Human skin substitutes expressing il-12

Abstract

The present invention relates generally to compositions for treating patients that have skin cancer or have recently had skin cancers removed. More specifically, the present invention provides human skin substitutes engineered to express exogenous IL-12 and compositions and methods for making human skin substitutes engineered to express exogenous IL-12. In addition, the present invention provides methods for treatment of sites on a patient where skin cancers have been removed with human skin substitutes engineered to express exogenous IL-12.

1 . A composition comprising host cells expressing heterologous Interleukin-12, wherein said host cells are selected from the group consisting of primary keratinocytes, keratinocyte precursors, transdifferentiated keratinocytes, and immortalized keratinocytes. 
     
     
         2 . The composition of  claim 1 , wherein said composition further comprises second host cells, wherein said second host cells express a second heterologous polypeptide. 
     
     
         3 . The composition of  claim 1 , wherein said host cells are selected from the group consisting of Near-Diploid Immortalized Keratinocytes cells and cells derived from Near-Diploid Immortalized Keratinocytes cells. 
     
     
         4 . The composition of  claim 3 , wherein said Interleukin-12 is selected from the group consisting of proteins having subunits comprising SEQ ID NO:1 and SEQ ID NO:2 and proteins having subunits sharing at least 95% identity with SEQ ID NO:1 and SEQ ID NO:2. 
     
     
         5 . The composition of  claim 4 , wherein said Interleukin-12 is a fusion of said subunits. 
     
     
         6 . The composition of  claim 1 , wherein a gene encoding said heterologous Interleukin-12 is operably linked to a promoter sequence that allows Interleukin-12 expression in said skin substitute. 
     
     
         7 . The composition of  claim 6 , wherein said promoter sequence is selected from the group consisting of a K14 promoter, an involucrin promoter, and an ubiquitin promoter. 
     
     
         8 . The composition of  claim 1 , wherein said composition is a cultured human skin substitute. 
     
     
         9 . The composition of  claim 1 , wherein said composition produces amounts of bioactive IL-12 which are capable of inducing the proliferation of the PHA-stimulated lymphoblasts. 
     
     
         10 . A method for providing a human skin substitute expressing heterologous Interleukin-12, comprising:
 a) providing keratinocytes selected from the group consisting of primary keratinocytes, keratinocyte precursors, transdifferentiated keratinocytes, and immortalized keratinocytes and an expression vector comprising a DNA sequence encoding Interleukin-12 operably linked to a regulatory sequence;   b) introducing said expression vector into said keratinocytes; and   c) organotypically culturing said keratinocytes to provide a human skin substitute.   
     
     
         11 . The method of  claim 10 , wherein said keratinocytes are Near-Diploid Immortalized Keratinocytes and said keratinocytes stratify into squamous epithelia. 
     
     
         12 . The method of  claim 11  further comprising co-culturing said Near-Diploid Immortalized Keratinocyte cells with cells derived from a patient. 
     
     
         13 . A method of treating a patient comprising:
 a) providing a human skin substitute comprising cells expressing heterologous Interleukin-12, wherein cells are selected from the group consisting of primary keratinocytes, keratinocyte precursors, transdifferentiated keratinocytes, and immortalized keratinocytes; and   b) contacting said patient with said human skin substitute.   
     
     
         14 . The method of  claim 13 , wherein said patient has skin cancer. 
     
     
         15 . The method of  claim 13 , wherein said skin cancer is selected from the group consisting of melanoma and basal cell carcinoma. 
     
     
         16 . The method  claim 13 , wherein said human skin substitute is applied to a site on said patient where a skin cancer has been removed. 
     
     
         17 . The method of  claim 16 , wherein said human skin substitute prevents or inhibits the spread of said skin cancer. 
     
     
         18 . The method of  claim 13 , wherein said human skin substitute produces amounts of bioactive IL-12 which are capable of inducing the proliferation of the PHA-stimulated lymphoblasts.