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. 2004 Jun 29;101(26):9763-8.
doi: 10.1073/pnas.0403235101. Epub 2004 Jun 21.

Pulmonary delivery of an erythropoietin Fc fusion protein in non-human primates through an immunoglobulin transport pathway

Alan J Bitonti  1 , Jennifer A Dumont, Susan C Low, Robert T Peters, Keith E Kropp, Vito J Palombella, James M Stattel, Yichun Lu, Cristina A Tan, Jeffrey J Song, Ana Maria Garcia, Neil E Simister, Gerburg M Spiekermann, Wayne I Lencer, Richard S Blumberg
Affiliations

Pulmonary delivery of an erythropoietin Fc fusion protein in non-human primates through an immunoglobulin transport pathway

Alan J Bitonti et al. Proc Natl Acad Sci U S A. .

Abstract

Administration of therapeutic proteins by methods other than injection is limited, in part, by inefficient penetration of epithelial barriers. Therefore, unique approaches to breaching these barriers are needed. The neonatal constant region fragment (Fc) receptor (FcRn), which is responsible for IgG transport across the intestinal epithelium in newborn rodents, is expressed in epithelial cells in adult humans and non-human primates. Here we show that FcRn-mediated transport is functional in the lung of non-human primates and that this transport system can be used to deliver erythropoietin (Epo) when it is conjugated to the Fc domain of IgG1. FcRn-dependent absorption was more efficient when the EpoFc fusion protein was deposited predominantly in the upper and central airways of the lung, where epithelial expression of FcRn was most prominently detected. To optimize fusion protein absorption in the lung, we created a recombinant "monomeric-Epo" Fc fusion protein comprised of a single molecule of Epo conjugated to a dimeric Fc. This fusion protein exhibited enhanced pharmacokinetic and pharmacodynamic properties. The bioavailability of the EpoFc monomer when delivered through the lung was approximately equal to that reported for unconjugated Epo delivered s.c. in humans. These studies show that FcRn can be harnessed to noninvasively deliver bioactive proteins into the systemic circulation in therapeutic quantities.

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Figures

Fig. 1.
Fig. 1.
Expression of FcRn in non-human primate lung. Immunohistochemical localization of FcRn in cynomolgus monkey bronchus (b) and alveoli (a) was performed by using immunoperoxidase staining after exposure of the tissue sections to nonspecific rabbit serum (A) or a rabbit serum containing a specific anti-FcRn antibody (B). The dark-brown deposits (on the right) indicate the presence of FcRn. Many similar sections of lung were examined from one male and one female monkey with similar staining patterns being observed. The section shown here is from a female monkey.
Fig. 2.
Fig. 2.
Surface plasmon resonance analysis of the interaction of human FcRn with EpoFc and human IgG1. Different concentrations of IgG1 or EpoFc fusion proteins (0.004-10 μM) were injected over a flow cell coupled with human FcRn (3,500-5,000 response units) at a flow rate of 10 μl/min. (A) Representative sensorgrams obtained with 4 μM EpoFc monomer and 4 μM EpoFc/IHH are shown. All sensorgrams were zero-adjusted and reference cell data subtracted. (B) Response units at equilibrium are plotted as a function of the log of IgG1 or EpoFc fusion protein concentrations. (C) Binding affinities were derived from the data in B by using biaevaluation software.
Fig. 3.
Fig. 3.
Effects of breathing pattern on absorption of EpoFc from the lungs of non-human primates. Approximately 0.3 mg/kg EpoFc or mutant EpoFc/IHH was deposited into the lungs of monkeys that were shallow breathing (EpoFc, n = 4; EpoFc/IHH, n = 3) or deep breathing (EpoFc, n = 4; EpoFc/IHH, n = 4). EpoFc and EpoFc/IHH in serum from each animal were measured over time by using a specific Epo ELISA. The AUC were calculated for each fusion protein, and the values shown are means ± SD.
Fig. 4.
Fig. 4.
Pulmonary delivery of EpoFc and EpoFc monomer. EpoFc and EpoFc monomer were aerosolized with an Aeroneb Pro nebulizer, and the aerosol was delivered directly into the lungs of two monkeys each via shallow breathing through an endotracheal tube. Blood was drawn at selected times after administration, and the serum concentrations of the EpoFc monomer and EpoFc were measured with an Epo ELISA.
Fig. 5.
Fig. 5.
Comparison of pulmonary absorption of Epogen and the EpoFc monomer in cynomolgus monkey lung. Equal molar doses of the EpoFc monomer and Epogen were aerosolized with an Aeroneb Pro nebulizer, and the aerosol was delivered directly into the lungs of three monkeys each. Serum concentrations of the two molecules were determined by using an Epospecific ELISA.
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