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Anti-human Tim-3 neutralized monoclonal antibody L3D and application thereof

A monoclonal antibody, the technology in the sequence list, applied in the direction of anti-animal/human immunoglobulin, antibody, anti-tumor drug, etc.

Active Publication Date: 2012-06-13
INST OF BASIC MEDICAL SCI ACAD OF MILITARY MEDICAL SCI OF PLA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although studies have shown that neutralizing antibodies to Tim-3 also play a good role in intervening in some disease models, there are currently no Tim-3 antibodies on the market at home and abroad, so the establishment and development of antibody-based antibodies with independent intellectual property rights It has important practical significance for the intervention of various related diseases

Method used

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  • Anti-human Tim-3 neutralized monoclonal antibody L3D and application thereof
  • Anti-human Tim-3 neutralized monoclonal antibody L3D and application thereof
  • Anti-human Tim-3 neutralized monoclonal antibody L3D and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Embodiment 1 Construction of anti-human Tim-3 monoclonal antibody hybridoma cell line

[0031] 1. Materials

[0032] Freund's complete adjuvant and Freund's incomplete adjuvant, chromogenic reagent TMB: product of Sigma company; 20% fetal bovine serum: product of Beijing Yuanheng Shengma Biotechnology Institute; serum-free RPMI 1640: product of Gibco company; SP2 / 0 cells: imported from ATCC and preserved by the Institute of Basic Medical Sciences of the Academy of Military Medical Sciences; Balb / c and C57BL / 6 mice: provided by the Experimental Animal Center of the Academy of Military Medical Sciences; other reagents were purchased from the market.

[0033] 2. Methods and results

[0034] (1) Immunization of Balb / c mice. Six 4- to 6-week-old female Balb / c mice were selected and immunized subcutaneously with 100 μg ricin in the groin. The first injection was added with Freund's complete adjuvant, and the second injection was added with Freund's incomplete adjuvant. The...

Embodiment 2

[0040] Example 2 Screening and Identification of Hybridoma Cell Lines with Human Tim-3 Neutralizing Monoclonal Antibody

[0041] 1. Materials

[0042] Same as embodiment one.

[0043] 2. Methods and results

[0044] (1) Use ELISA method to screen and identify the specific binding activity of human Tim-3 neutralizing monoclonal antibody L3D to antigen: respectively coat recombinant human Tim-3 protein and thioepyn (Trx) control protein, add different Diluted anti-human Tim-3 antibody and HRP-labeled anti-mouse IgG antibody were used to detect the antigen-binding activity and specificity of L3D by indirect ELISA. The results showed that L3D can specifically bind to human Tim-3 molecule with high titer.

[0045] (2) Flow cytometry (FACS) was used to detect the binding of monoclonal antibody L3D to Tim-3 on human U937 cells and the cross-binding activity to Tim-3 on mouse RAW264.7 cells. Experiments were carried out by conventional FACS method. The results showed that L3D can...

Embodiment 3

[0048] Example 3 Fishing of Human Tim-3 Neutralizing Monoclonal Antibody Hybridoma L3D Light and Heavy Chain Genes

[0049] 1. Materials

[0050] Primers: PHs1, see SEQ ID NO: 11 in the sequence listing; PHs2, see SEQ ID NO: 12 in the sequence listing; PHa1, see SEQ ID NO: 13 in the sequence listing; PLs1, see SEQ ID NO: 14 in the sequence listing, PLs2 See SEQ ID NO: 15 in the sequence listing; PLA1, see SEQ ID NO: 16; in the sequence listing. DNA fragment purification kit: product of OMEGA Biotechnology Company; T4DNA ligase: product of New England Biolabs; vector PGEM Teasy: product of Promega Company; competent bacteria JM109: purchased from Promega Company. All the other are with embodiment two.

[0051] 2. Method results

[0052] (1) Take 5×(10 6 ~10 7 ), centrifuge to remove the supernatant, and pop the cells evenly. Add 1ml TRIzol (Invitrogen) and pipette repeatedly to fully lyse the cells. After shaking for 5 minutes, add 0.2ml chloroform, shake for 15 seconds, ...

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Abstract

The invention discloses an anti-human Tim-3 neutralized monoclonal antibody L3D and an application thereof. Antibody light and heavy chain variable region genes are cloned from a prepared anti-human Tim-3 neutralized monoclonal antibody L3D hybridoma cell; and the obtained light chain and heavy chain variable region genes can be used for encoding a monoclonal antibody variable region. Based on the light and heavy chain variable region genes of the monoclonal antibody, a plurality of small molecular genetic engineering antibodies can be constructed and expressed. Polypeptides or proteins encoded on the basis of the genes can be cross-linked with a plurality of bioactive molecules, so that a Tim-3 expression level detection reagent is prepared for diagnosing and treating diseases caused by abnormal expression of Tim-3.

Description

technical field [0001] The present invention relates to a monoclonal antibody, in particular to an anti-human Tim-3 neutralizing monoclonal antibody L3D, and also relates to the use of the monoclonal antibody in preparing Tim-3 detection reagents and diagnosing and treating Tim-3 high expression applications that lead to disease. Background technique [0002] Structurally, Tim-3 belongs to the member of T cell immunoglobulin domain and Mucin domain protein (T cell Immunoglobulin domain and Mucin domain protein, Tim) family. Because the Tim family is widely involved in the immune regulation process of the body, its function is being paid more and more attention by people. Tim-3 is specifically expressed on the surface of activated Th1 and Th17 effector cells, but not on Th2 cells. It is now known that galactose-binding protein-9 (Galactin-9, Gal-9) is the natural ligand of Tim-3, and this molecule is widely expressed in the peripheral immune system. Gal-9 and Tim-3 molecule...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/18G01N33/577A61K39/395A61P31/18A61P31/14A61P35/00A61P37/02A61P19/04A61P11/06
Inventor 韩根成黎燕沈倍奋冯健男郎小玲蒋兴伟时凤敏杨小梅陈国江肖鹤侯春梅
Owner INST OF BASIC MEDICAL SCI ACAD OF MILITARY MEDICAL SCI OF PLA
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