' 
pulmonary metastases. IL-2 (15,000 u i.p. bid x 4 days) was started on the day of adoptive 
transfer. 
Table 2: Antitumor Efficacy of Anti-CD3/IL-2 Activated Cells Derived from B16 BL6 Draining I 
LN is Dependent upon Amount of C. parvum . 
Anti-CD3 
activated cells 
C. parvum (ugm) 
Mean no. pulmonary metastases (SEM) 
- 
- 
> 250 
+ 
0 
209 (38) 
+ 
12.5 
8(6)* 
+ 
25 
<1 (<ir 
+ 
50 
187 (30) 
+ 
100 
173 (20) 
p < 0.05 compared to group not receiving cells. 
We also examined the kinetics of pre-effector cell induction after vaccine-priming. In 
this experiment, mice were inoculated with 10 6 viable or 10 7 irradiated (4,000 cGy) B16 BL6 
tumor admixed with 12.5 ngm C. parvum s.c. every few days so that draining LN could be 
obtained on the same day for activation. The development of pre-effector cells in the draining 
LN was evident as early as day 4 after tumor priming but disappeared after day 14. These 
findings confirmed that irradiated B16BL6 tumor expressed appropriate tumor antigens and 
that the host immune response could be modulated to generate anti-CD3/IL-2 activated LN 
cells against this poorly immunogenic tumor. Based on these data, we are currently 
conducting a clinical study of similarly generated anti-CD3/IL-2 vaccine primed LN cells in 
patients with metastatic melanoma and renal cell cancer. 
3.2 Clinical experience with human tumor-primed LN cells. 
Extrapolating methods from our animal models, we initiated a clinical trial to evaluate 
the antitumor reactivity of tumor-primed IVS-LN cells (16). Patients with advanced melanoma 
or renal cell cancer were eligible. Patients were tumor-primed by the intradermal inoculation 
of 1-2 x 10 7 irradiated (2,500 cGy) autologous tumor admixed with fresh-frozen Tice BCG 
(1 0 7 cfu) in two separate sites. These sites were either on the anterior thigh or axilla in order 
that draining LN could be surgically removed under local anesthesia 10 days later for 
subsequent IVS culture. The use of BCG was employed since previous clinical studies 
demonstrated that it was an effective immune adjuvant in eliciting cellular immune responses 
when administered in conjunction with irradiated tumor (17,18). The yield of primed LN cells 
from 10 patients averaged 1.3 x 10 9 cells with removal of clinically hyperplastic LN. There 
were no complications associated with the tumor vaccination procedure nor removal of the 
primed LN. 
Upon retrieval of primed LN, the lymphocytes were isolated and placed in IVS culture. 
IVS cultures were established in gas-permeable culture bags with complete media (CM) 
containing 1-2 x 10 5 LN cells/ml and 1-4 x 10 5 irradiated tumor cells/ml. CM consisted of 
RPMI 1640 with 10% human AB sera, 100 Cetus u/ml IL-2, antibiotics, L-glutamine, non- 
essential amino acids and sodium pyruvate. Culture bags were incubated at 37°C in 5% 
CO 2 for 1 0-1 5 days. Cells were harvested after tumor cells cleared from the culture and the 
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Recombinant DNA Research, Volume 18 
