Schaeffer, MD, PhD (Co-PI) Sponsor This is an investigator -sponsored trial. Charles Drake, MD , PhD is the IND -holder. IRB # NA_00073453 Investigational agent s 1) GM-CSF–secreting cell-based ( PC3 /LNCaP ) prostate cancer immunotherapy 2) Cyclophosphamide , intravenous 3) Degarelix acetate , subcutaneous Phase Immunologic study (phase I/ II) Indication Men with high -risk localized prostate cancer , prior to radical prostatectom y Target population Inclusion criteria: • histologically confirmed prostate adenocarcinoma • clinical stage T1c –T3b, N0, M0 • Gleason sum 7 -10 • at least 2 positive cores • prior decision to undergo radical prostatectomy • adult male > 21 years of age • ECOG performance status 0-1, or Karnofsky score ≥ 70% • adequate kidney, liver, and bone marrow function • willingness to sign informed consent and adhere to study requirements Exclusion criteria: • presence of known lymph node involvement or distant metastases • prior radiation, hormon es, biologic s, or chemotherapy for prostate cancer • prior immunotherapy/vaccine therapy for prostate cancer • previous or concurrent use of cyclophosphamide • concomitant treatment with hormonal therapy or 5α-reductase inhibitors • use of experimental agents for prostate cancer with in the past 3 months • known allergy to cyclophosphamide or G- CSF/ GM-CSF • known sensitivity to materials of bovine origin • history of autoimmune disease requiring systemic immunosuppression • other concurrent malignancy • uncontrolled major infectious, cardiovascular , pulmonary , hematologic, or psychiatric illnesses that would make the patient a poor study candidate Start d ate/ Duration Initial pati ents are expected to be entered in October 2012 . With an estimated enrollment rate of 4 patients per month , accrual is expected to last 8 months . Androgen suppression with prostate GVAX and cyclophosphamide J1265 Johns Hopkins University 4 of 68 J1265 PROT (Vers) 1.4 113012 Sample size 32 evaluable patients (i.e. those that undergo prostatectomy) Rationale Cancer immunotherapy refers broadly to approaches which attempt to treat cancer by activating immune response s directed against malignant tissue . Prostate GVAX is an allogeneic cell -based prostate cancer vaccine composed of t wo irradiated cell lines (PC3 and LNCaP ) that have been genetically modified to secrete granulocyte -macrophage colony -stimulating factor (GM -CSF) . The release of GM -CSF by these modified tumor cells serves to recruit dendritic cells which then present tumor antigens to T- cells, thus initiating antitumor immune responses. However, abundant preclinical data show that, when used alone, c ell-based immunotherapy is unable to break specific T -cell tolerance in tumor -bearing hosts. Studies in an autochthonous prostate cancer mouse model have shown that giving low - dose cyclophosphamide pri or to a cell-based GM-CSF–secreting vaccine abrogates immune tolerance through augmentation of CD8+ T cell infiltration in the prostate , transient depletion of regulatory T cells ( Tregs), and increased expression of dendritic cell maturation markers . Enhan cement of antitumor immunity has also been observed in other preclinical models where cyclophosphamide was given in sequence with GM - CSF–secreting immunotherapy for the treatment of breast and pancreatic cancers . These preclinical data are supported by ear ly-phase clinical trials combining GVAX with low-dose cyclophosphamide in pancreatic and breast cancer s. Furthermore, emerging evidence suggests that androgen deprivation therapy (ADT) itself has profound effects on the host immune system, resulting in thymic regeneration and enhancement of antitumor immunity. In addition, preclinical and clinical studies demonstrate that ADT augments prostate cancer -specific immune responses induced by immunotherapy, suggesting that ADT may act synergistically with immunot herapy. Based on data from mouse models as well as human clinical trials , it has been suggested that prostate cancer immunotherapy may be most effective when admin istered in the setting of an androgen -suppressed environment . Building on these findings , we have designed a study to assess the use of ADT given alone or administered following immunization with low -dose cyclophosphamide and prostate GVAX , in patients undergoing radical prostatectomy. We a im (1) to determine whether ADT is immunogenic in men with localized prostate cancer by evaluating T- cell infiltration in harvested prostate glands; (2) to determine whether administering ADT after low-dose cyclophosphamide and prostate GVAX augments immune infiltration into the prostate gland ; and (3) to investi gate whethe r this combinatorial immuno -hormonal approach is safe and feasible. We hypothesize that the combination of ADT and cyclophosphamide/ GVAX will produce significantly greater antitumor immune responses than would ADT used alone . Objectives Primary : • To quantify the extent of CD8+ T cell infiltration into the prostate from harvested prostate glands in men with high -risk localized prostate cancer undergoing radical prostatectomy • To evaluate the safety and tolerability of ADT following vaccination with cyclophosphamide and GVAX 4 weeks prior to radical prostatectomy Androgen suppression with prostate GVAX and cyclophosphamide J1 265 Johns Hopkins University 5 of 68 J1265 PROT (Vers) 1.4 113012 Secondary: xTo evaluate the feasibility of administering ADT after immuniza tion with low-dose cyclophosphamide and prostate GVAX, beginning 4 weeks prior to radical prostatectomy xTo quantify the extent of CD4+ T cell and T reg infiltration into the prostate, and to quantify the CD8+/T reg ratio as well as the CD4+/T reg ratio in prostate specimens xTo quantify tissue androgen concentrations (testosterone, dihyd rotestosterone), and to quantify androgen receptor (AR) protein expression in pr ostate specimens xTo quantify markers of apoptosis (activated caspase 3) and prol iferation (Ki-67) in prostate tumor specimens xTo evaluate the proportion of pathological complete responses ( pCR) xTo evaluate the generation of novel antibodies to prostate-asso ciated antigens in the serum of patients, after the initiation of protocol therapy xTo determine the PSA response rate and time-to-PSA-recurrence a fter radical prostatectomy Study design This is a single-center, randomized, 2-arm, open-l abel, prospective clinical trial evaluating the immunogenicity and safety of a single administra tion of LHRH antagonist (degarelix) used alone, or degarelix administered following vac cination with low-dose cyclophosphamide and GVAX, given 4 weeks prior to radical prost atectomy in men with localized prostate cancer. Patients will be recruited from the outpatient Urology clinic. Eligible patients will be randomized in a 1:1 ratio ȋ  ǣζ͹ͺ -10) to receive one of two treatment regimens ( see Schema below ), each beginning 28 (±3) days prior to radical prostatectomy. In Arm B, cyclophosphamide will first be given a t a dose of 200 mg/m2 as a single intravenous infusion. One day later, prostate GVAX will be administered as five 0.8-mL intradermal injections of PC3 (2.5 × 108 cells) and five 0.5-mL intradermal injections of LNCaP (1.6 × 108 cells), for a total dose of 4.1 × 108 cells. On day 14, degarelix acetate will be administered as three 80-mg subcutane ous injections, for a total dose of 240 mg. Prostate glands will be harvested 14 (±3) days later, at the time of radical prostatectomy, and prostate tissue will be examined for the primary endpoint. In Arm A, an identical dose of degarelix acetate will be administe red, 14 (±3) days prior to surgery. A telephone follow-up evaluation (or an in-person vis it) for adverse events will occur 28 (±21) days after surgery. Patients will then be follow ed by their urologists according to standard institutional practices, but will require PSA evaluations every 3 (±1) months during year 1 and every 6 (±2) months during years 2-3. Treatment Schema: . Androgen suppression with prostate GVAX and cyclophosphamide J1265 Johns Hopkins University 6 of 68 J1265 PROT (Vers) 1.4 113012 Baseline evaluations: • Informed consent • Medical history and physical examination • Review of medications • Vital signs, including height and weight • Performance status • Central pathologic review of prostate core biopsies • CT (If allergic to CT scan contrast, obtain MRI with contrast) and/or bone scan, if clinically indicated • Hematology, coagulation, and chemistry laboratories • Serum PSA measurement • Sera for immunoassays (Pre -treatment) On day 28 (±3 days ): • Medical history and focused physical examination (including vital signs) • Review of medications • Performance status • Adverse event s/toxicity evaluation • Hematology and chemistry laboratories , as clinically indicated • Serum PSA • Sera for immunoassays • Radical prostatectomy • Pathologic review of surgical specimen according to standard procedures • Evaluation of harvested prostate gland for CD8+ T cell infiltration Post -operative day 28 (±21 days ): • Performance status (by telephone interview or clinic visit ) • Adverse events/toxicity evaluati on (by telephone interview or clinic visit ) • PSA every 3 months (post -op year 1), then every 6 months (post -op years 2 -3) Criteria for evaluation Primary endpoint s: • Mean CD8+ T cell density in tumor tissue from harvested prostate glands of patients in each of the 2 treatment groups • Frequency, type, and severity of adverse events in each of the 2 treatment groups Secondary endpoints: • Mean Treg density in tumor tissue from harvested prostate glands of patients in each of the 2 treatment groups • Mean CD8+/T reg and CD4+/T reg ratios in tumor tissue from harvested prostate glands of patients in each of the 2 treatment groups • Mean CD4+ T cell density in tumor tissue from harvested prostate glands of patients in each of the 2 treatment groups • Mean levels of apop totic markers (activated caspase 3) and proliferation markers (Ki-67) in prostate tumor specimens of patients in each of the 2 treatment groups • Mean tissue androgen concentrations (testosterone, dihydrotestosterone) and mean androgen receptor (AR) protein expression in prostate specimens • Proportion of pathological complete responses (pCR) in each treatment group Androgen suppression with prostate GVAX and cyclophosphamide J1265 Johns Hopkins University 7 of 68 J1265 PROT (Vers) 1.4 113012 • Proportion of patients generating of novel antibodies to prostate -associated antigens after the initiation of protocol therapy in each treatment gr oup • Proportion of patients achieving a PSA response rate (PSA <0.1 ng/mL) at 3 months after prostatectomy in each treatment group • Median time -to-PSA -recurrence (PSA ≥0.2 ng/mL) after radica l prostatectomy in each of the 2 treatment groups Statistical method s Our primary hypothe sis is that men receiving cyclophosphamide and GVAX followed by ADT (arm B) will have a 100 % or greater CD8+ T cell infiltr ation of the prostate than will men receiving ADT alone (arm A) . In the absence of any intervention prior to prostatectomy, we have previously determined that the mean staining percentage for CD8+ T cells in prostate tissue is 0.42% (sta ndard deviation [SD] 0.36%). With 16 patients per arm, and assuming an 86% coefficient of variation for the average CD8+ area/spot are a ratios, a one- sided 0.05 α-level t -test of the logarithms of these ratios would have 82% power to detect a 2- fold (100%) increase of CD8+ T cells in the cyclophosphamide/GVAX + ADT arm compared to the ADT alone arm . Treating Gleason score as a b lock factor reduces the variance, which would be adequate to offset the loss of power due to the 1 degree of freedom associated with the blocking factor. The primary efficacy endpoint will be expressed as the mean CD8+ T cell st aining percentage in prostate tumor tissues, reported separately for each treatment arm. High density (satu ration) t issue microarrays (TMAs) using the highest -grade/largest tumor per patient and sampling it with 100 -fold redundancy will be used to determine CD8 + T cell densities. Since the CD8+ T cell quantity is a ratio variable and the distribution is skewed, the log transformation will be used for this analysis. Log -transformed mean CD8+ T cell concentrations will be compared between treatment groups usi ng a two - way analysis of variance (ANOVA) with the stratification variable (Gleason score) treated as a block factor . Boxplots and descriptive summaries by treatment arm will also be provided. In other analyses, continuous variables will be summarized by descriptive statistics using the mean, standard deviation, median, and range , and 95% confidence intervals will be reported whe re appropriate . Two -way ANOVA will be used to investigate comparisons between the two treatment arms . Proportions will be compared using a Mantel- Haenszel test. Kaplan -Meier analysis will be used to det ermine median time- to- PSA -progression , and comparisons will be sought using the log -rank test , stratified by Gleason score . Statistical significance will be set at P <0.05. Safety analysis Frequency, types, and grades of adverse events in each treatment group will be measured using the NCI Co mmon Toxicity Criteria version 4 .0, and will be summarized using descriptive statistics. Formal safety assessments wil l be performed from the time of first administration of degarelix, cyclophosphamide or GVAX until the 28th postoperative day (±21 days) . In addition, all patients that received GVAX will be enrolled on a separate long -term follow -up protocol and will be evaluated for late GVAX toxicities ( e.g. autoimmune phenomena, second malignancies). These evaluations will occur annually for 3 years. Androgen suppression with prostate GVAX and cyclophosphamide J1265 Johns Hopkins University 8 of 68 J1265 PROT (Vers) 1.4 113012 TABLE OF CONTENTS 1. OBJECTIVES ...........................................................................................................................................................