g. Liver function tests: total bilirubin <2.0 mg.dl, SGOT <70 IU/L, SGPT <90 
IU/L. 
h. 18 years or older 
5.2 Patients will be excluded for: 
a. Prior second malignancy (except squamous or basal cell skin cancer) 
b. Previous severe reactions to any blood product. 
c. Chronic renal disease with creatinine > 2X normal 
d. Previous chemotherapy, radiation therapy, or steroids within 4 weeks. 
e. Brain metastasis. 
f. Active infection requiring treatment or unexplained febrile illness, any 
collagen disease or autoimmune process. 
g. History of ischemic or congestive cardiac disease requiring chronic 
medication (NYHA class III, IV) or evidence of ischemic change or 
venticular ectopy (>4/min) on electrocardiogram, or evidence of type II AV 
block. Any evidence of prior or current cardiac disease as determined by 
stress test and EKG. 
h. Functional respiratory impairment (FEV-| < 2.0 liters or < 75% predicted) 
i. Pregnancy, or men and women of procreation potential who refuse to 
take precautions to avoid pregnancy. 
j. Patients who are HIV positive. 
6.0 TREATMENT PLAN 
Patients with advanced melanoma will undergo surgical retrieval of tumor for use as a 
tumor vaccine. A tumor cell line will be established in culture and subsequently transduced 
with a retroviral vector containing the IL-4 gene. The transduced line will be screened for IL-4 
production and subsequently irradiated with 5,000 cGy. Patients will be vaccinated with 10 7 - 
10 8 irradiated transduced tumor cells intradermally in 1 or 2 sites. Surgical removal of 
draining LN 7 to 10 days after vaccination will be performed to obtain lymphocytes for in vitro 
activation with anti-CD3 monoclonal antibody (OKT3) and expansion in IL-2. After in vitro 
activation, these lymphocytes will be adoptively transferred intravenously to the patient 
followed by the administration of IL-2 (360,000 IU i.v. every 8 h. for 5 days). Fifteen patients 
will be treated to gain sufficient information regarding toxicity, antitumor reactivity and 
immunologic function. Retreatment will be considered if the patient has a response and 
cryopreserved LN cells are available. This retreatment will be performed 2 months after 
completion of the last therapy and will involve repeat infusion of activated lymphocytes and 
IL-2. The procedures for tumor preparation, LN cell preparation and anti- 
CD3/IL-2 activation have been previously reviewed and approved by the FDA 
(IND 3860) for prior clinical studies. 
6.1 Preparation of Single Cell Suspensions and Cultures from Solid Tumors 
Resected tumors will be collected at surgery and transported in cold (4°C) RPMI 1640 
media (GIBCO) until processing. Necrotic tumor and connective tissue will be trimmed and 
the remaining specimen will be minced to approximately 3-4 mm3 pieces with scissors in 
HBSS containing 2.5 units/ml hyaluronidase, 0.5 mg/ml collagenase and 0.05 mg/ml 
deoxyribonuclease. All enzymes are obtained from Sigma Chemical Co., St. Louis, MO in 
lyophilized form. The enzymatic digestion of solid tumors will be carried out at room 
temperature for 3-5 hours with constant stirring in sterile trypsinizing flasks containing 
magnetic stirring bars. The stirring plate will be set between 3-4 to allow turbulence without 
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