Em. Elder et al., SUCCESSFUL CULTURE AND SELECTION OF CYTOKINE GENE-MODIFIED HUMAN DERMAL FIBROBLASTS FOR THE BIOLOGIC THERAPY OF PATIENTS WITH CANCER, Human gene therapy, 7(4), 1996, pp. 479-487
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.