Targeted-MMSA-1-chimeric-antigen-receptor-modified T lymphocyte as well as preparation method and application thereof

A MMSA-1, chimeric antigen receptor technology, used in genetically modified cells, cells that are targeted for fusion of specific cells, and cells modified by the introduction of foreign genetic material, etc. Good anti-tumor effect

Active Publication Date: 2020-03-17
WUHAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the past ten years, although new targeted therapy drugs such as proteasome inhibitor bortezomib in clinical research and hematopoietic stem cell transplantation have achieved higher disease remission rate and quality of life than chemotherapy, bone destruction and bone marrow cell residual lesions still remain. major challenge in the treatment of multiple myeloma

Method used

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  • Targeted-MMSA-1-chimeric-antigen-receptor-modified T lymphocyte as well as preparation method and application thereof
  • Targeted-MMSA-1-chimeric-antigen-receptor-modified T lymphocyte as well as preparation method and application thereof
  • Targeted-MMSA-1-chimeric-antigen-receptor-modified T lymphocyte as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] [Example 1] Construction of MMSA-1 scFv and affinity flow detection

[0033] 1. Preparation of MMSA-1 CAR

[0034] After the wild-type antibody was humanized, MMSA-1 scFv was obtained. The MluI restriction site and CD8 transmembrane signal peptide were inserted before the fragment, and the BamHI restriction site was inserted after the fragment, which was then synthesized by a gene company (Jinweizhi). The pUC57-Amp plasmid containing MluI+CD8a-MMSA-1scFv+BamHI was synthesized by a gene company and was digested with MluI (NEB) and BamHI (NEB). The effect of the enzyme digestion was identified by agarose gel electrophoresis, and the modified gene was obtained by gel recovery. fragment. At the same time, the existing lentiviral backbone plasmid pHR containing the CD8 transmembrane region, 4-1BB co-stimulatory signal region and CD3ZetaTCR activation region was digested with MluI and BamHI, and the long fragments were recovered after identification by agarose gel electropho...

Embodiment 2

[0038] [Example 2] Preparation of MMSA-1 CAR-T cells and detection of infection efficiency

[0039] 1. Preparation of MMSA-1 CAR-T cells

[0040] Take 50 mL of fresh blood, and conduct density gradient centrifugation with lymphocyte separation medium (Tianjin Haoyang) to separate mononuclear cells. Divide mononuclear cells into 1-2 x 10 6 / mL resuspended in CTSTM AIM VTM SFM medium (GIBCO, Cat. No. A3021002). At the same time, 5% ICS (GIBCO, A2596101), CD3 monoclonal antibody (Ebioscience) 50ng / mL and CD28 monoclonal antibody (Ebioscience) 50ng / mL were added to activate T lymphocytes, and cultured at 37°C with 5% CO2 for 48 hours.

[0041] After 2 days of culture, collect the cells and resuspend the cells to 1x10 6 / mL, add the above-mentioned concentrated lentivirus according to MOI=5, add IL-2 (Quangang) and 4ug / mL polybrene (Sigma) at a final concentration of 200U / mL at the same time, mix well, and incubate at 37°C with 5% CO2 for 6- After 8 hours, centrifuge at 300g fo...

Embodiment 3

[0048] [Example 3] Detection of specific killing effect of MMSA-1 CAR-T cells in vitro by flow cytometry

[0049] Flow cytometry was used to detect the expression of MMSA-1 antigen in K562 cell line, RPMI-8226 cell line and primary tumor cells. Take K562 cell line, RPMI-8226 cell line and primary tumor cells 5×10 each 5 Add 1 μL of ZDHHC9 Polyclonal Antibody (Invitrogen) antibody and 1 μL of secondary antibody to each sample, incubate at 4°C in the dark for 15 minutes, centrifuge at 400 g for 5 minutes, wash once with 1 mL of PBS, resuspend the precipitate with 2% paraformaldehyde, and put it on a fascalibur (BD Bioscience) detection.

[0050] Such as image 3 As shown in A, K562 does not express MMSA-1 molecules on the surface, while RPMI-8226 cells and primary MM tumor cells express MMSA-1 molecules on the surface.

[0051] The two kinds of cells were seeded in a 96-well plate at a ratio of 1:1, 1×10 per well 5 For each species, MMSA-1 CAR-T cells and T cells cultured fo...

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Abstract

The invention provides a targeted-MMSA-1-chimeric-antigen-receptor-modified T lymphocyte as well as a preparation method and application thereof. A specific CAR-T cell line targeting MMSA-1 is constructed, the anti-myeloma activity of the specific CAR-T cell line is verified in vitro and vivo, and a new target is found for CAR-T immunotherapy of multiple myeloma. A specific MMSA-1 single-chain antibody (single-chain antibody fragment, scFv) is obtained by modifying a wild type MMSA-1 antibody in a humanized manner, the obtained MMSA-1 antibody gene fragment is modified and plasmid is loaded into the MMSA-1 antibody gene fragment, and an MMSA-1 chimeric receptor T cell is prepared by lentiviral transfection; and the expression of the CAR on the surface of the T cell is detected and a successfully transfected CAR-T cell is augmented in vitro, and then tests are carried out in vitro and vivo to detect an anti-myeloma effect of the CAR-T cell.

Description

technical field [0001] The invention belongs to the field of tumor immunotherapy, and specifically refers to a modified T lymphocyte targeting MMSA-1 chimeric antigen receptor and its preparation method and application. Background technique [0002] Multiple myeloma (MM) is a malignant clonal plasma cell disease, and myeloma bone disease (MBD) is one of its main clinical manifestations, and its bone destruction is characterized by unbalanced bone remodeling. feature. In the past ten years, although new targeted therapy drugs such as proteasome inhibitor bortezomib in clinical research and hematopoietic stem cell transplantation have achieved higher disease remission rate and quality of life than chemotherapy, bone destruction and bone marrow cell residual lesions still remain It is a major challenge in the treatment of multiple myeloma. Therefore, to improve the clinical efficacy of MM patients, it is very important to devote ourselves to finding precise treatment methods ...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/62C12N15/867C12N15/66A61K39/00A61P35/00
CPCA61K39/0011A61P35/00C07K14/7051C07K16/30C07K2319/02C07K2319/03C07K2319/33C12N5/0636C12N15/66C12N15/86C12N2510/00C12N2740/15043
Inventor 周芙玲童西琴魏永长金艳霞丁路邵亮江宏强灿灿商豫凤吴八路
Owner WUHAN UNIV
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