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Tumor binding specific gamma delta TCR gene modified alpha beta T cell and cancer suppression application thereof

A technology that combines specificity and genetic modification, applied in the field of adoptive immunotherapy of genetically modified lymphocytes, can solve the problems of low proportion of γδT cells, decreased immune activity, and reduced number of γδT cells, and achieve the best therapeutic effect.

Active Publication Date: 2016-02-03
北京佳德和细胞治疗技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] However, because the proportion of γδT cells in peripheral blood is very low, the adoptive therapy of natural γδT cell preparations for clinical tumors needs to be expanded in vitro for a long time to reach the dose required for treatment
However, immunosuppression in patients with advanced tumors or patients after chemotherapy leads to a decrease in the number of γδT cells and a decline in immune activity; the use of mouse-derived antibodies and bovine serum components will lead to many problems such as the in vivo immune response of the reinfused cells, resulting in the use of γδT cells for treatment. There are still some problems in the method of cell preparation that need to be solved urgently

Method used

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  • Tumor binding specific gamma delta TCR gene modified alpha beta T cell and cancer suppression application thereof
  • Tumor binding specific gamma delta TCR gene modified alpha beta T cell and cancer suppression application thereof
  • Tumor binding specific gamma delta TCR gene modified alpha beta T cell and cancer suppression application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0092] Example 1: Construction of recombinant lentiviral vector pCDH-δ1TCR-CMV-γ4TCR

[0093] Since the GTM sequence has tumor binding specificity, the present invention puts it into the complete δ1 chain to replace the original CDR3δ sequence, and then combines with the complete γ4 chain to form a complete γ4δ1 (GTM) TCR molecule ( Figure 1A ), so that it is expressed on the surface of αβT cells. This γ4δ1 (GTM) TCR gene-modified αβT lymphocyte containing tumor-binding specific CDR3δ sequence (GTM) will have tumor-binding specificity similar to GTM, and can be used for tumor Adoptive immunotherapy.

[0094] In order to make the expression levels of exogenous γ4 chain and δ1 chain in αβT lymphocytes comparable, so as to successfully combine to form a complete γ4δ1 (GTM) TCR molecule, the present invention will complete the genes of γ4 chain and δ1 chain (CDR3δ sequence is GTM) The fragments were connected in series on a lentiviral vector, because the vector contained a CMV p...

Embodiment 2

[0096] Example 2: Recombinant lentiviral vector pCDH-δ1TCR-CMV-γ4TCR corresponding target protein γ4δ1 (GTM) TCR molecule expression detection

[0097] The recombinant lentiviral vector pCDH-δ1TCR-CMV-γ4TCR obtained in Example 1 was transferred into 293T cells, the expression of GFP was observed with a fluorescence microscope, the cells were collected, lysed, and the expression of δ1TCR in the whole cell lysate was detected by Western blotting. The result is as Figure 1E As shown, the mouse anti-human δ1 monoclonal antibody was used as the primary antibody, and the HRP enzyme-labeled goat anti-mouse antibody was used as the secondary antibody. Compared with the empty vector control, the δ1 TCR protein was well expressed in 293T cells. Furthermore, it was proved that γ4δ1(GTM) TCR molecules were expressed in 293T cells.

Embodiment 3

[0098] Example 3: Identification of lentivirus infection of αβT cells and expression of γ4δ1(GTM) TCR in αβT cells after infection

[0099] The recombinant lentiviral vector pCDH-δ1TCR-CMV-γ4TCR obtained in Example 1 was packaged as a recombinant lentivirus.

[0100]PBMCs from healthy people were isolated, αβT cells were sorted, and infected with the above-mentioned recombinant lentivirus in an environment stimulated and activated by a solid-phase anti-CD3 antibody. On the 7th day after infection, the cells were collected, and the expression of γ4δ1(GTM) TCR on their surface was detected by flow cytometry. like Figure 1F As shown, compared with the empty vector-infected control, 35.9% of αβT cells simultaneously expressed γ4δ1(GTM) TCR, that is, successfully obtained αβT cells expressing tumor-binding specific γδTCR gene modification, namely γ4δ1(GTM)-αβT cells.

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Abstract

The invention relates to a tumor binding specific gamma delta TCR gene modified alpha beta T cell and cancer suppression application thereof. The alpha beta T cell expresses the tumor binding specific gamma delta TCR; the tumor binding specific gamma delta TCR comprises a gamma 4 chain and a delta 1 chain; the amino acid sequence of the delta 1 chain CDR3 is shown as SEQ ID NO: 1. Experiment results prove that the cell has cytotoxicity for a plurality of tumor cells, and a novel method and a novel strategy are provided for adoptive immunotherapy of tumors.

Description

technical field [0001] The invention relates to the field of gene-modified lymphocyte adoptive immunotherapy. In particular, it relates to a tumor-binding specific γδTCR gene-modified αβT cell and its tumor-suppressing application. Background technique [0002] Immunotherapy of tumors is highly expected by people. Its principle is to artificially regulate the immune function status of tumor patients and regulate the balance between various links of anti-tumor immunity through the use of modern biotechnology, so as to control tumors or reduce the risk of treatment. The purpose of related toxic side effects. Among all the currently developed immunotherapeutic approaches, the adoptive immunotherapy of cells, through in vitro activation and expansion of autologous or allogeneic immune effector cells and then infused into patients, is considered to be the most potential immunotherapy strategy. [0003] In adoptive immunotherapy, natural cells commonly used for adoptive reinfusi...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/867A61K35/17A61P35/02A61P35/00
Inventor 何维何康霞陈慧胡愉张建民
Owner 北京佳德和细胞治疗技术有限公司
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