Internet for Pharmaceutical and Biotech Communities
| Newsletter | Advertising |
 
 
 

  

Pharm/Biotech
Resources

Outsourcing Guide

Cont. Education

Software/Reports

Training Courses

Web Seminars

Jobs

Buyer's Guide

Home Page

Pharm Patents /
Licensing

Pharm News

Federal Register

Pharm Stocks

FDA Links

FDA Warning Letters

FDA Doc/cGMP

Pharm/Biotech Events

Consultants

Advertiser Info

Newsletter Subscription

Web Links

Suggestions

Site Map
 

 
   

 

  Pharmaceutical Patents  

 

Title:  Human antibodies that specifically recognize the toxin Cn2 from Centruroides noxius scorpion venom
United States Patent: 
7,381,802
Issued: 
June 3, 2008

Inventors: 
Riano-Umbarila; Lidia (Bogota, CO), Becerril Lujan; Baltazar (Morelos, MX), Possani Postay; Lourival Domingos (Morelos, MX)
Assignee: 
Universidad Nacional Autonoma De Mexico (UNAM) (MX)
Appl. No.: 
11/404,879
Filed: 
April 17, 2006


 

Pharm Bus Intell & Healthcare Studies


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 of the Invention

BRIEF SUMMARY OF THE INVENTION

Keeping in mind the need for new generation of safer antivenoms and the fact that BCF2 neutralizes both the Cn2 toxin and the whole venom of C. noxius, the inventors decided to obtain recombinant human antibodies specific for Cn2 toxin that are able to recognize the toxin and preferably to neutralize it. For this purpose, a human non-immune phage display library of 1.1.times.10.sup.8 members was constructed. Two specific scFv (3F and C1), which specifically bind the Cn2 toxin, were selected. The scFv 3F was affinity matured by directed evolution. After three cycles of maturation, several scFv clones were isolated which specifically recognize toxin Cn2 (6F, 610A and 6009F, 6D, 9C, 6003E, 6003G, 6010H, 6011G, 6105F and 6103E). Some of them showed an increment in the K.sub.d of 10.9 fold, 176 fold and 446 fold [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 Biacore analysis. All variants were monomeric. Although variants C1, 3F, 6F, 610A, 6D, 9C, 6003E, 6003G, 6010H and 6011G specifically recognized Cn2 did not neutralize the toxin nor the whole venom, while variants 6009F, 6105F and 6103E showed to be capable of neutralizing 2 LD50 of Cn2 toxin or 2 LD50 of whole venom, when a molar ratio of 1:5 toxin:antibody fragment, was used. Variant 6009F recognizes a different epitope than that of BCF2, a murine monoclonal antibody raised against scorpion toxin Cn2, which is capable of neutralizing both toxin Cn2 and the whole venom when tested in mice, and that of Alacramyn, a Mexican polyclonal antibody fragments antivenom from horse. The scFv 6009F is the first reported recombinant human antibody fragment capable of neutralizing 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.

DETAILED DESCRIPTION OF THE INVENTION

Obtaining Starting Parent Antibodies

In one embodiment of the present invention, two starting parent antibodies were generated, a human library of antibodies was constructed and phage displayed and two antibodies, C1 (SEQ. ID. NO: 18 for the coding DNA and SEQ. ID. NO: 19 for the amino acidic sequence) and 3F (SEQ. ID. NO: 24 for the coding DNA and SEQ. ID. NO: 25 for the amino acidic sequence), were isolated which specifically recognizes the toxin Cn2 from the venom of scorpion C. noxius.

The necessity to generate safer and more efficient antibodies to be used in human therapy has resulted in the development of recombinant antibodies from different sources. Ideally the source should be human itself. As detailed shown in example 1, the human scFv library of the present invention was generated by RT-PCR from total RNA purified from B lymphocytes of human peripheral blood. To avoid as much as possible a bias of antibody variable chain family representation improving the possibility of obtaining at least one scFv with affinity to the Cn2 toxin, each V family of variable regions (V.sub.H or V.sub.L), was amplified by independent PCR reactions. In a second step of PCR, the sequence of the linker peptide was added to each individual V family. A PCR overlapping process was performed in order to join both V domains (H and L). Every V.sub.H family was overlapped to every V.sub..kappa. or V.sub..lamda. families (a total of 72 V.sub.H-V.sub.L combinations). The DNA segments encoding the assembled products were fused to pIII gene of PSYN2 phagemid. The scFv library size was 1.2.times.10.sup.8 members. Twenty independent colonies were analyzed by PCR. Eighteen showed the right size and had different restriction patterns when digested with BstNI (FIG. 4, see Original Patent). The variability of the 18 different scFvs was confirmed by DNA sequence, which resulted finally in a library of 1.1.times.10.sup.8 variants. We found different combinations of variable domains, which included the majority of V families.

As detailed in example 2, after four rounds of biopanning of the human scFv library against the Cn2 toxin, only two anti-Cn2 scFvs were identified and named scFv 3F (SEQ. ID. NO: 24 for the coding DNA and SEQ. ID. NO: 25 for the amino acidic sequence) and scFv C1 (SEQ. ID. NO: 18 for the coding DNA and SEQ. ID. NO: 19 for the amino acidic sequence) (FIG. 1, see Original Patent), corresponding to human immunoglobulins. Clone 3F, comprises a V.sub.H3 heavy variable chain (SEQ. ID. NO: 26 for the coding DNA and SEQ. ID. NO: 27 for the amino acidic sequence) and VK3 light variable chain (SEQ. ID. NO: 28 for the coding DNA and SEQ. ID. NO: 29 for the amino acidic sequence), whereas clone C1 comprises a V.sub.H3 heavy variable chain (SEQ. ID. NO: 20 for the coding DNA and SEQ. ID. NO: 21 for the amino acidic sequence) and V.lamda.1 light variable chain (SEQ. ID. NO: 22 for the coding DNA and SEQ. ID. NO: 23 for the amino acidic sequence). These two clones showed to be highly specific to Cn2 despite the fact that Cn2 and the control toxins Cll1 and Cll2 have a high degree of identity (FIG. 2B, see Original Patent).

To know if the selected antibodies had the capacity to protect the mice against the toxic effect of Cn2, a neutralization assay was performed. The results revealed that both antibody fragments were unable to protect the mice from the effect of toxin Cn2. The affinity constants of both scFvs were similar, in the range of 10.sup.-7 M which are typical affinity values of the primary immune response (Lefranc, M. P. (2003) Nucleic Acids Res 31, 307-10., Foote, J. & Eisen, H. N. (1995) Proc Natl Acad Sci USA 92, 1254-6). Clones 3F and C1 showed a fast dissociation despite having good association, which suggest that the antibody fragments do not remain bound to the toxin enough time, to be neutralizing. Several reports have shown that some monomeric scFvs do not neutralize their targets, while their corresponding dimeric scFvs (diabody) do, as a consequence of an increase in their affinity (Aubrey, N., Devaux, C., Sizaret, P. Y., Rochat, H., Goyffon, M. & Billiald, P. (2003) Cell Mol Life Sci 60, 617-28., Lantto, J., Fletcher, J. M. & Ohlin, M. (2002) J Gen Virol 83, 2001-5). The dimeric forms of both scFvs were constructed but none of the diabodies 3F and C1 was able to neutralize the toxin in the protection assay.

III. Generation of Antibody Variants

In another embodiment of the present invention, antibody 3F was used as parent antibody to generate antibody variants by affinity maturation by directed evolution techniques, but it is possible to generate variants by other mutagenic techniques known to those skilled in the art, like cassette mutagenesis (Stemmer, W. P. C. et al., (1992) Biotechniques 14:256-265.; Arkin, A. and Youvan, D. C. (1992) Proc Natl Acad Sci USA 89:7811-7815.; Oliphant, A. R. et al., (1986) Gene 44:177-183.; Hermes, J. D. et al., (1990) Proc Natl 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, E. R. and Youvan D. C. (1992) Bio/Technology 10:1557-1561), in which the specific region to be optimized is replaced with a synthetically mutagenized oligonucleotide, gene shuffling and other mutagenesis procedures like directed evolution or random mutagenesis (see Neylon, C. (2004) Nucleic Acids Research 32, 1448-1459). The 3F scFv selected from the human non-immune scFv library of the present invention did not have the required affinity and/or stability to be neutralizing as it has been shown for most of the examples of neutralizing antibodies, which have affinities in the nanomolar range and lower (Maynard, J. A., Maassen, C. B., Leppla, S. H., Brasky, K., Patterson, J. L., Iverson, B. L. & Georgiou, G. (2002) Nat Biotechnol 20, 597-601., Sawada-Hirai, R., Jiang, I., Wang, F., Sun, S. M., Nedellec, R., Ruther, P., Alvarez, A., Millis, D., Morrow, P. R. & Kang, A. S. (2004) J Immune Based Ther Vaccines 2, 5., Devaux, C., 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 has a medium size. It has been learned that better binders can be selected from bigger libraries. (Sblattero, D. & Bradbury, A. (2000) Nat Biotechnol 18, 75-80., Vaughan, T. J., Williams, A. J., Pritchard, K., Osbourn, J. K., Pope, A. R., Earnshaw, J. C., McCafferty, J., Hodits, R. A., Wilton, J. & Johnson, K. S. (1996) Nat Biotechnol 14, 309-14., Sheets, M. D., Amersdorfer, P., Finnern, R., Sargent, P., Lindquist, E., Schier, R., Hemingsen, G., Wong, C., Gerhart, J. C., Marks, J. D. & Lindqvist, E. (1998) Proc Natl Acad Sci USA 95, 6157-62). The affinity of the toxin Cn2 for the sodium channels present in some cell preparations has been shown to be in the nM range (Garcia, C., Becerril, B., Selisko, B., Delepierre, M. & Possani, L. D. (1997) Comp Biochem Physiol B Biochem Mol Biol 116, 315-22., Sitges, M., Possani, L. D. & Bayon, A. (1987) J Neurochem 48, 1745-52) These results suggest that an antibody requires an affinity of at least in this range to neutralize the toxin. Taking this into consideration, and with the goal of having a human antibody able to neutralize the toxin Cn2 and the C. noxius venom, we decided to mature clones 3F and C1 by directed evolution and phage display. It has been shown that directed evolution of proteins has allowed increasing gradually a particular property of the protein. Usually it is necessary to perform several cycles of evolution in order to obtain the desired level of improvement. Each cycle starts with a parent antibody and generates one or more antibody variants which are expected to have improved kinetics qualities. Any of these resulting antibody variant may be used as a parent antibody for the next cycle of evolution or mutagenesis. As shown in examples 3, 4, 5 and 6, three cycles of evolution were necessary to obtain variants from scFv 3F (6009F, 6105F and 6103E) with an adequate level of affinity to be capable of neutralization. In the first cycle of maturation, the variant 6F (SEQ. ID. NO: 30 for the coding DNA and SEQ. ID. NO: 31 for the amino acidic sequence) was selected, which had an amino acid alteration, the substitution (Ser54Gly), at CDR2 of heavy chain which have a sequence SEQ. ID. NO: 32 for the coding DNA and SEQ. ID. NO: 33 for the amino acidic sequence (and a light chain with sequence SEQ. ID. NO: 34 for the coding DNA and SEQ. ID. NO: 35 for the amino acidic sequence), having an increment of one order of magnitude in the K.sub.D (from 183 nM to 16.8 nM; Table 1, see Original Patent). These results show that scFv 6F binds more efficiently to the toxin but it still detaches rapidly, indicating that residue at position 54 plays an important role in the interaction of the antibody with the toxin Cn2 (see example 3 for further details).

In the second cycle of maturation (example 4), the variant clone 610A (SEQ. ID. NO: 36 for the coding DNA and SEQ. ID. NO: 37 for the amino acidic sequence) was selected which have a second amino acid alteration at CDR3 of heavy chain, the substitution Val101Phe (sequences for the heavy and light chains are SEQ. ID. NO: 38 for the coding DNA and SEQ. ID. NO: 39 for the amino acidic sequence of the heavy chain and SEQ. ID. NO: 40 for the coding DNA and SEQ. ID. NO: 41 for the amino acidic sequence for the light chain). This mutation improved both the association constant but more importantly the dissociation constant. This result suggests that residue 109 in the CDR3 of heavy chain also plays an important role in the binding to the toxin. This change could result in a better interaction in terms of an increased area of contact. The accumulated changes at CDR2 and CDR3 in variant clone 610A had a synergistic effect on the affinity constant obtaining an increment of 176 fold (Table 1, see Original Patent).

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 acidic sequence) among others. As shown in examples 5 and 6, in this last maturation step, two alternative selection strategies were performed, the standard one and a modified procedure in order to create more stringent conditions intended to select improved variants in their affinity and stability. Those modifications were crucial for the selection of varieties of improved clones. Different strategies with the same purpose have been reported (Kotz, J. D., Bond, C. J. & Cochran, A. G. (2004) Eur J Biochem 271, 1623-9., Zhou, H. X., Hoess, R. H. & DeGrado, W. F. (1996) Nat Struct Biol 3, 446-51., Martin, A., Sieber, V. & Schmid, F. X. (2001) J Mol Biol 309, 717-26., Jung, S., Honegger, A. & Pluckthun, A. (1999) J Mol Biol 294, 163-80). The standard procedure of phage selection gave a low 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 coding DNA and amino acidic sequence of clone 6D and their V.sub.H and V.sub.L, 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 amino acidic sequence of clone 9C and their V.sub.H and V.sub.L, respectively) as compared to the more stringent 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 amino acidic sequence of whole clone and their V.sub.H and V.sub.L, 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 amino acidic sequence of whole clone and their V.sub.H and V.sub.L, 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 the coding DNA and amino acidic sequence of whole clone and their V.sub.H and V.sub.L, respectively), 6010H (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 amino acidic sequence of clone and their V.sub.H and V.sub.L, respectively) and clone 6009F. The number of nucleotide changes per variant in the selected clones from the two procedures was different. The DNA sequence of clone 6009F showed two silent mutations and four amino acid alterations with respect to clone 610A (Table 1). One of these amino acid alterations occurred at framework 3 of heavy chain, the substitution Asn74Asp, which have a sequence SEQ. ID. NO: 44 for the coding DNA and SEQ. ID. NO: 45 for the amino acidic sequence, and the other 3 substitutions at light chain which have a sequence SEQ. ID. NO: 46 for the coding DNA and SEQ. ID. NO: 47 for the amino acidic sequence. Two of them (Thr152Ile and Ser197Gly) occurred at frameworks 1 and 3 respectively and the third one (Tyr164Phe), at CDR1 (Table 1). The kinetic parameters shown in Table 1 reveal that both kinetic constants were improved about 2 fold as compared to clone 610A, resulting in an affinity constant K.sub.d of 410 pM.

The DNA sequence of clone 6105F showed one silent mutation and only two amino acid alterations with respect to clone 610A (Table 1). One of these amino acid alterations (Asn74Asp) occurred at framework 3 of heavy chain, which have a sequence SEQ. ID. NO: 88 for the coding DNA and SEQ. ID. NO: 89 for the amino acidic sequence, and the other substitution (Ala141Val) at framework 1 of the light chain, which have a sequence SEQ. ID. NO: 90 for the coding DNA and SEQ. ID. NO: 91 for the amino acidic sequence. The kinetic parameters shown in Table 1 reveal that both kinetic constants were improved about 1.5-2 fold as compared to clone 610A, resulting in an affinity constant K.sub.d of 590 pM.

The DNA sequence of clone 6103E showed one silent mutation and three amino acid alterations with respect to clone 610A (Table 1). Two of them (Asn74Asp) occurred at the heavy chain, one at framework 3, and the other (Thrr106Ser) occurred at CDR3 of the heavy chain, which have a sequence SEQ. ID. NO: 94 for the coding DNA and SEQ. ID. NO: 95 for the amino acidic sequence. The third substitution (Ala192Thr) occurred the framework3 of light chain, which have a sequence SEQ. ID. NO: 96 for the coding DNA and SEQ. ID. NO: 97 for the amino acidic sequence. The kinetic parameters shown in Table 1 reveal that both kinetic constants were improved about 1.5-2 fold as compared to clone 610A, resulting in an affinity constant K.sub.d of 630 pM.

It has been suggested that changes at CDRs are the most important to improve the affinity for the antigen (Cowell, L. G., Kim, H. J., Humaljoki, T., Berek, C. & Kepler, T. B. (1999) J Mol Evol 49, 23-6., Gonzalez-Fernandez, A., Gupta, S. K., Pannell, R., Neuberger, M. S. & Milstein, C. (1994) Proc Natl Acad Sci USA 91, 12614-8). However, recently it has been shown that changes at frameworks are determinant to improve not only affinity and stability (Daugherty, P. S., Chen, G., Iverson, B. L. & Georgiou, G. (2000) Proc Natl Acad Sci USA 97, 2029-34) but also the level of expression in cells (Graff, C. P., Chester, K., Begent, R. & Wittrup, K. D. (2004) Protein Eng Des Sel 17, 293-304). A similar phenomenon was observed during the maturation of clone 3F to variant clone 6009F, because scFv 6009F accumulated 3 changes at CDRS and 3 changes at the frameworks, similar considerations apply for variants 6105F and 6103E in which mutations occurred both at CDRs and Frameworks. The chromatographic elution profile of the antibody 6009F, showed a main peak corresponding to a monomeric form (FIG. 5, see Original Patent). We surmised that the changes in the frameworks contributed to reach a better affinity and functional stability. The analysis of affinity measurements [Table 1. and FIG. 6, see Original Patent], revealed that the clone 6009F, had a K.sub.d of 410 pM, which is comparable to affinities of other neutralizing antibodies of scorpion toxins (Aubrey, N., Devaux, C., Sizaret, P. Y., Rochat, H., Goyffon, M. & Billiald, P. (2003) Cell Mol Life Sci 60, 617-28). While clone 6105F had a K.sub.d of 590 pM and clone 6103E had a K.sub.d of 630 pM, which are comparable to affinities of other neutralizing antibodies of scorpion toxins too. The global synergistic improvement in the kinetic parameters with respect to 3F scFv shown in Table 1 leads to a 446 fold increment in K.sub.d for clone 6009F, while it leads to 310 for 6105F and 290 for 6103E.

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

As it shall be clear for those skilled in the art, other antibodies can be obtained by different combinations of the V.sub.H(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 V.sub.L(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) fragments. Accordingly, as long as these other antibodies retain the specific binding capacity to Cn2 toxin, they shall be considered within the scope of the present invention and thus shall be included in the term "antibodies of the present invention".

Similarly, it is clear for those skilled in the art that any of the antibodies of the present invention can be used as parent antibodies to generate further antibody variants. As long as these new antibody variants retain the specific binding capacity to Cn2 toxin, they shall be considered within the scope of the present invention and thus shall be considered functionally equivalents of the antibodies of the present invention.

Additionally, the V.sub.H(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 V.sub.L(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) antibody fragments clones can be used to generate not only the scFv antibody format but also any of the Fab, F(ab').sub.2 or even full length monoclonal antibody formats by the operably linked addition of part or the whole of the constant regions of the light and heavy chains, to be used in different applications.

IV. Expression of the Antibodies of the Present Invention

In general, antibodies of the invention may be produced by transformation of a suitable host cell with all or part of an antibody-encoding nucleic acid molecule or fragment thereof in a suitable expression vehicle.

Those skilled in the field of molecular biology will understand that any of a wide variety of expression systems may be used to provide the recombinant protein. The precise host cell used is not critical to the invention. An antibody of the invention may be produced in a prokaryotic host (e.g., E. coli) or in a eukaryotic host (e.g., Saccharomyces cerevisiae, insect cells, e.g., Sf21 cells, or mammalian cells, e.g., NIH 3T3, HeLa, or preferably COS cells). Such cells are available from a wide range of sources (e.g., the American Type Culture Collection, Rockland, Md.). The method of transformation or transfection and the choice of expression vehicle will depend on the host system selected and are well known to those skilled in the art. Expression vehicles may be chosen for instance from those provided, e.g., in Cloning Vectors: A Laboratory Manual (P. H. Pouwels et al). As shall be obvious for those skilled in the art, it will be important that the DNA sequences of the antibodies of the present invention are operably linked to the expression control sequences of the vector of the chosen expression system.

A variety of expression systems exists for the production of the antibodies of the present invention. Such vectors include, without limitation, chromosomal, episomal, and virus-derived vectors, e.g., vectors derived from bacterial plasmids, from bacteriophage, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses such as baculoviruses, papova viruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors derived from combinations thereof.

Particularly, for the purpose of providing enough material for the tests of the present invention, the antibodies were cloned in the vector PSYN1 and expressed in E. coli TG1 as detailed in example 7. But one particular bacterial expression system for antibody production is the E. coli pET expression system (Novagen, Inc., Madison, Wis.). According to this expression system, DNA encoding an antibody is inserted into a pET vector in an orientation designed to allow expression. Since the gene encoding such an antibody is under the control of the T7 regulatory signals, expression of the antibody is achieved by inducing the expression of T7 RNA polymerase in the host cell. This is typically achieved using host strains which express T7 RNA polymerase in response to IPTG induction. Once produced, recombinant antibody is then isolated according to standard methods known in the art.

Another bacterial expression system for antibody production is the pGEX expression system (Pharmacia). This system employs a GST gene fusion system which is designed for high-level expression of genes or gene fragments as fusion proteins with rapid purification and recovery of functional gene products. The protein of interest is fused to the carboxyl terminus of the glutathione S-transferase protein from Schistosoma japonicum and is readily purified from bacterial lysates by affinity chromatography using Glutathione Sepharose 4B. Fusion proteins can be recovered under mild conditions by elution with glutathione. Cleavage of the glutathione S-transferase domain from the fusion protein is facilitated by the presence of recognition sites for site-specific proteases upstream of this domain. For example, proteins expressed in pGEX-2T plasmids may be cleaved with thrombin; those expressed in pGEX-3X may be cleaved with factor Xa.

Once the recombinant antibody of the invention is expressed, it is isolated, e.g., using affinity chromatography. In one example, the antibodies were purified by Ni.sup.2+-NTA affinity chromatography (QIAGEN, Germany) (see example 7). In another example, toxin Cn2 may be attached to a column and used to isolate the recombinant antibodies. Lysis and fractionation of antibody-harboring cells prior to affinity chromatography may be performed by standard methods.

Once isolated, the recombinant antibody can, if desired, be further purified, e.g., 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.TM. 75 column (Pharmacia Biotech AB, Uppsala, Sweden) (see example 7).

Antibodies of the invention, can also be produced by chemical synthesis (e.g., by the methods described in Solid Phase Peptide Synthesis, 2 nd ed., 1984 The Pierce Chemical Co., Rockford, Ill.).

V. Comparing 6009F Antibody to BCF2 Monoclonal Antibody and Horse Polyclonal Antibody Fragments F(ab').sub.2

In order to know if the epitope recognized by clone 6009F was the same as the one recognized by BCF2, a displacement test using the Biacore was performed (FIG. 3, see Original Patent). As already mentioned, the monoclonal antibody BCF2 neutralizes toxin Cn2. The results showed that the F6009 antibody binds to Cn2 toxin in a site (epitope) different as the one recognized by monoclonal antibody BCF2. These results were confirmed by a competitive ELISA (FIG. 7, see Original Patent). See example 9 for major detail. It is important to mention that despite of being relatively small (66 amino acid residues), the toxin Cn2 seems to have several distinct epitopes (Zamudio, F., Saavedra, R., Martin, B. M., Gurrola-Briones, G., Herion, P. & Possani, L. D. (1992) Eur J Biochem 204, 281-92).

Some additional commentaries can be mentioned about the epitopes recognized by BCF2 and scFv 6009F. A dimeric scFv derived from BCF2 with an affinity constant of 75 pM (Juarez-Gonzalez, V. R. Riano-Umbarila, L. Quintero-Hernandez, V. Olamendi-Portugal, T. Ortiz-Leon, M. Ortiz, E. Possani, L. D. Becerril, B. (2005) J Mol Biol 346, 1287-1297.), was capable of neutralizing 1 DL50 of Cn2 toxin at a molar ratio 1:10 (toxin:scFv). However, a mild envenomation symptom was observed. These results suggest a differential effect of the toxin on its target (sodium channels) depending on the blocked epitope. This could explain why the clone 6009F with a lower affinity (200 pM) and at a lower molar ratio (1:5), was capable to completely neutralize 2 DL.sub.50 of the toxin without any symptom of intoxication observed. This indicates that the antibody 6009F is more efficient to block the interaction of the toxin with the channel as compared to matured scFv of BCF2; therefore the epitope recognized by 6009F seems to be more relevant than the recognized by BCF2. Similar results have been reported in other systems (Amersdorfer, P., Wong, C., Smith, T., Chen, S., Deshpande, S., Sheridan, R. & Marks, J. D. (2002) Vaccine 20, 1640-8).

Furthermore, as detailed in example 9, a displacement test using the Biacore was performed with scFv 6009F and a commercially available anti scorpion antivenom, Alacramyn from Instituto Bioclon S. A. de C. V. (Mexico), a polyclonal pool of horse antibody fragments (F(ab').sub.2) to determine if the epitope recognized by the scFv 6009F was also recognized by any of the antibody fragments present in the antivenom. The results (FIG. 9, see Original Patent) 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 suggest that the epitopes of the toxin Cn2 were not exactly the same when it was exposed to the naturally occurring immune system of the animals (mouse in the case of BCF2 or horses in the case of Alacramyn) than when it was exposed to our in vitro immune system.

VI. Use of the Antibodies of the Present Invention as Antivenoms

As shown in example 8, in contrast to variants 6F and 610A, antibody variant 6009F is able to neutralize toxin Cn2 (it is capable of neutralizing 2 LD50 of Cn2 toxin when a molar ratio of 1:5 is used, or 2 LD50 of whole venom). Accordingly, it can be used as a component of a pharmaceutical composition to treat animal and people in need of such treatment as consequence of being stung by a scorpion, particularly if the scorpion is determined to be Centruroides noxius. The pharmaceutical composition can comprise additionally other antibodies for instance polyclonal antibodies from horse or goat raised against scorpion venoms or other human or humanized antibodies against other toxins from C. noxius venom or against other toxin from the venom of different scorpions. The composition can additionally comprise several pharmaceutically acceptable carriers.

A method to treat an individual in need of such treatment, for instance after being stung by a scorpion, specially if the scorpion is a C. noxius scorpion, comprises the parenteral administration of said pharmaceutical composition which comprises the antibody 6009F 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, particularly 6009F can be used in a composition comprising the antibodies of the present invention adhered to a solid phase substrate such as glass (e.g. controlled pore glass), polysaccharides (e.g., agarose), polyacrylamides, polystyrene, polyvinyl alcohol, silicones, sepharose, carboximetil cellulose and nitrocellulose, in such a way that the composition can bind the Cn2 toxin, whether free or as part of C. noxius venom or as a contaminant of a blood or serum sample. In certain embodiments, depending on the context, the solid phase can comprise the well of an assay plate; in others it is a purification column (e.g. an affinity chromatography column) and in others it is part of a diagnostic kit. This term also includes a discontinuous solid phase of discrete particles, such as those described in U.S. Pat. No. 4,275,149. Then another embodiment of the present invention relates to a solid phase that comprises the antibodies 3F, 6F, 610A or preferably 6009F, 6105F or 6103E.

VIII. Use of Antibodies and or Solid Phase of the Present Invention in a Diagnostic System

There are some immunodiagnostic techniques available in the art, which commonly use specific antibodies to detect the presence of a particular antigen in a sample. For instance the Enzymatic Linked Immuno System Assay (ELISA) and Immunochromatographic Assay (hereinafter referred to as "ICA").

ICA is also referred to as "rapid test" due to its rapidity and simplicity. In such assay, tracer antibody molecules conjugated with gold particles bind to a particular antigen contained in a serum sample, after which the formed complexes pass through microspores of nitrocellulose (NC) membrane in terms of capillary phenomenon. The complexes finally bind to capture antibodies immobilized on the inner surface of microspore of the NC membrane and develop color of a positive line, whereby determining easily the existence of a particular antigen in the serum sample with the naked eye.

As noted above, the ICA, owing to simplicity of procedure and rapidity of the running result, has been widely used for the detection of various analytes such as antigens (Sato, K. et al. (1996) J Clin Microbiol 34, 1420-1423).

There are two major constituents in the ICA kit. One is the nitrocellulose membrane which has two invisible lines on the surface and the other is a glass fiber filter containing antibody-gold particle conjugates in a dry state on the surface. Two kinds of antibodies, that is, the monoclonal antibody being specific to antigen to be detected and Goat anti-mouse IgG, are immobilized on the lower line and the upper line of the nitrocellulose membrane, respectively.

A sample is added to a sample well of the ICA kit and then the antibody-gold particle conjugates on the surface of the NC membrane in a dry state are re-hydrated and then bound to antigens in the serum sample, after which the formed complexes pass through microspores of the NC membrane in terms of capillary phenomenon.

Thereinafter, the antigens of the complexes are reacted with the monoclonal antibodies immobilized on the lower line, resulting in developing a color. In addition, the upper line develops a color because the Goat anti-mouse IgG immobilized on the upper line may react with the antibody-gold particle conjugates although no antigen is present, thus the upper line always develops a color in each run of the test and may serve as a control line. In other words, when antigens exist in the serum sample, both the positive line and the control line of the ICA kit become visible. However, only the control line becomes visible, when no antigen is present.

The antibodies of the present invention, preferably 6009F, 6105F or 6103E, whether free or incorporated to the above described solid phase, can be used as part of a diagnostic kit to detect the presence or absence of the toxin Cn2 in a sample. They can be used as part of an ELISA or as part of an ICA. Polyclonal antibodies Goat anti-mouse shall be substituted by polyclonal antibodies anti-human, for instance Goat anti-human in order to use the antibodies of the present inventions.

Accordingly, another embodiment of the present invention relates to an immunodiagnostic kit comprising antibodies 3F, 6F, 610A or preferably 6009F, 6105F or 6103E. The Immunodiagnostic kit can be an ELISA or an Immunochromatographic Assay.

IX. The Use of Antibody 6009F to Treat the Envenomation with C. noxius Venom

Since antibody variant 6009F neutralizes the lethal effect of toxin Cn2 and whole C. noxius venom, as it is clearly shown in example 8, antibody variant 6009F and/or any functionally equivalent variants thereof (i.e. antibody variants of 6009F that neutralize the lethal effect of toxin Cn2 and C. noxius whole venom) may be used as part of a pharmaceutical composition to treat a patient that has been stung a scorpion, particularly if the scorpion is C. noxius. The pharmaceutical composition may include other antibodies like those horse polyclonal F(ab').sub.2 fragments already used as antivenoms. In this case the addition of antibody 6009F and/or functionally equivalent variants thereof is to strengthen its neutralizing effect, particularly against the C. noxius venom. Optionally, the pharmaceutical composition may also include a pharmaceutically acceptable carrier like those already mentioned.

Accordingly, another embodiment of the present invention is related to a pharmaceutical composition comprising the antibody 6009F, 6105F or 6103E and/or any functionally equivalent variant thereof to treat envenomation by a scorpion, particularly C. noxius scorpion.

In another embodiment, the present invention relates to a method to treat a mammal in need of such treatment, particularly patients who had been stung by a scorpion, especially if scorpion is C. noxius, comprising the step of administering a pharmaceutically effective amount of a pharmaceutical composition comprising the antibody variant 6009F, 6105F or 6103E and/or functionally equivalent variants thereof. The pharmaceutical composition can be administered locally and/or systemically through the conventional routes such as the intravenous, subcutaneous, intramuscular, intravaginal, intraperitoneal, intranasal, oral or other mucous routes to protect the patient against the lethal effect of toxin Cn2 of the venom of scorpions Centruroides.

X. Modified Biopanning Method for the Selection of Improved Clones

Standard biopanning method for selection of the affinity improved phage-antibody variants from a library obtained by a mutagenesis cycle generally include the steps of: 1) incubating the library in the presence of the target binding antigen, previously adhered to a solid phase like the immunotube (Nunc; Maxisorp), during a time to allow specific clones to bind to the immobilized objective antigen; 2) Extensively washing with PBS-Tween20 (1.times., 0-1%) and PBS to remove nonspecific phages-antibodies, and 3) recovering the bound phage-antibodies by the addition of either weak acid or base solution or a suspension of cells, particularly the cell strain the one that is the target of the phage used to phage-display the antibody repertory. Several rounds are commonly carried out to increase the number of positive clones to be screened. The resulting mutant clones of these standard procedures in general slightly improve the affinity of the clone. Commonly, standard procedures to evolve antibodies include several rounds of mutagenesis cycles followed by biopanning, before obtaining a satisfactory affinity improved clone.

In order to select antibody variants improved not only in its affinity but in its stability too, the inventors of the present invention have modified the standard procedure. Those modifications were crucial for the selection of a variety of improved antibody variants of clone 3F.

It is well know to those in the art, that the increase of the temperature during the incubation period increase the speed to which the bonds between the antibodies and the target antigen are formed and dissolved, helping the selection of strongly bond antibodies and the washing of weakly bond antibodies. Similarly, an increase in the time period of incubation tends to help to select strongly bond antibodies.

Additionally, it is a common practice to use weak acid or weak bases solutions to recover the bond antibodies. In the present invention, the inventors decided to take advantage of the above mentioned increase in the temperature and time of the period of incubation together with a diminution in the amount of the target antigen used to coat the immunotube (to increase the stringency of the biopannig), and additionally, they took the risk of detach the antibodies recovered through the use of a weak base and seek if there were any antibody that remain bond to the target antigen in the immunotube. Contrary to what it was expected, as is shown in examples 5 and 6, after a further recovery step with cells suspension, several antibodies were recovered from the immunotube. Those antibodies were improved in both affinity and stability, in respect to the parent antibody 610A, as compared with the antibodies recovered using the standard procedures which were only slightly improved in its affinity.

The method of the present invention to select antibody variants improved in affinity and stability, from a mutagenized antibody library comprises the steps of:

1) Incubating the library in the presence of the target binding antigen, previously adhered to a solid phase like the immunotube (for instance Nunc; Maxisorp), during a time to allow specific variants to bind to the immobilized target antigen at a temperature of at least (30.degree. C.), preferably 37.degree. C. and for a period of at least 5 Hr.

2) Extensively washing with PBS-Tween20 (1.times., 0.1%) and PBS to remove nonspecific phages-antibodies,

3) Washing with a weak acid or weak base solution (for instance 100 mM triethylamine) to remove the nonspecific and less stable or low binding phage-antibodies, follow by a neutralization; and

4) Recovering the bound phage-antibodies by the addition of a suspension of cells, particularly the cell strain that is the target of the phage used to phage-display the antibody repertory.

Optionally, the method can comprise an additional step of incubating the library in the presence of blocking agents before to the biopanning in order to eliminate as much as possible unspecific clones. The blocking agents are well known to those in the art and can include BSA, milk or gelatin for instance.

When used this modified method in comparison to the standard method to select the mutated clones, after a mutagenesis cycle of antibody variant 610A, the standard method of phage selection gave a lower number of positive variants (2 with medium signal of a total 88) as compared to the more stringent modified method (5 with high signal of a total 88) see FIGS. 8 and 2 (see Original Patent) more in a further procedure (2 with high signal of a total 88). The number of nucleotide changes in the selected clones from the two procedures was different. The clones selected from the standard procedure had a lower number of changes (usually one), while using the stringent strategy, the selected clones showed 2-6 changes. The affinity and stability of these 7 (5+2) clones was better than those of the 2 clones recovered by the standard method.
 

Claim 1 of 30 Claims

1. A composition comprising a human antibody that specifically recognizes the toxin Cn2, wherein said human antibody comprises a V.sub.H and a V.sub.L, and further wherein said V.sub.H and V.sub.L pairs are selected from the group consisting of: (a) SEQ ID NO: 45 and SEQ ID NO: 47, (b) SEQ ID NO: 89 and SEQ ID NO: 91, and (c) SEQ ID NO: 95 and SEQ ID NO: 97.

____________________________________________
If you want to learn more about this patent, please go directly to the U.S. Patent and Trademark Office Web site to access the full patent.

 

 

     
[ Outsourcing Guide ] [ Cont. Education ] [ Software/Reports ] [ Training Courses ]
[ Web Seminars ] [ Jobs ] [ Consultants ] [ Buyer's Guide ] [ Advertiser Info ]

[ Home ] [ Pharm Patents / Licensing ] [ Pharm News ] [ Federal Register ]
[ Pharm Stocks ] [ FDA Links ] [ FDA Warning Letters ] [ FDA Doc/cGMP ]
[ Pharm/Biotech Events ] [ Newsletter Subscription ] [ Web Links ] [ Suggestions ]
[ Site Map ]