mRNA Display selection of a novel activated leukocyte cell adhesion molecule (ALCAM) binding protein from a modified combinatorial protein library based on the tenth domain of human fibronection III (10FnIII)

Antibodies have served as the preeminent model for proteins tailored to exhibit specific binding properties. Unfortunately, the development and manufacturing of antibodies is an expensive and time-consuming process. Recently, however, the field of drug targeting has gravitated towards developing smaller, alternative binding proteins that harness the targeting power of antibodies, but are produced at more expedient rates and with lower costs. In vitro display platforms allow for the discovery of novel binding proteins from highly complex libraries of non-immunoglobulin scaffolds. The tenth domain of human fibronectin III (10FnIII) molecule is one of these extensively studied antibody mimics that demonstrate promise as an antibody alternative in targeted therapeutics and in diagnostic imaging. In this study, mRNA display was applied to screen for a novel binding protein specific to a known cancer biomarker, activated leukocyte cell adhesion molecule (ALCAM), in a previously modified combinatorial protein library based on the tenth domain of human fibronectin III (e10FnIII). Iterative rounds of affinity selection resulted in the discovery of a single e10FnIII variant, designated Fn16.3, which specifically binds ALCAM in vitro. In bacteria, Fn16.3 was robustly expressed but formed insoluble aggregates. These results demonstrate that in vitro selection can be used to isolate novel binding proteins, but that further evolution of functional clones may be required to generate a binding molecule that can also be expressed in a desirable expression system