Oncolytic/immunogenic complementary-adenoviral vector system

Abstract

This invention encompasses a composition for killing target cells, such as tumor cells. The composition comprises a first and a second adenoviral vector that have complementary function and are mutually dependent on each other for replication in a target cell. One of said adenoviral vectors has a target cell-activated promoter or a functional deletion that controls and limits propagation of the adenoviral vectors in the target cells which directly or indirectly kills the target cells. One of the adenoviral vectors comprises a gene encoding a protein which is expressed in the target cells and can induce anticancer immune responses. The target cells may be hepatoma, breast cancer, melanoma, colon cancer, or prostate cancer cells, for example. The vectors of this invention may also be utilized to treat other diseases such as restenosis, in which case the target cell may be a vascular smooth muscle cell, for example.

We claim:  
     
         1 . A composition comprising a first and a second adenoviral vector each having complementary gene sequences and being mutually dependent on each other for replication in a target cell wherein said first adenoviral vector has a target cell-activated promoter or a functional deletion, which controls and limits propagation of the first and the second adenoviral vectors to the target cell which directly or indirectly kills the target cell.  
     
     
         2 . A composition of  claim 1  wherein one of the adenoviral vectors encode a protein which directly or indirectly kills the target cell.  
     
     
         3 . The composition of  claim 1  wherein the target cell is a tumor cell.  
     
     
         4 . A composition according to  claim 1  wherein one of the adenoviral vectors is a controlled vector having a promoter activated in tumor cells.  
     
     
         5 . The composition of  claim 1  wherein one of the adenoviral vectors is a controlled vector having a functional deletion in a viral gene that is dispensable in the target cells.  
     
     
         6 . The composition of  claim 1  wherein one of the adenoviral vectors is a supplemental adenoviral vector that support the propagation of the con trolled adenoviral vector.  
     
     
         7 . The composition of  claim 1  wherein one or both of the adenoviral vectors comprises a gene encoding an immunomodulatory protein or other functional protein such that the protein is expressed in the target cells.  
     
     
         8 . The composition of  claim 1  wherein the tumor activated promoter is selected from the group consisting of α-fetoprotein promoter, DF-3 mucin enhancer, tyrosinase promoter, CEA promoter, PSA promoter, HI parvovirus promoter, inducible promoters, and synthetic promoters.  
     
     
         9 . The composition of  claim 1  wherein the immunomodulatory protein is selected from the group consisting of interferon-α, B7 co-stimulatory factor, interleukin, chemokine, and tumor-specific antigen.  
     
     
         10 . The composition of  claim 1  wherein the target cell is selected from the group consisting of hepatoma, breast cancer, melanoma, lung cancer, colon cancer, and prostate cancer.  
     
     
         11 . A composition comprising the adenoviral vector illustrated in FIG. 8 termed pGT8027.  
     
     
         12 . A composition comprising the adenoviral vector illustrated in FIG. 9 termed pGT8028.  
     
     
         13 . A method of preparing a composition of  claim 1  comprising: 
 transfecting a host cell with an adenoviral vector comprising a controlled-Ad vector sequence having an ITR sequence, a packaging sequence, a cell- or tissue-specific transcriptional control region operably linked to a gene encoding an AdE1 protein and a transcriptional control region operably linked to a gene encoding an immunomodulatory protein;  
 infecting the transfected host cells with a supplemental-Ad virus having a manipulated packaging signal; and  
 preparing a cell-free lysate from said host cells;  
 wherein the cell-free lysate comprises infective, replication-controlled recombinant controlled-Ad and supplemental-Ad vectors.  
 
     
     
         14 . A method of generating a complementary-Ad vector for treatment of a disease comprising: 
 infecting with a composition of  claim 1  a host cell having the ability to drive gene expression from the controlled-Ad vector tissue-specific promoter; and    preparing a cell-free lysate from said host cells; and    isolating the complementary-Ad vector comprising a mixture of the controlled-Ad vector and the supplementary-Ad vector.    
     
     
         15 . A method of preparing complementary-Ad vectors comprising: 
 infecting with a composition of  claim 1  a host cell having the ability to drive gene expression from the controlled-Ad vector tissue-specific promoter;    preparing a cell-free lysate from said host cells;    purifying the controlled-Ad vector and the supplemental-Ad vector; and    separating the controlled-Ad vector from the supplemental-Ad vector.    
     
     
         16 . A method of generating a complementary-Ad vector composition for treatment of a disease comprising: 
 co-transfecting a host cell with a controlled-Ad vector sequence having an ITR sequence, a packaging sequence, a cell- or tissue-specific transcriptional control region operably linked to a gene encoding an AdE1 protein and a transcriptional control region operably linked to a gene encoding an immunomodulatory protein and a supplemental-Ad vector sequence having an altered packaging signal; and    preparing a cell-free lysate from said host cells;    wherein the cell-free lysate contains infective, replication-impaired complementary adenoviral vector particles.    
     
     
         17 . An adenoviral vector useful for generating an infectious, replication-defective recombinant adenoviral particle comprising an adenoviral ITR, a packaging signal, and a first and second transcriptional control region, wherein each of said transcriptional control regions are operably linked to an effector or reporter gene.  
     
     
         18 . An adenoviral vector of  claim 17  wherein said packaging signal is a wild-type packaging signal.  
     
     
         19 . An adenoviral vector of  claim 17  wherein said first transcriptional specifically controlled region functions in a cell- or tissue-specific manner.  
     
     
         20 . An adenoviral vector of  claim 17  wherein said effector gene encodes an Ad E1 protein.  
     
     
         21 . An adenoviral vector of  claim 17  wherein said first specifically controlled transcriptional region functions in a cell- or tissue-specific or inducible manner that is operably linked to an effector gene which encodes an Ad E1 protein.  
     
     
         22 . An adenoviral vector of  claim 17  wherein said first transcriptional control region is the α-fetoprotein promoter/enhancer and is operably linked to an effector gene encoding an Ad E1 protein.  
     
     
         23 . An adenoviral vector of  claim 17  wherein said second transcriptional control region is operably linked to a gene encoding an immunomodulatory protein.  
     
     
         24 . An adenoviral vector of  claim 17  wherein said second transcriptional control region is operably linked to a gene encoding the B7.1 protein.  
     
     
         25 . An adenoviral vector of  claim 17  that further comprises a third transcriptional control region operably linked to a gene encoding an immunomodulatory protein.  
     
     
         26 . An adenoviral vector of  claim 17  that further comprises a third transcriptional control region operably linked to a gene encoding IFN-γ.  
     
     
         27 . An adenoviral vector of  claim 17  wherein said first transcriptional control region is the α-fetoprotein promoter/enhancer and is operably linked to an effector gene encoding an Ad E1 protein; said second transcriptional control region is operably linked to a gene encoding the B7.1 protein; said DNA molecule further comprising a third transcriptional control region operably linked to a gene encoding IFN-γ.  
     
     
         28 . The adenoviral vector illustrated in FIG. 8 termed pGT8027.  
     
     
         29 . The adenoviral vector illustrated in FIG. 9 termed pGT8028.  
     
     
         30 . A supplemental-Ad vector comprising a partial wild-type Ad genome and a modifier attenuated packaging signal.  
     
     
         31 . A supplemental-Ad vector of  claim 30  wherein the E1 region of the Ad genome is deleted or substituted.  
     
     
         32 . A supplemental-Ad vector of  claim 30  wherein the E2 region of the Ad genome is deleted or substituted.  
     
     
         33 . A supplemental-Ad vector of  claim 30  wherein the E4 region of the Ad genome is deleted or substituted.  
     
     
         34 . A supplemental-Ad vector of  claim 30  wherein the E1 region and the E2 region of the Ad genome are deleted or substituted.  
     
     
         35 . A supplemental-Ad vector of  claim 30  wherein the E1 region and the E4 region of the Ad genome are deleted or substituted.  
     
     
         36 . A supplemental-Ad vector of  claim 29  wherein the E1, E2 and E4 regions of the Ad genome are deleted or substituted.  
     
     
         37 . A method of treating cancer comprising local intratumoral administration or systemic administration of a controlled-Ad vector having a tumor-specific transcriptional regulatory region operably linked to a gene encoding an Ad E1 protein, a second transcriptional regulatory region operably linked to a gene encoding an immunomodulatory protein, and a supplemental-Ad vector having an altered packaging signal and a deleted E1 region to a patient whereby recombinant adenoviral vectors are propagated in tumor cells such that a local and a systemic anti-tumor immune responses are generated.  
     
     
         38 . A method of treating cancer with infection of complementary-Ad vectors comprising a controlled-Ad vector having a deletion in E1b or E1b-p55 gene from the Ad E1 genes, in which the E1 a genes are under control of a tissue-specific or tumor-activated transcriptional regulatory region, a second transcriptional regulatory region operably linked to a gene encoding an immunomodulatory protein, and a supplemental-Ad vector having an altered packaging signal and a deleted E1 region.  
     
     
         39 . A method of treating cancer comprising local intratumoral or systemic administration of a composition of  claim 3.