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Chimeric antigen receptor targeting to CD19-41BB-tEGFR, and application thereof

A receptor and single-chain antibody technology, applied in the NGF-receptor/TNF-receptor superfamily, for targeting specific cell fusion, polypeptides containing positioning/targeting motifs, etc., can solve problems beyond the treatment Need, T cell attack, high expansion, etc.

Active Publication Date: 2018-05-25
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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  • Chimeric antigen receptor targeting to CD19-41BB-tEGFR, and application thereof
  • Chimeric antigen receptor targeting to CD19-41BB-tEGFR, and application thereof
  • Chimeric antigen receptor targeting to CD19-41BB-tEGFR, and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0092] Example 1: Determination of CD19scFv-CD8-41BB-CD3ζ gene sequence

[0093] From the NCBI website database, the gene sequence information of the hinge region and transmembrane region of human CD8α, the intracellular region of human 41BB, and the intracellular region of human CD3ζ, the anti-CD19 single-chain antibody clone number is FMC63, and 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.

[0094] Using overlapping PCR, the above sequences were sequentially linked according to anti-CD19 scFv, human CD8α hinge region and transmembrane region, human 41BB intracellular region gene, and human CD3ζ intracellular region gene sequence, and different Restriction sites to form complete CD19-CAR gene sequence information.

[0095] The nucleotide sequence of the CAR molecule was double-digested with NotI (NEB)...

Embodiment 2

[0100] Example 2: Determination of CD19CAR-GMCSFR leader-tEGFR gene sequence

[0101] The human EGFR extracellular region gene sequence information 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 cells without changing the encoded amino acid sequence Express.

[0102] Using overlapping PCR, the above sequences were sequentially connected according to the CD19CAR, 2A, GMCSFR leader, and tEGFR in Example 1, and different restriction sites were introduced at the junctions of each sequence to form complete CD19CAR-tEGFR gene sequence information.

[0103] 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 intestine Bacillus (DH5α).

[0104] The recombinant plasmid was sent to Shanghai...

Embodiment 3

[0108] Embodiment 3: the construction of the viral vector comprising the nucleic acid sequence of CAR molecule

[0109] The nucleotide sequence of the CAR molecule prepared in Example 1 was double digested with NotI (NEB) and EcoRI (NEB), connected and inserted into the NotI-EcoRI site of the retroviral RV vector through T4 ligase (NEB), and transformed into After the competent E.coli (DH5α) was sequenced correctly, the plasmid was extracted and purified using the Qiagen company's plasmid purification kit, and the purified plasmid was transfected into 293T cells by the plasmid calcium phosphate method for retrovirus packaging experiments.

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PUM

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Abstract

The invention relates to a chimeric antigen receptor targeting to CD19-41BB-tEGFR, and application thereof. Specifically, the invention provides a polynucleotide sequence. The polynucleotide sequenceis selected from (1) a polynucleotide 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, a coding sequence of the hinge region of human CD8zeta and an optional coding sequence of an EGFR fragment containing an extracellular domain III and an extracellular domain IV; 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.

Description

technical field [0001] The invention belongs to the field of chimeric antigen receptors, and in particular relates to CD19-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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