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. 2015;7(5):922-30.
doi: 10.1080/19420862.2015.1067353.

Engineered antibody domains with significantly increased transcytosis and half-life in macaques mediated by FcRn

Tianlei Ying  1 , Yanping Wang, Yang Feng, Ponraj Prabakaran, Rui Gong, Lili Wang, Karalyne Crowder, Dimiter S Dimitrov
Affiliations

Engineered antibody domains with significantly increased transcytosis and half-life in macaques mediated by FcRn

Tianlei Ying et al. MAbs. 2015.

Abstract

Engineered antibody domains (eAds) are promising candidate therapeutics but their half-life is relatively short partly due to weak or absent binding to the neonatal Fc receptor (FcRn). We developed a novel approach to increase the eAd binding to FcRn based on a combination of structure-based design, computational modeling and phage display methodologies. By using this approach, we identified 2 IgG1 CH2-derived eAds fused to a short FcRn-binding motif derived from IgG1 CH3 that exhibited greatly enhanced FcRn binding with strict pH dependency. Importantly, the increased affinity resulted in significantly enhanced FcRn-mediated epithelial transcytosis and prolonged elimination half-life (mean 44.1 hours) in cynomolgus macaques. These results demonstrate for the first time that the half-life of isolated eAds can be prolonged (optimized) by increasing their binding to FcRn while maintaining their small size, which has important implications for development of therapeutics, including eAd-drug conjugates with enhanced penetration in solid tissues.

Keywords: CH2; CH3; FcRn; antibody domains; half-life; macaques; transcytosis.

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Figures

Figure 1.
Figure 1.
The native-like FcRn binding could be re-built by transplanting a short FcRn binding motif of IgG1 CH3 onto IgG1 CH2 domain. (a) The complex structure of rat FcRn with Fc (PDB entry 1FRT). The Fc CH2 domain was colored in orange and the Fc CH3 domain was colored in cyan. (b) A schematic diagram to describe the in silico design of CH2-CH3 hybrids. One chain of IgG1 Fc dimer compromising one CH2 and one CH3 domain was shown, and the FcRn binding motif in the CH3 domain was colored red. The CH2 domain and the FcRn binding motif of CH3 were joined by a randomized peptide loop. The residues considered to be important for FcRn binding (H433, H435 and Y436) in CH3 were highlighted.
Figure 2.
Figure 2.
Schematic representation of the engineering strategy to generate pH-dependent FcRn binders. (a) The overlapping view of 6 modeling structures of CH2-CH3 hybrids that possess randomly selected peptide linkers between m01s and FcRn binding motif of CH3. The molecule with a 4-mer linker was colored red, with a 6-mer linker was colored orange while all others have 8-mer linkers. Two histidines in the CH3 motif were drawn as bonds, showing different position and orientation of this motif with relative to m01s. (b) schematic representation of the panning method for the enrichment of only pH-dependent FcRn binders, as described in Experimental Procedures. (c) Plots of the change in fraction folded (calculated from CD molar ellipticity at 216 nm) for the isolated CH2 domain, 2C10, a2b and m01s.
Figure 3.
Figure 3.
Measurement of aggregation for IgG1 CH2, m01s, 2C10 and a2b by dynamic light scattering with the X-axis denoting protein particle size (diameter, nanometer) and the Y-axis intensity (%). Three different colors represent 3 measurements of the same protein.
Figure 4.
Figure 4.
FcRn binding of 2C10, a2b and m01s measured by BIAcore.
Figure 5.
Figure 5.
FcRn binding of measured by flow cytometry and ELISA. (a) Binding of MDCK-FcRn cells to 2C10, a2b, m01s and wild-type Fc (1 µM) at pH 6.0 (left) and 7.4 (right) analyzed by flow cytometry. (b) Schematic representation of the constructs with the FcRn binding motif or the conformational linker removed from 2C10 or a2b. (c) Binding of different antibody domain constructs to human single-chain soluble FcRn measured by ELISA at pH 6.0.
Figure 6.
Figure 6.
The improved FcRn binding correlates with significantly enhanced transcytosis and prolonged in vivo half-life. (a) In vitro transepithelial transport of m01s, a2b, 2C10 and Fc at 10, 25 ug/mL (pH 6.0) and 10 ug/mL (pH 7.4). (b) Pharmacokinetics of 2C10 dosed to individual primates numbered 1, 2 and 3. (c) Serum concentration-time course plot of pooled serum samples of the same 3 animals.
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