Chimeric antigen acceptor of target BCMA (B cell maturation antigen) and application of chimeric antigen acceptor

A chimeric antigen receptor, targeting technology, applied in the direction of targeting specific cell fusion, peptides containing localization/targeting motifs, applications, etc. Short half-life and other problems, to achieve the effects of small molecular weight and immunogenicity, high-efficiency specific treatment, and long retention time

Active Publication Date: 2019-07-23
上海科棋药业科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are currently no drugs targeting BCMA on the market, but there are a few therapies targeting BCMA to treat multiple myeloma through antibody-coupled drugs, CAR-T therapy, etc. The application in the field of tumors has achieved certain results. Data from European and American clinical trials show that the existing BCMA chimeric antigen receptor T (carT) technology has an objective cure rate (objective response, OBB) of about 50% in the treatment of multiple myeloma, but there are still The clinical shortcomings are as follows: the effective rate of treatment is lower than the typical CD19carT70-90% complete cure rate, and the effective rate

Method used

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  • Chimeric antigen acceptor of target BCMA (B cell maturation antigen) and application of chimeric antigen acceptor
  • Chimeric antigen acceptor of target BCMA (B cell maturation antigen) and application of chimeric antigen acceptor
  • Chimeric antigen acceptor of target BCMA (B cell maturation antigen) and application of chimeric antigen acceptor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] This example provides a method for constructing the above-mentioned CAR molecule into the lentiviral vector pHAGE.

[0051] According to the construction of BCMA CAR molecules in this example, according to the different antigen recognition regions and signal regions of CAR molecules, they can be divided into three types: B1-BBZ, B65-BBZ, and 11D5-3-28Z: the intracellular CAR molecules used in B1-BBZ The signal region is 4-1BB-CD3ζ, and the antigen recognition region adopts B1; the intracellular signal region used in B65-BBZ is 4-1BB-CD3ζ, and the antigen recognition region adopts B65; while the intracellular signal region adopted in 11D5-3-28Z The inner signal region is CD28-CD3ζ, and the antigen recognition region adopts 11D5-3; its molecular structure pattern is as follows figure 1 shown. The above CAR molecule was constructed into the lentiviral vector pHAGE by conventional molecular cloning methods.

[0052] Construction of the B1-BBZ vector: First, obtain the cor...

Embodiment 2

[0056] This example is the construction of BCMA CAR-T cells. The CAR molecules are packaged into lentiviruses, transduced into T cells, and the transfection efficiency is measured.

[0057] Packaging of CAR lentivirus: first, the plasmids prepared in Example 1 were respectively extracted using the QIAGEN endotoxin-free large-scale plasmid extraction kit.

[0058] The constructed lentiviral plasmids pHAGE-B1-BBZ, pHAGE-B65-BBZ, pHAGE-11D5-3-28Z and the lentiviral system auxiliary packaging plasmids pMD2.G and pSPAX2. Spread 1.8×10E7293T into T175 culture flasks one day before transfection. 1 hour before transfection, the medium of 293T cells was replaced with 30ml of serum-free medium. The plasmid was co-transfected into 293T cells using the calcium phosphate precipitation method, and the cell culture medium was replaced with 60ml of complete medium DMEM+10%FBS 24 hours after transfection. The cell supernatant was harvested 48 hours after transfection, and 60ml of fresh compl...

Embodiment 3

[0061] This example is for the construction of BCMA target cells and the determination of the activity of BCMA CAR-T cells.

[0062] Identification and construction of BCMA CAR-T target cells: Use flow cytometry to detect the expression of BCMA on cell lines such as U266 and A549, such as image 3 As shown, U266 cell line highly expresses BCMA antigen, while A549 hardly expresses BCMA antigen. A lentiviral vector encoding BCMA gene was used to screen with puromycin to construct A549 cell line A549-BCMA which highly expressed BCMA antigen. BCMA expression levels in the A549-BCMA cell line see image 3 . according to image 3 The results showed that the A549-BCMA and U266 cell lines were positive for BCMA, while the A549 cell line was negative for BCMA.

[0063] IFN-γ and IL2 secretion experiment: In a 96-well plate, three kinds of CART cells B1-BBZ, B65-BBZ and 11D5-3-28Z and four effector cells of control T cells and target cells U266 were used at a 5:1 effect-to-target ra...

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Abstract

The invention relates to a chimeric antigen acceptor of a target BCMA (B cell maturation antigen). The chimeric antigen acceptor comprises an extracellular identification region, a hinge region, a transmembrane region and an intracellular signal region, wherein the extracellular identification region has a BCMA resisting nanometer antibody sequence, and the BCMA resisting nanometer antibody sequence is a heavy-chain variable region sequence combining with BCMA and from alpacas. More specifically, the BCMA resisting nanometer antibody sequence is BCMA monoclonal antibody B1 or B65, the amino acid sequence of the B1 is as shown in SEQID NO.1, and the amino acid sequence of the B65 is as shown in SEQID NO.2. According to the chimeric antigen acceptor disclosed by the invention, compared witha traditional mouse-derived SCFV or humanized SCFV, the nanometer antibody from the alpacas is used, and has small molecule quantity and immunogenicity, the possibility that CAR-T cells prepared on the base of the nanometer antibody produce HAMA effects in vivo is smaller, the remaining time of the CAR-T cells in vivo can be longer, the CAR-T cells pass through BCMA protein on the surfaces of target tumor cells and activate signal channels at the downstream part of T cells, the capacity for killing tumor cells having BCMA target points is given to the T cells, and BCMA positive blood tumor canbe efficiently and specifically treated.

Description

technical field [0001] The invention relates to the technical field of cellular immunotherapy, in particular to a chimeric antigen receptor targeting BCMA and its application. Background technique [0002] B cell maturation antigen (BCMA) is an antigen expressed on plasma cells, plasmablasts and bone marrow plasma cells, but not on B cells or hematopoietic stem cells. The expression of BCMA is associated with many cancers, autoimmune diseases and infectious diseases. Cancers with increased BCMA expression include some blood cancers such as multiple myeloma, Hodgkin's and non-Hodgkin's lymphomas, various leukemias and glioblastoma, among others. This limited expression cell type makes BCMA a possible target for the treatment of hematological tumors to develop therapies such as monoclonal antibodies, antibody-drug conjugates, bispecific antibodies, and CAR-T. There are currently no drugs targeting BCMA on the market, but there are a few therapies targeting BCMA to treat mult...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N5/10C12N15/867A61K35/17A61P35/00A61P35/02
CPCA61K35/17A61P35/00A61P35/02C07K14/7051C07K16/2878C07K2319/03C07K2319/33C12N15/86C12N2740/15043
Inventor 郝瑞栋李彦涛刘根桃张大挺易桥勇红丽吴国祥
Owner 上海科棋药业科技有限公司
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