Establishment of a novel NFAT-GFP reporter platform useful for the functional avidity maturation of HLA class II-restricted TCRs.

CD4+ T cells that recognize antigenic peptides presented on HLA class II are essential for inducing an optimal anti-tumor immune response, and adoptive transfer of tumor antigen-specific TCR-transduced CD4+ T cells with high responsiveness against tumor is a promising strategy for cancer treatment. Whereas a precise evaluation method of functional avidity, an indicator of T cell responsiveness against tumors, has been established for HLA class I-restricted TCRs, it remains unestablished for HLA class II-restricted TCRs. In this study, we generated a novel platform cell line, CD4-2D3, in which GFP reporter was expressed by NFAT activation via TCR signaling, for correctly evaluating functional avidity of HLA class II-restricted TCRs. Furthermore, using this platform cell line, we succeeded in maturating functional avidity of an HLA class II-restricted TCR specific for a WT1-derived helper peptide by substituting amino acids in complementarity determining region 3 (CDR3) of the TCR. Importantly, we demonstrated that transduction of an avidity-maturated TCR conferred strong cytotoxicity against WT1-expressing leukemia cells on CD4+ T cells, compared to that of its original TCR. Thus, CD4-2D3 cell line should be useful not only to evaluate TCR functional avidity in HLA class II-restricted TCRs but also to screen appropriate TCRs for clinical applications such as cancer immunotherapy.

Enhanced antitumor activity of a novel, oral, helper epitope-containing WT1 protein vaccine in a model of murine leukemia.

BACKGROUND: A Wilms' tumor 1 (WT1) oral vaccine, Bifidobacterium longum (B. longum) 420, in which the bacterium is used as a vector for WT1 protein, triggers immune responses through cellular immunity consisting of cytotoxic T lymphocytes (CTLs) and other immunocompetent cells (e.g., helper T cells). We developed a novel, oral, helper epitope-containing WT1 protein vaccine (B. longum 2656) to examine whether or not B. longum 420/2656 combination further accelerates the CD4+ T cell help-enhanced antitumor activity in a model of murine leukemia. METHODS: C1498-murine WT1-a genetically-engineered, murine leukemia cell line to express murine WT1-was used as tumor cell. Female C57BL/6 J mice were allocated to the B. longum 420, 2656, and 420/2656 combination groups. The day of subcutaneous inoculation of tumor cells was considered as day 0, and successful engraftment was verified on day 7. The oral administration of the vaccine by gavage was initiated on day 8. Tumor volume, the frequency and phenotypes of WT1-specific CTLs in CD8+ T cells in peripheral blood (PB) and tumor-infiltrating lymphocytes (TILs), as well as the proportion of interferon-gamma (INF-γ)-producing CD3+CD4+ T cells pulsed with WT135-52 peptide in splenocytes and TILs were determined. RESULTS: Tumor volume was significantly smaller (p < 0.01) in the B. longum 420/2656 combination group than in the B. longum 420 group on day 24. WT1-specific CTL frequency in CD8+ T cells in PB was significantly greater in the B. longum 420/2656 combination group than in the B. longum 420 group at weeks 4 (p < 0.05) and 6 (p < 0.01). The proportion of WT1-specific, effector memory CTLs in PB increased significantly in the B. longum 420/2656 combination group than in the B. longum 420 group at weeks 4 and 6 (p < 0.05 each). WT1-specific CTL frequency in intratumoral CD8+ T cells and the proportion of IFN-γ-producing CD3+CD4+ T cells in intratumoral CD4+ T cells increased significantly (p < 0.05 each) in the B. longum 420/2656 combination group than in the 420 group. CONCLUSIONS: B. longum 420/2656 combination further accelerated antitumor activity that relies on WT1-specific CTLs in the tumor compared with B. longum 420.

WT1 Trio Peptide-Based Cancer Vaccine for Rare Cancers Expressing Shared Target WT1.

No standard treatment has been established for most rare cancers. Here, we report a clinical trial of a biweekly WT1 tri-peptide-based vaccine for recurrent or advanced rare cancers. Due to the insufficient number of patients available for a traditional clinical trial, the trial was designed for rare cancers expressing shared target molecule WT1. The recruitment criteria included WT1-expressing tumors as well as HLA-A*24:02 or 02:01. The primary endpoints were immunoglobulin G (IgG) antibody (Ab) production against the WT1-235 cytotoxic T lymphocyte (CTL) epitope and delayed-type hypersensitivity (DTH) skin reactions to targeted WT1 CTL epitopes. The secondary endpoints were safety and clinical efficacy. Forty-five patients received WT1 Trio, and 25 (55.6%) completed the 3-month protocol treatment. WT1-235 IgG Ab was positive in 88.0% of patients treated with WT1 Trio at 3 months, significantly higher than 62.5% of the weekly WT1-235 CTL peptide vaccine. The DTH positivity rate in WT1 Trio was 62.9%, which was not significantly different from 60.7% in the WT1-235 CTL peptide vaccine. The WT1 Trio safety was confirmed without severe treatment-related adverse events, except grade 3 myasthenia gravis-like symptoms observed in a patient with thymic cancer. Fifteen (33.3%) patients achieved stable disease after 3 months of treatment. In conclusion, the biweekly WT1 Trio vaccine containing the WT1-332 helper T lymphocyte peptide induced more robust immune responses targeting WT1 than the weekly WT1-235 CTL peptide vaccine. Therefore, WT1-targeted immunotherapy may be a potential therapeutic strategy for rare cancers.

An oral WT1 protein vaccine composed of WT1-anchored, genetically engineered Bifidobacterium longum allows for intestinal immunity in mice with acute myeloid leukemia.

Wilms' tumor 1 (WT1) is a promising tumor-associated antigen for cancer immunotherapy. We developed an oral protein vaccine platform composed of WT1-anchored, genetically engineered Bifidobacterium longum (B. longum) and conducted an in vivo study in mice to examine its anticancer activity. Mice were orally treated with phosphate-buffered saline, wild-type B. longum105-A, B. longum 2012 displaying only galacto-N-biose/lacto-N-biose I-binding protein (GLBP), and WT1 protein- and GLBP-expressing B. longum 420. Tumor size reduced significantly in the B. longum 420 group than in the B. longum 105-A and 2012 groups (P < 0.00 l each), indicating B. longum 420's antitumor activity via WT1-specific immune responses. CD8+ T cells played a major role in the antitumor activity of B. longum 420. The proportion of CD103+CD11b+CD11c+ dendritic cells (DCs) increased in the Peyer's patches (PPs) from mice in the B. longum 420 group, indicating the definite activation of DCs. In the PPs, the number and proportion of CD8+ T cells capable of producing interferon-gamma were significantly greater in the B. longum 420 group than in the B. longum 2012 group (P < 0.05 or < 0.01). The production of WT1-specific IgG antibody was significantly higher in the B. longum 420 group than in the 2012 group (P < 0.05). The B. longum 420 group showed the most intense intratumoral infiltration of CD4+ and CD8+ T cells primed by activated DCs in the PPs of mice in the B. longum 420 group. Our findings provide insights into a novel, intestinal bacterium-based, cancer immunotherapy through intestinal immunity.

T Cell-Intrinsic Vitamin A Metabolism and Its Signaling Are Targets for Memory T Cell-Based Cancer Immunotherapy.

Memory T cells play an essential role in infectious and tumor immunity. Vitamin A metabolites such as retinoic acid are immune modulators, but the role of vitamin A metabolism in memory T-cell differentiation is unclear. In this study, we identified retinol dehydrogenase 10 (Rdh10), which metabolizes vitamin A to retinal (RAL), as a key molecule for regulating T cell differentiation. T cell-specific Rdh10 deficiency enhanced memory T-cell formation through blocking RAL production in infection model. Epigenetic profiling revealed that retinoic acid receptor (RAR) signaling activated by vitamin A metabolites induced comprehensive epigenetic repression of memory T cell-associated genes, including TCF7, thereby promoting effector T-cell differentiation. Importantly, memory T cells generated by Rdh deficiency and blocking RAR signaling elicited potent anti-tumor responses in adoptive T-cell transfer setting. Thus, T cell differentiation is regulated by vitamin A metabolism and its signaling, which should be novel targets for memory T cell-based cancer immunotherapy.

Selective targeting of multiple myeloma cells with a monoclonal antibody recognizing the ubiquitous protein CD98 heavy chain.

Cancer-specific cell surface antigens are ideal therapeutic targets for monoclonal antibody (mAb)-based therapy. Here, we report that multiple myeloma (MM), an incurable hematological malignancy, can be specifically targeted by an mAb that recognizes a ubiquitously present protein, CD98 heavy chain (hc) (also known as SLC3A2). We screened more than 10,000 mAb clones raised against MM cells and identified R8H283, an mAb that bound MM cells but not normal hematopoietic or nonhematopoietic cells. R8H283 specifically recognized CD98hc. R8H283 did not react with monomers of CD98hc; instead, it bound CD98hc in heterodimers with a CD98 light chain (CD98lc), a complex that functions as an amino acid transporter. CD98 heterodimers were abundant on MM cells and took up amino acids for constitutive production of immunoglobulin. Although CD98 heterodimers were also present on normal leukocytes, R8H283 did not react with them. The glycoforms of CD98hc present on normal leukocytes were distinct from those present on MM cells, which may explain the lack of R8H283 reactivity to normal leukocytes. R8H283 exerted anti-MM effects without damaging normal hematopoietic cells. These findings suggested that R8H283 is a candidate for mAb-based therapies for MM. In addition, our findings showed that a cancer-specific conformational epitope in a ubiquitous protein, which cannot be identified by transcriptome or proteome analyses, can be found by extensive screening of primary human tumor samples.

WT1 epitope-specific IgG and IgM antibodies for immune-monitoring in patients with advanced sarcoma treated with a WT1 peptide cancer vaccine.

The Wilms' tumor gene WT1 is highly expressed in various malignancies and may be a common target antigen for cancer immunotherapy. In our group, peptide-based cancer vaccines targeting WT1 CTL epitopes were developed as an immunotherapy for these malignancies. In the present study, WT1 epitope-specific immune responses were analyzed in 31 patients with advanced sarcoma with human leukocyte antigen-A*24:02- and WT1-expressing tumors who received the WT1-235 peptide vaccine as monotherapy. The serum levels of IgG and IgM antibodies against the target epitope WT1-235 and the non-target epitopes WT1-332 and WT1-271 were measured using ELISA. IgM antibodies against WT1-235, WT1-332 and WT1-271 were detected in three (9.6%), four (12.9%) and 20 patients (64.5%), respectively, prior to vaccine administration, indicating immune recognition of the WT1 antigen prior to administering the vaccine. Of 15 patients who had completed the 3-month treatment protocol, WT1-235 IgG was positive in five (33.3%) patients. An enzyme-linked immunospot assay revealed that WT1-235 epitope-specific IL-10 production/secretion in peripheral blood mononuclear cells declined in the first month of vaccine administration in all three patients with positivity for WT1-235 IgM at the start of the vaccine. Furthermore, positivity for both WT1-235 and WT1-271 IgM antibodies at the start of treatment was associated with unfavorable tumor control at 3 months after vaccine administration. These results suggested that WT1 epitope-specific IgG and IgM antibodies may be utilized as immune-monitoring markers for WT1 peptide cancer vaccine immunotherapy. The trials were entered in the University hospital Medical Information Network (UMIN) Clinical Trials Registry (https://www.umin.ac.jp/ctr; no. UMIN000002001 on May 24, 2009 and no. UMIN000015997 on December 20, 2014).

Maintenance of WT1 expression in tumor cells is associated with a good prognosis in malignant glioma patients treated with WT1 peptide vaccine immunotherapy.

We have previously revealed the overexpression of Wilms' tumor gene 1 (WT1) in malignant glioma and developed WT1 peptide vaccine cancer immunotherapy. A phase II clinical trial indicated the clinical efficacy of the WT1 peptide vaccine for recurrent malignant glioma. Here, we aimed to investigate the immunological microenvironment in glioma tissues before and after WT1 peptide vaccine treatment. Paired tissue samples were obtained from 20 malignant glioma patients who had received the WT1 peptide vaccine for > 3 months and experienced tumor progression, confirmed radiographically and/or clinically, during vaccination. We discovered that the expression of WT1 and HLA class I antigens in the tumor cells significantly decreased after vaccination. Maintenance of WT1 expression, which is the target molecule of immunotherapy, in tumor cells during the vaccination period was significantly associated with a longer progression-free and overall survival. A high expression of HLA class I antigens and low CD4+/CD8+ tumor-infiltrating lymphocytes (TIL) ratio in pre-vaccination specimens, were also associated with a good prognosis. No statistically significant difference existed in the number of infiltrating CD3+ or CD8+ T cells between the pre- and post-vaccination specimens, whereas the number of infiltrating CD4+ T cells significantly decreased in the post-vaccination specimens. This study provides insight into the mechanisms of intra-tumoral immune reaction/escape during WT1 peptide vaccine treatment and suggests potential clinical strategies for cancer immunotherapy.

Cellular and Humoral Immune Responses Induced by an HLA Class I-restricted Peptide Cancer Vaccine Targeting WT1 Are Associated With Favorable Clinical Outcomes in Advanced Ovarian Cancer.

The HLA-A*24:02-restricted peptide vaccine targeting Wilms' tumor 1 (WT1) (WT1 vaccine) is a promising therapeutic strategy for ovarian cancer; however, its efficacy varies among patients. In this study, we analyzed WT1-specific immune responses in patients with advanced or recurrent ovarian cancer that was refractory to standard chemotherapies and their associations with clinical outcomes. In 25 patients, the WT1 vaccine was administered subcutaneously weekly for 3 months and biweekly thereafter until disease progression or severe adverse events. We assessed Wilms' tumor 1-specific cytotoxic T lymphocytes (WT1-CTLs) and Wilms' tumor 1 peptide-specific immunoglobulin G (WT1235-IgG). After vaccination, the percentage of tetramer high-avidity population of WT1-CTLs among CD8+ T lymphocytes (%tet-hi WT1-CTL) and the WT1235-IgG titer increased significantly, although the values were extremely low or below the limit of detection before vaccination (%tet-hi WT1-CTL: 0.003%-0.103%.; WT1235-IgG: <0.05-0.077 U/mL). Patients who had %tet-hi WT1-CTL of ≥0.25% (n=6) or WT1235-IgG of ≥0.10 U/mL (n=12) had a significantly longer progression-free survival than those of patients in the other groups. In addition, an increase in WT1235-IgG corresponded to a significantly longer progression-free survival (P=0.0496). In patients with systemic inflammation, as evidenced by elevated C-reactive protein levels, the induction of tet-hi WT1-CTL or WT1235-IgG was insufficient. Decreased serum albumin levels, multiple tumor lesions, poor performance status, and excess ascites negatively influenced the clinical effectiveness of the WT1 vaccine. In conclusion, the WT1 vaccine induced antigen-specific cellular and humoral immunity in patients with refractory ovarian cancer. Both %tet-hi WT1-CTL and WT1235-IgG levels are prognostic markers for the WT1 vaccine.

Polycythemia Vera Associated with Pulmonary Hypertension and Diffuse Large B-Cell Lymphoma: A Case Report.

BACKGROUND Myeloproliferative neoplasms (MPNs), including polycythemia vera (PV), are associated with pulmonary hypertension (PH) and malignant lymphomas. Although the underlying mechanisms have not been completely clarified, it has been suggested that the Janus kinase 2 (JAK2) mutation, which is frequently identified in PV, can be involved in the development and/or progression of these distinct diseases in patients with MPNs. However, no reports have described the coexistence of PH and malignant lymphoma in patients with MPNs. CASE REPORT A 79-year-old man being treated for PV for 27 years and PH for 5 years was hospitalized due to severe dyspnea at rest. His soluble interleukin-2 receptor levels gradually increased and the chest computed tomography showed remarkable progression of the lung lesions and an enlargement of the mediastinal and axillary lymph nodes. A lymph node biopsy was performed and the patient was diagnosed with diffuse large B-cell lymphoma (DLBCL). Owing to his poor condition, chemotherapy was not initiated, and he died on the 89th day of hospitalization. The pathological autopsy revealed the destruction of alveolar structures with neoplastic space-occupying lesions of DLBCL. Multifactorial features of PH associated with MPNs, including the intimal thickening of pulmonary arteries accompanied by megakaryocytes and obstructed pulmonary arteries with organized thrombi in the lung tissue specimens, were observed. We found a JAK2 mutation based on a genetic analysis of the patient's bone marrow. CONCLUSIONS We present the rare case of a patient who had PV with a JAK2 mutation, which coexisted with PH and DLBCL, and he developed severe refractory respiratory failure.

Distinct difference in tumor-infiltrating immune cells between Wilms' tumor gene 1 peptide vaccine and anti-programmed cell death-1 antibody therapies.

BACKGROUND: Wilms' tumor gene 1 (WT1) peptide vaccine and anti-programmed cell death-1 (anti-PD-1) antibody are expected as immunotherapies to improve the clinical outcome of glioblastoma. The aims of this study were to clarify how each immunotherapy affects tumor-infiltrating immune cells (TIIs) and to determine whether the combination of these two therapies could synergistically work. METHODS: Mice were transplanted with WT1 and programmed cell death-ligand 1 doubly expressing glioblastoma cells into brain followed by treatment with WT1 peptide vaccine, anti-PD-1 antibody, or the combination of the two, and survival of each therapy was compared. CD45+ cells were positively selected as TIIs from the brains with tumors, and TIIs were compared between WT1 peptide vaccine and anti-PD-1 antibody therapies. RESULTS: Most mice seemed to be cured by the combination therapy with WT1 peptide vaccine and anti-PD-1 antibody, which was much better survival than each monotherapy. A large number of CD4+ T cells, CD8+ T cells, and NK cells including WT1-specific CD8+ and CD4+ T cells infiltrated into the glioblastoma in WT1 peptide vaccine-treated mice. On the other hand, the number of TIIs did not increase, but instead PD-1 molecule expression was decreased on the majority of the tumor-infiltrating CD8+ T cells in the anti-PD-1 antibody-treated mice. CONCLUSION: Our results clearly demonstrated that WT1 peptide vaccine and anti-PD-1 antibody therapies worked in the different steps of cancer-immunity cycle and that the combination of the two therapies could work synergistically against glioblastoma.

Imaging Assessment of Tumor Response in the Era of Immunotherapy.

Assessment of tumor response during treatment is one of the most important purposes of imaging. Before the appearance of immunotherapy, response evaluation criteria in solid tumors (RECIST) and positron emission tomography response criteria in solid tumors (PERCIST) were, respectively, the established morphologic and metabolic response criteria, and cessation of treatment was recommended when progressive disease was detected according to these criteria. However, various types of immunotherapy have been developed over the past 20 years, which show novel false positive findings on images, as well as distinct response patterns from conventional therapies. Antitumor immune response itself causes 18F-fluorodeoxyglucose (FDG) uptake in tumor sites, known as "flare phenomenon", so that positron emission tomography using FDG can no longer accurately identify remaining tumors. Furthermore, tumors often initially increase, followed by stability or decrease resulting from immunotherapy, which is called "pseudoprogression", so that progressive disease cannot be confirmed by computed tomography or magnetic resonance imaging at a single time point. As a result, neither RECIST nor PERCIST can accurately predict the response to immunotherapy, and therefore several new response criteria fixed for immunotherapy have been proposed. However, these criteria are still controversial, and also require months for response confirmation. The establishment of optimal response criteria and the development of new imaging technologies other than FDG are therefore urgently needed. In this review, we summarize the false positive images and the revision of response criteria for each immunotherapy, in order to avoid discontinuation of a truly effective immunotherapy.

Identification of mouse helper epitopes for WT1-specific CD4+ T cells.

Helper T lymphocytes (HTLs) play a central role in cancer immunity because they can not only help the induction and proliferation of cytotoxic T lymphocytes (CTLs) but also their differentiation into cytotoxic CD4+ T cells and directly kill the target cells.This study describes the identification of three novel mouse Th epitope peptides, WT135-52, WT186-102 and WT1294-312, derived from WT1 protein, which is the most potent tumor-associated antigen. Compared to immunization with WT1 CTL peptide alone, immunization with the addition of these WT1-specific Th peptides strongly induced WT1-specific CTLs, continued to maintain them, and efficiently rejected the challenge of WT1-expressing tumor cells. Importantly, the majority of WT1-specific CTLs induced by the co-immunization with WT1 CTL and the WT1-specific Th peptides were CD44+CD62L- effector memory CD8+ T cells, which played a central role in tumor rejection. Establishment of mouse models suitable for the analysis of the detailed mechanism of these functions of HTLs is very important. These results clearly showed that WT1-specific HTLs perform an essential function in WT1-specific tumor immunity. Therefore, the WT1-specific Th peptides identified here should make a major contribution to elucidation of the mutual roles of WT1-specific CTLs and HTLs in cancer immunity in in vivo mouse models.

Identification of two distinct populations of WT1-specific cytotoxic T lymphocytes in co-vaccination of WT1 killer and helper peptides.

Simultaneous induction of tumor antigen-specific cytotoxic T lymphocytes (CTLs) and helper T lymphocytes (HTLs) is required for an optimal anti-tumor immune response. WT1332, a 16-mer WT1-derived helper peptide, induce HTLs in an HLA class II-restricted manner and enhance the induction of WT1-specific CTLs in vitro. However, in vivo immune reaction to WT1332 vaccination in tumor-bearing patients remained unclear. Here, a striking difference in WT1-specific T cell responses was shown between WT1 CTL + WT1 helper peptide and WT1 CTL peptide vaccines in patients with recurrent glioma. WT1-specific CTLs were more strongly induced in the patients who were immunized with WT1 CTL + WT1 helper peptide vaccine, compared to those who were immunized with WT1 CTL vaccine alone. Importantly, a clear correlation was demonstrated between WT1-specific CTL and WT1332-specific HTL responses. Interestingly, two novel distinct populations of WT1-tetramerlow WT1-TCRlow CD5low and WT1-tetramerhigh WT1-TCRhigh CD5high CTLs were dominantly detected in WT1 CTL + WT1 helper peptide vaccine. Although natural WT1 peptide-reactive CTLs in the latter population were evidently less than those in the former population, the latter population showed natural WT1 peptide-specific proliferation capacity comparable to the former population, suggesting that the latter population highly expressing CD5, a marker of resistance to activation-induced cell death, should strongly expand and persist for a long time in patients. These results demonstrated the advantage of WT1 helper peptide vaccine for the enhancement of WT1-specific CTL induction by WT1 CTL peptide vaccine.

Enhanced immune reaction resulting from co-vaccination of WT1 helper peptide assessed on PET-CT.

It has become evident that positron emission tomography/computed tomography (PET-CT) using 2-deoxy-2-[F-18]fluoro-D-glucose (FDG) (FDG PET-CT) can detect anti-tumor immune response induced by various immunotherapies. To evaluate whether FDG PET-CT could detect anti-cancer immune response caused by cancer vaccine therapy, we performed a retrospective analysis of FDG PET-CT imaging of patients who were treated with Wilms Tumor 1 (WT1) vaccine therapy in Osaka University during July 2008 and June 2018. Increased FDG uptakes were detected in WT1-vaccinated skin and their draining lymph nodes during the repeated vaccination. While the FDG uptakes seemed to decrease with time after the cessation of WT1 peptide vaccinations, persistence of FDG uptakes for years in WT1-vaccinated skin were also observed in 2 cases who showed good clinical course. Moreover, the FDG uptakes of patients treated with the combination vaccine of WT1 specific cytotoxic T cell (CTL) and helper peptides were significantly stronger than of those treated with the WT1 CTL peptide alone. Since it is evident that the combination vaccine can induce a more robust anti-tumor immunity than can CTL peptide vaccine alone, the FDG uptakes in WT1-vaccinated skin might reflect the degree of immune response. These results suggest that PET-CT might be a good tool for prediction of anti-tumor immune response induced by WT1 vaccine therapy. Larger scale prospective studies therefore seem to be warranted.

Encouraging Clinical Evolution of a Pediatric Patient With Relapsed Diffuse Midline Glioma Who Underwent WT1-Targeting Immunotherapy: A Case Report and Literature Review.

Diffuse midline glioma (DMG) in children is a highly aggressive, malignant brain tumor that is fatal when relapsed. Wilms tumor 1 (WT1) is a high-priority antigen target for cancer immunotherapy. We hereby report on a pediatric patient who had DMG that regrew after chemoradiotherapy and underwent WT1 peptide vaccination. A 13-year-old Japanese boy presented with vertigo, diplopia, and right hemiplegia at the initial visit to another hospital, where he was diagnosed with DMG by magnetic resonance imaging (MRI); DMG was categorized to histological grade IV glioma. The patient underwent radiotherapy and chemotherapy with temozolomide. After three cycles of chemotherapy, MRI revealed tumor regrowth that translated into deteriorated clinical manifestations. Immunohistochemically, the H3.3K27M mutation in the biopsy specimen was confirmed and the specimen was positive for WT1 protein. The patient underwent WT1-targeting immunotherapy with the WT1-specific peptide vaccine because of having HLA-A*24:02. Consequently, his quality of life drastically improved so much as to the extent that the patient became capable of conducting nearly normal daily activities at weeks 8 to 12 of vaccination. MRI at week 8 of vaccination revealed an obvious reduction in the signal intensity of the tumor. Furthermore, betamethasone dose could be reduced successively (4, 1, and 0.5 mg/day at weeks 4, 5, and 7, respectively) without deteriorating clinical manifestations. Best response among responses assessed according to the Response Assessment in Neuro-Oncology criteria was stable disease. Overall survival was 6.5 months after vaccination onset and was 8.3 months after relapse; the latter was markedly longer than the reported median OS of 3.2 months for pediatric patients with relapsed DMG in the literature. Modified WT1 tetramer staining revealed the WT1 peptide vaccine-induced production of WT1-specific cytotoxic T cells, and the interferon-γ (IFN-γ) ELISpot assay of peripheral blood mononuclear cells disclosed the production of IFN-γ. Delayed-type hypersensitivity test became positive. Any treatment-emergent adverse events did not occur except injection site erythema. Our pediatric patient exhibited an encouraging clinical evolution as manifested by stable disease, improved clinical manifestations, steroid dose reductions, a WT1-specific immune response, and a good safety profile. Therefore, WT1-targeting immunotherapy warrants further investigation in pediatric patients with relapsed DMG.

Reader-free ELISPOT assay for immuno-monitoring in peptide-based cancer vaccine immunotherapy.

Cancer vaccine immunotherapy is a therapy that induces cellular immune responses against a target molecule to elicit clinical anti-tumor effects. These cellular immune responses against the target molecule are monitored to evaluate whether the antigen-specific cellular immune responses are induced and maintained during the vaccination period. Enzyme-linked immunospot (ELISPOT) assay is widely performed to analyze not only the frequency of immune cells, but also their effector functions as determined by their cytokine production/secretion. The present study aimed to develop a reader-free ELISPOT assay using a handy membrane-punching device termed ELI 8. With the assistance of particle analysis by ImageJ software, the results of spot counting were reproducible with high inter-assay and inter-examiner concordance. Immune cells that produce and secrete Th1 cytokines without antigen-peptide stimulation of peripheral blood mononuclear cells (PBMCs) were detected, and their frequencies in patients with cancer were significantly higher compared with those in healthy individuals. These frequencies varied between individuals, as well as between time points during the course of cancer vaccine immunotherapy in each patient. Due to the variability in spontaneous cytokine production/secretion by PBMCs, an antigen-specific immune response (IR) index is proposed, which is a ratio of the number of spot-forming cells (SFCs) subjected to antigen-stimulation to that of SFCs with spontaneous cytokine secretion without antigen-stimulation. This index may be used as a marker for antigen-specific cellular immune responses in patients treated with cancer immunotherapy. The IR index successfully detected the induction of Wilms' tumor 1-specific cellular immune responses in patients with cancer treated with cancer vaccine immunotherapy.

Immune adjuvant therapy using Bacillus Calmette-Guérin cell wall skeleton (BCG-CWS) in advanced malignancies: A phase 1 study of safety and immunogenicity assessments.

The cell wall skeleton of Bacillus Calmette-Guérin (BCG-CWS) is a bioactive component that is a strong immune adjuvant for cancer immunotherapy. BCG-CWS activates the innate immune system through various pattern recognition receptors and is expected to elicit antigen-specific cellular immune responses when co-administered with tumor antigens. To determine the recommended dose (RD) of BCG-CWS based on its safety profile, we conducted a phase I dose-escalation study of BCG-CWS in combination with WT1 peptide for patients with advanced cancer.The primary endpoint was the proportion of treatment-related adverse events (AEs) at each BCG-CWS dose. The secondary endpoints were immune responses and clinical effects. A BCG-CWS dose of 50, 100, or 200 µg/body was administered intradermally on days 0, 7, 21, and 42, followed by 2 mg of WT1 peptide on the next day. For the escalation of a dose level, 3 + 3 design was used.Study subjects were 18 patients with advanced WT1-expressing cancers refractory to standard anti-cancer therapies (7 melanoma, 5 colorectal, 4 hepatobiliary, 1 ovarian, and 1 lung). Dose-limiting toxicity occurred in the form of local skin reactions in 2 patients at a dose of 200 µg although no serious treatment-related systemic AEs were observed. Neutrophils and monocytes transiently increased in response to BCG-CWS. Some patients demonstrated the induction of the CD4 T cell subset and its differentiation from the naïve to memory phenotype, resulting in a tumor response.The RD of BCG-CWS was determined to be 100 µg/body. This dose was well tolerated and showed promising clinical effects with the induction of an appropriate immune response.

A phase I clinical study of a cocktail vaccine of Wilms' tumor 1 (WT1) HLA class I and II peptides for recurrent malignant glioma.

PURPOSE: The safety and clinical efficacy of WT1 human leukocyte antigen (HLA) class I peptide vaccine have been established, but the safety of a cocktail vaccine of WT1 HLA class I and II peptides has not. To verify its safety, we performed a phase I clinical trial for patients with recurrent malignant gliomas and assessed the immunological responses and survival data. PATIENTS AND METHODS: Fourteen HLA-A*24:02-positive patients with recurrent malignant glioma (2 with grade 3, 12 with grade 4) were enrolled. Every week, the patients received alternately a vaccine containing 3 mg of WT1 HLA-A*24:02-restricted (HLA class I) peptide and a cocktail vaccine of the HLA class I peptide and one of 0.75, 1.5 or 3 mg of the WT1 HLA class II peptide. For patients who showed no significant adverse effects within 6 weeks, the WT1 vaccine was continued at 2-4-week intervals. RESULTS: Eleven of the 14 patients completed WT1 vaccination for 6 weeks, while 3 patients dropped out earlier due to disease progression. All patients showed grade I level of skin disorders at the injection sites. No grade III/IV toxicity or dose-limiting toxicity was observed for any dose of WT1 HLA class II peptide. Six of the 14 patients had stable disease at 6 weeks. Median OS and 1-year OS rates were 24.7 weeks and 36%, respectively. CONCLUSION: The safety of a cocktail vaccine of WT1 HLA class I and II peptides for malignant gliomas was verified. This vaccine is, therefore, considered promising for patients with recurrent malignant glioma.

Extremely strong infiltration of WT1-specific CTLs into mouse tumor by the combination vaccine with WT1-specific CTL and helper peptides.

In immunotherapy by cancer antigen-derived peptide vaccine, vaccination of cytotoxic T lymphocyte (CTL) peptide alone is common, while it remains unclear whether the addition of helper peptide vaccine to the CTL peptide vaccine is of great advantage for the enhancement of tumor immunity. In the present study, combination vaccine of Wilms' tumor gene 1(WT1) protein-derived CTL and helper peptides induced the strong infiltration of WT1-specific CD8+ T cells into mouse tumor at frequencies of 8.8%, resulting in the formation of multiple microscopic necrotic lesions in the tumor, whereas the frequencies of WT1-specific CD8+ T cell infiltration into the tumor in the vaccination of the CTL peptide alone were only 0.32%. The majority of the infiltrated WT1-specific CD8+ T cells was effector phenotype T cells, but importantly, WT1-specific CD8+CD44+CD62L+CD103+ resident memory T cells, which could differentiate into a lot of effector phenotype T cells, existed in the tumor of mice vaccinated with the both WT1 peptides. Furthermore, T-cell receptor repertoire analysis showed the oligoclonality of these tumor infiltrating WT1 tetramer+ CD8+ T cells, and 3 clones occupied about half of them. These results indicated that WT1-specific CD4+ T cells played an essential role not only in the priming and activation of WT1-specific CD8+ T cells, but also in trafficking and infiltration of the CD8+ T cells into tumors. These results should provide us with the concept that in the clinical setting, combination vaccine of WT1-specific CTL and helper peptides would be more advantageous than the CTL peptide vaccine alone.