approximately 2 mm 3 pieces in cold HBSS. Cells will be dissociated by pressing pieces of 
LN through a sterile steel mesh with the blunt end of a 10 ml syringe plunger. The resultant 
cell suspension will be filtered through a double layer of no. 100 nylon mesh. After washing 
the cells in HBSS x 3, they will be placed in anti-CD3 activation cultures immediately and the 
remaining LN cells will be cryopreserved in liquid nitrogen for subsequent analysis and 
possible use in retreatments. All procedures will be carried out under sterile conditions. 
6.5 Anti-CD3 Activation and Expansion 
Animal studies of adoptive immunotherapy have clearly demonstrated that the 
numbers of transferred effector cells are directly related to the antitumor effects which were 
observed. Clinical studies which have reported tumor responses from the adoptive transfer 
of LAK or TIL cells have generally administered 10 10 to 10 1 1 effector cells (1 ,2). From our 
current clinical studies, we have been able to generate these numbers of human activated 
LN cells utilizing the proposed anti-CD3/IL-2 activation procedure (see Section 2.2). This 
required culturing cells an average of 15 days and subjecting the draining LN cells to a 
primary and secondary culture procedures. The methods for the primary and secondary 
culture procedures utilized in this clinical study are described below: 
6. 5. a Primary Anti-CD3 Activation 
For primary anti-CD3 activation, 5 x 10 8 lymphocytes will be 
suspended in 250 ml of complete media (CM) and cultured in 24-well tissue 
culture plates pre-treated with OKT3. Plates are coated with mAb by adding 
400 |il of diluted OKT3 (1 |ig/ml) in sterile 0.05 M borate buffer, pH 8.6 per well 
and stored overnight at 4°C. Prior to use, the plates are washed of excess mAb 
with PBS. CM is X-Vivo-15 (Whittaker) supplemented with 10% human AB 
serum (Sigma). After 48 hours of activation the cells are harvested and 
expanded in IL-2. 
Expansion in IL-2 is accomplished by suspending 3 x 10 5 anti-CD3 
activated cells/ml by diluting the primary activation media with X-Vivo-15 
containing 5% human AB serum supplemented with 60 lU/ml of IL-2 (Cetus, 
Emeryville, CA). These cells are expanded in 3000 ml culture flasks (Lifecell 
3000, Fenwal, Deerfield, IL) each containing 500-850 ml of media. When cell 
density reaches approximately 10 6 cells/ml, an additional 2 liters of serum-free 
X-Vivo-15 is added to each flask. Cells are grown to a maximum density 
(approximately 2 x 10 6 cells/ml) which is anticipated to take 5 days based on 
our current experience. 
6.5. b Secondary Anti-CD3 Activation 
The procedure to perform the secondary anti-CD3 activation 
represents a slight modification of the primary culture procedure. Briefly, 2 x 
10^ anti-CD3 activated cells derived from the primary culture procedure will be 
suspended in 100 ml of CM in 150 cm 2 flasks with immobilized OKT3 mAb. 
After 1 day, the cells are harvested, washed and expanded in serum-free media 
(X-Vivo-15) containing IL-2 (60 lU/ml) using 3000 ml culture bags as previously 
described. Cells are harvested after a 3-4 day period when cells reach their 
maximum density. It is anticipated that each patient will require 50-100 L of CM 
to generate anti-CD3 activated cells from primary and secondary culture 
Recombinant DNA Research, Volume 18 
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