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BCMA and CD19-targeted duplex-specific chimeric antigen receptor and application thereof

A chimeric antigen receptor, bispecific technology, applied in the application of anti-tumor therapy, the construction of chimeric antigen receptor T cell technology, can solve the problems of reduced CD19 expression and easy escape from immune mechanisms, etc.

Active Publication Date: 2019-03-19
GUANGZHOU BIO GENE TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

CN 104788573A discloses a chimeric antigen receptor hCD19scFv-CD8α-CD28-CD3ζ and its use. After a CAR-T cell reinfusion of CD19 in this patent, the expression level of CD19 will decrease, and it is easy to escape immunity mechanism

Method used

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  • BCMA and CD19-targeted duplex-specific chimeric antigen receptor and application thereof
  • BCMA and CD19-targeted duplex-specific chimeric antigen receptor and application thereof
  • BCMA and CD19-targeted duplex-specific chimeric antigen receptor and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0070] Example 1: Design of chimeric antigen receptor

[0071] This example constructed anti-BCMA and anti-CD19 bispecific chimeric antigen receptors (B(G)19 and 19(G)B), anti-BCMA chimeric antigen receptors and anti-CD19 chimeric antigen receptors , The sequence diagram is as figure 1 As shown, the chimeric antigen receptor includes a CD8α signal peptide sequence (Leader), a bispecific antibody sequence that specifically binds to BCMA and CD19 antigens, an anti-BCMA single domain antibody and an anti-CD19 single domain antibody, CD8α The hinge region (Hinge) and transmembrane region sequence (Transmembrane), 4-1BB costimulatory domain sequence and CD3ζ signaling domain sequence, the specific partial sequences are as follows:

[0072] CD8α signal peptide (leader) amino acid sequence (SEQ ID NO.11): MALPVTALLLPLALLLHAARP;

[0073] CD8α signal peptide (leader) nucleotide sequence (SEQ ID NO.12):

[0074] ATGGCACTGCCAGTGACAGCCCTGCTGCTGCCACTGGCCCTGCTGCTGCACGCAGCACGCCCT;

[0075] The amino...

Embodiment 2

[0090] Example 2: Construction of anti-BCMA chimeric antigen receptor expression vector

[0091] (1) Complete gene synthesis of B(G)19CAR, 19(G)B CAR, BCMA CAR, and CD19CAR sequences. Use EcoRI and BamHI to digest the fully synthesized CAR and empty vector. After digestion in 37℃ water bath for 30 minutes , Use 1.5% agarose gel for DNA electrophoresis, and then use Tiangen's agarose gel kit for purification and recovery;

[0092] (2) Connection between pLVX-EF1-MCS vector and CAR gene fragment:

[0093] The connection system is as follows:

[0094]

[0095]

[0096] After ligation at 22°C for 1 hour, the ligation product was directly transformed into Stbl3 E. coli competent cells. 200 μl of the transformed product was coated on an ampicillin-resistant LB plate. The LB plate was cultured upside down in an incubator at 37°C overnight. Three single clones were randomly selected the next morning for colony PCR identification, and positive clones were sent for sequencing.

[0097] The elem...

Embodiment 3

[0098] Example 3: Lentivirus packaging

[0099] The lentiviral expression vectors in the examples were separately packaged with a four-plasmid system. The specific steps are as follows:

[0100] (1) The four-plasmid system expresses the gag / pol, Rev, VSV-G required for lentiviral vector packaging and the artificial chimeric antigen receptor composed of the engineered stable single-chain antibody of the present invention: the four plasmids are transiently transfected into 293T Cells, the total mass is 10μg;

[0101] (2) Add the above plasmid to a certain volume of serum-free DMEM, mix well and leave it for 15 minutes, add the above mixture to the T75 culture flask with 293T cells, mix gently, and incubate at 37℃, 5 %CO 2 Culture in a cell incubator for 6 hours;

[0102] (3) After 6 hours, replace the fresh medium, continue the culture, and add 10 mM sodium butyrate solution. After 72 hours, collect the culture supernatant of the lentivirus for purification detection.

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Abstract

The invention relates to a BCMA and CD19-targeted duplex-specific chimeric antigen receptor and application thereof. The chimeric antigen receptor comprises an extracellular structural domain, a membrane spanning structural domain and at least one intracellular structural domain which can be bound with an antigen, wherein the extracellular structural domain comprises an anti-BCMA antigen structural domain and an anti-CD-19 antigen structural domain. The chimeric antigen receptor has relatively small clinic side effects and relatively high safety, a solid tumor can be effectively shrunk, and the treatment effect of tumors can be effectively improved.

Description

Technical field [0001] The present invention relates to the field of cellular immunotherapy for tumors, in particular to a bispecific chimeric antigen receptor targeting BCMA and CD19 and its application, specifically a chimeric antigen receptor T based on specific targets BCMA and CD19 (CAR-T) cell technology construction method and its application in anti-tumor therapy. Background technique [0002] At present, T cells modified by chimeric antigen receptor (CAR) have achieved gratifying results in clinical trials for the treatment of B-cell malignancies, bringing CAR technology to the treatment of relapsed and refractory multiple myeloma (MM) The dawn. Multiple myeloma is a malignant disease that originates from plasma cells in the bone marrow, and plasma cells are cells that develop to the final functional stage of B lymphocytes. [0003] BCMA (CD269) is expressed in mature B cells and plasma cells, and is also widely expressed in MM. The ADC drug (GSK2857916) and dual-target...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/867C12N5/10C12N7/01A61K35/17A61P35/00A61P35/02
CPCA61K35/17A61P35/00A61P35/02C07K14/7051C07K16/2803C07K16/2878C07K2319/02C07K2319/33C12N7/00C12N15/86C12N2510/00C12N2740/15021C12N2740/15043
Inventor 李光超郭锦涛丁雯曾剑华罗敏莫文俊
Owner GUANGZHOU BIO GENE TECH CO LTD
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