Abstract B067: Agonistic CD137 (4-1BB) anchored immunotherapy (ANK-203) elicits potent 4-1BBL signaling <i>in vitro</i> and therapeutic responses against established tumors without systemic toxicity <i>in vivo</i>

Abstract Background: CD137 (4-1BB) is a costimulatory receptor expressed by activated CD8+ T cells, CD4+ T cells, and natural killer (NK) cells. CD137 agonistic monoclonal antibodies (mAbs) demonstrated preclinical anti-tumor activity, but clinical trials with first-generation CD137 mAbs were limited by dose-dependent hepatotoxicity (urelumab) or lack of efficacy (utomilumab). To overcome on-target, off-tumor toxicity of CD137 agonism, we anchored a fully humanized CD137 monoclonal antibody (mAb) and performed initial in vitro and in vivo characterization. Methods: Human CD137 agonistic mAb was genetically fused with an alum-binding peptide (ABP) and phosphorylated by co-expression with Fam20C protein kinase in permissive cells. Phosphorylation was confirmed by malachite green assay. The CD137-ABP was next mixed with aluminum hydroxide at various ratios and alum binding was measured in alum-retention assays. Alum-anchored CD137-ABP (designated ANK-203) was characterized for in vitro potency using FcR-independent NF-κB reporter cells. hCD137-transgenic C57BL/6 mice with bilateral flank MC38 tumors were used for in vivo therapeutic and immune testing. Primary tumors ∼100 mm3 size were injected intra-tumorally (IT) in the right flank tumor with two doses of ANK-203 (∼30 μg) 7 days apart and mice were evaluated for safety (body weight, liver enzymes) and efficacy (tumor measurements on both injected and noninjected flanks). Additional studies included serum PK, cytokine measurements, and immune cell infiltrates in the tumor by IHC and liver histology. Results: CD137-ABP demonstrated consistent phosphorylation and resulted in stable anchoring to alum. CD137-ABP potentiated NF-κB reporter activity alone and when complexed with alum (ANK-203). In a dual flank MC38 syngeneic tumor model in hCD137-C57BL/6 mice, ANK-203 was well tolerated with no signs of weight loss or significant elevations of ALT or AST. Two doses of ANK-203 induced significant tumor regression in established syngeneic MC38 tumors in hCD137-C57BL/6 mice. Locally administered ANK-203 was also associated with abscopal responses in contralateral noninjected tumors. Conclusions: ANK-203 is the first anchored immunotherapy using a fully humanized CD137 mAb. ANK-203 was associated with enhanced CD137 signaling in vitro and generated significant therapeutic activity in a bilateral MC38 tumor model without signs of systemic toxicity. Further studies to elucidate the impact on immune responses are in progress and ANK-203 may warrant further development for clinical investigation. Anchored immunotherapy represents a novel strategy for improving the therapeutic window of bioactive immune agonists. Citation Format: Robert G. Newman, Kanika Jain, Howard L. Kaufman, Sailaja Battula. Agonistic CD137 (4-1BB) anchored immunotherapy (ANK-203) elicits potent 4-1BBL signaling in vitro and therapeutic responses against established tumors without systemic toxicity in vivo [abstract]. In: Proceedings of the AACR Immuno-Oncology Conference (AACR IO): Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2026 Feb 18-21; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2026;14(2 Suppl):Abstract nr B067.