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Chimeric antigen receptor targeting novel BCMA (B-cell maturation antigen) and application thereof

A single-chain antibody, fusion protein technology, applied in the direction of targeting specific cell fusion, peptides containing localization/targeting motifs, antibody mimics/scaffolds, etc. Unsatisfactory efficacy and poor durability

Pending Publication Date: 2019-02-12
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 novel BCMA (B-cell maturation antigen) and application thereof
  • Chimeric antigen receptor targeting novel BCMA (B-cell maturation antigen) and application thereof
  • Chimeric antigen receptor targeting novel BCMA (B-cell maturation antigen) and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0076] Example 1: Determination of BCMA-scFv-CD8α-CD28-41BB-CD3ζ-mbIL15 gene sequence

[0077] The gene sequence information of human CD8α hinge region, human CD8α transmembrane region, 41BB intracellular region, human CD3ζ and mbIL15 intracellular region was searched from the NCBI website database. The anti-BCMA single-chain antibody clone number is C11D5.3. These sequences are available on the website http Codon optimization is performed on : / / sg.idtdna.com / site to ensure that it is more suitable for human cell expression without changing the encoded amino acid sequence.

[0078] Using overlapping PCR, the above sequences were sequentially connected according to anti-BCMA scFv, human CD8α hinge region gene, human CD8α transmembrane region gene, 41BB intracellular region gene, human CD3ζ intracellular region, and mbIL15 gene sequence, and introduced at the junction of each sequence Different enzyme cutting sites form complete BCMA-CAR gene sequence information.

[0079] The ...

Embodiment 2

[0085] Example 2: Retroviral Packaging

[0086] 1. Day 1: 293T cells should be less than 20 passages, not overgrown. Take 0.6×10 6 For cell / ml plating, add 10ml of DMEM medium to a 10cm dish, mix the cells well, and culture overnight at 37°C;

[0087] 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.

[0088] 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 preheated fresh DMEM medium. ...

Embodiment 3

[0089] Example 3: Retrovirus infection of human T cells

[0090] 1. Use Ficcol separation medium (Tianjin Haoyang) to separate and obtain relatively pure CD3+ T cells, and use X-VIVO (LONZA) medium containing 5% AB serum 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) Heron), the virus infection that prepares with embodiment 3 after stimulating culture for 48 hours;

[0091] 2. The next day after T cell activation culture, PBS was diluted to a final concentration of 15 μg / ml and Retronectin (Takara) was used to coat a non-tissue-treated culture plate, 250 μl per well of a 24-well plate. Protected from light, overnight at 4°C for later use.

[0092] 3. After T cell activation and culture for two days, two coated 24-well plates were taken out, the coating solution was disc...

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Abstract

The invention relates to a chimeric antigen receptor targeting a novel BCMA (B-cell maturation antigen) and application thereof. Specifically, the invention provides a polynucleotide sequence, which is chosen from: (1) a polynucleotide sequence of polynucleotide codding sequences containing the codding sequence of the anti-BCMA single-chain antibody, the coding sequence of the human CD8Alpha hingeregion, the coding sequence of the human CD8 transmembrane region, the coding sequence of the human 41BB intracellular region, the coding sequence of the human CD3Zeta intracellular region and the coding sequence of the mbIL15 structure connected in sequence; and (2) complementary sequences of the polynucleotide sequences (1). The invention further provides a related fusion protein, a vector containing the coding sequence and application of the fusion protein, the coding sequence 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): C07K19/00C12N15/62C12N15/867C12N5/10A61K35/17A61P35/00
CPCA61P35/00A61K35/17C12N5/0636C12N15/86C07K14/7051C07K16/2878C12N2740/10043C12N2510/00C07K2319/74C07K2319/33C07K2319/03C07K2319/02
Inventor 刘雅容邹雪梅
Owner HRAIN BIOTECHNOLOGY CO LTD
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