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T lymphocyte modified by Trop2 chimeric antigen receptor and application thereof

A chimeric antigen receptor and lymphocyte technology, applied in the field of T lymphocytes, can solve problems such as risks and achieve the effect of specific killing

Active Publication Date: 2016-09-07
秦皇岛未名健长幸医疗健康科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the function of TCR-modified T cells is limited by MHC and can only recognize protein antigens, and has no effect on sugar and glycolipid antigens
In addition, the transgenic TCR may be mismatched with the TCR gene endogenously produced by T cells, posing potential risks

Method used

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  • T lymphocyte modified by Trop2 chimeric antigen receptor and application thereof
  • T lymphocyte modified by Trop2 chimeric antigen receptor and application thereof
  • T lymphocyte modified by Trop2 chimeric antigen receptor and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1: Construction of Trop2-specific chimeric antigen receptor lentiviral vector

[0029] 1. According to the amino acid sequence of the VH chain and Vκ chain of the human Fab sequence of the anti-Trop2 extracellular region screened by the phage display technology in our laboratory, the codon sequence was optimized by OptimumGeneTM gene design software, and the amino acid sequence was used without changing the amino acid sequence. It is more suitable for human expression system. A connecting peptide was added between VH and Vκ to construct the ScFv partial structure of the obtained chimeric antigen receptor: Vκ-(Gly4Ser)3-VH (corresponding nucleic acid sequence such as SEQ ID NO.4), and the fragment was cloned after synthesis In the pUC57 vector, Xba I and Bam HI restriction sites are introduced at the junction, and the vector is named: pUC57-Trop2-ScFv

[0030] 2. Digest Trop2-scFv-pUC57 with restriction endonucleases NcoI and BamH I. The enzyme digestion system ...

Embodiment 2

[0033] Example 2: Identification of Trop2-specific Chimeric Antigen Receptor Expression

[0034] Extract the retroviral vector Trop2-scFv-CH2CH3-CD28-CD137-CD3ζ according to the operating instructions in the endotoxin-free plasmid extraction kit (Tiangen Biology), and transfect the extracted plasmid into human embryonic kidney with PI transfection reagent In 293T cells, after 48 hours, wash with PBS, lyse the cells with cell protein extraction reagent (RIPA), extract the protein of the transfected 293T cells, separate by 10% SDS-PAGE, and then flow at a constant flow (300mA, 1h) Transfer to PVDF membrane, incubate with anti-CD3ζ (1:1000) antibody, and incubate overnight at 4°C. After washing 3 times with PBST, they were incubated with HRP goat anti-mouse secondary antibody (1:5000) for 1 h at room temperature. After adding ECL for color development, the ChemiDoc XRS System of Bio-Rad was used for imaging, and the results were as follows: image 3 shown.

[0035] Depend on ...

Embodiment 3

[0036] Example 3: Preparation of T lymphocytes modified by Trop2-specific chimeric antigen receptor

[0037] 1. Package containing anti-Trop2 chimeric antigen receptor lentivirus

[0038] Extract the retroviral packaging plasmid pRD114 and the Trop2-scFv-CH2CH3-CD28-CD137-CD3ζ retroviral plasmid with the operating instructions in the endotoxin-free plasmid extraction kit (Tiangen Biology), and culture them in large quantities in LB medium. A large number of plasmids were extracted according to the operating instructions in the endotoxin-free plasmid large-scale extraction kit (Tiangen Biology). The plasmids were co-transfected into GP-293T cells, and the cell supernatant was collected 48 hours after transfection, and centrifuged at 4000 rpm for 10 minutes. The supernatant was collected, filtered through a 0.45 μm membrane filter, and frozen at -80°C.

[0039] 2. Preparation of T lymphocytes

[0040] Take 20mL of fresh anticoagulated blood from healthy volunteers, and separa...

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PUM

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Abstract

The invention discloses a T lymphocyte modified by a Trop2 specific chimeric antigen receptor. The Trop2 specific chimeric antigen receptor is expressed on the surface of the T lymphocyte, wherein the Trop2 specific chimeric antigen receptor is formed by connecting a human anti-Trop2 single-chain antibody, CH2CH3 of a human IgG1 (Immunoglobulin G 1) molecular FC (Crystalline Fragment) region, an intracellular region of CD28 (Cluster of Differentiation 28), an intracellular region of CD137 (Cluster of Differentiation 137) and an intracellular region of CD3[zeta] (Cluster of Differentiation 3[zeta]) in series. The invention also discloses an amino acid sequence and a nucleotide sequence of the Trop2 specific chimeric antigen receptor. The invention also discloses application of the T lymphocyte to the preparation of an antineoplastic drug.

Description

technical field [0001] The invention relates to the technical field of cellular immunity, in particular to a T lymphocyte modified by a Trop2 specific chimeric antigen receptor and an application thereof. Background technique [0002] The specific immune response against tumors mediated by T lymphocytes plays an important role in the process of eliminating tumor cells in the body. However, due to the host's immune tolerance and the local immunosuppressive microenvironment of the tumor, the killing effect of T cells on tumor cells is often limited. Studies have found that T cell technology modified by chimeric antigen receptor (CAR) can effectively improve the proliferation ability, survival time and targeted killing effect of T lymphocytes in the body, and has become a newly popular cellular immunotherapy technology. one. CAR includes an extracellular region, which is a specific receptor for tumor antigens, a transmembrane region, and an intracellular co-stimulatory signal...

Claims

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Application Information

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IPC IPC(8): C12N5/10A61K35/17A61P35/00
CPCA61K35/17A61K2039/5156A61K2039/5158C12N5/0636C12N2510/00
Inventor 朱进冯振卿许国贞刘振云唐小军唐奇徐亚如
Owner 秦皇岛未名健长幸医疗健康科技有限公司
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