Fully human anti-CTLA-4 monoclonal antibody, preparation method and application

A CTLA-4, monoclonal antibody technology, applied in the direction of antibodies, anti-receptor/cell surface antigen/cell surface determinant immunoglobulin, anti-infective drugs, etc., to achieve simple preparation method, inhibit tumor cell growth, and high-efficiency expression Effect

Active Publication Date: 2015-01-21
河南省健康伟业生物医药研究股份有限公司
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Problems solved by technology

However, it was found that blocking CTLA-4PB7 with anti-CTLA-4 antibody led to the occurrence of Th1 cell-mediated autoimmune diseases

Method used

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  • Fully human anti-CTLA-4 monoclonal antibody, preparation method and application
  • Fully human anti-CTLA-4 monoclonal antibody, preparation method and application
  • Fully human anti-CTLA-4 monoclonal antibody, preparation method and application

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[0032] Example 1

[0033] (1) Preparation of cDNA

[0034] Collect 10 ml of peripheral blood from 50 people with liver cancer, kidney cancer, melanoma, and healthy people, anticoagulate with sodium heparin, separate mononuclear cells (PBMC) by density gradient centrifugation, and sensitize with CTLA-4 antigen in vitro. After PBMC were sensitized in vitro, press 4×10 6 Add 1ml of EBV culture supernatant to PBMC, incubate at 37°C for 3 hours, discard the supernatant, add RPMI1640 complete medium containing 100ml / L FCS, and culture for 2 weeks, then replace with RPMI1640 complete medium containing 200ml / L NBS. The culture supernatant of transformed cells was screened by indirect ELISA method, CTLA-4 was used as the tumor cell antigen solid phase plate, the collected B cell culture supernatant was used as the primary antibody, and HRP-labeled goat anti-human IgG / M (HRP-GAH-IgG / M) as the secondary antibody. Cells from positive wells were expanded to 10 6 ~10 7 cells.

[0035...

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Abstract

The invention discloses a fully human anti-CTLA-4 monoclonal antibody, a preparation method and an application, and belongs to the technical field of antibody drug development and production. The fully human anti-CTLA-4 monoclonal antibody is screened out by constructing a high-capacity natural human phage antibody library, and has high affinity and low dissociation rate (low immunogenicity) for human CTLA-4 molecules. Compared with a chimeric or humanized antibody, the fully human anti-CTLA-4 monoclonal antibody can prevent a lot of side effects when being applied on a human body. The fully human anti-CTLA-4 monoclonal antibody is simple in preparation method, can be efficiently expressed in animal cells and is suitable for large-scale production. Besides, the fully human anti-CTLA-4 monoclonal antibody can suppress growth of tumor cells significantly, and has wide application prospects in resisting tumor, resisting infection and treating autoimmune diseases.

Description

technical field [0001] The invention relates to a fully human anti-CTLA-4 monoclonal antibody, a preparation method and application of the anti-CTLA-4 monoclonal antibody, and belongs to the technical field of antibody drug development and production. Background technique [0002] The activation of T cells requires two signals from extracellular stimulation, that is, the double signal effect of T lymphocyte activation. The first signal of T cell activation mainly comes from the specific combination of T cell antigen recognition receptor (TCR) and MHC molecule-antigen peptide complex, and this process is antigen recognition. The second signal of T cell activation, also known as co-stimulatory signal, is provided by the adhesion molecule pair on the surface of antigen presenting cells (APC) and T cells. These adhesion molecules are called co-stimulatory molecules, the most important of which are CD28 and CTLA-4 on the surface of T cells and the corresponding ligands B7 (inclu...

Claims

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Application Information

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IPC IPC(8): C07K16/28C12N15/63A61K39/395A61P35/00A61P31/00A61P37/00
Inventor 张胜华张守涛张一折郭亚楠杨艳丽郭奇根于振艳
Owner 河南省健康伟业生物医药研究股份有限公司
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