Four weeks after the first Inlection, tumor size will be examined and 
recorded. After recording, one of the injected tumors will be excisionally 
biopcied if possible and evaluated to determine the direct antitumor effect of 
the injection and the dose of IL-12 associated with the most intense 
mononuclear cell Infiltrate. If not possible to excise, 6 mm punch biopsies of 
the sites indicated by India ink will be performed. 
4.5 Preparation Testing: 
Prior to administration of the transduced fibroblast preparation, the 
following evaluations will be performed: 
A. Quantitation of IL-12 secretion by transfected fibroblasts- A minimum 
of 0.5 ng/ 10® cells /24 hrs and no more than 1000 ng / 10® cells /24 hrs of 
IL-12 by transfected fibroblasts will be used as criteria for treatment. IL-12 
expression from the transfected fibroblasts is predicted to be far less than 2 
mg/day total (0.04 mg/kg/day) , and Dose of up to 50 mg/kg/day have been well 
tolerated (see 10.1) (Data on file. Genetics Institute) . 
B. Microbiology testing for sterility- After each split of the 
fibroblasts (approximately once a week) , cultures will be sent to the 
microbiology laooratory to detect possible contamination with bacterial 
organisms. Mycoplasma testing will be performed one week before Infusion by a 
commercial testing service. 
C. S+/L- assay including 3T3 amplification will be performed following 
injection of transduced fibroblasts to detect replication competent 
retrovirus. 
4.6 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 quantitate systemic IL-12 and INF-g levels, respectively. Patients will 
be evaluated daily in the clinic for 1 week after each vaccine is 
administered. The sites will be assessed for the presense of induration, 
erythema, tenderness, ulceration, or infection. Patients' temperatures and 
vital signs will also be monitored. The area of erythema and induration will 
be measured. Injection sites will be excised as described above under local 
anesthesia in sterile fashion. The specimens will be divided into thirds. One 
third will be snap frozen in OCT medium and used for jin situ hybridization 
(appendix 19.5) and immunohistochemistry (appendix 19.6). Another portion 
(1/3) will be snap frozen for extraction of messenger RNA for RT-PCR (appendix 
19.7). The remaining third will be placed in tissue culture with 6000 lU/ml 
IL-2 and complete medium and expanded. Total lymphocytes and phenotype will be 
quantitated at 1 and 2 weeks. If cells expand well, cytotoxic assays and 
proliferation against autologous tumor will be evaluated. Serum will be tested 
for IL-12 levels and IFN-g levels. 
4.7 Periodic Monitoring for Evidence of Replication Competent Retrovirus. 
As recently required by the FDA, we will monitor this possibility 
periodically. We will perform serological assays for evidence of antibody to 
retroviral envelope proteins, assays for reverse transcriptase, and direct 
Recombinant DNA Research, Volume 19 
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