Exploiting Nanobodies' Singular Traits.

The unique class of heavy chain-only antibodies, present in Camelidae, can be shrunk to just the variable region of the heavy chain to yield VHHs, also called nanobodies. About one-tenth the size of their full-size counterparts, nanobodies can serve in applications similar to those for conventional antibodies, but they come with a number of signature advantages that find increasing application in biology. They not only function as crystallization chaperones but also can be expressed inside cells as such, or fused to other proteins to perturb the function of their targets, for example, by enforcing their localization or degradation. Their small size also affords advantages when applied in vivo, for example, in imaging applications. Here we review such applications, with particular emphasis on those areas where conventional antibodies would face a more challenging environment.

Structural insight into SARS-CoV-2 neutralizing antibodies and modulation of syncytia.

Infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is initiated by binding of the viral Spike protein to host receptor angiotensin-converting enzyme 2 (ACE2), followed by fusion of viral and host membranes. Although antibodies that block this interaction are in emergency use as early coronavirus disease 2019 (COVID-19) therapies, the precise determinants of neutralization potency remain unknown. We discovered a series of antibodies that potently block ACE2 binding but exhibit divergent neutralization efficacy against the live virus. Strikingly, these neutralizing antibodies can inhibit or enhance Spike-mediated membrane fusion and formation of syncytia, which are associated with chronic tissue damage in individuals with COVID-19. As revealed by cryoelectron microscopy, multiple structures of Spike-antibody complexes have distinct binding modes that not only block ACE2 binding but also alter the Spike protein conformational cycle triggered by ACE2 binding. We show that stabilization of different Spike conformations leads to modulation of Spike-mediated membrane fusion with profound implications for COVID-19 pathology and immunity.

High-resolution profiling of pathways of escape for SARS-CoV-2 spike-binding antibodies.

Defining long-term protective immunity to SARS-CoV-2 is one of the most pressing questions of our time and will require a detailed understanding of potential ways this virus can evolve to escape immune protection. Immune protection will most likely be mediated by antibodies that bind to the viral entry protein, spike (S). Here, we used Phage-DMS, an approach that comprehensively interrogates the effect of all possible mutations on binding to a protein of interest, to define the profile of antibody escape to the SARS-CoV-2 S protein using coronavirus disease 2019 (COVID-19) convalescent plasma. Antibody binding was common in two regions, the fusion peptide and the linker region upstream of the heptad repeat region 2. However, escape mutations were variable within these immunodominant regions. There was also individual variation in less commonly targeted epitopes. This study provides a granular view of potential antibody escape pathways and suggests there will be individual variation in antibody-mediated virus evolution.

Antigen-level resolution of commensal-specific B cell responses can be enabled by phage display screening coupled with B cell tetramers.

Induction of commensal-specific immunity contributes to tissue homeostasis, yet the mechanisms underlying induction of commensal-specific B cells remain poorly understood in part due to a lack of tools to identify these cells. Using phage display, we identified segmented filamentous bacteria (SFB) antigens targeted by serum and intestinal antibodies and generated B cell tetramers to track SFB-specific B cells in gut-associated lymphoid tissues. We revealed a compartmentalized response in SFB-specific B cell activation, with a gradient of immunoglobulin A (IgA), IgG1, and IgG2b isotype production along Peyer's patches contrasted by selective production of IgG2b within mesenteric lymph nodes. V(D)J sequencing and monoclonal antibody generation identified somatic hypermutation driven affinity maturation to SFB antigens under homeostatic conditions. Combining phage display and B cell tetramers will enable investigation of the ontogeny and function of commensal-specific B cell responses in tissue immunity, inflammation, and repair.

Allergenic food protein consumption is associated with systemic IgG antibody responses in non-allergic individuals.

Although food-directed immunoglobulin E (IgE) has been studied in the context of allergies, the prevalence and magnitude of IgG responses against dietary antigens are incompletely characterized in the general population. Here, we measured IgG binding against food and environmental antigens obtained from allergen databases and the immune epitope database (IEDB), represented in a phage displayed library of 58,233 peptides. By profiling blood samples of a large cohort representing the average adult Israeli population (n = 1,003), we showed that many food antigens elicited systemic IgG in up to 50% of individuals. Dietary intake of specific food protein correlated with antibody binding, suggesting that diet can shape the IgG epitope repertoire. Our work documents abundant systemic IgG responses against food antigens and provides a reference map of the exact immunogenic epitopes on a population scale, laying the foundation to unravel the role of food- and environmental antigen-directed antibody binding in disease contexts.

Selection-based discovery of druglike macrocyclic peptides.

Macrocyclic peptides are an emerging class of therapeutics that can modulate protein-protein interactions. In contrast to the heavily automated high-throughput screening systems traditionally used for the identification of chemically synthesized small-molecule drugs, peptide-based macrocycles can be synthesized by ribosomal translation and identified using in vitro selection techniques, allowing for extremely rapid (hours to days) screening of compound libraries comprising more than 10(13) different species. Furthermore, chemical modification of translated peptides and engineering of the genetic code have greatly expanded the structural diversity of the available peptide libraries. In this review, we discuss the use of these technologies for the identification of bioactive macrocyclic peptides, emphasizing recent developments.

Systematic Discovery of Short Linear Motifs Decodes Calcineurin Phosphatase Signaling.

Short linear motifs (SLiMs) drive dynamic protein-protein interactions essential for signaling, but sequence degeneracy and low binding affinities make them difficult to identify. We harnessed unbiased systematic approaches for SLiM discovery to elucidate the regulatory network of calcineurin (CN)/PP2B, the Ca2+-activated phosphatase that recognizes LxVP and PxIxIT motifs. In vitro proteome-wide detection of CN-binding peptides, in vivo SLiM-dependent proximity labeling, and in silico modeling of motif determinants uncovered unanticipated CN interactors, including NOTCH1, which we establish as a CN substrate. Unexpectedly, CN shows SLiM-dependent proximity to centrosomal and nuclear pore complex (NPC) proteins-structures where Ca2+ signaling is largely uncharacterized. CN dephosphorylates human and yeast NPC proteins and promotes accumulation of a nuclear transport reporter, suggesting conserved NPC regulation by CN. The CN network assembled here provides a resource to investigate Ca2+ and CN signaling and demonstrates synergy between experimental and computational methods, establishing a blueprint for examining SLiM-based networks.

A general approach for selection of epitope-directed binders to proteins.

Directing antibodies to a particular epitope among many possible on a target protein is a significant challenge. Here, we present a simple and general method for epitope-directed selection (EDS) using a differential phage selection strategy. This involves engineering the protein of interest (POI) with the epitope of interest (EOI) mutated using a systematic bioinformatics algorithm to guide the local design of an EOI decoy variant. Using several alternating rounds of negative selection with the EOI decoy variant followed by positive selection on the wild-type POI, we were able to identify highly specific and potent antibodies to five different EOI antigens that bind and functionally block known sites of proteolysis. Among these, we developed highly specific antibodies that target the proteolytic site on the CUB domain containing protein 1 (CDCP1) to prevent its proteolysis allowing us to study the cellular maturation of this event that triggers malignancy. We generated antibodies that recognize the junction between the pro- and catalytic domains for three different matrix metalloproteases (MMPs), MMP1, MMP3, and MMP9, that selectively block activation of each of these enzymes and impair cell migration. We targeted a proteolytic epitope on the cell surface receptor, EPH Receptor A2 (EphA2), that is known to transform it from a tumor suppressor to an oncoprotein. We believe that the EDS method greatly facilitates the generation of antibodies to specific EOIs on a wide range of proteins and enzymes for broad therapeutic and diagnostic applications.

Ceramide as an endothelial cell surface receptor and a lung-specific lipid vascular target for circulating ligands.

The vascular endothelium from individual organs is functionally specialized, and it displays a unique set of accessible molecular targets. These serve as endothelial cell receptors to affinity ligands. To date, all identified vascular receptors have been proteins. Here, we show that an endothelial lung-homing peptide (CGSPGWVRC) interacts with C16-ceramide, a bioactive sphingolipid that mediates several biological functions. Upon binding to cell surfaces, CGSPGWVRC triggers ceramide-rich platform formation, activates acid sphingomyelinase and ceramide production, without the associated downstream apoptotic signaling. We also show that the lung selectivity of CGSPGWVRC homing peptide is dependent on ceramide production in vivo. Finally, we demonstrate two potential applications for this lipid vascular targeting system: i) as a bioinorganic hydrogel for pulmonary imaging and ii) as a ligand-directed lung immunization tool against COVID-19. Thus, C16-ceramide is a unique example of a lipid-based receptor system in the lung vascular endothelium targeted in vivo by circulating ligands such as CGSPGWVRC.

Discovery of antibodies and cognate surface targets for ovarian cancer by surface profiling.

Although antibodies targeting specific tumor-expressed antigens are the standard of care for some cancers, the identification of cancer-specific targets amenable to antibody binding has remained a bottleneck in development of new therapeutics. To overcome this challenge, we developed a high-throughput platform that allows for the unbiased, simultaneous discovery of antibodies and targets based on phenotypic binding profiles. Applying this platform to ovarian cancer, we identified a wide diversity of cancer targets including receptor tyrosine kinases, adhesion and migration proteins, proteases and proteins regulating angiogenesis in a single round of screening using genomics, flow cytometry, and mass spectrometry. In particular, we identified BCAM as a promising candidate for targeted therapy in high-grade serous ovarian cancers. More generally, this approach provides a rapid and flexible framework to identify cancer targets and antibodies.

Phage display uncovers a sequence motif that drives polypeptide binding to a conserved regulatory exosite of O-GlcNAc transferase.

The modification of nucleocytoplasmic proteins by O-linked N-acetylglucosamine (O-GlcNAc) is an important regulator of cell physiology. O-GlcNAc is installed on over a thousand proteins by just one enzyme, O-GlcNAc transferase (OGT). How OGT is regulated is therefore a topic of interest. To gain insight into these questions, we used OGT to perform phage display selection from an unbiased library of ~109 peptides of 15 amino acids in length. Following rounds of selection and deep mutational panning, we identified a high-fidelity peptide consensus sequence, [Y/F]-x-P-x-Y-x-[I/M/F], that drives peptide binding to OGT. Peptides containing this sequence bind to OGT in the high nanomolar to low micromolar range and inhibit OGT in a noncompetitive manner with low micromolar potencies. X-ray structural analyses of OGT in complex with a peptide containing this motif surprisingly revealed binding to an exosite proximal to the active site of OGT. This structure defines the detailed molecular basis driving peptide binding and explains the need for specific residues within the sequence motif. Analysis of the human proteome revealed this motif within 52 nuclear and cytoplasmic proteins. Collectively, these data suggest a mode of regulation of OGT by which polypeptides can bind to this exosite to cause allosteric inhibition of OGT through steric occlusion of its active site. We expect that these insights will drive improved understanding of the regulation of OGT within cells and enable the development of new chemical tools to exert fine control over OGT activity.

The smallest functional antibody fragment: Ultralong CDR H3 antibody knob regions potently neutralize SARS-CoV-2.

Cows produce antibodies with a disulfide-bonded antigen-binding domain embedded within ultralong heavy chain third complementarity determining regions. This "knob" domain is analogous to natural cysteine-rich peptides such as knottins in that it is small and stable but can accommodate diverse loops and disulfide bonding patterns. We immunized cattle with SARS-CoV-2 spike and found ultralong CDR H3 antibodies that could neutralize several viral variants at picomolar IC50 potencies in vitro and could protect from disease in vivo. The independent CDR H3 peptide knobs were expressed and maintained the properties of the parent antibodies. The knob interaction with SARS-CoV-2 spike was revealed by electron microscopy, X-ray crystallography, NMR spectroscopy, and mass spectrometry and established ultralong CDR H3-derived knobs as the smallest known recombinant independent antigen-binding fragment. Unlike other vertebrate antibody fragments, these knobs are not reliant on the immunoglobulin domain and have potential as a new class of therapeutics.

Epitope-specific antibody fragments block aggregation of AGelD187N, an aberrant peptide in gelsolin amyloidosis.

Aggregation of aberrant fragment of plasma gelsolin, AGelD187N, is a crucial event underlying the pathophysiology of Finnish gelsolin amyloidosis, an inherited form of systemic amyloidosis. The amyloidogenic gelsolin fragment AGelD187N does not play any physiological role in the body, unlike most aggregating proteins related to other protein misfolding diseases. However, no therapeutic agents that specifically and effectively target and neutralize AGelD187N exist. We used phage display technology to identify novel single-chain variable fragments that bind to different epitopes in the monomeric AGelD187N that were further maturated by variable domain shuffling and converted to antigen-binding fragment (Fab) antibodies. The generated antibody fragments had nanomolar binding affinity for full-length AGelD187N, as evaluated by biolayer interferometry. Importantly, all four Fabs selected for functional studies efficiently inhibited the amyloid formation of full-length AGelD187N as examined by thioflavin fluorescence assay and transmission electron microscopy. Two Fabs, neither of which bound to the previously proposed fibril-forming region of AGelD187N, completely blocked the amyloid formation of AGelD187N. Moreover, no small soluble aggregates, which are considered pathogenic species in protein misfolding diseases, were formed after successful inhibition of amyloid formation by the most promising aggregation inhibitor, as investigated by size-exclusion chromatography combined with multiangle light scattering. We conclude that all regions of the full-length AGelD187N are important in modulating its assembly into fibrils and that the discovered epitope-specific anti-AGelD187N antibody fragments provide a promising starting point for a disease-modifying therapy for gelsolin amyloidosis, which is currently lacking.

Utility of protein-protein binding surfaces composed of anti-parallel alpha-helices and beta-sheets selected by phage display.

Over the past 3 decades, a diverse collection of small protein domains have been used as scaffolds to generate general purpose protein-binding reagents using a variety of protein display and enrichment technologies. To expand the repertoire of scaffolds and protein surfaces that might serve this purpose, we have explored the utility of (i) a pair of anti-parallel alpha-helices in a small highly disulfide-bonded 4-helix bundle, the CC4 domain from reversion-inducing Cysteine-rich Protein with Kazal Motifs and (ii) a concave beta-sheet surface and two adjacent loops in the human FN3 domain, the scaffold for the widely used monobody platform. Using M13 phage display and next generation sequencing, we observe that, in both systems, libraries of ∼30 million variants contain binding proteins with affinities in the low µM range for baits corresponding to the extracellular domains of multiple mammalian proteins. CC4- and FN3-based binding proteins were fused to the N- and/or C-termini of Fc domains and used for immunostaining of transfected cells. Additionally, FN3-based binding proteins were inserted into VP1 of AAV to direct AAV infection to cells expressing a defined surface receptor. Finally, FN3-based binding proteins were inserted into the Pvc13 tail fiber protein of an extracellular contractile injection system particle to direct protein cargo delivery to cells expressing a defined surface receptor. These experiments support the utility of CC4 helices B and C and of FN3 beta-strands C, D, and F together with adjacent loops CD and FG as surfaces for engineering general purpose protein-binding reagents.

KcsA-Kv1.x chimeras with complete ligand-binding sites provide improved predictivity for screening selective Kv1.x blockers.

Despite significant advances in the development of therapeutic interventions targeting autoimmune diseases and chronic inflammatory conditions, lack of effective treatment still poses a high unmet need. Modulating chronically activated T cells through the blockade of the Kv1.3 potassium channel is a promising therapeutic approach; however, developing selective Kv1.3 inhibitors is still an arduous task. Phage display-based high throughput peptide library screening is a rapid and robust approach to develop promising drug candidates; however, it requires solid-phase immobilization of target proteins with their binding site preserved. Historically, the KcsA bacterial channel chimera harboring only the turret region of the human Kv1.3 channel was used for screening campaigns. Nevertheless, literature data suggest that binding to this type of chimera does not correlate well with blocking potency on the native Kv1.3 channels. Therefore, we designed and successfully produced advanced KcsA-Kv1.3, KcsA-Kv1.1, and KcsA-Kv1.2 chimeric proteins in which both the turret and part of the filter regions of the human Kv1.x channels were transferred. These T+F (turret-filter) chimeras showed superior peptide ligand-binding predictivity compared to their T-only versions in novel phage ELISA assays. Phage ELISA binding and competition results supported with electrophysiological data confirmed that the filter region of KcsA-Kv1.x is essential for establishing adequate relative affinity order among selected peptide toxins (Vm24 toxin, Hongotoxin-1, Kaliotoxin-1, Maurotoxin, Stichodactyla toxin) and consequently obtaining more reliable selectivity data. These new findings provide a better screening tool for future drug development efforts and offer insight into the target-ligand interactions of these therapeutically relevant ion channels.

Proteome-scale characterisation of motif-based interactome rewiring by disease mutations.

Whole genome and exome sequencing are reporting on hundreds of thousands of missense mutations. Taking a pan-disease approach, we explored how mutations in intrinsically disordered regions (IDRs) break or generate protein interactions mediated by short linear motifs. We created a peptide-phage display library tiling ~57,000 peptides from the IDRs of the human proteome overlapping 12,301 single nucleotide variants associated with diverse phenotypes including cancer, metabolic diseases and neurological diseases. By screening 80 human proteins, we identified 366 mutation-modulated interactions, with half of the mutations diminishing binding, and half enhancing binding or creating novel interaction interfaces. The effects of the mutations were confirmed by affinity measurements. In cellular assays, the effects of motif-disruptive mutations were validated, including loss of a nuclear localisation signal in the cell division control protein CDC45 by a mutation associated with Meier-Gorlin syndrome. The study provides insights into how disease-associated mutations may perturb and rewire the motif-based interactome.

Discovery of Two Novel Immunoepitopes and Development of Peptide-based Sarcoidosis Immunoassay.

RATIONALE: Sarcoidosis is a systemic granulomatous disorder associated with hypergammaglobulinemia and the presence of autoantibodies. The specific antigens initiating granulomatous inflammation in sarcoidosis are unknown and there is no specific test available to diagnose sarcoidosis. To discover novel sarcoidosis antigens, we developed a high-throughput T7 phage display library derived from the sarcoidosis cDNA and identified numerous clones differentiating sarcoidosis from other respiratory diseases. After clone sequencing and homology search, we identified two epitopes (Cofilinµ and Chain A) that specifically bind to serum IgGs of sarcoidosis patients. OBJECTIVES: To develop and validate an epitope-specific IgG-based immunoassay specific for sarcoidosis. METHODS: We chemically synthesized both immunoepitopes (Cofilinµ and Chain A), and generated rabbit polyclonal antibodies against both neoantigens. After extensive standardization, we developed a direct peptide ELISA and measured epitope-specific IgG in sera of 386 subjects including, healthy controls (n=100), three sarcoidosis cohorts (n=186), pulmonary tuberculosis (n=70) and lung cancer (n=30). MEASUREMENTS AND MAIN RESULTS: To develop a model to classify sarcoidosis from other groups, data were analyzed using five-fold cross-validation when adjusting for confounders. The Cofilinµ IgGs model yielded a mean sensitivity, specificity, and positive and negative predictive value (PPV, NPV) of 0.97, 0.9, 0.9 and 0.96, respectively. Those same measures for Chain A IgG antibody were 0.9, 0.83, 0.84 and 0.9 respectively. Combining both biomarkers improved AUC, sensitivity, specificity, PPV and NPV. CONCLUSIONS: These results provide a novel immunoassay for sarcoidosis. The discovery of two neoantigens facilitates the development of biospecific drug discovery and the sarcoidosis-specific model.

Synergetic collision and space separation in microfluidic chip for efficient affinity-discriminated molecular selection.

Efficient molecular selection is a prerequisite for generating molecular tools used in diagnosis, pathology, vaccinology, and therapeutics. Selection efficiency is thermodynamically highly dependent on the dissociation equilibrium that can be reached in a single round. Extreme shifting of equilibrium towards dissociation favors the retention of high-affinity ligands over those with lower affinity, thus improving the selection efficiency. We propose to synergize dual effects by deterministic lateral-displacement microfluidics, including the collision-based force effect and the two-dimensional (2D) separation-based concentration effect, to greatly shift the equilibrium. Compared with previous approaches, this system can remove more low- or moderate-affinity ligands and maintain most high-affinity ligands, thereby improving affinity discrimination in selection. This strategy is demonstrated on phage display in both experiment and simulation, and two peptides against tumor markers ephrin type-A receptor 2 (EphA2) and CD71 were obtained with high affinity and specificity within a single round of selection, which offers a promising direction for discovery of robust binding ligands for a wide range of biomedical applications.

Directed evolution identifies high-affinity cystine-knot peptide agonists and antagonists of Wnt/ß-catenin signaling.

Developing peptide-based tools to fine-tune growth signaling pathways, in particular molecules with exquisite selectivity and high affinities, opens up opportunities for cellular reprogramming in tissue regeneration. Here, we present a library based on cystine-knot peptides (CKPs) that incorporate multiple loops for randomization and selection via directed evolution. Resulting binders could be assembled into multimeric structures to fine-tune cellular signaling. An example is presented for the Wnt pathway, which plays a key role in the homeostasis and regeneration of tissues such as lung, skin, and intestine. We discovered picomolar affinity CKP agonists of the human LPR6 receptor by exploring the limits of the topological manipulation of LRP6 dimerization. Structural analyses revealed that the agonists bind at the first ß-propeller domain of LRP6, mimicking the natural Wnt inhibitors DKK1 and SOST. However, the CKP agonists exhibit a different mode of action as they amplify the signaling of natural Wnt ligands but do not activate the pathway by themselves. In an alveolosphere organoid model, the CKP agonists induced alveolar stem cell activity. They also stimulated growth in primary human intestinal organoids. The approach described here advances the important frontier of next-generation agonist design and could be applied to other signaling pathways to discover tunable agonist ligands.

De novo mapping of α-helix recognition sites on protein surfaces using unbiased libraries.

The α-helix is one of the most common protein surface recognition motifs found in nature, and its unique amide-cloaking properties also enable α-helical polypeptide motifs to exist in membranes. Together, these properties have inspired the development of α-helically constrained (Helicon) therapeutics that can enter cells and bind targets that have been considered "undruggable", such as protein-protein interactions. To date, no general method for discovering α-helical binders to proteins has been reported, limiting Helicon drug discovery to only those proteins with previously characterized α-helix recognition sites, and restricting the starting chemical matter to those known α-helical binders. Here, we report a general and rapid screening method to empirically map the α-helix binding sites on a broad range of target proteins in parallel using large, unbiased Helicon phage display libraries and next-generation sequencing. We apply this method to screen six structurally diverse protein domains, only one of which had been previously reported to bind isolated α-helical peptides, discovering 20 families that collectively comprise several hundred individual Helicons. Analysis of 14 X-ray cocrystal structures reveals at least nine distinct α-helix recognition sites across these six proteins, and biochemical and biophysical studies show that these Helicons can block protein-protein interactions, inhibit enzymatic activity, induce conformational rearrangements, and cause protein dimerization. We anticipate that this method will prove broadly useful for the study of protein recognition and for the development of both biochemical tools and therapeutics for traditionally challenging protein targets.