Nanobody Binding Research Articles

Nanozyme and bifunctional nanobody-based colorimetric-SERS dual-mode Immunosensor for microcystin-LR detection

Microcystin-LR (MC-LR), a potent cyanotoxin in freshwater, poses a risk of severe liver damage and other health issues, making its detection vital. However, the detection capabilities of conventional antibodies are constrained, which limited their use in immunoassays. In this work, we designed a new bifunctional nanobody, named A2.3-SBP (comprised of nanobody and streptavidin binding peptide), capable of binding with MC-LR and streptavidin. Based on A2.3-SBP and Fe3O4@Au-Pt nanozyme, we introduced an enzyme-free immunosensor that operated in microplate with colorimetric and surface-enhanced Raman scattering (SERS) detection modes. The dual-mode assay showed color changes and SERS intensity directly correlating to MC-LR concentrations with a range from 1.0 to 500 ng/mL and a limit of detection of 0.26 and 0.032 ng/mL, respectively. This strategy eliminated the need for complex enzymatic reactions and realized dual-signal detection of MC-LR in 96 water samples (0.03 μg/kg) within 30 min, suggesting its potential in drinking water detection.

The development of single-domain VHH nanobodies that target the Candida albicans cell surface.

Candida albicans causes life-threatening invasive infections that are hard to diagnose and treat, with drug resistance leading to treatment failure. The goal of this study was to develop VHH (single variable domain on a heavy chain) nanobodies to detect drug-resistant infections. Llamas were immunized with a mixture of heat killed and fixed C. albicans cells of different morphologies. Llama lymphocyte RNA was used to generate phage display libraries that were tested for binding to C. albicans cells or cell wall fractions, and single antibody domains were isolated. The libraries were panned against echinocandin-resistant C. albicans isolates and counter-selected against echinocandin-susceptible isolates with the aim of isolating binding domains specific for antigens on drug-resistant cells. Thirty diverse VHH nanobodies were selected, and binding characteristics were assessed via dose-response ELISA. Binding was tested against a variety of C. albicans isolates and other Candida species, indicating that the VHHs were specific for C. albicans. The VHH nanobodies were sorted into four distinct groups based on their binding patterns. Two of the groups bound preferentially to the yeast cell poles and hyphae, respectively. Nanobody binding to C. albicans deletion mutants was tested by fluorescence microscopy and ELISA to identify the antigen targets. VHH19 nanobody, belonging to the largest group, recognized the Als4 adhesin. VHH14 antibody in the hyphae-specific group recognized Als3. None of the isolated VHH nanobodies was selective for drug-resistant clinical isolates. Our data indicate that this approach can generate valuable single-domain antibodies specific to C. albicans proteins.IMPORTANCEThe human fungal pathogen Candida albicans causes a range of diseases from superficial mucosal infections such as oral and vaginal thrush to life-threatening, systemic infections. Accurate and rapid diagnosis of these infections remains challenging, and currently, there are no rapid ways to diagnose drug-resistant infections without performing drug susceptibility testing from blood culture, which can take several days. In this proof-of-concept study, we have generated a diverse set of single domain VHH antibodies (nanobodies) from llamas that recognize and bind specifically to C. albicans cell surface. The nanobodies were classified into four groups based on their binding patterns, for example, cell poles or hyphae. Specific nanobodies were verified as recognizing the important adhesin Als4 or the hyphae associated invasin Als3, respectively. The data validate the approach that small VHH antibody domains hold future promise for diagnostic applications and as probes to study the fungal cell surface.

A nanobody interaction with SARS-COV-2 Spike allows the versatile targeting of lentivirus vectors.

While investigating methods to target gene delivery vectors to specific cell types, we examined the potential of using a nanobody against the SARS-CoV-2 Spike protein receptor-binding domain to direct lentivirus infection of Spike-expressing cells. Using four different approaches, we found that lentiviruses with surface-exposed nanobody domains selectively infect Spike-expressing cells. Targeting is dependent on the fusion function of the Spike protein, and conforms to a model in which nanobody binding to the Spike protein triggers the Spike fusion machinery. The nanobody-Spike interaction also is capable of directing cell-cell fusion and the selective infection of nanobody-expressing cells by Spike-pseudotyped lentivirus vectors. Significantly, cells infected with SARS-CoV-2 are efficiently and selectively infected by lentivirus vectors pseudotyped with a chimeric nanobody protein. Our results suggest that cells infected by any virus that forms syncytia may be targeted for gene delivery by using an appropriate nanobody or virus receptor mimic. Vectors modified in this fashion may prove useful in the delivery of immunomodulators to infected foci to mitigate the effects of viral infections.IMPORTANCEWe have discovered that lentiviruses decorated on their surfaces with a nanobody against the SARS-CoV-2 Spike protein selectively infect Spike-expressing cells. Infection is dependent on the specificity of the nanobody and the fusion function of the Spike protein and conforms to a reverse fusion model, in which nanobody binding to Spike triggers the Spike fusion machinery. The nanobody-Spike interaction also can drive cell-cell fusion and infection of nanobody-expressing cells with viruses carrying the Spike protein. Importantly, cells infected with SARS-CoV-2 are selectively infected with nanobody-decorated lentiviruses. These results suggest that cells infected by any virus that expresses an active receptor-binding fusion protein may be targeted by vectors for delivery of cargoes to mitigate infections.

Plug-and-play nucleic acid-mediated multimerization of biparatopic nanobodies for molecular imaging

In cancer molecular imaging, selecting binders with high specificity and affinity for biomarkers is paramount for achieving high-contrast imaging within clinical time frames. Nanobodies have emerged as potent candidates, surpassing antibodies in pre-clinical imaging due to their convenient production, rapid renal clearance, and deeper tissue penetration. Multimerization of nanobodies is a popular strategy to enhance their affinity and pharmacokinetics, however, traditional methods are laborious and may yield heterogeneous products. In this study, we employed a Holliday junction-like (HJ) nucleic acid-based scaffold to create homogeneous nanostructures with precise multivalent and multiparatopic nanobody displays. The plug-and-play assembly allowed the screening of several nanobody multimer configurations for the detection of the breast cancer biomarker, human epidermal growth factor receptor 2 (HER2). In vitro studies demonstrated significant improvements in binding avidity, particularly with the biparatopic construct exhibiting high sensitivity, surpassing that of traditional antibody-based cell binding. Furthermore, our HJ platform allowed for adaptation from fluorescence-based to nuclear imaging, as demonstrated in xenografted mice, thereby allowing for future in vivo applications. This work highlights the potential of nucleic acid-mediated multimerization to markedly enhance nanobody binding, by exploring synergistic combinations and offering versatility for both in vitro diagnostics and cancer molecular imaging with prospects for future theranostic applications.

Combining poly-epitope MoonTags and labeled nanobodies for signal amplification in cell-specific PET imaging in vivo

Immunorecognition provides an excellent basis for targeted imaging techniques covering a wide range from basic research to diagnostics and from single cells to whole organisms. Fluorescence- or radioisotope-labeled antibodies, antibody fragments or nanobodies enable a direct signal readout upon binding and allow for versatile imaging from microscopy to whole-body imaging. However, as the signal intensity directly correlates with the number of labeled antibodies bound to their epitopes (1:1 binding), sensitivity for low-expressing epitopes can be limiting for visualization. For the first time, we developed poly-epitope tags with multiple copies (1 to 7) of a short peptide epitope, specifically the MoonTag, that are recognized by a labeled nanobody and aimed at signal amplification in microscopy and cell-specific PET imaging. In transiently transfected HeLa cells or stably transduced A4573 cells we characterized complex formation and in vitro signal amplification. Indeed, using fluorescently and radioactively labeled nanobodies we found an approximately linear signal amplification with increasing numbers of epitope copies in vitro. To test the poly-epitope approach in vivo, A4573 tumor cells were injected subcutaneously into the shoulder of NSG mice, with A4573 tumor cells expressing a poly-epitope of 7 MoonTags on one side and WT cells on the other side. Using a [68Ga]-labeled NODAGA-conjugated MoonTag nanobody, we performed PET/CT imaging at day 8–9 after tumor implantation. Specific binding of a [68Ga]-labeled NODAGA-conjugated MoonTag nanobody was observed in 7xMoonTag tumors (1.7 ± 0.5%ID/mL) by PET imaging, showing significantly higher radiotracer accumulation compared to the WT tumors (1.1 ± 0.3%ID/mL; p < 0.01). Ex vivo gamma counter measurements confirmed significantly higher uptake in 7xMoonTag tumors compared to WT tumors (p < 0.001). In addition, MoonTag nanobody binding was detected by autoradiography which was spatially matched with histological analysis of the tumor tissues. In conclusion, we expect nanobody-based poly-epitope tag strategies to be widely applicable for multimodal imaging techniques given the advantageous properties of nanobodies and their amenability to genetic and chemical engineering.

A Nanobody Interaction with SARS-CoV-2 Spike Allows the Versatile Targeting of Lentivirus Vectors.

We have discovered that lentiviruses decorated on their surfaces with a nanobody against the SARS-CoV-2 Spike protein selectively infect Spike-expressing cells. Infection is dependent on the specificity of the nanobody and the fusion function of the Spike protein, and conforms to a reverse fusion model, in which nanobody binding to Spike triggers the Spike fusion machinery. The nanobody-Spike interaction also can drive cell-cell fusion, and infection of nanobody-expressing cells with viruses carrying the Spike protein. Importantly, cells infected with SARS-CoV-2 are selectively infected with nanobody-decorated lentiviruses. These results suggest that cells infected by any virus that expresses an active receptor-binding fusion protein may be targeted by vectors for delivery of cargoes to mitigate infections.

NanoBERTa-ASP: predicting nanobody paratope based on a pretrained RoBERTa model

BackgroundNanobodies, also known as VHH or single-domain antibodies, are unique antibody fragments derived solely from heavy chains. They offer advantages of small molecules and conventional antibodies, making them promising therapeutics. The paratope is the specific region on an antibody that binds to an antigen. Paratope prediction involves the identification and characterization of the antigen-binding site on an antibody. This process is crucial for understanding the specificity and affinity of antibody-antigen interactions. Various computational methods and experimental approaches have been developed to predict and analyze paratopes, contributing to advancements in antibody engineering, drug development, and immunotherapy. However, existing predictive models trained on traditional antibodies may not be suitable for nanobodies. Additionally, the limited availability of nanobody datasets poses challenges in constructing accurate models.MethodsTo address these challenges, we have developed a novel nanobody prediction model, named NanoBERTa-ASP (Antibody Specificity Prediction), which is specifically designed for predicting nanobody-antigen binding sites. The model adopts a training strategy more suitable for nanobodies, based on an advanced natural language processing (NLP) model called BERT (Bidirectional Encoder Representations from Transformers). To be more specific, the model utilizes a masked language modeling approach named RoBERTa (Robustly Optimized BERT Pretraining Approach) to learn the contextual information of the nanobody sequence and predict its binding site.ResultsNanoBERTa-ASP achieved exceptional performance in predicting nanobody binding sites, outperforming existing methods, indicating its proficiency in capturing sequence information specific to nanobodies and accurately identifying their binding sites. Furthermore, NanoBERTa-ASP provides insights into the interaction mechanisms between nanobodies and antigens, contributing to a better understanding of nanobodies and facilitating the design and development of nanobodies with therapeutic potential.ConclusionNanoBERTa-ASP represents a significant advancement in nanobody paratope prediction. Its superior performance highlights the potential of deep learning approaches in nanobody research. By leveraging the increasing volume of nanobody data, NanoBERTa-ASP can further refine its predictions, enhance its performance, and contribute to the development of novel nanobody-based therapeutics.Github repository: https://github.com/WangLabforComputationalBiology/NanoBERTa-ASP

Cryo-EM structures of type IV pili complexed with nanobodies reveal immune escape mechanisms

Type IV pili (T4P) are prevalent, polymeric surface structures in pathogenic bacteria, making them ideal targets for effective vaccines. However, bacteria have evolved efficient strategies to evade type IV pili-directed antibody responses. Neisseria meningitidis are prototypical type IV pili-expressing Gram-negative bacteria responsible for life threatening sepsis and meningitis. This species has evolved several genetic strategies to modify the surface of its type IV pili, changing pilin subunit amino acid sequence, nature of glycosylation and phosphoforms, but how these modifications affect antibody binding at the structural level is still unknown. Here, to explore this question, we determine cryo-electron microscopy (cryo-EM) structures of pili of different sequence types with sufficiently high resolution to visualize posttranslational modifications. We then generate nanobodies directed against type IV pili which alter pilus function in vitro and in vivo. Cryo-EM in combination with molecular dynamics simulation of the nanobody-pilus complexes reveals how the different types of pili surface modifications alter nanobody binding. Our findings shed light on the impressive complementarity between the different strategies used by bacteria to avoid antibody binding. Importantly, we also show that structural information can be used to make informed modifications in nanobodies as countermeasures to these immune evasion mechanisms.

Molecular mechanism of Kv1.3 inactivation: Insights from nanobody binding and mutational analysis

Molecular mechanism of Kv1.3 inactivation: Insights from nanobody binding and mutational analysis

The Variable Heavy Chain Antibody Syn-VWFA1 Inhibits the Interaction of Platelets to Von Willebrand Factor in Solution but Not to Immobilized Von Willebrand Factor Thereby Inhibiting 3-Dimensional but Not 2-Dimensional Thrombus Formation

Neutralizing the interaction between GP1bα and von Willebrand Factor (VWF) is an attractive strategy for treatment and prevention of platelet-VWF agglutinate formation in von Willebrand disease type 2B and thrombotic thrombocytopenic purpura. The aim of this study was to characterize a llama-derived variable heavy chain antibody (VHH) targeting the A1 domain of active VWF (Syn-VWFA1), in terms of its interference with platelet VWF interaction. Syn-VWFA1 (1.5 µM) abolished platelet binding to ristocetin-activated VWF (native and Haemate® P VWF) and R1306W VWF (VWF type 2B) in a flow-cytometric platelet function assay (Figure 1). Moreover, Syn-VWFA1 dose-dependently inhibited ristocetin-activated VWF and R1306W VWF platelet agglutination, but not collagen- and ADP-induced platelet aggregation. Under flow conditions, Syn-VWFA1 (1.8 µM) did not affect adhesion of platelets to collagen at high shear. In the shear- and collagen-dependent PFA-200, Syn-VWFA1 prolonged the closure time, but only at high concentrations (&amp;gt;3 µM), while GPIba binding to immobilized VWF remained unaffected by Syn-VWFA1. Syn-VWFA1 increased the cleavage of VWF multimers by ADAMTS13. Furthermore, by applying deuterium exchange, we found a small region in the β1 to α1 loop (1288-1293) in the A1 domain of VWF, which is likely to constitute the nanobody binding site (Figure 2) and binding of Syn-VWFA1 to the A1 domain results in a conformational change exposing regions in the α2 and α3 helices. In conclusion, Syn-VWFA1 dose-dependently blocks platelet-binding to activated VWF in solution, but not to (collagen-) immobilized active VWF. This implies that immobilization of VWF to collagen affects VWF conformation in such a way (1) that access of Syn-VWFA1 to its epitope in the A1 domain is hindered or (2) that conformational changes following Syn-VWFA1 binding are blocked, while allowing platelet binding. This implies that Syn-VWFA1 has potential treatment opportunities for patients with increased circulating active VWF, to prevent 3-dimensional thrombus formation (pathological platelet-VWF aggregate formation) but not 2-dimensional thrombus formation (leaving physiological hemostasis at sites of vascular injury intact).

Structural basis for the allosteric modulation of rhodopsin by nanobody binding to its extracellular domain

Rhodopsin is a prototypical G protein-coupled receptor (GPCR) critical for vertebrate vision. Research on GPCR signaling states has been facilitated using llama-derived nanobodies (Nbs), some of which bind to the intracellular surface to allosterically modulate the receptor. Extracellularly binding allosteric nanobodies have also been investigated, but the structural basis for their activity has not been resolved to date. Here, we report a library of Nbs that bind to the extracellular surface of rhodopsin and allosterically modulate the thermodynamics of its activation process. Crystal structures of Nb2 in complex with native rhodopsin reveal a mechanism of allosteric modulation involving extracellular loop 2 and native glycans. Nb2 binding suppresses Schiff base deprotonation and hydrolysis and prevents intracellular outward movement of helices five and six – a universal activation event for GPCRs. Nb2 also mitigates protein misfolding in a disease-associated mutant rhodopsin. Our data show the power of nanobodies to modulate the photoactivation of rhodopsin and potentially serve as therapeutic agents for disease-associated rhodopsin misfolding.

Enhanced in vivo blood brain barrier transcytosis of macromolecular cargo using an engineered pH-sensitive mouse transferrin receptor binding nanobody

BackgroundThe blood brain barrier limits entry of macromolecular diagnostic and therapeutic cargos. Blood brain barrier transcytosis via receptor mediated transport systems, such as the transferrin receptor, can be used to carry macromolecular cargos with variable efficiency. Transcytosis involves trafficking through acidified intracellular vesicles, but it is not known whether pH-dependent unbinding of transport shuttles can be used to improve blood brain barrier transport efficiency.MethodsA mouse transferrin receptor binding nanobody, NIH-mTfR-M1, was engineered to confer greater unbinding at pH 5.5 vs 7.4 by introducing multiple histidine mutations. The histidine mutant nanobodies were coupled to neurotensin for in vivo functional blood brain barrier transcytosis testing via central neurotensin-mediated hypothermia in wild-type mice. Multi-nanobody constructs including the mutant M1R56H, P96H, Y102H and two copies of the P2X7 receptor-binding 13A7 nanobody were produced to test proof-of-concept macromolecular cargo transport in vivo using quantitatively verified capillary depleted brain lysates and in situ histology.ResultsThe most effective histidine mutant, M1R56H, P96H, Y102H-neurotensin, caused > 8 °C hypothermia after 25 nmol/kg intravenous injection. Levels of the heterotrimeric construct M1R56H, P96H, Y102H-13A7-13A7 in capillary depleted brain lysates peaked at 1 h and were 60% retained at 8 h. A control construct with no brain targets was only 15% retained at 8 h. Addition of the albumin-binding Nb80 nanobody to make M1R56H, P96H, Y102H-13A7-13A7-Nb80 extended blood half-life from 21 min to 2.6 h. At 30–60 min, biotinylated M1R56H, P96H, Y102H-13A7-13A7-Nb80 was visualized in capillaries using in situ histochemistry, whereas at 2–16 h it was detected in diffuse hippocampal and cortical cellular structures. Levels of M1R56H, P96H, Y102H-13A7-13A7-Nb80 reached more than 3.5 percent injected dose/gram of brain tissue after 30 nmol/kg intravenous injection. However, higher injected concentrations did not result in higher brain levels, compatible with saturation and an apparent substrate inhibitory effect.ConclusionThe pH-sensitive mouse transferrin receptor binding nanobody M1R56H, P96H, Y102H may be a useful tool for rapid and efficient modular transport of diagnostic and therapeutic macromolecular cargos across the blood brain barrier in mouse models. Additional development will be required to determine whether this nanobody-based shuttle system will be useful for imaging and fast-acting therapeutic applications.

Allosteric activation or inhibition of PI3Kγ mediated through conformational changes in the p110γ helical domain

PI3Kγ is a critical immune signaling enzyme activated downstream of diverse cell surface molecules, including Ras, PKCβ activated by the IgE receptor, and Gβγ subunits released from activated GPCRs. PI3Kγ can form two distinct complexes, with the p110γ catalytic subunit binding to either a p101 or p84 regulatory subunit, with these complexes being differentially activated by upstream stimuli. Here, using a combination of cryo electron microscopy, HDX-MS, and biochemical assays, we have identified novel roles of the helical domain of p110γ in regulating lipid kinase activity of distinct PI3Kγ complexes. We defined the molecular basis for how an allosteric inhibitory nanobody potently inhibits kinase activity through rigidifying the helical domain and regulatory motif of the kinase domain. The nanobody did not block either p110γ membrane recruitment or Ras/Gβγ binding, but instead decreased ATP turnover. We also identified that p110γ can be activated by dual PKCβ helical domain phosphorylation leading to partial unfolding of an N-terminal region of the helical domain. PKCβ phosphorylation is selective for p110γ-p84 compared to p110γ-p101, driven by differential dynamics of the helical domain of these different complexes. Nanobody binding prevented PKCβ-mediated phosphorylation. Overall, this work shows an unexpected allosteric regulatory role of the helical domain of p110γ that is distinct between p110γ-p84 and p110γ-p101 and reveals how this can be modulated by either phosphorylation or allosteric inhibitory binding partners. This opens possibilities of future allosteric inhibitor development for therapeutic intervention.

Allosteric activation or inhibition of PI3Kγ mediated through conformational changes in the p110γ helical domain.

PI3Kγ is a critical immune signaling enzyme activated downstream of diverse cell surface molecules, including Ras, PKCβ activated by the IgE receptor, and Gβγ subunits released from activated GPCRs. PI3Kγ can form two distinct complexes, with the p110γ catalytic subunit binding to either a p101 or p84 regulatory subunit, with these complexes being differentially activated by upstream stimuli. Here, using a combination of cryo electron microscopy, HDX-MS, and biochemical assays, we have identified novel roles of the helical domain of p110γ in regulating lipid kinase activity of distinct PI3Kγ complexes. We defined the molecular basis for how an allosteric inhibitory nanobody potently inhibits kinase activity through rigidifying the helical domain and regulatory motif of the kinase domain. The nanobody did not block either p110γ membrane recruitment or Ras/Gβγ binding, but instead decreased ATP turnover. We also identified that p110γ can be activated by dual PKCβ helical domain phosphorylation leading to partial unfolding of an N-terminal region of the helical domain. PKCβ phosphorylation is selective for p110γ-p84 compared to p110γ-p101, driven by differential dynamics of the helical domain of these different complexes. Nanobody binding prevented PKCβ-mediated phosphorylation. Overall, this work shows an unexpected allosteric regulatory role of the helical domain of p110γ that is distinct between p110γ-p84 and p110γ-p101 and reveals how this can be modulated by either phosphorylation or allosteric inhibitory binding partners. This opens possibilities of future allosteric inhibitor development for therapeutic intervention.

Evaluation of 18F labeled glial fibrillary acidic protein binding nanobody and its brain shuttle peptide fusion proteins using a neuroinflammation rat model.

Astrogliosis is a crucial feature of neuroinflammation and is characterized by the significant upregulation of glial fibrillary acidic protein (GFAP) expression. Hence, visualizing GFAP in the living brain of patients with damaged central nervous system using positron emission tomography (PET) is of great importance, and it is expected to depict neuroinflammation more directly than existing neuroinflammation imaging markers. However, no PET radiotracers for GFAP are currently available. Therefore, neuroimaging with antibody-like affinity proteins could be a viable strategy for visualizing imaging targets that small molecules rarely recognize, such as GFAP, while we need to overcome the challenges of slow clearance and low brain permeability. The E9 nanobody, a small-affinity protein with high affinity and selectivity for GFAP, was utilized in this study. E9 was engineered by fusing a brain shuttle peptide that facilitates blood-brain barrier permeation via two different types of linker domains: E9-GS-ApoE (EGA) and E9-EAK-ApoE (EEA). E9, EGA and EEA were radiolabeled with fluorine-18 using cell-free protein radiosynthesis. In vitro autoradiography showed that all radiolabeled proteins exhibited a significant difference in neuroinflammation in the brain sections created from a rat model constructed by injecting lipopolysaccharide (LPS) into the unilateral striatum of wildtype rats, and an excess competitor displaced their binding. However, exploratory in vivo PET imaging and ex vivo biodistribution studies in the rat model failed to distinguish neuroinflammatory lesions within 3 h of 18F-EEA intravenous injection. This study contributes to a better understanding of the characteristics of small-affinity proteins fused with a brain shuttle peptide for further research into the use of protein molecules as PET tracers for imaging neuropathology.

Inactivation mechanism of an immunoglobulin modulator of the T-cell potassium channel Kv1.3.

Kv1.3 belongs to the Shaker family of voltage-activated potassium channels that are abundantly expressed in the T lymphocytes, where it plays an important role in T-cell activation. This role has made Kv1.3 a target for therapeutic immunomodulation for autoimmune diseases like multiple sclerosis, type 1 diabetes, and rheumatoid arthritis. Here, through cryo-electron microscopy (cryo-EM) and electrophysiology, we have investigated the modulatory mechanism of an inhibitory nanobody A0194009G09 (Ablynx/Sanofi). Functional studies reveal that the nanobody inhibits Kv1.3 by promoting slow C-type inactivation and that multiple nanobodies can bind to individual Kv1.3 channels. The structure of the human Kv1.3 in apo state (without modulators) showed a dynamic selectivity filter (SF) with two conformations (D1, D2) that are dilated when compared to reference structures where the SF is conducting. In the presence of the nanobody, the structure displays a SF in the D1 conformation, similar to the apo protein, but also a distinct conformation where the SF was further dilated (D3). Further, the nanobody-bound structure reveals how the nanobody bridges the turret loop within the pore domain and the S1-S2 loop within the voltage sensing domains to promote C-type inactivation. To explore how the nanobody promotes inactivation, we mutated residues at the interface between the nanobody and channel in both the S1-S2 loop and the turret loop, as well as a residue in the SF (H451) known to control inactivation in Kv1.3. Interestingly, the mutations that prevent inactivation in Kv1.3 largely attenuated the inhibitory action of the nanobody, similar to the mutations at the Kv1.3-nanobody binding interface. Taken together, our results demonstrate that the nanobody inhibits Kv1.3 by promoting inactivation, and we are currently solving mutant structures to elucidate the molecular mechanisms of how nanobody binding promotes inactivation.

Generation of Multivalent Nanobody-Based Proteins with Improved Neutralization of Long α-Neurotoxins from Elapid Snakes.

Recombinantly produced biotherapeutics hold promise for improving the current standard of care for snakebite envenoming over conventional serotherapy. Nanobodies have performed well in the clinic, and in the context of antivenom, they have shown the ability to neutralize long α-neurotoxins in vivo. Here, we showcase a protein engineering approach to increase the valence and hydrodynamic size of neutralizing nanobodies raised against a long α-neurotoxin (α-cobratoxin) from the venom of the monocled cobraNaja kaouthia. Based on the p53 tetramerization domain, a panel of anti-α-cobratoxin nanobody-p53 fusion proteins, termed Quads, were produced with different valences, inclusion or exclusion of Fc regions for endosomal recycling purposes, hydrodynamic sizes, and spatial arrangements, comprising up to 16 binding sites. Measurements of binding affinity and stoichiometry showed that the nanobody binding affinity was retained when incorporated into the Quad scaffold, and all nanobody domains were accessible for toxin binding, subsequently displaying increased blocking potency in vitro compared to the monomeric format. Moreover, functional assessment using automated patch-clamp assays demonstrated that the nanobody and Quads displayed neutralizing effects against long α-neurotoxins from both N. kaouthia and the forest cobra N. melanoleuca. This engineering approach offers a means of altering the valence, endosomal recyclability, and hydrodynamic size of existing nanobody-based therapeutics in a simple plug-and-play fashion and can thus serve as a technology for researchers tailoring therapeutic properties for improved neutralization of soluble targets such as snake toxins.

Nanoscale Features of Tunable Bacterial Outer Membrane Models Revealed by Correlative Microscopy.

The rise of antibiotic resistance is a growing worldwide human health issue, with major socioeconomic implications. An understanding of the interactions occurring at the bacterial membrane is crucial for the generation of new antibiotics. Supported lipid bilayers (SLBs) made from reconstituted lipid vesicles have been used to mimic these membranes, but their utility has been restricted by the simplistic nature of these systems. A breakthrough in the field has come with the use of outer membrane vesicles derived from Gram-negative bacteria to form SLBs, thus providing a more physiologically relevant system. These complex bilayer systems hold promise but have not yet been fully characterized in terms of their composition, ratio of natural to synthetic components, and membrane protein content. Here, we use correlative atomic force microscopy (AFM) with structured illumination microscopy (SIM) for the accurate mapping of complex lipid bilayers that consist of a synthetic fraction and a fraction of lipids derived from Escherichia coli outer membrane vesicles (OMVs). We exploit the high resolution and molecular specificity that SIM can offer to identify areas of interest in these bilayers and the enhanced resolution that AFM provides to create detailed topography maps of the bilayers. We are thus able to understand the way in which the two different lipid fractions (natural and synthetic) mix within the bilayers, and we can quantify the amount of bacterial membrane incorporated into the bilayer. We prove the system’s tunability by generating bilayers made using OMVs engineered to contain a green fluorescent protein (GFP) binding nanobody fused with the porin OmpA. We are able to directly visualize protein–protein interactions between GFP and the nanobody complex. Our work sets the foundation for accurately understanding the composition and properties of OMV-derived SLBs to generate a high-resolution platform for investigating bacterial membrane interactions for the development of next-generation antibiotics.

Structural insights into the function-modulating effects of nanobody binding to the integrin receptor αMβ2

The integrin receptor αMβ2 mediates phagocytosis of complement-opsonized objects, adhesion to the extracellular matrix, and transendothelial migration of leukocytes. However, the mechanistic aspects of αMβ2 signaling upon ligand binding are unclear. Here, we present the first atomic structure of the human αMβ2 headpiece fragment in complex with the nanobody (Nb) hCD11bNb1 at a resolution of 3.2 Å. We show that the receptor headpiece adopts the closed conformation expected to exhibit low ligand affinity. The crystal structure indicates that in the R77H αM variant, associated with systemic lupus erythematosus, the modified allosteric relationship between ligand binding and integrin outside–inside signaling is due to subtle conformational effects transmitted over a distance of 40 Å. Furthermore, we found the Nb binds to the αI domain of the αM subunit in an Mg2+-independent manner with low nanomolar affinity. Biochemical and biophysical experiments with purified proteins demonstrated that the Nb acts as a competitive inhibitor through steric hindrance exerted on the thioester domain of complement component iC3b attempting to bind the αM subunit. Surprisingly, we show that the Nb stimulates the interaction of cell-bound αMβ2 with iC3b, suggesting that it may represent a novel high-affinity proteinaceous αMβ2-specific agonist. Taken together, our data suggest that the iC3b–αMβ2 complex may be more dynamic than predicted from the crystal structure of the core complex. We propose a model based on the conformational spectrum of the receptor to reconcile these observations regarding the functional consequences of hCD11bNb1 binding to αMβ2.

Abstract 672: Development and validation of nanobodies specific to the oncogenic phosphatase protein tyrosine phosphatase 4A3 (PTP4A3 or PRL-3)

Abstract Protein Tyrosine Phosphatase 4A3 (PTP4A3 or PRL-3) is an oncogenic dual-specificity phosphatase that drives tumor metastasis, promotes cancer cell survival, and is correlated with poor patient prognosis in a variety of solid tumors and leukemias. The mechanisms that drive PRL-3’s oncogenic functions are not well understood, in part due to a lack of research tools available to study this protein. The development of such tools has proven difficult, as the PRL family is ~80% homologous and the PRL catalytic binding pocket is shallow and hydrophobic. Currently available small molecules do not exhibit binding specificity for PRL-3 over PRL family members, and the only research antibody specific for PRL-3 can only recognize denatured protein. To address the lack of tools available to study PRL-3, we have developed alpaca-derived single domain antibodies, or nanobodies, targeting PRL-3. Nanobodies have emerged as a valuable research tool and show promise as cancer therapeutics as they are ~15kD and lack light chains, allowing them to reach cavities within active sites that conventional antibodies cannot normally reach. Nanobodies also maintain high specificity and affinity for their antigens. We identified seven unique nanobodies that bind to PRL-3 with no activity towards PRL-1 and PRL-2, making our nanobodies one of the first tools to selectively target PRL-3 in its native state. We used biolayer interferometry and found the nanobody binding affinity for PRL-3 to be within a KD of 30 - 300 nM, similar to that of antibodies currently on the market. We identified PRL-3:nanobody interactions with hydrogen-deuterium exchange mass spectrometry (HDX-MS) and showed binding outside the active site. These data were confirmed by analyzing the effects of nanobodies on PRL-3 phosphatase activity and substrate binding. Our anti-PRL-3 nanobodies specifically pulled down PRL-3 over PRL-1/-2 in immunoprecipitation experiments. Finally, we used these nanobodies to analyze PRL-3 localization in fixed immunofluorescence experiments in human cancer cells. We found that a C-terminal tag on PRL-3, such as FLAG or GFP, enhanced PRL-3 localization to the membrane, compared to untagged protein, which may have confounded previous PRL-3 functional studies. We are currently utilizing these nanobodies in two ways to understand PRL-3’s role in cancer. First, we will use the nanobody to stabilize PRL-3 for X-ray crystallography to develop higher resolution structures that could contribute to substrate identification and drug design. Secondly, we will examine PRL-3 function and trafficking during various cancer processes, such as proliferation, invasion, and stress, to determine how PRL-3 localization contributes to cancer progression. Citation Format: Caroline Noel Smith, Kyle Kihn, Zachary A. Williamson, K. Martin Chow, Louis B. Hersh, Konstantin Korotkov, Daniel Deredge, Jessica S. Blackburn. Development and validation of nanobodies specific to the oncogenic phosphatase protein tyrosine phosphatase 4A3 (PTP4A3 or PRL-3) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 672.