Search

Niraparib (NIRA) is a highly selective inhibitor of poly (adenosine diphosphate-ribose) polymerase, PARP1 and PARP2, which play a role in DNA repair. The phase II QUEST study evaluated NIRA combinations in patients with metastatic castration-resistant prostate cancer who were positive for homologous recombination repair gene alterations and had progressed on 1 prior line of novel androgen receptor-targeted therapy. Results from the combination of NIRA with abiraterone acetate plus prednisone, which disrupts androgen axis signaling through inhibition of CYP17, showed promising efficacy and a manageable safety profile in this patient population.

Antibody responses to off-target cancer-associated proteins have been detected following immunotherapies for cancer, suggesting these may be the result of antigen spread. We have previously reported that serum antibodies to prostate cancer-associated proteins were detectable using a high-throughput peptide array. We hypothesized that the breadth of antibody responses elicited by a vaccine could serve as a measure of the magnitude of its induced antigen spread. Consequently, sera from patients with prostate cancer, treated prior to or after vaccination in one of four separate clinical trials, were evaluated for antibody responses to an array of 177,604 peptides derived from over 1600 prostate cancer-associated gene products. Antibody responses to the same group of 5680 peptides previously reported were identified following vaccinations in which patients were administered GM-CSF as an adjuvant, but not with vaccine in the absence of GM-CSF. Hence, antibody responses to off-target proteins following vaccination may not necessarily serve as evidence of antigen spread and must be interpreted with particular caution following vaccine strategies that use GM-CSF, as GM-CSF appears to have direct effects on the production of antibodies. The evaluation of T cell responses to non-target antigens is likely a preferred approach for detection of immune-mediated antigen spread.

Background The safety and immunogenicity of JNJ-64041809 (JNJ-809), a live-attenuated, double-deleted Listeria monocytogenes (LADD Lm)-based immunotherapy targeting 4 relevant prostate cancer antigens, was evaluated in a phase 1 study in patients with metastatic castration-resistant prostate cancer (mCRPC). Methods Men with progressive mCRPC who had received ≥2 prior approved therapies were enrolled. Primary study objectives were to determine the recommended phase 2 dose (RP2D) and to evaluate the safety and immunogenicity of JNJ-809. Results A total of 26 patients received JNJ-809 (1 × 108 CFU (n = 6); 1 × 109 CFU (n = 20)). No dose-limiting toxicities were reported, and 1 × 109 CFU was selected as the RP2D. The most common adverse events (AEs) reported were chills (92%), pyrexia (81%), and fatigue (62%). The most frequent grade ≥3 AEs were lymphopenia (27%) and hypertension (23%). Serious AEs were reported in 27% of patients including 1 patient with grade 3 intestinal obstruction. JNJ-809 transiently induced peripheral cytokines, including interferon-γ, interleukin-10, and tumor necrosis factor-α. Of the 7 patients evaluable for T cell responses at the 1 × 109 CFU dose, evidence of post-treatment antigenic responses were observed in 6 to the Listeria antigen listeriolysin O and in 5 to ≥1 of the 4 encoded tumor antigens. Best overall response was stable disease in 13/25 response-evaluable patients. The study was terminated early as data collected were considered sufficient to evaluate safety and immunogenicity. Conclusions JNJ-809 has manageable safety consistent with other LADD Lm-based therapies. Limited antigen-specific immune responses were observed, which did not translate into objective clinical responses.

Purpose: Preclinical studies demonstrated that a DNA vaccine (pTVG-AR, MVI-118) encoding the androgen receptor ligand-binding domain (AR LBD) augmented antigen-specific CD8+ T cells, delayed prostate cancer progression and emergence of castration-resistant disease, and prolonged survival of tumor-bearing mice. This vaccine was evaluated in a multicenter phase I trial. Patients and Methods: Patients with metastatic castration-sensitive prostate cancer (mCSPC) who had recently begun androgen deprivation therapy were randomly assigned to receive pTVG-AR on one of two treatment schedules over one year, and with or without GM-CSF as a vaccine adjuvant. Patients were followed for 18 months. Primary objectives were safety and immune response. Secondary objectives included median time to PSA progression, and 18-month PSA-PFS (PPFS). Results: Forty patients were enrolled at three centers. Twenty-seven patients completed treatment and 18 months of follow-up. Eleven patients (28%) had a PSA progression event before the 18-month time point. No grade 3 or 4 adverse events were observed. Of 30 patients with samples available for immune analysis, 14 (47%) developed Th1-type immunity to the AR LBD, as determined by IFNγ and/or granzyme B ELISPOT. Persistent IFNγ immune responses were observed irrespective of GM-CSF adjuvant. Patients who developed T-cell immunity had a significantly prolonged PPFS compared with patients without immunity (HR = 0.01; 95% CI, 0.0–0.21; P = 0.003). Conclusions: pTVG-AR was safe and immunologically active in patients with mCSPC. Association between immunity and PPFS suggests that treatment may delay the time to castration resistance, consistent with preclinical findings, and will be prospectively evaluated in future trials. See related commentary by Shenderov and Antonarakis, p. 5056

We previously found that activated CD8

BackgroundWhile checkpoint blockade has been unsuccessful in prostate cancer trials, the approval of Sipuleucel-T demonstrates the value of antigen-specific vaccination approaches for this disease. We have studied a DNA vaccine specific for the ligand-binding domain of the androgen receptor (pTVG-AR) as a more scalable vaccination approach, though its efficacy is likely limited by the immunosuppressive prostate microenvironment. External beam radiotherapy has been shown to sensitize poorly responsive tumors to immunotherapy, but is infeasible for patients with widely metastatic disease. Our group has developed a compound called NM600 that can deliver radiation to all cancer sites simultaneously, similar to other targeted radionuclide therapy (TRT) approaches. In this study, we used TRT in combination with pTVG-AR to improve anti-tumor efficacy in a murine prostate cancer model.Methods6-week old male C57BL/6 mice were implanted subcutaneously with TRAMP-C1 cells. pTVG-AR or the empty vector were administered weekly from the day after tumor implantation. An intravenous injection was administered of 50 (”low-dose”) or 250 μCi (”high dose”) of 90Y-NM600, estimated to deliver a dose of 3.1 Gy or 15.5 Gy to 300 mm3 tumors, respectively. In one study, this TRT treatment was repeated once after three weeks. Groups of mice (n=5) were euthanized at several time points for flow cytometry analysis of the tumors. Separate cohorts (n=7) were followed for survival.ResultsLow-dose TRT administered once in combination with pTVG-AR (median survival 91 days) significantly improved survival more than low-dose TRT alone (median survival 59 days; p=.049) or pTVG-AR alone (median survival 59 days; p=0.01). Low-dose TRT plus pTVG-AR was also superior to high-dose TRT plus pTVG-AR (median survival 67 days; p=0.05). We next examined the effect of giving high-dose TRT twice in combination pTVG-AR. We found that the combination of fractionated TRT and pTVG-AR greatly slowed tumor growth unlike fractionated TRT alone (p=0.03). High-dose TRT + pTVG-AR caused a two-fold increase in CD86 expression on dendritic cells (p=0.0009) on Day 3 and a 10% increase in effector memory CD8+ T cells (p=0.002) on Day 1 compared to TRT alone. This combination also resulted in T cells with 3-fold lower PD-1 expression (p=4e-7) and 2-fold lower TIGIT expression (p=0.01).ConclusionsThese data suggest that the combination of antigen-specific vaccination and TRT can be an effective treatment for cancers that are refractory to immunotherapy. This combination may act through increasing co-stimulation by dendritic cells, leading to a more active cytolytic CD8+ T cell population.

BackgroundWe previously reported a pilot clinical trial using a DNA vaccine encoding prostatic acid phosphatase (pTVG-HP), given over 12 weeks either concurrently or in sequence with pembrolizumab, in patients with mCRPC. We report here the final analysis of this trial following two additional treatment arms in which patients with mCRPC were treated beyond 12 weeks until progression.MethodsPatients with mCRPC were treated with pTVG-HP and pembrolizumab every 3 weeks (Arm 3, n=20), or pTVG-HP every 2 weeks and pembrolizumab every 4 weeks (Arm 4, n=20). The primary objectives were safety, 6-month PFS, median time to radiographic progression, and objective response rates. Secondary objectives included immunological evaluations.ResultsTreatment was without unexpected toxicity, and only 1 grade 4 event (hyperglycemia) was observed. Immune related adverse events (irAE) > grade 1 included adrenal insufficiency, hepatitis, colitis, thyroid dysfunction, pancreatitis, pneumonitis, and rash, occurring in 42% of patients overall. 10/25 patients with measurable disease experienced any decrease in tumor volume from baseline, with 1 confirmed PR and no CR. 23/66 (35%) experienced any PSA decline from baseline. Overall median TTP was 5.4 months (95% CI; 5.3–8.1 months); median TTP for Arm 3 was 5.3 months compared to 8.0 months for Arm 4. Overall, 41.7% of patients had no radiographic progression at 6 months (29.9% Arm 3, 57.9% Arm 4). Median overall survival was 22.9 months. IFNγ and/or granzyme B immune response to PAP was detected in 2/20 patients in Arm 3 and 6/20 patients in Arm 4. Cytokines associated with immune activation and CD8+ T cell recruitment were augmented in the plasma of patients at weeks 6 and 12. Increased IFNγ in the sera at week 6 trended with prolonged TTP (p=0.010) and overall survival (p=0.025). The development of irAE was associated with a prolonged TTP (HR=0.25, p=0.003).ConclusionsPD-1 pathway inhibitors have demonstrated little clinical activity to date as monotherapies for mCRPC. Our findings demonstrate that combining PD-1 blockade with tumor-targeted T-cell activation using pTVG-HP is safe, can augment tumor-specific T cells, and result in objective changes with longer time to progression than what has been observed in previous trials. The association of progression or survival with increased IFNγ, irAE, and vaccine schedule suggests T cell activation by vaccination is critical to the mechanism of action of this combination. This study suggests this approach should be further evaluated in randomized clinical trials for patients with advanced mCRPC.AcknowledgementsFunding for this trial was from a 2014 Movember Prostate Cancer Foundation Challenge Award and Madison Vaccines, Inc.Trial RegistrationNCT02499835Ethics ApprovalThis trial was reviewed and approved by the University of Wisconsin Human Subjects’ Review Committee (IRB), protocol 2015–0453. All participants provided IRB-approved written informed consent before taking part.

BackgroundSystemic radiation treatments that preferentially irradiate cancer cells over normal tissue, known as targeted radionuclide therapy (TRT), have shown significant potential for treating metastatic prostate cancer. Preclinical studies have demonstrated the ability of external beam radiation therapy (EBRT) to sensitize tumors to T cell checkpoint blockade. Combining TRT approaches with immunotherapy may be more feasible than combining with EBRT to treat widely metastatic disease, however the effects of TRT on the prostate tumor microenvironment alone and in combinfation with checkpoint blockade have not yet been studied.MethodsC57BL/6 mice-bearing TRAMP-C1 tumors and FVB/NJ mice-bearing Myc-CaP tumors were treated with a single intravenous administration of either low-dose or high-dose 90Y-NM600 TRT, and with or without anti-PD-1 therapy. Groups of mice were followed for tumor growth while others were used for tissue collection and immunophenotyping of the tumors via flow cytometry.Results90Y-NM600 TRT was safe at doses that elicited a moderate antitumor response. TRT had multiple effects on the tumor microenvironment including increasing CD8 +T cell infiltration, increasing checkpoint molecule expression on CD8 +T cells, and increasing PD-L1 expression on myeloid cells. However, PD-1 blockade with TRT treatment did not improve antitumor efficacy. Tregs remained functional up to 1 week following TRT, but CD8 +T cells were not, and the suppressive function of Tregs increased when anti-PD-1 was present in in vitro studies. The combination of anti-PD-1 and TRT was only effective in vivo when Tregs were depleted.ConclusionsOur data suggest that the combination of 90Y-NM600 TRT and PD-1 blockade therapy is ineffective in these prostate cancer models due to the activating effect of anti-PD-1 on Tregs. This finding underscores the importance of thorough understanding of the effects of TRT and immunotherapy combinations on the tumor immune microenvironment prior to clinical investigation.

Concurrent blockade of different checkpoint receptors, notably PD-1 and CTLA-4, elicits greater anti-tumor activity for some tumor types, and the combination of different checkpoint receptor inhibitors is an active area of clinical research. We have previously demonstrated that anti-tumor vaccination, by activating CD8 + T cells, increases the expression of PD-1, CTLA-4, LAG-3 and other inhibitory receptors, and the anti-tumor efficacy of vaccination can be increased with checkpoint blockade. In the current study, we sought to determine whether anti-tumor vaccination might be further improved with combined checkpoint blockade. Using an OVA-expressing mouse tumor model, we found that CD8 + T cells activated in the presence of professional antigen presenting cells (APC) expressed multiple checkpoint receptors; however, T cells activated without APCs expressed LAG-3 alone, suggesting that LAG-3 might be a preferred target in combination with vaccination. Using three different murine tumor models, and peptide or DNA vaccines targeting three tumor antigens, we assessed the effects of vaccines with blockade of PD-1 and/or LAG-3 on tumor growth. We report that, in each model, the anti-tumor efficacy of vaccination was increased with PD-1 and/or LAG-3 blockade. However, combined PD-1 and LAG-3 blockade elicited the greatest anti-tumor effect when combined with vaccination in a MycCaP prostate cancer model in which PD-1 blockade alone with vaccination targeting a “self” tumor antigen had less efficacy. These results suggest anti-tumor vaccination might best be combined with concurrent blockade of both PD-1 and LAG-3, and potentially other checkpoint receptors whose expression is increased on CD8 + T cells following vaccine-mediated activation.

e13554 Background: Quantitative 18F-NaF PET/CT imaging metrics have been shown to be prognostic in metastatic prostate cancer (mPC) patients. However, previous studies have shown conflicting results in which metrics could be prognostic. This study investigates if current methods from literature generalize to external datasets and explores which combination of features are necessary to for survival models to generalize across datasets. Methods: Imaging and progression-free survival (PFS) data from 118 patients with mPC from four separate prospective clinical trials were gathered retrospectively. Patients received 18F-NaF PET/CT imaging at baseline and at follow-up, between eight and thirteen weeks. TRAQinform IQ technology (AIQ Solutions) was used to identify, segment, and track individual lesions from baseline to follow-up. Eighty-four imaging features were extracted from each patient and sorted into baseline, follow-up, response, patient-level (no inter-lesion comparison), and intrapatient heterogeneity (comparisons between lesions). The data was split into two training and testing sets, 44 patients from one study and 73 patients from the remaining 3 studies. As they can utilize large number of inputs without overfitting, random survival forest (RSF) models were chosen to evaluate performance of feature sets in predicting PFS. Different combinations of features were used as inputs to RSF models to compare single timepoint features with response features and patient-level features with intrapatient heterogeneity features. The performance of the RSF models, together with other methods identified in literature, were evaluated in each dataset using Kaplan-Meier analysis for categorical variables and the c-index for continuous variables. Results: No patient-level imaging features highlighted by literature displayed significant association to PFS across all four clinical trials (c-index < 0.62 in at least one dataset). Other criteria from literature did not generalize across all datasets (P > 0.05). The RSF model trained with all features had high c-indices in all four datasets (range: 0.66-0.80). RSF models built with response features (min: 0.63) performed better on average than models built with features obtained from single timepoints (min: 0.55). Patient-level features (min: 0.56) were not sufficient in all testing scenarios as compared to intrapatient heterogeneity features (min: 0.63). Conclusions: The candidate imaging biomarkers from previous 18F-NaF PET/CT imaging studies of mPC patients did not generalize across all datasets. Incorporating response and heterogeneity features with single-timepoint and patient-level features resulted in RSF prediction models which were generalizable across all datasets. Use of such models hold promise for improving outcome prediction in mPC patients.

Background Previous studies of prostate cancer autoantibodies have largely focused on diagnostic applications. So far, there have been no reports attempting to more comprehensively profile the landscape of prostate cancer-associated antibodies. Specifically, it is unknown whether the quantity of antibodies or the types of proteins recognized change with disease progression or treatment. Methods A peptide microarray spanning the amino acid sequences of the gene products of 1611 prostate cancer-associated genes was synthesized. Serum samples from healthy male volunteers (n=15) and prostate cancer patients (n=85) were used to probe the array. These samples included patients with various clinical stages of disease: newly diagnosed localized prostate cancer, castration-sensitive non-metastatic prostate cancer (nmCSPC), castration-resistant non-metastatic prostate cancer (nmCRPC), and castration-resistant metastatic disease (mCRPC). Serial sera samples from individuals who received treatment with either standard androgen deprivation therapy (ADT) or an anti-tumor vaccine were also used to probe the array, to determine whether we could detect treatment-related changes. Results We demonstrated that this peptide array yielded highly reproducible measurements of serum IgG levels. We found that the overall number of antibody responses did not increase with disease burden. However, the composition of recognized proteins shifted with clinical stage of disease. Our analysis revealed that the largest difference was between patients with castration-sensitive and castration-resistant disease. Patients with castration-resistant disease recognized more proteins associated with nucleic acid binding and gene regulation compared to men in other groups. Our longitudinal data showed that vaccine-treated patients developed increased responses to more proteins over the course of treatment than did ADT-treated patients, consistent with the development of antigen spread. Conclusions This study represents the largest survey of prostate-cancer associated antibodies to date. We have been able to characterize the classes of proteins recognized by patients and determine how they change with disease burden. Our findings demonstrate the potential of this platform for measuring antigen spread and studying responses to immunomodulatory therapies. Acknowledgements UW-Madison Medical Scientist Training Program: GM008692UW-Madison Institute for Clinical and Translational Research Predoctoral TL1 Program: TR002375 Ethics Approval Study protocols that permitted collection and use of human blood samples were reviewed and approved the University of Wisconsin Human Subjects’ Review Board (IRB). All patients gave written informed consent for use of blood products for research.

Simple Summary The only vaccine approved by FDA as a treatment for cancer is sipuleucel-T, a therapy for patients with metastatic castration-resistant prostate cancer (mCRPC). Most investigators studying anti-tumor vaccines believe they will be most effective as parts of combination therapies, rather than used alone. Unfortunately, the cost and complexity of sipuleucel-T makes it difficult to feasibly be used in combination with many other agents. In this review article we discuss the use of DNA vaccines as a simpler vaccine approach that has demonstrated efficacy in several animal species. We discuss the use of DNA vaccines in combination with traditional treatments for mCRPC, and other immune-modulating treatments, in preclinical and early clinical trials for patients with mCRPC. Abstract Metastatic castration-resistant prostate cancer (mCRPC) is a challenging disease to treat, with poor outcomes for patients. One antitumor vaccine, sipuleucel-T, has been approved as a treatment for mCRPC. DNA vaccines are another form of immunotherapy under investigation. DNA immunizations elicit antigen-specific T cells that cause tumor cell lysis, which should translate to meaningful clinical responses. They are easily amenable to design alterations, scalable for large-scale manufacturing, and thermo-stable for easy transport and distribution. Hence, they offer advantages over other vaccine formulations. However, clinical trials with DNA vaccines as a monotherapy have shown only modest clinical effects against tumors. Standard therapies for CRPC including androgen-targeted therapies, radiation therapy and chemotherapy all have immunomodulatory effects, which combined with immunotherapies such as DNA vaccines, could potentially improve treatment. In addition, many investigational drugs are being developed which can augment antitumor immunity, and together with DNA vaccines can further enhance antitumor responses in preclinical models. We reviewed the literature available prior to July 2020 exploring the use of DNA vaccines in the treatment of prostate cancer. We also examined various approved and experimental therapies that could be combined with DNA vaccines to potentially improve their antitumor efficacy as treatments for mCRPC.

Sipuleucel-T is an autologous cellular immunotherapy that induces an immune response targeted against prostatic acid phosphatase (PAP) to treat asymptomatic or minimally symptomatic metastatic castration-resistant prostate cancer. In the phase III IMPACT study, sipuleucel-T was associated with a statistically significantly increased overall survival (OS) (median = 4.1 months) vs placebo. Patients with baseline prostate-specific antigen levels in the lowest quartile (≤22.1 ng/mL) exhibited a 13-month improvement in OS with sipuleucel-T. Together, this led sipuleucel-T to be approved and recommended as first-line therapy in various guidelines for treatment of metastatic castration-resistant prostate cancer. This review discusses the varied findings about the mechanisms of action of sipuleucel-T, bringing them together to form a more coherent picture. These pieces include inducing a statistically significant increase in antigen-presenting cell activation; inducing a peripheral immune response specific to the target (PAP) and/or immunizing (PA2024) antigens; stimulating systemic cytotoxic T-lymphocyte activity; and mediating antigen spread (ie, increased antibody responses to secondary proteins in addition to PAP and PA2024). Each of these pieces individually correlates with OS. Sipuleucel-T also traffics T cells to the prostate and is associated with long-term immune memory such that a second course of treatment induces an anamnestic immune response. Prostate cancer does not have a strongly inflamed microenvironment, thus its response to immune checkpoint inhibitors is limited. Because sipuleucel-T is able to traffic T cells to the tumor, it may be an ideal combination partner with immunotherapies including immune checkpoint inhibitors or with radiation therapy.

The hypoxia and profound inflammatory response associated with the pneumonitis observed with the severe acute respiratory virus coronavirus-2 (SARS-COV-2) virus responsible for the recent COVID-19 pandemic has overwhelmed intensive care facilities in the epicenters of infection including Wuhan

BackgroundWe previously reported a trial using a DNA vaccine encoding prostatic acid phosphatase (MVI-816, pTVG-HP), given over 12 weeks concurrently or sequentially with pembrolizumab, in patients with mCRPC. We report the final analysis of this trial following two additional treatment arms in which patients with mCRPC continued concurrent treatment until progression.Materials and methodsPatients with mCRPC were treated with MVI-816 and pembrolizumab every 3 weeks (arm 3, n=20) or MVI-816 every 2 weeks and pembrolizumab every 4 weeks (arm 4, n=20). The primary objectives were safety, 6-month progression-free survival (PFS), median time to radiographic progression, and objective response rates. Secondary objectives included immunological evaluations.ResultsIn 25 patients with measurable disease, there were no complete response and one confirmed partial response in a patient who subsequently found to have an MSIhitumor. 4/40 patients (10%) had a prostate-specific antigen decline >50%. The estimated overall radiographic PFS rate at 6 months was 47.2% (44.4% arm 3, 61.5% arm 4). Accounting for all off-study events, overall median time on treatment was 5.6 months (95% CI: 5.4 to 10.8 months), 5.6 months for arm 3 and 8.1 months for arm 4 (p=0.64). Thirty-two per cent of patients remained on trial beyond 6 months without progression. Median overall survival was 22.9 (95% CI: 16.2 to 25.6) months. One grade 4 event (hyperglycemia) was observed. Immune-related adverse events (irAEs) >grade 1 were observed in 42% of patients overall. Interferon-γ and/or granzyme B immune response to prostatic acid phosphatase was detected in 2/20 patients in arm 3 and 6/20 patients in arm 4. Plasma cytokines associated with immune activation and CD8+ T-cell recruitment were augmented at weeks 6 and 12. The development of irAE was significantly associated with a prolonged time on treatment (HR=0.42, p=0.003). Baseline DNA homologous recombination repair mutations were not associated with longer time to progression.ConclusionsFindings here demonstrate that combining programmed cell death 1 blockade with MVI-816 is safe, can augment tumor-specific T cells, and can result in a favorable 6-month disease control rate. Correlative studies suggest T-cell activation by vaccination is critical to the mechanism of action of this combination. Future randomized clinical trials are needed to validate these findings.Trial registration numberNCT02499835.

BackgroundProstate cancer (PC) responds to androgen deprivation therapy (ADT) usually in a transient fashion, progressing from hormone-sensitive PC (HSPC) to castration-resistant PC (CRPC). We investigated a mouse model of PC as well as specimens from PC patients to unravel an unsuspected contribution of thymus-derived T lymphocytes and the intestinal microbiota in the efficacy of ADT.MethodsPreclinical experiments were performed in PC-bearing mice, immunocompetent or immunodeficient. In parallel, we prospectively included 65 HSPC and CRPC patients (Oncobiotic trial) to analyze their feces and blood specimens.ResultsIn PC-bearing mice, ADT increased thymic cellularity and output. PC implanted in T lymphocyte-depleted or athymic mice responded less efficiently to ADT than in immunocompetent mice. Moreover, depletion of the intestinal microbiota by oral antibiotics reduced the efficacy of ADT. PC reduced the relative abundance of Akkermansia muciniphila in the gut, and this effect was reversed by ADT. Moreover, cohousing of PC-bearing mice with tumor-free mice or oral gavage with Akkermansia improved the efficacy of ADT. This appears to be applicable to PC patients because long-term ADT resulted in an increase of thymic output, as demonstrated by an increase in circulating recent thymic emigrant cells (sjTRECs). Moreover, as compared with HSPC controls, CRPC patients demonstrated a shift in their intestinal microbiota that significantly correlated with sjTRECs. While feces from healthy volunteers restored ADT efficacy, feces from PC patients failed to do so.ConclusionsThese findings suggest the potential clinical utility of reversing intestinal dysbiosis and repairing acquired immune defects in PC patients.

168 Background: Prostate cancer (PC) responds to androgen deprivation therapy (ADT) usually in a transient fashion, progressing from hormone-sensitive PC (HSPC) to castration-resistant PC (CRPC). While it is recognized that ADT has an immunomodulatory effect, little is known about the intestinal microbiome effect on therapeutic outcome of ADT. Methods: We investigated a mouse model of PC as well as specimens from PC patients to unravel an unsuspected contribution of thymus-derived T lymphocytes and the intestinal microbiota in the efficacy of ADT. Preclinical experiments were performed in PC-bearing mice, immunocompetent or immunodeficient. In parallel, we prospectively included 65 HSPC and CRPC patients to analyze their feces and blood specimen. Results: In PC-bearing mice, ADT increased thymic cellularity and output. PC implanted in T lymphocyte-depleted or athymic mice responded less efficiently to ADT than in immunocompetent mice. Moreover, depletion of the intestinal microbiota by oral antibiotics reduced the efficacy of ADT. PC reduced the relative abundance of Akkermansia muciniphila in the gut, and this effect was reversed by ADT. Moreover, cohousing of PC-bearing mice with tumor-free mice, or oral gavage with Akkermansia improved the efficacy of ADT. This appear to be applicable to PC patients because long-term ADT resulted in an increase of thymic output, as demonstrated by an increase in circulating recent thymic emigrant cells (sjTRECs). Moreover, as compared to HSPC controls, CRPC patients demonstrated a shift in their intestinal microbiota that significantly correlated with sjTRECs. While feces from healthy volunteers restored ADT efficacy. Conclusions: These findings suggest that reversing the intestinal dysbiosis and repairing acquired immune defects in PC patients have a potential impact on the therapeutic outcome of ADT.

Purpose: Preclinical data established IL15 as a homeostatic factor and powerful stimulator of NK and CD8+ T-cell function, the basis for clinical testing.Experimental Design: A first-in-human outpatient phase I dose escalation trial of subcutaneous (SC) rhIL15 was conducted in refractory solid tumor cancer patients. Therapy consisted of daily (Monday–Friday) subcutaneous injections of rhIL15 for two consecutive weeks (10 total doses/cycle). Clinical response was assessed by RECIST. Pharmacokinetics of rhIL15 and immune biomarkers were evaluated.Results: Nineteen patients were treated with rhIL15 at dose levels of 0.25, 0.5, 1, 2, and 3 mcg/kg/day. Fourteen patients completed ≥ 2 cycles of therapy that was well tolerated. One serious adverse event (SAE), grade 2 pancreatitis, required overnight hospitalization. Enrollment was halted after a patient receiving 3 mcg/kg/day developed a dose-limiting SAE of grade 3 cardiac chest pain associated with hypotension and increased troponin. No objective responses were observed; however, several patients had disease stabilization including a renal cell carcinoma patient who continued protocol treatment for 2 years. The treatment induced profound expansion of circulating NK cells, especially among the CD56bright subset. A proportional but less dramatic increase was found among circulating CD8+ T cells with maximal 3-fold expansion for the 2 and 3 mcg/kg patients.Conclusions: SC rhIL15 treatment was well tolerated, producing substantial increases in circulating NK and CD8+ T cells. This protocol establishes a safe outpatient SC rhIL15 regimen of 2 mcg/kg/day dosing amenable to self-injection and with potential as a combination immunotherapeutic agent. Clin Cancer Res; 24(7); 1525–35. ©2017 AACR.

Background Prostatic acid phosphatase (PAP) is a prostate tumor antigen, and the target of the only FDA-approved anti-tumor vaccine, sipuleucel-T. We have previously reported in two clinical trials that a DNA vaccine encoding PAP (pTVG-HP) could elicit PAP-specific, Th1-biased T cells in patients with PSA-recurrent prostate cancer. In the current pilot trial we sought to evaluate whether this vaccine could augment PAP-specific immunity when used as a booster to immunization with sipuleucel-T in patients with metastatic, castration-resistant prostate cancer (mCRPC). Methods Eigthteen patients with mCRPC were randomized to receive sipuleucel-T alone or followed by intradermal immunization with pTVG-HP DNA vaccine. Patients were followed for time to progression, and immune monitoring was conducted at defined intervals. Results Overall, patients were followed for a median of 24 months. 11/18 patients completed treatments as per protocol. No treatment-associated events > grade 2 were observed. Th1-biased PAP-specific T-cell responses were detected in 11/18 individuals, and were not statistically different between study arms. Higher titer antibody responses to PAP were detectable in patients who received pTVG-HP booster immunizations. Median time to progression was less than 6 months and not statistically different between study arms. The median overall survival for all patients was 28 months. Conclusions These findings suggest that prime-boost vaccination can augment and diversify the type of immunity elicited with anti-tumor vaccination in terms of T-cell and humoral immunity. Future studies will explore DNA as priming immunization rather than a booster immunization. Trial registration NCT01706458.

BackgroundT-cell checkpoint receptors are expressed when T-cell are activated, and activation of these receptors can impair the function of T-cells and their anti-tumor efficacy.1 We previously found that T-cells activated with cognate antigen increase the expression of PD-1, while this can be attenuated by the presence of specific Toll-like receptor (TLR) agonists.2 3 This effect was mediated by IL-12 secretion from professional antigen presenting cells and resulted in CD8+ T cells with greater anti-tumor activity. In the current report, we sought to determine whether combination of TLR agonists can further affect the expression of T-cell checkpoint receptors and improve T-cell anti-tumor immunity.MethodsOT-1 CD8+ T cells were stimulated with peptide (SIINFEKL) and dendritic cells (DC) in the presence of two different TLR agonists. The cells were collected and evaluated for the expression of T-cell checkpoint receptors (PD-1, CTLA-4, CD160, CD244, LAG-3, TIM-3, TIGIT and VISTA) by flow cytometry, and for transcriptional changes by RNA-seq. Purified DC were stimulated with TLR combinations and evaluated for cytokine release by ELISA. The anti-tumor efficacy of vaccination using peptide and TLR agonist combinations was evaluated in EG7-OVA tumor-bearing mice.ResultsActivation of CD8+ T cells in the presence of specific TLR ligands resulted in decreases in expression of PD-1 and/or CD160. These changes in T-cell checkpoint receptor expression were modestly affected when TLR ligands were used in combination, and notably with combinations of TLR1/2, TLR3, and TLR9 agonists. Immunization of tumor-bearing mice, co-administered with combinations of these agonists, showed greater anti-tumor effects. However, while the effect of TLR1/2 and/or TLR9 was abrogated in IL12KO mice, TLR3 demonstrated anti-tumor activity when co-administered with peptide vaccine. RNA sequencing of TLR-conditioned CD8+ T-cells revealed IL-12 pathway activation, and IFNß pathway activation following TLR3 stimulation. Stimulation of DC with TLR3 agonist, alone or in combination with other TLR agonists, resulted in increased IL-12 and IFNß secretion. Co-incubation of OT-1 splenocytes with rIL12 and/or rIFNß during peptide activation led to reduced expression of PD-1, and this could be reversed with antibodies blocking IL12R or IFNAR-1.ConclusionsMultiple TLR agonists can modulate the expression of T-cell checkpoint receptors, notably PD-1, by upregulating the secretion of IL-12 and IFNß. These data provide the mechanistic rationale for choosing optimal combinations of TLR ligands to use as adjuvants to improve the efficacy of anti-tumor vaccines.ReferencesJin H-T, et al. Cooperation of Tim-3 and PD-1 in CD8 T-cell exhaustion during chronic viral infection. Proceedings of the National Academy of Sciences 2010;107(33):14733–14738.Zahm CD, Colluru VT, McNeel DG. Vaccination with high-affinity epitopes impairs antitumor efficacy by increasing PD-1 expression on CD8+ T cells. Cancer Immunology Research 2017;5(8):630–641.Zahm CD, et al. TLR stimulation during T-cell activation lowers PD-1 expression on CD8+ T Cells. Cancer Immunology Research 2018;6(11):1364–1374.

Prostate cancer is the most common newly diagnosed malignancy in men, and the second most common cause of cancer-related death in the United States. The primary treatment for recurrent prostate cancer is androgen deprivation, and this therapy is typically continued lifelong for patients with metastatic prostate cancer. Androgens and androgen deprivation have profound effects on the immune system, a finding that has become more appreciated in an era where immune-based treatments for cancer are being increasingly explored. Preclinical studies suggest that androgen deprivation could potentially positively or negatively affect the use of approved immunotherapies, or those that are being developed for the treatment of prostate cancer. In this review, we provide a brief overview of the different types of androgen deprivation treatments used in the management of prostate cancer, discuss their effects on prostate tumors and the immune system and how they are being explored in combination with immunotherapy. Finally, we address some of the critical questions in the field that must be answered to identify the best approaches to combine androgen deprivation with immunotherapy for the treatment of prostate cancer.

PURPOSE We previously reported the safety and immunologic effects of a DNA vaccine (pTVG-HP [MVI-816]) encoding prostatic acid phosphatase (PAP) in patients with recurrent, nonmetastatic prostate cancer. The current trial evaluated the effects of this vaccine on metastatic progression. PATIENTS AND METHODS Ninety-nine patients with castration-sensitive prostate cancer and prostate-specific antigen (PSA) doubling time (DT) of less than 12 months were randomly assigned to treatment with either pTVG-HP co-administered intradermally with 200 μg granulocyte-macrophage colony-stimulating factor (GM-CSF) adjuvant or 200 μg GM-CSF alone six times at 14-day intervals and then quarterly for 2 years. The primary end point was 2-year metastasis-free survival (MFS). Secondary and exploratory end points were median MFS, changes in PSA DT, immunologic effects, and changes in quantitative 18F-sodium fluoride (NaF) positron emission tomography/computed tomography (PET/CT) imaging. RESULTS Two-year MFS was not different between study arms (41.8% vaccine v 42.3%; P = .97). Changes in PSA DT and median MFS were not different between study arms (18.9 v 18.3 months; hazard ratio [HR], 1.6; P = .13). Preplanned subset analysis identified longer MFS in vaccine-treated patients with rapid (< 3 months) pretreatment PSA DT (12.0 v 6.1 months; n = 21; HR, 4.4; P = .03). PAP-specific T cells were detected in both cohorts, including multifunctional PAP-specific T-helper 1–biased T cells. Changes in total activity (total standardized uptake value) on 18F-NaF PET/CT from months 3 to 6 increased 50% in patients treated with GM-CSF alone and decreased 23% in patients treated with pTVG-HP (n = 31; P = .07). CONCLUSION pTVG-HP treatment did not demonstrate an overall increase in 2-year MFS in patients with castration-sensitive prostate cancer, with the possible exception of a subgroup with rapidly progressive disease. Prespecified 18F-NaF PET/CT imaging conducted in a subset of patients suggests that vaccination had detectable effects on micrometastatic bone disease. Additional trials using pTVG-HP in combination with PD-1 blockade are under way.

// Erika Heninger 1, 3 , Timothy E.G. Krueger 3 , Stephanie M. Thiede 1, 3 , Jamie M. Sperger 1, 3 , Brianna L. Byers 3 , Madison R. Kircher 3 , David Kosoff 1, 3 , Bing Yang 2, 3 , David F. Jarrard 2, 3 , Douglas G. McNeel 1, 3 , Joshua M. Lang 1, 3 1 Department of Medicine, University of Wisconsin, Madison, Madison, WI 53705, USA 2 Department of Urology, University of Wisconsin, Madison, Madison, WI 53705, USA 3 University of Wisconsin Carbone Cancer Center, Madison, Madison, WI 53705, USA Correspondence to: Joshua M. Lang, email: jmlang@medicine.wisc.edu Keywords: cancer testis antigen, prostate cancer, epigenetics, tumor immunotherapy, methylation Received: April 22, 2016 Accepted: October 11, 2016 Published: October 17, 2016 ABSTRACT Immune tolerance to self-antigens can limit robust anti-tumor immune responses in the use of tumor vaccines. Expression of novel tumor associated antigens can improve immune recognition and lysis of tumor cells. The cancer-testis antigen (CTA) family of proteins has been hypothesized to be an ideal class of antigens due to tumor-restricted expression, a subset of which have been found to induce antibody responses in patients with prostate disease. We demonstrate that CTA expression is highly inducible in five different Prostate Cancer (PC) cell lines using a hypomethylating agent 5-Aza-2′-deoxycytidine (5AZA) and/or a histone deacetylase inhibitor LBH589. These CTAs include NY-ESO1, multiple members of the MAGE and SSX families and NY-SAR35. A subset of CTAs is synergistically induced by the combination of 5AZA and LBH589. We developed an ex vivo organ culture using human PC biopsies for ex vivo drug treatments to evaluate these agents in clinical samples. These assays found significant induction of SSX2 in 9/9 distinct patient samples and NY-SAR35 in 7/9 samples. Further, we identify expression of SSX2 in circulating tumor cells (CTC) from patients with advanced PC. These results indicate that epigenetic modifying agents can induce expression of a broad range of neoantigens in human PC and may serve as a useful adjunctive therapy with novel tumor vaccines and checkpoint inhibitors.

// Viswa Teja Colluru 1,2 and Douglas G. McNeel 1,2 1 Department of Medicine, University of Wisconsin-Madison, Madison, WI, USA 2 Carbone Cancer Center, University of Wisconsin-Madison, Madison, WI, USA Correspondence to: Douglas G. McNeel, email: // Keywords : DNA vaccine, B cells, dendritic cells, direct presentation, Immunology and Microbiology Section, Immune response, Immunity Received : July 26, 2016 Accepted : September 16, 2016 Published : September 21, 2016 Abstract In spite of remarkable preclinical efficacy, DNA vaccination has demonstrated low immunogenicity in humans. While efforts have focused on increasing cross-presentation of DNA-encoded antigens, efforts to increase DNA vaccine immunogenicity by targeting direct presentation have remained mostly unexplored. In these studies, we compared the ability of different APCs to present antigen to T cells after simple co-culture with plasmid DNA. We found that human primary peripheral B lymphocytes, and not monocytes or in vitro derived dendritic cells (DCs), were able to efficiently encode antigen mRNA and expand cognate tumor antigen-specific CD8 T cells ex vivo . Similarly, murine B lymphocytes co-cultured with plasmid DNA, and not DCs, were able to prime antigen-specific T cells in vivo. Moreover, B lymphocyte-mediated presentation of plasmid antigen led to greater Th1-biased immunity and was sufficient to elicit an anti-tumor effect in vivo . Surprisingly, increasing plasmid presentation by B cells, and not cross presentation of peptides by DCs, further augmented traditional plasmid vaccination. Together, these data suggest that targeting plasmid DNA to B lymphocytes, for example through transfer of ex vivo plasmidloaded B cells, may be novel means to achieve greater T cell immunity from DNA vaccines.

Prostate cancer is the most commonly diagnosed cancer, and the second leading cause of cancer-related death for men in the United States. Despite the approval of several new agents for advanced disease, each of these has prolonged survival by only a few months. Consequently, new therapies are sorely needed. For other cancer types, immunotherapy has demonstrated dramatic and durable treatment responses, causing many to hope that immunotherapies might provide an ideal treatment approach for patients with advanced prostate cancer. However, apart from sipuleucel-T, prostate cancer has been conspicuously absent from the list of malignancies for which immunotherapies have received recent approval from the US Food and Drug Administration. This has left some wondering whether immunotherapy will "work" for this disease. In this review, the authors describe current developments in immunotherapy, including approaches to engage tumor-targeting T cells, disrupt immune regulation, and alter the immunosuppressive tumor microenvironment. The authors then describe the recent application of these approaches to the treatment of prostate cancer. Given the Food and Drug Administration approval of 1 agent, and the finding that several others are in advanced stages of clinical testing, the authors believe that immunotherapies offer real hope to improve patient outcomes for men with prostate cancer, especially as investigators begin to explore rational combinations of immunotherapies and combine these therapies with other conventional therapies. Cancer 2016;122:3598-607. © 2016 American Cancer Society.

BackgroundPrevious studies of prostate cancer autoantibodies have largely focused on diagnostic applications. So far, there have been no reports attempting to more comprehensively profile the landscape of prostate cancer-associated antibodies. Specifically, it is unknown whether the quantity of antibodies or the types of proteins recognized change with disease progression.MethodsA peptide microarray spanning the amino acid sequences of the gene products of 1611 prostate cancer-associated genes was synthesized. Serum samples from healthy male volunteers (n=15) and patients with prostate cancer (n=85) were used to probe the array. These samples included patients with various clinical stages of disease: newly diagnosed localized prostate cancer (n=15), castration-sensitive non-metastatic prostate cancer (nmCSPC, n=40), castration-resistant non-metastatic prostate cancer (n=15) and castration-resistant metastatic disease (n=15). The patients with nmCSPC received treatment with either standard androgen deprivation therapy (ADT) or an antitumor DNA vaccine encoding prostatic acid phosphatase. Serial sera samples from these individuals were also used to probe the array, to secondarily determine whether this approach could be used to detect treatment-related changes.ResultsWe demonstrated that this peptide array yielded highly reproducible measurements of serum IgG levels. We found that the overall number of antibody responses did not increase with disease burden. However, the composition of recognized proteins shifted with clinical stage of disease. Our analysis revealed that the largest difference was between patients with castration-sensitive and castration-resistant disease. Patients with castration-resistant disease recognized more proteins associated with nucleic acid binding and gene regulation compared with men in other groups. Our longitudinal data showed that treatments can elicit antibodies detectable by this array, and notably vaccine-treated patients developed increased responses to more proteins over the course of treatment than did ADT-treated patients.ConclusionsThis study represents the largest survey of prostate cancer-associated antibodies to date. We have been able to characterize the classes of proteins recognized by patients and determine how they change with disease burden. Our findings further demonstrate the potential of this platform for measuring antigen spread and studying responses to immunomodulatory therapies.

Introduction: Prostate cancer remains the second leading cause of cancer-related death in American men because of its metastatic, incurable form. Prostate cancer responds poorly to checkpoint blockade likely due to its immunosuppressive microenvironment with relatively few infiltrating CD8+ T cells, limited mutational burden, and activity of myeloid-derived suppressor cells (MDSCs). External beam radiotherapy (EBRT) has been shown to help overcome this suppressive state, but EBRT is infeasible for patients with widely metastatic disease. Our group has developed a compound called NM600 that can be used to deliver radiation to all sites of disease simultaneously in an approach called Targeted radionuclide therapy (TRT). However, the effects of TRT on immune populations within prostate tumors are not yet well described. In this study, we examined the effects of TRT in the form of 90Y-NM600 on mice bearing Myc-CaP prostate tumors. Methods: 6-week old male FVB mice were implanted subcutaneously with Myc-CaP cells, then given a single intravenous injection of either 50 (“low-dose”) or 250 (“high-dose”) μCi of 90Y-NM600, estimated to deliver 3.1 Gy or 15.5 Gy to 300 mm3 tumors, respectively. Groups of mice (n=3) were then euthanized at several timepoints following TRT administration. Their tumors and spleens were collected for analysis by flow cytometry and Luminex cytokine analysis. A separate group (n=5) were followed for survival to 2000 mm3. Results: Flow cytometry of splenocytes revealed that high-dose, but not low-dose TRT caused a 6-fold increase in numbers of MDSCs (p = 0.002) at Day 21 compared to baseline. Additionally, low-dose TRT caused a 2-fold (p = 0.005) increase in CD8+ T cells at Day 21 compared to baseline, unlike in the high-dose condition. These CD8+ T cells in the low-dose TRT condition were predominantly proliferating naïve T cells. Flow cytometry of tumor infiltrating lymphocytes showed a 13% increase in CD8+ T cells in both dose conditions at Day 14 (p = 0.03). Returning CD8+ T cells displayed high expression of PD-1, CTLA-4, and LAG-3. Within the tumor, there was an 8-fold increase in chemokines including CXCL1, Rantes, and MIP2 at Day 21, suggesting that there may be further recruitment of tumor infiltrating cells even three weeks following treatment. Mice treated with high-dose TRT (median survival 37 days, p = .029) had significantly improved median survival compared to control (median survival 23 days), unlike low-dose treated mice (median survival 29 days, p =.250). Conclusions: These data suggest that low-dose TRT is superior for immunomodulation because, although it has little effect on tumor growth, it increases intratumoral CD8+ T cell infiltration while avoiding deleterious systemic immune effects. We hypothesize that immunomodulatory TRT combined with checkpoint blockade will improve anti-tumor efficacy compared to high-dose TRT. Citation Format: Hemanth Kumar Potluri, Reinier Hernandez, Christopher D. Zahm, Joseph Grudzinski, Christopher Massey, Jamey Weichert, Douglas G. McNeel. Low-dose targeted radionuclide therapy has favorable local and systemic effects on immune populations in a murine prostate cancer model [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2262.

TPS273 Background: Radical prostatectomy (RP) and radiation therapy (RT) are the gold standard, curative intent treatment for pts with presumed organ-confined PCa. However, about one third of these pts will have progressive or metastatic disease at 10 years (yr). Pts with PSA recurrence without radiographic evidence of metastatic (m) PCa (stage M0) will ultimately develop radiographically apparent mPCa within a median of 8 yr. Pts with rapid PSA doubling time (DT) have a markedly shorter time to mPCa and death. Utilizing an immunotherapeutic approach could utilize the sensitivity and specificity of the immune system to treat microscopic disease and potentially avoid or postpone androgen deprivation therapy (ADT) and its unwanted side effects. Methods: In this single arm, two-stage phase II trial, a total of 21-41 PCa pts with PSA only progression (4 rising PSA with final PSA ≥ 2 ng/mL) after initial RP for presumed organ-confined disease who have no radiographic evidence of mPCa in conventional imaging (CT and bone scan) will be enrolled. Pts with small cell or other variant PCa, or history of ADT other than concurrent with RT are excluded. All pts will be treated with pTVG-HP 100 μg intradermally (id) and Nivo 240 mg IV every (Q) 2 weeks (w) x6, and then pTVG-HP 100 μg id and Nivo 480 mg IV Q4w x9 beginning w12 on study. Pts with PSA on w4 > PSA on day 1 will additionally receive rhGM-CSF 208 μg id, as a vaccine adjuvant, Q2w x4 beginning w4 on study. Pts will be treated until progression or up to a total of 1 yr. Response will be monitored by CT and bone scan Q6 months or as clinically indicated. Primary endpoints are safety and tolerability of this combination and PSA complete response rate (PSA < 0.2 ng/mL). Key secondary endpoints are 2-yr metastasis-free survival, median radiographic progression-free survival, PSA response rate (≤50% of baseline), and changes in PSA DT. Elicited antigen-specific T-cell and/or IgG response and its correlation with PSA response will be explored. Bone mets not detected by conventional imaging and their association with response will be evaluated by Quantitative Total Bone Imaging (QTBI) by NaF PET/CT. Updated enrollment will be presented. Clinical trial information: NCT03600350.

Background Immunotherapy has demonstrated remarkable success in treating different cancers. Nonetheless, a large number of patients do not respond, many respond without immediate changes detectable with conventional imaging, and many have unusual immune-related adverse events that cannot be predicted in advance. In this exploratory study, we investigate how 3′-Deoxy-3’-18F-fluorothymidine (FLT) positron emission tomography (PET) measurements of tumor and immune cell proliferation might be utilized as biomarkers in immunotherapy. Methods Seventeen patients with metastatic castrate resistant prostate cancer were treated with combination pTVG-HP DNA vaccine and pembrolizumab. Patients underwent baseline and 12-week FLT PET/CT scans. FLT PET standardized uptake values (SUVs) were extracted from tumors, non-metastatic lymph nodes, spleen, bone marrow, pancreas, and thyroid to quantify cell proliferation in these tissues. Regional immune cell responses to pTVG-HP DNA vaccine were assessed by comparing FLT uptake changes in vaccine draining and non-draining lymph nodes. Cox proportional hazards regression was utilized to relate FLT uptake and other clinical markers (PSA and tumor size) to progression-free survival. Area under receiver operating characteristic (AUC) curves and concordance indices were used to assess the predictive capabilities of FLT uptake. Results Changes in FLT uptake in vaccine draining lymph nodes were significantly greater than changes in non-draining lymph nodes (P = 0.02), suggesting a regional immune response to vaccination. However, the changes in FLT uptake in lymph nodes were not significantly predictive of progression-free survival. Increases in tumor FLT uptake were significantly predictive of shorter progression-free survival (concordance index = 0.83, P

Anti-tumor vaccines have demonstrated efficacy in patients with castration-resistant metastatic prostate cancer. One vaccine, Prostvac-VF®, using a heterologous prime-boost strategy with vaccinia and fowlpox viral vectors encoding PSA, is currently being evaluated in a registration phase III multinational clinical trial. The current trial was planned to assess the clinical efficacy of this vaccine in patients with castration-resistant metastatic prostate cancer receiving subsequent docetaxel chemotherapy. 10 patients with metastatic castration-resistant prostate cancer, with a predicted survival of at least 18 months, were enrolled out of a planned 144 patients. Eight of 10 patients were treated and were randomized to receive docetaxel chemotherapy alone (Arm B, n = 2) versus treatment with Prostvac-VF (days 1, 15, 29, 43, 57) followed by docetaxel (Arm A, n = 6) chemotherapy beginning at month 3. The primary endpoint of the trial was overall survival, and secondary endpoints included time to radiographic progression and immunological response. The trial was opened within the Eastern Cooperative Oncology Group, but due to slow accrual was closed by CTEP after only 10 patients were enrolled within 13 months. Results: Presented here are the safety, clinical, and immunological results from 8 eligible patients who underwent treatment. Two of 6 patients treated on Arm A, with vaccine followed by docetaxel, had a >50% PSA response, with one of these patients experiencing a PSA decline during treatment with vaccine. Significant PSA-specific CD4+ and CD8+ T-cell responses and IgG antibody responses specific for PSA were not detected. The primary endpoint of overall survival cannot be assessed due to limited accrual. The lack of T-cell responses, even in this small cohort, suggests that further validation and development of immune biomarkers will be important for future studies. Other trials remain ongoing to evaluate the role of anti-tumor vaccination in sequence with other traditional anti-tumor therapies.

Background Immunotherapies have demonstrated clinical benefit for many types of cancers, however many patients do not respond, and treatment-related adverse effects can be severe. Hence many efforts are underway to identify treatment predictive biomarkers. We have reported the results of two phase I trials using a DNA vaccine encoding prostatic acid phosphatase (PAP) in patients with biochemically recurrent prostate cancer. In both trials, persistent PAP-specific Th1 immunity developed in some patients, and this was associated with favorable changes in serum PSA kinetics. In the current study, we sought to determine if measures of antigen-specific or antigen non-specific immunity were present prior to treatment, and associated with subsequent immune response, to identify possible predictive immune biomarkers. Methods Patients who developed persistent PAP-specific, IFNγ-secreting immune responses were defined as immune “responders.” The frequency of peripheral T cell and B cell lymphocytes, natural killer cells, monocytes, dendritic cells, myeloid derived suppressor cells, and regulatory T cells were assessed by flow cytometry and clinical laboratory values. PAP-specific immune responses were evaluated by cytokine secretion in vitro, and by antigen-specific suppression of delayed-type hypersensitivity to a recall antigen in an in vivo SCID mouse model. Results The frequency of peripheral blood cell types did not differ between the immune responder and non-responder groups. Non-responder patients tended to have higher PAP-specific IL-10 production pre-vaccination (p = 0.09). Responder patients had greater preexisting PAP-specific bystander regulatory responses that suppressed DTH to a recall antigen (p = 0.016). Conclusions While our study population was small (n = 38), these results suggest that different measures of antigen-specific tolerance or regulation might help predict immunological outcome from DNA vaccination. These will be prospectively evaluated in an ongoing randomized, phase II trial. Electronic supplementary material The online version of this article (doi:10.1186/s40425-017-0260-3) contains supplementary material, which is available to authorized users.

BACKGROUND The androgen receptor (AR) is a key oncogenic driver of prostate cancer, and has been the primary focus of prostate cancer treatment for several decades. We have previously demonstrated that the AR is also an immunological target antigen, recognized in patients with prostate cancer, and targetable by means of vaccines in rodent models with delays in prostate tumor growth. The current study was performed to determine the safety and immunological efficacy of a GMP-grade plasmid DNA vaccine encoding the ligand-binding domain (LBD) of the AR, pTVG-AR. METHODS Groups of male mice (n = 6–10 per group) were evaluated after four or seven immunizations, using different schedules and inclusion of GM-CSF as a vaccine adjuvant. Animals were assessed for toxicity using gross observations, pathological analysis, and analysis of serum chemistries. Animals were analyzed for evidence of vaccine-augmented immunity by tetramer analysis. Survival studies using different immunization schedules and inclusion of GM-CSF were conducted in an autochthonous genetically engineered mouse model. RESULTS No significant toxicities were observed in terms of animal weights, histopathology, hematological changes, or changes in serum chemistries, although there was a trend to lower serum glucose in animals treated with the vaccine. There was specifically no evidence of toxicity in other tissues that express AR, including liver, muscle, hematopoietic, and brain. Vaccination was found to elicit AR LBD-specific CD8+ T cells. In a subsequent study of tumor-bearing animals, animals treated with vaccine had prolonged survival compared with control-immunized mice. CONCLUSIONS These studies demonstrate that, in immunocompetent mice expressing the target antigen, immunization with the pTVG-AR vaccine was both safe and effective in eliciting AR-specific cellular immune responses, and prolonged the survival of prostate tumor-bearing mice. These findings support the clinical evaluation of pTVG-AR in patients with recurrent prostate cancer. Prostate 77:812–821, 2017. © 2017 Wiley Periodicals, Inc.

Sipuleucel-T is an autologous cellular immunotherapy used to treat asymptomatic or minimally symptomatic metastatic castration-resistant prostate cancer (mCRPC). Traditional short-term indicators of clinical response commonly used with chemotherapy have not correlated with survival in patients treated with sipuleucel-T. This retrospective study aimed to evaluate laboratory parameters as possible early biomarkers associated with clinical benefit following sipuleucel-T treatment. Patients treated with sipuleucel-T from three randomized, controlled, phase III clinical trials in mCRPC were considered: IMPACT (NCT00065442; n = 512), D9901 (NCT00005947; n = 127), and D9902A (NCT01133704; n = 98). Patients from these trials were included in this study if their samples were analyzed by the central laboratory and if data were available from baseline and ≥1 posttreatment time point (n = 377). We found that sipuleucel-T treatment was associated with a transient increase in serum eosinophil count at week 6 that resolved by week 14 in 28% of patients (105 of 377). This eosinophil increase correlated with induced immune response, longer prostate cancer–specific survival [HR, 0.713; 95% confidence interval (CI), 0.525–0.970; P = 0.031], and a trend in overall survival (HR, 0.753; 95% CI, 0.563–1.008; P = 0.057). Median serum globulin protein levels also increased transiently, which was associated with antigen-specific antibody responses; however, this finding did not correlate with longer survival. We conclude that transient increases in eosinophils at week 6 may be a useful, objective, short-term indicator of global immune activation and survival benefit with sipuleucel-T in patients with mCRPC. This observation warrants prospective evaluation in future clinical trials. Cancer Immunol Res; 2(10); 988–99. ©2014 AACR.

Background Preclinical studies have demonstrated that a DNA vaccine encoding the androgen receptor ligand-binding domain (AR LBD) can elicit CD8+ T cells specific for the AR LBD, delay disease progression and prolong survival of prostate tumor-bearing animals. Vaccination of tumor-bearing mice treated with androgen deprivation (AD) led to a delay in the emergence of castration-resistant disease. This same DNA vaccine (pTVG-AR, MVI-118) was evaluated in a phase I clinical trial in men with metastatic prostate cancer (mPC) on AD therapy. Methods 40 patients with mPC, within 6 months of beginning standard AD therapy, were randomly assigned to receive pTVG-AR on one of two different treatment schedules, and with or without GM-CSF used as an adjuvant. Ten total vaccinations were given over 1 year, and patients were followed for 18 months. The primary objectives were safety and immune activity. Secondary objectives were to evaluate different schedules, the requirement for GM-CSF, and whether treatment was associated with PSA progression-free survival (PSA PFS). Results Between 2015 and 2017, 40 patients were enrolled at 3 centers. 27 patients completed treatment and 18 months of follow-up. Eleven patients (28%) had a PSA progression event. No grade 3 or 4 adverse events were observed. Samples from 30 patients were available for immune analysis. 14/30 (47%) developed Th1 biased immunity to the AR LBD as determined by IFNγ or granzyme B ELISPOT that was detectable at least twice after beginning treatment. Differences in immune responses were observed with different schedules, but not with GM-CSF adjuvant. Patients who developed immunity had a significantly prolonged PSA PFS compared to patients without immunity (HR = 0.18, 95% CI: 0.04-0.92, p = 0.040). Conclusions This trial showed that pTVG-AR was safe and immunologically active in patients with mPC, and identified a preferred schedule of administration. Association between immunity and PSA PFS suggests treatment may delay the time to castration resistance, similar to observations in preclinical studies. Future studies will evaluate pTVG-AR in earlier stages of disease, and in combination with other agents. Clinical trial identification NCT02411786. Legal entity responsible for the study Douglas McNeel. Funding Madison Vaccines, Inc. Disclosure D.G. McNeel: Advisory / Consultancy, Research grant / Funding (self), Travel / Accommodation / Expenses, Shareholder / Stockholder / Stock options, Licensing / Royalties, Officer / Board of Directors: Madison Vaccines Inc; Research grant / Funding (institution): BMS; Research grant / Funding (institution): Janssen; Research grant / Funding (institution): Novartis; Research grant / Funding (institution): Merck. R. Lesniewski: Leadership role, Travel / Accommodation / Expenses, Shareholder / Stockholder / Stock options, Full / Part-time employment, Officer / Board of Directors: Madison Vaccines Inc. B. Olson: Licensing / Royalties: Madison Vaccines Inc. C. Kyriakopoulos: Research grant / Funding (institution): Madison Vaccines Inc. All other authors have declared no conflicts of interest.

5037 Background: Phase I/II studies of pTVG-HP showed safety and increases in prostatic acid phosphatase (PAP)-specific T cells. Based on these, we conducted a multi-center, randomized phase II trial using pTVG-HP with GM-CSF adjuvant vs GM-CSF alone in patients with PSA-recurrent, non-metastatic, non-castrate, prostate cancer (stage M0). Methods: 99 patients with M0 prostate cancer (negative CT and bone scan) with PSA doubling time (DT) of < 12m were randomly assigned to pTVG-HP + GM-CSF vs GM-CSF. Treatments were every 2 wk x 6, and then quarterly for up to 24m. Primary endpoint was 2-year MFS. Secondary endpoints included median MFS, changes in PSA DT, and immune response to PAP antigen. Additional analysis included quantitative changes in bone lesion activity by NaF PET/CT. Results: Few grade 3/4 events were observed and were not statistically different between arms. 2-year MFS between control and pTVG-HP was 42.3% and 41.8% (p = 0.97). Median MFS was not different (18.3m control versus 18.9m pTVG-HP, HR = 1.6, p = 0.13). Changes in PSA DT, and immunity to the PAP target, were not different between study arms. A pre-planned subset analysis showed median MFS was significantly longer in patients with rapid PSA DT ( < 3m) treated with pTVG-HP (6.1m versus 12.0m, n = 21, HR = 4.4, p = 0.03). In patients with baseline metastases detected on NaF PET/CT, total activity (SUVtotal) increased 17% with GM-CSF alone, but decreased 29% with pTVG-HP from baseline to month 6 (n = 27, p = 0.07). Conclusions: This trial did not demonstrate an overall increase in 2-year MFS following treatment with pTVG-HP, but suggested a modest effect in patients with rapidly progressive disease. NaF PET/CT imaging suggests that pTVG-HP has a detectable effect on decreasing metastatic activity in bone. A separate trial suggests that pTVG-HP is an immune activating agent that improves activity of PD-1 blockade. A trial using pTVG-HP in combination with nivolumab in patients with M0 prostate cancer is currently underway. Clinical trial information: NCT01341652.

e16509 Background: The safety and immunogenicity of JNJ-64041809 (JNJ-809), a live attenuated, double-deleted Listeria monocytogenes (LADD- Lm)-based immunotherapy targeting relevant prostate cancer antigens, was evaluated in a phase 1, multicenter, open-label study in patients (pts) with mCRPC. Methods: Men aged ≥18 years with progressive mCRPC who had received ≥2 prior approved therapies were included. The recommended phase 2 dose (RP2D) and the safety and immunogenicity of JNJ-809 were evaluated. Dose-limiting toxicities (DLTs), adverse events (AEs), T cell response, tumor response, and JNJ-809 bacterial shedding profile were assessed at 2 dose levels, 1 x 108 and 1 x 109 colony forming units (CFU). Results: 26 pts (median age 66.5 years, White 96%) were enrolled (1 x 108 CFU, n = 6; 1 x 109 CFU, n = 20). Median duration of treatment was 2.5 months (range 0-9.7). No DLTs occurred and 1 x 109 CFU was selected as the RP2D. Most common AEs were chills (n = 24, 92%), pyrexia (n = 21, 81%), and fatigue (n = 16, 62%). Grade ≥3 AEs were reported in 18 (69%) pts and serious AEs in 7 (27%) pts including 1 pt with investigator-assessed treatment-related grade 3 intestinal obstruction. At 1 x 108 CFU, all pts had negative blood cultures. At 1 x 109 CFU, 6 pts had LADD- Lm-positive blood cultures on day 1; blood cultures on all subsequent days were negative. JNJ-809 transiently induced peripheral cytokines including interferon-γ, interleukin-10, and tumor necrosis factor-α. Of 7 evaluable pts (1 x 109 CFU), post-treatment T cell responses were demonstrated in 6 pts to the Listeria antigen LLO and in 5 pts to ≥1 of the 4 encoded tumor antigens. Best overall response was stable disease (SD) in 13/25 evaluable pts with SD ≥16 weeks in 4 pts. The study was terminated early as the data collected from 26 pts were considered sufficient to evaluate safety and immune responses. Conclusions: JNJ-809 has a manageable safety profile consistent with that of other LADD- Lm-based therapies. Observed activation of immune responses with monotherapy did not translate into clinical activity, and further development of JNJ-809 will likely require a combination approach. Clinical trial information: NCT02625857.

Objective To assess the effects of timing and schedule of zoledronic acid (ZA) administration on bone mineral density (BMD) in patients beginning androgen deprivation therapy (ADT) for the treatment of recurrent prostate cancer. Patients and Methods In this randomized, 3-arm trial, we evaluated changes in BMD after 3 different ZA administration schedules in men with recurrent prostate cancer who were beginning ADT. Forty-four patients were enrolled and randomized to receive a single dose of ZA given 1 week before beginning ADT (arm 1), a single dose of ZA given 6 months after beginning ADT (arm 2), or monthly administration of ZA starting 6 months after beginning ADT, for a total of 6 doses (arm 3). Results Patients who received ZA before ADT had a significant improvement in BMD at the total proximal femur and trochanter after 6 months compared with the other groups. In addition, only patients in the arm that received multiple doses improved lumbar spine BMD while on ADT, with these findings persisting to 24 months. However, this group also experienced more grade 1 adverse events. Conclusions Analysis of these data suggests that ZA administration before initiation of ADT was superior to treatment 6 months after starting ADT in maintaining BMD. In addition, monthly ZA administration can increase BMD above baseline but is associated with more adverse events. Further study is needed to examine whether the timing and frequency of ZA therapy in patients on ADT can reduce fracture risk.

Prostate cancer is the most commonly diagnosed malignancy and second leading cause of cancer death among men in the United States. In recent years, several new agents, including cancer immunotherapies, have been approved or are currently being investigated in late-stage clinical trials for the management of advanced prostate cancer. Therefore, the Society for Immunotherapy of Cancer (SITC) convened a multidisciplinary panel, including physicians, nurses, and patient advocates, to develop consensus recommendations for the clinical application of immunotherapy for prostate cancer patients. To do so, a systematic literature search was performed to identify high-impact papers from 2006 until 2014 and was further supplemented with literature provided by the panel. Results from the consensus panel voting and discussion as well as the literature review were used to rate supporting evidence and generate recommendations for the use of immunotherapy in prostate cancer patients. Sipuleucel-T, an autologous dendritic cell vaccine, is the first and currently only immunotherapeutic agent approved for the clinical management of metastatic castrate resistant prostate cancer (mCRPC). The consensus panel utilized this model to discuss immunotherapy in the treatment of prostate cancer, issues related to patient selection, monitoring of patients during and post treatment, and sequence/combination with other anti-cancer treatments. Potential immunotherapies emerging from late-stage clinical trials are also discussed. As immunotherapy evolves as a therapeutic option for the treatment of prostate cancer, these recommendations will be updated accordingly. Electronic supplementary material The online version of this article (doi:10.1186/s40425-016-0198-x) contains supplementary material, which is available to authorized users.

Purpose: Nivolumab, an anti-PD-1 immune checkpoint inhibitor, improved overall survival versus everolimus in a phase 3 trial of previously treated patients with metastatic renal cell carcinoma (mRCC). We investigated immunomodulatory activity of nivolumab in a hypothesis-generating prospective mRCC trial. Experimental Design: Nivolumab was administered intravenously every 3 weeks at 0.3, 2, or 10 mg/kg to previously treated patients and 10 mg/kg to treatment-naïve patients with mRCC. Baseline and on-treatment biopsies and blood were obtained. Clinical activity, tumor-associated lymphocytes, PD-L1 expression (Dako immunohistochemistry; ≥5% vs. Results: In 91 treated patients, median overall survival [95% confidence interval (CI)] was 16.4 months [10.1 to not reached (NR)] for nivolumab 0.3 mg/kg, NR for 2 mg/kg, 25.2 months (12.0 to NR) for 10 mg/kg, and NR for treatment-naïve patients. Median percent change from baseline in tumor-associated lymphocytes was 69% (CD3+), 180% (CD4+), and 117% (CD8+). Of 56 baseline biopsies, 32% had ≥5% PD-L1 expression, and there was no consistent change from baseline to on-treatment biopsies. Transcriptional changes in tumors on treatment included upregulation of IFNγ-stimulated genes (e.g., CXCL9). Median increases in chemokine levels from baseline to C2D8 were 101% (CXCL9) and 37% (CXCL10) in peripheral blood. No new safety signals were identified. Conclusions: Immunomodulatory effects of PD-1 inhibition were demonstrated through multiple lines of evidence across nivolumab doses. Biomarker changes from baseline reflect nivolumab pharmacodynamics in the tumor microenvironment. These data may inform potential combinations. Clin Cancer Res; 22(22); 5461–71. ©2016 AACR.

O1 IL-15 primes an mTOR-regulated gene-expression program to prolong anti-tumor capacity of human natural killer cells #### Andreas Lundqvist1, Vincent van Hoef1, Xiaonan Zhang1, Erik Wennerberg2, Julie Lorent1, Kristina Witt1, Laia Masvidal Sanz1, Shuo Liang1, Shannon Murray3, Ola Larsson1

// Jordan E. Bloom 1 and Douglas G. McNeel 1,2 1 Department of Medicine, University of Wisconsin, Madison, WI, USA 2 University of Wisconsin Carbone Cancer Center, University of Wisconsin, Madison, WI, USA Correspondence to: Douglas G. McNeel, email: // Keywords : SSX, prostate cancer, CTA, focal adhesion, EMT Received : May 17, 2016 Accepted : May 26, 2016 Published : June 02, 2016 Abstract Prostate cancer is the most commonly diagnosed malignancy for men in the United States. Metastatic prostate cancer, the lethal form of the disease, has a life expectancy of approximately five years. Identification of factors associated with this transition to metastatic disease is crucial for future therapies. One such factor is the SSX gene family, a family of cancer/testis antigens (CTA) transcription factors which have been shown to be aberrantly expressed in other cancers and associated with the epithelial to mesenchymal transition (EMT). We have previously shown that SSX expression in prostate cancers was restricted to metastatic tissue and not primary tumors. In this study, we have identified SSX2 as the predominant SSX family member expressed in prostate cancer, and found its expression in the peripheral blood of 19 of 54 (35%) prostate cancer patients, with expression restricted to circulating tumor cells, and in 7 of 15 (47%) metastatic cDNA samples. Further, we examined SSX2 function in prostate cancer through knockdown and overexpression in prostate cancer cell lines. While overexpression had little effect on morphology or gene transcript changes, knockdown of SSX2 resulted in an epithelial morphology, increased cell proliferation, increased expression of genes involved in focal adhesion, decreased anchorage independent growth, increased invasion, and increased tumorigenicity in vivo . We conclude from these findings that SSX2 expression in prostate cancer is not a driver of EMT, but is involved in processes associated with EMT including loss of focal adhesion that may be related to tumor cell dissemination.

We have previously reported that tumor antigen-specific DNA vaccination in mice led to an increase in IFNγ-secreting T cells and an increase in tumor expression of PD-L1. Further, we demonstrated that increasing the encoded antigen's MHC-binding affinity led to increased PD-1 expression on antigen-specific CD8(+) T cells. Together these phenomena provided resistance to antitumor immunization that was abrogated with PD-1/PD-L1 blockade. We consequently sought to determine whether similar regulation occurred in human patients following antitumor immunization. Using clinical samples from prostate cancer patients who were previously immunized with a DNA vaccine, we analyzed changes in checkpoint receptor expression on antigen-specific CD8(+) T cells, the effect of PD-1 blockade on elicited immune responses, and for changes in checkpoint ligand expression on patients' circulating tumor cells (CTCs). We observed no significant changes in T-cell expression of PD-1 or other checkpoint receptors, but antigen-specific immune responses were detected and/or augmented with PD-1 blockade as detected by IFNγ and granzyme B secretion or trans vivo DTH testing. Moreover, PD-L1 expression was increased on CTCs following vaccination, and this PD-L1 upregulation was associated with the development of sustained T-cell immunity and longer progression-free survival. Finally, similar results were observed with patients treated with sipuleucel-T, another vaccine targeting the same prostate antigen. These findings provide in-human rationale for combining anticancer vaccines with PD-1 blocking antibodies, particularly for the treatment of prostate cancer, a disease for which vaccines have demonstrated benefit and yet PD-1 inhibitors have shown little clinical benefit to date as monotherapies.

Delivery of plasmid DNA encoding an antigen of interest has been demonstrated to be an effective means of immunization, capable of eliciting antigen-specific T cells. Plasmid DNA vaccines offer advantages over other anti-tumor vaccine approaches in terms of simplicity, manufacturing, and possibly safety. The primary disadvantage is their poor transfection efficiency and subsequent lower immunogenicity relative to other genetic vaccine approaches. However, multiple preclinical models demonstrate anti-tumor efficacy, and many efforts are underway to improve the immunogenicity and anti-tumor effect of these vaccines. Clinical trials using DNA vaccines as treatments for prostate cancer have begun, and to date have demonstrated safety and immunological effect. This review will focus on DNA vaccines as a specific means of antigen delivery, advantages and disadvantages of this type of immunization, previous experience in preclinical models and human trials specifically conducted for the treatment of prostate cancer, and future directions for the application of DNA vaccines to prostate cancer immunotherapy.

CTLA-4 blockade has demonstrated antitumor efficacy in human clinical trials. The antitumor mechanism is presumably mediated in part by the expansion of tumor-specific T cells. Androgen deprivation, the cornerstone of treatment for patients with metastatic prostate cancer, has been shown to elicit prostate tissue apoptosis and lymphocytic inflammation. We hypothesized that treatment with androgen deprivation, followed by an anti-CTLA-4 antibody, could augment a tumor-specific immune response elicited by androgen deprivation. We report here the results of a phase I trial evaluating a humanized monoclonal antibody targeting CTLA-4, CP-675,206 (tremelimumab), in combination with androgen deprivation using an antiandrogen. Eligible patients were those with PSA-recurrent prostate cancer after primary surgery and/or radiation therapy, not previously treated with androgen deprivation, and without radiographic evidence of metastatic disease. Subjects were treated in two cycles, 3 months apart, in which they received bicalutamide 150 mg daily days 1–28 and tremelimumab on day 29. The primary endpoint of the trial was safety. Secondary endpoints included measures of PSA kinetics and identification of a maximum tolerated dose. Eleven patients were enrolled and completed at least 1 year of follow-up. Dose-limiting toxicities included grade 3 diarrhea and skin rash. No favorable changes in PSA doubling time were observed in a period shortly after completing treatment; however, three patients experienced a prolongation in PSA doubling time detectable several months after completing treatment. The identification of delayed, prolonged favorable changes in serum PSA suggests that future studies could explore this combination in studies evaluating time to disease progression.

BACKGROUND T cells infiltrate the prostates of prostate cancer patients undergoing neoadjuvant androgen deprivation. These prostate-infiltrating T cells have an oligoclonal phenotype, suggesting the development of an antigen-specific T-cell response. We hypothesized that androgen deprivation might elicit a prostate tissue-specific T-cell response that could potentially be combined with other immune-active therapies, and consequently sought to investigate the nature and timing of this T-cell response following castration.

BACKGROUND Prostatic acid phosphatase (PAP) is a prostate cancer tumor antigen and is an immunological target in several active immunotherapy clinical trials for the treatment of prostate cancer. We and others have demonstrated that PAP-specific T-cell responses can be elicited and augmented following antigen-specific immunization in both humans and animal models. We have previously reported that prostate cancer patients immunized with a DNA vaccine encoding PAP (pTVG-HP) developed both CD4+ and CD8+ T-cell responses. PAP-specific, CD4+ T-cell proliferative responses were generated in three out of four HLA-DRB1*0101 patients suggesting the possibility that DR1-restricted epitopes exist. METHODS To identify PAP-specific HLA-DRB1*0101 restricted epitopes, we immunized HLA-A2.01/HLA-DRB1*0101 (A2/DR1) transgenic mice with the pTVG-HP DNA vaccine. To map DRB1*0101-restricted epitopes, splenocytes from immunized mice were screened against a library of overlapping 15-residue, PAP-derived peptides using an IFNγ ELISPOT assay. RESULTS We identified four HLA-DRB1*0101 epitopes for PAP in A2/DR1 mice (PAP161–175, PAP181–195, PAP191–205, and PAP 351–365). T cells specific for one epitope (PAP181–195) were found to be augmented after immunization in a HLA-DRB1*0101+ prostate cancer patient. CONCLUSIONS The identification of MHC class II epitopes may provide tools to directly monitor immune responses after vaccination and may be important for the design of future prostate cancer vaccines. Prostate 72:730–740, 2012. © 2011 Wiley Periodicals, Inc.

BACKGROUND We retrospectively explored changes in immunological parameters in men with biochemically recurrent prostate cancer treated with either 5 or 25 mg of lenalidomide in a randomized phase 2 trial, and determined whether those changes correlated with disease progression. METHODS Cytokine levels were compared for each patient at baseline and after 6 months of treatment with lenalidomide. Regression models for correlated data were used to assess associations of cytokine levels with lenalidomide treatment effect. Estimates were obtained using generalized estimating equations. Changes in circulating anti-prostate antibodies were evaluated using a high-throughput immunoblot technique. RESULTS Treatment with lenalidomide was associated with global changes in immunoreactivity to a number of prostate-associated antigens, as well as with changes in circulating levels of the TH2 cytokines IL-4, IL-5, IL-10, and IL-13. Disease progression in treated patients was associated with an increase in circulating IL-8 levels, while IL-8 levels decreased significantly in non-progressors. CONCLUSIONS Lenalidomide demonstrates immunomodulatory properties in patients with biochemically recurrent prostate cancer. The induction of novel anti-prostate antibodies is a potential mechanism for lenalidomide response. Changes in serum IL-8 levels may serve as a potential biomarker in treated patients. These hypotheses require formal testing in future prospective trials. Prostate 72:487–498, 2012. © 2011 Wiley Periodicals, Inc.

Prostate cancer continues to be one of the most serious afflictions of men of advanced age, remaining the most commonly diagnosed and second leading cause of cancer-related deaths in American men. The treatment options for patients with incurable metastatic, castrate-resistant disease have long focused on various chemotherapeutic approaches, which provide a slight survival benefit while being associated with potentially significant side effects. However, the recent approval of sipuleucel-T has given patients with advanced disease an additional treatment option that has demonstrated benefit without the side effects associated with chemotherapy. Sipuleucel-T is an antigen-presenting cell-based active immunotherapy that utilizes a patient's own immune cells, presumably to activate an antigen-specific immune response against tumor cells. This review focuses on the development and implementation of sipuleucel-T as a therapy for prostate cancer. Specifically, we present some of the issues associated with the management of advanced prostate cancer, the research and development that led to the approval of sipuleucel-T, how the approval of sipuleucel-T could change the clinical management of prostate cancer, and current and future areas of investigation that are being pursued with regard to sipuleucel-T and other treatments for advanced prostate cancer.

The androgen receptor (AR) is a hormone receptor that plays a critical role in prostate cancer, and depletion of its ligand has long been the cornerstone of treatment for metastatic disease. Here, we evaluate the AR ligand-binding domain (LBD) as an immunological target, seeking to identify HLA-A2-restricted epitopes recognized by T cells in prostate cancer patients. Ten AR LBD-derived, HLA-A2-binding peptides were identified and ranked with respect to HLA-A2 affinity and were used to culture peptide-specific T cells from HLA-A2+ prostate cancer patients. These T-cell cultures identified peptide-specific T cells specific for all ten peptides in at least one patient, and T cells specific for peptides AR805 and AR811 were detected in over half of patients. Peptide-specific CD8+ T-cell clones were then isolated and characterized for prostate cancer cytotoxicity and cytokine expression, identifying that AR805 and AR811 CD8+ T-cell clones could lyse prostate cancer cells in an HLA-A2-restricted fashion, but only AR811 CTL had polyfunctional cytokine expression. Epitopes were confirmed using immunization studies in HLA-A2 transgenic mice, in which the AR LBD is an autologous antigen with an identical protein sequence, which showed that mice immunized with AR811 developed peptide-specific CTL that lyse HLA-A2+ prostate cancer cells. These data show that AR805 and AR811 are HLA-A2-restricted epitopes for which CTL can be commonly detected in prostate cancer patients. Moreover, CTL responses specific for AR811 can be elicited by direct immunization of A2/DR1 mice. These findings suggest that it may be possible to elicit an anti-prostate tumor immune response by augmenting CTL populations using AR LBD-based vaccines.