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Specific chimeric antigen receptor T cell targeting nkg2dl, preparation method and application thereof

A chimeric antigen receptor and specific technology, applied in the field of tumor immunotherapy and biomedicine, can solve the problems of low killing efficiency and weak specificity, etc.

Active Publication Date: 2020-09-25
NANJING KAEDI BIOTHERAPEUTICS LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] In view of the above-mentioned problems and / or other problems in related technologies, the purpose of the present invention is to overcome the problems of low specificity and low killing efficiency of T cells in the tumor environment in the tumor environment that are faced in the existing clinical technology , providing chimeric antigen receptor targeting human NKG2DL, its gene and recombinant expression vector, engineered NKG2DL targeting chimeric antigen receptor modified immune response cells and its application

Method used

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  • Specific chimeric antigen receptor T cell targeting nkg2dl, preparation method and application thereof
  • Specific chimeric antigen receptor T cell targeting nkg2dl, preparation method and application thereof
  • Specific chimeric antigen receptor T cell targeting nkg2dl, preparation method and application thereof

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preparation example Construction

[0248] In the eighth aspect, the present application provides the method for preparing the isolated chimeric antigen receptor-modified immune response cells according to the sixth aspect of the present application, comprising the following steps:

[0249] Firstly, the nucleic acid molecule described in the third aspect is connected to an expression vector by means of molecular cloning to obtain an expression vector targeting NKG2DL-specific chimeric antigen receptor;

[0250] Then, transfect the obtained specific CAR expression vector targeting NKG2DL into 293T cells to obtain virus fluid;

[0251] Finally, the virus liquid is used to infect the immune response cells, and the modified immune response cells expressing the specific chimeric antigen receptor targeting NKG2DL are obtained from the infected cells.

[0252] In some non-limiting embodiments, the modified immune response cells of the present invention may be cells of the lymphoid lineage. The cells of the lymphoid li...

Embodiment 1

[0289] Preparation of expression plasmids targeting NKG2DL-specific chimeric antigen receptors

[0290] Step 1) Determination of the amino acid sequence of the specific chimeric antigen receptor targeting NKG2DL

[0291] First, the full-length amino acid sequence (as shown in SEQ ID No.1) and the full-length nucleotide sequence (as shown in SEQ ID No. .2).

[0292] Second, construct a specific chimeric antigen receptor targeting NKG2DL.

[0293] details as follows:

[0294] The amino acid sequence of the NKG2DL-specific CAR molecule: from the amino terminal to the carboxyl terminal, the amino acid sequence of the guide peptide (as shown in SEQ ID No.3), the human NKG2D amino acid sequence (as shown in SEQ ID No.4, , the amino acid sequence of the human CD8 hinge region (as shown in SEQ ID No.10), the amino acid sequence of the human CD8 transmembrane region (as shown in SEQ ID No.11), the amino acid sequence of the human 4-1BB intracellular domain ( shown in SEQ ID No.12),...

Embodiment 2

[0320] Example 2 Preparation of expression plasmids targeting NKG2DL-specific chimeric antigen receptors

[0321] In addition to step 1, the human NKG2D sequence (as shown in SEQ ID No.5) in the amino acid sequence of the CAR molecule targeting NKG2DL specificity, the amino acid sequence of the human CD3ζ domain is shown in SEQ ID No.15, targeting NKG2DL specificity The nucleotide sequence encoding the human NKG2D sequence (as shown in SEQ ID No.18) in the nucleotide sequence of the CAR molecule,

[0322] and step 2) construction and identification of a plasmid expressing a specific CAR molecule targeting NKG2DL

[0323] The nucleotide sequence of the specific CAR molecule targeting NKG2DL was synthesized from the whole gene, and connected to the lentiGuide-Puro lentiviral vector (Nanjing Yidao Bio, figure 1 ), constructing the full-length CAR sequence expression frame stage of a single coding frame, using the EFS promoter (SEQ ID No., 30 in the sequence table), and the rest ...

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Abstract

A specific chimeric antigen receptor targeting NKG2DL, its coding sequence, and modified immune response cells, as well as their preparation method and application. The modified immune response cells can effectively target and attack various tumor cells, especially positive tumor cells expressing NKG2DL, and can be used to prepare preparations for treating tumors.

Description

[0001] References to related applications [0002] This application claims the priority of the PCT patent application with the application number PCT / CN2017 / 076795 submitted on March 15, 2017, and the entire content of this patent document is incorporated herein by reference. technical field [0003] The invention belongs to the technical field of biomedicine for tumor immunotherapy, and relates to specific chimeric antigen receptor T cells, in particular to a specific chimeric antigen receptor (chimeric antigen receptor) targeting NKG2DL, comprising NKG2D protein or a variant thereof. receptor, CAR) and modified immune response cells, as well as their preparation methods and applications. Background technique [0004] With the rapid development of biotechnology, immune cell therapy has become the fourth largest therapy in the field of cancer treatment. [0005] Cancer immunotherapy mainly includes adoptive cell therapy, immunomodulators, tumor vaccines, and immune checkpoi...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/28C12N5/10C12N15/62C12N15/86A61K39/00A61P35/00
CPCC12N15/62C07K14/7056C07K2319/00C12N5/0636C12N2510/00C07K14/70578C07K2319/02C07K2319/03C07K2319/33C07K2319/32C12N2740/16043A61K2039/828A61K2039/892A61K2039/844A61K2039/852A61K2039/86A61K2039/80A61K2039/812A61K2039/884C07K14/7051C12N15/625A61K9/0019A61P35/00C07K14/70517A61P37/06A61K39/464412A61K39/464429A61K39/4611A61K2239/47A61K39/4631C12N15/111C07K14/4726C12N5/163C12N15/63
Inventor 代红久孙彬赵旭东
Owner NANJING KAEDI BIOTHERAPEUTICS LTD
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