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Preparation for anti human B7-H3 monoclonal antibody and application thereof

A monoclonal antibody, B7-H3 technology, applied in the direction of anti-animal/human immunoglobulin, antibody, measuring device, etc., can solve the difficulties of less cell surface, limited in-depth study of DC function, DC separation and purification and phenotypic identification And other issues

Inactive Publication Date: 2008-01-16
SUZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the extremely small number of DCs in tissues and the lack of characteristic markers on the cell surface, the in-depth study of DC functions is greatly limited.
Although the in vitro expansion method of DC has been established, the purification and phenotypic identification of DC isolation are still difficult due to the lack of characteristic surface markers

Method used

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  • Preparation for anti human B7-H3 monoclonal antibody and application thereof
  • Preparation for anti human B7-H3 monoclonal antibody and application thereof
  • Preparation for anti human B7-H3 monoclonal antibody and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1: Preparation of anti-human B7-H3 monoclonal antibody

[0043] This example describes the preparation of the anti-human B7-H3 monoclonal antibodies 4H7, and 21d4 of the present invention.

[0044] (1) Establishment of transgenic cells L929 / B7-H3 and L929 / mock

[0045] (a) Cloning of human B7-H3 gene: TRIZOL reagent (Gibco, BRL) was used to extract the total RNA of antigen-induced mature DC. According to the method recommended in the instruction manual of the MBI reverse transcription kit, the mRNA was reverse transcribed into cDNA. Take 5 μl of cDNA as a template, use the upstream primer 5'-AA CTG CAGATG GAA AGG GTC CAA CCC-3', and the downstream primer 5'-CG GGA TTC TCA AAGGAC ACA GAA TTC-3' for PCR amplification (denaturation at 94°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 1 min, and extension at 72°C for 5 min after 30 cycles). After purification, at T 4 The PCR product was directly connected to the pMD18-T vector under the action of ...

Embodiment 2

[0056] Example 2: Recognition ability of monoclonal antibody 21D4 to B7-H3 molecules expressed on the surface of Mo-DCs

[0057] 1) Mononuclear cells (PBMC) were routinely isolated from peripheral blood of healthy people. After washing twice with RPMI-1640, the cell density was adjusted to 3×10 with RPMI-1640 containing 10% FCS. 6 / ml, added to 6-well culture plate (2ml / well). then 5% CO 2 , 37°C for 2 hours. After aspirating the suspended cells, add GM-CSF (100ng / ml), IL-4 (50ng / ml), 10% FCS, 0.02mmol / LL-glutamine, 5×10 -5 / mol / L2-ME RPMI-1640 continued to be cultured, and the medium was changed every 3 days. After 7 days of culture, 5C11 (5 μg / ml) or TNF-α (10 ng / ml) was added for induction for 3 days.

[0058] 2) DCs in different developmental stages of each induction group were collected, and the expression of B7-H3 on the cell surface was analyzed by flow cytometry. The results showed that: B7-H3 showed extremely stable high-level expression on Mo-DCs. The expressio...

Embodiment 3

[0059] Example 3: Relationship between B7-H3 expression on tumor cells and tumor prognosis

[0060] 1) Case source

[0061]102 specimens of gastric cancer were obtained from gastric cancer surgery patients admitted in the Third Affiliated Hospital of Soochow University during 1998-1999. Among them, there were 75 males and 27 females. Aged 28-77 years old, with an average of 55 years old. Tumors were located in the mucosa and submucosa in 10 cases, located in the superficial muscular layer in 20 cases, and the tumor infiltration depth reached or exceeded the deep muscular layer in 72 cases. Histological types are divided into: differentiated adenocarcinoma (papillary adenocarcinoma, well-differentiated tubular adenocarcinoma, moderately differentiated tubular adenocarcinoma) and poorly differentiated adenocarcinoma (poorly differentiated adenocarcinoma, signet ring cell carcinoma, mucinous cell carcinoma). All patients did not receive chemotherapy or radiotherapy before oper...

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Abstract

The invention relates to binding proteins or polypeptides of a member of the immunoglobulin superfamily, specifically relates to an anti-human B7-H3 monoclonal antibodies 4H7 and 21D4, a preparation method of the monoclonal antibodies, and the applications of the monoclonal antibodies in tumor (gastric cancer) prognostic evaluation and in being used as a marked molecule for identification of human dendritic cells (DC).

Description

field of invention [0001] The present invention relates to binding proteins or polypeptides of members of the immunoglobulin superfamily, more specifically, the present invention relates to anti-human B7-H3 monoclonal antibodies 4H7 and 21D4, their preparation methods and the prognosis evaluation of these monoclonal antibodies in tumors (gastric cancer) And as a marker molecule in the identification of human dendritic cells (DC) in the application. background of the invention [0002] Many receptor-ligand interactions are known to be involved in the induction, establishment and regulation of antigen-specific immune responses. In order to effectively activate a T cell response, at least two signals are usually required. Among them, in addition to the T cell antigen receptor (TCR) recognizing the MHC-antigen complex on the antigen-presenting cell (APC) to provide the first signal, the antigen-specific signal, it is also necessary to obtain the interaction of costimulatory mol...

Claims

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Application Information

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IPC IPC(8): C07K16/18A61K39/395G01N33/577
Inventor 张学光张光波
Owner SUZHOU UNIV
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