MXPA06003909A - Human antibody variants that specifically recognize the toxin cn2 from centruroides noxius scorpion venom - Google Patents

Abstract

The present invention is directed to recombinant human antibodies specific for Cn2 toxin from C. noxius scorpion venom. The antibodies are able to recognize the toxin and preferably neutralize it as well as the whole venom of C. noxius scorpion. This invention is also directed to a human non-immune phage display library. One clone that specifically binds the Cn2 toxin was affinity matured by directed evolution. Three cycles of maturation were performed and several scFv clones were isolated which specifically recognize toxin Cn2 with increased Kd of 446 fold. All variants were monomeric and only variants 6009F, 6105F and 6103E showed to be capable of neutralizing toxin Cn2 and the whole venom. Variant 6009F recognizes a different epitope than that of BCF2, a murine monoclonal antibody raised against scorpion toxin Cn2 which is also capable of neutralizing both Cn2 toxin and the whole venom when tested in mice, as well as that of commercially available polyclonal antibody fragments antivenom from horse. The scFv 6009F is the first reported recombinant human antibody fragment capable of neutralizing a scorpion venom. These results pave the way for the generation of safer autologous recombinant neutralizing antivenom against scorpion stings. The antibodies of the present invention can be used as part of a composition to treat those in need of treatment including those already stung by one or more scorpions, particularly C. noxius scorpions.

Description

VARIANTS OF HUMAN ANTIBODIES THAT SPECIFICALLY RECOGNIZE THE TOXIN CN2 AND THE POISON OF THE ALACRAN Centruroides noxius SCOPE OF THE INVENTION The present invention is generally related to recombinant antibody fragments. In particular, variants derived from parental antibodies with one or more amino acid changes relative to said parent antibody and with binding affinity for the Cn2 toxin at least 10.5 times greater than the binding affinity of the parent antibody, for the toxin are disclosed. In another scope, the invention relates to antibody variants that neutralize the lethal effect of the Cn2 toxin and the entire C. noxius venom. The invention also relates to the DNAs encoding the antibody variants, to the molecular vectors containing these DNAs, to the cells containing these vectors and to the methods for producing the antibodies. It also relates to solid surfaces comprising the adhered antibody variants and diagnostic systems for detecting the presence of the Cn2 toxin in samples, including immunodiagnostic systems such as the ELISA assay and immunochromatographic assays, in which the antibodies of the present invention. Additionally, the present invention also relates to a method for selecting improved antibodies from a library of variants generated in vitro, where these antibodies are selected by improving not only their affinity but also their stability.

BACKGROUND Antibodies are proteins that bind specifically to an antigen. The native antibodies are heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light chains (L) and two identical heavy chains (H). Each light chain is attached to a heavy chain through a disulfide bridge. The number of disulfide bridges between heavy chains is variable in different immunoglobulin isotypes. Both chains, heavy and light, also have intra-chain disulfide bridges regularly spaced. Each heavy chain has at one end a variable domain (VH) followed by some constant domains. Each light chain has a variable domain at one end (VL) and a constant domain at its other end. The constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the The variable domain of the light chain is aligned with the variable domain of the heavy chain. It was believed that some amino acid residues of both chains form an interface between the variable domains of both chains. The term "variable" refers to the fact that certain portions of the variable domains differ widely in the sequence between antibodies and are responsible for the binding specificity of each particular antibody to its antigen. However, the variability is not distributed uniformly across the variable domains of the antibodies, it is concentrated in three segments called complementarity determining regions (CDRs) in the variable domains of both the light chain and the heavy chain. The most conserved portions of the variable domains are called framework regions (FR, by framework in English). The variable domains of the heavy and light native chains each comprise four FR regions, adopting mainly a "beta sandwich" configuration, connected by three CDRs, which form loops that connect, and in some cases are part of, the structure of beta sandwich. The CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs from the other chain, contribute to form the antigen binding site of the antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest , 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)).

The constant domains do not participate directly in the binding of an antibody to an antigen, but have several effector functions. Depending on the amino acid sequence of the constant region of its heavy chains, antibodies or immunoglobulins can be classified. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of them can further be divided into subclasses (isotypes), p. eg, lgG1, lgG2, lgG3, and lgG4; Ig1 and Ig2. The constant regions of the heavy chain corresponding to the different immunoglobulin classes are called (alpha), d (delta), e (epsilon), γ (gamma), μ (mu), respectively. It is known that of the various classes of human immunoglobulin, only IgG1, IgG2, IgG3 and IgM activate complement. In patent application WO92 / 01047 it is disclosed that antibody fragments can be displayed on the surface of bacteriophages and bind antigen. Using this feature, antibody fragments can be directly selected. The ability to isolate antibody fragments (F (ab ') 2, Fab, Fv, scFv and VH) by unfolding on the surface of a filamentous bacteriophage opened the possibility of isolating specific antibodies (ie, antibodies directed against a particular antigen). ) that were previously difficult or impossible to isolate. Application WO92 / 01047 showed in particular that specific antibodies can be isolated from humans to whom it has not been specifically immunized (non-immune), even for specific antigens such as 2-phenyl-5-oxazolone to which the human is normally not exposed. In vivo, the maturation of the affinity of the antibodies is carried out by selecting the antibody variants with greater affinity for their antigen, primarily by somatic hypermutation. Often a "repertoire update" is also observed in which the germline genes predominant in the secondary or tertiary response differ from those in the primary or secondary response. Several groups of researchers have attempted to simulate the maturation process of the affinity of the immune system by introducing mutations in the genes of the VH and VL antibodies in vitro and using affinity selection to isolate mutants with better affinity. These mutant antibodies can be displayed on the surface of a filamentous bacteriophage and select improved antibodies in terms of a greater affinity for the antigen, or more specifically, by its improvement in the antigen dissociation kinetics.

Alacranismo.

In Mexico, during the seventies and eighties, scorpion stings, reached more than 200,000 accidents per year in which 700 people died annually, approximately. For the decade of the nineties, the mortality reported was 300 and in 1998 136 people died. During 2002 fatal cases decreased to 70 (Weekly Epidemiological! Bulletin, Mexican Health Ministry). The decrease in the mortality rate coincided with a National Campaign for the use of antivenom, sponsored by the Mexican Institute of Social Security. During the last century serotherapy (administration of heterologous immune serum) has been used to treat poisonings caused by bites and stings of poisonous animals to humans (Choumet, V., Audebert, F., Riviere, G., Sorkine, M ., Urtizberea, M., Sabouraud, A., Scherrmann, JM &Bon, C. (1996) Adv Exp Med Biol 391, 515-20). The antivenom used today in Mexico consists of bivalent F (ab ') 2 purified fragments obtained by hyperimmunization of horses with aqueous extracts of poisonous glands of scorpions of the genus Centruroides (Calderon-Aranda, ES, Hozbor, D. &Possani , L D. (1993) Toxicon 31, 327-37). The polyclonal antibodies present in horse serum are generated against the components of the whole venom, although only a small number of toxic components they are important for poisoning. Of 221 species that inhabit Mexico, only eight are dangerous for humans (Dehesa-Davila, M. (1989) Toxicon 27, 281-6). The poisons of different species of scorpions of the genus Centruroides are very similar in terms of toxic components (Possani, LD, Becerril, B., Delepierre, M. &Tytgat, J. (1999) Eur J Biochem 264, 287-300) . It is important to mention that in the scorpion poisons there are short and long chain peptides with specific toxicity known for mammals. It has been shown that its deadly effect is due to the action on specific molecules called ion channels. There are several different ion channels that control the permeability of many ions, such as: Na +, K +, Ca2 +, CI. "These are integral membrane proteins that govern cellular excitability.The most important toxins in the scorpion venom are those that recognize Sodium channels (Possani, LD, Becerril, B., Delepierre, M. &Tytgat, J. (1999) Eur J Biochem 264, 287-300) .Therefore, identify the deadly components (especially toxins specific to channels of sodium) in those eight poisons can help to obtain neutralizing recombinant antibodies that will be elements of the next generation of antisera.A more specific antiserum would result in a safer drug in terms of a lower number of different antibodies present and use of human antibodies homologous, replacing the equine antibodies currently used, the murine monoclonal antibody BCF2, characterized in our laboratory (Zamudio, F., Sa avedra, R., Martin, B.M., Gurrola-Briones, G., Herion, P. & amp;; Possani, LD (1992) Eur J Biochem 204, 281-92), neutralizes the toxic effects of Cn2 (a specific toxin for mammalian sodium channels), one of the most abundant and toxic components in the scorpion venom Centruroides noxius, (Hoffmann), (6.8% of all venom; LD50 = 0.25 μg / 20 g of mouse weight). BCF2 can also neutralize the whole venom (LD50 = 2.5 μg / 20 g of mouse weight) (Ucea, A. F., Becerril, B. &Possani, L. D. (1996) Toxicon 34, 843-7). These data suggest the possibility of obtaining a recombinant antivenom of human origin composed exclusively of fragments of specific antibody, autologous, safer and more efficient for therapeutic application in humans. The expression of several antibody formats on the filamentous phage surface (phage display), has allowed to generate large repertoires with different purposes, revolutionizing among others the field of antibody engineering (Stockwin, LH &Holmes, S. (2003 ) Biochem Soc Trans 31, 433-6., Brekke, OH &Loset, GA (2003) Curr Opin Pharmacol 3, 544-50., Benhar, I. (2001) Biotechnol Adv 19, 1-33., Roque, A. C, Lowe, O R. &Taipa, MA (2004) Biotechnol Prog 20, 639-54). The separation of these repertoires by Sifting with different antigens is a selection step analogous to what occurs in the immune system (Hoogenboom, HR &Winter, G. (1992) J Mol Biol 227, 381-8., Winter, G., Griffiths, AD, Hawkins, RE & Hoogenboom, HR (1994) Annu Rev Immunol 12, 433-55), which allows to isolate fragments of different specificities.

BRIEF DESCRIPTION OF THE FIGURES Figure 1. Alignment of an amino acid sequence of scFvs selected from a human repertoire. These sequences include the carboxy-terminal C-myc sequence (nucleotides 251 to 267 in C1 and 241 to 257 in 3F) followed by a stretch of 6 histidines (H6). The complementarity determining regions (CDRs) of VH and VL are bounded by a rectangle. The closest germline, the diversity segment and the binding segments for the VH domain of clone C1 were IGHV3-30 * 18, IGHD2-21 * 01 and 1GHJ2 * 01 respectively. For VL, the germ line and the union segments corresponded to IGVL1-44 * 01 and IGLJ1 * 01. The closest germ line, the diversity segment and the union segments for the VH domain of clone 3F were IGHV3-9 * 01; IGHD2-8 * 02; IGHJ3 * 02. For VK, the germline and the binding segments corresponded to 1GVK3-11 * 01; IGKJ1 * 01. The sequences were deposited in GenBank under access numbers AY781342 (C1) and AY781338 (3F).

Figure 2. Specificity of phage-antibodies 3F and C1. TO). Cross reactivity: The 3F scFv is represented by shaded boxes and the C1 scFv with blank boxes. The binding to several antigens (mainly scorpion toxins) was determined by ELISA. Cn2, CII1, CII2, Pg7, Pg8, specific toxins for sodium channels and Pg5, specific toxin for potassium channel, all at a concentration of 3μg / ml; and FU (Toxic fraction ll of C. limpidus limpidus venom) at 20 μg / ml. The phage-antibodies were added at a concentration of 1 × 10 11 phages / ml. B). Amino acid sequence of the Cn2 toxin (C. noxius) and homologous toxins CII1 and CII2 (C. limpidus limpidus). The asterisk indicates identity, the single dots indicate a conserved "weak" group of residues, and the double dots indicate a "strong" group of conserved residues as defined in the ClustalX program (1.81).

Figure 3. Analysis by Biacore of competence (BCF2 and scFv 6009F). The first part of the sensorgram (up to 1200 seconds) shows the saturation of the sites recognized by BCF2 on the Cn2 toxin after a series of 6 injections. The second part shows the binding kinetics of scFv 6009F at a concentration of 5 nM.

Figure 4. Analysis of a human library. 1A. The inserts of the scFvs were amplified from 20 individual clones (lines 1-20). The band corresponding to the scFv is 850 bp. 1 B. Digestion pattern with SsÍN1 of the PCR products. The molecular weight marker corresponds to 100 bp multimers.

Figure 5. Purification by molecular exclusion. A) Chromatography by molecular exclusion in Superdex 75 of the 6009F antibody after its affinity purification on agarose-Ni2 +. B) Standard molecular weights: ovalbumin (44 Kd), trypsinogen (24Kd). The elution rate was 0.5 ml / min.

Figure 6. Affinity determination of scFv 6009F. TO). Kinetics of binding in BIACORE to the Cn2 toxin. The Langmuir binding model (1: 1) was used. B) The variation between the theoretical and experimental data (residual values), show the reliability of the data.

Figure 7. ELISA assays by competition. Plates were screened with scFv 6009F at a concentration of 10 μg / ml overnight. They were washed and saturated 2 hours with BSA (0.5% in PBS 1X). The Cn2 toxin (3 μg / ml) was then added, incubated 1 hour and the plates washed. The BCF2 antibody was added in different concentrations (0.1, 0.25, 0.5, 1.0, 2.5 and 5 μg / ml). The detection was carried out with a goat anti-mouse antibody labeled with horseradish peroxidase. The vertical lines indicate the standard deviation of the average, n = 3.

Figure 8. Soluble protein ELISA of the clones resulting from the third cycle of directed evolution and the third separation by sieving. The plate on the left contains clones obtained with the standard method and the plate on the right are clones obtained with the modified method of the present invention. It is clear that a greater number of positive clones was found, whose response was qualitatively better.

Figure 9. Analysis by competition in the Biacore (Alacramyn, BCF2 and scFv 6009F). The first part of the sensorgram (up to 2900 seconds) shows the saturation of the sites recognized by fragments of the polyclonal anti-poison Alacramyn on the Cn2 toxin after a series of 8 injections. In the next segment (2900 to 3300 seconds) it is shown the effect of the injection of BCF2. Finally, scFv 6009F was injected (starting at 3300 seconds and until the end). The lower curve represents the control of the binding kinetics of scFv 6009F (in concentration of 20 nM) on the Cn2 toxin.

Figure 10. Affinity determination of scFv 6105F. Binding kinetics (BIACORE) to the Cn2 toxin.

Figure 11. Affinity determination of scFv 6103E. Binding kinetics (BIACORE) to the Cn2 toxin.

DETAILED DESCRIPTION OF THE INVENTION Extract of the invention With the need always in mind of a new generation of safer antivenoms and the fact that the monoclonal antibody BCF2 neutralizes both the Cn2 toxin and the whole C. noxius venom, the researchers decided to obtain specific recombinant human antibodies for the Cn2 toxin that recognize it and ideally neutralize it. To this end, a non-immune repertoire was constructed to display a library of phage-antibodies with 1.1 X 108 members. Two specific scFvs (3F and C1) were selected, which bind specifically to the Cn2 toxin. The affinity of the 3F scFv was matured by directed evolution. After three cycles of maturation, several scFv clones that specifically recognize the Cn2 toxin were isolated (6F, 610A and 6009F, 6D, 9C, 6003E, 6003G, 601 OH, 6011G, 6105F and 6103E). Most of them showed increases in Kd of 10.9 times, 176 times and 446 times: [from 183 nM (3F) to 16.3 nM (6F), 1.04 nM (610A), 410 pM (6009F), 590 pM (6105F) ) and 630 pM (6103E)], respectively, as determined by analysis in Biacore. All the variants were monomeric. Although variants C1, 3F, 6F, 610A, 6D, 9C, 6003E, 6003E, 601 OH and 6011G specifically recognize Cn2, they do not neutralize toxin or whole venom, whereas variants 6009F, 6105F and 6103E showed the ability to neutralizing 2 LD50 of Cn2 toxin or 2 LD50 of the whole venom when a molar ratio of 1:10 to 1:14 was used; toxin: antibody fragment. The 6009F variant recognizes an epitope different from that recognized by BCF2, a monoclonal antibody selected against the Cn2 toxin of scorpion, with the ability to neutralize both said toxin and whole venom when tested in mice, and that of Alacramyn, a Mexican antivenom of fragments of equine polyclonal antibody. The scFv 6009F is the first human recombinant antibody fragment capable of neutralizing scorpion venom. These results pave the way for generating more reliable recombinant autologous neutralizing antivenoms against scorpion stings. The antibodies of the present invention can be used as part of a compound to treat those who require treatment including those who have already been stung by one or more scorpions, in particular of the C. noxius species.

I. Definitions The term "antibody" is used in the broadest sense and covers in particular monoclonal antibodies (including complete monoclonal antibodies), polyclonal antibodies and antibody fragments.

The term "antibody fragment" comprises a portion of a native antibody, in general the antigen binding segment or the variable regions thereof. Examples of antibody fragments include Fab, Fab ', F (ab') 2, and Fv fragments; diabodies; linear antibodies; and in particular single chain antibodies. (scFv).

The "single chain Fv" or "scFv" antibody fragments include the VH and VL domain of an antibody, in which these domains are present in a single polypeptide chain. Generally, the Fv polypeptide further comprises connecting peptide between the VH and VL domains that allows the scFv to form the desired structure to bind to the antigen. For a review of scFv see Pluckthun in: The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds. Springer- Verlag, New York, pp. 269-315 (1994).

The term "diabodies" refers to small fragments of antibody with two antigen binding sites, which comprise a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same polypeptide chain (VH) -VL). The use of short linker peptides (usually 10 amino acids or less) allows pairs to be formed between two domains on the same chain, domains forced to pair with the complementary domains of another chain and create two antigen binding sites. Diabodies are described more broadly in, for example, EP 404,097; WO 93/11161; and Hollinger et al., Proc. Nati Acad. Sci. USA 90: 6444-6448 (1993).

A "parent antibody" is an antibody that comprises an amino acid sequence used as a starting point for mutagenesis procedures such as directed evolution or random mutagenesis (see Neylon, O (2004), Nucleic Acids Research 32, 1448-1459), or shuffled of genes, to generate antibody variants in one or more cycles of mutagenesis that differ from said parent antibody by including one or more amino acid alterations in one or more hypervariable regions or adjacent to them. The parent polypeptide may comprise a native sequence (ie, a naturally occurring one) of antibody (including natural allelic variants) or an antibody with pre-existing amino acid sequence modifications (such as other insertions, deletions and / or substitutions) of a natural sequence. Preferably, the parent antibody is a human antibody. The parental antibodies of the present invention bind specifically to the Cn2 toxin of the scorpion venom C. noxius.

As used herein, "antibody variant" refers to an antibody generated by mutagenesis from a parent antibody of the present invention and possesses an amino acid sequence different from the amino acid sequence of said parent antibody. Preferably, the antibody variant comprises a heavy chain variable domain or a light chain variable domain with an amino acid sequence not present in nature. Said variants necessarily have less than 100% identity or similarity with the amino acid sequence of the parent antibody. The antibody variants of the present invention bind specifically to the Cn2 toxin of the C. noxius scorpion venom.

"Amino acid alteration" refers to a change in the amino acid sequence of a predetermined amino acid sequence. Examples of alterations include insertions, deletions and in particular substitutions. To refer a substitution, the amino acid present in the parental antibody is followed by a number corresponding to the position it occupies in said parent antibody (starting at the amino terminus) and followed by the amino acid that replaces it, now present in the variant of the amino acid. antibody.

"Neutralizing, neutralizing or neutralizing antibody" refers to the ability of an antibody of the present invention to bind to the Cn2 toxin and to abolish its lethal effect when administered to a mammal, either purified or as part of the complete venom, in particular the poison of the species C. noxius.

"Treatment" refers to the therapeutic treatment. The term Those in need of treatment includes those who have already been bitten by one or more scorpions of the C. noxius species.

"Isolated" nucleic acid molecule is a nucleic acid molecule identified and separated, at least, from another nucleic acid contaminating molecule that ordinarily accompanies it in the natural source of nucleic acids encoding some antibody. An isolated nucleic acid molecule is different in terms of the shape or arrangement found in nature. Therefore, the isolated nucleic acid molecules are distinguished from the nucleic acid molecules present in the cells that contain them. However, an isolated nucleic acid molecule is actually a molecule whose nucleotide sequence is present in the cells that ordinarily express the antibody, but this nucleic acid molecule when cloned is in a different context from the one it occupies in the cells natural The term "control sequences" refers to DNA sequences necessary to regulate the expression of an operably linked coding sequence in the context of a particular host organism. Suitable control sequences for prokaryotes include, for example, a promoter, optionally an operator sequence and a ribosome binding site. It is known that eukaryotic cells use promoters, polyadenylation signals and amplifiers.

A DNA sequence or fragment is "operably linked" when placed in a functional relationship with another nucleic acid sequence or fragment. For example, DNA encoding a presequence or a secretory leader are operably linked to the DNA of a polypeptide if it is expressed as a pre-protein that participates in the secretion of the polypeptide. A promoter or amplifier is operably linked to a functional sequence if it affects the transcription of said sequence; or a ribosome binding site is operably linked to a sequence if it is in position to facilitate translation. In general, "operably linked" means that the linked DNA sequences are contiguous, and, in the case of a leader secretor, contiguous and in reading phase.

The terms "cell" and "cell culture" as used herein are used as synonyms and all these designations include their progeny. Thus, the words "transformants" and "transformed cells" include the primary subject cell and cultures derived therefrom without considering the number of transfers. It is also understood that not all progeny must be precisely identical in their DNA content due to deliberate or inadvertent mutations. Mutant progeny that have the same function or biological activity as detected in the originally transformed cell are included. When different designations are attempted, they must be clear from the context.

The term "effective amount" or "pharmacologically effective amount" of a compound in unit dose of the mixture depends on several factors. These factors include amounts of the other ingredients if employed and tolerance of the active ingredient of the compound. The effective amount of the active ingredient ranges from about 8% to almost 35% by weight, based on the total weight of the compound. For compounds against scorpions, the preparation F (ab ') 2 contained in each bottle is the amount necessary to neutralize from 135 to almost 220 lethal doses 50% of the poison.

"Pharmaceutically acceptable carrier" means solid or liquid filler, diluent or substance that can be safely used in systemic or topical administration. According to the particular route of administration, several vehicles well known in the industry are pharmaceutically acceptable and include solids or fillers, diluents, hydrotropes, surfactants and encapsulating substances. The amount of vehicle employed together with the F (ab ') 2 fragments provides a manageable amount of material per unit dose of the compound. Pharmaceutically acceptable carriers for systemic administration which can be incorporated in the composition of the invention include sugar, starches, cellulose, vegetable oils, buffers, polyols and alginic acid. Specific pharmaceutically acceptable carriers are described in the following documents, all mentioned herein by reference: U.S. 4,401,663, Buckwaiter et al. issued on August 30, 1983; European Patent Application No. 089710, LaHann et al. published Sept. 28, 1983; and European Patent Application No. 0068592, Buckwaiter et al. published January. 5, 1983. Preferred carriers for parenteral administration include propylene glycol, pyrrolidone, ethyl oleate, aqueous ethanol, and combinations thereof. Representative carriers include acacia, agar, alginates, hydroxyalkylcellulose, hydroxypropylmethylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose, carrageenan, powdered cellulose, guar gum, cholesterol, gelatin, gum agar, gum arabic, karaya gum, ghatti gum, locust bean gum, octoxinol 9, oleyl alcohol, pectin, polyacrylic acid and its homologs, polyethylene glycol, polyvinyl alcohol, polyacrylamide, lauryl sodium sulfate, polyethylene oxide, polyvinylpyrrolidone, glycol monostearate, propylene glycol monostearate, xanthan gum, tragacanth, sorbitan esters, stearic alcohol, starch and its modifications. The appropriate ranges vary from 0.5% to 1%.

II. Obtaining initial parental antibodies. In a formulation of the present invention, two initial parental antibodies were generated, a human antibody library and a display phage were constructed and two antibodies, C1 (SEQ ID NO: 18 for coding DNA and SEQ ID NO: 18) were isolated. NO: 19 for the amino acid sequence) and 3F (SEQ ID NO: 24 for the coding DNA and SEQ ID NO: 25 for the amino acid sequence), which recognize in particular the Cn2 toxin from the scorpion venom C. noxius. The need to generate more efficient and safe antibodies for use in human therapy led to the development of recombinant antibodies from different sources. Under ideal conditions the source must be the human being itself. As shown in detail in Example 1, the human scFv library of the present invention was generated by RT-PCR from purified total RNA of human peripheral blood B-lymphocytes. To avoid as much as possible a bias in the family representation of the variable antibody chain and improve the possibility of obtaining at least one scFv with affinity for the Cn2 toxin, each family of variable regions (VH or V) was amplified by independent PCR reactions. In a second PCR step, the binding peptide sequence was added to each individual V family. A PCR process was carried out by overlapping in order to bind both domains V (H and L). Each VH family was overlapped to each V family? or V? (a total of 72 VH-VL combinations). The DNA segments encoding the assembled products were fused to the pIII gene of the phagemid pSyn2. The size of the scFv library was 1.2 X 108 members. Twenty independent colonies were analyzed by PCR. Eighteen presented the correct size and showed different restriction patterns when they were digested with ¿3síNI (Fig. 4). The variability of the 18 different scFvs was confirmed by DNA sequence, which resulted in a final library of 1.1 X 108 variants. Different combinations of variable domains were found that included most of the V families. As detailed in example 2, after four rounds of biological separation in the human scFv library against the Cn2 toxin, only two anti-Cn2 scFvs were identified. and they are termed scFv 3F (SEQ ID NO: 24 for the coding DNA and SEQ ID NO: 25 for the amino acid sequence) and scFv C1 (SEQ ID NO: 18 for the coding DNA and SEQ ID NO. : 19 for the amino acid sequence) (Fig. 1), corresponding to human immunoglobulins. Clone 3F comprises a heavy variable chain VH3 (SEQ ID NO: 26 for the coding DNA and SEQ ID NO: 27 for the amino acid sequence) and a light variable chain VK3 (SEQ ID NO: 28 for the coding DNA and SEQ ID NO: 29 for the amino acid sequence), while clone C1 comprises a VH3 heavy variable chain (SEQ ID NO: 20 for the coding DNA and SEQ ID NO: 21 for the amino acid sequence) and a light variable chain V? 1 (SEQ ID NO: 22 for the coding DNA and SEQ ID NO: 23 for the amino acid sequence). These two clones showed to be very specific to Cn2, although Cn2 and the control toxins CII1 and CII2 have a high degree of identity (Fig. 2B). To know whether the selected antibodies have the ability to protect mice against the toxic effect of Cn2, a neutralization assay was carried out. The results revealed that both fragments of the antibody lack the ability to protect mice from the effect of the Cn2 toxin. The affinity constants of both scFvs were similar, in the range of 10"7 M which are typically affinity values of the primary immune response (Lefranc, MP (2003) Nucleic Acids fies 31, 307-10., Foote, J. & Eisen, H. N. (1995) Proc Nati Acad Sci U S A 92, 1254-6). Clones 3F and C1 showed rapid dissociation, despite good affinity, this suggests that the antibody fragments do not remain bound to the toxin long enough to neutralize it. Several reports show that some monomeric scFvs do not neutralize their targets, while their corresponding scFvs (diabodies) dimers do so due to an ease in their affinity (Aubrey, N., Devaux, C, Sizaret, PY, Rochat , H., Goyffon, M. &Billiald, P. (2003) Cell Mol Life Sci 60, 617-28., Lantto, J., Fletcher, JM &Ohlin, M. (2002) J Gen Virol 83, 2001-5). The dimeric forms of both scFvs were constructed, but none of the 3F and C1 diabodies could neutralize the toxin in the protection assay.

III. Generation of antibody variants. In another area of the present invention, 3F parent antibody was used to generate antibody variants by affinity maturation through directed evolution techniques, but it is possible to generate variants by other mutagenic techniques known to those skilled in the art, such as mutagenesis of "cassete" (Stemmer, WPC et al., (1992) Biotechniques 14: 256-265; Arkin, A. and Youvan, D. O (1992) Proc Nati Acad Sci USA 89: 7811-7815; Oliphant, AR et al., (1986) Gene 44: 177-183; Hermes, JD et al., (1990) Proc Nati Acad Sci USA 87: 696-700; Delagrave et al. (1993) Protein Engineering 6: 327-331; Delgrave et al. (1993) Bio / Technology 11: 1548-1552; Goldman, ER and Youvan DC (1992) BioíTechnology 10: 1557-1561), in which the specific region to be optimized is replaced with a synthetically mutagenized oligonucleotide, shuffling of genes and other mutagenesis procedures such as directed evolution or random mutagenesis (see Neylon, O (2004) Nucleic Acids Research 32, 1448-1459). The 3F scFv selected from the library of non-immune human scFvs of the present invention does not have the affinity, stability or both, required to be neutralizing as has been demonstrated in most examples of neutralizing antibodies possessing affinities in the nanomolar range and lower (Maynard, JA, Maassen, OR B., Leppla, SH, Brasky, K., Patterson, J. L, Iverson, B. L &Georgiou, G. (2002) Nat Biotechnol 20, 597-601 ., Sawada-Hirai, R., Jiang, I., Wang, F., Sun, SM, Nedellec, R., Ruther, P., Alvarez, A., Millis, D., Morrow, PR &Kang, AS (2004) J Immune Based Ther Vaccines 2, 5, Devaux, O, Moreau, E., Goyffon, M., Rochat, H. &Billiald, P. (2001) Eur J Biochem 268, 694-702) . This result was expected, since the library is non-immune and of intermediate size. The teaching is that better segments of larger libraries can be selected. (Sblattero, D. &Bradbury, A. (2000) Nat Biotechnol 18, 75-80., Vaughan, TJ, Williams, AJ, Pritchard, K., Osbourn, JK, Pope, AR, Eamshaw, J. O. McCafferty, J., Hodits, RA, Wilton, J. &Johnson, KS (1996) Nat Biotechnol 14, 309-14., Sheets, MD, Amersdorfer, P., Finnern, R., Sargent, P., ündquist , E., Schier, R., Hemingsen, G., Wong, C, Gerhart, J. O, Marks, JD &ündqvist, E. (1998) Proc Nati Acad Sci USA 95, 6157-62). It has been shown that the affinity of the Cn2 toxin for sodium channels present in some cell preparations is in the nanomolar range (nM) (Garcia, O, Becerril, B., Selisko, B., Delepierre, M. &Possani , LD (1997) Comp Biochem Physiol B Biochem Mol Biol 116, 315-22., Sitges, M., Possani, LD &Bayon, A. (1987) J Neurochem 48, 1745-52). These results suggest that an antibody requires an affinity at least in this range to neutralize the toxin. Taking this into account, and in order to obtain a human antibody capable of neutralizing Cn2 toxin and C. noxius venom, it was decided to mature clones 3F and C1 by directed evolution and deployment on the surface of filamentous phages. It has been shown that the directed evolution of proteins allows to gradually increase a particular property of the protein. It is usually necessary to perform several cycles of evolution to obtain the desired level of improvement. Each cycle is started with a parent antibody and one or more antibody variants are generated with the expectation that they possess better kinetic qualities. Any of the resulting antibody variants can be used as a parent antibody for the next cycle of evolution or mutagenesis. As shown in examples 3, 4, 5 and 6, three evolution cycles were necessary to obtain the 3F scFv variants (6009F, 6105F and 6103E) with the appropriate affinity level to have neutralizing capacity. In the first maturation cycle the following were selected: variant 6F (SEQ ID NO: 30 for the coding DNA and SEQ ID NO: 31 for the amino acid sequence), with an alteration in the amino acid sequence that is the substitution (Ser54Gly), in the CDR2 of the heavy chain and that has a sequence SEQ. ID. NO: 32 for the coding DNA and SEQ. ID. NO: 33 for the sequence of amino acids (and a light chain with sequence SEQ ID NO: 34 for the coding DNA and SEQ ID NO: 35 for the amino acid sequence), with an increase of one order of magnitude in the KD (from 183 nM to 16.8 nM; Table 1). These results show that scFv 6F binds more efficiently to the toxin, but still breaks off as quickly, indicating that the residue at position 54 plays an important role in the interaction of the antibody with the Cn2 toxin (for more details see example 3). In the second maturation cycle (example 4), the variant 61 OA (SEQ ID NO: 36 for the coding DNA and SEQ ID NO: 37 for the amino acid sequence) having a second amino acid alteration was selected. in the CDR3 of the heavy chain consisting of the Va 01 Phe substitution (sequences for the light and heavy chains are SEQ ID NO: 38 for the coding DNA and SEQ ID NO: 39 for the amino acid sequence of the chain heavy and SEQ ID NO: 40 for the coding DNA and SEQ ID NO: 41 for the amino acid sequence of the light chain). This mutation improves the association constant, but even more importantly, it also improves the dissociation constant. This result suggests that residue 109 in the CDR3 of the heavy chain also plays an important role in binding to the toxin. This change could result in a better interaction in terms of greater contact area. The cumulative changes in the CDR2 and CDR3 in the 610A variant have a synergistic effect on the affinity constant, achieving an increase of 176 times (Table 1).

TABLE 1. KINETIC PARAMETERS OF DIFFERENT VARIANTS OF THE PRESENT INVENTION A third cycle of directed evolution allowed to select the clone 6009F (SEQ ID NO: 42 for the coding DNA and SEQ ID NO: 43 for the amino acid sequence) among others. As shown in examples 5 and 6, in this last stage of maturation, two selection strategies were carried out, one standard procedure and one modified to create more astringent conditions by selecting improved variants in affinity and stability. These modifications were decisive in selecting improved clone varieties. Different strategies have been reported for this same purpose (Kotz, JD, Bond, CJ &Cochran, AG (2004) Eur J Biochem 271, 1623-9., Zhou, HX, Hoess, RH &DeGrado, WF (1996) Nat Struct Biol Z, 446-51., Martin, A., Sieber, V. &Schmid, FX (2001) J Mol Biol 309, 717-26., Jung, S., Honegger, A. &Pluckthun, A. (1999) J Mol Biol 294, 163-80). The standard phage selection procedure produced a small number (2) of positive variants (SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52 and SEQ ID NO: 53 for the DNA encoding and the amino acid sequence of clone 6D and its VH and VL, respectively) and (SEQ ID NO: 54, SEQ ID. NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58 and SEQ ID NO: 59 for the coding DNA and the amino acid sequence of clone 9C and its VH and VL, respectively) compared to the more astringent procedure which gave 5 positive variants: 6003E (SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66 and SEQ. ID NO: 67 for the coding DNA and the amino acid sequence of the entire clone and its VH and VL, respectively), 6003G (SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO. : 70, SEQ ID NO: 71, SEQ ID NO: 72 and SEQ ID NO: 73 for the coding DNA and the amino acid sequence of the whole clone and its VH and V, respectively), 6011G ( SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78 and SEQ ID NO: 79 for DNA encoding and the amino acid sequence of the entire clone and its VH and VL, respectively), 601 OH (SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID. NO: 83, SEQ ID NO: 84 and SEQ ID NO: 85 for the coding DNA and the amino acid sequence of the clone and its VH and VL, respectively) and the clone 6009F. The number of changes in nucleotide per variant in the clones selected from the two procedures was different. The DNA sequence of clone 6009F showed two silent mutations and four alterations in amino acid with respect to clone 610A (Table 1). One of these alterations in amino acids occurred in the framework region 3 of the heavy chain, the Asn74Asp substitution, which has a sequence SEQ. ID. NO: 44 for the coding DNA and SEQ. ID. NO: 45 for the amino acid sequence, and the other 3 substitutions in the light chain that have a sequence SEQ. ID. NO: 46 for the coding DNA and SEQ. ID. NO: 47 for the amino acid sequence. Two of them (Thr152lle and Ser197Gly) occurred in frame regions 1 and 3 respectively and the third (Tyr164Phe), in CDR1 (Table 1). The kinetic parameters shown in Table 1 reveal that both kinetic constants improved about 2 fold compared to clone 610A, resulting in an affinity constant (K) of 410 pM. The DNA sequence of clone 6105F showed a silent mutation and only two alterations in amino acids with respect to clone 610A (Table 1). One of these alterations in amino acids (Asn74Asp) occurred in the framework region 3 of the heavy chain, which has a sequence SEQ. ID. NO: 88 for the coding DNA and SEQ. ID. NO: 89 for the amino acid sequence, and the other substitution (AIa141Val) in the framework region 1 of the light chain, which has a sequence SEQ. ID. NO: 90 for the coding DNA and SEQ. ID. NO: 91 for the amino acid sequence. The kinetic parameters shown in Table 1 reveal that both kinetic constants improved by about 1.5 - 2 times compared to clone 610A, resulting in an affinity constant (Ka) of 590 pM. The DNA sequence of clone 6103E showed a silent mutation and three alterations in amino acids with respect to clone 610A (Table 1). Two of them (Asn74Asp) occurred in the heavy chain, one in the framework region 3, and the other (Thr106Ser) occurred in the CDR3 of the heavy chain, which has a sequence SEQ. ID. NO: 94 for the coding DNA and SEQ. ID. NO: 95 for the amino acid sequence. The third substitution (Ala192Thr) occurred in the framework region 3 of the light chain, which possess a sequence SEQ. ID. NO: 96 for the coding DNA and SEQ. ID. NO: 97 for the amino acid sequence. The kinetic parameters shown in Table 1 reveal that both kinetic constants improved by about 1.5-2 times compared to clone 61 OA, resulting in an affinity constant (Kd) of 630 pM. It is suggested that the changes in CDRs are most important for improving antigen affinity (Cowell, L G., Kim, HJ, Humaljoki, T., Berek, C. &Kepler, TB (1999) J Mol Evo! 49, 23-6. , Gonzalez-Fernandez, A., Gupta, SK, Pannell, R., Neuberger, MS &Milstein, C. (1994) Proc Nati Acad Sci USA 91, 12614-8). However, it has recently been shown that changes in the framework regions are decisive for improving not only affinity and stability (Daugherty, PS, Chen, G., Iverson, B. L &Georgiou, G. (2000) Proc Nati Acad Sci USA 97, 2029-34) but also the level of expression in culture (Graff, CP, Chester, K., Begent, R. &Wittrup, KD (2004) Protein Eng Des Sel 17, 293-304). A similar phenomenon was observed during the maturation of clone 3F to obtain the variant 6009F, since the scFv 6009F accumulated 3 changes in the CDRs and 3 changes in the framework regions. Similar considerations apply for the 6105F and 6103E variants in which the mutations occurred in both CDRs and framework regions. The chromatographic elution profile of the 6009F antibody showed a major peak corresponding to a monomeric form (Fig. 5). It is assumed that the changes in the framework regions contributed to the achievement of a better affinity and functional stability. The analysis of the affinity measurements [Table 1. and Fig. 6], revealed that the clone 6009F, has a Kd of 410 pM, comparable to the affinity of other neutralizing antibodies of scorpion toxins (Aubrey, N., Devaux, O, Sizaret, PY, Rochat, H., Goyffon, M. &Billiald, P. (2003) Cell Mol Life Sci 60, 617-28). While clone 6105F possesses a Kd of 590 pM and clone 6103E possesses a Kd of 630 pM, comparable to the affinities of other antibodies that also neutralize scorpion toxins. The overall synergistic improvement in the kinetic parameters with respect to 3F scFv shown in Table 1 led to a 446-fold increase in Kd for clone 6009F, whereas it increased 310-fold for 6105F and 290-fold for 6103E.

It is important to emphasize that the three variants (6009F, 6105F and 6103E) contain a common mutation (Asn74Asp), which could be a key residue to improve the affinity and stability required to neutralize the Cn2 toxin.

As is easy to understand for those versed in this field of knowledge, other antibodies can be obtained by combining different VH fragments (SEQ ID NO: 20, SEQ ID NO: 26, SEQ ID NO: 32, SEQ: ID NO: 38, SEQ ID NO: 44, SEQ ID NO: 50, SEQ ID NO: 56, SEQ ID NO: 64, SEQ ID NO: 70, SEQ. ID NO: 76, SEQ ID NO: 82, SEQ ID NO: 88 and SEQ ID NO: 94) with the VL (SEQ ID NO: 22, SEQ ID NO: 28, SEQ ID NO: 34, SEQ ID NO: 40, SEQ ID NO: 46, SEQ ID NO: 52 SEQ ID NO: 58, SEQ ID NO: 66, SEQ ID NO: 72, SEQ ID NO: 78, SEQ ID NO: 84, SEQ ID NO: 90 and SEQ ID NO: 96). Accordingly, as long as these other antibodies retain the specific binding capacity to the Cn2 toxin, they should be considered within the scope of the present invention and will therefore be included in the term "antibodies of the present invention". Similarly, it is clear to those skilled in the art that any of the antibodies of the present invention can be used as a parent antibody to generate additional antibody variants. As long as these new antibody variants retain their specific binding capacity to the Cn2 toxin, they will be considered within the scope of the present invention and therefore functionally equivalent to the antibodies of the present invention.

In addition, the clones of the VH antibody fragments (SEQ ID NO: 20, SEQ ID NO: 26, SEQ ID NO: 32, SEQ: ID NO: 38, SEQ ID NO: 44, SEQ ID NO: 50, SEQ ID NO: 56, SEQ ID NO: 64, SEQ ID NO: 70, SEQ ID NO: 76, SEQ ID NO: 82, SEQ. ID NO: 88 and SEQ ID NO: 94) and VL (SEQ ID NO: 22, SEQ ID NO: 28, SEQ ID NO: 34, SEQ ID NO: 40, SEQ ID NO: 46, SEQ ID NO: 52 SEQ ID NO: 58, SEQ ID NO: 66, SEQ ID NO: 72, SEQ ID NO: 78, SEQ ID. NO: 84, SEQ ID NO: 90, and SEQ ID NO: 96) can be used to generate not only the scFv antibody format but also any of the Fab, F (ab ') 2 or even formats of full-length monoclonal antibody by addition, operationally linking part or all of the constant regions of light and heavy chains for use in different applications.

IV. Expression of the antibodies of the present invention. In general, the antibodies of this invention can be produced by transforming a suitable host cell with all or part of a nucleic acid molecule encoding antibody or a fragment thereof into a suitable expression vehicle. Those skilled in the field of molecular biology will understand that any of a wide variety of expression systems can be used to produce the recombinant protein. The precise host cell employed is not critical to the invention. An antibody of the invention can be produced in a prokaryotic host (e.g., E coli) or in a eukaryotic host (e.g., Saccharomyces cerevisiae, insect cells, e.g., Sf9 cells, or mammalian cells, p. eg, NIH 3T3, HeLa, or preferably COS cells). These cells are available from a wide range of sources (eg, the American Type Culture Collection, Rockland, Md.). The method of transformation or transfection and selection of the expression vehicle depends on the host system selected and still well known to those skilled in the art. For example, expression vehicles may be chosen from those provided, e.g. eg, in Cloning Vectors: A Laboratory Manual (P. H. Pouwels et al). As will be apparent to those skilled in the art, it is important that the DNA sequences of the antibodies of the present invention be operationally linked to the control sequences of the vector expression of the chosen expression system. A variety of expression systems exist to produce the antibodies of the present invention. Such vectors include, without limitation, chromosomal, episomal and virus derivatives, e.g. vectors derived from bacterial plasmids, bacteriophage, transposons, yeast episomes, insertion elements, yeast chromosomal elements, viruses such as baculovirus, papovavirus, such as SV40, virus vaccine, adenovirus, fowlpox virus , pseudorabies virus and retroviruses, and vectors derived from combinations thereof. In order to provide sufficient material for the tests of the present invention, the antibodies were cloned, in particular, on the pSynl vector and expressed in the TG1 strain of E coli as detailed in example 7. But a bacterial expression system Particular for antibody production is the E coli pET expression system (Novagen, Inc., Madison, Wl). According to this expression system, the DNA encoding an antibody is inserted into a pET vector in an orientation designed to allow its expression. Since the gene coding for said antibody is under the control of the T7 regulatory signals, expression of the antibody is achieved by inducing the expression of the T7 RNA polymerase in the host cell. Typically, this is achieved by using host strains expressing the RNA polymerase of T7 phage in response to induction by IPTG. Once produced, the recombinant antibody is isolated according to standard methods known in the art. Another bacterial expression system for the production of antibody is the expression system pGEX (Pharmacia). This system employs a fusion of the GST gene designed for the expression in high levels of genes or gene fragments in the form of fusion proteins with a rapid purification and recovery of their functional products. The protein of interest is fused to the carboxyl terminus of the Glutathione S-transferase protein of Schistosoma japonicum and is rapidly purified from bacterial lysates by affinity chromatography using Glutathione Sepharose 4B. The fusion proteins can be recovered under mild conditions by elution with glutathione. The separation of the glutathione S-transferase domain from the fusion protein is facilitated by the presence of recognition sites for site-specific proteases present upstream of this domain. For example, proteins expressed in pGEX-2T plasmids can be separated with thrombin; those expressed in pGEX-3X can be separated with factor Xa. Once the recombinant antibody of the invention is expressed, it is isolated, e.g. eg, using affinity chromatography. In one example, the antibodies were purified by affinity chromatography Ni2 + -NTA (QIAGEN, Germany) (see example 7). In another example, Cn2 toxin can be attached to a column and used to isolate the recombinant antibodies. Prior to affinity chromatography, lysis and fractionation of antibody-bearing cells can be performed by standard methods. Once isolated, the recombinant antibody can be further purified, if desired, e.g. eg, by high performance liquid chromatography (see, e.g., Fisher, Laboratory Techniques in Biochemistry and Molecular Biology, eds., Work and Burdon, Elsevier, 1980), or by gel filtration chromatography on a Superdex column. ™ 75 (Pharmacia Biotech AB, Uppsala, Sweden) (see example 7). The antibodies of the invention can also be produced by chemical synthesis (eg, the methods described in Solid Phase Peptide Synthesis, 2nd ed., 1984 The Pierce Chemical Co., Rockford, Ill).

V. Comparison of the 6009F antibody with the monoclonal antibody BCF2 and fragments of equine polyclonal antibody F (ab ') 2. In order to confirm whether the epitope recognized by the product of clone 6009F is the same as recognized by BCF2, a displacement test was carried out using a Biacore (Fig. 3). As already mentioned, the monoclonal antibody BCF2 neutralizes the Cn2 toxin. The results showed that the F6009 antibody binds to the Cn2 toxin at a site (epitope) different from that recognized by the monoclonal antibody BCF2. These results were confirmed by a competitive ELISA test (Fig. 7). See example 9 for more detail. It is worth mentioning that despite being relatively small (66 amino acid residues), the Cn2 toxin seems to have several distinct epitopes (Zamudio, F., Saavedra, R., Martin, BM, Gurrola-Briones, G., Herion, P . &Possani, L D. (1992) Eur J Biochem 204, 281-92). We can make some additional comments about the epitopes recognized by BCF2 and scFv 6009F. A dimeric scFv derived from BCF2 with an affinity of 75 pM (Juarez- González, V.R. Riano-Umbarila, L Quintero-Hernández, V. Olamendi-Portugal.T. Ortiz-Leon, M. Ortiz, E Possani, L.D. Becerril.B. (2005) J Mol Biol 346, 1287-1297.), Showed ability to neutralize 1 LD50 of the Cn2 toxin in molar ratio 1:10 (toxin: scFv). However, symptoms of mild poisoning were observed. These results suggest a different effect of the toxin on its target (sodium channels) according to the blocked epitope. This could explain why clone 6009F with lower affinity (200 pM) and lower molar ratio (1: 5), could completely neutralize 2 LD50 of the toxin without any symptoms of intoxication being observed. This indicates that the 6009F antibody is more efficient in blocking the interaction of the toxin with the channel compared to the matured scFv of BCF2; therefore, the epitope recognized by 6009F seems to be more relevant than that recognized by BCF2. Similar results have been reported in other systems (Amersdorfer, P., Wong, O, Smith, T., Chen, S., Deshpande, S., Sheridan, R. &Marks, JD (2002) Vaccine 20, 1640- 8). In addition, as detailed in Example 9, a displacement test was performed using the Biacore between the scFv 6009F and a commercial anti-lacune antivenom, Alacramyn from Instituto Bioclon S.A. of C.V. (Mexico), a source of antibody fragments Equine polyclonal (F (ab ') 2 to determine if the epitope recognized by scFv 6009F is also recognized by some of the fragments of the antibody present in the venom.The results (Fig. 9) showed that the epitope recognized by the scFv 6009F is totally different from those recognized by any of the antibody fragments present in the commercial antivenom.This observation suggests that the epitopes of the Cn2 toxin were not exactly the same when exposed to the natural immune system of animals (mouse in the case of BCF2). or horses in the case of Alacramyn) than when exposed to the in vitro immune system designed by the researchers.

SAW. Use of the antibodies of the present invention as antivenoms As shown in example 8, unlike the 6F and 610A variants, the 6009F antibody variant can neutralize the Cn2 toxin (it is capable of neutralizing 2 LD50 of Cn2 toxin when used in molar ratio of 1:10, or 1:14 when 2 LD50 of whole venom were tested). Accordingly, it can be used as an element of a pharmaceutical compound to treat animals and persons requiring such treatment as a result of scorpion stings, particularly if it is determined that the scorpion is Centruroides noxius. The pharmaceutical composition can also contain other antibodies, for example equine or goat polyclonal antibodies formed against scorpion poisons or other human or humanized antibodies against other toxins of C. noxius venom or against other poison of the poison of different scorpions. The compound may also comprise several pharmaceutically acceptable carriers. A method for treating an individual requiring this treatment, for example after suffering from a scorpion sting, especially if the scorpion is a C. noxius, includes parenteral administration of said pharmaceutical compound comprising the 6009F antibody of the present invention. .

VII Use of the antibodies of the present invention as part of a solid phase The antibodies of the present invention, in particular the 6009F antibody, can be employed in a compound containing the antibodies of the present invention adhered to a solid phase substrate such as a glass. (eg controlled pore glass), polysaccharides (eg, agarose), polyacrylamides, polystyrene, polyvinyl alcohol, silicones, sepharose, carboxymethyl cellulose and nitrocellulose, so that the compound can bind to the Cn2 toxin either in a free way, as part of the C. noxius poison or as a contaminant of a blood or serum sample. In certain environments, depending on the context, the solid phase may correspond to the well of a test plate; in others it is a purification column (eg, an affinity chromatography column) and in another it is part of a diagnostic kit. This term also includes a discontinuous solid phase of discrete particles, such as that described in U.S.

Pat. No. 4,275,149. In another context, the present invention relates to a solid phase comprising antibodies 3F, 6F, 61 OA or preferably 6009F, 6105F or 6103E.

VIII. Use of antibodies, a solid phase or both, of the present invention in a diagnostic system There are some immunodiagnostic techniques available in this field of knowledge, which commonly use specific antibodies to detect the presence of a particular antigen in a sample. For example, the Enzymatic Linked Immuno System Assay (ELISA) and the Immunochromatographic Assay (now referred to as "ICA"). ICA is also known as "rapid test" because of its speed and simplicity. In this assay, tracer antibody molecules conjugated with gold particles bind to a particular antigen present in a serum sample, after which the complexes formed are passed through a micropore nitrocellulose (NC) membrane based on a capillarity phenomenon. Finally, the complexes bind to capture immobilized antibodies on the inner surface of the NC micropore membrane and develop color in a line of positivity where the existence of a particular antigen in the serum sample can be easily determined with the naked eye. As noted before, the ICA test is used extensively to detect several analytes, for example antigens, due to the simplicity of the procedure and speed to run the test and obtain results (Sato, K. et al. (1996) J Clin Microbiol 34 , 1420-1423). There are two main elements in the ICA case. One is the nitrocellulose membrane that has two invisible lines on the surface and the other is a fiberglass filter that contains antibody-gold particle conjugates in a dry state on the surface. Two classes of antibodies, that is, the monoclonal antibody specific for the antigen to be detected and goat anti-murine IgG are immobilized on the lower line and the upper line of the nitrocellulose membrane, respectively. A sample is added to a well of the ICA kit and then the antibody-gold particle conjugates on the surface of the NC membrane in the dry state are rehydrated and then bind to the antigens in the serum sample, after which the complexes formed pass through the micropores of the NC membrane based on a capillary phenomenon. After this, the antigens of the complexes react with the monoclonal antibodies immobilized on the lower line, and as a result color develops. In addition, the upper line develops a color since the anti-murine goat IgG can react with the antibody-gold particle conjugates even though no antigen is present, so the upper line always develops color each time the test is run and can serve as a control line. In other words, when there are antigens in the serum sample, both the positive line and the control line of the ICA kit become visible. However, when there are no antigens, only the control line becomes visible. The antibodies of the present invention, preferably 6009F, 6105F or 6103E, either free or incorporated into the solid phase described above, can be used as part of a diagnostic kit to detect the presence or absence of the Cn2 toxin in a sample. They can be used as part of the ELISA or ICA tests. The goat anti-murine polyclonal antibodies are replaced by anti-human polyclonal antibodies, for example goat anti-human in order to use the antibodies of the present invention. Accordingly, another environment of the present invention relates to an immunodiagnostic kit comprising antibodies 3F, 6F, 61 OA or preferably 6009F, 6105F or 6103E. The immunodiagnostic kit can be an ELISA or Immunochromatographic Assay.

IX. Use of the 6009F antibody to treat poisoning with C. noxius venom Since the 6009F antibody variant neutralizes the lethal effect of the Cn2 toxin and the full C. noxius venom, as clearly demonstrated in example 8, the antibody variant 6009F and / or any functionally equivalent variants thereof (ie, 6009F antibody variants that neutralize the lethal effect of Cn2 toxin and C. noxius whole venom) can be used as part of a pharmaceutical compound to treat patients with Scorpion sting, particularly if the scorpion is C. noxius. The pharmaceutical compound can include other antibodies such as the F (ab ') 2 equine polyclonal fragments already used as antivenoms. In this case the addition of 6009F antibody and / or functionally equivalent variants thereof is to strengthen its neutralizing effect, in particular against C. noxius venom. The pharmaceutical compound may also include a pharmaceutically acceptable carrier such as those already mentioned, although this is optional. Accordingly, another environment of the present invention relates to a pharmaceutical compound comprising the antibodies 6009F, 6105F or 6103E and / or any functionally equivalent variant thereof to treat poisoning by some scorpion, in particular the scorpion C. noxius. In another environment, the present invention relates to a method for treating a mammal that requires this treatment, especially patients who have suffered scorpion stings, especially if the scorpion is C. noxius, including the step of administering a quantity pharmacologically. effective of a pharmaceutical compound comprising variants of antibodies 6009F, 6105F or 6103E and / or functionally equivalent variants thereof. The pharmaceutical compound can be administered via the local, systemic or both routes through the conventional routes such as intravenous, subcutaneous, intramuscular, intravaginal, intraperitoneal, intranasal, oral or other mucous to protect the patient against the lethal effect of the toxin Cn2 of the venom of Centruroides scorpions.

X. Modified bioseparation method for the selection of improved clones. The standard method of bioseparation to select variants with better affinity of the phage-antibodies obtained by a cycle of mutagenesis of a library, generally includes the steps of: 1) incubation of the library in the presence of the selected binding antigen, previously adhered to a solid phase such as the immunotube (Nunc; Maxisorp), for a time to allow the specific clones to bind to the immobilized antigen; 2) Wide wash with PBS-Tween20 (1X, 0 -1%) and PBS to remove nonspecific phage-antibodies, and 3) recovery of phage-antibodies bound by addition of either a weak acid or base solution or a cell suspension, in particular the white phage bacteria strain used as the deployment vehicle for the antibody repertoire. Commonly several rounds of sieving are carried out to increase the number of positive clones to be selected. The mutant clones resulting from these standard procedures generally slightly improve the affinity of the clone. It is common for standard procedures to evolve antibodies to include several rounds of mutagenesis cycles followed by bioselection, before achieving a clone with satisfactorily improved affinity. In order to select improved antibody variants not only in their affinity but also in their stability, the inventors of the present invention modified the standard procedure. Said modification was decisive for selecting some improved variants of clone 3F antibody. It is well known to those who work in this field that increasing the temperature during the incubation period increases the speed for formation and dissolution of bonds between antibodies and the selected antigen.. , this helps to select tightly bound antibodies and to wash weakly bound antibodies. Similarly, increasing the incubation time helps to select tightly bound antibodies. In addition, it is common practice to use weak acid or weak base solutions to recover bound antibodies. In the present invention, the inventors decided to take advantage of the aforementioned increases in temperature and time during the incubation period with a decrease in the amount of the selected antigen used to cover the immunotube (for more astringent biopanning) and also ran the risk of releasing the antibodies recovered through the use of a weak base and looking for antibodies bound to the selected antigen in the immunotube. Contrary to what would be expected, as shown in examples 5 and 6, after an additional step of recovery with cell suspension, several antibodies were recovered from the immunotube. Said antibodies had improved in affinity and stability, with respect to the parental antibody 61 OA, compared with antibodies recovered using the standard procedures that only slightly improved their affinity. In the present invention the method for selecting antibody variants improved affinity and stability of an antibody library mutagenized comprising the steps of: 1) Incubation of the library in the presence of the antigen selected bonding, previously adhered to a solid phase as immunotube (for example Nunc; Maxisorp), during a time to allow specific variants to bind to the immobilized selected antigen at a temperature of at least (30 ° C), preferably 37 ° C and for a minimum period of 5 hours. 2) Wide wash with PBS-Tween20 (1X, 0.1%) and PBS to remove nonspecific phage antibodies, 3) Wash with weak or weak base acid solution (eg 100 mM triethylamine) to remove non-specific phage antibodies. and less stable or less united, followed by a neutralization; and 4) Recovery of the phage-antibodies bound by the addition of a cell suspension, in particular the target cell strain of the phage used as the display phage for the antibody repertoire. The method may comprise an additional step of incubating the library in the presence of blocking agents before bioselection in order to eliminate as many nonspecific clones as possible, but this is optional. Blocking agents are well known to those working in this field and may include BSA, skim milk powder or gelatin, for example. When using this modified method compared to the standard method for selecting mutated clones after a cycle of mutagenesis of 61 OA antibody variants, the standard phage selection method yielded a lower number of positive variants (2 with intermediate signal from a total of 88) compared to the most strictly modified method (5 with high signal out of a total of 88) see Fig. 8 and 2 in an additional procedure (2 with high signal out of a total of 88). The number of nucleotide changes in the clones selected from the two procedures was different. The clones selected from the standard procedure showed a lower number of changes (usually one), while using the astringent strategy, the selected clones showed 2-6 changes. The affinity and stability of these 7 (5 + 2) clones was better than that of the 2 clones recovered by the standard method.

XI. scFv 6009F recognizes an epitope different from that recognized by the monoclonal antibody BCF2 or the whole equine polyclonal antivenom. As detailed in Example 9, the scFv 6009F recognized a different epitope as demonstrated by a competitive analysis in the BIACORE against the monoclonal antibody BCF2 (Fig 3). It was also confirmed by competitive ELISA (Fig 7). In addition, the poison Complete polyclonal equine antibody does not contain antibodies that compete with scFv 6009F for the same epitope as shown by an analysis in the BIACORE (Fig. 9).

Materials and Methods Antigens. The Cn2 toxin (formerly called 11-9.2.2), was purified from a venom obtained by electrical stimulation of Centruroides noxius Hoffmann scorpions. The venom was purified by gel filtration with Sephadex G-50 and chromatography with cation exchange (Zamudio, F., Saavedra, R., Martin, BM, Gurrola-Briones, G., Herion, P. &Possani, L D. (1992) Eur J Biochem 204, 281-92). The other toxins used (CII1, (Ramirez, AN, Martin, BM, Gurrola, GB &Possani, L D. (1994) Toxicon 32, 479-90), CII2 (Alagon, A. C, Guzman, HS, Martin , BM, Ramírez, AN, Carbone, E. &Possani, L D. (1988) Comp Biochem Physiol B 89, 153-61), Pg5, Pg7, Pg8, (unpublished data), FU (toxic fraction II of the Centruroides limpidus limpidus), (Alagon, A. O, Guzman, HS, Martin, BM, Ramirez, AN, Carbone, E. &Possani, LD (1988) Comp Biochem Physiol B 89, 153-61), were obtained by the same purification procedure from the poisons of the species Centruroides limpidus limpidus (CU) and Parabuthus granulatus (Pg).

Plasmid pSynl This vector allows the expression of the cloned segment under the control of the lac promoter. The expressed product contains a Cmyc peptide and a stretch of 6 histidines (His hexa) at the carboxyl terminus. (Schier, R., Marks, JD, Wolf, EJ, Apell, G., Wong, C, McCartney, JE, Bookman, MA, Huston, JS, Houston, L.L. &Weiner, M.L. (1995) Immunotechnology, 73-81., Bai, J., Sui, J., Zhu, RY, Tallarico, AS, Gennari, F., Zhang, D. &Marasco, WA (2003) J Biol Chem 278, 1433-42 ).

Plasmid pSyn2. This vector allows the cloned segment to be displayed under the control of the lac promoter fused to gene III (which codes for pIII). The expressed product contains a Cmyc peptide and a termination codon (amber) that allows expression of the free scFv in a suppressor strain.

Amplifications of the variable domains by PCR. For PCR reactions a GeneAmp PCR thermo-cycler (PERKIN ELMER 9600, Norwalk, USA) was used. The conditions for the amplifications were: three minutes of denaturation at 95 ° C, followed by 30 cycles at 95 ° C for 1 min, 55 ° C for 1 min and 72 ° C for 1 min, with a final extension cycle at 72 ° C for 10 min. The PCR products were purified with a rapid QIA purification kit (QIAGEN, Inc., Valencia, CA, USA).

Standard bioseparation method for anti-Cn2 scFv. Standard bioselection was carried out as described in (Marks, JD, Hoogenboom, HR, Bonnert, TP, McCafferty, J., Griffiths, AD &Winter, G. (1991) J Mol Biol 222, 581-97) with some modifications as follows: 1 ml of the library (1X1013 phage-antibodies) is incubated in the presence of different blocking agents (BSA or gelatin) before bioselection in order to eliminate as many nonspecific clones as possible. The rest of the library was taken to an immunotube (Nunc; Maxisorp) previously coated ovght (O / N) with 1 ml of Cn2 toxin at 50 μg / ml in NaHCO3 buffer, pH 9.4 at 4 ° C. Extensive washes were performed to remove nonspecific phages (20 washes with 1X PBS with 0.1% Tween20 and 20 washes with PBS). The phage-antibodies bound to the toxin were recovered by the addition of 1 ml of TG1 cells from a mid-log phase culture (OD600 = 0.7) (Lou, J., Marzari, R., Verzillo, V., Ferrero, F ., Pak, D., Sheng, M., Yang, O, Sblattero, D. &Bradbury, A. (2001) J Immunol Methods 253, 233-42., Sblattero, D. &Bradbury, A. ( 2000) Nat Biotechnol 18, 75-80) After four rounds of screening, phage-antibody clones were cultured in a random manner and analyzed for their specific binding to Cn2 by ELISA.

ELISA assays of phage-antibody clones in monovalent display. High capacity polystyrene ELISA plates (Corning, NY, USA) were coated ovght with 0.3 μg of Cn2 toxin (100 μl / well) in 50 mM bicarbonate buffer pH 9.4 at 4 ° C. Plates were washed three times with PBS and 0.1% Tween, and then blocked with 0.5% BSA in PBS for 2 h at 37 ° C. To each well the suptants with phage-antibodies were added, incubated for 1 hour at 37 ° C and then the plates were washed. The phage-antibodies bound to the toxin were detected with the conjugate horseradish peroxidase (HRP) -antibody anti-M13 (Amersham Pharmacia Biotech AB). The HRP activity was detected by adding the O-phenylenediamine substrate. The plates were read at 492 nm in an ELISA reader (Model Bio-RAD 2550). Clones that bound Cn2 with absorbance values above 2 were considered positive. The clones that recognized the toxin were specifically sequenced.

Cross-reactivity of the selected phage-antibodies. Phage antibodies selected for their specificity were analyzed for different antigens in ELISA assays.

High capacity polystyrene immunoplates were coated with various proteins (toxins Cn2, CII1, CII2, FU, Pg5, Pg7 and Pg8, and BSA, casein and gelatin) in 50 mM bicarbonate buffer pH 9.4 at 4 ° C ovght. 100 μl of each selected variant containing 1X1011 phage-antibodies / ml was added to the wells of the plate and detected as described. The phage-antibodies were detected with the horseradish peroxidase conjugate (HRP) -antibody anti-M13 (Amersham Pharmacia Biotech AB). The HRP activity was detected by adding the substrate O-ethylenediamine. Plates were read at 492 nm in an ELISA reader (Bio-RAD Model 2550). Clones bound to Cn2 with absorbance values above 2 were considered positive. The clones that specifically recognized the toxin were sequenced.

Directed evolution (random mutagenesis) by PCR susceptible to error. Selected clones of the library constructed after 4 rounds of bioselection, were subjected to mutagenesis. To construct scFvs libraries of random mutants with different mutation rates (low to medium and high), two standard PCR techniques susceptible to error were used (Cadwell, RC &Joyce, G. F, (1992) PCR Methods Appl 2 , 28-33) and 2% (Leung, DW, Chen, E. &Goeddel, DV (1989) Technique 1, 11-15). Both PCR products were mixed, digested with Sfi \ and? Fofl, gel purified and then ligated into the phagemid pSyn2. The ligated DNA was electroporated into electrocompetent E. co // TG1 cells. Three cycles of evolution were carried out and the variability (mutation rate) of each library was determined.

Indirect ELISA using scFv antibodies. Supernatants containing scFv antibodies (soluble proteins); They were transferred to a previously coated and blocked ELISA piaca. 100 μl / well of a 1: 2000 dilution of Anti-c-myc antibodies (Zymed Laboratories Inc., San Francisco, CA, USA) and HRP-Goat Anti-Mouse (Zymed Laboratories) were added consecutively and cultured 1 hour at 37 ° C. ). Detection of HRP activity and reading of the plate was carried out following standard procedures (Marks, JD, Hoogenboom, HR, Bonnert, TP, McCafferty, J., Griffiths, AD &Winter, G. (1991) J Mol Biol 222, 581-97).

Measurements by plasmon surface resonance. The kinetic constants of the interaction between scFv proteins and immobilized Cn2 toxin were determined in a biosensor system of molecular interactions in real time (BIACORE X). They joined. 24 μg of Cn2 toxin on a CM5 sensor chip using an equimolar mixture of N-hydroxysuccinimide (NHS) and N-ethyl-N- (dimethyl-aminopropyl-carbodiimide) (EDC) in 200 mM MES buffer pH 4.7. Nearly 400 resonance units (RU) were attached. The scFvs were diluted to various concentrations in HBS-EP buffer (Biacore) and 60 μl of immobilized Cn2 was injected at a rate of 30 μl / min with an injection delay of 600 seconds. The data (Kon, Koff and KD) were analyzed using the program BIAEVALUATION version 3.2.

Competency tests (between BCF2 and 6009F) by plasmon surface resonance. SPR binding assays (BIACORE) were used to find out if the matured scFv and the monoclonal BCF2 recognize the same epitope on the Cn2 toxin. It was carried out as described for an anti-chicken egg lysozyme antibody (HEL) (Donini, M., Morea, V., Desiderio, A., Pashkoulov, D., Villani, ME, Tramontano, A. & Benvenuto, E. (2003) J Mol Biol 330, 323-32). Six saturating injections (60 μl c / 1 of a 200 nM solution) of the BCF2 antibody were applied consecutively on a chip coated with Cn2 at a rate of 30 μl / min in HBS-EP buffer. After this 60 μl of scFv 6009F in 5 nM concentration was injected and the sensorgram was analyzed.

Competition (from Alacramyn, BCF2 and 6009F) by plasmon surface resonance.

To find out if the scFv 6009F and the polyclonal antibody fragments present in the Alacramyn share an epitope on the Cn2 toxin, SPR binding assays were used, which were carried out as described for an anti-chicken egg lysozyme antibody (HEL) ) (Donini, M., Morea, V., Desiderio, A., Pashkoulov, D., Villani, ME, Tramontano, A. &Benvenuto, E. (2003) J Mol Biol 330, 323-32). Eight saturating injections (40 μl of 200 nM) of Alacramyn F (ab ') 2 were applied consecutively on a chip coated with Cn2 at a rate of 10 μl / min in HBS-EP buffer. After this, 40 μl of BCF2 at 20 nM concentration was injected. Finally, 40μl of scFv 6009F was injected at a concentration of 20 nM and the sensorgram was analyzed.

EXAMPLES Example 1. Construction of the human scFv library. From a 400 ml sample of peripheral blood lymphocytes donated by a scorpion collector Centruroides limpidus limpidus a non-immune human scFvs library was prepared. The blood was centrifuged on a Ficoll gradient and separated and washed. lymphocytes Total RNA was isolated and purified with a KIT from Promega. The cDNA was synthesized from the total RNA by RT-PCR using random hexamers [Roche RT-PCR Kit (AMV), Indianapolis, IN, USA]. Immunoglobulin heavy chain variable domain repertoires were amplified from the cDNA using Vent DNA polymerase (New England Biolabs) in combination with each of the HuVHFOR primers and an equimolar mixture of HuJHBACK primers (Marks, JD, Hoogenboom, HR, Bonnert, TP, McCafferty, J., Griffiths, AD &Winter, 'G. (1991) J Mol Biol 222, 581-97) in independent reactions for each family. For the light chain variable domains, a similar procedure was carried out using each HUVKFOR and a mixture of HUJKBACK for chains and each HuV? FOR with a mixture of HuJ? BACK for? Chains. The resulting fragments were reamplified with oligonucleotides that allowed to include half of the splicing peptide [(Gly4-Ser) 3] in independent reactions. The connector primers, SEQ.ID.NO: 1, SEQ.ID.NO: 2, SEQ.ID.NO: 3, SEQ.ID.NO: 4, SEQ.ID.NO: 5, SEQ.ID.NO: 6, SEQ.ID.NO: 7, SEQ.ID.NO: 8, SEQ.ID.NO: 9, SEQ.ID.NO: 10, SEQ.ID.NO: 11, SEQ.ID.NO: 12, SEQ.ID.NO: 13, SEQ.ID.NO: 14, SEQ.ID.NO: 15, SEQ.ID.NO: 16 and SEQ.ID.NO: 17, were designed as already described (Hawlisch, H ., Meyer zu Vilsendorf, A., Bautsch, W., Klos, A. &Kohl, J. (2000) J Immunol Methods 236, 117-31). The PCR products were gel purified and overlaid by PCR as described by Marks (1991). Each overlaid product (72 in total) was amplified in the same overlapping reaction mixture with oligonucleotides as described by Marks (1991) allowing the incorporation of restriction sites Sffl and Non. The following program was used: denaturation at 95 ° C for 5 minutes followed by 7 cycles of 1 min at 95 ° C, 1.5 min at 64 ° C and 1 min at 72 ° C without external oligos. Next, external primers were added, followed by 30 cycles of 1 min at 95 ° C, 1 min at 64 ° C, and 1 min at 72 ° C and a final extension at 72 ° C for 10 min. Each PCR product was quantified and mixed in equimolar amounts to be digested. The DNA fragments were cut with Sffl and? Foi restriction enzymes and purified from an agarose gel. The resulting DNA fragments were ligated into the phagemid pSyn2 (kindly provided by J. D. Marks, UCSF, San Francisco, CA, USA) previously cut with the restriction enzymes Sfi \ and Non. The ligated DNA was electroporated into strain TG1 E. coli. Twenty individual clones chosen at random were analyzed by digestion with the BstN \ enzyme and sequenced. The sequences of the clones were determined with the direct oligonucleotides (5 ^ ATA CCT ATT GCC TAC GG C3 \ SEQ.ID.NO: 60) and reverse (5 ^ TTT CAA CAG TCT ATG CGG3 \ SEQ.ID.NO: 61) in the Applied BioSystems Model 3100 sequencer.

Example 2. Isolation of anti-Cn2 scFvs by screening the phage-antibody repertoires.

The library of human scFvs was deployed on the surface of filamentous phage infecting the cell culture with Helper phage (M13K07) (New England Biolabs, Benerly, MA USA) and was used to select antibodies against Cn2 toxin. Bioselection or screening was performed as described in the Methods. After four rounds of sieving, 88 clones isolated from phage-antibody were randomly taken, and their specific binding to Cn2 was evaluated by ELISA. High-capacity polystyrene ELISA plates for protein bonding (Corning, NY, USA) were coated overnight with 0.3 μg of Cn2 toxin (100 μl / well) in 50 mM bicarbonate buffer pH 9.4 at 4 ° C. The plates were washed three times with PBS and 0.1% Tween, and then blocked with 0.5% BSA in PBS for 2 h at 37 ° C. To each well, supernatant of the phage-antibody culture was added, incubated for 1 h at 37 ° C and the plates were washed. The phage-antibodies bound to the toxin were detected with the horseradish peroxidase conjugate (HRP) - anti-M13 antibody (Amersham Pharmacia Biotech AB). The HRP activity was detected by adding the O-phenylenediamine substrate. The plates were read at 492 nm in an ELISA reader (Bio-RAD Model 2550). Fifteen clones that bound Cn2 with absorbance values above 2 were considered positive and were sequenced and analyzed individually. Only two anti-Cn2 scFvs were identified and termed scFv 3F and scFv C1 (Fig. 1). The nucleotide sequences were compared to the database using the BLAST algorithm. The best grades corresponded to human immunoglobulins. They were also compared with the IMGT database (Lefranc, M. P. (2003) Nucleic Acids Res 31, 307-10) to determine the corresponding germ lineages. For clone 3F, the VH3-VK3 families were the closest for the VH and VL domains respectively. In the case of Cl, the VH3-V? 1 families had the highest scores. The specificity of these two scFvs was determined by phage ELISA (Fig. 2A). These two clones showed high specificity to Cn2 despite its strong identity of the latter with control toxins CII1 and CII2 (Fig. 2B). These scFvs were recloned into the expression vector pSynl to characterize them as soluble proteins. Both antibody fragments were unable to protect the mice from the effect of the Cn2 toxin. The affinity constants were determined in a biosensor of molecular interactions in real time (BIACORE). Table 2 shows the values obtained for the binding kinetic constants. The dimeric form of both scFvs was built by shortening of the connector peptide from 15 to 7 amino acid residues by PCR. None of the 3F and C1 diabodies could neutralize the toxin in the protection assay.

TABLE 2. Kinetic rates and affinity constants of soluble proteins corresponding to scFvs 3F and C1.

The kinetic and KD rates were calculated using the BIA-EVALUATION 3.2 software. SE means standard error.

Example 3. First maturation cycle. In the first cycle of maturation clone 3F was used as a parental antibody subject to evolution directed by PCR susceptible to error as described in Methods. A library of 1X106 variants (0.9% mutation rate), obtained from the 3F scFv, was evaluated by phage display against the Cn2 toxin. The 6F variant was selected, which showed a change (Ser54Gly) in the CDR2 of the heavy chain. The kinetic constants of this mutant (Table 1), showed that the association and dissociation constants improved approximately 7 times and 1.5 times respectively and as a result the KD was increased an order of magnitude (from 183 nM to 16.8 nM, Table 1). These results show that scFv 6F binds more efficiently to the toxin, but it is quickly released indicating that the residue at position 54 plays an important role in the interaction of the antibody with the Cn2 toxin.

Example 4. Second maturation cycle. In the second maturation cycle, the 6F variant selected in Example 3 was used as a parental antibody and subjected to PCR-directed evolution susceptible to error as described in Methods. A library of 1.6X106 variants of clone 6F (0.6% mutation rate) was obtained and was evaluated by phage display against Cn2 toxin and the 610A variant was selected. This variant showed a second change in the heavy chain CDR3 (Val101Phe). This mutation improved both constants, that of association but more importantly that of dissociation. This result suggests that residue 109 in the CDR3 of the heavy chain also plays an important role in binding to the toxin. The change of Val by Phe resulted in a better interaction in terms of greater contact area. The accumulated changes in the CDR2 and CDR3 in the clone variant 61 OA, showed a synergic effect on the affinity constant achieving a 176-fold increase [183 nM (clone 3F) at 1.04 nM (clone 61 OA); Table 1), as determined by means of BIACORE.

Example 5. Third cycle of maturation. In the third maturation cycle, the 61 OA variant selected in example 4 was used as a parental antibody and PCR-directed evolution susceptible to error was subjected as described in methods. A library of 1.0X107 variants of clone 61 OA (mutation rate 1%) was obtained and evaluated by phage display against the Cn2 toxin. In this last step of maturation, two alternative selection strategies were carried out. The first was the standard procedure and the second included some more astringent modifications in order to obtain better variants in terms of affinity and stability. The bioselection method was modified according to standard methods but with the following changes: the immunotube was coated with 1 ml of Cn2 at 5 μg / ml in NaHCO3 buffer, pH 9.4 at 4 ° C instead of 50 μg / ml used in the standard sieving procedure, the incubation time was increased from 2 hours to 5 hours and the temperature increased from 25 ° C to 37 ° C. After washing steps (20 washes with PBS-Tween20 (1x 0.1%) and 20 washes with PBS), 1 ml of 100 mM triethylamine (TEA from Pierce, Ill, USA) was added, to eliminate phage-less antibodies stable or more weakly united. The incubation time was 30 minutes, after which the eluted phages were eliminated. The immunotubes were rinsed with 1 ml of 1M Tris-HCl, pH 7 to neutralize the TEA and then washed three times with PBS. The phage-antibodies that remained bound to Cn2 were recovered with TG1 E Coli cells. The clones selected with this procedure were evaluated by ELISA as soluble proteins. The standard phage-antibody selection procedure yielded fewer positive variants compared to the more astringent procedure. In the selected clones, the number of nucleotide changes from the two procedures was different. From this screening procedure, the variant clone 6009F was selected. The DNA sequence of clone 6009F showed six mutations, two silent mutations, and four changes in amino acids in relation to clone 610A (Table 1). One of these amino acid changes took place in the framework region 3 (framework 3) of the heavy chain (Asn74Asp) and the other 3 changes in the light chain. Two of them (Thr152lle and Ser197Gly) occurred in frame regions 1 and 3 respectively and the third (Tyr164Phe), in CDR1 (Table 1). The analysis of Affinity measurements [Table 2 and Fig. 6], revealed that the variant clone 6009F showed a Kd of 410 pM, comparable to the affinities of other neutralizing antibodies of scorpion toxins (Aubrey, N., Devaux, C, Sizaret , PY, Rochat, H., Goyffon, M. &Billiald, P. (2003) Cell Mol Life Sci 60, 617-28). The kinetic parameters shown in Table 1 reveal that both kinetic constants improved about 2 times compared to clone 61 OA resulting in an affinity constant as mentioned above, in the picomolar range, leading to an increase of 446 times in Kd with respect to the 3F scFv.

EXAMPLE 6. Second screening for the third cycle of mutagenesis In a second screening procedure the same mutation library generated in example 5 was used. Again, the procedure included the astringent modifications in order to separate improved variants in its affinity and stability. The modified sieving method was performed as mentioned in example 5, but this time a smaller amount of toxin was used to coat the immunotube (2.5, 1 and 0.5 μg / ml for the first, second and third sieving cycles). After the washing steps, 1 ml of 100 mM TEA was added to remove the less stable or weakly bound phage-antibodies. The incubation time was 30 minutes, after which the eluted phages were eliminated. The immunotubes were rinsed with 1 ml of 1M Tris-HCl, pH 7 to neutralize the TEA and then washed three times with PBS. The phage-antibodies that remained bound to Cn2 were recovered with TG1 E Coli cells. Clones separated with this procedure were evaluated by ELISA as soluble proteins. From this modified sieving procedure, the variant clones 6105F and 6103E were obtained. The DNA sequence of clone 6105F showed 3 mutations: 1 silent mutation and 2 amino acid changes in relation to clone 610A (Table 1). One of these amino acid changes occurred in the framework region 3 of the heavy chain (Asn74Asp) and the other change in the light chain. The analysis of affinity measurements [Table 2. and Fig. 10], revealed that the 6105F variant presented a Kd of 590 pM, comparable to the affinities of other neutralizing antibodies to scorpion toxins (Aubrey, N., Devaux, C, Sizaret, PY, Rochat, H., Goyffon, M. &Billiald, P. (2003) Cell Mol Life Sci 60, 617-28). The kinetic parameters shown in Table 1 reveal that both kinetic constants improve by about 1.5-2 times compared to clone 610A, resulting in an affinity constant as already mentioned, in the picomolar range, which increases 310 times Kd in relation to the 3F scFv. On the other hand, the sequence of. DNA from clone 6103E showed 4 mutations, 1 silent mutation and 3 amino acid changes in relation to clone 610A (Table 1). Two of these amino acid changes occurred in the framework region 3 of the heavy chain (Asn74Asp) and the other changes in the light chain. The analysis of affinity measurements [Table 2 and Fig. 11], revealed that the 6103EF variant showed a Kd of 630 pM, comparable to the affinities of other scorpion toxin-neutralizing antibodies (Aubrey, N., Devaux, C, Sizaret, PY, Rochat, H., Goyffon, M. &Billiald, P. (2003) Cell Mol Life Sci 60, 617-28). The kinetic parameters shown in Table 1 reveal that both kinetic constants improved by about 2-3 times compared to the 610A variant, resulting in an affinity constant as already mentioned, in the picomolar range, leading to an increase in 290 times in Kd with respect to the 3F scFv Example 7. Expression of single chain antibodies (scFvs). To produce the free and soluble scFvs of the present invention, for evaluation in BIACORE and for its evaluation and use as neutralizing agents against Cn2 toxin and the total venom of C. noxius, the DNA fragments encoding the scFvs of some clones such as C1 (SEQ.ID.NO: 18), 3F (SEQ.ID.NO: 24), and variant clones 6F (SEQ.lD.NO: 30), 610A (SEQ.ID.NO: 36), 6009F (SEQ.ID.NO: 42), 6D ( SEQ.ID.NO: 48), 9C (SEQ.ID.NO: 54), 6003E (SEQ.ID.NO: 62), 6003G (SEQ.ID.NO: 68), 601 OH (SEQ.ID.NO : 74), 6011G (SEQ.ID.NO: 80), 6105F (SEQ.lD.NO:86) and 6103E (SEQ.ID.NO:92) were ligated into an expression vector, in this case pSynl. These constructions could be transformed into a competent host, in this case the strain E coli TG1 was used. 500 ml of recombinant cells were grown to reach OD600 = 0.7 (in YT2X, glucose 0.1%, Amp 200 μg / ml) (Marks, J. D., Hoogenboom, H. R., Bonnert, T. P., McCafferty, J., Griffiths, A. D. &; Winter, G. (1991) J Mol Biol 222, 581-97). The expression of the scFv was induced with 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) and the incubation prolongation 6 hours more. Then, the cells were harvested by centrifugation (6000 rpm, 10 min, at 4 ° C). To release the recombinant proteins, the pellet was resuspended in 12.5 ml of PPB extraction buffer (20% saccharose / 1 mM EDTA / 30 mM Tris HCl adjusted to pH 8) and the mixture was incubated on ice for 20 minutes. The cells were centrifuged at 6000 rpm at 4 ° C for 20 min. The soluble scFv protein present in the supernatant was collected. For purification, the pellet was resuspended in 5 mM MgSO4, kept on ice for 20 min and centrifuged at 6000 rpm at 4 ° C for 20 min. The supernatants PPB and MgSO4 were mixed and dialyzed twice against PBS 1X. The scFvs were finished purifying by Ni2 + -NTA affinity chromatography (QIAGEN, Germany) taking advantage of the stretch of 6 histidines that the vector added to the expressed product and eluted with 1 ml of 250 mM imidazole.

Finally, the scFvs preparations were further purified by gel filtration chromatography on a Superdex ™ 75 column (Pharmacia Biotech AB, Uppsala, Sweden).

EXAMPLE 8. Neutralization tests. Proteins purified in the form of scFvs were used to test their neutralization capacity against the toxic effects of Cn2 or total venom in mice. Groups of 10-20 female mice (strain CD1) were injected with a mixture of scFv and Cn2 toxin or venom. With each scFv one or two LD50 (0.25 or 0.5 μg / 20 g of mouse weight) of Cn2 toxin or two LD50 (5 μg / 20 g of mouse weight) of total venom were mixed in a final molecular ratio of 1 : 10 (toxin: scFv). The mixture was incubated for 30 min at 37 ° C and injected intraperitoneally. Three controls were used: venom, Cn2 or scFv alone were injected in independent trials. The number of animals was kept to a minimum but sufficient to validate the experiment. The protocols were approved by the Animal Care Ethics Committee of the Institute in which the researchers work.

TABLE 3. Neutralization tests. Results in groups of mice exposed to Cn2 toxin or total venom by intraperitoneal injection alone or in the presence of the indicated molar proportions of toxin: antibody.

Sample DL50 Molar ratio Protected / injected Cn2: 6009F 6009F 10/10 Cn2 1 none 6/10 Cn2 1 1: 10 20/20 Cn2 2 none 6/18 Cn2 2 1: 10 18/18 Poison intact 2 none 0/10 Poison integral 2 1: 14a 10/10 a = Estimated considering that Cn2 constitutes 6.8% of the complete poison. LD50 of Cn2 = 0.250 μg / 20 g of mouse weight Amount of whole venom used = 2.5 μg / 20 g of mouse weight. Sample DL50 Molar proportion Protected / injected Cn2: 6105F Cn2 1 none 6/10 Cn2 1 1:10 10/10 Cn2 2 1: 10 10/10 Sample DL50 Molar ratio Protected / injected Cn2: 6103E Cn2 1 none 6/10 Cn2 1 1: 10 10/10 Cn2 2 1: 10 10/10 The neutralization capacity against the Cn2 toxin of the purified soluble protein of clones 6F, 610A and 6009F was evaluated in CD1 mice. The scFv of clone 6009F was the only one that showed the ability to neutralize the toxin. When a lethal dose of toxin and 10 molar antibody was injected in excess, all mice survived compared to controls (Table 3). Worth mentioning is that the mice did not present any symptoms related to poisoning (Dehesa-Davila, M. &Possani, L. D. (1994) Toxicon 32, 1015-8). The next step was to use two lethal doses of toxin. The mice showed no signs of poisoning, demonstrating the effectiveness of the human antibody developed in the present invention (100% protection). In the case of total poison (only two lethal doses used with the same amount of scFv), the mice were protected, but had some symptoms such as respiratory distress from which they finally recovered 7 hours later. It seems convenient to emphasize that the 6009F antibody has the capacity to completely neutralize the lethal effect of two LD50 of Cn2 toxin and confers reasonably satisfactory protection against two lethal doses of total venom. The scFv 6009F is stable after 4 weeks of storage in PBS at 4 ° C, as demonstrated by evaluation of functional activity for 4 weeks. The scFv 6009F showed protective activity during this period, indicating that it is functionally stable, as would be expected from the astringent selection strategy (modified method) that was used.

EXAMPLE 9. Plasmon Surface Resonance (SPR) Competency Tests. Since the monoclonal antibody BCF2 also neutralizes the Cn2 toxin, in order to find out if the epitope recognized by clone 6009F is the same as the one recognizing BCF2, a displacement test was performed using the Biacore (Fig. 3). SPR binding assays were used to find out if the matured scFv and the monoclonal BCF2 recognized the same epitope on the Cn2 toxin. It was carried out as already described (Donini, M., Morea, V., Desiderio, A., Pashkoulov, D., Villani, ME, Tramontano, A. &Benvenuto, E. (2003) J Mol Biol 330 , 323-32). Six saturating injections (60 μl of 200 nM) of the BCF2 antibody were applied consecutively on a chip coated with Cn2 at a rate of 30 μl / min in HBS-EP buffer. After this, 60 μl of the scFv 6009F in 5 nM concentration were injected and the sensorgram was analyzed. The sensorogram showed that the 6009F antibody binds to the Cn2 toxin, in spite of being saturated the sites recognized by BCF2, this suggests that the clone 6009F recognizes a different site (epitope). These results were confirmed by competitive ELISA (see Fig. 7), in which scFv 6009F was first joined, then the toxin and the last BCF2. Both antibodies remain bound to the Cn2 toxin, confirming once again that they recognize different epitopes. In addition, the same procedure was used to determine whether the epitope recognized by clone 6009F is equal to any of the epitopes recognized by a polyclonal antibody present in the commercial antivenom F (ab ') produced against a group of scorpion venoms, which includes the total poison of the species C. noxius (Alacramyn, of the Bioclon Institute SA de CV, Mexico). The sensorgram (Fig. 9) showed no increase in the signal after the injection of BCF2, this means that BCF2 shares one of the epitopes recognized by Alacramyn, while the 6009F antibody injection gave a signal exactly like the control, that is, the binding kinetics of scFv 600F to the Cn2 toxin, showing that the 6009F variant recognizes a different epitope site than those recognized by antibodies produced in vivo (Alacramyn and BCF2).

References All the publications, patents and patent publications cited here are mentioned as a reference in their entirety in the exhibition. The above specification, including specific contexts and examples, are intended to be illustrative and not limiting. Numerous other variations and modifications may be made without departing from the true spirit and scope of the present invention.

Claims (25)

CLAIMS What is claimed as an invention is:

1. An isolated nucleic acid characterized in that it is selected from the group consisting of SEQ ID NO: 20, SEQ. ID. NO: 26, SEQ. ID, NO: 32, SEQ. ID. NO: 38, SEQ. ID. NO: 44, SEQ. ID. NO: 50 SEQ. ID. NO: 56, SEQ. ID. NO: 64, SEQ. ID. NO: 70, SEQ. ID. NO: 76, SEQ. ID. NO: 82, SEQ. ID. NO: 88 and SEQ. ID. NO.94

2. An isolated nucleic acid characterized in that it is selected from the group consisting of SEQ. ID. NO: 22, SEQ. ID. NO: 28, SEQ. ID. NO: 34, SEQ. ID. NO: 40, SEQ. ID. NO: 46, SEQ. ID. NO: 52 SEQ. ID. NO: 58, SEQ. ID. NO: 66, SEQ. ID. NO: 72, SEQ. ID. NO: 78, SEQ. ID. NO: 84, SEQ. ID. NO: 90 and SEQ. ID. NO: 96

3. An isolated nucleic acid characterized in that it comprises the isolated nucleic acids of claims 1 and 2.

4. The isolated nucleic acid of claim 3 characterized in that it has a sequence selected from the group consisting of SEQ. ID. NO: 18, SEQ. ID. NO: 24, SEQ. ID. NO: 30, SEQ. ID. NO: 36 SEQ. ID. NO: 42, SEQ. ID. NO: 48, SEQ. ID. NO: 54, SEQ. ID. NO: 62, SEQ. ID. NO: 68, SEQ. ID. NO: 74, SEQ. ID. NO: 80, SEQ. ID. NO: 86 and SEQ. ID. NO: 92

5. A molecular vector characterized in that it comprises the nucleic acid of the claim 3 operably linked to the vector control sequences.

6. A molecular vector characterized in that it comprises the nucleic acid of the claim 4 operationally linked to the vector control sequences.

7. The molecular vector of claims 5 or 6, characterized in that it is an expression vector

8. A cell characterized in that it comprises the molecular vector of claim 7.

9. The cell of claim 8 characterized in that it is a prokaryotic cell.

10. The cell of claim 9. characterized in that it is a bacterium.

11. An isolated polypeptide characterized in that it is encoded by a nucleic acid of claim 3.

12. An isolated polypeptide characterized in that it is encoded by a nucleic acid of claim 4.

13. A human antibody that recognizes in particular the Cn2 toxin, characterized in that it comprises a VH fragment with an amino acid sequence selected from the group consisting of SEQ. ID. NO: 21, SEQ. ID. NO: 27, SEQ. ID. NO: 33, SEQ. ID. NO: 39, SEQ. ID. NO: 45, SEQ. ID. NO: 51, SEQ. ID. NO: 57, SEQ. ID. NO: 65, SEQ. ID. NO: 71, SEQ. ID. NO: 77, SEQ. ID. NO: 83, SEQ. ID. NO: 89 and SEQ. ID. NO: 95 and a VL fragment with a sequence selected from the group consisting of SEQ. ID. NO: 23 SEQ. ID. NO: 29, SEQ. ID. NO: 35, SEQ. ID. NO: 41, SEQ. ID. NO: 47, SEQ. ID. NO: 53, SEQ. ID. NO: 59, SEQ. ID. NO: 67, SEQ. ID. NO: 73, SEQ. ID. NO: 79, SEQ. ID. NO: 85, SEQ. ID. NO: 91 and SEQ. ID. NO: 97

14. The human antibody of claim 13 selected from the group consisting of: an antibody comprising a VH fragment with an amino acid sequence SEQ. ID. NO: 45 or functionally equivalent variants thereof and a VL fragment with a sequence SEQ. ID. NO: 47; an antibody comprising a VH fragment with an amino acid sequence SEQ. ID. NO: 89 or functionally equivalent variants thereof and a VL fragment with a sequence SEQ. ID. NO: 91, and; an antibody comprising a VH fragment with an amino acid sequence SEQ. ID. NO: 95 or functionally equivalent variants thereof and a VL fragment with a sequence SEQ. ID. NO: 97, or functionally equivalent variants thereof, characterized in that it has the ability to neutralize the Cn2 toxin.

15. The human antibody of claim 13 characterized in that it has an amino acid sequence selected from the group consisting of SEQ. ID. NO: 19, SEQ. ID. NO: 25, SEQ. ID. NO: 31, SEQ. ID. NO: 37, SEQ. ID. NO: 43, SEQ. ID. NO: 49, SEQ. ID. NO: 55, SEQ. ID. NO: 63, SEQ. ID. NO: 69, SEQ. ID. NO: 75, SEQ. ID. NO: 81, SEQ. ID. NO: 87 and SEQ. ID. NO: 93

16. The human antibody of claim 15 characterized by having an amino acid sequence selected from the group consisting of SEQ ID NO: 43, SEQ. ID. NO: 87 and SEQ. ID. NO: 93 or functionally equivalent variants thereof characterized in that it shows ability to neutralize the Cn2 toxin.

17. A method for producing the human antibodies of claim 13, characterized in that it comprises the steps of: a) cultivating the cells of claim 8 in a suitable culture medium and conditions, for a period sufficient to allow the production of an amount of the antibody. b) releasing the human antibodies from the cell culture and optionally c) isolating and further purifying the antibodies.

18. A method for producing the human antibodies of claim 15 characterized in that it comprises the steps of: a) culturing the cells of claim 8 in a suitable culture medium and conditions for a period sufficient to allow the production of an amount of the antibody. b) releasing human antibodies from the cell culture and optionally c) isolating and further purifying the antibodies.

19. A pharmaceutical composition characterized in that it comprises the antibodies of claims 14 or 16 and pharmaceutically acceptable carriers, wherein said composition neutralizes in vivo the effect of the scorpion venom C. noxius.

20. A composition characterized in that it comprises the antibody of claim 13 bound to a substrate where said composition binds specifically to the Cn2 toxin of the scorpion venom Centruroides noxius.

21. A device for detecting the presence of the Cn2 toxin in a sample characterized in that it comprises the composition of claim 20.

22. The detection device of claim 21 characterized in that it is a detection device by ELISA.

23. The detection device of claim 21, characterized in that it is an immunochromatographic assay.

24. A method for selecting antibody variants from a mutagenized library of antibodies consisting of: 1) Incubation of the library in the presence of the selected binding antigen, previously adhered to a solid phase, for a period of at least 3 hours to allow variants Specific antibodies bind to the immobilized target antigen, preferably for a period of 5 hours, at a temperature selected from the range of 30 ° C to 39 ° C, preferably 37 ° C. 2) Extensive washing with a suitable buffer with and without Tween 20 (0.1%) to remove the nonspecific phage-antibodies, 3) Washing with an acid solution or weak base to remove the phage-antibodies unspecific and less stable or weakly bound, followed by a neutralization step, and 4) Recovery of the phage-antibodies bound by the addition of a cell suspension, in particular the phage target cell strain used for phage display of the antibody repertoire.

25. The method of claim 25 wherein the binding antigen selected from step 1) was pre-adhered to a solid phase in an initial concentration range from 1 to 10 μg / ml of said antigen in a suitable buffer.