Anti-gpiib/iiia antibodies and uses thereof

Abstract

Antibodies and antibody fragments that specifically bind to glycoprotein IIb/IIIa (GPIIb/IIIa) are disclosed. Chimeric molecules comprising such antibodies or antigen-binding fragments are also disclosed. In addition, methods of using the disclosed antibodies, antibody fragments, and chimeric molecules, e.g., to target agents to platelets and for the treatment or prevention of diseases or disorders are provided.

1 .- 14 . (canceled) 
     
     
         15 . An antibody or antigen-binding fragment thereof that specifically binds to GPIIb/IIIa, wherein the antibody or the antigen-binding fragment thereof comprises: (i) a heavy chain variable region that is at least 75% identical to the amino acid sequence set forth in any one of SEQ ID NOs:3, 5, 7, 9, 11, 12, or 197-218; and (ii) a light chain variable region that is at least 75% identical to the amino acid sequence set forth in any one of SEQ ID NOs:4, 6, 8, or 10. 
     
     
         16 .- 21 . (canceled) 
     
     
         22 . The antibody or antigen-binding fragment thereof of  claim 15 , wherein the heavy chain variable region comprises complementarity determining region (CDR)1, CDR2, and CDR3, consisting of the amino acid sequences
 (i) AYAMS (SEQ ID NO:25), SISSGGTTYYPDSVKR (SEQ ID NO:26), and GGDYGYALDY (SEQ ID NO:27), respectively;   (ii) AYAMS (SEQ ID NO:25), SISSGGTTYYPDSVKR (SEQ ID NO:26), and GGDYSYALDY (SEQ ID NO:245), respectively;   (iii) AYAMS (SEQ ID NO:25), SISSGGTTYYPDSVER (SEQ ID NO:241), and GGDYSYALDY (SEQ ID NO:245), respectively;   (iv) AYAMS (SEQ ID NO:25), SISSDGTTYYPDSVKR (SEQ ID NO:242), and GGDYSYALDY (SEQ ID NO:245), respectively;   (v) AYAMS (SEQ ID NO:25), SISSGGTTDYPDSVKR (SEQ ID NO:243), and GGDYGYALDY (SEQ ID NO:27), respectively; or   (vi) AYAMS (SEQ ID NO:25), GISSGGTTYYPDSVKR (SEQ ID NO:244), and GGDYGYALDY (SEQ ID NO:27), respectively.   
     
     
         23 .- 27 . (canceled) 
     
     
         28 . The antibody or antigen-binding fragment thereof of  claim 15 , wherein the light chain variable region comprises CDR1, CDR2, and CDR3, consisting of the amino acid sequences RASSSVNYMY (SEQ ID NO:28), YTSNLAP (SEQ ID NO:29), and QQFSSSPWT (SEQ ID NO:30), respectively. 
     
     
         29 . The antibody or antigen-binding fragment thereof of  claim 15 , wherein
 (i) the heavy chain variable region comprises complementarity determining region (CDR)1, CDR2, and CDR3, consisting of the amino acid sequences AYAMS (SEQ ID NO:25), SISSGGTTYYPDSVKR (SEQ ID NO:26), and GGDYGYALDY (SEQ ID NO:27), respectively, and the light chain variable region comprises CDR1, CDR2, and CDR3, consisting of the amino acid sequences RASSSVNYMY (SEQ ID NO:28), YTSNLAP (SEQ ID NO:29), and QQFSSSPWT (SEQ ID NO:30), respectively;   (ii) the heavy chain variable region comprises CDR1, CDR2, and CDR3, consisting of the amino acid sequences AYAMS (SEQ ID NO:25), SISSGGTTYYPDSVKR (SEQ ID NO:26), and GGDYSYALDY (SEQ ID NO:245), respectively, and the light chain variable region comprises CDR1, CDR2, and CDR3, consisting of the amino acid sequences RASSSVNYMY (SEQ ID NO:28), YTSNLAP (SEQ ID NO:29), and QQFSSSPWT (SEQ ID NO:30), respectively;   (iii) the heavy chain variable region comprises CDR1, CDR2, and CDR3, consisting of the amino acid sequences AYAMS (SEQ ID NO:25), SISSGGTTYYPDSVER (SEQ ID NO:241), and GGDYSYALDY (SEQ ID NO:245), respectively, and the light chain variable region comprises CDR1, CDR2, and CDR3, consisting of the amino acid sequences RASSSVNYMY (SEQ ID NO:28), YTSNLAP (SEQ ID NO:29), and QQFSSSPWT (SEQ ID NO:30), respectively;   (iv) the heavy chain variable region comprises CDR1, CDR2, and CDR3, consisting of the amino acid sequences AYAMS (SEQ ID NO:25), SISSDGTTYYPDSVKR (SEQ ID NO:242), and GGDYSYALDY (SEQ ID NO:245), respectively, and the light chain variable region comprises CDR1, CDR2, and CDR3, consisting of the amino acid sequences RASSSVNYMY (SEQ ID NO:28), YTSNLAP (SEQ ID NO:29), and QQFSSSPWT (SEQ ID NO:30), respectively;   (v) the heavy chain variable region comprises CDR1, CDR2, and CDR3, consisting of the amino acid sequences AYAMS (SEQ ID NO:25), SISSGGTTDYPDSVKR (SEQ ID NO:243), and GGDYGYALDY (SEQ ID NO:27), respectively, and the light chain variable region comprises CDR1, CDR2, and CDR3, consisting of the amino acid sequences RASSSVNYMY (SEQ ID NO:28), YTSNLAP (SEQ ID NO:29), and QQFSSSPWT (SEQ ID NO:30), respectively; or   (vi) the heavy chain variable region comprises CDR1, CDR2, and CDR3, consisting of the amino acid sequences AYAMS (SEQ ID NO:25), GISSGGTTYYPDSVKR (SEQ ID NO:244), and GGDYGYALDY (SEQ ID NO:27), respectively, and the light chain variable region comprises CDR1, CDR2, and CDR3, consisting of the amino acid sequences RASSSVNYMY (SEQ ID NO:28), YTSNLAP (SEQ ID NO:29), and QQFSSSPWT (SEQ ID NO:30), respectively.   
     
     
         30 .- 34 . (canceled) 
     
     
         35 . The antibody or antigen-binding fragment thereof of  claim 15 , comprising the heavy chain variable region and light chain variable region amino acid sequences set forth in (i) SEQ ID NO:7 and 4, respectively; (ii) SEQ ID NO:12 and 4, respectively; (iii) SEQ ID NO:197 and 4, respectively; (iv) SEQ ID NO:198 and 4, respectively; (v) SEQ ID NO:199 and 4, respectively; (vi) SEQ ID NO:200 and 4, respectively; (vii) SEQ ID NO:201 and 4, respectively; (viii) SEQ ID NO:202 and 4, respectively; (ix) SEQ ID NO:203 and 4, respectively; (x) SEQ ID NO:204 and 4, respectively; (xi) SEQ ID NO:205 and 4, respectively; (xii) SEQ ID NO:206 and 4, respectively; (xiii) SEQ ID NO:207 and 4, respectively; (xiv) SEQ ID NO:208 and 4, respectively; (xv) SEQ ID NO:209 and 4, respectively; (xvi) SEQ ID NO:210 and 4, respectively; (xvii) SEQ ID NO:211 and 4, respectively; (xviii) SEQ ID NO:212 and 4, respectively; (xix) SEQ ID NO:213 and 4, respectively; (xx) SEQ ID NO:214 and 4, respectively; (xxi) SEQ ID NO:215 and 4, respectively; (xxii) SEQ ID NO:216 and 4, respectively; (xxiii) SEQ ID NO:217 and 4, respectively; or (xxiv) SEQ ID NO:218 and 4, respectively. 
     
     
         36 . (canceled) 
     
     
         37 . An antibody or antigen-binding fragment thereof that specifically binds to glycoprotein IIb/IIIa (GPIIb/IIIa), wherein the antibody or the antigen-binding fragment thereof comprises:
 (i) a heavy chain variable region comprising complementarity determining region (CDR)1, CDR2, and CDR3, consisting of the amino acid sequences:
 (a) AYAMS (SEQ ID NO:25), SISSGGTTYYPDSVER (SEQ ID NO:26), and GGDYSYALDY (SEQ ID NO:245), respectively; 
 (b) AYAMS (SEQ ID NO:25), SISSGGTTYYPDSVER (SEQ ID NO:241), and GGDYSYALDY (SEQ ID NO:245), respectively; 
 (c) AYAMS (SEQ ID NO:25), SISSDGTTYYPDSVKR (SEQ ID NO:242), and GGDYSYALDY (SEQ ID NO:245), respectively; 
 (d) AYAMS (SEQ ID NO:25), SISSGGTTDYPDSVKR (SEQ ID NO:243), and GGDYGYALDY (SEQ ID NO:27), respectively; or 
 (e) AYAMS (SEQ ID NO:25), GISSGGTTYYPDSVKR (SEQ ID NO:244), and GGDYGYALDY (SEQ ID NO:27), respectively; and 
   (ii) a light chain variable region comprising complementarity determining region (CDR)1, CDR2, and CDR3, consisting of the amino acid sequences:
 (f) RASSSVNYMY (SEQ ID NO:28), YTSNLAP (SEQ ID NO:29), and QQFSSSPWT (SEQ ID NO:30), respectively. 
   
     
     
         38 . The antibody or antigen-binding fragment thereof of  claim 37 , wherein the antibody or antigen-binding fragment thereof has an apparent monovalent affinity that is about 1 to about 5×10 −8 M. 
     
     
         39 . The antibody or the antigen-binding fragment thereof of  claim 15 , wherein the antigen-binding fragment is selected from the group consisting of an Fab, an Fab′, an F(ab′)2, an Fv, an Fd, a diabody, an scFv, and an sc(Fv)2. 
     
     
         40 . A chimeric molecule comprising (i) the antibody or antigen-binding fragment thereof of  claim 15 , and (ii) a heterologous moiety. 
     
     
         41 . The chimeric molecule of  claim 40 , wherein the heterologous moiety comprises a clotting factor. 
     
     
         42 .- 44 . (canceled) 
     
     
         45 . The chimeric molecule of  claim 40 , further comprising a linker. 
     
     
         46 .- 48 . (canceled) 
     
     
         49 . The chimeric molecule of  claim 40 , further comprising a second heterologous moiety. 
     
     
         50 . The chimeric molecule according to  claim 49 , wherein the second heterologous moiety comprises a half-life extending moiety. 
     
     
         51 .- 52 . (canceled) 
     
     
         53 . A chimeric molecule comprising (i) the antibody or antigen-binding fragment thereof of  claim 15 , (ii) a Factor VII comprising a heavy chain and a light chain, and (iii) a half-life extending moiety. 
     
     
         54 . (canceled) 
     
     
         55 . The chimeric molecule of  claim 53 , wherein the heavy chain of the Factor VII is linked to the half-life extending moiety and the half-life extending moiety is linked to the antibody or antigen-binding fragment thereof. 
     
     
         56 .- 58 . (canceled) 
     
     
         59 . A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of  claim 15 , and a pharmaceutically acceptable carrier. 
     
     
         60 . A method of reducing the frequency or degree of a bleeding episode in a human subject in need thereof, comprising administering to the human subject an effective amount of the antibody or antigen-binding fragment thereof  claim 15 . 
     
     
         61 . The method of  claim 60 , wherein the subject has developed or has a tendency to develop an inhibitor against Factor VIII (“FVIII”), Factor IX (“FIX”), or both. 
     
     
         62 . The method of  claim 61 , wherein the inhibitor against FVIII or FIX is a neutralizing antibody against FVIII, FIX, or both. 
     
     
         63 . The method of  claim 60 , wherein the bleeding episode is a result of hemarthrosis, muscle bleed, oral bleed, hemorrhage, hemorrhage into muscles, oral hemorrhage, trauma, trauma capitis, gastrointestinal bleeding, intracranial hemorrhage, intra-abdominal hemorrhage, intrathoracic hemorrhage, bone fracture, central nervous system bleeding, bleeding in the retropharyngeal space, bleeding in the retroperitoneal space, bleeding in the illiopsoas sheath, or any combinations thereof. 
     
     
         64 . A method of treating a blood coagulation disorder in a human subject in need thereof, comprising administering to the human subject an effective amount of the antibody or antigen-binding fragment thereof of  claim 15 . 
     
     
         65 . The method of  claim 64 , wherein the blood coagulation disorder is hemophilia A or hemophilia B. 
     
     
         66 . (canceled) 
     
     
         67 . A method of detecting platelets, comprising:
 contacting a human blood preparation with the antibody or antigen-binding fragment thereof of  claim 15 ; and   detecting cells in the blood preparation to which the antibody or antigen-binding fragment thereof binds.   
     
     
         68 . A method for enriching platelets, comprising:
 contacting a human blood preparation with the antibody or antigen-binding fragment thereof of  claim 15 ; and   enriching cells to which the antibody or antigen-binding fragment thereof are bound as compared to those cells in the blood preparation that are not bound by the antibody or antigen-binding fragment thereof.   
     
     
         69 . An isolated nucleic acid comprising a nucleotide sequence that is identical to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 13-22, 59-68, and 219-240. 
     
     
         70 . (canceled) 
     
     
         71 . An isolated nucleic acid comprising a nucleotide sequence that encodes a polypeptide comprising an amino acid sequence that is identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 3 to 12, 74 to 77, and 197-218. 
     
     
         72 . (canceled) 
     
     
         73 . An isolated protein encoded by the nucleic acid of  claim 69 . 
     
     
         74 . A recombinant vector comprising the nucleic acid of  claim 69 . 
     
     
         75 . A host cell comprising the recombinant vector of  claim 74 . 
     
     
         76 . A method of preparing an antibody or antigen-binding fragment thereof, the method comprising culturing a host cell comprising a recombinant vector or recombinant vectors comprising:
 the nucleic acid sequences set forth in SEQ ID NOs: 14 and 19;   the nucleic acid sequences set forth in SEQ ID NOs: 15 and 19;   the nucleic acid sequences set forth in SEQ ID NOs: 16 and 19;   the nucleic acid sequences set forth in SEQ ID NOs: 17 and 19;   the nucleic acid sequences set forth in SEQ ID NOs: 18 and 19;   the nucleic acid sequences set forth in SEQ ID NOs: 13 and 22;   the nucleic acid sequences set forth in SEQ ID NOs: 14 and 22;   the nucleic acid sequences set forth in SEQ ID NOs: 15 and 22;   the nucleic acid sequences set forth in SEQ ID NOs: 16 and 22;   the nucleic acid sequences set forth in SEQ ID NOs: 17 and 22;   the nucleic acid sequences set forth in SEQ ID NOs: 18 and 22;   the nucleic acid sequences set forth in SEQ ID NOs: 18 and 21;   the nucleic acid sequences set forth in SEQ ID NOs: 219 and 19;   the nucleic acid sequences set forth in SEQ ID NOs: 220 and 19;   the nucleic acid sequences set forth in SEQ ID NOs: 221 and 19;   the nucleic acid sequences set forth in SEQ ID NOs: 222 and 19;   the nucleic acid sequences set forth in SEQ ID NOs: 223 and 19;   the nucleic acid sequences set forth in SEQ ID NOs: 224 and 19;   the nucleic acid sequences set forth in SEQ ID NOs: 225 and 19;   the nucleic acid sequences set forth in SEQ ID NOs: 226 and 19;   the nucleic acid sequences set forth in SEQ ID NOs: 227 and 19;   the nucleic acid sequences set forth in SEQ ID NOs: 228 and 19;   the nucleic acid sequences set forth in SEQ ID NOs: 229 and 19;   the nucleic acid sequences set forth in SEQ ID NOs: 230 and 19;   the nucleic acid sequences set forth in SEQ ID NOs: 231 and 19;   the nucleic acid sequences set forth in SEQ ID NOs: 232 and 19;   the nucleic acid sequences set forth in SEQ ID NOs: 233 and 19;   the nucleic acid sequences set forth in SEQ ID NOs: 234 and 19;   the nucleic acid sequences set forth in SEQ ID NOs: 235 and 19;   the nucleic acid sequences set forth in SEQ ID NOs: 236 and 19;   the nucleic acid sequences set forth in SEQ ID NOs: 237 and 19;   the nucleic acid sequences set forth in SEQ ID NOs: 238 and 19;   the nucleic acid sequences set forth in SEQ ID NOs: 239 and 19; or   the nucleic acid sequences set forth in SEQ ID NOs: 240 and 19,   under conditions appropriate for expression and production of the antibody or antigen-binding fragment thereof.   
     
     
         77 . The method of  claim 76 , further comprising isolating the antibody or antigen-binding fragment thereof. 
     
     
         78 . A method of preparing a chimeric molecule, the method comprising culturing a host cell comprising a recombinant vector or recombinant vectors comprising:
 the nucleic acid sequences encoding the amino acid sequences set forth in SEQ ID NOs: 77 and 75; or   the nucleic acid sequences encoding the amino acid sequences set forth in SEQ ID NOs: 77 and 76;   under conditions appropriate for expression and production of the chimeric molecule.   
     
     
         79 . The method of  claim 76 , wherein the host cell is a 293 cell, a CHO cell or a DG44i cell. 
     
     
         80 . A method of preparing a chimeric molecule, the method comprising culturing a host cell comprising a recombinant vector or vectors comprising the nucleic acid sequences encoding the amino acid sequences set forth in SEQ ID NOs: 247 and 75 under conditions appropriate for expression and production of the chimeric molecule. 
     
     
         81 . The method of  claim 80 , wherein the host cell is a 293 cell, a CHO cell or a DG44i cell. 
     
     
         82 . The antibody or the antigen-binding fragment thereof of  claim 37 , wherein the antigen-binding fragment is selected from the group consisting of an Fab, an Fab′, an F(ab′)2, an Fv, an Fd, a diabody, an scFv, and an sc(Fv)2. 
     
     
         83 . A chimeric molecule comprising (i) the antibody or antigen-binding fragment thereof of  claim 37 , and (ii) a heterologous moiety. 
     
     
         84 . A chimeric molecule comprising (i) the antibody or antigen-binding fragment thereof of  claim 37 , (ii) a Factor VII comprising a heavy chain and a light chain, and (iii) a half-life extending moiety. 
     
     
         85 . A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of  claim 37 , and a pharmaceutically acceptable carrier. 
     
     
         86 . A pharmaceutical composition comprising the chimeric molecule of  claim 40 , and a pharmaceutically acceptable carrier. 
     
     
         87 . A method of reducing the frequency or degree of a bleeding episode in a human subject in need thereof, comprising administering to the human subject an effective amount of the chimeric molecule of  claim 53 . 
     
     
         88 . A method of reducing the frequency or degree of a bleeding episode in a human subject in need thereof, comprising administering to the human subject an effective amount of the pharmaceutical composition of  claim 85 . 
     
     
         89 . A method of reducing the frequency or degree of a bleeding episode in a human subject in need thereof, comprising administering to the human subject an effective amount of the pharmaceutical composition of  claim 86 . 
     
     
         90 . A method of treating a blood coagulation disorder in a human subject in need thereof, comprising administering to the human subject an effective amount of the antibody or antigen-binding fragment thereof of  claim 37 . 
     
     
         91 . A method of treating a blood coagulation disorder in a human subject in need thereof, comprising administering to the human subject an effective amount of the chimeric molecule of  claim 53 . 
     
     
         92 . A method of treating a blood coagulation disorder in a human subject in need thereof, comprising administering to the human subject an effective amount of the pharmaceutical composition of  claim 85 . 
     
     
         93 . A method of treating a blood coagulation disorder in a human subject in need thereof, comprising administering to the human subject an effective amount of the pharmaceutical composition of  claim 86 . 
     
     
         94 . An isolated protein encoded by the nucleic acid of  claim 71 . 
     
     
         95 . A recombinant vector comprising the nucleic acid of  claim 71 . 
     
     
         96 . A host cell comprising the recombinant vector of  claim 95 . 
     
     
         97 . The method of  claim 78 , wherein the host cell is a 293 cell, a CHO cell or a DG44i cell.