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Chimeric antigen receptor cell library carrying gene element combination, preparation and screening method and application thereof

A chimeric antigen receptor and gene element technology, applied in the fields of biomedical engineering technology and synthetic biology, can solve problems such as changes in screening direction, antigen disease treatment products, and inability to directly target unknown antigens.

Pending Publication Date: 2020-04-07
PHARCHOICE THERAPEUTICS INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the limitation of this technology is that the screening direction of the CAR cell library cannot follow the change of the antigen, and can only be screened against a specific antigen.
Although this patent document describes a method for further screening of CAR libraries using general techniques, such as using iQue TM Screener (Intellicyt, Albuquerque, NM) (a high-throughput flow cytometer) for high-throughput testing of individual CAR molecules is on the one hand inefficient and on the other hand the system is designed to rapidly obtain usable CAR receptors. The body is not proposed for individualized treatment. For example, the methods of these CAR libraries in this document can obtain CAR receptors targeting specific antigens, but they cannot directly target unknown antigens, let alone serve as targets for heterogeneity, variability, and evolution. Therapeutic products for characteristic antigenic diseases

Method used

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  • Chimeric antigen receptor cell library carrying gene element combination, preparation and screening method and application thereof
  • Chimeric antigen receptor cell library carrying gene element combination, preparation and screening method and application thereof
  • Chimeric antigen receptor cell library carrying gene element combination, preparation and screening method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0107] Example 1. Fully synthetic murine chimeric antigen receptor T cell library

[0108] See the library construction and screening process figure 1 :

[0109] (A) Construction of phage antibody library: First, a fully synthetic murine phage single-chain antibody library is established using a total synthesis method. The method of antibody library preparation is well-known to those of ordinary skill in the art. The method of establishing a fully synthetic murine phage single-chain antibody library is the same as in the literature [Geuijen C et al.. European Journal of Cancer, 2005, 41(1): 178- 187; Noronha EJ, et al. Journal of Immunology, 1998, 161(6): 2968-2976.]. After the library capacity evaluation, the library capacity of the fully synthetic murine phage single-chain antibody library is 1×10 9 . The method of library capacity evaluation is the same as that in the literature [Ridgway J B B, et al. Cancer Research, 2013, 59(11): 2718-2723].

[0110] (B) Background eliminati...

Embodiment 2

[0119] Example 2. Treatment of breast cancer with fully synthetic mouse chimeric antigen receptor T cell library

[0120] This example was performed using the KRAB-iCasp9-CAR-T cell library described in Example 1.

[0121] Establishment of 4T1 mouse orthotopic breast cancer model: The method of model establishment is the same as that in the literature [Paschall A V, Liu K. JoVE2016(114):e54040.]. When the tumor volume in mice reached an average of 100mm 3 Mice were divided into control group, unrelated CAR-T cell group, KRAB-iCasp9-CAR-T cell library group 1, KRAB-iCasp9-CAR-T cell library group 2, and KRAB-iCasp9-CAR-T cell library group 3. Group; 4 groups of KRAB-iCasp9-CAR-T cell library.

[0122] The CAR-positive rate of CAR-T cells is standardized to 45%. The control group was given PBS treatment; the unrelated CAR-T cell group was given CD19-CAR-T cell treatment (gene control expression cassette such as figure 2 C), the dose is 5×10 6 Cells were injected intravenously, once ...

Embodiment 3

[0124] Example 3. In vivo screening of fully synthetic murine chimeric antigen receptor T cells targeting 4T1 breast cancer

[0125] All the experimental groups in Example 2 were sacrificed after the experiment, and the blood of the mice was separated and the CAR-positive cells were detected by flow cytometry. Figure 4 shows the ratio of CAR-positive cells obtained after the experiment in all treatment groups. The CAR-positive cells in each group were separated and cultured to complete the in vivo screening.

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Abstract

The invention provides a chimeric antigen receptor cell library carrying a gene element combination, preparation and screening methods and application. The chimeric antigen receptor cell library is formed by fusion of cells and a carrier assembly, the carrier assembly carries three gene elements, and the three gene elements are respectively as follows: a plurality of first gene elements for encoding one or more unique chimeric antigen receptors; a second genetic element carrying one or more genetic loops; and a third genetic element encoding one or more inducible proteins. The genetic loop ispreprogrammed and is a combination of a regulatory homeopathic factor and a transcription factor; and the inducible protein comprises one or two of drug resistance protein and suicide protein. By designing the chimeric antigen receptor library-genetic loop-inducible protein coupling scheme, the cell library and screening of complex and unknown disease target antigens are realized, so that the problems that antigens in abnormal antigen expression diseases are complex, diverse and easy to change, target identification is difficult and the like are solved. The method has a wide application prospect.

Description

Technical field [0001] The present invention relates to the fields of biomedical engineering technology and synthetic biology, in particular to a gene element combination and a chimeric antigen receptor cell library carrying the combination, and a method for preparing and constructing the gene element combination and the cell library for in vivo antigens And / or in vitro antigen screening methods and the use of cell libraries are described in detail. Background technique [0002] The use of genetically modified immune cells to treat diseases is a hot spot in the current technology. Through chimeric antigen receptor (Chimeric Antigen Receptor, CAR) expression, to the antigen expressed on tumor cells. CAR is an antigen receptor designed to recognize cell surface antigens in a human leukocyte antigen-independent manner. Attempts to treat these types of patients with genetically modified T cells expressing CAR have been successful to a certain extent (Molecular Therapy, 2010, 18: 4,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/867C40B50/06C40B40/02C40B30/06C12Q1/02A61K39/00A61P35/00A61P29/00A61P31/12A61P31/04A61P33/00A61P11/00A61P15/14A61P1/18A61P1/00A61P13/08A61P13/10A61P19/08A61P15/00A61P17/00A61P13/12A61P7/00A61P25/00A61P21/00A61P19/04A61P1/16A61P27/02A61P27/16A61P11/02A61P11/04A61P1/02A61P25/28A61P25/16
CPCC12N15/86C40B50/06C40B40/02C40B30/06G01N33/5038G01N33/505A61K39/0005A61K39/0007A61K39/001A61P35/00A61P29/00A61P31/12A61P31/04A61P33/00A61P11/00A61P15/14A61P1/18A61P1/00A61P13/08A61P13/10A61P19/08A61P15/00A61P17/00A61P13/12A61P7/00A61P25/00A61P21/00A61P19/04A61P1/16A61P27/02A61P27/16A61P11/02A61P11/04A61P1/02A61P25/28A61P25/16C12N2740/15043C12N2840/002A61K39/4643A61K2239/49A61K2239/38A61K39/46432A61K39/4613A61K2239/55C12N5/0636A61K39/464402A61K39/4611A61K2239/31A61K2239/54A61K39/464412A61K39/4631C12N2740/16043C07K14/7051A61K2039/812A61K2039/852C12N15/74C12N15/1055C07K2319/03C12N2510/00
Inventor 胡适傅文燕
Owner PHARCHOICE THERAPEUTICS INC
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