Multifunctional Cytokines

Abstract

The present invention relates to a novel fusion protein with the formula X-Y, or Y-X, wherein X represents a first immunoregulating polypeptide and Y represents a second immunoregulating polypeptide different from X. The present invention also relates to a nucleic acid molecule encoding such a fusion protein and a vector comprising such a nucleic acid molecule. The present invention also provides infectious viral particles and host cells comprising such a nucleic acid molecule or such a vector as well as a process for producing such infectious viral particles. The present invention also relates to a method for recombinantly producing such a fusion protein. Finally, the present invention also provides a pharmaceutical composition comprising such a fusion protein, a nucleic acid molecule, a vector, infectious viral particles and a host cell as well as the therapeutic use thereof.

1 . An infectious viral particle comprising a nucleic acid molecule encoding a fusion protein having the formula X-Y or Y-X, wherein:
 X represents a first immunoregulatory polypeptide;   Y represents a second immunoregulatory polypeptide; and   X is different from Y.   
     
     
         2 . The infectious viral particle of  claim 1 , wherein X and Y each represent a cytokine. 
     
     
         3 . The infectious viral particle of  claim 2 , wherein X represents a cytokine capable of enhancing a nonspecific immune response. 
     
     
         4 . The infectious viral particle of  claim 3 , wherein the nonspecific immune response is mediated by one or more of the effector cells selected from the group consisting of macrophages, dendritic cells, NK cells and NKT cells. 
     
     
         5 . The infectious viral particle of  claim 2 , wherein Y represents a cytokine capable of enhancing a specific immunity. 
     
     
         6 . The infectious viral particle of  claim 5 , wherein the specific immunity is mediated by the effector cells B and/or T lymphocytes. 
     
     
         7 . The infectious viral particle of  claim 2 , wherein X and Y independently are selected from the group consisting of IL-2, IL-7, IL-15, IL-18, IL-21, IL-27, IL-31 and IFNg. 
     
     
         8 . The infectious viral particle of  claim 7 , wherein:
 (a) X is IL-2 and Y is selected from the group consisting of IL-7, IL-15, IL-18, IL-21, IL-27, IL-31 and IFNg;   (b) X is IL-12 and Y is selected from the group consisting of IL-15, IL-18 and IL-21;   (c) X is IL-15 and Y is selected from the group consisting of IL-7, IL-18 and IL-21; or   (d) X is IL-18 and Y is IL-21.   
     
     
         9 . The infectious viral particle of  claim 8 , wherein the fusion protein:
 (a) has the formula Y-X, wherein X is IL-2 and Y is IL-7;   (b) has the formula X-Y or Y-X, wherein X is IL-2 and Y is IL-15;   (c) has the formula X-Y, wherein X is IL-2 and Y is IL-18;   (d) has the formula Y-X, wherein X is IL-2 and Y is IL-21;   (e) has the formula Y-X, wherein X is IL-2 and Y is IFN-g;   (f) has the formula X-Y, wherein X is IL-15 and Y is IL-7;   (g) has the formula X-Y or Y-X, wherein X is IL-15 and Y is IL-18;   (h) has the formula X-Y or Y-X, wherein X is IL-15 and Y is IL-21; and   (i) has the formula X-Y or Y-X, wherein X is IL-18 and Y is IL-21.   
     
     
         10 . The infectious viral particle of  claim 7 , wherein the IL-2 is an IL-2 variant which exhibits a reduced cytotoxicity as compared to the corresponding native IL-2. 
     
     
         11 . The infectious viral particle of  claim 10 , wherein the IL-2 variant is selected from the group consisting of:
 (a) the variant F42K having the phenyl alanine residue in position 42 of the native IL-2 substituted by a lysine residue;   (b) the variant R38A having the arginine residue in position 38 of the native IL-2 substituted by an alanine residue;   (c) the variant D20I having the aspartic acid residue in position 20 of the native IL-2 substituted by an isoleucine residue;   (d) the variant N88G having the asparagine residue in position 88 of the native IL-2 substituted by a glycine residue;   (e) the variant N88R having the asparagine residue in position 88 of the native IL-2 substituted by an arginine residue;   (f) the variant Q126M having the glutamine residue in position 126 of the native IL-2 substituted by a methionine residue; and   (g) any combination of (a) to (f).   
     
     
         12 . The infectious viral particle of  claim 7 , wherein the IL-18 is an IL-18 variant. 
     
     
         13 . The infectious viral particle of  claim 12 , wherein the IL-18 variant is the variant K89A having the lysine residue in position 89 of the corresponding native IL-18 substituted by an alanine residue. 
     
     
         14 . The infectious viral particle of  claim 7 , wherein the IL-18 is a proIL-18. 
     
     
         15 . The infectious viral particle of  claim 7 , wherein the fusion protein comprises an amino acid sequence which is at least 70% homologous to all or part of any of the amino acid sequences recited in SEQ ID NOS: 1-19. 
     
     
         16 . The infectious viral particle of  claim 15 , wherein the fusion protein comprises an amino acid sequence which is 100% homologous to all or part of any of the amino acid sequences recited in SEQ ID NOS: 1-19. 
     
     
         17 . The infectious viral particle of  claim 1 , wherein:
 X represents IL-2; and   Y represents IL-18.   
     
     
         18 . The infectious viral particle of  claim 17 , wherein the IL-18 is a pro-IL-18. 
     
     
         19 . The infectious viral particle of  claim 18 , wherein the pro-IL-18 is fused to the carboxy terminus of the IL-2. 
     
     
         20 . The infectious viral particle of  claim 17 , wherein the IL-18 is a K89A variant having the lysine residue in position 89 of the corresponding native IL-18 substituted by an alanine residue. 
     
     
         21 . The infectious viral particle of  claim 20 , wherein the fusion protein comprises the amino acid sequence of SEQ ID NO: 9. 
     
     
         22 . The infectious viral particle of  claim 1 , wherein the nucleic acid molecule is located on a vector. 
     
     
         23 . The infectious viral particle of  claim 22 , wherein the vector is a bacterial plasmid, bacteriophage, yeast episome, artificial chromosome, or a viral vector selected from the group consisting of baculoviral vectors, papovaviral vectors, herpes viral vectors, adenoviral vectors, adenovirus-associated viral (AAV) vectors, poxviral vectors, foamy viral vectors, and retroviral vectors. 
     
     
         24 . The infectious viral particle of  claim 23 , wherein the vector is an adenoviral vector. 
     
     
         25 . The infectious viral particle of  claim 24 , wherein the adenoviral vector is an E1- and E3-deleted replication-defective adenoviral vector comprising the nucleic acid molecule inserted in replacement of the E1 region and placed under the control of the CMV promoter. 
     
     
         26 . The infectious viral particle of  claim 22 , wherein the vector further comprises one or more transgenes encoding a polypeptide selected from the group consisting of tumor proliferation inhibitors and antigens against which an immune response is desired. 
     
     
         27 . The infectious viral particle of  claim 26 , wherein the tumor proliferation inhibitor is a polypeptide possessing both CDase (cytosine deaminase) and UPRTase (uracil phosphoribosyl transferase) activities of  Saccharomyces cerevisiae.    
     
     
         28 . The infectious viral particle of  claim 26 , wherein the antigen against which an immune response is desired is a human papilloma virus (HPV) antigen. 
     
     
         29 . The infectious viral particle of  claim 28 , wherein the HPV antigen is a membrane-anchored form of a non-oncogenic variant of the early HPV-16 E7 antigen. 
     
     
         30 . A method for producing an infectious viral particle according to  claim 1 , comprising:
 (a) introducing a viral vector comprising a nucleic acid molecule into a suitable cell line, wherein the nucleic acid molecule encodes a fusion protein having the formula X-Y or Y-X, wherein X represents a first immunoregulatory polypeptide, Y represents a second immunoregulatory polypeptide, and X is different from Y;   (b) culturing the cell line under suitable conditions so as to allow the production of the infectious viral particle;   (c) recovering the produced infectious viral particle from the culture of the cell line; and   (d) optionally purifying the recovered infectious viral particle.   
     
     
         31 . A host cell comprising the infectious viral particle of  claim 1 . 
     
     
         32 . A method for producing a fusion protein, comprising:
 (a) introducing an infectious viral particle according to  claim 1  into a suitable host cell to produce an infected host cell;   (b) culturing in-vitro the infected host cell under conditions suitable for growth of the host cell;   (c) recovering the fusion protein from the culture; and   (d) optionally purifying the recovered fusion protein.   
     
     
         33 . A composition comprising:
 (a) the infectious viral particle according to  claim 1 , a host cell comprising the infectious viral particle, or a combination thereof; and   (b) optionally a pharmaceutically acceptable vehicle.   
     
     
         34 . A method for the treatment of a human or animal organism, comprising administering to the organism a therapeutically effective amount of the composition of  claim 31 . 
     
     
         35 . A method for enhancing an immune response in an animal or human organism comprising introducing into the organism a therapeutically effective amount of the composition of  claim 31 , so as to enhance the immune response.