Bivalent Fv antibody fragments obtained by substituting the constant domains of a fab fragment with heterotetrameric molybdopterin synthase

The antibody Fv fragment is the smallest functional unit of an antibody but for practical use, the VH/VL interface requires stabilization, which is usually accomplished by a peptide linker that joins the two variable domains to form a single chain Fv fragment (scFv). An alternative format to scFv is proposed that (i) allows stabilization of the Fv fragment, and (ii) restores the bivalency of the antibody as a pseudo-F(ab')2 format. This new antibody fragment was constructed by replacing the CHI and CL domains of the Fab fragment with heterotetrameric molybdopterin synthase (MPTS). We found that this format, named MoaFv, improved significantly the cytoplasmic expression of the Fv as a soluble protein in BL21 or Origami Escherichia coli strains. This MoaFv format is expressed as a homogeneous heterotetrameric protein with a Mr value of 110 kDa containing two functional binding sites as revealed by active site titration. In its native condition at 37 degrees C or in the presence of urea, this format was nearly as stable as the corresponding scFv, indicating that non-covalent interactions between the MPTS subunits can replace the covalent peptide linker in scFv. Finally, this MoaFv construct could be a useful format when bivalency is desirable to improve the functional avidity.     

Antibody engineering using phage display with a coiled-coil heterodimeric Fv antibody fragment

A Fab-like antibody binding unit, ccFv, in which a pair of heterodimeric coiled-coil domains was fused to V(H) and V(L) for Fv stabilization, was constructed for an anti-VEGF antibody. The anti-VEGF ccFv showed the same binding affinity as scFv but significantly improved stability and phage display level. Furthermore, phage display libraries in the ccFv format were constructed for humanization and affinity maturation of the anti-VEGF antibody. A panel of V(H) frameworks and V(H)-CDR3 variants, with a significant improvement in affinity and expressibility in both E. coli and yeast systems, was isolated from the ccFv phage libraries. These results demonstrate the potential application of the ccFv antibody format in antibody engineering.     

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.     

Domain interactions in the Fab fragment: a comparative evaluation of the single-chain Fv and Fab format engineered with variable domains of different stability

Recombinant antibody fragments, most notably Fab and scFv, have become important tools in research, diagnostics and therapy. Since different recombinant antibody formats exist, it is crucial to understand the difference in their respective biophysical properties. We assessed the potential stability benefits of changing the scFv into the Fab format, the influence of the variable domains on the stability of the Fab fragment, and the influence of the interchain disulfide bond in the Fab fragment. To analyze domain interactions, the Fab fragment was broken down into its individual domains, several two-domain assemblies and one three-domain assembly. The equilibrium denaturation properties of these constructs were then compared to those of the Fab fragment. It was found that mutual stabilization occurred across the VH/VL and the CH1/CL interface, whereas the direct interaction between the V) and the CL domain had no influence on the stability of either domain. This observation can be explained by the different interfaces used for interaction. In contrast, the whole CH1CL and VHVL unit showed significant mutual stabilization, indicating a high degree of cooperation between the VH/VL and CH1/CL interface. The interchain disulfide bond in the Fab fragment plays an essential role in this stabilization. In addition to the effects of domain association on the thermodynamic (equilibrium) stability, Fab fragments differ from scFv fragments of similar equilibrium stability by having a very slow unfolding rate. This kinetic stabilization may increase significantly the resistance of Fab fragments against short time exposure to adverse conditions.     

Expression of a heterodimeric Fab antibody protein in one cloning step.

A new method is presented for creating antibody expression libraries in Escherichia coli. Rather than perform two cloning steps to express the heavy and light chains of the antigen binding domain, we have used a fusion-PCR method to link the coding regions for heavy and light chain sequences in a single DNA molecule prior to vector ligation. This greatly simplifies the construction of antibody expression clones.     

Structure-based stability engineering of the mouse IgG1 Fab fragment by modifying constant domains

A semi-rational approach based on structural data was exploited in a search for CH1 and CL domains with improved intrinsic thermodynamic stabilities. Structural and amino acid level comparisons were carried out against known biophysically well-behaving and thermodynamically beneficial scFv and Fab fragments. A number of mutant Fab fragments were constructed by site-directed mutagenesis of regions in the CH1 and CL domains expected to be most sensitive under physical stress conditions. These mutations were located on three sites in the Fab constant domains; a mobile loop in the CH1 domain, residues surrounding the two largest solvated hydrophobic cavities located in the interface of the CH1 and CL domains and the hydrophobic core regions of both CH1 and CL. Expression levels of functional Fab fragments, denaturant-induced unfolding equilibria and circular dichroism spectroscopy were used to evaluate the relative stabilities of the wild-type and the mutant Fab fragments. The highest thermodynamic stability was reached through the mutation strategy, where the hydrophobicity and the packing density of the solvated hydrophobic cavity in the CH1/CL interface was increased by the replacement of the hydrophilic Thr178 in the CL domain by a more hydrophobic residue, valine or isoleucine. The midpoint of the transition curve from native to unfolded states of the protein, measured by fluorescence emission, occurred at concentrations of guanidine hydrochloride of 2.4 M and 2.6 M for the wild-type Fab and the most stable mutants, respectively. Our results illustrate that point mutations targeted to the CH1/CL interface were advantageous for the overall thermodynamic stability of the Fab fragment.     

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.     

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.     

Recognition of DNA by VH and Fv domains of an IgG anti-poly(dC) antibody with a singly mutated VH domain

Secondary antigen stimulation usually produces IgG antibodies with hypermutated V segments. Studying a strong secondary response to the polynucleotide antigen poly(dC), however, we found a highly selective IgG antibody (mAb dC7) with only one mutation (a conservative Leu to Ileu substitution) throughout the whole VH domain. To investigate the roles of VH and VL domains in selective binding by this mAb, we prepared its VH, VL and single-chain Fv (scFv) fragments. A bacterial expression system produced soluble monomeric V region proteins. CD spectra confirmed that they had the beta-secondary structure expected for Ig domains. Both the scFv and VH fragments bound to single-stranded non-protonated poly(dC) and to ssDNA but not to protonated, more structured poly(dC) or dsDNA. The VL domain alone did not bind to nucleic acids, but VL association modified the VH binding, giving the scFv a 10-fold higher affinity than the VH for poly(dC) and greatly increasing the cytosine-dependent selectivity. Non-ionic interactions were prominent in the Fv reaction with a (dC)( n) sequence. Ionic interactions were revealed in Fv cross-reactions with ssDNA, and were more prominent in binding of either poly(dC) or ssDNA by VH alone, consistent with the lesser base selectivity of the VH. Thus, the Fv and VH alone bind to a single antigen, poly(dC), but mechanistic differences result from additional subsites in the Fv. Generation of a selective IgG with very few CDR mutations in either VH or VL, which was accompanied by IgM antibodies with unmutated V regions, also suggests that nucleic acid binding activity is a property of the B cell repertoire even before immunization.     

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.     

Purification of antibody Fab and F(ab')2 fragments using Gradiflow technology

The Gradiflow, a preparative electrophoresis instrument designed to separate molecules on the basis of their size and charge, was used to purify antibody Fab and F(ab')2 fragments. The method described is charge based, utilizing the difference in the pI between the antibody Fab/F(ab')2 fragments and antibody Fc fragments that occur after enzyme digestion of whole antibody molecules. This method of purification was successful across a range of monoclonal and polyclonal antibodies. In particular, F(ab')2 fragments were purified from a number of mouse monoclonal antibodies (both IgG1 and IgG2a isotypes) and Fab fragments were purified from egg yolk IgY polyclonal antibodies. This is a rapid purification method which has advantages over alternative methods that usually comprise ion exchange and gel filtration chromatography. This method may be applicable to most antibody digest preparations.     

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.     

Evidence for intermolecular domain exchange in the Fab domains of dimer and oligomers of an IgG1 monoclonal antibody

Recombinant protein therapeutics have become increasingly useful in combating human diseases, such as cancer and those of genetic origin. One quality concern for protein therapeutics is the content and the structure of the aggregated proteins in the product, due to the potential immunogenicity of these aggregates. Collective efforts have led to a better understanding of some types of protein aggregates, and have revealed the diversity in the structure and cause of protein aggregation. In this work we used a broad range of analytical techniques to characterize the quinary structure (complexes in which each composing unit maintains native quaternary structure) of the stable non-covalent dimer and oligomers of a monoclonal IgG1λ antibody. The results supported a mechanism of intermolecular domain exchange involving the Fab domains of 2 or more IgG molecules. This mechanism can account for the native-like higher order (secondary, tertiary and disulfide bonding) structure, the stability of the non-covalent multimers, and the previously observed partial loss of the antigen-binding sites without changing the antigen-binding affinity and kinetics of the remaining sites (Luo et al., 2009, mAbs 1:491). Furthermore, the previously observed increase in the apparent affinity to various Fcγ receptors (ibid), which may potentially promote immunogenicity, was also explained by the quinary structure proposed here. Several lines of evidence indicated that the formation of multimers by the mechanism of intermolecular domain exchange took place mostly during expression, not in the purified materials. The findings in this work will advance our knowledge of the mechanisms for aggregation in therapeutic monoclonal antibodies.     

Allosteric binding properties of a monoclonal antibody and its Fab fragment

Detailed equilibrium binding studies were conducted on a monoclonal antibody directed against Pb(II) complexed with a protein conjugate of diethylenetriaminepentaacetic acid (DTPA). Binding curves obtained with DTPA and a cyclohexyl derivative of DTPA in the presence and absence of metal ions were consistent with the anticipated one-site homogeneous binding model. Binding curves obtained with aminobenzyl-DTPA or its complexes with Ca(II), Sr(II), and Ba(II) were highly sigmoidal, characterized by Hill coefficients of 2.3-6.5. Binding curves obtained with the Pb(II) and In(III) complexes of aminobenzyl-DTPA were hyperbolic, but in each case the apparent affinity of the antibody for the chelator-metal complex was higher in the presence of excess chelator than it was in the presence of excess metal ion. In the presence of excess chelator, the equilibrium dissociation constant for the binding of aminobenzyl-DTPA-Pb(II) to the antibody was 9.5 x 10(-)(10) M. Binding curves obtained with the Hg(II) and Cd(II) complexes of aminobenzyl-DTPA were biphasic, indicative of negative cooperativity. Further binding studies demonstrated that aminobenzyl-DTPA-Hg(II) opposed the binding of additional chelator-metal complexes to the antibody more strongly than did aminobenzyl-DTPA-Cd(II). The Fab fragment differed from the intact antibody only in that the apparent affinity of the Fab was generally lower for a given chelator-metal complex. These data are interpreted in terms of a model in which (i) aminobenzyl-DTPA and its complexes bind both to the antigen binding site and to multiple charged sites on the surface of the compact immunoglobulin; and (ii) the bound, highly charged ligands interact in a complicated fashion through the apolar core of the folded antibody.     

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.     

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 studies of the Fab fragment of an anti-azophenylarsonate antibody

Single crystals of the Fab fragment of a murine A/J anti-azophenylarsonate monoclonal antibody have been prepared by the vapor diffusion method. Antibody 3A7 uses the same combination of variable region gene segments (VK, JK, VH, JH) as do anti-azophenylarsonate antibodies bearing a predominant cross-reactive idiotype, but utilizes a different D gene segment. The crystals grow in the presence of beta-octylglucoside as tetragonal bipyramids in the space group of either P4(1)2(1)2 or P4(3)3(1)2 and with unit cell dimensions of a = b = 77.9 A, and c = 146.7 A. They diffract X-rays to better than 2.7 A resolution. Data up to 2.7 A resolution have been collected.     

Production of functionalized single-chain Fv antibody fragments binding to the ED-B domain of the B-isoform of fibronectin in Pichia pastoris

The Pichia pastoris expression system was used to produce functionalized single-chain antibody fragments (scFv) directed against the ED-B domain of the B-fibronectin (B-Fn) isoform which was found to be present only in newly formed blood vessels during tumor angiogenesis. Therefore, scFv antibody fragments recognizing the ED-B domain are potential markers for angiogenesis. We constructed four functionalized scFv antibody fragments for direct labeling with radioactive molecules or toxins or for attachment to liposomes serving as carriers for cytotoxic or antiangiogenic compounds. The C-termini of the scFv antibody fragments contain 1-3 cysteine residues that are separated by a hydrophilic linker (GGSSGGSSGS) from the binding domain and are accessible for site-specific functionalization with thiol-reactive reagents. Plasmid expression, culture conditions, and purification were optimized in 1-L cultures. The scFv antibody fragments were purified by anion exchange chromatography. The yields were 5-20 mg/L culture medium. The large-scale production of one scFv antibody fragment in a 3.7-L fermenter gave a yield of 60 mg. The reactivity of the cyteines was demonstrated by labeling with the thiol-reactive fluorescent dye ABD-F. The four scFv antibody fragments bound specifically to ED-B-modified Sepharose and binding was further confirmed by immunofluorescence on cell cultures using ED-B-positive human Caco-2 tumor cells. Furthermore, we could demonstrate specific binding of scFv-modified liposomes to ED-B-positive tumor cells. Our results indicate that the P. pastoris expression system is useful for the large-scale production of cysteine-functionalized alpha-ED-B scFv antibody fragments.