RENAL 
SUBGROUP 
#PTS 
CELLS 
VECTOR 
BIOLOGICAL 
A 
5 
bulk TIL 
bulk PBL 
LNL6 
GIN 
IL-2 + IFNaA 
B 
5 
CD8( + )TIL 
CD8( + )PBL 
LNL6 
GIN 
IL-2 + IFNaA 
C 
5 
CD4( + )TIL 
CD4( + )PBL 
LNL6 
GIN 
IL-2 + IFNoA 
D 
5 
CD8( + )TIL 
CD4( + )TIL 
LNL6 
GIN 
IL-2 + IFNaA 
The preparation of bulk TIL, CD8( + ) TIL, CD4( + )TIL, bulk PBL, CD8(+) PBL and CD4( + ) 
PBL, and the protocol for IL-2 expansion are described in Appendix II. Briefly, metastatic 
tumor deposits are dissociated into a single cell suspension by mechanical and enzymatic means. 
Bulk TIL are generated by placing Ficoll-Hypaque enriched leukocytes into AIM V medium 
supplemented with IL-2 (1000 u/ml) . CD8( + )TIL and CD4( + )TIL are prepared by enrichment 
in Applied Immune Sciences (AIS) CELLector™ CD8 or CD4 Cell Culture Flasks and placement 
into AIM V + IL-2. Our ability to generate highly enriched CD8(+) and CD4(+) TIL is 
shown in Appendix II. PBL are prepared from 40 cc heparinized patient blood using 
centrifugation and expansion in AIM V + IL-2. PBL are selected in a similar fashion. After 
satisfactory growth has been confirmed, these cell populations will be transduced with the 
respective retroviral vectors LNL6 or GIN and then transferred to culture bags for large scale 
expansion for another 4-6 weeks. 
After achieving sufficient numbers of cells for treatment (10 9 - 10 n ), they will be sequentially 
infused into the patient and supported by IL-2 or IL-2/IFNoA. 
The LNL6 retroviral vector carries the neoR gene and is approved by the Food and Drug 
Administration for clinical use. The GIN vector is derived from LNL6 and differs only in the 
addition of polylinker sequences. FDA approval for use of this vector is pending. The 
additional poly linker sequence in GIN permits this vector to be distinguished from LNL6 using 
different PCR primers (see Appendix I). 
At intervals after TIL infusion samples of blood, skin, muscle and tumor will be obtained and 
subjected to semiquantitative PCR analysis for LNL6 and GIN sequences. This analysis will 
permit an analysis of TIL, TIL subset and PBL trafficking and lifespan in vivo . 
Recombinant DNA Research, Volume 15 
[521] 
