Properties of a single-chain antibody containing different linker peptides

Single-chain antibodies were constructed using six different linker peptides to join the VH and VL domains of an anti-2-phenyloxazolone (Ox) antibody. Four of the linker peptides originated from the interdomain linker region of the fungal cellulase CBHI and consisted of 28, 11, six and two amino acid residues. The two other linker peptides used were the (GGGGS)3 linker with 15 amino acid residues and a modified IgG2b hinge peptide with 22 residues. Proteolytic stability and Ox binding properties of the six different scFv derivatives produced in Escherichia coli were investigated and compared with those of the corresponding Fv fragment containing no joining peptide between the V domains. The hapten binding properties of different antibody fragments were studied by ELISA and BIAcoreTM. The interdomain linker peptide improved the hapten binding properties of the antibody fragment when compared with Fv fragment, but slightly increased its susceptibility to proteases. Single-chain antibodies with short CBHI linkers of 11, six and two residues had a tendency to form multimers which led to a higher apparent affinity. The fragments with linkers longer than 11 residues remained monomeric.     

Cloning and functional characterization of the 5''-flanking region of human methionine adenosyltransferase 2A gene

Electron microscopy of dimeric and trimeric single chain antibody Fv fragments (scFvs) complexed with anti-idiotype Fab fragments was used to reveal the orientation of antigen binding sites. This is the first structural analysis that discloses the multivalent binding orientation of scFv trimers (triabodies). Three different scFv molecules were used for the imaging analysis; NC10 scFv-5 and scFv-0, with five- and zero-residue linkers respectively between the VH and VL domains, were complexed with 3-2G12 anti-idiotype Fab fragments and 11-1G10 scFv-0 was complexed with NC41 anti-idiotype Fab fragments. The scFv-5 molecules formed bivalent dimers (diabodies) and the zero-linker scFv-0 molecules formed trivalent trimers (triabodies). The images of the NC10 diabody-Fab complex appear as boomerangs, not as a linear molecule, with a variable angle between the two Fab arms and the triabody-Fab complexes appear as tripods.     

Antibody VH domains as small recognition units

To develop immunoglobulin based recognition units of minimum size, a human heavy chain variable domain (VH) was designed for selection of phage displayed VH. Non-specific binding of the VH through its interface for the light chain variable domain (VL) was prevented through three mutations (G44E, L45R and W47G) in this interface. These mutations were introduced to mimic camelid antibody heavy chains naturally devoid of light chain partners. The third hypervariable loop of the modified VH was then randomised to yield a repertoire of 2 x 10(8) independent clones, which was displayed on phage and selected through antigen binding. VH clones specific for hapten and protein antigens were isolated. Soluble VH was expressed with an isoleucine residue at position 47 to improve expression and stability compared to VH containing a glycine residue at this position, which however was preferable for phage selection. Affinities of soluble VH for hapten were between 100 nM and 400 nM. The VH domains were highly specific, stable and well expressed in Escherichia coli. These positive biophysical properties and their small size make them attractive for biotechnological applications.     

Characterization of Fv fragments expressed on phage surface

We characterized a phage antibody in which an Fv fragment, namely, a free VH fragment noncovalently associated with a VL fragment that is fused with a truncated cpIII molecule (VL-DeltacpIII), is expressed on the phage surface. D1.3 antibody specific for hen egg-white lysozyme was used as a model system. Both VH and VL-DeltacpIII fragments were stably expressed and associated with each other to form a faithful antigen-binding site. The results of Western blotting indicated that more than 5% of phages expressed the Fv fragment on their surface. Analysis of the kinetics of binding of the phage antibody to the antigen suggested the possibility of presence of phages having multiple-binding sites on a single phage particle.     

A comparison of the crystallographic structures of two catalytic antibodies with esterase activity

The crystallographic structure of the Fab fragment of the catalytic antibody, 29G11, complexed with an (S)-norleucine phenyl phosphonate transition state analog was determined at 2.2 A resolution. The antibody catalyzes the hydrolysis of norleucine phenyl ester with (S)-enantioselectivity. The shape and charge complementarity of the binding pocket for the hapten account for the preferential binding of the (S)-enantiomer of the substrate. The structure is compared to that of the more catalytically efficient antibody, 17E8, induced by the same hapten transition state analog. 29G11 has different residues from 17E8 at eight positions in the heavy chain, including four substitutions in the hapten-binding pocket: A33V, S95G, S99R and Y100AN, and four substitutions at positions remote from the catalytic site, I28T, R40K, V65G and F91L. The two antibodies show large differences in the orientations of their variable and constant domains, reflected by a 32 degrees difference in their elbow angles. The VL and VH domains in the two antibodies differ by a rotation of 8.8 degrees. The hapten binds in similar orientations and locations in 29G11 and 17E8, which appear to have catalytic groups in common, though the changes in the association of the variable domains affect the precise positioning of residues in the hapten-binding pocket.     

Humanization of an anti-human IL-6 mouse monoclonal antibody glycosylated in its heavy chain variable region

Interleukin-6 (IL-6) inhibitors are good potential therapeutic agents in human patients, and anti-IL-6 antibodies are among the best candidates. Here, we have successfully humanized mouse monoclonal antibody SK2, which specifically binds to IL-6 and strongly inhibits IL-6 functions. Since this antibody possesses N-linked carbohydrates on Asn-30 of VH region, which seems to be very close to an antigen-binding site, influence of these carbohydrates on antigen-binding was investigated. A biosensor study showed that the mouse SK2 Fab and its deglycosylated fragments had almost equal Kd (Kon/Koff), 26.8 nM (1.05 x 10(6)/2.81 x 10(-2)) and 24.7 nM (1.28 x 10(6)/3.15 x 10(-2)), respectively. Furthermore, a mutant chimeric SK2 antibody, in which the N-glycosylation site was removed from the VH region, showed a Kd of 11 nM, almost similar to that of the original chimeric SK2 antibody, determined by Scatchard analysis with 125I-IL-6. These data indicate the carbohydrates of mouse SK2 VH region do not significantly influence antigen-binding activity. In the next step, two versions of each humanized SK2 VL and VH regions were carefully designed based on the amino acid sequences of human REI and DAW, respectively. Only one alteration, Tyr to Phe, was made at position 71 in the two light chains, according to the canonical residue for LI. A N-glycosylation site was introduced on the two heavy chains, by changing Ser to Asn at position 30. All four combinations of humanized light and heavy chains could bind to IL-6 as well as the chimeric SK2 antibody. The light chain first version, however, could not efficiently inhibit IL-6 binding to its receptor, indicating the importance of the LI loop conformation for the inhibitory activity of SK2 antibody. In contrast, both versions of the heavy chains were comparable, in yielding good humanized SK2 antibodies, suggesting that the glycosylation of the SK2 VH region has no influence in recreating a functional antigen-binding site in this humanization.     

Role of mouse VH10 and VL gene segments in the specific binding of antibody to Z-DNA, analyzed with recombinant single chain Fv molecules

A plasmid vector was constructed for the expression of a single chain Fv domain of mouse mAb to Z-DNA (antibody Z22), which is encoded by VH10 and V kappa 10 gene family members along with Dsp2, JH4, and J kappa 4 segments. The vector coded for a PhoA secretion signal, VH segment, flexible peptide linker, VL segment, (His)5, and a protein A domain. Unique restriction sites allowed exchange of the segments as cassettes. Bacteria transformed with the vector secreted soluble recombinant Fv with specific Z-DNA-binding activity. When the L chain of Z22 was replaced with a library of splenic VL cDNA from a mouse immunized with Z-DNA, only a light chain closely resembling that of the original Z22 (differing at six amino acid positions) yielded Fv with Z-DNA-binding activity. The Fv with this L chain replacement had a lowered affinity, but remained selective for Z-DNA. Replacement of the Z22 H chain with a mixture of 11 VH10-encoded H chains yielded two Z-DNA binding clones, but they bound B-DNA and denatured DNA as well as Z-DNA. The replacement clones indicate the importance of the H chain CDR3 and particular VH-VL combinations in formation of specific antibodies to Z-DNA.     

Hepatic leukostasis and hypoxic stress in adhesion molecule-deficient mice after gut ischemia/reperfusion

An anti-p185HER2/anti-CD3 humanized bispecific diabody was previously constructed from two cross-over single-chain Fv in which YH and VL domains of the parent antibodies are present on different polypeptides. Here this diabody is used to evaluate domain interface engineering strategies for enhancing the formation of functional heterodimers over inactive homodimers. A disulfide-stabilized diabody was obtained by introducing two cysteine mutations, VL L46C and VH D101C, at the anti-p185HER2.VL/VH interface. The fraction of recovered diabody that was functional following expression in Escherichia coli was improved for the disulfide-stabilized compared to the parent diabody (> 96% versus 72%), whereas the overall yield was > 60-fold lower. Eleven "knob-into-hole" diabodies were designed by molecular modeling of sterically complementary mutations at the two VL/VH interfaces. Replacements at either interface are sufficient to improve the fraction of functional heterodimer, while maintaining overall recoverable yields and affinity for both antigens close to that of the parent diabody. For example, diabody variant v5 containing the mutations VL Y87A:F98M and VH V37F:L45W at the anti-p185HER2 VL/VH interface was recovered as 92% functional heterodimer while maintaining overall recovered yield within twofold of the parent diabody. The binding affinity of v5 for p185HER2 extracellular domain and T cells is eightfold weaker and twofold stronger than for the parent diabody, respectively. Domain interface remodeling based upon either sterically complementary mutations or interchain disulfide bonds can facilitate the production of a functional diabody heterodimer. This study expands the scope of domain interface engineering by demonstrating the enhanced assembly of proteins interacting via two domain interfaces.     

Antibody fragments as inhibitors of Japanese radish acid phosphatase

VH (heavy-chain variable region) and VL (light-chain variable region) genes were amplified by PCR from hybridomas producing MAb-11 and MAb-18 which inhibited Japanese radish acid phosphatase. Nucleotide sequencing of the V genes demonstrates that the MAbs contained similar VH and identical VL domains. Initially, the VH and VL genes were expressed in Escherichia coli as single-chain Fv (ScFv) fragments. Fragments ScFv-11 and ScFv-18, named for MAb-11 and MAb-18, respectively, inhibited the enzyme activity to the same extent as the intact MAbs. Both of the antibody fragments widely cross-reacted with other phosphatases, including some phosphomonoesterases and phosphodiesterases from different sources. ScFv-18 also inhibited acid phosphatase from a different origin, but stimulated the activity of alkaline phosphatase from calf intestine. The PCR-amplified VH and VL genes were subsequently expressed separately in Escherichia coli as fusion products with glutathione S-transferase. The fusion proteins had little effect on Japanese radish acid phosphatase. Furthermore, a large number of recombinant ScFv fragments specific to the acid phosphatase were generated by using a bacteriophage expression system and a mouse ScFv gene library. These ScFv fragments had a range of effects on the enzyme activity, including inhibition, stimulation, and none. Among them, an ScFv fragment, designated ScFv-G7, inhibited more strongly than ScFv-11 and ScFv-18.     

Diffusing capacity of the lung for carbon monoxide

A phage-display technology was used to produce a single-chain Fv antibody fragment (scFv) from the 30AA5 hybridoma secreting anti-glycoprotein monoclonal antibody (MAb) that neutralizes rabies virus. ScFv was constructed and then cloned for expression as a protein fusion with the g3p minor coat protein of filamentous phage. The display of antibody fragment on the phage surface allows its selection by affinity using an enzyme-linked immunosorbent assay (ELISA); the selected scFv fragment was produced in a soluble form secreted by E. coli. The DNA fragment was sequenced to define the germline gene family and the amino-acid subgroups of the heavy (VH) and light (VL) chain variable regions. The specificity characteristics and neutralization capacity of phage-displayed and soluble scFv fragments were found to be identical to those of the parental 30AA5 MAb directed against antigenic site II of rabies glycoprotein. Phage-display technology allows the production of new antibody molecule forms able to neutralize the rabies virus specifically. The next step could be to engineer and produce multivalent and multispecific neutralizing antibody fragments. A cocktail of multispecific neutralizing antibodies could contain monovalent, bivalent or tetravalent scFv fragments, for passive immunoglobulin therapy.     

Evaluation of auditory discrimination in children with ADD and without ADD

BACKGROUND: Through antibody engineering, immunoglobulins can be tailored for their particular application. In this respect, small recognition units are desired for the targeting of antigens in obstructed locations like solid tumors. OBJECTIVES: To design efficient, minimum size recognition units, heavy chain variable regions (VH) had previously been modified for the use as antigen specific, single domain antibody fragments. To develop a rational approach to improve affinity, antigen binding is investigated here by analysing the effect of randomisations of CDR1 and 2 residues in VH domains specific for hapten and protein ligands. STUDY DESIGN: Randomised repertoires were displayed on phage and affinity selected to improve and analyse antigen binding. Affinities of newly selected VH domains were determined in their soluble format to assess the role of modified residues in binding. RESULTS: In four of five randomisation experiments, a new VH with an improved antigen affinity compared to the primary VH was selected. Dissociation constants decreased from 160 nM to 25 nM or 47 nM (CDR1 or CDR2 randomisation of an anti-Ox VH), from 300 nM to 31 nM (CDR2 randomisation of an anti-NIP VH) and from 3.1 microM to 1.6 microM (CDR2 randomisation of an anti-lysozyme VH). CONCLUSIONS: Thus the affinity of VH domains can be improved after site specific, secondary randomisations in CDR1 and CDR2, phage display and antigen selection. As differences in the CDR3 sequences had formed the only difference between the primary VH domains used in this study, the effect of CDR1 and CDR2 mutations of affinity is consistent with a participation of all three CDRs in antigen binding by single VH domains.     

Folding and assembly of an antibody Fv fragment, a heterodimer stabilized by antigen

The folding and assembly of the Fv fragment of the phosphorylcholine binding antibody McPC603, a non-covalent heterodimer of the variable domains VH and VL, was investigated. Since both domains, each engineered for stability and folding efficiency, could now be obtained in native and soluble form by themselves, fluorescence spectra of VH and VL in unfolded, folded and associated states can be reported. VH and VL only associate when they are native, and the stability of the heterodimer is strongly increased in the presence of antigen. VH rapidly folds into an hyperfluorescent intermediate, and the native state is reached in two parallel, proline-independent reactions. VL displays two fast refolding reactions, which are followed by two slower phases, limited by proline cis/trans-isomerization. The rate-limiting step for both the Fv and the scFv (single-chain Fv) fragment is the formation of the native VH-VL interface, which depends on ProL95 being in cis. The folding of the Fv fragment is fast after short-term denaturation or in the presence of proline cis/trans-isomerase catalysis, but the scFv fragment falls into a kinetic trap, observed by the persistence of the slow phases under all conditions. Furthermore, the scFv fragment, but not the Fv fragment, gives rise to premature interface formation, indicated by the fluorescence spectra and a much higher transient binding of 8-anilino-1-naphthalene sulfonate. The analysis of the folding pathway of the domains VH and VL in isolation and in non-covalent and covalent assemblies should provide helpful insights into the folding of multimeric proteins in general, and for the further engineering of stable and well-folding antibody fragments in particular.     

Xenoantibodies to pig endothelium are expressed in germline configuration and share a conserved immunoglobulin VH gene structure with antibodies to common infectious agents

BACKGROUND: The rejection of pig xenografts in humans is initiated by preformed antibodies that may be related to the natural antibodies that formulate a first line of defense against infectious agents. Immunoglobulin gene variable domains encoding the antibodies that react with similar epitopes expressed on xenoantigens and bacteria may share structurally similar antigen-binding site configurations. METHODS: We sequenced the VH immunoglobulin genes and germline progenitors of two rat monoclonal antibodies that recognize pig xenoantigens. Nucleic and amino acid sequences of these xenoantibodies were compared with immunoglobulin genes encoding antibodies that react with bacteria or viruses. RESULTS AND CONCLUSIONS: VH genes encoding rat anti-pig xenoantibodies are expressed in germline configuration and share structural similarities, including identical amino acids in key antigenic contact sites that define antibody canonical structural groups, with antibodies to infectious agents.     

Autoreactivity of human VH domains from cDNA libraries: analysis with a bacterial expression system

Previous studies showed that VH domains of several anti-DNA Abs can bind DNA in the absence of VL. In the current work, we tested the VH autoreactive potential more generally, examining VH domains that did not come from known autoantibodies. Using a bacterial expression system, we produced 11 fusion proteins, each containing a VH domain and a B domain of staphylococcal protein A. The VH domains were coded in cDNA libraries from circulating B cells of healthy young adult humans. Thus, binding properties of the Ig molecules from which they came were unknown. The B cells had not been stimulated in vitro. Seven cDNA clones combined the frequently expressed VH3-23 gene segment with varied DH and JH segments. The other clones contained unmutated VH3-7, VH3-9, VH3-53, and VH4-39 segments. We compared these bacterial expression products with single-chain Fv, VH and VL domains of IgM mAb 18/2, a VH3-23-encoded, DNA-binding autoantibody. Submicromolar concentrations of 5 of the 11 VH domains bound to ssDNA. Those and one more also bound to immobilized poly(dT), and two bound to circular plasmid dsDNA. Soluble poly(dT) was the most potent inhibitor in competitive ELISA. Seven of the VH domains also bound to immobilized nuclear ribonucleoprotein, four to histone and none to thyroglobulin. Two interacted with the matrix of a Sephacryl S-100 column. The polyreactive autoantigen-binding properties of these VH domains raise the question of whether these properties may play a role in the formation of the VH repertoire of circulating B cells.     

Open sandwich ELISA: a novel immunoassay based on the interchain interaction of antibody variable region

We describe an immunoassay that is based on the interchain interaction of separated VL and VH chains from a single chain antibody variable region. In the presence of antigen, the chains reassociate. VL fragments of anti-hen egg lysozyme (HEL) antibody HyHEL-10 were immobilized on microtiter plates. Samples were coincubated with an M13-displayed VH chain, and assayed with peroxidase-labeled anti-M13 antibody. Signal was detected in direct proportion to the amount of HEL in the sample. Wide dynamic range with < 15 ng/ml sensitivity was attained.     

Generation of rabbit monoclonal antibody fragments from a combinatorial phage display library and their production in the yeast Pichia pastoris

We have applied the combinatorial immunoglobulin library and phage display technologies to generate monoclonal rabbit single-chain Fv (scFv) antibody fragments specific for recombinant human leukemia inhibitory factor (rhLIF). The B cell immunoglobulin repertoire of an immunized rabbit was immortalized by the combinatorial cloning of the rearranged variable domains of light (VL) and heavy (VH) chains. Affinity selection of the library displaying the rabbit antibody domains on the phage surface resulted in the isolation of phage encoding scFv antibodies which specifically bind to the antigen. We utilized the methylotrophic yeast Pichia pastoris for high level secretion of soluble and functional scFv antibody fragment. More than 100 mg/L of pure and functional rabbit anti-rhLIF scFv antibody was obtained directly from the P. pastoris culture supernatant by one-step affinity chromatography.     

Specific V gene usage in anti-phosphorylcholine IgE antibodies

Three hybridomas from phosphorylcholine(PC)-KLH immunized BALB/c mice producing IgE antibodies against the PC hapten were investigated for their fine specificity to the hapten and usage of V gene segments in H- and L-chains. All three IgE antibodies recognize the entire azophenyl-PC hapten. They are T15 Id negative and do not bind to the natural PC determinant expressed by the Streptococcus carbohydrate R36A. T15 Id positive IgE antibodies could neither be elicited by immunization in detectable amounts nor generated by the cell fusion technique. By using the Southern blot technique and nucleotide sequence analysis of PCR amplified VHDJH and VLJL rearrangements, we have demonstrated that the three IgE anti-PC hybridomas use the VH1-DSP2-JH2, the VHOX1-DSP2-JH3 or the VH36-60-D-JH2 gene segment combinations for the H chain together with the V kappa 1C-J kappa 1, V kappa 1C-J kappa 2 or V lambda 1-J lambda 1 genes for the L chains. Except for the VH36-60, the same gene segments were found in different combinations in anti-PC antibodies of other Ig classes than IgE. However, high rates of somatic mutations are expressed in both VH1 of the H chain and in V kappa 1C of the L chain. The VH36-60 is expressed in antibodies with the major Id of the azophenyl-arsonate (Ars) response and VHOX1 generally contributes to the phenyl-oxazolone specificity. This suggests that these V genes are involved in the recognition of the azophenyl moiety of the coupled PC hapten. Thus PC-KLH specific IgE antibodies utilize mutated VH1 and/or VH/VL gene segment combinations which are involved in binding of the azophenyl spacer. These IgE are therefore specific for azophenyl-phosphorylcholine, unlike antibodies normally expressed against the Streptococcus PC determinant in mice. The genetic diversity and the high mutation rates indicate that the specific B cells develop later in the immune response. Thus, they represent newly generated specificities of so-called group II anti-PC antibodies and are not isotype-switch descendants from already existing T15 Id positive IgM antibodies.     

Improved tumour targeting by disulphide stabilized diabodies expressed in Pichia pastoris

Diabodies are dimeric antibody fragments held together by associated heavy and light chain variable domains present on different polypeptide chains. To improve their stability we have introduced cysteine residues into the V-domains to promote the disulphide crosslinking of the dimer. A crosslinked bivalent diabody against carcinoembryonic antigen (CEA) and a crosslinked bispecific diabody against CEA and the T-cell co-receptor CD3 were expressed from Pichia pastoris and Escherichia coli by secretion. From Pichia (but not E.coli) the chains were almost quantitatively crosslinked. Compared with the parent diabodies both crosslinked diabodies were more stable to heat (by >7 degrees C) and the crosslinked bivalent diabody showed improved localization to CEA+ human tumour xenografts in nude mice.     

Absolute conservation of residue 6 of immunoglobulin heavy chain variable regions of class IIA is required for correct folding

While studying the expression of single-chain antibodies (scFv) derived from several murine monoclonal antibodies, we found that residue 6 in Framework region 1 of the heavy chain variable domain plays a crucial role in antibody folding. Binding activity of three murine antibodies with a heavy chain variable region (VH) subgroup IIA was completely lost when at this position the wild-type residue glutamine (Q) was substituted by glutamate (E). Increased sensitivity towards trypsin digestion of soluble scFv suggested that the lack of binding activity was caused by incorrect folding of Q6E mutants. Grafting of the three additional class IA derived FR1 residues, based upon the comparison between both classes of VH sequences, on to the 'defect' subgroup IIA sequence, partially restored the antigen binding activity of the Q6E-containing scFv. Our results suggest that residue 6 of the heavy chain may be part of a folding nucleus, involving the first two beta-strands of Framework region 1. The evolutionary conservation of either glutamine or glutamate at position 6 in different antibody families may well indicate that within immunoglobulin VH domains, different family specific folding nuclei have evolved.