(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 + 1/1.4x 106 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 1/15,15 2 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 PSA Lubaroff Ad/PSA Phase II, protocol #2, page 11 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 nomogram s for patients with hormone refractory prostate cancer were applied to patients in this phase I trial (40,41). Table 5 show s 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 gr oups 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 Predicte d and Actual Patient 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 imm unotherapy clinical trials for p rostate cancer The last s everal 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 Lubaroff Ad/PSA Phase II, protocol #2, page 12 followed by vaccinia-PSA (rV-PSA) given by intradermal injection, or (C) rV -PSA followed 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 pat ients 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. ECO G is currently planning a phase III trial using the Vaccinia virus (PROSTVAC -V/TRICOM) followed by Folwlpox 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 notion s 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 with hormone-refractory metastatic disease into this Phase II clinical trial. This is similar to the population that constituted the majority of patients in our Phase I toxicity trial of the Ad/PSA vaccine. Patients in this trial will have low burden of disease, despite the fact that they are hormone refractory, i.e., have negative bone scans and/or Lubaroff Ad/PSA Phase II, protocol #2, page 13 low serum PSA. We will be comparing the clinical and immunologic response of these patients with two other patient populations in the trial. The latter populations, described in a separate protocol, will be patients with newly recurrent disease, either hormone naïve or during androgen depletion therapy. Although the patients with recent recurrences will have a smaller tumor burden than will the patients in this protocol that have metastatic disease, the observation period to detect a therapeutic effect of the vaccine will be shorter in the latter patient population than the former population. It is for this important reason that we will enroll patients with metastatic disease on this protocol. Patients will be eligible if the y have recent evidence of hormone refractory disease (D3) and either (a) have a positive bone scan or CT scan with a PSA doubling time of >6 months, and total PSA of <10 ng/ml, and asymptomatic; or (b) have a negative bone scan and CT scan with any PSA doubling time, asymptomatic, and not a candidate for chemotherapy. 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 (ICVAHCS ).