CONSTRUCTION AND EXPRESSION IN TUMOR-CELLS OF A RECOMBINANT VACCINIA VIRUS ENCODING HUMAN INTERLEUKIN-1-BETA

Background: Human interleukin-1 beta (hIL-1 beta) injected intratumora lly has demonstrated growth inhibition of transplanted subcutaneous tu mors in mice, regression of metastatic lesions, resistance to tumor re challenge, and increased survival. Vaccinia virus (VV) can be genetica lly engineered to produce cytokines and may be an effective vector for gene therapy of cancer. This study was designed to (a) construct a VV expressing hIL-1 beta, (b) assess tumor cell infection in vitro with this construct, (c) measure hIL-1 beta production, and (d) assess the bioactivity of the secreted cytokine. Methods: The hIL-1 beta gene was amplified from a plasmid clone using polymerase chain reaction (PCR) and then cloned into a homologous recombination (HR) and expression ve ctor, which was used to insert the hIL-1 beta gene into the VV genome. Selection of the recombinant VV (VMJ601hIL-1 beta) was based on inact ivation of viral TK and expression of beta-galactosidase. VMJ601hIL-1 beta infectivity and cytokine production was assessed by infecting tum or cell lines and analyzing culture supernatants for hIL-1 beta. Bioac tivity of the hIL-1 beta produced was demonstrated using an IL-1 depen dent T helper cell line. Results: The hIL-1 beta gene was successfully cloned into the VV genome by HR, which was confirmed by PCR. VMJ601hI L-1 beta efficiently infected tumor cells, as shown by increased hIL-1 beta secretion (0 to > 500 ng/ml) and morphologic evidence of viral c ytopathic effect, VMJ601hIL-1 beta-infected cells secreted large amoun ts of hIL-1 beta (mean 772 ng/10(6) cells/24 h). The secreted hIL-1 be ta was bioactive (mean bioactivity 6.8 x 10(8) U/mg of hIL-1 beta). Co nclusions: (a) hIL-1 beta can be cloned into VV, (b) VMJ601hIL-1 beta retains its infectivity, (c) a large amount of hIL-1 beta is secreted, and (d) the secreted hIL-1 beta is bioactive, Recombinant VV may allo w in situ cytokine gene delivery and expression in established tumors.