In vivo neutralization of coral snake venoms with an oligoclonal nanobody mixture

Abstract

The present invention relates to antigen-binding proteins capable of fully neutralizing snake venom PLA2 neurotoxin lethality, antigen-binding proteins capable of fully neutralizing snake venom short-chain alpha neurotoxin lethality, and oligoclonal antigen-binding mixtures capable of binding to, blocking, or neutralizing PLA2 neurotoxin and short-chain alpha neurotoxin from coral snake venoms. The invention also relates to pharmaceutical compositions, kit of parts and kits for the treatment and diagnosis of snake envenomation.

Description

[0001] In vivo neutralization of coral snake venoms with an oligoclonal nanobody mixture

[0002] Technical field

[0003] The present invention relates to antigen-binding proteins for use in treatment of coral snake envenomation. The antigen-binding proteins are for example capable of fully neutralizing snake venom PLA2 neurotoxin lethality or capable of fully neutralizing snake venom short-chain alpha neurotoxin. The invention also provides oligoclonal antigen-binding mixtures capable of binding to, blocking, or neutralizing PLA2 neurotoxin and short-chain alpha neurotoxin from coral snake venoms.

[0004] Background

[0005] Every year, over 2,400 people living in the Americas fall victim to coral snake envenomations. When the toxins of coral snake venoms are injected into mammalian prey and victims, they exert neurotoxic effects that manifest clinically as flaccid paralysis of skeletal muscles, which, if left untreated, can be fatal as this may progress to respiratory failure. Currently, the only available specific treatments for envenomed patients are antivenoms that consist of polyclonal antibodies isolated from the plasma of hyperimmunized animals. While these antivenoms have saved countless lives, they unfortunately suffer from several drawbacks, including a limited capacity to cross- neutralize venom from different coral snake species, batch-to-batch variation and a low content of therapeutically active antibodies. In comparison with many other antivenoms, such as those for viper envenomations, the latter drawback is even more relevant for coral snake antivenoms, as the low abundance and limited immunogenicity of the medically most relevant toxins in coral snake venoms ( / .e. PLA2S and 3FTxs) make it difficult to raise neutralizing antibodies via the animal immunization process used for traditional antivenom manufacture.

[0006] Summary

[0007] There is therefore a need for antigen-binding proteins capable of neutralizing the effects of the venom toxins, ideally fully neutralizing lethality of said toxins or whole venom. To date, no antigen-binding proteins, or mixtures thereof, have been disclosed that are able to fully neutralize the effects, a fortiori fully neutralize lethality, of the medically relevant toxins of coral snakes, namely phospholipases A2 (PLA2S) and short-chain alpha neurotoxins, let alone of whole coral snake venom.

[0008] Traditional approaches for envenomation treatment involve the use of polyclonal immunoglobulins from animal sources. An important limitation of these treatments is however that they often fail to neutralize many of medically relevant toxins. If they could neutralize all these toxins, it would improve the efficacy of the antivenoms.

[0009] This invention presents an antigen-binding protein capable of binding to, blocking, and / or neutralizing snake venom PLA2 neurotoxin lethality and an antigen-binding protein capable of binding to, blocking, and / or neutralizing venom short-chain a- neurotoxin lethality. Thus, by developing high-throughput pipelines for the generation of high affinity broadly-neutralizing antibodies, the present inventors were capable of developing antigen-binding proteins capable of neutralizing snake venom PLA2 neurotoxin and short-chain a-neurotoxin lethality. More importantly, the invention provide antigen-binding proteins and mixtures thereof capable of reducing or even neutralising whole snake venom lethality.

[0010] The antibodies of the invention have been developed by using phage display technology. It was surprisingly found that particularly useful nanobodies, broadly- neutralizing antigen-binding proteins targeting the medically most important toxins, namely phospholipases A2 (PLA2S) and short-chain alpha neurotoxins, in coral snake venoms could be isolated from a phage display library generated from one alpaca and one llama immunized with multiple elapid snake venoms. The phage display library was screened against the key venom toxins phospholipases A2 (PLA2S) and shortchain alpha neurotoxins and against recombinantly produced consensus antigens (i.e., antigens designed to represent the ‘average’ of several different proteins) (Rivera-de- torre et al., 2023). For the discovery of VHHS against these toxins, a cross-panning strategy was used (Ahmadi et al., 2018; Ledsgaard et al., 2023; Sorensen et al., 2023). The methodology described herein enabled the discovery of specific antibodies against the most medically relevant toxins, regardless of their abundance within the venoms or their immunogenicity (Roncolato et al., 2015) To date, work on recombinant monoclonal antibodies has primarily focused on using human monoclonal immunoglobulin G (IgG) antibodies, which are highly specific and have long serum half-lives, but they have limited stability ex vivo and are relatively expensive to manufacture compared to other types of recombinant binding proteins. Preferred antigen-binding proteins according to the present invention are camelid based single-domain antibodies (VHHS), which are also known as nanobodies. VHHS may be derived from heavy-chain-only antibodies present in Camelidae and are typically characterized by similar high affinity and specificity as IgG antibodies. They are significantly more stable at high temperatures and extreme pH than IgGs, and they can generally be expressed in large scale at lower cost. This invention discloses antigen-binding proteins, such as VHHS that can neutralize the lethality of coral snake neurotoxins.

[0011] Furthermore, the invention presents oligoclonal mixtures of antigen-binding proteins, e.g. HHS that can neutralize the lethality of the whole venoms.

[0012] A first aspect of the invention relates to an antigen-binding protein capable of binding to, blocking, and / or neutralizing snake venom PLA2 neurotoxin lethality.

[0013] A second aspect of the invention relates to an antigen-binding protein capable of binding to, blocking, and / or neutralizing venom short-chain a-neurotoxin lethality.

[0014] A third aspect of the invention relates to an oligoclonal antigen-binding protein mixture comprising at least two different antigen-binding proteins, wherein one of said antigenbinding proteins is capable of binding to, blocking, and / or neutralizing snake venom PLA2 neurotoxin and wherein another of the at least two different antigen-binding proteins is capable of binding to, blocking, and / or neutralizing snake venom short-chain alpha neurotoxin.

[0015] A fourth aspect of the invention relates to a multi-specific antigen-binding protein, one antigen-binding portion capable of binding to, blocking, and / or neutralizing snake venom PLA2 neurotoxin and another antigen-binding portion capable of binding to, blocking, and / or neutralizing snake venom short-chain alpha neurotoxin. A fifth aspect of the invention relates to a pharmaceutical composition comprising one or more antigen-binding proteins, the oligoclonal antigen-binding protein mixture or the multi-specific antigen-binding protein according to any one of the preceding items, and a pharmaceutically acceptable diluent, carrier and / or excipient.

[0016] A sixth aspect of the invention relates to a kit of parts comprising: a) one or more antigen-binding proteins, the oligoclonal antigen-binding protein mixture or the multi-specific antigen-binding protein as described herein; and b) another agent suitable for the treatment of snake envenomation.

[0017] A seventh aspect of the invention relates to an antigen binding protein, a oligoclonal antigen-binding protein mixture, a multi-specific antigen-binding protein, a pharmaceutical composition or a kit-of-parts as described herein, for use in the treatment of snake envenomation in a subject.

[0018] An eighth aspect of the invention relates to a method for treating snake envenomation in a subject, the method comprising administering a therapeutically effective amount of the antigen binding protein, the oligoclonal antigen-binding protein mixture, the multispecific antigen-binding protein, the pharmaceutical composition, or the kit of parts as described herein.

[0019] A ninth aspect of the invention relates to the use of the antigen binding protein, the oligoclonal antigen-binding protein mixture, the multi-specific antigen-binding protein, the pharmaceutical composition, or the kit of parts as described herein in a method of treatment of snake envenomation in a subject.

[0020] A tenth aspect of the invention relates to the use of the antigen binding protein, the oligoclonal antigen-binding protein mixture, the multi-specific antigen-binding protein, the pharmaceutical composition, or the kit of parts as described herein for the manufacture of a medicament for the treatment of snake envenomation.

[0021] An eleventh aspect of the invention relates to a kit for detection and / or quantification of snake venom in a sample comprising one or more of the antigen binding proteins, the oligoclonal antigen-binding protein mixtures or the multi-specific antigen-binding proteins as described herein.

[0022] A twelfth aspect of the invention relates to an antigen-binding protein, a oligoclonal antigen-binding protein mixture or a multi-specific antigen-binding protein as described herein for use in the diagnosis of snake envenomation in a subject.

[0023] A thirteenth aspect of the invention relates to the use of the antigen binding protein, the oligoclonal antigen-binding protein mixture or the multi-specific antigen-binding protein as described herein in an in vitro method for detection and / or diagnosis of snake envenomation in a subject.

[0024] The invention is defined in the attached claims.

[0025] Description of Drawings

[0026] Figure 1 : Antibody responses of two immunized camelids overtime. Antibody binding signals observed for samples collected at different time points from two camelids immunized with a mixture of 18 elapid venoms. A. Response of llama 0406. B. Response of alpaca 0541. Values correspond to the mean ± standard error (n = 2). RLU: Relative Light Units.

[0027] Figure 2 : Binding kinetics of monovalent VHHS constructs. Biotinylated toxins captured on streptavidin biosensors were dipped in decreasing concentrations of each of the HHS, followed by dissociation in kinetics buffer. Binding data were fitted using a 1 :1 model. A to C. Anti-PLA2 HHS binding PLA2N. D to F. Anti-aNTx VHHS binding DH.

[0028] Figure 3 : Binding kinetics of the VHH TPL0629_01_D11 as bivalent construct and binding kinetic parameters overview. G. Biotinylated toxin DH captured on streptavidin biosensors was dipped in decreasing concentrations of bivalent TPL0629_01_D11 , followed by dissociation in kinetics buffer. Binding data were fitted using a 1:1 model. H. Overview of the binding kinetic parameters obtained for the constructs tested in panels A to F of Fig. 2 and panel G of figure 3 respectively as indicated. The symbol used in the data of panel H is to be understood as a multiplicative sign e.g. “4.76-1O'10" and “4.76x1 O'10” are equivalents. Figure 4 : In vitro neutralization of PLA2 enzymatic activity by VHHS. Normalized enzymatic activity of PLA2N (100%) and PLA2N preincubated for 30 min with anti-PLA2 VHHS at 350 nM (1 :10 toxin to VHH molar ratio). TPL0629_01_D11 was included as a negative isotype control.

[0029] Figure 5 : Kaplan-Meier survival curves for mice challenged with PLA2N with or without the addition of VHHS. A. 3 LD50s of the toxin were preincubated at a 1 :1 toxin to HH molar ratio with either PBS or one of the VHHS and injected using the i.v. route. B. The mice were injected with 3 LD50s of toxin using the s.c. route followed by immediate i.v. injection with PBS or one of the VHHS using a 1 :2.5 toxin to VHH molar ratio.

[0030] Figure 6 : Kaplan-Meier survival curves for mice challenged with 3LDsoS of aNTx DH with or without VHH TPL0629_01_D11 in preincubation and rescue experiments. Preincubation and rescue experiments are shown for: A. and B., the monovalent, or C. and D., the bivalent VHH constructs. Molar ratios between the toxin and VHH binding sites are shown in parentheses.

[0031] Figure 7 : Kaplan-Meier survival curves for mice challenged with whole venoms in preincubation experiments. 3 LD50s of venom from A. M. diastema or B. M. fulvius, were preincubated with either PBS, the individual VHHS, or the VHH oligoclonal mixture. Toxin to VHH molar ratios were 1 :10.

[0032] Figure 8 : Binding of TPL0629_01_D11 to a-neurotoxins and TPL0637_01_A07 to PLA2s

[0033] DELFIA assay of the VHHS TPL0629_01_D11 (Left panel) and TPL0637_01_A07 (Right panel) were performed against different concentrations of a-neurotoxins and PLA2S, respectively. Alpha-cobratoxin is used as negative control. TRF: Time-Resolved Fluorescence.

[0034] Figure 9 : In silico modeling of VnH-toxin interactions.

[0035] In silico modelling was performed using the AlphaFold 3 Server (https: / / alphafoldserver.com / ) and processed using the software LICSF ChimeraX version: 1.8 (2024-06-10). A. Anti short chain alpha neurotoxin nanobodies interaction models. The interaction model of each VHH against (Vs) DHdia and scNTx toxins is presented. B. Anti PLA2 nanobodies interaction models. The interaction model of each VHH against (Vs) PLA2O toxin is presented. HHS are shown as ribbons (left side of each interaction model), and toxins are shown as surface models (right side of each interaction model). Contact regions between molecules are marked in dark grey on ribbons ( HHS) and surface models (toxins).

[0036] Detailed description

[0037] Definitions

[0038] 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.

[0039] As used herein, the term “antigen-binding protein” includes antibodies, antibody fragments and other antigen-binding protein constructs. The term encompasses 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 “sdAbs”, ScFv, heavy chain variable region fragment (referred to as HH or VHH), and singlechain antibodies. In preferred embodiments, the antigen-binding protein is a domain antibody. Preferably, the antigen binding molecule is a sdAb, i.e. an immunoglobulin single variable domain, such as a VH, VHH or VL.

[0040] The term “antigen binding molecule” as used herein refers to molecules capable of binding to target(s), such as two or more different targets. Antigen binding molecules may thus be dual targeting proteins. “Antigen binding molecule” may comprise of or consist of two or more antigen-binding proteins according to the invention. Thus, antigen-binding molecules may be a combination of any of the antibodies and antigen binding fragments described herein such as for example, one or more domain antibodies and / or one or more ScFvs linked to another antigen-binding molecule, for instance to a monoclonal antibody. The term “epitope” as used herein refers to a specific region or sequence of a macromolecule, typically a protein, 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. Which epitope is recognised by a given antigen binding protein may be determined using In silico modelling, for example 3D structures of antigen binding proteins bound to their antigens can be generated using AlphaFold3, referred to herein also as AlphaFold. Alphafold represents a state-of-the-art artificial intelligence (Al) system developed by DeepMind for predicting the three-dimensional (3D) structures of proteins from their amino acid sequences. This system is detailed in the publication: Jumper, J., Evans, R., Pritzel, A. et al. "Highly accurate protein structure prediction with AlphaFold." Nature 596, 583-589 (2021). https: / / doi.org / 10.1038 / s41586-021-03819-2. More specifically, In silico modelling may be performed as detailed herein in Example 8. Alternatively, which epitope is recognized by an antigen binding protein may be determined by a number of laboratory techniques well known to the skilled person, including, but not limited to peptide mapping, peptide scanning, site-directed mutagenesis methods, ELISA or Surface Plasmon Resonance.

[0041] “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 consta nt 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.

[0042] The terms “PLA2” and “phospholipase A2” are used herein as equivalent terms. The terms “PLA2 neurotoxin”, “PLA2-P neurotoxin” and “PLA2-beta neurotoxin” are used herein as equivalents. PLA2 neurotoxins include for instance PLA2O and PLA2N. The cytotoxic PLA2S Nn19 and Hh3 are also included to show broad binding of antigenbinding proteins of the present invention for PLA2S with different toxic activities. Nn19 and Hh3 refer to venom fractions originating from the venoms of the African snake species Naja nigricollis (black-necked spitting cobra) and Hemachatus haemachatus (rinkhal) described in Nguyen, G.T.T et al (2022). “PLA2O” as used herein refers to a wild-type PLA2 purified from fraction O of the venom of Micrurus fulvius as described in Vergara et al. 2014 and comprising the polypeptide sequence as set forth in SEQ. ID NO. 25. The mature native sequence of PLA2O can be found as GenBank accession number GAEP01002028 and set forth as SEQ ID NO. 34 herein. “PLA2N” as used herein refers to a wild-type PLA2 purified from fraction N of the venom of Micrurus fulvius as described in Vergara et al. 2014 and comprising the polypeptide sequence as set forth in SEQ. ID NO. 26.

[0043] The terms “short-chain a-neurotoxin”, “short-chain alpha neurotoxin” “short-chain neurotoxin”, “short neurotoxin”, and “aNTx” are used herein as equivalent terms. Shortchain alpha neurotoxins include for example Eurytoxin from Micruroides euryxanthus and its recombinant version rEury as described in Benard-Valle et al. 2021 , and the toxin D.H also referred to as “DH” or “DHdia” from Micrurus diastema and its recombinant version rDH, or“rDHdia”, as described in Guerrero-Garzon et al. 2018, scNTx, referring to the consensus short-chain alpha neurotoxin of polypeptide sequence as set forth in SEQ. ID. NO. 27, Hh1 and / or Nh2. Hh1 and Nh2 refer to venom fractions originating from the venoms of the African snakes Hemachatus haemachatus (rinkhal) and Naja haje (Egyptian cobra) described in Nguyen, G.T.T et al (2022). Mature native sequences of DH and scNTX without tags and signal peptides are provided as SEQ ID NO: 35 and SEQ ID NO: 36, respectively.

[0044] As used herein the term “binding to” refers to the formation of a non-covalent association between an antigen-binding protein and an antigen.

[0045] The term “blocking” as used herein refers to the binding of the antigen-binding protein to the antigen and interference with the antigen function, for example preventing the antigen from binding to other antigen-binding proteins and / or other target ligands of the antigen. The term “neutralizing” as used herein refers to an antigen-binding protein presenting the ability to substantially inhibit or eliminate the biological activity of a toxin, such as a venom toxin. Typically, a neutralizing anti-toxin antibody may inhibit or eliminate directly or indirectly, the activity of said toxin, such as for example enzymatic activity, signal transduction, induction of lysis, myotoxicity, neurotoxicity, anticoagulating activity, hypotensive activity, hemolytic activity, anti-platelet aggregation activity, bactericidal activity, proinflammatory activity and / or ultimately inhibit or eliminate in vivo lethality. In case of complete elimination of the biological activity (e.g. cytotoxicity, in vivo lethality), the term “fully neutralizing” of said biological activity is also used herein.

[0046] As used herein, the term “variant” refers to either a naturally occurring variation of a given peptide or a recombinantly prepared variation of a given peptide or protein in which one or more amino acid residues have been modified by amino acid substitution, addition, or deletion. The term “variant” may also define either a naturally occurring genetic mutant of a DNA sequence or its encoded RNA or protein product, or a recombinantly prepared variation of a DNA sequence or its encoded RNA or protein product.

[0047] As used herein, "KD" refers to the equilibrium dissociation constant for two binding molecules, e.g. the equilibrium dissociation constant for binding between an “antigenbinding protein” and its antigen. The KD value is expressed in Molar units (M), for example picomolar (pM) or nanomolar (nM), and is obtained by dividing the dissociation rate (kOff) by the association rate (kon). The association rate, the dissociation rate and the equilibrium dissociation constant are used to represent the binding affinity of an antibody to an antigen.

[0048] The term “EC50” or “half maximal effective concentration” as used herein, refers to the concentration of an agent, as described herein at which it induces a response halfway between the baseline and maximum response after some specified exposure time, such as maximum change in Relative Fluorescence Unit per second (RFU / s) rate; the EC50 of a graded dose response curve therefore represents the concentration of a compound at which 50% of its maximal effect is observed. 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).

[0049] Antigen-binding protein capable of binding to, blocking, and / or neutralizing snake venom PLA2 neurotoxin

[0050] The present invention relates to an antigen-binding protein capable of binding to, blocking, and / or neutralizing snake venom PLA2, preferably snake venom PLA2 neurotoxin. In some embodiments of the present invention, the antigen-binding protein is capable of binding to snake venom PLA2 neurotoxin. In some embodiments of the present invention, the antigen-binding protein is capable of blocking snake venom PLA2 neurotoxin. In preferred embodiments of the present invention, the antigen-binding protein is capable of neutralizing snake venom PLA2 neurotoxin.

[0051] In preferred embodiments, the antigen-binding proteins of the present invention are cross-reactive for several PLA2 neurotoxins.

[0052] Thus, in some embodiments, the antigen-binding protein is capable of binding to, blocking, and / or neutralizing one or more PLA2S, such as at least two, such as at least three, such as at least four PLA2S.

[0053] In other embodiments, the antigen-binding protein is capable of binding to, blocking, and / or neutralizing snake venom PLA2N.

[0054] In some embodiments, the antigen-binding protein is capable of binding to, blocking, and / or neutralizing snake venom is PLA2O.

[0055] In other embodiments, the antigen-binding protein is capable of binding to, blocking, and / or neutralizing snake venom Nn19.

[0056] In some embodiments, the antigen-binding protein is capable of binding to, blocking, and / or neutralizing snake venom Hh3.

[0057] In preferred embodiments, the antigen-binding protein is capable of binding to, blocking, and / or neutralizing snake venom PLA2N, PLA2O, Nn19, and / or Hh3, preferably the antigen-binding protein is capable of binding to, blocking, and / or all of neutralizing snake venoms PLA2N, PLA2O, Nn19, and Hh3.

[0058] The person skilled in the art will appreciate that the antigen-binding proteins recognize epitopes of the antigen. For example, an epitope can comprise or consist of a portion of a peptide (e.g., a PLA2 neurotoxin). An epitope can be a linear epitope, sequential epitope, or a conformational epitope.

[0059] The antigen binding proteins of the present invention may thus recognize epitopes within PLA2O sequences comprising the sequence as set forth in SEQ. ID NO. 25, such as the mature native PLA2O sequence as set forth in SEQ ID NO. 34.

[0060] In some embodiments, the antigen binding protein binds an epitope of PLA2O comprising one or more, preferably at least two, more preferably at least three amino acid residues selected from the group consisting of: L3, A22, G29, and S30 of SEQ ID NO. 34. In some embodiments, the antigen binding protein binds an epitope of PLA2O comprising all of L3, A22, G29, and S30 of SEQ ID NO. 34.

[0061] In other embodiments, the antigen binding protein binds an epitope of PLA2O comprising one or more, preferably at least 3, more preferably at least 5, more preferably at least 9, more preferably at least 12 amino acid residues selected from the group consisting of:

[0062] - N2, L3, I4, K7, S17, W18, W19, H20, A22, D23, G29, S30, Y110, and K111 of SEQ ID NO. 34; or

[0063] - L1 , L3, F6, A22, G29, S30, G31 , G32, H47, D48, Y51 , D52, E55, K56, G59, W61 , K63, and W64 of SEQ ID NO. 34; or

[0064] - L1, N2, L3, F6, K7, S17, W18, W19, H20, A22, D23, G29, S30, D107, Y110, and K111 of SEQ ID NO. 34.

[0065] In other embodiments, the antigen binding protein binds an epitope of PLA2O comprising:

[0066] - all of the amino acids N2, L3, I4, K7, S17, W18, W19, H20, A22, D23, G29, S30, Y110, and K111 of SEQ ID NO. 34; or

[0067] - all of the amino acids L1 , L3, F6, A22, G29, S30, G31, G32, H47, D48, Y51 , D52, E55, K56, G59, W61, K63, and W64 of SEQ ID NO. 34; or - all the amino acids L1 , N2, L3, F6, K7, S17, W18, W19, H20, A22, D23, G29, S30, D107, Y110, and K111 of SEQ ID NO. 34.

[0068] In some embodiments, the antigen-binding protein referred to as TPL0637_01_A07 binds an epitope of PLA2O comprising one or more amino acid residues selected from the group consisting of: N2, L3, I4, K7, S17, W18, W19, H20, A22, D23, G29, S30, Y110, and K111 of SEQ ID NO. 34.

[0069] In some embodiments, the antigen-binding protein referred to as TPL0637_01_A01 binds an epitope of PLA2O comprising one or more amino acid residues selected from the group consisting of: L1 , L3, F6, A22, G29, S30, G31 , G32, H47, D48, Y51 , D52, E55, K56, G59, W61, K63, and W64 of SEQ ID NO. 34.

[0070] In some embodiments, the antigen-binding protein referred to as TPL0638_01_C09 binds an epitope of PLA2O comprising one or more amino acid residues selected from the group consisting of: L1, N2, L3, F6, K7, S17, W18, W19, H20, A22, D23, G29, S30, D107, Y110, and K111 of SEQ ID NO. 34.

[0071] In yet other embodiments, the antigen binding protein binds an epitope of PLA2O comprising residues 2-3, 29-30, 17-20, 22-23, 29-30 and / or 110-111 of SEQ ID NO. 34.

[0072] In some embodiments of the present invention, the antigen-binding protein is capable of neutralizing snake venom PLA2 neurotoxin lethality.

[0073] Anti PLA2 CDRs

[0074] The antigen-binding proteins of the invention may preferably comprise the CDRs described in this section. In particular, antigen-binding proteins capable of binding to, blocking, and / or neutralizing snake venom PLA2 neurotoxin may comprise the CDRs described in this section.

[0075] In some embodiments of the present invention, the antigen-binding protein comprises a complementary determining region 1 (CDR1) comprising or consisting of CDR1 as set forth in any of SEQ ID NO: 2, 6, 10 or 31, or a sequence identical to any of the aforementioned except for up to 2, such as up to 1 amino acid substitution(s). In some embodiments of the present invention, the antigen-binding protein comprises a complementary determining region 2 (CDR2) comprising or consisting of CDR2 as set forth in any of SEQ ID NO: 3, 7, 11 or 32, or a sequence identical to any of the aforementioned except for up to 2, such as up to 1 amino acid substitution(s).

[0076] In some embodiments of the present invention, the antigen-binding protein comprises a complementary determining region 3 (CDR3) comprising or consisting of CDR3 as set forth in any of SEQ ID NO: 4, 8, 12 or 33, or a sequence identical to any of the aforementioned except for up to 2, such as up to 1 amino acid substitution(s).

[0077] In preferred embodiments of the present invention, the antigen-binding protein comprises complementary determining regions (CDRs) consisting of CDR1 as set forth by SEQ ID NO:31, CDR2 as set forth by SEQ ID NO: 32 and CDR3 as set forth by SEQ ID NO:33.

[0078] In some embodiments of the present invention, the antigen-binding protein comprises complementary determining regions (CDRs) comprising or consisting of a CDR1 as set forth by SEQ ID NO: 2 or a sequence identical thereto except for up to 2, such as up to 1 amino acid substitution(s); and a CDR2 as set forth by SEQ ID NO: 3 or a sequence identical thereto except for up to 2, such as up to 1 amino acid substitution(s); and a CDR3 as set forth by SEQ ID NO: 4 or a sequence identical thereto except for up to 2, such as up to 1 amino acid substitution(s).

[0079] In preferred embodiments of the present invention, the antigen-binding protein comprises complementary determining regions (CDRs) consisting of CDR1 as set forth by SEQ ID NO:2, CDR2 as set forth by SEQ ID NO: 3 and CDR3 as set forth by SEQ ID NO:4.

[0080] The antigen-binding protein may be the antigen-binding protein defined as TPL0637_01_A01.

[0081] In some embodiments of the present invention, the antigen-binding protein comprises complementary determining regions (CDRs) comprising or consisting of a CDR1 as set forth by SEQ ID NO: 6 or a sequence identical thereto except for up to 2, such as up to 1 amino acid substitution(s); and a CDR2 as set forth by SEQ ID NO: 7 or a sequence identical thereto except for up to 2, such as up to 1 amino acid substitution(s); and a CDR3 as set forth by SEQ ID NO: 8 or a sequence identical thereto except for up to 2, such as up to 1 amino acid substitution(s).

[0082] In preferred embodiments of the present invention, the antigen-binding protein comprises complementary determining regions (CDRs) consisting of CDR1 as set forth by SEQ ID NO:6, CDR2 as set forth by SEQ ID NO: 7 and CDR3 as set forth by SEQ ID NO:8.

[0083] The antigen-binding protein may be the antigen-binding protein defined as TPL0637_01_A07.

[0084] In some embodiments of the present invention, the antigen-binding protein comprises complementary determining regions (CDRs) comprising or consisting of a CDR1 as set forth by SEQ ID NO: 10 or a sequence identical thereto except for up to 2, such as up to 1 amino acid substitution(s); and a CDR2 as set forth by SEQ ID NO: 11 or a sequence identical thereto except for up to 2, such as up to 1 amino acid substitution(s); and a CDR3 as set forth by SEQ ID NO: 12 or a sequence identical thereto except for up to 2, such as up to 1 amino acid substitution(s).

[0085] In preferred embodiments of the present invention, the antigen-binding protein comprises complementary determining regions (CDRs) consisting of CDR1 as set forth by SEQ ID NQ.:10, CDR2 as set forth by SEQ ID NO.: 11 and CDR3 as set forth by SEQ ID NO.:12.

[0086] The antigen-binding protein may be the antigen-binding protein defined as TPL0638_01_C09. Anti PLA2VHHS

[0087] VHHS or also called nanobodies can bind antigens as effectively as full-length antibodies and are easy to clone and express.

[0088] The antigen-binding proteins of the invention may preferably comprise or even consist of the VHHS described in this section. In particular, antigen-binding proteins capable of binding to, blocking, and / or neutralizing snake venom PLA2neurotoxin may comprise or consist of the HHS described in this section.

[0089] In some embodiments of the present invention, the antigen-binding protein comprises or consists of a heavy chain variable (VH) region as set forth by any of SEQ ID NOs: 1, 5 or 9 or a functional homologue thereof having at least 70% sequence identity, such as at least 75% sequence identity, for example at least 80% sequence identity, such as at least 85% sequence identity, for example at least 90% sequence identity, such as at least 95% sequence identity thereto. In particular said VH region may comprise any of the CDRs mentioned herein above. In some embodiments of the present invention, the antigen-binding protein comprises or consists of a heavy chain variable (VH) region as set forth by SEQ ID NO. 1. In some embodiments of the present invention, the antigenbinding protein comprises or consists of a heavy chain variable (VH) region as set forth by SEQ ID NO.: 5. In some embodiments of the present invention, the antigen-binding protein comprises or consists of a heavy chain variable (VH) region as set forth by SEQ ID NO. 9.

[0090] Antigen-binding protein capable of binding to, blocking, and / or neutralizing snake venom short-chain a-neurotoxin

[0091] The present invention relates to an antigen-binding protein capable of binding to, blocking, and / or neutralizing snake venom short-chain a-neurotoxin. In some embodiments of the present invention, the antigen-binding protein is capable of binding to snake venom short-chain a-neurotoxin. In some embodiments of the present invention, the antigen-binding protein is capable of blocking snake venom short-chain a-neurotoxin. In preferred embodiments of the present invention, the antigen-binding protein is capable of neutralizing snake venom short-chain a-neurotoxin. In preferred embodiments, the antigen-binding proteins of the present invention are cross-reactive for several short-chain alpha neurotoxins. Thus, in some embodiments, the antigen-binding protein is capable of binding to, blocking, and / or neutralizing one or more short-chain a-neurotoxins, such as at least two, such as at least three, such as at least four, such as at least 5 short-chain a-neurotoxins.

[0092] In preferred embodiments, the antigen-binding protein is capable of binding to, blocking, and / or neutralizing scNTx, DH, Hh1 , rEury and / or Nh2, preferably scNTx, DH, Hh1 , rEury and Nh2, even more preferably DH and scNTX.

[0093] In some embodiments of the present invention, the antigen-binding protein is capable of neutralizing snake venom short-chain a-neurotoxin lethality.

[0094] The person skilled in the art will appreciate that the antigen-binding proteins recognize epitopes of the antigen. For example, an epitope can comprise or consist of a portion of a peptide (e.g., an short-chain a-neurotoxin). An epitope can be a linear epitope, sequential epitope, or a conformational epitope.

[0095] The antigen binding proteins of the present invention may thus recognize epitopes within short-chain alpha neurotoxin sequences such as scNTX, rEury, and rDH as set forth in SEQ. ID NO. 27, SEQ. ID NO. 28, and SEQ. ID NO. 29, respectively, and / or the sequence of the mature native DH sequence as set forth in SEQ ID NO. 35, and / or the mature native scNTX sequence as set forth in SEQ ID NO. 36.

[0096] In some embodiments, the antigen binding protein binds an epitope of DH comprising one or more, preferably at least 3, more preferably at least 6, more preferably at least 9 amino acid residues selected from the group consisting of: Q21, C41 , P42, T43, V44, P46, H49, I50, S51 , C52 and C53 of SEQ ID NO. 35 . In some embodiments, the antigen binding protein binds an epitope of DH comprising all of Q21 , C41 , P42, T43, V44, P46, H49, I50, S51 , C52 and C53 of SEQ ID NO. 35 .

[0097] In other embodiments, the antigen binding protein binds an epitope of scNTX comprising one or more, preferably at least two, more preferably at least three amino acid residues selected from the group consisting of: C41, K43, V44, P46, L50 of SEQ ID NO. 36. In some embodiments, the antigen binding protein binds an epitope of scNTX comprising all of C41 , K43, V44, P46, L50 of SEQ ID NO. 36.

[0098] In some embodiments, the antigen binding protein binds an epitope of DH comprising one or more, preferably at least 3, more preferably at least 5, more preferably at least 9 amino acid residues selected from the group consisting of:

[0099] - E19, Q21, C41 , P42, T43, V44, P46, G47, H49, I50, S51, C52, and C53 of SEQ ID NO. 35; or

[0100] - Q21, C41, P42, T43, V44, P46, H49, I50, S51, C52, and C53 of SEQ ID NO. 35; or

[0101] - Q21 , C41 , P42, T43, V44, P46, H49, I50, S51 , 052, 053, and 058 of SEQ ID NO. 35.

[0102] In other embodiments, the antigen binding protein binds an epitope of DH comprising:

[0103] - all of E19, Q21, 041 , P42, T43, V44, P46, G47, H49, I50, S51 , 052, and 053 of SEQ ID NO. 35; or

[0104] - all of Q21 , 041 , P42, T43, V44, P46, H49, I50, S51 , 052, and 053 of SEQ ID NO. 35; or

[0105] - all of Q21 , 041 , P42, T43, V44, P46, H49, I50, S51 , 052, 053, and 058 of SEQ ID NO. 35.

[0106] In some embodiments, the antigen binding protein referred to as TPL0629_01_D11 binds an epitope of DH comprising one or more amino acid residues selected from the group consisting of: E19, Q21, 041 , P42, T43, V44, P46, G47, H49, I50, S51 , 052, and 053 of SEQ ID NO. 35.

[0107] In some embodiments, the antigen binding protein referred to as TPL0629_01_A07 binds an epitope of DH comprising one or more amino acid residues selected from the group consisting of: Q21 , 041, P42, T43, V44, P46, H49, I50, S51 , 052, and 053 of SEQ ID NO. 35.

[0108] In some embodiments, the antigen binding protein referred to as TPL0629_01_G06 binds an epitope of DH comprising one or more amino acid residues selected from the group consisting of: Q21 , 041, P42, T43, V44, P46, H49, I50, S51, 052, 053, and 058 of SEQ ID NO. 35. In other embodiments, the antigen binding protein binds an epitope of scNTX comprising one or more, preferably at least 3, more preferably at least 5, more preferably at least 9 amino acid residues selected from the group consisting of:

[0109] - E19, T20, S21, C41 , K43, V44, P46, L50, R54, and T55 of SEQ ID NO. 36;

[0110] - C41, P42, K43, V44, P46, G47, L50, H51 , C52, and R54 of SEQ ID NO. 36; or

[0111] - E19, T20, S21, C41 , K43, V44, P46, L50, R54, and T55 of SEQ ID NO. 36.

[0112] In other embodiments, the antigen binding protein binds an epitope of scNTX comprising:

[0113] - all of E19, T20, S21, C41 , K43, V44, P46, L50, R54, and T55 of SEQ ID NO. 36;

[0114] - all of C41 , P42, K43, V44, P46, G47, L50, H51 , 052, and R54 of SEQ ID NO. 36; or

[0115] - all of E19, T20, S21, 041 , K43, V44, P46, L50, R54, and T55 of SEQ ID NO. 36.

[0116] In some embodiments, the antigen binding protein referred to as TPL0629_01_D11 binds an epitope of scNTX comprising one or more amino acid residues selected from the group consisting of: E19, T20, S21, 041 , K43, V44, P46, L50, R54, and T55 of SEQ ID NO. 36.

[0117] In some embodiments, the antigen binding protein referred to as TPL0629_01_A07 binds an epitope of scNTX comprising one or more amino acid residues selected from the group consisting of: 041, P42, K43, V44, P46, G47, L50, H51 , 052, and R54 of SEQ ID NO. 36

[0118] In some embodiments, the antigen binding protein referred to as TPL0629_01_G06 binds an epitope of scNTX comprising one or more amino acid residues selected from the group consisting of: E19, T20, S21, 041 , K43, V44, P46, L50, R54, and T55 of SEQ ID NO. 36 In other embodiments, the antigen binding protein binds an epitope of DH comprising residues 21 , 41-44, 46, and / or 49-53 of SEQ ID NO. 35.

[0119] In yet other embodiments, the antigen binding protein binds an epitope of scNTX comprising residues 2-3, 29-30, 17-20, 22-23, 29-30 and / or 110-111 of SEQ ID NO. 36.

[0120] Anti short-chain a-neurotoxin CDRs

[0121] The antigen-binding proteins of the invention may preferably comprise the CDRs described in this section. In particular, antigen-binding proteins capable of binding to, blocking, and / or neutralizing snake venom short-chain a-neurotoxin may comprise the CDRs described in this section.

[0122] In some embodiments of the present invention, the antigen-binding protein comprises complementary determining regions (CDRs) consisting of

[0123] - a CDR1 as set forth by any of SEQ ID NOs.: 14, 18 or 22 or a sequence identical to any of the aforementioned except for up to 2, such as up to 1 amino acid substitution(s); and

[0124] - a CDR2 as set forth by any of SEQ I D NOs. : 15, 19, or 23 or a sequence identical to any of the aforementioned except for up to 2, such as up to 1 amino acid substitution(s); and

[0125] - a CDR3 as set forth by any of SEQ ID NOs.: 16, 20, or 24 or a sequence identical to any of the aforementioned except for up to 2, such as up to 1 amino acid substitution(s).

[0126] In some embodiments of the present invention, the antigen-binding protein comprises complementary determining regions (CDRs) comprising or consisting of a CDR1 as set forth by SEQ ID NO: 14 or a sequence identical thereto except for up to 2, such as up to 1 amino acid substitution(s); and a CDR2 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 a CDR3 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). In some embodiments of the present invention, the antigen-binding protein comprises complementary determining regions (CDRs) consisting of CDR1 as set forth by SEQ ID NO.: 14, CDR2 as set forth by SEQ ID NO.: 15, and CDR3 as set forth by SEQ ID NO.: 16.

[0127] The antigen-binding protein may be the antigen-binding protein defined as TPL0629_01_D11.

[0128] In some embodiments of the present invention, the antigen-binding protein comprises complementary determining regions (CDRs) comprising or consisting of a CDR1 as set forth by SEQ ID NO: 18 or a sequence identical thereto except for up to 2, such as up to 1 amino acid substitution(s); and a CDR2 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 a CDR3 as set forth by SEQ ID NO: 20 or a sequence identical thereto except for up to 2, such as up to 1 amino acid substitution(s).

[0129] In some embodiments of the present invention, the antigen-binding protein comprises complementary determining regions (CDRs) consisting of CDR1 as set forth by SEQ ID NO: 18, CDR2 as set forth by SEQ ID NO: 19, and CDR3 as set forth by SEQ ID NO.: 20.

[0130] The antigen-binding protein may be the antigen-binding protein defined as TPL0629_01_A07.

[0131] In some embodiments of the present invention, the antigen-binding protein comprises complementary determining regions (CDRs) comprising or consisting of a CDR1 as set forth by SEQ ID NO: 22 or a sequence identical thereto except for up to 2, such as up to 1 amino acid substitution(s); and a CDR2 as set forth by SEQ ID NO: 23 or a sequence identical thereto except for up to 2, such as up to 1 amino acid substitution(s); and a CDR3 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). In some embodiments of the present invention, the antigen-binding protein comprises complementary determining regions (CDRs) consisting of CDR1 as set forth by SEQ ID NO.: 22, CDR2 as set forth by SEQ ID NO.: 23 and CDR3 as set forth by SEQ ID NO.: 24.

[0132] The antigen-binding protein may be the antigen-binding protein defined as TPL0629_01_G06.

[0133] Anti short-chain a-neurotoxin VHHS

[0134] The antigen-binding proteins of the invention may preferably comprise or even consist of the VHHS described in this section. In particular, antigen-binding proteins capable of binding to, blocking, and / or neutralizing snake venom short-chain a-neurotoxin may comprise or consist of the VHHS described in this section.

[0135] In some embodiments of the present invention, the antigen-binding protein comprises or consists of a heavy chain variable (VH) region as set forth by any of SEQ ID NOs.: 13, 17 and 21 or a functional homologue thereof having at least 70% sequence identity, such as at least 75% sequence identity, for example at least 80% sequence identity, such as at least 85% sequence identity, for example at least 90% sequence identity, such as at least 95% sequence identity thereto. In particular said VH region may comprise any of the CDRs mentioned herein above. In some embodiments of the present invention, the antigen-binding protein comprises or consists of a heavy chain variable (VH) region as set forth by SEQ ID NO.: 13. In some embodiments of the present invention, the antigen-binding protein comprises or consists of a heavy chain variable (VH) region as set forth by SEQ ID NO.: 17. In some embodiments of the present invention, the antigen-binding protein comprises or consists of a heavy chain variable (VH) region as set forth by SEQ ID NO.: 21.

[0136] Structures, sequences and modifications of antigen-binding proteins

[0137] The antigen-binding protein according to the present invention 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. In preferred embodiments of the present invention, the antigen-binding protein is a VHH.

[0138] In some embodiments of the present invention, the antigen-binding protein is monoclonal.

[0139] In some embodiments of the present invention, the antigen-binding protein is chimeric. In other embodiments of the present invention, the antigen-binding protein is humanized. In some embodiments of the present invention, 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.

[0140] In preferred embodiments, the antigen-binding protein is a single domain antibody, a nanobody, or a VHH, which preferably is of camelid origin.

[0141] In some embodiments of the present invention, 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 HH, linked to an immunoglobulin constant region.

[0142] In a preferred embodiment, the antigen-binding protein is a VHH-FC fusion protein.

[0143] In some embodiments of the present invention, 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 any of the antigen-binding proteins described herein, e.g. the polypeptide sequence as set forth by SEQ. ID. Nos: 1, 5, 9, 13, 17 or 21.

[0144] 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. 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 also 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 reduced antigen-binding turnover in vivo. The antigen-binding protein, in particular recombinant 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 antigenbinding protein, such as one or more N-glycosylations.

[0145] 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.

[0146] 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.

[0147] Thus, in one embodiment, the antigen-binding protein comprises a detection label.

[0148] In some applications, for instance spectroscopic assays, colorimetric assays, and light microscopy, it may be beneficial to use a colored 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 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. 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.

[0149] 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.

[0150] In some embodiments, the detection label on the antigen-binding protein is biotin.

[0151] 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.

[0152] In some embodiments of the present invention, the detection label is a gold nanoparticle.

[0153] The antigen-binding proteins according to the invention 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.

[0154] The antibodies are preferably identified using phase 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 and Laustsen et al., 2021. 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 the VHHS described herein, may be performed by any method known in the art. In some embodiments, the biopanning is performed against one or more snake toxins as antigens. These toxins may e.g. be recombinantly expressed.

[0155] In a preferred embodiment, the toxins are PLA2S and / or aNTxs and / or synthetic toxins consisting of a consensus sequence prepared based on the sequence from many different toxins. For example, the toxins used for the biopanning strategy may be one or more wild-type PLA2S, such as PLA2N (SEQ ID NO. 26) and / or PLA2O (SEQ ID NO. 25), one or more recombinantly expressed native aNTxs, such as rEury (SEQ ID NO. 28) and rDH (SEQ ID NO. 29), and / or a recombinantly expressed short-chain consensus aNTx (SEQ ID NO. 27) (scNTx, De la Rosa et al., 2018)

[0156] Preferably, the antigen-binding protein is discovered using the methods described in Example 1 and 2 below.

[0157] Absence of binding to long-chain alpha neurotoxins

[0158] In preferred embodiments, the antigen-binding proteins of the present invention do not bind long-chain alpha neurotoxins, such as alpha-cobratoxin.

[0159] This may for example be preferable when the antigen binding protein is used for diagnostic applications thereby enabling to differentiate between short and long alpha neurotoxins, and venoms comprising thereof.

[0160] The skilled person will appreciate that short and long-chain alpha neurotoxins are two different protein subfamilies that can generally not be recognized by the same antibodies. Engmark et al. 2016 demonstrates for example that existing antivenoms bind to very different parts of short vs. long-chain alpha-neurotoxins (Figure 4, 5 and 6 of Engmark et al. 2016) Said toxins are also known to the skilled person to have different receptor specificity. The skilled person will also appreciate that the main lethal components of coral snake venoms are short-chain (and not long-chain) alphaneurotoxins.

[0161] In embodiments of the oligoclonal antigen-binding protein mixture, the multi-specific antigen-binding protein, the composition, the kit of parts, the method, the use, or the kit according to the present disclosure, the antigen binding protein(s), the oligoclonal antigen-binding protein mixture(s) or the multi-specific antigen-binding protein(s) do not bind long-chain alpha neurotoxins, such as alpha-cobratoxin.

[0162] Oligoclonal mixture of antigen-binding protein capable of binding to, blocking, and / or neutralizing PLA2 neurotoxin and short-chain alpha neurotoxin

[0163] The present invention also provides oligoclonal mixture of antigen-binding proteins, e.g oligoclonal mixtures comprising a plurality of the antigen-binding proteins described herein. 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 monoclonal antibodies. In one embodiment, oligoclonal mixture of antigen-binding proteins consists of a predetermined mixture of antibodies against one or more epitopes present on one or more targets of coral snake venoms. Oligoclonal mixture of antigen-binding proteins are particularly useful when it is desired to target multiple epitopes. 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.

[0164] The present invention relates to an oligoclonal antigen-binding protein mixture comprising at least two different antigen-binding proteins, wherein one of said antigenbinding proteins is capable of binding to, blocking, and / or neutralizing snake venom PLA2 neurotoxin and wherein another of the at least two different antigen-binding proteins is capable of binding to, blocking, and / or neutralizing snake venom short-chain alpha neurotoxin. In preferred embodiments of the present invention, the oligoclonal antigen-binding protein mixture is capable of neutralizing snake venom short-chain alpha neurotoxin.

[0165] In some embodiments of the present invention, the at least two different antigenbinding proteins of the oligoclonal antigen-binding protein mixture comprise one or more antigen-binding proteins as described herein.

[0166] It is preferred that one 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 above in the sections “Anti PLA2 CDRs” and “Anti PLA2 VHHS”, respectively, and another antigen-binding protein comprising the CDRs and / or the VHHS as described herein above in the sections "Anti short-chain a-neurotoxin CDRs” and “Anti short-chain a-neurotoxin VHHS”, respectively.

[0167] In a preferred embodiment, the first of the at least two different antigen-binding proteins of the oligoclonal antigen-binding protein mixture comprises complementary determining regions (CDRs) consisting of:

[0168] (a) CDR1 as set forth by SEQ ID NO:6, CDR2 as set forth by SEQ ID NO:

[0169] 7 and CDR3 as set forth by SEQ ID NO:8, and; the other of the at least two different antigen-binding protein comprises complementary determining regions (CDRs) consisting of:

[0170] (b) CDR1 as set forth by SEQ ID NO.: 14, CDR2 as set forth by SEQ ID NO.: 15, and CDR3 as set forth by SEQ ID NO.: 16.

[0171] In other embodiments of the present invention, the first of the at least two different antigen-binding proteins of the oligoclonal antigen-binding protein mixture comprises or consists of:

[0172] (a) a heavy chain variable (VH) region as set forth by SEQ ID NO. 5, and; another of the at least two different antigen-binding protein comprises or consists of:

[0173] (b) a heavy chain variable (VH) region as set forth by SEQ ID NO.: 13.

[0174] In other embodiments of the present invention, the oligoclonal antigen-binding protein mixture consists of:

[0175] (a) an antigen-binding protein comprising complementary determining regions (CDRs) consisting of CDR1 as set forth by SEQ ID NO:6, CDR2 as set forth by SEQ ID NO: 7 and CDR3 as set forth by SEQ ID NO:8, and;

[0176] (b) an antigen-binding protein comprising complementary determining regions (CDRs) consisting of CDR1 as set forth by SEQ ID NO.: 14, CDR2 as set forth by SEQ ID NO.: 15, and CDR3 as set forth by SEQ ID NO.: 16.

[0177] In other embodiments of the present invention, the oligoclonal antigen-binding protein mixture consists of:

[0178] (a) an antigen-binding protein comprising or consisting of a heavy chain variable (VH) region as set forth by SEQ ID NO. 5, and;

[0179] (b) an antigen-binding protein comprising or consisting of a heavy chain variable (VH) region as set forth by SEQ ID NO.: 13. Multi-; antiqen binding

[0180] The term "multi-specific antigen binding molecule" or “multispecific antigen binding molecule” refers to an antibody comprising two or more variable regions binding to different epitopes. The epitopes may be on the same or different targets. The multispecific antigen binding protein binds to two or more kinds of antigens at the same time, wherein at least one of the antigen binding sites may include the snake venom PLA2 neurotoxin and / or short-chain a-neurotoxin.

[0181] The present invention relates to multi-specific antigen-binding molecules, wherein one antigen-binding portion is capable of binding to, blocking, and / or neutralizing snake venom PLA2 neurotoxin and another antigen-binding portion is capable of binding to, blocking, and / or neutralizing snake venom short-chain alpha neurotoxin. In preferred embodiments, the a multi-specific antigen-binding protein comprises or consist of one antigen-binding portion, which is capable of neutralizing snake venom PLA2 neurotoxin and another antigen-binding portion which is capable of neutralizing snake venom short-chain alpha neurotoxin. Each of said antigen-binding portions may be any of the antigen-binding proteins described herein.

[0182] In particular, the multi-specific antigen binding molecule may comprise the same CDRs and / or VHH, or combinations thereof, as described hereinabove for the oligoclonal mixtures of antigen-binding proteins.

[0183] Binding kinetics

[0184] The antigen-binding proteins of the present invention preferably have high affinity for the antigen, such as PLA2 and / or short-chain a-neurotoxin. 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. In some embodiments of the present invention, the antigen-binding protein or at least one of the antigen-binding portions binds PLA2 with a KD of at the most 100pM, for example at the most 150pM, such as at the most 200pM, for example at the most 250pM, for example at the most 300pM, for example at the most 350pM, for example at the most 400pM, such as at the most 410pM, for example at the most 420pM, such as at the most 430pM, for example at the most 440pM, such as at the most 450pM, for example at the most 460pM, such as at the most 470pM, for example at the most 480pM, such as at the most 490pM, for example at the most 500pM, such as at the most 510pM, for example at the most 520pM, such as at the most 530pM, for example at the most 540pM, such as at the most 550pM, for example at the most 50 nM.

[0185] In some embodiments of the present invention, the antigen-binding protein or at least one of the antigen-binding portions binds short-chain a-neurotoxin with a KD of at the most 500pM, such as at the most 510pM, for example at the most 520pM, such as at the most 530pM, for example at the most 540pM, such as at the most 550pM, for example at the most 1nM, such as at the most 10nM, for example at the most 15nM, such as at the most 20nM, for example at the most 30nM, such as at the most 40nM, for example at the most 50nM, such as at the most 60nM, for example at the most 65nM, such as at the most 66nM, for example at the most 67nM, such as at the most 68nM, for example at the most 69nM, such as at the most 70nM.

[0186] Pharmaceutical compositions and kits

[0187] Another aspect of the present invention relates to pharmaceutical composition comprising one or more antigen-binding proteins, the oligoclonal antigen-binding protein mixture or the multi-specific antigen-binding protein as described herein, and pharmaceutically acceptable diluents, carriers and / or excipients.

[0188] The antigen-binding proteins of the present invention may be combined in formulations comprising another agent suitable for the treatment of snake envenomation, for example, other antigen-binding protein(s) capable of binding to, blocking and / or neutralizing one or more snake venom antigen(s). 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 antigen-binding protein-based antivenoms, such as the antivenoms listed in Laustsen et al., 2016, Supplementary Table 1. In some embodiments of the present invention, the pharmaceutical composition comprises another agent suitable for the treatment of snake envenomation, for example one or more additional antigen-binding protein(s) capable of binding to, blocking, and / or neutralizing one or more snake venom antigen(s).

[0189] The present invention also relates to a kit of parts comprising: a) one or more antigen-binding proteins, the oligoclonal antigen-binding protein mixture or the multi-specific antigen-binding protein as described herein; and b) another agent suitable for the treatment of snake envenomation.

[0190] In some embodiments, the other agent suitable for the treatment of snake envenomation in the kit-of-parts is one or more additional antigen-binding protein(s), which e.g. may be selected from the group consisting of : IgG, IgM, scFv, F(ab)2, Fab, Fab’, domain antibodies, nanoboides, VHH antigen-binding protein(s) and fragments thereof.

[0191] The skilled person will know, as also mentioned in the background section of this document, that antivenom preparations, containing antigen-binding protein(s) capable of neutralizing one or more snake venom antigen(s) are traditionally obtained from hyperimmune equine, ovine, rabbit or camelid organisms. Several equine, ovine, rabbit or camelid-plasma derived antigen-binding proteins, or fragments thereof, may thus be combined with the antigen-binding protein as described herein.

[0192] In further embodiments, the other agent suitable for the treatment of snake envenomation is additional antigen-binding protein(s) capable of neutralizing one or more snake venom antigen(s), which may be selected from the group consisting of: Equine immunoglobulin(s) against snake venom, such as Equine IgG(s) or Equine Fab or Equine F(ab’)2fragment(s) against snake venom, Ovine immunoglobulin(s) against snake venom, such as Ovine IgG(s) or Ovine Fab or Ovine F(ab’)2 fragment(s) against snake venom, Rabbit immunoglobulin(s) against snake venom, such as Rabbit IgG(s) or Rabbit Fab or Rabbit F(ab’)2fragment(s) against snake venom, and Camelids immunoglobulin(s) against snake venom, such as Camelids IgG(s) or Camelids Fab or Camelids F(ab’)2fragment(s) against snake venom. In some other embodiments, 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 metalloproteinase (MMP) inhibitor, a snake venom metalloproteinase inhibitor (SVMP), a Kunitz-type trypsin inhibitor, an antimyotoxic factor, an antimyonecrotic factor, an antineurotoxic factor, an antihaemmoragic factor, an antioedematogenic factor and a glycoprotein, such as acid-a1 -glycoprotein.

[0193] A further type of agents used in the treatment of snake envenomation are small molecules, for instance as described in Laustsen et al., 2016.

[0194] In some embodiments, 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.

[0195] In preferred embodiments, the one or more small molecule(s) with inhibitory effect against snake toxins may be selected from the group consisting of : Suramin, Heparin, 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.

[0196] Medical uses of the antigen binding proteins, oligoclonal mixtures thereof and compositions of the present invention - Method of treatment of snake envenomation in a subject

[0197] Another aspect of the present invention relates to an antigen-binding protein as described herein, an oligoclonal antigen-binding protein mixture as described herein, a multi-specific antigen-binding protein as described herein, a pharmaceutical composition as described herein, or a kit-of-parts as described herein for use in a method of treatment of snake envenomation in a subject in need thereof.

[0198] In some embodiments of the present invention, the method for treating snake envenomation in a subject comprises administering a therapeutically effective amount of the antigen binding protein, the oligoclonal antigen-binding protein mixture, the multispecific antigen-binding protein, the pharmaceutical composition, or the kit of parts as described herein.

[0199] In a preferred embodiment, the antigen-binding protein, the oligoclonal antigen-binding protein mixture, the multi-specific antigen-binding protein, the pharmaceutical composition or the kit-of-parts for the use as described herein neutralizes venom- induced lethality.

[0200] In other embodiments, the antigen-binding protein, the oligoclonal antigen-binding protein mixture, the multi-specific antigen-binding protein, the pharmaceutical composition or the kit-of-parts for the use as described herein, prevents or delays envenomation symptoms.

[0201] The skilled person will appreciate that one of the main effects of snake envenomation, in particular snake envenomation by a venom containing a-neurotoxins, is paralysis. Several other direct and indirect symptoms can occur as a result of snake envenomation.

[0202] 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.

[0203] In preferred embodiments, the antigen-binding protein, the oligoclonal antigen-binding protein mixture, the multi-specific antigen-binding protein, the pharmaceutical composition or the kit-of-parts as described herein is administered parenterally or pulmonarily. Said parenteral administration may e.g. be intravenous, intramuscular and / or sub-cutaneous.

[0204] The skilled person will appreciate that antivenoms should ideally be provided to the subject in need thereof as soon as possible after the bite. Antivenoms may however not be available close to the location of the envenomation event, in particular when the snakebite occurs in remote geographical areas, for example in geographical areas with limited vehicle access, complicated logistics, and / or scarce healthcare center presence or healthcare resources. It may thus be of great benefit that the antigen-binding protein, the oligoclonal antigen-binding protein mixture, the multi-specific antigen-binding protein, the pharmaceutical composition or the kit-of-parts for use in the treatment of snake envenomation can be administered shortly after the bite but also may have a preventing or delaying action on the symptoms or a fortiori on lethality when administered relatively longer after the bite.

[0205] In some embodiments, the antigen-binding protein, the oligoclonal antigen-binding protein mixture, the multi-specific antigen-binding protein, the pharmaceutical composition or the kit-of-parts is administered no more than 5 min after envenomation, such as no more than 10 min after envenomation, for instance no more than 15 min after envenomation, such as no more than 20 min after envenomation, for instance no more than 30 min after envenomation, such as no more than 45 min after envenomation, for instance no more than 60 min after envenomation, such as no more than 90 min after envenomation, for instance no more than 120 min after envenomation, such as no more than 180 min after envenomation, for instance no more than 240 min after envenomation, such as no more than 6h after envenomation, for instance no more than 12h after envenomation, such as no more than 24h after envenomation, for instance no more than 48h after envenomation, such as no more than 72h after envenomation, for instance no more than 96h after envenomation, such as no more than 120h after envenomation for instance no more than 144h after envenomation.

[0206] In another aspect, the present invention relates to a method of treating snake envenomation which comprises administering to a subject an effective amount of the antigen-binding protein as described herein, the oligoclonal antigen-binding protein mixture as described herein, the multi-specific antigen-binding protein as described herein, the composition as described herein, or the kit-of-parts as described herein.

[0207] A further aspect of the present invention relates to the use of the antigen-binding protein as described herein, the oligoclonal antigen-binding protein mixture as described herein, the multi-specific antigen-binding protein as described herein, the composition as described herein, or the kit-of-parts as described herein, for the manufacture of a medicament for the treatment of snake envenomation.

[0208] In further embodiments, the antigen-binding protein, the oligoclonal antigen-binding protein mixture or the multispecific antigen-binding protein prevents or delays in vivo lethality of at least one toxin selected from the group consisting of: PLA2 neurotoxin and short-chain a-neurotoxin.

[0209] In further embodiments, the antigen-binding protein, the oligoclonal antigen-binding protein mixture or the multispecific antigen-binding protein prevents or delays in vivo lethality of PLA2 neurotoxin. In further embodiments, the antigen-binding protein, the oligoclonal antigen-binding protein mixture or the multispecific antigen-binding protein prevents or delays in vivo lethality of short-chain a-neurotoxin.

[0210] In further embodiments, the antigen-binding protein, the oligoclonal antigen-binding protein mixture, or the multi-specific antigen-binding protein, the kit of parts, the method or the use delays in vivo lethality of at least one toxin selected from the group consisting of: PLA2 neurotoxin and short-chain a-neurotoxin by at least 8h, such as at least 10h, for instance at least 12h, such as at least 1 day, for instance at least 2 days, such as at least 3 days after envenomation.

[0211] Diagnosis of snake envenomation

[0212] The antigen-binding protein of the present invention may be used for detection, diagnosis, and diagnostics purposes, for example in clinical settings. The antigenbinding protein may be used for instance as part of a companion diagnostic kit to support for example the clinical management of snakebites and identify therapeutic options. The invention also relates to an antigen-binding protein, an oligoclonal antigen-binding protein mixture or a multi-specific antigen-binding protein as described herein for use in the diagnosis of snake envenomation in a subject.

[0213] Another aspect of the invention relates to a kit for detection and / or quantification of snake venom in a sample comprising one or more of the antigen binding proteins, the oligoclonal antigen-binding protein mixtures or the multi-specific antigen-binding proteins as described herein.

[0214] In preferred embodiments, the sample is a blood sample , e.g. a plasma blood sample, a urine sample, or a smear of the bite site / wound.

[0215] The skilled person will appreciate that the kit may be an immunoassay, such as a method for detecting one or more analytes, such as snake venoms, or fractions thereof, for example a-neurotoxins or PLA2. Typically, the immunoassay may be a method based on the specific binding of the antigen-binding proteins of the present invention to one or more a-neurotoxins or PLA2. Suitable immunoassays include ELISA (enzyme-linked immunosorbent assay), indirect ELISA, sandwich ELISA, competitive ELISA, multiplex ELISA, radioimmunoassay (RIA), ELISPOT technologies, and other similar techniques known in the art.

[0216] In a further embodiment of the present invention, the kit is an immunoassay, such as a lateral flow assay, an ELISA, an electrochemical impedance spectroscopy-based assay, a Bilayer Interferometry (BLI)-based assay or a turbidimetric immunoassay.

[0217] The antigen-binding protein, the oligoclonal antigen-binding protein mixture, or a multispecific antigen-binding protein may be used for instance for the diagnosis of snake envenomation with a venom containing one or more a-neurotoxins and / or PLA2.

[0218] A further aspect of the present invention relates to the use of an antigen-binding protein as described herein in an in vitro method for detection and / or diagnosis of snake envenomation in a subject, e.g. a human.

[0219] The person skilled in the art will appreciate that the use of antivenoms may not be restricted to humans but can also be used on non-human animals, such as for instance pets, cattle, farm animals, or ornamental animals. The subject described herein may be a human or a non-human animal, for example the non-human animal subject may belong to the family of Equidae, Bovidae, Camelidae, Canidae, or Felidae.

[0220] In some embodiments, the non-human animal is a horse, a cow, an ox, a camel, a dog, or a cat.

[0221] An advantage of the antigen-binding proteins described herein in diagnostic application is the possibility to differentiate between short-chain alpha neurotoxins and long-chain alpha neurotoxins in venom samples. This is for example the case with TPL0629_01_D11 disclosed herein, which does not bind long-chain alpha neurotoxins, such as alpha-cobratoxin (TPL0629_01_D11 Figure 8). TPL0629_01_D11 on the other hand is able to bind all short-chain alpha neurotoxins tested (Figure 8).

[0222] Neutralization of snake venom toxins

[0223] This invention relates to antigen-binding proteins, oligoclonal antigen-binding protein mixtures, or multi-specific antigen binding proteins capable of neutralizing snake venom PLA2 neurotoxin lethality and / or snake venom short-chain a-neurotoxin lethality. The inventors have developed antigen-binding proteins capable of neutralizing snake venom PLA2 neurotoxin and short-chain a-neurotoxin lethality.

[0224] Any useful method may be used to determine the neutralization of lethality by the antigen-binding proteins, the oligoclonal antigen-binding protein mixtures or the multispecific antigen-binding proteins as described herein. More preferably, the neutralization of lethality may be performed using the methods described in Examples 4 and 5 below.

[0225] An assessment of the neutralizing capacity of the antigen-binding proteins, the oligoclonal antigen-binding protein mixtures, or the multi-specific antigen-binding proteins can be made by e.g. using the fluorometric EnzChek™ Phospholipase A2 Assay Kit. The kit provides rapid real-time monitoring of PLA2 enzyme activities. Therefore, the EC50 value and the neutralizing capacity of the antigen-binding proteins can be determined through the assay. In vivo neutralization capacity of the antigen-binding proteins, the oligoclonal antigen binding protein mixtures, or the multi-specific antigen binding proteins can be determined by preincubation experiments. In this regard, toxin(s) and / or venom(s) are preincubated with the oligoclonal antigen binding protein mixture, followed by intravenous injection (i.v) and monitoring the survival of the animals.

[0226] In vivo neutralization capacity of the antigen-binding proteins, the oligoclonal antigenbinding protein mixtures or the multi-specific antigen-binding proteins can be determined by rescue experiments. In such experiments, toxin can be subcutaneous (s.c.) injected into mice and intravenous (i.v.) injection of antigen-binding proteins can be used to rescue the animals.

[0227] In some embodiments of the present invention, the antigen-binding protein is capable of binding to, blocking, and / or neutralizing snake venom PLA2 neurotoxin, wherein said PLA2 neurotoxin is from group I A in Elapid snakes, preferably from group 1A in coral snake venoms.

[0228] In some embodiments of the present invention, the antigen-binding protein is capable of binding to, blocking, and / or neutralizing snake venom PLA2N and / or snake venom PLA2O.

[0229] In some embodiments of the present invention, the antigen-binding protein is capable of binding to, blocking, and / or neutralizing snake venom PLA2 neurotoxin, wherein the PLA2 neurotoxin comprises or consists of a polypeptide sequence selected from the group consisting of: SEQ. ID NO. 25, SEQ. ID NO. 26, or SEQ. ID NO. 34, or variant(s) thereof having at least 70% identity, such as at least 75% identity, for example at least 80% identity, such as at least 85% identity, for example at least 90% identity, such as at least 95% identity thereto.

[0230] In some embodiments, the antigen-binding protein, the oligoclonal antigen-binding protein mixture, or the composition as described herein neutralize the enzymatic activity of PLA2 neurotoxin and / or short-chain a-neurotoxin. The enzymatic activity and its neutralization may be determined as presented in the Examples herein, using any suitable kit known in the art for the direct or indirect measurement of enzymatic activities of said toxins, such as the EnzCheckTM Phospholipase A2 assay kit (Thermo Scientific).

[0231] In some embodiments of the present invention, the antigen-binding protein, the multispecific antigen-binding protein and / or the oligoclonal antigen-binding protein mixture is capable of neutralizing enzymatic PLA2 activity in vitro by at least 50%, such as by at least 60%, for example by at least 70%, such as by at least 80%, for example by at least 90% compared to without antigen-binding protein, as measured by any suitable enzymatic assay as described herein.

[0232] In some embodiments of the present invention, the antigen-binding protein, the oligoclonal antigen-binding protein mixture and / or the multi-specific antigen-binding protein is capable of binding to, blocking, and / or neutralizing snake venom short-chain a-neurotoxin, wherein the short-chain a-neurotoxin comprises or consists of a polypeptide sequence selected from the group consisting of: SEQ. ID NO. 27, SEQ. ID NO. 28, SEQ. ID NO. 29, SEQ. ID NO. 30, SEQ. ID NO. 35, SEQ. ID NO. 36, or variant(s) thereof having at least 70% identity, such as at least 75% identity, for example at least 80% identity, such as at least 85% identity, for example at least 90% identity, such as at least 95% identity thereto.

[0233] Neutralization of snake whole venom

[0234] This invention relates to antigen-binding proteins, oligoclonal antigen-binding protein mixtures, or multi-specific antigen binding proteins capable neutralizing whole snake venom lethality. Thus, by developing high-throughput pipelines for the generation of high affinity broadly-neutralizing antibodies, the present inventors were capable of developing antigen-binding proteins capable of neutralizing whole snake venom lethality.

[0235] Any useful method may be used to determine the neutralization of lethality by the antigen-binding proteins, the oligoclonal antigen-binding protein mixtures or the multispecific antigen-binding proteins as described herein. More preferably, the neutralization of whole snake venom lethality by the antigen-binding proteins, the oligoclonal antigen binding protein mixtures or the multi-specific antigen binding proteins as described herein is identified using the methods described in Example 6 below.

[0236] In vivo neutralization capacity of the antigen-binding proteins, the oligoclonal antigen binding protein mixtures, or the multi-specific antigen binding proteins can be determined by preincubation experiments. In this regard, toxin(s) and / or venom(s) are preincubated with the oligoclonal antigen binding protein mixture, followed by intravenous injection (i.v) and monitoring the survival of the animals.

[0237] In vivo neutralization capacity of the antigen-binding proteins, the oligoclonal antigen binding protein mixtures, or the multi-specific antigen binding proteins can also be determined by rescue experiments. In this regard, toxin(s) can be subcutaneous (s.c.) injected into mice and envenoming, whereupon and intravenous injection of oligoclonal mixtures can be used rescue the animals.

[0238] In a preferred embodiment, the antigen-binding protein, the pharmaceutical composition, the oligoclonal antigen-binding protein mixture, the multispecific antigenbinding protein or the kit-of-parts for the use as described herein prevents or delays in vivo lethality of whole snake venom, for example delays in vivo lethality of whole snake venom by at least 8h, such as at least 10h, for instance at least 12h, such as at least 1 day, for instance at least 2 days, such as at least 3 days after envenomation.

[0239] In some embodiments of the present invention, the antigen-binding protein, the oligoclonal antigen-binding protein mixture, the multi-specific antigen-binding protein, the composition, the kit of parts, the method, or the use fully neutralizes in vivo lethality of whole snake venom.

[0240] Elapid snakes

[0241] In some embodiments of the present invention, the snake is an elapid snake.

[0242] In preferred embodiments of the present invention, the snake is a coral snake.

[0243] In other embodiments, the snake is of a genus selected from the group consisting of: Micrurus, Naja, and Haemachatus. In other embodiments, the coral snake is of a genus selected from the group consisting of: Micrurus, Micruroides, Calliophis, Hemibungarus, Sinomicrurus, more preferably wherein the snake is of the genera Micrurus and Micruroides.

[0244] In preferred embodiments of the present invention, the coral snake is selected from the group consisting of: Micrurus fulvius or Micrurus diastema. In some embodiments of the present invention, the coral snake is Micrurus fulvius. In some embodiments of the present invention, the coral snake is Micrurus diastema.

[0245] Items

[0246] The invention may further be defined by anyone of the following items:

[0247] 1 . An antigen-binding protein capable of binding to, blocking, and / or neutralizing snake venom PLA2.

[0248] 2. An antigen-binding protein capable of binding to, blocking, and / or neutralizing at least two different snake venom PLA2S, such as at least three, such as at least four PLA2S, preferably wherein the snake venom PLA2 is PLA2N, PLA2O, Nn19, and / or Hh3, even more preferably PLA2N, PLA2O, Nn19, and Hh3.

[0249] 3. An antigen binding protein binding an epitope of PLA2O comprising one or more amino acid residues selected from the group consisting of:

[0250] - N2, L3, I4, K7, S17, W18, W19, H20, A22, D23, G29, S30, Y110, and K111 of SEQ ID NO. 34;

[0251] - L1 , L3, F6, A22, G29, S30, G31 , G32, H47, D48, Y51 , D52, E55, K56, G59, W61 , K63, and W64 of SEQ ID NO. 34; or

[0252] - L1 , N2, L3, F6, K7, S17, W18, W19, H20, A22, D23, G29, S30, D107, Y110, and K111 of SEQ ID NO. 34.

[0253] 4. The antigen-binding protein according to any one of the preceding items, wherein the antigen-binding protein is capable of neutralizing snake venom PLA2 neurotoxin lethality.

[0254] 5. The antigen binding protein according to any one of the preceding items, wherein the antigen-binding protein comprises a complementary determining region 1 (CDR1) comprising or consisting of CDR1 as set forth in any of SEQ ID NO: 2, 6, 10 or 31 , or a sequence identical to any of the aforementioned except for up to 2, such as up to 1 amino acid substitution(s).

[0255] 6. The antigen binding protein according to any one of the preceding items, wherein the antigen-binding protein comprises a complementary determining region 2 (CDR2) comprising or consisting of CDR2 as set forth in any of SEQ ID NO: 3, 7, 11 or 32, or a sequence identical to any of the aforementioned except for up to 2, such as up to 1 amino acid substitution(s).

[0256] 7. The antigen binding protein according to any one of the preceding items, wherein the antigen-binding protein comprises a complementary determining region 3 (CDR3) comprising or consisting of CDR3 as set forth in any of SEQ ID NO: 4, 8, 12 or 33, or a sequence identical to any of the aforementioned except for up to 2, such as up to 1 amino acid substitution(s).

[0257] 8. The antigen binding protein according to any one of the preceding items, wherein the antigen-binding protein comprises complementary determining regions (CDRs) consisting of: a CDR1 as set forth by any of SEQ ID NOs: 2, 6, or 10 or a sequence identical to any of the aforementioned except for up to 2, such as up to 1 amino acid substitution(s); and a CDR2 as set forth by any of SEQ ID NOs: 3, 7, or 11 , or a sequence identical to any of the aforementioned except for up to 2, such as up to 1 amino acid substitution(s); and a CDR3 as set forth by any of SEQ ID NOs: 4, 8, or 12 or a sequence identical to any of the aforementioned except for up to 2, such as up to 1 amino acid substitution(s).

[0258] 9. The antigen-binding protein according to any one of items 1 to 8, comprising complementary determining regions (CDRs) comprising or consisting of: a CDR1 as set forth by SEQ ID NO: 2 or a sequence identical thereto except for up to 2, such as up to 1 amino acid substitution(s); and a CDR2 as set forth by SEQ ID NO: 3 or a sequence identical thereto except for up to 2, such as up to 1 amino acid substitution(s); and a CDR3 as set forth by SEQ ID NO: 4 or a sequence identical thereto except for up to 2, such as up to 1 amino acid substitution(s). The antigen-binding protein according to any one of items 1 to 8, comprising complementary determining regions (CDRs) comprising or consisting of: a CDR1 as set forth by SEQ ID NO: 6 or a sequence identical thereto except for up to 2, such as up to 1 amino acid substitution(s); and a CDR2 as set forth by SEQ ID NO: 7 or a sequence identical thereto except for up to 2, such as up to 1 amino acid substitution(s); and a CDR3 as set forth by SEQ ID NO: 8 or a sequence identical thereto except for up to 2, such as up to 1 amino acid substitution(s). The antigen-binding protein according to any one of items 1 to 8, comprising complementary determining regions (CDRs) comprising or consisting of a CDR1 as set forth by SEQ ID NO: 10 or a sequence identical thereto except for up to 2, such as up to 1 amino acid substitution(s); and a CDR2 as set forth by SEQ ID NO: 11 or a sequence identical thereto except for up to 2, such as up to 1 amino acid substitution(s); and a CDR3 as set forth by SEQ ID NO: 12 or a sequence identical thereto except for up to 2, such as up to 1 amino acid substitution(s). The antigen-binding protein according to any one of the preceding items, wherein the antigen-binding protein comprises or consists of a heavy chain variable (VH) region as set forth by any of SEQ ID NOs: 1, 5 and 9, or a sequence identical thereto except for up to 2, such as up to 1 amino acid substitution(s). The antigen binding protein according to any one of the preceding items, wherein the antigen-binding protein comprises complementary determining regions (CDRs) consisting of CDR1 as set forth by SEQ ID NO:2, CDR2 as set forth by SEQ ID NO: 3 and CDR3 as set forth by SEQ ID NO:4. The antigen-binding protein according to any one of the preceding items, wherein the antigen-binding protein comprises or consists of a heavy chain variable (VH) region as set forth by SEQ ID NO. 1 15. The antigen binding protein according to any one of the preceding items, wherein the antigen-binding protein comprises complementary determining regions (CDRs) consisting of CDR1 as set forth by SEQ ID NO:6, CDR2 as set forth by SEQ ID NO: 7 and CDR3 as set forth by SEQ ID NO:8.

[0259] 16. The antigen-binding protein according to any one of the preceding items, wherein the antigen-binding protein comprises or consists of a heavy chain variable (VH) region as set forth by SEQ ID NO.: 5.

[0260] 17. The antigen binding protein according to any one of the preceding items, wherein the antigen-binding protein comprises complementary determining regions (CDRs) consisting of CDR1 as set forth by SEQ ID NO.: 10, CDR2 as set forth by SEQ I D NO. : 11 and CDR3 as set forth by SEQ I D NO. : 12.

[0261] 18. The antigen-binding protein according to any one of the preceding items, wherein the antigen-binding protein comprises or consists of a heavy chain variable (VH) region as set forth by SEQ ID NO. 9

[0262] 19. The antigen-binding protein according to any one of the preceding items, wherein the antigen-binding protein is capable of binding to, blocking, and / or neutralizing one or more PLA2S, such as at least two, such as at least three, such as at least four PLA2S.

[0263] 20. The antigen-binding protein according to any of the preceding items, wherein the antigen-binding protein is capable of binding to, blocking, and / or neutralizing snake venom PLA2 neurotoxin from group 1A.

[0264] 21. The antigen-binding protein according to any of the preceding items, wherein the antigen-binding protein is capable of binding to, blocking, and / or neutralizing snake venom PLA2 neurotoxin from group 1A from coral snake venoms.

[0265] 22. The antigen-binding protein according to any of the preceding items, wherein the antigen-binding protein is capable of binding to, blocking, and / or neutralizing snake venom PLA2N. 23. The antigen-binding protein according to any of the preceding items, wherein the antigen-binding protein is capable of binding to, blocking, and / or neutralizing snake venom PLA2O.

[0266] 24. The antigen-binding protein according to any of the preceding items, wherein the antigen-binding protein is capable of binding to, blocking, and / or neutralizing snake venom Nn19.

[0267] 25. The antigen-binding protein according to any of the preceding items, wherein the antigen-binding protein is capable of binding to, blocking, and / or neutralizing snake venom Hh3.

[0268] 26. The antigen-binding protein according to any of the preceding items, wherein the antigen-binding protein is capable of binding to, blocking, and / or neutralizing snake venom PLA2N, PLA2O, Nn19, and / or Hh3, preferably PLA2N, PLA2O, Nn19, and Hh3.

[0269] 27. The antigen-binding protein according to any one of the preceding items, wherein the antigen binding protein binds an epitope of PLA2O comprising one or more amino acid residues selected from the group consisting of: L3, A22, G29, and S30 of SEQ ID NO. 34.

[0270] 28. The antigen-binding protein according to any one of the preceding items, wherein the antigen binding protein binds an epitope of PLA2O comprising at least 2, preferably at least 3, even more preferably all of amino acid residues selected from the group consisting of: L3, A22, G29, and S30 of SEQ ID NO.

[0271] 34.

[0272] 29. The antigen-binding protein according to any one of the preceding items, wherein the antigen binding protein binds an epitope of PLA2O comprising one or more amino acid residues selected from the group consisting of:

[0273] - N2, L3, I4, K7, S17, W18, W19, H20, A22, D23, G29, S30, Y110, and K111 of SEQ ID NO. 34; - L1 , L3, F6, A22, G29, S30, G31 , G32, H47, D48, Y51 , D52, E55, K56, G59, W61 , K63, and W64 of SEQ ID NO. 34; or

[0274] - L1, N2, L3, F6, K7, S17, W18, W19, H20, A22, D23, G29, S30, D107, Y110, and K111 of SEQ ID NO. 34. The antigen-binding protein according to any one of the preceding items, wherein the antigen binding protein binds an epitope of PLA2O comprising at least 3, preferably at least 6, more preferably at least 9, for example all of the amino acid residues:

[0275] - N2, L3, I4, K7, S17, W18, W19, H20, A22, D23, G29, S30, Y110, and K111 of SEQ ID NO. 34; or

[0276] - L1 , L3, F6, A22, G29, S30, G31 , G32, H47, D48, Y51 , D52, E55, K56, G59, W61 , K63, and W64 of SEQ ID NO. 34; or

[0277] -L1, N2, L3, F6, K7, S17, W18, W19, H20, A22, D23, G29, S30, D107, Y110, and K111 of SEQ ID NO. 34. The antigen-binding protein according to any one of the preceding items, wherein the antigen binding protein binds an epitope of PLA2O comprising residues 2-3, 29-30, 17-20, 22-23, 29-30 and / or 110-111 of SEQ ID NO. 34. An antigen-binding protein capable of binding to, blocking, and / or neutralizing venom short-chain a-neurotoxin. An antigen-binding protein binding an epitope of DH comprising one or more amino acid residues selected from the group consisting of:

[0278] - E19, Q21, C41 , P42, T43, V44, P46, G47, H49, I50, S51, C52, and C53 of SEQ ID NO. 35; or

[0279] - Q21 , C41, P42, T43, V44, P46, H49, I50, S51, C52, and C53 of SEQ ID NO. 35; or

[0280] - Q21 , C41 , P42, T43, V44, P46, H49, I50, S51 , C52, C53, and C58 of SEQ ID NO. 35. An antigen-binding protein binding an epitope of scNTX comprising one or more amino acid residues selected from the group consisting of: - E19, T20, S21, C41 , K43, V44, P46, L50, R54, and T55 of SEQ ID NO. 36;

[0281] - C41, P42, K43, V44, P46, G47, L50, H51 , C52, and R54 of SEQ ID NO. 36; or

[0282] - E19, T20, S21, C41 , K43, V44, P46, L50, R54, and T55 of SEQ ID NO.

[0283] 36.

[0284] 35. The antigen-binding protein according to any one of items 32 to 34, wherein the antigen-binding protein is capable of neutralizing venom short-chain a- neurotoxin lethality.

[0285] 36. The antigen binding protein according to any one of items 32 to 35, wherein the antigen-binding protein comprises complementary determining regions (CDRs) consisting of

[0286] - a CDR1 as set forth by any of SEQ ID NOs.: 14, 18 or 22 or a sequence identical to any of the aforementioned except for up to 2, such as up to 1 amino acid substitution(s); and

[0287] - a CDR2 as set forth by any of SEQ I D NOs. : 15, 19, or 23 or a sequence identical to any of the aforementioned except for up to 2, such as up to 1 amino acid substitution(s); and

[0288] - a CDR3 as set forth by any of SEQ ID NOs.: 16, 20, or 24 or a sequence identical to any of the aforementioned except for up to 2, such as up to 1 amino acid substitution(s).

[0289] 37. The antigen-binding protein according to any one of items 32 to 36, comprising complementary determining regions (CDRs) comprising or consisting of a CDR1 as set forth by SEQ ID NO: 14 or a sequence identical thereto except for up to 2, such as up to 1 amino acid substitution(s); and a CDR2 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 a CDR3 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).

[0290] 38. The antigen-binding protein according to any one of items 32 to 36, comprising complementary determining regions (CDRs) comprising or consisting of a CDR1 as set forth by SEQ ID NO: 18 or a sequence identical thereto except for up to 2, such as up to 1 amino acid substitution(s); and a CDR2 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 a CDR3 as set forth by SEQ ID NO: 20 or a sequence identical thereto except for up to 2, such as up to 1 amino acid substitution(s). The antigen-binding protein according to any one of items 32 to 36, comprising complementary determining regions (CDRs) comprising or consisting of a CDR1 as set forth by SEQ ID NO: 22 or a sequence identical thereto except for up to 2, such as up to 1 amino acid substitution(s); and a CDR2 as set forth by SEQ ID NO: 23 or a sequence identical thereto except for up to 2, such as up to 1 amino acid substitution(s); and a CDR3 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). The antigen-binding protein according to any one of items 32 to 39, wherein the antigen-binding protein comprises or consists of a heavy chain variable (VH) region as set forth by any of SEQ ID NOs.: 13, 17 and 21 , or a sequence identical thereto except for up to 2, such as up to 1 amino acid substitution(s). The antigen binding protein according to any one of items 32 to 40, wherein the antigen-binding protein comprises complementary determining regions (CDRs) consisting of CDR1 as set forth by SEQ ID NO.: 14, CDR2 as set forth by SEQ ID NO.: 15, and CDR3 as set forth by SEQ ID NO.: 16. The antigen-binding protein according to any one of items 32 to 41 , wherein the antigen-binding protein comprises or consists of a heavy chain variable (VH) region as set forth by SEQ ID NO.: 13. The antigen binding protein according to any one of items 32 to 42, wherein the antigen-binding protein comprises complementary determining regions (CDRs) consisting of CDR1 as set forth by SEQ ID NO: 18, CDR2 as set forth by SEQ ID NO: 19, and CDR3 as set forth by SEQ ID NO.: 20. 44. The antigen-binding protein according to any one of items 32 to 43, wherein the antigen-binding protein comprises or consists of a heavy chain variable (VH) region as set forth by SEQ ID NO.: 17.

[0291] 45. The antigen binding protein according to any one of items 32 to 44, wherein the antigen-binding protein comprises complementary determining regions (CDRs) consisting of CDR1 as set forth by SEQ ID NO.: 22, CDR2 as set forth by SEQ ID NO.: 23 and CDR3 as set forth by SEQ ID NO.: 24.

[0292] 46. The antigen-binding protein according to any one of items 32 to 45, wherein the antigen-binding protein comprises or consists of a heavy chain variable (VH) region as set forth by SEQ ID NO.: 21.

[0293] 47. The antigen-binding protein according to any one of the preceding items, wherein the antigen-binding protein is capable of binding to, blocking, and / or neutralizing one or more short-chain a-neurotoxins, such as at least two, such as at least three, such as at least four, such as at least 5 short-chain a- neurotoxins.

[0294] 48. The antigen-binding protein according to any one of the preceding items, wherein the antigen-binding protein is capable of binding to, blocking, and / or neutralizing scNTx, DH, Hh1 , rEury and / or Nh2, preferably scNTx, DH, Hh1, rEury and Nh2, even more preferably DH and scNTX.

[0295] 49. The antigen-binding protein according to any one of the preceding items, wherein the antigen binding protein binds an epitope of DH comprising one or more amino acid residues selected from the group consisting of: Q21 , C41, P42, T43, V44, P46, H49, I50, S51 , C52 and C53 of SEQ ID NO. 35 .

[0296] 50. The antigen-binding protein according to any one of the preceding items, wherein the antigen binding protein binds an epitope of DH comprising at least 3, preferably at least 6, more preferably at least 9, for example all of the amino acid residues selected from the group consisting of: Q21, C41 , P42, T43, V44, P46, H49, I50, S51 , C52 and C53 of SEQ ID NO. 35 . The antigen-binding protein according to any one of the preceding items, wherein the antigen binding protein binds an epitope of scNTX comprising one or more amino acid residues selected from the group consisting of: C41, K43, V44, P46, L50 of SEQ ID NO. 36. The antigen-binding protein according to any one of the preceding items, wherein the antigen binding protein binds an epitope of scNTX comprising at least 3, preferably at least 6, more preferably at least 9, for example all amino acid residues selected from the group consisting of: C41, K43, V44, P46, L50 of SEQ ID NO. 36. The antigen-binding protein according to any one of the preceding items, wherein the antigen binding protein binds an epitope of DH comprising one or more amino acid residues selected from the group consisting of:

[0297] - E19, Q21, C41 , P42, T43, V44, P46, G47, H49, I50, S51 , C52, and C53 of SEQ ID NO. 35;

[0298] - Q21 , C41, P42, T43, V44, P46, H49, I50, S51 , C52, and C53 of SEQ ID NO. 35; or

[0299] - Q21 , C41, P42, T43, V44, P46, H49, I50, S51, C52, C53, and C58 of SEQ ID NO. 35. The antigen-binding protein according to any one of the preceding items, wherein the antigen binding protein binds an epitope of DH comprising at least 3, preferably at least 6, more preferably at least 9, for example all of amino acid residues:

[0300] - E19, Q21, C41 , P42, T43, V44, P46, G47, H49, I50, S51, C52, and C53 of SEQ ID NO. 35; or

[0301] - Q21 , C41, P42, T43, V44, P46, H49, I50, S51, C52, and C53 of SEQ ID NO. 35; or

[0302] - Q21 , C41 , P42, T43, V44, P46, H49, I50, S51 , C52, C53, and C58 of SEQ ID NO. 35. The antigen-binding protein according to any one of the preceding items, wherein the antigen binding protein binds an epitope of scNTX comprising one or more amino acid residues selected from the group consisting of: - E19, T20, S21, C41 , K43, V44, P46, L50, R54, and T55 of SEQ ID NO.

[0303] 36;

[0304] - C41, P42, K43, V44, P46, G47, L50, H51 , C52, and R54 of SEQ ID NO. 36; or

[0305] - E19, T20, S21, C41 , K43, V44, P46, L50, R54, and T55 of SEQ ID NO. 36. The antigen-binding protein according to any one of the preceding items, wherein the antigen binding protein binds an epitope of scNTX comprising at least 3, preferably at least 6, more preferably at least 9, for example all of amino acid residues:

[0306] - E19, T20, S21, C41 , K43, V44, P46, L50, R54, and T55 of SEQ ID NO. 36; or

[0307] - C41, P42, K43, V44, P46, G47, L50, H51 , 052, and R54 of SEQ ID NO. 36; or

[0308] - E19, T20, S21, 041 , K43, V44, P46, L50, R54, and T55 of SEQ ID NO. 36. The antigen-binding protein according to any one of the preceding items, wherein the antigen binding protein binds an epitope of DH comprising residues 21, 41-44, 46, and / or 49-53 of SEQ ID NO. 35. The antigen-binding protein according to any one of the preceding items, wherein the antigen binding protein binds an epitope of scNTX comprising residues 2-3, 29-30, 17-20, 22-23, 29-30 and / or 110-111 of SEQ ID NO. 36. The antigen-binding protein according to any one of the preceding items, wherein the antigen-binding protein does not bind long-chain alpha neurotoxins, such as alpha-cobratoxin. An oligoclonal antigen-binding protein mixture comprising at least two different antigen-binding proteins, wherein one of said antigen-binding proteins is capable of binding to, blocking, and / or neutralizing snake venom PLA2 and wherein another of the at least two different antigen-binding proteins is capable of binding to, blocking, and / or neutralizing snake venom short-chain alpha neurotoxin.

[0309] 61. The oligoclonal antigen-binding protein mixture according to item 60, wherein the at least two different antigen-binding proteins comprise one or more antigen-binding proteins according to any one of items 1 to 59.

[0310] 62. The oligoclonal antigen-binding protein mixture according to any one of items 60 to 61 , wherein one antigen-binding protein capable of binding to, blocking, and / or neutralizing snake venom PLA2 is the antigen-binding protein according to any one of items 1 to 31 and 59, and one antigen-binding protein capable of binding to, blocking, and / or neutralizing snake venom short-chain alpha neurotoxin is the antigen-binding protein according to any one of items 32 to 59.

[0311] 63. The oligoclonal antigen-binding protein mixture according to any one of items 60 to 62, wherein one of the at least two different antigen-binding proteins comprises complementary determining regions (CDRs) consisting of:

[0312] (a) CDR1 as set forth by SEQ ID NO:6, CDR2 as set forth by SEQ ID NO: 7 and CDR3 as set forth by SEQ ID NO:8, and; wherein another of the at least two different antigen-binding protein comprises complementary determining regions (CDRs) consisting of:

[0313] (b) CDR1 as set forth by SEQ ID NO.: 14, CDR2 as set forth by SEQ ID NO.:

[0314] 15, and CDR3 as set forth by SEQ I D NO. : 16.

[0315] 64. The oligoclonal antigen-binding protein mixture according to any one of items 60 to 63, wherein one of the at least two different antigen-binding protein comprises or consists of:

[0316] (a) a heavy chain variable (VH) region as set forth by SEQ ID NO. 5, and; wherein another of the at least two different antigen-binding protein comprises or consists of:

[0317] (b) a heavy chain variable (VH) region as set forth by SEQ ID NO.: 13.

[0318] 65. The oligoclonal antigen-binding protein mixture according to any one of items 60 to 64, consisting of: (a) an antigen-binding protein comprising complementary determining regions (CDRs) consisting of CDR1 as set forth by SEQ ID NO:6, CDR2 as set forth by SEQ ID NO: 7 and CDR3 as set forth by SEQ ID NO:8, and;

[0319] (b) an antigen-binding protein comprising complementary determining regions (CDRs) consisting of CDR1 as set forth by SEQ ID NO.: 14, CDR2 as set forth by SEQ ID NO.: 15, and CDR3 as set forth by SEQ ID NO.: 16.

[0320] 66. The oligoclonal antigen-binding protein mixture according to any one of items 60 to 65, consisting of:

[0321] (a) an antigen-binding protein comprising or consisting of a heavy chain variable (VH) region as set forth by SEQ ID NO. 5, and;

[0322] (b) an antigen-binding protein comprising or consisting of a heavy chain variable (VH) region as set forth by SEQ ID NO.: 13.

[0323] 67. A multi-specific antigen-binding protein, comprising or consisting of one antigen-binding portion capable of binding to, blocking, and / or neutralizing snake venom PLA2 and another antigen-binding portion capable of binding to, blocking, and / or neutralizing snake venom short-chain alpha neurotoxin.

[0324] 68. The multi-specific antigen-binding protein according to item 67, wherein one antigen-binding portion is as defined in any one of items 1 to 31 and 59, and another antigen-binding portion is as defined in any one of items 32 to 59.

[0325] 69. The multi-specific antigen binding protein according to any one of items 67 to 69 consisting of:

[0326] (a) an antigen-binding portion comprising complementary determining regions (CDRs) consisting of CDR1 as set forth by SEQ ID NO:6, CDR2 as set forth by SEQ ID NO: 7 and CDR3 as set forth by SEQ ID NO:8, and;

[0327] (b) an antigen-binding portion comprising complementary determining regions (CDRs) consisting of CDR1 as set forth by SEQ ID NO.: 14, CDR2 as set forth by SEQ ID NO.: 15, and CDR3 as set forth by SEQ ID NO.: 16.

[0328] 70. The multi-specific antigen binding protein according to any one of items 67 to 69, consisting of: (a) an antigen-binding portion comprising or consisting of a heavy chain variable (VH) region as set forth by SEQ ID NO. 5, and;

[0329] (b) an antigen-binding portion comprising or consisting of a heavy chain variable (VH) region as set forth by SEQ ID NO.: 13.

[0330] 71. The antigen-binding protein, the oligoclonal antigen-binding protein mixture or the multi-specific antigen-binding protein according to any one of the preceding items, wherein the PLA2 comprises or consists of a polypeptide sequence selected from the group consisting of: SEQ. ID NO. 25, SEQ. ID NO. 26, and sequence ID NO. 34, or variant(s) thereof having at least 70% identity, such as at least 75% identity, for example at least 80% identity, such as at least 85% identity, for example at least 90% identity, such as at least 95% identity thereto.

[0331] 72. The antigen-binding protein or the oligoclonal antigen-binding protein mixture or the multi-specific antigen-binding protein according to any one of the preceding items, wherein the short-chain a-neurotoxin comprises or consists of a polypeptide sequence selected from the group consisting of: SEQ. ID NO. 27, SEQ. ID NO. 28, SEQ. ID NO. 29, SEQ. ID NO. 30, SEQ. ID NO. 35, SEQ. ID NO. 36, or variant(s) thereof having at least 70% identity, such as at least 75% identity, for example at least 80% identity, such as at least 85% identity, for example at least 90% identity, such as at least 95% identity thereto.

[0332] 73. The antigen-binding protein, or the oligoclonal antigen-binding protein mixture or the multi-specific antigen-binding protein according to any one of the preceding items, wherein at least one of the antigen-binding protein, for example all of the antigen-binding proteins is (are) 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, and a triabody.

[0333] 74. The antigen-binding protein, or the oligoclonal antigen-binding protein mixture or the multi-specific antigen-binding protein according to any one of the preceding items, wherein at least one of the antigen-binding proteins, for example all of the antigen-binding proteins is (are) monoclonal. The antigen-binding protein, or the oligoclonal antigen-binding protein mixture or the multi-specific antigen-binding protein according to any one of the preceding items, wherein at least one of the antigen-binding proteins, for example all of the antigen-binding proteins is (are) chimeric. The antigen-binding protein, or the oligoclonal antigen-binding protein mixture or the multi-specific antigen-binding protein according to any one of the preceding items, wherein at least one of the antigen-binding proteins, for example all of the antigen-binding proteins is (are) humanized. The antigen-binding protein, or the oligoclonal antigen-binding protein mixture or the multi-specific antigen-binding protein according to any one of the preceding items, wherein at least one of the antigen-binding proteins, for example all of the antigen-binding proteins is (are) human. The oligoclonal antigen-binding protein mixture or the multi-specific antigenbinding protein comprising at least two different antigen-binding proteins according to any one of the preceding items, wherein at least one of the antigen-binding protein is selected from the group consisting of: IgG, IgM, IgA, IgD, and IgE. The oligoclonal antigen-binding protein mixture or the multi-specific antigenbinding protein comprising at least two different antigen-binding proteins according to any one of the preceding items, wherein at least one of the antigen-binding protein is a VHH. The antigen-binding protein, or the oligoclonal antigen-binding protein mixture or the multi-specific antigen-binding protein according to any one of the preceding items, wherein at least one of the antigen-binding proteins, for example all of the antigen-binding proteins is (are) VHH(S). The antigen-binding protein, or the oligoclonal antigen-binding protein mixture or the multi-specific antigen-binding protein according to any one of the preceding items, wherein at least one of the antigen-binding proteins, for example all of the antigen-binding proteins further comprise(s) an immunoglobulin constant region.

[0334] 82. The antigen-binding protein, or the oligoclonal antigen-binding protein mixture or the multi-specific antigen-binding protein according to any one of the preceding items, wherein at least one of the antigen-binding proteins, for example all of the antigen-binding proteins is (are) VHH-FC fusion(s).

[0335] 83. The antigen-binding protein, or the oligoclonal antigen-binding protein mixture or the multi-specific antigen-binding protein according to any one of the preceding items, wherein at least one of the antigen-binding proteins, for example all of the antigen-binding proteins is (are) multispecific.

[0336] 84. The antigen-binding protein, or the oligoclonal antigen-binding protein mixture according to any one of the preceding items, wherein at least one of the antigen-binding proteins, for example all of the antigen-binding proteins is (are) bispecific.

[0337] 85. The antigen-binding protein, or the oligoclonal antigen-binding protein mixture or the multi-specific antigen-binding protein according to any one of the preceding items, wherein at least one of the antigen-binding proteins is bivalent and wherein each monomer of the bivalent antigen-binding protein comprises or consists of the polypeptide sequence as set forth by SEQ. ID. NO: 13.

[0338] 86. The antigen-binding protein, or the oligoclonal antigen-binding protein mixture or the multi-specific antigen-binding protein according to any one of the preceding items, wherein at least one of the antigen-binding proteins is bivalent and wherein each monomer of the bivalent antigen-binding protein comprises a complementary determining region (CDR) consisting of CDR1 as set forth by SEQ ID NO.: 14, CDR2 as set forth by SEQ ID NO.: 15, and CDR3 as set forth by SEQ ID NO.: 16.

[0339] 87. The antigen-binding protein, or the oligoclonal antigen-binding protein mixture or the multi-specific antigen-binding protein according to any one of the preceding items, wherein 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.

[0340] 88. The antigen-binding protein, or the oligoclonal antigen-binding protein mixture or the multi-specific antigen-binding protein according to any one of the preceding items, wherein at least one of the antigen-binding proteins, for example all of the antigen-binding proteins comprise(s) a detection label.

[0341] 89. The antigen-binding protein, or the oligoclonal antigen-binding protein mixture or the multi-specific antigen-binding protein according to any one of the preceding items, wherein at least one of the antigen-binding proteins, for example all of the antigen-binding proteins comprise(s) a detection label selected from the group consisting of a colorimetric, a fluorescent, a luminescent, a magnetic, and a paramagnetic label.

[0342] 90. The antigen-binding protein, or the oligoclonal antigen-binding protein mixture or the multi-specific antigen-binding protein according to any one of the preceding items, wherein at least one of the antigen-binding proteins, for example all of the antigen-binding proteins comprise(s) a detection label, which is biotin or a gold nanoparticle.

[0343] 91. The antigen-binding protein, or the oligoclonal antigen-binding protein mixture or the multi-specific antigen-binding protein according to any one of the preceding items, wherein at least one of the antigen-binding proteins or at least one of the antigen-binding portions binds PLA2 with a KD of at the most 100pM, for example at the most 150pM, such as at the most 200pM, for example at the most 250pM, for example at the most 300pM, for example at the most 350pM, for example at the most 400pM, such as at the most 410pM, for example at the most 420pM, such as at the most 430pM, for example at the most 440pM, such as at the most 450pM, for example at the most 460pM, such as at the most 470pM, for example at the most 480pM, such as at the most 490pM, for example at the most 500pM, such as at the most 510pM, for example at the most 520pM, such as at the most 530pM, for example at the most 540pM, such as at the most 550pM, for example at the most 50nM. 92. The antigen-binding protein, or the oligoclonal antigen-binding protein mixture or the multi-specific antigen-binding protein according to any one of the preceding items, wherein at least one of the antigen-binding proteins or at least one of the antigen-binding portions binds short-chain a-neurotoxin with a KD of at the most 500pM, such as at the most 510pM, for example at the most 520pM, such as at the most 530pM, for example at the most 540pM, such as at the most 550pM, for example at the most 1nM, such as at the most 10nM, for example at the most 15nM, such as at the most 20nM, for example at the most 30nM, such as at the most 40nM, for example at the most 50nM, such as at the most 60nM, for example at the most 65nM, such as at the most 66nM, for example at the most 67nM, such as at the most 68nM, for example at the most 69nM, such as at the most 70nM.

[0344] 93. The antigen-binding protein, or the oligoclonal antigen-binding protein mixture or the multi-specific antigen-binding protein any one of the preceding items, wherein at least one of the antigen-binding proteins is capable of neutralizing enzymatic PLA2 activity in vitro by at least 50%, such as by at least 60%, for example by at least 70%, such as by at least 80%, for example by at least 90% compared to without antigen-binding protein, as measured by any suitable enzymatic assay as described herein.

[0345] 94. A pharmaceutical composition comprising one or more antigen-binding proteins, the oligoclonal antigen-binding protein mixture or the multi-specific antigenbinding protein according to any one of the preceding items, and a pharmaceutically acceptable diluent, carrier and / or excipient.

[0346] 95. The pharmaceutical composition of item 94, further comprising one or more additional antigen-binding protein(s) capable of binding to, blocking, or neutralizing one or more snake venom antigen(s).

[0347] 96. A kit-of-parts comprising: a) one or more antigen-binding proteins, the oligoclonal antigen-binding protein mixture or the multi-specific antigen-binding protein according to any one of items 1 to 95; and b) another agent suitable for the treatment of snake envenomation.

[0348] 97. The kit-of-parts according to item 96, wherein said other agent used for the treatment of snake envenomation is one or more additional antigen-binding protein(s) selected from the group consisting of: IgG, IgM, scFv, F(ab)2, Fab, VHH antigen-binding protein(s), and fragments thereof.

[0349] 98. The kit-of-parts according any one of items 96 to 97, wherein the another agent is one or more additional antigen-binding protein(s) capable of neutralizing one or more snake venom antigen(s), wherein said agent may be selected from the group consisting of: Equine immunoglobulin(s) against snake venom, such as Equine IgG(s) or Equine Fab or Equine F(ab’)2 fragment(s) against snake venom, Ovine immunoglobulin(s) against snake venom, such as Ovine IgG(s) or Ovine Fab or Ovine F(ab’)2 fragment(s) against snake venom, Rabbit immunoglobulin(s) against snake venom, such as Rabbit IgG(s) or Rabbit Fab or Rabbits F(ab’)2 fragment(s) against snake venom, and Camelids immunoglobulin(s) against snake venom, such as Camelids IgG(s) or Camelids Fab or Camelids F(ab’)2 fragment(s) against snake venom.

[0350] 99. The kit-of-parts according to any one of items 96 to 98, wherein said other agent used for the treatment of snake envenomation is one or more protein(s) displaying neutralization potential against snake venoms.

[0351] 100. The kit-of-parts according to any one of items 96 to 99, wherein the one or more agents is (are) protein(s) displaying neutralization potential against snake venoms selected from the group consisting of : a PLA2 inhibitor, a matrix metalloproteinase (MMP) inhibitor, a snake venom metalloproteinase inhibitor (SVMP), a Kunitz-type trypsin inhibitor, an antimyotoxic factor, an antimyonecrotic factor, an antineurotoxic factor, an antihaemmoragic factor, an antioedematogenic factor and a glycoprotein, such as acid-a1 -glycoprotein.

[0352] 101 . The kit-of-parts according to any one of items 96 to 100, wherein said other agent used for the treatment of snake envenomation is one or more small molecule(s) with inhibitory effect against snake toxins. The kit-of-parts according to item 101 , wherein the one or more small molecule(s) with inhibitory effect against snake toxins is (are) selected from the group consisting of: polyanions, polyphenolic compounds, and metalloproteinase inhibitors. The kit-of-parts according to any one of items 101 and 102, wherein the one or more small molecule(s) with inhibitory effect against snake toxins is (are) selected from the list consisting of : Suramin, Heparin, 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. An antigen binding protein, an oligoclonal antigen-binding protein mixture, a multi-specific antigen-binding protein, a pharmaceutical composition or a kit- of-parts according to any one of the preceding items, for use in the treatment of snake envenomation in a subject. A method for treating snake envenomation in a subject, the method comprising administering a therapeutically effective amount of the antigen binding protein, the oligoclonal antigen-binding protein mixture, the multispecific antigen-binding protein, the pharmaceutical composition, or the kit of parts according to any one of items 1 to 103. Use of the antigen binding protein, the oligoclonal antigen-binding protein mixture, the multi-specific antigen-binding protein, the pharmaceutical composition, or the kit of parts according to any one of items 1 to 103 in a method of treatment of snake envenomation in a subject. Use of the antigen binding protein, the oligoclonal antigen-binding protein mixture, the multi-specific antigen-binding protein, the pharmaceutical composition, or the kit of parts according to any one of items 1 to 103 for the manufacture of a medicament for the treatment of snake envenomation. The antigen binding protein, the oligoclonal antigen-binding protein mixture, the multi-specific antigen-binding protein, the pharmaceutical composition, the kit of parts, the method or the use according to any one of the preceding items, wherein the antigen-binding protein, the oligoclonal antigen-binding protein mixture or the multispecific antigen-binding protein prevents or delays in vivo lethality of at least one toxin selected from the group consisting of: PLA2 and short-chain a-neurotoxin. The antigen binding protein, the oligoclonal antigen-binding protein mixture, the multi-specific antigen-binding protein, the pharmaceutical composition, the kit of parts, the method or the use according to any one of the preceding items, wherein the antigen-binding protein, the oligoclonal antigen-binding protein mixture, or the multi-specific antigen-binding protein delays in vivo lethality of at least one toxin selected from the group consisting of: PLA2 neurotoxin and short-chain a-neurotoxin by at least 8h, such as at least 10h, for instance at least 12h, such as at least 1 day, for instance at least 2 days, such as at least 3 days after envenomation. The antigen binding protein, the oligoclonal antigen-binding protein mixture, the multi-specific antigen-binding protein, the pharmaceutical composition, the kit of parts, the method or the use according to any one of the preceding items, wherein the antigen-binding protein, the oligoclonal antigen-binding protein mixture, the multi-specific antigen-binding protein, the composition, the kit of parts, the method, or the use fully neutralizes in vivo lethality of at least one toxin selected from the group consisting of: PLA2 and short-chain a- neurotoxin. The antigen binding protein, the oligoclonal antigen-binding protein mixture, the multi-specific antigen-binding protein, the pharmaceutical composition, the kit of parts, the method or the use according to any one of the preceding items, wherein the antigen-binding protein, the oligoclonal antigen-binding protein mixture, the multi-specific antigen-binding protein, the composition, the kit of parts, the method, or the use prevents or delays in vivo lethality of whole snake venom. The antigen binding protein, the oligoclonal antigen-binding protein mixture, the multi-specific antigen-binding protein, the pharmaceutical composition, the kit of parts, the method or the use according to any one of the preceding items, wherein the antigen-binding protein, the oligoclonal antigen-binding protein mixture, the multi-specific antigen-binding protein, the composition, the kit of parts, the method, or the use delays in vivo lethality of whole snake venom by at least 8h, such as at least 10h, for instance at least 12h, such as at least 1 day, for instance at least 2 days, such as at least 3 days after envenomation. The antigen binding protein, the oligoclonal antigen-binding protein mixture, the multi-specific antigen-binding protein, the pharmaceutical composition, the kit of parts, the method or the use according to any one of the preceding items, wherein the antigen-binding protein, the oligoclonal antigen-binding protein mixture, the multi-specific antigen-binding protein, the composition, the kit of parts, the method, or the use fully neutralizes in vivo lethality of whole snake venom. The antigen binding protein, the oligoclonal antigen-binding protein mixture, the multi-specific antigen-binding protein, the pharmaceutical composition, the kit of parts, the method or the use according to any one of the preceding items, wherein the snake is an elapid snake. The antigen binding protein, the oligoclonal antigen-binding protein mixture, the multi-specific antigen-binding protein, the pharmaceutical composition, the kit of parts, the method or the use according to any one of the preceding items, wherein the snake is a coral snake. The antigen binding protein, the oligoclonal antigen-binding protein mixture, the multi-specific antigen-binding protein, the composition, the kit of parts, the method or the use according to any one of the preceding items, wherein the snake is of a genus selected from the group consisting of: Micrurus, Naja, and Haemachatus.

[0353] 117. The antigen binding protein, the oligoclonal antigen-binding protein mixture, the multi-specific antigen-binding protein, the composition, the kit of parts, the method or the use according to any one of items 115 and 116, wherein the coral snake is of a genus selected from the group consisting of: Micrurus, Micruroides, Calliophis, Hemibungarus, Sinomicrurus, more preferably wherein the snake is of the genera Micrurus and Micruroides.

[0354] 118. The antigen binding protein, the oligoclonal antigen-binding protein mixture, the composition, the kit of parts, the method or the use according to any one of items 115 to 117, wherein the coral snake is selected from the group consisting of: Micrurus fulvius or Micrurus diastema.

[0355] 119. The antigen binding protein, the oligoclonal antigen-binding protein mixture, the composition, the kit of parts, the method or the use according to item 118, wherein the coral snake is Micrurus fulvius.

[0356] 120. The antigen binding protein, the oligoclonal antigen-binding protein mixture, the composition, the kit of parts, the method or the use according to item 118, wherein the coral snake is Micrurus diastema.

[0357] 121. The antigen binding protein, the oligoclonal antigen-binding protein mixture, the multi-specific antigen-binding protein, the composition, the kit of parts, the method or the use according to any one of the preceding items, wherein the antigen-binding protein, the pharmaceutical composition or the kit-of-parts is administered parenterally or pulmonarily.

[0358] 122. The antigen binding protein, the oligoclonal antigen-binding protein mixture, the multi-specific antigen-binding protein, the composition, the kit of parts, the method or the use according to any one of the preceding items, wherein the administration is intravenous, intramuscular and / or sub-cutaneous. The antigen binding protein, the oligoclonal antigen-binding protein mixture, the multi-specific antigen-binding protein, the composition, the kit of parts, the method or the use according to any one of the preceding items, wherein the administration is performed no more than 5 min after envenomation, such as no more than 10 min after envenomation, for instance no more than 15 min after envenomation, such as no more than 20 min after envenomation, for instance no more than 30 min after envenomation, such as no more than 45 min after envenomation, for instance no more than 60 min after envenomation, such as no more than 90 min after envenomation, for instance no more than 120 min after envenomation, such as no more than 180 min after envenomation, for instance no more than 240 min after envenomation, such as no more than 6h after envenomation, for instance no more than 12h after envenomation, such as no more than 24h after envenomation, for instance no more than 48h after envenomation, such as no more than 72h after envenomation, for instance no more than 96h after envenomation, such as no more than 120h after envenomation for instance no more than 144h after envenomation. A kit for detection and / or quantification of snake venom / toxins in a sample comprising one or more of the antigen binding proteins, the oligoclonal antigen-binding protein mixtures or the multi-specific antigen-binding proteins according to any one of items 1 to 123. The kit according to item 124, wherein said snake is the snake of any one of items 114 to 120. The kit according to any one of items 124 to 125, wherein the sample is a blood sample, such as a plasma blood sample, a urine sample, a smear of the bite site, or a smear of the bite wound. The kit according to any one of items 124 to 126, wherein the kit is an immunoassay, such as a lateral flow assay, an ELISA, an electrochemical impedance spectroscopy-based assay, a Bilayer Interferometry (BLI)-based assay or a turbidimetric immunoassay. 128. An antigen-binding protein, an oligoclonal antigen-binding protein mixture or a multi-specific antigen-binding protein according to any one of items 1 to 123 for use in the diagnosis of snake envenomation in a subject.

[0359] 129. Use the antigen binding protein, the oligoclonal antigen-binding protein mixture or the multi-specific antigen-binding protein according to any one of items 1 to 123 in an in vitro method for detection and / or diagnosis of snake envenomation in a subject.

[0360] 130. The antigen binding protein, the oligoclonal antigen-binding protein mixture, the multi-specific antigen-binding protein, the composition, the kit of parts, the method or the use according to any one of the preceding items wherein the subject is a human.

[0361] 131. The antigen binding protein, the oligoclonal antigen-binding protein mixture, the multi-specific antigen-binding protein, the composition, the kit of parts, the method or the use according to any one of the preceding items, wherein the subject is a non-human animal, wherein said non-human animal for example may belong to the family of Equidae, Bovidae, Camelidae, Canidae or Felidae, for example the non-human animal is a horse, a cow, an ox, a camel, a dog or a cat.

[0362] 132. The antigen binding protein, the oligoclonal antigen-binding protein mixture, the multi-specific antigen-binding protein, the composition, the kit of parts, the method, the use, or the kit according to any one of the preceding items, wherein the antigen binding protein(s), the oligoclonal antigen-binding protein mixture(s) or the multi-specific antigen-binding protein(s) do not bind long- chain alpha neurotoxins, such as alpha-cobratoxin.

[0363] 133. The use according to any one of items 128 to 132 wherein said snake is the snake of any one of items 114 to 120. Examples

[0364] Example 1 : Camelid immunization and library generation

[0365] To generate VnH-displaying phage libraries, one alpaca and one llama were immunized with increasing doses of a mixture of 18 elapid venoms over a 16-week period (Table 1). Blood samples were taken from the animals on each injection day, and the antibody responses over time against all individual venoms used for immunization were analyzed using ELISA, as described hereafter.

[0366] Material and methods:

[0367] VHH library generation

[0368] A VnH-displaying phage library targeting elapid snake venoms was generated at the VIB Nanobody Core (Brussels, Belgium). For this, one alpaca and one llama were immunized with a mixture of venoms from 18 snake species (Dendroaspis angusticeps, D. jamesoni, D. polylepis, D. viridis, Naja anchietae, N. annulifera, N. ashei, N. haje, N. katiensis, N. melanoleuca, N. mossambica, N. nigricincta, N. nigricollis, N. nubiae, N. pallida, N. senegalensis, and Haemachatus haemachatus). Both camelids were injected subcutaneously (s.c.) bi-weekly at eight time points with increasing doses of the respective venom mixtures (for the immunization scheme, see Table 1). Venoms were mixed, diluted in phosphate-buffered saline (PBS: 137 mM NaCI, 3 mM KCI, 8 mM Na2HPO4.2H2O, 1.4 mM KH2PO4, pH 7.4) and mixed with Gerbu adjuvant P before injection. Blood samples were collected just before venom injection and on days 46, 49, 102, and 105 for library generation.

[0369] Table 1. Immunization scheme per animal.

[0370] The VHH phage display libraries were constructed as previously described (Pardon et al., 2014). Briefly, peripheral blood lymphocytes were prepared from the collected blood and used for total RNA extraction. Libraries were prepared by pooling the total RNA samples after 46 and 49 days to generate a first library and after 102 and 105 days to generate a second library. These were used as templates for first-strand synthesis of cDNA using oligo(dT) primers. Thereafter, VnH-encoding open reading frames were amplified by polymerase chain reaction (PCR), cloned into the phagemid vector pMECS, and transformed into electrocom petent E. coli TG1 cells.

[0371] Camel id antibody titer determination by enzyme-linked immunosorbent assay (ELISA)

[0372] White 96-Well Immuno Plates (GR-655074, In Vitro Scientific) were coated with 60 pL / well of whole venom diluted in PBS (0.5 pg / mL) and incubated overnight at 4 °C. The next day, the plates were washed 4 times with PBST (PBS + 0.1% Tween 20), blocked with 200 pL of 0.5% bovine serum albumin (BSA) in PBST for 1 h at room temperature (RT), and washed 4 times with PBST. Then, 60 pL / well of plasma samples diluted in 0.5% BSA-PBST (0.4% (v / v)) were added and incubated for 1 h at RT, followed by 4 washes with PBST. Bound heavy-chain-only IgGs were detected with 60 pL / well of HRP-conjugated anti-alpaca IgG HH domain (128-035-232, Jackson ImmunoResearch) diluted 1:10,000 in 0.5% BSA-PBST and incubated for 1 h at RT, followed by 4 washes with PBST. Finally, 60 pL / well of SuperSignal® ELISA Pico Chemiluminescent Substrate (37070, Thermo Fisher) was added, and the wells were incubated for 5 min at RT before the plates were read in a plate reader (VICTOR® Nivo™, PerkinElmer). Results:

[0373] Antibody binding signals increased over time for all venoms and both camelids showed similar immune responses against the same venoms. For example, lower binding signals were generally observed against Dendroaspis venoms compared to Naja venoms (Fig. 1).

[0374] Conclusions:

[0375] After 8 and 16 weeks of immunization, two VnH-displaying phage libraries were generated, both larger than 3x10A8 individual clones with insert rates >85%. As serum samples from the two animals showed a similar immune response, the phage libraries were mixed, resulting in one library from 8 weeks and one from 16 weeks of immunization.

[0376] Example 2: Phage display selection campaigns and screening of VHH binders

[0377] For the discovery of VHHS against the medically most relevant toxins in coral snake venoms, PLA2S and a-neurotoxins (aNTxs) from the 3FTx family, the 16-week library was panned against purified toxins. These included two wild-type PLA2S (PLA2N and PLA2O), two recombinantly expressed native aNTxs (rEury and rDH), and a recombinantly expressed short chain consensus aNTx (scNTx,). After three rounds of selection, the enriched libraries (TPL0637, TPL0638, TPL0622, TPL0623, and TPL0629) were eventually subcloned into a modified pHEN6 expression vector for soluble VHHS and transformed into BL21(DE) cells (Wouters et al., 2020). Monoclonal VHHS from individually picked clones were expressed and binding to their cognate target was assessed using an expression-normalized capture DELFIA.

[0378] Material and methods:

[0379] Venoms and purification of toxins

[0380] Venom from M. fulvius was obtained as a pool from 67 individual specimens, kindly donated by Jack Facente from “AGRITOXINS Venom Lab” (Florida, US). Venom from M. diastema was manually extracted from a single specimen collected in Los Tuxtlas, Veracruz, Mexico (Collection license # SGPS / DGVS / 03459 / 15, SEMARNAT, Mexico) and kept at the “Herpetario Cantil” at the Instituto de Biotecnologia, Universidad Nacional Autonoma de Mexico (IBt-UNAM), Cuernavaca, Mexico. The extracted venom was recovered using miliQ H2O, centrifuged at 14,000 rpm to remove cellular debris, lyophilized, and kept at 4 °C until use. The two main PLA2 neurotoxins from M. fulvius venom (PLA2N and PLA2O) and one short-chain aNTx from M. diastema were purified from whole venoms using reversed-phase high-performance liquid chromatography (RP-HPLC) with a C18 column, as previously described in Vergara et al., 2014 and Benard-Valle et al., 2021.

[0381] Expression and purification of recombinant toxins

[0382] Three short-chain aNTx from the three-finger toxin family (3FTx) were used as target antigens for HH discovery. Two of them, rEury and rDH, had previously been identified in coral snake venoms (Benard-Valle et al., 2021; Guerrero-Garzon et al., 2018) and the third one, scNTx, is a consensus protein, designed based on 11 sequences of aNTxs from venoms from different elapids De La Rosa et al., 2018. All three short chain aNTxs were recombinantly produced in E. coli, using the SHuffle® T7 strain (Biolabs) for rEury and Origami Gold DE3 (Novagen®) for rDH and scNTx. Glycerol stocks of E. coli cells transformed with the pQE30 vector containing the toxins, were kindly provided by Alejandro Olvera (rEury and rDH) and Dr Gerardo Corzo (scNTx) from I Bt, IINAM. The glycerol stocks were used to inoculate 50 mL LB medium supplemented with ampicillin (80 pg / mL) until an OD600 of 0.7 was reached. Next, 10 mL of these cultures were used to inoculate 1 L of LB medium and protein expression was induced with 0.1 mM IPTG for 24 h at 16 °C and 250 rpm. The toxins were purified from the supernatants by gravity flow purification using immobilized metal-ion affinity chromatography (Ni2+-NTA affinity resin, Thermo Fisher Scientific). The slurry was equilibrated with PBS before addition of the supernatant, after which the slurry was washed with PBS, and the toxins eluted using 250 mM imidazole. The imidazole was removed by dialysis against PBS. Further purification was achieved using RP-HPLC on a C18 column equilibrated with 0.1% trifluoroacetic acid (TFA) and eluted using a gradient towards acetonitrile with 0.1% TFA, as previously described5. All purified toxins were lyophilized and stored at 4°C until use. The identity and integrity of the toxins was verified using mass spectrometry with an electrospray ionization system (ESI-MS) on an LTQ-Orbitrap XLmass spectrometer (Thermo-Fisher). Biotinylation and MS analysis of toxins

[0383] Lyophilized toxins were resuspended in PBS to a final concentration of approximately 2 mg / mL for biotinylation. The toxins were biotinylated using EZ-Link™ NHS-PEG4-Biotin (A39259, Thermo Fisher) at a 1 :2 molar ratio (2 biotin for every toxin molecule) for 30 min at RT. Free biotin was removed using 2,000 Da MWCO filter tubes (Sartorius). The toxin concentrations were determined using absorbance at 280nm (NanoDrop, Thermo Scientific) and correcting for their molar extinction coefficients.

[0384] The molecular mass of all the toxins and the biotinylation ratio was determined by MALDI-TOF MS using a Proteomics Analyzer 4800 Plus mass spectrometer (Applied Biosystems).

[0385] Phage display selection campaigns and subcloning

[0386] To select for toxin-binding VHHS, phage display selection campaigns were performed using the VnH-displaying phage library, following a protocol similar to one previously described (Ahmadi et al., 2020; Ledsgaard et al., 2023). In short, three consecutive rounds of selection were performed for three aNTxs (scNTx, rDH, and rEury) and two PLA2S (PLA2N and PLA2O), incubating the phage library with final toxin concentrations of 50 nM in the first two rounds and 10 nM in the third round. After the third round, the VbiH-encoding genes were isolated and subcloned into an expression vector (a modified pHEN6 vector with an OmpA signal peptide and a 3xFLAG / 6xHis tag) and transformed into E. coli BL21 (DE3) cells, as previously described (Wouters et al., 2020). Subsequently, individual VHH clones were picked into 500 pL of 2xTY medium supplemented with kanamycin (50 pg / mL) and glucose (2%) in 96 well plates, and grown O / N at 30 °C and 800 rpm.

[0387] Screening for of VHHS for antigen binding using DELFI A

[0388] For VHH expression, 10 pL of each overnight culture was diluted into 1 mL of autoinduction medium (Studier et al., 2005) supplemented with kanamycin (50 pg / mL) in 96-deep-well plates and incubated O / N at 30 °C and 800 rpm. After that, the plates were centrifuged for 10 min at 3,000 g, and the pellets were frozen at -20 °C O / N. The next day, the pellets were resuspended in 110 pL PBS, centrifuged for 10 min at 4,500 g, and the supernatants containing the VHHS were transferred to a new 96-well plate and stored at -20 °C until use. The VHHS were screened for binding using an expression-normalized capture DELFI A as described previously (Laustsen et al., 2018).

[0389] The plasmids encoding binders with the highest signal against each target antigen were purified (GeneJET Plasmid MiniPrep kit, Thermo Scientific) according to the manufacturer’s protocol and sequenced using the M13rev-29 primer (Eurofin Genomics). The VHH frameworks and CDRs were annotated and analyzed to identify unique clones (CLC Main workbench v22.0.2).

[0390] Results:

[0391] In total, 24 and 49 clones with high binding signals to PLA2 and aNTxs, respectively, were selected for sequencing.

[0392] Conclusions:

[0393] Using the above sequencing approach, 19 unique anti-PLA2 and 17 unique anti-aNTxs VHH sequences clustered in 4 and 7 families, respectively, based on sequence similarity of the complementarity-determining regions (CDRs).

[0394] Example 3: Analysis of VHH binding kinetics with biolayer interferometry

[0395] Biolayer interferometry (BLI) experiments were performed to characterize the binding kinetics of the three unique VHH clones from each toxin group that showed the highest binding signals in the expression-normalized capture DELFIA. Also, to compare different VHH formats, TPL0629_01_D11 was produced as a bivalent construct by linking two identical VHHS to each other with a 15GS. The data were fitted to a 1 :1 binding model, which assumes a single VHH molecule binds to a single toxin molecule.

[0396] Next, the binding kinetics of the bivalent format of TPL0629_02_D11 (see Material and methods section hereafter) were determined by BLI, and the data were fitted using a 1 :2 kinetic model (toxin vs bivalent analyte) (Figure 3). Material and methods:

[0397] Expression and purification of VHHS

[0398] In total, six VHHS were selected for expression and purification: three from the PLA2 selection campaigns (TPL0637_01_A01, TPL0637_01_A07, and TPL0638_01_C09) and three from the aNTx selection campaigns (TPL0629_01_D11, TPL0629_01_A07 and TPL0629_01_G06). One VHH, TPL0629_01_D11 , was also expressed as a bivalent VHH construct where two identical copies of the HH were united by a 15GS linker for which the gene was purchased (Genscript). VHH expression was performed using E. coli BL21 (DE3) in TB medium supplemented with kanamycin (50 pg / mL), glucose (0.1% w / v), and MgSO4 (1 mM). The cells were grown at 37 °C and 220 rpm until OD600 reached 0.5. Subsequently, 0.5 mM IPTG was added to induce protein expression for 16 h at 30 °C and 220 rpm. The cells were collected by centrifugation at 4,000 g for 15 min at 4 °C, and stored at -20 °C.

[0399] To extract the periplasmic fractions, in which the majority of the VHH products was expected to exist, the frozen cells were resuspended in cold PBS supplemented with 10 mM imidazole and an EDTA-free protease inhibitor cocktail (Roche). Next, the supernatants containing the VHHS were collected by centrifugation at 20,000 g for 45 min at 4 °C. The His-tagged VHHS were captured on a Ni2+-NTA affinity resin by gravity flow (Thermo Fisher Scientific). Unbound proteins were washed away with wash buffer (PBS with 200 mM NaCI and 20 mM imidazole). The VHHS were eluted (PBS with 200 mM NaCI and 250 mM imidazole), after which the imidazole was removed by dialyzing against PBS. The purity of the VHHS was analyzed by SDS-PAGE and the VHH concentration was determined by absorbance at 280nm (NanoDrop, Thermo Fisher Scientific) by correcting for their molar extinction coefficients.

[0400] Binding analysis using bio-layer interferometry

[0401] Binding kinetics between the purified VHHS and their specific toxin targets were analyzed using bio-layer interferometry. Measurements were performed in kinetics buffer (PBS and 0.02% Tween 20; ForteBio) at 30 °C using an Octet RED 96 instrument (ForteBio). Biotinylated PL 2N or DH with a final concentration of 1 pg / mL were loaded onto streptavidin biosensors (Sartorius) until a thickness of approximately 0.9 nm was reached. Toxin-loaded biosensors were dipped into five different VHH concentrations (200, 66.7, 22.2, 7.4, 2.5, and 0.8 nM). The association of VHHS to the toxins was measured for 600 s, followed by the dissociation for 600 s by incubating the biosensors in kinetics buffer. Sensors were regenerated by two rounds of 5 s incubations in Glycine at pH 1.5 followed by kinetics buffer before measuring the binding kinetics of the next VHH -toxin pair. Two reference measurements, one without biotinylated toxin and the highest concentration of VHH, and one with biotinylated toxin but without any HH, were subtracted from all curves. All data were analyzed using Octet® Analysis Studio 12.2.2.26 (ForteBio).

[0402] Results:

[0403] Results of the binding kinetics of the antigen-binding proteins to the toxins are presented in Figs. 2 and 3.

[0404] Conclusions:

[0405] All the tested VHHS showed high affinity, with KD values in the pM range for the three anti-PLA2 VHHS and in the nM range for the three anti-aNTx VHHS (Fig. 3H). As expected, the apparent affinity of the bivalent VHH construct was significantly higher (KD = 5.25x1 O'10M) compared to the monovalent format (KD = 6.94x1 O'08M), which likely stems from avidity effects. The bivalent VHH construct, as well as four of the six monovalent VHHS showed very slow dissociation rates which indicates that a stable interaction takes place between them and their respective toxins (Figs. 2 and 3).

[0406] Example 4: Neutralization of PLA2 enzymatic activity

[0407] To obtain an initial assessment of the potential neutralizing capacity of the PLA2- targeting VHHS, an in vitro enzymatic PLA2 activity neutralization assay was performed using a commercial kit. First, the EC50 value of PLA2N was determined to be 35 nM under the assay conditions. Next, the three anti-PLA2 VHHS included for kinetic measurements in BLI were evaluated at a 1:10 toxin to VHH molar ratio (35 vs 350 nM). Material and methods:

[0408] In vitro neutralization of enzymatic PLA2 activity

[0409] Determination of PLA2 enzymatic activity was performed using the fluorometric EnzChek™ Phospholipase A2 Assay Kit (Invitrogen), according to manufacturer’s protocol. Fluorescence was measured using a plate reader (VICTOR® Nivo™, PerkinElmer) at an excitation wavelength of 480 nm and an emission wavelength of 530 nm. Measurements were made immediately after substrate addition and then every 30 seconds for 10 min. The slope was determined for the initial (linear) part of the curve, and units of enzymatic activity were defined as the change in relative fluorescence units per sec. The EC50 was then determined for the purified toxin PLA2N.

[0410] Neutralization of enzymatic activity was assessed by incubating 1 EC50 of PLA2N (35 nM) with a 1:10 toxin to HH molar ratio of each anti-PLA2 HHS for 30 min at RT. PLA2 activity was afterwards determined for each of the mixtures in triplicate. The PLA2N activity in the presence of the HHS was normalized by setting the activity of PLA2N without any HH addition to 100%.

[0411] Results:

[0412] Results of the neutralization of the enzymatic PLA2N activity are presented in Fig. 4.

[0413] Conclusions:

[0414] All three VHHS significantly reduced the enzymatic PLA2N activity, with the highest inhibition observed for TPL0637_01_A01. As expected, the anti-aNTx VHH TPL0629_01_D11 , included as a negative isotype control, did not show any neutralization of enzymatic PLA2 activity (Fig. 4).

[0415] Example 5: In vivo neutralization of purified toxins

[0416] Based on the in vitro binding and neutralization data, the most promising VHHS were assessed for neutralization of lethality with purified toxins via intravenous ( / .v.) injection of the preincubated toxin and VHH (preincubation experiments) and i.v. injection of VHH after envenoming using subcutaneous (s.c.) injection (rescue experiments). Three anti- PLA2 VHHS were selected for evaluation of their capacity to neutralize PLA2N-induced lethality. Also three anti-aNTx VHHS were selected to evaluate their capacity to neutralize the lethality induced by wild-type toxin aNTx DH. One of them, namely TPL0629_01_D11 , was evaluated using a monovalent and a bivalent VHH construct in all in vivo experiments. As negative controls, two groups of mice were injected with 3 median lethal doses (LD50s) of PLA2N or DH preincubated with an isotype VHH. AS expected, no neutralization or delay of time of death was observed in these groups (Data not shown). Also, a group of mice was injected i.v. with the highest dose evaluated of each HH. None of these mice showed any signs of adverse reactions.

[0417] Neutralization of PLA2S

[0418] Neutralization of PLA2-induced lethality was evaluated using 3 LD50s of PLA2N, purified from the venom of M. fulvius. The LD50 of this toxin was determined to be 10.3 pg / mouse and 34.6 pg / mouse using the i.v. and s.c. routes, respectively.

[0419] Neutralization of aNTx

[0420] To assess aNTx induced lethality, the LD50 of wild-type aNTx DH from M. diastema was determined to be 2.0 pg / mouse (i.v.) and 4.8 pg / mouse (s.c.). Neutralization of lethality by VHHS was evaluated using 3 LD50s of the toxin.

[0421] Material and methods:

[0422] In vivo neutralization experiments

[0423] All in vivo experiments were performed with groups of three CD1 mice between 18 and 20 g of total body weight and indistinct sex. The mice were kept under 12 h light and dark cycles with food and water ad libitum. For neutralization of whole coral snake venoms, two coral snake species (i.e. , M. fulvius and M. diastema), whose venoms contain identical or very similar toxins to the ones used as antigens in the phage selections, were selected. Toxin to VHH molar ratios were calculated based on the approximate abundance of PLA2S and 3FTxs in the venoms, obtained from proteomic data (Vergara et al., 2014). The number of 3FTx or PLA2 molecules present in 3 LD50s of venom was then used to calculate the necessary amount of VHHS in the mixture. For the bivalent TPL0629_01_D11 construct, where each molecule has two toxin-binding sites, a molar ratio of 1:5 toxin to VHH represents a 1 :10 ratio of toxin to binding sites.

[0424] Determination of LD50s for whole venoms and toxins

[0425] LD50s were determined for M. fulvius PLA2N and M. diastema DH toxins and the whole venoms of M. fulvius and M. diastema using intravenous ( / .v.) and subcutaneous (s.c.) routes. Groups of three mice were injected with varying doses of the toxin or whole venom in a final volume of 500 pL PBS for i.v. and 100 pL PBS for s.c. Percentage of survival was determined 24 h after injection, and the data were analyzed using a nonlinear regression (Semi-logarithmic dose-response curve) as previously described (Casasola et al., 2009).

[0426] Preincubation experiments

[0427] For preincubation experiments, 3 LD50S of each toxin or venom were combined with their corresponding VHH using a range of toxin to HH molar ratios going from 1 : 1 to 1 :10 in a total volume of 500 pL of PBS. Then, the mixtures were preincubated at 37 °C for 30 min and injected into groups of 3 mice using the i.v. route. The mice were observed during the first 3 h and then approximately every 6 h for appearance of envenomation signs. Percentage of survival was determined 24 h after the injection.

[0428] Rescue experiments

[0429] Rescue experiments were designed to better represent real envenomation where the venom or toxin is injected first and then the therapeutic molecule is administered using the i.v. route. In these experiments, mice were envenomed using the s.c. route with 3 LD50S of each toxin or venom in a final volume of 100 pL PBS. Immediately after toxin injection, the corresponding VHH was injected using the i.v. route in a total volume of 500 pL PBS. The experiments were performed using a range of toxin to VHH molar ratios going from 1:1 to 1:10. Mice were observed during the first 3 h and then approximately every 6 h for the appearance of envenomation signs. Percentage of survival was determined 24 h after the injection. Results:

[0430] Neutralization of PLA2S

[0431] In the preincubation experiments, TPL0637_01_A01 , TPL0637_01_A07, and TPL0638_01_C09 completely neutralized PLA2N-induced lethality at a toxin to VHH molar ratio of 1:1 (Fig. 5A).

[0432] In rescue experiments where the VHHS were injected i.v. immediately after s.c. injections of the toxin, TPL0637_01_A07 completely neutralized PLA2N-induced lethality in all three mice at a toxin to VHH molar ratio of 1:2.5. Using the same molar ratios, HHS TPL0637_01_A01 and TPL0638_01_C09 were able to rescue 1 of the 3 mice and the time of death was significantly delayed for the remaining two (Fig. 5B).

[0433] Neutralization ofaNTx

[0434] In preincubation experiments, both monovalent and bivalent TPL0629_01_D11 constructs prevented lethality at a 1:2.5 molar ratio between toxin and HH binding sites (Fig. 6A and 6C). TPL0629_01_A07 was equally successful at neutralizing lethality in preincubation experiments at a 1:2.5 molar ratio, whereas TPL0629_01_G06 did not neutralize lethality even when the molar ratio was increased to 1 :5 (Data not shown).

[0435] Due to mice availability, further rescue experiments, were only performed with TPL0629_01_D11 , including both the monovalent and the bivalent VHH constructs. Both TPL0629_01_D11 constructs delayed the time of death at a 1 :5 toxin to binding sites molar ratio, although the monovalent VHH extended the life of the mice for 10 hours and was able to save 1 of 3 mice, while the bivalent VHH delayed the time of death by 2 hours. The monovalent TPL0629_01_D11 rescued all mice at a 1 :10 toxin to binding sites molar ratio, while the bivalent construct only saved 1 of 3 mice (Fig. 6B and 6D).

[0436] All animals and in vivo methodologies used in the Examples were approved by the bioethics committee of the IBt, UNAM under project # 385 “Caracterizacion funcional y analisis de especificidad de venenos de coralillos Norteamericanos". Conclusions:

[0437] The antigen-binding proteins of the present invention are capable of neutralizing purified toxins in vivo, both in preincubation and rescue experiments.

[0438] Example 6: In vivo neutralization of whole coral snake venoms

[0439] After evaluating neutralization of individual purified toxins, the two VHHS that displayed the best neutralization of the purified toxins in rescue experiments (TPL0629_01_D11 for aNTxs and TPL0637_01_A07 for PLA2S) were combined into oligoclonal mixtures for neutralization of whole venoms from M. fulvius and M. diastema. The VHHS were mixed and molar ratios to their corresponding toxins were calculated based on the 3FTx and PLA2 abundance in the whole venoms for TPL0629_01_D11 and TPL0637_01_A07, respectively. The LD50s of the venoms were determined to be 6.0 pg / mouse and 5.7 pg / mouse for M. fulvius and M. diastema, respectively, using the i.v. route. Then, 3 LD50s of each venom were preincubated with the oligoclonal mixture prior to i.v. administration.

[0440] Material and methods:

[0441] Material and methods are according to described previously herein.

[0442] Results:

[0443] The results of the in vivo neutralization of whole coral snake venoms are presented in Fig. 7.

[0444] Conclusions:

[0445] The oligoclonal VHH mixture was able to prevent lethality in all the mice envenomed with M. fulvius venom and 2 of the 3 mice envenomed with M. diastema venom using a 1 :10 toxin to VHH molar ratio. Preincubation with either TPL0637_01_A07 (targeting PLA2S) or TPL0629_01_D11 (targeting aNTxs) alone did not prevent lethality or prolonged survival for any of the envenomed mice (Fig. 7). Example 7: Binding of TPL0629_01_D11 to a-neurotoxins and TPL0637_01_A07 to PLA2S

[0446] To measure the binding of the discovered VHHs, TPL0629_01_D11 and TPL0637_01_A07, the inventors performed an expression normalized DELFIA as described in Benard-Valle et al., (2024).

[0447] Material and methods:

[0448] Material and methods are according to described previously herein. Briefly, 96-well plates were coated with an anti-FLAG antibody and blocked with 3% skimmed milk in PBS buffer. The VHHs were added at 5 pg / mL followed by addition of the target toxins. The toxins were first diluted to 1 pM, and then titrated 1 :3 in 10 consecutive dilution steps. As target toxins for the anti-3FTx VHH, the inventors used 5 short chain a- neurotoxic 3FTxs (scNTx, rDHdia, Nh2, rEury, and Hh1), as well as a-cobratoxin, which is a long chain a-neurotoxic 3FTx (negative control). As target toxins for the anti-PLA2 VHH, 2 PLA2S from coral snake venoms (PLA2N and PLA2O) and 2 PLA2S from other elapid snake venoms (Hh3 and Nn19) were used.

[0449] Results:

[0450] The results of the DELFIA are presented in Figure 8.

[0451] Conclusions:

[0452] This experiment shows that the VHHs discovered in the present invention are able to recognize short chain a-neurotoxins from at least three genera of venomous elapids as well as PLA2S from cobras and coral snakes, illustrating their cross-reactive capabilities.

[0453] Example 8: In silico modeling of VnH-toxin interactions

[0454] Material and methods:

[0455] In silico modelling was performed using the AlphaFold 3 Server

[0456] (https: / / alphafoldserver.com / , Abramson et al. 2024) and processed using the software UCSF ChimeraX version: 1.8 (2024-06-10). VHHS are shown as ribbons, and toxins are shown as surface models. Contact regions between molecules are marked in dark grey on ribbons ( HHS) and surface models (toxins) (Figure 9).

[0457] Results:

[0458] The predicted template modelling (pTM) and the interface predicted Template Modelling (ipTM) scores (Zhang et al. 2004, Xu et al. 2010), which provide an evaluation of the accuracy of the models are shown in the following table (Table 2).

[0459] Table 2: predicted template modelling (pTM) and interface predicted Template Modelling (ipTM) scores

[0460] DHdia scNTx PLA2O ipTM pTM ipTM pTM ipTM pTM

[0461] TPL0629_01_Dll 0.77 0.86 0.77 0.85

[0462] TPL0629_01_A07 0.73 0.82 0.8 0.84

[0463] TPL0629_01_G06 0.79 0.87 0.18 0.67

[0464] TPL0637_01_A07 0.16 0.53

[0465] TPL0637_01_A01 0.23 0.56

[0466] TPL0638_01_C09 0.20 0.53

[0467] Using the models, amino acid residues in the toxins in contact with the VHHS of the present invention could be identified:

[0468] A.) Anti 3FTx VHHs

[0469] TPL0629 01 D11 vs DHdia

[0470] E19, Q21 , C41, P42, T43, V44, P46, G47, H49, I50, S51, C52, C53

[0471] TPL0629 01 D11 vs scNTx

[0472] E19, T20, S21, C41 , K43, V44, P46, L50, R54, T55

[0473] TPL0629 01 A07 vs DHdia

[0474] Q21 , C41, P42, T43, V44, P46, H49, I50, S51, C52, C53

[0475] TPL0629 01 A07 vs scNTx

[0476] C41, P42, K43, V44, P46, G47, L50, H51, C52, R54

[0477] TPL0629 01 G06 vs DHdia

[0478] Q21 , C41, P42, T43, V44, P46, H49, I50, S51 , C52, C53, C58

[0479] TPL0629 01 G06 vs scNTx

[0480] E19, T20, S21, C41 , K43, V44, P46, L50, R54, T55 B.) Anti PLA2VHHS

[0481] TPL0637 01 A07 vs PLA2O

[0482] N2, L3, I4, K7, S17, W18, W19, H20, A22, D23, G29, S30, Y110, K111

[0483] TPL0637 01 A01 vs PLA2O

[0484] L1, L3, F6, A22, G29, S30, G31 , G32, H47, D48, Y51 , D52, E55, K56, G59, W61, K63, W64

[0485] TPL0638 01 C09 vs PLA2O

[0486] L1, N2, L3, F6, K7, S17, W18, W19, H20, A22, D23, G29, S30, D107, Y110, and K111.

[0487] Conclusions:

[0488] Contact regions between the VHHS of the present invention and the toxins DHdia, scNTx and PLA2O could be modelled, and the amino acid residues in the toxins in contact with the VHHS of the present invention according to the models obtained could be determined.

[0489] Sequence overview

[0490] SEQ. ID. NO: 1: TPL0637_01_A01HH

[0491] QVQLQESGGGLVQSGGSLRLSCVVSGFTLNSYAMSWFRQGPGKEREWVAVITRSG DTAYVDSVKGRFTTSRDSAKNTFYLQMNSLTPEDTAVYYCNKAATDISLIDYWGQGT QVTVSSAAADYKDHDGDYKDHDIDYKDDDDKGAAHHHHHH

[0492] SEQ. ID. NO: 2: TPL0637_01_A01HH CDR1

[0493] GFTLNSYAMS

[0494] SEQ. ID. NO: 3: TPL0637_01_A01HH CDR2

[0495] VITRSGDTAYVDSVKG

[0496] SEQ. ID. NO: 4: TPL0637_01_A01HH CDR3

[0497] ATDISLIDY

[0498] SEQ. ID. NO: 5: TPL0637_01_A07HH

[0499] QVQLQESGGGLVQAGDSLRLSCAASGRTFSAYTMGWFRQAPGKEREMVAA QWS

[0500] SWGSVNTYYADSVKGRFTVSADNAQNTDYLQMNSLKPEDTAAYYCTVRRVTVDSN WSTLLSELREGPWGQGTQVTVSSAAADYKDHDGDYKDHDIDYKDDDDKGAAHHHH

[0501] HH

[0502] SEQ. ID. NO: 6: TPL0637_01_A07 VHH CDR1

[0503] GRTFSAYTMG

[0504] SEQ. ID. NO: 7: TPL0637_01_A07 VHH CDR2

[0505] AVQWSSWGSVNTYYADSVKG

[0506] SEQ. ID. NO: 8: TPL0637_01_A07 VHH CDR3

[0507] RVTVDSNWSTLLSELREGP

[0508] SEQ. ID. NO: 9: TPL0638_01_C09 VHH

[0509] QVQLQESGGGLVQAGNSLRLSCEASGRTFSAYVMGWFRQAPGKEREMVAAVQWS

[0510] SWGEVNTYYADSVKGRFTISRDNGKNTDYLQMNSLKPEDTATYYCTVRRVTLDSSW

[0511] SILLSGLREGPWGQGTQVTVSSAAADYKDHDGDYKDHDIDYKDDDDKGAAHHHHHH

[0512] SEQ. ID. NO: 10: TPL0638_01_C09 VHH CDR1

[0513] GRTFSAYVMG

[0514] SEQ. ID. NO: 11: TPL0638_01_C09 VHH CDR2

[0515] AVQWSSWGEVNTYYADSVKG

[0516] SEQ. ID. NO: 12: TPL0638_01_C09 VHH CDR3

[0517] RVTLDSSWSILLSGLREGP

[0518] SEQ. ID. NO: 13: TPL0629_01_D11 VHH

[0519] QVQLQESGGGLVQAGGSLRLSCAGSGDALGSYTMGWFRQAPGGGRDLVAQISVDG

[0520] SSTYHLDSVRGRFTASRDNAKNTVYLEMNSLNSEDTAVYYCAAAPLLRGNYDYWGQ

[0521] GTQVTVSSAAADYKDHDGDYKDHDIDYKDDDDKGAAHHHHHH

[0522] SEQ. ID. NO: 14: TPL0629_01_D11 VHH CDR1

[0523] GDALGSYTMG

[0524] SEQ. ID. NO: 15: TPL0629_01_D11 VHH CDR2 QISVDGSSTYHLDSVRG

[0525] SEQ. ID. NO: 16: TPL0629_01_D11 VHH CDR3

[0526] APLLRGNYDY

[0527] SEQ. ID. NO: 17: TPL0629_01_A07 VHH

[0528] QVQLQESGGGLVQAGGSLRLSCTASEGTVSYSTVGWFRQAPGGGRDLVAQSGSSD

[0529] EDTHVAGSVKGRFTISRDNTNNTVYLQMSSLKPEDSAVYYCAAAPGDQYKYWGQGT QVTVSSAAADYKDHDGDYKDHDIDYKDDDDKGAAHHHHHH

[0530] SEQ. ID. NO: 18: TPL0629_01_A07 VHH CDR1

[0531] EGTVSYSTVG

[0532] SEQ. ID. NO: 19: TPL0629_01_A07 VHH CDR2

[0533] QSGSSDEDTHVAGSVKG

[0534] SEQ. ID. NO: 20: TPL0629_01_A07 VHH CDR3

[0535] APGDQYKY

[0536] SEQ. ID. NO: 21 : TPL0629_01_G06 VHH

[0537] QVQLQESGGGLVQAGGSLRLSCAASGRAFSSYTMGWFRQAPGDMRELVAQITSSG SSTYHIDSVEGRFAISRDNAKNTVYLQMNSLQPEDAAIYYCAGGLNAYQYWGQGTQV TVSSAAADYKDHDGDYKDHDIDYKDDDDKGAAHHHHHH

[0538] SEQ. ID. NO: 22: TPL0629_01_G06 VHH CDR1

[0539] GRAFSSYTMG

[0540] SEQ. ID. NO: 23: TPL0629_01_G06 VHH CDR2

[0541] QITSSGSSTYHIDSVEG

[0542] SEQ. ID. NO: 24: TPL0629_01_G06 VHH CDR3

[0543] GLNAYQY

[0544] SEQ. ID NO. 25: PLA2O_PLA2 from fraction O of M. fulvius (partial)

[0545] N LI H FKN M I ECTTKRSWWH FADYGCYCGSG SEQ. ID NO. 26: PLA2N_PLA2 from fraction N of M. fulvius (partial)

[0546] N LI H KN M I CTTKRSWWH FADYGCYCGSGGSXTPV

[0547] SEQ. ID NO. 27: Recombinant scNTx consensus

[0548] MRGSHHHHHHGSIEGRMICYNQQSSQPPTTKTCSETSCYKKTWRDHRGTIIERGCG

[0549] CPKVKPGIKLHCCRTDKCNN

[0550] SEQ. ID NO. 28: Recombinant Eurytoxin (rEury)

[0551] MRGSHHHHHHGSMICYNQQSSEPPTTKTCPDGQCYKKNWSDHRGSKTERG

[0552] CGCPNVKPGIRINCCTTDKCNA

[0553] SEQ. ID NO. 29: Recombinant DH (rDH)

[0554] MRGSHHHHHHGSMICYNQQSSEPPTTKTCPDGQCYKKNWSDHRGSKTERG

[0555] CGCPNVKPGIRINCCTTDKCNA

[0556] SEQ. ID NO. 30: Native DH sequence (partial)

[0557] MICHNQQSSQPPTTKTCSEGQCYK

[0558] SEQ ID NO. 31

[0559] GX1TX2X3X4YX5MX6 wherein

[0560] Xi is F or R, preferably R; and

[0561] X2 is L or F, preferably F; and

[0562] X3 is N or S, preferably S; and

[0563] X4 is S or A. preferably A; and

[0564] X5 is any non-polar amino acid, preferably A, T or V; and

[0565] X6is S or G, preferably G.

[0566] SEQ ID NO: 32

[0567] TX7YX8DSVKG wherein

[0568] X7is A or Y; and

[0569] Xs is V or A.

[0570] SEQ ID NO: 33

[0571] RVTX9DSX10WSX11LLSX12LREGP wherein X9 is a nonpolar amino acid, preferably V or L; and X is a N or S; and

[0572] X11 is a nonpolar amino acid, preferably? or I; and X12 is E or G.

[0573] SEQ ID NO. 34: mature PLA2O sequence - GenBank accession number

[0574] GAEP01002028 - epitope determination

[0575] LN LI H FKNM I ECTTKRSWWH FADYGCYCGSGGSGTPVDDLDKCCQVH DECYDTAEK VHGCWPKWTLYSYDCSNGQLTCKDNNTKCKDFVCNCDRKAVTCFAKAPYNDKNYKI DPNRCQ

[0576] SEQ ID NO: 35 mature DH sequence - epitope determination

[0577] MICHNQQSSQPPTTKTCSEGQCYKKTWRDHRGTIIERGCGCPTVKPGIHISCCASDK CNA

[0578] SEQ ID NO: 36 mature scNTX sequence - Protein Data Bank (PDB) accession number 7Z14_F (chain F) - epitope determination

[0579] MICYNQQSSQPPTTKTCSETSCYKKTWRDHRGTIIERGCGCPKVKPGIKLHCCRTDK CNN

[0580] References

[0581] Abramson, J. et al. Accurate structure prediction of biomolecular interactions with

[0582] AlphaFold 3. Nature 630, 493-500 (2024).

[0583] Ahmadi, S. et al. An in vitro methodology for discovering broadly-neutralizing monoclonal antibodies. Nat. Sci. Reports 10, (2020).

[0584] Benard-Valle, M. et al. Protein composition and biochemical characterization of venom from Sonoran Coral Snakes (Micruroides euryxanthus). Biochimie 182, 206-216 (2021).

[0585] 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).

[0586] Benard-Valle, M. et al. Functional, proteomic and transcriptomic characterization of the venom from Micrurus browni browni: Identification of the first lethal multimeric neurotoxin in coral snake venom. Prep 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).

[0587] 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).

[0588] M. Engmark, M. et al. High-throughput immuno-profiling of mamba (Dendroaspis) venom toxin epitopes using high-density peptide microarrays. Sci Rep 6, 36629 (2016).

[0589] Guerrero-Garzon, J. F. et al. Cloning and sequencing of three-finger toxins from the venom glands of four Micrurus species from Mexico and heterologous expression of an alpha-neurotoxin from Micrurus diastema. Biochimie 147, 114-121 (2018).

[0590] Laustsen, A. H. Antivenom in the Age of Recombinant DNA Technology, in Handbook of Venoms and Toxins of Reptiles (CRC Press, 2021).

[0591] Laustsen, A. H. et al. In vivo neutralization of dendrotoxin-mediated neurotoxicity of black mamba venom by oligoclonal human IgG antibodies. Nat. Commun. 9, 3928 (2018).

[0592] Laustsen, A. H. et al. Animal Immunization, in Vitro Display Technologies, and Machine Learning for Antibody Discovery. Trends in Biotechnology. 39, 1263-1273 (2021)

[0593] Laustsen, A. H. et al. From Fangs to Pharmacology: The Future of Snakebite Envenoming Therapy. CPD. 22, 5270-5293 (2016).

[0594] 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) doi: 10.1101 / 2021.09.07.459075. 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).

[0595] Marks, J. D. et al. By-Passing Immunization: Building High Affinity Human Antibodies by Chain Shuffling. Bio / Technology 10, 779-783 (1992).

[0596] Nguyen, G.T.T et al. High-throughput proteomics and in vitro functional characterization of the 26 medically most important elapids and vipers from sub- Saharan Africa. GigaScience 11, giac121 (2022)

[0597] E. Pardon, T. Laeremans, S. Triest, S. G. F. Rasmussen, A. Wohlkdnig, A. Ruf, S. Muyldermans, W. G. J. Hol, B. K. Kobilka, J. Steyaert, A general protocol for the generation of Nanobodies for structural biology. Nat Protoc. 9, 674-693 (2014).

[0598] Rivera-de-torre, E., Lamparidou, S., Bohn, M. F. & Kazemi, S. M. Discovery of broadly neutralizing antibodies against brown recluse spider and Gadim scorpion sphingomyelinases using consensus toxins as antigens. bioRxiv Prepr. 1-29 (2023).

[0599] Roncolato, E. C. et al. Phage display as a novel promising antivenom therapy: A review. Toxicon 93, 79-84 (2015).

[0600] Sorensen, C. V. et al. Cross-reactivity trends when selecting scFv antibodies against snake toxins using a phage display-based cross-panning strategy. Sci. Rep. 13, 1-10 (2023).

[0601] Studier, F. W. Protein production by auto-induction in high density shaking cultures. Protein Expr. Purif. 41 , 207-234 (2005).

[0602] Vergara, I. et al. Eastern coral snake Micrurus fulvius venom toxicity in mice is mainly determined by neurotoxic phospholipases A2. J. Proteomics 105, 295-306 (2014)

[0603] Wouters, Y. et al. Identification and in vivo characterization of a brain-penetrating nanobody. Fluids Barriers CNS 17, 4-13 (2020) Xu, J. et al. How significant is a protein structure similarity with TM-score = 0.5? Bioinformatics 26, 889-895 (2010). Zhang, Y. et al. Scoring function for automated assessment of protein structure template quality. Proteins 57, 702-710 (2004).

Claims

Claims1. An antigen-binding protein capable of binding to, blocking, and / or neutralizing at least two different snake venom PLA2S, such as at least three, such as at least four PLA2S, preferably wherein the snake venom PLA2 is PLA2N, PLA2O, Nn19, and / or Hh3, even more preferably PLA2N, PLA2O, Nn19, and Hh3.

2. The antigen-binding protein according to claim 1 , wherein the antigen binding protein binds an epitope of PLA2O comprising one or more amino acid residues selected from the group consisting of:- N2, L3, I4, K7, S17, W18, W19, H20, A22, D23, G29, S30, Y110, and K111 of SEQ ID NO. 34; or- L1 , L3, F6, A22, G29, S30, G31 , G32, H47, D48, Y51 , D52, E55, K56, G59, W61 , K63, and W64 of SEQ ID NO. 34; or- L1 , N2, L3, F6, K7, S17, W18, W19, H20, A22, D23, G29, S30, D107, Y110, and K111 of SEQ ID NO. 34.

3. The antigen-binding protein according to any one of the preceding claims, comprising complementary determining regions (CDRs) comprising or consisting of:- a CDR1 as set forth by SEQ ID NO: 2 or a sequence identical thereto except for up to 2, such as up to 1 amino acid substitution(s); and- a CDR2 as set forth by SEQ ID NO: 3 or a sequence identical thereto except for up to 2, such as up to 1 amino acid substitution(s); and- a CDR3 as set forth by SEQ ID NO: 4 or a sequence identical thereto except for up to 2, such as up to 1 amino acid substitution(s); or- a CDR1 as set forth by SEQ ID NO: 6 or a sequence identical thereto except for up to 2, such as up to 1 amino acid substitution(s); and- a CDR2 as set forth by SEQ ID NO: 7 or a sequence identical thereto except for up to 2, such as up to 1 amino acid substitution(s); and- a CDR3 as set forth by SEQ ID NO: 8 or a sequence identical thereto except for up to 2, such as up to 1 amino acid substitution(s);or- a CDR1 as set forth by SEQ ID NO: 10 or a sequence identical thereto except for up to 2, such as up to 1 amino acid substitution(s); and- a CDR2 as set forth by SEQ ID NO: 11 or a sequence identical thereto except for up to 2, such as up to 1 amino acid substitution(s); and- a CDR3 as set forth by SEQ ID NO: 12 or a sequence identical thereto except for up to 2, such as up to 1 amino acid substitution(s).

4. The antigen-binding protein according to any one of the preceding claims, wherein the antigen-binding protein comprises or consists of a heavy chain variable (VH) region as set forth by any of SEQ ID NOs: 1 , 5 or 9, or a sequence identical thereto except for up to 2, such as up to 1 amino acid substitution(s).

5. An antigen-binding protein capable of binding to, blocking, and / or neutralizing venom short-chain a-neurotoxin.

6. The antigen-binding protein according to any one of the preceding claims, wherein the antigen binding protein binds an epitope of DH comprising one or more amino acid residues selected from the group consisting of:- E19, Q21, C41 , P42, T43, V44, P46, G47, H49, I50, S51, C52, and C53 of SEQ ID NO. 35; or- Q21 , C41, P42, T43, V44, P46, H49, I50, S51, C52, and C53 of SEQ ID NO. 35; or- Q21 , C41 , P42, T43, V44, P46, H49, I50, S51 , C52, C53, and C58 of SEQ ID NO. 35.

7. The antigen-binding protein according to any one of the preceding claims, wherein the antigen binding protein binds an epitope of scNTX comprising one or more amino acid residues selected from the group consisting of:- E19, T20, S21, C41 , K43, V44, P46, L50, R54, and T55 of SEQ ID NO. 36; or- C41, P42, K43, V44, P46, G47, L50, H51 , C52, and R54 of SEQ ID NO.36; orE19, T20, S21, C41 , K43, V44, P46, L50, R54, and T55 of SEQ ID NO.36.

8. The antigen-binding protein according to any one of the preceding claims, comprising complementary determining regions (CDRs) comprising or consisting of- a CDR1 as set forth by SEQ ID NO: 14 or a sequence identical thereto except for up to 2, such as up to 1 amino acid substitution(s); and- a CDR2 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- a CDR3 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); or- a CDR1 as set forth by SEQ ID NO: 18 or a sequence identical thereto except for up to 2, such as up to 1 amino acid substitution(s); and- a CDR2 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- a CDR3 as set forth by SEQ ID NO: 20 or a sequence identical thereto except for up to 2, such as up to 1 amino acid substitution(s); or- a CDR1 as set forth by SEQ ID NO: 22 or a sequence identical thereto except for up to 2, such as up to 1 amino acid substitution(s); and- a CDR2 as set forth by SEQ ID NO: 23 or a sequence identical thereto except for up to 2, such as up to 1 amino acid substitution(s); and- a CDR3 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).

9. The antigen-binding protein according to any one of the preceding claims, wherein the antigen-binding protein comprises or consists of a heavy chain variable (VH) region as set forth by any of SEQ ID NOs.: 13, 17 and 21 or a sequence identical thereto except for up to 2, such as up to 1 amino acid substitution(s).

10. An oligoclonal antigen-binding protein mixture comprising at least two different antigen-binding proteins, wherein one of said antigen-binding proteins is capable of binding to, blocking, and / or neutralizing snake venom PLA2, and wherein another of the at least two different antigen-binding proteins is capable of binding to, blocking, and / or neutralizing snake venom short-chain alpha neurotoxin, and wherein the at least two different antigen-binding proteins comprise one or more antigen-binding proteins according to any one of claims 1 to 9.11 . A multi-specific antigen-binding protein, comprising or consisting of one antigenbinding portion capable of binding to, blocking, and / or neutralizing snake venom PLA2 wherein said one antigen-binding portion is as defined in any one of claims 1 to 4, and of another antigen-binding portion capable of binding to, blocking, and / or neutralizing snake venom short-chain alpha neurotoxin, wherein said another antigen-binding portion is as defined in any one of claims 5 to 9.

12. A kit of parts comprising: a) one or more antigen-binding proteins, the oligoclonal antigen-binding protein mixture or the multi-specific antigen-binding protein according to any one of claims 1 to 11 ; and b) another agent suitable for the treatment of snake envenomation.

13. An antigen binding protein, an oligoclonal antigen-binding protein mixture, a multispecific antigen-binding protein, a pharmaceutical composition or a kit-of-parts according to any one of the preceding claims, for use in the treatment of snake envenomation in a subject.

14. A kit for detection and / or quantification of snake venom / toxins in a sample comprising one or more of the antigen binding proteins, the oligoclonal antigen-binding protein mixtures or the multi-specific antigen-binding proteins according to any one of claims 1 to 11.

15. The antigen binding protein, the oligoclonal antigen-binding protein mixture, the multi-specific antigen-binding protein, the kit of parts, the use, or the kit according to any one of the preceding claims, wherein the antigen binding protein(s), the oligoclonal antigen-binding protein mixture(s) or the multi-specific antigen-binding protein(s) do not bind long-chain alpha neurotoxins, such as alpha-cobratoxin.

Images