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Chimeric antigen receptor and use thereof

A chimeric antigen receptor and single-chain antibody technology, which is applied in the direction of hybrid peptides, antiviral agents, and antitumor drugs, can solve the problems of high cost, poor repeatability, and long preparation cycle, and achieve good repeatability and enhanced Effect, short cycle effect

Inactive Publication Date: 2014-06-11
SINOBIOWAY CELL THERAPY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The purpose of the present invention is to solve the problems of difficult preparation of antigen-presenting cells, high cost, long preparation period and poor repeatability in the current T cell therapy process.

Method used

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  • Chimeric antigen receptor and use thereof
  • Chimeric antigen receptor and use thereof
  • Chimeric antigen receptor and use thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0028] Example 1: Construction of chimeric antigen receptor LMP1-scFv-CH2CH3-CD28-CD134-CD3ζ retroviral vector.

[0029] 1. The amino acid sequences of the VH chain and VL chain of the human Fab sequence of the anti-LMP1 extracellular region screened according to the phage display technology of our laboratory (sequence reference patent "a human anti-nasopharyngeal carcinoma LMP1 extracellular region antibody and its Application", the patent application number is 201110364065), input the amino acid sequences of VH and VL in the OptimumGeneTM genetic design software, select the codon bias parameter, and the software calculates the optimized VH (SEQ ID NO 2) and VL (SEQ ID NO 2) 10) Nucleic acid sequence. Refer to Dutour, A., V. Marin, et al. (2012). "In Vitro and In Vivo Antitumor Effect of Anti-CD33 Chimeric Receptor-Expressing EBV-CTL against CD33 Acute Myeloid Leukemia." Adv Hematol 2012 in the construction of chimeric For antigen receptors, a signal peptide sequence (SEQ ID...

Embodiment 2

[0033] Expression identification of chimeric antigen receptor LMP1-scFv-CH2CH3-CD28-CD134-CD3ζ

[0034] The LMP1-scFv-CH2CH3-CD28-CD134-CD3ζ retroviral vector was extracted using the Dali kit (Tiangen Biology), and the plasmid was transfected into human embryonic kidney cell 293T with PI transfection reagent. After 24 hours, the plasmid was washed with PBS Wash once, add RIPA cell lysate (with Roche’s protease inhibitor Cocktail Tablets added), extract the protein of transfected 293T cells, conduct SDS-PAGE gel electrophoresis, semi-dry transfer membrane, use mouse anti-human CD3ζ antibody and rabbit Anti-human Fc antibody was incubated overnight at 4°C, and then incubated with horseradish peroxidase-labeled anti-mouse and anti-rabbit secondary antibodies at 37°C for 1 hour, and finally ECL color development solution was added to the membrane for color development. Results Both the anti-human CD3ζ antibody and the rabbit anti-human Fc antibody could detect the expression of th...

Embodiment 3

[0036] Preparation of Chimeric Antigen Receptor LMP1-scFv-CH2CH3-CD28-CD134-CD3ζ Modified T Lymphocytes

[0037] 1. Packaging preparation of retroviruses expressing CAR molecules

[0038] The pRD114 and Peq-pam3 plasmids (gifted by Dr. Xiaotong Song) and the LMP1-scFv-CH2CH3-CD28-CD134-CD3ζ retroviral vector were co-transfected into 293T cells using the Tiangen plasmid extraction kit 48h after transfection, the cell supernatant was collected, centrifuged at 4000prm for 10min, filtered through a 0.45μm filter membrane, and frozen at -80°C.

[0039] 2. Preparation of T lymphocytes

[0040] Take 20ml of fresh anticoagulated blood from healthy volunteers, and separate peripheral blood mononuclear cells (PBMC) with lymphatic separation fluid (purchased from GE). The isolated cells were stimulated for 48 hours with a plate coated with CD3 and CD28, and induced and cultured with T lymphocyte medium GT-T551 (purchased from TAKARA company) plus 3% autoplasma to obtain T lymphocytes. ...

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Abstract

The invention belongs to the biotechnical field of tumors, and discloses a preparation method of a chimeric antigen receptor and an application of the chimeric antigen receptor. The chimeric antigen receptor is formed through the structural series connection of a single chain antibody of a human anti-EB virus latent membrane protein 1, CH2CH3 of a human antibody IgG1, and intracellular signals of immune co-stimulating signal molecules CD28, CD134 and CD3zeta. The chimeric antigen receptor is used for modifying T lymphocytes, and the modified lymphocytes can be used for treating EB virus related tumors and preparing EB virus related tumor resisting medicines.

Description

technical field [0001] The invention belongs to the field of tumor biopharmaceuticals, and in particular relates to the preparation and application of a chimeric antigen receptor LMP1-scFv-CH2CH3-CD28-CD134-CD3ζ. Background technique [0002] Chimeric antigen receptor (CAR) is composed of antigen-specific receptors (such as single-chain antibody scFv), spacer sequence (spacer), transmembrane sequence (TM Domain) and intracellular co-stimulatory signal molecules. The spacer sequence can be hIgG1, hIgG4, hIgD, CD7 or CD8, etc., and the transmembrane sequence can be CD3ζ, CD4, CD7, CD8, FcεRIγ, H2-Kb, etc. The intracellular co-stimulatory signal molecule may be CD28, CD134, CD137, CD244, FcεRIγ, CD3ζ and the like. According to the differences in the composition of intracellular co-stimulatory signaling molecules, CARs are divided into three generations. The first-generation CARs contain only one intracellular signaling molecule (FcεRIγ or CD3ζ). By changing the signaling mol...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12N15/62A61K48/00A61P35/00A61P31/20
Inventor 冯振卿宋晓彤朱进唐小军陈仁杰
Owner SINOBIOWAY CELL THERAPY CO LTD
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