Fab Segments Research Articles

Quantitative Analysis of Therapeutic Antibody Interactions with Fcγ Receptors Using High-Speed Atomic Force Microscopy.

This study employed high-speed atomic force microscopy to quantitatively analyze the interactions between therapeutic antibodies and Fcγ receptors (FcγRs). Antibodies are essential components of the immune system and are integral to biopharmaceuticals. The focus of this study was on immunoglobulin G molecules, which are crucial for antigen binding via the Fab segments and cytotoxic functions through their Fc portions. We conducted real-time, label-free observations of the interactions of rituximab and mogamulizumab with the recombinant FcγRIIIa and FcγRIIa. The dwell times of FcγR binding were measured at the single-molecule level, which revealed an extended interaction duration of mogamulizumab with FcγRIIIa compared with that of rituximab. This is linked to enhanced antibody-dependent cellular cytotoxicity that is attributed to the absence of the core fucosylation of Fc-linked N-glycan. This study also emphasizes the crucial role of the Fab segments in the interaction with FcγRIIa as well as that with FcγRIIIa. This approach provided quantitative insight into therapeutic antibody interactions and exemplified kinetic proofreading, where cellular discrimination relies on ligand residence times. Observing the dwell times of antibodies on the effector molecules has emerged as a robust indicator of therapeutic antibody efficacy. Ultimately, these findings pave the way for the development of refined therapeutic antibodies with tailored interactions with specific FcγRs. This research contributes to the advancement of biopharmaceutical antibody design and optimizing antibody-based treatments for enhanced efficacy and precision.

Cascaded antibody directionality relying on a zinc-based nanocomposite for performance enhancement of multiplex lateral flow immunoassay

The quantity and orientation of antibodies are key elements affecting the bioactivity of immunoprobes, which have important influences on the sensitivity and specificity of immuno-biosensors. In this work, a multiplex lateral flow immunoassay (mLFIA) mediated by a powerful cascaded antibody directionality (CAbD) strategy was constructed for the simultaneous monitoring of nitrofurazone and furazolidone metabolites in food samples. Here the CAbD strategy innovatively integrated three elements: antibody staining by Coomassie brilliant blue R-250, directional anchoring of second antibodies (Ab2) by the amorphous ZnO-based nanocomposites (ZnO-CBB-Ab2, ZnO-NCMs), and orientation of detection antibodies (mAb1) by the Ab2. The ZnO-NCMs play a double role in antibody orientation and signaling to exhibit. Thanks to the difference in surface electrical properties of Fc and Fab segments in immunoglobulin G, the excessive R-250 stained Ab2 was anchored in ZnO-NCMs to form a multi-detection signal tag with colorimetric signal brightness. Compared with immobilization strategies of directly directional anchor-hold or post-adsorption of mAb1, this strategy can reduce the mAb1 consumption, better ensure the activity and utilization efficiency of mAb1, and shorten the construction time of the mLFIA system. As expected, the mLFIA is given satisfactory performances with a calculated sensitivity of 0.106 and 0.060 ng mL−1 for nitrofurazone and furazolidone metabolites, respectively. The developed multiplex immunosensor ultimately shows its application potential for point-of-care testing by performing well in fish and honey samples with accredited recoveries. It’s worth emphasizing that the CAbD strategy can be a universal and scatheless antibody-directed approach, especially in multiplex immunoassays for ultrasensitive food safety monitoring.

Elbow- and hinge-bending motions of IgG: Dielectric response and dynamic feature.

Immunoglobulin G (IgG) is a Y-shaped globular protein consisting of two Fab segments connecting to an Fc segment with a flexible hinge region, in which the Fab segments show secondary flexibility at an "elbow" region. In the present work, the hinge-bending and elbow-bending motions of aqueous solutions of IgG by microwave dielectric measurements below the freezing point of bulk water was observed. The presence of unfreezable water around the macromolecules reduced the effects of steric hindrance normally generated by ice and enabled the intramolecular motions of IgG. At the same time, the overall IgG molecule rotation was restricted by ice. Papain digestion and reduction of the disulfide linkage at the hinge region was used to generate Fab and Fc fragments. In solutions of these fragments, the dielectric relaxation process of the hinge-bending motion was absent, although the elbow-bending motion remained. Three relaxation processes were observed for papain-digested IgG. The high, middle, and low frequency processes were attributed to unfrozen water, local peptide motions cooperating with bound water, and the elbow-bending motion, respectively. In the case of the intact IgG, an additional relaxation process due to the hinge-bending motion was observed at frequencies lower than that of the elbow-bending motion. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 626-632, 2016.

Neutron reflectometry from poly (ethylene-glycol) brushes binding anti-PEG antibodies: Evidence of ternary adsorption

Neutron reflectometry provides evidence of ternary protein adsorption within polyethylene glycol (PEG) brushes. Anti-PEG Immunoglobulin G antibodies (Abs) binding the methoxy terminated PEG chain segment specifically adsorb onto PEG brushes grafted to lipid monolayers on a solid support. The Abs adsorb at the outer edge of the brush. The thickness and density of the adsorbed Ab layer, as well as its distance from the grafting surface grow with increasing brush density. At high densities most of the protein is excluded from the brush. The results are consistent with an inverted “Y” configuration with the two FAB segments facing the brush. They suggest that increasing the grafting density favors narrowing of the angle between the FAB segments as well as overall orientation of the bound Abs perpendicular to the surface.

Comparison of the conformations of two intact monoclonal antibodies with hinges.

Structures of two intact monoclonal antibodies were solved by X-ray diffraction analysis revealing, in both cases, the dispositions of all segments, as well as the structures of the hinge polypeptides. An IgG1, whose antigen is the drug phenobarbital, assumed a completely different conformation when compared with an IgG2a specific for canine lymphoma cells. Though neither IgG displays global two-fold symmetry, both maintain two pseudo dyad axes, one relating Fab segments, and the other the halves of the Fc. In both IgGs, the Fc segment is obliquely disposed with respect to the plane of the Fabs, making an angle of 128 degrees in the IgG2a, and 107 degrees in the IgG1. Hinge angles of the IgG1 are notably different at 78 degrees and 123 degrees, and unique as well from IgG2a values of 66 degrees and 113 degrees. Elbow angles within the IgG1 Fabs are the same at 155 degrees, but non-identical in IgG2a where they took on values of 143 degrees and 159 degrees. The IgG2a has an angle of 172 degrees between Fabs so that it exhibits a "distorted T" shape, whereas that angle in the IgG1 is a much more acute 115 degrees producing a "distorted Y". CH2 domains appear, in both antibodies, to be the most independently mobile of the paired IgG domains. This perhaps reflects their importance in modulating effector functions through exposure of binding loci on the CH2, at the CH2-CH3 interface, and on lower hinge polypeptides. Hinges in both antibodies contain disulfide-linked cores bounded by fluid regions above and below, which provide mobility to the Fabs and Fc respectively. The conformations seen in these two structures are undoubtedly only two among many but they illustrate the modes of flexibility inherent to the IgG architecture.

Crystallographic structure of an intact IgG1 monoclonal antibody

The structure of an intact monoclonal antibody for phenobarbital, subclass IgG1, has been determined to 3.2 Å resolution by X-ray crystallography. The molecule was visualized in a monoclinic unit cell having an entire immunoglobulin as the asymmetric unit. The two Fab segments, both with elbow angles of 155°, were related by a rotation of 179.7° plus a translation along the approximate dyad of 9 Å. This is the first observation of such an Fab translation in a structurally defined antibody. The approximate 2-fold of the Fc was independent of that relating Fabs, making an angle of 107° with the Fab dyad. The angle between long axes of the Fabs was 115°, the most acute angle yet observed, yielding a distorted Y shaped molecule. This is in contrast to the distorted T shape of the only other intact IgG (2a) whose complete structure is known. Primary lattice interactions arise through formation of VH antiparallel β ribbons whose strands are contributed by pseudo dyad related H2, and by L3 hypervariable loops from neighboring molecules. While one CH2 domain was mobile, Fabs and three domains of the Fc were well defined, as were hinge polypeptides connecting Fabs to the Fc, and the covalently attached oligosaccharides. Direct interactions are observed between hinge polypeptides, the glycosylated loop of one CH2 domain, and the oligosaccharide. Lattice interactions clearly influence, perhaps even determine the overall conformation of the antibody observed in this crystal. Comparison of this IgG1 with previously determined intact antibody structures extends the conformational range arising from segmental flexibility.

Refined structure of an intact IgG2a monoclonal antibody.

The structure of an intact, anti-canine lymphoma monoclonal antibody (Mab231) was determined by molecular replacement and refined in a triclinic cell to an R-value of 20.9%, using synchrotron diffraction data from 2.8 to 20 A resolution. All segments of the antibody, including the hinge region and carbohydrate component, are visible in electron density maps. There is no overall symmetry to the antibody, as the Fc is disposed in an entirely oblique manner with respect to the Fabs. The CH2 and CH3 domains do, however, possess a nearly exact, local 2-fold relationship. The Fab segments are related by a second, independent, local dyad axis, exact only with respect to constant domains. Variable domains exhibit no symmetry relationship as a consequence of the 16 degrees difference in Fab elbow angles. Variable domain pair associations VL:VH for the Fabs are virtually the same, and corresponding CDRs of the two Fabs also are nearly identical in structure. CDR-H3 displays the greatest difference. Hypervariable loops of both Fabs are involved in contacts with symmetry-related Fc segments at the CH2-CH3 switch junction, suggesting a "complex" structure. The hinge segment connecting Fabs with the Fc is quite extended and exhibits thermal factors indicative of a high degree of mobility. It consists of a well-defined upper hinge that partially maintains dyad symmetry and a fairly rigid core bounded above and below by fluid polypeptides that provide segmental flexibility. This structure represents the first visualization by X-ray analysis of a murine Fc segment, and its CH2 domains exhibit substantial rigid body conformational changes with respect to the human Fc used as an initial molecular replacement model. The oligosaccharides were found by difference Fourier syntheses to be very similar to those of the free human Fc fragment, although differences are present in the terminal residues. The detailed structure of the IgG presented here, and the distribution of effector binding sites, appears consistent with effector activation mechanisms involving translocation and/or aggregation of the Fc following antigen binding by the Fabs.

The three-dimensional structure of an intact monoclonal antibody for canine lymphoma.

Crystal structures of Fab antibody fragments determined by X-ray diffraction characteristically feature four-domain, beta-barrel arrangements. A human antibody Fc fragment has also been found to have four beta-barrel domains. The structures of a few intact antibodies have been solved: in two myeloma proteins, the flexible hinge regions that connect the Fc to the Fab segments were deleted so the molecules were non-functional, structurally restrained, T-shaped antibodies; a third antibody, Kol, had no hinge residues missing but the Fc region was sufficiently disordered that it was not possible to relate its disposition accurately with respect to the Fab components. Here we report the structure at 3.5 A resolution of an IgG2a antitumour monoclonal antibody which contains an intact hinge region and was solved in a triclinic crystal by molecular replacement using known Fc and Fab fragments. The antibody is asymmetric, reflecting its dynamic character. There are two local, apparently independent, dyads in the molecule. One relates the heavy chains in the Fc, the other relates the constant domains of the Fabs. The variable domains are not related by this 2-fold axis because of the different Fab elbow angles of 159 degrees and 143 degrees. The Fc has assumed an asymmetric, oblique orientation with respect to loosely tethered yet almost collinear Fabs. Our study enables the two antigen-binding segments as well as the Fc portion of a functional molecule to be visualized and illustrates the flexibility of these immune response proteins.

Conformations of IgE bound to its receptor Fc.epsilon.RI and in solution

Previous resonance energy transfer studies suggested that murine immunoglobulin E (IgE) is bent near the junction of its Fc and Fab segments when bound to its high-affinity receptor (Fc epsilon RI) on RBL cells. To examine further the conformations of IgE, both bound to this receptor and in solution, a mutant recombinant IgE (epsilon/C gamma 3*) was prepared that has a cysteine replacing a serine near the C-terminal ends of the heavy chain. The introduced cysteine residues provide a means for specific modification of IgE, and the sulfhydryl groups were selectively labeled with fluorescein-5-maleimide (FM-epsilon/C gamma 3*). This IgE also binds a 5-(dimethylamino)naphthalene-1-sulfonyl (DNS) group in the antigen-binding sites. Resonance energy transfer experiments carried out on receptor-bound FM-epsilon/C gamma 3* yielded a distance of 53 A between fluorescein near the C-terminal end of the Fc segment and amphipathic acceptor probes at the membrane surface. The average distance between this C-terminal fluorescein and acceptor eosin-DNS in the antigen-binding sites at the N-terminal ends of the Fab segments was found to be 69 A. These results combine with those from previous structural studies to provide an unprecedented detailed description of the bent geometry of IgE bound to its receptor on the membrane. Energy transfer measured for FM-epsilon/C gamma 3* in solution between fluorescein near the C-terminal end of the Fc segment and eosin-DNS at the N-terminal ends of the Fab segments indicates that the average distance between these probes is about 71 A.(ABSTRACT TRUNCATED AT 250 WORDS)

A nanosecond fluorescence depolarization study on the segmental flexibility of receptor-bound immunoglobulin E

Time-resolved fluorescence anisotropy measurements have been used to examine the segmental flexibility of anti-dansyl immunoglobulin E (IgE) bound to its high-affinity receptor on membrane vesicles from rat basophilic leukemia cells. Although IgE in this complex exhibits only a restricted angular range of segmental motion, much of this restricted motion occurs on a relatively rapid time scale. A fast component of motion with a rotational correlation time of 15-35 ns may correspond to the twisting of Fab segments about their major axis. Intermolecular cross-linking by a short bivalent ligand, N,N'-didansylcadaverine, results in complete loss of this segmental motion. Solubilization of monomeric IgE-receptor complexes using a zwitterionic detergent results in a time-dependent anisotropy decay that exhibits both a fast component and a slower component that is intermediate between the decay for soluble and membrane-bound forms of IgE at long times after excitation. These results are discussed in terms of a model in which binding of IgE to its membrane-bound receptor restricts not only its global rotation but also its slower modes of segmental flexibility as well, while allowing its Fab segments to undergo rapid reorientation within a limited angular range.

Structural mapping of Fc receptor bound immunoglobulin E: proximity to the membrane surface of the antibody combining site and another site in the Fab segments.

Resonance energy-transfer methods have been used to investigate the structure of immunoglobulin E (IgE) bound to its high-affinity receptor on plasma membrane vesicles derived from rat basophilic leukemia cells. The structural mapping of receptor-bound IgE was initiated in an earlier study [Holowka, D., & Baird, B. (1983) Biochemistry 22, 3475], and it is based on measuring the minimal distance from IgE sites that are selectively labeled with donor probes to a plane of amphipathic acceptors at the membrane surface. This paper describes the use of monoclonal IgE specific for 5-(dimethylamino)naphthalene-1-sulfonyl (DNS) to place a donor probe, DNS-L-Lys, in the antibody combining sites. The distance from these sites to the membrane surface was determined to be greater than 100 A with two different amphipathic acceptor probes. Another site in the Fab segments of monoclonal IgE (anti-dinitrophenyl) could be labeled selectively with N-[4-[7-(diethylamino)-4-methylcoumarin-3-yl]phenyl]maleimide (CPM) in the absence of reducing agents [CPM(-)], and the reaction could not be blocked by prereaction with N-ethylmaleimide. The pattern of CPM(-)-labeled proteolytic fragments and the lack of fluorescence quenching by (trinitrophenyl)lysine in the antibody combining sites suggested the CPM(-)-labeled site to be in the C epsilon 1 domain of IgE. The distance between this site on receptor-bound IgE and the membrane surface was determined to be 75-87 A with two different amphipathic acceptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Structural mapping of membrane-bound immunoglobulin E-receptor complexes: use of monoclonal anti-IgE antibodies to probe the conformation of receptor-bound IgE.

Previous resonance energy-transfer measurements have suggested that immunoglobulin E (IgE) may bend near the junction of its Fc and Fab segments in order to bind to its high-affinity receptor on rat basophilic leukemia cells. In order to test this possibility, two monoclonal antibodies were employed that bind specifically to rat IgE (IgER) when IgER is in solution and when it is bound to receptors on the plasma membrane. The F(ab')2 fragment of one monoclonal (B5) that is specific for the Fab region of IgER was labeled with donor probes and bound to IgER, and the quenching of the fluorescence of these donors due to simultaneous binding of the Fab' fragment of an anti-Fc monoclonal (A2) that was labeled with an acceptor probe at its interchain disulfide bond was measured. Significantly less energy transfer between these probes was observed when IgER was bound to its receptor on membrane vesicles than when it was free in solution, and this result is interpreted in light of other energy-transfer measurements using A2 and B5 that were preferentially labeled near their combining sites with donors and acceptors, respectively, as well as measurements of the distance of closest approach between these sites and the membrane surface. These results along with previous energy-transfer measurements and other biochemical information form the basis for a working model of the conformation and orientation of receptor-bound IgE. This study demonstrates the use of fluorescently labeled monoclonal antibodies as highly selective energy-transfer probes in assessing structures of macromolecular complexes on the plasma membrane.

Immunoglobulin G antibody bound to the C1q subcomponent of human complement exhibits segmental flexibility

The rotational dynamics of rabbit immunoglobulin G (IgG) anti-dansyl antibodies bound to the C1q subcomponent of human complement were studied by nanosecond fluorescence spectroscopy. Deconvoluted anisotropy decays of IgG-C1q mixtures were fitted to a two-exponential expression and were corrected for the effects of unbound IgG, which was determined with an analytical ultracentrifuge. Compared with the anisotropy parameters for free IgG, the pre-exponential weighting factors and the short correlation time of the C1q-bound antibody were nearly unchanged, and the long correlation time increased by only about 45 nanoseconds. These results, together with rotational diffusion calculations, indicate that the Fab arms of the C1q-bound antibody exhibited considerable flexibility. This finding may have biological relevance because it suggests that C1q can bind to the Fc segments of IgG molecules anchored in an immune complex, even though the angles between the two Fab arms of the different antibodies may vary. The results of this study also support our earlier interpretation that both the short and long correlation times of IgG principally represent flexible motions of the Fab segments.

Rotational dynamics of immunoglobulin G antibodies anchored in protein a soluble complexes

The rotational dynamics of rabbit IgG anti-dansyl antibodies anchored in staphylococcal protein A (SpA) soluble complexes were studied by both steady-state and nanosecond fluorescence spectroscopy. To aid in the interpretation of the anisotropy data, the results of recently reported hydrodynamic and electron microscopic studies of IgG-SpA complexes were used to calculate global tumbling times of the various complexes and to estimate the steric hindrance of the antibody Fab segments. The anisotropy decays, fitted to the sum of two exponentials, indicated that the Fab arms of antibodies bound to SpA by their Fc regions exhibit considerable flexibility. For the different IgG-SpA mixtures examined, changes in the IgG preexponential anisotropy weighting factors, ƒ S and ƒ L and the short rotational correlation time, φ S were relatively small. On the other hand, the long rotational correlation time, φ L , increased systematically when the percentage of larger IgG-SpA complexes in a mixture was increased. The greatest restriction of Fab flexibility was observed for antibodies anchored in the exceptionally compact IgG 4-SpA 2 complexes. Available electron microscopic data suggest that increases in φ L correlate with increased steric hindrance of the antibody segments. Both native and hinge-disulfide-cleaved IgG experienced similar percentage increases in φ L when bound in SpA complexes. In agreement with our earlier interpretation, the results of this study provide rather striking evidence that φ L mainly represents flexible motions of the Fab segments and not global tumbling: the φ L -values of IgG bound in the various SpA complexes ranged from 101 to 162 nsec, whereas the calculated global tumbling times of the different complexes ranged from about 300 to 3000 nsec.

Segmental flexibility of immunoglobulin G antibody molecules in solution: a new interpretation.

We propose a new model for the segmental flexibility of immunoglobulin G (IgG). The flexibility of native and mildly reduced anti-5-(dimethylamino)naphthalene-1-sulfonyl (anti-dansyl) antibody was reexamined by nanosecond fluorescence spectroscopy using deconvolution and lamp-shift corrections. The rabbit antibodies used for this study were purified of dimers and other aggregates. The original results indicated that the decay of fluorescence anisotropy involved two rotational correlation times. It was suggested that the short rotational correlation time, phi s, represented a flexible Fab arm motion over a restricted angle and that the long correlation time, phi L, represented global tumbling of the molecule [Yguerabide, J., Epstein, H. F., & Stryer, L. (1970) J. Mol. Biol. 51, 573--590]. Our new data indicate that the long correlation time primarily represents motions of the Fab segments and not global tumbling of the entire molecule. This interpretation implies a more flexible model for IgG. Thus, in solution the antibody arms appear to move over a wide angle and are not restricted to 33 degrees as was suggested in the earlier model. Simple diffusion calculations and other evidence suggest that phi s may represent V-module flexibility about the switch peptides or Fab twisting around its long axis, whereas phi L may represent wagging or wobbling motions of the Fab arms about the hinge region. The faster motions appear to occur over small angles whereas the slower wagging or wobbling motions responsible for most of the decay of anisotropy appear to be much less restricted. The biological function of IgG and anisotropy changes resulting from hinge disulfide cleavage are interpreted in terms of the proposed model. We also demonstrate a useful method for comparison of time-dependent and steady-state fluorescence polarization data.