A Hybrid Recombinant-Chemical Conjugation Approach to Fc Fusion Mimetics

H. Khalili, S. Brocchini, P. Khaw University College London Purpose We have recently shown that Fab-PEG-Fab (FpF) molecules are antibody (MAb) mimetics [1]. Site-specific conjugation of the Fab in the region where it is naturally bound to the hinge in a MAb to each terminus of a linear PEG molecule yields the FpF. Since the PEG in FpFs appears to allow good avidity of the Fabs, and better stability overall compared to MAbs, this study is to determine if we could extend our approach to examine the analogous Fc mimetics. Aflibercept (VEGF-trap) has two VEGF binding regions (rather than two Fabs) fused to an Fc fragment. The aim of this study was to determine if the corresponding ‘receptor fragment-PEG-receptor fragment’ (RpR) or RVEGF-PEG-RVEGF could be prepared by thiolselective bis-alkylation. Methods Aflibercept (VEGF-trap, Regeneron Pharmaceutical, 2.0 mg/mL, 1.0 mL) was proteolytically digested using FabRICATORE (IdeS enzyme, Genovis) for 30 minutes at ambient temperature. The VEGFR binding fragments were purified using a CaptureSelect column. The fragments were then conjugated by thiol-selective bis-alkylation and purified following the process used to prepare FpFs [1] to give RVEGF-PEG-RVEGF. Results Aflibercept was first incubated with DTT indicating that partial reduction yielded a band at approximately 50-55 kDa, which was presumed to be the individual heavy chains of the Fc with one copy of the VEGF-receptor fragment (VEGFR). Ellmans assay indicated there were 4 free thiols after treating aflibercet with DTT. The presumed VEGFR fragments (VEGFR-2) were obtained after digestion of aflibercept with the FabRICATORE enzyme and passing the digestion solution over a column to remove the Fc fragment (Fig. 1, lanes 1). The VEGFR fragments displayed binding to VEGF as determined by Biacore. The VEGFR fragment was treated with DTT (Fig 1, lane 2). After removal of DTT and incubation (Fig. 1, lane 3) with the bisalkylating reagent derived from PEG (10 kDa), the RVEGF-PEG10-RVEGF was obtained after IEX purification (Fig 1, lane 4). Conclusion A mimetic of a Fc fusion protein has been prepared from alfibercept to give RVEGF-PEG10-RVEGF. Comparative binding studies with aflibercept are currently under way.