In Vivo Selection of Mycophenolate Mofetil-Resistant T Cells for Adoptive Immunotherapy.
Authors: Griffin James Edward , Carpenter Ben , Nicholson Emma , Xue Shao-an , Pule Martin , Stauss Hans , Chakraverty Ronjon 
Published: October 2019

Abstract
Abstract 3008
In vivo selection of mycophenolate mofetil-resistant T cells for adoptive immunotherapy.

Background
Following allogeneic solid organ or hematopoietic stem cell transplantation, adoptive transfer of therapeutic T cells may be hindered by the requirement for immune suppressive drugs to prevent rejection or graft-versus-host disease. Mycophenolate mofetil (MMF) is a non-competitive inhibitor of inosine-5'-monophosphate dehydrogenase 2 (IMPDH2), an inducible enzyme that generate guanine nucleotides for DNA and RNA synthesis in T cells. In this study, we have evaluated the potential for gene transfer to T cells of a mutated IMPDH2 that confers >2000-fold resistance to MMF (IMPDH2R; T333I, S351Y).

Methods
Wild type IMPDH2WT, IMPDH2R and IMPDH2 with a catalytic site mutation (IMPDH2CS; C331A) were cloned into SFG retroviral vectors as fusions to eGFP reporter sequences. Murine thymoma (BW 5147) or CD8 T cells were transduced with each vector and their phenotype and function evaluated in the presence or absence of mycophenolic acid (MPA), the active metabolite of MMF.

Results
BW thymoma cells transduced with IMPDH2R exhibited less apoptosis than cells transduced with IMPDH2CS in response to MPA (ratio % Annexin V+ MPA: no MPA- IMPDH2R = 1.2; IMPDH2CS = 2.9, p=0.01). Cells transduced with IMPDH2R were also able to overcome the G1 cell-cycle arrest induced by MPA when compared to control IMPDH2CS cells (Ratio %cells in S-G2/M phases MPA: no MPA- IMPDH2R = 1.0; IMPDH2CS = 0.3, p=0.03). This led to selective enrichment of IMPDH2R transduced cells in the presence of MPA. At low dose MPA (450nM), IMPDH2R transduced cells enriched compared to IMPDH2CS but not IMPDH2WT (ratio % GFP MPA: no MPA- IMPDH2R = 1.6 vs IMPDH2CS = 1.1, p=0.04 and IMPDH2WT = 1.5 p= 0.14). However, at high dose (4500nM) IMPDH2R exhibited enhanced enrichment (ratio % GFP MPA: no MPA- IMPDH2R = 2.8 vs IMPDH2CS = 1.0 p=0.03 and vs IMPDH2WT= 1.5 p=0.03).
Gene transfer of IMPDH2R into murine CD8 T cells also led to selective enrichment compared to controls in the presence of MPA when cultured with proliferation-inducing common gamma-chain cytokines (ratio MPA: no MPA IMPDH2R = 4.4 vs. IMPDH2CS = 1.10, p=0.02). Strong selection for IMPDH2R-tranduced CD8 OT-1 T cells in the presence of MPA was also observed under conditions of antigen-induced proliferation (ratio IMPDH2R= 7.1, control = 0.8, p=0.002).
To assess in vivo selection, sub-lethally irradiated (2Gy) B6.PL (Thy1.1) mice were injected with a 1:1 mix of OT1 TCR transgenic Thy1.2 CD8 T cells transduced with IMPDH2R or IMPDH2CS that could be differentiated by the congenic markers CD45.1 and CD45.2. Transferred cells were stimulated by s.c. injection with cognate peptide (SIINFEKL) in IFA and MMF (200mg/kg/day) was given by daily ip injection. As in vitro, IMPDH2R-transduced OT1 cells were preferentially selected over IMPDH2CS-transduced cells following MMF treatment (day 14 ratio IMPDH2R to IMPDHCSwas 19.3 vs 1.2 in the absence of MMF).

Conclusions
T cells transduced with IMPDH2R are resistant to the anti-proliferative and apoptotic effects of MPA in vitro and demonstrate strong selection in vivo compared to controls at therapeutic levels of MMF. These data support the potential of conferring MMF resistance as a strategy to permit the survival of therapeutic T cells in immunosuppressed allograft recipients.

Disclosures:
Stauss: Cell Medica: Scientific Advisor Other.