Therapeutical polypeptides, their homologs, fragments, and application in thrombocyte-mediated aggregation modulation

FIELD: medicine.

SUBSTANCE: polypeptides include single-domain antibody against vWF, A1 domain of vWF, A1 domain of activated vWF, A3 domain of vWF, gp1b and/or collagen. Invention claims methods of obtaining indicated polypeptides, methods of coating devices applied in medical practice (e.g. in X-ray structural analysis, endoprosthetics) with indicated polypeptides.

EFFECT: obtainment of polypeptides for treatment of diseases requiring modulation of thrombocyte-mediated aggregation.

40 cl, 69 ex, 30 dwg, 32 tbl

 

The text descriptions are given in facsimile form.

1. The polypeptide or polypeptide design for the treatment, prevention and/or facilitate violations related to the adhesion of platelets and/or mediated platelet aggregation or dysfunction, comprising at least one single domain antibody directed against a factor von Willebrand (vWF).

2. The polypeptide or polypeptide construct according to claim 1, comprising two or more single domain antibodies directed against vWF.

3. The polypeptide or polypeptide construct according to claim 1, in which at least the bottom single-domain antibody against vWF directed against the A1 domain of vWF, A1 domain of activated vWF or A3 domain of vWF.

4. The polypeptide or polypeptide construct according to claim 1, comprising two or more single domain antibodies in which at least one single domain antibody against vWF directed against the A1 domain of the vWF A1 domain of activated vWF or A3 domain of vWF.

5. The polypeptide or polypeptide construct according to claim 1, in which at least one single domain antibody corresponds to a sequence represented by any of SEQ ID NO: 1 through 7, from 23 to 31 and 62 through 65, or:
a) a sequence homologous to any of SEQ ID NO: 1 through 7, from 23 to 31 and 62 through 65 with sequence identity greater than 70% with the parent sequence; or
b) a functional portion of any of SEQ ID NO: 1 through 7, from 23 to 31 and 62 through 65, which retains the interaction with the target with an affinity of 1·10-6M or above; or
c) a functional portion of any of SEQ ID NO: 1 through 7, from 23 to 31 and 62 through 65, which includes a partial deletion of the complete amino acid sequence and still maintains the binding site(s) and domain(s) protein required for binding and interaction with the target.

6. The polypeptide or polypeptide construct according to claim 1, in which at least one single domain antibody corresponds to a sequence represented by any of SEQ ID NO: 1 through 7, from 23 to 31 and 62 through 65, or
d) a sequence homologous Liu is th of SEQ ID NO: 1 through 7, from 23 to 31 and 62 through 65 with sequence identity greater than 70% with the parent sequence, and where this homologous sequence capable of inhibiting at least 50% of platelet aggregation under high shear stress (1600-1) at 1 µg/ml or at lower concentrations; or
e) a functional portion of any of SEQ ID NO: 1 through 7, from 23 to 31 and 62 through 65, which retains the interaction with the target with an affinity of 1·10-6M or above; or
f) a functional portion of any of SEQ ID NO: 1 through 7, from 23 to 31 and 62 through 65, which includes a partial deletion of the complete amino acid sequence and still maintains the binding site(s) and domain(s) protein required for binding and interaction with the target.

7. The polypeptide or polypeptide construct according to claim 1, in which at least one single domain antibody corresponds to a sequence represented by any of SEQ ID NO: 1 through 7, from 23 to 31 and 62 through 65, or
g) a sequence homologous to any of SEQ ID NO: 1 through 7, from 23 to 31 and 62 through 65 with sequence identity greater than 70% with the parent sequence, and where this homologous sequence (a) capable of inhibiting at least 50% of platelet aggregation under high shear stress (1600-1) at 1 µg/ml or more or the fir concentrations and b) not able to inhibit 50% of platelet aggregation at low shear stress (300 -1) at 1 µg/ml or at lower concentrations; or
h) a functional portion of any of SEQ ID NO: 1 through 7, from 23 to 31 and 62 through 65, which retains the interaction with the target with an affinity of 1·10-6M or above; or
i) a functional portion of any of SEQ ID NO: 1 through 7, from 23 to 31 and 62 through 65, which includes a partial deletion of the complete amino acid sequence and still maintains the binding site(s) and domain(s) protein required for binding and interaction with the target.

8. The polypeptide or polypeptide design for the treatment, prevention and/or facilitate violations related to the adhesion of platelets and/or mediated platelet aggregation or dysfunction, comprising at least one single domain antibody directed against a factor von Willebrand, in which at least one single domain antibody corresponds to a sequence represented by SEQ ID NO: 3, or a sequence homologous to SEQ ID NO: 3 with sequence identity greater than 70% with the parent sequence, and where this homologous sequence capable of inhibiting at least 50% aggregation platelets under high shear stress (1600-1) at 1 µg/ml or at lower concentrations.

9. The polypeptide or polypeptide design for the treatment, prevention and and/or facilitate violations associated with the adhesion of platelets and/or mediated platelet aggregation or dysfunction, comprising at least one single domain antibody directed against a factor von Willebrand, in which at least one single domain antibody corresponds to a sequence represented by SEQ ID NO: 5, or a sequence homologous to SEQ ID NO: 5 with sequence identity greater than 70% with the parent sequence, and where this homologous sequence capable of inhibiting at least 50% of platelet aggregation under high shear stress (1600-1) at 1 µg/ml or at lower concentrations.

10. The polypeptide or polypeptide design for the treatment, prevention and/or facilitate violations related to the adhesion of platelets and/or mediated platelet aggregation or dysfunction, comprising at least one single domain antibody directed against a factor von Willebrand, in which at least one single domain antibody corresponds to a sequence represented by SEQ ID NO: 7, or a sequence homologous to SEQ ID NO: 7 with sequence identity greater than 70% with the parent sequence, and where this homologous sequence capable of inhibiting at least 50 platelet aggregation under conditions of high shear stress (1600 -1) at 1 µg/ml or at lower concentrations.

11. The polypeptide or polypeptide design for the treatment, prevention and/or facilitate violations related to the adhesion of platelets and/or mediated platelet aggregation or dysfunction, comprising two or more single domain antibodies against factor von Willebrand, and
(i) in which two or more single domain antibodies correspond to the sequence represented by SEQ ID NO: 3, or a sequence homologous to SEQ ID NO: 3 with sequence identity greater than 70% with the parent sequence, and where this homologous sequence capable of inhibiting at least 50% of platelet aggregation under high shear stress (1600-1) at 1 µg/ml or at lower concentrations; or
(ii) in which two or more single domain antibodies correspond to the sequence represented by SEQ ID NO: 5, or a sequence homologous to SEQ ID NO: 5 with sequence identity greater than 70% with the parent sequence, and where this homologous sequence capable of inhibiting at least 50% of platelet aggregation under high shear stress (1600-1) at 1 µg/ml or at lower concentrations; or
iii) where two or more annodomini the antibody correspond to the sequence, represented by SEQ ID NO: 7, or a sequence homologous to SEQ ID NO: 7 with sequence identity greater than 70% with the parent sequence, and where this homologous sequence capable of inhibiting at least 50% of platelet aggregation under high shear stress (1600-1) at 1 µg/ml or at lower concentrations.

12. The polypeptide or polypeptide construct according to any one of claims 1, 8-10 or 11, in which at least one single domain antibody is a VHH domain.

13. The polypeptide or polypeptide construct according to any one of claims 1, 8-10 or 11, in which at least one single domain antibody is a VHH domain that includes the amino acid at position 45 according to the Kabat numbering selected from the group consisting of glycine, alanine, valine, leucine, isoleucine, Proline, phenylalanine, tyrosine, tryptophan, methionine, serine, threonine, asparagine and glutamine.

14. The polypeptide or polypeptide construct according to any one of claims 1, 8-10 or 11, in which at least one single domain antibody is a VHH domain that includes the amino acid at position 103 according to the Kabat numbering selected from the group consisting of arginine, serine or uncharged residue, optional glycine.

15. The polypeptide or polypeptide to the construction according to any one of claims 1, 8-10 or 11, in which at least one single domain antibody is a VHH domain, obtained by immunization of a camel and receive from him hybridoma, or by cloning libraries of single-domain antibodies and subsequent selection of VHH using phage display.

16. The polypeptide or polypeptide construct according to any one of claims 1, 8-10 or 11, in which at least one single domain antibody is humanized.

17. The polypeptide or polypeptide construct according to any one of claims 1, 8-10 or 11, in which at least one single domain antibody is humanitarianly VHH domain.

18. The polypeptide or polypeptide construct according to 17, in which at least one single domain antibody is humanized by replacing one or more amino acids Camelidae, the corresponding human equivalent, is found in the consensus sequence of a human.

19. The polypeptide or polypeptide construct according to 17, in which at least one single domain antibody is humanized by replacing any of the following residues or one or a combination of: provisions in FR1 1, 5, 28 and 30, the marker amino acids in FR2 at positions 37, 44, 45 and 47, the provisions in FR3 74, 75, 76, 83, 84, 93 and 94, and the provisions in FR4 103, 104, 108 and 111, and the numbering of the provisions listed according to the Kabat numbering.

20. The floor of the peptide or polypeptide construct according to claim 11, which includes two or more single domain antibodies directed against the A1 domain of vWF.

21. The polypeptide or polypeptide construct according to claim 11, in which two or more single domain antibodies have the same sequence.

22. The polypeptide or polypeptide construct according to claim 11, in which the end of the first single-domain antibodies can be connected to the N-end single-domain antibodies.

23. The polypeptide or polypeptide construct according to claim 11, which is not able to inhibit 50% or more of platelet aggregation at low shear stress (300-1) at 1 µg/ml or at lower concentrations.

24. Nucleic acid encoding a single domain antibody, corresponding to any of the sequences represented by SEQ ID NO: 1-7, 23-31 and 62-65.

25. The use of the polypeptide or polypeptide constructs according to any one of claims 1 to 23 or a nucleic acid according to paragraph 24 to obtain drugs for treatment, prevention and/or facilitate violations related to mediated platelet aggregation or dysfunction.

26. The use of the polypeptide or polypeptide constructs according to any one of claims 1 to 23 or a nucleic acid according to paragraph 24 to obtain drugs for the treatment of conditions requiring modulation mediated platelet aggregation.

27. The use of the polypeptide is whether the polypeptide constructs according to any one of claims 1 to 23 or a nucleic acid according to paragraph 24 for obtaining a medicinal product for the treatment or prevention of the first stages of the formation of a blood clot.

28. The use of the polypeptide or polypeptide constructs according to any one of claims 1 to 23 or a nucleic acid according to paragraph 24 to obtain drugs for treatment, prevention and/or facilitate violations related to mediated platelet aggregation or dysfunction, where these violations are any of the resulting transient ischemic brain damage, unstable or stable angina, angina pectoris, stroke, cerebral, myocardial infarction, occlusion of peripheral arteries, restenosis, coronary artery bypass, replacement heart valve or surgical procedures on the heart, such as angioplasty, hip replacement, carotid endarterectomy (CEA) or atherectomy.

29. The use of the polypeptide or polypeptide constructs according to any one of claims 1 to 23 or a nucleic acid according to paragraph 24 to obtain drugs for treatment, prevention and/or facilitate violations related to mediated platelet aggregation or dysfunction, where these violations are any of the educational reocclusion clots, education occlusal thrombus, the formation of blood clots, acute coronary occlusion, restenosis, restenosis after RST or knee replacement, blood clots in stenotic arteries, the ISU is blasii after angioplasty, atherectomy or replacement arteries, occlusal syndrome vascular system or loss of permeability of diseased arteries.

30. The use of the polypeptide or polypeptide constructs according to any one of claims 1 to 23 or a nucleic acid according to paragraph 24 to obtain drugs for treatment, prevention and/or relief of thrombotic thrombocytopenic purpura (TTP), transient ischemic brain damage, unstable or stable angina, stroke, cerebral, myocardial infarction, occlusion of peripheral arteries, restenosis or violations arising from the coronary vessels, heart valve replacement and surgical interventions on the heart, such as angioplasty, hip replacement, or atherectomy.

31. The use of the polypeptide or polypeptide constructs according to any one of claims 1 to 23 or a nucleic acid according to paragraph 24 to obtain drugs for treating, preventing and/or alleviating conditions associated with mediated platelet aggregation, including, but not limited to, unstable angina, stable angina, chest toad, education of the pitch, deep vein thrombosis, hemolytic uremic syndrome, hemolytic anemia, acute renal failure, thrombolytic complications, thrombotic thrombocytop the practical purple, disseminated intravascular congelatio, thrombosis, coronary heart disease, thromboembolic complications, myocardial infarction, restenosis, and the formation of a blood clot in the atrium in atrial fibrillation, chronic unstable angina, transient ischemic injuries and strokes, peripheral vascular disease, arterial thrombosis, pre-eclampsia, embolism, restenosis and/or thrombosis following angioplasty, carotid endarterectomy, anastomosis of vascular grafts, and chronic use of cardiovascular devices, and conditions arising out of thrombosis and vessel occlusion during and after thrombolytic therapy after angioplasty, and after coronary artery bypass grafting.

32. The use of the polypeptide or polypeptide constructs according to any one of claims 1 to 23 or a nucleic acid according to paragraph 24 to obtain drugs for treatment, prevention and/or facilitate in the formation of plaque or a blood clot in the conditions of high shear stress.

33. A method of obtaining a polypeptide or polypeptide constructs according to any one of claims 1 to 23, including:
(a) culturing host cells containing the nucleic acid according to paragraph 24, in conditions that ensure the expression of the polypeptide, and,
(b) extraction of the resulting polypeptide from the culture.

34. Ways is by p, where indicated cells-the hosts are bacterial cells, yeast cells, mammalian cells or insect cells.

35. The method according to p, where these cells-the hosts are bacterial cells or yeast cells.

36. The method according to p, where these cells are host cells are E. coli, S. cerevisiae cells or cells of P. pastoris.

37. The use of the polypeptide or polypeptide constructs according to any one of claims 1 to 23 for covering invasive medical devices to prevent mediated platelet aggregation around the site of intervention.

38. The application of clause 37, where invasive medical device is any of the implants, tubes, inflatable balloons, catheters, grafting materials, bridging devices or prostheses.

39. The use of the polypeptide or polypeptide constructs according to any one of claims 1 to 23 in the method of diagnosing diseases or disorders characterized by dysfunction mediated platelet aggregation, comprising the following stages:
a) contacting the sample with a polypeptide or polypeptide construct according to any one of claims 1 to 23, and
b) detecting binding of the aforementioned polypeptide or polypeptide structures with named sample, and
c) comparing binding, registered at the stage (b), with the standard, where the difference in the binding of p the relation to the said sample is a diagnostic indicator for diseases or disorders, characterized by dysfunction mediated platelet aggregation.

40. The use of the polypeptide or polypeptide constructs according to any one of claims 1 to 23 in the kit for screening for the diagnosis of diseases or disorders characterized by dysfunction mediated platelet aggregation.
Priorities for items:

10.01.2003 according to claims 1 to 4;

09.01.2004 on pp.5-7, 24-40;

01.12.2003 on PP-23.



 

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