Facile Display of Homomultivalent Proteins for In Vitro Selections

Abstract

Low-affinity protein binders are emerging as valuable domains for therapeutic applications because of their higher specificity when presented in multivalent ligands that increase the overall strength and selectivity of receptor binding. De novo discovery of low-affinity binders would be enhanced by the large library sizes attainable with in vitro selection systems, but these platforms generally maximize recovery of high-affinity monovalent binders. Here, we present a facile technology that uses rolling circle amplification to create homomultivalent libraries. We show proof of principle of this approach in ribosome display with off-rate selections of a bivalent ligand against monovalent and bivalent targets, thereby demonstrating high enrichment (up to 166-fold) against a low-affinity target that is bivalent but not monovalent. This approach to homomultivalent library construction can be applied to any binder tolerant of N- and C-terminal fusions and provides a platform for performing in vitro display selections with controlled protein valency and orientation.

Small Antibodies with Big Applications: Nanobody-Based Cancer Diagnostics and Therapeutics

“…There are not many studies using yeast surface display to generate nanobodies against cancer-related molecules. In 2022, Meltzer et al published a paper that created nanobodies against the extracellular domain of Tie1, an orphan receptor tyrosine kinase, via yeast-surface-displayed naïve and predesigned synthetic (non-immune) nanobody libraries [13,26]. The nanobody triggered a Tie1-dependent inhibition of RTK phosphorylation and angiogenesis in endothelial cells, as well as suppression of human glioblastoma cell viability and migration.…”

“…However, low-affinity nanobodies are also receiving attention. Ayako Ohoka et al have introduced a platform specifically targeting low-affinity multivalentspecific antibodies [26]. Ribosome display technology should be noted for its high cost, generation of nanobodies for many purposes as well as the poor stability of the "mRNAribosome-protein" ternary complexes, which may interfere with the proper performance of the display system.…”