1.2.2 Prostate Cancer Prostate cancer is the second leading cause of cancer deaths in men in the United States. According to the American Cancer Society, approximately 200,000 new diagnoses of, and 28,000 deaths from prostate cancer occur each year in the United States (Jemal 2009). Up to one- third of patients with localized disease will fail local therapy within 10 years (as evidenced by a rising PSA) (Pound 2001, Freedland 2007). Patients with recurrent disease generally undergo hormone suppression therapy, with medical or surgical castration. Unfortunately, in most cases, this is eventually followed (after good response for several years) by progressive disease. Castration- resistant prostate cancer (CRPC) is characterized by disease that is uncontrolled despite low levels of testosterone and/or the presence of androgen receptor antagonist treatment (Rini 2002). Upon the development of metastasis, overall survival is approximately 1– 3 years. Taxane- based chemotherapy (docetaxel) and Provenge™ (Provenge, sipuleucel -T) are the only approved agents for the first-line treatment of patients with metastatic disease that have shown a prolonged survival benefit. Prostate cancer has been shown to be generally poorly responsive to chemotherapy. Taxotere chemotherapy has a beneficial effect on survival of approximately 3 months (Tannock 2004, Petrylak 2004, Berthold 2008). Cabazitaxel -based chemotherapy also offers an apparent 2- 3 month improved survival in a second- line setting (after docetaxel chemotherapy) (DeBono 2010). However, as with many other cytolytic agents, the impact on the patient’s quality of life is significant. More recently, abiraterone (an androgen hormone synthesis inhibitor) has also been shown to prolong median survival by approximately 4 months, and has been approved for use in second- line setting (De Bono 2011). Provenge has shown a beneficial survival benefit of approximately 4 months (Kantoff 2010, NEJM). Provenge, while having a comparatively favorable safety profile with a slightly better survival benefit, requires extensive processing during the treatment regimen and, therefore, has practical limitations. Consequently, therapies that could significantly improve the therapeutic benefit (survival and safety) as well as practical use of that observed with chemotherapy and Provenge, respectively, are urgently needed. Protocol BNIT -PRV- 301; Amendment 7 PROSTVAC -V/F Version 2.7, 08 February 2017 CONFIDENTIAL AND RESTRICTED 18 Amend 7 Disclosure requires written consent from Bavarian Nordic , Inc. 1.2.3 Prostate Cancer Immunotherapy Therapeutic cancer vaccines have been investigated in both castration- sensitive and castration- resistant disease (Schlom 2007). Prostate cancer is an ideal candidate for treatments that stimulate the immune system to target cancer cells. Prostate cancer may progress slowly (even in advanced disease), allowing time for the immune system to be stimulated, and to mount an active immune response. It is a poorly chemosensitive disease, so patients are generally not heavily pretreated with cytotoxic agents, and their immune systems are more intact. Prostate cells have a unique and specific marker, prostate- specific antigen (PSA), which is secreted into the blood, and can be detected with even low levels of tumor burden. Several other gene products are unique to prostate cells, and prostate cancer cells over -express tumor -associated antigens (TAA), making for multiple targets suitable for immunotherapy responses (Cunha 2006, Bradford 2006). Many patients with prostate cancer have low levels of cytolytic T -cells capable of recognizing PSA, and this minimal response can be enhanced with immune stimulation by therapeutic cancer vaccines (Chakraborty 2003). Finally, because the prostate is a nonessential organ, targeting PSA - or TAA- specific immune cells to prostatic tissue is unlikely to have a significant negative clinical impact. The first FDA approved prostate cancer immunotherapy, Provenge (sipuleucel -T), is now available (approved 29 April 2010). However Provenge is available only in the United States. Provenge is a cell - based vaccine consisting of autologous PBMCs that have been activated ex -vivo with a recombinant protein representing a fusion of PAP (prostatic acid phosphatase) and GM- CSF. Early clinica l work demonstrated prolongation of overall survival (Small 2006), and up- regulations of cellular immunity and reductions in PSA and PAP levels (Patel 2008). The Phase 3 IMPACT study of 512 subjects with metastatic castration- resistant prostate cancer met its primary endpoint of prolongation of overall survival. Provenge was approved for use in metastatic castration- resistant prostate cancer based on an increase in median survival of 4.1 to 4.5 months (Kantoff 2010). 1.2.4 PSA as a Target Antigen PSA is a promising target antigen for immunotherapy of prostate cancer. PSA is a ~34,000 dalton glycoprotein that is produced in normal, benign, and cancerous prostate epithelia, but not in other normal tissues (Wang 1982, Lilja 2008). Because PSA is expressed essentially only in prostatic epithelial cells (normal and malignant), and the prostate gland is nonessential, this antigen is an enticing choice. The fact that PSA is secreted and not membrane- bound limits its use as a target for humoral immunity, but not its use as a target of specific cellular immune system attack. Cells, including tumor cells, present endogenously expressed proteins on their surface in the form of peptide- major histocompatibility complexes (MHC). Cytotoxic T -lymphocytes (CTLs) recognize and are activated by specific peptides in the context of the appropriate MHC class I molecule on antigen- presenting cells (APC). This activation can in turn lead to killing of tumor targets by the peptide- specific CTLs. The use of PSA as a target to elicit tumor -specific T -cell-mediated lysis has been validated in vitro . Correale et al. demonstrated in vitro killing of a PSA -peptide- pulsed HLA -A2+ human cell line by a PSA - Protocol BNIT -PRV- 301; Amendment 7 PROSTVAC -V/F Version 2.7, 08 February 2017 CONFIDENTIAL AND RESTRICTED 19 Amend 7 Disclosure requires written consent from Bavarian Nordic , Inc. specific human CTL cell line, and lysis was blocked by an antibody directed against MHC class I molecules (Correale 1997). It has also been shown that PSA -specific CTLs could be generated that lyse PSA- expressing prostate cancer cells (Correale 1997, 1998). The identification of these peptide epitopes further provides a means of identifying cytolytic T -cells resulting from immunization as an in vitro monitoring tool. 1.2.5 PSA-3-epitope modification Protein antigens are presented to CTLs as small peptides (approximately 9– 10 amino acids long) bound to class I molecules of the MHC. One strategy to increase the immunogenicity of a self -antigen such as PSA is to modify selected epitopes within the protein sequence to enhance their binding to MHC class I alleles. The stabilized binding enables more sustained and potent immune cell activation. One such epitope, designated PSA- 3 (amino acids 154 –163), which is specific for the MHC class I A2 allele, was modified by the introduction of a single amino acid change (I to L) at position 2 in the epitope (designated L155) (Correale 1998, Terasawa 2002). A number of in vitro studies suggest that PSA carrying this modification will be more immunogenic in HLA -A2 individuals than the native (unmodified) PSA polypeptide (Correale 1998, Terasawa 2002) . 1.2.6 Poxviral Vaccine Approach Poxviruses are large multi -enveloped DNA viruses which encode about 200 genes. Poxviral vectors are able to accommodate multiple transgenes, and are able to deliver PSA and multiple immunostimulatory genes directly or indirectly to APCs. The activated APCs process and present the PSA antigen, leading to PSA specific T -cell activation (Essajee 2004, Schlom 2008). PSA as a soluble protein is weakly immunogenic, especially in the tumor -bearing host. Vaccine strategies must induce greater activation of T-cells than is being achieved endogenously in the host. The poxviral system achieves this by generating a natural inflammatory context for facilitating activation of immune cells, and inducing PSA -specific immune responses. Extensive pre- clinical studies in experimental murine models have demonstrated the therapeutic utility of using poxviral vaccines to break tolerance (Bernards 1987, Irvine 1993, Kass 1999). Another advantage of poxviral vaccines is that they are well tolerated by the patients. The relative safety of PSA- based poxviral vectors has been well established in previous trials (Madan 2009, Kantoff 2010, Kaufman 2004, Arlen 2007). 1.2.7 Vaccinia Virus Vaccinia virus has been used for over 200 years as a vaccine for smallpox and has a well -established safety and adverse event (AE) profile (Casey 2005, Poland 2005). The virus actively replicates in human cells, and generates a strong inflammatory response, resulting in the presentation of high levels of antigen to the immune system over a period of one to two weeks, supporting a potent immune stimulation. Subsequently, the immune response specific to vaccinia then eliminates the virus. Host immune responses to vaccinia restrict its replication and thus limit its ability to be re- used for subsequent Protocol BNIT -PRV- 301; Amendment 7 PROSTVAC -V/F Version 2.7, 08 February 2017 CONFIDENTIAL AND RESTRICTED 20 Amend 7 Disclosure requires written consent from Bavarian Nordic , Inc. vaccinations. Consequently, vaccinia- based vaccines, although potent immunological priming agents, can be used to immunize an individual only once or twice. 1.2.8 Fowlpox Virus Fowlpox virus is a member of the genus Avipo x, which is evolutionarily divergent from vaccinia virus and serologically non cross- reactive (Taylor 1988, Beukema 2006). Immune responses to vaccinia are essentially non cross -reactive with fowlpox, and do not block infection and immunization with fowlpox- based vectors. Hence vaccinia- primed immune responses can be boosted with fowlpox vectors. In addition, fowlpox vectors do not replicate in human cells (only in avian cells), and are therefore much less of a safety risk than vaccinia- based vectors. Fowlpox vectors mediate a limited infection in human cells, with early viral and transgene expression, but late gene expression is blocked, and no infectious particles are produced. Thus minimal viral surface antigen is made, and minimal neutralizing antibody immune responses are induced. This enables multiple boosting with the fowlpox -based vectors. 1.2.9 Prime -Boost Recombinant vaccinia and fowlpox vectors are most effective when used in combination in prime- boost regimens. By priming with recombinant vaccinia virus and then boosting repeatedly with the corresponding recombinant fowlpox virus, maximum immune responses to the expressed tumor antigens can be obtained. This phenomenon has been demonstrated in animal models (Hodge 1997, Dale 2006) and has been supported by the results from the completed Phase 1 and Phase 2 trials conducted by the National Cancer Institute (NCI), Eastern Cooperative Oncology Group (ECOG), and Therion. 1.2.10 TRICOM - costimulatory molecules Destruction of immunological targets requires T -cell lymphocyte recognition, via the T -cell receptor, of antigenic peptides presented in the context of MHC molecules on APCs. In addition to this antigen-specific signal, a second, antigen- independent signal is required for T -cell activation (Boussiotis 1995, Hodge 2006). This second signal is provided by the interaction of specific ligands on the T -cell surface with “costimulatory” molecules expressed on APCs. The most extensively studied pathway of costimul ation is that involving the interaction of the costimulatory molecule B7.1 expressed on APCs with CD28 and CTLA 4 on the T -cell (Sharpe 2002, Riley 2005). A number of additional costimulatory molecules on APCs have been identified; these include ICAM- 1 and LFA-3, whose ligands are LFA -1 and CD2, respectively, on the surface of T -cells (Wingren 1995, Lebedeva 2005, Wang 2008). Proper engagement of the T -cell receptor and costimulatory receptor requires the expression of both antigen and costimulatory molecules, respectively, in the same cell. Therefore, co- expression of costimulatory molecules using a single recombinant vector presents the potential of cooperation among these proteins to enhance T -cell activation. Recombinant vectors co- expressing three costimulatory molecules, LFA- 3, ICAM- 1, and B7.1, designated TRICOM™ (TRICOM), have been shown to have synergistic effects on antitumor responses as compared to vectors expressing individual costimulatory molecules (Hodge 1999). Mice immunized with a recombinant vaccinia virus co- expressing Protocol BNIT -PRV- 301; Amendment 7 PROSTVAC -V/F Version 2.7, 08 February 2017 CONFIDENTIAL AND RESTRICTED 21 Amend 7 Disclosure requires written consent from Bavarian Nordic , Inc. carcinoembryonic antigen (CEA) and murine TRICOM exhibited greater immune responses and anti - tumor responses than mice immunized with a recombinant vaccinia virus co- expressing CEA and murine B7.1. Enhanced anti- tumor immunity was also observed in mice that were transgenic (tolerant) for CEA (Hodge 1999). PROSTVAC -V and PROSTVAC -F have, therefore, been designed to simultaneously express PSA together with B7.1, LFA -3, and ICAM- 1. 1.2.11 GM-CSF Granulocyte macrophage colony -stimulating factor (GM- CSF) has been shown to enhance antigen processing and presentation by dendritic cells (DC). GM- CSF affects man y immunologic processes, including increasing expression of class II MHC molecules, augmenting the primary antibody response, and inducing localized inflammation when administered by injection (Warren 2000, Chang 2004). GM- CSF has the ability to promote local DC differentiation, maturation, and subsequent migration to regional lymph nodes. Antigens released by target cells, such as those from tumors, are likely to be taken up and processed by DCs and presented to T -cells, thereby inducing the activation of a cellular immune response. These immunologic properties of GM- CSF have made this cytokine a potentially attractive adjuvant candidate for vaccines. Indeed, use of both recombinant human (rhu) GM- CSF and the recombinant poxviruses expressing GM- CSF has been shown to enhance immunological effects of vaccines in both non- clinical (Kass 2001) and clinical studies (Marshall 2000). GM- CSF was administered as an adjuvant to the PROSTVAC -V/F vaccine in the randomized, placebo controlled Phase 2 trial, and the positive outcome of this trial supports its use in this Phase 3 trial. More recent data have however, challenged the benefits of GM- CSF. A recent review of approximately 14 different human clinical cancer vaccine studies that employed GM- CSF as an adjuvant (P armiani, 2007) concluded that the studies reviewed provided limited evidence to justify the use of GM- CSF as an adjuvant and might be even detrimental under certain conditions. There is also growing evidence that systemic use of GM- CSF can compound tumor -mediated immune suppression by acting as a growth factor for myeloid- derived suppressor cells (MDSC) (Talmadge, 2007). Lastly, in a small Phase 2 trial of PROSTVAC -V/F conducted by the NCI, GM- CSF use was not associated with any dramatic clinical benefit. However, only eight subjects per arm were compared (GM- CSF, no GM -CSF, FPV -GM-CSF, FPV- GM-CSF high dose), making firm conclusions difficult. Together these data support going forward with a third arm of the trial comparing PROSTVAC -V/F without GM- CSF to c ontrol. 1.3 Placebo Rationale BNIT -PRV- 301 will utilize a placebo control in order to fulfill the double- blind design of the study. In a double- blind trial, neither the investigator nor the subject has knowledge of the treatment being administered. This study design is utilized in order to control variables that could be caused by knowledge of treatment assignment, either on the part of the investigator or the subject. In order to ensure a double-blind design, a study protocol must carefully account for all circumstances in which the Investigator or subject could obtain knowledge of treatment assignment. One of the most obvious requirements to maintain a blind is ensuring that study drug administration is perceived as identical across study arms. Protocol BNIT -PRV- 301; Amendment 7 PROSTVAC -V/F Version 2.7, 08 February 2017 CONFIDENTIAL AND RESTRICTED 22 Amend 7 Disclosure requires written consent from Bavarian Nordic , Inc. This protocol design minimizes the risk of bias and enhances the impartial analysis of the study drug versus placebo effect. However, it is important to note that in case this information is required for safety purposes it will be provided in an expedited manner. In minimally symptomatic metastatic prostate cancer there is no standard of care. Most patients and physicians utilize chemotherapy when the disease has become symptomatic, or has taken on accelerated kinetics. These patients are excluded from this trial (BN IT-PRV- 301). The side effect profile of chemotherapy and its associated risks have favored later use of chemotherapy, where the potential for pain relief shifts the risk -benefit profile. Immunotherapy is a promising approach in this early stage of metastatic prostate cancer. Although promising, sipuleucel -T is not widely available, and has logistic and cost concerns. In BNIT -PRV- 301 the placebo will be an empty viral vector. Tissue culture adapted fowlpox virus, the same host strain as used in PROSTVAC -F will be used. This vector is non- replicating in humans and has minimal safety risk. In addition, the cutaneous injection site reactions (and side effect profiles) are overlapping (and generally indistinguishable) with subcutaneously administered vaccinia (PROSTVAC -V). Thus this vector forms an ideal placebo. It has no known effects on cancer progression, and is not expected to have any significant effects on its own. It is manufactured by the same process as PROSTVAC -V and PROSTVAC -F, and has the same look and feel of the other investigational vaccine vectors. 1.4 Summary of Findings from Human Clinical Studies Clinical investigation of the PROSTVAC -V/F vaccine product was initiated in 2002 by Therion in the United States under BB -IND 10428. Therion manufactured all PROSTVAC -V/F product and conducted two clinical trials on PROSTVAC -V/F, including a Phase 1 trial evaluating the safety and immunogenicity of PROSTVAC -V/F in 10 subjects, and a randomized, placebo- controlled Phase 2 trial evaluating the safety and efficacy (as defined by time- to-progression; TTP) of PROSTVAC -V/F in approximately 125 men with castration- resistant metastatic prostate cancer. Both trials were conducted in the US only. Therion also manufactured the PROSTVAC -V/F product for all the NCI and ECOG trials. 1.4.1 Poxviral PSA Vaccine Clinical Trials The use of poxviral vectors to stimulate an immune response to PSA has been evaluated in several clinical trials. In a Phase 1 clinical trial using an earlier version of a PSA -based vaccine (devoid of an y costimulation), Eder et al . used three monthly vaccinations of recombinant vaccinia (rV) -PSA (Eder, 2000). Six out of 10 subjects with rising PSA following local therapy who received the highest tested dose, with GM- CSF, had a time of PSA progression of greater than six months. Four of these 6 subjects still showed no progression, with the longest time of follow- up greater than 24 months. This was suggested to be evidence of clinical activity (Eder 2000). Five of 7 HLA -A2+ subjects in this final dose level had at least a 2- fold increase in PSA -specific T -cells in peripheral blood mononuclear cells (PBMCs) as a result of vaccination, as measured by the ELISPOT assay, indicating that tolerance to self -antigens Protocol BNIT -PRV- 301; Amendment 7 PROSTVAC -V/F Version 2.7, 08 February 2017 CONFIDENTIAL AND RESTRICTED 23 Amend 7 Disclosure requires written consent from Bavarian Nordic , Inc. could be circumvented with these vaccine strategies. Another Phase 1 study of rV -PSA also demonstrated that immune responses could be elicited in subjects with metastatic hormone- refractory prostate cancer and that immune cells taken from these subjects could specifically lyse PSA -expressing cancer cel ls in vitro (Gulley 2002). ECOG previously conducted and reported a randomized Phase 2 study in which 64 subjects with rising PSA following definitive local therapy with no evidence of disease on imaging studies were randomized to receive four vaccines w ith rV -PSA (designated V) and/or rF -PSA (designated A for avipox) (Kaufman 2004). The arms were thus VAAA, AAAA and AAAV. PSA progression- free survival (PFS) was about 9 months in the latter arm, and about 18 months in the VAAA arm, lending further suppor t to the use of vaccinia priming and avipox vector boosting. Vaccinia and fowlpox vectors containing the gene for human PSA with a single amino acid substitution (designated PSA (L155)) along with genes encoding TRICOM have been tested in two fully accrued Phase 1 clinical trials with 25 subjects with metastatic prostate cancer (Arlen 2007, DiPaola 2006). There were no dose- limiting toxicities on these studies attributed to vaccine. One of these studies in subjects with mCRPC has s hown that subjects treated with PSA/TRICOM who were evaluable for immune response had an increase in PSA -specific T -cells after treatment; and 9 of 15 subjects had decreases in PSA velocity (Arlen 2007). The Therion- sponsored Phase 2 trial randomized subjects with mCRPC 2:1 in favor of vaccine vs. an empty fowlpox vector as control. 125 subjects with mCRPC and Gleason scores ≤ 7 were enrolled (122 treated). Subjects randomized to receive vaccine (82 subjects) were given subcutaneous rV -PSA- TRICOM prime with monthly boosts of rF -PSA- TRICOM, while control subjects (40 subjects) were given subcutaneous injections of fowlpox. Although no difference in TTP was seen in the short -term, in an interim analysis at two years of follow -up, median overall survival was 24.4 months in the vaccine arm compared to 16.6 months in the control arm, suggesting that although disease progression occurred at similar times in both groups, there appeared to be a long- term benefit for some subjects treated with PSA/TRICOM (Schlom 2 008, Kantoff 2006). The overall survival results after a final survey (median follow -up 4 years) were recently completed. The formal overall survival update revealed that PROSTVAC -V/F immunotherapy was associated with a significant survival benefit. PROS TVAC -V/F-treated subjects had a longer median survival (25.1 months versus 16.6 months controls), and an increased survival at 3 years post study (25 of 82 subjects, 30%; versus 7 of 40 control subjects, 17%). Statistical analysis of the Kaplan- Meier curves revealed an estimated hazard ratio of 0.56 (95% CI 0.37- 0.85), and a stratified log rank of P=0.006 (Kantoff 2010). In an NCI Phase 2 study (NCI no.5911) of PSA/TRICOM, 32 subjects with metastatic CRPC were treated with an rV -PSA- TRICOM prime and monthly boosts of rF -PSA- TRICOM. In that trial, 47% of subjects had a decrease in PSA velocity, 38% had a PSA decline. Median time to progression was 2.8 months. One subject at 8 months experienced a decrease in his hilar adenopathy associated with declining PSA (> 30%). He remained on study for 12 months. Another subject had a decrease of 29% in his adenopathy Protocol BNIT -PRV- 301; Amendment 7 PROSTVAC -V/F Version 2.7, 08 February 2017 CONFIDENTIAL AND RESTRICTED 24 Amend 7 Disclosure requires written consent from Bavarian Nordic , Inc. (by RECIST) associated with a decline in his PSA (> 30%). Another subject who enrolled on study with a super -scan on bone scan had a >70% decline in his PSA and remains on trial more than 4 years after enrollment. Overall, 13 of 29 evaluable subjects had a > 2- fold increase in PSA- specific T -cells. In addition, 5 subjects had a > 6- fold increase in PSA- specific T -cells, which was associated with a trend to improved overall survival (p = 0.055) (Madan 2008, Gulley 2009). 1.4.2 Clinical Safety of Recombinant Poxvirus -Based Vaccines Since 1991, 10 recombinant vaccinia- based vaccines and 8 recombinant fowlpox -based vaccines produced by Therion for the treatment of various cancers have been evaluated in human clinical trials sponsored by CTEP, DCTD, NCI. Over 1,000 cancer subjects, most with metastatic disease, have been treated to date with these poxvirus -based vaccines in 29 CTEP -sponsored or Therion- sponsored clinical trials (Kaufman 2004). These trials represent a large component of the relevant safety database that supports the initiation of the proposed Phase 3 trial of PROSTVAC -V/F. Significant safety experience in humans includes: (a) vaccinia- and/or fowl pox-based vaccines safely administered by a variety of routes including intradermal (by injection or scarification), subcutaneous (sc), intramuscular (im), intravenous (iv), and intra -tumoral at doses up to 2 x 10 9 Inf. U.