DNA vaccine encoding prostatic acid phosphatase (PAP) elicits long-term T-cell responses in patients with recurrent prostate cancer

Abstract

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-gamma-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-gamma-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.

FIGURE 1

DNA immunization augments the frequency of antigen-specific cytolytic T cells: peripheral blood mononuclear cells from 12 HLA-A2 expressing patients collected before immunization (grey) or after 6 immunizations (black) were cultured in the presence of HLA-A2 epitopes derived from the amino-acid sequence of prostatic acid phosphatase (p18-26, panel A; p112-120, panel B; and p299-307, panel C) or an influenza control 9-mer HLA-A2-specific epitope (GILGFVFTL, panel D). Beginning after 2 weekly in-vitro stimulations, cultures were evaluated for the presence of peptide-specific CTL. Columns indicate the first stimulation during which peptide-specific CTL were detectable. In panel D, CTL analysis was only conducted with pretreatment specimens (grey bars), and with selected patients. CTL indicates cytotoxic T lymphocytes; HLA, human leukocyte antigen; ND, patient samples not assessed.

FIGURE 2

Several DNA immunizations were necessary to elicit PAP-specific T cells: PBMC were collected from 4 patients (panel A, B, C, and D) before immunization, and after 2, 4, or 6 biweekly immunizations. Samples from one of the time points (preimmunization, panel A; and after 4 immunizations, panel C) were not available for 2 of these patients. PBMC were cultured in the presence of PAP protein, prostate-specific antigen (PSA) protein (negative control), tetanus toxoid (TET; immunization control), phytohemaglutinin (PHA; nonspecific mitogenic positive control), or media only for 48 hours, and IFNγ-secreting T cells under each culture condition were enumerated by enzyme-linked immunosorbent spot. Shown are the mean number and SD of antigen-specific IFNγ sfu (number of antigen-specific sfu–number of media-only sfu) per 10⁶ cells at each time point (from quadruplicate assays) for individual patients. Asterisks demonstrate significant responses (P < 0.05, t test) of antigen-specific sfu compared with media only. IFN indicates interferon; PAP, prostatic acid phosphatase; PBMC, peripheral blood mononuclear cells; sfu, spot-forming units.

FIGURE 3

DNA immunization elicited long-term PAP-specific T-cell immunity in multiple subjects: PBMC were collected before immunization, 2 weeks after 6 immunizations (post), and at 3-month intervals thereafter from patients observed to have a ≥200% increase in prostate-specific antigen (PSA) doubling time (panels B, C, E-H, J, K), and representative other patients (panels A, D, I, L). PBMC were cultured in the presence of PAP protein, TET, or PHA as in Figure 2 for 48 to 72 hours and were evaluated for antigen-specific immune responses by IFNγ enzyme-linked immunosorbent spot. Shown are the mean and SD of quadruplicate determinations of IFNγ sfu at each time point. Asterisks denote significant responses (P < 0.05, t test) of antigen-specific (PAP or TET-specific) sfu compared with media only. Data from individual samples for which the PHA-positive control was not demonstrably positive were not considered adequate for interpretation; however, these data are included for completeness. IFN indicates interferon; PAP, prostatic acid phosphatase; PBMC, peripheral blood mononuclear cells; PHA, phytohemaglutinin; sfu, spot-forming units; TET, tetanus toxoid.

FIGURE 4

DNA immunization can augment PAP-specific T-cell immunity previously elicited. Panel A: PBMC were collected immediately before, and 2 weeks after 2, 4, or 6 monthly booster immunizations. Cells were cultured in the presence of PAP, PSA, phytohemaglutinin (PHA), or media only, and assayed for proliferation after 5 days by BrdU incorporation. Shown are the fold increases of CD4/8⁺BrdU⁺ events (proliferative index) after antigen-specific culture over cells cultured in media only. Panel B: PBMC collected at multiple time points before and after initial immunization and booster immunization (indicated by checkered boxes) were thawed, stimulated in-vitro with peptides for 7 days, and then assayed for peptide-specific CD8⁺ T cells by pentamer staining. Shown is the percentage of pentamer⁺ cells among total CD8⁺ T cells. Panel C: For pentamer staining, samples were gated on CD3⁺ CD8⁺ events, with lymphocyte side scatter. Shown are representative staining dot plots from two of the time points indicated; x axis is CD8 staining, nd y axis is pentamer or lgG isotype control. The numbers indicate the percentage of events among CD3⁺ CD8⁺ cells. Panel D: Serum PSA values from this same patient. The timing of the initial immunization series and booster immunization series are indicated by the checkered boxes. BrdU indicates bromodeoxyuridine; IFN, interferon; PAP, prostatic acid phosphatase; PBMC, peripheral blood mononuclear cells; PSA, prostate-specific antigen.