AbstractFirst-generation 4-1BB (CD137) monoclonal antibody (mAb)-based agonists demonstrated significant potential in early preclinical and clinical studies by eliciting robust anti-tumor responses. However, their clinical development was hampered by dose-limiting hepatotoxicity, attributed to the systemic activation of 4-1BB receptors on monocytes, which in turn recruited CD8+ T cells to attack hepatocytes. To address this challenge, next-generation therapies have focused on decoupling the hepatotoxic effects of 4-1BB activation from its anti-tumor activity. These strategies utilize bispecific molecules to precisely target and activate 4-1BB receptors within the tumor microenvironment (TME). By binding to tumor-associated antigens, these molecules localize to TME, facilitating the hyperclustering and trimerization required for 4-1BB activation. This approach enhances tumor-specific immune responses while minimizing systemic activation and off-target effects. In this study, we introduce, to the best of our knowledge, the first PD-1-targeted 4-1BB agonist that leverages the PD-1 receptor on exhausted T cells to facilitate 4-1BB hyperclustering and activation. Three distinct murine-specific bispecific constructs were developed in silico, each employing an anti-PD-1 nanobody for crosslinking and trimerization of the 4-1BB receptor. Construct 1 was designed to function as a weak agonist in the absence of PD-1, transforming into a strong agonist in its presence. In contrast, constructs 2 and 3 are entirely dependent on PD-1 for hyperclustering and subsequent 4-1BB activation. The constructs were expressed and purified using Expi293F-based stable cell lines. Flow cytometry confirmed their ability to bind specifically to the cognate 4-1BB and PD-1 receptors. A 4-1BB luciferase bioassay was employed to demonstrate the PD-1 dependency of Constructs 2 and 3. As expected, constructs 2 and 3 exhibited no 4-1BB activation in the absence of PD1-expressing cells but transformed into potent agonists in their presence. Construct 1 activated the 4-1BB receptor regardless of PD-1, with its activity being markedly enhanced in its presence. Additionally, constructs 2 and 3 were shown to effectively block the PD-1/PD-L1 interaction. Preliminary preclinical evaluations confirmed that all three constructs function as designed. Following the completion of in vitro studies, we are currently conducting in vivo analyses using mouse xenograft models to evaluate the therapeutic efficacy, toxicity, and biodistribution of these constructs.Citation Format:Alana Kramer, Damien Ruiz, Jonathan Marable, Payal Agarwal, Deepa Bedi, Maninder Sandey. Engineering a PD-1 dependent 4-1BB agonist for combination immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6156.