Anti-LAG3 Bispecific Antibodies, Pharmaceutical Compositions, and Uses

Abstract

The present invention provides an anti-LAG3 antibody bispecific antibody, a pharmaceutical composition thereof, and uses thereof, which belong to the field of biomedicine. Specifically, the bispecific antibody comprises 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; the first protein functional domain is an anti-LAG3 antibody or an antigen-binding fragment thereof, which comprises a heavy chain variable region and a light chain variable region, and the heavy chain variable region comprises HCDR1 to HCDR3 having the amino acid sequences represented by SEQ ID NOs: 5 to 7, respectively. The light chain variable region comprises LCDR1 to LCDR3 having the amino acid sequences represented by SEQ ID NOs: 8 to 10, respectively. The bispecific antibody has excellent affinity and specificity, and is highly applicable.

Description

[Technical field]

[0001] The present invention relates to a bispecific antibody against LAG3 and its pharmaceutical composition and its use in the field of biomedicine. Specifically, the bispecific antibody is an anti-LAG3 / anti-PD-1 bispecific antibody. [Background technology]

[0002] Tumors, especially malignant tumors, are nowadays a serious health threat worldwide and the second leading cause of death among various diseases. In recent years, the incidence of these diseases has increased significantly. Malignant tumors are characterized by poor response to treatment, high rate of late metastasis, and poor prognosis. Although conventional treatment methods currently used clinically (e.g., radiation therapy, chemotherapy, and surgery) can significantly relieve pain and extend survival time, they have significant limitations and it is difficult to further improve their effectiveness.

[0003] Lymphocyte activation gene 3 (LAG3), or CD223, is a type I transmembrane protein of 498 amino acids and a member of the immunoglobulin superfamily (IgSF). LAG3 is primarily involved in the activation of activated CD4 + T cells and CD8 + It is expressed in T cells. LAG3 is also expressed in cells such as natural killer (NK) cells, B cells, regulatory T cells (Tregs), and plasmacytoid dendritic cells (pDCs) (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.).

[0004] The LAG3 gene is located adjacent to the CD4 gene on human chromosome 12 (20p13.3), and both molecules have the same exons and introns. Although LAG3 and CD4 molecules are structurally similar, the amino acid sequence homology between them is only about 20%. Major histocompatibility complex class II (MHC II) molecules, liver sinusoidal endothelial cell lectin (LSECtin) molecules, and galectin-3 molecules are relevant ligands for LAG3. MHC class II molecules are the major ligand for LAG3. The affinity of LAG3 for MHC class II molecules (Kd: 60 nmol L -1 ) was 100-fold higher than that of the CD4 molecule, indicating that the LAG3 molecule could effectively compete with the CD4 molecule for binding to MHC class II molecules and inhibit T cell activation.

[0005] In the tumor microenvironment, expression of the immunosuppressive molecule LAG3 can be detected 24 hours after T cell activation, which in turn leads to T cell dysfunction or apoptosis. The LAG3 molecule dimerizes via its D1 domain (which has a proline-rich loop structure) and binds to CD4 + It specifically binds to MHC class II molecules in the "CD3-TCR-MHCII" first signaling axis of T cell activation. On the one hand, this blocks the signaling pathway for T cell activation, and on the other hand, the intracellular segment of the LAG3 molecule (KIEELE motif) generates an immunosuppressive signal, suppressing the activation of CD4 +Downregulates T cell activity. LAG3 molecule promotes the differentiation of Treg cells and is involved in downstream signaling of signal transducer and activator of transcription 5, thereby enhancing the inhibitory effect of Treg cells. This is one of the mechanisms by which tumors avoid 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.).

[0006] Tumor-infiltrating CD8 in various malignant tumors + Several studies have shown that LAG3 is overexpressed in T cells. For example, in ovarian cancer, it is expressed by tumor-infiltrating CD8 T cells specific for the New York esophageal squamous cell carcinoma 1 (NY-ESO-1) antigen. + T cells highly express PD-1 and LAG3 and have a reduced ability to produce IFN-γ and TNF-α, which results in lymphocyte inactivation. Galectin-3 and LSECtin mainly interact with LAG3 and upregulate CD8 + It regulates T cell activation and function. Also, melanoma antigen-specific T cells isolated from patients with metastatic melanoma show significant upregulation of the expression of LAG3 as well as other immune checkpoint molecules CTLA-4 and TIM-3 (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.).

[0007] Currently, several LAG3 antibody drugs are in clinical research, of which Bristol Myers Squibb's relatolimab is the most advanced with 10 clinical studies ongoing. Most of these studies involve the combination of relatolimab and nivolumab to treat tumors such as hematological malignancies, melanoma, glioblastoma, renal cell carcinoma, non-small cell lung cancer, and the like.

[0008] The transmembrane receptor PD-1 (programmed cell death protein 1) is a member of the CD28 family and is expressed on activated T cells, B cells, and myeloid cells. The receptors for PD-1, PDL1, and PDL2 are members of the B7 superfamily. PDL1 is expressed on a variety of cells, including T cells, B cells, endothelial cells, and epithelial cells, whereas PDL2 is expressed only on antigen-presenting cells, such as dendritic cells and macrophages.

[0009] PD-1 plays a very important role in downregulating T cell activation, and PD-1-mediated T cell downregulation is one of the important mechanisms of tumor immune evasion. PD-L1 expressed on the tumor surface can bind to PD-1 on the immune cell surface and inhibit immune cell killing of tumor tissues via the PD-1 / PD-L1 signaling pathway. High PD-L1 expression in tumors is also associated with cancers that are difficult to detect (Hamanishi et al., Proc. Natl. Acad. Sci. USA, 2007; 104: 3360-5). One of the effective methods to antagonize PD-1 and inhibit the PD-1 / PD-L1 signaling pathway is the in vivo injection of anti-PD-1 antibodies.

[0010] Due to the broad anti-tumor potential and surprising efficacy of PD-1 antibodies, antibodies targeting the PD-1 pathway are believed to represent a breakthrough in the treatment of a variety of tumors, including non-small cell lung cancer, renal cell carcinoma, ovarian cancer, and melanoma (Homet MB, Parisi G., et al., Anti-PD-1 therapy in melanoma. Semin Oncol., 2015 Jun; 42(3): 466-473), as well as hematological malignancies and anemias (Held SA, Heine A, et al., Advances in immunotherapy of chronic myeloid leukemia CML. Curr Cancer Drug Targets, 2013 Sep; 13(7): 768-74).

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

[0012] Currently, there is a need to develop novel anti-LAG3 antibodies and bifunctional antibody drugs that simultaneously target PD-1 and LAG3. Summary of the Invention

[0013] The present inventors have conducted intensive research and creative efforts to obtain an anti-LAG3 antibody, and have developed an anti-LAG3 / anti-PD-1 bispecific antibody based on the obtained antibody. The present inventors have surprisingly found that the anti-LAG3 antibody of the present invention (hereinafter sometimes abbreviated as the antibody or the antibody of the present invention) and the anti-LAG3 / anti-PD-1 bispecific antibody of the present invention (hereinafter sometimes abbreviated as the bispecific antibody or the bispecific antibody of the present invention) have excellent affinity and / or specificity, and are superior in one or more aspects to positive control antibodies (e.g., nivolumab, pembrolizumab, relatolimab, and the like). The present invention is described in detail below.

[0014] One aspect of the present invention is an anti-LAG3 antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises HCDR1 to HCDR3 having the amino acid sequences set forth in SEQ ID NOs: 5 to 7, respectively, and the light chain variable region comprises LCDR1 to LCDR3 having the amino acid sequences set forth in SEQ ID NOs: 8 to 10, respectively; the heavy chain variable region comprises HCDR1 to HCDR3 having the amino acid sequences set forth in SEQ ID NOs: 5 to 7, respectively, and the light chain variable region comprises LCDR1 to LCDR3 having the amino acid sequences set forth in SEQ ID NOs: 8, 46, and 47, respectively; or The present invention relates to an anti-LAG3 antibody or an antigen-binding fragment thereof, wherein the heavy chain variable region comprises HCDR1 to HCDR3 having the amino acid sequences set forth in SEQ ID NOs: 5 to 7, respectively, and the light chain variable region comprises LCDR1 to LCDR3 having the amino acid sequences set forth in SEQ ID NOs: 48, 46, and 10, respectively.

[0015] In some embodiments of the present invention, the heavy chain variable region of the antibody has the amino acid sequence set forth in SEQ ID NO:2 and the light chain variable region of the antibody has the amino acid sequence set forth in SEQ ID NO:4; the heavy chain variable region of the antibody has the amino acid sequence set forth in SEQ ID NO:2 and the light chain variable region of the antibody has the amino acid sequence set forth in SEQ ID NO:42; or the heavy chain variable region of the antibody has the amino acid sequence set forth in SEQ ID NO:2, and the light chain variable region of the antibody has the amino acid sequence set forth in SEQ ID NO:44; The antibody or antigen-binding fragment thereof is provided.

[0016] In some embodiments of the invention, the antibody or antigen-binding fragment thereof is provided, which is selected from a Fab, a Fab', a F(ab')2, a Fd, a Fv, a dAb, a complementarity determining region fragment, a single chain fragment variable, a humanized antibody, a chimeric antibody, and a diabody.

[0017] In some embodiments of the invention, the antibody has an EC for human LAG3-mFc of less than 0.2 nM, e.g., less than 0.15 nM, less than 0.1 nM, less than 0.08 nM, less than 0.06 nM, or less than 0.05 nM, or less. 50 value; preferably said EC 50 The antibody or antigen-binding fragment thereof is provided, wherein the value is measured by indirect ELISA.

[0018] In some embodiments of the invention, there is provided an antibody or antigen-binding fragment thereof, wherein the antibody is derived from a species other than mouse, for example comprising non-CDR regions derived from a human antibody.

[0019] In some embodiments of the present invention, the antibody comprises a constant region derived from a human antibody; Preferably, the constant region of the antibody is selected from the constant region of human IgG1, IgG2, IgG3, or IgG4. The antibody or antigen-binding fragment thereof is provided.

[0020] In some embodiments of the present invention, The heavy chain constant region of the anti-LAG3 antibody is an Ig gamma-1 chain C region (e.g., as set forth in SEQ ID NO: 39) or an Ig gamma-4 chain C region (e.g., as set forth in SEQ ID NO: 45), and the light chain constant region of the anti-LAG3 antibody is an Ig kappa chain C region (e.g., as set forth in SEQ ID NO: 40); The antibody or antigen-binding fragment thereof is provided.

[0021] In some embodiments of the present invention, the antibody is of the human IgG1 subtype; wherein, according to the EU numbering system, the heavy chain constant region of said antibody comprises the following mutations: L234A and L235A; L234A and G237A; L235A and G237A; or L234A, L235A, and G237A, having; Preferably, the antibody comprises a heavy chain having the amino acid sequence set forth in SEQ ID NO:11, and a light chain having the amino acid sequence set forth in SEQ ID NO:12; The antibody or antigen-binding fragment thereof is provided.

[0022] In some embodiments of the present invention, the antibody is of the human IgG4 subtype; wherein, according to the EU numbering system, the heavy chain constant region of said antibody comprises the following mutations: F234A and L235A; F234A and G237A; L235A and G237A; or F234A, L235A, and G237A, having; Preferably, the antibody comprises a heavy chain having the amino acid sequence set forth in SEQ ID NO: 13, and a light chain having the amino acid sequence set forth in SEQ ID NO: 12; The antibody or antigen-binding fragment thereof is provided.

[0023] In some embodiments of the invention, the anti-LAG3 antibody is a monoclonal antibody.

[0024] In some embodiments of the invention, the anti-LAG3 antibody is in the form of an immunoglobulin.

[0025] In some embodiments of the invention, the anti-LAG3 antibody is a single chain variable region fragment.

[0026] Another aspect of the invention relates to an antibody drug conjugate (ADC) comprising an antibody or antigen-binding fragment thereof, and a small molecule drug, wherein said antibody or antigen-binding fragment thereof is an anti-LAG3 antibody or antigen-binding fragment thereof of any of the embodiments of the invention; preferably, said small molecule drug is a small molecule cytotoxic drug; more preferably, said small molecule drug is an anti-tumor chemotherapeutic drug.

[0027] The chemotherapeutic agent may be a conventional antitumor chemotherapeutic agent, such as alkylating agents, antimetabolites, antitumor antibiotics, plant-based anticancer agents, hormones, and immunological agents.

[0028] In one or more embodiments of the invention, the antibody-drug conjugate is provided, wherein the antibody or antigen-binding fragment thereof is linked to a small molecule drug via a linker; the linker may be any known to one of skill in the art, such as a hydrazone bond, a disulfide bond, or a peptide bond.

[0029] In one or more embodiments of the present invention, the antibody-drug conjugate is provided, wherein the molar ratio of the antibody or antigen-binding fragment thereof to the small molecule drug is 1:(2 to 4), for example, 1:2, 1:3, or 1:4.

[0030] Yet another aspect of the invention is a bispecific antibody comprising a first protein functional domain and a second protein functional domain, the first protein functional domain targets LAG3; the second protein functional region targets a target other than LAG3 (e.g., PD-1); the first protein functional domain is an antibody or antigen-binding fragment thereof of any of the embodiments of the present invention; Preferably, the bispecific antibody is in IgG-scFv format; Preferably, said first protein functional domain is an antibody of any embodiment of the invention and said second protein functional domain is a single chain variable domain fragment; or Preferably, the invention relates to a bispecific antibody, wherein said first protein functional domain is a single chain variable domain fragment and said second protein functional domain is an antibody of any of the embodiments of the invention.

[0031] The bispecific antibody of the invention is an anti-LAG3 / anti-PD-1 bispecific antibody.

[0032] In some embodiments of the present invention, the first protein functional domain and the second protein functional domain are linked directly or via a linker fragment; Preferably, the linker fragment is (GGGGS)m, where m is a positive integer, such as 1, 2, 3, 4, 5, or 6; or Preferably, the linker fragment is (GGGGS)nG, where n is a positive integer, such as 1, 2, 3, 4, 5, or 6; Said bispecific antibodies are provided.

[0033] In some embodiments of the invention, the bispecific antibody is provided, wherein the number of said first protein functional domain and said second protein functional domain is independently one, two or more.

[0034] In some embodiments of the invention, the bispecific antibody is provided, wherein the single chain variable region fragment is linked to the C-terminus of the heavy chain of the antibody.

[0035] In some embodiments of the present invention, A first protein functional domain that targets LAG3; and a second protein functional domain that targets PD-1; Including, said first protein functional domain is an anti-LAG3 antibody of any of the embodiments of the present invention, said anti-LAG3 antibody being in the form of an immunoglobulin; the second protein functional domain is an anti-PD-1 single chain variable domain fragment; Said bispecific antibodies are provided.

[0036] In some embodiments of the present invention, the anti-PD-1 single chain variable domain fragment comprises a heavy chain variable domain and a light chain variable domain; the heavy chain variable region comprises HCDR1 to HCDR3 having the amino acid sequences set forth in SEQ ID NOs: 26 to 28, respectively; and the light chain variable region comprises LCDR1 to LCDR3 having the amino acid sequences set forth in SEQ ID NOs: 29 to 31, respectively; Said bispecific antibodies are provided.

[0037] In some embodiments of the present invention, The anti-PD-1 single chain variable region fragment, the heavy chain variable region has the amino acid sequence set forth in SEQ ID NO: 15 and the light chain variable region has the amino acid sequence set forth in SEQ ID NO: 17; or The heavy chain variable region has the amino acid sequence set forth in SEQ ID NO: 19, and the light chain variable region has the amino acid sequence set forth in SEQ ID NO: 21 or SEQ ID NO: 38. Said bispecific antibodies are provided.

[0038] In some embodiments of the present invention, the heavy chain variable region and the light chain variable region of the anti-PD-1 single chain variable region fragment are linked directly or via a linker fragment; Preferably, the linker fragment is (GGGGS)m, where m is a positive integer, such as 1, 2, 3, 4, 5, or 6; or Preferably, the linker fragment is (GGGGS)nG, where n is a positive integer, such as 1, 2, 3, 4, 5, or 6. Said bispecific antibodies are provided.

[0039] In some embodiments of the present invention, a first protein functional region that targets LAG3; and a second protein functional domain that targets PD-1; Includes; the number of said first protein functional regions is 1 and the number of said second protein functional regions is 2; the first protein functional domain is an immunoglobulin and the second protein functional domain is a single chain variable domain fragment; the immunoglobulin comprises a heavy chain having the amino acid sequence set forth in SEQ ID NO:11 or SEQ ID NO:13, and a light chain having the amino acid sequence set forth in SEQ ID NO:12; the single chain variable region fragment comprises a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:19, and a light chain variable region having the amino acid sequence set forth in SEQ ID NO:21 or SEQ ID NO:38; the single chain variable region fragment is linked to the C-terminus of two heavy chains of the immunoglobulin; the first protein functional region is linked to the second protein functional region via a first linker fragment; the heavy chain variable region of the single chain variable region fragment is linked to the light chain variable region of the single chain variable region fragment via a second linker fragment; the first linker fragment and the second linker fragment are the same or different; Preferably, the first linker fragment and the second linker fragment have an amino acid sequence independently selected from SEQ ID NOs: 35 to 37; Preferably, the amino acid sequences of the first linker fragment and the second linker fragment are set forth in SEQ ID NO: 36. Said bispecific antibodies are provided.

[0040] In some embodiments of the present invention, a first protein functional region that targets LAG3; and a second protein functional domain that targets PD-1; Including, the first protein functional domain is an anti-LAG3 single chain variable domain fragment and the second protein functional domain is an anti-PD-1 antibody, the anti-PD-1 antibody being in the form of an immunoglobulin; the anti-LAG3 single chain variable region fragment comprises a heavy chain variable region and a light chain variable region; the heavy chain variable region comprises HCDR1 to HCDR3 having the amino acid sequences set forth in SEQ ID NOs: 5 to 7, respectively; the light chain variable region comprises LCDR1 to LCDR3 having the amino acid sequences set forth in SEQ ID NOs: 8 to 10, respectively; Said bispecific antibodies are provided.

[0041] In some embodiments of the present invention, The anti-LAG3 single chain variable region fragment, the heavy chain variable region has the amino acid sequence set forth in SEQ ID NO:2 and the light chain variable region has the amino acid sequence set forth in SEQ ID NO:4; the heavy chain variable region has the amino acid sequence set forth in SEQ ID NO:2 and the light chain variable region has the amino acid sequence set forth in SEQ ID NO:42; or The heavy chain variable region has the amino acid sequence set forth in SEQ ID NO: 2, and the light chain variable region has the amino acid sequence set forth in SEQ ID NO: 4. Said bispecific antibodies are provided.

[0042] In some embodiments of the present invention, the heavy chain variable region and the light chain variable region of the anti-LAG3 single chain variable region fragment are linked directly or via a linker fragment; Preferably, the linker fragment is (GGGGS)m, where m is a positive integer, such as 1, 2, 3, 4, 5, or 6; or Preferably, the linker fragment is (GGGGS)nG, where n is a positive integer, such as 1, 2, 3, 4, 5, or 6. Said bispecific antibodies are provided.

[0043] In some embodiments of the present invention, the anti-PD-1 antibody comprises a heavy chain variable region and a light chain variable region; the heavy chain variable region comprises HCDR1 to HCDR3 having the amino acid sequences set forth in SEQ ID NOs: 26 to 28, respectively; the light chain variable region comprises LCDR1 to LCDR3 having the amino acid sequences set forth in SEQ ID NOs: 29 to 31, respectively; Said bispecific antibodies are provided.

[0044] In some embodiments of the present invention, In the anti-PD-1 antibody, the heavy chain variable region has the amino acid sequence set forth in SEQ ID NO: 15 and the light chain variable region has the amino acid sequence set forth in SEQ ID NO: 17; or The heavy chain variable region has the amino acid sequence set forth in SEQ ID NO: 19, and the light chain variable region has the amino acid sequence set forth in SEQ ID NO: 21 or SEQ ID NO: 38. Said bispecific antibodies are provided.

[0045] In some embodiments of the invention, the bispecific antibody is provided, wherein the heavy chain constant region of the anti-PD-1 antibody is an Ig gamma-1 chain C region (e.g., as set forth in SEQ ID NO:39) or an Ig gamma-4 chain C region (e.g., as set forth in SEQ ID NO:45), and the light chain constant region of the anti-PD-1 antibody is an Ig kappa chain C region (e.g., as set forth in SEQ ID NO:40).

[0046] In some embodiments of the present invention, the anti-PD-1 antibody is a human IgG1 subtype; wherein, according to the EU numbering system, the anti-PD-1 antibody has the following mutation: L234A and L235A; L234A and G237A; L235A and G237A; or L234A, L235A, and G237A, having; Preferably, the anti-PD-1 antibody comprises a heavy chain having the amino acid sequence set forth in SEQ ID NO: 34, and a light chain having the amino acid sequence set forth in SEQ ID NO: 25. Said bispecific antibodies are provided.

[0047] In some embodiments of the present invention, the anti-PD-1 antibody is a human IgG4 subtype; wherein, according to the EU numbering system, the anti-PD-1 antibody has the following mutation: F234A and L235A; F234A and G237A; L235A and G237A; or F234A, L235A, and G237A, having; Preferably, the anti-PD-1 antibody comprises a heavy chain having the amino acid sequence set forth in SEQ ID NO: 32, and a light chain having the amino acid sequence set forth in SEQ ID NO: 25. Said bispecific antibodies are provided.

[0048] In some embodiments of the present invention, a first protein functional region that targets LAG3; and a second protein functional domain that targets PD-1; Includes; the number of said first protein functional regions is 1 and the number of said second protein functional regions is 2; the first protein functional domain is a single chain variable domain fragment and the second protein functional domain is an immunoglobulin; the single chain variable region fragment comprises a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:2 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO:4; the immunoglobulin comprises a heavy chain having the amino acid sequence set forth in SEQ ID NO:34 or SEQ ID NO:32, and a light chain having the amino acid sequence set forth in SEQ ID NO:25; the single chain variable region fragment is linked to the C-terminus of two heavy chains of the immunoglobulin; the first protein functional region is linked to the second protein functional region via a first linker fragment; the heavy chain variable region of the single chain variable region fragment is linked to the light chain variable region of the single chain variable region fragment via a second linker fragment; the first linker fragment and the second linker fragment are the same or different; Preferably, the first linker fragment and the second linker fragment have an amino acid sequence independently selected from SEQ ID NOs: 35 to 37; Preferably, the amino acid sequences of the first linker fragment and the second linker fragment are set forth in SEQ ID NO: 36. Said bispecific antibodies are provided.

[0049] In some embodiments of the present invention, one immunoglobulin molecule linked to two single chain variable fragment molecules; Preferably, the two single chain variable region fragment molecules are identical. Said bispecific antibodies are provided.

[0050] Yet another aspect of the invention pertains to an isolated nucleic acid molecule encoding an anti-LAG3 antibody of any of the embodiments of the invention, or a bispecific antibody of any of the embodiments of the invention.

[0051] Yet another aspect of the present invention pertains to a recombinant vector comprising the isolated nucleic acid molecule of the present invention.

[0052] Yet another aspect of the present invention relates to a host cell comprising the isolated nucleic acid molecule of the invention or the recombinant vector of the invention.

[0053] Yet another aspect of the invention relates to a method for preparing an antibody or antigen-binding fragment thereof of any of the embodiments of the invention, or a bispecific antibody of any of the embodiments of the invention, comprising culturing a host cell of the invention under suitable conditions and isolating said antibody or antigen-binding fragment thereof or bispecific antibody from the cell culture.

[0054] Yet another aspect of the present invention relates to a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of any of the embodiments of the present invention, the antibody-drug conjugate of any of the embodiments of the present invention, or the bispecific antibody of any of the embodiments of the present invention, said pharmaceutical composition optionally further comprising a pharmacologically acceptable excipient.

[0055] Yet another aspect of the invention is the use of an antibody or antigen-binding fragment thereof according to any of the embodiments of the invention, an antibody-drug conjugate according to any of the embodiments of the invention, or a bispecific antibody according to any of the embodiments of the invention in the preparation of a medicament for treating and / or preventing tumors or anemia, comprising: Preferably, the tumor is selected from one or more of ovarian cancer, esophageal cancer, melanoma, hematological malignancies, glioblastoma, renal cell carcinoma, lung cancer, prostate cancer, bladder cancer, colon cancer, rectal cancer, liver cancer, gastrointestinal cancer, breast cancer, brain cancer, pancreatic cancer, thyroid cancer, head and neck cancer, and kidney cancer; Preferably, the lung cancer is non-small cell lung cancer; Preferably, the hematological malignancy is leukemia; Preferably, the esophageal cancer is esophageal squamous cell carcinoma; The present invention relates to the use of an antibody or antigen-binding fragment thereof, an antibody-drug conjugate, or a bispecific antibody.

[0056] The antibody or antigen-binding fragment thereof according to any of the embodiments of the present invention, the antibody-drug conjugate according to any of the embodiments of the present invention, or the bispecific antibody according to any of the embodiments of the present invention is for use in the treatment and / or prevention of tumor or anemia, Preferably, the tumor is selected from one or more of ovarian cancer, esophageal cancer, melanoma, hematological malignancies, glioblastoma, renal cell carcinoma, lung cancer, prostate cancer, bladder cancer, colon cancer, rectal cancer, liver cancer, gastrointestinal cancer, breast cancer, brain cancer, pancreatic cancer, thyroid cancer, head and neck cancer, and kidney cancer; Preferably, the lung cancer is non-small cell lung cancer; Preferably, the hematological malignancy is leukemia; Preferably, the esophageal cancer is esophageal squamous cell carcinoma.

[0057] Yet another aspect of the present invention is a method for treating and / or preventing tumors or anemia, comprising administering to a subject in need thereof an effective amount of the antibody or antigen-binding fragment thereof of any of the embodiments of the present invention, the antibody-drug conjugate of any of the embodiments of the present invention, or the bispecific antibody of any of the embodiments of the present invention; Preferably, the tumor is selected from one or more of ovarian cancer, esophageal cancer, melanoma, hematological malignancies, glioblastoma, renal cell carcinoma, lung cancer, prostate cancer, bladder cancer, colon cancer, rectal cancer, liver cancer, gastrointestinal cancer, breast cancer, brain cancer, pancreatic cancer, thyroid cancer, head and neck cancer, and kidney cancer; Preferably, the lung cancer is non-small cell lung cancer; Preferably, the hematological malignancy is leukemia; Preferably, the esophageal cancer is esophageal squamous cell carcinoma.

[0058] In the present invention, unless otherwise defined, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Also, the laboratory procedures of cell culture, molecular genetics, nucleic acid chemistry, and immunology used herein are routine procedures widely used in the corresponding fields. Meanwhile, in order to facilitate understanding of the present invention, the definitions and explanations of relevant terms are provided below.

[0059] As used herein, EC 50 The term refers to the concentration for 50% of the maximal effect, i.e., the concentration capable of producing 50% of the maximal effect.

[0060] The term "antibody" as used herein refers to an immunoglobulin molecule that generally consists of two pairs of polypeptide chains, each pair having one "light" (L) chain and one "heavy" (H) chain. Antibody light chains are classified as kappa and lambda light chains. Heavy chains are classified as mu, delta, gamma, alpha, or epsilon. Antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE. In the light and heavy chains, the variable and constant regions are connected by a "J" region of about 12 or more amino acids, and the heavy chains further include a "D" region of about 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains, CH1, CH2, and CH3. Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain, CL. The constant region of an antibody can mediate the binding of immunoglobulins to host tissues or factors, including the binding of various cells of the immune system (e.g., effector cells) to the first component (C1q) of the classical complement system. The VH and VL regions can be further divided into hypervariable regions (called complementarity determining regions (CDRs)), with conserved regions called framework regions (FRs) distributed between the hypervariable regions. Each VH and VL consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of each heavy / light chain pair (VH and VL) form the antigen-binding site.The assignment of amino acids to regions or domains is based on Bethesda Md, Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, (1987 and 1991)) or Chothia & Lesk J. Mol. Biol., 1987; 196:901-917; Chothia et al., Nature, 1989; 342:878-883, or see the definitions in the IMGT numbering system, 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.

[0061] In particular, the heavy chain may further comprise more than three CDRs, for example six, nine or twelve etc. For example, in a bispecific antibody of the invention, the heavy chain may be the heavy chain of an IgG antibody having its C-terminus linked to one ScFv, in which case the heavy chain comprises nine CDRs.

[0062] The term "antibody" is not limited by any particular method for producing the antibody. For example, antibodies include recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. The antibody may be of various isotypes, such as IgG (e.g., subtypes IgG1, IgG2, IgG3, or IgG4), IgA1, IgA2, IgD, IgE, or IgM.

[0063] The terms "mAb" and "monoclonal antibody" as used herein refer to an antibody or antibody fragment that is derived from a group of highly homologous antibodies, i.e., from a group of identical antibody molecules with the exception of natural mutations that may occur naturally. Monoclonal antibodies are highly specific to a single epitope on an antigen. Unlike monoclonal antibodies, polyclonal antibodies usually contain at least two or more different antibodies that generally recognize different epitopes on an antigen. Monoclonal antibodies can generally be obtained using hybridoma technology, first reported by Kohler et al. (Kohler G, Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity [J]. Nature, 1975; 256(5517): 495), but can also be obtained using recombinant DNA technology (see, for example, U.S. Pat. No. 4,816,567).

[0064] The term "humanized antibody" as used herein refers to an antibody or antibody fragment obtained when the whole or part of the CDRs of a human immunoglobulin (receptor antibody) are replaced with the CDRs of a non-human antibody (donor antibody), which may be a non-human (e.g., mouse, rat, or rabbit) antibody with the expected specificity, affinity, or reactivity. Furthermore, some amino acid residues in the framework region (FR) of the receptor antibody can also be replaced with the corresponding amino acid residues of the non-human antibody or amino acid residues of other antibodies to further improve or optimize the performance of the antibody. For further details on humanized antibodies, see, e.g., Jones et al., Nature, 1986; 321:522-525; Reichmann et al., Nature, 1988; 332:323-329; Presta, Curr. Op. Struct. Biol., 1992; 2:593-596; and Clark, Immunol. Today, 2000; 21:397-402. In some cases, the antigen-binding fragment of an antibody is a diabody, H and V L The domains are expressed on a single polypeptide chain. However, due to the short linker used, pairing of the two domains on the same chain is not possible. Instead, the domains pair with complementary domains on the other chain, generating two antigen-binding sites (see, e.g., Holliger P. et al., Proc. Natl. Acad. Sci. USA, 1993; 90:6444-6448 and Poljak RJ et al., Structure, 1994; 2:1121-1123).

[0065] As used herein, the term "single chain variable fragment (ScFv)" refers to an antibody heavy chain variable fragment (V H ) and the antibody light chain variable region (V L ) linked by a linker. L and V HThe domains are paired to form monovalent molecules with a linker that allows them to produce a single polypeptide chain (see, e.g., Bird et al., Science, 1988; 242:423-426 and Huston et al., Proc. Natl. Acad. Sci. USA, 1988; 85:5879-5883). Such scFv molecules may have the following general structure: NH2-V L -Linker fragment-V H -COOH or NH2-V H -Linker fragment-V L -COOH. A suitable linker in the prior art consists of a repeat of the amino acid sequence GGGGS, or a variant thereof. For example, a linker having the amino acid sequence (GGGGS)4 may be used, or a variant thereof (Holliger et al., Proc. Natl. Acad. Sci. USA, 1993; 90:6444-6448). Other linkers that can be used in the present invention are described in Alfthan et al., Protein Eng., 1995; 8:725-731, Choi et al., Eur. J. Immunol., 2001; 31:94-106, Hu et al., Cancer Res., 1996; 56:3055-3061, Kipriyanov et al., J. Mol. Biol., 1999; 293:41-56, and Roovers et al., Cancer Immunology, Immunotherapy, 2001, 50(1):51-59.

[0066] The term "isolated" as used herein refers to being obtained by artificial means from a natural state. When a particular "isolated" substance or component occurs in nature, it may be that the change occurs in its natural environment, or that it is isolated from the natural environment, or both. For example, if a particular non-isolated polynucleotide or polypeptide naturally occurs in a particular animal organism, and the same polynucleotide or polypeptide is isolated in high purity from such a natural state, it 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] The term "vector" as used herein refers to a nucleic acid vehicle into which a polynucleotide can be inserted. If the vector allows expression of a protein encoded by the inserted polynucleotide, the vector is called an expression vector. The vector can be introduced into a host cell by transformation, transduction, or transfection so that the genetic material element carried by the vector can be expressed in the host cell. Vectors are well known to those skilled in the art, and examples include, but are not limited to, plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs); phages, such as lambda phage or M13 phage; and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex viruses), poxviruses, baculoviruses, papilloma viruses, and papova viruses (such as SV40). The vector may contain various elements for regulating expression, examples of which include, but are not limited to, a promoter sequence, a transcription initiation sequence, an enhancer sequence, a selection element, and a reporter gene. Furthermore, the vector may contain a replication origin.

[0068] The term "host cell" as used herein refers to a cell into which a vector can be introduced, including, but not limited to, a prokaryotic cell, such as E. coli or Bacillus subtilis; a fungal cell, such as a yeast cell or Aspergillus; an insect cell, such as an S2 Drosophila cell or Sf9; or an animal cell, such as a fibroblast, a CHO cell, a GS cell, a COS cell, an NSO cell, a HeLa cell, a BHK cell, a HEK293 cell, or a human cell.

[0069] As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and its target antigen. In some embodiments, an antibody that specifically binds to an antigen (or an antibody specific for an antigen) is one that binds to an antigen within about 10 -5 Less than m, e.g., about 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 Affinity (K D ) means that the antibody binds to the antigen.

[0070] As used herein, "K D The term "dissociation equilibrium constant" refers to the dissociation equilibrium constant of a specific antibody-antigen interaction and is used to describe the binding affinity between an antibody and an antigen. A small dissociation equilibrium constant indicates strong antibody-antigen binding and high affinity between the antibody and the antigen. In general, antibodies have a dissociation equilibrium constant of about 10 -5 Less than m, e.g., about 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 The dissociation equilibrium constant (K D ) to bind to an antigen (e.g., PD-1 protein). Dcan be determined using methods known to those of skill in the art, for example, using a Fortebio molecular interaction instrument.

[0071] As used herein, the terms "monoclonal antibody" and "mAb" have the same meaning and can be used interchangeably. The terms "polyclonal antibody" and "pAb" have the same meaning and can be used interchangeably. Also, herein, amino acids are generally represented using one-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.

[0072] The term "pharmacologically acceptable carrier and / or excipient" as used herein refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and active ingredient. Such carriers and / or excipients 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 examples include, but are not limited to, pH adjusters, surfactants, adjuvants, and ionic strength enhancers. For example, non-limiting examples of pH adjusters include phosphate buffers; non-limiting examples of surfactants include cationic, anionic, or nonionic surfactants, such as Tween-80; non-limiting examples of ionic strength enhancers include sodium chloride.

[0073] The term "effective amount" as used herein refers to an amount sufficient to obtain or at least partially obtain a desired effect. For example, a prophylactically effective amount for a disease (e.g., tumor) refers to an amount sufficient to prevent, inhibit or delay the onset of the disease (e.g., tumor), and a therapeutically effective amount refers to an amount sufficient to cure or at least partially inhibit the disease or its complications in a patient suffering from the disease. Determining such an effective amount is undoubtedly within the capabilities of a person skilled in the art. For example, an effective amount for therapeutic purposes depends on the severity of the disease to be treated; the overall state of the patient's own immune system; the general condition of the patient, such as age, weight and sex; the route of administration; and other treatments performed at the same time.

[0074] As used herein, reference to the amino acid sequence of PD-1 protein (NCBI GenBank: NM_005018) includes the full-length PD-1 protein, or the extracellular fragment of PD-1, PD-1 ECD, or a fragment containing PD-1 ECD, and also includes fusion proteins of the full-length PD-1 protein or fusion proteins of PD-1 ECD, such as fragments fused with Fc protein fragments (mFc or hFc) of mouse or human IgG. However, those skilled in the art will understand that mutations or alterations (including but not limited to substitutions, deletions, and / or additions) in the amino acid sequence of PD-1 protein may occur naturally or may be artificially introduced without affecting its biological function. Thus, in the present invention, the term "PD-1 protein" is intended to encompass all such sequences, including natural or artificial variants thereof. References to sequence fragments of PD-1 protein also include the corresponding sequence fragments in those natural or artificial variants.

[0075] In the present specification, the amino acid sequence of lymphocyte activation gene 3 (LAG3) includes the full-length LAG3 protein, or the extracellular fragment of LAG3, LAG3 ECD, or a fragment containing LAG3 ECD, and also includes a fusion protein of the full-length LAG3 protein or a fusion protein of LAG3 ECD, for example, a fragment fused with an Fc protein fragment (mFc or hFc) of mouse or human IgG. However, the skilled artisan will understand that mutations or alterations (including but not limited to substitutions, deletions, and / or additions) in the amino acid sequence of the LAG3 protein can occur naturally or can be artificially introduced without affecting its biological function. Thus, in the present invention, the term "LAG3 protein" is intended to include all such sequences, including their natural or artificial variants. Also, when describing a sequence fragment of the LAG3 protein, the corresponding sequence fragment in the natural or artificial variant is also included.

[0076] In the present invention, the terms "first" (e.g., first protein functional region) and "second" (e.g., second protein functional region) are used for expressive distinction or clarity, unless otherwise specified, and do not have a typical sequential meaning. Effect of the Invention

[0077] Beneficial Effects of the Invention The present invention achieves one or more of the following advantages: (1) The anti-LAG3 antibody of the present invention has excellent affinity and specificity; (2) the bispecific antibody of the present invention (e.g., BS-PL021A, BS-PL022B, or BS-PL023C) can specifically bind to LAG3, effectively block the binding of LAG3 to MHC II, and specifically alleviate the immunosuppression of LAG3 in an organism; (3) the bispecific antibody of the present invention (e.g., BS-PL021A, BS-PL022B, or BS-PL023C) can specifically bind to PD-1, effectively block the binding of PD-1 to PDL1, specifically relieve the immunosuppression of PD-1 in the organism, and activate the immune response; (4) the first protein functional region and the second protein functional region in the bispecific antibody of the present invention have a synergistic effect; (5) the bispecific antibody of the present invention, particularly BS-PL022B, has been completely deprived of its binding activity to the Fc receptors FcγRI, FcγRIIb, FcγRIIa_H131, FcγRIIIa_V158, and / or FcγRIIIa_F158, and has further been completely deprived of its ADCC activity or ADCP activity; and (6) The bispecific antibody of the present invention, particularly BS-PL022B, has been completely deprived of its binding activity to complement C1q and has further been deprived of its CDC activity. [Brief description of the drawings]

[0078] [Figure 1] Figure 1 shows the binding activity of BS-PL021A, BS-PL022B, BS-PL023C, Bs-PLV02, and 14C12H1L1 (hG1TM) to the antigen PD-1-mFc analyzed by indirect ELISA.

[0079] [Diagram 2] Figure 2 shows the results of analysis of the binding activity of BS-PL021A, BS-PL022B, BS-PL023C, BS-PLV02, relatolimab, and H7L8(hG1WT) to the antigen LAG3-mFc by indirect ELISA.

[0080] [Diagram 3] FIG. 3 shows the activity of anti-LAG3 / anti-PD-1 bispecific antibodies competing with human PDL1-mFc for binding to human PD-1-mFc-biotin by competitive ELISA.

[0081] [Figure 4] Figure 4 shows the binding activity of anti-LAG3 / anti-PD-1 bispecific antibodies to PD-1 on the membrane surface of 293T-PD1 cells analyzed by FACS.

[0082] [Diagram 5] Figure 5 shows the binding activity of anti-LAG3 / anti-PD-1 bispecific antibodies to LAG3 on the 293T-LAG3 membrane surface analyzed by FACS.

[0083] [Figure 6] FIG. 6 shows the activity of anti-LAG3 / anti-PD-1 bispecific antibodies competing with PDL1 for binding to the antigen PD-1 on the cell membrane surface, analyzed by competition flow cytometry.

[0084] [Figure 7] FIG. 7 shows the activity of anti-LAG3 / anti-PD-1 bispecific antibodies competing with LAG3 for binding to the antigen MHC II on the cell membrane surface, analyzed by competition flow cytometry.

[0085] [Figure 8A] FIG. 8A shows the results of an analysis of an anti-LAG3 / anti-PD-1 bispecific antibody that blocks LAG3 binding to MHC II.

[0086] [Figure 8B] Figure 8B shows the results of an analysis of an anti-LAG3 / anti-PD-1 bispecific antibody that blocks LAG3 binding to MHC II.

[0087] [Figure 9A] Figure 9A shows the results of an analysis of an anti-LAG3 / anti-PD-1 bispecific antibody that blocks PD-1 binding to PD-L1.

[0088] [Figure 9B]Figure 9B shows the results of an analysis of an anti-LAG3 / anti-PD-1 bispecific antibody that blocks PD-1 binding to PD-L1.

[0089] [Figure 10A] Figure 10A shows the results of an analysis of an anti-LAG3 / anti-PD-1 bispecific antibody that simultaneously blocks LAG3 binding to MHC II and PD-1 binding to PD-L1.

[0090] [Figure 10B] Figure 10B shows the results of an analysis of an anti-LAG3 / anti-PD-1 bispecific antibody that simultaneously blocks LAG3 binding to MHC II and PD-1 binding to PD-L1.

[0091] [Figure 11] FIG. 11 shows the results of a bridging assay of anti-LAG3 / anti-PD-1 bispecific antibodies.

[0092] [Figure 12A] FIG. 12A shows the biological activity of anti-LAG3 / anti-PD-1 bispecific antibodies in promoting IFN-γ secretion analyzed by mixed lymphocyte reaction (MLR).

[0093] [Figure 12B] Figure 12B shows the biological activity of anti-LAG3 / anti-PD-1 bispecific antibodies in promoting IL-2 secretion analyzed by mixed lymphocyte reaction (MLR).

[0094] [Figure 13] FIG. 13 shows the results of an analysis of the affinity constant of BS-PL022B for FcγRI.

[0095] [Figure 14] FIG. 14 shows the results of an analysis of the affinity constant of H7L8 (hG1WT) for FcγRI.

[0096] [Figure 15] FIG. 15 shows the results of an analysis of the affinity constant of BS-PL022B for FcγRIIIa_V158.

[0097] [Figure 16] FIG. 16 shows the results of an analysis of the affinity constant of H7L8 (hG1WT) for FcγRIIIa_V158.

[0098] [Figure 17] FIG. 17 shows the results of an analysis of the affinity constant of BS-PL022B for FcγRIIIa_F158.

[0099] [Figure 18] FIG. 18 shows the results of an analysis of the affinity constant of H7L8 (hG1WT) for FcγRIIIa_F158.

[0100] [Figure 19] FIG. 19 shows the results of an analysis of the affinity constant of BS-PL022B for FcγRIIa_H131.

[0101] [Figure 20] FIG. 20 shows the results of an analysis of the affinity constant of H7L8 (hG1WT) for FcγRIIa_H131.

[0102] [Figure 21] FIG. 21 shows the results of an analysis of the affinity constant of BS-PL022B for FcγRIIb.

[0103] [Figure 22] FIG. 22 shows the results of an analysis of the affinity constant of H7L8 (hG1WT) for FcγRIIb.

[0104] [Diagram 23] FIG. 23 shows the results of an analysis of the affinity constant of BS-PL022B for C1q.

[0105] [Figure 24] FIG. 24 shows the results of an analysis of the affinity constant of H7L8(hG1WT) for C1q.

[0106] [Diagram 25] Figure 25 shows the results of the ADCP effect analysis for BS-PL022B.

[0107] [Figure 26] Figure 26: Efficacy of anti-LAG3 / anti-PD-1 bispecific antibodies in a BALB / c-hPD1 / hLAG3 mouse model bearing CT26 tumors. *P<0.05, **P<0.01, ***P<0.001 compared to isotype control (2-way ANOVA).

[0108] [Figure 27] Figure 27. Effect of anti-LAG3 / anti-PD-1 bispecific antibody on body weight in a BALB / c-hPD1 / hLAG3 mouse model implanted with CT26 tumors. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0109] Detailed Description The embodiments of the present invention will be described in detail below with reference to examples. Those skilled in the art will understand that the following examples are merely illustrative of the present invention and should not be interpreted as limiting the scope of the present invention. Examples in which specific techniques or conditions are not described are carried out according to techniques or conditions described in the literature of the field (see, for example, Molecular Cloning: A Laboratory Manual, authored by J. Sambrook et al., and translated by Huang Peitang et al., third edition, Science Press) or according to the accompanying instructions. If the manufacturer of the reagent or equipment used is not described, they are commercially available conventional products. For example, MDA-MB-231 cells and U87-MG cells were available from ATCC.

[0110] BALB / c mice were purchased from Guangdong Medical Laboratory Animal Center.

[0111] Nivolumab was purchased from BMS under lot number ABA0330. Nivolumab is an anti-PD-1 antibody.

[0112] Pembrolizumab was purchased from MSD Ireland (Carlow) under lot number S023942. Pembrolizumab is an anti-PD-1 antibody.

[0113] The positive control antibody, leratolimab, has the sequence set forth in US Patent Publication No. 20160326248(A1), with the heavy chain amino acid sequence set forth in SEQ ID NO: 1 and the light chain amino acid sequence set forth in SEQ ID NO: 2. leratolimab is an anti-LAG-3 antibody.

[0114] The cell line 293T-PD1 was constructed by Akeso Biopharma. The cell line 293T-PD1 was produced by viral infection of HEK293T cells using a third generation lentivirus system (see, e.g., A Third Generation Lentivirus Vector with a Conditional Packaging System. Dull T, Zufferey R, Kelly M, Mandel RJ, Nguyen M, Trono D, and Naldini L., J Virol., 1998. 72(11): 8463-8471). The lentivirus expression vector used here was plenti6.3 / V5-PD1FL-BSD (PD1, Genebank ID: NM_005018; vector plenti6.3 / V5-BSD, purchased from Invitrogen, catalog number K5315-20).

[0115] The cell line 293T-LAG3 was constructed by Akeso Biopharma. The cell line 293T-LAG3 was produced by viral infection of HEK293T cells using a third generation lentivirus system (see, e.g., A Third Generation Lentivirus Vector with a Conditional Packaging System. Dull T, Zufferey R, Kelly M, Mandel RJ, Nguyen M, Trono D, and Naldini L., J Virol., 1998. 72(11): 8463-8471). The lentivirus expression vector used here was plenti6.3 / V5-huLAG3FL-BSD (LAG3, Genebank ID: NM_002277.4; vector plenti6.3 / V5-BSD, purchased from Invitrogen, catalog number K5315-20).

[0116] The cell line Raji-PDL1 was constructed by Akeso Biopharma. The cell line Raji-PDL1 was produced by viral infection of Raji cells using a third generation lentivirus system (see, e.g., A Third Generation Lentivirus Vector with a Conditional Packaging System. Dull T, Zufferey R, Kelly M, Mandel RJ, Nguyen M, Trono D, and Naldini L., J Virol., 1998. 72(11): 8463-8471). The lentivirus expression vector used here was plenti6.3 / V5-PDL1 (PDL1, Genebank ID: NP_054862.1; vector plenti6.3 / V5, purchased from Invitrogen, catalog number K5315-20).

[0117] The cell line Jurkat-NFAT-PD1-LAG3 was constructed by Akeso Biopharma. The cell line Jurkat-NFAT-PD1-LAG3 was prepared 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 lentiviral expression vector used here was pCDH-huLAG3FL-RFP-NEO (LAG3, Genebank ID: NM_002277.4; vector pCDH-CMV-MCS-EF1-RFP+Neo, purchased from Youbio, catalog number VT9005).

[0118] The cell line CHO-K1-PD1 was constructed by Akeso Biopharma. The cell line CHO-K1-PD1 was prepared by viral infection of CHO-K1 cells using a third generation lentivirus system (see, e.g., A Third Generation Lentivirus Vector with a Conditional Packaging System. Dull T, Zufferey R, Kelly M, Mandel RJ, Nguyen M, Trono D, and Naldini L., J Virol., 1998. 72(11): 8463-8471). The lentivirus expression vector used here was pCDH-CMV-PD-1FL-Puro (PD1, Genebank ID: NM_005018; vector pCDH-CMV-Puro, purchased from Youbio, catalog number VT1480).

[0119] The cell line CHO-K1-LAG3 was constructed by Akeso Biopharma. The cell line CHO-K1-LAG3 was produced by viral infection of CHO-K1 cells using a third generation lentivirus system (see, e.g., A Third Generation Lentivirus Vector with a Conditional Packaging System. Dull T, Zufferey R, Kelly M, Mandel RJ, Nguyen M, Trono D, and Naldini L., J Virol., 1998. 72(11): 8463-8471). The lentivirus expression vector used here was plenti6.3 / V5-huLAG3FL-BSD (LAG3, Genebank ID: NM_002277.4; vector plenti6.3 / V5-BSD, purchased from Invitrogen, catalog number K5315-20).

[0120] The cell line Jurkat-NFAT-CD64-CD32R was constructed by Akeso Biopharma Inc. The cell line Jurkat-NFAT-CD64-CD32R was prepared by viral infection of Jurkat cells using a third generation lentivirus system (see, e.g., A Third Generation Lentivirus Vector with a Conditional Packaging System. Dull T, Zufferey R, Kelly M, Mandel RJ, Nguyen M, Trono D, and Naldini L., J Virol., 1998. 72(11): 8463-8471). The lentiviral expression vectors used here were pCDH-NFAT-Hygro (obtained by modification in the laboratory of the present inventors based on the vector pCDH-Hygro, pCDH-CMV-MCS-EF1-Puro (purchased from Youbio, catalog number VT1480)), pcDH-hFCGR1AFL-Neo (obtained by modification in the laboratory of the present inventors based on the vector pCDH-Neo, pCDH-CMV-MCS-EF1-Puro (purchased from Youbio, catalog number VT1480)), and pCDH-hFCGR2A(H167)-puro (hFCGR2A(H167), Genebank ID: P12318; vector pCDH-CMV-MCS-EF1-Puro, purchased from Youbio, catalog number VT1480).

[0121] The cell line CHO-K1-PD1-LAG3 was constructed by Akeso Biopharma, Inc. The cell line CHO-K1-PD1-LAG3 was prepared by viral infection of CHO-K1 cells using a third generation lentivirus system (see, e.g., A Third Generation Lentivirus Vector with a Conditional Packaging System. Dull T, Zufferey R, Kelly M, Mandel RJ, Nguyen M, Trono D, and Naldini L., J Virol., 1998. 72(11): 8463-8471). The lentiviral expression vectors used here were pCDH-hPD1-FL-puro (PD-1, Genebank ID: NM_005018; vector pCDH-CMV-MCS-EF1-Puro, purchased from Youbio, catalog number VT1480) and plenti6.3 / V5-huLAG3FL-BSD (LAG3, Genebank ID: NM_002277.4; vector plenti6.3 / V5-BSD, purchased from Invitrogen, catalog number K5315-20). EXAMPLES

[0122] Preparation Example 1: Design and preparation of anti-LAG3 antibody 1. Antibody Design The inventors of the present application inventively designed a series of antibody sequences based on the known LAG3 protein sequence (NCBI reference sequence: NP_002277.4) and its three-dimensional crystal structure, etc. Through extensive screening and testing, humanized monoclonal antibodies that specifically bind to LAG3 were finally obtained and named H7L8, H7L9, and H7L10, respectively. The amino acid sequences of the heavy and light chain variable regions of the monoclonal antibodies and their coding sequences are as follows:

[0123] Nucleotide sequence of the heavy chain variable region H7v of H7L8 (360 bp): CAGGTGCAGCTGCAGCAGTGGGGAGCTGGACTGCTGAAACCTAGCGAGACACTGAGCCTGACCTGTGCTGTGTACGGCGGATCTATCAGCGATTACTACTGGAACTGGATCAGGCAGCCCCCTGGAAAGGGACTGGAATGGATCGGAGAGATCAACCACAGGGGCACCACCAACTCCAATCCC TCTCTGAAGAGCAGGGTGACACTGAGCCTCGACACAAGCAAGAATCAGTTCAGCCTGAAGCTGAGGTCCGTGACCGCTGCTGATACAGCTGTGTACTACTGTGCCTTCGGCTACAGCGATTACGAGTACGATTGGTTCGACCCTGGGGGCCAGGGAACACTGGTTACAGTGAGCTCC (SEQ ID NO: 1)

[0124] Amino acid sequence of the heavy chain variable region H7v of H7L8 (120 aa): QVQLQQWGAGLLKPSETLSLTCAVYGGSISDYYWNWIRQPPGKGLEWIGEINHRGTTNSNPSLKSRVTLSLDTSKNQFSLKLRSVTAADTAVYYCAFGYSDYEYDWFDPWGQGTLVTVSS (SEQ ID NO: 2)

[0125] Nucleotide sequence of the light chain variable region L8v of H7L8 (321 bp): GAGATCGTTCTGACCCAGAGCCCAGCTACACTGAGCCTGTCTCCTGGAGAGAGGGCTACACTGTCCTGCAGAGCTAGCCAGACCATCAGCAGCTACCTGGCTTGGTACCAGCAGAAGCCTGGCCAAGCTCCAAGGCTGCTGATCTACGACGCCTCTAATAGGGC CACCGGCATCCCTGCTAGATTCTCTGGAAGCGGCAGCGGAACCGACTTTACACTGACAATCAGCTCCCTGGAGCCCGAGGATTTCGCTGTTTACTACTGTCAGCAGCGCAGCAACTGGCCCATCACATTCGGACAGGGCACAAATCTGGAGATCAAG (SEQ ID NO: 3)

[0126] Amino acid sequence of the light chain variable region L8v of H7L8 (107 aa): EIVLTQSPATLSLSPGERATLSCRASQTISSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPITFGQGTNLEIK (SEQ ID NO: 4)

[0127] The nucleotide sequence of the heavy chain variable region H7v of H7L9 is identical to the nucleotide sequence of the heavy chain variable region H7v of H7L8 of SEQ ID NO:1.

[0128] The amino acid sequence of the heavy chain variable region H7v of H7L9 is identical to the amino acid sequence of the heavy chain variable region H7v of H7L8 in SEQ ID NO:2.

[0129] Nucleotide sequence of the light chain variable region L9v of H7L9 (321 bp): GAGATCGTTCTGACCCAGAGCCCAGCTACACTGAGCCTGTCTCCTGGAGAGAGGGCTACACTGTCCTGCAGAGCTAGCCAGACCATCAGCAGCTACCTGGCTTGGTACCAGCAGAAGCCTGGCCAAGCTCCAAGGCTGCTGATCTACGACGGCTCTAATAGGGC CACCGGCATCCCTGCTAGATTCTCTGGAAGCGGCAGCGGAACCGACTTTACACTGACAATCAGCTCCCTGGAGCCCGAGGATTTCGCTGTTTACTACTGTCAGCAGGCAGCAACTGGCCCCTCACATTCGGACAGGGCACAAATCTGGAGATCAAG (SEQ ID NO: 41)

[0130] Amino acid sequence of the light chain variable region L9v of H7L9 (107bp): EIVLTQSPATLSLSPGERATLSCRASQTISSYLAWYQQKPGQAPRLLIYDGSNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPLTFGQGTNLEIK (SEQ ID NO: 42)

[0131] The nucleotide sequence of the heavy chain variable region H7v of H7L10 is identical to the nucleotide sequence of the heavy chain variable region H7v of H7L8 of SEQ ID NO:1.

[0132] The amino acid sequence of the heavy chain variable region H7v of H7L10 is identical to the amino acid sequence of the heavy chain variable region H7v of H7L8 in SEQ ID NO:2.

[0133] Nucleotide sequence of the light chain variable region L10v of H7L10 (321 bp): GAGATCGTTCTGACCCAGAGCCCAGCTACACTGAGCCTGTCTCCTGGAGAGAGGGCTACACTGTCCTGCAGAGCTAGCCAGTCCATCAGCAGCTACCTGGCTTGGTACCAGCAGAAGCCTGGCCAAGCTCCAAGGCTGCTGATCTACGACGGCTCTAATAGGGC CACCGGCATCCCTGCTAGATTCTCTGGAAGCGGCAGCGGAACCGACTTTACACTGACAATCAGCTCCCTGGAGCCCGAGGATTTCGCTGTTTACTACTGTCAGCAGCGCAGCAACTGGCCCATCACATTCGGACAGGGCACAAATCTGGAGATCAAG (SEQ ID NO: 43)

[0134] Amino acid sequence of the light chain variable region L10v of H7L10 (107bp): EIVLTQSPATLSLSPGERATLSCRASQSISSYLAWYQQKPGQAPRLLIYDGSNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPITFGQGTNLEIK (SEQ ID NO: 44)

[0135] The amino acid sequences of the CDRs of antibody H7L8 are as follows (according to the IMGT numbering system): HCDR1: GGSISDYY (SEQ ID NO:5); HCDR2: INHRGTT (SEQ ID NO:6); HCDR3: AFGYSDYEYDWFDP (SEQ ID NO: 7); LCDR1: QTISSY (SEQ ID NO:8); LCDR2: DAS (SEQ ID NO: 9); and LCDR3: QQRSNWPIT (sequence number 10).

[0136] The amino acid sequences of the CDRs of antibody H7L9 are as follows (according to the IMGT numbering system): HCDR1: GGSISDYY (SEQ ID NO:5); HCDR2: INHRGTT (SEQ ID NO:6); HCDR3: AFGYSDYEYDWFDP (SEQ ID NO: 7); LCDR1: QTISSY (SEQ ID NO:8); LCDR2: DGS (SEQ ID NO: 46); and LCDR3: QQRSNWPLT (sequence number 47).

[0137] The amino acid sequences of the CDRs of antibody H7L10 are as follows (according to the IMGT numbering system): HCDR1: GGSISDYY (SEQ ID NO:5); HCDR2: INHRGTT (SEQ ID NO:6); HCDR3: AFGYSDYEYDWFDP (SEQ ID NO: 7); LCDR1: QSISSY (SEQ ID NO: 48); LCDR2: DGS (SEQ ID NO: 46); and LCDR3: QQRSNWPIT (sequence number 10).

[0138] 2. Expression and purification of humanized antibody H7L8 (hG1WT) The heavy chain cDNA sequence (the coding sequence of the variable region is shown in SEQ ID NO:1; the constant region was Ig gamma-1 chain C region, SEQ ID NO:39) and the light chain cDNA sequence (the coding sequence of the variable region is shown in SEQ ID NO:3; the constant region was P01834.1 (human Ig kappa chain C region, SEQ ID NO:40)) of H7L8 (hG1WT) were separately cloned into pUC57simple vector (GenScript) to obtain plasmids pUC57simple-H7 and pUC57simple-L8, respectively. The plasmids pUC57simple-H7 and pUC57simple-L8 were digested (HindIII and EcoRI), respectively. The heavy and light chains were isolated by electrophoresis and separately subcloned into pcDNA3.1 vector, and the recombinant plasmids were extracted and co-transfected into 293F cells. After 7 days of cell culture, the medium was separated by high-speed centrifugation, and the supernatant was concentrated and loaded onto a HiTrap MabSelect SuRe column. Proteins were eluted in one step with elution buffer: target samples were isolated and the buffer was exchanged into PBS.

[0139] Amino acid sequence of the heavy chain constant region of H7L8 (hG1WT) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 39)

[0140] Amino acid sequence of the light chain constant region of H7L8(hG1WT) RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 40)

[0141] 3. Design of humanized antibody H7L8 (hG1TM) The present inventors obtained humanized antibody H7L8(hG1TM) having mutations in the constant region by introducing a point mutation (L234A) from leucine to alanine at position 234 (according to the EU numbering system; the same applies below) in the heavy chain, a point mutation (L235A) from leucine to alanine at position 235, and a point mutation (G237A) from glycine to alanine at position 237 in the heavy chain of H7L8(hG1TM). The amino acid sequence of the heavy chain H7(hG1TM) of H7L8(hG1TM) is shown in SEQ ID NO:11, and the amino acid sequence of the light chain L8 is shown in SEQ ID NO:12.

[0142] Humanized antibody H7L8 (hG1TM) was prepared by the method described in step 2 above.

[0143] Amino acid sequence of heavy chain H7(hG1TM) of H7L8(hG1TM) QVQLQQWGAGLLKPSETLSLTCAVYGGSISDYYWNWIRQPPGKGLEWIGEINHRGTTNSNPSLKSRVTLSLDTSKNQFSLKLRSVTAADTAVYYCAFGYSDYEYDWFDPWGQGT LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTC PPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 11)

[0144] Amino acid sequence of the light chain L8 of H7L8(hG1TM) EIVLTQSPATLSLSPGERATLSCRASQTISSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPITFGQGTNLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 12)

[0145] 4. Design of humanized antibody H7L8 (hG4DM) The present inventors used the Ig gamma-4 chain C region as the heavy chain constant region based on H7L8(hG1WT), and introduced a point mutation (F234A) from phenylalanine to alanine at position 234 and a point mutation (L235A) from leucine to alanine at position 235 in the heavy chain constant region, while leaving the antibody variable region unchanged, to obtain humanized antibody H7L8(hG4DM) having a mutation in the constant region. The amino acid sequence of the heavy chain of H7L8(hG4DM) is shown in SEQ ID NO:13, and the amino acid sequence of the light chain is shown in SEQ ID NO:12.

[0146] Amino acid sequence of the heavy chain H7(hG4DM) of H7L8(hG4DM): QVQLQQWGAGLLKPSETLSLTCAVYGGSISDYYWNWIRQPPGKGLEWIGEINHRGTTNSNPSLKSRVTLSLDTSKNQFSLKLRSVTAADTAVYYCAFGYSDYEYDWFDPWGQG TLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCP PCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 13)

[0147] The amino acid sequence of the light chain L8 of H7L8 (hG4DM) is identical to the amino acid sequence of the light chain of H7L8 (hG1TM) in SEQ ID NO:12.

[0148] 5. Expression and Purification of Humanized Antibodies H7L8(hG4WT), H7L9(hG4WT), and H7L10(hG4WT) The heavy chain cDNA sequences of H7L8 (hG4WT), H7L9 (hG4WT), and H7L10 (hG4WT) (the coding sequence for the variable region is shown in SEQ ID NO: 1; the constant region was the Ig gamma-4 chain C region of SEQ ID NO: 45), the light chain cDNA sequence of H7L8 (hG4WT) (the coding sequence for the variable region is shown in SEQ ID NO: 3; the constant region was the human Ig kappa chain C region of SEQ ID NO: 40), the light chain cDNA sequence of H7L9 (hG4WT) (the coding sequence for the variable region is shown in SEQ ID NO: 42; the constant region was the human Ig kappa chain C region of SEQ ID NO: 45), The heavy and light chain cDNA sequence of H7L10 (hG4WT) (the coding sequence of the variable region is shown in SEQ ID NO: 44; the constant region was the human Ig kappa chain C region of SEQ ID NO: 40) and the light chain cDNA sequence of H7L10 (hG4WT) (the coding sequence of the variable region is shown in SEQ ID NO: 44; the constant region was the human Ig kappa chain C region of SEQ ID NO: 40) were separately cloned into pUC57simple vector (GenScript) to obtain plasmids pUC57simple-H7, pUC57simple-L8, pUC57simple-L9, and pUC57simple-L10, respectively. Plasmids pUC57simple-H7, pUC57simple-L8, pUC57simple-L9, and pUC57simple-L10 were each digested (HindIII and EcoRI). The heavy and light chains were isolated by electrophoresis and separately subcloned into pcDNA3.1 vector, and the recombinant plasmids were extracted and co-transfected into 293F cells. After 7 days of cell culture, the medium was separated by high-speed centrifugation, and the supernatant was concentrated and loaded onto a HiTrap MabSelect SuRe column. Proteins were eluted in one step with elution buffer. Target samples were isolated and the buffer was exchanged into PBS.

[0149] Amino acid sequence of the heavy chain constant region of H7L8 (hG4WT), H7L9 (hG4WT), or H7L10 (hG4WT) ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 45)

[0150] The amino acid sequence of the light chain constant region of H7L8 (hG4WT), H7L9 (hG4WT), or H7L10 (hG4WT): RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 40)

[0151] Preparation Example 2: Design and preparation of anti-PD-1 antibody 14C12 and its humanized antibody 14C12H1L1 The amino acid sequences and coding nucleotide sequences of the heavy and light chains of the anti-PD-1 antibody 14C12 and its humanized antibody 14C12H1L1 are identical to those of 14C12 and 14C12H1L1, respectively, in Chinese Patent Application Publication No. 106967172(A) (or No. 106977602(A)).

[0152] (1) 14C12 heavy and light chain variable region sequences Nucleotide sequence of the heavy chain variable region of 14C12: (354bp) GAGGTCAAACTGGTGGAGAGCGGCGGCGGGCTGGTGAAGCCCGGCGGGTCACTGAAACTGAGCTGCGCCGCTTCCGGCTTCGCCTTTAGCTCCTACGACATGTCATGGGTGAGGCAGACCCCTGAGAAGCGCCTGGAATGGGTCGCTACTATCAGCGGAGGCGGGCGATACACCTACTATC CTGACTCTGTCAAAGGGAGATTCACAATTAGTCGGGATAACGCCAGAAATACTCTGTATCTGCAGATGTCTAGTCTGCGGTCCGAGGATACAGCTCTGTACTATTGTGCAAACCGGTACGGCGAAGCATGGTTTGCCTATTGGGGACAGGGCACCCTGGTGACAGTCTCTGCC (SEQ ID NO: 14)

[0153] Amino acid sequence of the heavy chain variable region of 14C12: (118aa) EVKLVESGGGLVKPGGSLKLSCAASGFAFSSYDMSWVRQTPEKRLEWVATISGGGRYTYYPDSVKGRFTISRDNARNTLYLQMSSLRSEDTALYYCANRYGEAWFAYWGQGTLVTVSA (SEQ ID NO: 15)

[0154] Nucleotide sequence of the light chain variable region of 14C12: (321bp) GACATTAAGATGACACAGTCCCCTTCCTCAATGTACGCTAGCCTGGGCGAGCGAGTGACCTTCACATGCAAAGCATCCCAGGACATCAACACATACCTGTCTTGGTTTCAGCAGAAGCCAGGCAAAAGCCCCAAGACCCTGATCTACCGGGCCAAGACTGGT GGACGGGGTCCCCAGCAGATTCTCCGGATCTGGCAGTGGGCAGGATTACTCCCTGACCATCAGCTCCCTGGAGTATGAAGACATGGGCATCTACTATTGCCTGCAGTATGATGAGTTCCCTCTGACCTTTGGAGCAGGCACAAAACTGGAACTGAAG (SEQ ID NO: 16)

[0155] Amino acid sequence of the light chain variable region of 14C12: (107aa) DIKMTQSPSSMYASLGERVTFTCKASQDINTYLSWFQQKPGKSPKTLIYRANRLVDGVPSRFSGSGSGQDYSLTISSLEYEDMGIYYCLQYDEFPLTFGAGTKLELK (SEQ ID NO: 17)

[0156] (2) Heavy and light chain variable region sequences, and heavy and light chain sequences of humanized monoclonal antibody 14C12H1L1 Nucleotide sequence of the heavy chain variable region 14C12H1v of 14C12H1L1: (354 bp) GAAGTGCAGCTGGTCGAGTCTGGGGGAGGGCTGGTGCAGCCCGGCGGGTCACTGCGACTGAGCTGCGCAGCTTCCGGATTCGCCTTTAGCTCCTACGACATGTCCTGGGTGCGACAGGCACCAGGAAAGGGACTGGATTGGGTCGCTACTATCTCAGGAGGCGGGAGATACACCTACTATC CTGACAGCGTCAAGGGCCGGTTCACAATCTCTAGAGATAACAGTAAGAACAATCTGTATCTGCAGATGAACAGCCTGAGGGCTGAGGACACCGCACTGTACTATTGTGCCAACCGCTACGGGGAAGCATGGTTTGCCTATTGGGGGCAGGGAACCCTGGTGACAGTCTCTAGT (SEQ ID NO: 18)

[0157] Amino acid sequence of the heavy chain variable region 14C12H1v of 14C12H1L1: (118aa) EVQLVESGGGLVQPGGSLRLSCAASGFAFSSYDMSWVRQAPGKGLDWVATISGGGRYTYYPDSVKGRFTISRDNSKNNLYLQMNSLRAEDTALYYCANRYGEAWFAYWGQGTLVTVSS (SEQ ID NO: 19)

[0158] Nucleotide sequence of the light chain variable region 14C12L1v of 14C12H1L1: (321 bp) GACATTCAGATGACTCAGAGCCCCTCCTCCATGTCCGCCTCTGTGGGCGACAGGGTCACCTTCACATGCCGCGCTAGTCAGGATATCAACACCTACCTGAGCTGGTTTCAGCAGAAGCCAGGGAAAAGCCCCAAGACACTGATCTACCGGGCTAATAGACTGGT GTCTGGAGTCCCAAGTCGGTTCAGTGGCTCAGGGAGCGGACAGGACTACACTCTGACCATCAGCTCCCTGCAGCCTGAGGACATGGCAACCTACTATTGCCTGCAGTATGATGAGTTCCCACTGACCTTTGGCGCCGGGACAAAACTGGAGCTGAAG (SEQ ID NO: 20)

[0159] Amino acid sequence of the light chain variable region 14C12L1v of 14C12H1L1: (107aa) DIQMTQSPSSMSASVGDRVTFTCRASQDINTYLSWFQQKPGKSPKTLIYRANRLVSGVPSRFSGSGSGQDYTLTISSLQPEDMATYYCLQYDEFPLTFGAGTKLELK (SEQ ID NO: 21)

[0160] Nucleotide sequence of heavy chain 14C12H1 of 14C12H1L1: (1344 bp)

[0161] Heavy chain of 14C12H1L1 Amino acid sequence of 14C12H1: (448aa) EVQLVESGGGLVQPGGSLRLSCAASGFAFSSYDMSWVRQAPGKGLDWVATISGGGRYTYYPDSVKGRFTISRDNSKNNLYLQMNSLRAEDTALYYCANRYGEAWFAYWGQGTLV TVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCP PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 23)

[0162] Nucleotide sequence of the light chain of 14C12H1L1: (642 bp) GACATTCAGATGACTCAGAGCCCCTCCTCCATGTCCGCCTCTGTGGGCGACAGGGTCACCTTCACATGCCGCGCTAGTCAGGATATCAACACCTACCTGAGCTGGTTTCAGCAGAAGCCAGGGAAAAGCCCCAAGACACTGATCTACCGGGCTAATAGACTGGTGTCTGGAGTCCCAAGTCGGTTCAGTGGCTCAGGGAGCGGACAGGACTACACTCTGACCATCAGCTCCCTGCAGCCTGAGGACATGGCAACCTACTATTGCCTGCAGTATGATGAGTTCCCACTGACCTTTGGCGCCGGGACAAAACTGGAGCTGAAGCGAACTGTGGCCGCTCCCTCCGTCTTCATTTTTCCCCCTTCTGACGAACAGCTGAAATCAGGCACAGCCAGCGTGGTCTGTCTGCTGAACAATTTCTACCCTAGAGAGGCAAAAGTGCAGTGGAAGGTCGATAACGCCCTGCAGTCCGGCAACAGCCAGGAGAGTGTGACTGAACAGGACTCAAAAGATAGCACCTATTCCCTGTCTAGTACACTGACTCTGTCCAAGGCTGATTACGAGAAGCACAAAGTGTATGCATGCGAAGTGACACATCAGGGACTGTCAAGCCCCGTGACTAAGTCTTTTAACCGGGGCGAATGT (SEQ ID NO: 24)

[0163] Amino acid sequence of the light chain 14C12L1 of 14C12H1L1: (214 aa) DIQMTQSPSSMSASVGDRVTFTCRASQDINTYLSWFQQKPGKSPKTLIYRANRLVSGVPSRFSGSGSGQDYTLTISSLQPEDMATYYCLQYDEFPLTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 25)

[0164] The CDRs of antibodies 14C12 and 14C12H1L1 are identical to the following (according to the IMGT numbering system): HCDR1: GFAFSSYD (SEQ ID NO: 26) HCDR2: ISGGGRYT (SEQ ID NO: 27) HCDR3: ANRYGEAWFAY (SEQ ID NO: 28) LCDR1: QDINTY (sequence number 29) LCDR2: RAN (SEQ ID NO:30) LCDR3: LQYDEFPLT (SEQ ID NO:31)

[0165] Heavy and light chain variable region sequences of 14C12H1L1(M) 14C12H1L1(M) was obtained by mutating specific amino acids in the framework region (light chain) of 14C12H1L1.

[0166] The heavy chain variable region 14C12H1(M) of 14C12H1L1(M) is identical to the heavy chain variable region 14C12H1 of 14C12H1L1, i.e., both have the amino acid sequence of SEQ ID NO:19.

[0167] Light chain variable region of 14C12H1L1(M): DIQMTQSPSSMSASVGDRVTFTCRASQDINTYLSWFQQKPGKSPKTLIYRANRLVSGVPSRFSGSGSGQDYTLTISSLQPEDMATYYCLQYDEFPLTFGAGTKLELKR (SEQ ID NO: 38)

[0168] Preparation Example 3: Design of humanized antibody 14C12H1L1 (hG4DM) The present inventors used the Ig gamma-4 chain C region as the heavy chain constant region based on 14C12H1L1, introduced a point mutation (F234A) from phenylalanine to alanine at position 234 and a point mutation (L235A) from leucine to alanine at position 235 in the heavy chain constant region, and obtained a humanized antibody 14C12H1L1(hG4DM) having a mutation in the constant region, while leaving the antibody variable region unchanged. The amino acid sequence of the heavy chain 14C12H1(hG4DM) of 14C12H1L1(hG4DM) is shown in SEQ ID NO:32, and the amino acid sequence of the light chain is shown in SEQ ID NO:25.

[0169] Amino acid sequence of the heavy chain of 14C12H1L1 (hG4DM) EVQLVESGGGLVQPGGSLRLSCAASGFAFSSYDMSWVRQAPGKGLDWVATISGGGRYTYYPDSVKGRFTISRDNSKNNLYLQMNSLRAEDTALYYCANRYGEAWFAYWGQGTL VTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPP CPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 32)

[0170] The amino acid sequence of the light chain of 14C12H1L1 (hG4DM) is identical to the amino acid sequence of the light chain 14C12L1 of 14C12H1L1 of SEQ ID NO:25.

[0171] Preparation Example 4: Sequence design of humanized antibody 14C12H1L1 (hG1TM) The inventors of the present application obtained a humanized variant 14C12H1L1 (hG1TM) by introducing a point mutation from leucine to alanine at position 234 (L234A), a point mutation from leucine to alanine at position 235 (L235A), and a point mutation from glycine to alanine at position 237 (G237A) in accordance with the EU numbering system in the hinge region of the heavy chain based on the humanized antibody 14C12H1L1.

[0172] Heavy chain of 14C12H1L1(hG1TM) Nucleotide sequence of 14C12H1(hG1TM): (1344bp)

[0173] Heavy chain of 14C12H1L1(hG1TM) Amino acid sequence of 14C12H1(hG1TM): (448aa) EVQLVESGGGLVQPGGSLRLSCAASGFAFSSYDMSWVRQAPGKGLDWVATISGGGRYTYYPDSVKGRFTISRDNSKNNLYLQMNSLRAEDTALYYCANRYGEAWFAYWGQGTLV TVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCP PCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 34)

[0174] The nucleotide sequence of the light chain of 14C12H1L1 (hG1TM) is shown in SEQ ID NO:24.

[0175] The amino acid sequence of the light chain of 14C12H1L1 (hG1TM) is identical to the amino acid sequence of the light chain 14C12L1 of 14C12H1L1 of SEQ ID NO:25.

[0176] Preparation Example 5: Design and preparation of anti-LAG3 / PD-1 bifunctional antibody 1. Sequence Design The structures of the bifunctional antibodies BS-PL021A, Bs-PL022B, BS-PL023C, and Bs-PLV02 of the present invention are in the Morrison form (IgG-scFv), i.e., the C-terminus of each of the two heavy chains of an IgG antibody is linked to the scFv fragment of another antibody. The main compositional design of the heavy and light chains is shown in Table 1 below. [Table 1]

[0177] In Table 1 above: (1) Amino acid sequence of linker fragment (GGGGS)3: GGGGSGGGGSGGGGS (SEQ ID NO: 35) Amino acid sequence of the linker fragment (GGGGS)4: GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 36) Amino acid sequence of the linker fragment (GGGGS)4G: GGGGSGGGGSGGGGSGGGGSG (SEQ ID NO: 37) (2) The "v" in the lower right corner represents the variable region of the corresponding heavy chain or the variable region of the corresponding light chain. For those without the "v" designation, the corresponding heavy or light chain is full-length, including the constant region. The corresponding sequences described in the above preparation examples represent the amino acid sequences of these variable regions or full-length sequences, and the nucleotide sequences encoding them.

[0178] 2. Antibody Expression and Purification The heavy and light chain cDNA sequences of Bs-PL021A, Bs-PL022B, Bs-PL023C, and Bs-PLV02 were separately cloned into pUC57simple vector (GenScript) to obtain plasmids pUC57simple-Bs-PL021AH / pUC57simple-Bs-PL021AL, pUC57simple-Bs-PL022BH / pUC57simple-Bs-PL022BL, pUC57simple-Bs-PL023CH / pUC57simple-Bs-PL023CL, pUC57simple-Bs-PLV02H / pUC57simple-Bs-PLV02L, and pUC57simple-Bi-PGV02 / pUC57simple-Bi-PGV02, respectively.

[0179] The plasmids pUC57simple-Bs-PL021AH / pUC57simple-Bs-PL021AL, pUC57simple-Bs-PL022BH / pUC57simple-Bs-PL022BL, pUC57simple-Bs-PL023CH / pUC57simple-Bs-PL023CL, pUC57simple-Bs-PLV02H / pUC57simple-Bs-PLV02L, and pUC57simple-Bi-PGV02 / pUC57simple-Bi-PGV02 were digested with HindIII and EcoRI, respectively. The heavy and light chains were isolated by electrophoresis and subcloned separately into pcDNA3.1 vector, and the recombinant plasmids were extracted and co-transfected into 293F cells. After 7 days of cell culture, the medium was separated by high-speed centrifugation, and the supernatant was concentrated and loaded onto a HiTrap MabSelect SuRe column. Proteins were eluted in one step with elution buffer. Target samples were isolated and the buffer was exchanged into PBS.

[0180] Preparation Example 6: Preparation of fusion proteins PD-1-mFc, PD-1-hFc, and PDL1-hFc The preparation of fusion proteins PD-1-mFc, PD-1-hFc, and PDL1-hFc, and detection by SDS-PAGE electrophoresis were performed by referring to Preparation Example 1 of China Patent Publication No. 106632674(A) in its entirety.

[0181] The amino acid sequences and coding nucleotide sequences of the fusion proteins PD-1-mFc, PD-1-hFc, and PDL1-hFc in this preparation example are identical to those of PD-1-mFc, PD-1-hFc, and PDL1-hFc, respectively, in Preparation Example 1 of China Patent Publication No. 106632674(A).

[0182] The fusion proteins PD-1-mFc, PD-1-hFc, and PDL1-hFc were thus obtained.

[0183] Preparation Example 7: Preparation of human anti-egg lysozyme antibody The sequence of the human anti-hen egg lysozyme IgG (anti-HEL, or human IgG; abbreviated as hIgG) antibody was obtained from the variable region sequence of the Fab F10.6.6 sequence in the study reported by Acierno et al. entitled "Affinity maturation increases the stability and plasticity of the Fv domain of anti-protein antibodies" (Acierno et al., J Mol Biol., 2007; 374(1): 130-46). The preparation method was as follows:

[0184] Nanjing Genscript Biology was commissioned to carry out amino acid codon optimization and gene synthesis of the heavy and light chain (full sequence or variable region) genes of human IgG antibody. By referring to the standard techniques introduced in "Guide to Molecular Cloning Experiments (Third Edition)" and by using standard molecular cloning techniques, such as PCR, enzyme digestion, DNA gel extraction, ligation transformation, colony PCR, or enzyme digestion identification, the heavy and light chain genes were subcloned into antibody heavy chain expression vectors and antibody light chain expression vectors of a mammalian expression system, respectively. The heavy and light chain genes of the recombinant expression vectors were further sequenced and analyzed. After confirming that the sequences were accurate, large or medium amounts of endotoxin-free expression plasmids were prepared. To express the recombinant antibody, the heavy and light chain expression plasmids were transiently transfected into HEK293 cells. After 7 days of culture, the cell culture medium was collected and affinity purified using rProtein A column (GE). The quality of the obtained antibody samples was determined using standard analytical techniques of SDS-PAGE and SEC-HPLC.

[0185] Experimental Example 1: ELISA analysis of the binding activity of anti-LAG3 / anti-PD-1 bispecific antibodies to antigens 1. Analysis of the binding activity of BS-PL021A, BS-PL022B, BS-PL023C, Bs-PLV02, and 14C12H1L1 (hG1TM) to the antigen PD-1-mFc by indirect ELISA The procedure was as follows:

[0186] ELISA plates were coated with 0.5μg / mL human PD-1-mFc and incubated overnight at 4℃. The antigen-coated ELISA plates were then washed once with PBST and blocked for 2 hours at 37℃ using PBS solution containing 1% BSA as blocking solution. After blocking, the ELISA plates were washed three times with PBST. Antibodies serially diluted in PBST solution (antibody dilution gradient is shown in Table 2) were added. The ELISA plates containing the test antibodies were incubated at 37℃ for 30 minutes and then washed three times with PBST. After washing, a working solution of HRP-labeled goat anti-human IgG FC(H+L) (Jackson, Cat. No. 109-035-098) secondary antibody diluted at a ratio of 1:5000 was added, and the plates were then incubated at 37℃ for 30 minutes. After incubation, the plate was washed 4 times with PBST, and TMB (Neogen, 308177) was added in the dark for 5 min to develop the color, and then the stop solution was added to stop the color reaction. The ELISA plate was immediately placed in an ELISA plate reader, and the OD value of each well in the ELISA plate was read at 450 nm. Data were analyzed and processed using SoftMax Pro 6.2.1.

[0187] The analytical results are shown in Table 2 and Figure 1. [Table 2]

[0188] As can be seen from Figure 1, BS-PL021A, BS-PL022B, BS-PL023C, Bs-PLV02, and 14C12H1L1 (hG1TM) could effectively bind to the antigen human PD-1-mFc in a dose-dependent manner. Based on the quantitative analysis of the absorbance of the bound antibodies, the binding efficiency EC of antibodies BS-PL021A, BS-PL022B, BS-PL023C, Bs-PLV02, and 14C12H1L1 (hG1TM) (control) was obtained by curve fitting calculation. 50 The values ​​were 0.066 nM, 0.074 nM, 0.046 nM, 0.103 nM, and 0.02 nM, respectively.

[0189] The results showed that under the same experimental conditions, the binding activities of BS-PL021A, BS-PL022B, and BS-PL023C to PD-1-mFc were substantially comparable to that of the positive control 14C12H1L1 (hG1TM) to the same target, suggesting that BS-PL021A, BS-PL022B, BS-PL023C, and Bs-PLV02 had effective binding activity to PD-1-mFc.

[0190] 2. Analysis of the binding activity of BS-PL021A, BS-PL022B, BS-PL023C, Bs-PLV02, leratolimab, and H7L8 (hG1WT) to the antigen LAG3-mFc by indirect ELISA The procedure was as follows:

[0191] ELISA plates were coated with 2 μg / mL human LAG3-mFc (Akeso Biopharma, Lot No. 20200417) and incubated overnight at 4°C. The antigen-coated ELISA plates were then washed once with PBST and blocked for 2 hours at 37°C using PBS solution containing 1% BSA as blocking solution. After blocking, the ELISA plates were washed three times with PBST. Antibodies serially diluted in PBST solution (antibody dilution gradient is shown in Table 3) were added. The ELISA plates containing the test antibodies were incubated for 30 minutes at 37°C and then washed three times with PBST. After washing, a working solution of HRP-labeled goat anti-human IgG FC(H+L) (Jackson, Cat. No. 109-035-098) secondary antibody diluted at a ratio of 1:5000 was added, and the plates were then incubated for 30 minutes at 37°C. After incubation, the plate was washed 4 times with PBST, and TMB (Neogen, 308177) was added in the dark for 5 min to develop the color, and then the stop solution was added to stop the color reaction. The ELISA plate was immediately placed in an ELISA plate reader, and the OD value of each well in the ELISA plate was read at 450 nm. Data were analyzed and processed using SoftMax Pro 6.2.1.

[0192] The analysis results are shown in Table 3 and Figure 2. [Table 3]

[0193] As can be seen from FIG. 2, BS-PL021A, BS-PL022B, BS-PL023C, Bs-PLV02, leratolimab, and H7L8(hG1WT) could effectively bind to the antigen human LAG3-mFc in a dose-dependent manner. The absorbance for each dose is shown in Table 3. Based on the quantitative analysis of the absorbance of the bound antibodies, the binding efficiency EC of antibodies BS-PL021A, BS-PL022B, BS-PL023C, Bs-PLV02, leratolimab (positive control), and H7L8(hG1WT) (control) obtained by curve fitting calculations. 50The values ​​were 0.073 nM, 0.081 nM, 0.377 nM, 0.685 nM, 0.106 nM, and 0.045 nM, respectively.

[0194] The above experimental results show that under the same experimental conditions, BS-PL021A, BS-PL022B, and H7L8(hG1WT) had effective binding activity to LAG3-mFc, and that the binding activity of BS-PL021A, BS-PL022B, and H7L8(hG1WT) to human LAG3-mFc was stronger than the activity of the positive drug relatolimab against the same target, in particular, the binding activity of H7L8(hG1WT) to human LAG3-mFc was significantly stronger than the activity of the positive drug relatolimab against the same target.

[0195] Experimental Example 2: Analysis of the activity of anti-LAG3 / anti-PD-1 bispecific antibodies competing with human PDL1-mFc for binding to human PD-1-mFc-biotin by competitive ELISA An ELISA plate was coated with 2μg / mL human PDL1-mFc (PD-L1 Genbank ID: NP_054862.1, mFc SEQ ID NO:) and incubated overnight at 4℃. After incubation, the ELISA plate was blocked with PBS solution containing 1% BSA at 37℃ for 2 hours. After blocking, the plate was washed three times and dried. The antibody was serially diluted on a dilution plate at a 3-fold dilution rate to give seven concentrations starting from 80nM (final concentration 40nM). A blank control was also set up. Then, an equal amount of human PD-1-mFc-biotin solution at 1.2μg / mL (final concentration 0.6μg / mL) was added and mixed well with the diluted antibody. The mixture was then incubated at room temperature for 10 minutes. The reaction mixture was then added to the coated ELISA plate, and the ELISA plate was incubated at 37℃ for 30 minutes. After incubation, the plate was washed three times with PBST and dried. SA-HRP (KPL, 14-30-00) working solution was added and the plate was incubated at 37°C for 30 minutes. After incubation, the plate was washed four times and dried. Then, TMB (Neogen, 308177) was added in the dark to develop color for 5 minutes, and the color reaction was stopped by adding stop solution. The ELISA plate was immediately placed in an ELISA plate reader, and the OD value of each well in the ELISA plate was read at 450 nm. Data analysis and processing were performed with SoftMax Pro 6.2.1.

[0196] The analytical results are shown in Figure 3. The OD values ​​for all doses are shown in Table 4. Based on the quantitative analysis of the absorbance of the bound antibodies, curve fitting was performed to determine the competitive binding efficiency EC 50 The values ​​were obtained (Table 4). [Table 4]

[0197] The results show that BS-PL021A, BS-PL022B, BS-PL023C, Bs-PLV02, and 14C12H1L1(hG1TM) (control) could effectively block the binding of the antigen human PD-1-mFc-biotin to its ligand human PDL1-mFc in a dose-dependent manner, and the EC values ​​of BS-PL021A, BS-PL022B, BS-PL023C, Bs-PLV02, and 14C12H1L1(hG1TM) for blocking the binding of human PD-1-mFc-biotin to its ligand human PDL1-mFc were 50 The values ​​were 3.031 nM, 3.462 nM, 2.982 nM, 5.045 nM, and 2.606 nM, respectively. The efficacies of BS-PL021A, BS-PL022B, and BS-PL023C to block the binding of human PD-1-mFc-biotin to its ligand, human PDL1-mFc, were substantially comparable to that of 14C12H1L1 (hG1TM).

[0198] Experimental Example 3: FACS analysis of binding activity of anti-LAG3 / anti-PD-1 bispecific antibodies 1. Analysis of the binding activity of anti-LAG3 / anti-PD-1 bispecific antibodies to PD-1 on the 293T-PD1 membrane surface by FACS Harvest 293T-PD1 cells in logarithmic growth phase and plate 3 x 10 cells in a V-bottom 96-well plate. 5Cells / well were transferred. Then, 100 μL of 1% PBSA was added to each well, the mixture was centrifuged at 350×g for 5 minutes, and then the supernatant was removed. 100 μL of antibodies diluted in PBSA (final 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, respectively) were added. The mixture was gently mixed homogeneously and incubated on ice for 1 hour. 100 μL of 1% PBSA was added to each well, the mixture was centrifuged at 350×g for 5 minutes, and then the supernatant was removed. The plate was then washed twice with 200 μL of 1% PBSA. FITC-labeled goat anti-human IgG secondary antibody (Jackson, Cat. No. 109-095-098) diluted 400 times was added and resuspended. The mixture was mixed well and incubated on ice in the dark for 0.5 hours. 100 μL of 1% PBSA was added to each well, the mixture was centrifuged at 350×g for 5 minutes, and then the supernatant was removed. The plate was then washed twice with 200 μL of 1% PBSA. 400 μL of 1% PBSA was added to each well to resuspend the cell pellet, and the mixture was transferred to a flow cytometry tube for FACSCalibur assay.

[0199] The experimental results are shown in Table 5 and Figure 4. It was shown that 14C12H1L1 (hG1TM), BS-PL021A, BS-PL022B, Bs-PLV02, Bi-PGV02, nivolumab, and pembrolizumab could all specifically bind to the PD-1 receptor on the membrane surface of 293T-PD1 cells. [Table 5]

[0200] EC values ​​of binding of 14C12H1L1 (hG1TM), BS-PL021A, BS-PL022B, Bs-PLV02, Bi-PGV02, nivolumab, and pembrolizumab to 293T-PD1 cells under identical experimental conditions. 50The values ​​were 5.351 nM, 6.851 nM, 6.066 nM, 6.866 nM, 7.206 nM, 3.073 nM, and 3.970 nM, respectively. The above experimental results show that under the same experimental conditions, the binding activity of 14C12H1L1 (hG1TM), BS-PL021A, BS-PL022B, Bs-PLV02, and Bi-PGV02 to 293T-PD1 cells was comparable to that of the control antibodies nivolumab and pembrolizumab, suggesting that 14C12H1L1 (hG1TM), BS-PL021A, BS-PL022B, Bs-PLV02, and Bi-PGV02 have the activity of effectively binding to PD-1 on the membrane surface of 293T-PD1 cells.

[0201] 2. Analysis of binding activity of anti-LAG3 / anti-PD-1 bispecific antibodies to LAG3 on the membrane surface of 293T-LAG3 cells by FACS Logarithmic-phase 293T-LAG3 cells were digested with conventional pancreatin and plated in a V-bottom 96-well plate at 3 × 10 5Cells / well were transferred. Then, 100 μL of 1% PBSA was added to each well, the mixture was centrifuged at 350×g for 5 minutes, and then the supernatant was removed. 100 μL of antibody diluted in 1% PBSA (final concentrations of 300 nM, 100 nM, 33.3 nM, 11.1 nM, 3.7 nM, 1.23 nM, 0.123 nM, 0.0123 nM, and 0.00123 nM, respectively) was added. The mixture was mixed well and incubated on ice for 1 hour. 100 μL of 1% PBSA was added to each well, the mixture was centrifuged at 350×g for 5 minutes, and then the supernatant was removed. The plate was then washed twice with 200 μL of 1% PBSA. FITC-labeled goat anti-human IgG secondary antibody (Jackson, catalog number 109-095-098) diluted 300 times was added and resuspended. The mixture was mixed well and incubated on ice in the dark for 0.5 hours. 500 μL of PBSA was added to each well, the mixture was centrifuged at 350×g for 5 minutes, and then the supernatant was removed. The plate was then washed twice with 200 μL of 1% PBSA. 300 μL of 1% PBSA was added to each well to resuspend the cell pellet, and the mixture was transferred to a flow cytometry tube for FACSCalibur assay.

[0202] The experimental results are shown in Table 6 and FIG. [Table 6]

[0203] The results show that under identical experimental conditions, the EC values ​​of BS-PL022B and leratolimab for binding to LAG3 on the membrane surface of 293T-LAG3 cells were significantly higher than those of the control. 50 The values ​​were 3.213 nM and 4.113 nM, respectively, suggesting that the binding activity of BS-PL022B to LAG3 on the membrane surface of 293T-LAG3 cells was higher than that of leratolimab.

[0204] The above experimental results showed that BS-PL022B and the positive drug relatolimab, which targets the same target, were able to specifically and dose-dependently bind to LAG3 on the membrane surface of 293T-LAG3 cells, suggesting that BS-PL022B had the activity to effectively bind to LAG3 on the membrane surface of 293T-LAG3 cells and that its binding ability was stronger than that of relatolimab.

[0205] Experimental Example 4: Competitive binding of anti-LAG3 / anti-PD-1 bispecific antibodies to antigens on cell membrane surfaces 1. Analysis by competitive flow cytometry of the activity of anti-LAG3 / anti-PD-1 bispecific antibodies competing with PDL1 for binding to the antigen PD-1 on the cell membrane surface 293T-PD1 cells were digested using conventional methods and plated in a V-bottom 96-well plate at 3 × 10 5 Cells / well were transferred. Then, 100 μL of 1% PBSA was added to each well, and the mixture was centrifuged and washed. Corresponding serially diluted antibodies (final 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, respectively) were added at 100 μL per sample, and the mixture was incubated on ice for 30 min. 100 μL of PDL-1-mFc was added to each well, and the mixture was mixed well to a final concentration of 20 nM. Then, the wells were incubated on ice for 1 h and centrifuged at 350 × g for 5 min, after which the supernatant was removed and washed twice with 200 μL of 1% PBSA. 100 μL of 1:400 diluted FITC goat anti-mouse IgG / IgM antibody (BD Cat. No. 555988) was added while 100 μL of 1% PBSA was added to the blank sample. The mixture was mixed well and incubated on ice in the dark for 30 minutes, washed and centrifuged. The cells were resuspended and transferred to a sample loading tube for testing on the flow cytometer.

[0206] The results are shown in FIG. 6. 50 The values ​​are shown in Table 7. Fluorometric analysis and curve fitting revealed competitive binding EC values ​​for antibodies nivolumab, pembrolizumab, 14C12H1L1 (hG1TM), and BS-PL022B.50 The values ​​were calculated to be 3.608 nM, 2.769 nM, 2.511 nM, and 5.123 nM, respectively. [Table 7]

[0207] The results show that antibody BS-PL022B could effectively block the binding of PD-L1 to PD-1 on the surface of 293T-PD1 host cells in a dose-dependent manner.

[0208] 2. Analysis of the activity of anti-LAG3 / anti-PD-1 bispecific antibodies competing with LAG3-mG1Fc for binding to antigen MHC II on the cell membrane surface by competitive flow cytometry According to the experimental design, each antibody and LAG3-mG1Fc were diluted and mixed homogeneously at a 1:1 ratio to give a final concentration of 3 nM for LAG3-mG1Fc (Akeso Biopharma; Lot No. 20190508) and final antibody concentrations of 300 nM, 100 nM, 33.3 nM, 11.1 nM, 3.7 nM, 1.23 nM, 0.123 nM, 0.0123 nM, and 0.00123 nM. The mixture was then incubated on ice for 30 minutes. Raji cells (Cell Resource Center, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences; Catalog No. TCHu 44) were harvested and seeded into a V-bottom 96-well plate at 300,000 cells per sample, and 1% PBSA was added. The mixture was centrifuged at 500×g for 5 minutes, and the supernatant was removed. The cells in each well were resuspended in 100 μL of antibody-protein preincubation solution. A blank control (cells + PBSA + PBSA), a negative control (cells + PBSA + secondary antibody), and an isotype control were designed. The system was incubated on ice in the dark for 1 hour. After that, 100 μL of 1% PBSA was added, the mixture was centrifuged at 500×g for 5 minutes, and then the supernatant was removed. The cells were resuspended by adding 200 μL of 1% PBSA to each well, the suspension was centrifuged at 500×g for 5 minutes, and then the supernatant was removed to wash once. The cells were resuspended by adding 100 μL of APC goat anti-mouse IgG secondary antibody (BioLegend, Cat. No. 405308) (diluted at a ratio of 1:300) to each well, while the blank control was resuspended in 100 μL of 1% PBSA. The system was incubated on ice in the dark for 30 minutes. Then, 100 μL of 1% PBSA was added, the mixture was centrifuged at 500×g for 5 minutes, and the supernatant was then removed. The cells were washed once by adding 200 μL of 1% PBSA to each well to resuspend the cells, centrifuging the suspension at 500×g for 5 minutes, and then removing the supernatant. 200 μL of 1% PBSA was added to each well to resuspend the cells, and the suspension was transferred to a sample loading tube for testing on the flow cytometer.

[0209] The results are shown in FIG. 7 and Table 8. The EC 50 The values ​​are shown in the table. Fluorometric analysis and curve fitting determined the competitive binding EC 50 The values ​​were calculated to be 0.9689 nM and 1.306 nM, respectively. [Table 8]

[0210] The results show that antibody BS-PL022B was able to effectively block, in a dose-dependent manner, the binding of LAG-3 to MHC II on the surface of Raji host cells.

[0211] Experimental Example 5: Blocking analysis of anti-LAG3 / anti-PD-1 bispecific antibodies 1. Analysis of anti-LAG3 / anti-PD-1 bispecific antibodies that block LAG3 binding to MHC II Jurkat-NFAT-PD1-LAG3 cells (constructed by Akeso Biopharma, P9, viability: 97.75%) and Raji cells (Cell Resource Center, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences; Catalog No. TCHu 44) were harvested and centrifuged at 110×g for 5 min, and the supernatant was then removed. The cells were then resuspended in 1640 medium (containing 10% FBS) and counted. The Jurkat-NFAT-PD1-LAG3 cells were plated in a black-bottom 96-well plate (Corning; Model No. 3916) at 10×10 4Cells / well (25 μL / well) were seeded. According to the experimental design, antibodies (final concentrations of 900 nM, 300 nM, 100 nM, 33.3 nM, 3.3 nM, 0.3 nM, 0.03 nM, and 0.003 nM, respectively) were added at 30 μL / well, and the mixture was pre-incubated for 30 min at 37° C. in a 5% CO2 incubator. SEE (Staphylococcal Enterotoxin E) (final concentration 0.05 ng / mL, Toxin Technology, Cat. No. ET404) and Raji cells were incubated for 30 min at 37° C. in a 5% CO2 incubator. After 30 min of incubation, Raji cells were added at 2×10 4 Cells / well (25 μL / well) were added to a final volume of 80 μL in each well. The mixture was mixed well and incubated at 37°C in a 5% CO2 incubator for 16 hours. The plates were then removed and allowed to warm to room temperature. 80 μL / well of Bright-Glo™ Luciferase Assay System (Promega, Cat# E2650) was added and the mixture was incubated in the dark for 2 minutes. RLU values ​​were then read.

[0212] The results are shown in Figures 8A and 8B and in Table 9. [Table 9]

[0213] Results show that the EC values ​​of Bs-PLV02, Bi-PGV02, BS-PL022B, and leratolimab, which block LAG3 binding to MHC II, are 50 The values ​​(nM) were 1.21 nM, 1.483 nM, 0.9762 nM, and 8.563 nM, respectively. Bs-PLV02, Bi-PGV02, and BS-PL022B were more potent at blocking LAG3 binding to MHC II than the positive control antibody relatolimab.

[0214] 2. Analysis of anti-LAG3 / anti-PD-1 bispecific antibodies that block PD-1 binding to PD-L1 PDL1 aAPC / CHO-K1 cells (Promega, Cat. No. J1081A) were plated at 4 × 10 4 Cells were seeded per well (100 μL per well) and cultured overnight (in cell growth medium: Ham F-12 + 10% FBS). The next day, the medium in the plate was removed, and PD1 effector cells (Promega, Cat. No. J1121A) were added at 5 × 10 4 Cells / well (40 μL / well) (medium: 1640+10% FBS) were added. Antibodies (final concentrations of 1000 nM, 300 nM, 100 nM, 33.3 nM, 11.1 nM, 3.7 nM, 1.23 nM, 0.123 nM, and 0.0123 nM, respectively) were added at 40 μL / well, and isotype and negative controls were also set up. The final volume was 80 μL / well. The plate was placed in an incubator and incubated for 6 hours. Afterwards, the plate was removed and allowed to return to room temperature. Bright-Glo™ Luciferase Assay System (Promega, Cat. No. E2650) was added at 80 μL / well, and the mixture was incubated in the dark for 2 minutes. The RLU values ​​were then read.

[0215] The results are shown in Figures 9A and 9B and in Table 10. [Table 10]

[0216] Results showed that EC of nivolumab, pembrolizumab, 14C12H1L1 (hG1TM), and BS-PL022B, which block the binding of PD-1 to PD-L1, was significantly improved. 50 The values ​​(nM) were 4.089 nM, 1.281 nM, 5.219 nM, and 20.01 nM, respectively. The results show that nivolumab, pembrolizumab, 14C12H1L1 (hG1TM), and BS-PL022B were all able to block the binding of PD-1 to PD-L1.

[0217] 3. Analysis of anti-LAG3 / anti-PD-1 bispecific antibodies that simultaneously block LAG-3 binding to MHC II and PD-1 binding to PD-L1 Jurkat-NFAT-PD1-LAG3 and Raji-PDL1 cells were harvested and centrifuged at 110×g for 5 min, after which the supernatant was removed, and the cells were then resuspended in 1640 medium (containing 10% FBS) and counted.

[0218] The Jurkat-NFAT-PD1-LAG3 cells were cultured in a black-bottom 96-well plate (Corning; model number 3916) at 10 × 10 4 Cells / well (25 μL / well) were seeded. According to the experimental design, antibodies (final concentrations of 3000 nM, 1000 nM, 300 nM, 30 nM, 3 nM, 0.3 nM, 0.03 nM, and 0.003 nM, respectively) were added at 30 μL / well, and the mixture was preincubated for 30 min at 37° C. in a 5% CO2 incubator. SEE (Staphylococcal Enterotoxin E) (final concentration 0.1 ng / mL) was added to Raji-PDL1 cells, and the mixture was incubated for 30 min at 37° C. in a 5% CO2 incubator. After 30 min of incubation, Raji-PDL1 cells were added at 3×10 4 Cells / well (25 μL / well) were added to a final volume of 80 μL in each well. The mixture was mixed well and incubated at 37°C in a 5% CO2 incubator for 15 hours. The plates were then removed and allowed to warm to room temperature. Bright-Glo™ Luciferase Assay System (Promega, Cat# E2650) was added at 80 μL / well and the mixture was incubated in the dark for 5 minutes. RLU values ​​were then read.

[0219] The results are shown in Figures 10A and 10B and in Table 11. [Table 11]

[0220] Results showed that the EC of pembrolizumab, lelatolimab, 14C12H1L1(hG1TM), 14C12H1L1(hG1TM) + lelatolimab, and BS-PL022B, which simultaneously block PD1 binding to PD-L1 and LAG3 binding to MHC II, was significantly increased in patients with PD-L1-associated lelatolimab-resistant ... 50 The values ​​(nM) were 24.01 nM, 5.525 nM, 44.86 nM, 29.75 nM, and 16.21 nM, respectively. Pembrolizumab, relatolimab, 14C12H1L1(hG1TM), 14C12H1L1(hG1TM)+relatolimab, and Bs-PL022B could simultaneously block the binding of PD-1 to PD-L1 and the binding of LAG3 to MHC II, and the blocking ability of Bs-PL022B was higher than that of the other antibodies.

[0221] Experimental Example 6: Bridging assay of anti-LAG3 / anti-PD-1 bispecific antibodies CHO-K1 cells (Cell Resource Center, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, Cat. No. 3111C0001CCC000004), CHO-K1-PD1 cells (constructed by Akeso Biopharma), and CHO-K1-LAG3 cells (constructed by Akeso Biopharma) were conventionally digested and centrifuged at 170 × g for 5 min, after which the supernatant was removed. Cells were then resuspended in complete medium and counted to determine cell viability. CHO-K1-PD1 cells were incubated with CFSE (CFSE Cell Division Tracker Kit, Biolegend, Cat. No. 423801) (treatment concentration 1 μM, 1 mL / 10 × 10 6 CHO-K1-LAG3 cells were stained with Far red (Thermofisher, Cat. No. C34564) (treatment concentration 0.3 μM, 1 mL / 10 × 10 6Cells were stained with 1.5×10 GFP, and CHO-K1 cells were stained with Far red or CFSE. Cells were incubated in an incubator for 20 min for staining. Staining was stopped by adding complete medium, the mixture was centrifuged at 170×g for 5 min, and the supernatant was then removed. Additional complete medium was added, the mixture was incubated in an incubator for 10 min, centrifuged at 170×g for 5 min, the supernatant was then removed, and the cells were washed once. Cells were then resuspended in complete medium and counted. After staining, CHO-K1-PD1, CHO-K1-LAG3, and CHO-K1 cells were separately plated at 1.5×10 GFP in a V-bottom 96-well plate. 5 Cells / well were transferred and buffer (PBS+1% human serum) (human serum from ZhongKeChenYu Biotech; Cat. No. 168014-100mL) was added. The mixture was centrifuged and then the supernatant was removed. According to the experimental design, antibodies (30nM, 3nM, 1nM, 0.3nM, and 0.1nM final concentrations, respectively) were added to CHO-K1-PD1 cells at 100μL / well, buffer or antibodies (30nM, 3nM, 1nM, 0.3nM, and 0.1nM final concentrations, respectively) were added to CHO-K1-LAG3 cells at 100μL / well, and buffer was added to CHO-K1 cells at 100μL / well. The system was then incubated on ice for 60 minutes.

[0222] 100 μL of buffer was added, and the mixture was centrifuged at 350 × g for 5 min, after which the supernatant was removed and washed twice with 200 μL of buffer. CHO-K1-LAG3 and CHO-K1 cells in each well were separately resuspended in 100 μL of buffer, and the suspension was transferred to the corresponding CHO-K1-PD1 sample well at 1.5 × 10 5 The cells / well were transferred. The mixture was mixed well and then incubated on ice in the dark for 40 minutes. 200 μL of buffer was added to each well to resuspend the cells, and the suspension was transferred to a sample loading tube for testing on the flow cytometer.

[0223] The results are shown in Figure 11. Compared with the isotype control, the anti-LAG3 / anti-PD-1 bispecific antibody was able to simultaneously bind to CHO-K1-PD1 and CHO-K1-LAG3 cells and bridge the two cell types, whereas 14C12H1L1 (hG1TM) and leratolimab did not have this effect, even when used in combination.

[0224] Experimental Example 7: Analysis of the biological activity of anti-LAG3 / anti-PD-1 bispecific antibodies in promoting IFN-γ and IL-2 secretion by mixed lymphocyte reaction (MLR) 1. Analysis of the biological activity of anti-LAG3 / anti-PD-1 bispecific antibodies in promoting IFN-γ secretion in the Raji-PDL1 mixed lymphocyte reaction system Raji-PDL1 cells were subcultured as usual. PBMCs were thawed and cultured in 10 mL of 1640 complete medium and stimulated with 0.5 μg / mL of SEB (Staphylococcal Enterotoxin B) (Dianotech; Cat. No. S010201) for 2 days. The Raji-PDL1 cells were treated with MMC (Stressmarq; Cat. No. SIH-246-10MG) at a final concentration of 2 μg / mL and incubated at 37°C in a 5% CO2 incubator for 1 hour. PBMCs stimulated with SEB for 2 days and Raji-PDL1 cells treated with MMC for 1 hour were harvested and washed twice with PBS. The two types of cells were then resuspended in complete medium, counted, and plated at 10 × 10 cells in a U-shaped 96-well plate (Corning, Model No. 3799). 4 The cells were added at 1000 μg / well, respectively, and co-cultured. According to the experimental design, the antibodies (final concentrations of 300 nM, 30 nM, and 3 nM, respectively) were added and the cells were co-cultured for 3 days in an incubator. After 3 days, the cells were centrifuged at 1200 rpm for 5 minutes, and the cell culture supernatants were collected and analyzed for IFN-γ by ELISA.

[0225] As shown in Figure 12A, co-culture of human PBMCs and Raji-PDL1 cells significantly promoted IFN-γ secretion in PBMCs, and the addition of antibodies to the co-culture system significantly induced further IFN-γ secretion in PBMCs. The degree of activity of antibody BS-PL022B in promoting IFN-γ secretion was superior to that of antibody 14C12H1L1 (hG1TM) that targets PD-1 alone and control antibody relatolimab that targets LAG-3 alone. Even when compared with the combination of 14C12H1L1 (hG1TM) and relatolimab, BS-PL022B had a higher ability to promote IFN-γ secretion at two different antibody concentration levels (30 nM and 300 nM).

[0226] 2. Analysis of the biological activity of anti-LAG-3 / anti-PD-1 bispecific antibodies in promoting IL-2 secretion in the Raji-PDL1 mixed lymphocyte reaction system Raji-PDL1 cells were subcultured as usual. PBMCs were thawed and cultured in 10 mL of 1640 complete medium and stimulated with 0.5 μg / mL of SEB (Staphylococcal Enterotoxin B) (Dianotech; Cat. No. S010201) for 2 days. The Raji-PDL1 cells were treated with MMC (Stressmarq; Cat. No. SIH-246-10MG) at a final concentration of 2 μg / mL and incubated at 37°C in a 5% CO2 incubator for 1 hour. PBMCs stimulated with SEB for 2 days and Raji-PDL1 cells treated with MMC for 1 hour were harvested and washed twice with PBS. The two types of cells were then resuspended in complete medium, counted, and plated at 10 × 10 cells in a U-shaped 96-well plate (Corning, Model No. 3799). 4 The cells were added at 1000 x 1000 cells / well, respectively, and co-cultured. According to the experimental design, the antibodies (final concentrations of 300 nM, 30 nM, and 3 nM, respectively) were added and the cells were co-cultured for 3 days. After 3 days, the cells were centrifuged at 1200 rpm for 5 minutes, and the cell culture supernatants were collected and analyzed for IL-2 by ELISA.

[0227] As shown in Figure 12B, the co-culture of human PBMCs and Raji-PDL1 cells promoted IL-2 secretion in PBMCs to some extent. In addition, the addition of antibodies to the co-culture system could significantly induce further IL-2 secretion in PBMCs, which showed a significant dose-dependent relationship. In terms of the degree of activity to promote IL-2 secretion, BS-PL022B had a higher ability to promote IL-2 secretion at three different antibody concentration levels than the antibody 14C12H1L1 (hG1TM) that targets PD-1 alone and the control antibody relatolimab that targets LAG-3 alone. Even when compared with the combination of 14C12H1L1 (hG1TM) and relatolimab, BS-PL022B had a higher ability to promote IL-2 secretion at three different antibody concentration levels.

[0228] Experimental Example 8: Analysis of the affinity of BS-PL022B for the Fc receptor FcγRI The Fc receptor FcγRI (also known as CD64) can bind to the Fc fragment of IgG antibodies and is involved in antibody-dependent cell-mediated cytotoxicity (ADCC). The binding ability of therapeutic antibodies to Fc receptors may affect the safety and efficacy of the antibody. In this experiment, the affinity constant of BS-PL022B for FcγRI was measured using the Fortebio Octet system to evaluate the ADCC activity of the antibody.

[0229] The method for measuring the affinity constants of the corresponding antibodies to FcγRI by the Fortebio Octet system is briefly described below. The sample dilution buffer was a solution of 0.02% Tween-20 and 0.1% BSA in PBS at pH 7.4. A 1 μg / mL FcγRI solution (purchased from Sinobio) was added to the HIS1K sensor, and FcγRI was immobilized on the sensor surface for 50 seconds. Both the binding and dissociation constants of the antibodies to FcγRI were measured in buffer with antibody concentrations ranging from 3.12 to 50 nM (2-fold serial dilution). The shaking speed of the sample plate was 1000 rpm, the temperature was 30°C, and the frequency was 5.0 Hz. The data was analyzed by 1:1 model fitting to obtain the affinity constants.

[0230] The analysis results of the affinity constant of BS-PL022B for FcγRI are shown in Table 12 and Figures 13-14. [Table 12] N / A indicates that the antibody did not bind or the signal for the antigen was so weak that analysis of the results was not performed and no corresponding data was obtained.

[0231] The results show that H7L8(hG1WT) could bind to FcγRI with an affinity constant of 6.59E-09M, and that for BS-PL022B, it did not bind to FcγRI or the binding signal was extremely weak, so the results were not analyzed and no corresponding data was obtained.

[0232] The results show that the binding activity of BS-PL022B to FcγRI was effectively eliminated.

[0233] Experimental Example 9: Analysis of the affinity of BS-PL022B for the Fc receptor FcγRIIIa and its subtypes (1) Analysis of affinity constant of BS-PL022B for FcγRIIIa_V158 The Fc receptor FcγRIIIa_V158 (also known as CD16a_V158) can bind to the Fc fragment of IgG antibodies and mediate the ADCC effect. In this experiment, the affinity constant of BS-PL022B for FcγRIIIa_V158 was measured using the Fortebio Octet system to evaluate the ADCC activity of the antibody.

[0234] The method for measuring the affinity constant of the corresponding antibody by the Fortebio Octet system is briefly described below. The sample dilution buffer was a solution of 0.02% Tween-20 and 0.1% BSA in PBS at pH 7.4. 5 μg / mL of FcγRIIIa_V158 was immobilized on the HIS1K sensor for 60 seconds. The sensor was equilibrated in the buffer for 60 seconds, and the binding of the immobilized FcγRIIIa_V158 on the sensor to the antibody at a concentration of 31.25 to 500 nM (2-fold serial dilution) was measured for 60 seconds. The antibody was dissociated in the buffer for 60 seconds. The shaking speed of the sample plate was 1000 rpm, the temperature was 30 °C, and the frequency was 5.0 Hz. The data was analyzed with 1:1 model fitting to obtain the affinity constant.

[0235] The analysis results of the affinity constant of BS-PL022B for FcγRIIIa_V158 are shown in Table 13 and Figures 15 to 16. [Table 13] N / A indicates that the antibody did not bind or the signal for the antigen was so weak that analysis of the results was not performed and no corresponding data was obtained.

[0236] The results show that H7L8(hG1WT) was able to bind to FcγRIIIa_V158 with an affinity constant of 8.77E-08M, and that for BS-PL022B, it did not bind to FcγRIIIa_V158 or the binding signal was extremely weak, so the results were not analyzed.

[0237] The results show that the binding activity of BS-PL022B to FcγRIIIa_V158 was effectively eliminated.

[0238] (2) Analysis of affinity constant of BS-PL022B for FcγRIIIa_F158 The Fc receptor FcγRIIIa_F158 (also known as CD16a_F158) can bind to the Fc fragment of IgG antibodies and mediate the ADCC effect. In this experiment, the affinity constant of BS-PL022B for FcγRIIIa_F158 was measured using the Fortebio Octet system to evaluate the ADCC activity of the antibody.

[0239] The method for measuring the affinity constant of TF01 for FcγRIIIa_F158 by the Fortebio Octet system is briefly described below. The sample dilution buffer was a solution of 0.02% Tween-20 and 0.1% BSA in PBS at pH 7.4. 5 μg / mL of FcγRIIIa_F158 was immobilized on the HIS1K sensor for 120 seconds. The sensor was equilibrated in the buffer for 60 seconds, and the binding of the immobilized FcγRIIIa_F158 on the sensor to antibodies at concentrations of 31.25 to 500 nM (2-fold dilution) was measured for 60 seconds. The antibody was dissociated in the buffer for 60 seconds. The shaking speed of the sample plate was 1000 rpm, the temperature was 30°C, and the frequency was 5.0 Hz. The data was analyzed with a 1:1 model fitting to obtain the affinity constant.

[0240] The analysis results of the affinity constant of BS-PL022B for FcγRIIIa_F158 are shown in Table 14 and Figures 17 to 18. [Table 14] N / A indicates that the antibody did not bind or the signal for the antigen was so weak that analysis of the results was not performed and no corresponding data was obtained.

[0241] The results show that H7L8 (hG1WT) was able to bind to FcγRIIIa_F158 with an affinity constant of 3.64E-07M, and that for BS-PL022B, it did not bind to FcγRIIIa_F158 or the binding signal was extremely weak, so analysis of the results was not performed and no corresponding data was obtained.

[0242] The results show that the binding activity of BS-PL022B to FcγRIIIa_F158 was effectively eliminated.

[0243] Experimental Example 10: Analysis of the affinity of BS-PL022B for the Fc receptor FcγRIIa and its subtypes (1) Analysis of affinity constant of BS-PL022B for FcγRIIa_H131 The Fc receptor FcγRIIa_H131 (also known as CD32a_H131) can bind to the Fc fragment of IgG antibodies and is involved in antibody-dependent cellular phagocytosis (ADCP) or antibody-dependent cell-mediated cytotoxicity (ADCC). The binding ability of therapeutic antibodies to Fc receptors may affect the safety and efficacy of the antibodies. In this experiment, the affinity constant of BS-PL022B for FcγRIIa_H131 was measured using the Fortebio Octet system to evaluate the binding ability of the test antibodies to the Fc receptor.

[0244] The method for measuring the affinity constant of BS-PL022B for FcγRIIa_H131 by the Fortebio Octet system is briefly described below. The sample dilution buffer was a solution of 0.02% Tween-20 and 0.1% BSA in PBS at pH 7.4. 5 μg / mL of FcγRIIa_H131 was immobilized on the NTA sensor at an immobilization height of about 1.0 nm. The sensor was equilibrated in the buffer for 60 seconds, and the binding of the immobilized FcγRIIa_H131 on the sensor to antibodies at concentrations of 12.5 to 200 nM (2-fold serial dilution) was measured for 60 seconds. The antibody was dissociated in the buffer for 60 seconds. The shaking speed of the sample plate was 1000 rpm, the temperature was 30 °C, and the frequency was 5.0 Hz. The data was analyzed with 1:1 model fitting to obtain the affinity constant.

[0245] The analysis results of the affinity constant of BS-PL022B for FcγRIIa_H131 are shown in Table 15 and Figures 19 to 20. [Table 15] N / A indicates that the antibody did not bind or the signal for the antigen was so weak that analysis of the results was not performed and no corresponding data was obtained.

[0246] The results show that H7L8(hG1WT) could bind to FcγRIIa_H131 with an affinity constant of 1.78E-07M, and that for BS-PL022B, it did not bind to FcγRIIa_H131 or the binding signal was extremely weak, so the results were not analyzed and no corresponding data was obtained.

[0247] The results show that the binding activity of BS-PL022B to FcγRIIa_H131 was effectively eliminated.

[0248] Experimental Example 11: Analysis of affinity constant of BS-PL022B for FcγRIIb The Fc receptor FcγRIIb (also known as CD32b) can bind to the Fc fragment of IgG antibodies. In this experiment, the affinity constant of the test antibody for FcγRIIb was measured using the Fortebio Octet system, and the binding ability of BS-PL022B to the Fc receptor was evaluated.

[0249] The method for measuring the affinity constant of BS-PL022B for FcγRIIb by the Fortebio Octet system is briefly described below. The sample dilution buffer was a solution of 0.02% Tween-20 and 0.1% BSA in PBS at pH 7.4. 5 μg / mL of FcγRIIb was immobilized on the NTA sensor at an immobilization height of about 1.0 nm. The sensor was equilibrated in the buffer for 60 seconds, and the binding of the immobilized hFCGR2B-his on the sensor to antibodies at concentrations of 12.5 to 200 nM (2-fold serial dilution) was measured for 60 seconds. The antibodies were dissociated in the buffer for 60 seconds. The shaking speed of the sample plate was 1000 rpm, the temperature was 30°C, and the frequency was 5.0 Hz. The data was analyzed with a 1:1 model fitting to obtain the affinity constant.

[0250] The analysis results of the affinity constant of BS-PL022B for FcγRIIb are shown in Table 17 and Figures 21-22. [Table 16] N / A indicates that the antibody did not bind or the signal for the antigen was so weak that analysis of the results was not performed and no corresponding data was obtained.

[0251] The results show that H7L8(hG1WT) could bind to FcγRIIb with an affinity constant of 1.21E-07M, and that for BS-PL022B, it did not bind to FcγRIIb or the binding signal was extremely weak, so the results were not analyzed and no corresponding data was obtained.

[0252] The results show that the binding activity of BS-PL022B to FcγRIIb was effectively eliminated.

[0253] Experimental Example 12: Analysis of affinity of BS-PL022B for C1q Serum complement C1q can bind to the Fc fragment of IgG antibodies and mediate the CDC effect. The binding ability of therapeutic antibodies to C1q may affect the safety and efficacy of the antibodies. In this experiment, the affinity constant of BS-PL022B for C1q was measured using the Fortebio Octet system to evaluate the CDC activity of the antibody.

[0254] The method for measuring the affinity constants of the corresponding antibodies to C1q by the Fortebio Octet system is briefly described below. The sample dilution buffer was a solution of 0.02% Tween-20 and 0.1% BSA in PBS at pH 7.4. 50 μg / mL of each antibody was immobilized on the FAB2G sensor at an immobilization height of approximately 2.0 nm. The sensor was equilibrated in the buffer for 60 seconds, and the binding of the immobilized antibody on the sensor to the antigen C1q at concentrations of 0.625 to 10 nM (2-fold serial dilution) was measured for 60 seconds. The antigen-antibody was dissociated in the buffer for 60 seconds. The shaking speed of the sample plate was 1000 rpm, the temperature was 30 °C, and the frequency was 5.0 Hz. The data was analyzed with 1:1 model fitting to obtain the affinity constants. The data acquisition software was Fortebio Data Acquisition 7.0, and the data analysis software was Fortebio Data Analysis 7.0.

[0255] The results of the analysis of the affinity constant of BS-PL022B for C1q are shown in Table 18 and Figures 23-24. [Table 17] N / A indicates that the antibody did not bind or the signal for the antigen was so weak that analysis of the results was not performed and no corresponding data was obtained.

[0256] The results show that H7L8(hG1WT) could bind to C1q with an affinity constant of 1.75E-09M, and for BS-PL022B, it did not bind to C1q or the binding signal was extremely weak, so the results were not analyzed and no corresponding data was obtained.

[0257] The results show that the binding activity of BS-PL022B to C1q was effectively eliminated.

[0258] Experimental Example 13: Activity of BS-PL022B in antibody-dependent cellular phagocytosis of CHO-K1-PD1-LAG3 Jurkat-NFAT-CD64-CD32R cells (constructed by Akeso Biopharma) and CHO-K1-PD1-LAG3 cells (constructed by Akeso Biopharma) were harvested conventionally and centrifuged at 110×g for 5 min, after which the supernatant was removed. Cells were then resuspended in 1640+4% FBS and counted. Cell viability was determined and cell concentration was adjusted. According to the experimental design, test antibodies were diluted to 50 nM, 5 nM, and 0.5 nM (working concentration 10 nM, 1 nM, and 0.1 nM, or 5 nM, 0.5 nM, and 0.05 nM) with 1640+4% FBS, and control antibodies were diluted to 50 nM (working concentration 10 nM). Jurkat-NFAT-CD64-CD32R cell suspension was added to a 96-well black plate at 40 μL / sample (40,000 cells / well). To the samples already containing Jurkat-NFAT-CD64-CD32R cells, target cells CHO-K1-PD1-LAG3 were added at 40 μL / sample (40,000 cells / well). Antibodies were added to the corresponding samples at 20 μL / well and the mixture was mixed well. Blank and isotype controls were also set up. The plate was then incubated in an incubator for 5 hours and Bright-Glo™ Luciferase Assay System (Promega, Cat. No. E2650) was added to the samples at 50 μL / well. The mixture was mixed well and the plate was read.

[0259] The results are shown in Figure 25.

[0260] The results show that 14C12H1L1(G1WT)+H7L8(hG1WT) and nivolumab+leratolimab had ADCP effects at the same concentrations, whereas BS-PL022B had no ADCP effect.

[0261] Experimental Example 14: Pharmacodynamic evaluation of anti-LAG3 / anti-PD-1 bispecific antibodies in a mouse model implanted with subcutaneous tumor cells To measure the antitumor activity of the anti-LAG3 / anti-PD-1 bispecific antibody in vivo, CT26 colon cancer cells (purchased from GemPharmatech) were first subcutaneously inoculated into the right hind thigh of 7.1-7.3 week-old female BALB / c-hPD1 / hLAG3 mice (purchased from GemPharmatech). The day of implantation was defined as D0. The administration route was intraperitoneal injection (ip), and the administration was performed twice a week (BIW), for a total of six times. The modeling and specific administration regimen are shown in Table 18. After administration, the length and width of the tumor in each group were measured, and the tumor volume was calculated. [Table 18]

[0262] The results are shown in Figure 26. The results show that, compared with the isotype control antibody, both the anti-LAG3 / anti-PD-1 bispecific antibody BS-PL022B and the positive control antibody relatolimab were able to effectively inhibit tumor growth in mice. The results show that the anti-LAG3 / anti-PD-1 bispecific antibody BS-PL022B had a significantly superior anti-tumor effect than the positive control antibody relatolimab.

[0263] Furthermore, as shown in Figure 27, the test drug BS-PL022B was well tolerated by the tumor-bearing mice, and no effect on the body weight of the tumor-bearing mice was observed in any group.

[0264] Although the specific embodiments of the present invention have been described in detail, those skilled in the art will understand that, according to any teaching disclosed herein, various modifications and substitutions can be made to those details, and all of these modifications are included within the protection scope of the present invention. The full scope of the present invention is provided by the appended claims and any equivalents thereof.

Claims

1. 1. A bispecific antibody comprising a first protein functional region and a second protein functional region, the first protein functional domain targets LAG3; the second protein functional region targets a target other than LAG3 (e.g., PD-1); the first protein functional domain is an anti-LAG3 antibody or antigen-binding fragment thereof, comprising a heavy chain variable domain and a light chain variable domain; A bispecific antibody, wherein the heavy chain variable region comprises HCDR1 to HCDR3 having the amino acid sequences set forth in SEQ ID NOs: 5 to 7, respectively, and the light chain variable region comprises LCDR1 to LCDR3 having the amino acid sequences set forth in SEQ ID NOs: 8 to 10, respectively.

2. 2. The bispecific antibody of claim 1 , wherein the anti-LAG3 antibody comprises a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 2 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO:

4.

3. The following: (1) The anti-LAG3 antibody or antigen-binding fragment thereof is Fab, Fab', F(ab') ２ , Fd, Fv, dAb, complementarity determining region fragment, single chain variable region fragment, humanized antibody, chimeric antibody, and diabody; (2) the anti-LAG3 antibody binds to human LAG3-mFc with an EC 50 value of less than 0.2 nM, e.g., less than 0.15 nM, less than 0.1 nM, less than 0.08 nM, less than 0.06 nM, or less than 0.05 nM, or less; preferably, the EC 50 value is measured by indirect ELISA; (3) the anti-LAG3 antibody comprises a non-CDR region derived from a species other than mouse, e.g., a human antibody; (4) The anti-LAG3 antibody comprises a constant region derived from a human antibody; preferably, the constant region of the antibody is selected from the constant region of human IgG1, IgG2, IgG3, or IgG4; (5) The heavy chain constant region of the anti-LAG3 antibody is an Ig gamma-1 chain C region (e.g., as set forth in SEQ ID NO: 39) or an Ig gamma-4 chain C region (e.g., as set forth in SEQ ID NO: 45), and the light chain constant region of the anti-LAG3 antibody is an Ig kappa chain C region (e.g., as set forth in SEQ ID NO: 40); 2. The bispecific antibody of claim 1, characterized by one or more of:

4. 2. The bispecific antibody of claim 1 , (1) The anti-LAG3 antibody is a human IgG1 subtype; wherein, according to the EU numbering system, the heavy chain constant region of said antibody has the following mutations: L234A and L235A; L234A and G237A; L235A and G237A; or L234A, L235A, and G237A, having Preferably, the anti-LAG3 antibody comprises a heavy chain having the amino acid sequence set forth in SEQ ID NO: 11 and a light chain having the amino acid sequence set forth in SEQ ID NO: 12; or (2) the anti-LAG3 antibody is a human IgG4 subtype; wherein, according to the EU numbering system, the heavy chain constant region of said antibody has the following mutations: F234A and L235A; F234A and G237A; L235A and G237A; or F234A, L235A, and G237A, having Preferably, the anti-LAG3 antibody is a bispecific antibody comprising a heavy chain having the amino acid sequence set forth in SEQ ID NO: 13 and a light chain having the amino acid sequence set forth in SEQ ID NO:

12.

5. The following: (1) The bispecific antibody is in the form of an IgG-scFv; (2) the first protein functional domain is an anti-LAG3 antibody and the second protein functional domain is a single chain variable domain fragment; or the first protein functional domain is a single chain variable domain fragment and the second protein functional domain is an antibody that targets a target other than LAG3; (3) the first protein functional domain and the second protein functional domain are linked directly or via a linker fragment; Preferably, the linker fragment is (GGGGS)m, where m is a positive integer, such as 1, 2, 3, 4, 5, or 6; or Preferably, the linker fragment is (GGGGS)nG, where n is a positive integer, such as 1, 2, 3, 4, 5, or 6; (4) The number of the first protein functional domain and the second protein functional domain is independently 1, 2, or more. (5) the single-chain variable region fragment is linked to the C-terminus of the antibody heavy chain; 2. The bispecific antibody of claim 1, characterized by one or more of:

6. a first protein functional region that targets LAG3; and a second protein functional domain that targets PD-1; 2. The bispecific antibody of claim 1, comprising: the first protein functional domain is an anti-LAG3 antibody, and the anti-LAG3 antibody is in the form of an immunoglobulin; a bispecific antibody, wherein the second protein functional domain is an anti-PD-1 single chain variable domain fragment.

7. The following: (1) The anti-PD-1 single-chain variable region fragment comprises a heavy chain variable region and a light chain variable region; the heavy chain variable region comprises HCDR1 to HCDR3 having the amino acid sequences set forth in SEQ ID NOs:26 to 28, respectively; and the light chain variable region comprises LCDR1 to LCDR3 having the amino acid sequences set forth in SEQ ID NOs: 29 to 31, respectively; (2) The anti-PD-1 single-chain variable region fragment comprises a heavy chain variable region and a light chain variable region; the heavy chain variable region has the amino acid sequence set forth in SEQ ID NO: 15 and the light chain variable region has the amino acid sequence set forth in SEQ ID NO: 17; or the heavy chain variable region has the amino acid sequence set forth in SEQ ID NO: 19, and the light chain variable region has the amino acid sequence set forth in SEQ ID NO: 21 or SEQ ID NO: 38; (3) The anti-PD-1 single-chain variable region fragment comprises a heavy chain variable region and a light chain variable region, and the heavy chain variable region and the light chain variable region are linked directly or via a linker fragment; Preferably, the linker fragment is (GGGGS)m, where m is a positive integer, such as 1, 2, 3, 4, 5, or 6; or Preferably, the linker fragment is (GGGGS)nG, where n is a positive integer, such as 1, 2, 3, 4, 5, or 6; 7. The bispecific antibody of claim 6, characterized by one or more of:

8. 7. The bispecific antibody of claim 6, the bispecific antibody A first protein functional region that targets LAG3; and a second protein functional domain that targets PD-1; Including; the number of said first protein functional domains is 1 and the number of said second protein functional domains is 2; the first protein functional domain is an immunoglobulin and the second protein functional domain is a single chain variable domain fragment; the immunoglobulin comprises a heavy chain having the amino acid sequence set forth in SEQ ID NO: 11 or SEQ ID NO: 13, and a light chain having the amino acid sequence set forth in SEQ ID NO: 12; the single-chain variable region fragment comprises a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 19, and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 21 or SEQ ID NO: 38; the single-chain variable region fragment is linked to the C-terminus of two heavy chains of the immunoglobulin; the first protein functional region is linked to the second protein functional region via a first linker fragment; the heavy chain variable region of the single chain variable region fragment is linked to the light chain variable region of the single chain variable region fragment via a second linker fragment; the first linker fragment and the second linker fragment are the same or different; Preferably, the first linker fragment and the second linker fragment each have an amino acid sequence independently selected from SEQ ID NOs: 35 to 37; Preferably, the bispecific antibody, wherein the amino acid sequences of the first linker fragment and the second linker fragment are set forth in SEQ ID NO:

36.

9. a first protein functional region that targets LAG3; and a second protein functional domain that targets PD-1; 2. The bispecific antibody of claim 1, comprising: the first protein functional domain is an anti-LAG3 single chain variable domain fragment, the second protein functional domain is an anti-PD-1 antibody, and the anti-PD-1 antibody is in the form of an immunoglobulin; the anti-LAG3 single chain variable region fragment comprises a heavy chain variable region and a light chain variable region; the heavy chain variable region comprises HCDR1 to HCDR3 having the amino acid sequences set forth in SEQ ID NOs:5 to 7, respectively; and A bispecific antibody, wherein the light chain variable region comprises LCDR1 to LCDR3 having the amino acid sequences set forth in SEQ ID NOs: 8 to 10, respectively.

10. The following: (1) The anti-LAG3 single-chain variable region fragment, the heavy chain variable region has the amino acid sequence set forth in SEQ ID NO:2; the light chain variable region has the amino acid sequence set forth in SEQ ID NO:4; (2) The heavy chain variable region and the light chain variable region in the anti-LAG3 single-chain variable region fragment are linked directly or via a linker fragment; Preferably, the linker fragment is (GGGGS)m, where m is a positive integer, such as 1, 2, 3, 4, 5, or 6; or Preferably, the linker fragment is (GGGGS)nG, where n is a positive integer, such as 1, 2, 3, 4, 5, or 6; (3) The anti-PD-1 antibody comprises a heavy chain variable region and a light chain variable region; the heavy chain variable region comprises HCDR1 to HCDR3 having the amino acid sequences set forth in SEQ ID NOs:26 to 28, respectively; and the light chain variable region comprises LCDR1 to LCDR3 having the amino acid sequences set forth in SEQ ID NOs: 29 to 31, respectively; (4) The anti-PD-1 antibody, the heavy chain variable region has the amino acid sequence set forth in SEQ ID NO: 15 and the light chain variable region has the amino acid sequence set forth in SEQ ID NO: 17; or the heavy chain variable region has the amino acid sequence set forth in SEQ ID NO: 19, and the light chain variable region has the amino acid sequence set forth in SEQ ID NO: 21 or SEQ ID NO: 38; (5) The heavy chain constant region of the anti-PD-1 antibody is an Ig gamma-1 chain C region (e.g., as set forth in SEQ ID NO: 39) or an Ig gamma-4 chain C region (e.g., as set forth in SEQ ID NO: 45), and the light chain constant region of the anti-PD-1 antibody is an Ig kappa chain C region (e.g., as set forth in SEQ ID NO: 40); 10. The bispecific antibody of claim 9, characterized by one or more of:

11. 10. The bispecific antibody of claim 9, (1) The anti-PD-1 antibody is a human IgG1 subtype; wherein, according to the EU numbering system, the anti-PD-1 antibody has the following mutation: L234A and L235A; L234A and G237A; L235A and G237A; or L234A, L235A, and G237A, having Preferably, the anti-PD-1 antibody comprises a heavy chain having the amino acid sequence set forth in SEQ ID NO:34 and a light chain having the amino acid sequence set forth in SEQ ID NO:25; or (2) the anti-PD-1 antibody is a human IgG4 subtype; wherein, according to the EU numbering system, the anti-PD-1 antibody has the following mutation: F234A and L235A; F234A and G237A; L235A and G237A; or F234A, L235A, and G237A, having Preferably, the anti-PD-1 antibody is a bispecific antibody comprising a heavy chain having the amino acid sequence set forth in SEQ ID NO: 32 and a light chain having the amino acid sequence set forth in SEQ ID NO:

25.

12. 10. The bispecific antibody of claim 9, the bispecific antibody a first protein functional region that targets LAG3; and a second protein functional domain that targets PD-1; Including; the number of said first protein functional domains is two and the number of said second protein functional domains is one; the first protein functional domain is a single chain variable domain fragment and the second protein functional domain is an immunoglobulin; the single-chain variable region fragment comprises a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:2 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO:4; the immunoglobulin comprises a heavy chain having the amino acid sequence set forth in SEQ ID NO:34 or SEQ ID NO:32, and a light chain having the amino acid sequence set forth in SEQ ID NO:25; the single-chain variable region fragment is linked to the C-terminus of two heavy chains of the immunoglobulin; the first protein functional region is linked to the second protein functional region via a first linker fragment; the heavy chain variable region of the single chain variable region fragment is linked to the light chain variable region of the single chain variable region fragment via a second linker fragment; the first linker fragment and the second linker fragment are the same or different; Preferably, the first linker fragment and the second linker fragment each have an amino acid sequence independently selected from SEQ ID NOs: 35 to 37; Preferably, the amino acid sequences of the first linker fragment and the second linker fragment are set forth in SEQ ID NO: 36; Preferably, the bispecific antibody, wherein the amino acid sequences of the first linker fragment and the second linker fragment are set forth in SEQ ID NO:

37.

13. An isolated nucleic acid molecule encoding the bispecific antibody of any one of claims 1 to 12.

14. A recombinant vector comprising the isolated nucleic acid molecule of claim 13.

15. 14. A host cell comprising the isolated nucleic acid molecule or recombinant vector of claim 13, wherein the recombinant vector comprises the isolated nucleic acid molecule of claim 13.

16. 13. A pharmaceutical composition comprising the bispecific antibody of any one of claims 1 to 12, said pharmaceutical composition optionally further comprising a pharmacologically acceptable excipient.

17. Use of a bispecific antibody according to any one of claims 1 to 12 in the preparation of a medicament for treating and / or preventing tumors or anemia, comprising: Preferably, the tumor is selected from one or more of ovarian cancer, esophageal cancer, melanoma, hematological malignancies, glioblastoma, renal cell carcinoma, lung cancer, prostate cancer, bladder cancer, colon cancer, rectal cancer, liver cancer, gastrointestinal cancer, breast cancer, brain cancer, pancreatic cancer, thyroid cancer, head and neck cancer, and kidney cancer; Preferably, the lung cancer is non-small cell lung cancer; Preferably, the hematological malignancy is leukemia; Preferably, the use of a bispecific antibody, wherein said esophageal cancer is esophageal squamous cell carcinoma.

18. 13. A bispecific antibody according to any one of claims 1 to 12 for use in the treatment and / or prevention of tumors or anemia, Preferably, the tumor is selected from one or more of ovarian cancer, esophageal cancer, melanoma, hematological malignancies, glioblastoma, renal cell carcinoma, lung cancer, prostate cancer, bladder cancer, colon cancer, rectal cancer, liver cancer, gastrointestinal cancer, breast cancer, brain cancer, pancreatic cancer, thyroid cancer, head and neck cancer, and kidney cancer; Preferably, the lung cancer is non-small cell lung cancer; Preferably, the hematological malignancy is leukemia; Preferably, the bispecific antibody, wherein the esophageal cancer is esophageal squamous cell carcinoma.

19. A pharmaceutical composition for treating and / or preventing tumors or anemia, comprising an effective amount of a bispecific antibody according to any one of claims 1 to 12, Preferably, the tumor is selected from one or more of ovarian cancer, esophageal cancer, melanoma, hematological malignancies, glioblastoma, renal cell carcinoma, lung cancer, prostate cancer, bladder cancer, colon cancer, rectal cancer, liver cancer, gastrointestinal cancer, breast cancer, brain cancer, pancreatic cancer, thyroid cancer, head and neck cancer, and kidney cancer; Preferably, the lung cancer is non-small cell lung cancer; Preferably, the hematological malignancy is leukemia; Preferably, the pharmaceutical composition, wherein the esophageal cancer is esophageal squamous cell carcinoma.

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