Fig 1.
Cartoon representations of wildtype IgG Fc, monomeric Fc and fusion proteins.
(A) Cartoons of Fc homodimer in IgG and in a bivalent Fc fusion protein, as well as a one-arm mAb and a monovalent Fc fusion, supported by heterodimeric Fc (as shown) or tethered Fc. (B) Cartoons of a monomeric Fc, along with Fab- and scFv- fusion proteins with a monomeric Fc.
Fig 2.
Monomeric Fc library design, selection workflow and output analysis.
(A) Phage display library with mutations in CH2 and CH3 was built as indicated in the sequence alignment. X represents any amino acid and Z represents R, E or Q. The library selection was carried out first with Protein G binding, then several iterative rounds of thermal stress and FcRn binding. At the end of the library selection, randomly picked clones were sequenced and the frequency of amino acids at each position, in single letter codes, was plotted in the bar graphs. Parallel biopanning rounds with no thermal stress (B) and with thermal stress (C) showed the improved enrichment under thermal selection pressure.
Fig 3.
Analytical size-exclusion chromatograms of purified lead Fc clones 1, 2, 4 and 6.
The retention times of clones 1, 2 and 6 appear shorter than that of clone 4, suggesting a difference in their molecular masses.
Table 1.
Molecular weights of Fc fragments and fusion proteins determined by MALS.
Table 2.
X-ray data and model refinement statistics.
Fig 4.
Cartoon representation of the X-ray crystal structure of monomeric Fc C4n.
(A) The protein chain and carbohydrates attached to N297 are shown in light salmon color. Light blue spheres indicate Zn atoms. (B) The model of the carbohydrate moiety attached to N297 superimposed onto the electron density map. Both of the possible terminal sialic acids and one of the galactose residues have no corresponding electron density map. The fucose attached to the core GlcNAc showed only partial electron density. (C) CH3-CH3 interface of wildtype IgG4 Fc showing side chains of the amino acids targeted in the mutagenesis library. Two interacting CH3 domains are shown in light blue and blue colors. (D) Superposition of two C4n structures (shown in light salmon and brown colors) onto wildtype IgG4 Fc to illustrate the effects of the mutations at the CH3-CH3 interface.
Table 3.
Equilibrium binding of Fc variants to human FcRn in avidity binding format.
Fig 5.
Growth inhibition activities of anti-cMet-C4 fusion proteins and controls.
Lovo cells were treated with Onart-Fab-C4 (dark green), Onart-scFv-C4 (light purple), one-armed heterodimeric antibody Onartuzumab (light green) [6], bivalent anti-cMet IgG (red) and negative control antibody (blue) in triplicates and cell viability was measured by CellTiter-Glo assay after a 72-hour incubation. (A) Without ligand HGF treatment, there was a marked agonist effect when cells were exposed to bivalent anti-cMet IgG, an effect mostly absent in monovalent anti-cMet constructs. (B) With ligand treatment, anti-cMet targeting was shown to inhibit cell growth. Monomeric Fc fusions, Onart-Fab-C4 and Onart-scFc-C4 showed similar inhibitory effects as Onartuzumab.
Fig 6.
Pharmacokinetic profiles of monomeric fusion proteins in hFcRn transgenic mice.
Serum clearance curves are plotted for a wild-type IgG control (Motavizumab, red), Onart-Fab-C4 (blue), Onart-scFv-C4 (orange) and Fab control (purple) in hFcRn transgenic mice. Error bars, SE (standard error). C4 significantly extended the serum half-life of the fusion proteins. Compared to a wildtype IgG control, C4 fusions had an initial faster distribution clearance, possibly attributed to enhanced tissue distribution (Vss, Table 4) and then achieved similar serum half-life (T1/2, Table 4).
Table 4.
In vivo mouse pharmacokinetic analysis of monomeric Fc-fusion proteins.