An anti-human LAG-3 monoclonal antibody and a preparation method and application thereof

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to an anti-human LAG-3 monoclonal antibody, a preparation method and application thereof, and simultaneously provides a coding nucleic acid molecule, an expression vector, a host cell of the antibody, and a method for expressing the antibody, and further provides a pharmaceutical composition containing the antibody and use thereof. The anti-human LAG-3 monoclonal antibody has high binding affinity to human LAG-3, can block the combination of human LAG-3 and MHCII protein, but has differential characteristics, i.e. does not block or weakly blocks the combination of human LAG-3 and human FGL1 protein.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to an anti-human LAG-3 monoclonal antibody and a preparation method and application thereof. Background Art

[0002] Lymphocyte activation gene-3 or LAG-3 (also known as CD223) is a member of the immunoglobulin supergene family and is expressed on activated T cells (Huard et al. (1994) Immunogenetics 39:213), NK cells (Triebel et al. (1990) J. Exp. Med. 171:1393-1405), regulatory T cells (Huang et al. (2004) Immunity 21:503-513; Camisaschi et al. (2010) J Immunol. 184:6545-6551; Gagliani et al. (2013) Nat Med 19:739-746), and plasmacytoid dendritic cells (DCs) (Workman et al. (2009) J Immunol 182:1885-1891). LAG-3 is a membrane protein encoded by a gene located on chromosome 12 and is structurally and genetically related to CD4.

[0003] The role of LAG-3 as a negative regulator of T-cell responses is based on studies with LAG-3 knockout mice and the use of blocking anti-LAG-3 antibodies in in vitro and in vivo systemic models.

[0004] The most well-characterized ligand for LAG-3 is MHC II (Major Histocompatibility Complex class II). On the cell surface, LAG-3 is expressed as a dimer, which is required for the formation of a stable MHC class II binding site (Huard B et al. (1997) Proc Natl Acad Sci USA 94:5744-9). The MHC II transactivator (CIITA) has been shown to be a key regulator of LAG-3 ligands. CIITA induces the expression of MHC II and MHC II accessory molecules, including CD74 (invariant chain, Ii) and H2-DM.

[0005] Another ligand for LAG-3 is fibrinogen-like protein-1 (FGL1) (Jun Wang et al. (2019) Cell 176:1-14). FGL1 is a protein primarily expressed in the liver and is considered a liver protector and hepatocyte mitogen. Studies have found that mice lacking FGL1 (FGL1 knockout mice) are more than twice as likely to develop hepatocellular carcinoma (HCC) induced by a chemical carcinogen (diethylnitrosamine) as wild-type mice, indicating that FGL1 acts as a tumor suppressor gene in liver cancer through an Akt-dependent mechanism, supporting its potential as a target for liver cancer treatment (Hamed Nayeb et al. (2015) Biochemical and Biophysical Research Communications 465:167-173).

[0006] Currently, there are few clinical products, and there is a need to develop antigen-binding proteins that can bind to LAG-3 and block the interaction between LAG-3 and MHCII, while not blocking or weakly blocking the binding of LAG-3 and FGL1. Summary of the Invention

[0007] The present invention provides an anti-human LAG-3 monoclonal antibody, a preparation method, and an application thereof. The monoclonal antibody has a high binding affinity to human LAG-3 and can block the interaction between LAG-3 and MHCII while not blocking the interaction between LAG-3 and FGL1.

[0008] The technical solutions provided by the present invention are as follows:

[0009] The present invention provides an anti-human LAG-3 monoclonal antibody, wherein the antibody comprises a heavy chain variable region and a light chain variable region;

[0010] The heavy chain variable region comprises CDR-H1, CDR-H2 and CDR-H3, and the light chain variable region comprises CDR-L1, CDR-L2 and CDR-L3;

[0011] The amino acid sequence of the CDR-H1 is shown in SEQ ID NO: 3;

[0012] The amino acid sequence of the CDR-H2 is shown in SEQ ID NO: 4;

[0013] The amino acid sequence of the CDR-H3 is shown in SEQ ID NO: 5;

[0014] The amino acid sequence of the CDR-L1 is shown in SEQ ID NO: 6;

[0015] The amino acid sequence of the CDR-L2 is shown in SEQ ID NO: 7;

[0016] The amino acid sequence of the CDR-L3 is shown in SEQ ID NO: 8.

[0017] Preferably, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 1; and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 2.

[0018] Preferably, both the heavy chain and the light chain include a constant region, and the amino acid sequence of the heavy chain constant region is shown in SEQ ID NO: 9; the amino acid sequence of the light chain constant region is shown in SEQ ID NO: 10.

[0019] Preferably, both the heavy chain and the light chain further comprise a constant region, which is a constant region of mouse or human IgG, preferably a constant region of IgG1.

[0020] The present invention further provides a nucleotide molecule encoding the anti-human LAG-3 monoclonal antibody.

[0021] Preferably, the sequence of the nucleotide molecule is selected from SEQ ID NO: 11 and SEQ ID NO: 12;

[0022] The sequence SEQ ID NO: 11 encodes the heavy chain variable region of the antibody;

[0023] The sequence SEQ ID NO: 12 encodes the light chain variable region of the antibody.

[0024] The present invention further provides an expression vector containing the nucleotide molecule.

[0025] The present invention further provides a host cell containing the expression vector.

[0026] Preferably, the host cell is a eukaryotic cell, preferably a mammalian cell.

[0027] The present invention further provides a method for preparing an anti-human LAG-3 monoclonal antibody, comprising the following steps:

[0028] (1) preparing an expression vector containing a nucleotide molecule for expressing the anti-human LAG-3 monoclonal antibody;

[0029] (2) transfecting eukaryotic host cells with the expression vector obtained in step (1) and culturing the cells;

[0030] (3) Isolation and purification to obtain anti-human LAG-3 monoclonal antibody.

[0031] The present invention further provides an antibody immunoconjugate, a bispecific molecule, a chimeric antigen receptor or a pharmaceutical composition comprising the anti-human LAG-3 monoclonal antibody.

[0032] Furthermore, the pharmaceutical composition comprises a therapeutically effective amount of the anti-human LAG-3 monoclonal antibody and one or more pharmaceutically acceptable carriers, diluents or excipients.

[0033] The present invention further provides the use of the anti-human LAG-3 monoclonal antibody in the preparation of anti-tumor, anti-infection or autoimmune disease drugs.

[0034] Preferably, the autoimmune diseases include psoriasis, Crohn's disease, rheumatoid arthritis, primary biliary cirrhosis, systemic lupus erythematosus (SLE), syndrome, multiple sclerosis, ulcerative colitis, and autoimmune hepatitis.

[0035] Preferably, the tumor includes ovarian cancer, melanoma, prostate cancer, intestinal cancer, gastric cancer, esophageal cancer, breast cancer, lung cancer, kidney cancer, pancreatic cancer, uterine cancer, liver cancer, bladder cancer, cervical cancer, oral cancer, brain cancer, testicular cancer, skin cancer, and thyroid cancer.

[0036] Beneficial effects:

[0037] The anti-human LAG-3 monoclonal antibody of the present invention has high binding affinity for human LAG-3 and strong LAG-3-MHCII blocking activity. Furthermore, since FGL1 has anti-tumor effects, the antibody of the present invention's relatively weak blocking ability against LAG-3-FGL1 offers potential advantages in tumor treatment. It can be used to treat autoimmune diseases, infectious diseases, allergic diseases, and cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 Capture ELISA was used to determine the antibody's ability to bind to human LAG-3 protein;

[0039] Figure 2 Indirect ELISA was used to determine the antibody binding ability to mouse LAG-3 protein;

[0040] Figure 3 Indirect ELISA was used to determine the antibody binding ability to cynomolgus monkey LAG-3 protein;

[0041] Figure 4 Flow cytometry was used to evaluate the binding of antibodies to 293T cells overexpressing human LAG-3 on their surface;

[0042] Figure 5The ligand binding blocking ELISA was used to evaluate the antibody's ability to block the binding between human LAG-3 and human FGL1.

[0043] Figure 6 Flow cytometry was used to evaluate the ability of antibodies to block the binding of human LAG-3 to MHCII protein expressed on the surface of Daudi cells. DETAILED DESCRIPTION

[0044] the term

[0045] "LAG-3" refers to lymphocyte activation gene-3. The term "LAG-3" encompasses variants, isoforms, homologs, orthologs, and paralogs. For example, in certain instances, antibodies specific for human LAG-3 protein may cross-react with LAG-3 protein from species other than human. In other embodiments, antibodies specific for human LAG-3 protein may be completely specific for human LAG-3 protein and exhibit no species or other types of cross-reactivity, or may cross-react with LAG-3 from certain other species, but not all other species (e.g., cross-react with monkey LAG-3 but not with mouse LAG-3). The term "human LAG-3" refers to human sequence LAG-3, for example, human LAG-3 having the amino acid sequence of SEQ ID NO: P18627 (Leu23-Val450).

[0046] "Antigen binding site" refers to a discrete, three-dimensional site on an antigen that is recognized by the antibodies or antigen-binding fragments herein.

[0047] "Monoclonal antibody" refers to a preparation of antibody molecules having a single amino acid composition, and does not refer to the method by which they are produced. Monoclonal antibodies or antigen-binding fragments thereof can be produced, for example, by hybridoma technology, recombinant technology, phage display technology, synthetic techniques such as CDR grafting, or a combination of these or other techniques known in the art.

[0048] "Affinity" refers to the strength of the sum of all non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, herein, "binding affinity" refers to intrinsic binding affinity, which reflects a 1:1 interaction between an antibody and an antigen. Affinity can be measured by common methods known in the art, including those known in the art and described herein.

[0049] The term "compete" when used in the context of antigen-binding proteins (e.g., neutralizing antigen-binding proteins or neutralizing antibodies) that compete for the same epitope refers to competition between antigen-binding proteins, as determined by an assay in which the antigen-binding protein (e.g., an antibody or immunologically functional fragment thereof) to be tested prevents or inhibits (e.g., reduces) specific binding of a reference antigen-binding protein (e.g., a ligand or reference antibody) to a common antigen (e.g., LAG-3 or a fragment thereof). Numerous types of competitive binding assays can be used to determine whether one antigen-binding protein competes with another. Competitive inhibition is measured by measuring the amount of label bound to a solid surface or cell in the presence of the test antigen-binding protein. Typically, the test antigen-binding protein is present in excess. Antigen-binding proteins identified by competitive assays (competing antigen-binding proteins) include: antigen-binding proteins that bind to the same epitope as the reference antigen-binding protein; and antigen-binding proteins that bind to a neighboring epitope sufficiently close to the binding epitope of the reference antigen-binding protein that the two epitopes sterically hinder each other from binding.

[0050] Methods for producing and purifying antibodies and antigen-binding fragments are well known and disclosed in the art, such as the Cold Spring Harbor Laboratory Manual of Antibody Laboratory Techniques. For example, mice can be immunized with human LAG-3 or fragments thereof, and the resulting antibodies can be renatured, purified, and subjected to amino acid sequencing using conventional methods. Antigen-binding fragments can also be prepared using conventional methods.

[0051] "Treatment" means administering a therapeutic agent, such as a composition comprising a LAG-3 antibody or antigen-binding fragment thereof, to a patient experiencing one or more symptoms of a disease. Generally, the therapeutic agent is administered in an amount effective to alleviate one or more symptoms of the disease in the patient or population being treated, whether by inducing regression of such symptoms or inhibiting progression of such symptoms to any clinically measurable degree. The amount of therapeutic agent effective to alleviate any particular disease symptom (also referred to as a "therapeutically effective amount") can vary depending on a variety of factors, such as the patient's disease state, age, and weight, and the ability of the drug to produce the desired therapeutic effect in the patient. Whether a disease symptom has been alleviated can be assessed by any clinical test commonly used by a physician or other health care professional to assess the severity or progression of such symptoms.

[0052] An "effective amount" encompasses an amount sufficient to ameliorate or prevent the symptoms or conditions of a medical condition. An effective amount also means an amount sufficient to permit or facilitate diagnosis. The effective amount for a particular patient or veterinary subject may vary depending on factors such as the condition to be treated, the patient's overall health, the route and dosage of administration, and the severity of side effects. An effective amount can be the maximum dose or dosage regimen that avoids significant side effects or toxic effects.

[0053] A "pharmaceutical composition" refers to a mixture containing one or more LAG-3 antibodies or antigen-binding fragments thereof described herein and other pharmaceutical ingredients, such as physiologically / pharmaceutically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration to an organism, promote absorption of the active ingredient, and thereby exert its biological activity.

[0054] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the invention. The experimental methods in the following examples, for which specific conditions are not specified, are generally performed under conventional conditions or according to the conditions recommended by the manufacturer. Reagents for which specific sources are not specified are conventional reagents purchased from the market.

[0055] Example 1 Obtaining a specific anti-LAG-3 mouse monoclonal antibody by fusion hybridoma technology

[0056] 1.1 Animal immunization

[0057] LAG-3KO mice (Shanghai Model Organisms Science Co., Ltd.) were immunized according to the method described in the literature (E Harlow, D. Lane, Antibody: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1998). Human LAG-3 protein (prepared in-house, sequence number P18627, Leu23-Val450) was used as the immunogen.

[0058] To increase the immune response, Freund's complete adjuvant and Freund's incomplete adjuvant (Sigma, St. Louis, Mo., USA) were used for the first immunization and booster immunization, respectively. Briefly, the preparation of the adjuvant-antigen mixture first involves gently mixing the adjuvant in a vial using a vortex method. The required amount of adjuvant is removed from the vial and placed in an autoclaved 1.5 mL microcentrifuge tube. The antigen is prepared in PBS or normal saline at a concentration of 0.5-1.0 mg / ml. The calculated amount of antigen is added to the microcentrifuge tube together with the adjuvant, gently stirred for 2 minutes, and the emulsification mixture is repeated to form an oil-in-water solution. The adjuvant-antigen solution is then drawn into an appropriate syringe for animal injection. Each animal is immunized, and then 2 to 3 booster immunizations are performed depending on the antiserum titer. Animals with good titers are given a final immunization by intraperitoneal injection before fusion.

[0059] 1.2 Hybridoma fusion and screening

[0060] Prior to cell fusion, mouse myeloma cells (SP2 / 0-Ag14, ATCC #CRL-1581) were cultured in the logarithmic growth phase. Immunized mice were sacrificed and spleens were removed under sterile conditions and fused with myeloma cells according to the method described by Kohler G and Milstein C in "Continuous cultures of fused cells secreting antibodies of predefined specificity," Nature, 256:495-497 (1975).

[0061] The fused "hybrid cells" are then plated into 96-well cell plates containing HAT culture medium. Surviving hybridoma cells can typically be observed under a microscope 7-10 days after fusion. Two weeks after cell plating, the culture supernatant from each well is collected and hybridoma screening is performed using an ELISA assay using recombinant human LAG-3-Fc protein antigen. Briefly, ELISA plates are coated overnight at 4°C with human LAG-3-Fc protein (cat#16498-H02H, Sino Biological Inc., 2.0 μg / ml in PBS). The plates are washed four times with PBST and then blocked with blocking buffer (PBST containing 5% nonfat dry milk). Diluted mouse immune serum (for determination of mouse serum titer) or hybridoma supernatant is added to each well and incubated at 37°C for 40 minutes. The plate was washed four times with PBST and detected using GAM(Fc)-HRP (Jackson ImmunoResearch, cat# 115-035-071). The absorbance of each well was measured at 450 nm. Positive hybridomas secreting antibodies that bind to human LAG-3-Fc were then selected and transferred to a 24-well plate.

[0062] Hybridoma clones that produce antibodies that bind to human LAG-3 with high specificity and have LAG-3 / MHCII ligand blocking activity are subcloned by limiting dilution to ensure the monoclonality of the cell line, obtaining mouse monoclonal hybridoma cell lines. Mouse monoclonal antibodies are then expressed and secreted in cell culture and purified.

[0063] Example 2 Study on Binding Activity of Mouse Anti-LAG-3 Monoclonal Antibodies

[0064] The mouse anti-LAG-3 monoclonal antibodies (mAbs) produced by the hybridoma clones in Example 1 were further tested for their binding activity using the following method.

[0065] Relatlimab (BRISTOL-MYERS SQUIBB COMPANY, BMS) was used as a reference antibody (prepared in-house, the heavy chain amino acid sequence is shown in SEQ ID NO: 13, and the light chain amino acid sequence is shown in SEQ ID NO: 14).

[0066] 2.1 Determination of Antibody Binding Capacity Based on Capture ELSIA

[0067] A 96-well ELISA plate was coated with 100 μl / well of GAM-Fab (Jackson immunoResearch, cat#115-005-072) or GAH-Fab (Jackson immunoResearch, cat#109-005-097) in PBS at a final concentration of 2 μg / ml and incubated overnight at 4°C. The plate was washed once with elution buffer (PBS + 0.05% v / v Tween-20, PBST) and then blocked overnight at 4°C with 200 μl / well of 5% w / v nonfat dry milk in PBST. The plate was washed again and incubated with 100 μl / well of various concentrations of LAG-3 mouse monoclonal antibody or control antibody (starting at 66.7 nM, serially diluted 5-fold in PBST containing 2.5% nonfat dry milk) at 37°C for 40 minutes. The plate was then washed four times. The ELISA plate containing the capture LAG-3 antibody was incubated with 100 μl / well of biotinylated human LAG-3 protein (LAG-3-Fc-bio, cat# LA3-H82Fb, Acro) at 37°C for 40 minutes. The plate was then washed four times and incubated with streptavidin-conjugated horseradish peroxidase SA-HRP (1:10,000 dilution in PBST, Jackson Immuno Research, cat# 016-030-084, 100 μl / well) at 37°C for 40 minutes. After the final wash, the ELISA plate was incubated with 100 μl / well of ELISA substrate TMB (Innoreagents, #TMB-S-002). The reaction was terminated with 50 μl / well of 1 M H2SO4 at 25°C within 15 minutes, and the absorbance at 450-630 nm was measured. The results are shown in Table 1. Figure 1 and Table 1.

[0068] Figure 1 The results shown in Table 1 indicate that the antibody G1A7 of the present invention has good binding ability to human LAG-3 protein, and its binding ability is stronger than that of the reference antibody Relatlimab.

[0069] 2.2 Determination of antibody binding capacity based on indirect ELSIA

[0070] The cross-reactivity of LAG-3 antibodies with mouse LAG-3 protein and cynomolgus monkey LAG-3 protein was tested.

[0071] Briefly, 100 μl / well of 2 μg / ml mouse LAG-3-his (prepared in-house; mouse LAG-3 protein sequence referenced Uniprot database #Q61790, Gly24-Leu442) or 2 μg / ml monkey LAG-3-his (cat#90841-C08H, Sino Biological Inc.) was added to a 96-well ELISA plate and incubated at 37°C for 2 h. The plate was washed once with PBST (PBS + 0.05% Tween-20) and blocked with 200 μl of blocking buffer (5% w / v skim milk powder in PBST) at 37°C for 2 h. The plate was washed again and incubated with 100 μl / well of serially diluted anti-LAG-3 antibody or control antibody (starting at 66.7 nM, serially diluted 5-fold in PBST containing 2.5% skim milk powder) at 37°C for 2 h. The ELISA plate was washed four times and incubated at 37°C for 40 minutes with GAM (Fab)-HRP or GAH (Fab)-HRP (cat#115-035-006 or cat#109-035-097, Jackson ImmunoResearch Laboratories, Inc., 100 μl / well). After the final wash, 100 μl / well of TMB (cat#TMB-S-002, Innoreagents) was added and allowed to react at room temperature for 40 minutes. The reaction was then terminated with 1 M H2SO4 and the absorbance at 450-630 nm was measured. The data were analyzed using Graphpad Prism software to obtain the EC values. 50 For specific results, see Figure 2 、 Figure 3 and Table 1.

[0072] The results showed that the antibody G1A7 of the present invention did not bind to either mouse LAG-3 or cynomolgus monkey LAG-3 proteins, which was consistent with the performance of the reference antibody.

[0073] 2.3 Flow cytometry (FACS) analysis of the binding of LAG-3 monoclonal antibodies to 293T cell lines overexpressing human LAG-3

[0074] First, 293T cells were transfected with a plasmid containing the human LAG-3 protein coding sequence to construct a 293T cell line that overexpresses LAG-3 on its surface. Then, the stable 293T cell line overexpressing human LAG-3 on its surface was collected from the cell culture flask, washed twice, and resuspended in PBS phosphate buffered saline (FACS buffer) containing 2% v / v fetal bovine serum. 1×10 5Cells were incubated on ice for 40 minutes with FACS buffer containing different concentrations of LAG-3 antibodies or reference antibodies (starting from 10 μg / ml and serially diluted 5-fold). The cells were washed twice with FACS buffer and 100 μL / well of GAM-PE or GAH-PE (cat#115-116-146 or cat#109-115-098, Jackson Immunoresearch, diluted 1:1000 in FACS buffer) was added. After incubation at 4°C for 40 minutes, the cells were washed twice and resuspended in FACS buffer. Fluorescence measurements were performed using a Becton Dickinson FACS Canto II-HTS instrument. Data were analyzed using Graphpad Prism software to obtain the EC values ​​of antibody-bound cells. 50 The concentration value is the antibody concentration value corresponding to the maximum fluorescence binding signal of 50% when the LAG-3 antibody binds to the cells overexpressing LAG-3. The measurement results are shown in Figure 4 and Table 1.

[0075] The results showed that the antibody G1A7 of the present invention has stronger binding ability to 293T cells overexpressing human LAG-3 on their surface than the reference antibody Relatlimab.

[0076] Table 1. Binding activity of mouse anti-LAG-3 antibodies

[0077]

[0078] Example 3 Competitive Functional Blockade of Mouse Anti-LAG-3 Monoclonal Antibodies Against LAG-3-FGL1 or the Interaction of LAG-3 with Daudi Cells Expressing MHCII

[0079] 3.1 Ligand blocking ELISA

[0080] The ability of the anti-LAG-3 antibodies of the present invention to block the LAG-3-FGL1 interaction was assessed using a competitive ELISA. Briefly, plates were coated with 100 μg / well of human FGL1-Fc protein (prepared in-house; the amino acid sequence of FGL1 is referenced in the NCBI database #NP_004458.3, Glu19-Ile312) and incubated at 37°C for 2 hours. Plates were then washed with wash buffer (PBS + 0.05% Tween-20, PBST) and blocked with PBST containing 5% w / v skim milk powder for 2 hours at 37°C. LAG-3 antibody, hIgG, and Relatlimab were serially diluted four-fold in DMEM and mixed with 3 μg / ml of biotinylated human LAG-3 protein (LAG-3-Fc-bio) before preincubation at room temperature for 1 hour. The incubated mixture was then added to the FGL1-Fc-coated plate and incubated at 37°C for 1 hour. The plate was washed four times, and then SA-HRP was added and incubated at 37°C for 1 hour to detect the binding of biotinylated human LAG-3-Fc-bio to the bottom plate FGL1. The plate was then washed with wash buffer. Finally, TMB was added, and the reaction was terminated with 1M H2SO4. The absorbance at 450-630 nm was measured. The data were analyzed using Graphpad Prism software to obtain the IC 50 For specific results, see Figure 5 .

[0081] The results showed that the antibody G1A7 of the present invention had weak blocking activity against LAG-3-FGL1, while the reference antibody was able to block the LAG-3-FGL1 interaction. Because FGL1 has anti-tumor effects that can prevent rapid tumor growth, the antibody of the present invention's weak blocking ability against LAG-3-FGL1 has potential advantages in tumor treatment.

[0082] 3.2 Cell-based ligand blocking FACS

[0083] The ability of LAG-3 antibody to block the binding of human LAG-3 and MHCII protein expressed on the surface of Daudi cells was evaluated by flow cytometry (FACS) using Daudi cells expressing human MHCII on their surface (purchased from the Cell Bank of Shanghai Chinese Academy of Sciences).

[0084] Anti-LAG-3 antibody and reference antibody (antibody concentration starting at 66.67 nM, serially diluted 5-fold) were mixed with 0.4 μg / ml LAG-3-Fc (cat#16498-H02H, Sino Biological Inc.) and incubated at room temperature for 40 minutes. Daudi cells were harvested from cell culture flasks during logarithmic growth, washed twice, and resuspended in PBS containing 2% v / v fetal bovine serum (FACS buffer). 1 x 10 cells per well of a 96-well plate were used. 5 Daudi cells were then plated with 100 μl / well of a LAG-3 antibody / LAG-3-Fc mixture and incubated at 4°C for 40 minutes. The cells were washed twice with FACS buffer, and then 100 μl / well of GAH-PE (1:1000 dilution in FACS buffer) was added to detect LAG-3-Fc binding to Daudi cells and incubated at 4°C in the dark for 40 minutes. The cells were washed twice and resuspended in FACS buffer. Fluorescence measurements were performed using a Becton Dickinson FACS Canto II-HTS instrument. Data were analyzed using Graphpad Prism software, and IC values ​​were reported. 50 The result is as follows Figure 6 shown.

[0085] The results showed that the antibody G1A7 of the present invention can block the binding of human LAG-3 to the MHCII protein expressed on the surface of Daudi cells, and the blocking ability is comparable to that of the reference antibody Relatlimab.

[0086] Example 4 DNA cloning and sequencing, sequence analysis of anti-LAG-3 antibodies

[0087] Total RNA was extracted from the hybridoma cells of Example 1 using Trizol reagent (Invitrogen, catalog #15596-018).

[0088] The process is briefly described as follows: 5×10 6Place the cells in a 1.5ml centrifuge tube and aspirate the supernatant. Add 1ml of Trizol reagent and pipette repeatedly several times, then place at 25℃ for 5 minutes to lyse the cells. Next, add 0.2ml of chloroform solution to each tube, shake vigorously for 15 seconds, and place at room temperature for 3 minutes. Then, centrifuge the tube at 4℃ and 12000g for 10 minutes, remove the centrifuge tube, aspirate the upper aqueous phase solution into a new 1.5ml centrifuge tube, and add 0.4ml of isopropanol to precipitate RNA from the aqueous phase. Manually mix the EP tube and place it at 25℃ for 10 minutes, then centrifuge at 4℃ and 12000g for 10 minutes, and discard the supernatant. Add 1ml of 75% ethanol, centrifuge again at 4℃ and 7500rpm for 5 minutes, and discard the supernatant. After the RNA precipitate at the bottom of the tube is dried at room temperature for 10 minutes, add 30 to 50ul of sterile DEPC-treated water to dissolve the RNA sample.

[0089] Next, total RNA was converted to cDNA using Taraka's Reverse Transcription cDNA Kit (catalog #6110A). The experimental system was prepared as follows: 5 μl of total RNA, 0.5 μl of Oligo(dT), and 8.5 μl of RNase-free water (14 μl total) were pre-denatured at 65°C for 5 minutes, followed by 2 minutes on ice. 4 μl of 5× buffer, 1 μl of dNTP mix, 0.5 μl of RNase inhibitor, and 1 μl of reverse transcriptase (20.5 μl total) were added, mixed, and incubated at 40°C for 50 minutes, followed by 70°C for 10 minutes to complete cDNA synthesis. The cDNA was further added with poly-G at the 3' end. The reaction system was prepared as follows: 5μl of cDNA sample + 33.5μl of ddH2O + 5μl of 10×TdT buffer + 5μl of CoCl2 + 1μl of dGTP + 0.5μl of terminal deoxynucleotidyl transferase (total volume 50ul), incubated at 37°C for 30 minutes, and then at 70°C for 10 minutes to complete the poly-G tailing.

[0090] Furthermore, the tailed cDNA was used as a template for gene amplification of the antibody variable region. For amplifying the antibody heavy chain variable region sequence, the PCR reaction system was prepared: 5 μl of 10× Taq enzyme buffer + 0.5 μl of universal poly C primer (forward primer) + 0.5 μl of mouse IgG1 reverse primer + 1 μl of dNTP + 1 μl of Taq polymerase + 1 μl of cDNA + 41 μl of ddH2O. For amplifying the antibody light chain variable region sequence, the PCR reaction system was prepared: 5 μl of 10× Taq enzyme buffer + 0.5 μl of universal poly C primer (forward primer) + 0.5 μl of mouse IgG kappa chain reverse primer + 1 μl of dNTP + 1 μl of Taq polymerase + 1 μl of cDNA + 41 μl of ddH2O. The temperature cycle for PCR amplification of the antibody heavy and light chain variable regions is as follows (steps 2 to 4 are repeated 25 cycles):

[0091] 1) Pre-denaturation at 95°C for 5 min;

[0092] 2) Denaturation at 95°C for 20 seconds;

[0093] 3) Annealing at 56°C for 20 seconds;

[0094] 4) Extension at 72°C for 30 seconds;

[0095] 5) Store at 25°C for 60 minutes.

[0096] The PCR products were analyzed by 1% agarose gel electrophoresis, and DNA segments of corresponding sizes were cut out (VH approximately 600 bp, VK approximately 500 bp). DNA was extracted using the QIAquick Gel DNA Recovery Kit (catalog #28704). The procedure is as follows: the gel was weighed, 3 times the volume of QG buffer was added, and then incubated at 50°C for 10 minutes until the gel was completely dissolved. After adding 1 times the volume of isopropanol to mix, the sample was transferred to a QIA purification column and centrifuged at 13,000 rpm for 1 minute. 750 μl of PE buffer was added to the column, and then centrifuged at 13,000 rpm for 1 minute. The residual liquid in the column was removed by centrifugation again at 13,000 rpm. 30 μl of water was added and centrifuged at 13,000 rpm for 1 minute for elution to obtain the prepared DNA sample. The purified PCR product was sequenced to obtain the variable region sequence of the antibody.

[0097] The sequence information of the clones of the present invention is shown in Table 2.

[0098] Table 2. Amino acid sequence of anti-human LAG-3 antibody G1A7

[0099]

[0100] VH nucleotide sequence of anti-human LAG-3 antibody G1A7:

[0101] GAGGTCCAGCTGCAACAGTCTGGACCTGTGCTGGTGAAGCCTGGGGCTTCAGTGAAGATGTCCTGTAAGGCTTCTGGATACACTTTCACTCACTACTATATGAACTGGGTGAAACAGAGCCATGGAAAGAGCCTTGAATGGATTGGAGTTATTAATCCTTACAACGGTGATACTAGCTACAAGCAGAACTTCAAGGGCAAGGCCACATTGACTGTTGACAAGACCTCCAGCACAGCCTACATGGACCTCAACAGCCTGACATCTGAGGACTCTGCAGTCTATTACTGTGCAAGAGATGATGGTTACTACCAGTGGTACTTCGATGTCTGGGGCACAGGGACCACGGTCACCGTCTCCTCG(SEQ ID NO:

[0102] VL nucleotide sequence of anti-human LAG-3 antibody G1A7:

[0103] GACATCCAGCTGACCCAGTCTCCATCCTCCTTATCTGCCTCTCTGGGAGAAAGAGTCAGTCTCACTTGTCGGGCAAGTCAGGACATTGGTAGTAGGTTAATCTGGCTTCAGCAGGGACCAGATGGAACTATTAAACGCCTGATCTTCGCCACATCCAGTTTAGATTCTGGTGTCCCCAAAAGGTTCAGTGGCAGTAGGTCTGGGTCAGATTATTCTCTCACCATCAGCAGCCTTGAGTCTGAAGATTTTGTCGACTATTACTGTCTACAATGTGCTAGTTCTCCTCCGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA(SEQ ID NO:

[0104] Heavy chain amino acid sequence of Relatlimab:

[0105] QVQLQQWGAGLLKPSETLSLTCAVYGGSFSDYYWNWIRQPPGKGLEWIGEINHRGSTNSNPSLKSRVTLSLDTSKNQFSLKLRSVTAADTAVYYCAFGYSDYEYNWFDPWGQ GTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPP CPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISK AKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO:13)

[0106] Relatlimab light chain amino acid sequence:

[0107] EIVLTQSPATLSLSPGERATLSCRASQSISSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPLTFGQGTNLEIKRT VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ IDNO:14)

[0108] Although the present invention has been described in detail through the above preferred embodiments, it should be understood that the above description is not intended to limit the present invention. After reading the above description, various modifications and substitutions of the present invention will become apparent to those skilled in the art. Therefore, the scope of protection of the present invention should be defined by the appended claims. Sequence Listing <110> BioScience Biotechnology (Nanjing) Co., Ltd. <120> An anti-human LAG-3 monoclonal antibody and its preparation method and application <160> 14 <170> SIPO Sequence Listing 1.0 <210> 1 <211> 120 <212> PRT <213> Artificial Sequence <400> 1 Glu Val Gln Leu Gln Gln Ser Gly Pro Val Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr His Tyr 20 25 30 Tyr Met Asn Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile 35 40 45 Gly Val Ile Asn Pro Tyr Asn Gly Asp Thr Ser Tyr Lys Gln Asn Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Thr Ser Ser Thr Ala Tyr 65 70 75 80 Met Asp Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Tyr Met Asn Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile Ala Arg Asp Asp Gly Tyr Tyr Gln Trp Tyr Phe Asp Val Trp Gly Thr 100 105 110 Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 2 <211> 107<<212> PRT <213> Artificial Sequence <400> 2 Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly 1 5 10 15 Glu Arg Val Ser Leu Thr Cys Arg Ala Ser Gln Asp Ile Gly Ser Arg 20 25 30 Leu Ile Trp Leu Gln Gln Gly Pro Asp Gly Thr Ile Lys Arg Leu Ile 35 40 45 Phe Ala Thr Ser Ser Leu Asp Ser Gly Val Pro Lys Arg Phe Ser Gly 50 55 60 Ser Arg Ser Gly Ser Asp Tyr Ser Leu Thr Ile Ser Ser Leu Glu Ser 65 70 75 80 Glu Asp Phe Val Asp Tyr Tyr Cys Leu Gln Cys Ala Ser Ser Pro Pro 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 3 <211> 5 <212> PRT <213> Artificial Sequence <400> 3 His Tyr Tyr Met Asn 1 5 <210> 4 <211> 17 <212> PRT <213> Artificial Sequence <400> 4 Val Ile Asn Pro Tyr Asn Gly Asp Thr Ser Tyr Lys Gln Asn Phe Lys 1 5 10 15 Gly <210> 5 <211> 11 <212> PRT <213> Artificial Sequence <400> 5 Asp Asp Gly Tyr Tyr Gln Trp Tyr Phe Asp Val 1 5 10 <210> 6 <211> 11 <212> PRT <213> Artificial Sequence <400> 6 Arg Ala Ser Gln Asp Ile Gly Ser Arg Leu Ile 1 5 10 <210> 7 <211> 7 <212> PRT <213> Artificial Sequence <400> 7 Ala Thr Ser Ser Leu Asp Ser 1 5 <210> 8 <211> 9 <212> PRT <213> Artificial Sequence <400> 8 Leu Gln Cys Ala Ser Ser Pro Pro Thr 1 5 <210> 9 <211> 335 <212> PRT <213> Artificial Sequence <400> 9 Ala Lys Thr Thr Ala Pro Ser Val Tyr Pro Leu Ala Pro Val Cys Gly 1 5 10 15 Gly Thr Thr Gly Ser Ser Val Thr Leu Gly Cys Leu Val Lys Gly Tyr 20 25 30 Phe Pro Glu Pro Val Thr Leu Thr Trp Asn Ser Gly Ser Leu Ser Ser 35 40 45 Gly Val His Thr Phe Pro Ala Leu Leu Gln Ser Gly Leu Tyr Thr Leu 50 55 60 Ser Ser Ser Val Thr Val Thr Ser Asn Thr Trp Pro Ser Gln Thr Ile 65 70 75 80 Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys 85 90 95 Ile Glu Ser Arg Arg Pro Ile Pro Pro Asn Ser Cys Pro Pro Cys Lys 100 105 110 Glu Cys Ser Ile Phe Pro Ala Pro Asp Leu Leu Gly Gly Pro Ser Val 115 120 125 Phe Ile Phe Pro Pro Lys Ile Lys Asp Val Leu Met Ile Ser Leu Ser 130 135 140 Pro Ile Val Thr Cys Val Val Val Asp Val Ser Glu Asp Asp Pro Asp 145 150 155 160 Val Gln Ile Ser Trp Phe Val Asn Asn Val Glu Val His Thr Ala Gln 165 170 175 Thr Gln Thr His Arg Glu Asp Tyr Asn Ser Thr Leu Arg Val Val Ser 180 185 190 Ala Leu Pro Ile Gln His Gln Asp Trp Met Ser Gly Lys Glu Phe Lys 195 200 205 Cys Lys Val Asn Asn Arg Ala Leu Pro Ser Pro Ile Glu Lys Thr Ile 210 215 220 Ser Lys Pro Arg Gly Pro Val Arg Ala Pro Gln Val Tyr Val Leu Pro 225 230 235 240 Pro Pro Ala Glu Glu Met Thr Lys Lys Glu Phe Ser Leu Thr Cys Met 245 250 255 Ile Thr Asp Phe Leu Pro Ala Glu Ile Ala Val Asp Trp Thr Ser Asn 260 265 270 Gly His Lys Glu Leu Asn Tyr Lys Asn Thr Ala Pro Val Leu Asp Thr 275 280 285 Asp Gly Ser Tyr Phe Met Tyr Ser Lys Leu Arg Val Gln Lys Ser Thr 290 295 300 Trp Glu Lys Gly Ser Leu Phe Ala Cys Ser Val Val His Glu Gly Leu 305 310 315 320 His Asn His His Thr Thr Lys Thr Ile Ser Arg Ser Leu Gly Lys 325 330 335 <210> 10 <211> 107 <212> PRT <213> Artificial Sequence <400> 10 Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu<00​​​​​​​​​​​​​​​​​​​​​​​​​​<210> 11 <211> 360 <212> DNA <213> Artificial Sequence <400> 11 gaggtccagc tgcaacagtc tggacctgtg ctggtgaagc ctggggcttc agtgaagatg 60 tcctgtaagg cttctggata cactttcact cactactata tgaactgggt gaaacagagc 120 catggaaaga gccttgaatg gattggagtt attaatcctt acaacggtga tactagctac 180 aagcagaact tcaagggcaa ggccacattg actgttgaca agacctccag cacagcctac 240 atggacctca acagcctgac atctgaggac tctgcagtct attactgtgc aagagatgat 300 ggttactacc agtggtactt cgatgtctgg ggcacaggga ccacggtcac cgtctcctcg 360 <210> 12 <211> 321 <212> DNA <213> Artificial Sequence <400> 12 gacatccagc tgacccagtc tccatcctcc ttatctgcct ctctgggaga aagagtcagt 60 ctcacttgtc gggcaagtca ggacattggt agtaggttaa tctggcttca gcagggacca 120 gatggaacta ttaaacgcct gatcttcgcc acatccagtt tagattctgg tgtccccaaa 180 aggttcagtg gcagtaggtc tgggtcagat tattctctca ccatcagcag ccttgagtct 240 gaagattttg tcgactatta ctgtctacaa tgtgctagtt ctcctccgac gttcggtgga 300 ggcaccaagc tggaaatcaa a 321 <210> 13 <211> 447 <212> PRT <213> Artificial Sequence <400> 13 Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Asp Tyr 20 25 30 Tyr Trp Asn Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45<                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          &​​​​​​​​​​​​​​100 105 110 Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro 210 215 220 Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val 225 230 235 240 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 245 250 255 Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu 260 265 270 Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 275 280 285 Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser 290 295 300 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 305 310 315 320 Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile 325 330 335 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 340 345 350 Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 355 360 365 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375 380 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 385 390 395 400 Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg 405 410 415 Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 420 425 430 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 435 440 445 <210> 14 <211> 214 <212> PRT <213> Artificial Sequence <400> 14 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Leu [[ID= forty]]85 90 95 Thr Phe Gly Gln Gly Thr Asn Leu Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210

Claims

1. An anti-human LAG-3 monoclonal antibody, characterized in that: The antibody comprises a heavy chain variable region and a light chain variable region; The heavy chain variable region comprises CDR-H1, CDR-H2 and CDR-H3, and the light chain variable region comprises CDR-L1, CDR-L2 and CDR-L3; The amino acid sequence of the CDR-H1 is shown in SEQ ID NO: 3; The amino acid sequence of the CDR-H2 is shown in SEQ ID NO: 4; The amino acid sequence of the CDR-H3 is shown in SEQ ID NO: 5; The amino acid sequence of the CDR-L1 is shown in SEQ ID NO: 6; The amino acid sequence of the CDR-L2 is shown in SEQ ID NO: 7; The amino acid sequence of the CDR-L3 is shown in SEQ ID NO:

8.

2. The anti-human LAG-3 monoclonal antibody according to claim 1, wherein The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 1; the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

2.

3. The anti-human LAG-3 monoclonal antibody according to claim 1, wherein Both the heavy chain and the light chain include a constant region. The amino acid sequence of the heavy chain constant region is shown in SEQ ID NO: 9; the amino acid sequence of the light chain constant region is shown in SEQ ID NO:

10.

4. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the anti-human LAG-3 monoclonal antibody according to any one of claims 1 to 3.

5. The nucleic acid molecule according to claim 4, wherein The sequence of the nucleic acid molecule includes SEQ ID NO: 11 and SEQ ID NO: 12; The sequence SEQ ID NO: 11 encodes the heavy chain variable region of the antibody; The sequence SEQ ID NO: 12 encodes the light chain variable region of the antibody.

6. An expression vector, characterized in that The expression vector contains the nucleic acid molecule according to claim 4 or 5.

7. A host cell, characterized in that The host cell contains the expression vector according to claim 6.

8. The method for preparing the anti-human LAG-3 monoclonal antibody according to any one of claims 1 to 3, wherein: The following steps are included: preparing an expression vector containing a nucleic acid molecule for expressing the anti-human LAG-3 monoclonal antibody according to any one of claims 1 to 3; The obtained expression vector is transfected into eukaryotic host cells and cultured; The anti-human LAG-3 monoclonal antibody was isolated and purified.

9. A pharmaceutical composition comprising the anti-human LAG-3 monoclonal antibody according to any one of claims 1 to 3.

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