Recombinant DNA Advisory Committee - 10/7-8/91 
Dr. Freeman described his experimental animal model, the K-BALB murine 
fibrosarcoma cell fine. Cells that do not contain the TK gene are not affected by 
ganciclovir treatment. However, the two cell lines that do have the TK gene were killed 
by ganciclovir treatment. Experiments performed with human cells yielded the same 
results. In both cases, the transduced cells were killed by ganciclovir at levels that are 
achievable in patients. In one animal model experiment, the TK+ tumor cells were 
injected subcutaneously and the mice treated with ganciclovir, which resulted in tumor 
regression. The possible effect of in vivo killing of these cells by ganciclovir on 
immunization was discussed. The investigator found that cell killing in vivo by 
ganciclovir provided the ability to reject subsequent tumor challenge. Since it is not 
possible to get the TK gene into all of the patient's cells, the TK+ tumor cells must have 
some effect on the resident, un-modified tumor cells. Dr. Freeman showed data from in 
vitro mixing experiments where there was a mixture of modified and unmodified cells, in 
which the entire population of cells were killed by ganciclovir. He hypothesized that the 
TK+ cells may die, break up, and form vesicles. These vesicles may contain a toxic 
metabolite of ganciclovir that may be transferred to nearby TK- cells. However, the data 
is not conclusive and subsequent experiments will be directed at identifying the 
mechanism by which TK- cells die. 
Dr. Kelley said that it was important to determine the mechanism before proceeding to 
patients. If the killing of resident tumor cells requires cell to cell contact or contact with 
vesicles, then there have to be enough TK+ cells administered to the patient for 
effective therapy. 
Dr. Freeman said that fluorescent activated cell sorting (FACS) analysis showed that 
fragments of the plasma membrane from TK+ cells were present in or on the TK- cells. 
He cautioned that more experiments are necessary to prove this mechanism. He 
proposed doing electron microscopic studies on the cells to determine where the 
membrane vesicles are localized. 
Dr. Freeman restated that the TK+ cells can kill TK- cells in vitro. To assess this effect 
in vivo , TK+ and TK- cells were mixed and injected into animals subcutaneously and 
then treated with ganciclovir intraperitoneally. If at least 50% of the cells were TK+, 
the tumor did not grow. 
Dr. Haselkom asked if Dr. Freeman had ever treated the cells with ganciclovir before 
injecting them into animals. Dr. Freeman said he has not yet performed these 
experiments. 
Dr. Freeman presented data from another animal model experiment which he said has 
some relevance to the patients he will treat. Because these patients have tumor confined 
to their peritoneal cavity, he would like to inject the TK+ cells directly so they will be in 
[14] 
Recombinant DNA Research, Volume 15 
