Pairing mAbs 3B10 and 1C1 results in enhanced EphA2 detection sensitivity in conditioned media.

<p>(A) Binding of detection antibody mAb 3B2 plotted against EphA2 concentrations. (B) Logarithmic scale display with binding signals in ∼0.3–30 RU (or ∼0.03–3 ng/cm²) range. The bi-epitope 3B10-1C1 surface detected the lowest EphA2 concentration (15.6 pM at a binding signal of 6 RU or 0.6 ng/cm²), an ∼100- and 200-fold improvement in detection limits when compared with the corresponding 3B10 (1.3 nM) and 1C1 (3.1 nM), respectively, single-epitope surfaces.</p

EphA2 binding to individual mAbs 3B10 (A), 1C1 (B) and corresponding mixture (C) immobilized at high density levels.

<p>When using the single-epitope high density surfaces, dissociation rates were fast and similar to that of the corresponding low density surfaces. Surfaces immobilized with the antibody pair allowed for an ∼100-fold increase in the apparent dissociation rate (∼10⁻⁴ s⁻¹).</p

Generation and characterization of high density bi-epitope SPR sensor surfaces.

<p>(A) Immobilization sensorgrams of mAbs 3B10, 1C1 and 3B10-1C1 mixture. The immobilization profiles are comparable and yielded a high density surface (∼5,000–5,500 RU or ∼500–550 ng/cm²). (B) Confirmation of the co-existence of functional antibodies on the bi-epitope surfaces. Excess of mAbs 3B10 or 1C1 (1 µM) inhibited EphA2 binding to the single-epitope 3B10 or 1C1 surfaces, respectively, but not to the bi-epitope 3B10-1C1 surface.</p

Binding and epitope characterization of various anti-EphA2 mAbs.

<p>(A) Binding kinetics of mAbs 1C1, 3F2, 3B10 and 3B2. Measurements were conducted using a ProteOn XPR36. Each antibody was immobilized at low density (∼200–600 RU or ∼20–60 ng/cm²) using amine coupling and EphA2 injected over the resulting surfaces. All 4 antibodies exhibit fast dissociation rates in the 10⁻²−10⁻³ s⁻¹ range. (B) Epitope binning. Cross-competition binding studies between any pair of mAbs 1C1, 3F2, 3B10 and 3B2 was performed using a ProteOn XPR36 instrument. Injections are indicated by arrows. A response from the second injection indicated that each mAb in a given pair binds to a different epitope. (C) 3 distinct epitopes were identified, including 1 shared between mAbs 3B10 and 3F2.</p

Generation and Characterization of an IgG4 Monomeric Fc Platform

<div><p>The immunoglobulin Fc region is a homodimer consisted of two sets of CH2 and CH3 domains and has been exploited to generate two-arm protein fusions with high expression yields, simplified purification processes and extended serum half-life. However, attempts to generate one-arm fusion proteins with monomeric Fc, with one set of CH2 and CH3 domains, are often plagued with challenges such as weakened binding to FcRn or partial monomer formation. Here, we demonstrate the generation of a stable IgG4 Fc monomer with a unique combination of mutations at the CH3-CH3 interface using rational design combined with <i>in vitro</i> evolution methodologies. In addition to size-exclusion chromatography and analytical ultracentrifugation, we used multi-angle light scattering (MALS) to show that the engineered Fc monomer exhibits excellent monodispersity. Furthermore, crystal structure analysis (PDB ID: 5HVW) reveals monomeric properties supported by disrupted interactions at the CH3-CH3 interface. Monomeric Fc fusions with Fab or scFv achieved FcRn binding and serum half-life comparable to wildtype IgG. These results demonstrate that this monomeric IgG4 Fc is a promising therapeutic platform to extend the serum half-life of proteins in a monovalent format.</p></div

Cartoon representation of the X-ray crystal structure of monomeric Fc C4n.

<p>(<b>A</b>) The protein chain and carbohydrates attached to N297 are shown in light salmon color. Light blue spheres indicate Zn atoms. (<b>B</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. (<b>C</b>) 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. (<b>D</b>) 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.</p

Molecular weights of Fc fragments and fusion proteins determined by MALS.

<p>Molecular weights of Fc fragments and fusion proteins determined by MALS.</p

Cartoon representations of wildtype IgG Fc, monomeric Fc and fusion proteins.

<p>(<b>A</b>) 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>B</b>) Cartoons of a monomeric Fc, along with Fab- and scFv- fusion proteins with a monomeric Fc.</p

Monomeric Fc library design, selection workflow and output analysis.

<p>(<b>A</b>) 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>B</b>) and with thermal stress (<b>C</b>) showed the improved enrichment under thermal selection pressure.</p

Pharmacokinetic profiles of monomeric fusion proteins in hFcRn transgenic mice.

<p>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. <i>Error bars</i>, 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 (V_ss, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160345#pone.0160345.t004" target="_blank">Table 4</a>) and then achieved similar serum half-life (T_1/2, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160345#pone.0160345.t004" target="_blank">Table 4</a>).</p