Antibody framework engineering enables augmented cytokine receptor signaling capacities of bispecific single domain antibody-based IL-21 mimetics.

In this work, we developed bispecific antibody (bsAb)-derived surrogate agonists which mimic the function of IL-21 by targeting the IL-21 receptor composed of IL-21 R (CD360) and IL-2 Rγ (CD132). For this, antigen-specific VHHs (variable domains of the heavy chain of heavy-chain-only antibodies) were obtained by immunization of camelids and isolated using yeast surface display. Combinatorial reformatting of IL-21 R-specific single‑domain antibodies (sdAbs) and IL-2 Rγ-targeting paratopes into a monovalent bispecific antibody architecture enabled the identification of IL-21 mimetics displaying attenuated capacities in triggering STAT3 phosphorylation compared to the wild-type cytokine as demonstrated in NK-92 cells as well as peripheral blood mononuclear cells (PBMCs). Moreover, by applying different protein engineering strategies, we demonstrate that agonism capacities of the generated IL-21 mimetics, such as pSTAT3 induction or Granzyme B expression of cytotoxic T cells, can be significantly optimized. For this, framework mutations were introduced to engineer VHH:VHH interactions within the bispecific sdAb-Fc fusion geometry for a more rigid receptor targeting. Furthermore, we show that antibody format engineering, in which the VHHs were arranged in an IgG-like scaffold that replaces the conventional IgG VH and VL domains with the corresponding VHHs, combined with rigidifying mutations, enables IL-21 R agonism comparable to the wild-type cytokine. Taken together, these findings show that IL-21 receptor agonism can be substantially optimized by adapting the spatial orientation of paratopes targeting both receptor subunits via forced dimerization, without altering paratope valencies.