localization of the vaccine site for subsequent biopsy in the event a local 
reaction is not apparent. 0.1 ml of vaccine will be injected intradermally at 
each marked site using a 1 ml syringe and a 27 gauge needle. After 2 weeks, the 
5 sites will be excisionally biopsied and evaluated to determine the dose of IL-4 
associated with the most intense mononuclear cell infiltrate. 
This procedure will be repeated in 2 weeks provided that the patient has not 
suffered a severe local reaction such as abscess or ulceration. At this time, 
5 vaccines will be placed over the right lumbar region of the patient's back 
after the sites have been marked as before. Each vaccine will consist of 5 X 10 6 
tumor cells admixed with as many as 10 7 fibroblasts to provide the maximum dose 
of IL-4 that was found to be free of significant toxicity in the primary 
vaccination. All sites of secondary vaccination will be sequentially biopsied 
1, 2, 4, 7, and 14 days later to determine the kinetics of the immune response 
induced by the vaccine. This may help to define the proper time to begin 
systemic IL-2 therapy in subsequent protocols. We anticipate that concomitant 
IL-2 will be necessary to generate a systemic antitumor response from the local 
immune response at the vaccine site. If administered too early, IL-2 may 
attenuate the immune response to the vaccine in view of the previously documented 
ability of IL-2 to decrease DTH reactions to common recall antigens (19). 
3.5 Vaccine Testing: 
Prior to administration of the vaccine, the following evaluations will be 
performed: 
A. Quantitation of IL-4 secretion by transfected fibroblasts- At least 
10 U/10 7 cells/24 hours will need to be produced. Up to 10 7 cells will 
be delivered with the vaccine to give a maximum of 10 5 U/24 
hours/vaccine site. This upper limit is based on our previous IL-4 
protocol in which patients tolerated more than 10 6 U/day given as an IV 
bolus . 
B. Microbiology testing for sterility- After each split of the fibroblasts 
(approximately once a week) , cultures will be sent to the microbiology 
laboratory to detect possible contamination by bacterial organisms. 
Mycoplasma testing will be performed one week before infusion by a 
commercial testing service. 
C. S+/L- assay including 3T3 amplification 
D. Cell viability- Tumor preparation must be at least 50Z viable following 
thawing; if not, Ficoll -hypaque separation to eliminate dead cells may 
be carried out. 
3.6 Immunologic Monitoring: 
Fifty ml of blood will be collected in four green top tubes and 1 red top tube 
on days 0, 7, 14, 21, and 28. Peripheral blood mononuclear cells will be 
cryopreserved at -180° C and serum will be frozen at -20°C. These specimens will 
be used to detect alterations in the phenotype of circulating lymphocytes and to 
quatitate systemic IL-4 levels, respectively. Patients will be evaluated daily 
Recombinant DNA Research, Volume 16 
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