Anti-Claudin18A2 chimeric antigen receptor, T cell modified thereby, and preparation method and application of T cell

A chimeric antigen receptor, cell technology, applied in the field of genetic engineering, can solve the problem of difficult technical improvement

Active Publication Date: 2019-01-15
SHANDONG XINRUI BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The applicant found in long-term research that the gene drug prepared by simply knocking out the PD-1 gene in T cells needs to be further improved in the treatment of digestive system tumors, especially gastric cancer. However, the technical improvement at the bottleneck stage It is very difficult and also a technical problem in this field

Method used

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  • Anti-Claudin18A2 chimeric antigen receptor, T cell modified thereby, and preparation method and application of T cell
  • Anti-Claudin18A2 chimeric antigen receptor, T cell modified thereby, and preparation method and application of T cell
  • Anti-Claudin18A2 chimeric antigen receptor, T cell modified thereby, and preparation method and application of T cell

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Experimental program
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Effect test

Embodiment 1

[0044] Anti-Claudin18A2 chimeric antigen receptor, including sequentially linked CD8αLeader, Claudin18A2 antigen-binding region, CD8 hinge region and transmembrane region, 4-1BB and CD28 co-stimulatory region, CD3ζ signal transduction region, the complete nucleic acid sequence is shown in the appendix SEQ ID NO.1.

[0045] Module sequence of CAR-Claudin18A2

[0046] (1) CD8α Leader (SEQ ID NO.2)

[0047] (2) Claudin18A2 antigen-binding region (SEQ ID NO.3)

[0048] (3) CD8Hinge region (SEQ ID NO.4)

[0049] (4) CD8 transmembrane region (SEQ ID NO.5)

[0050] (5) CD28 co-stimulatory region (SEQ ID NO.6)

[0051] (6) 4-1BB co-stimulatory region (SEQ ID NO.7)

[0052] (7) CD3ζ signaling region (SEQ ID NO.8).

Embodiment 2

[0054] The anti-Claudin18A2 chimeric antigen receptor is prepared by a method comprising the following steps:

[0055] (1) According to the order of Example 1 CD8α Leader, Claudin18A2 antigen binding region, CD8 Hinge region and transmembrane region, 4-1BB and CD28 co-stimulatory region, CD3ζ signal transduction region entrusted to Beijing Biomed Gene Technology Co., Ltd. to synthesize Its entire expression cassette (Claudin18A2 module indicated figure 1 ), and inserted into the cloning vector pUC57, the resulting product was named pUC-Claudin18A2,;

[0056](2) Carry out double enzyme digestion of pUC-Claudin18A2, use agar electrophoresis to cut off the agar part containing the Claudin18A2 DNA fragment, use the DNA extraction kit sol solution to treat, pass through the DF column and discard the filtrate, rinse the DF column, empty and elute DF column, and the centrifuged matter was collected to obtain a purified Claudin18A2 DNA fragment. In this example, the detailed steps ...

Embodiment 3

[0058] Construction of pLent-EF1α-CAR-Claudin18A2 expression vector

[0059] The recombinant expression vector was prepared by the following steps: the entire expression frame was synthesized according to the CD8α Leader, Claudin18A2 antigen-binding region, CD8 Hinge region and transmembrane region, 4-1BB and CD28 co-stimulatory region, and CD3ζ signal transduction region and inserted into pLent -EF1α vector (purchased from Vigene company) BamHI-NotI site (such as figure 2 ), transformed into E.coli (Top10), after the sequence was correct, the plasmid was extracted using the plasmid extraction kit of OMEGA Company, and the recombinant expression vector pLent-EF1α-CAR-Claudin18A2 was obtained.

[0060] The detailed steps of recombinant plasmid pLent-EF1α-CAR-Claudin18A2 are as follows:

[0061] According to CD8αLeader, Claudin18A2 antigen-binding region, CD8 Hinge region and transmembrane region, 4-1BB and CD28 costimulatory region, CD3ζ signal transduction region commissioned...

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Abstract

The invention discloses an anti-Claudin18A2 chimeric antigen receptor, a T cell modified thereby, and a preparation method and application of the T cell. The anti-Claudin18A2 chimeric antigen receptorcomprises a CD8alpha leader, a Claudin18A2 antigen binding region, a Hinge region and a transmembrane region of CD8, a co-stimulation region of 4-1BB and CD28, and a signal transduction region of CD3zeta. After long-term experiments, the Claudin18A2 target is creatively selected and the knockout of PD-1 protein in a T cell is cooperatively implemented, so the modified T cell can be improved in the therapeutic effect on digestive system tumors, especially on gastric cancer.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to an anti-Claudin18A2 chimeric antigen receptor, a T cell modified by the same, and a method for preparing and using the T cell. Background technique [0002] PD-1 (programmed death receptor 1, programmed death 1) is a 55KD transmembrane protein that belongs to the immunoglobulin superfamily. There is only one IgV-like region in the extracellular region, two tyrosine residues in the cytoplasmic region, and one ITIM (immunoreceptor tryosine-based inhibitory motif) at the tail. PD-1 can be expressed on activated T cells, B cells and myeloid cells, as well as CD4-CD8-thymocytes. PD-1 has two ligands, PD-L1 (B7-H1) and PD-L2 (B7-DC), both of which are new members of the B7 family. PD-1 is an immunosuppressive receptor that interacts with its ligands PD-L1 and PD-L2 to transmit inhibitory signals and play a negative regulatory role in the immune response. The combination ...

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
CPCA61P35/00A61K35/17C12N15/86C07K14/7051C07K14/70521C07K16/28C07K2317/76C07K2319/33C12N2740/15043
Inventor 刘明录王立新韩庆梅万磊卢永灿张传鹏刘敏马洪华金海锋冯建海
Owner SHANDONG XINRUI BIOTECH CO LTD
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