Human single chain variable fragments antibody for recognizing human c-Met proteins, diagnostic reagent and CAR-T cell preparation thereof

A single-source, antibody-based technology, applied in animal/human proteins, anti-animal/human immunoglobulins, cells modified by introducing foreign genetic material, etc., can solve the problems of poor specificity of cancer treatment techniques and achieve Huge social and economic benefits, significant specific effects

Inactive Publication Date: 2018-10-26
李永海
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In order to solve the above-mentioned technical problems, the embodiment of the present invention provides a human single-chain antibody specifically recognizing human c-Met protein, a diagnostic reagent and its CAR-T cell preparation, aiming to solve the technical problems of cancer treatment in the prior art. Technical issues with poor specificity

Method used

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  • Human single chain variable fragments antibody for recognizing human c-Met proteins, diagnostic reagent and CAR-T cell preparation thereof
  • Human single chain variable fragments antibody for recognizing human c-Met proteins, diagnostic reagent and CAR-T cell preparation thereof
  • Human single chain variable fragments antibody for recognizing human c-Met proteins, diagnostic reagent and CAR-T cell preparation thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072] Example 1: Screening and verification of c-Met single chain antibody.

[0073] In Example 1, c-Met protein was used as an antigen to obtain a c-Met-specific single-chain antibody by screening a human single-chain antibody yeast display library.

[0074] Using prokaryotic expression vector, c-Met protein was produced by E.coli BL21DE3 as host cell. The complete c-Met protein contains an extracellular domain, a transmembrane domain and an intracellular domain.

[0075] Extracellular region genes were amplified and cloned into e.g. figure 1 in the vector shown. This prokaryotic expression vector contains GST domain, so c-Met-GST fusion protein is expressed. Then, the fusion protein can be purified by GST purification column.

[0076] Such as figure 2 As shown, after SDS-PAGE gel electrophoresis of the purified protein, a single protein band was presented by Coomassie blue staining. The purified fusion protein was further labeled with biotin. Then, the labeled c-Met...

Embodiment 2

[0081] Example 2: Expression, purification and specificity verification of single chain antibody-Fc fusion protein.

[0082] In Example 2, the restriction endonucleases EcoRI and Kpn1 used for molecular cloning and the HiFiDNA assembly master mix used for vector construction were purchased from Neb Company (Massachusetts, USA); DNA gel recovery kit and plasmid extraction kit were purchased from Axygen Company (Shanghai, China); Competent cells Dh5α for vector construction were purchased from Quanshijin Biotechnology Co., Ltd. (Beijing, China); Pro293 for protein extraction TM CD Serum-free Medium was purchased from Lonza Company (U.S.); the protein G affinity chromatography purification column used for protein purification was purchased from GE Company (U.S.); the ultrafiltration tube used for protein concentration was purchased from Millipore Company (U.S.); MET antibody used for positive control was purchased from CST Company (USA); goat anti-mouse AlexaFluor594 was purchase...

Embodiment 3

[0100] Example 3: In vitro functional verification of single-chain antibody-Fc fusion protein.

[0101] In Example 3, the functional verification of the fusion protein was further carried out, including using flow cytometry analysis and immunofluorescence to verify that the fusion protein specifically recognizes the c-Met protein expressed on the surface of tumor cells, and complement-dependent cytotoxicity, antibody-dependent Cytotoxicity test.

[0102] Among them, the process of flow cytometry experiment for detecting the specific recognition of c-Met by the single chain antibody-Fc fusion protein is as follows:

[0103] According to published literature, human lung adenocarcinoma A549 cells express c-Met protein. Commercialized anti-met antibody was incubated with A549 cells and combined with fluorescently labeled secondary antibody for flow staining. The results showed that there was migration of fluorescent signals, indicating that c-Met protein was expressed on the A54...

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Abstract

The invention relates to a nucleotide sequence and an amino acid sequence, which can specifically recognize human c-Met proteins, of a single chain antibody. Based on the single chain antibody, a fusion protein for specifically recognizing tumor cells with positive c-Met can be acquired. In vitro experiments prove that the fusion protein can mediate complement-dependent cytotoxicity effect and antibody-dependent cytotoxicity effect; the fusion protein can kill the tumor cells when being injected into a tumor-bearing mouse; and based on the single chain antibody, a chimeric antigen receptor (CAR) for recognizing the c-Met is constructed, and CAR-T cells are prepared and amplified. The c-Met CAR-T cells can specifically recognize human tumor cells with the positive c-Met and are effectivelykilled in vitro and expresses the treatment effects of specifically recognizing the c-Met and inhibiting growth of the tumor cells when being injected into the tumor-bearing mouse.

Description

【Technical field】 [0001] The invention relates to the technical field of biotechnology, in particular to a human single-chain antibody that recognizes human c-Met protein, a diagnostic reagent and its CAR-T cell preparation. 【Background technique】 [0002] Cancer is a common disease that threatens human health. Currently, cancer treatment methods commonly used clinically include surgery and radiotherapy / chemotherapy. Because these methods cannot accurately distinguish tumors from surrounding normal tissues. Therefore, it will inevitably cause loss and damage to the body, and cause serious side effects, especially some tumors that are in the advanced stage of development and metastasized. Human society is in urgent need of more effective, safe, and specific treatment methods and methods for tumor tissues. [0003] Receptor tyrosine kinase c-Met and its only known ligand HGF (hepatocyte growth factor) play an important role in cell proliferation, survival, invasion, tissue ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28C12N15/62C12N5/10G01N33/574
CPCC07K14/7051C07K16/2863C07K2317/622C07K2317/73C07K2317/732C07K2317/734C07K2319/30C07K2319/33G01N33/57484
Inventor 李永海储年生李正红
Owner 李永海
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