Mutational and biophysical robustness in a prestabilized monobody

Abstract

The fibronectin type III (FN3) monobody domain is a promising non-antibody scaffold, which features a less complex architecture than an antibody while maintaining analogous binding loops. We previously developed FN3Con, a hyperstable monobody derivative with diagnostic and therapeutic potential. Prestabilization of the scaffold mitigates the stability-function trade-off commonly associated with evolving a protein domain toward biological activity. Here, we aimed to examine if the FN3Con monobody could take on antibody-like binding to therapeutic targets, while retaining its extreme stability. We targeted the first of the Adnectin derivative of monobodies to reach clinical trials, which was engineered by directed evolution for binding to the therapeutic target VEGFR2; however, this function was gained at the expense of large losses in thermostability and increased oligomerization. In order to mitigate these losses, we grafted the binding loops from Adnectin-anti-VEGFR2 (CT-322) onto the prestabilized FN3Con scaffold to produce a domain that successfully bound with high affinity to the therapeutic target VEGFR2. This FN3Con-anti-VEGFR2 construct also maintains high thermostability, including remarkable long-term stability, retaining binding activity after 2 years of storage at 36 °C. Further investigations into buffer excipients doubled the presence of monomeric monobody in accelerated stability trials. These data suggest that loop grafting onto a prestabilized scaffold is a viable strategy for the development of monobody domains with desirable biophysical characteristics and that FN3Con is therefore well-suited to applications such as the evolution of multiple paratopes or shelf-stable diagnostics and therapeutics.

Keywords: adnectin; consensus design; fibronectin; loop grafting; mini proteins; monobodies; non-antibody scaffold; stability–function trade-off.

Figure 1

A and B, sequence-based grafting between monobody domains with loop sequences from (41). Affinity of FN3Con-anti-VEGFR2 to VEGFR2 was measured using (C) response units during steady-state of association in surface plasmon resonance (SPR), full data in Fig. S1, and (D) a general ELISA approach.

Figure 2

Circular dichroism (CD) thermal melts of (A) Adnectin-anti-VEGFR2 (T_mof 50 °C ± 0.4 °C) and (B) FN3Con-anti-VEGFR2 (T_mof 89 °C ± 0.2 °C), a reverse melt (blue) indicates reversible refolding in FN3Con-anti-VEGFR2.C, the Adnectin-anti-VEGFR2 sample aggregates completely over 1 month at 36 °C. D, more than 1/3 of FN3Con-anti-VEGFR2 sample is still present as a monomer after 2 years of 36 °C storage, with the remaining 2/3 present as higher-order oligomers. E, the graft also shows limited aggregation during 4 °C storage. F, ELISA of the 36 °C 24-month sample shows a matching threefold loss in affinity (K_D of 49–157 nM).

Figure 3

SEC elution profiles of FN3Con-anti-VEGFR2 after accelerated stability trials with a range of buffer excipients. All amino acid buffers slowed the loss of monomer. However, dimers appear at a similar rate between all samples, which could be linked to the generation of disulfide-bonded dimers due to degradation of BME. Tween80 was not significantly more destabilizing to the monomer than PBS buffer alone.

Figure 4

A, change in protein T_mas loops of the Adnectin scaffold were evolved for stronger binding to VEGFR2 (40, 41), transfer of binding loops to FN3Con retained similar affinity while heavily improving thermostability. Data in Table S1. B, this improvement in thermal stability produced a related increase in LTS under elevated temperatures. The Adnectin aggregated with no stable oligomers, so later monomer percentages were compared against the day 1 values (green). As FN3Con remained at ∼1 mg/ml over the course of each trial, monomer percentages were calculated from the total peak area within each timepoint (red, gray).