Taming interleukin‐12: Engineering of bispecific antibody‐based <scp>IL</scp>‐12 mimetics with biased agonism capacities

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.