Additional assays will be performed as recommended by the FDA and outlined in Appendix 12.5. 
5.13 Transduction Protocol 
The cultured tumor cells will be incubated with conditioned supernatant from the producer cell culture 
transfected with the retroviral vector plasmids as described (15). The tumor cells will be washed and then 
grown in culture media containing G418, (a neomycin analogue) to select for transduced cells expressing 
the neo^ gene. G418-resistant cells will be stored at -70° C until required. Prior to immunization, the 
G418-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). 
5.14 Preparation of Irradiated Cells 
After transduction and expansion, the genetically modified cells will be carried in continuous culture until 
there are sufficient cells for therapeutic applications and cyropreservation. The cyropreserved cells will 
be centrifuged and washed in Richter's zinc option media and then cryopreserved in a solution containing 
10% dimethyl sulphoxide and 50% fetal calf serum. The cells will be stored in liquid nitrogen until the 
time of administration. Prior to their use in subcutaneous immunizations, the cells Avill be irradiated and 
resuspended in lactated Ringer's solution prior to injection. The transduced tumor cells will be treated 
with 7,000 rads prior to their use in immunizations. This dose of radiation treatment to the glioblastoma 
tumor cells was based on discussions with Dr. Herman Suit, Chief of Radiation Oncology at 
Massachusetts General Hospital. Dr. Suit indicated that based on data generated in his laboratory with 
22 glioma cell lines, 7,000 rads would be the lowest radiation treatment sufficient to render the planned 
tumor cell doses incapable of proliferation and tumor formation. This dose was calculated from the 
following formula; 
TCDp=D0[LnM+Lnn-(Ln-Lnp)]; where D0=reciprocal of terminal slope of cell survival curve 
(D0=290 rads for the most resistant cell line tested); TCDp=the dose which inactivates all cells at 
probability p=99; M=the number of cells to be treated; and n=extrapolation number (n=10 for the most 
resistant cell line tested). It is our desire to utilize the lowest possible radiation dose for the transduced 
cells to optimize the level and duration of IL-2 secretion. We have evaluated the effects of the proposed 
7000 cGy dose on the GT9 cell line to be utilized in this study. This dose of radiation prevented the 
proliferation GT9 cells without inhibiting IL-2 secretion. The results of these studies are presented in 
more detail in Appendix 12.7 (Pre-Clnical Studies). 
5.2 Potential Toxicity 
Local toxicity at the sites of immunization are not expected to occur. If severe inflammation occurs at 
the site of injection, this will be treated by either topical steroid therapy and/or surgical excision of the 
inflamed tissues as indicated. 
Hypersensitivity reactions are unlikely to occur, since the injected material is comprised of minimally 
modified cells. Systemic toxicity related to IL-2 administration should not occur as the low levels of EL-2 
secreted by the genetically modified cells should not significantly effect systemic IL-2 concentrations. 
There are several theoretical toxicities related to the use of retroviral mediated gene transfer. These 
theoretical toxicities are not expected to occur as the retrovirus utilized for gene transfer has been 
modified to prevent these toxicities. The retroviral vector has been altered to prevent viral replication by 
the deletion of viral gag, pol and env genes. Hence, the IL-2 retroviral vector is replication defective and 
patients' cells are never exposed to a replication competent virus. Replication competent retrovirus may 
theoretically develop by recombination between the IL-2 vector and viral gene sequences in the 
packaging cell line utilized to produce the retroviral vector. This type of recombination has rarely been 
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Recombinant DNA Research, Volume 19 
