Tumor-selective activation of 4-1BB receptor via bispecific antibody combinations

INTRODUCTION: 4-1BB (CD137) is an inducible co-stimulatory molecule that plays crucial roles in immune activation. Upon ligand binding, 4-1BB forms trimeric signaling complexes triggering receptor activation. However, therapeutic monoclonal antibodies targeting 4-1BB often face challenges due to peripheral toxicities arising from systemic receptor activation. To address this, bispecific antibodies (bsAbs) have been developed to selectively activate 4-1BB in a tumor-associated antigen (TAA)-dependent manner. This report investigates whether a combination of bsAbs, pairing 4-1BB and TAA targeting arms, can further enhance 4-1BB clustering and activation in the tumor microenvironment. METHODS: A panel of Fc-silent bsAbs targeting different domains on 4-1BB and HER2 were generated and tested in combination for optimal 4-1BB signaling. For that purpose, 4-1BB reporter cells were co-cultured with HER2-expressing tumor cells. Functional in vitro assays were conducted using primary human T cells isolated from peripheral blood mononuclear cells to assess cytokine production and their tumoricidal activity. In vivo efficacy was evaluated in PBMC-engrafted NOG mice inoculated with JIMT-1, a HER2-expressing tumor cell line. RESULTS: The simultaneous binding of two bsAbs to the same epitope on 4-1BB, while targeting distinct epitopes on HER2, markedly enhances 4-1BB signaling, compared to the single bsAbs. 4-1BB signaling triggered by the bsAbs is driven by HER2 expression, with strong IL-2 release observed only in the presence of HER2-positive cells. In JIMT-1 xenograft models, combination of the bsAb pair with a T cell engager (TCE) resulted in superior antitumor activity and enhanced CD4⁺ and CD8⁺ T-cell infiltration within the tumor microenvironment, compared to either single bsAb plus TCE or the TCE alone. CONCLUSIONS: This study introduces a novel bsAb combination strategy that amplifies TAA-driven activation of 4-1BB⁺ T cells and may unlock similar opportunities to other TNFR superfamily targets such as OX40 and CD40.