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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 lack of effective TCR and the need to verify the therapeutic effect, and achieve the effect of good therapeutic effect and strong specificity.

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 are connected by furin cleavage site, SGSG linker and F2A sequence, the α chain and β chain genes are fully synthesized, and the TCR gene is cloned into lentivirus using restriction enzymes EcoRI and BamHI In the expression vector pCDH (purchased from SBI), a recombinant plasmid is obtained, which can express the amino acid sequences of the variable regions of the TCR α chain and β chain shown in sequences 6 and 8.

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

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

Embodiment 2

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

[0055] Peripheral blood of healthy volunteers was taken, and human peripheral blood mononuclear cells (PBMC) were obtained by using lymphocyte separation medium (Stemcell, 07861, USA). Dynabeads (Gibco, 11141D) and PBMC were mixed and incubated at room temperature for 20 min to isolate 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 48 hours of continuous culture in the incubator, adjust the cell density to 1x10 6 pcs / mL. Take out the lentivirus from the -80°C ultra-low temperature freezer, quickly thaw it in a 37°C water bath, add polybrene (SantaCruz, sc-134220) to the prepared T cells to a final concentration of 6 μg / mL, add the abo...

Embodiment 3

[0056] Example 3 Preparation of target cells

[0057] The lentiviral expression vector pCDH-A1101 overexpressing the HLA-A*1101 molecule was constructed, and 293T cells were transiently transfected to prepare a recombinant lentivirus. The method was the same as that in Example 1. Virus-transduced T2 cell line, according to the method of Example 2, to construct a T2 cell line expressing HLA-A*1101. After labeling with HLA molecule antibody, FACS was used to detect the expression of HLA-A*1101 on the surface of T2 cells, as shown in Figure 2. Figure 2A In the middle are T2 cells that were not transfected with HLA-A*1101, Figure 2B The middle is T2 cells transfected with HLA-A*1101, and the transfection efficiency of HLA-A*1101 was 96.6%.

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Abstract

The invention provides a specific TCR directed at EGFR L858R gene mutation and application of the specific TCR. The 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 onan HLA-A*1101 tumor cell of EGFR L858R gene mutation. In addition, the invention also provides a pharmaceutical composition for treating tumors related to the 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 fields 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 which 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 be greatly increased. In addition to rapidly killing tumors like cytotoxic chemotherapy and targeted therapy, this approach avoids the delayed effects of vaccines and immune checkpoint inhibitor therapy. TCR-T is one of the most promising tumor treatment technologies after tumor surgery, radiotherapy, chemotherapy, and targeted therapy. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/725C12N15/867C12N5/10A61K35/17A61P35/00
CPCC07K14/7051C12N15/86C12N5/0636A61K35/17A61P35/00C12N2740/15043C12N2800/107C12N2510/00
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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