Immune cell overexpressing CCR2 and application thereof

An immune cell and overexpression technology, applied in the field of immunology, can solve problems such as drug blocking, and achieve the effects of strong migration ability, tumor cell growth inhibition, and high secretion ability

Pending Publication Date: 2021-11-05
QINGDAO SINO-CELL BIOMEDICINE CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The purpose of the present invention is to provide an immune cell overexpressing CCR2, a pharmaceutical composition comprising the immune cell and its use in the preparation of immunotherapy products, aiming to give full ...

Method used

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  • Immune cell overexpressing CCR2 and application thereof
  • Immune cell overexpressing CCR2 and application thereof
  • Immune cell overexpressing CCR2 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Flow cytometric detection of CCR2 in TIL cells and PBMC after activation with anti-CD3 / CD28 magnetic beads + / - CD107a difference of cell population, the steps are as follows:

[0055] 1.1 Count the PBMC cell suspension by centrifugation, resuspend with AIM-V + 10% AB serum + double antibody complete medium and adjust the cell density to 1×10 6 / mL, spread 1 mL of cell suspension in each well of a 24-well plate, add CD3 monoclonal antibody 100 ng / mL, and IL-2 1000 U / mL overnight.

[0056] 1.2 Primary cultured TIL cells 3×10 6 cells, overnight.

[0057] 1.3 The above cells were grouped as shown in Table 1, with 3 replicates in each group, and activated with Miltenyi anti-CD3 / CD28 magnetic beads overnight.

[0058] Table 1

[0059] group 1 group 2 group 3 group 4 cell TIL TIL PBMC PBMC Stimulate + - + -

[0060] 1.4 After 18 hours of treatment, flow cytometric analysis: the ratio and quantity of each group of cells of CCR2, ...

Embodiment 2

[0063] CCR2 overexpression lentivirus packaging and preparation of CCR2 overexpression TIL cells, the steps are as follows:

[0064] 2.1 Lentivirus packaging and harvesting

[0065] first day:

[0066] 1) Replace the cultured 293FT (purchased from ATCC) with 9 mL of antibiotic-free DMEM+10% FBS before transfection;

[0067] 2) Set up a group of EP tubes;

[0068] 3) 2 EP tubes A: OPTi-MEM 1.5ml + main plasmid 20μg + pMDLg.PRRE 10μg + PRSV-Rev5μg + PMD2.G 5μg. A total of 40 μg of plasmid ratio (4:2:1:1); and corresponding 2 EP tubes B: OPti-MEM1.5ml+lipo300041μL. The plasmids in this step are all purchased from Addgene, and the main plasmid of the first EP tube A is empty plasmid, and the main plasmid of the second EP tube A is the main plasmid of the multiple cloning site inserted into the CCR2a DNA fragment.

[0069] 4) The two EP tubes B were mixed separately and left at room temperature for 5 minutes;

[0070] 5) Slowly drop the two EP tubes B into the corresponding two ...

Embodiment 3

[0094] CCR2a and CCR2b overexpression killing effect verification experiment, the steps are as follows:

[0095] 3.1 HLA-A of NL donor, the typing result is A*11:01 / A*33:03, HCC827 is homozygous for HLA*A locus A*11:01 / A*11:01; NL peripheral blood Separation of obtained PBMCs, magnetic bead sorting for CD8 + Cells, count the sorted cell suspension by centrifugation, resuspend with AIM-V + 10% AB serum + double antibody complete medium and adjust the cell density to 1 × 10 6 / mL, spread 1mL of cell suspension in each well of a 24-well plate, add OKT3 anti-CD3 monoclonal antibody 100ng / mL, IL-21000U / mL overnight.

[0096] 3.2 Take the CCR2a lentivirus concentrate and CCR2b lentivirus concentrate prepared in Example 2 out of the -80°C refrigerator, put them on ice and let them melt slowly.

[0097] 3.3 Infect PBMC cells with lentivirus at the ratio of MOI=10, that is, each cell corresponds to 10 viruses, and each well needs 1×10 viruses 7 indivual. Calculate the volume requir...

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Abstract

The invention belongs to the technical field of immunology, and particularly relates to an immune cell overexpressing CCR2 and application thereof. According to the invention, through over-expression of CCR2 in the immune cell, the CCR2 can be combined with CCL2; and the CCL2 can be inhibited to promote growth, migration and infiltration of tumour cells. Experiments prove that: through over-expression of the CCR2 in the immune cell, the migration ability of the immune cell is stronger; and the secretion ability of cell factors is also higher, so that the immune infiltration ability and tumour killing ability of the immune cell to a tumour microenvironment are improved. The invention provides a new choice for cellular immunotherapy, and has important significance for prevention and treatment of tumour diseases.

Description

technical field [0001] The invention relates to the field of immunology, in particular to an immune cell overexpressing CCR2, a pharmaceutical composition comprising the immune cell and its use in preparing immunotherapy products. Background technique [0002] C-C chemokine receptor 2 (C-C MotifChemokine Receptor 2, CCR2) is one of the 19 members of the human chemokine receptor family, located on chromosome 3, encoding a 355 amino acid residue protein, including two CCR2a and CCR2b Subtype. CCR2 is a G protein-coupled receptor found on various cell types. [0003] CCL2 is one of the important members of the CC subfamily of chemokines, and it is the main ligand that binds to CCR2. Although the combination of CCL2 and CCR2 is not specific, it can be speculated that CCL2 exerts its biological effects mainly by combining with CCR2 from the similar functional phenotypes of CCL2- / - and CCR2- / - knockout mice. In normal tissue cells, CCL2 can be produced by a variety of activated...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/12C12N15/867A61K35/17A61K35/15A61P35/00
CPCC12N5/0634C07K14/7158C12N15/86A61K35/17A61K35/15A61P35/00C12N2510/02C12N2740/15043
Inventor 龚拯李亚腾李斌李文静高青刁树青王泽瑶
Owner QINGDAO SINO-CELL BIOMEDICINE CO LTD
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