Fusion to an IgG Fc region is an established strategy to extend the half-life of therapeutic proteins. Most Fc fusion proteins, however, do not achieve the long half-life of IgGs. Based on findings that scFv-Fc fusion proteins exhibit a shorter half-life than the corresponding IgG molecules, we performed a comparative study of different antibodyderived Fc fusion proteins. We could confirm that fusion of single-chain Fv (scFv) and single-chain diabody (scDb) molecules to an Fc region yields in fusion proteins with substantially extended half-lives compared with the singlechain versions. However, even fusion proteins with a size similar to that of IgG, e.g., scDb-Fc, did not have a half-life as long as an IgG molecule. Binding to the neonatal Fc receptor (FcRn) under acidic and neutral conditions was similar for IgG and all Fc fusion proteins. However, we observed differences between IgG and the Fc fusion proteins for dissociation of FcRn-bound proteins induced by shifting from acidic to neutral pH, reflecting the physiological release mechanism, further supporting a contribution of the kinetics of pH-dependent release from FcRn to the pharmacokinetic properties of IgG and Fc fusion proteins.

The TNF-related apoptosis-inducing ligand (TRAIL) has been considered as a promising molecule for cancer treatment. However, clinical studies with soluble TRAIL failed to show therapeutic activity, which resulted in subsequent development of more potent TRAIL-based therapeutics. In this study, we applied defined oligomerization and tumor targeting as strategies to further improve the activity of a single-chain version of TRAIL (scTRAIL). We compared three different formats of EGF receptor (EGFR)-targeting dimeric scTRAIL fusion proteins [Diabody (Db)-scTRAIL, scFv-IgE heavy chain domain 2 (EHD2)-scTRAIL, scFv-Fc-scTRAIL] as well as two nontargeted dimeric scTRAIL molecules (EHD2-scTRAIL, Fc-scTRAIL) to reveal the influence of targeting and protein format on antitumor activity. All EGFR-targeted dimeric scTRAIL molecules showed similar binding properties and comparable cell death induction , exceeding the activity of the respective nontargeted dimeric format and monomeric scTRAIL. Superior properties were observed for the Fc fusion proteins with respect to production and half-life. studies using a Colo205 xenograft model revealed potent antitumor activity of all EGFR-targeting formats and Fc-scTRAIL and furthermore highlighted the higher efficacy of fusion proteins comprising an Fc part. Despite enhanced cell death induction of targeted scTRAIL molecules, however, comparable antitumor activities were found for the EGFR-targeting scFv-Fc-scTRAIL and the nontargeting Fc-scTRAIL .

Background: Half-life extension has become increasingly important for therapeutic proteins. Results: Fusion of different bacterial immunoglobulin-binding domains to small recombinant antibodies prolonged their half-life to varying extents. Conclusion: Fusion of domain C3 of streptococcal protein G showed the best effects, thus representing a promising module for half-life extension of small-sized therapeutics. Significance: This study further established immunoglobulin-binding domains as suitable half-life extension modules.

Fusion proteins combining oligomeric assemblies of a genetically obtained single-chain (sc) variant of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) with antibodies directed against tumorassociated antigens represent a promising strategy to overcome the limited therapeutic activity of conventional soluble TRAIL. To further improve the scTRAIL module in order to obtain a robust, thermostable molecule of high activity, we performed a comprehensive analysis of the minimal TNF homology domain (THD) and optimized linkers between the 3 TRAIL subunits constituting a scTRAIL. Through a stepwise mutagenesis of the N-and C-terminal region and the joining linker sequences, we generated bioactive scTRAIL molecules comprising a covalent linkage of the C-terminal Val280 and the Nterminal position 122 by only 2 amino acid residues in combination with conservative exchanges at positions 122 and 279. The increased thermal stability and solubility of such optimized scTRAIL molecules translated into increased bioactivity in the diabody-scTRAIL (Db-scTRAIL) format, exemplified here for an epidermal growth factor receptor-specific Db-scTRAIL. Additional modifications within the diabody linkers resulted in a fusion protein exerting high, target-dependent apoptosis induction in tumor cell lines in vitro and potent antitumor activity in vivo. Our results illustrate that protein engineering of scTRAIL and associated peptide linkers provides a promising strategy to develop antibody-scTRAIL fusion proteins as effective antitumor therapeutics.

Human epidermal growth factor receptor 3 (HER3, also known as ErbB3) has emerged as relevant target for antibody-mediated tumor therapy. Here, we describe a novel human antibody, IgG 3-43, recognizing a unique epitope formed by domain III and parts of domain IV of the extracellular region of HER3, conserved between HER3 and mouse ErbB3. An affinity of 11 nM was determined for the monovalent interaction. In the IgG format, the antibody bound recombinant bivalent HER3 with subnanomolar affinity (K = 220 pM) and HER3-expressing tumor cells with EC values in the low picomolar range (27 - 83 pM). The antibody competed with binding of heregulin to HER3-expressing cells, efficiently inhibited phosphorylation of HER3 as well as downstream signaling, and induced receptor internalization and degradation. Furthermore, IgG 3-43 inhibited heregulin-dependent proliferation of several HER3-positive cancer cell lines and heregulin-independent colony formation of HER2-overexpressing tumor cell lines. Importantly, inhibition of tumor growth and prolonged survival was demonstrated in a FaDu xenograft tumor model in SCID mice. These findings demonstrate that by binding to the membrane-proximal domains III and IV involved in ligand binding and receptor dimerization, IgG 3-43 efficiently inhibits activation of HER3, thereby blocking tumor cell growth both in vitro and in vivo.