Chimeric antigen receptor targeting B cell maturation antigens and uses thereof

A single-chain antibody, fusion protein technology, applied in the direction of targeting specific cell fusion, receptors/cell surface antigens/cell surface determinants, polypeptides containing localization/targeting motifs, etc. Short survival period cannot stimulate anti-tumor effect, poor persistence and other problems

Active Publication Date: 2018-05-08
HRAIN BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the first-generation CAR can see some specific cytotoxicity, when it was summarized in clinical trials in 2006, it was found that the curative effect was not satisfactory.
The reason is that the first-generation CAR-T cells will be exhausted soon in the patient's body, and their persistence is so poor that the CAR-T cells have already died before they have had time to contact a large number of tumor cells. CAR-T cells can stimulate anti-tumor cytotoxic effects, but the secretion of cytokines is relatively small, but their short survival period in vivo cannot stimulate long-lasting anti-tumor effects〔Chimeric NKG2D-modified T cells inhibit systemic T-cell lymphoma growth in a manner involving multiple cytokines and cytotoxic pathways, Cancer Res 2007, 67(22): 11029-11036〕

Method used

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  • Chimeric antigen receptor targeting B cell maturation antigens and uses thereof
  • Chimeric antigen receptor targeting B cell maturation antigens and uses thereof
  • Chimeric antigen receptor targeting B cell maturation antigens and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0078] Example 1: Determination of mBCMA(J22.9) scFv-CD8α-41BB-CD3ζ gene sequence

[0079]1.1 The human CD8α hinge region, human CD8α transmembrane region, human 41BB intracellular region and human CD3ζ intracellular region gene sequence information were searched from the NCBI website database. The anti-BCMA (J22.9) single-chain antibody clone number is J22.9, these sequences are on the website http: / / sg.idtdna.com / site Codon optimization is carried out to ensure that it is more suitable for expression in human cells under the condition that the encoded amino acid sequence remains unchanged.

[0080] See SEQENCE LISTING (SEQUENCE ID NO.1) for each amino acid and gene sequence information.

[0081] The above sequences were sequentially linked according to the anti-BCMA (J22.9) scFv, human CD8α hinge region gene, human CD8α transmembrane region gene, human 41BB intracellular region gene, and human CD3ζ intracellular region gene sequence, and the enzyme cutting site to form co...

Embodiment 2

[0087] Embodiment 2: the construction of the viral vector comprising the nucleic acid sequence of CAR molecule

[0088] The nucleotide sequence of the CAR molecule prepared in Example 1 was double digested with NotI (NEB) and EcoRI (NEB), inserted into the NotI-EcoRI site of the retroviral pRetro 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

[0089] Example 3: Retroviral packaging

[0090] 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;

[0091] 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 degrees for 4 hours, remove the medium, wash with PBS, and re-add the preheated fresh medium;

[0092] 3. Day 4: 48 hours after transfection, collect the supernatant and filter it with a 0.45um fil...

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PUM

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Abstract

The present invention relates to chimeric antigen receptor targeting BCMA(J22.9), and uses thereof, and particularly provides a polynucleotide sequence, which is selected from (1) a polynucleotide sequence containing the coding sequence of an anti-BCMA single chain antibody, the coding sequence of a human CD8 alpha hinge region, the coding sequence of a human CD8 transmembrane region, and the coding sequence of a human 41BB intracellular region, wherein the coding sequences are sequentially linked, and (2) a sequence complementary to the polynucleotide sequence (1). The present invention further provides a related fusion protein, a vector containing the coding sequence, and uses of the fusion protein, the coding sequences, and the vector.

Description

technical field [0001] The invention belongs to the field of chimeric antigen receptors, in particular to BCMA-targeted chimeric antigen receptors and applications thereof. Background technique [0002] Multiple myeloma is a malignant plasma cell disease, manifested as malignant clonal hyperplasia of bone marrow plasma cells, secreting monoclonal immunoglobulin or its fragments (M protein), resulting in bone, kidney and other related target organs or tissue damage, common clinical Manifested as bone pain, anemia, renal insufficiency, infection, etc. [Multiple myeloma.N Engl J Med,2011.364(11):p.1046-60.]. Currently, multiple myeloma is the second most malignant tumor of the hematological system, accounting for 10% of hematological malignancies, and it mostly occurs in men. Siegel, R., et al., Cancer statistics, 2014. CA Cancer J Clin, 2014.64(1): p.9-29.]. [0003] B-cell maturation antigen (BCMA), also known as CD269, consists of 184 amino acid residues, its intracellular...

Claims

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Application Information

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IPC IPC(8): C12N15/62C07K19/00C12N15/867C12N7/00A61K35/17A61P35/00
CPCA61K35/17C07K14/7051C07K14/70517C07K14/70596C07K2319/02C07K2319/03C07K2319/33C07K2319/74C12N15/86C12N2740/10043
Inventor 黄飞金涛王海鹰何凤史子啸
Owner HRAIN BIOTECHNOLOGY CO LTD
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