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BCMA specific chimeric antigen receptor T cell and application thereof

A chimeric antigen receptor, cell technology, applied in application, anti-receptor/cell surface antigen/cell surface determinant immunoglobulin, hybrid peptide, etc., can solve tissue and organ damage, CAR-T recognition error attack Normal cells, clinical side effects, etc.

Active Publication Date: 2019-05-14
广州因明生物医药科技股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In view of the limited number of cases at present, it is impossible to draw a systematic efficacy conclusion
And the results of existing cases have shown that some patients have more serious clinical side effects after reinfusion of CAR-T cells. What's more, some CAR-T recognition errors cause them to attack normal cells, which is the so-called "off-target effect".
So far, studies have confirmed that CAR-T cells with strong affinity and killing ability to the target antigen can also attack normal tissues while clearing tumors, causing damage to tissues and organs. In 2010, a case of colorectal cancer with liver cancer was reported. A patient with lung metastases died from off-target effects after receiving human epidermal growth factor receptor 2 CAR-T therapy

Method used

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  • BCMA specific chimeric antigen receptor T cell and application thereof
  • BCMA specific chimeric antigen receptor T cell and application thereof
  • BCMA specific chimeric antigen receptor T cell and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Example 1: Determination of anti-BCMA-CD8a-41BB-CD3ζ gene sequence

[0062] The gene sequence of the anti-human BCMA single-chain antibody is derived from the hybridoma cells of BCMA-immunized mice. The gene sequence information of human CD8α signal peptide region, hinge region and transmembrane region, human 4-1BB intracellular region and human CD3ζ intracellular region was searched from the NCBI website database. The above sequence was codon-optimized on the website http: / / www.jcat.de / to ensure that it is more suitable for human gene expression under the condition that the encoded amino acid sequence remains unchanged. A Kozak sequence and restriction site were introduced at the amino-terminal of the signal peptide gene sequence, and the nucleotide sequences of each segment were sent to Shanghai Sangon Biotechnology Co., Ltd. for synthesis. See SEQUENCE LISTING (SEQ ID NO: 1-22) for the sequence information of each gene. The structure of the successfully constructed...

Embodiment 2

[0063] Example 2: Construction of anti-BCMA-CD8a-41BB-CD3ζ lentiviral vector

[0064] Using overlapping PCR, the above-mentioned synthetic sequences were sequentially linked according to the sequence of signal peptide, anti-BCMAscFv, human CD8α hinge region, human CD8α transmembrane region, human 4-1BB intracellular region, and human CD3ζ intracellular region to form a complete anti- BCMA-CAR gene sequence information to obtain the CAR molecule (hereinafter referred to as "anti-BCMA-CAR"). The nucleotide sequence of anti-BCMA-CAR is shown in SEQ ID NO: 21 or SEQ ID NO: 22, and the encoded amino acid sequence is shown in SEQ ID NO: 19 or SEQ ID NO: 20.

[0065] The nucleotide sequence of anti-BCMA-CAR was double-digested with EcoR I and BamH I, and then inserted into the modified lentiviral vector pLVX-EF1a-GFP-N1 (Addgene) by T4 DNA ligase with EcoR I and BamH I. site, transformed into competent DH5α Escherichia coli. The obtained recombinant plasmid was sent to Shanghai San...

Embodiment 3

[0066] Example 3: Packaging and titer determination of anti-BCMA-CAR lentivirus

[0067] Extract the lentiviral packaging vectors pMD2.G, psPAX2 and anti-BCMA-CAR lentiviral vectors with the operating instructions in the endotoxin-free plasmid extraction kit (Tiangen Bio) and co-transfect them into 293T cells. After transfection, 48h and The cell supernatant was collected at 72 hours, centrifuged at 400 rcf for 10 minutes, and the cells and cell debris in the supernatant were removed. Filter the supernatant with a 0.45 μm filter membrane, aliquot and freeze for future use.

[0068] According to the expected virus titer (MOI), the virus supernatant was serially diluted and then infected 293T cells. The positive rate of GFP was detected by fluorescence microscope. According to the formula, the titers of pLVX-CAR and pLVX-empty virus stocks were calculated to be about 2 respectively. ×10 6 TU / mL and 5×10 6 TU / mL. After the concentrated solution is concentrated, the titer can ...

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Abstract

The invention discloses a specific chimeric antigen receptor (anti-BCMA scFv-CD8a-41BB-CD3 zeta) for human BCMA and application thereof. The chimeric antigen receptor is formed by a single-chain-antibody anti-BCMA scFv, a hinge region, a transmembrane region and an intracellular region in series. The chimeric antigen receptor is used for modifying human T lymphocyte, and the modified T lymphocyteis used for prevention and treatment of surface BCMA positive tumor and preparing of antitumor medicines.

Description

technical field [0001] The present invention relates to the field of tumor cell immunotherapy, in particular to a chimeric antigen receptor anti-BCMAscFv-CD8a-41BB-CD3ζ obtained by genetic engineering technology and T lymphocytes modified by the chimeric antigen receptor, the modified T lymphocytes can specifically recognize and kill tumor cells expressing BCMA on the surface, and the tumor cells are preferably multiple myeloma cells. Background technique [0002] Intact antibodies have a larger relative molecular mass. Single-chain antibody (single chain antibody fragment, scFv) is a small-molecule antibody formed by connecting only the V region (VH) of the antibody heavy chain and the V region (VL) of the light chain through a synthetic linker. The relative molecular mass of single-chain antibody is small, easy to construct and express, and its immunogenicity is weak, so it is not easy to cause hypersensitivity and rejection. Single-chain antibodies are currently one of ...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/867A61P35/00
CPCA61K48/00A61P35/00C07K16/28C07K19/00C12N15/62C12N15/867
Inventor 陈毓胡金芳李燕张丽琴龙飞
Owner 广州因明生物医药科技股份有限公司
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