Methods of treating inflammatory or thrombotic conditions with anti-P-selectin antibodies
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,
wherein the antibody or binding fragment thereof has the ability to block the binding of P-selectin glycoprotein ligand-1 (PSGL-1) to P-selectin and the ability to cause dissociation of a preformed P-selectin-PSGL-1 complex;
wherein the amino acids in amino acid positions 4, 14, 17, 21, and 22 are H, I, K, N, and R, respectively;
provided that the antibody or binding fragment thereof is not G1 antibody, the G1 antibody comprising variable light chains each individually having an amino acid sequence selected from the group consisting of SEQ ID NOs: 47, 51, 53, 55, and 67, and further comprising variable heavy chains each individually having an amino acid sequence selected from the group consisting of SEQ ID NOs: 61, 65, and 69;
further provided that the antibody or binding fragment thereof is not hSel001 antibody comprising variable light chains each individually having an amino acid sequence of SEQ ID NO:40 and variable heavy chains each individually having an amino acid sequence of SEQ ID NO:44; further provided that the antibody or binding fragment thereof is not a binding fragment of the G1 antibody; or a binding fragment of the hSel001 antibody.
2. The method of claim 1 wherein the conformational epitope is within amino acids 4-23 of SEQ ID NO:1.
3. The method of claim 1 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.
4. The method of claim 1 wherein the conformational epitope further comprises amino acid positions 20 and 23 of SEQ ID NO:1.
5. The method of claim 1 wherein the antibody or binding fragment thereof has the ability to cause dissociation of cell-cell binding between activated endothelial cells and leukocytes, lymphocytes, sickled red cells, and/or platelets.
6. The method of claim 1 wherein the antibody or binding fragment thereof has the ability to cause dissociation of cell-cell binding between activated platelets and leukocytes, lymphocytes, sickled red cells, and/or platelets.
7. The method of claim 1 wherein the antibody or binding fragment thereof has 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.
8. The method of claim 1 wherein the antibody or binding fragment thereof has 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.
9. The method of claim 1 wherein the antibody or binding fragment thereof is monoclonal.
10. The method of claim 1 wherein the antibody or fragment thereof is chimeric, human, or humanized.
11. The method of claim 1 wherein the antibody or binding fragment thereof comprises an immunoglobulin selected from the class consisting of IgA, IgD, IgE, IgG, and IgM.
12. The method of claim 11 wherein the antibody or fragment thereof is IgG1, IgG2, IgG3, IgG4, or an IgG2/G4 chimera.
13. The method of claim 1 wherein the binding fragment comprises at least one of a Fab, Fab′, F(ab) 2 , or scFv fragment.
14. The method of claim 1 wherein the antibody or binding fragment thereof binds to the conformational epitope with a Kd≦1000 μM, a Kd≦500 nM, a Kd≦100 nM, a Kd≦50 nM, a Kd≦25 nM, a Kd≦10 nM, a Kd≦5 nM, a Kd≦1 nM, or a Kd≦0.1 nM.
15. The method of claim 1 wherein the inflammatory or thrombotic condition or disorder is related to at least one of vasoocclusive sickle cell pain crisis, tumor metastasis, 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, type IV delayed hypersensitivity, systematic inflammatory response syndrome, or multiple organ failure.
16. The method of claim 15 wherein the ischemic reperfusion injury is caused by at least one of myocardial infarction, stroke, and organ transplantation.
17. The method of claim 1 wherein said antibody is administered to the subject parenterally, intramuscularly, intraperitoneally, epidurally, or orally, intravenously, subcutaneously, or in a nebulized form.
18. The method of claim 1 wherein said antibody or binding fragment is administered to the subject in an amount of 1 ng/kg to 100 mg/kg.