RU2732155C1 - Humanised antibody or its antigen-binding fragment (fab) against first and/or second type shiga like toxins (embodiments), composition for treating enterohaemorrhagic escherichia coli toxic conditions, containing said antibodies and/or fab - Google Patents

Abstract

FIELD: immunology.SUBSTANCE: present invention relates to immunology. There are presented humanised antibodies or their antigen-binding fragments selectively binding with shiga-like toxins of the first and/or second type and neutralizing them. What is also considered is a composition containing a mixture of the above antibodies.EFFECT: present invention can find further application in therapy of toxic conditions caused by enterohaemorrhagic Escherichia coli.6 cl, 1 tbl, 4 ex, 4 dwg

Description

Technology area

The present invention relates to antibodies, in particular to humanized antibodies or their antigen-binding fragments (Fab), which bind Shiga toxins Stx1 and / or Stx2 and neutralize them. Also, the present invention relates to a composition containing said antibodies or their Fabs for the treatment of toxic conditions caused by enterohaemorrhagic Escherichia coli.

Prior art

Every year in the world, including in economically prosperous countries, almost 3 million people are infected with enterohemorrhagic Escherichia coli, which produces shiga-like toxins of the first and / or second types (Stx1 and Stx2, respectively). 10% of those infected develop hemolytic uremic syndrome (HUS), often resulting in disability or death. The cause of HUS is the effect of shiga-like toxins on the epithelial cells of the vessels of the renal tubules and the brain. By binding to the gb3 receptor, Stx molecules penetrate into the cytoplasm of epithelial cells and inactivate ribosomes, which leads to an arrest of protein synthesis and cell death. As a result of vascular damage, life-threatening toxic conditions develop, such as acute renal failure and toxic encephalopathy. Currently, only symptomatic treatment is available to patients with hemolytic uremic syndrome, which does not always lead to a complete recovery. In hemolytic uremic syndrome, hemodialysis is used, and in severe cases, kidney transplantation, these measures can only mitigate the manifestations of HUS, but not stop its development. A more specific method of influencing the development of HUS is the use of Soliris (eculizumab), a monoclonal antibody against the C5 component of complement (Patheon Italia, Italy; Patheon Manufacturing Services, USA; WO 2016094834 A2). However, this drug allows you to affect only one of the components of HUS - inadequate development of the complement system reactions. Moreover, its use is severely limited by its high price.

A number of patent documents are known to date describing various variants of antibodies against shiga toxins.

WO9932645 A1 describes the preparation and use of biologically and immunologically active humanized monoclonal antibodies to Shiga toxin, an HC-related toxin and potentially life-threatening HUS transmitted by strains of pathogenic bacteria. In particular, human monoclonal antibodies derived from murine monoclonal antibodies 13C4 and 11E10 are described that specifically react with Stxl and Stx2, respectively. One aspect of the invention is a humanized monoclonal antibody that binds to Shiga toxin, wherein the constant regions are IG1-kappa and the variable regions are murine in origin.

WO2007143004 A2 includes methods, compositions and kits for treating a subject having Shiga toxin-associated disease with chimeric anti-ShigaToxin 1 (caStxl) and anti-ShigaToxin 2 (caStx2) antibodies, wherein said chimeric anti-Stxl and chimeric anti-Stx2 antibodies are co-administered ... A preparation containing a mixture of these two antibodies is known as ShigamAbs (Thallion Pharmaceuticals Inc., Canada).

WO2007124133 A2 relates to methods for producing anti-Stxl antibodies specific for the 13C4 epitope of the Stxl protein. In addition, the invention relates to methods of treating a subject at or at risk of developing associated with shiga toxin (e.g., hemolytic uremia syndrome and diseases associated with E. coli and S. dysenteriae infection) with a polypeptide that includes a 13C4 epitope or an anti-Stxl antibody developed using the methods of the invention.

WO2014191904 A1 refers to an antibody or antigen-binding fragment thereof that specifically binds the B subunit of Shiga toxin, provided that it contains the CDR3 sequence NLRGXaaNY (SEQIDNO: 6), where Xaa is S, T or A. Pharmaceutical compositions are also disclosed comprising antibodies or antigen binding fragments, a method for producing antibodies and antigen binding fragments, and a method for treating hemolytic uremic syndrome (HUS) in a subject.

US Pat. No. 9,310,368 B1 describes high-affinity monoclonal antibodies against Stx2 and hybridomas that produce such antibodies. The antibodies can be used in a kit for detecting Stx2 and its variants in a sample, as well as in neutralizing shiga toxin in vivo.

CN 101570574 B relates to an antibody against type I shiga toxin. The antibody has the effects of neutralizing the shiga toxin and effectively inhibiting the toxicity of the shiga toxin, can be used as an oral antitoxin for the prevention and treatment of complications caused by toxin-producing shigella, enterohemorrhagic E. coli O157, cholera vibrio, etc. and can be used simultaneously to detect and diagnose type I shiga toxin infection and its pathogen.

Patent CN 101357942 B relates to the technological field of bioengineering medicine, more specifically to monoclonal antibodies that neutralize enterohemorrhagic colitis O157: H7 shiga toxinII, Fab antibody sequences and uses. The monoclonal antibody is obtained by hybridoma cell strains while maintaining the CCTCC: C200822 amount. The antibody can be used to formulate drugs for the diagnosis and treatment of EHECO157 infections and their complicating diseases.

Patent CN103319593 B discloses an anti-StxII monoclonal antibody that has the heavy chain amino acid sequence shown in SEQ ID NO.2 table and the light chain amino acid sequence shown in SEQ ID NO.4 table, where the amino acid sequences of the complementary determining regions CDR1, CDR2 and CDR3 in the sequence of the variable region of the heavy chain are respectively represented by the tables of sequences of SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7 and the amino acid sequences of the complementary determining regions CDR1, CDR2 and CDR3 in the sequence of the variable region of the light chain are respectively shown in the tables sequences SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10.

Also known is the drug Urtoxazumab (Creative Biolabs, USA), which is a recombinant antibody only against Stx2 (Lopez EL et al., Safety and Pharmacokinetics of Urtoxazumab, a Humanized Monoclonal Antibody, against Shiga-Like Toxin 2 in Healthy Adults and in Pediatric Patients Infected with Shiga-Like Toxin-Producing Escherichia coli Antimicrob Agents Chemother 2010 Jan; 54 (1): 239-243).

None of the above documents disclose a cross-reactive antibody against Stx1 and Stx2.

The lack of effective means for the prevention and relief of hemolytic uremic syndrome (HUS) is currently a serious problem, and therefore the development of a new drug for the treatment of toxic conditions caused by enterohemorrhagic E. coli is an extremely urgent task.

Brief description of the invention

The aim of the present invention is to obtain antibodies, in particular humanized antibodies or their antigen-binding fragments (Fab), which bind and neutralize Shiga toxins Stx1 and / or Stx2. Also, the present invention relates to a composition containing said antibodies or their Fabs for the treatment of toxic conditions caused by enterohaemorrhagic Escherichia coli.

This goal was achieved by isolating two mouse antibodies, one of which is cross-reactive and has the ability to efficiently bind shiga toxins of both the first and second types (Stx1 and Stx2), and the second has the ability to effectively bind shiga toxins of the second type, determining the sequence of the variable regions of these antibodies and obtaining on their basis of two humanized antibodies that bind Shiga-toxins Stx1 and / or Stx2 and neutralize them. The obtained antibodies were used to create a composition for the treatment of toxic conditions caused by enterohemorrhagic E. coli.

Thus, the present invention is a humanized antibody or its antigen-binding fragment (Fab), selectively binds (s) to shiga-like toxins of the first and second types and neutralizes them , containing (s) CDR1 with the amino acid sequence SEQ ID NO: 1, CDR2 with amino acid sequence SEQ ID NO: 2, and CDR3 with amino acid sequence SEQ ID NO: 3 of the heavy chain, and CDR1 with amino acid sequence SEQ ID NO: 4, CDR2 with amino acid sequence SEQ ID NO: 5, and CDR3 with amino acid sequence SEQ ID NO: 6 light chain.

Also, the present invention is a humanized antibody as described above, containing the amino acid sequence SEQ ID NO: 7 of a heavy chain containing a variable domain and a constant region of human IgG1, and the amino acid sequence SEQ ID NO: 8 of a light chain containing a variable domain and a constant region C- kappa man.

Also, the present invention is a humanized antibody or antigen-binding fragment (Fab) thereof that selectively binds to and neutralizes a shig-like toxin of the second type , containing CDR1 with the amino acid sequence SEQ ID NO: 11, CDR2 with the amino acid sequence SEQ ID NO: 12, and CDR3 with the amino acid sequence SEQ ID NO: 13 of the heavy chain, and CDR1 with the amino acid sequence SEQ ID NO: 14, CDR2 with the amino acid sequence SEQ ID NO: 15, and CDR3 with the amino acid sequence SEQ ID NO: 16 light chain.

Also, the present invention is a humanized antibody as described above containing the amino acid sequence SEQ ID NO: 15 of the heavy chain containing the variable domain and the constant region of human IgG1, and the amino acid sequence SEQ ID NO: 16 of the light chain containing the variable domain and the constant region C- kappa man.

Also, the present invention provides a composition for the treatment of toxic conditions caused by enterohemorrhagic E. coli, containing a mixture of the above humanized antibodies or their Fab.

Brief Description of Figures

FIG. 1 shows the results of an enzyme-linked immunosorbent assay for the binding of humanized antibodies H8-hum and H4-hum to shiga toxin Stx2 in comparison with a control humanized antibody against rabies virus glycoprotein.

FIG. 2 shows the results of an enzyme-linked immunosorbent assay for the binding of humanized antibodies H8-hum and H4-hum to Shiga toxin Stx1 in comparison with a control humanized antibody against rabies virus glycoprotein.

FIG. 3 shows the results of in vitro neutralization of the toxic effect of Shiga-toxin Stx1 with humanized antibodies H8-hum and H4-hum.

FIG. 4 shows the results of in vitro neutralization of the toxic effect of Shiga-toxin Stx2 with humanized antibodies H8-hum and H4-hum.

In Tab. 1 shows the performance indicators of the composition of neutralizing antibodies against Stx1 and Stx2 in vivo.

Detailed description of the present invention

To prevent or relieve toxic conditions in patients infected with enterohaemorrhagic E. coli, it is necessary to neutralize the action of shiga-like toxins entering the bloodstream. There are no specific antidotes for shiga-like toxins in clinical practice.

Neutralizing antibodies against Stx are capable of preventing or arresting the development of HUS. For the most effective neutralization, at least two full-length antibodies directed to different Stx epitopes are required. The injected antibodies must be humanized in order to avoid unwanted immune responses of the patient's body. Due to the high mutational variability of shiga-like toxins, mainly Stx2, an important task is to find a universal neutralizing antibody that reacts with conserved epitopes of toxins. For clinical purposes, it is preferable to use a mixture of several antibodies that bind to conserved epitopes of various types of toxins.

Within the framework of the present invention, it was possible to obtain two such monoclonal mouse antibodies with a high degree of binding to neutralizing epitopes of toxins and effectively neutralizing them: one cross-reactive antibody against shiga toxins of both the first and second types, and the second against shiga toxins of the second type ...

Then the authors of the present invention cloned the genes encoding the variable domains of the heavy and light chains of mouse antibodies against shig-like toxins Stx1 and / or Stx2, and determined their sequences.

After that, the design and humanization of the indicated mouse antibodies against Shiga toxins Stx1 and / or Stx2 were carried out to obtain humanized antibodies, the heavy chain of which contains the variable domain and the constant region of human IgG1, and the light chain contains the variable domain and the constant region of human C-kappa.

On the basis of the obtained humanized antibodies, a composition was created for the treatment of toxic conditions caused by enterohaemorrhagic Escherichia coli. The model using Balb / c mice shows the high efficiency of the specified composition against the toxic effect of the second type of shiga toxin.

This is how the present invention was completed.

Antibodies usually consist of two heavy chains linked by disulfide bonds, and light chains associated with the N-terminus of each of the heavy chains. Each heavy chain contains a variable domain at the N-terminus with a constant domain at the other end. Each light chain also contains a constant domain variable domain at the N-terminus. The variable domains of each pair of light and heavy chains form an antigen-binding fragment (Fab). The variable domains of the light and heavy chains have a similar overall structure, and each domain includes a framework of four regions, the sequences of which are relatively conserved, linked by three complementarity determining regions (CDRs).

The term “CDR” or “complementarity determining region” refers to those portions of the heavy and light chains of an antibody that are located in close proximity to each other in three-dimensional space and form the antigen-binding surface of the antibody.

The four scaffolds form a beta-pleated conformation, and the CDRs form loops linking the beta-pleated construct. The CDRs are located in close proximity to each other due to the framework regions and contribute to the formation of the antigen-binding site. CDRs and antibody frameworks can be identified by reference to the Kabat numbering system (Kabat et al., (1987) "Sequences of Proteins of Immunological Interest", US Dept. of Health and Human Services, US Government Printing Office ) in conjunction with x-ray crystallography, as set forth in WO91 / 09967.

To obtain an antibody that can bind to a specific antigen, the method of Kohler and Milstein (Kohler et al., (1976) Nature 256: 495-497), called hybrid technology, is usually used. The essence of the method is to obtain a culture of "immortal" hybrids (hybridόm) by fusion of a B-lymphocyte producing antibodies and a tumor cell (myeloma or plasmacytoma). The resulting hybrid is capable of producing monoclonal antibodies (like a B-cell) and at the same time dividing infinitely (like a tumor cell), which allows for a sufficiently long time to secrete an antibody specific for the target antigen in vitro or in vivo.

Cell fusion occurs under the action of polyethylene glycol, isolecithin or the Sendai virus. At the next stage, it is necessary to select myeloma cells that have not fused with lymphocytes (there is no need to remove non-fused lymphocytes: after a short time they will die on their own). In the process of such selection, hybrid cells are planted on media containing substances that block the synthesis of nucleotides by myeloma cells. For this purpose, special mutant myeloma cell lines were deduced, defective in some enzymes involved in the synthesis of nucleotides.

The selection of hybrids secreting the desired antibodies is carried out by checking the environment in which the cells grew for the presence of antibodies of the required specificity.

Thus, cells are selected that secrete antibodies stably and in sufficient quantities. A large volume of antibodies is produced either in vitro, or by introducing hybridomas into the peritoneal cavity of mice to obtain ascites (Sveshnikov, P.G. Introduction to molecular immunology and hybridoma technology / P.G. Sveshnikov, V.V. Malaytsev, I.M. Bogdanova, O. N. Solopova, ed.MGU, M., 2006).

The preparation of material to be used as an antigen for injecting animals involves techniques well known in the art, for example, using a full-length protein, using a peptide selected from immunogenic regions of the protein, modifying the antigen by methods such as binding to dinitrophenol, binding with arsanilic acid, denaturation of the antigen, binding of the antigen to a carrier protein, such as, for example, hemocyanin of the snail, to peptides containing binding sites for class II T-cell receptors, to beads, as well as any other methods known from the prior art. See, for example, Harlow et al. Antibodies, a Laboratory Manual, Cold Spring Harbor Labs Press, 1988.

The authors of the present invention have obtained two humanized antibodies, H8 and H4, which have the ability to specifically bind shiga toxins Stx1 and / or Stx2 and effectively neutralize them, and the first antibody H8 is cross-reactive and has the ability to specifically bind shiga toxins as the first and the second type, and the second antibody H4 has the ability to specifically effectively bind shiga toxins of the second type .

The anti-shiga toxin antibody of both the first and second types according to the present invention comprises CDR1 with the amino acid sequence SEQ ID NO: 1, CDR2 with the amino acid sequence SEQ ID NO: 2, and CDR3 with the amino acid sequence SEQ ID NO: 3 of the heavy chain, and CDR1 with the amino acid sequence SEQ ID NO: 4, CDR2 with the amino acid sequence SEQ ID NO: 5, and CDR3 with the amino acid sequence SEQ ID NO: 6 of the light chain.

Antibody against shiga toxins of the second type according to the present invention contains CDR1 with the amino acid sequence SEQ ID NO: 11, CDR2 with the amino acid sequence SEQ ID NO: 12, and CDR3 with the amino acid sequence SEQ ID NO: 13 of the heavy chain, and CDR1 with the amino acid sequence SEQ ID NO: 14, CDR2 with the amino acid sequence SEQ ID NO: 15, and CDR3 with the amino acid sequence SEQ ID NO: 16 of the light chain.

The above antibodies are preferably humanized.

In particular, a specific embodiment of said humanized anti-shiga toxin antibody of both the first and second types is an H8 antibody comprising a heavy chain variable domain comprising the amino acid sequence SEQ ID NO: 7 and a light chain variable domain comprising the amino acid sequence SEQ ID NO: 9.

A specific embodiment of said humanized anti-Shiga toxin type II antibody is an H4 antibody comprising a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 17 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 19.

The nucleotide sequences encoding the heavy and light chains of said humanized antibodies H8 and H4 are shown in SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 18, SEQ ID NO: 20, respectively.

These humanized antibodies against shiga toxins of both the first and second types can be obtained from the indicated nucleotide sequence by methods well known to the person skilled in the art, for example, using recombinant DNA technologies and hybridoma technologies.

Methods for preparing plasmid DNA, digesting and ligating DNA, transforming, selecting oligonucleotides as primers, and similar methods can be standard methods well known to the person skilled in the art. These methods are described, for example, in Sambrook, J., Fritsch, E.F., and Maniatis, T., (“Molecular Cloning: A Laboratory Manual, Second Edition”, Cold Spring Harbor Laboratory Press (1989)).

In the present invention, the phrase “an antibody or Fab capable of binding and neutralizing shiga toxins Stx1 and / or Stx2” means a molecule that binds to shiga toxin Stx1 and / or Stx2, forms a stable complex and inhibits the activity of said shiga toxin ... A stable complex is a complex in which binding between partners occurs for a period of time sufficient to detect the indicated complex using the methods described herein. The term "selectively binds to shiga toxin Stx1 and / or Stx2" means the ability of the specified molecule to preferentially bind to shiga toxin Stx1 and / or Stx2, as opposed to binding to proteins not related to shiga toxins Stx1 and / or Stx2, or binding to non-protein components present in the sample or organism. An antibody or Fab that preferentially binds to the Shiga toxin Stx1 and / or Stx2 is an antibody or Fab that binds to the Shiga toxin Stx1 and / or Stx2 but does not substantially bind to other molecules or components that are present in the sample or the body.

The ability of an antibody or Fab to bind to an antigen can be determined by one of ordinary skill in the art using methods including, but not limited to, ELISA and equilibrium dialysis. Methods for determining the affinity and binding strength are well known to the person skilled in the art, described in detail by Janeway et al. (Immunobiology: The Immune System in Health and Disease (Garland Publishing Company, 1996)).

Antibodies and Fabs of murine monoclonal antibodies according to the present invention that selectively bind and neutralize shiga toxins of the first and / or second types can also be produced using a suitable hybrid. The hybrids can be grown in a nutrient medium and the accumulated antibodies can be isolated. Hereinafter, the term "growing cells" refers to hybrids or other cell lines that produce antibodies. Methods for obtaining and verifying such cultured cells are described by Harlow et al. (See above). Methods for preparing antigenic material for injection into an animal include methods known in the art, for example, using a full-length protein, peptides selected from an immunogenic region of this protein, modifying the antigen by methods such as, for example, binding to dinitrophenol, binding to arsanilic acid, antigen denaturation, antigen binding to a carrier protein, such as, for example, hemocyanin of the snail, to peptides containing binding sites for class II T-cell receptors, to beads, as well as any other methods known in the art. See Harlow et al. (See above).

Antibodies and Fabs that bind and neutralize Shiga toxin Stx1 and / or Stx2 according to the present invention may include multifunctional molecules, for example, bifunctional molecules containing at least one functional moiety that specifically binds to Shiga toxin Stx1 and / or Stx2 and neutralizes it. Such multifunctional molecules can include, for example, chimeric molecules, including a molecule that binds to the shiga toxin Stx1 and / or Stx2 and neutralizes it, and a second part that allows the chimeric molecule to bind to a substrate or allows it to be detected. in a way in which the binding with the shiga toxin Stx1 and / or Stx2 and its neutralization are not impaired. Examples of such second portions include, but are not limited to, fragments of an immunoglobulin molecule, a fluorescent protein, or an enzyme. Also, the antibodies and Fabs of the present invention that bind and neutralize the Shiga toxin Stx1 and / or Stx2 can be modified with polyethylene glycol, i.e. pegylated.

The term "interaction" as used herein means the introduction, addition of a sample suspected of containing shiga toxin Stx1 and / or Stx2 to an antibody or Fab that binds shiga toxin Stx1 and / or Stx2, for example, by combining or mixing the sample with the specified antibody or Fab. If shiga-toxin Stx1 and / or Stx2 is present in the sample, a complex of the antibody or Fab with shiga-toxin Stx1 and / or Stx2 is formed; the formation of such a complex means the ability of the Fab to bind selectively to the shiga toxin Stx1 and / or Stx2 to form a stable complex that can be detected. Detection can be qualitative, quantitative or semi-quantitative. Binding of the Shiga toxin Stx1 and / or Stx2 from the sample to the antibody or Fab occurs under conditions suitable for complex formation. Such conditions (eg, suitable concentrations, buffers, temperature, reaction time), as well as methods for optimizing such conditions, are well known to the person skilled in the art. Binding can be detected and measured using a variety of methods that are standard in the art, including, but not limited to, enzymatic immunochemistry methods (e.g., ELISA), immunoprecipitation, immunoblotting methods, and other immunochemistry methods described, for example, Sambrook et al. ((“Molecular Cloning: A Laboratory Manual, Second Edition”, Cold Spring Harbor Laboratory Press, 1989) and Harlow et al. (Supra) These references also describe examples of conditions under which the complex is formed.

The phrases "neutralizes the shiga toxin Stx1 and / or Stx2", "inhibits the activity of the shiga toxin Stx1 and / or Stx2" means that the specified antibody or Fab binds to the shiga toxin Stx1 and / or Stx2 in such a way that it neutralizes it, then eat inhibits the activity of shiga toxin Stx1 and / or Stx2 and blocks it. The ability of an antibody or Fab to neutralize (inhibit) the Shiga toxin Stx1 and / or Stx2 can be determined, for example, by the method described in Example 3.

Another object of the present invention is a composition for the treatment of toxic conditions caused by enterohaemorrhagic Escherichia coli, containing a mixture of humanized antibodies or their Fabs described above.

Preferred is a composition containing a mixture of two humanized antibodies, one of which contains the amino acid sequence SEQ ID NO: 7 of the heavy chain and the amino acid sequence SEQ ID NO: 9 of the light chain, and the second contains the amino acid sequence SEQ ID NO: 17 of the heavy chain and the amino acid sequence SEQ ID NO: 19 light chain.

The composition may also contain the necessary pharmaceutically acceptable auxiliary components such as carriers, excipients, preservatives and other components well known to those skilled in the art and necessary for the storage and administration of said composition to a subject in need of treatment.

The expression "pharmaceutically acceptable carrier" is conventional in the art and includes a pharmaceutically acceptable material, composition, or carrier suitable for administration to mammals, in particular humans. Carriers include a filler, diluent, diluent involved in carrying or transporting an agent from one organ or body part to another organ or body part. Each carrier must be "acceptable" in the sense that it must be compatible with the other ingredients of the composition and safe for the patient.

The term "filler" refers to an agent that can be added to a composition to provide a desired consistency, such as a change in bulk characteristics, to increase stability, and / or to adjust the osmotic concentration of a solution.

For example, for injectables, a pharmaceutically acceptable carrier may include buffering agents, preservatives, analgesics, solubilizers, isotonic agents, and stabilizers. For preparations administered by injection, the composition can be in the form of a unit dosage form, such as a vial containing drugs for multiple doses, or an ampoule for administration of a single dose.

The term "buffering agent" refers to a buffered solution that is resistant to changes in pH due to the action of acid-base coupled components. Buffers for pharmaceutical compositions typically have a pH in the range of about 4 to about 8; preferably from about 4.5 to about 7, and most preferably have a pH in the range from about 5.0 to about 6.5. Examples of buffers that will control the pH in this range include phosphate, acetate (eg sodium acetate), succinate (eg sodium succinate), gluconate, glutamate, histidine, citrate, and buffers including other organic acids. In one embodiment, a buffer suitable for use in a composition of the present invention is citrate and phosphate buffer.

On the other hand, examples of carriers, excipients and diluents suitable for the preparation of pharmaceuticals are lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose , methylcellulose, polyvinylpyrrolidone, water, mineral oils.

One embodiment of a composition according to the present invention, presented for illustrative purposes only, is the composition described in Example 4.

Doses of active substances, methods and modes of their administration are determined by the practitioner performing the treatment depending on the condition of the subject in need of treatment, the severity of the disease, the route of administration of the composition, body weight, sex, age and diet of the specified subject, general health, body reaction subject to the specified treatment, the duration of treatment and other factors known to specialists in this field and taken into account in the treatment.

The following examples are provided for purposes of explanation and do not limit the scope of the present invention in any way.

Examples of

Example 1. Obtaining murine antibodies against shiga toxins of the first and / or second types

To obtain antibodies, BALB / c mice were immunized twice with an interval of 2 weeks in the pads of the hind paws with an immunogen solution in complete (for the first immunization) or incomplete (for the second immunization) Freund's adjuvant. As an immunogen, a non-toxic analogue of type 2 shiga-like toxin (Stx2a) was used, provided by Yuri Vasilievich Kozlov, Federal State Budgetary Institution of Science, Institute of Molecular Biology named after V.I. V.A. Engelhardt Russian Academy of Sciences (IMB RAS) (Loukianov EV, Zacharova LA, Khasanova OS, Khasanov FK, Kozlov YV. Immunization with non-toxic variants of Shiga toxin type 2 (Stx2) generates high titers of protective antibodies. Dokl Biochem Biophys. 2015 ; 460: 23-5. Doi: 10.1134 / S160767291501007X. Epub 2015 Mar 13). For each round of immunization, 25 μg of immunogen was used. Hybridization of popliteal lymph node cells of immune mice with sp2 / 0 myeloma cells was carried out according to the standard method using polyethylene glycol with a molecular weight of 4000 (Monoclonal Antibody Production. National Research Council (US) Committee on Methods of Producing Monoclonal Antibodies. Washington (DC): National Academies Press (US); 1999.)

Screening of hybrid cell supernatants for antibodies against shiga toxins was performed three days after the second round of immunization by an indirect enzyme-linked immunosorbent assay (Engvall E, Perlmann P. Enzyme-linked immunosorbent assay (ELISA). Quantitative assay of immunoglobulin G., Immunochemistry. 1971, 8 (9): P.871-4).

As a result, 20 clones were obtained against shiga toxins, including clones H8 and H4.

Antibodies were accumulated in the ascites fluids of mice, for this mice, preliminarily primed with pristane, were injected intraperitoneally with 10 ⁶ cells of each clone. Purification of antibodies from ascites fluids was carried out by affinity chromatography on a column with protein-G sepharose according to the standard method (Jungbauer, A. et al. Comparison of protein A, protein G and copolymerized hydroxyapatite for the purification of human monoclonal antibodies, J. Chromatogr. 1989, 476: 257-268). The purity of the isolated antibodies was monitored by SDS-PAGE on 12% polyacrylamide gel electrophoresis in the presence of SDS and DTT in a Laemley step buffer system using the Mini PROTEAN 3 Electrophoresis System BIO-RAD, Catalog N 165-3301 (Laemmli, UK Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature 1970, 227: 680-685). The purity of all obtained antibodies was at least 95%.

Example 2. Obtaining humanized antibodies against shiga toxins of the first and / or second types

Then the authors of the present invention cloned the genes encoding the variable domains of the heavy and light chains of murine antibodies against shiga toxins Stx1 and Stx2, and determined their sequences.

The boundaries of the frame sections were determined according to Kabat, E.A., Wu, T.T., Reid-Miller, M., Perry, H.M. and Gottesman, K.S. (Sequences of Proteins of Immunological Interest, (1987) U.S. Department of Health and Human Services, NIH, Bethesda, MD) CDR sequences have been established.

Cross-reactive antibody H8 against shiga toxins of both the first and second types contains CDR1 with the amino acid sequence SEQ ID NO: 1, CDR2 with the amino acid sequence SEQ ID NO: 2, and CDR3 with the amino acid sequence SEQ ID NO: 3 of the heavy chain, and CDR1 with the amino acid sequence SEQ ID NO: 4, CDR2 with the amino acid sequence SEQ ID NO: 5, and CDR3 with the amino acid sequence SEQ ID NO: 6 of the light chain.

Antibody H4 against shiga toxins of the second type contains CDR1 with the amino acid sequence SEQ ID NO: 11, CDR2 with the amino acid sequence SEQ ID NO: 12, and CDR3 with the amino acid sequence SEQ ID NO: 13 of the heavy chain, and CDR1 with the amino acid sequence SEQ ID NO : 14, CDR2 with the amino acid sequence SEQ ID NO: 15, and CDR3 with the amino acid sequence SEQ ID NO: 16 of the light chain.

After that, the design and humanization of murine antibodies H8 and H4 against shiga toxins Stx1 and Stx2 were carried out.

The final sequences of the humanized variable domains of the anti-shiga toxin Stx1 and Stx2 antibodies were predominantly composed of the framework sequences of selected human variable domain genes and murine CDR sequences.

Numbering is in accordance with the Kabat nomenclature (Kabat numbering system, Kabat et al., 1987 “Sequences of Proteins of Immunological Interest,” US Dept. of Health and Human Services, US Government Printing Office).

Antibody H4

The method of Queen et al. (Queen et al. Proc. Natl. Acad. Sci. USA 86: 10029 (1989) and U.S. Pat. Nos. 5,585,089 and 5,693,762) was mainly used to preserve the affinity of the humanized murine H4 antibody. The choice of the source of the framework sequences for the humanized antibody can be critical to maintaining the affinity of the antibody. In principle, the framework sequences of any human antibody can serve as a template for grafting CDR regions. However, it has been shown that simple replacement of CDR regions in such a human antibody can lead to a significant decrease in affinity for antigen (Tempest et al., Biotechnology 9: 266 (1992)). The higher the homology between the sequences of the human and murine antibodies, the less likely human frameworks to introduce distortions in the structure of CDR sequences, leading to a decrease in affinity.

Using the IgBlast database, sequences of the functional variable regions of human germline antibodies were selected with the highest degree of homology to the amino acid sequences of the variable domains of the murine antibody H4 - IGHV4-4 * 08 - IGHJ6 * 01 and IGKV1-39 * 01- IGKJ4 * 01 for heavy and light chains, respectively. Based on the literature data and analysis of the spatial model of the variable fragments of the light and heavy chains of the mouse antibody H4, obtained using the Rosetta antibody modeling server, amino acid residues were selected in the sequences of human framework regions of the heavy chain, the reverse replacement of which with the corresponding residues of the mouse antibody H4 is likely to restore the affinity of the humanized H4-hum antibody. As a result of testing several variants of reverse substitutions, the sequences of the variable domains of the humanized H4-hum antibody were obtained, containing 7 reverse substitutions in the heavy chain: V67L, T68S, V71K, T73N, N76S, F78V and S79F. The genes of the variable domains of the light and heavy chains of the humanized antibody H4-hum were synthesized from a set of overlapping oligonucleotides using PCR. DNA fragments encoding the genes of the variable domains of the light and heavy chains of the humanized antibody H4 were combined by PCR with overlapping regions with the genes of the constant domain of the human light chain kappa C _κ and the constant domain of the heavy chain of human IgG1 C _H 1, respectively.

The nucleotide sequences encoding the heavy and light chains of said humanized H4-hum antibody are shown in SEQ ID NO: 18 and SEQ ID NO: 20, respectively.

Antibody H8

The procedure for humanizing the H8 antibody was carried out in the same way as for the antibody.

Using the IgBlast database, sequences of the functional variable regions of human germline antibodies were selected with the highest degree of homology to the amino acid sequences of the variable domains of the murine antibody H8 - IGHV1-3 * 01 - IGHJ4 * 03 and IGKV1-33 * 01 - IGKJ2 * 01 for heavy and light chain, respectively.

In the final sequence was introduced the reverse replacement R71V in the variable domain of the heavy chain of the humanized antibody H8-hum.

The nucleotide sequences encoding the heavy and light chains of said humanized H8-hum antibody are shown in SEQ ID NO: 8 and SEQ ID NO: 10, respectively.

Example 3. Study of the properties of humanized antibodies against shiga toxins of the first and / or second types

Immunoassay analysis .

Shiga toxins of types 1 and 2 (Stx1 and Stx2, respectively) were added to the wells of a 96-well ELISA plate in 0.1 M phosphate buffer pH 7.4 (PBS), incubated at 4 ° C overnight. The plates were washed three times with PBS with the addition of 0.05% Tween20 (PBS-T), then serial dilutions of antibodies in PBS-T with the addition of 0.2% bovine albumin (PBS-AT) were added. Each antibody dilution was taken in four replicates. Incubated for 1 hour at 37 ° C. The plates were washed, a solution of monoclonal antibodies against the kappa-light chain of human immunoglobulins conjugated with peroxidase (4G7-HRP) was added to the wells at a dilution of 1:75 thousand in PBS-AT. The plates were washed, a solution of TMB with hydrogen peroxide was added to the wells, and incubated for 20 minutes at room temperature until a color reaction developed. The reaction was stopped by adding a 0.5 M sulfuric acid solution. Optical density (OD) in the wells was measured at a wavelength of 450 nm. The mean values for the repetitions were found; from these values, a diagram of the dependence of the average OD on the concentration of antibodies was constructed. The results are shown in FIG. 1 and 2.

Conclusion: the H8 antibody is a cross-reactive antibody and is able to bind shiga toxins of both types, while the H4 antibody is able to effectively bind shiga toxins of the second type.

Neutralizing shiga-like toxins

The assay was performed on a Vero cell line.

The cells were seeded into the wells of a 96-well culture plate at a concentration of 5000 cells per well in complete growth medium DMEM supplemented with calf serum to a final concentration of 4%. Cultivated for 24 hours at 37 ° C in an atmosphere of 5% CO ₂ . The supernatants were then discarded and a mixture of Stx1 and Stx2 shiga toxins and humanized antibodies H8-hum and H4-hum were added to the wells in complete growth medium. Cultivated for 3 days at 37 ° C in an atmosphere of 5% CO ₂ . The MTT solution was added to the supernatants to a final concentration of 0.5%, incubated for 4 hours at 37 ° C and 5% CO ₂ . The supernatants were removed, and 100 μl of DMSO was added to the wells. The optical density in the wells was measured at a wavelength of 560 nm, the average values were calculated over 4 repetitions. Neutralization was evaluated as a percentage using the formula:

% neutralization = A _i / A ₀ x 100%,

where A _i - the average value of optical density in the wells with the i-th concentration of the antibody;

A ₀ is the average optical density in the wells without toxin.

The results are shown in FIG. 3 and 4.

Conclusion: antibody H8 effectively neutralizes shiga toxins of both types, and antibody H4 effectively neutralizes shiga toxins of the second type.

Example 4. Study of the effectiveness of the composition for the treatment of enterohemorrhagic infection caused by Escherichia coli

On the basis of the obtained humanized antibodies H4-hum and H8-hum, a composition against shiga toxins (finished dosage form, GLF) was obtained, intended for the treatment of enterohemorrhagic infection caused by Escherichia coli.

Used a composition of the following composition:

Active ingredients:

Antibody H4 (a recombinant humanized antibody against shigapid toxin of the second type Stx2) - 0.75 mg;

Antibody H8 (recombinant humanized antibody against shig-like toxins of the first and second types, Stxl and Stx2, respectively) - 2.25 mg.

Excipients:

Sodium citrate dihydrate 2.05 mg;

Polysorbate 80 - 2.1 mg;

Sodium chloride - 27.0 mg;

Dextran [cf. pier weight 40,000] 35-45 kDa, - 24.0 mg;

Water for injection - up to 3.0 ml.

The study was carried out on the basis of the vivarium of the All-Russian Scientific Center for Molecular Diagnostics and Treatment and the biotechnology laboratory of the All-Russian Scientific Center for Molecular Diagnostics and Treatment (117149, Moscow, Simferopolsky Boulevard, 8).

As a control solution, a buffer for GDF was used, which is a composition of excipients in the same concentrations as in the finished dosage form. For ethical reasons, control was used only in those experiments where the dosage of the injected toxins did not exceed the lethal doses of LD99.

The comparison drug was not used, since there are no drugs on the market that neutralize the effect of shiga toxins.

The studies were carried out on the finished dosage form (1x GDF) and 10-fold GDF (10x GDF).

As a reference drug, a buffer for GLF was used, which is a composition of excipients in the same concentrations as in the finished dosage form.

To simulate toxic conditions in animals, a shiga-like toxin of the second type was used in various dosages in physiological solution:

Shig-like toxin type 2 (complete toxin, holotoxin), subtype Stx2a,

(Toxin variant designation Stx2a-O157-EDL933, GenBank accession No. X07865).

The finished dosage form (GLF) for administration to experimental animals was obtained by diluting the concentrate of the drug solution (concentration for the active substance 1.0 mg / ml) with the buffer for GLF to a concentration of 1.3 mg / kg or 13 mg / kg for the active substance, which corresponds to human dosages of 0.1 mg / kg and 1 μ / kg, respectively, according to the Dose Conversion Table (Guidelines for the Experimental Study of New Pharmaceutical Substances. Ed. col. R.U. Khabriev et al. - Moscow: Medicine, 2005, p. . 49). The drug was injected intravenously into the tail vein in a volume of 0.25 ml.

Dosing of toxins to experimental animals was carried out in nanograms or micrograms of active substance per kilogram of body weight of experimental animals. Before the introduction, the volume of the preparation that should be administered to each individual animal was calculated depending on its body weight. Toxins were injected intraperitoneally in a volume of 0.5 ml.

Animal species: male Balb / c mice, 7-8 weeks old. Origin: Nursery "Stolbovaya" of the Federal State Budgetary Institution of Science "Scientific Center for Biomedical Technologies of the Federal Medical and Biological Agency"

Weight at the start of the study: 20-25 g.

Identification method: wool dyeing with fucorcin.

The animals were kept under conventional conditions with free access to water and food. The basic rules of maintenance and care were in accordance with the normative documents. All procedures for the routine care of animals were performed in accordance with the SOP of JSC VNTsMDL.

Accommodation: The animals were housed in polycarbonate cages with bedding, 6-8 cages in 26x17x12 cm cages. The cages are equipped with steel lids with a feeding recess, steel feed dividers and steel label holders.

Food: complete feed for keeping small laboratory rodents, autoclavable apathogenic production of OOO Assortiment-Agro.

Quarantine: at least 14 days.

The following parameters were recorded daily:

- survival rate;

- body mass;

- presence / absence of diarrhea;

- physical activity;

- the condition of the coat and skin.

The dosages of the Stx2 toxin were preliminarily determined, causing the death of 50% of animals (LD50) and 99% of animals (LD99). To determine these indicators, the express method according to Prozorovsky was used (Prozorovsky, V. B. Express method for determining the average effective dose and its error / V. B. Prozorovsky, M. P. Prozorovskaya, V. M. Demchenko // Pharmacology and toxicology - 1978. - T. 41, No. 4. P. 497 - 501), (Prozorovsky, VB Statistical processing of the results of pharmacological studies / VB Prozorovsky // Psychopharmacology and biological narcology. - 2007. - T. 7, No. 3-4. C 2090-2120) and Bliss probit analysis (Bliss, CI The method of probits. / CI Bliss // Science. 1934. - 12 Jan: Vol. 79, Issue 2037, pp. 38 -39.).

For experiments to determine the effectiveness of GLF in vivo, dosages in multiples of LD50 were used.

The toxin was administered intraperitoneally either simultaneously with the drug (the time interval between the administration of the toxin and the drug was less than 10 minutes), or 24 hours before the administration of the drug (delayed treatment).

The mice were monitored until they died, or for 14 days.

conclusions

With intravenous, intraperitoneal and subcutaneous administration of Stx2, a sequential dose increase always leads to the death of the animals. The lethal outcome occurs 3-6 days after the injection of the toxin.

For the intraperitoneal route of administration of the Stx2 toxin, the LD50 and LD99 values were 83 ± 8 ng / kg and 186 ng / kg, respectively.

With the introduction of sublethal doses of shiga toxins, external and internal signs of intoxication are observed, the severity of which correlates well with the loss of body weight in animals.

The effect of type II shiga toxin on the body of mice was investigated in a wide range of dosages, up to values corresponding to 100-fold LD50 or 44.6-fold LD99. When the toxin was administered simultaneously with the administration of the drug, no significant signs of intoxication were found in the mice, and no deaths were recorded. Therefore, the drug almost completely protects the body from the action of the toxin.

The effect of shiga-toxins on the body of mice was investigated after treatment with the study drug was delayed for 24 hours. It has been established that the effectiveness of protection with delayed treatment is lower than with timely treatment.

The indicators of the effectiveness of the composition in delayed treatment were calculated: survival, protection index (IZ), antidote power (AM) and guaranteed protection index (IGI).

The results of the experiment are shown in Table 1.

Table 1. The main quantitative indicators of the effectiveness of the composition

Survival at LD50 Survival at LD99 OF AM IGZ simultaneous administration of the toxin and the drug GLF one hundred% one hundred% > 100 > 44.6 > 44.6 10xGLF one hundred% one hundred% > 100 > 44.6 > 44.6 drug administration 24 hours after toxin administration GLF 95.8% 52% 2.29 1.022 0.553 10xGLF 95.2% 25% 1,777 0.793 0.508

Although the present invention has been described in detail with reference to preferred embodiments thereof, it will be apparent to those skilled in the art that various substitutions and equivalents may be used that do not depart from the scope of the present invention.

All documents cited in this description are part of this application and are fully incorporated into this description by reference.

--->

Sequence listing

<110> Open Joint Stock Company All-Russian Scientific Center

molecular diagnostics and treatment "(JSC VNTSMDL)

<120> Humanized antibody or antigen binding fragment (Fab)

against shiga toxins of the first and / or second types (options),

composition for the treatment of toxic conditions caused by

enterohaemorrhagic Escherichia coli containing the indicated

antibodies and / or Fab.

<130> mAbs against Stx1 and Stx2

<160> 20

<170> PatentIn version 3.5

<210> 1

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR1 VH of humanized mAb against Stx1 and Stx2

<400> 1

Glu Tyr Thr Met His

15

<210> 2

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR2 VH of humanized mAb against Stx1 and Stx2

<400> 2

Gly Ile Asn Pro Asn Asn Gly Gly Thr Ser Tyr Asn Gln Lys Phe Lys

1 5 10 15

Gly

<210> 3

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR3 VH of humanized mAb against Stx1 and Stx2

<400> 3

Val Tyr Ser Lys Ala Trp Phe Ala Tyr

15

<210> 4

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR1 VL of humanized mAb against Stx1 and Stx2

<400> 4

Lys Ala Ser Gln Ser Val Ser Asn Val Val Ala

1 5 10

<210> 5

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR2 VL of humanized mAb against Stx1 and Stx2

<400> 5

Phe Ala Ser Asn Arg Tyr Thr

15

<210> 6

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR3 VL of humanized mAb against Stx1 and Stx2

<400> 6

Gln His Asp Tyr Arg Ser Pro Tyr Thr

15

<210> 7

<211> 448

<212> PRT

<213> Artificial Sequence

<220>

<223> Heavy chain of humanized mAb against Stx1 and Stx2

<400> 7

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Glu Tyr

20 25 30

Thr Met His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met

35 40 45

Gly Gly Ile Asn Pro Asn Asn Gly Gly Thr Ser Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Arg Val Thr Ile Thr Val Asp Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Val Tyr Ser Lys Ala Trp Phe Ala Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr

210 215 220

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser

225 230 235 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

245 250 255

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

260 265 270

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

275 280 285

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

290 295 300

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

305 310 315 320

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

325 330 335

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

340 345 350

Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys

355 360 365

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

370 375 380

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

385 390 395 400

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

405 410 415

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

420 425 430

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 8

<211> 1344

<212> DNA

<213> Artificial Sequence

<220>

<223> ORF encoding VH of H8 mAb against Stx1 and Stx2

<400> 8

caggtccagc ttgtgcagtc tggggctgag gtgaagaagc ctggggcctc agtgaaggtt 60

tcctgcaagg cttctggata caccttcact gaatacacca tgcactgggt gcgccaggcc 120

cccggacaaa ggcttgagtg gatgggaggt attaatccta acaatggtgg tactagctac 180

aaccagaagt tcaagggcag agtcaccatt accgtagaca catccgcgag cacagcctac 240

atggagctga gcagcctgag atctgaagac acggctgtgt attactgtgc gagagtctac 300

agtaaggcct ggtttgctta ctggggccaa gggaccctgg tcaccgtctc ctcagcctcc 360

accaagggcc catcggtctt ccccctggca ccctcctcca agagcacctc tgggggcaca 420

gcggccctgg gctgcctggt caaggactac ttccccgaac cggtgacggt gtcgtggaac 480

tcaggcgccc tgaccagcgg cgtgcacacc ttcccggctg tcctacagtc ctcaggactc 540

tactccctca gcagcgtggt gaccgtgccc tccagcagct tgggcaccca gacctacatc 600

tgcaacgtga atcacaagcc cagcaacacc aaggtggaca agagagttga gcccaaatct 660

tgtgacaaaa ctcacacatg cccaccgtgc ccagcacctg aactcctggg gggaccgtca 720

gtcttcctct tccccccaaa acccaaggac accctcatga tctcccggac ccctgaggtc 780

acatgcgtgg tggtggacgt gagccacgaa gaccctgagg tcaagttcaa ctggtacgtg 840

gacggcgtgg aggtgcataa tgccaagaca aagccgcggg aggagcagta caacagcacg 900

taccgtgtgg tcagcgtcct caccgtcctg caccaggact ggctgaatgg caaggagtac 960

aagtgcaagg tctccaacaa agccctccca gcccccatcg agaaaaccat ctccaaagcc 1020

aaagggcagc cccgagaacc acaggtgtac accctgcccc catcccggga ggagatgacc 1080

aagaaccagg tcagcctgac ctgcctggtc aaaggcttct atcccagcga catcgccgtg 1140

gagtgggaga gcaatgggca gccggagaac aactacaaga ccacgcctcc cgtgctggac 1200

tccgacggct ccttcttcct ctatagcaag ctcaccgtgg acaagagcag gtggcagcag 1260

gggaacgtct tctcatgctc cgtgatgcat gaggctctgc acaaccacta cacgcagaag 1320

agcctctccc tgtctccggg taaa 1344

<210> 9

<211> 214

<212> PRT

<213> Artificial Sequence

<220>

<223> Light chain of humanized mAb against Stx1 and Stx2

<400> 9

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Ser Val Ser Asn Val

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Phe Ala Ser Asn Arg Tyr Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Gln His Asp Tyr Arg Ser Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 10

<211> 642

<212> DNA

<213> Artificial Sequence

<220>

<223> ORF encoding VL of H8 mAb against Stx1 and Stx2

<400> 10

gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60

atcacttgca aggccagtca gagtgtgagt aatgttgtag cttggtatca gcagaaacca 120

gggaaagccc ctaagctcct gatctacttt gcatccaatc gctacactgg ggtcccatca 180

aggttcagtg gaagtggatc tgggacagat tttactttca ccatcagcag cctgcagcct 240

gaagatattg caacatatta ctgtcagcac gattataggt ctccgtacac gtttggccag 300

gggaccaagc tggagatcaa acggactgtg gctgcaccat ctgtcttcat cttcccgcca 360

tctgatgagc agttgaaatc tggaactgcc tctgttgtgt gcctgctgaa taacttctat 420

cccagagagg ccaaagtaca gtggaaggtg gataacgccc tccaatcggg taactcccag 480

gagagtgtca cagagcagga cagcaaggac agcacctaca gcctcagcag caccctgacg 540

ctgagcaaag cagactacga gaaacacaaa gtctacgcct gcgaagtcac ccatcagggc 600

ctgagctcgc ccgtcacaaa gagcttcaac aggggagagt gt 642

<210> 11

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR1 VH of humanized mAb against Stx2

<400> 11

Thr Tyr Gly Val His

15

<210> 12

<211> 16

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR2 VH of humanized mAb against Stx2

<400> 12

Val Ile Trp Ala Gly Gly Ser Thr Asn Tyr Asn Ser Ala Leu Met Ser

1 5 10 15

<210> 13

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR3 VH of humanized mAb against Stx2

<400> 13

Gly Gly Arg Tyr Tyr Ala Met Asp Tyr

15

<210> 14

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR1 VL of humanized mAb against Stx2

<400> 14

Arg Ala Ser Glu Asn Ile Tyr Ser Tyr Leu Ala

1 5 10

<210> 15

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR2 VL of humanized mAb against Stx2

<400> 15

Asn Ala Lys Thr Leu Ala Glu

15

<210> 16

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR3 VL of humanized mAb against Stx2

<400> 16

Gln His His Tyr Leu Thr Pro Trp Thr

15

<210> 17

<211> 447

<212> PRT

<213> Artificial Sequence

<220>

<223> Heavy chain of humanized mAb against Stx2

<400> 17

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Thr Tyr

20 25 30

Gly Val His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Val Ile Trp Ala Gly Gly Ser Thr Asn Tyr Asn Ser Ala Leu Met

50 55 60

Ser Arg Leu Ser Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Phe Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Gly Gly Arg Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Thr

100 105 110

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

180 185 190

Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His

210 215 220

Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val

225 230 235 240

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

245 250 255

Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu

260 265 270

Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

275 280 285

Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser

290 295 300

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

305 310 315 320

Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile

325 330 335

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

340 345 350

Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

355 360 365

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

370 375 380

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

385 390 395 400

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

405 410 415

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

420 425 430

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 18

<211> 1341

<212> DNA

<213> Artificial Sequence

<220>

<223> ORF encoding VH of H4 mAb against Stx2

<400> 18

caggtgcagc tgcaggagtc gggaccagga ctggtgaagc cttcggagac cctgtccctc 60

acctgcactg tctctggtgg ctccatcagt acctatggtg tacactggat tcggcagccc 120

ccagggaagg gactggagtg gattggggta atatgggctg gtggaagcac aaattataat 180

tcggctctca tgtcccgact gagcatcagc aaagacaact ccaagagcca agttttcctg 240

aagctgagct ctgtgaccgc cgcagacacg gccgtgtatt actgtgcgag agggggccgt 300

tactatgcta tggactactg ggggcaaggg accacggtca ccgtctcctc agcctccacc 360

aagggcccat cggtcttccc cctggcaccc tcctccaaga gcacctctgg gggcacagcg 420

gccctgggct gcctggtcaa ggactacttc cccgaaccgg tgacggtgtc gtggaactca 480

ggcgccctga ccagcggcgt gcacaccttc ccggctgtcc tacagtcctc aggactctac 540

tccctcagca gcgtggtgac cgtgccctcc agcagcttgg gcacccagac ctacatctgc 600

aacgtgaatc acaagcccag caacaccaag gtggacaaga gagttgagcc caaatcttgt 660

gacaaaactc acacatgccc accgtgccca gcacctgaac tcctgggggg accgtcagtc 720

ttcctcttcc ccccaaaacc caaggacacc ctcatgatct cccggacccc tgaggtcaca 780

tgcgtggtgg tggacgtgag ccacgaagac cctgaggtca agttcaactg gtacgtggac 840

ggcgtggagg tgcataatgc caagacaaag ccgcgggagg agcagtacaa cagcacgtac 900

cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaatggcaa ggagtacaag 960

tgcaaggtct ccaacaaagc cctcccagcc cccatcgaga aaaccatctc caaagccaaa 1020

gggcagcccc gagaaccaca ggtgtacacc ctgcccccat cccgggagga gatgaccaag 1080

aaccaggtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag 1140

tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc 1200

gacggctcct tcttcctcta tagcaagctc accgtggaca agagcaggtg gcagcagggg 1260

aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac gcagaagagc 1320

ctctccctgt ctccgggtaa a 1341

<210> 19

<211> 214

<212> PRT

<213> Artificial Sequence

<220>

<223> Light chain of humanized mAb against Stx2

<400> 19

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Asn Ile Tyr Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Asn Ala Lys Thr Leu Ala Glu Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His His Tyr Leu Thr Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 20

<211> 642

<212> DNA

<213> Artificial Sequence

<220>

<223> ORF encoding VL of H4 mAb against Stx2

<400> 20

gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60

atcacttgcc gagcaagtga gaatatttac agttatttag catggtatca gcagaaacca 120

gggaaagccc ctaagctcct gatctataat gcaaaaacct tagcagaagg ggtcccatca 180

aggttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct 240

gaagattttg caacttacta ctgtcaacat cattatctta ctccgtggac gttcggcgga 300

gggaccaagg tggaaatcaa acggactgtg gctgcaccat ctgtcttcat cttcccgcca 360

tctgatgagc agttgaaatc tggaactgcc tctgttgtgt gcctgctgaa taacttctat 420

cccagagagg ccaaagtaca gtggaaggtg gataacgccc tccaatcggg taactcccag 480

gagagtgtca cagagcagga cagcaaggac agcacctaca gcctcagcag caccctgacg 540

ctgagcaaag cagactacga gaaacacaaa gtctacgcct gcgaagtcac ccatcagggc 600

ctgagctcgc ccgtcacaaa gagcttcaac aggggagagt gt 642

<---

Claims (6)

1. Humanized antibody or its antigen-binding fragment (Fab), selectively binds (s) to shiga-like toxins of the first and second types and neutralizes them , containing (s) CDR1 with the amino acid sequence SEQ ID NO: 1, CDR2 with the sequence amino acids SEQ ID NO: 2, and CDR3 with amino acid sequence SEQ ID NO: 3 of the heavy chain, and CDR1 with amino acid sequence SEQ ID NO: 4, CDR2 with amino acid sequence SEQ ID NO: 5, and CDR3 with amino acid sequence SEQ ID NO: 6 light chain. 2. The humanized antibody of claim 1, comprising the amino acid sequence of SEQ ID NO: 7 of the heavy chain and the amino acid sequence of SEQ ID NO: 9 of the light chain. 3. Humanized antibody or its antigen-binding fragment (Fab), selectively binds to and neutralizes shiga-like toxin of the second type , containing (s) CDR1 with the amino acid sequence SEQ ID NO: 11, CDR2 with the amino acid sequence SEQ ID NO : 12, and CDR3 with amino acid sequence SEQ ID NO: 13 of the heavy chain, and CDR1 with amino acid sequence SEQ ID NO: 14, CDR2 with amino acid sequence SEQ ID NO: 15, and CDR3 with amino acid sequence SEQ ID NO: 16 of the light chain. 4. The humanized antibody of claim 3 comprising the amino acid sequence of SEQ ID NO: 17 of the heavy chain and the amino acid sequence of SEQ ID NO: 19 of the light chain. 5. Composition for the treatment of toxic conditions caused by enterohemorrhagic Escherichia coli, containing a mixture of humanized antibodies or their Fab according to PP. 1 and 3, and a pharmaceutically acceptable carrier. 6. The composition according to claim 5, characterized in that said mixture of humanized antibodies is a mixture of humanized antibodies according to claims. 2 and 4.

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