Tumor neoantigen-based T cell sorting preparation technology and application thereof

A cell and new technology, applied in the field of molecular biology, can solve the problems of insufficient purity, affecting the therapeutic effect, and impurity of cells

Pending Publication Date: 2021-02-02
浙江格源致善生物医药科技有限公司
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  • Abstract
  • Description
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  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The purpose of the present invention is to provide a T cell sorting and preparation technology based on tumor neoantigens and its application to solve the problem that the existing NRT cell preparation process mentioned in the background technology is not pure enough to affect the therapeutic effect. For example, DC- The NRT cells prepared in the CIK mode are a kind of mixture, which contains NRT cells and other cells such as Treg cells, DC cells, etc. On the one hand, these cells bring uncertainty to the safety of the treatment, and the obtained cells are not pure. On the other hand, the cost of cell preparation is increased, and the effect of treatment is affected. Therefore, how to type and purify NRT cells is particularly important.

Method used

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  • Tumor neoantigen-based T cell sorting preparation technology and application thereof
  • Tumor neoantigen-based T cell sorting preparation technology and application thereof
  • Tumor neoantigen-based T cell sorting preparation technology and application thereof

Examples

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preparation example Construction

[0025] The present invention provides a method for preparing T cells based on tumor neoantigens, and the method for preparing T cells based on tumor neoantigens includes the following steps:

[0026] S1: Collect tumor specimens, analyze tumor gene mutations by gene sequencing, and collect patient tumor tissue by means of surgery, puncture, intervention, etc.; sequence and detect gene mutations by methods such as whole exome sequencing, probe recovery sequencing, etc.;

[0027] S2: Analyze tumor neoantigens based on gene mutation, and analyze tumor neoantigens based on gene mutation, HLA affinity, gene expression and other information;

[0028] S3: prepare a neoantigen vaccine according to the neoantigen, and prepare a polypeptide or RNA vaccine according to the neoantigen sequence;

[0029] S4: collect peripheral blood cells from patients, separate DC cells and T cells;

[0030] S5: Induce and stimulate DC cells with tumor neoantigen vaccine, and transfer polypeptide vaccine ...

Embodiment 1

[0038] This example is used to illustrate the method for sorting and preparing NRT according to the present invention;

[0039] Raw materials: tumor tissue, PBMC, DC cells, T cells, tumor neoantigen vaccine;

[0040] NRT sorting preparation method:

[0041] (1) Collect patient tissue samples, analyze gene mutations by gene sequencing, analyze tumor neoantigens according to the mutations, and prepare tumor neoantigen vaccines according to the results of neoantigen analysis;

[0042] (2) Isolation and culture of PBMC cells in peripheral blood of patients: after centrifuging 50ml of white blood cells collected by the apheresis machine to remove the plasma, add 50ml of normal saline to dilute, and mix gently;

[0043] Take two 50ml centrifuge tubes, first add 15ml Ficoll solution, and then gently add the diluted blood to the upper layer of Ficoll in the two centrifuge tubes, be gentle to avoid mixing the two solutions together, each centrifuge tube has 30ml of diluted blood ;2,000...

Embodiment 2

[0054] This example is used to illustrate the comparison of the killing effect of sorted NRT and non-sorted NRT:

[0055] 1: Prepare and sort 41BB positive T cells and T cells without sorting according to the above method;

[0056] 2: In vitro expansion of the above T cells;

[0057] 3: In vitro killing experiment: the T cells prepared above were co-incubated with tumor cells prepared from the patient's own tumor tissue, and the killing effect was detected by LDH release method.

[0058] Based on the above, the vaccine is prepared according to tumor neoantigens by sorting technology, and then the vaccine is transferred into antigen-presenting cells, and the activation of T cells is induced by co-incubation of antigen-presenting cells and T cells. The selected technology, the prepared cells can be used in the treatment or prevention of tumor drugs, and the NRT cells are sorted out based on tumor neoantigens to obtain pure NRT cells, which can be used for subsequent cell therap...

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Abstract

The invention discloses a tumor neoantigen-based T cell sorting preparation technology and application thereof in the technical field of molecular biology, and the T cell sorting preparation technology based on tumor neoantigen and application thereof comprise the following steps: S1: collecting a tumor specimen, analyzing tumor gene mutation through gene sequencing, and collecting tumor tissues of a patient by means of surgery, puncture, intervention and the like; performing sequencing and detecting gene mutation through methods such as full-exon sequencing and probe recovery method sequencing, S2: analyzing the tumor neoantigen according to the gene mutation, and analyzing the tumor neoantigen according to information such as the gene mutation, HLA affinity and gene expression. The T cell sorting preparation technology based on the tumor neoantigen and the application of the T cell sorting preparation technology are characterized in that NRT cells are sorted out based on tumor neoantigens, pure NRT cells are obtained, and preparations are made for subsequent cell treatment in the aspects of safety, cost and effectiveness.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to a T cell sorting and preparation technology based on tumor neoantigens and its application. Background technique [0002] At present, the comprehensive treatment effect of tumor treatment based on surgery, radiotherapy and chemotherapy is not satisfactory. Although the widespread application of molecular targeted therapy in recent years has significantly improved the quality of life of patients with advanced tumors, targeted therapy is mainly aimed at the population with mutations, and the benefit population is small, and eventually all face the problem of drug resistance. Tumor immunotherapy is a research hotspot in the field of tumor therapy in recent years. In 2013, the immunotherapy of tumors was rated as the top ten scientific breakthroughs of the year by "Science" magazine. In 2016, the American Society of Clinical Oncology (ASCO) declared immunotherapy the prem...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0783C12N5/10A61K35/17A61P35/00
CPCC12N5/0636C12N5/0639A61K35/17A61P35/00C12N2502/1121C12N2509/00
Inventor 苏小平李锐
Owner 浙江格源致善生物医药科技有限公司
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