Preliminary crystallographic study of a complex between an heteroclitic anti-hen egg-white lysozyme antibody and the heterologous antigen pheasant egg-white lysozyme

We report the preparation, crystallization and preliminary X-ray crystallographic study of the Fab fragment from a heteroclitic murine (BALB/c) monoclonal anti-hen egg-white lysozyme antibody complexed with a heterologous antigen, pheasant lysozyme. The complex between the heterologous antigen and the antibody has been crystallized from polyethylene glycol 8000 solutions in a form suitable for X-ray crystallographic studies. The crystals are monoclinic, space group C2 with a = 158.2 A, b = 49.1 A, c = 177.6 A, beta = 92.0 degrees (1 A = 0.1 nm).     

Crystal structure of an anti-Lewis alpha Fab determined by molecular replacement methods

The anti-Lewis alpha mouse immunoglobulin CF4C4 (IgGl, k) Fab has been crystallized from 58% saturated ammonium sulfate in space group Pl; unit cell dimensions a = 43.4 A b = 41.7 A, c = 62.0 A, a = 72.7 degrees, beta = 96.6 degrees, gamma = 100.1 degrees. X-ray diffraction data have been measured beyond 3.0 A Bragg spacing. The crystal structure has been determined by molecular replacement methods, using as search models the constant and variable domains of the mouse immunoglobulin McPC603 (IgA, kappa) Fab. The crystallographic residual for the data 5.0 to 4.0 A, is 0.47. The approximate 2-fold axis relating the VL and the VH domains forms an angle of 164 degrees with the 2-fold axis relating the constant domains. The crystal packing is reasonable.     

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.     

The crystal structure of the Fab fragment of a rat monoclonal antibody against the main immunogenic region of the human muscle acetylcholine receptor.

The crystal structure of the Fab fragment of a rat monoclonal antibody, number 192, with a very high affinity (Kd = 0.05 nM) for the main immunogenic region of the human muscle acetylcholine receptor (AChR), has been determined and refined to 2.4 A resolution by X-ray crystallographic methods. The overall structure is similar to a Fab (NC6.8) from a murine antibody, used as a search model in molecular replacement. Structural comparisons with known antibody structures showed that the conformations of the hypervariable regions H1, H2, L1, L2, L3 of Fab192 adopt the canonical structures 1, 1, 2, 1, and 1, respectively. The surface of the antigen-binding site is relatively planar, as expected for an antibody against a large protein antigen, with an accessible area of 2865 A2. Analysis of the electrostatic surface potential of the antigen-binding site shows that the bottom of the cleft formed in the center of the site appears to be negatively charged. The structure will be useful in the rational design of very high affinity humanized mutants of Fab192, appropriate for therapeutic approaches of the model autoimmune disease myasthenia gravis.     

Preliminary crystallographic study of the Fab fragments of two monoclonal anti-2-phenyloxazolone antibodies

We report on the preparation, crystallization, and preliminary x-ray crystallographic study of the Fab fragments of two monoclonal anti-2-phenyloxazolone antibodies obtained from the secondary response to this hapten. The Fab fragment from one of these (NQ10/12.5) has been crystallized from polyethylene glycol 8000 solutions in a form suitable for high-resolution x-ray crystallographic studies. These crystals are monoclinic, space group C2, with a = 129.2 A, b = 79.4 A, c = 57.7 A, beta = 96.2 degrees, and one Fab/asymmetric unit. Determination of the three-dimensional structure of Fab NQ10/12.5 should help clarify the role of somatic mutation in the maturation of an immune response. This antibody and an anti-lysozyme antibody also under study apparently use the same germ-line encoded VK and a similar VH gene, respectively, as the idiotypic anti-oxazolone antibodies characteristic of the primary response. A comparative study of the two structures should shed light on the role of the pairing of heavy and light chains in the antigen-binding function of antibodies.     

Crystallization of the Fab from a human monoclonal antibody against gp 41 of human immunodeficiency virus type I

A monoclonal IgG antibody directed against gp 41 from the human immunodeficiency virus (HIV-1) has been crystallized in both intact and Fab forms. Crystals of the intact antibody grow as tetragonal-like prisms too small for conventional X-ray analysis. However, the Fab portion of the antibody produces suitable plate-like crystals which belong to the space group P2(1)2(1)2(1) with unit cell constants of a = 66.5 A, b = 74.3 A and c = 105.3 A. There is one molecule of Fab in the asymmetric unit. The Fab crystals show diffraction to d-spacings less than 3.0 A.     

Crystallization and preliminary X-ray investigation of a complex between a Fab fragment and its antigen, cyclosporin

Preliminary crystallographic data are given for a complex between the cyclic undecapeptide cyclosporin and the Fab fragment of an anti-cyclosporin monoclonal antibody. Crystals of the complex are orthorhombic with space group P2(1)2(1)2(1) and diffract to 2.7 A resolution. The unit cell dimensions are a = 52.6 A, b = 70.2 A and c = 118.4 A. A native data set to 2.7 A resolution has been collected.     

Characterization of crystals of an Fab fragment of a murine monoclonal antibody.

The Fab fragment of an antibody, made against an E2-specific feline infectious peritonitis virus neutralizing antibody, has been crystallized in a form suitable for X-ray diffraction analysis from PEG 4000 using vapor diffusion methods. The Fab fragment crystals diffract to about 2.9 A resolution and are of triclinic space group P1. Unit cell dimensions, by which the reciprocal lattice can be indexed, are a = 57.16 A, b = 70.85 A, c = 75.81 A, alpha = 85.11 degrees, beta = 121.28 degrees and gamma = 116.33 degrees. There are two Fab fragments comprising the asymmetric unit of the crystals. The presence of a pseudo-mirror plane in the diffraction pattern suggests the presence of at least an approximate dyad axis relating the two Fab fragments within the asymmetric unit.     

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.     

Crystallization and preliminary X-ray analysis of the complex between a mouse Fab fragment and a single IgG-binding domain from streptococcal protein G.

The Fab fragment of a mouse immunoglobulin G1, complexed with a single IgG-binding domain from streptococcal protein G, has been crystallized in a form suitable for analysis by X-ray diffraction. The needle-shaped crystals were grown from polyethylene glycol 4000 using vapour diffusion methods and diffract to 2.3 A resolution. The space group is P2(1)2(1)2(1) (a = 64.5 A, b = 70.5 A and c = 120.1 A), with one Fab-protein G domain complex in the asymmetric unit. Solution of the three-dimensional structure of the complex will permit a detailed analysis of the molecular interactions between protein G and the Fab portion of IgG.     

Two routes for production and purification of Fab fragments in biopharmaceutical discovery research: Papain digestion of mAb and transient expression in mammalian cells

Fab (fragment that having the antigen binding site) of a monoclonal antibody (mAb) is widely required in biopharmaceutical research and development. At Centocor, two routes of Fab production and purification were used to enable a variety of research and development efforts, particularly, crystallographic studies of antibody–antigen interactions. One route utilizes papain digestion of an intact monoclonal antibody for Fab fragment production. After digestion, separation of the Fab fragment from the Fc (fragment that crystallizes) and residual intact antibody was achieved using protein A affinity chromatography. In another route, His-tagged Fab fragments were obtained by transient expression of an appropriate construct in mammalian cells, and typical yields are 1–20 mg of Fab fragment per liter of cell culture. The His-tagged Fab fragments were first captured using immobilized metal affinity chromatography (IMAC). To provide high quality protein sample for crystallization, Fabs from either proteolytic digestion or from direct expression were further purified using size-exclusion chromatography (SEC) and/or ion-exchange chromatography (IEC). The purified Fab fragments were characterized by mass spectrometry, SDS–PAGE, dynamic light scattering, and circular dichroism. Crystallization experiments demonstrated that the Fab fragments are of high quality to produce diffraction quality crystals suitable for X-ray crystallographic analysis.     

Crystallization and preliminary structural studies of a chorismate mutase catalytic antibody complexed with a transition state analog

The Fab′ fragment of a catalytic antibody with chorismate mutase activity has been crystallized as a complex with the transition-state analog hapten. The complex was crystallized by the vapor diffusion method using ammonium sulfate as the precipitant. The crystals belong to the orthorhombic space group P2₁2₁2₁ with unit cell dimensions a = 37.1 Å, b = 63.3 Å, c = 178.5 Å, and there is one Fab' molecule per asymmetric unit. The crystals diffract X-rays to at least 3.0 Å and are suitable for X-ray crystallographic studies. © 1994 John Wiley & Sons, Inc.     

Crystallization of the complexes between M315 idiotope and its monoclonal anti-idiotopic antibody Fab fragment

The Fab fragment of a monoclonal anti-idiotopic antibody against M315 has been isolated and its complexes with Fv and Fab' fragment of M315 have been crystallized by using poly(ethylene glycol) 6000 or ammonium sulfate. X-ray diffraction photographs showed that the crystal of the complex with Fv diffracts better than that with Fab'. The Fv-complexed crystal was shown to be tetragonal I4, with cell dimensions a = 152 A and c = 69 A, and to contain one complex molecule of about 75,000 molecular weight in the crystallographic asymmetric unit.     

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.     

Crystallization and preliminary X-ray diffraction studies of a monoclonal antibody Fab fragment specific for an influenza virus haemagglutinin and of an escape mutant of that haemagglutinin

Preliminary crystallographic data are given for two molecules involved in the interaction between the humoral immune response and the influenza virus. These molecules are the Fab fragment of an antibody specific for the haemagglutinin of influenza virus strain X31 (Hong Kong 1/68 (H3N2)) and a mutant of X31 haemagglutinin that escapes recognition by that antibody. Crystals of the haemagglutinin are isomorphous to those of X31, whose structure is known; they diffract to 3.4 A resolution. Crystals of the Fab fragment are trigonal with space group P3(1)21 (or P3(2)21) and diffract to 2.6 A resolution. The unit cell dimensions are a = b = 98.9 A, c = 89.2 A. A native data set has been collected for both proteins.     

Docking of a human rhinovirus neutralizing antibody onto the viral capsid

The structure of the complex between the Fab fragment of a human rhinovirus serotype 2 (HRV2) neutralizing antibody (8F5) and a cross-reactive synthetic peptide derived from the viral capsid protein VP2 has been recently determined by crystallographic methods.¹ The conformation adopted by the peptide was very similar to and could be superimposed onto the corresponding region of the viral protein VP2 of human rhinovirus 1A (HRV1A) whose three-dimensional structure is known.² The structure of the Fab fragment determined in the complex was docked onto the viral capsid using the superimposition transformation found for the peptide. In the resulting model the Fab protrudes almost radially to about 60 Å from the surface of the virion without any major steric problem. The Fab fragment was then placed on each one of the 60 equivalent epitopes using the T = 1 icosahedral symmetry of the virus. The closest pairs of Fab fragments are related by viral 2-fold axes and run almost parallel to each other without clashing. These axes of symmetry from the viral particle could thus be coincident with the dyad axes of the antibodies. Furthermore, comparison of the three-dimensional structure of the Fab/peptide complex with the structure of the Fab fragment alone³ indicates that the flexibility of the antibody's elbow would facilitate bivalent attachment to the same viral particle. In accordance with the docking results, experimental determination of the stoichiometry of binding yielded a ratio of 30 IgG molecules per virion also suggesting bivalent attachment of antibody 8F5 onto the viral particle. The neutralization of viral infectivity, being neither aggregation (this paper) nor inhibition of receptor binding,⁴ might be mainly achieved by reducing viral spread from cell to cell and/or inhibition of uncoating. © 1995 Wiley-Liss, Inc.     

Crystal structure of Fab198, an efficient protector of the acetylcholine receptor against myasthenogenic antibodies.

The crystal structure of the Fab fragment of the rat monoclonal antibody 198, with protective activity for the main immunogenic region of the human muscle acetylcholine receptor against the destructive action of myasthenic antibodies, has been determined and refined to 2.8 A resolution by X-ray crystallographic methods. The mouse anti-lysozyme Fab D1.3 was used as a search model in molecular replacement with the AMORE software. The complementarity determining regions (CDR)-L2, CDR-H1 and CDR-H2 belong to canonical groups. Loops CDR-L3, CDR-H2 and CDR-H3, which seem to make a major contribution to binding, were analyzed and residues of potential importance for antigen-binding are examined. The antigen-binding site was found to be a long crescent-shaped crevice. The structure should serve as a model in the rational design of very high affinity humanized mutants of Fab198, appropriate for therapeutic approaches in the model autoimmune disease myasthenia gravis.     

Crystallization of a soluble form of the rat T-cell surface glycoprotein CD4 complexed with Fab from the W3/25 monoclonal antibody

The structure of the T-lymphocyte cell surface glycoprotein CD4 is of considerable biological and medical interest. Recombinant rat CD4 expressed in soluble form in mammalian cells and complexed with W3/25 monoclonal Fab fragments formed crystals that diffract to 3.5 A and have the orthorhombic space group P2(1)2(1)2 or P2(1)2(1)2(1). The unit cell has dimensions a = 317 A, b = 161 A and c = 41.8 A and the asymmetric unit consists of two CD4:Fab complexes. These crystals are of suitable quality for X-ray diffraction analysis.     

Solution structure of human and mouse immunoglobulin M by synchrotron X-ray scattering and molecular graphics modelling. A possible mechanism for complement activation

The pentameric 71-domain structure of human and mouse immunoglobulin M (IgM) was investigated by synchrotron X-ray solution scattering and molecular graphics modelling. The radii of gyration RG of human IgM Quaife and its Fc5, IgM-S, Fab'2 and Fab fragments were determined as 12.2 nm, 6.1 nm, 6.1 nm, 4.9 nm and 2.9 nm in that order. The RG values were similar for mouse IgM P8 and its Fab'2 and Fab fragments, despite the presence of an additional carbohydrate site. The IgM scattering curves, to a nominal resolution of 5 nm, were compared with molecular graphics models based on published crystallographic alpha-carbon co-ordinates for the Fab and Fc structures of IgG. Good curve fits for Fab were obtained based on the crystal structure of Fab from IgG. A good curve fit was obtained for Fab'2, if the two Fab arms were positioned close together at their contact with the C mu 2 domains. The addition of the Fc fragment close to the C mu 2 domains of this Fab'2 model, to give a planar structure, accounted for the scattering curve of IgM-S. The Fc5 fragment was best modelled by a ring of five Fc monomers, constrained by packing considerations and disulphide bridge formation. A position for the J chain between two C mu 4 domains rather than at the centre of Fc5 was preferred. The intact IgM structure was best modelled using a planar arrangement of these Fab'2 and Fc5 models, with the side-to-side displacement of the Fab'2 arms in the plane of the IgM structure. All these models were consistent with hydrodynamic simulations of sedimentation data. The solution structure of IgM can therefore be reproduced quantitatively in terms of crystallographic structures for the fragments of IgG. Putative Clq binding sites have been identified on the C mu 3 domain. These would become accessible for interaction with Clq when the Fab'2 arms move out of the plane of the Fc5 disc in IgM, that is, a steric mechanism exposing pre-existing Clq sites. Comparison with a solution structure for Clq by neutron scattering shows that two or more of the six globular Clq heads in the hexameric head-and-stalk structure are readily able to make contacts with the putative Clq sites in the C mu 3 domains of free IgM if if the Clq arm-axis angle in solution is reduced from 40 degrees-45 degrees to 28 degrees. This could be the trigger for Cl activation.     

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.