Humanized monoclonal antibodies against activated protein c and uses thereof

Abstract

Provided are humanized antibodies that selectively bind to and inhibit activated protein C without binding to or inhibiting unactivated protein C. Methods of treatment employing these antibodies are described herein.

The invention claimed is: 
     
       1. A humanized antibody binding to activated protein C comprising:
 (a) a heavy light chain comprising heavy light chain CDRs represented by SEQ ID NOS: 1, 2 and 3; and wherein the light chain framework regions are represented by amino acid residues 1 to 23, 39 to 53, 61 to 92 and 102 to 112 of SEQ ID NO: 26, or having 5 or fewer conservative amino acid substitutions to SEQ ID NO: 26 framework regions, and 
 (b) a light heavy chain comprising light heavy chain CDRs represented by SEQ ID NOS: 4, 5 and 6 and wherein the heavy chain framework regions are represented by amino acid residues 1 to 30, 36 to 49, 69 to 100 and 108 to 118 of SEQ ID NO: 16, or having 5 or fewer conservative amino acid substitutions to SEQ ID NO: 16 framework regions. 
 
     
     
       2. The antibody of  claim 1 , wherein the heavy chain framework regions are represented by SEQ ID NOS: 7, 8, 9 and 10, or having 5 or fewer conservative amino acid substitutions. 
     
     
       3. The antibody of  claim 1 , wherein the light chain framework regions are represented by SEQ ID NOS: 11, 12, 13 and 14, or having 5 or fewer conservative amino acid substitutions. 
     
     
       4. The antibody of claim  2  1, wherein residue 14 49 of SEQ ID NO: 8 16 is substituted with Alanine. 
     
     
       5. The antibody of claim  2  1, wherein residues 11, 13 and 31 of SEQ ID NO: 9 79, 81 and 99 of SEQ ID NO: 16 are is substituted with one or more of Serine (residue 11 79), Valine (residue 13 81) and Isoleucine (residue 31 99). 
     
     
       6. The antibody of  claim 1 , wherein said heavy chain comprises SEQ ID NOS: 16-24 NO: 16, 17, 18, 19, 20, 21 or 22. 
     
     
       7. The antibody of claim  3  1, wherein residue 4 of SEQ ID NO: 11 26 is substituted with Leucine. 
     
     
       8. The antibody of claim  3  1, wherein residue 12 72 of SEQ ID NO: 13 26 is substituted with Arginine. 
     
     
       9. The antibody of  claim 1 , wherein said light chain comprises SEQ ID NOS: 26-30 NO: 26, 27, 28 or 29. 
     
     
       10. The antibody of  claim 1 , wherein the antibody is a single-chain antibody or antibody fragment binding activated protein C. 
     
     
       11. The antibody of  claim 10 , wherein the antibody fragment is further defined as Fab′, Fab, F(ab′) 2 , a single domain antibody, Fv, or scFv. 
     
     
       12. A cell or cell line comprising a nucleic acid encoding a humanized antibody binding to activated protein C comprising:
 (a) a heavy light chain comprising heavy light chain CDRs represented by SEQ ID NOS: 1, 2 and 3; and wherein the light chain framework regions are represented by amino acid residues 1 to 23, 39 to 53, 61 to 92 and 102 to 112 of SEQ ID NO: 26, or having 5 or fewer conservative amino acid substitutions to SEQ ID NO: 26 framework regions, and 
 (b) a light heavy chain comprising light heavy chain CDRs represented by SEQ ID NOS: 4, 5 and 6, wherein the heavy chain framework regions are represented by amino acid residues 1 to 30, 36 to 49, 69 to 100 and 108 to 118 of SEQ ID NO: 16, or having 5 or fewer conservative amino acid substitutions to SEQ ID NO: 16 framework regions. 
 
     
     
       13. The antibody of  claim 1 , dispersed in a pharmaceutically acceptable carrier. 
     
     
       14. A method of inhibiting activated protein C anticoagulant activity and/or amidolytic activity in a subject, comprising administering an effective amount of an antibody according to  claim 1  to said subject. 
     
     
       15. A method of treating a subject in need of blood coagulation comprising administering an effective amount of an antibody according to  claim 1  to said subject. 
     
     
       16. The method of  claim 15 , wherein said subject is suffering from hemophilia. 
     
     
       17. A method of promoting hemostasis or thrombosis in a subject, comprising administrating administering an effective amount of an antibody according to  claim 1  to said subject. 
     
     
       18. The method of  claim 17 , wherein the subject is a trauma patient. 
     
     
       19. A kit comprising an antibody according to  claim 1 .