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...

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), IFNa. 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 IFNa 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 IFNa 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 H uman tumor immunology research has advanced since the first human melanoma tumor antigen recognized by CD8+ cytotoxic T lymphocytes (CTL) was identified in 1992, (1) and more than 20 melanoma antigens have been reported. Some antigens and human leukocyte antigen (HLA) class Irestricted antigenic peptides underwent clinical trials, and their adverse effects and clinical and immunological responses were studied.(8-11) Rosenberg et al. (4) reported on a large number of melanoma patients and found that less than 5% of patients who received peptide vaccines such as gp100 and interleukin-2 (IL-2) had a complete response.Nevertheless, a UK-based pharmaceutical company reported that a 3-year-long observation after melanoma antigen family A, 3 (MAGE-A3) vaccine inoculation indicated a 33% reduction in the post-operative recurrence of non-small-cell lung cancers when compared with a placebo group.(12) This observation gives strong hope for future cancer immunotherapy and has prompted many different investigations for the establishment of human tumor immunotherapy.Meanwhile, human tumor antigens of non-melanoma tumors such as colon, lung, urinary tract and...

Background: Tumor necrosis factor-α (TNFα) is an important mediator of inflammatory and autoimmune diseases. Analysis of its pathophysiologic roles has been difficult because low concentrations of TNFα, including those in healthy controls, cannot be measured by existing methods. Methods: We developed a sensitive immuno-PCR assay for the detection of TNFα in human serum. The DNA label was generated by PCR amplification using biotinylated primer and was bound with streptavidin to the biotinylated third antibody. TNFα sandwiched by antibodies was detected by amplification of the DNA label using PCR. Results: The limit of detection of the assay was 0.001 ng/L, an ∼5 × 104-fold improvement compared with a conventional ELISA. The mean serum TNFα concentration (± SD) in healthy donors was 0.021 ± 0.044 ng/L in men (n = 29) and 0.033 ± 0.065 ng/L in women (n = 25). Conclusion: This method may be useful for analyzing the significance of TNFα concentration in various diseases.

We established the validity of a quantitative reverse transcription (RT)-PCR assay for the RNA component of human telomerase (hTR), using the TaqMan fluorogenic detection system. Using this assay, we quantified hTR expression in two human pancreatic cancer cell lines, ASPC-1 and MIAPaCa-2. Our results indicated that hTR expression in MIAPaCa-2 was 1.99-fold higher than that in ASPC-1 cells. This TaqMan RT-PCR assay appears to be useful in determining the amount of hTR in clinical specimens.

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