Bispecific antibodies, pharmaceutical compositions and uses

Abstract

Bispecific antibodies, pharmaceutical compositions, and applications. Specifically, the bispecific antibody comprises a first protein functional domain and a second protein functional domain, wherein the first protein functional domain targets LAG3, and the second protein functional domain targets a target other than LAG3.

Description

Technical Field

[0001] Reference to Related Applications This application claims priority based on Chinese Patent Application No. 202310327266.6 (filing date: March 29, 2023), and the entire content is incorporated herein by reference.

[0002] Technical Field This disclosure relates to the field of biomedicine, and relates to bispecific antibodies, pharmaceutical compositions containing bispecific antibodies, and uses thereof.

Background Art

[0003] Tumors, especially malignant tumors, are serious diseases threatening health in today's world and are the second leading cause of death among various diseases. In recent years, the incidence of this disease has been increasing significantly. Malignant tumors are characterized by low treatment efficacy, high late metastasis rate, and poor prognosis. Conventional treatment methods (e.g., radiotherapy, chemotherapy, surgical therapy) performed clinically can significantly relieve pain and extend the survival period, but there are significant limitations to this, and it is difficult to further improve its effectiveness.

[0004] Lymphocyte activation gene 3 (LAG3), that is, CD223, is a type I transmembrane protein consisting of 498 amino acids and belongs to the immunoglobulin superfamily (IgSF). LAG3 is mainly expressed on activated CD4 + T cells and CD8 + T cells. Furthermore, LAG3 is also expressed in cells such as natural killer cells (NK), B cells, regulatory T cells (Treg), plasmacytoid dendritic cells (pDC), etc. (Ruffo Elisa, Wu Richard C, Bruno Tullia C et al. Lymphocyte-activation gene 3 (LAG3): The next immune checkpoint receptor.[J]. Semin Immunol, 2019, 42: 101305).

[0005] The LAG3 gene is located on human chromosome 12 (12p13.3), adjacent to the CD4 gene, and their exons and introns are the same. Although the LAG3 molecule and the CD4 molecule have high structural similarity, their amino acid sequence homology is only about 20%. The major histocompatibility complex class II molecule (MHC-II), liver sinusoidal endothelial cell lectin (LSECtin) molecule, and galectin 3 molecule are related ligands of the LAG3 molecule. The MHC-II molecule is the main ligand of LAG3. The affinity of the LAG3 molecule for the MHC-II molecule (Kd: 60 nmol / L) is 100 times that of the CD4 molecule, indicating that the LAG3 molecule can effectively compete with the CD4 molecule for binding to the MHC-II molecule and inhibit T cell activation.

[0006] In the tumor microenvironment, the expression of the immunosuppressive molecule LAG3 is observed 24 hours after T cell activation, leading to T cell dysfunction or apoptosis. The LAG3 molecule forms a dimer molecule via its D1 domain (containing a proline-rich loop structure) in the first signal transduction axis of T cell activation, "CD3-TCR-MHC-II", and specifically binds to the MHC-II molecule. As a result, in one aspect, the signal transduction pathway of T cell activation is blocked, and in another aspect, the intracellular segment (KIEELE motif) of the LAG3 molecule generates an immunosuppressive signal to downregulate the activity of CD4 + T cells. In the first signal transduction axis of T cell activation, "CD3-TCR-MHC-II", the LAG3 molecule forms a dimer molecule and specifically binds to the MHC-II molecule. As a result, in one aspect, the signal transduction pathway of T cell activation is blocked, and in another aspect, the intracellular segment (KIEELE motif) of the LAG3 molecule generates an immunosuppressive signal to CD4 + T cell activity is downregulated. The LAG3 molecule can promote the differentiation of Treg cells and participate in the downstream signal transduction of signal transducer and activator of transcription 5 to enhance the inhibitory effect of Treg cells. This is one of the mechanisms by which tumors escape from immune killing (Andrews Lawrence P, Marciscano Ariel E, Drake Charles G, et al., LAG3 (CD223) as a cancer immunotherapy target. [J]. Immunol Rev, 2017, 276: 80-96).

[0007] LAG3 is expressed in tumor-infiltrating CD8 of various malignant tumors +Multiple studies have shown that it is overexpressed in T cells. For example, in ovarian cancer, CD8 is specific to tumor-invasive New York esophageal squamous cell carcinoma 1 (NY-ESO-1) antigen. + T cells express high levels of PD-1 and LAG3, leading to decreased production of IFNγ and TNFα, and thus to lymphocyte inactivation. Galectin 3 and LSECtin primarily interact with LAG3, and CD8 + It regulates T cell activation and function. Furthermore, melanoma antigen-specific T cells isolated from metastatic melanoma patients show significantly upregulated expression of LAG3 and other immune checkpoint molecules CTLA4 and TIM3 (Liu Hao, Li Xinying, Luo Longlong, et al., Research advances in biological function of lymphocyte activation gene-3 (LAG-3) molecule and clinical application of antibody drugs targeting LAG-3 [J]. Chinese Journal of Pharmacology and Toxicology, 2019, 33(01): 70-78).

[0008] Currently, several LAG3 antibody drugs are undergoing clinical trials, with Bristol Myers Squib's leratrimab being the most advanced, with 10 trials ongoing. The majority of these trials concern combination therapy with leratrimab and nivolumab for the treatment of tumors such as hematopoietic malignancies, melanoma, glioblastoma, renal cell carcinoma, and non-small cell lung cancer.

[0009] Ecto-5'-nucleotidase, or CD73 protein, is a multifunctional glycoprotein with a molecular weight of 70 kD, encoded by the NT5E gene. It is fixed to the cell membrane by glycosylphosphatidylinositol (GPI) (Zimmermann H. Biochem J. 1992; 285:345-365).

[0010] CD73 is widely distributed on the surface of human tissue cells, and studies have shown that it is highly expressed in various solid tumors, particularly cancer cells, dendritic cells, regulatory T cells (Tregs), natural killer cells (NK cells), myeloid-derived suppressor cells (MDSCs), and tumor-associated macrophages (TAMs), within the tumor microenvironment. A key characteristic of the tumor microenvironment is hypoxia. Hypoxia induces the upregulation of molecules such as hypoxia-inducible factor 1 (HIF-1), which leads to widespread expression of CD73 in the tumor microenvironment (Synnestvedt K, et al. J Clin Invest. 2002; 110:993-1002). Analysis of clinical tumor samples has shown that high CD73 expression is a potential biomarker and is closely associated with poor prognosis in various tumors, including breast cancer, lung cancer, ovarian cancer, kidney cancer, gastric cancer, and head and neck cancer.

[0011] CD73 possesses both hydrolase and non-hydrolase activity. The enzymatic and non-enzymatic functions of CD73 act simultaneously in related processes in tumors, mutually promoting each other and maintaining tumor progression. Further studies are revealing that CD73 is a crucial regulatory molecule for tumor cell proliferation, metastasis, and invasion in vitro, as well as for tumor angiogenesis and immune evasion mechanisms in vivo, and that important immunosuppressive mechanisms are mediated by the CD73-adenosine metabolic signaling pathway. CD39, upstream of CD73, can catalyze ATP to produce adenosine monophosphate (AMP), which is then converted to adenosine by CD73, and adenosine binds to the downstream adenosine receptor (A2AR). A2AR inhibits a series of signaling pathways related to immune activation, including LCK, MAPK, and PKC, and activates protein kinase A (PKA) and Csk kinase, thereby inhibiting the immune-killing effect of T cells and thus playing an immunosuppressive role that enables tumors to evade the immune system (Antonioli L, et al. Nat Rev Cancer. 2013; 13:842-857). CD73 expressed by immune and non-immune cells can promote immune evasion, progression, and metastasis of tumors, and preclinical animal models have shown that inhibition of cytotoxic T cell (CLT) and NK cell function by Treg cell-associated CD73-adenosine signaling is the most important factor.

[0012] In the treatment of solid tumors, one of the key aspects of overcoming drug resistance and improving therapeutic efficacy is blocking the inhibitory effects of immune effector cells on the tumor microenvironment (TME). The TME is a highly complex system composed of various cells, intercellular matrices, enzymes, cytokines, metabolites, and more. It is characterized by significantly low hydrogen, low pH, and high pressure, and is vastly different from normal tissue. The hypoxic state or ATP concentration caused by chemoradiotherapy that kills tumor cells promotes the cascade of CD39-CD73 adenosine signaling, 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. Clin. Exp. Immunol. 2013; 171:1-7).

[0013] The use of CD73-targeting antibodies and CD73 gene knockout animal models can effectively block tumor growth and metastasis. Recently, the use of CD73 monoclonal antibodies, small molecule interfering RNA technology, and specific inhibitors such as APCP has yielded remarkable therapeutic effects in animal studies, offering new approaches to antitumor therapy. Evidence from in vivo experiments indicates that CD73-targeted inhibition is an effective therapeutic tool for tumor patients.

[0014] The relationship between CD73 overexpression in patients and tumor subtype, prognosis, and response suggests that CD73 may be an important marker for future tumor treatment and individual detection. Therefore, research targeting CD73 is essential.

[0015] Currently, there is a need for the development of new anti-LAG3 antibody drugs and bispecific antibody drugs that target both CD73 and LAG3. [Overview of the Initiative]

[0016] Through intensive research and innovative efforts, the inventors obtained an anti-LAG3 anti-CD73 bispecific antibody. Surprisingly, the inventors found that the anti-LAG3 anti-CD73 bispecific antibody (also referred to simply as the antibody or the antibody in this disclosure) possesses excellent antitumor activity. Further details of this disclosure are as follows:

[0017] Aspects of this disclosure relate to bispecific antibodies comprising a first protein functional domain and a second protein functional domain, The first protein functional domain targets LAG3, and the second protein functional domain targets a target other than LAG3 (e.g., CD73, PD-1). The first protein functional region is an anti-LAG3 antibody or its antigen-binding fragment, the anti-LAG3 antibody comprises a heavy chain variable region and a light chain variable region; the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3; and the light chain variable region comprises LCDR1, LCDR2 and LCDR3. The amino acid sequence of HCDR1 is shown in SEQ ID NO: 9. The amino acid sequence of HCDR2 is shown in SEQ ID NO: 10, and, The amino acid sequence of HCDR3 is shown in SEQ ID NO: 11; The amino acid sequence of LCDR1 is shown in SEQ ID NO: 12 or SEQ ID NO: 17. The amino acid sequence of LCDR2 is shown in SEQ ID NO: 13 or SEQ ID NO: 15, and The amino acid sequence of LCDR3 is shown in SEQ ID NO: 14 or SEQ ID NO: 16.

[0018] In some aspects of this disclosure, The amino acid sequence of LCDR1 is shown in Sequence ID No. 12. The amino acid sequence of LCDR2 is shown in Sequence ID No. 13. The amino acid sequence of LCDR3 is shown in SEQ ID NO: 14; or, The amino acid sequence of LCDR1 is shown in Sequence ID No. 12. The amino acid sequence of LCDR2 is shown in Sequence ID No. 15. The amino acid sequence of LCDR3 is shown in SEQ ID NO: 16; or, The amino acid sequence of LCDR1 is shown in SEQ ID NO: 17. The amino acid sequence of LCDR2 is shown in Sequence ID No. 15. The amino acid sequence of LCDR3 is shown in SEQ ID NO: 14; or, The amino acid sequence of LCDR1 is shown in Sequence ID No. 12. The amino acid sequence of LCDR2 is shown in Sequence ID No. 13. The amino acid sequence of LCDR3 is shown in SEQ ID NO: 16; or, The amino acid sequence of LCDR1 is shown in Sequence ID No. 12. The amino acid sequence of LCDR2 is shown in Sequence ID No. 15. The amino acid sequence of LCDR3 is shown in SEQ ID NO: 14; or, The amino acid sequence of LCDR1 is shown in SEQ ID NO: 17. The amino acid sequence of LCDR2 is shown in Sequence ID No. 13. The amino acid sequence of LCDR3 is shown in SEQ ID NO: 14; or, The amino acid sequence of LCDR1 is shown in SEQ ID NO: 17. The amino acid sequence of LCDR2 is shown in Sequence ID No. 13. The amino acid sequence of LCDR3 is shown in SEQ ID NO: 16; or, The amino acid sequence of LCDR1 is shown in SEQ ID NO: 17. The amino acid sequence of LCDR2 is shown in Sequence ID No. 15. The amino acid sequence of LCDR3 is shown in SEQ ID NO: 16; Bispecific antibodies are provided.

[0019] In some aspects of this disclosure, The amino acid sequence of HCDR1 is shown in SEQ ID NO: 9. The amino acid sequence of HCDR2 is shown in SEQ ID NO: 10, and, The amino acid sequence of HCDR3 is shown in SEQ ID NO: 11; The amino acid sequence of LCDR1 is shown in Sequence ID No. 12. The amino acid sequence of LCDR2 is shown in SEQ ID NO: 13, and, The amino acid sequence of LCDR3 is shown in SEQ ID NO: 14; Bispecific antibodies are provided.

[0020] In some aspects of this disclosure, The amino acid sequence of HCDR1 is shown in SEQ ID NO: 9. The amino acid sequence of HCDR2 is shown in SEQ ID NO: 10, and, The amino acid sequence of HCDR3 is shown in SEQ ID NO: 11; The amino acid sequence of LCDR1 is shown in Sequence ID No. 12. The amino acid sequence of LCDR2 is shown in SEQ ID NO: 15, and The amino acid sequence of LCDR3 is shown in SEQ ID NO: 16; Bispecific antibodies are provided.

[0021] In some aspects of this disclosure, The amino acid sequence of HCDR1 is shown in SEQ ID NO: 9. The amino acid sequence of HCDR2 is shown in SEQ ID NO: 10, and, The amino acid sequence of HCDR3 is shown in SEQ ID NO: 11; The amino acid sequence of LCDR1 is shown in SEQ ID NO: 17. The amino acid sequence of LCDR2 is shown in SEQ ID NO: 15, and The amino acid sequence of LCDR3 is shown in SEQ ID NO: 14; Bispecific antibodies are provided.

[0022] In some aspects of this disclosure, The amino acid sequence of HCDR1 is shown in SEQ ID NO: 9. The amino acid sequence of HCDR2 is shown in SEQ ID NO: 10, and, The amino acid sequence of HCDR3 is shown in SEQ ID NO: 11; The amino acid sequence of LCDR1 is shown in Sequence ID No. 12. The amino acid sequence of LCDR2 is shown in SEQ ID NO: 13, and, The amino acid sequence of LCDR3 is shown in SEQ ID NO: 16; Bispecific antibodies are provided.

[0023] In some aspects of this disclosure, The amino acid sequence of HCDR1 is shown in SEQ ID NO: 9. The amino acid sequence of HCDR2 is shown in SEQ ID NO: 10, and, The amino acid sequence of HCDR3 is shown in SEQ ID NO: 11; The amino acid sequence of LCDR1 is shown in Sequence ID No. 12. The amino acid sequence of LCDR2 is shown in SEQ ID NO: 15, and The amino acid sequence of LCDR3 is shown in SEQ ID NO: 14; Bispecific antibodies are provided.

[0024] In some aspects of this disclosure, The amino acid sequence of HCDR1 is shown in SEQ ID NO: 9. The amino acid sequence of HCDR2 is shown in SEQ ID NO: 10, and, The amino acid sequence of HCDR3 is shown in SEQ ID NO: 11; The amino acid sequence of LCDR1 is shown in SEQ ID NO: 17. The amino acid sequence of LCDR2 is shown in SEQ ID NO: 13, and, The amino acid sequence of LCDR3 is shown in SEQ ID NO: 14; Bispecific antibodies are provided.

[0025] In some aspects of this disclosure, The amino acid sequence of HCDR1 is shown in SEQ ID NO: 9. The amino acid sequence of HCDR2 is shown in SEQ ID NO: 10, and, The amino acid sequence of HCDR3 is shown in SEQ ID NO: 11; The amino acid sequence of LCDR1 is shown in SEQ ID NO: 17. The amino acid sequence of LCDR2 is shown in SEQ ID NO: 13, and, The amino acid sequence of LCDR3 is shown in SEQ ID NO: 16; Bispecific antibodies are provided.

[0026] In some aspects of this disclosure, The amino acid sequence of HCDR1 is shown in SEQ ID NO: 9. The amino acid sequence of HCDR2 is shown in SEQ ID NO: 10, and, The amino acid sequence of HCDR3 is shown in SEQ ID NO: 11; The amino acid sequence of LCDR1 is shown in SEQ ID NO: 17. The amino acid sequence of LCDR2 is shown in SEQ ID NO: 15, and The amino acid sequence of LCDR3 is shown in SEQ ID NO: 16; Bispecific antibodies are provided.

[0027] In some aspects of this disclosure, The heavy chain variable region of the anti-LAG3 antibody is selected from the amino acid sequences shown in SEQ ID NO: 2 and SEQ ID NO: 44, and the light chain variable region of the anti-LAG3 antibody is selected from the amino acid sequences shown in SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 and SEQ ID NO: 45; Preferably, The amino acid sequence of the heavy chain variable region of the anti-LAG3 antibody is shown in SEQ ID NO: 2, and the amino acid sequence of the light chain variable region of the anti-LAG3 antibody is shown in SEQ ID NO: 4; The amino acid sequence of the heavy chain variable region of the anti-LAG3 antibody is shown in SEQ ID NO: 2, and the amino acid sequence of the light chain variable region of the anti-LAG3 antibody is shown in SEQ ID NO: 6; The amino acid sequence of the heavy chain variable region of the anti-LAG3 antibody is shown in SEQ ID NO: 2, and the amino acid sequence of the light chain variable region of the anti-LAG3 antibody is shown in SEQ ID NO: 8; The amino acid sequence of the heavy chain variable region of the anti-LAG3 antibody is shown in SEQ ID NO: 2, and the amino acid sequence of the light chain variable region of the anti-LAG3 antibody is shown in SEQ ID NO: 45; The amino acid sequence of the heavy chain variable region of the anti-LAG3 antibody is shown in SEQ ID NO: 44, and the amino acid sequence of the light chain variable region of the anti-LAG3 antibody is shown in SEQ ID NO: 4; The amino acid sequence of the heavy chain variable region of the anti-LAG3 antibody is shown in SEQ ID NO: 44, and the amino acid sequence of the light chain variable region of the anti-LAG3 antibody is shown in SEQ ID NO: 6; The amino acid sequence of the heavy chain variable region of the anti-LAG3 antibody is shown in SEQ ID NO: 44, and the amino acid sequence of the light chain variable region of the anti-LAG3 antibody is shown in SEQ ID NO: 8; or, The amino acid sequence of the heavy chain variable region of the anti-LAG3 antibody is shown in SEQ ID NO: 44, and the amino acid sequence of the light chain variable region of the anti-LAG3 antibody is shown in SEQ ID NO: 45; Bispecific antibodies are provided.

[0028] In some aspects of this disclosure, the anti-LAG3 antibody or its antigen-binding fragment is selected from Fab, Fab', F(ab')2, Fd, Fv, dAb, complementarity-determining region fragment, single-chain antibody, humanized antibody, and chimeric antibody. Bispecific antibodies are provided.

[0029] In some aspects of this disclosure, a bispecific antibody is provided in which the anti-LAG3 antibody comprises a non-CDR region derived from a human antibody.

[0030] In some aspects of this disclosure, a bispecific antibody is provided which is an anti-LAG3 anti-CD73 bispecific antibody.

[0031] In some aspects of this disclosure, The second protein functional region is an anti-CD73 antibody or its antigen-binding fragment, the anti-CD73 antibody includes a heavy chain variable region and a light chain variable region; the heavy chain variable region includes HCDR1 having the amino acid sequence shown in SEQ ID NO: 25, HCDR2 having the amino acid sequence shown in SEQ ID NO: 26, and HCDR3 having the amino acid sequence shown in SEQ ID NO: 27; and, The light chain variable region includes LCDR1 having the amino acid sequence shown in SEQ ID NO: 28, LCDR2 having the amino acid sequence shown in SEQ ID NO: 29, and LCDR3 having the amino acid sequence shown in SEQ ID NO: 30; Bispecific antibodies are provided.

[0032] In some aspects of this disclosure, The heavy chain variable region of the anti-CD73 antibody is selected from the amino acid sequences shown in SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, and SEQ ID NO: 46; the light chain variable region of the anti-CD73 antibody is selected from the amino acid sequences shown in SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 47, and SEQ ID NO: 62; Preferably, The amino acid sequence of the heavy chain variable region of the anti-CD73 antibody is shown in SEQ ID NO: 31, and the amino acid sequence of the light chain variable region of the anti-CD73 antibody is shown in SEQ ID NO: 32; The amino acid sequence of the heavy chain variable region of the anti-CD73 antibody is shown in SEQ ID NO: 31, and the amino acid sequence of the light chain variable region of the anti-CD73 antibody is shown in SEQ ID NO: 34; The amino acid sequence of the heavy chain variable region of the anti-CD73 antibody is shown in SEQ ID NO: 31, and the amino acid sequence of the light chain variable region of the anti-CD73 antibody is shown in SEQ ID NO: 36; The amino acid sequence of the heavy chain variable region of the anti-CD73 antibody is shown in SEQ ID NO: 31, and the amino acid sequence of the light chain variable region of the anti-CD73 antibody is shown in SEQ ID NO: 37; The amino acid sequence of the heavy chain variable region of the anti-CD73 antibody is shown in SEQ ID NO: 31, and the amino acid sequence of the light chain variable region of the anti-CD73 antibody is shown in SEQ ID NO: 47; The amino acid sequence of the heavy chain variable region of the anti-CD73 antibody is shown in SEQ ID NO: 31, and the amino acid sequence of the light chain variable region of the anti-CD73 antibody is shown in SEQ ID NO: 62; The amino acid sequence of the heavy chain variable region of the anti-CD73 antibody is shown in SEQ ID NO: 33, and the amino acid sequence of the light chain variable region of the anti-CD73 antibody is shown in SEQ ID NO: 32; The amino acid sequence of the heavy chain variable region of the anti-CD73 antibody is shown in SEQ ID NO: 33, and the amino acid sequence of the light chain variable region of the anti-CD73 antibody is shown in SEQ ID NO: 34; The amino acid sequence of the heavy chain variable region of the anti-CD73 antibody is shown in SEQ ID NO: 33, and the amino acid sequence of the light chain variable region of the anti-CD73 antibody is shown in SEQ ID NO: 36; The amino acid sequence of the heavy chain variable region of the anti-CD73 antibody is shown in SEQ ID NO: 33, and the amino acid sequence of the light chain variable region of the anti-CD73 antibody is shown in SEQ ID NO: 37; The amino acid sequence of the heavy chain variable region of the anti-CD73 antibody is shown in SEQ ID NO: 33, and the amino acid sequence of the light chain variable region of the anti-CD73 antibody is shown in SEQ ID NO: 47; The amino acid sequence of the heavy chain variable region of the anti-CD73 antibody is shown in SEQ ID NO: 33, and the amino acid sequence of the light chain variable region of the anti-CD73 antibody is shown in SEQ ID NO: 62; The amino acid sequence of the heavy chain variable region of the anti-CD73 antibody is shown in SEQ ID NO: 35, and the amino acid sequence of the light chain variable region of the anti-CD73 antibody is shown in SEQ ID NO: 32; The amino acid sequence of the heavy chain variable region of the anti-CD73 antibody is shown in SEQ ID NO: 35, and the amino acid sequence of the light chain variable region of the anti-CD73 antibody is shown in SEQ ID NO: 34; The amino acid sequence of the heavy chain variable region of the anti-CD73 antibody is shown in SEQ ID NO: 35, and the amino acid sequence of the light chain variable region of the anti-CD73 antibody is shown in SEQ ID NO: 36; The amino acid sequence of the heavy chain variable region of the anti-CD73 antibody is shown in SEQ ID NO: 35, and the amino acid sequence of the light chain variable region of the anti-CD73 antibody is shown in SEQ ID NO: 37; The amino acid sequence of the heavy chain variable region of the anti-CD73 antibody is shown in SEQ ID NO: 35, and the amino acid sequence of the light chain variable region of the anti-CD73 antibody is shown in SEQ ID NO: 47; The amino acid sequence of the heavy chain variable region of the anti-CD73 antibody is shown in SEQ ID NO: 35, and the amino acid sequence of the light chain variable region of the anti-CD73 antibody is shown in SEQ ID NO: 62; The amino acid sequence of the heavy chain variable region of the anti-CD73 antibody is shown in SEQ ID NO: 46, and the amino acid sequence of the light chain variable region of the anti-CD73 antibody is shown in SEQ ID NO: 32; The amino acid sequence of the heavy chain variable region of the anti-CD73 antibody is shown in SEQ ID NO: 46, and the amino acid sequence of the light chain variable region of the anti-CD73 antibody is shown in SEQ ID NO: 34; The amino acid sequence of the heavy chain variable region of the anti-CD73 antibody is shown in SEQ ID NO: 46, and the amino acid sequence of the light chain variable region of the anti-CD73 antibody is shown in SEQ ID NO: 36; The amino acid sequence of the heavy chain variable region of the anti-CD73 antibody is shown in SEQ ID NO: 46, and the amino acid sequence of the light chain variable region of the anti-CD73 antibody is shown in SEQ ID NO: 37; The amino acid sequence of the heavy chain variable region of the anti-CD73 antibody is shown in SEQ ID NO: 46, and the amino acid sequence of the light chain variable region of the anti-CD73 antibody is shown in SEQ ID NO: 47; or, The amino acid sequence of the heavy chain variable region of the anti-CD73 antibody is shown in SEQ ID NO: 46, and the amino acid sequence of the light chain variable region of the anti-CD73 antibody is shown in SEQ ID NO: 62; Bispecific antibodies are provided.

[0033] In some aspects of this disclosure, The anti-LAG3 antibody or anti-CD73 antibody further comprises a constant region derived from a human antibody; Preferably, the constant region of the anti-LAG3 antibody or the anti-CD73 antibody is selected from the constant regions of human IgG1, IgG2, IgG3, or IgG4; Preferably, the anti-LAG3 antibody comprises a heavy chain constant region and a light chain constant region, wherein the heavy chain constant region is an Igγ-1 chain C region (e.g., SEQ ID NO: 18) or an Igγ-4 chain C region (e.g., SEQ ID NO: 20), and the light chain constant region is an Igκ chain C region (e.g., SEQ ID NO: 19); Preferably, the anti-CD73 antibody comprises a heavy chain constant region and a light chain constant region, wherein the heavy chain constant region is an Igγ-1 chain C region (e.g., SEQ ID NO: 18) or an Igγ-4 chain C region (e.g., SEQ ID NO: 20), and the light chain constant region is an Igκ chain C region (e.g., SEQ ID NO: 19); Bispecific antibodies are provided.

[0034] Amino acid sequence of the constant region of the IgG1 heavy chain: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(Sequence ID 18)

[0035] In some aspects of this disclosure, (1) to (4) below: (1) The anti-LAG3 antibody subtype is human IgG1, and the heavy chain constant region of the antibody has the following mutation in the EU numbering system: L234A and L235A; L234A and G237A; L235A and G237A; or, L234A, L235A, and G237A; Having, (2) The anti-LAG3 antibody subtype is human IgG4, and the heavy chain constant region of the antibody has the following mutation in the EU numbering system: F234A and L235A; F234A and G237A; L235A and G237A; or, F234A, L235A, and G237A; Having, (3) The anti-CD73 antibody subtype is human IgG1, and the antibody's heavy chain constant region has the following mutation in the EU numbering system: L234A and L235A; L234A and G237A; L235A and G237A; or, L234A, L235A, and G237A; Having, (4) The anti-CD73 antibody subtype is human IgG4, and the antibody's heavy chain constant region has the following mutation in the EU numbering system: F234A and L235A; F234A and G237A; L235A and G237A; or, F234A, L235A, and G237A. Having, A bispecific antibody is provided, characterized by one of the following:

[0036] In some aspects of this disclosure, Bispecific antibodies are in the form of IgG-scFv; Preferably, the first protein functional region is an immunoglobulin and the second protein functional region is a single-chain antibody; or, Preferably, the first protein functional region is a single-chain antibody, and the second protein functional region is an immunoglobulin targeting a target other than LAG3 (e.g., CD73, PD-1); Bispecific antibodies are provided.

[0037] In some aspects of this disclosure, The first protein functional region targeting LAG3, and A second protein functional domain targeting CD73 A bispecific antibody containing the following is provided: The first protein functional region is an anti-LAG3 antibody, the anti-LAG3 antibody is an immunoglobulin, and the second protein functional region is an anti-CD73 single-chain antibody; or, The first protein functional region is an anti-LAG3 single-chain antibody, and the second protein functional region is an anti-CD73 antibody, and the anti-CD73 antibody is an immunoglobulin.

[0038] In some aspects of this disclosure, The heavy chain variable region of the immunoglobulin contains HCDR1 to HCDR3 of the amino acid sequences shown in SEQ ID NOs. 9 to 11, and the light chain variable region of the immunoglobulin contains LCDR1 to LCDR3 of the amino acid sequences shown in SEQ ID NOs. 12 to 14; the heavy chain variable region of the single-chain antibody contains HCDR1 to HCDR3 of the amino acid sequences shown in SEQ ID NOs. 25 to 27, and the light chain variable region of the single-chain antibody contains LCDR1 to LCDR3 of the amino acid sequences shown in SEQ ID NOs. 28 to 30; or, The heavy chain variable region of the single-chain antibody contains HCDR1 to HCDR3 of the amino acid sequences shown in SEQ ID NOs. 9 to 11, respectively, and the light chain variable region of the single-chain antibody contains LCDR1 to LCDR3 of the amino acid sequences shown in SEQ ID NOs. 12 to 14, respectively; the heavy chain variable region of the immunoglobulin contains HCDR1 to HCDR3 of the amino acid sequences shown in SEQ ID NOs. 25 to 27, respectively, and the light chain variable region of the immunoglobulin contains LCDR1 to LCDR3 of the amino acid sequences shown in SEQ ID NOs. 28 to 30, respectively; Bispecific antibodies are provided.

[0039] In some aspects of this disclosure, The heavy chain variable region of the immunoglobulin is selected from the amino acid sequences shown in SEQ ID NO: 2 and SEQ ID NO: 44, and the light chain variable region of the immunoglobulin is selected from the amino acid sequences shown in SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 and SEQ ID NO: 45; the heavy chain variable region of the single-chain antibody is selected from the amino acid sequences shown in SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35 and SEQ ID NO: 46, and the light chain variable region of the single-chain antibody is selected from the amino acid sequences shown in SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 47 and SEQ ID NO: 62; or, The heavy chain variable region of the single-chain antibody is selected from the amino acid sequences shown in SEQ ID NO: 2 and SEQ ID NO: 44, and the light chain variable region of the single-chain antibody is selected from the amino acid sequences shown in SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 and SEQ ID NO: 45; the heavy chain variable region of the immunoglobulin is selected from the amino acid sequences shown in SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35 and SEQ ID NO: 46, and the light chain variable region of the immunoglobulin is selected from the amino acid sequences shown in SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 47 and SEQ ID NO: 62; Bispecific antibodies are provided.

[0040] In some aspects of this disclosure, the following (1) to (24): (1) The amino acid sequence of the heavy chain variable region of immunoglobulin is shown in SEQ ID NO: 2, and the amino acid sequence of the light chain variable region of immunoglobulin is shown in SEQ ID NO: 4; the amino acid sequence of the heavy chain variable region of single-chain antibody is shown in SEQ ID NO: 31, and the amino acid sequence of the light chain variable region of single-chain antibody is shown in SEQ ID NO: 32; (2) The amino acid sequence of the heavy chain variable region of immunoglobulin is shown in SEQ ID NO: 2, and the amino acid sequence of the light chain variable region of immunoglobulin is shown in SEQ ID NO: 4; the amino acid sequence of the heavy chain variable region of single-chain antibody is shown in SEQ ID NO: 33, and the amino acid sequence of the light chain variable region of single-chain antibody is shown in SEQ ID NO: 34; (3) The amino acid sequence of the heavy chain variable region of immunoglobulin is shown in SEQ ID NO: 2, and the amino acid sequence of the light chain variable region of immunoglobulin is shown in SEQ ID NO: 4; the amino acid sequence of the heavy chain variable region of single-chain antibody is shown in SEQ ID NO: 35, and the amino acid sequence of the light chain variable region of single-chain antibody is shown in SEQ ID NO: 36; (4) The amino acid sequence of the heavy chain variable region of immunoglobulin is shown in SEQ ID NO: 2, and the amino acid sequence of the light chain variable region of immunoglobulin is shown in SEQ ID NO: 4; the amino acid sequence of the heavy chain variable region of single-chain antibody is shown in SEQ ID NO: 35, and the amino acid sequence of the light chain variable region of single-chain antibody is shown in SEQ ID NO: 37; (5) The amino acid sequence of the heavy chain variable region of immunoglobulin is shown in SEQ ID NO: 2, and the amino acid sequence of the light chain variable region of immunoglobulin is shown in SEQ ID NO: 4; the amino acid sequence of the heavy chain variable region of single-chain antibody is shown in SEQ ID NO: 46, and the amino acid sequence of the light chain variable region of single-chain antibody is shown in SEQ ID NO: 47; (6) The amino acid sequence of the heavy chain variable region of immunoglobulin is shown in SEQ ID NO: 2, and the amino acid sequence of the light chain variable region of immunoglobulin is shown in SEQ ID NO: 4; the amino acid sequence of the heavy chain variable region of single-chain antibody is shown in SEQ ID NO: 35, and the amino acid sequence of the light chain variable region of single-chain antibody is shown in SEQ ID NO: 62; (7) The amino acid sequence of the heavy chain variable region of immunoglobulin is shown in SEQ ID NO: 44, and the amino acid sequence of the light chain variable region of immunoglobulin is shown in SEQ ID NO: 45; the amino acid sequence of the heavy chain variable region of single-chain antibody is shown in SEQ ID NO: 31, and the amino acid sequence of the light chain variable region of single-chain antibody is shown in SEQ ID NO: 32; (8) The amino acid sequence of the heavy chain variable region of immunoglobulins is shown in SEQ ID NO: 44, and the amino acid sequence of the light chain variable region of immunoglobulins is shown in SEQ ID NO: 45; the amino acid sequence of the heavy chain variable region of single-chain antibodies is shown in SEQ ID NO: 33, and the amino acid sequence of the light chain variable region of single-chain antibodies is shown in SEQ ID NO: 34; (9) The amino acid sequence of the heavy chain variable region of immunoglobulin is shown in SEQ ID NO: 44, and the amino acid sequence of the light chain variable region of immunoglobulin is shown in SEQ ID NO: 45; the amino acid sequence of the heavy chain variable region of single-chain antibody is shown in SEQ ID NO: 35, and the amino acid sequence of the light chain variable region of single-chain antibody is shown in SEQ ID NO: 36; (10) The amino acid sequence of the heavy chain variable region of immunoglobulin is shown in SEQ ID NO: 44, and the amino acid sequence of the light chain variable region of immunoglobulin is shown in SEQ ID NO: 45; the amino acid sequence of the heavy chain variable region of single-chain antibody is shown in SEQ ID NO: 35, and the amino acid sequence of the light chain variable region of single-chain antibody is shown in SEQ ID NO: 37; (11) The amino acid sequence of the heavy chain variable region of immunoglobulin is shown in SEQ ID NO: 44, and the amino acid sequence of the light chain variable region of immunoglobulin is shown in SEQ ID NO: 45; the amino acid sequence of the heavy chain variable region of single-chain antibody is shown in SEQ ID NO: 46, and the amino acid sequence of the light chain variable region of single-chain antibody is shown in SEQ ID NO: 47; (12) The amino acid sequence of the heavy chain variable region of immunoglobulin is shown in SEQ ID NO: 44, and the amino acid sequence of the light chain variable region of immunoglobulin is shown in SEQ ID NO: 45; the amino acid sequence of the heavy chain variable region of single-chain antibody is shown in SEQ ID NO: 35, and the amino acid sequence of the light chain variable region of single-chain antibody is shown in SEQ ID NO: 62; (13) The amino acid sequence of the heavy chain variable region of a single-chain antibody is shown in SEQ ID NO: 2, and the amino acid sequence of the light chain variable region of a single-chain antibody is shown in SEQ ID NO: 4; the amino acid sequence of the heavy chain variable region of an immunoglobulin is shown in SEQ ID NO: 31, and the amino acid sequence of the light chain variable region of an immunoglobulin is shown in SEQ ID NO: 32; (14) The amino acid sequence of the heavy chain variable region of a single-chain antibody is shown in SEQ ID NO: 2, and the amino acid sequence of the light chain variable region of a single-chain antibody is shown in SEQ ID NO: 4; the amino acid sequence of the heavy chain variable region of an immunoglobulin is shown in SEQ ID NO: 33, and the amino acid sequence of the light chain variable region of an immunoglobulin is shown in SEQ ID NO: 34; (15) The amino acid sequence of the heavy chain variable region of a single-chain antibody is shown in SEQ ID NO: 2, and the amino acid sequence of the light chain variable region of a single-chain antibody is shown in SEQ ID NO: 4; the amino acid sequence of the heavy chain variable region of an immunoglobulin is shown in SEQ ID NO: 35, and the amino acid sequence of the light chain variable region of an immunoglobulin is shown in SEQ ID NO: 36; (16) The amino acid sequence of the heavy chain variable region of a single-chain antibody is shown in SEQ ID NO: 2, and the amino acid sequence of the light chain variable region of a single-chain antibody is shown in SEQ ID NO: 4; the amino acid sequence of the heavy chain variable region of an immunoglobulin is shown in SEQ ID NO: 35, and the amino acid sequence of the light chain variable region of an immunoglobulin is shown in SEQ ID NO: 37; (17) The amino acid sequence of the heavy chain variable region of a single-chain antibody is shown in SEQ ID NO: 2, and the amino acid sequence of the light chain variable region of a single-chain antibody is shown in SEQ ID NO: 4; the amino acid sequence of the heavy chain variable region of an immunoglobulin is shown in SEQ ID NO: 46, and the amino acid sequence of the light chain variable region of an immunoglobulin is shown in SEQ ID NO: 47; (18) The amino acid sequence of the heavy chain variable region of a single-chain antibody is shown in SEQ ID NO: 2, and the amino acid sequence of the light chain variable region of a single-chain antibody is shown in SEQ ID NO: 4; the amino acid sequence of the heavy chain variable region of an immunoglobulin is shown in SEQ ID NO: 35, and the amino acid sequence of the light chain variable region of an immunoglobulin is shown in SEQ ID NO: 62; (19) The amino acid sequence of the heavy chain variable region of a single-chain antibody is shown in SEQ ID NO: 44, and the amino acid sequence of the light chain variable region of a single-chain antibody is shown in SEQ ID NO: 45; the amino acid sequence of the heavy chain variable region of an immunoglobulin is shown in SEQ ID NO: 31, and the amino acid sequence of the light chain variable region of an immunoglobulin is shown in SEQ ID NO: 32; (20) The amino acid sequence of the heavy chain variable region of a single-chain antibody is shown in SEQ ID NO: 44, and the amino acid sequence of the light chain variable region of a single-chain antibody is shown in SEQ ID NO: 45; the amino acid sequence of the heavy chain variable region of an immunoglobulin is shown in SEQ ID NO: 33, and the amino acid sequence of the light chain variable region of an immunoglobulin is shown in SEQ ID NO: 34; (twenty one) The amino acid sequence of the heavy chain variable region of a single-chain antibody is shown in SEQ ID NO: 44, and the amino acid sequence of the light chain variable region of a single-chain antibody is shown in SEQ ID NO: 45; the amino acid sequence of the heavy chain variable region of an immunoglobulin is shown in SEQ ID NO: 35, and the amino acid sequence of the light chain variable region of an immunoglobulin is shown in SEQ ID NO: 36; (twenty two) The amino acid sequence of the heavy chain variable region of a single-chain antibody is shown in SEQ ID NO: 44, and the amino acid sequence of the light chain variable region of a single-chain antibody is shown in SEQ ID NO: 45; the amino acid sequence of the heavy chain variable region of an immunoglobulin is shown in SEQ ID NO: 35, and the amino acid sequence of the light chain variable region of an immunoglobulin is shown in SEQ ID NO: 37; (twenty three) The amino acid sequence of the heavy chain variable region of a single-chain antibody is shown in SEQ ID NO: 44, and the amino acid sequence of the light chain variable region of a single-chain antibody is shown in SEQ ID NO: 45; the amino acid sequence of the heavy chain variable region of an immunoglobulin is shown in SEQ ID NO: 46, and the amino acid sequence of the light chain variable region of an immunoglobulin is shown in SEQ ID NO: 47; and, (twenty four) The amino acid sequence of the heavy chain variable region of a single-chain antibody is shown in SEQ ID NO: 44, and the amino acid sequence of the light chain variable region of a single-chain antibody is shown in SEQ ID NO: 45; the amino acid sequence of the heavy chain variable region of an immunoglobulin is shown in SEQ ID NO: 35, and the amino acid sequence of the light chain variable region of an immunoglobulin is shown in SEQ ID NO: 62; A bispecific antibody is provided, selected from one of the following.

[0041] In some aspects of this disclosure, The immunoglobulin is human IgG1, and the constant region of the immunoglobulin's heavy chain has the following mutations in the EU numbering system: L234A and L235A; L234A and G237A; L235A and G237A; or, L234A, L235A, and G237A has Bispecific antibodies are provided.

[0042] In this disclosure, unless otherwise specified, the letter before the position number represents the amino acid before the mutation, and the letter after the position number represents the amino acid after the mutation.

[0043] In some aspects of this disclosure, The constant region of the heavy chain of immunoglobulins is numbered in the EU numbering system as follows: 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 It further has one or more selected from.

[0044] In some aspects of this disclosure, The immunoglobulin is human IgG4, and the constant region of the immunoglobulin's heavy chain has the following mutations in the EU numbering system: F234A and L235A; F234A and G237A; L235A and G237A; or, F234A, L235A, and G237A has Bispecific antibodies are provided.

[0045] In some aspects of this disclosure, The number of the first protein functional region and the second protein functional region is, independently, 1, 2, or more; Preferably, the first protein functional region and the second protein functional region are linked directly or via a linker; Preferably, the heavy chain variable region and the light chain variable region of the anti-LAG3 single-chain antibody are linked directly or via a linker; Preferably, the heavy chain variable region and the light chain variable region of the anti-CD73 single-chain antibody are linked directly or via a linker; Preferably, the linker is independently the polypeptide shown in Sequence ID No. 48 (GGGGS), or a polypeptide formed by linking multiple polypeptides (e.g., 2, 3, 4, 5, or 6) shown in Sequence ID No. 48; Preferably, the linker is a polypeptide independently having one or more glycines at the C-terminus of the polypeptide shown in SEQ ID NO: 48, or a polypeptide formed by linking multiple polypeptides (e.g., 2, 3, 4, 5, or 6) shown in SEQ ID NO: 48; Bispecific antibodies are provided.

[0046] In some aspects of this disclosure, bispecific antibodies are provided, in which each single-chain antibody is ligated to the C-terminus or N-terminus of one of the two heavy chains of an immunoglobulin.

[0047] In some aspects of this disclosure, The first protein functional region targeting LAG3, and A second protein functional domain targeting CD73 Includes, A bispecific antibody is provided, having one first protein functional region and two second protein functional regions. The first protein functional region is an immunoglobulin, and the second protein functional region is a single-chain antibody; The amino acid sequence of the heavy chain of the immunoglobulin is shown in SEQ ID NO: 38, and the amino acid sequence of the light chain of the immunoglobulin is shown in SEQ ID NO: 39; The amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 35, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 62; or, the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 46, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 47; Each single-chain antibody is ligated to the C-terminus of one of the two heavy chains of the immunoglobulin; The first protein functional region and the second protein functional region are linked via a first linker; the heavy chain variable region of the single-chain antibody and the light chain variable region of the single-chain antibody are linked via a second linker; the first linker and the second linker may be the same or different; Preferably, the amino acid sequences of the first linker and the second linker are independently selected from the sequences shown in SEQ ID NO: 42 and SEQ ID NO: 43, respectively; Preferably, the amino acid sequences of both the first and second linkers are shown in SEQ ID NO: 43; Preferably, the constant region of the heavy chain of the immunoglobulin (SEQ ID NO: 38) has the following mutation in the EU numbering system: L234A and L235A; L234A and G237A; L235A and G237A; or, L234A, L235A, and G237A. Includes.

[0048] In some aspects of this disclosure, The first protein functional region targeting LAG3, and A second protein functional domain targeting CD73 Includes, A bispecific antibody is provided having two first protein functional regions and one second protein functional region; The first protein functional region is a single-chain antibody, and the second protein functional region is an immunoglobulin; The amino acid sequence of the heavy chain of the immunoglobulin is shown in SEQ ID NO: 40, and the amino acid sequence of the light chain of the immunoglobulin is shown in SEQ ID NO: 41; The amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 2, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 4; or, the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 44, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 45; Each single-chain antibody is ligated to the C-terminus of one of the two heavy chains of the immunoglobulin; The first protein functional region and the second protein functional region are linked via a first linker; the heavy chain variable region of the single-chain antibody and the light chain variable region of the single-chain antibody are linked via a second linker; the first linker and the second linker may be the same or different; Preferably, the amino acid sequences of the first linker and the second linker are independently selected from the sequences shown in SEQ ID NO: 42 and SEQ ID NO: 43, respectively; Preferably, the amino acid sequences of both the first and second linkers are shown in SEQ ID NO: 43; Preferably, the constant region of the heavy chain of the immunoglobulin (SEQ ID NO: 40) has the following mutation in the EU numbering system: L234A and L235A; L234A and G237A; L235A and G237A; or, L234A, L235A, and G237A. Includes.

[0049] A bispecific antibody according to any aspect of this disclosure is provided for use in the treatment or prevention of tumors, Preferably, the tumor is selected from one or more of the following: ovarian cancer, esophageal cancer, melanoma, hematopoietic malignancies, glioblastoma, renal cell carcinoma, lung cancer, prostate cancer, bladder cancer, colon cancer, rectal cancer, liver cancer, stomach cancer, breast cancer, malignant brain tumors, pancreatic cancer, thyroid cancer, head and neck cancer, and kidney cancer; Preferably, the lung cancer is non-small cell lung cancer; Preferably, the hematopoietic malignancy is leukemia; Preferably, the esophageal cancer is esophageal squamous cell carcinoma.

[0050] Another aspect of this disclosure relates to isolated nucleic acid molecules encoding bispecific antibodies according to any aspect of this disclosure.

[0051] Another aspect of this disclosure relates to recombinant vectors containing isolated nucleic acid molecules of this disclosure.

[0052] Another aspect of this disclosure relates to host cells containing isolated nucleic acid molecules or recombinant vectors of this disclosure.

[0053] Another aspect of this disclosure relates to a method for producing a bispecific antibody according to any aspect of this disclosure, comprising the steps of culturing the host cells of this disclosure under appropriate conditions and isolating the bispecific antibody from the cell culture.

[0054] Another aspect of this disclosure relates to a pharmaceutical composition containing a bispecific antibody according to any aspect of this disclosure, wherein the pharmaceutical composition further optionally contains pharmaceutically acceptable auxiliary materials.

[0055] Another aspect of this disclosure relates to the use of bispecific antibodies in any aspect of this disclosure in the manufacture of a pharmaceutical product for treating or preventing tumors. Preferably, the tumor is selected from one or more of the following: ovarian cancer, esophageal cancer, melanoma, hematopoietic malignancies, glioblastoma, renal cell carcinoma, lung cancer, prostate cancer, bladder cancer, colon cancer, rectal cancer, liver cancer, stomach cancer, breast cancer, malignant brain tumors, pancreatic cancer, thyroid cancer, head and neck cancer, and kidney cancer; Preferably, the lung cancer is non-small cell lung cancer; Preferably, the hematopoietic malignancy is leukemia; Preferably, the esophageal cancer is esophageal squamous cell carcinoma.

[0056] Another aspect of this disclosure relates to a method for treating or preventing a tumor, comprising the step of administering an effective amount of a bispecific antibody according to any aspect of this disclosure to a subject requiring treatment. Preferably, the tumor is selected from one or more of the following: ovarian cancer, esophageal cancer, melanoma, hematopoietic malignancies, glioblastoma, renal cell carcinoma, lung cancer, prostate cancer, bladder cancer, colon cancer, rectal cancer, liver cancer, stomach cancer, breast cancer, malignant brain tumors, pancreatic cancer, thyroid cancer, head and neck cancer, and kidney cancer; Preferably, the lung cancer is non-small cell lung cancer; Preferably, the hematopoietic malignancy is leukemia; Preferably, the esophageal cancer is esophageal squamous cell carcinoma.

[0057] In one or more aspects of this disclosure, The anti-LAG3 anti-CD73 bispecific antibody is administered in a single dose of 0.1 to 100 mg per kg of body weight, preferably 1 to 15 mg, 1 to 12 mg, 1 to 10 mg (e.g., 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, or 10 mg) or 6 to 10 mg; or, 10 to 1000 mg of the anti-LAG3 anti-CD73 bispecific antibody is administered to each subject. (For example, approximately 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, or 1000 mg), preferably administered as a single dose of 50-500 mg, 100-400 mg, 150-300 mg, 150-250 mg, or 200 mg; Preferably, administration is performed once every 3, 4, 5, 6, 10 days, 1 week, 2 weeks, or 3 weeks; Preferably, the route of administration is intravenous infusion or intravenous injection; A method for treating or preventing tumors is provided.

[0058] In some embodiments, the anti-LAG3 anti-CD73 bispecific antibody is administered in 2-week (14-day) or 3-week (21-day) cycles, preferably intravenously on the first day (D1) of each cycle. For example, the anti-LAG3 anti-CD73 bispecific antibody is administered once every two weeks (q2w) or once every three weeks (q3w).

[0059] However, it should be recognized that the total daily dose of the drug (e.g., pharmaceutical composition) or active ingredient (e.g., anti-LAG3 anti-CD73 bispecific antibody) disclosed herein must be determined by the reliable medical judgment of a physician. In a particular patient, the specific therapeutically effective dose is determined based on a variety of factors, including the type and stage of the tumor being treated, the form of the drug formulation used, the patient's age, weight, overall health, sex and diet, time of administration, route of administration, elimination rate, duration of treatment, other drugs used concomitantly, and similar factors well known in the medical field. For example, in this field, it is common practice to start with a dose lower than the dose required to achieve the desired therapeutic effect and gradually increase the dose until the desired effect is achieved.

[0060] In this disclosure, unless otherwise defined, scientific and technical terms used in the specification have the meanings generally understood by those skilled in the art. Furthermore, the experimental procedures in cell culture, molecular genetics, nucleic acid chemistry, and immunology used in the specification are commonplace procedures widely practiced in their respective fields. For the purpose of understanding this disclosure, definitions and explanations of relevant terms are provided below.

[0061] This specification uses the term EC 50 This refers to the concentration that provides 50% of the maximum effect, i.e., the concentration that can produce 50% of the maximum effect.

[0062] In this specification, the term "antibody" generally refers to an immunoglobulin molecule consisting of two pairs of polypeptide chains (each pair having one "light" (L) chain and one "heavy" (H) chain). Antibody light chains are classified as κ light chains and λ light chains. Heavy chains are classified as μ, δ, γ, α, and ε. The isotypes of antibodies are defined as IgM, IgD, IgG, IgA, and IgE. In light and heavy chains, the variable region and the constant region are linked by a "J" region of about 12 amino acids or more, and the heavy chain further includes a "D" region of about 3 amino acids or more. Each heavy chain consists of a heavy chain variable region (V H ) and a heavy chain constant region (C H ). The heavy chain constant region consists of three domains (C H1 , C H2 , and C H3 ). Each light chain consists of a light chain variable region (V L ) and a light chain constant region (C L ). The light chain constant region consists of one domain C L . The constant region of an antibody can mediate the binding of an immunoglobulin to host tissues or factors, including binding to various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. V H and V L regions can be further subdivided into hypervariable regions (called complementarity-determining regions (CDRs)), between which conserved regions called framework regions (FRs) are distributed. Each V H and V L consists of three CDRs and four FRs arranged in the order of FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from the amino terminus to the carboxyl terminus. The variable regions (V H and V LThe ) forms the antibody binding site. The assignment of amino acids to the region or domain is based on Bethesda Md, Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, (1987 and 1991)) or Chothia & Lesk J. Mol. Biol., 1987; 196: 901-917; Chothia et al., Nature, 1989; 342: 878-883, or the definition of the IMGT numbering system (see the definition in Ehrenmann F, Kaas Q, Lefranc M P., IMGT / 3Dstructure-DB and IMGT / DomainGapAlign: a database and a tool for immunoglobulins or antibodies, T cell receptors, MHC, IgSF and MhcSF[J]., Nucleic acids research, 2009; 38(suppl_1): D301-D307.).

[0063] The term "antibody" is not limited to a specific method of manufacturing an antibody. For example, antibodies include recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. Antibodies may also be of different isotypes, such as IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtype), IgA1, IgA2, IgD, IgE, or IgM.

[0064] In this specification, the terms “mAb” and “monoclonal antibody” refer to an antibody or antibody fragment derived from a group of highly homologous antibodies, i.e., from a group of antibodies identical except for spontaneously occurring natural variations. Monoclonal antibodies are highly specific to a single epitope of an antigen. Polyclonal antibodies, compared to monoclonal antibodies, generally contain at least two different antibodies that generally recognize different epitopes of an antigen. Monoclonal antibodies can usually be obtained by the hybridoma method, first reported by Kohler et al. (Kohler G, Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity [J]. Nature, 1975; 256(5517): 495), but they can also be obtained by recombinant DNA (see, for example, U.S. Patent No. 4,816,567).

[0065] In this specification, the term "humanized antibody" refers to an antibody or antibody fragment obtained by substituting 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, rabbit) antibody having the expected specificity, affinity, or reactivity. Furthermore, to further improve or optimize the performance of the antibody, amino acid residues in the framework region (FR) of the recipient antibody may be substituted with corresponding amino acid residues of a non-human antibody or amino acid residues of another antibody. For 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.

[0066] In this specification, the term “isolated” refers to a substance obtained from its natural state by artificial means. If a “isolated” substance or component exists naturally, it may be isolated from its natural environment, or both. For example, an unisolated polynucleotide or polypeptide that exists naturally in a particular living animal, and the same polynucleotide or polypeptide isolated in high purity from such a natural state, is called an isolated polynucleotide or polypeptide. The term “isolated” does not exclude the presence of artificial or synthetic substances or other impurities that do not affect the activity of the substance.

[0067] In this specification, the term “vector” refers to a nucleic acid vehicle into which polynucleotides can be inserted. A vector is called an expression vector if it enables the expression of a protein encoded by the inserted polynucleotide. A vector can be introduced into a host cell by transformation, transduction, or transfection, resulting in the expression of the genetic material carried by the vector in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids; phagemids; cosmids; artificial chromosomes such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs); phages such as lambda phages or M13 phages; and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papovaviruses (e.g., SV40). The vector may contain, but is not limited to, a promoter sequence, a transcription start sequence, an enhancer sequence, a selection element, and a reporter gene, as well as various other elements that control expression. The vector may also further contain a replication start site.

[0068] In this specification, the term "host cell" refers to a cell into which a vector can be introduced, and includes, but is not limited to, prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as Drosophila S2 cells or Sf9 cells, or animal cells such as fibroblasts, CHO cells, GS cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK293 cells, or human cells.

[0069] In this specification, the term “specifically binding” refers to a non-random binding reaction between two molecules, such as a reaction between an antibody and the antigen it targets. In certain embodiments, an antibody that specifically binds to an antigen (or an antigen-specific antibody) is an antibody that binds to an antigen in a manner of approximately 10 -5 Less than M, for example, about 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 Affinity (K) less than or equal to M D This means that the antigen binds to the antigen.

[0070] In this specification, the term "K D The term "f(x)" refers to the dissociation equilibrium constant of a particular antibody-antigen interaction and is used to describe the binding affinity between an antibody and an antigen. A smaller dissociation equilibrium constant indicates stronger antibody-antigen binding and higher affinity between the antibody and the antigen. Generally, antibodies have a dissociation equilibrium constant of approximately 10. -5 Less than M, for example, about 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 Dissociation equilibrium constant (K) less than or equal to M D ) binds to antigens (e.g., PD-1 protein). K D This can be determined by methods known to those skilled in the art, for example, using the Fortebio intermolecular interaction apparatus.

[0071] In this specification, the terms “monoclonal antibody” and “mAb” are synonymous and used interchangeably; the terms “polyclonal antibody” and “pAb” are synonymous and used interchangeably. Furthermore, in this disclosure, amino acids are generally represented by one- and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.

[0072] In this specification, the term “pharmaceutically acceptable auxiliary materials” means carriers and / or excipients that are pharmacologically and / or physiologically compatible with the subject and active ingredients, and are well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and include, but are not limited to, pH modifiers, surfactants, adjuvants, and ionic strength enhancers. For example, pH modifiers include, but are not limited to, phosphate buffers; surfactants include, but are not limited to, cationic surfactants, anionic surfactants, or nonionic surfactants (e.g., Tween® 80); and ionic strength enhancers include, but are not limited to, sodium chloride.

[0073] In this specification, the term “effective dose” refers to an amount sufficient to obtain, or at least partially obtain, the desired effect. For example, a prophylactic effective dose for a disease (e.g., tumor) is an amount sufficient to prevent, inhibit, or delay the onset of the disease (e.g., tumor); a therapeutic effective dose is an amount sufficient to cure, or at least partially inhibit, the disease or its complications in a patient. There is no doubt that determining such an effective dose is within the scope of the skills of those skilled in the art. For example, a therapeutically effective dose depends on the severity of the disease being treated, the overall state of the patient’s immune system, the patient’s general circumstances such as age, weight, and sex, the route of administration, and other therapies used in combination.

[0074] The amino acid sequence of lymphocyte activation gene 3 (LAG3) includes the full-length LAG3 protein, the extracellular fragment of LAG3, LAG3 ECD, or fragments containing LAG3 ECD, as well as fusion proteins of LAG3 ECD, such as a fusion protein of the full-length LAG3 protein, or a fragment fused with a mouse or human IgG Fc protein fragment (mFc or hFc). However, those skilled in the art will understand that mutations or variations (including, but not limited to, substitutions, deletions, and / or additions) can be naturally generated or artificially introduced into the amino acid sequence of the LAG3 protein without affecting its biological function. Accordingly, in this disclosure, the terms “LAG3” or “LAG3 protein” include all such sequences, including their natural or artificial variants. Furthermore, sequence fragments of the LAG3 protein also include natural or artificial variants of the corresponding sequence fragment.

[0075] The amino acid sequence of CD73 includes the full-length CD73 protein, the extracellular fragment of CD73, CD73 ECD, or fragments containing CD73 ECD, as well as fusion proteins of CD73 ECD, such as fusion proteins of the full-length CD73 protein, or fragments fused with mouse or human IgG Fc protein fragments (mFc or hFc). However, those skilled in the art will understand that mutations or variations (including, but not limited to, substitutions, deletions, and / or additions) can be naturally generated or artificially introduced into the amino acid sequence of the CD73 protein without affecting its biological function. Accordingly, in this disclosure, the terms “CD73” or “CD73 protein” include all such sequences, including their natural or artificial variants. Furthermore, a sequence fragment of the CD73 protein also includes natural or artificial variants of the corresponding sequence fragment.

[0076] In this disclosure, the terms “first” (e.g., first protein functional region or first linker) and “second” (e.g., second protein functional region or second linker) are used to distinguish and clarify expressions, and unless otherwise specified, there is no specific order that is meaningful.

[0077] Beneficial effects of this disclosure This disclosure achieves one or more of the following technical effects: (1) The bispecific antibodies of this disclosure can bind well and specifically to LAG3, effectively block the binding of LAG3 to MHC-II, and specifically relieve the immunosuppression of LAG3 in the body; (2) The bispecific antibodies of this disclosure can effectively inhibit the enzymatic activity reaction of CD73; (3) The bispecific antibodies of this disclosure can promote INFγ secretion by T cells; (4) The first and second protein functional regions in the bispecific antibody of this disclosure exhibit a synergistic effect; (5) The bispecific antibodies of this disclosure can effectively inhibit tumor growth in a mouse xenograft tumor model; (6) The antibodies of this disclosure can effectively treat or prevent tumors. [Brief explanation of the drawing]

[0078] [Figure 1] Results of ELISA assays for the binding activity of H9L8 (hG4WT), H9L9 (hG4WT), and H9L10 (hG4WT) to human LAG3-mG1Fc antigen. [Figure 2] Results of FACS assays of the binding activity of H9L8 (hG4WT), H9L9 (hG4WT), and H9L10 (hG4WT) to human LAG3 antigen on the surface of 293T-LAG3 cells. [Figure 3] Results of competitive flow cytometry assays of H9L8 (hG4WT), H9L9 (hG4WT), and H9L10 (hG4WT) cells that compete with the MHC-II antigen on the Raji cell membrane surface for binding to human LAG3-mG1Fc. [Figure 4] Results of a mixed lymphocyte reaction (MLR) assay for the biological activity of anti-LAG3 antibodies in promoting IFNγ secretion. [Figure 5]Results of a mixed lymphocyte reaction (MLR) assay for the biological activity of anti-LAG3 antibodies in promoting IL2 secretion. [Figure 6] Results of an assay for the biological activity of an anti-LAG3 antibody in blocking the interaction between LAG3 and MHC-II. [Figure 7] Results of ELISA assays for the binding of LA5EV1 and LA5EV2 to the antigen NT5E-His. [Figure 8] Results of ELISA assays for the binding of NTLAV2 and NTLAV7 to the antigen NT5E-His. [Figure 9] Results of an ELISA assay for the binding of NTLAV8 to the antigen NT5E-His. [Figure 10] Results of ELISA assays for the binding of LA5EV1 and LA5EV2 to the antigen huLAG3-mG1Fc. [Figure 11] Results of ELISA assays for the binding of NTLAV2 and NTLAV7 to the antigen huLAG3-mG1Fc. [Figure 12] Results of an ELISA assay for the binding of NTLAV8 to the antigen huLAG3-mG1Fc. [Figure 13] Affinity constant of antibody LA5EV1 for hNT5E(1~552)-His. [Figure 14] Affinity constant of antibody LA5EV2 for hNT5E(1~552)-His. [Figure 15] Affinity constant of the antibody NTLAV2 for hNT5E(1~552)-His. [Figure 16] Affinity constant of antibody NTLAV7 for hNT5E(1~552)-His. [Figure 17] Affinity constant of antibody NTLAV8 for hNT5E(1~552)-His. [Figure 18] Affinity constant of antibody 19F3H2L3(hG1DM) for hNT5E(1~552)-His. [Figure 19] Affinity constant of antibody LA5EV1 for huLAG3-mG1Fc. [Figure 20]Affinity constant of antibody LA5EV2 for huLAG3-mG1Fc. [Figure 21] Affinity constant of antibody NTLAV2 for huLAG3-mG1Fc. [Figure 22] Affinity constant of antibody NTLAV7 for huLAG3-mG1Fc. [Figure 23] Affinity constant of antibody NTLAV8 for huLAG3-mG1Fc. [Figure 24] Affinity constant of antibody H9L8 (hG4WT) for huLAG3-mG1Fc. [Figure 25] Affinity constant of the antibody relatrimab for huLAG3-mG1Fc. [Figure 26] Results of FACS assays of the binding activity of NTLAV8, LA5EV2, H9L8 (hG4WT), and relatrimab to LAG3 on the surface of 293T-LAG3 cells. [Figure 27] Results of FACS assays of the binding activity of NTLAV8, LA5EV2, 19F3H2L3(hG1DM), and CPI-006 to CD73 on the surface of U87-MG cells. [Figure 28] Results of FACS assays for the binding of NTLAV8, LA5EV2, relatrimab, and H9L8 to human LAG3-mG1Fc antigen, which compete with MHC-II on the surface of Raji cells. [Figure 29] Results of blocking the interaction between LAG3 and MHC-II using antibodies NTLAV8 and H9L8 (hG4WT). [Figure 30] Results of an assay in which the enzymatic activity of CD73 on the cell membrane surface was inhibited by an anti-CD73 anti-LAG3 bispecific antibody. [Figure 31] Results of a mixed lymphocyte assay for the biological activity of anti-CD73 anti-LAG3 bispecific antibodies in promoting IFNγ secretion. [Figure 32] Results of an assay to assess the potential phagocytic activity of CHO-K1-LAG3-CD73 target cells using an anti-CD73 anti-LAG3 bispecific antibody. [Figure 33]Results of pharmacodynamic evaluation of anti-CD73 anti-LAG3 bispecific antibodies in a mouse model of tumor cells subcutaneously transplanted. [Figure 34] Results of the effect of anti-CD73 anti-LAG3 bispecific antibodies on body weight in tumor cell subcutaneous transplantation model mice. [Modes for carrying out the invention]

[0079] Detailed explanation The aspects of this disclosure will be described in detail below with reference to examples. Those skilled in the art will understand that the following examples are for illustrative purposes only and should not be construed as limiting the scope of this disclosure. Examples that do not specify specific techniques or conditions are carried out in accordance with techniques or conditions described in publications in the art (for example, see Molecular Cloning: A Laboratory Manual, authored by J. Sambrook et al., and translated by Huang Peitang et al., third edition, Science Press) or product descriptions. The reagents or equipment used are commercially available products unless the manufacturer is specified.

[0080] The sequence of the positive control antibody, relatrimab, is described in U.S. Patent Application Publication 2016 / 0326248. Refer to Sequence ID No. 1 in this publication for the heavy chain amino acid sequence, and Sequence ID No. 2 in this publication for the light chain amino acid sequence. Relatrimab is an anti-LAG3 antibody.

[0081] Amino acid sequence of the heavy chain of relatrimab: QVQLQQWGAGLLKPSETLSLTCAVYGGSFSDYYWNWIRQPPGKGLEWIGEINHRGSTNSNPSLKSRVTLSLDTSKNQFSLKLRSVTAADTAVYYCAFGYSDYEYNWFDPWGQG TLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCP PCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(Sequence ID 23)

[0082] Amino acid sequence of the light chain of relatrimab: EIVLTQSPATLSLSPGERATLSCRASQSISSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPLTFGQGTNLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (Sequence ID 24)

[0083] Antibody 14C12H1L1(hG1TM) is an anti-PD-1 antibody manufactured by Akeso Biopharma Inc.

[0084] The amino acid sequence of the heavy chain of 14C12H1L1(hG1TM): EVQLVESGGGLVQPGGSLRLSCAASGFAFSSYDMSWVRQAPGKGLDWVATISGGGRYTYYPDSVKGRFTISRDNSKNNLYLQMNSLRAEDTALYYCANRYGEAWFAYWGQGTLV TVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCP PCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (Sequence ID 21)

[0085] The amino acid sequence of the light chain of 14C12H1L1(hG1TM): DIQMTQSPSSMSASVGDRVTFTCRASQDINTYLSWFQQKPGKSPKTLIYRANRLVSGVPSRFSGSGSGQDYTLTISSLQPEDMATYYCLQYDEFPLTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (Sequence ID 22)

[0086] Amino acid sequence of human LAG3-mG1Fc: (Sequence ID 60)

[0087] Amino acid sequence of hNT5E(1~552)-His: (Sequence ID 61)

[0088] The cell line 293T-LAG3 was constructed by Akeso Biopharma Inc. The cell line 293T-LAG3 was generated by viral infection of HEK293T cells using a third-generation lentivirus system (see, for example, A Third Generation Lentivirus Vector with a Conditional Packaging System. Dull T, Zufferey R, Kelly M, Mandel RJ, Nguyen M, Trono D, and Naldini L., J Virol., 1998. 72(11): 8463-8471). The lentivirus expression vector used was plenti6.3 / V5-huLAG3FL-BSD (LAG3, Genebank ID: NP_002277.4; vector plenti6.3 / V5-BSD, purchased from Invitrogen (catalog number K5315-20)).

[0089] The cell line Raji-PDL1 was constructed by Akeso Biopharma Inc. The cell line Raji-PDL1 was generated by viral infection of Raji cells using a third-generation lentivirus system (see, for example, A Third Generation Lentivirus Vector with a Conditional Packaging System. Dull T, Zufferey R, Kelly M, Mandel RJ, Nguyen M, Trono D, and Naldini L., J Virol., 1998. 72(11): 8463-8471). The lentivirus expression vector used was plenti6.3 / V5-PDL1 (PDL1, Genebank ID: NP_054862.1; vector plenti6.3 / V5, purchased from Invitrogen (catalog number K5315-20)).

[0090] The cell line Jurkat-NFAT-PD1-LAG3 was constructed by Akeso Biopharma Inc. The cell line Jurkat-NFAT-PD1-LAG3 was generated by viral infection of PD-1 effector cells (CPM, manufacturer: Promega, catalog number J112A) using a third-generation lentivirus system (see, e.g., A Third Generation Lentivirus Vector with a Conditional Packaging System. Dull T, Zufferey R, Kelly M, Mandel RJ, Nguyen M, Trono D, and Naldini L., J Virol., 1998. 72(11): 8463-8471). The lentivirus expression vector used was pCDH-huLAG3FL-RFP-NEO (LAG3, Genebank ID: NP_002277.4; vector pCDH-CMV-MCS-EF1-RFP+Neo, purchased from Youbio (catalog number VT9005)).

[0091] The cell line CHO-K1-LAG3-CD73 was constructed by Akeso Biopharma Inc. The cell line CHO-K1-LAG3-CD73 was generated by viral infection of CHO-K1 cells (manufacturer: Institute of Basic Medical Sciences of the Chinese Academy of Medical Sciences, catalog number 3111C0001CCC000004) using a third-generation lentivirus system (see, for example, A Third Generation Lentivirus Vector with a Conditional Packaging System. Dull T, Zufferey R, Kelly M, Mandel RJ, Nguyen M, Trono D, and Naldini L., J Virol., 1998. 72(11): 8463-8471). The lentivirus expression vector used was plenti6.3 / V5-huLAG3FL-BSD (LAG3, Genebank ID: NP_002277.4; vector The software used was plenti6.3 / V5-BSD (purchased from Invitrogen, catalog number K5315-20) and pCDH-GFP-NT5EFL-puro (NT5E, Genebank ID: NP_002517.1; vector pCDH-puro, purchased from Youbio).

[0092] The sequence information for the positive control antibody MEDI9447 is as follows. MEDI9447 is an anti-CD73 antibody.

[0093] heavy chain amino acid sequence of MEDI9447 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAYSWVRQAPGKGLEWVSAISGSGGRTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARLGYGRVDEWGRGTLV TVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCP PCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (Sequence ID 56)

[0094] Light chain amino acid sequence of MEDI9447 QSVLTQPPSASGTPGQRVTISCSGSLSNIGRNPVNWYQQLPGTAPKLLIYLDNLRLSGVPDRFSGSKSGTSASLAISGLQSEDEADYYCATWDDSHPGWTFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS(Sequence ID 57)

[0095] The sequence information for the positive control antibody CPI-006 is as follows. CPI-006 is an anti-CD73 antibody.

[0096] Amino acid sequence of the heavy chain of CPI-006 QVQLVQSGAEVEKPGASVKVSCKASGYTFTSYWITWVRQAPGQGLEWMGDIYPGSGNTNYNEKFKTRVTITADKSTSTAYMELSSLRSEDTAVYYCAKEGGLTTEDYALDYWGQG TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (Sequence ID 58)

[0097] CPI-006 light chain amino acid sequence EIVLTQSPATLSLSPGERATLSCRASKNVSTSGYSYMHWYQQKPGQAPRLLIYLASNLESGIPPRFSGSGYGTDFTLTINNIESEDAAYYFCQHSRELPFTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (Sequence ID 59) [Examples]

[0098] Manufacturing Example 1: Design and Manufacturing of Anti-LAG3 Antibodies 1. Antibody design The inventors creatively designed a series of antibody sequences based on the known LAG3 protein sequence (NCBI reference sequence: NP_002277.4), its three-dimensional crystal structure, etc. Through extensive screening and testing, humanized monoclonal antibodies that specifically bind to LAG3 were ultimately obtained and named H9L8, H9L9, and H9L10, respectively. The amino acid sequences of the heavy and light chain variable regions of the monoclonal antibodies and the sequences encoding them are as follows:

[0099] Nucleotide sequence of the heavy chain variable region H9v of H9L8 (360 bp): CAGGTGCAGCTGCAGCAGTGGGGAGCTGGACTGCTGAAACCTAGCGAGACACTGAGCCTGACCTGTGCTGTGTACGGCGGATCTATCAGCGATTACTACTGGAACTGGATCAGGCAGCCCCCTGGAAAGGGACTGGAATGGATCGGAGAGATCAACTACAGGGGCACCACCAACTCCAATCCC TCTCTGAAGAGCAGGGTGACACTGAGCCTCGACACAAGCAAGAATCAGTTCAGCCTGAAGCTGAGGTCCGTGACCGCTGCTGATACAGCTGTGTACTACTGTGCCTTCGGCTACAGCGATTACGAGTACGATTGGTTCGACCCTGGGGGCCAGGGAACACTGGTTACAGTGAGCTCC (SEQ ID NO: 1)

[0100] Amino acid sequence of the heavy chain variable region H9v of H9L8 (120aa): QVQLQQWGAGLLKPSETLSLTCAVYGGSISDYYWNWIRQPPGKGLEWIGEINYRGTTNSNPSLKSRVTLSLDTSKNQFSLKLRSVTAADTAVYYCAFGYSDYEYDWFDPWGQGTLVTVSS (Sequence ID 2)

[0101] Nucleotide sequence of the light chain variable region L8v of H9L8 (321 bp): GAGATCGTTCTGACCCAGAGCCCAGCTACACTGAGCCTGTCTCCTGGAGAGAGGGCTACACTGTCCTGCAGAGCTAGCCAGACCATCAGCAGCTACCTGGCTTGGTACCAGCAGAAGCCTGGCCAAGCTCCAAGGCTGCTGATCTACGACGCCTCTAATAGGGC CACCGGCATCCCTGCTAGATTCTCTGGAAGCGGCAGCGGAACCGACTTTACACTGACAATCAGCTCCCTGGAGCCCGAGGATTTCGCTGTTTACTACTGTCAGCAGCGCAGCAACTGGCCCATCACATTCGGACAGGGCACAAATCTGGAGATCAAG (SEQ ID NO: 3)

[0102] Amino acid sequence of the light chain variable region L8v of H9L8 (107aa): EIVLTQSPATLSLSPGERATLSCRASQTISSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPITFGQGTNLEIK(Sequence ID 4)

[0103] The nucleotide sequence of the heavy chain variable region H9v of H9L9 is the same as the nucleotide sequence of the heavy chain variable region H9v of H9L8, as shown in SEQ ID NO: 1.

[0104] The amino acid sequence of the heavy chain variable region H9v of H9L9 is the same as the amino acid sequence of the heavy chain variable region H9v of H9L8, as shown in SEQ ID NO: 2.

[0105] Nucleotide sequence of the L9v light chain variable region of H9L9 (321 bp): GAGATCGTTCTGACCCAGAGCCCAGCTACACTGAGCCTGTCTCCTGGAGAGAGGGCTACACTGTCCTGCAGAGCTAGCCAGACCATCAGCAGCTACCTGGCTTGGTACCAGCAGAAGCCTGGCCAAGCTCCAAGGCTGCTGATCTACGACGGCTCTAATAGGGC CACCGGCATCCCTGCTAGATTCTCTGGAAGCGGCAGCGGAACCGACTTTACACTGACAATCAGCTCCCTGGAGCCCGAGGATTTCGCTGTTTACTACTGTCAGCAGGCAGCAACTGGCCCCTCACATTCGGACAGGGCACAAATCTGGAGATCAAG (SEQ ID NO: 5)

[0106] Amino acid sequence of the light chain variable region L9v of H9L9 (107 bp): EIVLTQSPATLSLSPGERATLSCRASQTISSYLAWYQQKPGQAPRLLIYDGSNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPLTFGQGTNLEIK (Sequence ID 6)

[0107] The nucleotide sequence of the heavy chain variable region H9v of H9L10 is the same as the nucleotide sequence of the heavy chain variable region H9v of H9L8, as shown in SEQ ID NO: 1.

[0108] The amino acid sequence of the heavy chain variable region H9v of H9L10 is the same as the amino acid sequence of the heavy chain variable region H9v of H9L8, as shown in SEQ ID NO: 2.

[0109] Nucleotide sequence of the light chain variable region L10v of H9L10 (321 bp): GAGATCGTTCTGACCCAGAGCCCAGCTACACTGAGCCTGTCTCCTGGAGAGAGGGCTACACTGTCCTGCAGAGCTAGCCAGTCCATCAGCAGCTACCTGGCTTGGTACCAGCAGAAGCCTGGCCAAGCTCCAAGGCTGCTGATCTACGACGGCTCTAATAGGGC CACCGGCATCCCTGCTAGATTCTCTGGAAGCGGCAGCGGAACCGACTTTACACTGACAATCAGCTCCCTGGAGCCCGAGGATTTCGCTGTTTACTACTGTCAGCAGCGCAGCAACTGGCCCATCACATTCGGACAGGGCACAAATCTGGAGATCAAG (SEQ ID NO: 7)

[0110] Amino acid sequence of the light chain variable region L10v of H9L10 (107 bp): EIVLTQSPATLSLSPGERATLSCRASQSISSYLAWYQQKPGQAPRLLIYDGSNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPITFGQGTNLEIK (Sequence ID 8)

[0111] The amino acid sequence of the H9L8 CDR is as follows (according to the IMGT numbering system): HCDR1: GGSISDYY(Sequence ID 9); HCDR2: INYRGTT(Sequence ID 10); HCDR3: AFGYSDYEYDWFDP(Sequence ID 11); LCDR1: QTISSY(array element 12); LCDR2: DAS(Sequence ID 13); LCDR3: QQRSNWPIT (Sequence ID 14).

[0112] The amino acid sequence of the H9L9 CDR is as follows (according to the IMGT numbering system): HCDR1: GGSISDYY(Sequence ID 9); HCDR2: INYRGTT(Sequence ID 10); HCDR3: AFGYSDYEYDWFDP(Sequence ID 11); LCDR1: QTISSY(array element 12); LCDR2: DGS(Sequence ID 15); LCDR3: QQRSNWPLT (Sequence ID 16).

[0113] The amino acid sequence of the H9L10 CDR is as follows (according to the IMGT numbering system): HCDR1: GGSISDYY(Sequence ID 9); HCDR2: INYRGTT(Sequence ID 10); HCDR3: AFGYSDYEYDWFDP(Sequence ID 11); LCDR1: QSISSY(sequence number 17); LCDR2: DGS(Sequence ID 15); LCDR3: QQRSNWPIT (Sequence ID 14).

[0114] 2. Expression and purification of humanized antibodies H9L8(hG4WT), H9L9(hG4WT), and H9L10(hG4WT) cDNA sequences of the heavy chains of H9L8(hG4WT), H9L9(hG4WT), and H9L10(hG4WT) (the sequence encoding the variable region is shown in SEQ ID NO: 1; the constant region is the Igγ4 chain C region), cDNA sequences of the light chain of H9L8(hG4WT) (the sequence encoding the variable region is shown in SEQ ID NO: 3; the constant region is the human Igκ chain C region), cDNA sequences of the light chain of H9L9(hG4WT) (the sequence encoding the variable region is shown in SEQ ID NO: 5; the constant region The constant region (the human Igκ chain C region) and the cDNA sequence of the light chain of H9L10 (hG4WT) (the sequence encoding the variable region is shown in SEQ ID NO: 7; the constant region is the human Igκ chain C region) were separately cloned into the pUC57simple vector (provided by GenScript), yielding plasmids pUC57simple-H9, pUC57simple-L8, pUC57simple-L9, and pUC57simple-L10, respectively. Each plasmid, pUC57simple-H9, pUC57simple-L8, pUC57simple-L9, and pUC57simple-L10, was digested (HindIII and EcoRI). The heavy and light chains isolated by electrophoresis were subcloned into pcDNA3.1 vectors, respectively, and the recombinant plasmids were extracted and co-transfected into 293F cells. After 7 days of incubation, the culture medium was separated using a high-speed centrifuge, the supernatant was concentrated, and filtered through a HiTrap MabSelect SuRe column. Proteins were eluted in a single step using elution buffer. The target sample was isolated, and the buffer was replaced with PBS.

[0115] Amino acid sequences of the heavy chain constant region of H9L8(hG4WT), H9L9(hG4WT), or H9L10(hG4WT): ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(Sequence ID 20)

[0116] Amino acid sequences of the H9L8(hG4WT), H9L9(hG4WT), or H9L10(hG4WT) light chain constant region: RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(Sequence ID 19)

[0117] 3. Expression and purification of humanized antibody H9L8(hG1WT) Based on the variable region of the above antibody H9L8(hG4WT), the antibody H9L8(hG1WT) was obtained using the Igγ-1 chain C region as the heavy chain constant region.

[0118] Amino acid sequence of the heavy chain of H9L8(hG1WT) QVQLQQWGAGLLKPSETLSLTCAVYGGSISDYYWNWIRQPPGKGLEWIGEINYRGTTNSNPSLKSRVTLSLDTSKNQFSLKLRSVTAADTAVYYCAFGYSDYEYDWFDPWGQGT LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTC PPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(Sequence ID 38)

[0119] Amino acid sequence of the light chain of H9L8(hG1WT) EIVLTQSPATLSLSPGERATLSCRASQTISSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPITFGQGTNLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (Sequence ID 39)

[0120] Manufacturing Example 2: Production of Human Anti-Egg Lysozyme Human Antibody The sequence of the human anti-egg lysozyme IgG (anti-HEL, or human IgG, abbreviated as hIgG) antibody was derived from the variable region of the Fab F10.6.6 sequence in the study report titled "Affinity maturation increases the stability and plasticity of the Fv domain of anti-protein antibodies" by Acierno et al. (Acierno et al., J Mol Biol., 2007; 374(1): 130-46). The manufacturing method was as follows: Codon optimization of amino acids and gene synthesis of the heavy and light chain (complete sequence or variable region) genes of the human IgG antibody were commissioned to Nanjing GenScript Biotech. The heavy and light chain genes were subcloned into mammalian expression system antibody heavy chain expression vectors and antibody light chain expression vectors, respectively, using standard molecular cloning techniques such as PCR, enzyme digestion, DNA gel extraction, ligation and transformation, colony PCR, or enzyme digestion identification, referencing the standard techniques described in Molecular Cloning: A Laboratory Manual (Third Edition). The heavy and light chain genes of the recombinant expression vector were further sequenced and analyzed. After confirming the correctness of the sequences, medium or large quantities of endotoxin-free expression plasmids were prepared, and the heavy and light chain expression plasmids were transiently co-transfected into HEK293 cells to express recombinant antibodies. After 7 days of culture, the cell culture medium was collected and affinity-purified using recombinant protein A column (GE), and the quality of the resulting antibody samples was measured by SDS-PAGE and SEC-HPLC standard analytical methods.

[0121] Unless otherwise specified, the hIgG1, hIgG1DM, and hIgG4WT used in this disclosure were anti-HEL isotype control antibodies prepared in the laboratories of Akeso Biopharma Inc., each containing the sequence of the constant region of hIgG1, hIgG1DM, and hIgG4WT, respectively.

[0122] Manufacturing Example 3: Design and Manufacturing of Anti-CD73 Antibodies 1. For the sequences and production methods of 19F3, 19F3H1L1, 19F3H2L2 and 19F3H2L3, which are anti-CD73 antibodies, refer to Chinese Patent Application CN113527489A, and a part of the sequences is listed below.

[0123] 19F3, 19F3H1L1, 19F3H2L2 and 19F3H2L3 have the same three heavy-chain CDRs and three light-chain CDRs (according to the IMGT numbering system): HCDR1: GYSFTGYT (SEQ ID NO: 25) HCDR2: INPYNAGT (SEQ ID NO: 26) HCDR3: ARSEYRYGGDYFDY (SEQ ID NO: 27) LCDR1: QSLLNSSNQKNY (SEQ ID NO: 28) LCDR2: FAS (SEQ ID NO: 29) LCDR3: QQHYDTPYT (SEQ ID NO: 30)

[0124] Amino acid sequence of the heavy-chain variable region of 19F3 (CDR sequences underlined): EVQLQQSGPELVKPGASMRMSCKAS GYSFTGYT MNWVKQSHGKNLEWIGL INPYNAGT SYNQKFKGKATLTVDKSSSTAYMELLSLTSEDSAVYYC ARSEYRYGGDYFDY WGQGTTLTVSS (SEQ ID NO: 31)

[0125] Amino acid sequence of the light-chain variable region of 19F3 (CDR sequences underlined): DIVMTQSPSSLAMSVGQKVTMSCKSS QSLLNSSNQKNY LAWYQQKPGQSPKLLVY FAS TRESGVPDRFIGSGSGTDFTLTISSVQAEDLADYFC QQHYDTPYT FGGGTKLEIK (SEQ ID NO: 32)

[0126] Amino acid sequence of the heavy-chain variable region of 19F3H1L1 (CDR sequences underlined): QVQLQQSGAEVVKPGASMKMSCKAS GYSFTGYTMNWVKQAHGQNLEWIGL INPYNAGT SYNQKFQGKATLTVDKSTSTAYMELSSLRSEDTAVYYC ARSEYRYGGDYFDY WGQGTTLTVSS(Sequence ID 33)

[0127] Amino acid sequence of the light chain variable region of 19F3H1L1 (CDR sequence is underlined): DIVMTQSPSSLAMSVGERVTMSCKSS QSLLNSSNQKNY LAWYQQKPGQAPKLLVY FAS TRESGVPDRFSGSGSGTDFTLTISSVQAEDLADYFC QQHYDTPYT FGGGTKLEIK (Sequence ID 34)

[0128] Amino acid sequence of the heavy chain variable region of 19F3H2L2 (CDR sequence is underlined): QVQLVQSGAEVVKPGASVKVSCKAS GYSFTGYT MNWVRQAPGQNLEWIGL INPYNAGT SYNQKFQGKVTLTVDKSTSTAYMELSSLRSEDTAVYYC ARSEYRYGGDYFDY WGQGTTLTVSS(Sequence ID 35)

[0129] Amino acid sequence of the light chain variable region of 19F3H2L2 (CDR sequence is underlined): DIVMTQSPSSLAVSVGERVTISCKSS QSLLNSSNQKNY LAWYQQKPGQAPKLLIY FAS TRESGVPDRFSGSGSGTDFTLTISSVQAEDVADYYC QQHYDTPYT FGGGTKLEIK (Sequence ID 36)

[0130] Amino acid sequence of the heavy chain variable region of 19F3H2L3 (CDR sequence is underlined): QVQLVQSGAEVVKPGASVKVSCKAS GYSFTGYT MNWVRQAPGQNLEWIGL INPYNAGT SYNQKFQGKVTLTVDKSTSTAYMELSSLRSEDTAVYYC ARSEYRYGGDYFDY WGQGTTLTVSS(Sequence ID 35)

[0131] Amino acid sequence of the light chain variable region of 19F3H2L3 (CDR sequence is underlined): DIVMTQSPSSLAVSVGERVTISCKSS QSLLNSSNQKNY LAWYQQKPGQAPKLLIY FAS TRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC QQHYDTPYT FGGGTKLEIK (Sequence ID 37)

[0132] 2. Production of humanized antibody 19F3H2L3 All of the heavy chain constant regions were Igγ-1 chain C regions, and all of the light chain constant regions were Igκ chain C regions.

[0133] The heavy chain cDNA and light chain cDNA of 19F3H2L3 were separately cloned into the pUC57simple vector (provided by GenScript) to obtain pUC57simple-19F3H2 and pUC57simple-19F3L3, respectively. Following the standard method described in Molecular Cloning: A Laboratory Manual (Third Edition), the full-length heavy chain and light chain genes synthesized by EcoRI and HindIII digestion were separately subcloned into the expression vector pcDNA3.1 by restriction enzyme digestion (EcoRI and HindIII) to obtain expression plasmids pcDNA3.1-19F3H2 and pcDNA3.1-19F3L3, and the heavy chain / light chain gene sequences of the recombinant expression plasmids were further analyzed. Subsequently, the designed gene combinations containing the corresponding light chain and heavy chain recombinant plasmids (pcDNA3.1-19F3H2 / pcDNA3.1-19F3L3) were simultaneously transfected into 293F cells, and the culture medium was collected and purified. After confirming the correctness of the sequence by sequencing, an endotoxin-free expression plasmid was prepared and transiently introduced into HEK293 cells to express the antibody. After 7 days, the cell culture medium was collected and affinity-purified using a protein A column to obtain the humanized antibody.

[0134] Amino acid sequence of the heavy chain of 19F3H2L3 QVQLVQSGAEVVKPGASVKVSCKASGYSFTGYTMNWVRQAPGQNLEWIGLINPYNAGTSYNQKFQGKVTLTVDKSTSTAYMELSSLRSEDTAVYYCARSEYRYGGDYFDYWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 40)

[0135] Amino acid sequence of the light chain of 19F3H2L3 DIVMTQSPSSLAVSVGERVTISCKSS QSLLNSSNQKNY LAWYQQKPGQAPKLLIY FAS TRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC QQHYDTPYT FGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 41)

[0136] 3. Production of humanized antibody 19F3H2L3 (hG1DM) The constant region of the light chain of antibody 19F3H2L3 (hG1DM) was the Igκ chain C region shown in SEQ ID NO: 19.

[0137] Based on the heavy chain constant region Igγ-1 chain C region (SEQ ID NO: 18), a humanized antibody was obtained by introducing a point mutation from leucine to alanine at position 234 (L234A) and a point mutation from leucine to alanine at position 235 (L235A), and named 19F3H2L3(hG1DM).

[0138] The heavy chain cDNA and light chain cDNA of 19F3H2L3(hG1DM) were separately cloned into the pUC57simple vector (provided by GenScript) to obtain pUC57simple-19F3H2(hG1DM) and pUC57simple-19F3L3, respectively. Following the standard method described in Molecular Cloning: A Laboratory Manual (Third Edition), the full-length heavy chain and light chain genes synthesized by EcoRI and HindIII digestion were separately subcloned into the expression vector pcDNA3.1 by restriction enzyme digestion (EcoRI and HindIII) to obtain expression plasmids pcDNA3.1-19F3H2(hG1DM) and pcDNA3.1-19F3L3, and the sequences of the heavy chain / light chain genes of the recombinant expression plasmids were further analyzed. Next, the designed gene combination, including the corresponding light-chain and heavy-chain recombinant plasmid (pcDNA3.1-19F3H2(hG1DM) / pcDNA3.1-19F3L3), was co-transfected into 293F cells, and the culture medium was collected and purified. After confirming the correctness of the sequence by sequencing, an endotoxin-free expression plasmid was prepared and transiently introduced into HEK293 cells to express the antibody. After 7 days, the cell culture medium was collected and affinity-purified using a protein A column to obtain the humanized antibody.

[0139] 19F3H2L3(hG1DM) full length heavy chain QVQLVQSGAEVVKPGASVKVSCKASGYSFTGYTMNWVRQAPGQNLEWIGLINPYNAGTSYNQKFQGKVTLTVDKSTSTAYMELSSLRSEDTAVYYCARSEYRYGGDYFDYWGQG TTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT CPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (Sequence ID 49)

[0140] 19F3H2L3(hG1DM) full length light chain DIVMTQSPSSLAVSVGERVTISCKSSQSLLNSSNQKNYLAWYQQKPGQAPKLLIYFASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQHYDTPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (Sequence ID 50)

[0141] Manufacturing Example 4: Design and Manufacturing of Anti-CD73 Anti-LAG3 Bispecific Antibodies 1. Array Design The structures of the bispecific antibodies NTLAV2, NTLAV7, NTLAV8, LA5EV1, and LA5EV2 in this disclosure are in Morrison format (IgG-scFv), meaning that the C-terminuses of two heavy chains of one IgG antibody are each linked to an scFv fragment of another antibody, and the main components of the heavy and light chains are shown in Table 1 below.

[0142] [Table 1]

[0143] Table 1 shows, (1) Amino acid sequence of linker (GGGGS)3: GGGGSGGGGSGGGGS (Sequence 42) (2) Amino acid sequence of linker (GGGGS) 4: GGGGSGGGGSGGGGSGGGGS(SEQ 43) That is the case.

[0144] Names with a "v" in the lower right corner indicate the variable region of the corresponding heavy chain or light chain. Names without a "v" indicate that the corresponding heavy chain or light chain is the full length including the constant region. Unless otherwise specified, all amino acid sequences and encoding nucleotide sequences of these variable regions or full length refer to the corresponding sequences described in the above manufacturing examples.

[0145] Table 1 above shows H9 in the scFv fragment of the antibody. V (M), L8 V (M), 19FH2 V (M), 19L3 V (M1) and 19L3 V (M2) is H9 V L8 V , 19FH2 V and 19L3 V It is generated by introducing specific amino acid mutations into the corresponding framework region, and therefore the antibody structure is effectively optimized and the antibody efficacy is enhanced.

[0146] H9 V (M) amino acid sequence QVQLQQWGAGLLKPSETLSLTCAVYGGSISDYYWNWIRQPPGKCLEWIGEINYRGTTNSNPSLKSRVTLSLDTSKNQFSLKLRSVTAADTAVYYCAFGYSDYEYDWFDPWGQGTLVTVSS (Sequence ID 44)

[0147] L8 V (M) amino acid sequence EIVLTQSPATLSLSPGERATLSCRASQTISSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPITFGCGTNLEIK (Sequence ID 45)

[0148] 19FH2 V (M) amino acid sequence QVQLVQSGAEVVKPGASVKVSCKASGYSFTGYTMNWVRQAPGQCLEWIGLINPYNAGTSYNQKFQGKVTLTVDKSTSTAYMELSSLRSEDTAVYYCARSEYRYGGDYFDYWGQGTTLTVSS(Sequence ID 46)

[0149] 19L3 V (M1) amino acid sequence DIVMTQSPSSLAVSVGERVTISCKSSQSLLNSSNQKNYLAWYQQKPGQAPKLLIYFASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQHYDTPYTFGCGTKLEIKR (Sequence ID 47)

[0150] 19L3 V (M2) amino acid sequence DIVMTQSPSSLAVSVGERVTISCKSSQSLLNSSNQKNYLAWYQQKPGQAPKLLIYFASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQHYDTPYTFGGGTKLEIKR (Sequence ID 62)

[0151] Based on the bispecific antibodies designed in Table 1 above, point mutations were introduced to change leucine to alanine at position 234 of the heavy chain (L234A), leucine to alanine at position 235 (L235A), and glycine to alanine at position 237 (G237A). These bispecific antibodies containing the above mutation sites were named NTLAV2(hG1TM), NTLAV7(hG1TM), NTLAV8(hG1TM), LA5EV1(hG1TM), and LA5EV2(hG1TM).

[0152] Amino acid sequence of the heavy chain of NTLAV2(hG1TM) (Sequence ID 51)

[0153] Amino acid sequence of the heavy chain of NTLAV7(hG1TM) (Sequence ID 52)

[0154] Amino acid sequence of the heavy chain of NTLAV8(hG1TM) (Sequence No. 53)

[0155] Amino acid sequence of the heavy chain of LA5EV1(hG1TM) (Sequence ID 54)

[0156] Amino acid sequence of the heavy chain of LA5EV2(hG1TM) (Sequence ID 55)

[0157] Unless otherwise specified, in the following experiments and examples, the bispecific antibodies NTLAV2(hG1TM), NTLAV7(hG1TM), NTLAV8(hG1TM), LA5EV1(hG1TM), and LA5EV2(hG1TM) having the above mutation site will be referred to as NTLAV2, NTLAV7, NTLAV8, LA5EV1, and LA5EV2, respectively.

[0158] 2. Antibody Expression and Purification The heavy and light chain cDNA sequences of NTLAV2, NTLAV7, NTLAV8, LA5EV1, and LA5EV2 were separately cloned into the pUC57simple vector (provided by GenScript), yielding the plasmids pUC57simple-NTLAV2H / pUC57simple-NTLAV2L, pUC57simple-NTLAV7H / pUC57simple-NTLAV7L, pUC57simple-NTLAV8H / pUC57simple-NTLAV8L, pUC57simple-LA5EV1H / pUC57simple-LA5EV1L, and pUC57simple-LA5EV2H / pUC57simple-LA5EV2H, respectively.

[0159] Each plasmid, pUC57simple-NTLAV2H / pUC57simple-NTLAV2L, pUC57simple-NTLAV7H / pUC57simple-NTLAV7L, pUC57simple-NTLAV8H / pUC57simple-NTLAV8L, pUC57simple-LA5EV1H / pUC57simple-LA5EV1L, and pUC57simple-LA5EV2H / pUC57simple-LA5EV2H, was digested (HindIII and EcoRI). The heavy and light chains isolated by electrophoresis were subcloned into pcDNA3.1 vectors, and the recombinant plasmids were extracted and co-transfected into 293F cells. After 7 days of culture, the culture medium was separated by high-speed centrifugation, the supernatant was concentrated, and filtered through a HiTrap MabSelect SuRe column. Proteins were eluted in a single step using elution buffer. The target sample was isolated, and the buffer solution was replaced with PBS.

[0160] Example 1: ELISA assay of the binding activity of anti-LAG3 antibody to antigen. ELISA plates were coated with 2 μg / mL human LAG3-mG1Fc and incubated overnight at 4°C. The antigen-coated ELISA plates were then washed once with PBST and blocked at 37°C for 2 hours with a PBS solution containing 1% BSA (blocking solution). After blocking, the ELISA plates were washed three times with PBST. Antibodies serially diluted in PBST solution (the antibody dilution gradient is shown in Table 2) were added. ELISA plates containing the test antibody were incubated at 37°C for 30 minutes and then washed three times with PBST. After washing, a 1:5000 diluted standard solution of HRP-labeled goat anti-human IgG secondary antibody (H+L) (Jackson, catalog no. 109-035-088) was added, and the plates were incubated at 37°C for 30 minutes. After incubation, the plates were washed four times with PBST. Next, TMB (Neogen, 308177) was used to allow the color to develop in the dark for 5 minutes, and then stop solution was added to halt the color reaction. Immediately afterward, the ELISA plate was placed in a microplate reader, and the OD (450 nm) of each well was measured. The data was analyzed using SoftMax Pro 6.2.1 software.

[0161] The assay results are shown in Table 2 and Figure 1.

[0162] [Table 2]

[0163] Antibodies H9L8(hG4WT), H9L9(hG4WT), and H9L10(hG4WT) all effectively bound to the antigen LAG3-mG1Fc in a dose-dependent manner, demonstrating binding activity equivalent to that of the positive control antibody reratrimab.

[0164] Example 2 Flow cytometry assay of the binding activity of anti-LAG3 antibody to human LAG3 antigen on the cell surface Antibody labeling and flow cytometry detection: Human LAG3-expressing 293T-LAG3 cells were digested with conventional pancreatin, and the number of cells in each recovery tube was reduced to 3 × 10⁶. 5The cells were divided into individual tubes. Using 1% PBSA (PBS containing 1% BSA), LAG3 antibody dilutions were prepared to final concentrations of 0.0123 nM, 0.123 nM, 1.23 nM, 3.7 nM, 11.1 nM, 33.3 nM, 100 nM, and 300 nM. Each of these was incubated with 293T-LAG3 cells expressing LAG3 on ice for 1 hour. The tubes were centrifuged, washed several times with 1% PBSA, and 100 μL of FITC goat anti-human IgG (purchased from Jackson (catalog no. 109-095-098)) (diluted 1:300) was added to each tube. The mixture was incubated on ice in the dark for 40 minutes. The cells were washed once with 1% PBSA and resuspended with 200 μL of 1% PBSA. Fluorescence signals were detected in the FITC channel of a flow cytometer.

[0165] Figure 2 shows the results of binding of humanized anti-LAG3 antibody to 293T-LAG3 cells. EC of the binding of anti-LAG3 antibody to antigen on the surface of 293T-LAG3 cells. 50 This is shown in Table 3.

[0166] [Table 3]

[0167] As can be seen from Figure 2, the anti-LAG3 antibodies were able to effectively bind to the LAG3 protein on the surface of the target 293T-LAG3 cells, and the binding activity of the anti-LAG3 antibodies H9L8(hG4WT), H9L9(hG4WT), and H9L10(hG4WT) to the antigen on the surface of 293T-LAG3 cells was equivalent to that of the positive control antibody reratrimab.

[0168] Example 3: Competitive flow cytometry assay of binding of anti-LAG3 antibody competing with MHC-II on the Raji cell membrane surface to human LAG3-mG1Fc antigen. Raji cells (culture medium: 1640 + 10% FBS) (Cell Resource Center, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, catalog number TCHu 44) were added to EP tubes at a rate of 300,000 cells per sample. 1000 μL of 1% PBSA (PBS containing 1% BSA) was added. The mixture was centrifuged at 600 × g for 5 minutes, and the supernatant was removed. 100 μL of 300 nM hIgG1 (prepared from Akeso Biopharma Inc. (batch number 20190410)) was added to each tube, and the mixture was incubated on ice for 1 hour; after incubation, 200 μL of 1% PBSA was added to the Raji cells, and the mixture was centrifuged at 600 × g for 5 minutes, and the supernatant was removed. According to the experimental design, 60 μL / tube of diluted antibody at the corresponding concentration was added to a separate clean EP tube. 60 μL of human LAG3-mG1Fc (prepared from Akeso Biopharma Inc. (batch number 20190508)) was added to each corresponding antibody tube, the mixture was thoroughly mixed, and pre-incubated on ice for 30 minutes to achieve final antibody concentrations of 300 nM, 100 nM, 33.3 nM, 11.1 nM, 3.7 nM, 1.23 nM, 0.123 nM, and 0.0123 nM. The final concentration of human LAG3-mG1Fc was 3 nM. 100 μL of the pre-incubated antibody-protein mixture was added to the cells. The resulting mixture was thoroughly mixed and incubated on ice in the dark for 1 hour; 200 μL of 1% PBSA was added, the mixture was centrifuged at 600 × g for 5 minutes, the supernatant was removed, and the pellet was washed twice; 100 μL of APC anti-mouse antibody (purchased from Biolegend (catalog no. 405308)) (diluted to 1:400) was added, the mixture was thoroughly mixed, and incubated on ice in the dark for 40 minutes; 200 μL of 1% PBSA was added, the mixture was centrifuged at 600 × g for 5 minutes, and the supernatant was removed; 200 μL of 1% PBSA was added to each tube to resuspend the cells, and the suspensions were transferred to sample loading tubes and analyzed by flow cytometry.

[0169] The results are shown in Figure 3 and Table 4. Fluorescence analysis and curve fitting revealed the competitive binding EC of the antibodies relatrimab, H9L8 (hG4WT), H9L9 (hG4WT), and H9L10 (hG4WT). 50 These values ​​were calculated to be 1.153 nM, 1.459 nM, 1.482 nM, and 1.435 nM, respectively.

[0170] [Table 4]

[0171] The antibodies H9L8(hG4WT), H9L9(hG4WT), and H9L10(hG4WT) competitively bound to LAG3 and effectively blocked the binding of LAG3 to MHC-II on the surface of Raji cells in a dose-dependent manner, exhibiting activity comparable to the positive control antibody reratrimab.

[0172] Example 4: Assay of the biological activity of anti-LAG3 antibody in promoting the secretion of IFNγ and IL2 using a mixed lymphocyte reaction (MLR). 1. Assay of the biological activity of anti-LAG3 antibodies that promote IFNγ secretion in the Raji-PDL1 mixed lymphocyte reaction system. Raji-PDL1 cells were subcultured using conventional methods. PBMCs (from healthy donors) were thawed, cultured in 10 mL of 1640 complete medium, and stimulated with 0.5 μg / mL of SEB (Staphylococcal Enterotoxin B) (Dianotech, catalog number S010201) for 2 days. Raji-PDL1 cells were treated with 2 μg / mL of MMC (Stressmarq, catalog number SIH-246-10MG) at a working concentration and incubated for 1 hour in a 37°C, 5% CO2 incubator; PBMCs stimulated with SEB for 2 days and Raji-PDL1 cells treated with MMC for 1 hour were collected, washed twice with PBS, then resuspended in complete medium (RPMI1640 + 10% FBS) and counted. PBMCs and Raji-PDL1 cells were separately placed in 10 × 10⁶ U-shaped 96-well plates (Corning, model number 3799). 4Cells were added to each well and co-cultured. According to the experimental design, antibodies (final concentrations of each antibody, whether used alone or in combination, were 300 nM, 30 nM, and 3 nM) were added and co-cultured with cells in an incubator for 3 days; after 3 days, the cells were centrifuged at 250 × g for 5 minutes, the cell culture supernatant was collected, and IFNγ was assayed by ELISA.

[0173] As shown in Figure 4, a mixed culture system of human PBMCs and Raji-PDL1 cells promoted IFNγ secretion in PBMCs, and the addition of antibodies to the mixed culture system further significantly induced IFNγ secretion in PBMCs. Regarding the activity of promoting IFNγ secretion, the anti-LAG3 antibodies H9L8(hG4WT), H9L9(hG4WT), and H9L10(hG4WT) combined with 14C12H1L1(hG1TM), as well as the positive control antibody relatrimab combined with 14C12H1L1(hG1TM), all promoted IFNγ secretion with comparable activity.

[0174] 2. Assay of the biological activity of anti-LAG3 antibodies that promote IL2 secretion in the Raji-PDL1 mixed lymphocyte reaction system. Raji-PDL1 cells were subcultured using conventional methods. PBMCs were thawed and cultured in 10 mL of 1640 complete medium, then stimulated with 0.5 μg / mL of SEB (Staphylococcal enterotoxin B, purchased from Dianotech (catalog number S010201)) for 2 days. Raji-PDL1 cells were treated with 2 μg / mL of MMC (Stressmarq, catalog number SIH-246-10MG) at a working concentration and incubated for 1 hour in a 37°C, 5% CO2 incubator. PBMCs stimulated with SEB for 2 days and Raji-PDL1 cells treated with MMC for 1 hour were collected, washed twice with PBS, then resuspended in complete medium (RPMI1640 + 10% FBS) and counted. PBMCs and Raji-PDL1 cells were separately placed in 10 × 10⁶ U-shaped 96-well plates (Corning, model number 3799). 4Cells were added to each well and co-cultured. According to the experimental design, antibodies (final concentrations of each antibody, whether used alone or in combination, were 300 nM, 30 nM, and 3 nM) were added and co-cultured with the cells for 3 days; after 3 days, the cells were centrifuged at 250 × g for 5 minutes, the cell culture supernatant was collected, and IL2 was assayed by ELISA.

[0175] As shown in Figure 5, a mixed culture system of human PBMCs (from healthy donors) and Raji-PDL1 cells promoted IL2 secretion in PBMCs to some extent, and the addition of antibodies to the mixed culture system further significantly induced IL2 secretion in PBMCs, showing a marked dose-dependent effect. Regarding the activity of promoting IL2 secretion, the anti-LAG3 antibodies H9L8(hG4WT), H9L9(hG4WT), and H9L10(hG4WT) combined with 14C12H1L1(hG1TM), as well as the positive control antibody relatrimab combined with 14C12H1L1(hG1TM), all promoted IL2 secretion with comparable activity.

[0176] Example 5: Assay for evaluating the biological activity of an anti-LAG3 antibody in blocking the interaction between LAG3 and MHC-II (reporter gene method) Jurkat-NFAT-PD1-LAG3 cells and Raji cells were used as reporter gene systems. After adding the superantigen SEE, the TCR-NFAT signaling pathway was activated to induce luciferase expression. LAG3 on Jurkat cells bound to MHC-II on Raji cells, inhibiting the NFAT signaling pathway and downregulating luciferase expression. The antibody specifically bound to LAG3, releasing this inhibition and upregulating luciferase expression.

[0177] Jurkat-NFAT-PD1-LAG3 cells and Raji cells (purchased from the Cell Resource Center, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences (catalog number TCHu 44)) were collected, centrifuged at 110×g for 5 minutes, and the supernatant was removed. The cells were then resuspended in 1640+10% FBS medium and counted. Jurkat-NFAT-PD1-LAG3 cells were divided into 105 Cells were seeded at 30 μL / well in a black-bottomed 96-well plate (Corning, model number 3916); according to the experimental design, antibodies (final concentrations of 0.3 nM, 3 nM, and 300 nM) were added at 10 μL / well, and the mixture was pre-incubated for 30 minutes in an incubator at 37°C and 5% CO2. Meanwhile, SEE (Staphylococcal Enterotoxin E, purchased from Toxin Technology (catalog number ET404)) (final concentration 0.05 ng / mL) was added to the Raji cells, and the mixture was incubated for 30 minutes in an incubator at 37°C and 5% CO2. After 30 minutes, the SEE-treated Raji cells were placed in the 96-well plate containing the Jurkat-NFAT-PD1-LAG3 cells described above, 2 × 10⁶ cells per well. 4 Cells were added at a rate of 40 μL / well, bringing the final volume of each well to 80 μL. The mixture was thoroughly mixed and incubated in a 37°C, 5% CO2 incubator for 6 hours. After incubation, the culture plates were removed and allowed to return to room temperature. Bright-Glo® luciferase assay system (purchased from Promega (catalog number E2650)) was added at a rate of 80 μL / well, and the mixture was incubated in the dark for 2 minutes. The RLU values ​​were then read. The isotype control hG4WT (hIgG4) was manufactured in-house by Akeso Biopharma Inc. (batch number 20190910).

[0178] As shown in Figure 6, the anti-LAG3 antibodies H9L8(hG4WT), H9L9(hG4WT), and H9L10(hG4WT), along with the positive control antibody reratrimab, were all able to block the interaction between LAG3 and MHC-II and upregulate luciferase expression. The activity of the anti-LAG3 antibodies H9L8(hG4WT), H9L9(hG4WT), and H9L10(hG4WT) was superior to that of the control antibody reratrimab.

[0179] Example 6: ELISA assay of the binding activity of anti-CD73 anti-LAG3 bispecific antibody to antigen. 1. Indirect ELISA assay of the binding activity of LA5EV1, LA5EV2, NTLAV2, NTLAV7, and NTLAV8 to the antigen NT5E-His. The specific method is as follows: ELISA plates were coated with 1 μg / mL NT5E-His and incubated overnight at 4°C. The antigen-coated ELISA plates were then washed once with PBST and blocked at 37°C for 2 hours with a PBS solution containing 1% BSA (blocking solution). After blocking, the ELISA plates were washed three times with PBST. Antibodies serially diluted with PBST solution were added (the antibody dilution gradient is shown in Tables 5-7). ELISA plates containing the test antibody were incubated at 37°C for 30 minutes and then washed three times with PBST. After washing, a 1:5000 diluted standard solution of HRP-labeled goat anti-human IgG FC secondary antibody (H+L) (Jackson, catalog number 109-035-098) was added, and the plates were incubated at 37°C for 30 minutes. After incubation, the plates were washed four times with PBST. Next, TMB (Neogen, 308177) was used to allow the color to develop in the dark for 5 minutes, and then stop solution was added to halt the color reaction. Immediately afterward, the ELISA plate was placed in a microplate reader, and the OD (450 nm) of each well was measured. The data was analyzed using SoftMax Pro 6.2.1 software.

[0180] The results are shown in Tables 5-7 and Figures 7-9.

[0181] Under the same experimental conditions, LA5EV1, LA5EV2, NTLAV2, NTLAV7, NTLAV8, and 19F3H2L3(hG1DM) were all able to effectively bind to the antigen NT5E-His in a dose-dependent manner.

[0182] [Table 5]

[0183] [Table 6]

[0184] [Table 7]

[0185] 2. Indirect ELISA assay of the binding activity of LA5EV1, LA5EV2, NTLAV2, NTLAV7, and NTLAV8 to the antigen huLAG3-mG1Fc. The specific method is as follows: ELISA plates were coated with 2 μg / mL huLAG3-mG1Fc and incubated overnight at 4°C. The antigen-coated ELISA plates were then washed once with PBST and blocked at 37°C for 2 hours with PBS solution containing 1% BSA (blocking solution). After blocking, the ELISA plates were washed three times with PBST. Antibodies serially diluted with PBST solution were added (the antibody dilution gradient is shown in Tables 8-10). ELISA plates containing the test antibody were incubated at 37°C for 30 minutes, followed by three washes with PBST. After washing, a 1:5000 diluted standard solution of HRP-labeled goat anti-human IgG FC secondary antibody (H+L) (Jackson, catalog number 109-035-098) was added, and the plates were incubated at 37°C for 30 minutes. After incubation, the plates were washed four times with PBST. Next, TMB (Neogen, 308177) was used to allow the color to develop in the dark for 5 minutes, and then stop solution was added to halt the color reaction. Immediately afterward, the ELISA plate was placed in a microplate reader, and the OD (450 nm) of each well was measured. The data was analyzed using SoftMax Pro 6.2.1 software.

[0186] The results are shown in Tables 8-10 and Figures 10-12.

[0187] Under the same experimental conditions, LA5EV1, LA5EV2, NTLAV2, NTLAV7, NTLAV8, and relatrimab all effectively bound to the antigen huLAG3-mG1Fc in a dose-dependent manner.

[0188] [Table 8]

[0189] [Table 9]

[0190] [Table 10]

[0191] Example 7: Determination of pharmacokinetic parameters of humanized antibodies LA5EV1, LA5EV2, NTLAV2, NTLAV7, and NTLAV8. 1. Determination of pharmacokinetic parameters of binding between humanized antibodies LA5EV1, LA5EV2, NTLAV2, NTLAV7, and NTLAV8 and hNT5E(1~552)-His. The sample dilution buffer was PBS (pH 7.4) containing 0.02% Tween® 20 and 0.1% BSA. 5 μg / mL of hNT5E(1~552)-His was immobilized on a HIS1K sensor at an immobilization height of 0.15 nm. The sensor was equilibrated in buffer for 60 seconds, and then the binding of the immobilized hNT5E(1~552)-His on the sensor to a 2.469~200 nM (3-fold dilution) antibody was measured for 120 seconds. The antibody was then dissociated in buffer for 300 seconds. The sensor was regenerated four times in 10 mM glycine (pH 1.7) for 5 seconds each time. The sample plate shaking speed was 1000 rpm, the measurement temperature was 37°C, and the frequency was 5.0 Hz. The data were analyzed by 1:1 model fitting to obtain affinity constants. The data acquisition software used was Fortebio Data Acquisition 12.0, and the data analysis software was Fortebio Data Analysis 12.0.

[0192] [Table 11]

[0193] The results are shown in Table 11 and Figures 13-18.

[0194] Humanized antibodies LA5EV1, LA5EV2, NTLAV2, NTLAV7, and NTLAV8 all showed good affinity for the antigen hNT5E(1~552)-His.

[0195] 2. Determination of pharmacokinetic parameters of binding between humanized antibodies LA5EV1, LA5EV2, NTLAV2, NTLAV7, and NTLAV8 and huLAG3-mG1Fc. The sample dilution buffer was PBS (pH 7.4) containing 0.02% Tween® 20 and 0.1% BSA. 10 μg / mL of huLAG3-mG1Fc was immobilized on an AMC sensor at an immobilization height of 0.8 nm. The sensor was equilibrated in buffer for 60 seconds, and then the binding of the immobilized huLAG3-mG1Fc on the sensor to 6.17–500 nM (3-fold dilution) antibody was measured for 100 seconds. The antibody was then dissociated in buffer for 400 seconds. The sensor was regenerated four times in 10 mM glycine (pH 1.7) for 5 seconds each time. The sample plate shaking speed was 1000 rpm, the measurement temperature was 30°C, and the frequency was 5.0 Hz. The data were analyzed by 1:1 model fitting to obtain affinity constants. The data acquisition software used was Fortebio Data Acquisition 12.0, and the data analysis software was Fortebio Data Analysis 12.0.

[0196] The results are shown in Table 12 and Figures 19-25.

[0197] Humanized antibodies LA5EV1, LA5EV2, NTLAV2, NTLAV7, and NTLAV8 all showed good affinity for the antigen huLAG3-mG1Fc.

[0198] [Table 12]

[0199] Example 8: FACS assay of the binding activity of an anti-CD73 anti-LAG3 bispecific antibody to the CD73 / LAG3 antigen. 1. FACS assay of the binding activity of an anti-CD73 anti-LAG3 bispecific antibody to LAG3 on the surface of a 293T-LAG3 membrane. Logarithmic growth phase 293T-LAG3 cells were collected and placed in 3 × 10⁶ wells of a 96-well flow cytometry plate. 5 Cells were transferred to wells. 100 μL of 1% PBSA was added, and the mixture was centrifuged at 350 × g for 5 minutes, with the supernatant removed. 100 μL of each antibody (final concentrations 300 nM, 100 nM, 33.33 nM, 11.11 nM, 3.7 nM, 1.23 nM, 0.41 nM, 0.041 nM, 0.0041 nM, and 0.00041 nM) diluted in 1% PBSA was added. The mixture was gently but thoroughly mixed, and then incubated on ice for 1 hour. 200 μL of 1% PBSA was added, and the mixture was centrifuged at 350 × g for 5 minutes, with the supernatant removed. The plate was washed twice. The 300-fold diluted Alexa Fluor® 647-labeled mouse anti-human IgG secondary antibody (Southern Biotech, catalog no. 9040-31) was added and the mixture was resuspended. The mixture was thoroughly mixed and incubated on ice in the dark for 0.5 hours. 200 μL of 1% PBSA was added, and the mixture was centrifuged at 350 × g for 5 minutes, with the supernatant removed. The plate was washed twice. The cell pellet was resuspended with 200 μL of 1% PBSA, and the suspension was assayed using a flow cytometer.

[0200] The experimental results are shown in Table 13 and Figure 26.

[0201] NTLAV8, LA5EV2, H9L8 (hG4WT), and relatrimab were all able to specifically bind to the LAG3 receptor on the surface of 293T-LAG3 cell membranes.

[0202] [Table 13]

[0203] 2. FACS assay of the binding activity of anti-CD73 anti-LAG3 bispecific antibody to CD73 on the surface of U87-MG membrane. Logarithmic growth phase U87-MG cells (ATCC, catalog number HTB-14) were collected and placed in 96-well plates in 3 × 10⁶ layers. 5Cells were transferred to wells. 200 μL of 1% PBSA was added, and the mixture was centrifuged at 750 × g for 5 minutes, with the supernatant removed. 100 μL of each antibody (final concentrations 300 nM, 100 nM, 33.33 nM, 11.11 nM, 3.7 nM, 1.23 nM, 0.41 nM, 0.041 nM, 0.0041 nM, and 0.00041 nM) diluted in 1% PBSA was added. The mixture was gently but thoroughly mixed, and then incubated on ice for 1 hour. 200 μL of 1% PBSA was added, and the mixture was centrifuged at 750 × g for 5 minutes, with the supernatant removed. The plate was washed twice. The 300-fold diluted Alexa Fluor® 647-labeled mouse anti-human IgG secondary antibody (Southern Biotech, catalog no. 9040-31) was added and the mixture was resuspended. The mixture was thoroughly mixed and incubated on ice in the dark for 0.5 hours. 100 μL of 1% PBSA was added, and the mixture was centrifuged at 750 × g for 5 minutes, with the supernatant removed. The plate was washed twice with 200 μL of 1% PBSA. The cell pellet was resuspended with 200 μL of 1% PBSA, and the suspension was assayed using a flow cytometer.

[0204] The experimental results are shown in Table 14 and Figure 27.

[0205] NTLAV8, LA5EV2, 19F3H2L3(hG1DM), and CPI-006 were all able to specifically bind to CD73 on the surface of U87-MG cells. The ability of NTLAV8 and LA5EV2 to bind to CD73 on the surface of U87-MG cells was stronger than that of the positive control CPI-006.

[0206] [Table 14]

[0207] Example 9: FACS assay of binding of an anti-CD73 anti-LAG3 bispecific antibody that competes with MHC-II on the Raji cell membrane surface to human LAG3-mG1Fc antigen. According to the experimental design, the antibody and LAG3-mG1Fc were diluted and thoroughly mixed in a 1:1 ratio to a final concentration of 3 nM for LAG3-mG1Fc and 300 nM, 100 nM, 33.33 nM, 11.11 nM, 3.7 nM, 1.23 nM, 0.41 nM, 0.041 nM, 0.0041 nM, or 0.00041 nM for the antibody. The mixtures were incubated on ice for 30 minutes. Raji cells were collected by conventional methods, and 3 × 10⁶ cells were collected. 5 Cells were seeded in a V-bottom 96-well plate at a rate of cells / well. 1% PBSA was added, and the mixture was centrifuged at 500×g for 5 minutes, with the supernatant removed. 150 μg / mL of mouse IgG isotype control (Thermofisher, catalog no. 10400C) was added, and the mixture was incubated on ice for 20 minutes, then centrifuged at 500×g for 5 minutes, with the supernatant removed. Cells were resuspended with 100 μL of the antibody-protein solution used before incubation to design blank controls, negative controls, and isotype controls. Plates were incubated on ice in the dark for 1 hour. 100 μL of 1% PBSA was added, and the mixture was centrifuged at 500×g for 5 minutes, with the supernatant removed. Plates were washed twice with 200 μL of 1% PBSA. Cells were resuspended with 100 μL of 300-fold diluted APC-labeled goat anti-mouse IgG secondary antibody (Biolegend, catalog number 405308), and a blank control was resuspended with 100 μL of 1% PBSA. The mixture was incubated on ice in the dark for 30 minutes. 100 μL of 1% PBSA was added, and the mixture was centrifuged at 500 × g for 5 minutes, and the supernatant was removed. The plate was washed twice with 200 μL of 1% PBSA. Cells were resuspended with 200 μL of 1% PBSA, and the mixture was assayed using a flow cytometer.

[0208] EC of the sample 50 The results are shown in Table 15. The results are shown in Figure 28. The antibodies NTLAV8, LA5EV2, relatrimab (positive control), and H9L8 (hG4WT) all competitively bound to human LAG3-mG1Fc and effectively blocked the binding of LAG3-mG1Fc to MHC-II on the surface of Raji cells in a dose-dependent manner.

[0209] [Table 15]

[0210] Example 10: Blocking assay of anti-CD73 anti-LAG3 bispecific antibody Jurkat-NFAT-PD1-LAG3 cells (constructed by Akeso Biopharma Inc.) and Raji cells (Cell Resource Center, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, catalog number TCHu 44) were collected and centrifuged at 110 × g for 5 minutes, and the supernatant was removed. The cells were resuspended in 1640 medium (containing 10% FBS) and counted. Jurkat-NFAT-PD1-LAG3 cells were divided into 10 × 10⁶ cells. 4 Cells / well were seeded into a black-bottomed 96-well plate (Corning, model number 3916). Following the design of experiment, antibodies (final concentrations 900 nM, 300 nM, 100 nM, 33.3 nM, 3.3 nM, 0.33 nM, 0.03 nM, and 0.003 nM) were added, and the mixture was pre-incubated for 30 minutes in an incubator at 37°C and 5% CO2. Raji cells were incubated with SEE (Staphylococcal Enterotoxin E) (final concentration 0.05 ng / mL, Toxin Technology, catalog number ET404) for 30 minutes in an incubator at 37°C and 5% CO2. After incubation, Raji cells were arranged in 2 × 10⁶ cells. 4 Cells / well (final system volume 80 μL) were added to a 96-well plate. The mixture was thoroughly mixed and incubated in a 37°C, 5% CO2 incubator for 16 hours. The culture plate was removed and allowed to return to room temperature. Firefly Glo Luciferase Reporter Gene Assay Kit (Yeasen, catalog no. 11404ES80) was added at 80 μL / well, and the mixture was incubated in the dark for 2 minutes. The RLU values ​​were then read.

[0211] The results are shown in Figure 29.

[0212] Both NTLAV8 and H9L8(hG4WT) were able to effectively block the inhibition of signaling pathways mediated by the interaction between LAG3 and MHC-II.

[0213] Example 11: Inhibition assay of CD73 enzymatic activity on the cell membrane surface using an anti-CD73 anti-LAG3 bispecific antibody. The experimental groups were set up as follows: ATP control group, AMP+ATP control group, negative control group (cells + AMP + ATP), APCP control group (cells + APCP + AMP + ATP), isotype control group (cells + isotype control antibody + AMP + ATP), and antibody group (cells + corresponding antibody + AMP + ATP).

[0214] ATP refers to adenosine triphosphate, AMP refers to adenosine monophosphate, and APCP refers to α,β-methyleneadenosine-5'-diphosphate, a specific inhibitor of CD73.

[0215] U87-MG cells (ATCC, catalog number HTB-14) in good condition during the logarithmic phase were collected, resuspended in the analytical medium (serum-free RPMI1640 culture solution), and counted. 2.5 × 10⁶ U87-MG cells were counted. 4Cells were seeded in a 96-well plate at 60 μL / well. 120 μM APCP and diluted antibody (initial concentration 300 nM, diluted in a 3-fold concentration gradient (the last two low concentrations were diluted 10-fold)) were prepared in analytical medium. APCP or the above diluted antibody was added to the 96-well plate at 60 μL / well, and the plate was incubated at 37°C for 1 hour. Next, AMP (purchased from TCI (catalog number A0158)) diluted to 600 μM in analytical medium was added at 60 μL / well, the mixture was thoroughly mixed, and incubated for 3 hours. After 3 hours, 100 μL of cell culture supernatant was collected from each well and transferred to a new 96-well plate to which 40 μL / well of CTG (CellTiter-Glo® One Solution Assay) (promega, catalog number G8461) had been added. The plate was gently tapped to mix thoroughly and left at room temperature in the dark for 5 minutes. After standing for 5 minutes, 10 μL of 300 μM ATP (5'-ATP 2Na hydrate, purchased from TCI (catalog number A0157)) prepared in TM buffer (prepared by Akeso Biopharma Inc.) was added to each well. The plates were gently tapped to mix thoroughly and left at room temperature in the dark for 5 minutes. Finally, the data were read using a multi-label microplate tester (PerkinElmer, Model No. 2140-0020).

[0216] The experimental results are shown in Table 16 and Figure 30. The anti-CD73-anti-LAG3 antibodies LA5EV2, NTLAV2, and NTLAV8, as well as the positive control drug MEDI9447 targeting CD73, all inhibited the enzymatic activity of CD73 endogenously expressed by U87-MG in a dose-dependent manner, thereby catalyzing the conversion of AMP to adenosine and consequently reducing the mean fluorescence intensity RLU in a dose-dependent manner; inhibition of CD73 enzymatic activity was more potent with LA5EV2 and NTLAV8 than with MEDI9447.

[0217] [Table 16]

[0218] Example 12: Assay of the biological activity of anti-CD73 anti-LAG3 bispecific antibody in promoting IFNγ secretion using mixed lymphocyte reaction (MLR). Raji-PDL1 cells (constructed by Akeso Biopharma Inc.) were subcultured using conventional methods; while PBMCs (from healthy donors) were thawed, cultured in 10 mL of 1640 complete medium, and stimulated with SEB (Staphylococcal Enterotoxin B, Toxin Technology, catalog number BT202) at a final concentration of 0.5 μg / mL for 2 days. After 2 days, Raji-PDL1 cells were collected using conventional methods, resuspended in analytical medium (RPMI1640 + 10% FBS), and then MMC (Mitomycin C, Stressmarq, catalog number SIH-246-10MG) at a final concentration of 2 μg / mL was added. The mixture was incubated in an incubator at 37°C and 5% CO2 for 1 hour. PBMCs stimulated with SEB for 2 days and Raji-PDL1 cells treated with MMC for 1 hour were collected using conventional methods and washed twice with analytical medium. Each of the two cell types was resuspended in the analysis medium and counted, resulting in 1 × 10⁻⁶ cells. 5 Cells were added to a U-shaped 96-well plate (Corning, model number 3799) at a concentration of one cell per well and co-cultured. According to the experimental design, AMP (5'-adenylate) (final concentration 200 μM, 200 TCI, catalog number A0158) and antibodies (final concentrations 300 nM, 30 nM, and 3 nM) were added to establish negative control groups (PBMC + Raji-PDL1 + AMP), isotype control groups, etc. The cells were co-cultured in an incubator for 3 days. After 3 days, the cells were centrifuged at 250 × g for 5 minutes, the cell culture supernatant was collected, and the IFNγ assay was performed by ELISA.

[0219] As shown in Figure 31, in a mixed culture system of human PBMCs and Raji-PDL1 cells, the addition of antibodies significantly induced IFNγ secretion in the system. The activity of the anti-CD73 anti-LAG3 antibodies LA5EV2 and NTLAV8 was shown to be superior to that of the antibody 19F3H2L3 (hG1DM), MEDI9447 which targets only CD73, the control antibody H9L8 (hG4WT), and relatrimab which targets only LAG-3.

[0220] Example 13: Potential for CHO-K1-LAG3-CD73 target cell phagocytosis activity by anti-CD73 anti-LAG3 bispecific antibody. Frozen MBMM (derived from C57 mice, purchased from Guangdong GemPharmatech Co., Ltd.) was thawed and cultured overnight in M-CSF (Murine M-CSF, peprotech, catalog number 315-02) medium with DMEM + 10% FBS + 100 ng / mL.

[0221] Target cells, CHO-K1-CD73-LAG3 (constructed by Akeso Biopharma Inc.), were collected, centrifuged at 170×g for 5 minutes, washed once with PBS, and counted. CFSE (Biolegend, catalog number 423801) was diluted to a final concentration of 2.5 μM with PBS. An appropriate amount of diluted CFSE was taken and the cells were resuspended (staining density: 1 × 10⁶). 7 Cells were incubated for 20 minutes with a solution of 1.5 × 10⁶ cells / mL. 6 mL of DMEM complete medium (containing 10% FBS) was added to stop staining. Cells were centrifuged at 170 × g for 5 minutes, and the supernatant was removed. 1 mL of DMEM complete medium was added, and the cells were incubated for 10 minutes. Antibodies were diluted in DMEM complete medium to final concentrations of 0.01 nM, 0.1 nM, 1 nM, 10 nM, and 100 nM to establish negative controls and isotype controls. Target cells were measured at 1.5 × 10⁶. 5 Cells were added to a V-bottom 96-well plate at the specified cell / well level, and antibody was added. The mixture was thoroughly mixed and incubated on ice for 40 minutes. The mixture was centrifuged at 170×g for 5 minutes and washed twice with DMEM complete medium. Macrophages (MBMMs) were collected, centrifuged at 750×g for 5 minutes, and the supernatant was removed. Cells were counted, resuspended in DMEM complete medium, and the concentration was increased to 5×10⁶. 4The cells were adjusted to 100 μL / cells and added to a V-bottom 96-well plate containing the target cells. The cells were resuspended, thoroughly mixed, and incubated in a 37°C incubator for 2 hours. 100 μL of 1% PBSA (room temperature) was added to each well, the mixture was centrifuged at 750 × g for 5 minutes, and the supernatant was removed. The plate was washed once with 200 μL of 1% PBSA. APC anti-mouse / human CD11b antibody (Biolegend, catalog no. 101212), diluted 500-fold in 1% PBSA, was added to the corresponding sample at a rate of 100 μL / well, the mixture was thoroughly mixed, and incubated on ice for 40 minutes. 100 μL of 1% PBSA was added to each well, the mixture was centrifuged at 750 × g for 5 minutes, and the supernatant was removed. The plate was washed once with 200 μL of 1% PBSA. The cells were resuspended in 200 μL of 1% PBSA and assayed using a flow cytometer.

[0222] The results are shown in Figure 32.

[0223] The antibody 19F3H2L2(G1WT), which targets only CD73, was able to promote phagocytosis of CHO-K1-CD73-LAG3 cells by macrophages (MBMMs) and showed an ADCP effect, but the anti-CD73 anti-LAG3 antibodies LA5EV2 and NTLAV8 did not show an ADCP effect.

[0224] Example 14 Pharmacodynamic evaluation of anti-CD73 anti-LAG3 bispecific antibody in a tumor cell subcutaneous transplantation model mouse To elucidate the in vivo antitumor activity of the anti-CD73 anti-LAG3 bispecific antibody, MIA-PaCa-2 cells (purchased from the China Center for Type Culture Collection Cell Bank) were first subcutaneously transplanted into 6-7 week old female NCG mice (purchased from Guangdong GemPharmatech Co., Ltd). The day on which the groups were divided was designated as D0. The administration route was intraperitoneal injection (IP), once a week for a total of four doses. The modeling and specific administration regimens are shown in Table 17. After administration, the length and width of the tumors in each group were measured, and the tumor volume was calculated. Tumor volume was calculated using the following formula: (Longest diameter of tumor × Shortest diameter of tumor) 2 ) / 2 The data was processed using GraphPad Prism 5 statistical processing software.

[0225] [Table 17]

[0226] The results are shown in Figure 33. Compared to isotype control antibodies, the anti-CD73 anti-LAG3 bispecific antibody NTLAV8, the control antibody relatrimab, and 19F3H2L3(hG1DM) all effectively inhibited tumor growth in mice. At the same dose, the antitumor effect of the anti-CD73 anti-LAG3 bispecific antibody NTLAV8 was superior to monotherapy with the control antibody relatrimab or 19F3H2L3(hG1DM), and to combination therapy with relatrimab and 19F3H2L3(hG1DM).

[0227] Furthermore, as shown in Figure 34, the tumor-bearing mice showed good resistance to all test drugs, and the test drugs in each group did not significantly affect the body weight of the tumor-bearing mice.

[0228] While specific aspects of this disclosure have been described in detail, those skilled in the art will understand that various modifications and substitutions are possible in accordance with all disclosed teachings, and that all such modifications fall within the scope of protection of this invention. The full scope of this disclosure is indicated by the claims and their equivalents.

Claims

1. A bispecific antibody comprising a first protein functional region and a second protein functional region, The first protein functional domain targets LAG3, and The second protein functional domain targets targets other than LAG3 (e.g., CD73, PD-1). The first protein functional region is an anti-LAG3 antibody or its antigen-binding fragment, the anti-LAG3 antibody comprising a heavy chain variable region and a light chain variable region; the heavy chain variable region comprising HCDR1, HCDR2, and HCDR3; and the light chain variable region comprising LCDR1, LCDR2, and LCDR3. The amino acid sequence of HCDR1 is shown in SEQ ID NO:

9. The amino acid sequence of HCDR2 is shown in SEQ ID NO: 10, and, The amino acid sequence of HCDR3 is shown in SEQ ID NO: 11; The amino acid sequence of LCDR1 is shown in SEQ ID NO: 12 or SEQ ID NO:

17. The amino acid sequence of LCDR2 is shown in SEQ ID NO: 13 or SEQ ID NO: 15, and, The amino acid sequence of LCDR3 is shown in SEQ ID NO: 14 or SEQ ID NO:

16. Bispecific antibodies.

2. The amino acid sequence of LCDR1 is shown in SEQ ID NO:

12. The amino acid sequence of LCDR2 is shown in SEQ ID NO:

13. The amino acid sequence of LCDR3 is shown in SEQ ID NO: 14; or, The amino acid sequence of LCDR1 is shown in SEQ ID NO:

12. The amino acid sequence of LCDR2 is shown in SEQ ID NO:

15. The amino acid sequence of LCDR3 is shown in SEQ ID NO: 16; or, The amino acid sequence of LCDR1 is shown in SEQ ID NO:

17. The amino acid sequence of LCDR2 is shown in SEQ ID NO:

15. The amino acid sequence of LCDR3 is shown in SEQ ID NO: 14; or, The amino acid sequence of LCDR1 is shown in SEQ ID NO:

12. The amino acid sequence of LCDR2 is shown in SEQ ID NO:

13. The amino acid sequence of LCDR3 is shown in SEQ ID NO: 16; or, The amino acid sequence of LCDR1 is shown in SEQ ID NO:

12. The amino acid sequence of LCDR2 is shown in SEQ ID NO:

15. The amino acid sequence of LCDR3 is shown in SEQ ID NO: 14; or, The amino acid sequence of LCDR1 is shown in SEQ ID NO:

17. The amino acid sequence of LCDR2 is shown in SEQ ID NO:

13. The amino acid sequence of LCDR3 is shown in SEQ ID NO: 14; or, The amino acid sequence of LCDR1 is shown in SEQ ID NO:

17. The amino acid sequence of LCDR2 is shown in SEQ ID NO:

13. The amino acid sequence of LCDR3 is shown in SEQ ID NO: 16; or, The amino acid sequence of LCDR1 is shown in SEQ ID NO:

17. The amino acid sequence of LCDR2 is shown in SEQ ID NO:

15. The amino acid sequence of LCDR3 is shown in SEQ ID NO: 16; The bispecific antibody according to claim 1.

3. The heavy chain variable region of the anti-LAG3 antibody is selected from the amino acid sequences shown in SEQ ID NO: 2 and SEQ ID NO: 44, and the light chain variable region of the anti-LAG3 antibody is selected from the amino acid sequences shown in SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 and SEQ ID NO: 45; Preferably, The amino acid sequence of the heavy chain variable region of the anti-LAG3 antibody is shown in SEQ ID NO: 2, and the amino acid sequence of the light chain variable region of the anti-LAG3 antibody is shown in SEQ ID NO: 4; The amino acid sequence of the heavy chain variable region of the anti-LAG3 antibody is shown in SEQ ID NO: 2, and the amino acid sequence of the light chain variable region of the anti-LAG3 antibody is shown in SEQ ID NO: 6; The amino acid sequence of the heavy chain variable region of the anti-LAG3 antibody is shown in SEQ ID NO: 2, and the amino acid sequence of the light chain variable region of the anti-LAG3 antibody is shown in SEQ ID NO: 8; The amino acid sequence of the heavy chain variable region of the anti-LAG3 antibody is shown in SEQ ID NO: 2, and the amino acid sequence of the light chain variable region of the anti-LAG3 antibody is shown in SEQ ID NO: 45; The amino acid sequence of the heavy chain variable region of the anti-LAG3 antibody is shown in SEQ ID NO: 44, and the amino acid sequence of the light chain variable region of the anti-LAG3 antibody is shown in SEQ ID NO: 4; The amino acid sequence of the heavy chain variable region of the anti-LAG3 antibody is shown in SEQ ID NO: 44, and the amino acid sequence of the light chain variable region of the anti-LAG3 antibody is shown in SEQ ID NO: 6; The amino acid sequence of the heavy chain variable region of the anti-LAG3 antibody is shown in SEQ ID NO: 44, and the amino acid sequence of the light chain variable region of the anti-LAG3 antibody is shown in SEQ ID NO: 8; or, The amino acid sequence of the heavy chain variable region of the anti-LAG3 antibody is shown in SEQ ID NO: 44, and the amino acid sequence of the light chain variable region of the anti-LAG3 antibody is shown in SEQ ID NO: 45; A bispecific antibody according to claim 1 or 2.

4. The anti-LAG3 antibody or its antigen-binding fragment is Fab, Fab', F(ab') ２ A bispecific antibody according to any one of claims 1 to 3, selected from Fd, Fv, dAb, complementarity-determining region fragment, single-chain antibody, humanized antibody, and chimeric antibody.

5. The bispecific antibody according to any one of claims 1 to 4, wherein the anti-LAG3 antibody includes a non-CDR region derived from a human antibody.

6. The second protein functional region is an anti-CD73 antibody or its antigen-binding fragment, the anti-CD73 antibody includes a heavy chain variable region and a light chain variable region; the heavy chain variable region includes HCDR1 having the amino acid sequence shown in SEQ ID NO: 25, HCDR2 having the amino acid sequence shown in SEQ ID NO: 26, and HCDR3 having the amino acid sequence shown in SEQ ID NO: 27; and, The light chain variable region includes LCDR1 having the amino acid sequence shown in SEQ ID NO: 28, LCDR2 having the amino acid sequence shown in SEQ ID NO: 29, and LCDR3 having the amino acid sequence shown in SEQ ID NO: 30; A bispecific antibody according to any one of claims 1 to 5.

7. The heavy chain variable region of the anti-CD73 antibody is selected from the amino acid sequences shown in SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, and SEQ ID NO: 46; the light chain variable region of the anti-CD73 antibody is selected from the amino acid sequences shown in SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 47, and SEQ ID NO: 62; Preferably, The amino acid sequence of the heavy chain variable region of the anti-CD73 antibody is shown in SEQ ID NO: 31, and the amino acid sequence of the light chain variable region of the anti-CD73 antibody is shown in SEQ ID NO: 32; The amino acid sequence of the heavy chain variable region of the anti-CD73 antibody is shown in SEQ ID NO: 31, and the amino acid sequence of the light chain variable region of the anti-CD73 antibody is shown in SEQ ID NO: 34; The amino acid sequence of the heavy chain variable region of the anti-CD73 antibody is shown in SEQ ID NO: 31, and the amino acid sequence of the light chain variable region of the anti-CD73 antibody is shown in SEQ ID NO: 36; The amino acid sequence of the heavy chain variable region of the anti-CD73 antibody is shown in SEQ ID NO: 31, and the amino acid sequence of the light chain variable region of the anti-CD73 antibody is shown in SEQ ID NO: 37; The amino acid sequence of the heavy chain variable region of the anti-CD73 antibody is shown in SEQ ID NO: 31, and the amino acid sequence of the light chain variable region of the anti-CD73 antibody is shown in SEQ ID NO: 47; The amino acid sequence of the heavy chain variable region of the anti-CD73 antibody is shown in SEQ ID NO: 31, and the amino acid sequence of the light chain variable region of the anti-CD73 antibody is shown in SEQ ID NO: 62; The amino acid sequence of the heavy chain variable region of the anti-CD73 antibody is shown in SEQ ID NO: 33, and the amino acid sequence of the light chain variable region of the anti-CD73 antibody is shown in SEQ ID NO: 32; The amino acid sequence of the heavy chain variable region of the anti-CD73 antibody is shown in SEQ ID NO: 33, and the amino acid sequence of the light chain variable region of the anti-CD73 antibody is shown in SEQ ID NO: 34; The amino acid sequence of the heavy chain variable region of the anti-CD73 antibody is shown in SEQ ID NO: 33, and the amino acid sequence of the light chain variable region of the anti-CD73 antibody is shown in SEQ ID NO: 36; The amino acid sequence of the heavy chain variable region of the anti-CD73 antibody is shown in SEQ ID NO: 33, and the amino acid sequence of the light chain variable region of the anti-CD73 antibody is shown in SEQ ID NO: 37; The amino acid sequence of the heavy chain variable region of the anti-CD73 antibody is shown in SEQ ID NO: 33, and the amino acid sequence of the light chain variable region of the anti-CD73 antibody is shown in SEQ ID NO: 47; The amino acid sequence of the heavy chain variable region of the anti-CD73 antibody is shown in SEQ ID NO: 33, and the amino acid sequence of the light chain variable region of the anti-CD73 antibody is shown in SEQ ID NO: 62; The amino acid sequence of the heavy chain variable region of the anti-CD73 antibody is shown in SEQ ID NO: 35, and the amino acid sequence of the light chain variable region of the anti-CD73 antibody is shown in SEQ ID NO: 32; The amino acid sequence of the heavy chain variable region of the anti-CD73 antibody is shown in SEQ ID NO: 35, and the amino acid sequence of the light chain variable region of the anti-CD73 antibody is shown in SEQ ID NO: 34; The amino acid sequence of the heavy chain variable region of the anti-CD73 antibody is shown in SEQ ID NO: 35, and the amino acid sequence of the light chain variable region of the anti-CD73 antibody is shown in SEQ ID NO: 36; The amino acid sequence of the heavy chain variable region of the anti-CD73 antibody is shown in SEQ ID NO: 35, and the amino acid sequence of the light chain variable region of the anti-CD73 antibody is shown in SEQ ID NO: 37; The amino acid sequence of the heavy chain variable region of the anti-CD73 antibody is shown in SEQ ID NO: 35, and the amino acid sequence of the light chain variable region of the anti-CD73 antibody is shown in SEQ ID NO: 47; The amino acid sequence of the heavy chain variable region of the anti-CD73 antibody is shown in SEQ ID NO: 35, and the amino acid sequence of the light chain variable region of the anti-CD73 antibody is shown in SEQ ID NO: 62; The amino acid sequence of the heavy chain variable region of the anti-CD73 antibody is shown in SEQ ID NO: 46, and the amino acid sequence of the light chain variable region of the anti-CD73 antibody is shown in SEQ ID NO: 32; The amino acid sequence of the heavy chain variable region of the anti-CD73 antibody is shown in SEQ ID NO: 46, and the amino acid sequence of the light chain variable region of the anti-CD73 antibody is shown in SEQ ID NO: 34; The amino acid sequence of the heavy chain variable region of the anti-CD73 antibody is shown in SEQ ID NO: 46, and the amino acid sequence of the light chain variable region of the anti-CD73 antibody is shown in SEQ ID NO: 36; The amino acid sequence of the heavy chain variable region of the anti-CD73 antibody is shown in SEQ ID NO: 46, and the amino acid sequence of the light chain variable region of the anti-CD73 antibody is shown in SEQ ID NO: 37; The amino acid sequence of the heavy chain variable region of the anti-CD73 antibody is shown in SEQ ID NO: 46, and the amino acid sequence of the light chain variable region of the anti-CD73 antibody is shown in SEQ ID NO: 47; or, The amino acid sequence of the heavy chain variable region of the anti-CD73 antibody is shown in SEQ ID NO: 46, and the amino acid sequence of the light chain variable region of the anti-CD73 antibody is shown in SEQ ID NO: 62; The bispecific antibody according to claim 6.

8. The anti-LAG3 antibody or the anti-CD73 antibody further comprises a constant region derived from a human antibody; Preferably, the constant region of the anti-LAG3 antibody or the anti-CD73 antibody is selected from the constant regions of human IgG1, IgG2, IgG3, or IgG4; Preferably, the anti-LAG3 antibody comprises a heavy chain constant region and a light chain constant region, wherein the heavy chain constant region is an Igγ-1 chain C region (e.g., SEQ ID NO: 18) or an Igγ-4 chain C region (e.g., SEQ ID NO: 20), and the light chain constant region is an Igκ chain C region (e.g., SEQ ID NO: 19); Preferably, the anti-CD73 antibody comprises a heavy chain constant region and a light chain constant region, wherein the heavy chain constant region is an Igγ-1 chain C region (e.g., SEQ ID NO: 18) or an Igγ-4 chain C region (e.g., SEQ ID NO: 20), and the light chain constant region is an Igκ chain C region (e.g., SEQ ID NO: 19); A bispecific antibody according to any one of claims 1 to 7.

9. The bispecific antibodies are as follows (1) to (4): （１） The subtype of the anti-LAG3 antibody is human IgG1, and the heavy chain constant region of the antibody has the following mutations in the EU numbering system: L234A and L235A; L234A and G237A; L235A and G237A; or L234A, L235A, and G237A; Having, （２） The subtype of the anti-LAG3 antibody is human IgG4, and the heavy chain constant region of the antibody has the following mutations in the EU numbering system: F234A and L235A; F234A and G237A; L235A and G237A; or F234A, L235A, and G237A; Having, （３） The subtype of the anti-CD73 antibody is human IgG1, and the heavy chain constant region of the antibody has the following mutation in the EU numbering system: L234A and L235A; L234A and G237A; L235A and G237A; or L234A, L235A, and G237A; has （４） The subtype of the anti-CD73 antibody is human IgG4, and the heavy chain constant region of the antibody has the following mutation in the EU numbering system: F234A and L235A; F234A and G237A; L235A and G237A; or F234A, L235A, and G237A. has A bispecific antibody according to any one of claims 1 to 8, characterized by any one of the above.

10. The aforementioned bispecific antibody is in the form of IgG-scFv; Preferably, the first protein functional region is an immunoglobulin, and the second protein functional region is a single-chain antibody; or, Preferably, the first protein functional region is a single-chain antibody, and the second protein functional region is an immunoglobulin targeting a target other than LAG3 (e.g., CD73, PD-1); A bispecific antibody according to any one of claims 1 to 9.

11. The first protein functional region targeting LAG3, and A second protein functional domain targeting CD73 including, Here, The first protein functional domain is an anti-LAG3 antibody, the anti-LAG3 antibody is an immunoglobulin, and the second protein functional domain is an anti-CD73 single-chain antibody; or, The first protein functional region is an anti-LAG3 single-chain antibody, the second protein functional region is an anti-CD73 antibody, and the anti-CD73 antibody is an immunoglobulin; A bispecific antibody according to any one of claims 1 to 10.

12. The heavy chain variable region of the immunoglobulin comprises HCDR1 to HCDR3 of the amino acid sequences shown in SEQ ID NOs: 9 to 11, and the light chain variable region of the immunoglobulin comprises LCDR1 to LCDR3 of the amino acid sequences shown in SEQ ID NOs: 12 to 14; the heavy chain variable region of the single-chain antibody comprises HCDR1 to HCDR3 of the amino acid sequences shown in SEQ ID NOs: 25 to 27, and the light chain variable region of the single-chain antibody comprises LCDR1 to LCDR3 of the amino acid sequences shown in SEQ ID NOs: 28 to 30; or, The heavy chain variable region of the single-chain antibody comprises HCDR1 to HCDR3 of the amino acid sequences shown in SEQ ID NOs: 9 to 11, respectively, and the light chain variable region of the single-chain antibody comprises LCDR1 to LCDR3 of the amino acid sequences shown in SEQ ID NOs: 12 to 14, respectively; the heavy chain variable region of the immunoglobulin comprises HCDR1 to HCDR3 of the amino acid sequences shown in SEQ ID NOs: 25 to 27, respectively, and the light chain variable region of the immunoglobulin comprises LCDR1 to LCDR3 of the amino acid sequences shown in SEQ ID NOs: 28 to 30, respectively; The bispecific antibody according to claim 10 or 11.

13. The heavy chain variable region of the immunoglobulin is selected from the amino acid sequences shown in SEQ ID NO: 2 and SEQ ID NO: 44, and the light chain variable region of the immunoglobulin is selected from the amino acid sequences shown in SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 and SEQ ID NO: 45; the heavy chain variable region of the single-chain antibody is selected from the amino acid sequences shown in SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35 and SEQ ID NO: 46, and the light chain variable region of the single-chain antibody is selected from the amino acid sequences shown in SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 47 and SEQ ID NO: 62; or, The heavy chain variable region of the single-chain antibody is selected from the amino acid sequences shown in SEQ ID NO: 2 and SEQ ID NO: 44, and the light chain variable region of the single-chain antibody is selected from the amino acid sequences shown in SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 and SEQ ID NO: 45; the heavy chain variable region of the immunoglobulin is selected from the amino acid sequences shown in SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35 and SEQ ID NO: 46, and the light chain variable region of the immunoglobulin is selected from the amino acid sequences shown in SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 47 and SEQ ID NO: 62; A bispecific antibody according to any one of claims 10 to 12.

14. The bispecific antibodies are (1) to (24) below: （１） The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 2, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 4; the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 31, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 32; （２） The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 2, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 4; the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 33, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 34; （３） The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 2, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 4; the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 35, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 36; （４） The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 2, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 4; the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 35, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 37; （５） The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 2, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 4; the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 46, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 47; （６） The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 2, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 4; the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 35, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 62; （７） The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 44, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 45; the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 31, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 32; （８） The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 44, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 45; the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 33, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 34; （９） The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 44, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 45; the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 35, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 36; （１０） The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 44, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 45; the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 35, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 37; （１１） The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 44, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 45; the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 46, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 47; （１２） The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 44, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 45; the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 35, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 62; （１３） The amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 2, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 4; the amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 31, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 32; （１４） The amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 2, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 4; the amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 33, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 34; （１５） The amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 2, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 4; the amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 35, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 36; （１６） The amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 2, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 4; the amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 35, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 37; （１７） The amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 2, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 4; the amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 46, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 47; （１８） The amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 2, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 4; the amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 35, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 62; （１９） The amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 44, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 45; the amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 31, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 32; （２０） The amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 44, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 45; the amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 33, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 34; （２１） The amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 44, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 45; the amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 35, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 36; （２２） The amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 44, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 45; the amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 35, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 37; （２３） The amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 44, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 45; the amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 46, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 47; and, （２４） The amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 44, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 45; the amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO: 35, and the amino acid sequence of the light chain variable region of the immunoglobulin is shown in SEQ ID NO: 62; A bispecific antibody according to any one of claims 10 to 13, selected from any one of the following.

15. The number of the first protein functional region and the second protein functional region is, independently, one, two, or more; Preferably, the first protein functional region and the second protein functional region are linked directly or via a linker; Preferably, the heavy chain variable region and the light chain variable region of the anti-LAG3 single-chain antibody are linked directly or via a linker; Preferably, the heavy chain variable region and the light chain variable region of the anti-CD73 single-chain antibody are linked directly or via a linker; Preferably, the linker is independently the polypeptide shown in Sequence ID No. 48, or a polypeptide formed by linking multiple polypeptides (e.g., 2, 3, 4, 5, or 6) shown in Sequence ID No. 48; Preferably, the linker is independently a polypeptide having one or more glycines at the C-terminus of the polypeptide shown in SEQ ID NO: 48, or a polypeptide formed by linking together multiple polypeptides (e.g., 2, 3, 4, 5, or 6) shown in SEQ ID NO: 48; A bispecific antibody according to any one of claims 1 to 14.

16. The bispecific antibody according to any one of claims 10 to 15, wherein each single-chain antibody is ligated to the C-terminus or N-terminus of one of the two heavy chains of the immunoglobulin.

17. The first protein functional region targeting LAG3, and A second protein functional domain targeting CD73 Includes, The number of the first protein functional regions is 1, and the number of the second protein functional regions is 2; Here, the first protein functional region is an immunoglobulin, and the second protein functional region is a single-chain antibody; The amino acid sequence of the heavy chain of the immunoglobulin is shown in SEQ ID NO: 38, and the amino acid sequence of the light chain of the immunoglobulin is shown in SEQ ID NO: 39; The amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 35, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 62; or, the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 46, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 47; Each single-chain antibody is ligated to the C-terminus of one of the two heavy chains of the immunoglobulin; The first protein functional region and the second protein functional region are linked via a first linker; the heavy chain variable region of the single-chain antibody and the light chain variable region of the single-chain antibody are linked via a second linker; the first linker and the second linker may be the same or different; Preferably, the amino acid sequences of the first linker and the second linker are independently selected from the sequences shown in SEQ ID NO: 42 and SEQ ID NO: 43, respectively; Preferably, the amino acid sequences of the first linker and the second linker are both shown in SEQ ID NO: 43; Preferably, the constant region of the heavy chain of the immunoglobulin has the following mutations in the EU numbering system: L234A and L235A; L234A and G237A; L235A and G237A; or, L234A, L235A, and G237A. including, A bispecific antibody according to any one of claims 1 to 16.

18. The first protein functional region targeting LAG3, and A second protein functional domain targeting CD73 Includes, The number of the first protein functional regions is 2, and the number of the second protein functional regions is 1; Here, the first protein functional region is a single-chain antibody, and the second protein functional region is an immunoglobulin; The amino acid sequence of the heavy chain of the immunoglobulin is shown in SEQ ID NO: 40, and the amino acid sequence of the light chain of the immunoglobulin is shown in SEQ ID NO: 41; The amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 2, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 4; or, the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO: 44, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO: 45; Each single-chain antibody is ligated to the C-terminus of one of the two heavy chains of the immunoglobulin; The first protein functional region and the second protein functional region are linked via a first linker; the heavy chain variable region of the single-chain antibody and the light chain variable region of the single-chain antibody are linked via a second linker; the first linker and the second linker may be the same or different; Preferably, the amino acid sequences of the first linker and the second linker are independently selected from the sequences shown in SEQ ID NO: 42 and SEQ ID NO: 43, respectively; Preferably, the amino acid sequences of the first linker and the second linker are both shown in SEQ ID NO: 43; Preferably, the constant region of the heavy chain of the immunoglobulin has the following mutations in the EU numbering system: L234A and L235A; L234A and G237A; L235A and G237A; or, L234A, L235A, and G237A including, A bispecific antibody according to any one of claims 1 to 16.

19. An isolated nucleic acid molecule encoding a bispecific antibody according to any one of claims 1 to 18.

20. A recombinant vector comprising an isolated nucleic acid molecule as described in claim 19.

21. A host cell comprising an isolated nucleic acid molecule according to claim 19 or a recombinant vector according to claim 20.

22. A pharmaceutical composition comprising a bispecific antibody according to any one of claims 1 to 18, further comprising, optionally, pharmaceutically acceptable auxiliary materials.

23. The use of a bispecific antibody according to any one of claims 1 to 18 in the manufacture of a pharmaceutical product for treating or preventing tumors, Preferably, the tumor is selected from one or more of the following: ovarian cancer, esophageal cancer, melanoma, hematopoietic malignancies, glioblastoma, renal cell carcinoma, lung cancer, prostate cancer, bladder cancer, colon cancer, rectal cancer, liver cancer, stomach cancer, breast cancer, malignant brain tumors, pancreatic cancer, thyroid cancer, head and neck cancer, and kidney cancer; Preferably, the lung cancer is non-small cell lung cancer; Preferably, the hematopoietic malignancy is leukemia; Preferably, the esophageal cancer is esophageal squamous cell carcinoma; use.

24. A bispecific antibody according to any one of claims 1 to 18, for use in the treatment or prevention of tumors, Preferably, the tumor is selected from one or more of the following: ovarian cancer, esophageal cancer, melanoma, hematopoietic malignancies, glioblastoma, renal cell carcinoma, lung cancer, prostate cancer, bladder cancer, colon cancer, rectal cancer, liver cancer, stomach cancer, breast cancer, malignant brain tumors, pancreatic cancer, thyroid cancer, head and neck cancer, and kidney cancer; Preferably, the lung cancer is non-small cell lung cancer; Preferably, the hematopoietic malignancy is leukemia; Preferably, the esophageal cancer is esophageal squamous cell carcinoma; Bispecific antibodies.

25. A method for treating or preventing a tumor, comprising the step of administering an effective amount of a bispecific antibody described in any one of claims 1 to 18 to a target subject, Preferably, the tumor is selected from one or more of the following: ovarian cancer, esophageal cancer, melanoma, hematopoietic malignancies, glioblastoma, renal cell carcinoma, lung cancer, prostate cancer, bladder cancer, colon cancer, rectal cancer, liver cancer, stomach cancer, breast cancer, malignant brain tumors, pancreatic cancer, thyroid cancer, head and neck cancer, and kidney cancer; Preferably, the lung cancer is non-small cell lung cancer; Preferably, the hematopoietic malignancy is leukemia; Preferably, the esophageal cancer is esophageal squamous cell carcinoma; Treatment or prevention methods.

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