Development and Differentiation in Monobodies Based on the Fibronectin Type 3 Domain

Abstract

As a non-antibody scaffold, monobodies based on the fibronectin type III (FN3) domain overcome antibody size and complexity while maintaining analogous binding loops. However, antibodies and their derivatives remain the gold standard for the design of new therapeutics. In response, clinical-stage therapeutic proteins based on the FN3 domain are beginning to use native fibronectin function as a point of differentiation. The small and simple structure of monomeric monobodies confers increased tissue distribution and reduced half-life, whilst the absence of disulphide bonds improves stability in cytosolic environments. Where multi-specificity is challenging with an antibody format that is prone to mis-pairing between chains, multiple FN3 domains in the fibronectin assembly already interact with a large number of molecules. As such, multiple monobodies engineered for interaction with therapeutic targets are being combined in a similar beads-on-a-string assembly which improves both efficacy and pharmacokinetics. Furthermore, full length fibronectin is able to fold into multiple conformations as part of its natural function and a greater understanding of how mechanical forces allow for the transition between states will lead to advanced applications that truly differentiate the FN3 domain as a therapeutic scaffold.

Reconstitution of an Anti‐HER2 Antibody Paratope by Grafting Dual CDR‐Derived Peptides onto a Small Protein Scaffold

“…[ 23 ] These monobodies were developed primarily using technologies such as phage display and mRNA display to select binders from libraries with randomized sequences in up to three loops present in the FN3 protein. [ 24,25 ] Our group has also succeeded in creating HER2‐binding small proteins based on the FN3 protein scaffold through conventional phage display library screening and subsequent affinity maturation. [ 18 ] In this study, an FN3‐based antibody mimetic was developed using an alternative approach that does not involve designing and creating libraries in vitro.…”

Mutational and biophysical robustness in a pre-stabilized monobody

“…Additionally, if there are already established antibodies in the clinic then there may be little need for an antibody mimic, unless they can provide meaningful advantages such as short half-life for radiolabelled imaging (5,54,55). Nevertheless, it may be more efficient instead to evolve new binding loops de novo with display technologies (56,57).…”

“…Critically, they exhibit comparable binding affinities to antibodies (2). Monobodies based on the Fibronectin Type 3 domain (FN3) are a popular scaffold for developing non-antibody therapeutics (2)(3)(4)(5). The FN3 domain has an Ig-like fold and thus retains three of the CDR-like loops of an antibody variable fragment, but is structurally simple enough to be engineered for advanced non-antibody functions as the monobody scaffold (4-7).…”

“…These formulation experiments are important for designing differentiated applications from the dominant antibody scaffold, where the FN3Con domain may instead find its place in niche applications (5). The improved thermostability of the FN3Con monobody could allow for greater control over selfassociation intermediates in order to create novel drug modalities (5). Furthermore, our demonstration of stability and activity over 1-2 years supports the use of consensus-designed Monobodies in diagnostic settings that cannot utilize cold-chain distribution, such as field applications of microfluidic devices (68)…”

FN3-based monobodies selective for the receptor binding domain of the SARS-CoV-2 spike protein

“…Several FN3 reagents, known as monobodies, have either reached clinical trial or shown promising results as diagnostic tools [ [29] , [30] , [31] , [32] ]. Here, the isolation of monobodies binding to the receptor binding domain (RBD) of SARS-CoV-2 is reported.…”