Abstract Background: Interleukin 12 (IL-12) is a proinflammatory cytokine that plays a central role in regulating both innate and adaptive antitumor immune responses. Clinically, administration of recombinant human IL-12 (rhIL-12) has been limited by a short half-life (t1/2) requiring frequent dosing, causing tachyphylaxis as well as toxic side effects. DF6002/BMS-986415 is an IL-12 Fc-fusion protein designed to have a prolonged t1/2 while retaining equivalent potency to rhIL-12 and promoting sustained IFN-γ response with reduced toxicity. Here we present the preclinical evaluation of DF6002/BMS-986415 ± anti–PD-1. Methods: DF6002/BMS-986415 and rhIL-12 stimulation of IFN-γ release from human and cynomolgus macaque (Cyno) peripheral blood mononuclear cells (PBMCs) and in vivo exposure in Cynos was assessed. As rhIL-12 is inactive in mice, pharmacokinetic, pharmacodynamic, and antitumor effects were evaluated with mDF6006, a mouse surrogate of DF6002/BMS-986415, and recombinant mouse IL-12 (rmIL-12). Tumor growth was evaluated in inflamed (CT26 and MC38) and immunosuppressed (4T1 and B16F10) mouse tumor models (staged ≥ 200 mm3). Results: DF6002/BMS-986415 induced IFN-γ production in human (EC50, 3.35 pM) and Cyno (EC50, 3.09 pM) PBMCs comparable to rhIL-12. DF6002/BMS-986415 was well tolerated at doses yielding greater exposure vs equimolar rhIL-12 in Cynos. In mice, the mouse surrogate mDF6006 vs rmIL-12 yielded a 5-fold increase in t1/2 and a 40-fold greater serum IFN-γ exposure. In contrast to rmIL-12, mDF6006 achieved protracted IFN-γ levels without peak effects associated with increased toxicity; serum IFN-γ Cmax occurred after day 6 and subsided by day 8. mDF6006 was efficacious across mouse tumor models tested. In the CT26 tumor model, mDF6006 (1 µg IP QW) monotherapy showed greater antitumor activity vs equimolar doses of rmIL-12 and led to complete regression (CR) in mice with large tumors (> 800 mm3); immunological memory response was observed in responders rechallenged with fresh tumor cells. In the B16F10 tumor model, mDF6006 (0.5 µg equivalent rmIL-12 SC QW) + anti–PD-1 (10 mg/kg IP Q2W) yielded a higher CR rate (mDF6006 + anti-PD-1, 4 of 10 CR; mDF6006, 1 of 10 CR) and increased survival vs either agent alone. An increase in the number and activation of CD4+ and CD8+ T cells as well as NK cells was observed 4 days after administration of mDF6006 (0.5 µg) in the B16F10 model. Conclusions: DF6002/BMS-986415, a novel IL-12 Fc-fusion protein, retained the full potency of rhIL-12 and showed improved exposure and tolerability; mDF6006 exhibited greater antitumor activity than rmIL-12. These data support the ongoing first-in-human phase 1/2 study evaluating DF6002/BMS-986415 as a novel therapeutic for the treatment of solid tumors in combination with nivolumab (NCT04423029). Citation Format: Eva Gutierrez, Mitchell Bigelow, Colin LaCroix, Patrick Kirby, Lynn Markowitz, Michael Naill, Steve O'Neil, Philip Ansumana Hull, John Engelhardt, Jean-Marie Cuillerott, Ann Cheung, Asya Grinberg, Nicolai Wagtmann. Preclinical characterization of DF6002/BMS-986415, a novel differentiated IL-12 Fc-fusion protein with robust antitumor activity as monotherapy or in combination with anti-PD-1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1714.