Anti-aggrus monoclonal antibody, domain in aggrus which is required for binding to clec-2, and method for screening for aggrus-clec-2 binding inhibitor

Abstract

A novel domain of Aggrus involved in the binding to CLEC-2 was searched for, and monoclonal antibodies recognizing the domain were obtained. The newly found PLAG4 domain is important for Aggrus binding to CLEC-2. Monoclonal antibodies recognizing this region were further developed. The present invention can provide novel Aggrus-CLEC-2 binding inhibitors, platelet aggregation inhibitors, cancer metastasis inhibitors, and tumor growth inhibitors using these antibodies.

1 . A neutralizing monoclonal antibody or a functional fragment thereof, which recognizes an Aggrus epitope consisting of an amino acid sequence represented by SEQ ID NO: 3 (RIEDL). 
     
     
         2 . The neutralizing monoclonal antibody or the functional fragment thereof, wherein the monoclonal antibody according to  claim 1  is chimeric or humanized. 
     
     
         3 . An Aggrus-CLEC-2 binding inhibitor comprising a neutralizing monoclonal antibody or a fragment consisting of a functional fragment thereof according to  claim 1 . 
     
     
         4 . A composition for inhibiting Aggrus-CLEC-2 binding comprising:
 a neutralizing monoclonal antibody or a fragment consisting of a functional fragment thereof according to  claim 1 , and   at least one monoclonal antibody, chimeric antibody thereof, humanized antibody thereof, and/or fragment consisting of a functional fragment thereof,   wherein the monoclonal antibody is produced by a hybridoma of deposition No. FERM BP-11446 (P2-0), FERM BP-11447 (MS-1), FERM BP-11448 (MS-3) and/or FERM BP-11449 (MS-4).   
     
     
         5 . A pharmaceutical composition for inhibition of platelet aggregation, inhibition of thrombus formation, inhibition of cancer progression or metastasis, or anti-inflammation, comprising a neutralizing monoclonal antibody or a fragment consisting of a functional fragment thereof according to  claim 1 . 
     
     
         6 . The pharmaceutical composition according to  claim 5 , further comprising at least one monoclonal antibody, chimeric antibody thereof, humanized antibody thereof, and/or fragment consisting of a functional fragment thereof,
 wherein the monoclonal antibody is produced by a hybridoma of deposition No. FERM BP-11446 (P2-0), FERM BP-11447 (MS-1), FERM BP-11448 (MS-3) and/or FERM BP-11449 (MS-4).   
     
     
         7 . A testing reagent for detection of Aggrus expression, comprising a neutralizing monoclonal antibody or a fragment consisting of a functional fragment thereof according to  claim 1 . 
     
     
         8 . The neutralizing monoclonal antibody or the functional fragment thereof, wherein the monoclonal antibody according to  claim 1  having the dissociation constants (K D ) for Aggrus is ≤3×10 −10  M. 
     
     
         9 . The neutralizing monoclonal antibody or the functional fragment thereof, wherein the monoclonal antibody according to  claim 1  is produced by using an amino acid sequence consisting region from a threonine residue at position 76 to a threonine residue at position 89 as an immunogen. 
     
     
         10 . A method for inhibiting Aggrus-CLEC-2 binding using antibody which recognizes an Aggrus epitope consisting of an amino acid sequence represented by SEQ ID NO: 3 (RIEDL). 
     
     
         11 . A method for inhibition of platelet aggregation, inhibition of thrombus formation, inhibition of cancer progression or metastasis, or anti-inflammation, wherein the method comprises administering the neutralizing antibody or the functional fragment thereof according  claim 1 .