A phase II trial of personalized peptide vaccination in castration-resistant prostate cancer patients: prolongation of prostate-specific antigen doubling time

Eligibility required a histological diagnosis of prostate adenocarcinoma and progressive disease (PD) defined as at least two consecutive increases in PSA, new metastatic lesion on radionuclide bone scan, or progressive tumor lesions on cross-sectional imaging, despite adequate androgen ablative therapy. Patients showed positive IgG responses to at least two of the 31 different candidate peptides (Table 1). Any number of previous hormonal therapies was allowed. Patients were required to wait at least four weeks for entry into the study after the completion of prior radiation therapy, chemotherapy, or a change in hormonal therapy. Other inclusion criteria included age ≥ 20 years; Eastern Cooperative Oncology Group (ECOG) performance status 0 or 1; life expectancy of at least 12 weeks; positive status for HLA-A2, -A24, -A3 supertype (−A3, -A11, -A31, and -A33), or -A26; adequate hematologic, hepatic, and renal function; and negative status for hepatitis virus B and C. Exclusion criteria included an acute infection; a history of severe allergic reactions; pulmonary, cardiac, or other systemic diseases; and other inappropriate conditions for enrollment as judged by clinicians.

This was a single institution, single arm, open-label, phase II study. The endpoints of this study were primarily safety and feasibility of PPV in patients with CRPC. Secondary endpoints were to assess the PSA kinetics and immune responses. In addition, we identified potential factors for predicting OS and selecting suitable patients for this treatment. This study protocol was approved by Kurume University Ethical Committee. Written informed consent was obtained from all patients before any study procedures.

In this study, 31 peptides, whose safety and immunological effects had been confirmed in previously conducted clinical studies [10‐18], were employed for vaccination [12 peptides for HLA-A2, 14 peptides for HLA-A24, 9 peptides for the HLA-A3 supertype (A3, A11, A31, or A33), and 4 peptides for HLA-A26] (Table 1). All peptides were prepared under conditions of Good Manufacturing Practice using a Multiple Peptide System (San Diego, CA). The selection of 2 to 4 peptides for vaccination to each patient was based on HLA typing and high titer level of peptide-specific IgG to candidate peptides. Each of the selected peptides was mixed with incomplete Freund’s adjuvant (Montanide ISA-51VG; Seppic, Paris, France) and emulsified in the 5 ml plastic syringe, and a maximum of four peptides of 1.5 ml emulsion (3 mg/peptide) were injected subcutaneously into the lateral thigh area once a week for 6 weeks. The peptides were re-selected according to peptide-specific IgG levels at every cycle of 6 vaccinations and administered at 2-, 3-, or 4-week intervals until withdrawal of consent or unacceptable toxicity.

Patients were monitored at each visit by history and physical examinations. Serum PSA test and routine laboratory studies were performed every 6 vaccinations for any adverse effects. Toxicity was graded according to the National Cancer Institute Common Terminology Criteria for Adverse Events version 3.0 (NCI-CTCAE Ver3).

All patients underwent relevant radiologic studies and bone scans every 6 months or at the progression of symptoms. PD was defined as radiographic progression evaluated by Response Evaluation Criteria in Solid Tumors (RECIST) criteria [19] or clinical progression.

To assess the PSA response for each patient, percent PSA change from baseline was calculated for each phase of the study (pre- and during vaccination). In addition, PSA doubling time (PSADT) was calculated using all serum PSA values for a specified period, and using a minimum of three PSA values by the formula log₂/b, where b denotes the least square estimate of the linear regression model of the log-transformed PSA values on time. For analytical purposes, negative PSADT estimates and high positive PSADT estimates (>36 months) were censored at 36 months.

To investigate biomarkers for OS that may allow patient selection and prediction of a response to PPV, serum amyloid A (SAA), C-reactive protein (CRP), and interleukin (IL)-6 in plasma at baseline were additionally examined by enzyme-linked immunosorbent assay (ELISA), respectively.

To study the humoral responses specific to the vaccinated peptides, peptide-specific IgG levels were measured by a Luminex system (Luminex, Austin, TX), as reported previously [20]. If the total titers of selected peptide-specific IgG in any cycles of post-vaccination plasma were more than 2-fold higher than those in the pre-vaccination plasma, the changes were considered to be a positive response.

Although T-cell subsets using flowcytometry was not analyzed in this study, T-cell responses specific to the vaccinated peptides were evaluated by IFN-γ ELISPOT assay using peripheral blood mononuclear cells (PBMCs), as reported previously [18]. Peptide-specific T-cell responses were evaluated by the differences between the numbers of spots per 10⁵ x PBMCs in response to the vaccine peptides and those to the control peptide at pre- and 6th vaccination; at least 2-fold more spots at the 6th vaccination than at pre-vaccination was considered positive.

All patients who received more than 6 vaccinations were considered evaluable for tumor response, and all patients entered were included in the survival analysis. Data were analyzed at the end of November, 2012 using commercially available computer software. The Student’s t-test and the chi-square test were used to compare quantitative and categorical variables, respectively. Survival was calculated from the date of first treatment until the date of any cause of death. Patients lost to follow-up were censored at the last known date of survival. The Kaplan-Meier method was used to estimate actuarial survival curves, and groups were compared using a log-rank test. Cox proportional hazards regression model was used for univariate and multivariate analyses to identify factors that had a significant impact on survival. All baseline parameters in the survival and proportional hazards regression analysis were analyzed as dichotomous variables using median or cut-off values. A two-sided significance level of 5% was considered statistically significant.

Between April 2009 and August 2011, 100 patients with CRPC were enrolled in this trial at Kurume University Hospital. All 100 patients received at least one vaccination with a median of 16 vaccinations (range, 1 to 40) and were included in the safety assessment and survival analysis. Three patients did not complete 6 vaccinations (1 cycle) and were excluded from the assessment of PSA response and immune responses. The reason for these failures to complete 6 vaccinations was withdrawal of consent. The median age of participants was 69 years (range, 51 to 92 years), and the ECOG performance status was 0 in 91of the patients and 1 in the remaining 9. The median PSA and pre-vaccination PSADT at the entry to the study was 29.8 ng/ml (range, 0.2 to 2481 ng/ml) and 2 months (range, 0.3 to 36+ months), respectively. Fifty-seven patients had a Gleason score of ≥ 8 and 86 patients had metastasis. All patients had experienced progression after androgen deprivation therapy as an initial or secondary therapy. Forty patients had received docetaxel based chemotherapy with a median cycle of 6.5 as a third line treatment. Baseline patient characteristics are shown in Table 2.

The overall toxicities are shown in Table 3. The most frequent adverse events were local redness and swelling at injection sites, bone pain, hypoalbuminemia, lymphocytopenia, appetite loss, fatigue, increased ALP, and anemia, which were grade 1 or 2 in most cases. There were no grade 4 toxicities and no treatment-related deaths. A total of 51 grade 3 toxicities including anemia, bone pain, increased ALP, lymphocytopenia, decreased white blood cells, increased creatinine, injection site reaction, and increased AST and ALT were observed during the study. All of these severe adverse events were concluded to be not directly associated with the vaccinations, but with cancer progression or other causes by the independent safety evaluation committee in this trial.

Forty-eight (49%) patients exhibited some decrease in PSA from baseline, ranging from 1.9% to 99.6% (Figure 1A). Confirmed ≥50% PSA decline at any point during PPV was observed in 21 patients (22%), with a median time of 4 months to ≥50% PSA decline and a median duration of ≥50% PSA decline of 3 months. Delayed PSA response was observed. Patients with ≥50% PSA decline during PPV showed longer survival than remaining patients ( p = 0.035) (Figure 1B). The median estimated PSADT pre- and during PPV were 2 and 3.89 months, respectively. Fifty-four (56%) patients displayed at least 2-fold increase over the pre-treatment PSADT (range, 2.1- to 75-fold), and these patients with a prolongation of PSADT showed longer survival than patients without a prolongation of PSADT (p = 0.013) (Figure 1C and D). To compare the difference in PSA responses with clinical outcomes, patients were divided into three groups: responder group with survival longer than 20 months after PPV, non-responder group with death within 12 months after PPV, and another group with the remaining patients. Average% PSA changes in the responder group were significantly lower than those in the non-responder group at 2 to 5 months (p < 0.005) and those in the other group at 5 to 10 months (p < 0.005) during the PPV. In addition, average% PSA changes in the responder group showed a trend of PSA plateau. Average% PSA changes from baseline among three groups before and during PPV are shown in Figure 1E.

There was no complete response or partial response in terms of measurable disease. The median time to disease progression, as defined by clinical and/or radiologic criteria, was 10.9 months (95% CI, 6 to 19 months). At the time of analysis with a median follow-up of 18 months (95% CI, 14.1 to 24 months), 64 deaths had occurred. Median survival time was 18.8 months (95% CI, 14.9 to 28.6 months) in all patients. Median survival time in chemotherapy naive patients and in patients after docetaxel chemotherapy were 21.6 months and 11.6 months, respectively.

The number of selected peptides were 4 peptides in 62 patients, 3 peptides in 17 patients and 2 peptides in 21 patients at the first screening. Same peptide at the first screening were only selected in 29 of 97 (30%) patients at second screening and in 10 of 66 (15%) patients at the third screening, remaining patients received at least 1 different peptide during the study. The most frequently selected peptides were Lck486 (40 patients), CypB129 (31 patients), PAP213 (24 patients), SART2-93 (21 patients), PSA248 (20 patients), Lck488 (17 patients) and WHSC2-123 (16 patients) at the first screening. All 31 peptides were selected at any screening in the study.

Total IgG responses specific to the vaccinated peptide were augmented in 42 of 97 (43%) patients, 62 of 66 (94%) patients, 36 of 36 (100%) patients, 16 of 16 (100%) patients, and 7 of 7 (100%) patients at the 6th, 12th, 18th, 24th, and 30th vaccinations, respectively. Finally, positive IgG responses during PPV were observed in 76/97 (79%) patients. PBMCs from 97 patients were available for IFN-γ Elispot assay at the pre- and 6th vaccination. Peptide-specific T-cell responses were detectable in 42 patients (43%) at the 6th vaccination. There was no obvious correlation between IgG and CTL responses. Positive immune responses of both IgG and CTL based on baseline characteristics including age, PS, HLA typing, PSA, Gleason score, presence of metastasis and prior chemotherapy are shown in Figure 2. There was no difference in positive immune responses among baseline characteristics. In comparing immune responses with PSA kinetics, although average PSA changes did not correlate with immune responses, average ratio of PSADT was significantly higher in patients with positive IgG (8 vs. 4, p < 0.0001) and CTL (8.8 vs. 6.1, p = 0.0007) responses (Figure 3).

Cox proportional hazards regression analysis was performed to determine factors that would predict disease death (Table 4). Univariate Cox analysis showed that good performance status (p < 0.0001), positive IgG response (p < 0.0001), low CRP (p = 0.012), prolongation of PSADT (p = 0.018), low PSA (p = 0.004), prior chemotherapy status (p = 0.037), positive T-cell response (p = 0.039), and presentation of ≥50% PSA decline (p = 0.046) were significantly associated with survival.

The factors showing p less than 0.05 in the univariate analysis were included in multivariate analysis of the model. Finally, positive IgG response (p = 0.001) and prolongation of PSADT (p = 0.004) during PPV, as well as baseline good performance status (p = 0.004), low CRP levels (p = 0.006), and low PSA levels (p = 0.008), were significantly favorable factors for OS (Table 4).