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Preparation and application of CXCL13 chemotaxis type CAR-T cells

A technology of immune cells and preparations, applied in the field of preparation and application of CXCL13 cytokine chemotactic CAR-T cells, which can solve the problem that CAR-T cells and tumor cells are difficult to contact closely

Pending Publication Date: 2020-07-07
EAST CHINA NORMAL UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] CAR-T therapy has shown significant therapeutic effects in the treatment of hematological tumors, but there are still many challenges in the application of CAR-T cells in solid tumors: 1. There are a large number of fibroblasts on the surface of tumor tissues, which act as a Physical barriers make it difficult for CAR-T cells and tumor cells to come into close contact

Method used

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  • Preparation and application of CXCL13 chemotaxis type CAR-T cells
  • Preparation and application of CXCL13 chemotaxis type CAR-T cells
  • Preparation and application of CXCL13 chemotaxis type CAR-T cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0285] Example 1 Construction of PCDH-NKG2D-CAR and PCDH-NKG2D-CXCR5-CAR vectors

[0286] The obtained NKG2D sequence (obtained from GenBank, NCBI Reference Sequence::AF461811.1) and CXCR5 sequence (obtained from GenBank, NCBI Reference Sequence: NM_032966.2) were linked together by overlapping PCR. After sequencing and identification, it was confirmed that the construction was successful, and the PLL3.7-NKG2D-CXCR5-CAR vector was obtained. The structure diagram is as follows figure 1 Shown: NKG2D-CXCR5-CAR was expressed using lentiviral expression system (the backbone plasmid is PLL3.7 plasmid), and the extracellular segment of NKG2D (containing exocrine signal peptide), CD8 transmembrane region and costimulatory factors were expressed using EF1a promoter 4-1BB is connected to the intracellular signal activation sequence CD3ζ, the back end is connected to T2A, and then connected to CXCR5, and the end uses PolyA stop codon.

Embodiment 2

[0287] Example 2 Preparation of CAR-T cells

[0288] Take human peripheral blood, use lymphocyte separation medium to separate human total lymphocytes, and use CD4 + , CD8 + Two kinds of magnetic beads were used to separate T lymphocytes, and CD3 and CD28 magnetic beads were used to activate T cells. After 48 hours, the medium was changed by centrifugation. Infect the lentivirus according to MOI=20:1, centrifuge and change the medium after 24 hours, add fresh medium, and detect the positive rate of T cells after staining with NKG2D antibody.

Embodiment 3

[0289] Example 3 CAR-T cell proliferation

[0290] T cells in the NC group, NKG2D-CAR-T (G2D) group and NKG2D-CXCR5-CAR-T (G5) group were labeled with eflour670 for 20 min, and then incubated with osteosarcoma cells U2OS respectively, and cell proliferation was detected by flow cytometry. After the cells are labeled with eflour670, the faster the cells proliferate, the lower the fluorescence intensity of the cells will be. The fluorescence intensity of the cells was detected by flow cytometry, and the stronger the fluorescence intensity, the frontier diagram would shift to the left. When CAR-T cells were co-incubated with target cells U2OS for 24 hours, the proliferation rate of the cells was significantly accelerated. Because T cells in the NC group cannot recognize specific tumor antigens, they cannot activate specific cell proliferation signals, and the fluorescence intensity of the cells is high, and the proliferation is slow ( figure 2 ). There was no significant diff...

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Abstract

The invention provides preparation and application of CXCL13 cytokine chemotaxis type CAR-T cells, and particularly engineering immunocyte. The engineering immunocyte can express chimeric antigen receptors CAR and CXCR5 targetting MICA. The engineering immunocyte can selectively kill tumor cells, such as tumor cells high in expression of NKG2D ligands or NKG2D ligands and CXCL13 at the same time,and besides, the CAR-T cells disclosed by the invention have notable killing effects on the tumor cells.

Description

technical field [0001] The invention belongs to the field of biotechnology. Specifically, the present invention relates to the preparation and application of a CXCL13 chemoattractant CAR-T cell, and more specifically, to the preparation and application of a CXCL13 cytokine chemoattractant CAR-T cell. Background technique [0002] As the most common primary malignant bone tumor, osteosarcoma mostly occurs in adolescents or children. Surgery and chemotherapy are the main treatment options. Due to the high rate of systemic metastases at initial diagnosis, cure with surgery alone is quite rare. For patients with metastatic osteosarcoma, the 5-year survival rate is less than 20%, and chemotherapy has no significant effect on lung metastasis. In addition, pulmonary metastases have a high recurrence rate after primary metastasectomy, and re-metastasis resection is sometimes required for treatment. [0003] CAR-T cells (Chimeric antigen receptor T cells, CAR-T cells) fuse single-...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/867C07K19/00C12N15/62
CPCC07K14/7051C12N15/86C07K2319/02C07K2319/03C07K2319/33C12N2740/15043
Inventor 江文正张利何聪段邑昕姚杰周滢刘明耀席在喜
Owner EAST CHINA NORMAL UNIV
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