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 (DHFRFS; L22F, F31S) and inosine monophosphate dehydrogenase II (IMPDH2IY; 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+DHFRFS+IMPDH2IY+ 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 DHFRFS/MTX and IMPDH2IY/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.

Chimeric Antigen Receptors With Mutated IgG4 Fc Spacer Avoid Fc Receptor Binding and Improve T Cell Persistence and Antitumor Efficacy

“…The EGFRt-T2A-DHFR FS -T2A-IMPDH2 IY _epHIV7 lentiviral vector used to generate EGFRt+ T cells was previously described [49]. The CD19R(L235E)28Z-T2A-EGFRt_epHIV7, CD19R(N297Q)28Z-T2A-EGFRt_epHIV7, and CD19R(EQ)28Z-T2A-EGFRt_epHIV7 vectors used to generate the CD19R(L235E)+, CD19R(N297Q)+ and CD19R(EQ)+ T cells respectively, were created by site directed mutagenesis using the QuikChange II XL kit (Agilent Technologies, Santa Clara, CA) of a codon optimized CD19R28Z_pGA plasmid that had been synthesized by Geneart (Life Technologies Corp., Grand Island, A c c e p t e d m a n u s c r i p t NY), digested with NheI/RsrII and ligated with a similarly digested CD19R28Z-T2A-EGFRt_epHIV7.…”

Lentivector Knockdown of CCR5 in Hematopoietic Stem and Progenitor Cells Confers Functional and Persistent HIV-1 Resistance in Humanized Mice

“…However, novel strategies for sorting of transduced CD34 ϩ cells based on the expression of truncated cellular surface receptors, such as CD25 (20), the epidermal growth factor receptor (44), or the nerve growth factor receptor (45), are very promising for achieving high numbers of engrafted gene-engineered cells. An alternative approach to pretransplantation sorting would be in vivo selection of transduced cells (46,47). Regrettably, current in vivo selection methods use potentially carcinogenic compounds, such as mycophenolate, methotrexate, or alkylating agents (i.e., O 6 -benzylguanine/bis-chloroethylnitrosourea), that offset their use in a disease, such as HIV, that is amenable to an efficient and well-tolerated cART.…”

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

“…The use of DHFR FS was later implemented in genetically modified T cells. T cells modified with DHFR FS are desired following bone marrow transplant for relapsed leukemia to overcome immune suppression of MTX, allowing genetically modified T cells to survive and target cancer (7,8). An advantage of DHFR FS is that it can be used to select for transgenes useful for therapeutic efficacy such as tumor targeting proteins such as chimeric antigen receptors (8), suicide genes (9), or imaging genes (10).…”