Mikael Åstrand

Engineering of bispecific affinity proteins with high affinity for ERBB2 and adaptable binding to albumin.

The epidermal growth factor receptor 2, ERBB2, is a well-validated target for cancer diagnostics and therapy. Recent studies suggest that the over-expression of this receptor in various cancers might also be exploited for antibody-based payload delivery, e.g. antibody drug conjugates. In such strategies, the full-length antibody format is probably not required for therapeutic effect and smaller tumor-specific affinity proteins might be an alternative. However, small proteins and peptides generally suffer from fast excretion through the kidneys, and thereby require frequent administration in order to maintain a therapeutic concentration. In an attempt aimed at combining ERBB2-targeting with antibody-like pharmacokinetic properties in a small protein format, we have engineered bispecific ERBB2-binding proteins that are based on a small albumin-binding domain. Phage display selection against ERBB2 was used for identification of a lead candidate, followed by affinity maturation using second-generation libraries. Cell surface display and flow-cytometric sorting allowed stringent selection of top candidates from pools pre-enriched by phage display. Several affinity-matured molecules were shown to bind human ERBB2 with sub-nanomolar affinity while retaining the interaction with human serum albumin. Moreover, parallel selections against ERBB2 in the presence of human serum albumin identified several amino acid substitutions that dramatically modulate the albumin affinity, which could provide a convenient means to control the pharmacokinetics. The new affinity proteins competed for ERBB2-binding with the monoclonal antibody trastuzumab and recognized the native receptor on a human cancer cell line. Hence, high affinity tumor targeting and tunable albumin binding were combined in one small adaptable protein.

Development and characterization of small bispecific albumin-binding domains with high affinity for ErbB3.

Affinity proteins based on small scaffolds are currently emerging as alternatives to antibodies for therapy. Similarly to antibodies, they can be engineered to have high affinity for specific proteins. A potential problem with small proteins and peptides is their short in vivo circulation time, which might limit the therapeutic efficacy. To circumvent this issue, we have engineered bispecificity into an albumin-binding domain (ABD) derived from streptococcal Protein G. The inherent albumin binding was preserved while the opposite side of the molecule was randomized for selection of high-affinity binders. Here we present novel ABD variants with the ability to bind to the epidermal growth factor receptor 3 (ErbB3). Isolated candidates were shown to have an extraordinary thermal stability and affinity for ErbB3 in the nanomolar range. Importantly, they were also shown to retain their affinity to albumin, hence demonstrating that the intended strategy to engineer bispecific single-domain proteins against a tumor-associated receptor was successful. Moreover, competition assays revealed that the new binders could block the natural ligand Neuregulin-1 from binding to ErbB3, indicating a potential anti-proliferative effect. These new binders thus represent promising candidates for further development into ErbB3-signaling inhibitors, where the albumin interaction could result in prolonged in vivo half-life.

ADAPT, a Novel Scaffold Protein-Based Probe for Radionuclide Imaging of Molecular Targets That Are Expressed in Disseminated Cancers.

Small engineered scaffold proteins have attracted attention as probes for radionuclide-based molecular imaging. One class of these imaging probes, termed ABD-Derived Affinity Proteins (ADAPT), has been created using the albumin-binding domain (ABD) of streptococcal protein G as a stable protein scaffold. In this study, we report the development of a clinical lead probe termed ADAPT6 that binds HER2, an oncoprotein overexpressed in many breast cancers that serves as a theranostic biomarker for several approved targeting therapies. Surface-exposed amino acids of ABD were randomized to create a combinatorial library enabling selection of high-affinity binders to various proteins. Furthermore, ABD was engineered to enable rapid purification, to eradicate its binding to albumin, and to enable rapid blood clearance. Incorporation of a unique cysteine allowed site-specific conjugation to a maleimido derivative of a DOTA chelator, enabling radionuclide labeling, ¹¹¹In for SPECT imaging and ⁶⁸Ga for PET imaging. Pharmacologic studies in mice demonstrated that the fully engineered molecule (111)In/⁶⁸Ga-DOTA-(HE)3-ADAPT6 was specifically bound and taken up by HER2-expressing tumors, with a high tumor-to-normal tissue ratio in xenograft models of human cancer. Unbound tracer underwent rapid renal clearance followed by high renal reabsorption. HER2-expressing xenografts were visualized by gamma-camera or PET at 1 hour after infusion. PET experiments demonstrated feasibility for discrimination of xenografts with high or low HER2 expression. Our results offer a preclinical proof of concept for the use of ADAPT probes for noninvasive in vivo imaging.

Influence of Histidine-Containing Tags on the Biodistribution of ADAPT Scaffold Proteins

Engineered scaffold proteins (ESP) are high-affinity binders that can be used as probes for radionuclide imaging. Histidine-containing tags enable both efficient purification of ESP and radiolabeling with (99m)Tc(CO)3. Earlier studies demonstrated that the use of a histidine-glutamate-histidine-glutamate-histidine-glutamate (HE)3-tag instead of the commonly used hexahistidine (H6)-tag reduces hepatic uptake of radiolabeled ESP and short peptides. Here, we investigated the influence of histidine-containing tags on the biodistribution of a novel type of ESP, ADAPTs. A series of anti-HER2 ADAPT probes having H6- or (HE)3-tags in the N-termini were prepared. The constructs, (HE)3-ADAPT6 and H6-ADAPT6, were labeled with two different nuclides, (99m)Tc or (111)In. The labeling with (99m)Tc(CO)3 utilized the histidine-containing tags, while (111)In was attached through a maleimido derivative of DOTA conjugated to the N-terminus. For (111)In-labeled ADAPTs, the use of (HE)3 provided a significantly (p < 0.05) lower hepatic uptake at 1 h after injection, but there was no significant difference in hepatic uptake of (111)In-(HE)3-ADAPT6 and H6-ADAPT6 at later time points. Interestingly, in the case of (99m)Tc, (99m)Tc(CO)3-H6-ADAPT6 provided significantly (p < 0.05) lower uptake in a number of normal tissues and was more suitable as an imaging probe. Thus, the influence of histidine-containing tags on the biodistribution of the novel ADAPT scaffold proteins was different compared to its influence on other ESPs studied so far. Apparently, the effect of a histidine-containing tag on the biodistribution is highly dependent on the scaffold composition of the ESP.

Investigating affinity-maturation strategies and reproducibility of fluorescence-activated cell sorting using a recombinant ADAPT library displayed on staphylococci

During the past decades, advances in protein engineering have resulted in the development of variousin vitroselection techniques (e.g. phage display) to facilitate discovery of new and improved proteins. The methods are based on linkage between genotype and phenotype and are often performed in successive rounds of selection. Since the resulting output depends on the selection pressures used and the applied strategy, parameters in each round must be carefully considered. In addition, studies have reported biases that can cause enrichment of unwanted clones and/or low correlation between abundance in output and affinity. We have recently developed a selection method based on display of protein libraries onStaphylococcus carnosusand isolation of affinity proteins by fluorescence-activated cell sorting. Here, we compared duplicate selections for affinity maturation using equilibrium binding at different target concentrations and kinetic off-rate selection. The results showed that kinetic selection is efficient for isolation of high-affinity binders and that equilibrium selection at subnanomolar concentrations should be avoided. Furthermore, the reproducibility of the selection was high and a clear correlation was observed between enrichment and affinity. This work reports on the reproducibility of bacterial display in combination with FACS and provides insights into selection design to help guide the development of new affinity proteins.

An Orthogonal Fusion Tag for Efficient Protein Purification

Protein fusion tags are important tools in research when robust methods for protein purification and detection are required. In this chapter we present an efficient method for stringent protein purification. A small domain, denoted ABDz1, with affinity for both human serum albumin and Protein A has been developed. The purification tag is based on an albumin-binding domain from Streptococcal Protein G that was engineered to bind Protein A. The ABDz1-tag can be fused to any protein of choice and the purification can be performed using standard laboratory equipment. In this chapter a method for purification of ABDz1-tagged proteins using two successive affinity purification steps is described.