Protocol ID93-008 
Page 8 
(c) Production of purified CD3 ~*~ CD8 ~^ TIL 
The T-150 CELLector devices will accommodate up to 9x10^ CD8'*' cells of interest 
(incorporated in Standard Operating Procedure from AIS) (detailed procedure also on 
ftle with Regulatory Affairs at NIH, Room 4B1 1). CD3'*' CD8+ TIL are purified from the 
expanding TIL culture by positive selection when the number of cells in culture reaches 
approximately 4.5x10®. In some cases negative selection may be required to remove 
double stained cells. An aliquot of CD8+ TIL harvested from the CELLector device are 
infected with GlNa - (neo^J vector as described in (d). The remaining cells that are not 
transduced will be transferred to one of two ACCS cartridges at 1x10^. 
(d) Preparation of neo^ marking vector and conditions of exposure to TIL cells 
(1) The safety-modified marking vector GlNa is a replication incompetent retroviral vector 
which has been extensively used in marking human hematopoietic cells for infusion 
into human subjects as part of therapeutic procedures. This vector is prepared by 
Genetic Therapy, Inc. of Gaithersburg. Maryland as a clinical grade cell-free 
supernatant from the PA317/GlNa40 producer cell line in DMEM medium at a titer of 
> 1x10® transducing units/cc. The procedures for preparing this vector have been 
previously reviewed and approved by the FDA.. 
The conditions of exposure of the CD8+ (and CD4+) T-cell lines to the marking vector 
GlNa are as follows: 
(2) The purified TIL cells detached from the CELLector flask (in the range of IxlO^ to 
2x10®) will be divided into two parts. One part of approximately 1x10® cells will be used 
for marking with neo^ . and these cells will be expanded in one ACCS cartridge.. The 
second part comprised of the remaining cells will not be infected but will be expanded 
directly in a second ACCS. 
(3) After thawing, the vector supernatant will be added in a laminar flow hood dedicated for 
clinical studies to the one portion of CD3+CD8+ cells in rIL-2 to reach a final 
concentration of 2-4 MOI per cell. This will require 2-4 cc of GlNa supernatant per 
Infection, assuming there are 1x10® cells. The viral supernatant is added with rIL-2 
(600 lU/ml) to the cells for 6 hours. The cells are pelleted and resuspended in 
conditioned complete AIM V medium up to 24 hours (1/3 conditioned AIM V and 2/3 
new AIM V + rIL-2 (600 lU/ml). The infections are repeated twice at 24 hour intervals. 
Between infections cells will be maintained in conditioned complete AIM V medium (i.e., 
containing rIL-2) after removing viral supernatant. rIL-2 concentration of 600 IU/10® 
cells will be maintained in the viral supernatant. 
(4) Preservative-free Protamine sulfate (I.V. grade, material). Elkins-Sinn, Inc. NDC# 0641- 
2554-41, at 10,000 ug/ml for I.V. use) will be added at 4 ug/ml during infection. 
(5) Following incubation, the cells will be collected from the supernatant and pelleted by 
spinning at 450 x g for five minutes, washed three times in complete AIM V and then 
counted. 
(6) The neoF transduced cells will undergo completed expansion in a parallel ACCS 
cartridge. 
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Recombinant DNA Research, Volume 19 
