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CLTX-NKG2D bispecific chimeric antigen receptor cell as well as preparation method and application thereof

A technology of chimeric antigen receptor and specificity, applied in antibacterial drugs, animal cells, antibody medical components, etc., can solve the problems of low specificity and low killing efficiency of tumor cells

Active Publication Date: 2021-03-16
NANJING KAEDI BIOTHERAPEUTICS LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] 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 effector cells in the intra-tumor environment in killing tumor cells faced in the existing tumor clinical technology, Provides bispecific targeting binding polypeptide domains targeting human CLTX-R and NKG2DL or functional variants thereof, bispecific chimeric antigen receptors targeting human CLTX-R and NKG2DL or functional variants thereof, Its encoding nucleotide sequence and its expression vector, engineered immune response cells modified by bispecific chimeric antigen receptors targeting CLTX-R and NKG2DL, and applications thereof

Method used

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  • CLTX-NKG2D bispecific chimeric antigen receptor cell as well as preparation method and application thereof
  • CLTX-NKG2D bispecific chimeric antigen receptor cell as well as preparation method and application thereof
  • CLTX-NKG2D bispecific chimeric antigen receptor cell as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0127] Example 1 Preparation of expression plasmids specifically targeting chimeric antigen receptors of human CLTX-R and NKG2DL

[0128] The overall design is as follows:

[0129] 1. Determination of the amino acid sequence of chimeric antigen receptors specifically targeting CLTX-R and NKG2DL

[0130] First, the full-length amino acid sequence of human NKG2D (NP_031386.2) was searched from the Genbank database of the National Library of Medicine (NCBI), and the full-length amino acid sequence of CLTX protein was searched from the Uniprot website (Uniprot: P45639).

[0131] Secondly, construct a chimeric antigen receptor specifically targeting human CLTX-R and NKG2DL, that is, determine the amino acid sequence of the chimeric antigen receptor molecule:

[0132] From the amino terminal to the carboxyl terminal, the amino acid sequence of the leader peptide (as shown in SEQ ID No.1), the amino acid sequence of CLTX (such as SEQ ID No.2, SEQ ID No.3 or SEQ ID No.4), human NKG2...

Embodiment 2

[0153] Example 2: Preparation of virus liquid of lentiviral vector

[0154] The recombinant plasmid (KD-618 expression plasmid) obtained in Example 1 specifically targeting human CLTX-R and NKG2DL chimeric antigen receptors and the packaging vectors psPAX2 and VSVG were used according to the ratio of 10:8:5 with Lipofectamine TM 6000 transfection reagents (purchased from Beyontien, product model C0526) were used to co-transfect 293T cells (see the transfection instructions for specific transfection procedures), and replaced with complete medium 6 hours after transfection (purchased from hyclone, The product model is SH30243.01), after 48 hours and 72 hours of culture, the cell supernatants rich in lentiviral particles were collected respectively, and the virus supernatants were concentrated by ultracentrifugation to obtain specific targeting human CLTX-R and The virus liquid of the lentiviral vector of the chimeric antigen receptor of NKG2DL (hereinafter referred to as KD-618...

Embodiment 3

[0155] Example 3: Isolation and culture of T cells

[0156] Fresh peripheral blood from healthy donors was taken, and fresh peripheral blood mononuclear cells were separated by density gradient centrifugation; paramagnetic beads coupled with anti-CD3 antibody and anti-CD28 antibody (purchased from Invitrogen, USA, product information for Human T-Activator CD3 / CD28) to enrich CD3+ T cells, specifically, dilute peripheral blood mononuclear cells to a concentration of (10-30)×10 6 single cells / ml, and then mixed the magnetic beads and cells in a culture dish at a ratio of 3:1, incubated at room temperature for 2-3 hours, and used a Magnetic particles concentrator (MPC for short, purchased from Invitrogen, USA) company) to enrich CD3+ T cells. Finally, the enriched CD3+T cells were resuspended in culture medium (purchased from Life Technologies, USA, product information is OpTmizer TM T-Cell Expansion SFM), adjusted to a cell concentration of 1×10 6 pcs / ml, finally at 37°C, ...

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Abstract

The invention belongs to the field of chimeric antigen receptor cells, and particularly relates to a coding amino acid sequence of a bispecific chimeric antigen receptor targeting human CLTX-R and NKG2DL, an immune response cell modified with the coding amino acid sequence as well as a preparation method and application of the immune response cell. The invention relates to a bispecific chimeric antigen receptor of a bispecific targeting human NKG2DL and chlorotoxin receptor (CLTX-R) protein or a functional variant thereof, an immune response cell modified with the bispecific chimeric antigen receptor, as well as a preparation method and application of the bispecific chimeric antigen receptor. The obtained novel functionalized immune response cell can effectively target and attack various tumors, and can be used for preparing a preparation for treating malignant tumors. The immune response cell modified with the bispecific chimeric antigen receptor targeting CLTX-R and NKG2DL can enhance the binding with tumor cells, and obviously enhances the anti-tumor activity.

Description

technical field [0001] The invention belongs to the field of chimeric antigen receptor cells, and relates to an amino acid coding sequence of a bispecific chimeric antigen receptor targeting human CLTX-R and NKG2DL, and modified immune response cells thereof, as well as their preparation methods and Application in pharmaceutical preparation. Background technique [0002] With the rapid development of biotechnology, immune cell therapy has become the fourth largest therapy in the field of cancer treatment. [0003] Cancer immunotherapy mainly includes adoptive cell therapy, immunomodulators, tumor vaccines, and immune junction blockade therapy. Among them, in the field of cell therapy, CAR-T therapy has undoubtedly become a star that research institutions and pharmaceutical companies are vying for. [0004] Immunotherapy represented by CAR-T (Chimeric Antigen Receptor T-Cell, Chimeric Antigen Receptor T-Cell) is based on the principle of modifying chimeric antigen receptors...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/867C12N15/861C12N15/864C12N7/01C12N5/10A61K39/00A61K35/17A61P35/00A61P35/02A61P37/02A61P31/04A61P31/12
CPCA61K35/17A61K39/0011A61P31/04A61P31/12A61P35/00A61P35/02A61P37/02C07K14/7051C07K16/18C07K16/2851C07K16/40C07K2319/02C07K2319/03C07K2319/33C12N5/0636C12N5/0638C12N5/0645C12N5/0646C12N7/00C12N15/86C12N2510/00C12N2710/10021C12N2710/10043C12N2740/10021C12N2740/10043C12N2740/15021C12N2740/15043C12N2750/14121C12N2750/14143
Inventor 代红久徐慧李晓宇朱靓婧
Owner NANJING KAEDI BIOTHERAPEUTICS LTD
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