Engineering of coiled-coil protein scaffolds as innovative tools for biosensing applications

2007/2008A new generation of protein scaffolds is becoming a valid alternative tool to recombinant antibodies of biotechnological, medical and pharmaceutical applications, where strong affinity and specificity are required. They share with antibodies important features (target affinity and specificity), but they have also some improvements (smaller size of molecule, tolerance to modification of the framework and the recognition site restricted to few residues), that can be exploited for biosensing application in nanotechnological platforms. Nanotechnology has been played an increasingly important role in the development of biosensors, improving the intrinsic features of biodevices. In this thesis work, we analyzed the coiled-coil domain, a widely spread dimerization domain shared by several protein scaffolds, and involved in protein-protein interaction in both eukaryotic and prokaryotic cells. The analysis of the coiled-coil structure allows a de novo design of new peptides, namely E and K, that can dimerize as a E/K coiled-coil system: the dimerization feature and the stability of the interaction makes this system an ideal platform to build up functional and customizable biosensors. A characterization of the E/K interaction was performed by using the protein complementation assay (PCA), a useful biological method to investigate the interaction between protein partners. With this in vivo method, we corroborate the interaction features determinate with circular dichroism, and we demonstrated that E and K coils effectively represent a protein scaffold, able to tolerate amino acid substitutions without altering its main structure. In addition, we create two libraries of K mutant coils, randomizing the peptide sequence, and with PCA we selected new K binders (Kran 5.17 and Krd F8) that showed a comparable interaction activity with the E-coil in preliminary in vitro tests. In the last part of this work, we generate a library of a new scaffold molecule (the single chain E-K) capable to bind small molecules as a single protein product containing both domains. Using the phage display selection system, we isolated scsE-K that can bind our analyte (the caffeine) with high specificity. This new molecules can be a powerful tool for analytical and biomedical applications.XXI Ciclo198

Mucosal tissue transglutaminase expression in celiac disease

Tissue transglutaminase (tTG) plays an important role in celiac disease pathogenesis and antibodies to tTG are a diagnostic marker of gluten-sensitive enteropathy. The aim of this study was to investigate the localization of tTG in the duodenal mucosa in control tissues and in different histological stages of celiac disease by using a commercial and a novel set of anti-tTG monoclonal antibodies, to see whether this assessment can be useful for diagnostic purpose. The distribution of tTG was firstly evaluated in 18 untreated celiac patients by using a commercial monoclonal antibody (CUB7402) against tissue transglutaminase enzyme and directed against the loop-core region of the enzyme. Thereafter, in further 30 untreated celiac patients we employed three newly characterized anti-tTG monoclonal antibodies produced against recombinant human-tTG. The epitopes recognized are located in three distinct domains of the protein corresponding to the core, C1 and C2 protein structure. Eleven age- and sex-matched patients with chronic duodenitis acted as controls. All subjects underwent upper endoscopy to obtain biopsy samples from the duodenum. Overall, we found that (i) tTG is equally expressed in CD at different stages of disease; (ii) tTG is expressed, at similar level, in CD and controls with duodenitis. Assessment of tTG level in biopsy samples by immunohistochemical methods is not useful in the clinical diagnostic work-up of CD.Facultad de Ciencias Exacta

Mucosal tissue transglutaminase expression in celiac disease

Tissue transglutaminase (tTG) plays an important role in celiac disease pathogenesis and antibodies to tTG are a diagnostic marker of gluten-sensitive enteropathy. The aim of this study was to investigate the localization of tTG in the duodenal mucosa in control tissues and in different histological stages of celiac disease by using a commercial and a novel set of anti-tTG monoclonal antibodies, to see whether this assessment can be useful for diagnostic purpose. The distribution of tTG was firstly evaluated in 18 untreated celiac patients by using a commercial monoclonal antibody (CUB7402) against tissue transglutaminase enzyme and directed against the loop-core region of the enzyme. Thereafter, in further 30 untreated celiac patients we employed three newly characterized anti-tTG monoclonal antibodies produced against recombinant human-tTG. The epitopes recognized are located in three distinct domains of the protein corresponding to the core, C1 and C2 protein structure. Eleven age- and sex-matched patients with chronic duodenitis acted as controls. All subjects underwent upper endoscopy to obtain biopsy samples from the duodenum. Overall, we found that (i) tTG is equally expressed in CD at different stages of disease; (ii) tTG is expressed, at similar level, in CD and controls with duodenitis. Assessment of tTG level in biopsy samples by immunohistochemical methods is not useful in the clinical diagnostic work-up of CD.Facultad de Ciencias Exacta

Mucosal tissue transglutaminase expression in celiac disease

Tissue transglutaminase (tTG) plays an important role in celiac disease pathogenesis and antibodies to tTG are a diagnostic marker of gluten-sensitive enteropathy. The aim of this study was to investigate the localization of tTG in the duodenal mucosa in control tissues and in different histological stages of celiac disease by using a commercial and a novel set of anti-tTG monoclonal antibodies, to see whether this assessment can be useful for diagnostic purpose. The distribution of tTG was firstly evaluated in 18 untreated celiac patients by using a commercial monoclonal antibody (CUB7402) against tissue transglutaminase enzyme and directed against the loop-core region of the enzyme. Thereafter, in further 30 untreated celiac patients we employed three newly characterized anti-tTG monoclonal antibodies produced against recombinant human-tTG. The epitopes recognized are located in three distinct domains of the protein corresponding to the core, C1 and C2 protein structure. Eleven age- and sex-matched patients with chronic duodenitis acted as controls. All subjects underwent upper endoscopy to obtain biopsy samples from the duodenum. Overall, we found that (i) tTG is equally expressed in CD at different stages of disease; (ii) tTG is expressed, at similar level, in CD and controls with duodenitis. Assessment of tTG level in biopsy samples by immunohistochemical methods is not useful in the clinical diagnostic work-up of CD.Facultad de Ciencias Exacta

Recombinant renewable polyclonal antibodies

Only a small fraction of the antibodies in a traditional polyclonal antibody mixture recognize the target of interest, frequently resulting in undesirable polyreactivity. Here, we show that high-quality recombinant polyclonals, in which hundreds of different antibodies are all directed toward a target of interest, can be easily generated in vitro by combining phage and yeast display. We show that, unlike traditional polyclonals, which are limited resources, recombinant polyclonal antibodies can be amplified over one hundred million-fold without losing representation or functionality. Our protocol was tested on 9 different targets to demonstrate how the strategy allows the selective amplification of antibodies directed toward desirable target specific epitopes, such as those found in one protein but not a closely related one, and the elimination of antibodies recognizing common epitopes, without significant loss of diversity. These recombinant renewable polyclonal antibodies are usable in different assays, and can be generated in high throughput. This approach could potentially be used to develop highly specific recombinant renewable antibodies against all human gene products

Rapid interactome profiling by massive sequencing

We have developed a high-throughput protein expression and interaction analysis platform that combines cDNA phage display library selection and massive gene sequencing using the 454 platform. A phage display library of open reading frame (ORF) fragments was created from mRNA derived from different tissues. This was used to study the interaction network of the enzyme transglutaminase 2 (TG2), a multifunctional enzyme involved in the regulation of cell growth, differentiation and apoptosis, associated with many different pathologies. After two rounds of panning with TG2 we assayed the frequency of ORFs within the selected phage population using 454 sequencing. Ranking and analysis of more than 120 000 sequences allowed us to identify several potential interactors, which were subsequently confirmed in functional assays. Within the identified clones, three had been previously described as interacting proteins (fibronectin, SMOC1 and GSTO2), while all the others were new. When compared with standard systems, such as microtiter enzyme-linked immunosorbant assay, the method described here is dramatically faster and yields far more information about the interaction under study, allowing better characterization of complex systems. For example, in the case of fibronectin, it was possible to identify the specific domains involved in the interaction

Cross-Neutralising Nanobodies Bind to a Conserved Pocket in the Hemagglutinin Stem Region Identified Using Yeast Display and Deep Mutational Scanning

<div><p>Cross-neutralising monoclonal antibodies against influenza hemagglutinin (HA) are of considerable interest as both therapeutics and diagnostic tools. We have recently described five different single domain antibodies (nanobodies) which share this cross-neutralising activity and suggest their small size, high stability, and cleft binding properties may present distinct advantages over equivalent conventional antibodies. We have used yeast display in combination with deep mutational scanning to give residue level resolution of positions in the antibody-HA interface which are crucial for binding. In addition, we have mapped positions within HA predicted to have minimal effect on antibody binding when mutated. Our cross-neutralising nanobodies were shown to bind to a highly conserved pocket in the HA2 domain of A(H1N1)pdm09 influenza virus overlapping with the fusion peptide suggesting their mechanism of action is through the inhibition of viral membrane fusion. We also note that the epitope overlaps with that of CR6261 and F10 which are human monoclonal antibodies in clinical development as immunotherapeutics. Although all five nanobodies mapped to the same highly conserved binding pocket we observed differences in the size of the epitope footprint which has implications in comparing the relative genetic barrier each nanobody presents to a rapidly evolving influenza virus. To further refine our epitope map, we have re-created naturally occurring mutations within this HA stem epitope and tested their effect on binding using yeast display. We have shown that a D46N mutation in the HA2 stem domain uniquely interferes with binding of R2b-E8. Further testing of this substitution in the context of full length purified HA from 1918 H1N1 pandemic (Spanish flu), 2009 H1N1 pandemic (swine flu) and highly pathogenic avian influenza H5N1 demonstrated binding which correlated with D46 whereas binding to seasonal H1N1 strains carrying N46 was absent. In addition, our deep sequence analysis predicted that binding to the emerging H1N1 strain (A/Christchurch/16/2010) carrying the HA2-E47K mutation would not affect binding was confirmed experimentally. This demonstrates yeast display, in combination with deep sequencing, may be able to predict antibody reactivity to emerging influenza strains so assisting in the preparation for future influenza pandemics.</p></div

A general strategy for high-throughput epitope mapping of single domain antibodies to hemagglutinin.

<p>Generation of panels of high affinity monoclonal single domain antibodies (sdAbs or nanobodies) to hemagglutinin using immunisation of alpacas with HA and phage display technology, (i) design of a library of HA variants, (ii) display library on yeast cell surface, (iii) selection using flow cytometric cell sorting to enrich hemagglutinin variants that lose binding to sdAbs but retain display of correctly folded HA on yeast cell surface, (iv) pools of enriched HA mutants are then analysed using deep sequencing, (v) mutations are enriched and their frequencies in the selected population relative to the non-selected population are identified using bioinformatic analysis. Functional loss of binding is experimentally determined to confirm residues are energetically important and contribute to the antibody epitope. This approach can be used to generate a database of epitopes corresponding to diverse collections of sdAbs specific for HA, which upon the emergence of a new viral strain can be used to predict which antibodies could be chosen as suitable binding reagents for applications in diagnosis, research, immune surveillance and vaccine potency testing.</p

Relative binding footprint of individual within the HA stem epitope.

<p>Surface structure models of hemagglutinin (HA) A(H1N1)pdm09 (PDB structure 3AL4) showing the two domains (HA1 in blue, HA2 in violet) and the key epitope residues of each sdAb. The key epitope residues of human antibody CR6261, defined by X-ray crystallography [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164296#pone.0164296.ref022" target="_blank">22</a>], is indicated with a dotted black line and demonstrates overlapping sdAb epitopes. The epitope footprint of each sdAb is shown in red relative to the HA stem and combines residues identified by deep mutational scanning (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164296#pone.0164296.g005" target="_blank">Fig 5</a>) and rational mutagenesis with naturally occurring subtype specific substitutions (Fig 7). The final panel shows a exploded view of the R1a-B6 epitope with key Gly²⁰, Trp²¹ and Ile⁴⁵ residues shown in red. Helix A and fusion peptide are highlighted in yellow and green respectively.</p

Experimental testing of naturally occurring mutations within epitope footprint.

<p>(A) Table showing binding activity of sdAbs to a panel of HA mutants carrying naturally occurring mutations. The extent of antibody binding was determined by dividing the MFI value of each antibody-mutant pair by the value of the wild-type H1N1 HA incubation, and the resulting ratio normalized to percentage values. Relative binding of sdAbs to each displayed mutant were categorized as no binding (-, red) and strong binding (+, green). (B) Flow cytometry histograms showing binding of R2a-G8 and R2b-E8 to wild-type H1N1 and panel of HA mutants. Absence of binding for D19A,Y34M, I45F and D46N mutation are indicated with a black arrow.</p