Antitoxin antibodies and uses thereof

Abstract

The present disclosure provides antibodies and antigen-binding fragments that can be administered to subjects bitten by venomous snakes.The antibodies or antigen-binding fragments herein can be capable of treating or curing subjects, and can provide protection from snake venom for up to several weeks.When the type of snake is unknown, or when a subject is bitten by more than one species of snake, a combination or population of antibodies and antigen-binding fragments can be administered to the subject.The present disclosure further provides a method for identifying such broadly neutralizing antibodies.

Description

[Technical field]

[0001] cross reference This application claims the benefit of U.S. Provisional Application No. 63 / 172,782, filed April 9, 2021, which is incorporated herein by reference in its entirety.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format, which is hereby incorporated by reference in its entirety. Said ASCII copy, created on __, 2022, is named 60333-704601_SL.txt and is __ bytes in size.

[0003] STATEMENT REGARDING FEDERALLY FUNDED RESEARCH This invention was made with United States Government support under Grant No. 1R43AI147898-01 awarded by the National Institutes of Health. The United States Government has certain rights in the invention. Summary of the Invention [Means for solving the problem]

[0004] Abstract The present application discloses universal, broadly neutralizing, fully human anti-venom antibodies and antibody cocktails that exhibit properties far superior to those currently available. References to antibodies herein may be interchanged with antigen-binding fragments. Anti-venom antibodies and antibody cocktails that can neutralize venoms from multiple snake species and have reduced potential for negative side effects are disclosed. Also disclosed are antibody populations that contain multiple copies of a single antibody that can broadly neutralize snake venoms from more than one snake species or from more than one snake genera. The present disclosure further provides methods for the identification of such broadly neutralizing antibodies.

[0005] Such broadly neutralizing monoclonal antibodies, effective against an entire class of venoms across multiple snake species, would enable the prospect of a single universal antivenom for all snake species. Broadly neutralizing antibodies (bnAbs) are necessary for antibody-based universal antivenoms because there are over 550 venomous snake species, each consisting of 10-70 unique toxic proteins. Thus, without antivenom bnAbs, generating 5,500-38,500 unique antibodies specific to each of the toxins from every species would be an unrealistically ambitious task, much less combining them so that they would be individually present in sufficient concentrations to provide any meaningful protection. However, with antivenom bnAbs, it is possible to have individual antibodies that neutralize the shared common variant toxin homologs found across all snake venoms from all species. Since snake venom toxins belong to 10 distinct toxin classes, and only the "big four" of these (long-chain neurotoxins, PLA2, SVSP, SVMP) cause morbidity and mortality in humans from snakebites, in principle a universal antivenin could be composed of a cocktail of just four broadly neutralizing antivenom monoclonal antibodies.

[0006] Our studies have recovered Centi-D9, a broadly neutralizing, ultra-high affinity antibody against long-chain neurotoxins that was able to neutralize and provide in vivo protection from long-chain neurotoxins from cobra, taipan and mamba. We obtained crystal structures of Centi-D9 in complex with cobra, taipan, krait and mamba long-chain neurotoxins, revealing a common broad-range neutralization mechanism for these evolutionarily related, yet distinct, toxin family members. Crucially, we have demonstrated that Centi-D9 as a monoclonal antibody can independently provide complete in vivo protection of mice from a total venom challenge for black mamba and multiple cobra species, as well as partial protection for taipan. The results of these studies demonstrate that a universal antivenin is possible.

[0007] In 2021, snakebite envenomation continues to rank on the World Health Organization's list of neglected tropical diseases, with between 81,000 and 138,000 people losing their lives annually and up to 300,000 people becoming amputees or permanently disabled each year (reported figures for 2018) due to its hemotoxic or neurotoxic effects. Snake envenomation affects every continent except Antarctica, with the highest incidence of envenomation in Asia, Africa and South America, disproportionately affecting rural populations (i.e., agricultural workers) and children. Snake envenomation also affects the United States, with 7,000-8,000 cases per year, including an average of 50 cases from imported exotic snake species, and 59.3% of these envenomations result in moderate to severe outcomes as well as an average of three deaths. Although it is a global problem, major pharmaceutical companies have not invested in developing envenoming treatments and have even abandoned the market in recent years due to the wide range of species involved, which fragments the market.

[0008] Unlike existing approaches, the strategy proposed by the inventors is to isolate and characterize broadly neutralizing fully human antitoxin antibodies that are stable and ready for use in the field without the need for refrigeration. Due to the low immunogenicity of fully human IgG, this approach will greatly reduce the chance of adverse effects such as anaphylactic shock that are common with existing therapies of animal-derived antitoxins. Importantly, this therapy targets multiple snake species locally, thus eliminating the need for snakebite victims to identify the envenoming species. These molecules can be optimized to have a better safety profile and potentially higher efficacy. They are also designed to be heat stable, thereby enabling on-site use for rapid treatment in the crucial minutes after a snakebite. Due to the looser storage requirements and longer shelf life, this therapy will make the treatment available at a more competitive price point, thereby greatly expanding the types of medical centers that can store and utilize it, making it accessible to a larger and more diverse population.

[0009] The technology described in this application utilizes a novel diverse immune library taken from a middle-aged man who has undergone dose-escalating autoimmunization (more than 700 boosts and 200 live bites) from the world's most venomous snakes, including mamba, taipan, krait, viper, rattlesnake and cobra, over the past 20 years. Venoms from multiple snake species were panned against our library over several rounds to identify cross-reactive clones. Preliminary studies revealed 282 unique clones enriched for VH CDR3 across different toxins, representing potential cross-reactive binders in these output pools. This is highly significant, as cross-reactive antibodies are essential for the development of broadly neutralizing antitoxins. With the discovery of Centi-D9, which targets long-chain alpha-neurotoxins, the inventors have already demonstrated successful completion of one therapeutic or prophylactic antibody capable of protecting against all venom in multiple types of elapid snakes (e.g., taipan, mamba, cobra).

[0010] The development of broad-spectrum vaccines has been limited by both the large number of different species and the diversity of toxins in each species' venom. For more than a century, treatment of snakebite envenomations has been equine or ovine serum therapy using animal-derived antivenom preparations containing either immunoglobulin G (IgG) or derived antigen-binding fragments (Fab), typically from a single venom. More recently, several ovine or equine-derived serum products have been created with some breadth in species coverage (CroFab for the American pit viper, EchiTAb for the West African saw-toothed snake or carpet viper). Although effective in reducing mortality and permanent disability in many cases, heterologous antivenom serum therapy presents several challenges. Depending on the antivenom, 6-59% of patients experience early adverse reactions to non-autologous reactions to non-human equine or ovine plasma-derived antivenoms, including early adverse anaphylactic reactions (by 24 hours). Antitoxins are composed of approximately 70% Fab or Ig against antigens encountered by the immunized animal, rather than toxin components, which reduces their potency and requires more extensive drug infusion, usually by intravenous infusion (IV) in a hospital environment that may not be near the envenomation site, resulting in significant logistical problems and death of the patient before reaching the hospital, or inability to treat by the time the patient reaches the hospital.

[0011] Polyclonal Fab-based preparations can lead to treatments with shortened half-life and inconsistent batch quality compared to monoclonal IgG. Digestion into Fab fragments mitigates serum sickness due to removal of the non-human Fc region, but these treatments shorten the half-life from weeks to hours and therefore require 8-10 doses of antivenom. However, patients cannot receive any additional future doses as they will have a severe reaction initiated against these antibodies. High concentrations of IgG can cause adverse reactions including inflammation and serum sickness 1-2 weeks after treatment. Finally, antivenoms developed for a single species require precise identification of the specific snake that bit the victim, which is extremely difficult for victims and medical personnel unfamiliar with the snake phenotype, and require clinics to stockpile the exact venom for many possible products for different envenomations.

[0012] The antivenom compositions described herein are broad-spectrum protective against major families of snakes with lethal venom. In addition, treatment does not require positive identification of the envenomed species and can be administered quickly after a snake bite or prophylactically to those at high risk of snake bite. This reduces costs by eliminating the need to stockpile multiple antivenoms and due to the extended half-life of Centivax's lyophilized formulation. Snake envenomation has traditionally been a highly unmet need that has been underinvested in, especially by big pharmaceutical companies, and is similar to a rare disease niche market. However, the key difference is that snake envenomation is not rare, with 5 million envenomations, 300,000 permanent disabilities, and 125,000 deaths per year. A single agent could consolidate and increase the existing market for antivenoms, as the market is currently fragmented between multiple venoms of snake species. Primary end users and customers will be the military forces of the top 25 countries where our antivenom can be implemented as an effective preventative or acute treatment option for deployed military personnel in austere environments. Further uses include stockpiling in many of the 28,600 hospitals worldwide, and with government subsidies to support distribution in severely affected countries.

[0013] Non-human immunotherapy. For more than a century, treatment of snakebite envenomation has been horse or sheep serum therapy using animal-derived antivenin preparations containing derivative antigen-binding fragments (Fabs), usually from a single venom. More recently, several sheep or horse serum products have been produced with some breadth of species coverage. CroFab (ovine-derived antivenin, polyvalent and lyophilized) and AnaVip (equine-derived Fab2, lyophilized powder for injection) are both specific for North American snakes: rattlesnakes, cottonmouths, and copperheads. EchiTAb-G is a sheep antiserum that is recommended for use in sub-Saharan Africa and is effective only against the West African saw snake or carpet viper. Although effective in reducing mortality and permanent disability in many cases, heterologous antivenin serum therapy presents several challenges. Depending on the antitoxin, 6-59% of patients experience early adverse reactions due to non-autologous reactions to the exogenous horse or sheep plasma-derived antitoxin. These adverse reactions may include early adverse anaphylactic reactions (up to 24 hours).

[0014] Older antitoxins require IV injections. Antitoxins are composed of approximately 70% Fab or Ig against other antigens rather than toxin components. This has multiple drawbacks as it reduces their potency and requires more extensive drug injections. Drugs are often delivered IV at locations other than the site of envenomation, thus leading to an increased number of deaths due to time delays in administration. IV drugs are highly limited in their availability for treatment due to time delays, medical personnel required, and treatment location. Polyclonal Fab-based formulations can lead to treatments with shortened half-lives and inconsistent batch quality compared to monoclonal IgG. Digestion into Fab fragments mitigates serum sickness due to removal of the non-human Fc region, but these treatments shorten the half-life from weeks to hours and therefore require 8-10 doses of antitoxin. Furthermore, additional administration of these antibodies cannot be done due to predicted serum toxicity. High concentrations of non-human IgG antitoxins can cause inflammation and serum sickness 1-2 weeks after treatment.

[0015] So far, antitoxins composed of monoclonal IgG have not been used in clinical settings. Other antitoxin companies, such as Venomyx, have demonstrated some cross-reactivity in preliminary studies, but still derive their molecules from immunized camels, thus requiring costly and time-consuming humanization. Furthermore, their single-domain nature may result in a shortened half-life compared to fully human IgG. If not adequately neutralized with a single dose, repeated injections are necessary due to their small size, which allows renal excretion.

[0016] In contrast, the technology described in this application utilizes a novel diverse immune library taken from a middle-aged man who over the past 17 years has undergone dose-escalating autoimmunization (over 700 boosts and 200 live bites) from the world's most venomous snakes, including mamba, taipan, krait, viper, rattlesnake and cobra.

[0017] Library construction utilized a unique next-generation sequencing (NGS) technique to amplify antibody variable domains from blood collected before and 7 days after the final venom immunization of male subjects, followed by deep sequencing and antibody phage display that allowed downstream toxin-binding factors to be traced back to the source blood collected. Antibody candidates against toxins from the black mamba, western diamondback rattlesnake, and coastal taipan were isolated during the initial library panning.

[0018] Heat-stabilized antibodies. Standard antivenoms require continuous refrigeration at 2-8°C to remain stable. This poses significant challenges for villages in arid regions, often with endemic venomous species, that may not have the infrastructure to properly store antivenom. Recent studies have investigated the stability of antivenoms at room temperature. This application will develop a heat-stabilized antibody with less stringent storage requirements, a longer shelf life, and is expected to be available at a more competitive price point, thus greatly expanding the types of medical centers that can store and utilize it. Antivenom cross-reactivity is a complex biochemical challenge, even though all medically relevant snakes belong to three families, Atractaspidae, Viperidae, and Elapidae, with the vast majority belonging to Viperidae or Elapidae. Within these groups, the toxin proteomes of their venoms are classified into 8-11 subfamilies, four of which are the major components across all venoms. Some existing antivenoms work well in certain snake lineages despite 8 million years of separation, while other, more closely related species exhibit poor neutralization against the same antivenoms because a single mutation in an amino acid in the toxin molecule is sufficient to interfere with antibody recognition.

[0019] Preliminary deep sequencing panning analysis of our human subject's antibodies under our control demonstrated VH CDR3 domain enrichment across multiple species, indicating potential cross-reactive candidates against multiple snake venom species. It is reasonable to assume that our subject has generated antibodies that can bind to epitopes that are conserved across related peptides between snake species, since he has been immunized with various snakes from the Viperidae and Elapidae families for almost 20 years. This application utilizes high-throughput surface plasmon resonance (SPR), full spectrum fluorescence and light scattering, and in vivo functional assays to isolate and characterize broadly neutralizing fully human anti-venom antibodies to develop broad-spectrum anti-venoms.

[0020] Broadly neutralizing antibodies (bnAbs). There are 550+ venomous snake species, and each venom consists of 10-70 unique toxic proteins. To develop a single universal antitoxin, a broadly neutralizing monoclonal antibody would need to be effective against the entire class of toxins across multiple snake species. Without antivenom bnAbs, generating 5,500-38,500 unique antibodies specific to each of the toxins from every species would be an unrealistically ambitious task, much less combining them so that they are individually present in sufficient concentrations to provide any meaningful protection. However, with antivenom bnAbs, it is possible to have individual antibodies that neutralize the shared common variant toxin homologs found across all snake venoms from all species. Since snake venom toxins belong to 10 distinct toxin classes, and only the "big four" of these (long-chain neurotoxins, PLA2, SVSP, SVMP) cause morbidity and mortality in humans from snakebites, a universal antivenin could be composed of a cocktail of only four broadly neutralizing antivenom monoclonal antibodies (Figure 11).

[0021] Broadly neutralizing antibodies will break the limitations of anti-venom therapy. 1) Broadly neutralizing antibodies can recognize the whole family of snake toxins, thus allowing a single anti-venom product for each lethal snake family. This will be dramatically beneficial in avoiding the risk of snake identification and incorrect anti-venom evaluation, and will ultimately strengthen the market economy by producing a single product for all regions of the world. 2) Since human monoclonal antibodies are not rejected as immunogens by the recipient's immune system, fully human antibodies can avoid serum toxicity by reducing the occurrence of adverse reactions. These molecules could be easily optimized to have a better safety profile and potentially higher efficacy. Monoclonal IgG has a favorable effect against snake toxins in in vivo lethality studies, neutralization tests, and in terms of neutralizing ability against multiple specific toxins involved in myonecrosis and proteolytic effects. ) Recombinant anti-venoms could be 100% pure anti-venom specific products and therefore much more potent, thereby reducing the volume and being able to be delivered by dual-chamber syringes outside the hospital environment. ) Due to its improved toxicity profile compared to sheep IgG, fully human IgG can be offered as a prophylactic with a period of protection of 3-6 weeks, which is suitable for individuals in areas and / or occupations with high exposure risk. Recombinant antibodies and antibody fragments of human origin are attractive alternatives for antivenom production.

[0022] Fully Human Antivenom Antibodies. This application describes the development of safer and more effective next generation antivenom biotherapeutics by isolating and characterizing a broadly neutralizing, fully human, antivenom antibody cocktail for the treatment of snakebite envenomation. Monoclonal fully human derived IgG greatly reduces the potential for adverse effects commonly seen with animal derived antivenoms and allows for greater optimization that is expected to be more cost effective than traditionally produced antivenoms.

[0023] Provided herein is a universal antitoxin composition comprising a population of antitoxin antibodies or antigen-binding fragments. The population of antitoxin antibodies or antigen-binding fragments can comprise one or more antibodies or antigen-binding fragments. Alternatively, the population of antitoxin antibodies or antigen-binding fragments can comprise two antibodies or antigen-binding fragments, three antibodies or antigen-binding fragments, four antibodies or antigen-binding fragments, five antibodies or antigen-binding fragments, six antibodies or antigen-binding fragments, or more antibodies or antigen-binding fragments.

[0024] The one or more antibodies or antigen-binding fragments may comprise, be derived from, or be a combination of IgG, IgM, IgE, IgA, or IgD. The one or more antibodies or antigen-binding fragments may comprise a monoclonal antibody, a grafted antibody, a chimeric antibody, a human antibody, or a humanized antibody. The antigen-binding fragment may comprise a Fab, Fab', F(ab')2, a variable fragment (Fv), a triabody, a tetrabody, a minibody, a bispecific F(ab')2, a trispecific F(ab')2, a diabody, a bispecific diabody, a single chain variable fragment (scFv), a scFv-Fc, a Fab-Fc, a VHH, or a bispecific scFv.

[0025] The one or more antibodies or antigen-binding fragments comprise a VH CDR1, a VH CDR2, a VH CDR3, a VL CDR1, a VL CDR2, and a VL CDR3. The one or more antibodies or antigen-binding fragments may comprise a VH CDR1 having an amino acid sequence that is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of the sequences in Table 2. Alternatively, or in addition, the one or more antibodies or antigen-binding fragments comprise a VH CDR2 having an amino acid sequence that is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of the sequences in Table 3. Alternatively, or in addition, the one or more antibodies or antigen-binding fragments comprise a VH CDR3 having an amino acid sequence that is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of the sequences in Table 1. Alternatively, or in addition, the one or more antibodies or antigen-binding fragments comprise a VL CDR1 having an amino acid sequence that is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of the sequences in Table 4. Alternatively, or in addition, the one or more antibodies or antigen-binding fragments comprise a VL CDR2 having an amino acid sequence that is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of the sequences in Table 5. Alternatively, or in addition, the one or more antibodies or antigen-binding fragments comprise a VL CDR3 having an amino acid sequence that is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of the sequences in Table 6.

[0026] Alternatively, or in addition, the one or more antibodies or antigen-binding fragments include a FW-H1 having an amino acid sequence at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of the sequences in Table 7. Alternatively, or in addition, the one or more antibodies or antigen-binding fragments include a FW-H2 having an amino acid sequence at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of the sequences in Table 8. Alternatively, or in addition, the one or more antibodies or antigen-binding fragments include a FW-H3 having an amino acid sequence at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of the sequences in Table 9. Alternatively, or in addition, the one or more antibodies or antigen-binding fragments include a FW-H4 having an amino acid sequence at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of the sequences in Table 10.

[0027] Alternatively, or in addition, the one or more antibodies or antigen-binding fragments include a FW-L1 having an amino acid sequence at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of the sequences in Table 11. Alternatively, or in addition, the one or more antibodies or antigen-binding fragments include a FW-L2 having an amino acid sequence at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of the sequences in Table 12. Alternatively, or in addition, the one or more antibodies or antigen-binding fragments include a FW-L3 having an amino acid sequence at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of the sequences in Table 13. Alternatively, or in addition, the one or more antibodies or antigen-binding fragments include a FW-L4 having an amino acid sequence at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of the sequences in Table 14.

[0028] Alternatively, or in addition, the one or more antibodies or antigen-binding fragments comprise a VH having an amino acid sequence that is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of the sequences in Table 15. Alternatively, or in addition, the one or more antibodies or antigen-binding fragments comprise a VL having an amino acid sequence that is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of the sequences in Table 16.

[0029] In another aspect, provided herein is a method of treating a subject suffering from envenomation, the method comprising administering to the subject a composition comprising an effective amount of a universal antitoxin comprising one or more antibodies or antigen-binding fragments. In some cases, the envenomation occurs by one or more snakes.

[0030] The method may include administering one or more antibodies or antigen-binding fragments to a subject. Alternatively, the method may include administering one antibody, two antibodies or antigen-binding fragments, three antibodies or antigen-binding fragments, four antibodies or antigen-binding fragments, five antibodies or antigen-binding fragments, six antibodies or antigen-binding fragments, seven antibodies or antigen-binding fragments, eight antibodies or antigen-binding fragments, nine antibodies or antigen-binding fragments, ten antibodies or antigen-binding fragments, or more antibodies or antigen-binding fragments to the subject. The one or more antibodies or antigen-binding fragments to be administered to the subject may include, be derived from, or be a combination of IgG, IgM, IgE, IgA, or IgD. The one or more antibodies or antigen-binding fragments to be administered to the subject may include a monoclonal antibody, a grafted antibody, a chimeric antibody, a human antibody, or a humanized antibody. The antigen-binding fragment administered to a subject may comprise a Fab, Fab', F(ab')2, variable fragment (Fv), triabody, tetrabody, minibody, bispecific F(ab')2, trispecific F(ab')2, diabody, bispecific diabody, single chain variable fragment (scFv), scFv-Fc, Fab-Fc, VHH, bispecific scFv, or combinations thereof.

[0031] In any of such methods, the subject can be administered one or more additional therapies or drugs to the subject.The additional therapies or drugs can include, for example, NSAIDs.Alternatively, or in addition, the additional therapies or drugs can include, for example, PLA2 inhibitors.PLA2 inhibitors include, but are not limited to, varespladib, methylvarespladib, or combinations thereof.Administration can be by any suitable means, for example, one or more injections.

[0032] In any of such methods, the one or more antibodies or antigen-binding fragments to be administered to the subject may comprise a VH CDR3 having an amino acid sequence that is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of the sequences in Table 1. Alternatively, or in addition, the one or more antibodies or antigen-binding fragments to be administered to the subject comprise a VH CDR1 having an amino acid sequence that is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of the sequences in Table 2. Alternatively, or in addition, the one or more antibodies or antigen-binding fragments to be administered to the subject comprise a VH CDR2 having an amino acid sequence that is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of the sequences in Table 3.

[0033] Alternatively, or in addition, the one or more antibodies or antigen-binding fragments to be administered to the subject comprise a VL CDR1 having an amino acid sequence that is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of the sequences in Table 4. Alternatively, or in addition, the one or more antibodies or antigen-binding fragments to be administered to the subject comprise a VL CDR2 having an amino acid sequence that is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of the sequences in Table 5. Alternatively, or in addition, the one or more antibodies or antigen-binding fragments to be administered to the subject comprise a VL CDR3 having an amino acid sequence that is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of the sequences in Table 6.

[0034] Alternatively, or in addition, the one or more antibodies or antigen-binding fragments to be administered to the subject comprise a FW-H1 having an amino acid sequence at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of the sequences in Table 7. Alternatively, or in addition, the one or more antibodies or antigen-binding fragments to be administered to the subject comprise a FW-H2 having an amino acid sequence at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of the sequences in Table 8. Alternatively, or in addition, the one or more antibodies or antigen-binding fragments to be administered to the subject include FW-H3 having an amino acid sequence at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of the sequences in Table 9. Alternatively, or in addition, the one or more antibodies or antigen-binding fragments to be administered to the subject include FW-H4 having an amino acid sequence at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of the sequences in Table 10.

[0035] Alternatively, or in addition, the one or more antibodies or antigen-binding fragments to be administered to the subject comprise a FW-L1 having an amino acid sequence at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of the sequences in Table 11. Alternatively, or in addition, the one or more antibodies or antigen-binding fragments to be administered to the subject comprise a FW-L2 having an amino acid sequence at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of the sequences in Table 12. Alternatively, or in addition, the one or more antibodies or antigen-binding fragments to be administered to the subject comprise FW-L3 having an amino acid sequence at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of the sequences in Table 13. Alternatively, or in addition, the one or more antibodies or antigen-binding fragments to be administered to the subject comprise FW-L4 having an amino acid sequence at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of the sequences in Table 14.

[0036] Alternatively, or in addition, the one or more antibodies or antigen-binding fragments to be administered to the subject comprise a VH having an amino acid sequence that is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of the sequences in Table 15. Alternatively, or in addition, the one or more antibodies or antigen-binding fragments to be administered to the subject comprise a VL having an amino acid sequence that is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of the sequences in Table 16.

[0037] In any of such examples of universal anti-venom compositions or methods, the one or more antibodies or antigen-binding fragments may be directed to or against any of the following snakes: Boiga irregularis (brown tree snake), Boiga cyanea (green tree snake), Boiga dendrophila (mangrove snake), Dispholidus typus (boomslang), Salvadora grahamiae (mountain patchnose snake), Spalerosophis diadema (crowned snake), Tantilla nigriceps (Plains blackhead snake), Thelotornis capensis (southern twig snake), Thelotornis kirtlandii (northern twig snake), Toxicodryas blandingii (Blanding's tree snake), Trimorphodon lambda (Sonoran lyre snake), Amphiesma stolatum (buff-striped scaly-back), Natrix scaly-back, tessellate(Dice snake), Rhabdophis subminiatus(Red collared burrowing asp), Rhabdophis tigrinus(Rabdophis tigrinus), Thamnophis elegans(Western land garter snake), Thamnophis sirtalis(Common garter snake), Ahaetulla nasuta(Long-legged burrowing asp), Atractaspis bibronii(Bibronzed burrowing asp), Atractaspis dahomeyensis(Dahomey burrowing asp), Atractaspis engaddensis(Palestinian mole snake), Atractaspis microlepidota(Small-scaled burrowing asp), Malpolon monspessulanus(Montpellier snake), Acanthophis antarcticus(Common death adder), Aipysurus laevis (Olive Brown Sea Snake), Aipysurus duboisii (Dubois Sea Snake), Austrelaps superbus (Lowland Copperhead), Cryptophis nigrescens (Small Ice Snake), Demansiaolivacea(Olive Whip Snake), Emydocephalus annulatus(Spotted White Sea Snake), Furina tristis(Stephen's Striped Snake), Hydrophis melanocephalus(Black Headed Sea Snake), Hydrophis curtus(Spiny Sea Snake), Hydrophis gracilis(Slender Sea Snake), Hydrophis elegans(Elegant Sea Snake), Hydrophis jerdonii(Corn Nosed Sea Snake), Hydrophis klossi(Llangol Sea Snake), Hydrophis peronii(Horned Sea Snake), Hydrophis belcheri(Belcher's Sea Snake), Hydrophis stricticollis(Bengal Sea Snake), Hydrophis major(Olive Headed Sea Snake), Hydrophis Hydrophis stokesii (large-headed sea snake), Hydrophis melanosoma (black-banded robust sea snake), Hydrophis hardwickii (dorsal-ventral sea snake), Hydrophis cyanocinctus (spotted sea snake), Hydrophis spiralis (narrow-banded sea snake), Hydrophis nigrocinctus (black-banded sea snake), Hydrophis platurus (back-striped sea snake), Hydrophis ornatus (black-spotted sea snake), Hydrophis viperinus (venomous sea snake), Hydrophis schistosus (rough-legged sea snake), Notechis scutatus (mainland tiger snake), Oxyuranus scutellatus (coastal taipan), Oxyuranus temporalis (central ranges taipan), Pseudechis australis (Margas Snake), Pseudechis butleri (Butler's Black Snake), Pseudechis colletti (Collet's Black Snake), Pseudechis guttatus (Blue-bellied Black Snake), Pseudechis papuanus (Papuan Black Snake), Pseudechisporphyriacus(Red-bellied Black Snake), Pseudonaja affinis(Dugite), Pseudonaja guttata(Speckled Brown Snake), Pseudonaja inframacula(Peninsula Brown Snake), Pseudonaja nuchalis(Western Brown Snake), Pseudonaja textilis(Eastern Brown Snake), Tropidechis carinatus(Clarence River Snake), Aspidelaps lubricus(Cape Coral Snake), Aspidelaps scutatus(Shield Nose Snake), Bungarus fasciatus(Round-tailed Krait), Bungarus caeruleus(Indian Krait), Bungarus candidus(Blue Krait), Bungarus flaviceps(Red-headed Krait), Bungarus multicinctus(Multi-banded Krait), Dendroaspis viridis(Western Green Mamba), Dendroaspis angusticeps(Eastern Green Mamba), Dendroaspis jamesoni(Jameson's Mamba), Dendroaspis polylepis(Black Mamba), Elapsoidea sundevallii(Sandevall's African Garter Snake), Hemachatus haemachatus(Lyncarus), Laticauda colubrina(Blue Spotted Sea Snake), Laticauda laticaudata(Broad-headed Sea Snake), Laticauda semifasciata(Black-spotted Sea Snake), Micrurus obscurus(Bolivian Coral Snake), Micrurus frontalis(Southern Coral Snake), Micrurus alleni(Allen's Coral Snake), Micrurus altirostris(Uruguay Coral Snake), Micrurus clarki(Clark's Coral Snake), Micrurus corallinus (Painted Coral Snake), Micrurus distans (West Mexican Coral Snake), Micrurus dumerilii (Dumerill's Coral Snake), Micrurusfulvius(Eastern coral snake), Micrurus hemprichii(Hemprich's coral snake), Micrurus ibiboboca(Cartinga coral snake), Micrurus lemniscatus(South American coral snake), Micrurus mipartitus(Red-tailed coral snake), Micrurus mosquitensis(Costa Rican coral snake), Micrurus multifasciatus(Multi-striped coral snake), Micrurus nigrocinctus(Central American coral snake), Micrurus pyrrhocryptus(Argentine coral snake), Micrurus spixii(Amazonian coral snake), Micrurus surinamensis(Aquatic coral snake), Micrurus tener(Texas coral snake), Micrurus tschudii(Desert coral snake), Naja siamensis(Indochinese spitting cobra), Naja annulata(Ring Water Cobra), Naja annulifera(Long-toed Cobra), Naja ashei(Giant Spitting Cobra), Naja atra(Taiwan Cobra), Naja christyi(Congo Cobra), Naja haje(Egyptian Cobra), Naja kaouthia(Thailand Cobra), Naja katiensis(Western African Spitting Cobra), Naja melanoleuca(Forest Cobra), Naja mossambica(Mozambican Spitting Cobra), Naja(Indian Cobra), Naja nigricollis(Black Necked Spitting Cobra), Naja nivea(Cape Cobra), Naja nubiae(Nubian Spitting Cobra), Naja oxiana(Caspian Cobra), Naja pallida(Red Spitting Cobra), Naja philippinensis(North Philippine Cobra), Naja samarensis (Samarcobra), Naja sputatrix (Indonesian Spitting Cobra), Naja sumatrana (Sumatran Spitting Cobra), Ophiophagus hannah (King Cobra), Walterinnesia aegyptia (Western Desert Black Cobra), Homalopsisbuccata(Linnaeus's water snake), Myrrophis chinensis(Chinese mud snake), Subsessor bocourti(Bocourt's water snake), Azemiops feae(Cobra viper), Agkistrodon bilineatus(Bear-eared pit viper), Agkistrodon contortrix(German copperhead), Agkistrodon piscivorus(Cotton murphy), Agkistrodon taylori(Castellana), Agkistrodon laticinctus(Broadband copperhead), Atropoides picadoi(Picado's jumping pit viper), Bothriechis lateralis(Side-stripe palm pit viper), Bothriechis nigroviridis(Black-spotted palm pit viper), Bothriechis schlegelii(Eyelash palm pit viper), Bothrops diporus(Chacolance head), Bothrops erythromelas (Curting Lancehead), Bothrops insularis (Golden Lancehead Viper), Bothrops jararaca (Hararaca), Bothrops neuwiedi (Neuwiedi's Lancehead), Bothrops pauloensis (Black-faced Lancehead), Bothrops asper (Terciopelo), Bothrops atrox (Kaikasa), Bothrops ayerbei (Ayerbe's Lancehead), Bothrops caribbaeus (Saint Lucia Lancehead), Bothrops jararacussu (Jararacussu), Bothrops lanceolatus (Martinie Lancehead), Bothrops leucurus (White-tailed Lancehead), Bothrops moojeni (Brazilian Lancehead), Bothrops alternatus (Urutu), Bothrops cotiara (Cotiara), Bothrops fonsecai (Fonseca's Lancehead), Bothrops itapetiningae (Sao Paulo Lancehead), Bothropstaeniatus (Speckled Forest Pit Viper), Bothrops mattogrossensis (Mato Grosso Lantzen Otter), Calloselasma rhodostoma (Malayan Pit Viper), Cerrophidion godmani (Godman Mountain Pit Viper), Cerrophidion sasai (Costa Rican Mountain Pit Viper), Crotalus viridis (Prairie Rattlesnake) , Crotalus atrox (Western diamondback rattlesnake), Crotalus adamanteus (Eastern diamondback rattlesnake), Crotalus basiliscus (West Coast Mexican rattlesnake), Crotalus catalinensis (Santa Catalina Island rattlesnake), Crotalus cerastes (Sidewalk rattlesnake), Crotalus Cerberus (Arizona black rattlesnake), Crotalus durissus (South American rattlesnake), Crotalus enyo (Baja California rattlesnake), Crotalus horridus (timber rattlesnake), Crotalus lepidus (mottled rock rattlesnake), Crotalus mitchellii (San Lucan spotted rattlesnake), Crotalus molossus (Northern black-tailed rattlesnake), Crotalus oreganus (Northern Pacific rattlesnake), Crotalus pricei(Western twin-spotted rattlesnake), Crotalus pusillus(Tassitalandusky rattlesnake), Crotalus ravus(Mexican pygmy rattlesnake), Crotalus ruber(Red diamond rattlesnake), Crotalus scutulatus(Mojave rattlesnake), Crotalus simus(Central American rattlesnake), Crotalus tigris(Tiger rattlesnake), Crotalus totonacus(Totonacan rattlesnake), Crotalus tzabcan(Yucatan neotropical rattlesnake), Crotalus willardi(Arizona ridgenose rattlesnake), Crotalus pyrrhus(Southwestern speckled rattlesnake), Crotalus vegrandis(Ulacoan rattlesnake), Deinagkistrodon acutus(Hypoda), Gloydius intermedius (intermediate pit viper), Gloydius blomhoffii (pit viper), Gloydius brevicaudus (striped pit viper), Gloydius halys (Siberian pit viper), Gloydius shedaoensis (Snake Island pit viper), Gloydiusussuriensis (Ussurian pit viper), Hypnale (Hump-nosed pit viper), Lachesis melanocephala (Black-headed bushmaster), Lachesis muta (Atlantic bushmaster), Ovophis okinavensis (Ovophis okinavensis), Porthidium nasutum (Rainforest pit viper), Porthidium ophryomegas (Slender pit viper), Protobothrops elegans (Sakishima pit viper), Protobothrops flavoviridis (Okinawa pit viper), Protobothrops mangshanensis (Mangshan pit viper), Protobothrops mucrosquamatus (Taiwan pit viper), Protobothrops tokarensis (Tokara pit viper), Sistrurus catenatus (Massasauga), Sistrurus miliarius (Pygmy rattlesnake), Trimeresurus stejnegeri(Taiwanese green pit viper), Trimeresurus albolabris(White-lipped blue pit viper), Trimeresurus erythrurus(Burmese green pit viper), Trimeresurus gramineus(Green pit viper), Trimeresurus labialis(Nicobar bamboo pit viper), Trimeresurus macrops(Large-eyed pit viper), Trimeresurus malabaricus(Malabar pit viper), Trimeresurus popeiorum(Pope's green pit viper), Trimeresurus purpureomaculatus(Mangrove pit viper), Trimeresurus sumatranus(Sumatran pit viper), Tropidolaemus wagleri(Wagler's pit viper),Metlapilcoatlus mexicanus(Mexican jumping pit viper),Metlapilcoatlus nummifer(Central American jumping pit viper), Atheris squamigera (Lizard Bush Viper), Bitis arietans (Puff Adder), Bitis Atropos (Cape Mountain Adder), Bitiscaudalis(Horned Puff Adder), Bitis cornuta(Western Horned Adder), Bitis gabonica(Central African Gabon Viper), Bitis nasicornis(Rhinoceros Adder), Bitis parviocula(Ethiopian Adder), Bitis rhinoceros(West African Gabon Viper), Causus rhombeatus(Common Night Adder), Cerastes(Horned Viper), Cerastes gasperettii(Arabian Horned Viper), Cerastes vipera(Saharan Viper), Daboia palaestinae(Palestinian Viper), Daboia russelii(Russell's Viper), Daboia siamensis(Eastern Russell's Viper), Daboia mauritanica(Moo Viper), Echis ocellatus(West African carpet viper), Echis carinatus(Japanese saw-tooth viper), Echis coloratus(Painted saw-tooth viper), Echis pyramidum(Egyptian saw-tooth viper), Macrovipera schweizeri(Milo's viper), Macrovipera lebetinus(Levantine viper), Montivipera bornmuelleri(Lebanese mountain viper), Montivipera latifii(Latifii viper), Montivipera raddei(Armenian mountain viper), Montivipera xanthine(Asian viper), Pseudocerastes fieldi(Field horned viper), Pseudocerastes persicus(Persian viper), Vipera ammodytes(European horned viper), Vipera aspis (Asp Viper), Vipera berus (European Viper), Vipera latastei (Ratastei Viper), Vipera lotievi (Caucasian Viper), Vipera seoanei (Basque Viper), Vipera ursinii (Viper), DiadophisThe envenomation binds to one or more toxins from one or more snakes selected from the group consisting of: Hydrodynastes gigas, Hypsiglena torquata, Hypsiglena jani, Leptodeira ashmeadii, Philodryas olfersii, and combinations thereof. In one instance, the envenomation is from a sawtooth snake, a water moccasin, a lancehead, a rattlesnake, a Russell's viper, a puff adder, or a combination thereof. In another instance, the envenomation is from a sawtooth snake, a water moccasin, a lancehead, a rattlesnake, a Russell's viper, and a puff adder. In other cases, the envenomation is from the common krait (Bungarus caeruleus), the black mamba (Dendroaspis polylepsis), the coastal taipan (Oxyuranus scutatellus), the Cape cobra (Naja nivea), or a combination thereof. In other cases, the envenomation is from the common krait (Bungarus caeruleus), the black mamba (Dendroaspis polylepsis), the coastal taipan (Oxyuranus scutatellus), and the Cape cobra (Naja nivea).

[0038] Provided herein is an antibody or antigen-binding fragment that is a broadly neutralizing antibody against a three-finger toxin (3FTx). In one embodiment, the 3FTx is a long-chain neurotoxin. The antibody or antigen-binding fragment can neutralize alpha-bungarotoxin (Japanese krait), alpha-elapitoxin (mamba), pseudonajatoxin (brown snake), alpha-cobratoxin (cobra), toxin B (king cobra), or a combination thereof. In one case, the antibody or antigen-binding fragment broadly neutralizes toxins from two, three, four, five, or more of these long-chain neurotoxins. In a preferred embodiment, the antibody or antigen-binding fragment neutralizes the effect of the long-chain neurotoxin. In a preferred embodiment, the antibody or antigen-binding fragment binds to one or more three-finger toxins in a manner that prevents the toxin from binding to human nicotinic acetylcholine receptors (nAChRs). In some embodiments, the antibody or antigen-binding fragment binds to at least 30%, 40%, 50%, 60%, 70%, 80% or 90% of the exposed area of ​​the toxin that is filled when the toxin binds to the nAChR. The antibody or antigen-binding fragment can be, for example, SNEURO_P01_D09. In one case, SNEURO_P01_D09 comprises a VH CDR1 of SEQ ID NO:28, a VH CDR2 of SEQ ID NO:41, a VH CDR3 of SEQ ID NO:12, a VL CDR1 of SEQ ID NO:46, a VL CDR2 of SEQ ID NO:80 and a VL CDR3 of SEQ ID NO:101. In another case, SNEURO_P01_D09 comprises a VH having an amino acid sequence of SEQ ID NO:242 and a VL having an amino acid sequence of SEQ ID NO:273.

[0039] Provided herein is a method for identifying broadly neutralizing antibodies or antigen-binding fragments that selectively bind to two or more snake venom toxins belonging to a family of homologous antigens, comprising: (a) immunizing a subject with two or more homologous antigens; and (b) performing an iterative selection process to specifically identify cross-reactive antibodies or antigen-binding fragments from the subject's B-cell repertoire, the iterative selection process using the two or more homologous antigens to down-select potential candidates. The subject may be, for example, a mammal (e.g., a human). In some embodiments, the family of homologous antigens includes three-finger toxins (3FTX), including but not limited to long-chain neurotoxins. The iterative selection process includes immunoprecipitation of antibodies from serum obtained from the subject, fluorescence-activated cell sorting (FACS) of B cells obtained from the subject, panning of libraries derived from B-cell RNA obtained from the subject displayed in phage or yeast, or any combination thereof.

[0040] In some examples, the subject is immunized once or multiple times with multiple homologous antigens from the same family. In some examples, the subject is immunized with 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more different snake venom toxins. In some examples, the method further comprises screening the identified antibody or antigen-binding fragment for binding to multiple homologous antigens. The screening can be any suitable assay, including but not limited to affinity assay, kinetic assay, or combinations thereof. In some examples, the affinity assay or kinetic assay comprises enzyme-linked immunosorbent assay (ELISA), Octet HTX assay, Biacore assay, or combinations thereof.

[0041] 16. The antibody or antigen-binding fragment described herein, which selectively binds to one or more snake toxins, comprising a VH CDR1, a VH CDR2, a VH CDR3, a VL CDR1, a VL CDR2, and a VL CDR3, wherein (i) the VH CDR1 comprises the amino acid sequence FX2X3X4X5X6DX8H, where X2 is selected from N, T, and S; X3 is selected from F, L; X4 is selected from R, G, and S; X5 is selected from N and T; X6 is selected from Y, F, L; and X8 is selected from M, I (SEQ ID NO: 525); and (ii) the VH CDR2 comprises the amino acid sequence X1X2X3X4X5X6X7GX9X 10 X 11 X 12 (Wherein, X1 is selected from P, S, and H; X2 is selected from V, G, and T; X3 is selected from V, F, L, and I; X4 is selected from D, G, and A; X5 is selected from Y, L, and H; X6 is selected from N, C, R, and T and S; X7 is selected from V, F, and G; X9 is selected from A, E; and 10 is selected from Q, H; X 11 is selected from S, D, Y, and H; X 12 (iii) the VH CDR3 comprises the amino acid sequence CX2RGTLYHYTSGSYX 15 SDAFDIW (wherein X2 is selected from V and A; X 15 is selected from Y and C (SEQ ID NO:527); (iv) the VL CDR1 comprises the amino acid sequence X1ASX4X5IX7X8X9LX 11 (Wherein, X1 is selected from Q and R; X4 is selected from Q and E; X5 is selected from D, G, T, and S; X7 is selected from R and S; X8 is selected from S, D, N, and I; X9 is selected from N, F, Y, W, and D; X 11(v) the VL CDR2 comprises the amino acid sequence X1ASX4X5X6X7, where X1 is selected from G and A; X4 is selected from N, T, and S; X5 is selected from L and S; X6 is selected from Q, L, and E; and X7 is selected from M and S (SEQ ID NO: 529); (vi) the VL CDR3 comprises the amino acid sequence CQQSYSTX8TF, where X8 is selected from I and H (SEQ ID NO: 530), or Provided herein is an antibody or antigen-binding fragment, wherein CDR3 comprises the amino acid sequence CQQX4X5X6X7PX9TF, where X4 is selected from A and S; X5 is selected from N and Y; X6 is selected from I, T, and S; X7 is selected from P, F, and T; and X9 is selected from P, Y, L, and W (SEQ ID NO:531).

[0042] Incorporation by Reference All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0043] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings, in which: [Brief description of the drawings]

[0044] [Figure 1A] FIG. 1A provides a pictorial illustration of clones SNEURO_P01_D09 light chain (D09LC) and SNEURO_P01_D09 heavy chain (D09HC) bound to toxin B.

[0045] [Figure 1B]FIG. 1B provides a pictorial illustration of the α-bungarotoxin / nicotinic acetylcholine receptor (nAChR) complex (PDB: 4UY2), in which the α9 ECD of the nAChR interacts with α-bungarotoxin in loop C.

[0046] [Figure 1C] FIG. 1C provides a pictorial illustration of footprint overlap of toxin B with D09 (dotted line) or nAChR (solid line).

[0047] [Diagram 2] FIG. 2 is a graph illustrating SNEURO_P01_D09 binding to Mamba-AVI-HIS at 0.8, 4, 20, 100 or 500 nM using Octect HTX BL1.

[0048] [Diagram 3] FIG. 3 is a graph illustrating SNEURO_P01_D09 binding to Taipan-AVI-HIS at 0.8, 4, 20, 100 or 500 nM using Octect HTX BL1.

[0049] [Figure 4] FIG. 4 is a graph illustrating SNEURO_P01_D09 binding to Cobra-AVI-HIS at 0.8, 4, 20, 100 or 500 nM using Octect HTX BL1.

[0050] [Diagram 5] Figure 5 is a graph illustrating SNEURO_P01_D09 binding to mamba-AVI-HIS at 0.8, 4, 20, or 100 nM at 25°C. The KD was determined to be <74 pM.

[0051] [Figure 6] Figure 6 is a graph illustrating SNEURO_P01_D09 binding to Taipan-AVI-HIS at 0.8, 4, 20, or 100 nM at 25°C. The KD was determined to be 490 pM.

[0052] [Figure 7] Figure 7 is a graph illustrating SNEURO_P01_D09 binding to Cobra-AVI-HIS at 0.8, 4, or 20 nM at 25°C. The KD was determined to be <37 pM.

[0053] [Figure 8] Figure 8 is a graph illustrating SNEURO_P01_D09 binding to mamba-AVI-HIS at 0.8, 4, 20, or 100 nM at 37°C. The KD was determined to be <57 pM.

[0054] [Figure 9] Figure 9 is a graph illustrating SNEURO_P01_D09 binding to Taipan-AVI-HIS at 0.8, 4, or 20 nM at 37°C. The KD was determined to be 960 pM.

[0055] [Figure 10] Figure 10 is a graph illustrating SNEURO_P01_D09 binding to Cobra-AVI-HIS at 0.8, 4, or 20 nM at 37°C. The KD was determined to be <37 pM.

[0056] [Figure 11] FIG. 11 illustrates the generation of a universal antitoxin from toxins derived from as few as four snake venoms.

[0057] [Figure 12] FIG. 12 illustrates the progress of the development of broad spectrum monoclonal antibody candidates against alpha-neurotoxin, dendrotoxin, and PLA2.

[0058] [Figure 13A]Figures 13A-13F provide the structure of the Neurotoxin B / Centi-LNX-D9 Fab complex at 2.1 Å, revealing the mechanism of neutralization. X-ray crystals of Centi-LNX-D9 against the coastal taipan (Figure 13A), Cape cobra (Figure 13B), mamba (Figure 13C) and common krait (Figure 13D) were obtained. The structure reveals a broad-spectrum neutralization mechanism by molecular mimicry of the native ligand epitope by the VH CDR3 and supporting residues (Figure 13E). High-resolution epitope mapping (Figure 13F) provides the basis for the broad-spectrum neutralization capacity of the Centi-LNX-D09 antibody observed from kinetics and in vivo protection. [Figure 13B] Figures 13A-13F provide the structure of the Neurotoxin B / Centi-LNX-D9 Fab complex at 2.1 Å, revealing the mechanism of neutralization. X-ray crystals of Centi-LNX-D9 against the coastal taipan (Figure 13A), Cape cobra (Figure 13B), mamba (Figure 13C) and common krait (Figure 13D) were obtained. The structure reveals a broad-spectrum neutralization mechanism by molecular mimicry of the native ligand epitope by the VH CDR3 and supporting residues (Figure 13E). High-resolution epitope mapping (Figure 13F) provides the basis for the broad-spectrum neutralization capacity of the Centi-LNX-D09 antibody observed from kinetics and in vivo protection. [Figure 13C] Figures 13A-13F provide the structure of the Neurotoxin B / Centi-LNX-D9 Fab complex at 2.1 Å, revealing the mechanism of neutralization. X-ray crystals of Centi-LNX-D9 against the coastal taipan (Figure 13A), Cape cobra (Figure 13B), mamba (Figure 13C) and common krait (Figure 13D) were obtained. The structure reveals a broad-spectrum neutralization mechanism by molecular mimicry of the native ligand epitope by the VH CDR3 and supporting residues (Figure 13E). High-resolution epitope mapping (Figure 13F) provides the basis for the broad-spectrum neutralization capacity of the Centi-LNX-D09 antibody observed from kinetics and in vivo protection. [Figure 13D]Figures 13A-13F provide the structure of the Neurotoxin B / Centi-LNX-D9 Fab complex at 2.1 Å, revealing the mechanism of neutralization. X-ray crystals of Centi-LNX-D9 against the coastal taipan (Figure 13A), Cape cobra (Figure 13B), mamba (Figure 13C) and common krait (Figure 13D) were obtained. The structure reveals a broad-spectrum neutralization mechanism by molecular mimicry of the native ligand epitope by the VH CDR3 and supporting residues (Figure 13E). High-resolution epitope mapping (Figure 13F) provides the basis for the broad-spectrum neutralization capacity of the Centi-LNX-D09 antibody observed from kinetics and in vivo protection. [Fig. 13E-13F] Figures 13A-13F provide the structure of the Neurotoxin B / Centi-LNX-D9 Fab complex at 2.1 Å, revealing the mechanism of neutralization. X-ray crystals of Centi-LNX-D9 against the coastal taipan (Figure 13A), Cape cobra (Figure 13B), mamba (Figure 13C) and common krait (Figure 13D) were obtained. The structure reveals a broad-spectrum neutralization mechanism by molecular mimicry of the native ligand epitope by the VH CDR3 and supporting residues (Figure 13E). High-resolution epitope mapping (Figure 13F) provides the basis for the broad-spectrum neutralization capacity of the Centi-LNX-D09 antibody observed from kinetics and in vivo protection.

[0059] [Figure 14A-14B] Figures 14A-14B illustrate the results of recombinant toxin challenge in mice. Figure 14A provides data demonstrating that Centi-D09 (D9 in the figure) provided complete broad-spectrum neutralizing protection against recombinant long-chain neurotoxins from black mamba, cape cobra, coastal taipan and Indian krait over a 2-hour period. Note: All lines for D9-treated animals overlap with 100% survival. Figure 14B provides data demonstrating complete protection against black mamba, coastal taipan and cape cobra that continues over a 25-hour period.

[0060] [Figure 15]Figure 15 illustrates the toxin survival percentage with bnAb Centi-D09. After obtaining LD100 for multiple toxins in mice, live challenge studies demonstrated that Centi-D09 (Centi-D09 in the figure) provided complete broad-spectrum neutralizing protection against all toxins from the Thai cobra (Naja kaouthia), black mamba (Dendroaspis polylepsis), and Cape cobra (Naja nivea). Note: All lines for D9-treated animals overlap with 100% survival.

[0061] [Figure 16] Figure 16 illustrates the ability of Centi-D09 to bind and neutralize 3FTX snake venom toxins from a variety of species (phylogeny shown in the left panel). Centi-D09 binds 3FTX long-chain neurotoxins from all species tested (right panel, left column). Centi-D09 protected against in vivo challenge with isolated long-chain neurotoxins from a variety of species (right panel, center column). Finally, Centi-D09 protected against in vivo whole-toxin challenge from a variety of species, and in several cases where the protection was incomplete, the effect was shown to be synergistic with the PLA2 inhibitor varespladib (right panel, right column). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0062] Detailed Description Provided herein is a universal anti-venom composition and a method of using the universal anti-venom composition to treat venomous snakebite.The universal anti-venom composition can comprise one or more antibodies or antigen-binding fragments.Antibody or antigen-binding fragment selectively binds to snake toxin described herein and comprises VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3.

[0063] Representative VH CDR3 sequences The antibody or antigen-binding fragment may comprise a VH CDR3 having an amino acid (AA) sequence that is at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to any one of the following sequences in Table 1. [Table 1-1] [Table 1-2] [Table 1-3]

[0064] Representative VH CDR1 sequences The antibody or antigen-binding fragment may comprise a VH CDR1 having an amino acid sequence that is at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to any one of the following sequences in Table 2. [Table 2-1] [Table 2-2] [Table 2-3]

[0065] Representative VH CDR2 sequences The antibody or antigen-binding fragment may comprise a VH CDR2 having an amino acid sequence that is at least 20%, 24%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to any one of the following sequences in Table 3. [Table 3-1] [Table 3-2] [Table 3-3]

[0066] Representative VL CDR1 sequences The antibody or antigen-binding fragment may comprise a VL CDR1 having an amino acid sequence that is at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to any one of the following sequences in Table 4. [Table 4-1] [Table 4-2]

[0067] Representative VL CDR2 sequences The antibody or antigen-binding fragment may comprise a VL CDR2 having an amino acid sequence that is at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to any one of the following sequences in Table 5. [Table 5-1] [Table 5-2]

[0068] Representative VL CDR3 sequences The antibody or antigen-binding fragment may comprise a VL CDR3 having an amino acid sequence that is at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to any one of the following sequences in Table 6. [Table 6-1] [Table 6-2] [Table 6-3]

[0069] Representative CDR combinations In one instance, an antibody or antigen-binding fragment that selectively binds to a snake toxin or combination of snake toxins comprises one of the following combinations of CDRs: [Table 7-1] [Table 7-2]

[0070] The antibody or antigen-binding fragment can be optimized to increase binding affinity. In one embodiment, such an antibody or antigen-binding fragment can have the heavy chain CDR combination shown in Table 7A: [Table 7A]

[0071] The antibody or antigen-binding fragment can be optimized to increase binding affinity. In one embodiment, such an antibody or antigen-binding fragment can have the light chain CDR combination shown in Table 7B: [Table 7B]

[0072] Representative FW-H1 sequences The antibody or antigen-binding fragment may comprise a VH framework (FW) 1 (FW-H1) having an amino acid sequence that is at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to any one of the following sequences in Table 8. [Table 8-1] [Table 8-2]

[0073] Representative FW-H2 sequences The antibody or antigen-binding fragment may comprise a VH framework (FW) 2 (FW-H2) having an amino acid sequence that is at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to any one of the following sequences in Table 9. [Table 9-1] [Table 9-2]

[0074] Typical FW-H3 arrangement The antibody or antigen-binding fragment may comprise a VH framework (FW) 3 (FW-H3) having an amino acid sequence that is at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to any one of the following sequences in Table 10. [Table 10-1] [Table 10-2]

[0075] Representative FW-H4 sequences The antibody or antigen-binding fragment may comprise a VH framework (FW) 4 (FW-H4) having an amino acid sequence that is at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to any one of the following sequences in Table 11. [Table 11-1] [Table 11-2]

[0076] Representative FW-L1 sequences The antibody or antigen-binding fragment may comprise a VL framework (FW) 1 (FW-L1) having an amino acid sequence that is at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to any one of the following sequences in Table 12. [Table 12-1] [Table 12-2]

[0077] Representative FW-L2 sequences The antibody or antigen-binding fragment may comprise a VL framework (FW) 2 (FW-L2) having an amino acid sequence that is at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to any one of the following sequences in Table 13. [Table 13-1] [Table 13-2]

[0078] Representative FW-L3 sequences The antibody or antigen-binding fragment may comprise a VL framework (FW) 3 (FW-L3) having an amino acid sequence that is at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to any one of the following sequences in Table 14. [Table 14-1] [Table 14-2]

[0079] Representative FW-L4 sequences The antibody or antigen-binding fragment may comprise a VL framework (FW) 4 (FW-L4) having an amino acid sequence that is at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to any one of the following sequences in Table 15. [Table 15-1] [Table 15-2]

[0080] Representative VH sequences The antibody or antigen-binding fragment may comprise a VH having an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to any one of the following sequences in Table 16. [Table 16-1] [Table 16-2] [Table 16-3] [Table 16-4] [Table 16-5] [Table 16-6] [Table 16-7] [Table 16-8]

[0081] Representative VL sequences The antibody or antigen-binding fragment may comprise a VL having an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to any one of the following sequences in Table 17. [Table 17-1] [Table 17-2] [Table 17-3] [Table 17-4] [Table 17-5] [Table 17-6]

[0082] Exemplary Antibodies The antibodies or antigen-binding fragments herein can comprise a variable heavy (VH) amino acid sequence and a variable light (VL) amino acid sequence described herein.

[0083] The antibody or antigen-binding fragment herein may comprise a VH having the amino acid sequence of SEQ ID NO: 224 and a VL having the amino acid sequence of SEQ ID NO: 249 (PLA2_P01_A04).

[0084] The antibody or antigen-binding fragment herein may comprise a VH having the amino acid sequence of SEQ ID NO: 225 and a VL having the amino acid sequence of SEQ ID NO: 250 (PLA2_P01_A08).

[0085] The antibody or antigen-binding fragment herein may comprise a VH having the amino acid sequence of SEQ ID NO: 226 and a VL having the amino acid sequence of SEQ ID NO: 251 (PLA2_P01_B02).

[0086] The antibody or antigen-binding fragment herein may comprise a VH having the amino acid sequence of SEQ ID NO: 227 and a VL having the amino acid sequence of SEQ ID NO: 252 (PLA2_P01_B08).

[0087] The antibody or antigen-binding fragment herein may comprise a VH having the amino acid sequence of SEQ ID NO: 228 and a VL having the amino acid sequence of SEQ ID NO: 253 (PLA2_P01_C02).

[0088] The antibody or antigen-binding fragment herein may comprise a VH having the amino acid sequence of SEQ ID NO: 229 and a VL having the amino acid sequence of SEQ ID NO: 254 (PLA2_P01_C03).

[0089] The antibody or antigen-binding fragment herein may comprise a VH having the amino acid sequence of SEQ ID NO: 230 and a VL having the amino acid sequence of SEQ ID NO: 255 (PLA2_P01_C04).

[0090] The antibody or antigen-binding fragment herein may comprise a VH having the amino acid sequence of SEQ ID NO: 231 and a VL having the amino acid sequence of SEQ ID NO: 256 (PLA2_P01_D06.1).

[0091] The antibody or antigen-binding fragment herein may comprise a VH having the amino acid sequence of SEQ ID NO: 231 and a VL having the amino acid sequence of SEQ ID NO: 257 (PLA2_P01_D06.2).

[0092] The antibody or antigen-binding fragment herein may comprise a VH having the amino acid sequence of SEQ ID NO: 232 and a VL having the amino acid sequence of SEQ ID NO: 258 (PLA2_P01_G05).

[0093] The antibody or antigen-binding fragment herein may comprise a VH having the amino acid sequence of SEQ ID NO: 233 and a VL having the amino acid sequence of SEQ ID NO: 259 (PLA2_P01_G07).

[0094] The antibody or antigen-binding fragment herein may comprise a VH having the amino acid sequence of SEQ ID NO: 224 and a VL having the amino acid sequence of SEQ ID NO: 249 (PLA2_P01_G12).

[0095] The antibody or antigen-binding fragment herein may comprise a VH having the amino acid sequence of SEQ ID NO: 234 and a VL having the amino acid sequence of SEQ ID NO: 260 (KUNITZ_P01_A03.1).

[0096] The antibody or antigen-binding fragment herein may comprise a VH having the amino acid sequence of SEQ ID NO: 234 and a VL having the amino acid sequence of SEQ ID NO: 261 (KUNITZ_P01_A03.2).

[0097] The antibody or antigen-binding fragment herein may comprise a VH having the amino acid sequence of SEQ ID NO: 235 and a VL having the amino acid sequence of SEQ ID NO: 262 (KUNITZ_P01_A07).

[0098] The antibody or antigen-binding fragment herein may comprise a VH having the amino acid sequence of SEQ ID NO: 236 and a VL having the amino acid sequence of SEQ ID NO: 263 (KUNITZ_P01_D07).

[0099] The antibody or antigen-binding fragment herein may comprise a VH having the amino acid sequence of SEQ ID NO: 237 and a VL having the amino acid sequence of SEQ ID NO: 264 (KUNITZ_P01_H05.1).

[0100] The antibody or antigen-binding fragment herein may comprise a VH having the amino acid sequence of SEQ ID NO: 237 and a VL having the amino acid sequence of SEQ ID NO: 265 (KUNITZ_P01_H05.2).

[0101] The antibody or antigen-binding fragment herein may comprise a VH having the amino acid sequence of SEQ ID NO: 238 and a VL having the amino acid sequence of SEQ ID NO: 266 (KUNITZ_P01_H07.1).

[0102] The antibody or antigen-binding fragment herein may comprise a VH having the amino acid sequence of SEQ ID NO: 238 and a VL having the amino acid sequence of SEQ ID NO: 267 (KUNITZ_P01_H07.2).

[0103] The antibody or antigen-binding fragment herein may comprise a VH having the amino acid sequence of SEQ ID NO: 239 and a VL having the amino acid sequence of SEQ ID NO: 268 (SNEURO_P01_A04).

[0104] The antibody or antigen-binding fragment herein may comprise a VH having the amino acid sequence of SEQ ID NO: 240 and a VL having the amino acid sequence of SEQ ID NO: 269 (SNEURO_P01_A08).

[0105] The antibody or antigen-binding fragment herein may comprise a VH having the amino acid sequence of SEQ ID NO: 241 and a VL having the amino acid sequence of SEQ ID NO: 270 (SNEURO_P01_A12).

[0106] The antibody or antigen-binding fragment herein may comprise a VH having the amino acid sequence of SEQ ID NO: 239 and a VL having the amino acid sequence of SEQ ID NO: 271 (SNEURO_P01_B05).

[0107] The antibody or antigen-binding fragment herein may comprise a VH having the amino acid sequence of SEQ ID NO: 239 and a VL having the amino acid sequence of SEQ ID NO: 272 (SNEURO_P01_B11).

[0108] The antibody or antigen-binding fragment herein may comprise a VH having the amino acid sequence of SEQ ID NO: 242 and a VL having the amino acid sequence of SEQ ID NO: 273 (SNEURO_P01_D09).

[0109] The antibody or antigen-binding fragment herein may comprise a VH having the amino acid sequence of SEQ ID NO: 243 and a VL having the amino acid sequence of SEQ ID NO: 274 (SNEURO_P01_E06).

[0110] The antibody or antigen-binding fragment herein may comprise a VH having the amino acid sequence of SEQ ID NO: 243 and a VL having the amino acid sequence of SEQ ID NO: 275 (SNEURO_P01_E09).

[0111] The antibody or antigen-binding fragment herein may comprise a VH having the amino acid sequence of SEQ ID NO: 244 and a VL having the amino acid sequence of SEQ ID NO: 276 (SNEURO_P01_F07).

[0112] The antibody or antigen-binding fragment herein may comprise a VH having the amino acid sequence of SEQ ID NO: 239 and a VL having the amino acid sequence of SEQ ID NO: 277 (SNEURO_P01_F09).

[0113] The antibody or antigen-binding fragment herein includes a VH having the amino acid sequence of SEQ ID NO: 245 and a VL having the amino acid sequence of SEQ ID NO: 278 (SNEURO_P01_H02).

[0114] The antibody or antigen-binding fragment herein may comprise a VH having the amino acid sequence of SEQ ID NO: 246 and a VL having the amino acid sequence of SEQ ID NO: 279 (VENM_M03_B12).

[0115] The antibody or antigen-binding fragment herein may comprise a VH having the amino acid sequence of SEQ ID NO: 247 and a VL having the amino acid sequence of SEQ ID NO: 280 (VENM_M12_G05).

[0116] The antibody or antigen-binding fragment herein may comprise a VH having the amino acid sequence of SEQ ID NO: 248 and a VL having the amino acid sequence of SEQ ID NO: 281 (VENM_M03_A06).

[0117] The antibody or antigen-binding fragment herein may comprise a VH having the amino acid sequence of SEQ ID NO: 235 and a VL having the amino acid sequence of SEQ ID NO: 282 (SNEURO_P01_H06).

[0118] The antibody or antigen-binding fragment herein may comprise a VH of SEQ ID NO: 429, and a VL of any one of SEQ ID NOs: 501-512.

[0119] The antibody or antigen-binding fragment herein may comprise a VH of SEQ ID NO: 430, and a VL of any one of SEQ ID NOs: 501-512.

[0120] The antibody or antigen-binding fragment herein may comprise a VH of SEQ ID NO: 431, and a VL of any one of SEQ ID NOs: 501-512.

[0121] The antibody or antigen-binding fragment herein may comprise a VH of SEQ ID NO: 432, and a VL of any one of SEQ ID NOs: 501-512.

[0122] The antibody or antigen-binding fragment herein may comprise a VH of SEQ ID NO: 433, and a VL of any one of SEQ ID NOs: 501-512.

[0123] The antibody or antigen-binding fragment herein may comprise a VH of SEQ ID NO: 434, and a VL of any one of SEQ ID NOs: 501-512.

[0124] The antibody or antigen-binding fragment herein may comprise a VH of SEQ ID NO: 435, and a VL of any one of SEQ ID NOs: 501-512.

[0125] The antibody or antigen-binding fragment herein may comprise a VH of SEQ ID NO: 436, and a VL of any one of SEQ ID NOs: 501-512.

[0126] The antibody or antigen-binding fragment herein may comprise a VH of SEQ ID NO: 437, and a VL of any one of SEQ ID NOs: 501-512.

[0127] The antibody or antigen-binding fragment herein may comprise a VH of SEQ ID NO: 438, and a VL of any one of SEQ ID NOs: 501-512.

[0128] The antibody or antigen-binding fragment herein may comprise a VH of SEQ ID NO: 439, and a VL of any one of SEQ ID NOs: 501-512.

[0129] The antibody or antigen-binding fragment herein may comprise a VH of SEQ ID NO: 440, and a VL of any one of SEQ ID NOs: 501-512.

[0130] The antibody or antigen-binding fragment herein may comprise a VH of SEQ ID NO: 441, and a VL of any one of SEQ ID NOs: 501-512.

[0131] The antibody or antigen-binding fragment herein may comprise a VH of SEQ ID NO: 442, and a VL of any one of SEQ ID NOs: 501-512.

[0132] The antibody or antigen-binding fragment herein may comprise a VH of SEQ ID NO: 443, and a VL of any one of SEQ ID NOs: 501-512.

[0133] The antibody or antigen-binding fragment herein may comprise a VH of SEQ ID NO: 444, and a VL of any one of SEQ ID NOs: 501-512.

[0134] The antibody or antigen-binding fragment herein may comprise a VH of SEQ ID NO: 445, and a VL of any one of SEQ ID NOs: 501-512.

[0135] The antibody or antigen-binding fragment herein may comprise a VH of SEQ ID NO: 446, and a VL of any one of SEQ ID NOs: 501-512.

[0136] The antibody or antigen-binding fragment herein may comprise a VH of SEQ ID NO: 447, and a VL of any one of SEQ ID NOs: 501-512.

[0137] The antibody or antigen-binding fragment herein may comprise a VH of SEQ ID NO:448, and a VL of any one of SEQ ID NOs:501-512.

[0138] The antibody or antigen-binding fragment herein may comprise a VH of SEQ ID NO: 449, and a VL of any one of SEQ ID NOs: 501-512.

[0139] The antibody or antigen-binding fragment herein may comprise a VH of SEQ ID NO: 450, and a VL of any one of SEQ ID NOs: 501-512.

[0140] The antibody or antigen-binding fragment herein may comprise a VH of SEQ ID NO: 513 and a VL of SEQ ID NO: 518. (SNEURO_P01_D09 VH germlined)

[0141] The antibody or antigen-binding fragment herein may comprise a VH of SEQ ID NO: 514 and a VL of SEQ ID NO: 519. (SNEURO_P01_D09 VK germlined)

[0142] The antibody or antigen-binding fragment herein may comprise a VH of SEQ ID NO: 515 and a VL of SEQ ID NO: 520. (SNEURO_P01_D09 FW germlined)

[0143] The antibody or antigen-binding fragment herein may comprise a VH of SEQ ID NO: 516 and a VL of SEQ ID NO: 521. (SNEURO_P01_D09 VK L3 NS->QS)

[0144] The antibody or antigen-binding fragment herein may comprise a VH of SEQ ID NO: 517 and a VL of SEQ ID NO: 522. (SNEURO_P01_D09 VK L3 NS->NT)

[0145] The antibody or antigen-binding fragment herein may comprise a VH of SEQ ID NO: 523 and a VL of SEQ ID NO: 524. (Centi-DTX-B03)

[0146] In one embodiment, the antibody or antigen-binding fragment herein comprises a VH CDR1, a VH CDR2, a VH CDR3, a VL CDR1, a VL CDR2, and a VL CDR3, wherein (i) the VH CDR1 comprises the amino acid sequence FX2X3X4X5X6DX8H, where X2 is selected from N, T, and S; X3 is selected from F, L; X4 is selected from R, G, and S; X5 is selected from N and T; X6 is selected from Y, F, L; and X8 is selected from M, I (SEQ ID NO: 525); and (ii) the VH CDR2 comprises the amino acid sequence X1X2X3X4X5X6X7GX9X 10 X 11 X 12(Wherein, X1 is selected from P, S, and H; X2 is selected from V, G, and T; X3 is selected from V, F, L, and I; X4 is selected from D, G, and A; X5 is selected from Y, L, and H; X6 is selected from N, C, R, and T and S; X7 is selected from V, F, and G; X9 is selected from A, E; and 10 is selected from Q, H; X 11 is selected from S, D, Y, and H; X 12 (iii) VH CDR3 comprises the amino acid sequence CX2RGTLYHYTSGSYX 15 SDAFDIW (wherein X2 is selected from V and A; X 15 is selected from Y and C (SEQ ID NO:527); (iv) VL CDR1 comprises the amino acid sequence X1ASX4X5IX7X8X9LX 11 (Wherein, X1 is selected from Q and R; X4 is selected from Q and E; X5 is selected from D, G, T, and S; X7 is selected from R and S; X8 is selected from S, D, N, and I; X9 is selected from N, F, Y, W, and D; X 11 (v) the VL CDR2 comprises the amino acid sequence X1ASX4X5X6X7, where X1 is selected from G and A; X4 is selected from N, T, and S; X5 is selected from L and S; X6 is selected from Q, L, and E; and X7 is selected from M and S (SEQ ID NO: 529); (vi) the VL CDR3 comprises the amino acid sequence CQQSYSTX8TF, where X8 is selected from I and H (SEQ ID NO: 530), or CDR3 comprises the amino acid sequence CQQX4X5X6X7PX9TF, where X4 is selected from A and S; X5 is selected from N and Y; X6 is selected from I, T, and S; X7 is selected from P, F, and T; and X9 is selected from P, Y, L, and W (SEQ ID NO:531).

[0147] In some embodiments, the antibody or antigen-binding fragment of the present invention specifically binds to multiple homologous members of the three-finger toxin (3FTX) family found in Elapidae. The antibody or antigen-binding fragment may specifically bind to one or more homologous members of the 3FTX family that are alpha-neurotoxins. The antibody or antigen-binding fragment may specifically bind to one or more homologous members of the 3FTX family that are long-chain neurotoxins. In certain embodiments, the antibody or antigen-binding fragment neutralizes the effect of the long-chain neurotoxin. In certain embodiments, the antibody or antigen-binding fragment binds to one or more three-finger toxins in a manner that prevents the toxin from binding to the human nicotinic acetylcholine receptor (nAChR). In some embodiments, the antibody or antigen-binding fragment binds to at least 30%, 40%, 50%, 60%, 70%, 80% or 90% of the exposed surface area of ​​the toxin that is filled when the toxin binds to the nAChR.

[0148] In certain embodiments, the antibody or antigen-binding fragment binds to two or more neurotoxin members of the 3FTX family using the same multispecific paratope (i.e., a single set of CDRs that can bind to two or more long-chain neurotoxin members of the 3FTX family).

[0149] In some embodiments, the antibody or antigen-binding fragment specifically binds to a member of the long-chain neurotoxin 3FTX family to which the hyperimmune subject from which the antibody was obtained has not been exposed.

[0150] The antibody or antigen-binding fragment may specifically bind to multiple members of the long-chain neurotoxin 3FTX family. The antibody or antigen-binding fragment may specifically bind to two three-fingered toxins that share less than 90%, 80%, 70%, 60% or 50% sequence identity. The antibody or antigen-binding fragment may specifically bind to three unpaired three-fingered toxins that share more than 90%, 80%, 70%, 60% or 50% sequence identity. The antibody or antigen-binding fragment may specifically bind to four, five or six unpaired three-fingered toxins that share more than 90%, 80%, 70%, 60% or 50% sequence identity.

[0151] In one embodiment, the antibody or antigen-binding fragment binds to alpha-bungarotoxin found in the Indian krait, alpha-elapitoxin found in the black mamba, and alpha-neurotoxin found in the Asian cobra. In one embodiment, the antibody or antigen-binding fragment may bind to the 3FTX family of long and short chain neurotoxins.

[0152] In one embodiment, the antibody or antigen-binding fragment binds to a long-chain neurotoxin three-finger type toxin found in two or three or more of the following: mamba species (in the genus dendroaspis), cobra species (in the genus naja), taipan species (in the genus oxyuranus), and krait species (in the genus bungarus). In one embodiment, the antibody or antigen-binding fragment binds to a long-chain neurotoxin three-finger type toxin found in at least one mamba species, at least one cobra species, at least one taipan species, and at least one krait species. In one embodiment, the antibody or antigen-binding fragment binds to long-chain neurotoxin three-finger toxins from two or three or more of the following species: black mamba (dendroaspis polylepis), naja kaouthia, coastal taipan (oxyuranus scutellatus), and bungarus caeruleus. In certain embodiments, the antibody or antigen-binding fragment binds to long-chain neurotoxin three-finger toxins from black mamba (dendroaspis polylepis), naja kaouthia, coastal taipan (oxyuranus scutellatus), and bungarus caeruleus.

[0153] In one embodiment, the antibody or antigen-binding fragment binds to long-chain neurotoxins from two or more species of cobras (in the naja genus). In one embodiment, the antibody or antigen-binding fragment binds to long-chain neurotoxins from two or more species of cobras found on different continents. The antibody or antigen-binding fragment may bind to 3FTX found in Asian snake species and 3FTX found in African snake species. For example, the antibody or antigen-binding fragment may bind to Indian cobra (naja naja) and Egyptian cobra (naja haje). The antibody or antigen-binding fragment may bind to 3FTX found in cobras found in India, cobras found in Africa, and cobras found in Indonesia.

[0154] In one embodiment, the antibody or antigen-binding fragment binds to a long-chain neurotoxin from one or more species of the Elapidae family found in Australia, a long-chain neurotoxin from one or more species of the Elapidae family found in India, and a long-chain neurotoxin from one or more species of the Elapidae family found in Africa. For example, the antibody or antigen-binding fragment may bind to a long-chain neurotoxin found in the coastal taipan (Australia), the Indian cobra (India), and the black mamba (Africa).

[0155] The antibody or antigen-binding fragment may bind to at least one long-chain neurotoxin found in cobras of the genus Naja and at least one long-chain neurotoxin found in cobras of the genus Ophiophagus.

[0156] In one embodiment, the antibody or antigen-binding fragment binds to at least one long-chain neurotoxin found in the venom of snakes of the family Elapidae and not found in the venom of snakes of the subfamily Hydrophiinae, and to at least one long-chain neurotoxin found in the venom of snakes of the subfamily Hydrophiinae. In one embodiment, the antibody or antigen-binding fragment binds to a long-chain neurotoxin from Laticauda colubrina and a long-chain neurotoxin from at least one species of the family Elapidae. In one embodiment, the antibody or antigen-binding fragment binds to a long-chain neurotoxin found in two or more of the following: aipysurus species, hydrophis species, and laticauda species.

[0157] The antibody or antigen-binding fragment combination or population herein may include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more different antibodies. In one case, the antibody or antigen-binding fragment population herein includes one antibody or antigen-binding fragment herein. In one case, the antibody or antigen-binding fragment combination or population herein includes two different antibodies or antigen-binding fragments. In one case, the antibody or antigen-binding fragment combination or population herein includes three different antibodies or antigen-binding fragments. In one case, the antibody or antigen-binding fragment combination or population herein includes four different antibodies or antigen-binding fragments. In one case, the antibody or antigen-binding fragment combination or population herein includes five different antibodies or antigen-binding fragments. In one case, the antibody or antigen-binding fragment combination or population herein includes six different antibodies or antigen-binding fragments. In one case, the antibody or antigen-binding fragment combination or population herein comprises seven different antibodies or antigen-binding fragments. In one case, the antibody or antigen-binding fragment combination or population herein comprises eight different antibodies or antigen-binding fragments. In one case, the antibody or antigen-binding fragment combination or population herein comprises nine different antibodies or antigen-binding fragments. In one case, the antibody or antigen-binding fragment combination or population herein comprises ten different antibodies or antigen-binding fragments. In one case, the antibody or antigen-binding fragment combination or population herein comprises more than ten different antibodies or antigen-binding fragments. The antibody or antigen-binding fragment combination or population herein may comprise an antibody that binds to snakes from a continent, country, or region. For example, the antibody or antigen-binding fragment combination or population herein may be useful for treating snakebites in North, Central America, South America, the Middle East, India, China, Africa, Asia, Australia, etc.Alternatively, combinations or populations of antibodies or antigen-binding fragments herein may be useful for treating snakebites in regions such as the Northeastern United States, the Southeast United States, the Southwestern United States, or the Northwestern United States. Such combinations or populations of antibodies or antigen-binding fragments herein are useful when a bitten subject is unable to identify the particular snake.

[0158] The present application also discloses methods for the identification of broadly neutralizing antibodies against a family of homologous antigens (e.g., snake venom toxins). Families of homologous antigens found in snake venoms include three-finger toxins (3FTX), long-chain neurotoxins, short-chain neurotoxins, snake venom metalloproteinases (SVMPs), snake venom serine proteases (SVSPs), and phospholipase A2 (PLA2). A family of homologous antigens can be another toxin or a family of toxins present in multiple species of snakes.

[0159] The method includes immunization of a human or another animal (a "hyperimmunized subject") with two or more homologous antigens. In some embodiments, the method may include immunization with 2, 3, 4, 5, 6, 7, 8, 9, or 10 homologous antigens. The method may include immunization with between 10 and 20 homologous antigens. The method may include immunization with between 20 and 30 homologous antigens. The method may include immunization with between 30 and 40 homologous antigens. In some embodiments, the immunization may include recombinant homologous antigens. These antigens can be selected to sample a diverse subset of the homologous antigens. In some embodiments, the immunization may include whole toxins from different species that contain the homologous toxin antigens. These species can be selected to be phylogenetically diverse.

[0160] Each of these immunizations can be repeated two or more times. In some embodiments, the method may include an immunization repeated 2, 3, 4, 5, 6, 7, 8, 9, or 10 times. The method may include an immunization repeated between 10 and 20 times. The method may include an immunization repeated between 20 and 30 times. The method may include an immunization repeated between 30 and 40 times. The method may include an immunization repeated more than 40 times. For example, an immunization schedule involving three homologous antigens, repeated twice, may be as follows: immunization with antigens A1, then A2, then A3, then again with A1, A2 and A3.

[0161] The method may further comprise identifying monoclonal antibodies from a subject that have cross-reactivity to two or more homologous antigens.In certain embodiments, the method comprises repeated selection rounds to identify such cross-reactive monoclonal antibodies.Examples of such methods for selection include immunoprecipitation of antibodies from serum; fluorescence-activated cell sorting (FACS) of B cells; or panning of libraries displayed in phage or yeast.

[0162] Regardless of the specific selection technique used, the method of the present invention involves iterative selection using multiple homologous antigens to specifically enrich for antibodies taken from a hyperimmunized subject that are cross-reactive and broadly neutralizing to multiple members of the homologous antigen family. The iterative selection rounds may utilize different homologous antigen sequences to preferentially select for antibodies that are cross-reactive. The iterative selection rounds may utilize members of the homologous antigen family with which the hyperimmunized subject is not immunized to preferentially select for antibodies that are cross-reactive. The selection process may utilize the use of 2, 3, 4, 5, 6, 7, 8, 9, or 10 different members of the homologous antigen family. Clones identified using iterative down-selection are guaranteed to be cross-reactive to all members of the homologous antigen family included in the iterative down-selection method.

[0163] An exemplary method for the identification of cross-reactive antibodies using iterative selection using phage display is given herein. The method may include isolation of PBMCs or B cells from said hyperimmune subject. The method may further include extraction of RNA. The method may further include reverse transcription of cDNA from the extracted RNA. The method may further include amplification (e.g., by PCR) of antibody variable domains. The method may further include construction of a DNA library of said antibody variable domains. The method may further include digestion of the DNA library and cloning into a phage display vector. The method may further include panning against one or more toxins. The method may include panning against 2, 3, 4, 5, 6, 7, 8, 9 or 10 different members of a homologous antigen family, so that the retained clones should be cross-reactive to all members utilized in the iterative panning process. The toxins may include toxins to which the hyperimmune subject has been previously exposed, in addition to homologous toxins to which the hyperimmune subject has not been previously exposed. The method may further include screening the antibody clones for binding, for example by ELISA or kinetics (eg Octet HTX or Biacore).

[0164] Engineered antibodies The present disclosure provides modified antibodies. Modified antibodies may include antibodies with one or more modifications that can improve their activity, binding, specificity, selectivity, or other characteristics. In one aspect, the present disclosure provides modified antibodies (which may include heteromultimers) including the antibodies described herein. Reference to modified antibodies herein also refers to modified antigen-binding fragments. Modified antibodies may include bispecific modified antibodies or antigen-binding fragments, trispecific modified antibodies or antigen-binding fragments, or tetraspecific modified antibodies or antigen-binding fragments.

[0165] The modified antibody may include a human modified antibody. Also included herein are amino acid sequence variants of the modified antibody, which may be prepared by introducing appropriate nucleotide changes into the modified antibody DNA or by synthesis of the desired modified antibody polypeptide. Such variants include, for example, deletion of residues from, insertion of residues into, and / or substitution of residues within the amino acid sequences of the first and second polypeptides forming the modified antibody. Any combination of deletion, insertion, and substitution may be made to arrive at the final construct, provided that the final construct has the desired antigen-binding properties. Amino acid changes may also alter post-translational processes of the modified antibody, such as changing the number or position of glycosylation sites.

[0166] "Alanine scanning mutagenesis" can be a useful method for identifying certain residues or regions of modified antibody polypeptides that may be preferred locations for mutagenesis. Here, one or a group of target residues is identified (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) and replaced with neutral or negatively charged amino acids (e.g., alanine or polyalanine) to affect the interaction of the amino acid with the surrounding aqueous environment inside or outside the cell. Domains that demonstrate the sensitivity of the functional group to the substitution are then refined by introducing additional or other variants at or to the substitution site. Thus, while the site for introducing the amino acid sequence variation is predetermined, the nature of the mutation itself need not be predetermined.

[0167] Typically, substitutions will involve conservative amino acid replacements in non-functional regions of the altered antibody. Exemplary substitutions are shown below. [Table 18]

[0168] Covalent modifications of antibodies, antigen-binding fragments, or modified antibody polypeptides are included within the scope of this disclosure. Covalent modifications of modified antibodies can be introduced into the molecule by reacting targeted amino acid residues of the modified antibody or fragments thereof with organic derivatizing agents capable of reacting with selected side chains or N- or C-terminal residues. Another type of covalent modification of modified antibody polypeptides can include altering the native glycosylation pattern of the polypeptide. As used herein, "altering" can mean deleting one or more carbohydrate moieties found in the original modified antibody and / or adding one or more glycosylation sites not present in the original modified antibody. Addition of glycosylation sites to the modified antibody polypeptide can be accomplished by altering the amino acid sequence such that it contains one or more N-linked glycosylation sites. Modifications can also be made by adding or substituting one or more serine or threonine residues (for O-linked glycosylation sites) to the original modified antibody sequence. Briefly, the modified antibody amino acid sequence can be altered by changes at the DNA level, specifically by mutating preselected bases in the DNA encoding the modified antibody polypeptide such that a codon is generated that will be translated into the desired amino acid. Another means of increasing the number of carbohydrate moieties on the modified antibody polypeptide is by chemical or enzymatic coupling of glycosides to the polypeptide. Removal of carbohydrate moieties present on the modified antibody can be accomplished chemically or enzymatically.

[0169] Another type of covalent modification of the altered antibodies involves linking the altered antibody polypeptide to one of a variety of nonproteinaceous polymers, eg, polyethylene glycol, polypropylene glycol, or polyoxyalkylenes.

[0170] Methods for conjugating the binding agent or antibody or its antigen-binding fragment with another agent herein are known in the art. Such methods may utilize one of several available heterobifunctional reagents used to couple or link molecules.

[0171] In one case, the Fc portion of an antibody can be modified to increase the half-life of the molecule circulating in the blood when administered to a subject.

[0172] In addition, antibodies can be produced or expressed such that they do not contain fucose on their complex N-glycoside linked sugar chains to increase effector function. Similarly, antibodies can be attached at their C-terminus to all or a portion of an immunoglobulin heavy chain from any of the antibody isotypes, e.g., IgG, IgA, IgE, IgD and IgM, and isotype subclasses, e.g., IgG1, IgG2b, IgG2a, IgG3 and IgG4.

[0173] Glycosylation of immunoglobulins has been shown to have a significant effect on their effector function, structural stability, and secretion rate from antibody-producing cells. The antibody or antigen-binding fragment herein can be glycosylated. Glycosylation in variable domain framework residues can change the binding interaction between antibody and antigen. The present disclosure includes criteria that can be selected for mutating a limited number of amino acids in the framework or CDR of immunoglobulin chains (e.g., by substitution, deletion, and / or addition of residues) to increase the affinity of the antibody.

[0174] Linkers for conjugating antibodies to other moieties are within the scope of this disclosure. Association (binding) of the antibody with the label includes, but is not limited to, covalent and non-covalent interactions, chemical conjugation, and recombinant techniques.

[0175] Antibodies, or antigen-binding fragments thereof, can be modified for various purposes, such as by the addition of polyethylene glycol (PEG). PEG modification (PEGylation) can result in one or more of improved circulation time, improved solubility, improved resistance to proteolysis, reduced antigenicity and immunogenicity, improved bioavailability, reduced toxicity, improved stability, and easier formulation.

[0176] The antibody or antigen-binding fragment can be conjugated to an affinity tag (e.g., a purification tag) or recombinantly engineered with said tag. Affinity tags have been described, such as, for example, the His6 tag (His-His-His-His-His-His) (SEQ ID NO: 283).

[0177] It will be appreciated that since it is often difficult to predict the properties of variant engineered antibodies in advance, some screening of the recovered variants may be required to select optimal variants. Exemplary methods for screening recovered variants are described in the Examples below.

[0178] Methods for expressing antibodies Methods for producing any of these antibodies or polypeptides are also provided herein. Polypeptides can be produced by proteolytic or other degradation of the antibodies, by recombinant methods as described above (i.e., single or fusion polypeptides), or by chemical synthesis. Antibody polypeptides, particularly shorter polypeptides up to about 50 amino acids, can be produced by chemical synthesis. Methods for chemical synthesis are commercially available. For example, antibodies could be produced by automated polypeptide synthesizers using the solid phase method.

[0179] Antibodies can be made recombinantly by first isolating the antibody and antibody-producing cells from a host animal, obtaining a gene sequence, and using the gene sequence to recombinantly express the antibody in a host cell (e.g., CHO cell). Another method that can be used is to express the antibody sequence in a plant (e.g., tobacco) or in transgenic milk. Methods for recombinantly expressing antibodies in plants or milk have been disclosed. Methods for making derivatives of antibodies, such as single chains, are also within the scope of this disclosure.

[0180] As used herein, "host cell" includes an individual cell or cell culture that can be or has been a recipient of a vector for the incorporation of a polynucleotide insert. A host cell includes the descendants of a single host cell, and the descendants may not necessarily be completely identical (in terms of morphology or genomic DNA complement) to the original parent cell due to natural, accidental or deliberate mutations. A host cell includes cells that are transfected with the polynucleotides of the present disclosure.

[0181] DNA encoding the antibody can be easily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that can specifically bind to genes encoding the heavy and light chains of the monoclonal antibody). Hybridoma cells can serve as a source of such DNA. Once isolated, the DNA can be placed into one or more expression vectors (e.g., expression vectors disclosed in PCT Publication No. WO87 / 04462), which are then transfected into host cells that do not otherwise produce immunoglobulin proteins, such as E. coli cells, monkey COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells, to achieve synthesis of monoclonal antibodies in recombinant host cells. DNA can also be modified, for example, by substituting coding sequences for human heavy and light chain constant domains in place of homologous mouse sequences, or by covalently linking all or part of the coding sequence of a non-immunoglobulin polypeptide to the immunoglobulin coding sequence. In this way, "chimeric" or "hybrid" antibodies with the binding specificity of the antibodies herein are prepared.

[0182] The vector that encodes one or more antibodies or antigen-binding fragments herein is contemplated herein.As used herein, "vector" refers to a construct that can deliver and possibly express one or more genes or sequences of interest in host cell.Examples of vector include, but are not limited to, virus vector; naked DNA or RNA expression vector; plasmid, cosmid, or phage vector; DNA or RNA expression vector with cationic condensing agent; DNA or RNA expression vector encapsulated in liposome; and certain eukaryotic cells, such as production cells.

[0183] As used herein, "expression control sequence" refers to a nucleic acid sequence that directs the transcription of a nucleic acid. The expression control sequence can be a promoter, for example, a constitutive or inducible promoter, or an enhancer. The expression control sequence is operably linked to the nucleic acid sequence that is to be transcribed. An expression vector can be used to direct the expression of an antibody. The expression vector can be administered to achieve the expression of a foreign protein in vivo.

[0184] A widely used mammalian expression system for high-level production utilizes Lonza's GS Gene Expression System™, which uses viral promoters and selection for glutamate metabolism to result in the generation of high-yielding, stable mammalian cell lines.

[0185] An alternative, widely used mammalian expression system for high-level production utilizes gene amplification with dihydrofolate reductase-deficient ("dhfr") Chinese hamster ovary cells. This system is based on the dihydrofolate reductase "dhfr" gene, which encodes the DHFR enzyme that catalyzes the conversion of dihydrofolate to tetrahydrofolate. To achieve high production, dhfr-CHO cells are transfected with an expression vector that contains a functional DHFR gene together with a gene encoding the desired protein. In this case, the desired protein is a recombinant antibody heavy and / or light chain.

[0186] By increasing the amount of the competitive DHFR inhibitor methotrexate (MTX), the recombinant cells develop resistance by amplifying the dhfr gene. In typical cases, the amplification unit utilized is much larger than the size of the dhfr gene, resulting in co-amplification of the antibody heavy chain.

[0187] When large-scale production of a protein such as an antibody chain is desired, both the expression level and stability of the cells to be utilized are considered.

[0188] The present application provides one or more isolated polynucleotides (nucleic acids) encoding the antibodies or antigen-binding fragments herein, vectors containing such polynucleotides, and host cells and expression systems for transcribing and translating such polynucleotides into polypeptides.

[0189] The present application also provides constructs in the form of plasmids, vectors, transcription or expression cassettes, comprising at least one polynucleotide as described above.

[0190] The present application also provides a recombinant host cell comprising one or more of the constructs described above. Any of the nucleic acids encoding the antibodies or antigen-binding fragments herein form an aspect of the present application, as do methods of producing the antibodies, including expression from the encoding nucleic acid therefrom. Expression can be achieved by culturing recombinant host cells containing the nucleic acid under appropriate conditions. After production by expression, the antibody or a portion thereof can be isolated and / or purified using any suitable technique and then used as appropriate.

[0191] Systems for cloning and expression of a polypeptide in a variety of different host cells are contemplated for use herein.

[0192] A further aspect provides a host cell containing the nucleic acid herein using any suitable method. Yet a further aspect provides a method comprising introducing such a nucleic acid into a host cell. Following introduction, expression from the nucleic acid can be caused or allowed, for example, by culturing the host cell under conditions for expression of the gene.

[0193] One or more polynucleotides encoding the antibody or antigen-binding fragment can be prepared recombinantly / synthetically in addition to or instead of being cloned. In further embodiments, the recombinant DNA molecules or entire DNA sequences of the cloned genes of the antibody or antigen-binding fragment herein can be operably linked to expression control sequences that can be introduced into a suitable host using any suitable method.

[0194] Nucleic acid sequences can be expressed by operably linking them to expression control sequences in an appropriate expression vector and transforming an appropriate host cell with that expression vector. Any of a wide variety of expression control sequences - sequences that control the expression of a nucleic acid sequence operably linked to it - can be used in these vectors to express the nucleic acid sequences.

[0195] A wide variety of host / expression vector combinations can be utilized in expressing the nucleic acid sequences of the present disclosure. It will be understood that not all vectors, expression control sequences, and hosts will function equally well to express a nucleic acid sequence. Not all hosts will function equally well in the same expression system. In some embodiments, when selecting a vector, the host is considered so that the vector can function therein. The copy number of the vector, the ability to control that copy number, and the expression of any other proteins encoded by the vector, such as antibiotic markers, may also be considered. In certain embodiments, when selecting a vector, the host is considered so that the vector can function therein. The copy number of the vector, the ability to control that copy number, and the expression of any other proteins encoded by the vector, such as antibiotic markers, may also be considered.

[0196] The present application also provides methods that include using the above-described constructs in an expression system to express the above-described antibodies (or portions thereof). Taking these and other factors into consideration, a variety of vector / expression control sequence / host combinations can be constructed that are capable of expressing nucleic acid sequences using fermentation or in large scale animal culture.

[0197] Co-incorporation of a nucleic acid encoding an antibody (or portion thereof) and a selected amino acid position alteration can be accomplished by a variety of suitable methods, including, for example, recombinant and chemical synthesis.

[0198] Provided herein is a method for expressing an antibody or antigen-binding fragment (e.g., an antibody or antigen-binding fragment) in a subject, the method comprising administering to the subject a composition comprising a polynucleotide (e.g., mRNA) encoding the antibody or antigen-binding fragment.

[0199] In some cases, administering a polynucleotide to a subject may include enteral, gastrointestinal, oral, transdermal, epicutaneous, intradermal, subcutaneous, nasal, intravenous, intraperitoneal, intraarterial, intramuscular, intraosseous injection, transmucosal, insufflation, or sublingual administration. In some cases, a polynucleotide may be administered by more than one route.

[0200] The antibody or antigen-binding fragment can be synthesized in the subject based at least in part on the polynucleotide encoding the antibody or antigen-binding fragment. For example, the polynucleotide can enter the subject's cells, and the antibody or antigen-binding fragment can be synthesized at least in part by using the transcription and / or translation machinery of the subject's cells. In some cases, for example, when the polynucleotide is an mRNA molecule, the antibody or antigen-binding fragment can be synthesized at least in part by using the translation machinery (e.g., ribosome, tRNA, etc.) of the subject's cells. In some cases, the antibody or antigen-binding fragment can be transported from the cell to the subject's plasma after translation.

[0201] composition Compositions comprising the antibodies or antigen-binding fragments herein can be prepared for storage in the form of lyophilized formulations or aqueous solutions by mixing the antibodies or antigen-binding fragments having the desired purity with pharma- ceutically acceptable carriers, excipients, or stabilizers as needed (Remington, The Science and Practice of Pharmacy 20th Ed. Mack Publishing (2000)).

[0202] As used herein, "pharmaceutical acceptable carrier" or "pharmaceutical acceptable excipient" includes any material that, when combined with active ingredient, allows the ingredient to retain biological activity and is non-reactive with the immune system of the subject. Examples include, but are not limited to, any of the standard pharmaceutical carriers, such as phosphate buffered saline solution, water, emulsions, such as oil / water emulsions, and various types of wetting agents. The preferred diluent for aerosol or parenteral administration is phosphate buffered saline or physiological (0.9%) saline. The composition containing such carriers is formulated by well-known conventional methods (see, for example, Remington's Pharmaceutical Sciences, 18th edition, A. Gennaro, Ed., Mack Publishing Co., Easton, Pa., 1990; and Remington, The Science and Practice of Pharmacy 20th Ed. Mack Publishing, 2000).

[0203] Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed and include, but are not limited to, buffers, such as phosphates, citrates, and other organic acids; salts, such as sodium chloride; antioxidants, including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl or benzyl alcohol, alkyl parabens, such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as For example, serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrins; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants, such as TWEEN®, PLURONICS®, or polyethylene glycol (PEG).

[0204] The composition to be used for in vivo administration can be sterilized.This can be achieved, for example, by filtration through a sterile filtration membrane or any other art-recognized sterilization method.Antibody compositions are generally placed in a container with a sterile outlet, for example, an intravenous solution bag or vial with a stopper that can be pierced by a hypodermic injection needle.Other methods for sterilization and filtration are known in the art and are contemplated herein.

[0205] In some embodiments of the present disclosure, the compositions are formulated to be pyrogen-free so that they are acceptable for administration to a subject.

[0206] Compositions according to the disclosure may be in unit dosage form, such as solutions or suspensions, tablets, pills, capsules, powders, granules, or suppositories, for intravenous, oral, parenteral or rectal administration, or by inhalation or insufflation.

[0207] The phrase "pharmacologically acceptable" refers to molecular entities and compositions that, when administered to a subject, are physiologically tolerable and do not normally produce allergic or similar adverse reactions, e.g., stomach upset, dizziness, and the like.

[0208] In some cases, the antibody or antigen-binding fragment can be bound to one or more carriers. The carrier can be active and / or inactive. Examples of well-known carriers include polypropylene, polystyrene, polyethylene, dextran, nylon, amylase, glass, natural and modified cellulose, polyacrylamide, agarose and magnetite. The nature of the carrier can be either soluble or insoluble for the purpose of this disclosure. Those skilled in the art will know or be able to ascertain, for example, by using routine experimentation, other carriers suitable for binding antibodies.

[0209] Various embodiments contemplate the use of the antibody or antigen-binding fragment to manufacture a medicament for treating a condition, disease or disorder described herein. The medicament can be formulated based on the physical characteristics of the subject needing treatment, and can be formulated into single or multiple formulations based on the stage of the condition, disease or disorder. The medicament can be packaged in a suitable package with appropriate labeling for distribution to hospitals and clinics, the labeling being for indicating that the subject having the disease described herein is treated. The medicament can be packaged as a single unit or as multiple units. Instructions for the dosage and administration of the composition can be included with the package as described below. The present disclosure further relates to a medicament of the antibody or antigen-binding fragment and a pharmaceutically acceptable carrier.

[0210] kit Provided herein is a kit comprising one or more antibodies or antigen-binding fragments herein. Provided herein is a container means comprising one or more antibodies or antigen-binding fragments herein. The container means can be any suitable container capable of containing a liquid or lyophilized composition, including but not limited to a vial, syringe, bottle, intravenous (IV) bag, ampoule or any other suitable container. The syringe can hold any volume of liquid suitable for injection into a subject, including but not limited to 0.5cc, 1cc, 2cc, 5cc, 10cc or more cc. In some embodiments, the antibody or antigen-binding fragment is lyophilized and the kit comprises one or more suitable buffers for reconstitution prior to injection.

[0211] The kit may include one or more instructions describing the use of the one or more antibodies or antigen-binding fragments. The kit may include one or more labels describing the contents and use of the one or more antibodies. Treatment

[0212] The present disclosure provides a method of preventing or treating a subject suffering from envenomation, comprising administering to the subject an antibody or antigen-binding fragment herein. In one case, the subject to be treated exhibits symptoms prior to administration of the antibody. In another case, the subject to be treated does not exhibit symptoms prior to administration of the antibody. "Subject" as used herein includes, but is not limited to, humans, rodents, primates, and the like. Treatment can result in partial or complete treatment of one or more symptoms of envenomation.

[0213] The antibody or antigen-binding fragment herein can be administered to a subject in an amount that achieves at least partial or complete relief of one or more symptoms. Relief can be, for example, about 5% or more reduction in one or more symptoms compared to before treatment. The composition can be formulated by methodologies known to those skilled in the art for administration to human patients. The amount of antibody required to provide therapeutic treatment of snakebite is essentially not fixed. The amount of antibody administered can vary depending on the widespreadness of the disease, the size of the person suffering from the snakebite. Treatment, in one case, reduces the infection rate within a population of subjects. Treatment can also result in a shorter recovery time, fewer symptoms, or less severe symptoms, or a combination thereof, compared to untreated subjects with snakebite.

[0214] "Administering" is considered herein to provide one or more compositions to a patient so that the compositions are present in the patient's body. Such administration can be by any route, including, but not limited to, by subcutaneous, intradermal, intravenous, intraarterial, intraperitoneal or intramuscular administration (e.g., injection), locally, regionally or systemically. In one case, administration is by intradermal injection. In another case, administration is by subcutaneous injection. In one embodiment, a subject is administered one or more times one of the antibodies or antigen-binding fragments herein. In another embodiment, a subject is administered one or more times two of the antibodies or antigen-binding fragments herein. In another embodiment, a subject is administered one or more times three of the antibodies or antigen-binding fragments herein. In another embodiment, a subject is administered one or more times four of the antibodies or antigen-binding fragments herein. Administration can be by any available route, including but not limited to enteral, gastrointestinal, oral, transdermal, epicutaneous, intradermal, subcutaneous, nasal, intravenous, intraperitoneal, intraarterial, intramuscular, intraosseous injection, transmucosal, insufflation or sublingual administration.

[0215] The actual dosage level of the antibody can be varied to obtain an amount of active ingredient that is effective to achieve the desired therapeutic response without being toxic to the patient. The dosage level selected will depend on a variety of factors, including the activity of the particular antibody employed, the route of administration, the number of administrations, the excretion rate of the particular antibody to be employed, the duration of treatment, other drugs, compounds and / or materials used in combination with the particular composition to be employed, the age, sex, weight, condition, general health and past medical history of the patient to be treated, and similar factors.

[0216] The antibodies or antigen-binding fragments herein can be administered to a subject in different dosages and over different time frames.

[0217] A physician or veterinarian can easily determine and prescribe the effective amount (ED50) of the antibody required. For example, the physician or veterinarian could start the dosage of the antibody used in the composition at a level lower than that required to achieve the desired therapeutic effect, and gradually increase the dosage until the desired effect is achieved. Alternatively, the dosage could remain constant.

[0218] The antibodies can be administered to the patient by any convenient route, such as those described above. Regardless of the route of administration selected, the antibodies of the present disclosure can be used in a suitable hydrated form and / or in a composition, and can be formulated into an acceptable dosage form.

[0219] The toxicity and therapeutic effectiveness of a compound can be measured, for example, by the LD50 (the dose lethal to 50% of the population) and ED 50The therapeutic index can be determined by standard procedures in cell cultures or experimental animals to determine the dose (therapeutically effective dose in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index, which can be expressed as the LD50 / ED50 ratio. Compounds that exhibit toxic side effects can be used, but care must be taken to design a delivery system that targets such compounds to affected tissues so as to minimize potential damage to healthy cells and thereby reduce side effects.

[0220] Data obtained from cell culture assays and / or animal studies can be used to formulate a dosage range for use in humans. The dosage of such compounds is preferably within a range of circulating concentrations that includes the ED50 with little or no toxicity. Dosages can vary within this range depending on the dosage form and route of administration used. For any compound, the therapeutically effective amount can be initially estimated from cell culture assays. A dose can be formulated in animal models to achieve a circulating plasma concentration that includes the IC50 (i.e., the concentration of the test compound that achieves half-maximal inhibition) determined in cell culture. Levels in plasma can be measured, for example, by high performance liquid chromatography. Such information can be used to more accurately determine useful doses in humans.

[0221] It will be understood that one or more administrations of antibody or antigen-binding fragment herein can be supplemented with one or more additional therapies or drugs, such as nonsteroidal anti-inflammatory drugs (NSAIDs).The most well-known NSAIDs are aspirin, acetaminophen, ibuprofen and naproxen, all of which are available over the counter (OTC) in most countries.Additional non-limiting examples of NSAIDs include, but are not limited to, diflunisal, dexibuprofen, fenoprofen, ketoprofen, dexketoprofen, flurbiprofen, oxaprozin, loxoprofen, indomethacin, tolmetin, sulindac, etodolac, ketorolac, diclofenac, aceclofenac, bromfenac, nabumetone, piroxicam, meloxicam, tenoxicam, droxicam, lornoxicam, naproxen sodium, acetylsalicylic acid, mefenamic acid, celecoxib, and phenylbutazone. The one or more additional therapies or drugs can include, for example, a PLA2 inhibitor, including, but not limited to, varespladib, methylvarespladib, or a combination thereof.

[0222] Illustrative Definitions The term "about," as used herein, generally refers to a range of 2%, 5%, 10%, 15% greater or less than (±) the stated numerical value in the context of the particular usage. For example, "about 10" would include a range of 8.5 to 11.5. As used herein, the terms "about" and "approximately," when used to modify a numerical value or numerical range, indicate deviations above and below the value or range of up to about 0.2%, about 0.5%, about 1%, about 2%, about 5%, about 7.5%, or about 10% (or any integer between about 1% and 10%) while remaining within the intended meaning of the recited value or range.

[0223] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a method" includes one or more methods and / or steps of the type herein and / or that will become apparent to those skilled in the art upon reading this disclosure.

[0224] An antigen can be a molecule that can be recognized by the immune system, which can include toxins, for example, of a snake. For example, an antigen can be a foreign substance that can induce an immune response. In some cases, the induced immune response can include the production of antibodies. In some cases, an antibody can recognize, attach to, or bind to an antigen.

[0225] "Snake", as referred to herein, refers to any venomous snake, including, but not limited to, the following: Boiga irregularis (brown tree snake), Boiga cyanea (green tree snake), Boiga dendrophila (mangrove snake), Dispholidus typus (boomslang), Salvadora grahamiae (mountain patchnose snake), Spalerosophis diadema (crowned crested snake), Tantilla nigriceps (Plains blackhead snake), Thelotornis capensis (southern twig snake), Thelotornis kirtlandii (northern twig snake), Toxicodryas blandingii (Blanding's tree snake), Trimorphodon lambda (Sonoran lyre snake), Amphiesma stolatum (buff-striped scaly-back), Natrix scaly-backed ... tessellate(Dice snake), Rhabdophis subminiatus(Red collared burrowing asp), Rhabdophis tigrinus(Rabdophis tigrinus), Thamnophis elegans(Western land garter snake), Thamnophis sirtalis(Common garter snake), Ahaetulla nasuta(Long-legged burrowing asp), Atractaspis bibronii(Bibronzed burrowing asp), Atractaspis dahomeyensis(Dahomey burrowing asp), Atractaspis engaddensis(Palestinian mole snake), Atractaspis microlepidota(Small-scaled burrowing asp), Malpolon monspessulanus(Montpellier snake), Acanthophis antarcticus(Common death adder), Aipysurus laevis (Olive Brown Sea Snake), Aipysurus duboisii (Dubois Sea Snake), Austrelaps superbus (Lowland Copperhead), Cryptophis nigrescens (Small Ice Snake), Demansiaolivacea(Olive Whip Snake), Emydocephalus annulatus(Spotted White Sea Snake), Furina tristis(Stephen's Striped Snake), Hydrophis melanocephalus(Black Headed Sea Snake), Hydrophis curtus(Spiny Sea Snake), Hydrophis gracilis(Slender Sea Snake), Hydrophis elegans(Elegant Sea Snake), Hydrophis jerdonii(Corn Nosed Sea Snake), Hydrophis klossi(Llangol Sea Snake), Hydrophis peronii(Horned Sea Snake), Hydrophis belcheri(Belcher's Sea Snake), Hydrophis stricticollis(Bengal Sea Snake), Hydrophis major(Olive Headed Sea Snake), Hydrophis Hydrophis stokesii (large-headed sea snake), Hydrophis melanosoma (black-banded robust sea snake), Hydrophis hardwickii (dorsal-ventral sea snake), Hydrophis cyanocinctus (spotted sea snake), Hydrophis spiralis (narrow-banded sea snake), Hydrophis nigrocinctus (black-banded sea snake), Hydrophis platurus (back-striped sea snake), Hydrophis ornatus (black-spotted sea snake), Hydrophis viperinus (venomous sea snake), Hydrophis schistosus (rough-legged sea snake), Notechis scutatus (mainland tiger snake), Oxyuranus scutellatus (coastal taipan), Oxyuranus temporalis (central ranges taipan), Pseudechis australis (Margas Snake), Pseudechis butleri (Butler's Black Snake), Pseudechis colletti (Collet's Black Snake), Pseudechis guttatus (Blue-bellied Black Snake), Pseudechis papuanus (Papuan Black Snake), Pseudechisporphyriacus(Red-bellied Black Snake), Pseudonaja affinis(Dugite), Pseudonaja guttata(Speckled Brown Snake), Pseudonaja inframacula(Peninsula Brown Snake), Pseudonaja nuchalis(Western Brown Snake), Pseudonaja textilis(Eastern Brown Snake), Tropidechis carinatus(Clarence River Snake), Aspidelaps lubricus(Cape Coral Snake), Aspidelaps scutatus(Shield Nose Snake), Bungarus fasciatus(Round-tailed Krait), Bungarus caeruleus(Indian Krait), Bungarus candidus(Blue Krait), Bungarus flaviceps(Red-headed Krait), Bungarus multicinctus(Multi-banded Krait), Dendroaspis viridis(Western Green Mamba), Dendroaspis angusticeps(Eastern Green Mamba), Dendroaspis jamesoni(Jameson's Mamba), Dendroaspis polylepis(Black Mamba), Elapsoidea sundevallii(Sandevall's African Garter Snake), Hemachatus haemachatus(Lyncarus), Laticauda colubrina(Blue Spotted Sea Snake), Laticauda laticaudata(Broad-headed Sea Snake), Laticauda semifasciata(Black-spotted Sea Snake), Micrurus obscurus(Bolivian Coral Snake), Micrurus frontalis(Southern Coral Snake), Micrurus alleni(Allen's Coral Snake), Micrurus altirostris(Uruguay Coral Snake), Micrurus clarki(Clark's Coral Snake), Micrurus corallinus (Painted Coral Snake), Micrurus distans (West Mexican Coral Snake), Micrurus dumerilii (Dumerill's Coral Snake), Micrurusfulvius(Eastern coral snake), Micrurus hemprichii(Hemprich's coral snake), Micrurus ibiboboca(Cartinga coral snake), Micrurus lemniscatus(South American coral snake), Micrurus mipartitus(Red-tailed coral snake), Micrurus mosquitensis(Costa Rican coral snake), Micrurus multifasciatus(Multi-striped coral snake), Micrurus nigrocinctus(Central American coral snake), Micrurus pyrrhocryptus(Argentine coral snake), Micrurus spixii(Amazonian coral snake), Micrurus surinamensis(Aquatic coral snake), Micrurus tener(Texas coral snake), Micrurus tschudii(Desert coral snake), Naja siamensis(Indochinese spitting cobra), Naja annulata(Ring Water Cobra), Naja annulifera(Long-toed Cobra), Naja ashei(Giant Spitting Cobra), Naja atra(Taiwan Cobra), Naja christyi(Congo Cobra), Naja haje(Egyptian Cobra), Naja kaouthia(Thailand Cobra), Naja katiensis(Western African Spitting Cobra), Naja melanoleuca(Forest Cobra), Naja mossambica(Mozambican Spitting Cobra), Naja naja(Indian Cobra), Naja nigricollis(Black Necked Spitting Cobra), Naja nivea(Cape Cobra), Naja nubiae(Nubian Spitting Cobra), Naja oxiana(Caspian Cobra), Naja pallida(Red Spitting Cobra), Naja philippinensis(North Philippine Cobra), Naja samarensis (Samarcobra), Naja sputatrix (Indonesian Spitting Cobra), Naja sumatrana (Sumatran Spitting Cobra), Ophiophagus hannah (King Cobra), Walterinnesia aegyptia (Western Desert Black Cobra), Homalopsisbuccata(Linnaeus's water snake), Myrrophis chinensis(Chinese mud snake), Subsessor bocourti(Bocourt's water snake), Azemiops feae(Cobra viper), Agkistrodon bilineatus(Bear-eared pit viper), Agkistrodon contortrix(German copperhead), Agkistrodon piscivorus(Cotton murphy), Agkistrodon taylori(Castellana), Agkistrodon laticinctus(Broadband copperhead), Atropoides picadoi(Picado's jumping pit viper), Bothriechis lateralis(Side-stripe palm pit viper), Bothriechis nigroviridis(Black-spotted palm pit viper), Bothriechis schlegelii(Eyelash palm pit viper), Bothrops diporus(Chacolance head), Bothrops erythromelas (Curting Lancehead), Bothrops insularis (Golden Lancehead Viper), Bothrops jararaca (Hararaca), Bothrops neuwiedi (Neuwiedi's Lancehead), Bothrops pauloensis (Black-faced Lancehead), Bothrops asper (Terciopelo), Bothrops atrox (Kaikasa), Bothrops ayerbei (Ayerbe's Lancehead), Bothrops caribbaeus (Saint Lucia Lancehead), Bothrops jararacussu (Jararacussu), Bothrops lanceolatus (Martinie Lancehead), Bothrops leucurus (White-tailed Lancehead), Bothrops moojeni (Brazilian Lancehead), Bothrops alternatus (Urutu), Bothrops cotiara (Cotiara), Bothrops fonsecai (Fonseca's Lancehead), Bothrops itapetiningae (Sao Paulo Lancehead), Bothropstaeniatus (Speckled Forest Pit Viper), Bothrops mattogrossensis (Mato Grosso Lantzen Otter), Calloselasma rhodostoma (Malayan Pit Viper), Cerrophidion godmani (Godman Mountain Pit Viper), Cerrophidion sasai (Costa Rican Mountain Pit Viper), Crotalus viridis (Prairie Rattlesnake) , Crotalus atrox (Western diamondback rattlesnake), Crotalus adamanteus (Eastern diamondback rattlesnake), Crotalus basiliscus (West Coast Mexican rattlesnake), Crotalus catalinensis (Santa Catalina Island rattlesnake), Crotalus cerastes (Sidewalk rattlesnake), Crotalus Cerberus (Arizona black rattlesnake), Crotalus durissus (South American rattlesnake), Crotalus enyo (Baja California rattlesnake), Crotalus horridus (timber rattlesnake), Crotalus lepidus (mottled rock rattlesnake), Crotalus mitchellii (San Lucan spotted rattlesnake), Crotalus molossus (Northern black-tailed rattlesnake), Crotalus oreganus (Northern Pacific rattlesnake), Crotalus pricei(Western twin-spotted rattlesnake), Crotalus pusillus(Tassitalandusky rattlesnake), Crotalus ravus(Mexican pygmy rattlesnake), Crotalus ruber(Red diamond rattlesnake), Crotalus scutulatus(Mojave rattlesnake), Crotalus simus(Central American rattlesnake), Crotalus tigris(Tiger rattlesnake), Crotalus totonacus(Totonacan rattlesnake), Crotalus tzabcan(Yucatan neotropical rattlesnake), Crotalus willardi(Arizona ridgenose rattlesnake), Crotalus pyrrhus(Southwestern speckled rattlesnake), Crotalus vegrandis(Ulacoan rattlesnake), Deinagkistrodon acutus(Hypoda), Gloydius intermedius (intermediate pit viper), Gloydius blomhoffii (pit viper), Gloydius brevicaudus (striped pit viper), Gloydius halys (Siberian pit viper), Gloydius shedaoensis (Snake Island pit viper), Gloydiusussuriensis (Ussurian pit viper), Hypnale hypnale (Hump-nosed pit viper), Lachesis melanocephala (Black-headed bushmaster), Lachesis muta (Atlantic bushmaster), Ovophis okinavensis (Ovophis okinavensis), Porthidium nasutum (Rainforest ash-striped pit viper), Porthidium ophryomegas (Slender ash-striped pit viper), Protobothrops elegans (Sakishima pit viper), Protobothrops flavoviridis (Okinawa pit viper), Protobothrops mangshanensis (Mangshan pit viper), Protobothrops mucrosquamatus (Taiwan pit viper), Protobothrops tokarensis (Tokara pit viper), Sistrurus catenatus (Massasauga), Sistrurus miliarius (Pygmy rattlesnake), Trimeresurus stejnegeri(Taiwanese green pit viper), Trimeresurus albolabris(White-lipped blue pit viper), Trimeresurus erythrurus(Burmese green pit viper), Trimeresurus gramineus(Green pit viper), Trimeresurus labialis(Nicobar bamboo pit viper), Trimeresurus macrops(Large-eyed pit viper), Trimeresurus malabaricus(Malabar pit viper), Trimeresurus popeiorum(Pope's green pit viper), Trimeresurus purpureomaculatus(Mangrove pit viper), Trimeresurus sumatranus(Sumatran pit viper), Tropidolaemus wagleri(Wagler's pit viper),Metlapilcoatlus mexicanus(Mexican jumping pit viper),Metlapilcoatlus nummifer(Central American jumping pit viper), Atheris squamigera (Lizard Bush Viper), Bitis arietans (Puff Adder), Bitis Atropos (Cape Mountain Adder), Bitiscaudalis(Horned Puff Adder), Bitis cornuta(Western Horned Adder), Bitis gabonica(Central African Gabon Viper), Bitis nasicornis(Rhinoceros Adder), Bitis parviocula(Ethiopian Adder), Bitis rhinoceros(West African Gabon Viper), Causus rhombeatus(Common Night Adder), Cerastes cerastes(Horned Viper), Cerastes gasperettii(Arabian Horned Viper), Cerastes vipera(Saharan Viper), Daboia palaestinae(Palestinian Viper), Daboia russelii(Russell's Viper), Daboia siamensis(Eastern Russell's Viper), Daboia mauritanica(Moo Viper), Echis ocellatus(West African carpet viper), Echis carinatus(Japanese saw-tooth viper), Echis coloratus(Painted saw-tooth viper), Echis pyramidum(Egyptian saw-tooth viper), Macrovipera schweizeri(Milo's viper), Macrovipera lebetinus(Levantine viper), Montivipera bornmuelleri(Lebanese mountain viper), Montivipera latifii(Latifii viper), Montivipera raddei(Armenian mountain viper), Montivipera xanthine(Asian viper), Pseudocerastes fieldi(Field horned viper), Pseudocerastes persicus(Persian viper), Vipera ammodytes(European horned viper), Vipera aspis (Asp Viper), Vipera berus (European Viper), Vipera latastei (Ratastei Viper), Vipera lotievi (Caucasian Viper), Vipera seoanei (Basque Viper), Vipera ursinii (Viper), Diadophispunctatus (Ring-necked snake), Hydrodynastes gigas (Water snake), Hypsiglena torquata (Night snake), Hypsiglena jani (San Luis toposi night snake), Leptodeira ashmeadii, or Philodryas olfersii (Liechtenstein green racer).

[0226] Toxins (e.g., snake venom) may be components of modified saliva that may contain animal toxins that can aid in the immobilization and / or digestion of prey or defense against dangerous entities. Toxins may be introduced into a subject, for example, by biting or spitting.

[0227] Toxins may include proteins or polypeptides. In some embodiments, toxins may include mixtures of proteins, enzymes, or other substances that have toxic or lethal properties that can affect a subject. The effects of a toxin on a subject may include effects on biological functions, such as blood clotting, blood pressure regulation, or the transmission of nerve or muscle impulses.

[0228] Proteins may constitute 90-95% of the toxin's dry weight and may be responsible for its biological effects. Toxins may include toxins (e.g., neurotoxins), non-toxic proteins (which may also have pharmacological properties), and / or enzymes, particularly hydrolytic ones. Enzymes may constitute 80-90% of Viperidae snake venoms and 25-70% of Elapidae venoms and may include digestive hydrolases, L-amino acid oxidases, phospholipases, thrombin-like procoagulants, and kallikrein-like serine proteases or metalloproteinases (hemorrhagic toxins), which may damage the vascular endothelium. Polypeptide toxins may include cytotoxins, cardiotoxins, and postsynaptic neurotoxins (e.g., α-bungarotoxin and α-cobratoxin), which may bind to acetylcholine receptors, for example at neuromuscular junctions. Compounds with low molecular weight (e.g., up to 1.5 KDa) include metals, peptides, lipids, nucleosides, carbohydrates, amines, or oligopeptides that inhibit angiotensin-converting enzyme (ACE) or enhance bradykinin (BPP). Inter- and intra-species differences in toxin chemical composition may be geographical and / or ontogenetic. Phosphodiesterases may interfere with the cardiac system, for example lowering blood pressure. Phospholipase A2 may cause hemolysis by dissolving the phospholipid cell membrane of red blood cells. Amino acid oxidases and proteases may be used in digestion. Amino acid oxidases may induce other enzymes and may be responsible for the yellow color of some species of toxins. Hyaluronidases may increase tissue permeability accelerating the absorption of other enzymes into tissues. Some snake venoms may have fasciculins, e.g., mamba (Dendroaspis), that may inhibit cholinesterases and cause loss of muscle control in prey. Examples of snake venom enzymes are provided in Table 19. [Table 19-1] [Table 19-2]

[0229] Toxins in toxins such as snake venoms can differ in function. Classes of toxins that can be found in snake venoms can include neurotoxins, hemotoxins, cytotoxins, myotoxins, dendrotoxins, cardiotoxins, fasciculins, neutrotoxins, sarafotoxins, hemorrhagic toxins, etc. Non-limiting examples of types of toxins that can be found in toxins such as snake venoms can be found in Table 20. [Table 20]

[0230] antibody As used herein, the term "antibody" refers to an immunoglobulin (Ig), which refers to a polypeptide or protein having a binding domain that is or is homologous to an antigen-binding domain. The term further includes "antigen-binding fragments" and other interchangeable terms for similar binding fragments, such as those described below. Native antibodies and native immunoglobulins (Ig) are usually heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light chains and two identical heavy chains. Each light chain is typically linked to a heavy chain by one covalent disulfide bond, although the number of disulfide linkages varies between the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced interchain disulfide bridges. Each heavy chain has a variable domain ("V") at one end and a variable domain ("V") at the other end. H "), followed by several constant domains ("C H Each light chain has at one end a variable domain ("V L " ) at its other end, and a constant domain ("C L "), in which the light chain constant domain is aligned with the first constant domain of the heavy chain and the light chain variable domain is aligned with the variable domain of the heavy chain. Particular amino acid residues are believed to form an interface between the light and heavy chain variable domains.

[0231] In some cases, the antibody or antigen-binding fragment thereof comprises an isolated antibody or antigen-binding fragment thereof, a purified antibody or antigen-binding fragment thereof, a recombinant antibody or antigen-binding fragment thereof, an engineered antibody or antigen-binding fragment thereof, or a synthetic antibody or antigen-binding fragment thereof. It will be understood that the antibodies described herein can be modified as described below or as known in the art.

[0232] The antibody and antigen-binding fragment herein can be partially or completely synthetically produced.Antibody or antigen-binding fragment can be a polypeptide or protein having a binding domain, which can be or be homologous to an antigen-binding domain.In one case, antibody or antigen-binding fragment thereof can be produced in a suitable in vivo animal model and then isolated and / or purified.

[0233] Depending on the amino acid sequence of the constant domain of its heavy chain, immunoglobulins (Ig) can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known in the art. An Ig or a portion thereof may in some cases be a human Ig. In some cases, the Ig may be a human Ig. H The three domains are from immunoglobulins. In some cases, the chains or portions of the antibody or antigen-binding fragment thereof, modified antibody or antigen-binding fragment thereof, or binding agent may be from Ig. In such cases, the Ig may be IgG, IgA, IgD, IgE, or IgM. If the Ig is an IgG, it may be a subtype of IgG, which may include IgG1, IgG2a, IgG2b, IgG3, and IgG4. In some cases, the C HThe three domains may be from an immunoglobulin selected from the group consisting of IgG, IgA, IgD, IgE, and IgM.

[0234] The "light chains" of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two clearly distinct types, called kappa ("κ" or "K") or lambda ("λ"), based on the amino acid sequences of their constant domains.

[0235] In this disclosure, where necessary, the following abbreviations (in parentheses) are used according to convention: heavy chain (H chain), light chain (L chain), heavy chain variable region (VH), light chain variable region (VL), complementarity determining region (CDR), first complementarity determining region (CDR1), second complementarity determining region (CDR2), third complementarity determining region (CDR3), heavy chain first complementarity determining region (VH CDR1), heavy chain second complementarity determining region (VH CDR2), heavy chain third complementarity determining region (VH CDR3), light chain first complementarity determining region (VL CDR1), light chain second complementarity determining region (VL CDR2), light chain third complementarity determining region (VL CDR3), first heavy chain framework region (FW-H1), second heavy chain framework region (FW-H2), third heavy chain framework region (FW-H3), fourth heavy chain framework region (FW-H4), first light chain framework region (FW-L1), second light chain framework region (FW-L2), third light chain framework region (FW-L3), fourth light chain framework region (FW-L4).

[0236] The "variable region" of an antibody refers to the variable region of the antibody light chain or the variable region of the antibody heavy chain, either alone or in combination. The variable regions of the heavy and light chains each consist of four framework regions (FWs) connected by three CDRs, also known as hypervariable regions. The CDRs in each chain are held together with the CDRs from the other chain in close proximity by the FWs and contribute to the formation of the antigen-binding site of the antibody.

[0237] With respect to antibodies, the term "variable domain" refers to the variable domain of an antibody that is used for the binding and specificity of each particular antibody to its particular antigen. However, variability is not evenly distributed throughout the variable domain of an antibody. Rather, it is concentrated in three segments called hypervariable regions (also known as CDRs) in both the light chain variable domain and the heavy chain variable domain. The more highly conserved parts of the variable domain are called "framework regions" or "FWs". Unmodified heavy and light chain variable domains each contain four FRs (FW1, FW2, FW3 and FW4) that mainly adopt a β-sheet configuration interspersed with three CDRs that form connecting loops and, in some cases, part of the β-sheet structure. The CDRs in each chain are held together with the CDRs from the other chain in close proximity by the FWs, which contribute to the formation of the antigen-binding site of the antibody.

[0238] The terms "hypervariable region" and "CDR" as used herein refer to the amino acid residues of an antibody that are involved in antigen binding. CDRs contain amino acid residues from three sequence regions that bind to an antigen in a complementary manner and are H and V L Each of the chains is known as CDR1, CDR2 and CDR3. It is understood that the CDRs of different antibodies may contain inserts and therefore may have different amino acid numbering. The CDR sequences of antibodies and their antigen-binding fragments are provided herein below.

[0239] As used herein, "framework region", "FW" or "FR" refers to framework amino acid residues that form part of an antigen-binding pocket or groove. In some embodiments, framework residues form loops that are part of the antigen-binding pocket or groove, and amino acid residues in the loops may or may not contact the antigen. Framework regions generally include the regions between the CDRs. The loop amino acids of the FRs can be evaluated and determined by examination of the three-dimensional structure of the antibody heavy chain and / or the antibody light chain. The three-dimensional structure can be analyzed for amino acid positions that are solvent exposed, as such positions are more likely to form loops and / or make contacts with the antigen within the antibody variable domain. Some of the solvent exposed positions can tolerate amino acid sequence diversity, while others (e.g., structural positions) generally have less diversity. The three-dimensional structure of the antibody variable domain can be derived from a crystal structure or protein modeling.

[0240] The term "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain. An "Fc region" may be a native sequence Fc region or a variant Fc region. Although the boundaries of the Fc region of an immunoglobulin heavy chain vary, the human IgG heavy chain Fc region is usually defined to extend from an amino acid residue at position Cys226, or from Pro230, to its carboxyl terminus. The numbering of residues within the Fc region is that of the EU index as in Kabat et al., (Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991). The Fc region of an immunoglobulin generally consists of two constant domains, the C and C-terminal constant domains, the C and C-terminal constant domains, and the C and C-terminal constant domains. H 2 and C H 3, including.

[0241] In one case, an antibody or antigen-binding fragment may comprise a variable region. The variable region may be the variable region of an antibody light chain or the variable region of an antibody heavy chain, either alone or in combination. Each of the variable regions of the heavy and light chains consists of four framework regions (FRs) connected by three complementarity determining regions (CDRs), also known as hypervariable regions. The CDRs in each chain are held together with the CDRs from the other chain in close proximity by the FRs, and contribute to the formation of the antigen-binding site of the antibody.

[0242] In one case, the antibody or antigen-binding fragment may comprise a light chain region, a heavy chain region, or a light and a heavy chain region that confers specific binding, for example to a toxin. In another case, the antibody or antigen-binding fragment may comprise a constant region. The constant region may comprise the constant region of an antibody light chain, either alone or in combination with the constant region of an antibody heavy chain.

[0243] "Antibodies" useful in the present disclosure include, but are not limited to, monoclonal antibodies, polyclonal antibodies, chimeric antibodies, bispecific antibodies, multispecific antibodies, heteroconjugate antibodies, humanized antibodies, human antibodies, deimmunized antibodies, mutants thereof, fusions thereof, immunoconjugates thereof, antigen-binding fragments thereof, and / or any other modified configuration of an immunoglobulin molecule that contains an antigen recognition site of the required specificity, including glycosylation variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies.

[0244] As used herein, "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies constituting the population are identical except for possible naturally occurring mutations that may be present in minor amounts. In contrast to polyclonal antibody preparations that typically contain different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen (epitope). The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and this modifier should not be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies to be used in accordance with the present disclosure can be produced by the hybridoma method first described by Kohler and Milstein, 1975, Nature, 256:495, or can be produced by recombinant DNA methods such as those described in U.S. Pat. No. 4,816,567. Monoclonal antibodies can also be isolated from phage libraries generated using the techniques described in, for example, McCafferty et al., 1990, Nature, 348:552-554. Other methods are known in the art and are contemplated for use herein.

[0245] As used herein, a "humanized" antibody refers to a form of a non-human (e.g., murine) antibody that is a particular chimeric immunoglobulin, immunoglobulin chain, or fragment thereof that contains minimal sequence derived from the non-human immunoglobulin. In most cases, a humanized antibody is a human immunoglobulin (recipient antibody) in which residues from a complementarity determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat, or rabbit that has the desired antibody specificity, affinity, and biological activity. In some cases, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, a humanized antibody may contain residues that are found neither in the recipient antibody nor in the imported CDR and framework sequences, but are included to further refine and optimize antibody performance. In general, a humanized antibody will contain substantially all of at least one, and usually two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are of a human immunoglobulin consensus sequence. A humanized antibody will also comprise at least a portion of an immunoglobulin constant region or domain (Fc), typically that of a human immunoglobulin. The antibody may have an altered Fc region, for example, as described in WO99 / 58572. Other forms of humanized antibodies have one or more CDRs (one, two, three, four, five or six) that are altered relative to the original antibody, also referred to as one or more CDRs "derived from" one or more CDRs from the original antibody.

[0246] If necessary, the antibodies or antigen-binding fragments thereof described herein can be evaluated for immunogenicity and, if necessary, deimmunized (i.e., the antibody is rendered less immunoreactive by altering one or more T cell epitopes). As used herein, a "deimmunized antibody" means that one or more T cell epitopes in the antibody sequence have been modified such that T cell responses are reduced after administration of the antibody to a subject compared to an antibody that has not been deimmunized. Analysis of the immunogenicity and T cell epitopes present in the antibodies and antigen-binding fragments described herein can be performed by using software and specific databases known in the art. Exemplary software and databases include iTope™, developed by Antitope of Cambridge, England. iTope™ is an in silico technology for the analysis of peptides that bind to human MHC class II alleles. The iTope™ software predicts peptides that bind to human MHC class II alleles, thereby providing an initial screening for the location of such "potential T cell epitopes". The iTope™ software predicts favorable interactions between peptide amino acid side chains and specific binding pockets in the binding grooves of the 34 human MHC class II alleles. The locations of key binding residues are obtained by in silico generation of 9-mer peptides that overlap by one amino acid across the test antibody variable region sequence. Each 9-mer peptide can be tested against each of the 34 MHC class II allotypes and scored based on their potential "fit" and interaction with the MHC class II binding groove. Peptides that give rise to a high average binding score (>0.55 in the iTope™ scoring function) for >50% of the MHC class II alleles are considered potential T-cell epitopes.In such regions, the core 9 amino acid sequence for peptide binding within the MHC class II groove is analyzed to determine the MHC class II pocket residues (P1, P4, P6, P7 and P9) and possible T cell receptor (TCR) contact residues (P-1, P2, P3, P5, P8). After identification of any T cell epitopes, amino acid residue changes, substitutions, additions and / or deletions can be introduced to eliminate the identified T cell epitopes. Such changes can be made to preserve antibody structure and function but still eliminate the identified epitopes. Exemplary changes can include, but are not limited to, conservative amino acid changes.

[0247] The antibody may be a human antibody. As used herein, "human antibody" refers to an antibody that has an amino acid sequence that corresponds to the amino acid sequence of an antibody produced by a human and / or that is produced using any of the techniques for producing human antibodies known in the art or disclosed herein. This definition of a human antibody includes an antibody that comprises at least one human heavy chain polypeptide or at least one human light chain polypeptide. One such example is an antibody that comprises a mouse light chain and a human heavy chain polypeptide. Human antibodies can be produced using a variety of techniques known in the art. In one embodiment, human antibodies are selected from phage libraries that express human antibodies (Vaughan et al., 1996, Nature Biotechnology, 14:309-314; Sheets et al., 1998, PNAS USA, 95:6157-6162; Hoogenboom and Winter, 1991, J. Mol. Biol., 227:381; Marks et al., 1991, J. Mol. Biol., 222:581). Human antibodies can also be produced by introducing human immunoglobulin loci into transgenic animals, e.g., mice, in which the endogenous immunoglobulin genes have been partially or completely inactivated. This approach is described in U.S. Patent Nos. 5,545,807, 5,545,806, 5,569,825, 5,625,126, 5,633,425, and 5,661,016. Alternatively, human antibodies can be prepared by immortalizing human B-lymphocytes that produce antibodies against target antigens (such B-lymphocytes can be retrieved from subjects or immunized in vitro). See, for example, Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985);Boerner et al., 1991, J. Immunol., 147 (1):86-95; and U.S. Patent No. 5,750,373.

[0248] Bispecific antibodies are antibodies that have binding specificities for at least two different antigens and can be prepared using the antibodies disclosed herein. Methods for making bispecific antibodies are known in the art (see, for example, Suresh et al., 1986, Methods in Enzymology 121:210). Traditionally, recombinant production of bispecific antibodies was based on the co-expression of two immunoglobulin heavy-light chain pairs, with the two heavy chains having different specificities (Millstein and Cuello, 1983, Nature, 305, 537-539). Bispecific antibodies can be composed of a hybrid immunoglobulin heavy chain with a first binding specificity in one arm and a hybrid immunoglobulin heavy-light chain pair (providing a second binding specificity) in the other arm. This asymmetric structure, with only one half of the bispecific molecule having an immunoglobulin light chain, facilitates the separation of the desired bispecific compound from undesired immunoglobulin chain combinations. This approach is described in PCT Publication No. WO94 / 04690.

[0249] According to one approach to making bispecific antibodies, antibody variable domains with the desired binding specificities (antibody-antigen combining sites) are fused to immunoglobulin constant domain sequences. Preferably, the fusion is with an immunoglobulin heavy chain constant domain, including at least a portion of the hinge, CH2 and CH3 regions. It is preferred to have the first heavy chain constant region (CH1) containing the site required for light chain binding present in at least one of the fusions. DNAs encoding the immunoglobulin heavy chain fusions and, if desired, the immunoglobulin light chain are inserted into separate expression vectors and co-transfected into a suitable host organism. This allows great flexibility in adjusting the mutual proportions of the three polypeptide fragments in embodiments when unequal ratios of the three polypeptide chains used in the construction result in optimal yields. However, it is possible to insert the coding sequences of two or all three polypeptide chains in one expression vector when expression of at least two polypeptide chains in the same ratio results in high yields or when the ratios are of no particular significance.

[0250] Heteroconjugate antibodies, which comprise two covalently linked antibodies, are also within the scope of this disclosure.Such antibodies have been used to target immune system cells to unwanted cells (US Pat. No. 4,676,980).Heteroconjugate antibodies can also be produced using any convenient cross-linking method.Suitable cross-linking agents and techniques are known in the art and are described, for example, in US Pat. No. 4,676,980.

[0251] "Chimeric" forms of non-human (e.g., murine) antibodies include chimeric antibodies that contain minimal sequence derived from non-human Ig. In most cases, chimeric antibodies are murine antibodies in which at least a portion of an immunoglobulin constant region (Fc), typically of a human immunoglobulin, has been inserted in place of the mouse Fc.

[0252] Chimeric or hybrid antibodies can also be prepared in vitro using known methods of synthetic protein chemistry, including those involving crosslinking agents. For example, immunotoxins can be constructed using a disulfide exchange reaction or by forming a thioether bond. Examples of suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate.

[0253] Provided herein are antibodies and antigen-binding fragments thereof, modified antibodies and antigen-binding fragments thereof, and binding agents that specifically bind to one or more epitopes on one or more target antigens. In one case, the binding agent specifically binds to an epitope on a single antigen. In another case, the binding agent is bivalent and specifically binds to two distinct epitopes on a single antigen, or specifically binds to two distinct epitopes on two distinct antigens. In another case, the binding agent is multivalent (i.e., trivalent, tetravalent, etc.), and the binding agent binds to three or more distinct epitopes on a single antigen, or binds to three or more distinct epitopes on two or more (multiple) antigens.

[0254] Functional fragments of any of the antibodies herein are also contemplated. The terms "antigen-binding portion," "antigen-binding fragment," "antigen-binding domain," "antibody fragment," or "functional fragment of an antibody" are used interchangeably herein to refer to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. Exemplary antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv, scFv, dsFv, variable heavy domain, variable light domain, variable NAR domain, bispecific scFv, bispecific Fab2, trispecific Fab3, AVIMER®, minibodies, diabodies, maxibodies, camelids, VHHs, intrabodies, fusion proteins comprising antibody moieties (e.g., domain antibodies), single-chain binding polypeptides, scFv-Fc, or Fab-Fc.

[0255] "F(ab')2" and "Fab" portions can be produced by treating Ig with proteases such as pepsin and papain, and include antigen-binding portions generated by digesting the immunoglobulin near the disulfide bond present between the hinge regions in each of the two heavy chains. For example, papain cleaves IgG upstream of the disulfide bond present between the hinge regions in each of the two heavy chains to produce V L and C L (light chain constant region) and V H and C Hγ1 Pepsin also cleaves IgG downstream of the disulfide bond between the hinge regions of each of the two heavy chains to produce an antibody fragment that is slightly larger than the fragment in which the two Fab's are connected at the hinge region. This antibody fragment is called F(ab')2.

[0256] The Fab fragment contains the constant domain of the light chain and the first constant domain of the heavy chain (C H 1). Fab' fragments also contain heavy chain C1 that contains one or more cysteines from the antibody hinge region. H They differ from Fab fragments by the addition of a few residues at the carboxyl terminus of one domain. Fab'-SH is the designation used herein for Fab' in which the cysteine ​​residues of the constant domains bear a free thiol group. F(ab')2 antibody fragments originally were produced as pairs of Fab' fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0257] "Fv" as used herein refers to an antibody fragment that contains a complete antigen recognition and binding site. This region consists of a dimer of one heavy and one light chain variable domain in tight non-covalent or covalent association (disulfide-linked Fvs are described in the art, Reiter et al. (1996) Nature Biotechnology 14:1239-1245). In this structure, the three CDRs of each variable domain interact to form a V H -V L Define the antigen-binding site on the surface of the dimer. H and V L The combination of one or more of the CDRs from each of the chains confers antigen-binding specificity to the antibody. For example, it will be understood that CDRH3 and CDRL3 are the V domain of a recipient antibody or antigen-binding fragment thereof. H and V L When transferred to a chain, these may be sufficient to confer antigen-binding specificity to the antibody, and this combination of CDRs can be tested for binding, specificity, affinity, etc. using any of the techniques described herein. Even a single variable domain (or half of an Fv containing only three CDRs specific for an antigen) will have the ability to recognize and bind to the antigen, although likely with less specificity or affinity than when combined with a second variable domain. Furthermore, the two domains of an Fv fragment (V L and V H ) are encoded by separate genes, but they can be joined using recombinant methods with synthetic linkers that allow them to be made into a single protein chain, thereby L Area and V HThe regions pair to form monovalent molecules (also known as single-chain Fvs (scFvs); Bird et al. (1988) Science 242:423-426; Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883; and Osbourn et al. (1998) Nat. Biotechnol. 16:778). Such scFvs are also intended to be encompassed within the term "antigen-binding portion" of an antibody. Any V of a particular scFv may be H and V L The sequences can be ligated to Fc region cDNA or genomic sequences to generate expression vectors encoding complete Ig (e.g., IgG) molecules or other isotypes. V is useful for generating Fab, Fv, or other fragments of Ig using either protein chemistry or recombinant DNA techniques. H and V L can also be used.

[0258] "Single-chain Fv" or "sFv" antibody fragments are fragments of the V H and V L In some embodiments, the Fv polypeptide comprises V domains, and these domains are present in a single polypeptide chain. In some embodiments, the Fv polypeptide comprises a polypeptide linker that enables the sFv to form the desired structure for antigen binding. H Domain and V L For a review of sFv, see, e.g., Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315 (1994).

[0259] The term "AVIMER®" refers to a class of therapeutic proteins of human origin unrelated to antibodies and antibody fragments, which are composed of several modules called A-domains (also called class A modules, complement-type repeats, or LDL-receptor class A domains) and reusable binding domains. They were developed from human extracellular receptor domains by in vitro exon shuffling and phage display (Silverman et al., 2005, Nat. Biotechnol. 23:1493-1494;Silverman et al., 2006, Nat. Biotechnol. 24:220). The resulting proteins may contain multiple independent binding domains that can exhibit improved affinity and / or specificity compared to single epitope binding proteins. Each of the 217 known human A-domains contains about 35 amino acids (about 4 kDa), and these domains are separated by linkers that average 5 amino acids in length. Native A-domains rapidly and efficiently fold into a uniform, stable structure mediated primarily by calcium binding and disulfide formation. A conserved scaffolding motif of only 12 amino acids is required for this consensus structure. The end result is a single protein chain containing multiple domains, each of which exhibits a separate function. Each domain of the protein binds independently, and the energetic contribution of each domain is additive.

[0260] Antigen-binding polypeptides also include heavy chain dimers, such as antibodies from camelids and sharks. Camelid and shark antibodies contain a homodimeric pair of two chains (neither of which has a light chain) of V-like and C-like domains. The V of heavy chain dimers in camelids IgG H The regions in the heavy chains that normally contact the light chains are altered to hydrophobic amino acid residues in camelids because they do not need to make hydrophobic interactions with the light chains. H The domain is V HHShark Ig-NAR contains a homodimer of one variable domain (termed the V-NAR domain) and five C-like constant domains (C-NAR domains). In camelids, the diversity of the antibody repertoire is largely determined by the V H or V HH The region is determined by CDRs 1, 2 and 3. HH The CDR3 in the region is characterized by its relatively long length, averaging 16 amino acids (Muyldermans et al., 1994, Protein Engineering, 7(9): 1129). This is quite different from the CDR3 regions of antibodies from many other species. For example, the CDR3 in mouse V H The CDR3 of has an average of 9 amino acids. Libraries of camelid-derived antibody variable regions that maintain the in vivo diversity of camelid variable regions can be generated, for example, by the methods disclosed in U.S. Patent Application No. 20050037421.

[0261] As used herein, "maxibody" refers to a bivalent scFv covalently attached to the Fc region of an immunoglobulin. See, e.g., Fredericks et al., Protein Engineering, Design & Selection, 17:95-106 (2004) and Powers et al., Journal of Immunological Methods, 251:123-135 (2001).

[0262] As used herein, "dsFv" may be an Fv fragment obtained by introducing a Cys residue into a suitable site in each of the heavy and light chain variable regions, and then stabilizing the heavy and light chain variable regions by disulfide bonds. The site in each chain to introduce a Cys residue can be determined based on the higher-order structure predicted by molecular modeling. In the present disclosure, for example, the higher-order structure is predicted from the amino acid sequence of the heavy and light chain variable regions of the antibody, and then DNA encoding each of the heavy and light chain variable regions into which mutations have been introduced based on such prediction is constructed. The DNA construct is then introduced into a suitable vector, and prepared from a transformant obtained by transformation with the aforementioned vector.

[0263] Single chain variable region fragments ("scFv") of antibodies are described herein. Single chain variable region fragments can be produced by linking light and / or heavy chain variable regions by using a short linking peptide. Bird et al. (1988) Science 242:423-426. Single chain variants can be produced recombinantly or synthetically. For synthetic production of scFv, an automatic synthesizer can be used. For recombinant production of scFv, a suitable plasmid containing a polynucleotide encoding the scFv can be introduced into a suitable host cell, either eukaryotic cells, such as yeast, plant, insect or mammalian cells, or prokaryotic cells, such as E. coli. A polynucleotide encoding the desired scFv can be produced by routine manipulation, such as ligation of polynucleotides. The resulting scFv can be isolated using standard protein purification techniques known in the art.

[0264] Diabodies can be single chain antibodies. Diabodies can be bivalent, bispecific antibodies, in which VH and VL domains are expressed on a single polypeptide chain, but the domains are paired with the complementary domains of another chain, creating two antigen-binding sites, by using a linker that is too short to allow pairing between the two domains on the same chain (see, for example, Holliger, P., et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993); and Poljak, RJ, et al., Structure, 2:1121-1123 (1994)).

[0265] As used herein, "minibody" refers to an scFv fused to CH3 via a peptide linker (hingeless) or via an IgG hinge as described in Olafsen, et al., Protein Eng Des Sel., April 2004; 17(4):315-23.

[0266] As used herein, "intrabody" refers to a single chain antibody that demonstrates intracellular expression and can manipulate intracellular protein function (Biocca, et al., EMBO J. 9:101-108, 1990; Colby et al., Proc Natl Acad. Sci. USA. 101:17616-21, 2004). Intrabodies that contain cellular signal sequences that retain the antibody construct within the intracellular domain can be produced as described in Mhashilkar et al., (EMBO J., 14:1542-51, 1995) and Wheeler et al. (FASEB J. 17:1733-5. 2003). Transbodies are cell-permeable antibodies in which a protein transduction domain (PTD) is fused to a single-chain variable fragment (scFv) antibody (Heng et al., Med Hypotheses. 64:1105-8, 2005).

[0267] Suitable linkers known in the art can be used to multimerize the binding agent.A non-limiting example of a linking peptide is (GGGGS)3 (SEQ ID NO:283), which bridges approximately 3.5 nm between the carboxy terminus of one variable region and the amino terminus of the other variable region.Linkers of other sequences have been designed and used.Bird et al. (ibid.).Linkers can be further modified for additional functions, such as attachment of drugs or attachment to solid support.

[0268] "Epitope" refers to that portion of an antigen or other macromolecule that can form a binding interaction with the variable region binding pocket of an antibody. Such binding interactions can be manifested as intermolecular contacts with one or more amino acid residues of one or more CDRs. Antigen binding can occur, for example, through a CDR3 or CDR3 pair, or in some cases, a V H and V L It may involve the interaction of up to all six CDRs of the chain. An epitope may be a linear peptide sequence ("continuous") or may be composed of non-contiguous amino acid sequences ("conformational" or "discontinuous"). An antibody may recognize one or more amino acid sequences, and therefore an epitope may define more than one distinct amino acid sequence. The epitope recognized by an antibody can be determined by peptide mapping and sequence analysis techniques well known to those skilled in the art. The binding interaction is manifested as an intermolecular contact between the epitope on the antigen and one or more amino acid residues of the CDRs.

[0269] An antibody selectively binds if it binds to a target with greater affinity, avidity, more readily, and / or with greater duration than it binds to other substances. For example, an antibody or antigen-binding fragment that selectively binds to, for example, a snake venom toxin is an antibody or antigen-binding fragment that binds to this epitope with greater affinity, avidity, more readily, and / or with greater duration than it binds to proteins that are not snake venom toxins.

[0270] The term "k" as used herein is intended to refer to the rate constant for the association of an antibody to an antigen.

[0271] The term "Koff," as used herein, is intended to refer to the rate constant for dissociation of an antibody from the antibody / antigen complex.

[0272] As used herein, the term "avidity" refers to the resistance of a complex of two or more agents to dissociation after dilution. Apparent affinity can be determined by methods such as enzyme-linked immunosorbent assay (ELISA) or any other technique well known to those skilled in the art. Avidity can be determined by methods such as Scatchard analysis or any other technique.

[0273] As used herein, the term "affinity" refers to the equilibrium constant for the reversible binding of two agents and is defined as the binding affinity (K D ) In some cases, K D k can refer to the rate constant for dissociation of an antibody from the antibody or antigen-binding fragment / antigen complex. off k can refer to the rate constant for the association of an antibody or antigen-binding fragment with an antigen, on Binding affinity can be expressed as a ratio to the binding affinity of the antibody or antigen-binding fragment described herein. Binding affinity can be determined using methods known in the art, including, for example, surface plasmon resonance (SPR; Biacore), Kinexa Biocensor, scintillation proximity assay, enzyme-linked immunosorbent assay (ELISA), ORIGEN immunoassay (IGEN), fluorescence quenching, fluorescence transfer, yeast display, or any combination thereof. Binding affinity can also be screened using a suitable bioassay. The binding affinity (K D) may be less than: 600nM, 590nM, 580nM, 570nM, 560nM, 550nM, 540nM, 530nM, 520nM, 510nM, 500nM, 490nM, 480nM, 470nM, 460nM, 450nM, 440nM, 430nM, 420nM, 410nM, 400nM, 390nM, 380nM, 370nM, 360nM, 350nM, 340nM, 330nM, 320nM, 310nM, 300nM, 290nM, 280nM, 270nM, 260nM, 250nM, 240nM, 230nM, 220nM, 210 nM, 200nM, 190nM, 180nM, 170nM, 160nM, 150nM, 140nM, 130nM, 120nM, 110nM , 100nM, 90nM, 80nM, 70nM, 50nM, 50nM, 49nM, 48nM, 47nM, 46nM, 45nM, 44nM, 4 3nM, 42nM, 41nM, 40nM, 39nM, 38nM, 37nM, 36nM, 35nM, 34nM, 33nM, 32nM, 31n M, 30nM, 29nM, 28nM, 27nM, 26nM, 25nM, 24nM, 23nM, 22nM, 21nM, 20nM, 19nM, 1 8nM, 17nM, 16nM, 15nM, 14nM, 13nM, 12nM, 11nM, 10nM, 9nM, 8nM, 7nM, 6nM, 5n M, 4nM, 3nM, 2nM, 1nM, 990pM, 980pM, 970pM, 960pM, 950pM, 940pM, 930pM, 920 pM, 910pM, 900pM, 890pM, 880pM, 870pM, 860pM, 850pM, 840pM, 830pM, 820pM , 810pM, 800pM, 790pM, 780pM, 770pM, 760pM, 750pM, 740pM, 730pM, 720pM, 71 0pM, 700pM, 690pM, 680pM, 670pM, 660pM, 650pM, 640pM, 630pM, 620pM, 610p M, 600pM, 590pM, 580pM, 570pM, 560pM, 550pM, 540pM, 530pM, 520pM, 510pM, 5 00pM, 490pM, 480pM, 470pM, 460pM, 450pM, 440pM, 430pM, 420pM, 410pM, 400 pM, 390pM, 380pM, 370pM, 360pM, 350pM, 340pM, 330pM, 320pM, 310pM, 300pM,290pM, 280pM, 270pM, 260pM, 250pM, 240pM, 230pM, 220pM, 210pM, 200pM, 190pM, 180pM, 170pM, or any integer between these.

[0274] The terms "polypeptide", "oligopeptide", "peptide", and "protein" are used interchangeably herein to generally refer to polymers of amino acids of any length. The polymers may be linear or branched, may contain modified amino acids, and may be interrupted by non-amino acids. These terms also encompass amino acid polymers that are modified naturally or by intervention, such as disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), as well as polypeptides containing other suitable modifications. It is understood that because the polypeptides of this disclosure are based on antibodies, the polypeptides can occur as single chains or as associated chains.

[0275] "Polynucleotide" or "nucleic acid", used interchangeably herein, generally refers to a polymer of nucleotides of any length, including DNA and RNA. Nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase. Polynucleotides can include modified nucleotides, such as methylated nucleotides and their analogs. If present, modifications to the nucleotide structure can be imparted prior to or after assembly of the polymer. The sequence of nucleotides can be interrupted by non-nucleotide components. Polynucleotides can be further modified after polymerization, such as by conjugation with a labeling component. Other types of modifications include, for example, "caps", substitution of one or more of the naturally occurring nucleotides with analogs, internucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.) and those with charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), those containing pendant moieties such as proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), those with intercalators (e.g., acridine, psoralen, etc.), those containing chelators (metals, radioactive metals, boron, metal oxides, etc.), those containing alkylators, those with modified linkages (e.g., alpha anomeric nucleic acids, etc.), as well as unmodified forms of polynucleotides.

[0276] Additionally, any of the hydroxyl groups originally present on the sugar can be replaced, for example, by phosphonate groups, phosphate groups, protected by standard protecting groups, or activated to prepare additional linkages to additional nucleotides, or conjugated to solid supports. The 5' and 3' terminal OH can be phosphorylated or replaced with amines, or organic capping group moieties having 1-20 carbon atoms. Other hydroxyls can also be derivatized to standard protecting groups. Polynucleotides can also contain analogous forms of ribose or deoxyribose sugars, including, for example, 2'-O-methyl-, 2'-O-allyl, 2'-fluoro-, or 2'-azido-ribose, carbocyclic sugar analogs, alpha-anomeric sugars, epimeric sugars, such as arabinose, xylose, or lyxose, pyranose sugars, furanose sugars, sedoheptulose, acyclic analogs, and abasic nucleoside analogs, such as methyl ribose. One or more phosphodiester linkages can be replaced with alternative linking groups. These alternative linking groups include, but are not limited to, embodiments in which phosphate is replaced with P(O)S ("thioate"), P(S)S ("dithioate"), (O)NR2 ("amidate"), P(O)R, P(O)OR', CO, or CH2 ("formacetal"), where R or R' is independently H or a substituted or unsubstituted alkyl (1-20C) optionally containing an ether (--O--) linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl, or aralkyl. Not all linkages in a polynucleotide need be identical. The foregoing description applies to all polynucleotides referred to herein, including RNA and DNA.

[0277] As used herein, "identity" can mean the percentage of identical nucleotides or amino acid residues at corresponding positions in two or more sequences when the sequences are aligned (taking into account gaps and insertions) to maximize sequence match. Identity can be readily calculated by known methods, including but not limited to those described in Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, AM, and Griffin, HG, eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48: 1073 (1988). Methods to determine identity are designed to give the largest match between the sequences tested. Moreover, methods to determine identity are codified in publicly available computer programs.Computer program methods for determining identity between two sequences include, but are not limited to, the GCG program package (Devereux, J., et al., Nucleic Acids Research 12(1): 387 (1984)), BLASTP, BLASTN and FASTA (Altschul, SF et al., J. Molec. Biol. 215: 403-410 (1990) and Altschul et al. Nuc. Acids Res. 25: 3389-3402 (1997)). BLAST X programs are publicly available from NCBI and other sources (BLAST Manual, Altschul, S., et al., NCBI NLM NIH Bethesda, Md. 20894; Altschul, S., et al., J. Mol. Biol. 215: 403-410 (1990). Identity can also be determined using the well-known Smith Waterman algorithm.

[0278] The desired range of sequence identity is about 80% to about 100% and integer values ​​therebetween. In general, the present disclosure encompasses sequences having about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identity with any sequence provided herein. The letter "X" or "Xaa", when used in an amino acid sequence herein, is intended to indicate that any of the 20 standard amino acids can be substituted at this position, unless otherwise specified. EXAMPLES

[0279] The present application can be better understood by reference to the following non-limiting examples, which are provided as exemplary embodiments of the present application. The following examples are presented to more fully illustrate the embodiments, but should not be construed in any way as limiting the broad scope of the present application.

[0280] Example 1 Antitoxin antibody kinetics Kinetic measurements were performed to calculate the binding affinity of the two antibodies to recombinant alpha-neurotoxins from mamba, taipan and cobra snakes. Alpha-neurotoxins were expressed and purified by affinity chromatography. Antibodies isolated from subjects with previous snake bites were incubated with alpha-neurotoxins, and the kinetics of binding of alpha-neurotoxins to the antibodies was measured. Kinetic data are provided in Tables 21A and 21B. [Table 21A] [Table 21B]

[0281] Example 2 Toxicity of venom toxins in mice The toxicity of different doses of neurotoxins in mice was determined. Recombinant alpha neurotoxins from various snake species were expressed and purified by affinity chromatography. Mice (5 mice per toxin group) were administered intraperitoneal doses greater than the LD50 (the dose at which 50 percent of the mice die after the compound) of each toxin. In addition to general observations, the weight and rectal temperature of each mouse were measured before toxin administration and again every 20 minutes after administration for up to 2 hours and then 24 hours after administration. Mice that recovered or were unaffected by a given dose of toxin showed complete recovery between approximately 2 and 4 hours after dosing and remained healthy for more than 24 hours after dosing. For a given dose listed, mice died by 60 minutes after dosing or were euthanized if a given dose of toxin was lethal. Survival data are presented in Table 22: [Table 22]

[0282] Example 3 Protective effect of antibodies against venom toxin in mice Individual antibody clones of the antibodies tested in Example 1 were tested for their ability to neutralize the toxins tested in Example 2 by premixing the neurotoxins with the antibodies in an Eppendorf tube and incubating at room temperature for 1 hour. The mixture was then administered to a group of mice (n=5) and lethality was observed as described above. Neutralization of lethal snake neurotoxins by the antibody clones is provided in Table 23. [Table 23]

[0283] The mice did not show any signs of morbidity. The molar ratio of toxin to antibody (taking into account bivalency) is shown. Example 4 Information on snakes, toxins and sequences [Table 24-1] [Table 24-2] [Table 25-1] [Table 25-2] [Table 25-3] [Table 26-1] [Table 26-2] [Table 26-3] [Table 26-4] [Table 26-5] [Table 26-6] [Table 26-7]

[0284] Example 5 Exemplary Treatment Methods A randomized double-blind controlled study was conducted to compare the effect of low dose to high dose. Patients who present hematological or neurological evidence of systemic envenomation within 24 hours of snake bite are included in the study. Patients are randomly assigned to receive either high dose or low dose treatment with the composition comprising one or more antibodies or antigen-binding fragments herein.

[0285] Primary endpoints include, but are not limited to, (1) a composite endpoint: the number of patients dying, requiring assisted ventilation, or showing worsening neurotoxicity defined as the appearance of two new or severe signs of neurotoxicity (e.g., loss of gag reflex or paradoxical breathing), and / or the number of patients with serious adverse events.

[0286] Example 6 Exemplary Mouse Model to Establish the MTD for Demonstrating that the Toxin was Effective Toxicity / tolerance for the toxin from each species was determined in mice by delivering an intraperitoneal dose sufficient to achieve greater than lethal dose 50 (LD50: 50% of the mice died after compound administration).

[0287] The weight and rectal temperature of each mouse were measured prior to toxin administration. Toxin was administered intraperitoneally to each mouse (5 mice per group). Rectal temperature measurements and general observations were taken every 20 minutes after dosing for up to 2 hours and then 24 hours after dosing. For a given toxin tested, mice that recovered or were unaffected by a given dose of toxin were found to show complete recovery 2-4 hours after dosing and remain healthy for more than 24 hours. If a given dose of toxin was lethal due to a sudden drop in rectal temperature (>2 degrees from baseline) for the given dose listed, the mice died by 60 minutes after dosing or were euthanized. Table 27 provides a description of the treatment groups. [Table 27]

[0288] Example 7 In vivo effects of antitoxin antibodies Experiments were conducted to determine the >LD50 of purified recombinant snake alpha neurotoxin when treated with (1) S-neurotoxin + SNEURO_P01_B11, (2) S-neurotoxin + SNEURO_P01_D9, or (3) no antibody control (S-neurotoxin). Survival rates are shown as mice that did not have any significant body temperature change or whose body temperature returned to normal by and beyond 2 hours (Table 28). [Table 28]

[0289] Example 8 Antibody Library This example demonstrates the production of an antibody library from a hyperimmune subject. Hyperimmune Subject (HiS). We collected B cell samples from a human subject with a 17-year history of immunization against snake venom by self-administration of dilute doses of venom from Taipan, Cobra, Rattlesnake, Coral Snake, Kanai, Tiger Snake, Eastern Brown Snake, and Mamba. The subject had acquired sufficient antibody titers to tolerate increasing concentrations of venom and to withstand the entire snake bite without succumbing to death or amputation. This HiS provides evidence of over 700 administrations of increasing doses of venom and over 200 live bites.

[0290] Antitoxin antibody library construction. In the passive study design, 20 ml of peripheral blood was collected on day 0 prior to and 28 days after a predefined immunization schedule used by hyperimmune subjects that had been established over a 17-year period to maintain hyperimmunity. PBMCs were isolated by Ficoll separation and cDNA was extracted. Antibody variable domain VH, Vκ, and Vλ diversity was extracted using primer sets designed for simultaneous amplification of the human repertoire for both high-throughput sequencing and antibody display library construction. Barcoded library samples were sequenced by MiSeq 2x300 paired-end reads to a depth of 500,000 reads per library. The same material was digested and cloned into scFv-pIII fusion display vectors and converted to a final library size of 2e9. The converted libraries were also sequence-confirmed by NGS to have a distribution of >1e8 heavy chains and >1e5 light chains. Heavy and light chains were cloned separately, but the size of the library was such that any starting clone with a frequency higher than 5e-4 would have its native pair present in the resulting library. The library was confirmed to contain all human native V genes. 2×10 9 were obtained during library conversion.

[0291] A set of 25 homologs was selected for toxin-Fc-AviTag fusions and toxin-10 His-AviTag fusions. An Fc tag and 10 His tags (His-His-His-His-His-His; SEQ ID NO: 326) were provided to aid in toxin purification, and an AviTag was provided to allow site-specific biotinylation to magnetic beads for automated soluble phase panning. When expression was induced in HEK293 cells, 21 of the 25 toxins produced measurable titers. All recombinant toxins were then exposed to serum from hyperimmune donors, and 15 of the 21 toxins, including multiple species against which the donors had never been directly immunized, had strong reactivity to the serum. These results established that (1) hyperimmune subjects were responsive to these particular toxins, and (2) the toxins were produced in a form that could be recognized by the donor's antibodies.

[0292] Isolation of a broadly neutralizing anti-alpha-neurotoxin antibody. We have demonstrated that the approach technology can successfully extract the essential proof of principle for the fruit of our labor: a fully human, broadly neutralizing antibody (Centi-D09) against alpha-neurotoxin, one of the "big four" toxins in venom. Through in vivo protection models in multiple snake species, crystallography of the bnAb in complex with alpha-neurotoxin from multiple snake species, and kinetic and biophysical characterization of the bnAb, we have established the molecular basis of the broadly neutralizing anti-venom antibody.

[0293] In certain aspects, the innovation of the embodiments described herein lies in three features of the next generation antitoxins: 1) the source of our antibodies; 2) technology development; and 3) storage of the final product.

[0294] The technology described herein utilizes a novel diverse immune library taken from a middle-aged man who has undergone dose-escalating autoimmunization (more than 700 boosts and 200 live bites) over the past 19 years from 17 diverse snake species, including the Western Diamondback, Mojave, Coral Snake, Black Mamba, Western Green Mamba, Eastern Green Mamba, Jameson Mamba, Tai Cobra, Egyptian Cobra, Forest Cobra, Water Cobra, Cape Cobra, Indian Krait, Round-headed Krait, Coastal Taipan, PNG Taipan, Tiger Snake, and Eastern Brown Snake. These antibodies are safe and effective in our middle-aged man, offering advantages over humanizing antibodies from animal sources or naive human libraries. Furthermore, their single-domain nature may result in a shortened half-life compared to fully human IgG. If not adequately neutralized with a single dose, repeated injections are warranted due to their small size allowing renal excretion.

[0295] Robotized cross-species phage panning. The technology described herein uses unique next-generation sequencing (NGS) to guide the amplification and tracking of antibody variable domain repertoires from blood collected before and 28 days after venom immunization of male subjects, followed by deep sequencing and antibody phage display that allows downstream toxin binding factors to be traced back to the source blood collected, allowing the generation of an immune library. This antibody library is enriched with high affinity mature anti-venom antibodies selected for their snake breadth and that have demonstrated the ability to protect against high doses of lethal snake venom in vivo. In addition, our robotized homolog cross-panning protocol allows for the rapid isolation of anti-venom broadly neutralizing antibodies. We have already demonstrated the value of this combination of both libraries and robotic cross-panning in the recovery of the in vivo protective anti-long chain neurotoxin bnAb Centi-D9.

[0296] Heat-stabilized lyophilized antibodies. Standard antivenoms require continuous refrigeration at 2-8°C to remain stable. This poses significant challenges to villages in arid regions, often with endemic venomous species, that may not have the infrastructure to properly store antivenom. In certain cases, the antibodies described herein are evaluated for room temperature stability in lyophilized or highly concentrated formulations of antivenom. This application describes heat-stabilized antibodies with less stringent storage requirements and longer shelf lives that will greatly expand the types of medical centers that can store and utilize them, allowing for on-site deployment of life-saving medications at envenomation sites.

[0297] The experiments described herein identified and characterized a pool of cross-reactive high affinity anti-toxin antibody candidates; (2) explored the mechanism of action through epitope characterization from the total toxin pool, and (3) determined unique protection patterns through in vivo challenge in mice.

[0298] We have demonstrated that broadly neutralizing antibodies (bnAbs) against snake venoms exist. A broadly neutralizing, ultra-high affinity antibody against long-chain neurotoxins, Centi-D9, was able to neutralize and confer in vivo protection against long-chain neurotoxins from cobras, taipans and mambas.

[0299] We also demonstrated that the biophysical properties of the top clone Centi-D9 can be optimized by mutating residues of Centi-D9 to germline residues at specific positions; and removing biochemical modification sites such as the N-linked glycosylation motif NS. The thermal stability and aggregation of these variants were tested for the modified clones based on the parent Centi-D9 in Table 29. Table 21B demonstrates that these modified clones retain affinity for multiple long-chain neurotoxins derived from snake venoms of the Elapidae family. [Table 29]

[0300] Identification of antibody candidates against three toxins: We identified 193, 97 and 190 antibody clones against neurotoxins, phospholipase A2 (PLA2) and E-dendrotoxin, respectively, by ELISA screening (Figure 12). Screening was performed by ELISA for long-chain neurotoxins. Thirteen clones were selected by ranked off-rates in periplasmic extracts (PPE) using Octet HTX, then reformatted and expressed as IgG. K ranged from 2.75e-09 to 2.63e-10 and 3.3e08 to 1.75e09 against various neurotoxins. D High-resolution kinetic data were generated for two candidates, Centi-D09 and Centi-B11, against three of the four toxins (long-chain neurotoxins from taipan, cobra, mamba, and krait), with β- and β-receptor domains, respectively. Centi-D9 is an ultra-high affinity (picomolar) bnAb against three snake species, taipan, cobra, and mamba, as assessed at both 25°C and 37°C with Octet HTX and BiaCORE 8K (490 pM, 37 pM, and 74 pM, respectively). For dendrotoxins, 10 of the original 190 epsilon (E) dendrotoxin positive clones were also ELISA positive against A and D-dendrotoxins. Six of these 10 with the broadest breadth against the three different dendrotoxins were reformatted into IgG. The binding kinetics of reformatted dendrotoxin antibody candidates were characterized, and one clone (Centi-DTX-B03) showed strong binding activity to dendrotoxins from four different species, demonstrating the neutralization breadth of the antibody. Crystallographic evidence of a complex was observed for one clone. Anti-PLA2 and anti-Kunitz clones were identified and characterized for breadth by ELISA.

[0301] We accomplished the following: 1) generation of kinetic data for each antibody tested against each toxin; 2) reformatting of antibody candidates to fully human IgG; and 3) characterization of the binding affinity of the reformatted IgG antibodies. Because the PLA2 enzyme was found to be toxic to mammalian cells expressing recombinant proteins, we adapted our protocol to use PLA2 isolated from whole snake venom, confirmed to be active by phospholipase enzyme assay, and biotinylated for panning. This product was then successfully used for panning and screening.

[0302] Kinetics and binding mode of antibody Centi-D9 (SNEURO_P01_D09). A crystal structure was generated to define its binding orientation and parameters (Figure 13). Four crystals mapped Centi-D09 in complex with long-chain neurotoxin (called alpha-bungarotoxin in this species) from the Japanese krait (Bungarus caeruleus), long-chain neurotoxin from the black mamba (Dendroaspis polylepsis), long-chain neurotoxin from the coastal taipan (Oxyuranus scutatellus), and long-chain neurotoxin from the Cape cobra (Naja nivea). The crystal structures demonstrated that Centi-D9 binds to the long-chain neurotoxins from all four species in a similar binding mode, thereby preventing binding to the nicotinic acetylcholine receptor (nAChR) by the long-chain neurotoxins. This is demonstrated by the binding footprints of Centi-D9 and nAChRs, which overlap almost completely on the surface of the long-chain neurotoxins (Figure 13F). Both Centi-D9 and Centi-B11 (SNEURO_P01_B11) bind toxins from these four Elapidae species, with Centi-D9 showing favorable kinetics compared to Centi-B11 in all Biacore800 and Octet HTX kinetic assays. The crystal structures of Centi-D9 in complex with these long-chain neurotoxins clearly demonstrate a binding mode / motif that is conserved across these diverse toxins (no pair has more than 65% sequence identity between them): the VH CDR3 of Centi-D9 contacts at least one residue in "finger" loop I and at least one residue in "finger" loop II.

[0303] Centi-D9 in vivo challenge studies in mouse models. The Centi-D9 in vivo challenge mouse model of envenomation was used to establish lethal dose ranges for four Elapidae species (Table 30) and to determine the functional activity and broad-spectrum efficacy of Centi-D9 against recombinant toxins and whole snake venom in vivo. [Table 30]

[0304] 1 LD50 Derived from experiments for all four snake venoms tested during a dose-ranging study to establish the LD100 LD100 .

[0305] Mice exposed to lethal doses of Tai cobra, Black mamba, and Cape cobra venom were completely protected from death by pretreatment with Centi-D9 with broad neutralizing activity, significantly exceeding expectations in terms of protection from a full venom challenge as a single antibody (as opposed to exposure to a cocktail) (Figure 15). We chose to focus on elapids based on the abundance of evidence pointing to long-chain neurotoxins as the primary driver of lethality in the majority of elapid snakes, and thus the essential basis of a universal elapid snake antivenom cocktail. Centi-D09 was demonstrated to be a broadly neutralizing antibody that protects in vivo against the full venom of six elapid species (Figure 16).

[0306] The presence of Centi-D09 provided evidence that broadly neutralizing antibodies against homologous snake toxins exist and can be isolated as the basis of a universal antivenom. Furthermore, the identification of Centi-D09 demonstrates that broadly neutralizing antibodies against snake venom toxins from a variety of species can be successfully isolated from a hyperimmune subject with a history of repeated initiation of humoral immune responses against a variety of such toxins.

[0307] Broad-spectrum neutralizing antivenoms may require a combination of agents that neutralize separate components found in two major classes of venomous snakes: Elapidae and Viperidae (Table 31). For neurotoxic Elapidae snakes, virulence of the venom is driven by long-chain neurotoxins (3FT) and PLA2. An anti-PLA2 bnAb from a panel of candidate binders can be combined with Centi-D9, an anti-long-chain neurotoxin bnAb, to create a two-component cocktail that provides protection against Elapidae snakes. [Table 31]

[0308] 1 The toxins listed can vary widely in abundance in specific venoms of individual snake species from both families, but these are the predominant toxins in the results and a general indication of relative abundance is noted by the number of check marks.

[0309] A single 3FT bnAb, Centi-D09, was found to be active against the black mamba (Dendroaspis polylepsis), the Cape cobra (Naja nivea), and the Thai cobra (Naja kaouthia). Centi-D09 was further found to be active against additional members of the Elapidae family, the Egyptian cobra (Naja haje), the Indian cobra (Naja naja), and the king cobra (Ophiphagus hannah). Centi-D09 was further shown to bind to the 3FTX long-chain neurotoxin in venom from tiger snake (Notechis scutatus scutatus), blue sea snake (Laticauda colubrina), green mamba (Dendoapsis viridis), Indonesian cobra (Naja sputatrix), inland taipan (Oxyuranus microlepidotus), and eastern brown snake (Pseudonaja textilis) by Gator BLI assay (Figure 16).

[0310] In some cases, Centi-D09 can be combined with an anti-PLA2 inhibitor. For example, Centi-D09 can be combined with the PLA2 small molecule inhibitor varespladib for coastal taipan. Alternatively, or in addition, the small molecule inhibitor can be replaced with a PLA2 bnAb. In other cases, Centi-D09 can be combined with an antibody against dendrotoxin (kunitz-like peptide) specific to the genus dendroaspis.

[0311] Production of recombinant toxins. The EXPi-293 mammalian expression system is used for expression of toxin antigens. Toxin proteins are purified with Protein G DYNABEADS™, biotinylated, and quality controlled for biotinylation and for binding to control antibodies. Some toxins are difficult to produce using this method because the toxin is lethal to the protein-producing cells. In these cases (e.g., PLA2), whole snake venom is obtained and fractionated using HPLC to obtain purified toxin for biotinylation.

[0312] Panning of the phage library against toxin antigens. The immune library (2 × 10 9 ) are heated for 10 minutes at 72°C and deselected against Protein G DYNABEADS™ (INVITROGEN®), M-280 Streptavidin DYNABEADS™ (INVITROGEN®), histones from calf thymus (Sigma), human IgG (Sigma) ssDNA-biotin NNK (Integrated DNA Technologies), and DNA-biotin NNK (Integrated DNA Technologies). The library is then sequentially panned against toxin antigens captured by M-280 Streptavidin DYNABEADS™ using an automated protocol on a Kingfisher FLEX (THERMO FISHER SCIENTIFIC®). Selected phages are acid eluted from the beads and neutralized using Tris-HCl pH 7.9 (TEKNOVA®). ER2738 cells are infected with the neutralized phage pool at a ratio of 1:10 at OD600=0.5, and after 40 min incubation at 37° C. and 100 rpm, the phage pool is centrifuged and incubated on agar overnight at 30° C. with antibiotic selection. Rescued phages are precipitated with PEG and subjected to an additional three rounds of soluble phase automated panning. The resulting antibody clones are screened by ELISA for binding activity against recombinant protein or whole snake venom.

[0313] Kinetic characterization of preliminary ELISA positives by SPR. A high-throughput SPR platform, CARTERRA® LSA (CSLA), is used to characterize the kinetic properties of the pool of ELISA positive antibodies against whole snake venom and fractionated toxins. Each antibody is tested against whole toxin or fractionated toxin for kinetic characterization and cross-reactive binding. Antibodies are further characterized for thermal stability and aggregation by fluorescence and light scattering techniques using Unchained Labs (UNcle). Selection of clones with the fastest on-rates (ka) and slowest off-rates (kd) minimizes trauma to the victim, as the toxin should be quickly neutralized and not released.

[0314] The binding kinetics of our ELISA positive antibody candidates are tested using CLSA. The data suggests that these candidates bind to the snake venoms they were panned against. Their binding affinity is characterized to determine their potential functionality in neutralizing the target toxin / toxin. Since cross-reactive molecules are used to achieve broad-spectrum antitoxin treatment, it is determined whether any of our candidates are able to bind to >2 toxin species or toxins. SPR technology allows for the collection of kinetic data (ka [association rate], kd [dissociation rate], K D Using the CARTERRA® software, which uses the maximum resonance ([A]Rmax) of antigen at a certain concentration bound to the scFv on the chip divided by the resonance equilibrium (KD=[A]Rmax / Req), the K DThe CLSA platform advantageously tests ≧4 unique antigens on 384 antibody candidates in ≦18 hours (h). This high-throughput technology determines the relative affinity of all candidates to the original toxin panned and ≧20 different whole toxins / fractionated peptide toxins in <7 days. Candidates with the greatest affinity (<10 nM) for the toxin / toxin are reformatted as IgG in our mammalian expression system. The IgG is purified and tested for thermostability and aggregation tendency. scFv expressed by bacterial cell line ER2738 are captured using an anti-V5 antibody coupled to a CLSA chip. Centivax's phagemid vector inserts a C-terminal V5 tag, which allows downstream scFv capture. Whole snake venom and toxin are run over every captured clone using an 8-point dilution scheme with a maximum concentration of 3 μM and a minimum concentration of 450 pM to determine binding kinetics and the presence of cross-reactivity. Anti-Her2 scFv is captured as a negative control for all toxins.

[0315] The CLSA platform is a versatile, high-throughput monoclonal antibody characterization method that combines patented continuous-flow microfluidics with array SPR detection, providing high-volume data generation in reduced time and at reduced consumable costs. We are the first to use this technique to determine antibody binding or cross-reactivity to snake species, and most snakebite laboratories do not have access to such SPR equipment.

[0316] For low affinity binders, proprietary affinity maturation POLISH technology can be utilized to generate millions of variants of these clones by CDRH3 mutagenesis and light chain shuffling. These clone sets are screened by CLSA for higher affinity (<10 nM). If poor cross-reactivity occurs, NGS data from the panning rounds can be used to reveal certain clones that were enriched through panning but did not appear during the initial ELISA screening, and these can be tested by ELISA and CARTERRA® in addition to the initial positives.

[0317] Kinetic data are generated for each antibody tested against each toxin. K<10 nM D Acceptable affinities, which are , are determined by an on-rate [ka] of 104 1 / Ms to 105 1 / Ms and an off-rate [kd] of 10-4 1 / s to 10-6 1 / s. Clones with the highest affinity (<10 nM) for ≧1 total toxin or toxins, and / or cross-reactive (as shown by binding to ≧1 species) antibodies are selected for IgG reformatting.

[0318] Reformatting of antibody candidates to human IgG. Identified antibody candidates are reformatted from scFv to fully human IgG by PCR. Since the goal is to bind and neutralize toxins and not to trigger downstream inflammatory immune responses, an IgG backbone with minimal effector function (e.g., LALA mutation) is used. Primers are designed to add compatible ends suitable for cloning into the mammalian expression vector, pTT5. Restriction sites suitable for the pTT5 vector are added by PCR to each scFv candidate for heavy and light chain V genes (previously characterized by SS). The resulting products are digested and cloned into DH5-alpha bacterial cells. Confirmation of proper cloning is performed by Sanger sequencing (Eton Bioscience). Using a commercially available kit (ZYMO RESEARCH®), clone DNA is midiprepped and transiently transfected into HEK293 cells for mammalian expression. Supernatants from each are harvested 5 days after transfection. IgG is purified and quantified by Protein A using an Octet QK with a minimum expression of 50 μg / mL, and expression purity is analyzed by SDS-PAGE. pTT5 vectors are used for reformatting and expression of antibody candidates.

[0319] Stability and kinetic characterization of IgG antibody candidates. The binding affinity of the reformatted antibody candidates in CLSA IgG format is determined against the previously tested toxins / peptides. The scFv contains the antibody domains that determine antigen binding, but affinity may change when complexed with the constant region of full IgG. Because full IgG contains two binding sites compared to one on scFv or Fab, binding efficiency may increase due to avidity. The stability of IgG candidates is characterized using UNchained Labs (UNcle). UNcle uses dynamic light scattering, static light scattering, and fluorescence to determine protein melting temperature, aggregation tendency, and isothermal stability, which are crucial to determining the overall stability of the IgG candidates and the quality of downstream antitoxins.

[0320] IgG is thermally unstable and prone to aggregation. To remedy this, IgG complementarity determining regions (CDRs) can be mutated and grafted onto more thermostable human germline frameworks. As a driving force between antigen binding, the scaffold on which the CDRs are loaded can be altered to retain binding and create a more thermostable molecule. This can be done by having a known thermostable human germline scaffold with the CDRs of our chosen antibody candidate synthesized by IDT or Twist and cloned into our pTT5 vector. The binding kinetics are retested to determine retention of binding.

[0321] IgG can bind with the same or higher affinity as it does in scFv format (<10 nM) with improved thermal stability and aggregation tendency by UNcle. Only IgG that is 95% monomeric with a melting temperature (Tm) of 68° C. or higher and a KD of <10 nM will be used for further purposes.

[0322] The antibody / antibody cocktail of the present invention can be lyophilized. The formulation for lyophilization can contain histidine, methionine, and polysorbate 80, for example, 50 mM histidine, 20 mM methionine, 0.05% polysorbate 80 (w / v), pH 6.8. The lyophilized formulation can further contain a combination of bulking agents / lyophilization stabilizers, including but not limited to sorbitol, mannitol, sucrose, dextrose, and glycine. The lyophilized dosage formulation is stored at 50°C and analyzed at regular time points over 28 days by the following methods: appearance, SEC-HPLC, optical density, pH, dynamic light scattering, T melt, T aggregation, particulates, water content, or any combination thereof.

[0323] In certain cases, multiple antibodies can be lyophilized in a single cocktail if a common set of lyophilization parameters allows all antibodies to be stably processed together. Alternatively, the antibodies can be lyophilized separately, the freeze-dried powders mixed by weight, and the combined powder reconstituted for downstream analysis.

[0324] In one aspect, a series of monoclonal antibodies against lethal toxins in snake venoms, i.e., a cocktail of antibodies that are broadly protective against vipers and elapids, are described herein. Each of these antibodies is individually optimized to broadly neutralize the entire family of polymorphic toxins (e.g., a single antibody that can neutralize dendrotoxins from elapid snakes worldwide). By combining several broad-spectrum anti-toxin antibodies, snakebite lethality can be abolished by neutralizing the most potent toxins present in the venoms of the world's most dangerous snakes. This unique combination of high potency and breadth of antibodies and cocktail strategy allows the creation of snakebite drugs that allow significantly faster treatment without the need for identification of the envenomed species. Furthermore, lyophilization of the antibody cocktail creates a drug that is ultra-stable and can be stored at ambient temperature for extended periods of time. Also described is a dual chamber autoinjector for use by untrained individuals or in field rescue settings that can be easily carried and used by travelers, medical personnel or military personnel in areas where fatal snakebites are a serious concern.

[0325] While preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, modifications and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein can be used in practicing the invention. It is intended that the following claims define the scope of the invention, and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

1. An antibody or antigen-binding fragment which is a broadly neutralizing antibody against two or more members of the family of three-finger toxins (3FTx). (a) the 3FTx is a long-chain neurotoxin; and / or (b) the antibody or antigen-binding fragment of claim 1, wherein the antibody neutralizes alpha-bungarotoxin (Japanese krait), alpha-elapitoxin (mamba), pseudonajatoxin (brown snake), alpha-cobratoxin (cobra), toxin B (king cobra), or a combination thereof, and optionally the antibody broadly neutralizes toxins from two, three, four, five or more of the long-chain neurotoxins.

3. The antibody or antigen-binding fragment thereof, comprising VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3; (i) said VH CDR1 comprises the amino acid sequence FX2X3X4X5X6DX8H, where X2 is selected from N, T, and S; X3 is selected from F, L; X4 is selected from R, G, and S; X5 is selected from N and T; X6 is selected from Y, F, L; and X8 is selected from M, I (SEQ ID NO:525); (ii) said VH CDR2 has the amino acid sequence X1X2X3X4X5X6X7GX9X10X11X12, wherein X1 is selected from P, S, and H; X2 is selected from V, G, and T; X3 is selected from V, F, L, and I; X4 is selected from D, G, and A; X5 is selected from Y, L, and H; X6 is selected from N, C, R, and T, and S; X7 is selected from V, F, and G; X9 is selected from A, E; X10 is selected from Q, H; X11 is selected from S, D, Y, and H; and X12 is selected from A and E) (SEQ ID NO:526); (iii) said VH CDR3 comprises the amino acid sequence CX2RGTLYHYTSGSYX15SDAFDIW, where X2 is selected from V and A; and X15 is selected from Y and C (SEQ ID NO:527); (iv) the VL CDR1 comprises the amino acid sequence X1ASX4X5IX7X8X9LX11, where Xi is selected from Q and R; X4 is selected from Q and E; X5 is selected from D, G, T, and S; X7 is selected from R and S; X8 is selected from S, D, N, and I; X9 is selected from N, F, Y, W, and D; and X11 is selected from N, G, and A (SEQ ID NO:528); (v) said VL CDR2 comprises the amino acid sequence X1ASX4X5X6X7, where X1 is selected from G and A; X4 is selected from N, T, and S; X5 is selected from L and S; X6 is selected from Q, L, and E; and X7 is selected from M and S (SEQ ID NO:529); (vi) the VL CDR3 comprises the amino acid sequence CQQSYSTX8TF, where X8 is selected from I and H (SEQ ID NO:530), or the VL CDR3 comprises the amino acid sequence CQQX4X5X6X7PX9TF, where X4 is selected from A and S; X5 is selected from N and Y; X6 is selected from I, T, and S; X7 is selected from P, F, and T; and X9 is selected from P, Y, L, and W (SEQ ID NO:531); The broadly neutralizing antibody or antigen-binding fragment of claim 1.

4. The antibody or antigen-binding fragment thereof, (c) a variable heavy (VH) complementarity determining region 3 (CDR3) having an amino acid sequence at least about 80% identical to any one of SEQ ID NOs: 12, or 327-348, a VH CDR2 having an amino acid sequence at least about 90% identical to any one of SEQ ID NOs: 41, or 371-385, and a VH CDR1 having an amino acid sequence at least about 80% identical to any one of SEQ ID NOs: 28, or 349-370; and / or (d) a variable light chain (VL) CDR1 having an amino acid sequence at least about 90% identical to any one of SEQ ID NOs: 46, or 393-404, a VL CDR2 having an amino acid sequence at least about 80% identical to any one of SEQ ID NOs: 80, or 405-416, and a VL CDR3 having an amino acid sequence at least about 90% identical to any one of SEQ ID NOs: 101, or 417-428. The broadly neutralizing antibody or antigen-binding fragment of claim 1 .

5. The antibody or antigen-binding fragment thereof, (e) a VH having an amino acid sequence at least about 90% identical to any one of SEQ ID NOs: 242, or 429-500; and / or (f) a VL having an amino acid sequence at least about 90% identical to any one of SEQ ID NOs: 273, or 501-512. The broadly neutralizing antibody or antigen-binding fragment of claim 1 .

6. The antibody or antigen-binding fragment thereof, (g) a VH CDR1 of SEQ ID NO:28, a VH CDR2 of SEQ ID NO:41, a VH CDR3 of SEQ ID NO:12, a VL CDR1 of SEQ ID NO:46, a VL CDR2 of SEQ ID NO:80, and a VL CDR3 of SEQ ID NO:101; or (h) a VH having the amino acid sequence of SEQ ID NO: 242, and a VL having the amino acid sequence of SEQ ID NO: 273; The broadly neutralizing antibody or antigen-binding fragment of claim 1 .

7. A universal antitoxin composition comprising one or more broadly neutralizing antibodies or antigen-binding fragments described in any one of claims 1 to 6.

8. A universal antitoxin composition as described in claim 7 for use in a method of treating a subject suffering from envenomation, said method comprising a step of administering said universal antitoxin composition to said subject.

9. A universal anti-venom composition for use as described in claim 8, wherein the envenomation occurs by one or more snakes.

10. The universal anti-venom composition for use according to claim 8 or 9, wherein the envenomation is from (a) a sawtooth snake, a water moccasin, a lancehead, a rattlesnake, a Russell's viper, a puff adder, or a combination thereof, or (b) a common krait (Bungarus caeruleus), a black mamba (Dendroaspis polylepsis), a coastal taipan (Oxyuranus scutatellus), a Cape cobra (Naja nivea), or a combination thereof.

11. A universal antitoxin composition for use as described in claim 8 or 9, wherein the method further comprises the step of administering an additional therapy or drug to the subject.

12. The universal antitoxin composition for use as described in claim 11, wherein the additional therapy or drug comprises a PLA2 inhibitor.

13. The universal antitoxin composition for use according to claim 12, wherein the PLA2 inhibitor comprises varespladib, methylvarespladib, or a combination thereof.

14. A composition for use in a method for identifying a broadly neutralizing antibody or antigen-binding fragment that selectively binds to two or more snake venom toxins belonging to a family of homologous antigens, said composition comprising two or more homologous antigens, said method comprising: (a) immunizing a subject with the composition; and (b) performing an iterative selection process to specifically identify cross-reactive antibodies or antigen-binding fragments from the subject's B-cell repertoire, the iterative selection process using two or more homologous antigens to downselect potential candidates. A composition comprising:

15. The composition of claim 14, wherein the iterative selection process comprises immunoprecipitation of antibodies from serum obtained from the subject, fluorescence activated cell sorting (FACS) of B cells obtained from the subject, panning of a library derived from B cell RNA obtained from the subject displayed on phage or yeast, or any combination thereof.

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