Anti-pd-1 antibody and medical use thereof

Abstract

Disclosed is a monoclonal antibody. The monoclonal antibody has a heavy chain variable region comprising a CDR with an amino acid sequence of SEQ ID NOs: 19-21 and / or a light chain variable region comprising a CDR with an amino acid sequence 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 to FcγRIIIa and / or C1q is lower than that before the mutation.

Description

An anti-PD-1 antibody and its pharmaceutical uses Technical Field

[0001] This invention belongs to the fields of tumor therapy and molecular immunology, and relates to an anti-PD-1 antibody and its pharmaceutical uses. Specifically, this invention relates to a mutated anti-PD-1 antibody. Background Technology

[0002] The transmembrane receptor PD-1 (programmed cell death 1) is a member of the CD28 gene family and is expressed in activated T cells, B cells, and myeloid cells. PD-1's ligands, PDL1 (Programmed cell death 1 ligand 1) and PDL2 (Programmed cell death 1 ligand 2), both belong to the B7 superfamily. PDL1 is expressed in various cell types, including T cells, B cells, endothelial cells, and epithelial cells, while PDL2 is expressed only in antigen-presenting cells such as dendritic cells and macrophages.

[0003] The PD-1 / PDL1 signaling pathway plays a crucial role in regulating immune tolerance, microbial infection, and tumor immune escape. PD-1 is primarily expressed on immune cells such as T cells, while its ligand, PDL1, is highly expressed in many 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 also promote the proliferation of tumor antigen-specific T cells, enabling them to kill tumor cells and thus inhibit local tumor growth (Julie R et al., 2012, N Engl J Med. 366:2455–2465).

[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 T cell immune responses. It inhibits TCR-mediated T cell activation, cytokine production, and T cell proliferation (Fife et al. (2011) Nature Immunology 10: 1185-1193); induces exhaustion or unresponsiveness in homologous antigen-specific T cells (Hofmeyer et al. (2011) Journal of Biomedicine and Biotechnology 2011: 1-9); promotes the differentiation of Th1 cells into Foxp3+ regulatory T cells (Armanath et al. (2011) Science TransMed 3: 1-13; Francisco et al. (2009) J. Exp. Med. 206: 3015-3029); and induces apoptosis of effector T cells. Disruption of the PD-L1 gene leads to upregulated T cell responses and the generation of autoreactive T cells (Latchman et al. (2004) PNAS 101:10691-10696). Antibody blockade of PD-1 or PD-L1 results in increased antitumor immunity (Iwai et al. (2002) PNAS 99:12293-12297).

[0005] For nearly two decades, researchers have strived to develop specific immune checkpoint inhibitors, hoping to provide new immunotherapies for cancer treatment. T lymphocytes, part of the innate immune system, possess powerful anti-cancer capabilities with broad and precise specificity, enabling responses to various tumor antigens. This emerging cancer immunotherapy enhances anti-tumor immune responses through adoptive transfer of activated effector cells, immunization against relevant antigens, or the delivery of non-specific immunostimulants. Therefore, PD-1 / PD-L1 specific immune checkpoint inhibitors hold the potential to treat related cancers.

[0006] The mechanism by which anti-PD-1 antibody drugs exert their therapeutic effects is to block the binding of the PD-1 protein on the surface of immune cells to its ligands PDL1 or PDL2, thereby activating immune cells and killing tumors. Currently, there is a need to develop new anti-PD-1 antibodies to reduce or eliminate antibody-mediated ADCC, ADCP, and / or CDC activities that cause damage to immune cells bound by anti-PD-1 antibodies, thus improving the efficacy of antibody drugs. ADCC (antibody-dependent cell-mediated cytotoxicity) refers to the binding of the antibody's Fab fragment to antigenic epitopes on virus-infected cells or tumor cells, and its Fc fragment binding to Fc receptors (FcRs) on the surface of killer cells (NK cells, macrophages, etc.), mediating the direct killing of target cells by killer cells.

[0007] CDC (complement-dependent cytotoxicity) refers to complement-dependent cytotoxicity. CDC action is caused by the initial binding of antibodies to the corresponding antigens on the cell membrane surface, followed by the binding of complement C1q. Subsequently, C2-C9 are activated to form the membrane attack complex, which exerts a lytic effect on the target cell.

[0008] Fc receptors are immunoglobulin family proteins expressed on the surface of specific immune cells, used to recognize antibody Fc regions and mediate immune responses. After the antibody Fab region recognizes an antigen, its antibody Fc region binds to the Fc receptor on immune cells (such as killer cells), initiating immune cell responses such as phagocytosis and ADCC.

[0009] Based on the type of antibody recognized by the Fc receptor and the different cells expressing it, Fc receptors are mainly divided into three types: FcγR, FcαR, and FcεR. FcγR can be further divided into four subtypes: FcγRI (also known as CD64), FcγRII (also known as CD32), FcγRIII (also known as CD16), and FcRn (also known as Neonatal Fc receptor). Among them, FcγRI, FcγRII, and FcγRIII are closely related to ADCC effects. FcγRIII is the most important molecule mediating ADCC, and it has two highly homologous subtypes, FcγRIIIa and FcγRIIIb, in different cell types. In the FcγRIIIa population, there are two subtypes: high-affinity FcγRIIIa caused by single nuclear stem polymorphism (SNP) sites, namely FcγRIIIa_V158 and low-affinity FcγRIIIa_F158. FcγRI has a high affinity for the Fc region of IgG and participates in the ADCC process. There are three subtypes of FcγRII: FcγRIIa, FcγRIIb, and FcγRIIc (also known as CD32a, CD32b, and CD32c, respectively). Among them, FcγRIIa has ADCC activity. There are two subtypes of FcγRIIa in the human population caused by single nucleotide mutations, namely FcγRIIa_H131 and FcγRIIa_R131. FcγRIIb is an inhibitory receptor. FcγRIIb is a typical inhibitory FcγR that can inhibit the nearby ITAM pathway. For example, after immune complexes bind to BCR, the Fc fragment binds to FcγRIIb on the same cell, negatively regulating B cell activation and reducing the secretion of antibodies and cytokines (Hogarth PM, Pietersz GA. 2012, NATURE REVIEWS DRUG DISCOVERY, 11(4):311-331).

[0010] The IgG family comprises four members: IgG1, IgG2, IgG3, and IgG4. The amino acid differences in the crystallizable (Fc) region of their heavy chain constant region result in varying affinities for FcγRs. IgG1 is the most abundant subtype in the human body and the most commonly used subtype in monoclonal antibody drugs. IgG1 can bind to various FcγRs and induce ADCC and CDC effects. IgG2 has the weakest affinity for FcγRs, but it can still induce monocyte-mediated ADCC by binding to FcγRIIa. IgG3 has the strongest binding affinity for FcγRs, inducing ADCC, and its CDC effect is stronger than that of IgG1. IgG4 molecules bind weakly to FcγRs other than FcγRIs, and the likelihood of IgG4 molecules inducing ADCC mediated by CDC and NK cells is low; however, IgG4 subtype antibodies can mediate ADCP effects by binding to FcγRIs. Antibody drugs targeting immune cells may cause immune cell damage due to ADCP effects, which have negative effects on drug pharmacology.

[0011] Studies by Zhang et al. (Zhang T et al. Cancer Immunol Immunother. 2018; 67(7):1079–1090.) and Dahan et al. (Dahan R et al. Cancer Cell. 2015; 28(3):285-95.) have shown that the binding of the Fc fragment of antibodies targeting immune checkpoints such as PD-1 and CTLA-4 to the Fc receptor can negatively affect antibody-mediated anticancer activity. This may be due to Fc-dependent effector function-induced immune cell damage, including antibody-dependent cell-mediated cytotoxicity. Antibody-dependent 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, most lung cancer patients are diagnosed at an advanced stage, and surgery and radiotherapy are not very effective. Chemotherapy has become an important treatment method. At present, platinum-based chemotherapy combined with other chemotherapy drugs remains the first-line chemotherapy regimen for lung cancer, including advanced sNSCLC and NSCLC (Pfister DG et al. J Clin Oncol. 2003; 22:330; De Ruysscher et al. (2006) Annals of Oncology 17:543-552.).

[0013] Chemotherapy drugs are currently mainly divided into the following nine categories (He Jie et al. Clinical Oncology. Beijing: People's Medical Publishing House, 2016: 230-237). The first category consists of drugs that directly bind to DNA and inhibit DNA replication, including various chemotherapeutic agents, mitomycin and bleomycin, dacarbazine, platinum-based drugs such as cisplatin and carboplatin, camptothecin-based drugs and their derivatives. The second category consists of drugs that inhibit nucleic acid biosynthesis. These drugs mainly affect the enzyme system of tumor cells, inhibiting the synthesis of DNA and RNA precursors, thereby inhibiting DNA or RNA formation. These drugs mainly include methotrexate, fluorouracil, 6-mercaptopurine, hydroxyurea, and cytarabine, which mainly act on S phase cells and belong to antimetabolite chemotherapeutic drugs, which are cell cycle-specific anticancer drugs. The third category consists of chemotherapeutic drugs that affect transcription. Their main pharmacological action is to insert into the DNA double helix and form a non-covalent binding with it, thereby interfering with the transcription of genetic information on DNA to DNA-dependent mRNA, resulting in impaired signaling function and transcriptional inhibition. These drugs mainly include... The fourth category consists of drugs that affect tubulin and mitosis, mainly including vinblastines, podophyllotoxins, and paclitaxel-based herbal medicines. The fifth category consists of drugs that affect ribosome function and inhibit protein synthesis, represented by cephalotaxine-based herbal medicines. These drugs inhibit the initiation of protein synthesis, causing ribosome breakdown and the release of nascent peptide chains, but they cannot prevent the binding of mRNA and tRNA to ribosomes. These drugs can reduce nuclear DNA and cytoplasmic RNA, depolymerize polyribosomes, and inhibit mitosis. The sixth category consists of drugs that affect the cell membrane, such as phytohemagglutinins (Con-A) and phytohemagglutinins (PHA), which can bind to glycoprotein receptors on the cell membrane. The first category consists of several types of chemotherapy drugs. The first category includes drugs that induce apoptosis, such as arsenic trioxide. The second category comprises drugs that regulate endocrine function to treat tumors, including estrogens, anti-estrogens, progestins, androgens, anti-androgens, adrenocortical hormones, and anti-adrenocortical hormones (including chlorobenzene dichloroethane and ammoniaglutide). The third category includes anti-tumor targeted therapy, including monoclonal antibodies, epidermal growth factor signaling inhibitors (such as targeted drugs against the receptor tyrosine kinase pathway), ubiquitin-proteasome inhibitors, and angiogenesis inhibitors. However, in addition to killing tumor cells, chemotherapy drugs also damage normal cells, often leading to severe toxic side effects in common chemotherapy regimens for cancer patients. More importantly, besides significant toxicity, many cancer patients still cannot achieve long-term disease control after chemotherapy, and the 5-year survival rate remains very low. Therefore, developing less toxic and more effective treatments or combination therapy regimens has significant clinical implications.

[0014] Anlotinib is a quinoline derivative tyrosine kinase inhibitor that acts as a multi-target tyrosine kinase inhibitor (TKI) by influencing tumor angiogenesis and proliferation signaling. Its main targets include: receptor tyrosine kinases vascular endothelial growth factor receptor (VEGFR) 1–3, epidermal growth factor receptor (EGFR), fibroblast growth factor receptor (FGFR) 1–4, platelet-derived growth factor receptor (PDGFR) α and β, and stem cell factor receptor (SCFR) 7, 8, and 9. A phase 2 trial showed that anlotinib improved progression-free survival and had a potential benefit in overall survival (Han B, et al. Br J Cancer. 2018; 118(5): 654-661). A multicenter, double-blind, phase 3 randomized clinical trial showed that anlotinib resulted in prolonged overall survival and progression-free survival in Chinese patients. This finding suggests that anlotinib is well-tolerated and is a potential third-line or further treatment for patients with advanced NSCLC (Han B, et al. JAMA Oncol. 2018 Nov; 4(11): 1569-1575).

[0015] Document WO2008112407 discloses a quinoline derivative tyrosine kinase inhibitor, 1-[[[4-(4-fluoro-2-methyl-1H-indol-5-yl)oxy-6-methoxyquinoline-7-yl]oxy]methyl]cyclopropylamine, and its preparation method in Example 24. Its structural formula is shown in Formula I below. Anlotinib hydrochloride is the hydrochloride salt of the compound of Formula I.

[0016]

[0017] Lenvatinib is an oral multi-tyrosine kinase inhibitor developed by Eisai Co., Ltd. of Japan. It is a multi-target 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 associated with pathogenic angiogenesis, tumor growth, and cancer progression, including fibroblast growth factor (FGF) receptors FGFR1, FGFR2, FGFR3, and FGFR4; transfection rearrangement receptor (RET), KIT, and platelet-derived growth factor receptor α (PDGFRα). Lenvatinib also exhibits antiproliferative activity in hepatocellular carcinoma cell lines, which depends on activated FGFR signaling and simultaneous inhibition of FGF receptor substrate 2α (FRS2α) phosphorylation.

[0018] Example 368 of U.S. Patent 7,612,208 discloses the compound structure of lenvatinib as 4-(3-chloro-4-(cyclopropylaminocarbonyl)aminophenoxy)-7-methoxy-6-quinoline carboxamide. U.S. Patent No. 7,253,286 discloses the methanesulfonate form of lenvatinib (i.e., methanesulfonate), with the chemical name 4-[3-chloro-4-(cyclopropylureoyl)phenoxy]-7-methoxyquinoline-6-carboxamide methanesulfonate. Its chemical structure (Formula II) is provided below:

[0019]

[0020] However, many cancer patients still cannot achieve long-term disease control after chemotherapy, and the 5-year survival rate remains very low. Therefore, developing less toxic and more effective treatments or combination therapy regimens has great clinical significance.

[0021] Summary of the Invention

[0022] Through in-depth research and creative work, the inventors have modified the Fc terminus of the anti-PD-1 antibody structure to reduce the binding of the Fc region to the Fc receptor, thereby reducing the toxic side effects on T cell ADCC, ADCP, and / or CDC, and increasing the efficacy of the anti-PD-1 antibody drug. This provides the following invention:

[0023] One aspect of the present invention relates to an antibody, wherein,

[0024] The heavy chain variable region of the antibody contains amino acid sequences such as HCDR1-HCDR3 as shown in SEQ ID NOs:19-21; and the light chain variable region of the antibody contains amino acid sequences such as LCDR1-LCDR3 as shown in SEQ ID NOs:22-24.

[0025] The antibody is a human IgG1 subtype;

[0026] Specifically, according to the EU numbering system, the heavy chain constant region of the antibody is mutated at any two or three sites among positions 234, 235 and 237, and the affinity constant of the mutated antibody is lower than that of FcγRIIIa and / or C1q before the mutation; preferably, the affinity constant is measured by a Fortebio Octet molecular interaction analyzer.

[0027] In one embodiment of the present invention, the antibody is a monoclonal antibody.

[0028] In one embodiment of the present invention, the antibody is an anti-PD-1 antibody, preferably an anti-PD-1 monoclonal antibody.

[0029] In some embodiments of the present invention, the antibody, according to the EU numbering system, has the following mutations in the heavy chain constant region at positions 234, 235, and / or 237:

[0030] L234A and L235A;

[0031] L234A and G237A;

[0032] L235A and G237A;

[0033] or

[0034] L234A, L235A, and G237A.

[0035] In this invention, unless otherwise specified, the letter before the site represents the amino acid before the mutation, and the letter after the site represents the amino acid after the mutation.

[0036] This invention also relates to an antibody, wherein,

[0037] The heavy chain variable region of the antibody contains amino acid sequences such as HCDR1-HCDR3 as shown in SEQ ID NOs:19-21; and the light chain variable region of the antibody contains amino acid sequences such as LCDR1-LCDR3 as shown in SEQ ID NOs:22-24.

[0038] The antibody is a human IgG1 subtype;

[0039] Specifically, according to the EU numbering system, the heavy chain constant region of the antibody has the following mutations at positions 234, 235, and / or 237:

[0040] L234A and L235A;

[0041] L234A and G237A;

[0042] L235A and G237A;

[0043] or

[0044] L234A, L235A, and G237A.

[0045] In some embodiments of the present invention, according to the EU numbering system, the heavy chain constant region of the antibody further has one or more mutations selected from the following:

[0046] 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.

[0047] In some embodiments of the present invention, the antibody, wherein...

[0048] The amino acid sequence of the heavy chain variable region of the antibody is selected from SEQ ID NO:2 and SEQ ID NO:6; and

[0049] The amino acid sequence of the variable region of the light chain of the antibody is selected from SEQ ID NO:4 and SEQ ID NO:8.

[0050] In some embodiments of the present invention, the antibody, wherein...

[0051] The amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO:2, and the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO:4.

[0052] The amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO:2, and the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO:8.

[0053] The amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO:6, and the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO:4; or

[0054] The amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO:6, and the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO:8.

[0055] In one embodiment of the present invention, the antibody:

[0056] Its heavy chain is shown in SEQ ID NO:16, and its light chain is shown in SEQ ID NO:12;

[0057] or

[0058] Its heavy chain is shown in SEQ ID NO:18, and its light chain is shown in SEQ ID NO:12.

[0059] The variable regions of the light and heavy chains determine antigen binding; each chain's variable region contains three hypervariable regions called complementarity-determining regions (CDRs) (the CDRs of the heavy chain (H) include HCDR1, HCDR2, and HCDR3, and the CDRs of the light chain (L) include LCDR1, LCDR2, and LCDR3; these were named by Kabat et al., see Sequences of Proteins of Immunological Interest, Fifth Edition (1991), Volumes 1-3, NIH Publication 91-3242, Bethesda Md).

[0060] Using techniques well known to those skilled in the art, such as analyzing the amino acid sequence of the CDR region of the monoclonal antibody sequence in items (1)-(3) above through the VBASE2 database:

[0061] The antibodies 14C12, 14C12H1L1 (hG1WT), 14C12H1L1 (hG1DM), and 14C12H1L1 (hG1TM) involved in this invention have the same CDR:

[0062] The amino acid sequences of the three CDR regions of its heavy chain variable region are as follows:

[0063] HCDR1:GFAFSSYD(SEQ ID NO:19),

[0064] HCDR2:ISGGGRYT(SEQ ID NO:20),

[0065] HCDR3:ANRYGEAWFAY(SEQ ID NO:21);

[0066] The amino acid sequences of the three CDR regions in the light chain variable region are as follows:

[0067] LCDR1:QDINTY(SEQ ID NO:22),

[0068] LCDR2:RAN(SEQ ID NO:23),

[0069] LCDR3: LQYDEFPLT (SEQ ID NO: 24).

[0070] In some embodiments of the invention, the antibody is at a concentration greater than about 10. -7 M, for example, greater than approximately 10 -6 M, 10-5 M, 10 -4 M or 10 -3 An affinity constant of M or greater is combined with FcγRIIIa_F158, FcγRI, FcγRIIa_H131, FcγRIIIa_V158 and / or FcγRIIb; preferably, the affinity constant is determined by a Fortebio Octet molecular interaction analyzer.

[0071] Preferably, the antibody has no binding signal or a binding signal less than 0.1 nm with FcγRIIIa_F158, FcγRI, FcγRIIa_H131, FcγRIIIa_V158 and / or FcγRIIb; preferably, the binding signal refers to the response value measured by a Fortebio Octet molecular interaction analyzer.

[0072] In some embodiments of the invention, the antibody is at a concentration greater than about 10. -9 M, for example, greater than approximately 10 -8 M, 10 -7 M, 10 -6 M or 10 -5 An affinity constant of M or greater is used to bind C1q; preferably, the affinity constant is measured using a Fortebio Octet molecular interaction analyzer;

[0073] Preferably, the antibody has no binding signal with C1q or the binding signal is less than 0.1 nm; preferably, the binding signal refers to the response value measured by a Fortebio Octet molecular interaction analyzer.

[0074] In some embodiments of the present invention, the antibody is a monoclonal antibody.

[0075] In some embodiments of the present invention, the antibody is a humanized antibody.

[0076] Another aspect of the present invention relates to an isolated nucleic acid molecule that encodes an antibody as described in any one of the present invention.

[0077] Another aspect of the invention relates to a carrier comprising the isolated nucleic acid molecules of the invention.

[0078] Another aspect of the invention relates to a host cell comprising the isolated nucleic acid molecule of the invention, or the vector of the invention.

[0079] Another aspect of the present invention relates to a conjugate comprising an antibody and a conjugation portion, wherein the antibody is any of the antibodies described in any one of the present invention, and the conjugation portion is a detectable marker; preferably, the conjugation portion is a radioactive isotope, a fluorescent substance, a luminescent substance, a colored substance, or an enzyme.

[0080] Another aspect of the present invention relates to a kit comprising the antibody as described in any one of the present invention, or comprising the conjugate of the present invention;

[0081] Preferably, the kit further includes a second antibody that specifically recognizes the antibody; optionally, the second antibody further includes a detectable label, such as a radioactive isotope, a fluorescent substance, a luminescent substance, a colored substance, or an enzyme.

[0082] Another aspect of the invention relates to the use of any of the antibodies or conjugates of the invention in the preparation of a kit for detecting the presence or level of PD-1 in a sample.

[0083] Another aspect of the invention relates to a pharmaceutical composition comprising an antibody or conjugate of any one of the present invention; optionally, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and / or excipient.

[0084] In one or more embodiments of the present invention, the drug further comprises one or more tumor chemotherapy drugs;

[0085] Preferably, the tumor chemotherapy drug is a tyrosine kinase inhibitor; more preferably, the tumor chemotherapy drug is anlotinib or a pharmaceutically acceptable salt thereof (e.g., hydrochloride), or lenvatinib or a pharmaceutically acceptable salt thereof (e.g., methanesulfonate).

[0086] In one or more embodiments of the present invention, the pharmaceutical composition wherein the unit dose of the pharmaceutical composition, calculated according to the mass of the antibody therein, is 100mg-1000mg, 200mg-800mg, 200mg-500mg, 300mg-600mg, 400mg-500mg, or 450mg.

[0087] Another aspect of the invention relates to a pharmaceutical combination comprising the antibody described in any one of the inventions, and at least one (e.g., one, two, or three) tumor chemotherapy drug.

[0088] In one or more embodiments of the present invention, the drug combination wherein the tumor chemotherapy drug is a tyrosine kinase inhibitor; more preferably, the tumor chemotherapy drug is anlotinib or a pharmaceutically acceptable salt thereof (e.g., hydrochloride), or lenvatinib or a pharmaceutically acceptable salt thereof (e.g., methanesulfonate).

[0089] In one or more embodiments of the present invention, the drug combination wherein the unit dose of the antibody is 100mg-1000mg, 200mg-800mg, 200mg-500mg, 300mg-600mg, 400mg-500mg, or 450mg.

[0090] In one or more embodiments of the present invention, the drug combination wherein the unit dose of the tumor chemotherapy drug is 0.1mg-100mg, 0.5mg-50mg, 0.5mg-10mg, 1mg-10mg, 2mg-8mg, or 1mg-5mg.

[0091] In one or more embodiments of the present invention, the drug combination wherein the unit dose of the tumor chemotherapy drug is 1 mg-20 mg, 2 mg-15 mg, 4 mg-12 mg, or 8 mg-12 mg.

[0092] In one or more embodiments of the present invention, the drug combination, wherein...

[0093] The drug combination is a fixed combination, for example, in the form of a solid drug composition or a liquid drug composition; or

[0094] The drug combination is a non-fixed combination; for example, the anti-PD-1 antibody and the tumor chemotherapy drug in the non-fixed combination are each in the form of a drug composition.

[0095] Another aspect of the present invention relates to a medicine box product comprising the pharmaceutical composition or pharmaceutical combination described in any one of the present invention, and product instructions.

[0096] Another aspect of the present invention relates to the use of any antibody, conjugate, pharmaceutical composition, or pharmaceutical combination described in any one of the present invention in the preparation of a medicament for treating and / or preventing tumors or anemia, or in the preparation of a medicament for diagnosing tumors or anemia; preferably, the tumor is selected from one or more of melanoma, renal tumor, prostate cancer, bladder cancer, colon cancer, rectal cancer, gastric cancer, liver cancer, lung cancer, ovarian cancer, leukemia, nasopharyngeal carcinoma, and endometrial cancer;

[0097] Preferably, the lung cancer is selected from one or more of non-small cell lung cancer, small cell lung cancer, and squamous cell carcinoma of the lung;

[0098] Preferably, the gastric cancer is gastric adenocarcinoma or esophageal junction adenocarcinoma;

[0099] Preferably, the tumor is a solid tumor with the MSI-H / dMMR phenotype; preferably, the tumor is selected from one or more of the following tumors with the MSI-H / dMMR phenotype:

[0100] Colon cancer, rectal cancer, endometrial cancer, gastric cancer, mesothelioma, sarcoma, adrenocortical carcinoma, malignant melanoma, or ovarian germ cell tumors.

[0101] In one or more embodiments of the present invention, the use is described in which the tumor is a recurrent, metastatic (e.g., lymph node metastasis, brain metastasis and / or bone metastasis) or refractory tumor.

[0102] MSI (microsatellite instability) refers to the instability of microsatellites. Microsatellites are short tandem repeats distributed throughout the human genome, consisting of single nucleotide, dinucleotide, or high-order nucleotide repeats, with repeat counts ranging from 10 to 50. Compared to normal cells, some abnormal tissue cells, such as tumors, exhibit altered microsatellite length due to the insertion or deletion of repeat units, a phenomenon known as MSI. Based on the degree of instability, MSI can be classified into highly unstable microsatellites (MSI-H), poorly unstable microsatellites (MSI-L), and stable microsatellites (MSS). The primary cause of MSI is a defect in DNA mismatch repair (MMR) function. Human mismatch repair genes (MMR genes) express corresponding mismatch repair proteins after transcription and translation. The absence of any MMR protein expression leads to a defect in the cell's mismatch repair function, resulting in the loss of repair function for base mismatches during DNA replication, causing accumulation and leading to microsatellite instability (MSI). Approximately 15% of colorectal cancers are caused by the MSI pathway. It was first discovered in colorectal cancer, but can also occur in gastric cancer, endometrial cancer, adrenocortical tumors, etc. (Baretti M et al. Pharmacol Ther. 2018; 189:45-62.). Subsequent studies have also found the MSI-H / dMMR feature in mesothelioma, sarcoma, adrenocortical carcinoma, malignant melanoma and ovarian germ cell tumors.

[0103] MSI-H and dMMR represent results from two different detection methods. dMMR and MSI-H are biologically consistent and are referred to as MSI-H / dMMR or MSI-high / dMMR. MSI-L and MSS represent the phenotype of normal MMR (proficient mismatch repair, pMMR). The dMMR detection method involves immunohistochemical protein detection of four mismatch genes—MSH2, MLH1, MSH6, and PMS2—in tumor specimens (including surgical and biopsy specimens). If any one of these four proteins is absent, it is dMMR; if all four proteins are positively expressed, the tumor is pMMR, meaning its mismatch repair function is intact. MSI detection involves pairing and comparing the lengths of repetitive DNA sequences (microsatellite sequences) from tumor cells and somatic cells. When using PCR to detect five standard loci based on the US NCI standards, if two or more loci are inconsistent, it is considered unstable and defined as MSI-H; if one locus is inconsistent, it is called MSI-L (microsatellite low instability); and if all five loci are consistent, it is called MSS. High-throughput sequencing (or next-generation sequencing technology, NGS) can also be used to detect microsatellite instability. When using more microsatellite loci, such as more than five or other microsatellite loci, for PCR detection, ≥30% inconsistency is usually called MSI-H, all consistent loci are defined as MSS, and inconsistency between 0% and 30% is MSI-L.

[0104] Another aspect of the present invention relates to the use of the antibody, conjugate, pharmaceutical composition, or pharmaceutical combination described in any one of the present invention in the preparation of the following pharmaceutical products:

[0105] Drugs that block the binding of PD-1 and PDL1

[0106] Drugs that downregulate PD-1 activity or levels

[0107] Drugs that relieve the immunosuppression caused by PD-1, or

[0108] Drugs that increase the expression of IFN-γ and / or IL-2 in T lymphocytes.

[0109] Interferon-γ (IFNγ) is primarily produced innately by natural killer (NK) cells and natural killer T cells (NKT), and by effector T cells such as CD4Th1 cells and CD8 cytotoxic T lymphocytes after stimulation by specific antigens. As an important innate and acquired immune cytokine, IFNγ plays a crucial role in combating or inhibiting viral, certain bacterial, and protozoal infections. Simultaneously, IFNγ can activate macrophages, induce the expression of major histocompatibility complex class II, and activate immune responses to control tumor development (Schoenborn JR, Wilson CB. Regulation of Interferon-γ During Innate and Adaptive Immune Responses. Advances in Immunology 2007; 96:41-101). In the in vitro experiments of this invention, the antibody of this invention can induce the secretion of IFNγ and activate immune responses.

[0110] Interleukin-2 (IL-2), produced by T cells, is a growth factor regulating T cell subsets and an important factor in regulating immune responses. It also promotes the proliferation of activated B cells and participates 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: pp. 14-27). In in vitro experiments, the antibody of this invention specifically relieves PD-1-induced immunosuppression, activates T cells, and induces IL-2 production, showing broad application prospects in the treatment of tumors and parasitic diseases.

[0111] Another aspect of the invention relates to an in vivo or in vitro method comprising the step of administering to cells or a subject in need an effective amount of any of the antibodies, conjugates, pharmaceutical compositions, or drug combinations described in any one of the inventions, the method being selected from the following:

[0112] Methods to block the binding of PD-1 and PDL1

[0113] Methods to downregulate PD-1 activity or levels

[0114] Methods to relieve the immunosuppression caused by PD-1, or

[0115] Methods to increase the expression of IFN-γ and / or IL-2 in T lymphocytes.

[0116] The antibody, conjugate, pharmaceutical composition, or pharmaceutical combination according to any one of the present invention is used to treat and / or prevent tumors or anemia, or to diagnose tumors or anemia; preferably, the tumor is selected from one or more of melanoma, kidney tumor, prostate cancer, bladder cancer, colon cancer, rectal cancer, stomach cancer, liver cancer, lung cancer, ovarian cancer, leukemia, nasopharyngeal carcinoma, and endometrial cancer;

[0117] Preferably, the lung cancer is selected from one or more of non-small cell lung cancer, small cell lung cancer, and squamous cell carcinoma of the lung;

[0118] Preferably, the gastric cancer is gastric adenocarcinoma or esophageal junction adenocarcinoma;

[0119] Preferably, the tumor is a solid tumor with the MSI-H / dMMR phenotype; preferably, the tumor is selected from one or more of the following tumors with the MSI-H / dMMR phenotype:

[0120] Colon cancer, rectal cancer, endometrial cancer, gastric cancer, mesothelioma, sarcoma, adrenocortical carcinoma, malignant melanoma, or ovarian germ cell tumors.

[0121] In one or more embodiments of the present invention, the antibody or conjugate of the present invention is wherein the tumor is a recurrent, metastatic (e.g., lymph node metastasis, brain metastasis and / or bone metastasis) or refractory tumor.

[0122] The antibody, conjugate, pharmaceutical composition, or pharmaceutical combination according to any one of the present invention is used for:

[0123] Blocking the binding of PD-1 to PDL1,

[0124] Downregulate PD-1 activity or level,

[0125] To relieve the immunosuppression caused by PD-1 in the body, or

[0126] Increase the expression of IFN-γ and / or IL-2 in T lymphocytes.

[0127] Another aspect of the present invention relates to a method for treating and / or preventing tumors or anemia, or a method for diagnosing tumors or anemia, comprising the step of administering to a subject in need an effective amount of any of the antibodies, conjugates, pharmaceutical compositions, or combinations of pharmaceuticals described in any one of the present invention; preferably, the tumor is selected from one or more of melanoma, kidney tumor, prostate cancer, bladder cancer, colon cancer, rectal cancer, stomach cancer, liver cancer, lung cancer, ovarian cancer, leukemia, nasopharyngeal carcinoma, and endometrial cancer;

[0128] Preferably, the lung cancer is selected from one or more of non-small cell lung cancer, small cell lung cancer, and squamous cell carcinoma of the lung;

[0129] Preferably, the gastric cancer is gastric adenocarcinoma or esophageal junction adenocarcinoma;

[0130] Preferably, the tumor is a solid tumor with the MSI-H / dMMR phenotype; preferably, the tumor is selected from one or more of the following tumors with the MSI-H / dMMR phenotype:

[0131] Colon cancer, rectal cancer, endometrial cancer, gastric cancer, mesothelioma, sarcoma, adrenocortical carcinoma, malignant melanoma, or ovarian germ cell tumors.

[0132] In one or more embodiments of the present invention, the method wherein the tumor is a recurrent, metastatic (e.g., lymph node metastasis, brain metastasis, and / or bone metastasis) or refractory tumor.

[0133] In one or more embodiments of the present invention, the method wherein the step of administering an effective amount of anti-PD-1 antibody to a subject in need is performed before or after surgical treatment, and / or before or after radiotherapy.

[0134] In one or more embodiments of the present invention, the method wherein...

[0135] The single dose of anti-PD-1 antibody is 0.1-100 mg per kilogram of body weight, preferably 1-10 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 single dose of anti-PD-1 antibody is 10-1000 mg per subject (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 50-500 mg, 100-400 mg, 150-300 mg, 150-250 mg or 200 mg;

[0136] Preferably, the drug is administered once every 3, 4, 5, 6, 10 days, 1 week, 2 weeks, or 3 weeks;

[0137] Preferably, the administration method is intravenous infusion or intravenous injection.

[0138] In some regimens, anti-PD-1 antibody treatment is administered in cycles of 2 weeks (14 days) or 3 weeks (21 days), preferably intravenously on the first day (D1) of each cycle. For example, the anti-PD-1 antibody may be administered every two weeks (q2w) or every three weeks (q3w).

[0139] In this invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the cell culture, molecular genetics, nucleic acid chemistry, and immunology laboratory procedures used herein are all standard procedures widely used in their respective fields. To better understand this invention, definitions and explanations of relevant terms are provided below.

[0140] 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, or the extracellular fragment PD-1ECD, or a fragment containing PD-1ECD; it also includes fusion proteins of PD-1ECD, such as fragments fused with a fragment of the Fc protein (mFc or hFc) of mouse or human IgG. However, those skilled in the art will understand that mutations or variations (including but not limited to substitutions, deletions, and / or additions) can be naturally generated or artificially introduced into the amino acid sequence of PD-1 protein without affecting its biological function. Therefore, in this invention, the term "PD-1 protein" should include all such sequences, and their natural or artificial variants. Furthermore, when describing a sequence fragment of PD-1 protein, it includes not only the sequence fragment itself but also the corresponding sequence fragments in its natural or artificial variants.

[0141] 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, or the extracellular fragment of PDL1, PDL1ECD, or a fragment containing PDL1ECD; it also includes fusion proteins of PDL1ECD, such as fragments fused with a fragment of the Fc protein (mFc or hFc) of mouse or human IgG. However, those skilled in the art will understand that mutations or variations (including, but not limited to, substitutions, deletions, and / or additions) can be naturally generated or artificially introduced into the amino acid sequence of the PDL1 protein without affecting its biological function. Therefore, in this invention, the term "PDL1 protein" should include all such sequences as well as their natural or artificial variants. Furthermore, when describing a sequence fragment of the PDL1 protein, it includes the PDL1 sequence fragment as well as corresponding sequence fragments from its natural or artificial variants.

[0142] As used in this article, the term EC 50 The half-maximal concentration (50% of maximal effect) refers to the concentration that produces a 50% maximal effect.

[0143] As used herein, the term "antibody" refers to an immunoglobulin molecule typically composed of two pairs of polypeptide chains (each pair consisting of a "light" (L) chain and a "heavy" (H) chain). Antibody light chains can be classified as κ and λ light chains. Heavy chains can be classified as μ, δ, γ, α, or ε, and antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE, respectively. Within both light and heavy chains, variable and constant regions are linked by "J" regions of approximately 12 or more amino acids, and heavy chains also contain "D" regions of approximately 3 or more amino acids. Each heavy chain is further divided by a heavy chain variable region (V... H ) and heavy chain constant region (C H The heavy chain constant region consists of three structural domains (C). H1 C H2 and C H3 It consists of ) light chains. Each light chain is composed of a light chain variable region (V L ) and light chain constant region (C L It consists of a light chain constant region composed of a structural domain C. L Composition. The constant region of an antibody mediates the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. V H and V L The region can be further subdivided into highly variable regions (called complementary determinant regions (CDRs)), interspersed with more conservative regions called framework regions (FRs). Each V H and V L It consists of three CDRs and four FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4, from the amino terminus to the carboxyl terminus. The variable region (V) of each heavy chain / light chain pair... H and V LEach amino acid is assigned to a specific region or domain to form an antibody binding site. The allocation of amino acids to these regions or domains follows the definitions in Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883. The term “antibody” is not limited to any particular method of antibody production. For example, it includes, in particular, recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. Antibodies can be different isotypes of antibodies, such as IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM antibodies.

[0144] As used herein, the terms “monoclonal antibody” and “monoclonal antibody” refer to an antibody or a fragment of an antibody derived from a group of highly homologous antibody molecules—that is, a group of identical antibody molecules except for the possibility of spontaneous natural mutations. Monoclonal antibodies exhibit high specificity for a single epitope on an antigen. Polyclonal antibodies, as opposed to monoclonal antibodies, typically contain at least two or more different antibodies that typically recognize different epitopes on an antigen. Monoclonal antibodies are usually obtained using hybridoma techniques first reported by Kohler et al. (Nature, 256:495, 1975), but can also be obtained using recombinant DNA techniques (see US Patent 4,816,567).

[0145] As used herein, the term "humanized antibody" refers to an antibody or antibody fragment obtained by replacing all or part of the CDR region of a human immunoglobulin (receptor antibody) with the CDR region of a non-human antibody (donor antibody), wherein the donor antibody can be a non-human antibody (e.g., mouse, rat, or rabbit) with the expected specificity, affinity, or reactivity. Furthermore, some amino acid residues in the framework region (FR) of the receptor antibody may also be replaced by amino acid residues of the corresponding non-human antibody, or by amino acid residues of other antibodies, to further improve or optimize the antibody's performance. For more detailed information on humanized antibodies, please refer to, for example, 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).

[0146] As used herein, the terms "separated" or "isolated" refer to substances obtained artificially from their natural state. If a substance or component is found in nature as a "separated" entity, it may be due to an alteration of its natural environment, the separation of the substance from its natural environment, or both. For example, a certain unseparated polynucleotide or polypeptide may naturally exist in the body of a living animal, and a high-purity identical polynucleotide or polypeptide separated from this natural state is called a separated one. The terms "separated" or "isolated" do not exclude the presence of artificial or synthetic substances, nor do they exclude the presence of other impurities that do not affect the substance's activity.

[0147] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which polynucleotides can be inserted. When a vector enables the expression of a protein encoded by the inserted polynucleotide, it is called an expression vector. Vectors can be introduced into host cells through transformation, transduction, or transfection, allowing the genetic material elements they carry to be expressed in the host cells. Vectors are well-known to those skilled in the art and include, but are not limited to: plasmids; phage particles; Cos plasmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage; and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retrotranscriptoviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40). A vector may contain multiple elements controlling expression, including but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, a vector may contain a replication initiation site.

[0148] As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including but not limited to prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells, or human cells.

[0149] 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 that is specific to an antigen) means that the antibody binds to an antigen at a concentration of less than about 10. -5 M, for example, less than approximately 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 M or lower affinity (K) D () binds to the antigen.

[0150] As used in this article, the term "K" D "" refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, which describes the binding affinity between the antibody and the antigen. The smaller the equilibrium dissociation constant, the stronger the antibody-antigen binding and the higher the affinity between the antibody and the antigen. Typically, antibodies have an equilibrium dissociation constant of less than approximately 10. -5 M, for example, less than approximately 10 -6 M, 10-7 M, 10 -8 M, 10 -9 M or 10 -10 M or a smaller dissociation equilibrium constant (K) D K binds to an antigen (e.g., PD-1 protein). K can be measured using methods known to those skilled in the art. D For example, measurements can be taken using a Fortebio molecular interaction analyzer.

[0151] As used herein, the terms "monoclonal antibody" and "monoclonal antibody" have the same meaning and are used interchangeably; the terms "polyclonal antibody" and "polyclonal antibody" have the same meaning and are used interchangeably; and the terms "peptide" and "protein" have the same meaning and are used interchangeably. Furthermore, in this invention, amino acids are generally represented by single-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.

[0152] As used herein, the term "pharmaceutically acceptable carrier and / or excipient" means a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, which is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to: pH adjusters, surfactants, adjuvants, and ionic strength enhancers. For example, pH adjusters include, but are not limited to, phosphate buffers; 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.

[0153] As used herein, the term "adjuvant" refers to a nonspecific immune enhancer that, when administered to the body along with or before an antigen, can enhance the body's immune response to the antigen or alter the type of immune response. There are many types of adjuvants, including but not limited to aluminum adjuvants (e.g., aluminum hydroxide), Freund's adjuvants (e.g., complete and incomplete Freund's adjuvants), Corynebacterium breve, lipopolysaccharides, and cytokines. Freund's adjuvant is currently the most commonly used adjuvant in animal studies. Aluminum hydroxide adjuvant is more frequently used in clinical trials.

[0154] As used herein, the term "effective amount" means an amount sufficient to achieve, or at least partially achieve, the desired effect. For example, an effective amount for preventing disease (e.g., cancer) means an amount sufficient to prevent, stop, or delay the onset of disease (e.g., cancer); an effective amount for treating disease means an amount sufficient to cure or at least partially stop the disease and its complications in a patient already suffering from the disease. Determining such an effective amount is entirely within the capabilities of those skilled in the art. For example, an effective amount for therapeutic purposes will depend on the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general characteristics such as age, weight, and sex, the manner of administration of the drug, and other concurrent treatments, etc.

[0155] As used herein, the term "complete elimination" means that no binding signal or an extremely low binding signal is detected by existing instrumentation (e.g., the Fortebio Octet molecular interaction analyzer). In one embodiment of the invention, no binding signal or an extremely low binding signal means a binding signal (i.e., a response value) of less than 0.1 nm.

[0156] "Recurrent" cancer is cancer that recurs at the initial site or a distant site after a response to initial treatment (such as surgery). "Locally recurrent" cancer is cancer that recurs at the same location as the previously treated cancer after treatment.

[0157] Metastatic cancer refers to cancer that has spread from one part of the body (such as the lungs) to another part of the body.

[0158] Beneficial effects of the invention

[0159] The present invention achieves one or more of the technical effects described in items (1) to (9) below:

[0160] (1) The antibodies of the present invention, especially 14C12H1L1 (hG1TM) and 14C12H1L1 (hG1WT), can effectively block the immunosuppression of immune cells induced by PD-1 / PDL1 binding and induce human peripheral blood mononuclear cells to secrete IFN-γ and IL-2.

[0161] (2) The antibody of the present invention, particularly 14C12H1L1 (hG1TM), completely eliminates its binding activity with Fc receptors FcγRI, FcγRIIa_H131, FcγRIIIa_V158 and / or FcγRIIIa_F158, thereby eliminating its ADCC or ADCP activity.

[0162] (3) The antibody of the present invention, especially 14C12H1L1 (hG1TM), completely eliminates its binding activity with complement C1q, thereby completely eliminating its CDC activity.

[0163] (4) The antibodies of the present invention, such as 14C12H1L1 (hG1DM), significantly weaken their binding to Fc receptors FcγRI, FcγRIIa_H131, FcγRIIa_R131 and / or FcγRIIIa_V158, and completely eliminate their binding to FcγRIIIa_F158 and / or FcγRIIb, thus significantly reducing their ADCC activity.

[0164] (5) The antibody of the present invention, particularly 14C12H1L1 (hG1DM), completely eliminates its binding activity with complement C1q, thereby completely eliminating its CDC activity.

[0165] (6) The monoclonal antibodies of the present invention, particularly 14C12H1L1 (hG1TM), 14C12H1L1 (hG1DM), and 14C12H1L1 (hG1WT), can bind specifically to PD-1 and effectively block the binding of PD-1 to PDL1, specifically relieving the immunosuppression caused by PD-1 and activating T lymphocytes. Among them, the PD-1 antibody 14C12H1L1 (hG1TM) shows a significantly stronger induction effect on IFN-γ and IL-2 secretion than the control anti-PD-1 antibody Nivolumab and the control anti-PDL1 antibody 5C10H2L2-IgG1mt. These antibodies have the potential to be used in the preparation of drugs for the prevention and treatment of tumors.

[0166] (7) It can effectively prevent and treat the aforementioned tumors;

[0167] (8) Low toxicity and side effects;

[0168] (9) The anti-PD-1 antibody of the present invention or the anti-PD-1 antibody in the drug combination of the present invention has a synergistic effect with the chemotherapy drug. Attached Figure Description

[0169] Figure 1: Affinity constant detection results of 14C12H1L1(hG1DM) and FcγRI. The antibody concentrations added to each pair of curves from top to bottom in the figure are 50 nM, 25 nM, 12.5 nM, 6.25 nM, and 3.12 nM, respectively.

[0170] Figure 2: Affinity constant detection results of 14C12H1L1(hG4) and FcγRI. The antibody concentrations added to each pair of curves from top to bottom in the figure are 50 nM, 25 nM, 12.5 nM, 6.25 nM, and 3.12 nM, respectively.

[0171] Figure 3: Affinity constant detection results of 14C12H1L1(hG1WT) and FcγRI. The antibody concentrations added to each pair of curves from top to bottom in the figure are 50 nM, 25 nM, 12.5 nM, 6.25 nM, and 3.12 nM, respectively.

[0172] Figure 4: Affinity constant detection results of 14C12H1L1(hG1TM) and FcγRI. The antibody concentrations added to each pair of curves from top to bottom in the figure are 50 nM, 25 nM, 12.5 nM, 6.25 nM, and 3.12 nM, respectively.

[0173] Figure 5: Affinity constant detection results of 5C10H2L2-IgG1mt and FcγRI. The antibody concentrations added to each pair of curves from top to bottom in the figure are 50 nM, 25 nM, 12.5 nM, 6.25 nM, and 3.12 nM, respectively.

[0174] Figure 6: Affinity constant detection results of 14C12H1L1(hG1DM) and FcγRIIIa_V158. The antibody concentrations added to each pair of curves from top to bottom in the figure are 500 nM, 250 nM, 125 nM, 62.5 nM, and 31.25 nM, respectively.

[0175] Figure 7: Affinity constant detection results of 14C12H1L1(hG4) and FcγRIIIa_V158. The antibody concentrations added to each pair of curves from top to bottom in the figure are 500 nM, 250 nM, 125 nM, 62.5 nM, and 31.25 nM, respectively.

[0176] Figure 8: Affinity constant detection results of 14C12H1L1(hG1WT) and FcγRIIIa_V158. The antibody concentrations added to each pair of curves from top to bottom in the figure are 500 nM, 250 nM, 125 nM, 62.5 nM, and 31.25 nM, respectively.

[0177] Figure 9: Affinity constant detection results of 14C12H1L1(hG1TM) and FcγRIIIa_V158. The antibody concentrations added to each pair of curves from top to bottom in the figure are 500 nM, 250 nM, 125 nM, 62.5 nM, and 31.25 nM, respectively.

[0178] Figure 10: Affinity constant detection results of 5C10H2L2-IgG1mt and FcγRIIIa_V158. The antibody concentrations added to each pair of curves from top to bottom in the figure are 500 nM, 250 nM, 125 nM, 62.5 nM, and 31.25 nM, respectively.

[0179] Figure 11: Affinity constant detection results of 14C12H1L1(hG1DM) and FcγRIIIa_F158. The concentrations of antigen added in each pair of curves from top to bottom in the figure are 500 nM, 250 nM, 125 nM, 62.5 nM, and 31.25 nM, respectively.

[0180] Figure 12: Affinity constant detection results of 14C12H1L1(hG4) and FcγRIIIa_F158. The antibody concentrations added to each pair of curves from top to bottom in the figure are 500 nM, 250 nM, 125 nM, 62.5 nM, and 31.25 nM, respectively.

[0181] Figure 13: Affinity constant detection results of 14C12H1L1(hG1WT) and FcγRIIIa_F158. The antibody concentrations added to each pair of curves from top to bottom in the figure are 500 nM, 250 nM, 125 nM, 62.5 nM, and 31.25 nM, respectively.

[0182] Figure 14: Affinity constant detection results of 14C12H1L1(hG1TM) and FcγRIIIa_F158. The antibody concentrations added to each pair of curves from top to bottom in the figure are 500 nM, 250 nM, 125 nM, 62.5 nM, and 31.25 nM, respectively.

[0183] Figure 15: Affinity constant detection results of 5C10H2L2-IgG1mt and FcγRIIa_F158. The antibody concentrations added to each pair of curves from top to bottom in the figure are 500 nM, 250 nM, 125 nM, 62.5 nM, and 31.25 nM, respectively.

[0184] Figure 16: Affinity constant detection results of 14C12H1L1(hG1DM) and FcγRIIa_H131. The antibody concentrations added to each pair of curves from top to bottom in the figure are 200 nM, 100 nM, 50 nM, 25 nM, and 12.5 nM, respectively.

[0185] Figure 17: Affinity constant detection results of 14C12H1L1(hG4) and FcγRIIa_H131. The antibody concentrations added to each pair of curves from top to bottom in the figure are 200 nM, 100 nM, 50 nM, 25 nM, and 12.5 nM, respectively.

[0186] Figure 18: Affinity constant detection results of 14C12H1L1(hG1WT) and FcγRIIa_H131. The antibody concentrations added to each pair of curves from top to bottom in the figure are 200 nM, 100 nM, 50 nM, 25 nM, and 12.5 nM, respectively.

[0187] Figure 19: Affinity constant detection results of 14C12H1L1(hG1TM) and FcγRIIa_H131. The antibody concentrations added to each pair of curves from top to bottom in the figure are 200 nM, 100 nM, 50 nM, 25 nM, and 12.5 nM, respectively.

[0188] Figure 20: Affinity constant detection results of 5C10H2L2-IgG1mt and FcγRIIa_H131. The antibody concentrations added to each pair of curves from top to bottom in the figure are 200 nM, 100 nM, 50 nM, 25 nM, and 12.5 nM, respectively.

[0189] Figure 21: Affinity constant detection results of 14C12H1L1(hG1DM) and FcγRIIa_R131. The antibody concentrations added to each pair of curves from top to bottom in the figure are 200 nM, 100 nM, 50 nM, 25 nM, and 12.5 nM, respectively.

[0190] Figure 22: Affinity constant detection results of 14C12H1L1(hG4) and FcγRIIa_R131. The antibody concentrations added to each pair of curves from top to bottom in the figure are 200 nM, 100 nM, 50 nM, 25 nM, and 12.5 nM, respectively.

[0191] Figure 23: Affinity constant detection results of 14C12H1L1(hG1WT) and FcγRIIa_R131. The antibody concentrations added to each pair of curves from top to bottom in the figure are 200 nM, 100 nM, 50 nM, 25 nM, and 12.5 nM, respectively.

[0192] Figure 24: Affinity constant detection results of 14C12H1L1(hG1TM) and FcγRIIa_R131. The antibody concentrations added to each pair of curves from top to bottom in the figure are 200 nM, 100 nM, 50 nM, 25 nM, and 12.5 nM, respectively.

[0193] Figure 25: Affinity constant detection results of 5C10H2L2-IgG1mt and FcγRIIa_R131. The antibody concentrations added to each pair of curves from top to bottom in the figure are 200 nM, 100 nM, 50 nM, 25 nM, and 12.5 nM, respectively.

[0194] Figure 26: Affinity constant detection results of 14C12H1L1(hG1DM) and FcγRIIb. The antibody concentrations added to each pair of curves from top to bottom in the figure are 200 nM, 100 nM, 50 nM, 25 nM, and 12.5 nM, respectively.

[0195] Figure 27: Affinity constant detection results of 14C12H1L1(hG4) and FcγRIIb. The antibody concentrations added to each pair of curves from top to bottom in the figure are 200 nM, 100 nM, 50 nM, 25 nM, and 12.5 nM, respectively.

[0196] Figure 28: Affinity constant detection results of 14C12H1L1(hG1WT) and FcγRIIb. The antibody concentrations added to each pair of curves from top to bottom in the figure are 200 nM, 100 nM, 50 nM, 25 nM, and 12.5 nM, respectively.

[0197] Figure 29: Affinity constant detection results of 14C12H1L1(hG1TM) and FcγRIIb. The antibody concentrations added to each pair of curves from top to bottom in the figure are 200 nM, 100 nM, 50 nM, 25 nM, and 12.5 nM, respectively.

[0198] Figure 30: Affinity constant detection results of 5C10H2L2-IgG1mt and FcγRIIb. The antibody concentrations added to each pair of curves from top to bottom in the figure are 200 nM, 100 nM, 50 nM, 25 nM, and 12.5 nM, respectively.

[0199] Figure 31: Detection results of the affinity constant between 14C12H1L1(hG1DM) and C1q. The antibody concentrations added to each pair of curves from top to bottom in the figure are 20 nM, 10 nM, 5 nM, 2.5 nM, and 1.25 nM, respectively.

[0200] Figure 32: Detection results of the affinity constant between 14C12H1L1(hG4) and C1q. The antibody concentrations added to each pair of curves from top to bottom in the figure are 20 nM, 10 nM, 5 nM, 2.5 nM, and 1.25 nM, respectively.

[0201] Figure 33: Detection results of the affinity constant between 14C12H1L1(hG1WT) and C1q. The antibody concentrations added to each pair of curves from top to bottom in the figure are 20 nM, 10 nM, 5 nM, 2.5 nM, and 1.25 nM, respectively.

[0202] Figure 34: Detection results of the affinity constant between 14C12H1L1(hG1TM) and C1q. The antibody concentrations added to each pair of curves from top to bottom in the figure are 20 nM, 10 nM, 5 nM, 2.5 nM, and 1.25 nM, respectively.

[0203] Figure 35: Affinity constant detection results of 5C10H2L2-IgG1mt and C1q. The concentrations of antigen added in each pair of curves from top to bottom in the figure are 20 nM, 10 nM, 5 nM, 2.5 nM, and 1.25 nM, respectively.

[0204] Figure 36: Results of IFN-γ secretion detection after mixed lymphocyte reaction with 14C12H1L1(hG1WT) and 14C12H1L1(hG1TM).

[0205] Figure 37: Results of IL-2 secretion detection after mixed lymphocyte reaction with 14C12H1L1(hG1WT) and 14C12H1L1(hG1TM).

[0206] Figure 38: ADCP effect results of 14C12H1L1(hG1WT), Nivolumab and 14C12H1L1(hG1TM).

[0207] Figure 39: Killing effect of 14C12H1L1(hG1TM) + anlotinib on human non-small cell lung cancer tumor cells.

[0208] Figure 40: 14C12H1L1 (hG1TM) inhibits the proliferation of mouse colorectal cancer MC38 cells.

[0209] Figure 41: 14C12H1L1 (hG1TM) effectively enhances the immune response of immune cells to human gastric cancer cells KATO III.

[0210] Figure 42: 14C12H1L1 (hG1TM) effectively enhances the immune response of immune cells to CNE-2Z nasopharyngeal carcinoma tumor cells.

[0211] Figure 43: 14C12H1L1 (hG1TM) effectively enhances the immune response of immune cells to NCI-H2452 mesothelioma tumor cells.

[0212] Figure 44: 14C12H1L1 (hG1TM) effectively enhances the immune response of immune cells to human small cell lung cancer cells NCI-H446 cells.

[0213] Figure 45: 14C12H1L1 (hG1TM) combined with anlotinib hydrochloride effectively enhances the immune response of immune cells to CNE-2Z nasopharyngeal carcinoma tumor cells.

[0214] Figure 46: 14C12H1L1 (hG1TM) combined with anlotinib significantly enhances the immune response of immune cells to the MSI-h / dMMR tumor cell line SW48.

[0215] Figure 47: 14C12H1L1 (hG1TM) significantly enhances the immune response of immune cells to human colorectal cancer cells SW837 that do not have the MSI-h / dMMR (i.e., MSS) phenotype.

[0216] Figure 48: 14: 14C12H1L1 (hG1TM) combined with anlotinib significantly enhanced the immune response of immune cells against human colorectal cancer cells SW837 cells with a non-MSI-h / dMMR (i.e., MSS) phenotype. Detailed Implementation

[0217] 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 for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the examples, they are performed according to the techniques or conditions described in the literature in the art (e.g., refer to J. Sambrook et al., *Molecular Cloning: A Laboratory Manual*, 3rd edition, Science Press, translated by Huang Peitang et al.) or according to the product instructions. Reagents or instruments whose manufacturers are not specified are conventional products that can be purchased commercially.

[0218] In the following experiments of this invention:

[0219] BALB / c mice were purchased from the Guangdong Provincial Medical Laboratory Animal Center.

[0220] The preparation method of anti-PDL1 antibody 5C10H2L2-IgG1mt is as described in PCT publication WO2017148424A1.

[0221] The anti-PD-1 antibody Nivolumab (trade name Opdivo) was purchased from Bristol-Myers Squibb.

[0222] All human peripheral blood mononuclear cells were isolated and prepared at Zhongshan Kangfang Biopharmaceutical Co., Ltd., with informed consent from the provider.

[0223] Raji-PDL1 is a cell expressing human PD-L1 constructed by Zhongshan Kangfang Biopharmaceutical Co., Ltd. based on the human B cell line Raji cells through transfection.

[0224] Ficoll-Paque TM PLUS (or Ficoll-Paque PLUS) was purchased from GE Healthcare.

[0225] The Human IL-2 ELISA Kit was purchased from Dakota Biotechnology Co., Ltd.

[0226] RPMI 1640 medium, DMEM medium, Trypsin-EDTA (0.25%), phenol red, and Blastidin were all derived from Gibco.

[0227] Staphylococcal enterotoxin antigen (SEB) is derived from Denotec.

[0228] FBS is derived from Excel bio.

[0229] Mitomycin C (MMC) is derived from Stressmarq.

[0230] The isotype control antibody is human anti-Hen Egg Lysozyme IgG (i.e., anti-HEL antibody, or human IgG, abbreviated as hIgG). The sequence is derived from the variable region of the Fab F10.6.6 sequence in the paper "Affinity maturation increases the stability and plasticity of the Fv domain of anti-protein antibodies" published by Acierno et al. (Acierno et al. J Mol Biol. 2007; 374(1):130-146.).

[0231] The anlotinib used in the examples is anlotinib hydrochloride, traded under the name Fucove. Its generic name is anlotinib hydrochloride, and it is sourced from Chia Tai Tianqing Pharmaceutical Group Co., Ltd.

[0232] Preparation Example 1: Sequence design of anti-PD-1 antibody 14C12 and its humanized antibody 14C12H1L1 (hG1WT)

[0233] The amino acid sequences of the heavy and light chains of the anti-PD-1 antibody 14C12 and its humanized antibody 14C12H1L1 (hG1WT), as well as the encoding nucleic acid sequences, are completely identical to 14C12 and 14C12H1L1 in Chinese Patent Publication CN 106967172A (or CN 106977602A).

[0234] (1) Heavy chain variable region sequence and light chain variable region sequence of 14C12

[0235] Nucleic acid sequence of the 14C12 heavy chain variable region: (354bp)

[0236]

[0237] The amino acid sequence of the variable region of the 14C12 heavy chain is: (118aa)

[0238]

[0239] Nucleic acid sequence of the variable region of the 14C12 light chain: (321bp)

[0240]

[0241] Amino acid sequence of the variable region of the 14C12 light chain: (107aa)

[0242]

[0243]

[0244] (2) Heavy chain variable region sequence and light chain variable region sequence, heavy chain sequence and light chain sequence of humanized monoclonal antibody 14C12H1L1(hG1WT)

[0245] Nucleic acid sequence of the variable region of the 14C12H1L1(hG1WT) heavy chain: (354bp)

[0246]

[0247] The amino acid sequence of the variable region of the 14C12H1L1(hG1WT) heavy chain is: (118aa)

[0248]

[0249] The nucleic acid sequence of the variable region of the 14C12H1L1(hG1WT) light chain is 321 bp.

[0250]

[0251] The amino acid sequence of the variable region of the 14C12H1L1(hG1WT) light chain is: (107 aa)

[0252]

[0253] The nucleic acid sequence of the 14C12H1L1(hG1WT) heavy chain: (1344bp)

[0254]

[0255] The amino acid sequence of the 14C12H1L1(hG1WT) heavy chain is: (448aa)

[0256]

[0257] Nucleic acid sequence of the 14C12H1L1(hG1WT) light chain: (642bp)

[0258]

[0259] The amino acid sequence of the 14C12H1L1(hG1WT) light chain is: (214aa)

[0260]

[0261] Preparation Example 2: Sequence Design of Humanized Antibody 14C12H1L1(hG4)

[0262] The heavy chain and light chain variable regions are identical to those of 14C12H1L1(hG1WT). Additionally, the Ig gamma-4 chain C region (ACCESSION: P01861.1) was used as the heavy chain constant region, and the Ig kappa chain C region (ACCESSION: P01834) was used as the light chain constant region, thus yielding the antibody 14C12H1L1(hG4). The sequence of 14C12H1L1(hG4) is as follows:

[0263] Nucleic acid sequence of the 14C12H1L1(hG4) heavy chain: (1335bp)

[0264]

[0265]

[0266] The amino acid sequence of the 14C12H1L1(hG4) heavy chain is: (445aa)

[0267]

[0268] The nucleic acid sequence of the 14C12H1L1(hG4) light chain is the same as that of SEQ ID NO:11.

[0269] The amino acid sequence of the 14C12H1L1(hG4) light chain is the same as that of SEQ ID NO:12.

[0270] Preparation Example 3: Sequence Design of Humanized Antibody 14C12H1L1 (hG1TM)

[0271] Based on the 14C12H1L1(hG1WT) obtained in Preparation Example 1, the inventors, following the EU numbering system, introduced a point mutation from leucine to alanine (L234A) at position 234, a point mutation from leucine to alanine (L235A) at position 235, and a point mutation from glycine to alanine (G237A) at position 237 in its heavy chain hinge region, thus obtaining a mutated humanized 14C12H1L1(hG1TM).

[0272] The nucleic acid sequence of the 14C12H1L1(hG1TM) heavy chain: (1344bp)

[0273]

[0274]

[0275] The amino acid sequence of the 14C12H1L1(hG1TM) heavy chain is: (448aa)

[0276]

[0277]

[0278] The nucleic acid sequence of the 14C12H1L1(hG1TM) light chain is the same as that of SEQ ID NO:11.

[0279] The amino acid sequence of the 14C12H1L1(hG1TM) light chain is the same as that of SEQ ID NO:12.

[0280] Preparation Example 4: Sequence Design of Humanized Antibody 14C12H1L1 (hG1DM)

[0281] Based on 14C12H1L1(hG1WT), the inventors obtained the mutated humanized antibody 14C12H1L1(hG1DM) by introducing a point mutation from leucine to alanine (L234A) at position 234 and a point mutation from leucine to alanine (L235A) at position 235 in the heavy chain hinge region.

[0282] Nucleic acid sequence of the 14C12H1L1(hG1DM) heavy chain: (1344bp)

[0283]

[0284]

[0285] The amino acid sequence of the 14C12H1L1(hG1DM) heavy chain is: (448aa)

[0286]

[0287] The nucleic acid sequence of the 14C12H1L1(hG1DM) light chain is the same as that of SEQ ID NO:11.

[0288] The amino acid sequence of the 14C12H1L1(hG1DM) light chain is the same as that of SEQ ID NO:12.

[0289] Experimental Example 1: Detection of Fc receptor FcγRI affinity of 14C12H1L1 (hG1DM), 14C12H1L1 (hG4), 14C12H1L1 (hG1WT), and 14C12H1L1 (hG1TM)

[0290] The Fc receptor FcγRI (also known as CD64) can bind to the Fc terminus of IgG antibodies, participating in antibody-dependent cell-mediated cytotoxicity (ADCC). The ability of therapeutic monoclonal antibodies to bind to the Fc receptor affects the safety and efficacy of the antibody. In this study, the affinity constants of 14C12H1L1 (hG1DM), 14C12H1L1 (hG4), 14C12H1L1 (hG1WT), and 14C12H1L1 (hG1TM) to FcγRI were detected using a Fortebio Octet molecular interaction analyzer to evaluate the ADCC activity of each antibody.

[0291] The experimental method for detecting the affinity constants of corresponding antibodies with FcγRI using the Fortebio Octet molecular interaction analyzer is briefly described below: The sample dilution buffer was PBS, 0.02% Tween-20, 0.1% BSA, pH 7.4. FcγRI (purchased from Sinobio) solution at a concentration of 1 μg / mL was added to the HIS1K sensor and immobilized for 50 s to allow FcγRI to adhere to the sensor surface. The binding and dissociation parameters of the antibody with FcγRI were measured in buffer solution at antibody concentrations ranging from 3.12 to 50 nM (two-fold serial dilution). After the sensor immobilized with antigen was equilibrated in buffer solution for 60 s, the binding time of FcγRI immobilized on the sensor with each antibody was measured at 120 s; the dissociation time of FcγRI from the antibody was also measured at 120 s. The detection temperature was 30℃, and the frequency was 0.3 Hz. Data were analyzed using a 1:1 model fitting to obtain the affinity constants of each antibody with FcγRI.

[0292] The affinity constants of FcγRI with 14C12H1L1(hG1DM), 14C12H1L1(hG4), 14C12H1L1(hG1WT), 14C12H1L1(hG1TM), and the control antibody 5C10H2L2-IgG1mt are shown in Table 1 and Figures 1-5.

[0293] Table 1: Kinetic parameters of binding of 14C12H1L1(hG1DM), 14C12H1L1(hG4), 14C12H1L1(hG1WT), 14C12H1L1(hG1TM), and control antibody 5C10H2L2-IgG1mt to FcγRI

[0294] Antibody K D(M)kon(1 / Ms)SE(kon)kdis(1 / s)SE(kdis)Rmax(nm)14C12H1L1(hG1DM)N / AN / AN / AN / AN / AN / A14C12H1L1(hG4)5.80E-096.37E+052.21E+043.69E-031.05E-040.45 -0.5414C12H1L1(hG1WT)2.52E-096.94E+052.19E+041.75E-039.14E-050.50-0. 5514C12H1L1(hG1TM)N / AN / AN / AN / AN / AN / A5C10H2L2-IgG1mtN / AN / AN / AN / AN / AN / A

[0295] N / A indicates that the antibody did not bind to the antigen or the binding signal was extremely low. The results were not analyzed, so no corresponding data was obtained.

[0296] The results showed that both 14C12H1L1(hG4) and 14C12H1L1(hG1WT) could bind to FcγRI, with affinity constants of 5.80E-09M and 2.52E-09M, respectively. 14C12H1L1(hG1TM) and 5C10H2L2-IgG1mt did not bind to FcγRI or had extremely low binding signals, so the results were not analyzed, and therefore no data were obtained.

[0297] The results showed that the binding activity of 14C12H1L1(hG1DM) and 14C12H1L1(hG1TM) to FcγRI was effectively eliminated compared with 14C12H1L1(hG4) and 14C12H1L1(hG1WT).

[0298] Experimental Example 2: Affinity determination of 14C12H1L1 (hG1DM), 14C12H1L1 (hG4), 14C12H1L1 (hG1WT) and 14C12H1L1 (hG1TM) with Fc receptor FcγRIIIa and its subtypes

[0299] (1) Determination of affinity constants of FcγRIIIa_V158 with 14C12H1L1(hG1DM), 14C12H1L1(hG4), 14C12H1L1(hG1WT) and 14C12H1L1(hG1TM)

[0300] The Fc receptor FcγRIIIa_V158 (also known as CD16a_V158) can bind to the Fc terminus of IgG antibodies, mediating ADCC effects. In this experiment, the affinity constants of 14C12H1L1 (hG1DM), 14C12H1L1 (hG4), 14C12H1L1 (hG1WT), and 14C12H1L1 (hG1TM) for FcγRIIIa_V158 were measured using a Fortebio Octet molecular interaction analyzer to evaluate the ADCC activity of each antibody.

[0301] The experimental method for detecting the affinity constants of the corresponding antibody and the control antibody 5C10H2L2-IgG1mt with FcγRIIIa_V158 using the Fortebio Octet molecular interaction analyzer is briefly described below: The sample dilution buffer was PBS, 0.02% Tween-20, 0.1% BSA, pH 7.4. 5 μg / mL of FcγRIIIa_V158 was immobilized on the HIS1K sensor for 120 s. The sensor equilibrated in the buffer for 60 s. The immobilized FcγRIIIa_V158 bound to each antibody (31.25-500 nM, 2-fold dilution) for 60 s. The antibody dissociated in the buffer for 60 s. The sensor was regenerated using 10 mM glycine at pH 1.5 for 5 s, repeated 4 times. The detection temperature was 30℃, and the frequency was 0.3 Hz. Data were analyzed using a 1:1 model to obtain the affinity constants.

[0302] The affinity constants of FcγRIIIa_V158 with 14C12H1L1(hG1DM), 14C12H1L1(hG4), 14C12H1L1(hG1WT), 14C12H1L1(hG1TM), and the control antibody 5C10H2L2-IgG1mt are shown in Table 2 and Figures 6-10.

[0303] Table 2: Kinetic parameters of binding of 14C12H1L1(hG1DM), 14C12H1L1(hG4), 14C12H1L1(hG1WT), 14C12H1L1(hG1TM), and control antibody 5C10H2L2-IgG1mt to FcγRIIIa_V158

[0304]

[0305] N / A indicates that the antibody did not bind to the antigen or the binding signal was extremely low. The results were not analyzed, so no corresponding data was obtained.

[0306] The results showed that 14C12H1L1(hG1DM) and 14C12H1L1(hG1WT) could both bind to FcγRIIIa_V158, with affinity constants of 6.21E-07M and 6.54E-08M, respectively; while 14C12H1L1(hG4), 14C12H1L1(hG1TM) and 5C10H2L2-IgG1mt were not analyzed because they did not bind to FcγRIIIa_V158 or had extremely low binding signals.

[0307] The results showed that, compared with 14C12H1L1(hG1DM) and 14C12H1L1(hG1WT), the binding activity of 14C12H1L1(hG4), 14C12H1L1(hG1TM) and the control antibody 5C10H2L2-IgG1mt to FcγR IIIa_V158 was effectively eliminated.

[0308] (2) Determination of affinity constants of FcγRIIIa_F158 with 14C12H1L1(hG1DM), 14C12H1L1(hG4), 14C12H1L1(hG1WT) and 14C12H1L1(hG1TM)

[0309] The Fc receptor FcγRIIIa_F158 (also known as CD16a_F158) can bind to the Fc terminus of IgG antibodies, mediating ADCC. In this experiment, a Fortebio Octet molecular interaction analyzer was used to detect the affinity constants of 14C12H1L1 (hG1DM), 14C12H1L1 (hG4), 14C12H1L1 (hG1WT), and 14C12H1L1 (hG1TM) and control antibodies with FcγRIIIa_F158 to evaluate the ADCC activity of each antibody.

[0310] The experimental method for detecting the affinity constants of 14C12H1L1(hG1DM), 14C12H1L1(hG4), 14C12H1L1(hG1WT), and 14C12H1L1(hG1TM) with FcγRIIIa_F158 using the Fortebio Octet molecular interaction analyzer is briefly described below: Sample dilution buffer was PBS, 0.02% Tween-20, 0.1% BSA, pH 7.4. 5 μg / mL FcγRIIIa_F158 was immobilized on the HIS1K sensor for 120 s. The sensor equilibrated in buffer for 60 s. The immobilized FcγRIIIa_F158 bound to the respective antibodies (31.25-500 nM, 2-fold dilution) for 60 s. The antibodies dissociated in buffer for 60 s. The sensor was regenerated using 10 mM glycine at pH 1.5 for 5 s, repeated 4 times. The detection temperature was 30℃, and the frequency was 0.3Hz. The data were analyzed using a 1:1 model fitting to obtain the affinity constant.

[0311] The affinity constants of FcγRIIIa_F158 with 14C12H1L1(hG1DM), 14C12H1L1(hG4), 14C12H1L1(hG1WT), 14C12H1L1(hG1TM), and the control antibody 5C10H2L2-IgG1mt are shown in Table 3 and Figures 11-15.

[0312] Table 3: Kinetic parameters of 14C12H1L1 antibody and its subtypes binding to FcγRIIIa_F158

[0313] Antibody K D (M)kon(1 / Ms)SE(kon)kdis(1 / s)SE(kdis)Rmax(nm)14C12H1L1(hG1DM)N / AN / AN / AN / AN / AN / A14C12H1L1(hG4)N / AN / AN / AN / AN / AN / A

[0314] 14C12H1L1(hG1WT)1.02E-072.52E+052.93E+042.56E-021.12E-030.34-0.571 4C12H1L1(hG1TM)N / AN / AN / AN / AN / AN / A5C10H2L2-IgG1mtN / AN / AN / AN / AN / AN / A

[0315] N / A indicates that the antibody did not bind to the antigen or the binding signal was extremely low. The results were not analyzed, so no corresponding data was obtained.

[0316] The results showed that 14C12H1L1(hG1WT) could bind to FcγRIIIa_F158 with an affinity constant of 1.02E-07M; however, 14C12H1L1(hG1DM), 14C12H1L1(hG4), 14C12H1L1(hG1TM), and 5C10H2L2-IgG1mt did not bind to FcγRIIIa_F158 or had extremely low binding signals, so the results were not analyzed and no corresponding data were obtained.

[0317] The results showed that, relative to 14C12H1L1(hG1WT), the binding activity of 14C12H1L1(hG1DM), 14C12H1L1(hG4), and 14C12H1L1(hG1TM) with FcγRIIIa_F1581 was effectively eliminated.

[0318] Experimental Example 3: Affinity determination of 14C12H1L1 (hG1DM), 14C12H1L1 (hG4), 14C12H1L1 (hG1WT), and 14C12H1L1 (hG1TM) with Fc receptor FcγRIIa and its subtypes

[0319] (1) Determination of affinity constants of FcγRIIa_H131 with 14C12H1L1(hG1DM), 14C12H1L1(hG4), 14C12H1L1(hG1WT) and 14C12H1L1(hG1TM)

[0320] The Fc receptor FcγRIIa_H131 (also known as CD32a_H131) can bind to the Fc terminus of IgG antibodies, participating in antibody-dependent cell-mediated cytotoxicity (ADCC). The ability of therapeutic monoclonal antibodies to bind to the Fc receptor affects the safety and efficacy of the antibody. In this study, the affinity constants of 14C12H1L1 (hG1DM), 14C12H1L1 (hG4), 14C12H1L1 (hG1WT), and 14C12H1L1 (hG1TM) with FcγRIIa_H131 were detected using a Fortebio Octet molecular interaction analyzer to evaluate the binding ability of each test antibody to the Fc receptor.

[0321] The experimental method for detecting the affinity constants of 14C12H1L1(hG1DM), 14C12H1L1(hG4), 14C12H1L1(hG1WT), and 14C12H1L1(hG1TM) with FcγRIIa_H131 using the Fortebio Octet molecular interaction analyzer is briefly described below: The solidified product dilution buffer was PBS, 0.02% Tween-20, 0.1% BSA, pH 7.4; the analyte dilution buffer was PBS, 0.02% Tween-20, 0.02% casein, 0.1% BSA. FcγRIIa_H131, at a concentration of 5 μg / mL, was immobilized on an NTA sensor to a height of approximately 1.0 nm using BSA, pH 7.4. The sensor was equilibrated for 300 s in a buffer solution of PBS, 0.02% Tween-20, 0.02% casein, 0.1% BSA, pH 7.4 to block the sensor. The immobilized FcγRIIa_H131 then bound to an antibody at concentrations ranging from 12.5 nM to 200 nM (two-fold serial dilution) for 60 s. The antibody was then dissociated in the buffer solution for 60 s. The sensor was regenerated using 10 mM Glycine, pH 1.7, and 10 mM Nickel Sulfate. The detection temperature was 30 °C, and the frequency was 0.6 Hz. Data were analyzed using a 1:1 model to obtain the affinity constant.

[0322] The affinity constants of FcγRIIa_H131 with 14C12H1L1(hG1DM), 14C12H1L1(hG4), 14C12H1L1(hG1WT), 14C12H1L1(hG1TM), and the control antibody 5C10H2L2-IgG1mt are shown in Table 4 and Figures 16-20.

[0323] Table 4: Kinetic parameters of 14C12H1L1 antibody and its subtypes binding to FcγRIIa_H131

[0324] Sample IDK D (M)kon(1 / Ms)SE(kon)kdis(1 / s)SE(kdis)Rmax(nm)14C12H1L1(hG1DM)N / AN / AN / AN / AN / AN / A14C12H1L1(hG4)5.07E-082.57E+052.37E+041.30E-026.47E-040.18 -0.3514C12H1L1(hG1WT)5.74E-084.65E+055.99E+042.67E-021.24E-030.82-1. 1214C12H1L1(hG1TM)N / AN / AN / AN / AN / AN / A5C10H2L2-IgG1mtN / AN / AN / AN / AN / AN / A

[0325] N / A indicates that the antibody did not bind to the antigen or the binding signal was extremely low. The results were not analyzed, so no corresponding data was obtained.

[0326] The results showed that 14C12H1L1(hG4) and 14C12H1L1(hG1WT) could both bind to FcγRIIa_H131, with affinity constants of 5.07E-08M and 5.74E-08M, respectively. However, 14C12H1L1(hG1DM), 14C12H1L1(hG1TM), and 5C10H2L2-IgG1mt did not bind to FcγRIIa_H131 or had extremely low binding signals, so the results were not analyzed and no corresponding data were obtained.

[0327] The results showed that the binding activity of 14C12H1L1(hG1DM) and 14C12H1L1(hG1TM) to FcγRIIa_H131 was effectively eliminated compared to 14C12H1L1(hG4) and 14C12H1L1(hG1WT).

[0328] (2) Determination of affinity constants of FcγRIIa_R131 with 14C12H1L1(hG1DM), 14C12H1L1(hG4), 14C12H1L1(hG1WT) and 14C12H1L1(hG1TM)

[0329] The Fc receptor FcγRIIa_R131 (also known as CD32a_R131) can bind to the Fc terminus of IgG antibodies, participating in antibody-dependent cell-mediated cytotoxicity (ADCC). The ability of therapeutic monoclonal antibodies to bind to the Fc receptor affects the safety and efficacy of the antibody. In this study, the affinity constants of 14C12H1L1(hG1DM), 14C12H1L1(hG4), 14C12H1L1(hG1WT), and 14C12H1L1(hG1TM) with FcγRIIa_R131 were detected using a Fortebio Octet molecular interaction analyzer to evaluate the binding ability of each test antibody to the Fc receptor.

[0330] The Fortebio Octet molecular interaction analyzer was used to detect the affinity constants of 14C12H1L1(hG1DM), 14C12H1L1(hG4), 14C12H1L1(hG1WT), and 14C12H1L1(hG1TM) with FcγRIIa_R131. The experimental method is briefly described below: The solidified product dilution buffer was PBS, 0.02% Tween-20, 0.1% BSA, pH 7.4; the analyte dilution buffer was PBS, 0.02% Tween-20, 0.02% casein, 0.1% BSA. 5 μg / mL FcγRIIa_R131 was immobilized on the NTA sensor at a height of approximately 1.0 nm using % BSA, pH 7.4. The sensor was equilibrated for 300 s in a buffer solution of PBS, 0.02% Tween-20, 0.02% casein, 0.1% BSA, pH 7.4 to block the sensor. The immobilized FcγRIIa_R131 then bound to the antibody at concentrations ranging from 12.5 to 200 nM (two-fold serial dilution) for 60 s. The antibody was then dissociated in the buffer solution for 60 s. The sensor was regenerated using 10 mM Glycine at pH 1.7 and 10 mM nickel sulfate. The detection temperature was 30 °C, and the frequency was 0.6 Hz. Data were analyzed using a 1:1 model to obtain the affinity constant.

[0331] The affinity constants of FcγRIIa_R131 with 14C12H1L1(hG1DM), 14C12H1L1(hG4), 14C12H1L1(hG1WT), 14C12H1L1(hG1TM), and the control antibody 5C10H2L2-IgG1mt are shown in Table 5 and Figures 21-25.

[0332] Table 5: Kinetic parameters of 14C12H1L1 antibody and its subtypes binding to FcγRIIa_R131

[0333] Antibody K D(M)kon(1 / Ms)SE(kon)kdis(1 / s)SE(kdis)Rmax(nm)14C12H1L1(hG1DM)N / AN / AN / AN / AN / AN / A14C12H1L1(hG4)3.13E-083.50E+053.05E+041.10E-026.43E-040.21-0.4614C12H1L1(h G1WT)3.46E-086.60E+059.46E+042.28E-021.33E-030.39-0.8314C12H1L1(hG1TM)2.32E- 074.04E+059.45E+049.38E-024.89E-030.19-0.355C10H2L2-IgG1mtN / AN / AN / AN / AN / AN / A

[0334] N / A indicates that the antibody did not bind to the antigen or the binding signal was extremely low. The results were not analyzed, so no corresponding data was obtained.

[0335] The results showed that 14C12H1L1(hG4), 14C12H1L1(hG1WT), and 14C12H1L1(hG1TM) could all bind 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 to FcγRIIa_R131 or had extremely low binding signals, so the results were not analyzed, and therefore no data were obtained.

[0336] The results showed that, compared with 14C12H1L1(hG4) and 14C12H1L1(hG1WT), among the antibodies with binding activity, 14C12H1L1(hG1TM) had the weakest binding force and the lowest binding activity with FcγRIIa_R131.

[0337] Experimental Example 4: Determination of the affinity constants of FcγRIIb with 14C12H1L1(hG1DM), 14C12H1L1(hG4), 14C12H1L1(hG1WT), and 14C12H1L1(hG1TM).

[0338] The Fc receptor FcγRIIb (also known as CD32b) can bind to the Fc terminus of IgG antibodies, negatively regulating the function of immune cells, inhibiting their activation and proliferation, and suppressing cytokine secretion. In this experiment, the affinity constants of each test antibody with FcγRIIb were detected using a Fortebio Octet molecular interaction analyzer to evaluate the binding affinity of 14C12H1L1 (hG1DM), 14C12H1L1 (hG4), 14C12H1L1 (hG1WT), and 14C12H1L1 (hG1TM) to the Fc receptor.

[0339] The Fortebio Octet molecular interaction analyzer was used to determine the affinity constants of 14C12H1L1(hG1DM), 14C12H1L1(hG4), 14C12H1L1(hG1WT), and 14C12H1L1(hG1TM) with FcγRIIb. The experimental method is briefly described below: The solidified product dilution buffer was PBS, 0.02% Tween-20, 0.1% BSA, pH 7.4; the analyte dilution buffer was PBS, 0.02% Tween-20, 0.02% casein, 0.1% BSA. At pH 7.4, 5 μg / mL hFcγRIIb-his was immobilized on an NTA sensor at a height of approximately 1.0 nm. The sensor was equilibrated for 300 s in a buffer solution of PBS, 0.02% Tween-20, 0.02% casein, and 0.1% BSA at pH 7.4 to block the sensor. The hFcγRIIb-his immobilized on the sensor then bound to an antibody at a concentration of 12.5 nM–200 nM (two-fold serial dilution) for 60 s. The antibody was then dissociated in the buffer solution for 60 s. The sensor was regenerated using 10 mM Glycine at pH 1.7 and 10 mM nickel sulfate. The detection temperature was 30 °C, and the frequency was 0.6 Hz. Data were analyzed using a 1:1 model to obtain the affinity constant.

[0340] The affinity constants of FcγRIIb with 14C12H1L1(hG1DM), 14C12H1L1(hG4), 14C12H1L1(hG1WT), 14C12H1L1(hG1TM), and the control antibody 5C10H2L2-IgG1mt are shown in Table 6 and Figures 26-30.

[0341] Table 6: Kinetic parameters of 14C12H1L1 antibody and its subtypes binding to FcγRIIb

[0342] Antibody K D(M)kon(1 / Ms)SE(kon)kdis(1 / s)SE(kdis)Rmax(nm)14C12H1L1(hG1DM)N / AN / AN / AN / AN / AN / A14C12H1L1(hG4)5.62E-082.88E+052.94E+041.62E-027.63E-040.22-0.33

[0343] 14C12H1L1(hG1WT)6.13E-083.18E+053.48E+041.95E-028.96E-040.16-0.371 4C12H1L1(hG1TM)N / AN / AN / AN / AN / AN / A5C10H2L2-IgG1mtN / AN / AN / AN / AN / AN / A

[0344] N / A indicates that the antibody did not bind to the antigen or the binding signal was extremely low. The results were not analyzed, so no corresponding data was obtained.

[0345] The results showed that 14C12H1L1(hG4) and 14C12H1L1(hG1WT) could both bind to FcγRIIb, with affinity constants of 5.62E-08M and 6.13E-08M, respectively. However, 14C12H1L1(hG1DM), 14C12H1L1(hG1TM), and 5C10H2L2-IgG1mt did not bind to FcγRIIb or had extremely low binding signals, so the results were not analyzed and no corresponding data were obtained.

[0346] The results showed that the binding activity of 14C12H1L1(hG1DM) and 14C12H1L1(hG1TM) to FcγRIIb was effectively eliminated compared to 14C12H1L1(hG4) and 14C12H1L1(hG1WT).

[0347] Experimental Example 5: Determination of the affinity of C1q with 14C12H1L1 (hG1DM), 14C12H1L1 (hG4), 14C12H1L1 (hG1WT), and 14C12H1L1 (hG1TM).

[0348] Serum complement C1q can bind to the Fc terminus of IgG antibodies, mediating the CDC effect. The ability of therapeutic monoclonal antibodies to bind to C1q affects the safety and efficacy of the antibody. In this study, the affinity constants of 14C12H1L1(hG1DM), 14C12H1L1(hG4), 14C12H1L1(hG1WT), and 14C12H1L1(hG1TM) with C1q were detected using a Fortebio Octet molecular interaction analyzer to evaluate the CDC activity of these antibodies.

[0349] The experimental method for detecting the affinity constant of the corresponding antibody with C1q using the Fortebio Octet molecular interaction analyzer is briefly described below: The sample dilution buffer was PBS, 0.02% Tween-20, 0.1% BSA, pH 7.4. 50 μg / mL antibody was immobilized on the FAB2G sensor at a height of approximately 2.0 nm. The sensor was equilibrated in the buffer for 60 s. The antibody immobilized on the sensor then bound to the antigen C1q at a concentration of 1.25 nM–20 nM (two-fold serial dilution) for 60 s. The antigen and antibody then dissociated in the buffer for 60 s. The sensor was regenerated using 10 mM glycine at pH 1.7 for 5 s, repeated 4 times. The sample plate vibration rate was 1000 rpm, the detection temperature was 30 °C, and the detection frequency was 0.6 Hz. Data were analyzed using a 1:1 model to obtain the affinity constant. Fortebio Data Acquisition 7.0 and Fortebio Data Analysis 7.0 were used for data acquisition and analysis.

[0350] The affinity constants of C1q with 14C12H1L1(hG1DM), 14C12H1L1(hG4), 14C12H1L1(hG1WT), 14C12H1L1(hG1TM), and the control 5C10H2L2-IgG1mt are shown in Table 7 and Figures 31-35.

[0351] Table 7: Kinetic parameters of 14C12H1L1 antibody and its subtypes binding to C1q

[0352]

[0353] N / A indicates that the antibody did not bind to the antigen or the binding signal was extremely low. The results were not analyzed, so no corresponding data was obtained.

[0354] The results showed that 14C12H1L1(hG1WT) could bind to C1q with an affinity constant of 1.35E-09M; however, 14C12H1L1(hG1DM), 14C12H1L1(hG4), and 14C12H1L1(hG1TM) did not bind to C1q or had extremely low binding signals, so no analysis was performed and no corresponding data were obtained.

[0355] The results also showed that 5C10H2L2-IgG1mt can bind to C1q with an affinity constant of 4.43E-09, indicating that it has binding activity to C1q and can induce the CDC effect.

[0356] Experimental Example 6: Detection of pharmacodynamic activities of 14C12H1L1 (hG1WT) and 14C12H1L1 (hG1TM) in a co-culture system of peripheral blood mononuclear cells and Raji-PDL1 cells.

[0357] In this experiment, the pharmacodynamic activity of anti-PD-1 antibodies 14C12H1L1 (hG1WT) and 14C12H1L1 (hG1TM), as well as control antibodies anti-PD-L1 antibody 5C10H2L2-IgG1mt and nivolumab in relieving PD-1 / PD-L1-mediated immunosuppression was detected using a co-culture system of peripheral blood mononuclear cells and Raji-PDL1 cells.

[0358] In mixed lymphocyte reactions, when isolated peripheral blood mononuclear cells (containing immune cells expressing immunologically active PD-1) are co-cultured with Raji-PDL1 cells expressing PD-L1, the interaction between PD-1 and PD-L1 can mediate the functional suppression of immune cells, manifested as a decrease in the secretion of cytokines IFN-γ and IL-2; while anti-PD-1 or PD-L1 antibodies can relieve the immunosuppression of immune cells, leading to an increase in cytokine secretion. Raji cells are B cell lineage cells, and as mentioned above, B cells can serve as antigen-presenting cells, mediating the immune response of immune cells against tumor cells; in this invention, the Raji-PDL1 and PBMCs co-culture system is used to evaluate the pharmacological activity of anti-PD-1 antibodies, and the pharmacological activity of PD-1 antibodies in different types of tumors is evaluated in the Raji-PDL1, PBMCs, and tumor cell co-culture system.

[0359] Peripheral blood mononuclear cells (PBMCs) were isolated using the Ficoll-Paque Plus method (GE Healthcare Cat No.: 171440-02). The isolated PBMCs were stimulated with SEB (0.5 μg / mL) for two days. Then, 1 x 10^6 mature PBMCs were added to each well of a 96-well plate. 5 Raji-PDL1 cells (1*10 cells / well) treated with MMC (Mito-mycin C treatment concentration 2 μg / mL) for 1 hour were also included. 5 Cells / well were added to 14C12H1L1 (hG1WT) or 14C12H1L1 (hG1TM) or control antibody Nivolumab or control antibody anti-PD-L1 antibody 5C10H2L2-IgG1mt, mixed thoroughly, and co-cultured. After 3 days, the cell supernatant was collected, and the secretion of IFN-γ and IL-2 was detected using an ELISA kit (purchased from Dakota).

[0360] Figure 36 shows the results of IFN-γ secretion in the mixed lymphocyte reaction. The results showed that, in the PBMC and Raji-PDL1 co-culture system, at the same dose level, the IFN-γ secretion induced by 14C12H1L1(hG1TM) was significantly higher than that 14C12H1L1(hG1WT), Nivolumab, or 5C10H2L2-IgG1mt.

[0361] 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, at the same dose level, the IL-2 secretion induced by 14C12H1L1(hG1TM) was significantly higher than that 14C12H1L1(hG1WT), Nivolumab, or 5C10H2L2-IgG1mt.

[0362] The results showed that 14C12H1L1(hG1TM) had significantly better pharmacodynamic activity than Nivolumab, 14C12H1L1(hG1WT) or 5C10H2L2-IgG1mt in relieving PD-1 / PD-L1-mediated immunosuppression.

[0363] Experimental Example 7: Antibody-mediated phagocytosis activity of Nivolumab, 14C12H1L1 (hG1WT) and 14C12H1L1 (hG1TM) against CHO-K1-PD1

[0364] To detect antibody-dependent cellular phagoxytosis (ADCP) activity, mouse macrophages were used as effector cells, and a cell line overexpressing PD1 was used as the target cell to examine its ADCP-mediated effect. First, femoral bone marrow was harvested from Blab / c mice (purchased from Guangdong Provincial Medical Laboratory Animal Center) under sterile conditions and lysed on ice for 5 min with erythrocyte lysis buffer. Lysis was terminated with DMEM complete medium (containing 10% FBS), and the cells were washed twice by centrifugation at 1000 rpm. The cell pellets were resuspended in 10 mL of DMEM complete medium, and M-CSF was added to a working concentration of 100 ng / mL. The cells were induced and cultured at 37°C and 5% CO2 for 7 days, with half the medium replaced and M-CSF added on days 3 and 5. Cell induction was completed on day 7. Macrophages were collected by digestion with 0.05% trypsin, centrifuged at 750xg for 5 min, the supernatant was discarded, and the cells were resuspended in DMEM complete medium (containing 10% FBS) and counted. The cell density was adjusted and the cells were aliquoted into sterile EP tubes for later use.

[0365] CHO-K1-PD1 cells (a cell line based on CHO-K1 cells overexpressing PD1) were centrifuged at 170*g for 5 min, washed once with PBS, and resuspended for cell counting and viability assessment. Carboxyfluorescein diacetate succinimidyl ester (CFSE) was diluted to 2.5 μM with PBS. An appropriate amount of diluted CFSE was used to resuspend the cells (staining density: 10 million cells / mL), and incubated in a cell culture incubator for 20 min. Staining was terminated by adding 6 mL of DMEM complete medium, centrifuged at 170*g for 5 min, and the supernatant was discarded. 1 mL of DMEM complete medium was added, and the cells were incubated for 10 min. The cell density was then adjusted to the experimental density, and the cells were named CHO-K-PD1-CFSE.

[0366] The antibody to be tested was diluted with DMEM complete medium to 20, 2, and 0.2 μg / mL (working concentrations of 10, 1, and 0.1 μg / mL). Anti-HEL IgG1 and culture medium were used as isotype control (anti-HEL antibody) and blank control, respectively. According to the experimental design, the diluted antibody and CHO-K1-PD1-CFSE cells were added to 1.5 mL EP tubes containing macrophages (final volume 100 μL, effector cell to target cell ratio 50,000:150,000), resuspended, mixed, and incubated at 37°C for 2 h. 800 μL of PBS solution containing 1% bovine serum albumin (BSA) was added to each tube at room temperature, centrifuged at 500 x g for 5 min, and the supernatant was discarded. The tubes were washed once with 800 μL of 1% PBSA. APC anti-mouse / human CD11b The antibody (Biolegend, catalog number: 101212) was diluted 400-fold with PBSA and added to the corresponding sample at a rate of 100 μL / sample. The mixture was incubated on ice for 40 min. Each sample was washed twice by centrifugation at 1200 x g for 5 min with 800 μL of 1% PBSA, and the supernatant was discarded. Cells were resuspended in 200 μL of 1% PBSA in each tube, transferred to flow cytometry tubes, and analyzed using a BD FACS Calibur flow cytometer. Macrophages in the flow cytometry system were APC+ positive, while macrophages that underwent phagocytosis were double-positive for both APC and CFSE. The ratio of double-positive cells to APC-positive cells was used as the phagocytosis rate, and this ratio was used to evaluate antibody-mediated ADCP activity. ADCP activity for each group was calculated using the following formula, expressed as P%:

[0367]

[0368] The results are shown in Figure 38.

[0369] The results showed that, at the same concentration, the phagocytosis rates of 14C12H1L1(hG1WT) and Nivolumab were 3.94 and 4.26 times that of the isotype control antibody group anti-HEL antibody, respectively, indicating that 14C12H1L1(hG1WT) and Nivolumab have ADCP effects; at the same concentration, the phagocytosis rate of 14C12H1L1(hG1TM) was comparable to that of the isotype control antibody group, indicating that 14C12H1L1(hG1TM) does not have ADCP effects.

[0370] The results show that the amino acid mutation introduced by 14C12H1L1(hG1TM) can effectively eliminate its ADCP effect, achieving unexpected technical results.

[0371] Experimental Example 8: Pharmacodynamic evaluation of 14C12H1L1 (hG1TM) + anlotinib hydrochloride in a subcutaneous transplantation model of human non-small cell lung cancer HCC827 cells from Scid / Beige immunodeficient mice.

[0372] Scid / Beige immunodeficient mice (purchased from Beijing Vital River Pharmaceuticals), 8 females per group. Staphylococcus aureus enterotoxin B (SEB) was added to a PBMC suspension of 1 million / ml and cultured for 3 days to activate PBMCs and increase PD1 expression on PBMC cells. On day 0, mice were subcutaneously inoculated with a mixture of 800,000 SEB-activated PBMC cells and 6 million HCC827 human non-small cell lung cancer cells (purchased from Guangzhou Genio Biotechnology). Two groups were formed: an isotype control antibody group (i.e., anti-HEL antibody, prepared by Zhongshan Kangfang Biopharmaceutical Co., Ltd., preparation method described above) and a 14C12H1L1 (hG1TM) + anlotinib hydrochloride group. 14C12H1L1 (hG1TM) was administered via tail vein once a week (the first dose was mixed with the cells and administered subcutaneously). Anlotinib was administered by gavage once daily for 30 consecutive days. Specific experimental protocols are shown in Table 8. The tumor was continuously measured during the experiment, and its volume was calculated using the formula a(tumor length)*b(tumor width)*b(tumor width) / 2.

[0373] Table 8: Experimental Plan and Grouping

[0374]

[0375] The experimental results are shown in Figure 39.

[0376] The results showed that 14C12H1L1(hG1TM) + anlotinib hydrochloride could significantly inhibit the growth of tumor volume in human non-small cell lung cancer cells, and had a good tumor killing effect.

[0377] Experimental Example 9: Pharmacodynamic evaluation of 14C12H1L1 (hG1TM) in a subcutaneous transplantation model of colon cancer MC38-hPDL1 / hCD73 in C57BL / 6-hPD1 / hPDL1 / hCD73 mice.

[0378] The mouse MC38 cell line is a mouse rectal cancer cell line, and it has been proven to be an effective model for studying human MSI-high / dMMR tumors (Efremova M et al. Nat Commun. 2018; 9(1):32.).

[0379] Female C57BL / 6-hPD1 / hPDL1 / hCD73 mice (purchased from Nanjing Jicui Yaokang Biotechnology Co., Ltd.), 8 mice per group, each mouse subcutaneously injected with colon cancer MC38-hPDL1 / hCD73 cells (purchased from Nanjing Jicui Yaokang Biotechnology Co., Ltd.) in the right forelimb (2*10). 6 Cells / 100μL / mouse). The day of vaccination is defined as day D0. The dosage volume is adjusted according to body weight: 10μL / g * mouse body weight (g). Anti-HEL antibody (preparation method and source same as in Example 8) or 14C12H1L1 (hG1TM) was administered intraperitoneally; twice a week for 3 weeks, for a total of 6 administrations. See Table 9 for the specific experimental protocol. Tumors were continuously measured during the experiment, and the tumor volume (mm²) was recorded as follows: 3 The volume is calculated using the formula (tumor length * (tumor width)²) / 2.

[0380] Table 9: Experimental Design and Grouping

[0381]

[0382] The experimental results are shown in Figure 40.

[0383] The results showed that, compared with the isotype control antibody, 14C12H1L1 (hG1TM) significantly inhibited the proliferation of MC38 cells, resulting in suppressed tumor volume growth, and could effectively treat solid tumors with the MSI-H / dMMR phenotype, such as colon cancer and / or rectal cancer.

[0384] Experimental Example 10: 14C12H1L1 (hG1TM) effectively enhances the immune response of immune cells against human gastric cancer cells KATO III.

[0385] Peripheral blood mononuclear cells (PBMCs) were isolated from healthy individuals according to the instructions of the Ficoll-Paque™ Plus reagent. The isolated PBMCs were counted and cryopreserved. Raji-PDL1 cells were routinely cultured in RPMI 1640 + 10% FBS complete medium, and KATO III cells (purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences) were routinely cultured in DMEM + 10% FBS complete medium. PBMCs were resuscitated and activated for two days with 0.5 μg / mL SEB. On the day of the experiment, Raji-PDL1 cells were treated with 2 μg / mL MMC for 1 hour. PBMCs activated with SEB for two days and Raji-PDL1 cells treated with MMC were collected, washed twice with PBS, resuspended in RPMI 1640 + 10% FBS complete medium, and counted. Both Raji-PDL1 and PBMC counts were 1*10. 5 5 x 10⁸ cells / well were seeded into 96-well plates; logarithmic growth phase KATO III cells were collected, with 5 x 10⁸ cells per well. 4 Cells were seeded per well in a 96-well plate; diluted antibody was added according to the experimental design, mixed well, and incubated at 37°C in a 5% CO2 incubator for 3 days. After 3 days, the cell culture supernatant was collected, and IL-2 was detected according to the ELISA KIT instructions. The experimental medium was 10% FBS + RPMI 1640.

[0386] The experimental results are shown in Figure 41.

[0387] The results showed that when 14C12H1L1(hG1TM) was co-cultured with human gastric cancer cells KATO III, it exhibited higher pharmacological activity compared with 14C12H1L1(hG1WT) or Nivolumab. Specifically, at the same concentration level, it could stimulate PBMCs to secrete more IL-2, indicating that 14C12H1L1(hG1TM) has the potential to treat gastric cancer.

[0388] Experimental Example 11: 14C12H1L1 (hG1TM) effectively enhances the immune response of immune cells to CNE-2Z nasopharyngeal carcinoma tumor cells.

[0389] Raji-PDL1 and CNE-2Z cells (from Guangzhou Genio Biotechnology Co., Ltd.) and PBMCs were resuscitated. PBMCs were stimulated with SEB (0.5 ug / ml) for two days after resuscitation. On the day of the experiment, Raji-PDL1 cells were treated with MMC (Mito-mycin C) at a concentration of 2 μg / mL, with a cell density of 200*10⁻⁶ cells / mL. 4 Treat with PBMCs ( / mL) for 1 hour. Collect PBMCs and treated Raji-PDL1 cells, wash twice with PBS, and collect 10*10^9 PBMCs and Raji-PDL1 cells respectively. 4 / hole, 10*10 each in PBMC and Raji-PDL1 4 Add 3*10 CNE-2Z cells to the existing well. 4 / well. Add antibody according to the experimental design (final antibody concentration 300 nM, final volume 200 μL), and co-culture for 3 days; collect cell supernatant for IL-2 detection. All experimental media were 10% FBS + RPMI 1640.

[0390] The results are shown in Figure 42.

[0391] The results showed that when 14C12H1L1(hG1TM) was co-cultured with human nasopharyngeal carcinoma cells CNE-2Z, it exhibited higher pharmacological activity compared with 14C12H1L1(hG1WT). Specifically, at the same concentration level, it could stimulate PBMCs to secrete more IL-2, indicating that 14C12H1L1(hG1TM) has the potential to treat nasopharyngeal carcinoma.

[0392] Experimental Example 12: 14C12H1L1 (hG1TM) effectively enhanced the immune response of immune cells against mesothelioma tumor cells NCI-H2452.

[0393] Raji-PDL1 and NCI-H2452 cells (derived from the Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences) and PBMCs were resuscitated. PBMCs were stimulated with SEB (0.5 μg / ml) for two days after resuscitation. On the day of the experiment, Raji-PDL1 cells were treated with MMC (Mito-mycin C) at a concentration of 2 μg / mL, with a cell density of 200*10⁻⁶ cells / mL. 4 Treat with PBMCs ( / mL) for 1 hour. Collect PBMCs and treated Raji-PDL1 cells, wash twice with PBS, and collect 10*10^9 PBMCs and Raji-PDL1 cells respectively. 4 / hole, 10*10 each in PBMC and Raji-PDL1 4 Add 3*10 NCI-H2452 cells to the existing wells. 4 / well. Add antibody according to the experimental design (final antibody concentration 300 nM, final volume 200 μL), and co-culture for 3 days; collect cell supernatant for IL-2 detection. All experimental media were 10% FBS + RPMI 1640.

[0394] The results are shown in Figure 43.

[0395] The results showed that when 14C12H1L1(hG1TM) was co-cultured with human mesothelioma cells NCI-H2452, it exhibited higher pharmacological activity compared with 14C12H1L1(hG1WT). Specifically, at the same concentration level, it could stimulate PBMCs to secrete more IL-2, indicating that 14C12H1L1(hG1TM) has the potential to treat mesothelioma.

[0396] Experimental Example 12: 14C12H1L1 (hG1TM) effectively enhances the immune response of immune cells against human small cell lung cancer cells NCI-H446 cells.

[0397] According to the separation liquid Ficoll-Paque TM The Plus reagent instructions describe the isolation of peripheral blood peripheral blood mononuclear cells (PBMCs) from healthy individuals. The isolated PBMCs were counted and cryopreserved. Raji-PDL1 and NCI-H446 cells (derived from the Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences) were routinely cultured in RPMI 1640 + 10% FBS complete medium. PBMCs were resuscitated and activated for two days with 0.5 μg / mL SEB. On the day of the experiment, Raji-PDL1 cells were treated with 2 μg / mL MMC for 1 hour. PBMCs activated with SEB for two days and Raji-PDL1 cells treated with MMC were collected, washed twice with PBS, resuspended in RPMI 1640 + 10% FBS complete medium, and counted. Both Raji-PDL1 and PBMC counts were 1*10. 5 8 cells / well were seeded into 96-well plates; NCI-H446 cells in logarithmic growth phase were collected, with each well containing 8 x 10 cells. 4 Cells were seeded per well in a 96-well plate; diluted antibody was added according to the experimental design, mixed well, and incubated at 37°C in a 5% CO2 incubator for 3 days. After 3 days, the cell culture supernatant was collected, and IL-2 was detected according to the ELISA KIT instructions. The experimental medium was 10% FBS + RPMI 1640.

[0398] The results are shown in Figure 44.

[0399] The results showed that when 14C12H1L1(hG1TM) was co-cultured with human small cell lung cancer cells NCI-H446, compared with 14C12H1L1(hG1WT) and nivolumab, 14C12H1L1(hG1TM) exhibited equal or higher pharmacological activity while effectively eliminating ADCC, CDC and ADCP activities. That is, at the same concentration level, it can stimulate PBMCs to secrete equal or more IL-2, indicating that 14C12H1L1(hG1TM) has the potential to treat human small cell lung cancer.

[0400] Experimental Example 13: 14C12H1L1 (hG1TM) combined with anlotinib hydrochloride effectively enhanced the immune response of immune cells against human nasopharyngeal carcinoma tumor cells CNE-2Z cells.

[0401] According to the separation liquid Ficoll-Paque TM The Plus reagent instructions describe the isolation of peripheral blood peripheral blood mononuclear cells (PBMCs) from healthy individuals. The isolated PBMCs were counted and cryopreserved. Raji-PDL1 and CNE-2Z cells (from Guangzhou Genio Biotechnology Co., Ltd.) were routinely cultured in RPMI 1640 + 10% FBS complete medium. PBMCs were resuscitated and activated for two days with 0.5 μg / mL SEB. On the day of the experiment, Raji-PDL1 cells were treated with 2 μg / mL MMC for 1 hour. PBMCs activated with SEB for two days and Raji-PDL1 cells treated with MMC were collected, washed twice with PBS, resuspended in RPMI 1640 + 10% FBS complete medium, and counted. Both Raji-PDL1 and PBMC counts were 1*10. 5 Cells were seeded per well in 96-well plates. Logarithmic growth phase CNE-2Z cells were collected at 3 x 10⁻⁶ cells / well. 4 Cells were seeded per well in a 96-well plate. Diluted antibody and anlotinib were added according to the experimental design, mixed well, and incubated at 37°C in a 5% CO2 incubator for 3 days. After 3 days, the cell culture supernatant was collected, and IL-2 levels were detected according to the ELISA kit instructions. The experimental medium was 10% FBS + RPMI 1640.

[0402] 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 response of immune cells to human nasopharyngeal carcinoma tumor cells CNE-2Z cells, as evidenced by a significant increase in IL-2 secretion levels. The pharmacological activity of 14C12H1L1(hG1TM) was superior to that of 14C12H1L1(hG1WT) and nivolumab.

[0403] Furthermore, the pharmacological activity of 14C12H1L1(hG1TM) in combination with anlotinib in stimulating immune cell activation is superior to that of 14C12H1L1(hG1TM) monotherapy, 14C12H1L1(hG1WT) monotherapy, and nivolumab monotherapy; it is also superior to 14C12H1L1(hG1WT) in combination with anlotinib and nivolumab in combination with anlotinib.

[0404] The results above show that 14C12H1L1(hG1TM) in combination with anlotinib has the potential to treat human nasopharyngeal carcinoma.

[0405] Experimental Example 14: 14C12H1L1 (hG1TM) combined with anlotinib significantly enhanced the immune response of immune cells against the MSI-h / dMMR tumor cell line SW48.

[0406] SW48 cells are a human rectal cancer cell line that has been identified as having the MSI-h / dMMR phenotype (Branch P et al. (1995). Cancer Res 55(11):2304–2309.) and have been used to detect the immune response of 14C12H1L1 (hG1TM) enhanced immune cells to tumors with the MSI-h / dMMR phenotype.

[0407] According to the separation liquid Ficoll-Paque TM The Plus reagent instructions describe the isolation of peripheral blood mononuclear cells (PBMCs) from healthy individuals. The isolated PBMCs were counted and cryopreserved. Raji-PDL1 cells were routinely cultured in RPMI 1640 10% FBS complete medium, and SW48 cells (from Guangzhou Genio Biotechnology Co., Ltd.) were routinely cultured in DMEM + 10% FBS complete medium. PBMCs were resuscitated and activated with 0.5 μg / mL SEB for two days. On the day of the experiment, Raji-PDL1 cells were treated with 2 μg / mL MMC for 1 hour. PBMCs activated with SEB for two days and Raji-PDL1 cells treated with MMC were collected, washed twice with PBS, resuspended in RPMI 1640 + 10% FBS complete medium, and counted. Both Raji-PDL1 and PBMC counts were 1*10. 5 SW48 cells were seeded per well in a 96-well plate; logarithmic growth phase SW48 cells were collected, with 2*10 cells per well. 5 Cells were seeded per well in a 96-well plate; diluted antibody and anlotinib were added according to the experimental design, mixed well, and incubated at 37°C in a 5% CO2 incubator for 3 days. After 3 days, the cell culture supernatant was collected, and IL-2 was detected according to the ELISA kit instructions. The experimental medium was 10% FBS + RPMI 1640.

[0408] The results are shown in Figure 46.

[0409] The results showed that, compared with anti-HEL antibody, 14C12H1L1(hG1TM), 14C12H1L1(hG1WT) and nivolumab significantly enhanced the immune response of immune cells to human colorectal cancer cells SW48 cells with the MSI-h / dMMR phenotype, as evidenced by a significant increase in IL-2 secretion levels. The pharmacological activity of 14C12H1L1(hG1TM) was superior to that of 14C12H1L1(hG1WT).

[0410] Furthermore, the pharmacological activity of 14C12H1L1(hG1TM) in combination with anlotinib in stimulating immune cell activation is superior to that of 14C12H1L1(hG1WT) monotherapy, 14C12H1L1(hG1TM) monotherapy, and nivolumab monotherapy; it is also superior to 14C12H1L1(hG1WT) in combination with anlotinib and nivolumab in combination with anlotinib.

[0411] The results above indicate that 14C12H1L1 (hG1TM) in combination with anlotinib has the potential to treat solid tumors with the MSI-h / dMMR phenotype, particularly colorectal and / or rectal cancers with the MSI-h / dMMR phenotype.

[0412] Experimental Example 15: 14C12H1L1 (hG1TM) significantly enhanced the immune response of immune cells against non-MSI-h / dMMR human colorectal cancer cells SW837.

[0413] SW 837 cells are human colorectal cancer cells with a non-MSI-h / dMMR (i.e., MSS) phenotype (Guo J et al. Cancer Res. 2011; 71(8):2978-2987.). In this example, they were used to detect the immune response of 14C12H1L1 (hG1TM) enhanced immune cells to tumors with a non-MSI-h / dMMR phenotype (i.e., MSS).

[0414] According to the separation liquid Ficoll-Paque TM The Plus reagent instructions describe the isolation of peripheral blood mononuclear cells (PBMCs) from healthy individuals. The isolated PBMCs were counted and cryopreserved. Raji-PDL1 cells were routinely cultured in RPMI 1640 + 10% FBS complete medium, while SW837 cells (from Shanghai Hongshun Biotechnology Co., Ltd.) were routinely cultured in 10% FBS + Leibovitz's L-15 complete medium (from Gibco). PBMCs were resuscitated and activated for two days with 0.5 μg / mL SEB. On the day of the experiment, Raji-PDL1 cells were treated with 2 μg / mL MMC for 1 hour. PBMCs activated with SEB for two days and Raji-PDL1 cells treated with MMC were collected, washed twice with PBS, resuspended in 1640 + 10% FBS complete medium, and counted. Both Raji-PDL1 and PBMC counts were 1*10. 5 SW837 cells were seeded at 5 x 10⁶ cells / well in a 96-well plate; logarithmic growth phase SW837 cells were collected, with 5 x 10⁶ cells per well. 4 Cells were seeded per well in a 96-well plate; diluted antibody was added according to the experimental design, mixed well, and incubated at 37°C in a 5% CO2 incubator for 3 days. After 3 days, the cell culture supernatant was collected, and IL-2 was detected according to the ELISA KIT instructions.

[0415] The results are shown in Figure 47.

[0416] The results showed that, compared with anti-HEL antibodies, 14C12H1L1(hG1TM), 14C12H1L1(hG1WT), and nivolumab all significantly enhanced the immune response of immune cells against non-MSI-h / dMMR human colorectal cancer cells SW837. Among them, in the medium and high dose groups, the pharmacological activity of 14C12H1L1(hG1TM) was superior to that of 14C12H1L1(hG1WT), manifested by a significant increase in IL-2 secretion levels.

[0417] The results above show that 14C12H1L1(hG1TM), in addition to effectively removing the effects of ADCC, CDC, or ADCP, has better or comparable pharmacological activity compared to 14C12H1L1(hG1WT) and nivolumab, indicating its potential for treating solid tumors with non-MSI-h / dMMR (i.e., MSS) phenotypes, particularly colorectal and / or rectal cancers with non-MSI-h / dMMR phenotypes.

[0418] Experimental Example 16: 14C12H1L1 (hG1TM) combined with anlotinib significantly enhanced the immune response of immune cells against non-MSI-h / dMMR human colorectal cancer cells SW837.

[0419] According to the separation liquid Ficoll-Paque TM The Plus reagent instructions describe the isolation of peripheral blood mononuclear cells (PBMCs) from healthy individuals. The isolated PBMCs were counted and cryopreserved. Raji-PDL1 cells were routinely cultured in RPMI 1640 + 10% FBS complete medium, and SW837 cells were routinely cultured in Leibovitz's L-15 + 10% FBS complete medium. PBMCs were resuscitated and activated with 0.5 μg / mL SEB for two days. On the day of the experiment, Raji-PDL1 cells were treated with 2 μg / mL MMC for 1 hour. PBMCs activated with SEB for two days and Raji-PDL1 cells treated with MMC were collected, washed twice with PBS, resuspended in 1640 + 10% FBS complete medium, and counted. Both Raji-PDL1 and PBMC counts were 1*102. 5 SW837 cells were seeded at 5 x 10⁶ cells / well in a 96-well plate; logarithmic growth phase SW837 cells were collected, with 5 x 10⁶ cells per well. 4 Cells were seeded per well in a 96-well plate; diluted antibody was added according to the experimental design, mixed well, and incubated at 37°C in a 5% CO2 incubator for 3 days. After 3 days, the cell culture supernatant was collected, and IL-2 was detected according to the ELISA KIT instructions.

[0420] The results are shown in Figure 48.

[0421] The results showed that, compared with 14C12H1L1(hG1WT) combined with anlotinib and nivolumab combined with anlotinib, 14C12H1L1(hG1TM) combined with anlotinib significantly enhanced the immune response of immune cells to non-MSI-h / dMMR human colorectal cancer cells SW837, manifested by an increase in IL-2 secretion levels, and had better efficacy in treating non-MSI-h / dMMR solid tumors, especially non-MSI-h / dMMR colorectal cancer and / or rectal cancer.

[0422] Although specific embodiments 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 based on all the teachings disclosed, and all such changes are 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 antibody, wherein, the heavy chain variable region of the antibody comprises HCDR1 - HCDR3 with amino acid sequences as shown in SEQ ID NOs: 19 - 21; and the light chain variable region of the antibody comprises LCDR1 - LCDR3 with amino acid sequences as shown in SEQ ID NOs: 22 - 24; the antibody is of human IgG1 subtype; wherein, according to the EU numbering system, the heavy chain constant region of the antibody has mutations at any 2 or 3 of positions 234, 235, and 237, and the affinity constant of the mutated antibody with FcγRIIIa and / or C1q is lower than that before mutation; preferably, the affinity constant is measured by a Fortebio Octet molecular interaction instrument.

2. The antibody according to claim 1, wherein, according to the EU numbering system, the heavy chain constant region of the antibody has the following mutations: L234A and L235A; L234A and G237A; L235A and G237A; or L234A, L235A, and G237A.

3. An antibody, wherein, the heavy chain variable region of the antibody comprises HCDR1 - HCDR3 with amino acid sequences as shown in SEQ ID NOs: 19 - 21; and the light chain variable region of the antibody comprises LCDR1 - LCDR3 with amino acid sequences as shown in SEQ ID NOs: 22 - 24; the antibody is of human IgG1 subtype; wherein, according to the EU numbering system, the heavy chain constant region of the antibody has the following mutations: L234A and L235A; L234A and G237A; L235A and G237A; or L234A, L235A, and G237A.

4. The antibody according to any one of claims 1 to 3, wherein, according to the EU numbering system, the heavy chain constant region of the antibody further has one or more mutations selected from the following: 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.

5. The antibody according to any one of claims 1 to 4, wherein, the amino acid sequence of the heavy chain variable region of the antibody is selected from SEQ ID NO: 2 and SEQ ID NO: 6; and the amino acid sequence of the light chain variable region of the antibody is selected from SEQ ID NO: 4 and SEQ ID NO:

8.

6. The antibody according to any one of claims 1 to 4, wherein, The amino acid sequence of the heavy chain variable region of the antibody is as shown in SEQ ID NO:2, and the amino acid sequence of the light chain variable region of the antibody is as shown in SEQ ID NO:4; The amino acid sequence of the heavy chain variable region of the antibody is as shown in SEQ ID NO:2, and the amino acid sequence of the light chain variable region of the antibody is as shown in SEQ ID NO:8; The amino acid sequence of the heavy chain variable region of the antibody is as shown in SEQ ID NO:6, and the amino acid sequence of the light chain variable region of the antibody is as shown in SEQ ID NO:4; Or The amino acid sequence of the heavy chain variable region of the antibody is as shown in SEQ ID NO:6, and the amino acid sequence of the light chain variable region of the antibody is as shown in SEQ ID NO:

8.

7. The antibody according to any one of claims 1 to 6: Its heavy chain is as shown in SEQ ID NO:16, and its light chain is as shown in SEQ ID NO:12; Or Its heavy chain is as shown in SEQ ID NO:18, and its light chain is as shown in SEQ ID NO:

12.

8. The antibody according to any one of claims 1 to 7, wherein, The antibody binds to FcγRIIIa_F158, FcγRI, FcγRIIa_H131, FcγRIIIa_V158, and / or FcγRIIb with an affinity constant greater than about 10 -7 M, such as greater than about 10 -6 M, 10 -5 M, 10 -4 M, or 10 -3 M or greater; preferably, the affinity constant is measured by a Fortebio Octet molecular interaction instrument; Preferably, the antibody has no binding signal or a binding signal less than 0.1 nm with FcγRIIIa_F158, FcγRI, FcγRIIa_H131, FcγRIIIa_V158 and / or FcγRIIb; preferably, the binding signal refers to the response value measured by a Fortebio Octet molecular interaction instrument.

9. The antibody according to any one of claims 1 to 8, wherein, The antibody binds to C1q with an affinity constant greater than about 10 -9 M, such as greater than about 10 -8 M, 10 -7 M, 10 -6 M or 10 -5 M or greater; preferably, the affinity constant is measured by a Fortebio Octet molecular interaction instrument; Preferably, the antibody has no binding signal or a binding signal less than 0.1 nm with C1q; preferably, the binding signal refers to the response value measured by a Fortebio Octet molecular interaction instrument.

10. An isolated nucleic acid molecule encoding the antibody according to any one of claims 1 to 9.

11. A vector comprising the isolated nucleic acid molecule of claim 10.

12. A host cell comprising the isolated nucleic acid molecule of claim 10, or the vector of claim 11.

13. A conjugate comprising an antibody and a conjugate moiety, wherein, The antibody is the antibody according to any one of claims 1 to 9, 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.

14. A kit comprising the antibody according to any one of claims 1 to 9, or comprising the conjugate of claim 13; 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.

15. Use of the antibody according to any one of claims 1 to 9 or the conjugate according to claim 13 in the preparation of a kit for detecting the presence or level of PD-1 in a sample.

16. A pharmaceutical composition comprising the antibody according to any one of claims 1 to 9 or the conjugate according to claim 13; optionally, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and / or excipient.

17. The pharmaceutical composition according to claim 16, further comprising one or more tumor chemotherapy drugs; Preferably, the tumor chemotherapy drug is a tyrosine kinase inhibitor; more preferably, the tumor chemotherapy drug is anlotinib or a pharmaceutically acceptable salt thereof (such as hydrochloride), or lenvatinib or a pharmaceutically acceptable salt thereof (such as mesylate).

18. The pharmaceutical composition according to claim 16 or 17, wherein, the unit dose of the pharmaceutical composition, calculated according to the mass of the antibody therein, is 100 mg - 1000 mg, 200 mg - 800 mg, 200 mg - 500 mg, 300 mg - 600 mg, 400 mg - 500 mg, or 450 mg.

19. A drug combination comprising the antibody according to any one of claims 1 to 9 and at least one (such as 1, 2, or 3) tumor chemotherapy drugs.

20. The drug combination according to claim 19, wherein, the tumor chemotherapy drug is a tyrosine kinase inhibitor; preferably, the tumor chemotherapy drug is anlotinib or a pharmaceutically acceptable salt thereof (such as hydrochloride), or lenvatinib or a pharmaceutically acceptable salt thereof (such as mesylate).

21. The drug combination according to claim 19 or 20, wherein, the unit dose of the antibody is 100 mg - 1000 mg, 200 mg - 800 mg, 200 mg - 500 mg, 300 mg - 600 mg, 400 mg - 500 mg, or 450 mg.

22. The drug combination according to claim 19 or 20, wherein, the unit dose of the tumor chemotherapy drug is 0.1 mg - 100 mg, 0.5 mg - 50 mg, 1 mg - 20 mg, 2 mg - 15 mg, 4 mg - 12 mg, or 8 mg - 12 mg.

23. The drug combination according to any one of claims 19 to 22, wherein, the drug combination is a fixed combination, such as in the form of a solid pharmaceutical composition or a liquid pharmaceutical composition; or the drug combination is a non-fixed combination, such as the anti-PD-1 antibody and the tumor chemotherapy drug in the non-fixed combination are each in the form of a pharmaceutical composition.

24. A medicine box product comprising the pharmaceutical composition according to any one of claims 16 to 18 or the drug combination according to any one of claims 19 to 23, and a product instruction manual.

25. Use of the antibody according to any one of claims 1 to 9, the conjugate according to claim 13, the pharmaceutical composition according to any one of claims 16 to 18, or the pharmaceutical combination according to any one of claims 19 to 23 in the preparation of a medicament for treating and / or preventing tumors or anemia, or in the preparation of a medicament for diagnosing tumors or anemia; preferably, the tumor is selected from one or more of melanoma, renal tumor, prostate cancer, bladder cancer, colon cancer, rectal cancer, gastric cancer, liver cancer, lung cancer, ovarian cancer, leukemia, nasopharyngeal carcinoma, and endometrial carcinoma; 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 adenocarcinoma of the gastroesophageal junction; Preferably, the tumor is a solid tumor with an MSI-H / dMMR phenotype; preferably, the tumor is selected from one or more of the following tumors with an MSI-H / dMMR phenotype: Colon cancer, rectal cancer, endometrial cancer, gastric cancer, mesothelioma, sarcoma, adrenocortical carcinoma, malignant melanoma, or ovarian germ cell tumor.

26. Use of the antibody according to any one of claims 1 to 9, the conjugate according to claim 13, the pharmaceutical composition according to any one of claims 16 to 18, or the pharmaceutical combination according to any one of claims 19 to 23 in the preparation of the following medicaments: A medicament for blocking the binding of PD-1 to PDL1, A medicament for downregulating the activity or level of PD-1, A medicament for relieving the immunosuppression of the body by PD-1, or A medicament for increasing the expression of IFN-γ and / or IL-2 in T lymphocytes.

27. The antibody according to any one of claims 1 to 9, the conjugate according to claim 13, the pharmaceutical composition according to any one of claims 16 to 18, or the pharmaceutical combination according to any one of claims 19 to 23, which is used for treating and / or preventing tumors or anemia, or for diagnosing tumors or anemia; preferably, the tumor is selected from one or more of melanoma, renal tumor, prostate cancer, bladder cancer, colon cancer, rectal cancer, gastric cancer, liver cancer, lung cancer, ovarian cancer, leukemia, nasopharyngeal carcinoma, and endometrial carcinoma; 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 adenocarcinoma of the gastroesophageal junction; Preferably, the tumor is a solid tumor with an MSI-H / dMMR phenotype; preferably, the tumor is selected from one or more of the following tumors with an MSI-H / dMMR phenotype: Colon cancer, rectal cancer, endometrial cancer, gastric cancer, mesothelioma, sarcoma, adrenocortical carcinoma, malignant melanoma, or ovarian germ cell tumor.

28. A method for treating and / or preventing tumors or anemia, or a method for diagnosing tumors or anemia, comprising the step of administering to a subject in need thereof an effective amount of the antibody according to any one of claims 1 to 9, the conjugate according to claim 13, the pharmaceutical composition according to any one of claims 16 to 18, or the pharmaceutical combination according to any one of claims 19 to 23; preferably, the tumor is selected from one or more of melanoma, renal tumor, prostate cancer, bladder cancer, colon cancer, rectal cancer, gastric cancer, liver cancer, lung cancer, ovarian cancer, leukemia, nasopharyngeal carcinoma, and endometrial carcinoma; 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 adenocarcinoma of the esophagogastric junction; Preferably, the tumor is a solid tumor with an MSI-H / dMMR phenotype; preferably, the tumor is selected from one or more of the following tumors with an MSI-H / dMMR phenotype: Colon cancer, rectal cancer, endometrial cancer, gastric cancer, mesothelioma, sarcoma, adrenocortical carcinoma, malignant melanoma, or ovarian germ cell tumor.

29. The method according to claim 28, wherein, An effective amount of the antibody is administered to a subject in need thereof before or after surgical treatment, and / or an effective amount of the antibody is administered to a subject in need thereof before or after radiotherapy.

30. The method according to any one of claims 28 to 29, wherein, The single-dose administration of the antibody is 0.1-100 mg per kilogram of body weight, preferably 1-10 mg; alternatively, the single-dose administration of the antibody is 10-1000 mg per subject, preferably 50-500 mg; Preferably, it is administered once every 3 days, 4 days, 5 days, 6 days, 10 days, 1 week, 2 weeks, or 3 weeks; Preferably, the administration method is intravenous drip or intravenous injection.

31. The method according to any one of claims 28 to 30, wherein, The administration of the antibody is in a cycle of 2 weeks or 3 weeks, preferably the antibody is intravenously administered on the first day of each cycle; preferably, the antibody is administered at a frequency of once every two weeks or once every three weeks.