Highly purified cytokine activating factor and methods of use

Abstract

Disclosed is an isolated Cytokine Activating Factor (CAF) protein, an isolated nucleic acid molecule encoding a CAF protein, an antibody that selectively binds to a CAF protein, a composition comprising a Cytokine Activating Factor (CAF), and a method for modulating the immune system using the composition. Also disclosed is a method for treating cancer using such a composition.

What is claimed is:  
     
         1 . An isolated protein comprising an amino acid sequence selected from the group consisting of: 
 a. an amino acid sequence selected from the group consisting of SEQ ID NO:1 and SEQ ID NO:6; and,    b. an amino acid sequence comprising at least 9 consecutive amino acid residues of an amino acid sequence of (a);    wherein said isolated protein upregulates expression of tumor necrosis factor α (TNFα), interleukin-1β (IL-1β) or interleukin-6 (IL-6), or downregulates expression of transforming growth factor β (TGFβ).    
     
     
         2 . The isolated protein of  claim 1 , wherein said protein comprises an amino acid sequence having at least about 15 consecutive amino acid residues of an amino acid sequence of (a).  
     
     
         3 . The isolated protein of  claim 1 , wherein said protein comprises an amino acid sequence having at least about 20 consecutive amino acid residues of an amino acid sequence of (a).  
     
     
         4 . The isolated protein of  claim 1 , wherein said protein comprises an amino acid sequence having at least about 25 consecutive amino acid residues of an amino acid sequence of (a).  
     
     
         5 . The isolated protein of ( claim 1 , wherein said protein comprises an amino acid sequence having at least about 50 consecutive amino acid residues of SEQ ID NO:6.  
     
     
         6 . The isolated protein of  claim 1 , wherein said protein comprises an amino acid sequence that is at least about 65% identical to an amino acid sequence of (a) over at least 15 amino acids of said amino acid sequence of (a).  
     
     
         7 . The isolated protein of  claim 1 , wherein said protein comprises an amino acid sequence that is at least about 75% identical to an amino acid sequence of (a) over at least 15 amino acids of said amino acid sequence of (a).  
     
     
         8 . The isolated protein of  claim 1 , wherein said protein comprises an amino acid sequence that is at least about 60% identical to SEQ ID NO:6 over 66 amino acids of SEQ ID NO:6.  
     
     
         9 . The isolated protein of  claim 1 , wherein said protein is encoded by a nucleic acid sequence that hybridizes under high stringency conditions to a nucleic acid sequence encoding an amino acid sequence selected from the group consisting of SEQ ID NO:1 and SEQ ID NO:6.  
     
     
         10 . The isolated protein of  claim 1 , wherein said protein comprises amino acid sequence selected from the group consisting of SEQ ID NO:1 and SEQ ID NO:6.  
     
     
         11 . The isolated protein of  claim 1 , wherein said protein comprises amino acid sequence SEQ ID NO:6.  
     
     
         12 . The isolated protein of  claim 1 , wherein said protein has at least one biologically active subunit which passes through a 3000 Dalton molecular weight cut-off ultrafiltration filter.  
     
     
         13 . The isolated protein of  claim 1 , wherein said protein is stable at a temperature up to at least about 50° C.  
     
     
         14 . The isolated protein of  claim 1 , wherein said protein is stable at pH of from about 2 to about 10.  
     
     
         15 . The isolated protein of  claim 1 , wherein said protein comprises an identifying characteristic selected from the group consisting of: 
 a. is water soluble;    b. is non-steroidal;    c. is negatively charged;    d. is substantially non-polar; and,    e. has a λ max  at about 254 nm.    
     
     
         16 . The isolated protein of  claim 1 , wherein said protein is biologically active when administered orally.  
     
     
         17 . The isolated protein of  claim 1 , wherein said protein is naturally present in both the egg white and egg yolk of avian eggs.  
     
     
         18 . The isolated protein of  claim 1 , wherein said protein upregulates expression of tumor necrosis factor α (TNFα), interleukin-1β (IL-1β) or interleukin-6 (IL-6).  
     
     
         19 . The isolated protein of  claim 1 , wherein said protein downregulates expression of transforming growth factor β (TGFβ).  
     
     
         20 . An isolated antibody that selectively binds to the isolated protein of  claim 1 .  
     
     
         21 . An isolated protein comprising an amino acid sequence selected from the group consisting of: 
 a. an amino acid sequence selected from the group consisting of SEQ ID NO:1 and SEQ ID NO:6; and,    b. an amino acid sequence that is at least 65% identical to an amino acid sequence of (a) over at least 15 amino acids of an amino acid sequence of (a);    wherein said isolated protein upregulates expression of tumor necrosis factor α (TNFα), interleukin-1β (IL-1β) or interleukin-6 (IL-6), or downregulates expression of transforming growth factor β (TGFβ).    
     
     
         22 . A composition comprising a pharmaceutically acceptable carrier and a cytokine activating factor (CAF) protein comprising an amino acid sequence selected from the group consisting of: 
 a. an amino acid sequence selected from the group consisting of SEQ ID NO:1 and SEQ ID NO:6; and,    b. an amino acid sequence comprising at least 9 consecutive amino acid residues of an amino acid sequence of (a);    wherein said CAF protein upregulates expression of tumor necrosis factor α (TNFα), interleukin-1β (IL-1β) or interleukin-6 (IL-6), or downregulates expression of transforming growth factor β (TGFβ).    
     
     
         23 . The composition of  claim 22 , wherein said pharmaceutically acceptable carrier is a food product selected from the group consisting of: 
 a. a hyperimmune egg product which is selected to be enriched for said CAF protein; and,    b. a food product produced with at least a fraction of a hyperimmune egg product, wherein said fraction comprises an enriched amount of said CAF protein as compared to said hyperimmune egg product.    
     
     
         24 . The composition of  claim 22 , wherein said pharmaceutically acceptable carrier comprises a fraction of a hyperimmune egg product containing an enriched amount of said CAF protein as compared to said hyperimmune egg product.  
     
     
         25 . The composition of  claim 24 , wherein said fraction is selected from the group consisting of: liquid egg yolk, liquid egg white, powdered egg yolk, powdered egg white, and a water soluble fraction of said hyperimmune egg product.  
     
     
         26 . The composition of  claim 22 , wherein said composition is in a form selected from the group consisting of a liquid, an aerosol, a capsule, a tablet, a pill, a powder, a gel and a granule.  
     
     
         27 . The composition of  claim 22 , wherein said pharmaceutically acceptable carrier comprises a controlled release formulation.  
     
     
         28 . The composition of  claim 22 , wherein said pharmaceutically acceptable carrier is selected from the group consisting of: water, phosphate buffered saline, Ringer's solution, dextrose solution, serum-containing solutions, Hank's solution, other aqueous physiologically balanced solutions, oils, esters, glycols, biocompatible polymers, polymeric matrices, capsules, microcapsules, microparticles, bolus preparations, osmotic pumps, diffusion devices, liposomes, lipospheres, cells, and cellular membranes.  
     
     
         29 . A method to regulate an immune response in an animal, comprising administering to said animal a composition comprising a cytokine activating factor (CAF) protein comprising an amino acid sequence selected from the group consisting of: 
 a. an amino acid sequence selected from the group consisting of SEQ ID NO:1 and SEQ ID NO:6; and,    b. an amino acid sequence comprising at least 9 consecutive amino acid residues of an amino acid sequence of (a);    wherein said CAF protein upregulates expression of tumor necrosis factor α (TNFα), interleukin-1β(IL-1β) or interleukin-6 (IL-6), or downregulates expression of transforming growth factor β (TGFβ).    
     
     
         30 . The method of  claim 29 , wherein said composition comprises a pharmaceutically acceptable carrier.  
     
     
         31 . The method of  claim 29 , wherein said composition is administered at a dose of from about 1 nanogram to about 400 milligrams of said CAF protein per kilogram body weight of said animal.  
     
     
         32 . The method of  claim 29 , wherein said composition is administered by a route selected from the group consisting of oral, intravenous administration, intraperitoneal administration, intramuscular administration, subcutaneous administration, transdermal delivery, intratracheal administration, inhalation, impregnation of a catheter, by suppository, and direct injection into a tissue.  
     
     
         33 . The method of  claim 29 , wherein said composition comprises a food product containing said CAF protein.  
     
     
         34 . The method of  claim 29 , wherein administration of said composition upregulates expression of tumor necrosis factor α (TNFα), interleukin-1β(IL-1β) or interleukin-6 (IL-6) by cells of said animal.  
     
     
         35 . The method of  claim 29 , wherein administration of said composition downregulates expression of transforming growth factor β (TGFβ) by cells of said animal.  
     
     
         36 . The method of  claim 29 , wherein animal is a mammal.  
     
     
         37 . A method of treating cancer in an animal, comprising administering to an animal that has or is at risk of developing cancer a composition comprising a cytokine activating factor (CAF) protein comprising an amino acid sequence selected from the group consisting of: 
 a. an amino acid sequence selected from the group consisting of SEQ ID NO:1 and SEQ ID NO:6; and,    b. an amino acid sequence comprising at least 9 consecutive amino acid residues of an amino acid sequence of (a);    wherein said CAF protein upregulates expression of tumor necrosis factor α (TNFα), interleukin-1β (IL-1β) or interleukin-6 (IL-6), or downregulates expression of transforming growth factor β (TGFβ).    
     
     
         38 . The method of  claim 37 , wherein said composition is administered at a dose of from about 1 nanogram to about 400 milligrams of said CAF protein per kilogram body weight of said animal.  
     
     
         39 . The method of  claim 37 , wherein said composition is administered by a route selected from the group consisting of oral, intravenous administration, intraperitoneal administration, intramuscular administration, subcutaneous administration, transdermal delivery, intratracheal administration, inhalation, impregnation of a catheter, by suppository, and direct injection into a tissue at or adjacent to said cancer.  
     
     
         40 . The method of  claim 37 , wherein said composition comprises a food product containing said CAF protein.  
     
     
         41 . The method of  claim 37 , wherein administration of said composition produces a result selected from the group consisting of: reduction in symptoms of the cancer, reduction of a tumor associated with the cancer, elimination of a tumor associated with the cancer, prevention of metastatic cancer, prevention of the cancer and stimulation of effector cell immunity against the cancer.  
     
     
         42 . An isolated nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of: 
 a. a nucleic acid sequence encoding a protein comprising an amino acid sequence selected from the group consisting of: 
 i. an amino acid sequence selected from the group consisting of SEQ ID NO:1 and SEQ ID NO:6; and,  
 ii. an amino acid sequence comprising at least 9 consecutive amino acid residues of an amino acid sequence of (i);  
   wherein said protein upregulates expression of tumor necrosis factor α (TNFα), interleukin-1β (IL-1β) or interleukin-6 (IL-6), or downregulates expression of transforming growth factor β (TGFβ); and,    b. a nucleic acid sequence that is fully complementary to said nucleic acid sequence of (a).    
     
     
         43 . The isolated nucleic acid molecule of  claim 42 , wherein said protein comprises an amino acid sequence selected from the group consisting of SEQ ID NO:1 and SEQ ID NO:6.  
     
     
         44 . A recombinant nucleic acid molecule comprising an isolated nucleic acid molecule as set forth in  claim 42 .  
     
     
         45 . A recombinant cell comprising an isolated nucleic acid molecule as set forth in  claim 42 , wherein said cell expresses said nucleic acid molecule.  
     
     
         46 . A recombinant virus comprising an isolated nucleic acid molecule as set forth in  claim 42.