Modified adenylate cyclase bordetella containing cd11b/cd18 interaction domain or cd11b/cd18 interaction domain deficient, and its application

FIELD: medicine, pharmaceutics.

SUBSTANCE: group of inventions refers to medicine, namely to infectious diseases, and can be used for treating whooping cough and/or for Bordetella infection protection. Polypeptide under the invention represents a fragment of adenylate cyclase Bordetella containing CD11b/CD18 interaction domain from an amino acid sequence extended from position 1166 to position 1281 SEQ ID NO:1. Said invention also concerns the specific fragments of adenylate cyclase Bordetella containing CD11b/CD18 interaction domain, and to their application, particularly for targeting of molecules of interest to CD11b expressing cells.

EFFECT: use of the inventions allows extending the range of products for treatment and prevention of the Bordetella infection.

26 cl, 9 dwg

 

The invention relates to modified adenylyl cyclase toxins Bordetella, which are defective in regard to the binding of CD11b/CD18, and to their use in the preparation of pharmaceutical compositions for the treatment of pertussis and/or to protect against Bordetella infection. This invention relates also to a specific fragments of Bordetella adenylate cyclase containing domain interaction CD11b/CD18, and to their use, in particular for targeting interest molecules on expressing CD11b-cells.

The genus Bordetella contains four types, namely: Bordetella pertussis, Bordetella parapertussis, Bordetella bronchiseptica and Bordetella avium.

Bordetella are gram coccobacillary responsible for respiratory infections. Bordetella pertussis and Bordetella parapertussis exactly are human pathogens. Bordetella bronchiseptica is pathogenic for a variety of mammals, and more rarely for people, and, unlike B.pertussis and Bordetella parapertussis, able to survive outside of the host. Bordetella avium is pathogenic for birds.

The most virulent species for humans is B.pertussis, which is the etiologic agent of whooping cough, a highly contagious respiratory disease of children characterized by bronchopneumonia and paroxysmal cough, interrupted breathing, convulsive breaths during a coughing fit.

Vaccination against pertussis is still spent on the more frequently with the use of inactivated whole bacteria. However, such vaccines are not always devoid of toxicity due to the fact that the virulence factors consist of proteins secreted by these bacteria, and not of the bacteria. Thus, these proteins can be severe pathological actions, even after the death of these bacteria.

European patent EP 0424158 (Pasteur Institute) describes the use of Bordetella adenylate cyclase as protective antigens against Bordetella pertussis and against Bordetella bronchiseptica.

European patent EP 0338169 (Pasteur Institute) describes the use of drugs active adenylate cyclase from Bordetella parapertussis as protective agents against whooping cough.

Were also developed other strategies, including the preparation of acellular vaccines using immunogenic detoxificating Bordetella toxins.

An example of a vaccine based on detoxificating pertussis toxin described in U.S. Patent SN 6040427 (Locht et al., 2000).

Among the various toxins produced by B.pertussis, adenylate cyclase (also referred to hereinafter by the term CyaA) is a decisive factor in the strategy of virulence of these bacteria during the early phases of colonization of the respiratory tract (Goodwin and Weiss, 1990; Khelef et al., 1992). This toxin allows the pathogen to avoid immune surveillance of the host, mainly through intoxication of neutrophils and macrophages, the induction of weakening phagocyte and induction of apoptosis of macrophages (Confer and Eaton, 1982; Gueirard et al., 1998; Harvill et al., 1999; Khelef and Guiso, 1995; Khelef et al., 1993). The role of CyaA in the pathogenesis of B.pertussis was clearly demonstrated in a murine respiratory model. Indeed, genetically modified B. Pertussis strains defective in the expression of CyaA were weakened in their ability to induce pulmonary damage and cause a lethal infection (Khelef et al., 1994; Weiss and Goodwin, 1989). On the other hand, it was shown that CyaA induces protective immunity against colonization of the lungs B.pertussis in the mouse model (Betsou et al., 1993; Betsou et al., 1995; Hormozi et al., 1999).

CyaA is a polypeptide with a length 1706 amino acid residues consisting of four functional domains; the domain of activity of adenylate cyclase (AC) (residues 1-400), hydrophobic channel-forming domain (residues 500-700), cellisvisible rich repeats of glycine/aspartate domain (residues 1000-1600) and C-terminal domain carrying the secretion signal (residues 1600-1706). CyaA able to enter eukaryotic cells and move its catalytic domain to the cytoplasm, where upon activation of the endogenous calmoduline, it catalyzes the conversion of ATP to camp (Ladant and Ullman, 1999). It is believed that the accumulation of camp in the cytosol of cells is responsible for the toxic action of this toxin (Rogel et al., 1991). The main consequences of this toxicity are apoptosis and change phagocytic JV is Sobota and the production of superoxide (Confer and Eaton, 1982; Friedman et al., 1987; Khelef et al., 1993; Njamkepo et al., 2000; Pearson et al., 1987).

The full sequence of the Bordetella pertussis adenylate cyclase shown in SEQ ID NO:1.

The full sequence of adenylate cyclase Bordetella bronchiseptica shown in SEQ ID NO:3.

CyaA need calcium to purchase specific to move conformation, which makes possible the delivery of the catalytic domain into the cytosol of cells (Regel and Hanski, 1992; Rose et al., 1995). Initially, CyaA is produced in the form of inactive protoxin, proCyaA, which, after post-translational modification by acyltransferase, the cyaC gene product is the active toxin. This covalent posttranslational acylation (atilov fatty acids) required to move the toxin through the membranes of target cells and deliver its catalytic domain of the AU, as well as for education hemolytic cation-selective channels. The acylation proCyaA occurs in two different positions, Lys-983 and Lys-860, which are located in conservative sites acylation RTX (Barry et al., 1991; Hackett et al., 1994). Although the acylation Lys-860, apparently, is not necessary for the activity of CyaA, it was shown that the acylation Lys-983 is decisive (Basar et al., 2001).

CyaA can penetrate in a wide range of cell types, including cells of mammals, devoid of membrane transport (Bellalou et al., 1990; Gray et al., 1999; Rogel and Hanski, 1992). In across the lagoon to the falsity of this effects the toxicity of CyaA, such as neutralization of phagocytic capacity and the induction of apoptosis, were mainly detected in immune cells, namely neutrophils and macrophages (Confer and Eaton, 1982; Khekef et al., 1993). In addition, in a murine respiratory infection has been shown that CyaA shows specific toxicity against alveolar macrophages (Gueirard et al., 1998). In Patent WO 93/21324 (Pasteur Institute, 1993) also described a vaccine containing recombinant adenylate cyclase toxin produced by B.pertussis attached to heterologous epitopes. Recently it was demonstrated that CyaA specifically binds to target cells via the integrin αMβ2(CD11b/CD18). This binding was saturable and completely inhibiting monoclonal anti-CD11b-antibody. CyaA showed selective toxicity against CD11b+cells, indicating that its interaction with CD11b is necessary to move the catalytic domain and the subsequent increase in cyclic amp and cell death. In addition, the sensitivity of cells SNO to the cytotoxicity of CyaA was markedly increased after expression of heterodimer CD11b/CD18. In addition, ions of CA2+that are required to move the catalytic domain into cells, were also strictly required for the interaction of CyaA with CD11b (Guermonprez et al., 2001). The importance of CD11b for the interaction of CyaA with cells was additional is but demonstrated in the system, where CyaA used as a vector to deliver foreign antigens in antigenpresenting cells, such as dendritic cells. Only dendritic cells of a subpopulation of CD11c+CD8α-CD11bhighwere actually able to present peptide complexes, MHC class I, corresponding to the epitope insertiona in recombinant CyaA (Guermonprez et al., 2002).

Protein CD11b is a member of a large family of β2-integrins, adhesion molecules of leukocytes, which contains LFA1 (CD11a), MAC-1 (CD11b) and P150,95 (CD11c). Members of this family differ in their α-chain, which is expressed in the form of obligatory heterodimer with β-chain (CD18) (Arnaout, 1990). CD11b, also known as receptor type 3 complement (CR3), is expressed on macrophages, neutrophils, dendritic cells, NK-cells, peritoneal cells In a-1 and a subpopulation of CD8+T cells (Arnaout, 1990; Bell et al., 1999). He plays a key role in adhesion function of leukocytes and initiates phagocytosis covered by the complement of the particles (Diamond and Springer, 1993). CD11b binds a variety of ligands, such as molecule intercellular adhesion ICAM-1, fibrinogen, coagulants the X factor and component inactivated C3b complement (iC3b) (Altieri and Edgington, 1988; Beller et al., 1982; Diamond et al., 1990; Wright et al., 1988).

Based on the binding properties of CyaA with CD11b/CD18, European patent application OR (Pasteur Institute) describes protein vectors, with the holding of recombinant adenylate cyclase type Bordetella, for the target of interest molecules, and in particular, the antigen on dendritic cells.

Now, the invention is based on the discovery that one or more areas of the Bordetella pertussis adenylate cyclase contained in the amino acid sequence extending from amino acid 1166 to amino acids 1281 (SEQ ID NO:2) are crucial for the interaction of the toxin with CD11b/CD18. This area is necessary to ensure CyaA binding capacity with CD11b/CD18 may be further combined with other areas of CyaA, acting as auxiliary areas.

This discovery provides the opportunity to obtain effective and multilateral vector delivery of molecules able to target interest molecule on dendritic cells. Alternatively, the deletion of the identified domain interaction CD11b/CD18 - cyclase can be used best way to design safer acellular vaccines for protection against Bordetella infection and, in particular, infection with Bordetella pertussis.

This invention also provides the use of the identified domain interaction CD11b/CD18 to obtain neutralizing antibodies that can block the interaction of native cyclase, produced infectious bacteria with cell receptors.

Thus the m the purpose of this invention is the provision of a protein consisting of Bordetella adenylate cyclase, which is modified in the domain of interaction of CD11b/CD18 by deletion, substitution or insertion of one or more amino acids, where this protein is defective in regard to the binding of CD11b/CD18, but is specifically reactive with antisera that recognizes the Bordetella adenylate cyclase wild-type.

The protein of this invention can be used as active principle in the pertussis vaccine. Thus, the mutation (mutation) in the domain of interaction CD11b/CD18 protects immune cells from the potentially negative effects, such as the transmission signal after seizing integrin-toxoid, and/or some functional interference due to competition for binding to CD11b with CyaA-toxodon, which also serves as a receptor CR3-complement.

In this context, the term "polypeptide" refers to a single chain of amino acids linked by peptide bonds, contain at least 6 amino acids, preferably at least 10 amino acids and more preferably at least 50 amino acids.

The term "protein" refers to a macromolecule, which essentially consists of one or more polypeptides.

The term "Bordetella adenylate cyclase" includes, in this invention, calmodulin is avishay adenylate cyclase, which natural synthesized in Bordetella species and which is the main virulence factor, required for the initial stages of bacterial colonization in the lung.

In one preferred embodiment, the protein of the present invention is obtained by modification of adenylate cyclase Bordetella pertussis, the agent of whooping cough in man.

In Bordetella pertussis this cyclase is synthesized and secreted in the form of the polypeptide from 1706 amino acids (SEQ ID NO:1). Calmodulinzawisimoy catalytic activity localized in the first 400 amino acids, and this domain is hereinafter referred to as "N-terminal catalytic domain. As previously reported, in order to be active, the specified adenylyl cyclase toxin do invasive and hemolytic after posttranslational modification by co-expression of gene product CyaC.

According to this invention the expression "domain interaction CD11b/CD18" refers to:

A. domain interaction CD11b/CD18 Bordetella pertussis, stretching from 1166 amino acids to amino acids 1281 Bordetella pertussis adenylate cyclase (SEQ ID NO:2) or

b. the domain of the adenylate cyclase of Bordetella species, appropriate domain interaction CD11b/CD18 Bordetella pertussis, as identified by mapping the sequence of adenylate cyclase these species Bordetella sequence of the Bordetella pertussis adenylate cyclase using algo is ITMA to find the best local mappings.

An example algorithm for finding the best local mapping is the BLAST algorithm (Altschul et al., 1990).

Domain interaction CD11b/CD18 Bordetella bronchiseptica represented by SEQ ID NO:4.

In this context, the expression “defective in regard to the binding of CD11b/CD18” means that the protein of the present invention does not compete with the adenylate cyclase wild type Bordetella for binding to expressing CD11b/CD18 αmβ2cells. "CD11b/CD18 αmβ2or CD11b/CD18" refers to the cellular receptor of Bordetella adenylate cyclase (Guermonprez et al., 2001). Examples of analyses linking to assess specific binding of recombinant toxin expressing CD11b/CD18 αmβ2the cells described in the following experimental part. The protein of the present invention preferably has less than 50% of the binding affinity against CD11b/CD18 αmβ2in comparison with the Bordetella adenylate cyclase wild type. Most preferably, the protein of the present invention is less than 10% and more preferably less than 5% of the analyzed binding affinity.

In this context, the expression “expressing CD11b-cells” refers to cells that Express CD11b/CD18 αmβ2on their surface. In particular, these cells are granulocytes/neutrophils, macrophages, NK-cells, a subpopulation of CD8+T-cells and b-cells is to and myeloid dendritic cells.

To ensure the protein of the present invention, the domain of interaction CD11b/CD18-Bordetella adenylate cyclase altered by insertion, deletion or replacement of one or several amino acids, and the obtained protein is defective in regard to the binding of CD11b/CD18.

In one embodiment of the present invention, the domain of interaction CD11b/CD18 modified by insertion of peptide. For example, a sequence consisting of residues 6-12, insertyour in the domain of interaction CD11b/CD18.

Specific options for implementation include Bordetella adenylate cyclase, modified by insertion between residues 1166-1167 or between residues 1281-1282 (the number indicates the position of these amino acids in the Bordetella pertussis adenylate cyclase wild-type) peptide containing 6 to 12 amino acids. Examples of the insertion of the FLAG epitope sequences in these provisions are described in the following experimental part, and hereinafter referred to as CyaA1166/FLAG and CyaA1281/FLAG.

Alternatively, residues, which, as shown, are involved in binding to CD11b/CD18, can be deleterows or replaced by non-functional remnants.

In one specific embodiment, the Bordetella adenylate cyclase altered by insertion, deletion or replacement of one or several amino acids in the region extending from residue 1208 to 1243 in the Bordetella pertussis adenylate cyclase or in the relevant area of the x adenylatecyclase other Bordetella species.

Preferred embodiments of the invention relate to protein and include Bordetella pertussis adenylate cyclase containing deletions of one or more amino acids or replacement of non-functional amino acids.

In one preferred embodiment, the Bordetella adenylate cyclase modify complete deletion of the domain interaction CD11b/CD18.

According to another particular variant of implementation of the present invention Bordetella pertussis adenylate cyclase modify a deletion of amino acids extending from position 1245 to position 1273, and these amino acids are optionally replaced by non-functional amino acids, for example oktapeptidom, as shown in the example of the experimental part, and this protein is hereinafter referred to as CyaAΔ1245-1273.

Additionally, for security guarantees for introduction into a living organism of the protein of the present invention Bordetella adenylate cyclase modify so that the catalytic activity is removed. According to one variant of implementation of the present invention Bordetella adenylate cyclase additionally modify the insertion, deletion or replacement of one or several amino acids in the N-terminal catalytic domain and the specified modified Bordetella adenylate cyclase has catalytic activity, which is reduced in comparison with kataliticheski the th activity of Bordetella adenylate cyclase wild type. Preferably this catalytic activity is less than 10% of the catalytic activity of Bordetella adenylate cyclase wild type, and more preferably is insignificant.

Examples of mutants in the N-terminal catalytic domain described in the art (for example, in WO 93/21324, Pasteur Institute).

Variants of the protein of the present invention include modified cyclase type Bordetella, devoid of at least amino acids 1-300 N-terminal catalytic domain, and preferably devoid of amino acids 1-373.

Alternatively, a dipeptide insertions can be performed in the ATP-binding site between residues 188 and 190 of the Bordetella pertussis adenylate cyclase or the corresponding residues in the cyclase from other Bordetella species.

In this invention it is also shown that the acylation of Bordetella adenylate cyclase is involved in the binding of CD11b/CD18 and the subsequent relocation of the toxin into the cell. Thus, in one preferred embodiment, the protein of the present invention the protein is acylated. In particular, the Bordetella adenylate cyclase additionally modified in the amino acids that allroots excision. These amino acids correspond to Lys-983 and Lys-860 Bordetella pertussis adenylate cyclase.

In this particular embodiment, the protein is not alleroed in position 983 and/or 860 on the sledovatelnot adenylate cyclase.

In another embodiment, the protein of the present invention is acylated.

The protein of the present invention is preferably immunogenic, but essentially non-toxic protein, i.e. a protein that is at least defective in respect of binding to a receptor of a cell and not necessarily in respect of adenylyl cyclase activity, but which is still specifically recognized by antibodies against adenylate cyclase toxin.

This invention relates also to pharmaceutical compositions containing the above-defined protein, in combination with a pharmaceutically acceptable carrier.

According to one variant of implementation of the specified composition is a vaccine suitable for human or animal. The vaccine is preferably capable of inducing immunity against pertussis. This vaccine contains immunoprotective and non-toxic amount of the protein of the present invention. This composition may further contain one or more suitable Primerose adjuvants, respectively. Other antigens known that they are preferably administered in conjugation with the protein of the present invention, may also be included in the vaccine of the present invention. Such additional components include other known protective antigen of Bordetella, a table is nochnogo of toxoid and/or diphtheria toxoid.

Of course, this invention relates also to a method of immunization of a human or animal against Bordetella infection and/or symptoms associated with a disease caused by Bordetella infection, providing for the introduction of the vaccine of the present invention to such person or animal.

The method of administration of the vaccine of this invention can be any suitable method that delivers immunoprotective the amount of protein of the present invention to the owner. However, this vaccine is administered preferably parenteral using intramuscular or subcutaneous route of administration. Other methods of introduction can also be used, when this is desirable, such as oral administration or introduction via other parenteral methods, i.e. intradermal through the nose or intravenously.

Another aspect of this invention relates to the use of the protein of this invention in the preparation of drugs for the treatment of human or animal disease symptoms associated with whooping cough, and/or to protect human or animal from symptoms associated with Bordetella infection.

Of course, this invention relates additionally to a method of treatment of the human or animal from Bordetella infection and/or symptoms associated with a disease caused by INPE is of Bordetella, introducing a drug of the present invention to such person or animal.

Another aspect of the present invention is a polypeptide that is capable of contacting the integrin CD11b/CD18, and the specified polypeptide is

A. a fragment of the Bordetella adenylate cyclase with 30-500 amino acids, preferably 50 to 300 and more preferably 50-150 amino acids, with the specified fragment contains a domain of interaction CD11b/CD18 specified Bordetella adenylate cyclase or contains a fragment of the specified domain interaction CD11b/CD18 wild type, sufficient to preserve the ability of binding to CD11b/CD18, or

b. option specified fragment having at least 70% identity, preferably at least 80% identity and more preferably at least 90% identity with the specified fragment, and this option retains the binding ability of CD11b/CD18.

The Bordetella adenylate cyclase is selected preferably from adenylatecyclase Bordetella pertussis, Bordetella parapertussis and Bordetella bronchiseptica and more preferably is Bordetella pertussis.

The polypeptides of the present invention will be selected from polypeptides that adopt a suitable conformation for binding to CD11b/CD18.

In specific embodiments, the implementation of the polypeptides of this invention may contain other auxiliary areas and is inelasticity Bordetella, involved in the optimal binding to CD11b/CD18. These areas include, more specifically, amino acid sequences containing region extending from amino acids 1416 to amino acids 1648.

In one preferred embodiment, the polypeptide of this invention is defined above in b., consisting of one or more fragments of 10-50 amino acids of domain interaction CD11b/CD18. For example, in one preferred embodiment, the specified polypeptide contains at least fragments of 10-50 amino acids district of adenylate cyclase B.pertussis, extending from amino acid 1208 to amino acids 1243 adenylate cyclase B.pertussis.

The percentage identity corresponds to the percentage of amino acid variant sequences that are identical to the sequence of wild-type mapping using the BLAST algorithm. The phrase "retains the ability to bind to CD11b/CD18 means that this variant retains at least 80% of the binding affinity against CD11b/CD18 in comparison with the corresponding fragment of the wild type, with which it could be mapped, and preferably at least 90% of the binding affinity against CD11b/CD18.

According to one preferred variant implementation of the specified polypeptide is specifically reactive is with antisera, recognizing adenylate cyclase wild type Bordetella, preferably Bordetella pertussis adenylate cyclase. More preferably, this polypeptide is capable, when administered to a mammal to induce antibodies that specifically recognize the Bordetella adenylate cyclase.

In one specific embodiment, the specified polypeptide is a fragment of the Bordetella pertussis adenylate cyclase. In another specific embodiment, the specified polypeptide essentially consists of a domain interaction CD11b/CD18 and, more specifically, domain interaction CD11b/CD18 B.pertussis, stretching from 1166 amino acids to amino acids 1281 adenylate cyclase B.pertussis (SEQ ID NO:2).

In other specific embodiments, the implementation of the specified polypeptide further comprises a domain acylation of Bordetella adenylate cyclase and/or hydrophobic domain. These domains acylation included in the relevant areas, extending from residue 700 to balance 1000 SEQ ID NO:1, as described in WO 93/21324, and contain Lys 983 and/or Lys 860. Hydrophobic domain corresponds to a region extending from residue 500 to balance 700 SEQ ID NO:1.

Preferably, this polypeptide is not toxic when introduced in vivo to a mammal.

The polypeptides of the present invention compete for binding to the integrin CD11b/CD18 with adenylate cyclase wild-type.

Thus, this invention relates to a note is to defined above polypeptide in the preparation of vaccines or medicines for the prevention or treatment in man or in animal disease symptoms associated with whooping cough, and/or to protect a person or animal from a disease associated with infection with Bordetella.

More specifically, this invention relates to the use of the specified polypeptide of the present invention to generate protective antibodies against Bordetella infection.

It was reported that the cyclase is an effective vector for the delivery of molecules able to target different antigens on dendritic cells, leading, in particular, to the generation of strong CD4+and CD8+T-cell reactions (ER, Pasteur Institute).

This invention relates also to the use of polypeptides of the present invention in obtaining a vector for targeting interest molecules, in particular, expressing CD11b-cells.

The term “specific” means in the context of this invention that the polypeptide when used as a vector for representing the interest of the molecule is directed mainly at expressing CD11b-cells in accordance with the high affinity binding domain interaction CD11b/CD18 with CD11b/CD18, thereby providing a means for targeting interest molecules on the surface of these cells or in cells in a selective manner relative to other glue is OK.

In particular, in one embodiment, the targeting of the specified molecule or peptide is effective in vivo. In other embodiments, implementation targeting the indicated molecule is effective in vitro or ex vivo. Under “in vitro” mean that the target cells are cells that are cultured in vitro. By “ex vivo” mean that the target cells are cells that have been extracted from a living organism, are cultured in vitro and are intended for re-introduction into a living organism.

Thus, this invention provides a means that is suitable for creating compositions suitable for administration to an animal or human host, requiring targeting specific cells and, in particular, myeloid dendritic cells, neutrophils or macrophages.

This invention more specifically relates to a vector for targeting interest molecules on expressing CD11b-cells, characterized by the fact that the vector contains the polypeptide has the ability to communicate with CD11b/CD18 defined above, associated with the specified interest molecule.

This invention relates also to a method for targeting in vitro interest molecules on expressing CD11b-cells, including:

A. ensuring expressing CD11b-cells, extragere the data of the living organism

b. the cultivation of these expressing CD11b-cells with the vector of this invention under conditions suitable for targeting the specified vector to the specified expressing CD11b-cells.

This invention relates to expressing CD11b-cells containing an interest in the molecule, obtained by the method described above.

According to this invention, the expression "interest molecule" refers to any molecule, preferably to a molecule that is not a fragment of adenylate cyclase type Bordetella.

Interest of the molecule can be selected from nucleic acids such as DNA, RNA, oligonucleotides, antisense DNA, plasmids and Comedy. They can also be selected from peptides or polypeptides and, in particular, enzymes, coenzymes, ligands, receptors, haptens, antigens, antibodies and their fragments. Of course, the person skilled in the art will select a suitable molecule depending on the desired use.

Interest of the molecule can be selected from active (current) start medicines, immunotoxins, antioxidants, antibiotics, growth factors, intracellular hormones, cytokines, toxins, neurotransmitters, antimicrobial agents, in particular antiviral, antibacterial, antiparasitical antitumor agents and, more generally, of any therapeutic or prophylactic interest agents.

In accordance with one particular embodiment of interest, the molecule selected from the group consisting of peptides, glycopeptides, lipopetides, polysaccharides, oligosaccharides, nucleic acids, lipids and chemicals.

In specific embodiments, the implementation of interest molecule is heterologous antigen or epitope, the term "heterologous" refers to the antigen or epitope other than antigenic determinants of adenylate cyclase contained in the vector.

Of interest, the molecule associated with the polypeptide of the present invention to obtain a vector of the present invention.

In this context, the term "associated" refers to any interaction, making it possible physical Association representing the interest of the molecule and the polypeptide. Preferably, this binding is covalent. It may be direct covalent binding or indirect binding using the binding (linker) the agent for the formation of the conjugate. The chemical binding methods well known in the field. The chemical bond may be selected, for example, from maleimides, peptide, disulfide or thioester linkages. For example, it may be used disulfide bonds, using N-pyridylmethylamine sulfhydryl.

One particular way is to add the linker to the polypeptide, and specified the linker comprises at least one cysteine, which can be easily used for disulfide bond. Another approach is to chemically binding biotinylate part of the molecule, which enables the binding of other molecules associated with streptavidin.

Multiple molecules can be chemically bonded to the polypeptide of the present invention via a disulfide bond with various cysteine residues, provided that this binding does not prevent interaction with CD11b/CD18.

In addition, functional properties expressing CD11b-cells determine the use of the polypeptides of this invention in the preparation of protein vector for targeting drugs to specific cells. In this context, in one particular embodiment, the so-called interest of the molecule is the active (current) start medicines. Specified, the active principle can be linked chemically or genetically with the polypeptide of the present invention. Preferably, interest molecule is an anti-inflammatory drug, which when binding the AI with adenylyl cyclase toxin is directed specifically to the cell surface, involved in the inflammatory response, such as neutrophils.

Because expressing CD11b-cells and, more specifically, myeloid dendritic cells, neutrophils and macrophages are involved in essential functions of the immune system and natural protection, in particular in inflammatory and specific immune responses, in a preferred embodiment of the present invention, the vector of the present invention design, more specifically, for the priming reaction CD4+- and CD8+cells, and this response follows the targeting of interest molecules on expressing CD11b-cells, particularly myeloid dendritic cells.

In this context of interest, the molecule is preferably an epitope or antigen or an epitope or antigen. More specifically, of interest, the molecule can be, in particular, the antigen is selected from the group consisting of poliovirus antigen, the antigen of the HIV virus, virus antigen of influenza virus, lymphocytic choriomeningitis, epitope, antigen human papillomavirus (HPV), bacterial antigen, for example, an antigen of Mycobacterium tuberculosis.

Thus, this invention provides a means for priming reaction CD4+- and CD8+cells in the patient, or targeting in vivo antigen or epitope on expressio the existing CD11b-cells, or ex vivo targeting of antigen or epitope on the extracted expressing CD11b-cells and the re-introduction of the received cells to the specified patient.

Thus, this invention relates to a method of targeting in vitro antigen or epitope on expressing CD11b-cells, providing

A. ensuring expressing CD11b-cells extracted from a living organism, and

b. the cultivation of these expressing CD11b-cells with the vector of the present invention, bearing antigen or epitope as the interest of the molecule under suitable conditions to target this vector to the specified expressing CD11b-cells.

Preferably, expressing CD11b-cells extracted from a living organism, are myeloid dendritic cells.

This invention also provides expressing CD11b-cells containing heterologous antigen or epitope derived as described above.

Thus, this invention relates to a product of cellular therapy for immunization of a human or animal against the antigen, characterized in that it contains an effective amount expressing CD11b-cells containing a heterologous antigen or epitope, obtained as described above, in combination with a pharmaceutically acceptable carrier.

D is more, this invention relates to the use of expressing CD11b-cells that contain the antigen or epitope, obtained as described above, in obtaining a product of cellular therapy for immunization of a human or animal against the antigen.

More specifically, this invention relates to a method of immunizing a patient against the antigen, providing

A. extraction expressing CD11b-cells from the specified patient,

b. the cultivation in vitro of these expressing CD11b-cells with the vector of the present invention, bearing antigen or epitope as the interest of the molecule, under conditions that are suitable for targeting the specified vector to the specified cell

C. re-introduction of an effective number of cells that contain the vector specified by the patient for priming CD4+and/or CD8+reactions

for immunization through this specified patient relative to a specific antigen.

According to a preferred variant implementation of the invention specified expressing CD11b-cells are myeloid dendritic cells.

Thus, this invention relates also to pharmaceutical compositions containing the vector of the present invention, bearing the epitope or antigen as interest mo is ecoli, in combination with a pharmaceutically acceptable carrier.

According to one variant of implementation of the said composition is a vaccine suitable for administration to a human or animal. Preferably, the vaccine is able to induce immunity against poliovirus, HIV or virus lymphocytic choriomeningitis. Of course, the type of induced immunity will depend on the selected antigen, which is transferred to the data vector. In another embodiment, the vaccine is able to induce immunity against whooping cough.

Such vaccines contain immunoprotective and non-toxic amount of the vector of the present invention. This composition may further contain suitable Primerose adjuvants, respectively.

This invention relates also to a method of immunization of a human or animal against infection by the pathogen, providing for the introduction of vaccines containing immunoprotective and non-toxic amount of the vector of the present invention, such a person or an animal.

This invention relates to means for obtaining a polypeptide, protein or vector of the present invention. In particular, these means include a nucleic acid encoding one of the following polypeptides:

A. the protein of the present invention, which is defective in respect of St. the statements CD11b/CD18;

b. the polypeptide of the present invention, which is able to contact the integrin CD11b/CD18; or

C. vector to target interest molecules on expressing CD11b-cells.

In particular, the nucleic acid of this invention can be obtained from the DNA that encodes adenylate cyclase wild-type any strain of Bordetella, using known methods, for example, the selection gene from the gene Bank, receiving complementary or cDNA of matrices mRNA or by polymerase chain reaction or from clinical isolates of species. Alternatively, DNA encoding adenylate cyclase wild type, can be synthesized by standard methods of DNA synthesis. Different strains of Bordetella are publicly available from commercial depositories.

Modification of the wild type DNA that encodes the Bordetella adenylate cyclase, can be obtained by genetic engineering DNA of the wild type using the conventional technology of molecular biology.

Another object of this invention is a recombinant nucleic acid comprising a nucleic acid that encodes a polypeptide, protein or vector of the present invention, expressing cloned in a vector suitable for expression of the encoded polypeptide or protein in the cell host. Optional, this recombinant DNA molecule contains the complement is inuu the coding sequence of the polypeptide carrier, which has immunostimulating properties, such as adjuvant, or which is useful in the expression, purification and/or preparation of the polypeptides of the present invention. This coding sequence can be placed in a reading frame with the coding sequence of the polypeptide, protein, or vector for targeting molecules of the present invention.

The choice of expressing the vector is, of course, depend on the used host cell.

Preferably, the specified expressing vector is a plasmid, cosmid, fahmida or viral DNA.

This invention relates also to a method for producing the protein of the present invention, defective in regard to the binding of CD11b/CD18; the polypeptide is able to bind to CD11b/CD18 described above; or vector for targeting interest molecules on expressing CD11b-cells, providing for the stage include recombinant nucleic acid described above, in a suitable cell host for expression of the respective interest of the polypeptide, protein or vector; culturing the transformed recombinant cells and extraction of the synthesized recombinant polypeptide, protein or vector of this invention.

Another aspect of the present invention is a host cell transformed by recombin nteu nucleic acid of this invention and, therefore, containing a nucleic acid or recombinant nucleic acid described above. In one embodiment, the recombinant nucleic acid may be integrated into the genome of the host cell by using common methods, including homologous recombination.

Preferred cells are the owners of this invention include cells belonging to the species E. coli and the genus Bordetella. Other cells of the host, which may be suitable include, but are not limited to, mammalian cells, insect cells, yeast cells and other bacterial cells.

The invention also includes polyclonal serum obtained by immunization of an animal or human polypeptide, protein, vector, or composition of this invention.

In one preferred embodiment, the polyclonal serum can be obtained by immunization of an animal or human polypeptide consisting of domain interaction CD11b/CD18-Bordetella adenylate cyclase, preferably domain interaction CD11b/CD18-Bordetella pertussis adenylate cyclase, stretching from 1166 amino acids to amino acids 1281.

This invention relates to a monoclonal antibody directed specifically against the polypeptides of the present invention, containing the domain of interaction CD11b/CD18.

p> In one preferred embodiment, a monoclonal antibody directed against an epitope located in the domain of interaction CD11b/CD18, preferably against an epitope located in the domain of interaction CD11b/CD18-Bordetella pertussis adenylate cyclase, stretching from 1166 amino acids to amino acids 1281.

Preferably, the specified polyclonal serum or monoclonal antibody capable of blocking the binding of adenylate cyclase wild-type CD11b/CD18. This block may be analyzed by assessing the ability of the mixture of these polyclonal serum or monoclonal antibodies with adenylate cyclase wild-type contact CD11b/CD18 in comparison with the ability of only the wild-type adenylate cyclase.

In one specific embodiment, the specified drug provides passive immunization against Bordetella infection.

For use in humans, the antibodies of this invention can be humanitarian, for example, replacement of the hypervariable portion of human immunoglobulin, which has no function of antibody hypervariable region of monoclonal immunoglobulin obtained from the above-described method.

For example, the methods of humanizing antibodies described Waldmann T., June 1991, Science, vol.252, p.1657-1662; G. Winter et al., 1993, Immunology Today, vol.14, No. 6, p.243-246; Carter et al., May 1992, Pro. Natl. Acad. Sci. USA, vol.89, p.4285-4289; Singer et al., 1 April 1993, the Journal of Immunology, vol.150, No. 7, p.2844-2857.

This invention relates also to pharmaceutical compositions containing polyclonal serum or monoclonal serum in combination with a pharmaceutically acceptable carrier.

This invention relates also to the use of polyclonal serum or monoclonal antibodies of this invention in the preparation of drugs for the treatment of human or animal disease symptoms associated with whooping cough, and/or to protect human or animal against disease symptoms associated with Bordetella infection.

The following experimental part shows the results of identification (i) the role of post-translational acylation in the interaction of CyaA with CD11b and (ii) domain interaction CD11b in the Bordetella pertussis adenylate cyclase.

CAPTIONS TO FIGURES

Figure 1. CyaA is associated specifically with CD11b-cells and inhibits the binding of both CyaA-Biotin, and monoclonal anti-CD11b antibodies to these cells

(A) Cells Cho or Cho-CD11b cells were then incubated with the indicated concentrations of CyaA. Surface-bound CyaA were detected using FACS with anti-CyaA-Mab (5G12). The results are expressed as ΔMFI = (the value of the MFI of cells incubated with CyaA) - (the value of the MFI of cells incubated without CyaA) and represent at least 2 is zavisimyh experiment.

(C) Cells Cho-CD11b was preincubator with the specified concentrations of CyaA. Then added separately CyaA-Biotin (30 nm) or anti-CD11b-Mab (2 μg/ml) in the continuous presence of the toxin, and their binding was measured using FACS.

(C) After predisturbance with the specified concentrations of CyaA, cells Cho-CD11b or cells SNO-CD11c were incubated with monoclonal or anti-CD11b or anti-CD11c-antibody, respectively, in the continuous presence of the toxin. Then the binding of the antibodies was determined using FACS.

For (b) and (C) results are expressed as% binding = (binding of the sample)/(maximum binding)×100 and represent at least 2 independent experiments.

Figure 2. Direct binding of CyaA or proCyaA with transfectants SNO

Cells Cho-CD11b (A) or Cho (C) were incubated with indicated concentrations of CyaA or proCyaA. Surface-bound CyaA were detected using anti-CyaA-Mab (5G12). The results are expressed as ΔMFI = (the value of the MFI of cells incubated with CyaA) - (the value of the MFI of cells incubated without CyaA) and represent at least 2 independent experiments.

Figure 3. The acylation CyaA is required for stable Association with cells Cho-CD11b

Cells Cho-CD11b was preincubator with the specified concentrations of CyaA or proCyaA. Then added CyaA-Biotin (A) or anti-CD11b-Mab (C) under continuous presence of CyaA or proCyaA. The surface is Resto-related CyaA-Biotin or anti-CD11b-Mab was measured using FACS. The results are expressed as% binding = (binding of the sample)/(maximum binding)×100 and represent at least 2 independent experiments.

Figure 4. The acylation CyaA is necessary for CyaA-induced accumulation of camp and cytotoxicity

Cells Cho-CD11b was preincubator or CyaA, or proCyaA at the indicated concentrations for 20 min at 37°C. Then cells were literally and measured camp (A). In parallel, the toxicity was determined by measuring the amount of lactate dehydrogenase released into the medium after incubation of cells Cho-CD11b for 4 hours at 37°C in the presence of the indicated concentrations of either CyaA, or proCyaA (In). Results represent at least 2 independent experiments.

Figure 5 Catalytic domain is not necessary for the interaction of CyaA with CD11b-cells

Cells Cho-CD11b was preincubator with the specified concentrations of CyaA, CyaA 1-384 or CyaA 373-1706. Then cells were incubated either with CyaA-Biotin (a), or with anti-CD11b-Mab (B). The binding of CyaA-Biotin and anti-CD11b-Mab was measured using FACS. The results are expressed as% binding = (binding of the sample)/(maximum binding)×100 and represent at least 2 independent experiments.

6. Direct binding fragments of CyaA with CD11b-cells

Cells Cho-CD11b (a, C) or Cho (b, D) were incubated with indicated concentrations of CyaA, CyaA 1-Yi CyaA 373-1706. Then surface-bound CyaA were detected with anti-CyaA-5G12-Mab, which recognizes the catalytic domain (a, b), or with anti-CyaA-6D7-Mab, which recognizes a domain repeats (C, D). The results are expressed as ΔMFI = (the value of the MFI of cells incubated with CyaA or fragments CyaA) - (the value of the MFI of cells incubated without CyaA or fragments CyaA) and represent at least 2 independent experiments.

7. The binding of CyaA-Biotin cells Cho-CD11b in the presence of the mutant CyaA-FLAG

Cells Cho-CD11b was preincubator with CyaA or mutant CyaA-FLAG (30 nm). Then added CyaA-Biotin in the continuous presence of CyaA or molecules CyaA-FLAG. Surface-bound CyaA-Biotin was detected using FACS with streptavidin-PE. The results are expressed as% binding = (binding of the sample)/(maximum binding)×100 and represent at least 2 independent experiments.

Fig. Analysis using electrophoresis in LTO-SDS page of purified preparations of CyaA and their invasive activity in erythrocytes

(A) Molecules CyaA/FLAG together with wild-type CyaA was purified from extracts of urea using DEAE - and phenyl-sepharose chromatography as described previously (Karimova et al., 1998). Approximately 3 µg of each protein was analyzed by 7.5% acrylamide gel, colored Kumasi blue. (C) Invasive activity of CyaA molecules/FLAG on sheep erythrocytes. Two micrograms of different be the Cove CyaA were incubated with 5×10 8washed sheep erythrocytes for 30 minutes, and the number of speakers-activity displaced by these cells was determined as described previously (Osicka et al., 2000). This value represents the average of three experiments performed in two iterations (n=6).

Fig.9. The binding of CyaA with cells Cho-CD11b in the presence of selected mutant CyaA-FLAG

Cells Cho-CD11b was preincubator with CyaA or mutant CyaA-FLAG at the indicated concentrations in the range of 7.5 nm to 240 nm. Added CyaA-Biotin in the continuous presence of CyaA molecules and were detected by surface-bound CyaA-Biotin. The results are expressed as% binding = (binding of the sample)/(maximum binding)×100 and represent at least 2 independent experiments.

EXPERIMENTAL PART

A.Materials and methods

A.1Receiving, clearing and modification produced from CyaA proteins

DNA manipulations were performed according to standard protocols (Sambrook et al., 1989) in the Escherichia coli strain XL1-Blue (Stratagene, Amsterdam, Netherlands) as host cells. Plasmids encoding reallyreally proCyaA wild-type (pACT7), acylated CyaA wild-type (pT7CACT1) and recombinant detoxified enduring CyaA-E5-CysOVA bearing a unique cysteine residue and the OVA epitope in its catalytic domain (pCACT-E5-CysOva), have already been described (Gmira et al., 2001; Guermonprez et al., 2001; Osicka et al., 2000; Sebo et al., 1991). A plasmid encoding CyA 373-1706 (pTRCyaAΔ1-373) is derived pTRCAG (Gmira et al., 2001), in which a DNA sequence encoding a catalytic domain of the toxin (contained between sites NdeI and BstBI), was demeterova and replaced by the corresponding synthetic a double-stranded oligonucleotide coding for the amino acid sequence: Met-Gly-Cys-Gly-Asn.

The Protocol for obtaining CyaA was already described elsewhere (Karimova et al., 1998). All the proteins expressed in E.coli strain BLR (Novagen, Merck KG, Darmstadt, Germany) and purified to homogeneity, greater than 95% (as determined by analysis of electrophoresis LTO-gel) of Taurus include a two-step procedure involving chromatography on DEAE-sepharose and chromatography on phenyl-sepharose, as described in Guermonprez et al., 2000. Purified protein CyaA-E5-CysOVA noted for its unique balance of cysteine sulfhydryl reagent N-(6-biotinamide)hexyl)-3'-(2'-pyridyldithio)propionamide (Biotin-HPDP) (Pierce, Bezons, France) according to the manufacturer's instructions. Biotinylated CyaA was re-purified on DEAE-sepharose to remove unreacted reagent Biotin-HPDP. CyaA 1-384 expressed and purified as described in Ladant et al., 1992.

The concentration of the toxin was determined spectrophotometrically from the absorbance at 278 nm using a ratio of the molecular extinction 141 mm-1·cm-1for full-CyaA toxins, 113 mm-1·cm-1for CyaA 373-1706 and 28 mm-1·cm-1

Molecules CyaA-FLAG was designed using predetermined permissive sites interturbine along CyaA molecules (Osicka et al., 2000). The authors created a series of 17 CyaA-structures, which are carried in individual permissive provisions synthetic octapeptide insert Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys for the FLAG epitope (Sigma, Saint Quentin Fallavier, France). To achieve this, three pairs of double-stranded synthetic oligonucleotides (5'-GTACTGATTATAAAGATGACGATGACAAATCAC + 5'-GTACGTGATTTGTCATCGTCATCTTTATAATCA, 5'-GTACTTATCGATTATAAAGATGACGATGACAAA + 5'-GTACTTTGTCATCGTCATCTTTATAATCGATAA and 5'-GTACGTGGATTATAAA GATGACGATGACAAAGC + 5'-GTACGCTTTGTCATCGTCATCTTTATAATCCAC, respectively) (SEQ ID nos:5-10), encoding the FLAG epitope on the required reading frames, was insertional in a unique BsrGI sites, previously entered into cyaA (Osicka et al., 2000). The correct insertion was verified by DNA sequencing, recombinant CyaA molecules expressed in E.coli and purified. Invasive ability of selected molecules CyaA/FLAG characterized using sheep erythrocytes as target cells, as described previously (Osicka et al., 2000).

A.2Obtaining monoclonal anti-CyaA-antibodies

BALB/c mice were first immunized intraperitoneally toxin CyaA (20 μg in alum). At intervals of about two weeks, mice were immunized again, 10 μg of CyaA in alum 3 times. Throughout the Protocol of immunization in mice took the blood, and their serum was tested on presets is of anti-CyaA antibodies using ELISA. For the detection of significant titers of sera had the last booster immunization of these mice and their splenocytes were merged with myeloma cells RH ATSS, Manassas, USA) after 3 days. Received hybridoma were subjected to screening for the production of CyaA-specific monoclonal antibodies using ELISA. Then selected high hybridoma and cloned by limiting dilutions of single cells and then used to obtain ascites in naked mice BALB/c mice to generate large quantities of monoclonal antibodies. Monoclonal antibodies were purified from ascites using the kit for treatment of T-Gel™ (Pierce, Bezone, France) according to the manufacturer's instructions. Antibody concentrations were measured using analysis of protein Bio-Rad (Bio-Rad, Marnes La Coquette, France). Two of these monoclonal antibodies used in this study: antibody 5G12, which reacts with an epitope located within amino acids 1-190, and antibody 6D7, which reacts with an epitope located within amino acids 1006-1706.

A.3Cells and cultivation

Cells of the ovary of the Chinese hamster, transfetsirovannyh CD11b/CD18 person (Cho-CD11b cells), CD11c/SNO man (SNO-CD11c-cells) or transfetsirovannyh one vector (Cho-cells), were the kind gift of D. Golenbock (Boston University School of Medicine, Boston, MA), and were cultured in the presence of neomycin is a, as described previously (Ingalls et al., 1998).

A.4Antibodies

Monoclonal antibodies specific against CD11b (ICRF44, murine IgG1, κ) and human CD11c (B-Ly6, murine IgG1, κ), purchased from BD Pharmingen (Le Pont de Claix, France).

A.5Analyses bind

These analyses were performed as described Guermonprez et al., 2001. Briefly, 2×105cells were incubated with indicated concentrations of CyaA molecules in DMEM containing 4.5 mg/ml glucose (Life Technologies, Cergy Pontoise, France) without serum in 96-well culture plates for 30 minutes on ice. After washing was added Mab against the catalytic domain of CyaA (5G12) or Mab against domain repeats CyaA (6D7) at 25 µg/ml In some experiments the cells were preincubator with the specified concentrations of CyaA molecules for 30 minutes on ice. Then added separately CyaA-Biotin (30 nm), anti-CD11b-Mab (2 μg/ml) or anti-CD11c-Mab (2 μg/ml) (BD Pharmingen) in the continuous presence of the toxins.

After washing and removal of the supernatant the cells were stained with goat antibodies against mouse IgG-PE (Caltag, Le Perray en Yvelines, France) or streptavidin-PE (BD Pharmingen) at a dilution of 1:300. After the last wash, the cells were analyzed by flow cytometry on a FACStar™ (Becton Dickinson, Le Pont de Claix, France) in the presence of 5 μg/ml of iodide of propecia. Aggregated and dead cells read discriminatory attitudes Windows based exceptions iodide of propecia. Dan is haunted binding transcribed from the average fluorescence intensity (MFI) and were expressed as ΔMFI = (the value of the MFI of cells, incubated with CyaA) - (the value of the MFI of cells incubated without CyaA) or as% binding = (binding of the sample)/(maximum binding)×100. The maximum binding corresponds to (the value of the MFI of cells incubated with CyaA or anti-CD11b in the absence of competitor) - (the value of the MFI of cells incubated only with the environment). Binding of the sample corresponds to (the value of the MFI of cells incubated with CyaA or anti-CD11b in the presence of competitor) - (the value of the MFI of cells incubated only with the environment).

A.6Analysis of camp

Cyclic AMP that is accumulated in the cells subjected to the action of toxin CyaA was determined essentially as described by Guermonprez et al., 2001. Briefly, 5×105cells were incubated with indicated concentrations of CyaA in DMEM + glucose for 20 minutes at 37°C. After washing camp, accumulated in the cytosol of cells, released by lysis of 0,1N. HCl and boiling for 5 minutes at 120°C. after neutralization with 0,1N. NaOH samples were added to microtiter plates, pre-coated with a conjugate of cyclic amp-BSA, and then incubated with the appropriate dilution of rabbit antisera against the camp. After washing, anti-camp antibodies were detected by antibodies against rabbit Ig associated with alkaline phosphatase. The content of camp each sample was determined from comparison with a standard curve obtained by adding known to the concentration camp.

A.7Invasive activity of CyaA

Invasive activity of CyaA molecules was determined as described previously Osicka et al., 2000. Briefly, sheep erythrocytes were incubated with the toxin for 30 minutes, and invasive activity was measured in the form of AC activity, moved in erythrocytes and protected against cleavage of the extracellular added trypsin.

CenturyResults

B.1CyaA specifically binds to CD11b+cells and inhibits the binding of CyaA-Biotin and anti-CD11b-CD11b antibodies+cells

To study the role of biological and structural properties of CyaA in its interaction with CD11b developed two complementary analyses: analysis of the binding and competitive analysis. The analysis of binding consisted of incubation of CyaA molecules with transfitsirovannykh cells SNO expressing CD11b/CD18 person (Cho-CD11b cells), or with false transfitsirovannykh cells SNO, and subsequent detection of cell-associated toxin using monoclonal anti-CyaA-antibodies (5G12), specifically in relation to the catalytic domain. As shown in figa, using this assay, the binding of CyaA was specifically detected in CD11b+-cells. In the competitive analysis of various molecules CyaA (mutants or fragments) can be tested for their ability to compete with the binding CyaA-bi is Tina or monoclonal anti-CD11b-antibody (Mab) with CD11b +-cells. In this case, the cells are Cho-CD11b cells were then incubated with CyaA at various concentrations for 30 minutes on ice. Then, when the continuous presence of CyaA was added CyaA-Biotin (30 nm) or anti-CD11b-Mab (2 μg/ml) and their binding to these cells was assessed using FACS. As shown in figv, CyaA effectively inhibited the binding of both CyaA-Biotin and anti-CD11b antibodies with cells Cho-CD11b-dependent dose-dependent manner. This inhibitory effect was specific against CD11b, as CyaA was quite unable to compete with another ligand (anti-CD11c-Mab) for its specific receptor (CD11c)expressing cells Cho (figs).

B.2The absence of acylation CyaA affects its binding to CD11b+cells

Because it is well established that CyaA needs in post-translational palmitoylation to perform its invasive activity and education hemolytic membrane channels, the inventors tested, does the absence of acylation on the interaction of CyaA with CD11b+-cells. In the analysis of the binding of Cho cells and Cho-CD11b cells were incubated with CyaA or palleroni proCyaA. Binding was assessed using a Mab against the catalytic domain of CyaA (5G12). As shown in figa, at low concentrations the binding of both CyaA molecules, and molecules proCyaA c CD11b+cells were labour is Ino comparable with a bit more efficient binding of acylated CyaA. This could be due to its increased interaction with the cellular membrane, best adapted conformation CyaA binding and/or higher affinity CyaA against CD11b-receptor. In fact, linking proCyaA reached saturation at much higher concentrations protoxin in comparison with the binding of CyaA. The simplest explanation for this observation could be that proCyaA binds to CD11b+cells with lower affinity than CyaA. At high concentrations of protoxin units and/or oligomers proCyaA associated with these cells and, consequently, a higher number proCyaA were found related to this system of detection antibodies. In contrast, very low binding of CyaA or proCyaA was detected with the control cells Cho (pigv).

B.3The acylation stabilizes the interaction of CyaA and CD11b+cells

For additional analysis of the role of acylation of CyaA in the interaction of the toxin with CD11b+cells the inventors tested the ability reallymoving proCyaA to compete with CyaA for binding to cells Cho-CD11b. As shown in figa, when compared with acylated CyaA reallyreally proCyaA showed significantly reduced ability to compete with biotinylated CyaA for binding to CD11b+-cells. For definition wide-angle is, was it the lack of inhibition due to inefficient interaction with CD11b, the inventors evaluated the ability proCyaA to block the binding of anti-CD11b antibodies with cells Cho-CD11b. Indeed, in comparison with CyaA proCyaA was not able to inhibit the binding of anti-CD11b antibodies with cells Cho-CD11b (pigv).

Because sverkhfizicheskoe the production of camp and the intoxication of the cells are the consequences of the interaction of CyaA with CD11b+cells, the inventors then analyzed using cells Cho-CD11b, depend on whether the function of the toxin from the acylation of CyaA. As expected, in contrast to the acylated toxin proCyaA not induced no increase of camp in cells Cho-CD11b (figa) and had no significant cytotoxic effect on these cells (pigv). Taken together, these results clearly demonstrate that the acylation CyaA is required for the functional interaction of the toxin with CD11b+cells and that the binding proCyaA with CD11b is not sufficient to run the cytotoxic action on expressing CD11b-cells.

B.4The catalytic domain is not necessary for the interaction of CyaA with CD11b

Functional CyaA consists of two main domains, supporting independent activity. N-terminal domain carries adenylyl cyclase activity (amino acids 1-400) whereas the carboxy-terminal gemolizinov part (amino acids 400-1706) is responsible for the delivery of this domain as in target cells and hemolytic activity of B.pertussis. To test the role of these two functional domains CyaA binding to CD11b+cells the inventors tested the ability of the catalytic domain encoded by residues 1-384, CyaA 1-384, and hemolytic part of the molecule encoded by residues 373-1706, CyaA 373-1706, to compete for binding to cells Cho-CD11b with CyaA-Biotin. As shown in figa, catalytic domain was not able to inhibit the binding of CyaA-Biotin cells Cho-CD11b, whereas CyaA 373-1706 found the same inhibition of binding, and full-CyaA. Similarly, the catalytic domain was not able to inhibit the binding of anti-CD11b-Mab with cells Cho-CD11b (pigv). In addition, direct assays of binding with anti-CyaA-Mab (5G12), specifically in relation to the catalytic domain, failed to reveal any significant Association CyaA 1-384 with the surface of the cells Cho-CD11b, whereas the binding of CyaA was easy detektivami (figa). Direct binding of CyaA 373-1706 cells Cho-CD11b could not be detected Mab 5G12, which recognizes an epitope located within the first 200 amino acids CyaA, but was clearly demonstrated with the use of other anti-CyaA-Mab (6D7), specific for the domain repeats (figs). Again, only weak binding of CyaA or CyaA 373-1706 was detected c Mab 6D7 on cells SNO deprived of CD11b (Fig. 6B and D). Taken together, these results clearly demo the illustrate, that the catalytic domain is not necessary for the interaction of CyaA with CD11b and that the domain of interaction of CyaA/CD11b localized in the fragment of CyaA 373-1706.

B.5The domain of CyaA, which interacts with CD11b, localized in the area of repetitions CyaA

To identify the area of CyaA, which interacts with CD11b, the inventors expressed and purified various subfragment C-terminal region of the CyaA 373-1706 (which includes the remains of 373-1490, or 700-1706, or 700-1490 or 1006-1706), which was tested in competitive analysis. However, none of these polypeptides was not able to compete significantly with the binding of CyaA-Biotin cells Cho-CD11b. This may be due to the fact that these selections take a modified conformation. Thus, the inventors used a mutational approach to determining the location of CD11b-binding domain of CyaA. Seventeen different modified CyaA molecules designed by insertional FLAG epitope (amino acid sequence: DYKDDDDK) at various specific locations throughout the polypeptide toxin, as described in section "Materials and methods". The authors hypothesized that the incorporation of heterologous and vysokopatogennogo peptide in certain provisions of CD11b-binding domain may impair the ability to interact with CD11b. These 17 LAG-labeled molecules CyaA expressed and purified to homogeneity and tested on the binding ability of CyaA-Biotin cells Cho-CD11b (you should pay attention to in two cases, CyaAΔ510-515/FLAG and CyaAΔ1245-1273/FLAG, amino acids 510-515 or 1245-1273 CyaA, respectively, were dellarovere and replaced insertional FLAG-epitope). As shown in Fig.7, the insertion FLAG-epitope in 3 different sites, localized between residues 1166-1281, eliminated the interaction with CD11b. The corresponding modified CyaA was essentially unable to compete with CyaA-Biotin for binding to CD11b when tested at concentrations of 30 nm. In contrast, all other FLAG-labeled recombinant CyaA were able to compete with CyaA-Biotin for binding to CD11b+cells, albeit with varying efficiency. It is noteworthy that these three recombinant constructs CyaA with FLAG-epitope, insertional near carboxykinase side of the protein (i.e. at position 1416, 1623 and 1648), were partially degraded in their ability to compete for binding to CD11b with CyaA-Biotin.

To further characterize the domain of CyaA, which interacts with CD11b, in addition to four other molecules CyaA/FLAG, the authors focused on three CyaA molecules/FLAG that could not inhibit the binding of CyaA-Biotin with CD11b+cells as effectively as intact CyaA. These molecules CyaA again expressed and purified almost to homogeneity (figa) and their cell-invasive activity experienced analisa is their ability to penetrate through the membrane of sheep red blood cells (RBC) and to deliver the catalytic domain in the compartment, not accessible to externally added trypsin. As shown in figv, except CyaA1387/FLAG invasive activity of all other tested molecules CyaA/FLAG insertion of FLAG-peptide was influenced to some extent. Invasive activity CyaA524/FLAG, which reflects the ability of CyaA to move the catalytic domain in erythrocytes, was completely eliminated by insertion of FLAG-peptide in the position of rest 524. However, the ability of other proteins, CyaA424/FLAG CyaA722/FLAG and CyaA1166/FLAG and, to a lesser extent, proteins CyaAΔ1245-1273/FLAG and CyaA1281/FLAG to penetrate RBC were comparable.

The ability of these molecules to compete with CyaA-Biotin for binding to cells Cho-CD11b felt dependent on the dose as shown in Fig.9. As expected, proteins CyaA1166/FLAG CyaAΔ1245-1273/FLAG and CyaA1281/FLAG were not able to inhibit the binding of CyaA-Biotin with CD11b+cells even at such high concentrations as 240 nm. In contrast, all other designs CyaA/FLAG inhibited binding of CyaA-Biotin dependent dose-dependent manner, similar to the intact CyaA. Thus, the lack of inhibition of binding CyaA1166/FLAG CyaAΔ1245-1273/FLAG and CyaA1281/FLAG could not be attributed to generalized conformational destruction of the toxin, caused by the insertion FLAG as invasive activity of these structures on RBC was comparable to the activity of the protein CyaA424/FLAG, which very effectively vzaimode is istue with CD11b +cells.

Thus, these results are undeniable proof that the domain portion of CyaA with RTX repeats, limited 1166 and 1281 residues and containing declared a loop that is located between two conservative blocks RTX repeats (Osicka et al., 2000), is crucial for the interaction of CyaA with CD11b+cells, and it most likely represents the main enterinvasive domain of CyaA.

C.Discussion

The biological activity of the adenylate cyclase toxin (act or CyaA) is completely dependent on covalent posttranslational acylation (acyl fatty acids). In the absence of acylation conservative residue Lys-983 CyaA cannot deliver its catalytic domain into the cytosol of erythrocytes and is not capable of forming hemolytic channels (Barry et al., 1991; Basar et al., 2001; Hackett et al., 1994). It is shown that CyaA penetrates with detectable efficiency in a wide variety of eukaryotic cells. For example, it was demonstrated that its primary target cells are myeloid cells such as neutrophils and pulmonary macrophages, which are particularly sensitive to CyaA and become paralyzed and commitlimit to apoptosis after exposure to the action of CyaA (Confer and Eaton, 1982; Khelef and Guiso, 1995; Khelef et al., 1993). The inventors have recently shown that this toxin has a specific cleoc the th receptor, αMβ2-integrin (CD11b/CD18), which is expressed exclusively on these immune cells like neutrophils, macrophages or dendritic cells, and that expression of CD11b, is most likely responsible for the high sensitivity of these cells to CyaA (Guermonprez et al., 2001). In this study, the inventors have shown that the activation of CyaA plays a major role in its interaction with CD11b+-cells. Indeed, although reallyreally proCyaA was able to contact CD11b+cells as effectively as CyaA, he was ineffective in competition with acylated CyaA for binding to Cho-CD11b+cells and was not able to block the binding of anti-CD11b-Mab with these cells. This suggests that, although he interacts still with CD11b, the nature of the interaction and, in particular, the affinity and/or stability of the complex proCyaA-CD11b differ materially from those properties involved in the interaction with CD11b Mature CyaA. In addition, although proCyaA still able to contact the receptor CD11b, this interaction does not allow protoxin to penetrate through the membrane. Thus, the acylation may be required to give competent in relation to the movement of the conformation of CyaA, which is necessary for the delivery of the catalytic domain in the cell cytosol, where it can catalyze the conversion of ATP into camp.

<> Functional CyaA can be divided into two main domains: one provided cyclase activity domain, localized between residues 1-400, and the one responsible for the haemolytic activity domain, localized between residues 400-1706 (Ladant and Ullman, 1999). After interaction of the toxin to target cells catalytic domain can be directly moved through the plasma membrane of erythrocytes. The data presented show that, although the catalytic domain plays a key role in the cytotoxic activity of CyaA through the catalysis of the conversion of ATP into camp, this domain is not required for binding of CyaA with its receptor. These results additionally show that the domain of interaction of CyaA/CD11b localized in hemolyzing part of the molecule and, more precisely, part of the region rich in glycine and aspartate RTX repeats containing residues 1166-1281, certain sites insertions FLAG-epitope structures with strongly manifested by binding to CD11b+-cells. In particular, the predicted loop structure, is inserted between the two blocks RTX repeats and containing the remains of 1208-1243 (Osicka et al., 2000), could play a crucial role in the interaction of CyaA with CD11b+-cells. Loss of interaction with CD11b structures CyaA1166/FLAG CyaAΔ1245-1273 and CyaA1281/FLAG, respectively, could be due to structural changes, selectionalism on functionally important segment, involved specifically in the interaction of CyaA protein with CD11b. It seems very likely, as all three designs, which could not bind CD11b, still showed significant cell-invasive activity (20%-50% of the activity of intact CyaA) in surrogate analysis system for red blood cells, where the activity of the toxin does not depend on interaction with CD11b. This suggests that the insertion of FLAG in the provisions 1166, 1281 1245 and not break the whole structure of CyaA, but rather selectively helps eliminate the ability of these structures to interact with CD11b+-cells. Together, these results suggest that the residues 1166-1281 CyaA outline the essential part enterinvasive domain involved in the interaction of the toxin with αMβ2-integrin (CD11b/CD18).

This conclusion is supported by results showing that all variants of CyaA with FLAG-peptide, insertions in the first 800 residues CyaA, fully competed for binding to CD11b with biotinylated intact CyaA. In contrast, CD11b-binding capacity was slightly reduced proteins CyaA1416/FLAG CyaA/FLAG1623 and CyaA/FLAG1648, which implies that the auxiliary CD11b-interacting domain of CyaA can be localized in the direction of carboxykinase side part RTX repeats this toxin.

The results that identify the area 1166-287 as the main CD11b-binding motif CyaA, provide an attractive explanation for earlier observations that binding of CyaA with CD11b was strictly Kalnyshevsky (Guermonprez et al., 2001). Because RTX-domain is involved in binding of calcium and undergoes a large structural rearrangeable after binding of calcium (Rose et al., 1995), suggest that CD11b binding motif located in the area 1166-1287, may be subject only to the influence of kaliswari conformation RTX domain. CD11b-binding motif identified here in the region of amino acids 1166-1287 CyaA, localized precisely between the second and third blocks RTX repeats. You can make the assumption that the α-helical structure of this segment is involved in the formation site of reception ("docking") for CD11b.

CyaA used in several protocols passive and active protection in mouse models of pertussis. Immunization with specific anti-CyaA-antibodies or purified CyaA reduced the time course of colonization of the respiratory tract B.pertussis and protected mice against a lethal intranasal infection (Guiso et al., 1989; Guiso et al., 1991). In addition, antibodies that are specific against CyaA, were detected in the sera of children (people)infected with B.pertussis (Arciniega et al., 1991; Guiso et al., 1993). These results suggest that the molecule CyaA, devoid, domain interaction of CyaA/CD11b, can be constructed for receipt is safe acellular vaccine to protect against infection with B.pertussis. The catalytic activity of such molecules can be easily inactivated by insertion of a dipeptide in the ATP binding site localized between residues 188 and 189 CyaA (Fayolle et al., 1996), whereas a deletion in the domain of interaction CD11b could prevent potentially negative effects of immune cells, such as signal transduction after binding of the integrin toxodon and/or a functional intervention, due to competition for binding to CD11b with toxodon CyaA, which also serves as an additional receptor CR3.

In conclusion, the authors believe that the presented results provide an important ability of penetration into the role of acylation and different domains of adenylate cyclase B.pertussis in its interaction with CD11b+cells, as well as in the subsequent biological activities triggered by this interaction.

1. The polypeptide which is a fragment of the Bordetella adenylate cyclase, where the specified polypeptide is able to communicate with CD11b/CD18 and contains a domain of interaction CD11b/CD18 specified Bordetella adenylate cyclase wild-type, consisting of:
(i) amino acid sequence, forgive audacia from position 1166 to position 1281 SEQ ID NO: 1; or
(ii) the amino acid sequence SEQ ID NO: 4; or
(iii) the respective domain of adenylate cyclase Bordetella species identified by comparison of the sequence of adenylate cyclase in the specified Bordetella sequence extending from amino acid 1166 to amino acids 1281 SEQ ID NO: 1 to achieve the best local mapping,
where the specified polypeptide has up to 500 amino acids.

2. The polypeptide according to claim 1, which is able to induce antibodies that recognize specific adenylate cyclase type Bordetella, preferably Bordetella pertussis adenylate cyclase.

3. The polypeptide according to claim 1, where the specified polypeptide:
(i) extends from amino acid 1166 to amino acids 1281 SEQ ID NO: 1;
(ii) is the appropriate domain of adenylate cyclase Bordetella species identified by comparison of the sequence of adenylate cyclase in the specified Bordetella sequence characterized in (i) to achieve the best local mappings.

4. The polypeptide according to claim 1, which additionally contains a domain acylation of adenylate cyclase and/or hydrophobic domain.

5. The polypeptide according to claim 1, where the specified polypeptide is non-toxic when introduced in vivo to a mammal.

6. The use of the polypeptide according to any one of claims 1 to 5 in the preparation of vectors for specific targeting expressing CD11b cells to provide the Commissioner of interest molecules, associated with the specified polypeptide.

7. Protein vector for targeting interest molecules on expressing CD11b/CD18 cells, characterized by the fact that the vector contains the polypeptide has the ability to communicate with CD11b/CD18 according to claim 1, associated with the interest of the molecule.

8. Protein vector for targeting interest molecules on expressing CD11b/CD18 cells, characterized by the fact that the vector contains the polypeptide has the ability to communicate with CD11b/CD18 according to any one of claim 2 to 5, associated with the interest of the molecule.

9. Protein vector for targeting interest molecules on expressing CD11b/CD18 cells according to claim 7, where this interest is a molecule selected from the group consisting of peptides, glycopeptides, lipopetides, polysaccharides, oligosaccharides, nucleic acids, lipids and chemicals.

10. Protein vector for targeting interest molecules on expressing CD11b/CD18 cells according to claim 7, where the specified interest molecule is the active (current) start medicines.

11. Protein vector for targeting interest molecules on expressing CD11b/CDIS cell according to claim 7, where the specified interest molecule is linked by a chemical bond.

12. Protein vector for which Eliane interest molecules on expressing CD11b/CD18 cells according to claim 7, where this interest, the molecule contains an antigen or epitope.

13. Protein vector for targeting interest molecules on expressing CD11b/CD18 cells according to claim 7, where the specified interest molecule is a peptide or polypeptide containing an antigen or epitope.

14. Nucleic acid which encodes one of the following polypeptides:
A. the polypeptide according to any one of claims 1 to 5;
b. protein vector according to claim 7.

15. Expressing the vector containing nucleic acid for 14 suitable for expression of the encoded polypeptide in the cell host.

16. Expressing the vector according to item 15, which is a plasmid, kosmidou, fahmida or viral DNA.

17. A host cell containing nucleic acid by 14.

18. A host cell containing expressing the vector according to item 16.

19. A composition comprising a protein vector according to claim 7, which is suitable for administration to an animal or human host, which requires targeting specific cells.

20. The composition according to claim 19, where these white cells are myeloid dendritic cells, neutrophils or macrophages.

21. Polyclonal serum obtained by immunization of an animal or human polypeptide according to claim 1 or a composition according to claim 19 or 20.

22. Polyclonal serum item 21, which is able to block the binding of hell is neutrilize with CD11b/CD18.

23. The method of targeting in vitro interest molecules on expressing CD11b cells, providing
A. ensuring expressing CD11b cells extracted from a living organism, and
b. the cultivation of these expressing CD11b cells with the protein vector according to claim 7, in suitable conditions, to target the specified vector to the specified expressing CD11b cells.

24. The method according to item 23, where this interest, the molecule contains an antigen or epitope.

25. The method according to item 23, where specified expressing CD11b cells are myeloid dendritic cells.

26. Expressing CD11b cells containing an interest in the molecule, obtained by the method of targeting according to any one of p-25.



 

Same patents:

FIELD: chemistry; biochemistry.

SUBSTANCE: invention relates to biotechnology. The Rhodococcus rhodochrous NCIMB 41164 strain produces nitrile hydrase. The strain is cultured in a culture medium containing urea or a urea derivative. Urea or derivative there of is added to the culture medium at least 6 hours after the microorganism begins to grow. Biocatalytic activity of the Rhodococcus rhodochrous NCIMB 41164 strain increases and is equal to 250-300000 mcmol/min/g of the dry biomass of cells. Nitrile hydrase obtained from said strain can convert (meth)acrylonitrile to (meth)acrylamide. The invention also discloses methods of producing amide from the corresponding nitrile. The methods involve reaction of nitrile hydrase in an aqueous medium in the presence of a biocatalyst which is a Rhodococcus rhodochrous NCIMB 41164 strain and the amide is (meth)acrylamide. The invention also discloses a suspension for storing biocatalyst, containing inactively growing cells of this strain, water and optionally residual components of the fermentation broth. The biocatalyst is stored at temperature higher than freezing point, preferably higher than 0°C and specifically from 4 to 30°C.

EFFECT: higher biocatalytic activity of the Rhodococcus rhodochrous NCIMB 41164 strain.

23 cl, 5 tbl, 7 ex

FIELD: agriculture.

SUBSTANCE: plant seeds are treated with ethylmethanesulphonate during 16 hours, then dried or greensprouted. The resulting plants M1 are self-pollinated and their seeds are obtained, which are used for growing the M2 wheat plants. The M2 plants are treated with imazamox, and plants with higher resistance to that as compared to the wild type plants are selected. The other version implies the transformation of plants with an expression vector containing one or more nucleic acids IMI.

EFFECT: higher resistance of plants to imidazolinone and possibility to use them when controlling weeds using herbicide.

44 cl, 8 dwg, 4 ex

FIELD: agriculture.

SUBSTANCE: plant seeds are treated with ethylmethanesulphonate during 6 hours, then dried or greensprouted. The resulting plants M1 are self-pollinated and their seeds are obtained, which are used for growing the M2 wheat plants. The M2 plants are treated with imazamox, and plants with higher resistance to that as compared to the wild type plants are selected. The other version implies the transformation of plants with an expression vector containing one or more nucleic acids IMI. The said nucleic acids are preferably located in different genomes or obtained from different genomes.

EFFECT: resulting plants have higher resistance to imidazolinone and may be used when controlling weeds using herbicide.

47 cl, 13 dwg, 5 ex

FIELD: organic chemistry, biotechnology.

SUBSTANCE: invention refers to biotechnology. Method describes nucleotide sequence coding polypeptide of acetooxyacids synthetase activity (AHAS) and coded polypeptide. Sequences are given in detailed description. Polypeptide application for enzymatic production of enantiomer-concentrated amino acids with branched chain is described. Vector of acetooxyacids synthetase (AHAS) expression in cells Corynebacterium and microorganism Corynebacterium, carrying specified vector are described. Specification for primer of coding nucleotide sequence by "ПЦР" method is provided. Transfer probe applied for specified nucleotide sequences detection is described. Method of recombinant polypeptides production is discovered implying that specified nucleotide sequence is exposed to mutagenesis. Then nucleotide sequence is cloned and transferred to proper expression system. Produced polypeptides are detected and separated. There is a detailed description provided of recombinant polypeptide produced by specified method and its application for enzymatic production of enantiomer-concentrated amino acids with branched chain. This invention enables to increase production of amino acids with branched chain by zymosis.

EFFECT: increased production of amino acids with branched chain.

16 cl, 2 dwg, 3 tbl, 7 ex

FIELD: biotechnology, in particular deep cultivation of Bacillus circulans.

SUBSTANCE: invention relates to deep cultivation of pectin-lyase producer such as Bacillus circulans, liquid standardization thereof and drying of cultural liquid or filtrate thereof. Cultural broth contains maize flour (2.75-3.0 %) or maize starch (2.75 %) or mixture of maize flour (3 % or less) with wheal bran (4 % or less). Method includes pH changing of filtrate or cultural liquid standardized with salts before dehydration. Changing of broth composition males it possible to obtain increased productivity of pectin-lyase biosynthesis process (up to 34100 U/cm3, to increase enzyme heat resistance up to 85.1 % (based on starting level); and changing of pH value (from 6.0 to 7.8) makes it possible to additionally increase of heat resistance up to 12.2 %.

EFFECT: bacterial enzyme with increased heat resistance.

2 tbl, 4 ex

The invention relates to biochemistry, can be used in biochemistry to study the regulatory effects of nitric oxide and mechanism of action of peroxide-based RHC sesquiterpenoids artemisininbased number of General formula I

The invention relates to new chemical compounds, in particular derived 1,4,2,5-deoxidizing General formula I, where R denotes the formula II and R1denotes the N3or NO2

The invention relates to biochemistry, in particular for use industeries derived maleimide General formula I, where if R=R1- H, R2- H or lower alkyl and R3--D-xylopyranosyl (D-Xyl),-D-galactopyranosyl (D-Gal),-L-arabinopyranosyl (L-Ara),-D-lactophenol (D-Lac) or - och2OCH2CH2HE, or if R+R1- simple connection, R2Is h or lower alkyl and R3--D-xylopyranosyl,-D-galactopyranosyl,-L-arabinopyranosyl or - och2OCH2CH2HE, as an inhibitor of the soluble form of guanylate cyclase (RGC)

The invention relates to biochemistry, in particular, to the use of known substituted N-(2-aminobenzyl)cyclohexylamine General formula, where R=Cl or Br, R1=H or HE, and their pharmacologically acceptable salts as an inhibitor of NO-dependent activation of soluble form of guanylate cyclase (RGC)

The invention relates to biochemistry, in particular to the application of known antibiotic bruneomitsina /streptonigrin, 5-amino-6-(7-amino-5,8-dihydro-6-methoxy-5,8-dioxoindoline-2-yl)-4-(2-hydroxy-3,4-acid)-3-methylpyridin-2-carboxylic acid and its known derivatives of the General formula where, if- simple connection, then X=NH and R=O or =NH, and, if- double bond, then X=N and R - carboxyl, (lower alkyl)oxycarbonyl, [bis(lower alkyl)amino] etoxycarbonyl or a group of General formula C(O)other1where R1=H, NH2, NHC(O)NH2, NHC(S)NH2, unsubstituted lower alkyl, lower alkyl containing as a substituent a hydroxyl, carboxyl or (lower alkyl) oxycarbonyl group, or-D-hexopyranosyl and biochemically acceptable salts as an inhibitor of NO-dependent activation of soluble form of guanylate cyclase (RGC)

FIELD: medicine, pharmaceutics.

SUBSTANCE: in invention described is hybridoma, producing monoclonal antibodies, which bind with protein 161P2F10B in a specific way. Invention describes versions of monoclonal antibodies, one of which includes amino acid sequence of heavy and light chain of antibody, produced by said hybridoma, and the other includes amono acid sequence of variable part of heavy and light chain of antibody, produced by said hybridoma, or amino acid sequence Fab, F(ab')2, Fv or Sfv of antibody fragment, which binds with protein 161P2F10B, 161P2F10B demonstrates tissue-specific expression in healthy, mature tissue and is abnormally expressed in case of oncologic diseases.

EFFECT: antibodies can be used as anti-cancer medication and in composition of pharmaceutical composition for treating cancer, in particular, kidney cancer, reducing tumour growth, as well as for detection of protein 161P2F10B in biological sample.

11 cl, 212 dwg, 8 tbl, 20 ex

FIELD: medicine.

SUBSTANCE: essence of invention includes contact of liquid sample, taken from mammal organism, with one or several monoclonal antibodies to Lawsonia intracellularis antigen, secreted by cell lines of ECACC hybridomes, which have registration numbers. Invention also includes diagnostic test-kit, containing antibodies specific for Lawsonia intracellularis.

EFFECT: increased specificity of antigen detection.

11 cl, 5 ex, 5 dwg

FIELD: medicine.

SUBSTANCE: invention aims at preparation of new strain of hybrid cells Mus. Musculus 6F3 - a producer of monoclonal antibody (MCA) to hemagglutinin protein of high-pathogen avian influenza virus A/duck/Novosibirsk/56/05. Strain 6F3 is prepared by fusing murine myeloma cells Sp2/0 with murine spleen cells BALB/c, immunised with a purified and inactivated preparation of avian influenza virus A/H5N1 (strain A/duck/Novosibirsk/56/05). Hybridoma produced MCA belong to IgA class. Strain 6F3 is deposited in the Collection of cell culture of Ivanovsky State Research Institution of Virology of the Russian Academy of Medical Sciences, No. 8/2/3. Using hybridoma allows producing specific monoclonal antibodies to hemagglutinin protein of avian influenza virus A/H5N1.

EFFECT: possibility to use antibodies to studying the antigenic structure of hemagglutinin for differential diagnostics of avian influenza virus A/H5 serotype.

1 dwg, 6 ex

FIELD: agriculture.

SUBSTANCE: invention relates to the field of genetic engineering and cloning. Transgenic cow is disclosed, having low activity of prion protein as a result of one or several genetically designed mutations. Specified transgenic cows are also genetically modified for expression of xenoantibodies.

EFFECT: as a result of their resistance to prion diseases, such as trembling disease of cattle, such cows are a safe source of human antibodies for use in pharmaceutics and a safe source of agricultural products.

12 cl, 112 dwg, 13 tbl, 19 ex

FIELD: medicine.

SUBSTANCE: invention refers to antibody specifically getting bound with PRO87299 version. In addition, the antibody according to the invention has ability to block interaction HVEM and PRO87299 and to function as PRO87299 agonist. The antibody of agonist nature is produced by hybridoma Btig5F5.1 or Btig3B1.9. For the antibody, there is established amino acid sequence given in the description. The invention discloses the methods of using the antibodies to stimulate or reduction of immune response in immune-associated diseases connected, to relieve lymphoma, and inflammatory disease in requiring mammal, to detect polypeptide PRO87299 in a sample and to manage rejection of grafted cells.

EFFECT: antibody is an immunomodulator that allows applying therapeutically identical medicinal agents both to intensify and reduce immune response.

16 cl, 34 dwg, 7 tbl, 20 ex

FIELD: chemistry.

SUBSTANCE: proposed is a chimeric or humanised monoclonal antibody against hepatocyte growth factor, produced from L2G7 antibody. Invented is a mouse antibody L2G7, produced by hybridoma ATCC PTA-5162, and the said hydbridoma. Described is a cell line, producing a chimeric or humanised monoclonal antibody against hepatocyte growth factor. Proposed is a pharmaceutical composition and a method of treating tumours based on the said antibody.

EFFECT: use of the invention provides for a neutralising antibody against hepatocyte growth factor, which can be used in treating human cancer.

7 cl, 12 dwg, 1 tbl, 3 ex

FIELD: medicine.

SUBSTANCE: invention concerns area of medicine and concerns the therapeutic agent for treatment of diseases related to children's chronic arthritic diseases, for example, actually children's chronic arthritic diseases, Still's disease and similar to it, including an antagonist of the interleukin-6 IL-6 receptor as an active ingredient, humanised antibody PM-1. Advantage of the invention consists in efficiency increase.

EFFECT: efficiency increase.

5 cl, 4 ex

FIELD: biotechnology, immunology.

SUBSTANCE: disclosed are variants of chimerical anti-IL-6 antibodies based on mice CLB-8 antibody. Each antibody contains constant region from one or more human antibodies. Described are variants of nuclear acids encoding anti-IL-6 antibody, vectors and host cells. Developed is method for production of anti-IL-6 antibody by using nuclear acid or vector. Described are variants of composition for application in method for modulation of malignant disease or immune disorder mediated with IL-6. Developed is method for treatment or modulation of malignant disease or immune disorder mediated with IL-6.

EFFECT: variant of chimerical anti-IL-6 antibody with high affinity of mice anti-IL-6 antibody and reduced immonogenicity.

26 cl, 16 dwg, 1 tbl, 8 ex

FIELD: medicine, biotechnology.

SUBSTANCE: invention proposes variants of antibodies showing specificity to peptide domain located by both side of hinged site R76S77 in pro-BNP(1-108). Indicated antibodies recognize specifically also circulating pro-BNP(1-108) in human serum or plasma samples but they don't recognize practically peptides BNP(1-76) or BNP(77-108). Also, invention describes variants of peptides used in preparing antibodies. Amino acid sequence is given in the invention description. Also, invention discloses methods for preparing indicated antibodies and among of them by using indicated peptides. Also, invention describes methods for preparing antibody-secreting hybridoma, and hybridoma is disclosed prepared by indicated method. Also, invention describes a monoclonal antibody secreted by hybridoma 3D4 and deposited at number CNCM I-3073. Also, invention discloses variants for diagnosis of cardiac insufficiency in vitro and by using antibodies proposed by the invention. Also, invention describes a set used for detecting pro-BNP(1-108) in a biological sample. Using this invention simplifies detection of pro-BNP(1-108) circulating in human serum or plasma samples and provides specific detection of pro-BNP(1-108) that can be used in early diagnosis of human cardiac insufficiency.

EFFECT: valuable medicinal properties of antibodies.

24 cl, 16 dwg, 5 tbl, 20 ex

FIELD: medicine, analytical immunology.

SUBSTANCE: invention relates to a set of reagents used in quantitative determination of secretory immunoglobulin A (sIgA) that provides assaying status of topical immunity and immunodeficient states in carrying out analysis of serum and secrets of human body. Proposed set comprises a plate with immobilized monoclonal antibodies, five calibrating samples containing 0; 1.0; 5.0; 10.0 or 20.0 mcg of sIgA/ml, conjugate of murine monoclonal antibodies with horse radish peroxidase and a reagent for carrying out the enzymatic reaction. Murine monoclonal antibodies produced by hybrid strain of animal Mus musculus L., № PKKK (P) 676D cultured cells, are immobilized on a carrier. Conjugate comprises murine monoclonal antibodies produced by another strain - Mus musculus L., № PKKK (P) 677D. The advantage of invention involves enhancing sensitivity in assay of sIgA.

EFFECT: improved assay method.

3 tbl, 5 ex

FIELD: biology.

SUBSTANCE: present invention pertains to biotechnology and is a mutant acetolactase synthase of bacteria (AHAS I), in which L-amino acid in positions 17, 30 and/or 33 in a small subunit of natural acetolactase synthase from Escherichia coli is substituted with another L-amino acid or several L-amino acids are integrated in the said position(s), and feedback inhibition with valine in the given subunit is weak. The invention also relates to a DNA fragment, encoding the acetolactase synthase, which is used to transform Escherichia bacteria so as to obtain branched L-amino acid.

EFFECT: invention allows for obtaining branched L-amino acid with high level of efficiency.

17 cl, 8 dwg, 5 tbl, 8 ex

Up!