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Antibody resisting lymphocyte activation gene 3 (LAG-3) and application

A technology of lymphocytes and genes, applied in the direction of antibodies, applications, anti-receptors/cell surface antigens/cell surface determinant immunoglobulins, etc., can solve the problems of lack of antibodies and achieve strong binding activity and excellent synergistic effects

Active Publication Date: 2020-05-29
北京国兆麦迪医学研究院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is still a lack of antibodies with outstanding target specificity, inhibitory efficiency and synergistic effect in this field

Method used

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  • Antibody resisting lymphocyte activation gene 3 (LAG-3) and application
  • Antibody resisting lymphocyte activation gene 3 (LAG-3) and application
  • Antibody resisting lymphocyte activation gene 3 (LAG-3) and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1: Screening of Anti-LAG-3 Antibody from Natural Phage Antibody Library

[0035] 1.1 Materials

[0036] Reagents: ampicillin, kanamycin, yeast extract, glucose, Tween 20, TMB, PBS, PEG, etc. were routine reagents, and anti-M13 HRP secondary antibody was purchased from Amersham Company.

[0037] Bacterial strains and phagemids: TG1 Escherichia coli strain and helper phage VCSM13 are commercial products.

[0038] 1.2 Method

[0039] 1.2.1 Antigen-specific phage display antibody screening

[0040] 1) Dilute the corresponding LAG-3 / Fc antigen to 10 μg / mL in sterile PBS, add 500 μL per tube into the immunotube, and coat overnight at 4°C.

[0041] 2) Discard the antigen solution, add 4% milk prepared in sterile PBST, and block for one hour at room temperature.

[0042] 3) Discard the milk, add 500 μL of blocked phage solution to the immunotube, and incubate at room temperature for one hour

[0043] 4) Wash the tube 15 times with PBST, and then wash the tube 10 ti...

Embodiment 2

[0071] Example 2: Construction and expression of anti-LAG-3 full-length antibody JY-001 expression vector

[0072] 2.1 Materials

[0073] Primers were synthesized by Sangon Bioengineering (Shanghai) Co., Ltd.; Q5 DNA polymerase was a product of NEB Company; T4 DNA ligase was a product of NEB Company; gene synthesis and Jetprime transfection reagent were purchased from French Polyplus Company; sequencing was provided by Sangon Bioengineering ( Shanghai) Co., Ltd.; other related reagents are commercially available.

[0074] 2.2 Method

[0075] 2.2.1 Construction of anti-LAG-3 full-length antibody expression vector

[0076] The light and heavy chain variable region sequence of the JY-001 antibody was obtained through Example 1, and the JY-001 variable region gene was cloned into the eukaryotic expression vector pJY-GS / KIgG4 containing the human IgG4 heavy chain constant region and the kappa light chain constant region , to construct the whole antibody expression vector, its ph...

Embodiment 3

[0084] Example 3: Analysis of Anti-LAG-3 Antibody Binding Activity

[0085] 3.1 Materials

[0086] LAG-3 / Fc antigen was purchased from Beijing Yiqiao Shenzhou Technology Co., Ltd.; goat anti-human Fab-HRP (horseradish peroxidase-labeled secondary antibody) was purchased from abcam company in the United States; casein, PBS, H 2 SO 4 For routine experimental reagents.

[0087] 3.2 Method

[0088] Dilute the LAG-3 / Fc antigen to 1 μg / mL in the coating solution, add 100 μL per well to the enzyme-linked plate, and place it in a humid box at 4°C overnight. Wash the enzyme-linked plate 3 times with a plate washer, block with 1.5% casein, 200 μL per well, and block for 1 hour at 37°C in a humid box. Dilute the LAG-3 antibody to 10 μg / mL with 1×PBS, and dilute it in a 2-fold gradient, add 100 μL per well to the enzyme-linked plate, react in a wet box at 37°C for 1 hour, wash the enzyme-linked plate 3 times, add sheep Anti-human Fab-HRP secondary antibody reacted at room temperature...

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Abstract

The invention discloses an antibody resisting a lymphocyte activation gene 3 (LAG-3). The antibody has a unique CDR region and a variable region sequence, has highly specific binding activity with a target antigen LAG-3, and can effectively block binding of the LAG-3 and an MHCII molecule and binding of the LAG-3 and an FGL1 molecule. The invention also provides a composition of the antibody and an anti-PD-1 antibody, and the antibody and the anti-PD-1 antibody show an excellent synergistic effect of inhibiting tumor growth through combination. The invention further provides application of theantibody in preparation of a drug for treating, preventing and diagnosing tumor or / and infectious diseases.

Description

technical field [0001] The invention discloses an anti-lymphocyte activation gene 3 (Lymphocyte Activation Gene 3, LAG-3) monoclonal antibody, a preparation method and application thereof, and belongs to the technical field of monoclonal antibodies. Background technique [0002] In 1990, Triebel et al. discovered that there was a new class of immunoglobulin superfamily molecules on the surface of activated NK and T cell membranes, and named them Lymphocyte-activation gene 3 (abbreviated as LAG-3; also known as CD223) molecules, namely anti-lymphocytes. Cell activation gene 3. This molecule is a type I transmembrane protein consisting of 498 amino acids. The gene of LAG-3 molecule is located on human chromosome 12 (20p13.3), adjacent to the gene of human CD4 molecule, and both have the same exons and introns, and have high structural similarity. [0003] LAG-3 molecule consists of extracellular region, transmembrane region and cytoplasmic region, with a molecular weight of ...

Claims

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Application Information

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IPC IPC(8): C07K16/28C12N15/13A61K39/395A61P35/00A61P35/02A61P35/04
CPCC07K16/2803C07K16/2818A61P35/00A61P35/02A61P35/04C07K2317/565C07K2317/56C07K2317/92C07K2317/76C07K2317/73A61K2039/505A61K2039/507
Inventor 许景峰宋丽雪许茜吴晓奇
Owner 北京国兆麦迪医学研究院
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