Christian Tiede

Adhiron: a stable and versatile peptide display scaffold for molecular recognition applications.

We have designed a novel non-antibody scaffold protein, termed Adhiron, based on a phytocystatin consensus sequence. The Adhiron scaffold shows high thermal stability (Tm ca. 101°C), and is expressed well in Escherichia coli. We have determined the X-ray crystal structure of the Adhiron scaffold to 1.75 Å resolution revealing a compact cystatin-like fold. We have constructed a phage-display library in this scaffold by insertion of two variable peptide regions. The library is of high quality and complexity comprising 1.3 × 1010 clones. To demonstrate library efficacy, we screened against the yeast Small Ubiquitin-like Modifier (SUMO). In selected clones, variable region 1 often contained sequences homologous to the known SUMO interactive motif (V/I-X-V/I-V/I). Four Adhirons were further characterised and displayed low nanomolar affinities and high specificity for yeast SUMO with essentially no cross-reactivity to human SUMO protein isoforms. We have identified binders against >100 target molecules to date including as examples, a fibroblast growth factor (FGF1), platelet endothelial cell adhesion molecule (PECAM-1; CD31), the SH2 domain Grb2 and a 12-aa peptide. Adhirons are highly stable and well expressed allowing highly specific binding reagents to be selected for use in molecular recognition applications.

Affimer proteins are versatile and renewable affinity reagents

Molecular recognition reagents are key tools for understanding biological processes and are used universally by scientists to study protein expression, localisation and interactions. Antibodies remain the most widely used of such reagents and many show excellent performance, although some are poorly characterised or have stability or batch variability issues, supporting the use of alternative binding proteins as complementary reagents for many applications. Here we report on the use of Affimer proteins as research reagents. We selected 12 diverse molecular targets for Affimer selection to exemplify their use in common molecular and cellular applications including the (a) selection against various target molecules; (b) modulation of protein function in vitro and in vivo; (c) labelling of tumour antigens in mouse models; and (d) use in affinity fluorescence and super-resolution microscopy. This work shows that Affimer proteins, as is the case for other alternative binding scaffolds, represent complementary affinity reagents to antibodies for various molecular and cell biology applications. DOI: http://dx.doi.org/10.7554/eLife.24903.001

Inhibition of complement C3 and fibrinogen interaction: a potential novel therapeutic target to reduce cardiovascular disease in diabetes.

BACKGROUND Enhanced complement C3 incorporation into the fibrin network in diabetes is one mechanism for impaired fibrinolysis and increased thrombosis risk in this condition. Our aim was to develop new strategies to modulate fibrinolysis in diabetes by interfering with fibrin-C3 interaction. METHODS To modulate interaction between fibrinogen and C3 we used a novel technique by screening fibrinogen with a phage display library of 3 billion random, conformational 9AA peptides (termed adhirons). The effect of high affinity fibrinogen binding adhirons, released by the addition of excess C3, on fibrin clot lysis and structure was assessed in turbidimetric assays. Fibrinogen-C3 interactions were further studied by peptide microarray techniques and modelled with the website PepSite2. FINDINGS Ten high affinity fibrinogen binding adhirons, released by C3, were available for turbidimetric analysis. One adhiron (A6) was found to have a sequence homology with C3 and studied further. In the absence of C3, adhiron A6 failed to modulate fibrin clot lysis time (mean 644 s [SE 13] and 620 [14] without and with adhiron A6, respectively). However, adhiron A6 abolished C3-induced prolongation of clot lysis, reducing mean lysis time from 728 s (SE 25) to 632 (24) (p=0·01). The peptide microarray screening of C3 identified two peptide motifs within the β chain of fibrinogen (residues 424-433, 435-445) that bound to C3. PepSite2 predicted that adhiron A6 binds to similar areas on the β chain of fibrinogen. INTERPRETATION Using a novel phage display system, we discovered an adhiron that shared sequence homology with C3 and abolished C3-induced prolongation of fibrin clot lysis by interfering with C3-fibrinogen interaction within the β chain. This technique offers a unique method to identify new therapeutic targets for the reduction of diabetes-specific thrombosis risk. FUNDING Sir Jules Thorn Charitable Trust.

Generation of specific inhibitors of SUMO-1– and SUMO-2/3–mediated protein-protein interactions using Affimer (Adhiron) technology

Artificial binding proteins are tools for exploring cellular processes dependent on SUMOylation. Artificial proteins target SUMO SUMOylation is the covalent attachment of SUMO-1, SUMO-2, SUMO-3, or combinations thereof to target proteins to control protein function and localization. Hughes et al. screened a library of artificial proteins called Affimers to identify those that bound to SUMO-1 or SUMO-2 (which is nearly identical to SUMO-3) and incorporated a negative selection step to remove SUMO-2–binding Affimers that also bound to SUMO-1. The authors identified Affimers that recognized SUMO-1, SUMO-2 and SUMO-3 (SUMO-2/3), or all three isoforms. Biochemical and cellular assays showed that these SUMO-specific Affimers (S-Affs) did not interfere with SUMO conjugation or deconjugation but inhibited a cellular stress response that required SUMO-mediated protein-protein interactions. In addition to generating S-Affs that will be useful tools for studying SUMO-dependent cellular processes, this study also shows the applicability of this technology for generating reagents that interfere with specific protein-protein interactions for basic research and potentially for clinical development (see the Protocol by Tang et al.). Because protein-protein interactions underpin most biological processes, developing tools that target them to understand their function or to inform the development of therapeutics is an important task. SUMOylation is the posttranslational covalent attachment of proteins in the SUMO family (SUMO-1, SUMO-2, or SUMO-3), and it regulates numerous cellular pathways. SUMOylated proteins are recognized by proteins with SUMO-interaction motifs (SIMs) that facilitate noncovalent interactions with SUMO. We describe the use of the Affimer system of peptide display for the rapid isolation of synthetic binding proteins that inhibit SUMO-dependent protein-protein interactions mediated by SIMs both in vitro and in cells. Crucially, these synthetic proteins did not prevent SUMO conjugation either in vitro or in cell-based systems, enabling the specific analysis of SUMO-mediated protein-protein interactions. Furthermore, through structural analysis and molecular modeling, we explored the molecular mechanisms that may underlie their specificity in interfering with either SUMO-1–mediated interactions or interactions mediated by either SUMO-2 or SUMO-3. Not only will these reagents enable investigation of the biological roles of SUMOylation, but the Affimer technology used to generate these synthetic binding proteins could also be exploited to design or validate reagents or therapeutics that target other protein-protein interactions.

Antibody Mimetics for the Detection of Small Organic Compounds Using a Quartz Crystal Microbalance

Conventional immunoassays rely on antibodies that provide high affinity, specificity, and selectivity against a target analyte. However, the use of antibodies for the detection of small-sized, nonimmunogenic targets, such as pharmaceuticals and environmental contaminants, presents a number of challenges. Recent advances in protein engineering have led to the emergence of antibody mimetics that offer the high affinity and specificity associated with antibodies, but with reduced batch-to-batch variability, high stability, and in vitro selection to ensure rapid discovery of binders against a wide range of targets. In this work we explore the potential of Affimers, a recent example of antibody mimetics, as suitable bioreceptors for the detection of small organic target compounds, here methylene blue. Target immobilization for Affimer characterization was achieved using long-chained alkanethiol linkers coupled with oligoethylene glycol (LCAT-OEG). Using quartz crystal microbalance with dissipation monitoring (QCM-D), we determine the affinity constant, KD, of the methylene blue Affimer to be comparable to that of antibodies. Further, we demonstrate the high selectivity of Affimers for its target in complex matrixes, here a limnetic sample. Finally, we demonstrate an Affimer-based competition assay, illustrating the potential of Affimers as bioreceptors in immunoassays for the detection of small-sized, nonimmunogenic compounds.

Ultraefficient Cap-Exchange Protocol To Compact Biofunctional Quantum Dots for Sensitive Ratiometric Biosensing and Cell Imaging

An ultraefficient cap-exchange protocol (UCEP) that can convert hydrophobic quantum dots (QDs) into stable, biocompatible, and aggregation-free water-dispersed ones at a ligand:QD molar ratio (LQMR) as low as 500, some 20–200-fold less than most literature methods, has been developed. The UCEP works conveniently with air-stable lipoic acid (LA)-based ligands by exploiting tris(2-carboxylethyl phosphine)-based rapid in situ reduction. The resulting QDs are compact (hydrodynamic radius, Rh, < 4.5 nm) and bright (retaining > 90% of original fluorescence), resist nonspecific adsorption of proteins, and display good stability in biological buffers even with high salt content (e.g., 2 M NaCl). These advantageous properties make them well suited for cellular imaging and ratiometric biosensing applications. The QDs prepared by UCEP using dihydrolipoic acid (DHLA)-zwitterion ligand can be readily conjugated with octa-histidine (His8)-tagged antibody mimetic proteins (known as Affimers). These QDs allow rapid, ratiometric detection of the Affimer target protein down to 10 pM via a QD-sensitized Förster resonance energy transfer (FRET) readout signal. Moreover, compact biotinylated QDs can be readily prepared by UCEP in a facile, one-step process. The resulting QDs have been further employed for ratiometric detection of protein, exemplified by neutravidin, down to 5 pM, as well as for fluorescence imaging of target cancer cells.

Affimer proteins inhibit immune complex binding to FcγRIIIa with high specificity through competitive and allosteric modes of action

Significance Autoimmune disease pathogenesis is driven by inflammation, induced partly by IgG autoantibody-containing immune complexes binding to Fc gamma receptors (FcγRs). These receptors are valid therapeutic targets in the treatment of autoimmunity. FcγRIIIa is one of a family of highly homologous receptors for IgG antibodies; previous attempts at therapeutic blockade have resulted in off-target effects involving cells that express the almost identical protein FcγRIIIb. Here we report the identification of functionally specific protein-based inhibitors (Affimer proteins) of FcγRIIIa and the structural/functional basis of their selectivity. As molecular research tools FcγRIIIa-specific Affimer proteins provide the ability to block IgG interaction with a single receptor. Our findings suggest that highly selective protein-based blocking agents that may have therapeutic applications can be readily produced. Protein–protein interactions are essential for the control of cellular functions and are critical for regulation of the immune system. One example is the binding of Fc regions of IgG to the Fc gamma receptors (FcγRs). High sequence identity (98%) between the genes encoding FcγRIIIa (expressed on macrophages and natural killer cells) and FcγRIIIb (expressed on neutrophils) has prevented the development of monospecific agents against these therapeutic targets. We now report the identification of FcγRIIIa-specific artificial binding proteins called “Affimer” that block IgG binding and abrogate FcγRIIIa-mediated downstream effector functions in macrophages, namely TNF release and phagocytosis. Cocrystal structures and molecular dynamics simulations have revealed the structural basis of this specificity for two Affimer proteins: One binds directly to the Fc binding site, whereas the other acts allosterically.

Alternative reagents to antibodies in imaging applications

Antibodies have been indispensable tools in molecular biology, biochemistry and medical research. However, a number of issues surrounding validation, specificity and batch variation of commercially available antibodies have prompted research groups to develop novel non-antibody binding reagents. The ability to select highly specific monoclonal non-antibody binding proteins without the need for animals, the ease of production and the ability to site-directly label has enabled a wide variety of applications to be tested, including imaging. In this review, we discuss the success of a number of non-antibody reagents in imaging applications, including the recently reported Affimer.

Sensitive and selective Affimer-functionalised interdigitated electrode-based capacitive biosensor for Her4 protein tumour biomarker detection

A novel Affimer-functionalised interdigitated electrode-based capacitive biosensor platform was developed for detection and estimation of Her4, a protein tumour biomarker, in undiluted serum. An anti-Her4 Affimer with a C-terminal cysteine was used to create the bio-recognition layer via self-assembly on gold interdigitated electrodes for the sensor fabrication. Electrochemical impedance spectroscopy (EIS) in the absence of redox markers was used to evaluate the sensor performance by monitoring the changes in capacitance. The Affimer sensor in buffer and in undiluted serum demonstrated high sensitivity with a broad dynamic range from 1 pM to 100 nM and a limit of detection lower than 1 pM both in buffer and in serum. Furthermore, the Affimer sensor demonstrated excellent specificity with negligible interference from serum proteins, suggesting resilience to non-specific binding. The sensing ability of the present Affimer sensor in spiked undiluted serum suggests its potential for a new range of Affimer-based sensors. The fabricated Affimer sensor can thus be further adapted with other probes having affinities to other biomarkers for a new range of biosensors.

Development of an Affimer-antibody combined immunological diagnosis kit for glypican-3

Glypican-3 (GPC3) is a promising new marker for hepatocellular carcinoma, but the reported values for serum GPC3 differ markedly between currently available kits. Here we isolated Affimer non-antibody binding proteins against GPC3 by phage display and developed a new sandwich chemiluminescence immunoassay (CLIA) combining an Affimer with a monoclonal antibody (Affimer-MAb CLIA). The proposed CLIA assay demonstrated a wide linear range  0.03–600 ng/mL) with a good linear correlation coefficient (0.9999), a high detection limitation (0.03 ng/mL) and specificity (0–0.002%) for detection of GPC3. The accuracy, hook effect and stability were demonstrated to be satisfactory. The mean level of GPC3 in serum was higher (>8.5 fold, P < 0.001) in hepatocellular carcinoma patients compared to healthy and other liver disease individuals. A poor correlation (correlation coefficients ranged from −0.286 to 0.478) was observed through pairwise comparison within different kits. However, only this newly developed CLIA test showed high specificity and correlated with the “gold standard” GPC3-immunohistochemistry. This study indicates that Affimer-MAb CLIA can be used to generate a sensitive immunodiagnostic kit, which offers the potential for a highly specific clinically-relevant detection system.