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

Abstract

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.

Keywords: Biomarker; DNA vaccine; Interleukin 10; Prostate cancer; Prostatic acid phosphatase.

Conflict of interest statement

Ethics approval and consent to participate

Samples were collected under University of Wisconsin IRB-approved protocols from two clinical trials (NCT00582140 and NCT00849121), and all patients gave written, informed consent for remaining samples to be used for research.

Consent for publication

Not applicable.

Competing interests

DGM has ownership interest, receives research support, and serves as consultant to Madison Vaccines, Inc. which has licensed material described in this manuscript. The authors declare that they have no competing interests.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Figures

Fig. 1

Schema of vaccination schedule of two phase I trials in which prostate cancer patients were treated with a DNA vaccine encoding PAP. Panel a: in Trial 1 (NCT00582140), patients received six biweekly immunization. Panel b: In Trial 2 (NCT00849121), patients received six biweekly immunizations followed by booster immunization every 3 months or based on immune monitoring. Grey arrows represent immunization time points. PAP-specific immune responses were monitored every 3 months after the initial six biweekly immunizations for each trial up to one year, as demonstrated by asterisks. Samples obtained before immunization were used for the biomarker analysis. Panel c: Shown are the number of immune responders and non-responders as defined for each trial

Fig. 2

Differences in absolute lymphocyte or monocyte counts in immune responding and non-responding patients were not detected. Panel a: Absolute lymphocyte and monocyte counts per μl of blood of responder and non-responder patients. Panel b: The lymphocyte-to-monocyte and neutrophil-to-lymphocyte ratios were calculated. Each dot represents the absolute lymphocyte or monocyte counts and lymphocyte ratios from individual subjects (closed squares, immune responders (n = 12); open squares, non-responders (n = 25)). Lines show median values

Fig. 3

Differences in peripheral blood cellular subsets were not detected in immune responding and non-responding patients. The percentage of T cells (CD3 + CD4+ T cells or CD3 + CD8+ T cells), B cells (CD3-CD19+), monocytes (CD14+), (Panel a); NK cells (CD56 + CD3-), NK T cells (CD56 + CD3+), (Panel b); dendritic cells (HLADR + CD11c + CD14-), (Panel c); and MDSCs (CD3-HLA-DRlow/negCD33 + CD11b + CD14+) and T regulatory cells (CD3 + CD4 + CD127-CD25 + FoxP3+) (Panel d) was evaluated in PBMCs from immune responding and non-responding patients. Each dot represents the percentage of each cell type among total live PBMCs from an individual subject (closed squares, immune-responders (n = 7); open squares, non-responders (n = 22)). Lines represent median values for each data set. Samples were analyzed in duplicate and averaged. Cryopreserved samples were not available for this analysis for 9 subjects

Fig. 4

Prostate cancer patients had preexisting immune responses to the PAP protein. PBMCs from responder or non-responder patients were stimulated 1 or 3 day(s) with PAP, PSA, AR LBD (AR), and Con A. Supernatants from cultures were collected and analyzed for cytokine concentrations (a) Granzyme B, (b) IFNγ, (c) IL-2, (d) IL-4, (e) IL-10, (f) IL-6, and (g) IL-17a using a cytokine bead array. Each dot represents the cytokine expression level for an individual prostate cancer patient (closed squares-responders (n = 7) and open square-non-responders (n = 23)). Samples were analyzed in triplicates and averaged. Lines represent median values for each data set. Statistical comparisons were made using a Mann Whitney t test. As in Fig. 3, cryopreserved samples were not available for 8 subjects

Fig. 5

Patients that were long term immune responders had preexisting PAP-specific regulatory responses. Pretreatment PBMCs from immune responder (n = 9) and non-responder patients (n = 23) were injected into the footpads of SCID mice with TT/D (recall antigen) alone or in combination with PAP or PSA. Each dot represents the net swelling (10−4 in.) after 24 h. This was defined as the antigen-specific swelling measurement minus the swelling due to the PBMCs and PBS alone. Shown is the net DTH immune response (10−4 in.) to TT/D alone (closed circles, responders; closed squares, non-responders) or in combination with PAP (a) or PSA (b) (circles, immune responders; squares, immune non-responders) for each patient. Statistical comparisons were made using a paired, non-parametric t test analysis. The log-transformed fold change is shown comparing c) TT/D PAP to TT/D and d) TT/D PSA to TT/D for immune responders and non-responders. Comparison of fold change was made using a Mann Whitney t test