title: Engineering human T cells for resistance to methotrexate and mycophenolate mofetil as an in vivo cell selection strategy.
abstract: Gene transfer and drug selection systems that enforce ongoing transgene expression in vitro and in vivo which are compatible with human pharmaceutical drugs are currently underdeveloped. Here, we report on the utility of incorporating human enzyme muteins that confer resistance to the lymphotoxic/immunosuppressive drugs methotrexate (MTX) and mycophenolate mofetil (MMF) in a multicistronic lentiviral vector for in vivo T lymphocyte selection. We found that co-expression of human dihydrofolate reductase (DHFR(FS); L22F, F31S) and inosine monophosphate dehydrogenase II (IMPDH2(IY); T333I, S351Y) conferred T cell resistance to the cytocidal and anti-proliferative effects of these drugs at concentrations that can be achieved clinically (up to 0.1 µM MTX and 1.0 µM MPA). Furthermore, using a immunodeficient mouse model that supports the engraftment of central memory derived human T cells, in vivo selection studies demonstrate that huEGFRt(+)DHFR(FS+)IMPDH2(IY+) T cells could be enriched following adoptive transfer either by systemic administration of MTX alone (4.4 -fold), MMF alone (2.9-fold), or combined MTX and MMF (4.9-fold). These findings demonstrate the utility of both DHFR(FS)/MTX and IMPDH2(IY)/MMF for in vivo selection of lentivirally transduced human T cells. Vectors incorporating these muteins in combination with other therapeutic transgenes may facilitate the selective engraftment of therapeutically active cells in recipients.
authors.affiliation: Departments of Cancer Immunotherapeutics & Tumor Immunology, and Hematology and Hematopoietic Cell Transplantation, Beckman Research Institute, City of Hope, Duarte, California, United States of America.
authors.forename: Mahesh
authors.initials: M
authors.lastname: Jonnalagadda
authors.forename: Christine E
authors.initials: CE
authors.lastname: Brown
authors.forename: Wen-Chung
authors.initials: WC
authors.lastname: Chang
authors.forename: Julie R
authors.initials: JR
authors.lastname: Ostberg
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Forman
authors.forename: Michael C
authors.initials: MC
authors.lastname: Jensen
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D016207:Cytokines / Q000378:metabolism; D005434:Flow Cytometry; D006801:Humans; D007168:IMP Dehydrogenase / Q000235:genetics / Q000378:metabolism; D019409:Interleukin-15 / Q000378:metabolism; D016086:Lentivirus / Q000235:genetics; D007963:Leukocytes, Mononuclear / Q000187:drug effects / Q000378:metabolism; D008727:Methotrexate / Q000494:pharmacology*; D051379:Mice; D009173:Mycophenolic Acid / Q000031:analogs & derivatives* / Q000494:pharmacology; D013601:T-Lymphocytes / Q000187:drug effects* / Q000378:metabolism*; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics / Q000378:metabolism
chemical_list: D016207:Cytokines; D019409:Interleukin-15; D007168:IMP Dehydrogenase; D013762:Tetrahydrofolate Dehydrogenase; D009173:Mycophenolic Acid; D008727:Methotrexate