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Claudin 18.2-targeting CAR molecule, immune cell modified by Claudin 18.2-targeting CAR molecule and application of Claudin 18.2-targeting CAR molecule

A nucleic acid construct and sequence technology, applied in the field of modified immune cells and CAR molecules, can solve the problems of poor immune cell effect

Pending Publication Date: 2020-10-30
L&L BIOPHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In order to solve the problem of poor effect of immune cells targeting Claudin18.2 in the prior art, a CAR molecule targeting Claudin18.2, modified immune cells and uses thereof are provided

Method used

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  • Claudin 18.2-targeting CAR molecule, immune cell modified by Claudin 18.2-targeting CAR molecule and application of Claudin 18.2-targeting CAR molecule
  • Claudin 18.2-targeting CAR molecule, immune cell modified by Claudin 18.2-targeting CAR molecule and application of Claudin 18.2-targeting CAR molecule
  • Claudin 18.2-targeting CAR molecule, immune cell modified by Claudin 18.2-targeting CAR molecule and application of Claudin 18.2-targeting CAR molecule

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0107] Cloning expression and purification of embodiment 1 recombinant protein, antibody

[0108] The recombinant proteins involved in the present invention, including the expression and purification of monoclonal antibodies, are obtained by cloning, expression and purification of the present invention. The vector used for expression cloning is pTT5 vector (Biovector, Cat#: 102762). All protein expressions (including antibody light and heavy chains) were purified after transiently transfecting HEK 293F cells (Life Technologies Cat. No. 11625019) with pTT5 vector.

[0109] 293F cells were expanded in Gibco FreeStyle 293Expression Medium (Gibco, Cat#12338018) medium. Before the start of the transient, adjust the cell concentration to 6-8×10 5 cell / ml, 1% FBS (Aus Gene X FBSExcellent supplier: AusGeneX, China, Cat#FBSSA 500-S), 37°C 8% CO 2 Cultured on a shaker for 24 hours, and the survival rate was >95% under the microscope again, and the cell concentration was 1.2×10 6 cel...

Embodiment 2

[0110] Example 2 Human CLDN18.2 binding ELISA experiment

[0111] The human CLDN18.2 high-expression cell line used in the present invention is completed through the company's stable cell line construction platform. Specific steps are as follows:

[0112] On the first day of the experiment, 293T cells (Cat#GNHu17, a cell bank of the Type Culture Collection Committee of the Chinese Academy of Sciences) were seeded in two 6cm culture dishes, and the number of cells in each culture dish reached 7.5×10 5 indivual. On the second day, the plasmids (pGag-pol, pVSV-G, pBabe, etc. BioVector, Plasmid Vector Strain Cell Gene Collection Center) and cloned human CLDN18.1 gene (NCBI Reference Sequence: NP_057453.1), human CLDN18.2 gene (NCBIReference Sequence: NP_001002026.1), mouse CLDN18.1 (NP_062789.1) or mouse CLDN18.2 (NP_001181850.1) gene plasmid pBabe each 4μg was added to OPTI-MEM (Thermofisher Scientific Cat#31985070), so that the final volume was 200 μl, prepare 200 μl OPTI-MEM...

Embodiment 3

[0114] Example 3 Discovery and effect identification of anti-human CLDN18.2 antibody

[0115] 1. Discovery of anti-human CLDN18.2 antibody

[0116] The anti-human CLDN18.2 monoclonal antibody of the present invention is a mouse immunized with the human CLDN18.2 high-expression cell line (hCLDN18.2+cell) obtained in Example 2, and the spleen of the immunized mouse is taken for hybridoma fusion, and obtained from several Screened and optimized from millions of hybridoma clones.

[0117] Experimental mice, female, 4 weeks old (SJL mice were purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd., animal production license number: SCXK (Beijing) 2016-0011; Balb / c mice were purchased from Shanghai West Poole-Bick Laboratory Animals Ltd). After purchase, they were raised in a laboratory environment for 1 week, with daytime light / night dark cycle adjustment, temperature 20-25°C, and humidity 40-60%. Mice were divided into 3 / group / cage.

[0118] Cultivate the...

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Abstract

The present invention discloses a nucleic acid construct. The nucleic acid construct is characterized in that the nucleic acid construct has a structure as shown in the formula car-[(IRES)-f]<m>, wherein the IRES is a sequence of an internal ribosome entry site, f encodes a functional protein F, m is 0 or a non-0 natural number, car encodes a CAR, the CAR comprises (a) an extracellular binding domain that specifically recognizes CLDN18.2, (b) a hinge domain, (c) a transmembrane domain, (d) a costimulating intracellular domain, and (e) a signal transduction domain, the extracellular binding domain comprises scFv, and the amino acid sequences and functions of the extracellular binding structural domain are described in the specification. The CAR molecule provided by the invention has excellent safety and effectiveness and shows a very good tumor inhibition effect.

Description

technical field [0001] The invention belongs to the field of biomedicine, and specifically relates to a CAR molecule targeting Claudin18.2, its modified immune cells and its application. Background technique [0002] Cell therapy is an emerging disease treatment model with remarkable curative effect. Those skilled in the art have been devoting themselves to developing new cellular immunotherapy in order to improve the effect of cellular immunotherapy and reduce its side effects. Chimeric antigen receptor T cells (CART or CAR-T for short) are T cells isolated from patients, and T cells are modified in vitro with chimeric antigen receptors (CAR) so that they can specifically recognize cancer cells, and then The modified CART cells are expanded and reinfused into the patient's body to achieve the effect of treating tumors. Chimeric antigen receptor NK cells (CARNK or CAR-NK for short) are NK cells isolated from patients, and the NK cells are modified in vitro by CAR to enable...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/867C12N5/10C12N7/01A61K39/00A61P35/00A61P35/02
CPCA61K35/14A61P35/00A61P35/02C07K14/7051C07K16/28C07K2317/24C07K2317/56C07K2317/565C07K2317/92C07K2319/33C12N2740/15021C12N2740/15043
Inventor 刘佳建
Owner L&L BIOPHARMA CO LTD
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