Phase II clinical trial of peptide cocktail therapy for patients with advanced pancreatic cancer: VENUS-PC study.

We previously conducted a phase I clinical trial combining the HLA-A*2402-restricted KIF20A-derived peptide vaccine with gemcitabine for advanced pancreatic cancer (PC) and confirmed its safety and immunogenicity in cancer patients. In this study, we conducted a multicenter, single-armed, phase II trial using two antiangiogenic cancer vaccines targeting VEGFR1 and VEGFR2 in addition to the KIF20A peptide. We attempted to evaluate the clinical benefit of the cancer vaccination in combination with gemcitabine. Chemotherapy naïve PC patients were enrolled to evaluate primarily the 1-year survival rate, and secondarily overall survival (OS), progression free survival (PFS), response rate (RR), disease control rate (DCR) and the peptide-specific immune responses. All enrolled patients received therapy without the HLA-A information, and the HLA genotypes were used for classification of the patients. Between June 2012 and May 2013, a total of 68 patients were enrolled. No severe systemic adverse effects of Grade 3 or higher related to these three peptides were observed. The 1-year survival rates between the HLA-A*2402-matched and -unmatched groups were not significantly different. In the HLA-A*2402 matched group, patients showing peptide-specific CTL induction for KIF20A or VEGFR1 showed a better prognosis compared to those without such induction (P = 0.023, P = 0.009, respectively). In the HLA-A*2402-matched group, the patients who showed a strong injection site reaction had a better survival rate (P = 0.017) compared to those with a weak or no injection site reaction. This phase II study demonstrated that this therapeutic peptide cocktail might be effective in patients who demonstrate peptide-specific immune reactions although predictive biomarkers are needed for patient selection in its further clinical application.

Pancreatic cancer is the fifth leading cause of cancer mortality in Japan,1 and the fourth leading cause in the United States.2 The prognosis for patients with pancreatic cancer is extremely poor, with an overall 5‐year survival of only 7% in Japan. The primary reason for this high mortality rate is the aggressive nature of the malignancy together with the difficulty of early detection. As a result, the majority of pancreatic cancers are unresectable.3 Gemcitabine has been one of the standard therapies in advanced pancreatic cancer for over a decade, although many chemotherapeutic agents have been tested in clinical trials over the past two decades.4, 5, 6 The overall survival rate has recently been significantly prolonged due to combination therapies such as the combination of gemcitabine and erlotinib, that of oxaliplatin, irinotecan, fluorouracil, and leucovorin, and that of gemcitabine and nab‐paclitaxel. However, as a consequence of using these combination therapies, many patients have experienced skin rash, febrile neutropenia, and peripheral neuropathy⁄ myelosuppression.7, 8, 9 Hence, these treatments can only be tolerated by a limited proportion of patients, mostly those with a good performance status.

The development of new treatment modalities, including specific immunotherapies, is thus required. Recent advances in molecular biology and cellular immunology in the field of tumor immunology have resulted in the identification of a large number of human leukocyte antigen (HLA) class I‐restricted antigens and epitopes that are recognized by cytotoxic T lymphocytes (CTL).10, 11, 12, 13, 14, 15 Using cDNA microarray technology coupled with laser microdissection, we previously identified novel HLA*A24‐restricted epitope peptides as targets for cancer vaccination for patients with pancreatic cancer.16, 17, 18 One of these peptides, KIF20A (RAB6KIFL), belongs to the kinesin superfamily of motor proteins, which have critical functions in the trafficking of molecules and organelles.19 Although immunotherapy using tumor infiltrating cells (TIL) or vaccine treatment is a promising modality for the treatment of cancer, recent reports have indicated several mechanisms in tumor tissues that allow cancer cells to escape from host immune attacks.20 Since the growth of solid neoplasms is almost always accompanied by neovascularization,21 which is associated with the expression of vascular endothelial growth factor receptor 1 (VEGFR1)22 and/or VEGFR2,23 our vaccine treatment also targeted peptides derived from VEGFR1 and VEGFR2 that are expressed in neovascular endothelial cells.

We conducted a phase II study of a cancer vaccine consisting of three peptides in combination with gemcitabine as a first‐line therapy for advanced pancreatic cancer, to evaluate the clinical benefit of this cancer vaccine treatment by adding to the standard therapy under the rules of ICH‐GCP.

Materials and Methods

Study design

This phase II, single‐arm, non‐randomized, HLA‐A‐status‐blind study was conducted to assess the efficacy of this combination therapy as first‐line treatment for advanced pancreatic cancer. This therapy consisted of a cocktail of three therapeutic epitope‐peptides in combination with gemcitabine. Chemotherapy naïve pancreatic cancer patients were enrolled to evaluate primarily the 1‐year survival rate, and secondarily overall survival (OS), progression free survival (PFS), response rate (RR), disease control rate (DCR) and peptide‐specific immune responses. Each of three peptides derived from KIF20A‐66 (3 mg/shot), VEGFR1‐1084 (2 mg/shot) and VEGFR2‐169 (2 mg/shot), was mixed with 1 mL of incomplete Freund's adjuvant (IFA) (Montanide ISA51; Seppic, Paris, France) and administered subcutaneously into the thigh or axilla regions once a week for the first 8 weeks, and then once every 2 weeks. Gemcitabine was administered at a dose of 1000 mg/m2 on days 1, 8, and 15 in a 28‐day cycle. All enrolled patients received therapy without the HLA‐A information, and the HLA genotypes were used for classification of the two groups for analysis. The endpoints were evaluated by comparison of the HLA‐A*2402‐matched group and the HLA‐A*2402‐unmatched group. The treatment was continued after the progression of disease by diagnostic imaging, until the disease progression was fully determined by the investigators in consideration of the patient's wishes and merits. The study treatment (vaccination + gemcitabine) was finalized according to proper discontinuance criteria. Written informed consent was obtained from each patient at the time of enrollment. The study was carried out in accordance with the Helsinki declaration on experimentation on human subjects, was approved by the Institutional Ethics Review Boards of Yamaguchi University (H24‐14) at each study site, and was registered in the UMIN Clinical Trials Registry as UMIN000008082.

Peptides

The KIF20A‐66 peptide (KVYLRVRPLL) restricted with HLA‐A*2402 was synthesized by BCN Peptides (Barcelona, Spain) and the VEGFR1‐1084 (SYGVLLWEI)24 and VEGFR2‐169 (RFVPDGNRI)25 peptides restricted with HLA‐A*2402 were synthesized by the American Peptide Company Inc. (Sunnyvale, CA, USA). These peptides were synthesized according to a standard solid‐phase synthesis method, and were then purified by reversed‐phase high‐performance liquid chromatography (HPLC). The purity (>95%) and identity of peptides were determined by analytical HPLC and mass spectrometry analysis, respectively. Endotoxin levels and the bio‐burden of these peptides were tested and confirmed to be within acceptable levels according to the Good Manufacturing Practice grade for vaccines.

Eligibility criteria

Eligible patients were 20 years of age or older, with locally advanced and/or metastatic pancreatic cancer that was histologically or cytologically diagnosed as adenocarcinoma, with no prior chemotherapy or radiotherapy for pancreatic cancer. If it was difficult to obtain histological or cytological data, image diagnosis was used to replace them. Entry criteria also included an Eastern Cooperative Oncology Group performance status of 0–1, a life expectancy of more than 3 months; and adequate hepatic, renal, bone marrow function, and lymphocyte percentage in the peripheral white blood cells of ≥15%. Eligible patients also had one or more measurable lesions according to the Response Evaluation Criteria in Solid Tumors version 1.1 (RECIST).

Adverse events and clinical responses

Adverse events were monitored according to the National Cancer Institute Common Terminology Criteria for Adverse Events version 3.0 (CTCAE). Clinical response was evaluated based on clinical observations and radiological findings. All known sites of disease were evaluated on a monthly basis by computed tomography (CT) or magnetic resonance imaging (MRI) before the treatment and after each cycle. Tumor size was estimated via direct measurement of the region of abnormal enhancement observed on CT or MRI. Patients were assigned a response category according to the Response Evaluation Criteria in Solid Tumors version 1.0 (RECIST). Overall survival (OS) was estimated from the date of the first vaccination to the date of death.

Enzyme‐linked immunoSpot assay

Specific CTL response was estimated by enzyme‐linked immunoSpot (ELISPOT) assay following in vitro sensitization.26 Frozen peripheral blood mononuclear cells (PBMCs) derived from the patient were thawed at the same time, and viability was confirmed as >90%. PBMCs (5 × 105/mL) were cultured with 10 μg/mL of the candidate peptide and 100 IU/mL of interleukin (IL)‐2 (Novartis, Emeryville, CA, USA) at 37°C for 2 weeks. Peptide was added into the culture on days 0 and 7. Following CD4+ cell depletion using a Dynal CD4‐positive isolation kit (Invitrogen, Carlsbad, CA, USA), an IFN‐γ ELISPOT assay was performed using a Human IFN‐γ ELISpot PLUS kit (MabTech, Nacka Strand, Sweden) according to instructions from the manufacturer. The number of peptide‐specific spots was calculated by subtracting the spot number in the control well from the spot number of a well with peptide‐pulsed stimulator cells. Peptide‐specific T‐cell response was classified into four grades (−, +, ++, or +++) according to the algorithm flow chart described in the previous report (Fig. S1).27 Sensitivity of this ELISPOT assay was estimated as being at an approximately average level by the ELISPOT panel of the Cancer Immunotherapy Consortium.28

Subgroup analysis

Some previous clinical trials of peptide vaccines with single‐arm treatment indicated better clinical outcomes in patients who showed a strong injection site reaction (ISR), suggesting that an ISR could be an indicator of immune response induced by peptide vaccines.29, 30 Subgroup analysis according to the degree of ISR was carried out. We defined ISR classification as follows: Grade 1; redness or induration, Grade 2; redness and induration, Grade 3; ulceration. We investigated ISR for all courses, and classified each grade.

Statistical analysis

Survival estimations were carried out using the Kaplan–Meier method. Log‐rank analysis was also carried out. Relations of treatment groups and each change were evaluated using the Mann–Whitney U‐test when changes were treated as continuous values. Relations between treatment groups and each change were evaluated using Fisher's exact test when changes were dichotomized into two groups at the median.

Results

Patient enrollment

Between June 2012 and May 2013, a total of 68 patients were enrolled in this study (Fig. 1); 38 patients had at least one allele of HLA‐A*2402 and 30 patients had no HLA‐A*2402 allele. The peptide vaccination was administered to all patients. One patient was excluded before treatment, and therefore safety was evaluated in 67 cases. One patient was excluded in a protocol violation; the other endpoints were therefore evaluated using the “full analysis set” of 66 cases (Fig. 1). The baseline characteristics showed no significant difference between the HLA‐matched and HLA‐unmatched groups (Table 1). Of 66 cases, 16 cases (24.2%) had locally advanced pancreatic cancer, 49 cases (74.2%) had metastatic pancreatic cancer, and the other case was not evaluated.

Figure 1

CONSORT diagram. Scheme showing an HLA‐A‐status double‐blind, biologically‐randomized phase ΙI study of three therapeutic epitope‐peptides combined with gemcitabine as a first‐line therapy for advanced pancreatic cancer (VENUS‐PC study).

Table 1

Baseline characteristics

	HLA‐A*24:02 matched (n = 37)	HLA‐A*24:02 unmatched (n = 29)	P‐value
Gender
Male	17 (45.9%)	19 (65.5%)
Female	20 (54.1%)	10 (34.5%)	NS
Age, years
Median (range)	64.0 (30–83)	63.0 (45–85)	NS
ECOG PS
PS0	33 (89.2%)	22 (75.9%)	NS
PS1	4 (10.8%)	7 (24.1%)
Primary tumor	32 (86.5%)	26 (89.7%)	NS
Recurrence	5 (13.5%)	3 (10.3%)
Pathology
Papillary adenoca.	0 (0.0%)	0 (0.0%)	NS
Tubular adenoca.	5 (13.5%)	2 (6.9%)	NS
Poorly diff. adenoca.	2 (5.4%)	1 (3.4%)	NS
Adenocarcinoma	23 (62.2%)	17 (58.6%)	NS
Other	0 (0.0%)	1 (3.4%)	NS
Not assessed	7 (18.9%)	8 (27.6%)	NS
Tumor marker
CA19‐9	397 (0.9–62500)	1012.7 (2.9–38106.7)	NS
CEA	5.41 (1–8027.3)	3.7 (1–38.4)	NS
CA125	48 (6–859.3)	46 (7.2–299.7)	NS
Extent of disease
Locally advanced	10 (27.0%)	6 (20.7%)	NS
Metastatic	27 (73.0%)	22 (75.9%)	NS
NE	0	1
Clinical stage (UICC)
III	10 (27.0%)	6 (20.7%)	NS
IV	27 (73.0%)	22 (75.9%)	NS

ECOG, Eastern Cooperative Oncology Group; HLA, human leukocyte antigens; NE, not evaluated; NS, not significant; PS, performance status; UICC, Unio Internationalis Contra Cancrum.

Clinical responses

The 1‐year survival rate was 27.0% and 34.5% in the HLA‐matched and HLA‐unmatched groups, respectively (Fig. 2a, P = 0.663). The median OS was 9.0 months for the HLA‐matched group and 10.0 months for the HLA‐unmatched group (Fig. 2a, P = 0.456). The median PFS was 4.7 months for the HLA‐matched group and 5.2 months for the HLA‐unmatched group (Fig. 2b, P = 0.275). The response rate was 10.8% in the HLA‐matched group and 13.8% in the HLA‐unmatched group (Table 2, P = 0.723). The disease control rate was 70.3% in the HLA‐matched group and 79.3% in the HLA‐unmatched group (Table 2, P = 0.572).

Figure 2

Kaplan–Meier estimates of overall survival (a) and progression‐free survival (b) in the full analysis set of pancreatic cancer patients treated with gemcitabine and peptide vaccination. N.S., not significant by the log‐rank test.

Table 2

Clinical response

	Total	HLA‐A*2402 matched	HLA‐A*2402 unmatched	P‐value
Number	66	37	29
CR	0	0	0
PR	8	4	4
SD	41	22	19
PD	15	10	5
NE	2	1	1
RR (%)	12.1	10.8	13.8	NS
RR 80% CI	7.2–18.9%	4.8–20.5%	6.2–25.7%
DCR (%)	74.20%	70.30%	79.30%	NS
DCR80% CI	66.1–81.2%	58.6–80.2%	66.5–88.8%

Clinical responses were evaluated according to Response Evaluation Criteria in Solid Tumors version 1.1. CR, complete response; DCR, disease control rate; HLA, human leukocyte antigens; PD, progressive disease; PR, partial response; RR, response rate; SD, stable disease.

Additional peptide treatment

The study treatment was continued until the disease progression was fully determined by the investigators in consideration of the patient's wishes and merits. The study treatment (vaccination + gemcitabine) was finalized according to proper discontinuance criteria. After the study treatment, the peptide injection was continued according to the patient's wishes under another protocol (IRB approved number; H24–41). A total of 24 patients were treated by the continuous administration of vaccination with or without any anti‐cancer drug (range: 0.4–27.7 months, median; 1.8 months).

Immunological monitoring

For each of the three peptides, we compared the positive CTL values (CTL+ or CTL++ or CTL+++) with the negative CTL values (CTL−). The patients with a peptide‐specific IFN‐γ response (CTL induction) for either KIF20A or VEGFR1 showed significantly better OS compared to those without an IFN‐γ response in the HLA‐A*2402 matched group (P = 0.023, P = 0.009, respectively). OS of patients with and without a KIF20A‐specific IFN‐γ response was 11.2 and 7.2 months, respectively (P = 0.023, Fig. 3a). OS of patients with and without a VEGFR1‐specific IFN‐γ response was 11.2 and 7.6 months, respectively (P = 0.009, Fig. 3b). On the other hand, OS of patients with and without a VEGFR2‐specific IFN‐γ response showed no statistical difference (P = 0.306, Fig. 3c). Although we also analyzed how the responses intensity (+ to +++) affected on the clinical outcomes, there was no association between the responses intensity and OS (data not shown).

Figure 3

Peptide‐specific IFN‐γ response (induction of CTLs) for KIF20A, VEGFR1 and VEGFR2, and its correlation with prognosis in the HLA‐A*2402 matched group. For each of the three peptides, we compared the positive CTL values (CTL+ or CTL++ or CTL+++) with the negative CTL values (CTL−). The patients with CTL induction specific to KIF20A and VEGFR1 showed significantly better OS compared to those without CTL induction in the HLA‐A*2402 matched group. IFN‐γ response was measured using the ELISPOT assay as described in Materials and Methods.

Safety

The incidence of hematological toxicity was high, but was not significantly different between the two groups except for Grade 3–4 thrombocytopenia. The incidence of non‐hematological toxicity was generally low in both groups, and was not significantly different between the two groups (Table 3). There were no deaths related to the protocol treatment.

Table 3

Summary of Grade 3 or worse adverse events related to the study drug

Drug related AE	HLA‐A*2402 matched (n = 38)		HLA‐A*2402 unmatched (n = 29)		P‐value
	All No. (%)	Grade 3–4 No. (%)	All No. (%)	Grade 3–4 No. (%)
Hematologic
Leukocytopenia	18 (47.4)	13 (34.2)	13 (44.8)	8 (27.6)	NS
Neutropenia	24 (63.2)	18 (47.4)	19 (65.5)	17 (58.6)	NS
Febrile neutropenia	1 (2.6)	1 (2.6)	0 (0.0)	0 (0.0)	NS
Thrombocytopenia	15 (39.5)	8 (21.1)	9 (31.0)	1 (3.4)	G3–4, P < 0.05
Anemia	8 (21.0)	1 (2.6)	9 (31.0)	0 (0.0)	NS
Non‐hematologic
ALT	2 (5.2)	1 (2.6)	1 (3.4)	1 (3.4)	NS
AST	1 (2.6)	1 (2.6)	1 (3.4)	1 (3.4)	NS
Cholangitis	7 (18.5)	5 (13.2)	4 (13.7)	3 (10.3)	NS
Bile duct stenosis	1 (2.6)	1 (2.6)	1 (3.4)	1 (3.4)	NS
Fatigue	13 (34.2)	3 (7.9)	5 (17.2)	0 (0.0)	NS
Anorexia	19 (50.0)	6 (15.8)	10 (34.4)	3 (10.8)	NS
Fever	13 (34.2)	0 (0.0)	9 (31)	1 (3.4)	NS
Diarrhea	8 (21.1)	2 (5.3)	4 (13.8)	0 (0.0)	NS
Nausea	19 (50.0)	1 (2.6)	14 (48.2)	3 (10.3)	NS
Vomiting	14 (36.8)	0 (0.0)	10 (34.5)	1 (3.4)	NS
Intestinal pneumonia	1 (2.6)	0 (0.0)	2 (6.9)	1 (3.4)	NS
Injection site reaction	35 (92.1)	3 (7.9)	24 (82.8)	1 (3.4)	NS

AE, adverse events; HLA, human leukocyte antigens.

In the subgroup analysis, the patients showing a strong injection site reaction (ISR) of Grade 2–3 showed significantly better OS compared to those with a weak ISR of Grade 0–1 in the HLA‐A*2402 matched group. The median survival time of patients with a strong ISR and a weak ISR was 10.8 and 6.3 months, respectively (log‐rank test, P = 0.017, Fig. 4a). On the other hand, there were no significant differences between a strong ISR and a weak ISR in the HLA‐A*2402 unmatched group (Fig. 4b). A representative case of a strong injection site reaction and clinical response is shown in Figure 5. This case showed a Grade 3 injection site reaction (Fig. 5a), and clinical response was evaluated as a partial response (Fig. 5b,c).

Figure 4

Injection site reaction (ISR) and its correlation with prognosis according to HLA‐genotypes. ISR Grade 2, 3, strong; ISR Grade 0, 1, weak.

Figure 5

A representative case of a strong injection site reaction and clinical response. This case showed a Grade 3 injection site reaction (a). The clinical response was evaluated as a partial response (white arrow) (b) before treatment, 27 × 19 mm and (c) after three courses, 17.9 × 13.1 mm.

Discussion

Following a phase I cancer vaccination trial using KIF20A, which determined its safety and immunogenicity in advanced pancreatic cancer patients,31 in this current study we conducted a phase II trial using a cocktail of KIF20A and antiangiogenic cancer vaccines targeting VEGFR1 (vascular endothelial growth factor receptor 1) and VEGFR2. The safety and immunogenicity of these two antiangiogenic peptides have been confirmed in advanced colorectal cancer.29, 32 We also previously reported that the survival period for patients in a study using a cocktail of five peptide vaccines for advanced colorectal cancer was significantly longer (P = 0.032) in patients who showed CTL induction against three or more peptides, compared with those with CTL induction against two peptides or fewer.29 Here we attempted to evaluate the clinical benefit of the cancer vaccination in combination with gemcitabine in advanced pancreatic cancer patients. The present study was an HLA‐A‐status‐blind, phase ΙI study using a cocktail of three epitope peptides (KIF20A, VEGFR1 and VEGFR2) with gemcitabine as a first‐line therapy for advanced pancreatic cancer.

The toxicity observed in the present study was feasible not only for the HLA‐A*2402 matched group but also for the HLA‐A*2402 unmatched group. However, Grade 3–4 thrombocytopenia was observed significantly more often in the HLA‐A*2402 matched group than the HLA‐A*2402 unmatched group. Since thrombocytopenia could be quickly improved by suspension of the administration of gemcitabine, we considered that thrombocytopenia was not related to the cancer vaccine treatment. The other adverse events were not significantly different between the two groups. Interstitial pneumonia was detected in one patient in the HLA‐A*2402‐matched group; we cannot exclude the possibility that this might be related to the cancer vaccine treatment. The patient recovered with appropriate treatment. In conclusion, this study is safe and well tolerated in this cohort.

In this study, we found no statistical difference in the 1‐year survival rate, OS rate or PFS rate between the HLA‐A*2402 matched group and the HLA‐A*2402 unmatched group. However, we did observe some interesting things.

Firstly, the patients with peptide‐specific CTL induction against either KIF20A or VEGFR1 showed significantly better OS compared to those without CTL induction in the HLA‐A*2402 matched group (Fig. 2). Treatment with cancer vaccines has been shown to cause an increase in circulating tumor antigen‐specific T cells.29, 33 In this respect, we in this study have demonstrated evidence of a positive correlation between the induction of peptide‐specific CTL responses and a better clinical outcome.

Secondly, the patients who showed a strong injection site reaction (ISR) of Grade 2–3 showed significantly better OS than those who showed an ISR of Grade 0–1 in the HLA‐A*2402 matched group (Fig. 3). In contrast, no significant difference was observed between strong and weak ISR groups in the HLA‐A*2402 unmatched patients. Some previous clinical trials of peptide vaccines with single‐arm treatment indicated better clinical outcomes in patients who showed a strong ISR, suggesting that ISR could be an indicator of immune response induced by peptide vaccines.30

One of the important reasons why we could not find any statistical differences in the 1‐year survival rate, OS rate or PFS rate between the HLA‐A*2402 matched group and the HLA‐A*2402 unmatched group, was the expression status of target antigens and HLA class I in tumor tissues. In our study, because we enrolled almost only unresectable pancreatic cancer patients, there was no chance to check the expression of VEGFR1, VEGFR2 and KIF20A. Expressions of HLA class I on the tumor cells were reported to be approximately 60–90%.34 Yamaue et al.35 found that immunohistochemically positive results for KIF20A were seen in seven out of the 30 cases (23.3%) of pancreatic cancer patients. VEGFR1 and VEGFR2 were analyzed in tumor cells and the tumor tissues with RT‐PCR; however, they could not obtain significant information with immunohistochemistry.24, 25 Based on these results, the selection of good target antigens might be also important for effective clinical vaccination in the future. Therefore, we have to research new tumor antigen candidates such as MUC‐1,36 WT137 and HSP7038 to induce strong immunization.

However, this phase II cancer vaccine therapy demonstrated that our therapeutic peptide cocktail might be effective in a subset of patients. It is also certainly important to find biomarkers such as serum IL6, NLR, and lymphocyte‐%29, 32, 39 in order to assess the response to the peptide vaccine and to select patients who are likely to have a better treatment outcome with the vaccination. It is also important to use a more effective adjuvant or to use peptide vaccines with a combination of molecular targeted drugs or radiation. We have previously shown in an experimental model that treatment with the combined adjuvant of poly(I:C) plus LAG‐3‐Ig profoundly enhanced peptide‐specific antitumor responses and led to complete regression of a pre‐established tumor in association with long‐term immunological memory.40 Several commonly‐used drugs, such as cyclophosphamide,41, 42 COX‐2 inhibitor,43 metformin,44 and cimetidine45 have shown an ability to modify the suppressive immune status in tumor microenvironments, and might enhance the immune responses induced by peptide vaccines. We are planning a combination peptide vaccine therapy and agents for immunomodulation against cancer in the near future.

Disclosure Statement

Yusuke Nakamura is a stock holder and a scientific advisor of OncoTherapy Science, Inc. The other authors have no potential conflicts of interest to disclose.

cytotoxic T lymphocyte

disease control rate

human leukocyte antigen

International Conference on Harmonization of Good Clinical Practice

injection site reaction

overall survival

progression free survival

response rate

Fig. S1. Positivity of antigen‐specific T cell response was quantitatively defined according to the evaluation tree algorithm.

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