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Anti-human B7-H4 monoclonal antibody, and preparation method and application thereof

A monoclonal antibody, B7-H4 technology, applied in the direction of antibodies, biochemical equipment and methods, anti-animal/human immunoglobulin, etc., can solve the problems of few tumor sites and undetectable B7-H4 expression, etc. Achieve the effect of low production cost, high cost performance and wide recognition spectrum

Active Publication Date: 2014-08-13
江苏澳洋生物医药研究院有限责任公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the prior art, anti-human B7-H4 monoclonal antibodies mainly include MIH43 (U.S., BioLegend) and H74 (U.S., eBioscience), but they recognize fewer tumor sites and cannot detect B7-H4 in tumor cells. expression on the strain

Method used

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  • Anti-human B7-H4 monoclonal antibody, and preparation method and application thereof
  • Anti-human B7-H4 monoclonal antibody, and preparation method and application thereof
  • Anti-human B7-H4 monoclonal antibody, and preparation method and application thereof

Examples

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Embodiment 1

[0039] Example 1: Preparation of anti-human B7-H4 monoclonal antibody

[0040] This example describes the preparation of the anti-human B7-H4 monoclonal antibody 2B7 of the present invention.

[0041] (1) Preparation of anti-human B7-H4 monoclonal antibody

[0042] Construct transgenic cells (293T / B7-H4) with high expression of B7-H4 molecules. For specific construction steps, refer to the literature published by Mao Yixiang et al.: Construction of human B7-H4 gene transfected cells and their co-stimulatory effects on T cells (Mao Yixiang, Wang Qin, Chen Jie, etc., Chinese Journal of Microbiology and Immunology, August 2006, Vol. 25, No. 8, pp. 609-613).

[0043] Using transgenic cells (293T / B7-H4) highly expressing B7-H4 molecules as immunogen, Balb / c mice (10 7 / 500 1 / piece) (interval 3 weeks). On the fourth day after the last immunization, mouse spleen cells were taken for cell fusion with Ag8 myeloma cell line (a total of 20 96-well plates). Using 293T / B7-H4 that highl...

Embodiment 2

[0054] Example 2: Blocking Effect of Monoclonal Antibody 2B7 on T Cell Proliferation Inhibition

[0055] 1) Isolate normal human peripheral blood PBMCs, sort T cells with magnetic beads, and inoculate them into 96-well plates (10 × 10 5 / well), add B7-H4 mouse Fc fusion protein (10ug / ml), B7-H4 monoclonal monomer (10ug / ml) or a mixture of the two, set 3 duplicate wells in each group, add CCK8 detection after 72h of culture Proliferation. Stimulating monoclonal antibodies CD3 and CD28 stimulated the proliferation of T cells, B7H4 mouse Fc fusion protein inhibited the proliferation of T cells, adding monoclonal antibody 2B7 to observe the blocking inhibitory effect. CCK8 results show that 2B7 can block the inhibitory effect of negative molecules to a certain extent; see attached Figure 9 , where 1 is unstimulated T cells, 2 is stimulated and activated T cells, 3 is activated T cells with B7-H4 mouse Fc fusion protein, 4 is activated T cells with B7-H4 mouse Fc fusion protein ...

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Abstract

The invention discloses an anti-human B7-H4 monoclonal antibody, and a preparation method and an application thereof, and concretely relates to a monoclonal antibody. The monoclonal antibody is generated by a hybridoma cell strain preserved in China General Microbiological Culture Collection Center with the preservation number of CGMCC No.8788. The anti-human B7-H4 monoclonal antibody 2B7 can identify the expression of B7-H4 molecules on the surfaces of different tumor cell strains, and can be applied in the tumor cell detection as a biologic detection index; and results of in vitro experiments show that the anti-human B7-H4 monoclonal antibody 2B7 can block the inhibition effect of the B7-H4 molecules on T cell proliferation.

Description

technical field [0001] The invention relates to a monoclonal antibody, in particular to an anti-human B7-H4 monoclonal antibody and a preparation method thereof, as well as the application of the monoclonal antibody in blocking B7-H4 molecules and inhibiting T cell proliferation. Background technique [0002] T cell activation requires dual signals. The first signal is the recognition of MHC complexes on the surface of APC cells by the T cell-specific receptor TCR, and the second signal is generated by the combination of co-stimulatory molecules with corresponding ligands or receptors. Lack of co-stimulatory signals leads to T cell anergy. The B7 family is a classic co-stimulatory signal provider. Members include CD80, CD86 and their ligands CD28 and CTL-4, which provide positive or negative signals during the activation of T cells. Recently, several homologues of the B7 family were identified, including B7-H1, B7-DC, B7-H2, B7-H3, B7-H4, B7-H6. B7-H4, also known as B7X o...

Claims

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Application Information

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IPC IPC(8): C12N5/20C07K16/18G01N33/577G01N33/574A61K39/395A61P35/00C12R1/91
Inventor 张学光虞培娟傅丰庆张光波
Owner 江苏澳洋生物医药研究院有限责任公司
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