Targeting insulin receptor in breast cancer using small engineered protein scaffolds

Insulin receptor (InsR) and the type I insulin-like growth factor (IGF1R) are homologous receptors necessary for signal transduction by their cognate ligands insulin, insulin-like growth factor-I and -II (IGF-I and IGF-II). IGF1R monoclonal antibodies, intended to inhibit malignant phenotypic signaling, failed to show benefit in patients with endocrine-resistant tumors in phase III clinical trials. Our previous work showed that in tamoxifen-resistant cells IGF1R expression was lacking but InsR inhibition effectively blocked growth. In endocrine-sensitive breast cancer cells, insulin was not growth stimulatory, likely due to the presence of hybrid InsR/IGF1R, which has high affinity for IGF-I, but not insulin. Combination inhibition of InsR and IGF1R showed complete suppression of the system in endocrine-sensitive breast cancer cells. To develop InsR-binding agents, we employed a small protein scaffold, T7 phage Gene 2 Protein (Gp2) with the long-term goal of creating effective InsR inhibitors and diagnostics. Using yeast display and directed evolution, we identified three Gp2 variants (Gp2 #1, #5 and #10) with low nanomolar affinity and specific binding to cell-surface InsR. These Gp2 variants inhibited insulin-mediated monolayer proliferation in both endocrine-sensitive and -resistant breast cancer, but did not downregulate InsR expression. Gp2 #5 and Gp2#10 disrupted InsR function by inhibiting ligand-induced receptor activation. In contrast, Gp2 #1 did not block InsR phosphorylation. Notably, Gp2 #1 binding was enhanced by pre-treatment of cells with insulin suggesting a unique receptor-ligand binding mode. These Gp2 variants are the first non-immunoglobulin protein scaffolds to target insulin receptor and present compelling opportunity for modulation of InsR signaling.

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