Anti-p-selectin antibodies and methods of their use and identification

Abstract

Antibodies are disclosed which bind specifically to P-selectin and which block the binding of PSGL-1 to P-selectin. These anti-P-selectin antibodies may also cause dissociation of preformed P-selectin/PSGL-1 complexes. The disclosure identifies a heretofore unrecognized, near N-terminal, antibody binding domain (a conformational epitope) of P-selectin to which the function-blocking antibodies (which may be chimeric, human or humanized antibodies for example) bind. Antibodies are disclosed which bind to the conformational epitope of P-selectin and which have a dual function in blocking binding of PSGL-1 to P-selectin, and in causing dissociation of preformed P-selectin/PSGL-1 complexes. Such single and dual function anti-P-selectin antibodies and binding fragments thereof may be used in the treatment of a variety of inflammatory and thrombotic disorders and conditions. Screening methods for identifying such antibodies are also disclosed.

1 . An isolated antibody or binding fragment thereof which specifically binds to a conformational epitope of P-selectin, wherein the conformational epitope is within amino acid positions 1-35 of SEQ ID NO:1. 
     
     
         2 . The isolated antibody or binding fragment of  claim 1  wherein the conformational epitope is within amino acids 4-23 of SEQ ID NO:1. 
     
     
         3 . The isolated antibody or binding fragment of  claim 1  wherein the conformational epitope comprises amino acid positions 4, 14, 17, 21, and 22 of SEQ ID NO:1. 
     
     
         4 . The isolated antibody or binding fragment of  claim 3  wherein the conformational epitope further comprises amino acid positions 20 and 23 of SEQ ID NO:1. 
     
     
         5 . The isolated antibody or binding fragment of  claim 3  wherein the amino acids in amino acid positions 4, 14, 17, 21, and 22 are H, I, K, N, and R, respectively. 
     
     
         6 . The isolated antibody or binding fragment thereof of  claim 3  wherein binding is abrogated when any one or more of amino acid positions 4, 14, 17, 21, or 22 is substituted with N,N, V, R, or H, respectively. 
     
     
         7 . The isolated antibody or binding fragment of  claim 1  comprising the ability to block the binding of P-selectin glycoprotein ligand-1 (PSGL-1) to P-selectin. 
     
     
         8 . The isolated antibody or binding fragment of  claim 5  further comprising the ability to cause dissociation of a preformed P-selectin-PSGL-1 complex. 
     
     
         9 . The isolated antibody or binding fragment of  claim 1  comprising the ability to block the function of P-selectin by inhibiting the binding of activated endothelial cells to leukocytes, lymphocytes, sickled red cells, and/or platelets. 
     
     
         10 . The isolated antibody or binding fragment of  claim 1  comprising the ability to block the function of P-selectin by inhibiting the binding of activated platelets to leukocytes, lymphocytes, sickled red cells, and/or platelets. 
     
     
         11 . The isolated antibody or binding fragment of  claim 1  comprising the ability to cause dissociation of cell-cell binding between activated endothelial cells and leukocytes, lymphocytes, sickled red cells, and/or platelets. 
     
     
         12 . The isolated antibody or binding fragment of  claim 1  comprising the ability to cause dissociation of cell-cell binding between activated platelets and leukocytes, lymphocytes, sickled red cells, and/or platelets. 
     
     
         13 . The isolated antibody or binding fragment of  claim 1  wherein the antibody or fragment thereof is monoclonal. 
     
     
         14 . The isolated antibody or binding fragment of  claim 1  wherein the antibody or fragment thereof is chimeric, human, or humanized. 
     
     
         15 . The isolated antibody or binding fragment of  claim 1  comprising an immunoglobulin selected from the class consisting of IgA, IgD, IgE, IgG, and IgM. 
     
     
         16 . The isolated antibody or binding fragment of  claim 15  wherein the isolated antibody or binding fragment thereof is an IgG selected from an isotype consisting of IgG1, IgG2, IgG3, IgG4, or an IgG2/G4 chimera. 
     
     
         17 . The binding fragment of  claim 1  comprising at least one of a Fab, Fab′, F(ab)2, or scFv fragment. 
     
     
         18 . The isolated antibody or binding fragment of  claim 1  which binds to the conformational epitope with a K d ≦1000 nM, a K d ≦500 nM, a K d ≦100 nM, a K d ≦50 nM, a K d ≦25 nM, a K d <10 nM, a K d ≦5 nM, a K d ≦1 nM, or a K d ≦0.1 nM. 
     
     
         19 . A composition comprising the isolated antibody or binding fragment of  claim 1  disposed within a pharmaceutically-acceptable carrier, vehicle, or diluent. 
     
     
         20 . A nucleic acid which encodes the antibody or binding fragment thereof of  claim 1 . 
     
     
         21 . A cell line which expresses the antibody or binding fragment of  claim 1 .