From November 2008 to March 2019, phase II studies of a PPV were conducted for 44 Japanese patients with cancers of different histologies at the Kurume University Cancer Vaccine Center or Kurume University Hospital. They were diagnosed as having no active tumor at the time of their entry for the PPV: lung adenocarcinoma (n=11), colon cancer (n=18), and breast cancer (n=15). The eligibility criteria were a pathologically confirmed diagnosis of lung adenocarcinoma, colon cancer, or breast cancer; positive IgG responses [≥10 fluorescence intensity units (FIU)] for ≥2 of the 31 warehouse peptides in their prevaccination plasma (Table SI); positive status for HLA-A2, -A24, -A3 supertypes (HLA-A3, -A11, -A31, or -A33) or -A26; age ≥20 years; Eastern Cooperative Oncology Group performance status of 0-2; a life expectancy of ≥12 weeks; and adequate bone marrow, hepatic, and renal functions.

The exclusion criteria were acute infection, a history of severe allergic reactions, or other systemic diseases as described (13-17). The 31 warehouse peptides consisted of peptides matched for 12 HLA-A2-positive, 14 for HLA-A24-positive, and nine for HLA-A3 supertype-positive patients, and four peptides for HLA-A26-positive cancer patients, respectively (Table SI). The peptides were prepared under Good Manufacturing Practice conditions with the use of a multiple peptide system (Multiple Peptide System, Inc.). The patients were vaccinated in a subcutaneous region with two to four peptides based on their HLA type and on their pre-existing immunity represented by their peptide-specific IgG levels as described (13-17).

The protocols were as follows: patients with lung cancer (under the protocol with UMIN registration no. 00002984), breast cancer (no. 000003081), or colon cancer (no. 000002987) received vaccinations consisting of a 1.5-ml emulsion (3 mg/each peptide) of 2-4 peptides across three cycles as follows: four visits at 1-week intervals, followed by four visits at 2-week intervals (the 1st cycle); four visits at 2-week intervals followed by four visits at 4-week intervals (the 2nd cycle); and eight visits at 4-week intervals (the 3rd cycle).

In addition, certain colon cancer patients from northern Japan or outside of Japan were entered in the protocols with UMIN registration nos. 000006927, 000011230 and 000001482. They received vaccinations consisting of a 3.0-ml emulsion (6 mg/each peptide) of two to four peptides at each visit (half the dose was injected into either side of the body) across three cycles as follows: four visits at 4-week intervals (the 1st cycle), followed by four visits at 4-to-8-week intervals (the 2nd cycle), and finally four visits at 8- to 12-week intervals (the 3rd cycle).

After the 3rd cycle, all patients who wished to continue received the vaccination at 4- to 8-week intervals until withdrawal of consent or unaccepTable toxicity. All protocols were approved by the ethical committee of Kurume University and by the regional ethical committee [Fukuoka Clinical Research Board (no. 718004)] and then registered in the UMIN Clinical Trials Registry of the Japanese government. All of these studies were in accordance with the Declaration of Helsinki and the International Conference on Harmonization of Good Clinical Practice guidelines and were conducted in an outpatient setting. Before their inclusion in the study, all participants gave written informed consent to participate in the clinical trial and to have their data used for research and publication purposes.

Toxicity and general conditions were monitored at the time of each visit. Toxicity was evaluated using the Common Terminology Criteria for Adverse Events ver. 4.0.

The patients' peripheral blood was collected at pre-treatment and at the end of the 1st, 2nd and 3rd cycles, and then at each visit after ≥3 months (range 3-45 months). Their IgG titers and cytotoxic T lymphocyte (CTL) activity specific to the peptide in plasma or peripheral blood mononuclear cells (PBMCs) were evaluated by a beadbased multiplexed Luminex assay (Luminex Platform LHC6003M; Invitrogen; Thermo Fisher Scientific, Inc.) and by an interferon (IFN)-γ ELISPOT (Immunocyte IFN-γ ELISPOT kit; Medical and Biological Laboratories), respectively (17). Pre-vaccination peptide-specific IgG levels with a cut-off level of 10 FIU were taken as detecTable levels of IgG. Patients were considered to have a positive IgG boost when the total sum of their post-vaccination IgG levels against the vaccinated peptides (2-4 peptides) was >5,000 FIUs compared to that of the pre-vaccination level.

The spreading of IgG boosting to non-vaccinated peptides was quantified by measuring the total sum of FIU in response to non-vaccinated peptides. The HLA-matched peptide-specific CTL activity in PBMCs was evaluated by the IFN-γ ELISPOT assay. Positive CTL boosting was defined as a >5-fold increase in the total sum of HLA-matched peptide-specific IFN-γ spots compared to the pre-vaccination level or >50 IFN-γ spots if the pre-vaccination CTL activity was undetectable. The CEF peptide pool (Mabtech, Cincinnati, OH) consisting of 23 HLA-class I-restricted peptides from human influenza virus, cytomegalovirus, and Epstein Barr virus was used as a control peptide.

The target cells used for the assay were T2 cells for HLA-A2 or HMy2.CIR (CRL-1993; ATCC) cells transfected with the HLA- A11, -A24, -A26, -A31, or -A33 gene as described (13-17). We calculated the spreading of CTL activity to non-vaccinated peptides by subtracting the sum of IFN-γ spots in response to all 31 peptide-mixtures from the sum of IFN-γ spots in response to the vaccinated peptides. Pre-vaccination samples at the screening time (14 days before the first vaccination) were provided for the measurement of blood cell counts and C-reactive protein (CRP) as described (17).

The corresponding author had full access to all of the study data and had final responsibility for the decision to submit this report for publication.

The Kaplan-Meier method was used for the statistical analyses. Recurrence was determined by the new appearance of tumor based on a radiological diagnosis or the increase of a tumor marker. Recurrence-free survival (RFS) and overall survival (OS) were calculated as the time in months from the day of the 1st vaccination (for events) or to the date of last contact. Mixed model analysis using Kenward-Roger F test was used for the patients' IgG level values after peptide vaccination. All statistical analyses were performed using JMP version 16.0 (SAS Institute Inc., Cary, NC, USA).

Table I summarizes the patients' characteristics and clinical outcomes. The 44 patients without an active tumor consisted of 11 patients with lung adenocarcinoma, 18 with colon cancer (origin of primary cancers: 13 rectal, two sigmoid colon, one each of cecum, ascending, and transverse colon); and 15 with breast cancer (eight luminal, two HER2+, four triple-negative types, and one apocrine cancer). Systemic therapies were combined with PPV in none of the patients with lung cancer, six of the patients with colon cancer (chemotherapy in six cases), and eight of the patients with breast cancer (chemotherapy and target therapy in one case each, and hormone therapy in six cases) (Table I). The median number of vaccinations was 20. The median follow-up time was 67.6 months (interquartile range (IQR): 45.6-82.8). Under these circumstances, 37 of the 44 patients (84%) had no recurrence. The remaining seven patients (one stage III lung cancer, two stage IV and two recurrent colon cancers, and two stage II breast cancers) had recurrence, and their median RFS and OS rates were 11.0 months [95% confidence interval (CI): 3.2-14.1] and 44.0 months (95% CI: 12.7-57.0), respectively (Fig. 1A and B).

The detailed information for each of the 44 enrolled patients from their first cancer surgery to the end of the clinical study (Table II) helps clarify the intervals from the surgery to the vaccination effect to the outcome of PPV-induced cancer prevention. The median period from surgery to the first vaccination was 14.5 months (IQR: 6.4-27.5 months). The median periods of the seven patients with recurrence from their surgery to their first vaccination were as follows: 1.5 months (stage IV colon cancer), 2.5 months (stage II triple-negative breast cancer), 6.5 months (stage III colon cancer), 13 months (stage III lung cancer), 17.5 months (stage II luminal type breast cancer), 17.5 months (stage II colon cancer), and 90 months (stage I colon cancer). These results indicate that the intervals from surgery to the first vaccination in the entire series of 44 patients were not very different from the intervals of the seven recurrent patients.

The majority of patients had grade I or II skin reactions at the injection sites. There were no PPV-related severe adverse events other than grade III injection site reactions. Details are given in Table SII.

The kinetics throughout the study for the patients without an active tumor are depicted in Fig. 2 (lung adenocarcinoma, n=11), (colon cancer, n=18), and (breast cancer, n=14), respectively. The plotted points of IgG levels (FIU) were pre-vaccination, the end of the 1st cycle (2-3 months later), the end of the 2nd cycle (7-9 months later), the end of the 3rd cycle (15-17 months later), and thereafter (every 3-34 months) during the follow-up. IgG boosting was observed in 36 of the 43 patients tested at the end of the 1st cycle, followed by an increase in IgG thereafter. In all 12 patients tested after the vaccine's termination, the IgG boosting levels were maintained at levels ranging from 23,165 FIU at 34 months to 1,601,706 FIU at 63 months post-termination.

Collectively, among the 44 patients without an active tumor, IgG boosting against the vaccinated peptides at the end of the 1st and 2nd cycles of PPV was observed in 36 of 43 patients and all 43 patients whose samples were available, respectively. Moreover, the PPV induced robust (>50,000 FIU) humoral immunity for 41 of the 43 tested cancer patients without an active tumor even after the follow-up period (Fig. 2A-C). IgG boosting at the end of the 1st cycle and the 2nd cycle was observed in three of the seven recurrent patients and in three of the four recurrent patients tested, respectively. These results suggest that post-vaccination IgG boosting was a prognostic marker in PPV patients, which is consistent with the reported observations (13,14,17).

The CTL activity against the vaccinated peptides at the end of the 1st cycle was boosted in 23 of the 30 tested patients. However, these elevated CTL activities declined at the end of the 2nd cycle in most of the tested cases (Fig. 3A). The CTL activity against non-vaccinated peptides in the 13 patients tested was no boosted at the 2nd cycle (Fig. 3B).

We investigated the IgG response to the Lck-486 peptide, which is capable of inducing HLA-A24-restricted CTL activity; we did so because this peptide was vaccinated for the majority of the present HLA-A24 patients (23 of 30). We speculated that Lck-486 peptide could thus be a suiTable representative for the evaluation of the kinetics of each peptide. This investigation was also conducted based on our previous study of a monoclonal anti-Lck-486 IgG (IgG 2b) peptide capable of inhibiting tumor growth in vivo with a suppression of tumor-infiltrating T regulatory cells in a murine model (18).

Among the 44 patients, 30 were HLA-A24+ and the remaining 14 were HLA-A24- (Table I). Twenty-three of the 30 HLA-A24+ patients received a vaccination with the Lck-486 peptide; the remaining seven did not. Robust IgG boosting (>50,000 FIU) against the Lck-486 peptide was observed in all 22 HLA-A24+ patients tested, and the boosting levels were maintained even after vaccine termination (Fig. 4A). The patients' median IgG levels were significantly higher than their pre-vaccination levels (P<0.0001) (Fig. 4B). In contrast, IgG boosting against the Lck-486 peptide was not observed in any of the seven HLA-A24+ patients without Lck-486 vaccination, whereas it occurred in two of the 14 HLA-A24- patients without Lck-486 vaccination (Fig. 4C).

We also assessed the clinical benefits of the vaccinations, as our earlier study revealed that the patients who received the Lck-486 vaccination had significantly longer survival compared to patients with advanced cancer (19). Three of the 23 HLA-A24+ patients (13%) developed cancer recurrence after receiving the Lck-486 peptide vaccination (among the 23 patients, the recurrence rate was 13%), whereas two of the HLA-A24+ patients (29%) developed cancer recurrence after Lck-486 peptide vaccination. The RFS values were 11.3, 14.1 and 20.5 months respectively in the former three patients, and 6.7 and 11.0 months in the latter two patients. The OS values were 66.7, 43.4 and 46.7 months in the former three patients, and 24.8 and 20.6 in the latter two, respectively. All measurements of clinical events (recurrence rate, RFS, and OS) were favorable in the patients who received the Lck-486 peptide.

We reported that pre-vaccination inflammatory signatures hampered the clinical benefits of PPV for patients with advanced cancer (13-17). Herein, we investigated the following pre-vaccination inflammatory signatures: 59.9% as the median percentage of neutrophils; 1.9 as the neutrophil-lymphocyte ratio; 4,800 as the white blood cell number; 31.2% as the percentage of lymphocytes; and 429x10⁴ as the red blood cell number. The median CRP level in the 17 tested patients without an active tumor was 0.09.