MXPA00011434A - Humanized antibodies that recognize verotoxin ii and cell line producing same - Google Patents

Abstract

The invention provides humanized antibodies that specifically bind to, and preferably, neutralize, verotoxin II (VT2). The antibodies are useful for treating patients suffering from, or at risk of suffering, toxic effects of verotoxin.

Description

HUMANIZED ANTIBODIES THAT RECOGNIZE VEROTOXIN II AND CELLULAR LINE THAT PRODUCES THE SAME FIELD OF THE INVENTION The present invention relates generally to the combination of recombinant DNA and monoclonal antibody technologies to develop novel biological products and, more particularly, for example, for the production of non-immunogenic immunoglobulins (in humans). specific for the antigen of Verotoxin II (VT2) and variant antigens of Verotoxin II (VT2V) and its uses in vi tro and in vi vo. The present invention also relates more specifically to humanized monoclonal antibodies against VT2 which are capable of neutralizing VT2 and VT2V, polynucleotide sequences encoding antibodies, a method for producing antibodies, pharmaceutical compositions containing the antibody as an active ingredient, therapeutic agents to treat E infection. coli that produces Verotoxin (VTEC) and the Hemolytic Uremic Syndrome (HUS) that comprise the antibody as an active ingredient, and with methods to treat such diseases.

BACKGROUND OF THE INVENTION It is known that Verotoxin (VT), also known as Shiga-like toxin (SLT), can cause hemorrhagic diarrhea and the development of hemolytic uremic syndrome in E. coli infection that produces Verotoxin (VTEC). One of the etiological agents of VTEC infection is virulent E. coli 0157. VT can also be produced by bacteria other than those that cause VTEC infection which can also result in a toxic syndrome in humans. In children or older adults with reduced immune responses, VT produced by bacteria that grow in the intestines can reach blood flow by breaking intestinal epithelial cells. This can induce Hemolytic Uremic Syndrome (HUS) which is characterized by renal dysfunction and sometimes brain damage (see, for example, M. et al., The Lancet, 1, 619-620 (1983); Siegler, R. , The Journal of Pediactrics, 125, 511-518 (1994)). To date, there is no effective drug for these toxic syndromes. Antibiotics have not shown efficacy in preventing the progress of toxic syndromes (see for example, Carter, A. et al., The New England Journal of Medicine, 316, 1496-1500 (1987); Griffen, P. et al., Annals of Interna! Medicine, 109, 705-712 (1988)). This may be due to the release of VT from killed bacteria by antibiotics and the ineffectiveness of antibiotics against VT. There are two types of Verotoxin, (or toxin similar to Shiga) Verotoxin I (VT1 or SLT-1) and Verotoxin II (VT2 or SLT-2). (See, O 'Brien et al., Curr. Top, Microbiol. Immunol, 180, '65 -94, (1992)). The E. coli that produces VT2 has been isolated from patients suffering from VTEC infection. (See, Russmann et al., J. Med. Microbiol., 40 (5), 338-343 (1994)). Ostroff et al., J. Infect. Dis. 160, 994-998 (1989) and Kleanthous et al., Arch. Dis. Child 65, 722-727 (1990) reports that strains of E. coli 0157 that contained VT2 but not VT1 were more frequently associated with HUS. There are additional variants of VT2 (VT2V) that have also been clinically isolated. (See, for example, Microb. Pathog., 8, 47-60 (1990), FEBS Lett., 79, 27-30 (1991), Microb. Pathoq., 5, 419-426 (1988), and J. Bacteriol., 170, 4223-4230 (1988)). Armstrong et al., J. Infect. Dis., 171 (4), 1042-1045 (1995), have tested an absorber of VT in clinical trials, however, this drug works only in the intestine and is not available to absorb the VT that has reached the blood flow. A β-globulin preparation showed a very low neutralization activity for VT compared to one of VT1 (Ashkenazi, S. et al., The Journal of Pediatrics, 113, 1008-1014 (1988); Morooka, T. et al., Acedia Paediatrica Japónica, 38, 294-295 (1996)). A mouse monoclonal antibody that neutralizes VT2 has been reported. Nevertheless, it was reported that this antibody shows a relatively low binding affinity for VT2V (, Schmitt, C. et al., Infection and I munity, 59, 1065-1073 (1991)). In addition, the use of murine monoclonal antibodies such as those described above have certain disadvantages in the treatment of humans, particularly in repeated therapeutic regimens as explained below. Mouse monoclonal antibodies, for example, tend to have a short half-life in humans, and lack other important functional characteristics of an immunoglobulin when used in humans. In addition, murine monoclonal antibodies contain substantial amino acid sequences that become immunogenic when injected into a human patient. Numerous studies have shown that, after injection of an external antibody, the immune response produced by a patient against the injected antibody can be very strong, essentially eliminating the therapeutic utility of the antibody after initial treatment. In addition, if antigenic monoclonal antibodies (for humans) from mouse or others are used to treat different human diseases, subsequent treatments with unrelated mouse antibodies may not be effective or even dangerous per se, due to cross-reactivity. Although the production of so-called "chimeric antibodies" (eg, mouse variable regions linked to human constant regions) has proven to be somewhat successful, a significant immunogenicity problem remains. In general, the production of human immunoglobulins reactive with the VT2 antigen with high affinity, as with many antigens, would be extremely difficult using typical human monoclonal antibody production techniques. Thus, there is a need for improved forms of humanized immunoglobulins specific for the VT2 antigen that are not substantially immunogenic in humans, since they can be produced easily and inexpensively in a form suitable for formulating therapeutic agents and other uses. The present invention satisfies these and other needs.

BRIEF DESCRIPTION OF THE INVENTION The invention provides humanized antibodies that bind specifically to VT2 and / or the variant of the VT2. Some of such antibodies bind specifically to subunit B of VT2 and / or subunit B of the variant of VT2. Some of these antibodies neutralize VT2 and / or the variant of VT2. Preferred antibodies neutralized VT2 and / or variant of VT2 to provide at least 50%, 75%, 95% and 100% protection against mice or other mammalian subjects challenged with an LD50 of 10 of VT2 or variant of VT2 . Some humanized antibodies are a humanized form of the mouse antibody VTml-1, the mouse antibody is characterized by a light chain variable region shown in Figure IB and a heavy chain variable region shown in Figure IA. The invention further provides antibodies that compete with the mouse antibody VTml-1 for specific binding to VT2 and / or variant of VT2. Some of the humanized antibodies, as described above, comprise regions that determine the complementarity of the mouse VTml-1 antibody and the heavy and light chain variable region structures of the heavy and light chain structures of the human GF4 antibody , provided that at least one position selected from a group consisting of L49, H29, H30, H49 and H98, is occupied by the amino acid present in the equivalent position of the heavy or light chain variable region of the VTml- antibody. 1 of mouse. Such humanized antibodies bind specifically to Verotoxin II with an affinity constant of between 107 M_1 and three, five or ten times the affinity of the mouse VTml-1 antibody. In some humanized antibodies described in the previous paragraph, each position selected from the group consisting of L49, H29, H30, H49 and H98 is occupied by the amino acid present in the equivalent position of the structure of the heavy or light chain variable region. of the mouse VTml-1 antibody. In some of the above humanized antibodies, at least one position selected from the group of L3, L4, L19, L76, L79, L85, H1, H4, H5, H79, H89 and H93 is occupied by an amino acid present in the equivalent position of a consensus sequence of a heavy chain of the human antibody. In some humanized antibodies each position selected from the group L3, L4, L19, L76, L79, L85, H1, H4, H5, H79, H89 and H93 is occupied by an amino acid present in the equivalent position of a consensus sequence of a heavy or light chain of the human antibody. Some humanized antibodies comprise a heavy chain variable region shown in Figure 2A and a light chain variable region shown in Figure 2B provided that one or more positions selected from the group consisting of L3, L4, L19, L76, L79, L85 , Hl, H4, H5, H79, H89 and H93 can be substituted as shown in Tables 2 and 3. Some humanized antibodies comprise a heavy chain variable region shown in Figure 2A and a variable region of light chain shown in Figure 2A. Figure 2B. Some humanized antibodies comprise a humanized heavy chain having an identity of at least 85% with the humanized heavy chain shown in Figure 2A and a humanized light chain having at least a sequence identity of 85% with the humanized light chain shown in Figure 2B, provided that at least one position selected from the group consisting of L49, H29, H30, H49 and H98, is occupied by the amino acid present in the equivalent position of the variable region structure of the heavy or light chain of the mouse VTml-1.4 antibody. Some of the humanized antibodies as described above comprise two pairs of light / heavy chain dimers where each chain comprises a variable region and a constant region. Some of the humanized antibodies as described above are a Fab fragment or an F (ab ') 2. Optionally, the humanized antibodies, as described above, are provided in purified form.

Some humanized antibodies, as described above, have an immunoglobulin IgG-isotype. The invention further provides methods for producing humanized VTml-1 antibody. Such methods comprise culturing a cell line, which codes for the heavy and light chains of any of the antibodies described above, whereby the humanized antibody is expressed; and recovered the humanized antibody expressed by the cell line. Some such methods further comprise mixing the antibody with a pharmaceutically acceptable carrier to produce a pharmaceutical composition. The invention further provides pharmaceutical compositions comprising any of the antibodies described above and a pharmaceutically acceptable carrier. One such preferred composition comprises a humanized antibody comprising a heavy chain variable region shown in Figure 2A and a light chain variable region shown in Figure 2B. In the invention it is further provided for the use of any of the antibodies described above in the manufacture of a medicament for the treatment of a patient suffering from or at risk of the toxic effects of a verotoxin. The invention further provides methods for treating a patient suffering from or at risk of the toxic effects of a verotoxin, which comprises administering to the patient an effective dose of a human or humanized antibody that specifically binds to verotoxin II and / or a variant of verotoxin II. In such methods, the antibody competes with the mouse antibody VTml-1 for specific binding to verotoxin II or a variant of verotoxin II. In some such methods, the humanized antibody binds specifically to VT2 and / or a variant of VT2. In some such methods, the humanized antibody binds specifically to subunit B of VT2 and / or variant of VT2. In some such methods, the humanized antibody binds specifically to VT2 and / or a variant of VT2 and neutralizes VT2 and / or a variant of VT2. In some such methods, the humanized antibody binds specifically to subunit B of VT2 and / or subunit B of the variant of VT2 and neutralizes VT2 and / or a variant of VT2. In some such methods the antibody is a humanized antibody, which is a humanized form of the mouse VTml-1 antibody. In some such methods, the antibody is a humanized antibody comprising a heavy chain variable region shown in Figure 2A and a light chain variable region shown in Figure 2B. In some such methods, the patient infected with E. coli produces verotoxin and the antibody is administered therapeutically. In some such methods, the patient is at risk for E. coli infection that produces verotoxin and the antibody is administered prophylactically. Some of these methods also include checking the recovery of the patient from the toxic effects of verotoxin II or a variant of verotoxin II. In a further aspect, the invention provides a cell line which produces any of the antibodies described above. The present invention provides novel compositions useful, for example, the treatment of verotoxin-producing E. coli infection (VTEC) and the Hemolytic Uremic Syndrome (HUS), the compositions containing humanized immunoglobulins capable of specifically binding to the B subunit of the antigen of VT2 and of neutralizing VT2 and variants of VT2. The immunoglobulins can have two pairs of light chain / heavy chain complexes, at least one chain comprises one or more regions that determine the complementarity of the mouse functionally linked to the segments of the region of the human structure. For example, regions that determine the complementarity of the mouse with or without residual mouse amino acids additionally naturally associated in regions of the human structure can be introduced to produce humanized immunoglobulins capable of binding the antigen at stronger affinity levels of approximately 107 M "1. These humanized immunoglobulins will also be able to block the binding of monoclonal antibody from CDR donor mice to VT2., including the binding fragments and other derivatives thereof, of the present invention can be easily produced by a variety of recombinant DNA techniques, with the final expression in transfected cells, preferably immortalized eukaryotic cells, such as myeloma or hybridoma. The polynucleotides comprise a first sequence coding for the regions of the structure of the humanized immunoglobulin and a second set of sequences coding for the regions determining the complementarity of the desired immunoglobulin or synthetically produced by a combination of the cDNA segments and Appropriate genomic DNA. Humanized immunoglobulins can be used in substantially pure form in the treatment of potentially toxic results of VT2 or VT2V such as those produced during infection by verotoxin-producing E. coli (VTEC) and Hemolytic Uremic Syndrome (HUS). The humanized immunoglobulins or their complexes can be prepared in a pharmaceutically acceptable dosage form, which will vary depending on the mode of administration.

BRIEF DESCRIPTION OF THE FIGURES Figure 1. The cDNA and the translated amino acid sequences of the heavy chain (A) and light chain variable regions (B) the mouse VTml .1 antibody (MuVTml.l). The regions that determine complementarity (CDR) are underlined and the first amino acids of the mature chains are doubly underlined. Figure 2. The cDNA and translated amino acid sequences of the heavy chain (A) and light chain (B) variable regions of the humanized VTml .1 antibody (HuVTml.l). The regions that determine complementarity (CDR) are underlined and the first amino acids of the mature chains are doubly underlined. Figure 3. Scheme for the synthesis of cDNA of the variable region of the humanized antibody. Figure 4. Competitive antibody binding MuVTml.l and HuVTml .1 to verotoxin II (VT2) of E. coli. Increasing concentrations of competing antibodies were tested with coated VT2 in the presence of biotinylated tracer MuVTml .1. Figure 5. Neutralization activity in vi tro of the HuVTml.l versus the MuVTml .1. Figure 6. Identification of the recognized antigen (subunit B of VT2) of HuVTml .1.

Figure 7. Neutralization activity of MuVTml .1 against VT2 and variant of VT2.

DEFINITIONS The phrase "substantially identical" in the context of nucleic acids or polypeptides (e.g., DNA encoding a humanized immunoglobulin or the amino acid sequence of humanized immunoglobulin) refers to two or more sequences or subsequences that have an identity. of at least 80%, preferably 85%, 90-95% or greater residual nucleotides or amino acids when compared and aligned for maximum correspondence, as measured using the following method of sequence comparison and / or inspection visual. Such "substantially identical" sequences are typically considered homologous. Preferably, the "substantial identity" exists over a region of the sequence that is at least 50 residues in length, more preferably over a region of at least about 100 residues, and most preferably the sequences are substantially identical over at least about 150 residues, or over the entire length of the two sequences to be compared.As described below, any two antibody sequences can be aligned only one way, using the Kabat feeding system. , for antibodies, the percent identity has a unique and well-defined meaning The amino acid sequences can be variables of the mature heavy and light chains of immunoglobulins are designated as Hx and Lx respectively, where x is a number designating the position of an amino acid according to the Kabat scheme, the Protein Sequences of Immunological Interest (National I nstitutes o Health, Bethesda MD, 1987 and 1991). Kabat lists many amino acid sequences for antibodies for each subgroup, and lists the amino acids that occur most frequently for each residue position in that subgroup to generate a consensus sequence. Kabat uses a method to assign a residue number to each amino acid in a listed sequence, and this method for evaluating the number of residues has become standard in the field. The Kabat scheme is extendable to other antibodies not included in the compendium by measuring the alignment of the antibody in question with one of the consensus sequences in Kabat with reference to the conserved amino acids. For example, an amino acid at the L50 position of the human antibody occupies the position equivalent to an amino acid at the L50 position of a mouse antibody. Conservative amino acid substitutions refer to the interchangeability of residues having similar side chains. For example, a group of amino acids that have aliphatic side chains is glycine, alanine, valine, leucine and isoleucine.; a group of amino acids that has aliphatic hydroxyl side chains is that of serine and threonine; a group of amino acids having side chains that have amide is asparagine and glutamine; a group of amino acids that have aromatic side chains and that of phenylalanine, tyrosine and tryptophan; a group of amino acids that has basic side chains is that of lysine, arginine, and histidine; A group of amino acids that have side chains that contain sulfur is that of cysteine and methionine. Preferred conservative amino acid substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine. Analogs of the exemplified antibody sequences can be separated by retention of binding activity using phage display methods according to those described by, for example, Cesareni, FEBS Lett 307: 66-70 (1992); Swimmer et al., Proc. Nati Acad. Sci. USA 89: 3756-60 (1992); Gram et al., Proc. Nati Acad. Sci. 89: 3576-80 (1992); Clakson et al., Nature 352; 624-8 (1991); Scott & Smith, Science 249: 386-90 (1990); Garrard et al., Bio / Techniques 9: 1373-1377 (1991), which were incorporated herein by reference in their entirety for all purposes. The term "substantially pure" means that an object species is the predominant species present (ie, on a molar basis, it is more abundant than any other individual species in the composition), and preferably a substantially purified composition is a composition where the target species comprises at least about 50% (on a molar basis) of all molecular species present. Generally, a substantially pure composition comprises no more than about 80 to 90 percent of all macromolecular species present in the composition. More preferably, the target species are purified essentially to homogeneity (contaminating species can not be detected in the composition by conventional detection methods),. where the composition consists essentially of a single macromolecular species. The competition between the antibodies is determined by an assay in which the immunoglobulin under test inhibits the specific binding of a reference antibody to an antigenic determinant. Numerous types of competitive binding assays are known, for example: direct or indirect solid phase radioimmunoassay (RIA), direct or indirect solid phase enzyme immunoassay (EIA), sandwich competition assay (see Sthali et al., Methods in Enzymology 9: 242-253 (1983)); EIA biotin-direct avidin in solid phase (see Kirkland et al., J. Immunol. 137: 3614-3619 (1986)); direct-label solid-phase assay, direct sandwich assay with direct labeling in solid phase (see Harlow and Lane, "Antibodies, A Laboratory Manual", Cold Spring Harbor Press' (1988)); RIA direct labeling in solid phase using as label 1-125 (see Morel et al., Molec.Immunol. 25 (1): 7-15 (1988)); RIA with direct biotin-avidin in solid phase (Cheung et al., Virology 176: 546-552 (1990)); and direct labeled RIA (Moldenhauer et al., Scand., J. Immunol., 32: 77-82 (1990)). Usually the test immunoglobulin is present in excess. Antibodies identified by competitive assay (competitive antibodies) include antibodies that bind to the same epitope as the reference antibody and antibodies that bind to an adjacent epitope sufficiently close to the epitope bound by the reference antibody for spherical hindrance to occur. Usually, when an excess competitive antibody is present, it will inhibit the specific binding of a reference antibody to an antigenic target designated by at least 50 or 75%.

DETAILED DESCRIPTION OF THE INVENTION In accordance with the present invention, humanized immunoglobulins that specifically react with the subunit of VT2. These immunoglobulins, which have binding affinities with the B subunit of VT2 of at least 107 M "1 to 1010 M" 1, preferably 1018 M "1 to 1010 M" 1 or stronger, are capable of, neutralize the toxicity of VT2 and VT2V (the antigens of VT2). The humanized immunoglobulins will have a human structure and will have one or more regions that determine the complementarity (CDR) of an immunoglobulin, typically a mouse immunoglobulin, specifically reactive to the VT2 antigens. In a preferred embodiment, one or more of the CDRs will prevent the MuVtml antibody .1. Thus, the immunoglobulins of the present invention, which can be produced economically in large quantities, find use, for example, in the treatment of the toxic results of E. coli infection that produces Verotoxin (VTEC) and the Hemolytic uremic syndrome (HUS) in human patients by a variety of techniques. It is known that the structural unit of the basic antibody comprises a tetramer. Each tetramer is composed of two identical pairs of two pairs of polypeptide chains. Each pair has a "light" chain (approximately 25 kD) and a "heavy" chain (approximately 50-70 kD). The amino terminal portion of each chain includes the variable region of approximately 100 to 110 amno acids primarily responsible for the recognition of the antigen. The carboxyterminal portion of each chain defines a constant region mainly responsible for the effector region. Light chains are classified as kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta or 'epsilon, and define the isotype of the antibody as IgG, IgM, IgA, IgD and IgE, respectively. Within the heavy and light chains, the variable and constant regions are linked by a "J" region and approximately 12 or more amino acids, with the heavy chain also including a "D" region or approximately 10 or more amino acids. (See, generally, Fundamental Immunology, Paul, W., Ed., Chapter 7, pp. 131-136, Raven Press, N.Y. (1984), which is incorporated herein by reference). The variable regions of each pair of light / heavy chains form the binding site of the antibody. All chains exhibit the same general structure of relatively conserved structural regions linked by three hypervariable regions, also known as complementarity determining regions or CDRs (see, "Sequences of proteins of Immunological Interest", Kabat, E., et al., US Department of Health and Human Services, (1987); and Chothia and Lesk, J. Mol. Biol., 196, 901-917 (1987), which are incorporated herein by reference). The CDRs of the two chains of each pair are aligned by the structural regions, allowing the binding to a specific epitope. As used herein, the term "immunoglobulin" refers to a protein that consists of one or more polypeptides substantially encoded by immunoglobulin genes. The recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as variable region genes of immunoglobulins. Immunoglobulins can exist in a variety of forms in addition to antibodies; including, for example, Fv, Fab, and F (ab ') 2 as well as bifunctional hybrid antibodies (for example, Lanzavecchia et al., Eur. J. Immunol., 17, 105 (1987)) and in single chains (e.g. , Houston et al., Proc. Nati, Acad. Sci. USA, 85, 5879-5883 (1988) and Bird et al., Science 242, 423-426 (1988), which are incorporated herein by reference). (See, generally, Hood et al., Immunology, Benjamin, NY, 2nd ed. (1984), Harlow and Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory (1988) and Hunkapiller and Hood, Nature, 323 , 15-16 (1986), which are incorporated herein by reference.). Chimeric antibodies are antibodies whose light and heavy chain genes have been constructed, typically by genetic engineering, from segments of immunoglobulin genes belonging to different species. For example, the variable segments (V) of the genes of a mouse monoclonal antibody can be linked to human constant segments (C), such as? I and? 3. A typical therapeutic chimeric antibody is thus a hybrid protein consisting of the V binding domain or the antigen of a mouse antibody and the C domain or effector of a human antibody, although other mammalian species may be used. As used herein, the term "structural region" refers to those portions of the variable regions of the immunoglobulin light and heavy chain that are relatively conserved (ie, other than the CDRs) among the different immunoglobulins in a single species. , as defined by Kabat, et al., op. cit. As used herein, a "human-structural region" is a structural region that is substantially identical (approximately 85% or more) to the structural region of a natural human antibody. As used herein, the term "humanized immunoglobulin" refers to an immunoglobulin comprising a human structure, at least one CDR of a non-human antibody, and in which any constant region present is substantially identical to a constant region of human immunoglobulin. , that is, at least about 85-90%, preferably at least 95% identical. Accordingly, all parts of a humanized immunoglobulin, except possibly the CDR, are substantially identical to the corresponding parts of one or more native human immunoglobulin sequences. For example, a humanized immunoglobulin encompasses a mouse variable region antibody and / or human constant region. The 'humanized antibodies have at least three potential advantages over the mouse and in some cases the chimeric antibodies for use in human therapy: Because the effector portion is human, it can interact better with the other parts of the human immune system (e.g. , destroy the target cells more efficiently by complement-dependent cytotoxicity (CDC) or antibody-dependent cellular cytotoxicity (ADCC)). The human immune system would not recognize the structure or C region of the humanized antibody as being foreign, and therefore the response of the antibody against such injected antibody would be less than against a totally foreign mouse antibody or a partially foreign chimeric antibody. It has been reported that injected mouse antibodies have a half-life in the human circulation much shorter than the half-life than normal antibodies (Shaw, D. et al., J. Im unol, 138, 4534-4538 (1987)). The humanized antibodies injected presumably will have a half life essentially identical to natural human antibodies, allowing smaller and less frequent doses to be given. In one aspect, the present invention is directed to recombinant polynucleotides encoding the heavy and / or light chain CDRs of an immunoglobulin capable of binding to subunit B of VT2, such as the monoclonal antibody MuVTml .1. The polynucleotides that code for those regions will typically bind polynucleotides that code for the appropriate human framework regions. As with the human structural region, an amino acid sequence of the structural or variable region of a non-human immunoglobulin that provides CDR is compared to the corresponding sequences in a human immunoglobulin sequence connection, and a sequence having high homology is selected. Exemplary polynucleotides, which upon expression encode the polypeptide chains comprising the CDR of the heavy and light chain of the monoclonal antibody MuVTml .1 are included in Figure 1. Due to codon degeneracy and non-critical amino acid substitutions, other polynucleotide sequences can be easily substituted by those sequences, as detailed below. The design of humanized immunoglobulins can be carried out as follows. When an amino acid falls under the following category, the structural amino acid of a human immunoglobulin to be used (acceptor immunoglobulin) is replaced by a structural amino acid of a human immunoglobulin that does not provide CDR (donor immunoglobulin): the amino acid in the human structural region of acceptor immunoglobulyria is unusual for human immunoglobulin in that position, whereas the corresponding amino acid in donor immunoglobulin is typical for human immunoglobulin in that position; the position of the amino acids is immediately adjacent to one of the CDRs; or the amino acid is within about 38 of a CDR in an immunoglobulin model of tertiary structure (see, Queen et al., op.cit., and Co et al., Proc.

Nati Acad. Sci. USA 88, 2869 (1991), respectively, both of which are incorporated herein by reference). When each of the amino acids in the human structural region of the immunoglobulin receptor and a corresponding amino acid in the donor immunoglobulin is unusual for the human immunoglobulin at that position, such an amino acid is replaced by an amino acid typical for the human immunoglobulin at that position.

For a detailed description of the production of humanized immunoglobulins see, Queen et al., Op. cit. , and co et al., op. cit. The polynucleotides will typically further include a polynucleotide sequence for the control of expression operably linked to the sequences encoding the humanized immunoglobulin, including the naturally associated heterologous promoter regions. Preferably, the sequences for the control of expression will be eukaryotic promoter systems in vectors capable of transforming or transfecting eukaryotic host cells, although control sequences for prokaryotic hosts may also be used. Once the vector has been incorporated into the appropriate host, the host is maintained under suitable control conditions for the high level of expression of the nucleotide sequences, and, when desired, the collection and purification of the light chains, heavy chains , light / heavy chain dimers or intact antibodies, binding fragments or other forms of immunoglobulin can follow. The nucleic acid sequences of the present invention capable of finally expressing the desired humanized antibodies can be formed from a variety of different polynucleotides (genomic or cDNA, RNA, synthetic oligonucleotides, etc.) and components (e.g., V regions, J, D, and C), as well as by a variety of different techniques. Genomic linkage and appropriate synthetic sequences is currently the most common production method, although DNA sequences can also be used (see, European Patent Application No. 0239400 and Riechmann, L. et al., Nature, 332, 323- 327 (1988), both of which are incorporated herein by reference). The DNA sequences of the human constant region can be isolated according to well known procedures from a variety of human cells, but preferably immortalized B cells (see, Kabat op.cit.and P 87/02371). The CDRs for producing the immunoglobulins of the present invention will similarly be derived from monoclonal antibodies capable of binding to the antigens of VT2 and produced in any mammalian source including, mice, rats, rabbits- or other vertebrates capable of producing antibodies by methods well known The original cells suitable for the polynucleotide sequences and the host cells for the expression and secretion of immunoglobulin can be obtained from a number of sources, such as the American Type Culture Collection Catalog of Cell Lines and Hybridomas, Fifth Edition (1985) Rockville, Maryland, US A., which is incorporated here as a reference.

In addition to the humanized immunoglobulins specifically described herein, other "substantially homologous" modified immunoglobulins can be designed and manufactured using the various recombinant DNA techniques well known to those skilled in the art. For example, structural regions may vary from the native sequences at the level of the primary structure by various amino acid substitutions, terminal and intermediate additions or deletions, and the like. In addition, a variety of different human structural regions can be used simply or in combination as a basis for the humanized immunoglobulins of the present invention. In general, modifications of genes can be easily effected by a variety of well-known techniques, such as site-directed mutagenesis (see, - Gillman and Smith, Gene 8, 81-97 (1979) and Roberts S. et al., Nature 328, 731-734 (1987), both of which are incorporated herein by reference. Alternatively, polypeptide fragments can be produced which comprise only a portion of the primary structure of the antibody, fragments which possess one or more immunoglobulin activities (e.g., complement fixation activity). Such polypeptide fragments can be produced by proteolytic incision in intact antibodies by methods well known in the art, or by insertion of stop codons at the desired locations in the vectors using site-directed mutagenesis, such as after CH1 to produce Fab fragments. or after the region of the hinge to produce the F (ab ') 2 fragment. Single chain antibodies can be produced by linking VL with VH with DNA linker (see Huston et al., Op.cit., And Bird et al., Op.cit.). Also because like many genes, the genes related to immunoglobulin contain separate functional regions, each having one or more different biological activities, genes can be fused to functional regions of other genes to produce fusion proteins that have properties Novelty As stated above, the polynucleotides will be expressed in the hosts after the sequences have been operably linked to (i.e., placed to ensure the functioning of) an expression control sequence. These expression vectors are typically reproducible in host organisms either as episomes or as an integral part of the chromosomal DNA of the host. Commonly, expression vectors will contain selection markers, for example, tetracycline or neomycin, to allow the detection of those cells transformed by the desired DNA sequences (see, for example, U.S. Patent 4,704,362 which is incorporated herein by reference. E. coli is a particularly useful prokaryotic host for cloning the polynucleotides of the present invention Other suitable microbial hosts to be used include bacilli, such as Bacillus subitulis, and other enterobacteria, such as Salmonella, Serratia and various species of Pseudomonas. prokaryotic hosts, expression vectors may also be produced, which will typically contain expression control sequences compatible with the host cell (e.g., a reproduction origin) in addition, any number of a variety of well-known promoters, such as the promoter system of the lactose, a tryptophan (trp) promoter system, a beta-lactamase promoter system, or a lamda phage promoter. Promoters will typically control expression, optionally with an operator sequence, and will have ribosomal binding site sequences and the like, to initiate and complete transcription and translation. Other microbes, such as yeast, can also be used for expression. Saccharomyces are a preferred host, with vectors having controls of expression sequences, such as promoters, including 3-phosphoglycerate kinase or other glycolytic reproduction enzymes, termination sequences and the like as desired. In addition to microorganism, cultures of mammalian tissue cells can also be used to express and produce the polypeptides of the present invention (see, Winnacker, From Genes to Clones, VCCH Publishers, N.Y., N.Y. (1987), which is incorporated herein by reference). Eukaryotic cells are really preferred, because they have been developed in the art in numerous lines of suitable host cells capable of secreting intact immunoglobulin, including CHO cell lines, variations of COS cell lines, HeLa cells, preferably lines. myeloma cells, etc., or B cells transformed from hybridomas. The expression vectors of these cells can include expression control sequences, such as a reproduction origin, a promoter, an amplifier (Queen et al., Immunol Rev. 89, 46-68 (1986), which is incorporated here for reference), and the necessary information processing sites, such as ribosomal binding sites, RNA splice sites, polyadenylation sites, and transcriptional terminator sequences. Preferred expression control sequences are promoters derived from immunoglobulin genes, SV40, Adenovirus, Bovine Papilloma Virus, cytomegalovirus and the like. The vectors containing the nucleotide sequences of interest (eg, the sequences encoding the heavy and light chain of the expression control sequences) can be transferred to the host cells by well-known methods, which vary according to the type of cellular host. For example, transfection with calcium chloride is commonly used for prokaryotic cells, while calcium phosphate treatment or electroporation can be used for other cellular hosts. (See, generally, Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press (1982), which is incorporated herein by reference). Once expressed, the complete antibodies, their dimers, individual light and heavy chains, or other forms of immunoglobulin of the present invention can be purified according to standard procedures in the art, including precipitation with ammonium sulfate, affinity columns , column chromatography, gel electrophoresis and the like (see, generally, Scopes, R., Protein Purification, Springer-Verlag, NY (1982), which is incorporated herein by reference). Substantially pure immunoglobulins with a homogeneity of at least about 90 to 95% are preferred, and a homogeneity of 98 to 99% or more preferred, for pharmaceutical uses. Once purified, partially or until homogeneous, as desired, the polypeptides can then be used therapeutically (including extracorporeally) or to develop and perform assay procedures, immunofluorescent staining and the like (see, generally, Immunological Methods, Vols. I and II, Lefkovits and Pernis, eds., Academic Press, New York, NY (1979 and 1981). The immunoglobulins of the present invention will typically find use individually in the treatment of the toxic effects of the E. coli infection that produces Verotoxin (VTEC) and the Hemolytic Uremic Syndrome (HUS) and / or in the neutralization of the VT2 antigens. By way of example but not limitation, some typical disease states suitable for treatment include hemorrhagic colitis locally in the intestine, kidney dysfunction and brain damage.The humanized immunoglobulins and the pharmaceutical compositions thereof are The invention is particularly useful for palenteral administration, i.e., subcutaneously, intramuscularly or intravenously. Compositions for palenteral administration will commonly comprise an immunoglobulin solution or a cocktail thereof dissolved in an acceptable carrier, preferably an aqueous carrier. A variety of aqueous carriers can be used, for example water, buffered water, 0.4% saline, 0.3% glycine, 5% glucose, human albumin solution and the like. These solutions are sterile and generally free of particulate matter. These compositions can be sterilized by conventional well-known sterilization techniques. The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting agents and buffers, tonicity agents, agents for adjusting toxicity and the like, eg, sodium acetate, sodium chloride , potassium chloride, calcium chloride, sodium lactate, sodium citrate, etc. The immunoglobulin concentration in these formulations can vary widely, ie, less than about 0.5%, usually at least about 1% up to 15 or 20% by weight and will be selected primarily based on fluid volumes, viscosities, etc. according to the particular mode of administration selected. Thus, a typical pharmaceutical composition for injection could be made to contain 1 ml of sterile buffered water, and 1-10 mg of immunoglobulin. A typical composition for intravenous infusion could be made to contain 250 ml of sterile Ringer's solution, and 150 mg of immunoglobulin. Current methods for preparing palenterally administrable compositions will be known or apparent to those skilled in the art and are described in greater detail in Remington's Pharmaceutical Science, 15th ed., Mack Publishing Company, Easton, Pennsylvania (1980), which is incorporated herein by reference. as reference. The immunoglobulins of this invention can be frozen or lyophilized to be stored and reconstituted in a suitable carrier before use. This technique has been shown to be effective with conventional immunoglobulins and the reconstitution lyophilization techniques known in the art can be employed. It will be appreciated by those skilled in the art that lyophilization and reconstitution can lead to various degrees of loss of immunoglobulin activity (eg, with conventional immune globulins, IgM antibodies tend to have a greater loss of activity than antibodies). IgG) and that the levels of use may have to be adjusted to compensate. The compositions comprising the humanized immunoglobulins present or a cocktail thereof can be administered for therapeutic or prophylactic treatments. In the therapeutic application, the compositions are administered to a patient who already suffers from an infection by E. coli that produces Verotoxin (BTEC) and Hemolytic Uremic Syndrome (HUS), or filles toxic manifestations of the antigens of the VT2, in a enough to cure or at least partially counteract the toxic syndrome and its complications. An adequate amount to achieve this is defined as a "therapeutically effective dose". In prophylactic applications, the compositions are administered to patients at risk of infection in an amount sufficient to prevent or detectably inhibit such infection and / or the toxic manifestations thereof due to VT2 antigens. The effective amounts for such uses depend on the severity of the disease and the general state of the patient's own immune system, but generally range from about 0.1 to 5 mg / kg of immunoglobulin per patient dose being commonly used. It should be borne in mind that the materials of this invention can be employed in general in serious disease states, which endanger life or potentially life-threatening situations. In such cases, in view of the minimization of foreign substances and the lower likelihood of rejection of "foreign substances" that is achieved with the humanized immunoglobulins present of this invention, it is possible and may be considered desirable by the treating physician to administer a substantial excess. of those immunoglobulins.

The single or multiple administration of the compositions can be carried out by levels and dosage patterns being selected by the attending physician. In such a case, the pharmaceutical formulations will provide an amount of the immunoglobulins of this invention sufficient to effectively treat the patient. In particular embodiments, compositions comprising humanized immunoglobulin of the present invention can be used to detect VT2 antigens in E. coli infections that produce Verotoxin (VTEC) and Hemolytic Uremic Syndrome (HUS) and / or in other infections that produce VT2 or VT2V. Thus, a humanized immunoglobulin of the present invention, such as an immunoglobulin that binds to the determinant antigen identified by the MuVTml .1 antibody, can be labeled and used to identify anatomical sites that contain significant concentrations of VT2 or VT2V. For example but without limitation, one or more marker portions for humanized immunoglobulin can be attached. Exemplary marker portions include, but are not limited to, radiopaque dyes, radiocontrast agents, fluorescent molecules, spin-labeled molecules, enzymes, or other marker portions of value in diagnosis, particularly in radiological or resonance imaging techniques. magnetic The humanized immunoglobulins of the present invention can also find a wide variety of uses in vi tro. By way of example, immunoglobulins can be used for the detection of VT2 antigens, or the like. For diagnostic purposes, the immunoglobulins may or may not be labeled. Unlabeled immunoglobulins can be used in combination with other labeled antibodies (second antibodies) that are relative to humanized immunoglobulin, such as antibodies specific for constant regions of human immunoglobulin. Alternatively, the inmunglobulins can be directly labeled. A wide variety of labels may be employed, such as radionucleotides, fluorine, enzymes, enzyme substrates, enzymatic cofactors, enzyme inhibitors, ligands (particularly haptens), etc. Numerous types of immunoassays are available and are well known to those skilled in the art. Equipment can also be provided for use with the subject immunoglobulins in the protection against or detection of cellular activity or for the presence of a selected antigen. In this way, the immunoglobulin composition object of the present invention can be provided, usually in lyophilized form in a container, either alone or in conjunction with additional antibodies specific for the desired cell type. Immunoglobulins, which can be conjugated with a brand or toxin, or unconjugated, are included in the equipment with buffers, such as Tris, phosphate, carbonate, etc., stabilizers, preservatives, biocides, inert proteins, for example, serum albumins , or similar, and a set of instructions for its use. Generally, those materials will be present in less than about 5% by weight based on the amount of active immunoglobulin, and usually present in a total amount of at least about 0.001% by weight based again on the concentration of the immunoglobulin. Frequently, it will be desirable to include an inert additive or excipient to dilute the active ingredients, wherein the excipient may be present from about 1 to 99% by weight of the total composition. Where a second antibody capable of binding immunoglobulin is used in an assay, this will usually be present in a separate vial. The second antibody is typically conjugated to a tag and formulated in a manner analogous to the immunoglobulin formulations described above.

Human Antibodies In another aspect of the invention, human antibodies competing with mouse Vtml-1 are provided by binding to verotoxin II or the variant of verotoxin II. to. Trioma Methodology The basic method and an exemplary cell fusion standard, SPAZ-4, for use in this method has been described by Oestberg et al., Hybridoma 2: 361-367 (1983); Oestberg, U.S. Patent No. 4,634,664; and Engleman et al., U.S. Patent No. 4,634,666 (each of which is incorporated herein by reference in its entirety for all purposes). The cell lines that produce antibodies obtained by this method are called triomas, because they descend from three cells - two human and one mouse. Initially, a line of mouse myeloma is fused with a human B-lymphocyte to obtain a hybrid xenogenetic cell that does not produce antibodies, such as the SPAZ-4 cell line described by Oestberg, supra. The xenogenetic cell is then fused with an immunized human B lymphocyte to obtain a trioma cell line that produces antibodies. It has been found that triomas produce antibodies more stably than ordinary hybridomas made from human cells. B lymphocytes are obtained from the blood, spleen, lymph nodes or bone marrow of a human donor. In vivo immunization of a living human with verotoxin II or a variant of verotoxin II is usually undesirable because of the risk of initiating a dangerous response. In this way, B lymphocytes are usually immunized in vi tro with those antigens or an antigenic fragment of any of these, or a cell carrying any of those. B lymphocytes are typically exposed to the antigen for a period of 7-14 days in media such as RPMI-1640 (see Engleman, supra) supplemented with 10% human serum. Immunized B lymphocytes are fused to a xenogenetic hybrid cell such as SPAZ-4 by well-known methods. For example, the cells are treated with 40-50% polyethylene glycol of MW of 1000-4000, at about 37 degrees for about 5-10 min. The cells are separated from the fusion mixture and propagated in selective media for the desired hybrids (eg, HAT or AH). Clones that secrete antibodies that have the required binding specificity are identified by assaying the trioma culture medium to determine the binding capacity of verotoxin II or a variant of verotoxin II. Triomas that produce human antibodies having the desired specificity are subcloned, for example, by the limiting dilution technique and grown in vitro in culture medium.

Although triomas are genetically stable they may not produce antibodies at very high levels. Expression levels can be increased by cloning trioma antibody genes in one or more expression vectors, and transforming the vector into a cell line such as the cell lines discussed, infra, for the expression of recombinant or humanized immunoglobulins. b. Non-Human Mammalian Transgenic Human antibodies reactive with verotoxin II and / or toxin of verotoxin II can also be produced from non-human transgenic mammals having transgenes encoding at least one segment of the human immunoglobulin site. Usually the site of the endogenous immunoglobulin of such transgenic mammals is functionally inactivated. Preferably, the site segment of the human immunoglobulin includes unfixed sequences of heavy and light chain components. Both the inactivation of the endogenous immunoglobulin genes and the introduction of exogenous immunoglobulin genes can be achieved by recombination of targeted homologs, or by the introduction of YAC chromosomes. Transgenic mammals resulting from this process are able to functionally rearrange the immunoglobulin component sequences, and express a repertoire of antibodies of various isotypes encoded by human immunoglobulin genes, without expressing endogenous immunoglobulin genes. The production and properties of mammals that have these properties are described in detail by Lonberg et al., W093 / 12227 (1993); Kucherlapati, WO 91/10741 (1991) (each of which is incorporated herein by reference in its entirety for all purposes.) Transgenic mice with particularly suitable antibodies are obtained by immunizing a transgenic non-human mammal, such as is described in Lonberg or Kucherlapati, supra.Monoclonal antibodies are prepared, for example, by fusion of B cells of such mammals with myeloma cell lines using Kohler-Milstein technology The following examples are offered by way of illustration, It will be understood that although the examples belong to the HuVTml.l antibody, the production of humanized antibodies with high binding affinity for the VT2 antigen subunit was also contemplated using the CDR of other monoclonal antibodies that bind to an epitope of the VT2.

EXPERIMENTAL PART Example 1. Immunization of mice with VT2 toxoid VT2 was prepared according to that described by Oku et al., Microb. Pathog., 1989, 6 (2), 113-122. VT2 toxoid was prepared by treating 1 mg of purified VT2 for 7 days at 37 ° C with 0.4% formaldehyde in phosphate buffer 0. 1 M, pH 7.6. Balb / C mice were immunized (Nippon Charles River) with VT2 toxoid (0.5μg) combined with Complete Freund's Adjuvant (Gibco RBL) with intraperitoneal injection (i.p.). After approximately 4 weeks, the mice received VT2 toxoid (1 μg) combined with Complete Freund's Adjuvant by ip injection, then mice received VT2 toxoid (1 μg twice, 4 μg twice, 5 μg once) ) combined with Freund's Incomplete Adjuvant by ip injection sequentially in an interval of 1 to 5 weeks. The mice were bled by cutting the tail vein and the serum was collected by incubation of the blood for 30 minutes at 37 ° C and by centrifugation at 300 rpm for 10 minutes. The titre of serum against VT2 was measured by ELISA as follows: Each well of a 96 well flat bottom plate (Falcon 3912, Becton Dickinson) was coated with 50 μl of 1 μg / ml VT2 diluted with 0.01 M phosphate buffered saline (PBS) and incubated at room temperature for 1 hour. Each well was washed three times with PBS - 0.05% Tween 20. Each well was blocked with PBS - 3% BSA at t.a. for 1 hour. Each well was washed three times with PBS - 0.05% Tween 20. 50 μl of diluted serum was added in series with PBS in each well and incubated at room temperature for 1 hour. Each well was washed three times with PBS - 0.05% Tween 20. To each well was added 50 μl of alkaline phosphatase conjugated with anti goat IgG (ZYMED) diluted (x 1000) with PBS - 3% BSA and incubated at room temperature for 1 hour. Each well was washed three times with PBS - 0.05% Tween 20. To each well was added p-nitrophenyl phosphate (PNPP-Wako Chemicals) and incubated at room temperature for 1 hour.

Absorption was measured and recorded at 405 nm for each well, Example 2. Construction of the hybridoma by the cell fusion method. The mice whose serum contained antibodies reactive against VT2 were chosen and deprived of the spleen. 5 X 107 spleen cells and 5 X 10 6 mouse myeloma cells 3 X 63 Ag8U.l (P2U1) were combined and washed with RPMI 1640 medium and centrifuged at 1500 rpm for 5 minutes. The cell mass was gently dispersed and resuspended in 1 ml of polyethylene glycol (PEG) solution (containing 5.75 ml of RPMI 1640 + 3.5 ml of PEG + 0.75 ml of dimethyl sulfoxide) and gently swirled for 2 minutes. Then 1 ml of RPMI medium was added and rotated for 2 minutes. After that, they added 2 ml of RPMI medium and spun it for 2 minutes. 4 ml of GIT-HAT medium (95 μM hypoxanthine, 0.4 μM aminopterin, 1.6 μM thymidine, 5% FCS) was added and rotated for 2 minutes. Then 8 ml of GIT-HAT medium was added and rotated for 2 minutes. After 30 minutes of incubation at 37 ° C the cell suspension was placed in each well of the 96-well flat bottom plate inoculated with 104 mouse / well peripherole macrophages. The plate was incubated at 37 ° C in an incubator with 5% CO 2 - 95% air for one week. Then, half of the middle of each well was replaced with fresh GIT-HAT medium (GIT-HAT medium without aminoferin) and the plate was incubated for about a week to allow growth of the hybridoma cells.

Example 3: Separation of mouse hybridoma cells secreting antibody against VT2 The separation was effected by selecting the hybridoma cells that secrete antibodies, which bind to VT2 and neutralize VT2. The supernatant of the hybridoma was used for separation. The binding activity was measured according to the ELISA method described in Example 1. The neutralization activity of VT2 was measured according to the following method. To each well of a 96-well plate were added μl of hybridoma supernatant and 30 μl of VT2 200 pg / ml in 10% FCS-MEM. The plate was incubated at 37 ° C for 1 hour. 50μl of the above solution was added to each well of a 96 well flat bottom plate on which a Vero cell (ATCC) (4 × 10 04 cells / well) was inoculated.

The plate was incubated at 37 ° C for 4 days in an incubator with 5% CO 2 - 95% air. 100 μl of a 0.014% neutral red solution was added to each well. The plate was incubated at 37 ° C for 1 hour to stain the living cells. After washing the well with PBS, 100 μl of 50% ethanol solution containing 1% acetic acid solution (ETOH-AcOH) was added. The absorption was measured and recorded at 550 nm for each well.

Example 4. Cloning of the hybridoma cells The cloning of the hybridoma cells was conducted by limiting the dilution. One or two hybridoma cells were added to each well of a 96-well flat bottom plate previously inoculated with 104 mouse periprodal macrophages as the commensal cells. After approximately two weeks, the supernatant was measured to determine binding to VT2 and neutralization against VT2 according to that described in Examples 1 and 3, respectively. Repeating this cloning procedure as described above, a hybridoma cell clone was established that secretes a monoclonal antibody (MuVTml.l), which neutralizes VT2.

Example 5: Purification of MuVTml .1 The hybridoma cells secreting MuVTml .1 were adapted to the free medium of basal serum eRDF containing insulin, transferrin, atenaolamin and selenite. The culture supernatant was passed over a Sepherose and Protein G column (Pharmacia) and the antibody was eluted using standard methods. The purity of the antibody was verified by electrophoresis on polyacrylamide gel (PAGE), and this concentration was determined by SRID. The purified MuVTml .1 was tested for its ability to neutralize the VT2 variants according to what is described in Example 3. The results are presented in the table below in Figure 7.

TABLE 1 Nautralizing Activation of MuVTml .1 Against Various Variants of VT2 Strains Origin Type of Toxin Neutralization E. Coli 0157 V50 Human VT2vh + E. Coli 0157 V354 Human VT2vn + the VL and VH cDNA. For the heavy chain, a single unique sequence was identified, typically from a variable region of the mouse heavy chain. For the light chain, two unique sequences were identified, both homologous to the sequences of the variable region of the mouse light chain. However, one sequence was not functional due to a missing nucleotide that caused a deviation of the frame at the V-J junction and was identified as the non-productive allele. The other sequence was typically from a variable region of the functional mouse kappa chain. The cDNA sequences of the variable region of the heavy chain and the functional light chain and the translated amino acid sequences are shown in Figure 1. The sequence V? of the mouse belongs to the subgroup V of the kappa chain of the mouse of Kabat. The mouse VH belongs to the subgroup VH III (D) of the Kabat heavy chain.

Example 7: Design of the variable regions of the humanized VTml .1 To retain the binding affinity of the mouse antibody in the human antibody, the general procedures of Queen et al. (Queen et al., Proc. Nati, Acad. Sci. USA 86: 10029 (1989) and U.S. Patent Nos. 5,585,089 and 5,693,762). The choice of structural residues can be critical to retain a high binding affinity. In principle, a structural sequence of any human antibody can serve as a standard for grafting CDRs; however, it has been shown that direct CDR replacement in such a structure can lead to a significant loss of binding affinity for the antigen (Glaser et al., J. Immunol., 149: 2606 (1992)).; Tempest et al., Biotechnology 9: 266 (1992); Shalaby et al., J. Exp. Med. 17: 217 (1992)). The more homologous a human antibody to the original mouse antibody, the less likely it is that the human structure will introduce distortions in the mouse CDR that could reduce affinity. Based on a sequence homology search against the Kabat database (Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., US Department of Health and Human Services, 1991), the human antibody GF4 was chosen as one that provides good structural homology with the MuVTml antibody .1. Other highly homologous human antibody chains would also be suitable for providing the structure of the humanized antibody, especially the kappa light chains of human subgroup III, and the heavy chains of human subgroup III as defined by Kabat. The computer programs ABMOD and ENCAD (Levitt et al., J. Mol. Biol. 168: 595 (1983)) were used to construct a molecular model of the variable domain of VTml.l, which was used to locate the amino acids in the structure of the VTml .1 that are sufficiently close to the CDRs to potentially interact with them. To design the heavy and light chain variable regions of the humanized VTml .1, the CDRs of the mouse VTml .1 antibody were grafted into the framework regions of the human GF4 antibody. At the positions of the structure where the computer model suggested significant contact with the CDR, the amino acids of the mouse antibody were replaced by the original human structural amino acids. For the humanized VTml .1, this was done at residues 29, 30, 49 and 98 of the heavy chain and at residue 49 of the light chain. In addition, the structural residues that occurred only rarely in their positions in the human antibody database were replaced by a consensus amino acid at those positions. For the humanized VTml .1, this was done in residues 1, 4, 5, 79, 89 and 93 of the heavy chain and in residues 3, 4, 19, 76, 79 and 85 of the light chain. The sequence of the heavy chain variable regions and the humanized VTml .1 antibody light chain, including the start codon and the signal peptide sequences, are shown in Figure 2. However, many other contact residues with the CDRs Potentials are susceptible to substitution by other amino acids that may still allow the antibody to retain a substantial affinity for the antigen. The following table lists a number of positions in the structure where the alternative amino acids may be suitable (LC = light chain, HC = heavy chain).

TABLE 2 Likewise, many of the structural residues that are not in contact with the CDR in the heavy and light chains of the humanized VTml .1 can accommodate amino acid substitutions of the corresponding positions of the human GF4 antibody, of other human antibodies, of the VTml antibody .1 of mouse, or other mouse antibodies, without significant loss of affinity or without immunogenicity of the humanized antibody. The following table lists a number of additional positions in the structure where the alternative amino acids may be suitable.

TABLE 3 The selection of the different alternative amino acids can be used to produce versions of humanized VTml .1 having various combinations of affinity, specificity, absence of immunogenicity, ease of manufacture, and other desirable properties. Thus, the examples in the above tables are offered by way of illustration, not limitation.

Example 8: Construction of Humanized VTml .1 Once the amino acid sequences of the humanized variable region had been designed as described above, genes were constructed to encode them, including signal peptides, splice donor signals and appropriate restriction sites ( Figure 2). The light and heavy chain variable region genes were constructed and amplified using eight superimposed synthetic oligonucleotides ranging in length from about 65 to 80 bases, as shown in Figure 3 (see He et al., J. Immunol. 1029 (1988)). The oligos were annealed in pairs and extended with the Klenow fragment, of DNA polymerase I, producing four double-stranded fragments. The resulting fragments were denatured, annealed, and extended with Klenow, producing two fragments. These fragments were denatured, annealed in pairs, and extended once more, producing a full-length gene. The resulting product was amplified by the polymerase chain reaction (PCR) using Taq polymerase, gel purified, Xbal digested, gel purified again, and subcloned into the XbaI site of the pVk or pVgl expression vector. The pVk and pVgl vectors for the expression of the respective light chain and heavy chain had previously been described (see Co et al., J. Immunol., 148: 1149 (1992)).

The structures of the final plasmids were verified by nucleotide sequencing and the restriction map trace. All the manipulations of DNA were made by standard methods well known to those taught in the art. To construct a cell line that produces humanized VTml .1, the heavy chain and light chain plasmids were transfected into the mouse myeloma cell line Sp2 / 0-Agl4 (ATTC CRL 1581). Before transfection, the plasmids containing the heavy and light chain were linearized using Fspl. About 20 μg of each plasmid was transfected into lxlO7 cells in PBS. The transfection was by electrophoration using a Gene Pulse apparatus (BioRad) at 360 V and a capacitance of 25 μFD according to the manufacturer's instructions. Cells from each transfection were grown in 96-well cell culture plates, and after two days, selection medium was applied (DMEM, 10% FCS, 1 x HT supplement) (Sigma), 0.25 mg / ml xanthine, 1 μg / ml mycophenolic acid). After approximately two weeks, the clones that appeared were separated by antibody production by ELISA. The antibody of a high production clone was prepared by growing the cells to confluence in a regular medium (DMEM with 10% FCS), then replacing the medium with serum free medium (Hybridoma SMF; Gibco) and cultivating until they were reached. the maximum antibody titers in the culture. The culture supernatant is run through a protein A-Sepharose column (Pharmacia); the antibody was eluted with 0.1 M glycine, 100 mM NaCl, pH 3, neutralized and subsequently exchanged in phosphate buffered saline (PBS). The purity of the antibody was verified by analyzing it on an acrylamide gel, and its concentration was determined by reading the D028o, assuming that 1.0 mg of antibody protein has an OD280 reading of 1.4.

Example 9: Properties of affinity measurements of humanized VTml .1 The affinity of MuVTml .1 antibody and HuVTml .1 for verotoxin II (VT2) was determined by a competitive binding with biotinylated MuVTml.l antibody. The procedure for the experiment is described below: Dynatech Immulon 2 96-well plates Coated Laboratories (parts # 0110103455) with 50 μl of VT2 solution (0.2 μg / ml in PBS) per well. Incubated at 37 ° C for 2 hours with gentle agitation.

Aspirated from the VT2 solution. Each well washed 4 times with 400 μl of wash buffer (0.1% Tween 20 in PBS). Each well blocked with 400 μl of Pierce SuperBlock Locking Buffer in PBS (cat # 37515) at room temperature for 30 minutes. Aspirate of the blocking solution. Each well washed 4 times with 400 μl of wash buffer. In each well, 20 ng of biotinylated MuVTml .1 antibody mixed with various concentrations of mouse VTml .1 or Hu antibodies labeled in a total volume of 100 μl of binding buffer (1% BSA, 0.1% Tween) was added to each well. in PBS). Incubate at 4 ° C during the night with gentle agitation. Aspirated from the antibody solution. Each well washed 4 times with 400 μl of wash buffer. Addition of 100 μl of strepavidin conjugated with peroxidase (Bioscource cat # 5NN2004) (1: 500 dilution in binding buffer) to each well. Incubate at 3 ° C for 1 hour with gentle shaking. Aspirate of estrepavidin solution. Each well washed 6 times with 400 μl of wash buffer. Addition of 100 μl / well of peroxidase substrate (BioRad # 172-1064). Incubated at room temperature until color developed. Reading the plate in an ELISA plate reader Molecular Devices at 15 nm. The result shown in Figure 4 demonstrates that the VTml-1 antibody competes well against the biotinylated mouse antibody in the competitive ELISA assay, with a factor of 2 when compared to the mouse antibody. The affinity of HuVTml .1 and MuVTml .1 was also calculated using the BIAcore method. The KD calculated for the HuVTml .1 is approximately 3.5 x 10 ~ 9 M against Mu Vtml .1 as shown in the Table below: TABLE 4 Example 10: In vitro neutralizing activity of HuVTml. 1 The neutralizing activity of HuVTml .1 was also measured by comparison with mouse MuVTml.l in an in vitro assay described below: The human kidney derived cell line (ACHN) was inoculated on a 96 well plate at 1 x 104 cell / well and incubated at 37 ° C for 24 hours.

The medium was aspirated and 50 μl of a mixed solution of 30 μl of 540 pg / ml of VT2 solution diluted with 10% FCS-MEM and 30 μl of test antibody diluted in series with 10% FCS-MEM (humanized mouse VTml .1) preincubated at 37 ° C for 1 hour in each well and incubated at 37 ° C for 4 days. 100 μl of 0.028% neutral red in the medium was added to each well and incubated at 37 ° C for 1 hour (Mullbacher, A. et al., 1984, Journal of Immunological Methods, 68, 205-215). The solution was aspirated. Each well was washed twice with 150 μl of PBS. 100 μl of 50% EtOH / 1% AcOH was added to each well and incubated at room temperature for 5-10 minutes. The absorption value was determined 550 nm for each well using an ELISA plate vector from Molecular Devices. The neutralizing activity was calculated according to the following formula: DO (VT2 + Ab) - DO (VT2 alone) Neutralization (%) = xlOO DO (medium only) - DO (VT2 alone) The neutralizing activity of HuVTml .1 compared to that of MuVTml .1 is shown in Figures 5. The ED 50 pair for HuVTMl .1 is 0.33 μg / ml versus 0.2 μg / ml for MuVTml.l. the neutralization activity of HuVTml .1 for the variants of VT2 is shown below.

TABLE 5 Neutralization assay of VT2 variants in vitro with HuVTml .1 Strains Origin Type of DE50 Toxin (μg / ml) E. coli 0157 Human VT2 0.33 E. coli 0157 (V50) Human VT2vh 0.36 E. coli 0157 (V354) Human VT2vh 0.39 E. coli 0157 (v601) Human VT2v 0.32 E. coli 0157: H7 (TK 40) Human VT2, VT2vx 0.35 + VT2vx: novel subtype of the variant of VT2 Example 11: Antigen analysis of recognized VT2 VT2 was reduced, subunits A and B were separated by a polyacrylamide gene containing SDS and transferred to a nitrocellulose membrane (Bio Rad) by the Western immunoblot method. The nitrocellulose membrane was blocked overnight in 3% BSA in PBS and then reacted with 10 μg / ml HuVTml .1 or rabbit anti-VT2 serum diluted with 3% BSA in PSb for one hour at room temperature. ambient. The membrane was washed 6 times with 1% BSA-PBS and reacted with 25μg / ml alkaline phosphatase conjugated with goat anti-human IgG (TAGO) or alkaline phosphatase conjugated with goat anti-rabbit IgG (TAGO) diluted with 3% BSA in PBS for one hour at room temperature. The membrane was washed 6 times with 1% BSA in PBS and reacted with NBT solution (nitro-blue tretrazolium chloride), BCIP (p-toluidine salt of 5-bromo-4-chloro-3'-indoliphosphate) ) [PIERCE] for 10 minutes at room temperature. The result shown in Figure 6 indicates that HuVTml .1 binds mainly to subunit B of VT2.

Example 12: In vivo activity of HuVTml .1 against VT2 Ddy mice (Nippon SLC) were injected via the intravenous route with 4.8 μg of HuVTml.l, MuVTml. 1 or sterile PBS. One hour later, the mice were injected via the intraperitoneal route (i.p.) with 46 mg of VT2 (corresponding to an LD50 of 10). After one week, the viability of the mice was measured.

The following table shows the results. HuVTml .1 completely protects mice from death caused by VT2.

TABLE 6 From the foregoing, it will be appreciated that the humanized immunoglobulins of the present invention offer numerous advantages over specific anti-VT antibodies. Compared with mouse monoclonal antibodies, the humanized immunoglobulins of the present invention contain substantially fewer non-human and potentially immunogenic amino acid sequences. This reduces the probability of antigenicity after injection of the human patient, which represents a significant therapeutic improvement. All publications and patent applications cited above are incorporated herein by reference to the same extent as if each individual patent application or publication was specifically and individually indicated as incorporated by reference. Although the present has been described in greater detail by way of illustration and example for purposes of clarity and understanding, it will be apparent that certain changes and modifications may be practiced within the scope of the appended claims.

Claims (33)

CHAPTER CLAIMING Having described the invention, it is considered as a novelty and, therefore, what is claimed is contained in the following CLAIMS:

1. A humanized antibody that binds specifically to VT2 and / or a variant of VT2.

2. A humanized antibody, characterized in that it binds specifically to subunit B of Vt2 and / or subunit B of a variant of VT2.

3. The humanized antibody according to claim 1, characterized in that it neutralizes the VT and / or a variant of the VT2.

4. The humanized antibody according to claim 2, characterized in that it neutralizes the VT and / or a variant of the VT2.

5. The humanized antibody which is a humanized form of the mouse antibody VTml.l, the antibody is characterized by a light chain variable region shown in the IB chain and a heavy chain variable region shown in Figure IA.

6. An antibody, characterized in that it competes with the mouse antibody VTml-1 for specific binding to VT2 and / or a variant of VT2.

The humanized antibody according to any one of claims 1-6, characterized in that it comprises regions that determine the complementarity of the mouse VTml-1 antibody and the heavy and light chain variable region structures of the heavy and light chain structures of the human GF4 antibody, provided that at least one position selected from the group consisting of L49, H29, H30, H49 and H98, is occupied by the amino acid present in the equivalent position of the heavy or light chain variable region structure of the antibody Mouse VTml-1, humanized antibody which binds specifically to verotoxin II with an affinity constant of between 107M_1 and ten times the affinity of the mouse VTml-1 antibody.

The humanized antibody according to claim 7, characterized in that each position selected from the group consisting of L49, H29, H30, H49 and H98 is occupied by the amino acid present in the equivalent position of the structure of the variable chain region heavy or light weight of the mouse VTml-1 antibody.

9. The humanized antibody according to claim 8, characterized in that at least one position selected from the group of L3, L4, L19, L76, L79, L85, H1, H4, H5, H79, H89 and H93 is occupied by an amino acid. present in the equivalent position of a consensus sequence of a human antibody heavy chain.

10. The humanized antibody according to claim 9, characterized in that each position selected from the group L3, L4, L19, L76, L79, L85, H1, H4, H5, H79, H89 and H93 is occupied by an amino acid present in the equivalent position of a consensus sequence of a heavy or light chain of the human antibody.

The humanized antibody according to any of claims 1-6, characterized in that it comprises a heavy chain variable region shown in Figure 2A and a light chain variable region shown in Figure 2B provided that one or more selected positions of the group consisting of L3, L4, L19, L76, L79, L85, H1, H4, H5, H79, H89 and H93 can be substituted as shown in Tables 2 and 3.

12. The humanized antibody in accordance with any of claims 1-6, characterized in that it comprises a heavy chain variable region shown in Figure 2A and a light chain variable region shown in Figure 2B.

The humanized antibody according to any of claims 1-6, characterized in that it comprises a humanized heavy chain having an identity of at least 85% with the humanized heavy chain shown in Figure 2A and a humanized light chain having the minus a sequence identity of 85% with the humanized light chain shown in Figure 2B, provided that at least one position selected from the group consisting of L49, H29, H30, H49 and H98, is occupied by the amino acid present in the equivalent position of the variable region structure of the heavy or light chain of the mouse VTml-1 antibody.

The humanized antibody according to any of claims 1-6, characterized in that the antibody comprises two pairs of light / heavy chain dimers, wherein each chain comprises a variable region and a constant region.

15. The humanized antibody according to any of claims 1-6, characterized in that it is a Fab fragment or an F (ab ') 2.

16. The humanized antibody according to any of claims 1-6, characterized in that it is in purified form.

17. The humanized antibody according to any of claims 1-6, characterized in that it has an IgGli immunoglobulin isotype.

18. A method for producing humanized VTml .1 antibody, characterized in that it comprises culturing a cell line, which codes for the heavy and light chains of the humanized antibody according to any of claims 1-6, whereby the humanized antibody is expressed; and recovering the humanized antibody expressed by the cell line.

The method according to claim 18, characterized in that it further comprises mixing the antibody with a pharmaceutically acceptable carrier to produce a pharmaceutical composition.

20. A pharmaceutical composition, characterized in that it comprises the humanized antibody according to any of claims 1-6 and a pharmaceutically acceptable carrier.

21. A pharmaceutical composition, characterized in that it comprises the humanized antibody according to claim 12 and a pharmaceutically acceptable carrier.

22. A method for treating a patient suffering from or at risk from the toxic effects of a verotoxin, characterized in that it comprises administering to the patient an effective dose of a human or humanized antibody that binds specifically to verotoxin II and / or a variant of verotoxin II.

23. The method according to claim 22, characterized in that the antibody competes with the mouse antibody VTml-1 for the specific binding to verotoxin II to a variant of verotoxin II.

24. The method of compliance with the claim 22, characterized in that the humanized antibody binds specifically to VT2 and / or a variant of VT2.

25. The method according to claim 22, characterized in that the humanized antibody binds specifically to subunit B of VT2 and / or a variant of VT2.

26. The method according to claim 22, characterized in that the humanized antibody binds specifically to VT2 and / or a variant of VT2 and neutralizes VT2 and / or a variant of VT2.

27. The method according to claim 22, characterized in that the humanized antibody binds specifically to subunit B of VT2 and / or subunit B of a variant of VT2 and neutralizes VT2 and / or a variant of VT2. .

28. The method according to claim 22, characterized in that the antibody is a humanized antibody, which is a humanized form of the mouse VTml-1 antibody.

29. The method according to claim 22, characterized in that the antibody is a humanized antibody, characterized in that it comprises a heavy chain variable region shown in Figure 2A and a light chain variable region shown in Figure 2B.

30. The method according to claim 22, characterized in that the patient is infected with E. coli which produces the verotoxin and the antibody is administered therapeutically.

31. The method of compliance with the claim 22, characterized in that the patient is at risk of E. coli infection which produces verotoxin and the antibody is administered prophylactically.

32. The method according to claim 30, characterized in that it comprises checking the patient to recover from the toxic effects of vero.toxin II or a variant of verotoxin II.

33. A cell line, characterized in that it produces an antibody according to any of claims 1-6.