Intrabody targeting of insulin receptor pY1150: A novel approach to modulate signaling pathways in colorectal cancer cells.

The activation of the insulin receptor (IR) is central to the regulation of physiological metabolism, growth, and proliferation, and is associated with various cancers, including colorectal cancer (CRC). Among the tyrosine residues in the intracellular kinase domain of IR, Tyr1150 plays a pivotal role in receptor activation by regulating substrate binding to the kinase active site. In this study, we introduce a novel approach for selectively modulating insulin signaling in CRC through the development of an intrabody targeting phosphorylated Tyr1150 (IR pY1150). Using phage display technology, we isolated a phosphorylation site-specific single-chain variable fragment (scFv), K109.1, from a human scFv antibody library, which specifically binds to IR pY1150. K109.1 was subsequently engineered as an intrabody designed to function within cells. Ectopic expression of K109.1 in CRC cells selectively inhibited insulin-mediated phosphorylation of key downstream effectors, including insulin receptor substrates and Akt, thereby leading to a significant reduction in insulin-dependent glucose uptake. Notably, K109.1 did not affect extracellular signal-regulated kinase phosphorylation or alter cell proliferation, migration, or invasion. We further evaluated K109.1 in BT-474 and HEK293 cells to assess its effects in additional cellular models. In BT-474 breast cancer cells, K109.1 selectively inhibited Akt phosphorylation, while in HEK293 cells it suppressed both Akt and ERK phosphorylation, indicating context-specific signaling responses. Taken together, these findings indicate that intrabody-mediated targeting of IR pY1150 is crucial for regulating glucose metabolism, suggesting that the developed antibody, K109.1, could serve as a tool for modulating insulin-mediated signaling pathways.