学位論文内容の要旨

博士の専攻分野の名称

博士（医 学）氏 名 塩浜 康雄

学 位 論 文 題 名Augmented induction of a novel cancer/testis antigen in demethylated cancer cells(脱メチル化処理がん細胞を用いた新規がん抗原の同定に関する研究)
Background and Objectives：Cancer immunotherapy including cancer vaccine has been considered
as an attractive therapy for cancer patients because of the fewer side effects. CT (cancer/testis)
antigens are widely expressed in various human cancers but their expressions in normal cells aretightly restricted in testis. Generally, it has been reported that cancer vaccine therapy using HLAclass I-binding short peptide derived from CT antigens induced the antigen-specific CTLs andmaintained the cancer patients at long stable disease. However, the vaccine therapy focused on only
CTL activation appears to be suboptimal to conquer cancer. To overcome this problem, it isnecessary to develop more efficient method to improve immunosuppression in cancer patients. It has
been demonstrated that the introduction of Th1-dominant immunity in tumor-bearing host isessential for inducing fully activated CTLs and the subsequent memory T cell generation.
5-Aza-2′-deoxycytidine (DAC) is well known to induce gene expression of CT antigens by thedemethylation of promoter CpG islands of the treated cancer cells. Therefore, we first used
DAC-treated demethylated cancer cells to identify novel CT antigens in the present study and thentried identification of the helper peptide epitopes, applicable to development of more efficient cancer
immunotherapy.
Methods：Human lung cancer cells, LC-OK, A549, and LC-MS cells were treated with or without
DAC for 3 days. Gene expression levels of the A549 cells were evaluated by microarray analysis.
The augmented gene in DAC-treated A549 cells were first selected as candidates, and the candidate
gene expression levels in normal tissues were further evaluated by the multiple tissue cDNA panels
including testis. C16orf73 gene, HP15 expressed in only testis, was selected as novel CT antigen inthis study. HP15-specific CD4+ T cells were induced from purified CD4+ T cells by using
HP15-overlapping peptides covering whole amino-acid sequence of HP15. HLA restriction of theHP15-specific CD4+ T cells was further determined by the IFN- production in response to thepeptide-loaded LCLs. HP15-specific T cells induced from PBMCs, were mixed with HLA-matchedor mismatched lung cancer cell lines, which were pre-treated with or without IFN- and DAC in thepresence or absence of anti-HLA class I and anti-HLA class II mAbs for 72 h. IFN- production by
the established T cells was detected by ELISA. HP15 gene expression levels in various cancer
tissues, primary cancer cells, and normal PBMCs were evaluated by quantitative- and RT-PCR.
Results: Gene expression levels of CT antigens, MAGE-A4, XAGE, and BAGE were augmented inhuman lung cancer cell lines, LC-OK, A549, or LC-MS cells after DAC-treatment. In addition,expression of a novel C16orf73 gene, HP15 was greatly enhanced in the demethylated lung cancercells. The HP15 expression was observed in only testis but not other normal tissues, suggesting that
HP15 was a novel CT antigen. HP15 gene expression was also detected in several cancer cell lines.
Next, HP15-specific CD4+ T cells were induced by using HP15-overlapping peptides. Then, helperepitopes of HP15 CT antigen and the HLA restriction were identified by HP15-specific CD4+ T cells
and the overlapping peptides. As a result, almost all regions of HP15 CT antigen contained helperepitopes. Furthermore, it was confirmed that HP15-specific T cells established from PBMCs
recognized HP15 antigen epitopes naturally processed and bond to HLA on human cancer cells in aHLA-class II dependent manner. In addition, the HP15-specific T cells did not respond toHLA-mismatched A549 cells, even if the cells were treated with DAC plus IFN- . HP15 gene waswidely expressed in colon, lung, gallbladder, head and neck, and renal tissues from cancer patients.
It was also confirmed that DAC-treatment enhanced HP15 gene expression level of primary cancer
cells established from colon cancer patients but did not affect on that of normal PBMCs from healthy
volunteers.
Discussion: In the present work, a novel CT antigen, HP15 was identified by DAC-treatment of
human lung cancer cells. HP15 gene expression was observed in various cancer tissues of cancer
patients as well as several cancer cell lines but not in normal tissues except testis. Therefore, thepresent strategy using demethylated cancer cells by treatment with DAC would be a useful tool tofind novel CT antigens applicable to cancer vaccine therapy in human. It was also demonstrated thatDAC-treatment caused the augmented induction of HP15 CT antigen in all cancer cells tested here
as well as other CT antigens such as MAGE-A4 and XAGE, and BAGE in various cancer cells.
These results suggested that DAC-treatment of cancer cells would increase their immunogenicity viaaugmented induction of CT antigens including our found HP15 CT antigen. To consider thispossibility, helper epitopes of HP15 antigen were further identified, which can induce
IFN- -producing CD4+ T cells. In the present experiments, it was confirmed that HP15-specificCD4+ T cells, induced by synthetic peptides, responded to the IFN- -treated and HLA class
II-induced target cancer cells in a HLA class II- but not class I-dependent manner. These datasuggest that such CD4+ T cells would at least recognize the helper peptide epitopes naturally
processed on HLA class II molecules of cancer cells. Recently, a clinical study for cancer patients
revealed that DAC treatment induced NY-ESO-1 gene expression in the tumor tissues. In fact, HP15gene expression was induced by DAC treatment in primary cancer cells from colorectal cancer
patient. It was indicated that HP15 CT antigen was widely expressed in human cancer tissues inaddition to human cancer cell lines. Taken together, the present results strongly indicate thatDAC-treatment will become a novel strategy to induce immunogenic CT antigen, which facilitate
the therapeutic efficacy of cancer vaccine treatment.
Conclusion: A novel CT antigen HP15 was identified in the present experiments and the gene
expression was enhanced by DAC-treatment or spontaneously expressed in some cancer cells. Inaddition, it was confirmed that HP15 was expressed in various cancer tissues of patients. Therefore,
demethylated cancer cells are useful tool to identify of novel CT antigens for cancer immunotherapy.
Then, helper epitopes of HP15 antigen were determined by using HP15 overlapping peptides. In fact,
HP15-specific CD4+ T cells were successfully induced from PBMCs by HP15-overlapping peptides
and the established T cells recognized HP15 and HLA-matched HLA-class II expressing cancer cells.
These results indicate that HP15 is applicable to T cell based cancer immunotherapy. HP15 waswidely expressed in colon, lung, gallbladder, head and neck, and renal tissues from cancer patients inaddition to various cancer cell lines. Thus, these findings suggested that demethylation drugs such as
DAC would be a useful tool not only for finding novel CT antigens but also for developing thecombined therapy with cancer vaccine.