A rational engineering strategy for designing protein a-binding camelid single-domain antibodies

Staphylococcal protein A (SpA) and streptococcal protein G (SpG) affinity chromatography are the gold standards for purifying monoclonal antibodies (mAbs) in therapeutic applications. However, camelid VHH single-domain Abs (sdAbs or VHHs) are not bound by SpG and only sporadically bound by SpA. Currently, VHHs require affinity tag-based purification, which limits their therapeutic potential and adds considerable complexity and cost to their production. Here we describe a simple and rapid mutagenesis-based approach designed to confer SpA binding upon a priori non-SpA-binding VHHs. We show that SpA binding of VHHs is determined primarily by the same set of residues as in human mAbs, albeit with an unexpected degree of tolerance to substitutions at certain core and non-core positions and some limited dependence on at least one residue outside the SpA interface, and that SpA binding could be successfully introduced into five VHHs against three different targets with no adverse effects on expression yield or antigen binding. Next-generation sequencing of llama, alpaca and dromedary VHH repertoires suggested that species differences in SpA binding may result from frequency variation in specific deleterious polymorphisms, especially Ile57. Thus, the SpA binding phenotype of camelid VHHs can be easily modulated to take advantage of tag-less purification techniques, although the frequency with which this is required may depend on the source species

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

Generation of recombinant single-chain antibodies neutralizing the cytolytic activity of vaginolysin, the main virulence factor of Gardnerella vaginalis

Generated scFvs is the first example of recombinant single-chain antibodies with VLY-neutralizing activity produced in prokaryote expression system. G. vaginalis caused infections continue to be a world-wide problem, therefore neutralizing recombinant antibodies may provide novel therapeutic agents useful in the treatment of bacterial vaginosis and other diseases caused by G. vaginalis

An update on antibody-based immunotherapies for Clostridium difficile infection

Greg Hussack,1 Jamshid Tanha1–3 1Human Health Therapeutics Portfolio, National Research Council Canada, Ottawa, 2School of Environmental Sciences, University of Guelph, Guelph, 3Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, ON, Canada Abstract: Clostridium difficile continues to be one of the most prevalent hospital-acquired bacterial infections in the developed world, despite the recent introduction of a novel and effective antibiotic agent (fidaxomicin). Alternative approaches under investigation to combat the anaerobic Gram-positive bacteria include fecal transplantation therapy, vaccines, and antibody-based immunotherapies. In this review, we catalog the recent advances in antibody-based approaches under development and in the clinic for the treatment of C. difficile infection. By and large, inhibitory antibodies that recognize the primary C. difficile virulence factors, toxin A and toxin B, are the most popular passive immunotherapies under investigation. We provide a detailed summary of the toxin epitopes recognized by various antitoxin antibodies and discuss general trends on toxin inhibition efficacy. In addition, antibodies to other C. difficile targets, such as surface-layer proteins, binary toxin, motility factors, and adherence and colonization factors, are introduced in this review. Keywords: antibody, Clostridium difficile, immunotherapy, toxi

Nickel nanoparticles synthesized by a modified polyol method for the purification of histidine-tagged single-domain antibody ToxA5.1

Nickel nanoparticles (NNPs) synthesized by a modified polyol method using ethylene glycol as a reducing agent, palladium chloride as a nucleating agent, and polyvinylpyrrolidone (PVP) as a protective agent were investigated as a potential magnetic adsorbent for the purification of hexahistidine-tagged (His6-tagged) recombinant proteins. The synthesis resulted in nanoparticles having an average diameter of 6828 nm. The x-ray diffraction pattern confirmed the presence of nickel metal, as well as the presence of unreacted nickel (II) hydroxide Ni(OH)2. Magnetic characterization showed that a magnetization saturation of 39.3 electromagnetic unit (emu)/g at 20,000 Oersted (Oe) was reached rapidly and that the material exhibited ferromagnetic behavior. Protein purification results showed that the synthesized NNPs were highly selective for binding to a His6-tagged recombinant protein single-domain antibody ToxA5.1. In addition, NNPs were used for four adsorption cycles without significant binding capacity losses. These particles have shown great potential such as being easily synthesized, cost-effective, and highly selective magnetic adsorbents for the purification of His6-tagged recombinant proteins. \ua9 Copyright \ua9 Materials Research Society 2012.Peer reviewed: YesNRC publication: Ye

Modulation of toxin production by the flagellar regulon in Clostridium difficile

We show in this study that toxin production in Clostridium difficile is altered in cells which can no longer form flagellar filaments. The impact of inactivation of fliC, CD0240, fliF, fliG, fliM, and flhB-fliR flagellar genes upon toxin levels in culture supernatants was assessed using cell-based cytotoxicity assay, proteomics, immunoassay, and immunoblotting approaches. Each of these showed that toxin levels in supernatants were significantly increased in a fliC mutant compared to that in the C. difficile 630 parent strain. In contrast, the toxin levels in supernatants secreted from other flagellar mutants were significantly reduced compared with that in the parental C. difficile 630 strain. Transcriptional analysis of the pathogenicity locus genes (tcdR, tcdB, tcdE, and tcdA) revealed a significant increase of all four genes in the fliC mutant strain, while transcription of all four genes was significantly reduced in fliM, fliF, fliG, and flhB-fliR mutants. These results demonstrate that toxin transcription in C. difficile is modulated by the flagellar regulon. More significantly, mutant strains showed a corresponding change in virulence compared to the 630 parent strain when tested in a hamster model of C. difficile infection. This is the first demonstration of differential flagellum-related transcriptional regulation of toxin production in C. difficile and provides evidence for elaborate regulatory networks for virulence genes in C. difficile. \ua9 2012, American Society for Microbiology.Peer reviewed: YesNRC publication: Ye

Structural basis for antibody recognition in the receptorbinding domains of toxins a and B from clostridium difficile

Background: TcdA and TcdB are the main virulence factors for Clostridium difficile infections. Results: X-ray crystallography, mass spectrometry, and size exclusion chromatography reveal the molecular basis of antibody recognition. Conclusion: Neutralizing antibodies do not directly block binding to known receptors, suggesting new mechanisms of neutralization. Significance: The molecular details of antibody recognition will assist with the development of novel therapeutics and diagnostics. \ua9 2014 by The American Society for Biochemistry and Molecular Biology, Inc.Peer reviewed: YesNRC publication: Ye