(39). Results of those analyses demonstrated that 57% of men immunized with the Ad/PSA vaccine developed measurable anti -PSA antibodies. ELISPOT assays were utilized to measure anti -PSA T cell responses. The results, depicted in Table 3, demonstrate that of the 32 patients, 18 (56.3%) developed anti -PSA T cell responses. The addition of Gelfoam did not appear to affect the development of anti -PSA responses, but in this Phase I study the numbers of patients in each group was too small to make statements of statistical significance of the data. These results demonstrate the ability of men with late stage metastatic prostate cancer, injected one time with Ad/PSA, to respond to the vaccine with the production of anti -PSA T cells. Table 3 ELISPOT Analysis of Anti -PSA T Cell Immune Responses Patient Number Dose/ Vehicle Response Frequency Pre-Immunization Post - Immunization AP-002 106-aqueous - 1/985,000 1/258,571 AP-004 106-aqueous - 0 1/311,111 AP-007 106-aqueous + 1/46,901 1/14,804 AP-003 106-matrix + 1/90,000 1/8075 AP-005 106-matrix + 1/1.2x106 1/152,353 AP-006 106-matrix + 1/93,103 1/15,762 AP-008 107-aqueous - 1/100,000 1/97,419 AP-010 107-aqueous - 0 0 AP-013 107-aqueous + 0 1/60,000 AP-009 107-matrix + 1/52,535 1/30,566 AP-012 107-matrix - 1/13,488 1/254,286 AP-014 107-matrix + 1/562,500 1/120,000 AP-015 108-aqueous + 1/1.4x106 1/780 AP-016 108-aqueous + 0 1/130,000 AP-017 108-aqueous + 0 1/124,375 AP-025 108-aqueous - 0 0 AP-026 108-aqueous - 0 1/560,000 AP-027 108-aqueous + 0 1/26,364 AP-029 108-aqueous + 0 1/333 AP-032 108-aqueous + 1/250,000 1/3793 AP-018 108-matrix - 0 0 AP-019 108-matrix + 1/14,235 1/336 AP-020 108-matrix + 1/8824 1/1802 AP-021 108-matrix + 1/689 1/431 AP-022 108-matrix + 0 1/180,000 AP-023 108-matrix - 1/320,000 0 AP-030 108-matrix + 1/666.667 1/32,000 AP-034 108-matrix + 1/80,000 1/15,152 AP-035 108-matrix + 1/5295 1/2459 AP-036 108-matrix + 1/62,000 1/9487 AP-037 108 matrix - 1/2.1x106 1/965,000 The effects of vaccination on serum PSA and on patient survival were also evaluated as a secondary endpoint in the phase I trial. Although there was no sustained decline in individual PSA levels, the PSA doubling times (PSADT, calculated based on 3 pre-enrollment consecutive PROTOCOL #1 Lubaroff, page 11 PSA measurements) were reduced in 54 percent of the patients compared to pre-vaccine administration, with the best responses occurring in patients immunized with the highest dose of the vaccine (Table 4). In addition, published survival nomograms for patients with hormone refractory prostate cancer were applied to patients in this phase I trial (40,41). Table 5 shows that 57% of all patients at all doses, whether injected with the vaccine as an aqueous suspension or in the collagen matrix, had a survival time longer than that predicted by the nomogram. The range of increased survivals in the different groups was 33% to 100%. Table 4 PSA Doubling Time (DT) in Phase I Patients Vaccine Dose & Vehicle Percent Increased PSA DT 106 aqueous 33% 106 matrix 33% 107 aqueous 67% 107 matrix 67% 108 aqueous 25% 108 matrix 62% Total 54% Table 5 Ad/PSA Phase I Trial Predicted and Actual Pati ent Survival Vaccine Dose Vehicle Percent with Longer Than Predicted Survival 106 Aqueous 33% 106 Matrix 67% 107 Aqueous 100% 107 Matrix 33% 108 Aqueous 56% 108 Matrix 50% Overall 57% 1.5 Other immunotherapy clinical trials for prostate cancer The last several years have seen an increase in the number of clinical trials using vaccine immunotherapy for the treatment of prostate cancer. The trials have used a variety of target antigens that have been shown to be associated with prostate and prostate cancer cells. These include PSA (39,42-49), prostatic acid phosphatase (PAP) (50-53), prostate specific membrane antigen (PSMA ) (54 -56), telomerase (hTERT) (57,58), T homsen-Friedenreich antigens (59), mucins (60), carbohydrates (61), and HLA-associated peptides (62). A variety of vectors have been used in the immunization process that include dendritic cells (45,50-58,63), vaccinia virus (39,42,43,47,49), fowlpox virus (39,47), liposomes (44), plasmids, (48), and chemical conjugates (59-61). Recently, Kaufman et al. recently reported the results of a phase II trial (ECOG 7897) with a prime/boost vaccine using vaccinia virus and fowlpox virus expressing human PSA in patients with hormone-dependent prostate cancer (64 ). Sixty -four eligible patients with biochemical progression after local therapy were randomly assigned to three treatment arms: (A) fowlpox - PSA (rF -PSA) by intramuscular injection every six weeks for four doses, (B) rF-PSA for three doses followed by vaccinia-PSA (rV -PSA) given by intradermal injection, or (C) rV -PSA followed PROTOCOL #1 Lubaroff, page 12 by three rF -PSA vaccines. Dreicer et al. reported a randomized phase II study with a recombinant Modified Vaccinia Ankara virus which expresses both MUC1 and IL2 (TG4010) (65). MUC1 is a glycoprotein associated with several malignancies. Eligible patients were required to have no evidence of metastatic disease following curative intent local therapy, evidence of PSA failure (PSA over 2 ng/ml) and PSA doubling time (PSA -DT) less than 10 months. Arm 1 had TG4010 injected sc weekly, at a dose of 108 pfu, for six weeks, then every three weeks. Arm 2 had 108 pfu TG4010 injected sc every three weeks. Therapy was continued to disease progression or to a maximum of 36 weeks. Lastly, Small et al. recently presented the results of a phase III trial with APC8015, an immunotherapy cellular product consisting of autologous peripheral blood mononuclear cells enriched for a dendritic cell fraction pulsed with PA2024, a Prostatic Acid Phosphatase (PAP) -GM-CSF construct (66). Patients with asymptomatic, metastatic hormone-refractory prostate cancer were randomized (2:1) to receive APC8015 (n=82) or placebo (n=45) every 2 weeks x 3. ECOG is currently planning a phase III trial using the Vaccinia virus (PROS TVAC- V/TRICOM) followed by Fowlpox virus vaccination (PROSTVAC -F/TRICOM) with GM -CSF, compared with placebo vaccine plus GM -CSF in patients with hormone-refractory prostate cancer with absence of metastatic disease (ECOG 1805, PARADIGM). In summary, the results from these trials vary in terms of patient populations studied (hormone dependent vs. independent) and in levels of positive results, which include the induction of antigen-specific immune responses, decreases in levels of serum PSA and in rates of change in PSA velocity, and measures of clinical responses. Thus far no single vaccine immunotherapy has proven to be definitely superior to others in terms of clinical benefit, and other phase II and III trials continue to be planned or conducted. The results of some of these vaccine trials raise the question that an increase in PSADT may in the future represent a possible surrogate marker for increased time to progression, or overall survival in immunotherapy studies, and that absolute PSA responses may not constitute an obligatory step for the ultimate demonstration of clinical benefit of immunotherapy approaches in prostate cancer. Furthermore, the T-cell stimulation index may have important correlation with clinical vaccine efficacy, as seen in the phase III trial by Small et al.(66). These developing notions further support the current proposal for clinical development of our Ad/PSA vaccine, also based on the results of our prior phase I trial. 1.6 Proposed phase II clinical trial : rationale Based on the significant pre-clinical activity of the Ad/PSA vaccine in generating tumor - specific T cells, and the encouraging safety and efficacy results from our phase I study, we propose to continue the clinical development of the Ad/PSA vaccine with the performance of the current phase II trial. It is our contention that the vaccine product and the method of immunization set this therapy apart from other ongoing prostate cancer investigational immunotherapeutic approaches. Specifically, the incorporation of Gelfoam, not present in other vaccine preparations, enhances the induction of strong anti -PSA responses. Immunization of mice with Ad/PSA in Gelfoam matrix was able to induce anti -PSA responses even in the presence of high-titer anti -adenovirus antibodies. Notably, most humans naturally possess high titers of anti -adenovirus antibodies due to natural exposure to adenoviruses. We plan to enroll prostate cancer patients into one of two arms (A & B) of the Phase II clinical trial. The ideal patient population to determine a therapeutic benefit of a new treatment, particularly immunotherapy, is one with minimal disease burden. The low tumor burden should allow therapies, particularly those relying on antigen-specific effector T lymphocytes, to destroy PROTOCOL #1 Lubaroff, page 13 all of the cancerous tissues and cells. The first therapeutic arm (Arm A) will enroll men with recent evidence of recurrence following surgery or radiation therapy for their primary tumor. Patients in the population will be eligible if they exhibit at least three separate rises in serum PSA, at least one month apart with differences > 0.03 ng/ml and a total PSA of >0.2 ng/ml; have a PSA doubling time of >6 months; are not at high risk or if they refuse treatment with Taxotere or radiation. A high risk patient is defined below . All patients will be hormone naïve, meaning that they may not have had prior systemic therapies for prostate cancer (hormonal therapy, including but not limited to LHRH agonists, antiandrogens, ketoconazole or chemotherapy – mitoxantrone/taxanes/estramustine, etc.) unless they stopped hormone therapy two or more years prior to enrollment and currently have normal testosterone levels.. Since standard therapy for these patients would be to postpone androgen ablation therapy until such time as there is a high serum PSA level ( >20 ng/ml), enrolling patients into this Phase II trial does not withhold accepted treatment. Patients will be excluded from the trial if they refuse salvage radiation therapy or had greater than two positive margins at s urgery, or positive lymph nodes. The second therapeutic arm (Arm B) will enroll men with recurrent disease who will undergo androgen depletion therapy. The choice of this additional patient population is based upon published documentation that inflammati on and the generation of immune responses are augmented by hormone withdrawal (67-69). Mercader, et al., in attempts to demonstrate an enhanced termination of tolerance to prostate associated antigens documented CD4+ and CD8+ T cell infiltrates in benign prostates and in prostate tumors of men undergoing androgen withdrawal (67). Roden and co-workers published data demonstrating that T cell levels and T cell proliferation were increased in mice following castration (68) while Drake, et al. reported breaki ng tolerance to antigens associated with the TRAMP prostate tumors in mice (69). Therefore, we propose to vaccinate men beginning 14 days after the initiation of androgen depletion therapy using the same three injection protocol. As part of consideration for enrollment, the study will only enroll subjects for whom androgen depletion therapy or deferral of such treatment are considered standard of care options. In addition, in order to be considered for the study, patients must be willing to be in either study group (Arm A or Arm 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 be printed for Arm A and 25 cards printed for Arm B and all 50 cards will be placed in a container. Following the determination of eligibility of each patient and administration of the Informed Consent document, a card will be drawn to determine which Arm of the study the patent is entered. We will enroll and treat patients at two affiliated and adjacent medical centers, the University of Iowa Hospitals and Clinics (UIHC) and the Iowa City Veterans Affairs Health Care System (IC VAHCS). There is a single Institutional Review Board (IRB) that approves protocols for both institutions. After the University of Iowa’s IRB has approved the protocols the Research and Development Committee of the IC VAHCS meets to provide their approval for the study. Patient Recruitment: The UIHC and IC VAHCS draw from a large catchment area that will ensure the enrollment of sufficient numbers of men into the protocol. We will use recruitment methods similar to the methods used to obtain subjects for our Phase I clinical trial. That includes contacting patients that are being or have been treated in the Departments of Urology, Internal Medicine, and Radiation Oncology at the UIHC and IC VAHCS and letters written to urologists, oncologists, and medical oncologists in Iowa and the neighboring states of Wisconsi n, Illinois, Missouri, Nebraska, Minnesota, and South Dakota. Drs.