SUCCESSFUL CULTURE AND SELECTION OF CYTOKINE GENE-MODIFIED HUMAN DERMAL FIBROBLASTS FOR THE BIOLOGIC THERAPY OF PATIENTS WITH CANCER

Human autologous dermal fibroblasts have been cultured, transduced wit h the interleukin-4 (IL-4) gene and used as a vaccine together with ir radiated autologous tumor cells in patients with cancer participating in a phase I/II clinical trial at the University of Pittsburgh Cancer Institute. In support of this clinical trial, methods have been devise d to facilitate isolation of fibroblasts from freshly harvested skin s pecimens, to enhance their outgrowth in large-scale cultures, and to a ssay cytokine (IL-4) production following transduction with the cytoki ne gene +/- irradiation. Fibroblasts were isolated from skin specimens by enzymatic digestion, grown in primary cultures, and transduced wit h a retroviral vector containing the gene for human IL-4 and the Neo(R ) gene as a selectable marker. Following selection in G418, the irradi ated, IL-4-producing fibroblasts were administered to patients in a va ccine containing irradiated autologous tumor cells. Seventy-eight spec imens of human skin were processed to obtain fibroblast suspensions. C ultures of fibroblasts were established from 68 of the 78 specimens (8 7%). Of 33 transduced and selected fibroblast cultures, 21 produced at least 1,000 units of IL-4/24 hours per 10(6) cells, as determined by ELISA, and 17/33 or 51% were used For therapy. The primary cultures we re typically maintained for up to seven or eight passages. The mean +/ - SD overall rime for obtaining a required number of transduced, selec ted cells was 53 +/- 4 days. The fibroblasts continued to produce IL-4 in culture for 3 weeks even after irradiation. Similar results have b een obtained with a retroviral vector encoding IL-12. This study shows that human dermal fibroblasts can be consistently and reproducibly ex panded and genetically modified to serve as a source of cytokines or o ther gene products for gene therapy trials.