Human Gene Therapy Subcommittee - 11/21-22/91 
administered, valuable information will be derived regarding the ability to ablate the T 
cell clones. The investigators will then have the option to retreat patients with non- 
transduced clones if their administration has been proven to be safe. 
Dr. Mclvor asked Dr. Greenberg to address the characterization of HyTK. What 
biochemical studies have been performed? Have inhibition studies been performed with 
ganciclovir? Have studies been performed on the enzymology of the fusion protein? Dr. 
Greenberg said that his collaborator, Dr. Bob Overell of Immunex, would present data 
regarding the biochemical characterization of HyTK. 
Dr. Leventhal asked if patients develop pneumonitis, how will the investigators 
determine the cause? Dr. Greenberg responded that patients with pneumonitis receive 
biopsies in order to evaluate the nature of the infection. These patients undergo lung 
bronchopulmonary lavage and lung biopsy. Dr. Greenberg stated that the patient would 
receive ganciclovir in the event that pneumonitis occurs. 
As a point of clarification. Dr. Epstein asked if the administration of the T cell clones is 
contingent on transduction with the HyTK gene. Dr. Greenberg affirmed that the T cell 
clones would only be administered to the patient if there is evidence of transduction. 
Based on the potential toxicities of T cell clones to recognized viral targets, there may be 
substantial risk to the patient in the absence of the ability to ablate them. The marker 
function of the gene is not the issue; the ablation function is critical. Dr. Epstein asked 
about the perceived risks. Dr. Greenberg responded that lymphocytic alveolitis and CNS 
dysfunction are the two major concerns. Dr. Epstein inquired as to the relative 
magnitude of the risk. What fraction of patients would require ablation? Dr. Greenberg 
stated that true indications for ablation are estimated to be approximately 1 in 4 
patients. 
Presentation-Dr. Overell 
Dr. Walters called on Dr. Overell to present the biochemical analysis data on HyTK. 
Dr. Overell described experiments using a panel of plasmid vectors expressing either the 
hygromycin phosphotransferase (hph) gene, the HSV-TK gene, or the HyTK gene under 
identical transcriptional control conditions. These plasmids were then transfected into 
rat cells that were selected in either hypoxanthine-aminopterin-thymidine (HAT) medium 
or hygromycin B. The ganciclovir sensitivity experiments that Dr. Mclvor inquired about 
have not been performed. However, data suggest that the HyTK protein is more 
effective at conferring ganciclovir sensitivity than the HSV-TK protein. To confirm this 
hypothesis, Dr. Overell presented data in which cells were transfected either with the 
hygromycin gene, the TK gene, or the HyTK gene. The cells were selected in either 
hygromycin B or HAT. Since these are TK(-) cells, HAT selection can be used, 
improving the ablation function. The data suggest that the HyTK is probably a superior 
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Recombinant DNA Research, Volume 15 
