Recombinant DMA Advisory Committee - 6/9-10/94 
the insert sequences; therefore, neo^ was inserted between these 2 elements to prevent 
splicing. Autologous fibroblasts will be transduced with the TFG vector, selected in 
G418, tested for IL-12 production, and injected into the patient. Southern blot analysis 
of a fibroblast cell line transduced with TFG demonstrated the appropriate 4 kb band, 
not a 2 kb band. These data indicate that there is no evidence of a splice event. 
Dr. LxDtze explained that the IL-12 cytokine was chosen based on promising preclinical 
data. Data suggests that IL-12 is involved in early immune response and rejection of 
subcutaneous tumors. The interleukins are part of a complex immunologici process in 
which significant knowledge is being gained. He hypothesized that positive results from 
both the IL-12 and IL-4 human trials could lead to a combination therapy of these 2 
cytokines. EL- 12 has been extensively tested in primates and has been demonstrated 
effective at doses much lower than those that are toxic, many orders of magnitude below 
toxic levels observed in primates. 
The lacZ mobilization assay has been used to determine the presence of helper vims. 
Helper vims has not been detected in a background of 1 x 10*^ vector particles. Dr. 
Lotze assured Dr. Miller that the supernatant from transduced autologous fibroblasts will 
be assayed for helper vims prior to administration to the patient. Vector production and 
testing will be performed at the University of Pittsburgh. 
Dr. Lotze summarized the preliminary results of his ongoing EL-4 protocol. A total of 9 
patients have been treated, 5 with melanoma, 1 with renal cell carcinoma, and 3 with 
colorectal carcinoma. Between 80,000 and 100,000 units of TLA are expressed by cells 
injected at the primary and secondary vaccine sites. Tumor specific T cell responses, 
increased numbers of infiltrating T lymphocytes, expression of vascular cell adhesion 
molecules (VCAM) by endothelial cells, and migration of infiltrating lymphocytes out of 
the vessels has been observed in response to this treatment. These responses are 
consistent with the proinflammatory effects of lE^ observed in preclinical experiments. 
Early expression of llAt gene has been demonstrated by RT/PCR assays. These assays 
demonstrate that 7 days after vaccination, the lE^ copy number per ng total RNA is 
substantially reduced in biopsies taken at the injection site. Only the EL-4 producing 
vaccine sites are associated with T cell growth. Patients entered on the TLA vaccine 
protocol have evidence of specific T cell proliferation in their tumors. Another 11 
patients will be entered on this protocol prior to completion of the study. 
Given the unique aspects of IL-12 and EL-4 biology, these 2 cytokines should be tested 
separately. The major difference between the EL-12 and lE^ studies is that the 11^12 
fibroblasts will be injected directly into the tumor. The II^ protocol involves 
subcutaneous injection of transduced cells into the subject's back. The proposed ID 12 
trial is a natural extension of the lE^ study. 
Dr. Lotze presented additional data demonstrating tumor regression in response to 
systemic IL-12 administration in mice. Dr. Miller noted that the vector construct used 
for the murine studies is different from the vector proposed for the human study. The 
murine vector has the neo^ gene at the end of the p40 and p35 subunit genes rather than 
Recombinant DNA Research, Volume 19 
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