employing IL-2 gene transfer, the treated animals also developed systemic anti-tumor immunity and 
were protected against subsequent tumor challenges with unmodified parental tumor (10,11). Similar 
inhibition of tumor growth and protective immunity were also demonstrated when immunizations were 
performed with a mixture of unmodified parental tumor cells and genetically modified tumor cells 
engineered to express the IL-2 gene. No toxicity associated with expression of the cytokine 
transgenes was reported in these animal tumor studies (10-13). 
We have successfully induced anti-tumor immunity in a model of colorectal carcinoma by 
immunization with a mixture of irradiated tumor cells and IL-2 transduced fibroblasts. Immunization 
with a mixture of irradiated tumor cells and IL-2 transduced cells induced systemic anti-tumor 
immunity capable of rejecting a subsequent live tumor cell challenge. Similar inhibition of tumor 
implantation and the induction of systemic anti-tumor immunity with tumor cells genetically modified 
to express IL-2 were demonstrated by Fearon et al (10) in the same colorectal carcinoma model 
utilized in our studies and by Gansbacher and co-workers (11) in a murine model of fibrosarcoma. 
Our study extends these observations by further demonstrating the ability of IL-2 gene therapy to 
eradicate established tumors. Repeated immunizations with a mixture of irradiated tumor cells and 
IL-2 transduced cells abolished visible tumors in a subset of the treated animals. The results of these 
pre-clinical animal studies are described in detail in Appendix 12.6. 
2.3 Immunotherapy of Colon Carcinoma 
Colorectal carcinoma is one of the most common cancers in the United States and Europe with an 
annual incidence of greater than 150,000 in the U.S. Most patients are treated with tumor resection 
and do not have clinically detectable tumor following surgery. However, the majority of patients 
have microscopic metastases and eventually relapse with clinically overt disease in the liver or 
abdominal cavity. The large number of patients and the small tumor burden following surgical 
resection makes colorectal carcinoma an attractive candidate for adjuvant immunotherapy trials. It is 
generally acknowledged that immunotherapies are likely to be most effective when the tumor burden 
is low. In this regard, the immunomodulator Levamisole is currently approved for the treatment of 
patients with Duke's C tumors (metastases to abdominal lymph nodes). In addition, encouraging 
results have been obtained with an autologous tumor vaccine as an adjuvant therapy following tumor 
resection (14). In this study, immunization with autologous tumor preparations and the adjuvant BCG 
resulted in a significant increase in disease free and total survival (14). 
2.4 Gene Transfer into Human Subjects 
The methods we will utilize to transfer cytokine genes into cells are virtually identical to those 
employed in previously approved human gene transfer studies in which tumor infiltrating lymphocytes 
(TILs) were modified by retroviral gene mediated transduction and administered to cancer patients 
(15). In this earlier Phase I study of retroviral mediated gene transfer, TILs were genetically 
modified to express the neomycin phosphotransferase (neo^) gene. Following intravenous infusion, 
polymerase chain reaction analysis consistently demonstrated the presence of genetically modified 
cells in the circulation for as long as two months after administration. No infectious retroviruses were 
identified in these patients and no side effects due to gene transfer were noted in any patients (15). 
Additional Phase I clinical protocols employing similar retroviral gene transfer methods have recently 
been approved by the Recombinant DNA Advisory Committee (RAC). Our Phase I study of human 
cancer patients will utilize the same gene transfer methods and safety testing procedures employed in 
these previously approved investigations. 
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Recombinant DNA Research, Volume 18 
