Glycosylated antibodies (variants) possessing enhanced antibody-dependent cellular cytotoxicity

FIELD: medicine, peptides, biochemistry, pharmacy.

SUBSTANCE: invention relates to modification of glycosylation of proteins for preparing polypeptides with improved therapeutic indices including antibodies with enhanced antibody-dependent cellular cytotoxicity. For preparing indicated polypeptides cell-host is used that is modified with nucleic acid encoding enzyme β-1,4-N-acetylglucosaminyltransferase III (GnTIII). Prepared polypeptide represents, in particular, IgG or its fragment. Invention discloses a method for preparing polypeptide and antibodies or its fragment and a fusion protein prepared by indicated method. Invention describes a pharmaceutical composition used for increasing Fc-mediated cellular cytotoxicity and comprising antibody and carrier, and its using in cancer treatment, and a method for treatment of disease associated with elevated amount or production of B cells using indicated antibody, in particular, against CD20, and representing antibody IDEC-C2B8 in the preferable variant. Invention provides preparing polypeptide and antibody possessing the enhanced Fc-mediated cellular cytotoxicity that decrease the content of B cells in a patient body.

EFFECT: improved preparing method, valuable medicinal properties of polypeptide and antibodies.

38 cl, 21 dwg, 4 ex

 

The text descriptions are given in facsimile form.

1. A host cell modified to obtain a polypeptide having increased Fc-mediated cytotoxicity by expression of at least one nucleic acid that encodes a β(1,4)-N-acetylglucosaminyltransferase III (GnT III), with the specified polypeptide is a whole antibody molecule, an antibody fragment, and a fusion protein that includes a region equivalent to the Fc region of immunoglobulin, and the specified GnT III is expressed in a quantity sufficient to increase the proportion of these polypeptides containing a bifurcated hybrid oligosaccharides or galactoglucomannan complex oligosaccharides or mixtures thereof in Fc region.

2. A host cell according to claim 1, characterized in that the polypeptide is an IgG or fragment.

3. A host cell according to claim 1, characterized in that the polypeptide is an IgGI or fragment.

4. A host cell according to claim 1, characterized in that said polypeptide is a fusion protein, of which the first contains the area equivalent to the Fc region of human IgG.

5. A host cell according to claim 1, characterized in that the nucleic acid molecule containing at least one gene encoding GnT III, enter into the specified cell of the host.

6. A host cell according to claim 1, characterized in that specified a host cell modified so that the gene is activated endogenous GnT III.

7. A host cell according to claim 6, characterized in that the specified endogenous GnT III is activated by an insert element DNA, which increases the expression of genes in the chromosome of the host.

8. A host cell according to claim 6, characterized in that the specified cell host selected so that it contained the mutation, stimulating the expression of endogenous GnT III.

9. A host cell of claim 8, characterized in that specified a host cell is a cell mutant lec 10 SNO.

10. A host cell according to claim 1, characterized in that specified a host cell is a cell SNO, cell KSS, a NSO cell, a cell SP2/0, the cell YO myeloma cell RSH myeloma mouse, PER cell, or a PER.C6 cell or a cell hybridoma.

11. A host cell of claim 10, characterized in that the polypeptide is an antibody against CD20.

12. A host cell of claim 10, characterized in that the antibody against CD20 is a IDEC-C2B8.

13. A host cell of claim 10, characterized in that the above cell-chose the n represents the cell SP2/0.

14. A host cell according to item 13, characterized in that said antibody is a chimeric monoclonal antibody chG250 against vaginal cancer man.

15. A host cell according to claim 5, characterized in that the at least one gene encoding GnT III, is introduced into the chromosome of the specified host cell.

16. A host cell according to claim 6, characterized in that the specified endogenous GnT III is activated by inserting a promoter element, a transposon, or a retroviral element in the chromosome of the host cell.

17. A host cell according to claim 1, characterized in that it also contains at least one transfected nucleic acid encoding the antibody molecule, antibody fragment or fusion protein that includes a region equivalent to the Fc region of immunoglobulin.

18. A host cell according to claim 1, characterized in that the at least one nucleic acid encoding GnT III, functionally linked to a constitutive promoter element.

19. A host cell according to 17, characterized in that specified a host cell contains at least one transfected nucleic acid encoding the antibody against CD-20, a chimeric monoclonal antibody chCE7 against human neuroblastoma, a chimeric monoclonal antibody chG250 against vaginal cancer human chimeric monoclonal antibody ING-1 against carcinoma of Obodo the big intestine, lung and breast cancer human humanitariannet monoclonal antibody (3622W94 against antigen 17-1A human humanitariannet antibody A33 against tumors of the colon, rectum, antibody R24 directed against a ganglioside GD3, against human melanoma, or chimeric monoclonal antibody SF-25 against squamous cell carcinoma human antibody against EGFR human antibody against EGFRvIII human antibody against human PSMA, PSCA antibody against human antibody against CD22 human antibody against human CD30. antibody against CD33 human antibody against CD38 human antibody against human CD40, antibody against CD45 human antibody against CD52 human antibody against human CD138, variant antibodies against HLA-DR human antibody against Arcam human antibody against CEA human antibody against MUC1 human antibody against the capsid protein MUC1 human antibody against aberrant glycosylated MUC1 human antibody against variants of human fibronectin containing the domain ED-B, and the antibody against HER2/neu human.

20. A method of obtaining a polypeptide containing a bifurcated hybrid oligosaccharides or galactoglucomannan complex or their mixture in the Fc-region, comprising culturing the host cell according to claims 1-19 under conditions which allow to obtain the specified polypeptide having at alicenew Fc-mediated cellular cytotoxicity.

21. The method according to claim 20, characterized in that it includes the selection of the specified polypeptide having increased Fc-mediated cellular cytotoxicity.

22. The method according to claim 20, characterized in that specified a host cell contains at least one nucleic acid encoding a fusion protein containing the region equivalent to the Fc region of immunoglobulin.

23. The method according to claim 20, characterized in that more than 50% of the oligosaccharides in the Fc-region of a specified polypeptide are forked.

24. The method according to claim 20, characterized in that more than 70% of the oligosaccharides in the Fc-region of a specified polypeptide are forked.

25. The method according to claim 20, characterized in that the content of the bifurcated hybrid oligosaccharides or galactoglucomannan complex oligosaccharides or mixtures thereof in the Fc-region exceeds the content forked complex oligosaccharides in the Fc-region of the indicated polypeptides.

26. The method according to claim 20, characterized in that the polypeptide is an antibody IDEC-C2B8 against CD20, and antibody IDEC-C2B8, educated specified the host-cell, according to the analysis of MALDI/TOF-MS have a range of glycosylation, which is almost equivalent to that shown in figa.

27. The method according to claim 20, characterized in that the polypeptide is a monoclonal antibody chG250, and antibodies chG250, educated specified the host-cell, which according to the analysis of MALDI/TOF-MS have a range of glycosylation which is almost equivalent to that shown in fig.7D.

28. Antibody with increased antibody-dependent cellular cytotoxicity (ADCC)obtained by the method according to item 21.

29. The antibody according p, characterized in that said antibody is selected from the group including IDEC-C2B8, chCE7, ch-G250, humanitariannet monoclonal antibody against HER2, ING-1, 3622W94, SF-25, A33 and R24.

30. The antibody fragment containing a region equivalent to the Fc region of immunoglobulin, having increased Fc-mediated cellular cytotoxicity obtained by the method according to item 21.

31. Protein fusion containing a region equivalent to the Fc region of immunoglobulin, having increased Fc-mediated cellular cytotoxicity and obtained by the method according to item 21.

32. Pharmaceutical composition for increased Fc-mediated cellular cytotoxicity, containing antibody at p, and a pharmaceutically acceptable carrier.

33. Pharmaceutical composition for increased Fc-mediated cellular cytotoxicity, containing a fragment of the antibody according to item 30, and a pharmaceutically acceptable carrier.

34. Pharmaceutical composition for increased Fc-mediated cellular cytotoxicity containing protein in p, and a pharmaceutically acceptable carrier.

35. A method of treating cancer, comprising introducing a therapeutically effective dose of the pharmaceutical composition is in PP-34 patient who needs such treatment.

36. The method of treatment of a disease associated with the increase in the number or production In cells, based on the decrease In cells and involving the introduction of a therapeutically effective dose of the antibody to a patient in need of such treatment, and the improvement consists in introducing a therapeutically effective to reduce the number of In-cell dose of the antibody obtained by the method according to p.

37. The method according to p, characterized in that said antibody is a monoclonal antibody against CD20.

38. The method according to clause 37, characterized in that the antibody against CD20 is an antibody IDEC-C2B8.



 

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1 tbl

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FIELD: biotechnology, immunology.

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EFFECT: valuable biological and medicinal properties of antibodies.

53 cl, 4 tbl, 10 dwg, 2 ex

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