Monoclonal antibody specifically binding to PD-1 and pharmaceutical use thereof

Abstract

The present invention relates to the field of immunotherapy and molecular immunology, and in particular to a monoclonal antibody specifically binding to PD-1 or an antigen-binding fragment thereof, and a pharmaceutical use thereof. The antibody or the antigen-binding fragment thereof can be particularly used for preventing or treating cancers, inflammation, and autoimmune diseases.

Description

Monoclonal antibodies specifically binding to PD-1 and their medical uses Technical Field

[0001] The present invention belongs to the fields of immunotherapy and molecular immunology, and specifically relates to a monoclonal antibody that specifically binds to PD-1 and its medical use. The antibody or its antigen-binding fragment can be particularly used to prevent or treat cancer, inflammation, and autoimmune diseases. Background Art

[0002] Programmed death receptor 1 (PD-1), also known as CD279, is encoded by the PDCD1 gene located on the long arm of chromosome 2, region 37, band 3 (2q37.3). It is a 55kD transmembrane protein (Agata et al., (1996), Int. Immunol. 8:765-772). PD-1 is a member of the immunoglobulin B7-CD28 family and consists of an extracellular domain, a hydrophobic transmembrane domain, and an intracellular domain. The intracellular domain contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) and an immunoreceptor tyrosine-based switch motif (ITSM). Activation of the ITSM is closely linked to effector T cell activity (Ishida et al., (1992) EMBO J 11:3887-3895). PD-1 protein is expressed in B lymphocytes, T lymphocytes, natural killer cells (NK), monocytes and dendritic cells (DC) (Duraiswamy, et al., (2013) Cancer Res. 73: 6900-12).

[0003] PD-1 has two ligands: PD-L1 (also known as B7-H1 and CD274) and PD-L2 (also known as B7-DC and CD273). Both are membrane proteins with IgV-like and IgC-like domains in their extracellular regions. PD-L1 is widely expressed on the surfaces of hematopoietic and non-hematopoietic cells, as well as tumor cells (Lesterhuis et al., (2011) Mol Immunol 49:1-3). PD-L1 is considered the primary ligand for PD-1, but PD-L2 has a 2- to 6-fold higher affinity for PD-1. PD-L2 is primarily expressed on antigen-presenting cells and Th2 cells, with low expression in other cells. Therefore, it generally has no effect on the binding of PD-L1 to PD-1 (Rozali et al., (2012) Clin. Dev. Immunol. 2012:656-340).

[0004] Activation of the PD-1 / PD-L1 signaling pathway can lead to immunosuppressive activity. It has been found that the aggregation of PD-1 induced by PD-L1 promotes the recruitment of SHP2 phosphatase, which then inhibits T cell activity through dephosphorylation of CD28 (Hui et al., Science, 355:1428-1433 (2017)). Conversely, the lack or lack of activity of PD-1 can lead to autoimmunity. For example, in C57BL / 6 mice, knockout of PD-1 leads to the development of a lupus-like syndrome (see, Nishimura et al., Immunity, 11:141-1151 (1999)). In humans, mutations in the PD-1 gene are highly correlated with the development and progression of systemic lupus erythematosus, type 1 diabetes, rheumatoid arthritis, and multiple sclerosis (see, Nielsen et al., Tisnie Antigens, d2(6):492-497 (2003); Bertsias et al., Arthritis & Rheumatism, 60(1):207-218 (2009); Ni et al., Hum Genet 121(2):223-232 (2007); Tahoori et al., Clin. Exp. Rheumatol, 29(5):763-767 (2011) and Kroner et al., Ann. Neurol, 58(1):50-57 (2005)). In this case, it would be beneficial to develop a PD-1 agonist, such as a PD-1 agonist antibody, to promote the immunosuppressive signal of PD-1, thereby inhibiting or treating some inflammatory or autoimmune diseases. This provides a new opportunity for the development of innovative antibody drugs targeting some immune checkpoints and new ideas for the treatment of some immune diseases. Summary of the Invention

[0005] The inventors of this application used hybridoma technology to obtain an anti-PD-1 agonist monoclonal antibody with excellent efficacy in promoting PD-1 activity and successfully humanized it. These antibodies have great potential for the preparation of drugs for activating PD-1 activity or levels, as well as for the treatment and adjuvant treatment of inflammatory and autoimmune diseases.

[0006] In one aspect, the present disclosure provides a monoclonal antibody or an antigen-binding fragment thereof that specifically binds to PD-1, comprising a heavy chain CDR and a light chain CDR, wherein

[0007] The amino acid sequence of CDR1 of the heavy chain comprises SEQ ID NO: 15, 21, 27, 33, 39, 50 or 56, or a sequence having two or fewer substituted amino acids compared to SEQ ID NO: 15, 21, 27, 33, 39, 50 or 56;

[0008] The amino acid sequence of the heavy chain CDR2 comprises SEQ ID NO: 16, 22, 28, 34, 40, 51 or 57, or a sequence having two or fewer substituted amino acids compared to SEQ ID NO: 16, 22, 28, 34, 40, 51 or 57, wherein X in SEQ ID NO: 40 is any amino acid;

[0009] The amino acid sequence of the heavy chain CDR3 comprises SEQ ID NO: 17, 23, 29, 35, 44, 52 or 58, or a sequence having two or fewer substituted amino acids compared to SEQ ID NO: 17, 23, 29, 35, 44, 52 or 58;

[0010] The amino acid sequence of CDR1 of the light chain comprises SEQ ID NO: 18, 24, 30, 36, 45, 53 or 59, or a sequence having two or fewer substituted amino acids compared to SEQ ID NO: 18, 24, 30, 36, 45, 53 or 59;

[0011] The amino acid sequence of CDR2 of the light chain comprises SEQ ID NO: 19, 25, 31, 37, 46, 54 or 60, or a sequence having two or fewer substituted amino acids compared to SEQ ID NO: 19, 25, 31, 37, 46, 54 or 60; and

[0012] The amino acid sequence of the light chain CDR3 comprises SEQ ID NO: 20, 26, 32, 38, 47, 55 or 61, or a sequence having two or fewer substituted amino acids compared to SEQ ID NO: 20, 26, 32, 38, 47, 55 or 61, wherein X in SEQ ID NO: 47 is any amino acid.

[0013] In some embodiments, X in the amino acid sequence of SEQ ID NO: 40 is N, Q, or V.

[0014] In some embodiments, X in the amino acid sequence of SEQ ID NO: 47 is N or T.

[0015] In some embodiments, the replacement amino acid is a conservatively substituted amino acid.

[0016] In some embodiments, the amino acid sequences of CDR1, CDR2, and CDR3 of the heavy chain, and the amino acid sequences of CDR1, CDR2, and CDR3 of the light chain are selected from one of the CDR combinations shown in ag:

[0017] a: The sequences of the heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 comprise SEQ ID NOs: 15-17, in order, and the sequences of the light chain CDR1, light chain CDR2 and light chain CDR3 comprise SEQ ID NOs: 18-20;

[0018] b: The sequences of the heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 comprise SEQ ID NOs: 21-23, in sequence; the sequences of the light chain CDR1, light chain CDR2, and light chain CDR3 comprise SEQ ID NOs: 24-26, in sequence;

[0019] c: The sequences of the heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 comprise SEQ ID NOs: 27-29, in sequence; the sequences of the light chain CDR1, light chain CDR2, and light chain CDR3 comprise SEQ ID NOs: 30-32, in sequence;

[0020] d: the sequences of the heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 comprise SEQ ID NOs: 33-35, in sequence; the sequences of the light chain CDR1, light chain CDR2, and light chain CDR3 comprise SEQ ID NOs: 36-38, in sequence;

[0021] e: The sequences of the heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 comprise SEQ ID NOs: 39, 40, and 44, respectively; the sequences of the light chain CDR1, light chain CDR2, and light chain CDR3 comprise SEQ ID NOs: 45, 46, and 47, respectively;

[0022] f: the sequences of the heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 comprise, in order, SEQ ID NOs: 50-52, and the sequences of the light chain CDR1, light chain CDR2, and light chain CDR3 comprise, in order, SEQ ID NOs: 53-55; or

[0023] g: The sequences of the heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 contain SEQ ID NOs: 56-58, in sequence; the sequences of the light chain CDR1, light chain CDR2 and light chain CDR3 contain SEQ ID NOs: 59-61, in sequence.

[0024] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is selected from any one of SEQ ID NO: 1, 3, 5, 7, 9, 11 and 13; and the amino acid sequence of the light chain variable region is selected from any one of SEQ ID NO: 2, 4, 6, 8, 10, 12 and 14.

[0025] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region are humanized.

[0026] In some embodiments, the amino acid sequence of the humanized heavy chain variable region is selected from any one of SEQ ID NO: 62, 64, 66, 68, 70, 71, 72 and 75; the amino acid sequence of the humanized light chain variable region is selected from any one of SEQ ID NO: 63, 65, 67, 69, 73, 74 and 76.

[0027] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain, wherein

[0028] The amino acid sequence of the heavy chain is SEQ ID NO: 77, and the amino acid sequence of the light chain is selected from SEQ ID NO: 80 or SEQ ID NO: 81; or

[0029] The amino acid sequence of the heavy chain is SEQ ID NO: 78 or SEQ ID NO: 79, and the amino acid sequence of the light chain is SEQ ID NO: 81; or

[0030] The amino acid sequence of the heavy chain is SEQ ID NO: 82, and the amino acid sequence of the light chain is SEQ ID NO: 83; or

[0031] The amino acid sequence of the heavy chain is SEQ ID NO: 84, and the amino acid sequence of the light chain is SEQ ID NO: 85; or

[0032] The amino acid sequence of the heavy chain is SEQ ID NO: 86, and the amino acid sequence of the light chain is SEQ ID NO: 87; or

[0033] The amino acid sequence of the heavy chain is SEQ ID NO: 88, and the amino acid sequence of the light chain is SEQ ID NO: 89; or

[0034] The amino acid sequence of the heavy chain is SEQ ID NO: 90, and the amino acid sequence of the light chain is SEQ ID NO: 91.

[0035] In one aspect, the present disclosure provides a pharmaceutical composition comprising a therapeutically effective amount of the antibody or antigen-binding fragment thereof and a pharmaceutically acceptable carrier.

[0036] In one aspect, the present disclosure provides the use of the antibody or its antigen-binding fragment in the preparation of a drug, which is used to activate PD-1 activity or PD-1 expression level, enhance PD-1's immunosuppression of the body, inhibit T lymphocytes, inhibit T lymphocytes from expressing cytokines such as INF-γ, and prevent, treat or assist in the treatment of inflammatory and autoimmune diseases.

[0037] In one aspect, the present disclosure provides use of the antibody or antigen-binding fragment thereof in the preparation of a medicament for treating a disease or disorder, wherein the disease or disorder is cancer or an autoimmune disease.

[0038] In some embodiments, the cancer is selected from the group consisting of colorectal cancer, colon cancer, renal cell carcinoma, breast cancer, epithelial squamous cell carcinoma, melanoma, myeloma, gastric cancer, brain cancer, lung cancer, pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, prostate cancer, testicular cancer, thyroid cancer, head and neck cancer, leukemia, and lymphoma.

[0039] In one aspect, the present disclosure provides a nucleic acid molecule, wherein the nucleic acid molecule encodes the above-mentioned antibody or antigen-binding fragment thereof.

[0040] In one aspect, the present disclosure provides an expression vector, wherein the vector comprises the sequence of the above-mentioned nucleic acid molecule and an expression control sequence related to the sequence.

[0041] In one aspect, the present disclosure provides a monoclonal antibody conjugate comprising a monoclonal antibody and a conjugated moiety, wherein the monoclonal antibody is the above-mentioned antibody or an antigen-binding fragment thereof, and the conjugated moiety is selected from one or more of a radionuclide, a pharmaceutical agent, a toxin, a cytokine, a cytokine receptor fragment, an enzyme, fluorescein, and biotin.

[0042] In one aspect, the present disclosure provides the use of the above-mentioned monoclonal antibody conjugate in the preparation of a drug, wherein the drug is used to activate PD-1 activity or PD-1 expression level, enhance the immunosuppressive effect of PD-1 on the body, inhibit T lymphocytes, inhibit T lymphocytes from expressing cytokines such as INF-γ, and prevent, treat or assist in the treatment of inflammatory and autoimmune diseases.

[0043] In one aspect, the present disclosure provides a method for activating PD-1 activity or PD-1 expression level, enhancing PD-1's immunosuppression of the body, inhibiting T lymphocytes, inhibiting T lymphocytes from expressing cytokines such as INF-γ, and preventing, treating or adjuvant treating inflammatory and autoimmune diseases, the method comprising administering a therapeutically effective amount of the above-mentioned antibody or its antigen-binding fragment or a therapeutically effective amount of a monoclonal antibody conjugate to a subject in need.

[0044] In one aspect, the present disclosure provides a method for treating a disease or condition in a subject in need thereof, comprising administering a therapeutically effective amount of the above-mentioned antibody or antigen-binding fragment thereof, or a therapeutically effective amount of a monoclonal antibody conjugate to a subject in need thereof, wherein the disease or condition is cancer or an autoimmune disease.

[0045] In some embodiments, the cancer is selected from the group consisting of colorectal cancer, colon cancer, renal cell carcinoma, breast cancer, epithelial squamous cell carcinoma, melanoma, myeloma, gastric cancer, brain cancer, lung cancer, pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, prostate cancer, testicular cancer, thyroid cancer, head and neck cancer, leukemia, and lymphoma.

[0046] In one aspect, the present disclosure provides a kit comprising

[0047] a) the antibody or the pharmaceutical composition; and

[0048] b) Instructions for use. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1: ELISA analysis of PD-1 agonist hybridoma antibody binding to hPD-1 antigen protein.

[0050] Figure 2: ELISA analysis of PD-1 agonist hybridoma antibodies binding to monkey (cyno)-PD-1 antigen protein.

[0051] Figure 3: Activation activity of hybridoma antibodies against PD-1 was measured using a CD3-mediated reporter gene assay.

[0052] FIG4 : Blocking activity of PD-1 agonist hybridoma antibodies against PD-L1 binding to PD-1 determined by ELISA.

[0053] Figure 5: ELISA analysis of binding of humanized PD1 agonistic antibodies to human PD-1.

[0054] Figure 6: ELISA analysis of binding of humanized PD1 agonistic antibodies to cyno PD-1.

[0055] Figure 7: CD3-induced reporter gene assay was used to detect the activation activity of humanized PD-1 agonist antibodies on PD-1 molecules.

[0056] FIG8 : ADCC activity of humanized PD1 agonist antibodies was measured using an ADCC reporter gene assay.

[0057] FIG9 : Effects of humanized PD-1 agonist antibodies on T cell cytokine release measured using MLR assay.

[0058] FIG10 : Detection of the effect of humanized PD-1 agonist antibody on SEB-induced total CD4+ T cell proliferation.

[0059] Figure 11: Detection of the effect of humanized PD-1 agonist antibody on CEFTA-induced CD4+ memory T cell proliferation.

[0060] FIG12 : ADCC activity of humanized PD1 agonist antibodies was measured using an ADCC reporter gene assay.

[0061] FIG13 : ELISA analysis of the binding of affinity-matured PD1 agonist antibodies to human PD1.

[0062] FIG14 : FACS analysis of affinity-matured PD1 agonist antibodies binding to Jurkat-PD-1 cells.

[0063] FIG15 : CD3 antibody-induced reporter gene assay to measure the activation of PD-1 molecules by PD-1 agonist antibodies after affinity maturation.

[0064] FIG16 : FACS analysis of the cell-killing activity of PD-1 agonist antibodies after affinity maturation.

[0065] FIG17 : Effects of affinity-matured PD-1 agonist antibodies on T cell cytokine release were measured using an MLR assay.

[0066] FIG18 : Determination of the effect of affinity-matured PD-1 agonist antibodies on SEB-induced total CD4+ T cell proliferation.

[0067] FIG19 : Determination of the effect of affinity-matured PD-1 agonist antibodies on CEFTA-induced CD4+ memory T cell proliferation. DETAILED DESCRIPTION

[0068] The inventors of this application used hybridoma technology to obtain an anti-PD-1 agonist monoclonal antibody with excellent efficacy in promoting PD-1 activity and successfully humanized it. These antibodies have great potential for the preparation of drugs for activating PD-1 activity or levels, as well as for the treatment and adjuvant treatment of inflammatory and autoimmune diseases.

[0069] The present disclosure provides monoclonal antibodies that specifically bind to PD-1. Administering a therapeutically effective amount of an anti-PD-1 antibody of the present disclosure to a subject in need thereof can be used to treat diseases or conditions, including inflammation, cancer, and autoimmune diseases.

[0070] The antibodies disclosed herein provide multiple advantages, such as specific binding to PD-1, activation of PD-1 activity or PD-1 expression level, enhancement of the immunosuppressive effect of PD-1 on the body, inhibition of T lymphocytes, inhibition of T lymphocyte expression of cytokines such as INF-γ, prevention, treatment or adjuvant treatment of inflammatory and autoimmune diseases, etc.

[0071] Unless otherwise defined, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. In the event of a conflict, the definitions in this specification shall prevail.

[0072] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the present invention. Although only certain exemplary materials and methods are described herein, many methods and materials similar or equivalent to those described herein can be used in the practice of the present disclosure.

[0073] Unless the context clearly indicates otherwise, the singular forms "a," "an," and "the" as used herein are intended to include the plural forms as well. In addition, the open-ended expressions "including" and "comprising" are interpreted as also including structural components or method steps that are not mentioned, but it should be noted that such open-ended expressions also cover situations where the composition consists only of the components and method steps (i.e., they cover situations where the closed-ended expression "consists of...").

[0074] In general, the term "about" is used herein to refer to a numerical value above and below the stated value with a modification of 5%.

[0075] As used in the entire text, range is used as a shorthand form for describing each numerical value and all numerical values ​​within the range. Any numerical value within the range, such as an integer value, a value that increases progressively by one tenth (when the end value of the range is one decimal place), or a value that increases progressively by one hundredth (when the end value of the range is two decimal places) can be selected as the endpoint of the range. For example, range 1-10 is used to describe all numerical values ​​within the range, such as 1, 2, 3, 4, 5, 6, 7, 8 ... 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, and 10 (values ​​that increase progressively by one tenth), and includes all subranges, such as 1-1.5, 2.0-3.0, 4.0-5.0, 6.0-7.0, 8.0-9.0, etc.

[0076] The term "antibody" as used herein may include whole antibodies and any antigen-binding fragments thereof (i.e., "antigen-binding portion") or single chains. In one embodiment, an "antibody" refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or an antigen-binding fragment thereof. Each heavy chain comprises a heavy chain variable region (herein referred to as a V H ) and heavy chain constant region. In some naturally occurring IgG, IgD and IgA antibodies, the heavy chain constant region comprises three domains, CH1, CH2 and CH3. In some naturally occurring antibodies, each light chain comprises a light chain variable region (herein referred to as V L ) and the light chain constant region. The light chain constant region comprises one domain, CL. V H and V L The V domains can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), alternating with regions of greater conserved expression, termed framework regions (FRs). H and V L It comprises three CDRs and four framework regions (FRs), arranged in the following order from amino terminus to carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains comprise a binding domain that interacts with an antigen. The constant region of an antibody mediates the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. Unless otherwise indicated, immunoglobulins may be from any commonly known isotype, including but not limited to IgA, secretory IgA, IgG, and IgM. IgG isotypes are divided into subclasses in certain species: IgG1, IgG2, IgG3, and IgG4 in humans, and IgG1, IgG2a, IgG2b, and IgG3 in mice. Immunoglobulins, for example, human IgG1, exist as several allotypes that differ from each other at most in a few amino acids.

[0077] Unless otherwise indicated, "antibody" may include, for example, whole antibodies, single-chain antibodies (scFv), heavy-chain antibodies, single-domain antibodies, Fab fragments, Fab' fragments, F(ab')2, Fv, Fd fragments, bispecific antibodies (BsAb), etc.; monoclonal and polyclonal antibodies; chimeric and humanized antibodies; human and non-human antibodies; fully synthetic antibodies, etc.

[0078] As used herein, an "antigen-binding fragment" of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to the antigen to which the intact antibody binds. An "antigen-binding fragment" is any proteinaceous structure that can exhibit binding affinity for a specific antigen. Antigen-binding fragments include those provided by any known technology such as enzymatic cleavage, peptide synthesis, and recombinant technology. Some antigen-binding fragments consist of portions of intact antibodies that retain the antigen-binding specificity of the parent antibody molecule. For example, an antigen-binding fragment may comprise at least one variable region (heavy chain variable region or light chain variable region) or one or more CDRs of an antibody known to bind to a specific antigen. Examples of suitable antigen-binding fragments include, but are not limited to, diabodies and single-chain molecules, as well as Fab, F(ab')2, Fc, Fabc, and Fv molecules; single-chain (Sc) antibodies; single antibody light chains; single antibody heavy chains; chimeric fusions between antibody chains or CDRs and other proteins; protein scaffolds; heavy chain monomers or dimers; light chain monomers or dimers; dimers consisting of one heavy chain and one light chain; monovalent fragments consisting of VL, VH, CL, and CH1 domains; or monovalent antibodies as described in WO2007059782; a bivalent fragment comprising two Fab fragments linked by a disulfide bond in the hinge region; an Fd fragment consisting essentially of the VH and CH1 domains; an Fv fragment consisting essentially of the VL and VH domains of a single arm of an antibody; a dAb fragment consisting essentially of a VH domain, also known as a domain antibody; alpacas or nanobodies; isolated complementarity-determining regions (CDRs), etc. All antibody isotypes can be used to generate antigen-binding fragments. Additionally, antigen-binding fragments may include non-antibody proteinaceous frameworks that can be successfully incorporated into polypeptide segments in an orientation that confers affinity for a given antigen of interest (such as a protein scaffold). Antigen-binding fragments may be produced recombinantly or by enzymatic or chemical cleavage of intact antibodies.

[0079] As used herein, the term sequence "identity" refers to the relationship between two or more polynucleotide sequences or two or more polypeptide sequences. When a position in a sequence is occupied by the same nucleic acid base or amino acid residue in the corresponding position of the comparison sequence, the sequence is called "identical" at that position. Percent sequence "identity" is calculated by determining the number of positions at which the same nucleic acid base or amino acid residue occurs in the two sequences to generate the number of "identical" positions. Then, the number of "identical" positions is divided by the total number of positions in the comparison window and multiplied by 100 to generate the percentage of sequence "identity". The percentage of "identity" is determined by comparing the two best aligned sequences in the comparison window. In order to optimally align sequences for comparison, the portion of the polynucleotide or polypeptide sequence in the comparison window can include additions or deletions called spaces, while the reference sequence remains constant. The best alignment is the alignment that produces the maximum possible number of "identical" positions between the reference sequence and the comparison sequence even with spaces. For example, the percent "identity" between two sequences can be determined using a version of the program "BLAST 2 Sequences" available from the National Center for Biotechnology Information (NCBI), which includes the programs BLASTN (for nucleotide sequence comparisons) and BLASTP (for polypeptide sequence comparisons), which are based on the algorithm of Karlin and Altschul (Proc. Natl. Acad. Sci. USA 90(12):5873-5877, 1993).

[0080] A "humanized" antibody refers to an antibody in which some, most, or all of the amino acids outside the CDR domain of a non-human antibody, such as a mouse antibody, are replaced by corresponding amino acids from a human immunoglobulin. In one embodiment of a humanized form of an antibody, some, most, or all of the amino acids outside the CDR domain are replaced by amino acids from a human immunoglobulin, while some, most, or all of the amino acids within one or more CDR regions are unchanged. Minor additions, deletions, insertions, substitutions, or modifications of amino acids are permitted as long as they do not abolish the ability of the antibody to bind to a specific antigen. A "humanized" antibody retains antigenic specificity similar to that of the original antibody.

[0081] A "chimeric antibody" refers to an antibody in which the variable region is derived from one species and the constant region is derived from another species, e.g., an antibody in which the variable region is derived from a mouse antibody and the constant region is derived from a human antibody. A "hybrid" antibody refers to an antibody having heavy and light chains of different types, e.g., a mouse (parent) heavy chain and a humanized light chain, or vice versa.

[0082] As used herein, the term "monoclonal antibody" refers to an antibody that displays a single binding specificity and affinity for a particular epitope, or an antibody composition in which all antibodies display a single binding specificity and affinity for a particular epitope.

[0083] As used herein, "isotype" refers to the antibody class encoded by the heavy chain constant region genes (eg, IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE antibodies).

[0084] As used herein, the terms "antibody-dependent cell-mediated cytotoxicity" and "ADCC" refer to a cell-mediated process in which nonspecific cytotoxic cells expressing FcγRs (e.g., monocytes, such as natural killer (NK) cells and macrophages) recognize bound antibodies (or other proteins capable of binding to FcγRs) on target cells and subsequently cause target cell lysis. In principle, any effector cell with an activating FcγR can be triggered to mediate ADCC. The primary cell mediating ADCC is NK cells, which express only FcγRIII, while monocytes can express FcγRI, FcγRII, and FcγRIII depending on their activation, localization, or differentiation state.

[0085] In one aspect, the present disclosure provides a monoclonal antibody or an antigen-binding fragment thereof that specifically binds to PD-1, comprising a heavy chain CDR and a light chain CDR, wherein

[0086] The amino acid sequence of CDR1 of the heavy chain comprises SEQ ID NO: 15, 21, 27, 33, 39, 50 or 56, or a sequence having two or fewer substituted amino acids compared to SEQ ID NO: 15, 21, 27, 33, 39, 50 or 56;

[0087] The amino acid sequence of the heavy chain CDR2 comprises SEQ ID NO: 16, 22, 28, 34, 40, 51 or 57, or a sequence having two or fewer substituted amino acids compared to SEQ ID NO: 16, 22, 28, 34, 40, 51 or 57, wherein X in SEQ ID NO: 40 is any amino acid;

[0088] The amino acid sequence of the heavy chain CDR3 comprises SEQ ID NO: 17, 23, 29, 35, 44, 52 or 58, or a sequence having two or fewer substituted amino acids compared to SEQ ID NO: 17, 23, 29, 35, 44, 52 or 58;

[0089] The amino acid sequence of CDR1 of the light chain comprises SEQ ID NO: 18, 24, 30, 36, 45, 53 or 59, or a sequence having two or fewer substituted amino acids compared to SEQ ID NO: 18, 24, 30, 36, 45, 53 or 59;

[0090] The amino acid sequence of CDR2 of the light chain comprises SEQ ID NO: 19, 25, 31, 37, 46, 54 or 60, or a sequence having two or fewer substituted amino acids compared to SEQ ID NO: 19, 25, 31, 37, 46, 54 or 60; and

[0091] The amino acid sequence of the light chain CDR3 comprises SEQ ID NO: 20, 26, 32, 38, 47, 55 or 61, or a sequence having two or fewer substituted amino acids compared to SEQ ID NO: 20, 26, 32, 38, 47, 55 or 61, wherein X in SEQ ID NO: 47 is any amino acid.

[0092] In some embodiments, the replacement amino acid is the amino acid of conservative substitution, for example, wherein the amino acid residue is replaced by the amino acid residue with similar side chain. The family of amino acid residues with similar side chain has been defined in the art. These families include amino acids with basic side chains (for example, lysine, arginine, histidine), acidic side chains (for example, aspartic acid, glutamic acid), uncharged polar side chains (for example, glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), non-polar side chains (for example, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), the side chains of β branches (for example, threonine, valine, isoleucine) and aromatic side chains (for example, tyrosine, phenylalanine, tryptophan, histidine).

[0093] In one aspect, the present disclosure provides an antibody that specifically binds to PD-1, wherein the antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3,

[0094] wherein the heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 respectively comprise an amino acid sequence that is at least 70%, 75%, 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% or 100% identical to:

[0095] (1) SEQ ID NO:15, SEQ ID NO:16 and SEQ ID NO:17;

[0096] (2) SEQ ID NO:21, SEQ ID NO:22 and SEQ ID NO:23;

[0097] (3) SEQ ID NO:27, SEQ ID NO:28 and SEQ ID NO:29;

[0098] (4) SEQ ID NO:33, SEQ ID NO:34 and SEQ ID NO:35;

[0099] (5) SEQ ID NO:39, SEQ ID NO:40 and SEQ ID NO:44;

[0100] (6) SEQ ID NO: 50, SEQ ID NO: 51 and SEQ ID NO: 52; or

[0101] (7) SEQ ID NO:56, SEQ ID NO:57 and SEQ ID NO:58; and

[0102] The light chain variable region comprises a light chain CDR1, a light chain CDR2, and a light chain CDR3, wherein the light chain CDR1, the light chain CDR2, and the light chain CDR3 each comprise an amino acid sequence that is at least 70%, 75%, 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% or 100% identical to:

[0103] (1) SEQ ID NO:18, SEQ ID NO:19 and SEQ ID NO:20;

[0104] (2) SEQ ID NO:24, SEQ ID NO:25 and SEQ ID NO:26;

[0105] (3) SEQ ID NO:30, SEQ ID NO:31 and SEQ ID NO:32;

[0106] (4) SEQ ID NO:36, SEQ ID NO:37 and SEQ ID NO:38;

[0107] (5) SEQ ID NO:45, SEQ ID NO:46 and SEQ ID NO:47;

[0108] (6) SEQ ID NO: 53, SEQ ID NO: 54 and SEQ ID NO: 55; or

[0109] (7) SEQ ID NO:59, SEQ ID NO:60 and SEQ ID NO:61.

[0110] In some embodiments, the amino acid sequences of CDR1, CDR2, and CDR3 of the heavy chain, and the amino acid sequences of CDR1, CDR2, and CDR3 of the light chain are selected from one of the CDR combinations shown in af:

[0111] a: The sequences of the heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 comprise SEQ ID NOs: 15-17, in order, and the sequences of the light chain CDR1, light chain CDR2 and light chain CDR3 comprise SEQ ID NOs: 18-20;

[0112] b: The sequences of the heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 comprise SEQ ID NOs: 21-23, in sequence; the sequences of the light chain CDR1, light chain CDR2, and light chain CDR3 comprise SEQ ID NOs: 24-26, in sequence;

[0113] c: The sequences of the heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 comprise SEQ ID NOs: 27-29, in sequence; the sequences of the light chain CDR1, light chain CDR2, and light chain CDR3 comprise SEQ ID NOs: 30-32, in sequence;

[0114] d: the sequences of the heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 comprise SEQ ID NOs: 33-35, in sequence; the sequences of the light chain CDR1, light chain CDR2, and light chain CDR3 comprise SEQ ID NOs: 36-38, in sequence;

[0115] e: The sequences of the heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 comprise SEQ ID NOs: 39, 40, and 44, respectively; the sequences of the light chain CDR1, light chain CDR2, and light chain CDR3 comprise SEQ ID NOs: 45, 46, and 47, respectively;

[0116] f: the sequences of the heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 comprise, in order, SEQ ID NOs: 50-52, and the sequences of the light chain CDR1, light chain CDR2, and light chain CDR3 comprise, in order, SEQ ID NOs: 53-55; or

[0117] g: The sequences of the heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 contain SEQ ID NOs: 56-58, in sequence; the sequences of the light chain CDR1, light chain CDR2 and light chain CDR3 contain SEQ ID NOs: 59-61, in sequence.

[0118] In some embodiments, the heavy chain variable region comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% or 100% identical to any one of SEQ ID NOs: 1, 3, 5, 7, 9, 11 and 13.

[0119] In some embodiments, the light chain variable region comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% or 100% identical to any one of SEQ ID NOs: 2, 4, 6, 8, 10, 12, and 14.

[0120] In some embodiments, the heavy chain variable region and light chain variable region are humanized.

[0121] In some embodiments, the humanized heavy chain variable region comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% or 100% identical to any one of SEQ ID NOs: 62, 64, 66, 68, 70, 71, 72, and 75.

[0122] In some embodiments, the humanized light chain variable region comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% or 100% identical to any one of SEQ ID NOs: 63, 65, 67, 69, 73, 74, and 76.

[0123] In some embodiments, the heavy chain comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO:77, 78, 79, 82, 84, 86, 88 or 90.

[0124] In some embodiments, the light chain comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO:80, 81, 83, 85, 87, 89 or 91.

[0125] In some embodiments, the antibody or antigen-binding fragment thereof is selected from the group consisting of an intact antibody, a single-chain antibody (scFv), a heavy chain antibody, a single-domain antibody, a Fab fragment, a Fab' fragment, a F(ab')2, a Fv, a Fd fragment, and a bispecific antibody (BsAb).

[0126] In some embodiments, the antibody is a human antibody, a humanized antibody, or a chimeric antibody.

[0127] In some embodiments, the antibody is isolated.

[0128] In some embodiments, the antibody is an isolated monoclonal antibody.

[0129] Also provided are "conservative sequence modifications" to the antibody sequences provided herein, i.e., nucleotide and amino acid sequence modifications that do not eliminate binding of the antibody encoded by the nucleotide sequence or comprising the amino acid sequence to the antigen. For example, modifications can be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative sequence modifications include conservative amino acid substitutions, in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0130] In some embodiments, the antibodies exhibit potent antibody-dependent cell-mediated cytotoxicity (ADCC).

[0131] In one aspect, the present disclosure provides a pharmaceutical composition comprising a therapeutically effective amount of the antibody and a pharmaceutically acceptable carrier, which may be an inert or physiologically active carrier. As used herein, the term "pharmaceutically acceptable carrier" includes any physiologically compatible solvent, dispersion medium, coating, antibacterial agent, antifungal agent, etc. Examples of suitable carriers include water, saline, phosphate-buffered saline, dextrose, glycerol, ethanol, etc., and any combination thereof.

[0132] In one aspect, the present disclosure provides an isolated nucleic acid molecule encoding the antibody.

[0133] In one aspect, the present disclosure provides an expression vector comprising the nucleic acid molecule.

[0134] In one aspect, the present disclosure provides a host cell, which expresses the antibody, and / or contains the nucleic acid molecule or the expression vector.

[0135] In one aspect, the present disclosure provides the antibody or the pharmaceutical composition for use in treating a disease or disorder, wherein the disease or disorder is cancer or an autoimmune disease.

[0136] In some embodiments, the cancer is selected from the group consisting of colorectal cancer, colon cancer, renal cell carcinoma, breast cancer, epithelial squamous cell carcinoma, melanoma, myeloma, gastric cancer, brain cancer, lung cancer, pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, prostate cancer, testicular cancer, thyroid cancer, head and neck cancer, leukemia, and lymphoma.

[0137] In some embodiments, the antibodies or pharmaceutical compositions are for administration in combination with one or more additional chemotherapeutic agents, radiotherapeutic agents, cytokines, or other antibodies.

[0138] In one aspect, the present disclosure provides use of the antibody or the pharmaceutical composition in the preparation of a medicament for treating a disease or disorder, wherein the disease or disorder is cancer or an autoimmune disease.

[0139] In some embodiments, the cancer is selected from the group consisting of colorectal cancer, colon cancer, renal cell carcinoma, breast cancer, epithelial squamous cell carcinoma, melanoma, myeloma, gastric cancer, brain cancer, lung cancer, pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, prostate cancer, testicular cancer, thyroid cancer, head and neck cancer, leukemia, and lymphoma.

[0140] In some embodiments, the antibodies or pharmaceutical compositions are for administration in combination with one or more additional chemotherapeutic agents, radiotherapeutic agents, cytokines, or other antibodies.

[0141] In one aspect, the present disclosure provides a method of treating a disease or condition in a subject in need thereof, comprising administering the antibody or pharmaceutical composition in a therapeutically effective amount, wherein the disease or condition is cancer or an autoimmune disease.

[0142] In some embodiments, the cancer is selected from the group consisting of colorectal cancer, colon cancer, renal cell carcinoma, breast cancer, epithelial squamous cell carcinoma, melanoma, myeloma, gastric cancer, brain cancer, lung cancer, pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, prostate cancer, testicular cancer, thyroid cancer, head and neck cancer, leukemia, and lymphoma.

[0143] In some embodiments, the antibody or pharmaceutical composition is administered in combination with one or more additional chemotherapeutic agents, radiotherapeutic agents, cytokines, or other antibodies.

[0144] "Administer" refers to the use of any of the various methods and delivery systems known to those skilled in the art to introduce a composition comprising a therapeutic agent into a subject. The route of administration of the pharmaceutical composition of the present disclosure includes intravenous, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral administration routes, such as by injection or infusion. The phrase "parenteral administration" used herein refers to the administration method generally injected except enteral and topical administration, and includes but is not limited to intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardial, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion and in vivo electroporation. In some embodiments, the composition is administered by non-parenteral route, in some embodiments, by oral administration. Other non-parenteral routes include topical, epidermal or mucosal administration routes, such as intranasal, vaginal, rectal, sublingual or topical. Administration can also, for example, be performed once, repeatedly and / or over one or more extended time periods.

[0145] As used herein, the term "effective amount" refers to the amount of an active agent required to provide a therapeutic and / or prophylactic benefit to a subject.

[0146] The "subject in need thereof" refers to any mammal, such as but not limited to humans, horses, cows, cats, mice, rabbits, rats, goats, etc. Preferably, the mammal is a human.

[0147] In one aspect, the present disclosure provides a kit comprising

[0148] a) the antibody or the pharmaceutical composition; and

[0149] b) Instructions for use.

[0150] In some specific embodiments, the amino acid sequence information of the heavy chain variable region, light chain variable region, heavy chain, light chain, heavy chain CDR and light chain CDR of the murine or humanized PD-1 agonist antibody or functional fragment provided by the present invention is shown in Table 1 below.

[0151] Table 1. Sequence information

[0152] The inventors of this application used a recombinant PD-1 extracellular domain fragment as an antigen to immunize mice. After that, the mouse spleen cells were fused with myeloma cells to obtain hybridoma cells. By cloning and screening a large number of hybridoma cells multiple times, multiple monoclonal hybridoma cell lines were obtained. These hybridoma cell lines can secrete and produce monoclonal antibodies that specifically bind to PD-1. These monoclonal antibodies can effectively activate PD-1 and thus inhibit the reporter gene signal of the immune response. These monoclonal antibodies cannot effectively block the binding of PD-L1 to PD-1; these monoclonal antibodies can effectively bind to monkey (cyno) PD-1 protein.

[0153] Furthermore, the genes encoding the variable regions of the antibody light and heavy chains were cloned by RT-PCR (Reverse Transcription-Polymerase Chain Reaction), and humanized antibodies were constructed using the complementarity-determining regions graft (CDR-graft) method in the form of human IgG1.

[0154] In vitro functional experiments have shown that humanized PD-1 agonist antibodies can specifically bind to human and monkey (cyno) PD-1 proteins and effectively activate PD-1, thereby inhibiting the reporter gene signal of the immune response; humanized PD-1 agonist antibodies can effectively inhibit T cell cytokine release, T cell proliferation, and the proliferation of memory T cells.

[0155] In addition, these humanized antibodies have a significant role in mediating ADCC. This application further modified the affinity maturation of the humanized 54H8 antibody and enhanced ADCC activity; the modified antibody (hu54H8-G1DE-M15) enhances the specific binding to the human PD-1 protein, increases the activation activity of PD-1, and enhances the inhibition of T cell cytokine release, T cell and memory T cell proliferation. Compared with the positive controls of Eli Lilly's Peresolimab (LY3462817) and Anaptysbio's Rosenilimab (ANB030), the modified hu54H8-G1DE-M15 in this application has a stronger inhibitory effect on T cell cytokine release, T cell and memory T cell proliferation. The sequence of the positive control antibody Peresolimab comes from Eli Lilly's patent US20190270818A1, and the sequence of the positive control antibody ANB030 comes from Anaptysbio's patent WO2020247648A2.

[0156] The present invention is further described with reference to the following non-limiting examples: The following exemplifies the development, characterization, and efficacy of the six antibodies described above: 65A12, 69H4, 39A9, 67B1, 54H8, and 48E7.

[0157] Example 1

[0158] Construction of mouse hybridoma antibodies

[0159] Mouse immunization and hybridoma cell fusion. Using the human PD-1 extracellular region and mouse Fc fusion protein (PD-1-ECD-mFc) as an antigen, after being fully emulsified with an equal volume of complete Freund's adjuvant (Sigma, Cat.No.: F5581), 6-8 week old Balb / c mice (purchased from Zhaoyan (Suzhou) New Drug Research Center Co., Ltd.) were subcutaneously immunized with an antigen immunization dose of 50 μg / mouse. Subsequently, every two weeks, the same dose of antigen was fully emulsified with incomplete Freund's adjuvant (Sigma, Cat.No.: F5506) and the mice were immunized subcutaneously three times. After three immunizations, the mouse serum titer was measured, and booster immunization was performed intraperitoneally 3 days before fusion. Using PEG Hybri-Max (Sigma, Cat.No.: 7181) as a fusion agent, mouse spleen cells and SP2 / 0 cells were mixed at a ratio of 4:1. The fused cells were added to a 96-well plate (1×10 5 Cells were plated at 400 nm (1 / well) with 0.1 mL of 1× HAT medium (Invitrogen, Cat. No. 21060-017) per well. On day 3, 0.1 mL of HT medium (Invitrogen, Cat. No. 11067-030) was added. On day 7, the culture medium in the 96-well plate was aspirated and replaced with 0.2 mL of fresh HT medium. On day 9, the supernatant was collected for ELISA and FACS analysis.

[0160] ELISA screening of antibodies binding to PD-1-ECD: 96-well plates (Corning, Cat. No.: 9018) were coated with the extracellular domain of human or monkey PD-1 and human Fc fusion protein (PD-1-ECD-hFc), incubated at 4 degrees overnight, washed three times with washing buffer (PBS + 0.05% Tween20), and then incubated with blocking buffer (PBS + 1% BSA) for 1 hour; the 96-well plates were washed three times; hybridoma supernatant was added and incubated for 1 hour, and washed three times; 100 μL of 1:10000 diluted goat anti-mouse IgG secondary antibody (Thermo, Cat. No.: 31432) was added to each well, incubated at room temperature for 1 hour, and then washed three times; 100 μL of TMB (Beijing Bioscience, Cat. No.: ES-002) was added to each well for color development for 3 minutes, and then 100 μL / well of stop solution (2N The reaction was terminated with H2SO4 and the OD450 signal of each sample was measured using a microplate reader.

[0161] Hybridoma PD-1 agonist antibody reporter gene detection in activating PD-1 response: The human PD-1 agonist antibody was serially diluted to prepare a 2-fold concentration of antibody dilution solution, and 50uL of the volume was transferred to a 96-well plate. Then, 50uL of cell mixture was added, including 1e5 / well Jurkat-PD-NFAT reporter gene cells, a final concentration of 10ng / mL stimulator OKT3 (ebioscience, cat.no:16-0037-85) and 5e4 / well THP-1. After incubation at 37°C for 5 hours, Bright Glo (Promega, Cat.No:E2620) was added to the cell wells to detect the signal intensity of the reporter gene.

[0162] ELISA screening of antibodies that block the binding of PD-L1 to PD-1. Hybridoma antibodies that block the binding of PD-L1 to PD-1 were screened using an ELISA method. A 96-well ELISA plate was coated with PD-1-ECD-hFc and incubated overnight. After washing three times with wash buffer (PBS + 0.05% Tween 20), 200 μL of blocking buffer was added and incubated for 1 hour. After washing three times, 100 μL of hybridoma supernatant was added and incubated at room temperature for 1 hour. 100 μL of PD-L1-Biotin was added to the sample wells and incubated at room temperature for 1 hour. After washing three times, the secondary antibody Avidin HRP (Invitrogen, Cat. No.: 18-4100-51) was added and incubated for 30 minutes. TMB was added for color development for 3 minutes. The reaction was terminated with 100 μL / well of stop solution (2N H2SO4). The OD450 signal of each sample was measured using a microplate reader.

[0163] Subcloning of hybridomas. Using the limiting dilution method, hybridomas that can block the binding of PD-1 to PD-L1 were subcloned, and then the ELISA and FACS methods were repeatedly used for detection and screening to obtain positive hybridoma monoclones. The positive monoclonal hybridoma was cultured in 50 mL of serum-free culture medium (Invitrogen, Cat. No.: 12045-076) for 9 days, and the supernatant was collected by centrifugation. The monoclonal antibody was purified by Protein A affinity chromatography. After the purified antibody sample was concentrated by ultrafiltration centrifuge tube (Millipore, Cat. No.: ACS500024), the protein concentration was determined by the BCA method, and the endotoxin content of the antibody sample was detected by Ao reagent (Xiamen Limulus Reagent Biotechnology Co., Ltd.).

[0164] Purified hybridoma antibody samples were tested for binding to human or monkey PD-1 using ELISA and FACS. Their PD-1 activation activity was assessed using a reporter gene assay. Their PD-L1 binding activity was also assessed using ELISA. The results are shown in Tables 2-4 and Figures 1-4. The figures indicate that the antibody samples exhibited good binding activity to PD-1 and potent PD-1 activation. Furthermore, five of the six purified antibodies showed no blocking activity against PD-L1 binding to PD-1, while 48E7 demonstrated blocking activity against PD-L1 binding to PD-1.

[0165] Table 2. Binding of PD-1 hybridoma antibodies to human PD-1 detected by ELSIA

[0166] Table 3. Binding of PD-1 hybridoma antibodies to monkey PD-1 detected by ELSIA

[0167] Table 4. Detection of PD-1 activation activity of hybridoma antibodies using reporter gene assay

[0168] Example 2

[0169] Cloning of the variable region gene of PD-1 antibody

[0170] The PD-1 monoclonal hybridoma cell line was lysed using TRIzon (Cwbiotech, Cat. No.: CW0580), and total RNA from the hybridoma cells was extracted. The hybridoma RNA was reverse-transcribed into cDNA using the HiFi Script cDNA Synthesis Kit (Cwbiotech, Cat. No.: CW2569). Using the cDNA as a template, the variable region genes of the heavy and light chains of the antibody were amplified using PCR (Kettleborough et al., (1993) Eur. J. Immunology 23:206-211; Strebe et al., (2010) Antibody Engineering 1:3-14) with degenerate primers. The PCR amplification product was ligated into a T / A vector, transformed into DH5a competent cells, plated, and incubated overnight at 37°C. A single clone was picked from the culture plate, expanded, and the plasmid was extracted, and the antibody gene sequence was determined. Based on the antibody gene sequence, the complementary determining regions (CDRs) and framework regions were analyzed. The amino acid sequences of the variable regions and CDRs of the heavy and light chains of some antibodies are shown in Table 1.

[0171] Example 3

[0172] Humanization of murine PD-1 antibody

[0173] The complementary determinant cluster grafting method was used to humanize the PD-1 antibody. First, the human germline antibody sequences with the highest homology to the light and heavy chain variable region sequences of the mouse 65A12, 69H4, 39A9, 67B1, 54H8 and 4H6 antibodies were searched in the IMGT database. The germline selected for the humanization of the light chain variable region of the 65A12 antibody was IGKV2-29*02, and the humanization of the heavy chain variable region was IGHV3-15*01. The germline selected for the humanization of the light chain variable region of the 69H4 antibody was IGKV4-1*01, and the humanization of the heavy chain variable region was IGHV2-70*4. The germline selected for the humanization of the light chain variable region of the 39A9 antibody was IGHV1-39*01, and the humanization of the heavy chain variable region was IGHV1-69-2* The germline used for humanizing the light chain variable region of the 67B1 antibody was IGKV2-29*02, and the germline used for humanizing the heavy chain variable region was IGHV1-3*01. The germline used for humanizing the light chain variable region of the 54H8 antibody was IGKV4-1*01, and the germline used for humanizing the heavy chain variable region was IGHV2-70*4. The germline used for humanizing the light chain variable region of the 4H6 antibody was IGHV4-4*08, and the germline used for humanizing the heavy chain variable region was IGKV2-29*02. The CDR regions of the mouse antibody were retained, and the framework region sequences of the mouse antibody were replaced with those of the human germline antibody. A structural model of the mouse antibody was established, and the amino acids at each position in the framework regions of the human and corresponding mouse antibodies were compared one by one. If the use of the human amino acid sequence at a certain position in the framework region did not disrupt or alter the spatial structure of the CDR region, the human amino acid sequence was used at that position. Otherwise, the corresponding mouse sequence was used at that position (i.e., the mutation was back to the mouse sequence).

[0174] Based on structural simulations, the 3rd Gln of the humanized heavy chain of the 65A12 antibody was backmutated to Lys, the 39th Gly was backmutated to Ala, and the 78th Leu was backmutated to Val. The 2nd Ile of the humanized light chain of the 65A12 antibody was backmutated to Val, the 3rd Val was backmutated to Leu, and the 49th Tyr was backmutated to Phe. The 77th Gln of the humanized heavy chain of the 69H4 antibody was backmutated to Leu. The 72nd Thr of the humanized light chain of the 69H4 antibody was backmutated to Ile. The 24th Val of the humanized heavy chain of the 39A9 antibody was backmutated to Ala, the 27th Tyr was backmutated to Phe, the 28th Thr was backmutated to Asn, the 29th Phe was backmutated to Val, the 30th Thr was backmutated to Lys, the 48th Met was backmutated to Ile, and the 67th Val was backmutated to Ala. In the humanized light chain of the 39A9 antibody, Asp at position 70 was backmutated to Gly, and Thr at position 72 was backmutated to Ser. In the humanized heavy chain of the 67B1 antibody, Gln at position 1 was backmutated to Glu, Tyr at position 27 was backmutated to Phe, Thr at position 28 was backmutated to Asn, Phe at position 29 was backmutated to Ile, Thr at position 30 was backmutated to Lys, Met at position 48 was backmutated to Ile, Val at position 67 was backmutated to Ala, and Arg at position 71 was backmutated to Ala. In the humanized light chain of the 67B1 antibody, Ile at position 2 was backmutated to Val, and Val at position 3 was backmutated to Leu. In the humanized heavy chain of the 54H8 antibody, Gln at position 77 was backmutated to Leu, and Val at position 79 was backmutated to Phe. No backmutations were found in the humanized light chain of the 54H8 antibody. In the humanized heavy chain of the 4H6 antibody, Gly at position 27 was backmutated to Phe, Ile at position 29 was backmutated to Leu, Ser at position 30 was backmutated to Thr, Ile at position 37 was backmutated to Val, Ile at position 48 was backmutated to Leu, Val at position 67 was backmutated to Leu, Val at position 71 was backmutated to Arg, Thr at position 73 was backmutated to Asn, and Phe at position 78 was backmutated to Val. In the humanized light chain of the 4H6 antibody, Ile at position 2 was backmutated to Val, and Tyr at position 49 was backmutated to Ser.

[0175] The amino acid sequence numbers of the heavy chain and light chain variable regions of the 65A12 humanized antibody are SEQ ID NO:62 and SEQ ID NO:63, respectively. The amino acid sequence numbers of the heavy chain and light chain variable regions of the 69H4 humanized antibody are SEQ ID NO:64 and SEQ ID NO:65, respectively. The amino acid sequence numbers of the heavy chain and light chain variable regions of the 39A9 humanized antibody are SEQ ID NO:66 and SEQ ID NO:67, respectively. The amino acid sequence numbers of the heavy chain and light chain variable regions of the 67B1 humanized antibody are SEQ ID NO:68 and SEQ ID NO:69, respectively. The amino acid sequence numbers of the heavy chain and light chain variable regions of the 54H8 humanized antibody are SEQ ID NO:70 / 71 / 72 and SEQ ID NO:73 / 74, respectively. The amino acid sequence numbers of the heavy chain and light chain variable regions of the 4H6 humanized antibody are SEQ ID NO:75 and SEQ ID NO:76, respectively. Humanized antibodies 65A12, 69H4, 39A9, 67B1, 54H8, and 4H6 were constructed as IgG1 subtype. Additionally, humanized antibody 54H8 was constructed as an IgG1 subtype with enhanced ADCC (containing S239D and I332E mutations in the heavy chain) (SEQ ID NO: 77).

[0176] The nucleic acid fragments encoding the light and heavy chains of humanized antibodies 65A12, 69H4, 39A9, 67B1, 54H8, and 4H6 were synthesized and inserted into the expression vector pcDNA3.1. 200 ml of 293 cells (cell density 1×10 6 ), cultured with shaking at 37 degrees for 6 days, and the supernatant was collected by centrifugation. The humanized antibody was purified with Protein A, and the purified humanized antibody was used for activity detection.

[0177] Example 4

[0178] Activity detection of humanized PD-1 antibodies

[0179] ELISA and FACS were used to detect the binding of purified humanized antibody samples to PD-1, and a reporter gene was used to detect the activation effect of humanized PD-1 antibodies on PD-1 (for specific methods, refer to Example 1). The results of the activity assay of the humanized antibodies are shown in Tables 5-7 and Figures 5-7. The results show that the PD-1 antigen binding activity of the humanized PD-1 antibodies in this patent is generally good. From the ELISA results, the EC50 range is from 31.29 ng / ml to 347.5 ng / ml. Among them, hu39A9, hu54H8, hu69H4 and hu4H6 have relatively strong antigen binding abilities; their EC50s are 31.29, 54.19, 48.69 and 35.93 ng / ml, respectively. The data of PD-1 antigen binding by FACS analysis showed that hu65A12 had poor binding, while hu39A9 and hu4H6 had relatively good binding (EC50 of 67.73 and 51.07 ng / ml, respectively); hu54H8 and hu69H4 were next, with EC50 of 164.2 ng / ml and 173.1 ng / ml, respectively).

[0180] Table 5. EC50 of PD-1 humanized antibodies binding to PD-1 detected by ELSIA

[0181] Table 6. Binding of humanized PD-1 antibodies to monkey PD-1 detected by ELSIA

[0182] Table 7. Detection of PD-1 activation activity of humanized antibodies using reporter gene assay

[0183] Example 5

[0184] Detection of ADCC activity of humanized PD-1 agonist antibodies

[0185] Reporter gene assay for ADCC activity of humanized PD-1 antibodies: A serial dilution of the humanized PD-1 agonist antibody was prepared at a 2x concentration of the antibody diluent. 50 μL of the dilution was transferred to a 96-well plate. Then, 50 μL of a cell mixture (1e5 / well Jurkat-PD cells and 1e5 / well Jurkat-CD16-NFAT cells) was added. After incubation at 37°C for 5 hours, Bright Glo (Promega, Cat. No. E2620) was added to the cell wells to measure the reporter gene signal intensity. The results are shown in Table 8 and Figure 8. The results show that the humanized PD-1 antibodies of this patent can induce ADCC, with hu39A9 being the most potent, followed by hu54H8, hu67B1, and hu69H4, and hu65A12 being relatively weak.

[0186] Table 8. Reporter gene assay for ADCC activity of humanized PD-1 antibodies

[0187] Example 6

[0188] Inhibitory effects of humanized PD-1 agonist antibodies on T cell cytokine release, proliferation, and memory T cell proliferation

[0189] Effect of humanized PD-1 agonist antibodies on cytokine secretion in mixed lymphocyte reactions: 2e5 activated human PBMCs were seeded per well in a 96-well plate in a total volume of 200 μL. 40 ng / mL OKT3 (ebioscience, cat. no. 16-0037-85) was added as the stimulator and cultured at 37°C for 2 days. The humanized PD-1 agonist antibody was serially diluted to a 4-fold concentration of the antibody diluent. 50 μL of the diluted antibody was transferred to a 96-well plate. 150 μL of the cell mixture, consisting of 2e5 activated PBMCs per well, 25 ng / mL OKT3, and 5e4 THP-1 cells per well, was added. The plate was cultured at 37°C for 2 days. The supernatant was collected and cytokine concentrations were measured using an IFN-γ ELISA kit. The results are shown in Figure 9. The results show that the humanized PD-1 antibody described in this patent exhibits a significant inhibitory effect on cytokine release from T cells.

[0190] Effect of humanized PD-1 agonist antibodies on T cell proliferation: Humanized PD-1 agonist antibodies were serially diluted to a 4-fold concentration of the antibody diluent. 50 μL of the antibody diluent was transferred to a 96-well plate. 150 μL of a cell mixture containing 1e5 CFSE-labeled humanized PBMCs per well and stimulatory factor SEB (Toxin Technology, Cat. No. BT202) at a final concentration of 1 ng / mL was then added. The cells were cultured at 37°C for 3-4 days. Activated PBMCs were harvested and 2.5 μL / well of CD4-PE-cy7 (Biolegend, Cat. No. 300512) was added. The cells were incubated at 4°C for 30 minutes, washed, and the percentage of T cell proliferation in CD4+ T cells was measured by flow cytometry. The results are shown in Figure 10. The results show that the humanized PD-1 antibodies described in this patent exhibited a significant inhibitory effect on T cell proliferation.

[0191] Effect of human PD-1 agonist antibodies on memory T cell proliferation: Human PD-1 agonist antibodies were serially diluted to a 4-fold concentration of the antibody diluent. 50 μL of the antibody diluent was transferred to a 96-well plate. 150 μL of a cell mixture containing 2e5 / well CFSE-labeled human PBMCs and a final concentration of 100 ng / mL of the stimulatory agent CEFTA (Mabtech, Cat. No. C34554) was then added. The cells were cultured at 37°C for 7-10 days. Activated PBMCs were harvested and 2.5 μL / well of CD4-PE-cy7 (Biolegend, Cat. No. 300512) was added. The cells were incubated at 4°C for 30 minutes, washed, and the percentage of memory T cell proliferation in CD4+ T cells was determined by flow cytometry. The results are shown in Figure 11. The results show that the humanized PD-1 antibody described in this patent exhibits a significant inhibitory effect on the proliferation of memory T cells.

[0192] Example 7

[0193] ADCC activity enhancement modification of humanized PD-1 agonist antibody hu54H8 and ADCC-enhanced version for further antibody construction

[0194] Based on comprehensive considerations, particularly those based on primary cell assays, hu54H8 was selected as a candidate antibody for affinity maturation. Furthermore, we recognized that enhanced ADCC activity could enhance the activation of PD-1 and T cell inhibition by humanized PD-1 agonist antibodies. Therefore, ADCC-enhancing modifications were performed on the hu54H8 antibody. We introduced two amino acid changes (S239D and I332E mutations) into the IgG1 heavy chain of 54H8 (SEQ ID NO: 77). These changes significantly enhanced the ADCC activity of hu54H8. As demonstrated by ADCC reporter gene assays (see Example 5 for specific methods), Figure 12 shows that the ADCC-enhanced mutant of hu54H8, hu54H8-G1DE (SEQ ID NO: 77 and SEQ ID NO: 80), exhibits enhanced ADCC activity compared to wild-type hu54H8-G1wt.

[0195] Example 8

[0196] Affinity maturation of humanized antibodies

[0197] (1) The humanized hu54H8 antibody was converted into the corresponding single-chain antibody (scFv) format. The nucleic acid sequence encoded by it was optimized using the codons preferred by E. coli. The nucleic acid sequence was then synthesized by a gene synthesis company and cloned into the phage display vector PAK100. The nucleic acid sequence encoded by it was randomly mutated by PCR using the GeneMorph II Random Mutagenesis Kit (Cat NO: 200550) from Aglient according to the instructions. The PCR product and the phage display vector PAK100 were digested with restriction endonucleases NcoI and EcoRI, the nucleic acid fragments were purified and recovered, and the PCR product fragments were ligated to the phage display vector using DNA ligase. The ligated product was then electroporated into competent XL-Blue E. coli to construct a random mutation library of the hu54H8 antibody. 10 μl of the transformation solution was plated by gradient dilution, and the remaining transformation solution was spread on a 15 cm plate and cultured overnight at 37°C. The next day, the colonies on the plate were scraped and frozen. Take part of the frozen bacteria and inoculate them into 200 ml of 2YT medium. When the OD600 reaches 0.5, add 1012M13KO7 for superinfection. Incubate at 32°C overnight and concentrate into 1 ml of phage library by PEG / NaCl precipitation the next day.

[0198] (2) 500 μl of phage library was mixed with biotin-labeled PD-1 protein and incubated at room temperature for 2 hours. Then, 50 μl of streptavidin magnetic beads blocked with bovine serum albumin were added and incubated at room temperature for 30 minutes. The mixture was placed on a magnetic stand, the liquid was aspirated, and the beads were repeatedly washed with 1 ml of PBST to wash off the weakly bound phages. Unlabeled PD-1 protein was then added to compete overnight. The next day, the beads were repeatedly washed with 1 ml of PBST. Finally, the phages adsorbed on the magnetic beads were digested with trypsin and infected with Escherichia coli XL-Blue in the logarithmic growth phase. After superinfection, a new round of phages was produced and concentrated for the next round of selection. Repeat the above steps for 5 rounds to enrich high-affinity clones.

[0199] (3) Screening of scFv phages using competitive ELISA. The monoclonal E. coli selected in the last round were inoculated into 96-well deep-well plates, and 300 μl of 2YT medium containing ampicillin and M13-KO7 helper phage was added to each well. The cells were cultured at 37°C overnight to prepare monoclonal scFv phages. The culture supernatant containing scFv phages was diluted 5 times with PBST buffer, 10 nM PD-1 antigen or BSA was added, and the cells were incubated at room temperature for 1 hour. The mixture was transferred to a 96-well ELISA plate embedded with PD-1 and blocked with BSA, incubated at room temperature for 15 minutes, washed 8 times with PBST, and anti-M13 antibody coupled with horseradish peroxidase was added and incubated for 30 minutes. The plates were washed with PBST, TMB was added for color development, and 1.0 M H2SO4 was used to stop the color development. The absorbance of the samples at a wavelength of 450 nm was measured. Monoclonal E. coli corresponding to low absorbance were selected for sequencing. Compared with the humanized antibody sequence, the final selected antibody heavy and light chain sequence mutations after affinity maturation are shown in Table 9.

[0200] Table 9. Heavy and light chain sequence mutations of humanized PD-L1 antibodies before and after affinity maturation

[0201] Example 9

[0202] Binding test of affinity-matured humanized agonist PD-1 antibody to PD-1

[0203] ELISA and FACS (for specific methods, see Example 1) were used to detect the binding of purified humanized antibody samples to PD-1. The results are shown in Table 10 and Figures 13 and 14. The results showed that compared with the humanized hu54H8 antibody, the mutation of Asn 99 to Thr in the light chain (light chain SEQ ID NO: 81) increased the antibody's antigen binding capacity by 1.6-fold (ELISA test) or 1.79-fold (FACS test).

[0204] Table 10. Binding activity of humanized and affinity-matured PD-1 antibodies to PD-1-mFc and activation activity on PD-1.

[0205] Example 10

[0206] Detection of PD-1 activation activity by affinity-matured humanized agonist PD-1 antibodies

[0207] The reporter gene assay (see Example 1 for specific methods) was used to detect the activation of PD-1 by humanized PD-1 antibodies. The results are shown in Figure 15 . The results demonstrate that the affinity-matured humanized PD-1 agonist antibody, hu54H8-G1DE-M15, exhibits stronger PD-1 activation and, consequently, more pronounced inhibition of T cell activation compared to the positive control antibodies Lilly-G1 (Peresolimab, LY3462817; Eli Lilly) and ANB030 (Anaptysbio).

[0208] Example 11

[0209] Detection of T cell cytotoxicity of affinity-matured humanized agonist PD-1 antibodies

[0210] FACS was used to detect cell killing. Donor A's T cells were induced to express PD1 using 10 ng / ml SEB (Toxin technology, cat. no: BT202). After 3-5 days, the cells were collected as target cells, or uninduced Jurkat-PD-1 cells were used as target cells and labeled with CFSE for later use. PD-1 agonist antibodies were then serially diluted to a 4-fold concentration of the antibody diluent. 50 μL of the resulting mixture was transferred to a 96-well plate and added to a 150 μL cell mixture. The ratio of effector Donor B PBMC to CFSE-labeled Donor A T cells or Jurkat-PD1 was 25:1. The cells were incubated at 37°C for 4 hours (Jurkat-PD-1 as target cells) or 16 hours (induced T cells as target cells). After incubation for 15 minutes with the apoptosis detection reagent 7-AAD (Biolegend, cat. no: 420404), the proportion of apoptotic cells in CFSE-positive cells was determined by flow cytometry. The results are shown in Figures 16A (Jurkat-PD-1 as target cells) and 16B (induced T cells as target cells). The results showed that the affinity-matured humanized PD-1 agonist antibody, hu54H8-G1DE-M15, showed stronger ADCC activity than the positive control antibodies Lilly-G1 (Peresolimab, LY3462817; Eli Lilly) and ANB030 (Anaptysbio). In particular, when induced T cells were used as target cells, hu54H8-G1DE-M15 showed significant ADCC activity, while Lilly-G1 and ANB030 had no significant ADCC activity.

[0211] Example 12

[0212] Inhibitory effects of affinity-matured humanized PD-1 agonist antibodies on T cell cytokine release, proliferation, and memory T cell proliferation

[0213] The effects of an affinity-matured humanized PD-1 agonist antibody, hu54H8-G1DE-M15, on cytokine secretion in a mixed lymphocyte reaction, the effects of affinity-matured humanized PD-1 agonist antibodies on T cell proliferation, and the inhibition of memory T cell proliferation by humanized PD-1 agonist antibodies were examined (see Example 6 for specific methods). In general, affinity-matured humanized PD-1 agonist antibodies have improvements in the above three functions (see Figures 17, 18, and 19).

[0214] Moreover, the humanized PD-1 agonist antibody of the present invention has a stronger and more significant effect on inhibiting T cell cytokine release, proliferation, and memory T cell proliferation compared with the positive control antibody Lilly-G1 (Peresolimab, LY3462817; Eli Lilly) and the positive control antibody ANB030 (Anaptysbio) (see Figures 15, 16, and 17).

[0215] Example 13

[0216] Amino acid position 59 in the CDR2 sequence of the heavy chain of hu54H8-G1DE-M15 is asparagine (N59, heavy chain sequence: SEQ ID NO: 77). Our in silico analysis suggests the possibility of glycosylation. To mitigate the effects of glycosylation, we introduced two point mutations, N59Q and N59V, at this site (corresponding heavy chain sequences: SEQ ID NO: 78 and SEQ ID NO: 79, respectively). Antigen binding and functional assays (see Example 1 for specific methods) revealed no significant changes in binding or functional activity (Table 11). Therefore, the three sequences, hu54H8-G1DE-M15, hu54H8-G1DE-M15-N59Q, and hu54H8-G1DE-M15-N59V, achieved comparable results.

[0217] Table 11. Antigen binding and functional activity analysis results of humanized hu54H8-G1DE-M15-N59Q and hu54H8-G1DE-M15-N59V mutants.

[0218] The results of the above examples show that the monoclonal antibody or antigen-binding fragment thereof of the present invention, or the monoclonal antibody conjugate comprising the monoclonal antibody or antigen-binding fragment thereof of the present invention has good application prospects in drugs that bind to PD-1, drugs that activate PD-1 activity or PD-1 expression level, drugs that enhance the immunosuppressive effect of PD-1 on the body, drugs that inhibit T lymphocytes, drugs that inhibit T lymphocytes from expressing cytokines such as INF-γ, and drugs that prevent, treat or assist in the treatment of inflammatory and autoimmune diseases.

[0219] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements can be made without departing from the principles of the present invention. These improvements should also be regarded as the scope of protection of the present invention.

Claims

1. A monoclonal antibody or antigen-binding fragment thereof that specifically binds to PD-1, comprising a heavy chain CDR and a light chain CDR, wherein The amino acid sequence of CDR1 of the heavy chain comprises SEQ ID NO: 15, 21, 27, 33, 39, 50 or 56, or a sequence having two or fewer substituted amino acids compared to SEQ ID NO: 15, 21, 27, 33, 39, 50 or 56; The amino acid sequence of the heavy chain CDR2 comprises SEQ ID NO: 16, 22, 28, 34, 40, 51 or 57, or a sequence having two or fewer substituted amino acids compared to SEQ ID NO: 16, 22, 28, 34, 40, 51 or 57, wherein X in SEQ ID NO: 40 is any amino acid; The amino acid sequence of the heavy chain CDR3 comprises SEQ ID NO: 17, 23, 29, 35, 44, 52 or 58, or a sequence having two or fewer substituted amino acids compared to SEQ ID NO: 17, 23, 29, 35, 44, 52 or 58; The amino acid sequence of CDR1 of the light chain comprises SEQ ID NO: 18, 24, 30, 36, 45, 53 or 59, or a sequence having two or fewer substituted amino acids compared to SEQ ID NO: 18, 24, 30, 36, 45, 53 or 59; The amino acid sequence of CDR2 of the light chain comprises SEQ ID NO: 19, 25, 31, 37, 46, 54 or 60, or a sequence having two or fewer substituted amino acids compared to SEQ ID NO: 19, 25, 31, 37, 46, 54 or 60; and The amino acid sequence of the light chain CDR3 comprises SEQ ID NO: 20, 26, 32, 38, 47, 55 or 61, or a sequence having two or fewer substituted amino acids compared to SEQ ID NO: 20, 26, 32, 38, 47, 55 or 61, wherein X in SEQ ID NO: 47 is any amino acid.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein X in the amino acid sequence of SEQ ID NO: 40 is N, Q or V.

3. The antibody or antigen-binding fragment thereof according to claim 1, wherein X in the amino acid sequence of SEQ ID NO: 47 is N or T.

4. The antibody or antigen-binding fragment thereof according to claim 1, wherein the amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain, and the amino acid sequences of CDR1, CDR2 and CDR3 of the light chain are selected from one of the CDR combinations shown in ag: a: The sequences of the heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 comprise SEQ ID NOs: 15-17, in order, and the sequences of the light chain CDR1, light chain CDR2 and light chain CDR3 comprise SEQ ID NOs: 18-20; b: The sequences of the heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 comprise SEQ ID NOs: 21-23, in sequence; the sequences of the light chain CDR1, light chain CDR2, and light chain CDR3 comprise SEQ ID NOs: 24-26, in sequence; c: The sequences of the heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 comprise SEQ ID NOs: 27-29, in sequence; the sequences of the light chain CDR1, light chain CDR2, and light chain CDR3 comprise SEQ ID NOs: 30-32, in sequence; d: the sequences of the heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 comprise SEQ ID NOs: 33-35, in sequence; the sequences of the light chain CDR1, light chain CDR2, and light chain CDR3 comprise SEQ ID NOs: 36-38, in sequence; e: The sequences of the heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 comprise SEQ ID NOs: 39, 40, and 44, respectively; the sequences of the light chain CDR1, light chain CDR2, and light chain CDR3 comprise SEQ ID NOs: 45, 46, and 47, respectively; f: the sequences of the heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 comprise, in order, SEQ ID NOs: 50-52, and the sequences of the light chain CDR1, light chain CDR2, and light chain CDR3 comprise, in order, SEQ ID NOs: 53-55; or g: The sequences of the heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 contain SEQ ID NOs: 56-58, in sequence; the sequences of the light chain CDR1, light chain CDR2 and light chain CDR3 contain SEQ ID NOs: 59-61, in sequence.

5. The antibody or antigen-binding fragment thereof according to claim 1, comprising a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is selected from any one of SEQ ID NOs: 1, 3, 5, 7, 9, 11 and 13; and the amino acid sequence of the light chain variable region is selected from any one of SEQ ID NOs: 2, 4, 6, 8, 10, 12 and 14. The antibody or antigen-binding fragment thereof according to claim 5 , comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region are humanized.

7. The antibody or antigen-binding fragment thereof according to claim 6, wherein the amino acid sequence of the humanized heavy chain variable region is selected from any one of SEQ ID NOs: 62, 64, 66, 68, 70, 71, 72 and 75; and the amino acid sequence of the humanized light chain variable region is selected from any one of SEQ ID NOs: 63, 65, 67, 69, 73, 74 and 76.

8. The antibody or antigen-binding fragment thereof according to claim 7, comprising a heavy chain and a light chain, wherein The amino acid sequence of the heavy chain is SEQ ID NO: 77, and the amino acid sequence of the light chain is selected from SEQ ID NO: 80 or SEQ ID NO: 81; or The amino acid sequence of the heavy chain is SEQ ID NO: 78 or SEQ ID NO: 79, and the amino acid sequence of the light chain is SEQ ID NO: 81; or The amino acid sequence of the heavy chain is SEQ ID NO: 82, and the amino acid sequence of the light chain is SEQ ID NO: 83; or The amino acid sequence of the heavy chain is SEQ ID NO: 84, and the amino acid sequence of the light chain is SEQ ID NO: 85; or The amino acid sequence of the heavy chain is SEQ ID NO: 86, and the amino acid sequence of the light chain is SEQ ID NO: 87; or The amino acid sequence of the heavy chain is SEQ ID NO: 88, and the amino acid sequence of the light chain is SEQ ID NO: 89; or The amino acid sequence of the heavy chain is SEQ ID NO: 90, and the amino acid sequence of the light chain is SEQ ID NO:

91.

9. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 8 in the preparation of a medicament for activating PD-1 activity or PD-1 expression level, enhancing PD-1 immunosuppression, inhibiting T lymphocytes, inhibiting T lymphocyte expression of cytokines such as INF-γ, and preventing, treating, or adjuvant treating inflammatory and autoimmune diseases.

10. A nucleic acid molecule, wherein the nucleic acid molecule encodes the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9.

11. An expression vector, wherein the vector comprises the sequence of the nucleic acid molecule according to claim 10 and an expression control sequence related to the sequence.

12. A monoclonal antibody conjugate comprising a monoclonal antibody and a conjugated moiety, wherein the monoclonal antibody is the antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, and the conjugated moiety is selected from one or more of a radionuclide, a pharmaceutical agent, a toxin, a cytokine, a cytokine receptor fragment, an enzyme, fluorescein, and biotin.

13. Use of the monoclonal antibody conjugate according to claim 12 in the preparation of a medicament for activating PD-1 activity or PD-1 expression level, enhancing the immunosuppressive effect of PD-1 on the body, inhibiting T lymphocytes, inhibiting T lymphocyte expression of cytokines, and preventing or treating inflammation, cancer, and autoimmune diseases.

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