Malignant glioma CAR-T therapeutic vector based on OCTS technology, and construction method and application thereof

An OCTS-based CAR-T vector for treating malignant glioma, comprising:

• a lentiviral skeleton plasmid, a human EF1α promoter, a sequence encoding an OCTS chimeric receptor structural domain, and a sequence encoding a PDL1 single-chain antibody;
• wherein
• the OCTS is one CAR with two ScFvs;
• the lentiviral skeleton plasmid comprises:
• an AmpR sequence containing an ampicillin resistance gene for a vast expansion of a target bacterial strain, as shown in SEQ ID NO:1;
• a prokaryotic replicon pUC Ori sequence for plasmid replication, as shown in SEQ ID NO:2;
• a viral replicator SV40 Ori sequence for enhancing replication in eukaryotic cells, as shown in SEQ ID NO:3;
• a lentiviral packaging cis-element for lentiviral packaging;
• a sequence coding a ZsGreen1 green fluorescent protein, as shown in SEQ ID NO:11;
• an IRES ribosome binding sequence, as shown in SEQ ID NO:12;
• and
• an eWPRE enhanced marmot hepatitis B virus post-transcriptional controlling element for enhancing transgenic expression efficiency, as shown in SEQ ID NO:13;
• a sequence of the human EF1α promoter is shown in SEQ ID NO:14;
• the sequence encoding OCTS chimeric receptor structural domain comprises:
• a sequence encoding a CD8 leader chimeric receptor signal peptide, as shown in SEQ ID NO:15,
• a sequence encoding a PDL1 single-chain antibody light chain VL, wherein the sequence encoding the PDL1 single-chain antibody light chain VL is humanized, a sequence encoding a PDL1 single-chain antibody heavy chain VH, wherein the sequence encoding the PDL1 single-chain antibody heavy chain VH is humanized,
• a sequence encoding a EGFRvIII single-chain antibody light chain VL, wherein the sequence encoding the EGFRvIII single-chain antibody light chain VL is humanized,
• a sequence encoding a EGFRvIII single-chain antibody heavy chain VH, wherein the sequence encoding the EGFRvIII single-chain antibody heavy chain VH is humanized,
• a sequence encoding an antibody Inner-Linker, as shown in SEQ ID NO:20,
• a sequence encoding a single-chain antibody Inter-Linker, as shown in SEQ ID NO:21,
• a sequence encoding a CD8 hinge chimeric receptor linker, as shown in SEQ ID NO:22,
• a sequence encoding a CD8 transmembrane chimeric receptor transmembrane domain, as shown in SEQ ID NO:23,
• a sequence encoding a chimeric receptor T cell activation domain of a T cell receptor (TCR), as shown in SEQ ID NO:26, and
• a sequence encoding a chimeric receptor co-stimulator domain wherein, the sequence encoding the chimeric receptor co-stimulator domain is a combination of CD 28 chimeric receptor co-stimulator as shown in SEQ ID NO:24 and CD 34 chimeric receptor co-stimulator as shown in SEQ ID NO:25.