Cytokine has been used as an immune stimulator and administered to patients for a treatment of cancer. Interleukin-12 (IL-12) is a potent cytokine which acts through a variety of functions including interferon-gamma production and cytotoxic T-cell activation. Considering the toxicity of high dose systemic IL-12 administration into human, local administration of low dose IL-12 can be a more efficient strategy. In ex vivo therapy, human dermal fibroblast has been considered as a useful vehicle for transferring genes, Here we show that human dermal fibroblast transduced with retrovirus containing IL-12 gene can be manipulated to produce reasonable amount of IL-12 protein. Human dermal fibroblast was isolated from freshly harvested skin specimens by collagenase digestion, grown in primary cultures, and transduced with a retroviral vector containing genes for human IL-12 and a selectable marker Neo(R). Following selection in G418, IL-12 producing fibroblasts were tested for secreted IL-12 level by ELISA. Six specimens of human skin were processed to obtain fibroblasts. ELISA results show that 40-150 units of IL-12 was produced for 24 h from 1x10(6) cells of transduced and selected fibroblast cultures. The primary cultures were maintained for up to nine passages about 108 days. The mean +/- overall time for obtaining enough number of cells was 49 +/- 2 days. The fibroblasts continued to produce IL-12 in culture for 90 days. These preliminary results can be used for the design of ex vivo gene therapy clinical trial using human dermal fibroblast.
Collagenases ; Digestion ; Enzyme-Linked Immunosorbent Assay ; Fibroblasts* ; Genes, Neoplasm* ; Genetic Therapy ; Humans* ; Interferon-gamma ; Interleukin-12* ; Retroviridae ; Skin ; T-Lymphocytes ; Zidovudine

Interleukin 12 (IL-12), a heterodimeric cytokine, promotes an effective antitumor response against tumors of various histological types when delivered systemically as a protein or locally by gene transfer. We investigated parameters that influenced the effectiveness of IL-12 retroviral-mediated gene therapy of cancer in animals using the murine breast cancer line TS/A. Syngeneic fibroblasts (TIB80), stably transduced with a retrovirus expressing murine IL-12, were used for peritumoral injection. Injection of fibroblasts into established tumors resulted in complete regression of tumor in 40 % of animals in a dose dependent manner when treated on day 4, and 20 % when treated on day 8. Significant inhibition of growth of day 21 and day 40 tumors was observed following peritumoral injection of IL-12-expressing fibroblasts in a dose-dependent manner. Delivery of IL-12 by syngeneic fibroblasts at a tumor site is effective in eradicating established, weakly immunogenic TS/A tumors.
Animals ; Breast Neoplasms ; Fibroblasts ; Genetic Therapy* ; Interleukin-12* ; Retroviridae

BACKGROUNDTo construct a DC-Ad-Ki-ras(V12) vaccine and investigate the anti-tumor activities of lung cancer dendritic cell vaccine modified by mutant Ki-ras gene in vitro.

METHODSKi-ras(V12) cDNA was transfected into cultured bone marrow-derived DC with the recombinant adenovirus [(Ad-Ki-ras(V12)] containing human mutant Ki-ras gene. Anti-tumor activity of the vaccine was studied in vitro by flow cytometry, PCR, MLR and cytotoxicity assay.

RESULTS(1) The DC vaccine was confirmed not only to express Ki-ras(V12) gene, but also to remarkably stimulate lymphocyte proliferation and improve CTL activity. (2) The DC vaccine modified by mutant Ki-ras gene could induce specifical CTL activity of immunized mice against Lewis lung carcinoma that could express Ki-ras(V12) gene, but not to B16.

CONCLUSIONSThe DC vaccine modified by mutant Ki-ras gene can induce obvious anti-tumor activities against Lewis lung carcinoma that can express Ki-ras(V12) gene.