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IL-12 is a pleiotropic cytokine that potently stimulates anti-tumor cytotoxic T and NK cell mediated immunity. Recombinant IL-12 reduces tumor growth in multiple mouse models, but its therapeutic application has been limited by severe toxicities. Protease dependent activation of therapeutics with high on-target, off-tumor toxicities may be used to localize activity to the tumor but achieving sufficient exposure of the activated therapeutic in the tumor microenvironment remains a challenge. We have previously shown our design strategy for masked, protease activated IL-12Fc that included optimization of blocking modules, fusion geometry, and linker design. Here we have further expanded our mechanistic evaluations by combining masked, protease activated IL-12Fc with attenuated potency engineering, and selected a lead candidate (ZW270) based on anti-tumor activity and non-human primate (NHP) tolerability. The relative activity of masked and unmasked IL-12Fc variants comprising wild type (wt) or attenuated (att) IL-12 potency was evaluated in CD8 T cell activity assays. The wt IL-12Fc displayed comparable potency to recombinant IL-12 and up to 100x reduced potency when masked. Introduction of attenuating mutations to the p40 subunit reduced the potency of the IL-12Fc by up to 20x and addition of the protease cleavable mask further reduced the potency to up to 5000x compared to wt IL-12Fc. In a single dose NHP study, masked att IL-12Fc was well tolerated up to 31.8 mg/kg, while wt IL-12Fc demonstrated a maximum tolerated dose of below 1.3 mg/kg. Despite the reduced potency of att IL-12Fc in vitro, in in vivo efficacy studies in a human PBMC engrafted xenograft model the masked att IL-12Fc was able to control tumor growth whereas masked wt IL-12Fc and wt IL-12Fc showed limited anti-tumor activity, suggesting the approach of masked, protease activated att IL-12Fc might be able to achieve a higher exposure of active cytokine in the tumor in comparison to the masked wt IL-12Fc and wt IL-12 comparator. To further investigate the potentially superior exposure response relationship of masked att IL-12Fc, we developed a quantitative systems pharmacology (QSP) model based on our experimental data and literature data. In this model the predicted ratio of IL-12-receptor occupancy by active IL-12Fc in the tumor vs. blood was 18x greater for masked att IL-12Fc than for wt IL-12Fc, whereas the ratio was under 10x greater for masked wt IL-12Fc vs. wt IL-12Fc. In summary, the affinity attenuated, masked IL-12Fc lead ZW270 has potent and superior anti-tumor activity to wt IL-12Fc and masked wt IL-12Fc, and is well tolerated in NHPs to >30 mg/kg. Our data suggests that the combined strategy of masked, protease activated IL-12Fc and attenuated IL-12 potency has the potential to widen the therapeutic index and to have superior activity to masked, protease cleavable wt IL-12 and unmasked wt IL-12 fusions. Citation Format: Maya C. Poffenberger, Jennifer L. Bishop, Ryan J. Blackler, Kevin G. Haworth, Steven Booth, Shalla Hanson, Jeff R. Proctor, I-Ting Shao, Nichole K. Escalante, Dayananda Siddappa, Joel Smith, Gursev Anmole, Saki Konomura, Nicholas A. Dawson, Sifa Arrafi, Desmond Lau, Gerry Rowse, Rupert H. Davies, Thomas Spreter von Kreudenstein. ZW270, a conditionally masked IL-12 cytokine fusion protein displaying potent anti-tumor activity absent systemic toxicity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2935. |
