Anti-CD73-Anti-PD-1 bispecific antibody and use thereof

Abstract

To provide a humanized bifunctional antibody for use in the preparation of a medicament for the prevention and treatment of solid tumors and hematological tumors.SOLUTION: The present invention provides an anti-CD73 / anti-PD-1 bispecific antibody, comprising a first protein functional region targeting PD-1 and a second protein functional region targeting CD73.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to the fields of tumor treatment and molecular immunology, and in particular to anti-CD73 / anti-PD-1 bispecific antibodies, pharmaceutical compositions thereof, and uses thereof. [Background technology]

[0002] Ecto-5'-nucleotidase, i.e., CD73 protein, is a multifunctional glycoprotein encoded by the NT5E gene, has a molecular weight of 70 KD, and is anchored on the cell membrane by glycosylphosphatidylinositol (GPI) (Zimmermann H., 5'-Nucleotidase: molecular structure and functional aspects., Biochem J., 1992; 285:345-365).

[0003] CD73 is widely distributed on the surface of human tissue cells, and studies have shown that it is highly expressed in various solid tumors, specifically in the tumor microenvironment, on cancer cells, dendritic cells, regulatory T cells (Tregs), natural killer cells (NK cells), myeloid-derived suppressor cells (MDSCs), tumor-associated macrophages (TAMs), etc. CD73 expression is regulated by TGF-β, EGFR, AKT, β-catenin, and other molecules, most importantly HIF-1, which functions as a transcription factor. An important feature of the tumor microenvironment is hypoxia, which induces upregulation of hypoxia-inducible factor-1 (HIF-1) and other molecules, thereby resulting in widespread expression of CD73 in the tumor microenvironment (Synnestvedt K, et al., Ecto-5'-nucleotidase (CD73) regulation by hypoxia-inducible factor-1 mediates permeability changes in intestinal epithelia. 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 associated with poor prognosis in various types of tumors, including breast cancer, lung cancer, ovarian cancer, kidney cancer, gastric cancer, head and neck cancer, etc.

[0004] CD73 has hydrolase and non-hydrolase activities. The enzymatic and non-enzymatic functions of CD73 act simultaneously in tumor-related processes and mutually promote and maintain tumor progression. More and more studies have found that CD73 is a key regulatory molecule in tumor cell proliferation, metastasis and invasion in vitro, and in tumor angiogenesis and tumor immune evasion mechanisms in vivo, and that an important mechanism of immune suppression is mediated by the CD73-adenosine metabolic signaling pathway. CD39, which is upstream of CD73, catalyzes ATP to generate adenosine monophosphate (AMP), which is converted to adenosine by CD73, and adenosine binds to the downstream adenosine receptor (A2AR). A2AR plays an immunosuppressive role by inhibiting a series of signaling pathways related to immune activation, such as LCK, MAPK, and PKC, and activating protein kinase A (PKA) and Csk kinase, thereby inhibiting the immune killing effect of T cells, allowing tumors to achieve immune evasion (Antonioli L, et al., Immunity, inflammation and cancer: a leading role for adenosine. Nat Rev Cancer., 2013; 13:842-857). Preclinical animal model studies have shown that CD73 expressed in immune and non-immune cells can promote immune evasion, development and metastasis of tumors, and the inhibition of cytotoxic T cell (CTL) and NK cell function by Treg cell-associated CD73-adenosine signaling is of utmost importance.

[0005] For the treatment of solid tumors, one of the important aspects to overcome drug resistance and improve therapeutic efficacy is to alleviate the inhibitory effect of the tumor microenvironment (TME) on immune effector cells. The TME is a very complex system composed of various cells, extracellular matrix, enzymes, cytokines, metabolic products, etc. The TME is characterized by extremely low hydrogen, low pH, and high pressure, and is significantly different from normal tissues. Hypoxia or ATP enrichment caused by chemoradiotherapy to kill tumor cells promotes the cascade reaction of CD39-CD73 adenosine signals, which is beneficial for the proliferation and function of various cancer-promoting cells, but not cancer-inhibiting cells (Regateiro, FS, Cobbold, SP & Waldmann, H., CD73 and adenosine generation in the creation of regulatory microenvironments. Clin. Exp. Immunol., 2013; 171:1-7).

[0006] The use of CD73-targeting antibodies or gene knockout of CD73 in animal models can effectively block tumor growth or metastasis, and recently, the use of CD73 monoclonal antibodies, small interfering RNA technology, specific inhibitor APCP, etc. has achieved remarkable therapeutic effects in antitumor treatment in animal experiments, providing a new avenue for antitumor treatment. Evidence from in vivo studies indicates that targeted blockade of CD73 can be an effective means of treatment for tumor patients.

[0007] The relationship between CD73 overexpression and tumor subtypes, prognosis and response of patients indicates that CD73 may be an important marker for future individual tumor treatment and detection. Therefore, research into CD73 targets is essential.

[0008] The transmembrane receptor PD-1 (programmed cell death protein 1) is a member of the CD28 family and is expressed on activated T cells, B cells, and myeloid cells. The receptors for PD-1, PDL-1 and PDL-2, are members of the B7 superfamily. PDL-1 is expressed on a variety of cells, including T cells, B cells, endothelial cells, and epithelial cells, whereas PDL-2 is expressed exclusively on antigen-presenting cells such as dendritic cells and macrophages.

[0009] PD-1 plays a very important role in downregulating T cell activation, and PD-1-mediated T cell downregulation is one of the important mechanisms of tumor immune escape. PDL-1 expressed on the surface of tumors can bind to PD-1 on the surface of immune cells, thereby inhibiting the killing of tumor tissues by immune cells through the PD-1 / PDL-1 signaling pathway, and tumors with high expression of PDL-1 are associated with cancers that are difficult to detect (Hamanishi et al., Proc. Natl. Acad. Sci. USA, 2007; 104:3360-5). An effective method of antagonizing PD-1 and thereby inhibiting the PD-1 / PDL-1 signaling pathway is the injection of anti-PDL-1 antibodies.

[0010] Due to the broad anti-tumor perspective and remarkable efficacy of PD-1 antibodies, it is widely accepted that antibodies targeting the PD-1 pathway offer breakthroughs in the treatment of various tumors: non-small cell lung cancer, renal cell carcinoma, ovarian cancer and melanoma (Homet MB, Parisi G., et al., Anti-PD-1 therapy in melanoma. Semin Oncol., 2015 Jun; 42(3):466-473), and hematological malignancies and anemias (Held SA, Heine A, et al., Advances in immunotherapy of chronic myeloid leukemia CML. Curr Cancer Drug Targets, 2013 Sep; 13(7):768-74).

[0011] Bifunctional antibodies, also known as bispecific antibodies, are specific antibody drugs that simultaneously target two different antigens, and can be produced by immunoselection and purification, or obtained by genetic engineering. Genetic engineering has flexibility in aspects such as optimization of binding sites, synthetic forms, and yield, and therefore has certain advantages. Currently, more than 45 types of bispecific antibody forms have been demonstrated (Muller D, Kontermann RE. Bispecific antibodies for cancer immunotherapy: current perspectives. BioDrugs 2010; 24:89-98). The IgG-ScFv format, or Morrison format (Coloma MJ, Morrison SL. Design and production of novel tetravalent bispecific antibodies. Nat Biotechnol. Nature Biotechnology, 1997; 15:159-163), has been demonstrated to be an ideal format for bifunctional antibodies due to its similarity to the naturally occurring IgG format and due to advantages in antibody engineering, expression, and production (Miller BR, Demarest SJ, et al., Stability engineering of scFvs for the development of bispecific and multivalent antibodies. Protein Eng Des Sel 2010; 23:549-57; Fitzgerald J, Lugovskoy A. Rational engineering of antibody therapeutics targeting multiple oncogene pathways. MAbs 2011; 3:299-309).

[0012] ADCC (Antibody-Dependent Cell-Mediated Cytotoxicity) refers to the killing of target cells by killer cells mediated by the binding of the Fab fragment of an antibody to an epitope on a virus-infected cell or tumor cell, and the binding of the Fc fragment of the antibody to the Fc receptor (FcR) on the surface of the killer cell (NK cell, macrophage, etc.).

[0013] CDC (complement-dependent cytotoxicity) refers to the lytic action against target cells by the membrane attack complex formed by an antibody binding to a corresponding antigen on the cell membrane surface, followed by binding to the complement C1q and activation of C2 to C9.

[0014] The IgG family includes four members, IgG1, IgG2, IgG3, and IgG4, which differ in amino acids in the fragment crystallizable (Fc) region of the heavy chain constant region, resulting in different affinities for FcγR. Wild-type IgG1 can bind to various FcγRs and induce ADCC and CDC. Zhang et al. reported that Fc-dependent effector function-induced immune cell damage, including antibody-dependent cell-mediated cytotoxicity, may be an important mechanism leading to immune cell damage, so that the binding of Fc fragments of antibodies targeting immune checkpoints such as PD-1 to Fc receptors negatively affects antibody-mediated anti-cancer activity (Zhang T et al, Cancer Immunol Immunother., 2018; 67(7):1079-1090.) and Dahan et al. (Dahan R et al., Cancer cell, 2015, 28(3):285-95.).

[0015] Interleukin-8 (IL-8) is a chemotactic cytokine, mainly secreted by monocytes. IL-8 plays an important role in promoting the proliferation of normal and tumor cells, especially tumor initiation and progression. Several studies have shown that IL-8 can promote tumor initiation, and tumor cells themselves can secrete IL-8 to promote tumor growth and metastasis (Lo MC et al., Cancer letters, 2013, 335(1):81-92). Therefore, IL-8 has become an essential and important inflammatory factor in the tumor microenvironment.

[0016] As a pro-inflammatory factor, IL-8 is closely related to tumor development and progression. During methylarsonate-induced malignant transformation of non-renal cancer cells, the expression of IL-8 gene is increased. Gene silencing of IL-8 can significantly inhibit the growth of transplanted tumors in mice, and furthermore, the reduction of IL-8 level can suppress the expression of matrix metalloproteinase-9, cyclin D1, proapoptotic protein Bcl-2 and vascular endothelial growth factor (VEGF) which are related to tumor growth and metastasis (Escudero-Lourdes C et al., Toxicology and applied pharmacology, 2012, 258(1):10-18). Inoue et al. found that IL-8 could induce malignant transformation of a non-tumor bladder cell line (233JP) and increase its aggressiveness, while the incidence of malignant transformation of 233JP cells was significantly reduced in IL-8 knockout mice (Inoue K et al., Cancer Res, 2000, 60(8):2290-2299). Furthermore, in prostate cancer, IL-8 can promote the development of castration-resistant prostate cancer (CRPC) in patients (Chen K et al., Cancer research, 2015, 75(10):1992-2004) and is associated with drug resistance to tumor treatment (Araki S et al., Cancer Res, 2007, 67(14):6854-6862); gene silencing of IL-8 or its receptor can induce cell cycle arrest in tumor cells and inhibit tumor growth (Singh RK, Lokeshwar BL., Molecul Cancer, 2009, 8:57). The above studies indicate that the level of IL-8 is closely related to tumor development and progression. Further studies (Mian BM et al. Clin Cancer Res, 2003, 9(8):3167-3175) indicate that IL-8 may be a new target for tumor treatment. In a bladder cancer tumor model, the use of anti-IL-8 antibodies can significantly suppress tumor growth.

[0017] IL-6 is rapidly produced by macrophages, mainly in response to pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs), and plays a protective role by eliminating infectious agents and inducing acute phase and immune responses to heal damaged tissues. Although IL-6 plays an important role in resistance and repair to infection and tissue damage, high levels of IL-6 can activate the coagulation pathway and vascular endothelial cells, thereby inhibiting myocardial function, and even cause a "cytokine storm" resulting in severe acute systemic inflammatory responses. Cytokine storm is a fatal complication and side effect in viral infections, tumor immunotherapy, etc.

[0018] Immune-related side effects are common and dangerous side effects in antitumor treatment with immune checkpoint inhibitors (ICIs) (Spain L et al., Cancer Treat Rev., 2016; 44:51-60). In recent years, immune checkpoint inhibitors have achieved great success in tumor immunotherapy, but they also bring about an entirely new toxicity profile due to off-target effects, among which severe immune-related adverse events (irAEs) in major organs (including the heart, lungs and brain) are particularly life-threatening (Bergqvist V, et al., Cancer Immunol Immunother., 2017; 66(5):581-592; Gomatou G et al., Respiration., 2020; 1:1-11; Joshi MN et al., Clin Endocrinol (Oxf)., 2016; 85(3):331-9; Prieux-Klotz C et al., Target Oncol., 2017; 12(3):301-308; Tajiri K et al., Jpn J Clin Oncol., 2018; 48(1):7-12). Existing data indicate that ICIs can induce off-target effects through four mechanisms, including direct binding to immune checkpoint molecules expressed on the surface of normal cells and activating complement hypersensitivity; the presence of homologous antigens / epitopes in normal tissues and tumor cells; producing autoantibodies; and increasing the levels of inflammatory cytokines such as IL-6 (Martins F et al., The Lancet Oncology, 20(1), e54-e64).

[0019] Currently, anti-IL-6 therapies such as tocilizumab, a recombinant humanized anti-IL-6R monoclonal antibody, are used to treat severe irAEs in the acute phase, severe / refractory arthritis, large vessel vasculitis, uveitis, myocarditis, pneumonia, and myasthenia gravis (Martins F et al., The Lancet Oncology, 20(1), e54-e64).

[0020] Binding of wild-type IgG1 or IgG4 antibodies to FcγRIa on macrophages can induce secretion of IL-8 and IL-6 by macrophages (Kinder M et al., mAbs., 2015), while induction of mutations in the Fc segment of antibodies and abolishing binding to FcγRIA can effectively inhibit IL-8 secretion, thereby improving the safety and efficacy of the antibodies. Summary of the Invention

[0021] The inventors used a mammalian cell expression system to express recombinant human CD73 and PD-1 as antigens for immunizing mice, and obtained hybridoma cells by fusing mouse spleen cells with myeloma cells. The inventors identified the following hybridoma cell lines by screening multiple samples: Hybridoma cell line LT014 (also called CD73-19F3): deposited at the China Culture Collection Center (CCTCC) on June 19, 2018 with accession number CTCCNO: C2018137; and Hybridoma cell line LT003 (also called PD-1-14C12): Deposited at the China Culture Collection Center (CCTCC) on June 16, 2015 with accession number CTCCNO: C2015105 Got

[0022] Surprisingly, the inventors discovered that: The hybridoma cell line LT014 can secrete a specific monoclonal antibody (named 19F3) that specifically binds to human CD73, and the monoclonal antibody can effectively inhibit the enzyme activity reaction of CD73 in a non-substrate competitive mode, reduce the production of adenosine, and promote the activity of T cells and the tumor-inhibiting effect; and The hybridoma cell line LT003 may secrete a specific monoclonal antibody (designated 14C12) that specifically binds to PD-1, and the monoclonal antibody can effectively block the binding of PD-1 to PDL-1.

[0023] Furthermore, the inventors have creatively prepared humanized anti-CD73 antibodies (designated as 19F3H2L2, 19F3H2L3, 19F3H2L3(hG1M) and 19F3H2L3(hG1TM) respectively) and humanized anti-PD-1 antibodies (designated as 14C12H1L1 and 14C12H1L1(hG1TM)).

[0024] Furthermore, the inventors creatively fused two kinds of humanized antibodies into a new antibody through protein recombination, and obtained a humanized bifunctional antibody (named P1D7V01, P1D7V03, NTPDV1, NTPDV2, NTPDV3 and NTPDV4, respectively (also referred to as NTPDV1(hG1TM), NTPDV2(hG1TM), NTPDV3(hG1TM) and NTPDV4(hG1TM) in this specification and in China Patent Application No. 202110270671.X)) that can bind to CD73 and PD-1, inhibit the activity of CD73, and block the binding of PD-1 to PDL-1, and has the potential to be used in the preparation of medicines for preventing and treating solid tumors and hematological tumors.

[0025] The present invention will be described in detail below.

[0026] One aspect of the present invention is a first protein functional region that targets PD-1; and Second protein functional domains that target CD73 The present invention relates to an anti-CD73 / anti-PD-1 bispecific antibody comprising the compound

[0027] In one embodiment of the invention, the bispecific antibody comprises The first protein functional region is HCDR1, HCDR2 and HCDR3 contained in a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 44 (wherein the amino acid sequences of HCDR1, HCDR2 and HCDR3 are preferably the sequences set forth in SEQ ID NOs: 45 to 47, respectively, or are sequences having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, sequence identity to the sequences set forth in SEQ ID NOs: 45 to 47, or are amino acid sequences having one or more (preferably, 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the sequences set forth in SEQ ID NOs: 45 to 47); and comprising LCDR1, LCDR2 and LCDR3 contained in a light chain variable region having the amino acid sequence set forth in SEQ ID NO:49 (wherein the amino acid sequences of LCDR1, LCDR2 and LCDR3 are preferably sequences set forth in SEQ ID NOs:50 to 52, respectively, or sequences having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, sequence identity to the sequences set forth in SEQ ID NOs:50 to 52, or amino acid sequences having one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the sequences set forth in SEQ ID NOs:50 to 52); The second protein functional region is HCDR1, HCDR2 and HCDR3 contained in a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 2 (wherein the amino acid sequences of HCDR1, HCDR2 and HCDR3 are preferably the sequences set forth in SEQ ID NOs: 3 to 5, or sequences having at least 80%, 81%, 82%, 83%, 84% 85%, 86%, 87%, 88%, 89% or 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, sequence identity to the sequences set forth in SEQ ID NOs: 3 to 5, or amino acid sequences having one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the sequences set forth in SEQ ID NOs: 3 to 5); and and LCDR1, LCDR2 and LCDR3 contained in a light chain variable region having the amino acid sequence set forth in SEQ ID NO:7 (wherein the amino acid sequences of LCDR1, LCDR2 and LCDR3 are preferably the sequences set forth in SEQ ID NOs:8 to 10, respectively, or are sequences having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, sequence identity to the sequences set forth in SEQ ID NOs:8 to 10, or are amino acid sequences having one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the sequences set forth in SEQ ID NOs:8 to 10).

[0028] In one embodiment of the invention, the anti-CD73 / anti-PD-1 bispecific antibody comprises: The first protein functional region is a sequence having an amino acid sequence as set forth in SEQ ID NO: 44 or SEQ ID NO: 62, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, sequence identity to said sequence as set forth in SEQ ID NO: 44 or 62, 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 as set forth in SEQ ID NO: 44 or 62; and a sequence having an amino acid sequence as set forth in SEQ ID NO: 49 or SEQ ID NO: 64, respectively, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to said sequence as set forth in SEQ ID NO: 49 or 64, 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 as set forth in SEQ ID NO: 49 or 64; and / or the second protein functional region comprises a sequence having an amino acid sequence as set forth in SEQ ID NO:2 or SEQ ID NO:20, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, sequence identity to said sequence as set forth in SEQ ID NO:2 or 20, 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 as set forth in SEQ ID NO:2 or 20; and a sequence having the amino acid sequence as set forth in SEQ ID NO: 7 or SEQ ID NO: 22, respectively, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, sequence identity to said sequence as set forth in SEQ ID NO: 7 or 22, or an amino acid sequence which has one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to said sequence as set forth in SEQ ID NO: 7 or 22; or the second protein functional region comprises an amino acid sequence as set forth in SEQ ID NO:20, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to said sequence as set forth in SEQ ID NO:20, 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 as set forth in SEQ ID NO:20; and The present invention relates to a sequence having an amino acid sequence as set forth in SEQ ID NO: 24, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, sequence identity to said sequence as set forth in SEQ ID NO: 24, 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 as set forth in SEQ ID NO: 24.

[0029] One aspect of the present invention is a first protein functional region that targets CD73; and A second protein functional domain that targets PD-1 The present invention relates to an anti-CD73 / anti-PD-1 bispecific antibody comprising the compound

[0030] In one embodiment of the invention, the bispecific antibody comprises the first protein functional region is HCDR1, HCDR2 and HCDR3 contained in a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:2 (wherein the amino acid sequences of HCDR1, HCDR2 and HCDR3 are preferably the sequences set forth in SEQ ID NOs:3 to 5, respectively, or have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, sequence identity to the sequences set forth in SEQ ID NOs:3 to 5, or have one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the sequences set forth in SEQ ID NOs:3 to 5); and comprising LCDR1, LCDR2 and LCDR3 contained in a light chain variable region having the amino acid sequence set forth in SEQ ID NO:7 (wherein the amino acid sequences of LCDR1, LCDR2 and LCDR3 are preferably the sequences set forth in SEQ ID NOs:8 to 10, respectively, or are sequences having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, sequence identity to the sequences set forth in SEQ ID NOs:8 to 10, or are amino acid sequences having one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the sequences set forth in SEQ ID NOs:8 to 10); the second protein functional region is HCDR1, HCDR2 and HCDR3 contained in a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 44 (wherein the amino acid sequences of HCDR1, HCDR2 and HCDR3 are preferably the sequences set forth in SEQ ID NOs: 45 to 47, respectively, or are sequences having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, sequence identity to the sequences set forth in SEQ ID NOs: 45 to 47, or are amino acid sequences having one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the sequences set forth in SEQ ID NOs: 45 to 47); and and LCDR1, LCDR2 and LCDR3 contained in a light chain variable region having the amino acid sequence set forth in SEQ ID NO:49 (wherein the amino acid sequences of LCDR1, LCDR2 and LCDR3 are preferably the sequences set forth in SEQ ID NOs:50 to 52, respectively, or are sequences having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, sequence identity to the sequences set forth in SEQ ID NOs:50 to 52, or are amino acid sequences having one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the sequences set forth in SEQ ID NOs:50 to 52).

[0031] In one embodiment of the invention, the anti-CD73 / anti-PD-1 bispecific antibody comprises: The first protein functional domain is a sequence having an amino acid sequence as set forth in SEQ ID NO: 2 or SEQ ID NO: 20, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, sequence identity to said sequence as set forth in SEQ ID NO: 2 or 20, 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 as set forth in SEQ ID NO: 2 or 20; and a sequence having an amino acid sequence as set forth in SEQ ID NO: 7, SEQ ID NO: 22 or SEQ ID NO: 24, respectively, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to said sequence as set forth in SEQ ID NO: 7, SEQ ID NO: 22 or SEQ ID NO: 24, respectively, 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 as set forth in SEQ ID NO: 7, 22 or 24; and / or The second protein functional domain is a sequence having an amino acid sequence as set forth in SEQ ID NO: 44 or SEQ ID NO: 62, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, sequence identity to said sequence as set forth in SEQ ID NO: 44 or SEQ ID NO: 62, 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 as set forth in SEQ ID NO: 44 or SEQ ID NO: 62; and The present invention relates to a sequence having an amino acid sequence as set forth in SEQ ID NO: 49 or 64, respectively, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, sequence identity to said sequence as set forth in SEQ ID NO: 49 or 64, 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 as set forth in SEQ ID NO: 49 or 64.

[0032] In one embodiment of the invention, in the anti-CD73 / anti-PD-1 bispecific antibody, the first protein functional domain and the second protein functional domain are linked directly or via a linker; preferably, the linker is (GGGGS)n, and n is a positive integer, such as 1, 2, 3, 4, 5 or 6.

[0033] In one embodiment of the invention, the first protein functional domain and the second protein functional domain in the anti-CD73 / anti-PD-1 bispecific antibody are independently an immunoglobulin or antigen-binding fragment, such as a half antibody, Fab, F(ab')2, or single chain variable domain fragment, preferably the first protein functional domain is an immunoglobulin and the second protein functional domain is an antigen-binding fragment, or the first protein functional domain is an antigen-binding fragment and the second protein functional domain is an immunoglobulin.

[0034] In one embodiment of the invention, the N-terminus of the heavy chain variable region of the antigen-binding fragment is linked directly (or via a linker) to the C-terminus of the CH1 of an immunoglobulin and the N-terminus of the light chain variable region of the antigen-binding fragment is linked directly (or via a linker) to the C-terminus of the light chain variable region CL of an immunoglobulin; or the N-terminus of the heavy chain variable region of the antigen-binding fragment is linked directly (or via a linker) to the C-terminus of the light chain variable region CL of an immunoglobulin and the N-terminus of the light chain variable region of the antigen-binding fragment is linked directly (or via a linker) to the C-terminus of the heavy chain variable region CH1 of an immunoglobulin.

[0035] In one embodiment of the invention, the C-terminus of the heavy chain variable region of the antigen-binding fragment is linked directly (or via a linker) to the N-terminus of an immunoglobulin heavy chain and the C-terminus of the light chain variable region of the antigen-binding fragment is linked directly (or via a linker) to the N-terminus of an immunoglobulin light chain; or the C-terminus of the heavy chain variable region of the antigen-binding fragment is linked directly (or via a linker) to the N-terminus of an immunoglobulin light chain and the C-terminus of the light chain variable region of the antigen-binding fragment is linked directly (or via a linker) to the N-terminus of an immunoglobulin heavy chain.

[0036] In one embodiment of the invention, the antigen-binding fragment is a single chain variable region fragment; preferably, the first protein functional region is an immunoglobulin and the second protein functional region is a single chain variable region fragment; or the first protein functional region is a single chain variable region fragment and the second protein functional region is an immunoglobulin.

[0037] In one embodiment of the invention said bispecific antibody is provided, wherein the number of said first protein functional domain and said second protein functional domain are each independently one, two or more.

[0038] In one embodiment of the present invention, in the anti-CD73 / anti-PD-1 bispecific antibody, the single chain variable region fragment is an antibody heavy chain variable region (V H ) and the antibody light chain variable region (V L ) via a linker; preferably, the single chain variable region fragment has the general structure: NH2-V L -Linker-V H -COOH or NH2-V H -Linker-V L It may have -COOH.

[0039] In one embodiment of the present invention, in the anti-CD73 / anti-PD-1 bispecific antibody, the single chain variable region fragment is a C-terminus (C H) (or the N-terminus of the heavy chain, the C-terminus of the CH1 of the heavy chain variable region) via a linker, H ) is linked first, or the antibody light chain variable region (V L ) may be concatenated first; Preferably, the single chain variable region fragment has the general structure: H -Linker-V H -Linker-V L -COOH or C H -Linker-V L -Linker-V H may have -COOH; Preferably, the heavy chain variable region of the immunoglobulin comprises a CDR having an amino acid sequence set forth in any one of SEQ ID NOs: 3 to 5, and the light chain variable region of the immunoglobulin comprises a CDR having an amino acid sequence set forth in any one of SEQ ID NOs: 8 to 10; the heavy chain variable region of the single-chain variable region fragment comprises CDRs having the amino acid sequences set forth in SEQ ID NOs: 45 to 47, and the light chain variable region of the single-chain variable region fragment comprises CDRs having the amino acid sequences set forth in SEQ ID NOs: 50 to 52; Preferably, a single chain variable region fragment (e.g., NH2-V L -Linker-V H -COOH or NH2-V H -Linker-V L When linked to the C-terminus of the heavy chain of an immunoglobulin via a linker, the antibody heavy chain variable region (V H ) is first linked to the antibody light chain variable region (V L ) may be concatenated first, Or preferably, the heavy chain variable region of the immunoglobulin comprises a CDR having an amino acid sequence as set forth in SEQ ID NOs: 45 to 47, and the light chain variable region of the immunoglobulin comprises a CDR having an amino acid sequence as set forth in SEQ ID NOs: 50 to 52; and / or the heavy chain variable region of the single-chain variable region fragment comprises CDRs having the amino acid sequences set forth in SEQ ID NOs: 3 to 5, and the light chain variable region of the single-chain variable region fragment comprises CDRs having the amino acid sequences set forth in SEQ ID NOs: 8 to 10; Here, the single chain variable region fragment (e.g., NH2-V L -Linker-V H -COOH or NH2-V H -Linker-V L When the C-terminus of the immunoglobulin heavy chain is linked to the C-terminus of the immunoglobulin heavy chain via a linker, the antibody heavy chain variable region (V H ) is linked first, or a single-chain variable region fragment of an antibody light chain variable region (V L ) may be concatenated first, Preferably, One immunoglobulin molecule is linked to two single chain variable region fragment molecules, and more preferably, the two single chain variable region fragment molecules are identical.

[0040] In one embodiment of the invention, in the anti-CD73 / anti-PD-1 bispecific antibody, the immunoglobulin is IgG, IgA, IgD, IgE or IgM, preferably IgG, such as IgG1, IgG2, IgG3 or IgG4.

[0041] In one embodiment of the present invention, in the anti-CD73 / anti-PD-1 bispecific antibody, the single-chain variable region fragment is linked to the C-terminus of the heavy chain of an immunoglobulin. Since an immunoglobulin consists of two heavy chains, two single-chain variable region molecules are bound to one immunoglobulin molecule. Preferably, the two single-chain variable region fragment molecules are identical.

[0042] In one embodiment of the invention, the anti-CD73 / anti-PD-1 bispecific antibody comprises: the heavy chain variable region of the immunoglobulin comprises CDRs having the amino acid sequences set forth in SEQ ID NOs: 3 to 5, and the light chain variable region of the immunoglobulin comprises CDRs having the amino acid sequences set forth in SEQ ID NOs: 8 to 10; and / or the heavy chain variable region of the single-chain variable region fragment comprises CDRs having the amino acid sequences set forth in SEQ ID NOs: 45 to 47, and the light chain variable region of the single-chain variable region fragment comprises CDRs having the amino acid sequences set forth in SEQ ID NOs: 50 to 52; Preferably, a single chain variable region fragment (e.g., NH2-V L -Linker-V H -COOH or NH2-V H -Linker-V L When the C-terminus of the immunoglobulin heavy chain is linked to the C-terminus of the immunoglobulin heavy chain via a linker, the antibody heavy chain variable region (V H ) is linked first, or a single-chain variable region fragment containing a CDR having the amino acid sequence set forth in SEQ ID NOs: 50 to 52 (V L ) may be concatenated first.

[0043] In another embodiment of the invention, the anti-CD73 / anti-PD-1 bispecific antibody comprises the heavy chain variable region of the immunoglobulin comprises a CDR having an amino acid sequence as set forth in SEQ ID NOs: 45 to 47, and the light chain variable region of the immunoglobulin comprises a CDR having an amino acid sequence as set forth in SEQ ID NOs: 50 to 52; and / or the heavy chain variable region of the single-chain variable region fragment comprises CDRs having the amino acid sequences set forth in SEQ ID NOs: 3 to 5, and the light chain variable region of the single-chain variable region fragment comprises CDRs having the amino acid sequences set forth in SEQ ID NOs: 8 to 10; Here, a single chain variable region fragment (e.g., NH2-V L -Linker-V H -COOH or NH2-V H -Linker-V LWhen the C-terminus of the immunoglobulin heavy chain is linked to the C-terminus of the immunoglobulin heavy chain via a linker, the antibody heavy chain variable region (V H ) is linked first, or a single-chain variable region fragment of an antibody light chain variable region (V L ) may be concatenated first.

[0044] In one embodiment of the invention, the anti-CD73 / anti-PD-1 bispecific antibody comprises: the heavy chain variable region of the immunoglobulin has an amino acid sequence selected from SEQ ID NO:2 and SEQ ID NO:20, and the light chain variable region of the immunoglobulin has an amino acid sequence selected from SEQ ID NO:7 and SEQ ID NO:22, respectively; or the heavy chain variable region of the immunoglobulin has an amino acid sequence set forth in SEQ ID NO:20, and the light chain variable region of the immunoglobulin has an amino acid sequence set forth in SEQ ID NO:24; and / or the heavy chain variable region of the single chain variable region fragment has an amino acid sequence selected from SEQ ID NO:44 and SEQ ID NO:62, and the light chain variable region of the single chain variable region fragment has an amino acid sequence selected from SEQ ID NO:49 and SEQ ID NO:64, respectively; Here, when the single-chain variable region fragment is linked to the C-terminus of the heavy chain of an immunoglobulin via a linker, the antibody heavy chain variable region (V H ) is linked to the antibody light chain variable region of the single chain variable region fragment (V L ) may be concatenated first.

[0045] In another embodiment of the invention, the anti-CD73 / anti-PD-1 bispecific antibody comprises the heavy chain variable region of the immunoglobulin has an amino acid sequence selected from SEQ ID NO:44 and SEQ ID NO:62; the light chain variable region of the immunoglobulin has an amino acid sequence selected from SEQ ID NO:49 and SEQ ID NO:64, respectively; or the heavy chain variable region of the single chain variable region fragment has an amino acid sequence selected from SEQ ID NO:2 and SEQ ID NO:20 and the light chain variable region of the single chain variable region fragment has an amino acid sequence selected from SEQ ID NO:7 and SEQ ID NO:22, respectively; or the heavy chain variable region of the single chain variable region fragment has the amino acid sequence set forth in SEQ ID NO:20 and the light chain variable region of the single chain variable region fragment has the amino acid sequence set forth in SEQ ID NO:24.

[0046] Another aspect of the invention relates to an isolated nucleic acid molecule comprising a nucleotide sequence capable of encoding a heavy chain variable region of a bispecific antibody, wherein The heavy chain variable region of the antibody comprises CDRs having the amino acid sequences of SEQ ID NOs: 3 to 5; CDRs having the amino acid sequences of SEQ ID NOs: 45 to 47; and CDRs having the amino acid sequences of SEQ ID NOs: 50 to 52; and the heavy chain variable region of the bispecific antibody specifically binds to CD73 and PD-1 antigens as part of the bispecific antibody, and the bispecific antibody further comprises a light chain variable region comprising CDRs having the amino acid sequences of SEQ ID NOs: 8 to 10; Preferably, the CDRs of the light chain variable region are different from the CDRs of the heavy chain variable region.

[0047] In one embodiment of the invention, the bispecific antibody comprises The immunoglobulin comprises non-CDR regions derived from a species other than mouse, such as from a human antibody.

[0048] In one embodiment of the invention, the immunoglobulin constant regions are humanized, for example, the heavy chain constant region is the Igγ1 chain C region, accession number P01857; and the light chain constant region is the Igκ chain C region, accession number P01834.

[0049] In one embodiment of the present invention, the immunoglobulin constant region is humanized, for example, the heavy chain constant region is Igγ1 chain C region, accession number P01857; and the light chain constant region is Igκ chain C region, accession number P01834; where the heavy chain constant region of the immunoglobulin comprises mutations at any two or three positions of 234, 235 and 237 according to the EU numbering system, and the affinity constant of the bispecific antibody to FcγRIa, FcγRIIIa and / or C1q is decreased after the mutations compared to that before the mutations; preferably, the affinity constant is measured by Fortebio's Octet system.

[0050] In one embodiment of the invention, for said bispecific antibody, the immunoglobulin heavy chain constant region has at positions 234, 235 and / or 237 the following, according to the EU numbering system: L234A and L235A; L234A and G237A; L235A and G237A; or L234A, L235A and G237A It has a mutation.

[0051] In one or more embodiments of the invention, unless otherwise specified, the letter before the position number indicates the amino acid before the mutation, and the letter after the position number indicates the amino acid after the mutation.

[0052] In one or more embodiments of the invention, the bispecific antibody comprises an immunoglobulin heavy chain constant region that is selected from the group consisting of: N297A, D265A, D270A, P238D, L328E, E233D, H268D, P271G, A330R, C226S, C229S, E233P, P331S, S267E, L328F, A330L, M252Y, S254T, T256E, N297Q, P238S, P238A, A327Q, A327G, P329A, K322A, T394D, G236R, G236A, L328R, A330S, P331S, H268A, E318A and K320A The present invention has one or more mutations selected from the following:

[0053] In a specific embodiment, the anti-CD73 / anti-PD-1 bispecific antibody has a structure shown as heavy chain-light chain-linker1-scFv, and the scFv is selected from 14C12H1V-linker2-14C12L1V, 14C12H1V-linker1-14C12L1V, 14C12H1V-linker2-14C12L1V and 14C12H1V-linker1-14C12L1V, specifically as follows: (1) NTPDV1, the heavy chain of which has the amino acid sequence set forth in SEQ ID NO: 85; the light chain of which has the amino acid sequence set forth in SEQ ID NO: 28, linker 1 has the amino acid sequence set forth in SEQ ID NO: 79, 14C12H1V has the amino acid sequence set forth in SEQ ID NO: 66, linker 2 has the amino acid sequence set forth in SEQ ID NO: 81, and 14C12L1V has the amino acid sequence set forth in SEQ ID NO: 68; (2) NTPDV2, the heavy chain of which has the amino acid sequence set forth in SEQ ID NO: 85, the light chain of which has the amino acid sequence set forth in SEQ ID NO: 28, linker 1 of which has the amino acid sequence set forth in SEQ ID NO: 79, 14C12H1V of which has the amino acid sequence set forth in SEQ ID NO: 66, linker 1 of which has the amino acid sequence set forth in SEQ ID NO: 79, and 14C12L1V of which has the amino acid sequence set forth in SEQ ID NO: 68; (3) NTPDV3, the heavy chain of which has the amino acid sequence set forth in SEQ ID NO: 85, the light chain of which has the amino acid sequence set forth in SEQ ID NO: 96, linker 1 of which has the amino acid sequence set forth in SEQ ID NO: 79, 14C12H1V of which has the amino acid sequence set forth in SEQ ID NO: 66, linker 2 of which has the amino acid sequence set forth in SEQ ID NO: 81, and 14C12L1V of which has the amino acid sequence set forth in SEQ ID NO: 68; and (4) NTPDV4, the heavy chain of which has the amino acid sequence set forth in SEQ ID NO: 85, the light chain of which has the amino acid sequence set forth in SEQ ID NO: 96, linker 1 having the amino acid sequence set forth in SEQ ID NO: 79, 14C12H1V having the amino acid sequence set forth in SEQ ID NO: 66, linker 1 having the amino acid sequence set forth in SEQ ID NO: 79, and 14C12L1V having the amino acid sequence set forth in SEQ ID NO: 68; is selected from the group consisting of:

[0054] In one embodiment of the invention, the bispecific antibody binds to the CD73 protein and / or the PD-1 protein at a concentration of about 10 -5 Less than M, e.g., about 10 -6 Less than M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 K below M D Combine with.

[0055] Yet another aspect of the present invention pertains to vectors comprising the isolated nucleic acid molecules disclosed herein.

[0056] Yet another aspect of the present invention pertains to a host cell comprising the isolated nucleic acid molecule or vector disclosed herein.

[0057] Yet another aspect of the present invention relates to a method for preparing a bispecific antibody as disclosed herein, comprising culturing a host cell as disclosed herein under suitable conditions and isolating the bispecific antibody from the cell culture.

[0058] Yet another aspect of the present invention relates to a conjugate comprising a bispecific antibody and a conjugated moiety, wherein the bispecific antibody is a bispecific antibody disclosed herein and said conjugated moiety is a detectable label; in particular, said conjugated moiety is a radioisotope, a fluorescent substance, a chemiluminescent substance, a colored substance or an enzyme.

[0059] Yet another aspect of the present invention relates to a kit comprising a bispecific antibody as disclosed herein or comprising a conjugate as disclosed herein; preferably, said kit further comprises a secondary antibody that specifically recognizes the bispecific antibody; optionally, said secondary antibody further comprises a detectable label, such as a radioisotope, a fluorescent substance, a chemiluminescent substance, a colored substance or an enzyme.

[0060] Yet another aspect of the present invention relates to the use of a bispecific antibody as disclosed herein in the preparation of a kit for detecting the presence or level of CD73 and / or PD-1 in a sample.

[0061] Yet another aspect of the present invention relates to a pharmaceutical composition comprising a bispecific antibody as disclosed herein or a conjugate as disclosed herein; optionally, further comprising a pharma- ceutically acceptable carrier and / or excipient.

[0062] Yet another aspect of the present invention relates to the use of a bispecific antibody as disclosed herein or a conjugate as disclosed herein in preventing and / or treating tumors or anemia or in diagnosing tumors or anemia.

[0063] Yet another aspect of the present invention relates to the use of a bispecific antibody as disclosed herein or a conjugate as disclosed herein in preparing a medicament for the prevention and / or treatment of a tumor or anemia or in preparing a medicament for the diagnosis of a tumor or anemia.

[0064] Yet another aspect of the present invention relates to the use of a bispecific antibody as disclosed herein, or an agent for detecting the level of CD73 in a sample; Agents for inhibiting the enzymatic activity of CD73; and / or an agent for blocking the binding of PD-1 to PDL-1; Agents for downregulating (e.g., downregulating) the activity or levels of PD-1; Agents for alleviating the immunosuppression of PD-1 in an organism; an agent for increasing IL-2 expression in T lymphocytes, or Agents for increasing IFN-γ expression in T lymphocytes The present invention relates to the use of the conjugates disclosed herein in the preparation of

[0065] Yet another aspect of the present invention relates to an in vivo or in vitro method comprising administering an effective amount of a bispecific antibody as disclosed herein or a conjugate as disclosed herein to a cell or to a subject in need thereof.

[0066] The anti-CD73 / anti-PD-1 bispecific antibodies described herein can inhibit the enzymatic activity of CD73 cell membrane surface and induce the secretion of IFNγ and IL-2 to activate the immune response.

[0067] The light and heavy chain variable regions determine the antigen binding; the variable region of each chain contains three hypervariable regions called complementarity determining regions (CDRs) (heavy (H) chain CDRs include HCDR1, HCDR2 and HCDR3, and light (L) chain 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.

[0068] Preferably, the CDRs may be defined by the IMGT numbering system, see Ehrenmann F, Kaas Q, and Lefranc M P., IMGT / 3Dstructure-DB and IMGT / DomainGapAlign: a database and a tool for immunoglobulins or antibodies, T cell receptors, MHC, IgSF and MhcSF[J]. Nucleic acids research 2009; 38(suppl_1): D301-D307.

[0069] The amino acid sequences of the CDRs of the monoclonal antibodies in (1) to (11) below were analyzed by technical means well known to those skilled in the art, for example, according to the definition of IMGT, and the results are as follows: (1) 19F3F The heavy chain variable region has the amino acid sequence set forth in SEQ ID NO:2, and the b light chain variable region has the amino acid sequence set forth in SEQ ID NO:7. The three CDRs of the heavy chain variable region have the following amino acid sequences: HCDR1: GYSFTGYT (SEQ ID NO: 3), HCDR2: INPYNAGT (SEQ ID NO: 4), and HCDR3: ARSEYRYGGDYFDY (SEQ ID NO:5); The three CDRs of the light chain variable region have the following amino acid sequences: LCDR1: QSLLNSSNQKNY (SEQ ID NO: 8), LCDR2:FAS (SEQ ID NO: 9), and LCDR3: has QQHYDTPYT (sequence number 10). (2)19F3H2L2 The heavy chain variable region has the amino acid sequence set forth in SEQ ID NO:20, and the light chain variable region has the amino acid sequence set forth in SEQ ID NO:22. The three CDRs of the heavy chain variable region have identical amino acid sequences to 19F3. The three CDRs of the light chain variable region have identical amino acid sequences to 19F3. (3)19F3H2L3 The heavy chain variable region has the amino acid sequence set forth in SEQ ID NO:20, and the light chain variable region has the amino acid sequence set forth in SEQ ID NO:24. The three CDRs of the heavy chain variable region have identical amino acid sequences to 19F3. The three CDRs of the light chain variable region have identical amino acid sequences to 19F3. (4)14C12 The heavy chain variable region has the amino acid sequence set forth in SEQ ID NO:44, and the light chain variable region has the amino acid sequence set forth in SEQ ID NO:49. The three CDRs of the heavy chain variable region have the following amino acid sequences: HCDR1: GFAFSSYD (SEQ ID NO: 45) HCDR2: ISGGGRYT (SEQ ID NO: 46) HCDR3:ANRYGEAWFAY (sequence number 47). The three CDRs of the light chain variable region have the following amino acid sequences: LCDR1: QDINTY (sequence number 50) LCDR2: RAN (SEQ ID NO: 51) LCDR3: Has LQYDEFPLT (sequence number 52). (5)14C12H1L1L1 The heavy chain variable region has the amino acid sequence set forth in SEQ ID NO:62, and the light chain variable region has the amino acid sequence set forth in SEQ ID NO:64. The three CDRs of the heavy chain variable region have identical amino acid sequences to 14C12. The three CDRs of the light chain variable region have identical amino acid sequences to 14C12. (6) The nine CDRs of the heavy chains of NTPDV1, NTPDV2, NTPDV3 and NTPDV4 have, from the N-terminus to the C-terminus, the same amino acid sequences as the CDRs of the 13F9 heavy chain, the 14C12 heavy chain, the 14C12 light chain and the 14C12, respectively. The sequence of the above is as follows: HCDR1: GYSFTGYT (SEQ ID NO: 3) HCDR2: INPYNAGT (SEQ ID NO: 4) HCDR3: ARSEYRYGGDYFDY (SEQ ID NO:5) HCDR4: GFAFSSYD (SEQ ID NO: 45) HCDR5: ISGGGRYT (SEQ ID NO: 46) HCDR6: ANRYGEAWFAY (SEQ ID NO: 47) HCDR7: QDINTY (SEQ ID NO: 50) HCDR8: RAN (SEQ ID NO: 51) HCDR9: LQYDEFPLT (SEQ ID NO:52) The three CDRs of the light chain have identical amino acid sequences to the three CDRs of the 19F3 light chain, and the sequences are as follows: LCDR1: QSLLNSSNQKNY (SEQ ID NO: 8) LCDR2: FAS (SEQ ID NO: 9) Lcdr3: QQHYDTPYT (sequence number 10).

[0070] Yet another aspect of the present invention relates to the hybridoma cell line LT014, deposited at the China Culture Collection Center (CCTCC) with collection number CCTCCNO:C2018137.

[0071] Yet another aspect of the present invention relates to the hybridoma cell line LT003, deposited at the China Culture Collection Center (CCTCC) with collection number CCTCCNO:C2015105.

[0072] 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 laboratory procedures of cell culture, molecular genetics, nucleic acid chemistry and immunology used herein are routine procedures widely used in the corresponding fields. In addition, in order to better understand the present invention, the definitions and explanations of related terms are provided below.

[0073] As used herein, the term "EC 50 " refers to the concentration of 50% of the maximal effect, i.e. the concentration capable of producing 50% of the maximal effect.

[0074] As used herein, the term "antibody" refers to an immunoglobulin molecule that generally consists of two pairs of polypeptide chains, each pair having one "light" (L) chain and one "heavy" (H) chain. Antibody light chains are classified as kappa and lambda light chains. Heavy chains are classified as mu, delta, gamma, alpha, or epsilon. Antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE. In the light and heavy chains, the variable and constant regions are joined by a "J" region of about 12 or more amino acids, and the heavy chains further include a "D" region of about 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains (CH1, CH2, and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain CL. The constant region of an antibody can mediate the binding of the immunoglobulin to host tissues or factors, including the binding of various cells of the immune system (e.g., effector cells) to the first component (C1q) of the classical complement system. The VH and VL regions can be further subdivided into highly variable regions (called complementarity determining regions (CDRs)) between which conserved regions called framework regions (FRs) are distributed. VH and VL each consist of three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of each heavy / light chain pair (VH and VL) form the antibody binding site.The assignment of amino acids to regions or domains is based on Bethesda Md, Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, (1987 and 1991)), or Chothia & Lesk J. Mol. Biol., 1987; 196:901-917; Chothia et al., Nature, 1989; 342:878-883, or the definition of the IMGT numbering system: the definition in Ehrenmann F, Kaas Q, Lefranc M P., IMGT / 3Dstructure-DB and IMGT / DomainGapAlign: a database and a tool for immunoglobulins or antibodies, T cell receptors, MHC, IgSF and MhcSF[J]., Nucleic acids research, 2009; 38(suppl_1): D301-D307.

[0075] In particular, the heavy chain may comprise three or more CDRs, such as 6, 9 or 12. For example, in the bispecific antibodies disclosed herein, the heavy chain may be the heavy chain of an IgG antibody linked at its C-terminus to one ScFv, in which case the heavy chain comprises nine CDRs.

[0076] The term "antibody" is not limited by any particular method for producing the antibody. For example, the antibody includes recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. The antibody may be of various isotypes, such as IgG (e.g., subtypes IgG1, IgG2, IgG3, or IgG4), IgA1, IgA2, IgD, IgE, or IgM.

[0077] As used herein, the terms "mAb" and "monoclonal antibody" refer to an antibody or antibody fragment that is derived from a group of highly homologous antibodies, i.e., from a group of identical antibody molecules, except for natural mutations that may occur naturally. Monoclonal antibodies are highly specific to a single epitope on an antigen. Polyclonal antibodies, in comparison to monoclonal antibodies, generally contain at least two or more different antibodies that usually recognize different epitopes on an antigen. Monoclonal antibodies can generally be obtained using hybridoma technology, first reported by Kohler et al. (Kohler 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. Pat. No. 4,816,567).

[0078] As used herein, the term "humanized antibody" refers to an antibody or antibody fragment obtained when all or part of the CDR regions of a human immunoglobulin (receptor antibody) are replaced by the CDR regions of a non-human antibody (donor antibody), where the donor antibody may be a non-human (e.g. mouse, rat or rabbit) antibody with the desired specificity, affinity or reactivity. Furthermore, some amino acid residues in the framework regions (FR) of the receptor antibody can also be replaced by the corresponding amino acid residues of the non-human antibody or by amino acid residues of other antibodies to further improve or optimize the performance of the antibody. For further 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, Immunol. Today, 2000; 21: 397-402. In some cases, the antigen-binding fragment of an antibody may be a V H Domain and V L Bispecific antibodies are diabodies in which the domains are expressed on a single polypeptide chain. However, the linker used is too short to pair the two domains on the same chain. This forces the domains to pair with complementary domains on the other chain, and creates two antigen-binding sites (see, for example, Holliger P. et al., Proc. Natl. Acad. Sci. USA, 1993; 90:6444-6448 and Poljak RJ et al., Structure, 1994; 2:1121-1123).

[0079] As used herein, the term "single chain variable region fragment (ScFv)" refers to an antibody heavy chain variable region (V H ) and the antibody light chain variable region (V L) linked by a linker. The VL and VH domains pair to form a monovalent molecule with the linker allowing them to generate a single peptide chain (see Bird et al, Science, 1988; 242:423-426 and Huston et al, Proc. Natl. Acad. Sci. USA, 1988; 85:5879-5883). Such scFv molecules have the general structure: NH2-V L -Linker-V H -COOH or NH2-V H -Linker-V L It may have -COOH. A suitable linker in the prior art consists of a repeated GGGGS amino acid sequence or a variant thereof. For example, a linker having the amino acid sequence (GGGGS)4 may be used, but a variant thereof may also be used (Holliger et al., Proc. Natl. Acad. Sci. USA, 1993; 90: 6444-6448). Other linkers that can be used in the present invention are described by Alfthan et al., Protein Eng., 1995; 8:725-731, Choi et al., Eur. J. Immunol., 2001; 31: 94-106, Hu et al., Cancer Res., 1996; 56:3055-3061, Kipriyanov et al., J. Mol. Biol., 1999; 293:41-56 and Roovers et al., Cancer Immunology, Immunotherapy, 2001, 50(1): 51-59.

[0080] As used herein, the term "isolated" means obtained from the natural state by artificial means. When a particular "isolated" substance or component occurs in nature, it may be that its natural environment is altered or that it is isolated from the natural environment, or both. For example, a particular non-isolated polynucleotide or polypeptide naturally occurs in a particular living animal, and the same polynucleotide or polypeptide of high purity isolated from such a natural state is referred to as an isolated polynucleotide or polypeptide. The term "isolated" does not exclude the presence of artificial or synthetic substances, or other impurities that do not affect the activity of the substance.

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

[0082] As used herein, the term "host cell" refers to a cell into which a vector can be introduced, 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 fibroblast cells, CHO cells, GS cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK293 cells, or human cells.

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

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

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

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

[0087] As used herein, the term "effective amount" refers to an amount sufficient to obtain a desired effect or at least partially obtain a desired effect. For example, a prophylactically effective amount for a disease (e.g., tumor) refers to an amount sufficient to prevent, stop or delay the onset of the disease (e.g., tumor); a therapeutically effective amount refers to an amount sufficient to cure or at least partially stop the disease and its complications in a patient suffering from the disease. It is certainly within the capabilities of a person skilled in the art to determine such an effective amount. For example, an effective amount for therapeutic purposes depends on the severity of the disease to be treated, the overall state of the patient's own immune system, the general condition of the patient, such as age, weight and sex, the route of administration, and other treatments used in combination, etc. Beneficial effects

[0088] The monoclonal antibody of the present invention (e.g., 13F9H2L3) can sufficiently and specifically bind to CD73, and can effectively inhibit the enzymatic activity reaction of CD73 in a non-substrate competitive mode, thereby reducing the production of adenosine and promoting the activity of T cells and tumor-inhibiting effect.

[0089] The bispecific antibodies disclosed herein, such as NTPDV1, NTPDV2, NTPDV3 and NTPDV4, can sufficiently and specifically bind to PD-1 and CD73, effectively block the binding of PD-1 to PDL-1, specifically relieve the immune suppression of PD-1 in the organism, inhibit the catalytic activity of CD73, relieve the inhibition of immune cells by adenosine, activate T lymphocytes, and do not cause the release of cytokines IL-8 and IL-6, effectively improving safety and efficacy.

[0090] The bifunctional antibodies disclosed herein have the potential to be used in the preparation of anti-tumor drugs. [Brief description of the drawings]

[0091] [Figure 1] Binding of P1D7V01, P1D7V02R, P1D7V03, P1D7V04R, 14C12H1L1 and nivolumab to PD-1-mFc measured by ELISA. [Diagram 2] Binding of P1D7V01, P1D7V02R, P1D7V03, P1D7V04R, 19F3H2L3 and MEDI9447 to human NT5E-biotin measured by ELISA. [Diagram 3] Activity of P1D7V01, P1D7V02R, P1D7V03, P1D7V04R, 14C12H1L1 and nivolumab in competing with human PDL-1-mFc for binding to human PD-1-mFc-biotin. [Figure 4] Affinity constant of P1D7V01 for PD-1-mFc. [Diagram 5]Affinity constant of 14C12H1L1 for PD-1-mFc. [Figure 6] Affinity constant of nivolumab for PD-1-mFc. [Figure 7] Affinity constant of P1D7V01 for human NT5E(1-552)-his. [Figure 8] Affinity constant of MEDI9447 for human NT5E(1-552)-his. [Figure 9] Binding activity of P1D7V01, P1D7V02R, P1D7V03, P1D7V04R and 14C12H1L1 to PD-1 on the surface of 293T-PD1 cells as measured by FACS. [Figure 10] Binding activity of P1D7V01, P1D7V03, MEDI9447 and 19F3H2L3 to CD73 on the surface of MDA-MB-231 cells as measured by FACS. [Figure 11] Inhibition of the enzymatic activity of CD73 on the MDA-MB-231 membrane surface by anti-CD73 / anti-PD-1 bispecific antibody. [Figure 12] Inhibition of the enzymatic activity of CD73 on the U87-MG membrane surface by anti-CD73 / anti-PD-1 bispecific antibody. [Figure 13] Biological activity of anti-CD73 / anti-PD-1 bispecific antibodies to promote IFN-γ secretion in the Raji-PDL-1 mixed lymphocyte reaction system. [Figure 14] Biological activity of anti-CD73 / anti-PD-1 bispecific antibodies to promote IL-2 secretion in the Raji-PDL-1 mixed lymphocyte reaction system. [Figure 15] Biological activity of anti-CD73 / anti-PD-1 bispecific antibody to promote IFN-γ secretion in DC mixed lymphocyte reaction system. [Figure 16] Biological activity of anti-CD73 / anti-PD-1 bispecific antibody to promote IL-2 secretion in DC mixed lymphocyte reaction system. [Figure 17] Affinity constant of 14C12H1L1(hG1TM) for PD-1-mFc. [Figure 18]Affinity constant of nivolumab for PD-1-mFc. [Figure 19] Affinity constant of NTPDV1 for PD-1-mFc. [Figure 20] Affinity constant of NTPDV2 for PD-1-mFc. [Figure 21] Affinity constant of NTPDV3 for PD-1-mFc. [Figure 22] Affinity constant of NTPDV4 for PD-1-mFc. [Diagram 23] Affinity constant of 19F3H2L3 (hG1M) for human NT5E(1-552)-his. [Figure 24] Affinity constant of NTPDV1 for human NT5E(1-552)-his. [Diagram 25] Affinity constant of NTPDV2 for human NT5E(1-552)-his. [Figure 26] Affinity constant of NTPDV3 for human NT5E(1-552)-his. [Figure 27] Affinity constant of NTPDV4 for human NT5E(1-552)-his. [Figure 28] Inhibition of the enzymatic activity of CD73 on the membrane surface of U87-MG cells by anti-CD73 / anti-PD-1 bispecific antibody. [Figure 29] Biological activity of anti-CD73 / anti-PD-1 bispecific antibodies to promote the secretion of IFN-γ and IL-2 as determined by mixed lymphocyte reaction (MLR). [Diagram 30] Effect of isotype control, 19F3H2L3(hG1M) and different doses of NTPDV2 on tumor volume in mice. [Diagram 31] Effect of isotype control, 19F3H2L3(hG1M) and different doses of NTPDV2 on mouse body weight. [Diagram 32] Effective ablation of PD-1 / CD73 bispecific antibody-mediated IL-8 secretion in human macrophages by amino acid mutations in the Fc segment in a co-culture system of CHO-K1-PD1 cells and human macrophages. [Diagram 33] Effective ablation of PD-1 / CD73 bispecific antibody-mediated IL-6 secretion in human macrophages by amino acid mutations in the Fc segment in a co-culture system of CHO-K1-PD1 cells and human macrophages. [Diagram 34] Effective ablation of PD-1 / CD73 bispecific antibody-mediated IL-8 secretion in human macrophages by amino acid mutations in the Fc segment in a co-culture system of U87-MG cells and human macrophages. [Diagram 35] Efficient ablation of PD-1 / CD73 bispecific antibody-mediated IL-6 secretion in human macrophages by amino acid mutations in the Fc segment in a co-culture system of U87-MG cells and human macrophages.

[0092] Biomaterials Retrieval Information: The hybridoma cell line LT003 (also called PD-1-14C12) was deposited at the China Culture Collection Center (CCTCC) on June 16, 2015, with collection number CTCCNO: C2015105, and the address of collection was Wuhan University, Wuhan, China, zip code: 430072.

[0093] The hybridoma cell line LT014 (also called CD73-19F3) was deposited at the China Culture Collection Center (CCTCC) on June 21, 2018, with collection number CTCCNO: C2018137, and the recovery address was Wuhan University, Wuhan, China, zip code: 430072. Detailed Description

[0094] The embodiments of the present invention are described in detail below with reference to the examples. Those skilled in the art will appreciate that the following examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention. If no techniques or conditions are specified, the examples may be modified from the techniques or conditions described in the literature in the art (e.g., Molecular Cloning: A Laboratory Manual, authored by J. Sambrook et al., and translated by Peitang Huang et al., 3 rd The experiments were carried out according to the International Publication No. 2002 (2002) (Ed., Science Press) or according to the product manuals. Reagents or equipment used were conventional products available from commercial sources unless the manufacturer was specified. For example, MDA-MB-231 cells and U87-MG cells can be purchased from ATCC.

[0095] In the following examples of the present invention, the BALB / c mice used were purchased from Guangdong Medical Experimental Animal Center.

[0096] In the following examples of the present invention, the positive control antibody MEDI9447 (generic name: oleculab) used is manufactured by Zhongshan Akesobio Co. Ltd., and its sequence is identical to antibody sequence numbers 21 to 24 described in U.S. Patent Publication No. 20160129108A1 of Medmmune Limited.

[0097] In the following examples of the present invention, a commercially available antibody against the same target, Nivolumab (trade name: Opdivo), purchased from Bristol-Myers Squibb, was used.

[0098] In the following examples of the present invention, the cell line 293T-PD1 used was constructed by Zhongshan Akesobio Co. Ltd. The cell line 293T-PD1 was prepared by viral infection of HEK293T cells using a third generation lentivirus system (see, for example, A Third Generation Lentivirus Vector with a Conditional Packaging System. Dull T, Zufferey R, Kelly M, Mandel RJ, Nguyenm, Trono D, and Naldini L., J Virol., 1998. 72(11):8463-8471), in which the lentivirus expression vector used was pCDH-CMV-PD-1FL-Puro (PD1, GenebankID:NM005018; vector pCDH-CMV-Puro, purchased from Youbio, catalog number VT1480).

[0099] In the following examples of the present invention, the cell line Raji-PDL-1 used was constructed by Zhongshan Akesobio Co. Ltd. The cell line Raji-PDL-1 was prepared by viral infection of Raji cells using a third generation lentivirus system (see, for example, A Third Generation Lentivirus Vector with a Conditional Packaging System. Dull T, Zufferey R, Kelly M, Mandel RJ, Nguyenm, Trono D, and Naldini L., J Virol., 1998. 72(11):8463-8471), in which the lentivirus expression vector used was plenti6.3-PDL-1 (PDL-1, GenebankID: NP54862.1; vector plenti6.3, purchased from Invitrogen, catalog number K5315-20).

[0100] In the following examples of the present invention, the cell line CHO-K1-PD1 used was constructed by Zhongshan Akesobio Co. Ltd. The cell line CHO-K1-PD1 was prepared by viral infection of CHO-K1 cells using a third generation lentivirus system (see, for example, A Third Generation Lentivirus Vector with a Conditional Packaging System. Dull T, Zufferey R, Kelly M, Mandel RJ, Nguyenm, Trono D, and Naldini L., J Virol., 1998. 72(11):8463-8471). Here, the lentivirus expression vector used was pCDH-CMV-PD-1FL-Puro (PD1, GenebankID:NM005018; vector pCDH-CMV-Puro, purchased from Youbio, catalog number VT1480).

[0101] Nivolumab (trade name: Opdivo), an anti-PD-1 antibody of the IgG4 subtype harboring the S228P mutation, was used as a control antibody in the samples and was purchased from Bristol-Myers Squibb.

[0102] In the following examples of the present invention, the isotype control antibody used, i.e., hIgG1, is an antibody targeting human anti-hen egg lysozyme (HEL), and the variable region sequence of the antibody is from the study titled "Affinity maturation increases the stability and plasticity of the Fv domain of anti-protein antibodies" reported by Acierno et al. (Acierno et al., J Mol biol., 2007; 374(1): 130-46). In the constant region fragment of hIgG1, the heavy chain constant region is Igγ-1 chain C region, accession number P01857, and the light chain constant region is Igκ chain C region, accession number P01834. hIgG1 was prepared in the laboratory of Zhongshan Akesobio Co.Ltd. Example 1

[0103] Preparation of anti-CD73 antibody 19F3 1. Preparation of Hybridoma Cell Line LT014 The antigen used to prepare anti-CD73 antibodies was human NT5E-his (for NT5E, GenbankID: NP002517.1, position: 1-552). Spleen cells of immunized mice were fused with mouse myeloma cells to prepare hybridoma cells. Using human NT5E-biotin (for NT5E, GenbankID: NP2517.1, position: 1-552) as an antigen, hybridoma cells were screened by indirect ELISA to obtain hybridoma cells capable of secreting antibodies capable of specifically binding to CD73. The hybridoma cells obtained by ELISA screening were subjected to limiting dilution to obtain stable hybridoma cell lines. The hybridoma cell line was named hybridoma cell line LT014, and the monoclonal antibody secreted therefrom was named 19F3. The hybridoma cell line LT014 (also called CD73-19F3) was deposited in the China Culture Collection Center (CCTCC) on June 21, 2018, with collection number CTCCNO: C2018137, and the collection address was Wuhan University, Wuhan, China, zip code: 430072.

[0104] 2. Preparation of Anti-CD73 Antibody 19F3 The cell line LT014 prepared above was cultured in a chemically defined medium (CD medium containing 1% penicillin-streptomycin) in an incubator at 37°C with 5% CO2. After 7 days, the supernatant was collected and purified by high-speed centrifugation, vacuum filtration through a microfiltration membrane and a HiTrap Protein A HP column to obtain antibody 19F3. Example 2

[0105] Sequence analysis of anti-CD73 antibody 19F3 According to the method described in the manual for RNApreppureCell / BacteriaKit (Tiangen, catalog number DP430), mRNA was extracted from the cell line LT014 prepared in Example 1. cDNA was synthesized according to the manual of Invitrogen SuperScript® III First-Strand Synthesis System for RT-PCR and amplified by PCR. The PCR-amplified product was subjected to direct TA cloning according to the manual for the pEASY-T1 Cloning Kit (Transgen CT101). The TA cloned product was directly sequenced and the sequence results are as follows: The nucleotide sequence (363 bp) of the 19F3 heavy chain variable region is set forth in SEQ ID NO:1, and the encoded amino acid sequence (121 aa) is set forth in SEQ ID NO:2. According to the IMGT numbering system, heavy chain CDR1 has the sequence set forth in SEQ ID NO:3, heavy chain CDR2 has the sequence set forth in SEQ ID NO:4, and heavy chain CDR3 has the sequence set forth in SEQ ID NO:5. The nucleotide sequence (339 bp) of the 19F3 light chain variable region is set forth in SEQ ID NO:6, and the encoded amino acid sequence (113 aa) is set forth in SEQ ID NO:7. According to the IMGT numbering system, the light chain CDR1 has the sequence set forth in SEQ ID NO:8, the light chain CDR2 has the sequence set forth in SEQ ID NO:9, and the light chain CDR3 has the sequence set forth in SEQ ID NO:10. The amino acid sequences of the four framework regions (FR-H1 to FR-H4) of the 19F3 heavy chain are set forth in SEQ ID NOs: 11 to 14, respectively. The amino acid sequences of the four framework regions (FR-H1 to FR-H4) of the 19F3 light chain are set forth in SEQ ID NOs: 15 to 18, respectively. Example 3

[0106] Design, preparation and determination of humanized anti-human CD73 antibodies 1. Design of the light and heavy chain sequences of humanized antibodies 19F3H2L3 and 19F3H2L2 Based on 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 mouse antibody 19F3 obtained in Example 2, the variable region sequences of antibodies 19F3H1L1, 19F3H2L3, and 19F3H2L3 were obtained by computer modeling and mutation design. The corresponding heavy chain variable region sequences were 19F3H1 and 19F3H2 (having the amino acid sequences set forth in SEQ ID NOs:93 and 97, respectively), and the light chain variable region sequences were 19F3L1, 19F3L2, and 19F3L3 (having the amino acid sequences set forth in SEQ ID NOs:95, 98, and 99, respectively). The antibody constant region sequences are from the NCBI database: the heavy chain constant region is Ig gamma-1 chain C region, accession number P01857; the light chain constant region is Ig kappa chain C region, accession number P01834. 19F3H2L3 is known as 19F3H2L3(hG1WT) in China Patent Application No. 202110270671.X, in which the light and heavy chain variable regions of 19F3H1L1, 19F3H2L2 and 19F3H2L3 are known as 19F3H1V (or 19F3H1 V ), 19F3H2V (or 19F3H2 V ), 19F3L1V (or 19F3L1 V ), 19F3L2V (or 19F3L2 V ) and 19F3L3V (or 19F3L3 V )

[0107] (1) The sequences of the heavy and light chain variable regions of humanized monoclonal antibody 19F3H1L1 are as follows: The nucleotide sequence of the heavy chain variable region (363 bp) is set forth in SEQ ID NO:92, and the encoded amino acid sequence (121 aa) is set forth in SEQ ID NO:93. The nucleotide sequence of the light chain variable region (339 bp) is set forth in SEQ ID NO:94, and the encoded amino acid sequence (113 aa) is set forth in SEQ ID NO:95. (2) The sequences of the heavy and light chain variable regions of humanized monoclonal antibody 19F3H2L2 are as follows: The nucleotide sequence (363 bp) of the heavy chain variable region 19F3H2 is set forth in SEQ ID NO:19, and the encoded amino acid sequence (121 aa) is set forth in SEQ ID NO:20. The nucleotide sequence (339 bp) of the light chain variable region 19F3L3 is set forth in SEQ ID NO:21, and the encoded amino acid sequence (113 aa) is set forth in SEQ ID NO:22. (3) The sequences of the heavy and light chain variable regions of humanized monoclonal antibody 19F3H2L3 are as follows: The nucleotide sequence (363 bp) of the heavy chain variable region 19F3H2 is set forth in SEQ ID NO:19, and the encoded amino acid sequence (121 aa) is set forth in SEQ ID NO:20. The nucleotide sequence (339 bp) of the light chain variable region 19F3L3 is set forth in SEQ ID NO:23, and the encoded amino acid sequence (113 aa) is set forth in SEQ ID NO:24.

[0108] 2. Preparation of humanized antibodies 19F3H1L1, 19F3H2L2, and 19F3H2L3 The heavy chain constant region used was the Igγ-1 chain C region, accession number P01857; the light chain constant region used was the Igκ chain C region, accession number P01834. The heavy and light chain cDNAs of 19F3H1L1, 19F3H2L2 and 19F3H2L3 were separately cloned into pUC57simple (provided by GenScript) vector to obtain pUC57simple-19F3H1, pUC57simple-19F3L1, pUC57simple-19F3H2, pUC57simple-19F3L2 and pUC57simple-19F3L3. With reference to the standard techniques described in Molecular Cloning: A Laboratory Manual (Second Edition), the heavy and light chain full-length genes synthesized by EcoRI & HindIII digestion were subcloned into the expression vector pcDNA3.1 through digestion with restriction enzymes (EcoRI & HindIII) to obtain expression plasmids pcDNA3.1-19F3H1, pcDNA3.1-19F3L1, pcDNA3.1-19F3H2, pcDNA3.1-19F3L2, and pcDNA3.1-19F3L3, and the heavy and light chain genes of the recombinant expression plasmids were subjected to sequence analysis. Then, the designed gene combinations containing the corresponding light and heavy chain recombinant plasmids (pcDNA3.1-19F3H1 / pcDNA3.1-19F3L1, pcDNA3.1-19F3H2 / pcDNA3.1-19F3L2, and pcDNA3.1-19F3H2 / pcDNA3.1-19F3L3) were separately co-transfected into 293F cells, and the culture medium was harvested and purified. After sequence confirmation, endotoxin-free expression plasmids were prepared and transiently transfected into HEK293 cells for antibody expression. The culture medium was harvested after 7 days and subjected to affinity purification via protein A column to obtain humanized antibodies.

[0109] 3. Design of the light and heavy chain sequences of humanized antibodies 19F3H2L3 (hG1M) and 19F3H2L3 (hG1TM) Based on 19F3H2L3 obtained in Example 3.1, a point mutation from leucine to alanine at position 234 (L234A) and a point mutation from leucine to alanine at position 235 (L235A) were introduced in the heavy chain to obtain 19F3H2L3(hG1M). The nucleotide and amino acid sequences of the heavy chain of 19F3H2L3(hG1M) are set forth in SEQ ID NO:25 and SEQ ID NO:26, respectively; the light chain constant region of 19F3H2L3(hG1M) is Ig κ chain C region accession number P01834, and the nucleotide and amino acid sequences of the light chain of 19F3H2L3(hG1M) are set forth in SEQ ID NO:27 and SEQ ID NO:28, respectively.

[0110] Based on 19F3H2L3 obtained in step 1 of Example 3, 19F3H2L3(hG1TM) was obtained by introducing a point mutation from leucine to alanine at position 234 (L234A), a point mutation from leucine to alanine at position 235 (L235A), and a point mutation from glycine to alanine at position 237 (G237A) in the heavy chain. The nucleotide sequence and amino acid sequence of the heavy chain are set forth in SEQ ID NO:29 and SEQ ID NO:30, respectively; the light chain is identical to 19F3H2L3(hG1M). The amino acid sequences of the four framework regions (FR-H1 to FR-H4) of 19F3H2 are set forth in SEQ ID NO:31 to SEQ ID NO:34, respectively; The amino acid sequences of the four framework regions (FR-H1 to FR-H4) of the 19F3L2 light chain are set forth in SEQ ID NO:35 to SEQ ID NO:38, respectively; and The amino acid sequences of the four framework regions (FR-H1 to FR-H4) of the 19F3L3 light chain are set forth in SEQ ID NO:39 to SEQ ID NO:42, respectively.

[0111] 4. Preparation of humanized antibody 19F3H2L3 (hG1M) The heavy and light chain cDNAs of 19F3H2L3 (hG1M) were separately cloned into vector pUC57Simple (provided by Genscript) to obtain pUC57Simple-19F3H2 (hG1M) and pUC57Simple-19F3L3, respectively. With reference to the standard techniques described in Molecular Cloning: A Laboratory Manual (Second Edition), the heavy and light chain full-length genes synthesized by EcoRI&HindIII digestion were subcloned into expression vector pcDNA3.1 through digestion with restriction enzymes (EcoRI&HindIII) to obtain expression plasmids pcDNA3.1-19F3H2 (hG1M) and pcDNA3.1-19F3L3, and the heavy / light chain genes of the recombinant expression plasmids were subjected to sequence analysis. The designed gene combinations, including the corresponding light and heavy chain recombinant plasmids pcDNA3.1-19F3H2(hG1M) / pcDNA3.1-19F3L3, were then co-transfected into 293F cells, and the culture medium was harvested and purified. After sequence confirmation, endotoxin-free expression plasmids were prepared and transiently transfected into HEK293 cells for antibody expression. The culture medium was harvested after 7 days and subjected to affinity purification via a protein A column to obtain the humanized antibody 19F3H2L3(hG1M). Example 4

[0112] Preparation of anti-PD-1 antibody 14C12 1. Preparation of Hybridoma Cell Line LT003 Spleen cells (purchased from Guangdong Medical Laboratory Animal Center) from immunized BALB / c mice and mouse myeloma cells were fused to hybridoma cells using PD-1-mFc fusion protein (PD-1, GenBank: NM005018, mFc SEQ ID NO: 89) as an antigen, and the hybridoma cells were cultured according to established methods (see, e.g., Stewart, SJ, "Monoclonal Antibody Production", in Basic Methods in Antibody Production and Characterization, Eds. GC Howard and DR Bethell, Boca Raton: CRC Press, 2000). For indirect ELISA, plates were coated with PD-1-hFc (PD-1, GenbankID: NM005018, hFc is human IgGFc purification tag, specifically Igγ-1 chain C region, GenbankID: P01857, position 114-330). Through screening, hybridoma cells secreting new antibodies that specifically bind to PD-1 were obtained. Hybridoma cell lines capable of secreting monoclonal antibodies competing with the ligand PDL-1-hFc (PDL-1, Genbank ID: NP54862.1) for binding to PD-1 were screened by competitive ELISA, and stable hybridoma cell lines were obtained by limiting dilution. LT003 stable cell line (PD-1-14C12) was obtained by limiting dilution, and the secreted monoclonal antibody was named 14C12. The hybridoma cell line LT003 (also called PD-1-14C12) was deposited at the China Culture Collection Center (CCTCC) on June 16, 2015, with collection number CTCCNO: C2015105, and the address of collection was Wuhan University, Wuhan, China, zip code: 430072.

[0113] 2. Preparation of Anti-PD-1 Antibody 14C12 The LT003 cells prepared above were cultured in IMDM medium containing 10% low IgG fetal bovine serum IMDM medium (IMDM medium containing 1% penicillin-streptomycin, cultured in a 5% CO2, 37°C cell incubator). After 7 days, the cell culture supernatant was collected and purified to obtain antibody 14C12. Example 5

[0114] Sequence analysis of anti-PD-1 antibody 14C12 mRNA was extracted from the hybridoma cell line LT003 prepared in Example 1 according to the method described in the manual for the RNAprep pure Cell / Bacteria Kit (Tiangen, catalog number DP430). cDNA was synthesized and amplified by PCR according to the manual of Invitrogen SuperScript® III First-Strand Synthesis System for RT-PCR. The PCR-amplified product was subjected to direct TA cloning according to the manual for the pEASY-T1 Cloning Kit (Transgen CT101). The TA cloned product was directly sequenced and the sequence results are as follows: The nucleotide sequence of the heavy chain variable region (354 bp) is set forth in SEQ ID NO:43, and the encoded amino acid sequence (118 aa) is set forth in SEQ ID NO:44. According to the IMGT numbering system, the heavy chain CDR1 has the sequence set forth in SEQ ID NO:45, the heavy chain CDR2 has the sequence set forth in SEQ ID NO:46, and the heavy chain CDR3 has the sequence set forth in SEQ ID NO:47. The nucleotide sequence of the light chain variable region (321 bp) has the sequence set forth in SEQ ID NO:48, and the encoded amino acid sequence (107 aa) is set forth in SEQ ID NO:49. According to the IMGT numbering system, the light chain CDR1 has the sequence set forth in SEQ ID NO:50, the light chain CDR2 has the sequence set forth in SEQ ID NO:51, and the light chain CDR3 has the sequence set forth in SEQ ID NO:52. The amino acid sequences of the four framework regions (FR-H1 to FR-H4) of the 14C12 heavy chain are set forth in SEQ ID NO:53 to SEQ ID NO:56, respectively; the amino acid sequences of the four framework regions (FR-H1 to FR-H4) of the 14C12 light chain are set forth in SEQ ID NO:57 to SEQ ID NO:60, respectively. Example 6

[0115] Design and preparation of humanized anti-PD-1 antibodies 14C12H1L1 and 14C12H1L1(hG1TM) 1.Design of humanized anti-PD-1 antibody 14C12H1L1 The light and heavy chain sequences of humanized antibody 14C12H1L1 were designed based on the three-dimensional crystal structure of PD-1 protein (Shinohara T, et al., Structure and chromosomal localization of the human PD-1 gene (PDCD1). Genomics 1995, 23 (3): 704-6). The variable region sequence of antibody 14C12H1L1 was obtained by subjecting the sequence of antibody 14C12 obtained in Example 5 to computer simulation of an antibody model and designing mutations in accordance with the model. The designed variable region sequences are as follows: The nucleotide sequence (354 bp) of the heavy chain variable region 14C12H1 of humanized monoclonal antibody 14C12H1L1 is set forth in SEQ ID NO:61, and the encoded amino acid sequence (118 aa) is set forth in SEQ ID NO:62. The nucleotide sequence (321 bp) of the light chain variable region 14C12L1 of humanized monoclonal antibody 14C12H1L1 is set forth in SEQ ID NO:63, and the encoded amino acid sequence (107 aa) is set forth in SEQ ID NO:64. The constant regions of antibody 14C12H1L1 are from the NCBI database (heavy chain constant region is Ig gamma-1 chain C region, accession number P01857; light chain constant region is Ig kappa chain C region, accession number P01834). The nucleotide and amino acid sequences of the 14C12H1L1 heavy chain are set forth in SEQ ID NOs: 65 and 66, respectively, and the nucleotide and amino acid sequences of the 14C12H1L1 light chain are set forth in SEQ ID NOs: 67 and 68, respectively. 14C12H1L1 is also known herein and in Chinese Patent Application No. 02110270671.X as 14C12H1L1(hG1WT), where the heavy and light chain variable regions of C12H1L1 are referred to herein and in Chinese Patent Application No. 02110270671.X as 14C12H1V (or 14C12H1 V ) and 14C12L1V (or 14C12L1 V ) is also known as

[0116] 2. Design of the light and heavy chain sequences of humanized antibody 14C12H1L1 (hG1TM) Based on 14C12H1L1 obtained in step 1 of Example 6, 14C12H1L1(hG1TM) was obtained by introducing a point mutation from leucine to alanine at position 234 (L234A), a point mutation from leucine to alanine at position 235 (L235A), and a point mutation from glycine to alanine at position 237 (G237A) in the heavy chain. The nucleotide and amino acid sequences of the heavy chain of 14C12H1L1(hG1TM) are set forth in SEQ ID NO:69 and SEQ ID NO:70, respectively. The light chain is identical to 14C12H1L1. The amino acid sequences of the four framework regions (FR-H1 to FR-H4) of 14C12H1 are set forth in SEQ ID NO: 71 to SEQ ID NO: 74, respectively. The amino acid sequences of the four framework regions (FR-H1 to FR-H4) of the 14C12L1 light chain are set forth in SEQ ID NO:75 to SEQ ID NO:78, respectively.

[0117] 3. Preparation of humanized antibodies 14C12H1L1 and 14C12H1L1(hG1TM) The heavy and light chain cDNAs of 14C12H1L1(hG1TM) and 14C12H1L1 were separately cloned into pUC57 simple (provided by GenScript) vector to obtain pUC57 simple-14C12H1, pUC57 simple-14C12L1 and pUC57 simple-14C12H1(hG1TM). With reference to the standard techniques described in Molecular Cloning: A Laboratory Manual (Second Edition), the heavy and light chain full-length genes synthesized by EcoRI&HindIII digestion were subcloned into expression vector pcDNA3.1 through digestion with restriction enzymes (EcoRI&HindIII) to obtain expression plasmids pcDNA3.1-14C12H1, pcDNA3.1-14C12L1 and pcDNA3.1-14C12H1(hG1TM), and the heavy / light chain genes of the recombinant expression plasmids were further subjected to sequence analysis. Then, the designed gene combinations containing the corresponding light and heavy chain recombinant plasmids (pcDNA3.1-14C12H1(hG1TM) / pcDNA3.1-14C12L1, and pcDNA3.1-14C12H1 / pcDNA3.1-14C12L1) were separately co-transfected into 293F cells, and the culture medium was harvested and purified. After sequence confirmation, endotoxin-free expression plasmids were prepared and transiently transfected into HEK293 cells for antibody expression. The culture medium was harvested after 7 days and subjected to affinity purification via protein A column to obtain humanized antibodies. Example 7

[0118] Sequence design and expression of anti-PD-1 / CD73 bifunctional antibodies 1. Sequence Design The structure of the bifunctional antibody described herein is the Morrison format (IgG-scFv), i.e., the C-terminus of the two heavy chains of an IgG antibody is each linked to an scFv fragment of another antibody, and the main compositional design of the heavy and light chains is shown in Table 1 below. [Table 1]

[0119] In Table 1 above: (1) Those labeled with a "V" in the lower right corner refer to the variable region of the corresponding heavy chain or the variable region of the corresponding light chain. For those without a "V" label, the corresponding heavy or light chain is full-length, constituting the constant region. The corresponding sequences described in the above examples refer to the amino acid sequences of these variable regions or full-lengths and the nucleotide sequences encoding them. (2) The amino acid sequence of linker 1 is (GGGGS)4 (the nucleotide sequence is SEQ ID NO:80 and the amino acid sequence is SEQ ID NO:79), and the amino acid sequence of linker 2 is (GGGGS)3 (the nucleotide sequence is SEQ ID NO:82 and the amino acid sequence is SEQ ID NO:81).

[0120] 2. Antibody Expression and Purification The heavy and light chain cDNA sequences of P1D7V01 were cloned into the vector pUC57Simple (provided by Genscript Inc.) to obtain the plasmids pUC57Simple-VP101H and pUC57Simple-VP101L, respectively. Plasmids pUC57simple-VP101H and pUC57simple-VP101L were enzymatically digested (HindIII & EcoRI), and the heavy and light chains isolated by electrophoresis were subcloned into vector pcDNA3.1, and the recombinant plasmids were extracted and co-transfected into 293F cells. After 7 days of cell culture, the culture medium was separated by high-speed centrifugation, and the supernatant was concentrated and loaded onto a HiTrapMabSelectSuRe column. Proteins were eluted in one step with elution buffer. Target samples were isolated and the buffer was exchanged into PBS. Purified antibodies P1D7V02R, P1D7V03, P1D7V04R, P1D7V07 and P1D7V08 were obtained by the expression and purification method of P1D7V01 described above. Example 8

[0121] Assay for antigen-binding activity of anti-CD73 / anti-PD-1 bispecific antibodies by ELISA 1. Binding activity of P1D7V01, P1D7V02R, P1D7V03 and P1D7V04R to the antigen PD-1-mFc measured by ELISA. The specific measurement method is as follows: The microplate was coated with 0.5 μg / mL PD-1-mFc and incubated overnight at 4°C. The antigen-coated microplate was then washed once with PBST and then blocked with PBS solution containing 1% BSA as blocking solution at 37°C for 2 hours. After blocking, the microplate was washed three times with PBST. Antibodies serially diluted in PBST solution (antibody dilution gradient is shown in Table 2) were added. The microplate containing the test antibodies was incubated at 37°C for 30 minutes and then washed three times with PBST. After washing, a secondary antibody working solution of goat anti-human IgG (H+L) labeled with HRP (Jackson, Cat. No. 09-035-088) diluted at a ratio of 1:5000 was added, and then the microplate was incubated at 37°C for 30 minutes. After incubation, the plate was washed 4 times with PBST, and TMB (Neogen, 308177) was added for 5 min in the dark to develop the color, and then stop solution was added to terminate the color reaction. The microplate was immediately placed in a microplate reader, and the OD value of each well in the microplate was read at 450 nm. The data was analyzed and processed by SoftMax Pro 6.2.1.

[0122] The results are shown in Table 2 and Figure 1. It can be seen that P1D7V01, P1D7V02R, P1D7V03 and P1D7V04R can effectively bind to the antigen PD-1-mFc in a dose-dependent manner. The absorbance intensity for each dose is shown in Table 2. Quantitative analysis of the absorbance of the bound antibodies gave the binding efficiency EC of antibodies P1D7V01, P1D7V02R, P1D7V03, P1D7V04R, 14C12H1L1 and nivolumab (as a control) obtained by curve fitting. 50 The values ​​were 0.078 nM, 0.078 nM, 0.075 nM, 0.089 nM, 0.033 nM and 0.051 nM, respectively.

[0123] The above experimental results showed that, under the same experimental conditions, the binding activities of P1D7V01, P1D7V02R, P1D7V03 and P1D7V04R to PD-1-mFc were equivalent to those of the control drugs 14C12H1L1 and nivolumab to the same targets, and suggested that P1D7V01, P1D7V02R, P1D7V03 and P1D7V04R have effective binding activity to PD-1-mFc. [Table 2]

[0124] 2. Binding activity of P1D7V01, P1D7V02R, P1D7V03 and P1D7V04R to the antigen human NT5E-biotin measured by ELISA The microplate was coated with 2 μg / mL streptavidin and then incubated overnight at 4° C. After incubation, the streptavidin-coated microplate was washed once with PBST and blocked with PBS solution containing 1% BSA as a microplate blocking solution for 2 hours at 37° C. After blocking, the microplate was washed three times with PBST. Then, the antigen human NT5E-biotin 0.5μg / mL was added and incubated at 37℃ for 30 minutes. The plate was then washed three times with PBST. Serially diluted antibodies in PBST solution (antibody dilution gradient is shown in Table 3) were added to the wells of the microplate. The microplate containing the test antibodies was incubated at 37℃ for 30 minutes and then washed three times with PBST. After washing, a secondary antibody working solution of goat anti-human IgG(H+L) labeled with HRP (Jackson, Cat. No. 09-035-088) diluted at a ratio of 1:5000 was added and the microplate was incubated at 37℃ for 30 minutes. After incubation, the plate was washed four times with PBST and TMB (Neogen, 308177) was added for 5 minutes in the dark to develop the color, and then stop solution was added to terminate the color reaction. The microplate was immediately placed in a microplate reader and the OD value of each well in the microplate was read at 450nm. Data were analyzed and processed with SoftMaxPro6.2.1.

[0125] The results are shown in Table 3 and Figure 2. It can be seen from the figure that P1D7V01, P1D7V02R, P1D7V03 and P1D7V04R can effectively bind to the antigen human NT5E-biotin in a dose-dependent manner. The absorbance intensity of each dose is shown in Table 3. Quantitative analysis of the absorbance of the bound antibodies gave the binding efficiency EC of antibodies P1D7V01, P1D7V02R, P1D7V03, P1D7V04R, 19F3H2L3 and MEDI9447 (as control antibody) obtained by curve fitting. 50 The values ​​were 0.063 nM, 0.230 nM, 0.068 nM, 0.439 nM, 0.045 nM and 0.042 nM, respectively.

[0126] The above experimental results showed that under the same experimental conditions, the binding activity of the bispecific antibodies P1D7V01, P1D7V02R, P1D7V03 and P1D7V04R to human NT5E-biotin was equivalent to that of the control drugs 19F3H2L3 and MEDI9447 to the same target, suggesting that P1D7V01, P1D7V02R, P1D7V03 and P1D7V04R have effective binding activity to human NT5E-biotin. [Table 3] Example 9

[0127] Activity of anti-CD73 / anti-PD-1 bispecific antibodies complemented with human PDL-1-mFc to bind human PD-1-mFc-biotin measured by competitive ELISA A microplate was coated with 2 μg / mL human PDL-1-mFc (PDL-1GenbankID: NP54862.1, mFc, SEQ ID NO: 143) and incubated overnight at 4°C. After incubation, the microplate was blocked with PBS solution containing 1% BSA at 37°C for 2 hours. After blocking, the plate was washed three times and dried. The antibody was serially diluted to a 7-fold concentration with a gradient ratio of 1:3 on a dilution plate starting at 10 μg / mL, and a blank control was set up. Then, an equal amount of human PD-1-mFc-biotin solution of 0.3 μg / mL was added, the system was mixed well, and incubated at room temperature for 20 minutes. The reaction mixture was then added to the coated microplate, and the microplate was incubated at 37°C for 30 minutes. After incubation, the plate was washed three times with PBST and dried. SA-HRP (KPL, 14-30-00) working solution was added and the plate was incubated at 37°C for 30 minutes. After incubation, the plate was washed 4 times and tapped dry. Then, TMB (Neogen, 308177) was added for 5 minutes in the dark to develop the color, and the stop solution was added to stop the color reaction. The microplate was then immediately placed in a microplate reader and the OD value of each well in the microplate was read at 450 nm. The data was analyzed and processed by SoftMaxPro6.2.1.

[0128] The results are shown in Figure 3. The OD values ​​of all doses are shown in Table 4. Quantitative analysis of the absorbance intensity of the bound antibody allowed curve simulation to determine the antibody binding efficiency, EC 50 was given (Table 4).

[0129] The results showed that P1D7V01, P1D7V02R, P1D7V03, P1D7V04R, 14C12H1L1 and nivolumab (as a control) could effectively block the binding of the antigen human PD-1-mFc-biotin to its receptor human PDL-1-mFc in a dose-dependent manner. The EC50 values ​​of P1D7V01, P1D7V02R, P1D7V03, P1D7V04R, 14C12H1L1 and nivolumab to inhibit the binding of human PD-1-mFc-biotin to its ligand human PDL-1-mFc were 1.115nM, 1.329nM, 1.154nM, 1.339nM, 1.459nM and 1.698nM, respectively. [Table 4] Example 10

[0130] Kinetic parameters of anti-CD73 / anti-PD-1 bispecific antibody binding to antigen human PD-1-mFc measured by the Fortebio system The dilution buffer for the samples was PBST, 0.1% BSA, pH 7.4. Antibodies were immobilized on the AHC sensor at a concentration of 5 μg / mL with an immobilization height of approximately 0.4 nm. The sensor was equilibrated in the buffer for 60 s, and the binding of the immobilized antibodies on the sensor at concentrations of 0.6-50 nM (3-fold dilution) with the antigen PD-1-mFc was measured in 120 s. The protein was dissociated in the buffer for 180 s. The detection temperature was 37 °C, the detection frequency was 0.3 Hz, and the vibration speed of the sample plate was 1000 rpm. The data was analyzed by 1:1 model fitting to obtain the affinity constants.

[0131] The affinity constants of humanized antibodies P1D7V01, 14C12H1L1 and nivolumab (as a control antibody) for human PD-1-mFc are shown in Table 5, and the detection results are shown in Figures 4, 5 and 6. The affinity constants of humanized antibodies P1D7V01, 14C12H1L1 and nivolumab for human PD-1-mFc were 1.76E-10M, 1.64E-10M and 2.32E-10M, respectively. The above experimental results suggested that the binding ability of P1D7V01 was equivalent to that of 14C12H1L1 and nivolumab, and that humanized antibody P1D7V01 had a stronger binding ability to human PD-1-mFc. [Table 5] Example 11

[0132] Kinetic parameters of binding of anti-CD73 / anti-PD-1 bispecific antibody to antigen human NT5E(1-552)his measured by the Fortebio system The dilution buffer for the samples was PBST, pH 7.4. Antibodies were immobilized on the protein A sensor at a concentration of 5 μg / mL with an immobilization time of approximately 15 seconds. The sensor was equilibrated in the buffer for 120 seconds and the binding of the antibodies immobilized on the sensor to the antigen human NT5E(1-552)-his at concentrations of 3.125-200 nM (two-fold dilution) was measured for 120 seconds. The protein was dissociated in the buffer for 600 seconds. The sensor was refreshed with a 10 mM Gly solution at pH 1.5. The detection temperature was 37 °C, the detection frequency was 0.6 Hz, and the vibration speed of the sample plate was 1000 rpm. The data was analyzed by 1:1 model fitting to obtain the affinity constants.

[0133] The affinity constants of the humanized antibodies P1D7V01 and MEDI9447 (control antibody) for human NT5E(1-552)-his are shown in Table 6, and the detection results are shown in Figures 7 and 8. The affinity constants of the humanized antibodies P1D7V01 and MEDI9447 for human NT5E(1-552)-his are 2.29E-10M and 1.04E-10M, respectively.

[0134] The above experimental results suggested that the binding ability of P1D7V01 was equivalent to that of MEDI9447, and that the humanized antibody P1D7V01 had stronger binding ability to human NT5E(1-552)-his. [Table 6] Example 12

[0135] Binding activity of anti-CD73 / anti-PD-1 bispecific antibodies measured by FACS 1. Binding activity of anti-CD73 / anti-PD-1 bispecific antibody to PD-1 on the surface of 293T-PD1 membrane measured by FACS Harvest 293T-PD1 cells in logarithmic growth phase and place them in 1.5 mL centrifuge tubes at 3 x 10 cells per tube. 5 The cells were transferred. 500 μL of PBSA was added, the mixture was centrifuged at 5600 rpm for 5 minutes, and the supernatant was removed. 100 μL of antibodies diluted in PBSA (final concentrations of 100 nM, 33.33 nM, 11.11 nM, 3.7 nM, 1.23 nM, 0.41 nM, 0.14 nM, and 0.05 nM) were added, respectively. The system was mixed gently to homogeneity, and then incubated on ice for 1 hour. After that, 500 μL of PBSA was added, the mixture was centrifuged at 5600 rpm for 5 minutes, and the supernatant was removed. FITC-labeled goat anti-human IgG secondary antibody (Jackson, Cat. No. 109-095-098) diluted 1:500 was added, resuspended, mixed well and incubated on ice in the dark for 0.5 h. 500 μL of PBSA was added and the mixture was centrifuged at 5600 rpm for 5 min and the supernatant was removed. Finally, 200 μL of PBSA was added to resuspend the cell pellet and the mixture was transferred to a flow tube for FACSCalibur detection.

[0136] The experimental results are shown in Table 7 and Figure 9. P1D7V01, P1D7V02R, P1D7V03 and P1D7V04R can specifically bind to PD-1 on the 293T-PD1 membrane surface in a dose-dependent manner, and compared with the PD1 single-targeting control antibody 14C12H1L1, the binding is stronger than that of 14C12H1L1.

[0137] EC of P1D7V01, P1D7V02R, P1D7V03, and P1D7V04R binding to 293T-PD1 under the same experimental conditions 50 The EC values ​​for 14C12H1L1 binding to 293T-PD1 were 1.000 nM, 1.075 nM, 1.377 nM, and 1.57 nM, respectively. 50 The value was 2.111nM.

[0138] The above experimental results indicated that under the same experimental conditions, P1D7V01, P1D7V02R, P1D7V03, P1D7V04R and 293T-PD1 all had better binding activity than the PD1 single-targeting control antibody 14C12H1L1, suggesting that P1D7V01, P1D7V02R, P1D7V03 and P1D7V04R have effective binding activity to PD-1 on the membrane surface of 293T-PD1. [Table 7]

[0139] 2. Binding activity of anti-CD73 / anti-PD-1 bispecific antibody to CD73 on the MDA-MB-231 membrane surface measured by FACS Digest MDA-MB-231 cells in log phase with regular trypsin and place in 1.5 mL centrifuge tubes at 3 x 10 cells per tube. 5The mixture was transferred to the wells at 4°C for 10 min. 500 μL of PBSA was added, the mixture was centrifuged at 5600 rpm for 5 min, and the supernatant was removed. 100 μL of antibody diluted in PBSA (final concentrations of 100 nM, 33.33 nM, 11.11 nM, 3.7 nM, 1.23 nM, 0.41 nM, 0.14 nM, 0.05 nM) was added. The system was gently mixed homogeneously and incubated on ice for 1 h. Then, 500 μL of PBSA was added, centrifuged at 5600 rpm for 5 min, and the supernatant was removed. A 500-fold diluted FITC-labeled goat anti-human IgG secondary antibody (Jackson, Cat. No. 109-095-098) was added, resuspended, mixed well, and the mixture was incubated in the dark on ice for 0.5 h. 500 μL of PBSA was added, centrifuged at 5600 rpm for 5 min, and the supernatant was removed. Finally, 200 μL of PBSA was added to resuspend the cell pellet, and the mixture was transferred to a flow tube for FACSCalibur detection.

[0140] The experimental results are shown in Table 8 and Figure 10. The binding activity of P1D7V01 and P1D7V03 to CD73 on the MDA-MB-231 membrane surface was superior to that of 19F3H2L3, where P1D7V01 was superior to the control drug MEDI9447 on the same target. Under the same experimental conditions, the EC of P1D7V01 and P1D7V03 binding to CD73 on the MDA-MB-231 membrane surface was 50 The EC values ​​for MEDI9447 and 19F3H2L3 binding to CD73 on the MDA-MB-231 membrane surface were 1.384 nM and 2.009 nM, respectively. 50 The values ​​were 1.589 nM and 2.773 nM, respectively.

[0141] The above experimental results showed that P1D7V01, P1D7V03, 19F3H2L3 and control drug MEDI9447 against the same target can specifically bind to CD73 on the MDA-MB-231 membrane surface in a dose-dependent manner. The binding activity of P1D7V01 and P1D7V03 is superior to that of 19F3H2L3, where P1D7V01 is superior to the control drug MEDI9447 against the same target. It was suggested that P1D7V01 and P1D7V03 have the activity of effectively binding to CD73 on the MDA-MB-231 membrane surface. [Table 8] Example 13

[0142] Detection of inhibition of the enzymatic activity of CD73 on the cell membrane surface by anti-CD73 / anti-PD-1 bispecific antibody 1. Detection of inhibition of the enzymatic activity of CD73 on the surface of MDA-MB-231 membrane by anti-CD73 / anti-PD-1 bispecific antibody The experimental procedure is as follows: MDA-MB-231 cells in logarithmic phase were well-dissolved, resuspended in serum-free RPMI-1640 medium, and then counted. MDA-MB-231 cells were plated at 2 × 10 per well. 4 Cells / 100μL were seeded into a 96-well plate. Antibodies were diluted in serum-free RPMI-1640 medium (serial 2.5-fold dilutions). Antibodies were added to the 96-well plate at 50μL per well, and the plate was incubated at 37°C for 1 hour. After 1 hour, 50μL of AMP (TCL, Cat. No. A0157) diluted in RPMI-1640 at 1200μM was added to each well. After 3 hours, 25μL of cell culture supernatant was removed and transferred to a new 96-well plate, and 25μL of 100μM ATP (TCL, Cat. No. A0158) was added to each well. 50μL of CTG (CellTiterGlo, Promega, Cat. No. G8641) color development solution was added to each well to develop the color, and the relative fluorescence intensity (RLU) was read using a multilabel microplate tester (PerkinElmer 2140-0020).

[0143] The experimental results are shown in Figure 11. The AMP amounts of P1D7V01, P1D7V02R, P1D7V03 and P1D7V04R are equivalent to the AMP amount of the positive control MEDI9447 in a concentration-dependent manner.

[0144] The above experimental results show that the added AMP can be converted to adenosine A through the enzymatic activity of CD73 on the surface of MDA-MB-231 cells without antibodies, and after the addition of antibodies, due to the binding affinity of antibodies P1D7V01, P1D7V02R, P1D7V03 and P1D7V04R to CD73, the enzymatic catalytic activity is reduced, so that AMP cannot be converted to adenosine A. It is suggested that the antibodies effectively inhibit the enzymatic activity reaction in a non-substrate competitive mode, reducing the production of adenosine.

[0145] 2. Detection of inhibition of the enzymatic activity of CD73 on the surface of U87-MG membrane by anti-CD73 / anti-PD-1 bispecific antibody U87-MG cells in good logarithmic phase were removed, resuspended in serum-free RPMI-1640 medium, and then counted. U87-MG cells were cultured at 2 × 10 per well. 4 Cells / 100μL were seeded into a 96-well plate. Antibodies were diluted in serum-free RPMI-1640 medium (serial 2.5-fold dilutions). Antibodies were added to the 96-well plate at 50μL per well, and the plate was incubated at 37°C for 1 hour. After 1 hour, 50μL of AMP diluted in RPMI-1640 at 1200μM was added to each well. After 3 hours, 25μL of cell culture supernatant was removed and transferred to a new 96-well plate, and 25μL of ATP at 100μM was added to each well. 50μL of CTG (CellTiterGlo) color development solution was added to each well to develop the color, and the relative fluorescence intensity (RLU) was read using a multi-label microplate tester (PerkinElmer 2140-0020).

[0146] The experimental results are shown in Figure 12. The AMP amounts of P1D7V01, P1D7V02R, P1D7V03 and P1D7V04R are equivalent to the AMP amount of the positive control MEDI9447 in a concentration-dependent manner.

[0147] The above experimental results indicate that the added AMP can be converted to adenosine A through the enzymatic activity of CD73 on the surface of U87-MG cells without antibodies, and after the antibodies are added, the antibodies P1D7V01, P1D7V02R, P1D7V03 and P1D7V04R bind to CD73, reducing the enzyme catalytic function, so that AMP cannot be converted to adenosine A. It is suggested that the antibodies effectively inhibit the enzymatic activity reaction in a non-substrate competitive mode, reducing the production of adenosine. Example 14

[0148] Biological activity of anti-CD73 / anti-PD-1 bispecific antibodies to promote IFN-γ and IL-2 secretion measured by mixed lymphocyte reaction (MLR) 1. Biological activity of anti-CD73 / anti-PD-1 bispecific antibody for promoting IFN-γ secretion in Raji-PDL-1 mixed lymphocyte reaction system Raji-PDL-1 cells were passaged as usual. PBMCs were thawed, incubated in 10 mL of 1640 complete medium, and stimulated with 0.5 μg / mL SEB (Dianotech, Cat. No. S010201) for 2 days. Raji-PDL-1 cells were treated with 25 μg / mL MMC (Sigma, Cat. No. M4287) and placed in a 37°C incubator for 1 hour. PBMCs (peripheral blood mononuclear cells) stimulated with SEB for 2 days and Raji-PDL-1 cells treated with MMC for 1 hour were harvested, washed twice with PBS, resuspended in complete medium, and counted. Cells were added separately in U-shaped 96-well plates at 100,000 cells per well. Antibodies were added according to the study design and cultured in the incubator for 3 days. After 3 days, cell culture supernatants were harvested and IFN-γ was measured by ELISA.

[0149] As shown in Figure 13, co-culture of human PBMCs and Raji-PDL-1 cells significantly promoted IFN-γ secretion from PBMCs, and addition of antibodies to the co-culture system significantly induced IFN-γ secretion in PBMCs. In terms of the level of promoting IFN-γ secretion activity, antibodies P1D7V01, P1D7V02R, P1D7V03 and P1D7V04R have activity equivalent to that of the parent PD-1 single targeting antibody 14C12H1L1.

[0150] 2. Biological activity of anti-CD73 / anti-PD-1 bispecific antibody to promote IL-2 secretion in Raji-PDL-1 mixed lymphocyte reaction system Raji-PDL-1 cells were subcultured as usual. PBMCs were thawed, incubated in 10 mL of 1640 complete medium, and stimulated with SEB (0.5 μg / mL) for 2 days. Raji-PDL-1 cells were treated with 25 μg / mL MMC and placed in a 37°C incubator for 1 h. PBMCs (peripheral blood mononuclear cells) stimulated with SEB for 2 days and Raji-PDL-1 cells treated with MMC for 1 h were harvested, washed twice with PBS, resuspended in complete medium, and counted. Cells were added separately to U-shaped 96-well plates at 100,000 cells per well. Antibodies were added and cultured for 3 days according to the study design. Cell culture supernatants were harvested and IL-γ was measured by ELISA.

[0151] As shown in Figure 14, the mixed culture of human PBMC and Raji-PDL-1 cells has a certain promoting effect on the secretion of IL-2 in PBMC, and the simultaneous addition of antibodies to the mixed culture system significantly induced the secretion of IL-2 in PBMC in a remarkable dose-dependent manner. In terms of the level of promoting IL-2 secretion activity, the bifunctional antibodies P1D7V01, P1D7V02R, P1D7V03 and P1D7V04R have slightly lower activity than the parent PD-1 single target antibody 14C12H1L1 at low concentrations, and have activity equivalent to that of the parent PD-1 single target antibody 14C12H1L1 at medium and high concentrations. Compared with the PD1 target positive control drug Nivolumab, P1D7V01, P1D7V02R, P1D7V03 and P1D7V04R have high IL-2 secretion promoting ability at three different antibody concentration levels.

[0152] 3. Biological activity of anti-CD73 / anti-PD-1 bispecific antibody that promotes DC mixed lymphoid reaction system and secretes IFN-γ PBMCs from normal human peripheral blood were isolated, resuspended in complete medium, and seeded on culture dishes. The culture dishes were placed in an incubator overnight for culture. The suspended PBMCs were collected and removed. The attached cells on the bottom of the dishes were washed with PBS buffer, and then DC maturation induction was performed. 10 mL of RPMI-1640 complete medium containing GM-CSF and IL-4 at a concentration of 1000 U / mL each was added to each dish. The dishes were cultured in an incubator at 37°C and 5% carbon dioxide for 3 days. Then, half of the medium was replaced, 1000 U / mL of GM-CSF and IL-4 were added, and the dishes were placed in a 37°C, 5% carbon dioxide incubator and cultured for 3 consecutive days. After 3 days, half of the medium was replaced again, 1000 U / mL of GM-CSF and IL-4, and 100 U / mL of TNF-α were added, and the dishes were cultured for another 2 days. PBMCs from other donors were freshly isolated and plated in 96-well plates at 100,000 cells per well after cell counting. DCs induced to mature were harvested and washed once with complete medium. Cells were counted and plated in 96-well plates containing PBMCs at 10,000 cells per well. Antibodies were added according to the study design. The system was mixed well and incubated in a 37°C, 5% carbon dioxide incubator for 5 days to perform co-culture. After 5 days, cell culture supernatants were harvested and IFN-γ was quantified by ELISA.

[0153] The results are shown in Figure 15. Compared with the culture of DCs or PBMCs alone, the co-culture of DCs and PBMCs significantly promoted the secretion of IFN-γ; and compared with the isotype control, the addition of anti-CD73 / anti-PD-1 bispecific antibody to the co-culture of DCs and PBMCs further significantly improved the IFN-γ secretion level.

[0154] Biological activity of anti-CD73 / anti-PD-1 bispecific antibody to promote IL-2 secretion in DC mixed lymphocyte reaction system PBMCs from normal human peripheral blood were isolated, resuspended in complete medium, and seeded on culture dishes. The culture dishes were placed in an incubator overnight for culture. The suspended PBMCs were collected and removed. The attached cells on the bottom of the dishes were washed with PBS buffer and then subjected to DC maturation induction. 10 mL of RPMI-1640 complete medium containing GM-CSF and IL-4 at a concentration of 2000 U / mL each was added to each dish. The dishes were cultured in an incubator at 37°C and 5% carbon dioxide for 3 days. Then, half of the medium was replaced, 50 ng / mL IFN-γ and 100 ng / mL LPS were added, and cultured for another 2 days. PBMCs from other donors were thawed, counted, and seeded on 96-well plates at 100,000 cells per well. DCs induced to mature were collected and washed once with complete medium. The cells were counted and PBMCs were seeded into 96-well plates containing 10,000 cells per well. Antibodies were added according to the study design. The systems were mixed well and co-cultured in a 37°C, 5% carbon dioxide incubator for 5 days. After 5 days, the cell culture supernatant was collected and IL-2 was quantified by ELISA. The results are shown in Figure 16. Compared with the culture of DCs or PBMCs alone, the co-culture of DCs and PBMCs significantly promoted the secretion of IL-2; and compared with the isotype control, the addition of anti-CD73 / anti-PD-1 bispecific antibody based on the co-culture of DCs and PBMCs further significantly improved the IL-2 secretion level. Example 15

[0155] Preparation of anti-PD-1 / CD73 bispecific antibodies NTPDV1, NTPDV2, NTPDV3 and NTPDV4 The structural pattern of the bispecific antibodies NTPDV1, NTPDV2, NTPDV3 and NTPDV4 is in the Morrison format (IgG-scFv), i.e. the C-terminus of the two heavy chains of one IgG antibody are separately linked to the scFv fragment of another antibody via a linker. The components of the heavy and light chain design are shown in Table 9 below.

[0156] NTPDV1, NTPDV2, NTPDV3 and NTPDV4 have amino acid mutations introduced into the constant regions of their immunoglobulin moieties to eliminate their binding activity to FcγR, and are therefore known as NTPDV1(hG1TM), NTPDV2(hG1TM), NTPDV3(hG1TM) and NTPDV4(hG1TM) in this specification and in Chinese Patent Application No. 202110270671.X. [Table 9]

[0157] In Table 9 above: Those labeled with a "V" at the bottom right refer to the variable region of the corresponding heavy chain or the variable region of the corresponding light chain. For those without a "V" label, the corresponding heavy or light chain is full length, including the constant region. The corresponding sequences set forth in the Examples above are referenced for the amino acid sequences of these variable regions or full length, and the nucleotide sequences encoding them. The amino acid sequence of linker 1 consists of four repeats of (GGGGS), i.e., (GGGGS)4 or (G4S)4 (nucleotide sequence SEQ ID NO:80 and amino acid sequence SEQ ID NO:79). The amino acid sequence of linker 2 consists of three repeats of (GGGGS), i.e., (GGGGS)4 or (G4S)3 (nucleotide sequence SEQ ID NO:82 and amino acid sequence SEQ ID NO:81). The three CDR sequences of the light chains 19F3L2 and 19F3L3 of the immunoglobulin portion in NTPDV1, NTPDV2, NTPDV3 and NTPDV4 are identical to the light chain CDR sequence of 19F3. The three CDR sequences of 19F3H2 (hG1TM) in the immunoglobulin portion in NTPDV1, NTPDV2, NTPDV3 and NTPDV4 are identical to the heavy chain CDR sequences of 19F3. The CDR sequences of 14C12H1V-linker2-14C12L1V and 14C12H1V-linker1-14C12L1V of the scFv portion in NTPDV1, NTPDV2, NTPDV3 and NTPDV4 are identical to the heavy chain CDRs and light chain CDRs of 14C12. The amino acid sequences of the heavy chains of NTPDV2 and NTPDV4 are identical and are marked as NTPDH2 / 4 (SEQ ID NO: 83), and the nucleotide sequence of the heavy chain of NTPDV2 or NTPDV4 is SEQ ID NO: 84. The amino acid sequences of the heavy chains of NTPDV1 and NTPDV3 are identical and are marked as NTPDH1 / 3 (SEQ ID NO: 85), and the nucleotide sequence of the heavy chain of NTPDV1 or NTPDV3 is SEQ ID NO: 86. The three CDR sequences of 19F3L3 and 19F3L2 of the immunoglobulin portion in NTPDV1, NTPDV2, NTPDV3 and NTPDV4 are identical to the three CDRs of the light chain of antibody 19F3. The amino acid sequence of the light chain 19F3L3 of the immunoglobulin portion in NTPDV1 or NTPDV2 is identical to the light chain sequence of antibody 19F3H2L3(G1M) (SEQ ID NO: 28), and the nucleotide sequence of the light chain 19F3L3 of the immunoglobulin portion in NTPDV1 or NTPDV2 is SEQ ID NO: 27. The amino acid sequence of the light chain 19F3L2 of the immunoglobulin portion in NTPDV3 or NTPDV4 is SEQ ID NO: 96, and the nucleotide sequence of the light chain 19F3L2 of the immunoglobulin portion in NTPDV3 or NTPDV4 is SEQ ID NO: 100.

[0158] (1) NTPDV1, the heavy chain of which has the amino acid sequence set forth in SEQ ID NO: 85, the light chain of which has the amino acid sequence set forth in SEQ ID NO: 28, linker 1 of which has the amino acid sequence set forth in SEQ ID NO: 79, 14C12H1V of which has the amino acid sequence set forth in SEQ ID NO: 66, linker 2 of which has the amino acid sequence set forth in SEQ ID NO: 81, and 14C12L1V of which has the amino acid sequence set forth in SEQ ID NO: 68; (2) NTPDV2, the heavy chain of which has the amino acid sequence set forth in SEQ ID NO: 85, the light chain of which has the amino acid sequence set forth in SEQ ID NO: 28, linker 1 of which has the amino acid sequence set forth in SEQ ID NO: 79, 14C12H1V of which has the amino acid sequence set forth in SEQ ID NO: 66, linker 1 of which has the amino acid sequence set forth in SEQ ID NO: 79, and 14C12L1V of which has the amino acid sequence set forth in SEQ ID NO: 68; (3) NTPDV3, the heavy chain of which has the amino acid sequence set forth in SEQ ID NO: 85, the light chain of which has the amino acid sequence set forth in SEQ ID NO: 96, linker 1 of which has the amino acid sequence set forth in SEQ ID NO: 79, 14C12H1V of which has the amino acid sequence set forth in SEQ ID NO: 66, linker 2 of which has the amino acid sequence set forth in SEQ ID NO: 81, and 14C12L1V of which has the amino acid sequence set forth in SEQ ID NO: 68; and (4) NTPDV4, the heavy chain of which has the amino acid sequence set forth in SEQ ID NO: 85, the light chain of which has the amino acid sequence set forth in SEQ ID NO: 96, linker 1 of which has the amino acid sequence set forth in SEQ ID NO: 79, 14C12H1V of which has the amino acid sequence set forth in SEQ ID NO: 66, linker 1 of which has the amino acid sequence set forth in SEQ ID NO: 79, and 14C12L1V of which has the amino acid sequence set forth in SEQ ID NO: 68.

[0159] Antibody expression and purification The heavy chain cDNA sequences of NTPDV1 and NTPDV3, the heavy chain cDNA sequences of NTPDV2 and NTPDV4, and the light chain cDNA sequences thereof were separately cloned into the vector pUC57Simple (provided by Genscript) to obtain the plasmids pUC57Simple-NTPDH2 / 4, pUC57Simple-NTPDH1 / 3, pUC57Simple-19F3L3, and pUC57Simple-19F3L2, respectively. Plasmid pUC57simple-NTPDH2 / 4 and plasmid pUC57simple-19F3L3, plasmid pUC57simple-NTPDH2 / 4 and plasmid pUC57simple-19F3L2, plasmid pUC57simple-NTPDH1 / 3 and plasmid pUC57simple-19F3L3, and plasmid pUC57simple-NTPDH1 / 3 and plasmid pUC57simple-19F3L2 were digested (HindIII & EcoRI). The recovered heavy and light chains were subcloned separately into vector pcDNA3.1 to obtain plasmids pcDNA3.1-NTPDH2 / 4 and pcDNA3.1-19F3L3, plasmids pcDNA3.1-NTPDH2 / 4 and pcDNA3.1-19F3L2, plasmids pcDNA3.1-NTPDH1 / 3 and pcDNA3.1-19F3L3, and plasmids pcDNA3.1-NTPDH1 / 3 and pcDNA3.1-19F3L2. The recombinant plasmids were extracted and co-transfected into 293F cells. After 7 days of cell culture, the medium was separated by centrifugation at high speed, and the supernatant was concentrated and loaded onto a HiTrapMabSelectSuRe column. The protein was eluted in one step with elution buffer. The target sample was isolated and the buffer was exchanged into PBS. The purified antibodies NTPDV1, NTPDV2, NTPDV3 and NTPDV4 were obtained according to the expression and purification methods described in the preparation examples above. Example 16

[0160] Assay for the binding activity of anti-CD73 / anti-PD-1 bispecific antibodies to antigens by ELISA 1. Binding activity of NTPDV1, NTPDV2, NTPDV3 and NTPDV4 to the antigen PD-1-mFc measured by ELISA The microplate was coated with PD-1-mFc (0.5 μg / mL) and incubated overnight at 4°C. The antigen-coated microplate was then washed once with PBST and then blocked with PBS solution containing 1% BSA as blocking solution at 37°C for 2 hours. After blocking, the microplate was washed three times with PBST. Antibodies serially diluted in PBST solution (antibody dilution gradient is shown in Table 2) were added. The microplate containing the test antibodies was incubated at 37°C for 30 minutes and then washed three times with PBST. After washing, HRP-labeled goat anti-human IgGFc (Jackson, Cat. No. 109-035-098) secondary antibody working solution diluted at a ratio of 1:5000 was added, and then the microplate was incubated at 37°C for 30 minutes. After incubation, the plate was washed 4 times with PBST, and TMB (Neogen, 308177) was added for 8 min in the dark to develop the color, and then the stop solution was added to terminate the color reaction. The microplate was immediately placed in a microplate reader, and the OD value of each well in the microplate was read at 450 nm. The data was analyzed and processed by SoftMaxPro6.2.1.

[0161] The results are shown in Table 10. It can be seen that NTPDV1, NTPDV2, NTPDV3 and NTPDV4 can effectively bind to the antigen PD-1-mFc in a dose-dependent manner. The absorbance intensity for each dose is shown in Table 10. Quantitative analysis of the absorbance of the bound antibodies gave the binding efficiency EC of antibodies NTPDV1, NTPDV2, NTPDV3, NTPDV4 and 14C12H1L1 (hG1TM) (as a control) obtained by curve fitting. 50 The values ​​were 0.101 nM, 0.119 nM, 0.110 nM, 0.123 nM and 0.031 nM, respectively.

[0162] The above experimental results indicated that under the same experimental conditions, the binding activity of NTPDV1, NTPDV2, NTPDV3 and NTPDV4 to PD-1-mFc was equivalent to that of the control drug 14C12H1L1 (hG1TM) to the same target, suggesting that NTPDV1, NTPDV2, NTPDV3 and NTPDV4 have effective binding activity to PD-1-mFc. [Table 10]

[0163] 2. Binding activity of NTPDV1, NTPDV2, NTPDV3 and NTPDV4 to the antigen human NT5E-biotin measured by ELISA The microplate was coated with streptavidin SA (2 μg / mL) and then incubated overnight at 4° C. After incubation, the streptavidin-coated microplate was washed once with PBST and blocked with PBS solution containing 1% BSA as microplate blocking solution at 37° C. for 2 hours. After blocking, the microplate was washed three times with PBST. Then, the antigen human NT5E-biotin 0.5 μg / mL was added and incubated at 37° C. for 30 minutes. Then, the plate was washed three times with PBST. The antibodies serially diluted in PBST solution (antibody dilution gradient is shown in Table 11) were added to the wells of the microplate. The microplate containing the test antibodies was incubated at 37° C. for 30 minutes and then washed three times with PBST. After washing, a working solution of HRP-labeled goat anti-human IgGFc (Jackson, Cat. No. 109-035-098) secondary antibody diluted at a ratio of 1:5000 was added, and the microplate was incubated at 37°C for 30 minutes. After incubation, the plate was washed 4 times with PBST, and TMB (Neogen, 308177) was added for 7 minutes in the dark to develop the color, and then stop solution was added to terminate the color reaction. The microplate was immediately placed in a microplate reader, and the OD value of each well in the microplate was read at 450 nm. The data was analyzed and processed by SoftMaxPro6.2.1.

[0164] The results are shown in Table 11. It can be seen that NTPDV1, NTPDV2, NTPDV3, NTPDV4 and 19F3H2L3 (hG1M) can effectively bind to the antigen human NT5E-biotin in a dose-dependent manner (the absorbance intensity for each dose is shown in Table 11). Quantitative analysis of the absorbance of the bound antibodies gave the binding efficiency EC of the antibodies NTPDV1, NTPDV2, NTPDV3, NTPDV4 and 19F3H2L3 (hG1M) (as a control antibody) obtained by curve fitting. 50 The values ​​were 0.079 nM, 0.082 nM, 0.084 nM, 0.077 nM and 0.029 nM, respectively.

[0165] The above experimental results showed that under the same experimental conditions, the binding activity of the bispecific antibodies NTPDV1, NTPDV2, NTPDV3 and NTPDV4 to human NT5E-biotin was equivalent to that of the control drug 19F3H2L3 (hG1M) to the same target, suggesting that NTPDV1, NTPDV2, NTPDV3 and NTPDV4 have the activity of effectively binding to human NT5E-biotin. [Table 11] Example 17

[0166] Activity of anti-CD73 / anti-PD-1 bispecific antibodies complemented with human PDL-1-mFc to bind human PD-1-mFc-biotin measured by competitive ELISA A microplate was coated with 2 μg / mL of human PDL-1-mFc (PDL-1, Genbank ID: NP54862.1, mFc SEQ ID NO: 89) and incubated overnight at 4 °C. After incubation, the microplate was blocked with PBS solution containing 1% BSA at 37 °C for 2 hours. After blocking, the plate was washed once and dried. The antibody was serially diluted to 7 concentrations with a gradient ratio of 1:3 on a dilution plate starting at 10 μg / mL, and a blank control was set up. Then, an equal amount of human PD-1-mFc-biotin solution of 0.3 μg / mL was added, the system was mixed well, and incubated at room temperature for 10 minutes. The reaction mixture was then added to the coated microplate, and the microplate was incubated at 37 °C for 30 minutes. After incubation, the plate was washed three times with PBST and dried. SA-HRP (KPL, 14-30-00) working solution was added, and the plate was incubated at 37 °C for 30 minutes. After incubation, the plate was washed four times and tapped dry. Then, TMB (Neogen, 308177) was added for 5 minutes in the dark to develop the color, and the color reaction was stopped by adding stop solution. Then, the microplate was immediately placed in a microplate reader, and the OD value of each well in the microplate was read at 450 nm. The data was analyzed and processed by SoftMaxPro6.2.1.

[0167] The OD values ​​of the total volume are shown in Table 12. Quantitative analysis of the absorbance intensity of the bound antibody allowed curve simulation to determine the antibody binding efficiency, EC 50 was given (Table 12).

[0168] The results showed that NTPDV1, NTPDV2, NTPDV3, NTPDV4 and 14C12H1L1(hG1TM) (as a control) could effectively block the binding of the antigen human PD-1-mFc-biotin to its receptor human PDL-1-mFc in a dose-dependent manner. EC values ​​of NTPDV1, NTPDV2, NTPDV3, NTPDV4 and 14C12H1L1(hG1TM) for blocking the binding of human PD-1-mFc-biotin to its ligand human PDL-1-mFc were 50 The values ​​were 2.249 nM, 2.253 nM, 2.332 nM, 2.398 nM, 2.216 nM and 2.231 nM, respectively. [Table 12] Example 18

[0169] Kinetic parameters for binding of anti-CD73 / anti-PD-1 bispecific antibodies to antigen human PD-1-mFc measured by the Fortebio system The sample dilution buffer was PBS (0.02% Tween-20, 0.1% BSA, pH 7.4). PD1-mFc was immobilized on the AMC sensor at a concentration of 5 μg / mL with an immobilization height of approximately 0.1 nM (time 60 s). The sensor was equilibrated in the buffer for 60 s and the binding of PD1-mFc immobilized on the sensor to antibodies at concentrations of 0.62 to 50 nM (three-fold dilution) was measured for 120 s. The protein was dissociated in the buffer for 300 s. The detection temperature was 30 °C, the detection frequency was 0.3 Hz, and the vibration speed of the sample plate was 1000 rpm. The data was analyzed by 1:1 model fitting to obtain the affinity constants.

[0170] The affinity constants of the humanized antibodies NTPDV1, NTPDV2, NTPDV3, NTPDV4 and nivolumab (as a control antibody) for human PD-1-mFc are shown in Table 13, and the detection results are shown in Figures 19, 20, 21, 22 and 18. The affinity constants of the humanized antibodies NTPDV1, NTPDV2, NTPDV3, NTPDV4 and nivolumab for human PD-1-mFc were 1.40E-10M, 7.39E-11M, 1.25E-10M, 1.13E-11M and 2.26E-10M, respectively. The above experimental results showed that the binding abilities of NTPDV1, NTPDV3 and nivolumab were equivalent, and the binding abilities of NTPDV2 and NTPDV4 were superior to that of nivolumab, suggesting that the humanized antibodies NTPDV1, NTPDV2, NTPDV3 and NTPDV4 have stronger binding abilities to human PD-1-mFc. [Table 13] Example 19

[0171] Kinetic parameters of binding of anti-CD73 / anti-PD-1 bispecific antibody to antigen human NT5E(1-552)-his measured by the Fortebio system The sample dilution buffer was PBS (0.02% Tween-20, 0.1% BSA, pH 7.4). HNT5E(1-552)-his was immobilized on the HIS1K sensor at a concentration of 5 μg / mL with an immobilization height of approximately 0.4 nM (time 50 s). The sensor was equilibrated in the buffer for 60 s, and the binding of HNT5E(1-552)-his immobilized on the sensor to antibodies at concentrations of 0.31 to 25 nM (triple dilution) was measured for 100 s. The protein was dissociated in the buffer for 180 s. The detection temperature was 30 °C, the detection frequency was 0.3 Hz, and the vibration speed of the sample plate was 1000 rpm. The data was analyzed with a 1:1 model fitting to obtain the affinity constants.

[0172] The measurement results of the affinity constants of the humanized antibodies 19F3H2L3 (hG1M) (as a control antibody), NTPDV1, NTPDV2, NTPDV3 and NTPDV4 against human NT5E (1-552)-his are shown in Table 14, and the detection results are shown in Figures 24 to 27. The affinity constants of the humanized antibodies NTPDV1, NTPDV2, NTPDV3 and NTPDV4 against human NT5E (1-552)-his were 3.29E-11M, 2.88E-11M, 7.92E-11M and 5.77E-11M, respectively.

[0173] The above experimental results suggested that the binding abilities of 19F3H2L3 (hG1M), NTPDV1, NTPDV2, NTPDV3 and NTPDV4 were equivalent, and that the humanized antibodies NTPDV1, NTPDV2, NTPDV3 and NTPDV4 had strong binding ability to human NT5E (1-552)-his. [Table 14] Example 20

[0174] Detection of inhibition of the enzymatic activity of CD73 on the membrane surface of U87-MG cells by anti-CD73 / anti-PD-1 bispecific antibody U87-MG cells in good logarithmic phase were removed, resuspended in serum-free RPMI-1640 medium, and then counted. U87-MG cells were cultured at 2.5 × 10 per well. 4Cells / 60μL were seeded into 96-well plates. Antibodies were diluted in serum-free RPMI-1640 medium according to the study design. Antibodies were added to the 96-well plates at 60μL per well, and the plates were incubated at 37℃ for 1 hour. After 1 hour, 60μL of AMP diluted in RPMI-1640 at 600μM was added to each well. After 3 hours, 100μL of cell culture supernatant was removed and transferred to a new 96-well plate, 40μL of CTG (CellTiterGlo) color development solution was added to each well, and the plate was placed at room temperature in the dark for 5 minutes. After 5 minutes, 10μL of 300μM ATP was added to each well to develop the color. The relative fluorescence intensity (RLU) was read by a multi-label microplate tester (PerkinElmer 2140-0020).

[0175] The experimental results are shown in Table 15 and FIG. 28. The AMP amount of NTPDV2 is equivalent to that of the positive control MEDI9447.

[0176] The above experimental results showed that the added AMP could be converted to adenosine A through the enzymatic activity of CD73 on the surface of U87-MG cells without antibody, and after the antibody was added, the antibody NTPDV2 bound to CD73, reducing the enzyme catalytic function, so that AMP could not be converted to adenosine A. This suggested that the antibody effectively inhibited the enzymatic activity reaction in a non-substrate competitive mode and reduced the production of adenosine. [Table 15] Example 21

[0177] Biological activity of anti-CD73 / anti-PD-1 bispecific antibodies to promote secretion of IFN-γ and IL-2 as measured by mixed lymphocyte reaction (MLR) 1. Biological activity of anti-CD73 / anti-PD-1 bispecific antibody to promote IFN-γ secretion in Raji-PDL-1 mixed lymphocyte reaction system Raji-PDL-1 cells were passaged as usual. PBMCs were thawed, incubated in 10 mL of 1640 complete medium and stimulated with 0.5 μg / mL SEB (Dianotech, Cat. No. S010201) for two days. Raji-PDL-1 cells were treated with 25 μg / mL MMC (mitomycin C, Stressmarq, Cat. SIH-246, Cat. No. SM286474) and placed in a 37°C incubator for 1 hour. PBMCs (peripheral blood mononuclear cells) stimulated with SEB for 2 days and Raji-PDL-1 cells treated with MMC for 1 hour were harvested, washed twice with PBS, resuspended in complete medium and counted. Cells were cultured at 1 × 10 per well. 5 The cells were separately added to a U-shaped 96-well plate. Antibodies were added according to the study design and cultured in an incubator for 3 days. After 3 days, the cell culture supernatants were collected and IFN-γ was measured by ELISA.

[0178] As shown in Figure 29, co-culture of human PBMCs and Raji-PDL-1 cells significantly promoted IFN-γ secretion from PBMCs, and addition of antibodies to the co-culture system significantly induced IFN-γ secretion in PBMCs. In terms of the level of promoting IFN-γ secretion activity, antibody NTPDV2 has activity equivalent to that of the parent PD-1 single targeting antibody 14C12H1L1.

[0179] 2. Biological activity of anti-CD73 / anti-PD-1 bispecific antibody to promote IL-2 secretion in Raji-PDL-1 mixed lymphocyte reaction system Raji-PDL-1 cells were passaged as usual. PBMCs were thawed, incubated in 10 mL of 1640 complete medium, and stimulated with SEB (0.5 μg / mL) for two days. Raji-PDL-1 cells were treated with 25 μg / mL MMC and placed in a 37°C incubator for 1 h. PBMCs (peripheral blood mononuclear cells) stimulated with SEB for 2 days and Raji-PDL-1 cells treated with MMC for 1 h were harvested, washed twice with PBS, resuspended in complete medium, and counted. Cells were cultured at 1 × 10 per well. 5Cells were added separately to U-shaped 96-well plates. Antibodies were added according to the study design and cultured for 3 days. Cell culture supernatants were harvested and IL-2 was measured by ELISA.

[0180] As shown in Figure 29, the mixed culture of human PBMC and Raji-PDL-1 cells had a certain promoting effect on IL-2 secretion from PBMC, and the simultaneous addition of antibodies to the mixed culture system significantly induced IL-2 secretion in PBMC in a remarkable dose-dependent manner. In terms of the promotion level of IL-2 secretion activity, the bifunctional antibody NTPDV2 has an activity equivalent to that of the parent PD-1 single targeting antibody 14C12H1L1 at low concentrations, and has a slightly lower activity than that of the parent PD-1 single targeting antibody 14C12H1L1 at medium and high concentrations. Example 22

[0181] In vivo tumor growth inhibition experiment using anti-CD73 / anti-PD-1 bispecific antibody To measure the in vivo antitumor activity of the anti-CD73 / anti-PD-1 bispecific antibody, MC38-hPDL-1 / hCD73 cells (GemPharmatech Co., Ltd.) were first subcutaneously inoculated into 5-7 week-old female C57BL6-hPD1hPDL-1hCD73 triple transgenic mice (GemPharmatech Co., Ltd.). Modeling and specific administration methods are shown in Table 16. After administration, the length and width of the tumors in each group were measured, and the tumor volume was calculated. [Table 16]

[0182] The experimental results are shown in Figures 30 and 31. The results show that, compared with the isotype control antibodies hIgG and 19F3H2L3 (hG1M), different doses of NTPDV2 can effectively inhibit the growth of mouse tumors, and high doses of NTPDV2 are superior to low doses in tumor inhibition. Moreover, NTPDV2 has no effect on the body weight of tumor-bearing mice. Example 23

[0183] Efficient ablation of bispecific immune checkpoint inhibitor PD-1 / CD73 bispecific antibody-mediated IL-8 and IL-6 secretion in human macrophages by amino acid mutations in the Fc segment HPMM were prepared by induction of PBMCs. The PBMCs used in this study were isolated and prepared by Zhongshan Akesobio Co. Ltd., with informed consent from the donors. Ficoll-Paque PLUS Lymphocyte Isolation Solution (GE, Cat. No. 17-1440-03); RPMI1640 (Gibco, Cat. No. 22400-105); CHO-K1-PD1 cells (constructed by Zhongshan Akesobio Co. Ltd.); U87-MG cells (cells from ATCC purchased from Beijing Zhongyuan Ltd.); FBS (Fetal Bovine Serum, Excell bio, Cat. No. FSP500); Human IFN-γ protein (Sinobio, Cat. No. 11725-HNAS-100); LPS (Lipopolysaccharide) (Sigma, Cat. No. L4391); 96-well cell culture plate (Corning).

[0184] Healthy human PBMCs were isolated according to the instructions of the isolation solution Ficoll-PaqueTMPlus reagent and resuspended in 1640 medium containing 2% FBS. The plates were incubated at 37°C in a cell incubator with 5% CO2. After 2 hours, the supernatant was removed and the attached cells were washed twice with PBS and induced with 1640 complete medium (containing 10% FBS) and 100ng / mL human M-CSF for 7 days. On days 3 and 5, the medium was replaced and M-CSF was added to induce HPMM. On day 7, after induction of HPMM, the cells were harvested, adjusted to a concentration of 100,000 cells / mL in complete medium, and loaded into 96-well plates. Recombinant human IFN-γ (50ng / mL) was added and incubated for 24 hours in an incubator. After 24 hours, CHO-K1-PD1 cells expressing human PD-1 or U87-MG cells expressing human CD73 constitutively in log phase were harvested. The cells were resuspended and then adjusted to a concentration of 300,000 cells / mL with complete medium. The antibodies were diluted to standard concentrations of 25 nM, 2.5 nM, and 0.25 nM with complete medium. Isotype control antibodies and blank controls were designed simultaneously. The supernatant in the 96-well plate was removed, and CHO-K1-PD1 or U87-MG cell suspension and antibodies were added (final volume 200 μL). The system was mixed well and incubated in an incubator for 24 hours. The mixture was centrifuged at 500 × g for 5 minutes, the supernatant was collected, and the secretion levels of IL-8 and IL-6 were measured using Dakewe kit. In the experiment, LPS was used as a positive control and adjusted to a concentration of 100 ng / mL with complete medium.

[0185] In this example, coculture of HPMM with CHO-K1-PD1 cells and U87-MG cells as target cells induced HPMM activation, and after the activated HPMM bound to the target cells via antibody Fab, the Fc fragment of the antibody interacted with FcγR on the HPMM, causing cytokine secretion by the HPMM.

[0186] 3. Experimental Results The results are shown in Figures 32 to 35. The results showed that compared with wild-type IgG1 subtype PD-1 antibody or CD73 antibody, the anti-PD-1 / CD73 bispecific antibody with Fc fragment mutations could effectively eliminate the secretion of IL-6 and IL-8 in immune cells.

[0187] Although the preferred embodiments of the present invention are described above in detail, the present invention is not limited to the above embodiments. Those skilled in the art can make various equivalent modifications or replacements without violating the spirit of the present invention. These equivalent modifications or replacements are included in the scope defined by the claims of this application.

[0188] Array List Nucleotide sequence of the 19F3 heavy chain variable region: [ka] (SEQ ID NO: 1 with CDR sequences underlined)

[0189] Amino acid sequence of the 19F3 heavy chain variable region: [ka] (SEQ ID NO:2, CDR sequences underlined) HCDR1 of 19F3: GYSFTGYT (SEQ ID NO: 3) HCDR2 of 19F3: INPYNAGT (SEQ ID NO: 4) HCDR3 of 19F3: ARSEYRYGGDYFDY (SEQ ID NO: 5)

[0190] Nucleotide sequence of the 19F3 light chain variable region: [ka] (SEQ ID NO:6, CDR sequences underlined)

[0191] Amino acid sequence of the 19F3 light chain variable region: [ka] (SEQ ID NO: 7, CDR sequences underlined) LCDR1 amino acid sequence of 19F3: QSLLNSSNQKNY (SEQ ID NO:8) LCDR2 amino acid sequence of 19F3: FAS (SEQ ID NO:9) LCDR3 amino acid sequence of 19F3: QQHYDTPYT (SEQ ID NO: 10)

[0192] Amino acid sequence of the 19F3 heavy chain framework region: FR-H1: EVQLQQSGPELVKPGASMRMSCKAS (SEQ ID NO: 11) FR-H2: MNWVKQSHGKNLEWIGL (SEQ ID NO: 12) FR-H3: SYNQKFKGKATLTVDKSSSTAYMELLSLTSEDSAVYYC (SEQ ID NO: 13) FR-H4:WGQGTTLTVSS (SEQ ID NO: 14)

[0193] Amino acid sequence of the 19F3 light chain framework region: FR-L1: DIVMTQSPSSLAMSVGQKVTMSCKSS (SEQ ID NO: 15) FR-L2: LAWYQQKPGQSPKLLVY (SEQ ID NO: 16) FR-L3: TRESGVPDRFIGSGSGTDFTLTISSVQAEDLADYFC (SEQ ID NO: 17) FR-L4: FGGGTKLEIK (SEQ ID NO: 18)

[0194] Nucleotide sequence of 19F3H2: (SEQ ID NO: 19, CDR sequences underlined) [ka]

[0195] Amino acid sequence of 19F3H2: (SEQ ID NO:20, CDR sequences underlined) [ka]

[0196] Nucleotide sequence of 19F3L2: (SEQ ID NO:21, CDR sequences underlined) [ka]

[0197] Amino acid sequence of 19F3L2: (SEQ ID NO:22, sequence underlined) [ka]

[0198] Nucleotide sequence of 19F3L3: (SEQ ID NO:23, CDR sequences underlined) [ka]

[0199] Amino acid sequence of 19F3L3: (SEQ ID NO:24, CDR sequences underlined) [ka]

[0200] 19F3H2L3(G1M) Heavy Chain (SEQ ID NO:25, non-variable region sequence underlined) [ka]

[0201] 19F3H2L3(G1M) heavy chain amino acid sequence (SEQ ID NO:26, non-variable region sequence underlined) [ka]

[0202] 19F3H2L3(G1M) light chain nucleotide sequence (SEQ ID NO:27, non-variable region sequence underlined): [ka]

[0203] 19F3H2L3(G1M) light chain amino acid sequence (SEQ ID NO:28, non-variable region sequence underlined) [ka]

[0204] 19F3H2L3 (hG1TM) heavy chain nucleotide sequence (SEQ ID NO:29, non-variable region sequence underlined): [ka]

[0205] Amino acid sequence of 19F3H2L3 (hG1TM) heavy chain variable region: (SEQ ID NO:30) [ka]

[0206] Amino acid sequence of the 19F3H2 framework region: FR-H1: QVQLVQSGAEVVKPGASVKVSCKAS (SEQ ID NO: 31) FR-H2: MNWVRQAPGQNLEWIGL (SEQ ID NO: 32) FR-H3: SYNQKFQGKVTLTVDKSTSTAYMELSSLRSEDTAVYYC (SEQ ID NO: 33) FR-H4: QVQLVQSGAEVVKPGASVKVSCKAS (SEQ ID NO: 34)

[0207] Amino acid sequence of the 19F3L2 framework region: FR-L1: DIVMTQSPSSLAVSVGERVTISCKSS (SEQ ID NO: 35) FR-L2: LAWYQQKPGQAPKLLIY (SEQ ID NO: 36) FR-L3: TRESGVPDRFSGSGSGTDFTLTISSVQAEDVADYYC (SEQ ID NO: 37) FR-L4: FGGGTKLEIK (SEQ ID NO: 38)

[0208] Amino acid sequence of the 19F3L3 framework region: FR-L1: DIVMTQSPSSLAVSVGERVTISCKSS (SEQ ID NO: 39) FR-L2: LAWYQQKPGQAPKLLIY (SEQ ID NO: 40) FR-L3: TRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC (SEQ ID NO: 41) FR-L4: FGGGTKLEIK (SEQ ID NO: 42)

[0209] Nucleotide sequence of the 14C12 heavy chain variable region: (SEQ ID NO:43) [ka]

[0210] Amino acid sequence of the 14C12 heavy chain variable region: (SEQ ID NO:44) [ka] HCDR1 amino acid sequence of 14C12: GFAFSSYD (SEQ ID NO: 45) HCDR2 amino acid sequence of 14C12: ISGGGRYT (SEQ ID NO:46) HCDR3 amino acid sequence of 14C12: ISGGGRYT ANRYGEAWFAY (SEQ ID NO: 47)

[0211] Nucleotide sequence of the 14C12 light chain variable region: (SEQ ID NO:48) [ka]

[0212] Amino acid sequence of the 14C12 light chain variable region: (SEQ ID NO:49) [ka] LCDR1 amino acid sequence of 14C12: QDINTY (SEQ ID NO:50) LCDR2 amino acid sequence of 14C12: RAN (SEQ ID NO:51) LCDR3 amino acid sequence of 14C12: LQYDEFPLT (SEQ ID NO:52)

[0213] Amino acid sequence of the 14C12 heavy chain framework region: FR-H1: EVKLVESGGGLVKPGGSLKLSCAAS (SEQ ID NO: 53) FR-H2: MSWVRQTPEKRLEWVAT (SEQ ID NO: 54) FR-H3: YYPDSVKGRFTISRDNARNTLYLQMSSLRSEDTALYYC (SEQ ID NO: 55) FR-H4: WGQGTLVTVSA (SEQ ID NO: 56)

[0214] Amino acid sequence of the 14C12 light chain framework region: HR-L1: DIKMTQSPSSMYASLGERVTFTCKAS (SEQ ID NO: 57) HR-L2: LSWFQQKPGKSPKTLIY (SEQ ID NO: 58) HR-L3: RLVDGVPSRFSGSGSGQDYSLTISSLEYEDMGIYYC (SEQ ID NO: 59) HR-L4: FGAGTKLELK (SEQ ID NO: 60)

[0215] Nucleotide sequence of 14C12H1: (SEQ ID NO:61) [ka]

[0216] Amino acid sequence of 14C12H1: (SEQ ID NO:62) [ka]

[0217] Nucleotide sequence of 14C12L1: (SEQ ID NO:63) [ka]

[0218] Amino acid sequence of 14C12L1: (SEQ ID NO:64) [ka]

[0219] Nucleotide sequence of 14C12H1L1 heavy chain (SEQ ID NO:65) [ka]

[0220] 14C12H1L1 heavy chain variable region amino acid sequence: (SEQ ID NO:66) [ka]

[0221] Nucleotide sequence of 14C12H1L1 light chain (SEQ ID NO:67) [ka]

[0222] 14C12H1L1 light chain amino acid sequence: (SEQ ID NO:68) [ka]

[0223] Nucleotide sequence of 14C12H1L1 (G1TM) heavy chain (SEQ ID NO:69) [ka]

[0224] Amino acid sequence of 14C12H1L1 (G1TM) heavy chain variable region: (SEQ ID NO: 70) [ka]

[0225] Amino acid sequence of the 14C12H1 heavy chain framework region: FR-H1: EVQLVESGGGLVQPGGSLRLSCAAS (SEQ ID NO: 71) FR-H2: MSWVRQAPGKGLDWVAT (SEQ ID NO: 72) FR-H3: YYPDSVKGRFTISRDNSKNNLYLQMNSLRAEDTALYYC (SEQ ID NO: 73) FR-H4: WGQGTLVTVSS (SEQ ID NO: 74)

[0226] 14C12L1 light chain framework region amino acid sequence: FR-L1: DIQMTQSPSSMSASVGDRVTFTCRAS (SEQ ID NO: 75) FR-L2: LSWFQQKPGKSPKTLIY (SEQ ID NO: 76) FR-L3: RLVSGVPSRFSGSGSGQDYTLTISSLQPEDMATYYC (SEQ ID NO: 77) FR-L4: FGAGTKLELK (SEQ ID NO: 78)

[0227] Amino acid sequence of linker 1: GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 79) Nucleotide sequence of linker 1: (SEQ ID NO:80) GGCGGCGGCGGCAGCGGCGGCGGCGGCTCCGGAGGAGGCGGCTCTGGCGGCGGCGGCAGC

[0228] Amino acid sequence of linker 2: GGGGSGGGSGGGGS: (SEQ ID NO: 81) Nucleotide sequence of linker 2: (SEQ ID NO:82) GGCGGCGGCGGCTCCGGAGGAGGCGGCTCTGGCGGCGGCGGCAGC

[0229] Amino acid sequence of the heavy chain of NTPDV2 and NTPDV4 (SEQ ID NO: 83): where the CDR regions of 19F3H2 (hG1TM) in the immunoglobulin portion are in bold, the CDR regions of 14C12H1V-linker1-14C12L1V in the scFv portion are in bold, mutated amino acids in the heavy chain region are in bold italics, and the linker region is in bold. [ka]

[0230] Nucleotide sequence of the heavy chain of NTPDV2 and NTPDV4: (SEQ ID NO:84) [ka]

[0231] Amino acid sequence of the heavy chain of NTPDV1 and NTPDV3 (SEQ ID NO:85): The CDR regions of 19F3H2 (hG1TM) in the immunoglobulin part are in bold, the CDR regions of 14C12H1V-linker2-14C12L1V in the scFv part are in bold, the mutated amino acids in the heavy chain region are in bold italics, and the linker region is in bold: [ka]

[0232] Nucleotide sequence of the heavy chain of NTPDV1 and NTPDV3: (SEQ ID NO:86) [ka]

[0233] Amino acid sequence of NT5E(1-552)-his: (SEQ ID NO:87) [ka]

[0234] Nucleotide sequence of NT5E(1-552)-his: (SEQ ID NO:88) [ka]

[0235] Amino acid sequence of mFc: (SEQ ID NO: 89) [ka]

[0236] Amino acid sequence of 19F3H1V-linker-19F3L2V: (SEQ ID NO: 90) [ka]

[0237] Nucleotide sequence of 19F3H1V-linker2-19F3L2V: (SEQ ID NO:91) [ka]

[0238] Nucleotide sequence of 19F3H1: (SEQ ID NO:92) [ka]

[0239] Amino acid sequence of 19F3H1: (SEQ ID NO:93) [ka]

[0240] Nucleotide sequence of 19F3L1: (SEQ ID NO:94) [ka]

[0241] Amino acid sequence of 19F3L1: (SEQ ID NO:95) [ka]

[0242] 19F3L2 Light Chain (Full Length) Amino Acids (SEQ ID NO:96, Non-Variable Region Sequence Underlined) [ka]

[0243] Amino acid sequence of 19F3H2 heavy chain variable region with CDR sequences underlined: (SEQ ID NO:97) [ka]

[0244] Amino acid sequence of the 19F3L2 heavy chain variable region with CDR sequences underlined: (SEQ ID NO:98) [ka]

[0245] Amino acid sequence of the 19F3L3 heavy chain variable region with CDR sequences underlined: (SEQ ID NO:99) [ka]

[0246] Nucleotide sequence of 19F3L2 light chain (full length): (SEQ ID NO: 100) [ka]

Claims

1. below: A first protein functional region that targets PD-1; and Second Protein Functional Domain Targeting CD73 1. An anti-CD73 / anti-PD-1 bispecific antibody comprising: The first protein functional region comprises HCDR1, HCDR2 and HCDR3 contained in a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO:44, wherein preferably the amino acid sequences of HCDR1, HCDR2 and HCDR3 are as set forth in SEQ ID NOs:45-47, respectively, or have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, sequence identity to the sequences as set forth in SEQ ID NOs:45-47, or have one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the sequences as set forth in SEQ ID NOs:45-47; and comprising LCDR1, LCDR2 and LCDR3 contained in a light chain variable region having an amino acid sequence as set forth in SEQ ID NO:49, wherein preferably the amino acid sequences of LCDR1, LCDR2 and LCDR3 are respectively set forth in SEQ ID NOs:50-52, or are sequences having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, sequence identity to said sequences as set forth in SEQ ID NOs:50-52, or are amino acid sequences having one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to said sequences as set forth in SEQ ID NOs:50-52; or said second protein functional region is HCDR1, HCDR2 and HCDR3 contained in a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO:2, wherein preferably said amino acid sequences of HCDR1, HCDR2 and HCDR3 are the amino acid sequences as set forth in SEQ ID NOs:3-5, respectively, or have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to said sequences as set forth in SEQ ID NOs:3-5, or have one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to said sequences as set forth in SEQ ID NOs:3-5; and comprising LCDR1, LCDR2 and LCDR3 contained in a light chain variable region having the amino acid sequence set forth in SEQ ID NO:7, wherein preferably the amino acid sequences of LCDR1, LCDR2 and LCDR3 are respectively set forth in SEQ ID NOs:8-10, or sequences having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, sequence identity to said sequences set forth in SEQ ID NOs:8-10, or amino acid sequences having one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to said sequences set forth in SEQ ID NOs:8-10; Anti-CD73 / anti-PD-1 bispecific antibody.

2. 2. The anti-CD73 / anti-PD-1 bispecific antibody of claim 1, The first protein functional region is a sequence having the amino acid sequence as set forth in SEQ ID NO: 44 or SEQ ID NO: 62, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, sequence identity to said sequence as set forth in SEQ ID NO: 44 or 62, 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 as set forth in SEQ ID NO: 44 or 62; and a sequence having the amino acid sequence as set forth in SEQ ID NO: 49 or SEQ ID NO: 64, respectively, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, sequence identity to said sequence as set forth in SEQ ID NO: 49 or 64, 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 as set forth in SEQ ID NO: 49 or 64; and / or the second protein functional domain has an amino acid sequence as set forth in SEQ ID NO:2 or SEQ ID NO:20, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, sequence identity to said sequence as set forth in SEQ ID NO:2 or SEQ ID NO:20, 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 as set forth in SEQ ID NO:2 or SEQ ID NO:20; and a sequence having the amino acid sequence as set forth in SEQ ID NO: 7 or SEQ ID NO: 22, respectively, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, sequence identity to said sequence as set forth in SEQ ID NO: 7 or SEQ ID NO: 22, respectively, or an amino acid sequence which has one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to said sequence as set forth in SEQ ID NO: 7 or SEQ ID NO: 22; Or, the second protein functional domain has an amino acid sequence as set forth in SEQ ID NO:20, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, sequence identity to said sequence as set forth in SEQ ID NO:20, 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 as set forth in SEQ ID NO:20; and It comprises an amino acid sequence having the amino acid sequence set forth in SEQ ID NO:24, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, sequence identity to said sequence set forth in SEQ ID NO:24, 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 set forth in SEQ ID NO:

24. Anti-CD73 / anti-PD-1 bispecific antibody.

3. 3. The anti-CD73 / anti-PD-1 bispecific antibody of claim 1 or 2, wherein the number of the first protein functional domain and the second protein functional domain is independently one, two or more.

4. The anti-CD73 / anti-PD-1 bispecific antibody according to any one of claims 1 to 3, wherein the first protein functional domain and the second protein functional domain are linked directly or via a linker, preferably the linker is (GGGGS)n, and n is a positive integer, e.g. 1, 2, 3, 4, 5 or 6. Anti-CD73 / anti-PD-1 bispecific antibody

5. The anti-CD73 / anti-PD-1 bispecific antibody of any one of claims 1 to 4, wherein the first protein functional domain and the second protein functional domain are independently an immunoglobulin or an antigen-binding fragment, such as a half antibody, Fab, F(ab') ２ or a single chain variable region fragment, preferably wherein the first protein functional region is an immunoglobulin and the second protein functional region is an antigen-binding fragment; or wherein the first protein functional region is an antigen-binding fragment and the second protein functional region is an immunoglobulin. Anti-CD73 / anti-PD-1 bispecific antibody.

6. 6. The anti-CD73 / anti-PD-1 bispecific antibody of any one of claims 1 to 5, wherein the N-terminus of the heavy chain variable region of the antigen-binding fragment is linked directly (or via a linker) to the C-terminus of CH1 of the immunoglobulin and the N-terminus of the light chain variable region of the antigen-binding fragment is linked directly (or via a linker) to the C-terminus of the light chain variable region CL of the immunoglobulin; or the N-terminus of the heavy chain variable region of the antigen-binding fragment is linked directly (or via a linker) to the C-terminus of the light chain variable region CL of the immunoglobulin and the N-terminus of the light chain variable region of the antigen-binding fragment is linked directly (or via a linker) to the C-terminus of the heavy chain variable region of the immunoglobulin; or the C-terminus of the heavy chain variable region of the antigen-binding fragment is linked directly (or via a linker) to the N-terminus of the heavy chain of the immunoglobulin, and the C-terminus of the light chain variable region of the antigen-binding fragment is linked directly (or via a linker) to the N-terminus of the light chain of the immunoglobulin; or the C-terminus of the heavy chain variable region of the antigen-binding fragment is linked directly (or via a linker) to the N-terminus of the light chain of the immunoglobulin, and the C-terminus of the light chain variable region of the antigen-binding fragment is linked directly (or via a linker) to the N-terminus of the heavy chain of the immunoglobulin. Anti-CD73 / anti-PD-1 bispecific antibody.

7. The anti-CD73 / anti-PD-1 bispecific antibody of any one of claims 1 to 6, wherein the antigen-binding fragment is a single chain variable region fragment; preferably, the first protein functional region is an immunoglobulin and the second protein functional region is a single chain variable region fragment; or, the first protein functional region is a single chain variable region fragment and the second protein functional region is an immunoglobulin. Anti-CD73 / anti-PD-1 bispecific antibody.

8. 8. The anti-CD73 / anti-PD-1 bispecific antibody of claim 7, wherein the single chain variable region fragment is an antibody heavy chain variable region (V Ｈ ) and an antibody light chain variable region (V Ｌ ) via a linker; preferably, the single chain variable region fragment has the following structure: NH ２ -V Ｌ -Linker-V Ｈ -COOH or NH ２ -V Ｈ -Linker-V Ｌ -COOH Anti-CD73 / anti-PD-1 bispecific antibody.

9. The anti-CD73 / anti-PD-1 bispecific antibody according to claim 7 or 8, wherein the single-chain variable region fragment is linked to the C-terminus (CH) of the heavy chain of the immunoglobulin (or the N-terminus of the heavy chain, the C-terminus of CH1 of the heavy chain variable region) via a linker, and the antibody heavy chain variable region (V Ｈ ) is linked first, or the antibody light chain variable region (V Ｌ ) is first linked; preferably, the single chain variable region fragment has the following structure: linker-V Ｈ -Linker-V Ｌ -COOH or linker-V Ｌ -Linker-V Ｈ may have -COOH; Preferably, the heavy chain variable region of said immunoglobulin comprises CDRs having the amino acid sequences set forth in SEQ ID NOs: 3-5, and the light chain variable region of said immunoglobulin comprises CDRs having the amino acid sequences set forth in SEQ ID NOs: 8-10; The heavy chain variable region of the single chain variable region fragment comprises a CDR having an amino acid sequence set forth in SEQ ID NOs: 45 to 47, and the light chain variable region of the single chain variable region fragment comprises a CDR having an amino acid sequence set forth in SEQ ID NOs: 50 to 52; Preferably, the single chain variable region fragment (NH ２ -V Ｌ -Linker-V Ｈ -COOH or NH ２ -V Ｈ -Linker-V Ｌ When a linker (such as —COOH) is linked to the C-terminus of the heavy chain of the immunoglobulin via a linker, the antibody heavy chain variable region (V Ｈ ) may be linked first, or a single chain variable region fragment of an antibody light chain variable region (V Ｌ ) may be linked first, Or preferably, the heavy chain variable region of said immunoglobulin comprises CDRs having the amino acid sequences set forth in SEQ ID NOs: 45-47, and the light chain variable region of said immunoglobulin comprises CDRs having the amino acid sequences set forth in SEQ ID NOs: 50-52; and / or The heavy chain variable region of the single chain variable region fragment comprises a CDR having an amino acid sequence set forth in SEQ ID NO: 3 to 5, and the light chain variable region of the single chain variable region fragment comprises a CDR having an amino acid sequence set forth in SEQ ID NO: 8 to 10; Here, the single chain variable region fragment (NH ２ -V Ｌ -Linker-V Ｈ -COOH or NH ２ -V Ｈ -Linker-V Ｌ When a linker (such as —COOH) is linked to the C-terminus of the heavy chain of the immunoglobulin via a linker, the antibody heavy chain variable region (V Ｈ ) may be linked first, or a single chain variable region fragment of an antibody light chain variable region (V Ｌ ) may be linked first, Preferably, One immunoglobulin molecule is linked to two single chain variable region fragment molecules, and more preferably, the two single chain variable region fragment molecules are identical. Anti-CD73 / anti-PD-1 bispecific antibody.

10. 3. The anti-CD73 / anti-PD-1 bispecific antibody of claim 1 or 2, wherein the immunoglobulin is IgG, IgA, IgD, IgE or IgM, preferably IgG, such as IgG1, IgG2, IgG3 or IgG4.

11. 3. The anti-CD73 / anti-PD-1 bispecific antibody of claim 1 or 2, wherein the single-chain variable region fragment is linked to the C-terminus of the heavy chain of the immunoglobulin, preferably, one immunoglobulin molecule is linked to two single-chain variable region fragment molecules, and more preferably, the two single-chain variable region fragment molecules are identical.

12. 3. The anti-CD73 / anti-PD-1 bispecific antibody of claim 1 or 2, wherein the immunoglobulin heavy chain variable region comprises CDRs having the amino acid sequences set forth in SEQ ID NOs: 45-47, and the immunoglobulin light chain variable region comprises CDRs having the amino acid sequences set forth in SEQ ID NOs: 50-52; and / or The heavy chain variable region of the single chain variable region fragment comprises a CDR having an amino acid sequence set forth in SEQ ID NO: 3 to 5, and the light chain variable region of the single chain variable region fragment comprises a CDR having an amino acid sequence set forth in SEQ ID NO: 8 to 10; Preferably, when the single-chain variable region fragment is linked to the C-terminus of the heavy chain of the immunoglobulin via a linker, the antibody heavy chain variable region (V Ｈ ) may be linked first, or a single chain variable region fragment of an antibody light chain variable region (V Ｌ ) may be concatenated first, Anti-CD73 / anti-PD-1 bispecific antibody.

13. 3. The anti-CD73 / anti-PD-1 bispecific antibody of claim 1 or 2, the heavy chain variable region of said immunoglobulin comprises CDRs having the amino acid sequences set forth in SEQ ID NOs: 3-5, and the light chain variable region of said immunoglobulin comprises CDRs having the amino acid sequences set forth in SEQ ID NOs: 8-10; and / or The heavy chain variable region of the single chain variable region fragment comprises a CDR having an amino acid sequence set forth in SEQ ID NOs: 45 to 47, and the light chain variable region of the single chain variable region fragment comprises a CDR having an amino acid sequence set forth in SEQ ID NOs: 50 to 52; Here, when the single-chain variable region fragment is linked to the C-terminus of the heavy chain of the immunoglobulin via a linker, the antibody heavy chain variable region (V Ｈ ) may be linked first, and a single chain variable region fragment of an antibody light chain variable region (V Ｌ ) may be concatenated first, Anti-CD73 / anti-PD-1 bispecific antibody.

14. 3. The anti-CD73 / anti-PD-1 bispecific antibody of claim 1 or 2, the heavy chain variable region of said immunoglobulin has an amino acid sequence selected from SEQ ID NO:44 and SEQ ID NO:62, and the light chain variable region of said immunoglobulin has an amino acid sequence selected from SEQ ID NO:49 and SEQ ID NO:64, respectively; and / or the heavy chain variable region of the single chain variable region fragment has an amino acid sequence selected from SEQ ID NO:2 and SEQ ID NO:20, and the light chain variable region of the single chain variable region fragment has an amino acid sequence selected from SEQ ID NO:7 and SEQ ID NO:22, respectively; or the heavy chain variable region of the single chain variable region fragment has the amino acid sequence set forth in SEQ ID NO:20, and the light chain variable region of the single chain variable region fragment has the amino acid sequence set forth in SEQ ID NO:24; Here, when the single-chain variable region fragment is linked to the C-terminus of the heavy chain of the immunoglobulin via a linker, the antibody heavy chain variable region (V Ｈ ) may be linked first, and the antibody light chain variable region (V Ｌ ) may be concatenated first, Anti-CD73 / anti-PD-1 bispecific antibody.

15. 3. The anti-CD73 / anti-PD-1 bispecific antibody of claim 1 or 2, the heavy chain variable region of the immunoglobulin has an amino acid sequence selected from SEQ ID NO:2 and SEQ ID NO:20, and the light chain variable region of the immunoglobulin has an amino acid sequence selected from SEQ ID NO:7 and SEQ ID NO:22; or the heavy chain variable region of the single chain variable region fragment has the amino acid sequence set forth in SEQ ID NO:20, and the light chain variable region of the single chain variable region fragment has the amino acid sequence set forth in SEQ ID NO:24; and / or the heavy chain variable region of the single chain variable region fragment has an amino acid sequence selected from SEQ ID NO: 44 and SEQ ID NO: 62, and the light chain variable region of the single chain variable region fragment has an amino acid sequence selected from SEQ ID NO: 49 and SEQ ID NO: 64; Here, when the single-chain variable region fragment is linked to the C-terminus of the heavy chain via a linker, the antibody heavy chain variable region (V Ｈ ) may be linked first, or the antibody light chain variable region (V Ｌ ) may be concatenated first, Anti-CD73 / anti-PD-1 bispecific antibody.

16. 15. The anti-CD73 / anti-PD-1 bispecific antibody of any one of claims 5 to 14, wherein the immunoglobulin comprises non-CDR regions, and the non-CDR regions are derived from a species other than mouse, such as from a human antibody; more preferably, the constant region of the immunoglobulin is humanized; for example, the heavy chain constant region is the Ig gamma-1 chain C region, Accession No. P01857; and the light chain constant region is the Ig kappa chain C region, Accession No. P01834; or the immunoglobulin heavy chain constant region is mutated at any two or three of positions 234, 235 and 237 based on the Igγ-1 chain C region, Accession No. P1857, and after the mutation, the bispecific antibody has a decreased affinity constant for FcγRIa, FcγRIIIa and / or C1q compared to the bispecific antibody before the mutation; More preferably, the heavy chain constant region comprises the following mutations at positions 234, 235 and / or 237 based on the Igγ-1 chain C region, Accession No. P01857, according to the EU numbering system: L234A and L235A; L234A and G237A; L235A and G237A; or having L234A, L235A and G237A, Even more preferably, the heavy chain constant region of said immunoglobulin also comprises the following mutation: N297A, D265A, D270A, P238D, L328E, E233D, H268D, P271G, A330R, C226S, C229S, E233P, P331S, S267E, L328F, A330L, M252Y, S254T, T256E, N297Q, P238S, P238A, A327Q, A327G, P329A, K322A, T394D, G236R, G236A, L328R, A330S, P331S, H268A, E318A and K320A, and having one or more mutations selected from Preferably, the anti-CD73 / anti-PD-1 bispecific antibody has a structure shown as heavy chain-light chain-linker1-scFv, and the scFv is selected from 14C12H1V-linker2-14C12L1V, 14C12H1V-linker1-14C12L1V, 14C12H1V-linker2-14C12L1V and 14C12H1V-linker1-14C12L1V, specifically: (1) NTPDV1, the heavy chain of which has the amino acid sequence set forth in SEQ ID NO: 85, the light chain of which has the amino acid sequence set forth in SEQ ID NO: 28, linker 1 having the amino acid sequence set forth in SEQ ID NO: 79, 14C12H1V having the amino acid sequence set forth in SEQ ID NO: 66, linker 2 having the amino acid sequence set forth in SEQ ID NO: 81, and 14C12L1V having the amino acid sequence set forth in SEQ ID NO: 68; (2) NTPDV2, the heavy chain of which has the amino acid sequence set forth in SEQ ID NO: 85, the light chain of which has the amino acid sequence set forth in SEQ ID NO: 28, and linker 1 of which has the amino acid sequence set forth in SEQ ID NO: 79, 14C12H1V of which has the amino acid sequence set forth in SEQ ID NO: 66, linker 1 of which has the amino acid sequence set forth in SEQ ID NO: 79, and 14C12L1V of which has the amino acid sequence set forth in SEQ ID NO: 68; (3) NTPDV3, the heavy chain having the amino acid sequence set forth in SEQ ID NO: 85, the light chain having the amino acid sequence set forth in SEQ ID NO: 96, linker 1 having the amino acid sequence set forth in SEQ ID NO: 79, 14C12H1V having the amino acid sequence set forth in SEQ ID NO: 66, linker 2 having the amino acid sequence set forth in SEQ ID NO: 81, and 14C12L1V having the amino acid sequence set forth in SEQ ID NO: 68; and (4) NTPDV4, the heavy chain of which has the amino acid sequence set forth in SEQ ID NO: 85, the light chain of which has the amino acid sequence set forth in SEQ ID NO: 96, and linker 1 of which has the amino acid sequence set forth in SEQ ID NO: 79, 14C12H1V of which has the amino acid sequence set forth in SEQ ID NO: 66, linker 1 of which has the amino acid sequence set forth in SEQ ID NO: 79, and 14C12L1V of which has the amino acid sequence set forth in SEQ ID NO: 68; Selected from the group consisting of: Anti-CD73 / anti-PD-1 bispecific antibody.

17. The anti-CD73 / anti-PD-1 bispecific antibody according to any one of claims 1 to 16, －５ Less than M, for example, about 10 －６ M, 10 －７ M, 10 －８ M, 10 －９ M, 10 －１０ K below M Ｄ An anti-CD73 / anti-PD-1 bispecific antibody that binds to CD73 protein and / or PD-1 protein.

18. An isolated nucleic acid molecule comprising a nucleotide sequence capable of encoding a heavy chain variable region of a bispecific antibody, The heavy chain variable region of the antibody comprises: CDRs having the amino acid sequences of SEQ ID NOs: 3-5, CDRs having the amino acid sequences of SEQ ID NOs: 45-47, and CDRs having the amino acid sequences of SEQ ID NOs: 50-52; or comprising a CDR having an amino acid sequence of SEQ ID NO:45-47, a CDR having an amino acid sequence of SEQ ID NO:3-5, and a CDR having an amino acid sequence of SEQ ID NO:8-10; and the heavy chain variable region of the bispecific antibody specifically binds to CD73 and PD-1 antigens as part of the bispecific antibody, and the bispecific antibody further comprises: CDRs having the amino acid sequences of SEQ ID NOs: 8-10; Or, a CDR having an amino acid sequence of SEQ ID NO: 50 to 52. and a light chain variable region comprising Preferably, the CDRs of the light chain variable region are different from the CDRs of the heavy chain variable region. An isolated nucleic acid molecule.

19. A vector comprising the isolated nucleic acid molecule of claim 18.

20. 20. A host cell comprising the isolated nucleic acid molecule of claim 18 or the vector of claim 19.

21. 21. A method for preparing an anti-CD73 / anti-PD-1 bispecific antibody according to any one of claims 1 to 17, comprising culturing a host cell according to claim 20 under suitable conditions and isolating said bispecific antibody from said cell culture.

22. 18. A conjugate comprising the anti-CD73 / anti-PD-1 bispecific antibody of any one of claims 1 to 17 and a conjugated moiety, wherein the conjugated moiety is a detectable label; in particular, the conjugated moiety is a radioisotope, a fluorescent substance, a chemiluminescent substance, a colored substance or an enzyme.

23. 27. A kit comprising an anti-CD73 / anti-PD-1 bispecific antibody according to any one of claims 1 to 17 or a conjugate according to claim 22, preferably said kit further comprising a secondary antibody specifically recognizing said bispecific antibody, optionally said secondary antibody further comprising a detectable label, such as a radioisotope, a fluorescent substance, a chemiluminescent substance, a colored substance or an enzyme.

24. 20. Use of an anti-CD73 / anti-PD-1 bispecific antibody according to any one of claims 1 to 17 in the preparation of a kit for detecting the presence or level of CD73 and / or PD-1 in a sample.

25. 27. A pharmaceutical composition comprising the anti-CD73 / anti-PD-1 bispecific antibody of any one of claims 1 to 17 or the conjugate of claim 22, optionally further comprising a pharma- ceutical acceptable carrier and / or excipient.

26. Use of an anti-CD73 / anti-PD-1 bispecific antibody according to any of claims 1 to 17 or a conjugate according to claim 22 in the prevention and / or treatment of tumors or anemia or in the diagnosis of tumors or anemia.

27. Use of an anti-CD73 / anti-PD-1 bispecific antibody according to any one of claims 1 to 17 or a conjugate according to claim 22 in the preparation of a medicament for preventing and / or treating a tumor or anemia, or in the preparation of a medicament for diagnosing a tumor or anemia.

28. The following drugs: an agent for detecting the level of CD73 in a sample; An agent for inhibiting the enzymatic activity of CD73; and / or Agents for blocking the binding of PD-1 to PDL-1; an agent for downregulating (e.g., downregulating) the activity or levels of PD-1; Agents for alleviating the immunosuppression of PD-1 in an organism; An agent for increasing IL-2 expression in T lymphocytes, or An agent for increasing IFN-γ expression in T lymphocytes.

23. Use of an anti-CD73 / anti-PD-1 bispecific antibody according to any one of claims 1 to 17 or a conjugate according to claim 22 in the preparation of

29. 27. An in vivo or in vitro method comprising administering to a cell or to a subject in need thereof an effective amount of the anti-CD73 / anti-PD-1 bispecific antibody of any one of claims 1 to 17 or the conjugate of claim 22.

30. below: Hybridoma cell line LT014 (also called CD73-19F3), deposited at the China Culture Collection Center (CCTCC) on June 19, 2018, with accession number CCTCCNO: C2018137; or Hybridoma cell line LT003 (also called PD-1-14C12), deposited at the China Culture Collection Center (CCTCC) on June 16, 2015 with accession number CCTCCNO: C2015105. A hybridoma cell line selected from the group consisting of

31. An anti-CD73 monoclonal antibody, wherein the antibody is 19F3H2L3 (hG1TM) and has a heavy chain amino acid sequence set forth in SEQ ID NO:30 and a light chain amino acid sequence set forth in SEQ ID NO:28.

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