Bicyclization and Tethering to Albumin Yields Long-Acting Peptide Antagonists

Abstract

Proteolytically stable peptide architectures are required for the development of long-acting peptide therapeutics. In this work, we found that a phage-selected bicyclic peptide antagonist exhibits an unusually high stability in vivo and subsequently deciphered the underlying mechanisms of peptide stabilization. We found that the bicyclic peptide was significantly more stable than its constituent rings synthesized as two individual macrocycles. The two rings protect each other from proteolysis when linked together, conceivably by constraining the conformation and/or by mutually shielding regions prone to proteolysis. A second stabilization mechanism was found when the bicyclic peptide was linked to an albumin-binding peptide to prevent its rapid renal clearance. The bicyclic peptide conjugate not only circulated 50-fold longer (t(1/2) = 24 h) but also became entirely resistant to proteolysis when tethered to the long-lived serum protein. The bicyclic peptide format overcomes a limitation faced by many peptide leads and appears to be suitable for the generation of long-acting peptide therapeutics.

Bicyclic Peptides Conjugated to an Albumin-Binding Tag Diffuse Efficiently into Solid Tumors

“…The peptide conjugate was most likely stabilized through binding to albumin. We found previously that tethering bicyclic peptides to albumin via SA21 increases much its proteolytic stability in blood in vitro and in vivo (22). The large quantity of intact peptide combined with a nanomolar K i for uPA (63.1 AE 8 nmol/L for albuminbound UK202-SA21 Alexa and 8.3 AE 3 nmol/L for free UK202-SA21 Alexa ) suggests that the activity of the target protein uPA is efficiently blocked.…”

“…On the basis of the binding affinity of UK18-SA21 for mouse albumin (K d ¼ 14 nmol/L; ref. 22), >99.9% of the conjugate is at any time bound to albumin in the blood stream. Most likely, the vast majority of peptide extravasates and diffuses into tissue in its albumin-bound form.…”

“…In a recent work, we conjugated a bicyclic peptide to the albumin-binding peptide SA21 developed by Dennis and colleagues (K d for mouse albumin ¼ 24 nmol/L; ref. 23) and showed that its plasma half-life was prolonged from 30 minutes to 24 hours in mice (22), but we did not investigate if the bicyclic peptide diffuses into tissue. Herein, we were interested to learn which concentrations can be reached and if this molecule format would access all regions of a tumor as well as distribute homogeneously.…”

“…In brief, billions of peptides having random sequences are displayed on the surface of filamentous phage, chemically cyclized and panned against immobilized protein targets (21). Because of their small size, bicyclic peptides are rapidly filtered out by the kidney, and delivery of large bicyclic peptide quantities into (tumor) tissue poses a great challenge (22).…”

“…In a recent study performed in our laboratory, the bicyclic peptide inhibitors UK18 and UK202 did not significantly reduce the growth rate of human MDA-MB-231 xenograft tumors in mice (unpublished observation), and this result raised the question whether the bicyclic peptides reached sufficiently high concentrations in the extracellular space of solid tumors to inhibit uPA. UK18 conjugated to the albumin-binding peptide SA21 (UK18-SA21) inhibits the protease with a K i of 132 nmol/L in the presence of mouse albumin (22). On the basis of the rather weak affinity and the relatively low expression level of uPA in tumors, we expected that the distribution of the bicyclic peptides UK18 and UK202 in mice is not influenced by their binding specificity and that their tissue distribution properties are representative for other bicyclic peptides.…”