Protocol ID93-008 
Page 9 
(7) Confirmation of transduction with the neo^ gene will be obtained by the polymerase 
chain reaction (PCR) using two neo^ gene specific oligonucleotide amplification primers 
(See Appendix) . 
1. 5’GGTGGAGAGGCTATTCGGCTATGA3’ 
2. 5ATCCTGATCGACAAGACCGGCTTC3’ 
(8) Determination of the percentage of neo^ transduced cultured CD3 + CD8+ T cells. 
The percentage of CD8‘*’ TIL that express the neo*^ gene product will be estimated by the 
PCR procedure and analysis as we have described (see Appendix 1 for validation data 
from our laboratory). DNA will be extracted by a standard proteinase K/phenol 
method. Neo^^ transduced K562 cells were obtained from Dr. A. Deisseroth. These 
K562 neo^ cells were cloned (clone 1). and the clone which expresses one copy of 
neof^/cell clone Is now established as a laboratory standard which is utilized to 
estimate the number of transduced CD8+ T cells in the cultured T-cell lines. Detection 
of the 424 b.p. gene product is performed by ethidium bromide (EtBr) staining. The 
neoamplification shows the expected product in the neo transduced TIL DNA and is 
appropriately negative in the blank and NT reactions. The relative intensity of the neo^^ 
bands was standardized by comparison of the actin bands obtained from TIL DNA in 
K562 DNA. 
(e) Tests Performed on Transduced TIL (Certificate of Analysis) 
The transduced cells from all patients will undergo the following testing; 
Test 
Method 
Limits 
Assay for residual 
tumor 
Cytology 
Negative 
Viability^ 
Tiypan Blue 
> 70% 
Microbiologic Studies* 
Gram stain 
No bacteria 
Bacterial culture 
and sensitivity 
Negative! 
Virology** 
S+/L-assay 
Negative! 
3T3 amplification 
Negative 
DNA analysis 
PCR or Southern 
Provirus 
Blot 
present! 
Autonomous growth 
IL-2 withdrawal 
Negative! 
Endotoxin* 
Limulus 
<5! 
Cytotoxicity* 
S^Cr release 
! 
Phenotype* 
FACS 
! 
•Described in detail in Methods and Validation studies submitted to Regulatory 
Affairs (see ID92-015, T92-0093. October 1. 1992), and June 15. 1993. 
♦•Will be done by GTI by methods used in previous studies. 
fResults will be available after treatment of the patient. 
4.0 PATIENT ELIGIBILITY 
4.1 Patients who participate in the marker protocol must have been registered 
under the treatment protocol ID92-015 (T92-0093). 
4.2 PaUents with histologically documented ovarian carcinoma who have persistent or 
recurrent disease after at least one platinum based chemotherapy regimen, and < 
cm residual. Patients with primary peritoneal carcinoma e.g., (serous or mucinous) 
are also eligible after failing cisplatin based chemotherapy. (In patients evaluated a 
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Recombinant DNA Research, Volume 19 
