Abstract 4067: Development of tumor-restricted IL-12 with antigen-dependent expression and localized IL-12 activity
Authors: Huang Szu-Han , Davenport Thaddeus M. , Moffett Howell F. , Weitzner Brian D. , Cassereau Luke , Baker Laura E. , Hammerson Bradley , Zhuang Summer , Saechao Christine , Song Lisa , Mimms Jade , Chian David , Sims Candace , Hiraragi Hajime , Lajoie Marc J. , Boyken Scott E. , Boldajipour Bijan 
Published: March 2024

Abstract
IL-12 is an immune-stimulatory cytokine that can modulate the tumor microenvironmnent (TME) to enhance the cytotoxic activity of T and NK cells; however, IL-12 expression by T cells caused severe toxicity in a previous clinical trial. Using Outpace’s OutSmart™ technology, we designed a tumor-restricted IL-12 (trIL-12) that is under control of an activation-inducible promoter and auto-inactivates within minutes after secretion.
T cells were engineered via lentiviral vectors (LVV) to express wild-type single-chain IL-12 (WT scIL-12) or trIL-12 under the control of an activation-inducible promoter; a second LVV introduces an NY-ESO-1 TCR. Kinetics of IL-12 expression was measured by qPCR and MSD technology; kinetics of IL-12p70 heterodimer half-life was measured using Octet bio-layer interferometry (BLI). T-cell cytotoxicity and cytokine production was evaluated in vitro after repeated stimulation with NY-ESO-1 expressing target cells using Incucyte and MSD. IL-12 activity in bystander cells was measured by detection of IFN-γ using flow cytometry. In vivo T-cell function of trIL-12-engineered NY-ESO-1 TCR T cells was measured in NSG MHCI/II KO mice bearing A375 xenografts. trIL-12 activity in a fully immune-competent mouse model was measured in B6 mice implanted with B16F10 tumor cells engineered to express murine surrogates of trIL-12.
Expression of WT scIL-12 under the control of an activation-inducible promoter peaks within 6 hours after activation and produces sufficient IL-12 to improve T-cell function in vitro and in vivo. trIL-12 was created by inclusion of a cleavable linker and elimination of the covalent disulfide bond between the p35 and p40 subunits of IL-12p70, resulting in dissociation of the functional cytokine within 10 minutes post-cleavage. Additional mutations further reduce the IL-12p70 half-life. trIL-12 activates the IL-12-producing T cells, as well as proximal bystander T-cells in direct co-culture, but it does not have the ability to activate distal bystander T-cells separated by Transwell membranes. In xenograft mouse models, trIL-12-expressing T cells have improved proliferation upon antigen recognition and display potent anti-tumor activity and cytokine production without showing systemic IL-12 accumulation. Furthermore, in fully immune competent mice, expression of a murine trIL-12 surrogate by tumor cells led to generation of potent anti-tumor responses without systemic trIL-12 accumulation and reduced systemic IFN-γ.
trIL-12-engineered T cells generate potent anti-tumor activity in vitro and in vivo. Unlike WT scIL-12, trIL-12 activity is localized to the region around the producing T cell and systemic IL-12 exposure is not observed in vivo. Collectively, these preclinical data suggest that trIL-12 may enable the development of potent T-cell therapeutics while maintaining an acceptable safety profile.
Citation Format: Szu-Han Huang, Thaddeus M. Davenport, Howell F. Moffett, Brian D. Weitzner, Luke Cassereau, Laura E. Baker, Bradley Hammerson, Summer Zhuang, Christine Saechao, Lisa Song, Jade Mimms, David Chian, Candace Sims, Hajime Hiraragi, Marc J. Lajoie, Scott E. Boyken, Bijan Boldajipour. Development of tumor-restricted IL-12 with antigen-dependent expression and localized IL-12 activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4067.