Protocol ID93-008 
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associated with objective clinical responses in patients with melanoma (12) and ovarian 
cancer (13). 
2.3 Ovarian tumor specific TIL studies conducted at M.D.A.C.C . 
We have developed T-cell lines from ascites (14,15) or from solid tumors (16,17) of EOC in 
low concentrations of rlL-2. These T-cell lines were either CD3+CD8+CD4'TCRap+ or 
CD3+CD4+CD8*TCRa(l+. Certain T-cell lines of the CD3+CD8+CD4' phenotype exhibited 
cytotoxicity primarily agciinst autologous ovarian tumor cells (14-16). Furthermore, we 
have shown that the T-cell receptor CD3 complex and MHC Class I antigens on tumor cells 
are involved in this tumor specific activity (15). CD3+CD8'CD4+, rIL-2 dependent T-cell 
lines exhibited MHC unrestricted cytotoxicity (15-17). Other investigators have also 
developed T-cell lines from ascites or from solid tumors of patients with EOC (18-22). A 
number of these T-cell lines were developed in higher concentrations of rIL-2 and expressed 
only nonspecific cytotoxicity (18-21). Other T-cell lines developed from 5 of 6 ovarian 
cancer patients by Peoples et al. (22) in low concentrations of rIL-2 exhibited autologous 
tumor specific activity. This is in agreement with our previous reports (14-16). 
2.4 Generation of large numbers flxlO ^Q to 1x10 of TIL- derived T cells for adoptive 
immunotherapy 
Large numbers of rIL-2 expanded TILs have been produced utilizing automated closed 
systems (23,24). We have produced large numbers (IxlO^o to 1x10 ^ M of TIL-derived T-cells 
from patients with ovarian carcinoma using a four-step expansion procedure (17). This 
procedure employs 24 well culture plates, T-flasks, polyolefin gas permeable bags (PGPB) 
(23) and a hollow fiber bioreactor (artificial capillaiy culture system) (ACCS) (17). Mixtures 
of TIL and autologous tumor cells were prepared either from malignant ascites by 
centrifugation on Ficoll/Hypaque or from solid tumor specimens by enzymatic dissociation. 
The mixtures were cultured in AIM V medium supplemented with 600 lU/ml rIL-2 (CETUS) 
in 24-well flat-bottomed tissue culture plates. After one week in culture the expanded TIL 
were transferred to T-75 Falcon plastic tissue culture flasks and after two to three weeks 
approximately 2.5 to 5.0x10® TIL were transferred into gas permeable polyolefin bags 
(Baxter). After 1x10® to 2x10® cells were generated (within a few days) the TIL were 
transferred with the media into a BRB-23B1 cartridge of the ACCS (17) (Cellco, 
Germantown. MD). The TIL were harvested at a mean of 47 days from inception of the 
culture with a mean fold of expansion of 8,044-fold (17). TIL expanded in the ACCS may be 
either CD3+CD4‘^TCRajJ+ or CD3+CD8'nX)Ra<J+. It has been shown (data submitted to 
Master File by Cellco, Germantown, MD) that a modified cellulose fiber with a 50% 
molecular weight cut-off of 30 Kd will support the expansion of T-cells (CD8+ and CD4+). 
We will utilize the AM-FP13E0 cartridge because it requires substantially lower 
concentrations of rIL-2 than that required by the BRB-23B1. 
2.5 A clinical trial in patients with ovarian carcinoma utilizing rlL-2 expanded TIL and low 
concentrations of rIL-2 
A pilot study was conducted in patients with advanced epithelial ovarian carcinoma, who 
were refractory to platinum based chemotherapy to determine the feasibility and clinical 
effects of a schedule of Intraperitoneal (IP) tumor infiltrating lymphocytes (TIL) expanded in 
recombinant interleukin-2 (rIL-2), and low dose rlL-2 IP. TIL were expanded from solid 
metastases or malignant effusions in serum free AIM V medium supplemented with low 
concentrations (600 lU/ml) of rIL-2 using the four-step expansion method described above. 
Patients received IP TIL suspended in 5% dextrose in 0.2% sodium chloride containing 
0.1% human albumin and 6x10^ IL rIL-2 on day 1. followed by 6x10^ lU rIL-2/m2 body 
surface area, administered daily by bolus injection, on days 2 to 4, 8 to 11. and 15 to 18. 
In the absence of disease progression two additional four day cycles of IP rIL-2 were 
administered. Patients (n = 3) whose TIL failed to grow in vitro received IP IL-2 alone. Eight 
patients received rIL-2 expanded TIL (10*®- 10* * range) plus rIL-2 followed by several cycles 
of rIL-2 alone (see above). One of these patients was treated twice with TIL plus cIL-2. 
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Recombinant DNA Research, Volume 19 
