Dendritic cells expressing chimeric antigen receptors and uses thereof
A technology of chimeric antigen receptors and dendritic cells, applied to receptors/cell surface antigens/cell surface determinants, anti-receptors/cell surface antigens/cell surface determinants immunoglobulins, and the introduction of foreign genetic material Modified cells and other directions can solve problems affecting the efficacy of cellular immunity and off-target toxicity
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Embodiment 1
[0073] 1. Determination of mesoCAR gene sequence
[0074] 1.1 The human GM-CSF signal peptide gene, human CD8α hinge region gene, human CD40 transmembrane region and Intracellular signal region gene sequence. Anti-mesothelin single-chain antibody (anti-SS1 scFv) gene sequence.
[0075] 1.2 The above gene sequences were sequentially connected according to the human GM-CSF signal peptide gene, anti-SS1scFv, human CD8α hinge region gene, human CD40 transmembrane region and intracellular signal region sequence to form the final complete GM-CSF-SS1scFv-CD8α- CD40 (mesoCAR) gene sequence SEQ ID NO.4.
[0076] 2. Construction of pCDH-mesoCAR expression plasmid
[0077] 2.1 Whole gene synthesis:
[0078] The complete mesoCAR gene sequence was synthesized by Shanghai Sangon, and restriction sites Xba I / EcoR I were added at both ends.
[0079] 2.2 Cloning the above sequence into pCDH-CMV-MCS-EF1-copGFP-T2A-puro (pCDH for short) lentiviral expression vector to obtain the expression ...
Embodiment 2
[0125] 1. Determination of EGFRvIIICAR gene sequence
[0126] 1.1 Obtain the human CD8α signal peptide gene, human CD8α hinge region gene, human CD40 transmembrane region and human CD40 signal domain from the GenBank database on the website of the National Library of Medicine (http: / / www.ncbi.nlm.nih.gov / entrez) gene sequence. Human CD8α signal peptide gene, EGFRvIII scFv light chain gene, linker region (Gly4-Ser) 3 , EGFRvIII scFv heavy chain gene, human CD8α hinge region gene, human CD40 transmembrane region and human CD40 signal domain gene sequence were connected to form the final complete EGFRvIIICAR gene sequence (SEQ ID NO.5) information.
[0127] 2. Construction of EGFRvIIICAR expression plasmid
[0128] 2.1 Whole gene synthesis:
[0129] The complete EGFRvIIICAR sequence was synthesized by Shanghai Sangon Biotechnology Co., Ltd., and an XbaI restriction site was added to its 5' end, and an EcoR I restriction site was added to the 3' end.
[0130] 2.2 Amplify the E...
Embodiment 3
[0169] 1. Construction of MOv19CAR expression plasmid
[0170] 1.1 The human CD8α signal peptide gene, human CD8α hinge region gene, human CD40 transmembrane region and intracellular Signal region gene sequence. MOv19 single chain antibody (anti-alpha folate receptor scFv) gene sequence.
[0171] 1.2 The above gene sequence was sequentially formed according to the human CD8α signal peptide gene, MOv19scFv, human CD8α hinge region gene, human CD40 transmembrane region and CD40 intracellular signal region sequence to form a complete MOv19CAR gene sequence (SEQ ID NO.6); in sequence 5 An EcoRI restriction site was introduced at the 'end of the sequence, and a BamHI restriction site was introduced at the 3' end of the sequence, which was handed over to Shanghai Sangon Biotechnology Co., Ltd. for synthesis. After the plasmid was synthesized, the MOv19-CAR was digested from the pUC57 plasmid, and at the same time the enzyme Cut pCDH empty plasmid, the enzyme cutting system is as f...
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