Background: We previously reported that survivin-2B, a splicing variant of survivin, was expressed in various types of tumors and that survivin-2B peptide might serve as a potent immunogenic cancer vaccine. The objective of this study was to examine the toxicity of and to clinically and immunologically evaluate survivin-2B peptide in a phase I clinical study for patients with advanced or recurrent breast cancer.

Cancer stem-like cells (CSCs) and tumor-initiating cells (TICs) are a small population of cancer cells that share three properties: tumor initiating ability, self-renewal, and differentiation. These properties suggest that CSCs/ TICs are essential for tumor maintenance, recurrence, and distant metastasis. Here, we show that cytotoxic T lymphocytes (CTLs) specific for the tumor-associated antigen CEP55 can efficiently recognize colon CSCs/ TICs both in vitro and in vivo. Using Hoechst 33342 dye staining, we isolated CSCs/TICs as side population (SP) cells from colon cancer cell lines SW480, HT29, and HCT15. The SP cells expressed high levels of the stem cell markers SOX2, POU5F1, LGR5, and ALDH1A1 and showed resistance to chemotherapeutic agents such as irinotecan or etoposide.To evaluate the susceptibility of SP cells to CTLs, we used CTL clone 41, which is specific for the CEP55-derived antigenic peptide Cep55/ c10orf3_193 ( Colon cancer is one of the most common malignancies worldwide. With recent progress in treatment, the prognosis has improved to some extent. In advanced disease, however, the prognosis remains unfavorable, because of recurrence, distant metastasis, and resistance to treatment. Thus, novel treatment modalities are needed.Cancers contain morphologically heterogeneous populations. This fact has led to the cancer stem cell theory, 1 the idea that cancers are composed of several types of cells, and that only a small population of cancer cells that can regenerate cancer tissues, much as normal tissue can be regenerated only by a small population of stemlike cells. Recently, cancer stem-like cells and tumorinitiating cells (CSCs/TICs) have been isolated from various types of malignancies, including colon cancer.2-6 In colon cancer, CSCs/TICs can reinitiate tumors that resemble mother colon cancer tissues morphologically when transplanted into immunodeficient mice.3 Furthermore, these CSCs/TICs have higher tumorigenic potential than do non-CSCs/TICs. Previous reports have shown that CSCs/TICs are resistant to a variety of treatments, including chemotherapy and radiotherapy, with varied mechanisms of resistance, including high expression of drug transporters, relative cell cycle quiescence, high levels of DNA repair machinery, and resistance to apoptosis. 7 These reports 3-6 support the hypothesis that malignant cancers comprise heterogeneous populations that organize in a hierarchical differentiation model. The CSCs/TICs are located at the top of this hierarchy, and targeting CSCs/TICs is essential to achieve efficient effects for treatment of malignant diseases. Recently, some trials targeting CSCs/TICs have been reported for hema-

Identification of tumor-associated antigens may facilitate vaccination strategies to treat patients with malignant diseases. We have found that the centrosomal protein, Cep55/c10orf3 acts as a novel breast carcinoma-associated tumor-associated antigen. Cep55/c10orf3 mRNA was detectable in a wide variety of tumor cell lines. Expression was barely detectable in normal tissues except for testis and thymus. Moreover, Cep55/c10orf3 protein could be detected by a monoclonal anti-Cep55/c10orf3 antibody (# 11-55) in 69.8% of breast carcinoma, 25% of colorectal carcinoma, and 57.8% of lung carcinoma tissues. The expression of Cep55/c10orf3 protein did not show any relationship with the hormone receptors such as estrogen receptor and progesterone receptor or expression patterns of p185 HER2/neu. We designed 11 peptides which displayed a human leukocyte antigen-A24 binding motif. One Cep55/c10orf3-peptide, Cep55/c10orf3_193(10) (VYVKGLLAKI), induced cytotoxic T lymphocytes (CTLs) in 3 of 3 patients with Cep55/c10orf3 (# 11-55)-positive breast carcinoma. A Cep55/c10orf3_193(10)-specific CTL clone could also recognize Cep55/c10orf3 (+) displayed on human leukocyte antigen-A24 (+) cancer cell lines. These data indicate that Cep55/c10orf3 peptides were naturally presented by breast cancer cells and can cause CTL clonal expansion in vivo. Monoclonal antibody # 11-55 and the Cep55/c10orf3_193(10) peptides may be useful as part of a therapeutic strategy for hormonal therapy or anti-p185 HER2/neu monoclonal antibody therapy-resistant breast carcinoma patients.

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.

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 Freund's adjuvant (IFA) and a-interferon (IFNa) 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 IFNa 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 IFNa 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) R ecent progress in human tumor immunology research has presented us with the possibility that immunotherapy could be established as an effective cancer therapy in the very near future.(1-6) Indeed, since the first discovery of a human tumor antigen in 1992, (7) many clinical trials for cancer vaccines have been carried out, and these studies have suggested that active immunization using HLA class I restricted tumor antigenic peptides and the whole or part of the tumor antigenic protein could work as activators of antigen-specific CTLs, at least in some cancer patients.(8-16) However, even in effective cases, vaccination with these molecules alone is not sufficient to evoke a potent and stable immune response and subsequent strong clinical effect. Thus, it is crucial to develop various methods for enhancing the immunological efficacy of tumor antigens.We have studied how tumor antigenicity can be efficiently enhanced in cancer patients since 2003. In our studies, the HLA-A24-restricted peptide survivin-2B80-88 was given s.c. to patients six times or more at biweekly intervals for colon, breast, lung, oral cavity, and urinary bladder cancers, and lymphomas. Clinically, certain patients with colon, lung, and urinary bladder ca...