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Chimeric antigen receptor targeting BCMA and application thereof

A chimeric antigen receptor, targeting technology, applied in the field of biomedicine, can solve problems such as recurrence or ineffectiveness, achieve strong killing effect, and prevent off-target effects.

Pending Publication Date: 2022-07-26
INST OF HEMATOLOGY & BLOOD DISEASES HOSPITAL CHINESE ACADEMY OF MEDICAL SCI & PEKING UNION MEDICAL COLLEGE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] One aspect of the present invention is to solve the problem of relapse or ineffectiveness due to the escape mechanism of MM during the application of traditional chemotherapy, hematopoietic stem cell transplantation and targeted drug therapy in the prior art, and provides a chimeric drug targeting BCMA Antigen receptors and their applications

Method used

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  • Chimeric antigen receptor targeting BCMA and application thereof
  • Chimeric antigen receptor targeting BCMA and application thereof
  • Chimeric antigen receptor targeting BCMA and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0102] Example 1: Preparation of antigen recognition region BCMA scFv in Chimeric Antigen Receptor

[0103] A. Commercial synthesis of the BCMA scFv based on the sequence.

[0104] Cloning of B.BCMA scFv

[0105] 1. Extract the total RNA of mouse anti-human BCMA monoclonal antibody hybridoma cell line: at 5×10 6 Add 1 ml of RNAiso Plus (Takara) to the cells and mix by pipetting. Add 200 μl of chloroform, turn upside down and vortex to mix. 4°C, 12000rpm, centrifugation for 5 minutes. Pipette the supernatant into a 1.5ml EP tube, add the same volume of isopropanol, and mix by gently inverting. 4°C, 12000 rpm, centrifugation for 15 minutes. Pre-chill 75% ethanol at 4°C to precipitate RNA, and dissolve total RNA in 50 μl DEPC water.

[0106] 2. Synthesize the first strand of cDNA by reverse transcription: prepare a PCR reaction system (20 μl) as follows: Oligo d(T)15Primers: 2 μl; M-MLV (200u / μl): 1 μl; dNTP (each 2.5 mM): 1 μl; DTT ( 0.1M): 2 μl; First strand buffer (5×):...

Embodiment 2

[0164] Example 2: Construction of Chimeric Antigen Receptor Vectors

[0165] 1. The plasmid containing the CD8α-4-1BB-CD3ζ fragment was digested with Nhe I and EcoR I endonucleases to obtain the CD8α-4-1BB-CD3ζ fragment, the amino acid sequence of which is shown in SEQ ID NO.18. The plasmid containing the CD8α-4-1BB-CD3ζ fragment can be prepared by any suitable method in the prior art.

[0166] 2. The BCMAscFv (mLH) and BCMAscFv (mHL) fragments obtained in Example 1 are connected with the target vector, and the constructed BCMA-VL-VH-CD8α-4-1BB-CD3ζCAR and BCMA-VH-VL-CD8α- The 4-1BB-CD3ζCAR destination vector was identified by restriction enzyme digestion with NheI and NotI. The result is as image 3 As shown, the results of enzyme digestion indicated that the positive clones contained the target band and were correctly identified by sequencing. The vector diagram is as Figure 4 shown.

[0167] 3. Commercially synthesize the humanized sequence after computer simulation, ...

Embodiment 3

[0168] Example 3: Preparation of Chimeric Antigen Receptor BCMA scFv-CD8α-4-1BB-CD3ζ Lentiviral Modified T Cells

[0169]1. The BCMAscFv-CD8α-4-1BB-CD3ζ (mLH, mHL, hLH, hHL) expression plasmid and packaging plasmids PRSV-Rev, pMDlg-PRRE, pMD were extracted with EndoFree Plasmid Maxi Kit (QIAGEN). 2G. The four plasmids were transfected with PEI transfection reagent (polyscience company) according to the ratio of 12.2:4.11:8.75:3.5 (see the instruction manual of PEI transfection reagent for specific methods). The fresh culture medium was replaced 12 hours after transfection, and the virus supernatant was collected 24 hours and 48 hours later, centrifuged at 4°C, 3000 rpm for 15 minutes, filtered through a 0.45 μm filter, and then ultracentrifuged at 50,000 g at 4°C for 2.5 hours. Concentrate 10 times and store at -80°C.

[0170] 2. Preparation of T cells: Take 10 ml of fresh healthy human peripheral blood, and use RosetteSep T cell enrichment Cocktail (Stemcell Company) and Fi...

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Abstract

The invention discloses a nucleic acid molecule for coding a chimeric antigen receptor targeting BCMA, the chimeric antigen receptor comprises an extracellular region, a transmembrane region and an intracellular signal transduction region, the extracellular region coded by the chimeric antigen receptor comprises a BCMA binding structural domain, the BCMA binding structural domain is a single-chain antibody fragment specifically binding to BCMA, and the intracellular signal transduction region is a single-chain antibody fragment specifically binding to BCMA. The single-chain antibody fragment comprises a light chain variable region amino acid sequence as shown in SEQ ID No. 1 and a heavy chain variable region amino acid sequence as shown in SEQ ID No. 2. The T cell modified by the chimeric antigen receptor has a very strong killing effect on multiple myeloma cells expressing BCMA, almost has no killing effect on cells not expressing BCMA, and effectively prevents an off-target effect. The chimeric antigen receptor disclosed by the invention can be used for treatment of BCMA + multiple myeloma cell hematologic tumors and combined treatment of CAR-T cells such as CD38, CD138 and CD19.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to a chimeric antigen receptor targeting BCMA and its application. Background technique [0002] Multiple myeloma (MM) is a clonal proliferative disease of malignant plasma cells of the bone marrow, resulting in suppressed bone marrow hematopoiesis and osteolytic disease. Although myeloma can be treated with traditional chemotherapy, hematopoietic stem cell transplantation and targeted drugs, relapsed and refractory MM cannot be completely cured. [0003] Cellular immunotherapy is becoming a promising new strategy. Chimeric antigen receptor-modified T cells (CAR-T cells) have MHC-independent tumor antigen-specific recognition, proliferation and killing capabilities. The chimeric antigen receptor is composed of an extracellular antigen-binding region, that is, a single-chain antibody (scFv) formed by linking the light chain variable region (VL) and heavy chain variable region (V...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/62C12N15/85C12N5/10A61K39/00A61P35/00
CPCC07K16/2878C07K14/7051C12N15/85A61K39/001117A61P35/00C07K2317/56C07K2319/02C07K2319/03C12N2800/107A61K2039/80
Inventor 王建祥王敏陈兆琪熊冬生卢杨饶青徐颖茜邢海燕
Owner INST OF HEMATOLOGY & BLOOD DISEASES HOSPITAL CHINESE ACADEMY OF MEDICAL SCI & PEKING UNION MEDICAL COLLEGE
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