Engineered Bivalent Ligands to Bias ErbB Receptor-mediated Signaling and Phenotypes

Abstract

The ErbB receptor family is dysregulated in many cancers, and its therapeutic manipulation by targeted antibodies and kinase inhibitors has resulted in effective chemotherapies. However, many malignancies remain refractory to current interventions. We describe a new approach that directs ErbB receptor interactions, resulting in biased signaling and phenotypes. Due to known receptor-ligand affinities and the necessity of ErbB receptors to dimerize to signal, bivalent ligands, formed by the synthetic linkage of two neuregulin-1β (NRG) moieties, two epidermal growth factor (EGF) moieties, or an EGF and a NRG moiety, can potentially drive homotypic receptor interactions and diminish formation of HER2-containing heterodimers, which are implicated in many malignancies and are a prevalent outcome of stimulation by native, monovalent EGF, or NRG. We demonstrate the therapeutic potential of this approach by showing that bivalent NRG (NN) can bias signaling in HER3-expressing cancer cells, resulting in some cases in decreased migration, inhibited proliferation, and increased apoptosis, whereas native NRG stimulation increased the malignant potential of the same cells. Hence, this new approach may have therapeutic relevance in ovarian, breast, lung, and other cancers in which HER3 has been implicated.

Engineered Multivalency Enhances Affibody-Based HER3 Inhibition and Downregulation in Cancer Cells

“…Thus, employing the Z 05413 affibody 27 as the HER3 binding domain and a flexible protease resistant peptide spacer 22 as a linker domain, bivalent and trivalent HER3 affibodies were designed (Figure 1A). Multivalent affibodies were expressed as recombinant proteins in Bl21 (DE3) E. coli and purified by immobilized metal ion chromatography and size-exclusion HPLC (Figure 1B).…”

“…17 The flexible, glycine rich, protease resistant spacer domain employed for the bivalent and trivalent affibodies described in Figure 1 contains three repeat units and has a predicted length of approximately 20 nm (nm), long enough to traverse the theoretical 10 nm distance required to sequester a cross-facing HER3 homodimer. 22 To systematically investigate the role of linker domain size on efficacy, a panel of bivalent affibodies was engineered by varying the number of repeat units in the spacer peptide from 1 to 4 (each unit having a predicted length of ~7 nm) (Figure 3A,B). Inhibitory efficacy against HER3 and Akt phosphorylation across multiple cell types with bivalent affibodies of various linker domain sizes was observed to be similar, with no consistent trends relating spacer domain length to inhibitory efficacy (Figures 3C and S7).…”

Emerging anti-cancer antibodies and combination therapies targeting HER3/ERBB3

“…Finally, an original strategy based on HER3 restricted ability to autophosphorylate was developed by Jay and colleagues, who made use of bivalent ligands. 90 In principle, the engineered ligands lock HER3/ERBB3 in a homodimeric conformation, thereby prevent HER3 from forming powerful heterodimers, such as HER2-HER3 or EGFR-HER3.…”

“…(2) Since HER3 homodimers are weakly active compared to the heterodimers HER3-HER2 and HER3-EGFR, a recombinant bivalent-NRG has been developed that locks HER3 in the homodimeric conformation and restricts its ability to form heterodimers. 90 (3) Several monoclonal and multispecific antibodies have been developed to target HER3 and its dimerization partners, leading them to degradation or/and avoiding their phosphorylation. 172 Some of these antibodies are able to trigger CDC or ADCC.…”

Tailoring Chimeric Ligands for Studying and Biasing ErbB Receptor Family Interactions

“…[3] ” Preliminary studies with cell lines treated with bivalent ligands for EGFR family members exhibit phenotypic and signaling behaviors consistent with a biasing of receptor dimerization or oligomerization away from those involving HER2, compared to treatment with monovalent ligands (as illustrated in Figure 1). Early prototypes of these bivalent ligands were created by recombinantly expressing individual ligands (either neuregulin-1β (NRG) or epidermal growth factor (EGF)) fused to one partner of a coiled-coil monomer motif and joining the desired ligands non-covalently, (later a bivalent single-chain construct was expressed) [4] with a fixed amino acid linker spacing.…”