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EGFR T790M neoantigen epitope peptide and application thereof in tumor treatment

An epitope, antigen technology, applied in the field of molecular immunology

Inactive Publication Date: 2020-10-16
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

The T790M mutation theory holds that exon 20 of the EGFR gene undergoes a secondary mutation during the treatment with EGFR-TKI, and as a result, the formation of steric hindrance affects the formation of hydrogen bonds between tyrosine kinase and EGFR-TKI, Ultimately, EGFR-TKI cannot be combined with tyrosine kinase, that is, first-line targeted drugs such as Iressa cannot continue to be effective. The mutation of T790M is the most important factor for secondary drug resistance of EGFR-TKI

Method used

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  • EGFR T790M neoantigen epitope peptide and application thereof in tumor treatment
  • EGFR T790M neoantigen epitope peptide and application thereof in tumor treatment
  • EGFR T790M neoantigen epitope peptide and application thereof in tumor treatment

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1 Isolation of peripheral blood mononuclear cells

[0032] Take 20ml of venous blood from HLA-C*1502 healthy donors, dilute the blood sample with an equal volume of sterile PBS, transfer the lymphocyte separation solution (Stemcell, 07861) into a new 50ml centrifuge tube, and ensure that the blood volume ratio is 3 : 4. Carefully add the diluted blood to the surface of the separation liquid, operate as gently as possible, avoid mixing, and clearly see the boundary between the two liquids. Centrifuge at 400g for 30min at room temperature. After centrifugation, suck out the mononuclear cell layer, transfer to a new sterile centrifuge tube, add PBS to wash twice. Cells were resuspended in RPMI-1640 medium and counted.

Embodiment 2

[0033] Example 2 Neoantigen epitope peptide activates specific T lymphocytes

[0034] Will 2.5×10 6 Each cell was suspended in 90% RPMI-1640 + 10% FBS (containing IL-2, 20ng / ml), adding 2.5×10 5 Dynabeads ® Human T-Activator CD3 / CD28 magnetic beads (Gibco, 11131D), inoculated into 6-well plate, 1.25×10 per well 6 cells, add neoantigen peptide LTSTVQLIM (2.5 μg / ml) and neoantigen peptide STVQLIMQL (2.5 μg / ml) to two wells respectively, at 37°C, 5% CO 2incubate. After 24 hours, resuspend the cells and magnetic beads, place them on the magnetic stand, discard the magnetic beads, collect the supernatant cells and continue to culture for a week, and detect the activated specific cytotoxic T lymphocytes (CTLs) using neoantigen-specific tetramers ),see picture 1, Figure 1A The neoantigen peptide LTSTVQLIM specifically caused CTL expression, and the expression rate was 31.4%. Figure 1B The neoantigen peptide STVQLIMQL can cause specific CTL expression, and the expression rate ...

Embodiment 3

[0035] Example 3 Killing of target cells by specific CTLs

[0036] 1. Construction of T2 target cells for HLA-C*1502

[0037] The full-length HLA-C*1502 gene was cloned into the lentiviral expression vector pCDH (purchased from SBI) using restriction endonucleases EcoRI and BamHI to obtain recombinant plasmids to construct the lentiviral expression vector pCDH overexpressing HLA-C*1502 molecules - C1502.

[0038] 293T cells in the logarithmic growth phase (purchased from the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences) were inoculated into T25 cell culture flasks containing 10% FBS in DMEM medium, and transfected when the cell confluence reached 80-90%. Mix the recombinant plasmid with the mixed packaging vector plasmid pPACKH1 (purchased from SBI), and add 500 μl 1 μg / μl lipofectamine transfection reagent Lip2000 (Invitrogen, 11668-027) after mixing thoroughly, mix immediately, and let stand at room temperature for 10 -15 minutes. Add the above...

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Abstract

The present invention provides an EGFR T790M neoantigen epitope peptide and application thereof in tumor treatment. The sequence of the EGFR T790M neoantigen epitope peptide contains one or two different amino acid sequences shown in SEQ ID No.1 and SEQ ID No.2, or an amino acid sequence formed by adding, deleting or substituting one or more amino acids on the basis of one or two different amino acid sequences in SEQ ID No.1 and SEQ ID No.2. The neoantigen epitope peptide can activate T lymphocytes, specifically kill HLA-C*1502 tumor cells with EGFR T790M mutation, and be used for preparing cancer drugs for treating non-small cell lung cancer.

Description

technical field [0001] The invention belongs to the field of molecular immunology and relates to an antigen epitope peptide, in particular to an EGFR T790M neoantigen epitope peptide and its application in treating tumors. Background technique [0002] Lung cancer is a highly malignant tumor that is common in clinical practice, with a very poor prognosis, and the five-year survival rate is less than 10%. According to histological classification, lung cancer can be divided into small cell lung cancer and non-small cell lung cancer, of which the incidence of non-small cell lung cancer accounts for about 80%. Surgery is the first choice and main treatment method for lung cancer. However, more than half of non-small cell lung cancer patients have already developed distant metastases when they were first diagnosed, and lost the opportunity for surgical treatment. The emergence of tumor-targeted drugs has greatly changed the traditional treatment model of tumors, greatly improve...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/71C07K19/00C12N5/0783C12N5/0784A61K39/00A61P35/00
CPCC07K14/71C07K14/70539C07K19/00C12N5/0638C12N5/0639C12N5/0636A61K39/001104A61K39/001111A61P35/00C12N2501/50A61K2039/5154A61K2039/5158
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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