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

Abstract

The present invention relates to anti-CD73-anti-PD-1 bispecific antibodies, pharmaceutical compositions and uses thereof.

Description

Technical Field

[0001] The present invention belongs to the field of tumor treatment and molecular immunology, and relates to an anti-CD73-anti-PD-1 bispecific antibody, a pharmaceutical composition thereof and uses thereof. Background Art

[0002] Ecto-5'-nucleotidase, also known as CD73 protein, is a multifunctional glycoprotein with a molecular weight of 70KD encoded by the NT5E gene. It is anchored to the cell membrane through glyocsyl phosphatidylinositol (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. Current studies have found that CD73 is highly expressed in a variety of solid tumors, such as cancer cells, dendritic cells, regulatory T cells (Treg), natural killer cells (NK cells), myeloid suppressor cells (MDSC), tumor-associated macrophages (TAM) and so on in the tumor microenvironment. The expression of CD73 is regulated by molecules such as TGF-β, EGFR, AKT, β-catenin, and especially HIF-1, which functions as a transcription factor, is the most critical. An important feature of the tumor microenvironment is hypoxia, and the upregulation of molecules such as hypoxia-inducible factor-1 (HIF-1) leads to the 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 showed that high expression of CD73 is a potential biomarker that is closely associated with poor prognosis in many types of tumors, including breast cancer, lung cancer, ovarian cancer, kidney cancer, gastric cancer, head and neck cancer, etc.

[0004] CD73 has both hydrolase activity and non-hydrolase function. The enzymatic and non-enzymatic functions of CD73 exist simultaneously in tumor-related processes and promote each other to maintain tumor progression. More and more studies have found that CD73 is a key regulatory molecule for tumor cell proliferation, metastasis and invasion in vitro, tumor angiogenesis and tumor immune escape in vivo. One of the important immunosuppressive mechanisms is mediated by the CD73-adenosine metabolic signaling pathway. CD39 upstream of CD73 can catalyze ATP to produce adenosine monophosphate (AMP), and the generated AMP is converted into adenosine by CD73, which binds to the downstream adenosine receptor (A2AR). A2AR activates protein kinase A (PKA) and Csk kinase, inhibits a series of signaling pathways related to immune activation such as LCK, MAPK, PKC, and inhibits the immune killing effect of T cells, thereby exerting an immunosuppressive effect and enabling tumors to achieve immune escape (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 by both immune cells and non-immune cells can promote tumor immune escape, development and metastasis, among which Treg cell-associated CD73-adenosine signals have the most significant inhibitory effect on CTL (cytotoxic T cells) and NK cell functions.

[0005] For the treatment of solid tumors, in order to overcome drug resistance and improve efficacy, a very important aspect is to relieve the inhibitory effect of the tumor microenvironment (TME) on immune effector cells. TME is a very complex system, composed of a variety of cells, extracellular matrix, enzymes, cytokines, metabolic products, etc., with significant characteristics of low hydrogen, low pH and high pressure, which is very different from normal tissues. Hypoxia or ATP enrichment caused by chemotherapy and radiotherapy to kill tumor cells will promote the cascade reaction of CD39-CD73 adenosine signals, which is beneficial to the proliferation and function of various cancer-promoting cells, but not to tumor suppressor 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] Using antibodies targeting CD73 or knocking out CD73 in animal models can effectively block tumor growth and metastasis. Recently, CD73 monoclonal antibodies, small interfering RNA technology, and specific inhibitor APCP have achieved significant therapeutic effects in animal experiments, providing a new approach for anti-tumor treatment. Evidence from in vivo experiments shows that targeted blocking of CD73 will be an effective treatment for tumor patients.

[0007] The association between CD73 overexpression and tumor subtypes, prognosis, and drug response in patients has shown that CD73 can be used as an important marker for future individual tumor treatment and detection. Therefore, the study of CD73 targets is indispensable.

[0008] The transmembrane receptor PD-1 (programmed cell death-1) is a member of the CD28 family and is expressed in activated T cells, B cells, and myeloid cells. PD-1 receptors PDL1 and PDL2 both belong to the B7 superfamily, of which PDL1 is expressed by a variety of cells, including T cells, B cells, endothelial cells, and epithelial cells, while PDL2 is only expressed in antigen-presenting cells such as dendritic cells and macrophages.

[0009] PD-1 plays a very important role in the activation process of negative regulatory T cells. PD-1-mediated negative regulation of T cells is one of the important mechanisms of tumor immune escape. PDL-1 expressed on the surface of tumors can bind to PD-1 on the surface of immune cells, thereby inhibiting immune cells from killing tumor tissues through the PD-1 / PDL-1 signaling pathway. Tumors with high expression of PD-L1 are accompanied by 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 thus inhibit the PD-1 / PDL-1 signaling pathway is to inject anti-PD-1 antibodies in vivo.

[0010] PD-1 antibodies have broad-spectrum anti-tumor prospects and amazing efficacy. Antibodies targeting the PD-1 pathway will bring breakthrough progress in the treatment of various tumors: for the treatment of non-small cell lung cancer, renal cell carcinoma, ovarian cancer, melanoma (Homet MB, Parisi G., et al., Anti-PD-1 Therapy in Melanoma. Semin Oncol. 2015 Jun; 42(3): 466-473), blood tumors and anemia (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 and bind to two different antigens. They can be produced by immunosorting and purification, or obtained by genetic engineering. Genetic engineering has corresponding flexibility in terms of binding site optimization, synthetic form considerations, and yield, so it has certain advantages. At present, more than 45 forms of bispecific antibodies have been proven (Müller D, Kontermann RE. Bispecific antibodies for cancer immunotherapy: Current perspectives. BioDrugs 2010; 24: 89-98). The IgG-ScFv format, also known as the Morrison model (Coloma MJ, Morrison SL. Design and production of novel tetravalent bispecific antibodies. Nat Biotechnol. Nature Biotechnology, 1997; 15: 159-163), has advantages in antibody engineering, expression and purification due to its similarity to the naturally occurring IgG format. It has been proven to be an ideal form of bifunctional antibodies (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) is an antibody-dependent cell-mediated cytotoxic effect, which refers to the Fab segment of the antibody binding to the antigenic epitope of virus-infected cells or tumor cells, and its Fc segment binding to the Fc receptor (Fc Receptor, FcR) on the surface of killer cells (NK cells, macrophages, etc.), mediating the killer cells to directly kill the target cells.

[0013] CDC (complement dependent cytotoxicity) refers to complement-dependent cytotoxicity. The CDC effect is caused by the first binding of antibodies to the corresponding antigens on the cell membrane surface, and then to complement C1q. Then C2-C9 is activated to form a membrane attack complex to exert a lytic effect on the target cell.

[0014] The IgG family consists of four members, IgG1, IgG2, IgG3, and IgG4. There are amino acid differences in the crystallizable fragment (Fc) region of their heavy chain constant regions, resulting in different affinities between them and FcγRs. Wild-type IgG1 can bind to various FcγRs and can induce ADCC and CDC effects. Studies by Zhang et al. (Zhang T et al. Cancer Immunol Immunother. 2018; 67(7): 1079-1090.) and Dahan et al. (Dahan R et al. Cancer Cell. 2015; 28(3): 285-95.) have shown that the binding of the Fc segment of antibodies targeting immune checkpoints such as PD-1 to Fc receptors can have a negative impact on the anti-cancer activity mediated by anti-antibodies, which may be due to the damage of immune cells induced by Fc-dependent effector functions, including antibody-dependent cell-mediated cytotoxicity, which is an important mechanism leading to immune cell damage.

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

[0016] As a pro-inflammatory factor, IL-8 is closely related to the occurrence and development of tumors. During the malignant transformation of non-renal cancer cells induced by methylarsonate, the expression of IL-8 gene increases, and IL-8 gene silencing can significantly inhibit the growth of transplanted tumors in mice. In addition, the reduction of IL-8 level can inhibit the expression of matrix metalloproteinase-9, cyclin D1, pro-apoptotic protein Bcl-2, and vascular endothelial growth factor (VEGF) related to tumor growth and metastasis (Escudero-Lourdes C et al. Toxicology and applied pharmacology, 2012, 258 (1): 10-18.). Inoue et al. found that IL-8 can induce malignant transformation and increased invasiveness of non-tumor bladder cell lines (233JP), and the probability of malignant transformation of 233JP cells in IL-8 knockout mice was significantly reduced (Inoue K et al. Cancer Res, 2000, 60 (8): 2290-2299.). In addition, in prostate cancer, IL-8 can promote the occurrence of castration-resistant prostate cancer (CRPC) in patients (Chen K et al. Cancer research, 2015, 75 (10): 1992-2004.), and is related to 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 proliferation (Singh RK, Lokeshwar BL. Molecul Cancer, 2009, 8: 57.). The above studies show that the level of IL-8 is closely related to the occurrence and development of tumors. Further studies (Mian BM et al. Clin Cancer Res, 2003, 9 (8): 3167-3175.) show that IL-8 can be used as a new target for tumor treatment. In a bladder cancer tumor model, the use of anti-IL-8 antibodies can significantly inhibit tumor growth.

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

[0018] Immune-related adverse reactions are a common and dangerous adverse reaction in anti-tumor therapy with immune checkpoint inhibitors (ICI) (Spain Let al. Cancer Treat Rev. 2016; 44: 51-60.). In recent years, immune checkpoint inhibitors have achieved great success in tumor immunotherapy, but they have also led to a new toxicity spectrum due to off-target effects. Among them, serious immune-related adverse events (irAEs) in major organs (including the heart, lungs and brain) may be life-threatening (Bergqvist V, et al. Cancer Immunol Immunother. 2017; 66(5): 581-592.; Gomatou G et al. Respiration. 2020; 1: 1-11.; JoshiMN 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 ICI may induce off-target effects through four mechanisms, including direct binding to immune checkpoint molecules expressed on the surface of normal cells, activating complement hypersensitivity reactions; the presence of homologous antigens / epitopes between normal tissues and tumor cells; the production of autoantibodies; and increased levels of pro-inflammatory cytokines, such as IL-6 (Martins F et al., The Lancet Oncology, 20(1), e54-e64.).

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

[0020] The binding of FcγRIa on macrophages to wild-type IgG1 or IgG4 antibodies can induce them to secrete IL-8 and IL-6 (Kinder M et al., mAbs. 2015). Mutating the Fc segment of the antibody and eliminating its binding to FcγRIa can effectively inhibit the secretion of IL-8, thereby increasing the safety and efficacy of the antibody. Summary of the invention

[0021] The inventors used a mammalian cell expression system to express recombinant CD73 and PD-1 as antigens to immunize mice, and obtained hybridoma cells by fusing mouse spleen cells with myeloma cells. The inventors screened a large number of samples and obtained the following hybridoma cell lines:

[0022] Hybridoma cell line LT014 (also known as CD73-19F3), which was deposited in China Center for Type Culture Collection (CCTCC) on June 19, 2018, with a deposit number of CCTCCNO: C2018137; and

[0023] The hybridoma cell line LT003 (also known as PD-1-14C12) was deposited in the China Center for Type Culture Collection (CCTCC) on June 16, 2015, with the deposit number CCTCCNO: C2015105.

[0024] The inventors surprisingly found that:

[0025] The hybridoma cell line LT014 can secrete a specific monoclonal antibody (named 19F3) that specifically binds to human CD73, and this monoclonal antibody can effectively inhibit the enzymatic activity of CD73 in a non-substrate competitive manner, reduce the production of adenosine, and promote T cell activity and tumor inhibition effects.

[0026] The hybridoma cell line LT003 can secrete a specific monoclonal antibody (named 14C12) that specifically binds to PD-1, and this monoclonal antibody can effectively block the binding of PD-1 to PDL1.

[0027] Furthermore, the present inventors creatively prepared humanized antibodies against CD73 (named 19F3H2L2, 19F3H2L3, 19F3H2L3 (hG1M), 19F3H2L3 (hG1TM) respectively) and humanized antibodies against PD-1 (named 14C12H1L1 and 14C12H1L1 (hG1TM)).

[0028] Furthermore, the inventors creatively fused the two types of humanized antibodies into new antibodies by protein recombination, and obtained humanized bifunctional antibodies that can bind to CD73 and PD-1, inhibit CD73 activity, and block the binding of PD-1 and PDL1 (respectively named P1D7V01, P1D7V03, NTPDV1, NTPDV2, NTPDV3, NTPDV4 (also written as NTPDV1 (hG1TM), NTPDV2 (hG1TM), NTPDV3 (hG1TM), NTPDV4 (hG1TM) in this article and in the Chinese invention patent with application number 202110270671.X), which have the potential to be used for the preparation of prevention and treatment of solid tumors and blood tumors.

[0029] This provides the following invention:

[0030] One aspect of the present invention relates to an anti-CD73-anti-PD-1 bispecific antibody comprising:

[0031] a first protein functional domain, wherein the first protein functional domain targets PD-1, and

[0032] A second protein functional domain, wherein the second protein functional domain targets CD73.

[0033] In one embodiment of the present invention, the bispecific antibody

[0034] The first protein functional region comprises an amino acid sequence such as HCDR1, HCDR2 and HCDR3 comprised by the heavy chain variable region as shown in SEQ ID NO:44, preferably the amino acid sequences of HCDR1, HCDR2 and HCDR3 are as shown in SEQ ID NOs:45-47, or sequences having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the sequences as shown in SEQ ID NOs:45-47, 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 sequences as shown in SEQ ID NOs:45-47, and comprises

[0035] The amino acid sequence of LCDR1, LCDR2 and LCDR3 comprised by the light chain variable region as shown in SEQ ID NO:49, preferably the amino acid sequences of LCDR1, LCDR2 and LCDR3 are as shown in SEQ ID NOs:50-52, respectively, or sequences having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the sequences as shown in SEQ ID NOs:50-52, 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 as shown in SEQ ID NOs:50-52;

[0036] The second protein functional region comprises an amino acid sequence such as HCDR1, HCDR2 and HCDR3 comprised by the heavy chain variable region as shown in SEQ ID NO: 2, preferably an amino acid sequence of HCDR1, HCDR2 and HCDR3 such as a sequence as shown in SEQ ID NOs: 3-5, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the sequence as shown in SEQ ID NOs: 3-5, 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 sequences as shown in SEQ ID NOs: 3-5, and comprises

[0037] The amino acid sequence of LCDR1, LCDR2 and LCDR3 comprised by the light chain variable region as shown in SEQ ID NO:7, preferably the amino acid sequences of LCDR1, LCDR2 and LCDR3 are as shown in SEQ ID NOs:8-10, respectively, or sequences having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the sequences shown in SEQ ID NOs:8-10, or amino acid sequences having one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the sequences shown in SEQ ID NOs:8-10.

[0038] In one embodiment of the present invention, in the anti-CD73-anti-PD-1 bispecific antibody:

[0039] The first protein functional region comprises

[0040] An amino acid sequence as shown 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%, 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the sequence shown in SEQ ID NOs: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 the sequence shown in SEQ ID NOs:44 or 62; and

[0041] corresponding to an amino acid sequence selected from the group consisting of SEQ ID NO:49 or SEQ ID NO:64, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence shown in SEQ ID NOs: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 the sequence shown in SEQ ID NOs:49 or 64;

[0042] and / or,

[0043] The second protein functional region comprises an amino acid sequence selected from the sequence shown in SEQ ID NO: 2, SEQ ID NO: 20, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the sequence shown in SEQ ID NO: 2, 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 the sequence shown in SEQ ID NO: 2, SEQ ID NO: 20; and

[0044] corresponding to SEQ ID NO:7, or SEQ ID NO:22, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, or an amino acid sequence having one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the sequence shown in SEQ ID NO:7 or SEQ ID NO:22;

[0045] or

[0046] The second protein functional region comprises an amino acid sequence as shown in SEQ ID NO: 20, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the sequence shown 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 the sequence shown in SEQ ID NO: 20, and comprises

[0047] An amino acid sequence as shown in SEQ ID NO:24, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the sequence shown 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 the sequence shown in SEQ ID NO:24.

[0048] One aspect of the present invention relates to an anti-CD73-anti-PD-1 bispecific antibody comprising:

[0049] a first protein domain, wherein the first protein domain targets CD73, and

[0050] The second protein functional domain targets PD-1.

[0051] In one embodiment of the present invention, in the bispecific antibody,

[0052] The first protein functional region comprises an amino acid sequence such as HCDR1, HCDR2 and HCDR3 comprised in the heavy chain variable region as shown in SEQ ID NO: 2, preferably the amino acid sequences of HCDR1, HCDR2 and HCDR3 are as shown in SEQ ID NOs: 3-5, respectively, or sequences having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the sequences as shown in SEQ ID NOs: 3-5, or an amino acid sequence having one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared with the sequences as shown in SEQ ID NOs: 3-5, and comprises

[0053] The amino acid sequence of LCDR1, LCDR2 and LCDR3 comprised by the light chain variable region as shown in SEQ ID NO:7, preferably the amino acid sequences of LCDR1, LCDR2 and LCDR3 are as shown in SEQ ID NOs:8-10, respectively, or sequences having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the sequences as shown in SEQ ID NOs:8-10, or amino acid sequences having one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the sequences as shown in SEQ ID NOs:8-10;

[0054] The second protein functional region comprises an amino acid sequence such as HCDR1, HCDR2 and HCDR3 comprised by the heavy chain variable region as shown in SEQ ID NO:44, preferably the amino acid sequences of HCDR1, HCDR2 and HCDR3 are respectively as shown in SEQ ID NOs:45-47, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the sequences as shown in SEQ ID NOs:45-47, 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 sequences as shown in SEQ ID NOs:45-47, and comprises

[0055] The amino acid sequence of LCDR1, LCDR2 and LCDR3 comprised by the light chain variable region as shown in SEQ ID NO:49, preferably the amino acid sequences of LCDR1, LCDR2 and LCDR3 are as shown in SEQ ID NOs:50-52, respectively, or sequences having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the sequences shown in SEQ ID NOs:50-52, 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 shown in SEQ ID NOs:50-52.

[0056] In one embodiment of the present invention, in the anti-CD73-anti-PD-1 bispecific antibody:

[0057] The first protein functional region comprises

[0058] An amino acid sequence as shown in SEQ ID NO: 2, SEQ ID NO: 20, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the sequence shown in SEQ ID NOs: 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 shown in SEQ ID NOs: 2 or 20; and

[0059] corresponding to an amino acid sequence selected from the group consisting of SEQ ID NO:7, SEQ ID NO:22, SEQ ID NO:24, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, or an amino acid sequence having one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the sequences shown in SEQ ID NOs:7, 22 or 24;

[0060] and / or,

[0061] The second protein functional region comprises an amino acid sequence selected from the sequence shown 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%, 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the sequence shown 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 the sequence shown in SEQ ID NO: 44 or SEQ ID NO: 62; and

[0062] The amino acid sequence corresponds to SEQ ID NOs: 49 or 64, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the sequence shown in SEQ ID NOs: 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 the sequence shown in SEQ ID NOs: 49 or 64.

[0063] In one embodiment of the present invention, in the anti-CD73-anti-PD-1 bispecific antibody, the first protein functional region and the second protein functional region are directly connected or connected through a connecting fragment; preferably, the connecting fragment is (GGGGS)n, where n is a positive integer, such as 1, 2, 3, 4, 5 or 6.

[0064] In one embodiment of the present invention, the first protein functional region and the second protein functional region in the anti-CD73-anti-PD-1 bispecific antibody are independently immunoglobulins or antigen-binding fragments, such as half antibodies, Fab, F(ab')2 or single-chain antibodies. 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.

[0065] In one embodiment of the present invention, the N-terminus of the heavy chain variable region of the antigen binding fragment is directly (or through a connecting fragment) connected 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 directly (or through a connecting fragment) connected to the C-terminus of the light chain variable region CL of the immunoglobulin; or the N-terminus of the heavy chain variable region of the antigen binding fragment is directly (or through a connecting fragment) connected to the C-terminus of the light chain variable region CL of the immunoglobulin, and the N-terminus of the light chain variable region of the antigen binding fragment is directly (or through a connecting fragment) connected to the C-terminus of the heavy chain variable region CH1 of the immunoglobulin.

[0066] In one embodiment of the present invention, the C-terminus of the heavy chain variable region of the antigen binding fragment is directly (or through a connecting fragment) connected 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 directly (or through a connecting fragment) connected 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 directly (or through a connecting fragment) connected 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 directly (or through a connecting fragment) connected to the N-terminus of the heavy chain of the immunoglobulin.

[0067] In one embodiment of the present invention, the antigen-binding fragment is a single-chain antibody. Preferably, the first protein functional region is an immunoglobulin and the second protein functional region is a single-chain antibody; or, the first protein functional region is a single-chain antibody and the second protein functional region is an immunoglobulin.

[0068] In one embodiment of the present invention, in the bispecific antibody, the first protein functional region and the second protein functional region are independently 1, 2 or more.

[0069] In one embodiment of the present invention, in the anti-CD73-anti-PD-1 bispecific antibody, the single-chain antibody is connected to the antibody heavy chain variable region (V H ) and antibody light chain variable region (V L ) molecules; preferably, may have the general structure: NH2-V L -Connection fragment-V H -COOH or NH2-V H -Connection fragment-V L -COOH.

[0070] In one embodiment of the present invention, in the anti-CD73-anti-PD-1 bispecific antibody, the single-chain antibody is connected to the C-terminus (C-terminus) of the heavy chain of the immunoglobulin via a linker fragment. H) (or the N-terminus of the heavy chain, the C-terminus of CH1 of the heavy chain variable region), the antibody heavy chain variable region (V H ), or, firstly, connecting the antibody light chain variable region (V L ); Preferably, the single-chain antibody may have a general structure: C H -Connection fragment-V H -Connection fragment-V L -COOH, or C H -Connection fragment-V L -Connection fragment-V H -COOH,

[0071] Preferably,

[0072] The immunoglobulin, wherein the heavy chain variable region comprises CDRs with amino acid sequences of SEQ ID NOs: 3-5, and the light chain variable region comprises CDRs with amino acid sequences of SEQ ID NOs: 8-10;

[0073] The single-chain antibody, wherein the heavy chain variable region comprises CDRs with amino acid sequences of SEQ ID NOs: 45-47, and the light chain variable region comprises CDRs with amino acid sequences of SEQ ID NOs: 50-52,

[0074] Preferably, the single-chain antibody (eg, NH2-V L -Connection fragment-V H -COOH or NH2-V H -Connection fragment-V L When the single-chain antibody is connected to the C-terminus of the heavy chain of the immunoglobulin through a linker fragment, the antibody heavy chain variable region (V H ), or, firstly, connecting the antibody light chain variable region (V L ),

[0075] or preferably,

[0076] The immunoglobulin, wherein the heavy chain variable region comprises CDRs with amino acid sequences of SEQ ID NOs: 45-47, and the light chain variable region comprises CDRs with amino acid sequences of SEQ ID NOs: 50-52; and / or,

[0077] The single-chain antibody, wherein the heavy chain variable region comprises CDRs with amino acid sequences of SEQ ID NOs: 3-5, and the light chain variable region comprises CDRs with amino acid sequences of SEQ ID NOs: 8-10,

[0078] Wherein, the single-chain antibody (e.g. NH2-V L -Connection fragment-V H -COOH or NH2-V H -Connection fragment-V L When the single-chain antibody is connected to the C-terminus of the heavy chain of the immunoglobulin through a linker fragment, the antibody heavy chain variable region (V H ), or, firstly, connecting the antibody light chain variable region (V L ),

[0079] Preferably,

[0080] One immunoglobulin molecule is linked to two single-chain antibody molecules, and more preferably, the two single-chain antibody molecules are identical.

[0081] In one embodiment of the present invention, in the anti-CD73-anti-PD-1 bispecific antibody, the immunoglobulin is IgG, IgA, IgD, IgE or IgM; preferably IgG, such as IgG1, IgG2, IgG3 or IgG4.

[0082] In one embodiment of the present invention, in the anti-CD73-anti-PD-1 bispecific antibody, the single-chain antibody is connected to the C-terminus of the heavy chain of the immunoglobulin. Since the immunoglobulin is composed of two heavy chains, one immunoglobulin molecule is connected to two single-chain antibody molecules. Preferably, the two single-chain antibody molecules are identical.

[0083] In one embodiment of the present invention, in the anti-CD73-anti-PD-1 bispecific antibody:

[0084] The immunoglobulin, wherein the heavy chain variable region comprises CDRs with amino acid sequences of SEQ ID NOs: 3-5, and the light chain variable region comprises CDRs with amino acid sequences of SEQ ID NOs: 8-10;

[0085] and / or,

[0086] The single-chain antibody, wherein the heavy chain variable region comprises CDRs with amino acid sequences of SEQ ID NOs: 45-47, and the light chain variable region comprises CDRs with amino acid sequences of SEQ ID NOs: 50-52,

[0087] Preferably, the single-chain antibody (eg, NH2-V L -Connection fragment-V H -COOH or NH2-VH -Connection fragment-V L When the single-chain antibody is connected to the C-terminus of the heavy chain of the immunoglobulin through a linker fragment, the antibody heavy chain variable region (V n ), or, firstly, connecting the antibody light chain variable region (V L ).

[0088] In another embodiment of the present invention, in the anti-CD73-anti-PD-1 bispecific antibody:

[0089] The immunoglobulin, wherein the heavy chain variable region comprises CDRs with amino acid sequences of SEQ ID NOs: 45-47, and the light chain variable region comprises CDRs with amino acid sequences of SEQ ID NOs: 50-52; and / or,

[0090] The single-chain antibody, wherein the heavy chain variable region comprises CDRs with amino acid sequences of SEQ ID NOs: 3-5, and the light chain variable region comprises CDRs with amino acid sequences of SEQ ID NOs: 8-10,

[0091] Wherein, the single-chain antibody (e.g. NH2-V L -Connection fragment-V H -COOH or NH2-V H -Connection fragment-V L When the single-chain antibody is connected to the C-terminus of the heavy chain of the immunoglobulin through a linker fragment, the antibody heavy chain variable region (V H ), or, firstly, connecting the antibody light chain variable region (V L ).

[0092] In one embodiment of the present invention, in the anti-CD73-anti-PD-1 bispecific antibody:

[0093] The amino acid sequence of the heavy chain variable region of the immunoglobulin is selected from SEQ ID NO: 2 or SEQ ID NO: 20; the amino acid sequence of the light chain variable region of the immunoglobulin is selected from SEQ ID NO: 7 or SEQ ID NO: 22, respectively, or the amino acid sequence of the heavy chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 20; the amino acid sequence of the light chain variable region of the immunoglobulin is the sequence shown in SEQ ID NO: 24;

[0094] and / or,

[0095] The amino acid sequence of the heavy chain variable region of the single-chain antibody is selected from SEQ ID NO: 44 or SEQ ID NO: 62; the amino acid sequence of the light chain variable region of the single-chain antibody is selected from SEQ ID NO: 49 or SEQ ID NO: 64, respectively;

[0096] Wherein, when the single-chain antibody is connected to the C-terminus of the heavy chain of the immunoglobulin through a linker, the antibody heavy chain variable region (V H ), or, firstly, connecting the antibody light chain variable region (V L ).

[0097] In another embodiment of the present invention, in the anti-CD73-anti-PD-1 bispecific antibody:

[0098] The amino acid sequence of the heavy chain variable region of the immunoglobulin is selected from SEQ ID NO: 44 or SEQ ID NO: 62; the amino acid sequence of the light chain variable region of the immunoglobulin is selected from SEQ ID NO: 49 or SEQ ID NO: 64, or the amino acid sequence of the heavy chain variable region of the single-chain antibody is selected from SEQ ID NO: 2 or SEQ ID NO: 20, and the amino acid sequence of the light chain variable region of the single-chain antibody is selected from SEQ ID NO: 7 or SEQ ID NO: 22, or the amino acid sequence of the heavy chain variable region of the single-chain antibody is the sequence shown in SEQ ID NO: 20, and the amino acid sequence of the light chain variable region of the single-chain antibody is the sequence shown in SEQ ID NO: 24.

[0099] Another aspect of the present invention relates to an isolated nucleic acid molecule comprising a nucleic acid sequence capable of encoding a heavy chain variable region of a bispecific antibody, wherein:

[0100] The heavy chain variable region of the antibody comprises:

[0101] CDRs with amino acid sequences of SEQ ID NOs: 3-5, CDRs with amino acid sequences of SEQ ID NOs: 45-47, and CDRs with amino acid sequences of SEQ ID NOs: 50-52;

[0102] And the bispecific antibody heavy chain variable region, as a part of the bispecific antibody, specifically binds to CD73 and PD-1 antigens, and the bispecific antibody further comprises a light chain variable region, and the light chain variable region comprises:

[0103] The amino acid sequences of CDRs are SEQ ID NOs: 8-10;

[0104] Preferably, the CDRs of the light chain variable region are different from the CDRs comprised by the heavy chain variable region.

[0105] In one embodiment of the present invention, in the bispecific antibody,

[0106] The immunoglobulin includes non-CDR regions, and the non-CDR regions are from a species other than murine, such as from a human antibody.

[0107] In one embodiment of the present invention, the constant region of the immunoglobulin is humanized, for example, the heavy chain constant region adopts Ig gamma-1 chain C region, ACCESSION: P01857; the light chain constant region adopts Ig kappa chain C region, ACCESSION: P01834.

[0108] In one embodiment of the present invention, the constant region of the immunoglobulin is humanized, for example, the heavy chain constant region adopts Ig gamma-1chain C region, ACCESSION: P01857; the light chain constant region adopts Ig kappachain C region, ACCESSION: P01834; wherein, according to the EU numbering system, the heavy chain constant region of the immunoglobulin is mutated at any two or three of sites 234, 235 and 237, and after mutation, the affinity constant of the bispecific antibody to FcγRIa, FcγRIIIa and / or C1q is reduced compared with before mutation; preferably, the affinity constant is measured by Fortebio Octet molecular interaction instrument.

[0109] In one or more embodiments of the present invention, the bispecific antibody, wherein, according to the EU numbering system, the heavy chain constant region of the immunoglobulin has the following mutations at positions 234, 235 and / or 237:

[0110] L234A and L235A; or

[0111] L234A and G237A; or

[0112] L235A and G237A;

[0113] or

[0114] L234A, L235A, G237A.

[0115] In the present invention, unless otherwise specified, the letters before the site represent the amino acid before mutation, and the letters after the site represent the amino acid after mutation.

[0116] In one or more embodiments of the present invention, the bispecific antibody, wherein, according to the EU numbering system, the heavy chain constant region of the immunoglobulin further has one or more mutations selected from the following:

[0117] 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.

[0118] In a specific embodiment, the anti-CD73-anti-PD-1 bispecific antibody structure is as shown in heavy chain-light chain-connecting fragment 1-scFv, and the scFv is selected from 14C12H1V-connecting fragment 2-14C12L1V, 14C12H1V-connecting fragment 1-14C12L1V, 14C12H1V-connecting fragment 2-14C12L1V and 14C12H1V-connecting fragment 1-14C12L1V, specifically selected from the group consisting of:

[0119] (1) NTPDV1, whose heavy chain amino acid sequence is shown in SEQ ID NO: 85, whose light chain amino acid sequence is shown in SEQ ID NO: 28, whose amino acid sequence of connecting segment 1 is shown in SEQ ID NO: 79, whose amino acid sequence of 14C12H1V is shown in SEQ ID NO: 62, whose amino acid sequence of connecting segment 2 is shown in SEQ ID NO: 81, whose amino acid sequence of 14C12L1V is shown in SEQ ID NO: 64,

[0120] (2) NTPDV2, whose heavy chain amino acid sequence is shown in SEQ ID NO: 83, whose light chain amino acid sequence is shown in SEQ ID NO: 28, whose amino acid sequence of connecting segment 1 is shown in SEQ ID NO: 79, whose amino acid sequence of 14C12H1V is shown in SEQ ID NO: 62, whose amino acid sequence of connecting segment 1 is shown in SEQ ID NO: 79, whose amino acid sequence of 14C12L1V is shown in SEQ ID NO: 64,

[0121] (3) NTPDV3, whose heavy chain amino acid sequence is shown in SEQ ID NO: 85, whose light chain amino acid sequence is shown in SEQ ID NO: 96, whose amino acid sequence of connecting segment 1 is shown in SEQ ID NO: 79, whose amino acid sequence of 14C12H1V is shown in SEQ ID NO: 62, whose amino acid sequence of connecting segment 2 is shown in SEQ ID NO: 81, whose amino acid sequence of 14C12L1V is shown in SEQ ID NO: 64, and

[0122] (4) NTPDV4, whose heavy chain amino acid sequence is shown in SEQ ID NO: 83, the light chain amino acid sequence is shown in SEQ ID NO: 96, the amino acid sequence of connecting fragment 1 is shown in SEQ ID NO: 79, the amino acid sequence of 14C12H1V is shown in SEQ ID NO: 62, the amino acid sequence of connecting fragment 1 is shown in SEQ ID NO: 79, and the amino acid sequence of 14C12L1V is shown in SEQ ID NO: 64.

[0123] In one embodiment of the invention, the bispecific antibody is expressed in an amount of less than about 10 -5 M, for example, less than about 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 M or smaller K D Binds to CD73 protein and / or PD-1 protein.

[0124] Yet another aspect of the present invention relates to a vector comprising the isolated nucleic acid molecule of the present invention.

[0125] Another aspect of the present invention relates to a host cell comprising the isolated nucleic acid molecule of the present invention, or the vector of the present invention.

[0126] Another aspect of the present invention relates to a method for preparing the bispecific antibody of the present invention, which comprises the steps of culturing the host cell of the present invention under suitable conditions, and recovering the bispecific antibody from the cell culture.

[0127] Another aspect of the present invention relates to a conjugate, which includes a bispecific antibody and a conjugated portion, wherein the bispecific antibody is the bispecific antibody of the present invention, and the conjugated portion is a detectable label; specifically, the conjugated portion is a radioactive isotope, a fluorescent substance, a chemiluminescent substance, a colored substance or an enzyme.

[0128] Another aspect of the present invention relates to a kit, which includes the bispecific antibody of the present invention, or includes the conjugate of the present invention; preferably, the kit also includes a second antibody, which specifically recognizes the bispecific antibody; optionally, the second antibody also includes a detectable label, such as a radioisotope, a fluorescent substance, a chemiluminescent substance, a colored substance or an enzyme.

[0129] Another aspect of the present invention relates to the use of the bispecific antibody of the present invention in the preparation of a kit for detecting the presence or level of CD73 and / or PD-1 in a sample.

[0130] Another aspect of the present invention relates to a pharmaceutical composition, which comprises the bispecific antibody of the present invention or the conjugate of the present invention; optionally, it also includes a pharmaceutically acceptable carrier and / or excipient.

[0131] Another aspect of the present invention relates to the use of the bispecific antibody or conjugate of the present invention in preventing and / or treating tumors or anemia, or in diagnosing tumors or anemia.

[0132] Another aspect of the present invention relates to the use of the bispecific antibody of the present invention or the conjugate of the present invention in the preparation of a drug for preventing and / or treating tumors or anemia, or in the preparation of a drug for diagnosing tumors or anemia.

[0133] Another aspect of the present invention relates to the use of the bispecific antibody of the present invention or the conjugate of the present invention in the preparation of the following drugs:

[0134] Drugs to detect CD73 levels in samples,

[0135] Inhibit the enzymatic activity of CD73;

[0136] and / or

[0137] Drugs that block the binding of PD-1 to PDL1,

[0138] Drugs that modulate (e.g., downregulate) PD-1 activity or levels,

[0139] Drugs that relieve the body's immune suppression by PD-1.

[0140] Drugs that increase IL-2 expression in T lymphocytes, or

[0141] Drugs that increase IFN-γ expression in T lymphocytes.

[0142] Another aspect of the present invention relates to an in vivo or in vitro method, comprising the step of applying an effective amount of the bispecific antibody of the present invention or the conjugate of the present invention to a cell or to a subject in need thereof,

[0143] The anti-CD73-anti-PD-1 bispecific antibodies involved in the present invention can inhibit the CD73 enzyme activity on the cell membrane surface, and can induce the secretion of IFNγ and IL-2, thereby activating the immune response.

[0144] The variable regions of the light and heavy chains determine antigen binding; the variable region of each chain contains three hypervariable regions, called complementarity determining regions (CDRs) (the CDRs of the heavy chain (H) include HCDR1, HCDR2, and HCDR3, and the CDRs of the light chain (L) include LCDR1, LCDR2, and LCDR3; they were named by Kabat et al., see Bethesda Md, Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 1991; 1-3: 91-3242.

[0145] Preferably, CDRs can also be defined by the IMGT numbering system, see Ehrenmann F, Kaas Q, Lefranc MP. 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.

[0146] By using technical means well known to those skilled in the art, for example, analyzing the amino acid sequences of the CDRs of the monoclonal antibody sequences in items (1) to (11) below according to the IMGT definition, the results are as follows:

[0147] (1) 19F3

[0148] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:2, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:7.

[0149] The amino acid sequences of the three CDRs in its heavy chain variable region are as follows:

[0150] HCDR1: GYSFTGYT (SEQ ID NO: 3),

[0151] HCDR2: INPYNAGT (SEQ ID NO: 4),

[0152] HCDR3: ARSEYRYGGDYFDY (SEQ ID NO: 5);

[0153] The amino acid sequences of the three CDRs in its light chain variable region are as follows:

[0154] LCDR1: QSLLNSSNQKNY (SEQ ID NO: 8),

[0155] LCDR2: FAS (SEQ ID NO: 9),

[0156] LCDR3: QQHYDTPYT (SEQ ID NO: 10).

[0157] (2) 19F3H2L2

[0158] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 20, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 22.

[0159] The amino acid sequences of the three CDRs in its heavy chain variable region are identical to those of 19F3.

[0160] The amino acid sequences of the three CDRs in its light chain variable region are identical to those of 19F3.

[0161] (3) 19F3H2L3

[0162] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 20, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 24.

[0163] The amino acid sequences of the three CDRs in its heavy chain variable region are identical to those of 19F3.

[0164] The amino acid sequences of the three CDRs in its light chain variable region are identical to those of 19F3.

[0165] (4) 14C12

[0166] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:44, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:49.

[0167] The amino acid sequences of the three CDRs in its heavy chain variable region are as follows:

[0168] HCDR1: GFAFSSYD (SEQ ID NO: 45)

[0169] HCDR2: ISGGGRYT (SEQ ID NO: 46)

[0170] HCDR3: ANRYGEAWFAY (SEQ ID NO: 47)

[0171] The amino acid sequences of the three CDR regions of its light chain variable region are as follows:

[0172] LCDR1: QDINTY (SEQ ID NO: 50)

[0173] LCDR2: RAN (SEQ ID NO: 51)

[0174] LCDR3: LQYDEFPLT (SEQ ID NO: 52)

[0175] (5) 14C12H1L1

[0176] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:62, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:64.

[0177] The amino acid sequence of the three CDRs in its heavy chain variable region is identical to that of 14C12.

[0178] The amino acid sequences of the three CDRs in its light chain variable region are identical to those of 14C12.

[0179] (6) NTPDV1, NTPDV2, NTPDV3, NTPDV4 The amino acid sequences of the 9 CDRs contained in the heavy chain are consistent with the CDR amino acid sequences of the 13F9 heavy chain, 14C12 heavy chain and 14C12 light chain regions in the order from N-terminal to C-terminal, and the sequences are arranged as follows:

[0180] HCDR1: GYSFTGYT (SEQ ID NO: 3)

[0181] HCDR2: INPYNAGT (SEQ ID NO: 4)

[0182] HCDR3: ARSEYRYGGDYFDY (SEQ ID NO: 5)

[0183] HCDR4: GFAFSSYD (SEQ ID NO: 45)

[0184] HCDR5: ISGGGRYT (SEQ ID NO: 46)

[0185] HCDR6: ANRYGEAWFAY (SEQ ID NO: 47)

[0186] HCDR7: QDINTY (SEQ ID NO: 50)

[0187] HCDR8: RAN (SEQ ID NO: 51)

[0188] HCDR9: LQYDEFPLT (SEQ ID NO: 52)

[0189] The amino acid sequences of the three CDRs of its light chain are consistent with the amino acid sequences of the three CDRs of the 19F3 light chain, and the sequences are as follows:

[0190] LCDR1: QSLLNSSNQKNY (SEQ ID NO: 8)

[0191] LCDR2:FAS (SEQ ID NO: 9)

[0192] LCDR3: QQHYDTPYT (SEQ ID NO: 10).

[0193] Another aspect of the present invention relates to a hybridoma cell line LT014, which is deposited in China Center for Type Culture Collection (CCTCC) with a deposit number of CCTCC NO: C2018137.

[0194] Another aspect of the present invention relates to a hybridoma cell line LT003, which is deposited in China Center for Type Culture Collection (CCTCC) with a deposit number of CCTCC NO: C2015105.

[0195] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the cell culture, molecular genetics, nucleic acid chemistry, and immunology laboratory operation steps used herein are conventional steps widely used in the corresponding fields. At the same time, in order to better understand the present invention, the definitions and explanations of the relevant terms are provided below.

[0196] As used herein, the term EC 50 It refers to the concentration for 50% of maximal effect, which is the concentration that can cause 50% of the maximum effect.

[0197] As used herein, the term "antibody" refers to an immunoglobulin molecule that is usually composed of two pairs of polypeptide chains, each pair having a "light" (L) chain and a "heavy" (H) chain. Antibody light chains can be classified as κ and λ light chains. Heavy chains can be classified as μ, δ, γ, α or ε, and the isotype of the antibody is defined as IgM, IgD, IgG, IgA and IgE, respectively. Within the light and heavy chains, the variable and constant regions are connected by a "J" region of about 12 or more amino acids, and the heavy chain also contains a "D" region of about 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains (CH1, CH2 and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain CL. The constant region of an antibody can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The VH and VL regions can also be subdivided into regions with high variability, called complementary determining regions (CDRs), interspersed with more conservative regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions (VH and VL) of each heavy chain / light chain pair form the antibody binding site, respectively. The assignment of amino acids to regions or domains follows 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, see Ehrenmann F, Kaas Q, Lefranc MP. 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.

[0198] In particular, the heavy chain may also contain more than 3 CDRs, such as 6, 9, or 12. For example, in the bispecific antibody of the present invention, the heavy chain may be an IgG antibody with a ScFv connected to the C-terminus of the heavy chain, in which case the heavy chain contains 9 CDRs.

[0199] The term "antibody" is not limited to any particular method of producing the antibody. For example, it includes recombinant antibodies, monoclonal antibodies and polyclonal antibodies. The antibody can be an antibody of different isotypes, for example, IgG (e.g., IgG1, IgG2, IgG3 or IgG4 subtype), IgA1, IgA2, IgD, IgE or IgM antibody.

[0200] As used herein, the terms "monoclonal antibody" and "monoclonal antibody" refer to an antibody or an antibody fragment from a group of highly homologous antibody molecules, that is, a group of identical antibody molecules except for natural mutations that may occur spontaneously. Monoclonal antibodies have high specificity for a single epitope on an antigen. Polyclonal antibodies are relative to monoclonal antibodies and usually contain at least two or more different antibodies, which usually recognize different epitopes on an antigen. Monoclonal antibodies can usually be obtained using the hybridoma technique first reported by Kohler et al. ( G, Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity [J]. Nature, 1975; 256 (5517): 495), but it can also be obtained by recombinant DNA technology (see, for example, US Patent 4, 816,567).

[0201] As used herein, the term "humanized antibody" refers to an antibody or antibody fragment obtained by replacing all or part of the CDR region of a human immunoglobulin (recipient antibody) with the CDR region of a non-human antibody (donor antibody), wherein the donor antibody may be a non-human (e.g., mouse, rat or rabbit) antibody with the desired specificity, affinity or reactivity. In addition, some amino acid residues in the framework region (FR) of the receptor antibody may also be replaced by amino acid residues of the corresponding non-human antibody, or by amino acid residues of other antibodies, to further improve or optimize the performance of the antibody. For more details on humanized antibodies, see, for example, Jones et al., Nature 1986; 321: 522-525; Reichmann et al., Nature, 1988; 332: 323-329; Presta, Curr. Op. Struct. Biol. 1992; 2: 593-596; and Clark, Immunol. Today 2000; 21: 397-402. In some cases, the antigen-binding fragment of an antibody is a diabodies, in which V H and V L The domains are expressed on a single polypeptide chain, but a linker is used that is too short to allow pairing between the two domains on the same chain, thereby forcing the domains to pair with the complementary domains of another chain and create two antigen binding sites (see, e.g., Holliger P. et al., Proc. Natl. Acad. Sci. USA 1993; 90: 6444-6448 and Poljak RJ et al., Structure 1994; 2: 1121-1123).

[0202] As used herein, the term "single chain antibody (ScFv)" refers to an antibody heavy chain variable region (V H ) and antibody light chain variable region (V L ) numerator. L and V H The domains are paired to form monovalent molecules via linkers that enable them to be generated as a single polypeptide chain (see, e.g., Birdet al, Science 1988; 242: 423-426 and Huston et al, Proc. Natl. Acad. Sci. USA 1988; 85: 5879-5883). Such scFv molecules may have the general structure: NH2-V L -Connection fragment-V H -COOH or NH2-V H-Connection fragment-V L -COOH. Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof. For example, linkers 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 useful 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.

[0203] As used herein, the term "isolated" or "isolated" refers to that obtained from the natural state by artificial means. If a certain "isolated" substance or component appears in nature, it may be that the natural environment in which it is located has changed, or the substance has been separated from the natural environment, or both. For example, a certain unisolated polynucleotide or polypeptide naturally exists in a living animal, and the same polynucleotide or polypeptide with high purity separated from this natural state is called isolated. The term "isolated" or "isolated" does not exclude the presence of artificial or synthetic substances, nor does it exclude the presence of other impure substances that do not affect the activity of the substance.

[0204] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector can express the protein encoded by the inserted polynucleotide, the vector is called an expression vector. The vector can be introduced into a host cell by transformation, transduction or transfection, so that the genetic material elements it carries are 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 (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC); bacteriophages such as lambda phage or M13 phage and animal viruses, etc. Animal viruses that can be used as vectors include but are not limited to retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomaviruses (such as SV40). A vector can contain a variety of elements that control expression, including but not limited to promoter sequences, transcription initiation sequences, enhancer sequences, selection elements and reporter genes. In addition, the vector may also contain a replication initiation site.

[0205] As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including but not limited to prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, GS cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.

[0206] As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as a reaction between an antibody and an antigen to which it is directed. In certain embodiments, an antibody that specifically binds to an antigen (or has specificity for an antigen) means that the antibody binds to the antigen with a specificity of less than about 10 -5 M, for example, less than about 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 M or less affinity (K D ) binds to the antigen.

[0207] As used herein, the term “K D " refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, which is used to describe the binding affinity between the antibody and the antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding and the higher the affinity between the antibody and the antigen. Generally, antibodies bind with a constant of less than about 10 -5 M, for example, less than about 10 -6M, 10 -7 M, 10 -8 M, 10 - 9 M or 10 -10 M or less dissociation equilibrium constant (K D ) binds to an antigen (e.g., PD-1 protein). K can be determined using methods known to those skilled in the art. D , for example, using the Fortebio molecular interaction instrument.

[0208] As used herein, the terms "monoclonal antibody" and "monoclonal antibody" have the same meaning and are used interchangeably; the terms "polyclonal antibody" and "polyclonal antibody" have the same meaning and are used interchangeably. And in the present invention, amino acids are generally represented by single-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.

[0209] 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 a subject and an active ingredient, which is well known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to, pH adjusters, surfactants, adjuvants, and ionic strength enhancers. For example, pH adjusters include, but are not limited to, phosphate buffers; surfactants include, but are not limited to, cationic, anionic or nonionic surfactants, such as Tween-80; ionic strength enhancers include, but are not limited to, sodium chloride.

[0210] As used herein, the term "effective amount" refers to an amount sufficient to obtain or at least partially obtain the desired effect. For example, an effective amount for preventing a disease (e.g., a tumor) refers to an amount sufficient to prevent, stop, or delay the occurrence of a disease (e.g., a tumor); an effective amount for treating a disease refers to an amount sufficient to cure or at least partially stop the disease and its complications in a patient who already has the disease. Determining such an effective amount is well within the capabilities of those skilled in the art. For example, an effective amount for therapeutic use will depend on the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general condition such as age, weight and sex, the mode of administration of the drug, and other treatments administered simultaneously, etc.

[0211] Advantageous Effects of the Invention

[0212] The monoclonal antibody of the present invention (e.g., 13F9H2L3) can bind specifically to CD73 very well, and can very effectively inhibit the enzymatic activity reaction of CD73 in a non-substrate competitive manner, reduce the production of adenosine, and promote T cell activity and tumor inhibition effects.

[0213] The bispecific antibodies involved in the present invention, such as NTPDV1, NTPDV2, NTPDV3, and NTPDV4, can bind specifically to PD-1 and CD73 very well, and can effectively block the binding of PD-1 and PDL1, specifically relieve the immunosuppression of PD-1 on the body, inhibit the catalytic activity of CD73, relieve the inhibition of adenosine on immune cells, activate T lymphocytes, and do not cause the release of cytokines IL-8 and IL-6, effectively increasing safety and effectiveness.

[0214] The bifunctional antibody of the present invention has the potential to be used in the preparation of anti-tumor drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0215] Figure 1 .ELISA detected the binding of P1D7V01, P1D7V02R, P1D7V03, P1D7V04R, 14C12H1L1, Nivolumab and PD-1-mFc.

[0216] Figure 2 .ELISA detected the binding of P1D7V01, P1D7V02R, P1D7V03, P1D7V04R, 19F3H2L3, MEDI9447 and human NT5E-Biotin.

[0217] Figure 3 .Activity detection results of P1D7V01, P1D7V02R, P1D7V03, P1D7V04R, 14C12H1L1 and Nivolumab competing with human PD-L1-mFc for binding to human PD-1-mFc-Biotin.

[0218] Figure 4 .Determination of affinity constant between P1D7V01 and PD-1-mFc.

[0219] Figure 5 .14C12H1L1 and PD-1-mFc affinity constant determination

[0220] Figure 6 .Determination of affinity constant between Nivolumab and PD-1-mFc.

[0221] Figure 7 .Determination of affinity constant between P1D7V01 and human NT5E(1-552)-his.

[0222] Figure 8 .Determination of affinity constant between MEDI 9447 and human NT5E(1-552)-his.

[0223] Fig. 9 .FACS was used to detect the binding activity of P1D7V01, P1D7V02R, P1D7V03, P1D7V04R and 14C12H1L1 to PD-1 on the surface of 293T-PD1 cells.

[0224] Fig.10 .FACS detection of the binding activity of P1D7V01, P1D7V03, MEDI9447 and 19F3H2L3 to CD73 on the surface of MDA-MB-231 cells

[0225] Fig.11 .Inhibitory effect of anti-CD73-anti-PD-1 bispecific antibody on CD73 enzyme activity on the membrane surface of MDA-MB-231.

[0226] Fig.12 .Inhibitory effect of anti-CD73-anti-PD-1 bispecific antibody on CD73 enzyme activity on the U87-MG membrane surface.

[0227] Fig.13 .Bioactivity detection of anti-CD73-anti-PD-1 bispecific antibody promoting IFN-γ secretion in Raji-PDL1 mixed lymphoid reaction system.

[0228] Fig.14 .Bioactivity detection of anti-CD73-anti-PD-1 bispecific antibody promoting IL-2 secretion in Raji-PDL1 mixed lympha reaction system.

[0229] Fig.15 .Bioactivity detection of anti-CD73-anti-PD-1 bispecific antibody-induced IFN-γ secretion in DC mixed lymphoproliferative reaction system.

[0230] Fig.16 .Bioactivity detection of anti-CD73-anti-PD-1 bispecific antibody promoting IL-2 secretion in DC mixed lymphoid reaction system.

[0231] Fig.17 .Determination of affinity constant between 14C12H1L1 (hG1TM) and PD-1-mFc.

[0232] Fig.18 .Determination of affinity constant between Nivolumab and PD-1-mFc

[0233] Fig.19 .Determination of affinity constant between NTPDV1 and PD-1-mFc.

[0234] Fig. 20 .Determination of affinity constant between NTPDV2 and PD-1-mFc.

[0235] Fig.21 .Determination of affinity constant between NTPDV3 and PD-1-mFc.

[0236] Fig. 22 .Determination of affinity constant between NTPDV4 and PD-1-mFc.

[0237] Fig.23 .Determination of affinity constant between 19F3H2L3 (hG1M) and human NT5E (1-552)-his.

[0238] Fig.24 .Determination of affinity constant between NTPDV1 and human NT5E(1-552)-his.

[0239] Fig.25 .Determination of affinity constant between NTPDV2 and human NT5E(1-552)-his.

[0240] Fig.26 .Determination of affinity constant between NTPDV3 and human NT5E(1-552)-his.

[0241] Fig. 27 .Determination of affinity constant between NTPDV4 and human NT5E(1-552)-his.

[0242] Fig.28 .Detection of the inhibition of CD73 enzyme activity on the membrane surface of U87-MG cells by anti-CD73-anti-PD-1 bispecific antibody.

[0243] Fig.29 .Mixed lymphocyte reaction (MLR) was used to detect the biological activity of anti-CD73-anti-PD-1 bispecific antibody in promoting IFN-γ and IL-2 secretion.

[0244] Fig.30 .Effects of isotype control, 19F3H2L3 (hG1M), and different doses of NTPDV2 on mouse tumor volume.

[0245] Fig.31 .Effects of isotype control, 19F3H2L3 (hG1M), and different doses of NTPDV2 on mouse body weight.

[0246] Fig.32 .In the co-culture system of CHO-K1-PD1 cells and human macrophages, Fc segment amino acid mutations effectively eliminated the secretion of IL-8 by human macrophages mediated by PD-1 / CD73 bispecific antibodies.

[0247] Fig.33 .In the co-culture system of CHO-K1-PD1 cells and human macrophages, Fc segment amino acid mutations effectively eliminated the secretion of IL-6 by human macrophages mediated by PD-1 / CD73 bispecific antibodies.

[0248] Fig.34 .In the co-culture system of U87-MG cells and human macrophages, Fc segment amino acid mutation effectively eliminated the secretion of IL-8 by human macrophages mediated by PD-1 / CD73 bispecific antibodies.

[0249] Fig.35 .In the co-culture system of U87-MG cells and human macrophages, Fc segment amino acid mutation effectively eliminated the secretion of IL-6 by human macrophages mediated by PD-1 / CD73 bispecific antibodies.

[0250] Biomaterial Retention Information:

[0251] The hybridoma cell line LT003 (also known as PD-1-14C12) was deposited in the China Center for Type Culture Collection (CCTCC) on June 16, 2015, with a deposit number of CCTCC NO: C2015105. The deposit address is Wuhan University, Wuhan, China, Postal Code: 430072.

[0252] The hybridoma cell line LT014 (also known as CD73-19F3) was deposited in the China Center for Type Culture Collection (CCTCC) on June 21, 2018, with the deposit number CCTCC NO: C2018137, and the deposit address is Wuhan University, Wuhan, China, Postal Code: 430072. DETAILED DESCRIPTION

[0253] The embodiments of the present invention will be described in detail below in conjunction with the examples. Those skilled in the art will appreciate that the following examples are only used to illustrate the present invention and should not be considered to limit the scope of the present invention. If the specific techniques or conditions are not specified in the examples, the techniques or conditions described in the literature in the art (for example, reference to "Molecular Cloning Experiment Guide" by J. Sambrook, Huang Peitang, etc., the third edition, Science Press) or according to the product instructions are carried out. If the manufacturer is not specified for the reagents or instruments used, they are conventional products that can be purchased on the market. For example, MDA-MB-231 cells and U87-MG cells can be purchased from ATCC.

[0254] In the following examples of the present invention, the BALB / c mice used were purchased from Guangdong Medical Experimental Animal Center.

[0255] In the following embodiments of the present invention, the positive control antibody MEDI9447 (generic name: Oleclumab) used is produced by Zhongshan Kangfang Biopharmaceutical Co., Ltd., and its sequence is the same as the antibody SEQ ID NOs: 21-24 described in the Medlmmune Limited patent publication number: US20160129108A1.

[0256] In the following embodiments of the present invention, Nivolumab, a drug antibody with the same target and marketed under the trade name Opdivo, was purchased from Bristol-Myers Squibb.

[0257] In the following examples of the present invention, the 293T-PD1 cell line used was constructed by Zhongshan Kangfang Biopharmaceutical Co., Ltd. The 293T-PD1 cell line was prepared by virus infection of HEK293T cells, and the virus preparation used was 3rd Generation Lentiviral Systems, see, for example, A Third Generation Lentivirus Vector with a Conditional Packaging System. Dull T, Zufferey R, Kelly M, Mandel RJ, Nguyen M, Trono D, and Naldini LJ Virol. 1998. 72 (11): 8463-8471. The lentiviral expression vector used was pCDH-CMV-PD-1FL-Puro (PD1, Genebank ID: NM_005018; vector pCDH-CMV-Puro, purchased from Youbao Bio, product number: VT1480).

[0258] In the following examples of the present invention, the Raji-PDL1 cell line used was constructed by Zhongshan Kangfang Biopharmaceutical Co., Ltd. The Raji-PDL1 cell line was prepared by virus infection of Raji cells, and the virus preparation used was 3rd Generation Lentiviral Systems, see, for example, A Third Generation Lentivirus Vector with a Conditional Packaging System. Dull T, Zufferey R, Kelly M, Mandel RJ, Nguyen M, Trono D, and Naldini L. J Virol. 1998. 72 (11): 8463-8471. The lentiviral expression vector used was plenti6.3-PDL1 (PDL1, Genebank ID: NP 054862.1; vector plenti6.3, purchased from Invitrogen, catalog number: K5315-20).

[0259] In the following examples of the present invention, the CHO-K1-PD1 cell line used was constructed by Zhongshan Kangfang Biopharmaceutical Co., Ltd. The CHO-K1-PD1 cell line was prepared by virus infection of CHO-K1 cells, and the virus preparation used was 3rd Generation Lentiviral Systems, see, for example, 3rd Generation Lentivirus Vector with a Conditional Packaging System. Dull T, Zufferey R, Kelly M, Mandel RJ, Nguyen M, Trono D, and Naldini L. J Virol. 1998. 72 (11): 8463-8471. The lentiviral expression vector used was pCDH-CMV-PD-1FL-Puro (PD1, Genebank ID: NM 005018; vector pCDH-CMV-Puro, purchased from Youbao Bio, product number: VT1480).

[0260] In the experimental example, Nivolumab (trade name Opdivo), an IgG4 subtype anti-PD-1 antibody carrying the S228P mutation, was used as a control antibody, both of which were purchased from Bristol-Myers Squibb.

[0261] In the following experimental examples of the present invention, the isotype control antibodies used, i.e., hIgG1, are antibodies targeting human anti-hen egg lysosomes (HEL), and the variable region sequences of the antibodies are from 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.) published by Acierno et al. The constant region fragment of hIgG1 uses Ig gamma-1chain C region, ACCESSION: P01857 as the heavy chain constant region, and Ig kappa chain C region, ACCESSION: P01834 as the light chain constant region; hIgG1 was prepared in the laboratory of Zhongshan Kangfang Biopharmaceutical Co., Ltd.

[0262] Example 1: Preparation of anti-CD73 antibody 19F3

[0263] 1. Preparation of hybridoma cell line LT014

[0264] The antigen used to prepare anti-CD73 antibodies is human NT5E-his (NT5E is Genbank ID: NP_002517.1, position: 1-552). The spleen cells of immunized mice were fused with mouse myeloma cells to make hybridoma cells. Human NT5E (NT5E is Genbank ID: NP_002517.1, position: 1-552)-Biotin was used as an antigen, and the hybridoma cells were screened by indirect ELISA to obtain hybridoma cells that can secrete antibodies that specifically bind to CD73. The hybridoma cells obtained by ELISA screening were subjected to a limiting dilution method to obtain a stable hybridoma cell line. The above hybridoma cell line was named hybridoma cell line LT014, and the monoclonal antibody secreted by it was named 19F3.

[0265] The hybridoma cell line LT014 (also known as CD73-19F3) was deposited in the China Center for Type Culture Collection (CCTCC) on June 21, 2018, with the deposit number CCTCC NO: C2018137, and the deposit address is Wuhan University, Wuhan, China, Postal Code: 430072.

[0266] 2. Preparation of anti-CD73 antibody 19F3

[0267] The LT014 cell line prepared above was cultured in CD medium (Chemical Defined Medium) (CD medium containing 1% penicillin-streptomycin, cultured in a 5% CO2, 37°C cell culture incubator). After 7 days, the cell culture supernatant was collected and purified by high-speed centrifugation, microporous membrane vacuum filtration and HiTrap protein A HP column to obtain antibody 19F3.

[0268] Example 2: Sequence analysis of anti-CD73 antibody 19F3

[0269] According to the method of the Cultured Cell Bacterial RNA Extraction Kit (Tiangen, Catalog No. DP430), mRNA was extracted from the LT014 cell line cultured in Example 1.

[0270] According to Invitrogen III First-Strand Synthesis System for RT-PCR Kit Instructions Synthesize cDNA and perform PCR amplification.

[0271] The PCR amplification product was directly subjected to TA cloning. For specific operations, refer to the instructions of the pEASY-T1 Cloning Kit (TransgenCT101).

[0272] The products of TA cloning were directly sequenced, and the sequencing results were as follows:

[0273] The nucleic acid sequence (363 bp) of the 19F3 heavy chain variable region is shown in SEQ ID NO: 1, and the amino acid sequence (121 aa) encoded by it is shown in SEQ ID NO: 2.

[0274] According to the IMGT numbering system, the sequence of the heavy chain CDR1 is shown in SEQ ID NO: 3, the sequence of the heavy chain CDR2 is shown in SEQ ID NO: 4, and the sequence of the heavy chain CDR3 is shown in SEQ ID NO: 5.

[0275] The nucleic acid sequence (339 bp) of the 19F3 light chain variable region is shown in SEQ ID NO:6, and the amino acid sequence (113 aa) encoded by it is shown in SEQ ID NO:7.

[0276] According to the IMGT numbering system, the sequence of the light chain CDR1 is shown in SEQ ID NO: 8, the sequence of the light chain CDR2 is shown in SEQ ID NO: 9, and the sequence of the light chain CDR3 is shown in SEQ ID NO: 10.

[0277] The amino acid sequences of the four framework regions (FR-H1 to FR-H4) of the 19F3 heavy chain are shown in SEQ ID NO: 11 to SEQ ID NO: 14, respectively; the amino acid sequences of the four framework regions (FR-H1 to FR-H4) of the 19F3 light chain are shown in SEQ ID NO: 15 to SEQ ID NO: 18, respectively.

[0278] Example 3: Design, preparation and detection of humanized antibodies against human CD73

[0279] 1. Design of light and heavy chain sequences of humanized antibodies 19F3H2L3 and 19F3H2L2

[0280] According to the three-dimensional crystal structure of human CD73 protein (Hage T, Reinemer P, Sebald W. Crystals of a1:1 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 the mouse antibody 19F3 obtained in Example 2, the antibody model was simulated by computer and mutations were designed according to the model to obtain the variable region sequences of antibodies 19F3H1L1, 19F3H2L3 and 19F3H2L3, whose corresponding heavy chain variable region sequences were 19F3H1 and 19F3H2 (amino acid sequences were shown in SEQ ID NO: 93 and SEQ ID NO: 97, respectively), and the light chain variable region sequences were 19F3L1, 19F3L2, 19F3L3 (amino acid sequences were shown in SEQ ID NO: 95, SEQ ID NO: 98, SEQ ID NO: 99, respectively). NO: 99), antibody constant region sequence, from NCBI database, heavy chain constant region uses Ig gamma-1chain C region, ACCESSION: P01857; light chain constant region uses Ig kappa chain C region, ACCESSION: P01834. Among them, 19F3H2L3 is also written as 19F3H2L3 (hG1WT) in the Chinese invention patent with application number 202110270671.X, and the light and heavy chain variable regions of 19F3H1L1, 19F3H2L2, and 19F3H2L3 can be recorded as 19F3H1V (or 19F3H1 V ), 19F3H2V (or 19F3H2 V ), 19F3L1V (or 19F3L1V ), 19F3L2V (or 19F3L2 V ), 19F3L3V (or 19F3L3 V ).

[0281] (1) Heavy chain variable region and light chain variable region sequences of humanized monoclonal antibody 19F3H1L1

[0282] The nucleic acid sequence (363 bp) of the heavy chain variable region is shown in SEQ ID NO:92, and the amino acid sequence (121 aa) encoded by it is shown in SEQ ID NO:93.

[0283] The nucleic acid sequence (339 bp) of the light chain variable region is shown in SEQ ID NO:94, and the amino acid sequence (113 aa) encoded by it is shown in SEQ ID NO:95.

[0284] (2) The sequences of the heavy chain variable region and light chain variable region of the humanized monoclonal antibody 19F3H2L2 are as follows:

[0285] The nucleic acid sequence (363 bp) of the heavy chain variable region 19F3H2 is shown in SEQ ID NO: 19, and the amino acid sequence (121 aa) encoded by it is shown in SEQ ID NO: 20.

[0286] The nucleic acid sequence (339 bp) of the light chain variable region 19F3L3 is shown in SEQ ID NO: 21, and the amino acid sequence (113 aa) encoded by it is shown in SEQ ID NO: 22.

[0287] (3) The heavy chain variable region and light chain variable region sequences of the humanized monoclonal antibody 19F3H2L3 are as follows:

[0288] The nucleic acid sequence (363 bp) of the heavy chain variable region 19F3H2 is shown in SEQ ID NO: 19, and the amino acid sequence (121 aa) encoded by it is shown in SEQ ID NO: 20.

[0289] The nucleic acid sequence (339 bp) of the light chain variable region 19F3L3 is shown in SEQ ID NO: 23, and the amino acid sequence (113 aa) encoded by it is shown in SEQ ID NO: 24.

[0290] 2. Preparation of humanized antibodies 19F3H1L1, 19F3H2L2 and 19F3H2L3

[0291] The heavy chain constant region used Ig gamma-1 chain C region, ACCESSION: P01857; the light chain constant region used Ig kappa chain C region, ACCESSION: P01834.

[0292] The heavy chain cDNA and light chain cDNA of 19F3H1L1, 19F3H2L2 and 19F3H2L3 were cloned into pUC57simple (provided by GenScript) vector to obtain pUC57simple-19F3H1, pUC57simple-19F3L1, pUC57simple-19F3H2, pUC57simple-19F3L2 and pUC57simple-19F3L3, respectively. Referring to the standard techniques introduced in the Molecular Cloning Experiment Guide (Second Edition), the full-length heavy and light chain genes synthesized by EcoRI&HindIII digestion were subcloned into the expression vector pcDNA3.1 by restriction enzyme digestion (EcoRI&HindIII) to obtain expression plasmids pcDNA3.1-19F3H1, pcDNA3.1-19F3L1, pcDNA3.1-19F3H2, pcDNA3.1-19F3L2, and pcDNA3.1-19F3L3, and the heavy and light chain genes of the recombinant expression plasmids were further sequenced and analyzed. Subsequently, the corresponding light and heavy chain recombinant plasmid design gene combinations (pcDNA3.1-19F3H1 / pcDNA3.1-19F3L1, pcDNA3.1-19F3H2 / pcDNA3.1-19F3L2, pcDNA3.1-19F3H2 / pcDNA3.1-19F3L3) were co-transfected into 293F cells and the culture fluid was collected for purification. After sequencing verification, the endotoxin-free expression plasmid was prepared and the plasmid was transiently transfected into HEK293 cells for antibody expression. After 7 days of culture, the cell culture fluid was collected and affinity purified using a Protein A column to obtain humanized antibodies.

[0293] 3. Design of light and heavy chain sequences of humanized antibodies 19F3H2L3 (hG1M) and 19F3H2L3 (hG1TM)

[0294] Based on the 19F3H2L3 obtained in Example 3, the inventors introduced a point mutation from leucine to alanine (L234A) at position 234 of the heavy chain and a point mutation from leucine to alanine (L235A) at position 235 to obtain 19F3H2L3 (hG1M). The nucleotide and amino acid sequences of the heavy chain of 19F3H2L3 (hG1M) are shown in SEQ ID NO: 25 and SEQ ID NO: 26, respectively; the light chain constant region of 19F3H2L3 (hG1M) is Ig kappa chain C region, ACCESSION: P01834, and the nucleotide and amino acid sequences of the light chain of 19F3H2L3 (hG1M) are shown in SEQ ID NO: 27 and SEQ ID NO: 28, respectively.

[0295] Based on 19F3H2L3 obtained in Example 3, the inventors introduced a point mutation from leucine to alanine (L234A) at position 234 of the heavy chain, a point mutation from leucine to alanine (L235A) at position 235, and a point mutation from glycine to alanine (G237A) at position 237, thereby obtaining 19F3H2L3 (hG1TM), whose heavy chain amino acid sequences are shown in SEQ ID NO: 29 and SEQ ID NO: 30, respectively; its light chain is consistent with 19F3H2L3 (hG1M).

[0296] The amino acid sequences of the four framework regions (FR-H1 to FR-H4) of 19F3H2 are shown in SEQ ID NO: 31 to SEQ ID NO: 34, respectively;

[0297] The amino acid sequences of the four framework regions (FR-H1 to FR-H4) of the 19F3L2 light chain are shown in SEQ ID NO: 35 to SEQ ID NO: 38, respectively;

[0298] The amino acid sequences of the four framework regions (FR-H1 to FR-H4) of the 19F3L3 light chain are shown in SEQ ID NO: 39 to SEQ ID NO: 42, respectively.

[0299] 4. Preparation of humanized antibody 19F3H2L3 (hG1M)

[0300] The heavy chain cDNA and light chain cDNA of 19F3H2L3 (hG1M) were cloned into pUC57simple (provided by GenScript) vector to obtain pUC57simple-19F3H2 (hG1M) and pUC57simple-19F3L3, respectively. According to the standard technique introduced in the Molecular Cloning Experiment Guide (Second Edition), the heavy and light chain full-length genes synthesized by EcoRI & HindIII were digested and subcloned into the expression vector pcDNA3.1 by restriction enzymes (EcoRI & HindIII) to obtain expression plasmids pcDNA3.1-19F3H2 (hG1M) and pcDNA3.1-19F3L3, and the heavy and light chain genes of the recombinant expression plasmids were further sequenced and analyzed. Subsequently, the corresponding light and heavy chain recombinant plasmid design gene combination pcDNA3.1-19F3H2 (hG1M) / pcDNA3.1-19F3L3 was co-transfected into 293F cells and the culture medium was collected for purification. After sequencing verification, the endotoxin-free expression plasmid was prepared and the plasmid was transiently transfected into HEK293 cells for antibody expression. After 7 days of culture, the cell culture medium was collected and affinity purified using a Protein A column to obtain the humanized antibody 19F3H2L3 (hG1M).

[0301] Example 4: Preparation of anti-PD-1 antibody 14C12

[0302] 1. Preparation of hybridoma cell line LT003

[0303] Using PD-1-mFc fusion protein (PD-1 GenBank: NM_005018, mFc SEQ ID NO: 89) as an antigen, spleen cells from immunized BALB / c mice (purchased from Guangdong Medical Experimental Animal Center) were fused with mouse myeloma cells to form hybridoma cells, referring to currently established methods (e.g., Stewart, SJ, "Monoclonal Antibody Production", in Basic Methods in antibody Production and Characterization, Eds. GC Howard and DRBethell, Boca Raton: CRC Press, 2000).

[0304] PD-1-hFc (PD-1 Genbank ID: NM_005018, hFc is a human IgG Fc purification tag, specifically Iggamma-1 chain C region, Genbank ID: P01857 position 114-330) was used as an antigen to coat an ELISA plate and screened by indirect ELISA to obtain hybridoma cells that secrete new antibodies that specifically bind to PD-1.

[0305] A hybridoma cell line that can secrete a monoclonal antibody that competes with the ligand PD-L1-hFc (PD-L1 Genbank ID: NP_054862.1) for binding to PD-1 was screened by competitive ELISA, and a stable hybridoma cell line was obtained by limiting dilution method. The LT003 stable cell line (PD-1-14C12) was obtained by limiting dilution method, and the monoclonal antibody secreted by it was named 14C12.

[0306] The hybridoma cell line LT003 (also known as PD-1-14C12) was deposited in the China Center for Type Culture Collection (CCTCC) on June 16, 2015, with a deposit number of CCTCC NO: C2015105. The deposit address is Wuhan University, Wuhan, China, Postal Code: 430072.

[0307] 2. Preparation of Anti-PD-1 Antibody 14C12

[0308] The LT003 cell line prepared above was cultured in IMDM medium containing 10% low IgG fetal bovine serum (IMDM medium, containing 1% penicillin-streptomycin, cultured in a 5% CO2, 37°C cell culture incubator). After 7 days, the cell culture supernatant was collected and purified to obtain antibody 14C12.

[0309] Example 5: Sequence analysis of anti-PD-1 antibody 14C12

[0310] According to the method of the total RNA extraction kit for cultured cells and bacteria (Tiangen, catalog number DP430), mRNA was extracted from the hybridoma cell line LT003 prepared in Example 1.

[0311] According to Invitrogen III First-Strand Synthesis System for RT-PCR Kit Instructions Synthesize cDNA and perform PCR amplification.

[0312] The PCR amplification product was directly subjected to TA cloning. For specific operations, refer to the instructions of the pEASY-T1 Cloning Kit (TransgenCT101).

[0313] The products of TA cloning were directly sequenced, and the sequencing results were as follows:

[0314] The nucleic acid sequence (354 bp) of the heavy chain variable region is shown in SEQ ID NO:43, and the amino acid sequence (118 aa) encoded by it is shown in SEQ ID NO:44.

[0315] According to the IMGT numbering system, the sequence of the heavy chain CDR1 is shown in SEQ ID NO: 45, the sequence of the heavy chain CDR2 is shown in SEQ ID NO: 46, and the sequence of the heavy chain CDR3 is shown in SEQ ID NO: 47.

[0316] The nucleic acid sequence (321 bp) of the light chain variable region is shown in SEQ ID NO:48, and the amino acid sequence (107aa) encoded by it is shown in SEQ ID NO:49.

[0317] According to the IMGT numbering system, the sequence of the light chain CDR1 is shown in SEQ ID NO: 50, the sequence of the light chain CDR2 is shown in SEQ ID NO: 51, and the sequence of the light chain CDR3 is shown in SEQ ID NO: 52.

[0318] The amino acid sequences of the four framework regions (FR-H1 to FR-H4) of the 14C12 heavy chain are shown 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 shown in SEQ ID NO: 57 to SEQ ID NO: 60, respectively.

[0319] Example 6: Design and preparation of anti-PD-1 humanized antibody 14C12H1L1 and 14C12H1L1 (hG1TM)

[0320] 1. Design of humanized anti-PD-1 antibody 14C12H1L1

[0321] The light chain and heavy chain sequences of the humanized antibody 14C12H1 L1 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) and the sequence of the antibody 14C12 obtained in Example 5. The antibody model was simulated by computer and mutations were designed according to the model to obtain the variable region sequence of the antibody 14C12H1L1.

[0322] The designed variable region sequences are as follows:

[0323] The nucleic acid sequence (354 bp) of the heavy chain variable region 14C12H1 of the humanized monoclonal antibody 14C12H1L1 is shown in SEQ ID NO: 61, and the amino acid sequence (118 aa) encoded by it is shown in SEQ ID NO: 62.

[0324] The nucleic acid sequence (321 bp) of the light chain variable region 14C12L1 of the humanized monoclonal antibody 14C12H1L1 is shown in SEQ ID NO: 63, and the amino acid sequence (107 aa) encoded by it is shown in SEQ ID NO: 64.

[0325] The antibody constant region sequence of antibody 14C12H1L1 is from the NCBI database, the heavy chain constant region adopts Ig gamma-1 chain C region, ACCESSION: P01857; the light chain constant region is Ig kappa chain C region, ACCESSION: P01834), the nucleotide sequence and amino acid sequence of the heavy chain of 14C12H1L1 are shown in SEQ ID NOs: 65 and 66, respectively, and the nucleotide sequence and amino acid sequence of the light chain of 14C12H1L1 are shown in SEQ ID NOs: 67 and 68, respectively. Among them, 14C12H1L1 is also written as 14C12H1L1(hG1WT) in this article and the Chinese invention patent with application number 202110270671.X, and the heavy chain variable region and light chain variable region of 14C12H1L1 are also written as 14C12H1V (or 14C12H1 V ), and 14C12L1 V (or 14C12L1 V ).

[0326] 2. Design of light and heavy chain sequences of humanized antibody 14C12H1L1 (hG1TM)

[0327] Based on 14C12H1L1 obtained in Example 6, the inventors obtained antibody 14C12H1L1 (hG1TM) by introducing a point mutation from leucine to alanine (L234A) at position 234 of the heavy chain, a point mutation from leucine to alanine (L235A) at position 235, and a point mutation from glycine to alanine (G237A) at position 237. The heavy chain nucleotide and amino acid sequences of 14C12H1L1 (hG1TM) are shown in SEQ ID NO: 69 and SEQ ID NO: 70, respectively; the amino acid sequence of its light chain variable region is consistent with the light chain variable region of antibody 14C12H1L1.

[0328] The amino acid sequences of the four framework regions (FR-H1 to FR-H4) of 14C12H1 are shown in SEQ ID NO: 71 to SEQ ID NO: 74, respectively;

[0329] The amino acid sequences of the four framework regions (FR-H1 to FR-H4) of the 14C12L1 light chain are shown in SEQ ID NO: 75 to SEQ ID NO: 78, respectively;

[0330] 3. Preparation of humanized antibodies 14C12H1L1, 14C12H1L1 (hG1TM)

[0331] The heavy chain cDNA and light chain cDNA of 14C12H1L1(hG1TM) and 14C12H1L1 were cloned into pUC57simple (provided by GenScript) vector to obtain pUC57simple-14C12H1, pUC57simple-14C12L1 and pUC57simple-14C12H1(hG1TM), respectively. According to the standard technique introduced in Molecular Cloning Experiment Guide (Second Edition), the heavy and light chain full-length genes synthesized by EcoRI&HindIII digestion were subcloned into the expression vector pcDNA3.1 by restriction enzyme (EcoRI&HindIII) digestion to obtain expression plasmids pcDNA3.1-14C12H1, pcDNA3.1-14C12L1, and pcDNA3.1-14C12H1(hG1TM), and the heavy and light chain genes of the recombinant expression plasmids were further sequenced and analyzed. Subsequently, the corresponding light and heavy chain recombinant plasmid design gene combinations (pcDNA3.1-14C12H1 (hG1TM) / pcDNA3.1-14C12L1, pcDNA3.1-14C12H1 / pcDNA3.1-14C12L1) were co-transfected into 293F cells and the culture fluid was collected for purification. After sequencing verification, the endotoxin-free expression plasmid was prepared and the plasmid was transiently transfected into HEK293 cells for antibody expression. After 7 days of culture, the cell culture fluid was collected and affinity purified using a Protein A column to obtain humanized antibodies.

[0332] Example 7: Sequence design and expression of anti-PD-1 / CD73 bifunctional antibody

[0333] 1. Sequence Design

[0334] The structural model of the bifunctional antibody in the present invention belongs to the Morrison model (IgG-scFv), that is, the C-termini of the two heavy chains of an IgG antibody are connected to the scFv fragment of another antibody, and the main composition design of the heavy chain and light chain is shown in the following Table 1. Table 1 Table 1: Composition design of the heavy chain and light chain of P1D7V01, P1D7V02R, P1D7V03, P1D7V04R, P1D7V07, and P1D7V08

[0335]

[0336]

[0337] In Table 1 above:

[0338] (1) The "V" in the lower right corner refers to the variable region of the corresponding heavy chain or the variable region of the corresponding light chain. If "V" is not marked, the corresponding heavy chain or light chain is the full length including the constant region. The amino acid sequences of these variable regions or full lengths and their encoding nucleic acid sequences are all referred to the corresponding sequences recorded in the above examples.

[0339] (2) The amino acid sequence of Linker1 is (GGGGS)4 ((nucleotide sequence SEQ ID NO: 80, amino acid sequence SEQ ID NO: 79), and the amino acid sequence of Linker2 is (GGGGS)3 ((nucleotide sequence SEQ ID NO: 82, amino acid sequence SEQ ID NO: 81).

[0340] 2. Antibody Expression and Purification

[0341] The heavy chain cDNA sequence and light chain cDNA sequence of P1D7V01 were cloned into pUC57simple (provided by GenScript) vector to obtain pUC57simple-VP101H and pUC57simple-VP101L plasmids, respectively.

[0342] Plasmids pUC57simple-VP101H and pUC57simple VP101L were digested (HindIII & EcoRI), and the heavy and light chains were recovered by electrophoresis and subcloned into pcDNA3.1 vectors, and the recombinant plasmids were extracted and co-transfected into 293F cells. After 7 days of cell culture, the culture medium was centrifuged at high speed, the supernatant was concentrated, and then loaded onto the HiTrap MabSelectSuRe column, and the protein was eluted in one step with Elution Buffer to recover the target sample and replace the solution with PBS.

[0343] Purified antibodies P1D7V02R, P1D7V03, P1D7V04R, P1D7V07, and P1D7V08 were obtained according to the above-mentioned expression and purification methods of P1D7V01.

[0344] Example 8: ELISA method to determine the binding activity of anti-CD73-anti-PD-1 bispecific antibody with antigen

[0345] 1. The ELISA method was used to determine the binding activity of P1D7V01, P1D7V02R, P1D7V03, and P1D7V04R with the antigen PD-1-mFc. The specific method is as follows:

[0346] The ELISA plate coated with PD-1-mFc, 0.5 μg / ml was incubated at 4°C overnight, and then the plate coated with antigen was washed once with PBST, and then the ELISA plate was blocked at 37°C for 2 hours using 1% BSA in PBS solution as a blocking solution. After the ELISA plate was blocked, it was washed 3 times with PBST, and the antibody diluted in gradient with PBST solution was added (the antibody dilution gradient is detailed in Table 2), and the ELISA plate with the antibody to be tested was incubated at 37°C for 30 minutes, and the plate was washed 3 times with PBST after the incubation was completed. After washing the plate, a 1:5000 dilution of HRP-labeled goat anti-human IgG (H+L) (Jackson, catalog number: 109-035-088) secondary antibody working solution was added, and then incubated at 37°C for 30 minutes. After the incubation was completed, the plate was washed 4 times with PBST, and then TMB (Neogen, 308177) was added to develop color in the dark for 5 minutes, and the color development reaction was terminated by adding the stop solution. Immediately place the ELISA plate into the ELISA reader, select 450nm wavelength to read the OD value of each well of the ELISA plate, and use SoftMax Pro 6.2.1 software to analyze and process the data.

[0347] The test results are shown in Table 2 and Figure 1 As shown in the figure, P1D7V01, P1D7V02R, P1D7V03, and P1D7V04R can effectively bind to the antigen PD-1-mFc, and the binding efficiency is dose-dependent. The absorbance intensity of each dose is shown in Table 2. By quantitatively analyzing the absorbance of the bound antibodies, curve simulation calculations were performed to obtain the binding efficiency EC50 of antibodies P1D7V01, P1D7V02R, P1D7V03, P1D7V04R, 14C12H1L1, and Nivolumab (as a control), which were 0.078nM, 0.078nM, 0.075nM and 0.089nM, 0.033nM, and 0.051nM, respectively.

[0348] The above experimental results show that under the same experimental conditions, the activity of P1D7V01, P1D7V02R, P1D7V03, and P1D7V04R in binding to PD-1-mFc is comparable to that of the positive drugs 14C12H1L1 and Nivolumab with the same target, indicating that P1D7V01, P1D7V02R, P1D7V03, and P1D7V04R have the activity of effectively binding to PD-1-mFc.

[0349] Table 2: ELISA detection of the binding of P1D7V01, P1D7V02R, P1D7V03, P1D7V04R, 14C12H1L1, Nivolumab and PD-1-mFc

[0350]

[0351]

[0352] 2. ELISA method was used to determine the binding activity of P1D7V01, P1D7V02R, P1D7V03, P1D7V04R with antigen human NT5E-Biotin

[0353] Coat the ELISA plate with streptavidin, 2 μg / ml, and incubate at 4°C overnight. After incubation, wash the ELISA plate coated with streptavidin once with PBST, and use 1% BSA in PBS as the blocking solution for the ELISA plate, and block the ELISA plate at 37°C for 2 hours. After the ELISA plate is blocked, wash the plate 3 times with PBST. Add the antigen human NT5E-Biotin 0.5 μg / ml and incubate at 37°C for 30 minutes, then wash the plate 3 times with PBST. Add antibodies diluted in gradients with PBST solution to the wells of the ELISA plate (see Table 3 for the antibody dilution gradient), add the ELISA plate with the antibody to be tested and incubate at 37°C for 30 minutes, and wash the plate 3 times with PBST after incubation. After washing, add HRP-labeled goat anti-human IgG (H+L) (Jackson, catalog number: 109-035-088) secondary antibody working solution diluted 1:5000 and incubate at 37°C for 30 minutes. After incubation, wash the plate 4 times with PBST, then add TMB (Neogen, 308177) to color for 5 minutes in the dark, and add stop solution to terminate the color reaction. Immediately put the ELISA plate into the ELISA reader, select 450nm light wavelength to read the OD value of each well of the ELISA plate. Use SoftMax Pro 6.2.1 software to analyze and process the data.

[0354] The test results are shown in Table 3 and Figure 2 As shown in the figure, P1D7V01, P1D7V02R, P1D7V03, and P1D7V04R can effectively bind to the antigen human NT5E-Biotin, and the binding efficiency is dose-dependent (the absorbance intensity of each dose is shown in Table 3). By quantitatively analyzing the absorbance of the bound antibodies, curve simulation calculations obtained the binding efficiency EC50 of antibodies P1D7V01, P1D7V02R, P1D7V03, P1D7V04R, 19F3H2L3 and MEDI9447 (as a control antibody) to be 0.063nM, 0.230nM, 0.068nM and 0.439nM, 0.045nM, 0.042nM, respectively.

[0355] The above experimental results show that under the same experimental conditions, the activity of bispecific antibodies P1D7V01, P1D7V02R, P1D7V03, and P1D7V04R in binding to human NT5E-Biotin is comparable to that of the positive drugs 19F3H2L3 and MEDI9447 with the same target, indicating that P1D7V01, P1D7V02R, P1D7V03, and P1D7V04R have the activity of effectively binding to human NT5E-Biotin.

[0356] Table 3: ELISA detection of the binding of P1D7V01, P1D7V02R, P1D7V03, P1D7V04R, 19F3H2L3, MEDI9447 and human NT5E-Biotin

[0357]

[0358] Example 9: Competitive ELISA method to determine the activity of the anti-CD73-anti-PD-1 bispecific antibody in competing with human PD-L1-mFc for binding to human PD-1-mFc-Biotin

[0359] After coating the ELISA plate with human PD-L1-mFc (PD-L1 Genbank ID: NP_054862.1, mFc SEQ ID NO: 143) at 2 μg / mL, incubate at 4°C overnight. After incubation, block the ELISA plate with 1% BSA in PBS at 37°C for 2 hours, wash the plate three times after blocking and pat dry. On the dilution plate, dilute the antibody to 7 concentrations at a dilution ratio of 1:3 with 10 μg / mL as the starting concentration, and set up a blank control. Then add an equal volume of 0.3 μg / mL human PD-1-mFc-Biotin solution, mix well and incubate at room temperature for 20 minutes. Then add the reaction mixture to the coated ELISA plate and incubate at 37°C for 30 minutes. After incubation, wash the plate three times with PBST and pat dry, add SA-HRP (KPL, 14-30-00) working solution, and incubate at 37°C for 30 minutes. After incubation, wash the plate four times and pat dry, then add TMB (Neogen, 308177) to develop color in the dark for 5 minutes, and add stop solution to terminate the color development reaction. Immediately put the ELISA plate into the ELISA reader, select 450nm light wavelength to read the OD value of each well of the ELISA plate, and use SoftMax Pro6.2.1 software to analyze and process the data.

[0360] Test results such as Figure 3 The OD values ​​of each dose are shown in Table 4. The absorbance intensity of the bound antibody was quantitatively analyzed, and the binding efficiency of the curve simulated the antibody to obtain the binding EC50 (Table 4).

[0361] The results showed that P1D7V01, P1D7V02R, P1D7V03, P1D7V04R, 14C12H1L1 and Nivolumab (as control) could effectively block the binding of antigen human PD-1-mFc-Biotin and its receptor human PD-L1-mFc, and the blocking efficiency was dose-dependent. The EC50 of P1D7V01, P1D7V02R, P1D7V03, P1D7V04R, 14C12H1L1 and Nivolumab in blocking the binding of human PD-1-mFc-Biotin and its ligand human PD-L1-mFc were 1.115nM, 1.329nM, 1.154nM, 1.339nM, 1.459nM and 1.698nM, respectively.

[0362] Table 4: Activity test results of P1D7V01, P1D7V02R, P1D7V03, P1D7V04R, 14C12H1L1 and Nivolumab competing with human PD-L1-mFc for binding to human PD-1-mFc-Biotin

[0363] Example 10: Determination of kinetic parameters of binding between anti-CD73-anti-PD-1 bispecific antibody and antigen human PD-1-mFc using the Fortebio molecular interaction instrument

[0364] The sample dilution buffer was PBST, 0.1% BSA, pH 7.4. The antibody was fixed on the AHC sensor at a concentration of 5 μg / mL, and the fixed height was about 0.4 nm. The sensor was balanced in the buffer for 60 seconds. The antibody fixed on the sensor was bound to the antigen PD-1-mFc, and the antigen concentration was 0.6-50nM (three-fold dilution). The binding time was 120 seconds, and the protein dissociated in the buffer for 180 seconds. The detection temperature was 37 degrees, the detection frequency was 0.3Hz, and the sample plate vibration rate was 1000rpm. The data was analyzed by fitting the 1:1 model to obtain the affinity constant.

[0365] The results of the affinity constant determination of humanized antibodies P1D7V01, 14C12H1L1 and Nivolumab (as a control antibody) with human PD-1-mFc are shown in Table 5. Figure 4 , Figure 5 and Figure 6As shown. The affinity constants of humanized antibodies P1D7V01, 14C12H1L1 and Nivolumab to human PD-1-mFc are 1.76E-10M, 1.64E-10M and 2.32E-10M, respectively. The above experimental results show that the binding ability of P1D7V01 to 14C12H1L1 and Nivolumab is comparable, indicating that the humanized antibody P1D7V01 has a strong binding ability to human PD-1-mFc.

[0366] Table 5: Determination of affinity constants of P1D7V01, 14C12H1L1, Nivolumab, and PD-1-mFc

[0367] Antibodies to be tested KD(M) kon(1 / Ms) SE(kon) kdis(1 / s) SE(kdis) Rmax(nm) P1D7V01 1.76E-10 4.19E+05 1.52E+04 7.37E-05 3.12E-05 0.13-0.17 14C12H1L1 1.64E-10 4.55E+05 1.61E+04 7.47E-05 2.98E-05 0.24-0.28 Nivolumab 2.32E-10 5.85E+05 2.03E+04 1.36E-04 3.47E-05 0.02-0.14

[0368] K D is the affinity constant; K D =kdis / kon

[0369] Example 11: The kinetic parameters of the binding of the anti-CD73-anti-PD-1 bispecific antibody to the antigen human NT5E(1-552)-his were determined using the Fortebio molecular interaction instrument.

[0370] The sample dilution buffer was PBST, pH 7.4. The antibody was fixed on the Protein A sensor at a concentration of 5 μg / mL for 15 seconds. The sensor was balanced in the buffer for 120 seconds. The antibody fixed on the sensor bound to the antigen human NT5E (1-552) -his, and the antigen concentration was 3.125-200nM (two-fold dilution). The binding time was 120 seconds, and the protein dissociated in the buffer for 600 seconds. The sensor was regenerated using 10mM Gly, pH 1.5 solution. The detection temperature was 37 degrees, the detection frequency was 0.6Hz, and the sample plate vibration rate was 1000rpm. The data was analyzed by fitting the 1:1 model to obtain the affinity constant

[0371] The affinity constants of humanized antibodies P1D7V01 and MEDI 9447 (as control antibody) and human NT5E(1-552)-his are shown in Table 6. Figure 7 and Figure 8 As shown, the affinity constants of humanized antibodies P1D7V01 and MEDI 9447 to human NT5E(1-552)-his are 2.29E-10M and 1.04E-10M, respectively.

[0372] The above experimental results show that the binding ability of P1D7V01 and MEDI 9447 is comparable, indicating that the humanized antibody P1D7V01 has a stronger binding ability with human NT5E(1-552)-his.

[0373] Table 6: Determination of affinity constants between P1D7V01, MEDI 9447 and human NT5E(1-552)-his

[0374] Antibodies to be tested KD(M) kon(1 / Ms) SE(kon) kdis(1 / s) SE(kdis) Rmax(nm) P1D7V01 2.29E-10 1.81E+05 2.30E+03 4.13E-05 4.54E-06 0.47-0.57 MEDI9447 1.04E-10 2.34E+05 3.20E+03 2.44E-05 5.02E-06 0.59-0.82

[0375] K D is the affinity constant; K D =kdis / kon

[0376] Example 12: FACS detection of binding activity of anti-CD73-anti-PD-1 bispecific antibodies

[0377] 1. FACS detection of the binding activity of anti-CD73-anti-PD-1 bispecific antibody to PD-1 on the surface of 293T-PD1 membrane

[0378] 293T-PD1 cells were collected in the logarithmic growth phase and cultured at 3x10 5 Transfer the cells to a 1.5 ml centrifuge tube, add 500 μL PBSA, centrifuge at 5600 rpm for 5 min, and remove the supernatant; add 100 μL of antibody diluted in PBSA (final concentrations are 100, 33.33, 11.11, 3.7, 1.23, 0.41, 0.14, and 0.05 nM, respectively), mix gently, and incubate on ice for 1 h; add 500 μL PBSA, centrifuge at 5600 rpm for 5 min, and remove the supernatant; add 500-fold diluted FITC-labeled goat anti-human IgG secondary antibody (Jackson, catalog number: 109-095-098), resuspend and mix, and incubate on ice in the dark for 0.5 h; add 500 μL PBSA, centrifuge at 5600 rpm for 5 min, and remove the supernatant; add 200 μL PBSA to resuspend the cell pellet, and transfer it to a flow tube for FACSCalibur detection.

[0379] The experimental results are shown in Table 7 and Fig. 9 As shown, P1D7V01, P1D7V02R, P1D7V03, and P1D7V04R can all specifically bind to PD-1 on the surface of 293T-PD1 membrane in a dose-dependent manner. Compared with the PD1 single-target control antibody 14C12H1L1, they are all stronger than 14C12H1 L1.

[0380] Under the same experimental conditions, the EC50 of P1D7V01, P1D7V02R, P1D7V03, and P1D7V04R binding to 293T-PD1 were 1.000nM, 1.075nM, 1.377nM, and 1.57nM, respectively, and the EC50 of 14C12H1L1 binding to 293T-PD1 was 2.111nM.

[0381] The above experimental results show that under the same experimental conditions, the binding activity of P1D7V01, P1D7V02R, P1D7V03, and P1D7V04R to 293T-PD1 is better than that of the PD1 single-target control antibody 14C12H1L1, indicating that P1D7V01, P1D7V02R, P1D7V03, and P1D7V04R have the activity of effectively binding to PD-1 on the membrane surface of 293T-PD1.

[0382] Table 7: FACS detection of the binding activity of P1D7V01, P1D7V02R, P1D7V03, P1D7V04R and 14C12H1L1 to PD-1 on the surface of 293T-PD1 cells

[0383] Concentration (nM) 0.05 0.14 0.41 1.23 3.70 11.11 33.33 100.00 EC50 14C12H1L1 35.69 57.70 129.42 437.62 974.97 1384.91 1174.75 1500.20 2.111 P1D7V01 26.65 58.65 150.57 307.19 530.77 577.76 479.34 531.15 1.000 P1D7V02R 23.44 42.98 108.44 217.56 404.33 426.06 405.29 301.40 1.075 P1D7V03 24.05 49.21 118.03 289.08 384.62 459.40 808.38 343.48 1.377 P1D7V04R 24.79 52.43 112.56 288.79 466.72 1284.73 400.33 360.79 1.57.0

[0384] 2. FACS detection of the binding activity of anti-CD73-anti-PD-1 bispecific antibody to CD73 on the surface of MDA-MB-231 membrane

[0385] Logarithmic phase MDA-MB-231 cells were digested with conventional trypsin and 3x10 5 Transfer the cells to a 1.5 ml centrifuge tube, add 500 μL PBSA, centrifuge at 5600 rpm for 5 min, and remove the supernatant; add 100 μL of antibody diluted in PBSA (final concentrations were 100, 33.33, 11.11, 3.7, 1.23, 0.41, 0.14, and 0.05 nM, respectively), mix gently, and incubate on ice for 1 h; add 500 μL PBSA, centrifuge at 5600 rpm for 5 min, and remove the supernatant; add 500-fold diluted FITC-labeled goat anti-human IgG secondary antibody (Jackson, catalog number: 109-095-098), resuspend and mix, and incubate on ice in the dark for 0.5 h; add 500 μL PBSA, centrifuge at 5600 rpm for 5 min, and remove the supernatant; add 200 μL PBSA to resuspend the cell pellet, and transfer it to a flow tube for FACSCalibur detection.

[0386] The experimental results are shown in Table 8 and Fig.10 As shown, the binding activity of P1D7V01 and P1D7V03 to CD73 on the surface of MDA-MB-231 membrane is better than 19F3H2L3, among which P1D7V01 is better than MEDI9447, a positive drug with the same target. Under the same experimental conditions, the EC50 of P1D7V01 and P1D7V03 binding to CD73 on the surface of MDA-MB-231 membrane were 1.384nM and 2.009nM, respectively, and the EC50 of MEDI9447 and 19F3H2L3 binding to CD73 on the surface of MDA-MB-231 membrane were 1.589nM and 2.773nM, respectively.

[0387] The above experimental results show that P1D7V01, P1D7V03, 19F3H2L3 and the positive drug MEDI9447 with the same target can specifically bind to CD73 on the surface of MDA-MB-231 membrane in a dose-dependent manner, and the binding activity of P1D7V01 and P1D7V03 is better than that of 19F3H2L3, and P1D7V01 is better than the positive drug MEDI9447 with the same target. It suggests that P1D7V01 and P1D7V03 have the activity of effectively binding to CD73 on the surface of MDA-MB-231 membrane.

[0388] Table 8: FACS detection of the binding activity of P1D7V01, P1D7V03, MEDI9447 and 19F3H2L3 to CD73 on the surface of MDA-MB-231 cells.

[0389] Concentration (nM) 0.05 0.14 0.41 1.23 3.70 11.11 33.33 100.00 EC50 MEDI9447 32.59 64.38 157.75 388.51 697.80 829.29 930.26 878.04 1.589 19F3H2L3 25.95 45.13 102.56 189.07 466.87 658.72 843.09 639.57 2.773 P1D7V01 23.37 38.02 76.84 271.72 571.56 560.81 525.91 705.50 1.384 P1D7V03 16.96 44.05 106.39 233.32 457.61 525.37 576.19 677.66 2.009

[0390] Example 13: Detection of the inhibition of CD73 enzyme activity on the cell membrane surface by anti-CD73-anti-PD-1 bispecific antibodies

[0391] 1. Detection of the inhibition of CD73 enzyme activity on the surface of MDA-MB-231 cell membrane by anti-CD73-anti-PD-1 bispecific antibody

[0392] The experimental steps are as follows: Take the logarithmic phase MDA-MB-231 cells in good condition, resuspend the cells in serum-free RPMI-1640 culture medium, and count the cells; inoculate the MDA-MB-231 cells into a 96-well plate, 2x10 4 cells / 100 μL / well; the antibody was diluted in a gradient of 2.5 times with serum-free RPMI-1640 culture medium; the antibody was added to a 96-well plate, 50 μL per well, incubated at 37°C for 1 hour, and after 1 hour, 50 μL of 1200 μM RPMI-1640-diluted AMP (TCL, catalog number: A0157) was added to each well; after 3 hours, 25 μL of cell culture supernatant was taken and transferred to a new 96-well plate, and 25 μL of 100 μM ATP (TCL, catalog number: A0158) was added to each well; 50 μL of CTG (CellTiterGlo, Promega, catalog number: G8641) colorimetric solution was added to each well for color development, and the relative fluorescence intensity RLU was read on a multi-label microplate reader (PerkinElmer 2140-0020).

[0393] The experimental results are as follows Fig.11 As shown, the AMP content of P1D7V01, P1D7V02R, P1D7V03, and P1D7V04R was comparable to that of the positive control drug MEDI9447 and was concentration-dependent.

[0394] The above experimental results show that the added AMP can be converted into adenosine A by the enzymatic activity of CD73 on the surface of MDA-MB-231 cells in the absence of antibodies. After adding antibodies, the P1D7V01, P1D7V02R, P1D7V03, and P1D7V04R antibodies bind to CD73, which reduces its enzymatic activity and prevents AMP from being converted into adenosine A. This suggests that antibodies effectively inhibit the enzymatic activity reaction in a non-substrate competitive manner and reduce the production of adenosine.

[0395] 2. Detection of the inhibition of CD73 enzyme activity on the surface of U87-MG cell membrane by anti-CD73-anti-PD-1 bispecific antibody

[0396] Take the logarithmic phase U87-MG cells in good condition, resuspend the cells in serum-free RPMI-1640 culture medium, and count them; inoculate U87-MG cells into 96-well plates, 2x10 4 cells / 100 μL / well; the antibody was diluted 2.5 times with serum-free RPMI-1640 culture medium, added to a 96-well plate, 50 μL per well, and incubated at 37°C for 1 hour; after 1 hour, 50 μL of 1200uM RPMI-1640-diluted AMP was added to each well; after 3 hours, 25 μL of cell culture supernatant was taken 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) colorimetric solution was added to each well, and after color development, the relative fluorescence intensity RLU was read on a multi-label microplate reader (PerkinElmer 2140-0020).

[0397] The experimental results are as follows Fig.12 As shown, the AMP content of P1D7V01, P1D7V02R, P1D7V03, and P1D7V04R was comparable to that of the positive control drug MEDI9447 and was concentration-dependent.

[0398] The above experimental results show that the added AMP can be converted into adenosine A by the enzymatic activity of CD73 on the surface of U87-MG cells in the absence of antibodies. However, after adding antibodies, the P1D7V01, P1D7V02R, P1D7V03, and P1D7V04R antibodies bind to CD73, which reduces its enzymatic function and prevents AMP from being converted into adenosine A. This suggests that antibodies effectively inhibit the enzymatic activity reaction in a non-substrate competitive manner and reduce the production of adenosine.

[0399] Example 14: Mixed lymphocyte reaction (MLR) detection of the biological activity of anti-CD73-anti-PD-1 bispecific antibody in promoting IFN-γ and IL-2 secretion

[0400] 1. Bioactivity detection of anti-CD73-anti-PD-1 bispecific antibody promoting IFN-γ secretion in Raji-PDL1 mixed lymphocyte reaction system

[0401] Raji-PDL1 cells were subcultured normally; PBMCs were resuscitated, cultured with 10 mL 1640 complete medium, and stimulated with 0.5 μg / mL SEB (Denotek, Catalog No.: S010201) for two days; Raji-PDL1 cells were treated with 25 μg / mL MMC (Sigma, Catalog No.: M4287) and placed in a 37°C incubator for 1 hour; PBMCs stimulated with SEB for 2 days and Raji-PDL1 cells treated with MMC for 1 hour were collected, washed twice with PBS, resuspended and counted in complete medium, and added to U-shaped 96-well plates, 100,000 cells / well; antibodies were added according to the experimental design, and co-cultured in an incubator for 3 days; after 3 days, the cell culture supernatant was collected, and IFN-γ was detected by ELISA.

[0402] like Fig.13 As shown in the results, mixed culture of human PBMC and Raji-PDL1 cells significantly promoted the secretion of IFN-γ by PBMC. Adding antibodies to the mixed culture system at the same time could significantly induce PBMC to further secrete IFN-γ. In terms of the activity level of promoting IFN-γ secretion, the activities of P1D7V01, P1D7V02R, P1D7V03, and P1D7V04R antibodies were comparable to those of their parent PD-1 single-target antibody 14C12H1L1.

[0403] 2. Bioactivity detection of anti-CD73-anti-PD-1 bispecific antibody promoting IL-2 secretion in Raji-PDL1 mixed lymphoid reaction system

[0404] Raji-PDL1 cells were cultured normally; PBMCs were revived, cultured with 10 mL of 1640 complete medium, and stimulated with SEB (0.5 μg / mL) for two days; Raji-PDL1 cells were treated with 25 μg / mL of MMC and placed in a 37°C incubator for 1 hour; PBMCs stimulated with SEB for 2 days and Raji-PDL1 cells treated with MMC for 1 hour were collected, washed twice with PBS, resuspended and counted in complete medium, and added to U-shaped 96-well plates, 100,000 cells / well; antibodies were added according to the experimental design and co-cultured for 3 days; cell culture supernatant was collected, and IL-2 was detected by ELISA.

[0405] like Fig.14As shown in the figure, mixed culture of human PBMC and Raji-PDL1 cells has a certain promoting effect on the secretion of IL-2 by PBMC. The simultaneous addition of antibodies in the mixed culture system can significantly induce PBMC to further secrete IL-2, with a significant dose-dependent relationship. In terms of the level of IL-2 secretion activity, the activity of the bifunctional antibodies P1D7V01, P1D7V02R, P1D7V03, and P1D7V04R at low concentrations is slightly lower than that of their parent PD-1 single-target antibody 14C12H1L1, while the medium and high concentrations are equivalent to it. Compared with the PD1 target positive control drug Nivolumab, P1D7V01, P1D7V02R, P1D7V03, and P1D7V04R have better IL-2 secretion potential at three different antibody concentration levels.

[0406] 3. Detection of the biological activity of anti-CD73-anti-PD-1 bispecific antibody in promoting the secretion of IFN-γ in DC mixed lymphoid reaction system

[0407] Normal human peripheral blood PBMCs were isolated, resuspended in complete medium, inoculated into culture dishes, and cultured in an incubator overnight. The suspended PBMCs were collected and removed, and the adherent cells at the bottom of the dish were washed with PBS buffer and then induced to mature DCs: 10 mL of RPMI1640 complete medium containing GM-CSF and IL-4 was added to each dish, and the concentrations of GM-CSF and IL-4 were both 1000 U / mL, and the dish was cultured in a 37°C, 5% carbon dioxide incubator for three days; half of the medium was replaced, and 1000 U / mL of GM-CSF and IL-4 were added, and the dish was cultured in a 37°C, 5% carbon dioxide incubator for three more days; three days Then, half of the medium was replaced again, and 1000U / mL of GM-CSF and IL-4 and 100U / mL of TNF-α were added, and the culture was continued for two days; PBMCs from another donor were freshly isolated, and the cells were counted and inoculated into a 96-well plate at 100,000 cells / well; induced mature DC cells were collected, washed once with complete medium, and the cells were counted and inoculated into a 96-well plate containing PBMC at 10,000 cells / well; antibodies were added according to the experimental design, mixed, and co-cultured in a 37°C, 5% carbon dioxide incubator for 5 days; after 5 days, the cell culture supernatant was collected, and IFN-γ was quantitatively detected by ELISA.

[0408] The results are as follows Fig.15 As shown in the figure, compared with the culture of DC or PBMC alone, the mixed culture of DC and PBMC can significantly promote the secretion of IFN-γ; compared with the isotype control, the addition of anti-CD73-anti-PD-1 bispecific antibody to the mixed culture of DC and PBMC can further significantly increase the level of IFN-γ secretion.

[0409] Bioactivity detection of anti-CD73-anti-PD-1 bispecific antibody promoting IL-2 secretion by DC mixed lymphoid reaction system. PBMCs from normal human peripheral blood were separated, resuspended in complete medium, inoculated in culture dishes, and cultured in an incubator overnight. The suspended PBMCs were collected and removed, and the adherent cells at the bottom of the dish were washed with PBS buffer and then induced to mature DCs. 10 mL of RPMI1640 complete medium containing GM-CSF and IL-4 was added to each dish. The concentrations of GM-CSF and IL-4 were both 2000 U / mL, and the dishes were cultured in a 37°C, 5% carbon dioxide incubator for three days. ; Replace half of the medium, add 50ng / mL IFN-γ and 100ng / mL LPS, and continue to culture for two days; revive the PBMC of another donor, count the cells and inoculate them into a 96-well plate, 100,000 cells / well; collect the induced mature DC cells, wash them once with complete medium, count the cells, and inoculate them into a 96-well plate containing PBMC, 10,000 cells / well; add antibodies according to the experimental design, mix well, and place in a 37°C, 5% carbon dioxide incubator for co-culture for 5 days; after 5 days, collect the cell culture supernatant, and perform ELISA to quantify IL-2. The results are as follows Fig.16 As shown in the figure, compared with the culture of DC or PBMC alone, the mixed culture of DC and PBMC can significantly promote the secretion of IL-2; compared with the isotype control, the addition of anti-CD73-anti-PD-1 bispecific antibody to the mixed culture of DC and PBMC can further significantly increase the IL-2 secretion level.

[0410] Example 15: Preparation of anti-PD-1 / CD73 bispecific antibodies NTPDV1, NTPDV2, NTPDV3, and NTPDV4

[0411] The structural model of the bispecific antibodies NTPDV1, NTPDV2, NTPDV3, and NTPDV4 belongs to the Morrison model (IgG-scFv), that is, the C-termini of the two heavy chains of an IgG antibody are connected to the scFv fragment of another antibody through a connecting fragment, and the design composition of the heavy chain and light chain is shown in Table 9 below.

[0412] Since amino acid mutations were introduced into the constant region of the immunoglobulin part to eliminate its binding activity with FcγR, NTPDV1, NTPDV2, NTPDV3, and NTPDV4 are also written as NTPDV1 (hG1TM), NTPDV2 (hG1TM), NTPDV3 (hG1TM), and NTPDV4 (hG1TM) in this article and in the Chinese invention patent with application number 202110270671.X.

[0413] Table 9: Sequence design of NTPDV1, NTPDV2, NTPDV3, and NTPDV4

[0414]

[0415] In Table 9 above:

[0416] The "V" in the lower right corner refers to the variable region of the corresponding heavy chain or the variable region of the corresponding light chain. If "V" is not marked, the corresponding heavy chain or light chain is the full length including the constant region. The amino acid sequences of these variable regions or full lengths and their encoding nucleic acid sequences are all referred to the corresponding sequences recorded in the above embodiments.

[0417] The amino acid sequence of Linker1 is a (GGGGS) structure repeated 4 times, i.e., (GGGGS)4 or (G4S)4 (nucleotide sequence SEQ ID NO: 80, amino acid sequence SEQ ID NO: 79);

[0418] The amino acid sequence of Linker2 is a (GGGGS) structure repeated three times, namely (GGGGS)4 or (G4S)3 (nucleotide sequence SEQ ID NO: 82, amino acid sequence SEQ ID NO: 81).

[0419] The three CDR sequences of the light chains 19F3L2 and 19F3L3 in the immunoglobulin part of NTPDV1, NTPDV2, NTPDV3, and NTPDV4 are consistent with the light chain CDR sequence of 19F3.

[0420] The three CDR sequences of 19F3H2 (hG1TM) in the immunoglobulin portion of NTPDV1, NTPDV2, NTPDV3, and NTPDV4 are consistent with the heavy chain CDR sequences of 19F3.

[0421] The CDR sequences of 14C12H1V-Linker2-14C12L1V and 14C12H1V-Linker1-14C12L1V of the scFv portion in NTPDV1, NTPDV2, NTPDV3, and NTPDV4 are consistent with the heavy chain CDR and light chain CDR in 14C12.

[0422] The amino acid sequences of the heavy chains of NTPDV2 and NTPDV4 are identical and are denoted as NTPDH2 / 4 (SEQ ID NO: 83). The nucleotide sequences of the heavy chains of NTPDV2 and NTPDV4 are SEQ ID NO: 84.

[0423] The amino acid sequences of the heavy chains of NTPDV1 and NTPDV3 are identical and are denoted as NTPDH1 / 3 (SEQ ID NO: 85). The nucleotide sequences of the heavy chains of NTPDV1 and NTPDV3 are SEQ ID NO: 86.

[0424] The three CDR sequences of 19F3L3 and 19F3L2 in the immunoglobulin part of NTPDV1, NTPDV2, NTPDV3, and NTPDV4 are consistent with the three CDRs of the antibody 19F3 light chain.

[0425] The amino acid sequence of the light chain 19F3L3 of the immunoglobulin portion in NTPDV1 and NTPDV2 is consistent with the light chain sequence of antibody 19F3H2L3 (G1 M) (SEQ ID NO: 28), and the nucleotide sequence of the light chain 19F3L3 of the immunoglobulin portion in NTPDV1 and NTPDV2 is SEQ ID NO: 27. The amino acid sequence of the light chain 19F3L2 of the immunoglobulin portion in NTPDV3 and NTPDV4 is SEQ ID NO: 96), and the nucleotide sequence of the light chain 19F3L2 of the immunoglobulin portion in NTPDV3 and NTPDV4 is SEQ ID NO: 100.

[0426] (1) NTPDV1, whose heavy chain amino acid sequence is shown in SEQ ID NO: 85, whose light chain amino acid sequence is shown in SEQ ID NO: 28, whose amino acid sequence of connecting segment 1 is shown in SEQ ID NO: 79, whose amino acid sequence of 14C12H1V is shown in SEQ ID NO: 62, whose amino acid sequence of connecting segment 2 is shown in SEQ ID NO: 81, whose amino acid sequence of 14C12L1V is shown in SEQ ID NO: 64,

[0427] (2) NTPDV2, whose heavy chain amino acid sequence is shown in SEQ ID NO: 83, whose light chain amino acid sequence is shown in SEQ ID NO: 28, whose amino acid sequence of connecting segment 1 is shown in SEQ ID NO: 79, whose amino acid sequence of 14C12H1V is shown in SEQ ID NO: 62, whose amino acid sequence of connecting segment 1 is shown in SEQ ID NO: 79, whose amino acid sequence of 14C12L1V is shown in SEQ ID NO: 64,

[0428] (3) NTPDV3, whose heavy chain amino acid sequence is shown in SEQ ID NO: 85, whose light chain amino acid sequence is shown in SEQ ID NO: 96, whose amino acid sequence of connecting segment 1 is shown in SEQ ID NO: 79, whose amino acid sequence of 14C12H1V is shown in SEQ ID NO: 62, whose amino acid sequence of connecting segment 2 is shown in SEQ ID NO: 81, whose amino acid sequence of 14C12L1V is shown in SEQ ID NO: 64, and

[0429] (4) NTPDV4, whose heavy chain amino acid sequence is shown in SEQ ID NO: 83, the light chain amino acid sequence is shown in SEQ ID NO: 96, the amino acid sequence of connecting fragment 1 is shown in SEQ ID NO: 79, the amino acid sequence of 14C12H1V is shown in SEQ ID NO: 62, the amino acid sequence of connecting fragment 1 is shown in SEQ ID NO: 79, and the amino acid sequence of 14C12L1V is shown in SEQ ID NO: 64.

[0430] Antibody expression and purification

[0431] The heavy chain cDNA of NTPDV1 and NTPDV3 and the heavy chain cDNA sequences of NTPDV2 and NTPDV4, as well as the cDNA sequences of their light chains were cloned into the pUC57simple (provided by GenScript) vector to obtain pUC57simple-NTPDH2 / 4, pUC57simple-NTPDH1 / 3, pUC57simple-19F3L3 plasmids and pUC57simple-19F3L2 plasmids, respectively.

[0432] The plasmids pUC57simple-NTPDH2 / 4 and pUC57simple-19F3L3, pUC57simple-NTPDH2 / 4 and pUC57simple-19F3L2, pUC57simple-NTPDH1 / 3 and pUC57simple-19F3L3, and pUC57simple-NTPDH1 / 3 and pUC57simple-19F3L2 were digested with HindIII and EcoRI. The heavy and light chains recovered by electrophoresis were subcloned into the pcDNA3.1 vector, and the pcDNA3.1-NTPDH2 / 4 and pcDNA3.1-19F3L3 plasmids, pcDNA3.1-NTPDH2 / 4 and pcDNA3.1-19F3L2 plasmids, pcDNA3.1-NTPDH1 / 3 and pcDNA3.1-19F3L3 plasmids, and pcDNA3.1-NTPDH1 / 3 and pcDNA3.1-19F3L2 plasmids were obtained. The recombinant plasmids were extracted and co-transfected into 293F cells. After 7 days of cell culture, the culture medium was centrifuged at high speed, the supernatant was concentrated, and then loaded onto the HiTrap MabSelectSuRe column, and the protein was eluted in one step with Elution Buffer, and the target sample was recovered and the solution was changed to PBS.

[0433] Purified antibodies NTPDV1, NTPDV2, NTPDV3, and NTPDV4 were obtained according to the expression and purification methods mentioned in the above preparation examples.

[0434] Example 16: ELISA method to determine the binding activity of anti-CD73-anti-PD-1 bispecific antibody with antigen

[0435] 1. The ELISA method was used to determine the binding activity of NTPDV1, NTPDV2, NTPDV3, NTPDV4 with the antigen PD-1-mFc.

[0436] The ELISA plate coated with PD-1-mFc (0.5 μg / ml) was incubated at 4°C overnight, and then the plate coated with antigen was washed once with PBST, and then the plate was blocked at 37°C for 2 hours using 1% BSA in PBS solution as a blocking solution. After the plate was blocked, the plate was washed 3 times with PBST, and the antibody diluted in gradient with PBST solution was added (the antibody dilution gradient is detailed in Table 2), and the plate with the antibody to be tested was incubated at 37°C for 30 minutes. After the incubation, the plate was washed 3 times with PBST. After washing the plate, a 1:5000 dilution of HRP-labeled goat anti-human IgG Fc (Jackson, catalog number: 109-035-098) secondary antibody working solution was added, and then incubated at 37°C for 30 minutes. After the incubation, the plate was washed 4 times with PBST, and then TMB (Neogen, 308177) was added to develop color in the dark for 8 minutes, and the color development reaction was terminated by adding the stop solution. Immediately place the ELISA plate into the ELISA reader, select 450nm wavelength to read the OD value of each well of the ELISA plate, and use SoftMax Pro 6.2.1 software to analyze and process the data.

[0437] The test 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, and the binding efficiency is dose-dependent. The absorbance intensity of each dose is shown in Table 10. By quantitatively analyzing the absorbance of the bound antibodies, the curve simulation calculation obtained that the binding efficiency EC50 of antibodies NTPDV1, NTPDV2, NTPDV3, NTPDV4, and 14C12H1L1 (hG1TM) (as a control) were 0.101nM, 0.119nM and 0.110nM, 0.123nM, and 0.031nM, respectively.

[0438] The above experimental results show that under the same experimental conditions, the activity of NTPDV1, NTPDV2, NTPDV3, and NTPDV4 in binding to PD-1-mFc is comparable to that of the positive drug 14C12H1L1 (hG1TM) with the same target, indicating that NTPDV1, NTPDV2, NTPDV3, and NTPDV4 have the activity of effectively binding to PD-1-mFc.

[0439] Table 10: ELISA detection of the binding of NTPDV1, NTPDV2, NTPDV3, NTPDV4, 14C12H1L1 (hG1TM) and PD-1-mFc

[0440]

[0441]

[0442] 2. ELISA method was used to determine the binding activity of NTPDV1, NTPDV2, NTPDV3, NTPDV4 and the antigen human NT5E-Biotin

[0443] After coating the ELISA plate with streptavidin SA (2 μg / ml), incubate it at 4°C overnight. After the incubation, wash the ELISA plate coated with streptavidin once with PBST, use 1% BSA in PBS as the blocking solution for the ELISA plate, and block the ELISA plate at 37°C for 2 hours. After the ELISA plate is blocked, wash the plate 3 times with PBST. Add 0.5 μg / ml of the antigen human NT5E-Biotin and incubate at 37°C for 30 minutes, then wash the plate 3 times with PBST. Add antibodies diluted in gradients with PBST solution to the wells of the ELISA plate (see Table 11 for the antibody dilution gradient), add the ELISA plate to the antibody to be tested and incubate it at 37°C for 30 minutes, and wash the plate 3 times with PBST after the incubation is completed. After washing, add HRP-labeled goat anti-human IgG Fc (Jackson, catalog number: 109-035-098) secondary antibody working solution diluted 1:5000 and incubate at 37°C for 30 minutes. After incubation, wash the plate 4 times with PBST, then add TMB (Neogen, 308177) to color for 7 minutes in the dark, and add stop solution to terminate the color reaction. Immediately put the ELISA plate into the ELISA reader, select 450nm light wavelength to read the OD value of each well of the ELISA plate. Use SoftMax Pro6.2.1 software to analyze and process the data.

[0444] The test results are shown in Table 11. It can be seen that NTPDV1, NTPDV2, NTPDV3, NTPDV4, 19F3H2L3 (hG1M) can effectively bind to the antigen human NT5E-Biotin, and its binding efficiency is dose-dependent (the absorbance intensity of each dose is shown in Table 11). By quantitatively analyzing the absorbance of the bound antibodies, the curve simulation calculation obtained the binding efficiency EC50 of the antibodies NTPDV1, NTPDV2, NTPDV3, NTPDV4, and 19F3H2L3 (hG1M) (as a control antibody) as 0.079nM, 0.082nM and 0.084nM, 0.077nM, 0.029nM, respectively.

[0445] The above experimental results show that under the same experimental conditions, the activity of bispecific antibodies NTPDV1, NTPDV2, NTPDV3, and NTPDV4 in binding to human NT5E-Biotin is comparable to that of the positive drug 19F3H2L3 (hG1M) with the same target, indicating that NTPDV1, NTPDV2, NTPDV3, and NTPDV4 have the activity of effectively binding to human NT5E-Biotin.

[0446] Table 11: ELISA detection of the binding of NTPDV1, NTPDV2, NTPDV3, NTPDV4, 19F3H2L3 (hG1M) and human NT5E-Biotin

[0447]

[0448] Example 17: Competitive ELISA method to determine the activity of anti-CD73-anti-PD-1 bispecific antibody in competing with human PD-L1-mFc for binding to human PD-1-mFc-Biotin

[0449] After coating the ELISA plate with human PD-L1-mFc (PD-L1 Genbank ID: NP_054862.1, mFc SEQ ID NO: 89) at 2 μg / mL, incubate at 4°C overnight. After incubation, block the ELISA plate with 1% BSA in PBS at 37°C for 2 hours, wash the plate once and pat dry after blocking. On the dilution plate, dilute the antibody to 7 concentrations at a dilution ratio of 1:3 with 10 μg / mL as the starting concentration, and set up a blank control. Then add an equal volume of 0.3 μg / mL human PD-1-mFc-Biotin solution, mix well and incubate at room temperature for 10 minutes. Then add the reaction mixture to the coated ELISA plate and incubate at 37°C for 30 minutes. After incubation, wash the plate three times with PBST and pat dry, add SA-HRP (KPL, 14-30-00) working solution, and incubate at 37°C for 30 minutes. After incubation, wash the plate four times and pat dry, then add TMB (Neogen, 308177) to develop color in the dark for 5 minutes, and add stop solution to terminate the color development reaction. Immediately put the ELISA plate into the ELISA reader, select 450nm light wavelength to read the OD value of each well of the ELISA plate, and use SoftMax Pro6.2.1 software to analyze and process the data.

[0450] The OD values ​​of each dose are shown in Table 12. The absorbance intensity of the bound antibody was quantitatively analyzed and the binding efficiency of the antibody was simulated by the curve to obtain the binding EC50 (Table 12).

[0451] The results showed that NTPDV1, NTPDV2, NTPDV3, NTPDV4, and 14C12H1L1 (hG1TM) (as control) could effectively block the binding of antigen human PD-1-mFc-Biotin and its receptor human PD-L1-mFc, and the blocking efficiency was dose-dependent. The EC50 of NTPDV1, NTPDV2, NTPDV3, NTPDV4, and 14C12H1L1 (hG1TM) in blocking the binding of human PD-1-mFc-Biotin and its ligand human PD-L1-mFc were 2.249nM, 2.253nM, 2.332nM, 2.398nM, 2.216nM, and 2.231nM, respectively.

[0452] Table 12: Activity test results of NTPDV1, NTPDV2, NTPDV3, NTPDV4, 14C12H1L1 (hG1TM) competing with human PD-L1-mFc for binding to human PD-1-mFc-Biotin

[0453]

[0454] Example 18: Determination of kinetic parameters of binding between anti-CD73-anti-PD-1 bispecific antibody and antigen human PD-1-mFc using the Fortebio molecular interaction instrument

[0455] The sample dilution buffer was PBS, 0.02% Tween-20, 0.1% BSA, pH 7.4. PD1-mFc was fixed on the AMC sensor at a concentration of 5 μg / mL, and the fixed height was about 0.1nm (time 60s). The sensor was balanced in the buffer for 60s. The PD1-mFc fixed on the sensor was bound to the antibody at a concentration of 0.62-50nM (three-fold dilution) for 120s. The protein was dissociated in the buffer for 300s. The detection temperature was 30 degrees, the detection frequency was 0.3Hz, and the sample plate vibration rate was 1000rpm. The data was fit analyzed with a 1:1 model to obtain the affinity constant.

[0456] The affinity constants of humanized antibodies NTPDV1, NTPDV2, NTPDV3, NTPDV4 and Nivolumab (as a control antibody) with human PD-1-mFc are shown in Table 13. Fig.19 , Fig. 20 , Fig.21 , Fig. 22 and Fig.18As shown. The affinity constants of humanized antibodies NTPDV1, NTPDV2, NTPDV3, NTPDV4 and Nivolumab to human PD-1-mFc are 1.40E-10M, 7.39E-11M, 1.25E-10M, 1.13E-11M, and 2.26E-10M, respectively. The above experimental results show that the binding abilities of NTPDV1, NTPDV3, and Nivolumab are comparable, and the binding abilities of NTPDV2 and NTPDV4 are better than those of Nivolumab, indicating that humanized antibodies NTPDV1, NTPDV2, NTPDV3, and NTPDV4 have strong binding abilities to human PD-1-mFc.

[0457] Table 13: Determination of affinity constants of 14C12H1L1 (hG1TM), NTPDV1, NTPDV2, NTPDV3, NTPDV4, Nivolumab and PD-1-mFc

[0458]

[0459] KD is the affinity constant; KD = kdis / kon

[0460] Example 19: The kinetic parameters of the binding of the anti-CD73-anti-PD-1 bispecific antibody to the antigen human NT5E(1-552)-his were determined using the Fortebio molecular interaction instrument.

[0461] The sample dilution buffer was PBS, 0.02% Tween-20, 0.1% BSA, pH 7.4. HNT5E (1-552) -His was fixed on the HIS1K sensor at a concentration of 5 μg / mL, and the fixed height was about 0.4nm (time 50s). The sensor was balanced in the buffer for 60s. The HNT5E (1-552) -His fixed on the sensor was bound to the antibody at a concentration of 0.31-25nM (three-fold dilution) for 100s. The protein was dissociated in the buffer for 180s. The detection temperature was 30 degrees, the detection frequency was 0.3Hz, and the sample plate vibration rate was 1000rpm. The data was analyzed by 1:1 model fitting to obtain the affinity constant.

[0462] The affinity constants of humanized antibody 19F3H2L3 (hG1M) (as control antibody), NTPDV1, NTPDV2, NTPDV3, NTPDV4 and human NT5E (1-552)-his are shown in Table 14. Figure 24-27As shown, the affinity constants of humanized antibodies NTPDV1, NTPDV2, NTPDV3, and NTPDV4 to human NT5E(1-552)-his are 3.29E-11M, 2.88E-11M, 7.92E-11M, and 5.77E-11M, respectively.

[0463] The above experimental results show that the binding abilities of 19F3H2L3 (hG1M), NTPDV1, NTPDV2, NTPDV3, and NTPDV4 are comparable, indicating that the humanized antibodies NTPDV1, NTPDV2, NTPDV3, and NTPDV4 have stronger binding abilities to human NT5E (1-552) -his.

[0464] Table 14: Determination of affinity constants between 19F3H2L3 (hG1M), NTPDV1, NTPDV2, NTPDV3, NTPDV4 and human NT5E (1-552)-his

[0465]

[0466] K D is the affinity constant; K D =kdis / kon

[0467] Example 20: Detection of the inhibition of CD73 enzyme activity on the surface of U87-MG cell membrane by anti-CD73-anti-PD-1 bispecific antibodies

[0468] Take the logarithmic phase U87-MG cells in good condition, resuspend the cells in serum-free RPMI-1640 culture medium, and count them; inoculate U87-MG cells into 96-well plates, 2.5x10 4 cells / 60μL / well; serum-free RPMI-1640 culture medium was used to grade dilute the antibody according to the experimental design concentration, and the antibody was added to a 96-well plate, 60μL per well, and incubated at 37°C for 1 hour; after 1 hour, 60μL 600uM RPMI-1640 diluted AMP was added to each well; after 3 hours, 100μL of cell culture supernatant was taken and transferred to a new 96-well plate, 40μL CTG (CellTiterGlo) colorimetric solution was added to each well, and it was placed at room temperature away from light for 5 minutes; after 5 minutes, 10μL 300μM ATP was added to each well, and the relative fluorescence intensity RLU was read on a multi-label microplate reader (PerkinElmer 2140-0020) after color development.

[0469] The experimental results are shown in Table 15 and Fig.28 As shown, the AMP content of NTPDV2 is comparable to that of the positive control drug MEDI9447.

[0470] The above experimental results show that the added AMP, in the absence of antibodies, can be converted into adenosine A by the enzymatic activity of CD73 on the surface of U87-MG cells. After adding antibodies, the NTPDV2 antibody binds to CD73, reducing its enzymatic function, so that AMP will not be converted into adenosine A. This suggests that antibodies effectively inhibit the enzymatic activity reaction in a non-substrate competitive manner and reduce the production of adenosine.

[0471] Table 15 Detection of the inhibition of CD73 enzyme activity on the surface of U87-MG cell membrane by anti-CD73-anti-PD-1 bispecific antibodies

[0472] Antibody Name MEDI9447 19F3H2L3(hG1M) NTPDV2 IC50(nM) 0.1189 0.3647 0.5551

[0473] Example 21: Mixed lymphocyte reaction (MLR) to detect the biological activity of anti-CD73-anti-PD-1 bispecific antibody in promoting IFN-γ and IL-2 secretion

[0474] 1. Bioactivity detection of IFN-γ secretion by anti-CD73-anti-PD-1 bispecific antibody-induced Raji-PDL1 mixed lymphazone reaction system

[0475] Raji-PDL1 cells were subcultured normally; PBMCs were revived, cultured with 10 mL 1640 complete medium, and stimulated with 0.5 μg / mL SEB (Denotek, catalog number: S010201) for two days; Raji-PDL1 cells were treated with 25 μg / mL MMC (mitomycin C, Stressmarq, catalog: SIH-246, batch number: SM286474) and placed in a 37°C incubator for 1 hour; PBMCs stimulated with SEB for 2 days and Raji-PDL1 cells treated with MMC for 1 hour were collected, washed twice with PBS, resuspended in complete medium, counted, and added to U-shaped 96-well plates, 1x10 5 cells / well; antibodies were added according to the experimental design and co-cultured in an incubator for 3 days; after 3 days, the cell culture supernatant was collected and IFN-γ was detected by ELISA.

[0476] like Fig.29 As shown, mixed culture of human PBMC and Raji-PDL1 cells significantly promoted the secretion of IFN-γ by PBMC. Adding antibodies to the mixed culture system at the same time could significantly induce PBMC to further secrete IFN-γ. In terms of the level of activity in promoting IFN-γ secretion, the activity of NTPDV2 antibody was comparable to that of its parent PD-1 single-target antibody 14C12H1L1.

[0477] 2. Bioactivity detection of anti-CD73-anti-PD-1 bispecific antibody promoting IL-2 secretion in Raji-PDL1 mixed lymphoid reaction system

[0478] Raji-PDL1 cells were subcultured normally; PBMCs were revived and cultured with 10 mL 1640 complete medium and stimulated with SEB (0.5 μg / mL) for two days; Raji-PDL1 cells were treated with 25 μg / mL MMC and placed in a 37°C incubator for 1 hour; PBMCs stimulated with SEB for 2 days and Raji-PDL1 cells treated with MMC for 1 hour were collected, washed twice with PBS, resuspended in complete medium, counted, and added to U-shaped 96-well plates, 1x10 5 cells / well; antibodies were added according to the experimental design and cultured for 3 days; cell culture supernatant was collected and IL-2 was detected by ELISA.

[0479] like Fig.29 As shown, the mixed culture of human PBMC and Raji-PDL1 cells has a certain promoting effect on the secretion of IL-2 by PBMC. The simultaneous addition of antibodies to the mixed culture system can significantly induce PBMC to further secrete IL-2, with a significant dose-dependent relationship. In terms of the level of IL-2 secretion promoting activity, the activity of the bifunctional antibody NTPDV2 at low concentrations is comparable to that of its parent PD-1 single-target antibody 14C12H1L1, while the medium and high concentrations are slightly lower than those of the parent PD-1 single-target antibody 14C12H1L1.

[0480] Example 22: In vivo tumor growth inhibition experiment of anti-CD73-anti-PD-1 bispecific antibody

[0481] To detect the in vivo anti-tumor activity of the anti-CD73-anti-PD-1 bispecific antibody, MC38-hPDL1 / hCD73 cells (from Jiangsu Jicui Pharmaceutical Kang Biotechnology Co., Ltd.) were first subcutaneously inoculated into 5-7 week old female C57BL6-hPD1hPDL1hCD73 triple transgenic mice (from Jiangsu Jicui Pharmaceutical Kang Biotechnology Co., Ltd.), and 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.

[0482] Table 16: Experimental design.

[0483]

[0484]

[0485] The experimental results are as follows Fig.30 , Fig.31 As shown, the results showed that compared with the isotype control antibody hIgG, 19F3H2L3 (hG1M), different doses of NTPDV2 can effectively inhibit the growth of mouse tumors, and high-dose NTPDV2 has better inhibition on tumors than low-dose NTPDV2. In addition, NTPDV2 has no effect on the body weight of tumor-bearing mice.

[0486] Experimental Example 23: Fc segment mutation can effectively eliminate the secretion of IL-8 and IL-6 mediated by the bispecific immune checkpoint inhibitor PD-1 / CD73 bispecific antibody

[0487] HPMM is induced by PBMC. The PBMC used in this study were isolated and prepared by Zhongshan Kangfang Biopharmaceutical Co., Ltd., and the informed consent was obtained from the providers.

[0488] Ficoll-Paque PLUS lymphocyte separation medium (GE, catalog number: 17-1440-03); RPMI 1640 (Gibco, catalog number: 22400-105); CHO-K1-PD1 cells (constructed by Zhongshan Kangfang Biotechnology Co., Ltd.); U87-MG cells (cells originated from ATCC, purchased from Beijing Zhongyuan Leading Technology Co., Ltd.); FBS (Fetal Bovine Serum, Excell bio, catalog number: FSP500); human IFN-γ protein (sinobio, catalog number: 11725-HNAS-100); LPS (Lipopolysaccharides), lipopolysaccharide (sigma, catalog number: L4391); 96-well cell culture plates (Corning).

[0489] According to the instructions of the separation medium Ficoll-PaqueTM Plus reagent, healthy human PBMCs were separated and resuspended in 1640 medium containing 2% FBS and placed in a cell culture incubator at 37°C and 5% CO2. After 2 hours, the supernatant was removed, the adherent cells were washed twice with PBS, and 1640 complete medium (containing 10% FBS) and 100ng / mL human M-CSF were added for induction for 7 days. On the 3rd and 5th days, the medium was changed and M-CSF was supplemented to induce HPMM. After HPMM induction was completed on the 7th day, the cells were collected, the concentration was adjusted to 100,000 / mL with complete medium and dispensed into 96-well plates, and recombinant human IFN-γ (50ng / mL) was added, and the cells were placed in the incubator for incubation for 24 hours. After 24 hours, the CHO-K1-PD1 cells expressing human PD-1 or the U87-MG cells constitutively expressing human CD73 in the logarithmic phase were collected, and the concentration was adjusted to 300,000 / mL with complete medium after resuspension. The antibody was diluted with complete medium to a working concentration of 25nM, 2.5nM, and 0.25nM. Isotype control antibodies and blank controls were designed at the same time. The supernatant in the 96-well plate was removed, and CHO-K1-PD1 or U87-MG cell suspension and antibody (final volume 200μL) were added and mixed, and placed in an incubator for 24h. After centrifugation at 500xg for 5min, the supernatant was collected and the secretion of IL-8 and IL-6 was detected using a Dako kit. LPS was used as a positive control drug, and the concentration was adjusted to 100ng / mL in the experiment using complete medium.

[0490] In this embodiment, CHO-K1-PD1 and U87-MG cells were used as target cells and co-cultured with HPMM to induce HPMM activation. After the activated HPMM was linked to the target cells through antibody Fab, the antibody Fc segment interacted with FcγR on HPMM, causing HPMM to secrete cytokines.

[0491] 3. Experimental results

[0492] like Figure 32-Figure 35 shown.

[0493] The results showed that compared with wild-type IgG1 subtype PD-1 antibody or CD73 antibody, the anti-PD-1 / CD73 bispecific antibody carrying Fc segment mutation can effectively eliminate the secretion of IL-6 and / or IL-8 by immune cells.

[0494] The preferred embodiments of the present invention are described in detail above, but the invention is not limited to the embodiments. Those skilled in the art may make various equivalent modifications or substitutions without violating the spirit of the invention. These equivalent modifications or substitutions are all included in the scope defined by the claims of this application.

[0495]

[0496]

[0497]

[0498]

[0499]

[0500]

[0501]

[0502]

[0503]

[0504]

[0505]

[0506]

[0507]

Claims

1. An anti-CD73-anti-PD-1 bispecific antibody comprising: a first protein functional domain, wherein the first protein functional domain targets PD-1, and A second protein functional domain, wherein the second protein functional domain targets CD73, Among them, according to the Kabat, Chothia or IMGT numbering system, The first protein functional region comprises HCDR1, HCDR2 and HCDR3 contained in the heavy chain variable region as shown in SEQ ID NO:44, and LCDR1, LCDR2 and LCDR3 contained in the light chain variable region as shown in SEQ ID NO:49; and the second protein functional region comprises HCDR1, HCDR2 and HCDR3 contained in the heavy chain variable region as shown in SEQ ID NO:2, and LCDR1, LCDR2 and LCDR3 contained in the light chain variable region as shown in SEQ ID NO:7, Wherein: 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; wherein the antigen binding fragment is Fab, F(ab')2 or a single-chain antibody.

2. The anti-CD73-anti-PD-1 bispecific antibody of claim 1, wherein the amino acid sequences of HCDR1, HCDR2 and HCDR3 comprised in the first protein functional region are the sequences shown in SEQ ID NOs: 45-47, respectively, according to the IMGT numbering system; The amino acid sequences of LCDR1, LCDR2 and LCDR3 contained in the first protein functional region are respectively as shown in SEQ ID NO: 50-52; or The amino acid sequences of HCDR1, HCDR2 and HCDR3 contained in the second protein functional region are respectively the sequences shown in SEQ ID NOs: 3-5; The amino acid sequences of LCDR1, LCDR2 and LCDR3 contained in the second protein functional region are the sequences shown in SEQ ID NOs: 8-10 respectively.

3. The anti-CD73-anti-PD-1 bispecific antibody of claim 1, wherein: The first protein functional region comprises an amino acid sequence as shown in SEQ ID NO:44 or a sequence having at least 89% sequence identity with the sequence shown in SEQ ID NO:44; and an amino acid sequence as shown in SEQ ID NO:49 or a sequence having at least 90% sequence identity with the sequence shown in SEQ ID NO:49, or The first protein functional region comprises the amino acid sequence of SEQ ID NO:62 or a sequence having at least 89% sequence identity with the sequence of SEQ ID NO:62; and comprises the amino acid sequence of SEQ ID NO:64 or a sequence having at least 90% sequence identity with the sequence of SEQ ID NO:64, and / or, The second protein functional region comprises the amino acid sequence of SEQ ID NO:2 or a sequence having at least 86% sequence identity with the sequence of SEQ ID NO:2; and comprises the amino acid sequence of SEQ ID NO:7 or a sequence having at least 90% sequence identity with the sequence of SEQ ID NO:7; and / or, The second protein functional region comprises the amino acid sequence of SEQ ID NO:20 or a sequence having at least 86% sequence identity with the sequence of SEQ ID NO:20; and comprises the sequence of SEQ ID NO:22 or a sequence having at least 92% sequence identity with the sequence of SEQ ID NO:22; and / or, The second protein functional region comprises the amino acid sequence of SEQ ID NO: 20 or a sequence having at least 86% sequence identity with the sequence of SEQ ID NO: 20, and comprises the amino acid sequence of SEQ ID NO: 24 or a sequence having at least 90% sequence identity with the sequence of SEQ ID NO: 24; and / or, The second protein functional region comprises the sequence shown in the amino acid sequence SEQ ID NO:93 or a sequence having at least 91% sequence identity with the sequence shown in SEQ ID NO:93, and comprises the sequence shown in the amino acid sequence SEQ ID NO:95 or a sequence having at least 94% sequence identity with the sequence shown in SEQ ID NO:

95.

4. The anti-CD73-anti-PD-1 bispecific antibody of claim 3, wherein the first protein functional region comprises a sequence having at least 90% sequence identity to the sequence shown in SEQ ID NO: 44; or the first protein functional region comprises a sequence having at least 91% sequence identity with the sequence shown in SEQ ID NO:49; or the first protein functional region comprises a sequence having at least 90% sequence identity with the sequence shown in SEQ ID NO:62; or the first protein functional region comprises a sequence having at least 91% sequence identity with the sequence shown in SEQ ID NO:64; or the second protein functional region comprises a sequence having at least 87% sequence identity with the sequence shown in SEQ ID NO: 2; or the second protein functional region comprises a sequence having at least 91% sequence identity with the sequence shown in SEQ ID NO:7; or the second protein functional region comprises a sequence having at least 87% sequence identity with the sequence shown in SEQ ID NO: 20; or the second protein functional region comprises a sequence having at least 93% sequence identity with the sequence shown in SEQ ID NO:22; or the second protein functional region comprises a sequence having at least 91% sequence identity with the sequence shown in SEQ ID NO:24; or the second protein functional region comprises a sequence having at least 92% sequence identity with the sequence shown in SEQ ID NO:93; Or the second protein functional region comprises a sequence having at least 95% sequence identity with the sequence shown in SEQ ID NO:

95.

5. The anti-CD73-anti-PD-1 bispecific antibody of claim 3, wherein the first protein functional region comprises a sequence having at least 91% sequence identity to the sequence shown in SEQ ID NO: 44; or the first protein functional region comprises a sequence having at least 92% sequence identity with the sequence shown in SEQ ID NO:49; or the first protein functional region comprises a sequence having at least 91% sequence identity with the sequence shown in SEQ ID NO:62; or the first protein functional region comprises a sequence having at least 92% sequence identity with the sequence shown in SEQ ID NO:64; or the second protein functional region comprises a sequence having at least 88% sequence identity with the sequence shown in SEQ ID NO: 2; or the second protein functional region comprises a sequence having at least 92% sequence identity with the sequence shown in SEQ ID NO:7; or the second protein functional region comprises a sequence having at least 88% sequence identity with the sequence shown in SEQ ID NO: 20; or the second protein functional region comprises a sequence having at least 94% sequence identity with the sequence shown in SEQ ID NO:22; or the second protein functional region comprises a sequence having at least 92% sequence identity with the sequence shown in SEQ ID NO:24; or the second protein functional region comprises a sequence having at least 93% sequence identity with the sequence shown in SEQ ID NO:93; Or the second protein functional region comprises a sequence having at least 96% sequence identity with the sequence shown in SEQ ID NO:

95.

6. The anti-CD73-anti-PD-1 bispecific antibody of claim 3, wherein the first protein functional region comprises a sequence having at least 92% sequence identity to the sequence shown in SEQ ID NO: 44; or the first protein functional region comprises a sequence having at least 93% sequence identity with the sequence shown in SEQ ID NO:49; or the first protein functional region comprises a sequence having at least 92% sequence identity with the sequence shown in SEQ ID NO:62; or the first protein functional region comprises a sequence having at least 93% sequence identity with the sequence shown in SEQ ID NO:64; or the second protein functional region comprises a sequence having at least 89% sequence identity with the sequence shown in SEQ ID NO: 2; or the second protein functional region comprises a sequence having at least 93% sequence identity with the sequence shown in SEQ ID NO:7; or the second protein functional region comprises a sequence having at least 89% sequence identity with the sequence shown in SEQ ID NO: 20; or the second protein functional region comprises a sequence having at least 95% sequence identity with the sequence shown in SEQ ID NO:22; or the second protein functional region comprises a sequence having at least 93% sequence identity with the sequence shown in SEQ ID NO:24; or the second protein functional region comprises a sequence having at least 94% sequence identity with the sequence shown in SEQ ID NO:93; Or the second protein functional region comprises a sequence having at least 97% sequence identity with the sequence shown in SEQ ID NO:

95.

7. The anti-CD73-anti-PD-1 bispecific antibody of claim 3, wherein the first protein functional region comprises a sequence having at least 93% sequence identity to the sequence shown in SEQ ID NO: 44; or the first protein functional region comprises a sequence having at least 94% sequence identity with the sequence shown in SEQ ID NO:49; or the first protein functional region comprises a sequence having at least 93% sequence identity with the sequence shown in SEQ ID NO:62; or the first protein functional region comprises a sequence having at least 94% sequence identity with the sequence shown in SEQ ID NO:64; or the second protein functional region comprises a sequence having at least 90% sequence identity with the sequence shown in SEQ ID NO: 2; or the second protein functional region comprises a sequence having at least 94% sequence identity with the sequence shown in SEQ ID NO:7; or the second protein functional region comprises a sequence having at least 90% sequence identity with the sequence shown in SEQ ID NO: 20; or the second protein functional region comprises a sequence having at least 96% sequence identity with the sequence shown in SEQ ID NO:22; or the second protein functional region comprises a sequence having at least 94% sequence identity with the sequence shown in SEQ ID NO:24; or the second protein functional region comprises a sequence having at least 95% sequence identity with the sequence shown in SEQ ID NO:93; Or the second protein functional region comprises a sequence having at least 98% sequence identity with the sequence shown in SEQ ID NO:

95.

8. The anti-CD73-anti-PD-1 bispecific antibody of claim 3, wherein the first protein functional region comprises a sequence having at least 94% sequence identity to the sequence shown in SEQ ID NO: 44; or the first protein functional region comprises a sequence having at least 95% sequence identity with the sequence shown in SEQ ID NO:49; or the first protein functional region comprises a sequence having at least 94% sequence identity with the sequence shown in SEQ ID NO:62; or the first protein functional region comprises a sequence having at least 95% sequence identity with the sequence shown in SEQ ID NO:64; or the second protein functional region comprises a sequence having at least 91% sequence identity with the sequence shown in SEQ ID NO: 2; or the second protein functional region comprises a sequence having at least 95% sequence identity with the sequence shown in SEQ ID NO:7; or the second protein functional region comprises a sequence having at least 91% sequence identity with the sequence shown in SEQ ID NO: 20; or the second protein functional region comprises a sequence having at least 97% sequence identity with the sequence shown in SEQ ID NO:22; or the second protein functional region comprises a sequence having at least 95% sequence identity with the sequence shown in SEQ ID NO:24; or the second protein functional region comprises a sequence having at least 96% sequence identity with the sequence shown in SEQ ID NO:93; Or the second protein functional region comprises a sequence having at least 99% sequence identity with the sequence shown in SEQ ID NO:

95.

9. The anti-CD73-anti-PD-1 bispecific antibody of claim 3, wherein the first protein functional region comprises a sequence having at least 95% sequence identity to the sequence shown in SEQ ID NO: 44; or the first protein functional region comprises a sequence having at least 96% sequence identity with the sequence shown in SEQ ID NO:49; or the first protein functional region comprises a sequence having at least 95% sequence identity with the sequence shown in SEQ ID NO:62; or comprising a sequence having at least 96% sequence identity to the sequence shown in SEQ ID NO:64; or the second protein functional region comprises a sequence having at least 92% sequence identity with the sequence shown in SEQ ID NO: 2; or the second protein functional region comprises a sequence having at least 96% sequence identity with the sequence shown in SEQ ID NO:7; or the second protein functional region comprises a sequence having at least 92% sequence identity with the sequence shown in SEQ ID NO: 20; or the second protein functional region comprises a sequence having at least 98% sequence identity with the sequence shown in SEQ ID NO:22; or the second protein functional region comprises a sequence having at least 96% sequence identity with the sequence shown in SEQ ID NO:24; Or the second protein functional region comprises a sequence having at least 97% sequence identity with the sequence shown in SEQ ID NO:

93.

10. The anti-CD73-anti-PD-1 bispecific antibody of claim 3, wherein the first protein functional region comprises a sequence having at least 96% sequence identity to the sequence shown in SEQ ID NO: 44; or the first protein functional region comprises a sequence having at least 97% sequence identity with the sequence shown in SEQ ID NO:49; or the first protein functional region comprises a sequence having at least 96% sequence identity with the sequence shown in SEQ ID NO:62; or the first protein functional region comprises a sequence having at least 97% sequence identity with the sequence shown in SEQ ID NO:64; or the second protein functional region comprises a sequence having at least 93% sequence identity with the sequence shown in SEQ ID NO: 2; or the second protein functional region comprises a sequence having at least 97% sequence identity with the sequence shown in SEQ ID NO:7; or the second protein functional region comprises a sequence having at least 93% sequence identity with the sequence shown in SEQ ID NO: 20; or the second protein functional region comprises a sequence having at least 99% sequence identity with the sequence shown in SEQ ID NO:22; or the second protein functional region comprises a sequence having at least 97% sequence identity with the sequence shown in SEQ ID NO:24; Or the second protein functional region comprises a sequence having at least 98% sequence identity with the sequence shown in SEQ ID NO:

93.

11. The anti-CD73-anti-PD-1 bispecific antibody of claim 3, wherein the first protein functional region comprises a sequence having at least 97% sequence identity to the sequence shown in SEQ ID NO: 44; or the first protein functional region comprises a sequence having at least 98% sequence identity with the sequence shown in SEQ ID NO:49; or the first protein functional region comprises a sequence having at least 97% sequence identity with the sequence shown in SEQ ID NO:62; or the first protein functional region comprises a sequence having at least 98% sequence identity with the sequence shown in SEQ ID NO:64; or the second protein functional region comprises a sequence having at least 94% sequence identity with the sequence shown in SEQ ID NO: 2; or the second protein functional region comprises a sequence having at least 98% sequence identity with the sequence shown in SEQ ID NO:7; or the second protein functional region comprises a sequence having at least 94% sequence identity with the sequence shown in SEQ ID NO: 20; or the second protein functional region comprises a sequence having at least 98% sequence identity with the sequence shown in SEQ ID NO:24; Or the second protein functional region comprises a sequence having at least 99% sequence identity with the sequence shown in SEQ ID NO:

93.

12. The anti-CD73-anti-PD-1 bispecific antibody of claim 3, wherein the first protein functional region comprises a sequence having at least 98% sequence identity to the sequence shown in SEQ ID NO: 44; or the first protein functional region comprises a sequence having at least 99% sequence identity with the sequence shown in SEQ ID NO:49; or the first protein functional region comprises a sequence having at least 98% sequence identity with the sequence shown in SEQ ID NO:62; or the first protein functional region comprises a sequence having at least 99% sequence identity with the sequence shown in SEQ ID NO:64; or the second protein functional region comprises a sequence having at least 95% sequence identity with the sequence shown in SEQ ID NO: 2; or the second protein functional region comprises a sequence having at least 99% sequence identity with the sequence shown in SEQ ID NO:7; or the second protein functional region comprises a sequence having at least 95% sequence identity with the sequence shown in SEQ ID NO: 20; Or the second protein functional region comprises a sequence having at least 99% sequence identity with the sequence shown in SEQ ID NO:

24.

13. The anti-CD73-anti-PD-1 bispecific antibody of claim 3, wherein the first protein functional region comprises a sequence having at least 99% sequence identity to the sequence shown in SEQ ID NO: 44; or the first protein functional region comprises a sequence having at least 99% sequence identity with the sequence shown in SEQ ID NO:62; or the second protein functional region comprises a sequence having at least 96% sequence identity with the sequence shown in SEQ ID NO: 2; Or the second protein functional region comprises a sequence having at least 96% sequence identity with the sequence shown in SEQ ID NO:

20.

14. The anti-CD73-anti-PD-1 bispecific antibody of claim 3, wherein the second protein functional region comprises a sequence having at least 97% sequence identity to the sequence shown in SEQ ID NO: 2; Or the second protein functional region comprises a sequence having at least 97% sequence identity with the sequence shown in SEQ ID NO:

20.

15. The anti-CD73-anti-PD-1 bispecific antibody of claim 3, wherein the second protein functional region comprises a sequence having at least 98% sequence identity to the sequence shown in SEQ ID NO: 2; Or the second protein functional region comprises a sequence having at least 98% sequence identity with the sequence shown in SEQ ID NO:

20.

16. The anti-CD73-anti-PD-1 bispecific antibody of claim 3, wherein the second protein functional region comprises a sequence having at least 99% sequence identity to the sequence shown in SEQ ID NO: 2; Or the second protein functional region comprises a sequence having at least 99% sequence identity with the sequence shown in SEQ ID NO:

20.

17. The anti-CD73-anti-PD-1 bispecific antibody of claim 1 or 2, wherein: The first protein functional region and the second protein functional region are independently one or more than two.

18. The anti-CD73-anti-PD-1 bispecific antibody of any one of claims 1-2, wherein: The first protein functional region and the second protein functional region are connected directly or through a connecting fragment.

19. The anti-CD73-anti-PD-1 bispecific antibody of claim 18, wherein: The connecting fragment is (GGGGS)n, where n is a positive integer.

20. The anti-CD73-anti-PD-1 bispecific antibody of claim 19, wherein n is 1, 2, 3, 4, 5 or 6.

21. The anti-CD73-anti-PD-1 bispecific antibody of any one of claims 1-2, wherein: The N-terminus of the heavy chain variable region of the antigen-binding fragment is directly or via a connecting fragment connected 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 directly or via a connecting fragment connected to the C-terminus of the light chain constant region CL of the immunoglobulin; or the N-terminus of the heavy chain variable region of the antigen-binding fragment is directly or via a connecting fragment connected to the C-terminus of the light chain constant region CL of the immunoglobulin, and the N-terminus of the light chain variable region of the antigen-binding fragment is directly or via a connecting fragment connected to the C-terminus of the heavy chain constant region CH1 of the immunoglobulin, or The C-terminus of the heavy chain variable region of the antigen-binding fragment is directly or via a connecting fragment connected 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 directly or via a connecting fragment connected 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 directly or via a connecting fragment connected 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 directly or via a connecting fragment connected to the N-terminus of the heavy chain of the immunoglobulin.

22. The anti-CD73-anti-PD-1 bispecific antibody of any one of claims 1-2, wherein: The antigen-binding fragment is a single-chain antibody.

23. The anti-CD73-anti-PD-1 bispecific antibody of any one of claims 1-2, wherein: The first protein functional region is an immunoglobulin, and the second protein functional region is a single-chain antibody; or, the first protein functional region is a single-chain antibody, and the second protein functional region is an immunoglobulin.

24. The anti-CD73-anti-PD-1 bispecific antibody of claim 22, wherein: The single-chain antibody is connected to the antibody heavy chain variable region (V H ) and antibody light chain variable region (V L ) molecules.

25. The anti-CD73-anti-PD-1 bispecific antibody of claim 24, wherein: The single-chain antibody has the structure: NH2-V L -Connection fragment-V H -COOH or NH2-V H -Connection fragment-V L -COOH.

26. The anti-CD73-anti-PD-1 bispecific antibody of claim 25, wherein: When the single-chain antibody is connected to the C-terminus of the heavy chain of the immunoglobulin, the N-terminus of the heavy chain, or the C-terminus of CH1 of the heavy chain constant region through a connecting fragment, the antibody heavy chain variable region (V H ), or, firstly, connecting the antibody light chain variable region (V L ).

27. The anti-CD73-anti-PD-1 bispecific antibody of claim 26, wherein: Single-chain antibody has the structure: Linker fragment-V H -Connection fragment-V L -COOH, or, linker fragment -V L -Connection fragment-V H -COOH.

28. The anti-CD73-anti-PD-1 bispecific antibody of claim 26, wherein: According to the IMGT numbering system, The immunoglobulin, wherein the heavy chain variable region comprises CDRs with amino acid sequences of SEQ ID NOs: 3-5, and the light chain variable region comprises CDRs with amino acid sequences of SEQ ID NOs: 8-10; The single-chain antibody has a heavy chain variable region comprising CDRs with amino acid sequences of SEQ ID NOs: 45-47, and a light chain variable region comprising CDRs with amino acid sequences of SEQ ID NOs: 50-52.

29. The anti-CD73-anti-PD-1 bispecific antibody of claim 26, according to the IMGT numbering system, wherein: When the single-chain antibody is connected to the C-terminus of the heavy chain of the immunoglobulin through the connecting fragment, the antibody heavy chain variable region (V H ), or, firstly, connecting the antibody light chain variable region (V L ).

30. The anti-CD73-anti-PD-1 bispecific antibody of claim 26, according to the IMGT numbering system, in: The immunoglobulin, wherein the heavy chain variable region comprises CDRs with amino acid sequences of SEQ ID NOs: 45-47, and the light chain variable region comprises CDRs with amino acid sequences of SEQ ID NOs: 50-52; and / or, The single-chain antibody, wherein the heavy chain variable region comprises CDRs with amino acid sequences of SEQ ID NOs: 3-5, and the light chain variable region comprises CDRs with amino acid sequences of SEQ ID NOs: 8-10, Wherein, when the single-chain antibody is connected to the C-terminus of the heavy chain of the immunoglobulin through the connecting fragment, the antibody heavy chain variable region (V H ), or, firstly, connecting the antibody light chain variable region (V L ).

31. The anti-CD73-anti-PD-1 bispecific antibody of claim 26 or 27, wherein: The single-chain antibody is NH2-V L -Connection fragment-V H -COOH or NH2-V H -Connection fragment-V L -COOH.

32. The anti-CD73-anti-PD-1 bispecific antibody of claim 26, wherein: One immunoglobulin molecule is linked to two single-chain antibody molecules.

33. The anti-CD73-anti-PD-1 bispecific antibody of claim 32, wherein: The two single-chain antibody molecules are identical.

34. The anti-CD73-anti-PD-1 bispecific antibody of claim 1 or 2, wherein: The immunoglobulin is IgG, IgA, IgD, IgE or IgM.

35. The anti-CD73-anti-PD-1 bispecific antibody of claim 34, wherein: The immunoglobulin is IgG.

36. The anti-CD73-anti-PD-1 bispecific antibody of claim 35, wherein: The immunoglobulin is IgG1, IgG2, IgG3 or IgG4.

37. The anti-CD73-anti-PD-1 bispecific antibody of claim 22, wherein: The single-chain antibody is linked to the C-terminus of the heavy chain of the immunoglobulin.

38. The anti-CD73-anti-PD-1 bispecific antibody of claim 37, wherein: One immunoglobulin molecule is linked to two single-chain antibody molecules.

39. The anti-CD73-anti-PD-1 bispecific antibody of claim 38, wherein: The two single-chain antibody molecules are identical.

40. The anti-CD73-anti-PD-1 bispecific antibody of claim 22, according to the IMGT numbering system, in: The immunoglobulin, wherein the heavy chain variable region comprises CDRs with amino acid sequences of SEQ ID NOs: 45-47, and the light chain variable region comprises CDRs with amino acid sequences of SEQ ID NOs: 50-52; and / or, The single-chain antibody has a heavy chain variable region comprising CDRs with amino acid sequences of SEQ ID NOs: 3-5, and a light chain variable region comprising CDRs with amino acid sequences of SEQ ID NOs: 8-10.

41. The anti-CD73-anti-PD-1 bispecific antibody of claim 40, according to the IMGT numbering system, in: When the single-chain antibody is connected to the C-terminus of the heavy chain of the immunoglobulin through the connecting fragment, the antibody heavy chain variable region (V H ), or, firstly, connecting the antibody light chain variable region (V L ).

42. The anti-CD73-anti-PD-1 bispecific antibody of claim 22, according to the IMGT numbering system, in: The immunoglobulin, wherein the heavy chain variable region comprises CDRs with amino acid sequences of SEQ ID NOs: 3-5, and the light chain variable region comprises CDRs with amino acid sequences of SEQ ID NOs: 8-10; and / or, The single-chain antibody, wherein the heavy chain variable region comprises CDRs with amino acid sequences of SEQ ID NOs: 45-47, and the light chain variable region comprises CDRs with amino acid sequences of SEQ ID NOs: 50-52, Wherein, when the single-chain antibody is connected to the C-terminus of the heavy chain of the immunoglobulin through the connecting fragment, the antibody heavy chain variable region (V) of the single-chain antibody comprising the CDR of the amino acid sequence of SEQ ID NO: 45-47 is first connected. H ), or, firstly, connecting the antibody light chain variable region (V L ).

43. The anti-CD73-anti-PD-1 bispecific antibody of claim 22, wherein: The amino acid sequence of the heavy chain variable region of the immunoglobulin is selected from SEQ ID NO: 44 or SEQ ID NO: 62; the amino acid sequence of the light chain variable region of the immunoglobulin is selected from SEQ ID NO: 49 or SEQ ID NO: 64, respectively; and / or, The amino acid sequence of the heavy chain variable region of the single-chain antibody is selected from SEQ ID NO: 2 or SEQ ID NO: 20; the amino acid sequence of the light chain variable region of the single-chain antibody is selected from SEQ ID NO: 7 and SEQ ID NO: 22 respectively; or the amino acid sequence of the heavy chain variable region of the single-chain antibody is SEQ ID NO: 20, and the amino acid sequence of the light chain variable region of the single-chain antibody is SEQ ID NO: 24; or the amino acid sequence of the heavy chain variable region of the single-chain antibody is SEQ ID NO: 93, and the amino acid sequence of the light chain variable region of the single-chain antibody is SEQ ID NO: 95; When the single-chain antibody is connected to the C-terminus of the heavy chain of the immunoglobulin through the connecting fragment, the antibody heavy chain variable region (V H ), or, firstly, connecting the antibody light chain variable region (V L ).

44. The anti-CD73-anti-PD-1 bispecific antibody of claim 22, wherein: The amino acid sequence of the heavy chain variable region of the immunoglobulin is selected from SEQ ID NO: 2 or SEQ ID NO: 20; the amino acid sequence of the light chain variable region of the immunoglobulin is selected from SEQ ID NO: 7 or SEQ ID NO: 22, or the amino acid sequence of the heavy chain variable region of the immunoglobulin is SEQ ID NO: 20, and the amino acid sequence of the light chain variable region of the immunoglobulin is SEQ ID NO: 24; or the amino acid sequence of the heavy chain variable region of the immunoglobulin is SEQ ID NO: 93, and the amino acid sequence of the light chain variable region of the immunoglobulin is SEQ ID NO: 95; and / or, The amino acid sequence of the heavy chain variable region of the single-chain antibody is selected from SEQ ID NO: 44 or SEQ ID NO: 62; the amino acid sequence of the light chain variable region of the single-chain antibody is selected from SEQ ID NO: 49 or SEQ ID NO: 64, When the single-chain antibody is connected to the C-terminus of the heavy chain via a linker, the antibody heavy chain variable region (V H ), or, first connect the antibody light chain variable region (V L ).

45. The anti-CD73-anti-PD-1 bispecific antibody of claim 1, wherein: The immunoglobulin includes non-CDR regions, and the non-CDR regions are from a species other than murine.

46. ​​The anti-CD73-anti-PD-1 bispecific antibody of claim 45, wherein: The non-CDR regions are from human antibodies.

47. The anti-CD73-anti-PD-1 bispecific antibody of claim 45, wherein: The constant regions of the immunoglobulins are humanized.

48. The anti-CD73-anti-PD-1 bispecific antibody of claim 45, wherein: The heavy chain constant region of the immunoglobulin adopts Ig gamma-1chain C region, ACCESSION: P01857; the light chain constant region adopts Ig kappachain C region, ACCESSION: P01834, or The heavy chain constant region of the immunoglobulin is mutated at any two or three of positions 234, 235 and 237 based on Ig gamma-1chain C region, ACCESSION: P01857, and after mutation, the affinity constant of the bispecific antibody to FcγRIa, FcγRIIIa and / or C1q is reduced compared with that before mutation.

49. The anti-CD73-anti-PD-1 bispecific antibody of claim 45, wherein: According to the EU numbering system, in Iggamma-1 chain C region, ACCESSION: P01857, the following mutations are present at positions 234, 235 and / or 237: L234A and L235A; or L234A and G237A; or L235A and G237A; or L234A, L235A, G237A.

50. The anti-CD73-anti-PD-1 bispecific antibody of claim 45, based on Ig gamma-1 chain C region, ACCESSION: P01857 according to the EU numbering system, in: The heavy chain constant region of the immunoglobulin further has one or more mutations selected from the following: N297A, D265A, D270A, P238D, L328E, E233D, H268D, P271G, A330R, C226S, C229S, E233P, P331S, S267E, L328F, A330L, M252Y, S254T, T256E, N297Q, P238S, P238A, A327Q, A327G, P329A, K322A, T394D, G236R, G236A, L328R, A330S, P331S, H268A, E318A, and K320A.

51. The anti-CD73-anti-PD-1 bispecific antibody of claim 45, wherein: The anti-CD73-anti-PD-1 bispecific antibody structure is shown as heavy chain-light chain-connecting fragment 1-scFv, and the scFv is selected from 14C12H1V-connecting fragment 2-14C12L1V, 14C12H1V-connecting fragment 1-14C12L1V, and is specifically selected from the group consisting of: (1) NTPDV1, whose heavy chain amino acid sequence is shown in SEQ ID NO:85, whose light chain amino acid sequence is shown in SEQ ID NO:28, whose amino acid sequence of connecting segment 1 is shown in SEQ ID NO:79, whose amino acid sequence of 14C12H1V is shown in SEQ ID NO:62, whose amino acid sequence of connecting segment 2 is shown in SEQ ID NO:81, whose amino acid sequence of 14C12L1V is shown in SEQ ID NO:64, (2) NTPDV2, whose heavy chain amino acid sequence is shown in SEQ ID NO:83, whose light chain amino acid sequence is shown in SEQ ID NO:28, whose amino acid sequence of connecting segment 1 is shown in SEQ ID NO:79, whose amino acid sequence of 14C12H1V is shown in SEQ ID NO:62, whose amino acid sequence of connecting segment 1 is shown in SEQ ID NO:79, whose amino acid sequence of 14C12L1V is shown in SEQ ID NO:64, (3) NTPDV3, whose heavy chain amino acid sequence is shown in SEQ ID NO:85, whose light chain amino acid sequence is shown in SEQ ID NO:96, whose amino acid sequence of connecting segment 1 is shown in SEQ ID NO:79, whose amino acid sequence of 14C12H1V is shown in SEQ ID NO:62, whose amino acid sequence of connecting segment 2 is shown in SEQ ID NO:81, whose amino acid sequence of 14C12L1V is shown in SEQ ID NO:64, and (4) NTPDV4, whose heavy chain amino acid sequence is shown in SEQ ID NO:83, the light chain amino acid sequence is shown in SEQ ID NO:96, the amino acid sequence of connecting segment 1 is shown in SEQ ID NO:79, the amino acid sequence of 14C12H1V is shown in SEQ ID NO:62, the amino acid sequence of connecting segment 1 is shown in SEQ ID NO:79, and the amino acid sequence of 14C12L1V is shown in SEQ ID NO:64; Or the anti-CD73-anti-PD-1 bispecific antibody structure is as shown in heavy chain-light chain-connecting fragment 1 or 2-scFv, specifically selected from the group consisting of: (i) P1D7V01, the amino acid sequence of the heavy chain of the immunoglobulin is shown in SEQ ID NO:66, the amino acid sequence of the light chain of the immunoglobulin is shown in SEQ ID NO:68, the amino acid sequence of the connecting fragment 1 is shown in SEQ ID NO:79, and the scFv is composed of the following sequences: the heavy chain variable region with the amino acid sequence shown in SEQ ID NO:20, the connecting fragment 1 with the amino acid sequence shown in SEQ ID NO:79, and the light chain variable region with the amino acid sequence shown in SEQ ID NO:24, (ii) P1D7V02R, the amino acid sequence of the heavy chain of the immunoglobulin is shown in SEQ ID NO:66, the amino acid sequence of the light chain of the immunoglobulin is shown in SEQ ID NO:68, the amino acid sequence of the connecting fragment 1 is shown in SEQ ID NO:79, and the scFv is composed of the following sequences: the light chain variable region with an amino acid sequence as shown in SEQ ID NO:24, the connecting fragment 1 with an amino acid sequence as shown in SEQ ID NO:79, and the heavy chain variable region with an amino acid sequence as shown in SEQ ID NO:20, (iii) P1D7V03, whose immunoglobulin heavy chain amino acid sequence is shown in SEQ ID NO:66, the immunoglobulin light chain amino acid sequence is shown in SEQ ID NO:68, the amino acid sequence of the connecting fragment 2 is shown in SEQ ID NO:81, and the scFv consists of the following sequences: the heavy chain variable region amino acid sequence shown in SEQ ID NO:20, the connecting fragment 1 amino acid sequence shown in SEQ ID NO:79, the light chain variable region amino acid sequence shown in SEQ ID NO:24, and (iv) P1D7V04R, whose immunoglobulin heavy chain amino acid sequence is shown in SEQ ID NO:66, the immunoglobulin light chain amino acid sequence is shown in SEQ ID NO:68, the amino acid sequence of connecting fragment 2 is shown in SEQ ID NO:81, and the scFv consists of the following sequence: the light chain variable region amino acid sequence shown in SEQ ID NO:24, the connecting fragment 1 amino acid sequence shown in SEQ ID NO:79, and the heavy chain variable region amino acid sequence shown in SEQ ID NO:

20.

52. The anti-CD73-anti-PD-1 bispecific antibody of any one of claims 1-2, wherein: The anti-CD73-anti-PD-1 bispecific antibody is less than 10 -5 M of K D Binds to CD73 protein and / or PD-1 protein.

53. The anti-CD73-anti-PD-1 bispecific antibody of any one of claims 1-2, wherein: The anti-CD73-anti-PD-1 bispecific antibody is less than 10 -6 M of K D Binds to CD73 protein and / or PD-1 protein.

54. The anti-CD73-anti-PD-1 bispecific antibody of any one of claims 1-2, wherein: The anti-CD73-anti-PD-1 bispecific antibody is less than 10 -7 M of K D Binds to CD73 protein and / or PD-1 protein.

55. The anti-CD73-anti-PD-1 bispecific antibody of any one of claims 1-2, wherein: The anti-CD73-anti-PD-1 bispecific antibody is less than 10 -8 M of K D Binds to CD73 protein and / or PD-1 protein.

56. The anti-CD73-anti-PD-1 bispecific antibody of any one of claims 1-2, wherein: The anti-CD73-anti-PD-1 bispecific antibody is less than 10 -9 M of K D Binds to CD73 protein and / or PD-1 protein.

57. The anti-CD73-anti-PD-1 bispecific antibody of any one of claims 1-2, wherein: The anti-CD73-anti-PD-1 bispecific antibody is less than 10 -10 M of K D Binds to CD73 protein and / or PD-1 protein.

58. An isolated nucleic acid molecule encoding the bispecific antibody of any one of claims 1-57.

59. A vector comprising the isolated nucleic acid molecule of claim 58.

60. A host cell comprising the isolated nucleic acid molecule of claim 58, or the vector of claim 59.

61. A method for preparing the anti-CD73-anti-PD-1 bispecific antibody according to any one of claims 1 to 57, comprising culturing the host cell of claim 60 under suitable conditions, and recovering the bispecific antibody from the cell culture.

62. A conjugate comprising the anti-CD73-anti-PD-1 bispecific antibody of any one of claims 1-57 and a conjugated moiety, wherein: The conjugated moiety is a detectable label.

63. The conjugate of claim 62, wherein the conjugated moiety is a radioisotope, a fluorescent substance, a chemiluminescent substance, a colored substance or an enzyme.

64. A kit comprising the anti-CD73-anti-PD-1 bispecific antibody of any one of claims 1-57, or the conjugate of claim 62 or 63.

65. The kit of claim 64, wherein the kit further comprises a second antibody that specifically recognizes the bispecific antibody.

66. The kit of claim 65, wherein the second antibody further comprises a detectable label.

67. The kit of claim 66, wherein the detectable label is a radioisotope, a fluorescent substance, a chemiluminescent substance, a colored substance or an enzyme.

68. Use of the anti-CD73-anti-PD-1 bispecific antibody according to any one of claims 1 to 57 in the preparation of a kit for detecting the presence or level of CD73 and / or PD-1 in a sample.

69. A pharmaceutical composition comprising the anti-CD73-anti-PD-1 bispecific antibody of any one of claims 1-57 or the conjugate of claim 62 or 63.

70. The pharmaceutical composition of claim 69, further comprising a pharmaceutically acceptable carrier and / or excipient.

71. Use of the anti-CD73-anti-PD-1 bispecific antibody of any one of claims 1-57 or the conjugate of claim 62 or 63 in the preparation of a medicament for treating tumors or anemia, or use of the anti-CD73-anti-PD-1 bispecific antibody of any one of claims 1-57 or the conjugate of claim 62 or 63 in the preparation of a medicament for diagnosing tumors or anemia.

72. A non-diagnostic or non-therapeutic in vitro method comprising the step of applying to cells an effective amount of the anti-CD73-anti-PD-1 bispecific antibody of any one of claims 1-57 or the conjugate of claim 62 or 63.

Images