OCTS technology-based pancreatic cancer and malignant mesothelioma CAR-T therapeutic vector, construction method therefor, and use thereof

An OCTS-based CAR-T vector for targeting malignant tumors expressing mesothelin, PDL1, or both of the mesothelin and the PDL1, comprising:

• a lentiviral skeleton plasmid, a human EF1α promoter, a first nucleotide sequence of a one CAR with two ScFvs (OCTS) chimeric receptor structural domain, and a second nucleotide sequence encoding a programmed cell death receptor ligand 1 (PDL1) single-chain antibody;
• wherein
• the lentiviral skeleton plasmid comprises:
• an AmpR sequence containing an ampicillin resistance gene for a vast expansion of a target bacterial strain, wherein the AmpR sequence is shown in SEQ ID NO:
• 1;
• a prokaryotic replicon pUC Ori sequence for plasmid replication, wherein the prokaryotic replicon pUC Ori sequence is shown in SEQ ID NO:
• 2;
• a viral replicator SV40 Ori sequence for enhancing replication in eukaryotic cells, wherein the viral replicator SV40 Ori sequence is shown in SEQ ID NO:
• 3;
• a lentiviral packaging cis-element for lentiviral packaging;
• a third nucleotide sequence encoding ZsGreen1 green fluorescent protein, wherein the third nucleotide sequence is shown in SEQ ID NO:
• 11,
• an internal ribosome entry site (IRES) ribosome binding sequence, wherein the IRES ribosome binding sequence is shown in SEQ ID NO:
• 12;
• and
• an eWPRE enhanced marmot hepatitis B virus post-transcriptional controlling element for enhancing transgenic expression efficiency, wherein the eWPRE enhanced marmot hepatitis B virus post-transcriptional controlling element is shown in SEQ ID NO:
• 13;
• a sequence of the human EF1αpromoter is shown in SEQ ID NO:
• 14;
• the first nucleotide sequence of the OCTS chimeric receptor structural domain comprises:
• a fourth nucleotide sequence encoding a cluster of differentiation 8 (CD8) leader chimeric receptor signal peptide, as shown in SEQ ID NO:
• 15,
• a fifth nucleotide sequence encoding a first PDL1 single-chain antibody light chain, as shown in SEQ ID NO:
• 16,
• a sixth nucleotide sequence encoding a second PDL1 single-chain antibody heavy chain, as shown in SEQ ID NO:
• 17,
• a seventh nucleotide sequence encoding a first mesothelin single-chain antibody light chain, as shown in SEQ ID NO:
• 18,
• an eighth nucleotide sequence encoding a second mesothelin single-chain antibody heavy chain, as shown in SEQ ID NO:
• 19,
• a ninth nucleotide sequence encoding an antibody Inner-Linker, as shown in SEQ ID NO:
• 20,
• a tenth nucleotide sequence encoding a single-chain antibody Inter-Linker, as shown in SEQ ID NO:
• 21,
• an eleventh nucleotide sequence encoding a CD8 hinge chimeric receptor linker, as shown in SEQ ID NO:
• 22,
• a twelfth nucleotide sequence encoding a CD8 transmembrane chimeric receptor transmembrane domain, as shown in SEQ ID NO:
• 23,
• a thirteenth nucleotide sequence of a chimeric receptor T cell activation domain of T cell receptor (TCR), as shown in SEQ ID NO:
• 26, and
• a fourteenth nucleotide sequence of a chimeric receptor co-stimulator domain;
• wherein the chimeric receptor co-stimulator domain is at least one selected from the group consisting of 4-1BB, ICOS, CD27, OX40 (CD134), CD28, MYD88, IL1R1, CD70, TNFRSF19L, TNFRSF27, TNFRSF1OD, TNFRSF13B, and TNFRSF18.