Abstract LB025: Spatiotemporally regulated expression of membrane-bound interleukin 12 (mbIL12) for armored adoptive cell therapy (ACT) shows strong antitumor activity in syngeneic solid tumor models without overt toxicity

Abstract ACTs armored with potent cytokines, such as IL12, have shown promising activity in solid tumors. However, clinical application of IL12 has been hindered by significant systemic toxicities (Zhang Cancer Res 2015), dictating precise control over its expression. cytoDRiVE® technology allows pharmacologic regulation of mbIL12 expressed as a fusion protein with a drug-responsive domain (DRD) derived from human dihydrofolate reductase, stabilized by the small molecule, trimethoprim (TMP). Combining activation-dependent (spatial) control using nuclear factor of activated T cells response elements (NFAT-RE) with pharmacologic (temporal) regulation using the cytoDRiVE platform provides tight regulation of mbIL12, improving safety and enabling anti-tumor activity and survival in preclinical models (Smith SITC 2024). Here, we show that this novel spatiotemporal control 1) regulates downstream IL12 effects in the tumor microenvironment (TME), and 2) is efficacious in a liver model of colon cancer. Syngeneic studies were used to assess the efficacy and pharmacodynamics of murine transgenic CD8+ pmel TCR T cells (gp100) expressing either NFAT-RE-inducible secreted IL12 (NFAT-sec-IL12) or NFAT-RE and cytoDRiVE-regulated mbIL12 (NFAT-cytoDRiVE-mbIL12). B16-F10 and gp100-engineered MC38 tumor cells were implanted subcutaneously or intrahepatically, respectively. B16-F10 tumor-bearing mice were randomized and lymphodepleted 24 hours before cell product infusion. Flow cytometry and cytokine analyses characterized the distinct phenotypes of the infused cells and corresponding host immune cells. B16-F10 tumor-bearing mice receiving pmel T cells expressing NFAT-sec-IL12 displayed significant body weight loss, unlike mice infused with pmel T cells expressing NFAT-cytoDRiVE-mbIL12 (p<0.05), while both groups showed tumor control (Smith SITC 2024). On Day 7 post-ACT, systemic cytokines were higher in the NFAT-sec-IL12 than the NFAT-cytoDRiVE-mbIL12 group, including 30× higher IFNγ (p<0.05), as well as higher TNFα (p<0.0001), IL6 (p<0.01), and IL10 (p<0.0001). In the tumor, NFAT-cytoDRiVE-mbIL12 pmel T cells demonstrated TMP-regulated mbIL12 expression leading to a proinflammatory TME with increased bulk CD3+CD8+ cells, pmel T cells, an M1 macrophage shift and a 10× increase of IFNγ. Moreover, in an MC38-gp100+ tumor model, without lymphodepletion, NFAT-cytoDRiVE-mbIL12 pmel T cells + TMP led to complete and durable responses in an immune-suppressive TME. Evidence of liver toxicity, measured by assessing systemic alanine aminotransferase (ALT) levels, was significantly higher in the NFAT-sec-IL12 group versus the NFAT-cytoDRiVE-mbIL12 + TMP group in both models (p<0.01, p<0.05, respectively), with ALT levels in the NFAT-cytoDRiVE-mbIL12 groups at or near those of the untreated control tumor groups. In summary, integrating both activation-dependent and cytoDRiVE-mediated pharmacologic regulation to govern the spatiotemporal expression of IL12 is a strategy for enhancing the safety of an IL12-armored ACT. By overcoming the barrier of systemic toxicity, this approach has the potential to enable clinical application of this potent cytokine in the form of a cell therapy. Citation Format: Theresa Ross, Patricia Timpug, Sean G. Smith, Meghan Langley, Benjamin Primack, Violet Young, Dexue Sun, Dan Jun Li, Dhruv K. Sethi, Jan ter Meulen, Michelle Ols, Jeremy Tchaicha. Spatiotemporally regulated expression of membrane-bound interleukin 12 (mbIL12) for armored adoptive cell therapy (ACT) shows strong antitumor activity in syngeneic solid tumor models without overt toxicity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_2):Abstract nr LB025.