Abstract4-1BB agonism represents a promising strategy in cancer immunotherapy, targeting the 4-1BB receptor (CD137, TNFRSF9) expressed on activated T cells and NK cells. This co-stimulatory receptor, when activated by receptor multimerization, enhances immune cell proliferation, survival and effector functions. Agonistic antibodies against 4-1BB, optimized for FcγR cross-linking, have demonstrated potent anti-tumor activity in preclinical models. However clinical development has been hindered by severe liver toxicity (i.e., urelumab), or insufficient single agent activity, as seen with utomilumab. Here we describe a novel MULTi-specific, multi-Affinity antiBODY (Multabody™, MB) platform that exploits multi-valency to deliver an innovative 4-1BB Multabody™ with broad therapeutic potential. To understand the impact of epitope on 4-1BB agonism, we generated a panel of 4-1BB Multabodies to unlock comprehensive comparisons of potency and maximal activity of these molecules in a suite of assays including a 4-1BB Jurkat reporter assay vs urelumab and utomilumab. We identified 4-1BB MBs that demonstrated a range of Fc-independent agonistic profiles, including those mimicking the natural ligand, 4-1BBL. Identified lead 4-1BB MB candidate, RBT101, was further evaluated in vivo using the syngeneic MC38 murine tumor model. RBT101 demonstrated robust in vivo efficacy resulting in durable complete tumor regressions. Unlike urelumab which demonstrated splenomegaly and liver inflammation, RBT101 showed no signs of spleen or liver toxicity. Furthermore, RBT101 also provided long-term protection in a tumor rechallenge study, suggesting generation of a durable immunological memory response driven by RBT101. These data suggest the Multabody™ platform provides a novel approach to delivering a 4-1BB therapeutic with a differentiated profile to address an unmet clinical need in cancer immunotherapy for patients with solid malignancies.Citation Format:Joanne Hulme, Peter Bayliss, Abilasha Rao, Shrayasee Saha, Xinwen He, Debbie Jin, Yvonne Tsao, Amy Berkley. Multabodies: A next-generation approach for cancer immunotherapy and 4-1BB agonist therapy [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 2889.