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CCR1 targeting chimeric antigen receptor and application thereof

A chimeric antigen receptor and targeting technology, applied in the direction of polypeptides containing positioning/targeting motifs, receptors/cell surface antigens/cell surface determinants, hybrid peptides, etc., can solve the problem of chimeric antigen receptors Lack of other issues

Active Publication Date: 2022-03-01
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, chimeric antigen receptors that target different tumor targets and have good tumor cell killing effects are still lacking.

Method used

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  • CCR1 targeting chimeric antigen receptor and application thereof
  • CCR1 targeting chimeric antigen receptor and application thereof
  • CCR1 targeting chimeric antigen receptor and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Embodiment 1 vector construction

[0058] The structure of the chimeric antigen receptor CCR1 CAR constructed in this example is as follows figure 1 shown. According to the amino acid sequence of the chimeric antigen receptor, the whole gene was synthesized in Guangzhou Aiji Biotechnology Co., Ltd. and constructed into a lentiviral expression vector. The vector structure of CCR1 CAR is as follows: figure 2 As shown, the sequence information is shown below.

[0059] CCR1 CAR amino acid sequence:

[0060]MDMRVPAQLLGLLLLWLRGARCDIQMTQSPSSLSASVGDRVTITCSASQGIRNYLNWYQQKPGKVPKLLIVYTSRLHSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQQYGKLPYTFGQGTKLEIKGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSDAWMDWVRQAPGKGLEWVGEIRSKVNNHETYYAESVKGRFTISRDDSKNSLYLQMNSLKTEDTAVYYCARNDYFDYWGQGTTVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTY...

Embodiment 2

[0063] Example 2 Flow cytometric detection of the expression of CCR1 on the surface of tumor cells

[0064] After co-incubating with target cells with APC anti-human CCR1 Antibody antibody (biolegend brand), the expression of CCR1 in U937, THP-1, HL60, KG1a, Jurkat, Daudi, RPMI8226, NCI-H929, Raji and K562 cells was detected by flow cytometry . Such as image 3 The results showed that U937, THP-1, RPMI8226 and NCI-H929 were CCR1 positive cells, and HL60, KG1a, Jurkat, Daudi, Raji and K562 were CCR1 negative cells.

Embodiment 3

[0065] Example 3 Preparation of anti-CCR1 CAR-T cells

[0066] (1) Separation of PBMCs

[0067] Collect 50mL of peripheral blood; add 15mL of lymphocyte separation medium to two 50mL sterilized centrifuge tubes in the ultra-clean workbench, slowly inject 25-30mL of peripheral blood into the centrifuge tubes containing lymphocyte separation Centrifuge the centrifuge tube at 700g for 20 minutes 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 30 minutes;

[0068] After centrifugation, the blood is divided into 4 layers, which are composed of plasma (upper layer), mononuclear cells between plasma and separation liquid (2nd layer), separation liquid (3rd layer) and red blood cells (bottom layer). Collect the mononuclear cells into a new centrifuge tube, add 20mL PBS to dilute the cell suspension, centrifuge at 500g for 10min; remove the supernatant, add 20mL PBS to dilute t...

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Abstract

The invention relates to a CCR1-targeting chimeric antigen receptor and application thereof. The CCR1-targeting chimeric antigen receptor comprises a CCR1-targeting single-chain antibody, a hinge region, a transmembrane structural domain, a costimulatory structural domain and a signal transduction structural domain which are sequentially connected from an amino terminal to a carboxyl terminal, the CCR1-targeting single-chain antibody comprises an anti-CCR1 antibody light chain variable region and an anti-CCR1 antibody heavy chain variable region, the amino acid sequence of the anti-CCR1 antibody light chain variable region is as shown in SEQ ID NO: 1, and the amino acid sequence of the anti-CCR1 antibody heavy chain variable region is as shown in SEQ ID NO: 2. The CAR-T cell constructed by using the CCR1 targeting chimeric antigen receptor has a specific targeting effect on CCR1 positive tumor cells, has a remarkable in-vivo and in-vitro killing effect, can effectively remove the CCR1 positive tumor cells, and has important significance in the field of tumor treatment.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to a chimeric antigen receptor targeting CCR1 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, that is, chimeric antigen receptor T cell therapy. Co-stimulatory molecules, expanded in vitro, are reinfused into the patient's body to recognize and attack their own tumor cells. T cells, also known as T lymphocytes, are a type of human white blood cells that originate from bone marrow hematopoietic stem cells, mature in the thymus, and then migrate to human blood, lymph and surrounding tissues and organs to perform immune functions, which can resist and eliminate infections and tumors , foreign bodies, etc. In the laboratory, technicians use genetic engineering technology to activate ...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/85C12N5/10A61K35/17A61P35/00
CPCC07K14/7158C07K14/7051C12N5/0636A61K35/17A61P35/00C12N2510/02C07K2319/02C07K2319/03C07K2317/56
Inventor 李光超罗敏王学俊刘劲松周兆
Owner GUANGZHOU BIO GENE TECH CO LTD