Specific chimeric antigen receptor cell targeting claudin18.2 and its preparation method and application

A chimeric antigen receptor, targeted binding technology, applied in genetically modified cells, cells modified by introducing foreign genetic material, animal cells, etc., can solve the problems of low tumor cell specificity and low safety, etc. To achieve the effect of high killing activity and high persistence

Active Publication Date: 2022-02-01
NANJING KAEDI BIOTHERAPEUTICS LTD
View PDF2 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] In view of the above-mentioned problems and / or other problems in related technologies, the purpose of the present invention is to overcome the low specificity and low safety of effector cells in the tumor microenvironment that tend to bind and kill tumor cells in the existing tumor clinical technology To solve the problem, provide a chimeric antigen receptor specifically targeting human Claudin18.2, its gene and recombinant expression vector, engineered immune response cells modified specifically targeting human Claudin18.2 chimeric antigen receptor and its application

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Specific chimeric antigen receptor cell targeting claudin18.2 and its preparation method and application
  • Specific chimeric antigen receptor cell targeting claudin18.2 and its preparation method and application
  • Specific chimeric antigen receptor cell targeting claudin18.2 and its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0120] Example 1 Preparation of expression plasmids specifically targeting chimeric antigen receptors of human Claudin18.2

[0121] The overall design is as follows:

[0122] 1. Acquisition of anti-human Claudin18.2 single-chain antibody (scFv) gene sequence

[0123] The full human amino acid sequence number of Claudin18.2 in the Genbank database of the National Library of Medicine (NCBI) is: NP_001002026.1. After immunizing mice with human Claudin18.2 protein, its antibody fragment was purified and humanized to obtain a humanized single-chain antibody (scFv) against human Claudin18.2; the single-chain antibody consists of a light chain and a heavy chain, and the light chain The connecting segment between chain and heavy chain is (G4S) n (3≤n≤8), finally obtained 4 scFv antibody fragments with high affinity to the target antigen Claudin18.2 after effect screening, the amino acid sequence of which is as SEQ ID No.2 or SEQ ID No.3 or SEQ ID No.4 or Shown in SEQ ID No.5:

[0...

Embodiment 2

[0145] Example 2: Preparation of virus liquid of lentiviral vector

[0146] The recombinant plasmid expressing the chimeric antigen receptor specifically targeting human Claudin18.2 obtained in Example 1 and the packaging vectors psPAX2 and VSVG were used according to the ratio of 10:8:5 with Lipofectamine TM 6000 transfection reagent (purchased from ThermoFisher, product model 11668019) co-transfected 293T cells (see ThermoFisher transfection manual for specific transfection operation process), and replaced with complete medium 6 hours after transfection (purchased from Life Technologies , the product model is 11995-065), 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 the expression-specific targeting human Claudin18. 2 chimeric antigen receptor lentiviral vector virus liquid (hereinafter referred to as KD-182-1 or KD-182-2 or...

Embodiment 3

[0147] Example 3: Isolation and culture of T cells

[0148] 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, peripheral blood mononuclear cells were diluted 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), adjust the cell solubility to 1×10 6 pcs / ml, finally at 3...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

Abstract

The present invention relates to a chimeric antigen receptor cell specifically targeting human Claudin18.2 and its preparation method and application. The immune response cell modified by the specific chimeric antigen receptor targeting human Claudin18.2 of the present invention can enhance and Combined with tumor cells, it has obvious anti-tumor activity. The chimeric antigen receptor-modified engineered immune cells specifically targeting human Claudin18.2 of the present invention have strong specificity and good safety.

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 chimeric antigen receptor specifically targeting human Claudin18.2, and modified immune response cells, as well as their preparation methods and pharmaceutical preparations in the application. 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] CAR-T (Chimeric Antigen Receptor T-Cell, Chimeric Antigen Receptor T-Cell) immunotherapy, its principle is mainly to take the patient's own T cells for genetic modification of ...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/867C12N5/10A61K39/00A61K35/17A61P35/00A61P37/02A61P31/12A61P31/04
CPCC07K16/28C07K14/70517C07K14/70521C07K14/70578C07K14/7051C12N15/86C12N5/0636A61K39/0011A61K35/17A61P35/00A61P37/02A61P31/12A61P31/04C07K2317/622C07K2317/24C07K2317/92C07K2319/02C07K2319/03C07K2319/33C07K2319/74C12N2740/15043C12N2800/107C12N2510/00A61K2039/5156
Inventor 代红久徐慧朱靓婧王梦瑶
Owner NANJING KAEDI BIOTHERAPEUTICS LTD
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap