Antigen-directed single domain antibody-based TNFR1 agonists elicit preferential killing of HER2-overexpressing cancer cells.

In this study, we present a strategy to uncouple tumor necrosis factor (TNF)-like cell death induction from TNFR2 agonism in a tumor-targeted fashion. Single-domain antibodies (sdAbs) targeting TNFR1 were generated by combining camelid immunization with yeast surface display. Reformatting of resulting paratopes as bispecific antibodies (bsAbs) in a 2 + 2 manner by employing an sdAb-based paratope targeting HER2 revealed the identification of an immunocytokine-like bsAb, referred to as immunocytokine mimetic (ICM), which triggered TNF-like tumor cell death of HER2-overexpressing cancer cells as well as robust caspase-1, -3, and -8 activation in a cis-targeted manner. By modulating the valency of the TNFR1-directed sdAb, killing capacities as well as caspase activities of HER2-targeted ICMs were significantly augmented, eventually resulting in enhanced cell death induction when compared with TNF. Moreover, HER2-targeted TNFR1 ICMs also displayed a beneficial, i.e., substantially reduced profile in inducing unconditional pro-inflammatory cytokine release from peripheral blood mononuclear cells (PBMCs).