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Abstract
Background We aimed to develop a chemoimmunotherapy regimen consisting of a novel Wilms’ tumor 1 (WT1) peptide-pulsed dendritic cell (WT1-DC) vaccine and multiagent chemotherapy and to investigate the safety, clinical outcomes, and WT1-specific immune responses of patients with unresectable advanced pancreatic ductal adenocarcinoma (UR-PDAC) who received this treatment.
Methods Patients with UR-PDAC with stage III disease (locally advanced (LA-PDAC; n=6)), stage IV disease (metastatic (M-PDAC; n=3)), or recurrent disease after surgery (n=1) were enrolled in this phase I study. The patients received one cycle of nab-paclitaxel plus gemcitabine alone followed by 15 doses of the WT1-DC vaccine independent of chemotherapy. The novel WT1 peptide cocktail was composed of a multifunctional helper peptide specific for major histocompatibility complex class II, human leukocyte antigen (HLA)-A*02:01, or HLA-A*02:06 and a killer peptide specific for HLA-A*24:02.
Results The chemoimmunotherapy regimen was well tolerated. In the nine patients for whom a prognostic analysis was feasible, the clinical outcomes of long-term WT1 peptide-specific delayed-type hypersensitivity (WT1-DTH)-positive patients (n=4) were significantly superior to those of short-term WT1-DTH-positive patients (n=5). During chemoimmunotherapy, eight patients were deemed eligible for conversion surgery and underwent R0 resection (four patients with LA-PDAC, one patient with M-PDAC, and one recurrence) or R1 resection (one patient with M-PDAC), and one patient with LA-PDAC was determined to be unresectable. Long-term WT1-DTH positivity was observed in three of the four patients with R0-resected LA-PDAC. These three patients exhibited notable infiltration of T cells and programmed cell death protein-1+ cells within the pancreatic tumor microenvironment (TME). All patients with long-term WT1-DTH positivity were alive for at least 4.5 years after starting therapy. In patients with long-term WT1-DTH positivity, the percentage of WT1-specific circulating CD4+ or CD8+ T cells that produced IFN-γ or TNF-α was significantly greater than that in patients with short-term WT1-DTH positivity after two vaccinations. Moreover, after 12 vaccinations, the percentages of both circulating regulatory T cells and myeloid-derived suppressor cells were significantly lower in patients with long-term WT1-DTH-positive PDAC than in short-term WT1-DTH-positive patients.
Conclusions Potent activation of WT1-specific immune responses through a combination chemoimmunotherapy regimen including the WT1-DC vaccine in patients with UR-PDAC may modulate the TME and enable conversion surgery, resulting in clinical benefits (Online supplemental file 1).
Trial registration number jRCTc030190195.
- Vaccine
- Tumor microenvironment - TME
- Tumor infiltrating lymphocyte - TIL
- Immunotherapy
- Dendritic
Data availability statement
Data are available upon reasonable request.
This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. See http://creativecommons.org/licenses/by-nc/4.0/.
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WHAT IS ALREADY KNOWN ON THIS TOPIC
Novel therapies that induce a Wilms’ tumor 1 (WT1)-specific immune response combined with chemotherapy may be important strategies to improve the clinical outcomes of patients with unresectable pancreatic ductal adenocarcinoma (UR-PDAC).
WHAT THIS STUDY ADDS
Mature dendritic cells (DCs) were pulsed with a novel WT1 peptide cocktail. The cocktail consisted of a multifunctional WT1-specific helper peptide designed to be specific for major histocompatibility complex class II, human leukocyte antigen (HLA)-A*02:01, or HLA-A*02:06, and a killer peptide specific for HLA-A*24:02. The DC vaccine was administered in combination with multiagent chemotherapy (nab-paclitaxel (Nab-P) plus gemcitabine (GEM): Nab-P/Gem).
HOW THIS STUDY MIGHT AFFECT RESEARCH, PRACTICE OR POLICY
7 of the 10 patients with UR-PDAC successfully underwent conversion surgery, including R0 resection (n=6) and R1 resection (n=1).
The induction and maintenance of long-term WT1-specific CD4+ and CD8+ immune responses are associated with favorable clinical outcomes. Super-responders exhibit a high density of WT1-specific tumor-infiltrating lymphocytes and programmed cell death protein-1+ cells but a low density of regulatory T cells in the tumor microenvironment (TME).
Potent activation of WT1-specific immune responses via chemoimmunotherapy in patients with UR-PDAC modulates the TME and enables conversion surgery, resulting in clinical benefits.
Introduction
Pancreatic ductal adenocarcinoma (PDAC) is a disease with a high fatality rate, characterized by inherent resistance to cytotoxic chemotherapy and radiation therapy.1 Most PDAC cases involve a stroma in which cells and molecules form a highly immunosuppressive and desmoplastic tumor microenvironment (TME) consisting of tumor-associated macrophages (TAMs), cancer-associated fibroblasts, myeloid-derived suppressor cells (MDSCs), tumor blood vessels, cytotoxic T lymphocytes (CTLs), T helper (Th) cells, natural killer cells, regulatory T cells (Tregs), and cancer stem cells.2 3 Therefore, novel therapies that target not only tumor cells but also the TME are urgently needed to improve the clinical outcomes of patients with unresectable PDAC (UR-PDAC).
Wilms’ tumor 1 (WT1) is a transcription factor that is closely associated with angiogenesis, tumor progression, invasion, and metastasis and functions as an oncogene in tumorigenesis.4–6 Importantly, WT1 is expressed not only in PDAC cells but also in cancer stem cells, MDSCs, and tumor blood vessels in the TME, indicating its potential as a viable target for cancer therapy.4 Previously, we conducted a clinical trial of patients with UR-PDAC who were treated with gemcitabine (GEM) combined with WT1-targeted vaccines.7 8 The induction of WT1-specific immunity was associated with positive clinical results in some patients. These findings suggest that inducing a stronger WT1-specific immune response through the administration of vaccines and chemotherapy is an important strategy for improving therapeutic efficacy in patients.
Dendritic cells (DCs) are professional antigen-presenting cells that play critical roles in the initiation and regulation of tumor-associated antigen-specific immune responses.9 We previously reported the clinical benefits of chemoimmunotherapy consisting of GEM and major histocompatibility complex (MHC) class I/II-restricted WT1 peptide-pulsed DC (WT1-DC) vaccines in patients with UR-PDAC.7 However, some patients with UR-PDAC do not show any clinical benefit. Recently, we developed a novel WT1 helper peptide (WT1-HP) for the simultaneous efficient induction of CD4+ Th1 cells and WT1-specific CTLs (WT1-CTLs).10 Interestingly, this novel WT1-HP contains human leukocyte antigen (HLA)-A*02:01-restricted or HLA-A*02:06-restricted peptide sequences and can activate not only WT1-specific CD4+ Th1 cells but also WT1-CTLs in an HLA-A*02:01-restricted or HLA-A*02:06-restricted manner.10
We report a clinical trial conducted to confirm the safety of this treatment strategy and further improve the outcomes of patients with UR-PDAC using mature DCs pulsed with a peptide cocktail containing an HLA-A*24:02-restricted WT1 killer peptide (WT1-24:02-KP) and a novel WT1-HP (WT1-DC) combined with nab-paclitaxel (Nab-P) plus GEM (Nab-P/Gem).11 12
Materials and methods
Study design
This study was a noncomparative, open-label, phase I study. The primary endpoints were safety and toxicity according to the Common Terminology Criteria for Adverse Events (V.5.0). The secondary endpoints were the WT1-specific immune response and clinical outcomes (progression-free survival (PFS) and overall survival (OS)).
Patient population
All patients exhibited pathologically confirmed adenocarcinoma with WT1 and MHC class I expression (Union for International Cancer Control (UICC) stage III (locally advanced (LA-PDAC); n=6), stage IV (metastatic (M-PDAC); n=3), or recurrent disease after surgery (n=1)). Patients were required to have a specific HLA type (online supplemental figure 1). Additionally, adult patients with a Karnofsky Performance Status of 80–100% and adequate hematologic and organ function who were a minimum of 6 months from the completion of any previous treatment were included. The exclusion criteria were pregnancy, serious infections, severe underlying disease, and severe allergic disease. All patients provided written informed consent.
Supplemental material
WT1-DC vaccine
A novel WT1 peptide cocktail (WT1-HP for MHC class II, HLA-A*02:01, or HLA-A*02:06: WAPVLDFAPPGASAYGSL, 34–51 and WT1-24:02-KP: CYTWNQMNL, 235–243) was used to generate the WT1-DC vaccine as described previously.13 To assess the phenotype of WT1-DCs, WT1-DCs were incubated with the following monoclonal antibodies (mAbs): Fluorescein isothiocyanate (FITC)-conjugated anti-human CD14 (61D3) (Thermo Fisher Scientific, Waltham, Massachusetts, USA), HLA-ABC (W6/32), CD80 (2D10), CD40 (5C3), phycoerythrin (PE)-conjugated anti-human CCR7 (150503) (all from R&D Systems, Minneapolis, Minnesota, USA), CD11c (3.9), HLA-DR (L243), CD83 (HB 15e), and CD86 (IT2.2) (all from BioLegend, San Diego, California, USA).
Chemoimmunotherapy
Nab-P 125 mg/m2 plus GEM 1,000 mg/m2 was administered on days 1, 8, and 15 of each 28-day cycle11 12 at the beginning of the experiment. After the first cycle of Nab-P/GEM administration, the patients were treated with Nab-P/GEM combined with the WT1-DC vaccine because it took approximately 1 month to prepare the WT1-DC vaccine (online supplemental figure 2). Approximately 1×107 WT1-DCs/dose were suspended in 0.5 mL of saline containing penicillin-killed and lyophilized preparations of a low-virulence strain of Streptococcus pyogenes (OK-432, Chugai Pharmaceutical, Tokyo, Japan), and the doses of OK-432 used were 0.2 Klinische Einheit (KE) once, 0.5 KE once, and then 1.0 KE continuously. The WT1-DC vaccine was intradermally administered at six sites (bilateral upper arms, lower abdomen, and femoral region) every 2 weeks for the first 6 doses and every 4 weeks thereafter regardless of chemotherapy. The number of doses was capped at 15 because one apheresis session can provide at least 15 doses of vaccine. Second-line chemotherapy treatment due to disease progression was not specified. After a minimum of 6 months of continued treatment, conversion surgery was deemed an acceptable option for patients who were able to undergo R0 resection. After surgical resection, the remaining vaccines were administered starting approximately 1 month after surgery. Adjuvant chemotherapy (S1, an oral fluoropyrimidine, which is the main chemotherapy regimen for PDAC in Japan,14 or Nab-P/Gem11 12) was also continued for at least 6 months to prevent recurrence.
Supplemental material
Clinical responses
Contrast-enhanced CT (CE-CT) was performed at intervals of approximately 4 weeks or more until the disease progressed. Treatment efficacy was evaluated according to the Response Evaluation Criteria in Solid Tumors. Patients who achieved stable disease (SD) for at least 6 months were defined as having long-term SD. Positron emission tomography with 2-deoxy-2-(fluorine-18)-fluoro-D-glucose integrated with CT (18F-FDG PET/CT)15 was also performed before and approximately 6 months after treatment, and the maximum standardized uptake value (SUVmax) was measured.
WT1 peptide-specific delayed-type hypersensitivity test
The WT1 peptide-specific delayed-type hypersensitivity (WT1-DTH) test was performed once before treatment and at the time of each vaccination. Briefly, 20 µg of the WT1 peptide (WT1-24:02-KP or WT1-HP) in saline or saline alone was intradermally injected into the forearm, and the maximum diameter of erythema was measured 48 hours after injection. WT1-DTH positivity was defined as erythema greater than 2 mm in diameter. An erythema size of 5 mm was used to discriminate between weak (2–5 mm) and strong (≥6 mm) WT1-DTH-positive reactions.7
Intracellular staining for IFN-γ and TNF-α
Peripheral blood mononuclear cells (PBMCs) were stimulated with 10 µg/mL HLA-A type-matched WT1-KPs and 20 µg/mL WT1-HP (both from Greiner Bio-One GmbH, Frickenhausen, Germany) in the presence of 10 U/mL rhIL-2 (Shionogi & Co, Osaka, Japan) and 10 ng/mL IL-7 (PeproTech, Rocky Hill, New Jersey, USA) for 9 days. HLA-A type-matched WT1-KPs were selected as described below: WT1-24:02-KP (235-243), HLA-A*02:01-restricted WT1-KP (VLDFAPPGASA, 37–47), and HLA-A*26:01- or HLA-A*26:03-restricted WT1-KP (FAPPGASAY, 40–48). HIV envelope peptides and matched isotype immunoglobulin G (IgG) (all from BioLegend) were used as negative controls. The cells were restimulated with 10 µg/mL HLA-A type-matched WT1-KPs, 20 µg/mL WT1-HP, and 10 µg/mL brefeldin A (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) for 5 hours. The cells were stained with the following mAbs: FITC-conjugated anti-human CD8 (T8) (Beckman Coulter, Brea, California, USA), APC-Cy7-conjugated anti-human CD4 (OKT4), APC-conjugated anti-human IFN-γ (B27), or APC-conjugated anti-human TNF-α (MAb11) (all from BioLegend). IFN-γ-producing or TNF-α-producing CD4+ or CD8+ T cells were analyzed. Moreover, all seven patients with HLA-A*24:02 were evaluated for the induction of HLA-A*24:02-restricted WT1-CTLs producing IFN-γ or TNF-α via PE-conjugated HLA-A*24:02-modified WT1-tetramer CYTWNQMNL (235-243) (MBL, Nagoya, Japan).
WT1-specific tumor-infiltrating lymphocytes
The resected fresh solid tumors were minced and passed through a cell strainer. Suspended cells were isolated via Ficoll-Paque density gradient centrifugation.16 The cells were stained with the following mAbs: PE-conjugated HLA-A*24:02-modified WT1-tetramer (MBL) and FITC-conjugated anti-human CD8 (RPA-T8) (BioLegend). Matched isotype IgG (BioLegend) was used as a negative control. The percentage of HLA-A*24:02-modified WT1-tetramer+cells among total CD8+ T cells was determined.
Detection of immunosuppressive cells
To assess CD4+ CD25+ forkhead box p3 (Foxp3) Tregs among total CD4+ T cells, PBMCs were stained with the following mAbs: APC/Cy7-conjugated anti-human CD4 (OKT4), APC-conjugated anti-human CD25 (BC96) (both from BioLegend), PE-conjugated anti-human Foxp3 (PCH101) (eBioscience, San Diego, California, USA) or matched isotype control IgG (BioLegend) using the Human/Non-Human Primate Regulatory T-Cell Staining Kit (Thermo Fisher Scientific). Moreover, to assess MDSCs, PBMCs were stained with the following mAbs: PE-conjugated anti-human CD14 (M5E2), FITC-conjugated anti-human CD11b (ICRF44), APC-conjugated anti-human CD33 (WM53), or matched isotype control IgG (all from BioLegend). The CD14−CD11b+CD33+ population among total PBMCs was defined as MDSCs.
Flow cytometric analysis
The phenotypes of the cells were analyzed via an Attune NxT Flow Cytometer (Thermo Fisher Scientific) and FlowJo analysis software (V.10.10.0. Tree Star, Ashland, Oregon, USA).
Immunohistochemical staining
Immunohistochemistry (IHC) was performed for WT1 and MHC class I in tumor cells obtained before treatment and for CD3, CD4, CD8, programmed cell death protein-1 (PD-1), Foxp3, CD68, and CD163 in six surgically resected during immunochemotherapy2 17–21 (online supplemental table 1). The cell nuclei were counterstained with hematoxylin. Negative control staining was performed with mouse IgG1 (Santa Cruz Biotechnology, Santa Cruz, California, USA) or rabbit IgG (diluted 1:590; DA1E; CST, Danvers, Massachusetts, USA). The immunohistochemical results were evaluated by three observers who were blinded to the clinical information. The density of immune-related cells (cell counts/mm2 tumor area), the percentage of Foxp3+/CD4+ cells, and the CD68+/CD163+ ratio were evaluated by counting the mean number of stained cells from 10 randomly selected tumor areas under 400×magnification. The intensity of WT1 expression was classified as weak (faint and barely perceptible cytoplasmic staining) or moderate (moderate complete cytoplasmic staining);19 the intensity of MHC class I staining was classified as moderate (less than 90% and more than 50% of PDAC cells) or strong (more than 90% of PDAC cells).17
Supplemental material
Statistical analysis
Statistical analyses of PFS or OS were performed via the Kaplan-Meier method and were evaluated via the log-rank test. Immunologic parameters were evaluated via Student’s t-test for two independent groups and one-way analysis of variance and Tukey’s multiple comparison test for multiple-group comparisons. P value<0.05 was considered to indicate a statistically significant difference. Statistical analyses were performed via GraphPad Prism V.10.2.3 software (GraphPad Software, San Diego, California, USA).
Results
Patient characteristics
We performed HLA typing of 14 patients to screen for eligibility, and all patients were matched for HLA type. However, 2 patients were not enrolled because a histologic diagnosis was not requested, and another 2 patients did not wish to participate in the treatment because of their distance from the hospital (online supplemental figure 1). Ultimately, 10 patients with UR-PDAC were enrolled between August 2018 and May 2020 (table 1).
Patient with UR-PDAC characteristics
WT1-DC vaccine quality
As shown in online supplemental table 2, WT1-DCs presented high levels of HLA-ABC, HLA-DR, CD80, CD86, CD83, CD40, and CD11c; moderate levels of CCR7; and low levels of CD14. The recovery rate of WT1-DCs from immature DCs was 84.52±21.72%. Trypan blue exclusion analysis confirmed the viability of the WT1-DCs.
Toxicity
As shown in online supplemental table 3, all patients experienced grade 1 skin reactions at the vaccination sites and grade 1–4 hematologic adverse events (neutropenia, leukocytopenia, lymphopenia, anemia, or thrombocytopenia) during three cycles of therapy. Other non-hematologic adverse events included grade 1–2 anorexia and nausea (six patients), grade 1–2 hepatic transaminase elevation (five patients), and grade 1 hypoalbuminemia (two patients), all of which have been previously reported as having Nab-P/GEM.11 12 The average total dose of Nab-P/GEM during three cycles of therapy was 67.67% of the standard dose due to chemotherapy-induced bone marrow suppression. Finally, all patients were able to complete 15 rounds of vaccination without toxicity. Administration of the WT1-DC vaccine in combination with Nab-P/GEM was well tolerated.
Clinical response
The best overall response was assessed by CE-CT. All patients had decreased tumor burdens (from 0.0% to −57.4%) (online supplemental figure 3). None of the patients achieved a complete response and seven patients (six LA-PDAC and one M-PDAC) achieved a partial response. The remaining three patients exhibited long-term SD. After approximately 6 months of treatment, the SUVmax significantly decreased (p=0.006) (figure 1 and online supplemental figure 4).
Supplemental material
Supplemental material
CE-CT and 18F-FDG PET/CT imaging. CE-CT and 18F-FDG PET/CT images of four patients with UR-PDAC (WT1-DC-01, WT1-DC-03, WT1-DC-05, and WT1-DC-09) are presented, demonstrating the changes observed before and after treatment. Yellow circles represent tumor lesions. CE-CT, contrast-enhanced CT; 18F-FDG PET/CT, positron emission tomography with 2-deoxy-2-(fluorine-18)-fluoro-D-glucose integrated with CT; UR-PDAC, unresectable pancreatic ductal adenocarcinoma; WT1-DC, Wilms’ tumor 1 peptide-pulsed dendritic cell.
Conversion surgery
During chemoimmunotherapy, four (WT1-DC-01, WT1-DC-03, WT1-DC-04, and WT1-DC-09) of the six patients with LA-PDAC, one (WT1-DC-05) of the three patients with M-PDAC, and one patient with a solitary lung metastasis in recurrence (WT1-DC-07) successfully underwent R0 surgical resection (online supplemental table 4). One patient with M-PDAC (WT1-DC-02) did not experience tumor shrinkage; however, the pretreatment SUVmax was 8.00 and decreased to 6.23 after 239 days of treatment (online supplemental figures 4 and 5). Therefore, we performed surgical resection under the expectation of R0 resection, resulting in R1 resection. The mean duration of treatment up to conversion surgery was 221 days in all seven patients for whom surgical resection was performed.
Supplemental material
Clinical outcomes
One patient with LA-PDAC (WT1-DC-10) had a remarkable reduction in tumor size (−57.4%), 18F-FDG uptake disappeared, and the patient was expected to undergo R0 resection. However, the tumor could not be removed from the major blood vessels surrounding the tumor. The patient was unable to receive sufficient treatment for some periods due to severe complications (lymphatic leakage and sepsis) from surgery, which affected OS and PFS. Therefore, the Data Safety and Monitoring Board excluded this patient (WT1-DC-10) from the assessment of secondary endpoints. As shown in figure 2A, the median PFS was 2.23 years, and the median OS was 3.52 years in all nine patients. Among the five patients with LA-PDAC, the median PFS and OS were 2.48 years and not reached, respectively. These values were significantly greater than the median PFS and OS for the three patients with M-PDAC (1.37 and 2.41 years, respectively) (p=0.046 and 0.022, respectively) (figure 2B).
K-M estimates of PFS and OS for patients with UR-PDAC. (A) PFS (left panel) or OS (right panel) of patients with UR-PDAC (n=9) who were treated with WT1-DC vaccines combined with chemotherapy. (B) PFS (left panel) or OS (right panel) for patients with LA-PDAC (n=5), patients with M-PDAC (n=3), and one patient with solitary lung recurrence (n=1). (C) PFS (left panel) or OS (right panel) in patients with UR-PDAC with long-term WT1-DTH positivity (n=4) or short-term WT1-DTH positivity (n=5). (D) PFS (left panel) and OS (right panel) of patients with LA-PDAC with long-term WT1-DTH positivity (n=3), patients with LA-PDAC with short-term WT1-DTH positivity (n=2), patients with M-PDAC with short-term WT1-DTH positivity (n=3), and one recurrent patient with long-term WT1-DTH positivity (n=1). K-M, Kaplan-Meier; LA-PDAC, locally advanced pancreatic ductal adenocarcinoma; M-PDAC, metastatic pancreatic ductal adenocarcinoma; OS, overall survival; PFS, progression-free survival; UR-PDAC, unresectable pancreatic ductal adenocarcinoma; WT1-DC, Wilms’ tumor 1 peptide-pulsed dendritic cell; WT1-DTH, Wilms’ tumor 1 peptide-specific delayed-type hypersensitivity.
WT1-DTH test and clinical prognosis
None of the patients exhibited positive WT1-DTH prior to treatment. During the vaccination periods, all nine patients were positive for WT1-DTH at least once, but the degree of positivity varied. The median number of positive WT1-DTH was 6 out of 15 tests. Therefore, the nine patients were first divided into two groups: The long-term WT1-DTH-positive group (>6 times) (WT1-DC-01, WT1-DC-03, WT1-DC-07, and WT1-DC-09) and the short-term WT1-DTH-positive group (≤6 times) (WT1-DC-02, WT1-DC-04, WT1-DC-05, WT1-DC-06, and WT1-DC-08) (table 2). In addition, two patients (WT1-DC-03 and WT1-DC-06) were negative for WT1-24:02-KP-DTH due to HLA-A*24:02 negativity. Interestingly, the clinical outcomes of long-term WT1-DTH-positive patients (median PFS and OS: Not reached) were significantly better than those of short-term WT1-DTH-positive patients (median PFS and OS: 1.37 years and 2.56 years, respectively) (p=0.005 and 0.005, respectively) (figure 2C). Moreover, among the five patients with LA-PDAC, those with long-term WT1-DTH positivity (n=3) had significantly longer clinical outcomes (median PFS and OS: Not reached) than did those with short-term WT1-DTH positivity (n=2) (median PFS and OS: 1.68 years and 3.04 years, respectively) (p=0.039 and 0.039, respectively) (figure 2D). All four long-term WT1-DTH-positive patients (three with LA-PDAC and one with lung recurrence) were alive for at least 4.5 years, and only one patient (WT1-DC-09) experienced relapse at 908 days after entry (figure 2D). We also evaluated the intensity of the WT1-DTH reaction. Strong WT1-24:02-KP-DTH or WT1-HP-DTH positivity was identified 6–12 times in long-term WT1-DTH-positive patients. In contrast, patients with short-term WT1-DTH positivity demonstrated strongly positive WT1-DTH test results, ranging from 0 to 4 times (table 2). The WT1-DTH test was also conducted approximately 3 years after the final vaccination in all four super-responders. One patient (WT1-DC-07) demonstrated weakly positive results for both WT1-24:02-KP-DTH and WT1-HP-DTH. Two patients (WT1-DC-01 and WT1-DC-03) presented weakly positive results for WT1-HP-DTH only, whereas both WT1-24:02-KP-DTH and WT1-HP-DTH were negative in one patient (WT1-DC-09). Furthermore, one patient (WT1-DC-03) was negative for WT1-24:02-KP-DTH due to HLA-A*24:02 negativity.
The number of WT1-DTH positive events during a total of 15 tests
WT1 and MHC class I expression in PDAC cells
The correlation between WT1 and MHC class I expression intensity in PDAC cells before treatment and the magnitude of the immune response to WT1 were evaluated in nine patients, excluding one patient (WT1-DC-10). As shown in the online supplemental figure 6, cytoplasmic WT1 expression in the PDAC cells of long-term WT1-DTH-positive patients was relatively weak compared with that in the PDAC cells of short-term WT1-DTH-positive patients. The clinical benefits (PFS and OS) of patients with weak cytoplasmic WT1 expression were significantly greater than those with moderate cytoplasmic WT1 expression (p=0.011 and p=0.011, respectively). In contrast, there was no correlation between WT1-DTH and MHC class I expression intensity in these small populations.
Supplemental material
Immune-related cells in the TME
The examination of immune-related cells in fine needle aspiration samples obtained prior to treatment was not appropriate because of the small size and heterogeneity of the immune-related cell population in the TME. Interestingly, six patients with PDAC underwent surgical PDAC resection during chemoimmunotherapy. The number of CD3+ tumor-infiltrating lymphocytes (TILs) in the TME of one patient (WT1-DC-01) was significantly greater than that in the other five patients (figure 3A). One patient (WT1-DC-02) had a significantly lower density of CD3+ TILs than did the other four patients (WT1-DC-01, WT1-DC-03, WT1-DC-05, and WT1-DC-09) (figure 3A). In addition, except for one patient (WT1-DC-02), the other five patients had detectable PD-1-positive cells to varying degrees (figure 3B). In particular, the number of PD-1+ cells was much greater in one patient (WT1-DC-01) than in four other patients (WT1-DC-02, WT1-DC-03, WT1-DC-04, and WT1-DC-09). Furthermore, there was no significant difference in PD-1+ cell density between patients WT1-DC-01 and WT1-DC-05. Specifically, in one patient (WT1-DC-01), CD3+ T cells appeared to form clusters in tertiary lymphoid aggregations, which may be associated with antigen presentation and T-cell activation (PD-1 expression).2
Immunohistochemical analysis of the pancreatic TME in patients with PDAC who underwent conversion surgery. The mean cell counts/mm2 tumor area for CD3+, CD4+, CD8+ (A) or PD-1+ (B) cells were analyzed in the pancreatic TME obtained from six surgically resected PDAC samples (four with LA-PDAC (WT1-DC-01, WT1-DC-03, WT1-DC-04, and WT1-DC-09) and two with M-PDAC (WT1-DC-02 and WT1-DC-05)). The mean percentage of Foxp3+/CD4+ cells (C) and the CD68+/CD163+ratio (D, E) in the pancreatic TME. Foxp3, forkhead box p3; IgG, immunoglobulin G; LA-PDAC, locally advanced pancreatic ductal adenocarcinoma; M-PDAC, metastatic pancreatic ductal adenocarcinoma; PD-1, programmed cell death protein-1; TME, tumor microenvironment; WT1-DC, Wilms’ tumor 1 peptide-pulsed dendritic cell.
Next, to assess Tregs, the percentage of Foxp3+ cells among total CD4+ cells was evaluated. One patient (WT1-DC-02) had a significantly greater percentage of Foxp3+/CD4+ cells than did the other five patients (p<0.05) (figure 3C). One patient (WT1-DC-01) had a significantly lower percentage of Foxp3+/CD4+ cells than did the other three patients (WT1-DC-02, WT1-DC-04, and WT1-DC-09) (p<0.02) (figure 3C). Moreover, to assess macrophage polarization in the TME, the M1 marker CD68 and the M2 marker CD163 were first quantified (data not shown). The number of CD68+ cells in the TME of one patient (WT1-DC-01) was significantly greater than that in the other five patients (p<0.0001). The number of CD163+ cells in the TME of one patient (WT1-DC-04) was significantly lower than that in the other five patients (p<0.002). The CD68/CD163 ratio, which is a biological indicator relevant to the prognosis of patients with PDAC due to the re-education of TAMs,22 23 was subsequently evaluated. In one patient (WT1-DC-01), the CD68/CD163 ratio was significantly greater than that in the other five patients (p<0.0001) (figure 3D,E). In one patient (WT1-DC-02), the CD68/CD163 ratio was significantly lower than that in the other five patients (p<0.002) (figure 3D,E).
Associations between circulating WT1-CTLs and clinical outcomes
After two vaccinations, significantly greater levels of IFN-γ or TNF-α production by CD4+ or CD8+ T cells were detected in all four long-term WT1-DTH-positive patients than in all five short-term WT1-DTH-positive patients (online supplemental figure 7). An increased percentage of WT1-CTLs producing IFN-γ or TNF-α, which is a hallmark of super-responders, may be associated with long-term WT1-DTH positivity (online supplemental figure 7). Next, to evaluate the details of the functional WT1-CTLs, we assessed the induction of HLA-A*24:02-restricted WT1-tetramer+CD8+ T cells in all 7 HLA-A*24:02-positive patients due to the unavailability of suitable tetramers other than HLA-A*24:02 (figure 4A,B). Following the administration of the four vaccine doses, patients with long-term WT1-DTH positivity continued to exhibit significantly greater percentages of HLA-A*24:02-restricted WT1-CTLs that produced IFN-γ or TNF-α than did those who exhibited short-term WT1-DTH positivity (figure 4C,D). Furthermore, these patients can be divided into two groups: Those with a sustained high percentage of functional WT1-CTLs producing IFN-γ or TNF-α (>median percentage: WT1-DC-01, WT1-DC-07, and WT1-DC-09) and those with relatively low percentages of these WT1-CTLs (≤median percentage: WT1-DC-02, WT1-DC-04, WT1-DC-05, and WT1-DC-08). Patients with HLA-A*24:02 who had a sustained presence of these WT1-CTLs were long-term survivors (>4.5 years) (figure 4E). In addition, the circulating cells from one patient (WT1-DC-02) had consistently low levels of HLA-A*24:02-restricted WT1-CTLs throughout the treatment period, and most of these cells did not produce IFN-γ or TNF-α (figure 4B,C).
Supplemental material
IFN-γ-producing or TNF-α-producing WT1-specific CD8+ T cells restricted to HLA-A*24:02. (A) IFN-γ (left panel) or TNF-α (right panel) levels in one WT1-DC-07 patient before and after the 4 vaccines are shown. (B) The left panel shows HLA-A*24:02-restricted WT1-tetramer+IFN-γ+ CD8+ T cells among total CD8+ T cells (red bar), whereas the right panel shows HLA-A*24:02-restricted WT1-tetramer+TNF-α+ CD8+ T cells among total CD8+ T cells (red bar). (C) Changes in IFN-γ-producing (upper panel) or TNF-α-producing (lower panel) WT1-CTLs among total CD8+ T cells restricted to HLA-A*24:02 during vaccination (0, 2, 4, 6, 8, and 10 vaccinations) are shown. Three patients (WT1-DC-01, WT1-DC-07, and WT1-DC-09) presented long-term DTH (blue line), whereas four patients (WT1-DC-02, WT1-DC-04, WT1-DC-05, and WT1-DC-08) presented short-term WT1-DTH positivity (red line). (D) CD8+ T cells from before and after vaccination (0, 2, 4, 6, 8, and 10 vaccinations) were analyzed via HLA-A*24:02-restricted WT1-tetramers and IFN-γ (upper panel) or TNF-α (lower panel) production on stimulation with WT1 peptides in vitro. Patients with UR-PDAC with long-term WT1-DTH positivity (blue bar) or short-term WT1-DTH positivity (red bar) are shown. Each data point is presented in scatter plots with mean bar graphs. (E) PFS (upper panel) or OS (lower panel) for seven patients with UR-PDAC with HLA-A*24:02. These patients were divided into two groups: Those with a sustained high percentage of functional WT1-CTLs producing IFN-γ or TNF-α (>median percentage: WT1-DC-01, WT1-DC-07, and WT1-DC-09) (blue line) and those with relatively low percentages of these WT1-CTLs (≤median percentage: WT1-DC-02, WT1-DC-04, WT1-DC-05, and WT1-DC-08) (red line). *p<0.05, **p<0.01. HLA, human leukocyte antigen; OS, overall survival; PFS, progression-free survival; UR-PDAC, unresectable pancreatic ductal adenocarcinoma; WT1, Wilms’ tumor 1; WT1-CTLs, WT1-specific cytotoxic T lymphocytes; WT1-DC, WT1 peptide-pulsed dendritic cell; WT1-DTH, WT1 peptide-specific delayed-type hypersensitivity.
Associations between circulating immunosuppressive cells and clinical responses
Before therapy, there were no significant differences in the percentages of Tregs among total CD4+ T cells or MDSCs among total PBMCs between long-term WT1-DTH-positive patients and short-term WT1-DTH-positive patients. However, after 12 vaccinations, the percentages of both Tregs among total CD4+ T cells and MDSCs among total PBMCs were significantly lower in long-term WT1-DTH-positive patients with PDAC than in short-term WT1-DTH-positive patients (p=0.004 and p=0.019, respectively) (online supplemental figure 8A,B).
Supplemental material
WT1-TILs
HLA-A*24:02-restricted WT1-TILs were detected among total CD8+ TILs (online supplemental figure 9A). The HLA-A*24:02-restricted WT1-TIL behavior was then divided into two groups: A high WT1-TIL group (≥median percentage: WT1-DC-01, WT1-DC-03, WT1-DC-07, and WT1-DC-09) and a low WT1-TIL group (<median percentage: WT1-DC-02, WT1-DC-04, and WT1-DC-05). Patients with PDAC with high WT1-TIL density had significantly longer PFS and OS than did those with low WT1-TIL density (p=0.0101 and 0.0101, respectively) (online supplemental figure 9B). All four patients with PDAC with high WT1-TIL levels were alive for at least 4.5 years, and one patient (WT1-DC-09) relapsed at 908 days after entry. In contrast, all patients with PDAC with low WT1-TIL numbers died (median PFS: 1.39 years; median OS: 2.77 years).
Supplemental material
Discussion
This clinical trial is the first to use mature DCs pulsed with a novel WT1 peptide cocktail in combination with Nab-P/Gem for patients with UR-PDAC. Nab-P/Gem alone has been reported to result in an OS of 18.8 months and 8.5 months in LA-PDAC12 and M-PDAC,11 respectively. Notably, a small percentage of patients (4.6%) underwent conversion surgical resection with Nab-P/Gem alone, and the median OS was 29.7 months.24–26 Therefore, the chemoimmunotherapy described here may be more efficacious than Nab-P/Gem alone. During chemoimmunotherapy, six patients (four LA-PDAC, one M-PDAC and one recurrence) underwent R0 surgical resection, and another patient with M-PDAC was resectable but had microscopic tumor infiltration (R1). The median OS of patients with LA-PDAC who underwent R0 resection was not reached (more than 4.5 years). R0 resection is the only treatment option that can improve long-term survival or even lead to full recovery in some individuals. Moreover, the survival rate of patients with PDAC who undergo R1 resection is significantly greater than that of patients with UR-PDAC.27 28 This finding highlights the clinical benefits of chemoimmunotherapy in an R1-resected patient (WT1-DC-02), with an OS of 843 days. Taken together, these results suggest that WT1-DC vaccines in combination with multiagent chemotherapy may provide clinical benefit by increasing the likelihood of conversion surgery, resulting in longer survival. There were patients who relapsed even after conversion surgery, but if the vaccine could be given for a long time after surgery, the time without relapse would be even longer.
Our previous report revealed that WT1-targeted cancer vaccines induce WT1-specific antitumor immunity, resulting in good clinical outcomes, but only for certain patients.7 8 In this clinical regimen, WT1-specific immunity is induced to a greater or lesser extent in all patients. Therefore, we attempted a detailed evaluation of WT1-DTH and clinical outcomes in nine patients, excluding one patient (WT1-DC-10) who could not be adequately treated. Importantly, compared with five patients with short-term WT1-DTH positivity, four patients with long-term WT1-DTH positivity exhibited significantly prolonged PFS and OS. A prerequisite for an effective cancer vaccine may be the ability not only to stimulate CD8+ CTL activity but also to stimulate CD4+ Th cells efficiently.7 Therefore, we next evaluated WT1-specific CD4+ or CD8+ T cells that produce IFN-γ or TNF-α when stimulated with WT1 peptides in vitro. Compared with patients with short-term WT1-DTH positivity, patients with long-term WT1-DTH positivity presented significantly increased levels of IFN-γ or TNF-α produced by CD4+ or CD8+ T cells after two vaccinations, and those levels were maintained throughout treatment. Our focus then shifted to assessing the functional activity of WT1-CTLs that produced IFN-γ or TNF-α and were restricted by HLA-A*24:02, as suitable tetramers other than HLA-A*24:02 were not available. In six of the seven HLA-A*24:02-positive patients, the percentages of IFN-γ+ or TNF-α+ WT1-tetramer+CD8+ T cells among total CD8+ T cells were detected and maintained after vaccination, even though there were differences in their percentages. Only one patient with M-PDAC (WT1-DC-02) exhibited little, if any, active immunity against WT1 peptides in an HLA-A*24:02-restricted manner. Patients with long-term WT1-DTH positivity, or super-responders, had a significantly greater percentage of WT1-tetramer+CD8+ T cells that produced IFN-γ or TNF-α among total CD8+ T cells than did those with short-term WT1-DTH positivity. Therefore, maintaining high levels of functional WT1-specific CD8+ CTLs and WT1-specific CD4+ Th cells may be important in patients receiving this regimen to prolong survival.
It is also important to assess the immunosuppressive status. After 12 vaccinations, the percentages of Tregs among total CD4+ T cells and MDSCs among total PBMCs in patients with long-term WT1-DTH positivity were significantly lower than those in patients with short-term WT1-DTH positivity. WT1 is expressed not only in PDAC cells but also in MDSCs within the TME.4 5 Therefore, WT1-DC vaccines may also eradicate WT1-expressing MDSCs. Long-term and high-level induction of functional WT1 immunity in patients may also be essential for maintaining a weak immunosuppressive status in the TME and the whole body, resulting in a prolonged prognosis. Taken together, these results indicate that in patients with persistently high levels of functional WT1 immunity, the immunosuppressive effects may be minimal, resulting in an excellent immune TME in which WT1-CTLs may be effective.
Blood samples were used to evaluate the induction of a systemic WT1-specific antitumor immune response by WT1-DC vaccines but, most importantly, to confirm the immune changes in the TME after therapy. In this study, three patients with LA-PDAC had a high density of WT1-TILs and survived for at least 4.5 years from entry. In particular, one patient with LA-PDAC (WT1-DC-01) had an extremely high density of T cells and PD-1+ cells in the TME, and these cells were mostly activated effector T cells,2 both of which are critical for regulating local antitumor cellular immunity and may be involved in coordinating responses to immunomodulatory therapies.29 Currently, the patient (WT1-DC-01) has survived well for more than 5 years since entry without recurrence. Another patient with LA-PDAC (WT1-DC-04) had a relatively low density of WT1-TILs in the TME compared with the other three patients with LA-PDAC, who eventually relapsed and died even after conversion surgery. Attempts have been made to categorize immune responses in the TME as either “cold” (non-T-cell-inflamed) or “hot” (T-cell-inflamed), according to the level of immune-related cell infiltration into the TME.30 WT1-targeted chemoimmunotherapy may remodel the TME of some patients with PDAC from “cold” to “hot”, resulting in significant tumor shrinkage. Therefore, conversion surgery after chemoimmunotherapy may be feasible for some patients with LA-PDAC, allowing for long-term survival and complete recovery. Although one patient with M-PDAC (WT1-DC-05) also had high infiltration of T cells and PD-1+ cells into the TME, this patient had bone metastases at the start of treatment. Therefore, the tumors in this patient may not have been completely eliminated by chemoimmunotherapy followed by surgical resection of the pancreatic lesions, resulting in relapse and death at 1013 days, after entry with a limited number of vaccinations (15 doses). Patients with M-PDAC have long been considered to have surgical contraindications because aggressive surgical resection of metastatic lesions has not shown a survival benefit.31 However, there is growing hope that surgery and chemoimmunotherapy targeting WT1 may lead to favorable outcomes in some patients with oligometastatic PDAC.
It is interesting to analyze the immunosuppressive status of the TME after chemoimmunotherapy. Three patients with PDAC (WT1-DC-02, WT1-DC-04, and WT1-DC-09) had relatively high percentages of Foxp3+/CD4+ cells and relapsed even after conversion surgery. A high CD68/CD163 ratio in one patient (WT1-DC-01) may be associated with prolonged survival. The interaction between immune-activating cells (CTLs and M1 macrophages) and immunosuppressive cells (Tregs and M2 macrophages) may determine whether the antitumor immune response is sustained or attenuated. WT1-DC vaccines may synergistically enhance systemic WT1-CTL responses that lead to improved control of local and disseminated disease or recurrent disease, resulting in prolonged survival in some patients. Moreover, multiagent chemotherapy may also be associated with an altered antitumorigenic TME, with a prognostically favorable immune cell infiltration pattern in patients with PDAC.32 33 Taken together, these findings indicate that chemoimmunotherapy consisting of a WT1-DC vaccine may modulate multiple aspects of the TME, resulting in significant therapeutic benefits, which may provide a new strategy for the treatment of advanced UR-PDAC.
The limitations of this study include the relatively small number of patients and the limited number of vaccine doses. Continued maintenance of WT1-specific immunity through repeated vaccination is essential to prevent relapse and achieve complete recovery in patients.34 Furthermore, weak WT1 expression in tumor cells may serve as a prognostic factor in patients with PDAC undergoing surgical resection19 or WT1-targeted chemoimmunotherapy.35 The correlation between WT1 overexpression and poor OS has been previously established in numerous types of solid tumors.4 36 Further comprehensive investigations into the immune response, such as the examination of memory T cells may be beneficial for advancing our understanding of this field.
Supplemental material
Data availability statement
Data are available upon reasonable request.
Ethics statements
Patient consent for publication
Ethics approval
This study was reviewed and approved by the Certified Committee of The Jikei University School of Medicine for Regenerative Medicine (No. NB3150036) and by the clinical study committee of Jikei University Kashiwa Hospital (No. 2018-01). This study was registered with the Japan Registry of Clinical Trials (Regenerative Medicine Provisioning Plan No. PC3180048) with the ethical principles of the Declaration of Helsinki. Participants gave informed consent to participate in the study before taking part.
Acknowledgments
We thank all the patients who participated in this study, their supporting families, and all the referring physicians and the supporting medical staff at all the clinical sites. We also thank Masami Ueda (Tokyo Midtown Center for Advanced Medical Science and Technology) for preparing the WT1-DC vaccines and Yoko Shimizu (Tokyo Midtown Center for Advanced Medical Science and Technology) for management.
References
Supplementary materials
Supplementary Data
This web only file has been produced by the BMJ Publishing Group from an electronic file supplied by the author(s) and has not been edited for content.
Footnotes
Contributors Conception and design: SKo, JT, SS, HS. Development of methodology: SKo, SKa, TB. Acquisition of data: SKo, JT, MSh, TM, ST. Analysis and interpretation of data: SKo, SKa, TB. Writing, review, and revision of the manuscript: SKo. Administrative, technical, or material support: SKo, JT, MSh. Study supervision: SKo, JT, MSh, ZI, KU, MSa, MSu, HY, NS, TO, SS, HS. Guarantor who takes full responsibility for the study and/or the conduct of the study, has access to the data and manages the decision to publish: SKo.
Funding This work was supported, in part, by Grants-in-Aid for Scientific Research (C) from the Ministry of Education, Cultures, Sports, Science and Technology of Japan, Grant Number 22K08088.
Competing interests None declared.
Provenance and peer review Not commissioned; externally peer reviewed.
Supplemental material This content has been supplied by the author(s). It has not been vetted by BMJ Publishing Group Limited (BMJ) and may not have been peer-reviewed. Any opinions or recommendations discussed are solely those of the author(s) and are not endorsed by BMJ. BMJ disclaims all liability and responsibility arising from any reliance placed on the content. Where the content includes any translated material, BMJ does not warrant the accuracy and reliability of the translations (including but not limited to local regulations, clinical guidelines, terminology, drug names and drug dosages), and is not responsible for any error and/or omissions arising from translation and adaptation or otherwise.