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. 2012 Oct 2;109(40):16095-100.
doi: 10.1073/pnas.1208857109. Epub 2012 Sep 18.

Engineering neonatal Fc receptor-mediated recycling and transcytosis in recombinant proteins by short terminal peptide extensions

Jonathan T Sockolosky  1 , Matthew R Tiffany, Francis C Szoka
Affiliations

Engineering neonatal Fc receptor-mediated recycling and transcytosis in recombinant proteins by short terminal peptide extensions

Jonathan T Sockolosky et al. Proc Natl Acad Sci U S A. .

Abstract

The importance of therapeutic recombinant proteins in medicine has led to a variety of tactics to increase their circulation time or to enable routes of administration other than injection. One clinically successful tactic to improve both protein circulation and delivery is to fuse the Fc domain of IgG to therapeutic proteins so that the resulting fusion proteins interact with the human neonatal Fc receptor (FcRn). As an alternative to grafting the high molecular weight Fc domain to therapeutic proteins, we have modified their N and/or C termini with a short peptide sequence that interacts with FcRn. Our strategy was motivated by results [Mezo AR, et al. (2008) Proc Natl Acad Sci USA 105:2337-2342] that identified peptides that compete with human IgG for FcRn. The small size and simple structure of the FcRn-binding peptide (FcBP) allows for expression of FcBP fusion proteins in Escherichia coli and results in their pH-dependent binding to FcRn with an affinity comparable to that of IgG. The FcBP fusion proteins are internalized, recycled, and transcytosed across cell monolayers that express FcRn. This strategy has the potential to improve protein transport across epithelial barriers, which could lead to noninvasive administration and also enable longer half-lives of therapeutic proteins.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Construction and characterization of FcBP-modified mKates. (A) Diagram of the gene sequences encoding mKate modified at its N and/or C termini with a cyclic or linear FcBP sequence. (B) Fluorescence emission spectra comparing equal molar concentrations (500 nM) of mKate (black line) and FcBP-modified mKates (grey lines). (C) SDS-PAGE analysis of purified mKate (lane 1), C-Term Linear FcBP mKate (lane 2), C-Term Cyclic FcBP mKate (lane 3), N-Term Cyclic FcBP mKate (lane 4), N-and-C-Term Cyclic FcBP mKate (lane 5), N-and-C-Term Cyclic FcBP mKate Y286H (lane 6), and N-Linear and C-Cyclic FcBP mKate (lane 7). In each lane, 7.5 μg of protein was loaded. (D) LC-MS/MS identification of the peptide containing the Gly4Ser linker and cyclic FcBP sequence from a LysC digest of C-Term Cyclic FcBP mKate. The blue and red peaks correspond to the identified y and b ions, respectively, within the peptide sequence.
Fig. 2.
Fig. 2.
In vitro characterization of FcBP-modified mKates in MDCK cells expressing hFcRn–EYFP by FACS. (A) Cellular accumulation of FcBP-modified mKates and labeled hIgG1 at pH 6 in MDCK hFcRn–EYFP/hβ2m cells. Solid lines represent data fit to a one-site total binding model in Prism. MFI for mKates and hIgG1 is shown on left and right axes, respectively. (B) Cellular accumulation of FcBP-modified mKates and labeled hIgG1 at pH 6 in the presence of 100 mol excess unlabeled hIgG or at pH 7.4. Cells were incubated with 2.5 μM mKate, 2.5 μM mKates modified with a single FcBP, 1 μM N-and-C-Term Cyclic FcBP mKate, or 1 μM labeled hIgG1 with or without 100 mol excess unlabeled hIgG1 as a competitor. The MFI for modified and unmodified mKates was normalized to the maximum MFI for N-and-C-Term Cyclic FcBP mKate. hIgG1 was normalized to itself, given that the fluorescent intensity between the two fluorophores (mKate and TAMRA) are different. Asterisk indicates significance with P < 0.001. The data shown in each panel are the mean (n = 3), and error bars indicate SD.
Fig. 3.
Fig. 3.
Fluorescence Imaging of FcBP modified mKates in MDCK cells expressing hFcRn–EYFP. (A) Wide-field epifluorescence image of N-and-C-Term Cyclic FcBP mKate colocalization with hFcRn–EYFP in MDCK cells. Scale bar, 5 μm. (B, i) Confocal images of MDCK hFcRn–EYFP/hβ2m cells showing the distribution of hFcRn–EYFP, N-and-C-Term Cyclic FcBP mKate, and 10 kDa dextran. (B, ii and iii) Wide-field epifluorescence images of MDCK hFcRn–EYFP/hβ2m cells showing the distribution of hFcRn–EYFP, N-and-C-Term Cyclic FcBP mKate, and LAMP1-mTurquoise after 1-h (ii) or 4-h (iii) chase at 37 °C in HBSS, pH 7.4, prior to imaging. Yellow boxes indicate areas of colocalization between N-and-C-Term Cyclic FcBP mKate and FcRn–EYFP; white boxes indicate LAMP1-mTurquoise compartments that do not contain N-and-C-Term Cyclic FcBP mKate or FcRn–EYFP; and pink boxes indicate areas of colocalization between N-and-C-Term Cyclic FcBP mKate and LAMP1-mTurquoise. Scale bars: (i) 10 μm; (ii and iii) 5 μm. The overlays are pseudocolored as follows: Green indicates hFcRn–EYFP; red indicates N-and-C-Term Cyclic FcBP mKate; blue indicates dextran or LAMP1-mTurquoise; yellow indicates colocalization between FcRn and N-and-C-Term Cyclic FcBP mKate; and pink indicates colocalization between N-and-C-Term Cyclic FcBP mKate and LAMP1.
Fig. 4.
Fig. 4.
FcBP fusion enables FcRn-mediated recycling and transcytosis. (A) In vitro FcRn-mediated recycling from MDCK hFcRn–EYFP/hβ2m cells. Asterisk indicates significance with P < 0.001. (B) In vitro FcRn-mediated transcytosis across MDCK hFcRn–EYFP/hβ2m or wild-type MDCK cell monolayers grown on transwell inserts. The data shown are the amount of protein transported to the basolateral compartment after a 2-h continuous incubation with 2.5 μM mKate or FcBP-modified mKates, and 1 μM labeled hIgG1 in the apical compartment. The apical chamber was equilibrated to pH 6, unless noted, and the basolateral to pH 7.4 in all cases. Double asterisks indicate that transcytosis is statistically significant between the specified groups, with P < 0.001. Single asterisk indicates significance with P < 0.01. Transport below the limit of quantification (LOQ) is indicated by the dashed line at 3.1 ng. (C) Transcytosis of 5 μM mKate or FcBP-modified mKates in the apical to basolateral direction after a 5-h continuous incubation at 37 °C with both compartments equilibrated to pH 7.4. Asterisk indicates significance with P < 0.001. The data shown for each panel are the mean (n = 3), and error bars indicate SD.
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