Abstract
The HER3 receptor is implicated in the progression of various cancers as well as in resistance to several currently used drugs, and is hence a potential target for development of new therapies. We have previously generated Affibody molecules that inhibit heregulin-induced signaling of the HER3 pathways. The aim of this study was to improve the affinity of the binders to hopefully increase receptor inhibition efficacy and enable a high receptor-mediated uptake in tumors. We explored a novel strategy for affinity maturation of Affibody molecules that is based on alanine scanning followed by design of library diversification to mimic the result from an error-prone PCR reaction, but with full control over mutated positions and thus less biases. Using bacterial surface display and flow-cytometric sorting of the maturation library, the affinity for HER3 was improved more than 30-fold down to 21 pM. The affinity is among the higher that has been reported for Affibody molecules and we believe that the maturation strategy should be generally applicable for improvement of affinity proteins. The new binders also demonstrated an improved thermal stability as well as complete refolding after denaturation. Moreover, inhibition of ligand-induced proliferation of HER3-positive breast cancer cells was improved more than two orders of magnitude compared to the previously best-performing clone. Radiolabeled Affibody molecules showed specific targeting of a number of HER3-positive cell lines in vitro as well as targeting of HER3 in in vivo mouse models and represent promising candidates for future development of targeted therapies and diagnostics.
Highlights
The HER3 receptor is part of the epidermal growth factor receptor family, which is a group of tyrosine kinase receptors that mediate normal cellular functions such as proliferation and cell-tocell interactions, but have been recognized as drivers of many different human cancers [1]
Variants with alanine mutations at positions 9, 10, and 17 demonstrated substantially lower signals compared to the control, indicating a reduced affinity for HER3 and that the corresponding original residues were contributing to the interaction (Figure 1A)
Prior to synthesizing the affinity maturation library, we wanted to confirm that the HER3-specific Affibody molecule was compatible with the eleven mutations in the framework
Summary
The HER3 receptor is part of the epidermal growth factor receptor family, which is a group of tyrosine kinase receptors that mediate normal cellular functions such as proliferation and cell-tocell interactions, but have been recognized as drivers of many different human cancers [1]. The importance of HER3 in human cancers is not limited to HER2-driven breast cancers, and HER3 has been shown to be involved in for example tumorigenicity of HER3-overexpressing prostate cancer xenografts in vivo, to maintain in vivo proliferation of a subset of ovarian cancers via an autocrine signaling loop, and to be involved in endocrine resistance of ER+ breast cancer cell lines [10,11,12] These findings demonstrate the potential of the HER3signalling pathway as an important therapeutic target in human cancers and several anti-HER3 antibodies are currently under clinical investigations [13].
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