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Preparation method and application of specific CTL cell of target KRAS frequent mutation tumor

A KRAS-12D and KRAS-12V technology, applied in the field of tumor treatment, can solve the problems that patients with KRAS mutations are difficult to benefit, the treatment effect is not satisfactory, and the survival period is short

Inactive Publication Date: 2019-08-30
BEIJING DCTY BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Therefore, it is difficult for patients with KRAS mutations to benefit from targeted drugs, and their survival is short. Therefore, it is urgent to develop immune cell therapy targeting KRAS mutations
Most of the CTLs in the prior art are CTLs targeting single-target KRAS mutations, but because the KRAS mutation site is not fixed, the therapeutic effect of CTLs single-targeting KRAS mutations is often unsatisfactory

Method used

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  • Preparation method and application of specific CTL cell of target KRAS frequent mutation tumor
  • Preparation method and application of specific CTL cell of target KRAS frequent mutation tumor
  • Preparation method and application of specific CTL cell of target KRAS frequent mutation tumor

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preparation example Construction

[0040] The present invention provides a preparation method of specific CTL targeting KRAS multiple mutation tumors, comprising the following steps:

[0041] 1) mixing the mutant polypeptides KRAS-12D, KRAS-12C, KRAS-12V, KRAS-12A, KRAS-12S, KRAS-13D and KRAS-61H to obtain a KRAS mutant polypeptide mixture;

[0042] 2) adding DCs to the KRAS mutant polypeptide mixture described in step 1), and culturing to obtain antigen-presenting cells;

[0043] 3) Co-culture the antigen-presenting cells described in step 2) with peripheral blood mononuclear cells to obtain specific CTLs targeting KRAS multiple mutation tumors;

[0044] The amino acid sequences of KRAS-12D, KRAS-12C, KRAS-12V, KRAS-12A, KRAS-12S, KRAS-13D and KRAS-61H are shown in SEQ ID NO.1-7.

[0045] In the present invention, firstly, the mutant polypeptides KRAS-12D, KRAS-12C, KRAS-12V, KRAS-12A, KRAS-12S, KRAS-13D and KRAS-61H are mixed to obtain the KRAS mutant polypeptide mixture.

[0046] In the present invention, ...

Embodiment 1

[0062] 1. Select the KRAS mutant sequence:

[0063] KRAS-12D: TEYKLVVVGADGVGKSALTIQ

[0064] KRAS-12C: TEYKLVVVGACGVGKSALTIQ

[0065] KRAS-12V: TEYKLVVVGAVGVGKSALTIQ

[0066] KRAS-12A: TEYKLVVVGAAGVGKSALTIQ

[0067] KRAS-12S: TEYKLVVVGASGVGKSALTIQ

[0068] KRAS-13D: EYKLVVVGAGDVGKSALTIQL

[0069] KRAS-61H: CLLDILDTAGHEEYSAMRDQY

[0070] 2. Synthesis of KRAS mutant polypeptide:

[0071] The peptides are synthesized by GenScript Biotechnology Co., Ltd. The requirements for synthetic peptides: 98% purity, with FITC fluorescent label, each peptide is dissolved in 1640, the concentration is 1mg / mL, and it is ready for use.

[0072] 3. Binding curve of KRAS mutant polypeptide to HLA on PBMC

[0073] 1) Take 30mL PBMC and centrifuge at 2000rpm for 5min;

[0074] 2) Cells were resuspended in 20mL PBS and counted, centrifuged at 2000rpm for 5min, resuspended in 1.5mL PBS to make the cell concentration 2×10 7 cells / mL;

[0075] 3) Take 50 μL cells (10 6 1) Add 0, 3.2, 6.3, 12...

Embodiment 2

[0081] Example 2 Loading DC with Mixed Antigen

[0082] 1) Blood collection and separation of PBMC;

[0083] 2) Adjust the PBMC to 1×10 medium 1640+10% FBS 6 Cells / mL, as in Petri dish, at 37°C 5% CO 2 Incubator, rest overnight;

[0084] 3) Pipette the cells attached to the bottom of the culture dish with culture medium X-VIVO+1% human serum albumin to collect the adherent monocytes; count and adjust to 1×10 6 Cells / mL, as for the dish, add 1000IU / mL IL-4 and 800IU / mL GM-CSF, at 37℃5%CO 2 incubator;

[0085] 4) After 3 days of culture, mature DCs were collected by centrifugation, re-selected with 5 mL KRAS mutant polypeptide mixture, and kept at 37°C in 5% CO 2 Incubator, impact 12h;

[0086] 5) Centrifuge at 2000 rpm for 5 minutes, collect DC, and resuspend in OKM100+12% FBS for use.

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Abstract

The invention provides a preparation method of specific CTL of a target KRAS frequent mutation tumor, and belongs to the technical field of tumor treatment. The preparation method comprises the following steps (1) mixing mutation polypeptide KRAS-12D with mutation polypeptide KRAS-12C, mutation polypeptide KRAS-12V, mutation polypeptide KRAS-12A, mutation polypeptide KRAS-12S, mutation polypeptideKRAS-13D and mutation polypeptide KRAS-61H, so as to obtain KRAS mutation polypeptide mixed liquor; (2) adding DC to the KRAS mutation polypeptide mixed liquor obtained in the step (1), and performing culturing so as to obtain antigen presenting cells; and (3) performing co-culturing on the antigen presenting cells and peripheral blood mononuclearcells so as to obtain the specific CTL of the target KRAS frequent mutation tumor. According to the method disclosed by the invention, 7 KRAS mutation antigens are presented at the same time, so that the prepared CTL can specifically recognize 7 KRASmutation antigens.

Description

technical field [0001] The invention relates to the technical field of tumor treatment, in particular to a preparation method and application of specific CTL cells targeting KRAS multiple mutation tumors. Background technique [0002] The ras gene family has three genes related to human tumors: HRAS, KRAS mutation and NRAS, which are located on chromosome 11, 12 and 1 respectively. The KRAS mutation is also known as the p21 gene because it encodes a 21kD ras protein. Among the ras genes, KRAS mutations have the greatest impact on human cancer. When KRAS mutations are normal, they can control the pathways that regulate cell growth; when abnormal, they cause cells to continue to grow and prevent cells from self-destruction. It participates in intracellular signal transmission. When the KRAS mutation gene is mutated, the gene will be permanently activated, unable to produce normal ras protein, causing intracellular signal transduction disorder, cell proliferation out of contro...

Claims

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Application Information

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IPC IPC(8): C12N5/0783C12N5/10A61K35/17A61P35/00
CPCA61K35/17A61P35/00C12N5/0638C12N2502/99
Inventor 焦顺昌张嵘周子珊
Owner BEIJING DCTY BIOTECH CO LTD
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