Chimeric antigen receptor comprising costimulatory receptor and application thereof

A chimeric antigen receptor and co-stimulation technology, which can be used in medical preparations containing active ingredients, fusion polypeptides, anti-tumor drugs, etc. It can solve the problems of immune side effects, poor efficacy, and impact on CAR-T.

Active Publication Date: 2019-12-27
SHANGHAI LONGYAO BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] However, the current clinically used CAR-T domain still has insufficient tumor killing ability and expansion ability, and has poor efficacy in controlling solid tumors / metastatic tumors. Some CAR-Ts use new regulatory molecules such as IL-12,4 -1BBL, etc. In addition to affecting CAR-T, these molecules also have a non-specific activation effect on other non-CAR-T cells, which has the potential to cause immune side effects

Method used

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  • Chimeric antigen receptor comprising costimulatory receptor and application thereof
  • Chimeric antigen receptor comprising costimulatory receptor and application thereof
  • Chimeric antigen receptor comprising costimulatory receptor and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1-20B

[0074] Example 1-20 Preparation of BBZ-2A-OX40CAR-T cells

[0075] The preparation of 20BBZ-2A-OX40CAR-T cells described in this example includes the following steps:

[0076] 1. Construction of lentiviral vector pCDH-MSCVEF-20BBZ-2A-OX40 and virus production

[0077] Add 2A (SEQ ID No.7) sequence to the middle of scFv-antihCD20-20BBZ (SEQ ID No.1) and OX40 (SEQ ID No.2) through overlap PCR, and add EcoRI and SalI restriction sites at both ends to clone pCDH - MSCVEF vector. The clones with correct sequencing were extracted without endotoxin, and co-transfected with lentiviral packaging plasmids (VSV-g, pMD Gag / Pol, RSV-REV) at 293X. After 48 and 72 hours, the supernatant was collected, filtered at 0.45uM, and used Beckman ultracentrifuge and SW28 rotor, 25000RPM centrifugation for 2 hours to concentrate the virus, which is pCDH-MSCVEF-20BBZ-2A-OX40 virus (abbreviated as 20BBZ-2A-OX40 virus), for subsequent CAR-T cell production. Produce contrast pCDH-MSCVEF-20BBZ virus (ab...

Embodiment 2-20B

[0080] Example 2-20 Preparation of BBZ-2A-HVEM CAR-T cells

[0081] The preparation of 20BBZ-2A-HVEM CAR-T cells described in this example includes the following steps:

[0082] 1. Construction of lentiviral vector pCDH-MSCVEF-20BBZ-2A-HVEM and virus production

[0083] Add 2A (SEQ ID No.8) sequence to the middle of scFv-antihCD20-20BBZ (SEQ ID No.1) and HVEM (SEQ ID No.3) through overlap PCR, and add EcoRI and SalI restriction sites at both ends to clone pCDH - MSCVEF vector. The clones with correct sequencing were extracted without endotoxin, and co-transfected with lentiviral packaging plasmids (VSV-g, pMD Gag / Pol, RSV-REV) at 293X. After 48 and 72 hours, the supernatant was collected, filtered at 0.45uM, and used Beckman ultracentrifuge and SW28 rotor, 25000RPM centrifugation for 2 hours to concentrate the virus, which is pCDH-MSCVEF-20BBZ-2A-HVEM virus (abbreviated as 20BBZ-2A-HVEM virus), for subsequent CAR-T cell production. Produce contrast pCDH-MSCVEF-20BBZ virus (...

Embodiment 3-20B

[0086] Example 3-20 Preparation of BBZ-2A-ICOS CAR-T cells

[0087] The preparation of 20BBZ-2A-ICOS CAR-T cells described in this example includes the following steps:

[0088] 1. Construction of lentiviral vector pCDH-MSCVEF-20BBZ-2A-ICOS and virus production

[0089] Add 2A (SEQ ID No.9) sequence to the middle of scFv-antihCD20-20BBZ (SEQ ID No.1) and ICOS (SEQ ID No.4) through overlap PCR, and add EcoRI and SalI restriction sites at both ends to clone pCDH - MSCVEF vector. The clones with correct sequencing were extracted without endotoxin, and co-transfected with lentiviral packaging plasmids (VSV-g, pMD Gag / Pol, RSV-REV) at 293X. After 48 and 72 hours, the supernatant was collected, filtered at 0.45uM, and used Beckman ultracentrifuge and SW28 rotor, 25000RPM centrifugation for 2 hours to concentrate the virus, which is pCDH-MSCVEF-20BBZ-2A-ICOS virus (abbreviated as 20BBZ-2A-ICOS virus), for subsequent CAR-T cell production. Produce contrast pCDH-MSCVEF-20BBZ virus (...

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PUM

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Abstract

The invention relates to a chimeric antigen receptor comprising a costimulatory receptor, wherein the structure of the chimeric antigen receptor is scFv (X)-(Y) CD3zeta-2A-(Z); among them, X includestumor-targeted antibodies or ligands and receptors that can specifically bind to tumors; Y is the intracellular region of the costimulatory receptor and Z is the costimulatory receptor selected from the group consisting of ICOS, CD28, CD27, HVEM, LIGHT, CD40L, 4-1BB, OX40, DR3, GITR, CD30, TIM1, SLAM, CD2, CD226. The invention also relates to a CAR-T cell constructed from the recombinant expression vector of the chimeric antigen receptor, a preparation method and an application thereof. The CAR-T cells significantly improve tumor killing ability and amplification ability, integrate costimulatory receptor signals into CAR-T, it has the function of potential enhancement effect and has the function of only acting on CAR-T cells play a role, reducing the risk of immune side effects.

Description

technical field [0001] The invention relates to the technical field of cellular immunotherapy, in particular to a chimeric antigen receptor comprising co-stimulatory receptors and an application thereof. Background technique [0002] The use of immunological treatment to overcome tumors has always been an important direction for the application of immunology in translational medicine. With the development of various omics (genomics, proteomics, etc.), the immunogenicity of tumor cells due to mutations has been widely recognized, which has laid a theoretical foundation for tumor immunotherapy. At the same time, with the accumulation of tumor immunology research itself, tumor immunotherapy has made great progress recently, and a series of new immunotherapy methods have gradually entered the clinic. The current tumor immunology research has established the central position of T cell killing in tumor immunotherapy, and chimeric antigen receptor T cells (CAR-T cells) combine the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N5/10C12N15/867A61K35/17A61P35/00
CPCC07K14/7051C07K16/30C12N5/0636C12N15/86A61K35/17A61P35/00C07K2317/622C07K2319/00C12N2510/00C12N2740/15043C07K2319/03A61K2039/5156C07K16/2887C07K14/70578C07K14/70596A61K39/001124A61K48/005C12N2740/16043A61K38/00A61K2039/505C07K14/7155
Inventor 杨选明傅阳心汪鑫叶圣勤李范林张会会
Owner SHANGHAI LONGYAO BIOTECH CO LTD
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