CN115340606B

CN115340606B - Antibody combined with human LAG-3 protein, encoding gene and application thereof

Info

Publication number: CN115340606B
Application number: CN202210739338.3A
Authority: CN
Inventors: 张鹏; 张蔓; 崔文俊
Original assignee: Reyoung Suzhou Biology Science & Technology Co ltd
Current assignee: Reyoung Suzhou Biology Science & Technology Co ltd
Priority date: 2019-05-31
Filing date: 2019-05-31
Publication date: 2023-12-19
Anticipated expiration: 2039-05-31
Also published as: CN115340606A; CN112010972A; CN112010972B

Abstract

The invention discloses an antibody combined with human LAG3 protein, and a coding gene and application thereof. The antibody combined with the human LAG-3 protein is a novel antibody molecule sequence which is different from the existing LAG-3 antibody, has an antigen combining site which is specifically combined with the human LAG3 protein, and shows good biological blocking activity. Meanwhile, the antibody provided by the invention can compete with BMS986016 for antigen epitope, can inhibit BMS986016 from binding to antigen, and can promote BMS986016 bound to antigen to be partially separated; this suggests that the binding of the antibody to human LAG-3 protein of the present invention and BMS986016 to the antigen are different in site, and that the binding of the antibody to human LAG-3 protein of the present invention can alter the antigen configuration, presumably to block the inhibition pathway of LAG-3 to T cells better than BMS986016.

Description

Antibody combined with human LAG-3 protein, encoding gene and application thereof

The application is as follows: 201910469160.3, the application date is 2019, 5 and 31 days, and the invention name is: antibodies that bind to human LAG-3 protein, and genes encoding and applications for the antibodies are described.

Technical Field

The invention belongs to the technical field of biological pharmacy, and particularly relates to an antibody combined with human LAG-3 protein, and an encoding amino acid sequence and application thereof.

Background

Normally, the human immune system recognizes and eliminates tumor cells in the tumor microenvironment, and the proposed "tumor-immune cycle" describes seven links of tumor production, tumor cell elimination by the human immune system: (1) tumor antigen release; (2) tumor antigen presentation; (3) activating and activating effector T cells; (4) T cell migration to tumor tissue; (5) tumor tissue T cell infiltration; (6) T cells recognize tumor cells; (7) tumor cell removal.

However, for survival and growth, tumor cells can adopt different strategies to suppress, abnormality and protect themselves from killing and clearance of the immune system of the human body, thereby surviving the stages of the anti-tumor immune response. Any abnormality in the seven links can lead to anti-tumor-immune circulatory failure and immune escape.

Tumor immunotherapy is a treatment that is achieved by restoring the anti-tumor immune response of the immune system to treat cancer using the immune system. Including monoclonal antibody immune checkpoint inhibitors, therapeutic antibodies, cancer vaccines, cytotherapeutic and small molecule inhibitors, and the like.

LAG-3 (Lymphocyte-activation gene 3) belongs to the immunoglobulin superfamily, whose domain consists of three parts, the extracellular region, the transmembrane region and the cytoplasmic region, and the LAG-3 gene is located on chromosome 12 (12P 13), similar to the location and structure of the CD4 molecule on the chromosome. Inhibiting LAG-3 allows T cells to regain cytotoxicity, thereby enhancing killing effect on tumors; meanwhile, the LAG-3 can be inhibited to reduce and regulate the function of T cells in inhibiting immune response. LAG-3 is therefore considered a more attractive target than other immune checkpoint proteins.

LAG-3 is a target with more clinical data and relatively determined patency in the second generation target of the current immune checkpoint, and an antibody medicine aiming at the target is likely to become an important anti-tumor medicine in the future.

Disclosure of Invention

The invention aims at providing an antibody combined with human LAG-3 protein, and a coding gene and application thereof.

In order to achieve the above object, the technical scheme of the present application is as follows:

an antibody that binds to human LAG-3 protein, comprising at least one amino acid fragment of a light chain variable region having a light chain CDR region comprising an amino acid sequence as set forth in (1), (2) or (3) and a heavy chain variable region:

(1) the method comprises the following steps An amino acid sequence having at least 50% homology with SEQ ID No.1, an amino acid sequence having at least 50% homology with SEQ ID No.2, and an amino acid sequence having at least 50% homology with SEQ ID No. 3;

(2) the method comprises the following steps An amino acid sequence having at least 50% homology with SEQ ID No.7, an amino acid sequence having at least 50% homology with SEQ ID No.8, and an amino acid sequence having at least 50% homology with SEQ ID No. 9;

(3) the method comprises the following steps An amino acid sequence having at least 50% homology with SEQ ID No.13, an amino acid sequence having at least 50% homology with SEQ ID No.14, and an amino acid sequence having at least 50% homology with SEQ ID No. 15;

the heavy chain variable region has a heavy chain CDR region comprising an amino acid sequence as set forth in (4) or (5):

(4) the method comprises the following steps An amino acid sequence having at least 50% homology with SEQ ID No.4, an amino acid sequence having at least 50% homology with SEQ ID No.5, and an amino acid sequence having at least 50% homology with SEQ ID No. 6;

(5) the method comprises the following steps An amino acid sequence having at least 50% homology with SEQ ID No.10, an amino acid sequence having at least 50% homology with SEQ ID No.11, and an amino acid sequence having at least 50% homology with SEQ ID No. 12.

The invention adopts the extracellular domain protein of the human LAG-3 protein with His tag to immunize mice, discovers that the serum titer of the immunized mice can reach 1:256000, then screens out hybridoma subclones with high affinity with the human LAG-3 by constructing hybridoma cell lines, and expresses and obtains the antibody combined with the human LAG-3 protein. The antibody combined with the human LAG-3 protein has the antigen affinity equivalent to BMS986016, and meanwhile, the antibody combined with the human LAG-3 protein can compete with BMS986016 for antigen epitope, can inhibit BMS986016 from combining with the antigen and can promote BMS986016 combined with the antigen to be partially separated; this suggests that the binding of the antibody to human LAG-3 protein of the present invention and BMS986016 to the antigen are different in site, and that the binding of the antibody to human LAG-3 protein of the present invention can alter the antigen configuration, presumably to block the inhibition pathway of LAG-3 to T cells better than BMS986016.

Preferably, the light chain variable region has the light chain CDR regions of the amino acid sequences shown in SEQ ID Nos. 13, 14 and 15 and the heavy chain variable region has the heavy chain CDR regions of the amino acid sequences shown in SEQ ID Nos. 4, 5 and 6.

Alternatively, the light chain variable region has the light chain CDR regions of the amino acid sequences shown in SEQ ID Nos. 7, 8 and 9 and the heavy chain variable region has the heavy chain CDR regions of the amino acid sequences shown in SEQ ID Nos. 10, 11 and 12.

The antibody binding to human LAG-3 protein of the invention may be a murine antibody, a human murine chimeric antibody or a humanized antibody. Both the human murine chimeric antibody and the humanized antibody are obtained by engineering on the basis of murine antibodies.

When the antibody combined with the human LAG-3 protein is a murine antibody or a human chimeric antibody, the heavy chain variable region has an amino acid sequence shown as SEQ ID No.16 or SEQ ID No.18, and the light chain variable region has an amino acid sequence shown as SEQ ID No.19 or SEQ ID No. 21.

When the antibody combined with the human LAG-3 protein is a human-mouse chimeric antibody, the light chain variable region of the human-mouse chimeric antibody combined with the human LAG-3 protein is the same as that described above, and the light chain constant region is the sequence of the human IgG kappa CL region; the heavy chain variable region is the same as described above, and the heavy chain constant region is the CH1, CH2 and CH3 region sequences of human IgG 4S 228P. When the antibody combined with the human LAG-3 protein is a fully humanized antibody, the heavy chain amino acid sequence of the fully humanized antibody is shown as SEQ ID No.23, and the light chain amino acid sequence of the fully humanized antibody is shown as SEQ ID No. 33.

Alternatively, the heavy chain amino acid sequence of the fully humanized antibody is shown as SEQ ID No.34, and the light chain amino acid sequence of the fully humanized antibody is shown as SEQ ID No. 37.

The invention also provides a coding gene of the antibody combined with the human LAG-3 protein and a recombinant vector containing the coding gene of the antibody combined with the human LAG-3 protein.

The invention also provides recombinant cells expressing the antibodies that bind to human LAG-3 protein.

The invention also provides application of the antibody combined with the human LAG-3 protein in preparing medicines for treating malignant tumors. The malignant tumor therapeutic agent can be the antibody which is combined with the human LAG-3 protein, or can be a conjugate or composition of the antibody which is combined with the human LAG-3 protein and other substances; malignant tumors for treatment are generally caused by T-cell failure by LAG-3.

Compared with the prior art, the invention has the beneficial effects that:

the invention adopts the extracellular domain of human LAG-3 protein with His tag to immunize mice, discovers that the serum titer of the immunized mice can reach 1:256000, then screens out hybridoma subclones with high affinity with human LAG-3 by constructing hybridoma cell lines, and expresses and obtains the antibody combined with human LAG-3 protein. The antibody combined with the human LAG-3 protein has the antigen affinity equivalent to BMS986016, has a good biological function of blocking the combination of the LAG3 and MHCII molecules, and can compete with BMS986016 for an epitope, inhibit the combination of BMS986016 and the antigen and promote the partial separation of the BMS986016 combined with the antigen; this suggests that the binding of the antibody to human LAG-3 protein of the present invention and BMS986016 to the antigen are different in site, and that the binding of the antibody to human LAG-3 protein of the present invention can alter the antigen configuration, presumably to block the inhibition pathway of LAG-3 to T cells better than BMS986016.

Drawings

FIG. 1a is a ligand binding blocking assay result 1 of subcloning of hybridoma cell lines;

FIG. 1b is a ligand binding blocking assay result 2 for subclones of hybridoma cell lines;

FIG. 2 shows the result of SDS-PAGE analysis of anti-human LAG-3 humanized antibodies;

FIG. 3 is a melting graph of an anti-human LAG-3 humanized antibody;

in the figure, temperature (. Degree. C.) represents the melting Temperature (. Degree. C.) of antibody Fab, and Derivative Reporter (-Rn') represents the detection report of data formation after analysis;

FIG. 4 is a Ka/Kd diagram of a humanized antibody against human LAG-3 No. 20;

FIG. 5 is a Ka/Kd diagram of a humanized antibody against human LAG-3 No. 22;

FIGS. 6a, 6b and 6c show the results of an epitope competition test between an anti-human LAG-3 humanized antibody and a BMS986016 antibody when the antigen-antibody is fed stepwise, wherein the BMS986016 antibody is fed in FIG. 6a, the 20-th anti-human LAG-3 humanized antibody is fed in FIG. 6b, and the 22-th anti-human LAG-3 humanized antibody is fed in FIG. 6 c;

wherein time(s) represents the time(s) for the analyte to flow through the chip and Response (Ru) represents the Response of the instrument to analyte antibody binding; "Ag followed by BMS-mAb" indicates that an antigen was flowed into the BMS986016 antibody, "Ag followed by 20mAb" indicates that an antigen was flowed into the BMS986016 antibody, and No. 20 anti-human LAG-3 humanized antibody was flowed into the BMS986016 antibody, "Ag followed by 22mAb" indicates that an antigen was flowed into the BMS986016 antibody, and No. 22 anti-human LAG-3 humanized antibody was flowed into the BMS antibody;

FIGS. 7a, 7b and 7c show the results of an epitope competition test of the anti-human LAG-3 humanized antibody and the BMS986016 antibody when the anti-human LAG-3 humanized antibody and the BMS986016 antibody are loaded after the antigen antibody is incubated, wherein the BMS986016 antibody and the human LAG-3 mixed incubation liquid is loaded in FIG. 6a, the 20 anti-human LAG-3 humanized antibody and the human LAG-3 mixed incubation liquid is loaded in FIG. 6b, and the 22 anti-human LAG-3 humanized antibody and the human LAG-3 mixed incubation liquid is loaded in FIG. 6 c;

wherein, "Ag and BMS-mAb co-incubate" means that the antigen flows in after mixed incubation with BMS986016 antibody, "Ag and 20mAb co-incubate" means that the antigen flows in after mixed incubation with 20 # anti-human LAG-3 humanized antibody, and "Ag and 22mAb co-incubate" means that the antigen flows in after mixed incubation with 22 # anti-human LAG-3 humanized antibody.

Detailed Description

The technical scheme of the invention is further described in detail below with reference to the attached drawings and the detailed description.

Example 1

1. Immunization of animals

Using the extracellular domain (Acro) of His-tagged human LAG-3 protein (hereinafter abbreviated as human LAG-3) as an antigen, 50ng of the antigen was mixed with Freund's incomplete adjuvant (Sigma-Titer Max) at a ratio of 1:1, female Balb/c mice (10 total) were immunized subcutaneously, the mice were immunized three times as described above, blood was taken from the 4 th tail vein after the third immunization, the serum Titer of the mice was measured, and the immune response of each immunized mouse was examined, thereby screening mice having the highest Titer against recombinant human IL-1. Beta. Immunoglobulin.

Serum titer detection method: ELISA plates were coated with 1. Mu.g/ml human LAG-3 and 100. Mu.l per well incubated overnight at 4 ℃; plates were washed with 200 μl/well PBS/Tween (0.1%) followed by blocking with 3% bovine serum albumin in 200 μl/well PBS/Tween (0.1%) for 1 hour; after washing the plates, serum dilutions from human LAG-3 immunized mice were added to each well, incubated at 37℃for 2 hours, and the plates were washed; then incubated with diluted HRP-bearing goat anti-mouse IgG (H+L) (BF 03001, beijing bloolone immunotechnology Co., ltd.) antibody at 37℃for 1 hour; after washing, each well was developed with 100. Mu.l of TMB developing solution (Biopanda, TMB-S-002); after the color change, 50. Mu.l of 2M sulfuric acid was added to terminate the reaction, and then analysis was performed by a microplate reader at OD450nm to 620nm, and the results are shown in Table 1.

TABLE 1 serum titers of individual immunized mice

According to the detection results in table 1, 10 mice immunized 3 times had OD values above 0.2, and were considered positive, and Negative Control (NC) OD was less than 0.06, with reliable experimental results. Thus, immunized mice numbered 7 were selected for fusion assays of hybridoma cells.

2. Hybridoma cell fusion

Mice were antigen-boosted prior to fusion, and after 3 days of continuous boosting, mice were sacrificed and spleens removed.

Culturing mouse myeloma SP2/0, regulating and ensuring its growth density not to exceed 1×10 ⁶ Suspending and collecting SP2/0 cells before fusion, extracting spleen of a mouse, homogenizing to generate single cell suspension, and ensuring that the number ratio of spleen cells or lymph node cells to SP2/0 cells after mixing is 3:1 to 1:1, fusing cells by an electrofusion apparatus.

Resuspension of the fused cells in RPIM1640/10% FBS medium containing hybridoma cell selective agent, hypoxanthine and aminopterin, to ensure spleen cell density of about 0.5X10 ⁶ Cell fluid was transferred to 96-well cell culture plates at 200 μl/well. The plates were exposed to 37℃and 5% CO ₂ After 7 days of fusion, the supernatant was harvested and the medium was changed, and the collected supernatant was screened out 2 positive hybridoma cell lines by ELISA binding method, flow cytometer ligand binding blocking assay.

Hybridoma cell subcloning screening

And (3) taking the screened positive hybridoma cell strain, blowing and uniformly mixing by using a gun, counting, transferring cell sap corresponding to 80 cells into 20mL of HT culture medium, uniformly mixing, and sucking the cell sap into a 96-well culture plate, wherein the cell sap is about 200 mu L per well, and ensuring about 0.8 cells per well. After culturing for 7 days, hybridoma clones grown singly were selected for ELISA detection (for detection method see step (1)), and the detection results are shown in Table 2.

TABLE 2 screening results for subclones of hybridoma cells

3. In vitro biological function assay for antibodies

Culturing and preparing Raji-MHC-II cells according to instructions, mixing the supernatant of the 59 hybridoma subclones screened in the step (3), control antibodies (BMS 986016, hIgG4 and mIgG4, the concentration of which is 10 mug/mL) and LAG-3 (about 100 mug), adding the mixture into a 96-well plate, arranging a double well for each sample, and incubating for 1h at room temperature; cells Raji cells were collected by centrifugation, washed twice with DPBS, resuspended in a suitable amount of DPBS buffer containing 1% BSA, and the cell density was adjusted to 1X 10 ⁶ Adding 100 mu L of cell suspension to each sample well, centrifuging and discarding the supernatant; adding the mixture of the incubated supernatant and LAG-3 into a cell hole, incubating for 30-45min at 4 ℃, centrifuging and discarding the supernatant; after washing cells 3 times with 250. Mu.L of DPBS buffer containing 1% BSA per well, 100. Mu.L of FITC-labeled goat anti-human IgG diluted with 1% BSA in DPBS buffer was added per well, incubated at 4℃for 1h in the dark, cells were washed three times with 1% BSA in DPBS buffer, pre-cooled at 4℃to 100. Mu.L of PBS was added to resuspend cells, and analyzed by flow cytometry in the dark

5. Preparation of anti-LAG-3 human-mouse chimeric antibody and affinity detection

Splicing the light chain variable region of the anti-LAG-3 murine antibody shown in Table 3 with the human IgG kappa light chain constant region sequence SEQ ID No.40 to obtain a full-length light chain sequence; the light chain expression vector is obtained by total gene synthesis and constructed on the pTT5 vector containing the nitrogen end signal peptide.

The heavy chain expression vector was obtained by splicing the heavy chain variable region sequence of the anti-LAG-3 murine antibody of table 3 with the heavy chain constant region SEQ ID No.39 of human IgG 4S 228P to form a full-length heavy chain sequence, and constructing the heavy chain expression vector onto the pTT5 vector containing the nitrogen-terminal signal peptide by total gene synthesis.

Co-transfecting HEK293F cells with a light chain expression vector and a heavy chain expression vector, culturing in a shake flask at 37 ℃ for 7 days, and collecting culture supernatants to obtain antibody supernatant 17E3VL-17E3VH, 29VL-17E3VH and 78H3VL-78H3VH; the ability of the anti-LAG-3 human murine chimeric antibody to bind to human LAG-3 protein was determined by SPR, and specifically comprises:

carrying out at 25 ℃, allowing human murine embedded antibody supernatant to flow through the surface of a Protein A chip at a certain flow rate, and allowing 100nM human LAG-3 Protein to flow through the surface of the chip on which the antibody is immobilized at a certain flow rate after the antibody is bound to the surface of the chip; taking the experiment buffer as a blank control, and setting a repeated concentration; the results of the experimental determination were fitted using a 1:1binding model. The detailed steps are as follows:

(1) And replacing the chip, and replacing the maintenance chip in the instrument with the protein A chip.

(2) The BIACORE instrument control software was turned on, the analysis sample program was set, and a cycle process and parameters were as follows:

a) Capture (capture, the process of capturing ligands by chip): the ligand flowed through the chip at a rate of 10. Mu.L/min for 300s.

b) Analysis (analysis):

binding (association, the process by which the analyte binds to the substance on the chip surface): the analyte flowed through the chip at a rate of 30. Mu.L/min for 300s;

dissociation (process of analyte separation from chip surface material): the buffer was flowed through the chip at a rate of 30. Mu.L/min for 300s.

c) Regeneration (regeneration process of chip): the chip regeneration buffer was flowed through the chip at a rate of 100. Mu.L/min for 60s.

(3) The ligands and analytes for this experimental cycle and the purposes are shown in table 4.

Table 4 design of experiments for affinity analysis of chimeric antibodies

SPR results for the five antibody supernatants are shown in Table 5.

TABLE 5 affinity analysis of anti-LAG-3 human murine chimeric antibodies

As can be seen in Table 5, the Rmax value of the 29VL-VH antibody in the five antibody supernatants was too small, indicating that it was not expressed. The other antibodies have normal affinity KD, binding constant ka, dissociation constant KD and Rmax, and several antibodies have affinity of 10 ^-10 M, the molecule belongs to the fast binding and slow dissociating type in the aspect of dynamics.

6. anti-LAG-3 murine antibody humanization and SPR screening

(1) Humanization

Analyzing the sequence of a variable region of a murine antibody, comparing the sequence with a human germ line (germline) gene, selecting the sequence of a framework region of a human light and heavy chain germ line gene with highest homology with the sequence of the framework region of the murine antibody as a basic framework, or selecting specific amino acids on the framework region for back mutation, then grafting the sequence of the CDR region of the murine antibody with the sequence of the framework region of the selected human germ line gene, and respectively constructing the sequence of the light and heavy chain variable region of the humanized antibody on pTT5 expression vectors comprising human IgG4 heavy chain Fc and human IgG kappa CL through gene synthesis after splicing.

The constructed humanized antibody (shown in Table 6) is co-transfected into HEK293F cells by pairing light and heavy chain plasmids one by one (the pairing combination is shown in Table 7), and supernatants are collected for later use after culturing in shake flasks at 37 ℃.

TABLE 6 humanized sequence correspondence table

TABLE 7 light and heavy chain combinations of humanized antibodies

(2) SPR screening

Humanized antibodies with high affinity were selected by SPR assay for affinity to LAG-3, see section 2 of this example, and the results are shown in Table 7.

TABLE 8 SPR screening results for humanized antibodies

As shown in the above table, the affinity of the humanized combination 20 and the humanized combination 22 corresponding to the parent molecule becomes very high, and the candidate selection combination 20 and the candidate combination 22 continue to perform expression and purification research in combination with the response value (Rmax), the expression level (Capture level) and the affinity (KD) detected by the binding instrument.

7. Expression purification and analysis of humanized antibodies

(1) Expression purification of humanized antibodies

The recombinant plasmids corresponding to the antibodies 20 and 22 screened by the experiment are transfected into HEK293 cells, and after shaking culture for 7 days, the supernatant is collected, filtered and sterilized for protein A chromatography purification. Before purification, the AKTA Start purification instrument line and the ProteinA column were depyrogenated with 0.1M NaOH and then purified with a solution containing 20mM Na ₂ HPO ₄ (pH 7.2) buffer equilibrated the column, the filtered supernatant was passed into the column at 1mL/min, and after completion, 20mM Na was used in succession ₂ HPO ₄ -1M NaCl (pH 6.5) and 20mM Na ₂ HPO ₄ The column was equilibrated and finally the IgG eluted using sterile 0.1M sodium citrate (ph 3.5), the eluate collected and neutralized with one tenth of the volume of sterile 1M Tris-HCl (ph 9.0). Under sterile conditions, the product buffer was exchanged for PBS (pH 7.4) and the antibodies were quantified by OD280nm using an extinction coefficient Ec of 1.43 (0.1%).

Purified antibodies were analyzed by SDS-PAGE using a 10% polyacrylamide gel using a BioRad electrophoresis system. The gel was stained with EstainL (GenScrip) and molecular size and purity were estimated by comparing the stained band with Protein Ladder (Thermo).

SDS-PAGE analysis results are shown in FIG. 2. The molecular weight of the complete antibody, the heavy chain and the light chain is estimated to be about 170kDa and 48kDa and 25kDa according to the protein Marker, the molecular weight of the complete antibody is larger than 150kDa in theory due to glycosylation, and the molecular weight of the light chain and the heavy chain of the antibody both accord with theory.

(2) Stability of

The folded state of the protein can be monitored by using SyproOrange (Sigma) fluorescent dye, the antibody is diluted to 1mg/mL, the dye is diluted by water and mixed with the antibody, the mixture is put into a fluorescence quantitative PCR (ABI 7500) instrument (ABI 7500) capable of detecting the change of fluorescent signals along with the temperature, a program is set, each sample is 30 mu L, each sample is repeated for 3 times, water is used as blank control, and the temperature is 25-99 ℃ and the scanning speed is 0.5 ℃/min. After the end, the data were analyzed in the ABI 7500 software.

The melting curve of the antibodies is shown in FIG. 3, and the melting curve can obviously reflect the melting point temperatures corresponding to Fab of the antibodies (a certain difference from the actual melting point is used for comparison between the antibodies, and not for quantitative measurement), namely the temperatures corresponding to the first peak, namely the temperatures of the antibodies 20 and 22 at 63 ℃ and 65 ℃ respectively, and BMS986016 at 68 ℃.

(3) Antibody affinity assay

The SPR method can analyze kinetic parameters Kd and Ka between the ligand and the analyte, and the measured affinity is more accurate by setting different concentrations of the analyte. The specific process is as follows:

(2) Editing method, setting analysis sample program, one cycle process and parameters are the same as those in the 2 nd part of this embodiment.

(3) HBS-EP+ dilutes the antibody to 5 μg/mL, HBS-EP+ dilutes the antigen to 2430nM, 810nM, 270nM, 90nM, 30nM, 10nM, 3nM, 1nM, 0.33nM, and the volume after dilution is given by instrumental methods; the sample to be tested was added to the corresponding position (running buffer: HBS-EP+, regeneration buffer: 10mM Glycine-HCl pH 1.7) with running buffer and regeneration buffer, running procedure.

(4) The data was analyzed Biacore Evaluation Software after the end of the run.

The SPR method measured the binding/dissociation of antibodies 20, 22 to human LAG-3 protein versus time as shown in fig. 4 and 5, and the affinity data corresponding to the figures is shown in table 8.

TABLE 9 affinity analysis of humanized antibodies

(4) SPR method for analyzing competition of humanized antibody and BMS986016 antibody recognition antigen epitope

In order to analyze whether the epitope recognized by the antibodies 20 and 22 is the same as the LAG-3 antibody BMS986016 which is the fastest in current research progress, one of the antibodies is immobilized on a chip by adopting an SPR method according to the principle of specific binding of the antibody epitope, the antigen is competitively bound with the other antibody, and meanwhile, the LAG-3 antigen is immobilized on the chip for comparison test.

The method comprises the following steps:

1) Ligand immobilization

(1) And opening BIACORE instrument control software, and editing a chip surface fixing method.

(2) The method is prepared before operation: diluting the ligand to 10 μg/mL with NaAc-HAc pH 5.5 solution;

and thawing EDC and NHS.

(3) The ligand immobilization process is as follows:

EDC, NHS at 1:1 mixing and activating the surface of the chip, 420s,10 mu L/min;

ligand loading, 10 mu L/min, stopping after reaching a required fixed level;

ethanolamine blocked the chip surface, 420s, 10. Mu.L/min.

(4) The chip processing results are shown in Table 9 below:

TABLE 10 chip processing results

2) Competitive assay for epitope analysis

Using the chip channel of immobilized BMS986016 ligand in the "ligand immobilization" experiment, two loading modes were analyzed for whether the epitope was identical or not:

(1) antigen-antibody step-by-step loading: BMS986016 antibody was immobilized on CM5 chip (GE), 10nM antigen was passed over the chip surface to bind to BMS986016 antibody, and then different concentrations of humanized antibody samples were flowed, respectively, to detect whether the antibody and antigen bound to on-chip BMS986016 were bound. The method specifically comprises the following steps:

10nM of antigen, analyte1 (analyte 1), and analyte2 (analyte 2) were passed through channels of the chip-immobilized BMS986016 with different concentrations (10 nM, 100nM, 1000 nM) of antibody, respectively, with the following parameters:

a) Analyte1 (analyte 1):

binding (association): the analyte flowed through the chip at a rate of 30. Mu.L/min for 300s;

dissociation (association): the buffer was flowed through the chip at a rate of 30. Mu.L/min for 10s.

b) Analyte2 (analyte 2):

dissociation (association): the buffer was flowed through the chip at a rate of 30. Mu.L/min for 600s.

c) Regeneration (regeneration): the chip regeneration buffer was flowed through the chip at a rate of 100. Mu.L/min for 60s.

The antigen LAG-3 and the antibodies BMS986016, 20 and 22 were diluted with HBS-EP+ according to the program setting, and the samples and buffers were placed in the corresponding positions to run the program.

d) After the operation is finished, the signal diagram obtained by Biacore Control Software is compared and analyzed.

(2) Loading after antigen-antibody incubation: humanized antibody samples with different concentrations are respectively incubated with antigens with certain concentrations for 1h at room temperature and then flow through a chip channel fixed with BMS986016, and whether the antigens combined with laboratory antibodies can be combined with BMS986016 is detected. The method specifically comprises the following steps:

10nM of Human-LAG-3-His was incubated with different concentrations (10 nM, 100nM, 1000 nM) of antibody for 30min as a channel for the analyte to flow through the immobilized chip BMS986016, with the following parameters:

a) Analysis (analysis):

b) Regeneration (regeneration): the chip regeneration buffer was flowed through the chip at a rate of 100. Mu.L/min for 60s.

The mixture of antigen LAG-3 and antibodies BMS986016, 20, 22 was diluted with HBS-EP+ and samples and buffers were placed in the corresponding positions, and the procedure was run.

c) After the operation is finished, the signal diagram obtained by Biacore Control Software is compared and analyzed.

And (3) mutually proving in the two modes (1) and (2) and comprehensively analyzing. The results of the stepwise loading of antigen-antibody are shown in FIGS. 6a, 6b and 6c, and the results of the loading after incubation of antigen-antibody are shown in FIGS. 7a, 7b and 7c.

As can be seen from fig. 6a, 6b and 6c, 10nM LAG-3 binds to on-chip BMS986016 within 300s after antigen loading, and the on-chip LAG-3 binding rate is saturated; whereas, after 300s later, the inflow of the different antibodies, antibodies No. 20 (fig. 6 b) and No. 22 (fig. 6 c) showed a signal binding to LAG-3, which showed a clear difference from the inflow of BMS986016 (fig. 6 a); in addition, as the concentrations of the antibodies No. 20 and No. 22 which flow in increase, the binding signals between the antibodies No. 20 and No. 22 and LAG-3 become stronger.

As can be seen from fig. 7a, 7b and 7c, the binding signal of LAG-3 (fig. 7 a) at 10nM to the on-chip BMS986016 antibody was stronger than the mixture of LAG-3 and antibody, and the binding signal of the mixed incubation with the on-chip BMS986016 antibody was weaker as the concentration of the 20 th antibody (fig. 7 b) and the 22 th antibody (fig. 7 c) incubated with LAG-3 was gradually increased.

The above analysis showed that antibody 20, antibody 22 and BMS986016 bind to different epitopes and that theory concluded that in the antigen-antibody mix incubation assay, the 10nM LAG-3 binding signal to BMS986016 on the chip should be comparable to that of 10nM LAG-3 mix mixed with antibody 20 or 22, but the results of figures 7a, 7b and c were different from that of the hypothesis that binding of antibody 20 or 22 to LAG-3 affected its binding to BMS986016, and that binding of antibody 20 or 22 resulted in a change in the conformational conformation of the antigen.

Sequence listing

<110> Ruiyang (su state) biotechnology Co., ltd

<120> antibody binding to human LAG-3 protein, encoding gene and use thereof

<160> 40

<170> SIPOSequenceListing 1.0

<210> 1

<211> 15

<212> PRT

<213> Balb/c mice (Mouse parvovirus 1 b)

<400> 1

Arg Thr Ser Glu Ser Ile Asp Asn Tyr Gly Val Ser Phe Met Asn

1 5 10 15

<210> 2

<211> 7

<212> PRT

<213> Balb/c mice (Mouse parvovirus 1 b)

<400> 2

Ala Ala Ser Asn Gln Gly Ser

1 5

<210> 3

<211> 9

<212> PRT

<213> Balb/c mice (Mouse parvovirus 1 b)

<400> 3

Gln Gln Ser Lys Glu Val Pro Phe Thr

1 5

<210> 4

<211> 10

<212> PRT

<213> Balb/c mice (Mouse parvovirus 1 b)

<400> 4

Gly Phe Thr Phe Ile Thr Tyr Ala Met Ser

1 5 10

<210> 5

<211> 15

<212> PRT

<213> Balb/c mice (Mouse parvovirus 1 b)

<400> 5

Ser Ile Asn Asn Gly Gly Thr His Tyr Pro Asp Thr Met Lys Gly

1 5 10 15

<210> 6

<211> 11

<212> PRT

<213> Balb/c mice (Mouse parvovirus 1 b)

<400> 6

Ser Tyr Asn Gly Ile Pro His Tyr Phe Asp Tyr

1 5 10

<210> 7

<211> 11

<212> PRT

<213> Balb/c mice (Mouse parvovirus 1 b)

<400> 7

Arg Ala Ser Gln Asp Ile Gly Asn Ser Leu Asn

1 5 10

<210> 8

<211> 7

<212> PRT

<213> Balb/c mice (Mouse parvovirus 1 b)

<400> 8

Ala Thr Ser Ser Leu Asp Ser

1 5

<210> 9

<211> 9

<212> PRT

<213> Balb/c mice (Mouse parvovirus 1 b)

<400> 9

Leu Gln His Ala Ser Ser Pro Pro Thr

1 5

<210> 10

<211> 10

<212> PRT

<213> Balb/c mice (Mouse parvovirus 1 b)

<400> 10

Gly Tyr Thr Leu Thr Asp Tyr Tyr Met Asn

1 5 10

<210> 11

<211> 17

<212> PRT

<213> Balb/c mice (Mouse parvovirus 1 b)

<400> 11

Ile Ile Asn Pro Tyr Asn Gly Asp Thr Ser Tyr Asn Gln Arg Phe Lys

1 5 10 15

Gly

<210> 12

<211> 11

<212> PRT

<213> Balb/c mice (Mouse parvovirus 1 b)

<400> 12

Asp Asp Gly Tyr Tyr Val Asp Tyr Phe Asp Tyr

1 5 10

<210> 13

<211> 15

<212> PRT

<213> Balb/c mice (Mouse parvovirus 1 b)

<400> 13

Arg Ala Ser Glu Ser Val Asp Asn Tyr Gly Val Ser Phe Val Asn

1 5 10 15

<210> 14

<211> 7

<212> PRT

<213> Balb/c mice (Mouse parvovirus 1 b)

<400> 14

Ala Ala Ser Asn Gln Gly Ser

1 5

<210> 15

<211> 9

<212> PRT

<213> Balb/c mice (Mouse parvovirus 1 b)

<400> 15

Gln Gln Ser Lys Glu Val Pro Phe Thr

1 5

<210> 16

<211> 118

<212> PRT

<213> Balb/c mice (Mouse parvovirus 1 b)

<400> 16

Glu Val Met Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ile Thr Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val

35 40 45

Ala Ser Ile Asn Asn Gly Gly Thr His Tyr Pro Asp Thr Met Lys Gly

50 55 60

Arg Phe Thr Ile Ser Arg Asp Asn Ala Arg Asn Ile Leu Tyr Leu Gln

65 70 75 80

Met Ser Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys Ala Arg

85 90 95

Ser Tyr Asn Gly Ile Pro His Tyr Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Thr Leu Thr Val Ser Ser

115

<210> 17

<211> 111

<212> PRT

<213> Balb/c mice (Mouse parvovirus 1 b)

<400> 17

Asp Ile Leu Met Thr Gln Thr Pro Ala Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Gln Arg Ala Thr Ile Ser Cys Arg Thr Ser Glu Ser Ile Asp Asn Tyr

20 25 30

Gly Val Ser Phe Met Asn Trp Phe Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Arg Leu Leu Ile Tyr Ala Ala Ser Asn Gln Gly Ser Gly Val Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Ala Asp Phe Ser Leu Asn Ile His

65 70 75 80

Pro Met Glu Glu Asp Asp Ser Ala Leu Tyr Phe Cys Gln Gln Ser Lys

85 90 95

Glu Val Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 18

<211> 120

<212> PRT

<213> Balb/c mice (Mouse parvovirus 1 b)

<400> 18

Glu Val Gln Leu Gln Gln Ser Gly Pro Val Leu Ala Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Thr Ser Gly Tyr Thr Leu Thr Asp Tyr

20 25 30

Tyr Met Asn Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile

35 40 45

Gly Ile Ile Asn Pro Tyr Asn Gly Asp Thr Ser Tyr Asn Gln Arg Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Asp Gly Tyr Tyr Val Asp Tyr Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Thr Leu Thr Val Ser Ser

115 120

<210> 19

<211> 107

<212> PRT

<213> Balb/c mice (Mouse parvovirus 1 b)

<400> 19

Asp Ile Gln Met 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 Asn Ser

20 25 30

Leu Asn Trp Leu Gln Gln Glu Pro Asp Gly Thr Ile Lys Arg Leu Ile

35 40 45

Tyr 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 His Ala Ser Ser Pro Pro

85 90 95

Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

100 105

<210> 20

<211> 118

<212> PRT

<213> Balb/c mice (Mouse parvovirus 1 b)

<400> 20

Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ile Thr Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val

35 40 45

Ala Ser Ile Asn Asn Gly Gly Thr His Tyr Pro Asp Ser Met Lys Gly

50 55 60

Arg Phe Thr Ile Ser Arg Asp Asn Ala Arg Asn Ile Leu Tyr Leu Gln

65 70 75 80

Met Ser Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys Ala Arg

85 90 95

Ser Tyr Asn Gly Ile Pro His Tyr Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Thr Leu Thr Val Ser Ser

115

<210> 21

<211> 111

<212> PRT

<213> Balb/c mice (Mouse parvovirus 1 b)

<400> 21

Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Asp Asn Tyr

20 25 30

Gly Val Ser Phe Val Asn Trp Phe Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Ala Ala Ser Asn Gln Gly Ser Gly Val Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Ser Leu Asn Ile His

65 70 75 80

Pro Met Glu Glu Asp Asp Ser Ala Leu Tyr Phe Cys Gln Gln Ser Lys

85 90 95

Glu Val Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 22

<211> 118

<212> PRT

<213> artificially synthesized sequence (Unknown)

<400> 22

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ile Thr Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Asn Asn Gly Gly Thr His Tyr Pro Asp Thr Met Lys Gly

50 55 60

Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln

65 70 75 80

Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Lys

85 90 95

Ser Tyr Asn Gly Ile Pro His Tyr Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Thr Val Thr Val Ser Ser

115

<210> 23

<211> 118

<212> PRT

<213> artificially synthesized sequence (Unknown)

<400> 23

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ile Thr Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Asn Asn Gly Gly Thr His Tyr Pro Asp Thr Met Lys Gly

50 55 60

Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln

65 70 75 80

Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg

85 90 95

Ser Tyr Asn Gly Ile Pro His Tyr Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Thr Val Thr Val Ser Ser

115

<210> 24

<211> 111

<212> PRT

<213> artificially synthesized sequence (Unknown)

<400> 24

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Thr Ser Glu Ser Ile Asp Asn Tyr

20 25 30

Gly Val Ser Phe Met Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro

35 40 45

Lys Leu Leu Ile Tyr Ala Ala Ser Asn Gln Gly Ser Gly Val Pro Ser

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Lys

85 90 95

Glu Val Pro Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 25

<211> 111

<212> PRT

<213> artificially synthesized sequence (Unknown)

<400> 25

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Thr Ser Glu Ser Ile Asp Asn Tyr

20 25 30

Gly Val Ser Phe Met Asn Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro

35 40 45

Lys Leu Leu Ile Tyr Ala Ala Ser Asn Gln Gly Ser Gly Val Pro Ser

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln Ser Lys

85 90 95

Glu Val Pro Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 27

<211> 111

<212> PRT

<213> artificially synthesized sequence (Unknown)

<400> 27

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Thr Ser Glu Ser Ile Asp Asn Tyr

20 25 30

Gly Val Ser Phe Met Asn Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro

35 40 45

Lys Leu Leu Ile Tyr Ala Ala Ser Asn Gln Gly Ser Gly Val Pro Ser

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Ser Leu Gln Pro Glu Asp Ser Ala Thr Tyr Phe Cys Gln Gln Ser Lys

85 90 95

Glu Val Pro Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 28

<211> 111

<212> PRT

<213> artificially synthesized sequence (Unknown)

<400> 28

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Thr Ser Glu Ser Ile Asp Asn Tyr

20 25 30

Gly Val Ser Phe Met Asn Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro

35 40 45

Lys Leu Leu Ile Tyr Ala Ala Ser Asn Gln Gly Ser Gly Val Pro Ser

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Ala Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln Ser Lys

85 90 95

Glu Val Pro Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 29

<211> 111

<212> PRT

<213> artificially synthesized sequence (Unknown)

<400> 29

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Thr Ser Glu Ser Ile Asp Asn Tyr

20 25 30

Gly Val Ser Phe Met Asn Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro

35 40 45

Lys Leu Leu Ile Tyr Ala Ala Ser Asn Gln Gly Ser Gly Val Pro Ser

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Ala Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Ser Leu Gln Pro Glu Asp Ser Ala Thr Tyr Phe Cys Gln Gln Ser Lys

85 90 95

Glu Val Pro Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 30

<211> 111

<212> PRT

<213> artificially synthesized sequence (Unknown)

<400> 30

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Asp Asn Tyr

20 25 30

Gly Val Ser Phe Val Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro

35 40 45

Lys Leu Leu Ile Tyr Ala Ala Ser Asn Gln Gly Ser Gly Val Pro Ser

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Lys

85 90 95

Glu Val Pro Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 31

<211> 111

<212> PRT

<213> artificially synthesized sequence (Unknown)

<400> 31

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Asp Asn Tyr

20 25 30

Gly Val Ser Phe Val Asn Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro

35 40 45

Lys Leu Leu Ile Tyr Ala Ala Ser Asn Gln Gly Ser Gly Val Pro Ser

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln Ser Lys

85 90 95

Glu Val Pro Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 32

<211> 111

<212> PRT

<213> artificially synthesized sequence (Unknown)

<400> 32

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Asp Asn Tyr

20 25 30

Gly Val Ser Phe Val Asn Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro

35 40 45

Lys Leu Leu Ile Tyr Ala Ala Ser Asn Gln Gly Ser Gly Val Pro Ser

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Ser Leu Gln Pro Glu Asp Ser Ala Thr Tyr Phe Cys Gln Gln Ser Lys

85 90 95

Glu Val Pro Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 33

<211> 111

<212> PRT

<213> artificially synthesized sequence (Unknown)

<400> 33

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Asp Asn Tyr

20 25 30

Gly Val Ser Phe Val Asn Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro

35 40 45

Lys Leu Leu Ile Tyr Ala Ala Ser Asn Gln Gly Ser Gly Val Pro Ser

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Ala Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln Ser Lys

85 90 95

Glu Val Pro Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 34

<211> 111

<212> PRT

<213> artificially synthesized sequence (Unknown)

<400> 34

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Asp Asn Tyr

20 25 30

Gly Val Ser Phe Val Asn Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro

35 40 45

Lys Leu Leu Ile Tyr Ala Ala Ser Asn Gln Gly Ser Gly Val Pro Ser

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Ala Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Ser Leu Gln Pro Glu Asp Ser Ala Thr Tyr Phe Cys Gln Gln Ser Lys

85 90 95

Glu Val Pro Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 35

<211> 120

<212> PRT

<213> artificially synthesized sequence (Unknown)

<400> 35

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Leu Thr Asp Tyr

20 25 30

Tyr Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Ile Ile Asn Pro Tyr Asn Gly Asp Thr Ser Tyr Asn Gln Arg Phe

50 55 60

Lys Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Asp Gly Tyr Tyr Val Asp Tyr Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 36

<211> 120

<212> PRT

<213> artificially synthesized sequence (Unknown)

<400> 36

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Thr Ser Gly Tyr Thr Leu Thr Asp Tyr

20 25 30

Tyr Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Ile Ile Asn Pro Tyr Asn Gly Asp Thr Ser Tyr Asn Gln Arg Phe

50 55 60

Lys Gly Arg Val Thr Met Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Asp Gly Tyr Tyr Val Asp Tyr Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 37

<211> 107

<212> PRT

<213> artificially synthesized sequence (Unknown)

<400> 37

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Gly Asn Ser

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Thr Ser Ser Leu Asp Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln His Ala Ser Ser Pro Pro

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 38

<211> 107

<212> PRT

<213> artificially synthesized sequence (Unknown)

<400> 38

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Gly Asn Ser

20 25 30

Leu Asn Trp Leu Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile

35 40 45

Tyr Ala Thr Ser Ser Leu Asp Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Ser Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln His Ala Ser Ser Pro Pro

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 39

<211> 107

<212> PRT

<213> artificially synthesized sequence (Unknown)

<400> 39

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Gly Asn Ser

20 25 30

Leu Asn Trp Leu Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile

35 40 45

Tyr Ala Thr Ser Ser Leu Asp Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Arg Ser Gly Ser Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln His Ala Ser Ser Pro Pro

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 40

<211> 327

<212> PRT

<213> person (Human)

<400> 40

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg

1 5 10 15

Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr

65 70 75 80

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro

100 105 110

Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

115 120 125

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

130 135 140

Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp

145 150 155 160

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe

165 170 175

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

180 185 190

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu

195 200 205

Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

210 215 220

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys

225 230 235 240

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

245 250 255

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

260 265 270

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

275 280 285

Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser

290 295 300

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

305 310 315 320

Leu Ser Leu Ser Leu Gly Lys

325

<210> 41

<211> 107

<212> PRT

<213> person (Human)

<400> 41

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

1 5 10 15

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

20 25 30

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

35 40 45

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

50 55 60

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

65 70 75 80

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

85 90 95

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

100 105

Claims

1. An antibody combined with human LAG-3 protein, comprising a light chain and a heavy chain, and is characterized in that the amino acid sequence of the light chain variable region of the light chain is shown as SEQ ID No.37 or SEQ ID No.38, and the amino acid sequence of the heavy chain variable region of the heavy chain is shown as SEQ ID No. 35.

2. A recombinant vector comprising the gene encoding the antibody that binds to human LAG-3 protein of claim 1.

3. A recombinant cell expressing the antibody of claim 1 that binds to human LAG-3 protein.