Antigen-binding proteins binding snake toxins

Abstract

The present disclosure provides for a composition comprising antigen-binding proteins capable of binding to, blocking, and / or neutralizing Type IA / IB cytotoxins, Orphan group XI toxins, Orphan group XIX toxins, aminergic toxins, Kunitz-type protease inhibitors, Type I α-neurotoxins (short-chain α-neurotoxins), Type II α-neurotoxins (long-chain α-neurotoxins), and phospholipase A2 (PLA2) toxins. The disclosure further provides for compositions comprising one or more of the disclosed antigen-binding proteins, which are capable of neutralizing snake venom lethality and dermonecrosis from medically relevant Sub-Saharan elapid snake species.

Description

[0001] P7584PC00

[0002] 1

[0003] Antigen-binding proteins binding snake toxins

[0004] Technical field

[0005] The present disclosure provides for a composition of antigen-binding proteins for use in the treatment of elapid snake envenomation. The antigen-binding proteins may for example be capable of fully neutralizing snake venom lethality from seven different toxin (sub)families across three genera, as well as capable of neutralizing snake venom dermonecrosis.

[0006] Background

[0007] Snakebite envenoming is a neglected tropical disease that disproportionately affects rural populations, with sub-Saharan Africa bearing a significant burden. Envenomation by elapid species can induce dermonecrosis and cause severe systemic effects, leading to thousands of deaths and amputations each year. The current primary treatment consists of antivenoms derived from the plasma of hyperimmunized animals, which, despite their therapeutic efficacy, have many drawbacks including batch-to-batch variation, high production costs, limited cross-neutralization, and potential immunological reactions. Additionally, they often contain low levels of therapeutically active antibodies, requiring large doses for effective treatment, and they may not prevent local tissue damage. The complexity and variability of elapid venoms further complicate treatment, as a single species' venom can contain numerous distinct toxins that vary between populations and regions. This diversity presents a significant challenge in developing broadly effective treatments capable of neutralizing medically relevant venoms across multiple elapid species.

[0008] Summary

[0009] Broadly neutralizing antibodies targeting specific toxin families have shown promise in preclinical models for preventing venom-induced toxicity, while combinations of recombinant monoclonal antibodies or camelid-derived VHHS (nanobodies) have demonstrated efficacy in neutralizing some snake toxins. However, for a recombinant antivenom to serve as a viable alternative to existing treatments, it should preferably be capable of neutralizing venoms from multiple medically significant elapid species. This remains particularly challenging due to the complexity and variability of elapid venoms, which contain a diverse array of toxin families that vary between species.P7584PC00

[0010] 2

[0011] Consequently, it has long been believed that neutralizing all clinically relevant toxins would require an impractically large number of antibodies.

[0012] To date, no antigen-binding proteins or combinations thereof have been described that can simultaneously neutralize the effects — let alone the lethality — of multiple medically significant elapid venom toxins. Existing polyclonal immunoglobulin-based treatments derived from hyperimmunized animals remain inconsistent in their ability to neutralize different medically relevant toxins, leading to variable efficacy across different venoms.

[0013] The present disclosure describes antigen-binding proteins capable of binding to, blocking, and / or neutralizing multiple medically significant elapid venom toxins, including cytotoxins, neurotoxins, Kunitz-type protease inhibitors, and phospholipase A2 (PLA2) toxins. The disclosure provides for compositions comprising one or more antigen-binding proteins that together effectively may neutralize key elapid venom toxins associated with lethality. More preferably, this disclosure provides for antigenbinding proteins and mixtures thereof capable of neutralizing whole venom lethality. Furthermore, the invention provides antigen-binding proteins and mixtures thereof capable of mitigating venom-induced dermonecrosis, marking a significant advancement in the development of next-generation snakebite therapeutics.

[0014] The disclosure further provides for broadly neutralizing antigen-binding proteins, such as nanobodies targeting key medically significant elapid venom toxins, including nanobodies against toxin subfamilies that have not been previously been targeted, such as Type IA / IB cytotoxins, Orphan group XI, Orphan group XIX toxins and / or aminergic toxins, in addition to Kunitz-type protease inhibitors, a-neurotoxins, and / or phospholipase A2 (PLA2) toxins. The disclosed therapeutic effect of combining these antigen-binding proteins has demonstrated that effective neutralization of venoms from a plurality of the most medically relevant elapid species in sub-Saharan Africa can be achieved with a remarkably small set of broadly neutralizing antigen binding proteins, herein exemplified by VHHS.

[0015] The present disclosure proves for the improved feasibility of developing a polyvalent recombinant antivenom with broad species coverage and offers a promising approach for the improvement of snakebite treatments. The disclosed nanobody-based antigen-P7584PC00

[0016] 3

[0017] binding proteins present a scalable, stable, and cost-effective alternative to traditional polyclonal antibody-based antivenoms.

[0018] In one aspect, the present disclosure, provides for a composition comprising:

[0019] • an antigen-binding protein capable of binding to, blocking, and / or neutralizing a snake venom Type I A or Type IB cytotoxin (herein referred to as antigenbinding protein ‘A’);

[0020] • an antigen-binding protein capable of binding to, blocking, and / or neutralizing a snake venom Orphan group XI toxin (herein referred to as antigen-binding protein ‘B’)

[0021] • an antigen-binding protein capable of binding to, blocking, and / or neutralizing a snake venom Orphan group XIX toxin and / or an aminergic toxin (herein referred to as antigen-binding protein ‘C’); and / or

[0022] • an antigen-binding protein capable of binding to, blocking, and / or neutralizing a snake venom Kunitz-type protease inhibitor toxin (herein referred to as antigenbinding protein ‘D’).

[0023] In a second aspect, the present disclosure provides for a composition comprising at least two of the following antigen-binding proteins:

[0024] • an antigen-binding protein capable of binding to, blocking, and / or neutralizing a snake venom Type IA or Type IB cytotoxin (herein referred to as antigenbinding protein ‘A’); and / or

[0025] • an antigen-binding protein capable of binding to, blocking, and / or neutralizing a snake venom Orphan group XI toxin (herein referred to as antigen-binding protein ‘B’); and / or

[0026] • an antigen-binding protein capable of binding to, blocking, and / or neutralizing a snake venom Orphan group XIX toxin and / or an aminergic toxin (herein referred to as antigen-binding protein ‘C’); and / or

[0027] • an antigen-binding protein capable of binding to, blocking, and / or neutralizing a snake venom Kunitz-type protease inhibitor (herein referred to as antigenbinding protein ‘D’); and / or

[0028] • an antigen-binding protein capable of binding to, blocking, and / or neutralizing a snake venom Type I a-neurotoxin (short-chain a-neurotoxin) (herein referred to as antigen-binding protein ‘E’); and / orP7584PC00

[0029] 4

[0030] • an antigen-binding protein capable of binding to, blocking, and / or neutralizing a snake venom Type II a-neurotoxin (long-chain a-neurotoxin) (herein referred to as antigen-binding protein ‘F’); and / or

[0031] • an antigen-binding protein capable of binding to, blocking, and / or neutralizing a snake venom phospholipase A2 (PLA2) toxin (herein referred to as antigenbinding protein ‘G’).

[0032] In a third aspect the present disclosure provides for a composition comprising an antigen-binding protein capable of binding to, blocking, and / or neutralizing a snake venom Type I A or Type IB cytotoxin (herein also referred to as antigen-binding protein ‘A’) and an antigen-binding protein capable of binding to, blocking, and / or neutralizing a snake venom phospholipase A2s (PLA2) toxin (herein also referred to as antigenbinding protein ‘G’).

[0033] In a fourth aspect the present disclosure provides for a multi-specific antigen-binding protein, comprising of consisting of at least two of the following:

[0034] • an antigen-binding protein capable of binding to, blocking, and / or neutralizing a snake venom Type I A or Type IB cytotoxin (herein referred to as antigen-binding protein ‘A’); and / or

[0035] • an antigen-binding protein capable of binding to, blocking, and / or neutralizing a snake venom Orphan group XI toxin (herein referred to as antigen-binding protein ‘B’); and / or

[0036] • an antigen-binding protein capable of binding to, blocking, and / or neutralizing a snake venom Orphan group XIX toxin and / or an aminergic toxin (herein referred to as antigen-binding protein ‘C’); and / or

[0037] • an antigen-binding protein capable of binding to, blocking, and / or neutralizing a snake venom Kunitz-type protease inhibitor (herein referred to as antigen-binding protein ‘D’); and / or

[0038] • an antigen-binding protein capable of binding to, blocking, and / or neutralizing a snake venom Type I a-neurotoxin (short-chain a-neurotoxin) (herein referred to as antigen-binding protein ‘E’); and / or

[0039] • an antigen-binding protein capable of binding to, blocking, and / or neutralizing a snake venom Type II a-neurotoxin (long-chain a-neurotoxin) (herein referred to as antigen-binding protein ‘F’); and / orP7584PC00

[0040] 5

[0041] • an antigen-binding protein capable of binding to, blocking, and / or neutralizing a snake venom phospholipase A2 (PLA2) toxin (herein referred to as antigenbinding protein ‘G’).

[0042] A fifth aspect of the present disclosure provides for a kit of parts comprising:

[0043] a) the composition as described herein; and

[0044] b) another agent suitable for the treatment of snake envenomation.

[0045] A sixth aspect of the present disclosure provides for a composition, a multi-specific antigen-binding protein or a kit-of-parts as described herein for use in a method of treating snakebite in an individual in need thereof.

[0046] A seventh aspect of the present disclosure provides for a method for treating snake envenomation in an individual in need thereof, the method comprising administering a therapeutically effective amount of the composition, the antigen binding protein, the multi-specific antigen-binding protein, or the kit of parts as described herein to said individual.

[0047] An eighth aspect of the present disclosure provides for the use of the antigen binding protein, the multi-specific antigen-binding protein, or the kit of parts described herein for the preparation of a medicament for treatment of snakebite in an individual in need thereof.

[0048] The invention is defined in the claims attached thereto.

[0049] Description of Drawings

[0050] FIG.1: Proteomics of venom fractions and clustering of toxins. Abundance of different toxin (sub)families in the venom fractions obtained through RP-HPLC and analyzed with LC-MS / MS.

[0051] FIG.2: Sequence similarity network (SSN) of the toxins present in the venoms of the 18 elapid snakes. Every circle represents a single toxin and a connecting line between two circles indicates a sequence homology of at least 70%. The most medically relevant toxins within the fractions used as target antigens in the phageP7584PC00

[0052] 6

[0053] display selection campaigns are outlined with a bold border and annotated with the accession number of the exact toxin or its closest known homolog.

[0054] FIG.3: Screening of monoclonal VHHS, dose-response binding curves, and isoaffinity plot of VHHS binding to toxins and venom fractions. Screening of over 3,000 VHH clones for binding to their cognate toxin in an expression-normalized capture DELFIA. Only VHHS with a signal intensity 10 times above the background level are shown, with the number of clones exceeding this threshold displayed on the x-axis.

[0055] FIG.4: Dose-response binding curves. 21 VHHs against a venom fraction or a toxin from each toxin (sub)family were measured using an expression-normalized capture DELFIA.

[0056] FIG.5: Isoaffinity plot of 15 cross-reactive VHHS binding to venom fractions and toxins from various snake (sub)genera measured with biolayer interferometry. Due to an instrument limit on dissociation rates (koff) at 10-7 s— 1, 4 points below this cutoff are plotted at <10-71 / s. The dotted diagonal lines represent specific KD values, as indicated in above them.

[0057] FIG.6: Neutralization of sNTx- and INTx-mediated blocking of the current through muscle-type nAChRs in whole-cell patch-clamp. Dose-response curves are shown with increasing concentrations of VHH to prevent the blocking of nAChRs by sNTx-1, sNTx-3, sNTx-6, INTx-3, INTx-5, and INTx-7. Experiments were performed using 8 replicates, and results are expressed as mean ± SD. The toxin concentrations are shown in parentheses, and the dotted line represents a 1:1 molar ratio of toxin: VHH.

[0058] FIG.7: In vitro neutralization of sNTx, INTx, CTx, and PLA2. Neutralization of venom-induced cytotoxicity using a cell viability assay with a N / TERT keratinocyte cell line. The positive control (PC) included media supplemented with PBS and was set to 100% cell viability. For the negative control (NC), media supplemented with triton X-100 was added, resulting in complete cell death. As an additional control, VHH without any venom was included (VHH). For all venoms, (-) indicates 2 IC50S of the venom without VHH addition, and (+) indicates venom incubated with a VHH at a 1:5 molar ratio of CTX: VHH. Experiments were conducted in triplicate, and results are expressed as mean ±SD.P7584PC00

[0059] 7

[0060] FIG.8: Neutralization of enzymatic activity caused by PLA2. The whole venoms of all Afronaja species were tested for neutralization by VHH20 a-PLA2 using a colorimetric enzymatic activity assay with the chromogenic substrate NOBA (4-nitro-3-(octanoyloxy)benzoic acid). Experiments were conducted in duplicates, and results are expressed as mean ± SD.

[0061] FIG.9: In vivo experiments to guide the design of a-sNTx and a-INTx nanobodies for the recombinant antivenom. Kaplan-Meier survival curves for mice challenged with sNTx-3 or INTx-7. Three LD50S of each venom fraction, toxin, or venom were preincubated with either PBS or different mixtures of VHHS and injected into mice using the i.v. route, n = 3. Molar ratio between toxin (sub)family and the corresponding VHH is 1:10, unless otherwise specified in parentheses.

[0062] FIG.10: In vivo experiments to guide the design of anti-elapid snake toxin nanobodies for the recombinant antivenom. Kaplan-Meier survival curves for mice challenged with elapid snake venoms. Three LD50S of each venom fraction, toxin, or venom were pre-incubated with either PBS or different mixtures of VHHS and injected into mice using the i.v. route, n = 3. Molar ratio between toxin (sub)family and the corresponding VHH is 1:10, unless otherwise specified in parentheses.

[0063] FIG.11: In vivo neutralization of venom-induced dermonecrosis. Neutralization of venom-induced dermonecrosis with a mixture of 3 HHS (VHH1 a-CTx, VHH4 a-CTx, and VHH20 a-PLA2) against 3 representative cytotoxic venoms from H. haemachatus, N. mossambica, and N. nigricollis, in both pre-incubation and rescue setups. In the preincubation setup, the VHHS and venom were pre-incubated before i.d. injection, and in the rescue setup, the VHH mixture was injected into the same region as the venom injection 15 minutes post venom injection. The lesion size was measured 72 hours post-injection.

[0064] FIG.12: The neutralization efficacy of the recombinant antivenom against dermonecrosis caused by the 3 venoms in another rescue setup. In this rescue setup, venom was injected i.d. and the recombinant antivenom was injected i.v. 15 minutes later. The lesion size was measured 48 hours post-injection. For N. nigricollisP7584PC00

[0065] 8

[0066] venom, in addition to the recombinant antivenom, Inoserp PAN-AFRICA antivenom was included for comparison.

[0067] FIG.13: In silico modeling of VnH-toxin interactions.

[0068] In silico modelling was performed using the AlphaFold 3 Server (https: / / alphafoldserver.com / ) and experimental determination was performed through X-ray crystallography. The data was processed using the software UCSF ChimeraX version: 1.8 (2024-06-10). The interactions between each VHH against (vs) three-finger toxins (3FTx) (A-C, E, F), Kunitz-type protease inhibitors toxins (D) and phospholipase A2(G) is shown. VHHS are shown as ribbons, and toxins are shown as surface models. Contact regions between molecules are marked in dark grey on ribbons (VHHS) and surface models (toxins).

[0069] FIG.14: Interactions in co-crystal structures and in the in silico predictions.

[0070] A. The biparatopic VHH1 a-CTx binding 2 different epitopes of cardiotoxin (P01468) and B. VHH5 a-sNTx with short neurotoxin 1 (P01426). C. Interactions of the relatively high-confidence in silico predictions of VHH4 a-CTx with cytotoxin 1 (P01456) and D. VHH13 a-AgTx (also referred to as a-S6C6 in this disclosure) with S2C4 (P01407), and E. VHH 15 a-Og XI (also referred to as a-S5C2 in this disclosure) with Toxin 4.9.6 (P01405). F. Interactions of the in silico predictions of HH9 a-INTx with a-cobratoxin (P01391), G. VHH17 a-KUN with dendrotoxin I (P00979) (green), and H, VHH20 a-PLA2with Basic phospholipase A2 nigexine (P14556). The close-ups illustrate the hydrogen, TT-TT, TT-CH and Van der Waals bonds (dotted lines). Sequence alignments of the verified target toxins of each respective VHH are shown under their structures.

[0071] FIG 15: A) Binding of representative individual VHHS to their corresponding toxin targets. LNTx is a-cobratoxin, SNTx is the venom fraction Nm3, and AgTx is the venom fraction Dj 10. B) Binding of the VHH mixture to non-toxin antigens, including Cardiolipin, dsDNA, lipopolysaccharide (LPS), human insulin, or human serum albumin (HSA), used to assess polyreactivity in DELFIA. Data are shown as relative fluorescence units (RFU).

[0072] Fig 16: Results from static and dynamic light scattering measurements of the VHH mixture at 20 mg / mL during isothermal incubations. A.) The light scattering intensity over time for three different incubations at either 40, 45, or 50 °C. B.) Intensity-weighedP7584PC00

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[0074] size distributions from Dynamic Light Scattering (DLS) at three timepoints (0, 5, and 16 h) of the incubation at 40 °C.

[0075] Detailed description

[0076] Definitions

[0077] As used herein, the singular forms “a”, “an” and “the” include plural referents unless the context clearly states otherwise. Thus, for example, reference to “an antigen-binding protein” includes a plurality of such antigen-binding proteins.

[0078] As used herein, the term “antigen-binding protein” refers to a protein which specifically binds an antigen. The antigen-binding proteins may in particular be antibodies, antibody fragments and other antigen-binding protein constructs. The term encompasses immunoglobulin-derived molecules, including intact antibodies that comprise at least two full-length heavy chains and two full-length light chains, as well as derivatives, variants, fragments, and mutations thereof, examples of which include Fab, Fab', F(ab')2, and Fv fragments. The antigen-binding proteins may also be domain antibodies such as nanobodies, single domain antibodies, heavy chain variable region fragments (referred to as VHH or VHH), and single-chain antibodies. In preferred embodiments, the antigen-binding protein is an immunoglobulin single variable domain, such as a VH, VHH or VL.

[0079] The terms " HH" and “nanobodies” are used herein as equivalent terms and refer to a single-domain antibody fragment derived from the variable domain of camelid heavy-chain-only antibodies. VHHS are capable of binding specifically to an antigen and are included within the definition of an antigen-binding protein. Due to their small size and stability, VHHS can penetrate tissues more efficiently and may provide advantages over conventional antibody formats.

[0080] The term "snake venom toxin" as used herein refers to a bioactive molecule present in the venom of snakes, which can contribute to the toxic effects of envenomation. Snake venom toxins include a diverse range of proteins and peptides that may exhibit enzymatic activity or receptor-binding properties, leading to systemic or local pathological effects such as neurotoxicity, cytotoxicity, coagulopathy, myotoxicity, and inflammation. These toxins can belong to various families, including but not limited toP7584PC00

[0081] 10

[0082] three-finger toxins, phospholipases A2, metalloproteinases, serine proteases, and neurotoxins. Individual snake venom toxins may be assigned designations comprising the toxin (sub)family name followed by a number, as described in section ‘Toxin overview’.

[0083] The term "individual" as used herein refers to a living organism, typically a human or an animal, preferably a mammal, more preferably a human being. The term may refer to subjects that may be affected by a condition, disorder, or external factor such as envenomation and may be in need of treatment, prevention, or therapeutic intervention to mitigate the effects of a harmful agent, including venom toxins. The term encompasses subjects of any age, sex, or health status, including those at risk of exposure or those already experiencing symptoms related to a specific condition.

[0084] The term "dermonecrosis" as used herein refers to a pathological condition characterized by localized tissue damage, including necrosis, inflammation, and cell death, which may result from exposure to certain toxins, infections, or other harmful agents. In the context of envenomation, dermonecrosis is associated with the action of venom components that disrupt cellular integrity and / or trigger inflammatory responses, potentially leading to swelling, blistering, hemorrhage, and / or ulceration at the affected site. Venom-induced dermonecrosis can be caused by a variety of bioactive molecules, including cytotoxins and enzymatic toxins such as phospholipase A2 (PLA2). These components may act individually or synergistically to damage cell membranes, degrade structural lipids, and induce inflammatory cascades that exacerbate tissue destruction. The extent and severity of dermonecrosis can vary depending on the nature of the toxin, the dose, the individual and the physiological response of the affected tissue.

[0085] The term "lethality" as used herein refers to the ability of a substance, condition, or agent to cause death in an individual. Lethality can be measured in various ways, including through lethal dose (LD) values, which quantify the amount of a substance required to cause mortality in a specified percentage of a population under defined conditions. In the context of venom toxicity, lethality refers to the capacity of venom components, such as neurotoxins, cytotoxins, or enzymatic toxins, to disrupt critical physiological functions, leading to fatal outcomes.P7584PC00

[0086] 11

[0087] The term “LD50” as used herein refers to the amount of a compound, such as a toxin, that has a lethal effect in 50% of a test population (e.g., rodents).

[0088] The term “epitope” as used herein refers to a specific region or sequence of a macromolecule, typically a protein (herein in general a toxin), that is recognized and bound by an antibody or an antigen binding protein. Epitopes can be linear, consisting of a continuous sequence of amino acids, or conformational, where the recognized region is formed by the spatial arrangement of amino acids that are not contiguous in the primary structure but are brought together in the three-dimensional structure of the antigen. Herein conformational epitopes are described by indicating some or all of the amino acids forming part of the epitope. Thus, epitopes may be defined by comprising a number of amino acids, which are not consecutive in the primary sequence.

[0089] Numerous methods are available for identifying and characterizing such epitopes which are not consecutive in the primary sequence. These methods include, without limitation, competition assays, cross-blocking experiments, binding inhibition studies employing point mutants, alanine scanning panels, and structural or biophysical approaches such as hydrogen-deuterium exchange mass spectrometry (HDX-MS) or cryo-electron microscopy (cryo-EM), as well as computational epitope mapping and related binding analyses.

[0090] As used herein, the term “apparent melting temperature” of a polypeptide, such as an antigen-binding protein, refers to the temperature at which a detectable change in a physicochemical property of the polypeptide, indicative of a thermal transition, is observed under defined experimental conditions. Such a transition may correspond, for example, to partial or complete unfolding, loss of secondary or tertiary structure, dissociation of oligomeric assemblies, or a change in binding or spectroscopic signal. The apparent melting temperature is typically defined as the temperature at which approximately 50% of the polypeptide population is unfolded or, conversely, remains structured, as determined by the particular measurement technique employed.

[0091] As used herein, the term “apparent onset of aggregation” of a polypeptide refers to the temperature or other stress condition at which a detectable increase in aggregation of the polypeptide is first observed under defined experimental conditions. Such aggregation may be evidenced, for example, by changes in light scattering, turbidity, particle size distribution, solubility, chromatographic behavior, or loss of monomeric species, as measured by the particular analytical technique employed. The apparentP7584PC00

[0092] 12

[0093] onset of aggregation is typically defined as the point at which aggregation becomes detectable above a predefined baseline or threshold, and is therefore determined operationally based on the sensitivity, measurement principle, and conditions of the assay used.

[0094] The term "complementary determining region" (CDR) as used herein refers to a specific region within the variable domain of an antibody or antigen-binding protein that is primarily responsible for recognizing and binding to an antigen. CDRs are highly variable in sequence and structure, allowing for the diversity necessary to recognize a wide range of antigens. Each antibody or antigen-binding protein typically contains three CDRs within the heavy chain variable region (CDR1, CDR2, and CDR3) and three within the light chain variable region, which together form the antigen-binding site. These regions interact with the antigen in a complementary manner, facilitating specific and high-affinity binding. Preferably, complementarity-determining regions (CDRs) are identified as described in Muyldermans et al. (1994), by aligning the variable domain amino acid sequences to standardized antibody sequence frameworks according to the Kabat numbering system (Kabat et al., 1991).

[0095] The term "sequence identity" as used herein refers to the degree of similarity between two nucleotide or amino acid sequences, expressed as a percentage, based on the number of identical residues at corresponding positions when the sequences are aligned. A specified percentage of sequence identity (e.g., 70%, 80%, 90%, or 95%) indicates the proportion of positions in a given sequence that are identical to a reference sequence over a defined length.

[0096] The term "functional homologue" as used herein refers to a molecule, such as a protein or peptide, that shares structural and sequence similarity with a reference molecule and retains at least one biological function of the reference. Functional homologues may exhibit at least a specified percentage of sequence identity (e.g., 70%, 80%, 90%, or 95%) while maintaining key functional properties, such as binding affinity, enzymatic activity, or interaction with molecular targets. Variations between a functional homologue and its reference molecule may include amino acid substitutions, deletions, or insertions that do not significantly alter its primary biological role.P7584PC00

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[0098] The terms "elapid," "elapid snake," and "elapid species" are used herein as equivalent terms. Elapids refer to a family of venomous snakes within the Elapidae family, characterized by their fixed, front-fanged venom delivery system and the production of highly potent venoms containing neurotoxic, cytotoxic, and enzymatic components. These snakes are widely distributed across Africa, Asia, Australia, and the Americas and include species such as Naja, including cobras (Naja spp.), mambas (Dendroaspis spp.), and the rinkhals (Hemachatus haemachatus). Elapid venoms often contain a diverse array of bioactive molecules, including three-finger toxins, phospholipases A2, and Kunitz-type protease inhibitors, which contribute to systemic and local toxicity following envenomation. For example, elapid venoms may comprise one or more the toxins provided in Table 1 in the section entitled “Toxin Overview”. Many elapid species are of medical significance due to their ability to cause life-threatening neurotoxicity, paralysis, coagulopathy, and tissue damage in envenomated individuals. The composition of elapid venoms can vary between species and populations, influencing the severity of envenomation and the effectiveness of treatment strategies.

[0099] The term "heavy chain variable" (VH) as used herein refers to the variable region of the heavy chain of an antibody or antigen-binding protein. The VH region is responsible for antigen recognition and contributes to the specificity and affinity of antigen binding. It is composed of framework regions (FRs) and three hypervariable loops known as complementarity-determining regions (CDRs), which directly interact with the antigen. The VH region is highly diverse due to genetic recombination and somatic hypermutation, allowing for the generation of a vast repertoire of antigen-binding specificities. In single-domain antibody fragments, such as VHHS from camelids, the heavy chain variable region functions independently without the need for a paired light chain.

[0100] The term "binding" as used herein refers to the specific interaction between a molecule, such as an antigen-binding protein, and its target, such as a toxin, receptor, or other biomolecule. Binding occurs through non-covalent interactions, including hydrogen bonding, electrostatic forces, van der Waals interactions, and hydrophobic interactions. The strength and specificity of binding are influenced by the structural complementarity between the binding molecule and its target, which can affect biological activity, stability, or signaling.P7584PC00

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[0102] The term "blocking" as used herein refers to the ability of a molecule, such as an antigen-binding protein, to interfere with the function or activity of a target by preventing its interaction with a biological receptor, substrate, or another molecular component. Blocking may occur through steric hindrance, allosteric modulation, or direct competition with a natural binding partner. By blocking a target, a molecule can inhibit downstream biological effects, such as receptor activation, enzymatic activity, or toxin-mediated damage.

[0103] The term "neutralizing" as used herein refers to the process of reducing, inhibiting, or counteracting the harmful effects of a substance, such as a toxin, virus, or other bioactive molecule. Neutralization may occur through direct binding, which prevents the target from interacting with its biological receptor, or through conformational changes that render the target inactive. Neutralization can reduce, mitigate or even eliminate toxic effects, prevent disease progression, and / or restore normal physiological function.

[0104] The term “Fc” as used herein refers to the fragment crystallizable region of an immunoglobulin, such as the fragment crystallizable region of an IgG. The Fc comprises the constant region or fragments of it. The Fc region may include constant regions composed of two identical protein fragments. Typically, the Fc region may be derived from the second and third constant domains of an antibody's heavy chains or composed of three heavy chain constant domains (CH domains 2-4) in each polypeptide chain. The Fc region can have different origin e.g. human, mouse, or pig. Fusion of the fragment crystallizable region of an antibody to the VHH domain results in a VHH-FC fusion protein.

[0105] Compositions

[0106] The present disclosure provides for a composition comprising one or more antigenbinding proteins capable of binding to, blocking, and / or neutralizing snake venom toxins. As used herein, the term "composition", “antivenom” and “recombinant antivenom” refer to a predetermined mixture of distinct antigen-binding proteins, each capable of binding to, blocking, and / or neutralizing specific snake venom toxins.

[0107] Traditional antivenoms are biological treatments derived from the plasma of hyperimmunized animals, such as horses or sheep, and contain polyclonal antibodies capable of binding to and neutralizing venom components. While these antivenomsP7584PC00

[0108] 15

[0109] have been effective in reducing envenomation-related mortality, they present several challenges, including high batch-to-batch variability, high production costs, potential immunogenic reactions, and limited cross-neutralization of venoms from different snake species. Recombinant antivenoms consist of monoclonal or oligoclonal mixtures of antigen-binding proteins engineered for enhanced specificity, stability, and broadspectrum neutralization.

[0110] Based on the assays and epitope characterizations disclosed herein, a person skilled in the art can select antigen-binding proteins or mixtures thereof that are suitable for the neutralization of venom lethality, the prevention of neurotoxic effects, or the mitigation of venom-induced dermonecrosis. In addition, a person skilled in the art can select antigen-binding proteins or mixtures thereof that are suitable for alleviating the aforementioned envenomation effects based on the specific elapid snake species responsible for said envenomation.

[0111] It may be preferred that the composition comprises an antigen-binding protein ‘A’ capable of binding to, blocking, and / or neutralizing a snake venom Type IA / IB cytotoxin, an antigen-binding protein ‘B’ capable of binding to, blocking, and / or neutralizing a snake venom Orphan group XI toxin, an antigen-binding protein ‘C’ capable of binding to, blocking, and / or neutralizing a snake venom Orphan group XIX toxin and / or an aminergic toxin, and / or an antigen-binding protein ‘D’ capable of binding to, blocking, and / or neutralizing a snake venom Kunitz-type protease inhibitor toxin.

[0112] In may also be preferred that the composition further comprises an antigen-binding protein ‘E’ capable of binding to, blocking, and / or neutralizing a snake venom Type I a-neurotoxin (short-chain a-neurotoxin), and / or an antigen-binding protein ‘F’ capable of binding to, blocking, and / or neutralizing a snake venom Type II a-neurotoxin (long-chain a-neurotoxin), and / or an antigen-binding protein ‘G’ capable of binding to, blocking, and / or neutralizing a snake venom phospholipase A2 (PLA2) toxin.

[0113] It may be preferred that the composition comprises at least two antigen-binding proteins selected from antigen-binding proteins ‘A’, ‘B’, ‘O’, ‘D’, ‘E’, ‘F’, and ‘G’, wherein each antigen-binding protein is capable of binding to, blocking, and / or neutralizing its respective aforementioned snake venom toxin. The composition may also compriseP7584PC00

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[0115] more than one of each type of antigen binding protein. Thus, the composition may comprise two or more different antigen binding proteins of each type. Antigen binding proteins may be different for several reasons, for example because they bind different epitopes and / or because they have a different primary sequence, for example they may comprise different CDRs.

[0116] In may also be preferred that the composition comprises at least two antigen-binding proteins ‘As’, wherein each antigen-binding protein ‘A’ is capable of binding to, blocking, and / or neutralizing different snake venom Type IA / IB cytotoxins.

[0117] Composition for the treatment of venom-induced dermonecrosis

[0118] Another aspect of the present disclosure provides for a composition that may comprise an antigen-binding protein ‘A’ and an antigen-binding protein ‘G’, wherein antigenbinding protein ‘A’ is capable of binding to, blocking, and / or neutralizing a snake venom Type IA / IB cytotoxin, and antigen-binding protein ‘G’ is capable of binding to, blocking, and / or neutralizing a snake venom phospholipase A2 (PLA2) toxin. A composition capable of neutralizing both Type IA / IB cytotoxins and PLA2 toxins, may help prevent dermonecrosis, reduce inflammation, and improve tissue recovery following envenomation by spitting cobras (Naja spp.) and the rinkhals (Hemachatus haemachatus).

[0119] In addition to said antigen-binding proteins ‘A’ and ‘G’, the composition may further comprise one or more of antigen-binding proteins ‘E’, ‘F’, ‘B’, ‘O’, and ‘D’, preferably all of antigen-binding proteins ‘E’, ‘F’, ‘B’, ‘O’, and ‘D’.

[0120] It is preferred that said composition comprises at least two different antigen-binding proteins ‘A’.

[0121] Compositions comprising various antigen-binding proteins

[0122] The present disclosure also provides compositions comprising several different antigen-binding proteins according to the disclosure. Such compositions may also be referred to as “oligoclonal mixture of antigen-binding proteins”. As used herein, the phrase "oligoclonal mixture of antigen-binding proteins" refers to a predetermined mixture of distinct antigen-binding proteins, e.g. of distinct nanobodies.P7584PC00

[0123] 17

[0124] In one embodiment, the composition comprises or consists of a predetermined mixture of antibodies against one or more epitopes present on one or more targets of snake venoms described herein. In view of the assays and epitopes disclosed herein, those skilled in the art can select antibodies or mixtures of antibodies that are applicable for the intended purpose and desired need e.g. neutralizing snake venom lethality. The present disclosure relates to compositions comprising at least two different antigenbinding proteins specifically binding type IA / IB cytotoxin, orphan group XI toxin, orphan group XIX toxin, aminergic toxin, Kunitz-type protease inhibitor toxin, type I a-neurotoxin, type II a-neurotoxin and / or phospholipase A2 toxin.

[0125] It may be preferred that the composition is capable of neutralizing snake venom type IA / IB cytotoxin. It may also be preferred that the composition is capable of neutralizing snake venom orphan group XI toxin.. It may also be preferred that the composition is capable of neutralizing snake venom orphan group XIX toxin. It may also be preferred that the composition is capable of neutralizing an aminergic toxin. It may also be preferred that the composition is capable of neutralizing snake venom Kunitz-type protease inhibitor toxin. It may also be preferred that the composition is capable of neutralizing snake venom type I a-neurotoxin. It may also be preferred that the composition is capable of neutralizing snake venom type II a-neurotoxin. It may also be preferred that the composition is capable of neutralizing snake venom phospholipase A2 toxin.

[0126] It is preferred that one or more of the antigen-binding proteins of the oligoclonal antigen-binding protein mixture comprises an antigen-binding protein comprising the CDRs and / or the VHHS as described herein below in the sections “Antigen-binding protein ‘A’ and Type IA / IB cytotoxins”, “Antigen-binding protein ‘B’ and Orphan Group XI cytotoxins”, “Antigen-binding protein ‘C’ and Orphan Group XIX or an aminergic toxin”, “Antigen-binding protein ‘D’ and Kunitz-type protease inhibitor toxin”, “Antigenbinding protein ‘E’ and Type I a-neurotoxin (short-chain a-neurotoxin)”, “Antigenbinding protein ‘F’ and Type II a-neurotoxin (long-chain a-neurotoxin)” and “Antigenbinding protein ‘G’ and Phospholipase A2 toxin”.

[0127] In particular, it may be preferred that the composition comprises antigen-binding proteins ‘A’, ‘B’, ‘O’, ‘D’, ‘E’, ‘F’, and ‘G’. It may be preferred that the composition comprises at least two different antigen-binding protein ‘A’s.P7584PC00

[0128] 18

[0129] It may be preferred that the composition comprises antigen-binding proteins 'A' and ' B'. It may be preferred that the composition comprises antigen-binding proteins 'A' and 'C. It may be preferred that the composition comprises antigen-binding proteins 'A' and 'D'. It may be preferred that the composition comprises antigen-binding proteins 'A' and ' E'. It may be preferred that the composition comprises antigen-binding proteins 'A' and 'F'. It may be preferred that the composition comprises antigen-binding proteins 'A' and 'G'. It may be preferred that the composition comprises antigen-binding proteins 'B' and 'C. It may be preferred that the composition comprises antigen-binding proteins ' B' and 'D'. It may be preferred that the composition comprises antigen-binding proteins ' B' and ' E'. It may be preferred that the composition comprises antigen-binding proteins ' B' and 'F'. It may be preferred that the composition comprises antigen-binding proteins ' B' and 'G'. It may be preferred that the composition comprises antigen-binding proteins 'C and 'D'. It may be preferred that the composition comprises antigen-binding proteins 'C and ' E'. It may be preferred that the composition comprises antigen-binding proteins 'C and 'F'. It may be preferred that the composition comprises antigen-binding proteins 'C and 'G'. It may be preferred that the composition comprises antigen-binding proteins 'D' and ' E'. It may be preferred that the composition comprises antigen-binding proteins ' D' and 'F'. It may be preferred that the composition comprises antigen-binding proteins ' D' and 'G'. It may be preferred that the composition comprises antigen-binding proteins ' E' and 'F'. It may be preferred that the composition comprises antigen-binding proteins ' E' and 'G'. It may be preferred that the composition comprises antigen-binding proteins 'F' and 'G'. It may be preferred that the composition comprises antigen-binding proteins 'A', ' B', and 'C. It may be preferred that the composition comprises antigen-binding proteins 'A', ' B', and 'D'. It may be preferred that the composition comprises antigen-binding proteins 'A', 'B', and ' E'. It may be preferred that the composition comprises antigen-binding proteins 'A', ' B', and 'F'. It may be preferred that the composition comprises antigenbinding proteins 'A', 'B', and 'G'. It may be preferred that the composition comprises antigen-binding proteins 'A', 'C, and 'D'. It may be preferred that the composition comprises antigen-binding proteins 'A', 'C, and 'E'. It may be preferred that the composition comprises antigen-binding proteins 'A', 'C, and 'F'. It may be preferred that the composition comprises antigen-binding proteins 'A', 'C, and 'G'. It may be preferred that the composition comprises antigen-binding proteins 'A', 'D', and 'E'. It may be preferred that the composition comprises antigen-binding proteins 'A', 'D', and 'F'. It may be preferred that the composition comprises antigen-binding proteins 'A', ' D', and 'G'. It may be preferred that the composition comprises antigen-binding proteins 'A', ' E',P7584PC00

[0130] 19

[0131] and 'F'. It may be preferred that the composition comprises antigen-binding proteins 'A', ' E', and 'G'. It may be preferred that the composition comprises antigen-binding proteins 'A', 'F', and 'G'. It may be preferred that the composition comprises antigenbinding proteins 'B', 'C, and 'D'. It may be preferred that the composition comprises antigen-binding proteins ' B', 'C, and ' E'. It may be preferred that the composition comprises antigen-binding proteins 'B', 'C, and 'F'. It may be preferred that the composition comprises antigen-binding proteins 'B', 'C, and 'G'. It may be preferred that the composition comprises antigen-binding proteins 'B', 'D', and 'E'. It may be preferred that the composition comprises antigen-binding proteins 'B', 'D', and 'F'. It may be preferred that the composition comprises antigen-binding proteins 'B', ' D', and 'G'. It may be preferred that the composition comprises antigen-binding proteins ' B', ' E', and 'F'. It may be preferred that the composition comprises antigen-binding proteins ' B', ' E', and 'G'. It may be preferred that the composition comprises antigen-binding proteins 'B', 'F', and 'G'. It may be preferred that the composition comprises antigenbinding proteins 'C, 'D', and 'E'. It may be preferred that the composition comprises antigen-binding proteins 'C, ' D', and 'F'. It may be preferred that the composition comprises antigen-binding proteins 'C, 'D', and 'G'. It may be preferred that the composition comprises antigen-binding proteins 'C, 'E', and 'F'. It may be preferred that the composition comprises antigen-binding proteins 'C, 'E', and 'G'. It may be preferred that the composition comprises antigen-binding proteins 'C, 'F', and 'G'. It may be preferred that the composition comprises antigen-binding proteins 'D', ' E', and 'F'. It may be preferred that the composition comprises antigen-binding proteins 'D', ' E', and 'G'. It may be preferred that the composition comprises antigen-binding proteins ' D', 'F', and 'G'. It may be preferred that the composition comprises antigen-binding proteins 'E', 'F', and 'G’.

[0132] In particular, it may be preferred that the composition comprises antigen-binding proteins ‘B’, ‘O’, ‘D’, ‘E’, and ‘F’.

[0133] In particular, it may be preferred that the composition comprises antigen-binding proteins ‘A’ and ‘E’.

[0134] In particular, it may be preferred that the composition comprises antigen-binding proteins ‘A’, ‘E’, and ‘F’.P7584PC00

[0135] 20

[0136] In particular, it may be preferred that the composition comprises antigen-binding proteins ‘D’, ‘E’, and ‘F’.

[0137] In particular, it may be preferred that the composition comprises antigen-binding proteins ‘B’, ‘C’, and ‘E’.

[0138] In particular, it may be preferred that the composition comprises antigen-binding proteins ‘E’ and ‘F’.

[0139] In particular, it may be preferred that the composition comprises antigen-binding proteins ‘B’, ‘E’, and ‘F’.

[0140] In particular, it may be preferred that the composition comprises antigen-binding proteins ‘B’, ‘C’, ‘D’, and ‘F’.

[0141] In particular, it may be preferred that the composition comprises antigen-binding proteins ‘B’, ‘C’, ‘E’, and ‘F’.

[0142] In particular, it may be preferred that the composition comprises antigen-binding proteins ‘A’, ‘B’, ‘C’, ‘E’, ‘D’, and ‘F’.

[0143] In particular, it may be preferred that the composition comprises antigen-binding proteins ‘A’ and ‘G’.

[0144] In particular, it may be preferred that the composition comprises antigen-binding proteins ‘F’, ‘A’, and ‘G’.

[0145] In particular, it may be preferred that the composition comprises antigen-binding proteins ‘F’, ‘E’, and ‘G’.

[0146] In particular, it may be preferred that the composition comprises antigen-binding proteins ‘E’, and ‘G’.

[0147] It may also be preferred that the composition comprises at least two, at least three, at least four, at least five, at least six, at least seven, or at least eight antigen-bindingP7584PC00

[0148] 21

[0149] proteins capable of binding to, blocking, and / or neutralizing at least two, at least three, at least four, at least five, at least six, at least seven, or at least eight different snake venom toxins.

[0150] In particular, it may be preferred that the composition is capable of neutralizing the lethality associated with Type IA / IB cytotoxins.

[0151] In particular, it may be preferred that the composition is capable of neutralizing the lethality of snake venom Orphan group XI toxins.

[0152] In particular, it may be preferred that the composition is capable of neutralizing the lethality of snake venom Orphan group XIX toxins.

[0153] In particular, it may be preferred that the composition is capable of neutralizing the lethality of aminergic toxins.

[0154] In particular, it may be preferred that the composition is capable of neutralizing the lethality associated with snake venom Kunitz-type protease inhibitor toxins.

[0155] In particular, it may be preferred that the composition is capable of neutralizing the lethality of snake venom Type I a-neurotoxins (short-chain a-neurotoxins).

[0156] In particular, it may be preferred that the composition is capable of neutralizing the lethality of snake venom Type II a-neurotoxins (long-chain a-neurotoxins).

[0157] In particular, it may be preferred that the composition is capable of neutralizing the lethality of snake venom phospholipase A2 (PLA2).

[0158] In some particularly preferred embodiments, the complementarity-determining regions (CDRs) were identified as described in Muyldermans et al. (1994), by aligning the variable domain amino acid sequences to standardized antibody sequence frameworks according to the Kabat numbering system (Kabat et al., 1991). Using this approach, the positions corresponding to CDR1, CDR2, and CDR3 were systematically delineated and extracted from the translated sequence data.P7584PC00

[0159] 22

[0160] Antigen-binding protein ‘A’ and Type IA / IB cytotoxins

[0161] The present disclosure provides for an antigen-binding protein, capable of binding to, blocking, and / or neutralizing a snake venom Type IA / IB cytotoxin. In the present disclosure, a Type IA / IB cytotoxin may refer to either a “Type I A cytotoxin” or a “Type IB cytotoxin”. Such antigen-binding proteins are referred to as “antigen-binding protein ‘A’” herein. It may be preferred that the antigen-binding protein ‘A’ is capable of binding to Type IA / IB cytotoxins. It may be preferred that the antigen-binding protein A is capable of blocking Type IA / IB cytotoxins. It may be preferred that the antigen-binding protein ‘A’ is capable of neutralizing Type IA / IB cytotoxins.

[0162] Cytotoxins (CTs) are a class of venom-derived proteins that disrupt the structure and function of cells, leading to cell damage or death. They often function by targeting cellular membranes, enzymes, or signalling pathways. These toxins are part of the three-finger toxin (3FTx) family and are amphipathic polypeptides that integrate into lipid bilayers, leading to cell lysis, tissue damage, and inflammation. Most of the CTs consist of 60 amino acid residues, classified as type IA / IB, with only four CTs characterized by the His4 insertion, as Type IB.

[0163] Antigen-binding protein ‘A’ may be capable of binding to Type IA / IB cytotoxins by recognizing specific epitopes. Said epitope may for example be critical to their toxic activity. The antigen-binding protein may interact with conserved structural motifs or key amino acid residues within the active regions of Type IA / IB cytotoxins, and may prevent the cytotoxins from integrating into and disrupting cellular membranes, thereby neutralizing them.

[0164] It may be preferred that the antigen-binding protein ‘A’ is capable of binding to, blocking, and / or neutralizing a Type IA / IB cytotoxin obtained from the venom of Hemachatus haemachatus. In other embodiments, the antigen-binding protein ‘A’ is capable of binding to, blocking, and / or neutralizing a Type IA / IB cytotoxin from an elapid snake of the genus Naja, such as Naja nigricollis, Naja mossambica, or Naja nivea. In some embodiments, the antigen-binding protein ‘A’ is capable of binding to, blocking, and / or neutralizing a Type IA / IB cytotoxin from Naja pallida, Naja nubiae, or Naja katiensis. In other embodiments, the antigen-binding protein ‘A’ is capable of binding to, blocking, and / or neutralizing a Type IA / IB cytotoxin from Naja ashei, Naja melanoleuca, or Naja annulifera.P7584PC00

[0165] 23

[0166] Type IA / IB cytotoxins may also be referred to as “CTx”. Thus, it may be preferred that the antigen-binding protein ‘A’ is capable of binding to a snake venom toxin CTx or a CTx fraction or a toxin comprised in a CTx fraction. Said CTx may be any CTx or any CTx fraction as described in Table 1 of section ‘Toxin overview’. Thus, the Type IA / IB cytotoxin may be a toxin comprised in a CTx fraction described in Table 1. Said CTx fraction may be CTx-1, CTx-2, CTx-3, CTx-4, CTx-5, CTx-6, CTx-7, CTx-8, CTx-9, CTx-10, CTx-11, CTx-12, CTx-13, CTx-14, CTx-15, CTx-16, CTx-17, CTx-18, Ctx-19 or CTx-20 of Table 1. It may also be preferred that the Type IA / IB cytotoxin is any of the Type IA / IB cytotoxin listed in Table 1. It may further be preferred that the antigenbinding protein ‘A’ binds to any snake venom that comprises a type IA / IB cytotoxin, a CTx toxin, or any fraction or designation thereof as described herein.

[0167] It may be preferred that the antigen-binding protein ‘A’ is capable of binding to a snake venom toxin as set forth in SEQ ID NO: 1 or a functional homologue thereof sharing at least 70%, such as at least 80%, for example at least 90%, such as at least 95% sequence identity therewith. Accordingly, said Type IA / IB cytotoxin may preferably be a snake venom toxin as set forth in SEQ ID NO: 1 or SEQ ID NO: 2 or a functional homologue of any of the aforementioned sharing at least 70%, such as at least 80%, for example at least 90%, such as at least 95% sequence identity therewith.

[0168] It may also be preferred that the antigen-binding protein ‘A’ is capable of binding to a snake venom toxin as set forth in SEQ ID NO: 2 or a functional homologue thereof sharing at least 70%, such as at least 80%, for example at least 90%, such as at least 95% sequence identity therewith.

[0169] In particular, it may preferred that the antigen-binding protein ‘A’ is capable of binding to an epitope of a snake venom Type I A or Type IB cytotoxin comprising the amino acid residues:

[0170] - 17, P8, P9, L6, and P33 of SEQ ID NO: 1;

[0171] - K44, R27, L48, S46, L47 and 151 of SEQ ID NO: 1; and / or

[0172] - P9, K5 and K35 of SEQ ID NO: 2.

[0173] It might further be preferred that the antigen-binding protein ‘A’ is capable of binding to an epitope of a snake venom Type IA or Type IB cytotoxin comprising the amino acid residues:

[0174] - 17, P8, P9, L6, and P33 of SEQ ID NO: 1; and / orP7584PC00

[0175] 24

[0176] -K44 R27, L48, S46 and L47 and 151 of SEQ ID NO: 1; and / or

[0177] - L6, I7, P8, P9, F10, R27, P33, K35, L47, L48 and K50 of SEQ ID NO: 1; and / or - L6, I7, P8, P9, F10, P33 and K35 of SEQ ID NO: 1; and / or

[0178] - R27, L47, L48 and K50 of SEQ ID NO: 1; and / or

[0179] - P9, K5 and K35 of SEQ ID NO: 2; and / or

[0180] - K5, L6, P9, W11, K12, T13, K18, K35, and C38 of SEQ ID NO: 2.

[0181] In one embodiment the antigen-binding protein ‘A’ may be the antibody referred to as VHH1 or VHH4. In preferred embodiments, the composition comprises both of those antibodies.

[0182] The antigen-binding protein ‘A’ may preferably comprise or consist of a heavy chain variable (VH) region as set forth in SEQ ID NO: 10 or SEQ ID NO: 26, or a sequence identical thereto except for up to 2, such as up to 1 amino acid substitution(s).

[0183] The antigen-binding protein ‘A’ may particularly preferably comprise or consist of a heavy chain variable (VH) region as set forth in SEQ ID NO: 57 or SEQ ID NO: 73, or a sequence identical thereto except for up to 2, such as up to 1 amino acid substitution(s). It may preferred that the antigen-binding protein ‘A’ may comprise or consist of a heavy chain variable (VH) region comprising complementary determining regions (CDRs) comprising or consisting of:

[0184] - a CDR1 as set forth by SEQ ID NO: 11; and

[0185] - a CDR2 as set forth by SEQ ID NO: 12; and

[0186] - a CDR3 as set forth by SEQ ID NO: 13.

[0187] It may also be preferred that the antigen-binding protein ‘A’ may comprise or consist of a heavy chain variable (VH) region comprising complementary determining regions (CDRs) comprising or consisting of:

[0188] - a CDR1 as set forth by SEQ ID NO: 27; and

[0189] - a CDR2 as set forth by SEQ ID NO: 28; and

[0190] - a CDR3 as set forth by SEQ ID NO: 29.

[0191] It may be particularly preferred that the antigen-binding protein ‘A’ may comprise or consist of a heavy chain variable (VH) region comprising complementary determining regions (CDRs) comprising or consisting of:P7584PC00

[0192] 25

[0193] - a CDR1 as set forth by SEQ ID NO: 58; and

[0194] - a CDR2 as set forth by SEQ ID NO: 59; and

[0195] - a CDR3 as set forth by SEQ ID NO: 60.

[0196] It may be particularly preferred that the antigen-binding protein ‘A’ may comprise or consist of a heavy chain variable (VH) region comprising complementary determining regions (CDRs) comprising or consisting of:

[0197] - a CDR1 as set forth by SEQ ID NO: 74; and

[0198] - a CDR2 as set forth by SEQ ID NO: 75; and

[0199] - a CDR3 as set forth by SEQ ID NO: 76.

[0200] The composition may in particular comprise two different antigen-binding protein ‘A’ comprising aforementioned CDRs.

[0201] Antigen-Binding Protein ‘B’ and Orphan Group XI toxins

[0202] The present disclosure provides for an antigen-binding protein capable of binding to, blocking, and / or neutralizing a snake venom Orphan group XI toxin. Such antigenbinding proteins are referred to as “antigen-binding protein ‘B’” herein. It may be preferred that the antigen-binding protein ‘B’ is capable of binding to Orphan group XI toxins It may be preferred that the antigen-binding protein ‘B’ is capable of blocking Orphan group XI toxins. It may be preferred that the antigen-binding protein ‘B’ is capable of neutralizing Orphan group XI toxins.

[0203] Orphan group XI toxins are a class of venom-derived proteins present in the venoms of certain elapid snakes, particularly mambas (Dendroaspis spp.). These toxins exhibit neurotoxic properties and contribute to systemic envenomation effects, including paralysis, respiratory distress, and cardiovascular dysfunction.

[0204] Antigen-binding protein ‘B’ may be capable of binding to Orphan group XI toxins by recognizing specific epitopes critical to their toxic activity. The antigen-binding protein may interact with conserved structural motifs or key amino acid residues within the active regions of Orphan group XI toxins and may prevent these toxins from interacting with neuronal receptors, thereby neutralizing their effects.P7584PC00

[0205] 26

[0206] It may preferred that the antigen-binding protein ‘B’ is capable of binding to, blocking, and / or neutralizing an Orphan group XI toxin obtained from the venom of Dendroaspis jamesoni or Dendroaspis viridis.

[0207] In the present disclosure, a snake venom Orphan group XI toxin may also be referred to as “Og XI toxin” or “Og XI”. The Orphan group XI toxin may be S5C4. Accordingly, in the present disclosure, the terms “Orphan group XI,” “Og XI toxin,” “Og XI” and “S5C4” are used interchangeably. Thus, it may be preferred that the antigen-binding protein ‘B’ is capable of binding to a snake venom toxin S5C4, which is classified as an Orphan group XI toxin or a S5C4 fraction or a toxin comprised in a S5C4 fraction. Said S5C4 may be any S5C4 or any S5C4 fraction as described in Table 1 of section ‘Toxin overview’. Thus, the Orphan group XI toxin may be a toxin comprised in a S5C4 fraction described in Table 1. Said S5C4 fraction may be Og XI-1 or Og XI-2 of Table 1. It may also be preferred that the Orphan group XI toxin is any of the toxins of Orphan group XI listed in Table 1. It may further be preferred that the antigen-binding protein ‘B’ binds to any snake venom that comprises an Orphan group XI toxin, a S5C4 toxin, or any fraction or designation thereof as described herein.

[0208] It may be preferred that the antigen-binding protein ‘B’ is capable of binding to a snake venom toxin as set forth in SEQ ID NO: 3 or a functional homologue thereof sharing at least 70%, such as at least 80%, for example at least 90%, such as at least 95% sequence identity therewith.

[0209] Accordingly, said Orphan group XI toxin may preferably be a snake venom toxin as set forth in SEQ ID NO: 3 or a functional homologue of any of the aforementioned sharing at least 70%, such as at least 80%, for example at least 90%, such as at least 95% sequence identity therewith.

[0210] In particular, it may preferred that the antigen-binding protein ‘B’ is capable of binding to an epitope of a snake venom Orphan group XI toxin comprising the amino acid residues: P9, Q10, K7 and K48 of SEQ ID NO: 3.

[0211] It might further be preferred that the antigen-binding protein ‘B’ is capable of binding to an epitope of a snake venom Orphan group XI toxin comprising the amino acid residues:P7584PC00

[0212] 27

[0213] - P9, Q10, K7 and K48 of SEQ ID NO: 3; and / or

[0214] - K7, Q10, K24, K28, G31, R36 and K48 of SEQ ID NO: 3.

[0215] In one embodiment the antigen-binding protein ‘B’ may be the antibody referred to as VHH15.

[0216] The antigen-binding protein ‘B’ may preferably comprise or consist of a heavy chain variable (VH) region as set forth in SEQ ID NO: 14, or a sequence identical thereto except for up to 2, such as up to 1 amino acid substitution(s).

[0217] The antigen-binding protein ‘B’ may particularly preferably comprise or consist of a heavy chain variable (VH) region as set forth in SEQ ID NO: 61, or a sequence identical thereto except for up to 2, such as up to 1 amino acid substitution(s).

[0218] In particular, it may preferred that the antigen-binding protein ‘B’ may comprise or consist of a heavy chain variable (VH) region comprising complementary determining regions (CDRs) comprising or consisting of:

[0219] - a CDR1 as set forth by SEQ ID NO: 15; and

[0220] - a CDR2 as set forth by SEQ ID NO: 16; and

[0221] - a CDR3 as set forth by SEQ ID NO: 17.

[0222] In particular, it may particularly preferred that the antigen-binding protein ‘B’ may comprise or consist of a heavy chain variable (VH) region comprising complementary determining regions (CDRs) comprising or consisting of:

[0223] - a CDR1 as set forth by SEQ ID NO: 62; and

[0224] - a CDR2 as set forth by SEQ ID NO: 63; and

[0225] - a CDR3 as set forth by SEQ ID NO: 64.

[0226] Antigen-binding protein ‘C’ and Orphan Group XIX toxins oraminergic toxins

[0227] The present disclosure provides for an antigen-binding protein capable of binding to, blocking, and / or neutralizing a snake venom Orphan group XIX toxin and / or an aminergic toxin. In the present disclosure, a snake venom Orphan Group XIX toxin may also be referred to as an “aminergic toxin,” and, conversely, an aminergic toxin may also be referred to as a “snake venom Orphan Group XIX toxin.” As used herein, the terms “snake venom Orphan Group XIX toxin” and “aminergic toxin” areP7584PC00

[0228] 28

[0229] interchangeable and may be abbreviated as “AgTx”. Such antigen- binding proteins are referred to as “antigen-binding protein ‘C’” herein. It may be preferred that the antigenbinding protein ‘C’ is capable of binding to Orphan group XIX toxins and / or an aminergic toxin. It may be preferred that the antigen-binding protein ‘C’ is capable of blocking Orphan group XIX toxins and / or an aminergic toxin. It may be preferred that the antigen-binding protein ‘C’ is capable of neutralizing Orphan group XIX toxins and / or an aminergic toxin.

[0230] Orphan group XIX toxins are venom-derived proteins found in the venoms of certain elapid snakes, particularly mambas (Dendroaspis spp.). These toxins contribute to the neurotoxic effects of envenomation by interfering with neuronal function, leading to paralysis, respiratory distress, and other severe systemic symptoms. Although classified as orphan toxins, they are believed to play a significant role in the overall potency of mamba venom.

[0231] Antigen-binding protein ‘C’ may capable of binding to Orphan group XIX toxins and / or an aminergic toxin by recognizing specific epitopes critical to their toxic activity. The antigen-binding protein may interact with conserved structural motifs or key amino acid residues within the active regions of Orphan group XIX toxins and / or an aminergic toxin, preventing their interaction with neuronal receptors and neutralizing their effects.

[0232] It may be preferred that the antigen-binding protein ‘C’ is capable of binding to, blocking, and / or neutralizing an Orphan group XIX toxin and / or an aminergic toxin obtained from the venom of Dendroaspis jamesoni or Dendroaspis viridis.

[0233] The Orphan group XIX toxin and / or aminergic toxin may for example be S6C6 or synergistic-type venom protein S2C4. Both S6C6 and S2C4 are members of Orphan Group XIX, herein also referred to as aminergic toxins or “AgTx.”

[0234] Thus, it may be preferred that the antigen-binding protein ‘C’ is capable of binding to a snake venom toxin S6C6, or a S6C6 fraction or a toxin comprised in a S6C6 fraction. Said S6C6 may be any S6C6 or any S6C6 fraction as described in Table 1 of section ‘Toxin overview’. Thus, the Orphan group XIX toxin and / or the aminergic toxin may be a toxin comprised in a S6C6 fraction described in Table 1. Said S6C6 fraction may be AgTx-1 or AgTx-2 of Table 1. It may also be preferred that the Orphan group XIX toxinP7584PC00

[0235] 29

[0236] is any of the toxins of Orphan group XIX listed in Table 1. It may further be preferred that the aminergic toxin is any of the aminergic toxins listed in Table 1.

[0237] Alternatively, it may be preferred that the antigen-binding protein ‘C’ is capable of binding to a synergistic-type venom protein S2C4, or an S2C4 fraction or a toxin comprised in an S2C4 fraction. Said S2C4 may be any S2C4 or any S2C4 fraction as described in Table 1 of section ‘Toxin overview’. Thus, the Orphan Group XIX toxin and / or the aminergic toxin may be a toxin comprised in an S2C4 fraction described in Table 1. Said S2C4 fraction may be AgTx-1 or AgTx-2 of Table 1. It may also be preferred that the Orphan Group XIX toxin is any of the toxins of Orphan Group XIX listed in Table 1. It may further be preferred that the aminergic toxin is any of the aminergic toxins listed in Table 1.

[0238] It may further be preferred that the antigen-binding protein ‘C’ binds to any snake venom that comprises an Orphan group XIX toxin, a AgTx toxin, an aminergic toxin or any fraction or designation thereof as described herein.

[0239] It may be preferred that the antigen-binding protein ‘C’ is capable of binding to a snake venom toxin as set forth in SEQ ID NO: 4 or a functional homologue thereof sharing at least 70%, such as at least 80%, for example at least 90%, such as at least 95% sequence identity therewith.

[0240] Accordingly, said aminergic toxin may preferably be a snake venom toxin as set forth in SEQ ID NO: 4 or a functional homologue of any of the aforementioned sharing at least 70%, such as at least 80%, for example at least 90%, such as at least 95% sequence identity therewith.

[0241] In particular, it may be preferred that the antigen-binding protein ‘C’ is capable of binding to an epitope of an aminergic toxin comprising the amino acid residues:

[0242] - C46, K22, P47, and E15 of SEQ ID NO: 4.

[0243] It might further be preferred that the antigen-binding protein ‘C’ is capable of binding to an epitope of an aminergic toxin comprising the amino acid residues:

[0244] - C46, K22, P47 and E15 of SEQ ID NO: 4; and / or

[0245] - E15, Q21, K22, A44, T45, C46 and K48 of SEQ ID NO: 4.P7584PC00

[0246] 30

[0247] In one embodiment the antigen-binding protein ‘C’ may be the antibody referred to as VHH13.

[0248] The antigen-binding protein ‘C’ may preferably comprise or consist of a heavy chain variable (VH) region as set forth in SEQ ID NO: 18, or a sequence identical thereto except for up to 2, such as up to 1 amino acid substitution(s).

[0249] The antigen-binding protein ‘C’ may particularly preferably comprise or consist of a heavy chain variable (VH) region as set forth in SEQ ID NO: 65, or a sequence identical thereto except for up to 2, such as up to 1 amino acid substitution(s).

[0250] In particular, it may be preferred that the antigen-binding protein ‘C’ may comprise or consist of a heavy chain variable (VH) region comprising complementary determining regions (CDRs) comprising or consisting of:

[0251] - a CDR1 as set forth by SEQ ID NO: 19; and

[0252] - a CDR2 as set forth by SEQ ID NO: 20; and

[0253] - a CDR3 as set forth by SEQ ID NO: 21.

[0254] In particular, it may be particularly preferred that the antigen-binding protein ‘C’ may comprise or consist of a heavy chain variable (VH) region comprising complementary determining regions (CDRs) comprising or consisting of:

[0255] - a CDR1 as set forth by SEQ ID NO: 66; and

[0256] - a CDR2 as set forth by SEQ ID NO: 67; and

[0257] - a CDR3 as set forth by SEQ ID NO: 68.

[0258] Antigen-binding protein ‘D’ and Kunitz-type protease inhibitor toxins

[0259] The present disclosure provides for an antigen-binding protein capable of binding to, blocking, and / or neutralizing a snake venom Kunitz-type protease inhibitor toxin. Such antigen- binding proteins are referred to as “antigen-binding protein ‘B’” herein. It may be preferred that the antigen-binding protein ‘D’ is capable of binding to Kunitz-type protease inhibitors. It may be preferred that the antigen-binding protein ‘D’ is capable of blocking Kunitz-type protease inhibitors. It may be preferred that the antigen-binding protein ‘D’ is capable of neutralizing Kunitz-type protease inhibitors.

[0260] Kunitz-type protease inhibitors (KUN) are venom-derived proteins that function as potent modulators of proteolytic activity, interfering with key physiological processesP7584PC00

[0261] 31

[0262] such as blood coagulation and neurotransmission. The Kunitz-type family includes both serine protease inhibitors and, importantly, neurotoxic components, such as dendrotoxins, which are known to block voltage-gated potassium channels, leading to neurotoxic effects including enhanced neurotransmitter release and neuromuscular disruption. The family is characterized by a conserved structural motif that allows them to effectively inhibit proteases such as trypsin, chymotrypsin, and plasmin. By targeting these enzymes or ion channels, Kunitz-type protease inhibitors can exert diverse toxic effects, including anticoagulation, disruption of normal haemostasis, inhibition of fibrinolysis, or interference with synaptic transmission, which results from the blockage of potassium channels.

[0263] Antigen-binding protein ‘D’ may be capable of binding to Kunitz-type protease inhibitors by recognizing specific structural motifs essential to their inhibitory function. By blocking their activity, antigen-binding protein ‘D’ may prevent venom-induced dysregulation of coagulation and neurotoxicity, reducing envenomation severity.

[0264] It may be preferred that the antigen-binding protein ‘D’ is capable of binding to, blocking, and / or neutralizing a Kunitz-type protease inhibitor toxin obtained from the venom of Dendroaspis jamesoni or Dendroaspis polylepis.

[0265] In the present disclosure, a Kunitz-type protease inhibitor toxin may also be referred to as “KUN”. It may thus be preferred that the antigen-binding protein ‘D’ is capable of binding to a snake venom toxin KUN, or a KUN fraction or a toxin comprised in a KUN fraction. Said KUN may be any KUN or any KUN fraction as described in Table 1 of section ‘Toxin overview’. Thus, the Kunitz-type protease inhibitor toxin may be a toxin comprised in a KUN fraction described in Table 1. Said KUN fraction may be KUN-1 or KUN-2 of Table 1. It may also be preferred that the Kunitz-type protease inhibitor toxin is any of the toxins of Venom Kunitz-type listed in Table 1. It may further be preferred that the antigen-binding protein ‘D’ binds to any snake venom that comprises a Kunitz-type inhibitor toxin or a KUN toxin, or any fraction or designation thereof as described herein.

[0266] It may also be preferred that the antigen-binding protein ‘D’ is capable of binding to a snake venom toxin as set forth in SEQ ID NO: 5 or a functional homologue thereofP7584PC00

[0267] 32

[0268] sharing at least 70%, such as at least 80%, for example at least 90%, such as at least 95% sequence identity therewith.

[0269] Accordingly, said Kunitz-type protease inhibitor toxin be a snake venom toxin as set forth in SEQ ID NO: 5 or a functional homologue thereof sharing at least 70%, such as at least 80%, for example at least 90%, such as at least 95% sequence identity therewith.

[0270] It might be preferred that the antigen-binding protein ‘D’ is capable of binding to an epitope of a snake venom Kunitz-type protease inhibitor comprising one or more amino acid residues selected from the group consisting of:

[0271] - R15, W37, R46, K48 and E52 of SEQ ID NO: 5.

[0272] The antigen-binding protein ‘D’ may preferably comprise or consist of a heavy chain variable (VH) region as set forth in SEQ ID NO: 22, or a sequence identical thereto except for up to 2, such as up to 1 amino acid substitution(s).

[0273] The antigen-binding protein ‘D’ may particularly preferably comprise or consist of a heavy chain variable (VH) region as set forth in SEQ ID NO: 69, or a sequence identical thereto except for up to 2, such as up to 1 amino acid substitution(s).

[0274] In one embodiment the antigen-binding protein ‘D’ may be the antibody referred to asHH17.

[0275] In particular, it may be preferred that the antigen-binding protein ‘D’ may comprise or consist of a heavy chain variable (VH) region comprising complementary determining regions (CDRs) comprising or consisting of:

[0276] - a CDR1 as set forth by SEQ ID NO: 23; and

[0277] - a CDR2 as set forth by SEQ ID NO: 24; and

[0278] - a CDR3 as set forth by SEQ ID NO: 25.

[0279] In particular, it may be particularly preferred that the antigen-binding protein ‘D’ may comprise or consist of a heavy chain variable (VH) region comprising complementary determining regions (CDRs) comprising or consisting of:

[0280] - a CDR1 as set forth by SEQ ID NO: 70; and

[0281] - a CDR2 as set forth by SEQ ID NO: 71; and

[0282] - a CDR3 as set forth by SEQ ID NO: 72.P7584PC00

[0283] 33

[0284] Antigen-Binding Protein E’ and Type I a-Neurotoxins (short-chain a-neurotoxins) The present disclosure provides for an antigen-binding protein capable of binding to, blocking, and / or neutralizing a snake venom Type I a-neurotoxin (short-chain a-neurotoxin). Such antigen-binding proteins are referred to as “antigen-binding protein ‘E’” herein. It may be preferred that the antigen-binding protein ‘E’ is capable of binding to Type I a-neurotoxins. It may be preferred that the antigen-binding protein ‘E’ is capable of blocking Type I a-neurotoxins. It may be preferred that the antigen-binding protein ‘E’ is capable of neutralizing Type I a-neurotoxins.

[0285] Type I a-neurotoxins, also referred to as short-chain a-neurotoxins, are venom-derived proteins that act as potent competitive antagonists of nicotinic acetylcholine receptors (nAChRs) at the neuromuscular junction. By binding to the same site as acetylcholine, these toxins effectively block receptor activation, preventing signal transmission between nerves and muscles. This disruption leads to progressive paralysis, beginning with muscle weakness and potentially resulting in fatal respiratory failure due to diaphragm paralysis.

[0286] Antigen-binding protein ‘E’ may be capable of binding to Type I a-neurotoxins by recognizing key structural motifs essential for receptor interaction. By blocking this interaction, antigen-binding protein ‘E’ may help prevent neuromuscular paralysis and respiratory failure.

[0287] It may be preferred that the antigen-binding protein ‘E’ is capable of binding to, blocking, and / or neutralizing a Type I a-neurotoxin obtained from the venom of Dendroaspis jamesoni, Dendroaspis polylepis, Dendroaspis viridis, Hemachatus haemachatus, or species of the Naja genus, such as Naja melanoleuca, Naja annulifera, Naja haje, Naja nivea, or Naja senegalensis.

[0288] In the present disclosure, type I a-neurotoxin may also be referred to as ”sNTx”. Thus, it may be preferred that the antigen-binding protein ‘E’ is capable of binding to a snake venom toxin sNTx, or a sNTx fraction or a toxin comprised in a sNTx fraction. Said sNTx may be any sNTx or any sNTx fraction as described in Table 1 of section ‘Toxin overview’. Thus, the type I a-neurotoxin may be a toxin comprised in a sNTx fractionP7584PC00

[0289] 34

[0290] described in Table 1. Said sNTx fraction may be sNTx-1, sNTx-2, sNTx-3, sNTx-4, sNTx-5, sNTx-6, sNTx-7, sNTx-8, sNTx-9, sNTx-10, or sNTx-11 of Table 1. It may also be preferred that the type I a-neurotoxin is any of the Type I alpha-neurotoxins listed in Table 1. It may further be preferred that the antigen-binding protein ‘E’ binds to any snake venom that comprises a type I a-neurotoxin toxin, a sNTx toxin, or any fraction or designation thereof as described herein.

[0291] It may also be preferred that the antigen-binding protein ‘E’ is capable of binding to a snake venom toxin as set forth in SEQ ID NO: 6 or SEQ ID NO:7 or a functional homologue thereof sharing at least 70%, such as at least 80%, for example at least 90%, such as at least 95% sequence identity therewith.

[0292] Accordingly, said type I a-neurotoxin may preferably be a snake venom toxin as set forth in SEQ ID NO: 6 or SEQ ID NO:7 or a functional homologue of any of the aforementioned sharing at least 70%, such as at least 80%, for example at least 90%, such as at least 95% sequence identity therewith.

[0293] In particular, it may be preferred that the antigen-binding protein ‘E’ is capable of binding to an epitope of a snake venom Type I a-neurotoxin toxin comprising the amino acid residues:

[0294] - Y52, C53, 151, K47, S44, E20 and P43 of SEQ ID NO: 7.

[0295] It might further be preferred that the antigen-binding protein ‘E’ is capable of binding to an epitope of a snake venom Type I a-neurotoxin (short-chain a-neurotoxin) comprising one or more amino acid residues selected from the group consisting of:

[0296] - Y52, C53, 151, K47, S44, E20 and P43 of SEQ ID NO: 7; and / or

[0297] - C42, T44, V45, L51, N52 and C53 of SEQ ID NO: 43.

[0298] The antigen-binding protein ‘E’ may preferably comprise or consist of a heavy chain variable (VH) region as set forth in SEQ ID NO: 30, or a sequence identical thereto except for up to 2, such as up to 1 amino acid substitution(s).

[0299] The antigen-binding protein ‘E’ may particularly preferably comprise or consist of a heavy chain variable (VH) region as set forth in SEQ ID NO: 77, or a sequence identical thereto except for up to 2, such as up to 1 amino acid substitution(s).P7584PC00

[0300] 35

[0301] In one embodiment the antigen-binding protein ‘E’ may be the antibody referred to as VHH5.

[0302] In particular, it may be preferred that the antigen-binding protein ‘E’ may comprise or consist of a heavy chain variable (VH) region comprising complementary determining regions (CDRs) comprising or consisting of:

[0303] - a CDR1 as set forth by SEQ ID NO: 31; and

[0304] - a CDR2 as set forth by SEQ ID NO: 32; and

[0305] - a CDR3 as set forth by SEQ ID NO: 33.

[0306] In particular, it may be particularly preferred that the antigen-binding protein ‘E’ may comprise or consist of a heavy chain variable (VH) region comprising complementary determining regions (CDRs) comprising or consisting of:

[0307] - a CDR1 as set forth by SEQ ID NO: 78; and

[0308] - a CDR2 as set forth by SEQ ID NO: 79; and

[0309] - a CDR3 as set forth by SEQ ID NO: 80.

[0310] Antigen-Binding Protein ‘F’ and Type II a-Neurotoxins (long-chain a-neurotoxins) The present disclosure provides for an antigen-binding protein capable of binding to, blocking, and / or neutralizing a snake venom Type II a-neurotoxin (long-chain a-neurotoxin). Such antigen- binding proteins are referred to as “antigen-binding protein ‘F’” herein. It may be preferred that the antigen-binding protein ‘F’ is capable of binding to Type II a-neurotoxins. It may be preferred that the antigen-binding protein ‘F’ is capable of blocking Type II a-neurotoxins. It may be preferred that the antigen-binding protein ‘F’ is capable of neutralizing Type II a-neurotoxins.

[0311] Type II a-neurotoxins, also known as long-chain a-neurotoxins, are venom-derived proteins structurally similar to Type I a-neurotoxins but containing an additional disulfide bridge. These extra bonds enhance stability and significantly increase binding affinity for nicotinic acetylcholine receptors (nAChRs), leading to more prolonged receptor inhibition at the neuromuscular junction, resulting in sustained neuromuscular paralysis. Their slower dissociation rate from receptors extends paralysis duration, making their effects more severe than those of short-chain a-neurotoxins.P7584PC00

[0312] 36

[0313] Envenomation by snakes producing Type II a-neurotoxins can cause progressive muscle weakness, respiratory distress, and, in severe cases, respiratory failure.

[0314] Antigen-binding protein ‘F’ may be capable of binding to Type II a-neurotoxins by targeting specific residues involved in receptor binding. By preventing this interaction, antigen-binding protein ‘F’ may help mitigate neurotoxic effects and improve patient outcomes.

[0315] It may be preferred that the antigen-binding protein ‘F’ is capable of binding to, blocking, and / or neutralizing a Type II a-neurotoxin obtained from the venom of Dendroaspis polylepis or species of the Naja genus, such as Naja melanoleuca, Naja haje, Naja nivea, Naja senegalensis, or Naja kaouthia.

[0316] Type II a-neurotoxin may also be referred to as ”INTx”. Thus, it may be preferred that the antigen-binding protein ‘F’ is capable of binding to a snake venom toxin INTx, or a INTx fraction or a toxin comprised in a INTx fraction. Said INTx may be any INTx or any INTx fraction as described in Table 1 of section ‘Toxin overview’. Thus, the Type II a-neurotoxin may be a toxin comprised in a INTx fraction described in Table 1. Said INTx fraction may be INTx-1, INTx-2, INTx-3, INTx-4, INTx-5, INTx-6, or INTx-7 of Table 1. It may also be preferred that the Type II a-neurotoxin is any of the Type II alphaneurotoxins listed in Table 1. It may further be preferred that the antigen-binding protein ‘F’ binds to any snake venom that comprises a type II a-neurotoxin toxin, a INTx toxin, or any fraction or designation thereof as described herein.

[0317] It may also be preferred that the antigen-binding protein ‘F’ is capable of binding to a snake venom toxin as set forth in SEQ ID NO: 8 or a functional homologue thereof sharing at least 70%, such as at least 80%, for example at least 90%, such as at least 95% sequence identity therewith.

[0318] Accordingly, said Type II a-neurotoxin may preferably be a snake venom toxin as set forth in SEQ ID NO: 8 or a functional homologue of any of the aforementioned sharing at least 70%, such as at least 80%, for example at least 90%, such as at least 95% sequence identity therewith.P7584PC00

[0319] 37

[0320] In particular, it may be preferred that the antigen-binding protein ‘F’ is capable of binding to an epitope of a snake venom Type II a-neurotoxin toxin comprising the amino acid residues:

[0321] - R36, P7, D27 and R33 of SEQ ID NO: 8.

[0322] It might further be preferred that the antigen-binding protein ‘F’ is capable of binding to an epitope of a snake venom Type II a-neurotoxin (long-chain a-neurotoxin) comprising one or more amino acid residues selected from the group consisting of:

[0323] - R36, P7, D27 and R33 of SEQ ID NO: 8; and / or

[0324] - 19, R33, R36, V37, F65 and R70 of SEQ ID NO: 8.

[0325] In one embodiment the antigen-binding protein ‘F’ may be the antibody referred to as VHH9.

[0326] The antigen-binding protein ‘F’ may preferably comprise or consist of a heavy chain variable (VH) region as set forth in SEQ ID NO: 34, or a sequence identical thereto except for up to 2, such as up to 1 amino acid substitution(s).

[0327] The antigen-binding protein ‘F’ may particularly preferably comprise or consist of a heavy chain variable (VH) region as set forth in SEQ ID NO: 81, or a sequence identical thereto except for up to 2, such as up to 1 amino acid substitution(s).

[0328] In particular, it may be preferred that the antigen-binding protein ‘F’ may comprise or consist of a heavy chain variable (VH) region comprising complementary determining regions (CDRs) comprising or consisting of:

[0329] - a CDR1 as set forth by SEQ ID NO: 35; and

[0330] - a CDR2 as set forth by SEQ ID NO: 36; and

[0331] - a CDR3 as set forth by SEQ ID NO: 37.

[0332] In particular, it may also be particularly preferred that the antigen-binding protein ‘F’ may comprise or consist of a heavy chain variable (VH) region comprising complementary determining regions (CDRs) comprising or consisting of:

[0333] - a CDR1 as set forth by SEQ ID NO: 82; and

[0334] - a CDR2 as set forth by SEQ ID NO: 83; and

[0335] - a CDR3 as set forth by SEQ ID NO: 84.P7584PC00

[0336] 38

[0337] Antigen-Binding Protein ‘G’ and Phospholipase A2 (PLA2) toxins

[0338] The present disclosure provides for an antigen-binding protein capable of binding to, blocking, and / or neutralizing a snake venom phospholipase A2 (PLA2) toxin. Such antigen- binding proteins are referred to as “antigen-binding protein ‘G’” herein. It may be preferred that the antigen-binding protein ‘G’ is capable of binding to PLA2 toxins. It may be preferred that the antigen-binding protein ‘G’ is capable of blocking PLA2 toxins. It may be preferred that the antigen-binding protein ‘G’ is capable of neutralizing PLA2 toxins.

[0339] Phospholipase A2 (PLA2) toxins are enzymes that hydrolyze phospholipids, leading to membrane disruption, inflammation, and cytotoxic effects. These enzymes target the sn-2 ester bond of glycerophospholipids, generating free fatty acids and lysophospholipids, which contribute to venom-induced tissue damage. PLA2 toxins are major contributors to venom-induced dermonecrosis, a severe complication of elapid snakebites that results in progressive tissue destruction.

[0340] Antigen-binding protein ‘G’ may be capable of binding to PLA2 toxins by targeting key residues involved in enzymatic activity and membrane interaction. By inhibiting PLA2 activity, antigen-binding protein ‘G’ may help prevent venom-induced tissue damage and neurotoxic effects.

[0341] It may be preferred that the antigen-binding protein ‘G’ is capable of binding to, blocking, and / or neutralizing a PLA2 toxin obtained from Naja nigricincta, Naja nigricollis, or Naja nubiae.

[0342] It may be preferred that the antigen-binding protein ‘G’ is capable of binding to a snake venom toxin PLA2, or a PLA2 fraction or a toxin comprised in a PLA2 fraction. Said PLA2 may be any PLA2 or any PLA2 fraction as described in Table 1 of section ‘Toxin overview’. Thus, the snake venom PLA2 toxin may be a toxin comprised in a PLA2 fraction described in Table 1. Said PLA2 fraction may be PLA2-1, PLA2-2 or PLA2-3 of Table 1. It may also be preferred that the snake venom PLA2 toxin is any of the Phospholipase A2s listed in Table 1. It may further be preferred that the antigenbinding protein ‘G’ binds to any snake venom that comprises a phospholipase 2 toxin, a PLA2 toxin, or any fraction or designation thereof as described herein.P7584PC00

[0343] 39

[0344] It may also be preferred that the antigen-binding protein ‘G’ is capable of binding to a snake venom toxin as set forth in SEQ ID NO: 9 or a functional homologue thereof sharing at least 70%, such as at least 80%, for example at least 90%, such as at least 95% sequence identity therewith.

[0345] Accordingly, said PLA2 toxin may preferably be a snake venom toxin as set forth in SEQ ID NO: 9 or a functional homologue of any of the aforementioned sharing at least 70%, such as at least 80%, for example at least 90%, such as at least 95% sequence identity therewith.

[0346] In particular, it may be preferred that the antigen-binding protein ‘G’ is capable of binding to an epitope of a snake venom PLA2toxin comprising the amino acid residues: - Y3, F63, W60, E52, D48 and Y62 of SEQ ID NO: 9.

[0347] It might further be preferred that the antigen-binding protein ‘G’ is capable of binding to an epitope of a snake venom phospholipase A2toxin comprising one or more amino acid residues selected from the group consisting of:

[0348] - Y3, F63, W60, E52, D48 and Y62 of SEQ ID NO: 9; and / or

[0349] - N1, R30, D48, W60, Y62 and F63 of SEQ ID NO: 9.

[0350] In one embodiment the antigen-binding protein ‘G’ may be the antibody referred to as VHH20.

[0351] The antigen-binding protein ‘G’ may preferably comprise or consist of a heavy chain variable (VH) region as set forth in SEQ ID NO: 38, or a sequence identical thereto except for up to 2, such as up to 1 amino acid substitution(s).

[0352] The antigen-binding protein ‘G’ may particularly preferably comprise or consist of a heavy chain variable (VH) region as set forth in SEQ ID NO: 85, or a sequence identical thereto except for up to 2, such as up to 1 amino acid substitution(s).

[0353] In particular, it may be preferred that the antigen-binding protein ‘G’ may comprise or consist of a heavy chain variable (VH) region comprising complementary determining regions (CDRs) comprising or consisting of:

[0354] - a CDR1 as set forth by SEQ ID NO: 39; and

[0355] - a CDR2 as set forth by SEQ ID NO: 40; and

[0356] - a CDR3 as set forth by SEQ ID NO: 41.P7584PC00

[0357] 40

[0358] In particular, it may be particularly preferred that the antigen-binding protein ‘G’ may comprise or consist of a heavy chain variable (VH) region comprising complementary determining regions (CDRs) comprising or consisting of:

[0359] - a CDR1 as set forth by SEQ ID NO: 86; and

[0360] - a CDR2 as set forth by SEQ ID NO: 87; and

[0361] - a CDR3 as set forth by SEQ ID NO: 88.

[0362] Structure, formats and modifications of antigen-binding proteins

[0363] The antigen-binding protein according to the present disclosure may have any useful structure or format. Preferably, the antigen-binding protein is selected from the group consisting of: a VHH, a full-length antibody, a Fab fragment, a F(ab’) fragment, a F(ab')2 fragment, an scFv, a diabody, or a triabody.

[0364] The skilled person understands that a variety of antibody formats can be engineered to recognize the same epitope and to exert the same molecular mechanism of action, provided that the paratope is preserved. Accordingly, the results and principles demonstrated herein can be extended to antigen-binding proteins selected from the group consisting of: VHH antibodies, full-length immunoglobulins, Fab fragments, F(ab') fragments, F(ab')2fragments, single-chain variable fragments (scFv), diabodies, and triabodies. These formats are well-established, structurally characterized variants that retain antigen specificity and can be readily generated by routine methods in the field. As such, the skilled person would recognize that the disclosed binding, blocking, and / or neutralizing functions are transferable to these related antibody formats without requiring undue experimentation.

[0365] It may be preferred that the antigen-binding protein is a VHH. It may be preferred that the antigen-binding protein is a full-length antibody. It may be preferred that the antigen-binding protein is a Fab fragment. It may be preferred that the antigen-binding protein is a F(ab’) fragment. It may be preferred that the antigen-binding protein is a F(ab')2 fragment. It may be preferred that the antigen-binding protein is an scFv. It may be preferred that the antigen-binding protein is a diabody. It may be preferred that the antigen-binding protein a triabody.P7584PC00

[0366] 41

[0367] It may be preferred that the antigen-binding protein is a VHH. It may also be preferred that the antigen-binding protein is monoclonal. It may also be preferred that the antigen-binding protein is humanized. It may also be preferred that the antigen-binding protein is human. The antigen-binding protein may for example be selected from the group consisting of: IgG, IgM, IgA, IgD, and IgE.

[0368] It may be preferred that the antigen-binding protein is a single domain antibody, a nanobody, or a VHH, which preferably is of camelid origin.

[0369] It may be preferred that the antigen-binding protein is not capable of binding to, blocking, and / or neutralizing non-toxin antigens.

[0370] It may be preferred that:

[0371] • the antigen-binding protein ‘A’ is not capable of binding to, blocking, and / or neutralizing a snake venom non-Type IA or non-Type IB cytotoxin; and / or

[0372] • the antigen-binding protein ‘B’ is not capable of binding to, blocking, and / or neutralizing a snake venom non-Orphan group XI toxin; and / or

[0373] • the antigen-binding protein ‘C’ is not capable of binding to, blocking, and / or neutralizing a snake venom non-Orphan group XIX toxin and / or an non-aminergic toxin; and / or

[0374] • the antigen-binding protein ‘D’ is not capable of binding to, blocking, and / or neutralizing a snake venom non-Kunitz-type protease inhibitor; and / or

[0375] • the antigen-binding protein ‘E’ is not capable of binding to, blocking, and / or neutralizing a snake venom non-Type I a-neurotoxin (short-chain a-neurotoxin); and / or • the antigen-binding protein ‘F’ is not capable of binding to, blocking, and / or neutralizing a snake venom non-Type II a-neurotoxin (long-chain a-neurotoxin); and / or • the antigen-binding protein ‘G’ is not capable of binding to, blocking, and / or neutralizing a snake venom non-phospholipase A2 (PLA2) toxin.

[0376] It may be preferred that:

[0377] • the antigen-binding protein ‘A’ does not bind, block, and / or neutralize a snake venom non-Type IA or non-Type IB cytotoxin; and / or

[0378] • the antigen-binding protein ‘B’ does not bind, block, and / or neutralize a snake venom non-Orphan group XI toxin; and / orP7584PC00

[0379] 42

[0380] • the antigen-binding protein ‘C’ does not bind, block, and / or neutralize a snake venom non-Orphan group XIX toxin and / or an non-aminergic toxin; and / or

[0381] • the antigen-binding protein ‘D’ does not bind, block, and / or neutralize a snake venom non-Kunitz-type protease inhibitor; and / or

[0382] • the antigen-binding protein ‘E’ does not bind, block, and / or neutralize a snake venom non-Type I a-neurotoxin (short-chain a-neurotoxin); and / or

[0383] • the antigen-binding protein ‘F’ does not bind, block, and / or neutralize a snake venom non-Type II a-neurotoxin (long-chain a-neurotoxin); and / or

[0384] • the antigen-binding protein ‘G’ does not bind, block, and / or neutralize a snake venom non-phospholipase A2 (PLA2) toxin.

[0385] It may be preferred that:

[0386] • the antigen-binding protein ‘A’ binds, blocks, and / or neutralizes a snake venom Type IA or Type IB cytotoxin; and / or

[0387] • the antigen-binding protein ‘B’ binds, blocks, and / or neutralizes a snake venom Orphan group XI toxin; and / or

[0388] • the antigen-binding protein ‘C’ binds, blocks, and / or neutralizes a snake venom Orphan group XIX toxin and / or an aminergic toxin; and / or

[0389] • the antigen-binding protein ‘D’ binds, blocks, and / or neutralizes a snake venom Kunitz-type protease inhibitor; and / or

[0390] • the antigen-binding protein ‘E’ binds, blocks, and / or neutralizes a snake venom Type I a-neurotoxin (short-chain a-neurotoxin); and / or

[0391] • the antigen-binding protein ‘F’ binds, blocks, and / or neutralizes a snake venom Type II a-neurotoxin (long-chain a-neurotoxin); and / or

[0392] • the antigen-binding protein ‘G’ binds, blocks, and / or neutralizes a snake venom phospholipase A2 (PLA2) toxin.

[0393] It may be preferred that:

[0394] • the antigen-binding protein ‘A’ is not capable of binding to, blocking, and / or neutralizing a non-snake venom polypeptide; and / or

[0395] • the antigen-binding protein ‘B’ is not capable of binding to, blocking, and / or neutralizing a non-snake venom polypeptide; and / or

[0396] • the antigen-binding protein ‘C’ is not capable of binding to, blocking, and / or neutralizing a non-snake venom polypeptide; and / orP7584PC00

[0397] 43

[0398] • the antigen-binding protein ‘D’ is not capable of binding to, blocking, and / or neutralizing a non-snake venom polypeptide; and / or

[0399] • the antigen-binding protein ‘E’ is not capable of binding to, blocking, and / or neutralizing a non-snake venom polypeptide; and / or

[0400] • the antigen-binding protein ‘F’ is not capable of binding to, blocking, and / or neutralizing a non-snake venom polypeptide; and / or

[0401] • the antigen-binding protein ‘G’ is not capable of binding to, blocking, and / or neutralizing a non-snake venom polypeptide.

[0402] It may be preferred that:

[0403] • the antigen-binding protein ‘A’ does not bind, block, and / or neutralize a non-snake venom polypeptide; and / or

[0404] • the antigen-binding protein ‘B’ does not bind, block, and / or neutralize a non-snake venom polypeptide; and / or

[0405] • the antigen-binding protein ‘C’ does not bind, block, and / or neutralize a non-snake venom polypeptide; and / or

[0406] • the antigen-binding protein ‘D’ does not bind, block, and / or neutralize a non-snake venom polypeptide; and / or

[0407] • the antigen-binding protein ‘E’ does not bind, block, and / or neutralize a non-snake venom polypeptide; and / or

[0408] • the antigen-binding protein ‘F’ does not bind, block, and / or neutralize a non-snake venom polypeptide; and / or

[0409] • the antigen-binding protein ‘G’ does not bind, block, and / or neutralize a non-snake venom polypeptide.

[0410] It may be preferred that the antigen-binding protein has an apparent melting temperature from 55 to 72 °C as determined by differential scanning fluorimetry.

[0411] It may be preferred that the antigen-binding protein has an apparent melting temperature above 55°C as determined by differential scanning fluorimetry.

[0412] It may be preferred that the antigen-binding protein has an apparent melting temperature of above 58 °C as determined by differential scanning fluorimetry.P7584PC00

[0413] 44

[0414] It may be preferred that apparent melting temperature is the temperature at which approximately 50% of the polypeptide population is unfolded or, conversely, remains structured, as determined by the particular measurement technique employed

[0415] It may be preferred that the antigen-binding protein has no apparent onset of aggregation below 65 °C as determined by light scattering, such as below 60°C, such as below 55°C, or such as below 50°C as determined by light scattering.

[0416] It may be preferred that the antigen-binding protein has no apparent onset of aggregation below 45 °C as determined by light scattering.

[0417] It may be preferred that the antigen-binding protein has an apparent onset of aggregation above 45°C as determined by light scattering, such as above 50°C, such as above 55°C, or such as above 60°C as determined by light scattering.

[0418] It may be preferred that the antigen-binding protein has an apparent onset of aggregation above 65 °C as determined by light scattering.

[0419] It may be preferred that the antigen-binding protein has an apparent onset of aggregation at 55 °C as determined by light scattering.

[0420] It may be preferred that the apparent onset of aggregation is the point at which aggregation becomes detectable above a predefined baseline or threshold, and is therefore determined operationally based on the sensitivity, measurement principle, and conditions of the assay used

[0421] It may be preferred that the antigen-binding protein that less than 5% of said antigenbinding protein aggregate upon incubation at 40 °C for 16 h as determined by dynamic light scattering, such as wherein less than 4% of said antigen-binding protein aggregate, such as wherein less than 3% of said antigen-binding protein aggregate, or such as wherein less than 2% of said antigen-binding protein aggregate.

[0422] It may be preferred that the antigen-binding protein that less than 1% of said antigenbinding protein aggregate upon incubation at 40 °C for 16 h as determined by dynamic light scattering.P7584PC00

[0423] 45

[0424] It may be preferred that the antigen-binding protein that less than 5% of said antigenbinding protein aggregate upon incubation at 40 °C for 5 h as determined by dynamic light scattering, such as wherein less than 4% of said antigen-binding protein aggregate, such as wherein less than 3% of said antigen-binding protein aggregate, or such as wherein less than 2% of said antigen-binding protein aggregate, or such as wherein less than 1% of said antigen-binding protein aggregate.

[0425] It may be preferred that the antigen-binding protein that less than 0.5% of said antigenbinding protein aggregate upon incubation at 40 °C for 5 h as determined by dynamic light scattering

[0426] It may also be preferred that the antigen-binding protein comprises an immunoglobulin constant region, for example the antigen-binding protein may be a single domain antibody, a nanobody or a VHH, linked to an immunoglobulin constant region.

[0427] It may be preferred that the antigen-binding protein is a VHH-FC fusion protein.

[0428] It may also be preferred that the antigen-binding protein is multivalent, e.g. bivalent, and each monomer of the bivalent antigen-binding protein may comprise or consist of any of the antigen-binding proteins described herein, e.g. the polypeptide sequence as set forth by SEQ. ID. NO: 10, 14, 18, 22, 26, 30, 34 or 38.

[0429] It may be preferred that the antigen-binding protein is a VHH-FC fusion protein.

[0430] It may also be particularly preferred that the antigen-binding protein is multivalent, e.g. bivalent, and each monomer of the bivalent antigen-binding protein may comprise or consist of any of the antigen-binding proteins described herein, e.g. the polypeptide sequence as set forth by SEQ ID NO: 57, 61, 65, 69, 73, 77, 81 or 85.

[0431] The skilled person will appreciate that antigen-binding proteins may be modified for improving their stability in vitro and / or in vivo. For example the antigen binding proteins may be modified to improve circulating half-life, such as for example when used in vivo.

[0432] In one embodiment, one or more antigen-binding proteins are multispecific antigen binding proteins. The term "multi-specific antigen binding protein" refers to an antibodyP7584PC00

[0433] 46

[0434] comprising two or more variable regions binding to different epitopes. The epitopes may be on the same or different targets. The multi-specific antigen binding protein binds to two or more antigens at the same time, wherein at least one of the antigens may include the type IA / IB cytotoxin, orphan group XI toxin, orphan group XIX toxin, aminergic toxin, Kunitz-type protease inhibitor toxin, type I a-neurotoxin, type II a-neurotoxin and / or phospholipase A2 toxin.

[0435] The multi-specific antigen binding protein may be a bi-specific antibody binding two different antigen, wherein the antigens are type IA / IB cytotoxin, orphan group XI toxin, orphan group XIX toxin, aminergic toxin, Kunitz-type protease inhibitor toxin, type I a-neurotoxin, type II a-neurotoxin and / or phospholipase A2 toxin.

[0436] In particular, the multi-specific antigen binding molecule may comprise the same CDRs and / or VHHS, or combinations thereof, as described herein below.

[0437] It may be preferred that the composition comprises at least one antigen-binding protein is an albumin-VHH fusion. The inclusion of albumin in the fusion construct may enhance the stability, half-life, and systemic retention of the antigen-binding proteins, thereby improving their therapeutic efficacy. Such fusions may offer several advantages, including increased serum persistence, reduced renal clearance, and enhanced biodistribution. Additionally, the albumin-VHH fusion may be designed for targeted delivery, improving specificity and reducing off-target effects. The fusion construct may be produced using recombinant expression system, such as bacterial, yeast, or mammalian cell cultures, ensuring high yield and stability.

[0438] It may be preferred that one or more, or even all, of the antigen-binding proteins in the composition are VHH fusions, where at least one VHH domain binds to albumin and at least one other VHH domain binds to any of the snake toxins described herein.

[0439] Increased antigen-binding protein half-life may improve for instance its distribution, efficacy, and duration of its functional effect. Such modifications include conjugation with molecules providing a higher molecular weight and / or hydrodynamic radius to the antigen-binding protein, such as PEGylation, polysialylation, or conjugation to any other protein or small molecule known to be suitable by the skilled person. The modification may also be associated with increased evasion from the immune system, thereby alsoP7584PC00

[0440] 47

[0441] increasing the circulatory time in vivo. Other approaches which may improve the antigen-binding protein half-life include engineering of the Fc region, such as for example a mutation of the Fc region associated with improved recycling and half-life. The composition, in particular the antigen-binding proteins, may also for instance be modified to improve their biological function and efficacy. The skilled person will appreciate that several post-translational modifications, such as glycosylations may have a critical impact on the efficacy of antigen-binding proteins, for example impacting their antigen binding capabilities or effector functions. For instance the glycosylations may be glycosylations of the Fc region or the Fab of the antigen-binding protein, such as one or more N-glycosylations. Thus in some embodiments, the antigen-binding protein has one or more modification(s) selected from the group consisting of PEGylation, polysialylation, Fc region mutation, and N-glycosylation.

[0442] The skilled person will appreciate that it may be beneficial to be able to detect and follow the antigen-binding protein for some applications. For instance conjugating the antigen-binding protein with a detection label, or detection agent, may be useful for diagnostic purposes, imaging purposes, multiplexing assays and other assays known to the skilled person. The label may be a primary label, a secondary label or any combinations thereof known to be suitable by the skilled person. Thus, it may be preferred that the antigen-binding protein comprises a detection label.

[0443] In some applications, for instance spectroscopic assays, colorimetric assays, and light microscopy, it may be beneficial to use a coloured or a luminescent dye, for instance chromophores and phosphors or any dye known to be suitable by the skilled person. Other applications such as for example fluorescent microscopic applications, flow 30 cytometry and fluorescence-activated cell sorting (FACS), hybridization assays, or any immunoassays known to the skilled person may require or benefit from an antigenbinding protein comprising a fluorescent label or moiety. The detection label may also be a magnetic or paramagnetic label, for instance for magnetic separation application or immunoassays known to the skilled person.

[0444] In some embodiments, the antigen-binding protein label is selected from the group consisting of a colorimetric, a fluorescent, a luminescent, a magnetic, and a paramagnetic label.P7584PC00

[0445] 48

[0446] The skilled person will also appreciate that other labels which can be used for instance for separation and purification, as well as for example detection in immunoassays, include the biotin-streptavidin pair. The antigen-binding protein may be biotinylated for instance for avidin (or streptavidin) detection.

[0447] In some embodiments, the detection label on the antigen-binding protein is biotin. For other applications, such as electron microscopy, flow cytometry and lateral flow immunoassays, the skilled person will appreciate that conjugation with gold nanoparticles can be used for the detection and / or visualisation of the presence or binding or the antigen-binding protein.

[0448] Preparation of the antigen-binding proteins

[0449] The antigen-binding proteins according to the present disclosure may be prepared by any useful method. In one embodiment, the antibodies are prepared by recombinant methods. Said methods may comprise cultivating a host cell comprising a nucleic acid encoding the antigen-binding protein under control of an element directing expression in said host cell or expressing said antigen-binding protein from a virus or a phage.

[0450] The antibodies are preferably identified using phage display technology, although other methods may also be employed. Useful methods include the methods described in Laustsen et al., (CRC Press, 2021), Laustsen et al., 2018, Ahmadi et al. 2020, Ledsgaard et al., 2021, Marks et al., 1992, Laustsen et al., 2021 and Benard-Valle, M. etal., (2023). For example, a phage display library may be screened by biopanning. The biopanning of phage display libraries for the selection and discovery of the antibodies, such as the VHHS described herein, may be performed by any method known in the art.

[0451] In some embodiments, the biopanning is performed against one or more snake toxins as antigens. It may be preferred that said snake toxins come from elapid snake venoms, for example Dendroaspis angusticeps, Dendroaspis jamesoni, Dendroaspis polylepis, Dendroaspis viridis and / or from species of the genus Naja, for example Naja anchietae, Naja annulifera, Naja ashei, Naja haje, Naja katiensis, for examples Naja melanoleuca, Naja mossambica, Naja nigricincta, Naja nigricollis, Naja nivea, NajaP7584PC00

[0452] 49

[0453] nubiae, Naja pallida and / or Naja senegalensis, and Hemachatus haemachatus. These toxins may for example be recombinantly expressed.

[0454] For example, the toxins used for the biopanning strategy may be one or more Type IA / IB cytotoxins, such as those as set forth in SEQ ID NO: 1 and / or 2, one or more orphan group XI toxins, such as those as set forth in SEQ ID NO: 3, one or more orphan group XIX toxins and / or an aminergic toxin, such as those as set forth in SEQ ID NO: 4, one or more Kunitz-type protease inhibitor toxins, such as those as set forth in SEQ ID NO: 5, one or more type I a-neurotoxin, such as those as set forth in SEQ ID NO: 6 and / or 7, one or more type II a-neurotoxins, such as those as set forth in SEQ ID NO: 8 and / or one or more phospholipase A2 toxins, such as those as set forth in SEQ ID NO: 9.

[0455] It may be further preferred that the toxins used for the biopanning strategy are any of the toxins, fractions or designations described in Table 1, section ‘Toxin overview’. For example the toxins may be one or more CTx, such as CTx-6 and / or CTx-9 and / or CTx-10 and / or CTx-11, one or more Og XI, such as Og XI-1 and / or Og XI-2, one or more AgTx, such as AgTx -2, one or more KUN, such as KUN-1, one or sNTx, such as sNTx-1, sNTx-3 and / or sNTx-6, one or more INTx, such as INTx-1, INTx-3 and / or INTx-7 and / or one or more PLA2, such as PLA2-3.

[0456] Preferably, the antigen-binding protein is prepared using the methods described in Example 1 and 3 below.

[0457] Binding kinetics

[0458] The antigen-binding proteins of the present disclosure preferably have high affinity for the antigen, such as type IA / IB cytotoxin, orphan group XI toxin, orphan group XIX toxin, aminergic toxin, Kunitz-type protease inhibitor toxin, type I a-neurotoxin, type II a-neurotoxin and / or phospholipase A2 toxin. As used herein “binding to” refers to the formation of a non-covalent association between the antigen-binding protein and the antigen, although such binding is not necessarily reversible. The KD mentioned hereinabove can be used to measure the affinity of the binding, wherein a lower relative KD value indicates a higher affinity for the antigen. Thus, in general a low KD value is preferred, meaning that the antigen binding protein has a high affinity.P7584PC00

[0459] 50

[0460] In some embodiments of the present disclosure, the antigen-binding protein binds their antigen with a KD of at the most 50 nM, for example at the most 10 nM, for example at the most 1 nM, for example at the most 0.1 nM, for example at the most 0.01 nM or for example at the most 0.001 nM.

[0461] Compositions and kits of parts

[0462] A further aspect of the present disclosure provides for a composition as described herein for use in a method of treatment of snake envenomation. The disclosure also provides a kit of parts that may comprise the composition as described herein and another agent that may be suitable for the treatment of snake envenomation.

[0463] The other antigen-binding protein(s) capable of binding to, blocking and / or neutralizing one or more snake venom antigen(s) may be for instance existing commercial antigenbinding protein-based antivenoms, such as the antivenoms listed in Laustsen et al., 2016, Supplementary Table 1.

[0464] It may be preferred that the another agent used for the treatment of snake envenomation may be one or more protein(s) displaying neutralization potential against snake venoms. The one or more protein(s) displaying neutralization potential against snake venoms may be selected from the group consisting of: a PLA2 inhibitor, a matrix 5 metalloproteinase (MMP) inhibitor, a snake venom metalloproteinase inhibitor (SVMP), a Kunitz-type trypsin inhibitor, an anti-myotoxic factor, an anti-myonecrotic factor, an anti-neurotoxic factor, an antihemorrhagic factor, an antioedematogenic factor and a glycoprotein, such as acid-a1 -glycoprotein.

[0465] A further type of agents used in the treatment of snake envenomation are small molecules, for instance as described in Laustsen et al., 2016.

[0466] It may be preferred that the another agent used for the treatment of snake envenomation is one or more small molecule(s) with inhibitory effect against snake toxins, which for example may be selected from the group consisting of: polyanions, polyphenolic compounds, and metalloproteinase inhibitors.

[0467] It may be preferred that the one or more small molecule(s) with inhibitory effect against snake toxins may be selected from the group consisting of: Suramin, Heparin,P7584PC00

[0468] 51

[0469] Wedelolcatone, Coumestrol, Glycyrrhizin, Fucoidan, Apigenin analogue, AG-3340, Aristolochic acid, Rosmarinic acid, Imidazopyridine, BAPTA, TPEN, EDTA, DMPS, dimercaprol, BAY-129566, Marimastat, CGS-27023A, Batimastat, Nafamostat, MV 8612 from Mandevilla velutina, MV 8608 from Mandevilla velutina, Koninginin, 4-nerolidylcatechol, Anisodamine, 12-methoxy-4-methylvoachalotine (MMV), Quinonoid xanthene ehretianone, Benzoylsalireposide, Salireposide, Chlorogenic acid, Caffeic acid, 2-hydroxy-4-methoxy benzoic acid, and Varespladib.

[0470] Method of treatment of snakebite

[0471] The present disclosure further provides for a composition, a multi-specific antigenbinding protein, or the kit of parts as described herein for use in a method of treating snakebite or snake envenomation in an individual in need thereof.

[0472] It may be preferred that the method for treating snakebite or snake envenomation in an individual in need thereof comprises administering a therapeutically effective amount of the composition, the antigen-binding protein, the multi-specific antigen-binding protein, or the kit of parts as described herein to said individual.

[0473] It may also be preferred that the antigen-binding protein, the multi-specific antigenbinding protein, or the kit of parts is used for the preparation of a medicament for the treatment of snakebite or snake envenomation in an individual in need thereof.

[0474] The skilled person will appreciate that one of the main effects of snakebite or snake envenomation, in particular snake envenomation by a venom containing a-neurotoxins, is paralysis. In some embodiments, the envenomation symptoms comprise limb paralysis, respiratory paralysis, redness, swelling, bruising, bleeding, blistering, severe pain at the site of the bite, tenderness at the site of the bite, nausea, vomiting, diarrhea, rapid heart rate, weak pulse, low blood pressure, disturbed vision, increased salivation, increased sweating, numbness, tingling and / or muscle twitching. Preferably, the antigen-binding proteins are useful for treatment of any of the aforementioned conditions, e.g. paralysis, such as limb paralysis, and / or respiratory paralysis caused by snake envenomation.

[0475] The skilled person will appreciate that one of the main effects of snakebite or snake envenomation, in particular snake envenomation by a venom containing type IA / IB andP7584PC00

[0476] 52

[0477] phospholipase A2 toxins, is dermonecrosis. In some embodiments, the envenomation symptoms comprise dermonecrosis, including tissue necrosis, skin ulceration, blistering, severe pain at the bite site, swelling, redness, hemorrhagic lesions, eschar formation, delayed wound healing, and secondary infections. Additional systemic effects may include fever, chills, nausea, vomiting, and general malaise. It may be preferred that the composition, method or use described herein, comprise neutralizing dermonecrosis associated with said snakebite. It may be preferred that the composition, method, or use is intended for the treatment of a snakebite from an elapid snake.

[0478] It may be preferred that the composition, method, or use described herein is for treating a snakebite or snake envenomation from an elapid snake selected from Hemachatus haemachatus, species of the Naja genus, such as Naja ashei, Naja haje, Naja katiensis, Naja kaouthia, Naja mossambica, Naja nigricincta, Naja nigricollis, Naja nubiae, Naja pallida, Naja melanoleuca, Naja anchietae, Naja annulifera, Naja nivea, or Naja senegalensis, as well as Dendroaspis jamesoni, Dendroaspis viridis, and Dendroaspis polylepis.

[0479] In some embodiments the elapid snake is Hemachatus haemachatus or Naja ashei or Naja haje or Naja katiensis or Naja kaouthia or Naja mossambica or Naja nigricincta or Naja nigricollis or Naja nubiae or Naja pallida or Naja melanoleuca or Naja anchietae or Naja annulifera or Naja nivea or Naja senegalensis or Dendroaspis angusticeps or Dendroaspis jamesoni or Dendroaspis viridis or Dendroaspis Polylepis.

[0480] In some embodiments the elapid snake is Hemachatus haemachatus or Naja ashei or Naja haje or Naja katiensis or Naja mossambica or Naja nigricollis or Naja nubiae or Naja pallida or Naja melanoleuca or Naja anchietae or Naja annulifera or Naja nivea or Naja senegalensis or Dendroaspis angusticeps or Dendroaspis jamesoni or Dendroaspis viridis or Dendroaspis polylepis.

[0481] In some embodiments the elapid snake is Dendroaspis jamesoni or Dendroaspis viridis or Naja haje or Naja melanoleuca or Hemachatus haemachatus.

[0482] In some embodiments, the elapid snake is Naja naja, Ophiophagus hannah, Ophiophagus kaalinga, Naja kaouthia or Bungarus spp.P7584PC00

[0483] 53

[0484] In some embodiments the snakebite is a bite from an elapid snake selected from Hemachatus haemachatus, species of the Naja genus, such as Naja ashei, Naja haje, Naja katiensis, Naja kaouthia, Naja mossambica, Naja nigricincta, Naja nigricollis, Naja nubiae, Naja pallida, Naja melanoleuca, Naja anchietae, Naja annulifera, Naja nivea, Naja naja, or Naja senegalensis, Dendroaspis jamesoni, Dendroaspis viridis, Dendroaspis polylepis, Ophiophagus hannah, Ophiophagus kaalinga, and Bungarus spp.

[0485] In some embodiments the elapid snake is Hemachatus haemachatus or Naja ashei or Naja haje or Naja katiensis or Naja kaouthia or Naja mossambica or Naja nigricincta or Naja nigricollis or Naja nubiae or Naja pallida or Naja naja or Naja melanoleuca or Naja anchietae or Naja annulifera or Naja nivea or Naja senegalensis or Dendroaspis angusticeps or Dendroaspis jamesoni or Dendroaspis viridis or Dendroaspis polylepis or Ophiophagus hannah or Ophiophagus kaalinga, or Bungarus spp.

[0486] In some embodiments the elapid snake is Hemachatus haemachatus or Naja ashei or Naja haje or Naja katiensis or Naja kaouthia or Naja mossambica or Naja nigricollis or Naja nubiae or Naja pallida or Naja melanoleuca or Naja anchietae or Naja annulifera or Naja nivea or Naja senegalensis or Naja naja or Dendroaspis angusticeps or Dendroaspis jamesoni or Dendroaspis viridis or Dendroaspis polylepis or Ophiophagus hannah or Ophiophagus kaalinga, or Bungarus spp.

[0487] In some embodiments the elapid snake is Dendroaspis jamesoni or Dendroaspis viridis or Naja haje or Naja melanoleuca or Hemachatus haemachatus or Naja naja or Ophiophagus hannah or Ophiophagus kaalinga or Naja kaouthia or Bungarus spp.

[0488] In some embodiments the elapid snake is Hemachatus haemachatus, species of the Naja genus, such as Naja ashei, Naja haje, Naja katiensis, Naja mossambica, Naja nigricincta, Naja nigricollis, Naja nubiae, Naja pallida, Naja melanoleuca, Naja anchietae, Naja annulifera, Naja nivea, or Naja senegalensis, or Dendroaspis jamesoni, Dendroaspis viridis, or Dendroaspis polylepis.

[0489] In some embodiments the elapid snake is Hemachatus haemachatus, species of the Naja genus, such as Naja ashei, Naja haje, Naja katiensis, Naja mossambica, NajaP7584PC00

[0490] 54

[0491] nigricincta, Naja nigricollis, Naja nubiae, Naja pallida, Naja melanoleuca, Naja anchietae, Naja annulifera, Naja nivea, or Naja senegalensis.

[0492] In some embodiments the elapid snake is Hemachatus haemachatus, species of the Naja genus, such as Naja ashei, Naja Naja, Naja kaouthia, Naja haje, Naja katiensis, Naja mossambica, Naja nigricincta, Naja nigricollis, Naja nubiae, Naja pallida, Naja melanoleuca, Naja anchietae, Naja annulifera, Naja nivea, or Naja senegalensis, or Dendroaspis jamesoni, Dendroaspis viridis, or Dendroaspis polylepis, or Ophiophagus hannah, or Ophiophagus kaalinga, or Bungarus spp.

[0493] In some embodiments the elapid snake is Hemachatus haemachatus, species of the Naja genus, such as Naja ashei, Naja Naja, Naja kaouthia, Naja haje, Naja katiensis, Naja mossambica, Naja nigricincta, Naja nigricollis, Naja nubiae, Naja pallida, Naja melanoleuca, Naja anchietae, Naja annulifera, Naja nivea, or Naja senegalensis or Ophiophagus hannah, or Ophiophagus kaalinga, or Bungarus spp.

[0494] Sequence overview

[0495] SEQ ID NO: 1: P01468, Snake three-finger toxin family / Short-chain subfamily / Type IA cytotoxin sub-subfamily LKCNQLIPPFWKTCPKGKNLCYKMTMRAAPMVPVKRGCIDVCPKSSLLIKYMCCNTD KCN SEQ ID NO: 2: P01456, Snake three-finger toxin family I Short-chain subfamily I Type IA cytotoxin sub-subfamily LKCHKLVPPVWKTCPEGKNLCYKMFMVSTSTVPVKRGCIDVCPKDSALVKYVCCSTD KCN SEQ ID NO: 3: P01405, Snake three-finger toxin family I Short-chain subfamily I Orphan group XI sub-subfamily MICYSHKTPQNSATITCEEKTCYKFVTKLPGVILARGCGCPKKEIFRKSIHCCRSDKCN E SEQ ID NO: 4: P01407, Snake three-finger toxin family I Short-chain subfamily I Aminergic toxin sub-subfamily LTCVTDKSFGGVNTEECAAGQKICFKNWKKMGPKLYDVKRGCTATCPKADDDGCVK CCNTDKP7584PC00

[0496] 55

[0497] SEQ ID NO: 5: P00979, Venom Kunitz-type family QPLRKLCILHRNPGRCYQKIPAFYYNQKKKQCEGFTWSGCGGNSNRFKTIEECRRTC IRK SEQ ID NO: 6: P01417, Snake three-finger toxin family I Short-chain subfamily I Type I alpha-neurotoxin sub-subfamily RICYNHQSTTPATTKSCGENSCYKKTWSDHRGTIIERGCGCPKVKQGIHLHCCQSDK CNN SEQ ID NO: 7: P01422, Snake three-finger toxin family I Short-chain subfamily I Type I alpha-neurotoxin sub-subfamily MICHNQQSSQPPTIKTCPGETNCYKKRWRDHRGTIIERGCGCPSVKKGVGIYCCKTN KCNR SEQ ID NO: 8: P01391, Snake three-finger toxin family I Long-chain subfamily I Type II alpha-neurotoxin sub-subfamily IRCFITPDITSKDCPNGHVCYTKTWCDAFCSIRGKRVDLGCAATCPTVKTGVDIQCCST DNCNPFPTRKRP SEQ ID NO: 9: P14556, Phospholipase A2 family I Group I subfamily I D49 subsubfamily NLYQFKNMIHCTVPSRPWWHFADYGCYCGRGGKGTPIDDLDRCCQVHDNCYEKAG KMGCWPYFTLYKYKCSKGTLTCNGRNGKCAAAVCNCDLVAANCFAGAPYINANYNID FKKRCQ SEQ ID NO: 10: TPL0870_01_G09HH (also referred to as VHH1) QVQLQESGGGLVQAGGSLRLSCAASGRTFSSYAMAWFRQAPGKEREFVASISWSG DSTYYADSVKGRFTISRDNAKNTWYLQMNSLKPEDTAVYYCNTEDESSGTYYEWGQ GTQVTVSSAAADYKDHDGDYKDHDIDYKDDDDKGAAHHHHHH SEQ ID NO: 11: TPL0870_01_G09HH (also referred to as VHH1) CDR1 GRTFSSYA SEQ ID NO: 12: TPL0870_01_G09HH (also referred to as VHH1) CDR2 ISWSGDST SEQ ID NO: 13: TPL0870_01_G09HH (also referred to as VHH1) CDR3 NTEDESSGTYYE SEQ ID NO: 14: TPL1039_01_C04HH (also referred to asHH15)P7584PC00

[0498] 56 QVQLQESGGGAVPAGGSLTLSCAASGRTLSDYTMGWFRQAPGKEREIVGAISWNDG YTYYDDSVKGRFTISRDNAKNTMYLQMNSLKPEDTAVYYCAAQAPEMVRSGNFGSW GPGTQVTVSSAAADYKDHDGDYKDHDIDYKDDDDKGAAHHHHHH SEQ ID NO: 15: TPL1039_01_C04 VHH (also referred to as VHH15) CDR1 GRTLSDYT SEQ ID NO: 16: TPL1039_01_C04 VHH (also referred to as VHH15) CDR2 ISWNDGYT SEQ ID NO: 17: TPL1039_01_C04 VHH (also referred to as VHH15) CDR3 AAQAPEMVRSGNFGS SEQ ID NO: 18: TPL1033_03_D05 VHH (also referred to as VHH13) QVQLQESGGGLVQAGGSLRLSCAASGLDFSTMPLTVGWFRQAPGKEREFVAAISWR GDSWNYEDGMEGRFTVSRDNAKNTVYLQMSSLHTEDTGVYYCAGDPTPYGTSSGG FSVWGQGTQVTVSSAAADYKDHDGDYKDHDIDYKDDDDKGAAHHHHHH SEQ ID NO: 19: TPL1033_03_D05 VHH (also referred to as VHH13) CDR1 DFSTMPLT SEQ ID NO: 20: TPL1033_03_D05 VHH (also referred to as VHH13) CDR2 ISWRGDSW SEQ ID NO: 21: TPL1033_03_D05 VHH (also referred to as VHH13) CDR3 AGDPTPYGTSSGGFSV SEQ ID NO: 22: TPL1013_03_F11 VHH (also referred to as VHH17) QVQLQESGGGLVQPGGSLRLSCITSGSNFDDLSVGWFRQAPGKEREGVSCFSSSDG STYYVDSVKGRFTMSRDNAKNTVYLQMNNLKPEDTAVYYCTADGFPYPSETMCSIPG GPDSVAWGQGTQVTVSSAAADYKDHDGDYKDHDIDYKDDDDKGAAHHHHHH SEQ ID NO: 23: TPL1013_03_F11 VHH (also referred to as VHH17) CDR1 GSNFDDLS SEQ ID NO: 24: TPL1013_03_F11 VHH (also referred to as VHH17) CDR2 FSSSDGST SEQ ID NO: 25: TPL1013_03_F11 VHH (also referred to as VHH17) CDR3 TADGFPYPSETMCSIPGGPDSVA SEQ ID NO: 26: TPL1054_01_A05 VHH (also referred to as VHH4)P7584PC00

[0499] 57 QVQLQESGGGLVQAGGSLRLSCAAPGFTFDNYAIGWFRQAPGKEREAVSMIDVNDG DTYYADSVKGRFATSIDNARITAYLQMNSLKPEDTAVYYCAAGEQGGIETEDDYGMD YYGKGTQVTVSSAAADYKDHDGDYKDHDIDYKDDDDKGAAHHHHHH SEQ ID NO: 27: TPL1054_01_A05 VHH (also referred to as VHH4) CDR1 GFTFDNYA SEQ ID NO: 28: TPL1054_01_A05 VHH (also referred to as VHH4) CDR2 IDVNDGDT SEQ ID NO: 29: TPL1054_01_A05 VHH (also referred to as VHH4) CDR3 AAGEQGGIETEDDYGMDY SEQ ID NO: 30: TPL0998_01_A05 VHH (also referred to as VHH5) QVQLQESGGGLVRPGGSLRLSCAVSGVSESIFANYVVGWFRQQDSGTGRDLVAQSS SDDEYNHVSGSVKGRFTISRDNAKNTVSLQMDNLKPEDTAIYICAAAIGPGDNYIYWG QGTQVTVSSAAADYKDHDGDYKDHDIDYKDDDDKGAAHHHHHH SEQ ID NO: 31: TPL0998_01_A05 (also referred to as VHH5) CDR1 GVSESIFANYV SEQ ID NO: 32: TPL0998_01_A05 (also referred to as VHH5) CDR2 QSSSDDEYN SEQ ID NO: 33: TPL0998_01_A05 (also referred to as VHH5) CDR3 AAAIGPGDNYIY SEQ ID NO: 34: TPL0877_01_A04 VHH (also referred to as VHH9) QVQLQESGGGLVQPGGSLRLSCTASGSIFSTVNTMGWYRQAPGKQRELVAAITGGG STNYADSVKGRFTISRDNVENTVHLRMNALKPEDTAVYYCNFQEYCSAYGCYELPHD YWGQGTQVTVSSAAADYKDHDGDYKDHDIDYKDDDDKGAAHHHHHH SEQ ID NO: 35: TPL0877_01_A04 VHH (also referred to as VHH9) CDR1 SIFSTVNT SEQ ID NO: 36: TPL0877_01_A04 VHH (also referred to as VHH9) CDR2 ITGGGST SEQ ID NO: 37: TPL0877_01_A04 VHH (also referred to as VHH9) CDR3 NFQEYCSAYGCYELPHDY SEQ ID NO: 38: TPL0599_01_C06 VHH (also referred to as VHH20)P7584PC00

[0500] QVQLQESGGGLAQAGGSLRLSCAASGLAFSNYSMGWARQAPGKEREFVAAISWSH QPTYYADSVKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCAADSRPNSYRLGGPGN YWGQGTQVTVSSAAADYKDHDGDYKDHDIDYKDDDDKGAAHHHHHH SEQ ID NO: 39: TPL0599_01_C06 VHH (also referred to as VHH20) CDR1 GLAFSNYS SEQ ID NO: 40: TPL0599_01_C06 VHH (also referred to as VHH20) CDR2 ISWSHQPT SEQ ID NO: 41: TPL0599_01_C06 VHH (also referred to as VHH20) CDR3 AADSRPNSYRLGGPGNY SEQ ID NO: 42: M13Rev primer

[0501] CAGGAAACAGCTATGAC SEQ ID NO: 43: P01426, Snake three-finger toxin family I Short-chain subfamily I Type I alpha-neurotoxin sub-subfamily LECHNQQSSQPPTTKTCPGETNCYKKVWRDHRGTIIERGCGCPTVKPGIKLNCCTTD KCNN SEQ ID NO: 44: P01463, Snake three-finger toxin family I Short-chain subfamily I Type IA cytotoxin sub-subfamily LKCHQLIPPFWKTCPEGKNLCYKMYMVATPMIPVKRGCIDVCPKNSALVKYMCCNTD K SEQ ID NO: 45: P01421, Snake three-finger toxin family / Short-chain subfamily / Type I alpha-neurotoxin sub-subfamily MICYKQRSLQFPITTVCPGEKNCYKKQWSGHRGTIIERGCGCPSVKKGIEINCCTTDK CNR SEQ ID NO: 46: P01423, Snake three-finger toxin family I Short-chain subfamily I Type I alpha-neurotoxin sub-subfamily MICHNQQSSQRPTIKTCPGETNCYKKRWRDHRGTIIERGCGCPSVKKGVGIYCCKTD KCNR SEQ ID NO: 47: P01424, Snake three-finger toxin family I Short-chain subfamily I Type I alpha-neurotoxin sub-subfamily MECHNQQSSQPPTTKTCPGETNCYKKQWSDHRGTIIERGCGCPSVKKGVKINCCTT DRCNNP7584PC00

[0502] 59

[0503] SEQ ID NO: 48: P01433, Snake three-finger toxin family I Short-chain subfamily I Type I alpha-neurotoxin sub-subfamily LECHNQQSSQTPTTQTCPGETNCYKKQWSDHRGSRTERGCGCPTVKPGIKLKCCTT DRCNK SEQ ID NO: 49: P68418, Snake three-finger toxin family I Short-chain subfamily I Type I alpha-neurotoxin sub-subfamily LECHNQQSSQPPTTKTCPGETNCYKKRWRDHRGSITERGCGCPSVKKGIEINCCTTD KCNN SEQ ID NO: 50: P01448, Snake three-finger toxin family I Short-chain subfamily I Type IA cytotoxin sub-subfamily LECNKLVPIAHKTCPAGKNLCYQMYMVSKSTIPVKRGCIDVCPKSSLLVKYVCCNTDR CN SEQ ID NO: 51: P0DQP2, Snake three-finger toxin family I Short-chain subfamily I Aminergic toxin sub-subfamily LTCVTDKSFGGVITEECAAGQKICFKNWKKMGPKLYDVKRGCTATCPKADDNGCVKC CNTDKCNK SEQ ID NO: 52: Q53B57, Snake three-finger toxin family I Long-chain subfamily I Type II alpha-neurotoxin sub-subfamily TKCYVTPDVTSQTCPDGQNICYTETWCDAWCGSRGKRVNLGCAATCPKVNPGVDIIC CSTDNCNPFPKRS SEQ ID NO: 53: C0HJD7, Snake three-finger toxin family I Long-chain subfamily I Type II alpha-neurotoxin sub-subfamily RTCNKTFSDQSKICPPGENICYTKTWCDAFCSQRGKRVELGCAATCPKVKAGVEIKC CSTDNCNKFQFGKPR SEQ ID NO: 54: P01390, Snake three-finger toxin family I Long-chain subfamily I Type II alpha-neurotoxin sub-subfamily IRCFITPDVTSQACPDGHVCYTKMWCDNFCGMRGKRVDLGCAATCPKVKPGVNIKC CSRDNCNPFPTRKRS SEQ ID NO: 55: P01389, Snake three-finger toxin family I Long-chain subfamily I Type II alpha-neurotoxin sub-subfamily

[0504] I RCFITPDVTSQACPDGQN ICYTKTWCDN FCGM RGKRVDLGCAATCPTVKPGVDI KC CSTDNCNPFPTRERSP7584PC00

[0505] 60

[0506] SEQ ID NO: 56: P00605, Phospholipase A2 family I Group I subfamily I D49 subsubfamily NLYQFKNMIHCTVPSRPWWHFADYGCYCGRGGKGTPVDDLDRCCQVHDNCYEKAG KMGCWPYLTLYKYKCSQGKLTCSGGNSKCGAAVCNCDLVAANCFAGARYIDANYNI NFKKRCQ

[0507] SEQ ID NO: 57: TPL0870_01_G09 VHH (also referred to as VHH1) QVQLQESGGGLVQAGGSLRLSCAASGRTFSSYAMAWFRQAPGKEREFVASISWNG DSTYYADSVKGRFTISGDNAKNTWYLQMKSLKPEDTAVYYCNTEDEGSGTYYEWGQ GTQVTVSS

[0508] SEQ ID NO: 58: TPL0870_01_G09 VHH (also referred to as VHH1) CDR1 as determined by aligning the variable domain amino acid sequences to standardized antibody sequence frameworks according to the Kabat numbering system SYAMA

[0509] SEQ ID NO: 59: TPL0870_01_G09 VHH (also referred to as VHH1) CDR2 as determined by aligning the variable domain amino acid sequences to standardized antibody sequence frameworks according to the Kabat numbering system SISWNGDSTYYADSVKG

[0510] SEQ ID NO: 60: TPL0870_01_G09 VHH (also referred to as VHH1) CDR3 as determined by aligning the variable domain amino acid sequences to standardized antibody sequence frameworks according to the Kabat numbering system EDEGSGTYYE

[0511] SEQ ID NO: 61: TPL1039_01_C04 VHH (also referred to as VHH15) QVQLQESGGGAVPAGGSLTLSCAASGRTLSDYTMGWFRQAPGKEREIVGAISWNDG YTYYDDSVKGRFTISRDNAKNTMYLQMNSLKPEDTAVYYCAAQAPEMVRSGNFGSW GPGTQVTVSS

[0512] SEQ ID NO: 62: TPL1039_01_C04 VHH (also referred to as VHH15) CDR1 as determined by aligning the variable domain amino acid sequences to standardized antibody sequence frameworks according to the Kabat numbering system

[0513] DYTMGP7584PC00

[0514] 61

[0515] SEQ ID NO: 63: TPL1039_01_C04 VHH (also referred to as VHH15) CDR2 as determined by aligning the variable domain amino acid sequences to standardized antibody sequence frameworks according to the Kabat numbering system AISWNDGYTYYDDSVKG

[0516] SEQ ID NO: 64: TPL1039_01_C04 VHH (also referred to as VHH15) CDR3 as determined by aligning the variable domain amino acid sequences to standardized antibody sequence frameworks according to the Kabat numbering system QAPEMVRSGNFGS

[0517] SEQ ID NO: 65: TPL1033_03_D05 VHH (also referred to as VHH13) QVQLQESGGGLVQAGGSLRLSCAASGLDFSTMPLTVGWFRQAPGKEREFVAAISWR GDSWNYEDGMEGRFTVSRDNAKNTVYLQMSSLHTEDTGVYYCAGDPTPYGTSSGG FSVWGQGTQVTVSS

[0518] SEQ ID NO: 66: TPL1033_03_D05 VHH (also referred to as VHH13) CDR1 as determined by aligning the variable domain amino acid sequences to standardized antibody sequence frameworks according to the Kabat numbering system TMPLTVG

[0519] SEQ ID NO: 67: TPL1033_03_D05 VHH (also referred to as VHH13) CDR2 as determined by aligning the variable domain amino acid sequences to standardized antibody sequence frameworks according to the Kabat numbering system AISWRGDSWNYEDGMEG

[0520] SEQ ID NO: 68: TPL1033_03_D05 VHH (also referred to as VHH13) CDR3 as determined by aligning the variable domain amino acid sequences to standardized antibody sequence frameworks according to the Kabat numbering system DPTPYGTSSGGFSV

[0521] SEQ ID NO: 69: TPL1013_03_F11 VHH (also referred to as VHH17) QVQLQESGGGLVQPGGSLRLSCITSGSNFDDLSVGWFRQAPGKEREGVSCFSSSDG STYYVDSVKGRFTMSRDNAKNTVYLQMNNLKPEDTAVYYCTADGFPYPSETMCSIPG GPDSVAWGQGTQVTVSS

[0522] SEQ ID NO: 70: TPL1013_03_F11 VHH (also referred to as VHH17) CDR1 as determined by aligning the variable domain amino acid sequences to standardized antibody sequence frameworks according to the Kabat numbering systemP7584PC00

[0523] 62

[0524] DLSVG

[0525] SEQ ID NO: 71: TPL1013_03_F11 VHH (also referred to as VHH17) CDR2 as determined by aligning the variable domain amino acid sequences to standardized antibody sequence frameworks according to the Kabat numbering system CFSSSDGSTYYVDSVKG

[0526] SEQ ID NO: 72: TPL1013_03_F11 VHH (also referred to as VHH17) CDR3 as determined by aligning the variable domain amino acid sequences to standardized antibody sequence frameworks according to the Kabat numbering system DGFPYPSETMCSIPGGPDSVA

[0527] SEQ ID NO: 73: TPL1054_01_A05 VHH (also referred to as VHH4) QVQLQESGGGLVQAGGSLRLSCAAPGFTFDNYAIGWFRQAPGKEREAVSMIDVNDG DTYYADSVKGRFATSIDNARITAYLQMNSLKPEDTAVYYCAAGEQGGIETEDDYGMD YYGKGTQVTVSS

[0528] SEQ ID NO: 74: TPL1054_01_A05 VHH (also referred to as VHH4) CDR1 as determined by aligning the variable domain amino acid sequences to standardized antibody sequence frameworks according to the Kabat numbering system

[0529] NYAIG

[0530] SEQ ID NO: 75: TPL1054_01_A05 VHH (also referred to as VHH4) CDR2 as determined by aligning the variable domain amino acid sequences to standardized antibody sequence frameworks according to the Kabat numbering system

[0531] M I DVN DGDTYYADSVKG

[0532] SEQ ID NO: 76: TPL1054_01_A05 VHH (also referred to as VHH4) CDR3 as determined by aligning the variable domain amino acid sequences to standardized antibody sequence frameworks according to the Kabat numbering system GEQGGIETEDDYGMDY

[0533] SEQ ID NO: 77: TPL0998_01_A05 VHH (also referred to as VHH5) QVQLQESGGGLVRPGGSLRLSCAVSGVSESIFANYVVGWFRQQDSGTGRDLVAQSS SDDEYNHVSGSVKGRFTISRDNAKNTVSLQMDNLKPEDTAIYICAAAIGPGDNYIYWG QGTQVTVSSP7584PC00

[0534] 63

[0535] SEQ ID NO: 78: TPL0998_01_A05 VHH (also referred to as VHH5) CDR1 as determined by aligning the variable domain amino acid sequences to standardized antibody sequence frameworks according to the Kabat numbering system IFANYVVG

[0536] SEQ ID NO: 79: TPL0998_01_A05 VHH (also referred to as VHH5) CDR2 as determined by aligning the variable domain amino acid sequences to standardized antibody sequence frameworks according to the Kabat numbering system QSSSDDEYNHVSGSVKG

[0537] SEQ ID NO: 80: TPL0998_01_A05 VHH (also referred to as VHH5) CDR3 as determined by aligning the variable domain amino acid sequences to standardized antibody sequence frameworks according to the Kabat numbering system AIGPGDNYIY

[0538] SEQ ID NO: 81: TPL0877_01_A04 VHH (also referred to as VHH9) QVQLQESGGGLVQPGGSLRLSCTASGSIFSTVNTMGWYRQAPGKQRELVAAITGGG STNYADSVKGRFTISRDNVENTVHLRMNALKPEDTAVYYCNFQEYCSAYGCYELPHD YWGQGTQVTVSS

[0539] SEQ ID NO: 82: TPL0877_01_A04 VHH (also referred to as VHH9) CDR1 as determined by aligning the variable domain amino acid sequences to standardized antibody sequence frameworks according to the Kabat numbering system TVNTMG

[0540] SEQ ID NO: 83: TPL0877_01_A04 VHH (also referred to as VHH9) CDR2 as determined by aligning the variable domain amino acid sequences to standardized antibody sequence frameworks according to the Kabat numbering system AITGGGSTNYADSVKG

[0541] SEQ ID NO: 84: TPL0877_01_A04 VHH (also referred to as VHH9) CDR3 as determined by aligning the variable domain amino acid sequences to standardized antibody sequence frameworks according to the Kabat numbering system QEYCSAYGCYELPHDY

[0542] SEQ ID NO: 85: TPL0599_01_C06 VHH (also referred to as VHH20)P7584PC00

[0543] 64 QVQLQESGGGLAQAGGSLRLSCAASGLAFSNYSMGWARQAPGKEREFVAAISWSH QPTYYADSVKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCAADSRPNSYRLGGPGN YWGQGTQVTVSS

[0544] SEQ ID NO: 86: TPL0599_01_C06 VHH (also referred to as VHH20) CDR1 as determined by aligning the variable domain amino acid sequences to standardized antibody sequence frameworks according to the Kabat numbering system

[0545] NYSMG

[0546] SEQ ID NO: 87: TPL0599_01_C06 VHH (also referred to as VHH20) CDR2 as determined by aligning the variable domain amino acid sequences to standardized antibody sequence frameworks according to the Kabat numbering system AISWSHQPTYYADSVKG

[0547] SEQ ID NO: 88: TPL0599_01_C06 VHH (also referred to as VHH20) CDR3 as determined by aligning the variable domain amino acid sequences to standardized antibody sequence frameworks according to the Kabat numbering system DSRPNSYRLGGPGNY

[0548] SEQ ID NO: 89: TPL0870_01_G09 VHH (also referred to as VHH1) comprising tag QVQLQESGGGLVQAGGSLRLSCAASGRTFSSYAMAWFRQAPGKEREFVASISWNG DSTYYADSVKGRFTISGDNAKNTWYLQMKSLKPEDTAVYYCNTEDEGSGTYYEWGQ GTQVTVSSAAADYKDHDGDYKDHDIDYKDDDDKGAAHHHHHH

[0549] Toxin overview

[0550] In the present disclosure, fractions and purified toxins are denoted by their main toxin (sub)family name and a number (e.g. sNTx-1). Table 1 shows an overview of the contents of each elapid snake venom.

[0551] Table 1: Overview of purified toxin fractions isolated from each species and UniProt accession number of toxins comprised in each fraction. UniProt accession numbers for each toxin refers to the UniProt sequence as they were listed in UniProt, Release 2025 01.

[0552] UniProt

[0553] Snake Fraction Prevalence

[0554] accession Toxin (Sub)Family species (Label) (%)

[0555]

[0556] numberP7584PC00

[0557] 65

[0558] Dendroaspis

[0559] angusticeps

[0560] X x x X

[0561] (L1307)

[0562] Tanzania

[0563] 74.26(77.00) P01417 Type I alpha-neurotoxin sNTx-1 14.46(14.59) P18328 Aminergic toxin 5.61(5.43) P60237 Type B muscarinic toxin 52.78 P01419 Type I alpha-neurotoxin Other

[0564] 35.63 P00979 Venom Kunitz-type 3FTx-1

[0565] 7.32 P01407 Aminergic toxin 57.71(57.97) P01407 Aminergic toxin Dendroaspis KUN-1 35.05(34.67) P00979 Venom Kunitz-type jamesoni

[0566] (L1308) 4.13(4.09) P0DQP2 Aminergic toxin Cameroon 41.38(43.46) P0DQP2 Aminergic toxin AgTx-1 30.27(31.78) P01407 Aminergic toxin 22.30(17.74) P25682 Orphan group XIX 89.89(71.41) P01405 Orphan group XI P01407(P0 Aminergic toxin(Orphan group Og XI-1 3.48(20.09)

[0567] 1406) XI) 1.18(3.01) C0HJB0(P0 Mambalgin toxin(Aminergic 1407) toxin) 72.37(72.25) P01416 Type I alpha-neurotoxin sNTx-2 19.15(19.07) Q53B57 Type II alpha-neurotoxin 4.16)4.15) P00981 Venom Kunitz-type 42.85(42.87) P00981 Venom Kunitz-type INTx-1 19.44(19.38) P01416 Type I alpha-neurotoxin 18.90(18.91) Q53B57 Type II alpha-neurotoxin Dendroaspis 31.52(31.48) P00981 Venom Kunitz-type polylepis INTx-2 26.51(26.48) P00984 Venom Kunitz-type (L1309)

[0568] Kenya, 20.26(20.22) P01390 Type II alpha-neurotoxin South Africa 30.71(30.66) C0HJD7 Type II alpha-neurotoxin INTx-3 24.57(24.45) P01390 Type II alpha-neurotoxin 16.81(16.74) P00981 Venom Kunitz-type 84.44(89.27) P00979 Venom Kunitz-type P14556(P0

[0569] KUN-2 5.55(3.32) Venom Kunitz-type 0981)

[0570] P00981(C0

[0571] 3.14(2.52) Type II alpha-neurotoxin HJD7)

[0572] 94.33(97.96) P01417 Type I alpha-neurotoxin sNTx-3

[0573] 3.66 P00986 Venom Kunitz-type 33.90(33.76) P01419 Type I alpha-neurotoxin Dendroaspis

[0574] viridis sNTx-4 28.65(28.62) C0HJD7 Type II alpha-neurotoxin (L1310) 24.48(24.46) A8N285 Type II alpha-neurotoxin Ghana

[0575] 80.61 P01407 Aminergic toxin AgTx-2 11.21 P25682 Ophan group XIX

[0576]

[0577] 2.37 C0HJD7 Type II alpha-neurotoxinP7584PC00

[0578] 66

[0579] 52.19(52.29) P25683 L-type calcium blocker Other

[0580] 3FTx-2 16.87(16.98) P18329 Orphan group X 16.88(7.89) P17696 Aminergic toxin 66.59(53.15) P01405 Orphan group XI P17696(P0 Aminergic toxin(Orphan group Og XI-2 10.77(14.40) 1406) XI)

[0581] 8.08(10.88) P01407(P1

[0582] 7696) Aminergic toxin 67.98(68.35) P01433 Type I alpha-neurotoxin sNTx-5 21.19(21.33) P14556 Phospholipase A2 Hemachatus

[0583] haemachatu 4.69(4.50) P01431 Type I alpha-neurotoxin s (L1311) 88.02 P24777 Type IB cytotoxin South Africa

[0584] CTx-1 2.94 P01473 Orphan group XV 2.70 P24778 Orphan group XIV Naja ashei 74.17(74.15) P01468 Type IA cytotoxin (L1375) CTx-2 18.30(18.28) P01456 Type IA cytotoxin Kenya 6.96(6.76) P0DSN1 Type IA cytotoxin Naja 44.83(42.24) P01468 Type IA cytotoxin katiensis 28.43(26.81)

[0585] (L1317) CTx-3 P01456 Type IA cytotoxin Burkina 10.08(14.15) P0DSN1 Type IA cytotoxin Faso

[0586] Naja 54.75(52.95) P01468 Type IA cytotoxin mossambica 16.15(15.61) P01456 Type IA cytotoxin (L1376) CTx-4

[0587] South Africa, 12.89(12.47) P25517 Type IA cytotoxin Tanzania

[0588] 66.13(79.49) P01468 Type IA cytotoxin P01400(P0 Orphan group ll(Type IA CTx-5 16.38(3.59) 1456) cytotoxin)

[0589] 2.98(2.93) P01456(P0

[0590] 1463) Type IA cytotoxin Naja 50.99(50.24) P00605 Phospholipase A2 nigricincta 26.36(26.01) P14556 Phospholipase A2 (L1368) PLA2-I

[0591] South Africa 10.11(8.21) P0DSN1(P0

[0592] 1470) Type IA cytotoxin 68.59(68.44) P01468 Type IA cytotoxin CTx-6 24.66(24.52) P01456 Type IA cytotoxin 3.08(3.18) P0DSN1(P0

[0593] 1470) Type IA cytotoxin 39.47(42.32) P01452 Type IA cytotoxin CTx-7 30.05(28.63) P25517 Type IA cytotoxin Naja 29.59(28.21) P01456 Type IA cytotoxin nigricollis 46.35

[0594] (L1327) P00605 Phospholipase A2 Cameroon, PLA2-2 23.32 P01465 Type IA cytotoxin Tanzania,

[0595] West Africa 13.36 P01468 Type IA cytotoxin 69.44(65.15) P01468 Type IA cytotoxin CTx-8

[0596]

[0597] 24.22(22.81) P01456 Type IA cytotoxinP7584PC00

[0598] 67

[0599] 5.81(11.36) P0DSN1 Type IA cytotoxin 40.84(40.23) P14556 Phospholipase A2 PLA2-3 22.11(19.42) P0DSN1P0

[0600] 1470 Type IA cytotoxin Naja nubiae

[0601] (L1342) 16.71(16.46) P01456 Type IA cytotoxin Egypt 81.72(80.85) P01468 Type IA cytotoxin CTx-9 14.61(14.46) P01456 Type IA cytotoxin 3.28(4.21) P0DSN1 Type IA cytotoxin Naja pallida

[0602] (L1321) CTx-9 Purity >99% P01468 Type IA cytotoxin Kenya

[0603] 35.18(35.95) P01424 Type I alpha-neurotoxin sNTx-6 33.49(34.22) Q9YGJ6 Type I alpha-neurotoxin 26.37(27.08) P68418 Type I alpha-neurotoxin 66.93(67.25) P0DQQ2 Type II alpha-neurotoxin Naja INTx-4 24.52(24.65) P01400 Ophan group II melanoleuca

[0604] (L1318) 3.43 P01388 Type II alpha-neurotoxin Cameroon, 32.62(29.21) P01448 Type IA cytotoxin Ghana,

[0605] Uganda CTx-11 22.27(19.94) P01456 Type IA cytotoxin 21.01(18.81) P00986 Venom Kunitz-type 58.61(58.57) P01448 Type IA cytotoxin CTx-12 31.07(31.05) P01456 Type IA cytotoxin 7.84 P00600 Phospholipase A2 Naja 96.49(97.01) P01456 Type IA cytotoxin anchietae

[0606] (L1374) CTx-13

[0607] Namibia 1.12 P82462 Type C muscarinic toxin sNTx-7 98.38(98.29) P01421 Type I alpha-neurotoxin CTx-14 97.22(97.28) P01456 Type IA cytotoxin 48.43(48.04) P01463 Type IA cytotoxin CTx-15 35.37(35.80) P01456 Type IA cytotoxin Naja

[0608] annulifera 4.60(4.65) P01468 Type IA cytotoxin (L1314) 37.59(37.17) P01456 Type IA cytotoxin Sub- Saharan CTx-16 28.85(27.89) P01463 Type IA cytotoxin Africa 22.15(21.97) P01462 Type IA cytotoxin 52.14(51.05) P01463 Type IA cytotoxin CTx-17 13.03(11.80) P62394(P1

[0609] 4541) Orphan group XV 10.24(10.49) P01456 Type IA cytotoxin 54.85(55.65) P68418 Type I alpha-neurotoxin 39.49(39.90) P01421 Type I alpha-neurotoxin Naja haje sNTx-8

[0610] (L1315) 2.81(2.19) P01422(P0 Type I alpha-neurotoxin Egypt, Mali 1423)

[0611] 38.48(38.43) P01422 Type I alpha-neurotoxin sNTx-9

[0612]

[0613] 28.24(28.20) P01423 Type I alpha-neurotoxinP7584PC00

[0614] 68

[0615] 26.74(26.69) P68418 Type I alpha-neurotoxin 68.30(68.29) P01389 Type II alpha-neurotoxin INTx-5 17.82(17.73) P0DQQ2 Type II alpha-neurotoxin 5.14(5.15) P01400 Ophan group II Other3FTx

[0616] -3 98.00 P25678 Orphan group XX 47.95(46.93) P01421 Type I alpha-neurotoxin sNTx-10 43.83(43.04) P01390 Type II alpha-neurotoxin Naja nivea 3.47(3.40) P01400 Ophan group II (L1328)

[0617] South Africa 46.32(44.94) P01463 Type IA cytotoxin CTx-18 26.26(25.54) P01456 Type IA cytotoxin 8.79(8.54) P01468 Type IA cytotoxin 41.34(44.40) P01400 Ophan group II sNTx-11 20.47(21.83) P01421 Type I alpha-neurotoxin 10.15(10.88) P01389 Type II alpha-neurotoxin 36.84(36.85) P01389 Type II alpha-neurotoxin INTx-6 36.12(36.19) P01390 Type II alpha-neurotoxin Naja 11.87(11.89) P01400 Ophan group II senegalensis

[0618] (L1350) 59.03(63.99) P01456 Type IA cytotoxin Mali

[0619] CTx-19 25.61(27.57) P01457 Type IA cytotoxin 9.52(2.37) P25687(P0 AVIT (prokinecticin) family(Type 1452) IA cytotoxin) 54.33(54.42) P01462 Type IA cytotoxin CTx-20 18.21(17.81) P01456 Type IA cytotoxin 5.11(4.74) P01468 Type IA cytotoxin Naja

[0620] kaouthia

[0621] Southeast INTx-7 Purity >99% P01391 Type II alpha-neurotoxin Asia

[0622]

[0623] * The prevalence numbers (%) outside parenthesis are based on the renaming and rearranging of certain fractions.

[0624] Items

[0625] The present disclosure is further defined by any of the following items:

[0626] 1. A composition comprising:

[0627] • an antigen-binding protein ‘A’ capable of binding to, blocking, and / or neutralizing a snake venom Type IA cytotoxin;

[0628] • an antigen-binding protein ‘B’ capable of binding to, blocking, and / or neutralizing a snake venom Orphan group XI toxin;P7584PC00

[0629] 69

[0630] • an antigen-binding protein ‘C’ capable of binding to, blocking, and / or neutralizing a snake venom Orphan group XIX toxin and / or an aminergic toxin; and / or

[0631] • an antigen-binding protein ‘D’ capable of binding to, blocking, and / or neutralizing a snake venom Kunitz-type protease inhibitor toxin.

[0632] 2. A composition comprising:

[0633] • an antigen-binding protein ‘A’ capable of binding to, blocking, and / or neutralizing a snake venom Type IA or Type IB cytotoxin;

[0634] • an antigen-binding protein ‘B’ capable of binding to, blocking, and / or neutralizing a snake venom Orphan group XI toxin;

[0635] • an antigen-binding protein ‘C’ capable of binding to, blocking, and / or neutralizing a snake venom Orphan group XIX toxin and / or an aminergic toxin; and / or

[0636] • an antigen-binding protein ‘D’ capable of binding to, blocking, and / or neutralizing a snake venom Kunitz-type protease inhibitor toxin.

[0637] 3. The composition according to any of the preceding items, further comprising:

[0638] • an antigen-binding protein ‘E’ capable of binding to, blocking, and / or neutralizing a snake venom Type I a-neurotoxin (short-chain a- neurotoxin); and / or

[0639] • an antigen-binding protein ‘F’ capable of binding to, blocking, and / or neutralizing a snake venom Type II a-neurotoxin (long-chain a- neurotoxin); and / or

[0640] • an antigen-binding protein ‘G’ capable of binding to, blocking, and / or neutralizing a snake venom phospholipase A2 (PLA2) toxin.

[0641] 4. A composition comprising at least two of the following antigen-binding proteins:

[0642] • an antigen-binding protein ‘A’ capable of binding to, blocking, and / or neutralizing a snake venom Type IA or Type IB cytotoxin; and / or

[0643] • an antigen-binding protein ‘B’ capable of binding to, blocking, and / or neutralizing a snake venom Orphan group XI toxin; and / or

[0644] • an antigen-binding protein ‘C’ capable of binding to, blocking, and / or neutralizing a snake venom Orphan group XIX toxin and / or an aminergic toxin; and / orP7584PC00

[0645] 70

[0646] • an antigen-binding protein ‘D’ capable of binding to, blocking, and / or neutralizing a snake venom Kunitz-type protease inhibitor; and / or

[0647] • an antigen-binding protein ‘E’ capable of binding to, blocking, and / or neutralizing a snake venom Type I a-neurotoxin (short-chain a- neurotoxin); and / or

[0648] • an antigen-binding protein ‘F’ capable of binding to, blocking, and / or neutralizing a snake venom Type II a-neurotoxin (long-chain a- neurotoxin); and / or

[0649] • an antigen-binding protein ‘G’ capable of binding to, blocking, and / or neutralizing a snake venom phospholipase A2 (PLA2) toxin.

[0650] 5. The composition according to any of the preceding items, wherein the composition comprises at least two antigen-binding protein ‘As’, wherein each antigen-binding protein ‘A’ is capable of binding to, blocking, and / or neutralizing different snake venom Type I A or type IB cytotoxins.

[0651] 6. A composition comprising an antigen-binding protein ‘A’ capable of binding to, blocking, and / or neutralizing a snake venom Type IA or type IB cytotoxin and an antigen-binding protein ‘G’ capable of binding to, blocking, and / or neutralizing a snake venom phospholipase A2s (PLA2) toxin.

[0652] 7. The composition according to item 6, further comprising:

[0653] • an antigen-binding protein ‘E’ capable of binding to, blocking, and / or neutralizing a snake venom Type I a-neurotoxin (short-chain a- neurotoxin); and / or

[0654] • an antigen-binding protein ‘F’ capable of binding to, blocking, and / or neutralizing a snake venom Type II a-neurotoxin (long-chain a- neurotoxin); and / or

[0655] • an antigen-binding protein ‘B’ capable of binding to, blocking, and / or neutralizing a snake venom Orphan group XI toxin;

[0656] • an antigen-binding protein ‘C’ capable of binding to, blocking, and / or neutralizing a snake venom Orphan group XIX toxin and / or an aminergic toxin; and / or

[0657] • an antigen-binding protein ‘D’ capable of binding to, blocking, and / or neutralizing a snake venom Kunitz-type protease inhibitor toxin.P7584PC00

[0658] 71

[0659] 8. The composition according to any one of the preceding items for use in a method of neutralizing dermonecrosis associated with snakebite or snake envenomation in an individual in need thereof.

[0660] 9. The composition according to any one of the preceding items, wherein the Type IA or Type IB cytotoxin (CTx) is obtained from the venom of an elapid snake selected from Hemachatus haemachatus or species of the Naja genus, such as Naja ashei, Naja katiensis, Naja mossambica, Naja nigricincta, Naja nigricollis, Naja nubiae, Naja pallida, Naja melanoleuca, Naja anchietae, Naja annulifera, Naja nivea or Naja senegalensis.

[0661] 10. The composition according to any one of the preceding items, wherein the Orphan group XI toxin is obtained from the venom of an elapid snake selected from Dendroaspis j a meson i or Dendroaspis viridis.

[0662] 11. The composition according to any one of the preceding items, wherein the Orphan group XIX toxin and / or the aminergic toxin is obtained from the venom of an elapid snake selected from Dendroaspis jamesoni or Dendroaspis viridis.

[0663] 12. The composition according to any one of the preceding items, wherein the Kunitz-type protease inhibitor (KUN) toxin is obtained from the venom of an elapid snake selected from Dendroaspis jamesoni or Dendroaspis polylepis.

[0664] 13. The composition according to any one of the preceding items, wherein the Type I a-neurotoxin (short-chain a-neurotoxin) is obtained from the venom of an elapid snake selected from Dendroaspis jamesoni, Dendroaspis polylepis, Dendroaspis viridis, Hemachatus haemachatus or species of the Naja genus, such as Naja melanoleuca, Naja annulifera, Naja haje, Naja nivea or Naja senegalensis.

[0665] 14. The composition according to any one of the preceding items, wherein the Type II a-neurotoxin (long-chain a-neurotoxin) is obtained from the venom of an elapid snake selected from Dendroaspis polylepis or species of the Naja genus, such as Naja melanoleuca, Naja haje, Naja nivea, Naja senegalensis or Naja kaouthia.P7584PC00

[0666] 72

[0667] 15. The composition according to any one of the preceding items, wherein the phospholipase 2 (PLA2) toxin is obtained from the venom of an elapid snake selected from species of the Naja genus, such as Naja nigricincta, Naja nigricollis or Naja nubiae.

[0668] 16. The composition according to any one of the preceding items, wherein the antigen-binding protein ‘A’ binds to a snake venom toxin as set forth in SEQ ID NO: 1 or a functional homologue thereof sharing at least 70%, such as at least 80%, for example at least 90%, such as at least 95% sequence identity therewith.

[0669] 17. The composition according to any one of the preceding items, wherein the antigen-binding protein ‘A’ binds to a snake venom toxin as set forth in SEQ ID NO: 2 or a functional homologue thereof sharing at least 70%, such as at least 80%, for example at least 90%, such as at least 95% sequence identity therewith.

[0670] 18. The composition according to any one of the preceding items, wherein the antigen-binding protein ‘B’ binds to a snake venom toxin as set forth in SEQ ID NO: 3 or a functional homologue thereof sharing at least 70%, such as at least 80%, for example at least 90%, such as at least 95% sequence identity therewith.

[0671] 19. The composition according to any one of the preceding items, wherein the antigen-binding protein ‘C’ binds to a snake venom toxin as set forth in SEQ ID NO: 4 or a functional homologue thereof sharing at least 70%, such as at least 80%, for example at least 90%, such as at least 95% sequence identity therewith.

[0672] 20. The composition according to any one of the preceding items, wherein the antigen-binding protein ‘D’ binds to a snake venom toxin as set forth in SEQ ID NO: 5 or a functional homologue thereof sharing at least 70%, such as at least 80%, for example at least 90%, such as at least 95% sequence identity therewith.

[0673] 21. The composition according to any one of the preceding items, wherein the antigen-binding protein ‘E’ binds to a snake venom toxin as set forth in SEQ ID NO: 6 or a functional homologue thereof sharing at least 70%, such as at least 80%, for example at least 90%, such as at least 95% sequence identity therewith.P7584PC00

[0674] 73

[0675] 22. The composition according to any one of the preceding items, wherein the antigen-binding protein ‘E’ binds to a snake venom toxin as set forth in SEQ ID NO 7 or a functional homologue thereof sharing at least 70%, such as at least 80%, for example at least 90%, such as at least 95% sequence identity therewith.

[0676] 23. The composition according to any one of the preceding items, wherein the antigen-binding protein ‘E’ binds to a snake venom toxin as set forth in SEQ ID NO: 43 or a functional homologue thereof sharing at least 70%, such as at least 80%, for example at least 90%, such as at least 95% sequence identity therewith.

[0677] 24. The composition according to any one of the preceding items, wherein the antigen-binding protein ‘F’ binds to a snake venom toxin as set forth in SEQ ID NO: 8 or a functional homologue thereof sharing at least 70%, such as at least 80%, for example at least 90%, such as at least 95% sequence identity therewith.

[0678] 25. The composition according to any one of the preceding items, wherein the antigen-binding protein ‘G’ binds to a snake venom toxin as set forth in SEQ ID NO: 9 or a functional homologue thereof sharing at least 70%, such as at least 80%, for example at least 90%, such as at least 95% sequence identity therewith.

[0679] 26. The composition according to any one of the preceding items, wherein the antigen-binding protein ‘A’ binds an epitope of a snake venom Type IA or Type IB cytotoxin comprising the amino acid residues:

[0680] - 17, P8, P9, L6, and P33 of SEQ ID NO: 1;

[0681] - K44, R27, L48, S46, L47 and 151 of SEQ ID NO: 1; and / or

[0682] - P9, K5 and K35 of SEQ ID NO: 2.

[0683] 27. The composition according to any one of the preceding items, wherein the antigen-binding protein ‘A’ binds an epitope of a snake venom Type IA or Type IB cytotoxin comprising the amino acid residues:

[0684] - 17,, P8, P9, L6, and P33 of SEQ ID NO: 1; and / or

[0685] - K44, R27, L48, S46, L47 and 151 of SEQ ID NO: 1; and / or

[0686] - L6, I7, P8, P9, F10, R27, P33, K35, L47, L48 and K50 of SEQ ID NO: 1; and / or - L6, I7, P8, P9, F10, P33 and K35 of SEQ ID NO: 1; and / or

[0687] - R27, L47, L48 and K50 of SEQ ID NO: 1; and / orP7584PC00

[0688] 74

[0689] - P9, K5 and K35 of SEQ ID NO: 2; and / or

[0690] - K5, L6, P9, W11, K12, T13, K18, K35, and C38 of SEQ ID NO: 2.

[0691] 28. The composition according to any one of the preceding items, wherein the antigen-binding protein ‘B’ binds an epitope of a snake venom Orphan group XI toxin comprising the amino acid residues:

[0692] - P9, Q10, K7 and K48 of SEQ ID NO: 3.

[0693] 29. The composition according to any one of the preceding items, wherein the antigen-binding protein ‘B’ binds an epitope of a snake venom Orphan group XI toxin comprising the amino acid residues:

[0694] - P9, Q10, K7 and K48 of SEQ ID NO: 3; and / or

[0695] - K7, Q10, K24, K28, G31, R36 and K48 of SEQ ID NO:3.

[0696] 30. The composition according to any one of the preceding items, wherein the antigen-binding protein ‘C’ binds an epitope of an aminergic toxin comprising the amino acid residues:

[0697] - C46, K22, P47 and E15 of SEQ ID NO: 4.

[0698] 31. The composition according to any one of the preceding items, wherein the antigen-binding protein ‘C’ binds an epitope of an aminergic toxin comprising one or more amino acid residues from the group consisting of:

[0699] - C46, K22, P47 and E15 of SEQ ID NO: 4;and / or

[0700] - E15, Q21, K22, A44, T45, C46 and K48 of SEQ ID NO:4.

[0701] 32. The composition according to any one of the preceding items, wherein the antigen-binding protein ‘D’ binds an epitope of a snake venom Kunitz-type protease inhibitor comprising one or more amino acid residues selected from the group consisting of:

[0702] - R15, W37, R46, K48 and E52 of SEQ ID NO: 5.

[0703] 33. The composition according to any one of the preceding items, wherein the antigen-binding protein ‘E’ binds an epitope of a snake venom Type I a-neurotoxin (short-chain a-neurotoxin) comprising one or more amino acid residues selectedP7584PC00

[0704] 75

[0705] from the group consisting of:

[0706] - Y52, C53, 151, K47, S44, E20 and P43 of SEQ ID NO: 7.

[0707] 34. The composition according to any one of the preceding items, wherein the antigen-binding protein ‘E’ binds an epitope of a snake venom Type I a-neurotoxin (short-chain a-neurotoxin) comprising one or more amino acid residues selected from the group consisting of:

[0708] - Y52, C53, 151, K47, S44, E20 and P43 of SEQ ID NO: 7; and / or

[0709] - C42, T44, V45, L51, N52 and C53 of SEQ ID NO: 43.

[0710] 35. The composition according to any one of the preceding items, wherein the antigen-binding protein ‘F’ binds an epitope of a snake venom Type II a-neurotoxin (long-chain a-neurotoxin) comprising one or more amino acid residues selected from the group consisting of:

[0711] - R36, P7, D27 and R33 of SEQ ID NO: 8.

[0712] 36. The composition according to any one of the preceding items, wherein the antigen-binding protein ‘F’ binds an epitope of a snake venom Type II a-neurotoxin (long-chain a-neurotoxin) comprising one or more amino acid residues selected from the group consisting of:

[0713] - R36, P7, D27 and R33 of SEQ ID NO: 8; and / or

[0714] - 19, R33, R36, V37, F65 and R70 of SEQ ID:8.

[0715] 37. The composition according to any one of the preceding items, wherein the antigen-binding protein ‘G’ binds an epitope of a snake venom phospholipase A2s toxin comprising one or more amino acid residues selected from the group consisting of:

[0716] - Y3, F63, W60, E52, D48 and Y62 of SEQ ID NO: 9.

[0717] 38. The composition according to any one of the preceding items, wherein the antigen-binding protein ‘G’ binds an epitope of a snake venom phospholipase A2s toxin comprising one or more amino acid residues selected from the group consisting of:

[0718] - Y3, F63, W60, E52, D48 and Y62 of SEQ ID NO: 9; and / or

[0719] - N1, R30, D48, W60, Y62 and F63 of SEQ ID NO:9.P7584PC00

[0720] 76

[0721] 39. The composition according to any one of the preceding items, wherein the antigen-binding protein ‘A’ comprises or consists of a heavy chain variable (VH) region as set forth in SEQ ID NO: 10 or SEQ ID NO: 26, or a sequence identical thereto except for up to 2, such as up to 1 amino acid substitution(s).

[0722] 40. The composition according to any one of the preceding items, wherein the antigen-binding protein ‘A’ comprises or consists of a heavy chain variable (VH) region as set forth in SEQ ID NO: 57 or SEQ ID: 73, or a sequence identical thereto except for up to 2, such as up to 1 amino acid substitution(s).

[0723] 41. The composition according to any one of the preceding items, wherein the antigen-binding protein ‘B’ comprises or consists of a heavy chain variable (VH) region as set forth in SEQ ID NO: 14, or a sequence identical thereto except for up to 2, such as up to 1 amino acid substitution(s).

[0724] 42. The composition according to any one of the preceding items, wherein the antigen-binding protein ‘B’ comprises or consists of a heavy chain variable (VH) region as set forth in SEQ ID NO: 61, or a sequence identical thereto except for up to 2, such as up to 1 amino acid substitution(s).

[0725] 43. The composition according to any one of the preceding items, wherein the antigen-binding protein ‘C’ comprises or consists of a heavy chain variable (VH) region as set forth in SEQ ID NO: 18, or a sequence identical thereto except for up to 2, such as up to 1 amino acid substitution(s).

[0726] 44. The composition according to any one of the preceding items, wherein the antigen-binding protein ‘C’ comprises or consists of a heavy chain variable (VH) region as set forth in SEQ ID NO: 65, or a sequence identical thereto except for up to 2, such as up to 1 amino acid substitution(s).

[0727] 45. The composition according to any one of the preceding items, wherein the antigen-binding protein ‘D’ comprises or consists of a heavy chain variable (VH) region as set forth in SEQ ID NO: 22, or a sequence identical thereto except for up to 2, such as up to 1 amino acid substitution(s).P7584PC00

[0728] 77

[0729] 46. The composition according to any one of the preceding items, wherein the antigen-binding protein ‘D’ comprises or consists of a heavy chain variable (VH) region as set forth in SEQ ID NO: 69, or a sequence identical thereto except for up to 2, such as up to 1 amino acid substitution(s).

[0730] 47. The composition according to any one of the preceding items, wherein the antigen-binding protein ‘E’ comprises or consists of a heavy chain variable (VH) region as set forth in SEQ ID NO: 30, or a sequence identical thereto except for up to 2, such as up to 1 amino acid substitution(s).

[0731] 48. The composition according to any one of the preceding items, wherein the antigen-binding protein ‘E’ comprises or consists of a heavy chain variable (VH) region as set forth in SEQ ID NO: 77, or a sequence identical thereto except for up to 2, such as up to 1 amino acid substitution(s).

[0732] 49. The composition according to any one of the preceding items, wherein the antigen-binding protein ‘F’ comprises or consists of a heavy chain variable (VH) region as set forth in SEQ ID NO: 34, or a sequence identical thereto except for up to 2, such as up to 1 amino acid substitution(s).

[0733] 50. The composition according to any one of the preceding items, wherein the antigen-binding protein ‘F’ comprises or consists of a heavy chain variable (VH) region as set forth in SEQ ID NO: 81, or a sequence identical thereto except for up to 2, such as up to 1 amino acid substitution(s).

[0734] 51. The composition according to any one of the preceding items, wherein the antigen-binding protein ‘G’ comprises or consists of a heavy chain variable (VH) region as set forth in SEQ ID NO: 38, or a sequence identical thereto except for up to 2, such as up to 1 amino acid substitution(s).

[0735] 52. The composition according to any one of the preceding items, wherein the antigen-binding protein ‘G’ comprises or consists of a heavy chain variable (VH) region as set forth in SEQ ID NO: 85, or a sequence identical thereto except for up to 2, such as up to 1 amino acid substitution(s).P7584PC00

[0736] 78

[0737] 53. The composition according to any one of the preceding items, wherein at least one antigen-binding protein ‘A’ comprises complementary determining regions (CDRs) comprising or consisting of:

[0738] - a CDR1 as set forth in SEQ ID NO: 11 or a sequence identical thereto except for up to 2, such as up to 1 amino acid substitution(s); and

[0739] - a CDR2 as set forth in SEQ ID NO: 12 or a sequence identical thereto except for up to 2, such as up to 1 amino acid substitution(s); and

[0740] - a CDR3 as set forth in SEQ ID NO: 13 or a sequence identical thereto except for up to 2, such as up to 1 amino acid substitution(s);

[0741] or

[0742] - a CDR1 as set forth in SEQ ID NO: 27 or a sequence identical thereto except for up to 2, such as up to 1 amino acid substitution(s); and

[0743] - a CDR2 as set forth in SEQ ID NO: 28 or a sequence identical thereto except for up to 2, such as up to 1 amino acid substitution(s); and

[0744] - a CDR3 as set forth in SEQ ID NO: 29 or a sequence identical thereto except for up to 2, such as up to 1 amino acid substitution(s).

[0745] 54. The composition according to any one of the preceding items, wherein at least one antigen-binding protein ‘A’ comprises complementary determining regions (CDRs) comprising or consisting of:

[0746] - a CDR1 as set forth in SEQ ID NO: 58 or a sequence identical thereto except for up to 2, such as up to 1 amino acid substitution(s); and

[0747] - a CDR2 as set forth in SEQ ID NO: 59 or a sequence identical thereto except for up to 2, such as up to 1 amino acid substitution(s); and

[0748] - a CDR3 as set forth in SEQ ID NO: 60 or a sequence identical thereto except for up to 2, such as up to 1 amino acid substitution(s);

[0749] or

[0750] - a CDR1 as set forth in SEQ ID NO: 74 or a sequence identical thereto except for up to 2, such as up to 1 amino acid substitution(s); and

[0751] - a CDR2 as set forth in SEQ ID NO: 75 or a sequence identical thereto except for up to 2, such as up to 1 amino acid substitution(s); and

[0752] - a CDR3 as set forth in SEQ ID NO: 76 or a sequence identical thereto except for up to 2, such as up to 1 amino acid substitution(s).P7584PC00

[0753] 79

[0754] 55. The composition according to any one of the preceding items, wherein at least one antigen-binding protein ‘A’ comprises or consists of a heavy chain variable (VH) region comprising complementary determining regions (CDRs) comprising or consisting of:

[0755] - a CDR1 as set forth by SEQ ID NO: 11; and

[0756] - a CDR2 as set forth by SEQ ID NO: 12; and

[0757] - a CDR3 as set forth by SEQ ID NO: 13;

[0758] or

[0759] - a CDR1 as set forth by SEQ ID NO: 27; and

[0760] - a CDR2 as set forth by SEQ ID NO: 28; and

[0761] - a CDR3 as set forth by SEQ ID NO: 29.

[0762] 56. The composition according to any one of the preceding items, wherein at least one antigen-binding protein ‘A’ comprises or consists of a heavy chain variable (VH) region comprising complementary determining regions (CDRs) comprising or consisting of:

[0763] - a CDR1 as set forth by SEQ ID NO: 58; and

[0764] - a CDR2 as set forth by SEQ ID NO: 59; and

[0765] - a CDR3 as set forth by SEQ ID NO: 60;

[0766] or

[0767] - a CDR1 as set forth by SEQ ID NO: 74; and

[0768] - a CDR2 as set forth by SEQ ID NO: 75; and

[0769] - a CDR3 as set forth by SEQ ID NO: 76.

[0770] 57. The composition according to any one of the preceding items, wherein the antigen-binding protein ‘B’ comprises complementary determining regions (CDRs) comprising or consisting of:

[0771] - a CDR1 as set forth by SEQ ID NO: 15 or a sequence identical thereto except for up to 2, such as up to 1 amino acid substitution(s); and

[0772] - a CDR2 as set forth by SEQ ID NO: 16 or a sequence identical thereto except for up to 2, such as up to 1 amino acid substitution(s); and

[0773] - a CDR3 as set forth by SEQ ID NO: 17 or a sequence identical thereto except forP7584PC00

[0774] 80

[0775] up to 2, such as up to 1 amino acid substitution(s).

[0776] 58. The composition according to any one of the preceding items, wherein the antigen-binding protein ‘B’ comprises complementary determining regions (CDRs) comprising or consisting of:

[0777] - a CDR1 as set forth by SEQ ID NO: 62 or a sequence identical thereto except for up to 2, such as up to 1 amino acid substitution(s); and

[0778] - a CDR2 as set forth by SEQ ID NO: 63 or a sequence identical thereto except for up to 2, such as up to 1 amino acid substitution(s); and

[0779] - a CDR3 as set forth by SEQ ID NO: 64 or a sequence identical thereto except for up to 2, such as up to 1 amino acid substitution(s).

[0780] 59. The composition according to any one of the preceding items, wherein the antigen-binding protein ‘B’ comprises or consists of a heavy chain variable (VH) region comprising complementary determining regions (CDRs) comprising or consisting of:

[0781] - a CDR1 as set forth by SEQ ID NO: 15; and

[0782] - a CDR2 as set forth by SEQ ID NO: 16; and

[0783] - a CDR3 as set forth by SEQ ID NO: 17.

[0784] 60. The composition according to any one of the preceding items, wherein the antigen-binding protein ‘B’ comprises or consists of a heavy chain variable (VH) region comprising complementary determining regions (CDRs) comprising or consisting of:

[0785] - a CDR1 as set forth by SEQ ID NO: 62; and

[0786] - a CDR2 as set forth by SEQ ID NO: 63; and

[0787] - a CDR3 as set forth by SEQ ID NO: 64.

[0788] 61. The composition according to any one of the preceding items, wherein the antigen-binding protein ‘C’ comprises complementary determining regions (CDRs) comprising or consisting of:

[0789] - a CDR1 as set forth by SEQ ID NO: 19 or a sequence identical thereto except for up to 2, such as up to 1 amino acid substitution(s); and

[0790] - a CDR2 as set forth by SEQ ID NO: 20 or a sequence identical thereto except forP7584PC00

[0791] 81

[0792] up to 2, such as up to 1 amino acid substitution(s); and

[0793] - a CDR3 as set forth by SEQ ID NO: 21 or a sequence identical thereto except for up to 2, such as up to 1 amino acid substitution(s).

[0794] 62. The composition according to any one of the preceding items, wherein the antigen-binding protein ‘C’ comprises complementary determining regions (CDRs) comprising or consisting of:

[0795] - a CDR1 as set forth by SEQ ID NO: 66 or a sequence identical thereto except for up to 2, such as up to 1 amino acid substitution(s); and

[0796] - a CDR2 as set forth by SEQ ID NO: 67 or a sequence identical thereto except for up to 2, such as up to 1 amino acid substitution(s); and

[0797] - a CDR3 as set forth by SEQ ID NO: 68 or a sequence identical thereto except for up to 2, such as up to 1 amino acid substitution(s).

[0798] 63. The composition according to any one of the preceding items, wherein the antigen-binding protein ‘C’ comprises or consists of a heavy chain variable (VH) region comprising complementary determining regions (CDRs) comprising or consisting of:

[0799] - a CDR1 as set forth by SEQ ID NO: 19; and

[0800] - a CDR2 as set forth by SEQ ID NO: 20; and

[0801] - a CDR3 as set forth by SEQ ID NO: 21.

[0802] 64. The composition according to any one of the preceding items, wherein the antigen-binding protein ‘C’ comprises or consists of a heavy chain variable (VH) region comprising complementary determining regions (CDRs) comprising or consisting of:

[0803] - a CDR1 as set forth by SEQ ID NO: 66; and

[0804] - a CDR2 as set forth by SEQ ID NO: 67; and

[0805] - a CDR3 as set forth by SEQ ID NO: 68.

[0806] 65. The composition according to any one of the preceding items, wherein the antigen-binding protein ‘D’ comprises complementary determining regions (CDRs) comprising or consisting of:

[0807] - a CDR1 as set forth by SEQ ID NO: 23 or a sequence identical thereto except forP7584PC00

[0808] 82

[0809] up to 2, such as up to 1 amino acid substitution(s); and

[0810] - a CDR2 as set forth by SEQ ID NO: 24 or a sequence identical thereto except for up to 2, such as up to 1 amino acid substitution(s); and

[0811] - a CDR3 as set forth by SEQ ID NO: 25 or a sequence identical thereto except for up to 2, such as up to 1 amino acid substitution(s).

[0812] 66. The composition according to any one of the preceding items, wherein the antigen-binding protein ‘D’ comprises complementary determining regions (CDRs) comprising or consisting of:

[0813] - a CDR1 as set forth by SEQ ID NO: 70 or a sequence identical thereto except for up to 2, such as up to 1 amino acid substitution(s); and

[0814] - a CDR2 as set forth by SEQ ID NO: 71 or a sequence identical thereto except for up to 2, such as up to 1 amino acid substitution(s); and

[0815] - a CDR3 as set forth by SEQ ID NO: 72 or a sequence identical thereto except for up to 2, such as up to 1 amino acid substitution(s).

[0816] 67. The composition according to any one of the preceding items, wherein the antigen-binding protein ‘D’ comprises or consists of a heavy chain variable (VH) region comprising complementary determining regions (CDRs) comprising or consisting of:

[0817] - a CDR1 as set forth by SEQ ID NO: 23; and

[0818] - a CDR2 as set forth by SEQ ID NO: 24; and

[0819] - a CDR3 as set forth by SEQ ID NO: 25.

[0820] 68. The composition according to any one of the preceding items, wherein the antigen-binding protein ‘D’ comprises or consists of a heavy chain variable (VH) region comprising complementary determining regions (CDRs) comprising or consisting of:

[0821] - a CDR1 as set forth by SEQ ID NO: 70; and

[0822] - a CDR2 as set forth by SEQ ID NO: 71; and

[0823] - a CDR3 as set forth by SEQ ID NO: 72.P7584PC00

[0824] 83

[0825] 69. The composition according to any one of the preceding items, wherein the antigen-binding protein ‘E’ comprises complementary determining regions (CDRs) comprising or consisting of:

[0826] - a CDR1 as set forth by SEQ ID NO: 31 or a sequence identical thereto except for up to 2, such as up to 1 amino acid substitution(s); and

[0827] - a CDR2 as set forth by SEQ ID NO: 32 or a sequence identical thereto except for up to 2, such as up to 1 amino acid substitution(s); and

[0828] - a CDR3 as set forth by SEQ ID NO: 33 or a sequence identical thereto except for up to 2, such as up to 1 amino acid substitution(s).

[0829] 70. The composition according to any one of the preceding items, wherein the antigen-binding protein ‘E’ comprises complementary determining regions (CDRs) comprising or consisting of:

[0830] - a CDR1 as set forth by SEQ ID NO: 78 or a sequence identical thereto except for up to 2, such as up to 1 amino acid substitution(s); and

[0831] - a CDR2 as set forth by SEQ ID NO: 79 or a sequence identical thereto except for up to 2, such as up to 1 amino acid substitution(s); and

[0832] - a CDR3 as set forth by SEQ ID NO: 80 or a sequence identical thereto except for up to 2, such as up to 1 amino acid substitution(s);

[0833] 71. The composition according to any one of the preceding items, wherein the antigen-binding protein ‘E’ comprises or consists of a heavy chain variable (VH) region comprising complementary determining regions (CDRs) comprising or consisting of:

[0834] - a CDR1 as set forth by SEQ ID NO: 31; and

[0835] - a CDR2 as set forth by SEQ ID NO: 32; and

[0836] - a CDR3 as set forth by SEQ ID NO: 33.

[0837] 72. The composition according to any one of the preceding items, wherein the antigen-binding protein ‘E’ comprises or consists of a heavy chain variable (VH) region comprising complementary determining regions (CDRs) comprising or consisting of:

[0838] - a CDR1 as set forth by SEQ ID NO: 78; and

[0839] - a CDR2 as set forth by SEQ ID NO: 79; and

[0840] - a CDR3 as set forth by SEQ ID NO: 80.P7584PC00

[0841] 84

[0842] 73. The composition according to any one of the preceding items, wherein the antigen-binding protein ‘F’ comprises complementary determining regions (CDRs) comprising or consisting of:

[0843] - a CDR1 as set forth by SEQ ID NO: 35 or a sequence identical thereto except for up to 2, such as up to 1 amino acid substitution(s); and

[0844] - a CDR2 as set forth by SEQ ID NO: 36 or a sequence identical thereto except for up to 2, such as up to 1 amino acid substitution(s); and

[0845] - a CDR3 as set forth by SEQ ID NO: 37 or a sequence identical thereto except for up to 2, such as up to 1 amino acid substitution(s).

[0846] 74. The composition according to any one of the preceding items, wherein the antigen-binding protein ‘F’ comprises complementary determining regions (CDRs) comprising or consisting of:

[0847] - a CDR1 as set forth by SEQ ID NO: 82 or a sequence identical thereto except for up to 2, such as up to 1 amino acid substitution(s); and

[0848] - a CDR2 as set forth by SEQ ID NO: 83 or a sequence identical thereto except for up to 2, such as up to 1 amino acid substitution(s); and

[0849] - a CDR3 as set forth by SEQ ID NO: 84 or a sequence identical thereto except for up to 2, such as up to 1 amino acid substitution(s).

[0850] 75. The composition according to any one of the preceding items, wherein the antigen-binding protein ‘F’ comprises or consists of a heavy chain variable (VH) region comprising complementary determining regions (CDRs) comprising or consisting of:

[0851] - a CDR1 as set forth by SEQ ID NO: 35; and

[0852] - a CDR2 as set forth by SEQ ID NO: 36; and

[0853] - a CDR3 as set forth by SEQ ID NO: 37.

[0854] 76. The composition according to any one of the preceding items, wherein the antigen-binding protein ‘F’ comprises or consists of a heavy chain variable (VH) region comprising complementary determining regions (CDRs) comprising or consisting of:

[0855] - a CDR1 as set forth by SEQ ID NO: 82; andP7584PC00

[0856] 85

[0857] - a CDR2 as set forth by SEQ ID NO: 83; and

[0858] - a CDR3 as set forth by SEQ ID NO: 84.

[0859] 77. The composition according to any one of the preceding items, wherein the antigen-binding protein ‘G’ comprises complementary determining regions (CDRs) comprising or consisting of:

[0860] - a CDR1 as set forth by SEQ ID NO: 39 or a sequence identical thereto except for up to 2, such as up to 1 amino acid substitution(s); and

[0861] - a CDR2 as set forth by SEQ ID NO: 40 or a sequence identical thereto except for up to 2, such as up to 1 amino acid substitution(s); and

[0862] - a CDR3 as set forth by SEQ ID NO: 41 or a sequence identical thereto except for up to 2, such as up to 1 amino acid substitution(s).

[0863] 78. The composition according to any one of the preceding items, wherein the antigen-binding protein ‘G’ comprises complementary determining regions (CDRs) comprising or consisting of:

[0864] - a CDR1 as set forth by SEQ ID NO: 86 or a sequence identical thereto except for up to 2, such as up to 1 amino acid substitution(s); and

[0865] - a CDR2 as set forth by SEQ ID NO: 87 or a sequence identical thereto except for up to 2, such as up to 1 amino acid substitution(s); and

[0866] - a CDR3 as set forth by SEQ ID NO: 88 or a sequence identical thereto except for up to 2, such as up to 1 amino acid substitution(s).

[0867] 79. The composition according to any one of the preceding items, wherein the antigen-binding protein ‘G’ comprises or consists of a heavy chain variable (VH) region comprising complementary determining regions (CDRs) comprising or consisting of:

[0868] - a CDR1 as set forth by SEQ ID NO: 39; and

[0869] - a CDR2 as set forth by SEQ ID NO: 40; and

[0870] - a CDR3 as set forth by SEQ ID NO: 41.

[0871] 80. The composition according to any one of the preceding items, wherein the antigen-binding protein ‘G’ comprises or consists of a heavy chain variable (VH) region comprising complementary determining regions (CDRs) comprising or consisting of:P7584PC00

[0872] 86

[0873] - a CDR1 as set forth by SEQ ID NO: 86; and

[0874] - a CDR2 as set forth by SEQ ID NO: 87; and

[0875] - a CDR3 as set forth by SEQ ID NO: 88.

[0876] 81. The composition according to any one of the preceding items, wherein the composition is capable of neutralizing snake venom Type IA or Type IB cytotoxin lethality.

[0877] 82. The composition according to any one of the preceding items, wherein the composition is capable of neutralizing snake venom Orphan group XI toxin lethality.

[0878] 83. The composition according to any one of the preceding items, wherein the composition is capable of neutralizing snake venom Orphan group XIX toxin and / or aminergic toxin lethality.

[0879] 84. The composition according to any one of the preceding items, wherein the composition is capable of neutralizing snake venom Kunitz-type protease inhibitor lethality.

[0880] 85. The composition according to any one of the preceding items, wherein the composition is capable of neutralizing snake venom Type I a-neurotoxin (shortchain a-neurotoxin) lethality.

[0881] 86. The composition according to any one of the preceding items, wherein the composition is capable of neutralizing snake venom Type II a-neurotoxin (long-chain a-neurotoxin) lethality.

[0882] 87. The composition according to any one of the preceding items, wherein the composition is capable of neutralizing snake venom phospholipase A2 lethality.

[0883] 88. The composition according to any of the preceding items comprising at least two, such as at least 3, for example at least 4, such as at least 5, for example at least 6, such as at least 7, for example at least 8 antigen-binding proteins capable of binding to at least two, such as at least 3, for example at least 4, such as at leastP7584PC00

[0884] 87

[0885] 5, for example at least 6, such as at least 7, for example at least 8 different snake venom toxins.

[0886] 89. The composition according to any of the preceding items comprising antigenbinding proteins ‘A’, ‘B’, ‘C’, ‘D’, ‘E’, ‘F’ and ‘G’.

[0887] 90. The composition according to any of the preceding items comprising antigenbinding proteins ‘B’, ‘O’, ‘D’, ‘E’ and ‘F’.

[0888] 91. The composition according to any of the preceding items comprising antigenbinding proteins ‘A’, and ‘E’.

[0889] 92. The composition according to any of the preceding items comprising antigenbinding proteins ‘A’, ‘E’ and ‘F’.

[0890] 93. The composition according to any of the preceding items comprising antigenbinding proteins ‘D’, ‘E’ and ‘F’.

[0891] 94. The composition according to any of the preceding items comprising antigenbinding proteins ‘B’, ‘C’ and ‘E’.

[0892] 95. The composition according to any of the preceding items comprising antigenbinding proteins ‘B’, ‘O’, ‘E’ and ‘F’.

[0893] 96. The composition according to any of the preceding items comprising antigenbinding proteins ‘A’, ‘B’, ‘O’, ‘E’, ‘D’ and ‘F’.

[0894] 97. The composition according to any of the preceding items comprising antigenbinding proteins ‘A’ and ‘G’.

[0895] 98. The composition according to any of the preceding items comprising antigenbinding proteins ‘E’ and ‘F’.

[0896] 99. The composition according to any one of the preceding items comprising antigen-binding proteins ‘B’, ‘E’, and ‘F’.P7584PC00

[0897] 88

[0898] 100. The composition according to any one of the preceding items comprising antigen-binding proteins ‘B’, ‘C’, ‘D’, and ‘F’.

[0899] 101. The composition according to any one of the preceding items, where at least one of the antigen-binding proteins, for example all of the antigen-binding proteins, is (are) monoclonal antigen binding protein(s).

[0900] 102. The composition according to any one of the preceding items, where at least one of the antigen-binding proteins, for example all of the antigen-binding proteins, is (are) human.

[0901] 103. The composition according to any one of the preceding items, where at least one of the antigen-binding proteins, for example all of the antigen-binding proteins, is (are) humanized.

[0902] 104. The composition according to any one of the preceding items, where at least one of the antigen-binding proteins, for example all of the antigen-binding proteins, is (are) a VHH(S), single domain antibodies or nanobodies.

[0903] 105. The composition according to any one of the preceding items, where at least one of the antigen-binding proteins, for example all of the antigen-binding proteins, is (are) VHH-FC fusion(s).

[0904] 106. The composition according to any one of the preceding items, where at least one of the antigen-binding proteins, for example all of the antigen-binding proteins, is (are) multi-specific.

[0905] 107. The composition according to any one of the preceding items, where at least one of the antigen-binding proteins, for example all of the antigen-binding proteins, is (are) albumin- VHH(S) fusions.P7584PC00

[0906] 89

[0907] 108. The composition according to any one of the preceding items, where at least one of the antigen-binding proteins, for example all of the antigen-binding proteins, is (are) VHHS fusions, wherein at least one VHH binds to albumin and at least one other VHH binds to a snake toxin.

[0908] 109. The composition according to any one of the preceding items, where at least one of the antigen-binding proteins, for example all of the antigen-binding proteins, has (have) one or more modification(s) selected from the group consisting of PEGylation, polysialylation, Fc region mutation, and N-glycosylation.

[0909] 110. The composition according to any one of the preceding items, where at least one of the antigen-binding proteins, for example all of the antigen-binding proteins, comprise(s) a detection label.

[0910] 111. The composition according to any one of the preceding items, wherein said antigen-binding protein ‘A’ is capable of binding said snake venom Type IA or Type IB cytotoxin; and / or

[0911] said antigen-binding protein ‘B’ is capable of binding said snake venom Orphan group XI toxin; and / or

[0912] said antigen-binding protein ‘C’ is capable of binding said snake venom Orphan group XIX toxin and / or an aminergic toxin; and / or

[0913] said antigen-binding protein ‘D’ is capable of binding said snake venom Kunitz- type protease inhibitor; and / or

[0914] said antigen-binding protein ‘E’ is capable of binding said snake venom Type I a-neurotoxin (short-chain a-neurotoxin); and / or

[0915] said antigen-binding protein ‘F’ is capable of binding said snake venom Type II a-neurotoxin (long-chain a-neurotoxin); and / or

[0916] said antigen-binding protein ‘G’ is capable of binding said snake venom phospholipase A2 (PLA2) toxin.

[0917] 112. The composition according to any one of the preceding items, wherein said antigen-binding protein ‘A’ is capable of blocking said snake venom Type IA or Type IB cytotoxin; and / or

[0918] said antigen-binding protein ‘B’ is capable of blocking said snake venom Orphan group XI toxin; and / orP7584PC00

[0919] 90

[0920] said antigen-binding protein ‘C’ is capable of blocking said snake venom Orphan group XIX toxin and / or an aminergic toxin; and / or

[0921] said antigen-binding protein ‘D’ is capable of blocking said snake venom Kunitz- type protease inhibitor; and / or

[0922] said antigen-binding protein ‘E’ is capable of blocking said snake venom Type I a-neurotoxin (short-chain a-neurotoxin); and / or

[0923] said antigen-binding protein ‘F’ is capable of blocking said snake venom Type II a-neurotoxin (long-chain a-neurotoxin); and / or

[0924] said antigen-binding protein ‘G’ is capable of blocking said snake venom phospholipase A2 (PLA2) toxin.

[0925] 113. The composition according to any one of the preceding items, wherein said antigen-binding protein ‘A’ is capable of neutralizing said snake venom Type IA or Type IB cytotoxin; and / or

[0926] said antigen-binding protein ‘B’ is capable of neutralizing said snake venom Orphan group XI toxin; and / or

[0927] said antigen-binding protein ‘C’ is capable of neutralizing said snake venom Orphan group XIX toxin and / or an aminergic toxin; and / or

[0928] said antigen-binding protein ‘D’ is capable of neutralizing said snake venom Kunitz-type protease inhibitor; and / or

[0929] said antigen-binding protein ‘E’ is capable of neutralizing said snake venom Type I a-neurotoxin (short-chain a-neurotoxin); and / or

[0930] said antigen-binding protein ‘F’ is capable of neutralizing said snake venom Type II a-neurotoxin (long-chain a-neurotoxin); and / or

[0931] said antigen-binding protein ‘G’ is capable of neutralizing said snake venom phospholipase A2 (PLA2) toxin.

[0932] 114. The composition according to any one of the preceding items, wherein said antigen-binding protein ‘A’ is capable of binding only said snake venom Type IA or Type IB cytotoxin; and / or

[0933] said antigen-binding protein ‘B’ is capable of binding only said snake venom Orphan group XI toxin; and / or

[0934] said antigen-binding protein ‘C’ is capable of binding only said snake venom Orphan group XIX toxin and / or an aminergic toxin; and / orP7584PC00

[0935] 91

[0936] said antigen-binding protein ‘D’ is capable of binding only said snake venom Kunitz-type protease inhibitor; and / or

[0937] said antigen-binding protein ‘E’ is capable of binding only said snake venom Type I a-neurotoxin (short-chain a-neurotoxin); and / or

[0938] said antigen-binding protein ‘F’ is capable of binding only said snake venom Type II a-neurotoxin (long-chain a-neurotoxin); and / or

[0939] said antigen-binding protein ‘G’ is capable of binding only said snake venom phospholipase A2 (PLA2) toxin.

[0940] 115. The composition according to any one of the preceding items, wherein said antigen-binding protein ‘A’ is capable of blocking only said snake venom Type IA or Type IB cytotoxin; and / or

[0941] said antigen-binding protein ‘B’ is capable of blocking only said snake venom Orphan group XI toxin; and / or

[0942] said antigen-binding protein ‘C’ is capable of blocking only said snake venom Orphan group XIX toxin and / or an aminergic toxin; and / or

[0943] said antigen-binding protein ‘D’ is capable of blocking only said snake venom Kunitz-type protease inhibitor; and / or

[0944] said antigen-binding protein ‘E’ is capable of blocking only said snake venom Type I a-neurotoxin (short-chain a-neurotoxin); and / or

[0945] said antigen-binding protein ‘F’ is capable of blocking only said snake venom Type II a-neurotoxin (long-chain a-neurotoxin); and / or

[0946] said antigen-binding protein ‘G’ is capable of blocking only said snake venom phospholipase A2 (PLA2) toxin.

[0947] 116. The composition according to any one of the preceding items, wherein said antigen-binding protein ‘A’ is capable of neutralizing only said snake venom Type IA or Type IB cytotoxin; and / or

[0948] said antigen-binding protein ‘B’ is capable of neutralizing only said snake venom Orphan group XI toxin; and / or

[0949] said antigen-binding protein ‘C’ is capable of neutralizing only said snake venom Orphan group XIX toxin and / or an aminergic toxin; and / or said antigen-binding protein ‘D’ is capable of neutralizing only said snake venom Kunitz-type protease inhibitor; and / orP7584PC00

[0950] 92

[0951] said antigen-binding protein ‘E’ is capable of neutralizing only said snake venom Type I a-neurotoxin (short-chain a-neurotoxin); and / or

[0952] said antigen-binding protein ‘F’ is capable of neutralizing only said snake venom Type II a-neurotoxin (long-chain a-neurotoxin); and / or

[0953] said antigen-binding protein ‘G’ is capable of neutralizing only said snake venom phospholipase A2 (PLA2) toxin.

[0954] 117. The composition according to any one of the preceding items,

[0955] a) wherein the antigen-binding protein ‘A’ is not capable of binding to, blocking, and / or neutralizing a non-snake venom polypeptide; and / or b) the antigen-binding protein ‘B’ is not capable of binding to, blocking, and / or neutralizing a non-snake venom polypeptide; and / or c) the antigen-binding protein ‘C’ is not capable of binding to, blocking, and / or neutralizing a non-snake venom polypeptide; and / or d) the antigen-binding protein ‘D’ is not capable of binding to, blocking, and / or neutralizing a non-snake venom polypeptide; and / or e) the antigen-binding protein ‘E’ is not capable of binding to, blocking, and / or neutralizing a non-snake venom polypeptide; and / or f) the antigen-binding protein ‘F’ is not capable of binding to, blocking, and / or neutralizing a non-snake venom polypeptide; and / or g) the antigen-binding protein ‘G’ is not capable of binding to, blocking, and / or neutralizing a non-snake venom polypeptide.

[0956] 118. The composition according to any one of the preceding items,

[0957] a) the antigen-binding protein ‘A’ does not bind, block, and / or neutralize a non-snake venom polypeptide; and / or

[0958] b) the antigen-binding protein ‘B’ does not bind, block, and / or neutralize a non-snake venom polypeptide; and / or

[0959] c) the antigen-binding protein ‘C’ does not bind, block, and / or neutralize a non-snake venom polypeptide; and / or

[0960] d) the antigen-binding protein ‘D’ does not bind, block, and / or neutralize a non-snake venom polypeptide; and / or

[0961] e) the antigen-binding protein ‘E’ does not bind, block, and / or neutralize a non-snake venom polypeptide; and / orP7584PC00

[0962] 93

[0963] f) the antigen-binding protein ‘F’ does not bind, block, and / or neutralize a non-snake venom polypeptide; and / or

[0964] g) the antigen-binding protein ‘G’ does not bind, block, and / or neutralize a non-snake venom polypeptide.

[0965] 119. The composition according to any one of the preceding items, where at least one of the antigen-binding proteins, for example all of the antigen-binding proteins, is (are) not capable of binding to, blocking, and / or neutralizing a non- snake venom polypeptide under conditions wherein said at least one of the antigen-binding proteins, for example all of the antigen-binding proteins, is (are) capable of binding to, blocking, and / or neutralizing the snake venom toxin.

[0966] 120. The composition according to any one of the preceding items comprising antigen-binding proteins ‘F’, ‘A’, and ‘G’.

[0967] 121. The composition according to any one of the preceding items comprising antigen-binding proteins ‘F’, ‘E’, and ‘G’.

[0968] 122. The composition according to any one of the preceding items comprising antigen-binding proteins ‘E’, and ‘G’.

[0969] 123. The composition according to any one of the preceding items, where at least one of the antigen-binding proteins, for example all of the antigen-binding proteins, is (are) not capable of binding to, blocking, and / or neutralizing nontoxin antigens.

[0970] 124. The composition according to any one of the preceding items, where at least one of the antigen-binding proteins, for example all of the antigen-binding proteins, has (have) an apparent melting temperature above 55°C as determined by differential scanning fluorimetry.

[0971] 125. The composition according to any one of the preceding items, where at least one of the antigen-binding proteins, for example all of the antigen-binding proteins, has (have) an apparent melting temperature of above 58 °C as determined by differential scanning fluorimetry.P7584PC00

[0972] 94

[0973] 126. The composition according to any one of the preceding items, where at least one of the antigen-binding proteins, for example all of the antigen-binding proteins, has (have) an apparent onset of aggregation above 45°C as determined by light scattering, such as above 50°C, such as above 55°C, or such as above 60°C as determined by light scattering.

[0974] 127. The composition according to any one of the preceding items, where at least one of the antigen-binding proteins, for example all of the antigen-binding proteins, has (have) an apparent onset of aggregation above 65 °C as determined by light scattering.

[0975] 128. The composition according to any one of the preceding items, wherein that less than 5% of said antigen-binding protein aggregate upon incubation at 40 °C for 16 h as determined by dynamic light scattering, such as wherein less than 4% of said antigen-binding protein aggregate, such as wherein less than 3% of said antigen-binding protein aggregate, or such as wherein less than 2% of said antigen-binding protein aggregate.

[0976] 129. The composition according to any one of the preceding items, wherein less than 5% of said antigen-binding protein aggregate upon incubation at 40 °C for 5 h as determined by dynamic light scattering, such as wherein less than 4% of said antigen-binding protein aggregate, such as wherein less than 3% of said antigen-binding protein aggregate, or such as wherein less than 2% of said antigen-binding protein aggregate, or such as wherein less than 1% of said antigen-binding protein aggregate.

[0977] 130. A multi-specific antigen-binding protein, comprising or consisting of at least two of the following:

[0978] a) an antigen-binding protein ‘A’ capable of binding to, blocking, and / or neutralizing a snake venom Type IA or Type IB cytotoxin; and / or

[0979] • an antigen-binding protein ‘B’ capable of binding to, blocking, and / or neutralizing a snake venom Orphan group XI toxin; and / or

[0980] • an antigen-binding protein ‘C’ capable of binding to, blocking, and / or neutralizing a snake venom Orphan group XIX toxin and / or an aminergic toxin; and / orP7584PC00

[0981] 95

[0982] • an antigen-binding protein ‘D’ capable of binding to, blocking, and / or neutralizing a snake venom Kunitz-type protease inhibitor; and / or

[0983] • an antigen-binding protein ‘E’ capable of binding to, blocking, and / or neutralizing a snake venom Type I a-neurotoxin (short-chain a- neurotoxin); and / or

[0984] • an antigen-binding protein ‘F’ capable of binding to, blocking, and / or neutralizing a snake venom Type II a-neurotoxin (long-chain a- neurotoxin); and / or

[0985] • an antigen-binding protein ‘G’ capable of binding to, blocking, and / or neutralizing a snake venom phospholipase A2 (PLA2) toxin.

[0986] 131. The multi-specific antigen-binding protein according to item 130, wherein: a) the antigen-binding protein ‘A’ is as defined in any one of items 1, 2, 4 to 6, 16, 17, 26, 27, 39, 40, 53, 54, 55 and 56,

[0987] b) the antigen-binding protein ‘B’ is as defined in any one of items 1, 2, 4, 7, 18, 28, 29, 41, 42, 57, 58, 59 and 60,

[0988] c) the antigen-binding protein ‘C’ is as defined in any one of items 1, 2, 4, 7, 19, 30, 31, 43, 43, 61, 62, 63, and 64,

[0989] d) the antigen-binding protein ‘D’ is as defined in any one of items 1, 2, 4, 7, 20, 32, 32, 45, 46, 65, 66, 67, and 68,

[0990] e) the antigen-binding protein ‘E’ is as defined in any one of items 3, 4, 7, 21, 22, 23, 33, 34, 47, 48, 69, 70, 71, and 72,

[0991] f) the antigen-binding protein ‘F’ is as defined in any one of items 3, 4, 7, 24, 35, 36, 49, 49, 73, 74, 75, and 76,

[0992] g) the antigen-binding protein ‘G’ is as defined in any one of items 3, 4,6, 7, 25, 37, 38, 50, 52, 77, 78, 79, and 79

[0993] 132. A kit of parts comprising:

[0994] a) the composition according to any one of items 1 to 131; and

[0995] b) another agent suitable for the treatment of snakebite or snake envenomation.

[0996] 133. A composition, a multi-specific antigen-binding protein or a kit-of-parts according to any one of the preceding items for use in a method of treating snakebite or snake envenomation in an individual in need thereof.P7584PC00

[0997] 96

[0998] 134. A method for treating snakebite or snake envenomation in an individual in need thereof, the method comprising administering a therapeutically effective amount of the composition, the multi-specific antigen-binding protein, or the kit of parts according to any one of items 1 to 133 to said individual.

[0999] 135. Use of the antigen binding protein, the multi-specific antigen-binding protein, or the kit of parts according to any one of items 1 to 132 for the preparation of a medicament for treatment of snakebite or snake envenomation in an individual in need thereof.

[1000] 136. The composition, method or use according to any one of items 133 to 135, wherein the method comprises neutralizing dermonecrosis associated with said snakebite or snake envenomation.

[1001] 137. The composition, method or use according to any one of items 1 to 136, wherein the snakebite is a bite from an elapid snake.

[1002] 138. The composition, method or use according to item 137, wherein the snakebite is a bite from an elapid snake selected from Hemachatus haemachatus, species of the Naja genus, such as Naja ashei, Naja haje, Naja katiensis, Naja kaouthia, Naja mossambica, Naja nigricincta, Naja nigricollis, Naja nubiae, Naja pallida, Naja melanoleuca, Naja anchietae, Naja annulifera, Naja nivea or Naja senegalensis, Dendroaspis jamesoni, Dendroaspis viridis and Dendroaspis polylepis.

[1003] 139. The composition, method, kit, use or multi-specific antigen-binding protein according to item 137, wherein the elapid snake is Hemachatus haemachatus or Naja ashei or Naja haje or Naja katiensis or Naja kaouthia or Naja mossambica or Naja nigricincta or Naja nigricollis or Naja nubiae or Naja pallida or Naja melanoleuca or Naja anchietae or Naja annulifera or Naja nivea or Naja senegalensis or Dendroaspis angusticeps or Dendroaspis jamesoni or Dendroaspis viridis or Dendroaspis polylepis.

[1004] 140. The composition, method, kit, use or multi-specific antigen-binding protein according to item 137, wherein the elapid snake is Hemachatus haemachatus or Naja ashei or Naja haje or Naja katiensis or Naja mossambica or Naja nigricollis or Naja nubiae or Naja pallida or Naja melanoleuca or Naja anchietae or NajaP7584PC00

[1005] 97

[1006] annulifera or Naja nivea or Naja senegalensis or Dendroaspis angusticeps or Dendroaspis jamesoni or Dendroaspis viridis or Dendroaspis polylepis.

[1007] 141. The composition, method, kit, use or multi-specific antigen-binding protein according to item 137, wherein the elapid snake is Dendroaspis jamesoni or Dendroaspis viridis or Naja haje or Naja melanoleuca or Hemachatus haemachatus.

[1008] 142. The composition, method or use according to item 137, wherein the snakebite is a bite from an elapid snake selected from Hemachatus haemachatus, species of the Naja genus, such as Naja ashei, Naja haje, Naja katiensis, Naja kaouthia, Naja mossambica, Naja nigricincta, Naja nigricollis, Naja nubiae, Naja pallida, Naja melanoleuca, Naja anchietae, Naja annulifera, Naja nivea, Naja naja, or Naja senegalensis, Dendroaspis jamesoni, Dendroaspis viridis, Dendroaspis polylepis, Ophiophagus hannah, Ophiophagus kaalinga, and Bungarus spp.

[1009] 143. The composition, method, kit, use or multi-specific antigen-binding protein according to item 137, wherein the elapid snake is Hemachatus haemachatus or Naja ashei or Naja haje or Naja katiensis or Naja kaouthia or Naja mossambica or Naja nigricincta or Naja nigricollis or Naja nubiae or Naja pallida or Naja naja or Naja melanoleuca or Naja anchietae or Naja annulifera or Naja nivea or Naja senegalensis or Dendroaspis angusticeps or Dendroaspis jamesoni or Dendroaspis viridis or Dendroaspis polylepis or Ophiophagus hannah or Ophiophagus kaalinga, or Bungarus spp.

[1010] 144. The composition, method, kit, use or multi-specific antigen-binding protein according to item 137, wherein the elapid snake is Hemachatus haemachatus or Naja ashei or Naja haje or Naja katiensis or Naja mossambica or Naja nigricollis or Naja nubiae or Naja pallida or Naja melanoleuca or Naja anchietae or Naja annulifera or Naja nivea or Naja senegalensis or Naja naja or Dendroaspis angusticeps or Dendroaspis jamesoni or Dendroaspis viridis or Dendroaspis polylepis or Ophiophagus hannah or Ophiophagus kaalinga, or Bungarus spp.

[1011] 145. The composition, method, kit, use or multi-specific antigen-binding protein according to item 137, wherein the elapid snake is Dendroaspis jamesoni or Dendroaspis viridis or Naja haje or Naja melanoleuca or Hemachatus haemachatusP7584PC00

[1012] 98

[1013] or Naja naja or Ophiophagus hannah or Ophiophagus kaalinga or Naja kaouthia or Bungarus spp.

[1014] 146. The composition, method, kit, use or multi-specific antigen-binding protein according to item 137, wherein the elapid snake is Naja naja, Ophiophagus hannah, Ophiophagus kaalinga, Naja kaouthia or Bungarus spp.

[1015] 147. The composition, method, kit, use or multi-specific antigen-binding protein according to item 137, wherein the elapid snake is Hemachatus haemachatus, species of the Naja genus, such as Naja ashei, Naja haje, Naja katiensis, Naja mossambica, Naja nigricincta, Naja nigricollis, Naja nubiae, Naja pallida, Naja melanoleuca, Naja anchietae, Naja annulifera, Naja nivea, or Naja senegalensis, or Dendroaspis jamesoni, Dendroaspis viridis, or Dendroaspis polylepis.

[1016] 148. The composition, method, kit, use or multi-specific antigen-binding protein according to item 137, wherein the elapid snake is Hemachatus haemachatus, species of the Naja genus, such as Naja ashei, Naja haje, Naja katiensis, Naja mossambica, Naja nigricincta, Naja nigricollis, Naja nubiae, Naja pallida, Naja melanoleuca, Naja anchietae, Naja annulifera, Naja nivea, or Naja senegalensis.

[1017] 149. The composition, method, kit, use or multi-specific antigen-binding protein according to item 137, wherein the elapid snake is Hemachatus haemachatus, species of the Naja genus, such as Naja ashei, Naja Naja, Naja kaouthia, Naja haje, Naja katiensis, Naja mossambica, Naja nigricincta, Naja nigricollis, Naja nubiae, Naja pallida, Naja melanoleuca, Naja anchietae, Naja annulifera, Naja nivea, or Naja senegalensis, or Dendroaspis jamesoni, Dendroaspis viridis, or Dendroaspis polylepis, or Ophiophagus hannah, or Ophiophagus kaalinga, or Bungarus spp.

[1018] 150. The composition, method, kit, use or multi-specific antigen-binding protein according to item 137, wherein the elapid snake is Hemachatus haemachatus, species of the Naja genus, such as Naja ashei, Naja Naja, Naja kaouthia, Naja haje, Naja katiensis, Naja mossambica, Naja nigricincta, Naja nigricollis, NajaP7584PC00

[1019] 99

[1020] nubiae, Naja pallida, Naja melanoleuca, Naja anchietae, Naja annulifera, Naja nivea, or Naja senegalensis or Ophiophagus hannah, or Ophiophagus kaalinga, or Bungarus spp.

[1021] Examples

[1022] Example 1: Camelid immunization and phage library generation

[1023] As outlined by the World Health Organization (WHO), within sub-Saharan Africa there are a total of 18 elapid snakes (Table 1, section entitled ‘Toxin overview’) that can be considered the most medically important, belonging to three genera: Dendroaspis (mambas; 4 species), Hemachatus (rinkhals; 1 species), and Naja (cobras; 13 species). In order to develop VHHS targeting the toxins of these species, immune VHH-displaying phage libraries were generated.

[1024] Materials and methods:

[1025] Immune VHH-displaying phage libraries targeting elapid snake venoms were constructed as described in Benard-Valle, M. etal., (2023). One alpaca and one llama were subcutaneously (s.c.) injected at bi-weekly intervals across 8 time points with increasing doses of venom mixtures from the 18 most medically relevant elapid snakes in sub-Saharan Africa, i.e. Dendroaspis angusticeps, Dendroaspis jamesoni, Dendroaspis polylepis, Dendroaspis viridis, Naja anchietae, Naja annulifera, Naja ashei, Naja haje, Naja katiensis, Naja melanoleuca, Naja mossambica, Naja nigricincta, Naja nigricollis, Naja nivea, Naja nubiae, Naja pallida, Naja senegalensis, and Hemachatus haemachatus. Following the initial series of injections, 3 additional booster injections were administered at 52, 54, and 60 weeks after the first immunization (Table 2).

[1026] Table 2. Immunization scheme per camelid animal including the time points for phage display library construction.P7584PC00

[1027] 100

[1028] Total venom Dose per Library

[1029] Day / week

[1030] dose (mg) venom (mg) generation

[1031] 0 d 0.27 0.015 No

[1032] 14 d 0.54 0.03 No

[1033] 28 d 0.72 0.04 No

[1034] 42 d 0.99 0.06 No

[1035] 46 d - - Yes (Library A)

[1036] 49 d - - 56 d 1.26 0.07 No

[1037] 70 d 1.44 0.08 No

[1038] 84 d 1.80 0.10 No

[1039] 98 d 2.16 0.12 No

[1040] 102 d - - Yes (Library B)

[1041] 105 d - - 52w 4.5 0.25 No

[1042] 54 w 4.5 0.25 No

[1043] 60 w 4.5 0.25 No

[1044] 60 w + 5 d

[1045] Yes (Library C)

[1046]

[1047] 60 w + 8 d

[1048] For the library generation, blood samples were collected on days 5 and 8 following the first set of 4 injections. The two blood samples from each animal were pooled separately, and individual libraries were prepared for each animal.

[1049] Results & Conclusions

[1050] A total of six VHH-displaying phage libraries, i.e. one library per time point and animal, were prepared by pooling the total RNA samples after days 46 and 49 (Library A), 102 and 105 (Library B), and days 5 and 8 following the final booster injections (Library C).

[1051] Example 2: Identification of most medically relevant elapid toxins

[1052] The most medically relevant elapid snake toxins were identified. These toxins belong to three distinct protein families: three-finger toxins (3FTx), phospholipase A2S (PLA2), and Kunitz-type serine protease inhibitors (KUN). The toxins were purified using reversed-phase high-performance liquid chromatography (RP-HPLC) followed by proteomic analysis of the 48 fractions in which the key toxins were expected to be present (Table 1, FIG.1), as described hereafter.

[1053] Materials and methods

[1054] Purification and biotinylation of the venom fractions and toxinsP7584PC00

[1055] 101

[1056] Cardiotoxin (P01468) from N. pallida, a-cobratoxin (P01391) from N. kaouthia, a-short chain neurotoxin from N. pallida, and whole venoms from the above-mentioned 18 elapid snakes were purchased in lyophilized form from Latoxan (Portes les Valence, France). Origin of the specimens can be found in Table 1 in section ‘Toxin overview’. Venom fractions containing short-chain neurotoxins (sNTx), long-chain neurotoxins (INTx), cytotoxins (CTx), Og XI (also referred to as S5C4), AgTx (also referred to as S6C6), phospholipase A2S (PLA2), and dendrotoxins (DTx) were isolated from the whole venoms using reversed-phase high-performance liquid chromatography, RP-HPLC, (Agilent 1200) with a C18 column (250 x 4.6 mm, 5 pm particle; Teknokroma). 1 mg of venom solubilized in 100 pL Solution A (MilliQ water supplemented with 0.1 % TFA) was applied to the column and elution was performed at a rate of 1 mL / min using Solution A and a gradient towards solution B (acetonitrile supplemented with 0.1% TFA): 0% B for 15 min, 0-15% B over 15 min, 15-45% B over 60 min, 45-70% B over 10 min, and 70% B over 9 min, as previously described.65 Fractions were collected and the solvent evaporated using a vacuum centrifuge. The venom fractions purified via RP-HPLC and toxins bought from Latoxan were dissolved in phosphate buffered saline (PBS: 137 mM NaCI, 3mM KCI, 8mM Na2HPO4-2H2O, 1.4 mM KH2PO4, pH 7.4) and biotinylated by amine coupling using a 1:1 to 1:3 molar ratio of venom fraction or toxin to EZ-Link™ NHS-PEG4-Biotin reagent (Thermo Scientific, A39259). Free biotin was removed using 2 or 4 kDa MWCO ultracentrifugation membranes (Vivacon® 500, VN01H91 and Amicon Ultra-4, UFC8000324, respectively) in accordance with the manufacturers’ guidelines. Following purification, the degree of biotinylation was analyzed by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS) using ProteoMass™ Protein MALDI-MS Calibration Kit (Sigma-Aldrich, MSCAL3) and an Ultraflex II TOF / TOF spectrometer (Bruker Daltonics).

[1057] Proteomics analysis of the selected venom fractions

[1058] From each venom fraction, 5 pg was diluted in 50 mM ammonium bicarbonate to a total volume of 25 pL. The samples were reduced and alkylated by 10 mM TCEP and 40 mM CAA before digestion with either GluC or trypsin in an enzyme-to-protein ratio of 1:100. Samples were incubated overnight at 37 °C, after which the digestion was stopped by addition of 2% TFA for a final concentration of 1%. The samples were desalted with SOLAp SPE plate (HRP, Thermo) C18 columns, following the sameP7584PC00

[1059] 102

[1060] procedure as previously described.67 Dried peptides were reconstituted in 12 pL 2% ACN, 1%TFA, and an estimated 500 ng of peptides was used for MS analysis.

[1061] Peptides were loaded onto a 2 cm C18 trap column (ThermoFisher 164946), connected in-line to a 15 cm C18 reverse-phase analytical column (Thermo EasySpray ES904) using 100% Buffer A (0.1% Formic acid in water) at 750 bar, using the Thermo EasyLC 1200 HPLC system, and the column oven operating at 35 °C. Peptides were eluted over a 35 min gradient ranging from 6 to 60% of Buffer B (80% acetonitrile, 0.1% formic acid) at 250 nL / min, and the Q-Exactive instrument (Thermo Fisher Scientific) was run in a DD-MS2 top10 method. Full MS spectra were collected at a resolution of 70,000, with an AGC target of 3x106 or maximum injection time of 20 ms and a scan range of 300-1750 m / z. The MS2 spectra were obtained at a resolution of 17,500, with an AGC target value of 1x106 or maximum injection time of 60 ms, a normalized collision energy of 25 and an intensity threshold of 1.7x104. Dynamic exclusion was set to 60 s, and ions with a charge state <2 or unknown were excluded.

[1062] The raw data from all fractions were analyzed with Proteome Discoverer v2.4. The data were searched against all snake venom proteins (retrieved from Uniprot, 2,263 sequences, accessed 09 / 11 / 2021). The trypsin-digested fractions were searched with tryptic specificity, while the GluC-digested fractions were searched with GluC specificity, with two maximum missed cleavages allowed for both proteases. Minimum and maximum peptide lengths were set to 7 and 40, respectively. Precursor mass tolerance was 10 ppm, and fragment mass tolerance was 0.02 Da. Methionine oxidation (+15.995 Da) was set as dynamic modification, while initiator methionine loss (-131.040 Da), acetylation (+42.011 Da), or the combination of methionine loss and acetylation (-89.030 Da) were included as dynamic modifications for the protein terminus. Cysteine carbamidomethylation (+57.021 Da) was added as a static modification. Peptide-spectrum matching was performed with Sequest HT, and FDR control with Percolator (0.01 strict and 0.05 relaxed target FDR). FDR was also controlled at the peptide and protein levels with the same target FDRs. Proteins were quantified based on the unique and razor peptides, using the Minora Feature Detector and the Precursor Ions Quantifier nodes with default settings, normalizing abundance to the total peptide amount in each MS run and scaling abundance values on the average of all runs.P7584PC00

[1063] 103

[1064] Clustering toxins based on sequence homology

[1065] A sequence similarity network (SSN) was made with the Enzyme Function Initiative -Enzyme Similarity Tool (EFI-EST). A fasta file containing the UniProt sequences of each discovered toxin from the whole venom of the included 18 elapid snakes was used by the tool to perform an all-by-all BLAST to obtain similarities between sequence pairs. Clustering of toxins with a minimum sequence homology of 70% was subsequently performed by using an alignment score threshold during SSN Finalization that corresponds to 70% identity in the “Percent Identity vs Alignment Score Box Plot” in the Dataset Analysis tab. The obtained SSN was visualized with Cytoscape.

[1066] Results

[1067] The 3FTx found in these fractions belonged to 5 different subfamilies, namely the Type I a-neurotoxin (sNTx), Type II a-neurotoxin (INTx), Type IA or Type IB cytotoxin (CTx), Orphan group XI (including S5C4), and Orphan group XIX (including S6C6) (FIG.2).

[1068] Conclusions

[1069] Fractions and purified toxins are denoted by their main toxin (sub)family name and a number (e.g. INTx-1, INTx-2, etc.) (Table 1, section entitled ‘Toxin overview’). Overall, 16 fractions (3 INTx, 3 sNTx, 2 KUN, 1 AgTx (also referred to as S6C6), 2 Og XI (also referred to as S5C4), 1 PLA2, and 4 CTx) (Table 1 and Table 3) were selected to be used as targets in subsequent phage display selections based on their abundance in the corresponding venom and their purity.

[1070] Table 3: Overview of venom fractions selected for subsequent experiments.

[1071] Toxin (sub)family Fractions

[1072] CTx CTx-6

[1073] CTx-9

[1074] CTx- 10

[1075] CTx- 11

[1076] sNTx sNTx-1

[1077] sNTx-3

[1078] sNTx-6

[1079] INTx INTx-1

[1080] INTx-3

[1081] INTx-7

[1082] AgTx AgTx-2

[1083] Og XI Og XI-1

[1084]

[1085] Og XI-2P7584PC00

[1086] 104

[1087] PLA2 PLA2-3

[1088]

[1089] KUN KUN-1

[1090] Example 3: Selection of VHHS

[1091] To minimize the number of neutralizing toxin-targeting VHHS, phage display campaigns were conducted. Following these phage display campaigns, 15 phage pool outputs were subcloned, expressed over 3,000 monoclonal VHHS in E. coli, and were screened for binding to their cognate target toxins using an expression-normalized capture Dissociation-Enhanced Lanthanide Fluorescence Immunoassay (DELFIA), as described hereafter.

[1092] Materials and methods

[1093] Solution-based phage display selections

[1094] VnH-displaying phage libraries were incubated with biotinylated venom fractions or toxins for 2 h at ambient temperature, with end over end rotation. Streptavidin coated Dynabeads (M-280, Fisher Scientific, 10465723) were blocked in PBS containing 3% non-fat dried milk powder for 1 h with end over end rotation, before addition to the target toxins mixed with the phage library. In each selection round, a background control was included where no antigen was mixed with the phage library.

[1095] Subsequently, a KingFisher Flex system (Thermo Scientific, 711-82573) was used to wash the beads 3 times with PBST (PBS + 0.1% Tween) and 3 times with PBS, before eluting the bound phages in 120 pL of 0.1 mg / mL trypsin (Sigma-Aldrich, T9201-500MG) in phage elution buffer (50 mM Tris, 1 mM CaCI2, pH 8.0). The eluted phages were amplified using the M13KO7 helper phage and concentrated by polyethylene glycol precipitation.

[1096] Subcloning, screening, and sequencing of VHHS

[1097] Phagemids from the chosen selection outputs were purified using the GeneJETTM Plasmid MiniPrep Kit (Thermo Fisher, K0503) according to the manufacturer’s protocol. The VnH-encoding genes were subcloned into the pBDS100 expression vector using the Pstl and Eco91l restriction enzymes (New England Biolabs). Following transformation into the E. coli strain BL21 (DE3) (New England Biolabs), at least 184 individual colonies were picked from each chosen selection output and used for the expression of soluble HHS. Auto-induction media was used to induce VHH expression for 16 h at 30 °C. Thereafter, periplasmic cell extracts, containing soluble expressed VHHS, were used for primary screenings in an previously described expression-P7584PC00

[1098] 105

[1099] normalized DELFIA using 25 nM of target toxin. Clones with a signal intensity 10 times higher than the background (no addition of biotinylated target), were cherry-picked and went through a second round of screening in the expression-normalized DELFIA, against multiple target toxins. For the cross-reactive clones, a dose-response experiment was performed, where the FLAG-tagged VHHS in the periplasmic extracts were captured onto the anti-FLAG coated wells as described above; however, instead of a single concentration, a serial dilution of target toxins (1:1,000 nM) was added. Clones displaying a signal intensity 50 times over the negative control, and / or a low EC50 value in the dose response curves, were Sanger sequenced (Eurofins Genomics sequencing service) using the M13Rev primer (CAGGAAACAGCTATGAC; SEQ ID NO: 42). The VHH frameworks and the complementarity determining regions (CDRs) were annotated using CLC Main Workbench (Qiagen) and the VHHS with unique CDR sequences were produced for in vitro and in vivo assays.

[1100] Production of VHHS for in vitro and in vivo experiments

[1101] For expression of VHHS at scales up to 100 mL, the periplasmic extracts containing HHS were produced as described in the screening section, and then purified using Ni-resin (Sigma-Aldrich, P6611) via gravity flow. For larger-scale expressions (>250 mL), BL21 (DE3) cells, containing the plasmid encoding for a unique VHH, were cultivated as previously described. Thereafter, the VnH-containing supernatants were purified using immobilized metal ion affinity chromatography with a 2 mL column volume of Ni-NTA resin (HIS-select Nickel Affinity Gel, Sigma-Aldrich, P6611) equilibrated with PBS supplemented with 200 mM NaCI and 20 mM imidazole, pH 8.0. Elution was performed with PBS containing 200 mM NaCI and 135 mM imidazole, pH 8.0, followed by an overnight dialysis in SnakeSkin Dialysis Tubings (10 kDa MWCO, ThermoFisher Scientific, 68100) against PBS. Subsequently, VHHS were concentrated using Amicon® Ultra-15 centrifugal filters (3 kDa MWCO, Fisher Scientific, 10781543).

[1102] Kinetic analysis of VHHS using biolayer interferometry

[1103] The binding of VHHS to the venom fractions and toxins was analyzed using biolayer interferometry (Octet-BLI; Octet RED 96, ForteBio). Biotinylated venom fractions and toxins at a concentration of 0.5 pg / mL were captured to a target spectral shift of 0.8 nanometer (nm) on a streptavidin coated BLI biosensor (Sartorius, 18-5020). A biosensor without antigen was included as a reference. VHHS were prepared in running buffer (10 mM HEPES, 150 mM NaCI, 3mM EDTA, 50 mM MES hydrate, and 0.05%P7584PC00

[1104] 106

[1105] P20 (MES-HEPES), pH 7.2). The toxin-loaded biosensors were dipped into 4 different VHH concentrations (7.5, 30, 120, 480 nM) and a control without any VHH. VHH association was measured for 600 sec, followed by measuring VHH dissociation in running buffer for 600 sec. Biosensors were regenerated by dipping into the regeneration buffer (10 mM Glycine, 4 M sodium chloride, pH 2.0) between each round, 5 times, for 10 sec each. For analysis, the reference BLI biosensor background was subtracted, a global model assuming a 1:1 interaction was used for fitting of the data, and calculations of kinetic parameters were all made in Octet Analysis Studio 12.2.2.26 (ForteBio).

[1106] Results

[1107] Throughout the phage display campaigns, cross-panning strategies were employed, including e.g. exposure to sNTx fractions from different snake species, and / or decreasing antigen concentrations in consecutive rounds to enrich for HHS with broad cross-reactivity and / or high affinity.

[1108] Approximately 60% of the HHS bound to their target toxins (FIG.3) and 25% of them were tested for cross-reactivity in a secondary DELFIA-based screening. Over 50% of the HHS bound multiple toxins from the same toxin (sub)family, which after sequencing revealed over 100 unique VHH clones. After the screening campaign, the top 21 unique cross-reactive VHHS were further evaluated against their corresponding toxin family in a dose-response DELFIA, yielding ECso values (half maximal effective concentrations) ranging from 1-15 nM (FIG.4).

[1109] Table 4. Affinity measurements between VHHS and venom fractions and toxins using BLI. x: no binding.

[1110] Toxin

[1111] (sub) Clone Target Kon ton koff KD(M) KD error Koff (l / s) R family (1 / Ms) error error2

[1112] CTx-1 X X X X X X X CTx-10 3.28-10'95.23-10'117.42-1045.10-1022.44- 10'43.51-1060.99 VHH1 a- CTx-12 1.91 10- CTx78.76- IO'92.31-1041.00-1034.40- IO36.68- IO50.9191 CTx- 13 X X X X X X X CTx

[1113] CTx- 18 8.22- IO'91. O3-1O105.64-1044.55-1024.64- 10'44.47- IO'60.9878 VHH2 a- CTx-10 3.81-10"94.28-10'111.47-1051.25-1035.61-1044.15-1060.9825 CTx CTx- 18 9.42- IO91. O2-1O'101.13-1051.09-1031.07-10'35.41-1060.9837

[1114]

[1115] CTx-1 X X X X X X XP7584PC00

[1116] 107

[1117] CTx-10 X X X X X X X CTx-12 6.64-1O103.57-10'111.02-1057.73-1026.74- IO53.58-10'60.9858 VHH4 a- CTx CTx- 13 2.42-10

[1118] 3.2O-1O'105.92-10'124.18-105+03 1.34 1042.35-10'60.9854 CTx- 18 X X X X X X X sNTx-1 1.90-10’91.41-1O102.49-1041.49-1024.72- IO'53.48-10'60.9956 sNTx-3 2.88-10'82.2O-1O'102.35-1041.28-1026.77- 10'43.61-1060.9965 sNTx-5 1.69-10'81.36-1O103.74-1042.15-1026.31-10"43.58-10'60.9966 VHH5 a- sNTx-6 0.9981 sNTx 5.46- 10'98.11-10112.98-1041.20-1021.63-1042.33-10'6sNTx-7 3.88-10'107.42-10'113.71-1041.85-1021.44-1052.75-10'60.9978 sNTx-8 <1.0-10121.16-1O102.24-1049.40-101<i.o-io-7- 0.9981 sNTx-9 1.70-10' 0.996 s97.32-10'113.96-1042.11-1026.75-10'52.88-10' 3 NTx6

[1119] sNTx-1 9.19-10108.91-10112.98-1041.38-1022.74- IO'52.65-10'60.9976 VHH6 a- sNTx-3 0.9966 sNTx 6.24- 10'84.27-1O'102.56-1041.56-1021.60-1034.97- IO'6sNTx-8 1.36-10"91.84-1O102.37-1041.89-1023.21-1054.34-10'60.989 sNTx-1 4.55-1097.39-10'115.01-1043.35-1022.28- 10'43.37-10'60.9967 VHH7 a- sNTx-3 <1.0-10126.03-10'118.07-1047.91-102<i.o-io-7- 0.9825 sNTx sNTx-8 3.37-10'83. O1-1O105.24-1044.29-1021.77-10'36.28- IO60.9923 sNTx-9 1.79-10'81.24-1O'107.45-1044.60-1021.33 1034.20- IO'60.9963 VHH9 a- lNTx-3 1.18 1091.50-10'111.73-1058.88-1022.03-10'42.38-10'60.9921 INTx

[1120] INTx lNTx-7 7.53-10'102.51-10113.60-1045.75-1012.71-10'59.03-10'70.9998 VHH12

[1121] lNTx-3 1.47-10'88.76-10'118.30-1044.41-1021.22-10'33.32-10'60.9953 a-lNTx

[1122] VHH13 AgTx-1 7.16-10'99.88-10114.91-1043.60-1023.52-10'44.12-1060.9946 a- AgTx AgTx-2 2.16-1096.67-10'117.47-1046.68-1021.61-10’ 4.77- IO' 0.99 AgTx4 6VHH14 AgTx-1 7. O2-1O'105.93-10'115.87-1043.79-1024.12-1053.47-10'60.9954 a- AgTx AgTx-2 7.47- IO'91.84-1O104.27-1045.26-1023.19-10"46.79- IO'60.9747 VHH15 Og XI-1 2.19-1094.10-10117.34-1043.95-1021.61-10’42.88-10'60.9963 a-Og XI Og XI-2 2.16-1095.42-10'115.67-1043.08-1021.22-10'43.00-10'60.9972 Og XI

[1123] VHH16 Og XI-1 2.40- IO'96.27-10'115.51-1043.46-1021.32-10"43.36-10'60.9938 a-Og XI Og XI-2 2.63-10'95.88-10'116.17-1043.93-1021.62-1043.48-10-60.9962 VHH17 KUN-1 4.05-10'114.13-1O102.08-1041.89-1028.43-10'78.60- IO60.9531 a-KUN KUN-2 <1.0-10122.66-10'118.69-1043.99-102<i.o-io-7- 0.998 VHH18

[1124] KUN KUN-1 2.07- IO'95.39-10'117.48-1045.70-1021.55-10"43.85-10'60.9875 a-KUN

[1125] KUN-1 3.51-10111.37-1051.53-103<1.0-10-7- 0.9887 VHH19 <1.0-1012

[1126] a-KUN 1.30x10 6.44x10 2.26x10- KUN-2 1.48xlO-101.75X10-115 2 1.92X10'56 0.9963

[1127]

[1128] Conclusions

[1129] The top 15 VHHS were selected, based on broad cross-reactivity and low ECso values, and evaluated their binding kinetics using biolayer interferometry (BLI). All of the 15 tested cross-reactive VHHS displayed low nanomolar affinity (KD) with slow dissociation rates (kOff <5.5x10-4 s-1) for most of their target toxins (Table 4, FIG.5).

[1130] Example 4: Evaluation of the neutralization abilities of the VHHS in vitro

[1131] To address the neutralization of INTx and sNTx, nicotinic acetylcholine receptors (nAChRs) currents were measured in patch-clamp assays, as INTx and sNTx exertP7584PC00

[1132] 108

[1133] their function by preventing acetylcholine binding and ion influx, which disrupts nervemuscle communication and often results in paralysis. Besides neurotoxicity, CTx and PLA2 toxins present in the venoms of 8 of the 18 elapid snakes, 7 Afronaja and 1 Hemachatus, can cause severe local tissue damage. Therefore, the ability of the anti-CTx VHHS to neutralize venom-induced cytotoxicity was assessed with cell-viability assays.

[1134] Materials and methods

[1135] Patch-clamp electrophysiology

[1136] Automated planar whole-cell patch-clamp experiments were performed as previously described.18 All experiments were performed on a Qube 384 automated patch-clamp platform (Sophion Bioscience) with 384-channel, 10X mode patch chips (10 patch holes / site, site resistance 0.2 ± 0.04 MQ). We used a human Rhabdomyosarcoma RD cell line (American Type Culture Collection, ATCC) endogenously expressing muscle type nAChRs ((a1)2piyb) and 70 pM ACh for receptor activation. We first determined the IC80 value for the included toxins or venom fractions (sNTX-1, sNTx-3, sNTx-6, INTx-3, INTx-5, and INTx-7) and used this concentration to evaluate the neutralization effect of the corresponding VHHS. The VHHS were used at molar ratios of 9:1 to 1:27 between toxin and VHH. Finally, the inhibitory effect of the toxins on the elicited ACh current was normalized to the full ACh response and averaged in each group (n = 8).

[1137] In vitro neutralization of cell cytotoxicity

[1138] A cell viability assay was performed as previously described.48 Briefly, the N / TERT keratinocyte cell line was seeded at 4,000 cells per well in 100 pL cell culture medium and incubated overnight under standard conditions. After determining the half-maximal inhibitory concentration (IC50) of each venom, the cells were subjected to a venom concentration of 2 IC50S, either in the absence or presence of a 1:5 molar ratio of CTx: VHH based on the cytotoxin contents of each venom, followed by a 24 h incubation step. Thereafter, the CellTiter-Glo luminescent cell viability assay (Promega, Madison, Wl, USA) was performed in triplicate according to the manufacturer’s protocol. A maximal cell death control was included, where cell culture medium was supplemented with 0.01% Tween 20 to disrupt the cells. In addition, a maximum cell viability control was included, with cell culture medium supplemented with PBS, as well as a HH control, where cells were incubated with the highest tested VHH concentration without venom, to confirm that the VHHS alone do not affect cell viability.P7584PC00

[1139] 109

[1140] In vitro neutralization of PLA2 enzymatic activity

[1141] Venom concentration inducing half of the maximum PLA2 enzymatic activity (EC50) was determined as previously described. For inhibitory dose-response curves, VHHs were diluted to 16 pM, followed by a 2-fold serial dilution in 10 steps. 50 pL of snake venom at a concentration of 4 EC50 values was mixed with the serial dilutions of the VHHs and then incubated at room temperature for 30 min. The enzymatic reaction was started by adding 100 pL of 0.5 mM 4-nitro-3-(octanoyloxy)benzoic acid (NOBA) into the mixture. Final concentrations of the individual components in the enzymatic activity assays were 0.25 mM NOBA, and a 2-fold serial dilution of the VHHs with the highest concentration set at 4 pM. After adding NOBA to the wells, plates were shaken at 300 rpm for 2 min, and then incubated at 37 °C for 40 min. Finally, the plates were centrifuged at 4,000 x g at 4 °C for 3 min, and absorbance was measured at 25 °C at 405 nm using a Multimode Microplate Reader (VICTOR Nivo, HH35000500). The experiments were performed in duplicate and the absorbance averages were determined after subtracting a blank control containing no venom.

[1142] Results

[1143] Pre-incubation of anti-INTx (a-INTx) VHHS with INTx-3, INTx-5, and INTx-7 or anti-sNTx VHHS with sNTx-1, sNTx-3, and sNTx-6 before addition to the cells protected the nAChR-mediated current, i.e. showing complete inhibition of neurotoxicity down to a 1:1 molar ratio between VHH and toxin (FIG.6). Pre-incubated venoms from the 8 snake species with two different anti-CTx (a-CTx) VHHS, VHH1 a-CTx and VHH4 a-CTx, before addition to a keratinocyte cell line, demonstrated broad neutralization, providing 75-95% and 50-85% protection, respectively, against the cytotoxic effects from the venoms of all 7 Afronaja (FIG.7). Assessment of the neutralizing effect of an anti-PLA2 VHH, in an enzymatic assay showed complete inhibition of PLA2 activity in the Afronaja venoms (FIG.8).

[1144] Conclusions

[1145] The results demonstrate that pre-incubation of specific VHHS effectively neutralizes key elapid venom toxins, with anti-INTx and anti-sNTx VHHS fully inhibiting neurotoxicity, anti-CTx VHHS providing substantial protection against venom-induced cytotoxicity, and anti-PLA2 VHHS completely inhibiting PLA2 enzymatic activity.P7584PC00

[1146] 110

[1147] Example 5: Selection of VHHS and evaluation of the recombinant antivenom

[1148] A series of WHO-recommended murine pre-incubation experiments (Guidelines for the production, control and regulation of snake antivenom immunoglobulins, 2013) were performed to examine whether these neutralizing effects would translate to in vivo neutralization. Aiming at having as few VHHS as possible in the recombinant antivenom, the most broadly-neutralizing VHHS were evaluated against each toxin (sub)family. The neutralization of individual pure toxins or toxin fractions was conducted first, followed by testing with simple whole venoms containing a few different toxin (sub)families, and finally, the neutralization of more complex venoms was evaluated.

[1149] Materials and methods

[1150] In vivo neutralization of venom-induced lethality

[1151] In vivo median lethal dose (LD50) determinations and lethality neutralization experiments were conducted using groups of mice weighing 18 to 20 grams, comprising both sexes. The CD1 mouse strain was used in the experiments performed for designing the recombinant antivenom, LD50 determinations, and rescue experiments. The NSA mouse strain was used for the pre-incubation assays. Time of death after administration of 3 LD50S of venoms was recorded in both strains to secure homogeneous results. All mice were kept under 12 h light and dark cycles with food and water ad libitum. LD50S were determined for selected toxins (INTx-7 and sNTx-3) and all the target venoms using the intravenous (i.v.) route. In the case of venoms selected for rescue assays, LD50S were also determined using the subcutaneous (s.c.) route.

[1152] Recombinant antivenom design experiments

[1153] To evaluate the neutralizing efficacy of the HHS and design a recombinant antivenom, neutralization of selected individual toxins, and selected whole venoms were performed in pre-incubation experiments (FIG. 3), as previously described.18 The mice were observed during the first 3 h and then approximately every 6 h for signs of envenoming. The percentage of survival was determined 24 h after the injection.

[1154] Results

[1155] Firstly, the median lethal dose (LD50) for venom fractions, toxins, and whole venoms when administered intravenously (i.v.) or subcutaneously (s.c.) was determined. The results are summarized in Table 5.P7584PC00

[1156] 111

[1157] Table 5. LD50S for venom fractions, toxins, and venoms using the s.c. and i.v. injection routes.

[1158] Sample LDso (pg / mouse)

[1159] i.v. s.c.

[1160] INTx-7 1-3 [-] ND

[1161] sNTx-3 1.3 [1.3 to 1.4] ND

[1162] D. angusticeps 38.6 [-] 41.6 [37.9 to 45.2] D. jamesoni 20.1 [-] 21.6 [20.2 to 22.9] Dendroaspis

[1163] D. polylepis 9.2 [-] 22.2 [19.1 to 25.4] D. viridis 9-8 [-] 12.9 [-] Hemachatus H. haemachatus 24.3 [-] 35.0 [20.4 to 24.1]

[1164] N. ashei 16.7 [-] ND

[1165] N. katiensis 23.2 [-] ND

[1166] N. mossambicci 22.1 [17.9 to 25.2] ND

[1167] Naja (Afronaja) N. nigricincta 16.5 [-] ND

[1168] N. nigricollis 17.1 [-] 79.3 [66.6 to 87.4] N. nubiae 8.7 [8.2 to 9.3] 9.6 [9.5 to 9.7] N. pallida 15.1 [-] ND

[1169] Naja

[1170] (Boulengerina) N. melanoleuca 19.9 [-] 41.8 [-]

[1171] N. anchietae 76.5 [72.3 to 80.6] >200

[1172] N. annulifera 119.2 [105.8 to 113.5] 122,6 [117.2 to 128.0] Naja (Uraeus) N. haje IO. I [-] 11.9 [-]

[1173] N. nivea 54.8 [54.1 to 55.5] 56.6 [53.8 to 59.2]

[1174]

[1175] N. senegalensis io.i [-] 12.2 [11.3 to 13.0]

[1176] After determination of the LD50S, venoms were pre-incubated with either single or multiple VHHs and administered to mice, with survival monitored for 24 hours.

[1177] The neutralization of sNTx-3 and INTx-7 was achieved using VHH5 a-sNTx and VHH9 a-INTx, respectively, resulting in the survival of all mice (FIG.9, Table 6). Thereafter, these two VHHS were combined and tested on the venoms from N. haje and N. melanoleuca, species known to produce venoms rich in sNTx and INTx. Pre-incubation of these venoms with the two VHHs led to complete prevention of lethality in all mice (FIG.10A and FIG.10B, Table 6).

[1178] The experiment was then extended to the more complex venom of D. viridis, which contains sNTx, INTx, AgTx, and Og XI. Pre-incubation of this venom with a mixture ofP7584PC00

[1179] 112

[1180] VHH5 a-sNTx, VVHH9 a-INTx, VHH15 a-Og XI, and VHH13 a- AgTx resulted in full protection of all mice (FIG.10C, Table 6), qualifying these four VHHS for inclusion in the recombinant antivenom. Next, the neutralization of mamba venom (D. polylepis), which is rich in dendrotoxins from the KUN family but also contains sNTx and INTx, was assessed. The addition of VHH17 a-KUN to the existing four-VHH mixture resulted in the survival of all mice when challenged with D. polylepis venom (FIG.10D, Table 6).

[1181] To evaluate the necessity of VHH15 a-Og XI and VHH13 a-AgTx, a mixture of VHH5 a-sNTx, VHH9 a-INTx, and VHH17 a-KUN was tested against D. viridis and D. jamesoni venom. While mice challenged with D. viridis venom all survived, they showed signs of lethargy, and only 2 mice survived after being challenged with D. jamesoni venom, illustrating that full neutralization is not achieved without VHH15 a-Og XI and VHH13 a- AgTx VHHS (FIG.10E, Table 6).

[1182] Lastly, the neutralization of PLA2 and CTx in N. nigricollis venom was evaluated. Preincubation with a combination of VHH20 a-PLA2 and two anti-CTx VHHS (VHH1 a-CTx and VHH4 a-CTx), which target different CTxs, provided complete protection from lethal venom effects in all mice (FIG.10F, Table 6).

[1183] Table 6. In vivo experiments for the design of the recombinant antivenom:P7584PC00

[1184] 113

[1185] Mouse Molar Amount Time of death (h) Venom / toxin VHHS

[1186] strain ratio (tig) Ml M2 M3 M4 M5 CD1 VHH7 a-sNTx 1:1 11.0 >24 >24 >24 - - CD1 sNTx-3 VHH5 a-sNTx 1:1 11.3 >24 >24 >24 - - CD1 VHH6 a-sNTx 1:1 11.4 12 13 >24 - - CD1 VHH9 a-lNTx 1:1 9.9 8 19 >24 - - lNTx-7

[1187] CD1 VHH9 a-lNTx 1:2.5 24.8 >24 >24 >24 - - VHH9 a-lNTx 1:10 130.8

[1188] CD1 N. haje >24 >24 >24 - - VHH5 a-sNTx 1:10 134.8

[1189] VHH9 a-lNTx 1:10 286.2

[1190] CD1 N. melanoleuca >24 >24 >24 - - VHH5 a-sNTx 1:10 185.5

[1191] VHH9 a-lNTx 1:10 293.2

[1192] CD1 D. viridis VHH5 a-sNTx 1:10 224.9 0,3 >24 >24 - - VHH15 a-Og XI 1:3 43.0

[1193] VHH9 a-lNTx 1:10 293,2

[1194] VHH5 a-sNTx 1:10 224.9

[1195] CD1 D. viridis >24 >24 >24 - - VHH15 a-Og XI 1:3 43.0

[1196] VHH13 a-AgTx 1:3 39.3

[1197] VHH9 a-lNTx >1:10 240.1*

[1198] VHH5 a-sNTx >1:10 413.1*

[1199] VVHH20 a-PLA2>1:10 291.4*

[1200] NSA D. viridis >24 >24 >24 >24 >24

[1201] VHH1 a-CTx >1:10 746.9*

[1202] VHH4 a-CTx >1:10 746.9*

[1203] VVHH17 a-KUN >1:10 479.7*

[1204] VHH9 a-lNTx >1:10 240.1*

[1205] VHH5 a-sNTx >1:10 413.1*

[1206] VVHH20 a-PLA2>1:10 291.4*

[1207] NSA D. jamesoni 0,3 0,4 0,5 >24 >24

[1208] VHH1 a-CTx >1:10 746.9*

[1209] VHH4 a-CTx >1:10 746.9*

[1210] VVHH17 a-KUN >1:10 479.7*

[1211] VHH9 a-lNTx 1:10 45.4

[1212] VHH5 a-sNTx 1:10 15.8

[1213] NSA D. polylepis VHH19 a-KUN 1:10 269.8 >24 >24 >24 3 to 24 3 to 24

[1214] VHH15 a-Og XI 1:10 13.8

[1215] VHH13 a-AgTx 1:10 14.4

[1216] VHH9 a-lNTx 1:10 45.4

[1217] VHH5 a-sNTx 1:10 15.8

[1218] NSA D. polylepis VVHH17 a-KUN 1:10 269.8 >24 >24 >24 >24 >24

[1219] VHH15 a-Og XI 1:10 13.8

[1220] VHH13 a-AgTx 1:10 14,4

[1221] CD1 N. nigricollis VVHH20 a-PLA21:10 159.9 0,3 2 >24 - - VHH1 a-CTx 1:10 525.2

[1222] CD1 N. nigricollis 9 h 10 >24 - - VVHH20 a-PLA21:10 159.9

[1223] VHH1 a-CTx 1:5 262.6

[1224] NSA N. nigricollis VHH4 a-CTx 1:5 262.6 >24 >24 >24 - -

[1225]

[1226] VVHH20 a-PLA21:10 159.9P7584PC00

[1227] 114

[1228] Table 7. Final composition of the recombinant antivenom:

[1229] Average SEQ ID nmoles of VHH VHH in Toxin MW of

[1230] MW Specific VHH NO VHH in cone. mixture (sub)family VHH (Da)

[1231] (kPa) mixture (mq / ml_) (kiq) CTx 8.0 VHH1 a-CTx 89 43.9 17.063 29.9 746.93

[1232] VHH4 a-CTx 26 43.9 17.397 39.0 746.93 sNTx 8.0 VHH5 a-sNTx 30 24.3 17.043 27.0 413.07 INTx 8.0 VVHH9 a-INTx 34 14.1 17.534 29.0 240.14 AgTx 7.5 VHH 13 a- AgTx 18 5.2 17.336 30.0 89.18 Og XI 7.0 VHH15 a-Og XI 14 35.0 17.069 30.0 594.60 PLA214.0 VVHH20 a-PLA238 17.1 17.329 28.7 291.40 KUN 7.0 VVHH17 a-KUN 22 28.2 17.849 28.8 479.66 Total 3601.9

[1233]

[1234] Alternatively, the composition of recombinant antivenom may comprise SEQ ID NO:

[1235] 10, 26, 30, 34, 18, 14, 38 and 22.

[1236] Alternatively, the composition of recombinant antivenom may comprise SEQ ID NO:

[1237] 57, 73, 77, 81, 65, 61, 85 and 69.

[1238] Conclusions

[1239] In total, eight VHHS targeting seven medically important toxin subfamilies were selected and combined into a recombinant antivenom, summarized in table 7.

[1240] Example 6: Prevention of venom-induced lethality from 17 elapid species

[1241] The recombinant antivenom prevents lethality

[1242] To assess whether the recombinant antivenom (i.e., the pool of 8 VHHS) could neutralize venom-induced lethality caused by all of the 18 most medically relevant snakes in sub-Saharan Africa, 3 LD50S of the venoms were pre-incubated with the same dose of recombinant antivenom and administered i.v. to groups of 5 mice.

[1243] Survival and signs of envenoming were monitored over a 24-hour period post-injection.

[1244] Materials and methods

[1245] For pre-incubation experiments of whole venoms, 3 LD50S of each venom (Table 4) were mixed with 3.6 mg (117 pL) of recombinant antivenom (Table 8) in a total volumeP7584PC00

[1246] 115

[1247] of 200 pL per mouse. This was then pre-incubated at 37 °C for 30 min before i.v. injection into groups of 5 mice. To compare the performance of the recombinant antivenom with a current plasma-derived commercial antivenom, 5 venoms were also tested for neutralization with the F(ab’)2 polyclonal antivenom Inoserp PAN-AFRICA (Lot# 5IT11003; expiration date November 2018) (INOSAN BioPharma, S. A.) which is currently recommended for the treatment of envenomings caused by 8 elapid and 5 viperid snakes from Africa. The antivenom was pre-incubated with the venom at 37 °C for 30 min, using the volume that neutralizes a minimum of 3 LD50S of venom from N. nigricollis and D. polylepis, according to the manufacturer’s product insert. This antivenom is also recommended for the treatment of bites by the elapid snakes D. viridis, D. angusticeps, D. jamesoni, N. haje, N. pallida, N. melanoleuca, N. nivea and N. katiensis. All mice were observed during the first 5 h and then approximately every 6 h for appearance of envenoming signs.

[1248] Table 8. Concentrations and LD50S in the recombinant antivenom.

[1249] Venom with

[1250] Toxin Abundance in Venom LD50 mmoles in 3 Volume highest nmoles in

[1251] (sub)family venom (%) (kiq / mouse) LDsos (kiL / mouse)

[1252] 3 LDsos

[1253] 4.39 25.0 CTx N. nigricollis 77.7 17.1

[1254] 4.39 19.2 sNTx D. jamesoni 31.3 20.1 2.43 15.3 INTx N. haje 40.5 10.2 1.41 8.3 AgTx D. viridis 14.4 9.7 0.52 3.0 Og XI D. jamesoni 45.0 20.1 3.50 19.8 PLA2N. melanoleuca 27.5 19.9 1.71 10.2 KUN D. polylepis 74.5 9.2 2.82 16.7 Total 117.3

[1255]

[1256] Results

[1257] The results observed in mice that were administered recombinant antivenom preincubated with venoms are summarised in table 9.

[1258] Table 9. Signs of envenoming in mice that were administered the recombinant antivenom pre-incubated with venoms.

[1259] Species venom Signs of envenoming in mice

[1260]

[1261] P7584PC00

[1262] 116

[1263] 1. D. jamesoni No signs

[1264] 2. H. haemachatus No signs

[1265] 3. N. ashei No signs

[1266] 4. N. katiensis No signs

[1267] 5. N. mossambica No signs

[1268] 6. N. nigricincta No signs

[1269] 7. N. nigricollis No signs

[1270] 8 N. nubiae No signs

[1271] 9. N. pallida No signs

[1272] 10. N. anchietae No signs

[1273] 11. N. haje No signs

[1274] 12. N. nivea No signs

[1275] 13. N. senegalensis No signs

[1276] 14. N. melanoleuca minor signs: closed eyes, lethargy, and excessive grooming

[1277] 15. D. viridis minor signs: closed eyes, lethargy, and excessive grooming

[1278] 16. N. annulifera minor signs, after 15h: closed eyes, lethargy, and excessive grooming

[1279] 17. D. polylepis initial state of severe lethargy for 1-2 minutes

[1280] 18. D. angusticeps prolonged survival from 3 to 6 hours

[1281]

[1282] Conclusions

[1283] The recombinant antivenom prevented venom-induced lethality for 17 out of 18 species.

[1284] Example 7: Rescue experiments

[1285] To better evaluate the efficacy of the recombinant antivenom in a scenario mimicking real snakebite envenoming, rescue experiments were performed using representative venoms from 4 different (sub)genera.

[1286] Materials and methods

[1287] The venoms of 5 elapid snakes (D. jamesoni, D. viridis, N. haje, N. melanoleuca, and H. haemachatus) were selected for their neutralization in a first rescue experiment. In a second experiment, the venoms of 11 elapid species (D. angusticeps, D. jamesoni, D.P7584PC00

[1288] 117

[1289] polylepis, D. viridis, N. annulifera, N. haje, N. melanoleuca, N. nivea, N. nubiae, N. senegalensis, and H. haemachatus) were selected. These were designed to better represent actual envenoming, where the venom is injected first (s.c.) and then the recombinant antivenom is administered using the i.v. route. In these experiments, 3 LD50 values of each of the selected venoms (Table 5) were injected in a final volume of 40 pL PBS. The final composition of the recombinant antivenom, as seen in Table 7, was injected 5 min later using the i.v. route in a total volume of 300 pL PBS. Since the recombinant antivenom was designed considering the LD50 value of each venom determined through i.v. administration, the dose of the recombinant antivenom used was adjusted based on the ratio between LD50 values determined through s.c. and i.v. injection for each venom. The mice were observed during the first 5 h and then approximately every 6 h for the appearance of envenoming signs. The percentage of survival was determined 24 h after the injection and plotted as Kaplan-Meier survival curves using GraphPad Prism v10.2.

[1290] To compare the performance of the recombinant antivenom with a current plasma-derived commercial antivenom, rescue experiments were performed for the same species used in the first experiment (D. jamesoni, D. viridis, N. haje, N. melanoleuca, and H. haemachatus) using Inoserp PAN-AFRICA (Lot# 5IT11003; expiration date November 2018) (INOSAN BioPharma, S. A.). Similar to the pre-incubation experiments, the antivenom dose was the volume that, according to the manufacturer, neutralizes a minimum of 3 LD50 values of venom adjusted based on the ratio between the LD50 value determined by i.v. or s.c. injection.

[1291] Due to the low availability of commercial antivenom, a vial from an expired batch of Inoserp PAN-AFRICA was used for all experiments.

[1292] Results

[1293] The effects on lethality of the rescue experiments observed in mice are summarised in tables 10 and 11.

[1294] Table 10. Effects on lethality in mice that were administered the recombinant antivenom 5 minutes after administration of 3 LD50S of the venom.

[1295] Species venom Lethality Signs of envenoming

[1296] H. haemachatus Complete prevention No signs

[1297] N. haje Complete prevention No signs

[1298]

[1299] P7584PC00

[1300] 118

[1301] N. melanoleuca Partial neutralization limited movement ~4h after venom injection and continuing throughout the experiment, swelling and haemorrhage of the eyeballs D. viridis Complete prevention limited movement ~4h after venom injection and continuing throughout the experiment, swelling and haemorrhage of the eyeballs, increased abdominal volume D. polylepis Prolonged survival

[1302] from 0.5 to 2 hours.

[1303]

[1304] Table 11. Summary of in vivo experiments to assess the recombinant antivenoms efficacy to prevent venom-induced lethality.P7584PC00

[1305] 119

[1306] AV Dose

[1307] Mouse strain Venom sp. Neutralizer (mg) Time of death (hours)*

[1308] Ml M2 M3 M4 M5 Recombinant AV- Preincubation setup

[1309] NSA N. haje Recombinant AV 3.6 >24 >24 >24 >24 >24 NSA D. polylepis Recombinant AV 3.6 >24 >24 >24 >24 NSA D. viridis Recombinant AV 3.6 >24 >24 >24 >24 NSA N. nigricollis Recombinant AV 3.6 >24 >24 >24 >24 NSA N. melanoleuca Recombinant AV 3.6 >24 >24 >24 >24 NSA D. jamesoni Recombinant AV 3.6 >24 >24 >24 >24 NSA D. angusticeps Recombinant AV 3.6 3 3 4.5 5 6.5 NSA H. haemachatus Recombinant AV 3.6 24 >24 >24 >24 NSA N. mossambica Recombinant AV 3.6 24 >24 >24 >24 NSA N. pallida Recombinant AV 3.6 24 >24 >24 >24 NSA N. ashei Recombinant AV 3.6 24 >24 >24 >24 NSA N. katiensis Recombinant AV 3.6 24 >24 >24 >24 NSA N. nigricincta Recombinant AV 3.6 24 >24 >24 >24 NSA N. nubiae Recombinant AV 3.6 24 >24 >24 >24 NSA N. anchietae Recombinant AV 3.6 24 >24 >24 >24 NSA N. annulifera Recombinant AV 3.6 24 >24 >24 >24 NSA N. nivea Recombinant AV 3.6 24 >24 >24 >24 NSA N. senegalensis Recombinant AV 3.6 > 74 7 Commercial AV - Preincubation setup

[1310] CD1 D. viridis I Inoserp PanAfnca I 2.2 1.5 5_ - - - - CD1 N. nigricollis i Inoserp PanAfrica I 2.2 7

[1311] CD1 0. polylepis i Inoserp PanAfrica I 2.2 >,x. > 74 7 7 Recombinant AV - Rescue setup

[1312] CD1 i Recombinant AV i > 74 > 74 > 74 CD1 N. haje | Recombinant AV; 4.8 > 74 7 < > 74 7 CD1 N. melanoleuca Recombinant AV 8.6 12 18 >24 >24 >24 CD1 D. viridis Recombinant AV 5.4 >24 >24 >24 >24 >24 CD1 D. polylepis Recombinant AV 9.9 2.5 3 2 0.75 2.20 CD1 H. haemachatus Recombinant AV 7.2 >24 >24 >24 >24 CD1 D. jamesoni Recombinant AV 4.4 0.67 1.33 0.83 0.92 1.08 CD1 N. annulifera Recombinant AV 4.6 >24 >24 >24 >24 CD1 N. nivea Recombinant AV 4.2 >24 >24 >24 >24 CD1 N. senegalensis Recombinant AV 4.9 >24 >24 >24 >24 CD1 N. nubiae Recombinant AV 4.5

[1313] CD1 1). angusticeps i Recombinant AV i 4.4 0.83 0.30 0.40 0.25 0.33 Commercial AV - Rescue Setup

[1314] CD1 I Inoserp PanAfrica I 5.4 >74

[1315] CD1 N. haje I Inoserp PanAfrica I 2.4 > 74 2.2 1.7 1.7 CD1 D. polylepis Inoserp PanAfrica 5.2 1.5 >24 20 7 7 CD1 N. melanoleuca Inoserp PanAfrica 4.5 11.5 2 7.5 2.2 7.5 CD1 N. melanoleuca Inoserp PanAfrica 9.1 >24 >24 >24 >24 >24 CD1 H. haemachatus Inoserp PanAfrica 3.4 >24 >24 0.8 1.4 2.5

[1316]

[1317] CD1 D. viridis Inoserp PanAfrica 2.8 3.8 5 12 12 24P7584PC00

[1318] 120

[1319] 3LD50 Venom Control (i.v.)

[1320] CD1 D. polylepis 0,5 - 0,7 0,5 - 0,7 0,5 - 0,7 - - NSA D. polylepis 0.18 0.22 0.28 0.33 0.42 NSA D. viridis 0.42 0.75 0.15 0.16 0.28 NSA N. nigricollis 0.15 0.15 0.14 0.16 0.29 NSA N. melanoleuca 0.05 0.05 0.20 0.22 0.25 NSA N. haje 0.08 0.14 0.17 0.15 0.19 NSA H. haemachatus 0.08 0.08 0.08 0.13 0.17 NSA D. angusticeps 0.05 0.05 0.08 - - NSA D. jamesoni 0.05 0.05 0.03 0.07 0.10 NSA N. pallida <0,08 <0,08 <0,08 <0,08 <0,08 NSA N. mossambica <0,08 <0,08 <0,08 <0,08 <0,08 NSA N. senegalensis <0,08 <0,08 <0,08 <0,08 <0,08 NSA M. katiensis <0,08 <0,08 <0,08 <0,08 0.13 NSA N. ashei <0,08 <0,08 <0,08 <0,08 0.25 NSA N. nubiae 0.32 0.32 0.33 0.35 0.38 NSA N. nigricincta 0.33 0.45 0.58 0.62 2.20 NSA N. anchietae <0,05 <0,05 <0,05 <0,05 <0,05 NSA N. nivea <0,05 <0,05 <0,05 <0,05 <0,05 NSA N. annulifera <0,05 <0,05 <0,05 <0,05 <0,05 3LD50 Venom Control (s.cj

[1321] CD1 D. polylepis 0.35 0.38 0.46 0.46 0.46 CD1 N. melanoleuca 0.78 0.68 1.27 1.10 0.77 CD1 N. haje 0,7 - 0,8 0,7 - 0,8 0,7 - 0,8 0,7 - 0,8 0,7 - 0,8 CD1 H. haemachatus 0,8 - 1,0 0,8 - 1,0 0,8 - 1,0 0,8 - 1,0 0,8 - 1,0 CD1 D. viridis 0.43 0.83 0.85 0.48 0.65 CD1 D. jamesoni 0.75 0.33 0.25 0.28 0.35 CD1 N. annulifera 1.42 1.50 1.58 2.00 1.50 CD1 N. nivea 1.50 1.50 1.50 1.50 1.50 CD1 N. senegalensis 1.33 1.33 1.33 2.33 2.33 CD1 N. nubiae 1.25 1.25 1.25 1.25 1.25

[1322]

[1323] CD1 D. angusticeps 0.58 0.25 0.22 0.23 0.33 *M1 to M5 represent an individual mice in each experiment

[1324] Note: The venom dose was 3 LD50values

[1325] In these rescue experiments, the dose of the recombinant antivenom was increased compared to the pre-incubation experiments relative to the increase in LD50 values that was observed when switching from i.v. to s.c. administration of venom (Table 5 and Table 11). For comparison, the commercial Inoserp PAN-AFRICA antivenom was included at a dose recommended by the manufacturer to neutralize 3 LD50 values of venom. The recombinant antivenom completely prevented lethality induced by the venoms from A / , haje, N. annulifera, N. nivea, N. senegalensis, N. nubiae, and H. haemachatus (Table 11), and the mice showed no signs of envenoming. Furthermore, the recombinant antivenom also prevented lethality induced by the venom of D. viridis (Table 11), although we observed signs of envenoming, including limited movement approximately 4 hours after venom injection and continuing throughout the experiment. In addition, after 20 hours, 4 out of 5 mice developed swelling and haemorrhage of theP7584PC00

[1326] 121

[1327] eyeballs, and 1 mouse developed an increased abdominal volume. For A / , melanoleuca venom, partial neutralization was observed, with 3 out of 5 mice surviving and the time of death substantially extended for the other 2 mice (Table 11). The surviving mice presented similar signs of envenoming as observed for D. viridis, but did not show an increased abdominal volume (Table 10). Finally, for D. polylepis venom, the recombinant antivenom delayed the time of death from approximately 0.5 hour in the venom-only control mice to 2 hours, for D. jamesoni the time of death was delayed from approximately 30 min to 60 min and no protective effect was seen with the venom from D. angusticeps (Table 11). Notably, in the rescue setting, the Inoserp PAN¬ AFRICA antivenom showed only partial neutralization of all of the tested venoms and an extension of time of death for the venom from A / , melanoleuca, demonstrating that, except for D. polylepis, the recombinant antivenom performed better than the commercial antivenom on all included venoms at the tested doses (Table 11).

[1328] Conclusions

[1329] The Inoserp PAN-AFRICA antivenom showed only partial neutralization of all of the tested venoms and an extension of time of death for the venom from A / , melanoleuca, demonstrating that, except for D. polylepis, the recombinant antivenom performed better than the commercial antivenom on all included venoms at the tested doses. In particular, the recombinant antivenom fully protected against multiple Naja species and H. haemachatus, prevented lethality but not all symptoms for D. viridis, and partially neutralized A / , melanoleuca. It also delayed death for D. polylepis and D. jamesoni, while showing no effect against D. angusticeps. Overall, these results confirm that the recombinant antivenom provided broader and more effective protection than the commercial antivenom in this clinically relevant setting.

[1330] Example 8: Prevention of dermonecrosis

[1331] Current plasma-derived antivenoms are typically poor at preventing local tissue damage, resulting in a high morbidity rate, including limbs lost, in snakebite victims.7-9 In elapid snakes in sub-Saharan Africa, local tissue damage is primarily associated with spitting snake species (i.e. Afronaja and Hemachatus spp.), and CTx and PLA2 play the most important role. Therefore, the ability of VHH1 a-CTx and VHH4 a-CTx and VHH20 a-PLA2 to prevent venom-induced dermonecrosis caused by N. mossambica, N. nigricollis, and H. haemachatus was assessed using an in vivo pre-incubation setup.P7584PC00

[1332] 122

[1333] After that, the recombinant antivenom (containing the 8 VHHS) was evaluated in another rescue assay where the recombinant antivenom was delivered i.v. 15 minutes after i.d. injection of the venom. Inoserp PAN-AFRICA antivenom was included for comparison with N. nigricol lis venom at a dose recommended by the manufacturer to neutralize 3 LD50S of venom.

[1334] Materials and methods

[1335] For venom challenges, mice were injected intradermally (i.d.) in the ventral abdominal region, with venoms from N. nigr / co / / / s (24 pg / mouse), N. mossambica (39 pg / mouse), and H. haemachatus (26 pg / mouse) dissolved in 50 pL PBS. This dose corresponds to one Minimum Necrotizing Dose (MND), i.e. the dose which induces an area of dermonecrosis of 5 mm in diameter, 72 h after injection. In pre-incubation models, one MND of venom from each of the three snakes was pre-incubated with 1.09 mg of a mixture of VHH1 a-CTx (450 pg / mouse), VHH4 a-CTx (450 pg / mouse), and VHH20 a-PLA2 (190 pg / mouse) at 37 °C for 30 min before i.d. injection. In the first rescue model, one MND dose of venom in 10 pL was injected i.d., followed by 1.09 mg of VHHS in 40 pL at the same region after 15 min. In an alternative rescue model, 1 MND dose of venom was injected i.d. in a 50 pL volume, followed by i.v. administration of 3.6 mg of recombinant antivenom (Table 5) in 200 pL 15 min later. For the control groups the same volume of PBS was administered instead of VHHS. AS a comparison, a group of mice received 1 MND of N. nigricollis venom followed by 4.2 mg of Inoserp PANAFRICA antivenom (I NOSAN Biopharma).

[1336] Mice were monitored continuously for the first 6 h post-injection, with additional checks every 3 h up to 12 h and then 3 times daily up to 72 h in pre-incubation and i.d. rescue models and up to 48 h in i.v. rescue studies. At the end of each experiment, animals were humanely euthanized via inhalational CO2. Lesions at injection sites were dissected, measured in 2 directions with digital calipers, and photographed with a camera and light ring.

[1337] Results

[1338] The HH mixture significantly reduced the dermonecrotic lesion areas, and all but one mouse per treatment group showed a complete absence of lesion for the 2 Naja venoms (FIG.11). In a rescue setup, the HH mixture was delivered i.d. to the same injection region 15 minutes after the venom. Despite observations of rapid discolorationP7584PC00

[1339] 123

[1340] at the injection site within the 15 minute treatment window, the VHH mixture significantly reduced the size of the dermonecrotic lesions caused by each of the three venoms (FIG.11).

[1341] The recombinant antivenom reduced the size of the lesions caused by each of the 3 venoms at the 48-hour experimental endpoint, although these results were only statistically significant for N. nigricollis (FIG.12). In comparison, less reduction of lesion size for Inoserp PAN-AFRICA antivenom on the tested venom from N. nigricollis was observed (FIG.12), demonstrating that the recombinant antivenom also outperforms existing treatment in this model at the tested doses.

[1342] Conclusions

[1343] The VHH mixture significantly reduced dermonecrotic lesion areas in response to Naja venoms, with most mice showing complete lesion absence, even in a rescue setup where treatment was administered 15 minutes post-venom injection; additionally, the recombinant antivenom effectively minimized lesion size for all three venoms at 48 hours, with statistically significant results for N. nigricollis, outperforming the Inoserp PAN-AFRICA antivenom at tested doses.

[1344] Example 9: In silico modeling and / or experimental determination of VHH-toxin interactions

[1345] Materials and methods

[1346] In silico modelling was performed using the AlphaFold 3 Server (https: / / alphafoldserver.com / ) and experimental determination was performed through X-ray crystallography. The data was processed using the software UCSF ChimeraX version: 1.8 (2024-06-10). HHS are shown as ribbons, and toxins are

[1347] shown as surface models. Contact regions between molecules are marked in dark grey on ribbons ( HHS) and surface models (toxins) (FIG.13)

[1348] Results

[1349] For the in silico modelling, the predicted template modelling (pTM) and the interface predicted Template Modelling (ipTM) scores, which provide an evaluation of the accuracy of the models are shown in the following table.P7584PC00

[1350] 124

[1351] Table 11: Predicted template modelling (pTM) and interface predicted Template Modelling (ipTM) scores

[1352] TPLO TPLO TPL1 TPLO TPL1 TPL1 TPL1 TPL1 599 870 158 998 013 033 039 054 _01_ 01 01 01 03 03 01 _01_ C06 G09 C09 A05 F11 D05 C04 A05 P14556 ipTM 0.46 - - - - - - - pTM 0.68 - - - - - - - P01468 ipTM - 0.89 - - - - - - pTM - 0.92 - - - - - - P01391 ipTM - - 0.83 - - - - - pTM - - 0.80 - - - - - P01422 ipTM - - - 0.87 - - - - pTM - - - 0.90 - - - - P00979 ipTM - - - - 0.25 - - - pTM - - - - 0.68 - - - P01407 ipTM - - - - - 0.8 - - pTM - - - - - 0.84 - - P01405 ipTM - - - - - - 0.86 - pTM - - - - - - 0.9 - P01456 ipTM - - - - - - - 0.87

[1353] pTM - - - - - - - 0.89

[1354]

[1355] Using the models, amino acid residues in the toxins in contact with the VHHs of the present disclosure could be identified:

[1356] Anti type IA / IB cytotoxin VHHS

[1357] TPL0870 01 G09 (SEQ ID NO: 57) vs P01468

[1358] I7, P8, P9, L6, P33;or

[1359] K44, R27, L48, S46, L47, 151

[1360] TPL1054 01 A05 vs P01456

[1361] P9, K5, K35

[1362] Alternatively, using the models, amino acid residues in the toxins in contact with the VHHs of the present disclosure could be identified:P7584PC00

[1363] 125

[1364] Anti type IA / IB cytotoxin VHHS TPL0870 01 G09 (SEQ ID NO: 10) vs P01468

[1365] I7, P8, P9, L6, P33;or

[1366] K44, R27, L48, S46, L47, 151

[1367] TPL1054 01 A05 vs P01456

[1368] P9, K5, K35

[1369] Anti Orphan group XI VHH

[1370] TPL1039 01 C04 vs P01405

[1371] P9, E10, K7, K48

[1372] Anti aminergic / Orphan group XIX VHH

[1373] TPL1033 03 D05 vs P01407

[1374] C46, K22, P47, E15

[1375] Anti type I alpha-neurotoxin HH

[1376] TPL0998 01 A05 vs P01422

[1377] Y52, C53, 151, K47, S44, E20, P43

[1378] Anti phospholipase A2 (PLA2) VHH

[1379] TPL0599 01 C06 vs P14556

[1380] Y3, F63, W60, E52, N48, Y62

[1381] Example 10: Structural basis of broad neutralization

[1382] To investigate the molecular basis of the broad neutralization observed for the eight preferred VHHs by examining their binding interactions with target toxins.

[1383] Materials and methods

[1384] Co-crystallization of VHHS and toxins

[1385] Lyophilized toxins and vacuum-dried venom fractions were reconstituted at 10 mg / mL in 5 mM Tris and 20 mM NaCI at pH 8.0. The toxins or venom fractions were then added to the VHHS at a 3-fold molar excess (VHH1 a-CTx: cardiotoxin (P01468), VHH5 a-sNTx: a-short chain neurotoxin (P01426)) and incubated overnight at 4 °C. The VnH:toxin complexes were purified using size-exclusion chromatography (Superdex 75 10 / 300GL column, Cytiva) on an NGC Quest™ 10 Plus Chromatography system (Bio-P7584PC00

[1386] 126

[1387] Rad) maintained at 4 °C, with the reconstitution buffer serving as the mobile phase. Before crystal screening, the VnH:toxin complexes were concentrated to 15.0 mg / mL using 3.0 kDa MWCO ultracentrifugation filters (UFC500324, Merck).

[1388] Crystallization trials were performed at 21 °C via the sitting drop vapour diffusion method. Drops (0.3 pL) were set up at reservoir-to-protein ratios of 2:1, 1:1, or 1:2 in a 96-well drop format on SWISSCI MRC 2 well crystallization plates (JENA) using LMB, BCS, Index, and Structure screening solutions (Hampton Research). The wells were sealed with crystal clear tape and equilibrated against 50 pL of reservoir solution. The VHH1 a-CTx co-crystal formed in 0.2 M ammonium acetate, 0.1 M sodium acetate, pH 4.6, 30% w / v PEG4000. The VHH5 a-sNTx co-crystal formed in 0.2 M Sodium Chloride, 0.1 M sodium acetate, pH 4.6, 30% v / v MPD. The developed crystals were harvested using mounted CryoLoops (Hampton Research) with cryoprotection performed by adding glycerol to a neighbour drop with no crystals to a final concentration of 25%. The loop edge was kept in contact with the cryo solution for approximately 5 s to equilibrate before flash freezing the crystal in liquid nitrogen and shipping to the beamline for remote data collection. Final structural models and corresponding structure factors have been deposited in the Protein Data Bank (PDB) under accession codes: 9RIT and 9RIU.

[1389] Data collection and structure determination

[1390] X-ray diffraction data for the VHH1 a-CTx and VHH5 a-sNTx crystals were obtained at the Biomax (MAX IV synchrotron facility, Lund, Sweden) beamline. Complete datasets were collected over a 360° rotation for the HH1 a-CTx and HH5 a-sNTx crystals. The data processing was performed with XDSAPP3, 79-81 and the data is summarized in Supplementary Tables 10 and 11. Structures of the HHS in complex with their respective toxins were determined by molecular replacement with Phaser-MR82 using an AlphaFold3 model for both the HH and the target toxin as a search model. Model building and refinement were performed with Phenix. refine81 and Coot.83

[1391] The structures were evaluated using MolProbity with final statistics presented in Supplementary Tables 10 and 11. Molecular graphics were presented with PyMOL Molecular Graphics System (Version 2.2r7pre, Schrodinger, LLC).

[1392] Coordinates / structure factors have been submitted to the PDB database with the accession codes 9RIT and 9RIU.

[1393] Generation of in silico predictions of VHH: toxin complexesP7584PC00

[1394] 127

[1395] For VnH:toxin complexes that did not yield protein crystals, protein sequences were submitted as input to AlphaFold3 for structure prediction.85 Multiple predictions were generated using randomized seeds for each VnH:toxin complex. The model exhibiting the highest confidence scores (per-residue confidence estimate (pLDDT), predicted template modelling (pTM), and interface predicted template modelling (ipTM)) were selected for further analysis. Molecular visualization and graphic preparation were presented with PyMOL Molecular Graphics System (Version 2.2r7pre, Schrodinger, LLC).

[1396] Results

[1397] Co-crystallization, cryo-EM, and in silico structural modelling of the HHS in complex with one of their target toxins revealed that the antibodies bind to residues predominantly conserved across the toxins they neutralize (FIG.14). The HH: SNTX and HHJNTX interaction modes were consistent with previous reports. Notably, HH1 a-CTx was found to be biparatopic, a feature that may account for its superior neutralization of cell cytotoxicity compared to HH4 a-CTx (FIG.14A and FIG.14C). The epitopes involved in the interactions of the VHHs with their target toxins, as determined by in silico predictions, are summarized in Table 12.

[1398] Table 12. Epitopes involved in the interactions of HHS with target toxins based on in silico predictions.

[1399] VHH Target Toxin (Sub)Family Epitopes

[1400] 1 Type IA cytotoxin L6, 17, P8, P9, F10, R27, P33, K35, L47, L48, K50 4 Type IA cytotoxin K5, L6, P9, W11, K12, T13, K18, K35, C38 5 Type 1 alpha-neurotoxin C42, T44, V45, L51, N52, C53

[1401] 9 Type II alpha-neurotoxin I9, R33, R36, V37, F65, R70

[1402] 13 Aminergic toxin E15, Q21, K22, A44, T45, C46, K48

[1403] 15 Orphan group XI K7, Q10, K24, K28, G31, R36, K48

[1404] 17 Venom Kunitz-type R15, W37, R46, K48, E52

[1405] 20 Phospholipase A2 N1, R30, D48, W60, Y62, F63

[1406]

[1407] Conclusions

[1408] The VHHS of the final composition achieve broad neutralization by targeting conserved residues across their toxin families.P7584PC00

[1409] 128

[1410] Example 11

[1411] Materials and methods

[1412] Thermal stability and aggregation

[1413] The thermal stability and aggregation of the mixture and each of the 8 VHHS in the mixture were measured by differential scanning fluorimetry (DSF), static light scattering (SLS), and dynamic light scattering (DLS), all using a Prometheus Panta instrument (Nanotemper Technologies GmbH, Munich Germany). Samples were loaded in standard grade capillaries (Nanotemper Technologies GmbH) at a total protein concentration of 20 mg / mL in PBS. Measurements were taken during a thermal ramp from 25 °C to 95 °C, and during isothermal scans at 40, 45, and 50 °C.

[1414] Polyspecificity assessment in DELFI A

[1415] Polyreactivity of VHHS was assessed using a DELFIA-based binding assay. MaxiSorp 96-well plates (Thermo Fisher Scientific) were coated overnight at 4 °C with dsDNA (10 pg / mL; Sigma-Aldrich, D4522), lipopolysaccharide (10 pg / mL; Sigma-Aldrich, L2880), human serum albumin (10 pg / mL; Sigma-Aldrich, A1653), human insulin (5 pg / mL; Sigma-Aldrich, I2643) in PBS, or cardiolipin (10 pg / mL; Sigma-Aldrich, C0563) in 96% ethanol. Plates were blocked with 0.1% (v / v) PBS containing Tween-20 (PBST) for 1 h at room temperature and washed three times with PBST. VHHS or the HH mixture were then applied in seven-point 1:3 serial dilutions starting at 1 pM (5 pM for the mixture) and incubated for 1 h at room temperature.

[1416] For positive controls, plates were coated overnight with streptavidin (2 pg / mL; Sigma-Aldrich, SAE0094), blocked with PBST, and incubated for 1 h with 50 nM biotinylated target antigens corresponding to the HHS prior to addition of the same VHH dilution series.

[1417] a-cobratoxin (L8114), cardiotoxin (L8102), and whole venoms from Dendroaspis jamesoni (L1308) and Naja melanoleuca (L1318) were obtained from Latoxan® (France). Whole venoms were fractionated as previously described (Ahmadi et al. 2025), yielding short neurotoxin (in fraction 3 of Naja melanoleuca venom, Nm3) and AgTx (fraction 10 of Dendroaspis jamesoni venom, Dj10). Toxins and venom fractions were biotinylated as described previously (Ahmadi et al. 2025).

[1418] Plates were washed six times with PBST and incubated for 1 h with europium-conjugated anti-FLAG antibody (Sigma-Aldrich, F3165) in DELFI A assay buffer (Revvity, 4002-0010). After six washes, DELFIA enhancement solution (Revvity, 4001-P7584PC00

[1419] 129

[1420] 0010) was added for 20 min, and europium fluorescence was measured using a Victor Nivo plate reader as described previously (Ahmadi et al. 2025). Experiments were done in duplicate.

[1421] Results

[1422] To assess developability and any potential polyreactivity, the binding of individual HHS and the HH mixture was analyzed in DELFIA against dsDNA, cardiolipin, lipopolysaccharide, insulin, and human serum albumin. No binding occurred, even at high antigen and HH concentrations (5-10 pg / mL and 1 pM, respectively). As expected, strong binding was evident when these VHHS were tested against their corresponding toxin targets at 50 nM (0.34-0.6 pg / mL). These results confirm the lack of polyreactivity of the VHHS against non-toxin antigens (Fig 15).

[1423] The mixture showed a single apparent melting temperature determined by differential scanning fluorimetry (DSF) of around 58 °C. This value is comparable to the apparent melting temperatures of the constituent VHHS, which range from 55 to 72 °C (Table 13). Upon heating, an apparent onset of aggregation of the mixture was observed at around 55 °C by light scattering. This is likewise comparable to the apparent temperatures for onset of aggregation of the constituent VHHS which range between 45 °C and 65 °C. Thus showing no indications of an increased propensity for aggregation in the cocktail.

[1424] Table 13: Apparent melting temperatures and temperatures for the onset of aggregation of the VHH mixture and each of the individual VHHS, all measured at a total protein concentration of 20 mg / mL.

[1425] VHH | App. TM (°C) Onset of Agg. (°C) Mixture | 60 57 TPL0877_01_A04 (INTX) I 72 I 49

[1426] TpL-_ -s-^ [ y‘2 j65TPL0599_01_C06 (PLA2) | 61 | 45 TPL0870_01_G09 (CTx) | 56 | 50 TPL1054_01_A05 (CTx) I 55

[1427] TPL1013_03_F11 (DTx) | 40 TPL1039_01_C04 (Og XI) 73 | 47 TPL1033_03_D05 (AgTx) j 69 | 60

[1428]

[1429] P7584PC00

[1430] 130

[1431] The aggregation of the mixture was also followed by light scattering during isothermal incubations at temperatures between 40 °C and 50 °C, showing minor signs of aggregation within hours (Fig. 16A). By dynamic light scattering, the mixture was found to contain both a small species with a hydrodynamic radius of around 2.5 nm consistent with monomeric VHHS, and larger species with hydrodynamic radius in the range from tens to hundreds of nm (Fig 16B). The small species was present in measurable quantities from start to finish of the 16 h incubation at 40 °C, indicating that the extent of aggregation is limited during this time-scale, and that the monomeric species persist and continue to dominate by numbers. Combined, this indicates good stability properties for the VHH mixture also at elevated temperatures and a similar behavior between the individual VHHS and the VHH mixture.

[1432] References

[1433] Ahmadi, S. et al. An in vitro methodology for discovering broadly-neutralizing monoclonal antibodies. Nat. Sci. Reports 10, (2020).

[1434] Ahmadi, S., Burlet, N. J., Benard-Valle, M. etal. Nanobody-based recombinant antivenom for cobra, mamba and rinkhals bites. Nature 647, 716-725 (2025).

[1435] Benard-Valle, M. et al. In vivo neutralization of coral snake venoms with an oligoclonal nanobody mixture in a murine challenge model. Nat Commun 15, 4310 (2024).

[1436] Casasola, A. et al. Paraspecific neutralization of the venom of African species of cobra by an equine antiserum against Naja melanoleuca: A comparative study. Toxicon 53, 602-608 (2009).

[1437] De La Rosa, G., Corrales-Garcia, L. L., Rodriguez-Ruiz, X., Estuardo Lopez-Vera, ■ & Corzo, G. Short-chain consensus alpha-neurotoxin: a synthetic 60-mer peptide with generic traits and enhanced immunogenic properties. 50, 885-895 (2018).

[1438] Guidelines for the production, control and regulation of snake antivenom immunoglobulins, Annex 5, TRS No 1004. 192 (World Health Organisation, 2013). Kabat, E. A. et al. Sequences of proteins of immunological interest. US Department of Health and Human Services, National Institutes of Health (1991).

[1439] Laustsen, A. H. Antivenom in the Age of Recombinant DNA Technology, in Handbook of Venoms and Toxins of Reptiles (CRC Press, 2021).P7584PC00

[1440] 131

[1441] Laustsen, A. H. et al. In vivo neutralization of dendrotoxin-mediated neurotoxicity of 20 black mamba venom by oligoclonal human IgG antibodies. Nat. Commun. 9, 3928 (2018).

[1442] Laustsen, A. H. et al. Animal Immunization, in Vitro Display Technologies, and Machine Learning for Antibody Discovery. Trends in Biotechnology. 39, 1263-1273 25 (2021)

[1443] Laustsen, A. H. et al. From Fangs to Pharmacology: The Future of Snakebite Envenoming Therapy. CPD. 22, 5270-5293 (2016).

[1444] Ledsgaard, L. et al. In vitro discovery and optimization of a human monoclonal antibody that neutralizes neurotoxicity and lethality of cobra snake venom. 2021.09.07.459075 https: / / www.biorxiv.org / content / 10.1101 / 2021.09.07.459075v1 (2021)

[1445] doi: 10.1101 / 2021.09.07.459075.

[1446] Ledsgaard, L. et al. Discovery and optimization of a broadly-neutralizing human monoclonal antibody against long-chain a-neurotoxins from snakes. Nat. Commun. 14, (2023).

[1447] Marks, J. D. et al. By-Passing Immunization: Building High Affinity Human Antibodies by Chain Shuffling. Bio / Technology 10, 779-783 (1992).

[1448] Muyldermans, S. et al. Sequence and structure of VH domain from naturally occurring camel heavy chain immunoglobulins lacking light chains. Protein Eng 7, 1129-1135 (1994).

Claims

P7584PC00132Claims1. A composition comprising at least two of the following antigen-binding proteins:• an antigen-binding protein ‘A’ capable of binding to, blocking, and / or neutralizing a snake venom Type IA or Type IB cytotoxin; and / or• an antigen-binding protein ‘B’ capable of binding to, blocking, and / or neutralizing a snake venom Orphan group XI toxin; and / or • an antigen-binding protein ‘C’ capable of binding to, blocking, and / or neutralizing a snake venom Orphan group XIX toxin and / or an aminergic toxin; and / or• an antigen-binding protein ‘D’ capable of binding to, blocking, and / or neutralizing a snake venom Kunitz-type protease inhibitor; and / or• an antigen-binding protein ‘E’ capable of binding to, blocking, and / or neutralizing a snake venom Type I a-neurotoxin (short-chain a- neurotoxin); and / or• an antigen-binding protein ‘F’ capable of binding to, blocking, and / or neutralizing a snake venom Type II a-neurotoxin (long-chain a- neurotoxin); and / or• an antigen-binding protein ‘G’ capable of binding to, blocking, and / or neutralizing a snake venom phospholipase A2 (PLA2) toxin.

2. A composition comprising:• an antigen-binding protein ‘A’ capable of binding to, blocking, and / or neutralizing a snake venom Type IA or Type IB cytotoxin;• an antigen-binding protein ‘B’ capable of binding to, blocking, and / or neutralizing a snake venom Orphan group XI toxin;• an antigen-binding protein ‘C’ capable of binding to, blocking, and / or neutralizing a snake venom Orphan group XIX toxin and / or an aminergic toxin; and / or• an antigen-binding protein ‘D’ capable of binding to, blocking, and / or neutralizing a snake venom Kunitz-type protease inhibitor toxin.

3. The composition according to any one of the preceding claims, furthercomprising:P7584PC00133• an antigen-binding protein ‘E’ capable of binding to, blocking, and / or neutralizing a snake venom Type I a-neurotoxin (short-chain a- neurotoxin); and / or• an antigen-binding protein ‘F’ capable of binding to, blocking, and / or neutralizing a snake venom Type II a-neurotoxin (long-chain a- neurotoxin); and / or• an antigen-binding protein ‘G’ capable of binding to, blocking, and / or neutralizing a snake venom phospholipase A2 (PLA2) toxin.

4. The composition according to any one of the preceding claims, wherein the antigen-binding protein ‘A’ binds an epitope of a snake venom Type IA or Type IB cytotoxin comprising the amino acid residues:- 17, P8, P9, L6, and P33 of SEQ ID NO. 1;- K44, R27, L48, S46, L47 and 151 of SEQ ID NO. 1; and / or- P9, K5 and K35 of SEQ ID NO. 2.

5. The composition according to any one of the preceding claims, wherein the antigen-binding protein ‘B’ binds an epitope of a snake venom Orphan group XI toxin comprising the amino acid residues:- P9, Q10, K7 and K48 of SEQ ID NO. 3.

6. The composition according to any one of the preceding claims, wherein the antigen-binding protein ‘C’ binds an epitope of an aminergic toxin comprising the amino acid residues:- C46, K22, P47 and E15 of SEQ ID NO. 4.

7. The composition according to any one of the preceding claims, wherein the antigen-binding protein ‘D’ binds an epitope of a snake venom Kunitz-type protease inhibitor comprising one or more amino acid residues selected from the group consisting of:- R15, W37, R46, L48 and E52 of SEQ ID NO: 5.

8. The composition according to any one of the preceding claims, wherein the antigen-binding protein ‘E’ binds an epitope of a snake venom Type I a- neurotoxin (short-chain a-neurotoxin) comprising one or more amino acid residues selected from the group consisting of:P7584PC00134- Y52, C53, 151, K47, S44, E20 and P43 of SEQ ID NO. 7 and / or- C42, T44, V45, L51, N52 and C53 of SEQ ID NO: 43.

9. The composition according to any one of the preceding claims, wherein the antigen-binding protein ‘F’ binds an epitope of a snake venom Type II a- neurotoxin (long-chain a-neurotoxin) comprising one or more amino acid residues selected from the group consisting of:- R36, P7, D27 and R33 of SEQ ID NO. 8.

10. The composition according to any one of the preceding claims, wherein the antigen-binding protein ‘G’ binds an epitope of a snake venom phospholipase A2s toxin comprising one or more amino acid residues selected from the group consisting of:- Y3, F63, W60, E52, D48 and Y62 of SEQ ID NO. 9.

11. The composition according to any one of the preceding claims, wherein at least one antigen-binding protein ‘A’ comprises or consists of a heavy chain variable (VH) region comprising complementary determining regions (CDRs) comprising or consisting of:- a CDR1 as set forth by SEQ ID NO: 58; and- a CDR2 as set forth by SEQ ID NO: 59; and- a CDR3 as set forth by SEQ ID NO: 60or- a CDR1 as set forth by SEQ ID NO: 74; and- a CDR2 as set forth by SEQ ID NO: 75; and- a CDR3 as set forth by SEQ ID NO: 76.

12. The composition according to any one of the preceding claims, wherein the antigen-binding protein ‘B’ comprises or consists of a heavy chain variable (VH) region comprising complementary determining regions (CDRs) comprising or consisting of:- a CDR1 as set forth by SEQ ID NO: 62; and- a CDR2 as set forth by SEQ ID NO: 63; and- a CDR3 as set forth by SEQ ID NO: 64.P7584PC0013513. The composition according to any one of the preceding claims, wherein the antigen-binding protein ‘C’ comprises or consists of a heavy chain variable (VH) region comprising complementary determining regions (CDRs) comprising or consisting of:- a CDR1 as set forth by SEQ ID NO: 66; and- a CDR2 as set forth by SEQ ID NO: 67; and- a CDR3 as set forth by SEQ ID NO: 68.

14. The composition according to any one of the preceding claims, wherein the antigen-binding protein ‘D’ comprises or consists of a heavy chain variable (VH) region comprising complementary determining regions (CDRs) comprising or consisting of:- a CDR1 as set forth by SEQ ID NO: 70; and- a CDR2 as set forth by SEQ ID NO: 71; and- a CDR3 as set forth by SEQ ID NO: 72.

15. The composition according to any one of the preceding claims, wherein the antigen-binding protein ‘E’ comprises or consists of a heavy chain variable (VH) region comprising complementary determining regions (CDRs) comprising or consisting of:- a CDR1 as set forth by SEQ ID NO: 78; and- a CDR2 as set forth by SEQ ID NO: 79; and- a CDR3 as set forth by SEQ ID NO: 80.

16. The composition according to any one of the preceding claims, wherein the antigen-binding protein ‘F’ comprises or consists of a heavy chain variable (VH) region comprising complementary determining regions (CDRs) comprising or consisting of:- a CDR1 as set forth by SEQ ID NO: 82; andP7584PC00136- a CDR2 as set forth by SEQ ID NO: 83; and- a CDR3 as set forth by SEQ ID NO: 84.

17. The composition according to any one of the preceding claims, wherein the antigen-binding protein ‘G’ comprises or consists of a heavy chain variable (VH) region comprising complementary determining regions (CDRs) comprising or consisting of:- a CDR1 as set forth by SEQ ID NO: 86; and- a CDR2 as set forth by SEQ ID NO: 87; and- a CDR3 as set forth by SEQ ID NO: 88.

18. A multi-specific antigen-binding protein, comprising or consisting of at least two of the following:• an antigen-binding protein ‘A’ capable of binding to, blocking, and / or neutralizing a snake venom Type IA or Type IB cytotoxin; and / or• an antigen-binding protein ‘B’ capable of binding to, blocking, and / or neutralizing a snake venom Orphan group XI toxin; and / or• an antigen-binding protein ‘C’ capable of binding to, blocking, and / or neutralizing a snake venom Orphan group XIX toxin and / or an aminergic toxin; and / or• an antigen-binding protein ‘D’ capable of binding to, blocking, and / or neutralizing a snake venom Kunitz-type protease inhibitor; and / or• an antigen-binding protein ‘E’ capable of binding to, blocking, and / or neutralizing a snake venom Type I a-neurotoxin (short-chain a- neurotoxin); and / or• an antigen-binding protein ‘F’ capable of binding to, blocking, and / or neutralizing a snake venom Type II a-neurotoxin (long-chain a- neurotoxin); and / or• an antigen-binding protein ‘G’ capable of binding to, blocking, and / or neutralizing a snake venom phospholipase A2 (PLA2) toxin.

19. A kit of parts comprising:a) the composition according to any one of claims 1 to 17; andb) another agent suitable for the treatment of snakebite or snake envenomation.P7584PC0013720. A composition, a multi-specific antigen-binding protein or a kit-of-parts according to any one of the preceding claims for use in a method of treating snakebite or snake envenomation in an individual in need thereof.

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