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Targeted ROR1 chimeric antigen receptor and application thereof

A single-chain antibody and fusion protein technology, applied in the field of cell therapy, can solve problems such as rare expression

Active Publication Date: 2018-08-24
HRAIN BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

ROR1 is expressed during embryonic development and in some leukemias, but is rarely expressed in normal adult tissues

Method used

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  • Targeted ROR1 chimeric antigen receptor and application thereof
  • Targeted ROR1 chimeric antigen receptor and application thereof
  • Targeted ROR1 chimeric antigen receptor and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] Example 1: Determination of the ROR1 scFv-CD8-41BB-CD3ζ gene sequence

[0067] 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-ROR1 single-chain antibody clone number is R12, 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.

[0068] Using overlapping PCR, the above sequences were sequentially linked according to anti-ROR1 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 ROR1-CAR gene sequence information.

[0069] The nucleotide sequence of the CAR molecule was double-digested with NotI (N...

Embodiment 2

[0074] Embodiment 2: the construction of the viral vector comprising the nucleic acid sequence of CAR molecule

[0075] 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.

Embodiment 3

[0076] Example 3: Retroviral Packaging

[0077] 1. On the first day, the 293T cells should be less than 20 passages and not overgrown. Plate with 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;

[0078] 2. On the second day, the 293T cell confluency reaches about 90% for transfection (usually about 14-18 hours after plating); prepare the plasmid complex, the amount of various plasmids is 12.5ug for RV-ROR1-BBz, and 12.5ug for Gag-pol 10ug, VSVg is 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 degrees for 4 hours, remove the medium, wash with PBS, and re-add the preheated fresh medium;

[0079] 3. Day 4: 48 hours after transfection, collect the supernatant and filter it with a 0.45um filter, ...

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Abstract

The invention relates to a targeted rabbit-source ROR1 chimeric antigen receptor and application thereof, and particularly provides polynucleotide sequences. The polynucleotide sequences are selectedfrom (1) polynucleotide sequences containing a coding sequence of an anti-ROR1 single-chain antibody, a coding sequence of a human CD8alpha hinge region, a coding sequence of a human CD8 transmembraneregion, a coding sequence of a human 41BB intracellular region and a coding sequence of a human CD3zeta intracellular region, wherein the coding sequences are sequentially connected; (2) complementary sequences of the polynucleotide sequences (1). The invention further provides relevant fusion protein, a carrier containing the coding sequences and application of the fusion protein, the coding sequences and the carrier. Prepared ROR1(R12)-BBz CAR-T cells have a strong killing function on specific tumor cells, and IFNgamma secretion is high.

Description

technical field [0001] The invention belongs to the field of cell therapy, and specifically relates to a chimeric antigen receptor targeting rabbit-derived ROR1 and its application. 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 ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/62C12N15/867C12N7/01C12N5/10C07K19/00A61K35/17A61P35/00A61P35/02
CPCA61P35/00A61P35/02A61K35/17C12N7/00C12N15/86C07K14/7051C07K16/2803C07K2317/622C07K2319/02C07K2319/03C07K2319/33C12N2740/10043C12N2740/10021C12N2510/02
Inventor 刘雅容金涛
Owner HRAIN BIOTECHNOLOGY CO LTD
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