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Method for cultivating T memory stem cells

A cell, amino acid technology, applied in the field of genetically modified T cells, can solve the problems of lack of antigen persistence and increased activity

Inactive Publication Date: 2018-06-29
HRAIN BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Memory cells generally have several characteristics: 1. Premature expansion and activation; 2. Lack of antigen persistence; 3. Increased activity when exposed to antigen again

Method used

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  • Method for cultivating T memory stem cells
  • Method for cultivating T memory stem cells
  • Method for cultivating T memory stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0084] Example 1: Determination of mCD19scFv-CD8α-CD28-CD3ζ gene sequence

[0085] From the NCBI website database, the gene sequence information of human CD28 transmembrane region, human CD28 intracellular region and human CD3ζ intracellular region was searched. The anti-CD19 single-chain antibody clone number is FMC63. These sequences are available on the website http: / / Codon optimization is performed on sg.idtdna.com / site to ensure that it is more suitable for expression in human cells without changing the encoded amino acid sequence.

[0086] Using overlapping PCR, the above sequences were sequentially connected according to anti-CD19 scFv, human CD8α hinge region gene, human CD28 transmembrane region gene, human CD28 intracellular region gene, and human CD3ζ intracellular region gene sequence, and different restriction enzymes were introduced at the junction of each sequence. site to form complete mCD19-CAR gene sequence information.

[0087] The nucleotide sequence of t...

Embodiment 2

[0092] Example 2: Retroviral packaging

[0093] 1. On the first day, the 293T cells should be less than 20 passages and not overgrown. Plate at 0.6*10^6 cells / ml, add 10ml of DMEM medium to a 10cm dish, mix the cells well, and culture overnight at 37 degrees.

[0094] 2. On the second day, the confluence of 293T cells reaches about 90% for transfection (usually about 14-18 hours after plating); prepare plasmid complexes, the amount of various plasmids is 12.5ug for Retro backbone, 10ug for Gag-pol, and 10ug for VSVg 6.25ug, CaCl2 250ul, H2O 1ml, the total volume is 1.25ml; add HBS equal to the volume of the plasmid complex in another tube, and vortex for 20s while adding the plasmid complex. Gently add the mixture to the 293T dish along the side, incubate at 37°C for 4 hours, remove the medium, wash it with PBS, and add pre-warmed fresh medium again.

[0095] 3. Day 4: 48 hours after transfection, collect the supernatant and filter it with a 0.45um filter, store in -80 degre...

Embodiment 3

[0096] Example 3: Retrovirus infection of human T cells

[0097] 1. Use Ficcol separation medium (Tianjin Haoyang) to separate relatively pure CD3+ T cells, and use 5% AB serum (GEMINI) X-VIVO (LONZA) medium to adjust the cell density to 1×106 / mL. Inoculate the cells with 1ml / well into the anti-human 50ng / ml CD3 antibody (Beijing Tongli Haiyuan) and 50ng / ml CD28 antibody (Beijing Tongli Haiyuan), and then add 100IU / ml interleukin 2 (Beijing Tongli Haiyuan) Shuanglu), 10ng / ml interleukin 7 (Peprotech), 5ng / ml interleukin 15 (Peprotech), virus infection after stimulating culture for 48 hours;

[0098] 2. The next day after T cell activation culture, PBS diluted to a final concentration of 15 μg / ml Retronectin (Takara) coated non-ti issue treated culture plate, 250 μl per well of a 24-well plate. Protected from light, overnight at 4°C for later use.

[0099] 3. After T cell activation and culture for two days, two coated 24-well plates were taken out, the coating solution was d...

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Abstract

The invention relates to a method for increasing memory stem cell phenotypes by using culture medium cultured CD19 CART cells containing IL7 and IL-15 cell factors, and belongs to the field of biotechnology. Specifically, the used IL7 and IL-15 culture medium cultivated CD19CART cells are more easily to be differentiated into memory stem cell phenotypes, and IL7 and IL-15 preteatment used for T cells indicates that the memory T cell function and CAR-T cell anti-tumor activity can be improved.

Description

technical field [0001] The invention belongs to the fields of immunology and tumor treatment, and more specifically relates to a method for cultivating T cells and genetically modified T cells prepared by the method. Background technique [0002] Chimeric Antigen Receptor-T cell (CAR-T) T cells refer to T cells that can recognize specific target antigens in an unrestricted manner by MHC after genetic modification, and continuously activate and expand T cells. In 2012, the annual meeting of the International Society for Cell Therapy pointed out that biological immune cell therapy has become the fourth means of tumor treatment besides surgery, radiotherapy, and chemotherapy, and will become a must-have means for future tumor treatment. CAR-T cell reinfusion therapy is the most clear and effective form of immunotherapy in current tumor treatment. A large number of studies have shown that CAR-T cells can effectively recognize tumor antigens, induce specific anti-tumor immune re...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/867C12N5/10C12N7/01A61K35/17A61P35/00A61P35/02
CPCA61K35/17C07K14/7051C07K14/70517C07K14/70521C07K16/2803C07K2317/622C07K2319/02C07K2319/33C07K2319/75C12N5/0636C12N7/00C12N15/86C12N2501/2302C12N2501/2307C12N2501/2315C12N2501/515C12N2740/10021C12N2740/10043
Inventor 黄飞史子啸金涛王海鹰何凤
Owner HRAIN BIOTECHNOLOGY CO LTD
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