Nanobody-based chimeric receptor gene integration in Jurkat cells mediated by PhiC31 integrase |
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| Authors: | Farnoush Jafari Iri-Sofla Fatemeh Rahbarizadeh Davoud Ahmadvand Mohammad J. Rasaee |
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| Affiliation: | aDepartment of Medical Biotechnology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran;bCenter of Pharmaceutical Nanotechnology and Nanotoxicology, Department of Pharmaceutics and Analytical Chemistry, University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen Ø, Denmark |
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| Abstract: | The crucial role of T lymphocytes in anti-tumor immunity has led to the development of novel strategies that can target and activate T cells against tumor cells. Recombinant DNA technology has been used to generate non-MHC-restricted chimeric antigen receptors (CARs). Here, we constructed a panel of recombinant CAR that harbors the anti-MUC1 nanobody and the signaling and co-signaling moieties (CD3ζ/CD28) with different spacer regions derived from human IgG3 with one or two repeats of the hinge sequence or the hinge region of FcγRII. The PhiC31 integrase system was employed to investigate if the recombination efficiency could be recruited for high and stable expression of T cell chimeric receptor genes. The effect of nuclear localization signal (NLS) and two different promoters (CMV and CAG) on efficacy of PhiC31 integrase in human T cell lines was evaluated. The presence of integrase in combination with NLS, mediated up to 7.6 and 8.5 fold increases in CAR expression in ZCHN-attB and ZCHHN-attB cassette integrated T cells, respectively. Our results showed that highly efficient and stable transduction of the Jurkat cell line by PhiC31 integrase is a feasible modality for generating anti-cancer chimeric T cells for use in cancer immunotherapy. |
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| Keywords: | Abbreviations: attB, phage attachment site BSA, bovine serum albumin CAR, chimeric antigen receptor CH3&ndash CH2-hinge, sequences coding for CH3&ndash CH2-hinge regions of human IgG3 CH3&ndash CH2-hinge-hinge, sequences coding for CH3&ndash CH2-hinge-hinge regions of human IgG3 CD28, cDNA coding for the transmembrane and intracellular part of CD28 (153&ndash 220 aa) CD3ζ, cDNA coding for the intracellular domain of CD3ζ (52&ndash 164 aa) CTL, cytolytic T cells ELISA, enzyme-linked immunosorbent assay DAXX, death-associated protein FcγRII, class II of cell surface receptor for the Fc portion of IgG HCAb, heavy chain antibodies of camels IgG, immunoglobulin G LB, Luria&ndash Bertani MW, molecular weight NLS, nuclear localization signal PBS, phosphate buffered saline pCAG-Int (NLS), plasmid which expresses PhiC31 integrase (under the control of CAG promoter) that is fused at C-terminal with nuclear localization signal of SV40 T antigen pCMVInt, plasmid which expresses PhiC31 integrase under the control of the cytomegalovirus (CMV) immediate-early promoter pCMV-Int (NLS), plasmid which expresses PhiC31 integrase (under the control of CMV promoter) that is fused at C-terminal with nuclear localization signal of SV40 T antigen pZCFN, CD3ζ/CD28/FcγRII/nanobody cassette inserted in pCDNA3.1/Hygro(+) plasmid pZCHHN, CD3ζ/CD28/CH3&ndash CH2-hinge-hinge/nanobody cassette inserted in pCDNA3.1/Hygro(+) plasmid pZCHN, CD3ζ/CD28/CH3&ndash CH2-hinge/nanobody cassette inserted in pCDNA3.1/Hygro(+) plasmid scFv, single-chain variable fragment TAA, tumor-associated antigen VHH, variable domain from a camel heavy chain antibody. |
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