Efficient Selection of Genetically Modified Human T Cells Using Methotrexate-Resistant Human Dihydrofolate Reductase

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 based on the pharmaceutical drug methotrexate (MTX), a potent allosteric inhibitor of human dihydrofolate reductase (DHFR). Here, we describe the development of SIN lentiviral vectors that direct the coordinated expression of a CD19-specific CAR, the human EGFRt tracking/suicide construct, and a methotrexate-resistant human DHFR mutein (huDHFR(FS); L22F, F31S). Our results demonstrate that huDHFR(FS) co-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 cDNA design insures that selected MTX-resistant T cells co-express functionally relevant levels of the CD19-specific CAR and EGFRt. This selection system based on huDHFR(FS) and MTX has considerable potential utility in the manufacturing of clinical-grade T cell products.