James A. Brown, MD Department of Urology Tel: 319-356-2273 e-mail: james -brown-2@uiowa.edu Mark C. Smith, MD Department of Radiation Oncology Tel: 319-384-6135 e-mail: mark-c-smith@uiowa.edu Michael O’Donnell, MD Department of Urology Tel: 319-384-6981 e-mail: michael -odonnell@uiowa.edu Sundeep Deorah , MD Department of Urology Tel: 319-384-5418 e-mail: sundeep-deora@uiowa.edu Amit Gupta, MD Department of Urology Tel: 319-384-5251 e-mail: amit-gupta-1@uiowa.edu Kenneth Nepple , MD Department of Urology Tel: 319-356-2114 e-mail: kenneth-nepple@uiowa.edu Mary Schall , RN, BSN Holden Comprehensive Cancer Center Tel: 319-356-3516 e-mail: mary-schall@uiowa.edu Karen Clark Griffith, RN, PhD Iowa City Veterans Affairs Health Care System Tel: 319-338-0581, ext. 7519 e-mail: karen.clarkgriffith@va.gov Erica Brown , ARNP Department of Urology Tel: 319-467-5072 e-mail: erica-brown@uiowa.edu Gideon Zamba, PhD Department of Biostatistics Tel: 319 384- 5020 e-mail: gideon-zamba@uiowa.edu Steven Varga, PhD Department of Microbiology Tel: 319-335-774 e-mail: steven-va rga@uiowa.edu Pamela Zehr, RN , MA Holden Comprehensive Cancer Center Tel: 319-353-8914 e-mail: pamela-zehr@uiowa.edu PROTOCOL #1 Lubaroff, page 2 Investigational Pharmacist Kristine Johnson, BS, RPh Pharmacy Department Tel: 319-353-7077 e-mail: kristine-johnson@uiowa.edu Medical Monitor Daniel Berg, MD Holden Comprehensive Cancer Center Tel: 319-353-7800 e-mail: daniel -j-berg@uiowa.edu The Principal Investigator is funding this clinical trial through an award from the Department of Defense Prostate Cancer Research Program . PROTOCOL #1 Lubaroff, page 3 INDEX Section Pages Introduction 4-14 Objectives 14 Patient Selection 15,16 Registration Procedures 16,17 Research Intervention Plan 17,18 Adverse Events 18-20 Measurement of Clinical and Immunological Effect 20-22 Study Parameters 22-26 Drug Formulation and Procurement 27-29 Statistical plan 29,30 Adden dum 30,31 Roles and Responsibilities of Study Personnel 31,32 Literature References 32-36 Footnote 36,37 PROTOCOL #1 Lubaroff, page 4 1. INTRODUCTION 1.1 Background - Immunotherapy in Prostate Cancer Prostate cancer is the second leading cause of cancer death among males in the United States. There will be an estimated 234,460 new diagnoses of prostate cancer made in the United States in 2006 (1) (the estimates for 2006 were not available at the time of the protocol completion). Treatments for organ- confined prostate cancer include radical prostatectomy and radiation therapy. When the cancer presents de novo, or recurs outside the prostate, first-line systemic treatments typically include hormonal blockade (with LHRH agonists or bilateral orchiectomy), which suppress testosterone levels, limit the growth of androgen-dependent cancer cells, and result in clinical tumor control. After a median time of 2 years, patients progress into a clinical hormone-refractory state, when the prostate specific antigen (PSA) levels rise despite castration, there is proliferation of androgen-independent cancer cells, and there is continued clinical tumor growth that becomes fatal. Therapeutic measures in this situation include further hormonal manipulations or the use of systemic chemotherapy, which has recently shown a small survival benefit in phase III trials. Approximately 30,000 Americans die from prostate cancer each year. Immunotherapeutic approaches against prostate cancer have been investigated for several years. Most of these studies have concentrated on active non-specific therapy and adoptive or passive therapy, with only recent focus on the induction of antigen-specific immune responses. Viral vectors have been used successfully in both gene transfer and vaccine therapy studies (2). Replication- competent and replication-deficient adenoviruses expressing foreign proteins have been used to elicit immune responses to a variety of tumor antigens (3-7). We have demonstrated that immunizations with adenovirus, carrying the human PSA gene, can induce vigorous anti -PSA T -cell responses and cause the destruction of PSA -secreting tumors in a pre-clinical mouse model of prostate cancer (8,9). Such active immunization against prostate-cancer associated antigens might be more effective than active non-specific or adoptive/passive immunotherapy. Therefore, we have pursued a vaccination strategy based on an adenovirus that carries the gene for prostate specific antigen (PSA). Results from our Phase I trial of adenovirus/PSA (Ad/PSA) vaccine (section 1.4, below) demonstrated that a single immunization of men with metastatic prostate cancer was able to induce anti -PSA T cell responses. The trial design was a dose escalation study with the vaccine administered subcutaneously (sc) either in an aqueous solution or in a collagen matrix (Gelfoam ®). We now propose a Phase II clinical trial using the Ad/PSA vaccine, administered in multiple injections to prostate cancer patients with minimal disease burden. 1.2 Adenovirus vectors Recombinant adenoviral vectors transduce a wide range of dividing and nondividing cells types, making this gene delivery system valuable as a tool for studying diseases, for vaccine therapy, and for potential clinical use (10). Recombinant adenovirus can be prepared and purified in high titers. In addition, wild-type adenovirus infections are extremely common in the general population, giving adenovirus a well -documented safety record (11). Moreover, adenoviruses are structurally stable and no adverse effects have been reported following the vaccination of US military recruits with wild types, demonstrating their safety for human use (11). Adenoviral vectors for gene therapy and vaccine therapy are adenoviruses which have been genetically modified to allow insertion of foreign genes and to render the virus replication-defective. Current vectors have a deletion in the E1 region or in both the E1 and E2 regions. PROTOCOL #1 Lubaroff, page 5 Adenoviral gene transfer has been used in a variety of experimental conditions that include transfers to the liver (12), lung (13), central nervous system (14,15), and to cancer cells (16). There is evidence that the introduction of foreign transgenes by adenovirus induces immune responses to the transgene product, which become ultimately responsible for the elimination of the virus (17,18). While this is disadvantageous for insertion of functional genes into host cells, it is advantageous in the use of viruses carrying foreign genes as immunogens. In the vaccine therapy of cancer, active immunization against a murine colon cancer, breast cancer, and melanoma antigens have been induced by adenoviral vaccines (19-25). The Ad/PSA vaccine used our laboratory and in our Phase I clinical trial was produced by inserting the gene for the full length pre-pro form of human PSA into a replication deficient adenovirus serotype 5. Replication deficiency was induced by deletion of the E1a and E1b genes of the virus. Details of the vaccine can be found in section 9 of this protocol. Approval for the use of the vaccine in the Phase I trial was obtained from the FDA under IND #9706. In pre-clinical studies, our group has demonstrated that the Ad/PSA vaccine was able to induce stronger anti -PSA immune responses than other viral PSA vaccines. These include vaccinia viruses, both replication competent and replication deficient, and to a canarypox vaccine (Table 1), The frequency of PSA -specific CD8+ cells T cells generated by the Ad/PSA vaccine was greater than were generated by any of the other vaccines tested.. In addition to the superior immunizing property of the Ad/PSA, the incorporation of Gelfoam, a collagen matrix (section 1.3), has been shown in pre-clinical studies to enhance the ability of the vaccine to induce strong anti -PSA immune responses (8). Lastly, immunization of mice with Ad/PSA in matrix can induce anti -PSA responses even in the presence of high titer anti -adenovirus antibodies (8). This latter finding is important in light of the fact that most humans have pre- existing levels of anti -adenovirus antibodies as a result of prior natural exposure to the virus. Table 1 Effector Cell Frequency Analysis (ELISPOT) Vaccine Virus Frequency of PSA -Specific CD8+ T Cells Ad/PSA* Replication deficient adenovirus 1/455 Prostvac Replication competent vacc inia 1/2028 NYVAC/PSA Replication deficient vaccinia 1/3597 ALVAC/PSA Canarypox 1/35,714 1.3 Gelfoam® Matrix Gelfoam (Pharmacia & Upjohn Company, Kalamazoo, MI) is a medical device intended for application to bleeding surfaces as a hemostatic agent. It is a water -insoluble, off -white, non- elastic, porous, pliable product prepared from purified pork skin. The Gelfoam gelatin preparation is available either as a cross -linked sponge or as non-cross linked beads. It is able to absorb and hold within its interstices approximately 45 times its weight of blood and other fluids (26). The absorptive capacity of Gelfoam is a function of its physical size, increasing with increasing gelatin volume (27). PROTOCOL #1 Lubaroff, page 6 The mechanism of action of surface-mediated hemostatic devices is supportive and mechanical (27). Surface-acting devices, when applied directly to bleeding surfaces, arrest bleeding by the formation of an artificial clot and by producing a mechanical matrix that facilitates clotting (28). Jenkins et al have theorized that the clotting effect of Gelfoam may be due to release of thromboplastin from platelets, occurring when platelets entering the Gelfoam become damaged by contact with its myriad of interstices (29). Thromboplastin interacts with prothrombin and calcium to produce thrombin, and this sequence of events initiates the clotting reaction. The authors suggest that the physiologic formation of thrombin in Gelfoam is sufficient to produce formation of a clot, by its action on the fibrinogen in blood (29). The spongy physical properties of Gelfoam hasten clot formation and provide structural support for the forming clot (28,30). Gelfoam has been used experimentally for the delivery of soluble proteins and drugs, including insulin, antibiotics , and growth factors (31-33). Gelfoam was used for sustained release of insulin in an ocular implant device (31). Delivery of insulin in solution had no effect on blood glucose levels. In contrast, the use of Gelfoam as a sustained release delivery agent provided measurable insulin activity for up to 10 hours after implantation. Glucose levels in the blood stabilized at 60% of the original value, whereas administration of insulin in eye drops had no effect. MacDonald and Mathews (34) studied Gelfoam implants in canine kidneys and reported that it assisted in healing, with no marked inflammatory or foreign-body reactions. Jenkins and Janda (35) studied the use of Gelfoam in canine liver resections and noted that Gelfoam appeared to offer a protective cover and provide structural support for the reparative process. Correll et al (36) studied the histology of Gelfoam when implanted in rat muscle and reported no significant tissue reaction. Gelfoam has been used as a hemostatic agent in dog prostate (37). In these studies no gross histological evidence of tissue damage or calcification was induced. In addition, these investigators demonstrated that placement of Gelfoam into the lumen of the bladder resulted in liquefaction of the Gelfoam without any evidence of calculogenesis. Finally, Bischoff and Goerttler (38) used Gelfoam in human prostate therapeutic embolization with success. Our laboratory, in collaboration with Dr. Timothy Ratliff, has demonstrated that administration of the Ad/PSA vaccine in Gelfoam induces a stronger anti -PSA immune response (Figure 1). In our pre-clinical studies, immunization with the vaccine in an aqueous suspension induces strong immunity with 10 9 pfu with weaker immunity induced with 108 and 107 pfu. Use of Gelfoam permits the induction of strong responses at the lower dose of 108 pfu. In addition, strong anti -PSA T cell responses could be induced by immunization with the Ad/PSA vaccine in Gelfoam even in mice pre-immunized to adenovirus (Figure 2). In the Phase I clinical trial (section 1.4), the addition of Gelfoam to the vaccine immunization did not result in excess serious adverse events. 1.26 3.125 6.25 12.5 25020406080100 Ad5-PSA 109 Ad5-PSA 108 Ad5-PSA 107 Ad5-lacZFluid Phase Effector Cells x 104/104 Target Cells%51Cr Release 1.56 3.125 6.25 12.5 25020406080100 Ad5-PSA 109 Ad5-PSA 108 Ad5-PSA 107 Ad5-lacZGelfoam Effector Cells x 104/104 Target Cells%51Cr Release Figure 1 – Anti- PSA cytotoxic activity of spleen cells obtained from mice immunized with Ad/PSA in aqueous (fluid phase) versus Gelfoam. PROTOCOL #1 Lubaroff, page 7 1.4 Phase I study A Phase I clinical trial of the Ad/PSA vaccine has been completed in men with measurable metastatic prostate cancer, with the primary objectives of determining the toxicity profile and maximal tolerated dose (MTD). The ability of the vaccine to induce anti -PSA immune responses and any clinical responses was also evaluated. Funding for the Phase I trial was provided by multiple sources that include the Holden Comprehensive Cancer Center, the Department of Urology, and the Carver College of Medicine at the University of Iowa. Eligible patients consisted of men with prostate cancer that had measurable metastatic disease, 90% of whom were stage D3. Prior therapies had included androgen withdrawal, ketaconazole, prednisone, Casodex, Taxotere, and external beam radiation, but the initiation of vaccine therapy was equal to, or greater than, 30 days after the most recent therapy. Patients were treated in successive dose levels and aqueous vs.