1 Reg. = Registration, the date patient eligibility approved by the study team. 2 Androgen deprivation therapy must continue for the duration of study participation. 3 If the PSA meets the definition of progression, partial or complete response, confirm the PSA results >/= 28 days. 4 will be repeated only if abnormal at screening or if patient develops a fever. 5 will be repeated only if abnormal at screening. 6 to be performed even if there is disease progression (up to 12 months from study entry) if the patient has consented to this in the informed consent (question in section “How long will I be on the study?).” 7 Follow -up for survival every 6 months by telephone. In the event of holidays or other extenuating circumstances the times on the table may vary by + 4 days for the first 90 days. Beyond that every effort will be made to see the patient within 2 weeks of the expected date of return, but the investigator is willing to allow for other arrangements for the convenience of the patient. Lubaroff Ad/PSA Phase II, protocol #2, page 27 9 DRUG FORMULATION AND PROCUREMENT 9.1 Drug Name Adenovirus/PSA (Ad/PSA) 9.2 Classification Vaccine 9.3 Mode of Action The adenovirus is a replication-deficient virus unable to produce virus progeny in the infected cells. The virus will infect cells in the location of the injection site, the PSA gene will produce the protein product which will be recognized as an antigen by the immune system and produce anti -PSA immune responses. Based upon our pre-clinical studies in an animal model of human prostate cancer, these responses, mainly the CD8+ CTL response, will cause the destruction of PSA -secreting prostate tumors. 9.4 Dose Specifics and Route of Administration The route of injection, vehicles for the vaccine, and dose schedules have been outlined in Section 5 .1 of this protocol. 9.5 Availability Produced and provided by Molecular Medicine, LLC, San Diego, CA 9.6 Storage The vaccine is stored in the UIHC’s Pharmacy Department in a -70oC temperature- monitored and controlled access freezer. Only the Investigational Pharmacist will remove the vaccine from the freezer and enter the amount removed in a trial -specific log. 9.7 Injection Procedures & Observation At the weekly meetings of the clinical trial team at which the eligibility of the patients is decided, orders for the initial vaccine injection will be written by clinician members of the team. Orders for the second and third vaccine injections will be written at the meetings as close to the date of the scheduled injections as possible. On the designated days of injections and immediately prior to vaccination, the Investigational Pharmacist will mix the Ad/PSA (10 8 pfu or 4.4 x 109 particles) with the Gelfoam powder (30 mg/ml). Each vial of the vaccine contains twice the volume needed for each injection. One half of the vial will contain the proper pfu and particles required for the vaccination. The injection material will be taken from the pharmacy in a 1 ml. syringe with 25 gauge needle and presented to staff in the Clinical Research Unit. Each patient will be injected with approximately 0.16 ml. of the vaccine/Gelfoam material subcutaneously in the thigh. The exact volume of the vaccination mixture ( Ad/PSA in Ge lfoam) is not as important as the exact number of pfu or particles and that is controlled by using the required volume from the dose vial. The empty syringe will be disposed of in a biohazard container. Lubaroff Ad/PSA Phase II, protocol #2, page 28 Patients will remain in the CRU for two hours after each injection to be monitored for signs of adverse events. 9.8 Manufacturing 9.8.1 The PSA cDNA provided by Donald Tindall, Mayo Clinic, Rochester, MN, was placed 3’ to the CMV promoter in a shuttle vector containing Ad5 DNA. The sequence inserted was the pre-pro form of PSA described by Lundwall (71 ) that encodes 262 amino acids with a predicted molecular weight of 28.8 kDa. Using methods previously described ( 72), the shuttle vector and E1a-E1b deletion mutant Ad5 DNA were transfected into HEK 293 cells, and recombination between the DNA species was allowed to occur. The amplification and purification of Ad/PSA was performed by the University of Iowa Gene Transfer Vector Core as previously described (73). Ad/lacZ used as a control was also obtai ned from the Gene Transfer Vector Core and is previously described (74). 9.8.2 The Principal Investigator provided the Ad/PSA vaccine used for the pre-clinical studies to Molecular Medicine, LLC of San Diego, CA for the production of the clinical grade product. Information on the manufacturing of the GMP Vaccine by Molecular Medicine, LLC is found in the accompanying documents supplied by the company. 10 STATISTICAL CONSIDERATIONS The ideal endpoint would be a clinical outcome that is of particular relevance to the patient such as increased time to tumor progression, increased time to death or reducing the proportion of death. This trial is using a surrogate endpoint as a substitute to the clinically meaningful outcome since the tumor s cannot be accessed directly. The association between the surrogate and survival rate had not been clearly established by any phase I & II trial. The trial consists of using Ad/PSA vaccine administered in multiple injections to prostate cancer patients with hormone—refrac tory metastatic prostate cancer —with the goal to induce anti -PSA T cells responses. Three injections of equal dose are proposed. The previous phase I trial consisting of a single injection in men with hormone-refractory metastatic prostate cancer was able to induce anti-PSA T cells responses. The Phase I consisting of a single injection using dose escalation protocol of the vaccine in an aqueous or matrix delivery vehicle did not show any significant AE. Additional pre-clinical pharmacology /toxicology studies required by the FDA did not show any significant side effects using the three-injection schedule. The primary endpoint is the serum PSADT. For efficacy purposes, we expect at least 50% of the patients to show 50% increase in PSADT. This proportion is judged clinically important and anything less than 30% can stop the trial for futility. The ideal design would be a two-stage design of Simon (1989) requiring 32 patients. After testing the research intervention on 12 patients, if 3 did not have a 50% increase in PSADT, the trial will be terminated. Otherwise, an additional 20 patients will be recruited for the trial. The expected sample size will be 19.73 and the probability of early termination, 0.72. If the research intervention is not effective, there is 0.1 probability of concluding that it is. If the research intervention is effective, there will be 0.2 probability of concluding that it is not. However, due to the complexity of the trial, we will not use Simon (1989) but a more conservative approach consisting of using 31 patients in a one-stage design at efficacy level and a two-stage design at toxicity level for stopping rules. By using a one-stage design at efficacy endpoint, we are making Lubaroff Ad/PSA Phase II, protocol #2, page 29 the probability of early termination for efficacy purposes to be zero. Early termination will be based on toxicity only.