This process will be completed within 15–30 minutes of faxing the eligibility checklist. CRO will keep track of all randomization data in Clinical Data Registry (CDR) of CRO. 2.5 B ASELINE EVALUATION A. Baseline electrocardiogram (EKG) on all patients, and appropr iate cardiologic evaluation, as clinically indicated, to provide baseline function and identify any patients who should be monitored closely fo r cardiac risks associ ated with vaccinia vaccination B. Apheresis (cohort 1 only) C. The following parameters will be obtained within 16 days prior to start of treatment: 1. Clinical evaluation xHistory and physical examination xECOG performance status (see Appendix A ) xHeight, weight 2. Laboratory studies xSerum PSA xCBC/differential, with platelet count xSerum testoste rone level xSerum chemistries (Na+, K+, Cl-, CO 2, glucose, BUN, creatinine, albumin, calcium, magnesium, phosphorus, alkaline phosphatase, ALT, AST, total bilirubin, LDH, CK, uric acid, total protein) xLymphocyte phenotyping CD3/CD4/CD8 3 STUDY IMPLEMENTATION 3.1 S TUDY DESIGN xThis is a randomized Pilot trial of enzalu tamide vs. enzalutamide and PSA-TRICOM in patients with nmCSPC prostate cancer. Thirty-four patients will be enrolled. xEnzalutamide will be given at the standard dose of 160 mg daily for 3 months (7). PSA-TRICOM will be administered identical to the Phase III dosing with vaccine given week 1 (vaccinia-PSA-TRICOM, 2x108infectious units subcutaneously) and then fowlpox-vaccine (1x109infectious units subcutaneously) on weeks 3 and 5 Version Date: 12/5/17 Abbreviated Title : Enzalutamide in non-met CSPC 28followed by monthly fowlpox-vaccine (1x109 infectious units subcutaneously) for a total of 5 months (including vaccinia, vaccine is administered over 6 months). xPatients will be followed for PSA recovery after enzalutamide has been discontinued. Patients who do not develop a 25% decline in PSA after 3 months will not be evaluated for tumor re-growth and additional patients will be enrolled to evaluate for that endpoint. xPatients who have been on study for at least 7 months without PSA equaling or exceeding the value at study entry, and whose PSA subsequently (beyond 7 months) returns to or exceeds the value at study entry, may be retreated with a 3-month course of Enzalutamide. xPatients who continue to be followed for rising PSA af ter completing either one or 2 courses of enzalutamide but have not required ADT may be followed after treatment. Such visits may occur at 4 week intervals based on logistics and clinical parameters (i.e. PSA velocity) once pa tients have exceeded their baseline PSA value. This approach is consistent with the clinical management of such non-metastatic patients (rising PSA and not on ADT.) xThe primary endpoint of this trial will be a comparison of the tumor re-growth rates at 7 months. This time frame should be suffici ent to evaluate immune effect on the re- growth rate given that the ECOG 9802 analysis demonstrated a significant change in tumor growth rate after starting PSA-TRICOM.Secondary endpoints include growth rate at points other than 7 months, toxicity, immunologic responses to vaccine with en zalutamide and to enzalutamide alone in men with normal testos terone, and response to second course of enzalutamide. When possible, patients will be followed for TTP. End of study period will be 12 months in each arm (24 months if patients are eligible and elect to receive a 2 ndcourse of enzalutamide). xPreliminary data from the first cohort of ra ndomized patients suggests that enzalutamide alone can induce an immunologic response. A second cohort of 15 patients will explore a lower dose of enzalutamide at 80 mg to de termine if similar immunol ogic responses can also be seen at a lower dose, where toxicity is less likely. 3.1.1 Protocol Stopping Rules This study will utilize the Common Terminology Criteria for Adverse Events (CTCAE) version 4.0 for grading systemic toxicity (see section 6.3). xOne occurrence of grade 5 toxicity by th e NCI-CTCAE version 4.0 attributable to the treatment regimen. xTwo occurrences of grade 4 toxicity by the NCI-CTCAE version 4.0 attributable to the treatment regimen. If either of the above occurs, the Principal Investigator will halt accrual to the trial and will discuss with the NCI Institutional Review Boar d (IRB) and Medivation and Astellas Inc. whether any changes need to be made to the protocol. Version Date: 12/5/17 Abbreviated Title : Enzalutamide in non-met CSPC 29All patients will receive vaccine treatments at the NIH Clinical Center, patients will receive enzalutamide primarily in the NCI outpatient setting. We estimate an accrual of approximately 2 patients per month, with full accrual of 34 patients within 18 months. 3.2 D RUG ADMINISTRATION All patients in cohort 1 (n=34) will receive enzalutamide 160 mg daily orally for 3 months. Patients randomized to vaccine arm (Arm B) w ill receive PSA-TRICOM identical to Phase III dosing, which consists of a single immunization of PROSTVAC-V (vaccinia-PSA-TRICOM, 2x108infectious units subcutaneously) in Week 1, followed by immunizations of PROSTVAC-F (fowlpox-PSA-TRICOM 1x109infectious units subcutaneously) administered in Weeks 3, 5, 9, 13, 17, and 21. 3.3 T REATMENT MODIFICATIONS 3.3.1 Vaccine 3.3.1.1 Dosing delay x3DWLHQWVPXVWKDYHUHFRYHUHGWRJUDGHWR[LFLW\UHODWHGWRY DFFLQHIRUWKH parameters used to assess levels of organ function required for eligibility (see Section 2.1.1) after each vaccination in order to receive a subsequent vaccination. x3DWLHQWVZLWKJUDGH WR[LFLW\QRWUHODWHGWRYDFFLQHWUHDWPH QW LH attributed to enzalutamide) will remain eligible to receive vaccine. xPatients that recover from > grade 2 toxicity unrelated to vaccine treatment (i.e. related to enzalutamide) may remain eligible for the vaccine. x,IJUDGHQRQDXWRLPPXQHWR[LFLW \DWWULEXWDEOHWRWKHYDFFLQH SHUVLVWVIRU!42 days, the patient will not receive furt her vaccine inoculations and will be removed from the protocol treatment and followed for resolution of toxicity and immune endpoints. xPatients who develop grade 3 injection site reactions will have their vaccine held until injection site reaction resolves to grade 2 or less. x3DWLHQWVZKRGHYHORSDJUD GHDOOHUJLFRUDXWRLPPXQ e disease that threatens YLWDORUJDQIXQFWLRQRUDQ\JUDGHDXWRLPPXQLW\QRWUHODWHGWRDWKHUDSHXWLF response, will be removed from the pr otocol treatment and followed for resolution of toxicity and immune endpoints. xPatients who develop any grade 4 toxicity attributable to the study drug(s) will be removed from the protocol treatment and followed for resolution of toxicity and immune endpoints. xIf a scheduled vaccine dose is missed due to scheduling or logistical issues, the vaccine may be given within 7 days of the appointed time 3.3.1.2 Dose modifications No dose modifications are allowed with this vaccine. Version Date: 12/5/17 Abbreviated Title : Enzalutamide in non-met CSPC 303.3.2 Enzalutamide ,IDSDWLHQWH[SHULH QFHVD*UDGHQRQ -hematologic toxicity attributable to enzalutamide or an intolerable side effect attributable to enza lutamide, withhold dosing for one week or until V\PSWRPVLPSURYHWR*UDGHWKH QUHVXPHDWWKHVDPHGRVHLI FOLQically appropriate. If toxicity recurs enzaluatmide will be held again using the above parameters or discontinued at the discretion of the investigator. Patients who develop any grade 4 toxicity attributable to the study drug(s) will be removed from the protocol treatment and followed for resolution of toxicity and immune endpoints. 3.4 P ROTOCOL EVALUATION (SEEAPPENDIX C) 3.4.1 All patients who are deemed eligible and who sign an info rmed consent will be enrolled in this trial. 3.4.2 A complete history and physical examina tion, including ECOG perfo rmance status, will be done within 16 days before enrollment. 3.4.3 Laboratory Studies (See also Appendix C ) xSerum PSA xCBC/differential with platelet count xSerum testoste rone level xSerum chemistries (Na+, K+, Cl-, CO 2, glucose, BUN, creatin ine, albumin, calcium, magnesium, phosphorus, alkaline phosphatase, ALT, AST, total bilirubin, LDH, CK, uric acid, total protein) xHIV/Hepatitis B/Hepatitis C tests (within 8 weeks prior to start of enrollment) xLymphocyte phenotyping CD3/CD4/CD8 xPAP (prostatic acid phosphatase) xThe following serum hormone levels will be drawn at each follow up visit after enrollment and pre-Enzalutamide administration, and 1 hour post, 24 hours post, 48 hours post, and 72 hours post administration, when logistically feasible. otestosterone odihydrotestosterone (DHT) osex hormone-binding globulin (SHBG) odehydroepiandrosterone (DHEA) oluteinizing hormone (LH) ofollicle-stimulating hormone (FSH) oandrostenedione oprolactin oestradiol xPlasma VEGF levels will be drawn at the st art and at the end of each patient’s second 12-week course of Enzalutamide. Version Date: 12/5/17 Abbreviated Title : Enzalutamide in non-met CSPC 313.4.4 Radiographic assessment Patients will not undergo scheduled radiographic assessment fo r metastatic disease unless clinically indicated for increasing PSA or symp toms. Radiologic studies consisting of bone scan, CT scan of chest, and CT scan of abdomen/p elvis will be performed at baseline. MRI may be substituted for CT scan at the discretion of the investigator. Patients will undergo a radiographic assessment with CT scan of chest, abdomen, and pelvis and bone scan once PSA returns to baseline at study entry or higher. These scans will be obtained prior to retreatment with a 3- month course of enzalutamide. For patients who are eligible and elect to receive a second course of enzalutamide therapy for 3 months, re-staging CT scan of chest, abdom en, and pelvis and bone scan will again be done when PSA reaches baseline. 3.4.5 Assessment for PSA-TRICO M administration (Arm B) Dosing and administration of PSA-TRICOM will be performed in the Day Hospital. Patients will be monitored with vital sign s (blood pressure, heart rate, resp iratory rate, temperature) prior to and within 1 hour after the initial vaccine treatment. On subse quent visits (where rF-PSA- TRICOM is administered) patients will have vital signs checked prior to vaccine and then be monitored for 30 minutes after vacci ne treatments. (Post-vaccine vital signs will not be required except for the first vaccination.) Documentation of any patient reported symptoms occurring between dosing will be included in the assessment. Laboratory assessments will be conducted as perAppendix C : On Study and Follow-Up Evaluations . 3.5 C ONCURRENT THERAPIES Concurrent hormonal therapy not otherwise stipulated in the protocol will not be allowed. Concurrent anticancer treatment with chemothe rapy, systemic glucoc orticoids (topical and inhaled steroids allowed), radia tion therapy, major surgical procedures for prostate cancer, and nonprotocol-related immunothera py will not be permitted. 3.6 C RITERIA FOR REMOVAL FROM PROTOCOL THERAPY AND OFFSTUDY CRITERIA Prior to removal from study, effort must be made to have all subjects complete a safety visit approximately 30 days following the last dose of study therapy. 3.6.1 Criteria for removal from protocol therapyPatients will be removed from treatment for the following: xClinical or radiographi c progression of disease. xGrade 3 or greater toxicity attributed to treatment that does not resolve to grade 1 within 42 days from time of scheduled treatment. xGrade 3 or greater toxicity not related to treatment that does not resolve to grade 2 within 42 days from time of scheduled treatment. xGrade 2 or greater autoimmune disease that threatens vitals organs. Version Date: 12/5/17 Abbreviated Title : Enzalutamide in non-met CSPC 32xAny Grade 4 toxicity that is possibly, probab ly or definitely rela ted to the protocol treatment will require a pa tient to be off-treatment xIntercurrent illness or medical circumstances. If at any time the constraints of this protocol are detrimental to the patient’s hea lth, the patient may be removed from protocol therapy and reasons for withdrawal will be documented. xAny grade of seizure will require a patient to be off-treatment. xRequirement for androgen deprivation therapy xCompletion of protocol therapy 3.6.2 Off-Study Criteria xPatient is off-treatment and has agreed to be followed on a long term therapy protocol as outlined in section 3.7 xPatient requests to be taken off study. Reasons for withdrawal will be documented. xNoncompliance with protocol guidelines (p atient removed at di scretion of Principal Investigator). xDeath 3.6.3 Off Protocol Therapy and Off-Study Procedure Authorized staff must notify Central Registrati on Office (CRO) when a subject is taken off protocol therapy and when a subject is taken off study. A Participant Status Update Form from the web site ( http://home.ccr.cancer.gov/intra/eligibility/welcome.htm ) main page must be completed and sent via encrypted email to: NCI Central Registration Office ncicentralregistration-l@mail.nih.gov. 3.7 P OST-STUDY EVALUATION (FOLLOW -UP) The Biologic Response Modifiers Advisory Committee has recommended that long-term follow-up extend over a period of 15 years. Information re garding the findings will be reported to the FDA. Patients randomized to receive PSA-TRICOM will be offered enrollment in the 04-C-0274“Follow-Up Study of Subjects Previously Enro lled in Immunotherapy Studies Utilizing Gene Transfer or other immunotherapeutic agents” once off study. 4 BIOSPECIMEN COLLECTION 4.1 C ORRELATIVE STUDIES FOR RESEARCH 4.1.1 Immunologic Parameters xIFN-gamma ELISPOT assay for PSA-specific T lymphocytes (HLA-A2 patients only) xAntibodies to PSA, vaccinia, fowlpox may be tested xLeukocyte CD3, CD4, CD8 subsets; CD4:CD8 ratio will be drawn at baseline and monthly prior to vaccination while the patient remains on trial. xThe results of the HIV antibody need to be available before treatment to determine eligibility Version Date: 12/5/17 Abbreviated Title : Enzalutamide in non-met CSPC 33xHLA class I expression (HLA typing) with A2 subtyping (obtain anytime prior to enrollment). Patients may refuse or test may be omitted for logistical purposes at the discretion of the investigator. xAdditional studies will include but are not limited to quantitative and qualitative assessments of regulatory T-cells, Natural Killers cells, Myeloid Derived Suppressor Cells, anti-glycan antibodi es and Naïve T-cell/new thymic emigrants. xAssessment of leve ls of cytokines. xImmunologic studies will be repeated more frequently if clinically indicated, and any abnormalities potentially related to treatment w ill be followed until they have resolved, or have been determined not to be treatment-related. 4.1.2 Collection of Research (Immunologic) Blood Samples Immunologic blood samples will be obt ained via apheresis when logis tically feasible at Baseline (prior to treatment), after 3 months and after 6 months in all HLA-A2 patients. (Additional apheresis could be considered at the discretion of the investigat or and with the consent of the patient.) We plan to examine the immune response in selected patients (HLA-A2 positive) when feasible and if patients consent. (The HLA-A2 requirement is based on the ability to assess immune response in these patients using immunologic response techniques (ELISPOT). This analysis is not limited to these patients due to any reason to believe that non-HLA-A2 will not have an immune response, but rather those immune responses can not be detected by our current ELISPOT methods.) In all patients undergoing apheresis, 5 x 10 8to 2 x 109mononuclear cells will be obtained by a single-access (single venipuncture) mononucl ear cell procedure on the Haemonetics V-50 instrument, during which 2.0 liters of whole blood w ould be process at a flow rate of about 70-80 ml/min. The total duration of the procedure is 90 min to 2 hours. Patient s will be required to have a minimum HCT of 28% and a platelet c ount of at least 75,000 to undergo a Haemonetics procedure. Apheresis will allow us to obtain sufficient PBMC samples from patients to be able to perform not only the standard Elispot using the PSA-3 peptid e, but to also look at other epitopes that may be important in evaluating an antigen cascade effect from the therapy administered. This will allow us to gain important immunologic info rmation to determine if patients are not only mounting immune responses to the antigens in th e vaccine, but to also immune responses to other antigens present on the tumor cells. At the time points when apheresis is not obtained, we will obtain 6 green top (10ml) tubes. These will be used for ELISPOT assays (as described inAppendix D ). In addition, 2 SST (tiger) top tubes will be drawn for antibody testing (as described in Appendix D ). If apheresis is not done, research samples will be obtained per Appendix C Blood samples may be used for other research studies which may include phenotypic and functional analysis of immune cell subsets, and analysis for cy tokines, chemokines, antibodies, tumor-associated antigens and / or other markers. Version Date: 12/5/17 Abbreviated Title : Enzalutamide in non-met CSPC 34Immunologic blood samples will be processed at: Clinical Services Program NCI Frederick Cancer Research and Development CenterPO Box BFrederick MD 21702301-846-1707On days samples are drawn, Jennifer Bang h at CSP should be notified (phone: [301] 846-5893; fax [301] 846-6222). She will arrange courier delivery of the specimens to the processing lab.The weekly patient lists of samples drawn will be emailed to Jen Bangh at banghj@mail.nih.gov and Theresa Burks burkst@mail.nih.gov . 4.1.3 Immunologic Assays 4.1.3.1 IFN-gamma ELISPOTWe plan to examine the immune response in se lected patients (HLA-A2-positive). Lymphocytes will be separated from heparinized blood using density gradient centrifugation. The lymphocytes will then be placed in human AB serum with 10% DMSO and stored in liquid nitrogen. When samples are available from pre- and post-treatment, the ELISPOT assay will be performed. The ELISPOT assay, measuring IFN-gamma produc tion, is used to determine CTL precursor frequency to peptides from PSA, PSMA, MUC-1, PSCA, CEA, and PAP in both pre- and post- vaccination peripheral blood mononuclear cells, as previously described (39-40). Briefly, 96- well mL HA plates (Millipore Corporation, Bedford, MA) are coated with 100 Pl/well of capture MAb against human IFN-gamma at a concentration of 10 Pg/mL for 12h at RT. Plates are blocked for 30 min with RPMI 1640 plus 10% human Ab serum 2 x 10 5 PBMC are added to each well. PSA-3A pulsed C1R-A2 cells are added into each well as antigen-presenting cells (APC) at an effector:APC ratio of 1:1. Unpulsed C1R-A2 ce lls are used as a negative control. HLA-A2 binding flu matrix peptide 59-66 is used as a positive peptide control ( 41). We also perform each sample with 6 replicates to control for variab ility. In addition, each sample is run with a flu peptide control (pre- and post-vaccine) as well as samples from a “normal” control HLA-A2-positive individual with previously determined levels of flu-specific T-cell precursors. Cells are incubated for 24 h and lysed with phosphate-buffered saline (PBS)-Tween (.05%). Biotinylated anti IFN-gamma antibody diluted to 2 Pg/mL in PBS-Tween containing 1% bovine serum albumin (BSA) is added and incubated overnight in 5% CO 2 at 37qC. Plates are washed 3 times and developed with avidin alkaline phosphatase (GIBCO/BRL, Grand Island, NY) for 45 min. After washing the plates 3 times, each well is examined for positive dots. This assay will be performed in the Laboratory of Tumor Immunolog y and Biology, NCI, NIH. The number of dots in each well will be counted by 2 separate investigators in a blinded manner , and the frequency of responding cells w ill be determined. 4.1.3.2 CD4 T Cell Pro liferation Assay It is planned that all patient s will undergo exploratory analysis of the ability to detect CD4- positive responses using a whole-protein PSA assay, as well as a peptide mix with 63 different 15-mer peptides by ELISPOT and/or ELISA. Version Date: 12/5/17 Abbreviated Title : Enzalutamide in non-met CSPC 354.1.3.3 Sera Antibody Analysis Serum will be stored at -80 degrees Celsius and there will be planned analysis for generation of antibodies to PSA, BCG, PAP, PSMA, PSCA, and/or MUC-1. 4.1.3.4 Flow cytometry analysis of thymic emigrant To determine recent thymic emigrants, flow cytometry analysis will be performed on peripheral blood mononuclear cells. Cells will be resuspended in staining buffer (phosphate-buffered saline containing 3% fetal bovine seru m) and stained for 30 minutes at 4°C with the combination of following antibodies: APC-H7-conjugated an ti-CD4, PE-CY7-conjugated anti-CD3; FITC- conjugated CD45RA, PE-c onjugated CD31, PerCP-CY5.5- conjugated Ki-67, AF-700- conjugated-CD197, V450-conjuga ted CD8, APC-conjugated CD 103, V500-conjugated CD27 all purchased from BD Pharmingen, San Diego, CA). After that, FoxP3 intra-cellular staining will be performed on the cells stained with anti-CD4 and anti-CD25. They will be fixed and permeabilyzed using a fix/perm kit (eBios cience, San Diego, CA) according to the manufacturer’s manual, and will be labeled with FITC-conjugated anti-Foxp3 antibody (236A/E7 clone) or its isotype control antibody (e Bioscience). Flow cytometry will be performed on a Becton Dickinson LSRII (BD Biosciences) device. 4.1.3.5 Natural Killer (NK) CELLS The number and phenotype of NK cells will be determined by phenotypic analysis of PMBCs stained for CD56, CD3, CD8, a nd CD16 by flow cytometry. 4.1.3.6 Regulatory T Cells Regulatory T cells have been shown to inhib it the activation and function of T cells that participate in antigen-specific imm une responses. Higher levels of regulatory T-cells have been reported in the peripheral blood m ononuclear cells of patients with se veral types of tumors. The number and phenotype of regulatory T-cells in peripheral blood mononucl ear cells from patients in this study will be determined by 7-color flow cytometry analysis. Cells will be resuspended in staining buffer (phosphate-buffered saline contai ning 3% fetal bovine serum) and stained for CD4, CD25, CD127, FoxP3, CTLA-4, CD45RA, and CD8. The ratios between regulatory T- cells and CD4 effector cells and the ratios between regulatory T-cells and CD8 Effector cells will also be analyzed. 4.1.4 Additional AssaysBlood samples may be used for additional research studies, which may include phenotypic and functional analysis of im mune-cell subsets and analyses for cytokines (IFN- Ȗ,/ -10, IL-12, IL-2, IL-4, etc.), chemokines, anti bodies, tumor associated antigens, and/or other markers All samples will be labeled with the following identifier system. xPatient’s enrollment # xTrial number xPatient’s initials Example: 01-ABC Version Date: 12/5/17 Abbreviated Title : Enzalutamide in non-met CSPC 36These labels are used only to send the samples from the NIH Clinical Center to the NCI Frederick Central Repository. The NCI Repository will process all samples, appropriatelydiscard the label on the blood tube, and then store the samples with unique identifiers, to which only NCI study personnel will have the code to link to patient specific clinical information.Samples will be tracked according to Section 4.2. If there are inadequate samples for analysis beca use of loss or destruction of those samples, the PI will report this to the IRB. 4.1.5 Plasma VEGF levelsSerum samples will be collected to measure vascular endothelial growth f actor (VEGF) levels. For this purpose, one 6mL EDTA tube will be obtained at baseline (prior to treatment), 1 month, 3 months, 3.5 months, 4 months, and 6 months. Th e analysis will be done with assays developed on electrochemiluminescence platform that provides ultra-high sensitivity and very large signal dynamic range. These studies will be done by the Molecular Pharmacology Program, under the direction of Dr.