Preparation method of cytotoxic T lymphocyte targeting to multiple KRAS mutant antigen epitopes of turmor

A technology for mutating antigens and lymphocytes, which is applied in the field of biotechnology development and application research, can solve the problems of prolonging the overall survival of patients, complicated operations, and low objective response rates, and achieves immune escape, broad-spectrum immune escape, and targeted highly specific effect

Pending Publication Date: 2019-07-02
上海尚泰生物技术有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the initial clinical trials confirmed its safety and effectiveness, the overall objective response rate was very low, and the overall survival of patients failed to be significantly prolonged
At the same time, these methods are complicated to operate and require a large amount of patient tumor tissue. Due to the uncertainty of tumor antigens, the clinical effect ...

Method used

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  • Preparation method of cytotoxic T lymphocyte targeting to multiple KRAS mutant antigen epitopes of turmor
  • Preparation method of cytotoxic T lymphocyte targeting to multiple KRAS mutant antigen epitopes of turmor
  • Preparation method of cytotoxic T lymphocyte targeting to multiple KRAS mutant antigen epitopes of turmor

Examples

Experimental program
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Effect test

Embodiment 1

[0029] Example 1. Construction of a lentiviral expression vector carrying an antigen coding sequence and viral packaging:

[0030] 1) Construction of lentiviral expression vector: The schematic diagram of the construction of lentiviral expression vector carrying the antigen coding sequence is shown in the attached figure 1 As shown, firstly, by using the Linker sequence, the epitope sequences (as shown in SEQ ID NO: 1-7) of the seven KRAS mutation sites (G12A, G12C, G12D, G12R, G12S, G12V and G13D) are connected in series Combining, and adding start and stop codons, creating a new artificial antigen coding sequence, as shown in SEQ ID NO: 9, the sequence size is 435bp; secondly, adding AscI and XbaI restriction enzyme cuts the site sequence and performs gene synthesis; finally, the synthesized antigen coding sequence fragment is double-digested by AscI and XbaI, and then connected into the lentiviral expression vector (pLVX) to construct the lentiviral expression vector (pLVX)...

Embodiment 2

[0032] Example 2. Induction culture and transfection of DC cells:

[0033] Collect peripheral blood, use Ficoll-Hypaque (polysucrose-diatrizoate meglumine) density gradient separation method to separate peripheral blood mononuclear cells PBMC, and use rhIL-4 (100ng / mL) and GM-CSF (1500U / mL) to stimulate and induce Culture DC cells. On the third day, immature DC cells were transfected with packaged and concentrated lentivirus particles, and the amount of lentivirus was calculated according to the optimal MOI value of the pre-experiment. After 24 hours of transfection, fresh DC medium was replaced and polyinosinic acid POLY (I:C) (10 μg / mL) was added to promote the expression of endogenous genes. Replenish fluid every other day, add tumor necrosis factor TNFα (10ng / mL) on the 6th day, and observe the maturation of the DC cells through a microscope on the 7th day, and at the same time detect the mature DC cells by flow cytometry and show that the markers CD83, CD86 and HLA- DR,...

Embodiment 3

[0034] Example 3. Preparation of antigen-specific CTL cells:

[0035] 1) Isolation and culture of CD8+T cells: PBMCs separated from peripheral blood by Ficoll, CD8+T cells were sorted by magnetic bead method. The sorted CD8+T cells were divided into 0.5-1×10 6 Inoculate in a T75 culture flask at a density of / mL, add cytokines IFN-γ (1000U / mL), rhIL-2 (1000U / mL) and autologous plasma (10%) to induce culture, and after 3 days of culture, add expansion in time Culture medium (1000U / mL rhIL-2). When the cells proliferated to an appropriate number, the cells were taken for flow cytometric analysis, as shown in the attached Figure 4 As shown, the expanded and cultured CD8+CTL cells have higher purity.

[0036] 2) Preparation of antigen-specific CTL cells: Co-culture antigen-loaded mature DCs and CTL cells at a ratio of 1:10 for 16 hours to sensitize the CTLs and generate specificity targeting seven major KRAS mutant epitopes

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Abstract

The invention relates to a preparation method of a cytotoxic T lymphocyte (CTL) targeting to multiple KRAS mutant antigen epitopes. The method comprises the steps: cloning a new artificial antigen coding sequence formed by series combination of 7 main KRAS mutant antigen epitope sequences into a lentivirus expression vector, transfecting DC, stimulating the proliferation and differentiation of activated CD8+T cells, and making the finally prepared cytotoxic T lymphocyte (CTL) having the specificity of targeting to seven main KRAS mutant antigen epitopes. The prepared KRAS mutant antigen specific cytotoxic T lymphocyte (CTL) has the advantages of high targeting specificity, no side effects and not easily causing of immune escape, and has great application prospects in treatment of KRAS mutant tumors.

Description

technical field [0001] The invention relates to the field of biotechnology development and application research, in particular to a method for preparing cytotoxic T lymphocytes (CTL) targeting multiple KRAS mutant antigen epitopes. Background technique [0002] KRAS is an important member of the RAS gene family, an important "molecular switch" in the intracellular signal transduction pathway, and plays an important role in the regulation of the MAPK (RAS-RAF-MEK-ERK) and PI3K (PI3K-AKT-mTOR) signal transduction pathways It is involved in a series of fundamental biological processes, including cell proliferation, survival and differentiation (Jancik et al., 2010). However, when KRAS is mutated, it cannot be hydrolyzed and inactivated by hydrolytic enzymes, but is in a continuously activated state, resulting in continuous transduction of downstream signals, resulting in excessive cell growth and proliferation, and promoting a variety of lethal human tumors. Today, KRAS mutati...

Claims

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Application Information

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IPC IPC(8): C12N15/867C12N5/10A61P35/00
CPCC12N15/86C12N5/0636A61K35/17C12N2740/15043C12N2510/00
Inventor 李晨蔚刘平磊
Owner 上海尚泰生物技术有限公司
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