The transfer of maternal gamma-globulin (IgG) provides the neonate with humoral immunity during early life. In humans, maternal IgG is transported across the placenta during the third trimester of pregnancy. The expression of the MHC class I-related receptor, FcRn, in the human placenta suggests that this Fc receptor might be involved in the delivery of maternal IgG, but direct evidence to support this is lacking. In the current study an ex vivo placental model has been used to analyze the maternofetal transfer of a recombinant, humanized (IgG1) antibody in which His435 has been mutated to alanine (H435A). In vitro binding studies using surface plasmon resonance indicate that the mutation ablates binding of the antibody to recombinant mouse and human FcRn. Relative to the wild-type antibody, the H435A mutant is deficient in transfer across the placenta. Significantly, the mutation does not affect binding to Fc gamma RIII, an FcR that has been suggested in earlier studies to mediate the transfer of maternal IgG. The analyses demonstrate that binding of an IgG to FcRn is a prerequisite for transport across the perfused placenta. FcRn therefore plays a central role in the maternofetal delivery of IgG and this has implications for the use of protein engineering to improve the properties of therapeutic antibodies.

II-2

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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.antibody engineering ͉ neonatal Fc receptor ͉ half-life ͉ human IgG1 ͉ maternofetal transfer R ecent developments in antibody engineering have resulted in approaches that target the Fc region with the goal of altering effector functions such as Fc␥ receptor-mediated cytotoxicity and in vivo persistence (1-6). As an IgG transporter, the neonatal Fc receptor (FcRn), serves to regulate the levels of IgG at diverse sites throughout the body by transporting IgGs within and across cellular barriers (7-13). The use of protein engineering, combined with knowledge of FcRn-IgG interactions at the molecular level, has resulted in approaches for the modulation of the persistence of antibodies in vivo (1,3,4,(14)(15)(16)(17), which has direct relevance for the successful application of therapeutic antibodies.Mice are routinely used as a readily accessible model for the preclinical evaluation of IgGs. Although human and mouse FcRn share sequence homology (18,19), leading to the belief that mice might serve as reliable models for FcRn function across species, mouse FcRn unexpectedly has a much broader binding specificity relative to human FcRn (20,21). In both humans and mice, FcRn-IgG interactions are characterized by pHdependent binding with relatively high affinity at pH 6.0 that becomes progressively weaker as pH 7.2 is approached (15,(22)(23)(24). The model for FcRn-mediated transport is that IgG molecules are taken up by fluid phase pinocytosis and, subsequently, interact with this Fc receptor in acidic endosomes (25-27). Receptor-bound IgGs then are recycled or transcytosed and released at the cell surface by exocytic events that involve FcRn (28). This cellular transport mechanism is responsible for the homeostasis and transport of IgGs. As a result, for (engineered) IgGs, there is a strong correlation between the IgGFcRn affinity (at pH Ϸ6.0), in vivo half-life, and transport across cellular barriers such as intestinal or lung epithelium and placental explants (1, 4, 13, 14, 17, 29, 30). However, this correlation breaks down if a significan...

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