Enhancing bioactivity, physicochemical, and pharmacokinetic properties of a nano-sized, anti-VEGFR2 Adnectin, through PASylation technology

Abstract

The crucial role of VEGF receptor 2 (VEGFR2) signaling in the angiogenesis and metastasis of solid tumors has prompted the development of inhibitors with minimal bystander effects. Recently, Adnectin C has attracted attention for cancer treatment. To overcome the problematic properties of Adnectin, a novel form of Adnectin C has been designed by its fusion to a biodegradable polymeric peptide containing Pro/Ala/Ser (PAS) repetitive residues. E. coli -expressed recombinant fused and unfused proteins were compared in terms of bioactivity, physicochemical, and pharmacokinetic properties using standard methods. Dynamic light scattering (DLS) analysis of PASylated adnectin C revealed an approximate 2-fold increase in particle size with a slight change in the net charge. Additionally, fusion of the PAS sequence improved its stability against the growth of thermo-induced aggregated forms. The high receptor-binding and improved binding kinetic parameters of PASylated Adnectin C was confirmed by ELISA and surface plasmon resonance assays, respectively. Pharmacokinetic studies showed a noticeable increase in the terminal half-life of Adnectin C-PAS#1(200) by a factor of 4.57 after single dose by intravenous injection into female BALB/c mice. The results suggest that PASylation could offer a superior delivery strategy for developing Adnectin-derived drugs with improved patient compliance.

Improvement of Certolizumab Fab′ properties by PASylation technology

“…This finding not only confirmed the ELISA results but also proved that PASylation did not have any considerable impact on the affinity of the Fab′ molecule in comparison to the PEGylation method. This finding was in accordance with the previous study reporting higher affinities for PASylated protein in comparison to the corresponding PEGylated form 29 .…”

“…Denaturation and initial decomposition temperatures of PASylated and Fab′ molecules were similar, confirming the protein thermal stability in the case of PASylation approach. On the other hand, the increased thermal stability of PEGylated protein may be due to the chemical nature of the PEG moiety and its strong conjugation to the antibody fragment 20 , 21 , 29 .…”

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

“…In spite of this clinical failure, the Adnectin-anti-VEGFR2 has become a model protein for development of technologies supporting the monobody scaffold, with recent studies aiming to enhance the Adenctin's pharmacokinetic properties through PASylation [50] or improve CMC through bacterial expression [51]. The wider lesson learnt from this first clinical attempt was primarily to select targets and applications that differentiate the FN3 scaffold instead of following established antibody methodologies, such as the re-purposing of the Adnectin-anti-VEGFR2 as an ultrasound imaging agent [52] or in CAR-T (Chimeric Antigen Receptor-T Cell) formats [53].…”

Injectables and Depots to Prolong Drug Action of Proteins and Peptides

“…Linkage to XTEN led to an increase in the plasma half-life of clotting factor VIIa (FVIIa) in haemophilia A mice models (8.9 h versus 1.2 h) [ 86 ], while a terminal half-life prolongation for FIX was noted (from 15 to 40 h). Aghaabdollahian et al [ 180 ] showed a 5-fold increased terminal half-life of a single IV dose of Adnectin-C-PAS compared to the unmodified protein in female BALB/c mice [ 180 ]. Schlapschy and co-workers [ 181 ] designed polypeptides with repeating glycine-serine units (HAPylation) to increase the hydrodynamic radius and half-life of an anti-human epidermal growth factor 2 (HER2) antibody fragment (Fab), noting a moderate extension in half-life and doubling in hydrodynamic radius [ 181 ].…”