Engineered immune cell for combined expression of CCR2b as well as preparation and application of engineered immune cell

An immune cell and engineering technology, applied in the field of tumor immunity and cell therapy, can solve problems such as excessive release of cytokines, recurrence, and failure to achieve therapeutic effects

Active Publication Date: 2021-11-09
GUANGZHOU BAIJI BIO PHARMACEUTICAL CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

On the basis of the second-generation CAR-T, the third-generation CAR-T adds a co-stimulatory domain. Although it can improve the killing activity of T cells, it may induce excessive release of cy

Method used

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  • Engineered immune cell for combined expression of CCR2b as well as preparation and application of engineered immune cell
  • Engineered immune cell for combined expression of CCR2b as well as preparation and application of engineered immune cell
  • Engineered immune cell for combined expression of CCR2b as well as preparation and application of engineered immune cell

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0259] Example 1 Lentivirus preparation

[0260] 1.1 Obtaining lentiviral vector plasmids

[0261] The nucleotide sequences of BN001, BN003, BC001, and BC006 were synthesized from the whole gene, and then connected to the lentiviral vector pCDH-EF1-MCS-T2A-copGFP plasmid by molecular cloning, so that it was in the human EF1α promoter and Kozak sequence. expression under control.

[0262] Transfection of lentiviral vector plasmid into 293T cells

[0263] Mix the above-mentioned lentiviral vector plasmids with lentiviral packaging plasmids pMD2.G, pRSV-Rev and pMDLg / pRRE with polyethyleneimine transfection reagent, and co-transfect 293T cells. After culturing for 48 h, the virus supernatant was collected separately, centrifuged at 4500 rpm at 4°C for 10-15 min, filtered through a filter membrane with a pore size of 0.5 μm, and then concentrated with a hollow fiber column ultrafiltration system, and then purified by chromatography. The lentivirus was purified, and finally fi...

Embodiment 2

[0268] Example 2 Preparation and detection of CAR-T cells

[0269] 2.1 T cell preparation

[0270] Adjust the density of peripheral blood mononuclear cells from healthy donors to 2 × 10 6 / ml, add 50 ng / ml anti-CD3 antibody, 50 ng / ml anti-CD28 antibody, and 200 IU / ml recombinant IL-2, and culture in a cell incubator for 24 h (cultivation temperature is 37°C, carbon dioxide concentration is 5%).

[0271] lentiviral transduction of T cells

[0272] Wash the obtained T cells and adjust the cell density to 2 × 10 6 / ml. Add lentivirus at MOI = 1-10 TU / cell for transduction, supplement 50 ng / ml anti-CD3 antibody, 50 ng / ml anti-CD28 antibody, and 200IU / ml recombinant IL-2, and place in the cell culture incubator Medium culture (cultivation temperature is 37°C, carbon dioxide concentration is 5%). After 24 h, adjust the cell density to 1.5-2 × 10 6 / ml, and supplemented with 300 IU / ml of IL-2. On the 4th day after transduction, wash the cells to remove residual lentiviral p...

Embodiment 3

[0282] Example 3 Target cell detection

[0283] 3.1 Target cell culture conditions

[0284] Tested colorectal cancer cell lines (also known as target cells or target cell lines): HCT 116 (McCoy's 5a medium + 10% fetal bovine serum + 100 U / ml penicillin + 100 μg / ml streptomycin), HT-29 (McCoy's 5a medium + 10% fetal bovine serum + 100 U / ml penicillin + 100 μg / ml streptomycin), LoVo (F-12K medium + 10% fetal bovine serum + 100 U / ml penicillin + 100 μg / ml streptomycin), SW480 (Leibovitz's L-15 medium + 10% fetal bovine serum + 100 U / ml penicillin + 100 μg / ml streptomycin), T84 (DMEM / F-12 medium + 5% fetal bovine serum + 100 U / ml penicillin + 100 μg / ml streptomycin), SK-OV-3 (McCoy's 5a medium + 10% fetal bovine serum + 100 U / ml penicillin + 100 μg / ml streptomycin).

[0285] Ligand (MICA / MICB) expression detection

[0286] The above target cells were washed twice with PBS and resuspended with FACS buffer. Add APC-labeled anti-human MICA / MICB antibody to each target cell sus...

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Abstract

The invention relates to an engineered immune cell for combined expression of CCR2b. In particular, the engineered immune cells of the present invention are T cells or NK cells. The CAR molecule containing the NKG2D extracellular domain and the CCR2b are jointly expressed in the CAR-T cell, so that the sensitivity of the CAR-T cell to chemotactic factors such as CCL2, CCL7 and the like can be improved through the CCR2b, the CAR-T cell efficiently migrates to focuses such as colorectal cancer, ovarian cancer, pancreatic cancer and the like, and the treatment efficiency is improved; and meanwhile, various target antigens on the surfaces of cells such as colorectal cancer, ovarian cancer, pancreatic cancer and the like can be recognized through NKG2D CAR molecules, the risk that the curative effect is reduced due to tumor heterogeneity or target antigen loss is reduced, and the tumor treatment effect is improved.

Description

technical field [0001] The invention belongs to the field of tumor immunity and cell therapy, and in particular relates to an engineered immune cell jointly expressing CCR2b. Background technique [0002] Cellular immunotherapy is an emerging tumor treatment mode with significant curative effect, and it is a new type of autoimmune anti-cancer treatment. It is a method of using biotechnology and biological agents to culture, modify and amplify immune cells collected from patients in vitro, and then infuse them back into the patient's body to stimulate and enhance the body's autoimmune function, so as to achieve the purpose of treating tumors. [0003] T cells are an important class of lymphocytes involved in cellular immunity, and can specifically recognize and kill tumor cells through the signal transmission of antigen-presenting cells. However, tumor cells can also be affected by the reduction or loss of antigenic epitopes, immunosuppression, and tumor heterogeneity (that ...

Claims

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Application Information

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IPC IPC(8): C12N5/10C07K19/00C07K16/28A61K39/00A61P35/00C12N15/62C12N15/13
CPCC12N5/0636C07K14/7158C07K16/2851C07K14/7051A61K39/001102A61P35/00C07K2319/33C07K2319/74C12N2510/00C07K2319/03A61K2039/5156
Inventor 张曦黄智宏韩德平燕妮王调霞朱晓娜王宁林伟雄卢永
Owner GUANGZHOU BAIJI BIO PHARMACEUTICAL CO LTD
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