- Research article
- Open Access
Prime-boost vaccination targeting prostatic acid phosphatase (PAP) in patients with metastatic castration-resistant prostate cancer (mCRPC) using Sipuleucel-T and a DNA vaccine
© The Author(s). 2018
- Received: 18 January 2018
- Accepted: 28 February 2018
- Published: 13 March 2018
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).
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.
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.
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.
- DNA vaccine
- Prostate cancer
- Prostatic acid phosphatase
- Immune monitoring
- Clinical trial
Sipuleucel-T was approved by the U.S. Food and Drug Administration (FDA) for the treatment of patients with metastatic, castration-resistant prostate cancer based on data from a randomized clinical trial demonstrating an improvement in overall survival compared to placebo . While the median improvement in overall survival was only 4 months, this is as comparable to other agents that have been approved for this stage of prostate cancer, including docetaxel [2, 3], cabazitaxel , abiraterone , radium-223 , and enzalutamide . Subsequent retrospective studies have suggested that patients with lower burdens of disease, and those who developed evidence of immunity to the prostatic acid phosphatase (PAP) target antigen with either antigen-specific IgG or T cells, might have had a superior outcome in terms of longer overall survival [8, 9]. Consequently, these findings suggest that the target of this vaccine, PAP, is a rational vaccine target antigen for prostate cancer treatment. Moreover, these findings suggest that using combination vaccine approaches to increase the immunological activity of sipuleucel-T to PAP might lead to superior clinical outcomes.
We have evaluated PAP-targeted vaccines using plasmid DNA as the means of antigen delivery . In two phase I trials evaluating dose and schedule in patients with non-metastatic prostate cancer (castration-sensitive and castration-resistant), we found vaccination to be safe and able to elicit PAP-specific CD4+ and CD8+ T cells with a Th1 phenotype [11, 12]. Unlike results from trials using sipuleucel-T, DNA vaccination did not elicit PAP-specific antibodies in either trial. The frequency of PAP-specific T cells was augmented with subsequent immunization, and the development of durable Th1-biased immune responses (detectable up to one year after treatment) appeared to be associated with favorable changes in PSA doubling time [12, 13]. Based on these results, a randomized phase II trial evaluating this vaccine is currently underway to determine whether treatment can delay the time to development of metastases in patients with biochemically recurrent prostate cancer (NCT01341652).
The ability of a DNA vaccine to elicit and augment Th1-biased immunity to PAP suggests it might be useful in a prime-boost strategy with sipuleucel-T, particularly since both vaccines target the same PAP antigen. Given that sipuleucel-T is an approved therapy delivered three times at two-week intervals, we sought to evaluate an approach in which DNA immunization was delivered after sipuleucel-T, as a booster immunization. We describe here the results of a pilot randomized clinical trial (NCT01706458) in which patients with mCRPC received sipuleucel-T alone (3 times at 2-week intervals), or sipuleucel-T (3 times at 2-week intervals) followed by DNA immunization 4 times at 2-week intervals, and then at months 6 and 9 after study initiation. The primary endpoint of the study was to determine if DNA vaccination could augment PAP-specific effector and memory T cells following treatment with sipuleucel-T. Secondary and exploratory objectives included effects on other measures of immunity, progression-free survival, and overall survival.
Investigational agent and regulatory information
pTVG-HP is a plasmid DNA encoding the full-length human PAP (ACPP gene) cDNA downstream of a eukaryotic promoter . The study protocol was reviewed and approved by all local (University of Wisconsin Human Subjects’ Review Board), and federal (FDA, NIH Recombinant DNA Advisory Committee) entities. All patients gave written informed consent for participation.
Male patients with a histological diagnosis of prostate adenocarcinoma and PSA recurrence following castration (surgical or ongoing luteinizing hormone-releasing hormone agonist therapy) were eligible, provided they had evidence of metastatic disease by CT of abdomen/pelvis and/or bone scintigraphy. Progressive disease following the last treatment was required, as per Prostate Cancer Working Group 2 criteria , and patients were required to be at least 4 weeks from prior treatment. A minimum of three PSA values, obtained from the same clinical laboratory over at least a 12-week period of time prior to registration, was required to calculate a PSA doubling time. Patients were required to have an Eastern Cooperative Oncology Group performance score of ≤ 2, and normal bone marrow, liver and renal function as defined by a WBC ≥ 2000/μL, ANC ≥ 1000 / mm3, hemoglobin ≥ 9.0 g/dL, platelet count ≥ 100,000/μL, AST and ALT ≤ 2.5× institutional upper limit of normal, and serum creatinine < 2.0 mg/dL. Patients were excluded if they had symptomatic disease (defined as requiring opioid analgesics for the treatment of pain attributed to a metastatic lesion), or been treated with chemotherapy within 6 months, or radiation therapy or systemic corticosteroid therapy (≥ 1 mg dose equivalent prednisone daily) within 4 weeks, of registration. Patients were further excluded if they had a history of HIV, hepatitis B, or hepatitis C infection, or if they had received prior sipuleucel-T treatment.
Study design and procedures
Demographics. Demographics for all patients enrolled
1 (n = 9)
2 (n = 9)
Race (n, %)
White / Caucasian
ECOG Performance Status (n, %)
Gleason score (n, %)
Metastatic sites (n, %)
Distant lymph nodes
Baseline PSA (ng/mL)
Baseline PSA doubling time (months)
For each time point, measures of antigen-specific T-cell immunity were performed with fresh (not cryopreserved) peripheral blood mononuclear cells (PBMC), and without in vitro stimulation prior to analysis. ELISPOT for IFNγ and granzyme B release were performed as previously described in 8-well replicates . For these analyses, protein antigens (PAP (Fitzgerald Industries, Acton, MA), PSA (Fitzgerald), tetanus toxoid (EMD Millipore, Billerica, MA), and GM-CSF (Leukine®, Sanofi, Bridgewater, NJ)) and human AB serum (Valley Biomedical, Winchester, VA) used were from the same lots to control for possible variation over time. A response resulting from immunization was defined as a PAP-specific response detectable more than once post-treatment that was both significant (compared to media only control), at least 3-fold higher than the pre-treatment value, and with a frequency > 1:100,000 PBMC. For peptide-specific evaluation over time, cryopreserved PBMC were thawed, stimulated in vitro with 0.5 μg/mL of peptide for 7–11 days, washed, and then evaluated by ELISPOT as indicated above. IgG specific for PAP, PSA, GM-CSF or tetanus toxoid were evaluated by indirect ELISA, as previously described . Peptide arrays (Roche-Nimblegen, Madison, WI) containing 16-mer peptides spanning the amino acid sequence of PAP, overlapping by 4 amino acids, were screened for IgG antibody responses, using sera diluted 1:100 from pre-treatment or 6-month blood collections, and assessed for mean fluorescence to each peptide, as previously reported .
Clinical response evaluation
Staging studies (CT of abdomen/pelvis and bone scintigraphy) were performed every 12 weeks, or as clinically indicated. PSA values were collected from the same clinical laboratory at 6–12 week intervals. A minimum of three PSA values collected over a 12-week period of time, with PSA values up to 6 months, and including the screening value, was used to determine the pre-treatment PSA DT. All values collected on study up to 6 months were used to determine the post-treatment PSA DT. PSA DT was calculated as log(2) divided by the slope parameter estimate of the linear regression model of the log-transformed PSA values on time.
Demographic characteristics and clinical outcomes were summarized in frequencies and percentages or medians and ranges. Immunological parameters were analyzed descriptively and displayed in graphic format using profile plots. Time to radiographic progression and overall survival were analyzed using the Kaplan-Meier method and compared between arms using the log-rank test. Statistical analyses were conducted using SAS software version 9.2 (SAS Institute Inc., Carey, NC).
Patient population and course of study
Adverse events. All adverse events at least possibly attributed to study treatment are shown. Numbers represent the number of patients per arm experience a particular event at any point during the treatment period, with the highest grade reported for any single individual. Adverse event grade is according to NCI CTCAE v.3
General / Constitutional
Injection site reaction
Pain in extremity
Heterologous prime-boost immunization strategies, in which two different vaccine types are used, each encoding the same antigen, have been demonstrated in many contexts to improve the immunological outcome of vaccination. Studies in preclinical models and human trials have shown increased antigen-specific T cells and/or antibodies using this approach in infectious disease and tumor systems [19–21]. Notably, in the case of viral or bacterial vector vaccines, the use of heterologous prime-boost approaches has been critical to avoid neutralizing immunity to the vector while augmenting immunity to the intended target antigen. This, in fact, was the approach used in the PSA-TRICOM vaccine targeting PSA as a prostate tumor antigen, using vaccinia virus encoding PSA as a priming immunization followed by booster immunizations with fowlpox encoding PSA . We have previously investigated a vaccinia vector encoding PAP and found that multiple immunization with that vector elicited a dominant response to the vector, not the target antigen, and this could be circumvented by booster immunization with either PAP protein or DNA encoding PAP . Given that DNA encoding PAP alone could elicit Th1-biased T cell immunity to PAP without eliciting vector-specific immunity, we have explored it in early clinical trials [11, 12]. At present, the only FDA-approved anti-tumor vaccine is sipuleucel-T, a treatment for advanced prostate cancer that similarly targets the prostate-specific antigen PAP. Given the availability of two vaccines each targeting this tumor antigen, the current trial evaluated whether T-cell responses to PAP could be augmented using them in a prime-boost approach. We found that with this sequence of administration, with sipuleucel-T followed by DNA immunization, there was no evidence of increased Th1-biased response to PAP.
This is the first trial to evaluate long-term effector and memory T cell immunity to PAP following sipuleucel-T treatment. We found that IFNγ- and granzyme B-secreting T cells specific for PAP were amplified with treatment, and could be detected up to at least one year following treatment in some individuals. Of note, PAP-specific granzyme B-secreting immunity was detected in half of patients at week 6 after completing the sipuleucel-T infusions. Antibody and Th1-biased cellular responses were also detected to GM-CSF. This is not surprising, as the PA2024 antigen used for activation of autologous cells in the manufacture of sipuleucel-T is a fusion protein of PAP and GM-CSF. The finding in our study that IFNγ-secreting response specific for GM-CSF were detectable in several individuals (5/16, 31%) who did not have an IFNγ-secreting response specific for PAP likely accounts for previous findings that the frequency of responses to the PA2024 antigen are higher than those detected to the native PAP antigen [1, 8]. That is, many patients likely develop immunity to the GM-CSF portion of the fusion protein. We have previously reported that immunity to GM-CSF can occur following immunization with GM-CSF protein, and to date there has been no evidence of adverse effect from immunity to GM-CSF .
This trial was not powered to detect differences in time to progression or overall survival, and no obvious trends were observed. Median overall survival was 28 months, which is consistent with previous trials conducted with sipuleucel-T in this patient population . Embedded within the trial design was an attempt to determine if treatment might slow the progression of disease. Specifically, in previous trials using anti-tumor vaccines conducted in this population, the median progression-free survival was about 12 weeks, at the first radiographic imaging time point [1, 24]. Treatment on the current trial was permitted beyond 12 weeks in order to determine if subsequent imaging showed stable disease, thus our results cannot be directly compared with previous studies using sipuleucel-T in terms of time to progression. Notwithstanding, using this approach, only two patients with evidence of progression at 3 months had stable disease after that, suggesting that delayed disease stability, if it occurs, is not common. Similarly, no differences were observed in pre-treatment and post-treatment PSA doubling times overall or for either treatment arm.
The trial was designed to test whether a DNA vaccine could boost cellular immunity elicited by sipuleucel-T. It was designed in this way given that sipuleucel-T is an approved therapy, and we did not want to potentially delay administration of an approved treatment. In addition, a prescribed course of sipuleucel-T involves three administrations at 2-week intervals, and is not amenable to large schedule interruptions or retreatment at later time points. Thus, the simplest design was to use a DNA vaccine after completing sipuleucel-T treatment. In retrospect, this was likely not the optimal design. First, the majority of patients had disease progression requiring study discontinuation before receiving multiple DNA immunizations, suggesting that, although safe, it may not be clinically feasible to sequence vaccines alone in this patient population when progression occurs quickly. In addition, nearly all studies to date evaluating DNA vaccines in heterologous prime-boost approaches, whether in preclinical models or human trials, have demonstrated that a preferred sequence of immunization is using DNA as the priming immunization [25–30]. In fact, an early study demonstrated that DNA priming followed by a booster immunization with a herpes simplex viral protein elicited a Th1-biased immune response, whereas the opposite sequence elicited a Th2-biased response . Given that sipuleucel-T elicits both Th1 and Th2 immunity to PAP, we suspect this Th2 response was preferentially boosted with DNA leading to an increased IgG response. A preferred approach may have been to use the DNA immunization prior to sipuleucel-T. In preclinical studies using the same DNA vaccine encoding PAP or a Listeria monocytogenes vector encoding PAP, we have found that priming with DNA followed by Listeria boost, and not the opposite sequence, elicited the most robust Th1-biased cellular immunity and anti-tumor response (manuscript in preparation). Consequently, future studies will explore heterologous prime-boost approaches using DNA as the priming immunization.
Our findings demonstrate that delivery of two vaccines encoding the same target antigen, using a DNA vaccine as a booster vaccine following treatment with sipuleucel-T, is safe and can augment and diversify the type of immunity elicited with anti-tumor vaccination.
We are grateful for the assistance of the research staff of the UWHC infusion center, UW pharmacy research center, clinical research coordinators and staff, treating physicians, and the participation of the patients and families.
This work was supported by an investigator-initiated clinical trial award from Dendreon Corporation, and by National Institutes of Health P30 CA014520.
Availability of data and materials
The data generated and/or analyzed during this study are available from the corresponding author on reasonable request.
EW, LEJ, and LD conducted and analyzed laboratory studies described; JCE was study biostatistician during design and analysis; MJS and GL led and supervised clinical trial and conduct; DGM designed protocol and oversaw analysis; all authors contributed to the writing and approval of the final manuscript.
Ethics approval and consent to participate
The study protocol was reviewed and approved by all local (University of Wisconsin Human Subjects’ Review Board (IRB), UW protocol CO11816), and federal (FDA, NIH Recombinant DNA Advisory Committee) entities. All patients gave written informed consent for participation. The trial national registration number is NCT01706458.
Consent for publication
Douglas G. McNeel has ownership interest, has received research support, and serves as consultant to Madison Vaccines, Inc. which has licensed intellectual property related to this content. None of the other authors have relevant potential conflicts of interest.
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