Methods of using human receptor protein 4-1BB

Abstract

Disclosed herein are the methods of using the H4-1BB protein, ligands to this protein, and various mAbs either directed against H4-1BB or other molecules that can be used therapeutically. The nature and importance of the H4-1BB molecule provides the ligands and related co-stimulatory molecules the ability to enhance or suppress T-cell activation and proliferation. By treating T-cells that have expressed receptor protein H4-1BB with one of the four anti-H4-1BB monoclonal antibodies disclosed herein activation or inhibition of the immune response is seen. Also disclosed herein is cDNA for the human receptor H4-1BB. The cDNA of the human receptor H4-1BB is about 65% homologous to the mouse cDNA 4-1BB and was isolated by using probes derived from murine cDNA 4-1BB. A fusion protein for detecting cell membrane ligands to human receptor protein H4-1BB was developed. It comprises the extracellular portion of the receptor protein H4-1BB and a detection protein, alkaline phosphatase, bound to the portion of the receptor protein H4-1BB. B-cells that have expressed a ligand to receptor protein H4-1BB can be treated with cells that have expressed receptor protein H4-1BB and B-cell proliferation may be induced. The use of H4-1BB to block H4-1BB ligand binding has practical application in the suppression of the immune system during organ transplantation or against autoimmune diseases including diabetes, rheumatoid arthritis, and lupus. Other applications of this technology include the development of therapeutic methods for the treatment of HIV-1 infected individuals, and the treatment of cancerous tumors.

1 . A method of enhancing T-cell activation comprising administering an effective amount of a first H4-1BB receptor ligand such that said receptor ligand comes into contact with at least one T-cell, thereby activating it.  
     
     
         2 . The method  claim 1  wherein said 144-1BB receptor ligand administered is an agonistic anti-4-1BB monoclonal antibody.  
     
     
         3 . The method  claim 1  wherein said H4-1BB receptor ligand administered is an antagonistic anti-4-1BB monoclonal antibody.  
     
     
         4 . The method of  claim 1  wherein said first H4-1BB receptor ligand is a H4-1BB protein.  
     
     
         5 . The method of  claim 1  wherein said first H4-1BB receptor ligand is administered at a dosage range equivalent to or greater than 0.20 mmol to 2.0 μmol, one to three times per day.  
     
     
         6 . The method of  claim 5  wherein the administration of said first H4-1BB receptor ligand is accomplished through an administration of a pharmaceutical formulation such as a tablet or intravenous injection, wherein administration of said first H4-1BB receptor ligand does not lessen the effectiveness of said first H4-1BB receptor ligand in activating said at least one T-cell.  
     
     
         7 . A method of enhancing T-cell activation of  claim 1  further comprised by administering a second stimulatory molecule, in conjunction with said first H4-1BB receptor ligand such that each of these compounds comes into contact with said at least one T-cell.  
     
     
         8 . The method of  claim 7 , wherein said second stimulatory molecule is selected from the group consisting essentially of: 
 a) an anti-CD3 antibody;    b) an anti-CD28 antibody; and    c) the CD28 protein.    
     
     
         9 . The method of  claim 5  wherein said first H4-1BB receptor ligand is administered at a dosage range equivalent to or greater than 0.20 μmol to 2.0 μmol, one to three times per day, and wherein said second stimulatory molecule is administered at a dosage range equivalent to or greater than 0.10 μmol to 2.0 μmol, one to three times per day.  
     
     
         10 . The method of  claim 5  wherein the administration of said first H4-1BB receptor ligand and said second stimulatory molecule is accomplished through an administration of a pharmaceutical formulation such as a tablet or intravenous injection.  
     
     
         11 . The method of  claim 9 , further comprising the use of a third stimulatory molecule, said second co-stimulatory molecule being an anti-CD3 antibody and said third stimulatory molecule being an anti-CD28 antibody.  
     
     
         12 . The method of  claim 11  wherein the administration of said first H4-1BB receptor ligand, said second stimulatory molecule, and said third stimulatory molecule is accomplished through an administration of a pharmaceutical formulation such as a tablet or intravenous injection.  
     
     
         13 . A method of treating cancerous tumors such that said cancerous tumors are reduced, comprising the administration of a first effective amount of H4-1BB receptor ligand such that said compound comes into contact with at least one T-cell, and wherein said H4-1BB protein works in conjunction with a second stimulatory molecule, such that both of these compounds come into contact with said at least one T-cell.  
     
     
         14 . The method of  claim 13 , wherein said second stimulatory molecule is selected from the group consisting essentially of: 
 a) an anti-CD3 antibody;    b) an anti-CD28 antibody; and    c) the CD28 protein.    
     
     
         15 . The method of  claim 13  wherein said first H4-1BB receptor ligand is administered at a dosage range equivalent to or greater than 2.0 μmol to 8.0 μmol, one to three times per day, and wherein said second stimulatory molecule is administered at a dosage range equivalent to or greater than 0.10 μmol to 2.0 μmol, one to three times per day.  
     
     
         16 . The method of  claim 13  wherein the administration of said first H4-1BB receptor ligand and said second-stimulatory molecule is accomplished through an oral administration of a pharmaceutical formulation such as a tablet or injection.  
     
     
         17 . A method of enhancing cytokine production in CD+4 and CD+8 T-cells comprising: 
 a) administering effective amounts of three compounds contemporaneously, said three compounds comprising: 
 i) an anti-H4-1BB antibody;  
 ii) an anti-CD3 antibody;  
 iii) an anti-CD28 antibody; and  
   wherein said administration is such that each of said three compounds comes into contact with said at least one T cell.    
     
     
         18 . The method of  claim 17  wherein the cytokine whose production is enhanced is selected from the group consisting of: 
 a) gamma interferon (IF);    b) interleukin 1 (IL-1);    c) interleukin 10 (IL-10);    d) B cell growth factor (BCGF);    e) B cell differentiating factor (BCDF); and    f) interleukin 2 (IL-2).    
     
     
         19 . A method of treating an autoimmune reaction comprising administering an effective amount of an antagonist to the H4-1BB protein, said antagonist being capable of preventing the H4-1BB protein from binding to a H4-1BB receptor, wherein said antagonist is itself incapable of activating CD4+ or CD8+ T cells.  
     
     
         20 . The method of  claim 19  wherein said first H4-1BB receptor ligand is administered at a dosage range equivalent to or greater than 0.20 μmol to 2.0 μmol, one to three times per day.  
     
     
         21 . The method of  claim 19  wherein the administration of said first H4-1BB receptor ligand is accomplished through an administration of a pharmaceutical formulation such as a tablet or intravenous injection.  
     
     
         22 . The method of  claim 19  wherein said autoimmune reaction treated is one associated with an autoimmune disease, wherein said autoimmune disease is selected from the group consisting of. 
 a) Diabetes Melitus;    b) Rheumatoid Arthritis; and    c) Systemic Lupus Erthyematosus.    
     
     
         23 . The method of  claim 19  wherein said method of preventing an autoimmune reaction is used to suppress an autoimmune response occurring after an organ transplantation.  
     
     
         24 . A method for monitoring the level of progression of Acquired Immune Deficiency caused by the pathogenic virus HIV-1 is accomplished by measuring the level of H4-1BB expression in a known quantity of tissue comprising: 
 a) collecting a sample of CD8+ T cells;    b) fractionating cells and retaining the lysate to test for the presence of the H4—1BB using a monoclonal antibody(s) directed against said H4-1BB protein;    c) attaching to said antibody(s) another molecule capable of being detected by a scintillation counter or fluorescent microscope or other means useful in measuring the degree of antibody binding; and    d) determining the level of H4-1BB expression in said sample of CD8+ T cells for comparison with a known measurement that reflects a normal level of expression of H4-1BB expression in a same size sample of an equivalent tissue type.    
     
     
         25 . A method of preventing an autoimmune reaction comprising administering an effective amount of an antagonist to the H4-1BB protein, said antagonist being capable of preventing the H4-1BB protein from binding to the H4-1BB receptor ligand, wherein said antagonist is itself incapable of activating CD4+ or CD8+ T cells.  
     
     
         26 . A method of interfering with HIV-1 progression comprising the step of administering an effective amount of an agent capable of binding said H4-1BB receptor protein on CD4+ T-lymphocytes, thereby blocking it.  
     
     
         27 . The method of  claim 26  wherein said agent is selected from the group consisting of: 
 a) a 4-1BB-Fc molecule,    b) a blocking anti-4-1BB monoclonal antibody; and    c) a fusion protein comprising a portion of said H4-1BB protein.    
     
     
         28 . An antibody that is immuno-reactive with a purified human 4-1BB polypeptide comprising the N-terminal amino acid sequence Phe-Glu-Arg-Thr-Arg-Ser-Leu-Gln-Asp-Pro-Cys-Ser-Asn-Cys-Pro-Ala-Gly-Thr.  
     
     
         29 . A method of blocking T cell activation comprising administering an effective amount of an H4-1BB protein antagonist such that said protein antagonist prevents the activation of the H4-1BB receptor.  
     
     
         30 . A method of treating Human Acquired Immune Deficiency caused by the viral pathogen HIV-1, comprising administering an effective amount of a first H4-1BB receptor ligand, such that said receptor ligand comes into contact with at least one T-cell thereby activating at least one CD8+ T cell.  
     
     
         31 . The method of  claim 30  wherein said first H4-1BB receptor ligand or agonistic mAb is administered at a dosage range equivalent to or greater than 0.20 μmol to 2.0 μmol, one to three times per day.  
     
     
         32 . The method of  claim 30  wherein said at least one CD8+ T cell is capable of killing HIV-1 infected cells seleceted from the group consisting of. 
 a) CD4+ cells;    b) astrocytes;    c) macrophages;    d) dendritic cells; and    e) microglial cells.    
     
     
         33 . The method of  claim 30  wherein the administration of said first H4-1BB receptor ligand is accomplished through an administration of a pharmaceutical formulation such as a tablet or intravenous injection.  
     
     
         34 . The method of  claim 2  wherein said agonistic anti-4-1BB monoclonal antibody is an monoclonal antibody designated BBK-1.  
     
     
         35 . The monoclonal antibody of  claim 34  further comprising a hybridoma capable of producing said monoclonal antibody designated BBK-1.  
     
     
         36 . The method of using the monoclonal antibody of  claim 34  to enhance T-cell activation comprising the step of treating T-cells that have expressed receptor protein H4-1BB with said monoclonal antibody designated BBK-1.  
     
     
         37 . The method of  claim 3  wherein said antagonistic anti-4-1BB monoclonal antibody is a monoclonal antibody designated BBK-2.  
     
     
         38 . The monoclonal antibody of  claim 37  further comprising a hybridoma capable of producing said monoclonal antibody designated BBK-2.  
     
     
         39 . The method of using the monoclonal antibody of  claim 37  to enhance T-cell activation comprising the step of treating T-cells that have expressed receptor protein H4-1BB with said monoclonal antibody designated BBK-2.  
     
     
         40 . The method of  claim 3  wherein said antagonistic anti-4-1BB monoclonal antibody is a monoclonal antibody designated BBK-3.  
     
     
         41 . The monoclonal antibody of  claim 40  further comprising a hybridoma capable of producing said monoclonal antibody designated BBK-3.  
     
     
         42 . The method of using the monoclonal antibody of  claim 40  to enhance T-cell activation comprising the step of treating T-cells that have expressed receptor protein H4-1BB with said monoclonal antibody designated BBK-3.  
     
     
         43 . The method of  claim 2  wherein said agonistic anti-4-1BB monoclonal antibody is an monoclonal antibody designated BBK4.  
     
     
         44 . The monoclonal antibody of  claim 43  further comprising a hybridoma capable of producing said monoclonal antibody designated BBK4.  
     
     
         45 . The method of using the monoclonal antibody of  claim 43  to enhance T-cell activation comprising the step of treating T-cells that have expressed receptor protein H4-1BB with said monoclonal antibody designated BBK-4.