LIF and let die: Generation, mechanistical and structural characterization of bispecific antibodies triggering leukemia inhibitory factor (LIF) receptor activation independent of the cytokine LIF.

Here, we report on the generation and characterization of single-domain antibody (sdAb)-derived bispecifics that mimic the function of LIF by triggering agonism on its heterodimeric receptor composed of the subunits LIF receptor (LIFR) and glycoprotein 130 (gp130). For this, LIFR-targeting sdAbs were identified by yeast surface display (YSD) after camelid immunization. Resulting 14 LIFR-specific paratopes were expressed in a bispecific antibody (bsAb) format all harboring the identical gp130-adressing sdAb. Profiling of bsAbs on Ba/F3 cells expressing human LIFR as well as gp130 revealed LIF-like functionality of one bsAb as characterized by the induction of cell proliferation. Cryo-EM structural analysis revealed that the bsAb targets a distinct LIFR epitope, indicating the formation of a receptor architecture that differs from previously reported LIF signaling complex structures. In addition, the identified sdAb-based bispecific LIF mimetic elicited STAT3 phosphorylation in HeLa as well as TF-1 cells naturally expressing the LIF receptor subunits. Moreover, agonism capabilities were further improved by antibody engineering. Taken together, we demonstrate that cytokine members of the IL-6 family can be mimicked by harnessing sdAbs as building blocks.