Anti-platelet membrane glycoprotein vi monoclonal antibody

Abstract

The present invention provides an antibody which has the following features, its active fragment, or a derivative thereof: a) It specifically binds to human platelet membrane glycoprotein VI (GPVI); b) The function to activate a platelet and/or the function to induce a thrombocytopenia in vivo are low; and c) It at least partially depletes GPVI on the platelet membrane by contacting with a platelet.

1 . An antibody, its active fragment, or a derivative thereof which:
 a) specifically binds to human platelet membrane glycoprotein VI (GPVI);   b) exhibits weak action to activate a platelet and/or the action to induce a thrombocytopenia in vivo are weak; and   c) at least partially depletes GPVI on the platelet membrane by contacting with a platelet.   
     
     
         2 . An antibody, its active fragment, or a derivative thereof which at least partially depletes GPVI on the platelet membrane by contacting with a platelet without shedding of platelet GPVI. 
     
     
         3 . The antibody of  claim 1 , its active fragment or a derivative thereof, which at least partially depletes GPVI on the platelet membrane by contacting with a platelet in vivo. 
     
     
         4 . The antibody of  claim 1 , its active fragment or a derivative thereof, which decreases or deletes an ability of platelet to aggregate responsive to collagen by contacting with a platelet by administering it in vivo. 
     
     
         5 . The antibody of  claim 1 , its active fragment, or a derivative thereof, wherein its prolonging action on bleeding time is weak. 
     
     
         6 . The antibody of  claim 1 , its active fragment or a derivative thereof, wherein the dissociation constant with GPVI is equal to or less than 4×10 −8  M. 
     
     
         7 . An antibody, its active fragment or a derivative thereof, which specifically recognizes an amino acid sequence or a structure on the GPVI comprising at least a part of loop 2, loop 3 and loop 5, or loop 4 and loop 5 in human GPVI domain 1, or loop 9, or loop 9 and loop 11 in domain 2. 
     
     
         8 . The antibody of  claim 7 , its active fragment or a derivative thereof, wherein at least a part of loop 2, loop 3 and loop 5, or loop 4 and loop 5 in human GPVI domain 1, or loop 9, or loop 9 and loop 11 in domain 2 are E21, K22 and P23 of loop 2, G33 of loop 3 and A57, K59 and L62 of loop 5, or S43, S44, S45, R46, and E48 of loop 4 and A57, K59 and L62 of loop 5, or T116, R117, G119 and Q122 of loop 9 or T116, R117, G119, and Q122 of loop 9 and R139 of loop 11. 
     
     
         9 . The antibody of  claim 7  or  8 , its active fragment or a derivative thereof, which specifically binds to loop 2, loop 3 and loop 5, or loop 4 and loop 5 in human GPVI domain 1, or loop 9, or loop 9 and loop 11 in domain 2. 
     
     
         10 . The antibody of  claim 1 , its active fragment or a derivative thereof, which recognizes an amino acid sequence or a structure on the GPVI comprising at least a part of loop 2, loop 3 and loop 5, or loop 4 and loop 5 in human GPVI domain 1, or loop 9, or loop 9 and loop 11 in domain 2. 
     
     
         11 . A heavy chain of antibody, or its active fragment, wherein the amino acid sequences of SEQ ID NOs: 15, 16, and 17, the amino acid sequences of SEQ ID NOs: 18, 19, and 20, the amino acid sequences of SEQ ID NOs: 21, 22, and 23, the amino acid sequences of SEQ ID NOs: 24, 25, and 26, the amino acid sequences of SEQ ID NOs: 27, 28, and 29, the amino acid sequences of SEQ ID NOs: 30, 31, and 32, the amino acid sequences of SEQ ID NOs: 33, 34, and 35, the amino acid sequences of SEQ ID NOs: 36, 37, and 38, the amino acid sequences of SEQ ID NOs: 39, 40, and 41, the amino acid sequences of SEQ ID NOs: 42, 43, and 44, the amino acid sequences of SEQ ID NOs: 45, 46, and 47, or the amino acid sequences of SEQ ID NOs: 48, 49, and 50, are comprised in VH CDR1, VH CDR2, and VH CDR3, respectively. 
     
     
         12 . A light chain of antibody, its active fragment, or a derivative thereof, wherein the amino acid sequences of SEQ ID NOs: 51, 52 and 53, the amino acid sequences of SEQ ID NOs: 54, 55 and 56, the amino acid sequences of SEQ ID NOs: 57, 58 and 59, the amino acid sequences of SEQ ID NOs: 60, 61 and 62, the amino acid sequences of SEQ ID NOs: 63, 64 and 65, the amino acid sequences of SEQ ID NOs: 66, 67 and 68, the amino acid sequences of SEQ ID NOs: 69, 70 and 71, the amino acid sequences of SEQ ID NOs: 72, 73 and 74, the amino acid sequences of SEQ ID NOs: 75, 76 and 77, the amino acid sequences of SEQ ID NOs: 78, 79 and 80, the amino acid sequences of SEQ ID NOs: 81, 82 and 83, or the amino acid sequences of SEQ ID NOs: 84, 85 and 86, are comprised in VL CDR1, VL CDR2 and VL CDR3, respectively. 
     
     
         13 . An antibody, its active fragment, or a derivative thereof, wherein the amino acid sequences of SEQ ID NOs: 15, 16, 17, 51, 52 and 53, the amino acid sequences of SEQ ID NOs: 18, 19, 20, 54, 55 and 56, the amino acid sequences of SEQ ID NOs: 21, 22, 23, 57, 58 and 59, the amino acid sequences of SEQ ID NOs: 24, 25, 26, 60, 61 and 62, the amino acid sequences of SEQ ID NOs: 27, 28, 29, 63, 64 and 65, the amino acid sequences of SEQ ID NOs: 30, 31, 32, 66, 67 and 68, the amino acid sequences of SEQ ID NOs: 33, 34, 35, 69, 70 and 71, the amino acid sequences of SEQ ID NOs: 36, 37, 38, 72, 73 and 74, the amino acid sequences of SEQ ID NOs: 39, 40, 41, 75, 76 and 77, the amino acid sequences of SEQ ID NOs: 42, 43, 44, 78, 79 and 80, the amino acid sequences of SEQ ID NOs: 45, 46, 47, 81, 82 and 83, or the amino acid sequences of SEQ ID NOs: 48, 49, 50, 84, 85 and 86, are comprised in VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3, respectively. 
     
     
         14 . A cell, which produces the antibody of  claim 1 , its active fragment or a derivative thereof. 
     
     
         15 . A polynucleotide, which comprises a base sequence encoding H-chain and/or L-chain of the antibody of  claim 1 , its active fragment or a derivative thereof. 
     
     
         16 . An anti-human GPVI antibody gene derived from an antibody heavy chain gene comprising any combination of mouse germ-line antibody gene segments V H , D H  and J H  listed in Table 16, or a heavy chain variable region gene thereof. 
     
     
         17 . An anti-human GPVI antibody gene comprising a nucleotide sequence encoding CDR amino acid sequence coded by an antibody heavy chain variable gene, or a heavy chain variable region gene thereof as claimed in  claim 16 . 
     
     
         18 . An anti-human GPVI antibody gene derived from an antibody light chain gene comprising any combination of mouse germ-line antibody gene segments V L  and J L  listed in Table 16, or a light chain variable region gene thereof. 
     
     
         19 . An anti-human GPVI antibody gene comprising a nucleotide sequence encoding CDR amino acid sequence coded by an antibody light chain variable gene, or a light chain variable region gene thereof as claimed in  claim 18 . 
     
     
         20 . An anti-human GPVI antibody or its heavy chain variable region polypeptide encoded by antibody gene of either  claim 16  or  17  or its heavy chain variable region gene. 
     
     
         21 . An anti-human GPVI antibody or its light chain variable region polypeptide encoded by antibody gene of either  claim 18  or  19  or its light chain variable region gene. 
     
     
         22 . The antibody of  claim 1 , which is polyethyleneglycolated (PEGylated), its active fragment, or a derivative thereof. 
     
     
         23 . A method of manufacturing an antibody, its active fragment or a derivative thereof, which is characterized by using the cell of  claim 14  or the polynucleotide of  claim 15 . 
     
     
         24 . A pharmaceutical composition, which comprises the antibody of  claim 1 , its active fragment or a derivative thereof as an active ingredient. 
     
     
         25 . A peptide, which comprises loop 2, loop 3 and loop 5, or loop 4 and loop 5 in human GPVI domain 1, or loop 9, or loop 9 and loop 11 in domain 2. 
     
     
         26 . A peptide, which consists of loop 2, loop 3 and loop 5, or loop 4 and loop 5 in human GPVI domain 1, or loop 9, or loop 9 and loop 11 in domain 2. 
     
     
         27 . A polypeptide, which comprises the amino acid sequence of SEQ ID NO: 135, 136, 137, 138 or 139. 
     
     
         28 . A method of screening an antibody, its active fragment or a derivative thereof, which comprises the following processes:
 a) a process to measure the binding property with human platelet membrane glycoprotein VI (GPVI);   b) a process to measure the function to activate a platelet and/or the function to induce thrombocytopenia in vivo; and   c) a process to measure the activity to at least partially deplete GPVI on the platelet membrane by contacting with a platelet.   
     
     
         29 . A method of identifying the recognition region of the antibody, which comprises the process to measure the reactivity with the peptide of  claim 16  or the polypeptide of  claim 17 . 
     
     
         30 . A method of manufacturing an antibody, which is characterized by using the peptide of  claim 16  or the polypeptide of  claim 17 .