Anti-CD73 antibodies and uses thereof

Abstract

The present invention relates to anti-CD73 antibodies and uses thereof, whose heavy chain variable regions comprise the amino acid sequences of HCDR1 to HCDR3 shown in SEQ ID NOs: 15 to 17, and whose light chain variable regions comprise the amino acid sequences of LCDR1 to LCDR3 shown in SEQ ID NOs: 18 to 20.

Description

[Technical Field]

[0001] The present invention relates to the field of immunology, specifically to anti-CD73 antibodies and uses thereof. [Background technology]

[0002] Ecto-5'-nucleotidase, or CD73 protein, is a multifunctional glycoprotein with a molecular weight of 70 kD, encoded by the NT5E gene, and anchored to the cell membrane by glycosylphosphatidylinositol (GPI) (Zimmermann H. Biochem J. 1992; 285: 345-365).

[0003] CD73 is widely distributed on the surface of human tissue cells, and previous studies have found that it is highly expressed in multiple solid tumors, including on cancer cells, dendritic cells, regulatory T cells (Tregs), natural killer cells (NK cells), myeloid-derived suppressor cells (MDSCs), and tumor-associated macrophages (TAMs) in the tumor microenvironment. Hypoxia induces upregulation of molecules such as hypoxia-inducible factor-1 (HIF-1), resulting in widespread expression of CD73 in the tumor microenvironment (Synnestvedt K, et al. J Clin Invest. 2002;110:993-1002). Analysis of clinical tumor samples has shown that high CD73 expression is a potential biological marker closely associated with poor prognosis in various tumor types, including breast, lung, ovarian, renal, gastric, and head and neck cancers.

[0004] CD73 has both hydrolase and non-hydrolase activities. The enzymatic and non-enzymatic functions of CD73 are present in tumor-associated processes and promote each other to maintain tumor progression. A growing body of research has demonstrated that CD73 is a key regulator of tumor cell proliferation, metastasis, and invasion in vitro, and of tumor angiogenesis and immune evasion in vivo. One of the most important immunosuppressive mechanisms is mediated by the CD73-adenosine metabolic signaling pathway. CD39, located upstream of CD73, catalyzes the synthesis of adenosine monophosphate (AMP) from ATP, which is then converted to adenosine by CD73. The resulting AMP then binds to the downstream adenosine receptor (A2AR). A2AR activates protein kinase A (PKA) and Csk kinase, thereby inhibiting a series of immune activation-related signaling pathways, including LCK, MAPK, and PKC, and thereby inhibiting the immune killing activity of T cells, thereby exerting immunosuppressive effects (Antonioli L, et al. Nat Rev Cancer. 2013;13:842-857).

[0005] The transmembrane receptor PD-1 (programmed cell death-1) is a member of the CD28 gene family and is expressed on activated T cells, B cells, and myeloid cells. PD-1's ligands, PDL1 (programmed cell death 1 ligand 1, also abbreviated as PDL-1) and PDL2 (programmed cell death 1 ligand 2, also abbreviated as PDL-2), both belong to the B7 superfamily. PDL1 is expressed on a variety of cells, including T cells, B cells, and endothelial and epithelial cells, whereas PDL2 is expressed exclusively on antigen-presenting cells, such as dendritic cells and macrophages.

[0006] The PD-1 / PDL1 signaling pathway plays an important role in regulating immune tolerance, microbial infection, and tumor immune evasion. PD-1 is primarily expressed on immune cells such as T cells, whereas its ligand, PDL1, is highly expressed in many human tumor tissues. Blocking the PD-1 / PDL1 signaling pathway activates suppressed T cells, which then attack cancer cells. Blocking the PD-1 / PDL1 signaling pathway promotes the proliferation of tumor antigen-specific T cells, which then kill tumor cells and ultimately suppress local tumor growth (Julie R et al., 2012, N Engl J Med. 366: 2455-2465). Furthermore, tumors with high PDL1 expression are associated with difficult-to-detect cancers (Hamanishi et al., 2007, Proc. Natl. Acad. Sci. USA 104: 3360-5). An effective method is to inject anti-PD-1 antibodies into the body to control the expression of PD-1. Antibodies against the PD-1 pathway are generally believed in the industry to bring about breakthrough progress in various tumors, such as non-small cell lung cancer, renal cell carcinoma, ovarian cancer, melanoma (Homet MB, Parisi G., et al., 2015, Semin Oncol. 42(3): 466-473), leukemia, and anemia (Held SA, Heine A, et al., 2013, Curr Cancer Drug Targets. 13(7): 768-74), due to the broad anti-tumor potential and remarkable efficacy of PD-1 antibodies.

[0007] Cytotoxic T lymphocyte-associated antigen 4 (CTLA4) is closely related to the CD28 molecule in terms of gene structure, chromosomal location, sequence homology, and gene expression. Both are receptors for the costimulatory molecule B7 and are expressed primarily on the surface of activated T cells. CTLA4 can inhibit the activation of mouse and human T cells by binding to B7, and plays a negative regulatory role in T cell activation.

[0008] CTLA4 antibodies (or anti-CTLA4 monoclonal antibodies) or CTLA4 ligands inhibit the binding of CTLA4 to its natural ligand, thereby blocking the transmission of negative regulatory signals from CTLA4 to T cells and enhancing T cell responsiveness to various antigens. In this regard, the results of in vivo and in vitro studies are generally consistent. Currently, CTLA4 monoclonal antibodies are already in clinical trials or approved for the treatment of prostate cancer, bladder cancer, colorectal cancer, gastrointestinal cancer, liver cancer, malignant melanoma, and other cancers (Grosso JF, Jure-Kunkel MN, 2013, Cancer Immun. 13: 5).

[0009] CTLA4 and CTLA4 antibodies, as important factors influencing the functional status of T cells, act by interfering with the immune microenvironment of the body. In vitro and in vivo studies have shown that CTLA4 antibodies can specifically reverse the immune suppression of CTLA4 in the body, activate T cells, and induce IL-2 production, which has the potential for widespread application in gene therapy for tumors and parasitic diseases. CTLA4 antibodies exhibit specific and highly effective therapeutic effects against diseases, complementing traditional drug therapy and opening up new avenues for gene therapy.

[0010] ADCC (antibody-dependent cell-mediated cytotoxicity), or antibody-dependent cell-mediated cytotoxicity, refers to the process in which the Fab fragment of an antibody binds to an antigen epitope on a virus-infected cell or tumor cell, and its Fc fragment binds to an Fc receptor (FcR) on the surface of a killer cell (NK cell, macrophage, etc.), directly killing the target cell via the killer cell.

[0011] Complement-dependent cytotoxicity (CDC) refers to the specific binding of antibodies to corresponding antigens on the cell membrane surface to form complexes, activating the complement system, which then forms MAC on the surface of target cells, resulting in their lysis. Complement lyses various bacteria and other pathogenic cells, and is an important defense mechanism for the body to resist infection by pathogens.

[0012] Fc receptors are immunoglobulin family proteins expressed on the surface of certain immune cells to mediate immune responses by recognizing the Fc region of antibodies. After the Fab region of an antibody recognizes an antigen, the Fc region of the antibody binds to the Fc receptor on the immune cell (e.g., killer cell) and initiates immune cell responses such as phagocytosis and ADCC. Depending on the type of antibody recognized by the Fc receptor and the cell expressing it, FcγRIIIa has been found to be closely related to the ADCC effect. FcγRIIIa is the primary molecule mediating ADCC (Hogarth PM, Pietersz GA. 2012, NATURE REVIEWS DRUG DISCOVERY, 11(4): 311-331).

[0013] The IgG family includes four members: IgG1, IgG2, IgG3, and IgG4. These members have different affinities for FcγRs due to amino acid differences in the fragment crystallizable (Fc) region of their heavy chain constant regions. IgG1 is the most abundant isoform in the human body and the most commonly used isoform in monoclonal antibody drugs. IgG1 can bind to various FcγRs and induce ADCC and CDC effects. The presence of ADCC and / or CDC in antibodies can cause unnecessary target tissue damage and adversely affect drug pharmacology. Summary of the Invention [Problem to be solved by the invention]

[0014] Through extensive research and creative efforts, the inventors have used a mammalian cell expression system to express recombinant human CD73 as an antigen, immunized mice, and obtained hybridoma cells by fusing mouse spleen cells with myeloma cells. After screening a large number of samples, the inventors have isolated the hybridoma cell line LT014 (deposit number: CCTCC NO: C2018137). [Means for solving the problem]

[0015] The present inventors have surprisingly found that the hybridoma cell line LTO14 can secrete a specific monoclonal antibody (designated 19F3) that specifically binds to human CD73, and that this monoclonal antibody can highly effectively inhibit the enzymatic activity of CD73 in a non-substrate competitive manner, suppress adenosine production, promote T cell activity, and suppress tumor growth.

[0016] Furthermore, the present inventors have creatively produced humanized anti-human CD73 antibodies (designated 19F3H1L1(hG1DM), 19F3H2L2(hG1DM), 19F3H2L3, and 19F3H2L3(hG1DM)), which have introduced amino acid mutations to eliminate ADCC and CDC effects, thereby avoiding unnecessary antibody-mediated toxic effects.

[0017] The inventors have surprisingly found that the combination of an antibody of the invention with an anti-PD-1 / CTLA-4 bispecific antibody has a more effective pharmacological effect in inhibiting tumor cell proliferation than the anti-PD-1 / CTLA-4 bispecific antibody or anti-CD73 antibody alone.

[0018] Another aspect of the present invention further relates to antibodies, wherein the anti-CD73 antibody is a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO: 2, SEQ ID NO: 6 or SEQ ID NO: 10, and a light chain variable region comprising LCDR1, LCDR2 and LCDR3 as set forth in SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 12 or SEQ ID NO: 14; Preferably, the anti-CD73 antibody is, according to the IMGT numbering system: an HCDR1 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 15, a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to said sequence, or an amino acid sequence having one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to said sequence; an HCDR2 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 16, a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to said sequence, or an amino acid sequence having one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to said sequence; an HCDR3 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 17, a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to said sequence, or an amino acid sequence having one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to said sequence; an LCDR1 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 18, a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to said sequence, or an amino acid sequence having one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to said sequence; an LCDR2 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 19, a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to said sequence, or an amino acid sequence having one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to said sequence; and an LCDR3 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 20, a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the above sequence, or an amino acid sequence having one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the above sequence.

[0019] In some embodiments of the present invention, The heavy chain variable region of the antibody is comprising or consisting of SEQ ID NO:2, SEQ ID NO:6 or SEQ ID NO:10, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:2, SEQ ID NO:6 or SEQ ID NO:10, or an amino acid sequence which has one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the amino acid sequence shown in SEQ ID NO:2, SEQ ID NO:6 or SEQ ID NO:10, and The light chain variable region of the antibody is or consisting of SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:12 or SEQ ID NO:14, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:12 or SEQ ID NO:14, or an amino acid sequence which has one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the amino acid sequence shown in SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:12 or SEQ ID NO:14.

[0020] In some embodiments of the invention, the amino acid sequence of the heavy chain variable region of the antibody is set forth in SEQ ID NO: 2 and the amino acid sequence of the light chain variable region of the antibody is set forth in SEQ ID NO: 4; The amino acid sequence of the heavy chain variable region of the antibody is set forth in SEQ ID NO: 6, and the amino acid sequence of the light chain variable region of the antibody is set forth in SEQ ID NO: 8; the amino acid sequence of the heavy chain variable region of the antibody is set forth in SEQ ID NO: 10, and the amino acid sequence of the light chain variable region of the antibody is set forth in SEQ ID NO: 12; or The amino acid sequence of the heavy chain variable region of the above antibody is shown in SEQ ID NO:10, and the amino acid sequence of the light chain variable region of the above antibody is shown in SEQ ID NO:14.

[0021] In some embodiments of the invention, the heavy chain constant region of the antibody is Ig gamma-1 chain C region, ACCESSION: P01857, and the light chain constant region is Ig kappa chain C region, ACCESSION: P01834.

[0022] In some embodiments of the present invention, the heavy chain constant region of the above-mentioned antibody is an Ig gamma-1 chain C region, ACCESSION: P01857, with a point mutation from leucine to alanine (L234A) introduced at position 234 and a point mutation from leucine to alanine (L235A) introduced at position 235; the light chain constant region is an Ig kappa chain C region, ACCESSION: P01834, and the amino acid sequence is set forth in SEQ ID NO: 22.

[0023] The variable regions of the light and heavy chains determine antigen binding, and each chain variable region contains three highly variable regions called complementarity-determining regions (CDRs) (heavy chain (H) CDRs include HCDR1, HCDR2, and HCDR3, and light chain (L) CDRs include LCDR1, LCDR2, and LCDR3, which were named by Kabat et al., Bethesda Md., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 1991; 1-3: 91-3242).

[0024] Preferably, the CDRs are defined by the IMGT numbering system, see Ehrenmann, Francois, Quentin Kaas, and Marie-Paule Lefranc. IMGT / 3Dstructure-DB and IMGT / DomainGapAlign: a database and a tool for immunoglobulins or antibodies, T cell receptors, MHC, IgSF and MhcSF. Nucleic acids research 2009; 38(suppl_1): D301-D307.

[0025] By technical means well known to those skilled in the art, the amino acid sequences of the CDR regions of the monoclonal antibody sequences were analyzed according to the IMGT definition, for example, in the VBASE2 database.

[0026] The antibodies 19F3, 19F3H1L1(hG1DM), 19F3H2L2(hG1DM), and 19F3H2L3(hG1DM) according to the present invention have the same CDRs.

[0027] The amino acid sequences of the three CDR regions of the heavy chain variable region are as follows: HCDR1: GYSFTGYT (SEQ ID NO: 15), HCDR2: INPYNAGT (SEQ ID NO: 16), HCDR3: ARSEYRYGGDYFDY (SEQ ID NO: 17),

[0028] The amino acid sequences of the three CDR regions of the light chain variable region are as follows: LCDR1: QSLLNSSNQKNY (SEQ ID NO: 18), LCDR2:FAS (SEQ ID NO: 19), LCDR3: QQHYDTPYT (sequence number 20).

[0029] In some embodiments of the invention, the antibody is a monoclonal antibody.

[0030] In some embodiments of the invention, the antibody is a humanized antibody, a chimeric antibody, or a multispecific antibody (eg, a bispecific antibody).

[0031] In some embodiments of the invention, the antigen-binding fragment is selected from Fab, Fab', F(ab')2, Fd, Fv, dAb, Fab / c, a complementarity-determining region fragment, a single-chain antibody (e.g., scFv), a humanized antibody, a chimeric antibody, or a bispecific antibody.

[0032] Another aspect of the present invention relates to an isolated polypeptide selected from the group consisting of:

[0033] (1) An isolated polypeptide comprising the sequences set forth in SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17, wherein the polypeptide specifically binds to CD73 as part of an anti-CD73 antibody, the antibody further comprising the sequences set forth in SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20.

[0034] (2) An isolated polypeptide comprising the sequence set forth in SEQ ID NO:18, SEQ ID NO:19, or SEQ ID NO:20, wherein the polypeptide specifically binds to CD73 as part of an anti-CD73 antibody, and the antibody further comprises the sequence set forth in SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17.

[0035] (3) An isolated polypeptide comprising the sequence set forth in SEQ ID NO: 2, SEQ ID NO: 6, or SEQ ID NO: 10, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the above sequences, or an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) conservative amino acid mutations (preferably substitutions, insertions, or deletions) compared to the above sequences, wherein the above polypeptide is an anti-CD73 and specifically binds to CD73 as part of an antibody, said antibody further correspondingly comprising the sequence set forth in SEQ ID NO:4, SEQ ID NO:8 or SEQ ID NO:12, respectively, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, or an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared thereto.

[0036] (4) An isolated polypeptide comprising the sequence set forth in SEQ ID NO: 4, SEQ ID NO: 8, or SEQ ID NO: 12, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the above sequences, or an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) conservative amino acid mutations (preferably substitutions, insertions, or deletions) compared to the above sequences, wherein the above polypeptide is an anti-CD73 and specifically binds to CD73 as part of an antibody, said antibody further correspondingly comprising the sequence set forth in SEQ ID NO:2, SEQ ID NO:6 or SEQ ID NO:10, respectively, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, or an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared thereto.

[0037] (5) An isolated polypeptide comprising the sequence set forth in SEQ ID NO: 10, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the above sequence, or an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) conservative amino acid mutations (preferably substitutions, insertions, or deletions) compared to the above sequence, wherein the above polypeptide is an anti-CD73 and specifically binds to CD73 as part of an antibody, said antibody further correspondingly comprising the sequence set forth in SEQ ID NO: 14, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, or an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared thereto.

[0038] (6) An isolated polypeptide comprising the sequence set forth in SEQ ID NO: 14, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the above sequence, or an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) conservative amino acid mutations (preferably substitutions, insertions, or deletions) compared to the above sequence, wherein the above polypeptide is an anti-CD73 and specifically binds to CD73 as part of an antibody, said antibody further correspondingly comprising the sequence set forth in SEQ ID NO: 10, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, or an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared thereto, respectively.

[0039] Another aspect of the invention relates to an isolated nucleic acid molecule encoding an antibody or antigen-binding fragment thereof according to any one of the invention, or an isolated polypeptide as described above.

[0040] A further aspect of the present invention relates to a vector comprising the isolated nucleic acid molecule of the present invention.

[0041] A further aspect of the invention relates to a host cell comprising the isolated nucleic acid molecule of the invention or the vector of the invention.

[0042] A further aspect of the present invention relates to a conjugate comprising an antibody and a conjugate moiety, wherein the antibody is an antibody or antigen-binding fragment thereof according to any one of the invention, and the conjugate moiety is a purification tag (e.g., a His tag), a detectable label, preferably a radioisotope, a fluorescent substance, a chemiluminescent substance, a colored substance, polyethylene glycol, or an enzyme.

[0043] A further aspect of the invention relates to a fusion protein or a multispecific antibody (preferably a bispecific antibody) comprising an antibody or antigen-binding fragment thereof according to any one of the claims of the invention.

[0044] A further aspect of the present invention relates to a kit comprising an antibody or antigen-binding fragment thereof, a conjugate, a fusion protein or a multispecific antibody according to any one of the claims of the present invention, preferably said kit further comprising a second antibody, which specifically recognizes said antibody, optionally said second antibody further comprising a detectable label, such as a radioisotope, a fluorescent substance, a chemiluminescent substance, a colored substance or an enzyme.

[0045] A further aspect of the invention relates to the use of an antibody or antigen-binding fragment thereof, a conjugate, a fusion protein or a multispecific antibody according to any one of the invention in the preparation of a kit for detecting the presence or level of CD73 in a sample.

[0046] A further aspect of the invention relates to a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof, a conjugate, a fusion protein or a multispecific antibody according to any one of the claims of the invention, optionally further comprising a pharmaceutically acceptable carrier and / or excipient. Preferably, the pharmaceutical composition is in a form suitable for administration by subcutaneous, intradermal, intravenous, intramuscular or intralesional injection.

[0047] A further aspect of the present invention relates to the use of an antibody or antigen-binding fragment thereof, a conjugate, a fusion protein or a multispecific antibody according to any one of the invention in the preparation of a medicament for the treatment and / or prevention of a tumor (e.g. a solid tumor, preferably non-small cell lung cancer, prostate cancer (including metastatic castration-resistant prostate cancer (mCRPC)), triple-negative breast cancer, ovarian cancer, colorectal cancer (including microsatellite stable (MSS) and mismatch repair deficient / microsatellite unstable (dMMR / MSI-high)), gastric cancer (including microsatellite stable (MSS) and mismatch repair deficient / microsatellite unstable (dMMR / MSI-high)), melanoma, head and neck cancer, renal cell carcinoma or pancreatic ductal adenocarcinoma, or in the preparation of a medicament for use in the diagnosis of a tumor.

[0048] A further aspect of the present invention relates to hybridoma cell line LT014, which has been deposited with the China Typical Culture Collection Center (CCTCC) and has deposit number CCTCC NO: C2018137.

[0049] A further aspect of the present invention relates to a kit comprising (1) an antibody or antigen-binding fragment thereof according to the invention, a conjugate according to the invention, or a fusion protein or multispecific antibody according to the invention, and (2) an anti-PD-1 / CTLA-4 bispecific antibody, and optionally, instructions for use.

[0050] A further aspect of the present invention relates to a method for treating and / or preventing tumors, comprising administering to a patient a therapeutically effective amount of drug A and a therapeutically effective amount of drug B, where drug A comprises an antibody or antigen-binding fragment thereof described in the present invention, a conjugate described in the present invention, or a fusion protein or multispecific antibody described in the present invention, and drug B comprises an anti-PD-1 / CTLA-4 bispecific antibody. Preferably, drug A and drug B are administered simultaneously or sequentially, where sequential administration refers to administering drug A first or drug B first.

[0051] In some embodiments of the invention, the heavy chain amino acid sequence of the PD-1 / CTLA-4 bispecific antibody is set forth in SEQ ID NO:35 and the light chain amino acid sequence is set forth in SEQ ID NO:36.

[0052] In the present invention, unless otherwise specified, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the cell culture, molecular genetics, nucleic acid chemistry, and immunological laboratory procedures used herein are common procedures widely used in the art. At the same time, in order to better understand the present invention, the following definitions and explanations of relevant terms are provided.

[0053] As used herein, the term EC 50 refers to the concentration for 50% of the maximal effect, which refers to the concentration that can produce 50% of the maximal effect.

[0054] As used herein, the term "antibody" refers to an immunoglobulin molecule generally consisting of two pairs of polypeptide chains, each pair having one "light" (L) chain and one "heavy" (H) chain. Antibody light chains can be classified as kappa and lambda light chains. Heavy chains are classified as mu, delta, gamma, alpha, or epsilon, and antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE, respectively. In light and heavy chains, the variable and constant regions are joined through a "J" region of about 12 or more amino acids, with heavy chains also including a "D" region of about 3 or more amino acids. Each heavy chain comprises a heavy chain variable region (V H ) and heavy chain constant region (C H The heavy chain constant region consists of three domains (C H1 , C H2 and C H3 Each light chain consists of a light chain variable region (V L ) and light chain constant region (C L The light chain constant region consists of one domain, C LThe antibody constant region mediates the binding of the immunoglobulin to host tissues or factors, including cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. H and V L The regions can be further subdivided into regions of high variability (called complementarity determining regions (CDRs)) interspersed with more conserved regions called framework regions (FRs). H and V L Each heavy / light chain pair consists of three CDRs and four FRs arranged from the amino to carboxyl terminals in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. H and V L), each forming an antigen-binding site. The assignment of amino acids to each region or domain is defined according to the Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, M.D. (1987 and 1991)), or Chothia & Lesk J. Mol. Biol. 1987; 196: 901-917; Chothia et al. Nature 1989; 342: 878-883, or the IMGT numbering system, and reference is made to the definition in Ehrenmann, Francois, Quentin Kaas, and Marie-Paule Lefranc. "IMGT / 3Dstructure-DB and IMGT / DomainGapAlign: a database and a tool for immunoglobulins or antibodies, T cell receptors, MHC, IgSF, and MhcSF." Nucleic acids research 2009; 38 (suppl_1): D301-D307. The term "antibody" is not limited by any particular method of producing the antibody. For example, it includes, among others, recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. The antibody may be of a different isotype, such as an IgG (e.g., an IgG1, IgG2, IgG3, or IgG4 isoform), IgA1, IgA2, IgD, IgE, or IgM antibody.

[0055] As used herein, the term "monoclonal antibody" or similar terms refers to an antibody or antibody fragment derived from a series of highly homologous antibody molecules, i.e., a series of antibody molecules that are completely identical except for spontaneously occurring natural mutations. Monoclonal antibodies have high specificity for a single epitope on an antigen. Compared to monoclonal antibodies, polyclonal antibodies generally contain at least two or more different antibodies that generally recognize different epitopes on an antigen. Monoclonal antibodies can generally be obtained using the hybridoma technology first described by Kohler et al. (Kohler G, Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity [J]. Nature, 1975; 256(5517): 495), or can also be obtained using recombinant DNA technology (see, e.g., U.S. Patent 4,816,567).

[0056] As used herein, the term "humanized antibody" refers to an antibody or antibody fragment obtained after all or part of the CDR regions of a human immunoglobulin (receptor antibody) are substituted with the CDR regions of a non-human antibody (donor antibody), where the donor antibody can be a non-human antibody (e.g., mouse, rat, or rabbit) having the desired specificity, affinity, or reactivity. Furthermore, some amino acid residues in the framework region (FR) of the receptor antibody can also be substituted with corresponding amino acid residues of the non-human antibody or other antibodies to further improve or optimize the performance of the antibody. For further details on humanized antibodies, see, for example, Jones et al., Nature 1986; 321:522-525; Reichmann et al., Nature 1988; 332:323-329; Presta, Curr. Op. Struct. Biol., 1992; 2:593-596; and Clark M. Antibody humanization: a case of the 'Emperor's new clothes'? [J]. Immunol. Today, 2000; 21(8): 397-402.

[0057] As used herein, the terms "isolate" or "isolated" refer to being obtained by hand from a natural state. When a particular "isolated" substance or component exists in nature, the natural environment in which it exists may have been altered, or the substance may have been isolated from its natural environment, or both. For example, a naturally occurring, non-isolated polynucleotide or polypeptide naturally occurs in the living body of an animal, and a highly purified version of the same polynucleotide or polypeptide isolated from such a natural state is referred to as isolated. The terms "isolate" or "isolated" do not exclude mixtures of artificial or synthetic substances, nor do they exclude the presence of other impurities that do not affect the activity of the substance.

[0058] As used herein, the term "vector" refers to a nucleic acid vehicle into which a polynucleotide can be inserted. A vector is called an expression vector when it allows for the expression of a protein encoded by the inserted polynucleotide. A vector can be introduced into a host cell by transformation, transduction, or transfection to allow the expression of the genetic material elements it carries in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids, phagemids, Cox plasmids, artificial chromosomes, such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs), bacteriophages, such as lambda phage or M13 phage, and animal viruses. Animal viruses useful as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex viruses), poxviruses, baculoviruses, papilloma viruses, and papilloma baculoviruses (such as SV40). A vector may contain multiple expression control elements, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. A vector may also contain an origin of replication.

[0059] As used herein, the term "host cell" refers to cells useful for introducing a vector, including, but not limited to, prokaryotic cells such as E. 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, GS cells, BHK cells, HEK 293 cells or human cells.

[0060] As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, e.g., a reaction between an antibody and an antigen to which it is directed. In one embodiment, an antibody that specifically binds to an antigen (or has specificity for an antigen) is one in which the antibody binds to an antigen at a concentration of about 10-5 Less than m, e.g., about 10 -6 Less than M, about 10 -7 Less than M, about 10 -8 Less than M, about 10 -9 Less than M, about 10 -10 Affinity (K D ) to bind to the antigen.

[0061] As used herein, the term "K D " refers to the dissociation equilibrium constant of a particular antibody-antigen interaction, which is used to describe the binding affinity between an antibody and an antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding and the higher the affinity between the antibody and the antigen. Generally, antibodies have a dissociation equilibrium constant of about 10 -5 Less than m, e.g., about 10 -6 Less than M, about 10 -7 Less than M, about 10 -8 Less than M, about 10 -9 Less than M, about 10 -10 The dissociation equilibrium constant (K D ) binds to an antigen (e.g., PD-1 protein). K can be produced by various methods known to those skilled in the art. D is measured, for example, using a Fortebio molecular interaction instrument.

[0062] 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 "polypeptide" and "protein" have the same meaning and are used interchangeably. Furthermore, in the present invention, amino acids are typically represented by abbreviations or abbreviation words well known in the art. For example, alanine can be represented by A or Ala.

[0063] As used herein, the term "pharmaceutically acceptable carrier and / or excipient" refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and active ingredient, and is well known in the art (see, e.g., 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.

[0064] As used herein, the term "effective amount" refers to an amount sufficient to achieve or at least partially achieve a desired effect. For example, an effective amount for preventing a disease (e.g., a tumor) refers to an amount sufficient to prevent, inhibit, or delay the onset of the disease (e.g., a tumor), and an effective amount for treating a disease refers to an amount sufficient to cure or at least partially inhibit the disease and its complications in a patient who has the disease. [Effects of the Invention]

[0065] The monoclonal antibodies of the present invention can bind specifically to CD73 and highly effectively inhibit the enzymatic activity of CD73 in a non-substrate competitive manner, suppressing adenosine production and promoting T cell activity and tumor growth suppression. At the same time, the combination of the antibodies of the present invention with the anti-PD-1 / CTLA-4 bispecific antibody has a more effective pharmacological effect on tumor cell proliferation than the anti-PD-1 / CTLA-4 bispecific antibody or the anti-CD73 antibody alone. [Brief explanation of the drawings]

[0066] [Figure 1]1 is a kinetic affinity data fitting curve for the binding of 19F3H2L3 (hG1DM) to C1q. [Figure 2] 1 is a fitting curve of the kinetic affinity data for the binding of MEDI9447 to C1q. [Figure 3] 1 is a fitting curve of kinetic affinity data for the binding of IgG1 wild-type antibody to C1q. [Figure 4] 1 shows the kinetic affinity data fitting curve for the binding of 19F3H2L3 (hG1DM) to FcγRIIIa. [Figure 5] Kinetic affinity data fitting curve for binding of MEDI9447 to FcγRIIIa. [Figure 6] 1 is a fitting curve of kinetic affinity data for the binding of IgG1 wild-type antibody to FcγRIIIa. [Figure 7] Tumor weights of mice in each group on day 23 after grouping. **P<0.01. [Figure 8] Compared to Day 0, this shows the weight change rate of animals in each group during the experiment. [Figure 9] Anti-CD73 antibodies effectively inhibit the enzymatic activity of CD73. DETAILED DESCRIPTION OF THE INVENTION

[0067] Hereinafter, the embodiments of the present invention will be described in detail with reference to examples. Those skilled in the art should understand that the following examples are merely for the purpose of illustrating the present invention and are not intended to limit the scope of the present invention. If specific techniques or conditions are not specified in the examples, they will be carried out according to the techniques or conditions described in the literature in this field (see, for example, Sambrook J, et al., Molecular Cloning: A Laboratory Manual, 3rd Ed., Cold Spring Harbor Laboratory Press) or according to the product instructions. If the manufacturer of the reagents or equipment used is not specified, they may be common commercially available products.

[0068] The BALB / c mice used in the following examples of the present invention are purchased from Guangdong Medical Experimental Animal Center.

[0069] The positive control antibody MEDI9447 (Oleclumab) used in the following Examples of the present invention is produced by Zhongshan Kangfang Biopharmaceutical Co., Ltd., and its sequence is the same as the antibody sequence listed in the International Nonproprietary Names for Pharmaceutical Substances (INN) published by Medimmune Limited on the WHO website (World Health Organization (2016). "International Nonproprietary Names for Pharmaceutical Substances (INN). Proposed INN: List 116" (PDF). WHO Drug Information. 30 (4), pp. 661-662.).

[0070] In the following examples of the present invention, the heavy chain constant region of the positive control wild-type IgG1 control antibody used is the Ig gamma-1 chain C region (Accession: P01857), and the light chain constant region is the Ig kappa chain C region (Accession: P01834).

[0071] In the following examples of the present invention, the C1q used was purchased from Fizgerald and has the product number A16050201.

[0072] In the following examples of the present invention, the FcγRIIIa-bio used was purchased from Sino Biological, with product number LC09JA0407.

[0073] In the following examples of the present invention, the CD73 (5'-nuclease) specific inhibitor APCP (alpha, beta-methylene adenosine-5'-diphosphate, 5'-α, β-methylene-adenosine diphosphate) used is from Sigma and has the product number M3763-10MG.

[0074] In the following Examples of the present invention, the isotype control antibody used is a human anti-hen egg lysozyme IgG (i.e., anti-HEL antibody, or human IgG, abbreviated as hIgG, or isotype control), the sequence of which is derived from the variable region sequence of Fab F10.6.6 in the Fv domain of antiprotein antibodies, which was reported by Acierno et al. to have affinity maturation that increases the stability and plasticity (Acierno et al., J Mol Biol. 2007; 374(1): 130-146). [Example]

[0075] Example 1: Preparation of anti-CD73 antibody 19F3 1. Preparation of hybridoma cell line LT014 The antigen used for the anti-CD73 antibody was human NT5E-His (NT5E is Genbank ID: NP_002517.1, location: 1-552, prepared by Zhongshan Kangfang Biopharmaceutical Co., Ltd.). After immunization, splenocytes were taken from the mice and fused with mouse myeloma cells to generate hybridoma cells. Using human NT5E (NT5E is Genbank ID: NP_002517.1, location: 1-552)-Biotin (NT5E-Biotin, prepared by Zhongshan Kangfang Biopharmaceutical Co., Ltd.), the hybridoma cells were screened using indirect ELISA to obtain hybridoma cells secreting antibodies that specifically bind to CD73. A stable hybridoma cell line was isolated using the limited dilution method. This hybridoma cell line was designated hybridoma cell line LT014, and the monoclonal antibody secreted by it was designated 19F3. The hybridoma cell line LT014 (also referred to as CD73-19F3) was deposited at the China Center for Type Culture Collection (CCTCC) on June 21, 2018, with the deposit number CTCCC NO: C2018137. The deposit address is Wuhan University, Wuhan, China, with the postal code 430072.

[0076] 2. Preparation of anti-CD73 antibody 19F3 The LT014 cell line was cultured in a chemically defined medium (CD medium) containing 1% penicillin and streptomycin at 37°C in a cell incubator with 5% CO2. After 7 days, the cell culture supernatant was collected and centrifuged at high speed. The supernatant was then filtered under vacuum using a microporous membrane. The antibody 19F3 was then purified using a HiTrap protein A HP column.

[0077] Example 2: Sequence analysis of anti-CD73 antibody 19F3 mRNA was extracted from the LT014 cell line cultured in Example 1 using a cultured cell / bacteria total RNA extraction kit (Tiangen, product number: DP430). cDNA is synthesized and PCR amplified according to the instructions of the Invitrogen SuperScript® III First-Strand Synthesis System for RT-PCR kit. TA cloning is performed directly on the PCR amplification product, and the specific procedure is performed by referring to the kit instructions for the pEASY-T1 Cloning Kit (Transgen CT101). The TA cloning product was directly sequenced, and the sequence analysis results of the anti-CD73 antibody 19F3 are as follows: The nucleic acid sequence of the heavy chain variable region is 363 bp in length as shown in SEQ ID NO:1. The amino acid sequence it encodes is 121 aa in length as shown in SEQ ID NO:2. Among them, the sequence of heavy chain CDR1 is shown in SEQ ID NO: 15, the sequence of heavy chain CDR2 is shown in SEQ ID NO: 16, and the sequence of heavy chain CDR3 is shown in SEQ ID NO: 17. The nucleic acid sequence of the light chain variable region is 339 bp in length as shown in SEQ ID NO:3. The amino acid sequence it encodes is 113 aa in length as shown in SEQ ID NO:4. Among them, the sequence of light chain CDR1 is shown in SEQ ID NO: 18, the sequence of light chain CDR2 is shown in SEQ ID NO: 19, and the sequence of light chain CDR3 is shown in SEQ ID NO: 20.

[0078] Example 3: Design and preparation of the light and heavy chains of a humanized anti-human CD73 antibody Using the three-dimensional crystal structure of human CD73 protein (Hage T, Reinemer P, Sebald W. Crystals of a 1:1 complex between human interleukin-4 and the extracellular domain of its receptor alpha chain. Eur J Biochem. 1998; 258(2):831-6.) and the sequence of antibody 19F3 obtained in Example 2, a computer-generated antibody model was simulated, and mutations were designed based on the model to obtain the variable region sequences of 19F3H1L1(hG1DM), 19F3H2L2(hG1DM), and 19F3H2L3(hG1DM). The designed variable region sequences of the above humanized antibodies are as follows:

[0079] (1) Heavy and light chain variable region sequences of humanized monoclonal antibody 19F3H1L1 (hG1DM) The nucleic acid sequence of the heavy chain variable region is 363 bp in length as shown in SEQ ID NO:5. The amino acid sequence it encodes is 121 aa in length as shown in SEQ ID NO: 6, of which the sequence of heavy chain CDR1 is shown in SEQ ID NO: 15, the sequence of heavy chain CDR2 is shown in SEQ ID NO: 16, and the sequence of heavy chain CDR3 is shown in SEQ ID NO: 17. The nucleic acid sequence of the light chain variable region is 339 bp in length as shown in SEQ ID NO:7. The amino acid sequence it encodes is 113 aa in length as shown in SEQ ID NO: 8, of which the sequence of light chain CDR1 is shown in SEQ ID NO: 18, the sequence of light chain CDR2 is shown in SEQ ID NO: 19, and the sequence of light chain CDR3 is shown in SEQ ID NO: 20.

[0080] (2) Heavy and light chain variable region sequences of humanized monoclonal antibody 19F3H2L2 (hG1DM) The nucleic acid sequence of the heavy chain variable region is 363 bp in length as shown in SEQ ID NO:9. The amino acid sequence it encodes is 121 aa in length as shown in SEQ ID NO: 10, of which the sequence of heavy chain CDR1 is shown in SEQ ID NO: 15, the sequence of heavy chain CDR2 is shown in SEQ ID NO: 16, and the sequence of heavy chain CDR3 is shown in SEQ ID NO: 17. The nucleic acid sequence of the light chain variable region is 339 bp in length as shown in SEQ ID NO:11. The amino acid sequence it encodes is 113 aa in length as shown in SEQ ID NO: 12, of which the sequence of light chain CDR1 is shown in SEQ ID NO: 18, the sequence of light chain CDR2 is shown in SEQ ID NO: 19, and the sequence of light chain CDR3 is shown in SEQ ID NO: 20.

[0081] (3) Heavy and light chain variable region sequences of humanized monoclonal antibody 19F3H2L3 (hG1DM) The nucleic acid sequence of the heavy chain variable region is 363 bp in length as shown in SEQ ID NO:9. The amino acid sequence it encodes is 121 aa in length as shown in SEQ ID NO: 10, of which the sequence of heavy chain CDR1 is shown in SEQ ID NO: 15, the sequence of heavy chain CDR2 is shown in SEQ ID NO: 16, and the sequence of heavy chain CDR3 is shown in SEQ ID NO: 17. The nucleic acid sequence of the light chain variable region is 339 bp in length as shown in SEQ ID NO:13. The amino acid sequence it encodes is 113 aa in length as shown in SEQ ID NO: 14, of which the sequence of light chain CDR1 is shown in SEQ ID NO: 18, the sequence of light chain CDR2 is shown in SEQ ID NO: 19, and the sequence of light chain CDR3 is shown in SEQ ID NO: 20.

[0082] 3. Preparation of humanized 19F3H1L1 (hG1DM), 19F3H2L2 (hG1DM), and 19F3H2L3 (hG1DM) The light chain constant region of the 19F3H1L1 (hG1DM), 19F3H2L2 (hG1DM) and 19F3H2L3 (hG1DM) antibodies is the Ig kappa chain C region, and the accession number is P01834. The heavy chain constant region was constructed by introducing a point mutation from leucine to alanine (L234A) at position 234 and a point mutation from leucine to alanine (L235A) (sequence number 21) at position 235 in the Ig gamma-1 chain C region, ACCESSION: P01857, to obtain humanized antibodies designated 19F3H1L1(hG1DM), 19F3H2L2(hG1DM), and 19F3H2L3(hG1DM). The heavy and light chain cDNAs of 19F3H1L1 (hG1DM), 19F3H2L2 (hG1DM), and 19F3H2L3 (hG1DM) were cloned into the pUC57simple vector (provided by GenScript) to obtain pUC57simple-19F3H1 (hG1DM), pUC57simple-19F3L1, pUC57simple-19F3H2 (hG1DM), pUC57simple-19F3L2, and pUC57simple-19F3L3, respectively. According to the standard techniques introduced in "Molecular Cloning: A Laboratory Manual, 2nd Edition," the synthesized full-length heavy and light chain genes were digested with EcoRI and HindIII and subcloned into the expression vector pcDNA3.1 by digestion with restriction enzymes (EcoRI and HindIII) to obtain expression plasmids pcDNA3.1-19F3H1(hG1DM), pcDNA3.1-19F3L1, pcDNA3.1-19F3H2(hG1DM), pcDNA3.1-19F3L2, and pcDNA3.1-19F3L3. The heavy and light chain genes of the recombinant expression plasmids were then sequenced. Next, recombinant plasmids containing the corresponding light and heavy chains were designed as gene combinations: pcDNA3.1-19F3H1(hG1DM) / pcDNA3.1-19F3L1, pcDNA3.1-19F3H2(hG1DM) / pcDNA3.1-19F3L2, and pcDNA3.1-19F3H2(hG1DM) / pcDNA3.1-19F3L3. These were then co-transfected into 293F cells and purified from the culture medium. After sequencing and verification, endotoxin-free expression plasmids were prepared and transiently co-transfected into HEK293 cells to express the antibodies. After 7 days of culture, the cell culture medium was harvested and affinity purified using a Protein A column to obtain the humanized antibodies.

[0083] Example 4: Kinetic affinity measurements of anti-CD73 antibodies to C1q and FcγRIIIa (1) Kinetic affinity measurement of anti-CD73 antibody and C1q The sample dilution buffer was PBS, 0.02% Tween-20, and 0.1% BSA, pH 7.4. 50 μg / mL of antibody was immobilized on the FAB2G sensor to an immobilization height of approximately 2.0 nm. The sensor was equilibrated in the buffer for 60 s. The antibody immobilized on the sensor bound to the antigen C1q, with antigen concentrations ranging from 0.63 to 10 nM (2-fold gradient dilution), for 60 s. The antigen-antibody dissociation was then performed in the buffer for 60 s. The sensor was regenerated with 10 mM glycine at pH 1.7 for 5 s, and this was repeated four times. The sample plate vibration speed was 1000 rpm, the detection temperature was 30°C, and the detection frequency was 0.6 Hz. The data was analyzed by fitting with a 1:1 model to obtain the affinity constant. The data acquisition software was Fortebio Data Acquisition 7.0, and the data analysis software was Fortebio Data Analysis 7.0. As a result, as shown in Table 1 and Figures 1 to 3, neither 19F3H2L3 (hG1DM) nor MEDI9447ha had binding activity to C1q.

[0084] [Table 1]

[0085] (2) Kinetic affinity measurement of anti-CD73 antibody and FcγRIIIa The sample dilution buffer was PBS, 0.02% Tween-20, and 0.1% BSA, pH 7.4. 0.5 μg / mL FcγRIIIa (from Sino Biological) was immobilized on the SA sensor for 120 s. The sensor was equilibrated with the buffer for 60 s. The CD16a immobilized on the sensor bound to each antibody, with antibody concentrations ranging from 31.3 to 500 nM (2-fold gradient dilution), for 60 s. The antibody-antigen dissociated in the buffer for 60 s. The sensor was regenerated with 10 mM NaOH. The detection temperature was 30 °C and the frequency was 0.6 Hz. The data were analyzed by fitting with a 1:1 model to obtain the affinity constants. The results, as shown in Table 2 and Figures 4 to 6, show that 19F3H2L3 (hG1DM) does not bind to FcγRIIIa, but MEDI9447 has binding activity to FcγRIIIa.

[0086] [Table 2]

[0087] Example 5: Effective blocking of tumor cell growth by the combination of anti-CD73 antibody and anti-CTLA-4 / PD-1 bispecific antibody This experiment investigated the pharmacological activity of the anti-CD73 antibody, 19F3H2L3 (hG1DM), in combination with the anti-PD-1 / CTLA-4 bispecific antibody BiAb004 (hG1TM) (the heavy chain amino acid sequence is set forth in SEQ ID NO: 35, the light chain amino acid sequence is set forth in SEQ ID NO: 36, and the light and heavy chain CDR amino acid sequences are set forth in SEQ ID NOs: 23 to 34) in suppressing tumor growth. As shown in Figure 7, the combination of anti-CD73 antibody and anti-PD-1 / CTLA-4 bispecific antibody showed superior tumor suppression compared to anti-CD73 antibody alone or anti-PD-1 / CTLA-4 bispecific antibody alone, and had no significant effect on mouse body weight (Figure 8).

[0088] [Table 3]

[0089] Murine colon cancer cells MC38-hPDL1 / hCD73 (provided by Jiangsu Jiaxing Pharmaceuticals Co., Ltd.) were resuscitated to the Pn+3 generation. MC38-hPDL1 / hCD73 cells in the logarithmic growth phase were harvested, the culture medium was removed, and the cells were washed twice with PBS. Then, they were inoculated into C57BL / 6-hPD1 / hPDL1 / hCD73 mice (provided by Jiangsu Jiaxing Pharmaceuticals Co., Ltd.) (the viability of MC38-hPDL1 / hCD73 cells before and after tumor formation was 99.1% and 96.4%, respectively). The inoculum size was 2 x 10 6 The injection site was the right front paw of each mouse. Five days after injection, the average tumor volume was 86.02 mm. 3When tumor volume reached 1000, the 32 mice were randomly divided into 4 groups based on tumor volume, with 8 mice per group. The day of grouping was designated Day 0, and administration began on Day 0 according to Table 3. After cell inoculation, the effects of tumors on the animals' normal behavior were monitored weekly. The administration days were Day 0, Day 3, Day 7, Day 10, Day 14, Day 17, and Day 22. Tumor size was observed and mice were weighed on Day 0, Day 3, Day 6, Day 10, Day 13, Day 17, Day 20, and Day 23. After the experiment was completed on Day 23, tumor tissue from each group was excised and weighed. Experimental results, including the rate of change in mouse weight and tumor weight, are expressed as mean ± standard error (Mean ± SEM). An independent sample T was used to verify whether there was a significant difference between the different treatment groups and the control group; a P < 0.05 was considered significant. Data were plotted using Graphpad or Excel.

[0090] Example 6: Detection of CD73 enzyme activity suppressed by anti-CD73 antibody The experimental steps were as follows: MDA-MB-231 cells in good logarithmic phase (derived from ATCC, HTB-26) were resuspended in serum-free RPMI-1640 medium and counted. MDA-MB-231 cells were plated in a 96-well plate (3 x 10 4Cells were seeded at 100 μL / well. Antibodies were diluted in serum-free RPMI-1640 medium to a starting concentration of 200 μg / mL, and then diluted 2.5-fold. The antibodies were added to a 96-well plate, 50 μL per well, and incubated at 37°C for 1 hour. After 1 hour, 50 μL of 600 μM AMP diluted in RPMI-1640 was added per well. After 3 hours, 25 μL of the cell culture supernatant was transferred to a new 96-well plate, and 25 μL of 100 μM ATP and 50 μL of CTG (CellTiter-Glo® One Solution Assay, Promega, Cat. G8461) color development solution were added per well. The color was developed, and the data were read using a multilabel microwell plate detector (PerkinElmer, Cat. No. 2140-0020). An isotype control antibody and a CD73-specific inhibitor APCP served as negative and positive controls, respectively. As shown in Figure 9, 19F3, 19F3H2L3(hG1DM), 19F3H2L3(hG1DM), and 19F3H2L3(hG1DM) dose-dependently inhibited the catalytic activity of endogenously expressed CD73 enzyme in MDA-MB-231 cells to convert AMP to adenosine A, thereby dose-dependently reducing the production of mean fluorescence intensity (RLU). These experimental results indicate that, in the absence of CD73 antibody treatment, added AMP is catalyzed by the CD73 enzyme endogenously expressed on the cell surface of MDA-MB-231 cells, converting it to adenosine and relieving the inhibition of luciferin enzyme activity. However, after antibody addition, CD73 is bound by the antibody, reducing its catalytic activity and preventing the conversion of AMP to adenosine. This suggests that anti-CD73 antibodies effectively inhibit the CD73 enzyme activity in a non-substrate competitive manner, reducing the production of adenosine.

Claims

1. 1. An anti-CD73 (e.g., human CD73) antibody or antigen-binding fragment thereof, wherein, according to the IMGT numbering system, the anti-CD73 antibody is comprising HCDR1, HCDR2 and HCDR3 of a heavy chain variable region as set forth in SEQ ID NO:2, SEQ ID NO:6 or SEQ ID NO:10, and LCDR1, LCDR2 and LCDR3 of a light chain variable region as set forth in SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:12 or SEQ ID NO:14; Preferably, according to the IMGT numbering system, said anti-CD73 antibody is HCDR1 comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 15, a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, or an amino acid sequence having one or more (preferably one, two or three) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to said sequence; HCDR2 comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 16, a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, or an amino acid sequence having one or more (preferably one, two or three) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to said sequence; an HCDR3 comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 17, a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, or an amino acid sequence having one or more (preferably one, two or three) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to said sequence; an LCDR1 comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 18, a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, or an amino acid sequence having one or more (preferably one, two or three) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to said sequence; an LCDR2 comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 19, a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, or an amino acid sequence having one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to said sequence; and an LCDR3 comprising or consisting of an amino acid sequence as set forth in SEQ ID NO:20, a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, or an amino acid sequence having one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to said sequence, An anti-CD73 (e.g., human CD73) antibody or an antigen-binding fragment thereof.

2. The heavy chain variable region of the antibody comprises comprising or consisting of SEQ ID NO:2, SEQ ID NO:6 or SEQ ID NO:10, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:2, SEQ ID NO:6 or SEQ ID NO:10, or an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the amino acid sequence shown in SEQ ID NO:2, SEQ ID NO:6 or SEQ ID NO:10, and The light chain variable region of the antibody comprises or comprising or consisting of an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the amino acid sequence shown in SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:12 or SEQ ID NO:14, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, and preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:12 or SEQ ID NO:14, 2. The anti-CD73 antibody or antigen-binding fragment thereof of claim 1.

3. The amino acid sequence of the heavy chain variable region of the antibody is set forth in SEQ ID NO: 2, and the amino acid sequence of the light chain variable region of the antibody is set forth in SEQ ID NO: 4; The amino acid sequence of the heavy chain variable region of the antibody is set forth in SEQ ID NO:6, and the amino acid sequence of the light chain variable region of the antibody is set forth in SEQ ID NO:8; the amino acid sequence of the heavy chain variable region of the antibody is set forth in SEQ ID NO: 10, and the amino acid sequence of the light chain variable region of the antibody is set forth in SEQ ID NO: 12; or The amino acid sequence of the heavy chain variable region of the antibody is set forth in SEQ ID NO: 10, and the amino acid sequence of the light chain variable region of the antibody is set forth in SEQ ID NO:

14.

3. An anti-CD73 antibody or an antigen-binding fragment thereof according to claim 1 or 2.

4. the heavy chain constant region of the antibody is an Ig gamma-1 chain C region, ACCESSION: P01857, and the light chain constant region is an Ig kappa chain C region, ACCESSION: P01834; Preferably, the heavy chain constant region of the antibody is represented by the sequence SEQ ID NO: 21, and the light chain constant region of the antibody is represented by the sequence SEQ ID NO:

22.

3. An anti-CD73 antibody or an antigen-binding fragment thereof according to claim 1 or 2.

5. The antibody is a monoclonal antibody, a humanized antibody, a chimeric antibody, a multispecific antibody (e.g., a bispecific antibody), 3. An anti-CD73 antibody or an antigen-binding fragment thereof according to claim 1 or 2.

6. The antigen-binding fragment may be Fab, Fab', F(ab') ２ , Fd, Fv, dAb, Fab / c, complementarity determining region fragment, single chain antibody (e.g., scFv), humanized antibody, chimeric antibody or bispecific antibody, 3. An anti-CD73 antibody or an antigen-binding fragment thereof according to claim 1 or 2.

7. below, (1) An isolated polypeptide comprising a sequence as set forth in SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17, wherein said polypeptide specifically binds to CD73 as part of an anti-CD73 antibody, said antibody further comprising a sequence as set forth in SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20; (2) An isolated polypeptide comprising a sequence as set forth in SEQ ID NO:18, SEQ ID NO:19, or SEQ ID NO:20, wherein said polypeptide specifically binds to CD73 as part of an anti-CD73 antibody, said antibody further comprising a sequence as set forth in SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17; (3) An isolated polypeptide comprising an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) conservative amino acid mutations (preferably substitutions, insertions, or deletions) compared to the sequence set forth in SEQ ID NO:2, SEQ ID NO:6, or SEQ ID NO:10, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, wherein the polypeptide is a portion of an anti-CD73 antibody. an isolated polypeptide which specifically binds to CD73 via a nucleotide sequence selected from the group consisting of nucleotides 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10; (4) An isolated polypeptide comprising an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the sequence set forth in SEQ ID NO: 4, SEQ ID NO: 8 or SEQ ID NO: 12, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, wherein the polypeptide is a portion of an anti-CD73 antibody. an isolated polypeptide which specifically binds to CD73 via a nucleotide sequence selected from the group consisting of nucleotides 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10, wherein the antibody further correspondingly comprises a sequence as set forth in SEQ ID NO:2, SEQ ID NO:6 or SEQ ID NO:10, respectively, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, or an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to said sequence; (5) An isolated polypeptide comprising an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) conservative amino acid mutations (preferably substitutions, insertions, or deletions) compared to the sequence set forth in SEQ ID NO: 10, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, wherein the polypeptide is a part of an anti-CD73 antibody. an isolated polypeptide which specifically binds to CD73, said antibody further correspondingly comprising the sequence set forth in SEQ ID NO: 14, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%,: 86%, 87%, 88%, 89%, 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, or an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to said sequence; and (6) An isolated polypeptide comprising a sequence as set forth in SEQ ID NO: 14, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to said sequence, or an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to said sequence, wherein said polypeptide is a portion of an anti-CD73 antibody. 10, wherein the antibody specifically binds to CD73 as set forth in SEQ ID NO: 11, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85% 86%, 87%, 88%, 89%, 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, or an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to said sequence. An isolated polypeptide selected from the group consisting of:

8. A nucleic acid sequence encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, or the isolated polypeptide according to claim 7. Nucleic acid molecule.

9. The nucleic acid molecule according to claim 8, vector.

10. 9. A nucleic acid molecule according to claim 8, or a vector according to claim 9. host cell.

11. A conjugate comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 6 and a conjugate moiety, wherein the conjugate moiety is a purification tag (e.g. His tag), a detectable label, preferably a radioisotope, a fluorescent substance, a chemiluminescent substance, a colored substance, polyethylene glycol or an enzyme. Conjugate.

12. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 6 is included. A fusion protein or a multispecific antibody (preferably a bispecific antibody).

13. A kit comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, a conjugate according to claim 11 or a fusion protein or a multispecific antibody according to claim 12, preferably said kit further comprising a second antibody which specifically recognises said antibody, optionally said second antibody further comprising a detectable label, such as a radioisotope, a fluorescent substance, a chemiluminescent substance, a coloured substance or an enzyme, preferably said kit being used for detecting the presence or level of CD73 in a sample, Preferably, the kit further comprises an anti-PD-1 / CTLA-4 bispecific antibody and, optionally, instructions for use. Preferably, the anti-PD-1 / CTLA-4 bispecific antibody comprises the following CDRs: HCDR1 comprising or consisting of the sequence shown in SEQ ID NO: 23; HCDR2 comprising or consisting of the sequence set forth in SEQ ID NO: 24; HCDR3 comprising or consisting of the sequence set forth in SEQ ID NO: 25; HCDR4 comprising or consisting of the sequence shown in SEQ ID NO: 26; HCDR5 comprising or consisting of the sequence set forth in SEQ ID NO: 27; HCDR6 comprising or consisting of the sequence shown in SEQ ID NO: 28; HCDR7 comprising or consisting of the sequence set forth in SEQ ID NO: 29; HCDR8 comprising or consisting of the sequence set forth in SEQ ID NO: 30; HCDR9 comprising or consisting of the sequence set forth in SEQ ID NO: 31; LCDR1 comprising or consisting of the sequence shown in SEQ ID NO: 32; LCDR2 comprising or consisting of the sequence shown in SEQ ID NO: 33; LCDR3 comprising or consisting of the sequence shown in SEQ ID NO: 34; More preferably, the anti-PD-1 / CTLA-4 bispecific antibody comprises a heavy chain amino acid sequence set forth in SEQ ID NO:35 and a light chain amino acid sequence set forth in SEQ ID NO:

36. kit.

14. A pharmaceutical composition comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, a conjugate according to claim 11 or a fusion protein or a multispecific antibody according to claim 12, optionally said pharmaceutical composition further comprising a pharma- ceutically acceptable carrier and / or excipient, preferably said pharmaceutical composition being in a form suitable for administration by subcutaneous, intradermal, intravenous, intramuscular or intralesional injection, Preferably, the pharmaceutical composition further comprises an anti-PD-1 / CTLA-4 bispecific antibody, Preferably, the anti-PD-1 / CTLA-4 bispecific antibody comprises the following CDRs: HCDR1 comprising or consisting of the sequence shown in SEQ ID NO: 23; HCDR2 comprising or consisting of the sequence set forth in SEQ ID NO: 24; HCDR3 comprising or consisting of the sequence set forth in SEQ ID NO: 25; HCDR4 comprising or consisting of the sequence shown in SEQ ID NO: 26; HCDR5 comprising or consisting of the sequence set forth in SEQ ID NO: 27; HCDR6 comprising or consisting of the sequence shown in SEQ ID NO: 28; HCDR7 comprising or consisting of the sequence set forth in SEQ ID NO: 29; HCDR8 comprising or consisting of the sequence set forth in SEQ ID NO: 30; HCDR9 comprising or consisting of the sequence set forth in SEQ ID NO: 31; LCDR1 comprising or consisting of the sequence shown in SEQ ID NO: 32; LCDR2 comprising or consisting of the sequence shown in SEQ ID NO: 33; LCDR3 comprising or consisting of the sequence shown in SEQ ID NO: 34; More preferably, the anti-PD-1 / CTLA-4 bispecific antibody comprises a heavy chain amino acid sequence set forth in SEQ ID NO:35 and a light chain amino acid sequence set forth in SEQ ID NO:

36. Pharmaceutical compositions.

15. In the manufacture of a drug for treating and / or preventing a tumor, or in the manufacture of a drug for use in diagnosing a tumor, Use of an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 6, a conjugate according to claim 11 or a fusion protein or a multispecific antibody according to claim 12, Preferably, the tumor is a solid tumor, preferably non-small cell lung cancer, prostate cancer (including metastatic castration-resistant prostate cancer (mCRPC)), triple-negative breast cancer, ovarian cancer, colorectal cancer (including microsatellite stable (MSS) and mismatch repair deficient / microsatellite unstable (dMMR / MSI-high)), gastric cancer (including microsatellite stable (MSS) and mismatch repair deficient / microsatellite unstable (dMMR / MSI-high)), melanoma, head and neck cancer, renal cell carcinoma or pancreatic ductal adenocarcinoma; use.

16. LT014, which has been deposited at the China Typical Culture Depository Center (CCTCC) and has the deposit number CCTCC NO: C2018137; Hybridoma cell lines.

17. A drug for treating and / or preventing a tumor, comprising drug A and drug B, wherein drug A comprises the antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, the conjugate according to claim 11, or the fusion protein or multispecific antibody according to claim 12, and drug B comprises an anti-PD-1 / CTLA-4 bispecific antibody; preferably, drug A and drug B are administered simultaneously or sequentially, wherein the sequential administration refers to administering drug A first or drug B first; Preferably, the anti-PD-1 / CTLA-4 bispecific antibody comprises the following CDRs: HCDR1 comprising or consisting of the sequence shown in SEQ ID NO: 23; HCDR2 comprising or consisting of the sequence set forth in SEQ ID NO: 24; HCDR3 comprising or consisting of the sequence set forth in SEQ ID NO: 25; HCDR4 comprising or consisting of the sequence shown in SEQ ID NO: 26; HCDR5 comprising or consisting of the sequence set forth in SEQ ID NO: 27; HCDR6 comprising or consisting of the sequence shown in SEQ ID NO: 28; HCDR7 comprising or consisting of the sequence set forth in SEQ ID NO: 29; HCDR8 comprising or consisting of the sequence set forth in SEQ ID NO: 30; HCDR9 comprising or consisting of the sequence set forth in SEQ ID NO: 31; LCDR1 comprising or consisting of the sequence shown in SEQ ID NO: 32; LCDR2 comprising or consisting of the sequence shown in SEQ ID NO: 33; LCDR3 comprising or consisting of the sequence shown in SEQ ID NO: 34; More preferably, the heavy chain amino acid sequence of the anti-PD-1 / CTLA-4 bispecific antibody is set forth in SEQ ID NO:35 and the light chain amino acid sequence is set forth in SEQ ID NO:36; Preferably, the tumor is a solid tumor, preferably non-small cell lung cancer, prostate cancer (including metastatic castration-resistant prostate cancer (mCRPC)), triple-negative breast cancer, ovarian cancer, colorectal cancer (including microsatellite stable (MSS) and mismatch repair deficient / microsatellite unstable (dMMR / MSI-high)), gastric cancer (including microsatellite stable (MSS) and mismatch repair deficient / microsatellite unstable (dMMR / MSI-high)), melanoma, head and neck cancer, renal cell carcinoma or pancreatic ductal adenocarcinoma; Drugs.

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