scalable-high-throughput-selection-from-phage-displayed-synthetic

Scalable High Throughput Selection From  Phage-displayed Synthetic Antibody Libraries

The demand for antibodies that fulfill the needs of both basic and clinical research applications is high and will dramatically increase in the future. ...

construction-synthetic-phage-displayed-fab-library-with-tailored

Construction of Synthetic Phage Displayed Fab Library with Tailored Diversity

Demand for monoclonal antibodies (mAbs) in basic research and medicine is increasing yearly. Hybridoma technology has been the dominant method for mAb ...

The Recombinant Antibody Network

University of Toronto

The interaction of nitric oxide with thiols is complex and still an active area of research. Herein, we provide an overview of the ways in which nitric oxide can be biologically transformed into species capable of adding an NO moiety to protein sulfhydryls, emphasizing how protein S-nitrosation differs from nitrosation of low molecular weight thiols. Protein S-nitrosation is being revealed as a post-translational means of chemically modifying and functionally altering proteins. Changes in protein function, which persist on a physiologically relevant time scale, effectively transmit biological signals and thus provide a framework for elucidating signaling networks. A description of recently developed methodology facilitating inquiry into this area is provided, along with a sketch of various proteins reported to be targets for nitrosation and the functional consequences therein. Protein denitrosation appears to be an active and perhaps enzymatically catalyzed process. Here, we summarize the evidence that suggests this and proffer a précis of proteins possessing denitrosation activity.

Protein S-nitrosation: biochemistry and characterization of protein thiol-NO interactions as cellular signals.

The objectives of this study were to determine the relationships among Type II diabetes (T2DM)-dependent elevations in platelet-derived reactive oxygen species (ROS), platelet-surface protein disulfide isomerase (psPDI) NO-releasing activity, and platelet aggregation and to evaluate the efficacy of rosuvastatin in normalizing these parameters in primary cells derived from a hamster model of prediabetic insulin resistance induced by fructose feeding. Platelets from rosuvastatin-treated non-fructose-fed (NFF) and fructose-fed (FF) hamsters were analyzed for aggregability and psPDI-denitrosation activity. Platelets from NFF animals treated with xanthine/xanthine oxidase (X/XO) were assessed for the same parameters and primary aortic endothelial cells (AEC) cultivated with a range of [rosuvastatin] +/- mevalonate were analyzed for ROS production. Platelets from FF hamsters displayed statistically significant enhanced ROS production, diminished psPDI-mediated NO-releasing activity, and hyperaggregability. Suggestively, platelets from NFF animals treated with X/XO displayed characteristics similar to platelets from FF animals. Rosuvastatin elicited a normalizing effect on all parameters measured in platelets from FF animals. Further, ROS production in primary AEC from FF animals could be blunted to that of NFF animals by concentrations of rosuvastatin in the range of those achieved in the bloodstream. Diminished psPDI-dependent NO-releasing activity and increased initial aggregation rates of FF platelets may result from elevated vascular ROS production under conditions of insulin resistance. Normalization of ROS production and platelet aggregation by rosuvastatin indicates its potential use as a vasculoprotective agent.

Antioxidant and antiplatelet effects of rosuvastatin in a hamster model of prediabetes.

Nitric oxide (NO) signaling is inextricably linked to both its physical and chemical properties. Due to its preferentially hydrophobic solubility, NO molecules tend to partition from the aqueous milieu into biological membranes. We hypothesized that plasma membrane ordering provided by cholesterol further couples the physics of NO diffusion with cellular signaling. Fluorescence lifetime quenching studies with pyrene liposome preparations showed that the presence of cholesterol decreased apparent diffusion coefficients of NO approximately 20-40%, depending on the phospholipid composition. Electrochemical measurements indicated that the diffusion rate of NO across artificial bilayer membranes were inversely related to cholesterol content. Sterol transport-defective Niemann-Pick type C1 (NPC1) fibroblasts exhibited increased plasma membrane cholesterol content but decreased activation of both intracellular soluble guanylyl cyclase and vasodilator-stimulated phosphoprotein (VASP) phosphorylation at Ser(239) induced by exogenous NO exposure relative to their normal human fibroblast (NHF) counterparts. Augmentation of plasma membrane cholesterol in NHF diminished production of both cGMP and VASP phosphorylation elicited by NO to NPC1-comparable levels. Conversely, decreasing membrane cholesterol in NPC1 resulted in the augmentation in both cGMP and VASP phosphorylation to a level similar to those observed in NHF. Increasing plasma membrane cholesterol contents in NHF, platelets, erythrocytes and tumor cells also resulted in an increased level of extracellular diaminofluorescein nitrosation following NO exposure. These findings suggest that the impact of cholesterol on membrane fluidity and microdomain structure contributes to the spatial heterogeneity of NO diffusion and signaling.

Plasma membrane cholesterol content affects nitric oxide diffusion dynamics and signaling.

Platelet-derived microparticles (pMP) have been shown to be pro-aggregatory and retain most of their platelet membrane markers. Recent studies have correlated elevated pMP levels with pathogenesis of diabetes mellitus and cardiovascular disease. The pro-aggregatory effect of pMP has been largely attributed to their negatively charged outer surface and activation of factor X by membrane associated Tissue factor (TF). Here we sought to investigate whether, like platelets, protein disulfide isomerase (PDI) is present on the surface of pMP and, if so, to analyze its contribution to platelet hyperaggregability and insulin degradation. Using a fluorescent assay based upon a novel pseudo-substrate of PDI, flow cytometry and immunological techniques, we have demonstrated the presence of PDI on the surface of pMP (termed msPDI) and its ability to influence insulin-mediated Akt phosphorylation (Thr308) in 3T3-L1 fibroblasts. Moreover, pMP are shown to contain catalytically active PDI, capable of both promoting platelet aggregation and disrupting insulin signaling. pMP increased initial rates of aggregation by 4-fold and the pro-aggregatory activity of pMPs could be attenuated with an anti-PDI antibody. The pMP insulin-reductase activity was further attributed to PDI based on the ability of anti-PDI antibodies to block the degradation of insulin, thereby restoring insulin signaling. Plasma pMP counts were also obtained from diabetic (n=10) and non-diabetic individuals (n=10) and found to be elevated in the diabetic state. Detection of increased levels of PDI-containing microparticles in patients with T2D raises the possibility that platelet hypersensitivity and insulin desensitization observed in diabetes can partially be attributed to msPDI activity.

Platelet microparticle-associated protein disulfide isomerase promotes platelet aggregation and inactivates insulin.

Protein microarrays offer a global perspective on the function of expressed gene products. However, technical issues related to the stability and dynamic range of microarrays printed with purified protein have hampered their widespread adoption. Taking an alternate approach, the Nucleic Acid Programmable Protein Array (NAPPA) is constructed by spotting protein-encoding plasmid DNA at high density, in addressable fashion, on an array surface. Proteins are subsequently generated in situ just prior to experimentation using cell-free expression systems. As such, the NAPPA platform offers a unique and viable alternative that circumvents many of the inherent limitations of spotted protein arrays, enabling diverse functional protein studies including protein-small molecule, protein-protein, antigen-antibody, and protein-nucleic acid interactions. It further offers a versatile and adaptable platform amenable to a variety of capture modalities and expression systems, and, most importantly, construction of the array is accessible to any lab with an array printer and laser slide scanner. This unit is intended to provide a reference for investigators wishing to generate arrays based on this platform, and details (1) the basic construction of cDNA-based protein microarrays from DNA isolation to printing and development, (2) quality-control efforts taken to ensure the usefulness and integrity of microarray data, and (3) a particular example of the application of self-assembling protein arrays to screen for blood-borne antibody biomarkers.

Nucleic Acid programmable protein arrays: versatile tools for array-based functional protein studies.

Proteomics aspires to elucidate the functions of all proteins. Protein microarrays provide an important step by enabling high-throughput studies of displayed proteins. However, many functional assays of proteins include untethered intermediates or products, which could frustrate the use of planar arrays at very high densities because of diffusion to neighboring features. The nucleic acid programmable protein array (NAPPA) is a robust in situ synthesis method for producing functional proteins just-in-time, which includes steps with diffusible intermediates. We determined that diffusion of expressed proteins led to cross-binding at neighboring spots at very high densities with reduced interspot spacing. To address this limitation, we have developed an innovative platform using photolithographically etched discrete silicon nanowells and used NAPPA as a test case. This arrested protein diffusion and cross-binding. We present confined high density protein expression and display, as well as functional protein-protein interactions, in 8000 nanowell arrays. This is the highest density of individual proteins in nanovessels demonstrated on a single slide. We further present proof of principle results on ultrahigh density protein arrays capable of up to 24000 nanowells on a single slide.

High density diffusion-free nanowell arrays.

Immunology: Evolving superkines for selectivity.

The need for biomarkers that illuminate the pathophysiology of type 1 diabetes (T1D), enhance early diagnosis and provide additional avenues for therapeutic intervention is well recognized in the scientific community. We conducted a proteome-scale, two-stage serological AAb screening followed by an independent validation study. In the first stage, the immunoreactivity was compared between T1D cases and healthy controls against ~6000 human proteins using the nucleic acid programmable protein array (NAPPA). Genes identified with higher signal intensities in patients were challenged with a larger sample set during the second stage. Statistical analysis revealed 26 novel autoantigens and a known T1D-associated autoantigen. During validation, we verified the presence of AAbs to dual specificity tyrosine-phosphorylation-regulated kinase 2 (DYRK2) using the Luciferase ImmunoPrecipitation System (LIPS) assay (36% sensitivity, 98% specificity). The AUC for a combination of DYRK2A and the classical T1D AAb IA-2A was 0.90 compared to 0.72 for DYRK2A and 0.64 for IA-2A alone. This is the first systematic screening for seroreactivity against a large number of human proteins in T1D patients. We demonstrated the application of protein microarrays to identify novel autoantigens in T1D, expanded the current T1D "autoantigenome" and help fulfill the goal of searching for novel biomarker candidates for T1D.

Serological autoantibody profiling of type 1 diabetes by protein arrays.

Antibodies are used in multiple cell biology applications, but there are no standardized methods to assess antibody quality-an absence that risks data integrity and reproducibility. We describe a mass spectrometry-based standard operating procedure for scoring immunoprecipitation antibody quality. We quantified the abundance of all the proteins in immunoprecipitates of 1,124 new recombinant antibodies for 152 chromatin-related human proteins by comparing normalized spectral abundance factors from the target antigen with those of all other proteins. We validated the performance of the standard operating procedure in blinded studies in five independent laboratories. Antibodies for which the target antigen or a member of its known protein complex was the most abundant protein were classified as 'IP gold standard'. This method generates quantitative outputs that can be stored and archived in public databases, and it represents a step toward a platform for community benchmarking of antibody quality. 

Assessment of a method to characterize antibody selectivity and specificity for use in immunoprecipitation.

Antibodies are key reagents in biology and medicine, but commercial sources are rarely recombinant and thus do not provide a permanent and renewable resource. Here, we describe an industrialized platform to generate antigens and validated recombinant antibodies for 346 transcription factors (TFs) and 211 epigenetic antigens. We describe an optimized automated phage display and antigen expression pipeline that in aggregate produced about 3000 sequenced Fragment antigen-binding domain that had high affinity (typically EC50<20 nm), high stability (Tm∼80 °C), good expression in E. coli (∼5 mg/L), and ability to bind antigen in complex cell lysates. We evaluated a subset of Fabs generated to homologous SCAN domains for binding specificities. These Fragment antigen-binding domains were monospecific to their target SCAN antigen except in rare cases where they cross-reacted with a few highly related antigens. Remarkably, immunofluorescence experiments in six cell lines for 270 of the TF antigens, each having multiple antibodies, show that ∼70% stain predominantly in the cytosol and ∼20% stain in the nucleus which reinforces the dominant role that translocation plays in TF biology. These cloned antibody reagents are being made available to the academic community through our web site recombinant-antibodies.org to allow a more system-wide analysis of TF and chromatin biology. We believe these platforms, infrastructure, and automated approaches will facilitate the next generation of renewable antibody reagents to the human proteome in the coming decade. 

A High Through-put Platform for Recombinant Antibodies to Folded Proteins.

Antibodies are indispensable tools in biochemical research and play an expanding role as therapeutics. While hybridoma technology is the dominant method for antibody production, phage display is an emerging technology. Here, we developed and employed a high-throughput pipeline that enables selection of antibodies against hundreds of antigens in parallel. Binding selections using a phage-displayed synthetic antigen-binding fragment (Fab) library against 110 human SH3 domains yielded hundreds of Fabs targeting 58 antigens. Affinity assays demonstrated that representative Fabs bind tightly and specifically to their targets. Furthermore, we developed an efficient affinity maturation strategy adaptable to high-throughput, which increased affinity dramatically but did not compromise specificity. Finally, we tested Fabs in common cell biology applications and confirmed recognition of the full-length antigen in immunoprecipitation, immunoblotting and immunofluorescence assays. In summary, we have established a rapid and robust high-throughput methodology that can be applied to generate highly functional and renewable antibodies targeting protein domains on a proteome-wide scale. 

Selection of recombinant anti-SH3 domain antibodies by high-throughput phage display.

Exogenous cytokine therapy can induce systemic toxicity, which might be prevented by activating endogenously produced cytokines in local cell niches. Here we developed antibody-based activators of cytokine signaling (AcCS), which recognize cytokines only when they are bound to their cell surface receptors. AcCS were developed for type I interferons (IFNs), which induce cellular activities by binding to cell surface receptors IFNAR1 and IFNAR2. As a potential alternative to exogenous IFN therapy, AcCS were shown to potentiate the biological activities of natural IFNs by ∼100-fold. Biochemical and structural characterization demonstrates that the AcCS stabilize the IFN-IFNAR2 binary complex by recognizing an IFN-induced conformational change in IFNAR2. Using IFN mutants that disrupt IFNAR1 binding, AcCS were able to enhance IFN antiviral potency without activating antiproliferative responses. This suggests AcCS can be used to manipulate cytokine signaling for basic science and possibly for therapeutic applications. 

Cytokine Activation by Antibody Fragments Targeted to Cytokine-Receptor Signaling Complexes.

Exploiting binding affinities between molecules is an established practice in many fields, including biochemical separations, diagnostics, and drug development; however, using these affinities to control biomolecule release is a more recent strategy. Affinity-controlled release takes advantage of the reversible nature of noncovalent interactions between a therapeutic protein and a binding partner to slow the diffusive release of the protein from a vehicle. This process, in contrast to degradation-controlled sustained-release formulations such as poly(lactic-co-glycolic acid) microspheres, is controlled through the strength of the binding interaction, the binding kinetics, and the concentration of binding partners. In the context of affinity-controlled release--and specifically the discovery or design of binding partners--we review advances in in vitro selection and directed evolution of proteins, peptides, and oligonucleotides (aptamers), aided by computational design.

Designer protein delivery: From natural to engineered affinity-controlled release systems.

If the isolation, production, and clinical use of insulin marked the inception of the age of biologics as therapeutics, the convergence of molecular biology and combinatorial engineering techniques marked its coming of age. The first wave of recombinant protein-based drugs in the 1980s demonstrated emphatically that proteins could be engineered, formulated, and employed for clinical advantage. Yet despite the successes of protein-based drugs such as antibodies, enzymes, and cytokines, the druggable target space for biologics is currently restricted to targets outside the cell. Insofar as estimates place the number of proteins either secreted or with extracellular domains in the range of 8000 to 9000, this represents only one-third of the proteome and circumscribes the pathways that can be targeted for therapeutic intervention. Clearly, a major objective for this field to reach maturity is to access, interrogate, and modulate the majority of proteins found inside the cell. However, owing to the large size, complex architecture, and general cellular impermeability of existing protein-based drugs, this poses a daunting challenge. In recent years, though, advances on the two related fronts of protein engineering and drug delivery are beginning to bring this goal within reach. First, prompted by the restrictions that limit the applicability of antibodies, intense efforts have been applied to identifying and engineering smaller alternative protein scaffolds for the modulation of intracellular targets. In parallel, innovative solutions for delivering proteins to the intracellular space while maintaining their stability and functional activity have begun to yield successes. This review provides an overview of bioactive intrabodies and alternative protein scaffolds amenable to engineering for intracellular targeting and also outlines advances in protein engineering and formulation for delivery of functional proteins to the interior of the cell to achieve therapeutic action. 

Intracellular targeting with engineered proteins.

We tested whether grafting an interaction domain into the hypervariable loop of a combinatorial antibody library could promote targeting to a specific epitope. Formation of the epidermal growth factor receptor (EGFR) signaling heterodimer involves extensive contacts mediated by a "dimerization loop." We grafted the dimerization loop into the third hypervariable loop of a synthetic antigen-binding fragment (Fab) library and diversified other loops using a tailored diversity strategy. This structure-directed Fab library and a naı̈ve synthetic Fab library were used to select Fabs against EGFR. Both libraries yielded high affinity Fabs that bound to overlapping epitopes on cell-surface EGFR, inhibited receptor activation, and targeted epitopes distinct from those of cetuximab and panitumumab. Epitope mapping experiments revealed complex sites of interaction, comprised of domains I and II but not exclusively localized to the receptor dimerization loop. These results validate the grafting approach for designing Fab libraries and also underscore the versatility of naı̈ve synthetic libraries.

ArticleTotal : 2	Year
Publication title Cited by 33	2015
Publication title Cited by 7	2018

Article	Year
Protein S-nitrosation: biochemistry and characterization of protein thiol-NO interactions as cellular signals. Clinical biochemistry\| PubMed ID: 16005861	2005
Antioxidant and antiplatelet effects of rosuvastatin in a hamster model of prediabetes. Free radical biology & medicine\| PubMed ID: 17189832	2007
Plasma membrane cholesterol content affects nitric oxide diffusion dynamics and signaling. The Journal of biological chemistry\| PubMed ID: 18445594	2008
Platelet microparticle-associated protein disulfide isomerase promotes platelet aggregation and inactivates insulin. Biochimica et biophysica acta\| PubMed ID: 18691554	2008
Nucleic Acid programmable protein arrays: versatile tools for array-based functional protein studies. Current protocols in protein science / editorial board, John E. Coligan ... [et al.]\| PubMed ID: 21488044	2011
High density diffusion-free nanowell arrays. Journal of proteome research\| PubMed ID: 22742968	2012
Immunology: Evolving superkines for selectivity. Nature chemical biology\| PubMed ID: 23183579	2012
Serological autoantibody profiling of type 1 diabetes by protein arrays. Journal of proteomics\| PubMed ID: 24148850	2013
Assessment of a method to characterize antibody selectivity and specificity for use in immunoprecipitation. Nature methods\| PubMed ID: 26121405	2015
A High Through-put Platform for Recombinant Antibodies to Folded Proteins. Molecular & cellular proteomics : MCP\| PubMed ID: 26290498	2015
Selection of recombinant anti-SH3 domain antibodies by high-throughput phage display. Protein science : a publication of the Protein Society\| PubMed ID: 26332758	2015
Cytokine Activation by Antibody Fragments Targeted to Cytokine-Receptor Signaling Complexes. The Journal of biological chemistry\| PubMed ID: 26546677	2016
Designer protein delivery: From natural to engineered affinity-controlled release systems. Science (New York, N.Y.)\| PubMed ID: 26989257	2016
Intracellular targeting with engineered proteins. F1000Research\| PubMed ID: 27547383	2016
Structure-Directed and Tailored Diversity Synthetic Antibody Libraries Yield Novel Anti-EGFR Antagonists. ACS chemical biology\| PubMed ID: 28375604	2017

Shane Miersch

Biography

JoVE Journal Publications

Other Publications