Griffin, JE; (2015) Generation of Mycophenolate Mofetil Resistant Lymphocytes for Adoptive Immunotherapy. Doctoral thesis , UCL (University College London).

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Abstract

Background Adoptive transfer of cells targeting viral infection or tumour following allogeneic transplantation is hindered by the requirement for immunosuppression. Reports of engineered resistance to calcineurin inhibitors have highlighted the potential of conferring immunosuppressive resistance in this setting. A commonly used immunosuppressant, mycophenolate mofetil (MMF) is a non-competitive inhibitor of inosine-5’-monophosphate dehydrogenase (IMPDH). I used T cells transduced with mutated IMPDH that confers >2000-fold resistance to MMF (IMPDH2R; T333I, S351Y). Methods IMPDH2R and IMPDH2 with a catalytic site mutation (IMPDH2CS; C331A) were cloned into retroviral vectors as fusions to eGFP reporter sequences. T cells were transduced with either vector, transferred to recipient mice and evaluated in the presence or absence of MMF. Results A selective advantage for IMPDH2R was demonstrated using a 1:1 mix of CD8 T cells transduced with IMPDH2R and IMPDH2CS. These were injected into recipients distinguishable from the transferred cells using congenic markers. Transferred cells were stimulated by cognate peptide and MMF (0- 200mg/kg/day) was given daily. Selection of IMPDH2R transduced cells occurred following stimulation even without drug (Mann-Whitney Ratio IMPDH2R:IMPDH2CS p=0.0356). MMF administered significantly increased selection (Mann-Whitney MMF 0 v 200 p=0.0365). To assess function, irradiated mice were injected with EL4.OVA tumour cells subcutaneously. After 10 days, either MMF or vehicle treatment was initiated and OT1 CD8 T cells transduced with either IMPDH2CS or IMPDH2R transferred. The combination of MMF and IMPDH2R transduced cells resulted in significantly improved survival over vehicle treated mice (log rank p<0.0001) with significantly smaller tumours at day 26 (Mann-Whitney p<0.0001). This effect was due to direct synergistic effects of both MMF and transferred T cells against tumour cells. Conclusions IMPDH2R confers cells with a selective advantage. Synergy with MMF resulted in improved tumour survival. In patients requiring on-going immunosuppression during adoptive immunotherapy, this combination has great therapeutic potential.

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