T cells with an APC is unknown and may result in apoptosis rather than further 
expansion of the desired T cell population. Despite these uncertainties, we 
believe that the existing data warrant initiation of the clinical trial. 
II. Objectives 
1. To determine the maximum number of B7-trans fected allogeneic melanoma cells 
(the maximum tolerated dose) that can be administered subcutaneously to 
patients with metastatic melanoma. 
2. To characterize the immune response to administration of B7-trans fected 
cells . 
3. To characterize the toxicity of the immunizations and to observe any 
antitumor responses. 
III. Cell Culture Methods and Preparation of Cells for Administration 
1. The cell culture medium is composed of Dulbecco's Modified Eagles Medium 
(DMEM) containing 10% fetal bovine serum (FBS), G418 (geneticin, Gibco/BRL, 
800 mcg/ml), penicillin G sodium (50 units/ml), streptomycin sulfate (50 
mg/ml), and sodium pyruvate (0.1 gm/liter) . The cells are frozen in cell 
culture medium containing 20% FBS and 10% DMSO. 
2. Frozen cells are stored in liquid nitrogen until needed. An ampule of the 
appropriate cell line is then removed and transported to the lab in dry ice. 
The ampule is rapidly thawed in a 379: water bath, and the cells in freeze 
medium are gently pipetted into a 15 ml conical tube. Cell culture medium (13 
ml) is added slowly to the cells. The tube is spun at 1000 rpm for 5 minutes, 
and the cell pellet is resuspended in culture medium and placed into a T25 or 
T75 flask and culture is begun at 379; in 5% C02- Viability of the cell lines 
are > 90% upon thawing. 
3. When the cells become confluent they are transferred into larger culture 
vessels. Culture medium is removed by aspiration and 4 ml of 0.25 % trypsin 
(Gibco/BRL) is added per T75 flask. When the cells have detached from the 
substratum, the cell suspension is passed into a vessel containing fresh 
culture medium. The cells will ultimately be expanded into Nunc triple flasks 
and grown until an appropriate number of cells is obtained. One Nunc triple 
flask supports the growth of greater than 5 x 10 7 of the human melanoma cell 
lines used in this protocol. 
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Recombinant DNA Research, Volume 18 
