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A kind of tcr-/pd-1- double negative T cell and its construction method

A PD-1 and double-negative technology, applied in the field of biomedicine, can solve the problems of increasing the burden on patients, the ineffectiveness of CART therapy, and low activity, and achieve the effects of reducing treatment costs, large-scale preparation in advance, and reducing immune damage

Active Publication Date: 2019-06-14
GUANGZHOU ANJIE BIOMEDICAL TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition to the side effects such as cytokine storm caused by CART, there are still three major problems: first, for some advanced patients with low lymphocyte count or poor quality, the chance of CART treatment is lost; second, the efficacy of CART therapy in solid tumors Still not significant, probably due to the influence of immunosuppressive checkpoint signaling pathways, resulting in poor survival rate and low activity of immune cells in tumor tissue; finally, since CART is an individualized treatment, it is expensive and increases the burden on patients
However, there are still some problems in the existing adoptive cell immunotherapy or immunodetection blockade using PD1 antibody to treat tumors: (1) For some advanced patients with low lymphocyte count or poor quality, they lose the opportunity of CART treatment; ( 2) The efficacy of CART therapy in solid tumors is still not significant due to the influence of immunosuppressive checkpoint signaling pathways; (3) Both adoptive cell immunotherapy and immune checkpoint therapy are individualized treatments, which are expensive; (4) ) PD1 antibody causes autoimmune damage due to systemic administration

Method used

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  • A kind of tcr-/pd-1- double negative T cell and its construction method
  • A kind of tcr-/pd-1- double negative T cell and its construction method
  • A kind of tcr-/pd-1- double negative T cell and its construction method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] Example 1 Construction of TCR based on CRISPR / Cas9 system - / PD-1 - sgRNA for double negative T cells

[0067] PD-1 is an inhibitory receptor on the surface of T cells. TCR (T cell receptor) is a molecular structure that T cells specifically recognize and bind to antigen peptide-MHC molecules. It usually exists in the form of a complex with CD3 molecules on T cells. Cell surface; the TCR of most T cells consists of alpha and beta peptide chains.

[0068] To construct TCR - / PD-1 - Double-negative T cells, in order to destroy the reading frame of the target gene as much as possible, the purpose of the present invention is to obtain the target gene TCR-α with PD1 sgRNA with knockdown activity. The present invention obtains 15 pairs of targeted genes TCR-α sgRNA, and 18 targeted genes PD-1 sgRNA.

[0069] Among them, the target TCR-α The sgRNA sequence of the gene is selected from any one of SEQ ID NO: 1-15; the reverse complementary DNA sequence of the sequenc...

Embodiment 2

[0072] Example 2 Construction of TCR based on CRISPR / Cas9 system - / PD-1 - DNA oligonucleotides for double negative T cells

[0073] According to the sgRNA designed in Example 1 above, synthesize the corresponding DNA oligonucleotide, add CACC to the 5' of the forward oligonucleotide, and add AAAC to the 5' of the reverse oligonucleotide; the sgRNA sequence of the above TCR-α The sequences of the forward oligonucleotides corresponding to SEQ ID NO: 1-4 are SEQ ID NO: 67, 69, 71, 73 respectively, and the sequences of the corresponding reverse oligonucleotides are SEQ ID NO: 68, 70, 72, 74; the sequences of the forward oligonucleotides corresponding to the sgRNA sequences of PD-1 SEQ ID NO: 16~19 are respectively SEQ ID NO: 75, 77, 79, 81, and the corresponding reverse oligonucleotides The nucleotide sequences are SEQ ID NO: 76, 78, 80, 82, respectively.

[0074] The above-mentioned synthetic forward oligonucleotide sequence and reverse oligonucleotide sequence are paired and...

Embodiment 3

[0077] Example 3 Construction method of CRISPR / Cas9-TCR-sgRNA plasmid

[0078] 1) The px601-AAV-CMV plasmid (map as figure 1 Shown, hereinafter are abbreviated as px601) for enzyme digestion to obtain a linearized px601 plasmid; the enzyme digestion system is as follows:

[0079] 1 μg px601 plasmid;

[0080] 2μl 10×FastDigest® buffer;

[0081] 1 μl FastDigest® BsaI (Thermo Scientific);

[0082] Replenish water to 20 μl, incubate at 37°C for 1 hour, then cut the gel and recover.

[0083] 2) connect

[0084] Ligate the double-stranded DNA oligonucleotides (TCR-DNA Oligos-1~4) obtained in Implementation 2 with the linearized px601 respectively, and the connection system is as follows:

[0085] 2.5 μl px601 plasmid;

[0086] 2.5 μl annealed double-stranded sgRNA;

[0087] 5 μl Solution I (Takara);

[0088] Incubate at 16°C for 1 hour.

[0089] 3) Transformation: Transform the above ligation product into Escherichia coli DH5α competent cells, incubate at 37°C for 16-18h, ...

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Abstract

The invention discloses a TCR- / PD-1-double negative T cell and its construction method. The present invention activates isolated peripheral blood mononuclear cells into T cells, knocks out TCR and PD-1 through CRISPR / Cas9 gene editing technology, and sorts out TCR- / PD-1-double negative T cells through magnetic beads. The TCR- / PD-1-double negative T cells prepared by the present invention can be used for adoptive cell immunotherapy of tumors, including DC-CIK, CTL, TIL, CART, etc. The TCR- / PD-1-double-negative T cells of the present invention can be derived from other healthy donors of the same type. Therefore, the application of the cells in adoptive cell therapy not only gets rid of the limitations of the patient's current disease status, but can also be used on a large scale in advance. preparation, reducing treatment costs.

Description

technical field [0001] The invention belongs to the field of biomedicine, and in particular relates to a TCR- / PD-1-double-negative T cell and a construction method thereof. Background technique [0002] With the development of immunology, tumor immunotherapy has made great progress. Tumor immunotherapy stimulates or mobilizes the patient's own immune system and enhances the anti-tumor immunity of the tumor microenvironment, thereby controlling and killing tumor cells. It is considered to be the fourth major tumor treatment technology after surgery, radiotherapy, and chemotherapy. In the past 20 years, tumor immunotherapy represented by adoptive cell therapy (ACT) and immune checkpoint therapy (immune checkpoint therapy) has made breakthrough progress, showing good application prospects, marking a new era of cancer treatment. The beginning of the era. [0003] Adoptive cellular immunotherapy (ACT) is to infuse activated and lethal immune cells to tumor patients, so that the...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/85C12N5/10A61K35/17A61P35/00A61P31/04A61P31/12A61P31/18A61P31/20
CPCC07K14/70503C07K14/70521C12N5/0636C12N15/1138C12N15/85C12N2310/10C12N2510/00A61K39/4632A61K39/4611A61K39/464411A61K39/464838A61K39/4648C12N5/10C12N15/113A61P31/04A61P31/20A61P35/00A61P31/18A61P31/12
Inventor 周超安鸿卢有德周玲巫春红彭涛尹海滨
Owner GUANGZHOU ANJIE BIOMEDICAL TECH CO LTD
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