Cell-targeted PD-1 agonists that mimic PD-L1 are potent T cell inhibitors.

The PD-1/PD-L1 pathway is a key immune checkpoint that regulates T cell activation. There is strong rationale to develop PD-1 agonists as therapeutics against autoimmunity, but progress in this area has been limited. Here, we generated T cell receptor (TCR) targeting, PD-1 agonist bispecifics called ImmTAAI molecules that mimic the ability of PD-L1 to facilitate the colocalization of PD-1 with the TCR complex at the target cell-T cell interface. PD-1 agonist ImmTAAI molecules specifically bound to target cells and were highly effective in activating the PD-1 receptor on interacting T cells to achieve immune suppression. Potent PD-1 antibody ImmTAAI molecules closely mimicked the mechanism of action of endogenously expressed PD-L1 in their localization to the target cell-T cell interface, inhibition of proximal TCR signaling events, and suppression of T cell function. At picomolar concentrations, these bispecifics suppressed cytokine production and inhibited CD8+ T cell-mediated cytotoxicity in vitro. Crucially, in soluble form, the PD-1 ImmTAAI molecules were inactive and, hence, could avoid systemic immunosuppression. This study outlines a promising new route to generate more effective, potent, tissue-targeted PD-1 agonists that can inhibit T cell function locally with the potential to treat autoimmune and chronic inflammatory diseases of high unmet need.

Measuring Antibody-Antigen Binding Kinetics Using Surface Plasmon Resonance.

Surface plasmon resonance (SPR) is now widely embraced as a technology for monitoring a diverse range of protein-protein interactions and is considered almost de rigueur for characterizing antibody-antigen interactions. The technique obviates the need to label either of the interacting species, and the binding event is visualized in real time. Thus, it is ideally suited for screening crude, unpurified antibody samples that dominate early candidate panels following antibody selection campaigns. SPR returns not only concentration and affinity data but when used correctly can resolve the discrete component kinetic parameters (association and dissociation rate constants) of the affinity interaction. Herein, we outline some SPR-based generic antibody screening configurations and methodologies in the context of expediting data-rich ranking of candidate antibody panels and ensuring that antibodies with the optimal kinetic binding characteristics are reliably identified.

Decoding Selection Bias Imparted by Unpaired Cysteines: a Tug of War Between Expression and Affinity.

In a recombinant antibody scFv format, the presence of an unpaired cysteine (Cys) is implicated in reduced soluble expression and inefficient presentation in phage display. Compared to other species, antibodies derived from rabbits are more likely to contain this unpaired Cys residue at position 80 (Cys80), when generated in a scFv format. In a screening campaign to isolate rabbit scFv against cardiac troponin I (cTnI), it was found that, a large proportion of isolated cTnI-specific clones contained unpaired Cys80. To analyze the factors that led to the selection of anti-cTnI Cys80 scFv, after five rounds of biopanning, the biopanning experiments were repeated with a Cys80 scFv (MG4Cys), its alanine variant (MG4Ala), and an irrelevant high expressing scFv control. It was found that the selection and subsequent enrichment of MG4Cys scFv was ousted by the superior expressing variant MG4Ala, indicating that the Cys80 scFv was selected primarily due to its affinity. It is evident that phage-based selection is influenced by specific sequence characteristics affecting the expression as well as the binding specificity and this needs to be taken into account for selection of optimal antibody derivatives.

Measuring Protein-Protein Interactions Using Biacore.

The use of optical biosensors for studying macromolecular interactions is gaining increasing popularity. In one study, 1514 papers that involved the application of biosensor data were identified for the year 2009 alone (Rich and Myszka, J Mol Recognit 24:892-914, 2011), the sheer volume and variety of which present a daunting task for the burgeoning biosensor user to accumulate and decipher. This chapter is designed to provide the reader with the tools necessary to prepare, design, and efficiently execute a kinetic experiment on Biacore. It is written to guide the Biacore user through basic theory, system maintenance, and assay setup while also offering some practical tips that we find useful for Biacore-based studies. Many kinetic-based screening assays require rigorous sample preparation and purification prior to analysis. To highlight these procedures, this protocol describes the kinetic characterization of single chain Fv (scFv) antibody fragments from crude bacterial lysates using an antibody affinity capture approach. Even though we specifically describe the capture of HA-tagged scFv antibody fragments to an anti-HA tag monoclonal antibody-immobilized surface prior to kinetic analysis, the same methodologies are universally applicable and can be used for practically any affinity pair and most Biacore systems.

Facile domain rearrangement abrogates expression recalcitrance in a rabbit scFv.

Rabbit-derived recombinant antibodies have traditionally been viewed as intractable molecules due to the presence of a cysteine in position 80 of the VL domain that becomes rendered 'aberrant' when present in the 'unpaired' context of a single chain Fv (scFv) and chimeric Fab formats. This aberrant Cys80 can severely impinge on the achievable expression levels when rabbit recombinant antibodies are produced in prokaryote systems. The unpaired Cys residue also renders purification problematic. Consequently, researchers often disregard rabbit antibody libraries due to perceived limitations in accessible repertoire diversity. We have shown that by switching the orientation of the VH and VL domains in an aberrant-Cys-containing rabbit scFv isolated in a bona fide screening campaign, it was possible to substantially increase the expression and purification yields of this clone. Furthermore, by incorporating a novel rabbit C-kappa constant fusion domain, we were able to potentiate a further increase in expression level and purify this antibody to a high degree of homogeneity, hitherto impossible to achieve using the aberrant-Cys-containing wild-type scFv. Cumulatively, these findings demonstrate that facile re-formatting can help make the rabbit antibody repertoire, a very valuable resource, more accessible to researchers in the field.

Targeted delivery of a SNARE protease to sensory neurons using a single chain antibody (scFv) against the extracellular domain of P2X(3) inhibits the release of a pain mediator.

P2X3 (P2X purinoceptor 3) is predominantly expressed on nociceptive sensory neurons and plays a crucial role in signalling leading to chronic inflammatory pain and some features of neuropathic pain. Thus it represents a potential target for pain therapeutics. BoNT/A (botulinum neurooxin type A) effectively relieves certain types of pain through inhibiting the neuronal release of pain peptides. A recombinant single-chain variable fragment (scFv) antibody designated MH7C was generated against the extracellular domain of P2X3 using phage display. The genes encoding the scFv and activated di-chain form of BoNT/A without the C-terminal-binding subdomain (LC-HN-HCN/A) were ligated and expressed in Escherichia coli cells as a composite fusion protein. The purified protein bound and entered P2X3-containing sensory neurons, cleaved synaptosomal-associated protein of 25 kDa and inhibited the release of a pain peptide. This novel fusion protein designated 'LC-HN-HCN/A-MH7C' has potential clinical applications in the treatment of chronic inflammatory and sympathetically maintained neuropathic pain.

Reconciling the structural attributes of avian antibodies.

Antibodies are high value therapeutic, diagnostic, biotechnological, and research tools. Combinatorial approaches to antibody discovery have facilitated access to unique antibodies by surpassing the diversity limitations of the natural repertoire, exploitation of immune repertoires from multiple species, and tailoring selections to isolate antibodies with desirable biophysical attributes. The V-gene repertoire of the chicken does not utilize highly diverse sequence and structures, which is in stark contrast to the mechanism employed by humans, mice, and primates. Recent exploitation of the avian immune system has generated high quality, high affinity antibodies to a wide range of antigens for a number of therapeutic, diagnostic and biotechnological applications. Furthermore, extensive examination of the amino acid characteristics of the chicken repertoire has provided significant insight into mechanisms employed by the avian immune system. A paucity of avian antibody crystal structures has limited our understanding of the structural consequences of these uniquely chicken features. This paper presents the crystal structure of two chicken single chain fragment variable (scFv) antibodies generated from large libraries by phage display against important human antigen targets, which capture two unique CDRL1 canonical classes in the presence and absence of a non-canonical disulfide constrained CDRH3. These structures cast light on the unique structural features of chicken antibodies and contribute further to our collective understanding of the unique mechanisms of diversity and biochemical attributes that render the chicken repertoire of particular value for antibody generation.

A highly stable, sensitive, regenerable and rapid immunoassay for detecting aflatoxin B1 in corn incorporating covalent AFB1 immobilization and a recombinant Fab antibody.

A highly robust immunoassay applicable for the detection of aflatoxin B1 (AFB1) using a Fab antibody fragment was developed. A key factor was the use of covalently immobilized AFB1 which allowed an almost three fold increase in sensitivity, reduced assay time and regeneration with retention of binding capacity. Various factors that might affect the sensitivity of the assay such as pH, organic solvents, storage stability and wash stringency were critically evaluated. It was also demonstrated that the assay was applicable for determination of AFB1 in corn samples at concentration within the European union regulatory limits.

At-line bioprocess monitoring by immunoassay with rotationally controlled serial siphoning and integrated supercritical angle fluorescence optics.

In this paper we report a centrifugal microfluidic "lab-on-a-disc" system for at-line monitoring of human immunoglobulin G (hIgG) in a typical bioprocess environment. The novelty of this device is the combination of a heterogeneous sandwich immunoassay on a serial siphon-enabled microfluidic disc with automated sequential reagent delivery and surface-confined supercritical angle fluorescence (SAF)-based detection. The device, which is compact, easy-to-use and inexpensive, enables rapid detection of hIgG from a bioprocess sample. This was achieved with, an injection moulded SAF lens that was functionalized with aminopropyltriethoxysilane (APTES) using plasma enhanced chemical vapour deposition (PECVD) for the immobilization of protein A, and a hybrid integration with a microfluidic disc substrate. Advanced flow control, including the time-sequenced release of on-board liquid reagents, was implemented by serial siphoning with ancillary capillary stops. The concentration of surfactant in each assay reagent was optimized to ensure proper functioning of the siphon-based flow control. The entire automated microfluidic assay process is completed in less than 30 min. The developed prototype system was used to accurately measure industrial bioprocess samples that contained 10 mg mL(-1) of hIgG.

Measuring antibody-antigen binding kinetics using surface plasmon resonance.

Surface plasmon resonance (SPR) is now widely embraced as a technology for monitoring a diverse range of protein-protein interactions and is considered almost de rigueur for characterizing antibody-antigen interactions. The technique obviates the need to label either of the interacting species and the binding event is visualized in real-time. Thus, it is ideally suited for screening crude, unpurified antibody samples that dominate early candidate panels following antibody selection campaigns. SPR returns both concentration and affinity data but when used correctly can also resolve the discrete component kinetic parameters (association and dissociation rate constants) of the affinity interaction. Herein, we outline some SPR-based generic antibody screening configurations and methodologies in the context of expediting data-rich ranking of candidate antibody panels and ensuring that antibodies with the optimal kinetic binding characteristics are reliably identified.

Exploiting recombinant antibodies in point-of-care (POC) diagnostics: the combinatorial advantage.

Antibodies are ubiquitously deployed on in vitro diagnostic (IVD) platforms for detecting a panoply of analytes indicative of environmental and food contamination, residue adulteration and both veterinary and medical diagnostics. In the clinical realm, rapid and accurate determination of disease status is paramount. The significance of immunodiagnostic performance cannot be overemphasized and in many cases reliable diagnosis informs medical intervention which can mean the difference between patient recovery and demise. Cardiovascular disease (CVD) is the single biggest cause of adult mortality in the western world and principal burden on the healthcare services. Although the troponin (Tn) family, in particular troponin I (TnI), are regarded as the gold standard for diagnosis of myocardial damage, over the last decade much research has focused on the identification of alternative cardiac biomarker molecules that can either supplant or complement TnI metrics to add value to cardiac risk stratification criteria. In particular, markers that appear earlier than TnI in the pathophyisiology of cardiac disease are highly sought after. The subject of this addendum represents part of a broader challenge to deliver novel rapid point-of-care (POC) diagnostics to provide a chip-based multi-plexed platform for more comprehensive profiling of cardiac status with additive diagnostic and prognostic value. Specifically, it outlines proof-of-concept direct myeloperoxidase (MPO) detection, demonstrates the benefits of using recombinant antibodies in POC diagnostics and describes optimized strategies for generation of superior candidate antibody panels. 

Novel disposable biochip platform employing supercritical angle fluorescence for enhanced fluorescence collection.

This paper presents an overview of development of a novel disposable plastic biochip for multiplexed clinical diagnostic applications. The disposable biochip is manufactured using a low-cost, rapid turn- around injection moulding process and consists of nine parabolic elements on a planar substrate. The optical elements are based on supercritical angle fluorescence (SAF) which provides substantial enhancement of the fluorescence collection efficiency but also confines the fluorescence detection volume strictly to the immediate proximity of the biochip surface, thereby having the potential to discriminate against background fluorescence from the analyte solution. An optical reader is also described that enables interrogation and fluorescence collection from the nine optical elements on the chip. The sensitivity of the system was determined with a biotin-avidin assay while its clinical utility was demonstrated in an assay for C-reactive protein (CRP), an inflammation marker.

Kinetics of immunoassays with particles as labels: effect of antibody coupling using dendrimers as linkers.

In this article, we report on poly(amidoamine) dendrimers (PAMAM) as coupling agents for recombinant single-chain (ScFv) antibodies to nanoparticle (NP) labels, for use in immunoassay. We present a simple theory for the kinetics of particle capture onto a surface by means of an antibody-antigen reaction, in which the important parameter is the fraction of the particle surface that is active for reaction. We describe how increasing the generation number of the linking dendrimers significantly increased the fraction of the NP surface that is active for antigen binding and consequently also increased the assay kinetic rates. Use of dendrimers for conjugation of the NP to the antibody resulted in a significantly higher surface coverage of active antibody, in comparison with mono-valent linker chemistry. As a direct consequence, the increase in effective avidity significantly out-weighed any effect of a decreased diffusion coefficient due to the NP, when compared to that of a molecular dye-labelled antibody. The signal to noise ratio of the G4.5 dendrimer-sensitised nanoparticles out-performed the dye-labelled antibody by approximately four-fold. Particle aggregation experiments with the multi-valent antigen CRP demonstrated reaction-limited aggregation whose rate increased significantly with increasing generation number of the dendrimer linker.

A high-affinity recombinant antibody permits rapid and sensitive direct detection of myeloperoxidase.

Over the past 10 years, a growing field of research supporting the value of myeloperoxidase (MPO) as a prognostic indicator in acute cardiac pathophysiologies has emerged. The availability of a rapid and disposable MPO detection platform would enable research clinicians to more readily assess MPO indications for guiding therapy and also facilitate clinicians at the patient interface to readily adopt MPO testing and potentially drive more informed prognoses. Here we describe the isolation of a high-affinity avian MPO-specific recombinant antibody panel using phage display. Rapid isolation of a suitable single-chain variable fragment (scFv) antibody was facilitated using a surface plasmon resonance (SPR)-based "off-rate ranking" screening process. The selected scFv was then successfully incorporated into a rapid, simple, and sensitive one-step lateral flow immunoassay (LFIA) for the detection of MPO. This "one-step" feature of the developed assay was made possible by the scFv's strong affinity for MPO, obviating the need for sandwich signal enhancement steps. The assay's rapid performance was also further enhanced by exploiting the intrinsic enzymatic properties of MPO in its final detection. Use of the optimized LFIA facilitated the sensitive detection of MPO in MPO-depleted serum within clinically relevant reference ranges.

Measuring protein-protein interactions using Biacore.

The use of optical biosensors for studying macromolecular interactions is gaining increasing popularity. In one study, 1,179 papers that involved the application of biosensor data were identified for the year 2007 alone (Rich and Myszka, J Mol Recognit 21:355-400, 2008), the sheer volume and variety of which present a daunting task for the burgeoning biosensor user to accumulate and decipher. This chapter is designed to provide the reader with the tools necessary to prepare, design, and efficiently execute a kinetic experiment on Biacore. It is written to guide the Biacore user through basic theory, system maintenance, and assay set-up while also offering some practical tips that we find useful for Biacore-based studies. Many kinetic-based screening assays require rigorous sample preparation and purification prior to analysis. To highlight these procedures, this protocol describes the kinetic characterisation of single chain Fv (scFv) antibody fragments from crude bacterial lysates using an antibody affinity capture approach. Even though we specifically describe the capture of HA-tagged scFv antibody fragments to an anti-HA tag monoclonal antibody-immobilised surface prior to kinetic analysis, the same methodologies are universally applicable and can be used for practically any affinity pair and most Biacore systems.

Highly sensitive recombinant antibodies capable of reliably differentiating heart-type fatty acid binding protein from noncardiac isoforms.

During recent times, heart-type fatty acid binding protein (hFABP) has gained increasing credence as a promising cardiac biomarker. This is largely due to its rapid myocardial release and subsequent clearance kinetics, which are superior to those of myoglobin and offer an earlier diagnostic window than the troponins. Realization of its full diagnostic and prognostic potential is dependent on accessibility to robust hFABP-specific assays. Here we describe a rational strategy for generation and screening of hFABP-specific avian-derived recombinant antibodies. These antibodies were confirmed to be exquisitely specific for hFABP, with no cross-reactivity observed in a representative panel of the most homologous non-heart-type FABP isoforms. All of the antibodies tested exhibited single-figure nanomolar affinities, and their analytical potential was demonstrated in a simple inhibition enzyme-linked immunosorbent assay (ELISA) format that returned an impressive limit of quantitation (LOQ) value of 1.9 ng/ml. The cumulative results underline the potential value of these antibodies as enabling reagents for use in a variety of immunodiagnostic configurations.

Surface plasmon resonance for vaccine design and efficacy studies: recent applications and future trends.

The lack of a clear correlation between design and protection continues to present a barrier to progress in vaccine research. In this article, we outline how surface plasmon resonance (SPR) biosensors are emerging as tools to help resolve some of the key biophysical determinants of protection and, thereby, facilitate more rational vaccine design campaigns. SPR technology has contributed significantly to our understanding of the complex biophysical determinants of HIV neutralization and offers a platform for preclinical evaluation of vaccine candidates. In particular, the concept of reverse-engineering HIV vaccine targets based on known broadly neutralizing antibody modalities is explored and extended to include other infectious diseases, such as malaria and influenza, and other diseases such as cancer. The analytical capacity afforded by SPR includes serum screening to monitor immune responses and highly efficient quality-control surveillance measures. These are discussed alongside key technological advances, such as developments in sample throughput, and a perspective predicting continued growth and diversification of the role of SPR in vaccine development is proposed.

Antibody production, design and use for biosensor-based applications.

Currently, the reliable detection and quantification of a multitude of different analytes is crucial in many applications and settings. Biosensors have revolutionised diagnostics for use in point-of-care testing (POC), the detection of food and environmental contaminants, biological warfare agents, illicit drugs and human/animal disease markers. Antibodies continue to play a pivotal role in many sensor devices due to their exquisite specificity for their cognate antigens. In this review current biosensor platforms employing antibodies for molecular recognition are briefly described. The use of molecular biological techniques for the generation and improvement of antibodies is critically examined. Such recombinant antibodies possess improved attributes for use in biosensor development in terms of design, stability, affinity and specificity.

Cardiac biomarkers and the case for point-of-care testing.

Cardiovascular disease (CVD) is the single greatest cause of adult mortality in the western world and, consequently, places a massive burden on healthcare services and the economy. Lifestyles, lack of clearly defined risk assessment criteria, consistently high incidences of misdiagnosis and inappropriate referrals, all contribute significantly to this problem. It also correlates directly with inefficient or non-accessible early detection systems. Over the last decade much research has focused on the identification of cardiac biomarkers that can be used for the detection of cardiac distress and that add value to current risk stratification criteria. An exposition of some of the most consistently cited biomarkers is provided and their current status and potential value as early CVD risk predictors, more accurate diagnostic markers of acute myocardial damage and as reliable prognostic indicators, is evaluated. The particular importance of early prediction and the integral role that point-of-care (POC) testing is expected to play in the future of cardiac care is critically discussed.