Preparing method and application of BTLA<-/->T lymphocytes resistant to tumor immunosuppression environment

A lymphocyte and immunosuppressive technology, applied in the intersection of life science and medicine, can solve the problems of tumor growth and immune escape, failure to inhibit tumor growth, and insufficient number of immune cells migrating into tumor sites.

Inactive Publication Date: 2019-01-04
北京中诚华科生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The resistance mechanisms of immunotherapy include the following: (1) the inhibitory microenvironment or the lack of antigenic stimulation/co-stimulation of immune cells, especially T cells, may

Method used

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  • Preparing method and application of BTLA&lt;-/-&gt;T lymphocytes resistant to tumor immunosuppression environment
  • Preparing method and application of BTLA&lt;-/-&gt;T lymphocytes resistant to tumor immunosuppression environment
  • Preparing method and application of BTLA&lt;-/-&gt;T lymphocytes resistant to tumor immunosuppression environment

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0016] Embodiment 1, construction and verification of BTLA-sgRNA-Cas9 plasmid;

[0017] 1. Synthesize two oligo sequences and anneal to form double-stranded DNA;

[0018] 1.1 Synthetic Oligo sequence:

[0019] BTLA-sgRNA1: 5'-GAGAGTTCATTTTGCTTTCC-3' and its complementary sequence;

[0020] BTLA-sgRNA2: 5'-GTGACTTGGTGCAAGCTCAA-3' and its complementary sequence;

[0021] BTLA-sgRNA3: 5'-GACTTAACTCCTCACACACATA-3' and its complementary sequence;

[0022] BTLA-sgRNA4: 5'-GGTCAGTTTACCTACCCCAG-3' and its complementary sequence;

[0023] BTLA-sgRNA5: 5'-GGAAAAACTGCTCAAGTAGA-3' and its complementary sequence;

[0024] 1.2 Anneal the synthesized Olig DNA to form DNA.

[0025] 2. Ligate the double-stranded DNA into the vector;

[0026] The Cas9 vector was linearized, and the Olig DNA and the vector were ligated using T4DNA ligase.

[0027] 3. Transformed DH5α cells, screened positive cells, extracted plasmids, and sequenced to verify the sequence;

[0028] The connected BTLA-sgRN...

Embodiment 2

[0035] Example 2, BTLA - / - Preparation of T lymphocytes;

[0036] 1. Collect peripheral blood from healthy people. After testing for no infectious disease contamination, dilute it with an equal volume of normal saline, use ficoll lymphocyte separation medium to separate mononuclear cells from the diluted peripheral blood, and separate mononuclear cells into magnetic beads Sorted to obtain CD3 + T lymphocytes;

[0037] 2. Use liposomes to transfer BTLA-sgRNA-Cas9 plasmid into T lymphocytes, and change the medium every other day;

[0038] 3. Put cells into X-VIVO15 medium containing 2000IU / mL IFN-γ, culture at 37°C, 5% carbon dioxide for 24h;

[0039] 4. Then add 20μg / mL CD3 monoclonal antibody, 500IU / mL IL-2, 50ng / mL IL-1α, 100ng / mL IL-4, 2000IU / mL GM-CSF, 5μg / mL Puromycin;

[0040] 5. After culturing for 3-5 days, add 500IU / mL IL-2 fresh medium, and keep the cell concentration at 1-2x10 6 / mL;

[0041] 6. Count the cells every 2-3 days, add fresh medium containing 500IU / ...

Embodiment 3

[0045] Example 3, BTLA - / - Determination of the killing activity of T lymphocytes to tumor cells;

[0046] 1. Add BTLA - / - T lymphocytes were co-cultured with A549 cells expressing HVEM;

[0047] 2. After 24 hours of co-culture, A549 cells were harvested, stained with AnnexinV-FITC and 7-AAD, and the apoptosis rate of A549 was determined by flow cytometry ( Figure 4 ).

[0048] The result is as Figure 4 As shown, relative to wild-type T lymphocytes, BTLA - / - T lymphocytes can cause increased apoptosis of A549 expressing HVEM.

[0049] Figure 1~4 showed that the BTLA gene of T lymphocytes can be knocked out using Cas9 technology, BTLA - / - T lymphocytes are able to resist the suppressive effect of HVEM.

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Abstract

The invention discloses a preparing method of BTLA<-/->T lymphocytes resistant to the tumor immunosuppression environment. The preparing method includes the steps that T lymphocytes are obtained through separation from peripheral blood of a tumor patient, BTLA is subjected to gene knockout in vitro with the CRISPR-Cas9 technology, then the obtained T lymphocytes are put into a serum-free medium and cultured, IFN-gamma, a CD3 monoclonal antibody, IL-2, IL-1alpha, IL-4, GM-CSF and Puromycin are added, the obtained T lymphocytes continue to be cultured for 10 days to 15 days, then centrifugationis carried out, and the BTLA<-/->T lymphocytes are obtained. The preparing method of the BTLA<-/->T lymphocytes is ingenious in design, the BTLA<-/->T lymphocytes prepared with the method have the characteristic resistant to the tumor immunosuppression environment, and have the better tumor killing effect, the clinical effect is improved, and a novel anti-tumor and tumor treatment mean is provided.

Description

technical field [0001] The present invention relates to the interdisciplinary technical field of life science and medicine, in particular to gene-edited immune cells, specifically to a BTLA resistant to tumor immunosuppressive environment - / - Preparation method and application of T lymphocytes. Background technique [0002] The immune system of T lymphocytes can recognize tumor antigens and kill tumor cells in vitro. T lymphocytes themselves should be the "fighters" against tumors in the human body, but due to various reasons, immune tolerance and escape can not be effectively killed in vivo. tumor cells. [0003] An established tumor is a complex tissue composed not only of tumor cells but also of stromal cells, inflammatory cells, vasculature and extracellular matrix, all of which together define the tumor microenvironment. The resistance mechanisms of immunotherapy include the following: (1) the inhibitory microenvironment or the lack of antigenic stimulation / co-stimula...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/113C12N15/90A61K35/17A61P35/00
CPCA61K35/17A61P35/00C07K14/70521C12N5/0636C12N15/113C12N15/907C12N2310/10C12N2310/20
Inventor 贾远航赵伟
Owner 北京中诚华科生物科技有限公司
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