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Sleeping Beauty (SB) transposon, a type of nonviral integrative vectors, provides an alternative to modify primary T cells. Creative biolabs has developed SB transposon CAR vector pSBCAR1 CEA (MFE23) h(28BBζ), which is constructed for the engineering of T cells to target human CEA. The T cells are genetically modified through transduction with a nonviral vector expressing scFv of anti-CEA antibody linked to a CD28 transmembrane domain/ endodomain and CD137 (4-1BB), CD3-zeta signaling domains. And the vector product was designed for the treatment of Pancreatic cancer.
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CAR Construction :
Fig.1 Immunoreactivity of purified MFE-23::CPG2 confirmed by ELISA: CEA reactive MFE-23::CPG2 detected with anti-CPG2 before (open circle) and after (closed circle) 125I-radiolabeling. No signal was obtained when CEA-coated wells were probed with anti-CPG2 after incubating with a mixture of (unfused) MFE-23 and Bhatia, J., Sharma, S. K., Chester, K. A., Pedley, R. B., Boden, R. W., Read, D. A., ... & Begent, R. H. (2000). Catalytic activity of an in vivo tumor targeted anti‐CEA scFv:: carboxypeptidase G2 fusion protein. International journal of cancer, 85(4), 571-577. |
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CAR Construction :
Fig.2 Flow cytometric analysis of binding of secreted two-chain αCD3xαCEA diabodies to the surface of HeLa cell. The numbers in the right corners of each histogram indicate the mean fluorescence intensity (MFI). Mølgaard, K., Compte, M., Nuñez-Prado, N., Harwood, S. L., Sanz, L., & Alvarez-Vallina, L. (2017). Balanced secretion of anti-CEA× anti-CD3 diabody chains using the 2A self-cleaving peptide maximizes diabody assembly and tumor-specific cytotoxicity. Gene Therapy, 24(4), 208-214. |
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CAR Construction :
Fig.3 CEA ELISA of the CPGz::MFE-23 SCFV fusion protein. Negative controls: chemically coupled SBlO anti-hCG F(ab')2-CPG2 and E. co/i TGI cell extract carrying pMTL20. L, denotes (Gly,Ser), linker. Michael, N. P., Chester, K. A., Melton, R. G., Robson, L., Nicholas, W., Boden, J. A., ... & Minton, N. P. (1996). In vitro and in vivo characterisation of a recombinant carboxypeptidase G2:: anti-CEA scFv fusion protein. Immunotechnology, 2(1), 47-57. |
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