A humanized anti-TNFR1 nanobody antagonist targeting the CRD3 region suppresses inflammation and necroptosis in vitro

Aberrant signaling through tumor necrosis factor receptor 1 (TNFR1) drives chronic inflammation and necroptotic cell death, yet selective therapeutic strategies remain limited. Here, humanized nanobodies targeting TNFR1 were developed and characterized. Biolayer interferometry and cell-based analyses showed that these nanobodies remained in the nanomolar affinity range to TNFR1 after humanization and showed no detectable binding to TNFR2 under the tested conditions. Functional assays demonstrated that the nanobodies inhibited TNF induced NF-κB activation and suppressed RIPK1-RIPK3-MLKL associated necroptotic signaling in vitro. Epitope mapping, competition binding, and docking analyses supported binding to a CRD3 containing region of TNFR1 that overlaps with the TNFα binding surface. Comparative analyses indicated that the Nb1 lineage showed slower dissociation and stronger antagonistic activity than the Nb21 lineage. Moreover, engineering Nb1 into a bivalent format yielded inhibitory activity in the same range as a clinical anti-TNFα antibody in vitro, while maintaining selectivity over the tested related receptors. These findings support TNFR1 selective nanobodies as a compact biologic format for further development against TNFR1 driven inflammatory and necroptotic responses. Humanized TNFR1-targeting nanobodies selectively block inflammatory and necroptotic signaling in vitro, offering a compact antagonist format for modulating pathological TNFR1 responses.