Isotope-edited nuclear magnetic resonance study of Fv fragment of anti-dansyl mouse monoclonal antibody: recognition of the dansyl hapten.

An isotope-edited proton nuclear magnetic resonance study is reported of Fv, which is the smallest antigen recognition unit composed of VH and VL domains. Fv has been obtained by clostripain digestion of a short-chain anti-dansyl mouse IgG2a monoclonal antibody [Igarashi, T., Sato, M., Katsube, Y., Takio, K., Tanaka, T., Nakanishi, M., & Arata, Y. (1990) Biochemistry 29, 5727-5733]. A variety of stable-isotope-labeled anti-dansyl Fv analogues have been prepared. The aromatic proton resonances for all Tyr residues of the Fv fragment have been assigned in the absence and presence of epsilon-dansyl-L-lysine by means of isotope-edited homonuclear and heteronuclear two-dimensional NMR experiments. On the basis of the established assignments, it has been concluded that the dansyl ring is bound through Tyr-96H and Tyr-104H to both ends of H3, the third hypervariable region of the heavy chain. We also suggest that the antigen binding results in the formation of a hydrophobic core comprising the dansyl ring and the aromatic rings of Tyr-96H and Tyr-104H.     

Preparation of the Fv fragment from a short-chain mouse IgG2a anti-dansyl monoclonal antibody and use of selectively deuterated Fv analogues for two-dimensional 1H NMR analyses of the antigen-antibody interactions

The Fv fragment, a univalent antigen-binding unit with a molecular weight of 25,000, has successfully been prepared in high yield by limited proteolysis with clostripain of a short-chain mouse IgG2a anti-dansyl monoclonal antibody in which the entire CH1 domain is deleted [Igarashi, T., Sato, M., Takio, K., Tanaka, T., Nakanishi, M., & Arata, Y. (1990) Biochemistry 29, 5727-5733]. The Fv fragment obtained is stable at room temperature and retains its full antigen-binding capability. It has been shown that selective deuterium labeling of the Fv fragment, which is half the size of the Fab fragment, provides 1H NMR spectral data at a sufficient resolution for a detailed structural analysis of the antigen-combining site. NOESY spectra of an Fv analogue, in which all aromatic protons except for His C2'-H and Tyr C3',5'-H had been deuterated, were measured in the presence of varying amounts of dansyl-L-lysine. On the basis of the NOESY data obtained, it was possible to assign all the ring proton resonances for the dansyl group that is bound to the Fv fragment. It was also possible to obtain information about His and Tyr residues of the Fv fragment in the absence and presence of the antigen. On the basis of the NMR data obtained, we have shown that at least two Tyr residues along with one of the amide groups are directly involved in antigen binding. The mode of interaction of the dansyl ring with these residues in the Fv fragment has briefly been discussed.     

Multinuclear NMR study of the structure of the Fv fragment of anti-dansyl mouse IgG2a antibody

A multinuclear NMR study is reported of Fv, which is a minimum antigen-binding unit of immunoglobulin. Fv has been prepared by clostripain digestion of a mouse anti-dansyl IgG2a monoclonal antibody that lacks the entire CH1 domain [Takahashi, H., Igarashi, T., Shimada, I., & Arata, Y. (1991) Biochemistry 30, 2840-2847]. A variety of Fv analogues labeled with 2H in the aromatic rings and with 13C and/or 15N in the peptide bonds have been prepared and used for multinuclear NMR analyses of Fv in the absence and presence of epsilon-dansyl-L-lysine (DNS-Lys). It has been shown that 1H-15N shift correlation spectra of Fv sensitively reflect the antigen binding and can be used along with 1H and 13C spectral data for the structural analyses of antigen-antibody interactions. Hydrogen-deuterium exchange of the amide protons has been followed in the absence and presence of DNS-Lys by using the 1H-15N shift correlation spectra. Use of the beta-shift observed for the carbonyl carbon resonances has also been helpful in following the hydrogen-deuterium exchange. On the basis of the NMR data obtained, the static and dynamic structure of the Fv fragment in the absence and presence of DNS-Lys has been discussed.     

Expression of an antibody Fv fragment in myeloma cells

The antigen binding site on antibodies is fashioned by loops at the tips of the beta-sheet framework of both heavy and light chain variable domains. A heterodimer of both variable domains (Fv fragment), incorporating loops from an anti-lysozyme antibody, was expressed and secreted from myeloma cells in good yield (8 mg/l in supernatant from roller bottles), and shown to bind lysozyme. The two subunits were found to be in dynamic equilibrium but are overwhelmingly associated at neutral pH. The small size of Fv fragments (25 x 10(3) Mr) make them attractive for structural studies, in vivo imaging, and therapy.     

Comparative thermodynamic analyses of the Fv, Fab* and Fab and Fab fragments of anti-dansyl mouse monoclonal antibody

In order to investigate the role of the constant domainson the antigen-binding property of the variable domains, we have carried out a comparative thermodynamic study of the anti-dansyl Fv, Fab* and Fab fragments that possess the identical amino acid sequence of the variable domains. The thermodynamic analyses have shown that binding constants, enthalphy changes and entropy changes are similar for the three antigen-binding fragments, whereas the thermal stability of Fab is much higher than that of Fv and Fab*. We have concluded that (i) the variable domains of the three antigen-binding fragments possess identical intrinsic capability for antigen binding and (ii) the two constant domains serve to improve the stability of the variable domains.     

Humanization of an anti-CD34 monoclonal antibody by complementarity-determining region grafting based on computer-assisted molecular modelling

4C8 is a new mouse anti-human CD34 monoclonal antibody (mAb), which recognizes class II CD34 epitopes and can be used for clinical hematopoietic stem/progenitor cell selection. In an attempt to improve its safety profiles, we have developed a humanized antibody of 4C8 by complementarity-determining region (CDR) grafting method in this study. Using a molecular model of 4C8 built by computer-assisted homology modelling, framework region (FR) residues of potential importance to the antigen binding were identified. A humanized version of 4C8, denoted as h4C8, was generated by transferring these key murine FR residues onto a human antibody framework that was selected based on homology to the mouse antibody framework, together with the mouse CDR residues. The resultant humanized antibody was shown to possess antigen-binding affinity and specificity similar to that of the original murine antibody, suggesting that it might be an alternative to mouse anti-CD34 antibodies routinely used clinically.     

A role of the third complementarity-determining region in the affinity maturation of an antibody

We recently found that there are two distinct antibody maturation pathways for the immune response of C57BL/6 mice to (4-hydroxy-3-nitrophenyl) acetyl and that a junctional amino acid introduced at a point far in advance of somatic hypermutation determined which pathway of affinity maturation was used. We describe here the structural basis for this aspect of maturation using recently developed H3 rules, which allow for reliable identification of the conformation of the third complementarity-determining region of the heavy chain (CDR-H3) from the primary amino acid sequences only. By the application of these rules, the anti-(4-hydroxy-3-nitrophenyl) acetyl antibodies examined here were classified into two major groups on the basis of their CDR-H3 structure, and these groups were found to be consistent with the maturation pathways. In addition, circular dichroism measurements revealed that the versatile nature of the antigen binding of the antibodies was significantly influenced by the pathway employed. We postulated in this study that flexibility in the CDR-H3 structure in the antigen-combining site could facilitate efficient antibody maturation supported by a plurality of possible antigen binding modes.     

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.     

Evidence for structural plasticity of heavy chain complementarity-determining region 3 in antibody-ssDNA recognition

Anti-DNA antibodies play important roles in the pathogenesis of autoimmune diseases. They also represent a unique and relatively unexplored class of DNA-binding protein. Here, we present a study of conformational changes induced by DNA binding to an anti-ssDNA Fab known as DNA-1. Three crystal structures are reported: a complex of DNA-1 bound to dT3, and two structures of the ligand-free Fab. One of the ligand-free structures was determined from crystals exhibiting perfect hemihedral twinning, and the details of structure determination are provided. Unexpectedly, five residues (H97-H100A) in the apex of heavy chain complementarity-determining region 3 (HCDR3) are disordered in both ligand-free structures. Ligand binding also caused a 2-4A shift of the backbone of Tyr L92 and ordering of the L92 side-chain. In contrast, these residues are highly ordered in the Fab/dT3 complex, where Tyr H100 and Tyr H100A form intimate stacking interactions with DNA bases, and L92 forms the 5' end of the binding site. The structures suggest that HCDR3 is very flexible and adopts multiple conformations in the ligand-free state. These results are discussed in terms of induced fit and pre-existing equilibrium theories of ligand binding. Our results allow new interpretations of existing thermodynamic and mutagenesis data in terms of conformational entropy and the volume of conformational space accessible to HCDR3 in the ligand-free state. In the context of autoimmune disease, plasticity of the ligand-free antibody could provide a mechanism by which anti-DNA antibodies bind diverse host ligands, and thereby contribute to pathogenicity.     

Structural and functional characterization of the single-chain Fv fragment from a unique HCV E1E2-specific monoclonal antibody

The nucleotide sequence of the unique neutralizing monoclonal antibody D32.10 raised against a conserved conformational epitope shared between E1 and E2 on the serum-derived hepatitis C virus (HCV) envelope was determined. Subsequently, the recombinant single-chain Fv fragment (scFv) was cloned and expressed in Escherichia coli, and its molecular characterization was assessed using multi-angle laser light scattering. The scFv mimicked the antibody in binding to the native serum-derived HCV particles from patients, as well as to envelope E1E2 complexes and E1, E2 glycoproteins carrying the viral epitope. The scFv D32.10 competed with the parental IgG for binding to antigen, and therefore could be a promising candidate for therapeutics and diagnostics.     

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     

Bivalency and epitope specificity of a high-affinity IgG3 monoclonal antibody to the Streptococcus group A carbohydrate antigen. Molecular modeling of a Fv fragment

The binding of Strep 9, a mouse monoclonal antibody (mAb) of the IgG3 subclass directed against the cell-wall polysaccharide of Group A Streptococcus (GAS), has been characterized. The intact antibody and proteolytic fragments of Strep 9 bind differently to GAS: the intact mAb and F(ab)2' have greater affinity for the carbohydrate epitope than the monomeric Fab or F(ab)'. A mode of binding in which Strep 9 binds bivalently to portions of the polysaccharide on adjacent chains on GAS is proposed. A competitive ELISA protocol using a panel of carbohydrate inhibitors shows that the branched trisaccharide, beta-D-GlcpNAc-(1-->3)-[alpha-L-Rhap-(1-->2)]-alpha-L-Rhap, and an extended surface are key components of the epitope recognized by Strep 9. Microcalorimetry measurements with the mAb and two synthetic haptens, a tetrasaccharide and a hexasaccharide, show enthalpy-entropy compensation as seen in other oligosaccharide-protein interactions. Molecular modeling of the antibody variable region by homology modeling techniques indicates a groove-shaped combining site that can readily accommodate extended surfaces. Visual docking of an oligosaccharide corresponding to the cell-wall polysaccharide into the site provides a putative model for the complex, in which a heptasaccharide unit occupies the site and the GlcpNAc residues of two adjacent branched trisaccharide units occupy binding pockets within the groove-shaped binding site.     

Symmetry of Fv architecture is conducive to grafting a second antibody binding site in the Fv region.

Molecular modeling studies on antibody Fv regions have been pursued to design a second antigen-binding site (chi-site) in a chimeric single-chain Fv (chi sFv) species of about 30 kDa. This analysis has uncovered an architectural basis common to many Fv regions that permits grafting a chi-site onto the Fv surface that diametrically opposes the normal combining site. By using molecular graphics analysis, chimeric complementarity-determining regions (chi CDRs) were defined that comprised most of the CDRs from an antibody binding site of interest. The chain directionality of chi CDRs was consistent with that of specific bottom loops of the sFv, which allowed for grafting of chi CDRs with an overall geometry approximating CDRs in the parent combining site. Analysis of 10 different Fv crystal structures indicates that the positions for inserting chi CDRs are very highly conserved, as are the corresponding chi CDR boundaries in the parent binding site. The results of this investigation suggest that it should be possible to generally apply this approach to the development of chimeric bispecific antibody binding site (chi BABS) proteins.     

The carbohydrate structures of a mouse monoclonal IgG antibody OKT3

A mouse monoclonal antibody OKT3, of IgG2a isotype, was isolated from hybridoma culture fluid. Sugar analysis showed the presence of sialic acid, galactose, mannose, fucose, and N-acetylglucosamine, i.e. sugars typical for N-glycosidically linked carbohydrate chains. The absence of N-acetylgalactosamine revealed that O-glycosidically linked carbohydrates were not present. The purified antibody was reduced, alkylated, and separated into heavy and light chains, and all carbohydrates were shown to be associated with the heavy chains. The N-linked carbohydrate chains were isolated as alditols using strong alkaline-borohydride degradation and further fractionated on a concanavalin A-Sepharose column and high performance ion exchange chromatography with pulsed amperometric detection. Structural analysis was carried out on the isolated oligosaccharide alditols by chemical analyses, fast atom bombardment mass spectrometry, and 500-MHz 1H NMR spectroscopy. Triantennary and biantenary types of structures were found. The triantennary structures were present as trisialo and tetrasialo forms without fucose; the tetrasialo forms were shown to contain a sequence of Neu5Ac alpha 2-3Gal beta 1-3[Neu5Ac alpha 2-6]GlcNAc beta 1- on one of the branches. The biantennary structures were present as completely sialylated nonfucosylated species and as asialo-, agalacto-, and partially fucosylated structures.     

Very high expression of an anti-carcinoembryonic antigen single chain Fv antibody fragment in the yeast Pichia pastoris

In this paper we report the development of a recombinant strain of the yeast Pichia pastoris, which secretes an anti-carcinoembryonic antigen single chain Fv (scFv) antibody fragment to the culture supernatant as a biologically active protein, at levels of 1.2 g l(-1). The yeast scFv was purified by IMAC, with a final yield of approximately 0.440 g of 93% pure scFv per liter of culture supernatant. The specific activity in ELISA of the yeast scFv was almost three times higher than that of a bacterial periplasmic counterpart. These results reaffirm that the yeast P. pastoris is a suitable host for high level production of scFv antibody fragments with potential in vivo diagnostic and therapeutic applications.     

The crystal structure of the fab fragment of the monoclonal antibody MAK33. Implications for folding and interaction with the chaperone bip

The Fab fragment of the murine monoclonal antibody, MAK33, directed against human creatine kinase of the muscle-type, was crystallized and the three-dimensional structure was determined to 2.9 A. The antigen-binding surface of MAK33 shows a convex overall shape typical for immunoglobulins binding large antigens. The structure allows us to analyze the environment of cis-prolyl-peptide bonds whose isomerization is of key importance in the folding process. These residues seem to be involved with not only domain stability but also seem to play a role in the association of heavy and light chains, reinforcing the importance of beta-strand recognition in antibody assembly. The structure also allows the localization of segments of primary sequence postulated to represent binding sites for the ER-specific chaperone BiP within the context of the entire Fab fragment. These sequences are found primarily in beta-strands that are necessary for interactions between the individual domains.     

Expression and characterization of a recombinant Fab fragment derived from an anti-human alpha-fetoprotein monoclonal antibody

Alpha-fetoprotein (AFP) is a well-known molecular marker indicating the development of cancer as well as fetal abnormalities such as open neural tube defects. Accordingly the measurement of serum AFP is important for the diagnosis of hepatocellular carcinoma (HCC) and other abnormalities. Monoclonal antibodies (McAb) to AFP were produced to develop an immunoassay kit, and to study the possibility of an antibody (Ab) therapy. The immunoglobulin genes were cloned from hybridoma cells, and expressed in E. coli as an Fab soluble into a culture medium. The Fab of anti-AFP McAb exhibited binding to AFP and similar affinity compared to the original IgG. This recombinant antibody can be studied further for in vivo imaging and immunotherapeutics.     

Pitfalls in the interpretation of structural changes in mutant proteins from crystal structures

PpcA is a small protein with 71 residues that contains three covalently bound hemes. The structures of single mutants at residue 58 have shown larger deviations in another part of the protein molecule than at the site of the mutation. Closer examination of the crystal packing has revealed the origin of this unexpected structural change. The site of mutation is within Van der Waals distance from another protein molecule related by a crystallographic twofold axis within the crystal. The structural changes occurred at or near the mutation site have led to a slight adjustment of the surface residues in contact. The observed deviations between the native and the mutant molecular structures are derived from the new crystal packing even though the two crystals are essentially isomorphous. Without careful consideration of the crystal lattice a non-expert looking at only the coordinates deposited in the Protein Data Bank could draw erroneous conclusion that mutation in one part of the molecule affected the structure of the protein in a distant part of the molecule.