Anti-PD-1 antibodies and their medical uses

Abstract

Disclosed is a monoclonal antibody having a heavy chain variable region comprising CDRs having the amino acid sequences of SEQ ID NOs: 19-21 and / or a light chain variable region comprising CDRs having the amino acid sequences of SEQ ID NOs: 22-24. Using the EU numbering system, the antibody has a heavy chain constant region mutated at any two or three of positions 234, 235, and 237. The affinity constant of the mutated antibody for FcγRIIIa and / or C1q is lower than that of the antibody before the mutation. TIFF2022543114000032.tif101165

Description

[Technical Field]

[0001] Technical Field The present invention relates to the fields of tumor therapy and molecular immunology, specifically to anti-PD-1 antibodies and their pharmaceutical uses. More specifically, the present invention relates to mutant anti-PD-1 antibodies. [Background technology]

[0002] background The transmembrane receptor PD-1 (programmed cell death protein 1) is a member of the CD28 family and is expressed on activated T cells, B cells, and myeloid cells. PD-1's ligands, PDL1 (programmed cell death 1 ligand 1, or PDL-1) and PDL2 (programmed cell death 1 ligand 2, or PDL-2), are both members of the B7 superfamily. PDL1 is expressed on a variety of cells, including T cells, B cells, endothelial cells, and epithelial cells, while PDL2 is expressed exclusively on antigen-presenting cells such as dendritic cells and macrophages.

[0003] The PD-1 / PDL1 signaling pathway plays an important role in regulating immune tolerance, microbial infection, and tumor immune evasion. PD-1 is primarily expressed in immune cells such as T cells, and its ligand, PDL1, is highly expressed in multiple human tumor tissues. Blocking the PD-1 / PDL1 signaling pathway can activate suppressed T cells, which then attack cancer cells. Blocking PD-1 / PDL1 signaling can promote the proliferation of tumor antigen-specific T cells, activate the tumor cell killing process, and inhibit local tumor growth (Julie R et al., 2012, N Engl J Med., 366:2455-2465 (Non-Patent Document 1)).

[0004] PD-1 / PD-L1 is an important specific immune checkpoint. The formation of the PD-1 / PD-L1 complex transmits inhibitory signals and negatively regulates the immune response of T cells. It suppresses TCR-mediated T cell activation, cytokine production, and T cell proliferation (Fife et al., (2011) Nature Immunology 10:1185-1193 (Non-Patent Document 2)), induces exhaustion or anergy in homologous antigen-specific T cells (Hofmeyer et al., (2011) Journal of Biomedicine and Biotechnology, 2011:1-9 (Non-Patent Document 3)), promotes the differentiation of Th1 cells into Foxp3+ regulatory T cells (Armanath et al., (2011) Science Trans. Med., 3:1-13 (Non-Patent Document 4); Francisco et al., (2009) J. Exp. Med., 206:3015-3029 (Non-Patent Document 5)), and induces apoptosis of effector T cells. Disruption of the PD-L1 gene resulted in upregulated T cell responses and the production of autoreactive T cells (Latchman et al., (2004) PNAS, 101:10691-10696 (Non-Patent Document 6)). Blockade of PD-1 or PD-L1 with antibodies led to increased anti-tumor immunity (Iwai et al., (2002) PNAS, 99:12293-12297 (Non-Patent Document 7)).

[0005] Over the past two decades, researchers have devoted considerable efforts to developing specific immune checkpoint inhibitors in the hope of providing new immunotherapeutic regimens for treating cancer. Among these, the innate T lymphocyte immune system, with its high anticancer potential and broad yet precise specificity, is able to respond to various tumor antigens. This newly emerging cancer immunotherapy enhances antitumor immune responses by adoptively transferring activated effector cells, immunizing against relevant antigens, or providing nonspecific immunostimulatory drugs. Therefore, PD-1 / PD-L1-specific immune checkpoint inhibitors hold promise for the treatment of related cancers.

[0006] The mechanism of action of anti-PD-1 antibodies is to block the binding of PD-1 protein on the surface of immune cells to its ligands, PDL1 or PDL2, activating immune cells to kill tumors. Currently, there is still a need to develop novel anti-PD-1 antibodies to reduce or eliminate the damage caused by antibody-mediated ADCC, ADCP, and / or CDC activity on immune cells to which anti-PD-1 antibodies bind, and to improve the efficacy of antibody therapy. ADCC (antibody-dependent cellular cytotoxicity) refers to the killing of target cells by killer cells (e.g., NK cells, macrophages) mediated by the binding of the Fab fragment of an antibody to an epitope on a virus-infected cell or tumor cell and the Fc fragment of the antibody to an Fc receptor (FcR) on the surface of the killer cell.

[0007] CDC (complement-dependent cytotoxicity) refers to the lytic effect on target cells by the membrane attack complex, which is formed by the sequential binding of antibodies and complement C1q to corresponding antigens on the surface of the cell membrane, and the activation of C2 to C9.

[0008] Fc receptors belong to the immunoglobulin family and are expressed on the surface of specific immune cells to recognize the antibody Fc region and mediate immune responses. After the Fab region recognizes an antigen, the Fc region of the antibody binds to the Fc receptor on the immune cell (e.g., killer cell) to initiate immune cell response functions, such as phagocytosis and ADCC.

[0009] Fc receptors are primarily classified into three types: FcγR, FcαR, and FcεR, depending on the type of antibody recognized by the Fc receptor and the cell type on which they are expressed. FcγR can be further classified into four subtypes: FcγRI (CD64), FcγRII (CD32), FcγRIII (CD16), and FcRn (neonatal Fc receptor). Among these, FcγRI, FcγRII, and FcγRIII are closely associated with ADCC. FcγRIII is the most predominant molecule mediating ADCC, and there are two highly homologous subtypes, FcγRIIIa and FcγRIIIb, in different cell types. Within the FcγRIIIa population, there are two subtypes, FcγRIIIa_V158 with high affinity and FcγRIIIa_F158 with low affinity, distinguished by a single nucleotide polymorphism (SNP). FcγRI has higher affinity for the Fc region of IgG and is involved in the ADCC process; FcγRII contains three subtypes, FcγRIIa, FcγRIIb, and FcγRIIc (also called CD32a, CD32b, and CD32c, respectively), of which FcγRIIa has ADCC activity; two subtypes of FcγRIIa, FcγRIIa_H131 and FcγRIIa_R131, exist in humans due to a single-nucleotide mutation; FcγRIIb is an inhibitory receptor and is a typical inhibitory FcγR that inhibits the nearby ITAM pathway. For example, after binding of an immune complex to the BCR, the Fc fragment binds to FcγRIIb on the same cell, negatively regulating B cell activation and reducing antibody and cytokine secretion (Hogarth PM, Pietersz GA., 2012, NATURE REVIEWS DRUG DISCOVERY, 11(4):311-331 (Non-Patent Document 8)).

[0010] The IgG family includes four members, IgG1, IgG2, IgG3, and IgG4, which differ in amino acids in the fragment crystallizable (Fc) region of the heavy chain constant region, resulting in different affinities for FcγR. IgG1 is the most abundant subtype in humans and the most common subtype used in monoclonal antibody drugs. IgG1 can bind to various FcγRs and induce ADCC and CDC effects. IgG2 has the lowest affinity for FcγRs but can still induce monocyte-mediated ADCC by binding to FcγRIIa. IgG3 is characterized by the highest binding ability to FcγRs and can induce greater CDC and ADCC than IgG1. IgG4 molecules exhibit weaker binding to FcγRs other than FcγRI, making them less likely to induce CDC and NK cell-mediated ADCC. However, IgG4 subtype antibodies can mediate the ADCP effect through binding to FcγRI, and the ADCP effect present in antibody therapies targeting immune cells can damage immune cells and cause adverse pharmacological effects.

[0011] Zhang et al. (Zhang T et al., Cancer Immunol Immunother., 2018; 67(7):1079-1090 (Non-Patent Document 9)) and Dahan et al. (Dahan R et al., Cancer cell, 2015, 28(3):285-95 (Non-Patent Document 10)) reported that binding of Fc fragments of antibodies targeting immune checkpoints such as PD-1 and CTLA-4 to Fc receptors adversely affects antibody-mediated anticancer activity, likely due to Fc-dependent effector function-induced immune cell damage, including antibody-dependent cellular cytotoxicity, in which antibody-dependent cellular phagocytosis (ADCP) is an important mechanism leading to immune cell damage.

[0012] Non-squamous non-small cell lung cancer (NSCLC) and squamous non-small cell lung cancer (sNSCLC) are both malignant tumors of the lung tissue. Current treatment strategies include early surgery. However, many lung cancer patients are diagnosed at advanced stages and show poor response to surgery and radiotherapy. Therefore, chemotherapy has become an important treatment. Currently, combination chemotherapy with platinum and other chemotherapeutic agents remains the first-line chemotherapy for lung cancer, including advanced sNSCLC and NSCLC (Pfister DG. et al., J. Clin. Oncol., 2003, 22:330 (Non-Patent Document 11); De Ruysscher et al., (2006) Annals of Oncology, 17:543-552 (Non-Patent Document 12)).

[0013] Chemotherapy is currently classified into nine main classes (He Jie, et al., Clinical Oncology, Beijing, People's Medical Publishing House, 2016:230-237). The first class is composed of drugs that directly bind to DNA and inhibit DNA replication, including various alkylating agents, mitomycin, bleomycin, dacarbazine, platinum-based drugs (e.g., cisplatin and carboplatin), camptothecin, and their derivatives. The second class is composed of drugs that inhibit nucleic acid biosynthesis, primarily affecting the enzyme systems of tumor cells and blocking the synthesis of DNA and RNA precursors, thereby inhibiting the formation of DNA or RNA. These include methotrexate, fluorouracil, 6-mercaptopurine, hydroxyurea, and cytarabine; such drugs primarily act on cells in the S phase and are antimetabolite chemotherapy agents and cell cycle-specific anticancer agents. The third class is chemotherapy drugs that affect transcription through a pharmacological mechanism in which the drug intercalates into the DNA double helix, forming non-covalent bonds with it, interfering with the transcription of genetic information on DNA into DNA-dependent mRNA, impairing template function and disrupting transcription. The fourth class is drugs that affect tubulin and mitosis, including vinca alkaloids, podophyllotoxins, and taxanes. The fifth class is drugs that affect ribosomal function and block protein synthesis; a representative example of such a drug is harringtonine, which inhibits the initiation of protein synthesis, disassembling ribosomes and releasing nascent peptide chains, but does not block the binding of mRNA and tRNA to ribosomes; such drugs cause a reduction in nuclear DNA and cytoplasmic RNA and depolymerization of polysomes, inhibiting mitosis. The sixth class is drugs that affect tumor cell membranes, such as concanavalin (Con-A) and phytohemagglutinin (PHA); they can bind to glycoprotein receptors on the cell membrane, thereby affecting DNA synthesis in tumor cells and preventing them from dividing.The seventh class is drugs that induce apoptosis, such as arsenic trioxide. The eighth class is hormones that treat tumors by regulating the endocrine system, including estrogens, antiestrogens, progestogens, androgens, antiandrogens, corticosteroids, and anticorticosteroids (including dichlorodiphenyldichloroethane and aminoglutethimide). The ninth class is targeted anticancer therapy, including monoclonal antibodies, epidermal growth factor signaling inhibitors (e.g., drugs targeting the receptor tyrosine kinase pathway), ubiquitin-proteasome inhibitors, and angiogenesis inhibitors. However, in addition to killing tumor cells, chemotherapy drugs also damage normal human cells, and therefore, conventional chemotherapy regimens for cancer patients often cause serious toxic side effects. More importantly, in addition to obvious toxicity, chemotherapy drugs only show short-term disease control and low 5-year survival rates in patients receiving chemotherapy drugs. Therefore, it is of great significance to develop drug therapies or combination therapies with lower toxicity and higher efficacy.

[0014] Anlotinib is a quinoline-derived tyrosine kinase inhibitor. As a multitarget tyrosine kinase inhibitor (TKI), it affects tumor angiogenesis and proliferation signaling. Its primary targets include receptor tyrosine kinases vascular endothelial growth factor receptors (VEGFR) 1-3, epidermal growth factor receptor (EGFR), fibroblast growth factor receptors (FGFR) 1-4, platelet-derived growth factor receptors (PDGFR) α and β, and stem cell factor receptors (SCFR) 7, 8, and 9. A phase 2 trial showed that anlotinib improved progression-free survival (PFS) with potential benefits for overall survival (Han B, et al., Br J Cancer, 2018; 118(5):654-661). A multicenter, double-blind, randomized, phase 3 clinical trial demonstrated that anlotinib prolonged overall survival and progression-free survival in Chinese patients. Findings suggested that anlotinib is well tolerated and is a potential third-line or additional treatment for patients with advanced NSCLC (Han B, et al., JAMA Oncol., 2018 Nov.; 4(11):1569-1575).

[0015] Example 24 of Patent No. WO2008112407 (Patent Document 1) discloses a quinoline-derived tyrosine kinase inhibitor, 1-[[[4-(4-fluoro-2-methyl-1H-indol-5-yl)oxy-6-methoxyquinolin-7-yl]oxy]methyl]cyclopropylamine, and a method for preparing the same. The structural formula of the quinoline-derived tyrosine kinase inhibitor is shown in Formula I. Anlotinib hydrochloride is the hydrochloride salt of the compound of Formula I. TIFF0007753186000001.tif61128

[0016] Lenvatinib, an oral multitargeted tyrosine kinase inhibitor developed by Eisai (Japan), is a multitargeted receptor tyrosine kinase inhibitor that inhibits the kinase activity of VEGFR1 (FLT1), VEGFR2 (KDR), and VEGFR3 (FLT4). In addition to normal cellular function, lenvatinib also inhibits other receptor tyrosine kinases involved in pathogenic angiogenesis, tumor growth, and cancer progression, including fibroblast growth factor (FGF) receptors FGFR1, FGFR2, FGFR3, and FGFR4, the "rearranged during transfection" (RET) receptor, KIT, and platelet-derived growth factor receptor α (PDGFRα). Lenvatinib also exhibits antiproliferative activity in hepatocellular carcinoma cell lines, which is dependent on the simultaneous inhibition of activated FGFR signaling and the phosphorylation of FGF receptor substrate 2α (FRS2α).

[0017] The structure of lenvatinib, 4-(3-chloro-4(cyclopropylaminocarbonyl)aminophenoxy)-7-methoxy-6-quinolinecarboxamide, is disclosed in U.S. Pat. No. 7,612,208 (Patent Document 2), Example 368. U.S. Pat. No. 7,253,286 (Patent Document 3) discloses a mesylate salt of lenvatinib (i.e., lenvatinib mesylate), named 4-[3-chloro-4-(cyclopropylureido)phenoxy]-7-methoxyquinoline-6-carboxamide mesylate, the chemical structure of which is provided below (Formula II). TIFF0007753186000002.tif52128

[0018] However, for various tumors, the disease remains uncontrollable for a long time after chemotherapy, and the 5-year survival rate remains very low. Therefore, it is of great significance to develop drug therapies or combination therapies with lower toxicity and higher efficacy. [Prior art documents] [Patent documents]

[0019]

Patent Document 1

Patent document 2

Patent Document 3

Non-licensed literature

[0020] [Non-licensed document 1] Julie R et al., 2012, N Engl J Med., 366:2455-2465 [Non-licensed document 2] Fife et al., (2011) Nature Immunology 10:1185-1193 [Non-licensed document 3] Hofmeyer et al., (2011) Journal of Biomedicine and Biotechnology, 2011:1-9

Non-licensed Document 4

Non-licensed Document 5

Non-licensed Document 6

Non-licensed Document 7

Non-licensed Document 8

Non-licensed literature 9

[0021] overview Through intensive research and creative efforts, the present inventors have correspondingly modified the Fc fragment of the anti-PD-1 antibody structure to reduce the binding ability of the Fc region to Fc receptors, thereby reducing the ADCC, ADCP, and / or CDC effects on T cells and increasing the efficacy of the anti-PD-1 antibody. The present invention is described in detail below.

[0022] One aspect of the invention relates to an antibody, wherein: the antibody heavy chain variable region comprises HCDR1 to HCDR3 having the amino acid sequences set forth in SEQ ID NOs: 19 to 21, respectively, and the antibody light chain variable region comprises LCDR1 to LCDR3 having the amino acid sequences set forth in SEQ ID NOs: 22 to 24, respectively; The antibody is of the human IgG1 subtype; wherein the heavy chain constant region of the antibody contains mutations at any two or three of positions 234, 235 and 237 according to the EU numbering system, and the affinity constant of the antibody for FcγRIIIa and / or C1q is decreased after the mutation compared to before the mutation; preferably, the affinity constant is measured by a Fortebio Octet system.

[0023] In one embodiment of the invention, the antibody is a monoclonal antibody.

[0024] In one embodiment of the invention, the antibody is an anti-PD-1 antibody, preferably an anti-PD-1 monoclonal antibody.

[0025] In some embodiments of the invention, for antibodies, the heavy chain constant region of the antibody comprises the following mutations at positions 234, 235 and / or 237, according to the EU numbering system: L234A and L235A; L234A and G237A; L235A and G237A; or L234A, L235A, and G237A.

[0026] In the present invention, unless otherwise specified, the letter before the position number indicates the amino acid before mutation, and the letter after the position number indicates the amino acid after mutation.

[0027] The present invention also relates to an antibody, wherein: the antibody heavy chain variable region comprises HCDR1 to HCDR3 having the amino acid sequences set forth in SEQ ID NOs: 19 to 21, respectively, and the antibody light chain variable region comprises LCDR1 to LCDR3 having the amino acid sequences set forth in SEQ ID NOs: 22 to 24, respectively; The antibody is of the human IgG1 subtype; wherein the heavy chain constant region of the antibody comprises the following mutations at positions 234, 235 and / or 237, according to the EU numbering system: L234A and L235A; L234A and G237A; L235A and G237A; or L234A, L235A, and G237A.

[0028] In some embodiments of the invention, the heavy chain constant region of the antibody is N297A, D265A, D270A, P238D, L328E, E233D, H268D, P271G, A330R, C226S, C229S, E233P, P331S, S267E, L328F, A330L, M252Y, S254T, T256E, N297Q, P238S, P238A, A327Q, A327G, P329A, K322A, T394D, G236R, G236A, L328R, A330S, P331S, H268A, E318A, and K320A The compound further comprises one or more mutations selected from:

[0029] In some embodiments of the invention, for an antibody: the heavy chain variable region of the antibody comprises an amino acid sequence selected from SEQ ID NO: 2 and SEQ ID NO: 6; and The light chain variable region of the antibody comprises an amino acid sequence selected from SEQ ID NO:4 and SEQ ID NO:8.

[0030] In some embodiments of the invention, for an antibody: the heavy chain variable region of the antibody comprises the amino acid sequence set forth in SEQ ID NO: 2, and the light chain variable region of the antibody comprises the amino acid sequence set forth in SEQ ID NO: 4; the heavy chain variable region of the antibody comprises the amino acid sequence set forth in SEQ ID NO: 2, and the light chain variable region of the antibody comprises the amino acid sequence set forth in SEQ ID NO: 8; the heavy chain variable region of the antibody comprises the amino acid sequence set forth in SEQ ID NO: 6, and the light chain variable region of the antibody comprises the amino acid sequence set forth in SEQ ID NO: 4; or The heavy chain variable region of the antibody comprises the amino acid sequence set forth in SEQ ID NO:6, and the light chain variable region of the antibody comprises the amino acid sequence set forth in SEQ ID NO:8.

[0031] In one aspect of the present invention, the antibody The heavy chain is set forth in SEQ ID NO: 16 and the light chain is set forth in SEQ ID NO: 12; or The heavy chain is set forth in SEQ ID NO:18 and the light chain is set forth in SEQ ID NO:12.

[0032] The variable regions of the light and heavy chains determine antigen binding; the variable region of each chain contains three hypervariable regions, or complementarity-determining regions (CDRs) (heavy chain (H) CDRs include HCDR1, HCDR2, and HCDR3, and light chain (L) CDRs include LCDR1, LCDR2, and LCDR3; these are defined by Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition (1991), Volumes 1-3, NIH Publication 91-3242, Bethesda, MD).

[0033] The amino acid sequences of the CDR regions of the monoclonal antibodies in (1) to (3) above are analyzed by technical means well known to those skilled in the art, for example, using the VBASE2 database.

[0034] The antibodies 14C12, 14C12H1L1(hG1WT), 14C12H1L1(hG1DM) and 14C12H1L1(hG1TM) involved in the present invention have the same CDRs.

[0035] The amino acid sequences of the three CDR regions of the heavy chain variable region are as follows: HCDR1: GFAFSSYD (SEQ ID NO: 19), HCDR2: ISGGGRYT (SEQ ID NO: 20), and HCDR3: ANRYGEAWFAY (SEQ ID NO: 21).

[0036] The amino acid sequences of the three CDR regions of the light chain variable region are as follows: LCDR1: QDINTY (SEQ ID NO: 22), LCDR2: RAN (SEQ ID NO: 23), and LCDR3: LQYDEFPLT (SEQ ID NO: 24).

[0037] In some embodiments of the invention, the antibody is -7 Larger than M, e.g., about 10 -6 M, 10 -5 M, 10 -4 M, or 10 -3 binds to FcγRIIIa_F158, FcγRI, FcγRIIa_H131, FcγRIIIa_V158, and / or FcγRIIb with an affinity constant of greater than M or greater; preferably, the affinity constant is measured by a Fortebio Octet system; Preferably, the antibody has no binding signal or a binding signal of less than 0.1 nm to FcγRIIIa_F158, FcγRI, FcγRIIa_H131, FcγRIIIa_V158, and / or FcγRIIb; preferably, the binding signal refers to the response measured by a Fortebio Octet system.

[0038] In some embodiments of the invention, the antibody is -9 Larger than M, e.g., about 10 -8 M, 10 -7 M, 10 -6 M, or 10 -5 binds to C1q with an affinity constant greater than M or greater; preferably, the affinity constant is measured by a Fortebio Octet system; Preferably, the antibody has no binding signal or a binding signal of less than 0.1 nm to C1q; preferably, the binding signal refers to the response measured by a Fortebio Octet system.

[0039] In some embodiments of the invention, the antibody is a monoclonal antibody.

[0040] In some embodiments of the invention, the antibody is a humanized antibody.

[0041] Another aspect of the invention pertains to an isolated nucleic acid molecule encoding an antibody according to any embodiment of the invention.

[0042] Yet another aspect of the present invention pertains to vectors comprising the isolated nucleic acid molecules disclosed herein.

[0043] Yet another aspect of the present invention pertains to host cells comprising the isolated nucleic acid molecules or vectors disclosed herein.

[0044] Yet another aspect of the present invention relates to a conjugate comprising an antibody and a conjugate moiety, wherein the antibody is an antibody according to any embodiment of the present invention and the conjugate moiety is a detectable label; preferably, the conjugate moiety is a radioisotope, a fluorescent substance, a luminescent substance, a colored substance, or an enzyme.

[0045] Yet another aspect of the present invention is a kit comprising an antibody according to any embodiment of the present invention or comprising a conjugate as disclosed herein; Preferably, the kit further comprises a second antibody that specifically recognizes the antibody; optionally, the second antibody further comprises a detectable label, such as a radioisotope, a fluorescent substance, a luminescent substance, a colored substance, or an enzyme.

[0046] Yet another aspect of the invention relates to the use of an antibody or conjugate according to any embodiment of the invention in preparing a kit for detecting the presence or level of PD-1 in a sample.

[0047] Yet another aspect of the present invention relates to a pharmaceutical composition comprising an antibody or conjugate according to any embodiment of the present invention; optionally, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and / or excipient.

[0048] In one or more embodiments of the present invention, the pharmaceutical composition further comprises one or more anti-tumor chemotherapeutic agents; Preferably, the antitumor chemotherapeutic agent is a tyrosine kinase inhibitor; more preferably, the antitumor chemotherapeutic agent is anlotinib or a pharmaceutically acceptable salt thereof (e.g., the hydrochloride salt), or lenvatinib or a pharmaceutically acceptable salt thereof (e.g., the mesylate salt).

[0049] In one or more embodiments of the present invention, the unit dose of the pharmaceutical composition is 100 to 1000 mg, 200 to 800 mg, 200 to 500 mg, 300 to 600 mg, 400 to 500 mg, or 450 mg based on the mass of the antibody.

[0050] Yet another aspect of the present invention relates to a therapeutic combination comprising an antibody according to any embodiment of the present invention and at least one (eg, one, two, or three) anti-tumor chemotherapeutic agent.

[0051] In one or more embodiments of the present invention, for the therapeutic combination, the anti-tumor chemotherapeutic agent is a tyrosine kinase inhibitor; preferably, the anti-tumor chemotherapeutic agent is anlotinib or a pharmaceutically acceptable salt thereof (e.g., the hydrochloride salt), or lenvatinib or a pharmaceutically acceptable salt thereof (e.g., the mesylate salt).

[0052] In one or more embodiments of the invention, for the therapeutic combination, the unit dose of the antibody is 100-1000 mg, 200-800 mg, 200-500 mg, 300-600 mg, 400-500 mg, or 450 mg.

[0053] In one or more embodiments of the present invention, for the therapeutic combination, the unit dose of the antitumor chemotherapeutic agent is 0.1 to 100 mg, 0.5 to 50 mg, 0.5 to 10 mg, 1 to 10 mg, 2 to 8 mg, or 1 to 5 mg.

[0054] In one or more embodiments of the present invention, for the therapeutic combination, the unit dose of the anti-tumor chemotherapeutic agent is 1-20 mg, 2-15 mg, 4-12 mg, or 8-12 mg.

[0055] In one or more embodiments of the present invention, the therapeutic combination comprises: The therapeutic combination is a fixed combination, e.g., in the form of a solid pharmaceutical composition or a liquid pharmaceutical composition; or The therapeutic combination is a non-fixed combination, for example, the anti-PD-1 antibody and the anti-tumor chemotherapeutic agent in the non-fixed combination are each in the form of a pharmaceutical composition.

[0056] Yet another aspect of the present invention relates to a kit product comprising a pharmaceutical composition according to any aspect of the present invention or a therapeutic combination according to any aspect of the present invention and a package insert.

[0057] Yet another aspect of the present invention is the use of an antibody according to any embodiment of the present invention, a conjugate disclosed herein, a pharmaceutical composition according to any embodiment of the present invention, or a therapeutic combination according to any embodiment of the present invention in preparing a medicament for treating and / or preventing a tumor or anemia, or in preparing a medicament for diagnosing a tumor or anemia, wherein preferably the tumor is selected from one or more of melanoma, kidney cancer, prostate cancer, bladder cancer, colon cancer, rectal cancer, gastric cancer, liver cancer, lung cancer, ovarian cancer, leukemia, nasopharyngeal cancer, and endometrial cancer; Preferably, the lung cancer is selected from one or more of non-small cell lung cancer, small cell lung cancer, and squamous cell lung cancer; Preferably, the gastric cancer is gastric adenocarcinoma or gastroesophageal junction adenocarcinoma; Preferably, the tumor is a solid tumor of MSI-H / dMMR phenotype; preferably, the tumor is one of the following tumors of MSI-H / dMMR phenotype: Colon cancer, rectal cancer, endometrial cancer, gastric cancer, mesothelioma, sarcoma, adrenocortical carcinoma, malignant melanoma, and ovarian germ cell tumors The use is selected from one or more of:

[0058] In one or more embodiments of the invention, for use, the tumor is a recurrent, metastatic (eg, lymphatic, brain, and / or bone metastasis), or refractory tumor.

[0059] MSI refers to microsatellite instability. Microsatellites are short tandem repeats throughout the human genome, containing 10–50 repeats of one, two, or more nucleotides. Microsatellites in certain abnormal cells, such as tumors, are altered in length by insertion or deletion of repeat units compared to normal cells. Such alterations are called MSI. Based on the level and extent of instability, MSI can be classified as high-frequency microsatellite instability (MSI-H), low-frequency microsatellite instability (MSI-L), and microsatellite stable (MSS). The primary cause of MSI is DNA mismatch repair (MMR) deficiency. Human mismatch repair genes (MMR genes) can express the corresponding mismatch repair proteins through transcription and translation. The absence of MMR proteins can lead to mismatch repair deficiency, which leads to the accumulation of base pair mismatches during DNA replication, ultimately resulting in MSI. Approximately 15% of colorectal cancers are caused by the MSI pathway. This was first reported in colorectal cancer and can also occur in gastric cancer, endometrial cancer, adrenocortical carcinoma, etc. (Baretti M et al., Pharmacol Ther., 2018; 189:45-62). MSI-H / dMMR signatures have also been found in subsequent studies in mesothelioma, sarcoma, adrenocortical carcinoma, malignant melanoma, and ovarian germ cell tumors.

[0060] MSI-H and dMMR, which represent biologically consistent results from two different assays, are referred to as MSI-H / dMMR or MSI-high / dMMR, while MSI-L and MSS represent a proficient MMR (pMMR) phenotype. dMMR detection involves immunohistochemical assay of the protein expression of four mismatched genes, MSH2, MLH1, MSH6, and PMS2, based on tumor specimens (including surgical and aspirate specimens). Absence of any of the four proteins confirms dMMR; positive results for all four proteins indicate pMMR, i.e., complete mismatch repair function. MSI detection involves matching and comparing the lengths of repeated DNA sequences (microsatellite sequences) in tumor cells and somatic cells. Using PCR to detect five standard loci based on the National Cancer Institute (NCI) standard, mismatches at two or more loci indicate instability defined as MSI-H, one mismatched locus indicates MSI-L, and five matched loci indicate MSS. High-throughput sequencing (also called next-generation sequencing, or NGS) can also be used as a method to detect microsatellite instability. For PCR assays, if more microsatellite loci are selected, such as more than five loci or additional microsatellite loci, a mismatch of ≥30% loci is defined as MSI-H, a match at all loci is defined as MSS, and a mismatch of 0–30% is defined as MSI-L.

[0061] Yet another aspect of the present invention is a method for producing a pharmaceutical composition comprising: a pharmaceutical agent for blocking the binding of PD-1 to PD-L1; A pharmaceutical agent for downregulating the activity or level of PD-1; a medicament for reducing immunosuppression of PD-1 in an organism; or Medicaments for increasing IFN-γ and / or IL-2 expression in T lymphocytes The present invention relates to the use of an antibody according to any aspect of the invention, a conjugate as described herein, a pharmaceutical composition according to any aspect of the invention, or a therapeutic combination according to any aspect of the invention in preparing a

[0062] Interferon-γ (IFN-γ) is primarily and naturally produced by natural killer cells (NK) and natural killer T cells (NKT), and also by effector T cells, such as CD4 Th1 cells and CD8 cytotoxic T lymphocytes, stimulated by specific antigens. As an important innate and adaptive immune cytokine, IFN-γ plays an important role in combating or suppressing viral infections and certain bacterial and protozoal infections. Meanwhile, IFN-γ can activate macrophages, induce the expression of type II major histocompatibility complex, activate immune responses, and control tumor progression (Schoenborn JR, Wilson CB., Regulation of Interferon-γ During Innate and Adaptive Immune Responses, Advances in Immunology, 2007, 96:41-101). In vitro experiments of the present invention demonstrated that the antibodies disclosed herein can induce IFN-γ secretion and activate immune responses.

[0063] Interleukin-2 (IL-2) is produced by T cells. It is a growth factor that regulates T cell subgroups and is an important factor in regulating immune responses. It promotes the proliferation of activated B cells and is involved in antibody responses, hematopoiesis, and tumor surveillance. Recombinant human IL-2 has been approved by the US FDA for the treatment of malignant tumors, including melanoma and renal tumors (Chavez, AR, et al., Pharmacologic administration of interleukin-2, Ann. NY Acad. Sci., 2009, 1182:14-27). In vitro studies have demonstrated that the antibodies disclosed herein can specifically alleviate PD-1 immunosuppression, activate T cells, and induce IL-2 production, potentially finding broad application in the therapy of diseases such as tumors and parasitic infections.

[0064] Yet another aspect of the present invention relates to an in vivo or in vitro method comprising administering to a subject in need thereof an effective amount of an antibody according to any embodiment of the invention, a conjugate as described herein, a pharmaceutical composition according to any embodiment of the invention, or a therapeutic combination according to any embodiment of the invention. The method is selected from: Methods for blocking the binding of PD-1 to PD-L1; Methods for downregulating PD-1 activity or levels, a method for reducing immunosuppression of PD-1 in an organism; or A method for increasing IFN-γ and / or IL-2 expression in T lymphocytes.

[0065] Also relates to an antibody according to any embodiment of the invention, a conjugate as described herein, a pharmaceutical composition according to any embodiment of the invention, or a therapeutic combination according to any embodiment of the invention for use in treating and / or preventing a tumor or anemia, or in diagnosing a tumor or anemia, wherein preferably the tumor is selected from one or more of melanoma, kidney cancer, prostate cancer, bladder cancer, colon cancer, rectal cancer, gastric cancer, liver cancer, lung cancer, ovarian cancer, leukemia, nasopharyngeal cancer, and endometrial cancer; Preferably, the lung cancer is selected from one or more of non-small cell lung cancer, small cell lung cancer, and squamous cell lung cancer; Preferably, the gastric cancer is gastric adenocarcinoma or gastroesophageal junction adenocarcinoma; Preferably, the tumor is a solid tumor of MSI-H / dMMR phenotype; preferably, the tumor is one of the following tumors of MSI-H / dMMR phenotype: Colon cancer, rectal cancer, endometrial cancer, gastric cancer, mesothelioma, sarcoma, adrenocortical carcinoma, malignant melanoma, and ovarian germ cell tumors is selected from one or more of:

[0066] In one or more embodiments of the invention, with respect to the antibodies or conjugates described herein, the tumor is a recurrent, metastatic (e.g., lymphatic, brain, and / or bone metastasis), or refractory tumor.

[0067] An antibody according to any embodiment of the invention, a conjugate described herein, a pharmaceutical composition according to any embodiment of the invention, or a therapeutic combination according to any embodiment of the invention may be used for: blocking the binding of PD-1 to PD-L1; downregulating PD-1 activity or levels; Reducing the immunosuppression of PD-1 in the organism; or Increasing IFN-γ and / or IL-2 expression in T lymphocytes.

[0068] Yet another aspect of the present invention is a method of treating and / or preventing a tumor or anemia, or a method of diagnosing a tumor or anemia, comprising administering to a subject in need thereof an effective amount of an antibody according to any embodiment of the invention, a conjugate described herein, a pharmaceutical composition according to any embodiment of the invention, or a therapeutic combination according to any embodiment of the invention, wherein preferably the tumor is selected from one or more of melanoma, kidney cancer, prostate cancer, bladder cancer, colon cancer, rectal cancer, gastric cancer, liver cancer, lung cancer, ovarian cancer, leukemia, nasopharyngeal cancer, and endometrial cancer; Preferably, the lung cancer is selected from one or more of non-small cell lung cancer, small cell lung cancer, and squamous cell lung cancer; Preferably, the gastric cancer is gastric adenocarcinoma or gastroesophageal junction adenocarcinoma; Preferably, the tumor is a solid tumor of MSI-H / dMMR phenotype; preferably, the tumor is one of the following tumors of MSI-H / dMMR phenotype: Colon cancer, rectal cancer, endometrial cancer, gastric cancer, mesothelioma, sarcoma, adrenocortical carcinoma, malignant melanoma, and ovarian germ cell tumors The method relates to a method selected from one or more of:

[0069] In one or more embodiments of the invention, with respect to the methods, the tumor is a recurrent, metastatic (eg, lymphatic, brain, and / or bone metastasis), or refractory tumor.

[0070] In one or more embodiments of the present invention, with respect to the method, administration occurs before or after surgery and / or before or after radiation therapy.

[0071] In one or more embodiments of the present invention, a method, wherein: The unit dose of the anti-PD-1 antibody is 0.1 to 100 mg per kg body weight, preferably 1 to 10 mg (e.g., 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, or 10 mg); or the unit dose of the anti-PD-1 antibody is 10 to 1000 mg (e.g., about 100 mg, about 150 mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg, about 400 mg, about 450 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg, or about 1000 mg), preferably 50 to 500 mg, 100 to 400 mg, 150 to 300 mg, 150 to 250 mg, or 200 mg, for each subject; Preferably, the dose is administered once every 3 days, 4 days, 5 days, 6 days, 10 days, 1 week, 2 weeks, or 3 weeks; Preferably, the route of administration is intravenous drip infusion or intravenous injection.

[0072] In some embodiments, the anti-PD-1 antibody is administered in a 2-week (14-day) or 3-week (21-day) cycle, and preferably, the anti-PD-1 antibody is administered intravenously on the first day (D1) of each cycle. For example, the anti-PD-1 antibody is administered once every 2 weeks (q2w) or 3 weeks (q3w).

[0073] In the present invention, unless otherwise defined, the scientific and technical terms used herein have the meanings that are commonly understood by those skilled in the art.In addition, the laboratory procedures of cell culture, molecular genetics, nucleic acid chemistry and immunology used herein are routine procedures that are widely used in the corresponding fields.Meanwhile, in order to better understand the present invention, the definitions and explanations of relevant terms are provided below.

[0074] As used herein, when referring to the amino acid sequence of PD-1 protein (programmed cell death protein 1, NCBI GenBank: NP_005009.2), it includes the full-length PD-1 protein, the extracellular fragment of PD-1, PD-1ECD, or fragments containing PD-1ECD. It also includes fusion proteins of PD-1ECD, such as fragments fused to the Fc protein fragments (mFc or hFc) of mouse or human IgG. However, those skilled in the art will recognize that mutations or modifications (including, but not limited to, substitutions, deletions, and / or additions) in the amino acid sequence of PD-1 protein can be naturally or artificially introduced without affecting its biological function. Therefore, in the present invention, the term "PD-1 protein" should encompass such sequences and all of their natural or artificial variants. Furthermore, when a sequence fragment of PD-1 protein is described, it encompasses not only the sequence fragment but also the corresponding sequence fragment in its natural or artificial variant.

[0075] As used herein, when referring to the amino acid sequence of the PDL1 protein (NCBI Genebank ID: NP_054862.1), it includes the full-length PDL1 protein, the extracellular fragment PDL1ECD of PDL1, or a fragment containing PDL1ECD; it also includes fusion proteins of PDL1ECD, such as fragments fused to the Fc protein fragment (mFc or hFc) of mouse or human IgG. However, those skilled in the art will recognize that mutations or modifications (including, but not limited to, substitutions, deletions, and / or additions) in the amino acid sequence of the PDL1 protein can be naturally or artificially introduced without affecting its biological function. Therefore, in the present invention, the term "PDL1 protein" includes such sequences and all of their natural or artificial variants. Furthermore, when a sequence fragment of the PDL1 protein is described, it includes not only the PDL1 sequence fragment but also the corresponding sequence fragment in its natural or artificial variant.

[0076] As used herein, EC 50 The term refers to half-maximal effective concentration.

[0077] As used herein, the term "antibody" refers to an immunoglobulin molecule generally consisting of two pairs of polypeptide chains, each pair having one "light" (L) chain and one "heavy" (H) chain. Antibody light chains are classified as kappa and lambda light chains. Heavy chains are classified as mu, delta, gamma, alpha, or epsilon. Antibody isotypes are defined as follows: IgM, IgD, IgG, IgA, and IgE. In the light and heavy chains, the variable and constant regions are joined by a "J" region of about 12 or more amino acids, with heavy chains further including a "D" region of about 3 or more amino acids. Each heavy chain comprises a heavy chain variable region (V H ) and the heavy chain constant region (C H The heavy chain constant region consists of three domains (C H1 , C H2 , and C H3 Each light chain consists of a light chain variable region (V L ) and the light chain constant region (C L The light chain constant region consists of one domain, C L The constant region of the antibody may mediate the binding of the immunoglobulin to host tissues and factors, including the binding of various cells of the immune system (e.g., effector cells) to the first component (C1q) of the classical complement system. H and V L The region can be further subdivided into hypervariable regions (called complementarity determining regions (CDRs)), interspersed with conserved regions called frame regions (FRs). H and V L The variable region (V) of each heavy / light chain pair consists of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. H and V L) form the antibody binding site. The assignment of amino acids to each region or domain follows the definitions in Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, MD. (1987 and 1991)), Chothia & Lesk, (1987) J. Mol. Biol., 196:901-917, or Chothia et al. (1989) Nature, 342:878-883. The term "antibody" is not limited by any particular method for producing the antibody. For example, antibodies include, inter alia, recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. Antibodies can be of different isotypes, for example, IgG (e.g., subtypes IgG1, IgG2, IgG3, or IgG4), IgA1, IgA2, IgD, IgE, or IgM.

[0078] As used herein, the terms "mAb" and "monoclonal antibody" refer to antibodies or fragments thereof derived from a population of highly homologous antibodies, i.e., a population of identical antibody molecules except for natural mutations that may occur naturally. Monoclonal antibodies are highly specific to a single epitope on an antigen. Polyclonal antibodies generally contain at least two or more different antibodies that generally recognize different epitopes on an antigen, compared to monoclonal antibodies. Monoclonal antibodies can generally be obtained by hybridoma technology, first described by Kohler et al. (Nature, 256:495, 1975), or by recombinant DNA technology (see, e.g., U.S. Pat. No. 4,816,567).

[0079] As used herein, the term "humanized antibody" refers to an antibody or antibody fragment obtained when all or part of the CDR regions of a human immunoglobulin (receptor antibody) are replaced by the CDR regions of a non-human antibody (donor antibody), where the donor antibody can be a non-human (e.g., mouse, rat, or rabbit) antibody with the desired specificity, affinity, or reactivity. Furthermore, some amino acid residues in the frame region (FR) of the receptor antibody can also be replaced by corresponding amino acid residues of a non-human antibody or by amino acid residues of other antibodies to further improve or optimize the performance of the antibody. For further details regarding humanized antibodies, see, e.g., Jones et al., Nature, 321:522-525 (1986); Reichmann et al., Nature, 332:323-329 (1988); Presta, Curr. Op. Struct. Biol., 2:593-596 (1992); and Clark, Immunol. Today, 21:397-402 (2000).

[0080] As used herein, the term "isolated" refers to being obtained by artificial means from a natural state.When a "isolated" substance or component exists in nature, it may be that the change occurs in its natural environment, or that it is isolated from the natural environment, or both.For example, when a non-isolated polynucleotide or polypeptide naturally exists in a living animal, such a polynucleotide or polypeptide with a higher purity than that isolated from such a natural state is called an isolated polynucleotide or polypeptide.The term "isolated" does not exclude the presence of artificial or synthetic substances or other impurities that do not affect the activity of the substance.

[0081] As used herein, the term "vector" refers to a nucleic acid vehicle into which a polynucleotide can be inserted. If the vector allows the expression of the protein encoded by the inserted polynucleotide, the vector is called an expression vector. The vector can be introduced into a host cell by transformation, transduction, or transfection so that the genetic material elements carried by the vector can be expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs); phages, such as lambda phage or M13 phage; and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex viruses), poxviruses, baculoviruses, papillomaviruses, and papovaviruses (such as SV40). Vectors may contain various elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, vectors may further contain an origin of replication.

[0082] As used herein, the term "host cell" refers to a cell into which a vector can be introduced, including, but not limited to, a prokaryotic cell, such as E. coli or Bacillus subtilis, a fungal cell, such as a yeast cell or Aspergillus, an insect cell, such as S2 Drosophila cell or Sf9, or an animal cell, such as a fibroblast, CHO cell, COS cell, NSO cell, HeLa cell, BHK cell, HEK 293 cell, or human cell.

[0083] As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and its target antigen. In some embodiments, an antibody that specifically binds to an antigen (or an antibody specific for an antigen) binds to an antigen when the antibody binds to an antigen within about 10 -5 Smaller than M, e.g., about 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 Affinity (K D ) means binding to an antigen.

[0084] As used herein, the term "K D " refers to the dissociation equilibrium constant for a specific antibody-antigen interaction, which is used to describe the binding affinity between an antibody and an antigen. A smaller equilibrium dissociation constant indicates stronger antibody-antigen binding and a higher affinity between the antibody and the antigen. Generally, antibodies have a dissociation equilibrium constant of about 10 -5 Smaller than M, e.g., about 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 M or smaller dissociation equilibrium constant (K D ) to bind to an antigen (e.g., PD-1 protein). D can be determined using methods known to those skilled in the art, for example, using the Fortebio system.

[0085] As used herein, the terms "monoclonal antibody" and "mAb" have the same meaning and can be used interchangeably; the terms "polyclonal antibody" and "pAb" have the same meaning and can be used interchangeably; the terms "polypeptide" and "protein" have the same meaning and can be used interchangeably. Furthermore, amino acids are generally represented herein by one-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.

[0086] As used herein, the term "pharmaceutically acceptable carrier and / or excipient" refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and active ingredient. Such carriers and / or excipients are well known in the art (e.g., Remington's Pharmaceutical Sciences, edited by Gennaro AR, 1999). th Ed., Pennsylvania, Mack Publishing Company, 1995), pH adjusters, surfactants, adjuvants, and ionic strength enhancers. For example, pH adjusters include, but are not limited to, phosphate buffer; surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80; and ionic strength enhancers include, but are not limited to, sodium chloride.

[0087] As used herein, the term "adjuvant" refers to a nonspecific immunopotentiator that, when delivered together with or prior to an antigen, can enhance or alter the type of immune response of the organism to the antigen. There are various adjuvants, including, but not limited to, aluminum adjuvants (e.g., aluminum hydroxide), Freund's adjuvants (e.g., complete Freund's adjuvant and incomplete Freund's adjuvant), Corynebacterium parvum, lipopolysaccharide, cytokines, and the like. Freund's adjuvant is the most commonly used adjuvant in animal experiments. Aluminum hydroxide adjuvant is more frequently used in clinical trials.

[0088] As used herein, the term "effective amount" refers to the amount that is sufficient to achieve or at least partially achieve the desired effect.For example, the preventively effective amount for a disease (such as RA) refers to the amount that is sufficient to prevent, stop or delay the onset of the disease (such as RA); the therapeutically effective amount refers to the amount that is sufficient to cure or partially stop the disease and its complications in the patient suffering from the disease.Determining such an effective amount is certainly within the scope of the ability of those skilled in the art.For example, the amount that is effective for therapeutic purposes depends on the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's overall condition such as age, weight and sex, the route of administration, and other treatments that are simultaneously performed, etc.

[0089] As used herein, the term "completely eliminated" refers to the absence of a binding signal or an extremely weak binding signal when detected by existing equipment (e.g., the Fortebio Octet system). In one embodiment of the present invention, the absence of a binding signal or an extremely weak binding signal refers to a binding signal (i.e., response) of less than 0.1 nm.

[0090] A "recurrent" cancer is one that regrows at the original site or a distant site after responding to previous treatment (e.g., surgery). A "locally recurrent" cancer is one that occurs after treatment in the same site as the previously treated cancer.

[0091] "Metastatic" cancer refers to cancer that spreads from one part of the body (eg, lungs) to another.

[0092] beneficial effects The present invention achieves one or more of the following technical effects (1) to (9): (1) The antibodies disclosed herein, particularly 14C12H1L1(hG1TM) and 14C12H1L1(hG1WT), can effectively block immune cell suppression induced by PD-1 / PDL1 binding and induce the secretion of IFN-γ and IL-2 in human peripheral blood mononuclear cells.

[0093] (2) The present invention completely eliminates the binding activity of antibodies, particularly 14C12H1L1 (hG1™), to Fc receptors, namely, FcγRI, FcγRIIa_H131, FcγRIIIa_V158, and / or FcγRIIIa_F158, thereby eliminating ADCC activity or ADCP activity.

[0094] (3) The present invention completely eliminates the binding activity of antibodies, particularly 14C12H1L1 (hG1™), to complement C1q, thereby eliminating CDC activity.

[0095] (4) The present invention significantly reduces the binding activity of antibodies, such as 14C12H1L1 (hG1DM), to Fc receptors, i.e., FcγRI, FcγRIIa_H131, FcγRIIa_R131 and / or FcγRIIIa_V158, and completely eliminates binding to FcγRIIIa_F158 and / or FcγRIIb, thereby significantly reducing ADCC activity.

[0096] (5) The present invention completely eliminates the binding activity of antibodies, particularly 14C12H1L1 (hG1DM), to complement C1q, thereby eliminating CDC activity.

[0097] (6) The monoclonal antibodies of the present invention, particularly 14C12H1L1(hG1TM), 14C12H1L1(hG1DM), and 14C12H1L1(hG1WT), can sufficiently and specifically bind to PD-1 and effectively block the binding of PD-1 to PDL1, thereby specifically alleviating PD-1-induced immunosuppression in living organisms and activating T lymphocytes. Among these, the PD-1 antibody 14C12H1L1(hG1TM) has a significantly stronger induction effect on IFN-γ and IL-2 secretion than those of the control anti-PD-1 antibody nivolumab and the control anti-PDL1 antibody 5C10H2L2-IgG1mt, indicating their potential use in preparing medicines for preventing and treating tumors.

[0098] (7) The antibodies disclosed herein have the ability to effectively prevent and treat the above-mentioned tumors.

[0099] (8) The antibodies disclosed herein have fewer toxic side effects.

[0100] (9) The anti-PD-1 antibodies disclosed herein, or the anti-PD-1 antibodies in the therapeutic combinations disclosed herein, have a synergistic effect with chemotherapeutic agents. [The present invention 1001] An antibody, the heavy chain variable region of the antibody comprises HCDR1 to HCDR3 having the amino acid sequences set forth in SEQ ID NOs: 19 to 21, respectively, and the light chain variable region of the antibody comprises LCDR1 to LCDR3 having the amino acid sequences set forth in SEQ ID NOs: 22 to 24, respectively; the antibody is of the human IgG1 subtype; the heavy chain constant region of the antibody comprises mutations at any two or three of positions 234, 235, and 237 according to the EU numbering system, and the affinity constant of the antibody for FcγRIIIa and / or C1q is decreased after the mutation compared to before the mutation; preferably, the affinity constant is measured by a Fortebio Octet system. antibody. [The present invention 1002] wherein the heavy chain constant region of said antibody comprises the following mutations according to the EU numbering system: L234A and L235A; L234A and G237A; L235A and G237A; or L234A, L235A, and G237A 1001. The antibody of the present invention, comprising: [The present invention 1003] An antibody, the heavy chain variable region of the antibody comprises HCDR1 to HCDR3 having the amino acid sequences set forth in SEQ ID NOs: 19 to 21, respectively, and the light chain variable region of the antibody comprises LCDR1 to LCDR3 having the amino acid sequences set forth in SEQ ID NOs: 22 to 24, respectively; the antibody is of the human IgG1 subtype; wherein the heavy chain constant region of said antibody contains the following mutations according to the EU numbering system: L234A and L235A; L234A and G237A; L235A and G237A; or L234A, L235A, and G237A Including, antibody. [The present invention 1004] the heavy chain constant region of said antibody is N297A, D265A, D270A, P238D, L328E, E233D, H268D, P271G, A330R, C226S, C229S, E233P, P331S, S267E, L328F, A330L, M252Y, S254T, T256E, N297Q, P238S, P238A, A327Q, A327G, P329A, K322A, T394D, G236R, G236A, L328R, A330S, P331S, H268A, E318A, and K320A The antibody of any of claims 1001 to 1003, further comprising one or more mutations selected from the following: [The present invention 1005] the heavy chain variable region of the antibody comprises an amino acid sequence selected from SEQ ID NO: 2 and SEQ ID NO: 6; and the light chain variable region of the antibody comprises an amino acid sequence selected from SEQ ID NO: 4 and SEQ ID NO: 8; The antibody of any one of 1001 to 1004 of the present invention. [The present invention 1006] the heavy chain variable region of the antibody comprises the amino acid sequence set forth in SEQ ID NO: 2, and the light chain variable region of the antibody comprises the amino acid sequence set forth in SEQ ID NO: 4; the heavy chain variable region of the antibody comprises the amino acid sequence set forth in SEQ ID NO: 2, and the light chain variable region of the antibody comprises the amino acid sequence set forth in SEQ ID NO: 8; the heavy chain variable region of the antibody comprises the amino acid sequence set forth in SEQ ID NO: 6, and the light chain variable region of the antibody comprises the amino acid sequence set forth in SEQ ID NO: 4; or the heavy chain variable region of the antibody comprises the amino acid sequence set forth in SEQ ID NO: 6, and the light chain variable region of the antibody comprises the amino acid sequence set forth in SEQ ID NO: 8; The antibody of any one of 1001 to 1004 of the present invention. [The present invention 1007] The heavy chain is set forth in SEQ ID NO: 16 and the light chain is set forth in SEQ ID NO: 12; or The heavy chain is set forth in SEQ ID NO: 18 and the light chain is set forth in SEQ ID NO: 12. Any one of the antibodies 1001 to 1006 of the present invention. [The present invention 1008] The antibody is about 10 -7 Larger than M, e.g., about 10 -6 M、10 -5 M、10 -4 M, or 10 -3 binds to FcγRIIIa_F158, FcγRI, FcγRIIa_H131, FcγRIIIa_V158, and / or FcγRIIb with an affinity constant of greater than M or greater; preferably, the affinity constant is measured by a Fortebio Octet system; Preferably, the antibody has no binding signal or a binding signal of less than 0.1 nm to FcγRIIIa_F158, FcγRI, FcγRIIa_H131, FcγRIIIa_V158, and / or FcγRIIb; preferably, the binding signal refers to the response measured by a Fortebio Octet system. Any one of the antibodies 1001 to 1007 of the present invention. [The present invention 1009] The antibody is about 10 -9 Larger than M, e.g., about 10 -8 M、10 -7 M、10 -6 M, or 10 -5 binds to C1q with an affinity constant greater than M or greater; preferably, the affinity constant is measured by a Fortebio Octet system; Preferably, the antibody has no binding signal or a binding signal of less than 0.1 nm to C1q; preferably, the binding signal refers to the response measured by the Fortebio Octet system. Any one of the antibodies 1001 to 1008 of the present invention. [The present invention 1010] An isolated nucleic acid molecule encoding any one of the antibodies of the present invention 1001 to 1009. [The present invention 1011] A vector comprising the isolated nucleic acid molecule of the present invention. [The present invention 1012] A host cell comprising an isolated nucleic acid molecule of the invention 1010 or a vector of the invention 1011. [The present invention 1013] A conjugate comprising any one of the antibodies of the present invention 1001 to 1009 and a conjugate moiety, The conjugated moiety is a detectable label; preferably, the conjugated moiety is a radioisotope, a fluorescent substance, a luminescent substance, a colored substance, or an enzyme. Conjugates. [The present invention 1014] A kit comprising any one of the antibodies of the present invention 1001 to 1009 or the conjugate of the present invention 1013, Preferably, the kit further comprises a second antibody that specifically recognizes the antibody; optionally, the second antibody further comprises a detectable label, such as a radioisotope, a fluorescent substance, a luminescent substance, a colored substance, or an enzyme. kit. [The present invention 1015] Use of any of the antibodies of the present inventions 1001 to 1009 or the conjugate of the present invention 1013 in preparing a kit for detecting the presence or level of PD-1 in a sample. [The present invention 1016] A pharmaceutical composition comprising any one of the antibodies 1001 to 1009 of the present invention or the conjugate 1013 of the present invention, optionally further comprising a pharmaceutically acceptable carrier and / or excipient. [The present invention 1017] further comprising one or more anti-tumor chemotherapeutic agents; Preferably, the antitumor chemotherapeutic agent is a tyrosine kinase inhibitor; more preferably, the antitumor chemotherapeutic agent is anlotinib or a pharmaceutically acceptable salt thereof (e.g., hydrochloride), or lenvatinib or a pharmaceutically acceptable salt thereof (e.g., mesylate). The pharmaceutical composition of the present invention. [The present invention 1018] The pharmaceutical composition of claim 1016 or 1017, wherein the unit dose of the pharmaceutical composition is 100 to 1000 mg, 200 to 800 mg, 200 to 500 mg, 300 to 600 mg, 400 to 500 mg, or 450 mg based on the mass of the antibody. [The present invention 1019] A therapeutic combination comprising any of the antibodies of the present inventions 1001 to 1009 and at least one (eg, one, two, or three) anti-tumor chemotherapeutic agent. [The present invention 1020] The therapeutic combination of the present invention 1019, wherein said antitumor chemotherapeutic drug is a tyrosine kinase inhibitor; preferably, said antitumor chemotherapeutic drug is anlotinib or a pharmaceutically acceptable salt thereof (e.g., hydrochloride), or lenvatinib or a pharmaceutically acceptable salt thereof (e.g., mesylate). [The present invention 1021] The therapeutic combination of claim 1019 or 1020, wherein the unit dose of the antibody is 100 to 1000 mg, 200 to 800 mg, 200 to 500 mg, 300 to 600 mg, 400 to 500 mg, or 450 mg. [The present invention 1022] The therapeutic combination of claim 1019 or 1020, wherein the unit dose of the antitumor chemotherapeutic agent is 0.1 to 100 mg, 0.5 to 50 mg, 1 to 20 mg, 2 to 15 mg, 4 to 12 mg, or 8 to 12 mg. [The present invention 1023] the therapeutic combination is a fixed combination, e.g., in the form of a solid pharmaceutical composition or a liquid pharmaceutical composition; or the therapeutic combination is a non-fixed combination, for example, the anti-PD-1 antibody and the anti-tumor chemotherapeutic agent in the non-fixed combination are each in the form of a pharmaceutical composition; The therapeutic combination of any one of 1019 to 1022 of the present invention. [The present invention 1024] A kit product comprising any one of the pharmaceutical compositions of the present inventions 1016 to 1018 or any one of the therapeutic combinations of the present inventions 1019 to 1023, and an attached document. [The present invention 1025] Use of any of the antibodies of the present inventions 1001 to 1009, the conjugate of the present invention 1013, the pharmaceutical composition of the present inventions 1016 to 1018, or the therapeutic combination of any of the present inventions 1019 to 1023 in preparing a medicament for treating and / or preventing a tumor or anemia, or in preparing a medicament for diagnosing a tumor or anemia, Preferably, the tumor is selected from one or more of melanoma, kidney cancer, prostate cancer, bladder cancer, colon cancer, rectal cancer, gastric cancer, liver cancer, lung cancer, ovarian cancer, leukemia, nasopharyngeal cancer, and endometrial cancer; Preferably, the lung cancer is selected from one or more of non-small cell lung cancer, small cell lung cancer, and squamous cell lung cancer; Preferably, the gastric cancer is gastric adenocarcinoma or gastroesophageal junction adenocarcinoma; Preferably, the tumor is a solid tumor of MSI-H / dMMR phenotype; preferably, the tumor is one of the following tumors of MSI-H / dMMR phenotype: Colon cancer, rectal cancer, endometrial cancer, gastric cancer, mesothelioma, sarcoma, adrenocortical carcinoma, malignant melanoma, and ovarian germ cell tumors selected from one or more of: use. [The present invention 1026] a pharmaceutical agent for blocking the binding of PD-1 to PD-L1; A pharmaceutical agent for downregulating the activity or level of PD-1; a medicament for reducing immunosuppression of PD-1 in an organism; or Medicaments for increasing IFN-γ and / or IL-2 expression in T lymphocytes Use of any of the antibodies of the present inventions 1001 to 1009, the conjugate of the present invention 1013, the pharmaceutical composition of the present inventions 1016 to 1018, or the therapeutic combination of any of the present inventions 1019 to 1023 in preparing a therapeutic agent. [The present invention 1027] Preferably, the tumor is selected from one or more of melanoma, kidney cancer, prostate cancer, bladder cancer, colon cancer, rectal cancer, gastric cancer, liver cancer, lung cancer, ovarian cancer, leukemia, nasopharyngeal cancer, and endometrial cancer; Preferably, the lung cancer is selected from one or more of non-small cell lung cancer, small cell lung cancer, and squamous cell lung cancer; Preferably, the gastric cancer is gastric adenocarcinoma or gastroesophageal junction adenocarcinoma; Preferably, said tumor is a solid tumor of MSI-H / dMMR phenotype; preferably, said tumor is one of the following tumors of MSI-H / dMMR phenotype: Colon cancer, rectal cancer, endometrial cancer, gastric cancer, mesothelioma, sarcoma, adrenocortical carcinoma, malignant melanoma, and ovarian germ cell tumors selected from one or more of: An antibody of any of claims 1001 to 1009, a conjugate of claim 1013, a pharmaceutical composition of any of claims 1016 to 1018, or a therapeutic combination of any of claims 1019 to 1023, for use in treating and / or preventing tumors or anemia, or for use in diagnosing tumors or anemia. [The present invention 1028] A method for treating and / or preventing a tumor or anemia, or a method for diagnosing a tumor or anemia, comprising administering an effective amount of an antibody of any of the present inventions 1001 to 1009, a conjugate of the present invention 1013, a pharmaceutical composition of any of the present inventions 1016 to 1018, or a therapeutic combination of any of the present inventions 1019 to 1023 to a subject in need thereof, Preferably, the tumor is selected from one or more of melanoma, kidney cancer, prostate cancer, bladder cancer, colon cancer, rectal cancer, gastric cancer, liver cancer, lung cancer, ovarian cancer, leukemia, nasopharyngeal cancer, and endometrial cancer; Preferably, the lung cancer is selected from one or more of non-small cell lung cancer, small cell lung cancer, and squamous cell lung cancer; Preferably, the gastric cancer is gastric adenocarcinoma or gastroesophageal junction adenocarcinoma; Preferably, the tumor is a solid tumor of MSI-H / dMMR phenotype; preferably, the tumor is one of the following tumors of MSI-H / dMMR phenotype: Colon cancer, rectal cancer, endometrial cancer, gastric cancer, mesothelioma, sarcoma, adrenocortical carcinoma, malignant melanoma, and ovarian germ cell tumors selected from one or more of: method. [The present invention 1029] The method of claim 1028, wherein an effective amount of said antibody is administered to a subject in need thereof before or after a surgical procedure and / or before or after radiation therapy. [The present invention 1030] the unit dose of the antibody is 0.1 to 100 mg per kg body weight, preferably 1 to 10 mg per kg body weight; or the unit dose of the antibody is 10 to 1000 mg, preferably 50 to 500 mg for each subject; Preferably, the dose is administered once every 3 days, 4 days, 5 days, 6 days, 10 days, 1 week, 2 weeks, or 3 weeks; Preferably, the route of administration is intravenous infusion or intravenous injection. 1028 or 1029. [The present invention 1031] Any of the methods of claims 1028 to 1030, wherein the antibody is administered in a 2- or 3-week cycle, and preferably the antibody is administered intravenously on the first day of each cycle; preferably the antibody is administered once every 2 or 3 weeks. [Brief explanation of the drawings]

[0101] [Figure 1] Affinity constant assay of 14C12H1L1 (hG1DM) for FcγRI. Antibody concentrations for the top and bottom curve pairs are 50 nM, 25 nM, 12.5 nM, 6.25 nM, and 3.12 nM, respectively. [Figure 2] Affinity constant assay of 14C12H1L1 (hG4) for FcγRI. Antibody concentrations for the top and bottom curve pairs are 50 nM, 25 nM, 12.5 nM, 6.25 nM, and 3.12 nM, respectively. [Figure 3] Affinity constant assay of 14C12H1L1 (hG1WT) for FcγRI. Antibody concentrations for the top and bottom curve pairs are 50 nM, 25 nM, 12.5 nM, 6.25 nM, and 3.12 nM, respectively. [Figure 4] Affinity constant assay of 14C12H1L1 (hG1™) for FcγRI. Antibody concentrations for the top and bottom curve pairs are 50 nM, 25 nM, 12.5 nM, 6.25 nM, and 3.12 nM, respectively. [Figure 5] Affinity constant assay of 5C10H2L2-IgG1mt for FcγRI. Antibody concentrations for the top and bottom curve pairs are 50 nM, 25 nM, 12.5 nM, 6.25 nM, and 3.12 nM, respectively. [Figure 6] Affinity constant assay of 14C12H1L1 (hG1DM) for FcγRIIIa_V158. Antibody concentrations for the top to bottom curve pairs are 500 nM, 250 nM, 125 nM, 62.5 nM, and 31.25 nM, respectively. [Figure 7] Affinity constant assay of 14C12H1L1 (hG4) for FcγRIIIa_V158. Antibody concentrations for the top to bottom curve pairs are 500 nM, 250 nM, 125 nM, 62.5 nM, and 31.25 nM, respectively. [Figure 8]Affinity constant assay of 14C12H1L1 (hG1WT) for FcγRIIIa_V158. Antibody concentrations for the top to bottom curve pairs are 500 nM, 250 nM, 125 nM, 62.5 nM, and 31.25 nM, respectively. [Figure 9] Affinity constant assay of 14C12H1L1 (hG1™) for FcγRIIIa_V158. Antibody concentrations for the top and bottom curve pairs are 500 nM, 250 nM, 125 nM, 62.5 nM, and 31.25 nM, respectively. [Figure 10] Affinity constant assay of 5C10H2L2-IgG1mt for FcγRIIIa_V158. Antibody concentrations for the top to bottom curve pairs are 500 nM, 250 nM, 125 nM, 62.5 nM, and 31.25 nM, respectively. [Figure 11] Affinity constant assay of 14C12H1L1 (hG1DM) for FcγRIIIa_F158. The antigen concentrations for the top and bottom curve pairs are 500 nM, 250 nM, 125 nM, 62.5 nM, and 31.25 nM, respectively. [Figure 12] Affinity constant assay of 14C12H1L1 (hG4) for FcγRIIIa_F158. Antibody concentrations for the top to bottom curve pairs are 500 nM, 250 nM, 125 nM, 62.5 nM, and 31.25 nM, respectively. [Figure 13] Affinity constant assay of 14C12H1L1 (hG1WT) for FcγRIIIa_F158. Antibody concentrations for the top to bottom curve pairs are 500 nM, 250 nM, 125 nM, 62.5 nM, and 31.25 nM, respectively. [Figure 14] Affinity constant assay of 14C12H1L1 (hG1™) for FcγRIIIa_F158. Antibody concentrations for the top and bottom curve pairs are 500 nM, 250 nM, 125 nM, 62.5 nM, and 31.25 nM, respectively. [Figure 15]Affinity constant assay of 5C10H2L2-IgG1mt for FcγRIIa_F158. Antibody concentrations for the top to bottom curve pairs are 500 nM, 250 nM, 125 nM, 62.5 nM, and 31.25 nM, respectively. [Figure 16] Affinity constant assay of 14C12H1L1 (hG1DM) for FcγRIIa_H131. Antibody concentrations for the top and bottom curve pairs are 200 nM, 100 nM, 50 nM, 25 nM, and 12.5 nM, respectively. [Figure 17] Affinity constant assay of 14C12H1L1 (hG4) for FcγRIIa_H131. Antibody concentrations for the top and bottom curve pairs are 200 nM, 100 nM, 50 nM, 25 nM, and 12.5 nM, respectively. [Figure 18] Affinity constant assay of 14C12H1L1 (hG1WT) for FcγRIIa_H131. Antibody concentrations for the top to bottom curve pairs are 200 nM, 100 nM, 50 nM, 25 nM, and 12.5 nM, respectively. [Figure 19] Affinity constant assay of 14C12H1L1 (hG1™) for FcγRIIa_H131. Antibody concentrations for the top and bottom curve pairs are 200 nM, 100 nM, 50 nM, 25 nM, and 12.5 nM, respectively. [Figure 20] Affinity constant assay of 5C10H2L2-IgG1mt for FcγRIIa_H131. Antibody concentrations for the top and bottom curve pairs are 200 nM, 100 nM, 50 nM, 25 nM, and 12.5 nM, respectively. [Figure 21] Affinity constant assay of 14C12H1L1 (hG1DM) for FcγRIIa_R131. Antibody concentrations for the top and bottom curve pairs are 200 nM, 100 nM, 50 nM, 25 nM, and 12.5 nM, respectively. [Figure 22]Affinity constant assay of 14C12H1L1 (hG4) for FcγRIIa_R131. Antibody concentrations for the top to bottom curve pairs are 200 nM, 100 nM, 50 nM, 25 nM, and 12.5 nM, respectively. [Figure 23] Affinity constant assay of 14C12H1L1 (hG1WT) for FcγRIIa_R131. Antibody concentrations for the top to bottom curve pairs are 200 nM, 100 nM, 50 nM, 25 nM, and 12.5 nM, respectively. [Figure 24] Affinity constant assay of 14C12H1L1 (hG1™) for FcγRIIa_R131. Antibody concentrations for the top and bottom curve pairs are 200 nM, 100 nM, 50 nM, 25 nM, and 12.5 nM, respectively. [Figure 25] Affinity constant assay of 5C10H2L2-IgG1mt for FcγRIIa_R131. Antibody concentrations for the top and bottom curve pairs are 200 nM, 100 nM, 50 nM, 25 nM, and 12.5 nM, respectively. [Figure 26] Affinity constant assay of 14C12H1L1 (hG1DM) for FcγRIIb. Antibody concentrations for the top and bottom curve pairs are 200 nM, 100 nM, 50 nM, 25 nM, and 12.5 nM, respectively. [Figure 27] Affinity constant assay of 14C12H1L1 (hG4) for FcγRIIb. Antibody concentrations for the top and bottom curve pairs are 200 nM, 100 nM, 50 nM, 25 nM, and 12.5 nM, respectively. [Figure 28] Affinity constant assay of 14C12H1L1 (hG1WT) for FcγRIIb. Antibody concentrations for the top and bottom curve pairs are 200 nM, 100 nM, 50 nM, 25 nM, and 12.5 nM, respectively. [Figure 29]Affinity constant assay of 14C12H1L1 (hG1™) for FcγRIIb. Antibody concentrations for the top and bottom curve pairs are 200 nM, 100 nM, 50 nM, 25 nM, and 12.5 nM, respectively. [Figure 30] Affinity constant assay of 5C10H2L2-IgG1mt for FcγRIIb. Antibody concentrations for the top and bottom curve pairs are 200 nM, 100 nM, 50 nM, 25 nM, and 12.5 nM, respectively. [Figure 31] Affinity constant assay of 14C12H1L1 (hG1DM) for C1q. Antibody concentrations for the top and bottom curve pairs are 20 nM, 10 nM, 5 nM, 2.5 nM, and 1.25 nM, respectively. [Figure 32] Affinity constant assay of 14C12H1L1 (hG4) for C1q. Antibody concentrations for the top and bottom curve pairs are 20 nM, 10 nM, 5 nM, 2.5 nM, and 1.25 nM, respectively. [Figure 33] Affinity constant assay of 14C12H1L1 (hG1WT) for C1q. Antibody concentrations for the top and bottom curve pairs are 20 nM, 10 nM, 5 nM, 2.5 nM, and 1.25 nM, respectively. [Figure 34] Affinity constant assay of 14C12H1L1 (hG1™) for C1q. Antibody concentrations for the top and bottom curve pairs are 20 nM, 10 nM, 5 nM, 2.5 nM, and 1.25 nM, respectively. [Figure 35] Affinity constant assay of 5C10H2L2-IgG1mt for C1q. The antigen concentrations for the top and bottom curve pairs are 20 nM, 10 nM, 5 nM, 2.5 nM, and 1.25 nM, respectively. [Figure 36] IFN-γ secretion assay by adding 14C12H1L1(hG1WT) and 14C12H1L1(hG1TM) to a mixed lymphocyte reaction. [Figure 37]IL-2 secretion assay by adding 14C12H1L1(hG1WT) and 14C12H1L1(hG1TM) to a mixed lymphocyte reaction. [Figure 38] ADCP efficacy assay of 14C12H1L1(hG1WT), nivolumab, and 14C12H1L1(hG1TM). [Figure 39] Assay of the killing effect of 14C12H1L1(hG1™) + anlotinib on human non-small cell lung cancer cells. [Figure 40] Growth inhibition of mouse colorectal cancer MC38 cells by 14C12H1L1 (hG1™). [Figure 41] 14C12H1L1 (hG1TM) effectively enhanced immune responses of immune cells against human gastric cancer KATO III cells. [Figure 42] Effectively enhanced immune response of immune cells against nasopharyngeal carcinoma CNE-2Z cells by 14C12H1L1 (hG1TM). [Figure 43] 14C12H1L1 (hG1TM) effectively enhanced immune response of immune cells against mesothelioma NCI-H2452 cells. [Figure 44] 14C12H1L1 (hG1™) effectively enhanced immune responses of immune cells against human small cell lung cancer NCI-H446 cells. [Figure 45] Effectively enhanced immune response of immune cells against nasopharyngeal carcinoma CNE-2Z cells by 14C12H1L1 (hG1TM) in combination with anlotinib hydrochloride. [Figure 46] Significantly enhanced immune response of immune cells against MSI-H / dMMR tumor SW48 cells by 14C12H1L1 (hG1TM) in combination with anlotinib. [Figure 47] Significantly enhanced immune response of immune cells against human colorectal cancer SW837 cells of non-MSI-H / dMMR (i.e., MSS) phenotype by 14C12H1L1 (hG1TM). [Figure 48]Significantly enhanced immune response of immune cells against human colorectal cancer SW837 cells of non-MSI-H / dMMR (i.e., MSS) phenotype by 14C12H1L1 (hG1TM) in combination with anlotinib. DETAILED DESCRIPTION OF THE INVENTION

[0102] Detailed Description The embodiments of the present invention will be described in detail below with reference to examples. Those skilled in the art will understand that the following examples are merely for illustrating the present invention and are not to be construed as limiting the scope of the present invention. If techniques or conditions are not specified, the examples are carried out according to the techniques or conditions described in the literature in the art (see, for example, Molecular Cloning: A Laboratory Manual, J. Sambrook et al., translated by Huang Peitang et al., 3rd Edition, Science Press) or according to the product manual. If the reagents or equipment used are not specified by their manufacturers, they are conventional products available on the market.

[0103] In the following experiments of the present invention: BALB / c mice were purchased from Guangdong Medical Laboratory Animal Center.

[0104] The anti-PDL1 antibody 5C10H2L2-IgG1mt was prepared by the method described in PCT Publication No. WO2017148424A1.

[0105] The anti-PD-1 antibody nivolumab (trade name: Opdivo) was purchased from Bristol-Myers Squibb.

[0106] Human peripheral blood mononuclear cells were isolated and prepared at Akeso Biopharma, Inc. with informed consent of the donors.

[0107] Raji-PDL1 is a cell line expressing human PD-L1 that was constructed by Akeso Biopharma based on the human B cell line Raji by transfection.

[0108] Ficoll-Paque™ PLUS (or Ficoll-Paque PLUS) was purchased from GE Healthcare.

[0109] Human IL-2 ELISA kit was purchased from Dakewe Biotech Co., Ltd.

[0110] RPMI 1640 medium, DMEM medium, trypsin-EDTA (0.25%), phenol red, and blasticidin were all purchased from Gibco.

[0111] Staphylococcus aureus enterotoxin B (SEB) was purchased from Dianotech.

[0112] FBS was purchased from Excell bio.

[0113] Mitomycin C (MMC) was purchased from Stressmarq.

[0114] The sequence of the isotype control, human anti-hen egg white lysozyme IgG (anti-HEL antibody, or human IgG, abbreviated as hIgG), was derived from the variable region sequence of the Fab F10.6.6 sequence in the study reported by Acierno et al., entitled "Affinity maturation increases the stability and plasticity of the Fv domain of anti-protein antibodies" (Acierno et al., J Mol Biol., 2007; 374(1):130-146).

[0115] The anlotinib used in the examples is the hydrochloride salt of anlotinib under the trade name Fukewei®, generic name anlotinib hydrochloride, and was purchased from CTTQ Pharma. [Example]

[0116] Preliminary Example 1: Sequence design of anti-PD-1 antibody 14C12 and its humanized antibody 14C12H1L1 (hG1WT) The amino acid sequences and coding nucleotide sequences of the heavy and light chains of anti-PD-1 antibody 14C12 and its humanized antibody 14C12H1L1 (hG1WT) are identical to those of 14C12 and 14C12H1L1, respectively, in Chinese Patent Publication No. CN106967172A (or No. CN106977602A).

[0117] (1) 14C12 heavy and light chain variable region sequences Nucleotide sequence of the heavy chain variable region of 14C12: (354 bp) TIFF0007753186000003.tif63165

[0118] Amino acid sequence of the heavy chain variable region of 14C12: (118 aa) TIFF0007753186000004.tif21164

[0119] Nucleotide sequence encoding the light chain variable region of 14C12: (321 bp) TIFF0007753186000005.tif55165

[0120] Amino acid sequence of the light chain variable region of 14C12: (107 aa) TIFF0007753186000006.tif22164

[0121] (2) Heavy and light chain variable regions and heavy and light chain sequences of humanized monoclonal antibody 14C12H1L1 (hG1WT) Nucleotide sequence of the heavy chain variable region of 14C12H1L1 (hG1WT): (354 bp) TIFF0007753186000007.tif63163

[0122] Amino acid sequence of the heavy chain variable region of 14C12H1L1 (hG1WT): (118 aa) TIFF0007753186000008.tif21162

[0123] Nucleotide sequence encoding the light chain variable region of 14C12H1L1 (hG1WT): (321 bp) TIFF0007753186000009.tif55164

[0124] Amino acid sequence of the light chain variable region of 14C12H1L1 (hG1WT): (107 aa) TIFF0007753186000010.tif21162

[0125] Nucleotide sequence of the heavy chain of 14C12H1L1 (hG1WT): (1344 bp) TIFF0007753186000011.tif224165

[0126] Amino acid sequence of the heavy chain of 14C12H1L1 (hG1WT): (448 aa) TIFF0007753186000012.tif72165

[0127] Nucleotide sequence of the light chain of 14C12H1L1 (hG1WT): (642 bp) TIFF0007753186000013.tif106165

[0128] Amino acid sequence of the light chain of 14C12H1L1 (hG1WT): (214 aa) TIFF0007753186000014.tif38165

[0129] Preliminary Example 2: Sequence design of humanized antibody 14C12H1L1 (hG4) The heavy and light chain variable regions are identical to those of 14C12H1L1 (hG1WT). The Igγ4 chain C region (ACCESSION: P01861.1) was used as the heavy chain constant region, and the Igκ chain C region (ACCESSION: P01834) was used as the light chain constant region, thus obtaining antibody 14C12H1L1 (hG4). The sequence of 14C12H1L1 (hG4) is as follows:

[0130] Nucleotide sequence of the heavy chain of 14C12H1L1 (hG4): (1335 bp) TIFF0007753186000015.tif224165

[0131] Amino acid sequence of the heavy chain of 14C12H1L1 (hG4): (445 aa) TIFF0007753186000016.tif72165

[0132] The nucleotide sequence of the 14C12H1L1 (hG4) light chain is identical to SEQ ID NO:11.

[0133] The amino acid sequence of the 14C12H1L1 (hG4) light chain is identical to SEQ ID NO:12.

[0134] Preliminary Example 3: Sequence design of humanized antibody 14C12H1L1 (hG1™) Based on 14C12H1L1 (hG1WT) obtained in Preliminary Example 1, the humanized variant 14C12H1L1 (hG1TM) was obtained by introducing a point mutation from leucine to alanine at position 234 (L234A), a point mutation from leucine to alanine at position 235 (L235A), and a point mutation from glycine to alanine at position 237 (G237A) in the hinge region of the heavy chain according to the EU numbering system.

[0135] Nucleotide sequence of the heavy chain of 14C12H1L1 (hG1™): (1344 bp) TIFF0007753186000017.tif224165

[0136] Amino acid sequence of the heavy chain of 14C12H1L1 (hG1™): (448 aa) TIFF0007753186000018.tif72165

[0137] The nucleotide sequence of the 14C12H1L1 (hG1™) light chain is identical to SEQ ID NO:11.

[0138] The amino acid sequence of the 14C12H1L1 (hG1™) light chain is identical to SEQ ID NO:12.

[0139] Preliminary Example 4: Sequence design of humanized antibody 14C12H1L1 (hG1DM) Based on 14C12H1L1(hG1WT), the humanized mutant antibody 14C12H1L1(hG1DM) was obtained by introducing a point mutation from leucine to alanine at position 234 (L234A) and a point mutation from leucine to alanine at position 235 (L235A) in the hinge region of the heavy chain.

[0140] Nucleotide sequence of the heavy chain of 14C12H1L1 (hG1DM): (1344 bp) TIFF0007753186000019.tif224165

[0141] Amino acid sequence of the heavy chain of 14C12H1L1 (hG1DM): (448 aa) TIFF0007753186000020.tif72165

[0142] The nucleotide sequence of the 14C12H1L1 (hG1DM) light chain is identical to SEQ ID NO:11.

[0143] The amino acid sequence of the 14C12H1L1 (hG1DM) light chain is identical to SEQ ID NO:12.

[0144] Experimental Example 1: Affinity assay of 14C12H1L1(hG1DM), 14C12H1L1(hG4), 14C12H1L1(hG1WT) and 14C12H1L1(hG1TM) for the Fc receptor FcγRI The Fc receptor FcγRI, also known as CD64, can bind to the Fc fragment of IgG antibodies and participate in antibody-dependent cellular cytotoxicity (ADCC). The binding ability of therapeutic monoclonal antibodies to Fc receptors affects the safety and efficacy of the antibody. The affinity constants of 14C12H1L1(hG1DM), 14C12H1L1(hG4), 14C12H1L1(hG1WT), and 14C12H1L1(hG1TM) for FcγRI were measured in this experiment using the Fortebio Octet system to evaluate the ADCC activity of the antibodies.

[0145] The method for determining the affinity constant of an antibody to FcγRI using the Fortebio Octet system is briefly described as follows: The sample dilution buffer was a solution of 0.02% Tween-20 and 0.1% BSA in PBS, pH 7.4. A 1 μg / mL FcγRI solution (Sinobio) was added to the HIS1K sensor, and FcγRI was immobilized on the sensor surface for 50 seconds. Both the binding and dissociation constants of the antibody to FcγRI were determined in buffer, with antibody concentrations ranging from 3.12 to 50 nM (two-fold serial dilution). The sensor with the immobilized antigen was equilibrated in buffer for 60 seconds, and then the binding of the immobilized FcγRI on the sensor to the antibody was determined for 120 seconds; the dissociation of FcγRI from the antibody was determined for 120 seconds. The temperature was 30°C, and the frequency was 0.3 Hz. The data were fitted and analyzed with a 1:1 model to obtain the affinity constant of the antibody to FcγRI.

[0146] The results of affinity constant assays of 14C12H1L1(hG1DM), 14C12H1L1(hG4), 14C12H1L1(hG1WT), and 14C12H1L1(hG1TM) and the control antibody 5C10H2L2-IgG1mt for FcγRI are shown in Table 1 and Figures 1 to 5.

[0147] Table 1. Kinetics of binding of 14C12H1L1(hG1DM), 14C12H1L1(hG4), 14C12H1L1(hG1WT), and 14C12H1L1(hG1TM), as well as control antibody 5C10H2L2-IgG1mt, to FcγRI. TIFF0007753186000021.tif62165

[0148] N / A indicates that the antibody did not bind to the antigen or had a very weak binding signal; therefore, the results were not analyzed and no corresponding data was obtained.

[0149] The results showed that both 14C12H1L1(hG4) and 14C12H1L1(hG1WT) bound to FcγRI with affinity constants of 5.80E-09 M and 2.52E-09 M, respectively; 14C12H1L1(hG1TM) and 5C10H2L2-IgG1mt did not bind or had very weak binding signals to FcγRI, and therefore the results were not analyzed and no corresponding data were obtained.

[0150] The results suggested that the binding activity of 14C12H1L1(hG1DM) and 14C12H1L1(hG1TM) to FcγRI was effectively abolished compared to 14C12H1L1(hG4) and 14C12H1L1(hG1WT).

[0151] Experimental Example 2: Affinity assay of 14C12H1L1(hG1DM), 14C12H1L1(hG4), 14C12H1L1(hG1WT), and 14C12H1L1(hG1TM) for the Fc receptor FcγRIIIa and its subtypes (1) Affinity constant assay of 14C12H1L1(hG1DM), 14C12H1L1(hG4), 14C12H1L1(hG1WT), and 14C12H1L1(hG1TM) for FcγRIIIa_V158 The Fc receptor FcγRIIIa_V158 (also known as CD16a_V158) binds to the Fc fragment of IgG antibodies and can mediate ADCC effects. The affinity constants of 14C12H1L1(hG1DM), 14C12H1L1(hG4), 14C12H1L1(hG1WT), and 14C12H1L1(hG1TM) for FcγRIIIa_V158 were measured in this experiment using the Fortebio Octet system to evaluate the ADCC activity of the antibodies.

[0152] The method for determining the affinity constants of antibodies against FcγRIIIa_V158 and the control antibody 5C10H2L2-IgG1mt using the Fortebio Octet system is briefly described as follows: The sample dilution buffer was a solution of 0.02% Tween-20 and 0.1% BSA in PBS, pH 7.4. 5 μg / mL FcγRIIIa_V158 was immobilized on the HIS1K sensor for 120 seconds. The sensor was equilibrated in the buffer for 60 seconds, and binding of the immobilized FcγRIIIa_V158 on the sensor to antibodies at concentrations ranging from 31.25 to 500 nM (two-fold serial dilutions) was determined for 60 seconds. The antibody was allowed to dissociate in the buffer for 60 seconds. The sensor was refreshed four times in 10 mM glycine, pH 1.5, for 5 seconds each. The temperature was 30°C, and the frequency was 0.3 Hz. To obtain the affinity constants, the data were analyzed by 1:1 model fitting.

[0153] The results of affinity constant assays of 14C12H1L1(hG1DM), 14C12H1L1(hG4), 14C12H1L1(hG1WT), and 14C12H1L1(hG1TM) and the control antibody 5C10H2L2-IgG1mt for FcγRIIIa_V158 are shown in Table 2 and Figures 6 to 10.

[0154] Table 2: Kinetics of binding of 14C12H1L1(hG1DM), 14C12H1L1(hG4), 14C12H1L1(hG1WT), and 14C12H1L1(hG1TM), as well as control antibody 5C10H2L2-IgG1mt, to FcγRIIIa_V158 TIFF0007753186000022.tif69163

[0155] N / A indicates that the antibody did not bind to the antigen or had a very weak binding signal; therefore, the results were not analyzed and no corresponding data was obtained.

[0156] The results showed that both 14C12H1L1(hG1DM) and 14C12H1L1(hG1WT) bound to FcγRIIIa_V158 with affinity constants of 6.21E-07M and 6.54E-08M, respectively; 14C12H1L1(hG4), 14C12H1L1(hG1TM) and 5C10H2L2-IgG1mt did not bind or had very weak binding signals to FcγRIIIa_V158, and therefore the results were not analyzed.

[0157] The results indicated that the binding activity of 14C12H1L1(hG4), 14C12H1L1(hG1TM) and the control antibody 5C10H2L2-IgG1mt to FcγR IIIa_V158 was effectively abolished compared to 14C12H1L1(hG1DM) and 14C12H1L1(hG1WT).

[0158] (2) Affinity constant assay of 14C12H1L1(hG1DM), 14C12H1L1(hG4), 14C12H1L1(hG1WT), and 14C12H1L1(hG1TM) for FcγRIIIa_F158 The Fc receptor FcγRIIIa_F158 (also known as CD16a_F158) binds to the Fc fragment of IgG antibodies and can mediate ADCC effects. The affinity constants of 14C12H1L1(hG1DM), 14C12H1L1(hG4), 14C12H1L1(hG1WT), 14C12H1L1(hG1TM) and control antibodies for FcγRIIIa_F158 were measured in this experiment using the Fortebio Octet system to evaluate the ADCC activity of the antibodies.

[0159] The method for determining the affinity constants of 14C12H1L1(hG1DM), 14C12H1L1(hG4), 14C12H1L1(hG1WT), and 14C12H1L1(hG1TM) for FcγRIIIa_F158 using the Fortebio Octet system is briefly described as follows: The sample dilution buffer was a solution of 0.02% Tween-20 and 0.1% BSA in PBS, pH 7.4. 5 μg / mL FcγRIIIa_F158 was immobilized on the HIS1K sensor for 120 seconds. The sensor was equilibrated in buffer for 60 seconds, and binding of the immobilized FcγRIIIa_F158 on the sensor to antibodies at concentrations ranging from 31.25 to 500 nM (two-fold serial dilutions) was determined for 60 seconds. The antibodies were allowed to dissociate in buffer for 60 seconds. The sensor was refreshed four times for 5 seconds each in 10 mM glycine pH 1.5. The temperature was 30°C and the frequency was 0.3 Hz. The data were analyzed by 1:1 model fitting to obtain affinity constants.

[0160] The results of affinity constant assays of 14C12H1L1(hG1DM), 14C12H1L1(hG4), 14C12H1L1(hG1WT), and 14C12H1L1(hG1TM) and the control antibody 5C10H2L2-IgG1mt for FcγRIIIa_F158 are shown in Table 3 and Figures 11 to 15.

[0161] Table 3. Kinetics of binding of 14C12H1L1 antibody and its isotype to FcγRIIIa_F158 TIFF0007753186000023.tif69166

[0162] N / A indicates that the antibody did not bind to the antigen or had a very weak binding signal; therefore, the results were not analyzed and no corresponding data was obtained.

[0163] The results showed that 14C12H1L1(hG1WT) bound to FcγRIIIa_F158 with an affinity constant of 1.02E-07M; 14C12H1L1(hG1DM), 14C12H1L1(hG4), 14C12H1L1(hG1TM) and 5C10H2L2-IgG1mt did not bind or had very weak binding signals to FcγRIIIa_F158; therefore, the results were not analyzed and no corresponding data were obtained.

[0164] The results suggested that the binding activity of 14C12H1L1(hG1DM), 14C12H1L1(hG4) and 14C12H1L1(hG1TM) to FcγRIIIa_F158 was effectively abolished compared to 14C12H1L1(hG1WT).

[0165] Experimental Example 3: Affinity assay of 14C12H1L1(hG1DM), 14C12H1L1(hG4), 14C12H1L1(hG1WT) and 14C12H1L1(hG1TM) for the Fc receptor FcγRIIa and its subtypes (1) Affinity constant assay of 14C12H1L1(hG1DM), 14C12H1L1(hG4), 14C12H1L1(hG1WT), and 14C12H1L1(hG1TM) for FcγRIIa_H131 The Fc receptor FcγRIIa_H131, also known as CD32a_H131, can bind to the Fc fragment of IgG antibodies and participate in antibody-dependent cellular cytotoxicity (ADCC). The binding ability of therapeutic monoclonal antibodies to Fc receptors affects the safety and efficacy of the antibody. The affinity constants of 14C12H1L1 (hG1DM), 14C12H1L1 (hG4), 14C12H1L1 (hG1WT), and 14C12H1L1 (hG1TM) for FcγRIIa_H131 were measured in this experiment using the Fortebio Octet system to evaluate the binding ability of antibodies to Fc receptors.

[0166] The method for determining the affinity constants of 14C12H1L1(hG1DM), 14C12H1L1(hG4), 14C12H1L1(hG1WT), and 14C12H1L1(hG1TM) for FcγRIIa_H131 using the Fortebio Octet system is briefly described as follows: the immobilization dilution buffer was a solution of PBS, 0.02% Tween-20, and 0.1% BSA, pH 7.4, and the analyte dilution buffer was a solution of 0.02% Tween-20, 0.02% casein, and 0.1% BSA in PBS, pH 7.4. 5 μg / mL FcγRIIa_H131 was immobilized on the NTA sensor at an immobilization height of approximately 1.0 nm. The sensor was equilibrated for 300 seconds in a buffer solution of 0.02% Tween-20, 0.02% casein, and 0.1% BSA in PBS, pH 7.4, for blocking. Binding of the immobilized FcγRIIa_H131 antibody on the sensor to antibodies at concentrations ranging from 12.5 to 200 nM (two-fold serial dilutions) was determined for 60 seconds. The antibody was allowed to dissociate in the buffer for 60 seconds. The sensor was refreshed in 10 mM glycine, pH 1.7, and 10 mM nickel sulfate. The temperature was 30°C, and the frequency was 0.6 Hz. The data were analyzed by 1:1 model fitting to obtain affinity constants.

[0167] The results of affinity constant assays of 14C12H1L1(hG1DM), 14C12H1L1(hG4), 14C12H1L1(hG1WT), and 14C12H1L1(hG1TM) and the control antibody 5C10H2L2-IgG1mt for FcγRIIa_H131 are shown in Table 4 and Figures 16 to 20.

[0168] Table 4. Kinetics of binding of 14C12H1L1 antibody and its isotype to FcγRIIa_H131 TIFF0007753186000024.tif65167

[0169] N / A indicates that the antibody did not bind to the antigen or had a very weak binding signal; therefore, the results were not analyzed and no corresponding data was obtained.

[0170] The results showed that both 14C12H1L1(hG4) and 14C12H1L1(hG1WT) bound to FcγRIIa_H131 with affinity constants of 5.07E-08M and 5.74E-08M, respectively; 14C12H1L1(hG1DM), 14C12H1L1(hG1TM) and 5C10H2L2-IgG1mt did not bind or had very weak binding signals to FcγRIIa_H131, and therefore the results were not analyzed and no corresponding data were obtained.

[0171] This suggests that the binding activity of 14C12H1L1(hG1DM) and 14C12H1L1(hG1TM) to FcγRIIa_H131 is effectively abolished compared to 14C12H1L1(hG4) and 14C12H1L1(hG1WT).

[0172] (2) Affinity constant assay of 14C12H1L1(hG1DM), 14C12H1L1(hG4), 14C12H1L1(hG1WT), and 14C12H1L1(hG1TM) for FcγRIIa_R131 The Fc receptor FcγRIIa_R131, also known as CD32a_R131, can bind to the Fc fragment of IgG antibodies and participate in antibody-dependent cellular cytotoxicity (ADCC). The binding ability of therapeutic monoclonal antibodies to Fc receptors affects the safety and efficacy of the antibody. The affinity constants of 14C12H1L1 (hG1DM), 14C12H1L1 (hG4), 14C12H1L1 (hG1WT), and 14C12H1L1 (hG1TM) for FcγRIIa_R131 were measured in this experiment using the Fortebio Octet system to evaluate the binding ability of antibodies to Fc receptors.

[0173] The method for determining the affinity constants of 14C12H1L1(hG1DM), 14C12H1L1(hG4), 14C12H1L1(hG1WT), and 14C12H1L1(hG1TM) for FcγRIIa_R131 using the Fortebio Octet system is briefly described as follows: the immobilization dilution buffer was a solution of PBS, 0.02% Tween-20, and 0.1% BSA, pH 7.4, and the analyte dilution buffer was a solution of 0.02% Tween-20, 0.02% casein, and 0.1% BSA in PBS, pH 7.4. 5 μg / mL FcγRIIa_R131 was immobilized on the NTA sensor at an immobilization height of approximately 1.0 nm. The sensor was equilibrated for 300 seconds in a buffer solution of 0.02% Tween-20, 0.02% casein, and 0.1% BSA in PBS, pH 7.4, for blocking. Binding of the immobilized FcγRIIa_R131 on the sensor to antibodies at concentrations ranging from 12.5 to 200 nM (two-fold serial dilutions) was determined for 60 seconds. The antibody was allowed to dissociate in the buffer for 60 seconds. The sensor was refreshed in 10 mM glycine, pH 1.7, and 10 mM nickel sulfate. The temperature was 30°C, and the frequency was 0.6 Hz. The data were analyzed by 1:1 model fitting to obtain affinity constants.

[0174] The results of affinity constant assays of 14C12H1L1(hG1DM), 14C12H1L1(hG4), 14C12H1L1(hG1WT), and 14C12H1L1(hG1TM) and the control antibody 5C10H2L2-IgG1mt for FcγRIIa_R131 are shown in Table 5 and Figures 21 to 25.

[0175] Table 5. Kinetics of binding of 14C12H1L1 antibody and its isotype to FcγRIIa_R131 TIFF0007753186000025.tif72165

[0176] N / A indicates that the antibody did not bind to the antigen or had a very weak binding signal; therefore, the results were not analyzed and no corresponding data was obtained.

[0177] The results showed that 14C12H1L1(hG4), 14C12H1L1(hG1WT) and 14C12H1L1(hG1TM) bound to FcγRIIa_R131 with affinity constants of 3.13E-08M, 3.46E-08M and 2.32E-07M, respectively; 14C12H1L1(hG1DM) and 5C10H2L2-IgG1mt did not bind or had very weak binding signals to FcγRIIa_R131, and therefore the results were not analyzed and no corresponding data were obtained.

[0178] The results suggest that among the antibodies with binding activity, 14C12H1L1(hG1TM) has the weakest binding ability and lowest binding activity to FcγRIIa_R131 compared to 14C12H1L1(hG4) and 14C12H1L1(hG1WT).

[0179] Experimental Example 4: Affinity constant assay of 14C12H1L1(hG1DM), 14C12H1L1(hG4), 14C12H1L1(hG1WT), and 14C12H1L1(hG1TM) for FcγRIIb The Fc receptor FcγRIIb (also known as CD32b) binds to the Fc fragment of IgG antibodies and can downregulate immune cell function, inhibit immune cell activation and proliferation, and inhibit cytokine secretion. The affinity constants of antibodies to FcγRIIb were measured in this experiment using the Fortebio Octet system to evaluate the binding ability of 14C12H1L1(hG1DM), 14C12H1L1(hG4), 14C12H1L1(hG1WT), and 14C12H1L1(hG1TM) to the Fc receptor.

[0180] The method for determining the affinity constants of 14C12H1L1(hG1DM), 14C12H1L1(hG4), 14C12H1L1(hG1WT), and 14C12H1L1(hG1TM) for FcγRIIb using the Fortebio Octet system is briefly described as follows: the immobilization dilution buffer was a solution of PBS, 0.02% Tween-20, and 0.1% BSA, pH 7.4, and the analyte dilution buffer was a solution of 0.02% Tween-20, 0.02% casein, and 0.1% BSA in PBS, pH 7.4. 5 μg / mL hFcγRIIb-his was immobilized on the NTA sensor at an immobilization height of approximately 1.0 nm. The sensor was equilibrated for 300 seconds in a buffer solution of 0.02% Tween-20, 0.02% casein, and 0.1% BSA in PBS, pH 7.4, for blocking. Binding of immobilized hFcγRIIb-his to antibodies at concentrations ranging from 12.5 to 200 nM (two-fold serial dilutions) was determined for 60 seconds. The antibodies were allowed to dissociate in the buffer for 60 seconds. The sensor was refreshed in 10 mM glycine, pH 1.7, and 10 mM nickel sulfate. The temperature was 30°C, and the frequency was 0.6 Hz. The data were analyzed by 1:1 model fitting to obtain affinity constants.

[0181] The results of affinity constant assays of 14C12H1L1(hG1DM), 14C12H1L1(hG4), 14C12H1L1(hG1WT), and 14C12H1L1(hG1TM) and the control antibody 5C10H2L2-IgG1mt for FcγRIIb are shown in Table 6 and Figures 26 to 30.

[0182] Table 6. Kinetics of binding of 14C12H1L1 antibody and its isotypes to FcγRIIb TIFF0007753186000026.tif65167

[0183] N / A indicates that the antibody did not bind to the antigen or had a very weak binding signal; therefore, the results were not analyzed and no corresponding data was obtained.

[0184] The results showed that both 14C12H1L1(hG4) and 14C12H1L1(hG1WT) bound to FcγRIIb with affinity constants of 5.62E-08M and 6.13E-08M, respectively; 14C12H1L1(hG1DM), 14C12H1L1(hG1TM) and 5C10H2L2-IgG1mt did not bind or had very weak binding signals to FcγRIIb, and therefore the results were not analyzed and no corresponding data were obtained.

[0185] The results suggested that the binding activity of 14C12H1L1(hG1DM) and 14C12H1L1(hG1TM) to FcγRIIb was effectively abolished compared to 14C12H1L1(hG4) and 14C12H1L1(hG1WT).

[0186] Experimental Example 5: Affinity assay of 14C12H1L1(hG1DM), 14C12H1L1(hG4), 14C12H1L1(hG1WT) and 14C12H1L1(hG1TM) for C1q Serum complement C1q can bind to the Fc fragment of IgG antibodies and mediate the CDC effect. The binding ability of therapeutic monoclonal antibodies to C1q affects the safety and efficacy of the antibody. The affinity constants of 14C12H1L1(hG1DM), 14C12H1L1(hG4), 14C12H1L1(hG1WT), and 14C12H1L1(hG1TM) for C1q were measured in this experiment using the Fortebio Octet system to evaluate the CDC activity of the antibodies.

[0187] The method for determining the affinity constant of an antibody for C1q using the Fortebio Octet system is briefly described as follows: The sample dilution buffer was a solution of 0.02% Tween-20 and 0.1% BSA in PBS, pH 7.4. 50 μg / mL antibody was immobilized on a FAB2G sensor at an immobilization height of approximately 2.0 nm. The sensor was equilibrated in the buffer for 60 seconds for blocking, and binding of the immobilized antibody on the sensor to the antigen C1q at concentrations ranging from 1.25 to 20 nM (two-fold serial dilutions) was determined for 60 seconds. The antigen and antibody were dissociated in the buffer for 60 seconds. The sensor was refreshed four times in 10 mM glycine, pH 1.7, for 5 seconds each. The shaking speed of the sample plate was 1000 rpm, the temperature was 30 °C, and the frequency was 0.6 Hz. To obtain the affinity constant, the data were analyzed by 1:1 model fitting. The data acquisition software was Fortebio Data Acquisition 7.0, and the data analysis software was Fortebio Data Analysis 7.0.

[0188] The results of affinity constant assays of 14C12H1L1(hG1DM), 14C12H1L1(hG4), 14C12H1L1(hG1WT), and 14C12H1L1(hG1TM) and the control 5C10H2L2-IgG1mt for C1q are shown in Table 7 and Figures 31-35.

[0189] Table 7. Kinetics of binding of 14C12H1L1 antibody and its isotypes to C1q TIFF0007753186000027.tif65168

[0190] N / A indicates that the antibody did not bind to the antigen or had a very weak binding signal; therefore, the results were not analyzed and no corresponding data was obtained.

[0191] The results showed that 14C12H1L1(hG1WT) bound to C1q with an affinity constant of 1.35E-09M; 14C12H1L1(hG1DM), 14C12H1L1(hG4) and 14C12H1L1(hG1TM) did not bind to C1q or had very weak binding signals, therefore, the results were not analyzed and no corresponding data were obtained.

[0192] The results also showed that 5C10H2L2-IgG1mt bound to C1q with an affinity constant of 4.43E-09, indicating that it has binding activity to C1q and can induce the CDC effect.

[0193] Experimental Example 6: Pharmacodynamic activity of 14C12H1L1 (hG1WT) and 14C12H1L1 (hG1TM) in a co-culture system of peripheral blood mononuclear cells and Raji-PDL1 cells In this experiment, the pharmacodynamic activities of the anti-PD-1 antibodies 14C12H1L1(hG1WT) and 14C12H1L1(hG1TM), as well as the control anti-PD-L1 antibodies 5C10H2L2-IgG1mt and nivolumab in alleviating PD-1 / PD-L1-mediated immunosuppression, were detected in a co-culture system of peripheral blood mononuclear cells and Raji-PDL1 cells.

[0194] When isolated peripheral blood mononuclear cells (containing immunocompetent PD-1-expressing immune cells) and PD-L1-expressing Raji-PDL1 cells are cocultured in a mixed lymphocyte reaction, the interaction of PD-1 and PD-L1 can mediate immune cell function inhibition, as evidenced by reduced secretion of the cytokines IFN-γ and IL-2. Anti-PD-1 or anti-PD-L1 antibodies can alleviate such immunosuppression and increase cytokine secretion. Raji is a B cell line. As mentioned above, B cells can be used as antigen-presenting cells to mediate immune cell responses against tumor cells. In this study, the Raji-PDL1 and PBMC coculture system was used to evaluate the pharmacological activity of anti-PD-1 antibodies, and the Raji-PDL1, PBMC, and tumor cell coculture system was used to evaluate the pharmacological activity of anti-PD-1 antibodies in various tumors.

[0195] Peripheral blood mononuclear cells were isolated using Ficoll-Paque Plus (GE Healthcare, Cat No.: 171440-02) and then stimulated with SEB (0.5 μg / mL) for 2 days. Stimulated mature peripheral blood mononuclear cells (1 × 10 5 cells / well), and Raji-PDL1 cells (1 × 10 ) treated with MMC (mitomycin C with a treatment concentration of 2 μg / mL) for 1 h. 5 Cells (0.133 cells / well) were added to a 96-well plate, followed by the addition of 14C12H1L1(hG1WT), 14C12H1L1(hG1TM), control antibody nivolumab, or control anti-PD-L1 antibody 5C10H2L2-IgG1mt. The mixture was mixed thoroughly and incubated. After 3 days, the culture supernatants were collected and tested for IFN-γ and IL-2 secretion using ELISA kits (purchased from Dakewe Biotech Co., Ltd.).

[0196] The results of IFN-γ secretion in the mixed lymphocyte reaction are shown in Figure 36. The results showed that in the PBMC and Raji-PDL1 co-culture system, 14C12H1L1(hG1TM) induced significantly higher IFN-γ secretion than that induced by 14C12H1L1(hG1WT), nivolumab, or 5C10H2L2-IgG1mt at the same dose level.

[0197] The results of IL-2 secretion in the mixed lymphocyte reaction are shown in Figure 37. The results showed that in the PBMC and Raji-PDL1 co-culture system, 14C12H1L1(hG1TM) induced significantly higher IL-2 secretion than that induced by 14C12H1L1(hG1WT), nivolumab, or 5C10H2L2-IgG1mt at the same dose level.

[0198] The results suggested that the pharmacodynamic activity of 14C12H1L1(hG1TM) in alleviating PD-1 / PD-L1-mediated immunosuppression was significantly superior to that of nivolumab, 14C12H1L1(hG1WT), or 5C10H2L2-IgG1mt.

[0199] Experimental Example 7: Antibody-mediated phagocytic activity of nivolumab, 14C12H1L1(hG1WT), and 14C12H1L1(hG1TM) against CHO-K1-PD1 To detect antibody-dependent cellular phagocytosis (ADCP) activity, mouse macrophages were used as effector cells and a PD1-overexpressing cell line was used as target cells. Femoral bone marrow from Blab / c mice (purchased from the Guangdong Medical Laboratory Animal Center) was first aseptically collected and lysed with red blood cell lysis buffer on ice for 5 minutes. Lysis was terminated with DMEM complete medium (containing 10% FBS), and the lysate was centrifuged at 1000 rpm and washed twice. The cell pellet was resuspended in 10 mL of DMEM complete medium, and M-CSF was added to a working concentration of 100 ng / mL. Cells were cultured in a cell culture chamber at 37°C and 5% CO2 for 7 days for induction. Half of the medium was replaced, and M-CSF was added on days 3 and 5. Cell induction was completed on day 7. Cells were digested with 0.05% trypsin. Macrophages were collected and centrifuged at 750 × g for 5 minutes. The supernatant was discarded and the cells were suspended in DMEM complete medium (containing 10% FBS) and counted. The cells were adjusted to the appropriate density and placed in a sterile EP tube for further use.

[0200] CHO-K1-PD1 cells (a CHO-K1 cell line overexpressing PD1) were centrifuged at 170 × g for 5 minutes, washed once with PBS, resuspended, and counted. Viability was determined. Carboxyfluorescein diacetate succinimidyl ester (CFSE) was diluted to 2.5 μM in PBS, and the cells were resuspended (staining density: 10 million cells / mL). An appropriate amount of cells was incubated in a cell incubator for 20 minutes. To stop the staining, 6 mL of DMEM complete medium was added. The cells were centrifuged at 170 × g for 5 minutes, and the supernatant was discarded. 1 mL of DMEM complete medium was added. The cells were incubated in an incubator for 10 minutes and adjusted to the experimental density. The cells were coded as CHO-K-PD1-CFSE.

[0201] The test antibodies were diluted to 20, 2, and 0.2 μg / mL in DMEM complete medium (working concentrations were 10, 1, and 0.1 μg / mL). Anti-HEL IgG1 antibody and medium were used as isotype and blank controls. According to the study design, the diluted antibodies and CHO-K1-PD1-CFSE cells were added to 1.5-mL EP tubes containing macrophages (final volume was 100 μL, effector-to-target ratio was 50,000:150,000). The mixtures were mixed thoroughly for resuspension and incubated at 37°C in an incubator for 2 hours. 800 μL of PBS containing 1% bovine serum albumin (BSA) was added to each tube at room temperature. The mixtures were centrifuged at 500 × g for 5 minutes, and the supernatant was discarded. The cells were washed once with 800 μL of 1% PBSA. APC anti-mouse / human CD11b antibody (Biolegend, Cat. No.: 101212) was diluted 400-fold with PBSA and added to the corresponding samples at 100 μL / sample. The mixture was mixed thoroughly, incubated on ice for 40 minutes, washed with 800 μL of 1% PBSA, centrifuged twice at 1200 × g for 5 minutes, and the supernatant was discarded. 200 μL of 1% PBSA was added to each tube to resuspend the cells. The cells were transferred to a loading tube and analyzed using a BD FACS Calibur flow cytometer. Macrophages in the system were APC. + Macrophages that were positive and involved in phagocytosis were APC and CFSE double positive. The phagocytosis rate was determined as the ratio of the number of double positive cells to the number of APC positive cells, and antibody-mediated ADCP activity was evaluated. The ADCP activity of each group, indicated by P%, was calculated according to the following formula: TIFF0007753186000028.tif11128

[0202] The results are shown in Figure 38.

[0203] The results showed that at the same concentrations, the phagocytosis rates of 14C12H1L1(hG1WT) and nivolumab were 3.94-fold and 4.26-fold, respectively, that of the isotype control anti-HEL antibody, indicating that 14C12H1L1(hG1WT) and nivolumab have ADCP effects; at the same concentrations, the phagocytosis rate of 14C12H1L1(hG1TM) was comparable to that of the isotype control antibody, indicating that 14C12H1L1(hG1TM) does not have ADCP effects.

[0204] The results suggest that the amino acid mutations introduced by 14C12H1L1 (hG1™) can effectively eliminate the ADCP effect, resulting in a surprising technical effect.

[0205] Experimental Example 8: Pharmacodynamic evaluation of 14C12H1L1 (hG1™) + anlotinib hydrochloride in a Scid / beige immunodeficient mouse model bearing human non-small cell lung cancer HCC827 subcutaneous xenograft tumors Female Scid / beige immunodeficient mice (purchased from Vital River) were divided into eight groups. 0.2 μg / mL Staphylococcus aureus enterotoxin B (SEB) was added to a PBMC suspension at 1 million cells / mL. PBMCs were incubated for 3 days for activation to increase PD1 expression on PBMCs. On day 0, mice were subcutaneously implanted with a mixture of 800,000 SEB-activated PBMCs and 6,000,000 HCC827 human non-small cell lung cancer cells (purchased from GuangZhou Jennio Biotech Co., Ltd.) and divided into two groups: an isotype control antibody group (i.e., an anti-HEL antibody prepared by Zhongshan Akeso Biopharma as described above) and an 14C12H1L1 (hG1™) + anlotinib hydrochloride group. For 30 days, 14C12H1L1 (hG1™) was administered weekly via the tail vein (the initial dose was co-administered subcutaneously with the cells), and anlotinib was administered orally by gavage once daily. The specific protocol is shown in Table 8. Tumors were measured continuously throughout the experiment, and the volume was calculated according to the following formula: (tumor length) × b (tumor width) × b (tumor width) / 2.

[0206] Table 8: Experimental design and grouping TIFF0007753186000029.tif63164

[0207] The experimental results are shown in FIG.

[0208] The results showed that 14C12H1L1(hG1™) + anlotinib hydrochloride significantly inhibited the increase in tumor volume of human non-small cell lung cancer cells, indicating a good tumor-killing effect.

[0209] Experimental Example 9: Pharmacodynamic evaluation of 14C12H1L1 (hG1™) in a C57BL / 6-hPD1 / hPDL1 / hCD73 mouse model bearing colon cancer MC38-hPDL1 / hCD73 subcutaneously transplanted tumors The murine MC38 cell line is a murine colorectal cancer cell line. The MC38 cell line has been demonstrated to be a useful model for studying human MSI-H / dMMR tumors (Efremova M et al., Nat Commun., 2018; 9(1):32).

[0210] Female C57BL / 6-hPD1 / hPDL1 / hCD73 mice (purchased from Nanjing GemPharmatech Co., Ltd.) were divided into eight groups and injected with colon cancer MC38-hPDL1 / hCD73 cells (purchased from Nanjing GemPharmatech Co., Ltd.) (2 × 10 6 Cells / 100 μL / mouse) were subcutaneously implanted onto the right forelimb. The implantation day was defined as D0. The administration volume was adjusted according to body weight: 10 μL / g mouse body weight (g). Anti-HEL antibody (preparation and source are the same as those in Experimental Example 8) or 14C12H1L1 (hG1™) was intraperitoneally administered twice a week for 3 weeks, for a total of 6 doses. The specific protocol is shown in Table 9. The tumors were measured continuously in the experiment, and the volume was calculated as follows: tumor volume (mm 3 ) = (tumor length × (tumor width) 2 ) / 2.

[0211] Table 9. Protocols and groupings TIFF0007753186000030.tif34168

[0212] The experimental results are shown in FIG.

[0213] The results showed that tumor growth was inhibited compared to the isotype control antibody, indicating that 14C12H1L1 (hG1™) can significantly inhibit the proliferation of MC38 cells and can effectively treat solid tumors of the MSI-H / dMMR phenotype, such as colon and / or rectal cancer.

[0214] Experimental Example 10: 14C12H1L1 (hG1™) effectively enhanced immune cell responses against human gastric cancer KATO III cells PBMCs were isolated from healthy human peripheral blood according to the Ficoll-Paque™ Plus reagent instructions, and the isolated PBMCs were counted and frozen. Raji-PDL1 cells were cultured in RPMI 1640 + 10% FBS complete medium, and KATO III cells (purchased from the Chinese Academy of Sciences Shanghai Cell Bank) were cultured in DMEM + 10% FBS complete medium. PBMCs were thawed and activated with 0.5 μg / mL SEB for 2 days. On the day of the experiment, Raji-PDL1 cells were treated with 2 μg / mL MMC for 1 hour. SEB-activated PBMCs and MMC-treated Raji-PDL1 cells were harvested, washed twice with PBS, resuspended in RPMI 1640 + 10% FBS complete medium, and counted. Raji-PDL1 and PBMC cells were collected at 1 × 10 5 KATO III cells were harvested in the logarithmic growth phase and seeded at 5 × 10 cells / well onto a 96-well plate. 4 Cells were seeded onto a 96-well plate at 1000 x g / well. Diluted antibodies were added according to the study design. The mixture was mixed evenly and incubated at 37°C in a 5% CO2 incubator for 3 days. After 3 days, cell culture supernatants were collected and tested for IL-2 according to the ELISA kit instructions. The media used in this experiment were all 10% FBS + RPMI 1640.

[0215] The experimental results are shown in FIG.

[0216] The results showed that 14C12H1L1(hG1TM) co-cultured with human gastric cancer KATO III cells exhibited higher pharmacological activity than 14C12H1L1(hG1WT) or nivolumab. 14C12H1L1(hG1TM) could stimulate PBMCs to secrete more IL-2 at the same concentration level, indicating its potential for the treatment of gastric cancer.

[0217] Experimental Example 11: 14C12H1L1 (hG1™) effectively enhanced immune cell responses against nasopharyngeal carcinoma CNE-2Z cells Raji-PDL1, CNE-2Z cells (purchased from GuangZhou Jennio Biotech Co., Ltd.) and PBMCs were thawed, where PBMCs were stimulated with SEB (0.5 μg / mL) for 2 days after thawing for 2 hours. On the day of the experiment, Raji-PDL1 cells were treated with MMC (mitomycin C with a treatment concentration of 2 μg / mL) and 200 × 10 4 The PBMCs were harvested and the treated Raji-PDL1 cells were washed twice with PBS. PBMCs and Raji-PDL1 cells were incubated at 10 × 10 4 Add 3 × 10 CNE-2Z cells / well to the cell plate. 4 Antibodies (with a final concentration of 300 nM and a final volume of 200 μL) were added according to the experimental design and co-cultured with the cells for 3 days. The culture supernatants were collected and assayed for IL-2. The media in all experiments was 10% FBS + RPMI 1640.

[0218] The results are shown in Figure 42.

[0219] The results showed that 14C12H1L1(hG1™) co-cultured with human nasopharyngeal carcinoma CNE-2Z cells exhibited higher pharmacological activity than 14C12H1L1(hG1™). 14C12H1L1(hG1™) could stimulate PBMCs to secrete more IL-2 at the same concentration level, indicating its potential for the treatment of nasopharyngeal carcinoma.

[0220] Experimental Example 12: 14C12H1L1 (hG1™) effectively enhanced immune cell responses against mesothelioma NCI-H2452 cells Raji-PDL1, NCI-H2452 cells (purchased from the Chinese Academy of Sciences, Shanghai Institutes for Biological Sciences) and PBMCs were thawed, where PBMCs were stimulated with SEB (0.5 μg / mL) for 2 days after thawing for 2 hours. On the day of the experiment, Raji-PDL1 cells were incubated with MMC (mitomycin C with a treatment concentration of 2 μg / mL) and 200 × 10 4 The PBMCs were harvested and the treated Raji-PDL1 cells were washed twice with PBS. PBMCs and Raji-PDL1 cells were incubated at 10 × 10 4 Add 3 × 10 NCI-H2452 cells / well to the cell plate. 4 Antibodies (with a final concentration of 300 nM and a final volume of 200 μL) were added according to the experimental design and co-cultured with the cells for 3 days. The culture supernatants were collected and assayed for IL-2. The media in all experiments was 10% FBS + RPMI 1640.

[0221] The results are shown in Figure 43.

[0222] The results showed that 14C12H1L1(hG1™) co-cultured with human mesothelioma NCI-H2452 cells exhibited higher pharmacological activity than 14C12H1L1(hG1™), which could stimulate PBMCs to secrete more IL-2 at the same concentration level, indicating its potential for the treatment of mesothelioma.

[0223] Experimental Example 12: 14C12H1L1 (hG1™) effectively enhanced immune cell responses against small cell lung cancer NCI-H446 cells PBMCs were isolated from healthy human peripheral blood according to the Ficoll-Paque™ Plus reagent instructions, and the isolated PBMCs were counted and frozen. Raji-PDL1 and NCI-H446 cells (purchased from the Chinese Academy of Sciences, Shanghai Institutes for Biological Sciences) were cultured in RPMI 1640 + 10% FBS complete medium. PBMCs were thawed and activated with 0.5 μg / mL SEB for 2 days. On the day of the experiment, Raji-PDL1 cells were treated with 2 μg / mL MMC for 1 hour. SEB-activated PBMCs and MMC-treated Raji-PDL1 cells were harvested, washed twice with PBS, resuspended in RPMI 1640 + 10% FBS complete medium, and counted. Raji-PDL1 and PBMC cells were collected at 1 × 10 5 NCI-H446 cells in the logarithmic growth phase were harvested and seeded at 8 × 10 cells / well onto a 96-well plate. 4 Cells were seeded onto a 96-well plate at 1000 x g / well. Diluted antibodies were added according to the study design. The mixture was mixed evenly and incubated at 37°C in a 5% CO2 incubator for 3 days. After 3 days, cell culture supernatants were collected and tested for IL-2 according to the ELISA kit instructions. The media used in this experiment were all 10% FBS + RPMI 1640.

[0224] The results are shown in Figure 44.

[0225] The results showed that 14C12H1L1(hG1™) co-cultured with human small cell lung cancer NCI-H446 cells exhibited equivalent or greater pharmacological activity compared with 14C12H1L1(hG1WT) and nivolumab based on effectively abolished ADCC, CDC, and ADCP activities. 14C12H1L1(hG1™) could stimulate PBMCs to secrete equivalent or more IL-2 at the same concentration level, demonstrating its potential for the treatment of small cell lung cancer.

[0226] Experimental Example 13: Effectively enhanced immune response of immune cells against human nasopharyngeal carcinoma CNE-2Z cells by 14C12H1L1 (hG1™) in combination with anlotinib hydrochloride PBMCs were isolated from healthy human peripheral blood according to the Ficoll-Paque™ Plus reagent instructions, and the isolated PBMCs were counted and frozen. Raji-PDL1 and CNE-2Z cells (purchased from GuangZhou Jennio Biotech Co., Ltd.) were cultured in RPMI 1640 + 10% FBS complete medium. PBMCs were thawed and activated with 0.5 μg / mL SEB for 2 days. On the day of the experiment, Raji-PDL1 cells were treated with 2 μg / mL MMC for 1 hour. SEB-activated PBMCs and MMC-treated Raji-PDL1 cells were harvested, washed twice with PBS, resuspended in RPMI 1640 + 10% FBS complete medium, and counted. Raji-PDL1 and PBMC cells were collected at 1 × 10 5 CNE-2Z cells in the logarithmic growth phase were harvested and seeded at 3 × 10 cells / well onto a 96-well plate. 4 Cells were seeded onto a 96-well plate at 1000 x g / well. Diluted antibodies and anlotinib were added according to the study design. The mixture was mixed evenly and incubated at 37°C in a 5% CO2 incubator for 3 days. After 3 days, the cell culture supernatant was collected and tested for IL-2 according to the ELISA kit instructions. The media used in this experiment were all 10% FBS + RPMI 1640.

[0227] The results are shown in Figure 45. Compared with anti-HEL antibody and anlotinib monotherapy, 14C12H1L1 (hG1TM), 14C12H1L1 (hG1WT) and nivolumab significantly enhanced the immune cell response to human nasopharyngeal carcinoma CNE-2Z cells, which was characterized by significantly increased IL-2 secretion levels. 14C12H1L1 (hG1TM) has superior pharmacological activity to those of 14C12H1L1 (hG1WT) and nivolumab.

[0228] Furthermore, the pharmacological activity of 14C12H1L1(hG1TM) in combination with anlotinib in stimulating immune cell activation was superior to that of 14C12H1L1(hG1TM) monotherapy, 14C12H1L1(hG1WT) monotherapy, and nivolumab monotherapy, and also superior to that of 14C12H1L1(hG1WT) in combination with anlotinib and nivolumab in combination with anlotinib.

[0229] The above results indicated that 14C12H1L1 (hG1TM) in combination with anlotinib has potential for the treatment of human nasopharyngeal carcinoma.

[0230] Experimental Example 14: Significantly enhanced immune response of immune cells against MSI-H / dMMR tumor SW48 cells by 14C12H1L1 (hG1™) in combination with anlotinib SW48 is a human colorectal cancer cell line identified as an MSI-H / dMMR phenotype (Branch P et al., (1995), Cancer Res, 55(11): 2304-2309.) It was used to detect enhanced immune cell responses against MSI-H / dMMR tumors induced by 14C12H1L1 (hG1™).

[0231] PBMCs were isolated from healthy human peripheral blood according to the Ficoll-Paque™ Plus reagent instructions, and the isolated PBMCs were counted and frozen. Raji-PDL1 cells were cultured in RPMI 1640 + 10% FBS complete medium, and SW48 cells (purchased from GuangZhou Jennio Biotech Co., Ltd.) were cultured in DMEM + 10% FBS complete medium. PBMCs were thawed and activated with 0.5 μg / mL SEB for 2 days. On the day of the experiment, Raji-PDL1 cells were treated with 2 μg / mL MMC for 1 hour. SEB-activated PBMCs and MMC-treated Raji-PDL1 cells were harvested, washed twice with PBS, resuspended in RPMI 1640 + 10% FBS complete medium, and counted. Raji-PDL1 and PBMC cells were collected at 1 × 105 SW48 cells in logarithmic growth phase were harvested and seeded at 2 × 10 cells / well onto a 96-well plate. 5 Cells were seeded onto a 96-well plate at 1000 x g / well. Diluted antibodies and anlotinib were added according to the study design. The mixture was mixed evenly and incubated at 37°C in a 5% CO2 incubator for 3 days. After 3 days, the cell culture supernatant was collected and tested for IL-2 according to the ELISA kit instructions. The media used in this experiment were all 10% FBS + RPMI 1640.

[0232] The results are shown in Figure 46.

[0233] The results showed that 14C12H1L1(hG1TM), 14C12H1L1(hG1WT), and nivolumab significantly enhanced immune cell responses against human colorectal cancer SW48 cells with an MSI-H / dMMR phenotype, characterized by significantly increased levels of IL-2 secretion, compared with anti-HEL antibody. 14C12H1L1(hG1TM) has superior pharmacological activity to that of 14C12H1L1(hG1WT).

[0234] Furthermore, the pharmacological activity of 14C12H1L1(hG1TM) in combination with anlotinib in stimulating immune cell activation was superior to that of 14C12H1L1(hG1WT) monotherapy, 14C12H1L1(hG1TM) monotherapy, and nivolumab monotherapy, and also superior to that of 14C12H1L1(hG1WT) in combination with anlotinib and nivolumab in combination with anlotinib.

[0235] The above results indicated that 14C12H1L1 (hG1™) in combination with anlotinib has potential for the treatment of solid tumors with MSI-H / dMMR phenotype, particularly colon and / or rectal cancer with MSI-H / dMMR phenotype.

[0236] Experimental Example 15: 14C12H1L1 (hG1™) significantly enhanced immune cell responses against non-MSI-H / dMMR phenotype human colorectal cancer SW837 cells SW837 is a human colorectal cancer cell line with a non-MSI-H / dMMR (i.e., MSS) phenotype (Guo J et al., Cancer Res., 2011;71(8):2978-2987.), and in this example, it was used to detect enhanced immune cell responses by 14C12H1L1 (hG1™) against tumors with a non-MSI-H / dMMR (i.e., MSS) phenotype.

[0237] PBMCs were isolated from healthy human peripheral blood according to the Ficoll-Paque™ Plus reagent instructions, and the isolated PBMCs were counted and frozen. Raji-PDL1 cells were cultured in RPMI 1640 + 10% FBS complete medium, and SW837 cells (purchased from Shanghai Honsun Biological Technology Co., Ltd.) were cultured in 10% FBS + Leibovitz's L-15 complete medium (purchased from Gibco). PBMCs were thawed and activated with 0.5 μg / mL SEB for 2 days. On the day of the experiment, Raji-PDL1 cells were treated with 2 μg / mL MMC for 1 hour. SEB-activated PBMCs and MMC-treated Raji-PDL1 cells were harvested, washed twice with PBS, resuspended in RPMI 1640 + 10% FBS complete medium, and counted. Raji-PDL1 and PBMC cells were cultured at 1 × 10 5 SW837 cells in logarithmic growth phase were harvested and seeded at 5 × 10 cells / well onto a 96-well plate. 4 Cells / well were seeded onto a 96-well plate. Diluted antibodies were added according to the study design. The mixture was mixed evenly and incubated at 37°C in a 5% CO2 incubator for 3 days. After 3 days, the cell culture supernatant was collected and tested for IL-2 according to the ELISA kit instructions.

[0238] The results are shown in Figure 47.

[0239] Results showed that 14C12H1L1(hG1TM), 14C12H1L1(hG1WT), and nivolumab significantly enhanced immune cell responses against SW837 human colorectal cancer cells with a non-MSI-H / dMMR phenotype. The pharmacological activity of 14C12H1L1(hG1TM) in the medium and high dose groups was superior to that of 14C12H1L1(hG1WT) and was characterized by significantly increased levels of IL-2 secretion.

[0240] The above results indicate that 14C12H1L1(hG1TM) had better or equivalent pharmacological activity compared to 14C12H1L1(hG1WT) and nivolumab based on effectively abolishing ADCC, CDC, or ADCP effects, indicating its potential for the treatment of solid tumors with non-MSI-H / dMMR (i.e., MSS) phenotype, particularly colon and / or rectal cancer with non-MSI-H / dMMR phenotype.

[0241] Experimental Example 16: Significantly enhanced immune response of immune cells against non-MSI-H / dMMR phenotype human colorectal cancer SW837 cells by 14C12H1L1 (hG1™) in combination with anlotinib PBMCs were isolated from healthy human peripheral blood according to the Ficoll-Paque™ Plus reagent instructions, and the isolated PBMCs were counted and frozen. Raji-PDL1 cells were cultured in RPMI 1640 + 10% FBS complete medium, and SW837 cells were cultured in Leibovitz's L-15 + 10% FBS complete medium. PBMCs were thawed and activated with 0.5 μg / mL SEB for 2 days. On the day of the experiment, Raji-PDL1 cells were treated with 2 μg / mL MMC for 1 hour. SEB-activated PBMCs and MMC-treated Raji-PDL1 cells were harvested, washed twice with PBS, resuspended in RPMI 1640 + 10% FBS complete medium, and counted. Raji-PDL1 and PBMC cells were collected at 1 × 10 5 SW837 cells in logarithmic growth phase were harvested and seeded at 5 × 10 cells / well onto a 96-well plate. 4Cells / well were seeded onto a 96-well plate. Diluted antibodies were added according to the study design. The mixture was mixed evenly and incubated at 37°C in a 5% CO2 incubator for 3 days. After 3 days, the cell culture supernatant was collected and tested for IL-2 according to the ELISA kit instructions.

[0242] The results are shown in Figure 48.

[0243] The results showed that compared with 14C12H1L1(hG1WT) in combination with anlotinib and nivolumab in combination with anlotinib, 14C12H1L1(hG1TM) in combination with anlotinib significantly enhanced the immune cell immune response against non-MSI-H / dMMR phenotype human colorectal cancer SW837 cells, which are characterized by significantly increased IL-2 secretion levels, indicating superior therapeutic efficacy against non-MSI-H / dMMR phenotype solid tumors, especially non-MSI-H / dMMR phenotype colon and / or rectal cancer.

[0244] Although specific aspects of the present invention have been described in detail, those skilled in the art will understand that various modifications and substitutions can be made to those details in accordance with all of the teachings disclosed, and all of these changes fall within the scope of protection of the present invention. The full scope of the present invention is given by the appended claims and any equivalents thereof.

Claims

1. An anti-PD-1 antibody, the heavy chain is set forth in SEQ ID NO: 16 and the light chain is set forth in SEQ ID NO: 12; or the heavy chain is set forth in SEQ ID NO: 18 and the light chain is set forth in SEQ ID NO: 12; and the anti-PD-1 antibody is of the human IgG1 subtype; The anti-PD-1 antibody.

2. 10. An isolated nucleic acid molecule encoding the anti-PD-1 antibody of claim 1.

3. A vector comprising the isolated nucleic acid molecule of claim 2.

4. A host cell comprising the isolated nucleic acid molecule of claim 2 or the vector of claim 3.

5. A conjugate comprising the anti-PD-1 antibody of claim 1 and a conjugate moiety, the conjugated moiety is a detectable label; The conjugate.

6. 6. The conjugate of claim 5, wherein the conjugate moiety is a radioisotope, a fluorescent substance, a luminescent substance, a colored substance, or an enzyme.

7. 7. A kit comprising the anti-PD-1 antibody of claim 1 or the conjugate of any one of claims 5-6.

8. The kit of claim 7, further comprising a second antibody that specifically recognizes the anti-PD-1 antibody.

9. 9. The kit of claim 8, wherein the second antibody further comprises a radioisotope, a fluorescent substance, a luminescent substance, a coloring substance, or an enzyme.

10. Use of the anti-PD-1 antibody of claim 1 or the conjugate of any one of claims 5 to 6 in preparing a kit for detecting the presence or level of PD-1 in a sample.

11. 7. A pharmaceutical composition comprising the anti-PD-1 antibody of claim 1 or the conjugate of any one of claims 5-6.

12. 12. The pharmaceutical composition of claim 11, further comprising a pharmaceutically acceptable carrier and / or excipient.

13. 13. The pharmaceutical composition of claim 11 or 12, further comprising one or more anti-tumor chemotherapeutic agents.

14. 14. The pharmaceutical composition of claim 13, wherein the antitumor chemotherapeutic agent is a tyrosine kinase inhibitor.

15. The pharmaceutical composition according to any one of claims 13 to 14, wherein the antitumor chemotherapeutic drug is anlotinib or a pharmaceutically acceptable salt thereof, or lenvatinib or a pharmaceutically acceptable salt thereof.

16. 16. The pharmaceutical composition of any one of claims 11 to 15, wherein the unit dose of the pharmaceutical composition is 100 to 1000 mg, 200 to 800 mg, 200 to 500 mg, 300 to 600 mg, 400 to 500 mg, or 450 mg, based on the mass of the anti-PD-1 antibody.

17. 17. The pharmaceutical composition of any one of claims 11-16, wherein the unit dose of the anti-PD-1 antibody is 100-1000 mg, 200-800 mg, 200-500 mg, 300-600 mg, 400-500 mg, or 450 mg.

18. 15. The pharmaceutical composition of any one of claims 13-14, wherein the unit dose of the anti-tumor chemotherapeutic agent is 0.1-100 mg, 0.5-50 mg, 1-20 mg, 2-15 mg, 4-12 mg, or 8-12 mg.

19. the anti-PD-1 antibodies are in a fixed combination in the form of a solid pharmaceutical composition or a liquid pharmaceutical composition; or the anti-PD-1 antibody is a non-fixed combination, and the anti-PD-1 antibody and the anti-tumor chemotherapeutic agent in the non-fixed combination are each in the form of a pharmaceutical composition; 19. The pharmaceutical composition of any one of claims 11 to 18.

20. A kit product comprising the pharmaceutical composition of any one of claims 11 to 19 and an attached document.

21. 20. Use of the anti-PD-1 antibody of claim 1, the conjugate of any one of claims 5-6, or the pharmaceutical composition of any one of claims 11-19 in preparing a medicament for treating and / or preventing a tumor or anemia, or in preparing a medicament for diagnosing a tumor or anemia.

22. 22. The use of claim 21, wherein the tumor is selected from one or more of melanoma, kidney cancer, prostate cancer, bladder cancer, colon cancer, rectal cancer, gastric cancer, liver cancer, lung cancer, ovarian cancer, leukemia, nasopharyngeal cancer, and endometrial cancer.

23. 23. The use of claim 22, wherein the lung cancer is selected from one or more of non-small cell lung cancer, small cell lung cancer, and squamous cell lung cancer.

24. 23. The use according to claim 22, wherein the gastric cancer is gastric adenocarcinoma or gastroesophageal junction adenocarcinoma.

25. 25. The use according to any one of claims 21 to 24, wherein the tumor is a solid tumor of MSI-H / dMMR phenotype.

26. The tumor has an MSI-H / dMMR phenotype: Colon cancer, rectal cancer, endometrial cancer, gastric cancer, mesothelioma, sarcoma, adrenocortical carcinoma, malignant melanoma, and ovarian germ cell tumors 26. The use of claim 25, wherein the compound is selected from one or more of:

27. a pharmaceutical agent for blocking the binding of PD-1 to PD-L1; a pharmaceutical agent for downregulating the activity or level of PD-1; a medicament for reducing immunosuppression of PD-1 in an organism; or Medicaments for increasing IFN-γ and / or IL-2 expression in T lymphocytes 20. Use of the anti-PD-1 antibody of claim 1, the conjugate of any one of claims 5-6, or the pharmaceutical composition of any one of claims 11-19 in preparing

28. 20. The anti-PD-1 antibody of claim 1, the conjugate of any one of claims 5-6, or the pharmaceutical composition of any one of claims 11-19, for use in treating and / or preventing tumors or anemia, or for use in diagnosing tumors or anemia.

29. 29. The anti-PD-1 antibody of claim 1, the conjugate of any one of claims 5-6, or the pharmaceutical composition of any one of claims 11-19, for use according to claim 28, wherein the tumor is selected from one or more of melanoma, kidney cancer, prostate cancer, bladder cancer, colon cancer, rectal cancer, gastric cancer, liver cancer, lung cancer, ovarian cancer, leukemia, nasopharyngeal cancer, and endometrial cancer.

30. 29. The anti-PD-1 antibody of claim 1, the conjugate of any one of claims 5-6, or the pharmaceutical composition of any one of claims 11-19, for use according to claim 29, wherein the lung cancer is selected from one or more of non-small cell lung cancer, small cell lung cancer, and squamous cell lung cancer.

31. 29. The anti-PD-1 antibody of claim 1, the conjugate of any one of claims 5-6, or the pharmaceutical composition of any one of claims 11-19, for use according to claim 29, wherein the gastric cancer is gastric adenocarcinoma or gastroesophageal junction adenocarcinoma.

32. 30. The anti-PD-1 antibody of claim 1, the conjugate of any one of claims 5-6, or the pharmaceutical composition of any one of claims 11-19, for use according to any one of claims 28-29, wherein the tumor is a solid tumor of MSI-H / dMMR phenotype.

33. The tumor is of the MSI-H / dMMR phenotype: Colon cancer, rectal cancer, endometrial cancer, gastric cancer, mesothelioma, sarcoma, adrenocortical carcinoma, malignant melanoma, and ovarian germ cell tumors 33. The anti-PD-1 antibody of claim 1, the conjugate of any one of claims 5-6, or the pharmaceutical composition of any one of claims 11-19, for use according to claim 32, selected from one or more of:

34. 34. The anti-PD-1 antibody of claim 1, the conjugate of any one of claims 5-6, or the pharmaceutical composition of any one of claims 11-19, for the use of any one of claims 28 to 33, wherein an effective amount thereof is administered to a subject in need thereof before or after a surgical procedure and / or before or after radiation therapy.

35. 35. The anti-PD-1 antibody of claim 1, the conjugate of any one of claims 5-6, or the pharmaceutical composition of any one of claims 11-19, for use according to claim 34, wherein said effective amount is a unit dose of said anti-PD-1 antibody, said unit dose being 0.1 to 100 mg per kg body weight.

36. 36. The anti-PD-1 antibody of claim 1, the conjugate of any one of claims 5-6, or the pharmaceutical composition of any one of claims 11-19, for use according to claim 35, wherein the unit dose of the anti-PD-1 antibody is 1 to 10 mg per kg body weight, or the unit dose of the anti-PD-1 antibody is 10 to 1000 mg, or the unit dose of the anti-PD-1 antibody is 50 to 500 mg in each subject.

37. 37. The anti-PD-1 antibody of claim 1, the conjugate of any one of claims 5-6, or the pharmaceutical composition of any one of claims 11-19, for the use of any one of claims 35-36, wherein said unit dose is administered once every 3 days, 4 days, 5 days, 6 days, 10 days, 1 week, 2 weeks, or 3 weeks.

38. An anti-PD-1 antibody according to claim 1, a conjugate according to any one of claims 5-6, or a pharmaceutical composition according to any one of claims 11-19, for use according to any one of claims 28-37, wherein the route of administration is intravenous infusion or intravenous injection.

39. 39. The anti-PD-1 antibody of claim 1, the conjugate of any one of claims 5-6, or the pharmaceutical composition of any one of claims 11-19, for the use of any one of claims 28-38, wherein administration of the anti-PD-1 antibody is in a 2- or 3-week cycle.

40. 40. The anti-PD-1 antibody of claim 1, the conjugate of any one of claims 5-6, or the pharmaceutical composition of any one of claims 11-19, for the use of claim 39, wherein said anti-PD-1 antibody is administered intravenously on the first day of each cycle.

41. 41. The anti-PD-1 antibody of claim 1, the conjugate of any one of claims 5-6, or the pharmaceutical composition of any one of claims 11-19, for the use of any one of claims 39-40, wherein said anti-PD-1 antibody is administered once every two or three weeks.

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