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Chimeric antigen receptor targeting CD19, method for co-expression of variable region of anti-PD1 antibody and application of chimeric antigen receptor

A technology of variable regions and antigens, which can be used to target specific cell fusion, polypeptides containing positioning/targeting motifs, receptors/cell surface antigens/cell surface determinants, etc., which can solve the problem of discounting anti-tumor ability And other issues

Active Publication Date: 2018-11-23
HRAIN BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

CAR-T cells have strong targeting and high specificity, and can proliferate rapidly and massively after being stimulated by tumor antigens, which may be limited by inhibitory signals, thus greatly reducing their anti-tumor ability

Method used

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  • Chimeric antigen receptor targeting CD19, method for co-expression of variable region of anti-PD1 antibody and application of chimeric antigen receptor
  • Chimeric antigen receptor targeting CD19, method for co-expression of variable region of anti-PD1 antibody and application of chimeric antigen receptor
  • Chimeric antigen receptor targeting CD19, method for co-expression of variable region of anti-PD1 antibody and application of chimeric antigen receptor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0085] Example 1: Determination of CD19scFv-CD8α-CD28-CD3ζ-P2A-aPD1 scFv gene sequence

[0086] Searched from the NCBI website database to find anti-CD19 antibody light chain and heavy chain variable region, human CD8α hinge region, human CD28 transmembrane region and intracellular region, human CD3ζ intracellular region, anti-human PD1 antibody heavy chain and light Chain variable region gene sequence information, these sequences are codon-optimized on the website http: / / sg.idtdna.com / CodonOpt to ensure that the encoded amino acid sequence is more suitable for expression in human cells.

[0087] Using overlapping PCR, the above sequences were sequenced according to anti-CD19 scFv, human CD8α hinge region gene, human CD28 transmembrane region gene, human CD28 intracellular region gene, human CD3ζ intracellular region gene, anti-PD1 antibody heavy chain and light chain variable region genes The sequences were connected, and different enzyme cutting sites were introduced at the ...

Embodiment 2

[0094] Example 2: Retroviral packaging

[0095] 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.

[0096] 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.

[0097]3. Day 4: 48 hours after transfection, collect the supernatant and filter it with a 0.45um filter, s...

Embodiment 3

[0098] Example 3: Retrovirus infection of human T cells

[0099] 1. Use Ficcol separation medium (Tianjin Haoyang) to separate and obtain relatively pure CD3+ T cells, and use 5% AB serum (GEMINI) X-VIVO (LONZA) medium to adjust the cell density to 1×10 6 / 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), virus infection after stimulation and culture for 48 hours.

[0100] 2. The next day after T cell activation culture, Retronectin (Takara) diluted in PBS to a final concentration of 15 μg / ml was coated on a 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.

[0101] 3. After T cell activation and culture for two days, two coated 24-well plates were taken out, the coating solution was discarded, and HBSS containing 2% BSA was...

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Abstract

The invention discloses a chimeric antigen receptor CD19 scFV-CD8hinge-CD28TM-CD28-CD3zeta joint aPD1 scFV CAR-T cell and use thereof. Specifically, the invention provides a polynucleotide sequence selected from: (1) a coding sequence containing sequentially linked anti-CD19 single chain antibodies, a coding sequence of a human CD8alpha hinge region, a coding sequence of a human CD28 transmembraneregion, a coding sequence of a human CD28 intracellular region, a coding sequence of a human CD3zeta intracellular region, a coding sequence of a P2A polypeptide, a human IL2 signal peptide coding sequence, and an anti-human PD1 monoclonal antibody coding sequence; and (2) a complementary sequence of the polynucleotide sequence in (1). The invention further provides a relevant fusion protein, a vector containing the coding sequences, and use of the fusion protein, the coding sequences and the vector. A prepared CD19-28z-aPD1 CAR-T cell has a very strong killing function on specific tumor cells. The prepared CD19-28z-aPD1 CAR-T cell has the function of secreting a PD1 antibody, and can block the site of PD1-PDL1 to prevent CAR-T cells from being inhibited by PD1.

Description

technical field [0001] The invention belongs to the field of chimeric antigen receptors, and specifically relates to CD19 CAR-targeted cells expressing anti-PD1 antibody variable region and its use. 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 signi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/867C12N7/01C12N5/10A61P35/00A61P35/02
CPCA61K35/17C12N5/0636C12N7/00C12N15/86C07K14/7051C07K16/2803C07K16/2818C07K2317/622C07K2317/56C07K2319/02C07K2319/33C12N2501/2302C12N2501/515C12N2510/00C12N2740/10043C12N2740/10021
Inventor 黄飞金涛王海鹰何凤史子啸
Owner HRAIN BIOTECHNOLOGY CO LTD
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