In this work, we have generated bispecific interleukin (IL)‐12 surrogate agonists based on camelid‐derived single‐domain antibodies (sdAbs) targeting the IL‐12 receptor (IL‐12R) subunits IL‐12Rβ1 and IL‐12Rβ2. Following immunization and antibody display‐based paratope isolation, respective sdAbs were combinatorially reformatted into a monovalent bispecific architecture by grafting resulting paratopes onto the hinge region of a heterodimeric Fc region. Functional characterization using NK‐92 cells enabled the identification of multiple different sdAb‐based bispecifics displaying divergent IL‐12R agonism capacities as analyzed by STAT4 phosphorylation. Further investigations by harnessing peripheral blood mononuclear cells (PBMCs) from healthy donors revealed attenuated pSTAT4 activation compared to recombinant human (rh) wild‐type IL‐12 regarding both natural killer (NK)‐cell and T‐cell activation but robust IL‐12R agonism on stimulated T cells. While several sdAb‐based IL‐12 mimetics were nearly inactive on NK cells as well as T cells obtained from PBMCs, they elicited significant STAT4 phosphorylation and interferon (IFN)‐γ release on stimulated T cells as well as an IL‐12‐like transcriptional signature. Furthermore, we demonstrate that the activity of receptor agonism of generated bispecific IL‐12 mimetics can also be biased towards stimulated T cells by changing the spatial orientation of the individual sdAbs within the molecular design architecture. Taken together, we present an alternative strategy to generate IL‐12‐like biologics with tailor‐made characteristics.
