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 . A method of treating or inhibiting an inflammatory or thrombotic condition or disorder in a subject in need of such therapy, comprising: administering to the subject a therapeutically-effective amount of an antibody or binding fragment thereof which specifically binds to a conformational epitope of P-selectin which is within amino acid positions 1-35 of SEQ ID NO:1. 
     
     
         2 . The method of  claim 1  wherein the conformational epitope is within amino acid positions 4-23 of SEQ ID NO: 1. 
     
     
         3 . The method of  claim 1  wherein the conformational epitope comprises amino acid positions 4, 14, 17, 21, and 22 of SEQ ID NO: 1. 
     
     
         4 . The method 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. 
     
     
         5 . The method of  claim 4  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. 
     
     
         6 . The method of  claim 3  wherein the conformational epitope further comprises amino acid positions 20 and 23 of SEQ ID NO: 1. 
     
     
         7 . The method of  claim 1  wherein the antibody or binding fragment thereof comprises the ability to block the binding of P-selectin glycoprotein ligand-1 (PSGL-1) to P-selectin. 
     
     
         8 . The method of  claim 7  wherein the antibody or binding fragment thereof further comprises the ability to cause dissociation of a preformed P-selectin-PSGL-1. 
     
     
         9 . The method of  claim 8  wherein the antibody or binding fragment of  claim 1  comprises the ability to cause dissociation of cell-cell binding between activated endothelial cells and leukocytes, lymphocytes, sickled red cells, and/or platelets. 
     
     
         10 . The method of  claim 8  wherein the antibody or binding fragment of  claim 1  comprises the ability to cause dissociation of cell-cell binding between activated platelets and leukocytes, lymphocytes, sickled red cells, and/or platelets. 
     
     
         11 .- 18 . (canceled) 
     
     
         19 . The method of  claim 1  wherein the inflammatory or thrombotic condition or disorder is related to at least one of vasoocculsive sickle cell pain crisis, thrombosis, atherosclerosis, allergic reactions, thyroiditis, psoriasis, dermatitis, nephritis, lupus erythematosis, scleroderma, sepsis, rhinitis, anaphylaxis, diabetes, multiple sclerosis, graft rejection, graft vs. host disease, asthma, chronic obstructive pulmonary disease, inflammatory bowel disease, arthritis, and ischemic reperfusion\ injury conditions associated with extensive trauma, or chronic inflammation, such as for example, type IV delayed hypersensitivity, associated for example with infection by Tubercle bacilli, or systematic inflammatory response syndrome, or multiple organ failure. 
     
     
         20 . The method of  claim 19  wherein the ischemic reperfusion injury is caused by at least one of myocardial infarction, stroke, and organ transplantation. 
     
     
         21 . The method of  claim 1  wherein said antibody is administered to the subject parenterally, intramuscularly, intra peritoneally, epidurally, or orally, intravenously, subcutaneously, or in a nebulized form. 
     
     
         22 . The method of  claim 1  wherein said antibody or binding fragment is administered to the subject an amount of 1 ng/kg to 100 mg/kg.