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Development of a human antibody fragment cross-neutralizing scorpion toxins
Abstract
Previously, it was demonstrated that from the single chain fragment variable (scFv) 3F it is possible to generate variants capable of neutralizing the Cn2 and Css2 toxins, as well as their respective venoms (Centruroides noxius and Centruroides suffusus). Despite this success, it has not been easy to modify the recognition of this family of scFvs toward other dangerous scorpion toxins. The analysis of toxin-scFv interactions and in vitro maturation strategies allowed us to propose a new maturation pathway for scFv 3F to broaden recognition toward other Mexican scorpion toxins. From maturation processes against toxins CeII9 from C. elegans and Ct1a from C. tecomanus, the scFv RAS27 was developed. This scFv showed an increased affinity and cross-reactivity for at least 9 different toxins while maintaining recognition for its original target, the Cn2 toxin. In addition, it was confirmed that it can neutralize at least three different toxins. These results constitute an important advance since it was possible to improve the cross-reactivity and neutralizing capacity of the scFv 3F family of antibodies.
Centruroides huichol scorpion venom is lethal to mammals. Analysis of the venom allowed the characterization of four lethal toxins named Chui2, Chui3, Chui4, and Chui5. scFv 10FG2 recognized well all toxins except Chui5 toxin, therefore a partial neutralization of the venom was observed. Thus, scFv 10FG2 was subjected to three processes of directed evolution and phage display against Chui5 toxin until obtaining scFv HV. Interaction kinetic constants of these scFvs with the toxins were determined by surface plasmon resonance (SPR) as well as thermodynamic parameters of scFv variants bound to Chui5. In silico models allowed to analyze the molecular interactions that favor the increase in affinity. In a rescue trial, scFv HV protected 100% of the mice injected with three lethal doses 50 (LD50) of venom. Moreover, in mix-type neutralization assays, a combination of scFvs HV and 10FG2 protected 100% of mice injected with 5 LD50 of venom with moderate signs of intoxication. The ability of scFv HV to neutralize different toxins is a significant achievement, considering the diversity of the species of Mexican venomous scorpions, so this scFv is a candidate to be part of a recombinant anti-venom against scorpion stings in Mexico.
Antibody drugs exert therapeutic effects via a range of mechanisms, including competitive inhibition, allosteric modulation, and immune effector mechanisms. Facilitated dissociation is an additional mechanism where antibody-mediated “disruption” of stable high-affinity macromolecular complexes can potentially enhance therapeutic efficacy. However, this mechanism is not well understood or utilized therapeutically. Here, we investigate and engineer the weak disruptive activity of an existing therapeutic antibody, omalizumab, which targets IgE antibodies to block the allergic response. We develop a yeast display approach to select for and engineer antibody disruptive efficiency and generate potent omalizumab variants that dissociate receptor-bound IgE. We determine a low resolution cryo-EM structure of a transient disruption intermediate containing the IgE-Fc, its partially dissociated receptor and an antibody inhibitor. Our results provide a conceptual framework for engineering disruptive inhibitors for other targets, insights into the failure in clinical trials of the previous high affinity omalizumab HAE variant and anti-IgE antibodies that safely and rapidly disarm allergic effector cells.
A fundamental issue of the characterization of single-chain variable fragments (scFvs), capable of neutralizing scorpion toxins, is their cross-neutralizing ability. This aspect is very important in Mexico because all scorpions dangerous to humans belong to the Centruroides genus, where toxin sequences show high identity. Among toxin-neutralizing antibodies that were generated in a previous study, scFv 10FG2 showed a broad cross-reactivity against several Centruroides toxins, while the one of scFv LR is more limited. Both neutralizing scFvs recognize independent epitopes of the toxins. In the present work, the neutralization capacity of these two scFvs against two medically important toxins of the venom of Centruroides sculpturatus Ewing was evaluated. The results showed that these toxins are recognized by both scFvs with affinities between 1.8 × 10⁻⁹ and 6.1 × 10⁻¹¹ M. For this reason, their ability to neutralize the venom was evaluated in mice, where scFv 10FG2 showed a better protective capacity. A combination of both scFvs at a molar ratio of 1:5:5 (toxins: scFv 10FG2: scFv LR) neutralized the venom without the appearance of any signs of intoxication. These results indicate a complementary activity of these two scFvs during venom neutralization.
Antibodies are essential molecules for diagnosis and treatment of diseases caused by pathogens and their toxins. Antibodies were integrated in our medical repertoire against infectious diseases more than hundred years ago by using animal sera to treat tetanus and diphtheria. In these days, most developed therapeutic antibodies target cancer or autoimmune diseases. The COVID-19 pandemic was a reminder about the importance of antibodies for therapy against infectious diseases. While monoclonal antibodies could be generated by hybridoma technology since the 70ies of the former century, nowadays antibody phage display, among other display technologies, is robustly established to discover new human monoclonal antibodies. Phage display is an in vitro technology which confers the potential for generating antibodies from universal libraries against any conceivable molecule of sufficient size and omits the limitations of the immune systems. If convalescent patients or immunized/infected animals are available, it is possible to construct immune phage display libraries to select in vivo affinity-matured antibodies. A further advantage is the availability of the DNA sequence encoding the phage displayed antibody fragment, which is packaged in the phage particles. Therefore, the selected antibody fragments can be rapidly further engineered in any needed antibody format according to the requirements of the final application. In this review, we present an overview of phage display derived recombinant antibodies against bacterial, viral and eukaryotic pathogens, as well as microbial toxins, intended for diagnostic and therapeutic applications.
For years, a discussion has persevered on the benefits and drawbacks of antibody discovery using animal immunization versus in vitro selection from non-animal-derived recombinant repertoires using display technologies. While it has been argued that using recombinant display libraries can reduce animal consumption, we hold that the number of animals used in immunization campaigns is dwarfed by the number sacrificed during preclinical studies. Thus, improving quality control of antibodies before entering in vivo studies will have a larger impact on animal consumption. Both animal immunization and recombinant repertoires present unique advantages for discovering antibodies that are fit for purpose. Furthermore, we anticipate that machine learning will play a significant role within discovery workflows, refining current antibody discovery practices.
The aggregation of amyloidogenic polypeptides is strongly linked to several neurodegenerative disorders, including Alzheimer’s and Parkinson’s diseases. Conformational antibodies that selectively recognize protein aggregates are leading therapeutic agents for selectively neutralizing toxic aggregates, diagnostic and imaging agents for detecting disease, and biomedical reagents for elucidating disease mechanisms. Despite their importance, it is challenging to generate high-quality conformational antibodies in a systematic and site-specific manner due to the properties of protein aggregates (hydrophobic, multivalent and heterogeneous) and limitations of immunization (uncontrolled antigen presentation and immunodominant linear epitopes). Toward addressing these challenges, we have developed a systematic directed evolution procedure for affinity maturing antibodies against Alzheimer’s Aβ fibrils and selecting variants with strict conformational and sequence specificity. We first designed a library based on a lead conformational antibody by sampling combinations of amino acids in the antigen-binding site predicted to lead to high antibody specificity. Next, we displayed this library on the surface of yeast, sorted it against Aβ aggregates, and identified promising clones using deep sequencing. We identified several antibodies with similar or higher affinities than clinical-stage Aβ antibodies (aducanumab and crenezumab). Moreover, the affinity-matured antibodies retain high conformational specificity for Aβ aggregates, as observed for aducanumab and unlike crenezumab. Notably, the affinity-maturated antibodies display extremely low levels of non-specific interactions, as observed for crenezumab and unlike aducanumab. We expect that our systematic methods for generating antibodies with unique combinations of desirable properties will improve the generation of high-quality conformational antibodies specific for diverse types of aggregated conformers.
Broadly-neutralizing monoclonal antibodies are of high therapeutic utility against infectious diseases caused by bacteria and viruses, as well as different types of intoxications. Snakebite envenoming is one such debilitating pathology, which is currently treated with polyclonal antibodies derived from immunized animals. For the development of novel envenoming therapies based on monoclonal antibodies with improved therapeutic benefits, new discovery approaches for broadly-neutralizing antibodies are needed. Here, we present a methodology based on phage display technology and a cross-panning strategy that enables the selection of cross-reactive monoclonal antibodies that can broadly neutralize toxins from different snake species. This simple in vitro methodology is immediately useful for the development of broadly-neutralizing (polyvalent) recombinant antivenoms with broad species coverage, but may also find application in the development of broadly-neutralizing antibodies against bacterial, viral, and parasitic agents that are known for evading therapy via resistance mechanisms and antigen variation.
Significance
The accurate quantification of the amounts of small oligomeric assemblies formed by the amyloid β (Aβ) peptide represents a major challenge in the Alzheimer’s field. There is therefore great interest in the development of methods to specifically detect these oligomers by distinguishing them from larger aggregates. The availability of these methods will enable the development of effective diagnostic and therapeutic interventions for this and other diseases related to protein misfolding and aggregation. We describe here a single-domain antibody able to selectively quantify oligomers of the Aβ peptide in isolation and in complex protein mixtures from animal models of disease.
In this communication the isolation, chemical and physiological characterization of three new toxins from the scorpion Centruroides baergi are reported. Their immunoreactive properties with scFvs generated in our group are described. The three new peptides, named Cb1, Cb2 and Cb3 affect voltage-dependent Na⁺ channels in a differential manner. These characteristics, explain the toxicity of this venom. Molecular interactions in real-time among these toxins and the best recombinant antibodies generated in our group, revealed that one of them was able to neutralize the main toxin of this venom (Cb1). These results represent an important advance for the neutralization of this venom and serve as the basis for generating new scFvs that will allow the neutralization of similar toxins from other venoms that have no yet been neutralized.
It has been more than three decades since the first monoclonal antibody was approved by the United States Food and Drug Administration (US FDA) in 1986, and during this time, antibody engineering has dramatically evolved. Current antibody drugs have increasingly fewer adverse effects due to their high specificity. As a result, therapeutic antibodies have become the predominant class of new drugs developed in recent years. Over the past five years, antibodies have become the best-selling drugs in the pharmaceutical market, and in 2018, eight of the top ten bestselling drugs worldwide were biologics. The global therapeutic monoclonal antibody market was valued at approximately US150 billion by the end of 2019 and $300 billion by 2025. Thus, the market for therapeutic antibody drugs has experienced explosive growth as new drugs have been approved for treating various human diseases, including many cancers, autoimmune, metabolic and infectious diseases. As of December 2019, 79 therapeutic mAbs have been approved by the US FDA, but there is still significant growth potential. This review summarizes the latest market trends and outlines the preeminent antibody engineering technologies used in the development of therapeutic antibody drugs, such as humanization of monoclonal antibodies, phage display, the human antibody mouse, single B cell antibody technology, and affinity maturation. Finally, future applications and perspectives are also discussed.
The recombinant antibody fragments generated against the toxic components of scorpion venoms are considered a promising alternative for obtaining new antivenoms for therapy. Using directed evolution and site-directed mutagenesis, it was possible to generate a human single-chain antibody fragment with a broad cross-reactivity that retained recognition for its original antigen. This variant is the first antibody fragment that neutralizes the effect of an estimated 13 neurotoxins present in the venom of nine species of Mexican scorpions. This single antibody fragment showed the properties of a polyvalent antivenom. These results represent a significant advance in the development of new antivenoms against scorpion stings, since the number of components would be minimized due to their broad cross-neutralization capacity, while at the same time bypassing animal immunization.
Chemokine receptors typically have multiple ligands. Consequently, treatment with a blocking antibody against a single chemokine is expected to be insufficient for efficacy. Here we show single-chain antibodies can be engineered for broad crossreactivity toward multiple human and mouse proinflammatory ELR+ CXC chemokines. The engineered molecules recognize functional epitopes of ELR+ CXC chemokines and inhibit neutrophil activation ex vivo. Furthermore, an albumin fusion of the most crossreactive single-chain antibody prevents and reverses inflammation in the K/BxN mouse model of arthritis. Thus, we report an approach for the molecular evolution and selection of broadly crossreactive antibodies towards a family of structurally related, yet sequence-diverse protein targets, with general implications for the development of novel therapeutics.
Antibody technologies are being increasingly applied in the field of toxinology. Fuelled by the many advances in immunology, synthetic biology, and antibody research, different approaches and antibody formats are being investigated for the ability to neutralize animal toxins. These different molecular formats each have their own therapeutic characteristics. In this review, we provide an overview of the advances made in the development of toxin-targeting antibodies, and discuss the benefits and drawbacks of different antibody formats in relation to their ability to neutralize toxins, pharmacokinetic features, propensity to cause adverse reactions, formulation, and expression for research and development (R&D) purposes and large-scale manufacturing. A research trend seems to be emerging towards the use of human antibody formats as well as camelid heavy-domain antibody fragments due to their compatibility with the human immune system, beneficial therapeutic properties, and the ability to manufacture these molecules cost-effectively.
The current trend of using recombinant antibody fragments in research to develop novel antidotes against scorpion stings has achieved excellent results. The polyclonal character of commercial
antivenoms, obtained through the immunization of animals and which contain several neutralizing antibodies that recognize different epitopes on the toxins, guarantees the neutralization
of the venoms. To avoid the use of animals, we aimed to develop an equivalent recombinant antivenom composed of a few neutralizing single chain antibody fragments (scFvs) that bind to two different epitopes on the scorpion toxins. In this study, we obtained scFv RU1 derived from scFv C1. RU1 showed a good capacity to neutralize the Cn2 toxin and whole venom of the scorpion Centruroides noxius. Previously, we had produced scFv LR, obtained from a different parental fragment (scFv 3F). LR also showed a similar neutralizing capacity. The simultaneous administration of both scFvs resulted in improved protection, which was translated as a rapid recovery of previously poisoned animals. The crystallographic structure of the ternary complex scFv LR-Cn2-scFv RU1 allowed us to identify the areas of interaction of both scFvs with the toxin, which correspond to
non-overlapping sites. The epitope recognized by scFv RU1 seems to be related to a greater efficiency in the neutralization of the whole venom. In addition, the structural analysis of the complex helped us to explain the cross-reactivity of these scFvs and how they neutralize the venom.
Fish allergy is associated with moderate to severe IgE-mediated reactions to the calcium binding parvalbumins present in fish muscle. Allergy to multiple fish species is caused by parvalbumin-specific cross-reactive IgE recognizing conserved epitopes. In this study, we aimed to produce cross-reactive single chain variable fragment (scFv) antibodies for the detection of parvalbumins in fish extracts and the identification of IgE epitopes. Parvalbumin-specific phage clones were isolated from the human ETH-2 phage display library by three rounds of biopanning either against cod parvalbumin or by sequential biopanning against cod (Gad m 1), carp (Cyp c 1) and rainbow trout (Onc m 1) parvalbumins. While biopanning against Gad m 1 resulted in the selection of clones specific exclusively for Gad m 1, the second approach resulted in the selection of clones cross-reacting with all three parvalbumins. Two clones, scFv-gco9 recognizing all three parvalbumins, and scFv-goo8 recognizing only Gad m 1 were expressed in the E. coli non-suppressor strain HB2151 and purified from the periplasm. scFv-gco9 showed highly selective binding to parvalbumins in processed fish products such as breaded cod sticks, fried carp and smoked trout in Western blots. In addition, the scFv-gco9-AP produced as alkaline phosphatase fusion protein, allowed a single-step detection of the parvalbumins. In competitive ELISA, scFv-gco9 was able to inhibit binding of IgE from fish allergic patients' sera to all three β-parvalbumins by up to 80%, whereas inhibition by scFv-goo8 was up to 20%. 1H/15N HSQC NMR analysis of the rGad m 1:scFv-gco9 complex showed participation of amino acid residues conserved among these three parvalbumins explaining their cross-reactivity on a molecular level. In this study, we have demonstrated an approach for the selection of cross-reactive parvalbumin-specific antibodies that can be used for allergen detection and for mapping of conserved epitopes.
Background:
Botulism is a naturally occurring disease, mainly caused by the ingestion of food contaminated by the botulinum neurotoxins (BoNTs). Botulinum neurotoxins are the most lethal. They are classified among the six major biological warfare agents by the Centers for Disease Control. BoNTs act on the cholinergic motoneurons, where they cleave proteins implicated in acetylcholine vesicle exocytosis. This exocytosis inhibition induces a flaccid paralysis progressively affecting all the muscles and generally engendering a respiratory distress. BoNTs are also utilized in medicine, mainly for the treatment of neuromuscular disorders, preventing large scale vaccination. Botulism specific treatment requires injections of antitoxins, usually of equine origin and thus poorly tolerated. Therefore, development of human or human-like neutralizing antibodies is of a major interest, and it is the subject of the European framework project called "AntiBotABE".
Results:
In this study, starting from a macaque immunized with the recombinant heavy chain of BoNT/A1 (BoNT/A1-HC), an immune antibody phage-display library was generated and antibody fragments (single chain Fragment variable) with nanomolar affinity were isolated and further characterized. The neutralization capacities of these scFvs were analyzed in the mouse phrenic nerve-hemidiaphragm assay.
Conclusions:
After a three-round panning, 24 antibody fragments with affinity better than 10 nM were isolated. Three of them neutralized BoNT/A1 efficiently and two cross-neutralized BoNT/A1 and BoNT/A2 subtypes in the mouse phrenic nerve-hemidiaphragm assay. These are the first monoclonal human-like antibodies cross-neutralizing both BoNT/A1 and BoNT/A2. The antibody A1HC38 was selected for further development, and could be clinically developed for the prophylaxis and treatment of botulism.
ABSTRACT Staphylococcus aureus is a major human pathogen associated with high mortality. The emergence of antibiotic resistance and the inability of antibiotics to counteract bacterial cytotoxins involved in the pathogenesis of S. aureus call for novel therapeutic approaches, such as passive immunization with monoclonal antibodies (mAbs). The complexity of staphylococcal pathogenesis and past failures with single mAb products represent considerable barriers for antibody-based therapeutics. Over the past few years, efforts have focused on neutralizing alpha-hemolysin. Recent findings suggest that the concerted actions of several cytotoxins, including the bi-component leukocidins play important roles in staphylococcal pathogenesis. Therefore, we aimed to isolate mAbs that bind to multiple cytolysins by employing high diversity human IgG1 libraries presented on the surface of yeast cells. Here we describe cross-reactive antibodies with picomolar affinity for alpha-hemolysin and four different bi-component leukocidins that share only ∼26% overall amino acid sequence identity. The molecular basis of cross-reactivity is the recognition of a conformational epitope shared by alpha-hemolysin and F-components of gamma-hemolysin (HlgAB and HlgCB), LukED and LukSF (Panton-Valentine Leukocidin). The amino acids predicted to form the epitope are conserved and known to be important for cytotoxic activity. We found that a single cross-reactive antibody prevented lysis of human phagocytes, epithelial and red blood cells induced by alpha-hemolysin and leukocidins in vitro, and therefore had superior effectiveness compared to alpha-hemolysin specific antibodies to protect from the combined cytolytic effect of secreted S. aureus toxins. Such mAb afforded high levels of protection in murine models of pneumonia and sepsis.
Norovirus infections are a common cause of gastroenteritis and new methods to rapidly diagnose norovirus infections are needed.
The goal of this study was to identify antibodies that have broad reactivity of binding to various genogroups of norovirus.
A human scFv phage display library was used to identify two antibodies, HJT-R3-A9 and HJT-R3-F7, which bind to both genogroups
I and II norovirus virus-like particles (VLPs). Mapping experiments indicated that the HJT-R3-A9 clone binds to the S-domain
while the HJT-R3-F7 clone binds the P-domain of the VP1 capsid protein. In addition, a family of scFv antibodies was identified
by elution of phage libraries from the GII.4 VLP target using a carbohydrate that serves as an attachment factor for norovirus
on human cells. These antibodies were also found to recognize both GI and GII VLPs in enzyme-linked immunosorbent assay (ELISA)
experiments. The HJT-R3-A9, HJT-R3-F7 and scFv antibodies identified with carbohydrate elution were shown to detect antigen
from a clinical sample known to contain GII.4 norovirus but not a negative control sample. Finally, phages displaying the
HJT-R3-A9 scFv can be used directly to detect both GI.1 and GII.4 norovirus from stool samples, which has the potential to
simplify and reduce the cost of diagnostics based on antibody-based ELISA methods.
Significance
This study demonstrated that cross-neutralizing anti-poliovirus antibodies bind the site on poliovirus capsid surface that significantly overlaps the binding site of the cellular receptor. A second antibody with similar specificity was isolated by sequential phage display panning, suggesting that cross-reactive anti-poliovirus antibodies may be more prevalent in primates than previously recognized. Binding to the receptor recognition site explains unusually broad specificity of the antibodies. The antibodies bind type 1 and type 2 polioviruses at a slightly different angle, indicating that molecular details of virus–antibody interaction are different and suggesting that further screening or engineering may produce an antibody neutralizing all three serotypes of poliovirus. These results may be used for developing new antiviral strategies for the polio eradication campaign.
Even though the effect of antibody affinity on neutralization potency is well documented, surprisingly, its impact on neutralization
breadth and escape has not been systematically determined. Here, random mutagenesis and DNA shuffling of the single-chain
variable fragment of the neutralizing antibody 80R followed by bacterial display screening using anchored periplasmic expression
(APEx) were used to generate a number of higher-affinity variants of the severe acute respiratory syndrome coronavirus (SARS-CoV)-neutralizing
antibody 80R with equilibrium dissociation constants (KD) as low as 37 pM, a >270-fold improvement relative to that of the parental 80R single-chain variable fragment (scFv). As
expected, antigen affinity was shown to correlate directly with neutralization potency toward the icUrbani strain of SARS-CoV.
Additionally, the highest-affinity antibody fragment displayed 10-fold-increased broad neutralization in vitro and completely protected against several SARS-CoV strains containing substitutions associated with antibody escape. Importantly,
higher affinity also led to the suppression of viral escape mutants in vitro. Escape from the highest-affinity variant required reduced selective pressure and multiple substitutions in the binding epitope.
Collectively, these results support the hypothesis that engineered antibodies with picomolar dissociation constants for a
neutralizing epitope can confer escape-resistant protection.
The Old World scorpion Androctonus australis hector (Aah) produces one of the most lethal venoms for humans. Peptidic α-toxins AahI to AahIV are responsible for its potency, with AahII accounting for half of it. All four toxins are high affinity blockers of the fast inactivation phase of mammalian voltage-activated Na(+) channels. However, the high antigenic polymorphism of α-toxins prevents production of a polyvalent neutralizing antiserum, whereas the determinants dictating their trapping by neutralizing antibodies remain elusive. From an anti-AahII mAb, we generated an antigen binding fragment (Fab) with high affinity and selectivity for AahII and solved a 2.3 Å-resolution crystal structure of the complex. Sequestering of the C-terminal region of the bound toxin within a groove formed by the Fab combining loops is associated with a toxin orientation and main and side chain conformations that dictate the AahII antigenic specificity and efficient neutralization. From an anti-AahI mAb, we also preformed and crystallized a high affinity AahI-Fab complex. The 1.6 Å-resolution structure solved revealed a Fab molecule devoid of a bound AahI and with combining loops involved in packing interactions, denoting expulsion of the bound antigen upon crystal formation. Comparative analysis of the groove-like combining site of the toxin-bound anti-AahII Fab and planar combining surface of the unbound anti-AahI Fab along with complementary data from a flexible docking approach suggests occurrence of distinctive trapping orientations for the two toxins relative to their respective Fab. This study provides complementary templates for designing new molecules aimed at capturing Aah α-toxins and suitable for immunotherapy.
It has previously been reported that several single-chain antibody fragments of human origin (scFv) neutralize the effects of two different scorpion venoms through interactions with the primary toxins of Centruroides noxius Hoffmann (Cn2) and Centruroides suffusus suffusus (Css2). Here we present the crystal structure of the complex formed between one scFv (9004G) and the Cn2 toxin, determined in two crystal forms at 2.5 and 1.9 Å resolution. A 15-residue span of the toxin is recognized by the antibody through a cleft formed by residues from five of the complementarity-determining regions of the scFv. Analysis of the interface of the complex reveals three features. First, the epitope of toxin Cn2 overlaps with essential residues for the binding of β-toxins to its Na(+) channel receptor site. Second, the putative recognition of Css2 involves mainly residues that are present in both Cn2 and Css2 toxins. Finally, the effect on the increase of affinity of previously reported key residues during the maturation process of different scFvs can be inferred from the structure. Taken together, these results provide the structural basis that explain the mechanism of the 9004G neutralizing activity and give insight into the process of directed evolution that gave rise to this family of neutralizing scFvs.
Flaviviruses are a group of human pathogenic, enveloped RNA viruses that includes dengue (DENV), yellow fever (YFV), West Nile (WNV), and Japanese encephalitis (JEV) viruses. Cross-reactive antibodies against Flavivirus have been described, but most of them are generally weakly neutralizing. In this study, a novel monoclonal antibody, designated mAb 2A10G6, was determined to have broad cross-reactivity with DENV 1-4, YFV, WNV, JEV, and TBEV. Phage-display biopanning and structure modeling mapped 2A10G6 to a new epitope within the highly conserved flavivirus fusion loop peptide, the (98)DRXW(101) motif. Moreover, in vitro and in vivo experiments demonstrated that 2A10G6 potently neutralizes DENV 1-4, YFV, and WNV and confers protection from lethal challenge with DENV 1-4 and WNV in murine model. Furthermore, functional studies revealed that 2A10G6 blocks infection at a step after viral attachment. These results define a novel broadly flavivirus cross-reactive mAb with highly neutralizing activity that can be further developed as a therapeutic agent against severe flavivirus infections in humans.
We report the optimization of a family of human single chain antibody fragments (scFv) for neutralizing two scorpion venoms. The parental scFv 3F recognizes the main toxins of Centruroides noxius Hoffmann (Cn2) and Centruroides suffusus suffusus (Css2), albeit with low affinity. This scFv was subjected to independent processes of directed evolution to improve its recognition toward Cn2 (Riaño-Umbarila, L., Juárez-González, V. R., Olamendi-Portugal, T., Ortíz-León, M., Possani, L. D., and Becerril, B. (2005) FEBS J. 272, 2591-2601) and Css2 (this work). Each evolved variant showed strong cross-reactivity against several toxins, and was capable of neutralizing Cn2 and Css2. Furthermore, each variant neutralized the whole venoms of the above species. As far as we know, this is the first report of antibodies with such characteristics. Maturation processes revealed key residue changes to attain expression, stability, and affinity improvements as compared with the parental scFv. Combination of these changes resulted in the scFv LR, which is capable of rescuing mice from severe envenomation by 3 LD(50) of freshly prepared whole venom of C. noxius (7.5 μg/20 g of mouse) and C. suffusus (26.25 μg/20 g of mouse), with surviving rates between 90 and 100%. Our research is leading to the formulation of an antivenom consisting of a discrete number of human scFvs endowed with strong cross-reactivity and low immunogenicity.
Chemokines are important mediators of the immune response that are responsible for the trafficking of immune cells between lymphoid organs and migration towards sites of inflammation.Using phage display selection and a functional screening approach, we have isolated a panel of single-chain fragment variable (scFv) capable of neutralizing the activity of the human chemokine CXCL10 (hCXCL10). One of the isolated scFv was weakly cross-reactive against another human chemokine CXCL9,but was unable to block its biological activity. We diversified the complementarity determining region 3 (CDR3) of the light chain variable domain (VL) of this scFv and combined phage display with high throughput antibody array screening to identify variants capable of neutralizing both chemokines. Using this approach it is therefore possible to engineer pan-specific antibodies that could prove very useful to antagonize redundant signaling pathways such as the chemokine signaling network.
Directed evolution circumvents our profound ignorance of how a protein's sequence encodes its function by using iterative rounds of random mutation and artificial selection to discover new and useful proteins. Proteins can be tuned to adapt to new functions or environments by simple adaptive walks involving small numbers of mutations. Directed evolution studies have shown how rapidly some proteins can evolve under strong selection pressures and, because the entire 'fossil record' of evolutionary intermediates is available for detailed study, they have provided new insight into the relationship between sequence and function. Directed evolution has also shown how mutations that are functionally neutral can set the stage for further adaptation.
Na+-channel specific scorpion toxins are peptides of 60-76 amino acid residues in length, tightly bound by four disulfide bridges. The complete amino acid sequence of 85 distinct peptides are presently known. For some toxins, the three-dimensional structure has been solved by X-ray diffraction and NMR spectroscopy. A constant structural motif has been found in all of them, consisting of one or two short segments of alpha-helix plus a triple-stranded beta-sheet, connected by variable regions forming loops (turns). Physiological experiments have shown that these toxins are modifiers of the gating mechanism of the Na+-channel function, affecting either the inactivation (alpha-toxins) or the activation (beta-toxins) kinetics of the channels. Many functional variations of these peptides have been demonstrated, which include not only the classical alpha- and beta-types, but also the species specificity of their action. There are peptides that bind or affect the function of Na+-channels from different species (mammals, insects or crustaceans) or are toxic to more than one group of animals. Based on functional and structural features of the known toxins, a classification containing 10 different groups of toxins is proposed in this review. Attempts have been made to correlate the presence of certain amino acid residues or 'active sites' of these peptides with Na+-channel functions. Segments containing positively charged residues in special locations, such as the five-residue turn, the turn between the second and the third beta-strands, the C-terminal residues and a segment of the N-terminal region from residues 2-11, seems to be implicated in the activity of these toxins. However, the uncertainty, and the limited success obtained in the search for the site through which these peptides bind to the channels, are mainly due to the lack of an easy method for expression of cloned genes to produce a well-folded, active peptide. Many scorpion toxin coding genes have been obtained from cDNA libraries and from polymerase chain reactions using fragments of scorpion DNAs, as templates. The presence of an intron at the DNA level, situated in the middle of the signal peptide, has been demonstrated.
An scFv has been engineered to bind carcinoembryonic antigen (CEA) with a dissociation half-time >4 days at 37°C. Two mutations
responsible for this affinity increase were isolated by screening yeast surface-displayed mutant libraries by flow cytometry.
Soluble expression of the mutant scFv in a yeast secretion system was increased 100-fold by screening mutant libraries for
improved yeast surface display level. This scFv will be useful as a limiting case for evaluating the significance of affinity
in tumor targeting to non-internalizing antigens.
There is widespread interest in facile methods for generating potent neutralizing antibodies, nanobodies, and other affinity proteins against SARS-CoV-2 and related viruses to address current and future pandemics. While isolating antibodies from animals and humans are proven approaches, these methods are limited to the affinities, specificities, and functional activities of antibodies generated by the immune system. Here we report a surprisingly simple directed evolution method for generating nanobodies with high affinities and neutralization activities against SARS-CoV-2. We demonstrate that complementarity-determining region swapping between low-affinity lead nanobodies, which we discovered unintentionally but find is simple to implement systematically, results in matured nanobodies with unusually large increases in affinity. Importantly, the matured nanobodies potently neutralize both SARS-CoV-2 pseudovirus and live virus, and possess drug-like biophysical properties. We expect that our methods will improve in vitro nanobody discovery and accelerate the generation of potent neutralizing nanobodies against diverse coronaviruses.
Bushmasters (Lachesis spp) and lancehead vipers (Bothrops spp) are two of the most dangerous snakes found in Latin America. Victims of envenoming by these snakes require urgent administration of antivenom. Here, we report the identification of a small set of broadly neutralizing human monoclonal single-chain variable fragment (scFv) antibodies targeting key phospholipases A2 from Lachesis and Bothrops spp using phage display technology and demonstrate their in vitro efficacy using a hemolysis assay.
Scorpionism is a severe threat to public health in North America. Historically, few species of Centruroides have been considered to be the offending taxa, but we know now that their diversity is greater and our knowledge incomplete. Current distribution maps are inadequate for some species. Epidemiologic studies are sporadic and local, and a complete synthesis for North America is missing. We analyze historical and recent knowledge about the identity, distribution and epidemiology of species of medical importance in North America. PubMed, Google Scholar, the National Collection of Arachnids, and results of recent field work were consulted in the preparation of our analysis. We recognized 21 species and one subspecies of medically important scorpions in need of precise geographical delimitation. All these species are found in Mexico, which is clearly a hotspot for scorpionism. Although mortality has been steadily decreasing, deaths still occur, and morbidity remains high. Mortality is most common at age classes of 0-10 years and >50. Morbidity is highest in age class 15-50 years, including the most economically active segment of the population. The season of the highest incidence of scorpion sting peaks between spring and summer but there appears to be a second, lower peak at the end of the summer. Although the systematics of the genus Centruroides has advanced considerably, our knowledge of its diversity remains fragmentary. There is a disconnection between the actual distribution of the scorpions and the incidence maps constructed from scorpion sting records. Despite a historically robust knowledge of the distribution of well-known species, most recently described species are known from only a few localities. Some of the epidemiological parameters are consistent among studies reported herein.
The increment in the number of scorpion envenoming cases in Mexico is mainly associated to the rapid growth of the urban areas, and consequently, to the invasion of natural habitats of these arachnids. On the other hand, there is a great diversity of scorpion species, so it is indispensable to identify those of medical importance, which we now know are many more than the 7-8 previously reported as dangerous to humans. Because different LD50 values have been reported for the venom of the same species, probably due to variations in the experimental conditions used, in this work we determined the LD50 values for the venoms of 13 different species of scorpions using simple but systematic procedures. This information constitutes a referent on the level of toxicity of medically important scorpion species from Mexico and establishes the bases for a more comprehensive assessment of the neutralizing capacity of current and developing antivenoms.
Directed evolution has proved to be an effective strategy for improving or altering the activity of biomolecules for industrial, research and therapeutic applications. The evolution of proteins in the laboratory requires methods for generating genetic diversity and for identifying protein variants with desired properties. This Review describes some of the tools used to diversify genes, as well as informative examples of screening and selection methods that identify or isolate evolved proteins. We highlight recent cases in which directed evolution generated enzymatic activities and substrate specificities not known to exist in nature.
Immune recognition of protein antigens relies on the combined interaction of multiple antibody loops, which provide a fairly large footprint and constrain the size and shape of protein surfaces that can be targeted. Single protein loops can mediate extremely high-affinity binding, but it is unclear whether such a mechanism is available to antibodies. Here we report the isolation and characterization of an antibody called C05, which neutralizes strains from multiple subtypes of influenza A virus, including H1, H2 and H3. X-ray and electron microscopy structures show that C05 recognizes conserved elements of the receptor-binding site on the haemagglutinin surface glycoprotein. Recognition of the haemagglutinin receptor-binding site is dominated by a single heavy-chain complementarity-determining region 3 loop, with minor contacts from heavy-chain complementarity-determining region 1, and is sufficient to achieve nanomolar binding with a minimal footprint. Thus, binding predominantly with a single loop can allow antibodies to target small, conserved functional sites on otherwise hypervariable antigens.
Excellent results regarding improved therapeutic properties have been often obtained through the conversion of a single-chain variable fragment (scFv) into a noncovalent dimeric antibody (diabody) via peptide linker shortening. We utilized this approach to obtain a dimeric version of the human scFv 6009F, which was originally engineered to neutralize the Cn2 toxin of Centruroides noxius scorpion venom. However, some envenoming symptoms remained with diabody 6009F. Diabody 6009F was subjected to directed evolution to obtain a variant capable of eliminating envenoming symptoms. After two rounds of biopanning, diabody D4 was isolated. It exhibited a single mutation (E43G) in framework 2 of the heavy-chain variable domain. Diabody D4 displayed an increase in T(m) (thermal transition midpoint temperature) of 6.3°C compared with its dimeric precursor. The importance of the E43G mutation was tested in the context of the human scFv LR, a highly efficient antibody against Cn2, which was previously generated by our group [Riaño-Umbarila, L., Contreras-Ferrat, G., Olamendi-Portugal, T., Morelos-Juárez, C., Corzo, G., Possani, L. D. and Becerril, B. (2011). J. Biol. Chem.286, 6143-6151]. The new variant, scFv LER, displayed an increase in T(m) of 3.4°C and was capable of neutralizing 2 LD(50) of Cn2 toxin with no detectable symptoms when injected into mice at a 1:1 toxin-to-antibody molar ratio. These results showed that the E43G mutation might increase the therapeutic properties of these antibody fragments. Molecular modeling and dynamics results suggest that the rearrangement of the hydrogen-bonding network near the E43G mutation could explain the improved functional stability and neutralization properties of both the diabody D4 and scFv LER.
A correct alignment is an essential requirement in homology modeling. Yet in order to bridge the structural gap between template and target, which may not only involve loop rearrangements, but also shifts of secondary structure elements and repacking of core residues, high-resolution refinement methods with full atomic details are needed. Here, we describe four approaches that address this “last mile of the protein folding problem” and have performed well during CASP8, yielding physically realistic models: YASARA, which runs molecular dynamics simulations of models in explicit solvent, using a new partly knowledge-based all atom force field derived from Amber, whose parameters have been optimized to minimize the damage done to protein crystal structures. The LEE-SERVER, which makes extensive use of conformational space annealing to create alignments, to help Modeller build physically realistic models while satisfying input restraints from templates and CHARMM stereochemistry, and to remodel the side-chains. ROSETTA, whose high resolution refinement protocol combines a physically realistic all atom force field with Monte Carlo minimization to allow the large conformational space to be sampled quickly. And finally UNDERTAKER, which creates a pool of candidate models from various templates and then optimizes them with an adaptive genetic algorithm, using a primarily empirical cost function that does not include bond angle, bond length, or other physics-like terms. Proteins 2009. © 2009 Wiley-Liss, Inc.
Two toxins, which we propose to call toxins 2 and 3, were purified to homogeneity from the venom of the scorpion Centruroides noxius Hoffmann. The full primary structures of both peptides (66 amino acid residues each) was determined. Sequence comparison indicates that the two new toxins display 79% identity and present a high similarity to previously characterized Centruroides toxins, the most similar toxins being Centruroides-suffusus toxin 2 and Centruroides limpidus tecomanus toxin 1. Six monoclonal antibodies (mAb) directed against purified fraction II–9.2 (which contains toxins 2 and 3) were isolated in order to carry out the immunochemical characterization of these toxins. mAb BCF2, BCF3, BCF7 and BCF9 reacted only with toxin 2, whereas BCF1 and BCF8 reacted with both toxins 2 and 3 with the same affinity. Simultaneous binding of mAb pairs to the toxin and cross-reactivity of the venoms of different scorpions with the mAb were examined. The results of these experiments showed that the mAb define four different epitopes (A–D). Epitope A (BCF8) is topographically unrelated to epitopes B (BCF2 and BCF7), C (BCF3) and D (BCF9) but the latter three appear to be more closely related or in close proximity to each other. Epitope A was found in all Centruroides venoms tested as well as on four different purified toxins of C. noxius, and thus seems to correspond to a highly conserved structure. Based on the cross-reactivity of their venoms with the mAb, Centruroides species could be classified in the following order: Centruroides elegans, Centruroides suffusus suffusus = Centruroides infamatus infamatus, Centruroides limpidus tecomanus, Centruroides limpidus limpidus, and Centruroides limpidus acatlanensis, according to increasing immunochemical relatedness of their toxins to those of Centruroides noxius. All six mAb inhibited the binding of toxin 2 to rat brain synaptosomal membranes, but only mAb BCF2, which belongs to the IgG2a subclass, displayed a clear neutralizing activity in vivo.
Two novel toxins containing 66 amino acid residues each were isolated from the venom of the scorpions Centruroides infamatus infamatus and Centruroides limpidus limpidus, respectively. Their full amino acid sequences were determined. Comparison of primary structures showed that they share 97% similarity among themselves and 83% to that of toxin 2 from Centruroides noxius. The three toxins studied compete with each other for the same binding sites on membranes prepared from rat brain synaptosomes, suggesting that they are all β-scorpion toxins. Toxin action was assayed into the μI-2 rat skeletal muscle Na+ channel heterologously expressed into Xenopus oocytes. All three toxins block this Na+ channel in a similar fashion, without affecting inactivation, and showed IC50 values in the micromolar concentration range.
The complete amino acid sequence of the major toxic component (II.20.3.4), named toxin 1, from the venom of the Mexican scorpion C. l. tecomanus is reported. The sequence (66 amino acids) was obtained by direct Edman degradation of reduced and alkylated toxin, followed by sequence determination of selected peptides separated after enzymatic cleavage with S. aureus V8 protease. In cultured chick dorsal root ganglion cells, 0.5 μM toxin 1 slowed down specifically the time course of Na+ current inactivation, while Ca2+ currents from the same preparation were little affected. In neonatal rat ventricular heart cells, toxin 1, at concentrations between 0.1 and 0.5 μM, reduced Na+ currents without changing the kinetics and Ca2+ currents were unaffected. Comparative analysis of the primary structure of this toxin with other scorpion toxins shows a high degree of similarity with the North American scorpion toxins. This analysis suggests that the ‘fine tuning’ of the molecular mechanism of action of these toxins is related to variations in the primary structure as well as to the type of membrane under study (tissue specificity).
Scorpion beta-toxins represent a particular pharmacological group of voltage-gated sodium channel (VGSC) neurotoxins. They typically shift the voltage dependence of activation to more hyperpolarizing potentials and reduce the peak current amplitude by binding to receptor-site 4. Here, we report the purification and functional characterization of the first voltage-gated sodium channel toxins, CeII8 and CeII9, isolated from the scorpion Centruroides elegans (Thorell, 1876), which is responsible for deadly cases of intoxication in Mexico. The soluble venom was fractionated by gel filtration and ion-exchange chromatography, followed by reversed-phase HPLC. The toxins CeII8 and CeII9 were further purified and both their amino acid sequence and molecular weight were determined. Both toxins were electrophysiologically characterized on four mammalian VGSCs (rNa(v)1.2, rNa(v)1.4, hNa(v)1.5 and rNa(v)1.7) expressed heterologously in Xenopus laevis oocytes, using the two-electrode voltage-clamp technique. Although CeII8 has the highest sequence similarity with scorpion alpha-toxins, inhibiting the inactivation of VGSCs, 300 nM toxin had a clear beta-toxin effect and was selective towards Na(v)1.7, involved in short-term and inflammatory pain. To the best of our knowledge, CeII8 is the first beta-toxin active on Na(v)1.7. CeII9, a typical anti-mammalian beta-toxin, selectively modulated Na(v)1.4 at a concentration of 700 nM and was, in contrast to CeII8, found to be lethal to mice. Interestingly, both toxins, despite their differences in amino acid sequence, only altered the biophysical properties of a fraction of the expressed sodium channels. Since these effects have also been reported for the beta-toxin CssIV, the bioactive surfaces of the toxins have been compared to each other.
This communication revises the state of the art concerning antivenoms against snakes, spiders and scorpions. An overview of the historical facts that preceded the therapeutic use of antibodies is mentioned. A brief list of the major protein components of these venomous animals is revised with a short discussion of what is known on the proteomic analysis of their venoms, but the emphasis is placed on the type of antivenoms available commercially, including pertinent literature and addresses of the companies that prepare these antivenoms. The final section revises and discusses current research on the field and new potential applications that are being developed geared at obtaining new therapeutic antibodies or fragments of antibodies for neutralization of toxic components of venomous animals.
Although fewer antibody fragments have entered the clinic than full-length monoclonal antibodies, proof-of-concept studies for these therapeutics remain the main hurdle.
A modified polymerase chain reaction (PCR) was developed to introduce random point mutations into cloned genes. The modifications were made to decrease the fidelity of Taq polymerase during DNA synthesis without significantly decreasing the level of amplification achieved in the PCR. The resulting PCR products can be cloned to produce random mutant libraries or transcribed directly if a T7 promoter is incorporated within the appropriate PCR primer. We used this method to mutagenize the gene that encodes the Tetrahymena ribozyme with a mutation rate of 0.66% +/- 0.13% (95% C.I.) per position per PCR, as determined by sequence analysis. There are no strong preferneces with respect to the type of base substituion. The number of mutations per DNA sequence follows a Poisson distribution and the mutations are randomly distributed throughout the amplified sequence.
The soluble venom of the scorpion of Colima, Mexico, was fractionated by Sephadex G-50 gel filtration followed by separation of the toxic fraction (number II) with carboxymethyl-cellulose columns in 20 mM ammonium acetate buffer, pH 4.7. Of 24 fractions five were toxic to mice and were further separated in 50 mM phosphate buffer, pH 6.0 using the same ion exchange resin. Final separation included chromatography of the toxic subfractions in the same resin, but in 75 mM ammonium acetate buffer, pH 5.0. By this procedure at least five distinct toxins were obtained in pure form according to gel electrophoresis analysis. Amino acid composition of toxin 1 (component II.20.3.4) and toxic component II.22.5 is included. The N-terminal amino acid sequence of component II.22.5 was shown to be: Lys-Glu-Gly-Tyr-Ile-Val-Asn-Tyr-His-Thr-Gly-Cys-Lys-Tyr-Thr-Cys-Ala-Lys- Leu-Gly - Asp-Asn-Asp-Tyr-Cys-Leu-Arg-Glu-Cys-Lys-.
Single-chain antibody mutants have been evolved in vitro with antigen-binding equilibrium dissociation constant K(d) = 48 fM and slower dissociation kinetics (half-time > 5 days) than those for the streptavidin-biotin complex. These mutants possess the highest monovalent ligand-binding affinity yet reported for an engineered protein by over two orders of magnitude. Optimal kinetic screening of randomly mutagenized libraries of 10(5)-10(7) yeast surface-displayed antibodies enabled a >1,000-fold decrease in the rate of dissociation after four cycles of affinity mutagenesis and screening. The consensus mutations are generally nonconservative by comparison with naturally occurring mouse Fv sequences and with residues that do not contact the fluorescein antigen in the wild-type complex. The existence of these mutants demonstrates that the antibody Fv architecture is not intrinsically responsible for an antigen-binding affinity ceiling during in vivo affinity maturation.
BCF2, a monoclonal antibody raised against scorpion toxin Cn2, is capable of neutralizing both, the toxin and the whole venom of the Mexican scorpion Centruroides noxius Hoffmann. The single chain antibody fragment (scFv) of BCF2 was constructed and expressed in Escherichia coli. Although its affinity for the Cn2 toxin was shown to be in the nanomolar range, it was non-neutralizing in vivo due to a low stability. In order to recover the neutralizing capacity, the scFv of BCF2 was evolved by error-prone PCR and the variants were panned by phage display. Seven improved mutants were isolated from three different libraries. One of these mutants, called G5 with one mutation at CDR1 and another at CDR2 of the light chain, showed an increased affinity to Cn2, as compared to the parental scFv. A second mutant, called B7 with a single change at framework 2 of heavy chain, also had a higher affinity. Mutants G5 and B7 were also improved in their stability but they were unable to neutralize the toxin. Finally, we constructed a variant containing the changes present in G5 and B7. The purpose of this construction was to combine the increments in affinity and stability borne by these mutants. The result was a triple mutant capable of neutralizing the Cn2 toxin. This variant showed the best affinity constant (KD=7.5x10(-11) M), as determined by surface plasmon resonance (BIAcore). The k(on) and k(off) were improved threefold and fivefold, respectively, leading to 15-fold affinity improvement. Functional stability determinations by ELISA in the presence of different concentrations of guanidinium hydrochloride (Gdn-HCl) revealed that the triple mutant is significantly more stable than the parental scFv. These results suggest that not only improving the affinity but also the stability of our scFv were important for recovering its neutralization capacity. These findings pave the way for the generation of recombinant neutralizing antisera against scorpion stings based on scFvs.
This study describes the construction of a library of single-chain antibody fragments (scFvs) from a single human donor by individual amplification of all heavy and light variable domains (1.1 x 10(8) recombinants). The library was panned using the phage display technique, which allowed selection of specific scFvs (3F and C1) capable of recognizing Cn2, the major toxic component of Centruroides noxius scorpion venom. The scFv 3F was matured in vitro by three cycles of directed evolution. The use of stringent conditions in the third cycle allowed the selection of several improved clones. The best scFv obtained (6009F) was improved in terms of its affinity by 446-fold, from 183 nm (3F) to 410 pm. This scFv 6009F was able to neutralize 2 LD(50) of Cn2 toxin when a 1 : 10 molar ratio of toxin-to-antibody fragment was used. It was also able to neutralize 2 LD(50) of the whole venom. These results pave the way for the future generation of recombinant human antivenoms.