Anti-CD73 / anti-PD-1 bispecific antibodies and uses thereof

Abstract

Provided are anti-CD73 / anti-PD-1 bispecific antibodies, pharmaceutical compositions thereof, and uses thereof.

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 to 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, particularly 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, with HIF-1 acting as a transcription factor being the most important. An important feature of the tumor microenvironment is hypoxia, which induces upregulation of hypoxia-inducible factor-1 (HIF-1) and other molecules, 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 has shown 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, and head and neck cancer.

[0004] CD73 possesses both 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. Increasingly, research has revealed that CD73 is a key regulatory molecule for tumor cell proliferation, metastasis, and invasion in vitro, and for tumor angiogenesis and tumor immune evasion in vivo. An important mechanism of immunosuppression is mediated by the CD73-adenosine metabolic signaling pathway. CD39, located 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 immune activation-related signaling pathways, including LCK, MAPK, and PKC, and by activating protein kinase A (PKA) and Csk kinase, inhibiting the immune killing effect of T cells, thereby enabling 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 on immune and non-immune cells, can promote tumor immune evasion, development, and metastasis, with the most important role being the inhibition of cytotoxic T cell (CTL) and NK cell function by Treg cell-associated CD73-adenosine signaling.

[0005] For the treatment of solid tumors, one of the key aspects to overcoming drug resistance and improving therapeutic efficacy is alleviating the inhibitory effects of the tumor microenvironment (TME) on immune effector cells. The TME is a highly complex system composed of various cells, extracellular matrices, enzymes, cytokines, metabolites, and other components. The TME is characterized by significantly low hydrogen, low pH, and high pressure, significantly different from normal tissues. Hypoxia or ATP enrichment induced by chemoradiotherapy to kill tumor cells promotes the CD39-CD73 adenosine signaling cascade, which is beneficial to 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 CD73 gene knockout in animal models can effectively block tumor growth and metastasis. Recently, the use of CD73 monoclonal antibodies, small interfering RNA technology, and the specific inhibitor APCP have achieved remarkable therapeutic effects in animal experiments, providing a new avenue for antitumor treatment. Evidence from in vivo studies indicates that targeted blockade of CD73 may be an effective treatment for tumor patients.

[0007] The relationship between CD73 overexpression and patient tumor subtype, prognosis, and response 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 crucial role in downregulating T cell activation, and PD-1-mediated downregulation of T cells is one of the key 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 immune cell killing of tumor tissue through the PD-1 / PDL-1 signaling pathway. Tumors with high PDL-1 expression are associated with cancers that are difficult to detect (Hamanishi et al., Proc. Natl. Acad. Sci. USA, 2007;104:3360-5). An effective method to antagonize PD-1 and thereby inhibit the PD-1 / PDL-1 signaling pathway is the injection of anti-PDL-1 antibodies.

[0010] Due to the broad anti-tumor prospects 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, including 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 by genetic engineering. Genetic engineering offers 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, i.e., the 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 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 an Fc receptor (FcR) on the surface of the killer cell (NK cell, macrophage, etc.).

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

[0014] The IgG family includes four members: IgG1, IgG2, IgG3, and IgG4. These members differ in amino acids in the heavy chain constant region (Fc) fragment, known as the crystallizable (Fc) region, resulting in different affinities for FcγRs. 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, and that binding of the Fc fragment of antibodies targeting immune checkpoints such as PD-1 to Fc receptors negatively impacts antibody-mediated anticancer 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 secreted primarily by monocytes. IL-8 plays an important role in promoting the proliferation of normal and tumor cells, particularly tumor initiation and progression. Several studies have shown that IL-8 can promote tumor initiation, and tumor cells themselves secrete IL-8 to promote tumor growth and metastasis (Lo MC et al., Cancer Letters, 2013, 335(1):81-92). Therefore, IL-8 is an essential inflammatory factor in the tumor microenvironment.

[0016] As a pro-inflammatory factor, IL-8 is closely related to tumor development and progression. IL-8 gene expression increases during methylarsonate-induced malignant transformation of non-renal cancer cells. IL-8 gene silencing can significantly inhibit the growth of transplanted tumors in mice, and reduced IL-8 levels can suppress the expression of matrix metalloproteinase-9, cyclin D1, the proapoptotic protein Bcl-2, and vascular endothelial growth factor (VEGF), which are associated with 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 can induce malignant transformation of a non-tumorous 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). These studies indicate that IL-8 levels are 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 tumor model of bladder cancer, the use of anti-IL-8 antibodies can significantly suppress tumor growth.

[0017] IL-6 is rapidly produced by macrophages, primarily 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 against infection and tissue injury, high levels of IL-6 can activate the coagulation pathway and vascular endothelial cells, thereby inhibiting myocardial function and even causing a "cytokine storm," resulting in a severe acute systemic inflammatory response. Cytokine storms are a potentially fatal complication and side effect of viral infections, tumor immunotherapy, and other treatments.

[0018] Immune-related side effects are common and dangerous side effects of antitumor treatment using 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. However, off-target effects have also led to novel toxicity profiles, including severe immune-mediated adverse events (irAEs) in major organs (including the heart, lungs, and brain), which 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 the secretion of IL-8 and IL-6 by macrophages (Kinder M et al., mAbs., 2015). However, mutations in the Fc segment of antibodies that eliminate FcγRIA binding 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 a large number of samples: Hybridoma cell line LT014 (also called CD73-19F3): deposited with the China Culture Collection Center (CCTCC) on June 19, 2018, with accession number CCTCCNO: 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 CCTCCNO: C2015105 got

[0022] Surprisingly, the inventors discovered that: The hybridoma cell line LTO14 can secrete a specific monoclonal antibody (designated 19F3) that specifically binds to human CD73, and the monoclonal antibody can effectively inhibit the enzymatic activity reaction of CD73 in a non-substrate competitive manner, reduce the production of adenosine, and promote the activation of T cells and 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 present inventors have creatively prepared humanized anti-CD73 antibodies (designated 19F3H2L2, 19F3H2L3, 19F3H2L3(hG1M), and 19F3H2L3(hG1™), respectively) and humanized anti-PD-1 antibodies (designated 14C12H1L1 and 14C12H1L1(hG1™)).

[0024] Furthermore, the inventors creatively fused two kinds of humanized antibodies into a new antibody through protein recombination, and obtained humanized bifunctional antibodies (named P1D7V01, P1D7V03, NTPDV1, NTPDV2, NTPDV3 and NTPDV4, respectively (also referred to as NTPDV1(hG1™), NTPDV2(hG1™), NTPDV3(hG1™) and NTPDV4(hG1™) 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 have 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 domain that targets PD-1; and A second protein functional domain that targets 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 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 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 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 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 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 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 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 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 the amino acid sequence 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 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 set forth in SEQ ID NO: 44 or 62; and a sequence having the amino acid sequence 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 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 set forth in SEQ ID NO: 49 or 64; and / or the second protein functional region comprises a sequence having the amino acid sequence 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 the sequence 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 the sequence set forth in SEQ ID NO:2 or 20; and a sequence having the amino acid sequence 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 set forth in SEQ ID NO: 7 or 22, 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: 7 or 22; or the second protein functional region comprises an amino acid sequence having the amino acid sequence 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 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 set forth in SEQ ID NO:20; and The present invention relates to a 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.

[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 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 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 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 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 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 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 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 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 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).

[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 the amino acid sequence 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 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 set forth in SEQ ID NO: 2 or 20; and a sequence having the amino acid sequence 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 set forth in SEQ ID NO: 7, SEQ ID NO: 22 or 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: 7, 22 or 24; and / or The second protein functional domain is a sequence having the amino acid sequence 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 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 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 present 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, where 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 region and the second protein functional region 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 region fragment; preferably, the first protein functional region is an immunoglobulin and the second protein functional region is an antigen-binding fragment, or the first protein functional region is an antigen-binding fragment and the second protein functional region is an immunoglobulin.

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

[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 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.

[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, the bispecific antibody is provided, wherein the number of first protein functional domains and second protein functional domains is 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 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 located at the C-terminus (CH) of the heavy chain of an immunoglobulin (or the N-terminus, Steady When the antibody is linked to the C-terminus of the CH1 of the single-chain variable region fragment via a linker, the antibody heavy chain variable region (VH) of the single-chain variable region fragment may be linked first, or the antibody light chain variable region (VL) of the single-chain variable region fragment may be linked first; Preferably, the single chain variable region fragment may have the general structure: CH-linker-VH-linker-VL-COOH, or CH-linker-VL-linker-VH-COOH; Preferably, 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; the heavy chain variable region of the single-chain variable region fragment comprises CDRs having the amino acid sequences of 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 of SEQ ID NOs: 50 to 52; Preferably, when a single-chain variable region fragment (e.g., NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH) is linked to the C-terminus of an immunoglobulin heavy chain via a linker, the antibody heavy chain variable region (VH) of the single-chain variable region fragment comprising a CDR having the amino acid sequence set forth in any of SEQ ID NOS: 45 to 47 may be linked first, or the antibody light chain variable region (VL) of the single-chain variable region fragment comprising a CDR having the amino acid sequence set forth in any of SEQ ID NOS: 50 to 52 may be linked first. Or preferably, the heavy chain variable region of the immunoglobulin 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 immunoglobulin comprises a CDR having an amino acid sequence 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, when the single-chain variable region fragment (e.g., NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH) is linked to the C-terminus of the heavy chain of an immunoglobulin via a linker, the antibody heavy chain variable region (VH) of the single-chain variable region fragment comprising a CDR having the amino acid sequence set forth in SEQ ID NOs: 3 to 5 may be linked first, or the antibody light chain variable region (VL) of the single-chain variable region fragment comprising a CDR having the amino acid sequence set forth in SEQ ID NOs: 8 to 10 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.

[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, for example 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. Because 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 heavy chain of an immunoglobulin is linked to the C-terminus of the heavy chain of the immunoglobulin via a linker, the antibody heavy chain variable region (V H ) is first linked to a single-chain variable region fragment of an antibody light chain variable region (V) containing a CDR having the amino acid sequence set forth in SEQ ID NOs: 50 to 52. 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 CDRs having the amino acid sequences set forth in SEQ ID NOS: 45 to 47, and the light chain variable region of the immunoglobulin comprises CDRs having the amino acid sequences 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 L When the C-terminus of the heavy chain of an immunoglobulin is linked to the C-terminus of the heavy chain of the immunoglobulin via a linker, the antibody heavy chain variable region (V H ) is first linked to a single-chain variable region fragment of an antibody light chain variable region (V) containing a CDR having the amino acid sequence set forth in SEQ ID NOs: 8 to 10. 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 the amino acid sequence set forth in SEQ ID NO:20, and the light chain variable region of the immunoglobulin 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, 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 (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 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 present invention, the constant region of the immunoglobulin is humanized. For example, the heavy chain constant region is the Igγ1 chain C region, Accession No. P01857 (SEQ ID NO: 102) and the light chain constant region is Ig kappa chain C region, accession number P01834 (SEQ ID NO: 101) is.

[0049] In one embodiment of the present invention, the immunoglobulin constant region is 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; wherein the immunoglobulin heavy chain constant region comprises mutations at any two or three of positions 234, 235, and 237 according to the EU numbering system, and the affinity constant of the 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 using the Fortebio Octet system.

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

[0051] In one or more embodiments of the present 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, for the bispecific antibody, the heavy chain constant region of the immunoglobulin is one of the following, according to the EU numbering system: N297A, D265A, D270A, P238D, L328E, E233D, H268D, P271G, A330R, C226S, C229S, E233P, P331S, S267E, L328F, A330L, M252Y, S254T, T256E, N297Q, P238S, P238A, A327Q, A327G, P329A, K322A, T394D, G236R, G236A, L328R, A330S, P331S, H268A, E318A and K320A The compound has one or more mutations selected from the group consisting of:

[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: (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; and 14C12H1V of which has the amino acid sequence set forth in SEQ ID NO: 62 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:64 having the amino acid sequence set forth in (2) NTPDV2, the heavy chain of which is represented by SEQ ID NO: 83 the light chain 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; and 14C12H1V has the amino acid sequence set forth in SEQ ID NO: 62 Linker 1 has the amino acid sequence set forth in SEQ ID NO: 79, and 14C12L1V has the amino acid sequence set forth in SEQ ID NO: 64 having the amino acid sequence set forth in (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, and linker 1 of which has the amino acid sequence set forth in SEQ ID NO: 79; and 14C12H1V of which has the amino acid sequence set forth in SEQ ID NO: 62 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: 64 having the amino acid sequence set forth in (4) NTPDV4, the heavy chain of which is represented by SEQ ID NO: 83 the light chain has the amino acid sequence set forth in SEQ ID NO: 96; linker 1 has the amino acid sequence set forth in SEQ ID NO: 79; and 14C12H1V has the amino acid sequence set forth in SEQ ID NO: 62 Linker 1 has the amino acid sequence set forth in SEQ ID NO: 79, and 14C12L1V has the amino acid sequence set forth in SEQ ID NO: 64 having the amino acid sequence set forth in 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 Under 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 host cells comprising the isolated nucleic acid molecules or vectors 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; particularly, 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 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 pharmaceutically 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 the bispecific antibodies disclosed herein or the following: an agent for detecting the level of CD73 in a sample; Drugs for inhibiting the enzymatic activity of CD73; and / or an agent for blocking the binding of PD-1 to PDL-1; an agent for downregulating (e.g., downregulating) the activity or level of PD-1; Drugs for alleviating the immunosuppression of PD-1 in organisms; 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 to a cell, or to a subject in need thereof, an effective amount of a bispecific antibody disclosed herein or a conjugate disclosed herein.

[0066] The anti-CD73 / anti-PD-1 bispecific antibodies described herein can inhibit the enzymatic activity of CD73 on the 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 chain (H) CDRs include HCDR1, HCDR2, and HCDR3, and light chain (L) CDRs include LCDR1, LCDR2, and LCDR3), which were named by Kabat et al., Bethesda Md., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 1991; 1-3:91-3242.

[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: has 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 (SEQ ID NO: 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 the same amino acid sequences as 19F3. The three CDRs of the light chain variable region have the same amino acid sequences as 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 the same amino acid sequences as 19F3. The three CDRs of the light chain variable region have the same amino acid sequences as 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 (SEQ ID NO: 47). The three CDRs of the light chain variable region have the following amino acid sequences: LCDR1:QDINTY (SEQ ID NO:50) LCDR2:RAN (SEQ ID NO: 51) LCDR3: Has LQYDEFPLT (SEQ ID NO: 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 the same amino acid sequences as 14C12. The three CDRs of the light chain variable region have amino acid sequences identical to those of 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 the same amino acid sequences as 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 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 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 order to better understand the present invention, the definitions and explanations of relevant terms are provided below.

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

[0074] As used herein, the term "antibody" refers to an immunoglobulin molecule generally composed 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 light and heavy chains, the variable and constant regions are connected by a "J" region of about 12 or more amino acids, and 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 regions of antibodies can mediate the binding of immunoglobulins 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)), with conserved regions called framework regions (FRs) distributed between these regions. VH and VL each consist of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, 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 IMGT numbering system definition: 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] Specifically, 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 C-terminally linked 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 derived from a group of highly homologous antibodies, i.e., a group of identical antibody molecules, excluding natural mutations that may occur naturally. Monoclonal antibodies are highly specific for a single epitope on an antigen. Compared to monoclonal antibodies, polyclonal 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 described by Kohler et al. (Kohler G, Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity [J]. Nature, 1975; 256(5517): 495), 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 with 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 having the desired specificity, affinity, or reactivity. Furthermore, some amino acid residues in the framework region (FR) of the receptor antibody can be replaced with corresponding amino acid residues of a non-human antibody or with 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 Domains and V L Bispecific antibodies (diabodies) are antibodies in which two domains are expressed on a single polypeptide chain. However, the linker used is too short to pair two domains on the same chain. This forces the domains to pair with complementary domains on the other chain, and two antigen-binding sites are generated (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 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 The linker may have -COOH. Suitable linkers in the prior art consist of repeated GGGGS amino acid sequences or variants thereof. For example, a linker having the amino acid sequence (GGGGS)4 may be used, but variants 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, its natural environment may be altered, it may be isolated from its natural environment, or both. For example, a particular non-isolated polynucleotide or polypeptide naturally occurs in a particular living animal, and a highly purified version of the same polynucleotide or polypeptide isolated from such a natural state would be 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. A vector can be introduced into a host cell by transformation, transduction, or transfection so that the genetic material elements carried by the vector can be expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids; phagemids; cosmids; artificial chromosomes such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs); phages such as lambda phage or M13 phage; and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex viruses), poxviruses, baculoviruses, papillomaviruses, and papovaviruses (such as SV40). 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 fibroblasts, 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 its target antigen. In some embodiments, an antibody that specifically binds to an antigen (or an antibody specific for an antigen) is one in which the antibody binds to an antigen within about 10 -6 Under 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 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 ) binds 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 "pharmaceutically acceptable carrier and / or excipient" refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient. 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 buffer; surfactants include, but are not limited to, cationic surfactants, anionic surfactants, and nonionic 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 achieve a desired effect or at least partially achieve a desired effect. For example, a prophylactically effective amount for a disease (e.g., a tumor) refers to an amount sufficient to prevent, stop, or delay the onset of the disease (e.g., a 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. Determining such effective amounts is certainly within the capabilities of those skilled in the art. For example, an effective amount for therapeutic purposes depends on the severity of the disease being treated, the overall state of the patient's own immune system, the patient's general condition, such as age, weight, and sex, the route of administration, and other treatments used in combination. Beneficial effects

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

[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 immunosuppression of PD-1 in the body, 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, thereby 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 explanation of the drawings]

[0091] [Figure 1] Binding of P1D7V01, P1D7V02R, P1D7V03, P1D7V04R, 14C12H1L1, and nivolumab to PD-1-mFc measured by ELISA. [Figure 2] Binding of P1D7V01, P1D7V02R, P1D7V03, P1D7V04R, 19F3H2L3 and MEDI9447 to human NT5E-biotin measured by ELISA. [Figure 3] Activity of P1D7V01, P1D7V02R, P1D7V03, P1D7V04R, 14C12H1L1, and nivolumab to compete with human PDL-1-mFc for binding to human PD-1-mFc-biotin. [Figure 4] Affinity constant of P1D7V01 for PD-1-mFc. [Figure 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, measured by FACS. [Figure 10] Binding activity of P1D7V01, P1D7V03, MEDI9447, and 19F3H2L3 to CD73 on the surface of MDA-MB-231 cells, measured by FACS. [Figure 11] Inhibition of the enzymatic activity of CD73 on the surface of MDA-MB-231 membranes by anti-CD73 / anti-PD-1 bispecific antibodies. [Figure 12] Inhibition of the enzymatic activity of CD73 on the surface of U87-MG membrane by anti-CD73 / anti-PD-1 bispecific antibodies. [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 antibodies to promote IFN-γ secretion in a DC mixed lymphocyte reaction system. [Figure 16] Biological activity of anti-CD73 / anti-PD-1 bispecific antibodies to promote IL-2 secretion in a DC mixed lymphocyte reaction system. [Figure 17] Affinity constant of 14C12H1L1 (hG1™) 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. [Figure 23] Affinity constant of 19F3H2L3 (hG1M) for human NT5E(1-552)-his. [Figure 24] Affinity constant of NTPDV1 for human NT5E(1-552)-his. [Figure 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 antibodies. [Figure 29] Biological activity of anti-CD73 / anti-PD-1 bispecific antibodies to promote IFN-γ and IL-2 secretion as determined by mixed lymphocyte reaction (MLR). [Figure 30] Effect of isotype control, 19F3H2L3 (hG1M) and different doses of NTPDV2 on tumor volume in mice. [Figure 31] Effect of isotype control, 19F3H2L3(hG1M) and different doses of NTPDV2 on mouse body weight. [Figure 32] Efficient ablation of PD-1 / CD73 bispecific antibody-mediated IL-8 secretion in human macrophages by amino acid mutation in the Fc segment in a co-culture system of CHO-K1-PD1 cells and human macrophages. [Figure 33] Efficient ablation of PD-1 / CD73 bispecific antibody-mediated IL-6 secretion in human macrophages by amino acid mutation in the Fc segment in a co-culture system of CHO-K1-PD1 cells and human macrophages. [Figure 34] Efficient ablation of PD-1 / CD73 bispecific antibody-mediated IL-8 secretion in human macrophages by amino acid mutation in the Fc segment in a co-culture system of U87-MG cells and human macrophages. [Figure 35] Effective ablation of PD-1 / CD73 bispecific antibody-mediated IL-6 secretion in human macrophages by amino acid mutation 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 CCTCCNO: C2015105, and the address of collection was Wuhan University, Wuhan, China, postal 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 CCTCCNO: C2018137, and the recovery address was Wuhan University, Wuhan, China, with postal code: 430072. Detailed Description

[0094] Embodiments of the present invention are described in detail below with reference to examples. Those skilled in the art will understand that the following examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention. When techniques or conditions are not specified, the examples may be applied to 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 American Society of Microbiology (ASMI) (Scientific Press, 2002) or according to the product manuals. Reagents or equipment used were commercially available conventional products unless the manufacturer is 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, 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: olecurab) used is manufactured by Zhongshan Akesobio Co. Ltd., and its sequence is identical to antibody SEQ ID NOs: 21 to 24 described in U.S. Patent Publication No. 20160129108 A1 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, e.g., 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 lentiviral expression vector used was pCDH-CMV-PD-1FL-Puro (PD1, Genebank ID: 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 lentiviral expression vector used was plenti6.3-PDL-1 (PDL-1, Genebank ID: 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 lentiviral system (see, e.g., 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). The lentiviral expression vector used here was pCDH-CMV-PD-1FL-Puro (PD1, Genebank ID: 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 carrying 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 a study reported by Acierno et al. titled "Affinity maturation increases the stability and plasticity of the Fv domain of anti-protein antibodies" (Acierno et al., J Mol Biol., 2007; 374(1): 130-46). In the constant region fragment of hIgG1, the heavy chain constant region is the Ig γ-1 chain C region, accession number P01857, and the light chain constant region is the Ig κ 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 (Genbank ID: NP002517.1, position: 1-552 for NT5E). Spleen cells from immunized mice were fused with mouse myeloma cells to prepare hybridoma cells. Using human NT5E-biotin (Genbank ID: NP2517.1, position: 1-552 for NT5E) as the antigen, the 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 at the China Culture Collection Center (CCTCC) on June 21, 2018, with collection number CCTCCNO: C2018137, and the retrieval 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 with 5% CO2 at 37°C. After 7 days, the supernatant was collected and purified by high-speed centrifugation, microfiltration, and vacuum filtration through a HiTrap Protein A HP column to obtain antibody 19F3. Example 2

[0105] Sequence analysis of anti-CD73 antibody 19F3 mRNA was extracted from the cell line LT014 prepared in Example 1 according to the method described in the manual for RNApreppureCell / BacteriaKit (Tiangen, catalog number DP430). cDNA was synthesized according to the manual of the 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, light chain CDR1 has the sequence set forth in SEQ ID NO:8, light chain CDR2 has the sequence set forth in SEQ ID NO:9, and 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 characterization 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 the Ig gamma-1 chain C region, accession number P01857; the light chain constant region is the Ig kappa chain C region, accession number P01834. 19F3H2L3 is known as 19F3H2L3 (hG1WT) in Chinese Patent Application No. 202110270671.X, where the light and heavy chain variable regions of 19F3H1L1, 19F3H2L2, and 19F3H2L3 are 19F3H1V (or 19F3H1 V ), 19F3H2V (or 19F3H2 V ), 19F3L1V (or 19F3L1 V ), 19F3L2V (or 19F3L2 V ) and 19F3L3V (or 19F3L3 V ) can be further noted.

[0107] (1) The sequences of the heavy chain variable region and light chain variable region 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 chain variable region and light chain variable region 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 chain variable region and light chain variable region 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 the pUC57Simple (provided by GenScript) vector to obtain pUC57Simple-19F3H1, pUC57Simple-19F3L1, pUC57Simple-19F3H2, pUC57Simple-19F3L2, and pUC57Simple-19F3L3. Following the standard techniques described in Molecular Cloning: A Laboratory Manual (Second Edition), the full-length heavy and light chain genes synthesized by EcoRI and HindIII digestion were subcloned into the expression vector pcDNA3.1 via digestion with restriction enzymes (EcoRI and HindIII) to obtain expression plasmids pcDNA3.1-19F3H1, pcDNA3.1-19F3L1, pcDNA3.1-19F3H2, pcDNA3.1-19F3L2, and pcDNA3.1-19F3L3. The heavy and light chain genes of the recombinant expression plasmids were subjected to sequence analysis. Next, 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 verification, endotoxin-free expression plasmids were prepared and transiently transfected into HEK293 cells for antibody expression. After 7 days, the culture medium was harvested and subjected to affinity purification via a protein A column to obtain the humanized antibody.

[0109] 3. Design of the light and heavy chain sequences of humanized antibodies 19F3H2L3 (hG1M) and 19F3H2L3 (hG1™) Based on the 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 NOs:25 and 26, respectively; the light chain constant region of 19F3H2L3(hG1M) is Igκ 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 NOs:27 and 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 the vector pUC57Simple (provided by Genscript) to obtain pUC57Simple-19F3H2(hG1M) and pUC57Simple-19F3L3, respectively. Following the standard techniques described in Molecular Cloning: A Laboratory Manual (Second Edition), the synthesized full-length heavy and light chain genes were subcloned into the expression vector pcDNA3.1 via digestion with restriction enzymes (EcoRI and HindIII) to obtain the expression plasmids pcDNA3.1-19F3H2(hG1M) and pcDNA3.1-19F3L3. The heavy and light chain genes of the recombinant expression plasmids were then subjected to sequence analysis. Next, the engineered gene combination containing the corresponding light and heavy chain recombinant plasmids pcDNA3.1-19F3H2(hG1M) / pcDNA3.1-19F3L3 was co-transfected into 293F cells, and the culture medium was harvested and purified. After sequence verification, an endotoxin-free expression plasmid was prepared and transiently transfected into HEK293 cells for antibody expression. After 7 days, the culture medium was harvested 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 from immunized BALB / c mice (purchased from the Guangdong Medical Laboratory Animal Center) and mouse myeloma cells were fused to hybridoma cells using a PD-1-mFc fusion protein (PD-1, GenBank: NM005018, mFc SEQ ID NO: 89) as an antigen, following established procedures (e.g., Stewart, SJ, "Monoclonal Antibody Production," in Basic Methods in Antibody Production and Characterization, Eds. G.C. Howard and D.R. Bethell, Boca Raton: CRC Press, 2000). For indirect ELISA, plates were coated with PD-1-hFc (PD-1, Genbank ID: NM005018, hFc is a human IgG Fc purification tag, specifically the Igγ-1 chain C region, Genbank ID: P01857, positions 114-330). Hybridoma cells secreting new antibodies that specifically bind to PD-1 were obtained through screening. Hybridoma cell lines capable of secreting monoclonal antibodies that compete 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. The LT003 stable cell line (PD-1-14C12) was obtained by limiting dilution, and the secreted monoclonal antibody was designated 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 CCTCCNO: C2015105, and the address of collection was Wuhan University, Wuhan, China, postal 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 containing 1% penicillin-streptomycin, 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 the 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, heavy chain CDR1 has the sequence set forth in SEQ ID NO: 45, heavy chain CDR2 has the sequence set forth in SEQ ID NO: 46, and heavy chain CDR3 has the sequence of 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, light chain CDR1 has the sequence set forth in SEQ ID NO: 50, light chain CDR2 has the sequence set forth in SEQ ID NO: 51, and 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(hG1™) 1. Design of the 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 the 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 sequences of antibody 14C12H1L1 were obtained by computer simulation of the antibody model of the sequence of antibody 14C12 obtained in Example 5 and designing mutations according to the model. The designed variable region sequences are as follows: The nucleotide sequence (354 bp) of the heavy chain variable region 14C12H1 of the 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 the 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 were 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 as 14C12H1L1(hG1WT) herein and in Chinese Patent Application No. 02110270671.X, 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 (hG1™) Based on 14C12H1L1 obtained in Step 1 of Example 6, 14C12H1L1(hG1™) 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(hG1™) 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 NOs: 71 to 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(hG1™) The heavy and light chain cDNAs of 14C12H1L1(hG1™) and 14C12H1L1 were separately cloned into pUC57Simple (provided by GenScript) vectors to obtain pUC57Simple-14C12H1, pUC57Simple-14C12L1, and pUC57Simple-14C12H1(hG1™). Following the standard techniques described in Molecular Cloning: A Laboratory Manual (Second Edition), the synthesized full-length heavy and light chain genes were subcloned into the expression vector pcDNA3.1 through digestion with restriction enzymes (EcoRI and HindIII) to obtain expression plasmids pcDNA3.1-14C12H1, pcDNA3.1-14C12L1, and pcDNA3.1-14C12H1(hG1™). The heavy and light chain genes of the recombinant expression plasmids were further subjected to sequence analysis. The designed gene combinations containing the corresponding light and heavy chain recombinant plasmids (pcDNA3.1-14C12H1(hG1™) / pcDNA3.1-14C12L1 and pcDNA3.1-14C12H1 / pcDNA3.1-14C12L1) were then separately co-transfected into 293F cells, and the culture medium was harvested and purified. After sequence verification, endotoxin-free expression plasmids were prepared and transiently transfected into HEK293 cells for antibody expression. After 7 days, the culture medium was harvested and subjected to affinity purification via a protein A column to obtain the humanized antibody. Example 7

[0118] Sequence design and expression of anti-PD-1 / CD73 bifunctional antibodies 1. Sequence Design The bifunctional antibody structure described herein is the Morrison format (IgG-scFv), i.e., the C-terminus of each of the two heavy chains of an IgG antibody is linked to the scFv fragment of another antibody. 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 marked 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" marking, the corresponding heavy or light chain is the full-length chain constituting the constant region. The corresponding sequences described in the above examples refer to the amino acid sequences of these variable regions or full-length sequences 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) to obtain the plasmids pUC57Simple-VP101H and pUC57Simple-VP101L, respectively. The plasmids pUC57Simple-VP101H and pUC57Simple-VP101L were enzymatically digested (HindIII and EcoRI), and the heavy and light chains isolated by electrophoresis were subcloned into the vector pcDNA3.1. 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. The protein was eluted in one step with elution buffer. The target sample was isolated, and the buffer was exchanged for PBS. Purified antibodies P1D7V02R, P1D7V03, P1D7V04R, P1D7V07 and P1D7V08 were obtained by the expression and purification method for 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: A 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 for 2 hours at 37°C with a PBS solution containing 1% BSA as a blocking solution. After blocking, the microplate was washed three times with PBST. A serially diluted antibody (antibody dilution gradient shown in Table 2) was added in PBST. The microplate containing the test antibody was incubated for 30 minutes at 37°C and then washed three times with PBST. After washing, a 1:5000 diluted working solution of HRP-labeled goat anti-human IgG (H+L) (Jackson, catalog number 09-035-088) secondary antibody was added, and the microplate was then incubated for 30 minutes at 37°C. 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, followed by the addition of stop solution 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. Data were analyzed and processed using SoftMax Pro 6.2.1.

[0122] The results are shown in Table 2 and Figure 1. The figure shows 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 yielded the binding efficiencies EC of the 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 comparable to those of the control drugs 14C12H1L1 and nivolumab to the same targets, suggesting 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 A 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 for 2 hours at 37°C with a PBS solution containing 1% BSA as a microplate blocking solution. After blocking, the microplate was washed three times with PBST. The antigen, human NT5E-biotin (0.5 μg / mL), was then added and incubated at 37°C for 30 minutes. The plate was then washed three times with PBST. Serially diluted antibodies (antibody dilution gradients are shown in Table 3) in PBST were added to the wells of the microplate. The microplate containing the test antibody was incubated at 37°C for 30 minutes and then washed three times with PBST. After washing, a 1:5000 dilution of HRP-labeled goat anti-human IgG (H+L) (Jackson, catalog no. 09-035-088) secondary antibody working solution was added, and the microplate was incubated at 37°C 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, followed by the addition of stop solution to terminate the color development reaction. The microplate was immediately placed in a microplate reader, and the OD of each well in the microplate was read at 450 nm. Data were analyzed and processed with SoftMaxPro 6.2.1.

[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 for each dose is shown in Table 3. Quantitative analysis of the absorbance of the bound antibodies yielded the binding efficiencies EC of the antibodies P1D7V01, P1D7V02R, P1D7V03, P1D7V04R, 19F3H2L3, and MEDI9447 (as a 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-1 Genbank ID: NP54862.1, mFc, SEQ ID NO: 143) and incubated overnight at 4°C. After incubation, the microplate was blocked with 1% BSA in PBS at 37°C for 2 hours. After blocking, the plate was washed three times and dried. Starting at 10 μg / mL, the antibody was serially diluted to 7x concentrations at a 1:3 gradient on a dilution plate to set up a blank control. An equal volume of 0.3 μg / mL human PD-1-mFc-biotin solution was then added, the mixture was mixed thoroughly, 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 four times and gently tapped dry. TMB (Neogen, 308177) was then added for 5 minutes in the dark to develop the color, and a 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. Data were analyzed and processed using SoftMaxPro 6.2.1.

[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 1.115 nM, 1.329 nM, 1.154 nM, 1.339 nM, 1.459 nM, and 1.698 nM, respectively. [Table 4] Example 10

[0130] Kinetic parameters of anti-CD73 / anti-PD-1 bispecific antibody binding to the antigen human PD-1-mFc measured by the Fortebio system The sample dilution buffer was PBST, 0.1% BSA, pH 7.4. The antibody was 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 seconds, and binding of the immobilized antibody on the sensor to the antigen PD-1-mFc at concentrations ranging from 0.6 to 50 nM (3-fold dilution) was measured over 120 seconds. The protein was allowed to dissociate in the buffer for 180 seconds. The detection temperature was 37°C, the detection frequency was 0.3 Hz, and the sample plate vibration speed was 1000 rpm. The data were analyzed using a 1:1 model fitting to obtain affinity constants.

[0131] The affinity constants of the humanized antibodies P1D7V01, 14C12H1L1, and nivolumab (as a control antibody) for human PD-1-mFc are measured in Table 5, and the detection results are shown in Figures 4, 5, and 6. The affinity constants of the humanized antibodies P1D7V01, 14C12H1L1, and nivolumab for human PD-1-mFc were 1.76E-10 M, 1.64E-10 M, and 2.32E-10 M, respectively. These experimental results suggest that the binding ability of P1D7V01 is comparable to that of 14C12H1L1 and nivolumab, and that the humanized antibody P1D7V01 has 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 the antigen human NT5E(1-552)his measured by the Fortebio system The sample dilution buffer was PBST, pH 7.4. The antibody was immobilized on the protein A sensor at a concentration of 5 μg / mL for a fixation time of approximately 15 seconds. The sensor was equilibrated in the buffer for 120 seconds, and the binding of the antibody immobilized on the sensor to the antigen human NT5E(1-552)-his was measured for 120 seconds at concentrations ranging from 3.125 to 200 nM (two-fold dilution). 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 sample plate vibration speed was 1000 rpm. The data were analyzed by 1:1 model fitting to obtain the affinity constant.

[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-10 M and 1.04E-10 M, 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 antibodies to PD-1 on the surface of 293T-PD1 membranes 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 to the cells. 500 μL of PBSA was added, the mixture was centrifuged at 5600 rpm for 5 minutes, 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, and 0.05 nM) was added to each well. The system was gently mixed uniformly and then incubated on ice for 1 hour. 500 μL of PBSA was then added, the mixture was centrifuged at 5600 rpm for 5 minutes, and the supernatant was removed. A 500-fold diluted FITC-labeled goat anti-human IgG secondary antibody (Jackson, catalog no. 109-095-098) was added, resuspended, mixed well, and incubated on ice in the dark for 0.5 hours. 500 μL of PBSA was added, and the mixture was centrifuged at 5600 rpm for 5 minutes, 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 were able to specifically bind to PD-1 on the 293T-PD1 membrane surface in a dose-dependent manner, and compared with the PD1 single-target control antibody 14C12H1L1, the binding was 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 2.111 nM.

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

[0139] 2. Binding activity of anti-CD73 / anti-PD-1 bispecific antibodies to CD73 on the MDA-MB-231 membrane surface as measured by FACS MDA-MB-231 cells in logarithmic phase were digested with regular trypsin and placed in 1.5 mL centrifuge tubes at 3 x 10 cells per tube. 5The mixture was transferred to the wells at 100°C. 500 μL of PBSA was added, and the mixture was centrifuged at 5600 rpm for 5 minutes, followed by removal of the supernatant. 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, and 0.05 nM) was added. The mixture was gently mixed uniformly and incubated on ice for 1 hour. 500 μL of PBSA was then added, and the mixture was centrifuged at 5600 rpm for 5 minutes, followed by removal of the supernatant. 500-fold diluted FITC-labeled goat anti-human IgG secondary antibody (Jackson, catalog no. 109-095-098) was added, resuspended, and mixed well. The mixture was then incubated in the dark on ice for 0.5 hours. 500 μL of PBSA was added, and the mixture was centrifuged at 5600 rpm for 5 minutes, followed by removal of the supernatant. 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, and P1D7V01 was superior to the control drug MEDI9447 against the same target. Under the same experimental conditions, the EC values ​​of P1D7V01 and P1D7V03 binding to CD73 on the MDA-MB-231 membrane surface were 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 the control drug MEDI9447, which are targeted to the same target, were able to specifically bind to CD73 on the MDA-MB-231 membrane surface in a dose-dependent manner. The binding activity of P1D7V01 and P1D7V03 was superior to that of 19F3H2L3, and P1D7V01 was superior to that of the control drug MEDI9447 against the same target. These results suggest that P1D7V01 and P1D7V03 have effective binding activity 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 antibodies 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 antibodies The experimental procedure was as follows: MDA-MB-231 cells in logarithmic phase were harvested in good condition, resuspended in serum-free RPMI-1640 medium, and then counted. 2 × 10 MDA-MB-231 cells were plated per well. 4 Cells were seeded at 100 μL per well in a 96-well plate. Antibodies were diluted in serum-free RPMI-1640 medium (serial 2.5-fold dilutions). 50 μL of antibody was added per well to the 96-well plate, and the plate was incubated at 37°C for 1 hour. After 1 hour, 50 μL of AMP (TCL, catalog no. A0157) diluted to 1200 μM in RPMI-1640 was added to each well. After 3 hours, 25 μL of cell culture supernatant was removed and transferred to a new 96-well plate, and 25 μL of 100 μM ATP (TCL, catalog no. A0158) was added to each well. 50 μL of CTG (CellTiterGlo, Promega, catalog no. G8641) color development solution was added to each well for color development, 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 were concentration-dependently equivalent to the AMP amount of the positive control MEDI9447.

[0144] The above experimental results indicate that added AMP can be converted to adenosine A through the enzymatic activity of CD73 on the surface of MDA-MB-231 cells without antibody, but after antibody addition, the binding affinity of antibodies P1D7V01, P1D7V02R, P1D7V03, and P1D7V04R to CD73 reduces the enzymatic catalytic activity, resulting in the inability of AMP to be converted to adenosine A. These results suggest that the antibodies effectively inhibit the enzymatic activity reaction in a non-substrate competitive manner, 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 antibodies U87-MG cells in good logarithmic phase were harvested, resuspended in serum-free RPMI-1640 medium, and then counted. U87-MG cells were plated at 2 × 10 per well. 4 Cells were seeded at 100 μL per well in a 96-well plate. Antibodies were diluted in serum-free RPMI-1640 medium (serial 2.5-fold dilutions). 50 μL of antibody was added per well to the 96-well plate, and the plate was incubated at 37°C for 1 hour. After 1 hour, 50 μL of AMP diluted in RPMI-1640 at 1200 μM was added to each well. After 3 hours, 25 μL of cell culture supernatant was removed and transferred to a new 96-well plate, and 25 μL of 100 μM ATP was added to each well. 50 μL of CTG (CellTiterGlo) color development solution was added to each well for color development, and the relative fluorescence intensity (RLU) was read using a multilabel microplate tester (PerkinElmer 2140-0020).

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

[0147] The above experimental results indicate that added AMP can be converted to adenosine A by the enzymatic activity of CD73 on the surface of U87-MG cells without antibody, but after antibody addition, antibodies P1D7V01, P1D7V02R, P1D7V03, and P1D7V04R bind to CD73, reducing the catalytic activity of the enzyme and preventing the conversion of AMP to adenosine A. These results suggest that the antibodies effectively inhibit the enzymatic activity reaction in a non-substrate competitive manner, reducing the production of adenosine. Example 14

[0148] Biological activity of anti-CD73 / anti-PD-1 bispecific antibodies to stimulate IFN-γ and IL-2 secretion measured by mixed lymphocyte reaction (MLR) 1. Biological activity of anti-CD73 / anti-PD-1 bispecific antibodies for promoting IFN-γ secretion in the 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, catalog no. S010201) for 2 days. Raji-PDL-1 cells were treated with 25 μg / mL MMC (Sigma, catalog 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 separately plated into a U-shaped 96-well plate at 100,000 cells per well. Antibodies were added according to the study design, and the cells were cultured in an incubator for 3 days. After 3 days, cell culture supernatants were collected and IFN-γ was measured by ELISA.

[0149] As shown in Figure 13, coculture of human PBMCs and Raji-PDL-1 cells significantly promoted IFN-γ secretion from PBMCs, and addition of antibodies to the coculture system significantly induced IFN-γ secretion in PBMCs. Antibodies P1D7V01, P1D7V02R, P1D7V03, and P1D7V04R have comparable IFN-γ secretion-promoting activity to that of the parent PD-1 monotargeting antibody 14C12H1L1.

[0150] 2. Biological activity of anti-CD73 / anti-PD-1 bispecific antibodies to enhance IL-2 secretion in the 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 2 days. Raji-PDL-1 cells were treated with 25 μg / mL MMC 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 separately plated into a U-shaped 96-well plate at 100,000 cells per well. Antibodies were added according to the study design and cultured for 3 days. Cell culture supernatants were collected, and IL-γ was measured by ELISA.

[0151] As shown in Figure 14, co-culture of human PBMCs and Raji-PDL-1 cells had a specific promoting effect on IL-2 secretion in PBMCs, and the simultaneous addition of antibodies to the co-culture system significantly induced IL-2 secretion in PBMCs in a dose-dependent manner. In terms of the level of IL-2 secretion-promoting activity, the bifunctional antibodies P1D7V01, P1D7V02R, P1D7V03, and P1D7V04R had slightly lower activity than the parent PD-1 monotargeting antibody 14C12H1L1 at low concentrations, but had activity equivalent to that of the parent PD-1 monotargeting antibody 14C12H1L1 at medium and high concentrations. Compared to the PD1-targeting positive control drug nivolumab, P1D7V01, P1D7V02R, P1D7V03, and P1D7V04R had 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 stimulates DC mixed lymphoid reaction system and secretes IFN-γ PBMCs were isolated from normal human peripheral blood, resuspended in complete medium, and seeded onto culture dishes. The culture dishes were placed in an incubator overnight for culture. The suspended PBMCs were collected and removed. Adherent 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 1000 U / mL each was added to each dish. The dishes were cultured at 37°C in a 5% carbon dioxide incubator for 3 days. After that, half of the medium was replaced, and 1000 U / mL of GM-CSF and IL-4 were added. 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, and 1000 U / mL of GM-CSF and IL-4, and 100 U / mL of TNF-α were added, and the dishes were cultured for an additional 2 days. PBMCs from another donor were freshly isolated, counted, and seeded into 96-well plates at 100,000 cells per well. Matured DCs were collected and washed once with complete medium. The cells were counted and seeded into the 96-well plates containing PBMCs at 10,000 cells per well. Antibodies were added according to the study design. The mixture was thoroughly mixed and co-cultured for 5 days in a 37°C, 5% carbon dioxide incubator. After 5 days, cell culture supernatants were collected 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 IFN-γ secretion; 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 antibodies to enhance IL-2 secretion in DC mixed lymphocyte reaction system PBMCs were isolated from normal human peripheral blood, resuspended in complete medium, and seeded onto culture dishes. The culture dishes were placed in an incubator overnight for culture. The suspended PBMCs were collected and removed. Adherent 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 concentrations of 2000 U / mL each was added to each dish. The dishes were cultured in a 37°C, 5% carbon dioxide incubator for 3 days. After that, half of the medium was replaced, 50 ng / mL IFN-γ and 100 ng / mL LPS were added, and the culture was continued for another 2 days. PBMCs from another donor were thawed, counted, and seeded onto a 96-well plate at 100,000 cells per well. The matured DCs 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 mixture was thoroughly mixed and co-cultured for 5 days in a 37°C, 5% carbon dioxide incubator. 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 cultures of DCs or PBMCs alone, co-culture of DCs and PBMCs significantly promoted IL-2 secretion; 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 IL-2 secretion levels. 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 is 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 portions to eliminate their binding activity to FcγR, and are therefore known as NTPDV1(hG1™), NTPDV2(hG1™), NTPDV3(hG1™) and NTPDV4(hG1™) in this specification and Chinese Patent Application No. 202110270671.X. [Table 9]

[0157] In Table 9 above: 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. 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-lengths, 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 portions in NTPDV1, NTPDV2, NTPDV3 and NTPDV4 are identical to the light chain CDR sequence of 19F3. The three CDR sequences of 19F3H2 (hG1™) in the immunoglobulin portion of 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 CDR and light chain CDR 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 portions 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 has a heavy chain having the amino acid sequence set forth in SEQ ID NO: 85, a light chain having 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, and 14C12H1V having the amino acid sequence set forth in SEQ ID NO: 62 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: 64 having the amino acid sequence set forth; (2) NTPDV2, whose heavy chain is represented by SEQ ID NO: 83 wherein the light chain 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, and 14C12H1V has the amino acid sequence set forth in SEQ ID NO: 62 14C12L1V has the amino acid sequence set forth in SEQ ID NO: 79, and linker 1 has the amino acid sequence set forth in SEQ ID NO: 79.64 having the amino acid sequence set forth in (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, and 14C12H1V of which has the amino acid sequence set forth in SEQ ID NO: 62 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: 64 having the amino acid sequence set forth in (4) NTPDV4, whose heavy chain is represented by SEQ ID NO: 83 wherein the light chain has the amino acid sequence set forth in SEQ ID NO: 96, linker 1 has the amino acid sequence set forth in SEQ ID NO: 79, and 14C12H1V has the amino acid sequence set forth in SEQ ID NO: 62 14C12L1V has the amino acid sequence set forth in SEQ ID NO: 79, and linker 1 has the amino acid sequence set forth in SEQ ID NO: 79. 64 It has the amino acid sequence set forth in

[0159] Antibody expression and purification The heavy chain cDNA sequences of NTPDV1 and NTPDV3, the heavy chain cDNA sequences of NTPDV2 and NTPDV4, and their light chain cDNA sequences 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 separately subcloned into the vector pcDNA3.1 to obtain the plasmids pcDNA3.1-NTPDH2 / 4 and pcDNA3.1-19F3L3, pcDNA3.1-NTPDH2 / 4 and pcDNA3.1-19F3L2, pcDNA3.1-NTPDH1 / 3 and pcDNA3.1-19F3L3, and 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 high-speed centrifugation, 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 for 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 antigen-binding activity of anti-CD73 / anti-PD-1 bispecific antibodies by ELISA 1. Binding activity of NTPDV1, NTPDV2, NTPDV3, and NTPDV4 to the antigen PD-1-mFc measured by ELISA A 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 for 2 hours at 37°C with a PBS solution containing 1% BSA as a blocking solution. After blocking, the microplate was washed three times with PBST. A serially diluted antibody (antibody dilution gradient shown in Table 2) was added in PBST. The microplate containing the test antibody was incubated for 30 minutes at 37°C and then washed three times with PBST. After washing, a 1:5000 diluted HRP-labeled goat anti-human IgGFc (Jackson, catalog no. 109-035-098) secondary antibody working solution was added, and the microplate was then incubated for 30 minutes at 37°C. After incubation, the plate was washed four times with PBST and TMB (Neogen, 308177) was added for 8 minutes in the dark to develop the color, followed by the addition of stop solution to terminate the color reaction. The microplate was immediately placed in a microplate reader, and the OD of each well in the microplate was read at 450 nm. Data were analyzed and processed using SoftMaxPro 6.2.1.

[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 yielded the binding efficiencies EC of the antibodies NTPDV1, NTPDV2, NTPDV3, NTPDV4, and 14C12H1L1 (hG1™) (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 (hG1™) 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 A 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 for 2 hours at 37°C with a PBS solution containing 1% BSA as a microplate blocking solution. After blocking, the microplate was washed three times with PBST. Then, 0.5 μg / mL of the antigen human NT5E-biotin was added and incubated for 30 minutes at 37°C. The plate was then washed three times with PBST. Serially diluted antibodies (the antibody dilution gradient is shown in Table 11) in PBST solution were added to the wells of the microplate. The microplate containing the test antibody was incubated for 30 minutes at 37°C and then washed three times with PBST. After washing, a 1:5000 dilution of HRP-conjugated goat anti-human IgGFc (Jackson, catalog no. 109-035-098) secondary antibody working solution was added, and the microplate was incubated at 37°C for 30 minutes. After incubation, the plate was washed four times with PBST, and TMB (Neogen, 308177) was added for 7 minutes in the dark to develop the color, followed by the addition of stop solution to terminate the color reaction. The microplate was immediately placed in a microplate reader, and the OD of each well in the microplate was read at 450 nm. Data were analyzed and processed using SoftMaxPro 6.2.1.

[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 yielded the binding efficiencies 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 effective binding activity 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 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 1% BSA in PBS at 37°C for 2 hours. After blocking, the plate was washed once and dried. Starting at 10 μg / mL, the antibody was serially diluted to seven concentrations in a 1:3 gradient on a dilution plate to set up a blank control. An equal volume of 0.3 μg / mL human PD-1-mFc-biotin solution was then added, the mixture was thoroughly mixed, 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. A working dilution of SA-HRP (KPL, 14-30-00) was added, and the plate was incubated at 37°C for 30 minutes. After incubation, the plate was washed four times and gently tapped dry. Then, TMB (Neogen, 308177) was added for 5 minutes in the dark to develop the color, and 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. Data were analyzed and processed using SoftMaxPro 6.2.1.

[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(hG1™) (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 EC values ​​of NTPDV1, NTPDV2, NTPDV3, NTPDV4, and 14C12H1L1(hG1™) 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 the 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 ranging from 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 sample plate vibration speed was 1000 rpm. The data were analyzed by 1:1 model fitting to obtain 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-10 M, 7.39E-11 M, 1.25E-10 M, 1.13E-11 M, and 2.26E-10 M, respectively. The above experimental results showed that the binding abilities of NTPDV1, NTPDV3, and nivolumab were comparable, and that 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 antibodies to the 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 0.31–25 nM (three-fold dilution) was measured for 100 s. The protein was allowed to dissociate in the buffer for 180 s. The detection temperature was 30 °C, the detection frequency was 0.3 Hz, and the sample plate vibration speed was 1000 rpm. The data were analyzed using a 1:1 model fitting to obtain affinity constants.

[0172] 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-11 M, 2.88E-11 M, 7.92E-11 M, and 5.77E-11 M, 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 harvested, resuspended in serum-free RPMI-1640 medium, and then counted. U87-MG cells were plated at 2.5 × 10 per well. 4Cells were seeded into 96-well plates at 60 μL per well. Antibodies were diluted in serum-free RPMI-1640 medium according to the study design. Antibodies were added to the 96-well plate at 60 μL per well, and the plate was incubated at 37°C for 1 hour. After 1 hour, 60 μL of AMP diluted in RPMI-1640 at 600 μM was added to each well. After 3 hours, 100 μL of cell culture supernatant was removed and transferred to a new 96-well plate. 40 μL of CTG (CellTiterGlo) color development solution was added to each well, and the plate was placed in the dark at room temperature for 5 minutes. After 5 minutes, 10 μL of 300 μM ATP was added to each well to develop the color. Relative fluorescence intensity (RLU) was read using a multilabel microplate tester (PerkinElmer 2140-0020).

[0175] The experimental results are shown in Table 15 and Figure 28, and 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 the need for antibodies. However, after the addition of antibodies, the binding of antibody NTPDV2 to CD73 reduced the catalytic function of the enzyme, resulting in the inability to convert AMP to adenosine A. This suggests that the antibody effectively inhibits the enzymatic activity reaction in a non-substrate competitive manner, reducing the production of adenosine. [Table 15] Example 21

[0177] 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 antibodies to enhance IFN-γ secretion in the 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 0.5 μg / mL SEB (Dianotech, catalog number S010201) for two days. Raji-PDL-1 cells were treated with 25 μg / mL MMC (mitomycin C, Stressmarq, catalog number SIH-246, catalog number 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 placed at 1 × 10 per well. 5 Cells were separately added to a U-shaped 96-well plate. Antibodies were added according to the study design, and the cells were 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, coculture of human PBMCs and Raji-PDL-1 cells significantly promoted IFN-γ secretion from PBMCs, and addition of antibodies to the coculture system significantly induced IFN-γ secretion in PBMCs. Antibody NTPDV2 has activity equivalent to that of the parent PD-1 monotargeting antibody 14C12H1L1 in terms of the level of IFN-γ secretion promotion activity.

[0179] 2. Biological activity of anti-CD73 / anti-PD-1 bispecific antibodies to enhance IL-2 secretion in the 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 two days. Raji-PDL-1 cells were treated with 25 μg / mL of MMC 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 at 1 × 10 per well. 5Cells were separately plated in a U-shaped 96-well plate. Antibodies were added according to the study design and cultured for 3 days. Cell culture supernatants were collected and IL-2 was measured by ELISA.

[0180] As shown in Figure 29, co-culture of human PBMCs and Raji-PDL-1 cells had a specific promoting effect on IL-2 secretion from PBMCs, and the simultaneous addition of antibodies to the co-culture system significantly induced IL-2 secretion in PBMCs in a dose-dependent manner. In terms of the level of promoting IL-2 secretion activity, the bifunctional antibody NTPDV2 had activity equivalent to that of the parental PD-1 monotargeting antibody 14C12H1L1 at low concentrations, and slightly lower activity than that of the parental PD-1 monotargeting 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 anti-tumor activity of the anti-CD73 / anti-PD-1 bispecific antibody, MC38-hPDL-1 / hCD73 cells (GemPharmatech Co., Ltd.) were first subcutaneously inoculated into 5- to 7-week-old female C57BL6-hPD1hPDL-1hCD73 triple transgenic mice (GemPharmatech Co., Ltd.). The 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 showed that, compared with the isotype control antibodies hIgG and 19F3H2L3 (hG1M), different doses of NTPDV2 could effectively inhibit tumor growth in mice, and that high doses of NTPDV2 were superior to low doses in tumor inhibition. Furthermore, NTPDV2 did not affect 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 was 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 Separation Medium (GE, Catalog No. 17-1440-03); RPMI1640 (Gibco, Catalog 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, Catalog No. FSP500); Human IFN-γ Protein (Sinobio, Catalog No. 11725-HNAS-100); LPS (Lipopolysaccharide) (Sigma, Catalog No. L4391); 96-well cell culture plate (Corning).

[0184] Healthy human PBMCs were isolated according to the Ficoll-Paque™ Plus reagent instructions and resuspended in 1640 medium containing 2% FBS. The plates were incubated at 37°C in a 5% CO2 cell incubator. After 2 hours, the supernatant was removed, and adherent cells were washed twice with PBS and induced with 1640 complete medium (containing 10% FBS) and 100 ng / 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 HPMM induction, cells were harvested, adjusted to a concentration of 100,000 cells / mL in complete medium, and plated into a 96-well plate. Recombinant human IFN-γ (50 ng / mL) was added and the plate was incubated for 24 hours in the incubator. After 24 hours, CHO-K1-PD1 cells expressing human PD-1 or U87-MG cells constitutively expressing human CD73 in logarithmic phase were harvested. The cells were resuspended and then adjusted to a concentration of 300,000 cells / mL using complete medium. Antibodies were diluted to standard concentrations of 25 nM, 2.5 nM, and 0.25 nM using complete medium. Isotype control antibodies and blank controls were also designed. The supernatant in the 96-well plate was removed, and CHO-K1-PD1 or U87-MG cell suspension and antibody 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, and the supernatant was collected. IL-8 and IL-6 secretion levels were measured using a Dakewe kit. LPS was used as a positive control in the experiment and adjusted to a concentration of 100 ng / mL in complete medium.

[0185] In this example, co-culture of HPMM with CHO-K1-PD1 and U87-MG cells as target cells induced the activation of HPMM. 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 or CD73 antibodies, the anti-PD-1 / CD73 bispecific antibody with Fc fragment mutations could effectively eliminate IL-6 and IL-8 secretion in immune cells.

[0187] Although the preferred embodiments of the present invention have been described in detail above, 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 (hG1™) heavy chain nucleotide sequence (SEQ ID NO: 29, non-variable region sequence underlined) [ka]

[0205] Amino acid sequence of 19F3H2L3 (hG1™) 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 14C12 heavy chain variable region: (SEQ ID NO: 43) [ka]

[0210] Amino acid sequence of 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 14C12 light chain variable region: (SEQ ID NO: 48) [ka]

[0212] Amino acid sequence of 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(G1™) heavy chain (SEQ ID NO: 69) [ka]

[0224] Amino acid sequence of 14C12H1L1(G1™) 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): wherein the CDR regions of 19F3H2 (hG1™) in the immunoglobulin portion are in bold, the CDR regions of 14C12H1V-linker1-14C12L1V in the scFv portion are in bold, the 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 sequences of the heavy chains of NTPDV1 and NTPDV3 (SEQ ID NO: 85): CDR regions of 19F3H2 (hG1™) in the immunoglobulin portion are in bold, CDR regions of 14C12H1V-linker2-14C12L1V 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]

[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 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 that targets CD73 an anti-CD73 / anti-PD-1 bispecific antibody comprising: The first protein functional region comprises HCDR1, HCDR2 and HCDR3, wherein, according to the IMGT numbering system, the amino acid sequences of HCDR1, HCDR2 and HCDR3 are the sequences set forth in SEQ ID NOs: 45 to 47, respectively; and comprising LCDR1, LCDR2 and LCDR3, wherein, according to the IMGT numbering system, the amino acid sequences of LCDR1, LCDR2 and LCDR3 are the sequences set forth in SEQ ID NOs: 50 to 52, respectively; and the second protein functional region comprises HCDR1, HCDR2 and HCDR3, wherein, according to the IMGT numbering system, the amino acid sequences of HCDR1, HCDR2 and HCDR3 are the amino acid sequences set forth in SEQ ID NOs: 3 to 5, respectively; and and LCDR1, LCDR2, and LCDR3, wherein, according to the IMGT numbering system, the amino acid sequences of LCDR1, LCDR2, and LCDR3 are the sequences set forth in SEQ ID NOs: 8 to 10, respectively. 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 set forth in SEQ ID NO: 44 or SEQ ID NO: 62; and comprising a sequence having the amino acid sequence set forth in SEQ ID NO: 49 or SEQ ID NO: 64; and / or The second protein functional domain has the amino acid sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 20; and comprising a sequence having the amino acid sequence set forth in SEQ ID NO: 7 or SEQ ID NO: 22; Or, The second protein functional domain has the amino acid sequence set forth in SEQ ID NO: 20; and or comprising a sequence having the amino acid sequence set forth in SEQ ID NO: 24; or The second protein functional domain has the amino acid sequence set forth in SEQ ID NO: 93; and Contains a sequence having the amino acid sequence set forth in SEQ ID NO: 95 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. Anti-CD73 / anti-PD-1 bispecific antibody.

5. 5. The anti-CD73 / anti-PD-1 bispecific antibody of claim 4, wherein the linker is (GGGGS)n, and n is a positive integer.

6. The anti-CD73 / anti-PD-1 bispecific antibody of claim 5, wherein n is 1, 2, 3, 4, 5, or 6.

7. 7. The anti-CD73 / anti-PD-1 bispecific antibody of any one of claims 1 to 6, wherein the anti-CD73 / anti-PD-1 bispecific antibody has any one of the following characteristics: (a) the first protein functional domain and the second protein functional domain are independently an immunoglobulin or antigen-binding fragment; (b) the first protein functional domain is an immunoglobulin and the second protein functional domain is an antigen-binding fragment; and (c) the first protein functional domain is an antigen-binding fragment and the second protein functional domain is an immunoglobulin; Anti-CD73 / anti-PD-1 bispecific antibody.

8. The antigen-binding fragments include half antibodies, Fab, F(ab') ２ or a single-chain variable region fragment.

9. The anti-CD73 / anti-PD-1 bispecific antibody of claim 7 or 8, wherein the N-terminus of the heavy chain variable region of the antigen-binding fragment is linked to the C-terminus of CH1 of the immunoglobulin, either directly or via a linker, and the N-terminus of the light chain variable region of the antigen-binding fragment is linked to the C-terminus of the light chain constant region CL of the immunoglobulin, either directly or via a linker; or the N-terminus of the heavy chain variable region of the antigen-binding fragment is linked to the C-terminus of the light chain constant region CL of the immunoglobulin, either directly or via a linker, and the N-terminus of the light chain variable region of the antigen-binding fragment is linked to the C-terminus of the heavy chain constant region CHI of the immunoglobulin, either directly or via a linker; or the C-terminus of the heavy chain variable region of the antigen-binding fragment is linked to the N-terminus of the heavy chain of the immunoglobulin directly or via a linker, and the C-terminus of the light chain variable region of the antigen-binding fragment is linked to the N-terminus of the light chain of the immunoglobulin directly or via a linker; or the C-terminus of the heavy chain variable region of the antigen-binding fragment is linked to the N-terminus of the light chain of the immunoglobulin directly or via a linker, and the C-terminus of the light chain variable region of the antigen-binding fragment is linked to the N-terminus of the heavy chain of the immunoglobulin directly or via a linker. Anti-CD73 / anti-PD-1 bispecific antibody.

10. The anti-CD73 / anti-PD-1 bispecific antibody of claim 7 or 8, wherein the antigen-binding fragment is a single-chain variable region fragment. Anti-CD73 / anti-PD-1 bispecific antibody.

11. 11. The anti-CD73 / anti-PD-1 bispecific antibody of claim 10, 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. Anti-CD73 / anti-PD-1 bispecific antibody.

12. 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 The anti-CD73 / anti-PD-1 bispecific antibody of claim 11, which has -COOH.

13. 12. The anti-CD73 / anti-PD-1 bispecific antibody of claim 10 or 11, wherein the anti-CD73 / anti-PD-1 bispecific antibody has any one of the following characteristics: (a) the single-chain variable region fragment is a heavy chain (C) of the immunoglobulin via a linker; Ｈ ), or the N-terminus of the heavy chain, or the C-terminus of CH1 of the heavy chain constant region, Ｈ ), or the antibody light chain variable region (V Ｌ ) are connected; (b) the single-chain variable region fragment has the following structure: linker-V Ｈ -Linker-V Ｌ -COOH or linker-V Ｌ -Linker-V Ｈ having —COOH; (c) the immunoglobulin heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1, HCDR2, and HCDR3 have the amino acid sequences set forth in SEQ ID NOs: 3, 4, and 5, respectively, and the immunoglobulin light chain variable region comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1, LCDR2, and LCDR3 have the amino acid sequences set forth in SEQ ID NOs: 8, 9, and 10, respectively; (d) the heavy chain variable region of the single-chain variable region fragment comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1, HCDR2, and HCDR3 have the amino acid sequences set forth in SEQ ID NOs: 45, 46, and 47, respectively; and the light chain variable region of the single-chain variable region fragment comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1, LCDR2, and LCDR3 have the amino acid sequences set forth in SEQ ID NOs: 50, 51, and 52, respectively; (e) When the single-chain variable region fragment is linked to the C-terminus of the heavy chain of the immunoglobulin via a linker, the single-chain variable region fragment comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1, HCDR2, and HCDR3 have the amino acid sequences set forth in SEQ ID NOs: 45, 46, and 47, respectively. Ｈ ), or a single-chain variable region fragment of an antibody light chain variable region (V) comprising LCDR1, LCDR2 and LCDR3, wherein LCDR1, LCDR2 and LCDR3 have the amino acid sequences set forth in SEQ ID NOs: 50, 51 and 52, respectively. Ｌ ) are connected; (f) the immunoglobulin heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1, HCDR2, and HCDR3 have the amino acid sequences set forth in SEQ ID NOs: 45, 46, and 47, respectively, and the immunoglobulin light chain variable region comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1, LCDR2, and LCDR3 have the amino acid sequences set forth in SEQ ID NOs: 50, 51, and 52, respectively; (g) the heavy chain variable region of the single-chain variable region fragment comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1, HCDR2, and HCDR3 have the amino acid sequences set forth in SEQ ID NOs: 3, 4, and 5, respectively; and the light chain variable region of the single-chain variable region fragment comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1, LCDR2, and LCDR3 have the amino acid sequences set forth in SEQ ID NOs: 8, 9, and 10, respectively; (h) When the single-chain variable region fragment is linked to the C-terminus of the heavy chain of the immunoglobulin via a linker, the single-chain variable region fragment comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1, HCDR2, and HCDR3 have the amino acid sequences set forth in SEQ ID NOs: 3, 4, and 5, respectively. Ｈ ), or a single-chain variable region fragment of an antibody light chain variable region (V) comprising LCDR1, LCDR2 and LCDR3, wherein LCDR1, LCDR2 and LCDR3 have the amino acid sequences set forth in SEQ ID NOs: 8, 9 and 10, respectively. Ｌ ) are connected; (i) One immunoglobulin molecule is linked to two single-chain variable region fragment molecules. Anti-CD73 / anti-PD-1 bispecific antibody.

14. 14. The anti-CD73 / anti-PD-1 bispecific antibody of claim 13, In the above (e) or (h), the single-chain variable region fragment is ２ -V Ｌ -Linker-V Ｈ -COOH or NH ２ -V Ｈ -Linker-V Ｌ represented by —COOH; or In (i), the two single-chain variable region fragment molecules are identical. Anti-CD73 / anti-PD-1 bispecific antibody.

15. 9. The anti-CD73 / anti-PD-1 bispecific antibody of claim 7 or 8, wherein the immunoglobulin is IgG, IgA, IgD, IgE, or IgM.

16. 16. The anti-CD73 / anti-PD-1 bispecific antibody of claim 15, wherein the immunoglobulin is IgG1, IgG2, IgG3, or IgG4.

17. 9. The anti-CD73 / anti-PD-1 bispecific antibody of claim 7 or 8, wherein the single-chain variable region fragment is linked to the C-terminus of the immunoglobulin heavy chain.

18. 18. The anti-CD73 / anti-PD-1 bispecific antibody of claim 17, wherein one immunoglobulin molecule is linked to two single-chain variable region fragment molecules.

19. 19. The anti-CD73 / anti-PD-1 bispecific antibody of claim 18, wherein the two single-chain variable region fragment molecules are identical.

20. 9. The anti-CD73 / anti-PD-1 bispecific antibody of claim 8, the heavy chain variable region of the immunoglobulin comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1, HCDR2, and HCDR3 have the amino acid sequences set forth in SEQ ID NOs: 45, 46, and 47, respectively; and the light chain variable region of the immunoglobulin comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1, LCDR2, and LCDR3 have the amino acid sequences set forth in SEQ ID NOs: 50, 51, and 52, respectively; and / or the heavy chain variable region of the single-chain variable region fragment comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1, HCDR2, and HCDR3 have the amino acid sequences set forth in SEQ ID NOs: 3, 4, and 5, respectively; and the light chain variable region of the single-chain variable region fragment comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1, LCDR2, and LCDR3 have the amino acid sequences set forth in SEQ ID NOs: 8, 9, and 10, respectively. Anti-CD73 / anti-PD-1 bispecific antibody.

21. 21. The anti-CD73 / anti-PD-1 bispecific antibody of claim 20, When the single-chain variable region fragment is linked to the C-terminus of the heavy chain of the immunoglobulin via a linker, the single-chain variable region fragment comprises HCDR1, HCDR2, and HCDR3, and HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 3, 4, and 5, respectively. Ｈ ), or a single-chain variable region fragment of an antibody light chain variable region (V) comprising LCDR1, LCDR2 and LCDR3, wherein LCDR1, LCDR2 and LCDR3 have the amino acid sequences set forth in SEQ ID NOs: 8, 9 and 10, respectively. Ｌ ) are connected, Anti-CD73 / anti-PD-1 bispecific antibody.

22. 9. The anti-CD73 / anti-PD-1 bispecific antibody of claim 8, the immunoglobulin heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, wherein HCDR1, HCDR2 and HCDR3 have the amino acid sequences set forth in SEQ ID NOs: 3, 4 and 5, respectively, and the immunoglobulin light chain variable region comprises LCDR1, LCDR2 and LCDR3, wherein LCDR1, LCDR2 and LCDR3 have the amino acid sequences set forth in SEQ ID NOs: 8, 9 and 10, respectively; and / or the heavy chain variable region of the single-chain variable region fragment comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1, HCDR2, and HCDR3 have the amino acid sequences set forth in SEQ ID NOs: 45, 46, and 47, respectively; and the light chain variable region of the single-chain variable region fragment comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1, LCDR2, and LCDR3 have the amino acid sequences set forth in SEQ ID NOs: 50, 51, and 52, respectively; 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) of the single-chain variable region fragment comprises HCDR1, HCDR2, and HCDR3, and HCDR1, HCDR2, and HCDR3 have the amino acid sequences set forth in SEQ ID NOs: 45, 46, and 47, respectively. Ｈ ), or a single-chain variable region fragment of an antibody light chain variable region (V) comprising LCDR1, LCDR2 and LCDR3, wherein LCDR1, LCDR2 and LCDR3 have the amino acid sequences set forth in SEQ ID NOs: 50, 51 and 52, respectively. Ｌ ) are connected, Anti-CD73 / anti-PD-1 bispecific antibody.

23. 9. The anti-CD73 / anti-PD-1 bispecific antibody of claim 8, the heavy chain variable region of the immunoglobulin has an amino acid sequence selected from SEQ ID NO:44 and SEQ ID NO:62, and 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; 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; 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; or the heavy chain variable region of the single-chain variable region fragment has the amino acid sequence set forth in SEQ ID NO:93, and the light chain variable region of the single-chain variable region fragment has the amino acid sequence set forth in SEQ ID NO:95; 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 ), or the antibody light chain variable region (V Ｌ ) are connected, Anti-CD73 / anti-PD-1 bispecific antibody.

24. 9. The anti-CD73 / anti-PD-1 bispecific antibody of claim 8, 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; or the heavy chain variable region of the immunoglobulin has the amino acid sequence set forth in SEQ ID NO:93, and the light chain variable region of the immunoglobulin has the amino acid sequence set forth in SEQ ID NO:95, 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 ), or the antibody light chain variable region (V Ｌ ) are connected, Anti-CD73 / anti-PD-1 bispecific antibody.

25. 24. The anti-CD73 / anti-PD-1 bispecific antibody of any one of claims 7 to 23, wherein the anti-CD73 / anti-PD-1 bispecific antibody has any one of the following characteristics: (a) the immunoglobulin comprises non-CDR regions, and the non-CDR regions are derived from a species other than mouse; (b) the constant region of said immunoglobulin is humanized; (c) the heavy chain constant region is an Igγ-1 chain C region consisting of the amino acid sequence set forth in SEQ ID NO: 102 (Accession No. P01857), and the light chain constant region is an Igκ chain C region consisting of the amino acid sequence set forth in SEQ ID NO: 101 (Accession No. P01834); (d) 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 consisting of the amino acid sequence set forth in SEQ ID NO: 102 (Accession No. P01857), 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; (e) the heavy chain constant region comprises the following mutations at positions 234, 235, and / or 237 based on the Igγ-1 chain C region consisting of the amino acid sequence set forth in SEQ ID NO: 102 (Accession No. P01857), according to the EU numbering system: L234A and L235A; L234A and G237A; L235A and G237A; or having L234A, L235A and G237A, (f) the heavy chain constant region of said immunoglobulin also contains 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, having one or more mutations selected from: (g) the anti-CD73 / anti-PD-1 bispecific antibody is selected from the group consisting of: (i) P1D7V01, the immunoglobulin heavy chain having the amino acid sequence set forth in SEQ ID NO: 66, the immunoglobulin light chain having the amino acid sequence set forth in SEQ ID NO: 68, and linker 1 having the amino acid sequence set forth in SEQ ID NO: 79, and the scFv consisting of a heavy chain variable region having the amino acids set forth in SEQ ID NO: 20, linker 1 having the amino acid sequence set forth in SEQ ID NO: 79, and a light chain variable region having the amino acids set forth in SEQ ID NO: 24; (ii) P1D7V02R, the immunoglobulin heavy chain having the amino acid sequence set forth in SEQ ID NO: 66, the immunoglobulin light chain having the amino acid sequence set forth in SEQ ID NO: 68, and linker 1 having the amino acid sequence set forth in SEQ ID NO: 79, and the scFv consisting of a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 24, linker 1 having the amino acid sequence set forth in SEQ ID NO: 79, and a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 20; (iii) P1D7V03, the immunoglobulin heavy chain having the amino acid sequence set forth in SEQ ID NO: 66, the immunoglobulin light chain having the amino acid sequence set forth in SEQ ID NO: 68, linker 1 having the amino acid sequence set forth in SEQ ID NO: 81, and the scFv consisting of a heavy chain variable region having the amino acids set forth in SEQ ID NO: 20, linker 1 having the amino acid sequence set forth in SEQ ID NO: 79, and a light chain variable region having the amino acids set forth in SEQ ID NO: 24; (iv) P1D7V04R, the immunoglobulin heavy chain having the amino acid sequence set forth in SEQ ID NO: 66, the immunoglobulin light chain having the amino acid sequence set forth in SEQ ID NO: 68, linker 1 having the amino acid sequence set forth in SEQ ID NO: 81, and the scFv consisting of a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 24, linker 1 having the amino acid sequence set forth in SEQ ID NO: 79, and a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 20; selected from the group consisting of: (h) 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) NTP DV1, 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: 62, 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: 64; (2) NTP DV2, the heavy chain of which has the amino acid sequence set forth in SEQ ID NO: 83, 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: 62, 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: 64; (3) NTP DV3, 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: 62, 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: 64; and (4) NTP DV4, the heavy chain of which has the amino acid sequence set forth in SEQ ID NO: 83, 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: 62, 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: 64; selected from the group consisting of Anti-CD73 / anti-PD-1 bispecific antibody.

26. 26. The anti-CD73 / anti-PD-1 bispecific antibody of claim 25, wherein the non-CDR regions are derived from a human antibody.

27. The anti-CD73 / anti-PD-1 bispecific antibody according to any one of claims 1 to 26, －５ K measured by Fortevio's octet system less than M Ｄ An anti-CD73 / anti-PD-1 bispecific antibody that binds to CD73 protein and / or PD-1 protein.

28. The anti-CD73 / anti-PD-1 bispecific antibody according to any one of claims 1 to 26, －６ K measured by Fortevio's octet system below M Ｄ An anti-CD73 / anti-PD-1 bispecific antibody that binds to CD73 protein and / or PD-1 protein.

29. The anti-CD73 / anti-PD-1 bispecific antibody according to any one of claims 1 to 26, －７ K measured by Fortevio's octet system below M Ｄ An anti-CD73 / anti-PD-1 bispecific antibody that binds to CD73 protein and / or PD-1 protein.

30. The anti-CD73 / anti-PD-1 bispecific antibody according to any one of claims 1 to 26, －８ K measured by Fortevio's octet system below M Ｄ An anti-CD73 / anti-PD-1 bispecific antibody that binds to CD73 protein and / or PD-1 protein.

31. The anti-CD73 / anti-PD-1 bispecific antibody according to any one of claims 1 to 26, －９ K measured by Fortevio's octet system below M Ｄ An anti-CD73 / anti-PD-1 bispecific antibody that binds to CD73 protein and / or PD-1 protein.

32. The anti-CD73 / anti-PD-1 bispecific antibody according to any one of claims 1 to 26, －１０ K measured by Fortevio's octet system below M Ｄ An anti-CD73 / anti-PD-1 bispecific antibody that binds to CD73 protein and / or PD-1 protein.

33. 33. An isolated nucleic acid molecule comprising a nucleotide sequence capable of encoding the anti-CD73 / anti-PD-1 bispecific antibody of any one of claims 1 to 32.

34. 34. A vector comprising the isolated nucleic acid molecule of claim 33.

35. 35. A host cell comprising the isolated nucleic acid molecule of claim 33 or the vector of claim 34.

36. 33. A method for preparing an anti-CD73 / anti-PD-1 bispecific antibody according to any one of claims 1 to 32, comprising culturing under suitable conditions a host cell according to claim 35, and isolating said bispecific antibody from said cell culture.

37. 33. A conjugate comprising the anti-CD73 / anti-PD-1 bispecific antibody of any one of claims 1 to 32 and a conjugated moiety, wherein the conjugated moiety is a detectable label.

38. 38. The conjugate of claim 37, wherein the conjugated moiety is a radioisotope, a fluorescent substance, a chemiluminescent substance, a colored substance, or an enzyme.

39. A kit comprising the anti-CD73 / anti-PD-1 bispecific antibody of any one of claims 1 to 32 or the conjugate of claim 37.

40. 40. The kit of claim 39, further comprising a secondary antibody that specifically recognizes the bispecific antibody.

41. 41. The kit of claim 40, wherein the secondary antibody further comprises a detectable label.

42. 42. The kit of claim 41, wherein the detectable label is a radioisotope, a fluorescent substance, a chemiluminescent substance, a colored substance, or an enzyme.

43. 33. Use of an anti-CD73 / anti-PD-1 bispecific antibody according to any one of claims 1 to 32 in the preparation of a kit for detecting the presence or level of CD73 and / or PD-1 in a sample.

44. 39. A pharmaceutical composition comprising the anti-CD73 / anti-PD-1 bispecific antibody of any one of claims 1 to 32 or the conjugate of claim 37 or 38.

45. 45. The pharmaceutical composition of claim 44, further comprising a pharmaceutically acceptable carrier and / or excipient.

46. 39. An anti-CD73 / anti-PD-1 bispecific antibody according to any one of claims 1 to 32 or a conjugate according to claim 37 or 38 for use as an active ingredient in the prevention and / or treatment of tumors or anemia, or in the diagnosis of tumors or anemia.

47. 39. Use of an anti-CD73 / anti-PD-1 bispecific antibody according to any one of claims 1 to 32 or a conjugate according to claim 37 or 38 in the preparation of a medicament for preventing and / or treating tumors or anemia, or in the preparation of a medicament for diagnosing tumors or anemia.

48. 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 an agent for blocking the binding of PD-1 to PDL-1; agents for downregulating the activity or levels of PD-1; Agents for alleviating PD-1 immunosuppression in an organism; an agent for increasing IL-2 expression in T lymphocytes, or Agent for increasing IFN-γ expression in T lymphocytes 39. Use of an anti-CD73 / anti-PD-1 bispecific antibody according to any one of claims 1 to 32 or a conjugate according to claim 37 or 38 in the preparation of

49. 46. ​​The pharmaceutical composition according to claim 44 or 45, for the prevention and / or treatment of tumors or anemia.

Images