Method for culturing cd19car-inkt cells and use thereof

A cell and amino acid technology, applied in DNA/RNA fragments, tissue culture, genetically modified cells, etc., can solve problems such as limited variability

Active Publication Date: 2020-12-08
HRAIN BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

iNKT cells express constant TCRα chains (TCRVα14-Jα18 for mice, TCRVα24-Jα18 for humans), and the pairing of constant TCRα chains and TCRβ chains is diverse, but compared with the TCRβ chains of traditional MHC-restricted T cells, their limited variability

Method used

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  • Method for culturing cd19car-inkt cells and use thereof
  • Method for culturing cd19car-inkt cells and use thereof
  • Method for culturing cd19car-inkt cells and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0079] Example 1: Determination of CD19scFv-CD8α-41BB-CD3ζ gene sequence

[0080] From the NCBI website database, the gene sequence information of human CD8 transmembrane region, human 41BB 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.

[0081] Using overlapping PCR, the above sequences were sequentially connected according to anti-CD19 scFv, human CD8α hinge region gene, human CD8 transmembrane region gene, human 41BB intracellular region gene, and human CD3ζ intracellular region gene sequence, and different restriction enzymes were introduced at the junction of each sequence. site, forming a complete CD19-CAR gene sequence.

[0082] The nucleotide sequence of the CAR molecu...

Embodiment 2

[0088] Example 2: Retroviral packaging

[0089] 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.

[0090] 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, CaCl 2 250ul,H 2 O is 1ml and the total volume is 1.25ml; add HBS equal to the volume of the plasmid complex in another tube, and vortex for 20 seconds 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.

[0091] 3. Day 4: 48 hours after transfection, collect the supernatant and filter it with a 0.45um filter, ...

Embodiment 3

[0092] Example 3: Activation of iNKT cells

[0093] 1. After separating PBMC, use Miltenyi Biotec's iNKT sorting kit (Cat.130-091-221) to obtain iNKT cells, resuspend in iNKT medium [X-VIVO M 15 (LONZA) + 1% double antibody (GIBCO) + 1% GlutaMax (GIBCO) + 1% HEPES (GIBCO) + 2% N-acetyl-cysteine ​​(Meilun Bio) + 5% inactivated human AB serum ( GEMINI)+200IU / mlIL-2 (Beijing Shuanglu)].

[0094] 2. Take the PBMC irradiated with 40Gy γ-rays, centrifuge and resuspend the cells in iNKT medium, add 5 μg / ml αGalcer (Avanti), mix well, and put the cells at 37°C, 5% CO2 for culture Pretreatment in the box for 2h.

[0095] 3. After pretreatment, mix iNKT: γ-ray irradiated PBMC = 1:5, put it in a 37°C, 5% CO2 incubator for culture, add IL-2 every other day, the added concentration 200IU / ml.

[0096] 4. After 10 days, the iNKT cells were activated for the second time according to the same operation method.

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Abstract

The invention relates to a targeting CD19CAR-iNKT cell and application thereof. Specifically, the invention provides a polynucleotide sequence, and activation culture and application thereof. The polynucleotide sequence is selected from (1) a coding sequence containing, successively connected, a coding sequence of an anti-CD19 single-chain antibody, a coding sequence of the hinge region of human CD8alpha, a coding sequence of the transmembrane region of human CD8, a coding sequence of the intracellular region of human 41BB and a coding sequence of the intracellular region of human CD3zeta; and(2) complementary sequences of the polynucleotide sequence described in (1). The invention also provides related fusion proteins, vectors containing the coding sequences, and application of the fusion proteins, the coding sequences and the vectors. The invention also provides activation and culture methods and the application scope of iNKT cells.

Description

technical field [0001] The invention belongs to the field of chimeric antigen receptors, and specifically relates to targeting CD19CAR-iNKT cells and uses thereof. 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 responses, and significantly improve the survival of pa...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/62C12N15/867C12N5/10C12N7/01C07K19/00A61K35/17A61P35/00A61P35/02
CPCA61K35/17C07K14/7051C07K14/70517C07K14/70578C07K16/2896C07K2317/622C07K2319/02C12N7/00C12N15/86C12N2510/00C12N2740/10021C12N2740/10043
Inventor 黄飞何凤赵晓楠金涛王海鹰史子啸
Owner HRAIN BIOTECHNOLOGY CO LTD
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