CN111205371B

CN111205371B - Antibody for resisting lymphocyte activating gene 3 and application

Info

Publication number: CN111205371B
Application number: CN202010075504.5A
Authority: CN
Inventors: 许景峰; 宋丽雪; 许茜; 吴晓奇
Original assignee: Beijing Jilmadi Biomedical Technology Co ltd
Current assignee: Beijing Guozhao Medical Research Institute
Priority date: 2020-01-22
Filing date: 2020-01-22
Publication date: 2022-03-29
Anticipated expiration: 2040-01-22
Also published as: CN111205371A

Abstract

The invention discloses an antibody against lymphocyte activation gene 3(LAG-3), which has unique CDR (complementary deoxyribonucleic acid) regions and variable region sequences, has high specific binding activity with a target antigen LAG-3 and can effectively block the binding of the LAG-3 and MHCII (cytokine induced immune) molecules and the binding of the LAG-3 and FGL1 molecules. The invention also provides a composition of the antibody and the anti-PD-1 antibody, and the antibody and the anti-PD-1 antibody show excellent synergistic effect in the aspect of inhibiting tumor growth. The invention also provides the application of the antibody in the preparation of medicaments for treating, preventing and diagnosing tumors or/and infectious diseases.

Description

Antibody for resisting lymphocyte activating gene 3 and application

Technical Field

The invention discloses a monoclonal antibody of Lymphocyte Activation Gene 3(LAG-3) and a preparation method and application thereof, belonging to the technical field of monoclonal antibodies.

Background

In 1990, Triebel et al found that a new class of immunoglobulin superfamily molecules existed on the surface of activated NK and T cell membranes, and named as Lymphocyte-activation gene 3 (abbreviated as LAG-3; also named as CD223) molecule, i.e., anti-Lymphocyte activation gene 3. The molecule is a type I transmembrane protein consisting of 498 amino acids. The gene of the LAG-3 molecule is located on human chromosome 12 (20p13.3), is adjacent to the human CD4 molecule gene, and has the same exons and introns and high structural similarity.

The LAG-3 molecule consists of an extracellular domain, a transmembrane domain and a cytoplasmic domain, and has a molecular weight of 70 KDa. Among them, the extracellular region is composed of four immunoglobulin superfamily-like domains (D1-D4). D1 belongs to the V-series immunoglobulin superfamily, while D2, D3 and D4 belong to the C-series immunoglobulin superfamily. The D1 region contains a proline-rich loop (about 30 amino acid residues) that specifically binds to MHCII molecules. There are some repeats of the amino acid sequence between the D1 and D3 regions and between the D2 and D4 regions. In stark contrast to other immunosuppressive molecules, the LAG-3 cytoplasmic domain does not contain an immunotyrosine inhibitory motif (ITAM) or an immunotyrosine switching motif (ITSM). The cytoplasmic domain consists of 3 parts: (1) serine phosphorylation sites (human LAG-3 molecule contains two serines and mouse contains one serine site) can serve as a substrate for PKC phosphorylation. (2) A plurality of glutamic acid-proline repeating units (EP). (3) The conserved KIEELE motif is found in all primates, mice, rats. Preliminary results suggest that the conserved KIEELE motif may be closely related to the downstream inhibitory signal of LAG-3.

The homology of LAG-3 to CD4 molecules determines that MHCII molecules are also ligands for LAG-3 molecules. MHCII molecules are mainly expressed on antigen presenting cells such as B cells, monocyte-macrophages and dendritic cells, and mainly have the function of presenting treated antigen fragments to CD4 in the initial stage of immune response⁺T cells, in turn, generate a specific cellular immune response. The binding site of LAG-3 to MHCII molecules is located in the proline-rich loop structure of the D1 domain. Moreover, the affinity (Kd: 60nM) of LAG-3 to MHCII molecules was 100 times that of CD4 molecules to MHCII molecules, which also demonstrates that LAG-3 molecules can effectively compete for the binding of CD4 to MHCII molecules, thereby negatively feeding back the inhibition of T cell activation. Further studies revealed that there are three other ligand molecules for the LAG-3 molecule as well: LSEtin molecule, galectin-3 molecule and Fibrinogen-related Protein 1(Fibrinogen-like Protein 1, FGL 1). LSECtins are a novel class of C-type lectin family carbohydrate recognition molecules, belong to DC-SIGN family members, and are generally expressed on the surface of liver or melanoma cells to inhibit T cell-dependent antitumor activity. In a melanoma microenvironment, LSECtin combined with LAG-3 molecules can inhibit the secretion of antigen-specific T cell IFN-gamma, thereby inhibiting the anti-tumor activity of T cells. galectin-3 molecules are further ligands of the LAG-3 molecule, belonging to members of the lectin familySince the molecule participates in important biological processes such as cell adhesion, cell growth and differentiation, cell cycle and apoptosis, a great deal of research shows that the molecule is closely related to diseases such as cancer, inflammation, heart disease, stroke and the like. Highly glycosylated LAG-3 molecules are capable of interacting with CD8⁺Binding of galectin-3 molecules associated with T cell activation, resulting in CD8⁺Inhibition of IFN-gamma secretion by T cells, and thus inhibition of CD8⁺T cell immune response. Another important ligand molecule FGL1 of LAG-3 was reported in 2018, and binding of LAG-3 to inhibit T cell activation, and release of both effects could generate potential antitumor activity. When the monoclonal antibody is used for inhibiting FGL1 protein or LAG-3 protein of a tumor mouse, the monoclonal antibody also has the effect of inhibiting tumors; however, after T cells are eliminated, the inhibition is not present. After the mouse with the knockout of LAG-3 or FGL1 gene is transplanted with a tumor, the growth of the tumor is obviously inhibited, which indicates that the FGL1-LAG-3 pathway plays a role in the development of the tumor, and further proves that the tumor can activate the LAG-3 receptor of T cells through FGL1 protein, inhibit the T cells and realize immune escape; the process is independent of known immune escape mechanisms (such as PD-1-PD-L1 and the like), and is a brand new immune escape mechanism.

In view of the excellent properties of anti-LAG-3 antibodies in inhibiting tumor growth, the development of target-specific, predominantly inhibitory anti-LAG-3 antibodies is a technical need in the art, and the development of antibody drugs against LAG-3 is still in the early clinical stage, including MGD013 by Macrogenics (PD-1/LAG-3 bispecific antibody), TSR-033 by Tesaro, Relatimab by Bethes Baishigui (also known as BMS-986016, see WO 0020148218), GSK2831781 by Kurarin Steek, LAG525 by Nowa, MK-4280 by Merck, and REGN3767 by regenerant. Clinical studies with LAG-3 antibody most have been performed in combination with PD-1 antibody. Wherein Relatimab and Nivolumab antibody of Baishiqianbao are used in combination for treating tumors such as hemangioma, melanoma, glioblastoma, renal cell carcinoma, and non-small cell lung cancer. In a clinical trial of solid tumors, in which RELALIMAB was used in combination with Nivolumab to treat melanoma, LAG-3 expressing patients in peritumoral immune cells had an Objective Remission Rate (ORR) of 18% for the combined therapy with RELALIMAB and Nivolumab, while ORR was 5% in less than 1% of patients with peritumoral immune cells expressing LAG-3. This result confirmed that the combination of Relatlimab and Opdivo had superior therapeutic effects on patients carrying LAG-3 expressing immune cell tumors. Meanwhile, the safety of Relatlimab and Opdivo combination therapy is similar to that of Opdivo monotherapy. In addition, LAG-3 antibodies from major companies such as Kurarian Schker, Nowa, Rezaoge and Merck are also in clinical research, and clinical development strategies of drug combination with PD-1 antibody are adopted. It is worth mentioning that the GSK2831781 constant region developed by Kurarian Schker selects IgG1 structure, and simultaneously adopts fucose engineering to modify mammalian cell expression, kills immune cells expressing LAG-3 by enhancing ADCC effect, treats autoimmune diseases, at present, the antibody drug is in phase I clinic, and is used for treating plaque psoriasis. IMP761 from Prima BioMed is a LAG-3 activating antibody that, in autoimmune disease states, down-regulates the LAG-3+ T cell activity of the autoimmune response, preventing inflammatory responses and tissue damage. In the development of the PD-1/LAG-3 bispecific antibody, two companies, MacroGenics and F-star, show unique innovative development strategies. Macrogenetics utilizes DART (Dual-Affinity Re-Targeting) platform to develop tetravalent bifunctional antibody molecules that specifically bind both PD-1 and LAG-3 molecules. Preliminary preclinical data indicate that the in vivo and in vitro biological activities of MGD013 are significantly better than the single drug effects of nivolumab (anti-PD-1) and 25F7(anti-LAG-3, disclosed in US20110150892A1), and better than the combined use of the two antibodies in activating T cells. At present, the medicine is in phase I clinic and is used for treating malignant tumors. British Biotechnology corporation F-star uses a unique antibody development platform to introduce a novel LAG-3 antigen binding site (Fcab) at the Fc end of the constant region of an antibody, while the variable region of the antibody recognizes the PD-L1 antigen. The ingenious structural design introduces another antigen binding site at the Fc end under the condition of ensuring that the Y-shaped structure of the antibody is not changed, so as to form the bifunctional antibody with a trivalent structure. The results before clinical application show that the in vitro tumor inhibiting effect of the medicine molecule is superior to that of the combined use of two monoclonal antibodies.

The target specificity of anti-LAG-3 antibodies, whether multispecific antibodies or combinations of antibodies, plays a very important role therein, which is a key technical parameter that determines whether an antibody is capable of specifically inhibiting the specific binding of LAG-3 to a ligand and the synergistic inhibitory effect of the combined antibodies. At present, antibodies which show outstanding target specificity, inhibition efficiency, synergistic effect and the like are still lacking in the field. Therefore, the invention aims to obtain the anti-LAG-3 humanized antibody which has high affinity to antigen, can specifically recognize LAG-3 protein, can effectively block the combination of MHCII, FGL1 and LAG-3 molecules, and has potential tumor inhibition activity and synergistic effect by screening natural phage antibody library.

Disclosure of Invention

Based on the above objects, the present invention firstly provides a monoclonal antibody against lymphocyte activation gene 3(LAG-3), in the present invention a specific embodiment is monoclonal antibody JY-001, the heavy chain variable region CDR1, CDR2 and CDR3 region amino acid sequences of the antibody are respectively shown as SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO: 3; the amino acid sequences of the CDR1, CDR2 and CDR3 regions in the light chain variable region of the antibody are respectively shown in SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6. Antibody variable region CDR amino acid sequence assignments were divided by HTTP:// www.abysis.org according to the KABAT classification scheme.

In a preferred embodiment, the amino acid sequence of the heavy chain variable region of the antibody JY-001 is shown as SEQ ID NO. 7, and the amino acid sequence of the light chain variable region of the antibody is shown as SEQ ID NO. 9.

In a more preferred embodiment, the amino acid sequence of the heavy chain of the antibody JY-001 is shown as SEQ ID NO.11, and the amino acid sequence of the light chain of the antibody is shown as SEQ ID NO. 13.

More preferably, the monoclonal antibody is of the IgG1 or IgG4 isotype.

The present invention also provides an antigen-binding fragment comprising the light chain variable region and the heavy chain variable region of the monoclonal antibody, wherein the antigen-binding fragment is an Fab fragment, an F (ab') 2 fragment, or a single chain antibody. The Fab fragment is a single-arm fragment left after complete digestion of the IgG molecule by papain to remove the Fc and hinge regions, is monovalent, and has only one antigen binding site. The F (ab') 2 fragment is formed by hydrolyzing the disulfide bond of the IgG hinge region near the C end by pepsin, is bivalent, can simultaneously combine 2 antigen epitopes, and can perform agglutination reaction and precipitation reaction. The single chain antibody (scFv) is an antibody formed by connecting an antibody heavy chain variable region and an antibody light chain variable region through a short peptide (linker) with 15-20 amino acids.

Thirdly, the invention also provides a polynucleotide for coding the heavy chain and/or the light chain of the monoclonal antibody, wherein the sequence of the polynucleotide for coding the heavy chain variable region of the antibody is shown as SEQ ID NO. 8, and the sequence of the polynucleotide for coding the light chain variable region is shown as SEQ ID NO. 10.

In a preferred embodiment, the polynucleotide encoding the heavy chain of the antibody has the sequence shown in SEQ ID NO.12 and the polynucleotide encoding the light chain of the antibody has the sequence shown in SEQ ID NO. 14.

Fourth, the present invention also provides a vector encoding the above polynucleotide encoding the heavy chain and/or the light chain of the monoclonal antibody. The expression vector of the present invention is not particularly limited as long as it can express the antibody claimed in the present invention. In a preferred embodiment, the vector is pJY-GS/KIgG 4.

Fifth, the present invention also provides a host cell comprising the above vector.

In a preferred embodiment, the host cell is a mammalian cell expression system. In one embodiment, the mammalian cell is a Chinese Hamster Ovary (CHO) cell, a mouse kidney (BHK) cell, a COS cell, a mouse NSO thymoma cell, a mouse myeloma SP2/0 cell, a human kidney epithelial cell 293T, and the like. In one embodiment, the mammalian cell is a CHO cell.

Sixth, the present invention provides a method for producing the monoclonal antibody described above, which comprises the steps of: the above-mentioned host cells are expressed under culture conditions suitable for the expression of the anti-lymphocyte activating gene 3 antibody.

Seventh, the present invention provides a composition comprising the monoclonal antibody.

In a preferred embodiment, the composition further comprises an anti-PD-1 antibody.

Eighth, the present invention also provides a chimeric molecule comprising the heavy chain and/or light chain variable region of the above monoclonal antibody or a fragment thereof.

In a preferred embodiment, the chimeric molecule is a bispecific antibody, an immunoconjugate, a chimeric antigen receptor, a genetically engineered T cell receptor, or an oncolytic virus. The bispecific antibody is an artificial antibody containing 2 specific antigen binding sites; the immune cross-linked product is a targeted therapeutic and diagnostic preparation generated by cross-linking an antibody and preparations such as compounds, toxins, radionuclides, drug-loaded microspheres and the like; the Chimeric Antigen Receptor (CAR) is a cell surface Receptor that can recognize a specific protein (Antigen), and is composed of a tumor-associated Antigen (TAA) binding region (generally, scFV fragment derived from an Antigen binding region of a monoclonal antibody), an extracellular Hinge region (hindge area), a Transmembrane region (Transmembrane region), and an intracellular Immunoreceptor tyrosine-based activation motif (ITAM); the Chimeric Antigen Receptor is a core component of Chimeric Antigen Receptor T-Cell Immunotherapy (CAR-T); genetically modified T cell receptors refer to receptors that express a specific T cell receptor (TCR; recognition of a specific tumor antigen) or chimeric antigen receptor as described above by genetic engineering; oncolytic viruses (oncolytics) are a class of viruses that tend to infect tumor cells, multiply in large numbers in the tumor cells, eventually lyse the tumor cells and further elicit an anti-tumor immune response in the body. The heavy chain and/or light chain variable region of the monoclonal antibody or the fragment thereof provided by the invention can be used as a part of the chimeric molecule structure to specifically recognize and bind with a tumor and enable the chimeric molecule to exert subsequent therapeutic effects.

Finally, the invention provides the application of the monoclonal antibody in the preparation of medicines for treating, preventing and diagnosing tumors or/and infectious diseases.

In a preferred embodiment, the tumor is selected from melanoma, non-small cell lung cancer, colorectal cancer, prostate cancer, breast cancer, head and neck cancer, colorectal cancer, pancreatic cancer, hematologic cancer, non-hodgkin lymphoma or leukemia or a metastatic lesion of the cancer.

The antibody against lymphocyte activation gene 3 provided by the invention has unique CDR region and variable region sequences, has high specific binding activity with target antigen LAG-3, and compared with the antibody Relatlimab in the prior art, the EC of the antibody is₅₀The value was 0.68 times that of Relatlimab, and the affinity was 11.82 times that of Relatlimab, which showed a stronger binding activity. The antibody provided by the invention can effectively block the combination of LAG-3 and MHCII molecules and the combination of LAG-3 and FGL1 molecules, shows the application prospect in the aspect of inhibiting tumor growth, and shows excellent synergistic effect with an anti-PD-1 antibody in the tumor growth inhibition experiment of combined application of the antibody and the anti-PD-1 antibody. Through the pJY-GS/KIgG4 expression system and 293T transient expression, the transient expression quantity of the antibody is 77.46 +/-8.04 mu g/mL, and the expression quantity of the contrast antibody Relatimab is 52.88 +/-0.76 mu g/mL, so that the antibody can realize the expression preparation of a eukaryotic system, can be used for industrial production and stable strain construction, and has industrial practicability.

Drawings

FIG. 1 is a graph showing the comparison of the specific binding activity of ELISA-detected antibody clones to LAG-3 antigen;

FIG. 2 is a schematic structural view of expression vector pJY-GS/KIgG 4;

FIG. 3 is a control chart showing the analysis of the expression level of monoclonal antibody after transient transfection;

FIG. 4 is a graph of the binding activity of JY-001 antibody to LAG-3/Fc antigen;

FIG. 5 is a graph showing the binding dissociation curves of JY-001 and Relatimab positive antibody with antigen; wherein, A.Relatimab and LAG-3 antigen combination dissociation curve, B.JY-001 and LAG-3 antigen combination dissociation curve;

FIG. 6 is a graph of flow cytometry analysis of JY-001 and Relatimab positive antibodies blocking MHCII binding to LAG-3;

FIG. 7, bar graph of the effect of JY-001 and Relatimab positive antibodies blocking the binding of FGL1 to LAG-3;

FIG. 8 is a graph showing the effect of JY-001 and Relatimab positive antibody in combination with anti-mouse PD-1 antibody on the inhibition of tumor growth time.

Detailed Description

The invention will be further described with reference to specific embodiments, and the advantages and features of the invention will become apparent as the description proceeds. These examples are only illustrative and do not limit the scope of protection defined by the claims of the present invention.

Example 1: screening of anti-LAG-3 antibodies from natural phage antibody library

1.1 materials

Reagent: ampicillin, kanamycin, yeast extract, glucose, tween 20, TMB, PBS, PEG, etc. were used as conventional reagents, and anti-M13 HRP secondary antibody was purchased from Amersham.

Strain, phagemid: TG1 E.coli strain and the helper phage VCSM13 were all commercial products.

1.2 methods

1.2.1 antigen-specific phage display antibody screening

1) The corresponding LAG-3/Fc antigen diluted in sterile PBS to 10. mu.g/mL was added to the immunization tubes at 500. mu.L per tube and coated overnight at 4 ℃.

2) The antigen solution was discarded, 4% milk prepared with sterile PBST was added and blocked for one hour at room temperature.

3) Milk was discarded, 500. mu.L of the blocked phage solution was added to the immune tube and incubated at room temperature for one hour

4) The tubes were washed 15 times with PBST and 10 times with PBS.

5) The phage were eluted with 500. mu.L of 0.1M TEA for ten minutes at room temperature. 500 μ L of 1M Tris-HCl was added to adjust the pH of the eluate to 7.5.

6) The phage was eluted again using 500. mu.L of 0.1M TEA for ten minutes at room temperature, and the second eluent was added to the mixed solution of the first eluent and Tris.

1.2.2 phage regeneration and amplification

1) Inoculating 200. mu.L TG1 to 50mL 2YT, culturing to OD₆₀₀About 0.6 to about 0.8.

2) mu.L of phage was added to 10mL of TG1 in the logarithmic growth phase, and incubated at 37 ℃ for 0.5 hour.

3) Centrifuging at 4000rpm for 15min at 20 ℃.

4) The supernatant was discarded and all the pellet was plated on 2YTAG (100. mu.g/mL Amp in 2YT medium plus 2% glucose) plates and incubated overnight at 37 ℃.

5) All TG1 strains were scraped from the plates and added to 50mL of 2YTAG (100. mu.g/mL Amp in 2YT medium and 2% glucose) medium to make OD₆₀₀The value reaches about 0.1.

6) Incubated at 37 ℃ for 2.5 hours at 220 rpm.

7) And after the strain grows to a logarithmic phase, taking 10mL, adding 20-200 mu L M13ko7 helper phage into the strain, and performing static culture at 37 ℃ for 0.5 hour.

8) Centrifuge at 4000rpm for 15 minutes at 20 ℃.

9) The supernatant was discarded and 50mL of 2YTAK (100. mu.g/mL Amp and 70. mu.g/mL Kana in 2YT medium) was resuspended.

10) Shake culture overnight at 28 ℃.

11) Phage were pelleted for the next round of panning as described above.

1.2.3 post-screening analysis of phage antigen specificity

1) The ELISA plates were coated with the corresponding antigen overnight at 4 ℃.

2) Add 200. mu.L of PBSM (4% skim milk in PBS) per well and block for 0.5 hours at room temperature.

3) Adjusting the phage supernatant to the same initial concentration by comparing the absorbance values of the phage supernatant, and performing gradient dilution on the phage supernatant according to 1X, 5X, 25X and 125X to obtain four concentration gradients.

4) After discarding the solution in the wells, 100. mu.L of phage was added to each well and incubated for 1 hour at room temperature.

5) Wash plates were washed five times with 0.1% Tween-20 deionized water.

6) mu.L of anti-M13/HRP antibody (1:5000 dilution in PBSM) was added to each well and incubated for 1 hour at room temperature.

7) The plate was washed 8 times.

8) Color was developed by adding 100. mu.L of TMB to each well.

9) Adding the neutralization solution and then measuring OD by an enzyme-linked instrument₄₅₀。

10) The positive well clones were sequenced to obtain candidate antibody sequences.

1.3 results

After three rounds of screening and enrichment of LAG-3 antigen, 50 clones of the natural phage antibody library are selected for phage antibody display and ELISA identification. FIG. 1 shows that 14 of the clones specifically bind LAG-3 antigen. Among them, JY-001 had the best binding activity. JY-001 clone was sequenced, and the obtained variable region gene was analyzed on line by abYsis (http:// www.abysis.org /), following Kabat classification model, JY-001 heavy chain variable region amino acid sequence as shown in SEQ ID NO:7, wherein the amino acid sequences of the three CDR regions are respectively SEQ ID NO: 1. SEQ ID NO: 2. SEQ ID NO: 3; the amino acid sequence of the light chain variable region is SEQ ID NO:9, wherein the amino acid sequences of the three CDR regions are respectively SEQ ID NO: 4. SEQ ID NO: 5. SEQ ID NO: 6. the nucleic acid sequence for encoding the heavy chain variable region is shown as SEQ ID NO: 8; the nucleic acid sequence for coding the light chain variable region is shown as SEQ ID NO: shown at 10.

Example 2: construction and expression of expression vector of full-length anti-LAG-3 antibody JY-001

2.1 materials

The primers were synthesized by Biotechnology engineering (Shanghai) GmbH; q5 DNA polymerase is a product of NEB company; t4 DNA ligase is a product of NEB company; gene synthesis and Jetprime transfection reagent were purchased from the company Polyplus, France; sequencing was performed by Biotechnology engineering (Shanghai) Inc.; other relevant reagents are commercially available.

2.2 methods

2.2.1 anti-LAG-3 full-Length antibody expression vector construction

The JY-001 antibody light-heavy chain variable region sequence was obtained by example 1, and JY-001 variable region genes were cloned into eukaryotic expression vector pJY-GS/KIgG4 containing human IgG4 heavy chain constant region and kappa light chain constant region, to construct a whole antibody expression vector, the physical map of which is shown in FIG. 2 (each component of pJY-GS/KIgG4 is a component known in the art, and is recombined in the order shown). The whole antibody is named as JY-001 antibody (heavy chain amino acid sequence is SEQ ID NO.11, nucleotide sequence is SEQ ID NO.12, light chain amino acid sequence is SEQ ID NO.13, nucleotide sequence is SEQ ID NO. 14). In order to compare the characteristics of the antibody JY-001, the invention constructs the light-heavy chain gene of the similar clinical in-process research drug Relatimab of Baisimei Guibao into an pJY-GS/KIgG4 vector as a positive control drug.

2.2.2 measurement of expression level of anti-LAG-3 antibody JY-001

1) Transient transfection

293T cells in logarithmic growth phase at 5X 10⁵The culture medium is inoculated into a 6-well plate at a density of one well per well, and the fresh culture medium is discarded after 12 hours. Adding 2 mu g of antibody expression plasmid (JY-001 full-antibody expression plasmid or Relatimab full-antibody expression plasmid) into 200 mu L of buffer culture medium, fully mixing uniformly, adding 4 mu L of Jetprime transfection reagent, mixing uniformly, standing at room temperature for 10min, adding the mixture into a corresponding 6-well plate, replacing a serum-free antibody special expression culture medium after 6 hours, and culturing for 72 hours to obtain a supernatant.

2) Determination of antibody expression level in transient expression supernatant by double-sandwich ELISA

The GAH-IgG antibody was diluted with the coating solution to 2. mu.g/mL, 100. mu.L per well was added to the plate, and placed in a wet box overnight at 4 ℃. The plate washer washes the enzyme-linked plate 3 times, 1.5% casein block, 200. mu.L per well, wet box 37 ℃ block for L hours. Herceptin (anti-Her 2 therapeutic antibody, control antibody) was diluted 1 × PBS to 1 μ g/mL, on the basis of which 2-fold dilutions were made to obtain 8 concentration gradients, while transient expression supernatants were diluted 625-fold, 3125-fold, 15625-fold with 1 × PBS. Adding Herceptin and transient expression supernatant into an enzyme-linked plate according to 100 muL/each hole for reaction for 1 hour at 37 ℃, cleaning the enzyme-linked plate for 3 times, adding goat anti-human enzyme labeled secondary antibody for reaction for 45min at room temperature, cleaning the enzyme-linked plate for 5 times, adding 100 muL TMB substrate for color development, reacting for 1min, and using 100 muL 2N H₂SO₄The reaction was stopped and the ELISA read at 450 nm. Taking the LN value of the standard substance concentration as the abscissa and the corresponding OD value as the ordinate, drawing a standard curve and adding linear regressionCurve and its correlation coefficient R²(R²>0.95) and the concentration of antibody in the sample was calculated using the y ═ kx + b linear equation.

2.3 results

A schematic representation of the antibody expression vector is shown in FIG. 2. The expression vector is used for expressing JY-001 transiently, the expression level is shown in figure 3, the anti-LAG-3 full-length antibody can be expressed and prepared by the method, and the JY-001 and the Relatlimab antibody transient expression levels obtained by double sandwich ELISA measurement are 77.46 +/-8.04 mu g/mL and 52.88 +/-0.76 mu g/mL respectively. JY-001 can realize full-length expression under the same expression vector, cell and expression condition, and the expression quantity is slightly superior to that of positive control Relatimab. Sufficient supernatant was collected and the antibody was affinity purified using conventional ProteinA.

Example 3: anti-LAG-3 antibody binding Activity assay

3.1 materials

LAG-3/Fc antigen was purchased from Beijing Yi Qiao Shen science and technology Co., Ltd; goat anti-human Fab-HRP (horseradish peroxidase labeled secondary antibody) was purchased from Abcam, USA; casein, PBS, H₂SO₄Is a routine experimental reagent.

3.2 methods

The coating solution diluted LAG-3/Fc antigen to 1. mu.g/mL, 100. mu.L per well was added to the ELISA plate and placed in a wet box overnight at 4 ℃. The plate washer washes the enzyme-linked plate 3 times, 1.5% casein block, 200. mu.L per well, block for 1 hour at 37 ℃ in a wet box. Diluting LAG-3 antibody to 10 μ g/mL with 1 XPBS, diluting with 2-fold gradient, adding 100 μ L of LAG to each well of an ELISA plate, reacting at 37 deg.C for 1 hr in a wet box, washing the ELISA plate 3 times, adding goat anti-human Fab-HRP secondary antibody, reacting at room temperature for 45min, washing the ELISA plate 3 times, adding 100 μ L of 2N H, developing, reacting for 3min, and adding 100 μ L of 2N H₂SO₄The reaction was stopped and the ELISA read at 450 nm. Relatlimab was set during the experiment as a positive antibody control. And an antibody-antigen binding curve is drawn by taking the antibody concentration as an abscissa and the OD value as an ordinate. Fitting a four parameter equation curve using Graphpad analysis software, equation y ═ a-D)/(1+ (X/C)^{^B}) + D, wherein B represents the slope and C represents EC₅₀。

3.3 results

ELISA binding Activity of LAG-3 antibody with LAG-3/Fc antigen As shown in FIG. 4, the antibody was able to specifically recognize the antigen and the binding was dose-dependent. Wherein, JY-001 antibody and positive antibody Relatimab are combined with LAG-3 antigen EC₅₀0.1084 μ g/mL and 0.1580 μ g/mL, respectively. EC for JY-001 compared to the positive control antibody Relatimab₅₀The value was 0.68 times that of the control antibody, and the binding activity was stronger.

Example 4: anti-LAG-3 antibody affinity assay

4.1 materials

Anti-Human IgG Fc Capture (AHC) chip purchased from PALL company, LAG-3-HIS antigen purchased from Beijing Yi Qian Shen science and technology limited, glycine, PBS, Tween-20, BSA are all conventional biochemical reagents.

4.2 methods

Binding kinetics and affinity of the antibody to LAG-3 antigen were measured using Fortebio biofilm layer interferometry. Capturing an antibody to be detected by an Anti-Human IgG Fc Capture (AHC) chip, diluting the concentration of the antibody to be detected to 10 mu g/mL by using a running buffer solution (0.1% Tween-20 and 0.02% BSA are added into a PBS solution), and loading for 90 s; the analyte LAG-3-HIS was likewise diluted with a running buffer gradient to the corresponding concentrations (250nM, 125nM, 62.5nM, 31.25nM, 15.625nM, 7.812nM, 3.906nM and 0nM), the binding time of the test antibody to the analyte was 300s, and the dissociation time was 600 s; the chip was regenerated by repeating 3 pulses of 10mM glycine HCl, pH 1.7 solution for 5 seconds. Determination of data to fit to a 1:1 binding model to determine the equilibrium dissociation constant K_D. Relatimab was set as a positive antibody control during the experiment

4.3 results

The binding dissociation curves of JY-001 antibody and positive antibody Relatimab to LAG-3 antigen are shown in FIG. 5, in which the binding dissociation curves of A.Relatimab to LAG-3 antigen and B.JY-001 to LAG-3 antigen are shown, and their binding constants (K)_on) Are respectively 1.28 multiplied by 10⁵/Ms、8.00×10⁴(Ms); dissociation constant (K)_dis) Are respectively 1.18 multiplied by 10^-4/s、6.23×10^-6And s. Equilibrium dissociation constant K according to the formula_D＝K_dis/K_onThe calculated affinity of the two is 9.21 multiplied by 10 respectively^-10M and 7.78X 10^-11And M. The comparison shows that the affinity of JY-001 is 11.82 times higher than that of a control antibody Relatimab, and is obviously better than that of a positive control antibody. Equilibrium dissociation constant K corresponding to candidate antibody_DThe results are shown in Table 1.

TABLE 1 equilibrium dissociation constant K for candidate antibodies_D

	K_on(1/Ms)	K_dis(1/s)	K_D(M)
				JY-001	8.00×10⁴	6.23×10^-6	7.78×10^-11
Relatlimab	1.28×10⁵	1.18×10^-4	9.21×10^-10

Example 5: functional epitope validation of anti-LAG-3 antibodies

5.1 materials

Raji cells were purchased from ATCC; RPMI-1640, FBS, purchased from Gibco; LAG-3/Fc antigen is purchased from Beijing Yi Qian Shen science and technology Limited, and is purchased from Beijing Jia intelligent Rui biological science and technology Limited APC fluorescence and biotin labeling, FGL1/HIS from Yi Qian Shen; Streptomyces-HRP was purchased from BIOSS; PBS and paraformaldehyde are both conventional biochemical reagents.

5.2 methods

5.2.1 flow cytometric competition method

Raji cells were cultured in RPMI-1640 complete medium (10% FBS) to logarithmic growth phase, cells were gently homogenized by pipette and counted. Adjust cells to 3X 10⁵piece/mL, 1mL into 1.5mL EP tube. Centrifuge at 7000rpm for 1min, discard supernatant, flick tube bottom and resuspend with 1mL FACS (2% FBS in PBS) wash solution, and clean cells once the same procedure. Meanwhile, 5. mu.g/mL of LAG-3/Fc-APC fluorescent antigen was prepared with FACS wash solution, and competitive samples were added to 100. mu.g/mL, respectively, based on the prepared solution. The positive control group and the negative control group are combined in the experimental process, the cells are washed twice by FACS washing liquor after the reaction is carried out for 30 minutes at 4 ℃, and the washing steps are the same as the above. Finally, fixing the cells by using 1% paraformaldehyde, and carrying out sample loading detection by using a flow cytometer. Relatlimab was set as a positive control antibody during the experiment.

5.2.2 competitive ELISA method

FGL1/HIS antigen was diluted to 1. mu.g/mL in the coating solution, 100. mu.L per well was added to the plate, and placed in a wet box overnight at 4 ℃. The plate washer washes the enzyme-linked plate 3 times, 1.5% casein block, 200. mu.L per well, block for 1 hour at 37 ℃ in a wet box. Diluting LAG-3-Biotin with 1 XPBS to 0.3. mu.g/mL, adding the corresponding anti-LAG-3 antibody to the above solution to a final concentration of 6. mu.g/mL, adding 100. mu.L of the antibody to each well of an ELISA plate, reacting at 37 ℃ for 1 hour in a wet box, washing the ELISA plate 3 times, adding a Streptomyces-HRP secondary antibody to the reaction mixture at room temperature for 45min, washing the ELISA plate 3 times, adding 100. mu.L of TMB substrate to the reaction mixture, developing the color, reacting for 3min, and reacting with 100. mu.L of 2N H₂SO₄The reaction was stopped and the ELISA read at 450 nm.

5.3 results

5.3.1 anti-LAG-3 antibodies block the effects of MHCII and LAG-3

LAG-3/Fc-APC can be specifically combined with MHCII molecules on the surface of Raji cells, the experimental result is shown in figure 6, the positive control LAG-3 labeled Raji cell positive rate Gate value is 84.68%, and the Mean fluorescence intensity Mean value is 1193.47. When JY-001 or a control antibody Relatimab is added into the system, both the Gate value and the Mean value are reduced to the background value, which shows that the antibody can effectively compete the interaction of MHCII and LAG-3,

5.3.2 anti-LAG-3 antibodies block the effects of FGL1 and LAG-3

As shown in FIG. 7, LAG-3 molecule also specifically binds to its other ligand FGL1, JY-001 and Relatimab antibody both effectively block the binding of them, and irrelevant antibody Herceptin has no blocking effect. The above results demonstrate that JY-001 antibody can exert effector functions by specifically blocking the action of LAG-3 with the above two important ligand molecules.

Example 6: experiment of combination of antibody LAG-3 antibody and anti-PD-1 for inhibiting tumor growth

6.1 materials

MC38 cells were purchased from ATCC; DMEM and FBS are purchased from Gibco; humanized LAG-3 mice (LAG-3B6.129-Lag3tm1(hLAG3) Smoc) were purchased from southern model animal centers; murine PD-1 monoclonal antibody (Anti-mPD-1) was purchased from InvivoGen.

6.2 methods

LAG-3 humanized mice, 6-8 weeks old, were purchased and placed in SPF-grade animal houses for one week for observation. Simultaneously recovering MC38 cells, subculturing in DMEM complete medium (containing 10% FBS), after the cell density reaches 80% confluence rate, digesting the cells with pancreatin, and resuspending and counting. Washing the cells with physiological saline 1 time, and adjusting the cells to 1X 10⁷one/mL. 100 μ L of MC38 cells were inoculated subcutaneously into mice. When the tumor grows to 100-150mm³Mice were randomly assigned to each experimental group (n-5 mice per group) according to the random number table, and the experimental groups included: a physiological saline solution group; 0.5mg/kg Anti-mPD-1 monotherapy group; 10mg/kg JY-001 antibody and 0.5mg/kg Anti-mPD-1 combination group; 10mg/kg positive antibody, Relatimab, was combined with 0.5mg/kg Anti-mPD-1. The dosing cycle was 2 times per week for a total of 6 doses. The administration is carried out simultaneously with the measurement of the length and the short diameter of the tumor. According to the tumor calculation formula (V ═ LW)²2; v represents tumor volume, L represents tumorMajor axis, W represents tumor minor axis) and tumor proliferation curves were plotted with time as abscissa and tumor size as ordinate.

6.3 results

As shown in FIG. 8, the Anti-mPD-1 group had no antitumor effect at a low dose compared with the saline group. The addition of the LAG-3 antibody to Anti-mPD-1 can inhibit tumor growth to some extent. The LAG-3 antibody and the PD-1 antibody have certain synergistic effect. Statistical analysis of tumor size at the experimental end-point was performed using paired t-test. The results show that LAG-3 antibody JY-001 is statistically different from the Anti-mPD-1 combination group compared to the Anti-mPD-1 single drug group (P ═ 0.046). The effect of this combination was even slightly superior to that of the combination of the positive antibody Relatlimab.

Sequence listing

<110> Beijing Gill Maydi biomedical science and technology Co., Ltd

<120> antibody for resisting lymphocyte activation gene 3 and application

<160> 14

<170> SIPOSequenceListing 1.0

<210> 1

<211> 5

<212> PRT

<213> Homo sapiens

<400> 1

Glu Tyr Tyr Trp Asn

1 5

<210> 2

<211> 16

<212> PRT

<213> Homo sapiens

<400> 2

Glu Ile Ser His Arg Gly Ser Thr Asn Ser Asn Pro Ser Leu Lys Ser

1 5 10 15

<210> 3

<211> 12

<212> PRT

<213> Homo sapiens

<400> 3

Gly Tyr Ser Asp Tyr Ser Tyr Asn Trp Phe Asp Pro

1 5 10

<210> 4

<211> 11

<212> PRT

<213> Homo sapiens

<400> 4

Arg Ala Ser Gln Ser Ile Ser Ser Tyr Leu Ala

1 5 10

<210> 5

<211> 7

<212> PRT

<213> Homo sapiens

<400> 5

Asn Ala Ser Asn Arg Ala Asn

1 5

<210> 6

<211> 9

<212> PRT

<213> Homo sapiens

<400> 6

Gln Gln Arg Ser Asp Trp Pro Leu Thr

1 5

<210> 7

<211> 120

<212> PRT

<213> Homo sapiens

<400> 7

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 Gln Tyr Ser Glu Tyr

20 25 30

Tyr Trp Asn Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Ser His Arg Gly Ser Thr Asn Ser Asn Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Leu Ser Leu Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

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

85 90 95

Phe Gly Tyr Ser Asp Tyr Ser Tyr Asn Trp Phe Asp Pro Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 8

<211> 360

<212> DNA

<213> Homo sapiens

<400> 8

caggtgcagc tgcagcagtg gggcgccggc ctgctgaagc ccagcgagac cctgagcctg 60

acctgcgccg tgtatggcgg ccagtacagc gagtattatt ggaactggat ccgccagccc 120

cccggcaagg gcctggagtg gatcggcgag atcagccacc gcggcagcac caacagcaac 180

cccagcctga agagccgcgt gaccctgagc ctggatacca gcaagaacca gtttagcctg 240

aagctgcgca gcgtgaccgc cgccgatacc gccgtgtatt attgcgcctt tggctatagc 300

gattatagct ataactggtt tgatccctgg ggccagggca ccctggtgac cgtgagcagc 360

<210> 9

<211> 107

<212> PRT

<213> Homo sapiens

<400> 9

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 Asn Ala Ser Asn Arg Ala Asn 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 Asp Trp Pro Leu

85 90 95

Thr Phe Gly Gln Gly Thr Asn Leu Glu Ile Lys

100 105

<210> 10

<211> 321

<212> DNA

<213> Homo sapiens

<400> 10

gagatcgtgc tgacccagag ccccgccacc ctgagcctga gccccggcga gcgcgccacc 60

ctgagctgcc gcgccagcca gagcatcagc agctatctgg cctggtatca gcagaagccc 120

ggccaggccc cccgcctgct gatctataac gccagcaacc gcgccaacgg catccccgcc 180

cgctttagcg gcagcggcag cggcaccgat tttaccctga ccatcagcag cctggagccc 240

gaggattttg ccgtgtatta ttgccagcag cgcagcgact ggcccctgac ctttggccag 300

ggcaccaacc tggagatcaa g 321

<210> 11

<211> 446

<212> PRT

<213> Homo sapiens

<400> 11

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 Gln Tyr Ser Glu Tyr

20 25 30

Tyr Trp Asn Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Ser His Arg Gly Ser Thr Asn Ser Asn Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Leu Ser Leu Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

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

85 90 95

Phe Gly Tyr Ser Asp Tyr Ser Tyr Asn Trp Phe Asp Pro Trp Gly Gln

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

435 440 445

<210> 12

<211> 1338

<212> DNA

<213> Homo sapiens

<400> 12

caggtgcagc tgcagcagtg gggcgccggc ctgctgaagc ccagcgagac cctgagcctg 60

acctgcgccg tgtatggcgg ccagtacagc gagtattatt ggaactggat ccgccagccc 120

cccggcaagg gcctggagtg gatcggcgag atcagccacc gcggcagcac caacagcaac 180

cccagcctga agagccgcgt gaccctgagc ctggatacca gcaagaacca gtttagcctg 240

aagctgcgca gcgtgaccgc cgccgatacc gccgtgtatt attgcgcctt tggctatagc 300

gattatagct ataactggtt tgatccctgg ggccagggca ccctggtgac cgtgagcagc 360

gctagcacaa agggaccttc cgtgttccca ctggccccat gctccagaag cacatctgag 420

tccaccgccg ctctgggctg tctggtgaag gactacttcc ctgagccagt gaccgtgtcc 480

tggaacagcg gcgccctgac atccggagtg cacacctttc cagctgtgct gcagtccagc 540

ggcctgtaca gcctgtcttc cgtggtgaca gtgcccagct cttccctggg caccaagaca 600

tatacctgca acgtggacca taagcctagc aataccaagg tggataagag ggtggagtct 660

aagtacggac caccttgccc accatgtcca gctcctgagt ttctgggagg accatccgtg 720

ttcctgtttc ctccaaagcc taaggacacc ctgatgatct ctcggacacc tgaggtgacc 780

tgcgtggtgg tggacgtgtc ccaggaggat ccagaggtgc agttcaactg gtacgtggat 840

ggcgtggagg tgcacaatgc taagaccaag ccaagagagg agcagtttaa tagcacatac 900

cgcgtggtgt ctgtgctgac cgtgctgcat caggattggc tgaacggcaa ggagtataag 960

tgcaaggtga gcaataaggg cctgcccagc tctatcgaga agacaatctc taaggctaag 1020

ggacagccta gggagccaca ggtgtacacc ctgccccctt cccaggagga gatgacaaag 1080

aaccaggtga gcctgacctg tctggtgaag ggcttctatc catctgacat cgctgtggag 1140

tgggagtcca atggccagcc cgagaacaat tacaagacca caccacccgt gctggactct 1200

gatggctcct tctttctgta ttccaggctg acagtggata agagccggtg gcaggagggc 1260

aacgtgttta gctgctctgt gatgcacgag gctctgcaca atcattatac ccagaagtcc 1320

ctgagcctgt ctctgggc 1338

<210> 13

<211> 214

<212> PRT

<213> Homo sapiens

<400> 13

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 Asn Ala Ser Asn Arg Ala Asn 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 Asp Trp Pro Leu

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

<210> 14

<211> 642

<212> DNA

<213> Homo sapiens

<400> 14

gagatcgtgc tgacccagag ccccgccacc ctgagcctga gccccggcga gcgcgccacc 60

ctgagctgcc gcgccagcca gagcatcagc agctatctgg cctggtatca gcagaagccc 120

ggccaggccc cccgcctgct gatctataac gccagcaacc gcgccaacgg catccccgcc 180

cgctttagcg gcagcggcag cggcaccgat tttaccctga ccatcagcag cctggagccc 240

gaggattttg ccgtgtatta ttgccagcag cgcagcgact ggcccctgac ctttggccag 300

ggcaccaacc tggagatcaa gcgtacggtg gctgctccat ctgtcttcat cttcccacca 360

tctgatgagc agctgaagtc tggaactgcc tctgtggtgt gcctgctgaa taacttctat 420

cccagagagg ccaaagtgca gtggaaggtg gataacgccc tgcagtccgg caactcccag 480

gagagtgtca cagagcagga cagcaaggac agcacctaca gcctgagcag caccctgacc 540

ctgagcaaag ccgactacga gaagcacaaa gtctacgcct gcgaggtcac ccatcagggc 600

ctgagcagcc ccgtcacaaa gagcttcaac aggggagagt gt 642

Claims

1. The monoclonal antibody of anti lymphocyte activation gene 3 is characterized in that amino acid sequences of CDR1, CDR2 and CDR3 regions in a heavy chain variable region of the antibody are respectively shown as SEQ ID NO 1, SEQ ID NO 2 and SEQ ID NO 3; the amino acid sequences of the CDR1, CDR2 and CDR3 regions of the antibody light chain variable region are respectively shown as SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6.

2. The monoclonal antibody according to claim 1, wherein the amino acid sequence of the antibody heavy chain variable region is represented by SEQ ID NO. 7, and the amino acid sequence of the antibody light chain variable region is represented by SEQ ID NO. 9.

3. The monoclonal antibody of claim 2, wherein the amino acid sequence of the antibody heavy chain is set forth in SEQ ID NO.11 and the amino acid sequence of the antibody light chain is set forth in SEQ ID NO. 13.

4. The monoclonal antibody of any one of claims 1-3, wherein the monoclonal antibody is of the IgG1 or IgG4 isotype.

5. An antigen-binding fragment comprising the variable regions of the light and heavy chains of the monoclonal antibody of any one of claims 1-3, wherein the antigen-binding fragment is a Fab fragment, a F (ab') 2 fragment, or a single chain antibody.

6. A polynucleotide encoding the monoclonal antibody of any one of claims 1-3, wherein the polynucleotide encoding the heavy chain variable region of the antibody has the sequence shown in SEQ ID No. 8, and the polynucleotide encoding the light chain variable region of the antibody has the sequence shown in SEQ ID No. 10.

7. The polynucleotide of claim 6, wherein the polynucleotide encoding the heavy chain of the antibody has the sequence shown in SEQ ID NO.12, and the nucleotide encoding the light chain of the antibody has the sequence shown in SEQ ID NO. 14.

8. An expression vector comprising the polynucleotide encoding the monoclonal antibody of claim 7.

9. The vector of claim 8, wherein the vector is pJY-GS/KIgG 4.

10. A host cell comprising the vector of claim 9.

11. A method of producing a monoclonal antibody according to any one of claims 1-3, comprising the steps of: expressing the host cell of claim 10 under culture conditions suitable for expression of the anti-lymphocyte activating gene 3 antibody.

12. A composition comprising the monoclonal antibody of any one of claims 1-3.

13. The composition of claim 12, wherein the composition further comprises an anti-PD-1 antibody.

14. A chimeric molecule comprising a heavy chain variable region and a light chain variable region of the monoclonal antibody of any one of claims 1-3.

15. The chimeric molecule according to claim 14, wherein said chimeric molecule comprises a bispecific antibody, an immunoconjugate, a chimeric antigen receptor, a genetically engineered T cell receptor, or an oncolytic virus.

16. Use of a monoclonal antibody according to any one of claims 1 to 3 for the preparation of a medicament for the treatment, prevention, diagnosis of a tumor and/or an infectious disease.

17. The use according to claim 16, wherein the tumor is selected from melanoma, non-small cell lung cancer, colorectal cancer, prostate cancer, breast cancer, head and neck cancer, colorectal cancer, pancreatic cancer, non-hodgkin's lymphoma.