Rational design of a repeated-dosing schedule for KNP-101 guided by IFN-gamma regulation

Background: Recombinant IL-12 demonstrated potent antitumor efficacy in early clinical trials; however, its development was hampered by severe systemic toxicities. To overcome these limitations, multiple approaches have been investigated, including strategies to prolong IL-12 half-life while modulating its activity, tumor-localized delivery using an immunocytokine modality, and selective activation through prodrug or masking technologies. While overcoming systemic toxicity has been the central focus in developing cytokines as effective anticancer agents, IL-12 continues to pose unique challenges that require further resolution. IL-12 exerts its antitumor effects primarily through induction of IFN-gamma. However, clinical observations indicated that prior exposure to single-injection of IL-12 dramatically regulated toxicity upon subsequent dosing, likely due to negative feedback of IFN-gamma expression, known as tachyphylaxis. In addition to tachyphylaxis, due to the delayed induction of IFN-gamma following immune activation, pharmacokinetic (PK) and pharmacodynamic (PD) profiles are often uncoupled upon IL-12 treatment, posing additional challenges for clinical development. Experiments and Unpublished data: We have focused on how IFN-gamma is regulated upon repeated-dose of KNP-101, a novel FAP-targeting IL-12-based therapeutic that we have developed. By adjusting dosing intervals and amounts, we characterized IFN-gamma dynamics in multiple murine tumor models including CT26, MC38, and EMT6. Repeated dosing of KNP-101 suppressed tumor growth, and analysis of IFN-gamma levels revealed that while systemic IFN-gamma induction exhibited strong negative feedback, this effect was markedly attenuated within the tumor microenvironment, where IFN-gamma function is critical for antitumor immunity. In addition, systemic IFN-gamma suppression by repeated KNP-101 treatment could be relieved by anti-PD1 combination. In non-tumor-bearing mice, repeated KNP-101 dosing led to pronounced negative feedback of IFN-gamma expression in serum after 3 weeks, which gradually recovered by week 8. In human PBMC systems, recovery occurred more rapidly (~6 weeks). Interestingly, humanized CD34+ mice did not display the same degree of IFN-gamma suppression by repeated KNP-101 treatment, suggesting that there is differential regulation of IFN-gamma, likely due to differences between mouse and human immune systems such as memory T/NK cell compositions. These findings suggest that optimizing dosing and its frequency in clinical trials will be crucial to achieve sustained efficacy while minimizing cytokine-related toxicity. Conclusion: Our results demonstrate that the kinetics of IFN-gamma regulation differ between systemic and tumor compartments, as well as between species, providing valuable insight for the rational design of IL-12-based immunotherapy.