Jamshid Tanha

Selection of phage-displayed llama single-domain antibodies that transmigrate across human blood-brain barrier endothelium

Delivery to the brain of drugs, peptides, and genes depends on the availability of brain‐specific delivery vectors. We used a phage‐displayed library of llama single‐domain antibodies (sdAbs) to enrich for species that selectively bind to and are internalized by human cerebromicrovascular endothelial cells (HCEC). Two sdAbs (FC5 and FC44) were selected, sequenced, subcloned, and expressed as fusion proteins with c‐Myc‐His5 tags. Similar to phage‐displayed sdAbs, soluble FC5 and FC44 were shown to selectively bind HCEC and to transmigrate across an in vitro human blood‐brain barrier (BBB) model. Both FC5 and FC44, in contrast to an unrelated llama sdAb, were also detected in the brain after i.v. injection into mice. These small (~14 kDa) antibodies have characteristics essential for a carrier‐vector and can be used to facilitate drug transport across the BBB.

Selection by phage display of llama conventional VH fragments with heavy chain antibody VHH properties

A llama single domain antibody (dAb) library designed and constructed to contain only heavy chain antibody variable domains (V(H)Hs) also contained a substantial number of typical conventional antibody heavy chain variable sequences (V(H)s). Panning the library against two carbohydrate-specific antibodies yielded anti-idiotypic dAbs and enriched solely for sequences from the V(H) subpopulation of the library. The conventional antibody origin of these V(H)s was confirmed by using oligonucleotide probes, specific for the enriched V(H)s, to identify the parental sequences in the message employed in library construction. Surprisingly, these V(H) dAbs, which are produced in high yield in Escherichia coli, are highly soluble, have excellent temperature stability profiles and do not display any aggregation tendencies. The very close similarity of these molecules to human V(H)s makes them potentially very useful as therapeutic dAbs.

Prokaryotic expression of antibodies.

SummaryMaximizing the expression yields of recombinant whole antibodies and antibody fragments such as Fabs, single-chain Fvs and single-domain antibodies is highly desirable since it leads to lower production costs. Various eukaryotic and prokaryotic expression systems have been exploited to accommodate antibody expression but Escherichia coli systems have enjoyed popularity, in particular with respect to antibody fragments, because of their low cost and convenience. In many instances, product yields have been less than adequate and intrinsic and extrinsic variables have been investigated in an effort to improve yields. This review deals with various aspects of antibody expression in E. coli with a particular focus on single-domain antibodies.

Neutralization of Clostridium difficile Toxin A with Single-domain Antibodies Targeting the Cell Receptor Binding Domain

Clostridium difficile is a leading cause of nosocomial infection in North America and a considerable challenge to healthcare professionals in hospitals and nursing homes. The Gram-positive bacterium produces two high molecular weight exotoxins, toxin A (TcdA) and toxin B (TcdB), which are the major virulence factors responsible for C. difficile-associated disease and are targets for C. difficile-associated disease therapy. Here, recombinant single-domain antibody fragments (VHHs), which specifically target the cell receptor binding domains of TcdA or TcdB, were isolated from an immune llama phage display library and characterized. Four VHHs (A4.2, A5.1, A20.1, and A26.8), all shown to recognize conformational epitopes, were potent neutralizers of the cytopathic effects of toxin A on fibroblast cells in an in vitro assay. The neutralizing potency was further enhanced when VHHs were administered in paired or triplet combinations at the same overall VHH concentration, suggesting recognition of nonoverlapping TcdA epitopes. Biacore epitope mapping experiments revealed that some synergistic combinations consisted of VHHs recognizing overlapping epitopes, an indication that factors other than mere epitope blocking are responsible for the increased neutralization. Further binding assays revealed TcdA-specific VHHs neutralized toxin A by binding to sites other than the carbohydrate binding pocket of the toxin. With favorable characteristics such as high production yield, potent toxin neutralization, and intrinsic stability, these VHHs are attractive systemic therapeutics but are more so as oral therapeutics in the destabilizing environment of the gastrointestinal tract.

Optimal Design Features of Camelized Human Single-domain Antibody Libraries

We have constructed a human VHlibrary based on a camelized VH sequence. The library was constructed with complete randomization of 19 of the 23 CDR3 residues and was panned against two monoclonal antibody targets to generate VH sequences for determination of the antigen contact residue positions. Furthermore, the feasibility and desirability of introducing a disulfide bridge between CDR1 and CDR3 was investigated. Sequences derived from the library showed a bias toward the use of C-terminal CDR3 residues as antigen contact residues. Mass spectrometric analyses indicated that CDR1-CDR3 disulfide formation was universal. However, surface plasmon resonance and NMR data showed that the CDR3 constraint imposed by the disulfide bridge was not always desirable. Very high yields of soluble protein products and lack of protein aggregation, as demonstrated by the quality of the1H-15N HSQC spectra, indicated that the VH sequence for library construction was a good choice. These results should be useful in the design of VHlibraries with optimal features.

Bacteriophage tailspike proteins as molecular probes for sensitive and selective bacterial detection.

We report the use of genetically engineered tailspike proteins (TSPs) from the P22 bacteriophage for the sensitive and selective detection of Salmonella enterica serovar Typhimurium. High yields of two mutant TSPs, one with an N-terminal cysteine (N-Cys) and another with a C-terminal cysteine (C-Cys), have been obtained using recombinant protein expression and purification in Escherichia coli. The mutant TSPs did not have the native endorhamnosidase enzymatic activity of intact P22 phage as well as wild type TSPs (wtTSPs). We have used the Cys-tag to immobilize these TSPs onto gold coated surfaces using thiol-chemistry. Our results demonstrate that the N-Cys configuration of TSPs gives a bacterial capture density of 25.87 ± 0.61 bacteria/100 μm(2) while the C-Cys configuration shows a density of 8.57 ± 0.19 bacteria/100 μm(2). This confirms that the appropriate orientation of the TSPs on the surface is important for efficient capture of the host bacteria. The bacterial capture density of the mutant N-Cys TSP was also 6-fold better than that obtained for intact P22 phage as well as wtTSPs. Bovine-serum albumin was used as a protective layer to prevent any non-specific binding of the bacteria onto the gold substrate. The recognition specificity was confirmed using 3 strains of E. coli which showed negligible binding. In addition, the host bacteria did not show any binding in the absence of the TSPs on the surface. We further show a selective real-time analytical detection of Salmonella by N-Cys mTSP-immobilized on gold coated SF-10 glass plates using surface plasmon resonance. The sensitivity of detection was found to be 10(3)cfu/ml of bacteria.

Isolation of Monomeric Human VHS by a Phage Selection

Human VH domains are promising molecules in applications involving antibodies, in particular, immunotherapy because of their human origin. However, they are, in general, prone to aggregation. Therefore, various strategies have been employed to acquire monomeric human VHs. We had previously discovered that filamentous phages displaying engineered monomeric VH domains gave rise to significantly larger plaques on bacterial lawns than phages displaying wild type VHs with aggregation tendencies. Using plaque size as the selection criterion and a phage-displayed naïve human VH library we identified 15 VHs that were monomeric. Additionally, the VHs demonstrated good expression yields, good refolding properties following thermal denaturation, resistance to aggregation during long incubation at 37 °C, and to trypsin at 37 °C. These 15 VHs should serve as good scaffolds for developing immunotherapeutics, and the selection method employed here should have general utility for isolating proteins with desirable biophysical properties.

Modulation of Toxin Production by the Flagellar Regulon in Clostridium difficile

ABSTRACT 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.

Single‐Domain Antibody‐Conjugated Nanoaggregate‐Embedded Beads for Targeted Detection of Pathogenic Bacteria

The rapid screening of pathogenic bacteria remains a key issue in the diagnosis of infectious diseases, food safety, and public health assurance. In particular, the emergence of drug-resistant bacteria presents great challenges to the health care sector. Methicillin-resistant Staphylococcus aureus (MRSA) is responsible for one of the better-known hospital-acquired infections. S. aureus is a common pathogen that can colonize various areas of the human anatomy. Healthy individuals may carry MRSA asymptomatically, but patients with a compromised immune system are at a greater risk of symptomatic secondary infection. Due to the drug-resistant nature of S. aureus, preventive measures, such as routine patient screening, remain the most effective way to control the spread of this bacterium in clinical environments. In the clinical setting, routine analysis of pathogenic bacteria typically involves biochemical characterization of cultured microorganisms taken from contaminated sources. Standardized procedures are time consuming, which thus highlights the need for a rapid and targeted detection methodology. Advances in nanotechnology and biotechnology offer new possibilities for the rapid screening of harmful microorganisms. Surface-enhanced Raman scattering (SERS) has been demonstrated to achieve ultra-high sensitivity detection in many bioanalytical assays. Herein, we combine the high sensitivity of a newly developed SERS nanoprobe with the high specificity of single-domain antibody (sdAb) to achieve the targeted detection of a single bacterial pathogen, S. aureus. The ability of metallic nanostructures to localized surface plasmon resonance (LSPR) under appropriate electromagnetic field excitation is largely responsible for the SERS effect. The LSPR is strongly dependent on the size and shape of the nanostructures. It has been demonstrated that extremely high SERS enhancement can be achieved when nanostructures are closely spaced, which allows their LSPR to couple. At the optimal interparticle spacing, LSPR coupling results in the capacitive enhancement of the Raman effect for molecules located between particles in the SERS “hot sites”, which thus enables the detection of very few molecules under optimal excitation conditions. A specially designed SERS nanoprobe called a nanoaggregateembedded bead (NAEB) was fabricated with this optimization in mind. NAEBs are fabricated by controlled formation of small Au nanoparticle (NP) aggregates that are subsequently encapsulated in a protective silica shell (Scheme 1a), and fully utilize the advantage of LSPR coupling of a small NP aggregate; as a result each nanosized bead is an ultrahigh sensitivity SERS nanoprobe. Raman reporter molecules (R6G) were incorporated into the nanoaggregate during the formation process to give each NAEB a unique Raman signature. Compared with other SERS-based bioanalytical applications that utilize Ag or Au NPs, NAEBs have the added advantage of stability. Without the protective silica shell, even passivated Au or Ag NPs immersed in biological buffers are prone to parasitic signals from adsorption of the molecules in the biological fluids or loss of signals due to the desorption of the Raman reporter molecule. This problem was recognized by the groups of Liz-Marzan, Natan, Nie, and Brown, who pioneered the work on encapsulating Au NPs with a protective silica shell to improve their stability. In these earlier works, encapsulation was done without deliberate aggregation, which resulted in a relatively low [a] P.-J. Huang, Prof. L.-K. Chau Department of Chemistry and Biochemistry National Chung Cheng University 168 University Road Min-Hsiung, Chia-Yi (Taiwan) Fax: (+886)5-2721040 E-mail : chelkc@ccu.edu.tw [b] Dr. L.-L. Tay, Dr. J. Tanha, S. Ryan Institute for Microstructural Sciences and Institute for Biological Sciences National Research Council Canada Ottawa, ON K1A 0R6 (Canada) Fax: (+1) 952-6337 E-mail : lilin.tay@nrc-cnrc.gc.ca Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/chem.200901397.

Identification of single-domain, Bax-specific intrabodies that confer resistance to mammalian cells against oxidative-stress-induced apoptosis

Bax is a proapoptotic protein implicated in cell death involved in several neurodegenerative diseases. Intracellularly expressed antibody (Ab) fragments (intrabodies) inhibiting Bax function would have potential for developing therapeutics for the aforementioned diseases and can serve as research tools. We report identification, cloning, and functional characterization of several Bax‐specific single‐domain antibodies (sdAbs). These minimal size Ab fragments, which were isolated from a llama VHH phage display library by panning, inhibited Bax function in in vitro assays. Importantly, as intrabodies, these sdAbs, which were stably expressed in mammalian cells, were nontoxic to their host cells and rendered them highly resistant to oxidative‐stress‐induced apoptosis. The intrabodies prevented mitochondrial membrane potential collapse and apoptosis after oxidative stress in the host cells. These anti‐Bax VHHs could be used as tools for studying the role of Bax in oxidative‐stress‐induced apoptosis and for developing novel therapeutics for the degenerative diseases involving oxidative stress.—Gueorguieva, D., Li, S., Walsh, N., Mukerji, A., Tanha, J., Pandey, S. Identification of single‐domain, Bax‐specific intrabodies that confer resistance to mammalian cells against oxidative‐stress‐induced apoptosis. FASEB J. 20, E2209–E2219 (2006)