Abstract 4794: Anti-VEGF x 4-1BB bifunctional AND-Body combines VEGF blockade and allosteric 4-1BB mechanism for sustained activation of adaptive and innate anti-tumor immunity

Abstract Background: Bifunctional therapeutics that combine synergistic mechanisms in a single molecule (e.g., anti-PD-1/VEGF) have recently shown great promise in the clinic. The potent co-stimulatory receptor 4-1BB represents a powerful pathway to apply these bifunctional mechanisms, where growing evidence supports a unique ability to drive sustained activation of both adaptive and innate immunity. We leveraged our AND-Body platform to develop a novel anti-VEGF x 4-1BB bifunctional therapeutic, which simultaneously blocks VEGF and triggers allosteric activation of the 4-1BB pathway in the tumor, thereby increasing the therapeutic window. In preclinical models, tumor VEGF drives potent 4-1BB agonism in a cis/paracrine mechanism, enhancing tumor killing and mitigating systemic activity in tissues where VEGF levels are low. This represents a new approach to bifunctional cancer therapeutics. Methods: The VEGF x 4-1BB bifunctional was evaluated in humanized 4-1BB transgenic mice harboring MC38 or B16F10 tumors and compared to aflibercept and urelumab. Tumor growth, survival rates, immune cell infiltration, and adverse events were assessed, including scRNA sequencing of tumor-infiltrating lymphocytes. The molecule was also tested in combination with anti-PD-1. Results: The AND-Body robustly activated 4-1BB signaling only at high concentrations of VEGF in vitro. Monotherapy induced durable, localized anti-tumor immunity in MC38 and B16F10 models, leading to enhanced survival, and 90% of animals were tumor-free upon re-challenge with MC38 vs. 47% in the urelumab group. Combination therapy with anti-PD-1 showed additive benefit with 100% tumor-free survival in the MC38 model and extended survival in B16F10 tumor-bearing mice. AND-Body treatment mitigated hepatotoxicity and dermatitis compared to urelumab. Mechanistically, VEGF x 4-1BB bifunctional promoted progenitor exhausted T cell expansion and enhanced M1 macrophage differentiation distinct from other immune checkpoint inhibitors. Treatment normalized the TME, promoting aerobic respiration, preventing T cell exhaustion, and supporting sustained anti-tumor immunity. Conclusions: Anti-VEGF x 4-1BB AND-Body represents a novel bifunctional agent, combining established benefits of VEGF blockade with an allosteric 4-1BB agonist. Potent anti-tumor activity with high therapeutic index was achieved in preclinical models by localizing immune responses via a unique mechanism of action. These studies support advancement to the clinic as a potentially first-in-class candidate for cancer immunotherapy, both as a monotherapy and in combinations. Citation Format: Xiaofei Zhou, Michelle Yen, Joseph R. Brady, Oliver Yvon, Marwa Bsat, Kyriakos D. Economides, Daniël Blom. Anti-VEGF x 4-1BB bifunctional AND-Body combines VEGF blockade and allosteric 4-1BB mechanism for sustained activation of adaptive and innate anti-tumor immunity [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 4794.