Laura E. Johnson

Safety and Immunological Efficacy of a DNA Vaccine Encoding Prostatic Acid Phosphatase in Patients With Stage D0 Prostate Cancer

PURPOSE Prostatic acid phosphatase (PAP) is a prostate tumor antigen. We have previously demonstrated that a DNA vaccine encoding PAP can elicit antigen-specific CD8+ T cells in rodents. We report here the results of a phase I/IIa trial conducted with a DNA vaccine encoding human PAP in patients with stage D0 prostate cancer. PATIENTS AND METHODS Twenty-two patients were treated in a dose-escalation trial with 100 microg, 500 microg, or 1,500 microg plasmid DNA, coadministered intradermally with 200 microg granulocyte-macrophage colony-stimulating factor as a vaccine adjuvant, six times at 14-day intervals. All patients were observed for 1 year after treatment. RESULTS No significant adverse events were observed. Three (14%) of 22 patients developed PAP-specific IFN gamma-secreting CD8+ T-cells immediately after the treatment course, as determined by enzyme-linked immunospot. Nine (41%) of 22 patients developed PAP-specific CD4+ and/or CD8+ T-cell proliferation. Antibody responses to PAP were not detected. Overall, the prostate-specific antigen (PSA) doubling time was observed to increase from a median 6.5 months pretreatment to 8.5 months on-treatment (P = .033), and 9.3 months in the 1-year post-treatment period (P = .054). CONCLUSION The demonstration that a DNA vaccine encoding PAP is safe, elicits an antigen-specific T-cell response, and may be associated with an increased PSA doubling time suggests that a multi-institutional phase II trial designed to evaluate clinical efficacy is warranted.

DNA Vaccine Encoding Prostatic Acid Phosphatase (PAP) Elicits Long-term T-cell Responses in Patients With Recurrent Prostate Cancer

Prostatic acid phosphatase (PAP) is a tumor antigen in prostate cancer and the target of several anti-tumor vaccines in earlier clinical trials. Ultimately, the goal of anti-tumor vaccines is to elicit a sustainable immune response, able to eradicate a tumor, or at least restrain its growth. We have investigated plasmid DNA vaccines and have previously conducted a phase 1 trial in which patients with recurrent prostate cancer were vaccinated with a DNA vaccine encoding PAP. In this study, we investigated the immunologic efficacy of subsequent booster immunizations, and conducted more detailed longitudinal immune analysis, to answer several questions aimed at guiding optimal schedules of vaccine administration for future clinical trials. We report that antigen-specific cytolytic T-cell responses were amplified after immunization in 7 of 12 human leukocyte antigen-A2-expressing individuals, and that multiple immunizations seemed necessary to elicit PAP-specific interferon-γ-secreting immune responses detectable by enzyme-linked immunosorbent spot assay. Moreover, among individuals who experienced a ≥200% increase in prostate-specific antigen doubling time, long-term PAP-specific interferon-γ-secreting T-cell responses were detectable in 6 of 8, but in only 1 of 14 individuals without an observed change in prostate-specific antigen doubling time (P=0.001). Finally, we identified that immune responses elicited could be further amplified by subsequent booster immunizations. These results suggest that future trials using this DNA vaccine, and potentially other anti-tumor DNA vaccines, could investigate ongoing schedules of administration with periodic booster immunizations. Moreover, these results suggest that DNA vaccines targeting PAP could potentially be combined in heterologous immunization strategies with other vaccines to further augment PAP-specific T-cell immunity.

Plasmid DNA vaccine encoding prostatic acid phosphatase is effective in eliciting autologous antigen-specific CD8+ T cells

Prostatic acid phosphatase (PAP) is a prostate cancer tumor antigen and a prostate-specific protein shared by rats and humans. Previous studies indicated that Copenhagen rats immunized with a recombinant vaccinia virus expressing human PAP (hPAP) developed PAP-specific cytotoxic T cells (CTL) with cross reactivity to rat PAP (rPAP) and evidence of prostate inflammation. Viral delivery of vaccine antigens is an active area of clinical investigation. However, a potential difficulty with viral-based immunizations is that immune responses elicited to the viral vector might limit the possibility of multiple immunizations. In this paper, we investigate the ability of another genetic immunization method, a DNA vaccine encoding PAP, to elicit antigen-specific CD8+ T cell immune responses. Specifically, Lewis rats were immunized with either a plasmid DNA-based (pTVG-HP) or vaccinia-based (VV-HP) vaccine each encoding hPAP. We determined that rats immunized with a DNA vaccine encoding hPAP developed a Th1-biased immune response as indicated by proliferating PAP-specific CD4+ and CD8+ cells and IFNγ production. Rats immunized with vaccinia virus encoding PAP did not develop a PAP-specific response unless boosted with a heterologous vaccination scheme. Most importantly, multiple immunizations with a DNA vaccine encoding the rat PAP homologue (pTVG-RP) could overcome peripheral self-tolerance against rPAP and generate a Th1-biased antigen-specific CD4+ and CD8+ T cell response. Overall, DNA vaccines provide a safe and effective method of generating prostate antigen-specific T cell responses. These findings support the investigation of PAP-specific DNA vaccines in human clinical trials.

HLA-A2-restricted T-cell epitopes specific for prostatic acid phosphatase

Prostatic acid phosphatase (PAP) has been investigated as the target of several antigen-specific anti-prostate tumor vaccines. The goal of antigen-specific active immunotherapies targeting PAP would ideally be to elicit PAP-specific CD8+ effector T cells. The identification of PAP-specific CD8+ T-cell epitopes should provide a means of monitoring the immunological efficacy of vaccines targeting PAP, and these epitopes might themselves be developed as vaccine antigens. In the current report, we hypothesized that PAP-specific epitopes might be identified by direct identification of pre-existing CD8+ T cells specific for HLA-A2-restricted peptides derived from PAP in the blood of HLA-A2-expressing individuals. 11 nonamer peptides derived from the amino acid sequence of PAP were used as stimulator antigens in functional ELISPOT assays with peripheral blood mononuclear cells from 20 HLA-A2+ patients with prostate cancer or ten healthy blood donors. Peptide-specific T cells were frequently identified in both groups for three of the peptides, p18–26, p112–120, and p135–143. CD8+ T-cell clones specific for three peptides, p18–26, p112–120, and p299–307, confirmed that these are HLA-A2-restricted T-cell epitopes. Moreover, HLA-A2 transgenic mice immunized with a DNA vaccine encoding PAP developed epitope-specific responses for one or more of these three peptide epitopes. We propose that this method to first identify epitopes for which there are pre-existing epitope-specific T cells could be used to prioritize MHC class I-specific epitopes for other antigens. In addition, we propose that the epitopes identified here could be used to monitor immune responses in HLA-A2+ patients receiving vaccines targeting PAP to identify potentially therapeutic immune responses.

Real-time immune monitoring to guide plasmid DNA vaccination schedule targeting prostatic acid phosphatase in patients with castration-resistant prostate cancer

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.

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.

Immunization with a prostate cancer xenoantigen elicits a xenoantigen epitope-specific T-cell response

Vaccines encoding xenoantigens, “non-self” proteins that are highly homologous to their autologous counterparts, have been investigated as a means to increase immunogenicity and overcome tolerance to “self” antigens. We have previously shown that DNA vaccines encoding native prostatic acid phosphatase (PAP) were able to elicit PAP-specific T cells in both rats and humans, but required multiple immunization courses. In this study, we investigated in a preclinical model whether immunizations with a DNA vaccine encoding a xenoantigen could elicit a cross-reactive immune response to the native protein, potentially requiring fewer immunizations. Lewis rats were immunized with a DNA vaccine encoding human PAP and splenocytes from immunized rats were screened with a human peptide library containing overlapping, 15-mer PAP-derived peptides using T-cell proliferation and interferon γ (IFNγ) release as measures of the immune response. One dominant PAP-specific, RT1.Al-restricted, epitope was identified. Direct immunization with the immunodominant peptide (HP201–215) containing this epitope demonstrated that it included a naturally presented MHC Class I epitope recognized by CD8+ T cells in Lewis rats. However, no cross-reactive immune response was elicited to the corresponding rat peptide despite a difference of only three amino acids. Immunization with DNA vaccines encoding rat PAP (rPAP) in which this foreign dominant epitope was included as well as with DNA vaccines coding for a variant of the xenoantigen from which this epitope was deleted, did not elicit responses to the native antigen. Overall, these results indicate that the immunization with a xenoantigen-coding DNA vaccine can lead to an immune response which potentially favors foreign epitopes and hence limits any cross-reactive response to the native antigen.

Identification of prostatic acid phosphatase (PAP) specific HLA-DR1-restricted t-cell epitopes†

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.

DNA Vaccines for Prostate Cancer

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.

Pretreatment antigen-specific immunity and regulation - association with subsequent immune response to anti-tumor DNA vaccination

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.