Ten days later TDLN were harvested and activated by the anti-CD3/IL-2 method. Antitumor 
reactivity was assessed by the adoptive immunotherapy of A9 pulmonary metastases as 
previously described. As shown in Table 5, A9PLJ1 did not elicit therapeutic TDLN, however, 
the use of C. parvum plus A9PLJ1 resulted in activated TDLN with antitumor efficacy 
confirming our previous findings. In comparison, activated TDLN from mice inoculated with 
A94-3 alone had comparable antitumor reactivity compared to cells from mice inoculated with 
A9PLJ1 plus C. parvum . In the same experiment, we examined if there was an additive effect 
of C. parvum with A94-3 cells in the stimulation of pre-effector TDLN cells. The addition of 
parvum to A94-3 did not enhance nor abrogate the antitumor reactivity of the activated TDLN 
cells in adoptive immunotherapy. 
Table 5: Immunotherapy of 3-Day BL6 Lung Metastases with Activated TDLN Cells: Effect of 
C. parvum as an Adjuvant 
Source of TDLN 
Tumor C. oarvum 3 
Cells 
Transferred 15 
Mean Pulmonary 
Metastases (SEMI 
- 
- 
- 
> 250 
A9PLJ1 
- 
+ 
224 (21) 
A9PLJ1 
+ 
+ 
115 (21 ) c 
A94-3 
- 
+ 
125 (29) c 
m3 
+ 
+ 
123 (35) 
a C. parvum (13 meg) admixed with 10 6 tumor cells followed by TDLN harvest 10 days later. 
b 5 x 10 7 cells were transferred per mouse. All mice received 15,000 u IL-2 i.p. bid for 4 days 
after cell transfer. 
c p < 0.05 compared to no cells and A9PLJ1 control groups. 
Our group has already established considerable experience in utilizing autologous 
tumor cells plus the bacterial adjuvant, BCG, to stimulate LN cells in cancer patients for 
subsequent adoptive immunotherapy. The preliminary results of this experience appears to 
be very encouraging for patients with renal cell cancer; however, the need to develop 
improved methods to treat melanoma is apparent. The studies summarized in this section 
indicate that IL-4 transfection of the B16BL6 melanoma can upregulate T cell sensitization in 
draining LN. These draining LN can be further activated in vitro to successfully eradicate 
established disseminated parental tumors. We therefore propose in this study to vaccinate 
patients with autologous tumor cells which are transduced with the IL-4 gene. Upon 
documentation of IL-4 production by the tumor cells, they will be irradiated and inoculated i.d. 
into patients near LN bearing areas. Approximately 7 to 10 days later, draining LN will be 
removed for anti-CD3/IL-2 activation. The activated cells will be subsequently infused i.v. into 
patients followed by the concomitant administration of IL-2 (360,000 lU/kg q8h for 5 days). 
4.0 DRUG INFORMATION 
4.1 lnt erle uKin-2 
The recombinant IL-2 (Proleukin R ) used in this protocol is manufactured by Cetus 
Corporation, Emeryville, CA and will be supplied by the Cancer Treatment Evaluation 
Program (CTEP), NCI. Proleukin R is a protein manufactured by recombinant technology and 
is similar to the native IL-2 protein found in humans. Proleukin R is supplied as a lyophilized 
cake in 5 ml vials containing 1 mg of protein. Each vial should be reconstituted with 1.2 ml of 
sterile water and gently swirled without excess foaming. When reconstituted each ml 
contains 1 mg (18 million IU) of Proleukin R . For intravenous injection, Proleukin R can be 
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Recombinant DNA Research, Volume 18 
