463 Antigen-responsive promoters coupled with cytoDRiVE^® technology provide tight spatiotemporal regulation for tumor-infiltrating lymphocytes (TIL) expressing membrane-bound IL12

<h3>Background</h3> TIL engineered to express the Th1-polarizing cytokine interleukin IL12 (IL12) have the potential to be a powerful cell therapy for recalcitrant solid tumors. TIL modified to secrete IL12 upon antigen stimulation showed a promising response rate in patients with metastatic melanoma receiving low cell doses of TIL, but also unacceptable cytokine-related systemic toxicities,¹ highlighting the need for additional means of regulating IL12 expression. We have developed a membrane-bound IL12 (mbIL12) with enhanced regulation of expression by combining novel nuclear factor of activated T cells response elements (NFAT-RE) with the drug-inducible cytoDRiVE platform, which requires both T-cell receptor engagement (spatial) and presence of a small-molecule drug (temporal) for spatiotemporal regulation of IL12 expression. <h3>Methods</h3> TIL were expanded and engineered with vectors containing different cytokines. NFAT-RE were tested for their ability to drive antigen stimulation-dependent expression of mbIL12 and shed IL12, with and without additional regulation using a cytoDRiVE platform drug-responsive domain (DRD) derived from human dihydrofolate reductase (hDHFR), stabilized by the FDA-approved small-molecule drug trimethoprim. cytoTIL15^TM cells expressing mbIL15 with a constitutive promoter, regulated with the DRD carbonic anhydrase 2 and stabilized by acetazolamide, were used as an experimental control. Antitumor activity was assessed against autologous patient-derived xenograft tumors (PDX) from a colorectal cancer (CRC) metastasis grown in NSG mice. <h3>Results</h3> TIL expressing IL12 regulated by both NFAT-RE promoters and hDHFR showed significantly greater regulation of mbIL12 and shed IL12 than with either system alone, with IL12 levels slightly above limit of detection in the absence of antigen and DRD ligand, and ~20-fold upregulation only after both antigen stimulation and DRD ligand exposure. NFAT-RE/mbIL12-hDHFR regulation reduced shed IL12 levels from activated TIL by 10× compared to IL12 regulated by only NFAT-RE. TIL expressing NFAT-RE/mbIL12-hDHFR delayed growth of an autologous CRC PDX tumor <i>in vivo</i> (44% reduction 44 days post-cell transfer vs no transfer experimental control, p&lt;0.05, Mann-Whitney, n=8), and persisted in the tumor for 8 weeks without any T-cell growth factor support. No IL12 was detectable in the plasma, and markedly less systemic IFNγ was detected than with mbIL15-engineered TIL, suggesting IL12 TIL activity was focused at the tumor site. <h3>Conclusions</h3> Combining antigen activation-dependent and pharmacologic regulation for spatiotemporal control of IL12 expression is a promising approach to increase the safety of TIL cell therapy armored with IL12, potentially decreasing the systemic side effects that have been a long-standing obstacle to the clinical use of IL12 in cell therapies. <h3>Reference</h3> Zhang L, <i>et al</i>. Tumor-infiltrating lymphocytes genetically engineered with an inducible gene encoding interleukin-12 for the immunotherapy of metastatic melanoma. <i>Clin Cancer Res</i> 2015;<b>21</b>(10):2278–2288.