Cell-Surface Expression Levels Are Important for Fine-Tuning the Performance of Receptor Tyrosine Kinase-Based Signalobodies

Abstract

As receptor tyrosine kinases (RTKs) play important roles in cell-fate control of various cell types, engineered RTKs that could respond to inexpensive ligands might drastically reduce the cost of producing desired cells for various applications in regenerative medicine. We developed several engineered RTKs named "signalobodies" in which the ligand-recognition domain of RTKs is replaced by single-chain Fv for enabling recognition of a specific antigen. However, the remaining concern was the dysregulation of antigen-dependent on/off signaling of the signalobodies. This study aims at fine-tuning the performance of the signalobodies based on three RTKs (fibroblast growth factor receptor 1, insulin receptor, and c-fms). To this end, the cell-surface expression levels of the RTK-based signalobodies were altered by locating their genes either upstream or downstream of the internal ribosomal entry site, and by inserting 1 to 3 alanine residue(s) at the intracellular juxtamembrane region. As a result, while the signaling response was different among the three signalobodies, the antigen-dependent on/off regulation became tighter when the cell-surface expression levels of the signalobodies were lowered. Therefore, we successfully developed a method to diminish the leaky signaling of RTK-based signalobodies, which will be important for establishing the signalobody-based platform technology that can produce cells of interest for regenerative medicine.

Keywords: antibody; cell proliferation; chimeric protein; mammalian cell; receptor tyrosine kinase.