Antibodies against CD73 and their uses

Abstract

The present invention relates to anti-CD73 antibodies and their applications. Specifically, the heavy chain variable region of the antibody comprises HCDR1-HCDR3 having amino acid sequences as shown in SEQ ID NOs: 15-17; and the light chain variable region of the antibody comprises LCDR1-LCDR3 having amino acid sequences as shown in SEQ ID NOs: 18-20.

Description

Technical Field

[0001] The present invention relates to the field of immunology. Specifically, it relates to anti-CD73 antibodies and their uses. Background Art

[0002] Ecto-5'-nucleotidase, namely CD73 protein, is a multifunctional glycoprotein with a molecular weight of 70KD encoded by the NT5E gene, which is anchored to the cell membrane through glyocsyl phosphatidylinositol (GPI) (Zimmermann H. Biochem J. 1992; 285: 345–365).

[0003] CD73 is widely distributed on the surface of human tissue cells. Current studies have found that CD73 is highly expressed in a variety of solid tumors, such as cancer cells, dendritic cells, regulatory T cells (Treg), natural killer cells (NK cells), myeloid-derived suppressor cells (MDSC), tumor-associated macrophages (TAM), etc. in the tumor microenvironment. Hypoxia induces the upregulation of molecules such as hypoxia-inducible factor-1 (HIF-1), which in turn leads to the widespread expression of CD73 in the tumor microenvironment (Synnestvedt K, et al. J Clin Invest. 2002; 110: 993–1002.). Analysis of clinical tumor samples shows that high expression of CD73 is a potential biomarker and is closely related to the poor prognosis of various types of tumors, including breast cancer, lung cancer, ovarian cancer, kidney cancer, gastric cancer, head and neck cancer, etc.

[0004] CD73 has both hydrolase activity and non-hydrolytic enzyme functions. The enzymatic and non-enzymatic functions of CD73 coexist in the relevant processes of tumors and promote each other to maintain tumor progression. More and more studies have found that CD73 is a key regulatory molecule for tumor cell proliferation, metastasis and invasion in vitro, and tumor angiogenesis and tumor immune escape mechanisms in vivo. One of the important immunosuppressive mechanisms is mediated by the CD73-adenosine metabolic signaling pathway. CD39 upstream of CD73 can catalyze ATP to produce adenosine monophosphate (AMP), and the produced AMP is converted to adenosine by CD73. Adenosine then binds to the downstream adenosine receptor (A2AR). A2AR inhibits a series of immune activation-related signaling pathways such as LCK, MAPK, and PKC by activating protein kinase A (PKA) and Csk kinase, and inhibits the immune killing effect of T cells, thereby playing an immunosuppressive role (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 in activated T cells, B cells, and myeloid cells. The ligands of PD-1, 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. Among them, PDL1 is expressed in a variety of cells, including T cells, B cells, endothelial cells, and epithelial cells, while PDL2 is only expressed in 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 escape. The expression of PD-1 is mainly in immune cells such as T cells, while the ligand of PD-1, PDL1, is highly expressed in many human tumor tissues. Blocking the PD-1 / PDL1 signaling pathway can activate inhibited T cells, which then attack cancer cells. Blocking the PD-1 / PDL1 signal can promote the proliferation of tumor antigen-specific T cells, play a role in killing tumor cells, and thus inhibit local tumor growth (Julie R et al., 2012, N Engl J Med. 366:2455–2465). In addition, tumors with high expression of PDL1 are accompanied by cancers that are difficult to detect (Hamanishi et al., 2007, Proc. Natl. Acad. Sci. USA 104:3360-5). An effective method is to regulate the expression of PD-1 by in vivo injection of anti-PD-1 antibodies. Due to the broad-spectrum anti-tumor prospect and amazing efficacy of PD-1 antibodies, the industry generally believes that antibodies targeting the PD-1 pathway will bring breakthrough progress in the treatment of various tumors: for the treatment of non-small cell lung cancer, renal cell carcinoma, ovarian cancer, melanoma (Homet M.B., 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).

[0007] Cytotoxic T lymphocyte-associated antigen-4 (cytotoxic T lymphocyte sociated antigen4, also simply referred to as CTLA4) and the CD28 molecule have a very similar relationship in terms of gene structure, chromosomal localization, sequence homology, and gene expression. They are both co-stimulatory molecule B7 receptors and are mainly expressed on the surface of activated T cells. After CTLA4 binds to B7, it can inhibit the activation of mouse and human T cells and plays a negative regulatory role in T cell activation.

[0008] CTLA4 antibodies (or anti-CTLA4 monoclonal antibodies) or CTLA4 ligands can prevent CTLA4 from binding to its natural ligand, thereby blocking the transmission of negative regulatory signals of CTLA4 to T cells and enhancing the reactivity of T cells to various antigens. In this regard, the in vivo and in vitro research results are basically the same. Currently, there are CTLA4 monoclonal antibodies in the clinical trial stage or approved for the treatment of prostate cancer, bladder cancer, colorectal cancer, gastrointestinal cancer, liver cancer, malignant melanoma, etc. (Grosso JF., Jure-Kunkel MN., 2013, Cancer Immun. 13:5.).

[0009] As important influencing factors of T cell function status, CTLA4 and CTLA4 antibodies play a role by intervening in the body's immune microenvironment. In in vitro and in vivo experiments, CTLA4 antibodies can specifically relieve the immune suppression of CTLA4 on the body, activate T cells, and induce the production of IL-2, showing broad application prospects in gene therapy for diseases such as anti-tumor and anti-parasite. CTLA4 antibodies can have a specific therapeutic effect on diseases and exert high efficacy, supplementing the deficiencies of traditional medications, thus opening up a new way for gene therapy.

[0010] ADCC (antibody-dependent cell-mediated cytotoxicity) refers to the antibody-dependent cell-mediated cytotoxic effect, which means that the Fab segment of the antibody binds to the antigen epitope of virus-infected cells or tumor cells, and its Fc segment binds to the Fc receptor (Fc Receptor, FcR) on the surface of killer cells (NK cells, macrophages, etc.), mediating the direct killing of target cells by killer cells.

[0011] CDC (complement dependent cytotoxicity) refers to the complement-dependent cytotoxicity, which means that when the antibody specifically binds to the corresponding antigen on the cell membrane surface, a complex is formed to activate the complement system, and then MAC is formed on the surface of the target cell, resulting in the lysis of the target cell. Complement can cause the lysis of a variety of bacteria and other pathogenic biological cells and is an important defense mechanism for the body to resist pathogenic biological infections.

[0012] Fc receptors are immunoglobulin family proteins expressed on the surface of specific immune cells and used to recognize the Fc region of antibodies to mediate immune responses. After the Fab region of an antibody recognizes an antigen, the Fc region of the antibody binds to the Fc receptor on immune cells (such as killer cells), initiating the response function of immune cells, such as phagocytosis and ADCC. According to the different types of antibodies recognized by Fc receptors and the expressing cells, it is found that FcγRIIIa is closely related to the ADCC effect. FcγRIIIa is the most important molecule mediating ADCC (Hogarth PM, Pietersz GA. 2012, NATURE REVIEWS DRUG DISCOVERY, 11(4):311-331).

[0013] The IgG family contains four members, IgG1, IgG2, IgG3, and IgG4. There are amino acid differences in the crystallizable fragment (Fc) region of their heavy chain constant regions, resulting in different affinities for FcγRs. IgG1 is the most abundant subtype in the human body and is also the most used subtype in monoclonal antibody drugs. IgG1 can bind various FcγRs and can trigger ADCC and CDC effects. The presence of ADCC and / or CDC in antibodies may cause unnecessary targeted tissue damage and have a negative impact on the pharmacology of the drug. Summary of the Invention

[0014] Through in-depth research and creative work, the present inventors used a mammalian cell expression system to express recombinant human CD73 as an antigen to immunize mice, and obtained hybridoma cells by fusing mouse spleen cells with myeloma cells. Through the screening of a large number of samples, the inventors obtained the hybridoma cell line LT014 (deposit number: CCTCC NO: C2018137).

[0015] The present inventors surprisingly found that the hybridoma cell line LT014 can secrete a specific monoclonal antibody (named 19F3) that specifically binds to human CD73, and this monoclonal antibody can very effectively inhibit the enzymatic activity reaction of CD73 in a non-substrate competitive manner, reduce the production of adenosine, promote T cell activity, and exert an effect of inhibiting tumor growth.

[0016] Furthermore, the present inventors creatively prepared humanized antibodies against human CD73 (named 19F3H1L1 (hG1DM), 19F3H2L2 (hG1DM), 19F3H2L3, and 19F3H2L3 (hG1DM)). In addition, amino acid mutations were introduced into the anti-CD73 antibodies to eliminate ADCC and CDC effects, avoiding unnecessary toxic effects mediated by the antibodies.

[0017] The inventor also surprisingly found that the antibody of the present invention in combination with an anti-PD-1 / CTLA-4 bispecific antibody has a pharmacological effect of effectively inhibiting the proliferation of tumor cells, which is superior to that of the anti-PD-1 / CTLA-4 bispecific antibody alone or the anti-CD73 antibody alone.

[0018] Another aspect of the present invention also relates to an antibody, wherein the anti-CD73 antibody comprises:

[0019] HCDR1, HCDR2 and HCDR3 of the heavy chain variable region shown in SEQ ID NO:2, SEQ ID NO:6 or SEQ ID NO:10; and LCDR1, LCDR2 and LCDR3 of the light chain variable region shown in SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:12 or SEQ ID NO:14;

[0020] Preferably, according to the IMGT numbering system, the anti-CD73 antibody comprises

[0021] HCDR1, which comprises the amino acid sequence shown 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 with the 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 with the sequence, or consisting of the same,

[0022] HCDR2, which comprises the amino acid sequence shown 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 with the 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 with the sequence, or consisting of the same,

[0023] HCDR3, which comprises the amino acid sequence shown in SEQ ID NO:17, and sequences 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 with said sequence, or amino acid sequences having one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared with said sequence, or consisting thereof,

[0024] LCDR1, which comprises the amino acid sequence shown in SEQ ID NO:18, and sequences 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 with said sequence, or amino acid sequences having one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared with said sequence, or consisting thereof,

[0025] LCDR2, which comprises the amino acid sequence shown in SEQ ID NO:19, and sequences 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 with said sequence, or amino acid sequences having one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared with said sequence, or consisting thereof, and

[0026] LCDR3, which comprises the amino acid sequence shown in SEQ ID NO:20, and sequences 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 with said sequence, or amino acid sequences having one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared with said sequence, or consisting thereof.

[0027] In some embodiments of the present invention,

[0028] the heavy chain variable region of the antibody comprises the following sequence, or consists of the following sequence:

[0029] SEQ ID NO:2, SEQ ID NO:6 or SEQ ID NO:10, 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 with 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

[0030] The light chain variable region of the antibody comprises the following sequence, or consists of the following sequence:

[0031] SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:12 or SEQ ID NO:14, 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 with SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:12 or SEQ ID NO:14, 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:4, SEQ ID NO:8, SEQ ID NO:12 or SEQ ID NO:14.

[0032] In some embodiments of the invention, 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;

[0033] 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;

[0034] The amino acid sequence of the heavy chain variable region of the antibody is as shown in SEQ ID NO:10, and the amino acid sequence of the light chain variable region of the antibody is as shown in SEQ ID NO:12; or

[0035] The amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO: 10, and the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO: 14.

[0036] In some embodiments of the present invention, the heavy chain constant region of the antibody is the C region of the Ig gamma-1 chain, ACCESSION: P01857; the light chain constant region is the C region of the Ig kappa chain, ACCESSION: P01834.

[0037] In some embodiments of the present invention, the heavy chain constant region of the antibody is based on the C region of the Ig gamma-1 chain, ACCESSION: P01857, with a point mutation of leucine to alanine (L234A) introduced at position 234 and a point mutation of leucine to alanine (L235A) introduced at position 235; the light chain constant region is the C region of the Ig kappa chain, ACCESSION: P01834, and the amino acid sequence is shown in SEQ ID NO: 22.

[0038] The variable regions of the light and heavy chains determine antigen binding; each variable region of the chain contains three hypervariable regions, called complementarity-determining regions (CDRs) (the CDRs of the heavy chain (H) include HCDR1, HCDR2, HCDR3, and the CDRs of the light chain (L) include LCDR1, LCDR2, LCDR3; they were named by Kabat et al., see Bethesda M.d., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 1991; 1-3: 91-3242).

[0039] Preferably, the CDRs can also be 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.

[0040] By means well known to those skilled in the art, for example, the amino acid sequences of the CDR regions of monoclonal antibody sequences are analyzed according to the IMGT definition through the VBASE2 database.

[0041] The antibodies 19F3, 19F3H1L1 (hG1DM), 19F3H2L2 (hG1DM), and 19F3H2L3 (hG1DM) involved in the present invention have the same CDRs:

[0042] The amino acid sequences of the three CDR regions of their heavy chain variable regions are as follows:

[0043] HCDR1: GYSFTGYT (SEQ ID NO: 15),

[0044] HCDR2: INPYNAGT (SEQ ID NO: 16),

[0045] HCDR3: ARSEYRYGGDYFDY (SEQ ID NO: 17);

[0046] The amino acid sequences of the three CDR regions of their light chain variable regions are as follows:

[0047] LCDR1: QSLLNSSNQKNY (SEQ ID NO: 18),

[0048] LCDR2: FAS (SEQ ID NO: 19),

[0049] LCDR3: QQHYDTPYT (SEQ ID NO: 20).

[0050] In some embodiments of the present invention, the antibody is a monoclonal antibody.

[0051] In some embodiments of the present invention, the antibody is a humanized antibody, a chimeric antibody, or a multispecific antibody (such as a bispecific antibody).

[0052] In some embodiments of the present invention, the antigen-binding fragment is selected from 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.

[0053] Another aspect of the present invention relates to an isolated polypeptide selected from the group consisting of:

[0054] (1) An isolated polypeptide comprising the sequences set forth in SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17, wherein said polypeptide specifically binds CD73 as part of an anti-CD73 antibody, and said antibody further comprises the sequences set forth in SEQ ID NO:18; SEQ ID NO:19; SEQ ID NO:20.

[0055] (2) An isolated polypeptide comprising the sequences set forth in SEQ ID NO:18; SEQ ID NO:19; SEQ ID NO:20, wherein said polypeptide specifically binds CD73 as part of an anti-CD73 antibody, and said antibody further comprises the sequences set forth in SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17.

[0056] (3) An isolated polypeptide comprising the sequences 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, 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 specifically binds CD73 as part of an anti-CD73 antibody, and said antibody further comprises the sequences 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 to said sequence.

[0057] (4) An isolated polypeptide comprising the sequence shown 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 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 specifically binds CD73 as part of an anti-CD73 antibody, and said antibody further comprises the sequence shown 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 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.

[0058] (5) An isolated polypeptide comprising the sequence shown 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 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 specifically binds CD73 as part of an anti-CD73 antibody, and said antibody further comprises the sequence shown in SEQ ID NO:14 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 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.

[0059] (6) An isolated polypeptide comprising the sequence shown 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 specifically binds CD73 as part of an anti-CD73 antibody, and said antibody further comprises the sequence shown 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 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.

[0060] Another aspect of the present invention relates to an isolated nucleic acid molecule encoding the antibody or an antigen-binding fragment thereof according to any one of the present invention, or said isolated polypeptide.

[0061] Yet another aspect of the present invention relates to a vector comprising the isolated nucleic acid molecule of the present invention.

[0062] Yet another aspect of the present invention relates to a host cell comprising the isolated nucleic acid molecule of the present invention, or the vector of the present invention.

[0063] Yet another aspect of the present invention relates to a conjugate comprising an antibody and a conjugate moiety, wherein said antibody is the antibody or an antigen-binding fragment thereof according to any one of the present invention, and said conjugate moiety is a purification tag (such as a His tag), a detectable label; preferably, said conjugate moiety is a radioisotope, a fluorescent substance, a chemiluminescent substance, a colored substance, polyethylene glycol or an enzyme.

[0064] Yet another aspect of the present invention relates to a fusion protein or a multispecific antibody (preferably a bispecific antibody) comprising the antibody or an antigen-binding fragment thereof according to any one of the present invention.

[0065] Another aspect of the present invention relates to a kit, which comprises the antibody or its antigen-binding fragment according to any one of the present invention, the conjugate, fusion protein or multispecific antibody of the present invention; 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 chemiluminescent substance, a colored substance or an enzyme.

[0066] Another aspect of the present invention relates to the use of the antibody or its antigen-binding fragment according to any one of the present invention, the conjugate, fusion protein or multispecific antibody of the present invention in the preparation of a kit for detecting the presence or level of CD73 in a sample.

[0067] Another aspect of the present invention relates to a pharmaceutical composition, which comprises the antibody or its antigen-binding fragment according to any one of the present invention, the conjugate, fusion protein or multispecific antibody of the present invention; optionally, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and / or excipient. Preferably, the pharmaceutical composition is in a form suitable for administration by subcutaneous injection, intradermal injection, intravenous injection, intramuscular injection or intralesional injection.

[0068] Another aspect of the present invention relates to the use of the antibody or its antigen-binding fragment according to any one of the present invention, the conjugate, fusion protein or multispecific antibody of the present invention in the preparation of a drug for treating and / or preventing tumors (such as solid tumors, 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) types), gastric cancer (including microsatellite stable (MSS) and mismatch repair-deficient / microsatellite unstable (dMMR / MSI-high) types), melanoma, head and neck cancer, renal cell carcinoma or pancreatic ductal adenocarcinoma), or in the preparation of a drug for diagnosing tumors.

[0069] Another aspect of the present invention relates to the hybridoma cell line LT014, which is deposited with the China Center for Type Culture Collection (CCTCC), and the deposit number is CCTCC NO: C2018137.

[0070] Another aspect of the present invention relates to a kit, which comprises (1) the antibody or its antigen-binding fragment according to the present invention, the conjugate according to the present invention or the fusion protein or multispecific antibody according to the present invention, and (2) an anti-PD-1 / CTLA-4 bispecific antibody, and optionally, an instruction manual.

[0071] Another aspect of the present invention relates to a method for treating and / or preventing tumors, which comprises administering to a patient a therapeutically effective amount of drug A and a therapeutically effective amount of drug B, wherein drug A comprises the antibody or its antigen-binding fragment described in the present invention, the conjugate described in the present invention, the 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, and in the case of sequential administration, drug A or drug B is administered first.

[0072] In some embodiments of the present invention, the amino acid sequence of the heavy chain of the anti-PD-1 / CTLA-4 bispecific antibody is as shown in SEQ ID NO:35, and the amino acid sequence of the light chain is as shown in SEQ ID NO:36.

[0073] In the present invention, unless otherwise specified, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Moreover, the cell culture, molecular genetics, nucleic acid chemistry, and immunology laboratory procedures used herein are all conventional procedures widely used in the relevant fields. Meanwhile, for a better understanding of the present invention, the definitions and explanations of relevant terms are provided below.

[0074] As used herein, the term EC 50 refers to the concentration for 50% of maximal effect, which is the concentration that can cause 50% of the maximal effect.

[0075] As used herein, the term "antibody" refers to an immunoglobulin molecule that typically consists 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 κ and λ light chains. Heavy chains can be classified as μ, δ, γ, α, or ε, and the isotypes of antibodies are defined as IgM, IgD, IgG, IgA, and IgE, respectively. Within the light and heavy chains, the variable region and the constant region are connected by a "J" region of about 12 or more amino acids, and the heavy chain also contains a "D" region of about 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (V H ) and a 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 a light chain constant region (C L ). The light chain constant region consists of one domain C LComposition. The constant region of an antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (C1q). V H and V L regions can also be subdivided into regions of high variability (termed complementarity-determining regions (CDRs)), interspersed with more conserved regions called framework regions (FRs). Each V H and V L is composed of three CDRs and four FRs arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions (V H and V L ) of each heavy chain / light chain pair form the antigen-binding site respectively. The assignment of amino acids to each region or domain follows the definition of 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, 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. The term "antibody" is not restricted to any particular method of antibody production. For example, it includes, in particular, recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. Antibodies can be antibodies of different isotypes, e.g., IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM antibodies.

[0076] As used herein, the terms "monoclonal antibody" and "mAb" refer to an antibody or a fragment of an antibody from a group of highly homologous antibody molecules, i.e., a group of identical antibody molecules except for possible naturally occurring spontaneous mutations. A monoclonal antibody has high specificity for a single epitope on an antigen. Polyclonal antibodies, in contrast to monoclonal antibodies, generally contain at least two or more different antibodies that typically recognize different epitopes on an antigen. Monoclonal antibodies can generally be obtained using the hybridoma technology first reported by Kohler et al.( G. Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity[J]. nature, 1975; 256(5517): 495), but can also be obtained using recombinant DNA technology (see, e.g., U.S. Patent 4,816,567).

[0077] As used herein, the term "humanized antibody" refers to an antibody or antibody fragment in which all or part of the CDR regions of a human immunoglobulin (recipient antibody) have been replaced with the CDR regions of a non-human antibody (donor antibody), where the donor antibody can be a non-human (e.g., murine, rat, or rabbit) antibody having the desired specificity, affinity, or reactivity. In addition, some amino acid residues in the framework regions (FRs) of the recipient antibody can also be replaced with the amino acid residues of the corresponding non-human antibody or with the amino acid residues of other antibodies to further improve or optimize the properties of the antibody. For more details on humanized antibodies, see, e.g., 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.

[0078] As used herein, the term "isolated" or "separated" refers to being obtained by artificial means from its natural state. If a "separated" substance or component occurs in nature, it may be that its natural environment has changed, or the substance has been separated from its natural environment, or both. For example, a certain unseparated polynucleotide or polypeptide naturally exists in a living animal body, and the highly purified same polynucleotide or polypeptide separated from this natural state is called isolated. The term "isolated" or "separated" does not exclude the admixture of artificial or synthetic substances, nor does it exclude the presence of other impure substances that do not affect the activity of the substance.

[0079] As used herein, the term "vector" refers to a nucleic acid vehicle into which a polynucleotide can be inserted. When the vector enables the expression of the protein encoded by the inserted polynucleotide, the vector is called an expression vector. The vector can be introduced into a host cell by transformation, transduction or transfection, so that the genetic element it carries can be expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to: plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs) or P1-derived artificial chromosomes (PACs); phages such as λ phage or M13 phage and animal viruses, etc. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, polyomaviruses (such as SV40). A vector can contain various elements for controlling expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements and reporter genes. In addition, the vector can also contain an origin of replication.

[0080] As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, and it includes, but is 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 fibroblast cells, CHO cells, COS cells, NSO cells, HeLa cells, GS cells, BHK cells, HEK 293 cells or human cells.

[0081] 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 the antigen it targets. In certain embodiments, an antibody that specifically binds to an antigen (or an antibody specific for an antigen) means that the antibody binds with a dissociation constant of less than about 10 -5 M, for example less than about 10 -6 M, 10 -7 M, 10-8 M, 10 -9 M or 10 -10 An affinity of M or less (K D ) binds to the antigen.

[0082] As used herein, the term "K D " refers to the dissociation equilibrium constant of a specific 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, an antibody binds to an antigen (e.g., the PD-1 protein) with a dissociation equilibrium constant of less than about 10 -5 M, such as less than about 10 -6 M, 10 -7 M, 10 -8 M, 10 - 9 M or 10 -10 M or less dissociation equilibrium constant (K D ). K D can be measured by methods known to those skilled in the art, such as using a Fortebio molecular interaction instrument.

[0083] As used herein, the terms "monoclonal antibody" and "mAb" have the same meaning and are used interchangeably; the terms "polyclonal antibody" and "pAb" have the same meaning and are used interchangeably; the terms "polypeptide" and "protein" have the same meaning and are used interchangeably. And in the present invention, amino acids are generally represented by single-letter and three-letter abbreviations well-known in the art. For example, alanine can be represented by A or Ala.

[0084] 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 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 regulators, surfactants, adjuvants, ionic strength enhancers. For example, pH regulators include but are not limited to phosphate buffer; surfactants include but are not limited to cationic, anionic or non-ionic surfactants, such as Tween-80; ionic strength enhancers include but are not limited to sodium chloride.

[0085] As used herein, the term "effective amount" refers to an amount sufficient to achieve or at least partially achieve the desired effect. For example, an effective amount for preventing a disease (such as a tumor) refers to an amount sufficient to prevent, inhibit, or delay the occurrence of the disease (such as a tumor); 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 already suffering from the disease.

[0086] Advantages of the invention:

[0087] The monoclonal antibody of the present invention can specifically bind to CD73 well, and can very effectively inhibit the enzymatic activity of CD73 in a non-substrate competitive manner, reduce the production of adenosine, and promote T cell activity and tumor suppression effect. At the same time, the antibody of the present invention in combination with an anti-PD-1 / CTLA-4 bispecific antibody has a pharmacological effect of effectively inhibiting tumor cell proliferation, which is superior to the anti-PD-1 / CTLA-4 bispecific antibody alone or the anti-CD73 antibody alone. Brief description of the drawings

[0088] Figure 1. Fitting curve of the kinetic affinity data of 19F3H2L3 (hG1DM) binding to C1q.

[0089] Figure 2. Fitting curve of the kinetic affinity data of MEDI9447 binding to C1q.

[0090] Figure 3. Fitting curve of the kinetic affinity data of IgG1 wild-type antibody binding to C1q.

[0091] Figure 4. Fitting curve of the kinetic affinity data of 19F3H2L3 (hG1DM) binding to FcγRIIIa.

[0092] Figure 5. Fitting curve of the kinetic affinity data of MEDI9447 binding to FcγRIIIa.

[0093] Figure 6. Fitting curve of the kinetic affinity data of IgG1 wild-type antibody binding to FcγRIIIa.

[0094] Figure 7. Tumor weights of mice in each group on the 23rd day after grouping. **P<0.01.

[0095] Figure 8. Body weight change rate of each group of animals during the animal experiment, compared with Day 0.

[0096] Figure 9. The anti-CD73 antibody effectively inhibits the enzymatic activity of CD73. Detailed implementation manners

[0097] The embodiments of the present invention will be described in detail below in conjunction with examples. Those skilled in the art will understand that the following examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention. For those without specific technical or conditions noted in the examples, the techniques or conditions described in the literature in this field (for example, referring to "Molecular Cloning: A Laboratory Manual" by J. Sambrook et al., translated by Huang Peitang et al., Third Edition, Science Press) or according to the product specifications are followed. For reagents or instruments without the manufacturer noted, they are conventional products that can be obtained through market purchase.

[0098] In the following embodiments of the present invention, the BALB / c mice used were purchased from the Guangdong Provincial Medical Experimental Animal Center.

[0099] In the following embodiments of the present invention, the positive control antibody MEDI9447 (Oleclumab) used was produced by Zhongshan Kangfang Biopharmaceutical Co., Ltd., and its sequence is the same as the antibody sequence recorded in the International Nonproprietary Names for Pharmaceutical Substances (INN) publicly published by Medlmmune 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), P661 - 662.).

[0100] In the following embodiments of the present invention, the heavy chain constant region of the wild IgG1 control antibody used as the positive control antibody adopts Ig gamma - 1 chain C region, ACCESSION: P01857; the light chain constant region adopts Ig kappa chain C region, ACCESSION: P01834.

[0101] In the following embodiments of the present invention, the C1q used was purchased from Fitzgerald, catalog number: A16050201;

[0102] In the following embodiments of the present invention, the FcγRIIIa - bio used was purchased from Sino Biological, catalog number: LC09JA0407;

[0103] In the following embodiments of the present invention, the CD73 (5'-nuclease) specific inhibitor APCP (alpha, beta-methylene adenosine-5'-diphosphate) is from sigma, catalog number: M3763-10MG.

[0104] In the following embodiments of the present invention, the sequence of the isotype control antibody, human anti-Hen Egg Lysozyme IgG (i.e., anti-HEL antibody, or human IgG, abbreviated as hIgG, or isotype control), is from the variable region sequence of the Fab F10.6.6 sequence published by Acierno et al. in Affinity maturation increases the stability and plasticity of the Fv domain of anti-protein antibodies (Acierno et al. J Mol Biol. 2007; 374(1): 130-146.).

[0105] Example 1: Preparation of anti-CD73 antibody 19F3

[0106] 1. Preparation of hybridoma cell line LT014

[0107] The antigen used for preparing the anti-CD73 antibody is human NT5E-His (NT5E is Genbank ID: NP_002517.1, position: 1-552, prepared by Zhongshan Kangfang Biopharmaceutical Co., Ltd.). The spleen cells of immunized mice were fused with mouse myeloma cells to prepare hybridoma cells. Using human NT5E (NT5E is Genbank ID: NP_002517.1, position: 1-552)-Biotin (NT5E-Biotin, prepared by Zhongshan Kangfang Biopharmaceutical Co., Ltd.) as the antigen, the hybridoma cells were screened by indirect ELISA method to obtain hybridoma cells that can secrete antibodies specifically binding to CD73. The screened hybridoma cells were obtained as a stable hybridoma cell line by the limiting dilution method. The above hybridoma cell line was named hybridoma cell line LT014, and the monoclonal antibody secreted by it was named 19F3.

[0108] The hybridoma cell line LT014 (also known as CD73-19F3) was deposited with the China Center for Type Culture Collection (CCTCC) on June 21, 2018, with the deposit number CCTCC NO: C2018137, and the deposit address is Wuhan University, Wuhan, China, Zip Code: 430072.

[0109] 2. Preparation of Anti-CD73 Antibody 19F3

[0110] The LT014 cell line prepared above was cultured separately using CD medium (Chemical Defined Medium) (the CD medium contains 1% penicillin-streptomycin and was cultured in a 5% CO 2 , 37 °C cell culture incubator). After 7 days, the cell culture supernatant was collected, centrifuged at high speed, filtered by vacuum through a microporous filter membrane, and purified using a HiTrap protein A HP column to obtain antibody 19F3.

[0111] Example 2: Sequence Analysis of Anti-CD73 Antibody 19F3

[0112] mRNA was extracted from the LT014 cell line cultured in Example 1 according to the method of the Total RNA Extraction Kit for Cultured Cells Bacteria (Tiangen, product number DP430).

[0113] According to Invitrogen III First-Strand Synthesis System for RT-PCR kit instructions, cDNA was synthesized and PCR amplification was performed.

[0114] The PCR amplification product was directly subjected to TA cloning, and the specific operation was referred to the pEASY-T1 Cloning Kit (Transgen CT101) kit instructions.

[0115] The product of TA cloning was directly sequenced, and the sequencing results of the sequence analysis of anti-CD73 antibody 19F3 are as follows:

[0116] The nucleic acid sequence of the heavy chain variable region is shown in SEQ ID NO:1, with a length of 363 bp.

[0117] The encoded amino acid sequence is shown in SEQ ID NO:2, with a length of 121 aa;

[0118] 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.

[0119] The nucleic acid sequence of the light chain variable region is shown in SEQ ID NO:3, with a length of 339 bp.

[0120] The encoded amino acid sequence is shown in SEQ ID NO:4, with a length of 113 aa;

[0121] The sequence of the light chain CDR1 is shown in SEQ ID NO:18, the sequence of the light chain CDR2 is shown in SEQ ID NO:19, and the sequence of the light chain CDR3 is shown in SEQ ID NO:20.

[0122] Example 3: Design and Preparation of the Light Chain and Heavy Chain of a Humanized Antibody Against Human CD73

[0123] Based on the three-dimensional crystal structure of the 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, the antibody model was simulated by computer, and mutations were designed according to the model to obtain the variable region sequences of 19F3H1L1 (hG1DM), 19F3H2L2 (hG1DM), and 19F3H2L3 (hG1DM);

[0124] The designed variable region sequences of the above humanized antibodies are as follows:

[0125] (1) Sequences of the heavy chain variable region and light chain variable region of the humanized monoclonal antibody 19F3H1L1 (hG1DM)

[0126] The nucleic acid sequence of the heavy chain variable region is shown in SEQ ID NO:5, with a length of 363bp.

[0127] The encoded amino acid sequence is shown in SEQ ID NO:6, with a length of 121aa. Among them, the sequence of the heavy chain CDR1 is shown in SEQ ID NO:15, the sequence of the heavy chain CDR2 is shown in SEQ ID NO:16, and the sequence of the heavy chain CDR3 is shown in SEQ ID NO:17.

[0128] The nucleic acid sequence of the light chain variable region is shown in SEQ ID NO:7, with a length of 339bp.

[0129] The encoded amino acid sequence is shown in SEQ ID NO:8, with a length of 113aa. Among them, the sequence of the light chain CDR1 is shown in SEQ ID NO:18, the sequence of the light chain CDR2 is shown in SEQ ID NO:19, and the sequence of the light chain CDR3 is shown in SEQ ID NO:20.

[0130] (2) Nucleotide sequences of the heavy chain variable region and the light chain variable region of the humanized monoclonal antibody 19F3H2L2 (hG1DM)

[0131] The nucleotide sequence of the heavy chain variable region is shown in SEQ ID NO:9, with a length of 363bp.

[0132] The amino acid sequence encoded by it is shown in SEQ ID NO:10, with a length of 121aa. 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.

[0133] The nucleotide sequence of the light chain variable region is shown in SEQ ID NO:11, with a length of 339bp.

[0134] The amino acid sequence encoded by it is shown in SEQ ID NO:12, with a length of 113aa. 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.

[0135] (3) Nucleotide sequences of the heavy chain variable region and the light chain variable region of the humanized monoclonal antibody 19F3H2L3 (hG1DM)

[0136] The nucleotide sequence of the heavy chain variable region is shown in SEQ ID NO:9, with a length of 363bp.

[0137] The amino acid sequence encoded by it is shown in SEQ ID NO:10, with a length of 121aa. 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.

[0138] The nucleotide sequence of the light chain variable region is shown in SEQ ID NO:13, with a length of 339bp.

[0139] The amino acid sequence encoded by it is shown in SEQ ID NO:14, with a length of 113aa. 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.

[0140] 3. Preparation of humanized 19F3H1L1 (hG1DM), 19F3H2L2 (hG1DM) and 19F3H2L3 (hG1DM)

[0141] The light chain constant regions of the antibodies 19F3H1L1 (hG1DM), 19F3H2L2 (hG1DM), and 19F3H2L3 (hG1DM) are the Ig kappa chain C region, ACCESSION: P01834.

[0142] Based on the Ig gamma-1 chain C region, ACCESSION: P01857, a point mutation of leucine to alanine (L234A) was introduced at position 234 and a point mutation of leucine to alanine (L235A) was introduced at position 235 (SEQ ID NO: 21), resulting in the humanized antibodies named 19F3H1L1 (hG1DM), 19F3H2L2 (hG1DM), and 19F3H2L3 (hG1DM).

[0143] The cDNA of the heavy chain and the cDNA of the light chain of 19F3H1L1 (hG1DM), the cDNA of the heavy chain and the cDNA of the light chain of 19F3H2L2 (hG1DM), and the cDNA of the heavy chain and the cDNA of the light chain of 19F3H2L3 (hG1DM) were respectively cloned into the pUC57simple vector (provided by GenScript Corporation) to obtain pUC57simple-19F3H1 (hG1DM), pUC57simple-19F3L1; pUC57simple-19F3H2 (hG1DM), pUC57simple-19F3L2, and pUC57simple-19F3L3. Referring to the standard techniques described in "Molecular Cloning: A Laboratory Manual (Second Edition)", the full-length genes of the synthesized heavy and light chains were digested with EcoRI & HindIII, and subcloned into the expression vector pcDNA3.1 by restriction enzyme (EcoRI & HindIII) digestion to obtain the expression plasmids pcDNA3.1-19F3H1 (hG1DM), pcDNA3.1-19F3L1, pcDNA3.1-19F3H2 (hG1DM), pcDNA3.1-19F3L2, and pcDNA3.1-19F3L3, and the heavy / light chain genes of the recombinant expression plasmids were further sequenced and analyzed. Subsequently, the gene combinations of the corresponding light and heavy chain recombinant plasmids (pcDNA3.1-19F3H1 (hG1DM) / pcDNA3.1-19F3L1, pcDNA3.1-19F3H2 (hG1DM) / pcDNA3.1-19F3L2, and pcDNA3.1-19F3H2 (hG1DM) / pcDNA3.1-19F3L3) were co-transfected into 293F cells respectively, and the culture medium was collected for purification after transfection. After correct sequencing verification, endotoxin-free expression plasmids were prepared and the plasmids were transiently transfected into HEK293 cells for antibody expression. After culturing for 7 days, the cell culture medium was collected and affinity purified using a Protein A column to obtain the humanized antibody.

[0144] Example 4: Determination of the kinetic affinity of the anti-CD73 antibody for C1q and FcγRIIIa.

[0145] (1) Determination of the kinetic affinity of the anti-CD73 antibody for C1q

[0146] The sample dilution buffer was PBS, 0.02% Tween-20, 0.1% BSA, pH 7.4. Antibodies at 50 μg / mL were immobilized on the FAB2G sensor, with an immobilization height of approximately 2.0 nm. The sensor was equilibrated in the buffer for 60 s. The antibodies immobilized on the sensor were bound to antigen C1q, with the antigen concentration at 0.63 - 10 nM (two-fold serial dilution) for 60 s. The antigen-antibody complex was dissociated in the buffer for 60 s. The sensor was regenerated using 10 mM glycine, pH 1.7 for 5 s, and repeated 4 times. The shaking rate of the sample plate was 1000 rpm, the detection temperature was 30 °C, and the detection frequency was 0.6 Hz. The data was analyzed by fitting to a 1:1 model to obtain the affinity constant. The data acquisition software was Fortebio Data Acqμisition 7.0, and the data analysis software was Fortebio DataAnalysis 7.0.

[0147] The results are shown in Table 1 and Figures 1 - 3. Neither 19F3H2L3 (hG1DM) nor MEDI9447 had binding activity to C1q.

[0148]

[0149] KD = kdis / kon

[0150] (2) Determination of the kinetic affinity of anti-CD73 antibody to FcγRIIIa

[0151] The sample dilution buffer was PBS, 0.02% Tween-20, 0.1% BSA, pH 7.4. FcγRIIIa at 0.5 μg / mL (from Sino Biological) was immobilized on the SA sensor for 120 s. The sensor was equilibrated in the buffer for 60 s. CD16a immobilized on the sensor was bound to each antibody, with the antibody concentration at 31.3 - 500 nM (two-fold serial dilution) for 60 s. The antibody-antigen complex was dissociated in the buffer for 60 s. The sensor was regenerated using 10 mM NaOH. The detection temperature was 30 °C, and the frequency was 0.6 Hz. The data was analyzed by fitting to a 1:1 model to obtain the affinity constant.

[0152] The results are shown in Table 2 and Figures 4 - 6. 19F3H2L3 (hG1DM) did not bind to FcγRIIIa, while MEDI9447 had binding activity to FcγRIIIa.

[0153]

[0154] KD = kdis / kon

[0155] Example 5: The combination of anti-CD73 antibody and anti-CTLA-4 / PD-1 bispecific antibody effectively blocks the growth of tumor cells

[0156] 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 shown in SEQ ID NO: 35, the light chain amino acid sequence is shown in SEQ ID NO: 36, and the light chain and heavy chain CDR amino acid sequences are shown in SEQ ID NOs: 23 to 34) in inhibiting tumor growth.

[0157] The results are as as shown in Figure 7 shown. The combination of the anti-CD73 antibody and the anti-PD-1 / CTLA-4 bispecific antibody showed more excellent tumor inhibitory effects compared to the anti-CD73 antibody alone and the anti-PD-1 / CTLA-4 bispecific antibody alone, and had no significant effect on the body weight of mice ( (Figure 8) .

[0158] Table 3. Experimental design

[0159]

[0160] Mouse colon cancer cells, MC38-hPDL1 / hCD73 (provided by Jiangsu Genscript Biotech Co., Ltd.), were resuscitated at the Pn+3 passage. Logarithmically growing MC38-hPDL1 / hCD73 cells were collected, the culture medium was removed, and the cells were washed twice with PBS and then inoculated into C57BL / 6-hPD1 / hPDL1 / hCD73 mice (provided by Jiangsu Genscript Biotech Co., Ltd.) (the survival rates of MC38-hPDL1 / hCD73 cells before and after tumor inoculation were 99.1% and 96.4%, respectively). Inoculation dose: 2*10 6 / 100 μL / mouse, inoculation site: the right forelimb of the mouse. On the 5th day after inoculation, when the average tumor volume reached 86.02 mm 3 , 32 mice were randomly divided into 4 groups of 8 mice each according to the tumor volume. The day of grouping was defined as Day 0, and drug administration was started on Day 0 according to Table 3.

[0161] After cell seeding, the effects of the tumor on the normal behavior of the animals were routinely monitored every week. Administration dates: Day 0, Day 3, Day 7, Day 10, Day 14, Day 17, Day 22. The tumor size was observed and the body weights of the mice were measured on Day 0, Day 3, Day 6, Day 10, Day 13, Day 17, Day 20, and Day 23. After the experiment ended on Day 23, the tumor tissues of the animals in each group were excised and weighed. The experimental results such as the change rate of the mouse body weight and the tumor weight were expressed as mean ± standard error (Mean±SEM). An independent samples T-test was used to compare whether there were significant differences between the different treatment groups and the control group. A P value < 0.05 was considered to be significantly different. The data was graphed using Graphpad or Excel.

[0162] Example 6: The experimental procedure for detecting the inhibitory effect of anti-CD73 antibody on the endogenous expression of CD73 enzyme activity in cells is as follows: Take logarithmically growing MDA-MB-231 cells (from ATCC, HTB-26) in good condition, resuspend and count the cells with serum-free RPMI-1640 culture medium; inoculate the MDA-MB-231 cells into a 96-well plate, 3*10 4 cells / 100 μl / well; dilute the antibody with serum-free RPMI-1640 culture medium, with an initial concentration of 200 μg / ml, and perform serial dilutions at a 2.5-fold dilution; add the antibody to the 96-well plate, 50 μl per well, and incubate at 37 °C for 1 hour. After 1 hour, add 50 μl of 600 μM AMP diluted with RPMI-1640 to each well; after 3 hours, take 25 μl of the cell culture supernatant and transfer it to a new 96-well plate, and add 25 μl of 100 μM ATP to each well; and 50 μl of CTG ( One Solution Assay, promega, Cat: G8461) chromogenic solution for color development, and read the data with a multi-label microplate reader (PerkinElmer, catalog number: 2140-0020). Isotype control antibody and CD73-specific inhibitor APCP were used as negative control and positive control, respectively.

[0163] Experimental results: The results are as as shown in Figure 9 shown. 19F3, 19F3H2L3 (hG1DM), 19F3H2L3 (hG1DM), and 19F3H2L3 (hG1DM) can all dose-dependently inhibit the activity of the CD73 enzyme that endogenously expresses in MDA-MB-231 to catalyze AMP into adenosine A, thereby dose-dependently reducing the average fluorescence intensity RLU generated.

[0164] The above experimental results indicate that in the absence of CD73 antibody treatment, the added AMP can be catalyzed by the CD73 enzyme endogenously expressed on the cell surface of MDA-MB-231 and converted into adenosine, thus relieving the inhibition of luciferase activity. After adding the antibody, since CD73 is bound by the antibody, its enzymatic catalytic activity is reduced, and AMP cannot be converted into adenosine. This suggests that the anti-CD73 antibody effectively inhibits the enzymatic reaction of CD73 in a non-substrate competitive manner and reduces the production of adenosine.

[0165] Sequence Listing

[0166] Nucleic acid sequence of the heavy chain variable region of 19F3: (SEQ ID NO:1)

[0167]

[0168] Amino acid sequence of the heavy chain variable region of 19F3: (SEQ ID NO:2)

[0169]

[0170] Nucleic acid sequence of the light chain variable region of 19F3: (SEQ ID NO:3)

[0171]

[0172] Amino acid sequence of the light chain variable region of 19F3: (SEQ ID NO:4)

[0173]

[0174] Nucleic acid sequence of the heavy chain variable region of 19F3H1L1 (hG1DM): (SEQ ID NO:5)

[0175]

[0176] Amino acid sequence of the heavy chain variable region of 19F3H1L1 (hG1DM): (SEQ ID NO:6)

[0177]

[0178] Nucleic acid sequence of the light chain variable region of 19F3H1L1 (hG1DM): (SEQ ID NO:7)

[0179]

[0180] Amino acid sequence of the light chain variable region of 19F3H1L1 (hG1DM): (SEQ ID NO:8)

[0181]

[0182] Nucleic acid sequences of the heavy chain variable regions of 19F3H2L2(hG1DM) and 19F3H2L3(hG1DM): (SEQ ID NO:9)

[0183]

[0184] Amino acid sequences of the heavy chain variable regions of 19F3H2L2(hG1DM) and 19F3H2L3(hG1DM): (SEQ ID NO:10)

[0185]

[0186] Nucleic acid sequence of the light chain variable region of 19F3H2L2(hG1DM): (SEQ ID NO:11)

[0187]

[0188] Amino acid sequence of the light chain variable region of 19F3H2L2(hG1DM): (SEQ ID NO:12)

[0189]

[0190] Nucleic acid sequence of the light chain variable region of 19F3H2L3(hG1DM): (SEQ ID NO:13)

[0191]

[0192] Amino acid sequence of the light chain variable region of 19F3H2L3(hG1DM): (SEQ ID NO:14)

[0193]

[0194] CDR regions of 19F3 and 19F3H1L1(hG1DM), 19F3H2L2(hG1DM), 19F3H2L3(hG1DM)

[0195] HCDR1: GYSFTGYT (SEQ ID NO:15)

[0196] HCDR2: INPYNAGT (SEQ ID NO:16)

[0197] HCDR3: ARSEYRYGGDYFDY (SEQ ID NO:17)

[0198] LCDR1: QSLLNSSNQKNY (SEQ ID NO:18)

[0199] LCDR2: FAS (SEQ ID NO: 19)

[0200] LCDR3: QQHYDTPYT (SEQ ID NO: 20)

[0201] Heavy chain constant region sequences of 19F3H1L1 (hG1DM), 19F3H2L2 (hG1DM) and 19F3H2L3 (hG1DM) (330 aa, mutation sites are underlined):

[0202]

[0203] Light chain constant region sequences of 19F3H1L1 (hG1DM), 19F3H2L2 (hG1DM) and 19F3H2L3 (hG1DM) (107 aa):

[0204]

[0205] Amino acid sequences of 9 CDR regions involved in the variable region of the heavy chain of BiAb004 (hG1TM) are as follows:

[0206] HCDR1: GFAFSSYD (SEQ ID NO: 23)

[0207] HCDR2: ISGGGRYT (SEQ ID NO: 24)

[0208] HCDR3: ANRYGEAWFAY (SEQ ID NO: 25)

[0209] HCDR4: GYSFTGYT (SEQ ID NO: 26)

[0210] HCDR5: INPYNNIT (SEQ ID NO: 27)

[0211] HCDR6: ARLDYRSY (SEQ ID NO: 28)

[0212] HCDR7: TGAVTTSNF (SEQ ID NO: 29)

[0213] HCDR8: GTN (SEQ ID NO: 30)

[0214] HCDR9: ALWYSNHWV (SEQ ID NO: 31)

[0215] Amino acid sequences of 3 CDR regions involved in the variable region of the light chain of BiAb004 (hG1TM) are as follows:

[0216] LCDR1:QDINTY(SEQ ID NO:32)

[0217] LCDR2:RAN(SEQ ID NO:33)

[0218] LCDR3:LQYDEFPLT(SEQ ID NO:34)

[0219] Amino acid sequence of the heavy chain of BiAb004(hG1TM) (713aa, mutation sites are underlined)

[0220]

[0221] Amino acid sequence of the light chain of BiAb004(hG1TM) (214aa)

[0222]

Claims

1. A kit comprising (1) an anti-CD73 antibody or an antigen-binding fragment thereof, a conjugate or fusion protein or multispecific antibody comprising the anti-CD73 antibody or an antigen-binding fragment thereof, and (2) an anti-PD-1 / CTLA-4 bispecific antibody, wherein the heavy chain amino acid sequence of the anti-PD-1 / CTLA-4 bispecific antibody is as shown in SEQ ID NO: 35 and the light chain amino acid sequence is as shown in SEQ ID NO: 36, wherein according to the IMGT numbering system: the anti-CD73 antibody or an antigen-binding fragment thereof comprises HCDR1, HCDR2 and HCDR3 of the heavy chain variable region shown in SEQ ID NO: 2; and LCDR1, LCDR2 and LCDR3 of the light chain variable region shown in SEQ ID NO: 4, Wherein the antigen-binding fragment is selected from Fab, Fab', F(ab') 2 , Fv, Fab / c, or single-chain antibody.

2. The kit according to claim 1, wherein CD73 is human CD73.

3. The kit according to claim 1, wherein according to the IMGT numbering system: the anti-CD73 antibody or an antigen-binding fragment thereof comprises HCDR1, which consists of the amino acid sequence shown in SEQ ID NO: 15, HCDR2, which consists of the amino acid sequence shown in SEQ ID NO: 16, HCDR3, which consists of the amino acid sequence shown in SEQ ID NO: 17, LCDR1, which consists of the amino acid sequence shown in SEQ ID NO: 18, LCDR2, which consists of the amino acid sequence shown in SEQ ID NO: 19, and LCDR3, which consists of the amino acid sequence shown in SEQ ID NO:

20.

4. The kit according to claim 1, wherein the amino acid sequence of the heavy chain variable region of the anti-CD73 antibody is as shown in a sequence having at least 85% sequence identity with the sequence shown in SEQ ID NO: 2; and the amino acid sequence of the light chain variable region of the anti-CD73 antibody is as shown in a sequence having at least 90% sequence identity with the sequence shown in SEQ ID NO: 4; the amino acid sequence of the heavy chain variable region of the anti-CD73 antibody is as shown in a sequence having at least 90% sequence identity with the sequence shown in SEQ ID NO: 6, and the amino acid sequence of the light chain variable region of the anti-CD73 antibody is as shown in a sequence having at least 93% sequence identity with the sequence shown in SEQ ID NO: 8; the amino acid sequence of the heavy chain variable region of the anti-CD73 antibody is as shown in a sequence having at least 85% sequence identity with the sequence shown in SEQ ID NO: 10, and the amino acid sequence of the light chain variable region of the anti-CD73 antibody is as shown in a sequence having at least 92% sequence identity with the sequence shown in SEQ ID NO: 12; or The amino acid sequence of the heavy chain variable region of the anti-CD73 antibody is shown as a sequence having at least 85% sequence identity with the sequence shown in SEQ ID NO: 10, and the amino acid sequence of the light chain variable region of the anti-CD73 antibody is shown as a sequence having at least 90% identity with the sequence shown in SEQ ID NO:

14.

5. The kit according to claim 4, wherein the amino acid sequence of the heavy chain variable region of the anti-CD73 antibody is shown as a sequence having at least 86% sequence identity with the sequence shown in SEQ ID NO: 2, or the amino acid sequence of the light chain variable region of the anti-CD73 antibody is shown as a sequence having at least 91% sequence identity with the sequence shown in SEQ ID NO: 4; or the amino acid sequence of the heavy chain variable region of the anti-CD73 antibody is shown as a sequence having at least 91% sequence identity with the sequence shown in SEQ ID NO: 6, or the amino acid sequence of the light chain variable region of the anti-CD73 antibody is shown as a sequence having at least 94% sequence identity with the sequence shown in SEQ ID NO: 8; or the amino acid sequence of the heavy chain variable region of the anti-CD73 antibody is shown as a sequence having at least 86% sequence identity with the sequence shown in SEQ ID NO: 10, or the amino acid sequence of the light chain variable region of the anti-CD73 antibody is shown as a sequence having at least 93% sequence identity with the sequence shown in SEQ ID NO: 12; or the amino acid sequence of the light chain variable region of the anti-CD73 antibody is shown as a sequence having at least 91% identity with the sequence shown in SEQ ID NO:

14.

6. The kit according to claim 4, wherein the amino acid sequence of the heavy chain variable region of the anti-CD73 antibody is shown as a sequence having at least 87% sequence identity with the sequence shown in SEQ ID NO: 2, or the amino acid sequence of the light chain variable region of the anti-CD73 antibody is shown as a sequence having at least 92% sequence identity with the sequence shown in SEQ ID NO: 4; or the amino acid sequence of the heavy chain variable region of the anti-CD73 antibody is shown as a sequence having at least 92% sequence identity with the sequence shown in SEQ ID NO: 6, or the amino acid sequence of the light chain variable region of the anti-CD73 antibody is shown as a sequence having at least 95% sequence identity with the sequence shown in SEQ ID NO: 8; or the amino acid sequence of the heavy chain variable region of the anti-CD73 antibody is shown as a sequence having at least 87% sequence identity with the sequence shown in SEQ ID NO: 10, or the amino acid sequence of the light chain variable region of the anti-CD73 antibody is shown as a sequence having at least 94% sequence identity with the sequence shown in SEQ ID NO: 12; or the amino acid sequence of the light chain variable region of the anti-CD73 antibody is shown as a sequence having at least 92% identity with the sequence shown in SEQ ID NO:

14.

7. The kit according to claim 4, wherein The amino acid sequence of the heavy chain variable region of the anti-CD73 antibody is as shown by a sequence having at least 88% sequence identity with the sequence shown in SEQ ID NO: 2, or The amino acid sequence of the light chain variable region of the anti-CD73 antibody is as shown by a sequence having at least 93% sequence identity with the sequence shown in SEQ ID NO: 4; or The amino acid sequence of the heavy chain variable region of the anti-CD73 antibody is as shown by a sequence having at least 93% sequence identity with the sequence shown in SEQ ID NO: 6, or The amino acid sequence of the light chain variable region of the anti-CD73 antibody is as shown by a sequence having at least 96% sequence identity with the sequence shown in SEQ ID NO: 8; or The amino acid sequence of the heavy chain variable region of the anti-CD73 antibody is as shown by a sequence having at least 88% sequence identity with the sequence shown in SEQ ID NO: 10, or The amino acid sequence of the light chain variable region of the anti-CD73 antibody is as shown by a sequence having at least 95% sequence identity with the sequence shown in SEQ ID NO: 12; or The amino acid sequence of the light chain variable region of the anti-CD73 antibody is as shown by a sequence having at least 93% identity with the sequence shown in SEQ ID NO:

14.

8. The kit according to claim 4, wherein The amino acid sequence of the heavy chain variable region of the anti-CD73 antibody is as shown by a sequence having at least 89% sequence identity with the sequence shown in SEQ ID NO: 2, or The amino acid sequence of the light chain variable region of the anti-CD73 antibody is as shown by a sequence having at least 94% sequence identity with the sequence shown in SEQ ID NO: 4; or The amino acid sequence of the heavy chain variable region of the anti-CD73 antibody is as shown by a sequence having at least 94% sequence identity with the sequence shown in SEQ ID NO: 6, or The amino acid sequence of the light chain variable region of the anti-CD73 antibody is as shown by a sequence having at least 97% sequence identity with the sequence shown in SEQ ID NO: 8; or The amino acid sequence of the heavy chain variable region of the anti-CD73 antibody is as shown by a sequence having at least 89% sequence identity with the sequence shown in SEQ ID NO: 10, or The amino acid sequence of the light chain variable region of the anti-CD73 antibody is as shown by a sequence having at least 96% sequence identity with the sequence shown in SEQ ID NO: 12; or The amino acid sequence of the light chain variable region of the anti-CD73 antibody is as shown by a sequence having at least 94% identity with the sequence shown in SEQ ID NO:

14.

9. The kit according to claim 4, wherein The amino acid sequence of the heavy chain variable region of the anti-CD73 antibody is as shown by a sequence having at least 90% sequence identity with the sequence shown in SEQ ID NO: 2, or The amino acid sequence of the light chain variable region of the anti-CD73 antibody is as shown by a sequence having at least 95% sequence identity with the sequence shown in SEQ ID NO: 4; or The amino acid sequence of the heavy chain variable region of the anti-CD73 antibody is shown as a sequence having at least 95% sequence identity with the sequence shown in SEQ ID NO: 6, or The amino acid sequence of the light chain variable region of the anti-CD73 antibody is shown as a sequence having at least 98% sequence identity with the sequence shown in SEQ ID NO: 8; or The amino acid sequence of the heavy chain variable region of the anti-CD73 antibody is shown as a sequence having at least 90% sequence identity with the sequence shown in SEQ ID NO: 10, or The amino acid sequence of the light chain variable region of the anti-CD73 antibody is shown as a sequence having at least 97% sequence identity with the sequence shown in SEQ ID NO: 12; or The amino acid sequence of the light chain variable region of the anti-CD73 antibody is shown as a sequence having at least 95% identity with the sequence shown in SEQ ID NO:

14.

10. The kit according to claim 4, wherein The amino acid sequence of the heavy chain variable region of the anti-CD73 antibody is shown as a sequence having at least 91% sequence identity with the sequence shown in SEQ ID NO: 2, or The amino acid sequence of the light chain variable region of the anti-CD73 antibody is shown as a sequence having at least 96% sequence identity with the sequence shown in SEQ ID NO: 4; or The amino acid sequence of the heavy chain variable region of the anti-CD73 antibody is shown as a sequence having at least 96% sequence identity with the sequence shown in SEQ ID NO: 6, or The amino acid sequence of the light chain variable region of the anti-CD73 antibody is shown as a sequence having at least 99% sequence identity with the sequence shown in SEQ ID NO: 8; or The amino acid sequence of the heavy chain variable region of the anti-CD73 antibody is shown as a sequence having at least 91% sequence identity with the sequence shown in SEQ ID NO: 10, or The amino acid sequence of the light chain variable region of the anti-CD73 antibody is shown as a sequence having at least 98% sequence identity with the sequence shown in SEQ ID NO: 12; or The amino acid sequence of the light chain variable region of the anti-CD73 antibody is shown as a sequence having at least 96% identity with the sequence shown in SEQ ID NO:

14.

11. The kit according to claim 4, wherein The amino acid sequence of the heavy chain variable region of the anti-CD73 antibody is shown as a sequence having at least 92% sequence identity with the sequence shown in SEQ ID NO: 2, or The amino acid sequence of the light chain variable region of the anti-CD73 antibody is shown as a sequence having at least 97% sequence identity with the sequence shown in SEQ ID NO: 4; or The amino acid sequence of the heavy chain variable region of the anti-CD73 antibody is shown as a sequence having at least 97% sequence identity with the sequence shown in SEQ ID NO: 6, or The amino acid sequence of the heavy chain variable region of the anti-CD73 antibody is shown as a sequence having at least 92% sequence identity with the sequence shown in SEQ ID NO: 10, or The amino acid sequence of the light chain variable region of the anti-CD73 antibody is shown by a sequence having at least 99% sequence identity with the sequence shown in SEQ ID NO: 12; or The amino acid sequence of the light chain variable region of the anti-CD73 antibody is shown by a sequence having at least 97% identity with the sequence shown in SEQ ID NO:

14.

12. The kit according to claim 4, wherein The amino acid sequence of the heavy chain variable region of the anti-CD73 antibody is shown by a sequence having at least 93% sequence identity with the sequence shown in SEQ ID NO: 2, or The amino acid sequence of the light chain variable region of the anti-CD73 antibody is shown by a sequence having at least 98% sequence identity with the sequence shown in SEQ ID NO: 4; or The amino acid sequence of the heavy chain variable region of the anti-CD73 antibody is shown by a sequence having at least 98% sequence identity with the sequence shown in SEQ ID NO: 6, or The amino acid sequence of the heavy chain variable region of the anti-CD73 antibody is shown by a sequence having at least 93% sequence identity with the sequence shown in SEQ ID NO: 10, or The amino acid sequence of the light chain variable region of the anti-CD73 antibody is shown by a sequence having at least 98% identity with the sequence shown in SEQ ID NO:

14.

13. The kit according to claim 4, wherein The amino acid sequence of the heavy chain variable region of the anti-CD73 antibody is shown by a sequence having at least 94% sequence identity with the sequence shown in SEQ ID NO: 2, or The amino acid sequence of the light chain variable region of the anti-CD73 antibody is shown by a sequence having at least 99% sequence identity with the sequence shown in SEQ ID NO: 4; or The amino acid sequence of the heavy chain variable region of the anti-CD73 antibody is shown by a sequence having at least 99% sequence identity with the sequence shown in SEQ ID NO: 6, or The amino acid sequence of the heavy chain variable region of the anti-CD73 antibody is shown by a sequence having at least 94% sequence identity with the sequence shown in SEQ ID NO: 10, or The amino acid sequence of the light chain variable region of the anti-CD73 antibody is shown by a sequence having at least 99% identity with the sequence shown in SEQ ID NO:

14.

14. The kit according to claim 4, wherein The amino acid sequence of the heavy chain variable region of the anti-CD73 antibody is shown by a sequence having at least 95% sequence identity with the sequence shown in SEQ ID NO: 2, or The amino acid sequence of the heavy chain variable region of the anti-CD73 antibody is shown by a sequence having at least 95% sequence identity with the sequence shown in SEQ ID NO:

10.

15. The kit according to claim 4, wherein The amino acid sequence of the heavy chain variable region of the anti-CD73 antibody is shown by a sequence having at least 96% sequence identity with the sequence shown in SEQ ID NO: 2, or The amino acid sequence of the heavy chain variable region of the anti-CD73 antibody is shown by a sequence having at least 96% sequence identity with the sequence shown in SEQ ID NO:

10.

16. The kit according to claim 4, wherein the amino acid sequence of the heavy chain variable region of the anti-CD73 antibody is shown by a sequence having at least 97% sequence identity with the sequence shown in SEQ ID NO: 2, or the amino acid sequence of the heavy chain variable region of the anti-CD73 antibody is shown by a sequence having at least 97% sequence identity with the sequence shown in SEQ ID NO:

10.

17. The kit according to claim 4, wherein the amino acid sequence of the heavy chain variable region of the anti-CD73 antibody is shown by a sequence having at least 98% sequence identity with the sequence shown in SEQ ID NO: 2, or the amino acid sequence of the heavy chain variable region of the anti-CD73 antibody is shown by a sequence having at least 98% sequence identity with the sequence shown in SEQ ID NO:

10.

18. The kit according to claim 4, wherein the amino acid sequence of the heavy chain variable region of the anti-CD73 antibody is shown by a sequence having at least 99% sequence identity with the sequence shown in SEQ ID NO: 2, or the amino acid sequence of the heavy chain variable region of the anti-CD73 antibody is shown by a sequence having at least 99% sequence identity with the sequence shown in SEQ ID NO:

10.

19. The kit according to claim 1 or 2, wherein the amino acid sequence of the heavy chain variable region of the anti-CD73 antibody is as shown in SEQ ID NO: 2, and the amino acid sequence of the light chain variable region of the anti-CD73 antibody is as shown in SEQ ID NO: 4; the amino acid sequence of the heavy chain variable region of the anti-CD73 antibody is as shown in SEQ ID NO: 6, and the amino acid sequence of the light chain variable region of the anti-CD73 antibody is as shown in SEQ ID NO: 8; the amino acid sequence of the heavy chain variable region of the anti-CD73 antibody is as shown in SEQ ID NO: 10, and the amino acid sequence of the light chain variable region of the anti-CD73 antibody is as shown in SEQ ID NO: 12; or the amino acid sequence of the heavy chain variable region of the anti-CD73 antibody is as shown in SEQ ID NO: 10, and the amino acid sequence of the light chain variable region of the anti-CD73 antibody is as shown in SEQ ID NO:

14.

20. The kit according to claim 1 or 2, wherein the heavy chain constant region of the anti-CD73 antibody is the C region of Ig gamma-1 chain, ACCESSION: P01857; the light chain constant region is the C region of Ig kappa chain, ACCESSION: P01834.

21. The kit according to claim 1 or 2, wherein the anti-CD73 antibody is a monoclonal antibody, a humanized antibody, a chimeric antibody, a multispecific antibody.

22. The kit according to claim 1 or 2, wherein the anti-CD73 antibody is a bispecific antibody.

23. The kit according to claim 1 or 2, wherein the antigen-binding fragment is scFv.

24. The kit according to claim 1 or 2, wherein the conjugate comprises the anti-CD73 antibody or its antigen-binding fragment as described above, and a conjugation moiety, wherein, the conjugation moiety is a purification tag or a detectable label.

25. The kit according to claim 24, wherein the purification tag is a His tag.

26. The kit according to claim 24, wherein the conjugation moiety is a radioisotope, a fluorescent substance, a chemiluminescent substance, a colored substance, polyethylene glycol or an enzyme.

27. Use of a therapeutically effective amount of drug A and a therapeutically effective amount of drug B in the preparation of a kit for treating and / or preventing tumors, wherein drug A comprises the anti-CD73 antibody or its antigen-binding fragment, the conjugate, the fusion protein or the multispecific antibody as defined in any one of claims 1-23, drug B comprises an anti-PD-1 / CTLA-4 bispecific antibody, the heavy chain amino acid sequence of the anti-PD-1 / CTLA-4 bispecific antibody is as shown in SEQ ID NO:35, and the light chain amino acid sequence is as shown in SEQ ID NO:36, wherein the tumor is positive for CD73, PD1 and CTLA-4.

28. The use according to claim 27, wherein the tumor is a solid tumor.

29. The use according to claim 27, wherein the tumor is non-small cell lung cancer, prostate cancer, triple-negative breast cancer, ovarian cancer, colorectal cancer, gastric cancer, melanoma, head and neck cancer, colon cancer, renal cell carcinoma or pancreatic ductal adenocarcinoma.

30. The use according to claim 27, wherein the tumor is metastatic castration-resistant prostate cancer (mCRPC).

31. The use according to claim 29, wherein the colorectal cancer is of the microsatellite stable (MSS) and defective mismatch repair / microsatellite instability (dMMR / MSI-high) type.

32. The use according to claim 29, wherein the gastric cancer is of the microsatellite stable (MSS) and defective mismatch repair / microsatellite instability (dMMR / MSI-high) type.

Images