3. Viral Shedding: Patients will have samples from nasopharyngeal swabs, sputum, and stools sent 
for viral culture. In addition, any pleural fluid obtained will be sent for viral cultures. 
4. Efficacy: Although this is a Phase 1 study, patients will, of course, be followed closely after 
treatment for any signs of efficacy as evidenced by improvement on chest radiograph or CAT/MRI 
scan. If patients undergo further surgical procedures (as clinically indicated), careful evaluation of 
tumor extent will be obtained. 
5. Immunological Responses: A major aspect of this protocol will be to evaluate the immunologic 
response of the patient to intrapleurally delivered adenovirus. We will monitor both the generation 
of cellular immune responses and the generation of cytotoxic T-lymphocytes (CTLs), as well the 
development of neutralizing antibodies. 
Cellular Immunity. In order to detect virally directed CTLs, we will study the ability of patients T- 
lymphocytes to lyse and to proliferate in response to autologous cells which have been transfected 
with adenovirus. These assays are in routine use in Dr. Wilson's labs (i.e. see Yang et al., 1994). 
To conduct the CTL assays, we will first infect the autologous cells (lymphocytes or skin fibroblasts 
-target cells) with no virus, H5.01 ORSVTK, or H5.01 ORSV/acZ adenovirus. Infection will be 
accomplished by exposing the cells to virus (MOI 100) for two hours in tissue culture medium and 
used two days after infection. Cells derived from peripheral blood or pleural fluid (effector cells) 
will be tested for their ability to lyse ^Icr-labeled target cells. 
Humoral Immunity: We will evaluate for the presence of neutralizing antibodies using a protocol 
previously described in detail by our group (Kozarsky et al., 1994). The highest dilution of serum 
at which cell viability is above 50% as determined by crystal violet intensity will be designated as 
the neutralizing titer. 
6. Efficiency and stability of gene transfer - One key feature of the protocol is the inclusion of a 
biopsy procedure 3 days after viral instillation to provide an opportunity to assess gene transfer and 
HSV7K expression. In order to evaluate the success or failure of this protocol, it will be critical to 
know if successful gene tranfer occurred and to what extent We will use multiple approaches to 
assess gene transfer including analysis of HSVT/C message by RT-PCR, Northern blotting, and in situ 
hybridization. We will also look for TK protein using our polyclonal anti-HS VTK antiserum. 
V.C.5. Toxicity Parameters and Dose Escalation 
The proposed clinical protocol is a Phase I dose escalation study to evaluate toxicity of a one-time 
administration of recombinant adenovirus. Specific criteria used to assess toxicity are summarized 
in Appendix F. Continuation of the protocol will be reevaluated if major toxicity occurs. 
Definition of Dose Limiting Toxicity (DLT) 
Any toxicity requiring removal of the patient from study 
Any grade; 3 or 4 non-hematological toxicity which might be attributable to Ad.RSVtk virus. Grade 
4 myelosuppressive toxicity for >5 days. 
Dose Escalation 
If none of the patients treated at a given dose level experiences a dose limiting toxicity (DLT), then 
Ad.RSVtk virus is escalated to the next higher dose in the 3 subsequent patients. If one of the three 
patients experiences a DLT at a dose level, then three more patients will be accrued to the same dose 
level. If none of these additional patients suffers DLT, then the dose will escalated in subsequent 
patients. If at least one of the additional patients does have DLT, the MTD has been exceeded and three 
more patients are treated at the prior dose level (if only 3 patients were previously treated at that 
prior dose). The MTD is the dose level at which 0/6 or 1/6 patients experience DLT; and at least 
2/3 or 2/6 patients treated with the next higher dose will have had DLT. Dose escalation will not 
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Recombinant DNA Research, Volume 20 
