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Recombination chimeric antigen receptor gene and application thereof

A technology of recombining genes and antigens, which is applied in the field of tumor treatment, can solve the problems of low specificity, insufficient proliferation ability of T cells, and insufficient effect, and achieve the effect of high transfection rate, high specificity, and good proliferation ability

Active Publication Date: 2018-11-06
明慧南京基因生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] The purpose of the present invention is to provide a recombinant chimeric CAR-T cell therapy for Hodgkin's lymphoma or non-Hodgkin's lymphoma with insufficient effect, low specificity and insufficient proliferation ability of T cells in existing CAR-T cell therapy. Antigen receptor gene and its application

Method used

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  • Recombination chimeric antigen receptor gene and application thereof
  • Recombination chimeric antigen receptor gene and application thereof
  • Recombination chimeric antigen receptor gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0088] Embodiment 1: Construction of CART-30scFv plasmid

[0089] The method for obtaining the CART-30 scFv plasmid: design the coding nucleotide sequences of the scFv of 3 CD30 antibodies, including the CD8a signal peptide coding sequence whose sequence is SEQ ID NO: 26 and shown by SEQ ID NO: 23, 24 and 25 respectively The coding sequence of the scFv fragment is connected with the sequence shown in SEQ ID NO: 11, 12 and 13 to form 3 CAR genes:

[0090] CAR1: consists of SEQ ID NO: 26, 23, 11, 12 and 13;

[0091] CAR2: consists of SEQ ID NO: 26, 24, 11, 12 and 13;

[0092] CAR3: consists of SEQ ID NO: 26, 25, 11, 12 and 13;

[0093] The sequence of CAR3 is SEQ ID NO: 21;

[0094] The complete sequences of the recombinant genes CAR1, CAR2 and CAR3 were synthesized (completed by GenScript Biotechnology Co., Ltd.), and respectively ligated into the lentiviral plasmid vector pLent-EF1a (provided by Weizhen Biotechnology Co., Ltd.) by AsisI / NsiI (NEB Company). Technology Co., ...

Embodiment 2

[0095] Example 2: T lymphocyte transfection

[0096] The CART-30scFv plasmid constructed in Example 1 was packaged and purified by lentivirus, and then using lentiviral vector technology (references: Tumaini B, Lee DW, Lin T, Castiello L, et al. Simplified process for the production of anti-CD19 -CAR engineered T cells.Cytotherapy.2013; 15(11):1406-1415) expressed three recombinant CAR genes in T lymphocytes, and used flow cytometry to detect the positive rate of infection. The results are shown in image 3 and 4 , the transfection efficiency of lentivirus-infected T lymphocytes is about 60%.

Embodiment 3

[0097] Embodiment 3: Detection of killing rate in vitro

[0098] 1. Construction of CAR-T cytotoxicity indicator vector

[0099] According to the method described in the applicant's previous Chinese patent 201610537806.3, it specifically includes the following steps:

[0100] (1) Composite code The nucleotide sequences of Luciferase, P2A and CD30 are SEQ ID NO: 30, and Asis I and MluI enzyme cutting sites are respectively introduced upstream and downstream;

[0101] (2) Use Asis I and MluI to digest Plent-EF1α-Puro-CMV vector and step (1) at 37°C Luciferase-P2A-CD30 sequence, use DNA gel extraction kit to recover enzyme-digested Luciferase-P2A-CD30 sequence and Plent-EF1α-Puro-CMV vector;

[0102] (3) Digested The Luciferase-P2A-CD30 sequence and the Plent-EF1α-Puro-CMV vector were ligated at 22°C for 2 hours, and the ligation product was transformed into Escherichia coli DH5α competent cells by chemical method to obtain the cytotoxicity indicator vector pLent-EF1a-Nl...

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Abstract

The invention belongs to the field of immunotherapy of tumor cells, and provides a recombination chimeric antigen receptor (CAR) gene, a carrier comprising the CAR gene, a CAR-T cell and application thereof. The recombination CAR gene comprises nucleotide sequences encoding the following parts: an antigen binding part of a CD30 antibody, a transmembrane part, a CD137 cytoplasm functional area anda CD3zeta cytoplasm functional area, the CD137 cytoplasm functional area and the CD3zeta cytoplasm functional area are connected in arbitrary order; the invention further provides the application, andthe application applies a separated recombinant nucleic acid, a CAR fusion protein or a recombination T-lymphocyte to preparing drugs used for the treatment or adjuvant treatment of Hodgkin disease lymphoma or anaplastic large cell lymphoma or other CD3O positive tumors.

Description

technical field [0001] The invention belongs to the field of tumor treatment, and specifically relates to a recombinant chimeric antigen receptor CAR gene and its carrier and its preparation for the treatment or adjuvant treatment of Hodgkin's lymphoma or anaplastic large cell lymphoma or other CD30-positive tumors. in the application. Background technique [0002] Hodgkin's lymphoma (HL) is a unique type of lymphoma, and it is one of the most common malignant tumors among young people. According to epidemiological surveys, the incidence of Hodgkin's lymphoma varies greatly around the world. It occurs frequently in European and American countries, accounting for about 45% of lymphomas, ranking first in lymphomas, while China and Japan have a lower incidence rate. Classical Hodgkin's lymphoma has positive expression of CD30 antigen on RS cells, which is an important immune marker for identifying RS cells . [0003] Anaplastic large cell lymphoma (ALCL), which is an independ...

Claims

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Application Information

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IPC IPC(8): C12N15/62C12N5/10C12N15/867C07K19/00A61K35/17A61P35/00
CPCA61K35/17A61P35/00C07K14/7051C07K16/2878C07K2319/02C07K2319/33C12N15/86C12N2740/15043
Inventor 孙秀莲
Owner 明慧南京基因生物技术有限公司
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