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Duplex-specific chimeric antigen receptor and application thereof

A chimeric antigen receptor and bispecific technology, applied in the field of Trop2/PD-L1 bispecific chimeric antigen receptor, can solve the loss, weakening of anti-tumor effect, and weakening of the therapeutic effect of antigen-specific T cell infusion And other issues

Active Publication Date: 2019-05-14
NANJING FIRST HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, antigen-specific T cells will be regulated by negative regulators in patients, so that their anti-tumor effects will be lost or weakened
Studies have found that this immunosuppressive environment in the body mainly comes from the tumor microenvironment, and a large number of immunosuppressive molecules and cells can partially or completely inhibit the proliferation and activation of tumor-specific T cells, exert anti-tumor effects, and then make antigen-specific T cells The effect of cell reinfusion therapy has been significantly weakened. For example, programmed death receptor-1 (programmed death-1, PD-1) and its ligand PD-L1 (programmed deathligand 1, PD-L1) have been studied more

Method used

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  • Duplex-specific chimeric antigen receptor and application thereof
  • Duplex-specific chimeric antigen receptor and application thereof
  • Duplex-specific chimeric antigen receptor and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Construction of Trop2 / PD-L1 bispecific chimeric antigen receptor retroviral plasmid:

[0033] 1. According to the phage display technology of our laboratory, the V of the human Fab sequence of anti-Trop2 and anti-PD-L1 extracellular region H and V L Amino acid sequence of Trop2: V H (SEQ ID NO.3), V L (SEQ ID NO.4); PD-L1: V H (SEQ ID NO.7), V L (SEQ ID NO.8), using Optimum Gene TM gene design software to optimize the codon sequence, making it more suitable for human expression system without changing the amino acid sequence. at V H and V L A connecting peptide (G 4 S) 3 , plus the signal peptide CD8α in front, the scFv partial structure of the obtained bispecific chimeric antigen receptor is CD8α-Trop2 V H -(G 4 S) 3 -Trop2V L -(G 4 S) 3 -PD-L1 V H -(G 4 S) 3 -Trop2V L , the connection introduces Bam H I and Xho I Restriction sites. Digest the Psfg-CD8TM-CD28-CD137-CD3ζ vector in the same way, and use agarose gel electrophoresis to recover the ta...

Embodiment 2

[0037] Identification of bispecific chimeric antigen receptor CD8α-Trop2 / PD-L1 scFv-CD8TM- CD28- CD137- CD3ζ expression:

[0038] Extract the bispecific chimeric antigen receptor retroviral plasmid according to the operation manual of the endotoxin-free plasmid extraction kit (Tiangen Biology), and transfect it into human embryonic kidney cell 293T cells with PI transfection reagent for 48 hours Afterwards, wash with PBS, lyse the cells with cell protein extraction reagent RIPA, extract the protein of the transfected 293T cells, perform gel electrophoresis on 10% SDS-PAGE, transfer to the membrane, and incubate overnight at 4°C with mouse anti-human CD3ζ antibody , and then incubated with horseradish peroxidase-labeled anti-mouse secondary antibody for 1 h, and ECL developed the color. The result is as image 3 As shown, the anti-human CD3ζ antibody can detect the expression of CAR molecules, and the protein molecular size is consistent with the theory, which is 75kDa (such a...

Embodiment 3

[0040] Preparation of bispecific chimeric antigen receptor CD8α-Trop2 / PD-L1 scFv-CD8TM- CD28- CD137- CD3ζ modified T lymphocytes:

[0041] 1. Packaging preparation of retroviruses expressing CAR molecules

[0042]Plasmid extraction kit extracts retroviral packaging plasmids pRD114 and Peg-pam3 plasmids, co-transfects 293T cells with CD8α-Trop2 / PD-L1 scFv-CD8TM-CD28-CD137-CD3ζ retroviral plasmids, and collects cells after incubation for 48 hours The supernatant was centrifuged at 4000rpm for 10min, then filtered through a 0.45μm filter membrane, subpackaged and stored at -80°C.

[0043] 2. Preparation of T Lymphocytes

[0044] Take 30mL of fresh anticoagulated blood from healthy volunteers, and use lymphocyte separation medium (GE Company) to separate peripheral blood mononuclear cells (PBMC). The isolated cells were stimulated for 48 hours with a well plate coated with CD3 and CD28, and then induced and cultured with T lymphocyte medium GT-T551 (Takara Company) plus 3% autop...

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Abstract

The invention relates to a duplex-specific chimeric antigen receptor and application thereof. The duplex-specific chimeric antigen receptor is composed of CD8alpha, a humanization anti-Trop2 and anti-PD-L1 single-chain antibody, a transmembrane region Cd8TM, a notch intracellular domain CD28, a notch intracellular domain CD137 and a notch intracellular domain CD3zeta in serial connection. According to the chimeric antigen receptor CD8alpha-Trop2 / PD-L1 scFv-CD8TM-CD28-CD137-CD3zeta, by adopting a retrovirus technology, T lymphocyte of the human can be infected in vitro, obtained CAR-T cells cansimultaneously express tumor cells of Trop 2 and PD-L1 antigens through partial specific identification of the single-chain antibody of CAR, the CAR-T cells are activated and release multiple kinds of cell factors, and the specific killing effect on the tumor cells is achieved.

Description

technical field [0001] The invention belongs to cellular immunity technology, and in particular relates to a Trop2 / PD-L1 bispecific chimeric antigen receptor and its application. Background technique [0002] Chimeric Antigen Receptor T-cell Immunotherapy (CAR-T) has made great progress in the treatment of tumor immune cells, especially in the treatment of hematological tumors. . CAR-T cells combine the high affinity of antibodies with the killing effect of T lymphocytes. By constructing a specific chimeric antigen receptor carrier and genetically modifying T cells to express this chimeric antigen receptor, you can Specific recognition of target antigens to kill tumor cells. The structure of CAR includes a single-chain antibody sequence (scFv) capable of recognizing tumor antigens, an intracellular immunoreceptor tyrosine activation motif (ITMA, usually CD3ζ) and co-stimulatory molecular signals (CM, usually CD28, CD134, CD137 etc.), the CAR-T cells prepared in this way c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N5/10C12N15/867A61K35/17A61P35/00
Inventor 赵薇冯振卿朱进唐奇贾立周章明炯黄骁辰
Owner NANJING FIRST HOSPITAL
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