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Specific T cell receptor (TCR) directed at EGFR L858R gene mutation and application thereof

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.

Inactive Publication Date: 2020-10-30
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 very scarce
At the same time, the effective patient population of TCR-T and the therapeutic effect of the new structure of TCR-T have yet to be verified

Method used

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  • Specific T cell receptor (TCR) directed at EGFR L858R gene mutation and application thereof
  • Specific T cell receptor (TCR) directed at EGFR L858R gene mutation and application thereof
  • Specific T cell receptor (TCR) directed at EGFR L858R gene mutation and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Example 1 Preparation of TCR lentivirus

[0051] 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 lentivirus using restriction endonucleases EcoRI and BamHI In the expression vector pCDH (purchased from SBI), a recombinant plasmid was obtained, which was capable of expressing the amino acid sequences of the TCRα chain and β chain variable region shown in sequence 6 and 8.

[0052] The recombinant plasmid was transformed into XL-10 competent cells, spread evenly on the LB solid medium plate containing ampicillin, cultured at 37°C for 12 h, picked a single colony into the LB liquid medium containing ampicillin, and incubated at 37°C. Cultivate with shaking at 220 rpm / min for 14-16 h, and extract the plasmid.

[0053] Packaging of recombinant plasmids: 293T cells in the logarithmic growth phase (purchased from the Basic...

Embodiment 2

[0054] Example 2 Preparation of TCR-T cells and analysis of TCR expression in TCR-T cells by flow cytometry

[0055] Peripheral blood from healthy volunteers was collected, and human peripheral blood mononuclear cells (PBMC) were obtained by separating human peripheral blood mononuclear cells (PBMC) using lymphocyte separation medium (Stemcell, USA, 07861). 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 1×10 6 individual / mL. Take out the lentivirus from the ultra-low temperature refrigerator at -80°C, thaw it quickly in a 37°C water bath, add polybrene (Santa Cru...

Embodiment 3

[0056] Example 3 Preparation of target cells

[0057] 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 The middle is T2 cells not transfected with HLA-A*1101, Figure 2B The middle is the T2 cells transfected with HLA-A*1101, and the transfection efficiency of HLA-A*1101 is 96.6%.

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Abstract

The invention provides a specific TCR directed at EGFR L858R gene mutation and application thereof. The specific TCR has a property of binding to a neoantigen peptide KITDFGRAK-HLA-A*1101 complex derived from the EGFR L858R gene mutation, and the TCR comprises variable regions and constant regions of an alpha chain and a beta chain. A T cell modified by the TCR has a specific killing effect on anHLA-A*1101 tumor cell of EGFR L858R gene mutation. In addition, the invention also provides a pharmaceutical composition for treating tumors related to expression of the gene mutation. The pharmaceutical composition has the characteristics of strong specificity and good individualized treatment effect.

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): C07K14/725C12N15/867C12N5/10A61K35/17A61P35/00
CPCC07K14/7051C12N15/86C12N5/0636A61K35/17A61P35/00C12N2740/15043C12N2510/00
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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