(Central Time). Patients at the University of Iowa Clinical Cancer Center will be registered by calling Pamela Zehr, RN, MA at 319-353-8914. Patients at the ICVAHCS will be registered by calling Karen Clark Griffith, RN, PhD at 319 -338-0 581, ext. 7519. Information from the eligibility form will be provided by the investigator or the investigator's research staff to the University of Iowa Cancer Center at this time, and the patient will be registered and assigned a unique patient number. 4.5 No patient may be enrolled or begin research intervention prior to registration and assignment of a patient number. As a follow -up, University of Iowa Cancer Center will provide the investigator with written confirmation of each patient's registrati on. 4.6 All investigators will be notified by the Chair of the Protocol Review and Monitoring Committee or by the trial’s Data and Safety Monitoring Board if the study is placed on administrative hold, and when the study is completed or closed to further patient enrollment. 4.7 Patients must begin the vaccine protocol within 30 days of registration (date the study committee approves patient eligibility) . 5. RESEARCH INTERVENTION PLAN 5.1 Administration Schedule Ad/PSA All patients will receive three injection s of 0.16 ml. (0.125 ml. vaccine + 0.035 ml. Gelfoam) of the Ad/PSA subcutaneously in the right thigh unless patient condition precludes the use of that leg, in which case the left thigh will be used for all three injections. The dose of the vaccine, based upon our results from the Phase I trial, will be 1 x 10 8 pfu (4.4 x 109 particles) in the Gelfoam matrix. The Gelfoam comes in sterile patient-ready packages. The virus will be suspended in sterile saline and the Gelfoam powder added in a ratio of 30 mg of powder per ml. of virus suspension. Injections will be spaced apart by 30 days, such that each patient will receive the vaccine on days 1, 30, and 60. The use of the matrix has been shown in collaborative pre-clinical experiments to enhance infection of host cells by the virus. Results from the Phase I trial indicated that the injection of the vaccine in Gelfoam did not produce any adverse events greater than those produced by the vaccine in an aqueous suspension. The vaccine induced anti-PSA immune response in patients injected as an aqueous or Gelfoam vaccination. Injections will be carried out in the University of Iowa Clinical Research Unit (CRU ). Each subject will be required to stay in the CRU for two hours and observed for early signs of toxicities. Lubaroff Ad/PSA Phase II, protocol #2, page 17 5.2 5.2 Design and Stages The ideal design would be a two-stage design of Simon (1989) requiring 32 patients. But due to the complexity of the trial, a more conservative approach consisting of using 32 patients in a one-stage design at effic acy level and a two looks at toxicity level as means of stopping rules will be carried out. By using a one-stage design at efficacy endpoint, we are making the probability of early termination for efficacy purposes to be zero. The reason for this design is due to the nature of the research intervention as outlined in section 10. Stopping design based on toxicity is as follow: one look after 17 subjects are treated and another look after 25 subjects are treated. If 5 out of 17 show vaccine-related grade 3 toxicity or higher, the trial will stop. Otherwise, an additional 8 subjects will enter the trial. If 7 out of 25 show grade 3 toxicity or higher, the trial will stop otherwise we will proceed to full registration of 32 patients to assess efficacy. 6. Adverse Events Toxicity will be graded according to the NCI common toxicity criteria “Common Terminology Criteria for Adverse Events” (NCI-CTCAE v3.0 can be accessed at website: http://ctep.cancer.gov/forms/CTCAE_Index.pdf). Non-hematologic and Hematologic dose limiting toxicity (DLT) will be defined as described in Section 8.5.1. 6.1 Definitions Adverse Event (AE) is any untoward medical occurrence in a patient or clinical investigation subject administered a pharmaceutical product and that does not necessarily have a causal relationship with this research intervention. An AE can therefore be any unfavorable and unintended sign (including an abnormal laboratory finding), symptom, or disease temporally associated with the use of a medicinal (investigational) product, whether or not related to the medicinal (investigational) product. This will also include intercurrent diseases and accidents observed during the research intervention period as well as corresponding events during drug-free, pre- and post- intervention periods, under placebo or in a reference group receiving drug or non-drug therapy. Serious adverse event (SAE) is any untoward medical occurrence that: a. results in death b. is life -threatening B c. requires inpatient hospitalization or prolongation of existing hospitalization d. results in persistent or significant disability or incapacity e. is a congenital anomaly / birth defect or f. is another medically important condition.C B The term “life -threatening” in the definition of “serious” refers to an event in which the patient is at risk of death at the time of the event; it does not refer to an event, which hypothetically might have caused death if it were more severe. C Medically important conditions that may not result in death, be life- threatening or require hospitalization may be considered as SAE when, based upon appropriate medical judgment, they may jeopardize the patient or may require intervention to prevent one of the outcomes listed in the definition above. Examples of such events are intensive treatment in an emergency room or at home for allergic Lubaroff Ad/PSA Phase II, protocol #2, page 18 6.2 Procedures of documentation of AEs All AEs occurring during the study must be documented, regardless of the assumption of a causal relationship, on the respective AE CRF. All events which occurred after signed informed consent should be documented. The investigator should ensure that all events are recorded that occurred within at least 4 weeks after the last exposure to the study drug (through Day 90) . Documentation of AEs includes: date of onset and offset, intensity, frequency, seriousness, related interventions and outcome. The investigator will also evaluate the probabi lity of a causal relationship of the adverse event to the study medication as being: “definite, probable, possible, unlikely, or unrelated.” All unanticipated problems involving risk to subjects or others, serious adverse events related to participation in the study and subject deaths related to participation in the study will be promptly reported by phone (301-619-2165), by email (hsrrb@det.amedd.army.mil ), or by facsimile (301-619-7803) to the USAMRMC, Office of Research Protections, Human Research Protection Office. A complete written report will follow the initial notification. In addition to the methods above, the complete report will be sent to the U.S. Army Medical Research and Materiel Command, ATTN: MCMR -ZB-PH, 504 Scott Street, Fort Detrick, Maryland 21702-5012. The medical monitor is required to review all unanticipated problems involving risk to volunteers or others, serious adverse events , and all volunteer deaths associated with the protocol and provide an unbiased written report of the event. At a minimum, the medical monitor should comment on the outcomes of the event or problem, and in the case of a serious adverse event or death, comment on the relationship to participation in the study. The medical monitor should also indicate whether he/she concurs with the details of the report provided by the study investigator. Reports for events determined by either the investigator or medical monitor to be possibly or definitely related to participation and reports of events resulting in death should be promptly forwarded to the HSRRB. Expedited reporting The investigator must immediately report serious adverse events (SAE) occurring or observed during the course of the study and within 4 weeks of last administration of the study drug to the FDA, IRB, OBA, and CRU. The order of reporting of SAEs is the following: reported to the Principal Investigator within 24 hours of the event; the PI will then report the event to the FDA , OBA, Institutional Biosa fety Committee (IBC), and DOD within 7 days of the event and to the IRB within 10 days of the event. After notifying the proper agencies by telephone of an SAE the “Serious Adverse Event Report ”must also be sent by fax to the agencies whether or not complete information is available at the time. If complete information is unavailable the investigator must provide follow -up information to the agencies as soon as it is known. bronchospasm; blood dyscrasias or convulsions that do not result in hospitalization; or development of drug dependency or drug abuse. N.B.: The term “severe” is often used to describe the intensity (severity) of an event (such as: mild, moderate, or severe e.g., pain). The event itself may be of relatively minor medical significance (such as severe headache). This is not the same as “serious”, which is based on patient/event outcome or action criteria usually associated with events that pose a threat to patient’s life or vital functions. Seriousness (not severity) serves as a guide for defining regulatory reporting obligations. Lubaroff Ad/PSA Phase II, protocol #2, page 19 In particular, the investigator must inform the agencies and committees by phone and fax using the timeline described above of occurrence of immediately life-threatening SAEs or SAEs with fatal outcome. SAEs must be reported to the site’s IRB according to the IRB’s requirements. Important: The investigator must report any SAE to the FDA, IRB, OBA, and CRU, regardless of causality. Reports will be evaluated by the Medical Monitor/Sponsor. FDA/HPB and investigators will be informed as required by the regulations. The same information will also be made available to all participating investigators as well as to other investigators participating in different clinical trials utilizing the same study medication. It should be noted that, although the Ad/PSA vaccine contains the gene for PSA, there is no need for patients to utilize contraceptive practices. The adenovirus will not be transmitted from patient to his partner. 7 MEASUREMENT OF CLINICAL AND IMMUNOLOGICAL EFFICACY 7.1 Methods of Malignant Disease Evaluation - Each patient will have a baseline evaluation prior to the injection of the Ad/PSA vaccine. The measurements will include physical exam, temperature, weight, serum PSA, blood chemistries, urinalysis, a quantitative bone scan for bone metastases, chest x-ray, and CT for soft tissue metastases, and performance status for qual ity of life. Patients will be seen in the Urology and Oncology Clinics (see Table 6 for schedule) . The injection site will be examined for evidence of erythema, induration and necrosis and patients will have their temperature and weight recorded and the patients will be interviewed to determine whether they experienced any adverse reactions. Blood samples will also be taken for standard laboratory testing and measurement of PSA and anti-PSA immune responses (see Table 6). Patients recruited from the DM MCC will return to that facility for their follow-up examinations and laboratory testing on days 44, 74, and at 6, 9, 12 months and all subsequent visits. At the 6 month, 9 month, 12 month, and subsequent semi -annual visits each patient will be evaluated comparing the baseline visit measurements to those obtained at the specific visit. Because changes to the clinical status of the patients will be prolonged, continued follow up will be important. Therefore, patients will return to the clinic for two additional visits, at 18 and 24 months, unless travel distance is a prohibitive factor. Follow -up of patients for survival will occur every 6 months by telephone. 7.2 Scans – Bone scans will be performed at 90 days, 6 months, and 12 months during the first y ear as well as the 24 month visit and annually until progression. If there is measurable disease by CT scan at registration then CT scans will also be performed using the same schedule as the bone scans. 7.3 Use of Serum PSA for Disease Evaluation – Base d upon our pre-clinical experiments and the results from the Phase I clinical trial we expect the immunized men to produce anti - PSA antibodies. The levels of antibody will be measured by a flow cytometry assay as described by Cavacini, et al. used in our Phase I clinical trial (40). We will also explore the use of a second serum marker for prostate cancer, hK2 in collaboration with Donald Tindall, Charles Young, and George Clee at the Mayo Clinic. Investigators at Mayo, along Lubaroff Ad/PSA Phase II, protocol #2, page 20 with Hybritech, Inc. have been exploring hK2 and published a number of papers in recent years on the subject ( 67-70). Patient sera from each clinic visit will be sent to Mayo where they will measure the levels of hK2. We will use the data to evaluate the effect of anti - PSA antibodies on both PSA and hK2 in the sera of vaccinated patients. PSA measurements, CT and bone scans are routinely used to follow disease recurrence and/or progression in individual prostate cancer patients and as such would be considered standard of care. Laboratory measurements such as hematology, liver function and kidney function chemistries, while are routinely used to follow the health of a prostate cancer patient, would not normally be performed at the frequency proposed in this trial to assess possible vaccination toxicities. Therefore, they would be considered part of the research protocol. 7.4 Experimental Evaluation of the Ad/PSA Vaccination 7.4.1 Blood will be collected prior to each injection of the Ad/PSA vaccine and at return dates indicated in Table 6, page 26. Two separate samples will be collected; one in red top tubes to allow collection of serum from coagulated blood and a second in heparinized tubes to permit collection of lymphocytes. 7.4.2 Levels of PSA, hK2, anti -PSA antibodies, and anti-adenovirus antibodies will be measured in the serum. 7.4.3 Anti-PSA T cell immune responses will be measured by ELISPOT analysis using the methods developed for, and used in, our Phase I clinical trial. In addition to measuring the anti -PSA T cell activity, we will also measure anti -adenovirus T cell activity as well as reactivity to stimulation with cytomegalovirus (CMV). A non-specific stimulus will be provided by PMA and ionomycin for each patient’s lymphocytes. 7.5 Definitions of Response – 7.5.1 Primary Endpoint - PSA doubling-time response 7.5.1.1 Definition: a 50% increase in the PSADT compared to pre-enrollment PSADT. 7.5.1.2 PSADT will be calculated based on the MSKCC calculator, available at http://www.mskcc.org/mskcc/html/10088.cfm . 7.5.1.3 PSADT response will be measured at 9 and 18 months after initiation of the research intervention . 7.5.1.4 Three measurements of PSA, spaced at least 2 weeks apart, will be required prior to study enrollment. Post-intervention PSADT will be based on PSA levels at 3, 6 and 9 months (9 month PSADT calculation) and 3, 6, 9,12,15,18 month levels (18 month PSADT calculation). 7.5.2 Secondary Endpoint – PSA response Lubaroff Ad/PSA Phase II, protocol #2, page 21 7.5.2.1 Definition: a 50% reduction in the pre-research intervention PSA value, verified with a second measurement >28 days later. 7.5.3 Progression : Progression of disease is defined for 3 different categories of disease: measurable lesions; non -measurable/non-target lesions ; and PSA-only disease. The first category of disease to progress will meet the definition of progression in this trial, as follows: 7.5.3.1 Progression for measurable disease: Definition: Measurable lesions are defined as those that can be accurately measured in at least one dimension (longest diameter to be recorded) and is equal or greater than 20 mm with conventional techniques (CT, MRI, x -ray) or equal or greater than 10 mm with spiral CT scan. All tumor measurements must be recorded in millimeters (or decimal fractions of centimeters) All measurable lesions (up to a maximum of 10) representative of all involved organs should be identified as target lesions and will be recorded and measured at baseline. Target lesions should be selected on the basis of their size (lesions with the longest diameter) and their suitability for accurate repetitive measurements (either by imaging techniques or clinically). A sum of the longest diameter (LD) for all target lesions will be calculated and reported as the baseline sum LD. The baseline sum LD will be used as reference to further characterize the objective tumor response of the measurable dimension of the disease. All other lesions, including non-measurable small lesions as defined above and bone metastasis, w ill be identified as non -target lesions (see definition of progression for non-target lesions below). Progression is defined if any of the criteria below are met: • at least a 20% increase in the sum of the LDs of target lesions (taking as a reference the smallest sum LD recorded since the treatment started) or the appearance of two or more new lesions • development of an indication for radiation therapy while on treatment 7.5.3.2 Progression for non-measurable disease/non-target lesions: Non-measurable di sease/non-target lesions: progression of disease is defined as any of the following: 7.5.3.2.1 - worsening bone scan as evidenced by the appearance of more than two new lesions, not felt to be consistent with a tumor flare; worsening of pre-existing lesions (increase in intensity or size of a lesion) may be difficult to interpret and in such a case will not be considered the evidence of progressive disease; or 7.5.3.2.2 - appearance of new metastatic lesions outside the bone; or unequivocal progression of existing non-target lesions. Because a clear progression Lubaroff Ad/PSA Phase II, protocol #2, page 22 of non-target lesions only is exceptional, such circumstances should be discussed with the study co-investigator's; or 7.5.3.2.3 - development of an indication for radiation therapy while on treatment 7.5.3.3 Progression for patients with PSA-only disease PSA progression will be evaluated as follows: 7.5.3.3.1 In patients that had any PSA decline from baseline: 7.5.3.3.1.1 - increasing PSA by 25% above the nadir plus 7.5.3.3.1.2 - an increase in PSA by a minimum of 2 ng/ml from the nadir plus 7.5.3.3.1.3 - confirmation by second consecutive rising PSA >28 days later 7.5.3.3.2 - in patients whose PSA has not dec lined, progression is defined as 7.5.3.3.2.1 - an increase in PSA by 25% above the pretreatment level plus 7.5.3.3.2.2 - an increasing PSA by a minimum of 2 ng/ml relative to the study entry PSA value, plus 7.5.3.3.2.3 - confirmation by second consecutive rising PSA >28 days later . 7.5.4 Onset of Response – I n patients that present with radiologic evidence of metastases onset of response is the onset of PR or CR . For patients with biochemical disease only the onset of response is defined by the first drop in serum PSA. 7.5.5 Duration of Response – In patients that present with radiologic evidence of metastases, duration of response is time from onset of PR or CR, whichever occurs first, (even if the patient later has a CR) until objective evidence of progression. In patients with PSA -only disease, duration of response is time from first drop in serum to objective evidence of progression. Progression is defined in Section 7.5.3, beginning on page 20. 7.6 Timing of Toxicity Assessments - Toxicity assessment will occur as stated in the calendar. Lubaroff Ad/PSA Phase II, protocol #2, page 23 8 STUDY PARAMETERS 8.1 Scans or x -rays used to document measurable or evaluable disease must be done with 6 weeks prior to the initiation of treatment. 8.2 CBC with differential, LFT’s must be done < 2 weeks before study entry. Castrate levels of testosterone must be drawn at initiation of treatment. 8.3 All chemistries must be done <2 weeks before the study entry, unless specifically required on day 1 as per protocol. If abnormal, they can be repeated within 48 hours prior to the initiation of treatment. 8.4 Hgb, Hct, WBC, Plt must be done < 2 weeks before study entry but, if abnormal, they can be repeated <48 hours prior to initiation of treatment. 8.5 REMOVAL OF PATIENTS FROM STUDY (Criteria for discontinuation of a patient's study participation) 8.5.1 Adverse events: Study subjects will be monitored for adverse events for 30 days beyond the final study injection, or longer if existing unresolved AEs are being monitored. In the event of a vaccine-associated unmanageable or irreversible toxicity, that would include hematologi cal and non-hematological toxicities, the investigator will withdraw a patient from further research intervention and notify the Study Chair immediately. In addition, the FDA and the IRB and other agencies listed on page 17 will be notified of the adverse events. If unmanageable or irreversible toxicities occur the patients will receive the best possible medical care according to the recommendations of the treating physicians. The treatment plan will depend upon what unmanageable or irreversible toxiciti es may occur. In the event of an emergency the patient will be instructed to seek immediate care by calling 911, his local physician, the University of Iowa or Iowa City VA Health Care System urology staff on call. 8.5.1.1 Management of Toxicities 8.5.1.1.1 Hema tological Toxicity Management 8.5.1.1.1.1 Patients with grade 2 or 3 hematological toxicity will not receive the subsequent dose of vaccine until there is normalization of cbc parameters until grade 1 or less toxicity is reached. An exception to withholding a dose of vaccine would be if the cbc parameters were stable (at or near the pre-vaccination values). 8.5.1.1.1.2 In case of grade 2 or 3 hematological toxicity detected any time during protocol therapy, CBC with differential will be checked on a weekly basis until grade 1 or less toxicity is reached. 8.5.1.1.1.3 If the subsequent vaccine dose needs to be postponed for more than 2 weeks, those patients will be removed from protocol therapy. Lubaroff Ad/PSA Phase II, protocol #2, page 24 8.5.1.1.1.4 Patients with grade 4 hematological toxicity at any time will be permanently removed from protocol therapy. 8.5.1.1.2 Non-Hematological Toxicity Management 8.5.1.1.2.1 Patients with grade 2 or 3 non-hematological toxicity will not have the subsequent dose of vaccine administered until the toxicity ameliorates down to a grade 1 level. 8.5.1.1.2.2 Patients will be evaluated once a week with study team assessments, if a grade 3 non-hematological toxicity occurs. 8.5.1.1.2.3 If the subsequent dose of vaccine needs to be postponed for more than 2 weeks, those patients will be permanently removed from protocol therapy. 8.5.1.1.2.4 Patients with grade 4 non-hematological toxicity at any time will be permanently removed from protocol therapy. Patients may also be removed from protocol therapy at any time based on assessment of any other risks, at the discretion of the study investigators. 8.5.2 Disease Progression: Patients will be taken off-study if they have progressive disease (PD) or clinically significant deterioration at any time during the study if the investigator feels that (a) alternative prostate cancer therapy might benefit the patient, or (b) to continue on study might be unsafe for the patient. Patients receiving alternative prostate cancer therapies will be followed for toxicity for 30 days beyond the third AdPSA injection only unless existing unresolved AEs are being monitored. Patients will be asked in the Informed Consent document whether they would return every three months up to 12 months from study entry for immunologic evaluations. Patients who are being treated with prednisone at doses higher than 10 mg daily (or equipotent steroid doses) will be excluded from these every three month returns . 8.5.3 Allergic Reactions: Patients must be removed from the study should they develop grade II allergic reactions. 8.5.4 Personal Reasons: As stated in the informed consent, patients may withdraw from the study at any time. 8.5.5 Clinical Judgment: A patient may be withdrawn from the study, if, in the opinion of the investigators, it is not in the patient's best interest to continue (e.g. an adverse experience, intercurrent illness, non-compliance, etc.) 8.5.6 The date of discontinuation and the reason(s) for patient discontinuation from the study will be recorded in the CRF. All efforts will be made to conduct evaluations that are required at the follow -up for each patient who discontinues research intervention, regardless of the reason. Lubaroff Ad/PSA Phase II, protocol #2, page 25 Regulatory and Reporting Requirements The Data and Safety Monitoring Committee (DSMC) of the Holden Comprehensive Cancer Center will provide data and safety monitoring for this study. “The Data and Safety Monitoring Plan of the Holden Comprehensive Cancer Center” provides standard operating procedures to monitor all clinical cancer trials at the UIHC. All investigator -initiated trials are automatically monitored by the DSMC. A detailed data and safety monitoring plan for this study is on file with the DSMC and the Clinical Research Safety Officer (CRSO). Data Management, Quality Control and Data Security In order to protect confidentiality the subject will be assigned an identification number. This number will be used on all specimens from the subject and will be used for documentation purposes . Data management for the optimal entry, processing, storage, and retrieval for this protocol's data will be accomplished by the principal investigator. The database will be located on a computer or in a locked cabinet in a locked office. This computer will be secured, accessible only by the research team. There will be more than one copy of the database. The second, secured, copy of the protocol data will be stored in a locked room accessible only by the research team. For quality control, auditing, and checking data for integrity, there will be a regular accounting of data periodically performed. The medical record and research record will be linked by the study identification number. The data managers in this trial will be responsible for verification of the accuracy of all data transferred from the medical record to the research record. These records will be audited quarterly by the Data Safety Monitoring Committee. All Data Safety Monitoring Committee reports will be provided to the USAMRMC Office of Research Protections, Human Research Protection Office as they become available. Data will be kept on file on each patient for at least two years past the termination of that patient’s participation in the trial. All records will be stored long-term in a secure location. UIHC records will be kept for at least 15 years and then confidentially destroyed. VAHCS records will be retained until disposition instructions are approved by National Archives and Records Administration. Information from the medical records of patients referred by physicians outside the University of Iowa and the VA Health Care System required to determine eligibility for this protocol will be placed in a research folder, identified by the patient code and will be available only to members of the clinical trial team (identified on pages 30 and 31). The signed HIPAA forms will be kept in the patient’s folder in the office of the referring physician. The records of such patients that are deemed ineligible following a screening procedure will have thei r signed Release of Information forms, informed consent document, and eligibility form kept in a research folder under similar security conditions. The records for ineligible subjects will be stored with the other research records as indicated above. Lubaroff Ad/PSA Phase II, protocol #2, page 26 Table 6 Study Design and Testing Std. of Care or Res. Prior to Reg.1 Day 1 First Inj. 30 d. 2nd Inj. 44 d. 60 d. 3rd Inj. 74 d. 90 d. 6 mo 9 mo 12 mo 18 mo. 24 mo.