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Chimeric antigen receptor (CAR) containing third signal receptor and application of CAR

A chimeric antigen receptor and receptor technology, which can be used in medical preparations, carriers, and fusion polypeptides containing active ingredients, and 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 (CAR) containing third signal receptor and application of CAR
  • Chimeric antigen receptor (CAR) containing third signal receptor and application of CAR
  • Chimeric antigen receptor (CAR) containing third signal receptor and application of CAR

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1-20B

[0074] Example 1-20 Preparation of BBZIL2RbIL2Rg CAR-T cells

[0075] The preparation of 20BBZIL2RbIL2Rg CAR-T cells described in this example includes the following steps:

[0076] 1. Construction of lentiviral vector pCDH-MSCVEF-20BBZIL2RbIL2Rg and virus production

[0077] The scFv-antihCD20-20BBZ (SEQ ID No.1), IL2Rb intracellular region (SEQ ID No.3) and IL2Rg intracellular region (SEQ ID No.7) were formed into a fusion protein by overlapping PCR, and EcoRI and BamHI were added at both ends Restriction site cloning 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 the pCDH-MSCVEF-20BBZIL2RbIL2Rg virus (referred to as 20BBZIL2RbIL2Rg virus), which wil...

Embodiment 2-20B

[0080] Example 2-20 Preparation of BBZIL4RaIL2Rg CAR-T cells

[0081] The preparation of the 20BBZIL4RaIL2Rg CAR-T cells described in this example includes the following steps:

[0082] 1. Construction of lentiviral vector pCDH-MSCVEF-20BBZIL4RaIL2Rg and virus production

[0083] The fusion protein of scFv-antihCD20-20BBZ (SEQ ID No.1), IL4Ra intracellular region (SEQ ID No.4) and IL2Rg intracellular region (SEQ ID No.7) was formed by overlapping PCR, and EcoRI and BamHI were added at both ends Restriction site cloning 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 the pCDH-MSCVEF-20BBZIL4RaIL2Rg virus (referred to as 20BBZIL4RaIL2Rg virus), for subseq...

Embodiment 3-20B

[0086] Example 3-20 Preparation of BBZIL7RaIL2Rg CAR-T cells

[0087] The preparation of the 20BBZIL7RaIL2Rg CAR-T cells described in this example includes the following steps:

[0088] 1. Construction of lentiviral vector pCDH-MSCVEF-20BBZIL7RaIL2Rg and virus production

[0089]The scFv-antihCD20-20BBZ (SEQ ID No.1), IL7Ra intracellular region (SEQ ID No.2) and IL2Rg intracellular region (SEQ ID No.7) were formed into a fusion protein by overlapping PCR, and EcoRI and BamHI were added at both ends Restriction site cloning 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, centrifuge at 25000RPM for 2 hours to concentrate the virus, which is the pCDH-MSCVEF-20BBZIL7RaIL2Rg virus (referred to as 20BBZIL7RaIL2Rg virus), which w...

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PUM

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Abstract

The invention relates to a chimeric antigen receptor (CAR) containing a third signal receptor. The structure of the CAR is scFv(X)-(Y)CD3zeta-MN, wherein X comprises a tumor-targeting antibody or a ligand and receptor capable of being specifically combined with tumors, Y is an intracellular region of a costimulatory receptor, the costimulatory receptor is selected from ICOS, CD28, CD27, HVEM, LIGHT, CD40L, 4-1BB, OX40, DR3, GITR, CD30, TIM1, SLAM, CD2 and CD226, M is an intracellular region of a cytokine receptor of a gamma chain family, the cytokine receptor is selected from IL2Ra, IL2Rb, IL4Ra, IL7Ra, IL9Ra, IL15Ra and IL21Ra, and N is an IL2Rg intracellular region. The invention further relates to a CAR-T cell constructed from a recombinant expression vector of the CAR and a preparationmethod and application of the CAR-T cell. Through the CAR-T cell, the tumor killing ability and the amplification ability are significantly improved, the CAR-T cell contains the third signal receptor, the third signal receptor has the potential function of effect enhancement and only has the effect on the CAR-T cell, and the risk of causing immune side effects is reduced.

Description

technical field [0001] The invention relates to the technical field of cellular immunotherapy, in particular to a chimeric antigen receptor comprising a third signal receptor and its application. 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 target ...

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
CPCC07K14/7051C07K16/2887C12N5/0636C12N15/86A61P35/00C07K2317/622C07K2319/00C07K2319/33C07K2319/74C12N2510/00C12N2740/15043C12N2800/107A61K39/4631A61K39/4611A61K39/464424A61K48/00C07K2319/03C07K14/7155C07K14/70521C07K14/70578A61K2039/585A61K38/00A61K2039/505A61K35/17C07K16/28C07K16/2851C07K16/2863C07K16/2869C07K16/2878C07K2319/033
Inventor 杨选明傅阳心汪鑫叶圣勤李民
Owner SHANGHAI LONGYAO BIOTECH CO LTD
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