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IL-12 is a cytokine produced by antigen-presenting cells that increases T cell proliferation, IFN gamma mediated Th1 effector functions and T and NK cell cytotoxicity. While these effects generate potent anti-tumor immunity in mouse models, the high toxicity of IL-12 in cancer patients has limited its clinical utility. Potency attenuation and intratumoral cytokine localization may improve IL-12 tolerability, but these approaches can reduce efficacy or be limited by intratumoral delivery or tumor antigen expression. To improve both tolerability and efficacy of IL-12, we engineered IL-12Fc fusions with anti-IL-12 antibodies to block IL-12 potency. Using linkers designed to be cleaved by highly active intratumoral proteases, blocking antibodies are released specifically in the tumor microenvironment, thereby increasing intratumoral IL-12 activity. Single chain IL-12 was fused to one C-termini of an Azymetric™ Fc heterodimer. To the other Fc C-termini, an anti-IL-12 scFv was fused via a protease-cleavable linker in order to block IL-12 activity. In addition to antibody blockade, modifications of IL-12Fc fusions were used to further limit IL-12Fc activity. In vitro potency was determined by CD8T cell IFN gamma release. Anti-IL-12 scFv cleavage in the presence of recombinant enzyme or human tumor material was assessed by reducing CE-SDS and LC/MS, respectively. All IL-12Fc fusions were produced with favorable biophysical characteristics. Modified IL-12Fc molecules had up to 500x reduced potency, while antibody blocked IL-12Fc molecules had up to 100,000x reduced potency compared to IL-12Fc control. In vitro cleavage of the anti-IL-12 scFv from IL-12Fc recovered potency to that of the corresponding non-masked IL-12Fc molecules. Reducing CE-SDS confirmed that anti-IL-12 scFvs were cleaved from IL-12Fc in the presence of recombinant enzyme. Cleavage of anti-IL-12 scFvs also occurred when variants were incubated in pancreatic tumor cell supernatant or human tumor tissue lysate. These results indicate that antibody blockade and re-activation by protease cleavage is a promising strategy to localize the activity of IL-12 to the tumor microenvironment while potentially limiting off-tumor toxicities. Further modifying antibody blocked IL-12 has the potential to better fine tune therapeutic index. We are pursuing tumor-specific IL-12 fusions for clinical application in tumors with high intratumoral protease activity. Citation Format: Jennifer Leah Bishop, Ryan Blackler, Gesa Volkers, Maya Poffenberger, Irene Yu, Joel Smith, Akram Khodabandehloo, Sifa Arrafi, Desmond Lau, Liz Stangle, Leisa Stenberg, Patricia Zwierzchowski, Iulia Dude, David Douda, Grant Wickman, Jeff Proctor, Gerry Rowse, Laurence Madera, Genevieve Desjardins, Nicole Afacan, Stuart Barnscher, David Mills, Thomas Spreter, Surjit Dixit. Increasing the therapeutic index of IL12 by engineering for tumor specific protease activation [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 1788.