Niraparib with Abiraterone Acetate and Prednisone for Metastatic Castration-Resistant Prostate Cancer: Phase II QUEST Study Results.

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.

Optimizing Flow Cytometric Analysis of Immune Cells in Samples Requiring Cryopreservation from Tumor-Bearing Mice.

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.

GM-CSF elicits antibodies to tumor-associated proteins when used as a prostate cancer vaccine adjuvant.

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.

Increased indoleamine 2,3-dioxygenase activity and expression in prostate cancer following targeted immunotherapy.

BACKGROUND: We previously found that PD-L1 expression is increased on tumor cells following vaccination treatments that lead to increased tumor-specific T cells that secrete IFNγ. Indoleamine 2,3-dioxygenase (IDO) is another IFNγ inducible gene that has potent immunosuppressive effects. There have been reports of IDO expression in prostate cancer; however, it is unknown whether IDO expression might similarly increase in prostate tumors following T-cell-based immunotherapy. METHODS: Blood samples from normal male blood donors (n = 12) and patients with different stages of prostate cancer (n = 89), including patients with metastatic, castration-resistant prostate cancer treated with a DNA vaccine and/or pembrolizumab, were evaluated for IDO activity by kynurenine and tryptophan levels. Metastatic tissue biopsies obtained pre- and post-treatments were evaluated for IDO expression. IDO suppression of vaccine-induced T-cell function was assessed by ELISPOT. RESULTS: Overall, IDO activity was increased in patients with more advanced prostate cancer. This activity, and IDO expression as detected immunohistochemically, increased following treatment with either a DNA vaccine encoding the prostatic acid phosphatase (PAP) tumor antigen or PD-1 blockade with pembrolizumab. Increased IDO activity after treatment was associated with the absence of clinical effect, as assessed by lack of PSA decline following treatment. Increased antigen-specific T-cell response, as measured by IFNγ release, to the vaccine target antigen was detected following in vitro stimulation of peripheral blood cells with 1-methyltryptophan. CONCLUSIONS: These findings suggest that IDO expression is a mechanism of immune evasion used by prostate cancer and that future clinical trials using T-cell-based immune strategies might best include IDO inhibition.

Noninvasive Imaging and Quantification of Radiotherapy-Induced PD-L1 Upregulation with 89Zr-Df-Atezolizumab.

Immune checkpoint expression is highly dynamic, and combination treatments including radiotherapy can particularly modulate this expression. PET imaging using 89Zr-Df-atezolizumab can provide insight into the levels of PD-L1 variation following radiotherapy treatments. In vitro screening was used to monitor PD-L1 expression by lung cancer cells following radiotherapy. Mice bearing PD-L1+ (H460) or PD-L1- (A549) tumors were subjected to various external beam radiotherapy regimens and then imaged using 89Zr-Df-atezolizumab PET. ROI analysis and ex vivo biodistribution studies were employed to quantify tracer accumulations. H460 cells were found to have PD-L1 expression at baseline, and this expression increased following daily radiotherapy of 5 fractions of 2 Gy. PD-L1 expression could not be induced on A549 cells, regardless of radiotherapy regimen. The increase in PD-L1 expression in H460 tumors following fractionated radiotherapy could be imaged in vivo using 89Zr-Df-atezolizumab, with statistically significant higher tracer accumulation noted in fractionated H460 tumors over that in all other H460 or A549 groups after 72 h postinjection of the tracer. Significant accumulation of the tracer was also noted in other PD-L1+ organs, including the spleen and lymph nodes. Ex vivo staining of tumor tissues verified that tumor cells as well as tumor-infiltrating immune cells were responsible for increased PD-L1 expression after radiotherapy in tumor tissues. Overall, PD-L1 expression can be modulated with radiotherapy interventions, and 89Zr-Df-atezolizumab is able to noninvasively monitor these changes in preclinical models.

89Zr-labeled nivolumab for imaging of T-cell infiltration in a humanized murine model of lung cancer.

PURPOSE: Nivolumab is a human monoclonal antibody specific for programmed cell death-1 (PD-1), a negative regulator of T-cell activation and response. Acting as an immune checkpoint inhibitor, nivolumab binds to PD-1 expressed on the surface of many immune cells and prevents ligation by its natural ligands. Nivolumab is only effective in a subset of patients, and there is limited evidence supporting its use for diagnostic, monitoring, or stratification purposes. METHODS: 89Zr-Df-nivolumab was synthesized to map the biodistribution of PD-1-expressing tumor infiltrating T-cells in vivo using a humanized murine model of lung cancer. The tracer was developed by radiolabeling the antibody with the positron emitter zirconium-89 (89Zr). Imaging results were validated by ex vivo biodistribution studies, and PD-1 expression was validated by immunohistochemistry. Data obtained from PET imaging were used to determine human dosimetry estimations. RESULTS: The tracer showed elevated binding to stimulated PD-1 expressing T-cells in vitro and in vivo. PET imaging of 89Zr-Df-nivolumab allowed for clear delineation of subcutaneous tumors through targeting of localized activated T-cells expressing PD-1 in the tumors and salivary glands of humanized A549 tumor-bearing mice. In addition to tumor uptake, salivary and lacrimal gland infiltration of T-cells was noticeably visible and confirmed via histological analysis. CONCLUSIONS: These data support our claim that PD-1-targeted agents allow for tumor imaging in vivo, which may assist in the design and development of new immunotherapies. In the future, noninvasive imaging of immunotherapy biomarkers may assist in disease diagnostics, disease monitoring, and patient stratification.

Safety and Immunological Efficacy of a DNA Vaccine Encoding the Androgen Receptor Ligand-Binding Domain (AR-LBD).

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.

ImmunoPET Imaging of CTLA-4 Expression in Mouse Models of Non-small Cell Lung Cancer.

Cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) is expressed on the surface of activated T cells and some tumor cells, and is the target of the clinically approved monoclonal antibody ipilimumab. In this study, we investigate specific binding of radiolabeled ipilimumab to CTLA-4 expressed by human non-small cell lung cancer cells in vivo using positron emission tomography (PET). Ipilimumab was radiolabeled with 64Cu (t1/2 = 12.7 h) through the use of the chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) to formulate 64Cu-DOTA-ipilimumab. CTLA-4 expression in three non-small cell lung cancer (NSCLC) cell lines (A549, H460, and H358) was verified and quantified by Western blot and enzyme-linked immunosorbent assays (ELISA). A receptor binding assay was utilized to monitor the binding and internalization of 64Cu-DOTA-ipilimumab in the NSCLC cell lines. Next, the biodistribution of 64Cu-DOTA-ipilimumab was mapped by longitudinal PET imaging up to 48 h after injection. Ex vivo biodistribution and histological studies were employed to verify PET results. By in vitro analysis, CTLA-4 was found to be expressed on all three NSCLC cell lines with A549 and H358 showing the highest and lowest level of expression, respectively. PET imaging and quantification verified these findings as the tracer accumulated highest in the A549 tumor model (9.80 ± 0.22%ID/g at 48 h after injection; n = 4), followed by H460 and H358 tumors with uptakes of 9.37 ± 0.26%ID/g and 7.43 ± 0.05%ID/g, respectively (n = 4). The specificity of the tracer was verified by injecting excess ipilimumab in A549 tumor-bearing mice, which decreased tracer uptake to 6.90 ± 0.51%ID/g at 48 after injection (n = 4). Ex vivo analysis following the last imaging session also corroborated these findings. 64Cu-DOTA-ipilimumab showed enhanced and persistent accumulation in CTLA-4-expressing tissues, which will enable researchers further insight into CTLA-4 targeted therapies in the future.

Immunotherapy for prostate cancer: False promises or true hope?

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.

Human prostate tumor antigen-specific CD8+ regulatory T cells are inhibited by CTLA-4 or IL-35 blockade.

Regulatory T cells play important roles in cancer development and progression by limiting the generation of innate and adaptive anti-tumor immunity. We hypothesized that in addition to natural CD4(+)CD25(+) regulatory T cells (Tregs) and myeloid-derived suppressor cells, tumor Ag-specific Tregs interfere with the detection of anti-tumor immunity after immunotherapy. Using samples from prostate cancer patients immunized with a DNA vaccine encoding prostatic acid phosphatase (PAP) and a trans-vivo delayed-type hypersensitivity (tvDTH) assay, we found that the detection of PAP-specific effector responses after immunization was prevented by the activity of PAP-specific regulatory cells. These regulatory cells were CD8(+)CTLA-4(+), and their suppression was relieved by blockade of CTLA-4, but not IL-10 or TGF-ß. Moreover, Ag-specific CD8(+) Tregs were detected prior to immunization in the absence of PAP-specific effector responses. These PAP-specific CD8(+)CTLA-4(+) suppressor T cells expressed IL-35, which was decreased after blockade of CTLA-4, and inhibition of either CTLA-4 or IL-35 reversed PAP-specific suppression of tvDTH response. PAP-specific CD8(+)CTLA-4(+) T cells also suppressed T cell proliferation in an IL-35-dependent, contact-independent fashion. Taken together, these findings suggest a novel population of CD8(+)CTLA-4(+) IL-35-secreting tumor Ag-specific Tregs arise spontaneously in some prostate cancer patients, persist during immunization, and can prevent the detection of Ag-specific effector responses by an IL-35-dependent mechanism.

A negative selection methodology using a microfluidic platform for the isolation and enumeration of circulating tumor cells.

Circulating tumor cells (CTCs) exist in the peripheral blood stream of metastatic cancer patients at rates of approximately 1 CTC per billion background cells. In order to capture and analyze this rare cell population, various techniques exist that range from antibody-based surface marker positive selection to methods that use physical properties of CTCs to negatively exclude background cells from a CTC population. However, methods to capture cells for functional downstream analyses are limited due to inaccessibility of the captured sample or labeling techniques that may be prohibitive to cell function. Here, we present a negative selection method that leverages a Microfluidic Cell Concentrator (MCC) to allow collection and analysis of this rare cell population without needing cell adhesion or other labeling techniques to keep the cells within the chamber. Because the MCC is designed to allow collection and analysis of non-adherent cell populations, multiple staining steps can be applied in parallel to a given CTC population without losing any of the population. The ability of the MCC for patient sample processing of CTCs for enumeration was demonstrated with five patient samples, revealing an average of 0.31 CTCs/mL. The technique was compared to a previously published method - the ELISPOT - that showed similar CTC levels among the five patient samples tested. Because the MCC method does not use positive selection, the method can be applied across a variety of tumor types with no changes to the process.

Toll-like receptor agonists as cancer vaccine adjuvants.

Cancer immunotherapy has emerged as a promising strategy to treat cancer patients. Among the wide range of immunological approaches, cancer vaccines have been investigated to activate and expand tumor-reactive T cells. However, most cancer vaccines have not shown significant clinical benefit as monotherapies. This is likely due to the antigen targets of vaccines, "self" proteins to which there is tolerance, as well as to the immunosuppressive tumor microenvironment. To help circumvent immune tolerance and generate effective immune responses, adjuvants for cancer vaccines are necessary. One representative adjuvant family is Toll-Like receptor (TLR) agonists, synthetic molecules that stimulate TLRs. TLRs are the largest family of pattern recognition receptors (PRRs) that serve as the sensors of pathogens or cellular damage. They recognize conserved foreign molecules from pathogens or internal molecules from cellular damage and propel innate immune responses. When used with vaccines, activation of TLRs signals an innate damage response that can facilitate the development of a strong adaptive immune response against the target antigen. The ability of TLR agonists to modulate innate immune responses has positioned them to serve as adjuvants for vaccines targeting infectious diseases and cancers. This review provides a summary of various TLRs, including their expression patterns, their functions in the immune system, as well as their ligands and synthetic molecules developed as TLR agonists. In addition, it presents a comprehensive overview of recent strategies employing different TLR agonists as adjuvants in cancer vaccine development, both in pre-clinical models and ongoing clinical trials.

Combining toll-like receptor agonists with immune checkpoint blockade affects antitumor vaccine efficacy.

BACKGROUND: T cell checkpoint receptors are expressed when T cells are activated, and modulation of the expression or signaling of these receptors can alter the function of T cells and their antitumor efficacy. We previously found that T cells activated with cognate antigen had increases in the expression of PD-1, and this was attenuated in the presence of multiple toll-like receptor (TLR) agonists, notably TLR3 plus TLR9. In the current report, we sought to investigate whether combining TLR agonists with immune checkpoint blockade can further augment vaccine-mediated T cell antitumor immunity in murine tumor models. METHODS: TLR agonists (TLR3 plus TLR9) and immune checkpoint inhibitors (antibodies targeting PD-1, CTLA-4, LAG-3, TIM-3 or VISTA) were combined and delivered with vaccines or vaccine-activated CD8+T cells to E.G7-OVA or MyC-CaP tumor-bearing mice. Tumors were assessed for growth and then collected and analyzed by flow cytometry. RESULTS: Immunization of E.G7-OVA tumor-bearing mice with SIINFEKL peptide vaccine, coadministered with TLR agonists and αCTLA-4, demonstrated greater antitumor efficacy than immunization with TLR agonists or αCTLA-4 alone. Conversely, the antitumor efficacy was abrogated when vaccine and TLR agonists were combined with αPD-1. TLR agonists suppressed PD-1 expression on regulatory T cells (Tregs) and activated this population. Depletion of Tregs in tumor-bearing mice led to greater antitumor efficacy of this combination therapy, even in the presence of αPD-1. Combining vaccination with TLR agonists and αCTLA-4 or αLAG-3 showed greater antitumor than with combinations with αTIM-3 or αVISTA. CONCLUSION: The combination of TLR agonists and αCTLA-4 or αLAG-3 can further improve the efficacy of a cancer vaccine, an effect not observed using αPD-1 due to activation of Tregs when αPD-1 was combined with TLR3 and TLR9 agonists. These data suggest that optimal combinations of TLR agonists and immune checkpoint blockade may improve the efficacy of human anticancer vaccines.

Sequence of androgen receptor-targeted vaccination with androgen deprivation therapy affects anti-prostate tumor efficacy.

RATIONALE: Androgen deprivation therapy (ADT) is the primary treatment for recurrent and metastatic prostate cancer. In addition to direct antitumor effects, ADT has immunomodulatory effects such as promoting T-cell infiltration and enhancing antigen processing/presentation. Previous studies in our laboratory have demonstrated that ADT also leads to increased expression of the androgen receptor (AR) and increased recognition of prostate tumor cells by AR-specific CD8+T cells. We have also demonstrated that ADT combined with a DNA vaccine encoding the AR significantly slowed tumor growth and improved the survival of prostate tumor-bearing mice. The current study aimed to investigate the impact of the timing and sequencing of ADT with vaccination on the tumor immune microenvironment in murine prostate cancer models to further increase the antitumor efficacy of vaccines. METHODS: Male FVB mice implanted with Myc-CaP tumor cells, or male C57BL/6 mice implanted with TRAMP-C1 prostate tumor cells, were treated with a DNA vaccine encoding AR (pTVG-AR) and ADT. The sequence of administration was evaluated for its effect on tumor growth, and tumor-infiltrating immune populations were characterized. RESULTS: Vaccination prior to ADT (pTVG-AR → ADT) significantly enhanced antitumor responses and survival. This was associated with increased tumor infiltration by CD4+ and CD8+ T cells, including AR-specific CD8+T cells. Depletion of CD8+T cells prior to ADT significantly worsened overall survival. Following ADT treatment, however, Gr1+ myeloid-derived suppressor cells (MDSCs) increased, and this was associated with fewer infiltrating T cells and reduced tumor growth. Inhibiting Gr1+MDSCs recruitment, either by using a CXCR2 antagonist or by cycling androgen deprivation with testosterone replacement, improved antitumor responses and overall survival. CONCLUSION: Vaccination prior to ADT significantly improved antitumor responses, mediated in part by increased infiltration of CD8+T cells following ADT. Targeting MDSC recruitment following ADT further enhanced antitumor responses. These findings suggest logical directions for future clinical trials to improve the efficacy of prostate cancer vaccines.

Myeloid-derived suppressor cells attenuate the antitumor efficacy of radiopharmaceutical therapy using 90Y-NM600 in combination with androgen deprivation therapy in murine prostate tumors.

RATIONALE: Androgen deprivation therapy (ADT) is pivotal in treating recurrent prostate cancer and is often combined with external beam radiation therapy (EBRT) for localized disease. However, for metastatic castration-resistant prostate cancer, EBRT is typically only used in the palliative setting, because of the inability to radiate all sites of disease. Systemic radiation treatments that preferentially irradiate cancer cells, known as radiopharmaceutical therapy or targeted radionuclide therapy (TRT), have demonstrable benefits for treating metastatic prostate cancer. Here, we explored the use of a novel TRT, 90Y-NM600, specifically in combination with ADT, in murine prostate tumor models. METHODS: 6-week-old male FVB mice were implanted subcutaneously with Myc-CaP tumor cells and given a single intravenous injection of 90Y-NM600, in combination with ADT (degarelix). The combination and sequence of administration were evaluated for effect on tumor growth and infiltrating immune populations were analyzed by flow cytometry. Sera were assessed to determine treatment effects on cytokine profiles. RESULTS: ADT delivered prior to TRT (ADT→TRT) resulted in significantly greater antitumor response and overall survival than if delivered after TRT (TRT→ADT). Studies conducted in immunodeficient NRG mice failed to show a difference in treatment sequence, suggesting an immunological mechanism. Myeloid-derived suppressor cells (MDSCs) significantly accumulated in tumors following TRT→ADT treatment and retained immune suppressive function. However, CD4+ and CD8+ T cells with an activated and memory phenotype were more prevalent in the ADT→TRT group. Depletion of Gr1+MDSCs led to greater antitumor response following either treatment sequence. Chemotaxis assays suggested that tumor cells secreted chemokines that recruited MDSCs, notably CXCL1 and CXCL2. The use of a selective CXCR2 antagonist, reparixin, further improved antitumor responses and overall survival when used in tumor-bearing mice treated with TRT→ADT. CONCLUSION: The combination of ADT and TRT improved antitumor responses in murine models of prostate cancer, however, this was dependent on the order of administration. This was found to be associated with one treatment sequence leading to an increase in infiltrating MDSCs. Combining treatment with a CXCR2 antagonist improved the antitumor effect of this combination, suggesting a possible approach for treating advanced human prostate cancer.

The androgen receptor: a biologically relevant vaccine target for the treatment of prostate cancer.

The androgen receptor (AR) plays an essential role in the development and progression of prostate cancer. However, while it has long been the primary molecular target of metastatic prostate cancer therapies, it has not been explored as an immunotherapeutic target. In particular, the AR ligand-binding domain (LBD) is a potentially attractive target, as it has an identical sequence among humans as well as among multiple species, providing a logical candidate for preclinical evaluation. In this report, we evaluated the immune and anti-tumor efficacy of a DNA vaccine targeting the AR LBD (pTVG-AR) in relevant rodent preclinical models. We found immunization of HHDII-DR1 mice, which express human HLA-A2 and HLA-DR1, with pTVG-AR augmented AR LBD HLA-A2-restricted peptide-specific, cytotoxic immune responses in vivo that could lyse human prostate cancer cells. Using an HLA-A2-expressing autochthonous model of prostate cancer, immunization with pTVG-AR augmented HLA-A2-restricted immune responses that could lyse syngeneic prostate tumor cells and led to a decrease in tumor burden and an increase in overall survival of tumor-bearing animals. Finally, immunization decreased prostate tumor growth in Copenhagen rats that was associated with a Th1-type immune response. These data show that the AR is as a prostate cancer immunological target antigen and that a DNA vaccine targeting the AR LBD is an attractive candidate for clinical evaluation.

B cells require licensing by dendritic cells to serve as primary antigen-presenting cells for plasmid DNA.

DNA vaccines have been an attractive approach for treating cancer patients, however have demonstrated modest immunogenicity in human clinical trials. Dendritic cells (DCs) are known to cross-present DNA-encoded antigens expressed in bystander cells. However, we have previously reported that B cells, and not DCs, serve as primary antigen-presenting cells (APCs) following passive uptake of plasmid DNA. Here we sought to understand the requirements for B cells to present DNA-encoded antigens, to ultimately increase the immunogenicity of plasmid DNA vaccines. Using ovalbumin-specific OT-1 CD8+ T cells and isolated APC populations, we demonstrated that following passive uptake of plasmid DNA, B cells but not DC, can translate the encoded antigen. However, CD8 T cells were only activated by B cells when they were co-cultured with DCs. We found that a cell-cell contact is required between B cells and DCs. Using MHCI KO and re-purification studies, we demonstrated that B cells were the primary APCs and DCs serve to license this function. We further identified that the gene expression profiles of B cells that have been licensed by DCs, compared to the B cells that have not, are vastly different and have signatures similar to B cells activated with a TLR7/8 agonist. Our data demonstrate that B cells transcribe and translate antigens encoded by plasmid DNA following passive uptake, however require licensing by live DC to present antigen to CD8 T cells. Further study of the role of B cells as APCs will be important to improve the immunological efficacy of DNA vaccines.

Vaccines as treatments for prostate cancer.

Prostate cancer is a leading cause of death in men worldwide. For over 30 years, growing interest has focused on the development of vaccines as treatments for prostate cancer, with the goal of using vaccines to activate immune cells capable of targeting prostate cancer to either eradicate recurrent disease or at least delay disease progression. This interest has been prompted by the prevalence and long natural history of the disease and by the fact that the prostate is an expendable organ. Thus, an immune response elicited by vaccination might not need to target the tumour uniquely but could theoretically target any prostate tissue. To date, different vaccine approaches and targets for prostate cancer have been evaluated in clinical trials. Overall, five approaches have been assessed in randomized phase III trials and sipuleucel-T was approved as a treatment for metastatic castration-resistant prostate cancer, being the only vaccine approved to date by the FDA as a treatment for cancer. Most vaccine approaches showed safety and some evidence of immunological activity but had poor clinical activity when used as monotherapies. However, increased activity has been observed when these vaccines were used in combination with other immune-modulating therapies. This evidence suggests that, in the future, prostate cancer vaccines might be used to activate and expand tumour-specific T cells as part of combination approaches with agents that target tumour-associated immune mechanisms of resistance.

Targeted Radiation and Immune Therapies-Advances and Opportunities for the Treatment of Prostate Cancer.

Prostate cancer is the most diagnosed malignancy in men in the United States and the second leading cause of cancer-related death. For localized disease, radiation therapy is a standard treatment that is often curative. For metastatic disease, radiation therapy has been primarily used for palliation, however, several newer systemic radiation therapies have been demonstrated to significantly improve patient outcomes and improve survival. In particular, several targeted radionuclide therapies have been approved for the treatment of advanced-stage cancer, including strontium-89, samarium-153, and radium-223 for bone-metastatic disease, and lutetium-177-labeled PSMA-617 for patients with prostate-specific membrane antigen (PSMA)-expressing metastatic castration-resistant prostate cancer (mCRPC). Contrarily, immune-based treatments have generally demonstrated little activity in advanced prostate cancer, with the exception of the autologous cellular vaccine, sipuleucel-T. This has been attributed to the presence of an immune-suppressive prostate cancer microenvironment. The ability of radiation therapy to not only eradicate tumor cells but also potentially other immune-regulatory cells within the tumor immune microenvironment suggests that targeted radionuclide therapies may be well poised to combine with immune-targeted therapies to eliminate prostate cancer metastases more effectively. This review provides an overview of the recent advances of targeted radiation agents currently approved for prostate cancer, and those being investigated in combination with immunotherapy, and discusses the challenges as well as the opportunities in this field.

Phase 2 trial of a DNA vaccine (pTVG-HP) and nivolumab in patients with castration-sensitive non-metastatic (M0) prostate cancer.

PURPOSE: We have previously reported that a plasmid DNA vaccine encoding prostatic acid phosphatase (pTVG-HP) had greater clinical activity when given in combination with pembrolizumab to patients with metastatic, castration-resistant prostate cancer. The current trial was conducted to evaluate vaccination with PD-1 blockade, using nivolumab, in patients with early, recurrent (M0) prostate cancer. METHODS: Patients with M0 prostate cancer were treated with pTVG-HP (100 µg administered intradermally) and nivolumab (240 mg intravenous infusion) every 2 weeks for 3 months, and then every 4 weeks for 1 year of total treatment. Patients were then followed for an additional year off treatment. The primary objectives were safety and complete prostate-specific antigen (PSA) response (PSA<0.2 ng/mL). RESULTS: 19 patients were enrolled. No patients met the primary endpoint of complete PSA response; however, 4/19 (21%) patients had a PSA decline >50%. Median PSA doubling times were 5.9 months pretreatment, 25.6 months on-treatment (p=0.001), and 9.0 months in the subsequent year off-treatment. The overall median radiographic progression-free survival was not reached. Grade 3 or 4 events included adrenal insufficiency, fatigue, lymphopenia, and increased amylase/lipase. 9/19 (47%) patients developed immune-related adverse effects (irAE). The development of irAE and increased CXCL9 were associated with increased PSA doubling time. Quantitative NaF PET/CT imaging showed the resolution of subclinical lesions along with the development of new lesions at each time point. CONCLUSIONS: In this population, combining nivolumab with pTVG-HP vaccination was safe, and immunologically active, prolonged the time to disease progression, but did not eradicate disease. Quantitative imaging suggested that additional treatments targeting mechanisms of resistance may be required to eliminate tumors. TRIAL REGISTRATION NUMBER: NCT03600350.