Specific T cell receptor (TCR) specific to EGFR L858R gene mutation and application of TCR

A cell receptor and specific technology, applied in the field of genetic engineering and tumor immunotherapy, can solve the problems of unverified therapeutic effect and lack of effective TCR, etc., and achieve good therapeutic effect and strong specificity

Inactive Publication Date: 2019-08-27
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

However, since the development of TCR-T is still in its infancy, effective TCRs for specific mutations and corresponding patient populations are still

Method used

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  • Specific T cell receptor (TCR) specific to EGFR L858R gene mutation and application of TCR
  • Specific T cell receptor (TCR) specific to EGFR L858R gene mutation and application of TCR
  • Specific T cell receptor (TCR) specific to EGFR L858R gene mutation and application of TCR

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Embodiment 1 TCR lentivirus preparation

[0053] The coding sequences of TCR α chain and β chain were connected by furin cleavage site, SGSG linker and F2A sequence, the α chain and β chain genes were fully synthesized, and the TCR gene was cloned into lentiviral expression using restriction endonucleases EcoRI and BamHI In the vector pCDH (purchased from SBI), a recombinant plasmid capable of expressing the amino acid sequences of the TCRα chain and β chain variable regions shown in Sequences 6 and 8 was obtained.

[0054] 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 the plasmid.

[0055] Packaging of recombinant plasmids: Take 293T cells in the logarithmic growth phase (purchased from the Basic Medical Cell Center o...

Embodiment 2

[0056] Example 2 Preparation of TCR-T cells and analysis of TCR expression in TCR-T cells by flow cytometry

[0057] Peripheral blood from healthy volunteers was collected, and human peripheral blood mononuclear cells (PBMC) were separated using a lymphocyte separation medium (Stemcell, 07861, USA). Dynabeads (Gibco, 11141D) and PBMC were mixed and incubated at room temperature for 20 min to separate activated T cells. Add 3 mL of X-Vivo 15 medium (Lonza, DL-201) to T cells, resuspend the mixture of cells and Dynabeads (Thermo, 11141D) with a pipette, and adjust the cell density to 0.5-1×10 6 cells / mL to obtain a cell suspension. Place the cell suspension at 37°C, 5% CO 2 After continuous culture in the incubator for 48 h, the cell density was adjusted to 1x10 6 individual / mL. Take out the lentivirus from the -80°C ultra-low temperature refrigerator, thaw it quickly in a 37°C water bath, add polybrene (Santa Cruz, sc-134220) to the prepared T cells to a final concentration...

Embodiment 3

[0058] The preparation of embodiment 3 target cells

[0059] The lentiviral expression vector pCDH-A1101 overexpressing the HLA-A*1101 molecule was constructed, transiently transfected into 293T cells, and the recombinant lentivirus was prepared. The method was the same as in Example 1. The T2 cell line was transduced with the virus, and the T2 cell line expressing HLA-A*1101 was constructed according to the method in Example 2. After labeling with an antibody against HLA molecules, FACS was used to detect the expression of HLA-A*1101 on the surface of T2 cells, as shown in FIG. 2 . Figure 2A shows T2 cells not transfected with HLA-A*1101, and Figure 2B shows T2 cells transfected with HLA-A*1101, and the transfection efficiency of HLA-A*1101 obtained by comparison is 96.6%.

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Abstract

The invention provides a specific T cell receptor (TCR) specific to EGFR L858R gene mutation and application of the TCR. The TCR has the characteristic of combining a new antigen peptide KITDFGRAK-HLA-A*1101 compound derived from the EGFR L858R gene mutation, and comprises variable regions and constant regions of an alpha chain and a beta chain. T cells modified by the TCR has specific killing effect on HLA-A*1101 tumor cells with the EGFR L858R gene mutation. In addition, the invention further provides a drug combination for treating tumors involved in expressing the gene mutation; the characteristics of strong specificity and good individualized treatment effect are realized.

Description

technical field [0001] The invention relates to the field of genetic engineering and tumor immunotherapy, in particular to a specific T cell receptor 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 targ...

Claims

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Application Information

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IPC IPC(8): C07K14/725C12N15/12C12N15/867C12N5/10A61K39/00A61P35/00
CPCA61K39/001102A61P35/00C07K14/7051C12N5/0636C12N15/86C12N2510/00C12N2740/15043C12N2800/107
Inventor 杜学明李凤娥霍冲邓丽刚邹庆薇王亚玲
Owner 天津亨佳生物科技发展有限公司
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