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T cell antigen receptor gene and application thereof

A technology of receptor gene and cell antigen, applied in the field of genetic engineering, can solve the problem of off-target effect of antigen receptor

Active Publication Date: 2016-08-03
河北格尔泰生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The risk of CART therapy lies in the off-target effec

Method used

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  • T cell antigen receptor gene and application thereof
  • T cell antigen receptor gene and application thereof
  • T cell antigen receptor gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1, Design and Construction of CAR-EGFR

[0033] 1-1. Design description of CAR. According to statistics, the CAR structures most used in clinical trials include second-generation and third-generation CARs (see attached figure 1 ), among the relevant clinical reports, there are many second-generation CARs, especially the CAR structure led by the University of Pennsylvania (NCT01626495, NCT01029366), so we adopted this second-generation CAR frame structure.

[0034] 1-2. The structure and construction of CAR. The CAR-EGFR sequence includes the following parts: CD8-pre, EGFR-scFv, CD8 hinge + transmembrane, 4-1BB (CD137), CD3ζ. See SEQ ID NO: 1 for its complete sequence.

[0035] 1-3. The structure and construction of the CAR control group. The sequence structure of CAR-control group includes: CD8-pre, EGFR-scFv, CD8-ΔCD3ζ. See SEQ ID NO:2 for its complete sequence.

Embodiment 2

[0036] Example 2. Preparation and phenotypic verification of CAR-EGFR modified T cells (CART-EGFR)

[0037] 2-1. Preparation of CART-EGFR and CART-control group. The preparation process of CART-EGFR and CART-control group is attached figure 2 shown. Specifically include the following:

[0038] 2-1-1. Experimental reagents and experimental instruments:

[0039] Experimental reagents include:

[0040] Ficoll-PaquePLUS (GE, cat#17-1440-02)

[0041] Complete medium TexMACS (MiltenyiBiotechnolog, cat#170-076-309)+IL-2 (MiltenyiBiotechnolog, cat#130-097-748)

[0042] PerCP5.5 anti-human CD3 (BD, cat#552852)

[0043] PEanti-human CD4 (BD, cat#555347)

[0044] APCanti-humanCD8 (BD, cat#555369)

[0045] FACS buffer: PBS+2.5%FBS

[0046] TRANSB.

[0047] Experimental equipment includes:

[0048] Centrifuge: Xiangyi L550

[0049] Flow cytometer: BDC6

[0050] CO2 incubator: ESCOMCO-20AIC

[0051] Cell counter: Cellometer Auto1000.

[0052] 2-1-2. PBMC separation: transfer...

Embodiment 3

[0066] Example 3. Functional verification of CART-EGFR—verification of target cells

[0067] Experimental purpose: To detect the expression of EGFR on target cells and non-target cells. Experimental design: The target cells (Eca109) and control cells (MCF7) were detected using the flow cytometry antibody of EGFR. Experimental results and analysis: The results of flow cytometry detection of Eca109 and MCF7EGFR expression are as follows: Image 6 , the results showed that the expression of EGFR was low in the negative control MCF7, but the expression of EGFR was high in the target cell Eca109.

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Abstract

The invention discloses a T cell antigen receptor gene which is used for preparing the T cell of a chimeric antigen receptor so that the T cell shows specific esophagus cancer related functional activity. The T cell antigen receptor gene is of important potential and practical value to the treatment of esophagus cancer, and a new technical direction is provided for the treatment of esophagus cancer.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to an esophageal cancer-associated T cell antigen receptor gene and its application. Background technique [0002] Cancer has always been a huge problem plaguing mankind. There are about 3.12 million new cancer cases in my country every year, and an average of 8,550 people per day means that 6 people are diagnosed with malignant tumors every minute. These data show that cancer is still a major threat to health. Over the years, the main methods of cancer treatment are surgery, chemotherapy and radiotherapy, and the effective rate of clinical treatment for tumor patients is still very low. According to statistics, the effective rate of the same anti-tumor drug is only 25%. For the same tumor, with the same drug and standard dose of treatment, the curative effect and side effects are very different in different patients. The main reason for this result is that tumor is a ...

Claims

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Application Information

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IPC IPC(8): C12N15/62C12N15/12C12N5/10C12N5/0783C12N15/85C07K19/00A61K35/17A61P35/00
CPCA61K35/17C07K14/7051C07K14/70517C07K14/70596C07K16/2863C07K2317/622C07K2319/03C12N15/85
Inventor 汪治宇
Owner 河北格尔泰生物科技有限公司
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