reached one to two centimeters and an attempt will be made to grow tumor 
infiltrating lymphocytes from this site. Lymphocytes either from draining 
lymph nodes or from the tumor site itself (whichever become available first) 
will be grown in vitro by the standard techniques used in many previous 
protocols and assays published detail. These lymphocytes will then be 
adoptively transferred to the patient along with 720,000 IU/kg of IL-2, 
exactly as in our previous protocols and as previously published in detail. 
The impact of the immunization procedure on established tumor at other sites 
will also be monitored to evaluate whether this immunization procedure itself 
has anti-tumor effects. Biopsies of cutaneous or subcutaneous lesions may be 
performed. 
2 . Preparation of the IL-2-NeoR vector containing supernatant. The 
IL-2-NeoR vector was constructed by modifying the Moloney murine leukemia 
vector by techniques similar to those previously described. Retroviral vector 
supernatant is produced by harvesting the cell culture medium from the PA317 
packaging line developed by Dr. A. Dusty Miller (36,37). This line has been 
extensively characterized and was used by us in our previous studies of the 
infusion of TIL modified by the LNL6 vector (11,12). The IL-2-Neo vector 
preparations from PA317 will be extensively tested to assure that no 
detectable replication competent virus is present. Tests for replication ■ 
competent virus will be conducted on both the vector supernatant and on the 
tumor cells after transduction. The vector includes the retroviral LTR 
p romoting the IL-2 gene followed by the SV-40 early promoter and the gene 
coding for neomycin phosphotransferase. Testing will be the same as 
previously approved for the LNL6 supernatants used to introduce the NeoR gene 
into TIL (protocol 86-C-183c). The following tests will be run on the 
producer line and/or the viral supernatant: 
1) The viral titer will be determined on 3T3 cells. Viral 
preparations with titers greater than 5 x 10 4 colony forming units/ml will be 
used. 
2) Southern blots will be run on the producer line to detect the 
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