Takayuki Kanaseki

A Potent Immunogenic General Cancer Vaccine That Targets Survivin, an Inhibitor of Apoptosis Proteins

We reported previously a HLA-A24-restricted antigenic peptide, survivin-2B80-88 (AYACNTSTL), recognized by CD8+ CTL. This peptide was derived from survivin protein, an inhibitor of apoptosis proteins, expressed in a variety of tumors, such as adenocarcinoma, squamous cell carcinoma, and malignant melanoma. In this report, we provide further evidence that survivin-2B80-88 peptide might serve as a potent immunogenic cancer vaccine for various cancer patients. Overexpression of survivin was detected in surgically resected primary tumor specimens of most breast and colorectal cancers and some gastric cancers as assessed by immunohistochemical study. HLA-A24/survivin-2B80-88 tetramer analysis revealed that there existed an increased number of CTL precursors in peripheral blood mononuclear cells (PBMC) of HLA-A24+ cancer patients, and in vitro stimulation of PBMCs from six breast cancer patients with survivin-2B80-88 peptide could lead to increases of the CTL precursor frequency. Furthermore, CTLs specific for this peptide were successfully induced from PBMCs in all 7 (100%) patients with breast cancers, 6 of 7 (83%) patients with colorectal cancers, and 4 of 7 (57%) patients with gastric cancers. These data indicate that survivin expressed in tumor tissues is antigenic in cancer patients, and survivin-2B80-88-specific CTLs are present in PBMCs of various cancer patients. Our study raises the possibility that this peptide may be applicable as a general cancer vaccine to a large proportion of HLA-A24+ cancer patients.

Epigenetic inactivation of class II transactivator (CIITA) is associated with the absence of interferon-gamma-induced HLA-DR expression in colorectal and gastric cancer cells

Tightly regulated at the level of transcription, expression of MHC class II molecules varies significantly among gastrointestinal cancers. High levels of MHC class II expression are often associated with a better prognosis, which is indicative of the involvement of CD4+ lymphocytes in tumor suppression, but the molecular mechanism by which MHC class II expression is regulated remains unclear. In the present study, we investigated the expression of one inducible MHC class II molecule, HLA-DR, and its coactivators in a panel of colorectal and gastric cancer cell lines. Interferon-γ induced expression of HLA-DR in 14 of 20 cell lines tested; the remaining six cell lines did not express HLA-DR. Analysis of the expression of transcription factors and coactivators associated with HLA-DR revealed that the loss of CIITA expression was closely associated with the absence of HLA-DR induction. Moreover, DNA methylation of the 5′ CpG island of CIITA-PIV was detected in all cancer cells that lacked CIITA. The methylation and resultant silencing of CIITA-PIV depended on the activities of two DNA methyltransferases, DNMT1 and DNMT3B, and their genetic inactivation restored CIITA-PIV expression. It thus appears that CIITA methylation is a key mechanism that enables some gastrointestinal cancer cells to escape immune surveillance.

Endoplasmic Reticulum Aminopeptidase Associated with Antigen Processing Defines the Composition and Structure of MHC Class I Peptide Repertoire in Normal and Virus-Infected Cells

The MHC class I (MHC-I) molecules ferry a cargo of peptides to the cell surface as potential ligands for CD8+ cytotoxic T cells. For nearly 20 years, the cargo has been described as a collection of short 8-9 mer peptides, whose length and sequences were believed to be primarily determined by the peptide-binding groove of MHC-I molecules. Yet the mechanisms for producing peptides of such optimal length and composition have remained unclear. In this study, using mass spectrometry, we determined the amino acid sequences of a large number of naturally processed peptides in mice lacking the endoplasmic reticulum aminopeptidase associated with Ag processing (ERAAP). We find that ERAAP-deficiency changed the oeuvre and caused a marked increase in the length of peptides normally presented by MHC-I. Furthermore, we observed similar changes in the length of viral peptides recognized by CD8+ T cells in mouse CMV-infected ERAAP-deficient mice. In these mice, a distinct CD8+ T cell population was elicited with specificity for an N-terminally extended epitope. Thus, the characteristic length, as well as the composition of MHC-I peptide cargo, is determined not only by the MHC-I peptide-binding groove but also by ERAAP proteolysis in the endoplasmic reticulum.

HSP DNAJB8 Controls Tumor-Initiating Ability in Renal Cancer Stem-like Cells

Cancer stem-like cells (CSC) are a small population of cancer cells with superior tumor initiating, self-renewal, and differentiation properties. In this study, we show that the cancer-testis antigen and HSP40 family member DNAJB8 contributes to the CSC phenotype in renal cell carcinoma (RCC). DNAJB8 overexpression increased the percentage of side population (SP) cells representing CSCs in RCC cells, enhancing their tumor-initiating ability. Conversely, attenuation of DNAJB8 decreased SP cells and reduced tumor-initiating ability. The utility of DNAJB8 as an immunologic target was established in DNA vaccination experiments. Compared with immunization with the tumor-associated antigen survivin, which was expressed in both CSCs and non-CSCs in RCC, immunization with Dnajb8 expression plasmids yielded stronger antitumor effects. Together, our findings suggest that DNAJB8 plays a role in CSC maintenance and that it offers a candidate for CSC-targeting immunotherapy in RCC.

Immunotherapeutic benefit of α-interferon (IFNα) in survivin2B-derived peptide vaccination for advanced pancreatic cancer patients

Survivin, a member of the inhibitor of apoptosis protein (IAP) family containing a single baculovirus IAP repeat domain, is highly expressed in cancerous tissues but not in normal counterparts. Our group identified an HLA‐A24‐restricted antigenic peptide, survivin‐2B80–88 (AYACNTSTL), that is recognized by CD8 + CTLs and functions as an immunogenic molecule in patients with cancers of various histological origins such as colon, breast, lung, oral, and urogenital malignancies. Subsequent clinical trials with this epitope peptide alone resulted in clinical and immunological responses. However, these were not strong enough for routine clinical use as a therapeutic cancer vaccine, and our previous study of colon cancer patients indicated that treatment with a vaccination protocol of survivin‐2B80–88 plus incomplete Freunds adjuvant (IFA) and α‐interferon (IFNα) conferred overt clinical improvement and enhanced the immunological responses of patients. In the current study, we further investigated whether this vaccination protocol could efficiently provide not only improved immune responses but also better clinical outcomes for advanced pancreatic cancers. Tetramer and enzyme‐linked immunosorbent spot analysis data indicated that more than 50% of the patients had positive clinical and immunological responses. In contrast, assessment of treatment with IFNα only to another group of cancer patients resulted in no obvious increase in the frequency of survivin‐2B80‐88 peptide‐specific CTLs. Taken together, our data clearly indicate that a vaccination protocol of survivin‐2B80‐88 plus IFA and IFNα is very effective and useful in immunotherapy for this type of poor‐prognosis neoplasm. This trial was registered with the UMIN Clinical Trials Registry, no. UMIN000000905. (Cancer Sci 2013; 104: 124–129)

Immunogenic enhancement and clinical effect by type-I interferon of anti-apoptotic protein, survivin-derived peptide vaccine, in advanced colorectal cancer patients

We previously identified a human leukocyte antigen (HLA)‐A24‐restricted antigenic peptide, survivin‐2B80‐88, recognized by CD8+ cytotoxic T lymphocytes (CTL). Subsequently, we attempted clinical trials with this epitope peptide alone for some malignancies, resulting in clinical and immunological responses, although their potential was not strong enough for routine clinical use as a cancer vaccine. In the current study, to assess whether immunogenicity of the survivin‐2B80‐88 peptide could be enhanced with other vaccination protocols, we performed clinical trials in advanced colon cancer patients with two vaccination protocols: (i) survivin‐2B80‐88 plus incomplete Freund’s adjuvant (IFA); and (ii) survivin‐2B80‐88 plus IFA and a type‐I interferon (IFN), IFNα. Our data clearly indicated that, although the effect of survivin‐2B80‐88 plus IFA was not significantly different from that with survivin‐2B80‐88 alone, treatment with the vaccination protocol of survivin‐2B80‐88 plus IFA and IFNα resulted in clinical improvement and enhanced immunological responses of patients. Tetramer analysis of survivin‐2B80‐88 peptide‐specific CTL demonstrated that such CTL were increased at least twofold after vaccination with this protocol in four of eight patients. In these patients, enzyme‐linked immunosorbent spot (ELISPOT) results were also enhanced. Subsequent study of single‐cell clone separation by cell sorting of peptide‐specific CTL showed that each CTL clone was indeed not only peptide‐specific but also cytotoxic against human cancer cells in the context of the expression of both HLA‐A24 and survivin molecules. Taken together, these results indicate that vaccination of colon cancer patients with survivin‐2B80‐88 plus IFA and IFNα can be considered to be a very potent immunotherapeutic regimen, and that this protocol might work for other cancers. (Cancer Sci 2011; 102: 1181–1187)

Preferential expression of cancer/testis genes in cancer stem‐like cells: proposal of a novel sub‐category, cancer/testis/stem gene

Cancer/testis (CT) antigens encoded by CT genes are immunogenic antigens, and the expression of CT gene is strictly restricted to only the testis among mature organs. Therefore, CT antigens are promising candidates for cancer immunotherapy. In a previous study, we identified a novel CT antigen, DNAJB8. DNAJB8 was found to be preferentially expressed in cancer stem-like cells (CSCs)/cancer-initiating cells (CICs), and it is thus a novel CSC antigen. In this study, we hypothesized that CT genes are preferentially expressed in CSCs/CICs rather than in non-CSCs/-CICs and we examined the expression of CT genes in CSCs/CICs. The expression of 74 CT genes was evaluated in side population (SP) cells (=CSC) and main population (MP) cells (=non-CSC) derived from LHK2 lung adenocarcinoma cells, SW480 colon adenocarcinoma cells and MCF7 breast adenocarcinoma cells by RT-PCR and real-time PCR. Eighteen genes (MAGEA2, MAGEA3, MAGEA4, MAGEA6, MAGEA12, MAGEB2, GAGE1, GAGE8, SPANXA1, SPANXB1, SPANXC, XAGE2, SPA17, BORIS, PLU-1, SGY-1, TEX15 and CT45A1) showed higher expression levels in SP cells than in MP cells, whereas 10 genes (BAGE1, BAGE2, BAGE4, BAGE5, XAGE1, LIP1, D40, HCA661, TDRD1 and TPTE) showed similar expression levels in SP cells and MP cells. Thus, considerable numbers of CT genes showed preferential expression in CSCs/CICs. We therefore propose a novel sub-category of CT genes in this report: cancer/testis/stem (CTS) genes.

Histone Deacetylation, But Not Hypermethylation, Modifies Class II Transactivator and MHC Class II Gene Expression in Squamous Cell Carcinomas

In this study, we first categorized nine squamous cell carcinoma (SCC) cell lines into two groups in terms of the expression of HLA-DR, -DP, and -DQ molecules. Subsequently, the expression of class II transactivator (CIITA) was studied in these cell lines, because it is widely accepted that the expression of MHC class II molecules is regulated by different types of CIITA transcripts that are initiated by distinct promoters. The majority of the SCC cell lines (six of nine) expressed HLA-DR molecules and CIITA promoter IV (pIV) transcripts in the presence of IFN-γ. In contrast, three of the nine SCC cell lines were completely negative for class II molecules and all types of CIITA, suggesting epigenetic changes in the promoter region in these cells. Previously, methylation of CIITA pIV was reported to silence CIITA gene expression. We extensively studied the methylation status of CIITA pIV using a panel of 22 SCC cell lines. Remarkably, none of the SCC cell lines demonstrated hypermethylation at the site. In contrast, treatment with a histone deacetylation inhibitor in combination with IFN-γ clearly restored the expression of the CIITA type IV gene in the HLA-DR-negative SCC cell lines, and the acetylation status of histone H3 examined by chromatin immunoprecipitation analysis was closely associated with the gene expression. Moreover, stable transfection of the CIITA gene into an HLA-DR-negative cell line restored constitutive expression of MHC class II molecules. Therefore, histone deacetylation, but not hypermethylation, modifies CIITA DNA and class II gene expression in SCC.

Heat shock protein DNAJB8 is a novel target for immunotherapy of colon cancer-initiating cells

The aim of the present study was to establish cancer stem‐like cell/cancer‐initiating cell (CSC/CIC)‐targeting immunotherapy. The CSC/CIC are thought to be essential for tumor maintenance, recurrence and distant metastasis. Therefore they are reasonable targets for cancer therapy. In the present study, we found that a heat shock protein (HSP) 40 family member, DnaJ (Hsp40) homolog, subfamily B, member 8 (DNAJB8), is preferentially expressed in CSC/CIC derived from colorectal cancer (CRC) cells rather than in non‐CSC/CIC. Overexpression of DNAJB8 enhanced the expression of stem cell markers and tumorigenicity, indicating that DNAJB8 has a role in CRC CSC/CIC. A DNAJB8‐specific cytotoxic T lymphocyte (CTL) response could be induced by a DNAJB8‐derived antigenic peptide. A CTL clone specific for DNAJB8 peptide showed higher killing activity to CRC CSC/CIC compared with non‐CSC/CIC, and CTL adoptive transfer into CRC CSC/CIC showed an antitumor effect in vivo. Taken together, the results indicate that DNAJB8 is expressed and has role in CRC CSC/CIC and that DNAJB8 is a novel target of CRC CSC/CIC‐targeting immunotherapy.

Immune responses to human cancer stem-like cells/cancer-initiating cells

Cancer stem‐like cells (CSC)/cancer‐initiating cells (CIC) are defined as minor subpopulations of cancer cells that are endowed with properties of higher tumor‐initiating ability, self‐renewal ability and differentiation ability. Accumulating results of recent studies have revealed that CSC/CIC are resistant to standard cancer therapies, including chemotherapy, radiotherapy and molecular targeting therapy, and eradiation of CSC/CIC is, thus, critical to cure cancer. Cancer immunotherapy is expected to become the “fourth” cancer therapy. Cytotoxic T lymphocytes (CTL) play an essential role in immune responses to cancers, and CTL can recognize CSC/CIC in an antigen‐specific manner. CSC/CIC express several tumor‐associated antigens (TAA), and cancer testis (CT) antigens are reasonable sources for CSC/CIC‐targeting immunotherapy. In this review article, we discuss CSC/CIC recognition by CTL, regulation of immune systems by CSC/CIC, TAA expression in CSC/CIC, and the advantages of CSC/CIC‐targeting immunotherapy.