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. 2006 Dec 5;103(49):18709-14.
doi: 10.1073/pnas.0606304103. Epub 2006 Nov 20.

Divergent activities of an engineered antibody in murine and human systems have implications for therapeutic antibodies

Carlos Vaccaro  1 , Roger Bawdon, Sylvia Wanjie, Raimund J Ober, E Sally Ward
Affiliations

Divergent activities of an engineered antibody in murine and human systems have implications for therapeutic antibodies

Carlos Vaccaro et al. Proc Natl Acad Sci U S A. .

Abstract

The MHC class I-related receptor, neonatal Fc receptor (FcRn), plays a central role in regulating the transport and in vivo persistence of immunoglobulin G (IgG). IgG-FcRn interactions can be targeted for engineering to modulate the in vivo longevity and transport of an antibody, and this has implications for the successful application of therapeutic IgGs. Although mice are widely used to preclinically test antibodies, human and mouse FcRn have significant differences in binding specificity. Here we show that an engineered human IgG1 has disparate properties in murine and human systems. The mutant shows improved transport relative to wild-type human IgG1 in assays of human FcRn function but has short in vivo persistence and competitively inhibits FcRn activity in mice. These studies indicate potential limitations of using mice as preclinical models for the analysis of engineered antibodies. Alternative assays are proposed that serve as indicators of the properties of IgGs in humans.

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Conflict of interest statement

Conflict of interest statement: There is a pending patent that describes the mutated human IgG1 used in the studies.

Figures

Fig. 1.
Fig. 1.
The location of the mutations that constitute the HN mutant and binding of this mutant to human FcRn at pH 6.0 and 7.2. (a) α-carbon trace of human IgG1 (Fc region; ref. 48) with location of His-433 and Asn-434 shown. Figure was drawn by using Rasmol (courtesy of Roger Sayle, Bioinformatics Research Institute, University of Edinburgh, Edinburgh, U.K.). (b) Sensorgrams showing interaction of human FcRn with HN mutant at pH 6.0 and 7.2. Flow cells were coupled with HN mutant (622 RU) and recombinant human FcRn injected at a concentration of 250 nM in PBS plus 0.01% Tween 20, pH 6.0 or 7.2. All data are representative of duplicate injections and were zero adjusted, and reference cell data (flow cell coupled with buffer only during coupling cycle) was subtracted.
Fig. 2.
Fig. 2.
The HN mutant has a relatively short half-life and enhances the clearance of 125I-labeled wild-type human IgG1 in mice. (a) Swiss–Webster mice (five per group) were injected with 125I-labeled IgGs, and radioactivity levels were assessed by whole-body counting. Data shown are mean values for each group, and bars indicate standard deviations. (b) Swiss–Webster mice (five per group) were injected with 125I-labeled wild-type human IgG1, and 72 h later (indicated by arrow), were injected with 500 μg of wild-type human IgG1 or 500 μg of HN mutant. Levels of radioactivity in the mice were determined at the indicated times by whole-body counting. The data shown are means of remaining radioactivity in each group of mice. Error bars indicate standard deviations. ∗, data for these time points are significantly different, with P < 0.02 (Student's t test). Data are representative of two independent experiments.
Fig. 3.
Fig. 3.
The HN mutant shows different recycling behavior in human microvascular endothelial cell 1 cells transfected with human-GFP (hFcRn-GFP) or mouse FcRn-GFP (mFcRn-GFP). (a) Cells were pulsed with Alexa 647-labeled wild-type (WT) human IgG1 or HN mutant and then chased for 0, 15, or 30 min. Cell-associated fluorescence (Alexa 647) levels in GFP+ cells were quantitated by flow cytometry and are expressed as mean fluorescence intensities. (b) Transfected cells were pulsed with Alexa 555-labeled HN mutant, washed, fixed, and imaged by fluorescence microscopy. Alexa 555-labeled HN mutant was imaged at the same exposure times for both mouse and human FcRn-GFP transfected cells. (c) As in b, except that a longer exposure time was used for imaging Alexa 555-labeled HN mutant. GFP and Alexa 555 are shown in green and red, respectively. (Scale bars: 5 μm.) Data shown are representative of at least two independent experiments.
Fig. 4.
Fig. 4.
The HN mutant is transported more efficiently relative to wild-type human IgG1 across the ex vivo placenta. Biotinylated and iodinated IgGs were premixed and added to the maternal compartment. Transport of each IgG into the fetal compartment was assessed from 30 min to 240 min after the addition. Data from two experiments in which the IgGs were differently labeled are shown.
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