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Application of vitamin D acceptor and ligand thereof to improvement of antitumor capacity of immunocyte

A technology of immune cells and vitamins, applied in the direction of blood/immune system cells, receptors/cell surface antigens/cell surface determinants, anti-tumor drugs, etc., can solve the problems of expensive and side effects, achieve good safety performance, and promote secretion , well tolerated effect

Active Publication Date: 2019-12-20
北京德衍科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this receptor (VDR) has not been reported in the regulation of immune checkpoints, cytokine production and anti-tumor effects of immune cells.
In addition, since PD-1 / PD-L1 monoclonal antibody and CAR-T therapy are extremely expensive and accompanied by serious side effects, related inhibitors for vitamin D receptors are developed or used to transform immune T cells to promote immune T cells. Antitumor activity has broad application prospects

Method used

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  • Application of vitamin D acceptor and ligand thereof to improvement of antitumor capacity of immunocyte
  • Application of vitamin D acceptor and ligand thereof to improvement of antitumor capacity of immunocyte
  • Application of vitamin D acceptor and ligand thereof to improvement of antitumor capacity of immunocyte

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] Example 1: The ligand structural formula of the vitamin D receptor is as follows:

[0069]

[0070] The above compounds (vitamin D 3 (VD 3 ), 25(OH)D 3 , 1α,25(OH) 2 D. 3 ) as an activator of the vitamin D receptor. This compound is commercially available from Selleck Corporation.

Embodiment 2

[0071] Example 2: Isolation and culture of CD8T and CD4T cells

[0072] CD8T and CD4T cells are derived from peripheral blood mononuclear cells (PBMCs), and PBMCs are derived from the peripheral blood of healthy people. Will 1×10 7 For every 50 μL of PBMCs cells (according to the instructions, every 50 μL of sorting magnetic beads liquid, add 1×10 7 1 PBMCs) sorting magnetic beads CD8 and CD4 (both magnetic beads were purchased from BD Company), and incubated at 4°C for 25 min. Add 950 μL of PBS to resuspend in the flow tube, put it into the magnetic stand for 5 minutes, remove the supernatant, and repeat this step 2 times. Count and plate, add the sorted CD8T or CD4T cells to the 48 wells of the pre-coated plate (αCD3, 5 μg / mL, Biolegend) in advance, add 1 μg / mL (final concentration) αCD28 (Biolegend) to stimulate for 48 h, two Add rhIL-2 (final concentration: 40 ng / mL) to cells available one day later (γδT / CD3>90% detected by flow cytometry).

Embodiment 3

[0073] Embodiment 3: recombinant vitamin D receptor gene cloning

[0074] (1) Overexpression of VDR gene

[0075] The vitamin D receptor (vitamin D receptor, VDR) gene was cloned from CD8 T cells (obtained in Example 2). RNA (Trizol) in CD8 T cells was extracted, cDNA library was obtained by reverse transcription (Takara), and primers were designed to clone the full-length fragment of VDR (upstream primer sequence: 5'-ATGGAGGCAATGGCGGC-3', downstream primer sequence: 5' -TCAGGAGATCTCATTGCCAAACA-3'), double digested with XbaI and EcoRI (NEB), and cloned into the plasmid pTSB-CMV-MCS-SBP-3Flag-copGFP-F2A-PuroR (TranSheepBio) to obtain the plasmid pTSB-CMV-MCS -SBP-3Flag-copGFP-F2A-PuroR-VDR, and then transform it into Escherichia coli DH5α (Takara), after BGI sequencing and NCBI database comparison, the cloned gene is indeed the vitamin D receptor gene, The sequence is as follows (SEQ ID NO.1):

[0076] atggaggcaatggcggccagcacttccctgcctgaccctggagactttgaccggaacgtgccccggatctgtg...

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Abstract

The invention discloses an application of a vitamin D acceptor and a ligand thereof to improvement of antitumor capacity of immunocyte. The inventor finds that the vitamin D acceptor and the dependentligand thereof can effectively reduce expression of PD-1, Tim-3 and TIGIT on the surface of the immunocyte. In addition, restraining of the vitamin D acceptor in the immunocyte may lead to decrease of cytokine secretion, and gene overexpression of the vitamin D acceptor can reinforce effective release of the cytokine under activation of TCR, so as to raise the sensibility of T cells to tumors andpromote the killing capacity of the T cells to tumors. The vitamin D acceptor and the ligand thereof can improve the function of the immunocyte, the sensibility of the immunocyte is improved, and thevitamin D acceptor and the ligand thereof have the characteristics of being small in toxic and side effects, large in tolerance range and high in safety performance. Therefore, the vitamin D acceptorand the ligand can be applied to respects of united treatment of immunization inspection points, and cell reimportation treatment.

Description

technical field [0001] The invention belongs to the biomedical technical field of anti-tumor immunotherapy, and relates to the application of a vitamin D receptor and its ligand in improving the anti-tumor ability of immune cells, in particular to the application of a vitamin D receptor and its ligand in the preparation of inhibitory Application of immune checkpoint expressed products. Background technique [0002] According to the latest literature reports, global cancer incidence and mortality are increasing rapidly, with 18.1 million new cancer cases and 9.6 million cancer deaths worldwide in 2018. In both sexes, the cancers with the highest incidence rates were lung cancer, female breast cancer, prostate cancer, and colorectal cancer (Bray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA: a cancer journal forclinicians 2018,68(6):394-424....

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N5/078C12N5/0783C12P21/00A61K45/00A61P35/00
CPCA61K45/00A61P35/00C07K14/705C12N5/0634C12N5/0636C12P21/00
Inventor 高云飞黎鹏尹芝南王笑吴阮
Owner 北京德衍科技有限公司
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