FastL-CAR fusion protein, T cell expressing fusion protein, and preparation method and application thereof

A fusion protein and protein technology, applied in the field of FasL-CAR fusion protein and T cells expressing fusion protein and their preparation, can solve the problems of large tumors, incomplete tumor cell removal and high cost, and achieve strong killing ability and prevent tumors. effect of recurrence

Active Publication Date: 2020-02-14
贵州康钦承平生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] However, after the CAR-T cells in the prior art use CAR to recognize and combine with tumor cells, they only rely on the T cell's own mechanism to eliminate tumor cells, and its ability to kill tumor cells is low
During the treatment process, it is necessary to deliver a large number of chimeric antigen receptor T cells to the patient, or to perform repeated CAR-T cell therapy multiple times to achieve the elimination of tumor cells. The existing CAR-T cell therapy has poor efficacy, low efficiency, and The process is complicated and the cost is high; in addition, due to the low tumor lethality of the existing CAR-T cells, after tumor treatment with this technology, the tumor cells are not completely removed, and there is a greater risk of tumor recurrence

Method used

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  • FastL-CAR fusion protein, T cell expressing fusion protein, and preparation method and application thereof
  • FastL-CAR fusion protein, T cell expressing fusion protein, and preparation method and application thereof
  • FastL-CAR fusion protein, T cell expressing fusion protein, and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1: Preparation of Chimeric Antigen Receptor T Cells Enhancing FasL / Fas Signaling Pathway

[0047] The schematic diagram of the structure of T cells (CAR-FasL-T cells) prepared in this example is as follows figure 1 As shown, VH and VL in the figure respectively represent the heavy chain variable region and the light chain variable region of the antibody, the extracellular antigen binding region includes the heavy chain variable region and the light chain variable region, and FasL represents the FasL protein. The specific construction process of CAR-FasL-T cells is as follows:

[0048] (1): Construction of expression plasmids:

[0049] The original lentiviral expression vector is pLVX-IRES-ZsGreen1 (product number: 632187; Baoriyi Biotechnology Co., Ltd.). pLVX-IRES-ZsGreen1 was transformed, and CAR gene and FasL gene were inserted at the same time. The CAR gene includes the extracellular antigen binding region gene (SEQ ID NO:1), CD8 transmembrane region gene...

Embodiment 2

[0075] Example 2: Preparation of Chimeric Antigen Receptor T Cells (without enhancing FasL / Fas signaling pathway function, CAR-T cells)

[0076] This comparative example is basically the same as in Example 1, except that the expression vector is not connected to the FasL gene, but is a lentiviral expression vector (pLVX-CAR-IRES-ZsGreen1 plasmid vector) with only the CAR gene.

experiment example 1

[0079] Experimental Example 1: Cell Immunofluorescence Detection Experiment

[0080] The experimental method is as follows:

[0081] 1. Use a histochemical pen to draw a circle on the glass slide (the inner circle is marked with a non-fading marker pen), and then use a small amount of PBS to resuspend the cell droplet, and dry it in a 37°C incubator without washing.

[0082] 2. Fix with 10 times the volume of 4% paraformaldehyde fixative at room temperature for 10 minutes, and soak in PBS for 10 minutes.

[0083] 3. Membrane rupture: Use PBS containing 0.2% Triton X-100 to rupture the membrane at room temperature for 20 minutes, then block with PBST solution containing 2% BSA for 50 minutes at room temperature, without washing, discard the excess blocking solution and directly add primary antibody.

[0084] 4. His primary antibody staining: Dilute the primary antibody with PBST containing 1% BSA, block overnight at 4°C in a wet box (not more than 16h), and then soak in PBST f...

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PUM

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Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to a FasL-CAR fusion protein, a T cell containing the FasL-CAR fusion protein, and a preparation method and an application thereof. The T cell expressing the FasL-CAR fusion protein has the property of enhancing FasL protein expression, and the ability of a FasL / Fas signal pathway of the T cell to inducetumor cell apoptosis is enhanced, so that the ability of the CAR-T cell to kill tumor cells is enhanced, and the possibility of tumor recurrence after treatment is reduced. When the FastL-CAR fusionprotein and the T cell expressing the FastL-CAR fusion protein are applied to a medicine for treating tumors, an effective way for treating cancers can be provided.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to a FasL-CAR fusion protein, a T cell expressing the fusion protein, a preparation method and application thereof. Background technique [0002] Chimeric Antigen Receptor T Cell (CAR-T) immunotherapy is an efficient and novel tumor immunotherapy. Infused back into the body to remove tumor cells. At present, most of the techniques for transforming and preparing CAR-T cells focus on changing the type of chimeric antigen receptor (CAR) to enhance the ability of T cells to recognize tumor cells, and then use T cells to eliminate tumor cells. mechanism to kill tumor cells. CAR is composed of intracellular signaling region (such as CD3zeta activation region and CD28 / 4-1BB co-stimulatory region), transmembrane region (such as CD8 transmembrane region) and extracellular antigen binding region. The extracellular antigen-binding region has the function of recognizing a spe...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N5/10C12N15/867A61K39/00A61P35/00
CPCA61K2039/5158A61P35/00A61K39/001129C07K14/7051C07K14/70575C07K16/30C07K2319/03C07K2319/33C12N5/0636C12N15/86C12N2510/00C12N2740/15043
Inventor 胡以国孙媛媛潘聪
Owner 贵州康钦承平生物科技有限公司
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