Proteins binding antigen, growth factor similar to heparin-binding epidermal growth factor

FIELD: biotechnologies.

SUBSTANCE: invention proposes an antibody that specifically binds heparin-binding EGF-like growth factor (HB-EGF) and its antigen-binding fragment. Invention describes a nucleic acid molecule, an expressing vector, a host cell and a method for obtaining an antibody or its antigen-binding fragment, as well as use of antibody or its antigen-binding fragment for obtaining pharmaceutical composition for diagnostics, prevention or treatment of hyperproliferation disease, methods and sets for diagnostics and prevention or treatment of the state associated with HB-EGF expression. This invention can be further found in therapy of diseases determined with or related to HB-EGF expression.

EFFECT: improving efficiency of composition and treatment method.

34 cl, 43 dwg, 28 ex, 12 tbl

 

The text descriptions are listed faxing

1. An isolated antibody or antigennegative fragment that specifically binds HB-EGF, including:
a) a CDRL1 of SEQ ID NO:207, CDRL2 of SEQ ID NO:225, CDRL3 of SEQ ID NO:257, and
CDRH1 of SEQ ID NO:292, CDRH2 of SEQ ID NO:323, and a CDRH3 of SEQ ID NO:361 or
b) a CDRL1 of SEQ ID NO:210, CDRL2 of SEQ ID NO:221, CDRL3 of SEQ ID NO:260, and
CDRH1 of SEQ ID NO:292, CDRH2 of SEQ ID NO:320, and a CDRH3 of SEQ ID NO:362, or
c) a CDRL1 of SEQ ID NO:213, CDRL2 of SEQ ID NO:230, CDRL3 of SEQ ID NO:263, and
CDRH1 of SEQ ID NO:293, CDRH2 of SEQ ID NO:324, and a CDRH3 of SEQ ID NO:364.

2. An isolated antibody or antigennegative fragment according to claim 1, including:
a) VLof SEQ ID NO:121 and VHof SEQ ID NO:172, or
b) VLof SEQ ID NO:124 and VHof SEQ ID NO:175, or
c) VLof SEQ ID NO:127 and VHof SEQ ID NO:178.

3. An isolated antibody or antigennegative fragment according to claim 1, which specifically recognizes at least IHGE-containing epitope and/or EGF-like domain of HB-EGF.

4. An isolated antibody or antigennegative fragment according to claim 1, which competes for binding with the antibody according to claim 1.

5. An isolated antibody or antigennegative fragment according to claim 1, which represents a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a human antibody, humanitariannet antibody or chimeric antibody.

6. An isolated antibody or antigennegative fragment according to claim 5, where the specified antibody fragment is a Fab fragment, a fragment Fab', the fragment F(AB')2the Fv fragment, dianthicola or single-stranded molecule of the antibody.

7. An isolated antibody or antigennegative fragment according to claim 5, representing a human antibody.

8. An isolated antibody or antigennegative fragment according to claim 5, representing monoclonalnoe antibody.

9. An isolated antibody or antigennegative fragment according to claim 1, which represents the type IgG1, IgG2, IgG3 or IgG4.

10. An isolated antibody or antigennegative fragment according to claim 9, representing the type IgG2 or IgG4.

11. An isolated antibody or antigennegative fragment according to claim 1, combined with group-label.

12. An isolated antibody or antigennegative fragment according to claim 11, where the group-label is a radioisotope, radionuclide, a fluorescent group, enzyme group, a chemiluminescent group, or biotinyl group.

13. An isolated antibody or antigennegative fragment according to claim 1, combined with effector group.

14. An isolated antibody or antigennegative fragment according to item 13, where the indicated effector group is a radioisotope, radionuclide, a toxin, a therapeutic group or chemotherapeutic group.

15. An isolated antibody or antigennegative fragment on 14 where the specified therapeutic group or chemotherapy group represents calicheamicin, auristatin-RE, geldanamycin or mechanisin.

16. An isolated antibody or antigennegative fragment according to claim 1, reduce, at least partially, HB-EGF-mediated signal transduction.

17. The molecule of nucleic acid, Cody the respective isolated antibody or antigennegative fragment according to any one of claims 1 to 4.

18. The nucleic acid molecule according to 17, functionally connected with the control sequence.

19. Expressing a vector comprising the nucleic acid molecule by 17.

20. Expressing a vector comprising the nucleic acid molecule according p.

21. A host cell expressing the antibody according to claims 1 to 4, comprising the vector according p.

22. A host cell expressing the antibody according to claims 1 to 4, comprising the vector according to any one of p or 20.

23. A method of obtaining antibodies or antigennegative fragment according to any one of claims 1 to 4, comprising a step for the specified antibodies or antigennegative fragment from the host cell, which secretes a specified antibody or a fragment of such antibody.

24. The use of at least one antibody or its antigennegative fragment according to claims 1 to 4 to obtain pharmaceutical compositions for the diagnosis, prevention or treatment of hyperproliferative diseases.

25. The application of paragraph 24, the specified hyperproliferative disease associated with the expression of HB-EGF.

26. The way to diagnose the condition, associated with expression of HB-EGF, including the stage of contacting the sample with the antibody or antigennegative fragment according to claims 1-4, and determining the presence of HB-EGF in the specified pattern.

27. The method according to p, where the state represents the Wallpaper hyperproliferative disease by A.25.

28. The method of prevention or treatment of a patient condition associated with expression of HB-EGF, including placing a patient in need, an effective amount of at least one antibody or its antigennegative fragment according to claims 1-4.

29. The method according to p, where the condition is a hyperproliferative disease by A.25.

30. The method according to p, where the patient is a mammal.

31. A kit for the diagnosis of hyperproliferative diseases associated with expression of HB-EGF, comprising the antibody or antigennegative fragment according to claims 1-4.

32. Set for the prevention or treatment of hyperproliferative diseases associated with expression of HB-EGF, comprising the antibody or antigennegative fragment according to claims 1-4.

33. Set according to any one of p or 32, comprising at least one additional active agent.

34. Set p, where the additional active agent is an antitumor agent.



 

Same patents:

FIELD: biotechnologies.

SUBSTANCE: invention describes polynucleotide, expression vector, host cell and production method of humanised antibody together with their use, as well as medical preparation against rheumatoid arthritis, prophylaxis or treatment method of rheumatoid arthritis and use of humanised antibody at production of pharmaceutical preparation for prophylaxis or treatment of rheumatoid arthritis. This invention can be used in therapy of human diseases associated with α9 integrin.

EFFECT: improved activity and thermal stability.

14 cl, 6 dwg, 6 tbl, 11 ex

Organic compounds // 2502802

FIELD: biotechnologies.

SUBSTANCE: invention refers to eucariotic vector for expression of target recombinant product in a mammal cell and to its use, to a mammal cell for production of target recombinant product and to a method for its production, a method of a mammal cell selection and a method for obtaining a target recombinant product. Vector includes the first polynucleotide coding a functional folate receptor bound to a membrane as a selective marker and the second polynucleotide coding the target product that is expressed in a recombinant manner. Target product represents a pharmaceutically active, therapeutically active or diagnostic polypeptide. Functional folate receptor bound to the membrane and target product are expressed from the above expression vector. Sampling system is based on introduction of a gene of exogenic functional folate receptor bound to the membrane to a mammal cell.

EFFECT: invention allows effective selection of transformed cells and high yield of target product.

26 cl, 3 tbl, 2 ex

FIELD: biotechnologies.

SUBSTANCE: expression vector includes: (a) replication origin OriP obtained from Epstein-Barr virus (EBV), where replication origin contains: 1) symmetry element of the second order (DS); and 2) duplication section (FR) that contains fixation point EBNA; (b) replication origin SV40; (c) insertion section for inserting a gene of concern; (d) promoter EF-1b functionally bound to the insertion section; (e) poly-A signal; (f) bacterial replication origin; (g) selected marker; and unnecessarily containing (h) sequence of nucleic acid, which codes constant area of heavy or light chain of antibody, which is functionally bound to the insertion section. With that, replication origin OriP is bound to an initiation factor of replication EBNA 1, which acts from outside and is not coded with an expression vector.

EFFECT: use of an expression vector in an extracted host cell, a set and a method for obtaining recombinant protein provides production of abundant protein expression.

26 cl, 25 dwg, 3 tbl, 4 ex

FIELD: biotechnologies.

SUBSTANCE: invention can be used for obtaining recombinant human blood coagulability factor VIII with deletion of B-domain (hFVIII-BDD). Recombinant plasmid DNA pAP227 coding polypeptide with sequence hFVIII-BDD also including MAR - binding area to nuclear matrix of lysozyme gene of birds, virus transmission enhancer CMV, internal translation initiation site IRES of encephalomyocarditis virus, gene DHFR of a mouse, a polyadenylation signal of virus SV40, gene of aminoglycoside-3'-phosphotransferase providing stability to geneticin (Neo) and a cassette for expression in bacteria cells of gene of β-lactamase providing stability to ampicillin, cells of line Cricetulus griseus CHO DHFR(-) are obtained so that there produced is cell line Cricetulus griseus CHO 2H5 producing recombinant hFVIII-BDD with highly stable yield at the level of about 20 IU/ml/24 h. Cultivation of cells-producers is performed in medium DME/F12 containing 2-4% of Fetal Bovine Serum, 1% of dimethylsulphoxide and 50 IU/l of insulin.

EFFECT: improvement of the method.

4 cl, 5 dwg, 9 ex

FIELD: biotechnologies.

SUBSTANCE: recombinant plasmid DNA pBK415 coding polypeptide with sequence of tissular activator of human plasminogen, also including MAR - binding area to nuclear matrix of lysozyme gene of birds, virus transmission enhancer CMV, internal translation initiation site IRES of encephalomyocarditis virus, gene DHFR of a mouse, a polyadenylation signal of virus SV40, gene of aminoglycoside-3'-phosphotransferase providing stability to geneticin (Neo) and a cassette for expression in bacteria cells of gene of β-lactamase providing stability to ampicillin, cells of line Cricetulus griseus CHO DHFR(-) are obtained so that there produced is cell line Cricetulus griseus CHO 1F8 producing recombinant protein of tissular activator of plasminogen with highly stable yield at the level of up to 190 mg/l. Cultivation of cells-producers is performed under perfusion conditions in presence of a mixture consisting of additive CHO Bioreactor supplement and sodium butyrate or dimethylsulphoxide with further separation of a target product.

EFFECT: improvement of the method.

5 cl, 5 dwg, 3 tbl, 8 ex

FIELD: biotechnologies.

SUBSTANCE: invention can be used for obtaining recombinant blood coagulability factor IX of human being (hFIX). Recombinant plasmid DNA pAK380 containing gene of protein rhFIX, MAR - binding area to nuclear matrix of lysozyme gene of birds, virus transcription enhancer CMV and an internal translation initiation site IRES of encephalomyocarditis virus, gene DHFR of a mouse, a polyadenylation signal of virus SV40, gene of aminoglycoside-3'-phosphotransferase for stability to geneticin (Neo), a cassette for expression in bacteria cells of gene β-lactamase for stability to ampicillin, is used for obtaining recombinant factor hFIX in cells of line Cricetulus griseus CHO 1E6. By transformation of cell line C. griseus CHO DHFR - recombinant plasmid DNA pAK380 there obtained is cell line C. griseus CHO 1E6 producing recombinant hFIX with stable high yield at the level of 50 mg/l/24 h. After cultivation of cells-producers there extracted is hFIX by pseudoaffine chromatography on Q Sepharose with elution of 10mM CaCl2; then, on Heparin-Sepharose FF with elution of 600 mM NaCl, and chromatography on hydroxyapatite of type I with elution of 600 mM K3PO3 and chromatography on Source 30Q with elution of 600 mM with ammonium acetate.

EFFECT: improvement of the method.

4 cl, 5 dwg, 7 ex, 3 tbl

FIELD: biotechnologies.

SUBSTANCE: invention proposes an antibody that specifically connects segment M1' IgE and that induces apoptosis in IgE-expressing B-cells and its antigen-binding fragment. Besides, compositions and curing methods of IgE-mediated abnormalitiy, an item, a specific elimination method of IgE-producing B-cells, methods for prophylaxis and reduction of IgE products induced with an allergen, as well as isolated nucleic acid, an expression vector, a host cell and a method for obtaining an antibody as per the invention together with their use are considered.

EFFECT: invention can be further used in therapy of diseases associated with IgE.

46 cl, 19 dwg, 5 tbl, 13 ex

FIELD: biotechnologies.

SUBSTANCE: method involves introduction to a plant, some part of the plant or a plant cell of nucleotide sequence for 80-100% of identical nucleotide sequence determined in SEQ ID NO: 17, and coding a composite protein containing a cytoplasmic end segment, a transmembrane domain, a steam area (CTS domain) of N-acetylglucosaminyl transferase (GNT1), which is merged with catalytic domain of beta-1,4-galactosyl transferase (GalT); with that, the above first nucleotide sequence is functionally connected to the first regulatory area being active in the plant; and the second nucleotide sequence for coding of a target protein; with that, the above second nucleotide sequence is functionally connected to the second regulatory area being active in the plant, as well as transient co-expression of the first and the second nucleotide sequences with synthesis of the target protein containing glycans, with reduced xylosylation, reduced fucosylation or their combination at comparison to the same target protein obtained from a wild plant. The invention described nucleic acid coding the protein that modifies glycosylation of target protein, a composite protein for modification of glycosylation of target protein; nucleic acid that codes it, as well as a plant, a plant cell and a seed, which contain the above nucleic acid or the above composite protein.

EFFECT: invention allows effective production of a target protein with reduced xylosylation, reduced fucosylation or their combination.

20 cl, 7 dwg, 9 ex

Vns-met-histones // 2498997

FIELD: biotechnologies.

SUBSTANCE: nucleic acid molecule codes a polypeptide consisting of two residues of methionine as the first and the second N-end amino-acid residues connected through a peptide link to a mature eucariotic histone. Polypeptide is obtained by cultivation of a host cell transformed by an expression vector including the above molecule of nucleic acid. Polypeptide is used as part of pharmaceutical composition for therapy of cancer, bacterial, virus or fusarium infections. Besides, polypeptide is used as part of composition for diagnostics of a patient in relation to response to pharmaceutical composition containing the above polypeptide, or in relation to curability using it.

EFFECT: invention allows improving efficiency of recombinant expression and simplifying determination of the above polypeptide in presence of endogenic histones at preservation of biologic activity of mature eucariotic histone.

17 cl, 3 dwg, 6 tbl, 7 ex

FIELD: biotechnologies.

SUBSTANCE: proposed chimeric protein with SEQ ID NO:02 is fluorescent biosensor, built on the basis of HyPer protein and mutant of PH-domain of Btk tyrosine kinase.

EFFECT: proposed inventions allow performing simultaneous monitoring of product of hydrogen peroxide and phosphatidyl inositol-3,4,5-triphosphate in a living cell.

4 cl, 4 dwg, 3 ex

FIELD: biotechnologies.

SUBSTANCE: invention describes polynucleotide, expression vector, host cell and production method of humanised antibody together with their use, as well as medical preparation against rheumatoid arthritis, prophylaxis or treatment method of rheumatoid arthritis and use of humanised antibody at production of pharmaceutical preparation for prophylaxis or treatment of rheumatoid arthritis. This invention can be used in therapy of human diseases associated with α9 integrin.

EFFECT: improved activity and thermal stability.

14 cl, 6 dwg, 6 tbl, 11 ex

FIELD: biotechnologies.

SUBSTANCE: invention represents a method for obtaining recombinant DNAse I of a human or its mutein, as well as their conjugates with polyethylene glycol, using a bacterium belonging to Escherichia class, transformed with expression plasmid, containing a promoter functioning in a bacterial cell, DNA fragment coding a hexahistidine cluster, a fragment coding enterokinase recognition sequence amalgamated in frame with human DNAse I or its functionally active mutein containing replacements of asparagine with cysteine, transcription termination section, vector pET28a(+) fragment containing initiation section of replication of bacteriophage fl, sequence coding aminoglycoside-3'-phosphotransferase, area of beginning of plasmid pBR322 replication, gene RNA-organising protein Rop, sequence coding lactose operon repressor.

EFFECT: invention allows obtaining recombinant human DNAse I or its mutein with high yield.

18 cl, 7 dwg, 1 tbl, 12 ex

Organic compounds // 2502802

FIELD: biotechnologies.

SUBSTANCE: invention refers to eucariotic vector for expression of target recombinant product in a mammal cell and to its use, to a mammal cell for production of target recombinant product and to a method for its production, a method of a mammal cell selection and a method for obtaining a target recombinant product. Vector includes the first polynucleotide coding a functional folate receptor bound to a membrane as a selective marker and the second polynucleotide coding the target product that is expressed in a recombinant manner. Target product represents a pharmaceutically active, therapeutically active or diagnostic polypeptide. Functional folate receptor bound to the membrane and target product are expressed from the above expression vector. Sampling system is based on introduction of a gene of exogenic functional folate receptor bound to the membrane to a mammal cell.

EFFECT: invention allows effective selection of transformed cells and high yield of target product.

26 cl, 3 tbl, 2 ex

FIELD: chemistry.

SUBSTANCE: invention relates to a peptide capable of binding with scurfin and inhibiting biological activity of scurfin, which is selected from a peptide consisting of an amino acid sequence Arg-Asp-Phe-Gln-Ser-Phe-Arg-Lys-Met- Trp-Pro-Phe-Phe-X, where X is absent or X is present and represents X14 or X14-X15, where X14 and X15 independently denote an amino acid, a version of said peptide and a pharmaceutically acceptable salt thereof. The invention also discloses a fused protein and a pharmaceutical composition, which involves use of said peptide and fused protein, as well as use thereof to produce and treat pathologies which require transient regulation or inhibition of immunosuppressive activity of regulatory T lymphocytes, such as a neoplastic disease or infectious disease. The invention also relates to a method of producing said peptide and fused protein, including a protein or peptide coding nucleic acid, a DNA construct, an expression vector and a host cell.

EFFECT: invention provides effective treatment of infectious and neoplastic diseases which require transient regulation or inhibition of immunosuppressive activity of regulatory T lymphocytes.

26 cl, 10 dwg, 5 ex

FIELD: biotechnologies.

SUBSTANCE: invention can be used for obtaining recombinant human blood coagulability factor VIII with deletion of B-domain (hFVIII-BDD). Recombinant plasmid DNA pAP227 coding polypeptide with sequence hFVIII-BDD also including MAR - binding area to nuclear matrix of lysozyme gene of birds, virus transmission enhancer CMV, internal translation initiation site IRES of encephalomyocarditis virus, gene DHFR of a mouse, a polyadenylation signal of virus SV40, gene of aminoglycoside-3'-phosphotransferase providing stability to geneticin (Neo) and a cassette for expression in bacteria cells of gene of β-lactamase providing stability to ampicillin, cells of line Cricetulus griseus CHO DHFR(-) are obtained so that there produced is cell line Cricetulus griseus CHO 2H5 producing recombinant hFVIII-BDD with highly stable yield at the level of about 20 IU/ml/24 h. Cultivation of cells-producers is performed in medium DME/F12 containing 2-4% of Fetal Bovine Serum, 1% of dimethylsulphoxide and 50 IU/l of insulin.

EFFECT: improvement of the method.

4 cl, 5 dwg, 9 ex

FIELD: biotechnologies.

SUBSTANCE: recombinant plasmid DNA pBK415 coding polypeptide with sequence of tissular activator of human plasminogen, also including MAR - binding area to nuclear matrix of lysozyme gene of birds, virus transmission enhancer CMV, internal translation initiation site IRES of encephalomyocarditis virus, gene DHFR of a mouse, a polyadenylation signal of virus SV40, gene of aminoglycoside-3'-phosphotransferase providing stability to geneticin (Neo) and a cassette for expression in bacteria cells of gene of β-lactamase providing stability to ampicillin, cells of line Cricetulus griseus CHO DHFR(-) are obtained so that there produced is cell line Cricetulus griseus CHO 1F8 producing recombinant protein of tissular activator of plasminogen with highly stable yield at the level of up to 190 mg/l. Cultivation of cells-producers is performed under perfusion conditions in presence of a mixture consisting of additive CHO Bioreactor supplement and sodium butyrate or dimethylsulphoxide with further separation of a target product.

EFFECT: improvement of the method.

5 cl, 5 dwg, 3 tbl, 8 ex

FIELD: biotechnologies.

SUBSTANCE: invention can be used for obtaining recombinant blood coagulability factor IX of human being (hFIX). Recombinant plasmid DNA pAK380 containing gene of protein rhFIX, MAR - binding area to nuclear matrix of lysozyme gene of birds, virus transcription enhancer CMV and an internal translation initiation site IRES of encephalomyocarditis virus, gene DHFR of a mouse, a polyadenylation signal of virus SV40, gene of aminoglycoside-3'-phosphotransferase for stability to geneticin (Neo), a cassette for expression in bacteria cells of gene β-lactamase for stability to ampicillin, is used for obtaining recombinant factor hFIX in cells of line Cricetulus griseus CHO 1E6. By transformation of cell line C. griseus CHO DHFR - recombinant plasmid DNA pAK380 there obtained is cell line C. griseus CHO 1E6 producing recombinant hFIX with stable high yield at the level of 50 mg/l/24 h. After cultivation of cells-producers there extracted is hFIX by pseudoaffine chromatography on Q Sepharose with elution of 10mM CaCl2; then, on Heparin-Sepharose FF with elution of 600 mM NaCl, and chromatography on hydroxyapatite of type I with elution of 600 mM K3PO3 and chromatography on Source 30Q with elution of 600 mM with ammonium acetate.

EFFECT: improvement of the method.

4 cl, 5 dwg, 7 ex, 3 tbl

FIELD: biotechnologies.

SUBSTANCE: invention proposes an antibody that specifically connects segment M1' IgE and that induces apoptosis in IgE-expressing B-cells and its antigen-binding fragment. Besides, compositions and curing methods of IgE-mediated abnormalitiy, an item, a specific elimination method of IgE-producing B-cells, methods for prophylaxis and reduction of IgE products induced with an allergen, as well as isolated nucleic acid, an expression vector, a host cell and a method for obtaining an antibody as per the invention together with their use are considered.

EFFECT: invention can be further used in therapy of diseases associated with IgE.

46 cl, 19 dwg, 5 tbl, 13 ex

FIELD: biotechnologies.

SUBSTANCE: invention represents recombinant BMPRIA-CBD plasmid, and E.coli strain transformed with that plasmid. The invention also refers to recombinant BMPRIA-CBD protein that is used for production of BMP-2 protein.

EFFECT: invention allows producing chromatographic clean fractions of biologically active dimeric form of BMP-2 protein, which can be used as osteoinductive components of osteoplastic materials of new generation.

4 cl, 2 dwg, 6 ex

FIELD: biotechnologies.

SUBSTANCE: invention represents recombinant BMPRIB-CBD plasmid, and E.coli strain transformed with that plasmid. The invention also refers to recombinant BMPRIB-CBD protein that is used for production of BMP-7 protein.

EFFECT: invention allows producing chromatographic clean fractions of biologically active dimeric form of BMP-7 protein, which can be used as osteoinductive components of osteoplastic materials of new generation.

4 cl, 2 dwg, 6 ex

FIELD: biotechnology.

SUBSTANCE: method comprises the following stages: preparing cells in the first set comprising at least 96 receivers, adding an agent at least to one receiver; incubating the agent with the cells, lysing the cells by adding a buffer which is hypotonic or comprises detergent, and contains an inhibitor of DNAase; removal of RNA from the cell lysate with ribonuclease; centrifugation of the first set of receivers at low speed; transfer of a supernatant containing DNA fragments to the second set of receivers; adding a detectable compound capable of intercalation with DNA fragments in a buffer for detection containing DNAase inhibitor to at least one receiver; measuring the amount of intercalated detectable compound; and comparing the amount of intercalated detectable compound to the control for determining the difference that enables to identify the said agent as a modifying agent if the difference exceeds a predetermined limit.

EFFECT: invention enables to detect agents that affect the formation of DNA fragments in cells using high performance screening applications.

10 cl, 14 dwg, 2 tbl, 11 ex

Up!