Minute virus of mice, a parvovirus, in complex with the Fab fragment of a neutralizing monoclonal antibody

The structure of virus-like particles of the lymphotropic, immunosuppressive strain of minute virus of mice (MVMi) in complex with the neutralizing Fab fragment of the mouse monoclonal antibody (MAb) B7 was determined by cryo-electron microscopy to 7-A resolution. The Fab molecule recognizes a conformational epitope at the vertex of a three-fold protrusion on the viral surface, thereby simultaneously engaging three symmetry-related viral proteins in binding. The location of the epitope close to the three-fold axis is consistent with the previous analysis of MVMi mutants able to escape from the B7 antibody. The binding site close to the symmetry axes sterically forbids the binding of more than one Fab molecule per spike. MAb as well as the Fab molecules inhibits the binding of the minute virus of mice (MVM) to permissive cells but can also neutralize MVM postattachment. This finding suggests that the interaction of B7 with three symmetry-related viral subunits at each spike hinders structural transitions in the viral capsid essential during viral entry.     

Structure of human rhinovirus complexed with Fab fragments from a neutralizing antibody.

We have determined the structure of a human rhinovirus (HRV)-Fab complex by using cryoelectron microscopy and image reconstruction techniques. This is the first view of an intact human virus complexed with a monoclonal Fab (Fab17-IA) for which both atomic structures are known. The surface area on HRV type 14 (HRV14) in contact with Fab17-IA was approximately 500 A2 (5 nm2), which is much larger than the area that constitutes the NIm-IA epitope (on viral protein VP1) defined by natural escape mutants. From modeling studies and electrostatic potential calculations, charged residues outside the neutralizing immunogenic site IA (NIm-IA) were also predicted to be involved in antibody recognition. These predictions were confirmed by site-specific mutations and analysis of the Fab17-IA-HRV14 complex, along with knowledge of the crystallographic structures of HRV14 and Fab17-IA. The bound Fab17-IA reaches across a surface depression (the canyon) and meets a related Fab at the nearest icosahedral twofold axis. By adjusting the elbow angles of the bound Fab fragments from 162 degrees to 198 degrees, an intact antibody molecule can be easily modeled. This, along with aggregation and binding stoichiometry results, supports the earlier proposal that this antibody binds bivalently to the surface of HRV14 across icosahedral twofold axes. One prediction of this model, that the intact canyon-spanning immunoglobulin G molecule would block attachment of the virus to HeLa cells, was confirmed experimentally.     

Three-dimensional structure of the Fab fragment of a neutralizing antibody to human rhinovirus serotype 2.

The crystal structure of the antigen-binding fragment of a monoclonal antibody (8F5) that neutralizes human rhinovirus serotype 2 has been determined by X-ray diffraction studies. Antibody 8F5, obtained by immunization with native HRV2 virions, cross-reacts with peptides of the viral capsid protein VP2, which contribute to the neutralizing immunogenic site B in this serotype. The structure was solved by the molecular replacement method and has been refined to an R-factor of 18.9% at 2.8 A resolution. The elbow angle, relating the variable and constant modules of the molecule is 127 degrees, representing the smallest elbow angle observed so far in an Fab fragment. Furthermore, the charged residues of the epitope can be well accommodated in the antigen-binding site. This is the first crystal structure reported for an antibody directed against an icosahedral virus.     

Anchoring a cationic ligand: the structure of the Fab fragment of the anti-morphine antibody 9B1 and its complex with morphine

The crystal structures of an anti-morphine antibody 9B1 (to 1.6A resolution) and its complex with morphine (to 2.0 A resolution) are reported. The morphine-binding site is described as a shallow depression on the protein surface, an unusual topology for a high-affinity ( Ka approximately 10(9) M(-1)) antibody against a small antigen. The polar part of the ligand is exposed to solvent, and the cationic nitrogen atom of the morphine molecule is anchored at the bottom of the binding site by a salt-bridge to a glutamate side-chain. Additional affinity is provided by a double cation-pi interaction with two tryptophan residues. Comparison of the morphine complex with the structure of the free Fab shows that a domain closure occurs upon binding of the ligand.     

Crystal structure of vascular endothelial growth factor-B in complex with a neutralising antibody Fab fragment

Vascular endothelial growth factor (VEGF) B effects blood vessel formation by binding to VEGF receptor 1. To study the specifics of the biological profile of VEGF-B in both physiological and pathological angiogenesis, a neutralising anti-VEGF-B antibody (2H10) that functions by inhibiting the binding of VEGF-B to VEGF receptor 1 was developed. Here, we present the structural features of the ‘highly ordered’ interaction of the Fab fragment of this antibody (Fab-2H10) with VEGF-B. Two molecules of Fab-2H10 bind to symmetrical binding sites located at each pole of the VEGF-B homodimer, giving a unique U-shaped topology to the complex that has not been previously observed in the VEGF family. VEGF-B residues essential for binding to the antibody are contributed by both monomers of the cytokine. Our detailed analysis reveals that the neutralising effect of the antibody occurs by virtue of the steric hindrance of the receptor-binding interface. These findings suggest that functional complementarity between VEGF-B and 2H10 can be harnessed both in analysing the therapeutic potential of VEGF-B and as an antagonist of receptor activation.     

Solution conformation of an immunogenic peptide from HRV2: comparison with the conformation found in a complex with a Fab fragment of an anti-HRV2 neutralizing antibody

The conformation of a [15]-peptide (H-VKAETRLNPDLQPTE-NH₂) from VP2 of rhinovirus HRV2 complexed with a Fab fragment was previously shown by X-ray crystallographic studies to be similar to the one found in the corresponding region of HRV1A. Antibodies raised against this peptide bind to and neutralize HRV2. In order to identify structural features preserved in solution that may explain the ability of this short peptide to mimic the structure of the protein surface, the peptide has been studied by NMR in aqueous solution as well as under denaturing conditions. The peptide is shown to be a random coil in solution. However, the sequence forming a 3₁₀ helix in the complex is biased into a helical conformation according to NOE intensity data as well as from urea and pH titrations. This sequence adopts the same conformation in an unrelated protein. NOE data suggest that a β-turn found in the complex may be sampled in solution. Also, Glu4, interacting with Arg6 in the crystal, has a reduced pK_a value in solution. It is concluded that the local structure present in the random coil state of VP2(156–170) contains enough information to direct the production of antibodies that bind to and neutralize HRV2. © 1998 European Peptide Society and John Wiley & Sons, Ltd.     

Preliminary crystallographic study of a complex between the Fab fragment of a monoclonal anti-lysozyme antibody (D1.3) and the Fab fragment from an anti-idiotopic antibody against D1.3

An anti-lysozyme antibody, D1.3, was used as immunogen to obtain syngeneic (Balb/c) monoclonal anti-idiotopic antibodies. The complex between Fab D1.3 and the Fab fragment from the anti-idiotopic antibody E225 has been crystallized. The crystals are monoclinic, space group P2(1), with a = 75.7 A, b = 77.4 A, c = 97.2 A, beta = 111.90 degrees and one molecule of the complex in the asymmetric unit. X-ray photographs show reflections extending to a resolution of about 3 A. Although twinning occurs frequently in the large crystals obtained, this material is suitable for high-resolution X-ray analysis.     

Crystal structure of the TCR co-receptor CD8alphaalpha in complex with monoclonal antibody YTS 105.18 Fab fragment at 2.88 A resolution

The CD8 glycoprotein functions as an essential element in the control of T-cell selection, maturation and the TCR-mediated response to peptide antigen. CD8 is expressed as both heterodimeric CD8alphabeta and homodimeric CD8alphaalpha isoforms, which have distinct physiological roles and exhibit tissue-specific expression patterns. CD8alphaalpha has previously been crystallized in complex with class I pMHC and, more recently, with the mouse class Ib thymic leukemia antigen (TL). Here, we present the crystal structure of a soluble form of mouse CD8alphaalpha in complex with rat monoclonal antibody YTS 105.18 Fab fragment at 2.88 A resolution. YTS 105.18, which is commonly used in the blockade of CD8+ T-cell activation in response to peptide antigen, is specific for mouse CD8alpha. The YTS 105.18 Fab is one of only five rat IgG Fab structures to have been reported to date. Analysis of the YTS 105.18 Fab epitope on CD8alpha reveals that this antibody blocks CD8 activity by hydrogen bonding to residues that are critical for interaction with both class I pMHC and TL. Structural comparison of the liganded and unliganded forms of soluble CD8alphaalpha indicates that the mouse CD8alphaalpha immunoglobulin-domain dimer does not undergo significant structural alteration upon interaction either with class I pMHC or TL.     

Germline humanization of a murine Aβ antibody and crystal structure of the humanized recombinant Fab fragment

Alzheimer's disease is the most common form of dementia, affecting 26 million people worldwide. The Aβ peptide (39–43 amino acids) derived from the proteolytic cleavage of the amyloid precursor protein is one of the main constituents of amyloid plaques associated with disease pathogenesis and therefore a validated target for therapy. Recently, we characterized antibody fragments (Fab and scFvs) derived from the murine monoclonal antibody WO‐2, which bind the immunodominant epitope (₃EFRH₆) in the Aβ peptide at the N‐terminus. In vitro, these fragments are able to inhibit fibril formation, disaggregate preformed amyloid fibrils, and protect neuroblastoma cells against oligomer‐mediated toxicity. In this study, we describe the humanization of WO‐2 using complementary determining region loop grafting onto the human germline gene and the determination of the three‐dimensional structure by X‐ray crystallography. This humanized version retains a high affinity for the Aβ peptide and therefore is a potential candidate for passive immunotherapy of Alzheimer's disease.     

Structure of an anti-Lewis X Fab fragment in complex with its Lewis X antigen

The Lewis X trisaccharide is pivotal in mediating specific cell-cell interactions. Monoclonal antibody 291-2G3-A, which was generated from mice infected with schistosomes, has been shown to recognize the Lewis X trisaccharide. Here we describe the structure of the Fab fragment of 291-2G3-A, with Lewis X, to 1.8 A resolution. The crystallographic analysis revealed that the antigen binding site is a rather shallow binding pocket, and residues from all six complementary determining regions of the antibody contact all sugar residues. The high specificity of the binding pocket does not result in high affinity; the K(D) determined by isothermal calorimetry is 11 microM. However, this affinity is in the same range as for other sugar-antibody complexes. The detailed understanding of the antibody-Lewis X interaction revealed by the crystal structure may be helpful in the design of better diagnostic tools for schistosomiasis and for studying Lewis X-mediated cell-cell interactions by antibody interference.     

Crystallization and preliminary x-ray studies of the Fab fragment from a humanized version of the mouse anti-human fas antibody HFE7a

A humanized version of the apoptosis-inducing mouse anti-human Fas monoclonal antibody, HFE7A, is under further development for the treatment of autoimmune diseases such as rheumatoid arthritis. We have crystallized the antigen-binding fragment (Fab) of the humanized HFE7A. The crystals belong to the orthorhombic space group P2(1)2(1)2(1) with cell dimensions a = 54.4 A, b = 82.7 A, c = 104.9 A and contain one Fab molecule in the asymmetric unit. X-ray diffraction data were collected to 2.8 A resolution.     

Radioiodinated iodobenzoyl conjugates of a monoclonal antibody Fab fragment. In vivo comparisons with chloramine-T-labeled Fab

A comparative investigation of the biodistributions of radioiodinated p- and m-iodobenzoyl conjugates of a monoclonal antibody Fab fragment, NR-LU-10 Fab, and the same antibody Fab fragment radioiodinated by the chloramine-T (ChT) method has been carried out in mice. Coinjected, dual-isotope studies in athymic mice with tumor xenografts have demonstrated that there are only minor differences in the in vivo distributions of the iodobenzoyl-labeled Fabs, except in the excretory organs, kidneys, and intestines, where major differences were observed. Similarly, coinjection of either the p-iodobenzoyl or m-iodobenzoyl conjugate of NR-LU-10 Fab with the Fab radioiodinated with ChT/radioiodide into BALB/c mice provided additional data that indicated that the two iodobenzoyl conjugates distributed similar in a number of selected tissues. The tissue-distribution differences of the regioisomeric iodobenzoyl conjugates in relation to the ChT-radioiodinated Fab were large for the stomach and neck, consistent with previous studies. The most notable difference between the two iodobenzoyl conjugates was the kidney activity, where the m-iodobenzoyl conjugate was similar to the directly labeled Fab, but the p-iodobenzoyl-conjugated Fab was higher by nearly a factor of 2.     

Structure of a glycoconjugate in solution and in complex with an antibody Fv fragment

By use of heteronuclear (¹³c, ¹H) NMR methods, the threedimensionalstructure and dynamia of the glycoconjugate estrone-3-glucuronide(E3G) uniformly ¹³c enriched in the glucuronic acid moiety hasbeen probed both in free solution and in association with ananti-E3G antibody singlechain Fv fragment. The glycan is foundto exist in multiple conformations in free solution, with particularlylarge torsional fluctuations about the glycosidic linkage .Resonance assignments and distance restraints for the glycococonjugatein the bound state were obtained from heteronuclear protonarbon-carbon-proton-COSYand isotopeedited NOESY techniques, respectively. Quantitationof the NOE data with a full-relaxation matrix approach showedthat the antibody selects a conformation from the solution repertoirewhich does not correspond with either of the two lowest energyconformations of the free glycan, and the internal energy ofthe glycan in the bound state is estimated to be at most 15kcal/mol higher than the global minimum energy conformation.The glucuronide moiety undergoes a stacking interaction withan aromatic ring in the binding site, and both ring-currentshifts and nuclear Overhauser effects computed from the predictedboundstate conformation are in good agreement with experiment.The bound-state conformation is also in goad agreement withpreliminary x-ray data on a related complex. NMR estrone antibody ring current shifts     

Expression, purification, and characterization of humanized anti-HBs Fab fragment

Anti-HBs Fab fragment has considerable potential for use in the prevention and treatment of liver diseases by HBV. Here we established a high-level expression system to directly produce anti-HBs Fab fragment in Pichia pastoris. This was achieved by co-integration of the genes encoding the heavy and light chains both under the genome of the yeast cells. The Fab fragment was efficiently secreted into medium at a concentration of 50 mg/liter. The authenticity of the Fab fragment was confirmed by immunoblot analysis, which yielded one band of approximately 50 kDa under nonreducing conditions and two bands of approximately 28 kDa under reducing conditions. The anti-HBs Fab fragment was prepared with a purity of 95% by affinity chromatography. The affinity activity of the recombinant Fab was detected by ELISA, which indicated that 1 mg of recombinant Fab was equivalent to 40 IU HBIG (20 IU/mg). The results demonstrated that the recombinant Fab fragment could sufficiently neutralize the HBsAg.     

Crystal structure of the alpha1beta1 integrin I domain in complex with an antibody Fab fragment

The alpha1beta1 (VLA-1) integrin is a cell-surface receptor for collagen and laminin and has been implicated in biological pathways involved in several pathological processes. These processes may be inhibited by the monoclonal antibody AQC2, which binds with high affinity to human alpha1beta1 integrin. To understand the structural basis of the inhibition we determined the crystal structure of the complex of a chimeric rat/human I domain of the alpha1beta1 integrin and the Fab fragment of humanized AQC2 antibody. The structure of the complex shows that the antibody blocks the collagen binding site of the I domain. An aspartate residue, from the CDR3 loop of the antibody heavy chain, coordinates the MIDAS metal ion in a manner similar to that of a glutamate residue from collagen. Substitution of the aspartate residue by alanine or arginine results in significant reduction of antibody binding affinity. Interestingly, although the mode of metal ion coordination resembles that of the open conformation, the I domain maintains an overall closed conformation previously observed only for unliganded I domains.     

Preliminary crystallographic study of the complex between the Fab fragment of a monoclonal anti-lysozyme antibody and its antigen

Preliminary crystallographic data are given for the complex between the Fab fragment of a monoclonal anti-lysozyme antibody and its antigen. This crystalline complex was found by screening a number of Fab-lysozyme complexes prepared from monoclonal anti-lysozyme antibodies produced by hybrids of BALB/c immune spleen cells with a non-secreting mouse hybrid myeloma line. The complex crystallizes in the monoclinic space group P21 with a = 55.5 (+/- 0.1) A, b = 143.5 (+/- 0.3) A, c = 49.1 (+/- 0.1) A, beta = 120 degrees 20' (+/- 10'). X-ray photographs show reflections extending to a resolution of 2.7 A. The crystals are suitable for high-resolution X-ray diffraction studies.     

Crystallization and preliminary x-ray diffraction studies of the Fab fragment of a neutralizing monoclonal antibody directed against human rhinovirus serotype 2

We report on the preparation, crystallization, and preliminary x-ray diffraction analysis of the Fab fragment of the monoclonal antibody 8F5 that neutralizes infectivity of human rhinovirus serotype 2 (HRV2). Fab fragments prepared from this antibody by papain digestion were purified to isoelectric homogeneity by ion exchange chromatography and chromatofocusing. Crystals were obtained by the hanging drop vapor diffusion method using ammonium sulfate as precipitant. The crystals belong to the orthorhombic space group P2(1)2(1)2(1) with unit cell dimensions a = 59.9 A, b = 86.3 A, c = 128.2 A and diffract to at least 2.8-A resolution. The cell volume suggests the presence of one molecule per asymmetric unit, and the solvent content is estimated to be 61%.     

The crystal structure of an octapeptide repeat of the Prion protein in complex with a Fab fragment of the POM2 antibody

Prion diseases are progressive, infectious neurodegenerative disorders caused primarily by the misfolding of the cellular prion protein (PrP^c) into an insoluble, protease‐resistant, aggregated isoform termed PrP^sc. In native conditions, PrP^c has a structured C‐terminal domain and a highly flexible N‐terminal domain. A part of this N‐terminal domain consists of 4–5 repeats of an unusual glycine‐rich, eight amino acids long peptide known as the octapeptide repeat (OR) domain. In this article, we successfully report the first crystal structure of an OR of PrP^c bound to the Fab fragment of the POM2 antibody. The structure was solved at a resolution of 2.3 Å by molecular replacement. Although several studies have previously predicted a β‐turn‐like structure of the unbound ORs, our structure shows an extended conformation of the OR when bound to a molecule of the POM2 Fab indicating that the bound Fab disrupts any putative native β turn conformation of the ORs. Encouraging results from several recent studies have shown that administering small molecule ligands or antibodies targeting the OR domain of PrP result in arresting the progress of peripheral prion infections both in ex vivo and in in vivo models. This makes the structural study of the interactions of POM2 Fab with the OR domain very important as it would help us to design smaller and tighter binding OR ligands.     

Production of recombinant humanized anti-HBsAg Fab fragment from Pichia pastoris by fermentation

In this report, we describe the high-yield secretory expression of the recombinant human anti-HBsAg Fab fragment from Pichia pastoris that was achieved by co-integration of the genes encoding the heavy and light chains (both under the control of alcohol oxidase promoter) into the genome of the yeast cells. The fed-batch fermentations were carried out in a 5 L scale. Both chains of the Fab were successfully expressed upon methanol induction. The absorbance (OD600) of the broth can reach 350 approximately 500 at the end of fed-batch phase. After the induction, the expression level of the recombinant Fab (soluble) reached 420 approximately 458 mg/L. The recombinant Fab fragment was purified from the crude culture supernatant by ion exchange chromatography and the purity of the recombinant Fab fragment was over 95%. The affinity activities of the crude fermentation supernatant and the purified Fab were analyzed by indirect ELISA, which showed that the purified recombinant Fab fragment had high affinity activity with hepatitis B surface antigen.