in SEQ ID NO:7 or 15, and the VL can comprise the amino acid sequence set forth in SEQ ID NO:8 or 10.

[0160] In some embodiments, the VH can comprise the amino acid sequence set forth in SEQ ID NO:7, and the VL can comprise the amino acid sequence set forth in SEQ ID NO:8.

[0161] For example, the HCDR1, HCDR2, and HCDR3 of the antigen binding protein can comprise, in order, the amino acid sequences set forth in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively, and the LCDR1, LCDR2, and LCDR3 of the antigen binding protein can comprise, in order, the amino acid sequences set forth in SEQ ID NO: 4, SEQ ID NO: 5 (YAS), and SEQ ID NO: 6, respectively, and the HFR1, HFR2, HFR3, and HFR4 of the antigen binding protein can comprise, in order, the amino acid sequences set forth in SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14, respectively, and the LFR1, LFR2, LFR3, and LFR4 of the antigen binding protein can comprise, in order, the amino acid sequences set forth in SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18, respectively.

[0162] In some embodiments, the VH can comprise the amino acid sequence set forth in SEQ ID NO: 9, and the VL can comprise the amino acid sequence set forth in SEQ ID NO: 10.

[0163] For example, the HCDR1, HCDR2, and HCDR3 of the antigen binding protein can comprise, in order, the amino acid sequences set forth in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively, and the LCDR1, LCDR2, and LCDR3 of the antigen binding protein can comprise, in order, the amino acid sequences set forth in SEQ ID NO: 4, SEQ ID NO: 5 (YAS), and SEQ ID NO: 6, respectively, and the HFR1, HFR2, HFR3, and HFR4 of the antigen binding protein can comprise, in order, the amino acid sequences set forth in SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22, respectively. The LFR1, LFR2, LFR3, and LFR4 of the antigen binding protein can comprise, in order, the amino acid sequences set forth in SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, and SEQ ID NO: 26, respectively.

[0164] In the present application, the antigen binding protein can comprise a heavy chain constant region, and the heavy chain constant region can comprise the amino acid sequence set forth in any one of SEQ ID NO: 27 or SEQ ID NO: 28.

[0165] In some embodiments, the VH can comprise the amino acid sequence set forth in SEQ ID NO: 7, and the VL can comprise the amino acid sequence set forth in SEQ ID NO: 8, and the heavy chain constant region of the antigen binding protein can comprise the amino acid sequence set forth in any one of SEQ ID NO: 27 or SEQ ID NO: 28.

[0166] For example, the HCDR1, HCDR2, and HCDR3 of the antigen binding protein can comprise, in order, the amino acid sequences set forth in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively, and the LCDR1, LCDR2, and LCDR3 of the antigen binding protein can comprise, in order, the amino acid sequences set forth in SEQ ID NO: 4, SEQ ID NO: 5 (YAS), and SEQ ID NO: 6, respectively, and the HFR1, HFR2, HFR3, and HFR4 of the antigen binding protein can comprise, in order, the amino acid sequences set forth in SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14, respectively, and the LFR1, LFR2, LFR3, and LFR4 of the antigen binding protein can comprise, in order, the amino acid sequences set forth in SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18, respectively, and the VH can comprise the amino acid sequence set forth in SEQ ID NO: 7, and the VL can comprise the amino acid sequence set forth in SEQ ID NO: 8, and the heavy chain constant region of the antigen binding protein can comprise the amino acid sequence set forth in any one of SEQ ID NO: 27 or SEQ ID NO: 28.

[0167] In some embodiments, the VH can comprise the amino acid sequence set forth in SEQ ID NO: 9, and the VL can comprise the amino acid sequence set forth in SEQ ID NO: 10.

[0168] For example, the HCDR1, HCDR2, and HCDR3 of the antigen binding protein can comprise, in order, the amino acid sequences set forth in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively, and the LCDR1, LCDR2, and LCDR3 of the antigen binding protein can comprise, in order, the amino acid sequences set forth in SEQ ID NO: 4, SEQ ID NO: 5 (YAS), and SEQ ID NO: 6, respectively, and the HFR1, HFR2, HFR3, and HFR4 of the antigen binding protein can comprise, in order, the amino acid sequences set forth in SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22, respectively, and the LFR1, LFR2, LFR3, and LFR4 of the antigen binding protein can comprise, in order, the amino acid sequences set forth in SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, and SEQ ID NO: 26, respectively, and the VH can comprise the amino acid sequence set forth in SEQ ID NO: 15, and the VL can comprise the amino acid sequence set forth in SEQ ID NO: 16, and the heavy chain constant region of the antigen binding protein can comprise the amino acid sequence set forth in any one of SEQ ID NO: 27 or SEQ ID NO: 28.

[0169] In another aspect, the application provides an isolated antigen binding protein comprising a light chain variable region and a heavy chain variable region, the heavy chain variable region comprising a HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1; a HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 2; and a HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 3; the light chain variable region comprising a LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 4; a LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 5 (YAS); and a LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 6.

[0170] For example, the heavy chain variable region of an antigen binding protein of the application comprises at least one FR in an antibody heavy chain variable region VH comprising the amino acid sequence set forth in any one of SEQ ID NOs: 54-56.

[0171] For example, the antigen binding protein of the present application comprises at least one FR in the antibody heavy chain variable region VH set forth in SEQ ID NO: 67. For example, the antigen binding protein of the present application comprises at least one FR in the antibody heavy chain variable region VH set forth in any one of SEQ ID NOs: 54 to 56. For example, the antigen binding protein of the present application comprises 1, 2, 3, or 4 FRs in the above-mentioned VH. For example, the antigen binding protein of the present application comprises 4 H-FRs in the above-mentioned VH. For example, the antigen binding protein of the present application comprises H-FR1 to 4 in the above-mentioned same or different VH.

[0172] Further, the light chain variable region of the antigen binding protein of the present application comprises at least one FR in the antibody light chain variable region VL comprising the amino acid sequence set forth in SEQ ID NO: 68. For example, the light chain variable region of the antigen binding protein of the present application comprises at least one FR in the antibody light chain variable region VL comprising the amino acid sequence set forth in any one of SEQ ID NOs: 57 to 59.

[0173] For example, the antigen binding protein of the present application comprises at least one FR in the antibody light chain variable region VL set forth in SEQ ID NO: 68. For example, the antigen binding protein of the present application comprises at least one FR in the antibody light chain variable region VL set forth in any one of SEQ ID NOs: 57 to 59. For example, the antigen binding protein of the present application comprises 1, 2, 3, or 4 FRs in the above-mentioned VL. For example, the antigen binding protein of the present application comprises 4 L-FRs in the above-mentioned VL. For example, the antigen binding protein of the present application comprises L-FR1 to 4 in the above-mentioned same or different VL.

[0174] For example, the antigen binding protein of the present application comprises at least one FR in the antibody heavy chain variable region VH set forth in SEQ ID NO: 67, and comprises at least one FR in the antibody light chain variable region VL set forth in SEQ ID NO: 68. For example, the antigen binding protein of the present application comprises at least one FR in the antibody heavy chain variable region VH set forth in any one of SEQ ID NOs: 54 to 56, and comprises at least one FR in the antibody light chain variable region VL set forth in any one of SEQ ID NOs: 57 to 59.

[0175] For example, the heavy chain variable region of the antigen binding protein of the present application can comprise H-FR1, H-FR2, H-FR3, and H-FR4, and the H-FR4 can comprise the amino acid sequence set forth in SEQ ID NO: 45.

[0176] For example, the H-FR3 of the present application can comprise the amino acid sequence set forth in SEQ ID NO: 63.

[0177] For example, the H-FR3 of the present application can comprise the amino acid sequence set forth in any one of SEQ ID NOs: 42-44.

[0178] For example, the H-FR2 of the present application can comprise the amino acid sequence set forth in SEQ ID NO: 41.

[0179] For example, the H-FR1 of the present application can comprise the amino acid sequence set forth in SEQ ID NO: 62.

[0180] For example, the H-FR1 of the present application can comprise the amino acid sequence set forth in any one of SEQ ID NOs: 39-40.

[0181] For example, the heavy chain variable region of the antigen binding protein of the present application can comprise H-FR1, H-FR2, H-FR3, and H-FR4, the H-FR4 can comprise the amino acid sequence set forth in SEQ ID NO: 45, the H-FR3 can comprise the amino acid sequence set forth in SEQ ID NO: 63, the H-FR2 can comprise the amino acid sequence set forth in SEQ ID NO: 41, and the H-FR1 can comprise the amino acid sequence set forth in SEQ ID NO: 62.

[0182] For example, the heavy chain variable region of the antigen binding protein of the present application can comprise H-FR1, H-FR2, H-FR3, and H-FR4, the H-FR4 can comprise the amino acid sequence set forth in SEQ ID NO: 45, the H-FR3 can comprise the amino acid sequence set forth in any one of SEQ ID NOs: 42-44, the H-FR2 can comprise the amino acid sequence set forth in SEQ ID NO: 41, and the H-FR1 can comprise the amino acid sequence set forth in any one of SEQ ID NOs: 39-40.

[0183] For example, the heavy chain variable region of the antigen binding protein of the present application can comprise H-FR1, H-FR2, H-FR3, and H-FR4, the H-FR4 can comprise the amino acid sequence set forth in SEQ ID NO: 45, the H-FR3 can comprise the amino acid sequence set forth in SEQ ID NO: 42, the H-FR2 can comprise the amino acid sequence set forth in SEQ ID NO: 41, and the H-FR1 can comprise the amino acid sequence set forth in SEQ ID NO: 39.

[0184] For example, the heavy chain variable region of an antigen binding protein of the present application can comprise a H-FR1, a H-FR2, a H-FR3, and a H-FR4, the H-FR4 can comprise the amino acid sequence set forth in SEQ ID NO: 45, the H-FR3 can comprise the amino acid sequence set forth in SEQ ID NO: 43, the H-FR2 can comprise the amino acid sequence set forth in SEQ ID NO: 41, and the H-FR1 can comprise the amino acid sequence set forth in SEQ ID NO: 40.

[0185] For example, the heavy chain variable region of an antigen binding protein of the present application can comprise a H-FR1, a H-FR2, a H-FR3, and a H-FR4, the H-FR4 can comprise the amino acid sequence set forth in SEQ ID NO: 45, the H-FR3 can comprise the amino acid sequence set forth in SEQ ID NO: 44, the H-FR2 can comprise the amino acid sequence set forth in SEQ ID NO: 41, and the H-FR1 can comprise the amino acid sequence set forth in SEQ ID NO: 40.

[0186] For example, the light chain variable region of an antigen binding protein of the present application can comprise a L-FR1, a L-FR2, a L-FR3, and a L-FR4, the L-FR4 can comprise the amino acid sequence set forth in SEQ ID NO: 53.

[0187] For example, the L-FR3 of the present application can comprise the amino acid sequence set forth in SEQ ID NO: 66.

[0188] For example, the L-FR3 of the present application can comprise the amino acid sequence set forth in any one of SEQ ID NOs: 51-52.

[0189] For example, the L-FR2 of the present application can comprise the amino acid sequence set forth in SEQ ID NO: 65.

[0190] For example, the L-FR2 of the present application can comprise the amino acid sequence set forth in any one of SEQ ID NOs: 49-50.

[0191] For example, the L-FR1 of the present application can comprise the amino acid sequence set forth in SEQ ID NO: 64.

[0192] For example, the L-FR1 of the present application can comprise the amino acid sequence set forth in any one of SEQ ID NOs: 46-48.

[0193] For example, the light chain variable region of an antigen binding protein of the present application can comprise a L-FR1, a L-FR2, a L-FR3, and a L-FR4, which L-FR4 can comprise the amino acid sequence set forth in SEQ ID NO: 53, which L-FR3 can comprise the amino acid sequence set forth in SEQ ID NO: 66, which L-FR2 can comprise the amino acid sequence set forth in SEQ ID NO: 65, and which L-FR1 can comprise the amino acid sequence set forth in SEQ ID NO: 64.

[0194] For example, the light chain variable region of an antigen binding protein of the present application can comprise a L-FR1, a L-FR2, a L-FR3, and a L-FR4, which L-FR4 can comprise the amino acid sequence set forth in SEQ ID NO: 53, which L-FR3 can comprise the amino acid sequence set forth in any one of SEQ ID NOs: 51-52, which L-FR2 can comprise the amino acid sequence set forth in any one of SEQ ID NOs: 49-50, and which L-FR1 can comprise the amino acid sequence set forth in any one of SEQ ID NOs: 46-48.

[0195] For example, the light chain variable region of an antigen binding protein of the present application can comprise a L-FR1, a L-FR2, a L-FR3, and a L-FR4, which L-FR4 can comprise the amino acid sequence set forth in SEQ ID NO: 53, which L-FR3 can comprise the amino acid sequence set forth in SEQ ID NO: 51, which L-FR2 can comprise the amino acid sequence set forth in SEQ ID NO: 49, and which L-FR1 can comprise the amino acid sequence set forth in SEQ ID NO: 46.

[0196] For example, the light chain variable region of an antigen binding protein of the present application can comprise a L-FR1, a L-FR2, a L-FR3, and a L-FR4, which L-FR4 can comprise the amino acid sequence set forth in SEQ ID NO: 53, which L-FR3 can comprise the amino acid sequence set forth in SEQ ID NO: 52, which L-FR2 can comprise the amino acid sequence set forth in SEQ ID NO: 50, and which L-FR1 can comprise the amino acid sequence set forth in SEQ ID NO: 47.

[0197] For example, the light chain variable region of an antigen binding protein of the present application can comprise a L-FR1, a L-FR2, a L-FR3, and a L-FR4, which L-FR4 can comprise the amino acid sequence set forth in SEQ ID NO: 53, which L-FR3 can comprise the amino acid sequence set forth in SEQ ID NO: 52, which L-FR2 can comprise the amino acid sequence set forth in SEQ ID NO: 40, and which L-FR1 can comprise the amino acid sequence set forth in SEQ ID NO: 48.

[0198] For example, the heavy chain variable region of an antigen binding protein of the present application can comprise H-FR1, H-FR2, H-FR3, and H-FR4, which H-FR4 can comprise the amino acid sequence set forth in SEQ ID NO: 45, which H-FR3 can comprise the amino acid sequence set forth in SEQ ID NO: 63, which H-FR2 can comprise the amino acid sequence set forth in SEQ ID NO: 41, and which H-FR1 can comprise the amino acid sequence set forth in SEQ ID NO: 62; and the light chain variable region of an antigen binding protein of the present application can comprise L-FR1, L-FR2, L-FR3, and L-FR4, which L-FR4 can comprise the amino acid sequence set forth in SEQ ID NO: 53, which L-FR3 can comprise the amino acid sequence set forth in SEQ ID NO: 66, which L-FR2 can comprise the amino acid sequence set forth in SEQ ID NO: 65, and which L-FR1 can comprise the amino acid sequence set forth in SEQ ID NO: 64.

[0199] For example, the heavy chain variable region of an antigen binding protein of the present application can comprise H-FR1, H-FR2, H-FR3, and H-FR4, which H-FR4 can comprise the amino acid sequence set forth in SEQ ID NO: 45, which H-FR3 can comprise the amino acid sequence set forth in any one of SEQ ID NOs: 42-44, which H-FR2 can comprise the amino acid sequence set forth in SEQ ID NO: 41, and which H-FR1 can comprise the amino acid sequence set forth in any one of SEQ ID NOs: 39-40; and the light chain variable region of an antigen binding protein of the present application can comprise L-FR1, L-FR2, L-FR3, and L-FR4, which L-FR4 can comprise the amino acid sequence set forth in SEQ ID NO: 53, which L-FR3 can comprise the amino acid sequence set forth in any one of SEQ ID NOs: 51-52, which L-FR2 can comprise the amino acid sequence set forth in any one of SEQ ID NOs: 49-50, and which L-FR1 can comprise the amino acid sequence set forth in any one of SEQ ID NOs: 46-48.

[0200] For example, the heavy chain variable region of an antigen binding protein of the present application can comprise H-FR1, H-FR2, H-FR3, and H-FR4, which H-FR4 can comprise the amino acid sequence set forth in SEQ ID NO: 45, which H-FR3 can comprise the amino acid sequence set forth in SEQ ID NO: 42, which H-FR2 can comprise the amino acid sequence set forth in SEQ ID NO: 41, and which H-FR1 can comprise the amino acid sequence set forth in SEQ ID NO: 39; and the light chain variable region of an antigen binding protein of the present application can comprise L-FR1, L-FR2, L-FR3, and L-FR4, which L-FR4 can comprise the amino acid sequence set forth in SEQ ID NO: 53, which L-FR3 can comprise the amino acid sequence set forth in SEQ ID NO: 51, which L-FR2 can comprise the amino acid sequence set forth in SEQ ID NO: 49, and which L-FR1 can comprise the amino acid sequence set forth in SEQ ID NO: 46.

[0201] For example, the heavy chain variable region of an antigen binding protein of the present application can comprise H-FR1, H-FR2, H-FR3, and H-FR4, which H-FR4 can comprise the amino acid sequence set forth in SEQ ID NO: 45, which H-FR3 can comprise the amino acid sequence set forth in SEQ ID NO: 43, which H-FR2 can comprise the amino acid sequence set forth in SEQ ID NO: 41, and which H-FR1 can comprise the amino acid sequence set forth in SEQ ID NO: 40; and the light chain variable region of an antigen binding protein of the present application can comprise L-FR1, L-FR2, L-FR3, and L-FR4, which L-FR4 can comprise the amino acid sequence set forth in SEQ ID NO: 53, which L-FR3 of the present application can comprise the amino acid sequence set forth in SEQ ID NO: 52, which L-FR2 can comprise the amino acid sequence set forth in SEQ ID NO: 50, and which L-FR1 can comprise the amino acid sequence set forth in SEQ ID NO: 47.

[0202] For example, the heavy chain variable region of an antigen binding protein of the present application can comprise H-FR1, H-FR2, H-FR3, and H-FR4, which H-FR4 can comprise the amino acid sequence set forth in SEQ ID NO: 45, which H-FR3 can comprise the amino acid sequence set forth in SEQ ID NO: 44, which H-FR2 can comprise the amino acid sequence set forth in SEQ ID NO: 41, and which H-FR1 can comprise the amino acid sequence set forth in SEQ ID NO: 40; and the light chain variable region of an antigen binding protein of the present application can comprise L-FR1, L-FR2, L-FR3, and L-FR4, which L-FR4 can comprise the amino acid sequence set forth in SEQ ID NO: 53, which L-FR3 can comprise the amino acid sequence set forth in SEQ ID NO: 52, which L-FR2 can comprise the amino acid sequence set forth in SEQ ID NO: 40, and which L-FR1 can comprise the amino acid sequence set forth in SEQ ID NO: 48.

[0203] For example, the heavy chain variable region of an antigen binding protein of the present application comprises the amino acid sequence set forth in SEQ ID NO: 67. For example, the heavy chain variable region of an antigen binding protein of the present application comprises the amino acid sequence set forth in any one of SEQ ID NOs: 54-56.

[0204] For example, the light chain variable region of an antigen binding protein of the present application comprises the amino acid sequence set forth in SEQ ID NO: 68. For example, the light chain variable region of an antigen binding protein of the present application comprises the amino acid sequence set forth in any one of SEQ ID NOs: 57-59.

[0205] For example, the heavy chain variable region of an antigen binding protein of the present application comprises the amino acid sequence set forth in SEQ ID NO: 67, and the light chain variable region of an antigen binding protein of the present application comprises the amino acid sequence set forth in SEQ ID NO: 68. For example, the heavy chain variable region of an antigen binding protein of the present application comprises the amino acid sequence set forth in any one of SEQ ID NOs: 54-56, and the light chain variable region of an antigen binding protein of the present application comprises the amino acid sequence set forth in any one of SEQ ID NOs: 57-59.

[0206] For example, the heavy chain variable region of an antigen binding protein of the present application comprises the amino acid sequence set forth in SEQ ID NO: 54, and the light chain variable region of an antigen binding protein of the present application comprises the amino acid sequence set forth in SEQ ID NO: 57.

[0207] For example, the heavy chain variable region of an antigen binding protein of the present application comprises the amino acid sequence set forth in SEQ ID NO: 55, and the light chain variable region of an antigen binding protein of the present application comprises the amino acid sequence set forth in SEQ ID NO: 58.

[0208] For example, the heavy chain variable region of an antigen binding protein of the present application comprises the amino acid sequence set forth in SEQ ID NO: 56, and the light chain variable region of an antigen binding protein of the present application comprises the amino acid sequence set forth in SEQ ID NO: 59.

[0209] Further, an antigen binding protein of the present application comprises an antibody heavy chain constant region. For example, an antigen binding protein of the present application comprises an antibody heavy chain constant region derived from a human IgG constant region. For example, the antibody heavy chain constant region of an antigen binding protein of the present application comprises the amino acid sequence set forth in SEQ ID NO: 60.

[0210] Further, an antigen binding protein of the present application comprises an antibody light chain constant region. For example, the antibody light chain constant region of an antigen binding protein of the present application comprises the amino acid sequence set forth in SEQ ID NO: 61.

[0211] For example, an antigen binding protein of the present application comprises an antibody or an antigen binding fragment thereof. For example, an antibody of the present application comprises a Fab, Fab', Fv fragment, F(ab')2, scFv, di-scFv, and / or dAb. For example, an antigen binding fragment of the present application is selected from the group consisting of a humanized antibody and a fully human antibody.

[0212] The physical / chemical properties and / or biological activities of the PD-1 antigen binding proteins described herein can be identified, screened or characterized by various assays known in the art.

[0213] In one aspect, antigen binding activity of an antigen binding protein or fusion protein of the application can be tested, for example, by known methods such as enzyme-linked immunosorbent assay (ELISA), immunoblotting (e.g., Western blot), flow cytometry (e.g., FACS), immunohistochemistry, immunofluorescence, and the like. An antigen binding protein (e.g., a PD-1 antibody) described herein is capable of specifically binding to a PD-1 antigen. An antigen binding protein (e.g., a PD-1 antibody) that "specifically binds" to a PD-1 antigen can typically bind to PD-1, but not to other proteins that lack the sequence of PD-1. An antigen binding protein (e.g., a PD-1 antibody) described herein is capable of specifically binding to a PD-1 antigen or a labeled form thereof (e.g., a fluorescently labeled PD-1 antigen), but not to other proteins that lack the PD-1 epitope. Whether an antigen binding protein (e.g., an antibody) binds to a PD-1 antigen can be determined using any assay known in the art. Examples of assays known in the art to analyze binding affinity include Bio-Layer Interferometry (BLI).

[0214] An antigen binding protein described herein can bind to a human PD-1 protein. In certain cases, an antigen binding protein described herein can also cross-react with PD-1 of a monkey (e.g., cynomolgus monkey). For example, as detected by flow analysis techniques and enzyme-linked immunoassays. In the present application, "cross-react" refers to the ability of an antibody to react with a homologous protein from another species.

[0215] In certain cases, the binding activity of an antigen binding protein described herein to PD-1 can be detected using an enzyme-linked immunoassay. For example, in an ELISA using a human PD-1 antigen protein, the EC50 value of the PD-1 antigen binding protein to PD-1 can be between about 0.0001 nM to about 100 nM, for example, can be between about 0.001 nM to about 10 nM, can be between about 0.001 nM to about 5 nM, can be between about 0.001 nM to about 1 nM, or can be between about 0.01 nM to about 1 nM.

[0216] In another aspect, an antigen binding protein described herein is capable of blocking the binding of PD-1 to PD-L1 and or PD-L2. In certain cases, the antigen binding protein blocks the binding of PD-1 to PD-L1 and or PD-L2 in an enzyme-linked immunoassay, ELISA. For example, a PD-L1 and or PD-L2 antigen protein is first coated on a plate, and a mixture of decreasing amounts of unlabeled antigen binding protein and biotin-labeled PD-1 protein is incubated together. Then, the cells are analyzed using ELISA to demonstrate that the antigen binding protein can block the binding of PD-1 to PD-L1 and or PD-L2.

[0217] The antigen binding proteins described herein can stimulate the proportion of CD07a cells expressing immune cells. The antigen binding proteins described herein can stimulate the secretion of IFN-γ, TNF-a, and / or IL2 from immune cells. For example, the stimulating ability of the antigen binding proteins described herein can increase the effect of immune cell activation on the basis of activation by known activators in the art (e.g., CD3 antibodies, CD28 antibodies, or nanomaterials TransAct TM ) containing CD3 antibodies and CD28 antibodies. The immune cells can include lymphocytes, such as B cells, T cells, natural killer cells, myeloid cells, such as monocytes, macrophages, mast cells, basophils, and granulocytes. For example, the immune cells can comprise tumor infiltrating lymphocytes (TILs). For example, the immune cells can comprise artificially modified immune cells. For example, the immune cells can comprise TCR-T cells. Secretion of cytokines from immune cells can be determined using any method known to one of skill in the art, for example, by enzyme-linked immunoassay (ELISA) to quantitatively determine immune cell (e.g., T cell) proliferation or cytokine production (e.g., IFN-γ or IL-2 production by T cells) by immune cells. For example, the stimulatory effect of PD-1 antibodies on T lymphocytes can be detected by mixed lymphocyte reaction (MLR) assays.

[0218] Nucleic acid molecules, vectors, and cells

[0219] In another aspect, the present application provides one or more nucleic acid molecules that can encode the isolated antigen binding proteins described herein. The nucleic acid molecules described herein can be isolated. For example, they can be produced or synthesized by (i) amplification in vitro, such as by polymerase chain reaction (PCR) amplification, (ii) recombinant production by cloning, (iii) purification, such as by enzymatic cleavage and gel electrophoresis fractionation, or (iv) synthesis, such as by chemical synthesis. In certain embodiments, the isolated nucleic acid is a nucleic acid molecule prepared by recombinant DNA technology.

[0220] In another aspect, the present application provides a vector that can comprise the nucleic acid molecules described herein. In addition, the vector can comprise other genes, such as marker genes that allow selection of the vector in appropriate host cells and under appropriate conditions. In addition, the vector can comprise expression control elements that allow proper expression of the coding region in appropriate hosts. Such control elements are well known to those skilled in the art, for example, and can include promoters, ribosome binding sites, enhancers, and other control elements that regulate transcription or mRNA translation of genes. The vector can comprise, for example, a plasmid, cosmid, virus, bacteriophage, or other vector commonly used in, for example, genetic engineering. For example, the vector is an expression vector.

[0221] In another aspect, the present application provides a cell, which can comprise a nucleic acid molecule described herein or a vector described herein. In certain embodiments, each or every host cell can comprise one or a nucleic acid molecule or vector described herein. In certain embodiments, each or every host cell can comprise a plurality (e.g., 2 or more) or a plurality (e.g., 2 or more) of nucleic acid molecules or vectors described herein. For example, a vector described herein can be introduced into the host cell, e.g., a eukaryotic cell, such as a cell from a plant, a fungal or yeast cell, etc. The vector described herein can be introduced into the host cell by methods known in the art, such as electroporation, lipofectine transfection, lipofectamin transfection, etc.

[0222] Pharmaceutical composition

[0223] In another aspect, the present application provides a pharmaceutical composition, which can comprise an antigen binding protein described herein and / or the nucleic acid molecule, the vector, the host cell described herein, and optionally a pharmaceutically acceptable adjuvant. The pharmaceutically acceptable adjuvant is non-toxic to a recipient at the dosages and concentrations employed, and can include buffers, antioxidants, preservatives, low molecular weight (less than about 10 residues) polypeptides, proteins, hydrophilic polymers, amino acids, carbohydrates, salt-forming counterions, metal complexes, and / or non-ionic surfactants. The pharmaceutical composition herein can also contain more than one active compound, generally those with complementary activities that do not adversely affect each other. The type and effective amount of such pharmaceuticals depend, for example, on the amount and type of antagonist present in the formulation, and on the clinical parameters of the subject.

[0224] The pharmaceutical composition described herein can comprise a prophylactically and / or therapeutically effective amount of the antigen binding protein. The prophylactically and / or therapeutically effective amount is the amount required to prevent and / or treat (at least partially treat) a disease or disorder and / or any complications thereof in a subject suffering therefrom or at risk of developing it.

[0225] Method of preparation

[0226] In another aspect, the present application provides a method of preparing the antigen binding protein described herein. The method can comprise culturing the host cell described herein under conditions such that the antigen binding protein is expressed. For example, the method can be carried out by using an appropriate medium, an appropriate temperature and culturing time, etc., which are known to those of ordinary skill in the art.

[0227] Any method suitable for producing monoclonal antibodies can be used to produce the antigen binding proteins of the present application. For example, an animal can be immunized with a linked or naturally occurring PD-1 protein or fragment thereof. Suitable immunization protocols can be used, including adjuvants, immunostimulants, repeated booster immunizations, and one or more routes of administration can be used.

[0228] Any suitable form of PD-1 can be used as an immunogen (antigen) for the production of non-human antibodies specific for PD-1, and for screening the biological activity of the antibodies. The immunogen can be full-length, mature human PD-1, including the native homodimer, or a peptide containing one or more epitopes. The immunogen can be used alone or in combination with one or more immunogenicity enhancers known in the art.

[0229] The humanized antibodies can be selected from any class of immunoglobulin, including IgM, IgD, IgG, IgA, and IgE. In the present application, the antibodies are IgG antibodies, and the IgG1 subtype is used. Optimization of the necessary constant domain sequences to produce the desired biological activity can be achieved by screening the antibodies using the biological assays described in the Examples below. Likewise, any class of light chain can be used in the compounds and methods of the present application. In particular, kappa, lambda chains, or variants thereof, are useful in the compounds and methods of the present application.

[0230] The sequence of the DNA molecule of the antigen binding proteins of the present application, or fragments thereof, can be obtained using conventional techniques, such as PCR amplification or screening of genomic libraries. In addition, the coding sequences for the light and heavy chains can be fused together to form a single chain antibody.

[0231] Once the relevant sequences have been obtained, they can be obtained in large quantities using recombinant methods. This is typically done by cloning them into vectors, which are then introduced into cells, and then isolating the relevant sequences from the propagated host cells using conventional methods. In addition, the relevant sequences can be synthesized using artificial synthesis methods, particularly for shorter fragments. Typically, longer fragments are obtained by first synthesizing a number of smaller fragments, which are then ligated together. The nucleic acid molecule can then be introduced into a variety of existing DNA molecules (or vectors, for example) and cells known in the art.

[0232] The present application also relates to vectors comprising the appropriate nucleic acid molecules described above, as well as appropriate promoters or control sequences. These vectors can be used to transform appropriate host cells, so that they are able to express the proteins. The host cells can be prokaryotic cells, such as bacterial cells, or lower eukaryotic cells, such as yeast cells, or higher eukaryotic cells, such as mammalian cells. For example, the animal cells can include (but are not limited to) 293T cells.

[0233] The steps for transforming a host cell with recombinant DNA described herein can be performed using techniques well known in the art. The resulting transformant can be cultured using conventional procedures and the polypeptide encoded by the nucleic acid molecule of the application expressed by the transformant. The host cells are typically cultured under conditions suitable for expression of the antigen binding protein of the application. The antigen binding protein of the application can then be purified using conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, size exclusion chromatography, or affinity chromatography steps well known to those skilled in the art.

[0234] The resulting monoclonal antibodies can be identified using conventional means. For example, the binding specificity of the monoclonal antibodies can be determined using immunoprecipitation or an in vitro binding assay such as flow cytometry sorting (FACS) or enzyme linked immunosorbent assay (ELISA).

[0235] Methods and uses

[0236] In another aspect, the application provides the use of the antigen binding protein described herein in the manufacture of a medicament. The antigen binding protein can be administered alone or in combination with one or more additional therapies, such as chemotherapy, radiotherapy, immunotherapy, surgical intervention, or any combination of these. The medicament can be used to treat a PD-1 mediated disease or disorder, for example, to treat cancer, to inhibit tumor growth, and / or to inhibit tumor cell proliferation.

[0237] In another aspect, the application provides the antigen binding protein described herein for use in the prophylaxis or treatment of a PD-1 mediated disease or disorder. The prophylaxis or treatment of the disease or disorder can mean inhibiting or delaying the development or progression of the disease or disorder. For example, the development or progression of a tumor can be inhibited. For example, tumor growth or tumor cell proliferation can be inhibited.

[0238] In another aspect, the application provides the antigen binding protein described herein for use in the prophylaxis or treatment of a PD-1 mediated disease or disorder.

[0239] In another aspect, the application provides a method of inhibiting the binding of PD-1 to PD-L1 and or PD-L2 comprising administering the antigen binding protein described herein. The method can be an ex vivo or in vitro method. In certain embodiments, the method can comprise contacting a biological sample with the antigen binding protein described herein and / or PD-L1 and or PD-L2 under conditions permissive for the antigen binding protein and / or PD-1 to bind to PD-L1 and or PD-L2, detecting whether a complex is formed between the antigen binding protein and PD-1, and detecting whether a complex is formed between PD-1 and PD-L1 and or PD-L2. In another aspect, the application provides a method of inhibiting the binding of PD-1 to PD-L1 and or PD-L2 comprising administering the antigen binding protein described herein. The method can be an ex vivo or in vitro method. In certain embodiments, the method can comprise contacting a biological sample with the antigen binding protein described herein and / or PD-L1 and or PD-L2 under conditions permissive for the antigen binding protein and / or PD-1 to bind to PD-L1 and or PD-L2, detecting whether a complex is formed between the antigen binding protein and PD-1, and detecting whether a complex is formed between PD-1 and PD-L1 and or PD-L2.

[0240] In another aspect, the present application provides a method for preventing, alleviating or treating a PD-1 mediated disease or disorder, comprising administering to a subject in need thereof an antibody or antigen-binding fragment thereof described herein, a molecular nucleic acid described herein, a vector described herein, a host cell described herein and / or a pharmaceutical composition described herein. For example, the method can be used to inhibit the development or progression of a tumor. For example, the method can inhibit tumor growth or tumor cell proliferation.

[0241] Without wishing to be bound by any theory, the following examples are merely intended to illustrate the proteins, preparation methods and uses of the present application, and are not intended to limit the scope of the present application. The experimental methods in the following examples, for which specific conditions are not indicated, are generally carried out according to conventional conditions, or according to the conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are percentages by weight and parts by weight.

[0242] Example

[0243] Example 1: Construction of an expression vector for a fusion protein of the 25th to 167th amino acid sequence of recombinant human programmed death receptor 1 (PD-1) and a human IgG1 Fc region (PD-1-huIgG1 Fc) and eukaryotic expression

[0244] 1. Synthesis of the gene sequence of the 25th to 167th amino acid sequence of PD-1 and construction of an expression vector for a PD-1-huIgG1 Fc fusion protein

[0245] The gene sequence of the 25th isoleucine to the 167th glutamine of programmed death receptor 1 (PD-1) (NCBI accession No. NP_005009.2) was synthesized by chemical synthesis, which encodes the amino acid sequence shown in SEQ ID NO: 30. The gene sequence of the 100th proline to the 330th lysine of the human IgG1 heavy chain constant region was synthesized by chemical synthesis. An upstream primer containing a mouse Igkv3-10 signal peptide gene sequence was chemically synthesized for expression vector construction. The PD-1 gene fragment and the human IgG1 Fc gene fragment were spliced by molecular cloning. The splicing product was cloned into pCDNA3.1 (Thermo) using the TaKaRa Seamless Cloning Kit.

[0246] 2. Expression and purification of recombinant PD-1-huIgG1 Fc fusion protein

[0247] After transfecting 293T cells (ATCC) with the expression vector for 5 days, the culture supernatant was collected and the recombinant PD-1-huIgG1 Fc fusion protein was purified using AKTA explorer 100 (GE). Due to glycosylation modification and other reasons, the recombinant PD-1-huIgG1 Fc fusion protein showed a size of about 60 kDa by Coomassie blue staining after reduced SDS-PAGE electrophoresis.

[0248] Example 2: Construction of expression vector for recombinant human Programmed Death Receptor 1 (PD-1) 25th to 167th amino acid sequence fused with poly-histidine tag fusion protein (PD-1-his) and eukaryotic expression

[0249] 1. Synthesis of gene sequence of PD-1 25th to 167th amino acid interval and construction of expression vector for poly-histidine tag fusion protein

[0250] The gene sequence of the 25th isoleucine to the 167th glutamine interval of Programmed Death Receptor 1 (PD-1) (NCBI accession No. NP_005009.2) was synthesized by chemical synthesis. The upstream primer containing the mouse Igkv3-10 signal peptide gene sequence and the downstream primer containing the poly-histidine tag (having the amino acid sequence shown in SEQ ID NO: 32) gene sequence (SEQ ID NO: 31) were used to construct the pCDNA3.1 vector for expressing PD-1-his.

[0251] 2. Expression and purification of recombinant PD-1-his protein

[0252] After transfecting 293T cells (ATCC) with the expression vector for 5 days, the culture supernatant was collected and the recombinant PD-1-his protein was purified using AKTA explorer 100 (GE). Due to glycosylation modification and other reasons, the recombinant PD-1-his protein showed a size of about 40 kDa by Coomassie blue staining after reduced SDS-PAGE electrophoresis.

[0253] Example 3: Construction of expression vector for anti-human PD-1 antibody (Nivolumab) and eukaryotic expression

[0254] 1. Acquisition of Nivolumab antibody variable region gene and construction of expression vector

[0255] The Nivolumab antibody light and heavy chain variable region genes were synthesized by chemical synthesis, and the sequences were SEQ ID NO: 33 and SEQ ID NO: 34, respectively. The Nivolumab antibody light chain variable region had an amino acid sequence as shown in SEQ ID NO: 35, and the Nivolumab antibody heavy chain variable region had an amino acid sequence as shown in SEQ ID NO: 36. The heavy chain variable region fragment was amplified by PCR using the heavy chain variable region gene as a template, and the amplification product was cloned into pFUSEss-CHIg-hG1 (invivogen) containing a human IL-2 signal peptide gene and a human IgG1 heavy chain constant region gene using a TaKaRa Seamless Cloning Kit. The light chain variable region fragment was amplified by PCR using the light chain variable region gene as a template, and the amplification product was cloned into pFUSE2ss-CLIg-hK (invivogen) containing a human IL-2 signal peptide gene and a human kappa light chain constant region gene using a TaKaRa Seamless Cloning Kit.

[0256] 2. Expression and purification of Nivolumab antibody

[0257] After co-transfecting 293T cells (ATCC) with the double plasmids at a ratio of 1:1 for 5 days, the culture supernatant was collected, and the Nivolumab antibody was purified using an AKTA explorer 100 (GE). After non-reducing SDS-PAGE electrophoresis, the Nivolumab antibody was stained with Coomassie blue, and the size was about 150 kDa.

[0258] Example 4: ELISA detection of recombinant human PD-1 (PD-1-huIgG1 Fc or PD-1-his) binding to Nivolumab antibody

[0259] The enzyme-linked immunosorbent assay (ELISA) was used to detect the binding of recombinant human PD-1 (PD-1-huIgG1 Fc or PD-1-his) to Nivolumab antibody. The ELISA experiment was performed as follows: 100 ng / well of the PD-1-huIgG1 Fc or PD-1-his fusion protein prepared above was added to a microplate and coated at 4°C overnight. After washing three times with PBS, 1% BSA / PBS was added at 200 uL / well, and the plate was blocked at 37°C for 1 hour. After washing the plate with 100 ul PBS, 100 ng / well of Nivolumab chimeric antibody was added, and the plate was combined at 37°C for 1 hour. After washing three times with PBST, 100 ul of 1:5000 diluted HRP-goat anti-human IgG (Fab specific) was added, and the plate was combined at 37°C for 1 hour. After washing three times with PBST, 100 uL / well of TMB color developing solution was added, and the plate was developed at 37°C for 10 minutes. 100 uL / well of ELISA stop solution was added, and the OD450 value was read on a microplate reader. The OD450 value reflects the binding of Nivolumab antibody to recombinant human PD-1.

[0260] Example 5: Expression and affinity determination of tetravalent human cell programed death-ligand 1 (PD-L1) extracellular segment mutant to human PD-1 molecule

[0261] 1. Construction of expression vector for tetravalent human PD-L1 extracellular segment mutant

[0262] The gene sequence of two human PD-L1 extracellular segment mutants connected by a flexible peptide (SEQ ID NO: 37) was synthesized by chemical synthesis, which encodes the amino acid sequence shown in SEQ ID NO: 38. The gene sequence of the amino acid region from the 100th proline to the 330th lysine of the human IgG1 heavy chain constant region (UniProtKB / Swiss-Prot accession No. P01857.1) was synthesized by chemical synthesis. An upstream primer containing a mouse Igkv3-10 signal peptide gene sequence was chemically synthesized for expression vector construction. By molecular cloning, the gene fragment of the PD-L1 extracellular segment mutant repeat structure was spliced with the human IgG1 Fc gene fragment. The splicing product was cloned into pCDNA3.1 (Thermo) using the TaKaRa Seamless Cloning Kit.

[0263] 2. Expression and purification of tetravalent human PD-L1 extracellular segment mutant

[0264] After transfecting 293T cells (ATCC) with the expression vector for 5 days, the culture supernatant was collected and the tetravalent human PD-L1 ectodomain mutant protein was purified using an AKTA explorer 100 (GE). Due to glycosylation modification and other reasons, the tetravalent human PD-L1 ectodomain mutant protein showed a size of about 85 kDa by Coomassie blue staining after reduction SDS-PAGE electrophoresis.

[0265] 3. Biotinylation of PD-1-huIgG1 Fc fusion protein

[0266] The PD-1-huIgG1 Fc fusion protein was randomly biotinylated using the standard operating procedure provided by EZ-Link Sulfo-NHS-LC-Biotin (Thermo). The binding activity of the biotinylated PD-1-huIgG1 Fc fusion protein to Nivolumab antibody was verified by ELISA.

[0267] 4. Determination of the avidity of tetravalent human PD-L1 ectodomain mutant to PD-1-huIgG1 Fc

[0268] The avidity of the tetravalent human PD-L1 ectodomain mutant to PD-1-huIgG1 Fc was determined using an Octet K2 (ForteBio) molecular interaction analyzer. According to the standard operating procedure of Octet K2, 100 nM of biotinylated PD-1-huIgG1 Fc fusion protein was configured and immobilized on SA probes (ForteBio) with an immobilization height of 1 nM. The tetravalent human PD-L1 ectodomain mutant was diluted by two-fold gradient starting from 50 nM as the analyte for the experiment, and the avidity of the tetravalent human PD-L1 ectodomain mutant to PD-1-huIgG1 Fc was determined to be 9.4 nM. The avidity determination results are shown in Figure 1 . It is known that the binding affinity of wild-type PD-1 and PD-L1 is 8.2 uM (see Cheng, X. et al., J. Biol. Chem., 288(17): 11771-85, 2013).

[0269] Example 6: Preparation of anti-human PD-1 murine antibody, murine humanization

[0270] 1. Immunization of animals

[0271] Take 2 mg / mL of the PD-1-huIgG1 Fc fusion protein prepared in Example 1 as an antigen, mix and emulsify with an equal volume of complete Freund's adjuvant (Sigma-Aldrich), take 10 six-week-old female Balb / c mice (subcutaneous immunization, 100 ug antigen per mouse. After the first immunization, perform a booster immunization every ten days, a total of four subcutaneous immunizations, and the fifth immunization is a direct spleen pulse immunization with the PD-1-huIgG1 Fc fusion protein.

[0272] 2. Serum titer detection

[0273] Take 50 uL of blood from the tail vein before each booster immunization, centrifuge to remove cells, and retain the serum. Add PD-1-his (ACRO Biosystems) 50 ng / well to the ELISA microplate, and coat overnight at 4°C. Wash three times with PBS, add 1% BSA / PBS, 200 uL / well, and block at 37°C for 1 hour. Add gradient-diluted mouse serum, and bind at 37°C for 1 hour. Wash three times with PBST, add 100 ul of 1:5000 diluted HRP-goat anti-mouse IgG (Shanghai Yisheng), and bind at 37°C for 1 hour. Wash three times with PBST, add 100 uL / well TMB developing solution, and develop at 37°C for 10 minutes. Add 100 uL / well ELISA stop solution, and read the OD450 value on a microplate reader.

[0274] 3. Construction of immune library

[0275] 3.1 Total cDNA acquisition of mouse spleen cells

[0276] Four days after pulse immunization by direct intraperitoneal injection with the PD-1-huIgG1 Fc fusion protein, the mice were sacrificed, and the spleen was removed. The entire spleen was ground with a 70-micron cell screen (BD) to obtain spleen cells. After washing twice with PBS, the spleen cells were centrifuged at 1000g for 10 minutes. Total RNA was extracted using the Trizol RNA extraction kit.

[0277] Using the RNA as a template, the first-strand cDNA was synthesized using the SuperScript TM IV First-Strand Synthesis System kit.

[0278] 3.2 Amplification of antibody genes and splicing of light and heavy chains

[0279] The cDNA was used as a template, and the antibody amplification primers described in the literature (Schaefer J.V., Honegger A., Pluckthun A. (2010) Construction of scFv Fragments from Hybridoma or Spleen Cells by PCR Assembly. In: Kontermann R., Dubel S. (eds) Antibody Engineering. Springer Protocols Handbooks. Springer, Berlin, Heidelberg) were used to PCR amplify the heavy chain variable domain gene using the heavy chain variable domain upstream primer and the downstream primer, and to PCR amplify the kappa chain variable domain gene using the light chain variable domain upstream primer and the downstream primer. In a 50 uL reaction system, 25 uL phusion master mix (Thermo), 2.5 uL upstream primer (25 pmol), 2.5 uL downstream primer (25 pmol), 1.5 uL DMSO, 0.5 uL cDNA and 18 uL ddH2O were added, respectively. The PCR reaction was performed according to the following program: 98°C pre-denaturation for 1 minute, followed by temperature cycling, 98°C denaturation for 30 seconds, 58°C annealing for 30 seconds, 72°C extension for 1 minute, 30 cycles, 72°C final extension for 10 minutes.

[0280] The amplified VH and VL genes were recovered using a DNA gel recovery kit. An equal amount of VH and VL genes were mixed as a template, and the scFv gene was amplified by overlap PCR using the upstream primer scFv-F and the downstream primer scFv-R. In a 50 uL reaction system, 25 uL phusion master mix, 2.5 uL upstream primer (25 pmol), 2.5 uL downstream primer (25 pmol), 1.5 uL DMSO, 0.5 uL cDNA and 18 uL ddH2O were added, respectively. The PCR reaction was performed according to the following program: 98°C pre-denaturation for 1 minute, followed by temperature cycling, 98°C denaturation for 30 seconds, 58°C annealing for 30 seconds, 72°C extension for 1 minute, 30 cycles, 72°C final extension for 10 minutes.

[0281] The amplified scFv gene fragment was recovered using a DNA gel recovery kit.

[0282] 3.3 Construction of immune library

[0283] Digest scFv gene fragment and pcomb3 XTT vector (Scripps Institute, USA) with Sfil DNA endonuclease respectively. In 50 uL reaction system, add Sfil 2 uL, 10x buffer 5 uL, DNA 3 ug, and ddH2O to 50 uL respectively. After mixing thoroughly, incubate at 50°C for 3 hours.

[0284] Recover the digested scFv gene fragment and pcomb3 XTT vector using DNA gel recovery kit. Use T4 ligase to circularize the digested scFv gene fragment and the digested pcomb3 XTT vector. In 50 uL reaction system, add T4 ligase 1 uL, 10x buffer 5 uL, scFv gene 100 ng, pComb3 XTT vector 500 ng, and ddH2O to 50 uL respectively. After mixing thoroughly, incubate at 4°C for 16 hours. Take a small amount of product to verify the ligation efficiency by agarose gel electrophoresis.

[0285] Add 10 uL of the above-mentioned circularization ligation product to self-made TG1 electrotransformation competent cells, and then perform electric shock transformation by using an electrotransformation instrument. Take 10 ul of the electrotransformed bacteria, dilute them reasonably, and streak them on a plate containing ampicillin, so as to count and count the size of the phage antibody library. The remaining electrotransformed bacteria are added to 2xYT medium containing 100 ug / mL ampicillin and 2% glucose, and placed in a heating incubator for culture. After the culture is completed, centrifuge at 4000G for 10 minutes at 4°C, and supplement the precipitated bacteria with an appropriate amount of glycerol and store them at -80°C as an antibody bacterial seed library. Through multiple electrotransformation accumulation, an scFv immune library with a capacity of more than 3E9 is obtained.

[0286] 4. Screening and identification of murine immune antibody phage library

[0287] 4.1 Biotinylation of PD-1-huIgG1 Fc fusion protein

[0288] Randomly biotinylate the PD-1-huIgG1 Fc fusion protein according to the standard operating procedure provided by EZ-Link Sulfo-NHS-LC-Biotin. Verify the binding activity of the biotinylated PD-1-huIgG1 Fc fusion protein to Nivolumab antibody by ELISA method.

[0289] 4.2 Bio-panning

[0290] PD-1-huIgG1 Fc fusion protein as the target protein, the above-mentioned murine immunized antibody library was biopanned to obtain antibodies binding to PD-1-huIgG1 Fc fusion protein (especially the extracellular domain of PD-1). 100 OD bacteria were taken from the antibody bacterial library to start OD600=0.1 density recovery and growth to logarithmic phase, and then M13KO7 helper phage was used to rescue the antibody library. After centrifugation, the supernatant was resuspended with 2xYT medium containing ampicillin and kanamycin and amplified overnight at 30°C. The phage was precipitated by PEG / NaCl, and the phage precipitate was dissolved with glycerol / PBST to obtain the phage suspension of the immunized library. The casein-blocked phage was incubated in a co-incubation system of casein-blocked biotinylated huIgG1 Fc (ACRO Biosystems) fusion protein and casein-blocked Dynabeads M-270 streptavidin, and the supernatant phage suspension was collected. Further, the collected phage suspension was incubated in a co-incubation system of casein-blocked biotinylated PD-1-huIgG1 Fc fusion protein and casein-blocked Dynabeads M-270 streptavidin, and the magnetic beads were washed with PBST to remove phage that could not bind to PD-1-huIgG1 Fc fusion protein. Under appropriate elution conditions, the phage was competitively eluted with a self-made tetravalent PD-L1 extracellular segment mutant. 10 ul of eluted phage solution was taken for determination of the total output phage amount, and the remaining phage solution was used to infect logarithmically growing TG1, and after overnight amplification, it was considered as the antibody library used in the next round of panning. The biopanning was performed for three rounds, and the panning experiment parameters are shown in Table 2.

[0291] Table 2 Antibody library biopanning experiment parameters

[0292]

[0293] 4.3 Screening of clones specifically binding to PD-1 extracellular domain and having PD-1-tetravalent PD-L1 blocking effect

[0294] The antibody library obtained after the third round of biopanning was diluted and plated on plates containing ampicillin to obtain monoclonal antibodies, and the monoclonal antibodies were selected for overnight culture in a deep well plate. The next day, the deep well plate was subjected to repeated freezing and thawing three times, and the supernatant was centrifuged for subsequent two types of ELISA reactions.

[0295] ELISA reaction to detect binding activity: 50 ng PD-1-his was used to coat overnight, washed with PBS for three times, 1% BSA / PBS was added, 200 uL / well, 37°C blocking for 1 hour. After washing with PBS for three times, 100 ul centrifugal supernatant and 20 ng tetravalent PD-L1 mixture were added and incubated at 37°C for 1 hour, then washed with PBST for three times, 100 ul 1:5000 diluted HRP conjugated goat anti-mouse IgG (Fab specific) (Thermo) was added. Washed with PBST for three times, 100 uL / well TMB color developing liquid was added, color developed at 37°C for 10 minutes, 100 uL / well ELISA stop solution was added, and the OD450 value was read by a microplate reader. The screening step was repeated twice for independent experiments to ensure data accuracy, and clones with OD450 numerical average greater than 0.15 were selected for subsequent analysis.

[0296] ELISA reaction to detect blocking activity: PD-1-his was used to coat overnight, washed with PBS for three times, 1% BSA / PBS was added, 200 uL / well, 37°C blocking for 1 hour. After washing with PBS for three times, 100 ul centrifugal supernatant and 20 ng tetravalent PD-L1 mixture were added and incubated at 37°C for 1 hour, then washed with PBST for three times, 100 ul 1:5000 diluted HRP conjugated goat anti-mouse IgG (Fab specific) (Thermo) was added. Washed with PBST for three times, 100 uL / well TMB color developing liquid was added, color developed at 37°C for 10 minutes, 100 uL / well ELISA stop solution was added, and the OD450 value was read by a microplate reader. The screening step was repeated twice for independent experiments to ensure data accuracy, and clones with OD450 numerical average less than 1 were selected for subsequent analysis.

[0297] Further, the candidate clones that meet both of the above conditions were sequenced to obtain the antibody light chain variable domain and heavy chain variable domain sequences.

[0298] 4.4 Rescreening of clones with PD-1 extracellular domain specific binding and PD-1-tetravalent PD-L1 blocking effect

[0299] The candidate clones obtained by preliminary screening were expanded in 50 ml shake flasks (thermo), and the periplasmic cavity extract was obtained by using lysozyme (Shanghai Biotech) combined with three freeze-thaw methods, and the binding and blocking activities of the clones were identified again by two ELISA. 50 ng of PD-1-his was coated overnight, washed with PBS three times, 1% BSA / PBS was added, 200 uL / well, 37°C blocking for 1 hour. After washing with PBS three times, 100 ul of centrifugal supernatant was added and incubated at 37°C for 1 hour, washed with PBST three times, and then 100 ul of 1:5000 diluted HRP conjugated goat anti-mouse IgG (Fab specific) (Thermo) was added. PBST was washed three times, 100 uL / well TMB color developing liquid was added, and color development was carried out at 37°C for 10 minutes, 100 uL / well ELISA stop solution was added, and the OD450 value was read by a microplate reader to verify the binding activity. PD-1-his was coated overnight, washed with PBS three times, 1% BSA / PBS was added, 200 uL / well, 37°C blocking for 1 hour. After washing with PBS three times, 100 ul of centrifugal supernatant and different amounts of four-valent PD-L1 mixed solution were added and incubated at 37°C for 1 hour, washed with PBST three times, and then 100 ul of 1:5000 diluted HRP conjugated goat anti-mouse IgG (Fab specific) (Thermo) was added. PBST was washed three times, 100 uL / well TMB color developing liquid was added, and color development was carried out at 37°C for 10 minutes, 100 uL / well ELISA stop solution was added, and the OD450 value was read by a microplate reader to verify the blocking activity. The clones with low ELISA read values (i.e. strong blocking effect) were selected for sequencing to obtain the antibody sequence, and the 6H6 with the strongest blocking effect was selected for subsequent analysis. The amino acid sequences of the heavy chain variable region and the light chain variable region of the 6H6 antibody are SEQ ID NO. 7 and SEQ ID NO. 8, respectively.

[0300] 4.5 Expression of 6H6 clone hIgG1 form antibody

[0301] The heavy chain variable region sequence of 6H6 was cloned into pFUSEss-CHIg-hG1, respectively. The light chain variable region sequence of 6H6 was cloned into pFUSE2ss-CLIg-hK, respectively. After co-transfecting 293T cells (ATCC) with the double plasmids at a ratio of 1:1 for 5 days, the culture supernatant was collected, and the 6H6 antibody was purified by AKTAexplorer 100 (GE). After non-reducing SDS-PAGE electrophoresis of the 6H6 antibody, Coomassie blue staining showed that its size was about 150 kDa.

[0302] 4.6 Detection of the molecular level blocking effect of each hIgG1 antibody (6H6 and Nivolumab)

[0303] Competitive ELISA was performed with 6H6, Nivolumab and hlgG1 isotype control (R&D Systems, USA). Briefly, 100 ng of PD-1-huIgG1 Fc fusion protein was coated overnight per well, and 50 ul of 2 ng / ul of different hlgG1 antibodies (6H6, Nivolumab and isotype control antibody) and 50 ul of different concentrations of PD-L1-his (ACROBiosystems) were sequentially added. 100 ul of 1:5000 mouse anti-his tag-HRP (Thermo) was used for color development to detect the blocking effect of each antibody molecule at the molecular level. The detection steps contained three replicate wells, and the average values of the ELISA results are shown in Table 3. The blocking effect of 6H6 at the molecular level was better than that of Nivolumab.

[0304] Table 3. Blocking effect of 6H6, Nivolumab and hlgG1 isotype control antibody at the molecular level

[0305]

[0306] 5. Humanization of anti-human PD-1 murine antibody 6H6

[0307] In view of the better blocking activity at the molecular level of 6H6 molecule, humanization was carried out for 6H6. First, antibody humanization based on complementarity determining region transplantation was carried out, and IGKV6-21*02 and IGKJ4*01 were selected to replace the corresponding murine light chain and heavy chain germline genes of 6H6, respectively. IGHV3-23*04 and IGHJ4*01 human germline genes were selected. Second, key amino acids contributing to antibody affinity were predicted and back mutation was completed: the first type of amino acid residues are located on the interface between VL and VH, which play a key role in the packing of the two domains, the second type of amino acid residues are located near the CDR region and embedded in the protein, and the third type of amino acid residues are directly interacted with the CDR region, including hydrophobic interaction / hydrogen bond / salt bridge, etc. Based on the above rules, three candidate molecules were constructed, and Hu_6H6 molecule was selected as the humanized 6H6 antibody version by in vitro affinity determination. The antibody heavy chain variable region and light chain variable region amino acid sequences of Hu_6H6 are SEQ ID NO. 9 and SEQ ID NO. 10, respectively.

[0308] Example 7: Preliminary in vitro evaluation of anti-PD-1 murine antibody

[0309] 1. In vitro neutralization test of huIgG1 type PD-1 antibody

[0310] An in vitro neutralization assay of PD-1 antibodies was performed using ELISA to verify the ability of 6H6 and pembrolizumab huIgG1 antibodies to block the binding of PD-L1 to PD-1: PD-L1-huIgG1 Fc (ACRO Biosystems) was coated at 1 ug / ml 100 ul per well for four overnight, after washing and tapping dry, blocking was performed using 1% BSA in PBS 200 ul / well for 1 hour at 37°C. After washing 3 times with 0.1% PBST and tapping dry, a mixture of scFv-huIgG1 Fc type antibodies and Biotinylated PD-1-huIgG1 Fc (Kactus Biosystems) was added and incubated for 1 hour at room temperature (2 ug / ml 50 ul of PD-1-huIgG1 Fc mixed with 50 ul of antibodies starting from 160 ug / ml with two-fold gradient dilution, a total of eight gradients). After washing 3 times with 0.1% PBST, Streptavidin-HRP (R&D Systems) was added at 100 ul per well (1:200 dilution) and incubated for 1 hour at room temperature. After washing 6 times with 0.1% PBST, TMB color developing solution was added at 100 ul per well and incubated for 10 minutes at room temperature, after adding stop solution at 100 ul per well, OD450 readings were taken using a microplate reader. The data were analyzed and calculated to obtain the half inhibitory concentration IC50 of 6H6h and pembrolizumab huIgG1 antibodies to PD-1 and PD-L1 binding, respectively, as shown in Figure 2

[0311] 2. In vitro binding assay of huIgG1 PD-1 antibodies

[0312] ​PD-1 antibody affinity assay was performed by ELISA to verify the affinity of huIgG1 antibody of 6H6 and pembrolizumab: PD-1 Protein, Human, Recombinant (His Tag) (Beijing Yiqiao Shenzhou) 0.5 ug / ml 100ul per well was coated overnight, after washing and drying, 1% BSA in PBS 200ul / well was used for blocking at 37℃ for 1 hour. After washing 3 times with 0.1% PBST and drying, scFv-huIgG1 Fc type antibody was added at room temperature for 1 hour (starting from 10ug / ml, two-fold gradient dilution, a total of eight gradients). After washing 3 times with 0.1% PBST, Goat anti-Mouse IgG F(ab')2 Secondary Antibody, HRP (Thermo) / Goat Anti-Human IgG Secondary Antibody (HRP) (Beijing Yiqiao Shenzhou) 100ul per well (1:10000 dilution) was added at room temperature for 1 hour. After washing 6 times with 0.1% PBST, TMB color developing liquid 100ul per well was added at room temperature for 10 minutes, and after adding stop solution 100ul per well, OD450 reading was performed by using a microplate reader. The data was analyzed and processed, and the EC50 of huIgG1 antibody of 6H6 and pembrolizumab binding to PD-1 was calculated to be 0.01723ug / ml and 0.01106ug / ml, respectively, as shown in Figure 3 .

[0313] Example 8: PD-1 antibody stimulates tumor infiltrating lymphocytes (TIL)

[0314] The stimulating effect of the antibody of the present application on TIL cells was verified by adding PD-1 antibody in the cell culture medium of tumor infiltrating lymphocytes (TIL). Two batches of TIL cells (donor A and donor B) were recovered and cultured in medium containing IL-2 for 48 hours. After the end of the culture, IL-2 was removed by washing, and 1E5 / well cells were added to a 96-well plate. The test group (Transact+PD-1) added T Cell TransAct TM (Miltenyi Biotec) and 1ug / ml of 6H6 PD-1 antibody according to the instructions at a ratio of 1:1000, and the control group only added T Cell TransAct TM , and the proportion of CD107a cells and cytokine secretion were detected after incubation overnight.

[0315] Figure 4Figure 8A shows the results of CD 107a cell proportion in the presence of PD-1 antibody in tumor infiltrating lymphocyte (TIL) cell culture medium derived from donor A. Figure 4 Figure 8B shows the results of CD 107a cell proportion in the presence of PD-1 antibody in tumor infiltrating lymphocyte (TIL) cell culture medium derived from donor B. The results show that the PD-1 antibody of the present application can increase the proportion of CD 107a-expressing cells.

[0316] Figure 5 Figure 9 shows the results of cytokine IL-2, TNF-a and / or IFN-g secretion in the presence of PD-1 antibody in tumor infiltrating lymphocyte (TIL) cell culture medium derived from donors A and B. The results show that the PD-1 antibody of the present application can increase IL-2, TNF-a and / or IFN-g secretion; the PD-1 antibody of the present application has the ability to activate immune cells.

[0317] Example 9: Mixed lymphocyte reaction (MLR) experiment to detect the stimulation of T lymphocytes by PD-1 antibody

[0318] The mixed lymphocyte reaction (MLR) experiment was used to detect the ability of the PD-1 antibody to stimulate T lymphocytes to secrete IL-2 and IFN-g. Human PBMC was adjusted to a cell density of 2E7 cells / ml, and monocytes were sorted using CD14 MicroBeads, human (Miltenyi Biotec). 50 ng / ml of GM-CSF (ACRO Biosystems) and 50 ng / ml of IL-4 (ACRO Biosystems) were added for 5 days of culture. 10 ng / ml of LPS (Sigma) and 20 ng / ml of TNF-a (ACRO Biosystems) were added to induce DC cell maturation. Naive CD4+ T Cell Isolation Kit II, human (Miltenyi Biotec) was used to isolate CD4+ T cells from PBMC of different human sources. In a 96-well plate, each well contained 250 μl of culture medium containing 1.0E5 isolated T cells, 2E4 induced mature DC cells, and a series of concentration gradients of pembrolizumab or the PD-1 antibody 6H6 of the present application. Human IgG1 isotype control was used as a negative control. The mixed lymphocytes were cultured in a 37°C, 5% CO2 cell incubator for 3 days, and then 100 ul of culture supernatant was taken from each well of the 96-well plate for IL-2 and IFN-g determination.

[0319] Figure 6The results of the mixed lymphocyte reaction (MLR) assay for detecting the stimulation of IL-2 (A) and IFN-γ (B) secretion by T lymphocytes by the PD-1 antibody are shown. The results show that the PD-1 antibody of the present application has the ability to activate immune cells.

[0320] Example 10: PD-1 antibody enhances the killing ability of immune cells

[0321] To verify whether the PD-1 antibody enhances the killing ability of TCR-T, the PD-1 antibody was added to the killing of A375 cells by NYESO-1 TCR-T cells. CD3-positive T cells were sorted from freshly isolated human PBMC cells by CD3 MicroBeads, human (Miltenyi Biotec 130-050-101). The TCR-T cells can be obtained by any transduction method, for example, a NY-ESO-1 TCR (Kite Pharma) lentivirus carrying the NY-ESO-1 TCR was used to transduce the sorted CD3-positive T cells, and the transduction efficiency was 40% as detected by flow cytometry. A375 cells were plated at 2E4 cells per well in a 96-well plate, and 2E4 / well of transduced NYESO-1 TCR cells were added, together with 10 μg / ml of 6H6 antibody and control antibody pembrolizumab as test groups, and the same amount of PBMC cells were added to the A375 cells as negative controls, and only A375 cells as blank controls, and all groups were added with fluorescent dye SuperView 488 Caspase-3 at the same time. The 96-well plate was transferred to the IncuCyte S3, and the killing results were analyzed after automatic photographing every 3 hours for 33 hours. TM 488 Caspase-3. The 96-well plate was transferred to the IncuCyte S3, and the killing results were analyzed after automatic photographing every 3 hours for 33 hours.

[0322] Figure 7 The results of the killing ability enhancement of TCR-T cells by the addition of the PD-1 antibody are shown. The results show that the PD-1 antibody of the present application can enhance the killing ability of immune cells.

[0323] Example 11: Humanization of anti-human PD-1 murine antibody

[0324] (1) Design of humanization of antibody

[0325] The 6H6 was humanized. First, the antibody humanization based on complementarity determining region transplantation was carried out, the 6H6 murine anti-gene was compared with the human embryonic gene, the key amino acids contributing to the affinity of the antibody were obtained and the back mutation was completed: the first type of amino acid residues were the residues on the VL and VH binding interface, which played a key role in the packing of the two domains, the second type of amino acid residues were the residues close to the CDR region and embedded in the protein, and the third type of amino acid residues were the residues directly interacting with the CDR region, including hydrophobic interaction / hydrogen bond / salt bridge, etc. Based on the above rules, candidate molecules were constructed, sequences were synthesized and expression vectors were constructed, and the antibodies were purified by suspension expression in 293F cells, and 6H6-5, 6H6-25 and 6H6-29 antibody molecules were selected as the humanized 6H6 antibody versions by in vitro affinity determination. The heavy chain constant region of the antibody can be the sequence shown in SEQ ID NO: 60; the light chain constant region of the antibody can be the sequence shown in SEQ ID NO: 61.

[0326]

[0327] (2) Binding affinity test

[0328] The binding affinity of the huIgG1 type PD-1 antibody was measured by surface plasmon resonance (SPR) using a Biacore T200 biosensor equipped with a pre-fixed protein A sensor chip. The humanized antibody (1 ug / ml) was injected into the Biacore T200 biosensor at 10 ul / min for about 24-33 seconds, and the desired protein density (about 44-57 response units) was reached. Then the human PD1 protein (PD-1 Protein, Human, Recombinant His Tag) was injected at a concentration of 50 ug / ml, 25 ug / ml, 12.5 ug / ml, 6.25 ug / ml, 3.125 ug / ml, 1.5625 ug / ml at 30 ul / min for 300 seconds, and the dissociation was detected for 250 seconds. Figure 8 、 9 , 10 and 11 respectively show the antigen binding affinity results of 6H6, 6H6-5, 6H6-25, 6H6-29 antibodies.

[0329] Antibody 6H6 6H6-5 6H6-25 6H6-29 Affinity (KD) 4.50E-08 1.09E-08 8.07E-09 1.78E-08

[0330] (3) In vitro neutralization test of antibody

[0331] An in vitro neutralization assay was performed using ELISA to verify the ability of the humanized 6H6 huIgG1 antibody to block PD-L1 binding to PD-1 after humanization: PD-L1-huIgG1 Fc (Kactus Biosystems) was coated at 1 ug / ml 100ul per well at 4 degrees Celsius overnight, plates were washed and tapped dry before blocking with 1% BSA in PBS 200ul / well for 1 hour at 37 degrees Celsius. Plates were washed 3 times with 0.1% PBST and tapped dry before incubating with a mixture of scFv-huIgG1 Fc antibody and Biotinylated PD-1-huIgG1 Fc (Kactus Biosystems) for 1 hour at room temperature (2ug / ml 50ul of PD-1-huIgG1 Fc mixed with 50ul of antibody starting at 160ug / ml and doubling dilutions, for a total of 15 dilutions). Plates were washed 3 times with 0.1% PBST and Streptavidin-HRP (R&D Systems) was added at 100ul per well (1:200 dilution) for 1 hour at room temperature. Plates were washed 6 times with 0.1% PBST before adding TMB developing solution at 100ul per well for 10 minutes at room temperature, and stopping solution was added at 100ul per well before reading OD450 using a microplate reader. Data was analyzed and the half maximal inhibitory concentration IC50 of 6H6 and humanized 6H6-5, 6H6-25, 6H6-29 huIgG1 antibodies for PD-1 and PD-L1 binding was calculated 50 As shown in Figure 12

[0332] Antibody 6H6 6H6-5 6H6-25 6H6-29 IC 50 (ug / ml) 2.6477 2.9335 0.8004 1.9073

[0333] The foregoing detailed description has been presented for purposes of illustrations and description. It is not intended to be exhaustive or to limit the claims to the precise form disclosed. Numerous modifications and variations are possible in light of the above teachings. It is intended that the subj ect matter claimed comprise any such modifications and variations as fall within the scope of the claims and their equivalents.​

Claims

1. An antigen binding protein that binds PD-1, the antigen binding protein comprising a light chain variable region and a heavy chain variable region, the heavy chain variable region comprising a HCDR1, a HCDR2, and a HCDR3, the amino acid sequence of the HCDR1 is set forth in SEQ ID NO: 1; the amino acid sequence of the HCDR2 is set forth in SEQ ID NO: 2; the amino acid sequence of the HCDR3 is set forth in SEQ ID NO: 3; the light chain variable region comprising a LCDR1, a LCDR2, and a LCDR3, the amino acid sequence of the LCDR1 is set forth in SEQ ID NO: 4; the sequence of the LCDR2 is set forth in SEQ ID NO: 5; the amino acid sequence of the LCDR3 is set forth in SEQ ID NO: 6; and, the heavy chain variable region of the antigen binding protein comprises FR1 to 4 in an antibody heavy chain variable region VH, the VH comprising an amino acid sequence set forth in SEQ ID NO: 67; and the light chain variable region of the antigen binding protein comprises FR1 to 4 in an antibody light chain variable region VL, the VL comprising an amino acid sequence set forth in SEQ ID NO:

68.

2. The antigen binding protein of claim 1, the amino acid sequence of the heavy chain variable region of the antigen binding protein is set forth in any one of SEQ ID NO: 54 to 56.

3. The antigen binding protein of claim 1, the amino acid sequence of the light chain variable region of the antigen binding protein is set forth in any one of SEQ ID NO: 57 to 59.

4. The antigen binding protein of claim 1, the heavy chain variable region of the antigen binding protein comprises an amino acid sequence set forth in SEQ ID NO: 54, and the light chain variable region of the antigen binding protein comprises an amino acid sequence set forth in SEQ ID NO:

57.

5. The antigen binding protein of claim 1, the heavy chain variable region of the antigen binding protein comprises an amino acid sequence set forth in SEQ ID NO: 55, and the light chain variable region of the antigen binding protein comprises an amino acid sequence set forth in SEQ ID NO:

58.

6. The antigen binding protein of claim 1, the heavy chain variable region of the antigen binding protein comprises an amino acid sequence set forth in SEQ ID NO: 56, and the light chain variable region of the antigen binding protein comprises an amino acid sequence set forth in SEQ ID NO:

59.

7. The antigen binding protein of claim 1, the antigen binding protein further comprises an antibody heavy chain constant region.

8. The antigen binding protein of claim 7, the antibody heavy chain constant region of the antigen binding protein comprises an amino acid sequence set forth in SEQ ID NO:

60.

9. The antigen binding protein of claim 1, the antigen binding protein further comprises an antibody light chain constant region.

10. The antigen binding protein of claim 9, the antibody light chain constant region of the antigen binding protein comprises an amino acid sequence set forth in SEQ ID NO:

61.

11. The antigen binding protein of any one of claims 1-10, which is an antibody or an antigen binding fragment thereof.

12. The antigen binding protein of any one of claims 1-10, which has one or more of the properties selected from the group consisting of: the ability to bind human PD-1; the ability to block PD-1 and PD-L1 binding; the ability to block PD-1 and PD-L2 binding; the ability to stimulate secretion of IL-2, TNF-a, and / or IFN-g in immune cells; the ability to inhibit tumor growth and / or tumor cell proliferation; and the ability to increase the killing capacity of immune cells.

13. A nucleic acid encoding the antigen binding protein of any one of claims 1-10.

14. A vector comprising the nucleic acid molecule of claim 13.

15. A cell expressing the antigen binding protein of any one of claims 1-10, and / or comprising the nucleic acid of claim 13.

16. A pharmaceutical composition comprising the antigen binding protein of any one of claims 1-10, the nucleic acid molecule of claim 13, the vector of claim 14, and / or the cell of claim 15, and optionally a pharmaceutically acceptable adjuvant.

17. An immunoconjugate comprising the antigen binding protein of any one of claims 1-10, and a conjugating moiety selected from the group consisting of a detectable label, a radionuclide, or an enzyme.

18. Use of the antigen binding protein of any one of claims 1-10, the nucleic acid molecule of claim 13, the vector of claim 14, the cell of claim 15, the pharmaceutical composition of claim 16, and / or the immunoconjugate of claim 17 in the manufacture of a medicament for treating a PD-1 mediated disease or disorder; wherein the disease or disorder is a solid tumor.

19. A method for affecting cytokine production by target cells for non-therapeutic and / or diagnostic purposes, the method comprising administering the antigen binding protein of any one of claims 1-10, the nucleic acid of claim 13, a vector comprising the nucleic acid, and / or a cell expressing the antigen binding protein or comprising the nucleic acid and / or the vector.

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