ABSTRACT
Immunoglobulin G (IgG) antibodies that bind their cognate antigen in a pH-dependent manner (acid-switched antibodies) can release their bound antigen for degradation in the acidic environment of endosomes, while the IgGs are rescued by the neonatal Fc receptor (FcRn). Thus, such IgGs can neutralize multiple antigens over time and therefore be used at lower doses than their non-pH-responsive counterparts. Here, we show that light-chain shuffling combined with phage display technology can be used to discover IgG1 antibodies with increased pH-dependent antigen binding properties, using the snake venom toxins, myotoxin II and α-cobratoxin, as examples. We reveal differences in how the selected IgG1s engage their antigens and human FcRn and show how these differences translate into distinct cellular handling properties related to their pH-dependent antigen binding phenotypes and Fc-engineering for improved FcRn binding. Our study showcases the complexity of engineering pH-dependent antigen binding IgG1s and demonstrates the effects on cellular antibody-antigen recycling.
ABSTRACT
Broadly-neutralizing monoclonal antibodies are becoming increasingly important tools for treating infectious diseases and animal envenomings. However, designing and developing broadly-neutralizing antibodies can be cumbersome using traditional low-throughput iterative protein engineering methods. Here, we present a new high-throughput approach for the standardized discovery of broadly-neutralizing monoclonal antibodies relying on phage display technology and consensus antigens representing average sequences of related proteins. We showcase the utility of this approach by applying it to toxic sphingomyelinases from the venoms of species from very distant orders of the animal kingdom, the recluse spider and Gadim scorpion. First, we designed a consensus sphingomyelinase and performed three rounds of phage display selection, followed by DELFIA-based screening and ranking, and benchmarked this to a similar campaign involving cross-panning against recombinant versions of the native toxins. Second, we identified two scFvs that not only bind the consensus toxins, but which can also neutralize sphingomyelinase activity of native whole venom in vitro. Finally, we conclude that the phage display campaign involving the use of the consensus toxin was more successful in yielding cross-neutralizing scFvs than the phage display campaign involving cross-panning.
Subject(s)
Sphingomyelin Phosphodiesterase , Spider Venoms , Animals , Brown Recluse Spider , Scorpions , Broadly Neutralizing Antibodies , Consensus , Antibodies, MonoclonalABSTRACT
Protein structure determination is a critical aspect of biological research, enabling us to understand protein function and potential applications. Recent advances in deep learning and artificial intelligence have led to the development of several protein structure prediction tools, such as AlphaFold2 and ColabFold. However, their performance has primarily been evaluated on well-characterised proteins and their ability to predict sturtctures of proteins lacking experimental structures, such as many snake venom toxins, has been less scrutinised. In this study, we evaluated three modelling tools on their prediction of over 1000 snake venom toxin structures for which no experimental structures exist. Our findings show that AlphaFold2 (AF2) performed the best across all assessed parameters. We also observed that ColabFold (CF) only scored slightly worse than AF2, while being computationally less intensive. All tools struggled with regions of intrinsic disorder, such as loops and propeptide regions, and performed well in predicting the structure of functional domains. Overall, our study highlights the importance of exercising caution when working with proteins with no experimental structures available, particularly those that are large and contain flexible regions. Nonetheless, leveraging computational structure prediction tools can provide valuable insights into the modelling of protein interactions with different targets and reveal potential binding sites, active sites, and conformational changes, as well as into the design of potential molecular binders for reagent, diagnostic, or therapeutic purposes.
Subject(s)
Artificial Intelligence , Snake Venoms , Binding Sites , Furylfuramide , Proteins/chemistry , Snake Venoms/chemistryABSTRACT
Recycling IgG antibodies bind to their target antigen at physiological pH in the blood stream and release them upon endocytosis when pH levels drop, allowing the IgG antibodies to be recycled into circulation via FcRn-mediated cellular pathways, while the antigens undergo lysosomal degradation. This enables recycling antibodies to achieve comparable therapeutic effect at lower doses than their non-recycling counterparts. The development of such antibodies is typically achieved by histidine doping of their variable regions or by performing in vitro antibody selection campaigns utilizing histidine doped libraries. Both are strategies that may introduce sequence liabilities. Here, we present a methodology that employs a naïve antibody phage display library, consisting of natural variable domains, to discover antibodies that bind α-cobratoxin from the venom of Naja kaouthia in a pH-dependent manner. As a result, an antibody was discovered that exhibits a 7-fold higher off-rate at pH 5.5 than pH 7.4 in bio-layer interferometry experiments. Interestingly, no histidine residues were found in its variable domains, and in addition, the antibody showed pH-dependent binding to a histidine-devoid antigen mutant. As such, the results demonstrate that pH-dependent antigen-antibody binding may not always be driven by histidine residues. By employing molecular dynamics simulations, different protonation states of titratable residues were found, which potentially could be responsible for the observed pH-dependent antigen binding properties of the antibody. Finally, given the typically high diversity of naïve antibody libraries, the methodology presented here can likely be applied to discover recycling antibodies against different targets ab initio without the need for histidine doping.
Subject(s)
Bacteriophages , Histidine , Histidine/metabolism , Antigens/metabolism , Immunoglobulin G/genetics , Hydrogen-Ion Concentration , Bacteriophages/metabolism , Peptide LibraryABSTRACT
Despite the considerable global impact of snakebite envenoming, available treatments remain suboptimal. Here, we report the discovery of a broadly-neutralizing human monoclonal antibody, using a phage display-based cross-panning strategy, capable of reducing the cytotoxic effects of venom phospholipase A2s from three different snake genera from different continents. This highlights the potential of utilizing monoclonal antibodies to develop more effective, safer, and globally accessible polyvalent antivenoms that can be widely used to treat snakebite envenoming.
Subject(s)
Snake Bites , Animals , Humans , Venoms , Antibodies, Monoclonal , Antivenins/pharmacology , Snakes , Phospholipases A2 , Snake VenomsABSTRACT
Firefly luciferase is homologous to fatty acyl-CoA synthetases from insects that are not bioluminescent. Here, we determined the crystal structure of the fruit fly fatty acyl-CoA synthetase CG6178 to 2.5 Å. Based on this structure, we mutated a steric protrusion in the active site to create the artificial luciferase FruitFire, which prefers the synthetic luciferin CycLuc2 to d-luciferin by >1000-fold. FruitFire enabled in vivo bioluminescence imaging in the brains of mice using the pro-luciferin CycLuc2-amide. The conversion of a fruit fly enzyme into a luciferase capable of in vivo imaging underscores the potential for bioluminescence with a range of adenylating enzymes from nonluminescent organisms, and the possibilities for application-focused design of enzyme-substrate pairs.
ABSTRACT
Antibodies with cross-reactive binding and broad toxin-neutralizing capabilities are advantageous for treating indications such as infectious diseases and animal envenomings. Such antibodies have been successfully selected against closely related antigens using phage display technology. However, the mechanisms driving antibody cross-reactivity typically remain to be elucidated. Therefore, we sought to explore how a previously reported phage display-based cross-panning strategy drives the selection of cross-reactive antibodies using seven different snake toxins belonging to three protein (sub-)families: phospholipases A2, long-chain α-neurotoxins, and short-chain α-neurotoxins. We showcase how cross-panning can increase the chances of discovering cross-reactive single-chain variable fragments (scFvs) from phage display campaigns. Further, we find that the feasibility of discovering cross-reactive antibodies using cross-panning cannot easily be predicted by analyzing the sequence, structural, or surface similarity of the antigens alone. However, when antigens share the (exact) same functions, this seems to increase the chances of selecting cross-reactive antibodies, which may possibly be due to the existence of structurally similar motifs on the antigens.
Subject(s)
Bacteriophages , Single-Chain Antibodies , Animals , Peptide Library , Neurotoxins , Antigens , Bacteriophages/genetics , Snake VenomsABSTRACT
Phage display technology is a powerful tool for selecting monoclonal antibodies against a diverse set of antigens. Within toxinology, however, it remains challenging to generate monoclonal antibodies against many animal toxins, as they are difficult to obtain from venom. Recombinant toxins have been proposed as a solution to overcome this challenge, but so far, few have been used as antigens to generate neutralizing antibodies. Here, we describe the recombinant expression of α-cobratoxin in E. coli and its successful application as an antigen in a phage display selection campaign. From this campaign, an scFv (single-chain variable fragment) was isolated with similar binding affinity to a control scFv generated against the native toxin. The selected scFv recognizes a structural epitope, enabling it to inhibit the interaction between the acetylcholine receptor and the native toxin in vitro. This approach represents the first entirely in vitro antibody selection strategy for generating neutralizing monoclonal antibodies against a snake toxin.
Subject(s)
Bacteriophages , Single-Chain Antibodies , Animals , Single-Chain Antibodies/genetics , Epitopes , Peptide Library , Escherichia coli/genetics , Escherichia coli/metabolism , Antibodies, Monoclonal , Snake Venoms/metabolism , Bacteriophages/metabolismABSTRACT
The urokinase-type plasminogen activator receptor (uPAR) is an essential regulator for cell signaling in tumor cell proliferation, adhesion, and metastasis. The ubiquitous nature of uPAR in many aggressive cancer types makes uPAR an attractive target for immunotherapy. Here, we present a rapid and successful workflow for developing cross-reactive anti-uPAR recombinant antibodies (rAbs) using high-throughput optofluidic screening of single B-cells from human uPAR-immunized mice. A total of 80 human and cynomolgus uPAR cross-reactive plasma cells were identified, and selected mouse VH/VL domains were linked to the trastuzumab (Herceptin®) constant domains for the expression of mouse-human chimeric antibodies. The resulting rAbs were characterized by their tumor-cell recognition, binding activity, and cell adhesion inhibition on triple-negative breast cancer cells. In addition, the rAbs were shown to enact antibody-dependent cellular cytotoxicity (ADCC) in the presence of either human natural killer cells or peripheral blood mononuclear cells, and were evaluated for the potential use of uPAR-targeting antibody-drug conjugates (ADCs). Three lead antibodies (11857, 8163, and 3159) were evaluated for their therapeutic efficacy in vivo and were shown to suppress tumor growth. Finally, the binding epitopes of the lead antibodies were characterized, providing information on their unique binding modes to uPAR. Altogether, the strategy identified unique cross-reactive antibodies with ADCC, ADC, and functional inhibitory effects by targeting cell-surface uPAR, that can be tested in safety studies and serve as potential immunotherapeutics.
Subject(s)
Leukocytes, Mononuclear , Receptors, Urokinase Plasminogen Activator , Humans , Animals , Mice , Antibodies , Signal Transduction , B-LymphocytesABSTRACT
Single-molecule electron sources of fullerenes driven via constant electric fields, approximately 1 nm in size, produce peculiar emission patterns, such as a cross or a two-leaf pattern. By illuminating the electron sources with femtosecond light pulses, we discovered that largely modulated emission patterns appeared from single molecules. Our simulations revealed that emission patterns, which have been an intractable question for over seven decades, represent single-molecule molecular orbitals. Furthermore, the observed modulations originated from variations of single-molecule molecular orbitals, practically achieving the subnanometric optical modulation of an electron source.
ABSTRACT
Immunotargeting of tumor-specific antigens is a powerful therapeutic strategy. Immunotherapies directed at MHC-I complexes have expanded the scope of antigens and enabled the direct targeting of intracellular oncoproteins at the cell surface. We asked whether covalent drugs that alkylate mutated residues on oncoproteins could act as haptens to generate unique MHC-I-restricted neoantigens. Here, we report that KRAS G12C mutant cells treated with the covalent inhibitor ARS1620 present ARS1620-modified peptides in MHC-I complexes. Using ARS1620-specific antibodies identified by phage display, we show that these haptenated MHC-I complexes can serve as tumor-specific neoantigens and that a bispecific T cell engager construct based on a hapten-specific antibody elicits a cytotoxic T cell response against KRAS G12C cells, including those resistant to direct KRAS G12C inhibition. With multiple K-RAS G12C inhibitors in clinical use or undergoing clinical trials, our results present a strategy to enhance their efficacy and overcome the rapidly arising tumor resistance.
Subject(s)
Antineoplastic Agents , Histocompatibility Antigens Class I/immunology , Neoplasms , Proto-Oncogene Proteins p21(ras)/antagonists & inhibitors , Antibodies , Antineoplastic Agents/pharmacology , Humans , Immunologic Factors , Immunotherapy , Peptides/pharmacology , Proto-Oncogene Proteins p21(ras)/geneticsABSTRACT
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.
Subject(s)
Elapidae , Single-Domain Antibodies , Animals , Elapid Venoms/chemistry , Elapid Venoms/metabolism , Elapidae/metabolism , Neurotoxins/chemistry , Neurotoxins/metabolism , Single-Domain Antibodies/metabolism , Tumor Suppressor Protein p53/metabolismABSTRACT
Viruses are responsible for some of the most deadly human diseases, yet available vaccines and antivirals address only a fraction of the potential viral human pathogens. Here, we provide a methodology for managing human herpesvirus (HHV) infection by covalently inactivating the HHV maturational protease via a conserved, non-catalytic cysteine (C161). Using human cytomegalovirus protease (HCMV Pr) as a model, we screened a library of disulfides to identify molecules that tether to C161 and inhibit proteolysis, then elaborated hits into irreversible HCMV Pr inhibitors that exhibit broad-spectrum inhibition of other HHV Pr homologs. We further developed an optimized tool compound targeted toward HCMV Pr and used an integrative structural biology and biochemical approach to demonstrate inhibitor stabilization of HCMV Pr homodimerization, exploiting a conformational equilibrium to block proteolysis. Irreversible HCMV Pr inhibition disrupts HCMV infectivity in cells, providing proof of principle for targeting proteolysis via a non-catalytic cysteine to manage viral infection.
Subject(s)
Cytomegalovirus Infections , Cytomegalovirus , Cysteine , Cytomegalovirus/physiology , Humans , Peptide Hydrolases , Viral ProteasesABSTRACT
Applying strong direct current (DC) electric fields on the apex of a sharp metallic tip, electrons can be radially emitted from the apex to vacuum. Subsequently, they magnify the nanoscopic information on the apex, which serves as a field emission microscope (FEM). When depositing molecules on such a tip, peculiar electron emission patterns such as clover leaves appear. These phenomena were first observed seventy years ago. However, the source of these emission patterns has not yet been identified owing to the limited experimental information about molecular configurations on a tip. Here, we used fullerene molecules and characterized the molecule-covered tip by an FEM. In addition to the experiments, simulations were performed to obtain optimized molecular configurations on a tip. Both results indicate that the molecules, the source of the peculiar emission patterns, appear on a molecule layer formed on the tip under strong DC electric fields. Furthermore, the simulations revealed that these molecules are mostly isolated single molecules forming single-molecule-terminated protrusions. Upon the excellent agreements in both results, we concluded that each emission pattern originates from a single molecule. Our work should pave the way to revive old-fashioned electron microscopy as a powerful tool for investigating a single molecule.
ABSTRACT
Inorganic nanocrystals such as quantum dots (QDs) and upconverting nanoparticles (UCNPs) are uniquely suited for quantitative live-cell imaging and are typically functionalized with ligands to study specific receptors or cellular targets. Antibodies (Ab) are among the most useful targeting reagents owing to their high affinities and specificities, but common nanocrystal labeling methods may orient Ab incorrectly, be reversible or denaturing, or lead to Ab-NP complexes too large for some applications. Here, we show that SpyCatcher proteins, which bind and spontaneously form covalent isopeptide bonds with cognate SpyTag peptides, can conjugate engineered Ab to nanoparticle surfaces with control over stability, orientation, and stoichiometry. Compact SpyCatcher-functionalized QDs and UCNPs may be labeled with short-chain variable fragment Ab (scFv) engineered to bind urokinase-type plasminogen activator receptors (uPAR) that are overexpressed in many human cancers. Confocal imaging of anti-uPAR scFv-QD conjugates shows the antibody mediates specific binding and internalization by breast cancer cells expressing uPAR. Time-lapse imaging of photostable scFv-UCNP conjugates shows that Ab binding causes uPAR internalization with a â¼20 min half-life on the cell surface, and uPAR is internalized to endolysosomal compartments distinct from general membrane stains and without significant recycling to the cell surface. The controlled and stable conjugation of engineered Ab to NPs enables targeting of diverse receptors for live-cell study of their distribution, trafficking, and physiology.
Subject(s)
Nanoparticles , Quantum Dots , Humans , Receptors, Urokinase Plasminogen Activator/metabolism , Antibodies/metabolism , Cell Membrane/metabolism , Urokinase-Type Plasminogen Activator/metabolismABSTRACT
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.
Subject(s)
Antibodies, Neutralizing/chemistry , Giant Cells/metabolism , Spike Glycoprotein, Coronavirus/chemistry , Angiotensin-Converting Enzyme 2/chemistry , Angiotensin-Converting Enzyme 2/genetics , Angiotensin-Converting Enzyme 2/immunology , Angiotensin-Converting Enzyme 2/metabolism , Animals , Antibodies, Neutralizing/immunology , Antibodies, Neutralizing/metabolism , Antigen-Antibody Complex/chemistry , Antigen-Antibody Complex/metabolism , Binding Sites , CHO Cells , COVID-19/pathology , COVID-19/virology , Cricetinae , Cricetulus , Cryoelectron Microscopy , Giant Cells/cytology , Humans , Membrane Fusion , Peptide Library , Protein Binding , Protein Domains , Protein Structure, Quaternary , SARS-CoV-2/isolation & purification , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/immunology , Spike Glycoprotein, Coronavirus/metabolismABSTRACT
In vitro antibody selection against pathogens from naïve combinatorial libraries can yield various classes of antigen-specific binders that are distinct from those evolved from natural infection1-4. Also, rapid neutralizing antibody discovery can be made possible by a strategy that selects for those interfering with pathogen and host interaction5. Here we report the discovery of antibodies that neutralize SARS-CoV-2, the virus responsible for the COVID-19 pandemic, from a highly diverse naïve human Fab library. Lead antibody 5A6 blocks the receptor binding domain (RBD) of the viral spike from binding to the host receptor angiotensin converting enzyme 2 (ACE2), neutralizes SARS-CoV-2 infection of Vero E6 cells, and reduces viral replication in reconstituted human nasal and bronchial epithelium models. 5A6 has a high occupancy on the viral surface and exerts its neutralization activity via a bivalent binding mode to the tip of two neighbouring RBDs at the ACE2 interaction interface, one in the "up" and the other in the "down" position, explaining its superior neutralization capacity. Furthermore, 5A6 is insensitive to several spike mutations identified in clinical isolates, including the D614G mutant that has become dominant worldwide. Our results suggest that 5A6 could be an effective prophylactic and therapeutic treatment of COVID-19.
ABSTRACT
Protein-protein interactions between E3 ubiquitin ligases and protein termini help shape the proteome. These interactions are sensitive to proteolysis, which alters the ensemble of cellular N and C termini. Here we describe a mechanism wherein caspase activity reveals latent C termini that are then recognized by the E3 ubiquitin ligase CHIP. Using expanded knowledge of CHIP's binding specificity, we predicted hundreds of putative interactions arising from caspase activity. Subsequent validation experiments confirmed that CHIP binds the latent C termini at tauD421 and caspase-6D179. CHIP binding to tauD421, but not tauFL, promoted its ubiquitination, while binding to caspase-6D179 mediated ubiquitin-independent inhibition. Given that caspase activity generates tauD421 in Alzheimer's disease (AD), these results suggested a concise model for CHIP regulation of tau homeostasis. Indeed, we find that loss of CHIP expression in AD coincides with the accumulation of tauD421 and caspase-6D179. These results illustrate an unanticipated link between caspases and protein homeostasis.
Subject(s)
Caspases/metabolism , Ubiquitin-Protein Ligases/metabolism , Caspases/genetics , Cell Line, Tumor , Crystallography, X-Ray , Escherichia coli/metabolism , Gene Expression Regulation , Humans , Protein Binding , Ubiquitin/genetics , Ubiquitin/metabolism , Ubiquitin-Activating Enzymes/genetics , Ubiquitin-Activating Enzymes/metabolism , UbiquitinationABSTRACT
Enzymes that modify the proteome, referred to as post-translational modifying (PTM) enzymes, are central regulators of cellular signaling. Determining the substrate specificity of PTM enzymes is a critical step in unraveling their biological functions both in normal physiological processes and in disease states. Advances in peptide chemistry over the last century have enabled the rapid generation of peptide libraries for querying substrate recognition by PTM enzymes. In this article, we highlight various peptide-based approaches for analysis of PTM enzyme substrate specificity. We focus on the application of these technologies to proteases and also discuss specific examples in which they have been used to uncover the substrate specificity of other types of PTM enzymes, such as kinases. In particular, we highlight our multiplex substrate profiling by mass spectrometry (MSP-MS) assay, which uses a rationally designed, physicochemically diverse library of tetradecapeptides. We show how this method has been applied to PTM enzymes to uncover biological function, and guide substrate and inhibitor design. We also briefly discuss how this technique can be combined with other methods to gain a systems-level understanding of PTM enzyme regulation and function.