Methods for Treating Diseases and HSV Using Antibodies to Aminophospholipids

Abstract

Disclosed are surprising discoveries concerning the role of anionic phospholipids and aminophospholipids in tumor vasculature and in viral entry and spread, and compositions and methods for utilizing these findings in the treatment of cancer and viral infections. Also disclosed are advantageous antibody, immunoconjugate and duramycin-based compositions and combinations that bind and inhibit anionic phospholipids and aminophospholipids, for use in the safe and effective treatment of cancer, viral infections and related diseases.

What is claimed is: 
     
         1 . A method of quantifying viral load in a biological sample, comprising contacting said biological sample with a first antibody, or an antigen-binding fragment thereof, in an amount effective to bind viruses in said biological sample and quantifying the bound viruses; wherein said first antibody binds to phosphatidylserine on the luminal surface of tumor vascular endothelial cells when administered to an animal with a solid tumor and wherein said first antibody exhibits significant binding to an ELISA plate coated with phosphatidylserine in an ELISA conducted in the presence of serum, but no detectable binding to an ELISA plate coated with phosphatidylcholine in an ELISA conducted in the presence of serum, wherein said ELISA conducted in the presence of serum comprises the steps of:
 (a) coating a first ELISA plate with phosphatidylserine to prepare a PS-coated ELISA plate and coating a second ELISA plate with phosphatidylcholine to prepare a PC-coated ELISA plate;   (b) blocking said PS-coated ELISA plate and said PC-coated ELISA plate with a blocking buffer comprising 10% serum;   (c) adding said first antibody diluted in said blocking buffer to said PS-coated ELISA plate and said PC-coated ELISA plate under conditions effective to allow binding of said first antibody to said PS-coated ELISA plate and said PC-coated ELISA plate in the presence of serum; and   (d) detecting the binding of said first antibody to said PS-coated ELISA plate and said PC-coated ELISA plate in the presence of serum using a secondary antibody that binds to said first antibody; wherein said first antibody exhibits significant binding to said PS-coated ELISA plate, but no detectable binding to said PC-coated ELISA plate.   
     
     
         2 . The method of  claim 1 , wherein said first antibody effectively competes with the 3G4 antibody (monoclonal antibody 3G4 deposited as ATCC PTA 4545) in binding to an ELISA plate coated with phosphatidylserine in a competition ELISA conducted in the presence of serum, wherein said competition ELISA comprises:
 (a) coating an ELISA plate with phosphatidylserine to prepare a PS-coated ELISA plate;   (b) blocking said PS-coated ELISA plate with a blocking buffer comprising 10% serum;   (c) adding said 3G4 antibody diluted in said blocking buffer to said PS-coated ELISA plate under conditions effective to allow binding of said 3G4 antibody to said PS-coated ELISA plate in the presence of serum;   (d) detecting the binding of said 3G4 antibody to said PS-coated ELISA plate in the presence of serum using a secondary antibody that binds to said 3G4 antibody; and   (e) identifying a first antibody that effectively competes with said 3G4 antibody in said competition ELISA by selecting a first antibody that substantially reduces the binding of said 3G4 antibody to said PS-coated ELISA plate in the presence of serum.   
     
     
         3 . The method of  claim 1 , wherein said antibody is bound to a solid support. 
     
     
         4 . The method of  claim 1 , wherein said biological sample is a blood sample. 
     
     
         5 . A method of purging a virus from a biological sample, comprising contacting said biological sample with a first antibody, or an antigen-binding fragment thereof, in an amount effective to bind and remove said virus from said biological sample; wherein said first antibody binds to phosphatidylserine on the luminal surface of tumor vascular endothelial cells when administered to an animal with a solid tumor and wherein said first antibody exhibits significant binding to an ELISA plate coated with phosphatidylserine in an ELISA conducted in the presence of serum, but no detectable binding to an ELISA plate coated with phosphatidylcholine in an ELISA conducted in the presence of serum, wherein said ELISA conducted in the presence of serum comprises the steps of:
 (a) coating a first ELISA plate with phosphatidylserine to prepare a PS-coated ELISA plate and coating a second ELISA plate with phosphatidylcholine to prepare a PC-coated ELISA plate;   (b) blocking said PS-coated ELISA plate and said PC-coated ELISA plate with a blocking buffer comprising 10% serum;   (c) adding said first antibody diluted in said blocking buffer to said PS-coated ELISA plate and said PC-coated ELISA plate under conditions effective to allow binding of said first antibody to said PS-coated ELISA plate and said PC-coated ELISA plate in the presence of serum; and   (d) detecting the binding of said first antibody to said PS-coated ELISA plate and said PC-coated ELISA plate in the presence of serum using a secondary antibody that binds to said first antibody; wherein said first antibody exhibits significant binding to said PS-coated ELISA plate, but no detectable binding to said PC-coated ELISA plate.   
     
     
         6 . The method of  claim 5 , wherein said first antibody effectively competes with the 3G4 antibody (monoclonal antibody 3G4 deposited as ATCC PTA 4545) in binding to an ELISA plate coated with phosphatidylserine in a competition ELISA conducted in the presence of serum, wherein said competition ELISA comprises:
 (a) coating an ELISA plate with phosphatidylserine to prepare a PS-coated ELISA plate;   (b) blocking said PS-coated ELISA plate with a blocking buffer comprising 10% serum;   (c) adding said 3G4 antibody diluted in said blocking buffer to said PS-coated ELISA plate under conditions effective to allow binding of said 3G4 antibody to said PS-coated ELISA plate in the presence of serum;   (d) detecting the binding of said 3G4 antibody to said PS-coated ELISA plate in the presence of serum using a secondary antibody that binds to said 3G4 antibody; and   (e) identifying a first antibody that effectively competes with said 3G4 antibody in said competition ELISA by selecting a first antibody that substantially reduces the binding of said 3G4 antibody to said PS-coated ELISA plate in the presence of serum.   
     
     
         7 . The method of  claim 5 , wherein said antibody is bound to a solid support. 
     
     
         8 . The method of  claim 7 , wherein said method is an affinity chromatography method. 
     
     
         9 . The method of  claim 5 , wherein said biological sample is a blood sample. 
     
     
         10 . A method for treating a mammal with septic shock, comprising administering to said mammal a pharmaceutical composition comprising a first antibody, or an antigen-binding fragment thereof, in an amount effective to treat said septic shock; wherein said first antibody effectively competes with the 3G4 antibody (monoclonal antibody 3G4 deposited as ATCC PTA 4545) in binding to an ELISA plate coated with phosphatidylserine in a competition ELISA conducted in the presence of serum, wherein said competition ELISA comprises:
 (a) coating an ELISA plate with phosphatidylserine to prepare a PS-coated ELISA plate;   (b) blocking said PS-coated ELISA plate with a blocking buffer comprising 10% serum;   (c) adding said 3G4 antibody diluted in said blocking buffer to said PS-coated ELISA plate under conditions effective to allow binding of said 3G4 antibody to said PS-coated ELISA plate in the presence of serum;   (d) detecting the binding of said 3G4 antibody to said PS-coated ELISA plate in the presence of serum using a secondary antibody that binds to said 3G4 antibody; and   (e) identifying a first antibody that effectively competes with said 3G4 antibody in said competition ELISA by selecting a first antibody that substantially reduces the binding of said 3G4 antibody to said PS-coated ELISA plate in the presence of serum.   
     
     
         11 . The method of  claim 10 , wherein said first antibody is a human, humanized, part-human, chimeric, recombinant or engineered antibody. 
     
     
         12 . The method of  claim 10 , wherein said antibody is an Fab dimer of said 3G4 antibody. 
     
     
         13 . The method of  claim 10 , wherein said mammal is a human patient. 
     
     
         14 . A method for treating a mammal with sickle cell anaemia, comprising administering to said mammal a pharmaceutical composition comprising a first antibody, or an antigen-binding fragment thereof, in an amount effective to treat said sickle cell anaemia; wherein said first antibody effectively competes with the 3G4 antibody (monoclonal antibody 3G4 deposited as ATCC PTA 4545) in binding to an ELISA plate coated with phosphatidylserine in a competition ELISA conducted in the presence of serum, wherein said competition ELISA comprises:
 (a) coating an ELISA plate with phosphatidylserine to prepare a PS-coated ELISA plate;   (b) blocking said PS-coated ELISA plate with a blocking buffer comprising 10% serum;   (c) adding said 3G4 antibody diluted in said blocking buffer to said PS-coated ELISA plate under conditions effective to allow binding of said 3G4 antibody to said PS-coated ELISA plate in the presence of serum;   (d) detecting the binding of said 3G4 antibody to said PS-coated ELISA plate in the presence of serum using a secondary antibody that binds to said 3G4 antibody; and   (e) identifying a first antibody that effectively competes with said 3G4 antibody in said competition ELISA by selecting a first antibody that substantially reduces the binding of said 3G4 antibody to said PS-coated ELISA plate in the presence of serum.   
     
     
         15 . The method of  claim 14 , wherein said first antibody is a human, humanized, part-human, chimeric, recombinant or engineered antibody. 
     
     
         16 . The method of  claim 14 , wherein said antibody is an Fab dimer of said 3G4 antibody. 
     
     
         17 . The method of  claim 14 , wherein said mammal is a human patient. 
     
     
         18 . A method for treating a mammal with a protozoan infection, comprising administering to said mammal a pharmaceutical composition comprising a first antibody, or an antigen-binding fragment thereof, in an amount effective to treat said protozoan infection; wherein said first antibody effectively competes with the 3G4 antibody (monoclonal antibody 3G4 deposited as ATCC PTA 4545) in binding to an ELISA plate coated with phosphatidylserine in a competition ELISA conducted in the presence of serum, wherein said competition ELISA comprises:
 (a) coating an ELISA plate with phosphatidylserine to prepare a PS-coated ELISA plate;   (b) blocking said PS-coated ELISA plate with a blocking buffer comprising 10% serum;   (c) adding said 3G4 antibody diluted in said blocking buffer to said PS-coated ELISA plate under conditions effective to allow binding of said 3G4 antibody to said PS-coated ELISA plate in the presence of serum;   (d) detecting the binding of said 3G4 antibody to said PS-coated ELISA plate in the presence of serum using a secondary antibody that hinds to said 3G4 antibody; and   (e) identifying a first antibody that effectively competes with said 3G4 antibody in said competition ELISA by selecting a first antibody that substantially reduces the binding of said 3G4 antibody to said PS-coated ELISA plate in the presence of serum.   
     
     
         19 . The method of  claim 18 , wherein said first antibody is a human, humanized, part-human, chimeric, recombinant or engineered antibody. 
     
     
         20 . The method of  claim 18 , wherein said mammal is a human patient. 
     
     
         21 . A method for treating a mammal with antiphospholipid syndrome, comprising administering to said mammal a pharmaceutical composition comprising a first antibody, or an antigen-binding fragment thereof, in an amount effective to treat said antiphospholipid syndrome; wherein said first antibody effectively competes with the 3G4 antibody (monoclonal antibody 3G4 deposited as ATCC PTA 4545) in binding to an ELISA plate coated with phosphatidylserine in a competition ELISA conducted in the presence of serum, wherein said competition ELISA comprises:
 (a) coating an ELISA plate with phosphatidylserine to prepare a PS-coated ELISA plate;   (b) blocking said PS-coated ELISA plate with a blocking buffer comprising 10% serum;   (c) adding said 3G4 antibody diluted in said blocking buffer to said PS-coated ELISA plate under conditions effective to allow binding of said 3G4 antibody to said PS-coated ELISA plate in the presence of serum;   (d) detecting the binding of said 3G4 antibody to said PS-coated ELISA plate in the presence of serum using a secondary antibody that binds to said 3G4 antibody; and   (e) identifying a first antibody that effectively competes with said 3G4 antibody in said competition ELISA by selecting a first antibody that substantially reduces the binding of said 3G4 antibody to said PS-coated ELISA plate in the presence of serum.   
     
     
         22 . The method of  claim 21 , wherein said first antibody is a human, humanized, part-human, chimeric, recombinant or engineered antibody. 
     
     
         23 . The method of  claim 21 , wherein said mammal is a human patient. 
     
     
         24 . A method for competing with a pathogenic antibody from a patient with antiphospholipid syndrome for binding to its phospholipid-protein target, comprising contacting said pathogenic antibody bound to said phospholipid-protein target with a first antibody, or an antigen-binding fragment thereof, which displaces said pathogenic antibody from said phospholipid-protein target; wherein said phospholipid-protein target is a phosphatidylserine (PS)-β 2 -glycoprotein I target and wherein said first antibody effectively competes with the 3G4 antibody (monoclonal antibody 3G4 deposited as ATCC PTA 4545) in binding to an ELISA plate coated with phosphatidylserine in a competition ELISA conducted in the presence of serum, wherein said competition ELISA comprises:
 (a) coating an ELISA plate with phosphatidylserine to prepare a PS-coated ELISA plate; 
 (b) blocking said PS-coated ELISA plate with a blocking buffer comprising 10% serum; 
 (c) adding said 3G4 antibody diluted in said blocking buffer to said PS-coated ELISA plate under conditions effective to allow binding of said 3G4 antibody to said PS-coated ELISA plate in the presence of serum; 
 (d) detecting the binding of said 3G4 antibody to said PS-coated ELISA plate in the presence of serum using a secondary antibody that binds to said 3G4 antibody; and 
 (e) identifying a first antibody that effectively competes with said 3G4 antibody in said competition ELISA by selecting a first antibody that substantially reduces the binding of said 3G4 antibody to said PS-coated ELISA plate in the presence of serum. 
 
     
     
         25 . The method of  claim 24 , wherein said first antibody is a human, humanized, part-human, chimeric, recombinant or engineered antibody. 
     
     
         26 . A method for treating a mammal with a herpes virus infection, comprising administering to said mammal a pharmaceutical composition comprising at least a first anti-viral agent in an amount effective to treat said herpes virus infection; wherein said at least a first anti-viral agent is an antibody, or antigen-binding fragment thereof, that binds to an aminophospholipid. 
     
     
         27 . The method of  claim 26 , wherein at least a second, distinct anti-viral agent is administered to said mammal. 
     
     
         28 . The method of  claim 27 , wherein said second, distinct anti-viral agent is gancyclovir. 
     
     
         29 . The method of  claim 26 , wherein said herpes virus infection is associated with the eye. 
     
     
         30 . The method of  claim 29 , wherein said at least a first anti-viral agent is formulated as an ophthalmic preparation and administered topically to the eye. 
     
     
         31 . The method of  claim 30 , wherein said at least a first anti-viral agent is administered topically to the eye in a daily dose of about 1 mg, about 2 mgs or about 4 mgs. 
     
     
         32 . The method of  claim 26 , wherein said mammal is a human patient. 
     
     
         33 . A method for treating a mammal with a herpes virus infection associated with the eye, comprising administering to said mammal at least a first and second anti-viral agent in an amount effective to treat said herpes virus infection; wherein said first anti-viral agent is an antibody that binds to an aminophospholipid and wherein said second anti-viral agent is gancyclovir. 
     
     
         34 . The method of  claim 33 , wherein said first and second anti-viral agents are administered topically to the eye. 
     
     
         35 . The method of  claim 34 , wherein said at least a first anti-viral agent is administered topically to the eye in a daily dose of about 1 mg, about 2 mgs or about 4 mgs. 
     
     
         36 . The method of  claim 33 , wherein said mammal is a human patient.