Anti-psgl-1 inhibitors and screening methods

Abstract

The present invention is directed to antibodies and binding fragments thereof, which bind with high affinity and specificity to human P-selectin glycoprotein ligand 1 (PSGL-1) and which block both selectin and chemokine binding to PSGL-1 expressed on leukocytes, lymphocytes and endothelial cells and thus which inhibit migration and/or rolling of these cells and to methods for screening for inhibitory compounds including such antibodies and binding fragments thereof and to methods of therapeutic use thereof.

1 . A method of screening for an inhibitory compound that specifically binds to P-selectin glycoprotein ligand-1 (PSGL-1) and blocks binding of P-selectin and/or L-selectin thereto and blocks binding of at least one chemokine thereto, comprising:
 (a) providing a polypeptide, fusion protein, or protein fragment having a binding epitope comprising at least a sulfated portion of amino acids 42-62 of SEQ ID NO:1;   (b) combining the polypeptide, fusion protein, or protein fragment with a test compound to form a treated epitope comprising the polypeptide, fusion protein, or protein fragment which has been exposed to the test compound;   (c) contacting the treated epitope with P-selectin and/or L-selectin, and simultaneously or separately with at least one chemokine; and   (d) assessing the binding of the P-selectin and/or L-selectin and the at least one chemokine to the treated epitope thereby characterizing the ability of the test compound to inhibit binding of the P-selectin and/or L-selectin, and of the at least one chemokine thereto.   
     
     
         2 . The method of  claim 1  wherein the test compound is a human monoclonal antibody or fragment thereof or a humanized antibody or fragment thereof. 
     
     
         3 . The method of  claim 1  wherein the binding epitope comprises a portion of a polypeptide expressed on a surface of a leukocyte, lymphocyte or endothelial cell. 
     
     
         4 . The method of  claim 1  wherein the test compound is determined to have substantially blocked binding of the P-selectin and/or L-selectin and the at least one chemokine to the treated epitope when binding of the P-selectin and/or L-selectin, and the at least one chemokine is at least 75% less than the binding thereof in the presence of a control PSGL-1 binding antibody which binds to PSGL-1 at a site other than said binding epitope. 
     
     
         5 . The method of  claim 1  further comprising a step of selecting the test compound for further evaluation when the binding of the P-selectin and/or L-selectin, and the at least one chemokine to the treated epitope is determined to have been substantially blocked. 
     
     
         6 . The method of  claim 5  comprising the additional step of measuring selectin-mediated adhesion and chemokine-mediated chemotaxis of a leukocyte, lymphocyte, or endothelial cell after the leukocyte, lymphocyte or endothelial cell has been exposed to the test compound. 
     
     
         7 . The method of  claim 1  wherein the inhibitory compound binds to PSGL-1 with a K d  of ≦100 nM. 
     
     
         8 . The method of  claim 1  wherein the inhibitory compound does not activate complement via the classical pathway by interacting with C1Q. 
     
     
         9 . The method of  claim 1  wherein the inhibitory compound does not bind to Fc receptors. 
     
     
         10 . The method of  claim 1  wherein the inhibitory compound does not express effector function as defined by activation of complement or binding of Fc receptors. 
     
     
         11 . The method of  claim 1  wherein the at least one chemokine is selected from the group consisting of CCL19, CCL21, CCL27, and CCL28. 
     
     
         12 . The method of  claim 1  wherein the inhibitory compound is able to block chemotaxis of leukocytes, lymphocytes, or endothelial cells which is mediated by chemokines CCL19, CCL21, CCL27, and/or CCL28.