Inhibition of platelet aggregation using anti-human GPVI antibodies

1. A method for treating a cardiovascular disease or event associated with inflammation and/or thrombosis in a subject in need thereof comprising administering an isolated humanized protein binding to human Glycoprotein VI (hGPVI), wherein said isolated humanized protein is administered for at least 2 hours, wherein said isolated humanized protein is a monovalent antibody fragment, wherein

the variable region of the heavy chain of the antibody fragment comprises the following CDRs:

VH-CDR1:

(SEQ ID NO: 1)

GYTFTSYNMH;

VH-CDR2:

(SEQ ID NO: 2)

GIYPGNGDTSYNQKFQG;

and

VH-CDR3:

(SEQ ID NO: 3)

GTVVGDWYFDV;

and

the variable region of the light chain of the antibody fragment comprises the following CDRs:

VL-CDR1:

(SEQ ID NO: 4)

RSSQSLENSNGNTYLN;

VL-CDR2:

(SEQ ID NO: 5)

RVSNRFS;

and

VL-CDR3:

(SEQ ID NO: 6)

LQLTHVPWT.

2. The method according to claim 1 , wherein said isolated humanized protein is administered during at least 4 to 6 hours.

3. The method according to claim 1 , wherein the isolated humanized protein is injected.

4. The method according to claim 1 , wherein the isolated humanized protein is administered by intravenous infusion.

5. The method according to claim 1 , wherein a dose of humanized protein ranging from about 0.5 mg/kg to about 50 mg/kg is administered to the patient.

6. The method according to claim 1 , wherein a dose of humanized protein ranging from about 1 mg/kg to about 32 mg/kg is administered to the patient.

7. The method according to claim 1 , wherein a dose of humanized protein of about 8 mg/kg is administered to the patient.

8. The method according to claim 1 , wherein a dose of humanized protein ranging from about 125 mg to about 2000 mg is administered to the patient.

9. The method according to claim 1 , wherein a first bolus is administered.

10. The method according to claim 9 , wherein the first bolus comprises about 10 to 50% of the total dosage of the isolated humanized protein to be administered.

11. The method according to claim 9 , wherein said first bolus is administered in about 5 to 30 minutes.

12. The method according to claim 1 , wherein said protein binds to a conformational epitope comprising:

at least one amino acid residue from amino acid residues 114 to 142 of hGPVI (SEQ ID NO: 13) or from a sequence sharing at least 60% of identity over amino acid residues 114 to 142 of hGPVI (SEQ ID NO: 13); and

at least one amino acid residue from amino acid residues 165 to 187 of hGPVI (SEQ ID NO: 13) or from a sequence sharing at least 60% of identity over amino acid residues 165 to 187 of hGPVI (SEQ ID NO: 13).

13. The method according to claim 12 , wherein said conformational epitope comprises at least one amino acid residue from amino acid residues 121 to 135 or from 121 to 136 of hGPVI (SEQ ID NO: 13) or from a sequence sharing at least 60% of identity over amino acid residues 121 to 135 or from 121 to 136 of hGPVI (SEQ ID NO: 13); and at least one amino acid residue from amino acid residues 169 to 183 of hGPVI (SEQ ID NO: 13) or from a sequence sharing at least 60% of identity over amino acid residues 169 to 183 of hGPVI (SEQ ID NO: 13).

14. The method according to claim 1 , wherein said protein has a KD for binding to hGPVI less than 15 nM, wherein said K D is measured by surface plasmon resonance using 960 to 1071 RU of soluble human GPVI and using PBS pH 7.4 as running buffer and wherein said isolated humanized protein does not induce a GPVI depletion phenotype in vivo.

15. The method according to claim 1 , wherein said protein is a monovalent antibody fragment selected from the group consisting of a single chain antibody, a Fv, a Fab; and a unibody.

16. The method according to claim 1 , wherein the amino acid sequence encoding the heavy chain variable region of the antibody fragment is SEQ ID NO:

7 and the amino acid sequence encoding the light variable region of the antibody fragment is SEQ ID NO: 8, or any sequence having an amino acid sequence that shares at least 60% of identity with said SEQ ID NO: 7 or 8.

17. The method according to claim 15 , wherein the amino acid sequence encoding the heavy chain variable region of the antibody is SEQ ID NO: 7 and the amino acid sequence encoding the light variable region of the antibody is SEQ ID NO: 9, or any sequence having an amino acid sequence that shares at least 60% of identity with said SEQ ID NO: 7 or 9.

18. The method according to claim 1 , wherein said cardiovascular disease or event associated with inflammation and/or thrombosis is selected from arterial and venous thrombosis, restenosis, acute coronary syndrome, cerebrovascular accidents due to atherosclerosis, critical limb ischemia, cerebral vascular diseases, ischemic stroke, venous thromboembolism diseases, thrombotic microangiopathies and vascular purpura.

19. The method according to claim 1 , wherein said cardiovascular disease or event associated with inflammation and/or thrombosis is selected from coronary artery and cerebral artery diseases.

20. The method according to claim 1 , wherein said cardiovascular disease or event associated with inflammation and/or thrombosis is selected from atherothrombosis, ischemic events, acute coronary artery syndrome, myocardial infarction, stroke, percutaneous coronary intervention, stenting thrombosis, ischemic restenosis, acute ischemia, chronic ischemia, diseases of the aorta and its branches, peripheral artery disease, venous thrombosis, acute phlebitis, pulmonary embolism, cancer-associated thrombosis, inflammatory thrombosis and thrombosis associated to infection.