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Efficient selection of genetically modified human T cells using methotrexate-resistant human dihydrofolate reductase

Gene Therapy volume 20, pages 853–860 (2013)Cite this article

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Abstract

Genetic modification of human T cells to express transgene-encoded polypeptides, such as tumor targeting chimeric antigen receptors, is an emerging therapeutic modality showing promise in clinical trials. The development of simple and efficient techniques for purifying transgene+ T cells is needed to facilitate the derivation of cell products with uniform potency and purity. Unlike selection platforms that utilize physical methods (immunomagnetic or sorting) that are technically cumbersome and limited by the expense and availability of clinical-grade components, we focused on designing a selection system on the basis of the pharmaceutical drug methotrexate (MTX), a potent allosteric inhibitor of human dihydrofolate reductase (DHFR). Here, we describe the development of self inactivating (SIN) lentiviral vectors that direct the coordinated expression of a CD19-specific chimeric antigen receptor (CAR), the human EGFRt tracking/suicide construct, and a methotrexate-resistant human DHFR mutein (huDHFRFS; L22F, F31S). Our results demonstrate that huDHFRFS expression renders lentivirally transduced primary human CD45RO+CD62L+ central memory T cells resistant to lymphotoxic concentrations of MTX up to 0.1 μM. Our modular complementary DNA (cDNA) design insures that selected MTX-resistant T cells co-express functionally relevant levels of the CD19-specific CAR and EGFRt. This selection system on the basis of huDHFRFS and MTX has considerable potential utility in the manufacturing of clinical-grade T cell products.

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Acknowledgements

We would like to thank Renate Starr, Araceli Hamlett, Winnie Wong and Sean Cho for their technical assistance, Xiuli Wang for the helpful discussions, and Xueli Liu for statistical analysis. This work was funded by NIH P50 CA107399, PO1 CA030206, the Lymphoma Research Foundation, the Tim Nesvig Lymphoma Research Foundation, the Neidorf Foundation, the Marcus Foundation, the Skirball Foundation, and the Timothy Lindenfelser Foundation.

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Authors and Affiliations

  1. Departments of Cancer Immunotherapeutics and Tumor Immunology, and Hematology and Hematopoietic Cell Transplantation, Beckman Research Institute, City of Hope National Medical Center, Duarte, 91010, CA, USA

    M Jonnalagadda, C E Brown, W C Chang, J R Ostberg, S J Forman & M C Jensen

  2. Ben Towne Center for Childhood Cancer Research, Seattle Children’s Research Institute, Seattle, WA, USA

    M C Jensen

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  1. M Jonnalagadda
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  2. C E Brown
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Correspondence to M C Jensen.

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Competing interests

MCJ is an inventor of licensed intellectual property and a co-founder/equity of ZetaRx Biosciences, Inc. All other authors have no conflicts of interest to disclose.

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Jonnalagadda, M., Brown, C., Chang, W. et al. Efficient selection of genetically modified human T cells using methotrexate-resistant human dihydrofolate reductase. Gene Ther 20, 853–860 (2013). https://doi.org/10.1038/gt.2012.97

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