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.

Keywords: adnectin; biosensor; fibronectin; monobody; non-antibody scaffold; therapeutic.

Figure 1

(A) The Type 3 Fibronectin domains 7 to 10 from human fibronectin with the original RGD binding sequence highlighted in red. (B) Antibody domains use a set of three hypervariable binding loops to form a complementary region to a target binding site. (C) Fibronectin type III (FN3) domains have a comparable set of analogous loops which can be engineered for similar binding function, as well as an expanded binding footprint in the side and ‘south’ loops. Six derivatives of the FN3 domain under development have similar size and structure but can vary widely in (D) amino acid sequence, sharing only the F-Strand sequence across the domains, which leads to (E) a large variation in overall sequence identity between derivatives. Colouring: (D) Sequence alignment: */green—identical amino-acid,:/purple—strongly similar,/blue—weakly similar.; (E) Sequence pairwise identity matrix: green—highly identical sequences, pale green—strongly identical sequences, pink—weakly identical, red—low identical amino-acid matches.

Figure 2

Applications of monomeric antibody domains. (A) Antibody Drug Conjugate (ADC) drug delivery [58]. Tumour imaging with monobodies conjugated to (B) radioisotopes [62] and (C) microbubbles [52]. (D) Targeted degradation of endogenous intracellular proteins [63] and (E) targeted intracellular fluorescence reporters for endogenous proteins [64].

Figure 3

Multimeric applications. (A) FN3 domains on the fibronectin chain exhibit binding to multiple partners. Mimicking this beads-on-a-string approach quickly produces (B) bivalent, (C) tetravalent and (D) bispecific monobody constructs. (E) Furthering this fusion approach, tandem monobodies are fused with domains which confer longer circulating half-life or (F) greater avidity.

Figure 4

Applying monobody advantages to the antibody scaffold. Monobodies are fused with antibody fragments to extend half-life and generate bivalency. Replacing either (A) Antigen-binding fragments or (B) individual variable domains. (C) mAbtyrins extend this combination through developing by specifics by fusing monobodies to 1 of 4 positions on the C- or N-terminal ends of either chain.

Figure 5

(A) monobody fragment is generated from fragments involved in binding (orange), where the original monobody undergoes loss-of-function mutations in residues to remove affinity to the target and loss-of-stability mutations to disrupt beta-sheet packing (Red). (B) The presence of ligand and fragment allow a fragment-exchange complex to form that brings a Forster Resonance Energy Transfer (FRET) pair together, providing biosensors for the presence of a ligand.