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Chimeric antigen receptor, construction method thereof and application of chimeric antigen receptor

A chimeric antigen receptor and antigen technology, applied in the field of chimeric antigen receptor and its construction, can solve the problems of no in vivo data, few combinations, single screening data, etc., so as to improve the survival rate and improve the tumor inhibition effect in vivo. Effect

Active Publication Date: 2021-02-23
FUNDAMENTA THERAPEUTICS INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The patent invention of WO2016 / 102965A1 conducts research on tumor targets CD19 and CD22. This patent compares the in vitro tumoricidal activity of four intracellular stimulatory factor combinations (including 41BBz-41BBz, OX40z-OX40z , 41BBz-28z and OX40z-28z), but the screening data of this patent is single, the combination is less, and there is no relevant in vivo data

Method used

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  • Chimeric antigen receptor, construction method thereof and application of chimeric antigen receptor
  • Chimeric antigen receptor, construction method thereof and application of chimeric antigen receptor
  • Chimeric antigen receptor, construction method thereof and application of chimeric antigen receptor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Example 1: Design of bispecific chimeric antigen receptors CAR19-CAR20 and CAR19-CAR22 in parallel

[0055] The present inventors designed seven different parallel forms of bispecific chimeric antigen receptor CAR19-CAR20, in which the structure of CAR19 was kept fixed, that is, all selected ones with the structure of FMC63-CD8hinge-CD8TM-4-1BB-CD3z CAR19 (see CN105392888A), and combine it with 7 different CAR20 structures. Wherein, the amino acid sequence of the antigen-binding domain FMC63 in the above-mentioned CAR19 is shown in SEQ ID NO: 5, and the nucleotide sequence encoding the amino acid sequence is shown in SEQ ID NO: 6. The amino acid sequences of CD8hinge, CD8TM, 4-1BB and CD3z in the above-mentioned CAR19 are respectively shown in SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13 and SEQ ID NO: 15; the nucleotides encoding the above amino acid sequences The acid sequences are shown in SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14 and SEQ ID NO:16.

[0056] The seven ...

Embodiment 2

[0069] Example 2: Comparison of the killing effect of CAR19-CAR20-T cells prepared by bispecific chimeric antigen receptors with different structures on target cells

[0070] The dual-target CAR-T cells were prepared by using the parallel-connected bispecific chimeric antigen receptor CAR19-CAR20 described in Example 1, and then the dual-target CAR-T cells were combined with CD19 + K562-luc-GFP, CD20 + K562-luc-GFP two different target cells according to different effector cell (E): target cell (T) ratio, that is, E / T=1:1, 2.5:1, 5:1, 10:1, 20 : 1 ratio and incubated for 18-24 hours, using T cells without genetic modification (ie, T cells without lentivirus infection, hereinafter referred to as NC-T cells) as the background control, the constructed target cell line had There is luciferase, which detects the killing effect of effector cells on target cells through the principle of chemiluminescence. The specific operation is as follows:

[0071] (1) Isolation of peripheral b...

Embodiment 3

[0083] Example 3: T cell phenotypes displayed by CAR19-CAR20-T cells prepared from bispecific chimeric antigen receptors of different structures

[0084] The dual-target CAR-T cells were prepared by using the parallel bispecific chimeric antigen receptor CAR19-CAR20 described in Example 1, and differentiated with conventional T cells on the 7th-10th day after lentiviral transfection Antigen-antibody analysis of differentiated populations of cells by flow cytometry.

[0085] Test method: The seven bispecific chimeric antigen receptors CAR19-CAR20 prepared in Example 1 were selected as test materials, and the corresponding dual-target CAR-T cells were prepared according to the preparation method of effector cells described in Example 2. For the prepared 7 kinds of dual-target CAR-T cells, 1×10 6 After washing the CAT-T cells with PBS, incubate CD62L-PE-Cy5 antibody (BD, catalog number: 555545) and CD45RO-FITC antibody (BD, catalog number: 555492), and incubate at 4°C for 30 min...

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Abstract

The invention provides a chimeric antigen receptor, a construction method thereof and application of the chimeric antigen receptor. Specifically, the chimeric antigen receptor provided by the invention consists of an antigen binding region, an extracellular hinge region, a transmembrane region, a co-stimulatory domain and a CD3z signal structural domain. The CAR-T cell including two chimeric antigen receptors including different antigen binding regions provided by the invention has bispecificity, and shows increased cell killing efficiency and a better in-vivo tumor inhibition effect.

Description

technical field [0001] The invention belongs to the field of biotechnology, and specifically relates to a chimeric antigen receptor and its construction method and application. Background technique [0002] With the development of tumor treatment, chimeric antigen receptor T (Chimeric Antigen Receptor-T, CAR-T) cell immunotherapy has gradually become a treatment method that has attracted much attention. The CAR expressed by CAR-T cells generally includes an extracellular antigen-binding domain, a transmembrane region, a co-stimulatory factor domain, and an intracellular signaling domain. Usually, CAR-T cells are transduced and expanded by the patient's T cells through the CAR gene, and finally reinfused into the patient. CAR-T cells can effectively recognize tumor antigens and induce specific anti-tumor immune responses without being restricted by the Major Histocompatibility Complex (MHC). At present, the US FDA has approved the marketing of two autologous CAR-T cell prod...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N5/10C12N15/62C12N15/85A61K39/00A61P35/00
CPCC07K16/2887C12N5/0636C07K16/2803C12N15/85A61P35/00C07K2317/622C07K2317/24C12N2510/00C07K2319/02C07K2319/03A61K2239/29A61K39/464412A61K39/4631A61K2239/48A61K2239/28A61K39/464424A61K39/4611A61K39/464413C07K19/00C12N5/10C12N15/62C07K14/705C12N15/63A61P35/02C07K2317/31C12N2740/15043C07K16/2815C07K16/2818
Inventor 李俊张鹏潮徐昭陈影钟林茂江雨辰何玲
Owner FUNDAMENTA THERAPEUTICS INC
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