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CD22 targeted chimeric antigen receptor and use thereof

A receptor and single-chain antibody technology, applied in the field of CD22-targeted chimeric antigen receptors, can solve problems beyond the treatment requirements, T cell attack, and high amplification

Active Publication Date: 2018-04-27
上海恒润达生生物制药有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this treatment has not been perfected, and T cells will go off target and attack other tissues, or expand too much, beyond the treatment needs

Method used

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  • CD22 targeted chimeric antigen receptor and use thereof
  • CD22 targeted chimeric antigen receptor and use thereof
  • CD22 targeted chimeric antigen receptor and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0088] Example 1: Determination of CD22-scFv-CD8α-CD28-41BB-CD3ζ gene sequence

[0089] The gene sequence information of human CD8α hinge region, human CD8α transmembrane region, 41BB intracellular region and human CD3ζ intracellular region was searched from the NCBI website database. The anti-CD22 single-chain antibody clone number is m971. These sequences are available on the website http: / / sg Codon optimization is performed on .idtdna.com / site to ensure that it is more suitable for expression in human cells without changing the encoded amino acid sequence.

[0090] Using overlapping PCR, the above sequences were sequentially connected according to anti-CD22 scFv, human CD8α hinge region gene, human CD8α transmembrane region gene, 41BB intracellular region gene, and human CD3ζ intracellular region gene sequence, and different enzyme cutting sites were introduced at the junction of each sequence Click to form the complete CD22CAR gene sequence information.

[0091] The nucle...

Embodiment 2

[0097] Example 2: Determination of CD22CAR-GMCSFR leader sequence-tEGFR gene sequence

[0098] The gene sequence information of the extracellular region of human EGFR was searched from the NCBI website database, and the sequence was codon-optimized on the website http: / / sg.idtdna.com / site to ensure that it is more suitable for human cell expression under the condition that the encoded amino acid sequence remains unchanged .

[0099] Using overlapping PCR, the above sequences were sequentially connected according to the CD22CAR, GMCSFR leader sequence, and tEGFR in Example 1, and different restriction sites were introduced at the junction of each sequence to form a complete CD22CAR-GMCSFR leader sequence-tEGFR gene sequence information.

[0100] The nucleotide sequence of the CAR molecule was double-digested with NotI (NEB) and EcoRI (NEB), connected and inserted into the NotI-EcoRI site of retrovirus MSCV (Addgene) by T4 ligase (NEB), and transformed into competent large intes...

Embodiment 3

[0106] Example 3: Retroviral packaging

[0107] 1. Day 1: 293T cells should be less than 20 passages, not overgrown. Take 0.6×10 6 Cells / ml were plated, and 10ml of DMEM medium was added to a 10cm dish, the cells were thoroughly mixed, and cultured overnight at 37°C.

[0108] 2. Day 2: 293T cell confluency reaches about 90% for transfection (usually about 14-18 hours after plating); prepare plasmid complexes, the amount of various plasmids is MSCV backbone 12.5ug, Gag-pol 10ug, VSVg 6.25 Ug, CaCl 2 250ul,H 2 O 1ml, 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 with PBS, and add pre-warmed fresh medium again.

[0109] 3. Day 4: 48 hours after transfection, collect the supernatant and filter it with a 0.45um filter, store in -80°C, and continue to add prehea...

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Abstract

The invention relates to a CD22 targeted chimeric antigen receptor and a use thereof, and concretely provides a polynucleotide sequence. The polynucleotide sequence is selected from (1) an anti-CD22 single chain antibody polynucleotide sequence, a human CD8-alpha hinge region coding sequence, a human CD8 transmembrane region coding sequence, a human 41 BB intracellular region coding sequence, a human CD3 intracellular region coding sequence, and a coding sequence of a fragment containing an extracellular domain III and an extracellular domain IV of optional EGFR, which are sequentially linked; and (2) a complementary sequence of the polynucleotide sequence (1). The invention also provides a related fusion protein, a vector containing the coding sequences, and a use of the fusion protein,the coding sequences and the vector.

Description

technical field [0001] The invention belongs to the field of chimeric antigen receptors, and in particular relates to CD22-targeting chimeric antigen receptors 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 ...

Claims

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Application Information

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IPC IPC(8): C12N15/62C12N15/867C12N7/01C12N5/10C07K19/00A61K35/17A61P35/00A61P35/02
CPCA61K35/17C07K14/7051C07K14/70517C07K14/70578C07K14/71C07K16/2896C07K2317/56C07K2317/622C07K2319/02C07K2319/33C07K2319/74C12N7/00C12N15/86C12N2510/00C12N2740/10021C12N2740/10043
Inventor 黄飞金涛王海鹰何凤史子啸
Owner 上海恒润达生生物制药有限公司
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