Combination Of Bispecific Antibodies Enable Targeted TNFRSF Agonism and Effective Antitumor Activity.

4-1BB is an inducible costimulatory molecule that requires receptor clustering to boost anti-cancer immune response. However, the clinical development of monoclonal antibodies targeting 4-1BB has been constrained by toxicities driven by systemic activation. To address this, bispecific antibodies (bsAbs) have been developed to selectively activate 4-1BB in a HER2-dependent manner. 4-1BB activation was assessed using 4-1BB-GFP and reporter cells, before evaluating the cytokine production and tumoricidal activity of primary T cells. In vivo antitumor efficacy was assessed in PBMC-xenograft and syngeneic h4-1BB/h4-1BBL transgenic (Tg) models, the latter enabling comparison of systemic inflammatory responses with urelumab. Combining two 4-1BB x HER2 bsAbs that share a 4-1BB epitope but bind non-overlapping HER2 epitopes markedly enhanced 4-1BB clustering and signaling, and correlated with HER2 expression levels, with strong T-cell activation and tumor growth inhibition. In the PBMC-xenograft model, combining the bsAb pair with a T-cell engager potentiated antitumor activity and increased T-cell infiltration within the tumor microenvironment. In the syngeneic Tg model, the paired bsAbs maintained robust efficacy while mitigating peripheral inflammation compared to urelumab. Overall, we established a bsAb combination strategy that amplifies HER2-driven 4-1BB+ T-cell activation and provides an applicable framework for targeting other tumor necrosis factor receptor superfamily members.