Engineering a Monomeric Fc Domain Modality by N-Glycosylation for the Half-life Extension of Biotherapeutics

Human IgG is a bivalent molecule that has two identical Fab domains connected by a dimeric Fc domain. For therapeutic purposes, however, the bivalency of IgG and Fc fusion proteins could cause undesired properties. We therefore engineered the conversion of the natural dimeric Fc domain to a highly soluble monomer by introducing two Asn-linked glycans onto the hydrophobic C_H3-C_H3 dimer interface. The monomeric Fc (monoFc) maintained the binding affinity for neonatal Fc receptor (FcRn) in a pH-dependent manner. We solved the crystal structure of monoFc, which explains how the carbohydrates can stabilize the protein surface and provides the rationale for molecular recognition between monoFc and FcRn. The monoFc prolonged the in vivo half-life of an antibody Fab domain, and a tandem repeat of the monoFc further prolonged the half-life. This monoFc modality can be used to improve the pharmacokinetics of monomeric therapeutic proteins with an option to modulate the degree of half-life extension.     

Binding site on human immunoglobulin G for the affinity ligand HWRGWV

Affinity ligand HWRGWV has demonstrated the ability to isolate human immunoglobulin G (hIgG) from mammalian cell culture media. The ligand specifically binds hIgG through its Fc portion. This work shows that deglycosylation of hIgG has no influence on its binding to the HWRGWV ligand and the ligand does not compete with Protein A or Protein G in binding hIgG. It is suggested by the mass spectrometry (MS) data and docking simulation that HWRGWV binds to the pFc portion of hIgG and interacts with the amino acids in the loop Ser383–Asn389 (SNGQPEN) located in the C_H3 domain. Subsequent modeling has suggested a possible three‐dimensional minimized solution structure for the interaction of hIgG and the HWRGWV ligand. The results support the fact that a peptide as small as a hexamer can have specific interactions with large proteins such as hIgG. Copyright © 2009 John Wiley & Sons, Ltd.     

Structure of the Murine Unglycosylated IgG1 Fc Fragment

A prototypic IgG antibody can be divided into two major structural units: the antigen-binding fragment (Fab) and the Fc fragment that mediates effector functions. The IgG Fc fragment is a homodimer of the two C-terminal domains (C_H2 and C_H3) of the heavy chains. Characteristic of the Fc part is the presence of a sugar moiety at the inner face of the C_H2 domains. The structure of this complex branched oligosaccharide is generally resolved in crystal structures of Fc fragments due to numerous well-defined sugar-protein interactions and a small number of sugar-sugar interactions. This suggested that sugars play an important role in the structure of the Fc fragment. To address this question directly, we determined the crystal structure of the unglycosylated Fc fragment of the murine IgG1 MAK33. The structures of the C_H3 domains of the unglycosylated Fc fragment superimpose perfectly with the structure of the isolated MAK33 C_H3 domain. The unglycosylated C_H2 domains, in contrast, approach each other much more closely compared to known structures of partly deglycosylated Fc fragments with rigid-body motions between 10 and 14 Å, leading to a strongly “closed” conformation of the unglycosylated Fc fragment. The glycosylation sites in the C′E loop and the BC and FG loops are well defined in the unglycosylated C_H2 domain, however, with increased mobility and with a significant displacement of about 4.9 Å for the unglycosylated Asn residue compared to the glycosylated structure. Thus, glycosylation both stabilizes the C′E-loop conformation within the C_H2 domain and also helps to ensure an “open” conformation, as seen upon Fc receptor binding. These structural data provide a rationale for the observation that deglycosylation of antibodies often compromises their ability to bind and activate Fcγ receptors.     

CH2 domain orientation of human immunoglobulin G in solution: Structural comparison of glycosylated and aglycosylated Fc regions using small-angle X-ray scattering

The N-linked glycan in immunoglobulin G is critical for the stability and function of the crystallizable fragment (Fc) region. Alteration of these protein properties upon the removal of the N-linked glycan has often been explained by the alteration of the C_H2 domain orientation in the Fc region. To confirm this hypothesis, we examined the small-angle X-ray scattering (SAXS) profile of the glycosylated Fc region (gFc) and aglycosylated Fc region (aFc) in solution. Conformational characteristics of the C_H2 domain orientation were validated by comparison with SAXS profiles theoretically calculated from multiple crystal structures of the Fc region with different C_H2 domain orientations. The reduced chi-square values from the fitting analyses of gFc and aFc associated with the degree of openness or closure of each crystal structure, as determined from the first principal component that partially governed the variation of the C_H2 domain orientation extracted by a singular value decomposition analysis. For both gFc and aFc, the best-fitted SAXS profiles corresponded to ones calculated based on the crystal structure of gFc that formed a “semi-closed” C_H2 domain orientation. Collectively, the data indicated that the removal of the N-linked glycan only negligibly affected the C_H2 domain orientation in solution. These findings will guide the development of methodology for the production of highly refined functional Fc variants.     

Structural Characterization of Anti-Inflammatory Immunoglobulin G Fc Proteins

Immunoglobulin G (IgG) is a central mediator of host defense due to its ability to recognize and eliminate pathogens. The recognition and effector responses are encoded on distinct regions of IgGs. The diversity of the antigen recognition Fab domains accounts for IgG's ability to bind with high specificity to essentially any antigen. Recent studies have indicated that the Fc effector domain also displays considerable heterogeneity, accounting for its complex effector functions of inflammation, modulation, and immune suppression. Therapeutic anti-tumor antibodies, for example, require the pro-inflammatory properties of the IgG Fc to eliminate tumor cells, while the anti-inflammatory activity of intravenous IgG requires specific Fc glycans for activity. In particular, the anti-inflammatory activity of intravenous IgG is ascribed to a small population of IgGs in which the Asn297-linked complex N-glycans attached to each Fc C_H2 domain include terminal α2,6-linked sialic acids. We used chemoenzymatic glycoengineering to prepare fully disialylated IgG Fc and solved its crystal structure. Comparison of the structures of asialylated Fc, sialylated Fc, and F241A Fc, a mutant that displays increased glycan sialylation, suggests that increased conformational flexibility of the C_H2 domain is associated with the switch from pro-inflammatory to anti-inflammatory activity of the Fc.     

Structural Determinants of Unique Properties of Human IgG4-Fc

Human IgG4, normally the least abundant of the four subclasses of IgG in serum, displays a number of unique biological properties. It can undergo heavy-chain exchange, also known as Fab-arm exchange, leading to the formation of monovalent but bispecific antibodies, and it interacts poorly with FcγRII and FcγRIII, and complement. These properties render IgG4 relatively “non-inflammatory” and have made it a suitable format for therapeutic monoclonal antibody production. However, IgG4 is also known to undergo Fc-mediated aggregation and has been implicated in auto-immune disease pathology. We report here the high-resolution crystal structures, at 1.9 and 2.35 Å, respectively, of human recombinant and serum-derived IgG4-Fc. These structures reveal conformational variability at the C_H3–C_H3 interface that may promote Fab-arm exchange, and a unique conformation for the FG loop in the C_H2 domain that would explain the poor FcγRII, FcγRIII and C1q binding properties of IgG4 compared with IgG1 and -3. In contrast to other IgG subclasses, this unique conformation folds the FG loop away from the C_H2 domain, precluding any interaction with the lower hinge region, which may further facilitate Fab-arm exchange by destabilisation of the hinge. The crystals of IgG4-Fc also display Fc–Fc packing contacts with very extensive interaction surfaces, involving both a consensus binding site in IgG-Fc at the C_H2–C_H3 interface and known hydrophobic aggregation motifs. These Fc–Fc interactions are compatible with intact IgG4 molecules and may provide a model for the formation of aggregates of IgG4 that can cause disease pathology in the absence of antigen.     

Some Peculiarities of the Dynamics of the Immunoglobulin M Structure

Abstract

The isotropic mobility of separate regions of the intact molecule of immunoglobulin M (IgM) and its Fab and (Fc)₅ fragments was studied using spin-labeling of carbohydrate (2,2,6,6-tetramethyl-4-aminopiperidine-l-oxyl) and peptide (2,2,5,5-tetramethyl-3-dichloro-triazinylaminopyrrolidine-l-oxyl) moieties.

The spin-labeled oligosaccharide groups (OGs) in the Fab region are shown to have much more amplitude of anisotropic motion than those in the (Fc)₅ region. The spin label in the latter is evidently attached in the Cμ3 domain to one of its OGs which is probably stabilized by ionic contacts between terminal N-acetylneuraminic acid residue and the peptide moiety of the IgM molecule.

When the amount of the glycosidase-cleaved carbohydrate does not exceed 10–15%, most OGs affected are of the Fab region. Upon profound splitting (≥50%) the OGs of the (Fc)₅region are also affected; that results evidently in loosening the ionic contacts between the shortened OGs and the peptide moiety of IgM, and consequently in increasing mobility of the former.

The structure of the (Fc)₅ region of IgM is labile; after detaching this moiety from the intact IgM molecule, its structure is stabilized, but one of its domains (Cμ3) becomes more mobile than it is in the intact IgM molecule; at the same time the amplitude of anisotropic motion of OG bound here is decreased. In the latter case, this decrease depends on the sequence of spin-labeling and fragmentation.

The most probable cause of stabilization of the (Fc)₅ fragment is the heating of IgM solution to 56°C during fragmentation with trypsin. At this temperature the τ value for the (Fc)₅ fragment is unusually low, equaling 23 ns.

The spin-labeling in the peptide part of IgM occurs mostly in the Fab region which is a rather rigid moiety as expected.     

Constant domain-exchanged Fab enables specific light chain pairing in heterodimeric bispecific SEED-antibodies

BackgroundBispecific antibodies promise to broadly expand the clinical utility of monoclonal antibody technology. Several approaches for heterodimerization of heavy chains have been established to produce antibodies with two different Fab arms, but promiscuous pairing of heavy and light chains remains a challenge for their manufacturing.MethodsWe have designed a solution in which the C_H1 and C_L domain pair in one of the Fab fragments is replaced with a C_H3-domain pair and heterodimerized to facilitate correct modified Fab-chain pairing in bispecific heterodimeric antibodies based on a strand-exchange engineered domain (SEED) scaffold with specificity for epithelial growth factor receptor and either CD3 or CD16 (FcγRIII).ResultsBispecific antibodies retained binding to their target antigens and redirected primary T cells or NK cells to induce potent killing of target cells. All antibodies were expressed at a high yield in Expi293F cells, were detected as single sharp symmetrical peaks in size exclusion chromatography and retained high thermostability. Mass spectrometric analysis revealed specific heavy-to-light chain pairing for the bispecific SEED antibodies as well as for one-armed SEED antibodies co-expressed with two different competing light chains.ConclusionIncorporation of a constant domain-exchanged Fab fragment into a SEED antibody yields functional molecules with favorable biophysical properties.General significanceOur results show that the novel engineered bispecific SEED antibody scaffold with an incorporated Fab fragment with C_H3-exchanged constant domains is a promising tool for the generation of complete heterodimeric bispecific antibodies with correct light chain pairing.     

The amino acid sequence of “heavy chain disease” protein ZUC. Structure of the Fc fragment of immunoglobulin G3

The sequence of the Fc fragment of human IgG3 was studied, using a naturally-occurring γ3 heavy chain variant (ZUC). Though the molecule is internally deleted, it contains 248 residues, including the entire Fc fragment. The almost complete sequence of the C_H2 and C_H3 domains (position 234 to 446) indicates an extremely close evolutionary relationship with γ1 and γ4 chains. There is a 95% homology between IgG3 and IgGl and 92% between IgG3 and IgG4 in the C_H2 in the C_H3 domains.     

Charge-mediated Fab-Fc interactions in an IgG1 antibody induce reversible self-association,cluster formation,and elevated viscosity

Concentration-dependent reversible self-association (RSA) of monoclonal antibodies (mAbs) poses a challenge to their pharmaceutical development as viable candidates for subcutaneous delivery. While the role of the antigen-binding fragment (Fab) in initiating RSA is well-established, little evidence supports the involvement of the crystallizable fragment (Fc). In this report, a variety of biophysical tools, including hydrogen exchange mass spectrometry, are used to elucidate the protein interface of such non-covalent protein-protein interactions. Using dynamic and static light scattering combined with viscosity measurements, we find that an IgG1 mAb (mAb-J) undergoes RSA primarily through electrostatic interactions and forms a monomer-dimer-tetramer equilibrium. We provide the first direct experimental mapping of the interface formed between the Fab and Fc domains of an antibody at high protein concentrations. Charge distribution heterogeneity between the positively charged interface spanning complementarity-determining regions CDR3H and CDR2L in the Fab and a negatively charged region in C_H3/Fc domain mediates the RSA of mAb-J. When arginine and NaCl are added, they disrupt RSA of mAb-J and decrease the solution viscosity. Fab-Fc domain interactions between mAb monomers may promote the formation of large transient antibody complexes that ultimately cause increases in solution viscosity. Our findings illustrate how limited specific arrangements of amino-acid residues can cause mAbs to undergo RSA at high protein concentrations and how conserved regions in the Fc portion of the antibody can also play an important role in initiating weak and transient protein-protein interactions.     

A comparison of biophysical characterization techniques in predicting monoclonal antibody stability

With the rapid growth of biopharmaceutical product development, knowledge of therapeutic protein stability has become increasingly important. We evaluated assays that measure solution-mediated interactions and key molecular characteristics of 9 formulated monoclonal antibody (mAb) therapeutics, to predict their stability behavior. Colloidal interactions, self-association propensity and conformational stability were measured using effective surface charge via zeta potential, diffusion interaction parameter (k_D) and differential scanning calorimetry (DSC), respectively. The molecular features of all 9 mAbs were compared to their stability at accelerated (25°C and 40°C) and long-term storage conditions (2–8°C) as measured by size exclusion chromatography. At accelerated storage conditions, the majority of the mAbs in this study degraded via fragmentation rather than aggregation. Our results show that colloidal stability, self-association propensity and conformational characteristics (exposed tryptophan) provide reasonable prediction of accelerated stability, with limited predictive value at 2–8°C stability. While no correlations to stability behavior were observed with onset-of-melting temperatures or domain unfolding temperatures, by DSC, melting of the Fab domain with the C_H2 domain suggests lower stability at stressed conditions. The relevance of identifying appropriate biophysical assays based on the primary degradation pathways is discussed.     

Novel asymmetrically engineered antibody Fc variant with superior FcγR binding affinity and specificity compared with afucosylated Fc variant

Fc engineering is a promising approach to enhance the antitumor efficacy of monoclonal antibodies (mAbs) through antibody-dependent cell-mediated cytotoxicity (ADCC). Glyco- and protein-Fc engineering have been employed to enhance FcγR binding and ADCC activity of mAbs; the drawbacks of previous approaches lie in their binding affinity to both FcγRIIIa allotypes, the ratio of activating FcγR binding to inhibitory FcγR binding (A/I ratio) or the melting temperature (T_M) of the C_H2 domain. To date, no engineered Fc variant has been reported that satisfies all these points. Herein, we present a novel Fc engineering approach that introduces different substitutions in each Fc domain asymmetrically, conferring optimal binding affinity to FcγR and specificity to the activating FcγR without impairing the stability. We successfully designed an asymmetric Fc variant with the highest binding affinity for both FcγRIIIa allotypes and the highest A/I ratio compared with previously reported symmetrically engineered Fc variants, and superior or at least comparable in vitro ADCC activity compared with afucosylated Fc variants. In addition, the asymmetric Fc engineering approach offered higher stability by minimizing the use of substitutions that reduce the T_M of the C_H2 domain compared with the symmetric approach. These results demonstrate that the asymmetric Fc engineering platform provides best-in-class effector function for therapeutic antibodies against tumor antigens.     

Integrated fragmentation of human IgG and purification of Fab using a reactant adsorptive membrane bioreactor separator system

This article discusses an integrated separation–reaction–separation scheme for producing Fab fragment directly from human immunoglobulin G (hIgG) present in serum feed. The novel reactant adsorptive membrane bioreactor separator (or RAMBS) system used in the current study consisted of a stack of microporous adsorptive membranes held within a temperature controlled module. The membrane stack, in the presence of salt, selectively and reversibly adsorbed hIgG by hydrophobic interaction while allowing most other serum proteins to flow through. The bound hIgG was then fragmented by pumping a solution of papain through the reactor at controlled temperature and flow rate. The salt concentration and pH for reaction and separation were systematically optimized using pure hIgG as reactant. The Fab fragment was separated from undigested hIgG and other byproducts such as Fc fragment based on their differences in hydrophobicity. Under optimal conditions, Fab was obtained in the reaction flow through while the other proteins remained bound to the membrane, these being subsequently eluted by lowering the salt concentration. The RAMBS system in addition to being convenient from process integration and intensification points of view also showed higher catalytic efficiency of papain in comparison to that in liquid phase reactions. Biotechnol. Bioeng. 2009; 104: 152–161 © 2009 Wiley Periodicals, Inc.     

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.     

Crystal Structure of a Bivalent Antibody Fab Fragment

We determined the crystal structure to 1.8 Å resolution of the Fab fragment of an affinity-matured human monoclonal antibody (HC84.26.5D) that recognizes the E2 envelope glycoprotein of hepatitis C virus (HCV). Unlike conventional Fabs, which are monovalent monomers, Fab HC84.26.5D assembles into a bivalent domain-swapped dimer in which the two V_L/V_H modules are separated by ~25 Å. In solution, Fab HC84.26.5D exists predominantly as a dimer (~80%) in equilibrium with the monomeric form of the Fab (~20%). Dimerization is mediated entirely by deletion of a single residue, V_HSer113 (Kabat numbering), in the elbow region linking the V_H and C_H1 domains. In agreement with the crystal structure, dimeric Fab HC84.26.5D is able to bind two HCV E2 molecules in solution. This is only the second example of a domain-swapped Fab dimer from among >3000 Fab crystal structures determined to date. Moreover, the architecture of the doughnut-shaped Fab HC84.26.5D dimer is completely different from that of the previously reported Fab 2G12 dimer. We demonstrate that the highly identifiable shape of dimeric Fab HC84.26.5D makes it useful as a fiducial marker for single-particle cryoEM analysis of HCV E2. Bivalent domain-swapped Fab dimers engineered on the basis of HC84.26.5D may also serve as a means of doubling the effective size of conventional Fab–protein complexes for cryoEM.     

Anti-TNFα domain antibody construct CEP-37247

We report preclinical data for CEP-37247, the first human framework domain antibody construct to enter the clinic. At approximately 11 – 13kDa, domain antibodies or dAbs are the smallest antibody domain able to demonstrate the antigen-recognition function of an antibody, e.g. high selectivity and affinity for target antigen. CEP-37247 is a bivalent anti-tumor necrosis factor (TNF)α domain antibody protein construct combining the antigen-recognition function of a dAb with the pharmacological advantages of an antibody Fc region. As a homodimer, with each chain comprising V_L dAb, truncated C_H1, hinge, C_H2 and C_H3 domains, CEP-37247 has a molecular mass of approximately 78kDa, which is about half the size of a conventional IgG molecule. Surface plasmon resonance data demonstrate that CEP-37247 possesses high selectivity and affinity for TNFα. CEP-37247 is a potent neutralizer of TNFα activity in vitro in the L929 TNF-mediated cytotoxicity assay. In a human TNFα-over-expressing mouse model of polyarthritis, CEP-37247 prevents development of disease, and is at least as effective as the marketed product etanercept. Fc functionality is intact – CEP-37247 is capable of mediating antibody-dependent cell-mediated cytotoxicity and has a circulating half-life of approximately 4.5 days in cynomolgus macaques. Given the favorable properties outlined above, and its high expression levels (approaching 7 g/L) in a CHOK1 based-expression system, CEP-37247 is progressing into the clinic, where other potential advantages such as enhanced efficacy due to improved tissue distribution, and beneficial immunogenicity profile, will be evaluated.     

Structure of Fab fragment of malaria transmission blocking antibody 2A8 against P. vivax P25 protein

Understanding the structural basis of recognition between antigen and antibody requires the structural comparison of free and complexed components. Previously, we have reported the crystal structure of the complex between Fab fragment of murine monoclonal antibody 2A8 (Fab2A8) and Plasmodium vivax P25 protein (Pvs25) at 3.2 Å resolution. We report here the crystallization and X-ray structure of native Fab2A8 at 4.0 Å resolution. The 2A8 antibody generated against Pvs25 prevents the formation of P. vivax oocysts in the mosquito, when assayed in membrane feeding experiment.Comparison of native Fab2A8 structure with antigen bound Fab2A8 structure indicates the significant conformational changes in CDR-H1 and CDR-H3 regions of V_H domain and CDR-L3 region of V_L domain of Fab2A8. Upon complex formation, the relative orientation between V_L and V_H domains of Fab2A8 is conserved, while significant differences are observed in elbow angles of heavy and light chains. The combing site residues of complexed Fab2A8 exhibited the reduced temperature factor compared to native Fab2A8, suggesting a loss of conformational entropy upon antigen binding.     

Anti-TNFα domain antibody construct CEP-37247: Full antibody functionality at half the size

We report preclinical data for CEP-37247, the first human framework domain antibody construct to enter the clinic. At approximately 11–13 kDa, domain antibodies or dAbs are the smallest antibody domain able to demonstrate the antigen-recognition function of an antibody, e.g., high selectivity and affinity for target antigen. CEP-37247 is a bivalent anti-tumor necrosis factor (TNF)α domain antibody protein construct combining the antigen-recognition function of a dAb with the pharmacological advantages of an antibody Fc region. As a homodimer, with each chain comprising V_L dAb, truncated C_H1, hinge, C_H2 and C_H3 domains, CEP-37247 has a molecular mass of approximately 78 kDa, which is about half the size of a conventional IgG molecule. Surface plasmon resonance data demonstrate that CEP-37247 possesses high selectivity and affinity for TNFα. CEP-37247 is a potent neutralizer of TNFα activity in vitro in the L929 TNF mediated cytotoxicity assay. In a human TNFα-overexpressing mouse model of polyarthritis, CEP-37247 prevents development of disease and is at least as effective as the marketed product etanercept. Fc functionality is intact—CEP-37247 is capable of mediating antibody-dependent cell-mediated cytotoxicity and has a circulating half-life of approximately 4.5 days in cynomolgus macaques. Given the favorable properties outlined above and its high expression levels (approaching 7 g/L) in a CHOK1 based-expression system, CEP-37247 is progressing into the clinic where other potential advantages, such as enhanced efficacy due to improved tissue distribution and beneficial immunogenicity profile, will be evaluated.Key words: CEP-37247, ART621, domain antibody, dAbs, anti-TNFα, Fc construct     

Disulfide linkage in the coiled-coil domain of subunit H of A1AO ATP synthase from Methanocaldococcus jannaschii and the NMR structure of the C-terminal segment H85-104

The C-terminal residues 98-104 are important for structure stability of subunit H of A₁A_O ATP synthases as well as its interaction with subunit A. Here we determined the structure of the segment H_85-104 of H from Methanocaldococcus jannaschii, showing a helix between residues Lys90 to Glu100 and flexible tails at both ends. The helix-helix arrangement in the C-terminus was investigated by exchange of hydrophobic residues to single cysteine in mutants of the entire subunit H (H_I93C, H_L96C and H_L98C). Together with the surface charge distribution of H_85-104, these results shine light into the A-H assembly of this enzyme.