Anti-LAG3 antibody, pharmaceutical composition and uses

Abstract

This invention relates to an anti-LAG3 antibody, a pharmaceutical composition, and its uses. Specifically, this invention relates to an anti-LAG3 antibody or its antigen-binding fragment. The anti-LAG3 antibody includes a heavy chain variable region and a light chain variable region. The heavy chain variable region includes HCDR1, HCDR2, and HCDR3, and the light chain variable region includes 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. This anti-LAG3 antibody exhibits excellent affinity and specificity, and is expected to have antitumor applications.

Description

[Technical Field]

[0001] References to related applications This application is based on Chinese Patent Application No. 202310327266.6 (Filing Date: March 29, 2023), claiming priority thereunder, which is incorporated in its entirety by reference.

[0002] Technical field This disclosure relates to the field of biomedicine, to pharmaceutical compositions and uses comprising anti-LAG3 antibodies, anti-LAG3 antibodies, or antigen-binding fragments thereof. [Background technology]

[0003] Tumors, especially malignant tumors, are serious health threats in today's world and the second leading cause of death among various diseases. In recent years, the incidence of these diseases has increased dramatically. Malignant tumors are characterized by low treatment efficacy, a high rate of late-stage metastasis, and a poor prognosis. While conventional clinical treatments (e.g., radiation therapy, chemotherapy, surgery) significantly reduce pain and extend survival, they have significant limitations, and further improvement in their effectiveness is difficult.

[0004] Lymphocyte-activation gene 3 (LAG3), also known as CD223, is a type I transmembrane protein consisting of 498 amino acids and belongs to the immunoglobulin superfamily (IgSF). LAG3 is primarily expressed in 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), and plasmacytoid dendritic cells (pDC) (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 adjacent to the CD4 gene on human chromosome 12 (12p13.3), and both genes share the same exons and introns. While the LAG3 and CD4 molecules are highly structurally similar, their amino acid sequence homology is only about 20%. Major histocompatibility complex class II (MHC-II), hepatic sinusoidal endothelial cell lectin (LSECtin), and galectin 3 are associated ligands for the LAG3 molecule. The MHC-II molecule is the primary ligand for 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 effectively competes with the CD4 molecule for binding to the MHC-II molecule, thereby suppressing 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, via its D1 domain (which contains a single proline-rich loop structure), interacts with CD4 + In the first signaling pathway for T cell activation, "CD3-TCR-MHC-II," a dimeric molecule is formed and specifically binds to the MHC-II molecule. As a result, in one aspect, the signaling pathway for T cell activation is blocked, and in another aspect, the intracellular segment of the LAG3 molecule (KIEELE motif) generates an immunosuppressive signal to CD4 + It downregulates T cell activity. The LAG3 molecule can promote Treg cell differentiation and enhance the inhibitory effect on Treg cells by participating in downstream signaling of signaling transcription factor 5. This is one of the mechanisms by which tumors evade killing by the immune system (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 a tumor infiltration of various malignant tumors CD8 +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 liraglutide and nivolumab for the treatment of tumors such as hematopoietic malignancies, melanoma, glioblastoma, renal cell carcinoma, and non-small cell lung cancer.

[0009] Currently, there is a need to develop new anti-LAG3 antibody drugs. [Overview of the project]

[0010] As a result of intensive research and creative efforts, the inventors have obtained anti-LAG3 antibodies. The inventors have surprisingly found that the anti-LAG3 antibodies of this disclosure (also abbreviated as antibodies or the antibodies of this disclosure) have excellent affinity and / or specificity and are equivalent to or even superior to the positive control antibody (e.g., Relatlimab) in one or more aspects. The details of this disclosure are as follows: One aspect of this disclosure relates to an anti-LAG3 antibody or an antigen-binding fragment thereof, wherein 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, HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 9, HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 10, HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 11, LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 12 or SEQ ID NO: 17, LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 13 or SEQ ID NO: 15, LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 14 or SEQ ID NO: 16.

[0011] In some embodiments 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, 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, the amino acid sequence of LCDR3 is shown in SEQ ID NO: 14 or SEQ ID NO: 16, An anti-LAG3 antibody or an antigen-binding fragment thereof is provided.

[0012] In some embodiments of this disclosure, LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 12, LCDR2 contains the amino acid sequence shown in SEQ ID NO: 13, LCDR3 contains the amino acid sequence shown in SEQ ID NO: 14; or, LCDR1 contains the amino acid sequence shown in SEQ ID NO: 12. LCDR2 contains the amino acid sequence shown in SEQ ID NO: 15. LCDR3 contains the amino acid sequence shown in SEQ ID NO: 16; or, LCDR1 contains the amino acid sequence shown in SEQ ID NO: 17, LCDR2 contains the amino acid sequence shown in SEQ ID NO: 15. LCDR3 contains the amino acid sequence shown in SEQ ID NO: 14; or, LCDR1 contains the amino acid sequence shown in SEQ ID NO: 12. LCDR2 contains the amino acid sequence shown in SEQ ID NO: 13, LCDR3 contains the amino acid sequence shown in SEQ ID NO: 16; or, LCDR1 contains the amino acid sequence shown in SEQ ID NO: 12. LCDR2 contains the amino acid sequence shown in SEQ ID NO: 15. LCDR3 contains the amino acid sequence shown in SEQ ID NO: 14; or, LCDR1 contains the amino acid sequence shown in SEQ ID NO: 17, LCDR2 contains the amino acid sequence shown in SEQ ID NO: 13, LCDR3 contains the amino acid sequence shown in SEQ ID NO: 14; or, LCDR1 contains the amino acid sequence shown in SEQ ID NO: 17, LCDR2 contains the amino acid sequence shown in SEQ ID NO: 13, LCDR3 contains the amino acid sequence shown in SEQ ID NO: 16; or, LCDR1 contains the amino acid sequence shown in SEQ ID NO: 17, LCDR2 contains the amino acid sequence shown in SEQ ID NO: 15. LCDR3 contains the amino acid sequence shown in SEQ ID NO: 16. An anti-LAG3 antibody or its antigen-binding fragment is provided.

[0013] 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 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 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: 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 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 Sequence ID No. 16. An anti-LAG3 antibody or its antigen-binding fragment is provided.

[0014] In some aspects of this disclosure, HCDR1 contains the amino acid sequence shown in SEQ ID NO: 9, HCDR2 contains the amino acid sequence shown in SEQ ID NO: 10. HCDR3 contains the amino acid sequence shown in SEQ ID NO: 11, LCDR1 contains the amino acid sequence shown in SEQ ID NO: 12. LCDR2 contains the amino acid sequence shown in SEQ ID NO: 13, LCDR3 contains the amino acid sequence shown in SEQ ID NO: 14. An anti-LAG3 antibody or its antigen-binding fragment is provided.

[0015] In some aspects of this disclosure, HCDR1 contains the amino acid sequence shown in SEQ ID NO: 9, HCDR2 contains the amino acid sequence shown in SEQ ID NO: 10. HCDR3 contains the amino acid sequence shown in SEQ ID NO: 11, LCDR1 contains the amino acid sequence shown in SEQ ID NO: 12. LCDR2 contains the amino acid sequence shown in SEQ ID NO: 15. LCDR3 contains the amino acid sequence shown in SEQ ID NO: 16. An anti-LAG3 antibody or its antigen-binding fragment is provided.

[0016] In some aspects of this disclosure, HCDR1 contains the amino acid sequence shown in SEQ ID NO: 9, HCDR2 contains the amino acid sequence shown in SEQ ID NO: 10. HCDR3 contains the amino acid sequence shown in SEQ ID NO: 11, LCDR1 contains the amino acid sequence shown in SEQ ID NO: 17, LCDR2 contains the amino acid sequence shown in SEQ ID NO: 15. LCDR3 contains the amino acid sequence shown in SEQ ID NO: 14. An anti-LAG3 antibody or its antigen-binding fragment is provided.

[0017] In some aspects of this disclosure, HCDR1 contains the amino acid sequence shown in SEQ ID NO: 9, HCDR2 contains the amino acid sequence shown in SEQ ID NO: 10. HCDR3 contains the amino acid sequence shown in SEQ ID NO: 11, LCDR1 contains the amino acid sequence shown in SEQ ID NO: 12. LCDR2 contains the amino acid sequence shown in SEQ ID NO: 13, LCDR3 contains the amino acid sequence shown in SEQ ID NO: 16. An anti-LAG3 antibody or its antigen-binding fragment is provided.

[0018] In some aspects of this disclosure, HCDR1 contains the amino acid sequence shown in SEQ ID NO: 9, HCDR2 contains the amino acid sequence shown in SEQ ID NO: 10. HCDR3 contains the amino acid sequence shown in SEQ ID NO: 11, LCDR1 contains the amino acid sequence shown in SEQ ID NO: 12. LCDR2 contains the amino acid sequence shown in SEQ ID NO: 15. LCDR3 contains the amino acid sequence shown in SEQ ID NO: 14. An anti-LAG3 antibody or its antigen-binding fragment is provided.

[0019] In some aspects of this disclosure, HCDR1 contains the amino acid sequence shown in SEQ ID NO: 9, HCDR2 contains the amino acid sequence shown in SEQ ID NO: 10. HCDR3 contains the amino acid sequence shown in SEQ ID NO: 11, LCDR1 contains the amino acid sequence shown in SEQ ID NO: 17, LCDR2 contains the amino acid sequence shown in SEQ ID NO: 13, LCDR3 contains the amino acid sequence shown in SEQ ID NO: 14. An anti-LAG3 antibody or its antigen-binding fragment is provided.

[0020] In some aspects of this disclosure, HCDR1 contains the amino acid sequence shown in SEQ ID NO: 9, HCDR2 contains the amino acid sequence shown in SEQ ID NO: 10. HCDR3 contains the amino acid sequence shown in SEQ ID NO: 11, LCDR1 contains the amino acid sequence shown in SEQ ID NO: 17, LCDR2 contains the amino acid sequence shown in SEQ ID NO: 13, LCDR3 contains the amino acid sequence shown in SEQ ID NO: 16. An anti-LAG3 antibody or its antigen-binding fragment is provided.

[0021] In some aspects of this disclosure, HCDR1 contains the amino acid sequence shown in SEQ ID NO: 9, HCDR2 contains the amino acid sequence shown in SEQ ID NO: 10. HCDR3 contains the amino acid sequence shown in SEQ ID NO: 11, LCDR1 contains the amino acid sequence shown in SEQ ID NO: 17, LCDR2 contains the amino acid sequence shown in SEQ ID NO: 15. LCDR3 contains the amino acid sequence shown in SEQ ID NO: 16. An anti-LAG3 antibody or its antigen-binding cleavage is 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. 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. 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. An anti-LAG3 antibody or its antigen-binding fragment is 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. 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. The amino acid sequence of LCDR2 is shown in Sequence ID No. 15. The amino acid sequence of LCDR3 is shown in Sequence ID No. 16. An anti-LAG3 antibody or its antigen-binding fragment is 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. 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 Sequence ID No. 15. The amino acid sequence of LCDR3 is shown in SEQ ID NO: 14. An anti-LAG3 antibody or its antigen-binding fragment is 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. 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. The amino acid sequence of LCDR2 is shown in Sequence ID No. 13. The amino acid sequence of LCDR3 is shown in Sequence ID No. 16. An anti-LAG3 antibody or its antigen-binding fragment is 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. 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. 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. An anti-LAG3 antibody or its antigen-binding fragment is provided.

[0027] 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. 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 Sequence ID No. 13. The amino acid sequence of LCDR3 is shown in SEQ ID NO: 14. An anti-LAG3 antibody or its antigen-binding fragment is provided.

[0028] 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. 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 Sequence ID No. 13. The amino acid sequence of LCDR3 is shown in Sequence ID No. 16. An anti-LAG3 antibody or its antigen-binding fragment is provided.

[0029] 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. 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 Sequence ID No. 15. The amino acid sequence of LCDR3 is shown in Sequence ID No. 16. An anti-LAG3 antibody or its antigen-binding fragment is provided.

[0030] In some aspects of this disclosure, The heavy chain variable region of the anti-LAG3 antibody contains the amino acid sequence shown in SEQ ID NO: 2, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO: 4; The heavy chain variable region of the anti-LAG3 antibody contains the amino acid sequence shown in SEQ ID NO: 2, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO: 6; or, The heavy chain variable region of the anti-LAG3 antibody contains the amino acid sequence shown in SEQ ID NO: 2, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO: 8; An anti-LAG3 antibody or its antigen-binding fragment is provided.

[0031] In some aspects of this disclosure, 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; or, 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; An anti-LAG3 antibody or its antigen-binding fragment is provided.

[0032] In some aspects of this disclosure, an anti-LAG3 antibody or an antigen-binding fragment thereof is provided, wherein the anti-LAG3 antibody or the antigen-binding fragment thereof is selected from Fab, Fab', F(ab')2, Fd, Fv, dAb, complementarity-determining region fragment, single-chain antibody, humanized antibody, and chimeric antibody.

[0033] In some aspects of this disclosure, an anti-LAG3 antibody or an antigen-binding fragment thereof is provided, wherein the anti-LAG3 antibody comprises a non-CDR region derived from a human antibody.

[0034] In some aspects of this disclosure, The anti-LAG3 antibody contains a constant region derived from a human antibody; Preferably, the anti-LAG3 antibody includes a constant region selected from the constant regions of human IgG1, IgG2, IgG3, or IgG4; An anti-LAG3 antibody or its antigen-binding fragment is provided.

[0035] In some aspects of this disclosure, an anti-LAG3 antibody or an antigen-binding fragment thereof is provided, wherein the heavy chain constant region of the anti-LAG3 antibody is the Igγ-1 chain C region (e.g., SEQ ID NO: 18) or the Igγ-4 chain C region (e.g., SEQ ID NO: 20), and the light chain constant region is the Igκ chain C region (e.g., SEQ ID NO: 19).

[0036] Amino acid sequence of the constant region of the IgG1 heavy chain: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(Sequence ID 18)

[0037] In certain aspects of this disclosure, 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; It holds.

[0038] In certain aspects of this disclosure, 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; It holds.

[0039] 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.

[0040] In some aspects of this disclosure, the heavy chain constant region of immunoglobulins is, in the EU numbering system, N297A, D265A, D270A, P238D, L328E, E233D, H268D, P271G, A330R, C226S, C229S, E233P, P331S, S267E, L328F, A330L, M252Y, S254T, T256E, N297Q, P238S, P238A, A327Q, A327G, P329A, K322A, T394D, G236R, G236A, L328R, A330S, P331S, H268A, E318A and K320A It further possesses one or more mutations selected from the following.

[0041] In certain aspects of this disclosure, the anti-LAG3 antibody is a monoclonal antibody.

[0042] In certain aspects of this disclosure, the anti-LAG3 antibody is an immunoglobulin.

[0043] In certain aspects of this disclosure, the anti-LAG3 antibody is a single-chain antibody.

[0044] Another aspect of this disclosure relates to antibody-drug conjugates (ADCs) comprising an antibody or its antigen-binding fragment and a small molecule drug, wherein the antibody or its antigen-binding fragment is the anti-LAG3 antibody or its antigen-binding fragment as described in this invention; preferably, the small molecule drug is a small molecule cytotoxic agent or a cell agonist; more preferably, the small molecule drug is a tumor chemotherapy agent, and the cell agonist is an immune cell agonist, such as a T cell agonist or an NK cell agonist.

[0045] Chemotherapy drugs may include conventional oncological chemotherapy drugs such as alkylating agents, antimetabolites, antitumor antibiotics, plant-derived anticancer drugs, hormones, and immunomodulators.

[0046] In some aspects of this disclosure, an antibody-drug conjugate is provided in which an anti-LAG3 antibody or an antigen-binding fragment thereof is linked to a small molecule drug via a linker; the linker may be a linker known to those skilled in the art, such as a hydrazone bond, a disulfide bond, or a peptide bond.

[0047] In some aspects of this disclosure, an antibody-drug conjugate is provided in which the molar ratio of an anti-LAG3 antibody or its antigen-binding fragment to a small molecule drug is 1:(2-4), for example, 1:2, 1:3, or 1:4.

[0048] Another aspect of this disclosure relates to isolated nucleic acid molecules encoding the anti-LAG3 antibody or its antigen-binding fragment as described in this disclosure.

[0049] Another aspect of this disclosure relates to recombinant vectors containing isolated nucleic acid molecules of this disclosure.

[0050] Another aspect of this disclosure relates to host cells containing isolated nucleic acid molecules or recombinant vectors of this disclosure.

[0051] Another aspect of this disclosure relates to a method for producing an anti-LAG3 antibody or its antigen-binding fragment according to this disclosure, comprising the steps of culturing host cells under appropriate conditions and recovering an anti-LAG3 antibody or its antigen-binding fragment from the cell culture.

[0052] Another aspect of this disclosure relates to pharmaceutical compositions containing the anti-LAG3 antibody or its antigen-binding fragment or the antibody-drug conjugate described herein, and optionally further contain pharmaceutically acceptable excipients.

[0053] Another aspect of this disclosure relates to a combined product comprising a first product and a second product packaged separately, hereby, The first product described above comprises an anti-LAG3 antibody or its antigen-binding fragment or antibody-drug conjugate as described herein; and, The second product comprises at least an anti-PD-1 antibody or an anti-CD73 antibody; Preferably, the first product and the second product each independently comprise one or more pharmaceutically acceptable excipients: Preferably, the combined product further includes a product description.

[0054] In some aspects of this disclosure, combination products are provided in which the molar ratio of an anti-LAG3 antibody or its antigen-binding fragment to an anti-PD-1 antibody or anti-CD73 antibody is (1:5) to (5:1), for example, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, or 5:1.

[0055] In some aspects of this disclosure, the anti-CD73 antibody is a combination product in which the anti-CD73 antibody is one of the above anti-CD73 antibodies.

[0056] Another aspect of this disclosure relates to an anti-LAG3 antibody or its antigen-binding fragment or antibody-drug conjugate in any aspect of this disclosure for use in the treatment or prevention of tumors or anemia. Preferably, the tumor is one or more selected from the group consisting of 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] An anti-LAG3 antibody or its antigen-binding fragment or antibody-drug conjugate is provided in any aspect of this disclosure for use in the treatment or prevention of tumors or anemia. Preferably, the tumor is one or more selected from the group consisting of 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.

[0058] Another aspect of this disclosure relates to a method for treating or preventing tumors or anemia, comprising the step of administering to a subject requiring treatment an effective amount of an anti-LAG3 antibody or its antigen-binding fragment or antibody-drug conjugate according to any aspect of this disclosure. Preferably, the tumor is one or more selected from the group consisting of 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.

[0059] In one or more aspects of this disclosure, The anti-LAG3 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 the anti-LAG3 antibody is administered in a single dose of 10 to 1000 mg (e.g., about 100 mg) per subject. It is administered in single doses of approximately 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 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.

[0060] In some embodiments, the anti-LAG3 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 antibody is administered once every two weeks (q2w) or once every three weeks (q3w).

[0061] However, it should be recognized that the total daily dose of the drug (e.g., pharmaceutical composition) or active ingredient (e.g., anti-LAG3 antibody or its antigen-binding fragment) 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.

[0062] Unless otherwise defined, scientific and technical terms used in this disclosure have the meanings commonly understood by those skilled in the art. Also, experimental procedures in cell culture, molecular genetics, nucleic acid chemistry, and immunology used in this disclosure are routine procedures widely practiced in the corresponding fields. For better understanding of this disclosure, definitions and explanations of related terms are provided below.

[0063] In this specification, the term EC 50 refers to the concentration at 50% of the maximum effect, i.e., the concentration that can cause 50% of the maximum effect.

[0064] In this specification, the term "antibody" generally refers to an immunoglobulin molecule composed of two pairs of polypeptide chains (each pair consisting of one "light" (L) chain and one "heavy" (H) chain). Antibody light chains are classified into κ light chains and λ light chains. Heavy chains are classified into 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 contains 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 differentiated into hypervariable regions (called complementarity-determining regions (CDRs)), between which conserved regions called framework regions (FRs) are distributed. Each V H and V LIt consists of three CDRs and four FRs arranged in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from the amino terminus to the carboxyl terminus. The variable region (V) of each heavy chain / light chain pair H and V L The ) 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.).

[0065] 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.

[0066] 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).

[0067] 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.

[0068] 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.

[0069] 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.

[0070] 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.

[0071] 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.

[0072] 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.

[0073] 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.

[0074] In this specification, the term “pharmaceutically acceptable carriers and / or excipients” 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.

[0075] 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.

[0076] 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.

[0077] In this disclosure, the terms “First” (e.g., First Product) and “Second” (e.g., Second Product) are used to distinguish and clarify expressions, and unless otherwise specified, there is no particular order that is meaningful.

[0078] Beneficial effects of this disclosure This disclosure achieves one or more of the following effects: (1) The anti-LAG3 antibody disclosed herein exhibits excellent affinity and specificity. (2) The anti-LAG3 antibody of this disclosure effectively blocks the interaction between LAG3 and MHC-II and specifically reduces LAG3 immunosuppression in organisms. (3) The antibodies of this disclosure can effectively treat or prevent tumors. [Brief explanation of the drawing]

[0079] [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 the 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. [Modes for carrying out the invention]

[0080] 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.

[0081] 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.

[0082] Amino acid sequence of the heavy chain of relatrimab: QVQLQQWGAGLLKPSETLSLTCAVYGGSFSDYYWNWIRQPPGKGLEWIGEINHRGSTNSNPSLKSRVTLSLDTSKNQFSLKLRSVTAADTAVYYCAFGYSDYEYNWFDPWGQG TLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCP PCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(Sequence ID 23)

[0083] Amino acid sequence of the light chain of relatrimab: EIVLTQSPATLSLSPGERATLSCRASQSISSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPLTFGQGTNLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (Sequence ID 24)

[0084] Antibody 14C12H1L1(hG1TM) is an anti-PD-1 antibody prepared by Akeso Biopharma Inc.

[0085] The amino acid sequence of the heavy chain of 14C12H1L1(hG1TM): EVQLVESGGGLVQPGGSLRLSCAASGFAFSSYDMSWVRQAPGKGLDWVATISGGGRYTYYPDSVKGRFTISRDNSKNNLYLQMNSLRAEDTALYYCANRYGEAWFAYWGQGTLV TVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCP PCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (Sequence ID 21)

[0086] The amino acid sequence of the heavy chain of 14C12H1L1(hG1TM): DIQMTQSPSSMSASVGDRVTFTCRASQDINTYLSWFQQKPGKSPKTLIYRANRLVSGVPSRFSGSGSGQDYTLTISSLQPEDMATYYCLQYDEFPLTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (Sequence ID 22)

[0087] Amino acid sequence of human LAG3-mG1Fc: (Sequence ID 25)

[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, 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 pCDH-huLAG3FL-RFP-NEO (LAG3, Genebank ID: NP_002277.4; vector pCDH-CMV-MCS-EF1-RFP+Neo, purchased from Youbio (catalog number VT9005)). [Examples]

[0091] 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:

[0092] Nucleotide sequence of the heavy chain variable region H9v of H9L8 (360 bp): CAGGTGCAGCTGCAGCAGTGGGGAGCTGGACTGCTGAAACCTAGCGAGACACTGAGCCTGACCTGTGCTGTGTACGGCGGATCTATCAGCGATTACTACTGGAACTGGATCAGGCAGCCCCCTGGAAAGGGACTGGAATGGATCGGAGAGATCAACTACAGGGGCACCACCAACTCCAATCCC TCTCTGAAGAGCAGGGTGACACTGAGCCTCGACACAAGCAAGAATCAGTTCAGCCTGAAGCTGAGGTCCGTGACCGCTGCTGATACAGCTGTGTACTACTGTGCCTTCGGCTACAGCGATTACGAGTACGATTGGTTCGACCCTGGGGGCCAGGGAACACTGGTTACAGTGAGCTCC (SEQ ID NO: 1)

[0093] Amino acid sequence of the heavy chain variable region H9v of H9L8 (120aa): QVQLQQWGAGLLKPSETLSLTCAVYGGSISDYYWNWIRQPPGKGLEWIGEINYRGTTNSNPSLKSRVTLSLDTSKNQFSLKLRSVTAADTAVYYCAFGYSDYEYDWFDPWGQGTLVTVSS (Sequence ID 2)

[0094] Nucleotide sequence of the light chain variable region L8v of H9L8 (321 bp): GAGATCGTTCTGACCCAGAGCCCAGCTACACTGAGCCTGTCTCCTGGAGAGAGGGCTACACTGTCCTGCAGAGCTAGCCAGACCATCAGCAGCTACCTGGCTTGGTACCAGCAGAAGCCTGGCCAAGCTCCAAGGCTGCTGATCTACGACGCCTCTAATAGGGC CACCGGCATCCCTGCTAGATTCTCTGGAAGCGGCAGCGGAACCGACTTTACACTGACAATCAGCTCCCTGGAGCCCGAGGATTTCGCTGTTTACTACTGTCAGCAGCGCAGCAACTGGCCCATCACATTCGGACAGGGCACAAATCTGGAGATCAAG (SEQ ID NO: 3)

[0095] Amino acid sequence of the light chain variable region L8v of H9L8 (107aa): EIVLTQSPATLSLSPGERATLSCRASQTISSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPITFGQGTNLEIK(Sequence ID 4).

[0096] 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.

[0097] 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.

[0098] Nucleotide sequence of the L9v light chain variable region of H9L9 (321 bp): GAGATCGTTCTGACCCAGAGCCCAGCTACACTGAGCCTGTCTCCTGGAGAGAGGGCTACACTGTCCTGCAGAGCTAGCCAGACCATCAGCAGCTACCTGGCTTGGTACCAGCAGAAGCCTGGCCAAGCTCCAAGGCTGCTGATCTACGACGGCTCTAATAGGGC CACCGGCATCCCTGCTAGATTCTCTGGAAGCGGCAGCGGAACCGACTTTACACTGACAATCAGCTCCCTGGAGCCCGAGGATTTCGCTGTTTACTACTGTCAGCAGGCAGCAACTGGCCCCTCACATTCGGACAGGGCACAAATCTGGAGATCAAG (SEQ ID NO: 5)

[0099] Amino acid sequence of the light chain variable region L9v of H9L9 (107 bp): EIVLTQSPATLSLSPGERATLSCRASQTISSYLAWYQQKPGQAPRLLIYDGSNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPLTFGQGTNLEIK (Sequence ID 6)

[0100] 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.

[0101] 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.

[0102] Nucleotide sequence of the light chain variable region L10v of H9L10 (321 bp): GAGATCGTTCTGACCCAGAGCCCAGCTACACTGAGCCTGTCTCCTGGAGAGAGGGCTACACTGTCCTGCAGAGCTAGCCAGTCCATCAGCAGCTACCTGGCTTGGTACCAGCAGAAGCCTGGCCAAGCTCCAAGGCTGCTGATCTACGACGGCTCTAATAGGGC CACCGGCATCCCTGCTAGATTCTCTGGAAGCGGCAGCGGAACCGACTTTACACTGACAATCAGCTCCCTGGAGCCCGAGGATTTCGCTGTTTACTACTGTCAGCAGCGCAGCAACTGGCCCATCACATTCGGACAGGGCACAAATCTGGAGATCAAG (SEQ ID NO: 7)

[0103] Amino acid sequence of the light chain variable region L10v of H9L10 (107 bp): EIVLTQSPATLSLSPGERATLSCRASQSISSYLAWYQQKPGQAPRLLIYDGSNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPITFGQGTNLEIK (Sequence ID 8)

[0104] 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).

[0105] 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).

[0106] 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).

[0107] 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 cDNA sequences of the human Igκ chain C region and 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. Plasmids pUC57simple-H9, pUC57simple-L8, pUC57simple-L9, and pUC57simple-L10 were 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 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.

[0108] Amino acid sequences of the heavy chain constant region of H9L8(hG4WT), H9L9(hG4WT), or H9L10(hG4WT): ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(Sequence ID 20)

[0109] Amino acid sequences of the light chain constant region of H9L8(hG4WT), H9L9(hG4WT), or H9L10(hG4WT): RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(Sequence ID 19)

[0110] 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 antibody" by Acierno et al. (Acierno et al., J Mol Biol., 2007; 374(1): 130-46). The manufacturing method is as follows:

[0111] The amino acid codon optimization and gene synthesis of the heavy and light chain (complete sequence or variable region) genes of human IgG antibodies were commissioned to Nanjing GenScript Biotech. Following the standard techniques described in Molecular Cloning: A Laboratory Manual (Third Edition), the heavy and light chain genes were subcloned into mammalian antibody heavy chain and antibody light chain expression vectors, respectively, using standard molecular cloning techniques such as PCR, enzyme digestion, DNA gel extraction, ligation and transformation, and colony PCR or enzyme digestion identification. The heavy and light chain genes of the recombinant expression vectors 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, 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.

[0112] 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 1) 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, the ELISA plate was allowed to develop color in the dark for 5 minutes using TMB (Neogen, 308177), and the color reaction was stopped by adding stop solution. 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.

[0113] The assay results are shown in Table 1 and Figure 1.

[0114] [Table 1]

[0115] The 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.

[0116] 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 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.

[0117] 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 The values ​​are shown in Table 2.

[0118] [Table 2]

[0119] As can be seen from Table 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.

[0120] 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 obtain 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.

[0121] The results are shown in Figure 3 and Table 3. Fluorescence analysis and curve fitting revealed the competitive binding EC of the antibodies relatrimab, H9L8 (hG4WT), H9L9 (hG4WT), and H9L10 (hG4WT). 50 The calculated values ​​were 1.153 nM, 1.459 nM, 1.482 nM, and 1.435 nM, respectively.

[0122] [Table 3]

[0123] The antibodies H9L8(hG4WT), H9L9(hG4WT), and H9L10(hG4WT) competitively bind to LAG3 and effectively block the binding of LAG3 to MHC-II on the surface of Raji cells in a dose-dependent manner, demonstrating activity equivalent to that of the positive control antibody relatrimab.

[0124] 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 no. S010201)) for 2 days. Raji-PDL1 cells were treated with 2 μg / mL of MMC (Stressmarq (catalog no. SIH-246-10MG)) at a working concentration and incubated in a 37°C, 5% CO2 incubator for 1 hour; PBMCs stimulated with SEB for 2 days and Raji-PDL1 cells treated with MMC for 1 hour were collected, washed twice with PBS, then resuspended in complete medium (i.e., RPMI1640 + 10% FBS) and counted. The PBMCs and Raji-PDL1 cells were separately placed in 10 × 10⁶ U-shaped 96-well plates (Corning (model no. 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.

[0125] 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.

[0126] 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 (i.e., 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.

[0127] 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.

[0128] 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.

[0129] 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).

[0130] 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.

[0131] 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. An anti-LAG3 antibody or its antigen-binding fragment, wherein 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. HCDR1 contains the amino acid sequence shown in SEQ ID NO: 9, HCDR2 contains the amino acid sequence shown in SEQ ID NO:

10. HCDR3 contains the amino acid sequence shown in SEQ ID NO: 11; LCDR1 contains the amino acid sequence shown in SEQ ID NO: 12 or SEQ ID NO:

17. LCDR2 contains the amino acid sequence shown in SEQ ID NO: 13 or SEQ ID NO:

15. LCDR3 contains the amino acid sequence shown in SEQ ID NO: 14 or SEQ ID NO:

16. Anti-LAG3 antibody or its antigen-binding fragment.

2. LCDR1 contains the amino acid sequence shown in SEQ ID NO:

12. LCDR2 contains the amino acid sequence shown in SEQ ID NO:

13. LCDR3 contains the amino acid sequence shown in SEQ ID NO: 14; or, LCDR1 contains the amino acid sequence shown in SEQ ID NO:

12. LCDR2 contains the amino acid sequence shown in SEQ ID NO:

15. LCDR3 contains the amino acid sequence shown in SEQ ID NO: 16; or, LCDR1 contains the amino acid sequence shown in SEQ ID NO:

17. LCDR2 contains the amino acid sequence shown in SEQ ID NO:

15. LCDR3 contains the amino acid sequence shown in SEQ ID NO: 14; or, LCDR1 contains the amino acid sequence shown in SEQ ID NO:

12. LCDR2 contains the amino acid sequence shown in SEQ ID NO:

13. LCDR3 contains the amino acid sequence shown in SEQ ID NO: 16; or, LCDR1 contains the amino acid sequence shown in SEQ ID NO:

12. LCDR2 contains the amino acid sequence shown in SEQ ID NO:

15. LCDR3 contains the amino acid sequence shown in SEQ ID NO: 14; or, LCDR1 contains the amino acid sequence shown in SEQ ID NO:

17. LCDR2 contains the amino acid sequence shown in SEQ ID NO:

13. LCDR3 contains the amino acid sequence shown in SEQ ID NO: 14; or, LCDR1 contains the amino acid sequence shown in SEQ ID NO:

17. LCDR2 contains the amino acid sequence shown in SEQ ID NO:

13. LCDR3 contains the amino acid sequence shown in SEQ ID NO: 16; or, LCDR1 contains the amino acid sequence shown in SEQ ID NO:

17. LCDR2 contains the amino acid sequence shown in SEQ ID NO:

15. LCDR3 contains the amino acid sequence shown in SEQ ID NO: 16; The anti-LAG3 antibody or antigen-binding fragment thereof as described in claim 1.

3. 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 the aforementioned 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 the aforementioned LCDR3 is shown in SEQ ID NO: 16; or, The amino acid sequence of LCDR1 is shown in Sequence ID No.

17. The amino acid sequence of LCDR2 is shown in Sequence ID No.

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

17. The amino acid sequence of LCDR2 is shown in Sequence ID No.

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

17. The amino acid sequence of LCDR2 is shown in Sequence ID No.

13. The amino acid sequence of the aforementioned LCDR3 is shown in SEQ ID NO: 16; or, The amino acid sequence of LCDR1 is shown in Sequence 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 Sequence ID No. 16; The anti-LAG3 antibody or antigen-binding fragment thereof according to claim 1 or 2.

4. The heavy chain variable region of the anti-LAG3 antibody includes the amino acid sequence shown in SEQ ID NO: 2, and the light chain variable region of the anti-LAG3 antibody includes the amino acid sequence shown in SEQ ID NO: 4; The heavy chain variable region of the anti-LAG3 antibody includes the amino acid sequence shown in SEQ ID NO: 2, and the light chain variable region of the anti-LAG3 antibody includes the amino acid sequence shown in SEQ ID NO: 6; or, The heavy chain variable region of the anti-LAG3 antibody includes the amino acid sequence shown in SEQ ID NO: 2, and the light chain variable region of the anti-LAG3 antibody includes the amino acid sequence shown in SEQ ID NO: 8; An anti-LAG3 antibody or antigen-binding fragment thereof according to any one of claims 1 to 3.

5. 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; Or, 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; An anti-LAG3 antibody or antigen-binding fragment thereof according to any one of claims 1 to 4.

6. The anti-LAG3 antibody or its antigen-binding fragment is Fab, Fab', F(ab') ２ An anti-LAG3 antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, selected from Fd, Fv, dAb, complementarity-determining region fragment, single-chain antibody, humanized antibody, and chimeric antibody.

7. The anti-LAG3 antibody includes a non-CDR region derived from a human antibody; Preferably, the constant region of the anti-LAG3 antibody is derived from a human antibody; Preferably, the constant region of the anti-LAG3 antibody is selected from the constant regions of human IgG1, IgG2, IgG3, or IgG4; The heavy chain constant region of the anti-LAG3 antibody is the Igγ-1 chain C region (e.g., SEQ ID NO: 18) or the Igγ-4 chain C region (e.g., SEQ ID NO: 20), and the light chain constant region is the Igκ chain C region (e.g., SEQ ID NO: 19); An anti-LAG3 antibody or antigen-binding fragment thereof according to any one of claims 1 to 6.

8. 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; or, 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; An anti-LAG3 antibody or antigen-binding fragment thereof according to any one of claims 1 to 7.

9. An antibody-drug conjugate comprising an antibody or an antigen-binding fragment thereof and a small molecule drug, wherein the antibody or the antigen-binding fragment thereof is the antibody or the antigen-binding fragment thereof according to any one of claims 1 to 8; preferably, the small molecule drug is a small molecule cytotoxic agent or a cell agonist; more preferably, the small molecule drug is a chemotherapy agent for tumors, and the cell agonist is an agonist of immune cells, such as a T cell or an NK cell agonist.

10. The anti-LAG3 antibody or its antigen-binding fragment is linked to the small molecule drug via a linker; for example, the linker is a hydrazone bond, a disulfide bond, or a peptide bond; Preferably, the molar ratio of the anti-LAG3 antibody or its antigen-binding fragment to the low-molecular-weight drug is 1:(2-4); The antibody-drug conjugate according to claim 9.

11. An isolated nucleic acid molecule encoding an anti-LAG3 antibody according to any one of claims 1 to 8.

12. A recombinant vector comprising an isolated nucleic acid molecule as described in claim 11.

13. A host cell comprising an isolated nucleic acid molecule according to claim 11 or a recombinant vector according to claim 12.

14. A method for producing an anti-LAG3 antibody or its antigen-binding fragment according to any one of claims 1 to 8, comprising the step of culturing the host cell according to claim 13 under appropriate conditions and recovering the anti-LAG3 antibody or its antigen-binding fragment from the cell culture.

15. A pharmaceutical composition comprising an anti-LAG3 antibody or an antigen-binding fragment thereof according to any one of claims 1 to 8, or an antibody-drug conjugate according to claim 9 or 10, further comprising, optionally, a pharmaceutically acceptable excipient.

16. A combination product comprising a first product and a second product packaged separately, The first product comprises an anti-LAG3 antibody or its antigen-binding fragment according to any one of claims 1 to 8, or an antibody-drug conjugate according to claim 9 or 10. The second product comprises at least an anti-PD-1 antibody or an anti-CD73 antibody; Preferably, the first and second products each independently comprise one or more pharmaceutically acceptable excipients: Preferably, the combined product further includes a product description. Combination product.

17. The combination product according to claim 16, wherein the molar ratio of the anti-LAG3 antibody or its antigen-binding fragment to the anti-PD-1 antibody or anti-CD73 antibody is (1:5) to (5:1).

18. The use of an anti-LAG3 antibody or its antigen-binding fragment according to any one of claims 1 to 8, or an antibody-drug conjugate according to claim 9 or 10, in the manufacture of a pharmaceutical product for treating or preventing tumors, Preferably, the tumor is one or more selected from the group consisting of 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 tumor, 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.

19. An anti-LAG3 antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, or an antibody-drug conjugate according to claim 9 or 10, for use in the treatment or prevention of tumors, Preferably, the tumor is one or more selected from the group consisting of 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 tumor, 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; Anti-LAG3 antibody or its antigen-binding fragment or antibody-drug conjugate.

20. A method for treating or preventing a tumor, comprising the step of administering an effective amount of an anti-LAG3 antibody or its antigen-binding fragment, or an antibody-drug conjugate, according to any one of claims 1 to 8, or according to claim 9 or 10, to a target subject, Preferably, the tumor is one or more selected from the group consisting of 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 tumor, 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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