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Manufacture of T cells using the Sleeping Beauty system to enforce expression of a CD19-specific chimeric antigen receptor

Abstract

T cells can be reprogrammed to redirect specificity to tumor-associated antigens (TAAs) through the enforced expression of chimeric antigen receptors (CARs). The prototypical CAR is a single-chain molecule that docks with TAA expressed on the cell surface and, in contrast to the T-cell receptor complex, recognizes target cells independent of human leukocyte antigen. The bioprocessing to generate CAR+ T cells has been reduced to clinical practice based on two common steps that are accomplished in compliance with current good manufacturing practice. These are (1) gene transfer to stably integrate the CAR using viral and nonviral approaches and (2) activating the T cells for proliferation by crosslinking CD3 or antigen-driven numeric expansion using activating and propagating cells (AaPCs). Here, we outline our approach to nonviral gene transfer using the Sleeping Beauty system and the selective propagation of CD19-specific CAR+ T cells on AaPCs.

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Acknowledgements

This study received grant support from: Cancer Center Core Grant (CA16672); RO1 (CA124782, CA120956, CA141303; CA141303); R33 (CA116127); P01 (CA148600); Burroughs Wellcome Fund; Cancer Prevention and Research Institute of Texas; CLL Global Research Foundation; Estate of Noelan L Bibler; Gillson Longenbaugh Foundation; Harry T Mangurian, Jr, Fund for Leukemia Immunotherapy; Institute of Personalized Cancer Therapy; Leukemia and Lymphoma Society; Lymphoma Research Foundation; MDACC’s Sister Institution Network Fund; Miller Foundation; Moon Shot program of MDACC; Mr Herb Simons; Mr and Mrs Joe H Scales; Mr Thomas Scott; National Foundation for Cancer Research; Pediatric Cancer Research Foundation; William Lawrence and Blanche Hughes Children’s Foundation.

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Correspondence to L J N Cooper.

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Dr Cooper founded and owns InCellerate. He has patents with Sangamo BioSciences with artificial nucleases. He consults with Targazyme (formerly American Stem Cells), GE Healthcare, Ferring Pharmaceuticals, Fate Therapeutics, Janssen Pharmaceuticals and Bristol-Myers Squibb. He is on the Scientific Advisory Board of Cellectis. He receives honoraria from Miltenyi Biotec.

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Singh, H., Moyes, J., Huls, M. et al. Manufacture of T cells using the Sleeping Beauty system to enforce expression of a CD19-specific chimeric antigen receptor. Cancer Gene Ther 22, 95–100 (2015). https://doi.org/10.1038/cgt.2014.69

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