Anti-lag3 antibodies, pharmaceutical compositions, and uses

Abstract

The present application belongs to the field of biological medicine, and relates to an anti-LAG3 antibody, a pharmaceutical composition containing the anti-LAG3 antibody and purposes. Specifically, the present application relates to an anti-LAG3 antibody or an antigen binding fragment thereof, containing a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region contains HCDR1-HCDR3 with the amino acid sequences shown in SEQ ID NOs: 9-11 respectively; and the light chain variable region contains LCDR1-LCDR3 with the amino acid sequences shown in SEQ ID NOs: 12-14 respectively. The anti-LAG3 antibody of the present application has superior affinity and specificity, and has a good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and relates to an anti-LAG3 antibody, a pharmaceutical composition containing the anti-LAG3 antibody, and its uses. Background Technology

[0002] Cancer, especially malignant cancer, is a serious threat to human health worldwide, ranking second among all causes of death. Moreover, its incidence has shown a significant upward trend in recent years. Malignant cancers have poor treatment outcomes, high rates of late-stage metastasis, and generally poor prognoses. While current clinical treatments such as radiotherapy, chemotherapy, and surgery have significantly alleviated pain and prolonged survival, these methods have considerable limitations, making further improvements in efficacy difficult.

[0003] Lymphocyte-activation gene 3 (LAG3), also known as CD223, is a type I transmembrane protein composed of 498 amino acids and belongs to the immunoglobulin superfamily (IgSF). LAG3 is mainly expressed on activated CD4+. + T cells and CD8 + T cells, as well as natural killer (NK) cells, B cells, regulatory T cells (Treg), and plasmacytoid dendritic cells (pDC) also express LAG3. (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 on human chromosome 12 (20p13.3), adjacent to the CD4 gene, and both share the same exons and introns. Although their amino acid sequence homology is only about 20%, LAG3 and CD4 molecules show high structural similarity. Major histocompatibility complex class II (MHC II), hepatic sinusoidal endothelial cell lectin (LSECtin), and galectin-3 are related ligands of LAG3. MHC II molecules are the major ligands of LAG3, and their affinity for MHC II molecules (Kd: 60 nmol·L⁻¹) is 100 times that of CD4, indicating that LAG3 can effectively compete with CD4 for binding to MHC II molecules and inhibit T cell activation.

[0005] In the tumor microenvironment, the expression of the immunosuppressive molecule LAG3 can be detected 24 hours after T cell activation, leading to T cell dysfunction or apoptosis. LAG3 forms a dimer with CD4 through its D1 domain (containing a proline-rich ring structure). + The specific binding of MHCII molecules in the first signaling axis of T cell activation, "CD3-TCR-MHCII," blocks the signal transduction pathway of T cell activation on the one hand, and on the other hand, the intracellular segment of LAG3 (Keele motif) generates an immunosuppressive signal that downregulates CD4. + T cell activity. LAG3 molecules can promote Treg cell differentiation, participate in signal transduction and downstream signaling of transcription activator factor 5, thereby enhancing the inhibitory effect of Treg cells. This is one of the mechanisms by which tumors escape the killing effect of 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] Multiple studies have shown that LAG3 plays a role in the tumor-invasive CD8 complex of various malignant tumors. + Overexpression in T cells. For example, in ovarian cancer, tumor-infiltrating New York esophageal squamous cell carcinoma 1 antigen (NY-ESO-1)-specific CD8+. + T cells express high levels of PD-1 and LAG3, resulting in reduced ability to produce IFN-γ and TNF-α, which in turn leads to lymphocyte inactivation. Galectin-3 and LSECtin primarily interact with LAG3 to regulate CD8. +T cell activation and function. Furthermore, melanoma antigen-specific T cells isolated from melanoma metastasis patients showed significant upregulation of LAG3 and other immune checkpoint molecules CTLA-4 and TIM-3. (Liu Hao, Li Xinying, Luo Longlong, et al. Research progress on molecular biological function of lymphocyte activation gene 3 and its clinical application as an antibody drug [J]. Chinese Journal of Pharmacology and Toxicology, 2019, 33(01):70-78.).

[0007] Currently, several LAG3 antibody drugs have entered the clinical research stage. Among them, Bristol-Myers Squibb's Relatlimab is progressing the fastest, with 10 clinical studies underway. The vast majority of these studies involve the combination of Relatlimab and nivolumab for the treatment of hematologic malignancies, melanoma, glioblastoma, renal cell carcinoma, and non-small cell lung cancer.

[0008] Currently, there is still a need to develop new anti-LAG3 antibody drugs. Summary of the Invention

[0009] Through in-depth research and creative labor, the inventors have obtained an anti-LAG3 antibody. The inventors have surprisingly discovered that the anti-LAG3 antibody of this invention (also simply referred to as the antibody or the antibody of this invention) possesses superior affinity and / or specificity, even surpassing positive control antibodies (e.g., Relatlimab) in one or more aspects. This provides the following invention:

[0010] One aspect of the present invention relates to an anti-LAG3 antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein,

[0011] The heavy chain variable region comprises: amino acid sequences as shown in SEQ ID NOs:9-11, namely HCDR1-HCDR3; and the light chain variable region comprises: amino acid sequences as shown in SEQ ID NOs:12-14, namely LCDR1-LCDR3.

[0012] The heavy chain variable region comprises: amino acid sequences as shown in SEQ ID NOs:9-11, respectively, HCDR1-HCDR3; and the light chain variable region comprises: amino acid sequences as shown in SEQ ID NO:12, SEQ ID NO:15 and SEQ ID NO:16, respectively, LCDR1-LCDR3;

[0013] or

[0014] The heavy chain variable region comprises: amino acid sequences as shown in SEQ ID NOs:9-11, namely HCDR1-HCDR3; and the light chain variable region comprises: amino acid sequences as shown in SEQ ID NO:17, SEQ ID NO:15 and SEQ ID NO:14, namely LCDR1-LCDR3.

[0015] In some embodiments of the present invention, the antibody or its antigen-binding fragment, wherein...

[0016] The amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO:2, and the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO:4.

[0017] The amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO:2, and the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO:6.

[0018] or

[0019] The amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO:2, and the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO:8.

[0020] In some embodiments of the present invention, the antibody or its antigen-binding fragment is selected from Fab, Fab', F(ab')2, Fd, Fv, dAb, complementarity-determining region fragment, single-chain antibody, humanized antibody or chimeric antibody.

[0021] In some embodiments of the present invention, the antibody or its antigen-binding fragment is wherein the antibody is in an EC50 concentration of less than 0.2 nM, for example less than 0.15 nM, less than 0.1 nM, less than 0.08 nM, 0.06 nM, or less than 0.05 nM or smaller. 50 Combined with human LAG3-mFc; preferably, the EC 50 Measured by indirect ELISA method.

[0022] In some embodiments of the present invention, the antibody or its antigen-binding fragment, wherein...

[0023] The antibody includes a non-CDR region, and the non-CDR region is derived from a species other than rodents, such as from human antibodies.

[0024] In some embodiments of the present invention, the antibody or its antigen-binding fragment, wherein...

[0025] The antibody described herein has a constant region derived from human antibodies;

[0026] Preferably, the constant region of the antibody is selected from the constant regions of human IgG1, IgG2, IgG3 or IgG4.

[0027] In some embodiments of the present invention, the antibody or its antigen-binding fragment, wherein...

[0028] The heavy chain constant region of the anti-LAG3 antibody is the Ig gamma-1 chain C region (e.g., as shown in SEQ ID NO:18) or the Ig gamma-4 chain C region (e.g., as shown in SEQ ID NO:20); the light chain constant region is the Ig kappa chain C region (e.g., as shown in SEQ ID NO:19).

[0029] In some embodiments of the present invention, the anti-LAG3 antibody is a monoclonal antibody.

[0030] In some embodiments of the present invention, the anti-LAG3 antibody is in the form of an immunoglobulin.

[0031] In some embodiments of the present invention, the anti-LAG3 antibody is a single-chain antibody.

[0032] Another aspect of the present invention relates to antibody-drug conjugates (ADCs), comprising an antibody or an antigen-binding fragment thereof and a small molecule drug, wherein the antibody or antigen-binding fragment thereof is any one of the anti-LAG3 antibodies or antigen-binding fragments thereof described in the present invention; preferably, the small molecule drug is a small molecule cytotoxic drug; more preferably, the small molecule drug is an anti-tumor chemotherapy drug.

[0033] The chemotherapy drugs can be conventional tumor chemotherapy drugs, such as alkylating agents, antimetabolites, antitumor antibiotics, herbal anticancer drugs, hormones, immunomodulators, etc.

[0034] In one or more embodiments of the present invention, the antibody-drug conjugate wherein the antibody or its antigen-binding fragment is linked to a small molecule drug via a linker; the linker may be a linker known to those skilled in the art, for example, the linker may be a hydrazone bond, a disulfide bond or a peptide bond.

[0035] In one or more embodiments of the present invention, the antibody-drug conjugate wherein the molar ratio of the antibody or its antigen-binding fragment to the small molecule drug is 1:(2-4), for example 1:2, 1:3 or 1:4.

[0036] Another aspect of the invention relates to an isolated nucleic acid molecule encoding the anti-LAG3 antibody described in any one of the inventions.

[0037] Another aspect of the invention relates to a recombinant vector comprising the isolated nucleic acid molecules of the invention.

[0038] Another aspect of the invention relates to a host cell comprising the isolated nucleic acid molecules of the invention, or comprising the recombinant vector of the invention.

[0039] Another aspect of the invention relates to a method for preparing the antibody or antigen-binding fragment thereof as described in any one of the invention, comprising culturing the host cells of the invention under suitable conditions, and recovering the antibody or antigen-binding fragment thereof from the cell culture.

[0040] Another aspect of the present invention relates to a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof as described in any one of the present invention, or an antibody-drug conjugate as described in any one of the present invention; optionally, it further comprises pharmaceutically acceptable excipients.

[0041] Another aspect of the present invention relates to the use of any antibody or antigen-binding fragment thereof described in any one of the present invention, or any antibody-drug conjugate described in any one of the present invention, in the preparation of medicaments for treating and / or preventing tumors or anemia;

[0042] Preferably, the tumor is selected from one or more of the following: ovarian cancer, esophageal cancer, melanoma, hematologic malignancy, 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.

[0043] Preferably, the lung cancer is non-small cell lung cancer;

[0044] Preferably, the hematologic malignancy is leukemia;

[0045] Preferably, the esophageal cancer is esophageal squamous cell carcinoma.

[0046] The antibody or its antigen-binding fragment according to any one of the present invention, and the antibody-drug conjugate according to any one of the present invention, are used for the treatment and / or prevention of tumors or anemia;

[0047] Preferably, the tumor is selected from one or more of the following: ovarian cancer, esophageal cancer, melanoma, hematologic malignancy, 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.

[0048] Preferably, the lung cancer is non-small cell lung cancer;

[0049] Preferably, the hematologic malignancy is leukemia;

[0050] Preferably, the esophageal cancer is esophageal squamous cell carcinoma.

[0051] Another aspect of the present invention relates to a method for treating and / or preventing tumors or anemia, comprising the step of administering to a subject in need an effective amount of any antibody or antigen-binding fragment thereof described in any one of the present invention, or any antibody-drug conjugate described in any one of the present invention;

[0052] Preferably, the tumor is selected from one or more of the following: ovarian cancer, esophageal cancer, melanoma, hematologic malignancy, 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.

[0053] Preferably, the lung cancer is non-small cell lung cancer;

[0054] Preferably, the hematologic malignancy is leukemia;

[0055] Preferably, the esophageal cancer is esophageal squamous cell carcinoma.

[0056] In this invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the cell culture, molecular genetics, nucleic acid chemistry, and immunology laboratory procedures used herein are all standard procedures widely used in their respective fields. To better understand this invention, definitions and explanations of relevant terms are provided below.

[0057] As used in this article, the term EC 50 The half-maximal concentration (WMC) is the concentration that produces a 50% maximum effect.

[0058] As used herein, the term "antibody" refers to an immunoglobulin molecule typically composed of two pairs of polypeptide chains (each pair consisting of one "light" (L) chain and one "heavy" (H) chain). Antibody light chains can be classified as κ and λ light chains. Heavy chains can be classified as μ, δ, γ, α, or ε, and antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE, respectively. Within both light and heavy chains, variable and constant regions are linked by "J" regions of approximately 12 or more amino acids, and the heavy chain also contains "D" regions of approximately 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains (CH1, CH2, and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain, CL. The constant regions of antibodies mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The VH and VL regions can be further subdivided into highly degenerated regions (called complementarity-determining regions (CDRs)) interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4, from the amino terminus to the carboxyl terminus. The variable regions (VH and VL) of each heavy / light chain pair form the antibody binding sites. The allocation of amino acids to various regions or domains follows Bethesda Md, Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, (1987 and 1991)), or Chothia & Lesk J. Mol. Biol. 1987; 196: 901-917; Chothia et al. Nature 1989; 342: 878-883, or the definition of the IMGT numbering system, see 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.

[0059] The term "antibody" is not limited to any particular method of producing antibodies. For example, it includes recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. Antibodies can be different isotypes of antibodies, such as IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM antibodies.

[0060] As used herein, the terms "monoclonal antibody" and "monoclonal antibody" refer to an antibody or a fragment of an antibody derived from a group of highly homologous antibody molecules—that is, a group of identical antibody molecules except for the possibility of spontaneous natural mutations. Monoclonal antibodies exhibit high specificity for a single epitope on an antigen. Polyclonal antibodies, as opposed to monoclonal antibodies, typically contain at least two or more different antibodies that typically recognize different epitopes on an antigen. Monoclonal antibodies can usually be obtained using the hybridoma technique first reported by Kohler et al. G, Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity[J]. Nature, 1975; 256(5517):495), but it can also be obtained using recombinant DNA technology (see USPatent 4,816,567).

[0061] As used herein, the term "humanized antibody" refers to an antibody or antibody fragment obtained by replacing all or part of the CDR region of a human immunoglobulin (receptor antibody) with the CDR region of a non-human antibody (donor antibody), wherein the donor antibody can be a non-human antibody (e.g., mouse, rat, or rabbit) with the expected specificity, affinity, or reactivity. Furthermore, some amino acid residues in the framework region (FR) of the receptor antibody may also be replaced by amino acid residues of the corresponding non-human antibody, or by amino acid residues of other antibodies, to further improve or optimize the antibody's performance. For more detailed information on humanized antibodies, please refer to, for example, Jones et al., Nature 1986; 321:522-525; Reichmann et al., Nature, 1988; 332:323-329; Presta, Curr. Op. Struct. Biol. 1992; 2:593-596; and Clark, Immunol. Today 2000; 21:397-402.

[0062] As used herein, the terms "separated" or "isolated" refer to substances obtained artificially from their natural state. If a substance or component is found in nature as a "separated" entity, it may be due to an alteration of its natural environment, the separation of the substance from its natural environment, or both. For example, a certain unseparated polynucleotide or polypeptide may naturally exist in the body of a living animal, and a high-purity identical polynucleotide or polypeptide separated from this natural state is called a separated one. The terms "separated" or "isolated" do not exclude the presence of artificial or synthetic substances, nor do they exclude the presence of other impurities that do not affect the substance's activity.

[0063] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which polynucleotides can be inserted. When a vector enables the expression of a protein encoded by the inserted polynucleotide, it is called an expression vector. Vectors can be introduced into host cells through transformation, transduction, or transfection, allowing the genetic material elements they carry to be expressed in the host cells. Vectors are well-known to those skilled in the art and include, but are not limited to: plasmids; phage particles; Cos plasmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage; and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retrotranscriptoviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40). A vector may contain multiple elements controlling expression, including but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, a vector may contain a replication initiation site.

[0064] As used herein, the term “host cell” refers to a cell that can be used to introduce a vector, including but not limited to prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, GS cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells, or human cells.

[0065] 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 that is specific to an antigen) means that the antibody binds to an antigen at a concentration of less than about 10. -5 M, for example, less than approximately 10 -6 M, 10 -7 M, 10-8 M, 10 -9 M or 10 -10 M or lower affinity (K) D () binds to the antigen.

[0066] As used in this article, the term "K" D "" refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, which describes the binding affinity between the antibody and the antigen. The smaller the equilibrium dissociation constant, the stronger the antibody-antigen binding and the higher the affinity between the antibody and the antigen. Typically, antibodies have an equilibrium dissociation constant of less than approximately 10. -5 M, for example, less than approximately 10 -6 M, 10 -7 M, 10 -8 M, 10 - 9 M or 10 -10 M or a smaller dissociation equilibrium constant (K) D K binds to an antigen (e.g., PD-1 protein). K can be measured using methods known to those skilled in the art. D For example, measurements can be taken using a Fortebio molecular interaction analyzer.

[0067] As used herein, the terms "monoclonal antibody" and "monoclonal antibody" have the same meaning and are used interchangeably; the terms "polyclonal antibody" and "polyclonal antibody" have the same meaning and are used interchangeably. Furthermore, in this invention, amino acids are generally represented by single-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.

[0068] As used herein, the term "pharmaceutically acceptable carrier and / or excipient" means a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, which is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to: pH adjusters, surfactants, adjuvants, and ionic strength enhancers. For example, pH adjusters include, but are not limited to, phosphate buffers; surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80; and ionic strength enhancers include, but are not limited to, sodium chloride.

[0069] As used herein, the term "effective amount" means an amount sufficient to achieve, or at least partially achieve, the desired effect. For example, an effective amount for preventing disease (e.g., cancer) means an amount sufficient to prevent, stop, or delay the onset of disease (e.g., cancer); an effective amount for treating disease means an amount sufficient to cure or at least partially stop the disease and its complications in a patient already suffering from the disease. Determining such an effective amount is entirely within the capabilities of those skilled in the art. For example, an effective amount for therapeutic purposes will depend on the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general characteristics such as age, weight, and sex, the manner of administration of the drug, and other concurrent treatments, etc.

[0070] As used herein, when referring to the amino acid sequence of lymphocyte-activation gene 3 (LAG3), it includes the full-length LAG3 protein, or the extracellular fragment LAG3 ECD, or a fragment containing LAG3 ECD; it also includes fusion proteins of the full-length LAG3 protein or fusion proteins of LAG3 ECD, such as fragments fused with a fragment of the Fc protein (mFc or hFc) of mouse or human IgG. However, those skilled in the art will understand that mutations or variations (including, but not limited to, substitutions, deletions, and / or additions) can be naturally generated or artificially introduced into the amino acid sequence of the LAG3 protein without affecting its biological function. Therefore, in this invention, the term "addition," "protein," should include all such sequences, including their natural or artificial variants. Furthermore, when describing a sequence fragment of the LAG3 protein, it also includes the corresponding sequence fragment from its natural or artificial variants.

[0071] Beneficial effects of the invention

[0072] This invention achieves one or more of the following effects:

[0073] (1) The anti-LAG3 antibody of the present invention has superior affinity and specificity;

[0074] (2) The anti-LAG3 antibody of the present invention can effectively block the interaction between LAG3 and MHC-II and specifically relieve the immunosuppression of the body by LAG3. Attached Figure Description

[0075] Figure 1 Results of indirect ELISA method for determining the binding activity of H7L8 (hG1WT) to antigen LAG3-mFc.

[0076] Figure 2 The binding activity of H7L8(hG4WT), H7L9(hG4WT), and H7L10(hG4WT) to human LAG3-mFc antigen was determined by ELISA.

[0077] Figure 3 FACS was used to detect the binding activity of H7L8(hG4WT), H7L9(hG4WT), and H7L10(hG4WT) to the 293T-LAG3 cell surface antigen LAG3.

[0078] Figure 4 Competitive flow cytometry was used to determine the results of the competitive binding of H7L8(hG4WT), H7L9(hG4WT), and H7L10(hG4WT) to LAG3-mFc for the 293T-LAG3 cell membrane surface antigen MHC II.

[0079] Figure 5 Results of the bioactivity assay of anti-LAG3 antibody in promoting IFN-γ secretion by mixed lymphocyte reaction (MLR).

[0080] Figure 6 Results of the bioactivity assay of anti-LAG3 antibody in promoting IL-2 secretion by mixed lymphocyte reaction (MLR).

[0081] Figure 7 Results of biological activity assay for anti-LAG antibody blocking the interaction between LAG-3 and MHC-II. Detailed Implementation

[0082] 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 for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the examples, they are performed according to the techniques or conditions described in the literature in the art (e.g., refer to J. Sambrook et al., *Molecular Cloning: A Laboratory Manual*, 3rd edition, Science Press, translated by Huang Peitang et al.) or according to the product instructions. Reagents or instruments whose manufacturers are not specified are conventional products that can be purchased commercially.

[0083] The positive control antibody, Relatlimab, has its sequence referenced in US Patent Publication US20160326248A1. The heavy chain amino acid sequence refers to SEQ ID NO:1 and the light chain amino acid sequence refers to SEQ ID NO:2 in that patent publication. Relatlimab is an anti-LAG-3 antibody.

[0084] The heavy chain amino acid sequence of Relatlimab:

[0085] QVQLQQWGAGLLKPSETLSLTCAVYGGSFSDYYWNWIRQPPGKGLEWIGEINHRGSTNSNPSLKSRVTLSLDTSKNQFSLKLRSVTAADTAVYYCAFGYSDYEYNWFDPWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO:23)

[0086] The amino acid sequence of the light chain of Relatlimab:

[0087] EIVLTQSPATLSLSPGERATLSCRASQSISSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPLTFGQGTNLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ IDNO:24)

[0088] The control antibody 14C12H1L1 (hG1TM) is an anti-PD-1 antibody prepared by Akeso Biopharma, lot number B105Y2080601.

[0089] The amino acid sequence of the heavy chain of 14C12H1L1(hG1TM):

[0090] EVQLVESGGGLVQPGGSLRLSCAASGFAFSSYDMSWVRQAPGKGLDWVATISGGGRYTYYPDSVKGRFTISRDNSKNNLYLQMNSLRAEDTALYYCANRYGEAWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:21)

[0091] Amino acid sequence of the light chain of 14C12H1L1 (hG1TM)

[0092] DIQMTQSPSSMSASVGDRVTFTCRASQDINTYLSWFQQKPGKSPKTLIYRANRLVSGVPSRFSGSGSGQDYTLTISSLQPEDMATYYCLQYDEFPLTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ IDNO:22)

[0093] The 293T-LAG3 cell line was constructed by Zhongshan Kangfang Biopharmaceutical Co., Ltd. The 293T-LAG3 cell line was obtained by viral infection of HEK293T cells. The virus preparation used 3rd Generation Lentiviral Systems, see, for example, A Third Generation Lentivirus Vector with a Conditional Packaging System. Dull T, Zufferey R, Kelly M, Mandel RJ, Nguyen M, Trono D, and Naldini L. J Virol. 1998. 72(11): 8463-8471. The lentiviral expression vector used was plenti6.3 / V5-huLAG3FL-BSD (LAG3, Genebank ID: NM_002277.4; vector plenti6.3 / V5-BSD, purchased from Invitrogen, product number: K5315-20).

[0094] The Raji-PDL1 cell line was constructed by Zhongshan Kangfang Biopharmaceutical Co., Ltd. The Raji-PDL1 cell line was obtained by viral infection of Raji cells. The virus preparation used 3rd Generation Lentiviral Systems, see, for example, AThird Generation Lentivirus Vector with a Conditional Packaging System. Dull T, Zufferey R, Kelly M, Mandel RJ, Nguyen M, Trono D, and Naldini LJ Virol. 1998. 72(11): 8463-8471. The lentiviral expression vector used was plenti6.3 / V5-PDL1 (where PDL1, Genebank ID: NP_054862.1; vector plenti6.3 / V5, purchased from Invitrogen, catalog number: K5315-20).

[0095] The Jurkat-NFAT-PD1-LAG3 cell line was constructed by Zhongshan Kangfang Biopharmaceutical Co., Ltd. The Jurkat-NFAT-PD1-LAG3 cell line was prepared by viral infection of PD-1 effector cells (CPM, Promega, catalog number J112A). The virus preparation used 3rd Generation Lentiviral Systems, see, for example, A Third Generation Lentivirus Vector with a Conditional Packaging System. Dull T, Zufferey R, Kelly M, Mandel RJ, Nguyen M, Trono D, and Naldini L. J Virol. 1998. 72(11): 8463-8471. The lentiviral expression vector used was pCDH-huLAG3FL-RFP-NEO (where LAG3, Genebank ID: NM_002277.4; vector pCDH-CMV-MCS-EF1-RFP+Neo, purchased from UBO Biotechnology, product number: VT9005).

[0096] Preparation Example 1: Design and Preparation of Anti-LAG3 Antibody

[0097] 1. Antibody design

[0098] Based on the existing LAG3 protein sequence (NCBI Reference Sequence: NP_002277.4) and its three-dimensional crystal structure, the inventors creatively designed a series of antibody sequences. Through extensive screening and testing, humanized monoclonal antibodies that specifically bind to LAG3 were finally obtained, named H7L8, H7L9, and H7L10, respectively. The amino acid sequences and their coding sequences of the heavy and light chain variable regions of these monoclonal antibodies are as follows.

[0099] The nucleic acid sequence (360bp) of the H7L8 heavy chain variable region H7v:

[0100] CAGGTGCAGCTGCAGCAGTGGGGAGCTGGACTGCTGAAACCTAGCGAGACACTGAGCCTGACCTGTGCTGTGTACGGCGGATCTATCAGCGATTACTACTGGAACTGGATCAGGCAGCCCCCTGGAAAGGGACTGGAATGGATCGGAGAGATCAACCACAGGGGCACCACCAACTCCAATCCCTCTCTGAAGAGCAGGGTGACACTGAGCCTCGACACAAGCAAGAATCAGTTCAGCCTGAAGCTGAGGTCCGTGACCGCTGCTGATACAGCTGTGTACTACTGTGCCTTCGGCTACAGCGATTACGAGTACGATTGGTTCGACCCTTGGGGCCAGGGAACACTGGTTACAGTGAGCTCC(SEQ ID NO:1)

[0101] Amino acid sequence (120 aa) of the heavy chain variable region H7v of H7L8:

[0102] QVQLQQWGAGLLKPSETLSLTCAVYGGSISDYYWNWIRQPPGKGLEWIGEINHRGTTNSNPSLKSRVTLSLDTSKNQFSLKLRSVTAADTAVYYCAFGYSDYEYDWFDPWGQGTLVTVSS(SEQ ID NO:2)

[0103] Nucleic acid sequence (321 bp) of the light chain variable region L8v of H7L8:

[0104] GAGATCGTTCTGACCCAGAGCCCAGCTACACTGAGCCTGTCTCCTGGAGAGAGGGCTACACTGTCCTGCAGAGCTAGCCAGACCATCAGCAGCTACCTGGCTTGGTACCAGCAGAAGCCTGGCCAAGCTCCAAGGCTGCTGATCTACGACGCCTCTAATAGGGCCACCGGCATCCCTGCTAGATTCTCTGGAAGCGGCAGCGGAACCGACTTTACACTGACAATCAGCTCCCTGGAGCCCGAGGATTTCGCTGTTTACTACTGTCAGCAGCGCAGCAACTGGCCCATCACATTCGGACAGGGCACAAATCTGGAGATCAAG(SEQ ID NO:3)

[0105] The amino acid sequence (107aa) of the L8v variable region of the H7L8 light chain:

[0106] EIVLTQSPATLSLSPGERATLSCRASQTISSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPITFGQGTNLEIK(SEQID NO:4)

[0107] The nucleic acid sequence of the heavy chain variable region H7v of H7L9 is the same as that of the heavy chain variable region H7v of H7L8, i.e., SEQ ID NO:1.

[0108] The amino acid sequence of the heavy chain variable region H7v of H7L9 is the same as that of the heavy chain variable region H7v of H7L8, i.e., SEQ ID NO:2.

[0109] The nucleic acid sequence (321 bp) of the L9v variable region of the H7L9 light chain:

[0110] GAGATCGTTCTGACCCAGAGCCCAGCTACACTGAGCCTGTCTCCTGGAGAGAGGGCTACACTGTCCTGCAGAGCTAGCCAGACATCAGCAGCTACCTGGCTTGGTACCAGCAGAAGCCTGGCCAAGCTCCAAGGCTGCTGATCTACGACGGCTCTAATAGG GCCACCGGCATCCCTGCTAGATTCTCTGGAAGCGGCAGCGGAACCGACTTTACACTGACAATCAGCTCCCTGGAGCCCGAGGATTTCGCTGTTTACTACTGTCAGCAGCGCAGCAACTGGCCCCTCACATTCGGACAGGGCACAAATCTGGAGATCAAG(SEQ ID NO:5)

[0111] The amino acid sequence (107 bp) of the L9v variable region of the light chain of H7L9:

[0112] EIVLTQSPATLSLSPGERATLSCRASQTISSYLAWYQQKPGQAPRLLIYDGSNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPLTFGQGTNLEIK(SEQ ID NO:6)

[0113] The nucleic acid sequence of the heavy chain variable region H7v of H7L10 is the same as that of the heavy chain variable region H7v of H7L8, i.e., SEQ ID NO:1.

[0114] The amino acid sequence of the heavy chain variable region H7v of H7L10 is the same as that of the heavy chain variable region H7v of H7L8, i.e., SEQ ID NO:2.

[0115] The nucleic acid sequence (321 bp) of the L10v variable region of the H7L10 light chain:

[0116] GAGATCGTTCTGACCCAGAGCCCAGCTACACTGAGCCTGTCTCCTGGAGAGAGGGCTACACTGTCCTGCAGAGCTAGCCAGTCCATCAGCAGCTACCTGGCTTGGTACCAGCAGAAGCCTGGCCAAGCTCCAAGGCTGCTGATCTACGACGGCTCTAATAGG GCCACCGGCATCCCTGCTAGATTCTCTGGAAGCGGCAGCGGAACCGACTTTACACTGACAATCAGCTCCCTGGAGCCCGAGGATTTCGCTGTTTACTACTGTCAGCAGCGCAGCAACTGGCCCATCACATTCGGACAGGGCACAAATCTGGAGATCAAG(SEQ ID NO:7)

[0117] The amino acid sequence (107 bp) of the L10v variable region of the light chain of H7L10:

[0118] EIVLTQSPATLSLSPGERATLSCRASQSISSYLAWYQQKPGQAPRLLIYDGSNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPITFGQGTNLEIK(SEQ ID NO:8)

[0119] The amino acid sequence of the CDR of antibody H7L8 is as follows (according to the IMGT numbering system):

[0120] HCDR1:GGSISDYY(SEQ ID NO:9);

[0121] HCDR2: INHRGTT (SEQ ID NO: 10);

[0122] HCDR3: AFGYSDYEYDWFDP (SEQ ID NO: 11);

[0123] LCDR1: QTISSY(SEQ ID NO:12);

[0124] LCDR2: DAS (SEQ ID NO:13);

[0125] LCDR3: QQRSNWPIT (SEQ ID NO: 14).

[0126] The amino acid sequence of the CDR of antibody H7L9 is as follows (according to the IMGT numbering system):

[0127] HCDR1:GGSISDYY(SEQ ID NO:9);

[0128] HCDR2: INHRGTT (SEQ ID NO: 10);

[0129] HCDR3: AFGYSDYEYDWFDP (SEQ ID NO: 11);

[0130] LCDR1: QTISSY(SEQ ID NO:12);

[0131] LCDR2: DGS (SEQ ID NO:15);

[0132] LCDR3: QQRSNWPLT (SEQ ID NO: 16).

[0133] The amino acid sequence of the CDR of antibody H7L10 is as follows (according to the IMGT numbering system):

[0134] HCDR1:GGSISDYY(SEQ ID NO:9);

[0135] HCDR2: INHRGTT (SEQ ID NO: 10);

[0136] HCDR3: AFGYSDYEYDWFDP (SEQ ID NO: 11);

[0137] LCDR1: QSISSY(SEQ ID NO:17);

[0138] LCDR2: DGS (SEQ ID NO:15);

[0139] LCDR3: QQRSNWPIT (SEQ ID NO: 14).

[0140] 2. Expression and purification of humanized antibody H7L8 (hG1WT)

[0141] The heavy chain cDNA sequence of H7L8 (hG1WT) (the variable region coding sequence is shown in SEQ ID NO:1; the constant region is the Ig gamma-1 chain C region) and the light chain cDNA sequence (the variable region coding sequence is shown in SEQ ID NO:3; the constant region is the human Ig kappa chain C region) were cloned into the pUC57simple vector (provided by GenScript) to obtain pUC57simple-H7 and pUC57simple-L8 plasmids, respectively. The pUC57simple-H7 and pUC57simple-L8 plasmids were digested with HindIII and EcoRI, and the heavy and light chains were recovered by electrophoresis and subcloned into the pcDNA3.1 vector, respectively. The recombinant plasmids were extracted and co-transfected into 293F cells. After 7 days of cell culture, the culture medium was centrifuged at high speed, the supernatant was concentrated, and the sample was loaded onto a HiTrap MabSelect SuRe column. Proteins were eluted with Elution Buffer in one step, the target sample was recovered, and the medium was changed to PBS.

[0142] H7L8(hG1WT) heavy chain constant region amino acid sequence

[0143] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:18)

[0144] H7L8(hG1WT) light chain constant region amino acid sequence

[0145] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:19)

[0146] 3. Expression and purification of humanized antibodies H7L8(hG4WT), H7L9(hG4WT), and H7L10(hG4WT)

[0147] The heavy chain cDNA sequences of H7L8(hG4WT), H7L9(hG4WT), and H7L10(hG4WT) (variable region coding sequence as shown in SEQ ID NO:1; constant region is Iggamma-4chain C region) and the light chain cDNA sequences of H7L8(hG4WT) (variable region coding sequence as shown in SEQ ID NO:3; constant region is human Ig kappa chain C region), H7L9(hG4WT) light chain (variable region coding sequence as shown in SEQ ID NO:5; constant region is human Ig kappa chain C region), and H7L10(hG4WT) light chain (variable region coding sequence as shown in SEQ ID NO:7; constant region is human Ig kappa chain C region) were obtained. The regions were cloned into the pUC57simple vector (provided by GenScript) to obtain pUC57simple-H7, pUC57simple-L8, pUC57simple-L9, and pUC57simple-L10 plasmids, respectively. The pUC57simple-H7, pUC57simple-L8, pUC57simple-L9, and pUC57simple-L10 plasmids were digested with HindIII and EcoRI, and the heavy and light chains were recovered by electrophoresis and subcloned into the pcDNA3.1 vector, respectively. The recombinant plasmids were extracted and co-transfected into 293F cells. After 7 days of cell culture, the culture medium was centrifuged at high speed, the supernatant was concentrated, and the sample was loaded onto a HiTrapMabSelect SuRe column. Proteins were eluted with Elution Buffer in one step, the target sample was recovered, and the medium was changed to PBS.

[0148] H7L8(hG4WT), H7L9(hG4WT), or H7L10(hG4WT) heavy chain constant region amino acid sequence:

[0149] ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVH NAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO:20)

[0150] The amino acid sequence of the light chain constant region of H7L8(hG4WT), H7L9(hG4WT), or H7L10(hG4WT):

[0151] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:19)

[0152] Preparation Example 2: Preparation of Human Anti-Egg Lysosomal Antibody

[0153] Human anti-Hen Egg Lysozyme IgG (anti-HEL, i.e., human IgG, abbreviated as hIgG) has a sequence derived from the variable region of the FabF10.6.6 sequence in Acierno et al.'s study on 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 is as follows:

[0154] Human IgG was codon-optimized and gene synthesized for the heavy and light chain (full sequence or variable region) genes of the antibody by Nanjing GenScript Biotech. Following standard techniques described in *Molecular Cloning: A Laboratory Manual (3rd Edition)*, standard molecular cloning techniques including PCR, enzyme digestion, DNA gel recovery, ligation transformation, and colony PCR or enzyme digestion identification were used to subclone the heavy and light chain genes into antibody heavy chain expression vectors and antibody light chain expression vectors in mammalian expression systems. Further sequencing analysis of the heavy and light chain genes in the recombinant expression vectors was performed. After sequencing verification, endotoxin-free expression plasmids were prepared in large quantities and transiently co-transfected into HEK293 cells for recombinant antibody expression. After 7 days of culture, cell culture medium was collected and purified using rProtein A column (GE). The harvested antibody samples were quality-assessed using standard analytical techniques such as SDS-PAGE and SEC-HPLC.

[0155] Experimental Example 1: ELISA method for determining the binding activity of anti-LAG3 antibody to antigen.

[0156] Human LAG3-mFc (self-made by Kangfang Biotechnology, batch number: 20200417), 0.5 μg / mL, was coated onto an ELISA plate and incubated overnight at 4°C. The plate was then washed once with PBST, followed by blocking with 1% BSA in PBS solution at 37°C for 2 hours. After blocking, the plate was washed three times with PBST. Serially diluted antibody (antibody dilution gradient detailed in Table 1) was added, and the plate was incubated at 37°C for 30 minutes. After incubation, the plate was washed three times with PBST. Finally, HRP-labeled goat anti-human IgG FC(H+L) (Jackson, catalog number: 109-035-098) secondary antibody working solution diluted 1:5000 was added, and the plate was incubated at 37°C for 30 minutes. After incubation, wash the plate four times with PBST, then add TMB (Neogen, 308177) for 5 min in the dark, and finally add stop solution to terminate the reaction. Immediately place the plate in a microplate reader and read the OD values ​​of each well at 450 nm. Analyze the data using SoftMax Pro 6.2.1 software.

[0157] The test results are shown in Table 1 and Figure 1 As shown.

[0158] Table 1: ELISA detection of the binding of Relatlimab, H7L8 (hG1WT) to LAG3-mFc

[0159]

[0160] Depend on Figure 1It was found that Relatlimab, H7L8(hG1WT), and human LAG3-mFc antigen could bind effectively, and the binding efficiency was dose-dependent. The absorbance intensities at each dose are shown in Table 1. By performing quantitative absorbance analysis on the bound antibodies, the binding efficiency EC50 of antibodies Relatlimab (as a positive control) and H7L8(hG1WT) was obtained through curve simulation calculation. 50 The values ​​are 0.106 nM and 0.045 nM, respectively.

[0161] The above experimental results show that, under the same experimental conditions, H7L8(hG1WT) has effective binding activity against human LAG3-mFc, and the binding activity of H7L8(hG1WT) against human LAG3-mFc is stronger than that of the target-positive drug Relatlimab.

[0162] Example 2: ELISA method for determining the binding activity of anti-LAG3 antibody to antigen.

[0163] Human LAG3-mFc, 2 μg / mL, was coated onto an ELISA plate and incubated overnight at 4°C. The plate was then washed once with PBST, followed by blocking with 1% BSA in PBS at 37°C for 2 hours. After blocking, the plate was washed three times with PBST. Serially diluted antibodies (see Table 1 for antibody dilution gradients) were added, and the plate was incubated at 37°C for 30 minutes. After incubation, the plate was washed three times with PBST. Following washing, a 1:5000 dilution of HRP-labeled goat anti-human IgG (H+L) (Jackson, catalog number: 109-035-088) secondary antibody working solution was added, and the plate was incubated at 37°C for 30 minutes. After incubation, the plate was washed four times with PBST, followed by TMB (Neogen, 308177) for 5 minutes in the dark. The colorimetric reaction was then terminated with stop solution. Immediately place the ELISA plate into the microplate reader and select a 450nm wavelength to read the OD values ​​of each well. Analyze the data using SoftMax Pro 6.2.1 software.

[0164] The test results are shown in Table 2 and Figure 2 As shown.

[0165] Table 2: ELISA detection of the binding of H7L8 (hG4WT), H7L9 (hG4WT), and H7L10 (hG4WT) to human LAG3-mFc antigen

[0166]

[0167]

[0168] The results showed that antibodies H7L8 (hG4WT), H7L9 (hG4WT), and H7L10 (hG4WT) could effectively bind to human LAG3-mFc antigen, with binding efficiency showing a dose-dependent relationship, and their binding activity was comparable to that of the positive control antibody Relatlimab.

[0169] Example 3: Flow cytometry method for detecting the binding activity of anti-LAG3 antibody to cell surface antigen LAG3.

[0170] 1. Construction of host cell 293T expressing LAG3 antigen

[0171] The specific steps are as follows:

[0172] Construction of host cells expressing LAG3 antigen 293T: 293T cells were transfected with the vector pLenti6.3 / V5-huLAG3FL-BSD (pLenti6.3 vector purchased from Invitrogen) containing LAG3 according to the lipofectamin transfection kit (purchased from Invitrogen). After screening, a stable clonal population of 293T-LAG3 expressing LAG3 was obtained.

[0173] 2. Binding of antibodies to 293T-LAG3 cell surface antigens

[0174] Antibody labeling and flow cytometry: 293T-LAG3 host cells expressing the LAG3 antigen were obtained using the above steps via conventional trypsin digestion, with each collection tube containing 3 × 10⁶ cells. 5 LAG3 antibody dilutions were prepared with 1% PBSA (PBS containing 1% BSA) to final concentrations of 0.0123 nM, 0.123 nM, 1.23 nM, 3.7 nM, 11.1 nM, 33.3 nM, 100 nM, and 300 nM, and incubated with LAG3-expressing 293T cells on ice for 1 hour. After washing several times with 1% PBSA by centrifugation, 100 μL of FITC-concentrated goat anti-human IgG (purchased from Jackson, catalog number: 109-095-098) (1:500 dilution) was added to each tube, and the cells were incubated on ice in the dark for 40 min. After washing once with 1% PBSA, the cells were resuspended in 200 μL of 1% PBSA, and the fluorescence signal was detected using the FITC channel on a flow cytometer.

[0175] The binding results of humanized anti-LAG3 antibody to 293T-LAG3 cells are as follows: Figure 3 As shown. The binding efficiency (EC50) of each anti-LAG3 antibody to the 293T-LAG3 surface antigen is shown. 50 As shown in Table 3.

[0176] Table 3: Flow cytometry detection of the binding activity of anti-LAG3 antibody to 293T-LAG3 surface antigen

[0177] <![CDATA[EC 50 (nM)]]> Relatlimab 4.289 H7L8(hG4WT) 4.929 H7L9(hG4WT) 4.809 H7L10(hG4WT) 4.168

[0178] Depend on Figure 3 It is evident that anti-LAG3 antibodies can effectively bind to the target LAG3 protein on the surface of host cells 293T-LAG3, and the binding activity of anti-LAG3 antibodies H7L8 (hG4WT), H7L9 (hG4WT), and H7L10 (hG4WT) to the 293T-LAG3 surface antigen is comparable to that of the positive control antibody Relatlimab.

[0179] Example 4 Competitive flow cytometry was used to determine the competitive binding of anti-LAG3 antibodies to cell membrane surface antigens by LAG3-mFc. Competitive binding activity of MHC II

[0180] Raji cells (culture medium: 1640 + 10% FBS) (Cell Resource Center, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, catalog number: THu 44) were added to EP tubes at a rate of 300,000 cells / sample. 1000 μL of 1% PBSA (PBS containing 1% BSA) was added, and the tubes were centrifuged at 600 × g for 5 min, discarding the supernatant. According to the experimental design, 100 μL of hIgG1 (prepared by Kangfang Biotechnology, batch number: 20190410) was added to each tube to a final concentration of 300 nM, and the tubes were incubated on ice for 1 h. After incubation, 200 μL of 1% PBSA was added to the Raji cells, and the tubes were centrifuged at 600 × g for 5 min, discarding the supernatant. Meanwhile, according to the experimental design, clean EP tubes were prepared and 60 μL of antibodies diluted to the corresponding concentrations (300 nM, 100 nM, 33.3 nM, 11.1 nM, 3.7 nM, 1.23 nM, 0.123 nM, 0.0123 nM) were added to each tube, and Blank (PBSA+ cells) was designed. Then, 60 μL of LAG3-mFc (prepared by Kangfang Biotechnology, batch number: 20190508) (final concentration of 3 nM) was added to the corresponding antibody tubes, mixed well, and pre-incubated on ice for 30 min. Add 100 μL of pre-incubated antibody and protein mixture to the sample, mix well, and incubate on ice in the dark for 1 h; add 200 μL of 1% PBSA, centrifuge at 600×g for 5 min, discard the supernatant, and wash twice; add 100 μL of APC anti-mouse antibody (purchased from Biolegend, catalog number: 405308) (1:400 dilution), mix well, and incubate on ice in the dark for 40 min; add 200 μL of 1% PBSA, centrifuge at 600×g for 5 min, and discard the supernatant; resuspend the cells in 200 μL of Washing Buffer / tube, transfer to a flow cytometry tube, and perform flow cytometry testing.

[0181] The results are as follows Figure 4Table 4 shows the competitive binding EC50 values ​​of antibodies Relalimab, H7L8(hG4WT), H7L9(hG4WT), and H7L10(hG4WT) as calculated using quantitative fluorescence analysis and curve fitting. 50 The values ​​are 1.153 nM, 1.342 nM, 1.317 nM, and 1.267 nM, respectively.

[0182] Table 4: Fluorescence intensity analysis of Relalimab, H7L8(hG4WT), H7L9(hG4WT), and H7L10(hG4WT) competitive binding to Raji cell surface antigen by FACS detection

[0183] <![CDATA[EC 50 (nM)]]> Relatlimab 1.153 H7L8(hG4WT) 1.342 H7L9(hG4WT) 1.317 H7L10(hG4WT) 1.267

[0184] The results showed that antibodies H7L8 (hG4WT), H7L9 (hG4WT), and H7L10 (hG4WT) could effectively block the binding of LAG-3 to MHC II on the surface of Raji host cells in a dose-dependent manner, and their activity was comparable to that of the positive control antibody Relatlimab.

[0185] Experimental Example 5: Detection of the biological activity of anti-LAG3 antibody in promoting IFN-γ and IL-2 secretion by mixed lymphocyte reaction (MLR)

[0186] 1. Detection of the bioactivity of anti-LAG3 antibody in promoting the secretion of IFN-γ by the Raji-PDL1 mixed lymphocyte reaction system.

[0187] Raji-PDL1 cells were routinely passaged and cultured; PBMCs were revived and cultured in 10 mL of RPMI 1640 complete medium, and stimulated for two days with 0.5 μg / mL SEB (Staphylococcal enterotoxin B) (Denotec, catalog number: S010201). Raji-PDL1 cells were treated with 25 μg / mL MMC (Stressmarq, catalog number: SIH-246-10MG) 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 collected, washed twice with PBS, and resuspended in complete medium (i.e., RPMI 1640 + 10% FBS) for counting. PBMCs and Raji-PDL1 cells were divided into 10 × 10⁻⁶ cells each. 4 Cells / well were added to a 96-well U-shaped plate (Corning, model: 3799) for co-culture. Antibodies were added according to the experimental design (final concentrations of each antibody were 300 nM, 30 nM, and 3 nM for both single and combined use), and the cells were co-cultured in an incubator for 3 days. After 3 days, the cells were centrifuged at 1200 rpm for 5 min, and the cell culture supernatant was collected for IFN-γ detection by ELISA.

[0188] like Figure 5As shown, co-culture of human PBMCs and Raji-PDL1 cells promotes IFN-γ secretion in PBMCs. The addition of antibodies to the co-culture system significantly induces further IFN-γ secretion in PBMCs. In terms of IFN-γ secretion-promoting activity, anti-LAG3 antibodies H7L8 (hG4WT), H7L9 (hG4WT), and H7L10 (hG4WT) combined with 14C12H1L1 (hG1TM), and positive control antibody Relatlimab combined with 14C12H1L1 (hG1TM) all promoted IFN-γ secretion, and their activities were comparable.

[0189] 2. Detection of the bioactivity of anti-LAG antibody in promoting IL-2 secretion by the Raji-PDL1 mixed lymphocyte reaction system

[0190] Raji-PDL1 cells were routinely passaged and cultured. PBMCs were revived and cultured in 10 mL of RPMI 1640 complete medium, followed by stimulation with 0.5 μg / mL SEB (Staphylococcal enterotoxin B, purchased from Denotech, catalog number: S010201) for two days. Raji-PDL1 cells were then treated with 25 μg / mL MMC (Stressmarq, catalog number: SIH-246-10MG) 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 collected, washed twice with PBS, and resuspended in complete medium (RPMI 1640 + 10% FBS) for counting. PBMCs and Raji-PDL1 cells were divided into 10 × 10⁻⁶ cells each. 4 Cells / well were added to a U-shaped 96-well plate (Corning, model: 3799) for co-culture. Antibodies were added according to the experimental design (the final concentrations of each antibody were 300 nM, 30 nM, and 3 nM for both single and combined use), and the cells were co-cultured for 3 days. After 3 days, the cells were centrifuged at 1200 rpm for 5 min, and the cell culture supernatant was collected for IL-2 detection by ELISA.

[0191] like Figure 6 As shown, co-culture of human PBMCs (from healthy donors) and Raji-PDL1 cells has a certain promoting effect on IL-2 secretion in PBMCs. The addition of antibodies to the co-culture system can significantly induce PBMCs to further secrete IL-2 in a significant dose-dependent manner. In terms of IL-2 secretion-promoting activity, anti-LAG3 antibodies H7L8 (hG4WT), H7L9 (hG4WT), and H7L10 (hG4WT) combined with 14C12H1L1 (hG1TM), and the positive control Relatlimab combined with 14C12H1L1 (hG1TM) can all promote IL-2 secretion, and their activities are comparable.

[0192] Experimental Example 6: Evaluation of the biological activity of anti-LAG antibodies in blocking the interaction between LAG-3 and MHC-II (reporter gene) Law)

[0193] Using Jurkat-NFAT-PD1-LAG3 cells and Raji cells as reporter gene systems, the addition of SEE superantigen activated the TCR-NFAT signaling pathway, inducing luciferase expression. LAG-3 on Jurkat cells binds to MHC-II on Raji cells, inhibiting the NFAT signaling pathway and downregulating luciferase expression. The antibody, by specifically binding to LAG-3, relieved the inhibition and upregulated luciferase expression.

[0194] Jurkat-NFAT-PD1-LAG3 cells and Raji cells (purchased from the Cell Resource Center of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, catalog number: TCHU 44) were collected, centrifuged at 110xg for 5 min, the supernatant was discarded, and the cells were resuspended in 1640+10% FBS medium and counted. Jurkat-NFAT-PD1-LAG3 cells were divided into groups of 10... 5 30 μL of cells / well was seeded into a 96-well black-background plate (Corning, model: 3916). Antibodies (final concentrations of 0.3 nM, 3 nM, and 300 nM) were added according to the experimental design, 10 μL / well, and the plates were pre-incubated at 37°C in a 5% CO2 incubator for 30 min. Simultaneously, SEE (Staphylococcal Enterotoxins E, purchased from Toxin Technology, catalog number: ET404) (final concentration 0.05 ng / mL) was added to Raji cells and incubated at 37°C in a 5% CO2 incubator for 30 min. After 30 min, the SEE-treated Raji cells were seeded at a rate of 2 × 10⁶ cells / well. 4 Add 40 μL / well (1 cell / well) to a 96-well plate containing Jurkat-NFAT-PD1-LAG3 cells, bringing the final volume to 80 μL. Mix well and incubate at 37°C for 6 hours in a 5% CO2 incubator. After incubation, remove the plate, allow it to equilibrate to room temperature, and add Bright-Glo... TM The Luciferase Assay System (purchased from Promega, catalog number: E2650) was 80 μL / well. After incubation in the dark for 2 min, the RLU value was read. The isotype control hIgG1DM was prepared by Akeso Biopharma, batch number: 20181107; the isotype control hG4WT was prepared by Akeso Biopharma, batch number: 20190910.

[0195] The results are as follows Figure 7As shown, anti-LAG antibodies H7L8 (hG4WT), H7L9 (hG4WT), H7L10 (hG4WT), and the positive control antibody Relatlimab can all block the interaction between LAG-3 and MHC-II and upregulate the expression of luciferase. Moreover, the activities of anti-LAG antibodies H7L8 (hG4WT), H7L9 (hG4WT), and H7L10 (hG4WT) are all superior to those of the control antibody Relatlimab.

[0196] Although specific embodiments of the invention have been described in detail, those skilled in the art will understand that various modifications and substitutions can be made to those details based on all the teachings disclosed, and all such changes are within the scope of protection of this invention. The full scope of this invention is given by the appended claims and any equivalents thereof.

Claims

1. An anti-LAG3 antibody or its antigen-binding fragment, comprising a heavy chain variable region and a light chain variable region, wherein, The heavy chain variable region comprises: amino acid sequences as shown in SEQ ID NOs: 9-11, namely HCDR1-HCDR3; and the light chain variable region comprises: amino acid sequences as shown in SEQ ID NOs: 12-14, namely LCDR1-LCDR3. The heavy chain variable region comprises: amino acid sequences as shown in SEQ ID NOs: 9-11, namely HCDR1-HCDR3; and the light chain variable region comprises: amino acid sequences as shown in SEQ ID NO: 12, SEQ ID NO: 15 and SEQ ID NO: 16, namely LCDR1-LCDR3; or The heavy chain variable region comprises: amino acid sequences HCDR1-HCDR3 as shown in SEQ ID NOs: 9-11 respectively; and the light chain variable region comprises: amino acid sequences LCDR1-LCDR3 as shown in SEQ ID NO: 17, SEQ ID NO: 15 and SEQ ID NO: 14 respectively.

2. The anti-LAG3 antibody or its antigen-binding fragment according to claim 1, wherein, The amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO: 2, and the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO: 4; The amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO: 2, and the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO:

6. or The amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO: 2, and the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO:

8.

3. The anti-LAG3 antibody or its antigen-binding fragment according to claim 1, wherein, The anti-LAG3 antibody or its antigen-binding fragment is selected from Fab, Fab', F(ab')2, Fv, single-chain antibody, humanized antibody or chimeric antibody.

4. The anti-LAG3 antibody or its antigen-binding fragment according to any one of claims 1 to 3, wherein, The anti-LAG3 antibody was expressed at an EC50 concentration of less than 0.2 nM. 50 Combined with human LAG3-mFc; wherein, the EC 50 Measured by indirect ELISA method.

5. The anti-LAG3 antibody or its antigen-binding fragment according to claim 4, wherein, The anti-LAG3 antibody was expressed at an EC50 concentration of less than 0.15 nM. 50 Combined with human LAG3-mFc.

6. The anti-LAG3 antibody or its antigen-binding fragment according to claim 4, wherein, The anti-LAG3 antibody was expressed at an EC50 concentration of less than 0.1 nM. 50 Combined with human LAG3-mFc.

7. The anti-LAG3 antibody or its antigen-binding fragment according to claim 4, wherein, The anti-LAG3 antibody was expressed at an EC50 concentration of less than 0.08 nM. 50 Combined with human LAG3-mFc.

8. The anti-LAG3 antibody or its antigen-binding fragment according to claim 4, wherein, The anti-LAG3 antibody was expressed at an EC50 concentration of less than 0.06 nM. 50 Combined with human LAG3-mFc.

9. The anti-LAG3 antibody or its antigen-binding fragment according to claim 4, wherein, The anti-LAG3 antibody was prepared at an EC50 concentration of less than 0.05 nM. 50 Combined with human LAG3-mFc.

10. The anti-LAG3 antibody or its antigen-binding fragment according to any one of claims 1 to 3, wherein, The anti-LAG3 antibody includes a non-CDR region, and the non-CDR region is derived from a human antibody.

11. The anti-LAG3 antibody or its antigen-binding fragment according to any one of claims 1 to 3, wherein, The anti-LAG3 antibody described herein has a constant region derived from human antibodies.

12. The anti-LAG3 antibody or its antigen-binding fragment according to any one of claims 1 to 3, wherein, The constant region of the anti-LAG3 antibody is selected from the constant region of human IgG1, IgG2, IgG3 or IgG4.

13. The anti-LAG3 antibody or its antigen-binding fragment according to any one of claims 1 to 3, wherein, The heavy chain constant region of the anti-LAG3 antibody is either the Ig gamma-1 chain C region or the Ig gamma-4 chain C region; the light chain constant region is the Ig kappa chain C region.

14. The anti-LAG3 antibody or its antigen-binding fragment according to claim 13, wherein, The amino acid sequence of the Ig gamma-1 chain C region is shown in SEQ ID NO:

18.

15. The anti-LAG3 antibody or its antigen-binding fragment according to claim 13, wherein, The amino acid sequence of the Ig gamma-4 chain C region is shown in SEQ ID NO:

20.

16. The anti-LAG3 antibody or its antigen-binding fragment according to claim 13, wherein, The amino acid sequence of the Ig kappa chain C region is shown in SEQ ID NO:

19.

17. An isolated nucleic acid molecule encoding the anti-LAG3 antibody as described in any one of claims 1 to 16.

18. A recombinant vector comprising the isolated nucleic acid molecule of claim 17.

19. A host cell comprising the isolated nucleic acid molecule of claim 17, or comprising the recombinant vector of claim 18.

20. A method for preparing the anti-LAG3 antibody or antigen-binding fragment thereof according to any one of claims 1 to 16, comprising culturing the host cell of claim 19 under suitable conditions, and recovering the antibody or antigen-binding fragment thereof from the cell culture.

21. A pharmaceutical composition comprising the anti-LAG3 antibody or its antigen-binding fragment as described in any one of claims 1 to 16.

22. The pharmaceutical composition of claim 21, further comprising pharmaceutically acceptable excipients.

23. Use of the anti-LAG3 antibody or its antigen-binding fragment as described in any one of claims 1 to 16 in the preparation of a medicament for treating or preventing tumors.

24. The use according to claim 23, wherein, The tumor is selected from one or more of the following: ovarian cancer, melanoma, hematologic malignancy, renal cell carcinoma, lung cancer, prostate cancer, bladder cancer, liver cancer, gastrointestinal cancer, breast cancer, brain cancer, pancreatic cancer, head and neck cancer, and kidney cancer.

25. The use according to claim 24, wherein, The lung cancer in question is non-small cell lung cancer.

26. The use according to claim 24, wherein, The hematologic tumor is leukemia.

27. The use according to claim 23, wherein, The tumor is selected from one or more of glioblastoma, thyroid cancer, esophageal cancer, colon cancer, and rectal cancer.

28. The use according to claim 27, wherein, The esophageal cancer mentioned is esophageal squamous cell carcinoma.

Images