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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.

Flow cytometry gating strategies for data presented in Figures 1-6. Supplementary Figure S1. Representative dot plots and gating strategy for Figure 1. Supplementary Figure S2. Representative dot plots and gating strategy for Figure 2.

Representative video of OT-1 T cells interacting with SIINFEKL (OVA) loaded APC.

Supplementary Figures and Legends

Supplementary Table S1. Baseline demographics and disease characteristics in patients with mCRPC from the phase III sipuleucel-T trials included in the eosinophil analysis and the overall pooled population. Supplementary Figure S1. Overview of the study designs of three phase III clinical trials from which data were pooled for this retrospective analysis. Supplementary Figure S2. Changes in eosinophil counts for sipuleucel-T-treated patients with or without an elevated eosinophil count in randomized phase III trials, versus the control group. Supplementary Figure S3. Association of immune response with change in eosinophil counts for sipuleucel-T-treated patients in randomized phase III trials.

Antitumor vaccines encoding self-antigens generally have low immunogenicity in clinical trials. Several approaches are aimed at improving vaccine immunogenicity, including efforts to alter encoded epitopes. Immunization with epitopes altered for increased affinity for the major histocompatibility complex (MHC) or T-cell receptor (TCR) elicits greater numbers of CD8 T cells but inferior antitumor responses. Our previous results suggested that programmed death 1 (PD-1) and its ligand (PD-L1) increased on antigen-specific CD8 T cells and tumor cells, respectively, after high-affinity vaccination. In this report, we use two murine models to investigate whether the dose, MHC affinity, or TCR affinity of an epitope affected the antitumor response via the PD-1/PD-L1 axis. T cells activated with high-affinity epitopes resulted in prolonged APC:T-cell contact time that led to elevated, persistent PD-1 expression, and expression of other checkpoint molecules, in vitro and in vivo. Immunization with high-affinity epitopes also decreased antitumor efficacy in the absence of PD-1 blockade. Thus, APC:T-cell contact time can be altered by epitope affinity and lead to therapeutically relevant changes in vaccine efficacy mediated by changes in PD-1 expression. These findings have implications for the use of agents targeting PD-1 expression or function whenever high-affinity CD8 T cells are elicited or supplied by means of vaccination or adoptive transfer. Cancer Immunol Res; 5(8); 630–41. ©2017 AACR.

Expression of T-cell checkpoint receptors can compromise antitumor immunity. Blockade of these receptors, notably PD-1 and LAG-3, which become expressed during T-cell activation with vaccination, can improve antitumor immunity. We evaluated whether T-cell checkpoint expression could be separated from T-cell activation in the context of innate immune stimulation with TLR agonists. We found that ligands for TLR1/2, TLR7, and TLR9 led to a decrease in expression of PD-1 on antigen-activated CD8+ T cells. These effects were mediated by IL12 released by professional antigen-presenting cells. In two separate tumor models, treatment with antitumor vaccines combined with TLR1/2 or TLR7 ligands induced antigen-specific CD8+ T cells with lower PD-1 expression and improved antitumor immunity. These findings highlight the role of innate immune activation during effector T-cell development and suggest that at least one mechanism by which specific TLR agonists can be strategically used as vaccine adjuvants is by modulating the expression of PD-1 during CD8+ T-cell activation. Cancer Immunol Res; 6(11); 1364–74. ©2018 AACR.

Androgen deprivation is the primary therapy for recurrent prostate cancer, and agents targeting the androgen receptor (AR) pathway continue to be developed. Because androgen-deprivation therapy (ADT) has immmunostimulatory effects as well as direct antitumor effects, AR-targeted therapies have been combined with other anticancer therapies, including immunotherapies. Here, we sought to study whether an antigen-specific mechanism of resistance to ADT (overexpression of the AR) may result in enhanced AR-specific T-cell immune recognition, and whether this might be strategically combined with an antitumor vaccine targeting the AR. Androgen deprivation increased AR expression in human and murine prostate tumor cells in vitro and in vivo. The increased expression persisted over time. Increased AR expression was associated with recognition and cytolytic activity by AR-specific T cells. Furthermore, ADT combined with vaccination, specifically a DNA vaccine encoding the ligand-binding domain of the AR, led to improved antitumor responses as measured by tumor volumes and delays in the emergence of castrate-resistant prostate tumors in two murine prostate cancer models (Myc-CaP and prostate-specific PTEN-deficient mice). Together, these data suggest that ADT combined with AR-directed immunotherapy targets a major mechanism of resistance, overexpression of the AR. This combination may be more effective than ADT combined with other immunotherapeutic approaches. Cancer Immunol Res; 5(12); 1074–85. ©2017 AACR.

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.

S1. Phenotypic validation of HLA-A2-transfected 22Rv1/FCS and 22Rv1/CSS cells. S2. AR-specific T cells obtained following peptide-immunization have increased recognition and lysis of androgen-deprived prostate tumor cells. S3. Immunization with pTVG-AR delays tumor growth, in a CD8-dependent fashion, in the presence or absence of ADT. S4. Immunization with pTVG-AR delays tumor growth in the presence or absence of ADT, and results in increased tumor-infiltrating T cells.

Supplementary Figure 2: T-cell immunological response to tetanus toxoid over time: Panel A: IFNγ ELISPOT data, similar to that as shown in Figure 3, were plotted over time, subtracting the pre-treatment value, to evaluate the magnitude and durability of immunity over time. Panel B: Immune response over time was plotted as "area under the curve," and evaluated with respect to treatment arm. Lines show median and interquartile range.

59 probesets from an analysis of blood These are probesets for which the null hypothesis was rejected and the change over time averaged over treatment groups was {greater than or equal to}1.2-fold.

CONSORT diagram.

Supplementary Information

PDF file - 26KB

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

Supplementary Data from Treatment of Chronic Lymphocytic Leukemia with a Hypomethylating Agent Induces Expression of NXF2, an Immunogenic Cancer Testis Antigen

Supplemental Figure 1: T-cell immunological response to PSA over time: Panel A: IFNγ ELISPOT data, similar to that as shown in Figure 3, were plotted over time, subtracting the pre-treatment value, to evaluate the magnitude and durability of immunity over time. Panel B: Immune response over time was plotted as "area under the curve," and evaluated with respect to treatment arm. Lines show median and interquartile range.

Purpose: Critical to the success of active immunotherapy against cancer is the identification of immunologically recognized cancer-specific proteins with low tolerogenic potential. Cancer testis antigens (CTA), in particular, fulfill this requirement as a result of their aberrant expression restricted to cancer cells and lack of expression in normal tissues bypassing tolerogenic mechanisms against self. Although CTAs have been extensively studied in solid malignancies, little is known regarding their expression in chronic lymphocytic leukemia (CLL).Experimental Design: Using a two-pronged approach we evaluated the immunogenicity of 29 CTAs in 22 patients with CLL and correlated these results to reverse transcriptase PCR data from CLL cell lines and patient cells.Results: We identified IgG-specific antibodies for one antigen, NXF2, and confirmed this response by ELISA and Western blot. We found that treatment of CLL with 5-aza-2′-deoxycytidine can induce expression of NXF2 that lasted for several weeks after treatment. Treatment also increased levels of MHC and costimulatory molecules (CD80, CD86, and CD40) necessary for antigen presentation. In addition, we identified other promising antigens that may have potential immunotherapeutic application.Conclusions: Our findings suggest that NXF2 could be further pursued as an immunotherapeutic target in CLL, and that treatment with demethylating agents could be exploited to specifically modulate CTA expression and effective antigen presentation in malignant B cells.

Supplementary Figure 3: IgG immunological response: IgG responses to AR LBD protein (panel A), PSA protein (panel B), and tetanus toxoid (panel C) were evaluated by Luminex. Sera samples were assessed at a 1:100 dilution from the time points indicated. Data were plotted over time, subtracting the pre-treatment value to evaluate change in post-treatment specimens.

PDF file - 729KB, Supplementary Table 1: Adverse events. Supplementary Figure 1: Patient allocation. Supplementary Figure 2: DNA immunization elicits PAP-specific cellular immune responses. Supplementary Figure 3: Immune responses to PSA and tetanus evaluated by intracellular cytokine staining. Supplementary Figure 4: Immune responses to PAP, PSA and tetanus evaluated by cytokine secretion.

Purpose: We have previously reported that a DNA vaccine encoding prostatic acid phosphatase (PAP) could elicit PAP-specific T cells in patients with early recurrent prostate cancer. In the current pilot trial, we sought to evaluate whether prolonged immunization with regular booster immunizations, or “personalized” schedules of immunization determined using real-time immune monitoring, could elicit persistent, antigen-specific T cells, and whether treatment was associated with changes in PSA doubling time (PSA DT).Experimental Design: Sixteen patients with castration-resistant, nonmetastatic prostate cancer received six immunizations at 2-week intervals and then either quarterly (arm 1) or as determined by multiparameter immune monitoring (arm 2).Results: Patients were on study a median of 16 months; four received 24 vaccinations. Only one event associated with treatment >grade 2 was observed. Six of 16 (38%) remained metastasis-free at 2 years. PAP-specific T cells were elicited in 12 of 16 (75%), predominantly of a Th1 phenotype, which persisted in frequency and phenotype for at least 1 year. IFNγ-secreting T-cell responses measured by ELISPOT were detectable in 5 of 13 individuals at 1 year, and this was not statistically different between study arms. The overall median fold change in PSA DT from pretreatment to posttreatment was 1.6 (range, 0.6–7.0; P = 0.036).Conclusions: Repetitive immunization with a plasmid DNA vaccine was safe and elicited Th1-biased antigen-specific T cells that persisted over time. Modifications in the immunization schedule based on real-time immune monitoring did not increase the frequency of patients developing effector and memory T-cell responses with this DNA vaccine. Clin Cancer Res; 20(14); 3692–704. ©2014 AACR.

Supplementary figure legends

PDF file - 114K, Additional prostate cancer biopsy cores from microarray showing punctate SSX staining Panel A: Benign core stained with 1A4 mAb, B01P pAb, or IgG isotype control showing punctate B01P cytoplasmic staining. Panel B: Metastatic tissue core showing punctate B01P (nuclear staining).

PDF file - 160K, Sequence alignment and primer design for SSX1-10 SSX1-10 mRNA sequence alignment with the location of primers used in these studies in red and published primers not used in these experiments in green. Start and stop codons are shown in bold lettering.

Recent U.S. Food and Drug Administration approval of the first immunotherapy for prostate cancer encourages efforts to improve immune targeting of this disease. The synovial sarcoma X chromosome breakpoint (SSX) proteins comprise a set of cancer–testis antigens that are upregulated in MHC class I–deficient germline cells and in various types of advanced cancers with a poor prognosis. Humoral and cell-mediated immune responses to the SSX family member SSX2 can arise spontaneously in prostate cancer patients. Thus, SSX2 and other proteins of the SSX family may offer useful targets for tumor immunotherapy. In this study, we evaluated the expression of SSX family members in prostate cancer cell lines and tumor biopsies to identify which members might be most appropriate for immune targeting. We found that SSX2 was expressed most frequently in prostate cell lines, but that SSX1 and SSX5 were also expressed after treatment with the DNA demethylating agent 5-aza-2′-deoxycytidine. Immunohistochemical analysis of microarrayed tissue biopsies confirmed a differential level of SSX protein expression in human prostate cancers. Notably, SSX expression in patient tumor samples was restricted to metastatic lesions (5/22; 23%) and no expression was detected in primary prostate tumors examined (0/73; P < 0.001). We determined that cross-reactive immune responses to a dominant HLA-A2–specific SSX epitope (p103-111) could be elicited by immunization of A2/DR1 transgenic mice with SSX vaccines. Our findings suggest that multiple SSX family members are expressed in metastatic prostate cancers which are amenable to simultaneous targeting. Cancer Res; 71(21); 6785–95. ©2011 AACR.

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.

An immunosuppressive tumor microenvironment has hampered the efficacy of immunotherapy in prostate cancer. However, radiation-induced immunological effects can partly mediate anti-tumor effects by promoting a pro-inflammatory environment potentially responsive to immunotherapy. Herein, we examined the immunomodulatory properties of a radiopharmaceutical therapy (RPT) with NM600 radiolabeled with either a beta or alpha emitter in two prostate cancer models. 225Ac-NM600, but not 177Lu-NM600, promoted significant anti-tumor effects and improved overall survival. Immunomodulatory effects were dose, radionuclide, and tumor type-dependent. 225Ac-NM600 elicited an array of immunomodulatory effects such as increased CD8/Treg ratio, activation of effector and memory T cells, abrogation of infiltrating suppressor cells (e.g., Tregs and MDSCs), and increased levels of Th1 cytokine and pro-inflammatory chemokines. Importantly, we demonstrate the need to carefully characterize the immune responses elicited by RPT both pre-clinically and clinically to maximize tumor control and avoid potential counterproductive immunosuppressive effects.TeaserTargeted alpha therapy can create a pro-inflammatory tumor micro-environment that partly explains stronger anti-tumor responses in prostate cancer

Most shared resource flow cytometry facilities do not permit analysis of radioactive samples. We are investigating low-dose molecular targeted radionuclide therapy (MTRT) as an immunomodulator in combination with in situ tumor vaccines and need to analyze radioactive samples from MTRT-treated mice using flow cytometry. Further, the sudden shutdown of core facilities in response to the COVID-19 pandemic has created an unprecedented work stoppage. In these and other research settings, a robust and reliable means of cryopreservation of immune samples is required. We evaluated different fixation and cryopreservation protocols of disaggregated tumor cells with the aim of identifying a protocol for subsequent flow cytometry of the thawed sample, which most accurately reflects the flow cytometric analysis of the tumor immune microenvironment of a freshly disaggregated and analyzed sample. Cohorts of C57BL/6 mice bearing B78 melanoma tumors were evaluated using dual lymphoid and myeloid immunophenotyping panels involving fixation and cryopreservation at three distinct points during the workflow. Results demonstrate that freezing samples after all staining and fixation are completed most accurately matches the results from noncryopreserved equivalent samples. We observed that cryopreservation of living, unfixed cells introduces a nonuniform alteration to PD1 expression. We confirm the utility of our cryopreservation protocol by comparing tumors treated with in situ tumor vaccines, analyzing both fresh and cryopreserved tumor samples with similar results. Last, we use this cryopreservation protocol with radioactive specimens to demonstrate potentially beneficial effector cell changes to the tumor immune microenvironment following administration of a novel MTRT in a dose- and time-dependent manner.

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.

Systemic 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.C57BL/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-doseOur data suggest that the combination of

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