Novel anti-a2ap antibodies and uses thereof

Abstract

The present invention relates to an isolated antibody or antigen-binding fragment thereof that binds to human A2AP. The isolated antibody or antigen-binding fragment according to the present invention i) cross-reacts with rabbit and/or cynomolgus A2AP, ii) does not inhibit human plasmin activity, and iii) increases plasmin mediated clot lysis in the presence of A2AP.

What is claimed is: 
     
         1 . An isolated antibody or antigen-binding fragment thereof capable of binding to A2AP and inhibiting the activity of A2AP, wherein said isolated antibody or antigen-binding fragment thereof does not inhibit plasmin activity. 
     
     
         2 . The isolated antibody or antigen-binding fragment thereof according to  claim 1 , wherein said isolated antibody or antigen-binding fragment thereof binds to human A2AP of the sequence of amino acid 40-491 of SEQ ID NO: 1 with a dissociation constant (K D ) selected from the group consisting of ≤100 nM, ≤50 nM, ≤25 nM, ≤10 nM, ≤1 nM, and ≤0.5 nM; and wherein said isolated antibody or antigen-binding fragment thereof inhibits the activity of human A2AP of the sequence of amino acid 40-491 of SEQ ID NO: 1 with an EC 50  selected from the group consisting of ≤500 nM, ≤250 nM, ≤100 nM, ≤50 nM, ≤25 nM, ≤10 nM, ≤1 nM, and ≤0.5 nM in an in vitro A2AP function blocking assay. 
     
     
         3 . The isolated antibody or antigen-binding fragment thereof according to  claim 1 , wherein said isolated antibody or antigen-binding fragment thereof does not inhibit plasmin activity up to a concentration of said isolated antibody or antigen-binding fragment thereof selected from the group consisting of 1 μM, 2 μM, 5 μM, and 10 μM in an in vitro plasmin inhibition assay. 
     
     
         4 . The isolated antibody or antigen-binding fragment thereof according to  claim 1 , wherein said plasmin is human plasmin comprising SEQ ID NO: 118 and SEQ ID NO: 119. 
     
     
         5 . The isolated antibody or antigen-binding fragment thereof according to  claim 1 , comprising:
 i) a heavy chain antigen-binding region that comprises a heavy chain complementarity determining region 1 (H-CDR11 comprising SEQ ID NO: 6, an H-CDR2 comprising SEQ ID NO: 8, and an H-CDR3 comprising SEQ ID NO: 13 and a light chain antigen-binding region that comprises a light chain complementarity determining region 1 (L-CDR11 comprising SEQ ID NO: 9, an L-CDR2 comprising SEQ ID NO: 10, and an L-CDR3 comprising SEQ ID NO:18;   ii) a heavy chain antigen-binding region that comprises an H-CDR1 comprising SEQ ID NO: 6, an H-CDR2 comprising SEQ ID NO: 7, and an H-CDR3 comprising SEQ ID NO: 13 and a light chain antigen-binding region that comprises an L-CDR1 comprising SEQ ID NO: 9, an L-CDR2 comprising SEQ ID NO: 10, and an L-CDR3 comprising SEQ ID NO:18;   iii) a heavy chain antigen-binding region that comprises an H-CDR1 comprising SEQ ID NO: 6, an H-CDR2 comprising SEQ ID NO: 7, and an H-CDR3 comprising SEQ ID NO: 11 and a light chain antigen-binding region that comprises an L-CDR1 comprising SEQ ID NO: 9, an L-CDR2 comprising SEQ ID NO: 10, and an L-CDR3 comprising SEQ ID NO:17;   iv) a heavy chain antigen-binding region that comprises an H-CDR1 comprising SEQ ID NO: 6, an H-CDR2 comprising SEQ ID NO: 7, and an H-CDR3 comprising SEQ ID NO: 11 and a light chain antigen-binding region that comprises an L-CDR1 comprising SEQ ID NO: 9, an L-CDR2 comprising SEQ ID NO: 10, and an L-CDR3 comprising SEQ ID NO:18;   v) a heavy chain antigen-binding region that comprises an H-CDR1 comprising SEQ ID NO: 6, an H-CDR2 comprising SEQ ID NO: 7, and an H-CDR3 comprising SEQ ID NO: 12 and a light chain antigen-binding region that comprises an L-CDR1 comprising SEQ ID NO: 9, an L-CDR2 comprising SEQ ID NO: 10, and an L-CDR3 comprising SEQ ID NO:18;   vi) a heavy chain antigen-binding region that comprises an H-CDR1 comprising SEQ ID NO: 6, an H-CDR2 comprising SEQ ID NO: 7, and an H-CDR3 comprising SEQ ID NO: 13 and a light chain antigen-binding region that comprises an L-CDR1 comprising SEQ ID NO: 9, an L-CDR2 comprising SEQ ID NO: 10, and an L-CDR3 comprising SEQ ID NO:19;   vii) a heavy chain antigen-binding region that comprises an H-CDR1 comprising SEQ ID NO: 6, an H-CDR2 comprising SEQ ID NO: 7, and an H-CDR3 comprising SEQ ID NO: 14 and a light chain antigen-binding region that comprises an L-CDR1 comprising SEQ ID NO: 9, an L-CDR2 comprising SEQ ID NO: 10, and an L-CDR3 comprising SEQ ID NO:18;   viii) a heavy chain antigen-binding region that comprises an H-CDR1 comprising SEQ ID NO: 6, an H-CDR2 comprising SEQ ID NO: 7, and an H-CDR3 comprising SEQ ID NO: 14 and a light chain antigen-binding region that comprises an L-CDR1 comprising SEQ ID NO: 9, an L-CDR2 comprising SEQ ID NO: 10, and an L-CDR3 comprising SEQ ID NO:20;   ix) a heavy chain antigen-binding region that comprises an H-CDR1 comprising SEQ ID NO: 6, an H-CDR2 comprising SEQ ID NO: 7, and an H-CDR3 comprising SEQ ID NO: 15 and a light chain antigen-binding region that comprises an L-CDR1 comprising SEQ ID NO: 9, an L-CDR2 comprising SEQ ID NO: 10, and an L-CDR3 comprising SEQ ID NO:18;   x) a heavy chain antigen-binding region that comprises an H-CDR1 comprising SEQ ID NO: 6, an H-CDR2 comprising SEQ ID NO: 7, and an H-CDR3 comprising SEQ ID NO: 16 and a light chain antigen-binding region that comprises an L-CDR1 comprising SEQ ID NO: 9, an L-CDR2 comprising SEQ ID NO: 10, and an L-CDR3 comprising SEQ ID NO:18;   xi) a heavy chain antigen-binding region that comprises an H-CDR1 comprising SEQ ID NO: 21, an H-CDR2 comprising SEQ ID NO: 8, and an H-CDR3 comprising SEQ ID NO: 13 and a light chain antigen-binding region that comprises an L-CDR1 comprising SEQ ID NO: 9, an L-CDR2 comprising SEQ ID NO: 10, and an L-CDR3 comprising SEQ ID NO:18; or   xii) a heavy chain antigen-binding region that comprises an H-CDR1 comprising SEQ ID NO: 6, an H-CDR2 comprising SEQ ID NO: 22, and an H-CDR3 comprising SEQ ID NO: 13 and a light chain antigen-binding region that comprises an L-CDR1 comprising SEQ ID NO: 9, an L-CDR2 comprising SEQ ID NO: 10, and an L-CDR3 comprising SEQ ID NO:18.   
     
     
         6 . The isolated antibody or antigen-binding fragment thereof according to  claim 1 , comprising:
 xiii) a variable heavy chain domain comprising SEQ ID NO: 32 and a variable light chain domain comprising SEQ ID NO: 38;   xiv) a variable heavy chain domain comprising SEQ ID NO: 23 and a variable light chain domain comprising SEQ ID NO: 37;   xv) a variable heavy chain domain comprising SEQ ID NO: 24 and a variable light chain domain comprising SEQ ID NO: 38;   xvi) a variable heavy chain domain comprising SEQ ID NO: 25 and a variable light chain domain comprising SEQ ID NO: 38;   xvii) a variable heavy chain domain comprising SEQ ID NO: 26 and a variable light chain domain comprising SEQ ID NO: 38;   xviii) a variable heavy chain domain comprising SEQ ID NO: 27 and a variable light chain domain comprising SEQ ID NO: 39;   xix) a variable heavy chain domain comprising SEQ ID NO: 27 and a variable light chain domain comprising SEQ ID NO: 38;   xx) a variable heavy chain domain comprising SEQ ID NO: 28 and a variable light chain domain comprising SEQ ID NO: 38;   xxi) a variable heavy chain domain comprising SEQ ID NO: 28 and a variable light chain domain comprising SEQ ID NO: 40;   xxii) a variable heavy chain domain comprising SEQ ID NO: 29 and a variable light chain domain comprising SEQ ID NO: 38;   xxiii) a variable heavy chain domain comprising SEQ ID NO: 30 and a variable light chain domain comprising SEQ ID NO: 38;   xxiv) a variable heavy chain domain comprising SEQ ID NO: 31 and a variable light chain domain comprising SEQ ID NO: 38;   xxv) a variable heavy chain domain comprising SEQ ID NO: 33 and a variable light chain domain comprising SEQ ID NO: 38;   xxvi) a variable heavy chain domain comprising SEQ ID NO: 34 and a variable light chain domain comprising SEQ ID NO: 38;   xxvii) a variable heavy chain domain comprising SEQ ID NO: 35 and a variable light chain domain comprising SEQ ID NO: 38; or   xxviii) a variable heavy chain domain comprising SEQ ID NO: 36 and a variable light chain domain comprising SEQ ID NO: 38.   
     
     
         7 . The isolated antibody or antigen-binding fragment thereof according to  claim 1 , wherein the isolated antibody is an IgG antibody. 
     
     
         8 . The isolated antibody according to  claim 7  comprising:
 xxix) a heavy chain comprising SEQ ID NO: 55 and a light chain comprising SEQ ID NO: 57; 
 xxx) a heavy chain comprising SEQ ID NO: 41 and a light chain comprising SEQ ID NO: 56; 
 xxxi) a heavy chain comprising SEQ ID NO: 42 and a light chain comprising SEQ ID NO: 57; 
 xxxii) a heavy chain comprising SEQ ID NO: 43 and a light chain comprising SEQ ID NO: 57; 
 xxxiii) a heavy chain comprising SEQ ID NO: 44 and a light chain comprising SEQ ID NO: 57; 
 xxxiv) a heavy chain comprising SEQ ID NO: 45 and a light chain comprising SEQ ID NO: 58; 
 xxxv) a heavy chain comprising SEQ ID NO: 45 and a light chain comprising SEQ ID NO: 57; 
 xxxvi) a heavy chain comprising SEQ ID NO: 46 and a light chain comprising SEQ ID NO: 57; 
 xxxvii) a heavy chain comprising SEQ ID NO: 46 and a light chain comprising SEQ ID NO: 59; 
 xxxviii) a heavy chain comprising SEQ ID NO: 47 and a light chain comprising SEQ ID NO: 57; 
 xxxix) a heavy chain comprising SEQ ID NO: 48 and a light chain comprising SEQ ID NO: 57; 
 xl) a heavy chain comprising SEQ ID NO: 49 and a light chain comprising SEQ ID NO: 57; 
 xli) a heavy chain comprising SEQ ID NO: 50 and a light chain comprising SEQ ID NO: 57; 
 xlii) a heavy chain comprising SEQ ID NO: 51 and a light chain comprising SEQ ID NO: 57; 
 xliii) a heavy chain comprising SEQ ID NO: 52 and a light chain comprising SEQ ID NO: 57; 
 xliv) a heavy chain comprising SEQ ID NO: 53 and a light chain comprising SEQ ID NO: 57; or 
 xlv) a heavy chain comprising SEQ ID NO: 54 and a light chain comprising SEQ ID NO: 57. 
 
     
     
         9 . The isolated antibody or antigen-binding fragment thereof according to  claim 1 , wherein the antigen-binding fragment is selected from the group consisting of scFv, Fab, Fab′ fragment, and a F(ab′)2 fragment. 
     
     
         10 . The isolated antibody or antigen-binding fragment thereof according to  claim 1 , which is a monoclonal antibody or antigen-binding fragment thereof. 
     
     
         11 . The isolated antibody or antigen-binding fragment according to  claim 10 , which is a human, humanized or chimeric antibody or antigen-binding fragment thereof. 
     
     
         12 . An isolated antibody or antigen-binding fragment thereof that competes with the isolated antibody or antigen-binding fragment thereof according to  claim 1  for binding to A2AP. 
     
     
         13 . An antibody conjugate, comprising the isolated antibody or antigen binding fragment thereof according to  claim 1 . 
     
     
         14 . An isolated nucleic acid sequence that encodes the isolated antibody or antigen-binding fragment thereof according to  claim 1 . 
     
     
         15 . A vector comprising the nucleic acid sequence according to  claim 14 . 
     
     
         16 . An isolated cell expressing the isolated antibody or antigen-binding fragment thereof according to  claim 1 , a nucleic acid encoding the isolated antibody or antigen-binding fragment thereof, a vector comprising the nucleic acid, or a combination thereof. 
     
     
         17 . (canceled) 
     
     
         18 . A method of producing the isolated antibody or antigen-binding fragment according to  claim 1 , comprising culturing of a cell expressing the isolated antibody or antigen-binding fragment thereof, a nucleic acid encoding the isolated antibody or antigen-binding fragment thereof, a vector comprising the nucleic acid, or a combination thereof and purifying the antibody or antigen-binding fragment thereof. 
     
     
         19 . A pharmaceutical composition comprising the isolated antibody or antigen-binding fragment thereof according  claim 1  or an antibody conjugate comprising the isolated antibody or antigen binding fragment thereof, and one or more pharmaceutically acceptable excipients. 
     
     
         20 . (canceled) 
     
     
         21 . (canceled) 
     
     
         22 . A method for the treatment or prophylaxis of disorders or diseases associated with ischemic events due to partial or complete vessel occlusion, the method comprising administration of a therapeutically effective amount of the isolated antibody or antigen-binding fragment thereof according to  claim 1 . 
     
     
         23 . (canceled) 
     
     
         24 . A kit comprising the isolated antibody or antigen-binding fragment thereof according to  claim 1 , an antibody conjugate comprising the isolated antibody or antigen binding fragment thereof, or the pharmaceutical composition thereof and instructions for use.