A universal pro-immunocytokine platform using a spatial hindrance strategy to overcome MHC resistance and enhance efficacy

Immune checkpoint blockade (ICB) drugs are regarded as advanced approaches in cancer immuno-oncology (IO) therapy. (Background) However, they still face clinical limitations due to insufficient tumor selectivity, which can cause immune-related damage to normal tissues, and limited efficacy, particularly in tumors with major histocompatibility complex (MHC) loss that leads to immunotherapy resistance. While the interferon (IFN) family promotes immune activation and upregulates MHC expression, systemic administration often results in severe toxicities that restrict clinical application. (Methods) To address these challenges, we developed an innovative pro-immunocytokine (IFN-ICB) platform using a spatial hindrance strategy, in which IFN and ICB are linked via a protease-cleavable linker that mutually masks their activities. Upon protease-mediated cleavage within the tumor microenvironment, both IFN and ICB restore their activities and upregulate tumor MHC expression, thereby enhancing therapeutic efficacy while mitigating systemic toxicity. (Results) In our project, we initially demonstrated the proof of concept for pro-immunocytokine engineering by fusing IFNs to several ICBs, including anti-CTLA-4, anti-PD-1, and anti-PD-L1 antibodies. To optimize the blocking efficiency of IFN-ICBs, AI-based simulations were employed to predict spatial hindrance structures and calculate relevant blocking parameters. In vitro assessments confirmed that IFN-ICBs enhance tumor MHC expression and increase cytotoxicity. In vivo studies further demonstrated that IFN-ICBs efficiently improve antitumor efficacy in melanoma and colon cancer mouse models, indicating stronger immune responses within the tumor compared with either IFN or ICB monotherapy. Importantly, IFN-ICBs also reduced systemic toxicity and were safer than the combined administration of IFN and ICB drugs. (Conclusion) Our innovative pro-immunocytokine platform possesses the universal capability to transform any ICB or antibody into a pro-immunocytokine via AI-based prediction, enhancing therapeutic efficacy while improving safety, and thus holds promise to impact the next generation of IO therapeutics.