IN-SITU INTERLEUKIN-4 GENE-EXPRESSION IN CANCER-PATIENTS TREATED WITHGENETICALLY-MODIFIED TUMOR VACCINE

Patients with advanced malignancies, participating in our ongoing phas e I interleukin-4 (IL-4) gene therapy protocol at the Pittsburgh Cance r Institute, were vaccinated with irradiated autologous tumor cells to gether with IL-4 gene-transduced irradiated autologous fibroblasts. Th e level of expression of the IL-4 gene in cultured transduced and sele cted fibroblasts and in biopsies obtained from vaccination sites was e valuated using quantitative reverse transcription-polymerase chain rea ction (RT-PCR), The number of copies of IL-4 mRNA/ng of total cellular RNA was determined in the transduced fibroblasts. Good agreement was observed between IL-4 message expression, as determined by RT-PCR, and IL-4 production, as determined by enzyme-linked immunosorbent assay ( ELISA) in the fibroblast supernatants. Tissue biopsies of multiple vac cination sites were obtained from the patients to determine the level of gene expression in situ for IL-4 and Neo-r. The Neo-r gene was used as a marker for transduced fibroblasts. Two weeks after the first vac cination, mRNA for the IL-4 gene was still detectable in all tissue bi opsies. The Neo-r gene was also detectable, indicating the presence of transduced fibroblasts in the biopsy. After the second vaccination, e xpression of the IL-4 and Neo-r genes was generally the highest on day 1 after vaccine administration and was considerably lower but still d etectable on day 14 in all biopsies tested. These data indicate that a utologous dermal fibroblasts transduced with the IL-4 and Neo-r genes and used as a source of IL-4 in tumor vaccine are able to express the IL-4 gene in vivo.