Chimeric antigen receptors targeting Ccr1 and Nkg2d ligands and their applications

A technology of chimeric antigen receptors and ligands, which can be applied to polypeptides containing localization/targeting motifs, applications, retro RNA viruses, etc., and can solve problems such as affecting tumor treatment effects and large antigen escape

Active Publication Date: 2022-07-15
GUANGZHOU BIO GENE TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] However, the current chimeric antigen receptors have a large antigen escape problem, which affects the effect of tumor therapy

Method used

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  • Chimeric antigen receptors targeting Ccr1 and Nkg2d ligands and their applications
  • Chimeric antigen receptors targeting Ccr1 and Nkg2d ligands and their applications
  • Chimeric antigen receptors targeting Ccr1 and Nkg2d ligands and their applications

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Example 1 Vector construction

[0060] The structures of the two chimeric antigen receptors constructed in this example are as follows: figure 1 As shown, A is NKG2D-CCR1 CAR and B is CCR1-NKG2D CAR. According to the amino acid sequence of the chimeric antigen receptor, the whole gene was synthesized in Guangzhou Aike Biotechnology Co., Ltd. and constructed into a lentiviral expression vector. The vector structures of NKG2D-CCR1CAR and CCR1-NKG2D CAR are respectively as follows figure 2 and image 3 shown, the sequence information is shown below.

[0061] NKG2D-CCR1 CAR (sp-NKG2D-Linker-antiCCR1 VL-(G4S) 3- VH-CD8hinge-CD8TM-4-1BB-CD3Z)氨基酸序列:MDMRVPAQLLGLLLLWLRGARCLFNQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQDLLKLVKSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENCSTPNTYICMQRTVGSTSGSGKPGSGEGSTKGDVVMTQTPRSLPVSLGDQASISCRSRQSLIHSNGITFLHWYLQKAGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLRISRVEAEDLGVYFCSQGTHVPPTFGGGTKLEIKGGGGSGGGGSGGGGSQVQLKQSGPGLVQPSQSLSITCTVSGFS...

Embodiment 2

[0065] Example 2 Detection of the expression of CCR1 and NKG2D ligands by tumor cells

[0066] First, CCR1 was detected by flow cytometry in U937, THP-1, HL60, KG1a, Jurkat, Daudi, RPMI8226, NCI-H929, Raji, and K562 cells after co-incubating with target cells with APC anti-human CCR1 Antibody (biolegend brand). expression. The result is as Figure 4 As indicated, U937, THP-1, RPMI8226 and NCI-H929 were CCR1 positive cells and HL60, KG1a, Jurkat, Daudi, Raji and K562 were CCR1 negative cells.

[0067] Target cells K562, NCI-H929, NCI-H929, U937 expresses antigen status. The result is as Figure 5 and Image 6 As shown, K562 cells negatively expressed CCR1, and NKG2DL, MICA, ULBP2 / 5 / 6 were positive; NCI-H929 cells were strongly positive for CCR1, and NKG2DL, MICA, ULBP2 / 5 / 6 were all negatively expressed; U937 cells were CCR1 positive, Positive expression of NKG2DL, ULBP2 / 5 / 6, negative expression of MICA.

[0068] Table 1 Expression of CCR1 and NKG2D ligand (NKG2DL) in tum...

Embodiment 3

[0070] Example 3 Preparation of anti-CCR1, NKG2D dual-target CAR-T cells

[0071] (1) Separation of PBMC

[0072] Collect 50 mL of peripheral blood; add 15 mL of lymphocyte separation solution to two 50 mL sterilized centrifuge tubes on the ultra-clean workbench, and slowly pour 25-30 mL of peripheral blood into the centrifuge tube containing lymphocyte separation solution. According to the instructions for use, centrifuge the centrifuge tube at 700g for 20min at room temperature, increase the speed by 1 and decrease the speed by 2. If the blood is stored for more than 2 hours, increase the centrifugation time to 30min;

[0073] After centrifugation, the blood is divided into 4 layers, consisting of plasma (upper layer), mononuclear cells between plasma and separating fluid (layer 2), separating fluid (layer 3), and red blood cells (bottom layer). The mononuclear cells were collected into a new centrifuge tube, 20 mL of PBS was added to dilute the cell suspension, and centrif...

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PUM

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Abstract

The present invention relates to a chimeric antigen receptor targeting CCR1 and NKG2D ligands and its application, comprising an antigen binding domain, a hinge region, a transmembrane domain, a costimulatory domain and a Signal transduction domain; the antigen binding domain includes anti-CCR1 single chain antibody and NKG2D. The CAR-T cells constructed by using the chimeric antigen receptors of the present invention that simultaneously target CCR1 and NKG2D ligands have specific targeting effects on CCR1 and NKG2D ligand-positive tumor cells, have significant killing effects in vitro, and can effectively remove CCR1 And NKG2D ligand single / double positive tumor cells, reducing antigen escape, is of great significance in the field of tumor therapy.

Description

technical field [0001] The present invention relates to the technical field of molecular biology, in particular to a chimeric antigen receptor targeting CCR1 and NKG2D ligands and its application. Background technique [0002] In recent years, chimeric antigen receptor T cell (CAR T) therapy has shown very good efficacy in tumor treatment. The full name of CAR-T therapy is Chimeric antigen receptor T cell therapy, which is chimeric antigen receptor T cell therapy. The costimulatory molecules, amplified in vitro, are then infused back into the patient to recognize and attack their own tumor cells. T cells, also known as T lymphocytes, are a type of human white blood cells, derived from bone marrow hematopoietic stem cells, mature in the thymus, and then migrate to the human blood, lymph and surrounding tissues and organs to exert immune function, which can resist and eliminate infections, tumors. , foreign bodies, etc. In the laboratory, technicians activate T cells throug...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12N15/62C12N5/10C12N15/867A61K39/00A61P35/00A61P35/02
CPCC07K16/2866C07K16/2851C12N5/0636C12N15/86A61K39/001116A61K39/001102A61P35/00A61P35/02C07K2317/53C07K2317/56C07K2317/565C07K2319/03C07K2319/71C07K2319/02C12N2740/15043C12N2510/00A61K2039/5156A61K2039/804
Inventor 李光超罗敏周兆王学俊
Owner GUANGZHOU BIO GENE TECH CO LTD
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