Antithymidylate resistance enables transgene selection and cell survival for T cells in the presence of 5-fluorouracil and antifolates

Abstract

Antithymidylates (AThy) constitute a class of drugs used in the treatment of cancers such as lung, colon, breast and pancreas. These drugs inhibit DNA synthesis by targeting the enzymes dihydrofolate reductase (DHFR) and/or thymidylate synthase (TYMS). AThys effectively inhibit cancer cells, and also inhibit T cells, preventing anticancer immunity, which might otherwise develop from AThy-induced cancer destruction. We establish that T cells expressing mutant DHFR--DHFR L22F, F31S (DHFR(FS))--and/or mutant TYMS--TYMS T51S, G52S (TYMS(SS))-effectively survive in toxic concentrations of AThys methotrexate, pemetrexed and 5-fluorouracil. Furthermore, we show that DHFR(FS) permitted rapid selection of an inducible suicide transgene in T cells. These findings demonstrate that AThy resistances prevent AThy cytotoxicity to T cells while permitting selection of important transgenes. This technological development could enhance in vitro and in vivo survival and selection of T-cell therapeutics being designed for a broad range of cancers.

Dihydrofolate Reductase and Thymidylate Synthase Transgenes Resistant to Methotrexate Interact to Permit Novel Transgene Regulation

“…Cell line origin and attributes have been previously discussed (9). T cells were derived from healthy donor-derived peripheral blood mononuclear cells (PBMC) from the MDACC Blood Bank, Houston, TX.…”

“…T cells were derived from healthy donor-derived peripheral blood mononuclear cells (PBMC) from the MDACC Blood Bank, Houston, TX. PBMC were isolated and cryopreserved until use (9).…”

“…Culture Conditions-Both Jurkat cells and human PBMC were electroporated, maintained, and selected as described previously (9). Briefly, Jurkat cells were electroporated with transgene, and 48 h later, drug was applied for 2 weeks.…”

“…Propagation of PBMC with activating and propagating cells and IL-2 selectively expanded T cells and was repeated weekly. The T cells surviving drug treatment from days 2 to 14 were co-cultured without drug for another 3 weeks (9,20,21). On day 35 of propagation, T cells were stimulated with anti-CD3, anti-CD28, and IL-2 (9).…”

“…In an effort to broaden the application of DHFR FS , our group added a human TYMS mutant, discovered in a bacterial screen, resistant to 5-fluorouracil (5-FU) (11 (12,13). We suggested that the addition of TYMS SS restores thymidine synthesis in MTX-treated T cells (9).…”