Crystal Structure of a Human Single Domain Antibody Dimer Formed through VH-VH Non-Covalent Interactions

Single-domain antibodies (sdAbs) derived from human VH are considered to be less soluble and prone to aggregate which makes it difficult to determine the crystal structures. In this study, we isolated and characterized two anti-human epidermal growth factor receptor-2 (HER2) sdAbs, Gr3 and Gr6, from a synthetic human VH phage display library. Size exclusion chromatography and surface plasmon resonance analyses demonstrated that Gr3 is a monomer, but that Gr6 is a strict dimer. To understand this different molecular behavior, we solved the crystal structure of Gr6 to 1.6 Å resolution. The crystal structure revealed that the homodimer assembly of Gr6 closely mimics the VH-VL heterodimer of immunoglobulin variable domains and the dimerization interface is dominated by hydrophobic interactions

Multivalent Anchoring and Oriented Display of Single-Domain Antibodies on Cellulose

Antibody engineering has allowed for the rapid generation of binding agents against virtually any antigen of interest, predominantly for therapeutic applications. Considerably less attention has been given to the development of diagnostic reagents and biosensors using engineered antibodies. Recently, we produced a novel pentavalent bispecific antibody (i.e., decabody) by pentamerizing two single-domain antibodies (sdAbs) through the verotoxin B subunit (VTB) and found both fusion partners to be functional. Using a similar approach, we have engineered a bispecific pentameric fusion protein consisting of five sdAbs and five cellulose-binding modules (CBMs) linked via VTB. To find an optimal design format, we constructed six bispecific pentamers consisting of three different CBMs, fused to the Staphylococcus aureus-specific human sdAb HVHP428, in both orientations. One bispecific pentamer, containing an N-terminal CBM9 and C-terminal HVHP428, was soluble, non-aggregating, and did not degrade upon storage at 4 °C for over six months. This molecule was dually functional as it bound to cellulose-based filters as well as S. aureus cells. When impregnated in cellulose filters, the bispecific pentamer recognized S. aureus cells in a flow-through detection assay. The ability of pentamerized CBMs to bind cellulose may form the basis of an immobilization platform for multivalent display of high-avidity binding reagents on cellulosic filters for sensing of pathogens, biomarkers and environmental pollutants

Toxin-Specific Antibodies for the Treatment of Clostridium difficile: Current Status and Future Perspectives †

Therapeutic agents targeting bacterial virulence factors are gaining interest as non-antibiotic alternatives for the treatment of infectious diseases. Clostridium difficile is a Gram-positive pathogen that produces two primary virulence factors, enterotoxins A and B (TcdA and TcdB), which are responsible for Clostridium difficile-associated disease (CDAD) and are targets for CDAD therapy. Antibodies specific for TcdA and TcdB have been shown to effectively treat CDAD and prevent disease relapse in animal models and in humans. This review summarizes the various toxin-specific antibody formats and strategies under development, and discusses future directions for CDAD immunotherapy, including the use of engineered antibody fragments with robust biophysical properties for systemic and oral delivery

Engineered Single-Domain Antibodies with High Protease Resistance and Thermal Stability

The extreme pH and protease-rich environment of the upper gastrointestinal tract is a major obstacle facing orally-administered protein therapeutics, including antibodies. Through protein engineering, several Clostridium difficile toxin A-specific heavy chain antibody variable domains (VHHs) were expressed with an additional disulfide bond by introducing Ala/Gly54Cys and Ile78Cys mutations. Mutant antibodies were compared to their wild-type counterparts with respect to expression yield, non-aggregation status, affinity for toxin A, circular dichroism (CD) structural signatures, thermal stability, protease resistance, and toxin A-neutralizing capacity. The mutant VHHs were found to be well expressed, although with lower yields compared to wild-type counterparts, were non-aggregating monomers, retained low nM affinity for toxin A, albeit the majority showed somewhat reduced affinity compared to wild-type counterparts, and were capable of in vitro toxin A neutralization in cell-based assays. Far-UV and near-UV CD spectroscopy consistently showed shifts in peak intensity and selective peak minima for wild-type and mutant VHH pairs; however, the overall CD profile remained very similar. A significant increase in the thermal unfolding midpoint temperature was observed for all mutants at both neutral and acidic pH. Digestion of the VHHs with the major gastrointestinal proteases, at biologically relevant concentrations, revealed a significant increase in pepsin resistance for all mutants and an increase in chymotrypsin resistance for the majority of mutants. Mutant VHH trypsin resistance was similar to that of wild-type VHHs, although the trypsin resistance of one VHH mutant was significantly reduced. Therefore, the introduction of a second disulfide bond in the hydrophobic core not only increases VHH thermal stability at neutral pH, as previously shown, but also represents a generic strategy to increase VHH stability at low pH and impart protease resistance, with only minor perturbations in target binding affinities. These are all desirable characteristics for the design of protein-based oral therapeutics

Orally Administered P22 Phage Tailspike Protein Reduces Salmonella Colonization in Chickens: Prospects of a Novel Therapy against Bacterial Infections

One of the major causes of morbidity and mortality in man and economically important animals is bacterial infections of the gastrointestinal (GI) tract. The emergence of difficult-to-treat infections, primarily caused by antibiotic resistant bacteria, demands for alternatives to antibiotic therapy. Currently, one of the emerging therapeutic alternatives is the use of lytic bacteriophages. In an effort to exploit the target specificity and therapeutic potential of bacteriophages, we examined the utility of bacteriophage tailspike proteins (Tsps). Among the best-characterized Tsps is that from the Podoviridae P22 bacteriophage, which recognizes the lipopolysaccharides of Salmonella enterica serovar Typhimurium. In this study, we utilized a truncated, functionally equivalent version of the P22 tailspike protein, P22sTsp, as a prototype to demonstrate the therapeutic potential of Tsps in the GI tract of chickens. Bacterial agglutination assays showed that P22sTsp was capable of agglutinating S. Typhimurium at levels similar to antibodies and incubating the Tsp with chicken GI fluids showed no proteolytic activity against the Tsp. Testing P22sTsp against the three major GI proteases showed that P22sTsp was resistant to trypsin and partially to chymotrypsin, but sensitive to pepsin. However, in formulated form for oral administration, P22sTsp was resistant to all three proteases. When administered orally to chickens, P22sTsp significantly reduced Salmonella colonization in the gut and its further penetration into internal organs. In in vitro assays, P22sTsp effectively retarded Salmonella motility, a factor implicated in bacterial colonization and invasion, suggesting that the in vivo decolonization ability of P22sTsp may, at least in part, be due to its ability to interfere with motility… Our findings show promise in terms of opening novel Tsp-based oral therapeutic approaches against bacterial infections in production animals and potentially in humans

Preparation and analysis of anti-DNA antigen binding fragments

Three DNA-binding antibodies, Jel 72, Jel 274 and Jel 241, were studied to understand their binding characteristics. Binding constant measurements of Jel 274 and Jel 241 IgG, as a function of salt concentration, by fluorescence polarimetry revealed that in both cases the interaction with duplexes was very dependent on ionic strength and 4 to 6 ion pairs were involved in complex formation. A van't Hoff analysis revealed that for both antibodies, the standard heat capacity change was dependent on ionic strength and unlike the case with DNA-binding proteins, a large negative value did not correlate with sequence specific interactions. Jel 72 and Jel 274 single-chain Fv fragments (scFv) were expressed in Escherichia coli (E. coli) and subsequently analyzed. Both scFv, in particular Jel 72, showed a considerable change of specificity compared to the parent IgG examined previously at high ionic strength (Lee et al., 1984b). Detailed analysis of Jel 72 Fab and IgG by fluorescence polarimetry at low ionic strength revealed that similar to its scFv derivative, Jel 72 IgG was not specific for dG•dC sequence. These results demonstrates that the specificity may be derived from salt dependent DNA conformational changes. In separate experiments, the effect of three parameters on Jel 72 scFv specificity was tested: linker length, chain polarity, and two non-CDR (CDR = complementary determining region) positively charged amino acids in the vicinity of the antibody combining site. Thus, six different scFv were constructed and analyzed. Binding to (dG)20•(dC)20 and (dA)20•(dT)20 showed that none of the three parameters had any effect on antibody affinity or specificity. The role of CDR H3 (H = heavy chain) arginine residues at positions 98, 100, 100a in Jel 72 affinity and specificity was also examined. Mutation to asparagine or glutamine did not result in any significant specificity for (dA)20•(dT)20 as suggested on the basis of structural and biochemical studies (Seeman et al., 1976; Mol et al., 1994b; Choo and Klug, 1994a). As well, substitution of all three arginine with asparagine, glutamine, serine, and lysine, surprisingly, did not change the affinity and specificity of Jel 72, demonstrating that these arginine are not important in antigen binding. Further studies were performed with Jel 274 scFv. Experiments in which the light chain of Jel 274 scFv was replaced by those from Jel 72 and Jel 318 (a triplex-binding antibody) revealed that, in both cases, the specificity and, in the case of latter, the affinity was altered, demonstrating the importance of the light chain in antibody binding. To search for DNA-binding antibodies, a phage display library was panned against various duplexes and many positive clones were selected against poly (d(GC)), poly(dA)•poly(dT), poly(rA)•poly(dT), and a few against calf thymus DNA and the triplex DNA. Poly(dA)•poly(dT)-, and poly(rA)•poly(dT)-binders, in general, did not bind to calf thymus DNA, suggesting that structure and sequence specific antibodies are present in phage display libraries

SDS-PAGE for screening non-aggregating human VH domains

Antibody heavy chain variable domains (VHs) form a significant class of biologics. With VH display libraries\u2014the primary source of VH binders\u2014unwanted aggregating VHs are coselected, sometimes overwhelmingly, alongside nonaggregating VHs. Thus, methods enabling efficient screening for nonaggregating VHs are highly valuable. Here, we found that on nonreducing sodium dodecyl sulfate\u2013polyacrylamide gel electrophoresis (SDS\u2013PAGE) gels, nonaggregating VHs migrate faster than expected, giving underestimated molecular weights (MWs), whereas aggregating ones migrate slower, giving overestimated MWs. Our finding can be applied to large-scale screening for nonaggregating VHs and possibly other proteins, in particular in display library settings, by SDS\u2013PAGE.Peer reviewed: YesNRC publication: Ye

Phage display technology for identifying specific antigens on brain endothelial cells

NRC publication: Ye

Phage display technology for identifying selective antigens and address molecules on brain endothelial cells. In Blood Barrier: Biology and Research Protocols (Ed. S. Nag)

NRC publication: Ye