Investigating the Interaction between the Neonatal Fc Receptor and Monoclonal Antibody Variants by Hydrogen/Deuterium Exchange Mass Spectrometry

The recycling of immunoglobulins by the neonatal Fc receptor (FcRn) is of crucial importance in the maintenance of antibody levels in plasma and is responsible for the long half-lives of endogenous and recombinant monoclonal antibodies. From a therapeutic point of view there is great interest in understanding and modulating the IgG–FcRn interaction to optimize antibody pharmacokinetics and ultimately improve efficacy and safety. Here we studied the interaction between a full-length human IgG₁ and human FcRn via hydrogen/deuterium exchange mass spectrometry and targeted electron transfer dissociation to map sites perturbed by binding on both partners of the IgG–FcRn complex. Several regions in the antibody Fc region and the FcRn were protected from exchange upon complex formation, in good agreement with previous crystallographic studies of FcRn in complex with the Fc fragment. Interestingly, we found that several regions in the IgG Fab region also showed reduced deuterium uptake. Our findings indicate the presence of hitherto unknown FcRn interaction sites in the Fab region or a possible conformational link between the IgG Fc and Fab regions upon FcRn binding. Further, we investigated the role of IgG glycosylation in the conformational response of the IgG–FcRn interaction. Removal of antibody glycans increased the flexibility of the FcRn binding site in the Fc region. Consequently, FcRn binding did not induce a similar conformational stabilization of deglycosylated IgG as observed for the wild-type glycosylated IgG. Our results provide new molecular insight into the IgG–FcRn interaction and illustrate the capability of hydrogen/deuterium exchange mass spectrometry to advance structural proteomics by providing detailed information on the conformation and dynamics of large protein complexes in solution.Antibodies and variants thereof constitute the fastest growing category of therapeutic agents, and currently more than 30 immunoglobulins (Igs)¹ have been approved for the treatment of cancer, immunological diseases, and infectious diseases (1). The success of therapeutic monoclonal antibodies (mAbs) is based on the ability to specifically target diverse antigens and activate immunological effector responses. An Ig is a “dimer of a dimer” consisting of light chains and heavy chains in which each light chain is linked to a heavy chain and the light–heavy dimers are connected by disulfide bridges to form the intact antibody. IgG is the most prevalent Ig isotype in plasma and is the most commonly used isotype for therapeutic antibodies because of its strong ability to induce antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity (2). The IgG₁ subtype is a 150 kDa Y-shaped glycoprotein. Its stem and arms are referred to as the fragment crystallizable (Fc) and fragment antigen binding (Fab) regions, respectively. The Fab region is composed of a variable (V) and constant (C) domain from both the light chain and the heavy chain (V_L, C_L, V_H, C_H1). Antigen binding is achieved through three highly variable complementary determining regions in each variable domain (V_L and V_H) of the Fab region. The Fc region is composed of additional constant domains of the heavy chain (C_H2 and C_H3); it mediates antibody-dependent cellular cytotoxicity through interaction with Fcγ receptors (3, 4) and activates complement-dependent cytotoxicity through interaction with C1q (5). The Fc region also interacts with the neonatal Fc receptor (FcRn), which regulates the maintenance of antibody levels in plasma and thus the half-life of endogenous and recombinant monoclonal antibodies (6). The interaction between IgG and FcRn displays a characteristic pH dependence that is the basis for the function of FcRn in IgG recycling (7). FcRn rescues and recycles IgG from lysosomal degradation by binding with low micromolar affinity to internalized IgG in the slightly acidic late endosome of, for example, vascular endothelial cells (pH < 6.5). The IgG is rescued from intracellular degradation as the IgG–FcRn complex returns to the cell surface, where the IgG is released into circulation as FcRn binding is abolished in the neutral pH of plasma (6). FcRn-mediated IgG recycling contributes to the long catabolic half-life of endogenous and therapeutic antibodies of ∼22 days (8).The FcRn is a heterodimer of an MHC-class-I-like heavy chain and a β₂-microglobulin (β₂m) light chain. The FcRn heavy chain (α-chain) is composed of three structural domains, α₁, α₂, and α₃, followed by a transmembrane region and a cytoplasmic domain. The three-dimensional structure of FcRn is similar to that of MHC class I molecules in which domains α₁ and α₂ are stacked against domain α₃ and β₂m (9, 10). The pH dependence of the IgG–FcRn interaction is attributed to highly conserved residues in both FcRn and IgG (10). The first crystal structures of rat FcRn and rat Fc revealed that FcRn binds to the C_H2 and C_H3 domains of the IgG Fc region—specifically, C_H2 residues 252–254 and 309–311, as well as C_H3 residues 434–436 (11, 12). Several positively charged histidines in the IgG C_H2 and C_H3 domains (H310, H433, H435, and H436; the latter is not found in humans) interact with acidic residues E117, E132, W133, E135, and D137 in the FcRn α₂ domain, accounting for the pH-sensitive nature of the IgG–FcRn interaction. The interface is also composed of a hydrophobic core around Fc I253 that interacts with FcRn W133 and the N-terminal I1 residue of the β₂m, which has been proposed to contact Fc residues 309–311. The interaction of FcRn and IgG occurs in a 2:1 stoichiometry, where two FcRn molecules bind to one IgG through binding sites on each heavy chain (12). Two distinct binding modes have been suggested in which the FcRn molecules bind in a symmetric or asymmetric fashion to the Fc. In symmetric models FcRns bind to opposite sites on the Fc, whereas in the asymmetric models two FcRn molecules form a homodimer with only one FcRn molecule binding the Fc directly (6, 11). The extracellular domains of rat and human FcRn have 68% sequence identity and are structurally similar (9, 10). The first crystal structure of human FcRn in complex with an engineered human Fc fragment (Fc-YTE) as well as human serum albumin was published recently (13) and showed a binding mode similar to that of rodent IgG–FcRn variants, with the exception of the additional interaction sites caused by substitutions in the Fc domain. To the best of our knowledge, no crystal structures of full-length human IgG and human FcRn are currently available.From a therapeutic point of view there is great interest in understanding and modulating the IgG–FcRn interaction to optimize the pharmacokinetics and thus ultimately the efficacy of therapeutic monoclonal antibodies. The goal of FcRn modulation is typically prolongation of the in vivo half-life in order to reduce dosing frequency and ultimately the cost of treatment. However, a shorter half-life can also be desirable, for example, for antibody–toxin conjugates or antibodies used in bioimaging (6). Several engineered therapeutic mAb variants with improved in vitro FcRn binding affinity and extended in vivo half-life have been generated via mutation of residues in the Fc domain (14–19). For example, the engineered variants of palivizumab (M252Y/S254T/T256E) (15, 16) and bevacizumab (M428L/N434S) (17) show 10- and 11-fold increases in relative FcRn affinity that result in increases of the in vivo half-life in cynomolgus monkeys of 4- and 3-fold, respectively. Mutation can also impact half-life negatively: mAb engineering can improve FcRn affinity at both pH 6 and 7.5 such that the pH-dependent release of IgGs is prohibited, leading to increased IgG clearance (16). Interestingly, post-translational modifications such as oxidation of conserved methionines in the C_H2 and C_H3 domains of IgG₁ and IgG₂ have been shown to affect FcRn affinity negatively. Antibody oxidation that can occur during production or storage significantly reduces FcRn binding in vitro (20, 21), which also translates to a reduced in vivo half-life in human FcRn transgenic mice models (22). The molecular origins of the effect of post-translational modifications on the IgG–FcRn interaction are, however, unclear. Further, the impact of FcRn binding on the conformational properties and dynamics of IgG in solution is currently not well understood.In this study we investigated the interaction between human FcRn and two variants of a full-length IgG₁ by means of hydrogen/deuterium exchange monitored by mass spectrometry (HDX-MS). HDX-MS has become a popular approach for studying protein dynamics and interactions (23–27), as the technique provides access to proteins at native solution conditions with modest sample requirements. Amide HDX rates in native proteins are highly influenced by higher order structure: fully solvated (non-hydrogen-bonded) amides exchange rapidly, whereas structurally protected (hydrogen-bonded) amides exchange up to 7 orders of magnitude slower (28, 29). Protein interactions can be studied and mapped via HDX-MS, as binding events can perturb HDX rates as solvation and hydrogen bonding changes directly in the binding interface or indirectly in conformationally linked regions. The structural resolution of a classic peptide-level HDX-MS experiment is dependent on the generation of overlapping peptides by acid-stable proteases, such as pepsin, typically used in HDX-MS workflows. More recently, the use of gas-phase fragmentation of deuterated peptides with ETD (30–33) has become a viable option for sublocalizing deuterium uptake to short peptide stretches or even individual amino acids, thus increasing the spatial resolution of the classical bottom-up HDX-MS method.Here, we used HDX-MS to probe the solution-phase interactions of human FcRn with a full-length recombinant human IgG₁ and its deglycosylated variant. Our results allowed us to map antibody and FcRn regions that displayed changes in HDX upon complex formation and examine the impact of antibody glycosylation on FcRn binding. Additionally, by coupling ETD to the HDX-MS workflow in a targeted manner, we obtained high-resolution information on the HDX of individual sites that became protected upon IgG₁–FcRn complex formation.