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.
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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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