I! 
standardized vaccine such as can be achieved with allogeneic cell lines. Both the 
clinical experience by Mitchell (5) in melanoma and the animal studies employing 
allogeneic lines (15, 16) indicate that the use of allogeneic vaccines would be 
possible if there is sufficient diversity in the vaccines to represent a broad spectrum of 
antigens that can contribute to the immunogenicity of the vaccines. 
3.3 Gene transfer clinical studies 
"Gene Transfer and Therapy" protocols have been approved for human trials 
in the past few years (17). The initial Gene Transfer study was a gene marking study 
by Rosenberg and colleagues to mark tumor infiltrating lymphocytes (TIL) in vitro with 
the neo R gene. These marked TIL were injected back into the patient in order to 
determine the in vivo fate of these cells. This was accomplished by using the 
polymerase chain reaction (PCR) technique to analyze cells isolated from the patient 
over time. These cultured TIL frequently demonstrate in vitro cytotoxicity to the tumor 
from which the cells were isolated and, cause necrosis of tumor deposits when 
injected into the patient (18). While melanoma patients have had up to a 10% 
complete response, the lack of significant response in the majority of treated patients 
has raised questions about the fate of infused TIL. Studies to determine cell survival 
have been limited by the instability of the radionucleotide used to label TIL. To avoid 
this limitation TIL cells were permanently marked with the LNL-6 retroviral vector, 
which contains the neomycin resistance gene as a marker (17). TIL transduced in 
vitro with the LNL-6 retroviral vector have been infused into patients and, 
subsequently, polymerase chain reaction (PCR) was used to detect the neo R DNA 
sequence within a tissues (17). Retrovirally marked cells from 4 of 5 evaluable 
patients could be reisolated and grown in culture in the presence of G418, the 
neomycin analog toxic to mammalian cells. Using PCR, neo R TIL could be 
consistently detected in the circulation for three to eight weeks post-infusion. The 
neo^ DNA could be detected by PCR in biopsies of patient tumor deposits for up to 64 
days (17). 
There have been two gene therapy protocols approved (19, 20). One protocol 
by Blaese and colleagues has genetically altered lymphocytes from adenosine 
deaminase deficiency patients. They have inserted the gene for adenosine 
deaminase (ADA), the defective gene in this disease, into these patients’ lymphocytes 
The transduced cells are g-own to large numbers and then infused into the patient. 
The hope is that these cells with a normal ADA gene will also function normally and 
cure the immunodeficiency found in these patients. The second gene therapy protocol 
also involves the use of TIL. Clinical studies using recombinant TNF to infuse into 
patients have been unsuccessful possibly due to the toxic side effects of TNF. 
TNF producing TIL may be a more effective method to deliver the TNF to the tumor 
deposits. The TIL are genetically modified with a gene encoding the tumor necrosis 
factor (TNF) protein. These TNF producing TIL when in|ected into the patient may 
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