Stability Enhancement of a Dimeric HER2-Specific Affibody Molecule through Sortase A-Catalyzed Head-to-Tail Cyclization

Abstract

Natural backbone-cyclized proteins have an increased thermostability and resistance towards proteases, characteristics that have sparked interest in head-to-tail cyclization as a method to stability-enhance proteins used in diagnostics and therapeutic applications, for example. In this proof-of principle study, we have produced and investigated a head-to-tail cyclized and HER2-specific ZHER2:342 Affibody dimer. The sortase A-mediated cyclization reaction is highly efficient (>95%) under optimized conditions, and renders a cyclic ZHER3:342-dimer with an apparent melting temperature, Tm, of 68 °C, which is 3 °C higher than that of its linear counterpart. Circular dichroism spectra of the linear and cyclic dimers looked very similar in the far-UV range, both before and after thermal unfolding to 90 °C, which suggests that cyclization does not negatively impact the helicity or folding of the cyclic protein. The cyclic dimer had an apparent sub-nanomolar affinity (Kd ~750 pM) to the HER2-receptor, which is a ~150-fold reduction in affinity relative to the linear dimer (Kd ~5 pM), but the anti-HER2 Affibody dimer remained a high-affinity binder even after cyclization. No apparent difference in proteolytic stability was detected in an endopeptidase degradation assay for the cyclic and linear dimers. In contrast, in an exopeptidase degradation assay, the linear dimer was shown to be completely degraded after 5 min, while the cyclic dimer showed no detectable degradation even after 60 min. We further demonstrate that a site-specifically DyLight 594-labeled cyclic dimer shows specific binding to HER2-overexpressing cells. Taken together, the results presented here demonstrate that head-to-tail cyclization can be an effective strategy to increase the stability of an Affibody dimer.

The Chemical Synthesis of Knob Domain Antibody Fragments

“…Our data suggest that knob domains are highly resistant to plasma proteolysis in vitro, potentially due to their network of disulfide bonds. We have shown that the proximity of the N and C termini can give rise to fully cyclic antibody fragments, which, on the basis of observations with other peptides, , may provide a route to reduce proteolysis even further, especially when used in conjunction with other chemical approaches.…”

“…For the development of peptide drug candidates, head-to-tail cyclization can extend exposure in vivo by preventing exopeptidase cleavage. 19,30,31 To create small, cyclic antibody fragments, we synthesized head-to-tail cyclized forms of our knob domains, which are subsequently termed K8 chem FE cyclic , K57 chem FE cyclic , K92 chem FE cyclic , and K149 chem FE cyclic . All cyclic knob domains bound C5 (Figure 7A−D and Supporting Information S16).…”

Expression and Characterization of Intein-Cyclized Trimer of Staphylococcus aureus Protein A Domain Z

“…Previous reports demonstrated chemically synthesized cyclic two-helix Z domain protein, high affinity bivalent intein-circularized 2Z protein, and a sortase-mediated cyclized dimer of Z domain affibodies [ 31 , 32 , 33 ]. In this study, we constructed a cyclic trimer of the Z domain (cyclic Z3) of protein A using gp41-1-based split-intein circular ligation of peptides and proteins (SICLOPPS) [ 34 ].…”