Human monoclonal antibodies to receptor of epidermal growth factor (egfr), method of their preparation and usage, hybridome, transfectome, transgene animal, expression vector

FIELD: medicine; pharmacology.

SUBSTANCE: allocated human monoclonal antibodies which specifically bind a receptor of the epidermal growth factor (EGFR), and also corresponding compositions on the basis of antibodies and a biospecific molecule are described. Human antibodies can be received with use of the transgenic mouse capable to formation of set of isotypes of human monoclonal antibodies by recombination V-D-J and switching of isotypes. The pharmaceutical compositions containing human antibodies for treatment or prevention of diseases, mediated by expression EGFR, the transgenic animals distinct from a human, the specified expressing antibodies, hybridomes and transfectomes which produce human antibodies are also presented. Ways of therapy and diagnostics of the diseases mediated by expression EGFR, with use of human antibodies or their antigen-binding of fragments, and also methods of growth suppression of the cells expressing EGFR, and an induction of cytolysis of the specified cells are described.

EFFECT: invention allows obtaining therapeutic and diagnostic preparations of antibodies with improved properties.

53 cl, 22 dwg, 4 tbl, 11 ex

 

The text descriptions are given in facsimile form.

1. Selected human monoclonal antibody that binds to human receptor for epidermal growth factor (EGFR), selected from the group including IgGI antibodies, IgA, IgE, IgM, IgG4, and IgD, and when this antibody contains: frame segments of the human heavy chain CDR1 plot of the human heavy chain CDR2 area of human heavy chain and CDR3 of the area of the human heavy chain, which is a CDR3 area of human heavy the th chain monoclonal antibodies 2F8, presented at Fig and 22; and a frame segments of the human light chain, CDR1 area of human light chain CDR2 area of human light chain and CDR3 area of human light chain, which is a CDR3 area of human light chain monoclonal antibodies 2F8, presented at Fig and 22.

2. Selected human monoclonal antibody according to claim 1, characterized in that it is represented by IgGI antibody.

3. The human antibody according to claim 1, characterized in that it inhibits the binding of the ligand EGFR with human EGFR.

4. The human antibody according to claim 3, characterized in that the EGFR ligand represented by EGF or TGF-α.

5. The human antibody according to claim 1, characterized in that it binds to human EGFR with an equilibrium constant of Association (KAndat least approximately 108M-1.

6. The human antibody according to claim 1, characterized in that it binds to human EGFR with an equilibrium constant of Association (KAndat least approximately 109M-1.

7. The human antibody according to claim 3, characterized in that it blocks the binding of the ligand with human EGFR EGFR of at least approximately 50%.

8. The human antibody according to claim 2, characterized in that it comprises a variable region from an IgGl heavy chain and the variable region of the Kappa light CE is I.

9. The human antibody according to claim 1, characterized in that it is encoded by nucleic acids of the human heavy chain and human Kappa-light chain containing in their variable regions of the nucleotide sequence represented in SEQ ID NO:1 and SEQ ID NO:3, respectively, and conservative modifications of these sequences.

10. The human antibody according to claim 1, characterized in that it has a variable region heavy chain and Kappa light chains, which contain the amino acid sequence represented in SEQ ID NO:2 and SEQ ID NO:4, respectively, and conservative modifications of these sequences.

11. The human antibody according to claim 1, characterized in that it has a variable region heavy chain and Kappa light chains, which contain the amino acid sequence represented in SEQ ID NO:2 and SEQ ID NO:4, respectively.

12. The human antibody according to claim 1, characterized in that it binds to EGFR and inhibits induced by EGF or TGF-α autophosphorylation of EGFR.

13. The human antibody according to claim 1, characterized in that it binds and inhibits the growth of cells expressing EGFR.

14. The human antibody according to item 13, characterized in that it binds to the cell, which is a tumor cell selected from the group comprising cell bladder, clickomania gland, cell, colon cell tumors of the kidney, cell, ovarian cell, prostate cell renal cell tumors, squamous cell and nemiscau cell lung.

15. The human antibody according to item 13, characterized in that it binds to the cell that is selected from the group consisting of cells of the synovial fibroblast and keratinocyte.

16. The human antibody according to claim 1, characterized in that it binds to cells expressing EGFR and induces lysis of the cells in the presence of human effector cells.

17. The human antibody according to claim 1, characterized in that it binds to cells expressing EGFR, but does not induce complement-mediated lysis of the cells.

18. The selected human antibody according to claim 1, characterized in that it is a Fab fragment or single-chain antibody.

19. The selected human antibody according to claim 1, characterized in that it is produced by hybridomas, which includes In-cell obtained from a transgenic animal other than human, and having a genome that contains the transgene, human heavy chain and human transgene light chain, merged with immortalizing cell.

20. The selected human antibody according to claim 1, characterized in that it is produced by transfective containing nucleic acids encoding a human heavy is EPI and a human light chain.

21. Selected human monoclonal antibody, which binds to the epitope on EGFR, a specific antibody according to item 11.

22. Hybridoma containing a cell obtained from a transgenic animal other than human, and having a genome that contains the transgene, human heavy chain and human transgene light chain, merged with immortalizing cell, while hybridoma produces detectable quantity of the monoclonal antibody according to claim 1 or an antigen-binding part.

23. Transfactual containing nucleic acids encoding a human heavy chain and human light chain, while transfactual produces detectable quantity of the monoclonal antibody according to claim 1 or an antigen-binding part.

24. Transfactual according to item 23, characterized in that it contains a nucleic acid encoding a human heavy chain and human light chain, which in their variable regions contain nucleotide sequences shown in SEQ ID NO:1 and SEQ ID NO:3, respectively, or conservative modifications.

25. Transgenic animal other than human, which expresses the antibody according to claim 1, in this transgenic animal other than human, has a genome containing the transgene of human heavy chain and human transgene light chain.

26. The way the floor is for the antibody according to claim 1, characterized in that carry out the immunization of non-human transgenic animal having a genome containing the transgene of human heavy chain and human transgene light chain, using EGFR or cells expressing EGFR, therefore the production of antibodies by b-cells of the animal, isolated b cells of the animal and carry out the fusion of b cells with myeloma cells with the formation of immortalized cells hybridoma that secrete antibody.

27. Bespecifically molecule containing human antibody according to claim 1 and a second binding specificity against human antigen-presenting cells.

28. Bespecifically molecule containing human antibody according to claim 1 and a second binding specificity against human Fc receptor.

29. Bespecifically molecule on p, characterized in that the said Fc receptor is a human receptor FcγRI or human Fc receptorα.

30. Bespecifically molecule on p, characterized in that it binds the Fc-receptor on the center, which is different from the binding site of the receptor and immunoglobulin.

31. Pharmaceutical composition for treatment or prevention of diseases mediated by the expression of EGFR containing human antibody according to claim 1 and pharmaceutically acceptable wear the ü.

32. Pharmaceutical composition for treatment or prevention of diseases mediated by EGFR expression containing a combination of two or more human antibodies according to claim 1, or antigen-binding fragments, where each of the above antibodies or antigen-binding fragments binds a single epitope of EGFR.

33. Pharmaceutical composition for treatment or prevention of diseases mediated by the expression of EGFR containing human antibody according to claim 1 and a chemotherapeutic agent.

34. Immunotoxin containing human antibody according to claim 1, associated with the cytotoxic agent.

35. The method of suppressing the growth of cells expressing EGFR, characterized in that exercise contacting cells with an effective amount of the antibody of claim 1 that inhibits binding of a ligand of EGFR with human EGFR, and thus inhibit the growth of cells expressing EGFR.

36. The method according to p, wherein the cell is chosen from the group comprising cell, bladder cell, breast cancer cell, colon cell, kidney cell, ovarian, prostate cell, squamous cell, nemelka cell lung cell synovial fibroblast and keratinocyte.

37. Method of induction of cytolysis of cells expressing EGFR, characterized in that exercise contacting cells expressing EGF, with the antibody according to claim 1 in the presence of effector cells with the provision of cytolysis of cells expressing EGFR.

38. The method according to clause 37, wherein the cell is chosen from the group comprising cell, bladder cell, breast cancer cell, colon cell, kidney cell, ovarian, prostate cell, squamous cell, nemelka cell lung cell synovial fibroblast and keratinocyte.

39. The method of treatment or prophylaxis of a disease mediated by expression of EGFR, wherein the subject is administered a pharmaceutical composition according to any one of p-33 in amounts effective for the treatment or prophylaxis of a disease mediated by expression of EGFR.

40. The method according to § 39, wherein the disease is a cancer.

41. The method according to § 39, wherein the disease is an autoimmune disease.

42. The method according to § 39, wherein the subject is administered a human antibody conjugated with a binding specificity for an Fc receptor.

43. The method according to § 39, wherein the subject is administered a human antibody conjugated to a cytotoxin.

44. The method according to § 39, characterized in that it further carry out joint introduction of therapeutic agent.

45. The method according to item 44, wherein therapeutic agent is chosen from the group, ostoja of doxorubicin (adriamycin), cisplatin, bleomycin sulfate, carmustine, hlorambuzila and cyclophosphamideinduced.

46. The method according to p, wherein the disease is a cancer selected from the group including bladder cancer, breast cancer, colon cancer, kidney cancer, ovarian cancer, prostate cancer, renal cell cancer, head and neck cancer.

47. The method according to paragraph 41, wherein the disease includes hyperproliferative epithelium.

48. The method according to § 39, wherein the disease presents inflammatory arthritis.

49. The method of detecting the presence of EGFR antigen or cells expressing EGFR in the sample, characterized in that carry out the contacting of the sample with the antibody of claim 1 under conditions ensuring formation of a complex between the antibody or a part of it and EGFR, and make the detection of complex formation.

50. The expression vector containing the nucleotide sequence encoding the variable region of the light chain and/or heavy chain of the human monoclonal antibody according to any one of claims 1 to 21.

51. The expression vector according to item 50, characterized in that it further comprises a nucleotide sequence encoding a constant region of light chain and/or heavy chain of human monoclonal antibody that binds EGFR.

52. Expression of vecto is, containing the nucleotide sequence encoding the variable region of the heavy chain and light chain, which contain amino acid sequences shown in SEQ ID NO:2 and SEQ ID NO:4, respectively, and conservative modifications of those sequences.

53. Transfactual containing the expression vector according to item 50.



 

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1 tbl, 4 ex

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3 cl, 3 tbl, 4 ex

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53 cl, 4 tbl, 10 dwg, 2 ex

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13 cl, 5 dwg, 4 tbl, 2 ex

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45 cl, 14 tbl, 28 ex

FIELD: microbiological and immunological methods.

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2 cl, 6 dwg

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12 cl, 6 dwg, 8 tbl, 9 ex

FIELD: biotechnology, hybridoma technology.

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EFFECT: valuable properties of strain.

3 tbl, 1 ex

FIELD: biotechnology and virology.

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EFFECT: vaccines for prophylaxis and therapy of diseases associated with pathogenic for human orthopoxviruses.

2 tbl, 5 ex

FIELD: immunology.

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EFFECT: improved method for analysis.

9 cl, 1 tbl, 2 dwg, 3 ex

FIELD: biotechnology, immunology, medicine, oncology.

SUBSTANCE: strain of hybrid cultured mammalian cells Mus musculus VKPM H-98 is prepared by the hybridoma technology method. This strain is a producer of monoclonal antibodies possessing individual specificity to hypoglycosidated and deglycosidated isoforms of tumor-associated human antigen Muc I. Productivity of the strain and specificity of produced antibodies is estimated based on the immunoenzyme assay using some markers of specificity: natural purified antigen Muc I isolated from human milk; VNTR22-polypeptide; synthetic monomeric polypeptide (TR1); deglycosidated antigen Muc I (de-Muc I) prepared by chemical oxidation of natural Muc I; hypoglycosidated antigen Muc I (o-Muc I) prepared by periodate oxidation of natural Muc I. Monoclonal antibodies produced by the claimed strain recognize clinically significant isoforms of antigen Muc I and allows assaying its concentrations in human serum blood in carrying out the early diagnosis of tumors. Invention can be used in preparing monoclonal antibodies to tumor-associated human antigen Muc I.

EFFECT: valuable properties of strain.

2 dwg, 3 ex

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