Abstract
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.
Highlights
Numerous studies have demonstrated that smaller affinity proteins can be engineered for high affinity and specificity and are expected to be excellent alternatives to antibodies for clinical applications
Phage display selection of ABD-Derived Affinity ProTeins (ADAPTs) was performed in three rounds using recombinant human ERBB2 as target
Circular dichroism (CD) spectroscopy demonstrated that all variants shared a spectrum similar to what has been measured previously for albumin-binding domain (ABD), indicating a retained three-helical structure [23]
Summary
Numerous studies have demonstrated that smaller affinity proteins can be engineered for high affinity and specificity and are expected to be excellent alternatives to antibodies for clinical applications. Several non-immunoglobulin binding proteins have been generated against different biomarkers, for example designed ankyrin repeat proteins [1,2] and Affibody molecules [3,4] Such small binding proteins have shown quick biodistribution, good penetration into tumor tissue and fast elimination from serum and nondiseased tissues [5,6]. Small size is generally attractive for therapy, while a longer serum half-life is usually necessary to extend the exposure time of a drug and to limit the dosing frequency. Both experiments and modeling approaches have identified two groups of affinity reagents with a high potential for solid tumor targeting [7,8,9]. The first group constitutes very small molecules with a high affinity for the tumor target, but generally with short half-lives
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