= Registration, the date patient eligibility approved by the study team. 2 If the PSA meets the definition of progression, partial or complete response, confirm the PSA results >/= 28 days. 3 For Arm B only 4 First ADT injection 14 days prior to vaccine injection #1. Second dose 12 -16 weeks later (depending on formulation; see package insert) 5 Will be repeated only if patient develops a fever. 6 Will be repeated only if t he patient demonstrates a rise in PSA (50% increase above baseline on 2 consecutive measurements, 30 days apart) 7 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?).” 8 Follow -up for survival every 6 months by telephone. 9 Obtained at 18 and 24 months only 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. PROTOCOL #1 Lubaroff, 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 resp onses. 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. syr inge with 25 gauge needle and presented to staff in the General Clinical Research Center. Each patient will be injected with approximately 0.125 ml. of the vaccine/Gelfoam material subcutaneously in the thigh. The exact volume of the vaccination mixture (Ad/PSA in Gelfoam) is not as important as the exact number of pfu or particles and that PROTOCOL #1 Lubaroff, page 28 is controlled by using the required volume from the dose vial. The empty syringe will be disposed of in a biohazard container. Patients wi ll remain in the CRU for 2 hours after each injection to be monitored for signs of adverse events. 9.8 Assignment of Patients to Arms A or B Randomization of patients to Arm A (vaccine only) or Arm B (ADT plus vaccine) will be 1:1. Fifty (50) patients will be enrolled, 25 in each Arm of the study. Twenty five (25) cards will in printed for Arm A and 25 cards printed for Arm B and all 50 cards will be placed in a container. Following the administration of the Informed Consent document and determination of eligibility of each patient, a card will be drawn to determine which Arm of the study the patent is entered. Patients that choose not to be placed on ADT will not be enrolled in the study . For this study, ADT will consist of LHRH or GNRH agonists, such as leuprolide (Lupron) or goserelin (Zoladex). ADT will be continued for the first nine months of the study. Anti-androgens will be used initially to avoid testosterone flair, but then discontinued. Aromatase inhibitors will not be used. Patients in Arm B will be vaccinated 14 days after the initiation of ADT. To insure that all patients in Arm B reach castrate levels of testosterone (T) (<50 ng/ml), serum T levels will be measured on their 60 day visit, prior to their third vaccination. This will be approximately 10 weeks after the initiation of androgen withdrawal, a time at which T levels should reach their nadir . 9.9 Manufacturing 9.9.1 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 (75) that encodes 262 amino acids with a predicted molecular weight of 28.8 kDa. Using methods previously described (75), 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 (76). Ad/lacZ used as a control was also obtained from the Gene Transfer Vector Core and is previously described (75). 9.9.2 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. The trial is using a surrogate endpoint as a substitute to the clinically meaningful outcome since the tumor 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 minimal disease burden—with the goal to induce anti -PSA T cells responses. Three injections of equal dose are proposed. A previous phase I trial consisting of a single injection in PROTOCOL #1 Lubaroff, page 29 men with metastatic prostate cancer was able to induce anti- PSA T cells responses. The Phase I consisting of a single injection using a 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 development of anti -PSA immune response— defined as an increase of greater than 200% above pre-immunization level of anti -PSA T cells as measured by ELISPOT analysis, measured 14 days after the 3 rd immunization. This endpoint will be the same for both arms considered in the protocol —the vaccine only and the vaccine plus androgen deprivation therapy arms. During the Phase I trial consisting of vaccine alone, patients that developed anti -PSA T cell responses had an increase in the frequency of anti -PSA T cells. We decided to express the anti-T cell response as percent change in antigen-specific T cell frequency compared to the frequency prior to immunization. Based on our phase I results, we judge 70% of the patients developing anti -PSA immune response as being clinically important while anything less than 40% in both arms will be judged unworthy. The ideal design would be a two-stage design of Simon (1989). After testing the research intervention on 10 patients, if 3 did not have the desired increase in anti -PSA T cells, the trial will be terminated. Otherwise, an additional 15 patients will be recruited for the trial for total of 25 patients with the goal that more than 13 will show the desired increase. If 8 or more show the desired increase, then the research intervention will be judged worthy for further considerations. In case the number is less than 8, the research intervention will be rejected. The expected sample size will be 15.14 and the probability of early termination, 0.63. If the research intervention is not effective, there is 0.05 probability of concluding that it is. If the research intervention is effective, there will be 0.09 probability of concluding that it is not. We will be conservative and use the same design and sample size considerations for both arms (25 per arm for a total of 50 patients) even though we expect the vaccine plus androgen deprivation therapy arm to yield better results. This brings a natural question of why two different research interventions and how one would assess the superiority of one research intervention over the other. We postulate that men in Arm B, those receiving ADT plus vaccine, will generate a stronger anti -PSA immune response, as measured by the production of PSA - specific T cells, than will men in Arm A, those receiving the vaccine alone. For patients in Arm B, there will be a quantitative and/or qualitative difference in the generation of anti -PSA T cells. That is, a higher percentage of men will demonstrate antigen specific T cells and/or the magnitude of the T cell response, as indicated by the precursor frequency, will be greater in this population. The differences will be most evident 14 days after the third and last vaccination. A test for comparison of these two proportions will reveal the superiority of ADT plus Vaccine over Vaccine alone. The secondary endpoint is the PSADT that will be assessed 18 to 20 months after initial patient accrual. Points estimate and exact confidence interval for the proportion of subjects who show 50% increase in PSADT will be reported. Monitoring toxicity: Stopping rules for toxicity purpose are based on testing the null that toxicity level is less than 15% versus the alternative that it is actually greater than 35%. In this setting, we will carry two different but dependent looks. If 3 out of 10 show grade 3 vaccine -related toxicity or higher, the trial will stop (this will correspond to testing the toxicity hypotheses at level alpha=0.1 with a power of 68%); otherwise, another test for toxicity after we have a total of 17 PROTOCOL #1 Lubaroff, page 30 subjects will be considered. If 5 out of 17 show a grade 3 vaccine-related toxicity or higher, or if 2 out 17 show a grade 4 toxicity the trial will stop (this will be equivalent to testing the toxicity hypotheses at level alpha =0.1 with a power 77%); otherwise we will proceed to full registration. The overall type I error for testing toxicity level is around 0.19. In addition, the trial will stop if any vaccine -related deaths occur. The determination of a death related to the vaccine will be made by the clinical trial team and an audit by the Data and Safety Monitoring Board. Randomization to clinical arm: one-to-one. Simon, Richard. “Optimal Two-Stage Designs for Phase II Clinical Trials,” Controlled Clinical Trials, 1989, Volume 10, pages 1-10. Addendum PHASE II STUDY OF ADENOVIRUS/ PSA VACCINE IN MEN WITH RECURRENT PROSTATE CANCER AFTER LOCAL THERAPY Food and Drug Administration (FDA) Investigational New Drug (IND) #9706 Department of Defense, Prostate Cancer Research Program #A-14059.1 The following are reporting requirements and responsibilities of the Principal Investigator to the United States Army Medical Research and Materiel Command’s (USAMRMC) Office of Research Protections (ORP), Human Research Protection Office (HRPO). 1.