Conditional Agonism: PD1-Dependent Activation of 4-1BB for Enhanced Safety - PROJECT SUMMARY Immune checkpoint therapies targeting PD-1, PD-L1, and CTLA-4 have revolutionized cancer treatment over the past 15 years. Antibody-mediated inhibition of these checkpoints can lead to significant tumor reduction, durable responses, and prolonged survival. Unfortunately, only 10-20% of cancer patients experience these therapeutic benefits, and even among responders, relapse remains a challenge. Targeted therapies that activate co-stimulatory molecules, such as 4-1BB, are being explored to enhance the clinical effectiveness of immune checkpoint blockade. Several preclinical studies have highlighted the potential of combining anti-PD-1 monoclonal antibodies (mAbs) and 4-1BB agonists, demonstrating robust and durable tumor regression by activating potent antitumor immunity. However, the initial enthusiasm surrounding these therapies is tempered by concerns regarding their safety profile, particularly evident in recent phase II clinical trials where first- generation 4-1BB agonistic mAbs caused severe hepatotoxicity, resulting in two fatalities. Targeting strategies that confine the co-stimulatory activity to the tumor site is an attractive approach for developing 4-1BB agonists with improved safety profiles. To this end, we have engineered a bispecific fusion protein, aPD1-4-1BBL, designed to disrupt PD-1/PD-L1 interaction while harnessing its binding to the PD-1 receptor for localized 4-1BB hyperclustering. We have developed three murinized (murine-specific) and caninized (canine-specific) aPD1-4- 1BBL constructs with varying biophysical properties to identify the construct with the most optimal combination of therapeutic efficacy and safety profile. Our preliminary data confirm that all aPD1-4-1BBL constructs induce PD-1-dependent and FcγR-independent 4-1BB hyperclustering. We hypothesize that the PD-1 blockade and 4- 1BB co-stimulation induced by aPD1-4-1BBL will cause significant tumor regression while avoiding immune- related hepatotoxicity. Our specific aims will: (Aim 1a) to evaluate the therapeutic efficacy, safety, and toxicity profiles of murine-specific aPD1-4-1BBL constructs in the syngeneic B16-F0 and B16-F10 mouse melanoma models and (Aim 1b) to assess the safety and toxicity profiles of canine-specific aPD1-4-1BBL constructs in healthy beagle dogs. We will employ a cross-species approach to identify an aPD1-4-1BBL construct that demonstrates superior tumor growth inhibition and has high safety and low toxicity profiles. We will perform clinical correlative studies on tumor tissues, tumor-draining lymph nodes (TDLNs), and peripheral blood samples to elucidate the cellular and molecular mechanisms underlying the aPD1-4-1BBL-mediated activation of antitumor immune responses. Our long-term goal is to utilize the insights gathered from this study to perform clinical trials in tumor-bearing canine patients using a caninized aPD1-4-1BBL in support of our ultimate goal of developing an aPD1-4-1BBL construct for human patients that maintains potent antitumor activity without causing hepatotoxicity.
