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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: 2014-05-28
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] Example 1: Construction of anti-human Tim-3 monoclonal antibody hybridoma cell line

[0031] 1. Material

[0032] Freund's complete adjuvant and Freund's incomplete adjuvant, color reagent TMB: product of Sigma; 20% fetal bovine serum: product of Beijing Yuanheng Shengma Biotechnology Research Institute; serum-free RPMI 1640: product of Gibco; SP2 / 0 cells: introduced by ATCC and preserved by the Institute of Basic Medicine, Academy of Military Medical Sciences; Balb / c and C57BL / 6 mice: provided by the Laboratory Animal Center of Academy of Military Medical Sciences; other reagents are commercially available.

[0033] 2. Methods and results

[0034] (1) Balb / c mice immunization. Six female Balb / c mice aged 4 to 6 weeks were used for subcutaneous immunization with 100 μg ricin in the groin. The first injection was supplemented with Freund’s complete adjuvant, and the second injection with Freund’s incomplete adjuvant. Immunization once every 3 weeks. A total of 3 immunizations...

Embodiment 2

[0040] Example 2 Screening and identification of hybridoma cell lines with human Tim-3 neutralizing monoclonal antibody

[0041] 1. Material

[0042] The same as in Example 1.

[0043] 2. Methods and results

[0044] (1) Screen and identify the specific binding activity of human Tim-3 neutralizing monoclonal antibody L3D with antigen by ELISA method: respectively coat the recombinant human Tim-3 protein and the thioglycerin (Trx) control protein, add different Diluted anti-human Tim-3 antibodies and HRP-labeled anti-mouse IgG antibodies were used to detect the antigen binding activity and specificity of L3D by indirect ELISA. The results show that L3D can specifically bind to human Tim-3 molecules 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 with Tim-3 on mouse RAW264.7 cells. The experiment was carried out according to the conventional FACS method. The result...

Embodiment 3

[0048] Example three people Tim-3 neutralizing monoclonal antibody hybridoma cell L3D light and heavy chain genes

[0049] 1. Material

[0050] Primer: 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, PLs2 in the sequence listing 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; T4DNA ligase: product of New England Biolabs; carrier PGEM Teasy: product of Promega; competent bacteria JM109: purchased from Promega. The rest is the same as the second embodiment.

[0051] 2. Method results

[0052] (1) Take the neutralizing monoclonal antibody L3D cell 5×(10 6 ~10 7 ), centrifuge to remove the supernatant and bounce the cells evenly. Add 1ml of TRIzol (Invitrogen) and repeatedly pipette to fully lyse the cells. After shaking for 5 minutes, add 0.2ml of chloroform...

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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 the anti-human Tim-3 neutralizing monoclonal antibody L3D, and also relates to the use of the monoclonal antibody in the preparation of Tim-3 detection reagents, and the diagnosis and treatment of Tim-3 high expression. Lead to the application of diseases. Background technique [0002] Tim-3 is structurally a member of the T cell immunoglobulin domain and mucin domain protein (Tim) family. Because the Tim family is widely involved in the body's immune regulation process, its function is getting more and more attention. 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 Gal-9 (Galectin-9, Gal-9) is the natural ligand of Tim-3. This molecule is widely expressed in the peripheral immune system. Gal-9 and Tim-3 molecules on Th1 and Th17 cells Specific binding can trigger the apoptosis of the latter, down-re...

Claims

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Application Information

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Patent Type & Authority Patents(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