Abstract
Anticalin® proteins have been proven as versatile clinical stage biotherapeutics. Due to their small size (∼20 kDa), they harbor a short intrinsic plasma half-life which can be extended, e.g., by fusion with IgG or Fc. However, for antagonism of co-immunostimulatory Tumor Necrosis Factor Receptor Superfamily (TNFRSF) members in therapy of autoimmune and inflammatory diseases, a monovalent, pharmacokinetically optimized Anticalin protein format that avoids receptor clustering and therefore potential activation is favored. We investigated the suitability of an affinity-improved streptococcal Albumin-Binding Domain (ABD) and the engineered Fab-selective Immunoglobulin-Binding Domain (IgBD) SpGC3Fab for plasma Half-Life Extension (HLE) of an OX40-specific Anticalin and bispecific Duocalin proteins, neutralizing OX40 and a second co-immunostimulatory TNFRSF member. The higher affinity of ABD fusion proteins to human serum albumin (HSA) and Mouse Serum Albumin (MSA), with a 4 to 5-order of magnitude lower KD compared with the binding affinity of IgBD fusions to human/mouse IgG, translated into longer terminal plasma half-lives (t 1/2). Hence, the anti-OX40 Anticalin-ABD protein reached t 1/2 values of ∼40 h in wild-type mice and 110 h in hSA/hFcRn double humanized mice, in contrast to ∼7 h observed for anti-OX40 Anticalin-IgBD in wild-type mice. The pharmacokinetics of an anti-OX40 Anticalin-Fc fusion protein was the longest in both models (t 1/2 of 130 h and 146 h, respectively). Protein formats composed of two ABDs or IgBDs instead of one single HLE domain clearly showed longer presence in the circulation. Importantly, Anticalin-ABD and -IgBD fusions showed OX40 receptor binding and functional competition with OX40L-induced cellular reactivity in the presence of albumin or IgG, respectively. Our results suggest that fusion to ABD or IgBD can be a versatile platform to tune the plasma half-life of Anticalin proteins in response to therapeutic needs.
Highlights
The receptor/ligand pairs of the Tumor Necrosis Factor Receptor Superfamily (TNFRSF) including OX40/OX40L have a prominent role as controllers of T lymphocyte activation and survival, but are associated with exacerbating effects in inflammatory processes and autoimmunity (Webb et al, 2016)
The Anticalin protein binding to human OX40 (OX40 Ac), the parental scaffold protein (NGAL Ac) and a bispecific Duocalin protein composed of two Anticalin proteins were fused at their C-termini either with Albumin-Binding Domain (ABD) or Immunoglobulin-Binding Domain (IgBD) using Gly/Ser peptide linkers with a length of 10 amino acid residues
Duocalin proteins with the capability to antagonize two receptor species were created by joining OX40 Ac via a 15residue peptide linker with an Anticalin protein directed against a second, non-disclosed co-immunoregulatory TNFRSF member, designated as Rec2 Ac
Summary
The receptor/ligand pairs of the Tumor Necrosis Factor Receptor Superfamily (TNFRSF) including OX40/OX40L have a prominent role as controllers of T lymphocyte activation and survival, but are associated with exacerbating effects in inflammatory processes and autoimmunity (Webb et al, 2016). Anticalin libraries generated by introducing amino acid diversification at certain positions of two lipocalin scaffolds, Neutrophil Gelatinase-Associated Lipocalin (NGAL) or Tear lipocalin (Tlc), can be applied in standard display technologies to select Anticalin proteins with specific binding properties for use in diagnostics or therapy (Skerra, 2008). Due to their small size of ∼20 kDa, Anticalin proteins are rapidly cleared via renal filtration when systemically administered, sharing the fate of most proteins with a mass below ∼60 kDa or a small hydrodynamic radius (Kontermann, 2011). In light of biopharmaceutical development, pharmacokinetic engineering of naked Anticalin proteins can be applied to adjust and extend the inherently short in vivo circulation to the specific program need and even reach an antibody-like plasma half-life if necessary
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