Human interleukin-13 antibodies and their application

FIELD: medicine.

SUBSTANCE: there are offered versions of antibodies and their antigen-binding IL-13, particularly human IL-13 specific fragments. There are described: a pharmaceutical composition, a pharmaceutical compound of the antibody, versions of coding and hybridising nucleic acids and expression vectors. There are offered versions of: cells and methods of producing the antibody, methods of treating IL-13 associated disorders. A method of IL-13 detection in a sample is described.

EFFECT: use of the invention provides new IL-13 antibodies with KD about 10-10 M which can be used for diagnosing, preventing or treating one or more IL-13 associated diseases.

87 cl, 37 dwg, 5 tbl, 6 ex

 

The text descriptions are given in facsimile form.

1. The antibody or antigennegative fragment containing the variable region of the heavy chain of an antibody and a variable region light chain immunoglobulin that bind IL-13 with KDless than 10-7M, with the specified variable region light chain contains an amino acid sequence:
(i) KASESVDNYGKSLMH (SEQ ID NO:19), in CDR1,
(ii) RASNLES (SEQ ID NO: 20), in CDR2, and
(iii) QQSNEDPWT (SEQ ID NO: 21), in CDR3;
and the variable region of the heavy chain contains an amino acid sequence:
(i) SYAMS (SEQ ID NO: 22), in CDR1,
(ii) SISSGGNTYYPDSVKG (SEQ ID NO: 23), in CDR2, and
(iii) LDGYYFGFAY (SEQ ID NO: 24), in CDR3,
or an amino acid sequence that differs by less than three conservative substitutions of amino acids from amino acid sequence selected from SEQ ID nos: 19-24.

2. The antibody or antigennegative fragment containing the variable region of the heavy chain of an antibody and a variable region light chain immunoglobulin that bind IL-13 with KDless than 10-7M, with the specified variable region light chain contains an amino acid sequence:
(i) KASESVDNYGKSLMH (SEQ ID NO: 19), in CDR1,
(ii) RASNLES (SEQ ID NO: 20), in CDR2, and
(iii) QQSNEDPWT (SEQ ID NO: 21), in CDR3;
and the variable region of the heavy chain contains an amino acid sequence:
(i) SYAMS (SEQ ID NO: 22), in CDR1,
(ii) SISSGGNTYYPDSVKG (SEQ ID NO: 23), in CDR2, and
(iii) LDGYYFGFAY (SEQ ID NO: 24), in CDR3,
or an amino acid sequence that differs by less than three conservative substitutions of amino acids from amino acid sequence selected from SEQ ID nos: 19-24,
while the antibody or antigennegative fragment connected to: another antibody, a toxin, a radioisotope, cytotoxic agent or cytostatic agent is M.

3. The antibody or antigennegative fragment according to claim 1, in which the variable region of light chain of immunoglobulin is at least 95% identical to the sequence of SEQ ID NO: 11, 12, or 35.

4. The antibody or antigennegative fragment according to claim 1, in which the variable region of the heavy chain immunoglobulin contains the amino acid sequence of SEQ ID NO: 15, 16, or 36.

5. The antibody or antigennegative fragment according to claim 1 or 4, in which the variable region light chain immunoglobulin contains the amino acid sequence of SEQ ID NO: 11, 12, or 35.

6. The antibody or antigennegative fragment according to claim 1, which additionally contain a constant region of light chain of immunoglobulin is at least 95% identical to the amino acid sequence SEQ ID NO: 18.

7. The antibody or antigennegative fragment according to claim 1, which additionally contain a constant region of a heavy chain immunoglobulin which comprises the amino acid sequence of SEQ ID NO: 17.

8. The antibody or antigennegative fragment according to claim 1 or 7, which additionally contain a constant region light chain immunoglobulin which comprises the amino acid sequence of SEQ ID NO: 18.

9. The antibody or antigennegative fragment containing the variable region of the heavy chain of an antibody and variable region l is gcoi chain immunoglobulin, which bind IL-13 with KDless than 10-7M variable region light chain contains an amino acid sequence:
(i) xxxxxxxNYxKxxxx (SEQ ID NO: 25), in CDR1,
(ii) Rxxxxxx (SEQ ID NO: 26), in CDR2, and
(iii) xxxNxDxWx (SEQ ID NO: 27), in CDR3;
and the variable region of the heavy chain contains an amino acid sequence:
(i) SxAMx (SEQ ID NO: 28), in CDR1,
(ii) SxSSxxxxxYxxSVKG (SEQ ID NO: 29), in CDR2, and
(iii) LDGYYFxxxx (SEQ ID NO: 30), in CDR3,
and x represents an amino acid having the charge and hydrophobicity similar to the amino acid in the corresponding position in the amino acid sequence of SEQ ID NO: 19-24.

10. The antibody or antigennegative fragment according to claim 1, which has one or more of the following properties:
(a) the variable region of the heavy chain immunoglobulin contains a sequence of CDR3 of the heavy chain of the monoclonal antibody mAb 13.2 (SEQ ID NO: 24 or a sequence of CDR3 of the heavy chain, which differs by less than three conservative substitutions of amino acids from the corresponding sequence of CDR3 of the heavy chain of the monoclonal antibody mAb 13.2;
(b) variable region light chain immunoglobulin contains a sequence of light chain CDR1 of monoclonal antibody mAb 13.2 (SEQ ID NO: 19) or a sequence of light chain CDR1 that differs by less than 3 conservative substitutions of amino acids from the corresponding sequences CDR1 became the first chain of the monoclonal antibody mAb 13.2;
(c) variable region of the heavy chain immunoglobulin contains a sequence encoded by a nucleic acid which hybridizes in conditions of high stringency to the sequence complementary to the nucleic acid sequence that encodes a variable domain of the heavy chain h13.2 (SEQ ID NO: 15, 16 or 36);
(d) variable region of the heavy chain immunoglobulin contains the amino acid sequence encoded by a nucleic acid which hybridizes in conditions of high stringency to a nucleic acid which is complementary to a nucleic acid that contains a nucleotide sequence SEQ ID NO: 7, 8, or 34;
(e) variable region light chain immunoglobulin contains a sequence encoded by a nucleic acid which hybridizes in conditions of high stringency to the sequence complementary to the nucleic acid sequence that encodes a variable domain light chain of h13.2 (SEQ ID NO: 11,12 35);
(f) variable region light chain immunoglobulin contains the amino acid sequence encoded by a nucleic acid which hybridizes in conditions of high stringency to a nucleic acid which is complementary to a nucleic acid that contains a nucleotide sequence SEQ ID NO: 3, 4, or 33;
(g) variable about the art of the heavy chain of immunoglobulin is at least 90% identical to the variable domain of the heavy chain h13.2 (SEQ ID NO: 15, 16 or 36);
(h) variable region light chain of immunoglobulin is at least 90% identical to the variable domain of the light chain of h13.2 (SEQ ID NO: 11, 12, or 35);
(i) the antibody or antigennegative fragment competes with the monoclonal antibody mAb 13.2 for binding to IL-13 human;
(j) the antibody or antigennegative domain contacts with IL-13, in terms of amino acid residues: 68, 72, 88, 91, 92, 93 and 105 of the sequence SEQ ID NO: 31;
(k) the variable region of the heavy chain has the same canonical structure as the monoclonal antibody mAb13.2;
(l) the variable region of the light chain has the same canonical structure as monoclonalnoe antibody mAb13.2;
(m) the variable region of the heavy chain and/or variable region light chain have the frame region FR1, FR2 and FR3 of the VH segments encoded by genes in the germ line of human DP-54 and DPK-9, respectively, or a sequence at least 95% identical to the VH segments encoded by genes in the germ line of human DP-54 and DPK-9;
(n) gives the post-injection protective effect against antigen Roundworm model sheep for at least 6 weeks after injection.

11. Isolated recombinant antibody IgG, which binds IL-13 and contains two chains of immunoglobulins: light chain, which contains the amino acid sequence that is appropriate to atstumu SEQ ID NO: 9, 10, 11, 12, or 35 and a heavy chain which contains an amino acid sequence corresponding to SEQ ID NO: 13, 14, 15, 16, or 36, or an amino acid sequence at least 95% identical to SEQ ID NO: 9, 10, 11, 12, 13, 14, 15, 16, 35 or 36.

12. Isolated recombinant antibody IgG no claim 5, in which the heavy chain further comprises the amino acid sequence of SEQ ID NO: 17 and a light chain further comprises the amino acid sequence of SEQ ID NO: 18 or an amino acid sequence at least 95% identical to SEQ ID NO: 17 or 18.

13. The antibody or antigennegative fragment according to claim 1, characterized in that have one or more of the following properties:
(a) specifically associated with epitope containing residues 81-93 or 114-132 IL-13 human (SEQ ID NO: 31), or conservative amino acid substitutions;
(b) specific contact epitope of IL-13 human, containing the following amino acid residues: glutamate at position 49, the asparagine at position 53; glycine in position 69, Proline at position 72, the histidine at position 73, the lysine at position 74 and arginine at position 86 of the sequence SEQ ID NO: 32 or a conservative amino acid substitution;
(c) associated with a complex of IL-13 and IL13Rα1;
(d) inhibit the binding interaction between IL-13 and IL-4Rα;
(e) inhibit the binding interaction between IL-13/IL-13Rα1 and IL-4Rα; and
(f) specific Saint is called with IL-13 human and competitively inhibit the binding of the second antibody with the above antigen person, moreover, the above-mentioned second antibody contains a variable region heavy and light chain selected from the mAbl3.2 (SEQ ID NO: 13 and SEQ ID NO: 9, respectively), ch13.2 (SEQ ID NO: 14 and SEQ ID NO: 10, respectively), h13.2v1 (SEQ ID NO: 15 and SEQ ID NO: 14, respectively), h13.2v2 (SEQ ID NO: 16 and SEQ ID NO: 12, respectively), or h13.2v3 (SEQ ID NO: 36 and SEQ ID NO: 35, respectively); and/or
(g) the antibody contains a variable region light chain immunoglobulin sequence SEQ ID NO: 11, 12, or 35, amino acid residues which: ASN31 (CDR1), TYR32 (CDR1), LYS34 (CDR1), ARG54 (CDR2), ASN96 (CDR3), ASP98 (CDR3), and TRP100 (CDR3) in contact with IL-13; the variable region of the heavy chain immunoglobulin sequence SEQ ID NO: 15, 16, or 36, amino acid residues which: ILE30, SER31 (CDR1), ALA33 (CDR1), TRP47, SER50 (CDR2), SER52 (CDR2), SER53 (CDR2), TYR58 (CDR2), LEU98 (CDR3), ASP99 (CDR3), GLY100 (CDR3), TYR101 (CDR3), TYR102 (CDR3) and RNA (CDR3), contact with IL-13.

14. The antibody or antigennegative fragment according to item 13, which are connected to: another antibody, a toxin, a radioisotope, cytotoxic agent or cytostatic agent.

15. The antibody or antigennegative fragment according to item 13, which contain a constant region is modified in such a way as to reduce: binding to Fc receptor glycosylation of the antibody, the number of cysteine residues, the function of effector cells and complement.

16. The antibody or antigennegative fragment according to item 13, which are more the tion contain a constant region of human IgG1, in which the modified one or more residues 116 and 119 of the sequence SEQ ID NO: 17 to reduce binding to Fc receptor.

17. The antibody or antigennegative fragment according to item 13, which additionally contain light chain Kappa immunoglobulin.

18. The antibody or antigennegative fragment according to item 13, which contain at least one CDR region containing the amino acid sequence selected from the group comprising: the amino acid sequence of SEQ ID NO: 19, amino acid sequence SEQ ID NO: 20, amino acid sequence SEQ ID NO: 21, amino acid sequence SEQ ID NO: 22, amino acid sequence SEQ ID NO: 23 and the amino acid sequence of SEQ ID NO: 24.

19. The antibody or antigennegative fragment according to item 13, wherein the fragment is a scFv fragment, Fab, Fd, dAb, or F(ab')2.

20. The antibody or antigennegative fragment according to item 13, which additionally contain a light chain containing the constant region of the Kappa immunoglobulin or its active fragment and a heavy chain containing a constant region of human IgG or its active fragment.

21. The antibody or antigennegative fragment according to claim 20, in which the constant region of Kappa above the light chain of human immunoglobulin contains amino acid p is the sequence of SEQ ID NO: 18 or its active fragment.

22. The antibody or antigennegative fragment according to claim 20, in which the constant region of the aforementioned heavy chain of human IgG modified to reduce binding of the FcR and complement.

23. The antibody or antigennegative fragment according to claim 20, in which a modified constant region above the heavy chain of IgG person contains the amino acid sequence of SEQ ID NO: 17 or its active fragment.

24. The antibody or antigennegative fragment according to claim 1 or 13, which contain the variable region of the heavy chain of an antibody and a variable region light chain immunoglobulin that bind IL-13 with KDless than 10-7M, with the specified variable region light chain contains an amino acid sequence:
(i) KASESVDNYGKSLMH (SEQ ID NO: 19), in CDR1,
(ii) RASNLES (SEQ ID NO: 20), in CDR2, and
(iii) QQSNEDPWT (SEQ ID NO: 21), in CDR3;
and the variable region of the heavy chain contains an amino acid sequence:
(i) SYAMS (SEQ ID NO: 22), in CDR1,
(ii) SISSGGNTYYPDSVKG (SEQ ID NO: 23), in CDR2, and
(iii) LDGYYFGFAY (SEQ ID NO: 24), in CDR3.

25. The antibody or antigennegative fragment according to claim 1 or 24, in which the variable region light chain immunoglobulin contains the sequence of SEQ ID NO: 11, 12, or 35, amino acid residues which: ASN31 (CDR1), TYR32 (CDR1), EYS34 (CDR1), ARG54 (CDR2), ASN96 (CDR3), ASP98 (CDR3), and TRP100 (CDR3) in contact with IL-13; the variable region of the heavy chain of immunoglobulin is on with the sequence SEQ ID NO:15, 16, or 36, amino acid residues which: ILE30, SER31 (CDR1), ALA33 (CDR1), TRP47, SER50 (CDR2), SER52 (CDR2), SER53 (CDR2), TYR58 (CDR2), LEU98 (CDR3), ASP99 (CDR3), GEY100 (CDR3), TYR101 (CDR3), TYR102 (CDR3) and RNA (CDR3), contact with IE-13.

26. The antibody or antigennegative fragment according to claim 1, in which the variable region of the heavy chain of immunoglobulin is at least 95% identical to the sequence of SEQ IDNO: 15, 16, or 36.

27. The antibody or antigennegative fragment on p, in which the variable region of the heavy chain of immunoglobulin is at least 95% identical to the sequence of SEQ ID NO: 11, 12, or 35.

28. The antibody or antigennegative fragment of claim 1, wherein the constant region is modified to reduce one or more of the following properties: a binding to the Fc receptor, glycosylation of the antibody, the number of cysteine residues, the function of effector cells and complement.

29. The antibody or antigennegative fragment according to claim 1, which additionally contain a constant region of human lgG1, in which the modified one or more residue at position 116 and 119 of SEQ ID NO: 17 to reduce binding to Fc receptor.

30. The antibody or antigennegative fragment by clause 29, which further contain a light chain type Kappa immunoglobulin.

31. The antibody or antigennegative fragment according to claim 1, which additionally contain a constant region is agelou chain immunoglobulin at least 95% identical to the amino acid sequence SEQ ID NO: 17.

32. The antibody or antigennegative fragment according to item 30, which additionally contain a constant region of light chain of immunoglobulin is at least 95% identical to the amino acid sequence SEQ ID NO: 18.

33. The antibody or antigennegative fragment according to claim 1, which contain the amino acid sequence of variable region of the heavy chain, which encodes a nucleic acid, hybridization under conditions of high stringency to a nucleic acid which is complementary nucleic acid, which encodes the variable region of the heavy chain containing the amino acid sequence of SEQ ID NO: 15, 16, or 36; or a nucleic acid which is complementary to a nucleic acid that contains a nucleotide sequence SEQ ID NO: 7, 8, or 34.

34. The antibody or antigennegative fragment on p that contain the amino acid sequence of variable region of the light chain, which encodes a nucleic acid, hybridization under conditions of high stringency to a nucleic acid which is complementary to a nucleic acid that encodes the variable region of the light chain containing the amino acid sequence of SEQ ID NO: 11, 12, or 35; or nucleic acid that is complementary to a nucleic acid that contains a nucleotide sequence SEQ ID NO: , 4, or 33.

35. The antibody or antigennegative fragment according to claim 1, variable region of the heavy chain which contains an amino acid sequence that encodes a nucleic acid containing the sequence of SEQ ID NO: 7, 8, or 34.

36. The antibody or antigennegative fragment on p, the variable region of the light chain containing the amino acid sequence, which encodes a nucleic acid containing the sequence of SEQ ID NO:3, 4, or 33.

37. The antibody or antigennegative fragment of claim 10, which is connected to: another antibody, a toxin, a radioisotope, cytotoxic agent or cytostatic agent.

38. The antibody or antigennegative fragment according to any one of claims 1, 10, 11, 13, which are purified or recombinant.

39. The antibody or antigennegative fragment according to any one of claims 1, 10, 11, 13, which are recombinant full-size IgG.

40. The antibody or antigennegative fragment according to any one of claims 1, 10, 11, 13, which are Fab, F(ab')2, Fd, dAb, or scFv.

41. The antibody or antigennegative fragment according to any one of claims 1, 10, 11, 13, which contain the frame region, which is at least 90% identical to the framework region of the germline of the person.

42. The antibody or antigennegative fragment according to any one of claims 1, 0, 11, 13, which contain the frame region of human antibodies, the Fc region of human antibodies, or both of these areas.

43. The antibody or antigennegative fragment according to any one of claims 1, 10, 11, 13, in which the variable region of the heavy chain forms a hydrogen bond with IL-13 with the participation of amino acids SER50 (CDR2), SER53 (CDR2), TYR101 (CDR3) and TYR102 (CDR3) sequence SEQ ID NO:15, 16, or 36; and a variable region light chain immunoglobulin forms a hydrogen bond with IL-13 with the participation of amino acids ASN31, TYR32 (CDR1), LYS34 (CDR1), ASN96 (CDR3) and ASP98 (CDR3) sequence SEQ ID NO: 11, 12, or 35.

44. The antibody or antigennegative fragment according to item 43, in which the variable region light chain immunoglobulin contains the sequence of SEQ ID NO: 11, 12, or 35, amino acid residues which: ASN31 (CDR1), TYR32 (CDR1), LYS34 (CDR1), ARG54 (CDR2), ASN96 (CDR3), ASP98 (CDR3), and TRP100 (CDR3) in contact through the interaction of van der Waals and IL-13;
variable region of the heavy chain immunoglobulin sequence SEQ ID NO: 15, 16 or 36, amino acid residues which: ILE30, SER31 (CDR1), ALA33 (CDR1), TRP47, SER50 (CDR2), SER52 (CDR2), SER53 (CDR2), TYR58 (CDR2), LEU98 (CDR3), ASP99 (CDR3), GLY100 (CDR3), TYR101 (CDR3), TYR102 (CDR3) and RNA (CDR3), contact through the interaction of van der Waals forces with IL-13.

45. The antibody or antigennegative fragment according to any one of claims 1, 10, 11, 13, which bind IL-13 man with a KD of 90 to 120 gr.

46. The antibody or antigennegative fragment according to any of the C claims 1, 10. 11, 13, which bind IL-13 human with Koffless than 1×10-4with-1.

47. The antibody or antigennegative fragment according to any one of claims 1, 10, 11, 13, which bind IL-13 human with Kon5×104up to 8×105M-lcl.

48. The antibody or antigennegative fragment according to any one of claims 1, 10, 11, 13, which reduce the ability of IL-13 to contact with IL-4Rα.

49. The antibody or antigennegative fragment according to any one of claims 1, 10, 11, 13 that are associated with IL-13, which is in complex with IL-13Rα1 in vitro.

50. Selected recombinant IgG antibody, which binds IL-13 and contains two chains of the immunoglobulin light chain containing an amino acid sequence that encodes SEQ ID NO: 1, 2, 3, 4, or 33, or a sequence that hybridizes in conditions of high stringency to the sequence of SEQ ID NO: 1, 2, 3, 4, or 33; and a heavy chain containing an amino acid sequence that encodes SEQ ID NO: 5, 6, 7, 8, or 34, or a sequence that hybridizes in conditions of high hardness with the sequence SEQ ID NO: 5, 6, 7, 8, or 34.

51. Selected recombinant IgG antibody, which binds IL-13 and contains two chains of the immunoglobulin light chain containing an amino acid sequence that encodes SEQ ID NO: 4, or a nucleotide sequence that hybridizes in the conditions of high stringency to the sequence of SEQ ID NO: 4; and a heavy chain containing an amino acid sequence that encodes SEQ ID NO: 8, or the nucleotide sequence, which hybridizes in conditions of high stringency to the sequence of SEQ ID NO: 8.

52. Selected recombinant IgG antibody, which binds IL-13 and contains two chains of the immunoglobulin light chain containing the amino acid sequence of SEQ ID NO: 12; and a heavy chain containing the amino acid sequence of SEQ ID NO: 16.

53. Selected recombinant IgG antibody according to paragraph 52, in which the specified heavy chain further comprises a sequence of SEQ ID NO: 17; and the light chain further comprises a sequence of SEQ ID NO: 18.

54. Pharmaceutical composition for binding IL-13, containing the antibody or antigennegative fragment according to any one of claims 1 to 53, which contains a sequence of variable regions of the heavy chain of immunoglobulin and sequence of the variable region of the light chain of immunoglobulins that bind IL-13 with KDless than 10-7and pharmaceutically acceptable carrier in combination with one or more additional therapeutic agent selected from the group comprising: inhalation steroids, beta-agonists, antagonists of leukotrienes or leukotriene receptors, inhibitors of IgE inhibitors fosfodiesterasa, xantina, anticalin licencie drugs agents, stabilizing mast cells, inhibitors of IL-4, inhibitors of IL-5 inhibitors eotaxin/ CCR3, antihistamines, TNF antagonists, antagonists of TNF enzyme antagonists TGFα, interferon gamma, preventon, chemotherapeutic agents, non-steroidal anti-inflammatory agents, immunomodulators, inhibitors R and NFκB inhibitors.

55. The pharmaceutical composition intended for binding IL-13, containing an effective amount of the antibody or its antigennegative fragment according to any one of claims 1 to 53, and a pharmaceutically acceptable carrier.

56. The pharmaceutical composition according to § 55, which is suitable for subcutaneous injection, inhalation or topical application.

57. Nucleic acid that contains a nucleotide sequence that:
(i) encodes a polypeptide containing the variable region of the heavy chain of immunoglobulin antibodies or antigennegative fragment that bind IL-13, which is:
(a) contains the amino acid sequence:
(i) SYAMS (SEQ ID NO: 22), in CDR1, (ii) SISSGGNTYYPDSVKG (SEQ ID NO: 23), in CDR2, and (iii) LDGYYFGFAY (SEQ ID NO: 24), in CDR3,
or an amino acid sequence that differs by less than three conservative substitutions of amino acids from amino acid sequence selected from: SEQ ID nos: 22-24; or
(b) identical variable domain of the heavy chain h13.2 (SEQ ID NO: 15, 16, or 36) of at least 90%; or
(ii) Hybride is : in conditions of high stringency to the sequence complementary nucleic acid sequence (SEQ ID NO: 15, 16, or 36)that encodes a variable domain of the heavy chain h13.2.

58. Nucleic acid that contains a nucleic acid sequence that:
(i) encodes a polypeptide that contains the variable region of the heavy chain of immunoglobulin antibodies or antigennegative fragment that bind IL-13, which is:
(a) contains the amino acid sequence:
(i) KASESVDNYGKSLMH (SEQ ID NO: 19), in CDR1, (ii) RASNLES (SEQ ID NO: 20), in CDR2, and (iii) QQSNEDPWT (SEQ ID NO: 21), in CDR3;
or an amino acid sequence that differs by less than three conservative substitutions of amino acids from amino acid sequence selected from: SEQ ID nos: 19-21; or
(b) identical variable domain of the light chain of h13.2 (SEQ ID NO: 11,12 or 35) of at least 90%; or
(ii) hybridizes in conditions of high stringency to the sequence complementary to the nucleic acid sequence that encodes a variable domain light chain of h13.2 (SEQ ID NO: 11, 12, or 35).

59. Nucleic acid containing a nucleotide sequence that hybridizes in conditions of high stringency to a nucleic acid complementary to a nucleic acid that encodes the variable region of the heavy chain of the antibody or its antigennegative fragment that bind IL-13, which contains the serial is inost of amino acid residues SEQ ID NO: 15, 16 or 36 or with a nucleic acid complementary to a nucleic acid that contains a nucleotide sequence SEQ ID NO: 7, 8 or 34.

60. Nucleic acid p, wherein said nucleotide sequence encodes the variable region of the heavy chain that contains the sequence of amino acid residues SEQ ID NO: 15, 16, or 36, or contains a nucleotide sequence SEQ ID NO: 7, 8 or 34.

61. Nucleic acid p, wherein said nucleotide sequence encodes the variable region of the heavy chain that contains the sequence of amino acid residues SEQ ID NO: 16, or contains a nucleotide sequence SEQ ID NO: 8.

62. Nucleic acid containing a nucleotide sequence that hybridizes in conditions of high stringency to a nucleic acid complementary to a nucleic acid that encodes the variable region of the light chain of the antibody or its antigennegative fragment that bind IL-13, which contains a sequence of amino acid residues SEQ ID NO: 11, 12, or 35, or nucleic acid complementary to a nucleic acid that contains a nucleotide sequence SEQ ID NO: 3, 4, or 33.

63. Nucleic acid according to item 62, wherein said nucleotide sequence encodes rebellow region light chain, which contains the sequence of amino acid residues SEQ ID NO: 11, 12, or 35, or contains a nucleotide sequence SEQ ID NO: 3, 4, or 33.

64. Nucleic acid p, wherein said nucleotide sequence encodes the variable region of light chain, which contains the sequence of amino acid residues SEQ ID NO: 12, or contains a nucleotide sequence SEQ ID NO: 4.

65. Expression vector that contains a nucleic acid according to any one of p-64.

66. Expression vector that contains a nucleic acid encoding a variable region of the heavy chain and the variable region of the light chain according to any one of p-64.

67. A host cell to obtain antibodies that bind IL-13 or its antigennegative fragment, characterized in that specified a host cell contains a nucleic acid according to any one of p-64.

68. A host cell to obtain antibodies that bind IL-13 or its antigennegative fragment, characterized in that specified a host cell contains a nucleic acid encoding a variable region of the heavy chain and the variable region of the light chain according to any one of p-64.

69. A host cell according p, characterized in that it is a cell of a mammal.

70. A host cell according p, characterized in that it is a cell m is capitalware.

71. A host cell to obtain antibodies or antigennegative fragment that contains the sequence of the nucleic acid encoding the antibody or antigennegative fragment according to any one of claims 1 to 53.

72. A method of obtaining a recombinant antibody that binds IL-13, comprising: providing a host cell according p or 68 and maintenance of specified cells under conditions in which expression of the indicated antibodies or antigennegative fragment.

73. A method of obtaining a recombinant antibody that binds IL-13, including:
providing the host cell sequence which includes a nucleic acid sequence that encodes the antibody or antigennegative fragment according to any one of claims 1 to 53, and the maintenance of specified cells under conditions in which the expression of antibodies or antigennegative fragment.

74. The method according to p which further includes the selection of the protein from the specified host cell or from the medium in which is supported a host cell.

75. The method according to p which further includes the preparation of the specified selected protein in the form of a pharmaceutical composition.

76. A method of treating disorders associated with IL-13, comprising the administration to a mammal with a specified disorder or with an increased risk of the buildings permanently, it is of a specified disorder effective amount of the antibody or its antigennegative fragment according to any one of claims 1 to 53.

77. The method according to p, wherein the disorder associated with IL-13 selected from the group comprising: an asthmatic disorder, atopic disorder chronic obstructive pulmonary disease (COPD), a condition involving airway inflammation, eosinophilia, fibrosis and excess secretion of mucus, inflammatory condition, an autoimmune condition, a tumor or a cancer, a viral infection and suppression of manifestations of a protective immune response in type 1.

78. The method according to p, wherein the disorder is an asthmatic disorders or allergic rhinitis.

79. The method according to p, wherein the disorder is an inflammatory bowel disease.

80. The method according to p, wherein the disorder is a chronic obstructive pulmonary disease (COPD).

81. The method according to p, wherein the disorder is an atopic disorder.

82. The method according to p, in which the protein is injected subcutaneously, via inhalation, or topically.

83. The method according to p, characterized in that the inflammatory bowel disease is a Crohn's disease or ulcerative colitis.

84. A method of treating disorders associated with IL-13, comprising the administration to a mammal an effective amount of the antibody or its antigennegative the slice l is the Boma one of claims 1 to 53, moreover, the specified antibody has one or more of the following properties:
(i) gives the post-injection protective effect against antigen Roundworm model sheep for at least 6 weeks after injection; or
(ii) inhibits the binding of IL-13 with IL-4Rα, but does not prevent the binding of IL-13cIL-13Rα1.

85. The method of detecting the presence of IL-13 in the sample, including:
(i) bringing into contact of the sample and antibodies against IL-13 or its fragment according to any one of claims 1 to 53; and
(ii) detecting formation of a complex between the antibody against IL-13 or its fragment and the sample, and the formation of the complex in the sample indicates the presence of IL-13 mammal.

86. The method according to p, characterized in that the sample receiving from a mammal.

87. A method of treating a mammal having asthma symptoms selected from the group including wheezing, shortness of breath, bronchostenosis, hyperactivity of the Airways, lung-volume reduction, fibrosis, airway inflammation and mucus secretion, and the above method includes the step of introducing the patient the antibody according to any one of claims 1 to 53, and the antibody binds to IL-13 and prevents the formation of a functional signaling complex of IL-13.



 

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SUBSTANCE: method includes analysing aliquots of said sample by one or more methods of protein description specified in the chromatography. The method is based on genetic analysis techniques specified in RFLP and T-RFLP. These methods can be applied both separately, and in a combination. The offered methods allow obtaining the information on the presence and fractions of various individual proteins or coding sequences. The obtained information can be used for evaluating stability of a polyclonal cell line in process, and also estimating a structure of various parties of end polyclonal products.

EFFECT: methods allow describing the composition consisting more than of 10, 20 or greater number of antibodies.

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SUBSTANCE: composition includes at least three oligonucleotide probes and enables simultaneously determining a level of PSMB4, FCER2 and POU2F2 genes expression. The oligonucleotide composition under the invention is presented to be used, including as a part of a microchip, in a method for prediction of a developing disease in a subject suffering chronic lymphatic leukemia that involves analysing a level of expression of at least three named genes in patient's blood samples.

EFFECT: higher efficacy of the composition.

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EFFECT: invention allows quick, effective and reliable differentiation of the strains.

1 tbl, 3 ex

FIELD: medicine.

SUBSTANCE: what is offered is a method of marking a human 7th chromosome providing in situ hybridisation of metaphasic or interphasic cell chromosomes of a tested sample and a DNA probe presented by a marker plasmid alpha R1-13 which consists of EcoR 1-EcoR l DNA fragment of a pBR 325 vector of the value 5966 base pairs and EcoRl-EcoRl alphoid DNA fragment of the human 7th chromosome of the value 680 base pairs. The testing environment in which the used DNA-probe specifically interacts with a centromeric region of the 7th chromosome without cross hybridisation with other human chromosomes are developed.

EFFECT: more efficient identification of the presented chromosome and enabled application of the new method in medical diagnostics.

4 ex

FIELD: medicine.

SUBSTANCE: reactions are conducted in the same PCR-microtube on walls of which there is a probe immobilised. In comparison with standard, the PCR-tubes used have a feature of construction: an extended internal surface, and as consequence - a great sorption capacity. A process of selective amplificate sorption on the test tube walls occurs after each anneal stage: temperature decreasing leads to hybridisation of amplicon chain not only among themselves, and with probes preliminary immobilised on the test tube walls. Using the similar PCR-microtubes enables increasing sensitivity of an immune-enzyme assay of amplicons and decreasing a number of polymerase chain reaction cycles. The fixed analytical signal will characterise both the presence of the required DNA, and show its concentration in the sample.

EFFECT: offered test system can be adapted for any existing PCR-technique, does not require special instrumentation and is reliable, simple and efficient for all parameters.

6 cl, 1 dwg, 1 tbl, 1 ex

New corn plants // 2423528

FIELD: agriculture.

SUBSTANCE: during selection by markers, a set of alleles related to loci of quantity tokens (QTL) that contribute to expression of certain phenes of economic value is introduced into a corn idioplasm. The criteria are selected among grain crop capacity, grain moisture when picked, early and late root fit, stem drowning, frequency of common rust, frequency of ear rotting caused by Fusarium (ear wilt), resistance to Sulcotrione and panicle structure. Invention also relates to the method of such plants production, and also to methods of analysis and screening to identify plants with a required profile of alleles.

EFFECT: improved method of new corn plants production.

33 cl, 1 dwg, 19 tbl

FIELD: medicine.

SUBSTANCE: synthetic oligonucleotides for indentifying DNA of Torque teno virus of all known genotypes are disclosed. Primers are combined in a set for DNA identification in blood and other biomaterials of the infectious agent of the latent viral infection Torque teno virus of Circoviridae family by polymerase chain reaction.

EFFECT: invention allows reliable identification of said virus in a biological material.

FIELD: medicine.

SUBSTANCE: analysed sample is studied simultaneously by two methods: the first one is a indirect immunofluorescence (IIMF) reaction, while the second one involves a polymerase chain reaction (PCR); the IIMF provides using monoclonal antibodies "BCKK" (P-384D and 434D). The antibodies interact with the capsular antigen F1 specific for Y.pestis species, or the plasmid-temperature-independent surface protein PFV which is found in all Y.pestis strains and rare R-form Y.pseudotuberculosis strains. The bacteria luminescence shows the presence of native or fraction-less Y.pestis bacteria, or typical and PFV-atypical Y.pseudotuberculosis strains in the sample. The PCR is conducted by two pairs of primers vlm33for/ISrevl754 - specific for Y.pestis species, and JS - specific for Y. pseudotuberculosis species. The values derived with the first pair of the primers are estimated as positive if observing the amplicons in 400 bps, and with the second pair if observing the amplicons in 223 bps; the analysed sample is identified by the matching the IIMF and PCR values with the reference strains.

EFFECT: method provides quick identification and high-reliability differentiation of all strains.

7 tbl, 6 ex

FIELD: physics.

SUBSTANCE: method for bioindication of water bodies involves collecting samples of planktons inhabiting in a water body, determining the contamination level by analysing said samples and assessing the analysis results. The contamination level is determined via phylogenetic analysis of ribosomal RNA genes (18S rRNA) of planktons in the sample. Phylogenetic trees built from the conservative 18S rRNA gene are determined and evolutionary relationships of the analysed object with other saprobionts are identified. Analysis results are assessed as follows: at high (over 85%) value of bootstrap support of clusters containing the analysed planktons and resistant saprobionts, the following conclusions are made: resistant indicator organisms xeno- or oligosaprobic (or exclusively xenosaprobic) of water bodies and the analysed plankton merge into one cluster, it is concluded that the water body is in a safe ecological state and there is no threat of negative anthropogenic action, if resistant indicator organisms oligo- and mesosaprobic (or exclusively oligosaprobic) of the water bodies and the analysed plankton merge into one cluster, it is concluded that the water body is in an unstable (transition from safe to unsafe state) ecological state, is under insignificant anthropologic load, is capable of self-recovery and does not need additional environmental protection measures, if resistant indicator organisms meso- and polysaprobic (or exclusively mesosaprobic) of water bodies and the analysed plankton merge into one cluster, it is concluded that the water body is in an unsafe state and is under considerable anthropologic load, natural capability of self-recovery is insufficient and the water body needs environmental protection measures, if resistant indicator organisms of polysaprobic water bodies and the analysed plankton merge into one cluster, it is concluded that there is a local ecological disaster and there is need for urgent recovery measures.

EFFECT: high reliability of the biomonitoring result for use without territorial limit, independent of the geographical location of the investigated water body.

3 ex

FIELD: medicine.

SUBSTANCE: bacteria freely immobilised on a slide are processed with a lysis solution containing an ionogenic protein-denaturing detergent and EDTA. Further, DNA-nucleoid is dry heating and microwave incubation stabilised. DNA is stained by high-sensitivity DNA-specific fluorochrome, in conclusion DNA integrity is evaluated. Offered is a kit for evaluating the DNA bacteria integrity which comprises all the components required.

EFFECT: invention can be used for determining the bacterial DNA fragmentation levels by a precise express technique.

16 cl, 11 dwg, 5 tbl, 9 ex

Glypican-3 antibody // 2427588

FIELD: medicine.

SUBSTANCE: versions of antibodies bound with glypican-3 in a site with amino acid residues 1-563 are offered. Each version is characterised by the fact that it contains three CDRs of a light chain and three CDRs of a heavy chain. There are described: coding polynucleotide, and also a based expression and a host cell on the basis of the vector. There are disclosed: a method of producing the antibody with using a host cell, a cell growth inhibitor on the basis of the antibody, versions of application of the antibody for treating cancer or hepatoma. There is described peptide for producing glypican-3 antibodies containing residues 546-551 of glypican-3. The offered new antibodies exhibit higher cytotoxicity as compared with known glypican-3 antibodies and are specific to a certain site of glypican-3.

EFFECT: invention use can find further application in cancer therapy.

16 cl, 20 dwg, 2 tbl, 27 ex

FIELD: medicine.

SUBSTANCE: polypeptide contains extracellular and transmembrane domains of a human tissue factor. A DNA fragment coding this polypeptide, and a plasmid pET28a(+) are used to produce a genetic make-up enabling biosynthesis of the recombinant human tissue factor. Also, the E coli BL21 [DE3]/p6E-tTF strain that is a producer of the recombinant human tissue factor is offered.

EFFECT: high-yield recombinant protein and simplified recovery and purification procedures.

2 cl, 4 dwg, 3 ex

FIELD: medicine.

SUBSTANCE: what is offered is a human OX40L antibody containing a light chain and a heavy chain each of which contains respectively three CDRs of the light chain and three CDRs of the heavy chain. There are described: a coding polynucleotide, and also an expression vector and a host cell including coding polynucleotide. There are disclosed: a method of producing and a method of treating with using the antibody.

EFFECT: use of the invention can find further application in therapy of the OX40L mediated immune disorders.

28 cl, 8 dwg, 1 tbl, 7 ex

FIELD: medicine.

SUBSTANCE: versions of the CD38 specific antibodies and their functional fragments are offered. Each version is characterised by the fact that it contains three CDRs of a light chain and three CDRs of a heavy chain. There are described: a coding polynucleotide, and also an expression vector and a host cell including coding polynucleotide. There are disclosed: a pharmaceutical and diagnostic compositions, a method of treating, a method of detecting CD38 in erythrocyte, a method of inducing specific CD38 expressing tumour cell killing with using the antibody. The offered new antibodies exhibit the unexpected properties: to bind minipig's CD38 and to cause cross-linked specific CD38 expressing cell killing.

EFFECT: use of the invention can find further application in therapy of the CD38 mediated disorders.

87 cl, 37 dwg, 4 tbl, 6 ex

FIELD: medicine.

SUBSTANCE: what is described in a cartridge genetic maker based on GeneClip-Ul-neo vector producing three siRNA - reproduction inhibitors of human immunodeficiency virus type 1 and human CCR5 gene. The maker contains a fragment of the length 27 bp related to a conservative region of a HIV-1 genome reverse transcriptase domain, a fragment of the length 27 bp related to the other conservative region of the HIV-1 genome reverse transcriptase domain, as well as a fragment 19 bp related to CCR5 gene mRNA. The invention can be used in medicine and scientific research.

EFFECT: invention allows producing more effective anti-HIV preparations based on interfering RNA produced in cells by means of the introduced cartridge genetic maker containing palindromes for three siRNA production.

3 dwg, 2 tbl, 2 ex

FIELD: medicine.

SUBSTANCE: recovered polynucleotide coding aminopeptidase containing a nucleotide sequence presented in the description is presented. Also, polynucleotide hybridised with said polynucleotide in the high stiffness environment is presented. An expression vector containing said polynucleotide, and an applicable host cell are described. Polypeptide related to the sequence presented in the description and being aminopeptidase is presented. A method of producing said polypeptide involving the stages of applicable host cell transformation by said polynucleotide or vector, cell cultivations in the medium adequate for said polynucleotide expression, and optional polypeptide purification from said cell or culture medium is offered. Besides, there has been described diagnostic technique for Aspergillus-infection in an organism involving the stages: a) recovery of a biological sample from said organism which is supposed to be Aspergillus infected, b) recovery of nucleic acid from said sample, c) determination whether said recovered nucleic acid contains polynucleotides hybridised with polynucleotide recovered from Aspergillus niger.

EFFECT: invention allows diagnosing Aspergillus-infection in the organism.

13 cl, 3 tbl, 11 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology and immunology. Versions of a human CD37 specific antibody are presented, each containing a variable site of a light and heavy chain. A coding nucleic acid and a based expression vector are described. There are disclosed: a host cell containing a vector, a method of producing the antibody with using the cell, and also a composition for decreasing the B-cell count and a method of treating diseases associated with aberrant B-cell activity, with using the CD37-specific antibodies.

EFFECT: use of the invention provides specific loss of 80% BJAB cells at the antibody concentration 10 mcg/ml, and also increases the survival rate of Daudi mice as compared with Rhithuximab therapy that can find application for treating various tumours.

39 cl, 39 dwg, 7 tbl, 18 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to field of biotechnology and deals with conjugates "caliheamicin derivative-carrier". Essence of invention includes methods of obtaining conjugates "monomer cytotoxic medication-caliheamicin/carrier" with considerably higher loading with medication than in earlier described methods, with low aggregation degree and with low content of low-conjugated fraction (LCF), as well as conjugates "cytotoxic medication-caliheamicin derivative/anti-CD22-antibody". Invention also includes compositions, which contain conjugate with CD22 antibody and application of said conjugates.

EFFECT: invention makes it possible to create conjugates with high loading of cytotoxic medication.

181 cl, 11 ex, 13 tbl, 29 dwg

FIELD: chemistry.

SUBSTANCE: described is a polypeptide, having phytase activity, selected from: a polypeptide with an amino acid sequence given in the description of the invention, a fragment of that polypeptide, and a polypeptide which exhibits at least 90% identity with the said polypeptide. Described also is a polynucleotide which codes the said polypeptide. An expression cassette and an expression vector which contain the described polynucleotides are disclosed. A host organism producing the described polypeptide is disclosed. Furthermore, a feed additive and animal feed containing the said polypeptide, host organism or metabolites thereof is obtained. The invention discloses use of the said polypeptide to prepare a feed additive or animal feed and for hydrolysis of myoinositol hexakisphosphate to inorganic monophosphate, to myoinositol with lower degree of phosphorylation and to free myoinositol.

EFFECT: invention enables to obtain inorganic monophosphate, myoinositol with lower degree of phosphorylation or free myoinositol from a complex compound of myoinositol hexakisphosphate and vary the ration of domestic animals.

14 cl, 11 dwg, 9 tbl, 2 ex

FIELD: medicine.

SUBSTANCE: offered are versions of antibodies each of which is specifically bound with IGF-IR, inhibits its activity and is its antagonist, not exhibiting substantially IGF-IR agonist activity. Each of the antibodies is characterised at least by the presence of a variable area of a heavy and easy chain. There are described: antibody conjugates with cytotoxic agents, and also versions of a pharmaceutical composition for cancer diagnosing and therapy, methods of cancer treatment and diagnosing, a cancer diagnosing reagent - on the basis of the antibodies. There are disclosed: a method of producing the antibodies; nucleic acid (NA) coding the antibody; an expression vector containing NA. Offered is a hybridoma EM 164 producing the antibody under the invention, deposited in ATCC, No. PTA-4457, and also the use of the antibody for IGF-IR linkage. The use of the invention presents the antibodies which allow inhibiting MCF cell growth approximately in 12 times that is higher approximately in 5 times than hen using the antibody IR3, and can be used for cancer diagnosing and treatment expressing higher levels of receptor IGF-I, such as breast cancer, colon cancer, lung cancer, prostate cancer, ovarian cancer, synovial carcinoma and pancreatic cancer.

EFFECT: more efficient diagnosing and treatment of said cancers.

58 cl, 28 dwg, 10 tbl, 1 ex

Glypican-3 antibody // 2427588

FIELD: medicine.

SUBSTANCE: versions of antibodies bound with glypican-3 in a site with amino acid residues 1-563 are offered. Each version is characterised by the fact that it contains three CDRs of a light chain and three CDRs of a heavy chain. There are described: coding polynucleotide, and also a based expression and a host cell on the basis of the vector. There are disclosed: a method of producing the antibody with using a host cell, a cell growth inhibitor on the basis of the antibody, versions of application of the antibody for treating cancer or hepatoma. There is described peptide for producing glypican-3 antibodies containing residues 546-551 of glypican-3. The offered new antibodies exhibit higher cytotoxicity as compared with known glypican-3 antibodies and are specific to a certain site of glypican-3.

EFFECT: invention use can find further application in cancer therapy.

16 cl, 20 dwg, 2 tbl, 27 ex

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