Scientific Abstract 
1. A one page scientific abstract of the protocol 
The incidence of breast cancer is steadily increasing. Approximately 1 in 8 American 
women will develop breast cancer. At the present time, the metastatic form of breast 
cancer that has failed all conventional types of therapy has a poor prognosis. The 
ineffectiveness of hormonal therapy and chemotherapy has led investigators to evaluate a 
variety of immunotherapy regimens. The ultimate goal has been to consistently augment 
the immunological reactivity of breast cancer patients to their own tumor. In other human 
tumor systems, active specific immunotherapy (treatment with autologous tumor cells) has 
been shown to be effective in some patients with metastatic disease. 
Berd et. al. immunized patients with irradiated autologous tumor cells and noted clinical 
responses in 25% of the patients treated. Other investigators have reported similar response 
rates in patients with renal cell carcinoma and colon carcinoma. Mitchell et. al. has 
reported clinical responses and stimulation of specific cytotoxic T-lymphocytes (CTL) in 
patients immunized with autologous melanoma cells. Seigler et. al. have serially injected 
high risk Stage I and Stage II melanoma patients using either x-irradiated melanoma cells 
or a vaccinia viral oncolysate of the tumor cells and have demonstrated improved survival 
in patients receiving adjuvant active treatment. 
We have demonstrated that tumor specific CTLs can be generated in vitro by exposing 
patient lymphocytes to autologous tumor cells in the presence of a low dose interleukin-2 
(unpublished data). The specific CTLs have effected cell mediated killing both in vitro and 
in vivo (unpublished data). Specific CTLs can also be generated against breast cancer 
specific antigens such as oncoproteins and peptides . In addition, CTLs were shown to 
recognize endogenously synthesized proteins and presented on the surface together with 
major histocompatibility (MHC) class I molecules (Maryanski JL, Pala P, et. al. 1986; 
Townsend ARM, Rothbard J, et. al. 1986; Moore MW, Carbone FR, et. al. 1988). 
Consequently, antigens include any primary amino acid sequence in any cellular proteins, 
either membrane bound or intracellular. 
A variety of lymphokine genes have been introduced into tumor cells using retroviral 
vectors as well as non-viral delivery methods. The transduced cells express the inserted 
gene and secrete the specific lymphokine. We have used cationic liposomes to facilitate 
adeno-associated virus (AAV) plasmid transfections of primary and cultured cell types. 
AAV plasmid DNA complexed with liposomes (Avectin™) showed levels of expression 
several fold higher than those of complexes with standard plasmids. In addition, long-term 
expression (>30 days) of the gene, unlike the transient expression demonstrated by typical 
liposome-medicated transfection with standard plasmids, was observed. Primary breast, 
ovarian, and lung cultured tumor cells were transfectable with the AAV plasmid DNA- 
liposome complexes. Transfected primary and cultured tumor cells were able to express 
transgene product even after lethal irradiation. Transfection efficiency ranged from 10 to 
50% as assessed by intracellular IL-2 levels in IL-2 transfected cells. 
The primary objective of this protocol will be to evaluate safety, biological response, and 
survival in patients serially treated with autologous breast cancer cells transduced with the 
gene for human Interleukin-2 (IL-2) via the Avectin™ method. We will monitor the safety 
and toxicity of the treatment and will attempt to determine its immunological effects on the 
patient. We will also evaluate the clinical responses and duration of any such response to 
this treatment. 
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Recombinant DNA Research, Volume 20 
