5.0 Therapeutic Modalities 
5.1 Preparation and Characterization of Biological Agents 
5.1.1 Genetically Modified Autologous Tumor Cells and Fibroblasts 
Skin punch biopsies and clinically indicated colon tumor resections will be obtained under sterile 
conditions. The tumor tissue will be minced and prepared as a single cell suspension for 
cryopreservation as described for the large prospective, randomized trial of adjuvant active 
immunotherapy of colon cancer with autologous tumor (14). The skin biopsy tissues will be minced 
and placed in DMEM media containing 10% fetal calf serum to establish growth of skin fibroblasts in 
culture. The cultured cells will be genetically modified by retroviral mediated gene transfer to 
express the IL-2 gene product. 
5.1.2 Retroviral Vector 
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l 
The retroviral vector utilized in this study is derived from a murine retrovirus similar to the vectors 
employed by a number of investigators for in vitro and in vivo studies including recently approved 
investigations with human subjects (15). The cytokine vector used in this investigation (LNCX/IL-2) 
contains a recombinant human IL-2 cDNA under the control of an internal CMV promoter. Before 
utilization in human studies, the transduced cells will be certified to be free of contaminating 
replication competent virus and other adventitious agents by criteria recommended by the FDA. The 
assays to be performed will include the following: 
Sterility testing by bacterial and fungal culture 
Mycoplasma testing by Gen-Probe kit assay 
Replication competent retrovirus testing by PG-4 S+L- assay 
Additional safety and quality assurance assays will be performed as described in Appendix 12.5 
(Laboratory Procedures and Safety Testing) 
5.1.3 Transduction Protocol 
The cultured skin fibroblasts will be incubated with conditioned supernatant from the producer cell 
culture transfected with the retroviral vector plasmid as described (16). The fibroblasts will be 
washed and then grown in culture media containing G418, (a neomycin analogue) to select for 
transduced cells expressing the neo R gene. G418-resistant cells will be stored at -70° C until 
required. Prior to immunization, the G4 1 8-resistant cells will be tested for expression of the IL-2 
gene by measurements of the concentration of IL-2 in the culture supernatant by an enzyme-linked 
immunoabsorbent assay (ELISA). 
5.1.4 Preparation of Irradiated Cells 
After transduction and expansion, the genetically modified cells will be carried in continuous culture 
until there are sufficient cells for use in therapeutic applications or for cyropreservation. The 
cyropreserved cells will be centrifuged and washed in DMEM media and then cryopreserved in a 
solution containing 10% dimethyl sulphoxide and 50% fetal calf serum in DMEM media. The cells 
will be stored in liquid nitrogen until the time of administration. Non-transduced tumor cells utilized 
for immunizations will be treated with 10,000 rads as described for the adjuvant active 
immunotherapy trial of colon cancer with autologous tumor (14) and resuspend in lactated Ringer's 
solution prior to injection. Transduced fibroblasts will be irradiated with 3,000 rads to minimize the 
risk of chronic local inflammatory reactions at the site of immunization due to continued secretion of 
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Recombinant DNA Research, Volume 18 
