Specific TCR pointing to EGFR L858R gene mutation and application of specific TCR

A specific, KITDFGRAK-HLA-A1101 technology, applied in the field of genetic engineering and tumor immunotherapy, can solve the problems of clinical treatment effect to be verified, lack of clinical trial data, etc., and achieve good therapeutic effect and strong specificity

Inactive Publication Date: 2020-05-08
天津亨佳生物科技发展有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although TCR-T has achieved some success in preclinical testing and treatment of a small number of patients, clinical trial data for TCR-T are still scarce
At the same time, since the research and development of TCR-T is still in its infancy, TCR-T and T cells of clinical therapeutic quality still need to be improved. More importantly, the effective patient population of TCR-T and the clinical therapeutic effect of the newly constructed TCR-T yet to be verified

Method used

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  • Specific TCR pointing to EGFR L858R gene mutation and application of specific TCR
  • Specific TCR pointing to EGFR L858R gene mutation and application of specific TCR
  • Specific TCR pointing to EGFR L858R gene mutation and application of specific TCR

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041]Example 1 Flow cytometry analysis of the TCR expression of the constructed TCR-T

[0042] According to the TCR gene sequence obtained by sequencing, the TCR α chain and β chain are fully synthesized. Digest the pMX-IRES-GFP plasmid with Not1 and EcoR1 restriction endonucleases, recover the empty vector gene fragment from the gel; dephosphorylate the virus plasmid with alkaline phosphatase (CIAP), and the reaction system is 50ul: plasmid 20ul, 10×Buffer 5ul , CIAP 2ul, sterile water 23ul, incubated at 37°C for 30min, and purified plasmid DNA by ethanol precipitation. The TCR full-length gene was ligated into pMX-IRES-GFP empty vector plasmid, and ligated overnight at 16°C. Transform the recombinant plasmid into XL-10 competent cells, evenly spread it on the LB solid medium plate containing ampicillin, culture at 37°C for 12 hours, pick a single colony into the LB liquid medium containing ampicillin, and incubate at 37°C , 220rpm / min shaking culture for 14-16h, extract t...

Embodiment 2

[0045] Example 2 Flow Cytometry Analysis of the Ability of Specific TCR-T to Recognize Specific Antigen Peptides

[0046] Take peripheral blood from healthy volunteers, and separate PBMCs by density gradient centrifugation: Peripheral blood is diluted with PBS at a ratio of 1:1, and the diluted blood is carefully added to the lymphocyte separation medium at a ratio of 1:1 to form an obvious layer. Centrifuge horizontally at 1600rpm / min for 25min. Aspirate the mononuclear cell layer carefully with a pipette, wash the cells twice with serum-free 1640 medium, resuspend the cells and count them. Human peripheral blood mononuclear cells (PBMCs) were activated for 2 days with anti-CD3 antibody (20 ng / mL) and interleukin 2 (IL-2; 300 IU / ml). The day before the infection, the PBMC cells in the logarithmic growth phase were pipetted into a single cell suspension and counted 5×10 5 2 cells were seeded in a 6-well plate coated with fibronectin. On the day of infection, 200ul concentrat...

Embodiment 3

[0047] Example 3 Flow Cytometry Analysis of the Killing Effect of Specific TCR-T Cells on Target Cells

[0048] The T2 cell line of HLA-A1101 is constructed. The T2 cell line lacks transporters related to antigen processing (TAP), so it can effectively load foreign peptides, and as antigen presenting cells, present the loaded antigen peptides to T cells for recognition. The artificially synthesized antigen peptide KITDFGRAK was mixed with T2 cells at 37°C, 5% CO 2 Incubate for 24 h under the conditions (the concentration of polypeptide is 50 μg / ml, the concentration of T2 cells is 1×10 6 cells / ml), washed to remove unbound antigenic peptides, and then the cells were collected, which were T2 cells loaded with antigenic peptide KITDFGRAK.

[0049] Specific TCR-T cells and T2 cells loaded with antigen peptide KITDFGRAK were incubated at 37°C, 5% CO 2 Incubate for 24 h under the condition (the cell concentration is 1×10 6 pieces / ml). Control cells were T2 cells not loaded with...

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Abstract

The invention provides a specific TCR pointing to EGFR L858R gene mutation and application of the specific TCR. The TCR has the characteristic of bonding to a new antigen peptide KITDFGRAK-HLA-A1101 complex derived from the EGFR L858R gene mutation and comprises variable and constant regions of alpha and beta chains. T cells modified with the TCR have a specific killing action on tumor cells HLA-A1101 expressing EGFR L858R gene mutation. In addition, the invention further provides a pharmaceutical composition for treating tumors involving expression of gene mutation. The pharmaceutical composition has the characteristics of high specificity and good individualized treatment effect.

Description

technical field [0001] The invention relates to the fields of genetic engineering and tumor immunotherapy, in particular to a specific TCR for EGFR L858R gene mutation and its application. Background technique [0002] Tumor immunotherapy is a treatment method to control and eliminate tumors by restarting and maintaining the tumor-immune cycle and restoring the body's normal anti-tumor immune response. TCR (T cell receptor) therapy is T cell receptor therapy. In this therapy, endogenous T cells are isolated, engineered, and infused back into the human body. In this way, the number of T cells with the ability to target cancer cells will greatly increase. In addition to killing tumors quickly like cytotoxic chemotherapy and targeted therapy, this approach also avoids the delayed effects of vaccines and immune checkpoint inhibitor therapies. TCR-T is one of the most promising tumor treatment technologies following tumor surgery, radiotherapy, chemotherapy, and targeted therap...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N5/10A61K35/17A61P35/00
CPCC07K14/7051C07K16/2863A61K35/17A61P35/00C07K2319/30C12N2510/00
Inventor 杜学明霍冲邓丽刚邹庆薇
Owner 天津亨佳生物科技发展有限公司
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