47 10. STATISTICAL CONSIDER ATIONS ...................................................................................................................... 48 10.1 Study Design and Sample Size ............................................................................................................................... 48 10.2 Study Endpoints ......................................................................................................................................................... 49 10.3 Analysis Populations ................................................................................................................................................ 52 10.4 Safety Analysis ............................................................................................................................................................ 52 10.5 Statistical Procedures .............................................................................................................................................. 53 11. PROTECTION OF HUMAN SUBJECTS ............................................................................................................... 53 11.1 Ethical Considerations ............................................................................................................................................. 53 11.2 Protocol Amendments ............................................................................................................................................. 54 11.3 Written Informed Consent ..................................................................................................................................... 54 11.4 Protection of Privacy ................................................................................................................................................ 54 11.5 Study Termination or Modification ..................................................................................................................... 55 12. REFERENCES ......................................................................................................................................................... 56 APPENDIX A: P erformance Status Criteria ............................................................................................................. 59 APPENDIX B: C ommon T oxicity Criteria, V ersion 4.0 .......................................................................................... 62 APPENDIX C: S erious Adverse E vent (SAE) R eporting Form.............................................................................. 63 APPENDIX D: A bbreviations and A cronyms ........................................................................................................... 65 APPENDIX E: D ata and S afety Monitoring Plan (DSMP) ...................................................................................... 68 LIST O F TABLES Table 1 Study Calendars 24 Table 2 Intensity of Adverse Event 37 Table 3 Relationship of Adverse Event to Study Drug 38 LIST OF FIGURES Figure 1 Clinical States of Prostate Cancer 11 Figure 2 Cyclophosphamide Augments Antitumor Immunity Produced by GVAX 16 Figure 3 Study Schema 23 Androgen suppression with prostate GVAX and cyclophosphamide J1265 Johns Hopkins University 10 of 68 J1265 PROT (Vers) 1.4 113012 1. OBJECTIVES 1.1 Primary Objective s • To quantify the extent of CD8+ T cell infiltration into the prostate from harvested prostate glands in men with localized prostate cancer receiving neoadjuvant ADT alone (2 weeks prior to surgery), or cyclophosphamide and GVAX followed by ADT, (with CY/GVAX administered 4 weeks prior to prostatectomy, and ADT administered 2 weeks prior to prostatectomy ). • To evaluate the safety and tolerability of ADT used alone (2 weeks prior to surgery), or cyclophosphamide and GVAX followed by ADT (with CY/GVAX administered 4 weeks prior to prostatectomy, and ADT administered 2 weeks prior to prostatectomy). 1.2 Secondary Objectives • To evaluate the feasibility of administering ADT used alone (2 weeks prior to surgery), or cyclophosphamide and GVAX followed by ADT (with CY/GVAX administered 4 weeks prior to prostatectomy, and ADT administered 2 weeks prior to prostatectomy). • To quantify and compare the extent of CD4 + T cell and Treg infiltratio n into the prostate in prostate specimens of patients in each of the 2 treatment groups. • To quantify and compare CD8+/T reg and CD4+/T reg ratios in prostate specimens of patients in each of the 2 treatment groups. • To quantify and compare tissue androgen concentrations (testosterone, dihydrotestosterone), and to quantify androgen receptor (AR) protein expression in prostate specimens of patients in each of the 2 treatment groups. • To quantify markers of apoptosis (activated caspase 3) and markers of cell proliferation (Ki -67) in prostate tumor specimens of patients in each of the 2 treatment groups. • To evaluate and compare the proportion of pathological complete responses (pCR) in prostate tumor specimens of patients in each of the 2 treatment groups . • To evaluate and compare the generation of novel antibody specificities to prostate - associated antigens in the serum of patients in each group, after the receipt of protocol therapy . • To evaluate and compare PSA response rates and the time -to-PSA -recurrence after radical prostatectomy in each of the 2 treatment groups . 2. BACKGROUND AND RATIONALE 2.1 Disease Background Prostate cancer is the second leading cause of cancer deaths in men. According to American Cancer Society estimates in 2008, as many as 186,320 American men will be diagnosed with prostate cancer, and nearly 28,660 will die of the disease (Jemal et al 2008 ). Yet, localized pros tate cancer is often curable and even metastatic diseas e can respond to treatment. Androgen suppression with prostate GVAX and cyclophosphamide J1265 Johns Hopkins University 11 of 68 J1265 PROT (Vers) 1.4 113012 The course of prostate cancer from diagnosis to death is best categorized as a series of clinical states (Figure 1). These clinical states involve the complex interplay of a network of signaling molecules that collectively promote net cell proliferation relative to cell death. Based on the extent of disease, hormonal status, and absence or presence of detectable metastases on imaging studies, the states are: localized disease, rising PSA after radiation therapy or surgery with no detectable metastases, and clinical metastases in the non- castrate or castrate settings . Treatment of localized prostate cancer usually consists of surgery or radiation therapy o r both. For patients with high -risk or locally advanced disease, a combination of radiation therapy and hormonal therapy is often used. However, even after definitive local therapy, approximately 30 -50% of patients will have a local or distant recurrence ( Pound et al 1999, D’Amico et al 2000). The risk of recurrence depends on multiple factors including tumor stage, tumor Gleason score, and serum PSA at the time of diagnosis. Recently, PSA doubling time at recurrence has been shown to be a powerful predicto r of distant metastases and survival (Antonarakis et al 2012 ; Freedland et al 2007). Figure 1 Clinical States of Prostate C ancer Patients with metastatic prostate cancer have a median survival of 3 -7 years, and most die of the disease. Treatment for metastatic disease involves surgical castration or hormonal manipulation using luteinizing hormone -releasing hormone (LHRH) agonists /antagonists , antiandrogens, or both. Although the majority of these patients initially respond to androgen deprivation ther apy, all eventually progress to a state of castration -resistant prostate cancer (CRPC). Treatment options for these men are limited. Secondary hormonal manipulations, such as ketoconazole, are often used but these produce short -lived respons es and do not i mprove survival (Small et al 2004). Moreover, these agents have unfavorable side effect profiles. Other acceptable approaches in these men include watchful waiting until the development of symptoms, or the initiation of cytotoxic chemotherapy. In this rega rd, docetaxel has been shown to improve overall survival in patients with CRPC, but only by a median of 2.9 months (Tannock et al 2004; Berthold et al 2008). However, because therapies for metastatic prostate cancer are palliative and not curative, there r emains an urgent need to improve the outcome of primary therapies for prostate cancer. 2.2 Treatment Background 2.2.1 GM-CSF– secreting allogeneic cell -based vaccine as immunotherapy for prostate cancer Cancer immunotherapy refers broadly to approaches whic h attempt to treat or cure cancer by activating an immune response direc ted against malignant tissue (Pardoll 2002 ). A number of different approaches fall broadly into this group, including dendritic cell vaccination, DNA vaccination, peptide vaccination, and others. Each of these methodologies has its own advantages and disadvantages. In particular, the use of granulocyte -macrophage colony -stimulating factor (GM -CSF) as an adjuvant has been widely evaluated in both murine models and in patients with cancer (Borrello and Androgen suppression with prostate GVAX and cyclophosphamide J1265 Johns Hopkins University 12 of 68 J1265 PROT (Vers) 1.4 113012 Pardoll 2002 ). These studies suggest that GM -CSF based immunotherapy approaches function by recruiting and activating dendritic cells, now fairly well accepted as the most potent antigen presenting cells. Activated dendritic cells take up antigens from the tumor cell line or autologous tumor used in the biological formulation, and traffic to draining lymph nodes. In the lymphoid tissue, these activated dendritic cells present antigens derived from tumor peptides to T -cells, which then circulate to the site s of disease and (if successful) initiate a lytic immune response against the malignant tissue. After initial evaluation in murine models (Dranoff et al 1993 ), the first clinical applications of GM -CSF transduced tumor cells employed a utologous tumor in the setting of metastatic renal cell carcinoma (Simons et al 1997 ). These studies demonstrated the relative safety of this approach, but also underscored the technical difficulty of using autologous tumor as a source of antigens for immu notherapy. Recently , two significant findings have impact ed the use of GM- CSF immunotherapies . First, it has been shown that GM-CSF need not come directly from the immunizing tumor cells, and that irradiated bystander erythroleukemia cells tr ansduced to produce GM - CSF can provide identical response rates to directly transduced immunothe rapy lines in murine models (Borrello et al 1999 ). Perhaps more significant, though, was the recognition that many common tumor antigens may indeed be shared between patients with the same tumor type, and that immortalized cell lines could thus be transduced with GM- CSF to serve as immunotherapies. The use of immortalized GM -CSF–secreting cell lines has been evaluated in patients with pancreatic cancer (Jaffee et al 2001 ), wit h preliminary results showing the safety of this immunotherapy , in addition to suggesting some degree of efficacy especially at higher immunotherapy doses. In prostate cancer, the initial immunotherapy approach also utilized irradiated autologous tumor cel ls transduced in vitro to secrete GM -CSF (Simons et al 1999; Dummer 2001). In a p hase I trial, prostate tumor cells were harvested surgically an d transduced with a replication -defective retrovirus. Doses of 10 million tumor cells were administered without significant adverse affects, and new delayed- type hypersensitivity (DTH ) responses were documented in two out of eight treated patients. For phase II evaluation, an immunotherapy comprised of two common prostate cancer cell lines (LNCaP and PC3) has been d eveloped (Higano et al 2008 ). LNCaP was originally isolated from a lymph node metastasis and expresses a number of prostate- restricted differentiation antigens including PSA and prostate- specifi c membrane antigen (PSMA) (Horoszewicz et al 1987; Israeli et al 1994 ). It also contains a mutant androgen receptor as well as the most common epi genetic abnormality in human prostate cancer: methylation of the glutathione -S-transferase gene (Lee et al 1994 ). The PC 3 cell line was developed from a bone metastasis and expresses significant levels of proteases associated with metastatic prostate cancer. While LNCaP is androgen -sensitive, PC 3 is considered androgen insensitive. T ogether , these 2 cell lines are likely to serve as a diverse source of prostate- and prostate tumor -specific antigens. Two p hase II studies have been performed u sing this product, officially registered as PC3/ LNCaP immunotherapy for prostate c ancer (or GVAX ). In one of these studies, 41 hormone -naïve patients were p rimed with a single 500 million -cell immunotherapy dose, then boosted with 12 doses of 100 million cells at 2 week intervals. No autoimmunity was detected, and no life -threatening toxicities were observed. The most common adverse event was a reaction at the injection si te. One patient ha d a partial PSA response (Simons et al 2006) . In a se cond phase II trial, 55 hormone -refractory patients were treated with a 500 million -cell immunotherapy dose, then boosted with 12 doses of either 100 million or 300 million cells at 2 week intervals . One patient had a PSA Androgen suppression with prostate GVAX and cyclophosphamide J1265 Johns Hopkins University 13 of 68 J1265 PROT (Vers) 1.4 113012 response, and there was a non -significant trend towards a longer median time to bone scan progression (140 days vs. 85 days, P = 0.095) in the high -dose arm (Small et al 2007) . Taken together, these phase II data demonstrate the safety o f this immunotherapy approach, but suggest a relatively low overall response rate. The most common adverse events in both studies were injection -site erythema, fever, fatigue, malaise, myalgias, and arthralgias. Two large randomized phase III studies of GM -CSF–secreting cell -based immunotherapy for prostate cancer ( GVAX ) have been conducted: VITAL -1 and VITAL -2. The first study, VITAL -1, enrolled 626 men with asymptomatic chemotherapy -naïve castration -resistant prostate cancer, and randomized them to receive GVAX or docetaxel/prednisone, with the primary endpoint being overall survival (Higano et al 2009) . The second study, VITAL -2, was planned ini tially to enroll 600 patients with symptomatic metastatic castration -resistant prostate cancer, randomizing them to docetaxel/prednisone or docetaxel/ GVAX (Small et al 2009). Both trials were terminated early because of data observed at the time of interim analyses suggesting that the survival improvements initially hypothesized were unlikely to be observed if the trials were to be continued (Higano et al 2009 ; Small et al, 2009) . In both studies, overall survival was not statistically superior in patients receiving GVAX . While a large number of potential reasons for this low activity may be postulated, perhaps the simplest is the well -known immunological observation that persistent antigen exposure tends to induce immune tolerance (Ramsdell and Fowlkes 1992 ; Rocha et al 1993; den Boer et al 2004). In addition , cancer immunotherapy might prove more efficacious in a minimal disease state, as has been observed in multiple experimental systems (Migita and Ochi 1993 ). 2.2.2 Cyclophosphamide as a potent enhancer of immune responses to GVAX Efficient immunization against cancer requires a vaccine capable of eliciting a potent CD4+ and CD8+ T cell immune response. However, tumors have evolved several mechanisms to escape immune surveillance, incl uding immune tolerance mediated by immunosuppressive T lymphocytes (Drake et al 2006). T o this end, tumors have been shown to induce rapid expansion of CD4+CD25+ regulatory T cells (T regs) in humans and mice, leading to delayed rejection of immu nogenic cancer s (Liyanag e et al 2002; Woo et al 2001; Curiel et al 2004). Conversely, elimination of these T regs, which constitute about 2-5% of the peripheral CD4+ T cells in naïve mice, elicits potent antitumor immune responses leading to tu mor eradication (Shimizu et al 1999; Sutmuller et al 2001). For many years, c yclophosphamide has been stud ied for its role in breaking immune tolerance. It is known that low -dose and high -dose cyclophosphamide have different biological activities (Motoyoshi et al 2006). High -dose cyclo phosphamide has cytotoxic activity, whereas low -dose cyclophosphamide shows predominantly immune modulating effect s. Cyclophosphamide, as an FDA -approved cytotoxic chemotherapy agent, is used in treating multiple types of cancer including breast cancer, lymphoma, leukemia, and sarcoma. In ad dition, cyclophosphamide is used as an immune suppressor in many autoimmune disorders . Immune modulating doses of cyclophosphamide -containing chemotherapy have been studied in mouse tumor models in conjunction with vaccination. For example, Dr. Jaffee and colleagu es have studied a HER -2/neu transgenic mouse model of mammary tumors , representing a clinically relevant model of human breast cancer. These mice develop spontaneous HER -2/neu –expressing mammary tumors due to activation of the transgene by the murine mammary tumor virus (MMTV) promoter in normal mammary tissue. It has been demonstrated that vaccination of th ese mice induces weak HER - 2/neu –specific B cell and T cell responses that are not capable of controlling mammary Androgen suppression with prostate GVAX and cyclophosphamide J1265 Johns Hopkins University 14 of 68 J1265 PROT (Vers) 1.4 113012 tumor growth re gardless of the vaccine type. However, treatment of these mice with low-dose cyclophosphamide prior to vaccination resulted in t umor rejection in 10 -30% of animals, while mice receiving vaccination alone all be came toleran t to tumor growth (Ercolini et al 2005). In this study, it was shown that the immune enhanci ng effect of cyclophosphamide was mediated through selective inhibition of the cycling population of CD4+CD25+ Treg cells and recruitment of high- avidit y antitumor effector T cells. These findings were confirmed in a separate study using the same HER -2/neu transgenic mouse model of breast cancer. In that study, combined treatment with immune - modulating doses of cyclophosphamide and a GM -CSF–secreting whol e-cell vaccine delayed tumor growth by overcoming immune tolerance and inducing antigen -specific antitumor immune responses (Machiels et al 2001). Importantly , because cyclophosphamide exerts its effect on the regulatory T cells of the immune system rather than on the cancer cells themselves , this approach can be applied to treat any type of cancer. These animal studies have led to a phase I/II clinical trial testing the feasibili ty, safety, and immunogenicity of cyclophosphamide when given in sequence with a GM -CSF– secreting cell -based vaccine in breast cancer patients (Emens et al 2004). In this study, 21 women with metastatic breast cancer received one of three doses of cyclophosphamide (200, 250, or 350 mg/m2) in conjunction with do xorubicin and an allogeneic, HER -2/neu+, GM -CSF–secreting breast tumor vaccine. I n addition, 6 women received immunotherapy alone. Patients on the combined- modality arm s received three monthly immunizations and a boost er 6 to 8 months later according to the following treatment schedule: c yclophosphamide was given on day –1, vaccination on day 0, and doxorubicin on day +7. Total Her-2–specific immune responses were observed in 33%, 50%, 21%, and 25% of patients receiving 0, 200, 250, and 350 mg/m2 of cycloph osphamide respectively (Emens et al 2008). These clinical data suggest that addition of cyclophosphamide enhances Her-2–specific vaccine responses in patients with breast cancer, but that increasing doses of cyclophosphamide may abrogate these immune respo nses. A second pilot study borrowing from these princip les has recently been conducted in patients with metastatic pancreatic cancer (Laheru et al 2008). In this open -label study, 30 patients received up to six doses of a GM -CSF–secreting cell -based pancreatic cancer vaccine at 21 -day intervals, while 20 patients received 250mg/m2 of intravenous cyclophosphamide 1 day prior to the same vaccine. After 3 cycles of therapy, p ancreatic cancer -specific (mesothelin -specific) CD8+ T cell responses were augmented in only 50% of patients treated with vaccine alone compared to 90% of patients treated with cyclophosphamide plus vaccine. Mean immune response augmentation (as judged by mesothelin -specific CD8 + T cell effects ) after 3 cyc les of treatment was 39% in patients receiving immunotherapy alone and 131% in patients receiving cyclophosphamide plus immunotherapy (Laheru et al 2008) . In addition, median survival in patients on the vaccine arm was 2.3 months while survival on the cycl ophosphamide/vaccine arm was 4.3 months. The administration of this cell -based immunotherapy in sequence with cyclophosphamide showed minimal treatment -related toxicity, although all patients developed erythema, induration and/or pain, soreness at the treatment sites following immunotherapy. Other common adverse events included fever, chills, fatigue, malaise, skin rash, arthralgias, and nausea . 2.2.3 Cyclophosphamide combined with GVAX in prostate cancer Recently , data evaluating the effect of cyclophospha mide on ant i-tumor immune response s to a cell-based GM -CSF–secreting prostate cancer vaccine ( GVAX ) was Androgen suppression with prostate GVAX and cyclophosphamide J1265 Johns Hopkins University 15 of 68 J1265 PROT (Vers) 1.4 113012 published by Dr. Drake and colleagues (Wada et al 2009). In this study, an autochthonous animal model was created using the transgenic adenocarcinoma of the mouse prostate (TRAMP) mouse crossed with analogous transgenic mice expressing the model antigen hemagglutinin (HA) in a prostate- restricted manner (ProHA) . This clinically relevant murine model allowed investigation of immune responses to tissue - tumor –restricted HA as a function of immunotherapy and/or cyclophosphamid e administration . In this model ( ProHA × TRAMP ), giving cyclophosphamide 1 day prior to GVA X immunotherapy induced maximal augmentation of HA -specific CD8 + T cell responses both in the prostate and the prostate -draining lymph nodes (Figure 2 a). Administering cyclophosphamide on day –4, day –2, or day +1 in relation to GVAX was not as effective. Using this treatment sequence, the optimal dose of cyclophosphamide with respect to antitumor immune responses was found to be 5 0 mg/kg given intraperitoneally. This dose produces serum levels equivalent to those achieved in humans using a cyclophosphamide dose of 200 mg/m2 given intravenously. Importantly, higher doses of cyclophosphamide in this animal model resulted in a loss of immunotherapy efficacy and a suppression of specific CD8+ T cell responses . When comparing the antitumor imm une responses of different permutations of GVAX and cyclophosphamide in ProHA × TRAMP mice (Wada et al 2009) , it was shown that the combination of these two agents produced markedly superior HA -specific CD8+ T cell responses in both the prostate and prostate -draining lymph nodes than either agent used alone (Figure 2b). This finding is particularly im portant, as it further demonstrates the weak antitumor immunogenicity of GVAX when used alone against prostate cancer. Further mechanistic experiments showed that immune augmentation by cyclophosphamide was associated with a transient depletion of regulatory (CD4 +FOXP3+) T cells in the prostate and prostate- draining lymph nodes ( Figure 2c ) but not in the peripheral circulation. In addition, combined treatment resulted in increased ratios of CD4+ and CD8+ effector T cells to regulatory T cells (T eff/T reg ratio) in the prostate glands of these mice (Figure 2d). Alterations in T reg function with cyclophosphamide use were not seen . Finally, combination of GVAX and cyclophosphamide resulted in increased expression of dendritic cell maturation markers , although a dramatic change in dendritic cell numbers was n ot observed. Taken together, t hese data provide a strong rationale for a strategy combining low -dose cyclophosphamide with allogeneic GVAX immunotherapy in patients with prostate cancer . Androgen suppression with prostate GVAX and cyclophosphamide J1 265 Johns Hopkins University 16 of 68 J1265 PROT (Vers) 1.4 113012 Figure 2 Cyclophosphamide Augments Antitumor Immunity Produced by GVAX 2.2.4 Immunological effects of androgen deprivation It is now appreciated that androgen deprivation therapy (ADT) h as profound effects on the host immune system, resulting in enhancement of antitumo r immune responses (Aragon-Ching et al 2007). Initial observations in th is regard came from a study demonstrating that ADT administered before prostatectomy resulted in profuse CD4+ T cell infiltration into human prostate glands, and that these T cells were oligoclonal suggesting an antigen-specific response (Merca der et al 2001). In a similar study of androgen-deprived prostate cancer patients, an increased CD8+ T cell infiltrate was noted as well (Gannon et al 2009). Also sup porting a pro- immunogenic role for ADT are recent data demonstrating inductio n of new antibody specificities in castrated patients (Morse et al 2010). In the laboratory, using an autocthonous prostate cancer mouse model, our group has previou sly shown that ADT decreases CD4+ T cell tolerance to prostate cancer-associated antigens, Androgen suppression with prostate GVAX and cyclophosphamide J1265 Johns Hopkins University 17 of 68 J1265 PROT (Vers) 1.4 113012 mitigating immune evasion (Drak e et al 2005). Intriguingly, additional studies in both mice and humans have shown that ADT reverses age -related thymic involution, resulting in renewed thymopoiesis and increased output of naïve T cells (Sutherland et al 2005). Finally, expansion of the p re-B cell population is observed in the bone marrow after castration in mice, a process that may improve antigen pres entation and T cell responses (Wilson et al 1995). Taken together, these data support the notion that ADT, through its augmentation of pros tate-specific immune responses, may act synergistically with immunotherapy in the treatment of prostate cancer. 2.2.5 Combining androgen deprivation with immunotherapy The addition of ADT to immunotherapy has been examined in several preclinical and clinic al studies. Using our autocthonous prostate cancer mouse model, our group noted prostate -specific effector T cell expansion when a recombinant vaccinia virus vaccine was administered to castrated tumor -bearing mice, but this did not occur in non -castrated mice (Drake et al 2005). In a more recent study of tumor -free mice, vaccination with a prostate stem cell antigen (PSCA) -containing DNA -based vaccine produced greater numbers of PSCA -specific IFN -γ-secreting T cells in mice that were castrated compared to those that were not castrated (Koh et al 2009). Notably, this expansion of effector T cells was only seen when vaccination was performed prior to castration, and not when this treatment order was reversed. Another study using a PSA -expressing transgenic mouse model demonstrated proliferation of PSA -specific cytotoxic T cell in response to immunization with a PSA -based recombinant vaccinia virus but this effect was only noted in castrated mice ( Arredouani et al 2010). A small randomized clinical trial in me n with non- metastatic castration -resistant prostate cancer examining the combination of antiandrogen therapy and a PSA - based recombinant poxviral vaccine showed a higher frequency of PSA -specific T cell responses in men who received vaccination before antiandrogen than in those receiving antiandro gen before vaccination (Arlen et al 2005). Intriguingly, longer follow up of this same study revealed that overall survival was increased in men who received vaccine followed by antiandrogen than in those who recei ved this combination in the reverse order (median survival 6.2 vs 3.7 years, P = 0.045) (Madan et al 2008). Finally, a larger randomized study of sipuleucel -T versus placebo combined with ADT in men with non -castrate PSA -recurrent prostate cancer showed a slower post -treatment PSA doubling time (PSADT) in men receiving the combination therapy than in those receiving ADT alone (PSADT 5.1 vs 3.4 months, P = 0.038) ( Beer et al 2011 ). In summary, these studies suggest that combined immuno -hormonal therapy may provide additive clinical benefit and is worthy of further evaluation. 2.3 Rationale 2.3.1 Rationale for c onducting the s tudy In both animal models and humans with cancer (including prostate cancer) , allogeneic cell - based GM -CSF–secreting immunotherap y is often unable to break specific T cell tolerance when this modality is used alone. Preclinical studies in an autochthonous prostate cancer mouse model have shown that giving low -dose cyclophosphamide prior to GVAX abrogates immun e tolerance through enhancement of CD8+ T cell infiltration into the prostate, transient depletion of regulatory T cells (T regs), and increased expression of dendritic cell Androgen suppression with prostate GVAX and cyclophosphamide J1265 Johns Hopkins University 18 of 68 J1265 PROT (Vers) 1.4 113012 maturation markers. Augmentation of antitumor immunity has also been observed in early human clinical trials where cyclophosphamide has been given in sequence with GM -CSF– secreting vaccines for the treatment of breast a nd pancreatic cancers. 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 immunotherapy. Based on data from mouse models as well as human clinical trials, it has b een suggested that prostate cancer immunotherapy may be most effective when administered in the setting of an androgen -suppressed environment . The sequencing of androgen- ablation after immunotherapy is supported by preclinical data (Koh et al 2009) and more importantly by clinical data (Aragon -Ching et al 2007) . In this sequence, the additive effect of these two treatment modalities likely represents a classical prime -boost regimen in which the well -established immune effects of androgen -ablation boost T cell responses initiated by vaccination. It should be noted that other sequences are possible; in fact a randomized phase II of s ipuleucel -T testing two alternative sequences (NCT01431391) is currently accruing patients . Building on these findings, we ha ve designed a study to assess the use of ADT given alone or in sequence with low -dose cyclophosphamide and prostate GVAX, in patients scheduled to undergo radical prostatectomy. We aim (1) to determine whether ADT is immunogenic in men with localized prost ate cancer by evaluating T -cell infiltration in harvested prostate glands; (2) to determine whether administering ADT in sequence with low -dose cyclophosphamide and prostate GVAX augments immune infiltration into the prostate gland; and (3) to investigate whether this combinatorial immuno -hormonal approach is safe and feasible. We hypothesize that the combination of ADT and cyclophosphamide/GVAX will produce significantly greater antitu mor immune responses than ADT used alone . If this study shows significan t enhancement of antitumor immunity when using ADT in sequence with low -cyclophosphamide and prostate GVAX , and this combination is feasible and safe, then future studies would aim to use this combinatorial approach in both the adjuvant and the salvage set ting s. For example, a trial could be designed to assess the efficacy of ADT combined with cyclophosphamide/ GVAX given before and after radical prostatectomy , using PSA recurrence as the primary endpoint. Alternatively, a trial of ADT combined with cyclopho sphamide/ GVAX for patients with PSA recurrence could be designed , with metastatic progression as the primary endpoint. 2.3.2 Rationale for dosage selection The dose of prostate GVAX in this study will be the same as the priming dose used in two large phase III studies evaluating this immunotherapy in patients with castration -resistant metastatic prostate cancer (Higano et al 2009; Small et al, 2009). Five 0.8-mL intradermal injections of PC3 (2.5 × 10 8 cells) and five 0.5-mL intradermal injections of LNCaP (1.6 × 108 cells) wi ll be administered, for a total dose of 4.1 × 108 cells. In contrast to previous trials , the present study will use only a single dose of immunotherapy rather than a ser ies of doses spread out over time. A single dose s hould be sufficient since we will be evaluating T cell infiltration in resected prostate glands (harvested three weeks later) as our primary endpoint. Cyclopho sphamide will be administered at 200 mg/m2, as a single intravenous infusion. In the cyclophosphamide/ GVAX treatment arm, cyclophosphamide will be given one day prior to GVAX , which i tself will be administered two weeks prior to pr ostatectomy. We hypothesize that 20 0 mg/m2 of cyclophosphamide will be the optimal immune priming dose in humans because this dose produces serum levels equivalent to those achieved using 50 Androgen suppression with prostate GVAX and cyclophosphamide J1265 Johns Hopkins University 19 of 68 J1265 PROT (Vers) 1.4 113012 mg/kg intraperitoneally in the ProHA × TRAMP mouse model, the dose which produced the largest antitumor immune responses (Wada et al 2009). In addition, the 20 0 mg/m2 dose of cyclophosphamide was recently used in a human pancreatic cancer study, where it was shown to augment the immune effects of an allogeneic cell -based GM -CSF–secreting pancreatic cancer vaccine (Laheru et al 2008). In another study of breast cancer patients, 200 mg/m2 of cyclophosphamide given i n sequence with a cell -based GM-CSF–secreting breast cancer vaccine seemed to be the most immunogenic dose, while doses of 250 mg/m2 and 350 mg/m2 appeared to suppress H ER-2–specific immune responses (Emens et al 2008) . Degarelix acetate will be administered as three 8 mg subcutaneous injections, for a total dose of 240 mg. This is the standard loading dose of degarelix, and is expected to induce castrate levels of serum testosterone for at least 4 weeks. Degarelix, an LHRH antagonist, was chosen rather th an an LHRH agonist in order to produce rapid testosterone suppression without causing an initial testosterone surge. This rapid testosterone ablation more closely resembles the effect of surgical castration in preclinical animal models. 2.3.3 Rationale for immunobiologic endpoints The primary goal of this study is to assess the degree of immune infiltration into the prostate after administering the protocol therapy, and to determine whether the combination of ADT and cyclophosphamide /GVAX may enhance intra prostatic immune infiltration compared to ADT administered alone . In studies using the ProHA × TRAMP mouse model, the primary endpoint was hemagglutinin (HA) -specific CD8 + T cell responses in harvested prostate glands (Wada et al 2009). However, in humans with prostate cancer receiving GVAX , the exact immunogenic antigen is not known. Therefore, the primary endpoint for the present study will be assessment of total CD8+ T cell infiltration into the prostate. Our previous studies have shown that the mean sta ining percentage for CD8+ T cells in human prostate tumor tissue is 0.42% (standard deviation 0.36%, interquartile range 0.07% –0.71%). In the present study, we seek to augment the intratumoral CD8+ T cell response by 100% in the experimental arm compared t o the control arm. A 100% increase in tumor -infiltrating CD8+ T cells was sought because in patients with advanced melanoma, adoptive T cell transfer studies have shown that a 100% increase in tumor -infiltrating CD8 + T cells was required to cause regressio n of metastatic melanoma lesions (Rosenberg et al 2011); increases in tumor - infiltrating CD8+ T cells that were less than 100% did not result in tumor regressions. In the present study, CD8+ T cell density will serve as a surrogate measure of prostate- /pro state cancer -specific T cell responses. By comparison, the human breast cancer trial used Her-2–specific immune responses as its primary endpoint (Emens et al 2008), while the pancreatic cancer trial used mesothelin -specific T cell responses as its primary endpoint (Laheru et al 2008). One of our goals in the future will be to identify a useful tumor - associated antigen in prostate cancer patients receiving GVAX , and to use this as a more rational marker of immunologic effects. In addit ion to augmenting antitumor immune responses in ProHA × TRAMP mice, the combination of cyclophosphamide/ GVAX also decreased the wet weight of the urogenital tract (a gross surrogate for tumor burden) compared to either agent used alone. Moreover, histologic tumo r scores were lower and apoptotic bodies were more frequent in the combined treatment group compared with either single treatment alone (Wada et al 2009). To evaluate potential direct antitumor effects in the present study, we will measure tumor apoptosis and proliferation using validated markers such as activated caspase 3 ( Marcelli et al 2000) and Ki -67 (Rubin et al 2002). These will be assessed by immunohistochemical staining of formalin -fixed tissue samples . Ki-67 is a nuclear antigen that is found in proliferating cells , whose expression correlates with the degree of apoptosis and cellular turnover in prost ate cancer (Berges et al 1995). Apoptotic cells will also be identified histologically and quantified by the method of terminal deoxynucleotidyl transferase (TdT)- mediated deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL assay) (Furuya et al Androgen suppression with prostate GVAX and cyclophosphamide J1265 Johns Hopkins University 20 of 68 J1265 PROT (Vers) 1.4 113012 1995). We will also evaluate the proportion of patients achieving a pathological complete response (pCR) in each of the 2 treatment groups. 3. PATIENT SELECTION 3.1 Target Population Subjects will include men with multifocal, Gleason 7 or greater, clinically localized prostate cancer for whom the decision has been made to perform radical prostatectomy at Johns Hopkins Hospital. Subjects will be identifie d and recruited through the Outpatient Clinic of the Department of Urology and from the Multidisciplinary Prostate Cancer Clinic . 3.2 Inclusion Criteria To be eligible for this study, patients must meet all of the following criteria: • Histologically confirm ed adenocarcinoma of the prostate ( clinical stage T1c –T3b, N0, M0) without involvement of lymph nodes, bone, or visceral organs • Initial prostate biopsy is available for central pathologic review, and is confirmed to show at least 2 positive cores and a max imum Gleason sum of ≥ 7 • Radical prostatectomy has been scheduled at Johns Hopkins Hospital • Age ≥21 years • ECOG performance status 0 -1, or Karnofsky score ≥ 70% (see Append ix A) • Adequate bone marrow, hepatic, and renal function : o WBC >3,000 cells/mm3 o ANC > 1,500 cells/mm3 o Hemoglobin > 9.0 g/dL o Platelet count >100,000 cells/mm3 o Serum creatinine <2.0 mg/dL o Serum bilirubin < 2 mg/dL o ALT < 2 × upper limit of normal (ULN) o AST < 2 × ULN o Alkaline phosphatase <2 × ULN • Willing ness to provide written informed consent and HIPAA authorization for the release of personal health information , and the ability to comply with the study requirements (note : HIPAA authorization will be included in the informed consent ) • Willing ness to use barrier contraception from the time of cyclophosphamide and/or GVAX administration until the time of prostatectomy. 3.3 Exclusion Criteria To be eligible for this study, p atients should not mee t any of the following criteria : • Presence of known lymph node involvement or distant metastases • Other histologic types of prostate cancers such as ductal, sarcomatous, lymphoma, small cell, and neuroendocrine tumors • Prior radiation therapy, hormonal therapy, biologic therapy, or chemotherapy for prostate cancer Androgen suppression with prostate GVAX and cyclophosphamide J1265 Johns Hopkins University 21 of 68 J1265 PROT (Vers) 1.4 113012 • Prior immunotherapy/vaccine therapy for prost ate cancer • Previous or concurrent use of cyclophosphamide • Concomitant treatment with other hormonal therapy or 5 α-reductase inhibitors • Current use of systemic corticosteroids or use of corticosteroids within 4 weeks of enrollment (inhaled corticosteroids for asthma or COPD are permitted) • Use of experimental agents for prostate cancer within the past 3 months • Known allergy to cyclophosphamide or G -CSF/GM -CSF • Known hyper sensitivity to materials of bovine origin (e.g. fetal bovine serum), or other components of GVAX which include DMSO and hydroxyethyl starch as well as small amoun ts of porcine trypsin and DNase • History or presence of autoimmune disease requiring systemic immunosuppression (including but not limited to : inflammatory bowel disease, systemic lupus erythematosus, vasculitis, rheumatoid arthritis, scleroderma, multiple sclerosis, hemolytic anemia, Sjögren syndrome, and sarcoidosis) • Other concurrent malignancies, with the exception of non -melanoma skin cancers and superficial bladder cancer • Uncontrolled major active infectious, cardiovascular, pulmonary, hematologic, or psychiatric illnesses that would make the patient a poor study candidate • Known prior or current history of HIV and/or hepatitis B/C 3.4 Inclusion of Minorities Men of all races and ethnic groups will be considered for study participation. Candidates must confor m to all eligibility criteria to be accepted into the study.