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NY-ESO-1 is a cancer/testis antigen expressed in normal adult tissues solely in the testicular germ cells of normal adults and in various cancers. It induces specific humoral and cellular immunity in patients with NY-ESO-1-expressing cancer. The aim of this study was to determine the frequency of NY-ESO-1 mRNA and protein expression in malignant and benign breast tumors. NY-ESO-1 mRNA expression was detected by conventional reverse transcription-PCR and real-time PCR, and that of the protein expression by immunohistochemistry and Western blot analysis. Expression of NY-ESO-1 mRNA was detected in 37 of 88 (42%) cancer specimens, whereas that of the NY-ESO-1 protein was detected only in 1 mRNA-positive specimen. In the latter case, expression level of NY-ESO-1 mRNA relative to that in the testis was relatively high (75% of testicular expression) and to the other among breast cancer specimens. In benign breast lesions, 21 of 31 (68%) specimens expressed low levels of NY-ESO-1 mRNA. In 1 case of fibroadenoma, NY-ESO-1 mRNA was 8% of the testicular level, and protein was detected by Western blot analysis. Only 1 breast cancer patient had detectable antibody at time of surgery, which disappeared within 2 years. Tumor specimen from this patient was both NY-ESO-1 mRNA and protein positive, and NY-ESO-1-specific CD8 T cells were detected in this patient by IFN-γ enzyme-linked immunospot assay using NY-ESO-1 recombinant adeno and vaccinia virus. A higher rate of NY-ESO-1 expression was noted in breast cancer with high histological grade and negative hormone receptor status, suggesting NY-ESO-1 as a potential tumor antigen for immunotherapy in patients with breast cancer and poor prognosis.

Introduction and Purpose: Recently, immune checkpoint therapy has been incorporated into treatment of solid cancers including lung cancer, resulted in prolonged survival benefit. Checkpoint inhibitors target on PD-1/PD-L1 or CTLA-4 immuno-inhibitory molecules in immune or cancer cells. However, checkpoint therapy in lung adenocarcinoma (LAD) showed less than 20% response rate, suggesting existence of other immuno-inhibitory pathways. Therefore, we focused on T cell immunoglobulin and mucin domain 3 (TIM-3) on the CD4 and CD8 T-cell surface and its ligand Galectin-9 in tumor cells, and analyzed the expression and function in LAD cells and tissues. Materials and Methods: TILs and PBMCs were obtained from 11 patients with LAD, and 194 LAD tissues resected surgically were analyzed. Expression of immuno-inhibitory molecules (PD-1, TIM-3, BTLA, LAG-3) was examined by flow cytometry using FACS on TILs and PBMCs, and PD-L1 and Galectin-9 expressions in LAD tissues was analyzed by immunohistochemistry using tissue microarray. To investigate soluble Galectin-9 released from LAD cells, EGFR-mutated PC-9 and EGFR-wild-type OU-LC-SK cells were treated with EGFR-TKI afatinib at a concentration of 10 nM for 3 days. Then, the amount of Galectin-9 in culture supernatant was measured by ELISA. To investigate T-cell apoptosis induction by Galectin-9, established XAGE1-specifiic CD8 cloned T-cells were incubated with Galectin-9 protein for 8 hrs. Results: Increased expression in TILs compared to PBMCs was observed on PD-1 and TIM-3, but not on BTLA or LAG-3 in CD4 and CD8 T-cells. In LAD tissues, the frequencies of high PD-L1 and Galectin-9 expression in the tumor cell membrane or cytoplasm were 49% and 31%, respectively (in squamous cell carcinoma, the frequencies were 32% and 16%, respectively). Furthermore, correlated expression of PD-L1, Galectin-9 and CD3 (T-cell infiltration) was observed at the periphery of the tumor nest. Those findings suggest the relevance of the PD-1/PD-L1 and TIM-3/Galectin-9 pathways in the tumor microenvironment of LAD. Next, soluble Galectin-9 released from the LAD cells was measured. Galectin-9 was detected only in the medium of EGFR-mutated LAD cells following treatment with afatinib. Moreover, apoptosis was induced in TIM-3-positive CD8 T-cell clones following interaction with Galectin-9 protein and this was inhibited by the addition of anti-Galectin-9 or an anti-TIM-3 antibody. The findings suggested that a significant amount of Galectin-9 could be released from LAD cells and induced T-cell apoptosis in tumor microenvironment. Conclusions: Our results suggested the relevance of the PD-1/PD-L1 and TIM-3/Galectin-9 immuno-inhibitory pathways in the tumor microenvironment of LAD, and that release of soluble Galectin-9 from LAD cells could negatively regulate T-cell function. For successful immune checkpoint therapy in LAD, simultaneous inhibition of TIM-3/Galectin-9 pathway may be needed. Citation Format: Mikio Oka, Yoshihiro Ohue, Koji Kurose, Yumi Nishio, Midori Isobe, Eiichi Nakayama. Immuno-inhibitory pathway, TIM-3/Galectin-9, in lung adenocarcinoma: clinical and in vitro analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2932. doi:10.1158/1538-7445.AM2017-2932

We showed that pRL1a multiple antigen peptide (MAP)-sensitized dendritic cell (DC) and P815 cell lysis by pRL1a-specific B-24 CTL was blocked by incubating target cells at 4 degrees C during sensitization. The finding suggested that pRL1a MAP was mostly internalized in DC and P815 cells and produced pRL1a peptide epitopes for presentation with H-2L(d). Furthermore, we showed that sensitization with pRL1a MAP was inhibited by the addition of chloroquine, cycloheximide, and brefeldin A to the culture, but not by the addition of inhibitors for lysosomal proteases or proteasome. Inhibition of sensitization by the addition of chloroquine to the culture suggested the requirement of acidification of the endosomal compartment for pRL1a MAP processing. Inhibition of sensitization by the addition of cycloheximide and brefeldin A to the culture indicated the requirement of newly generated MHC class I antigen molecules and the involvement of transport of the peptide MHC class I complex from the endoplasmic reticulum to the Golgi. The findings suggested that pRL1a MAP in the endosomal compartment leaked to the cytosol, and degraded, and the pRL1a peptide produced was presented by the MHC class I pathway.

Objectives: Tregs down-regulate immune responses against various antigens including tumor cells. Impairment of Tregs cause autoimmune responses. CC chemokine receptor 4 (CCR4) is expressed selectively on Foxp3+CD4 T-cells, so that the treatment of PBMCs with KM2760, anti-human CCR4 mAb with a defucosylated Fc region, could deplete CCR4+Tregs by ADCC. In this study, we analyzed depletion of Tregs phenotypically and functionally after treatment of PBMCs with KM2760. Methods and main results: CCR4 expression was analyzed on CD4 T-cells purified from healthy donor PBMCs by FACS. CCR4 positivecells were 21% in CD4 T-cells, and 71% in Foxp3+cells. Depletion of CCR4 positive cells by treatment with KM2760 was examined. PBMCs were incubated with KM2760 (0, 0.01, 0.1, 1, 10 μg/ml) for 4 or 20 hours, and analyzed by FACS. Efficient depletion was observed with higher antibody concentration and longer incubation time. Then we examined inhibition of CFSE dilution of labeled CD4 or CD8 T cells stimulated with anti-CD3 mAb after culture with an equal number of CD4+25+127low Tregs and CD4-8- cells pretreated with or without KM2760. Proliferating responder cells were 68% in culture with CD4+25+127low Tregs pretreated with KM2760, whereas those cells with CD4+25+127low Tregs without pretreatment was 38% in CD4 T-cells, 77%and 40% in CD8 T-cells,respectively. Conclusions: Efficient Treg depletion was observed in vitro by treatment of PBMCs with KM2760. In vivo treatment with the same mAb may induce tumor immune responses by Treg depletion. Citation Format: Koji Kurose, Shingo Eikawa, Yu Mizote, Yoshihiro Ohue, Midori Isobe, Akiko Uenaka, Mikio Oka, Eiichi Nakayama. Depletion of Tregs from PBMCs by treatment with defucosylated anti-CCR4 mAb. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4732. doi:10.1158/1538-7445.AM2013-4732

BACKGROUND:XAGE-1bis a cancer/testis (CT) antigen expressed highly in non-small cell lung cancer (NSCLC) with restricted expressiononly in testis in normal tissues. In this study, we investigated correlation of intensity of humoral and cellular immune responses against XAGE-1b and itsclinical relevance in NSCLC patients. MATERIALS AND METHODS:Peripheral bloodwas obtained from NSCLC patients who visited Kawasaki Medical School Hospital between 2005 and 2012 under written informed consent. Antibody response to XAGE-1bwas analyzed by ELISA using synthesized XAGE-1b protein. CD4 and CD8 T-cell responses were examined by IFN-γ ELISA and/or IFN-γcapture assay by FACS using 12-mer or 16-merXAGE-1b-overlapping peptides (OLPs) spanning the entire XAGE-1b (GAGED2a) protein. RESULTS: Antibody positive frequencies of total 362 NSCLC, 220 lung adenocarcinoma and 85 lung squamous cell carcinoma patients were 8.8% (32/362), 12.7% (28/220) and 1.2% (1/85), respectively. With antibody positive NSCLC patients, the patients showing high, intermediate and low antibody response was 7, 20 and 5, respectively. The frequency of XAGE-1b-reactive CD4 T-cells in patients showing high, intermediate and low antibody response was 5.5 ±2.1 x 10−5, 1.5 ±0.7 x 10−5 and < 1.1 x 10−5, respectively. The frequency of XAGE-1b-reactive CD8 T-cells was 6.9 ±1.6 x 10−6, 4.6 ±1.6 x 10−6 and < 1.1 x 10−6, respectively. The frequency of cytotoxic CD8 T-cells in IFN-γ secreting CD8 T-cells in response to XAGE-1b-peptide-pulsed autologous EBV-B cells in patients showing high and intermediate antibody responses was 59.0% and 4.8%, respectively.We evaluated overall survival (OS) time with 120 stage IIIB/IV lung adenocarcinoma patients. Median OS were 32.3 and 14.4 months in antibody-positive and negative patients(P-.039). There was no significant differencewith 1-year survival rate in antibody-positive (68.7%) and negative (55.6%)patients, but was significant with 3-year survival rate in 34.8% and 14.1% respectively. Furthermore, univariate and multivariable analyses showed XAGE-1b antibody response was independent prognostic factor. CONCLUSION:Our findings indicate that CT antigen XAGE-1b is highly immunogenic in NSCLC patients inducing antibody, and CD4 and CD8 T-cell responses and the immune response against XAGE-1b is beneficial for prognosis. Citation Format: Yoshihiro Ohue, Koji Kurose, Shingo Eikawa, Yu Mizote, Hirofumi Matsumoto, Midori Isobe, Akiko Uenaka, Minoru Fukuda, Eiichi Nakayama, Mikio Oka. Immune response to XAGE-1b (GAGED2a) in NSCLC patients and its clinical relevance. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 495. doi:10.1158/1538-7445.AM2013-495

BACKGROUND: Cancer/testis (CT) antigens expresses in various cancers and normal testis. Because of no expression of HLA class-I antigen in normal testis, CT antigens are promising molecular target for specific cancer immunotherapy. XAGE-1 gene has been identified as a CT-like antigen, and XAGE-1 gene has four alternative splice variants: XAGE-1a, XAGE-1b, XAGE-1c, and XAGE-1d. We previously reported that the transcript of XAGE-1a and XAGE-1b coded the same protein (GAGED2, isoform a, 81 amino acids) and the transcript of XAGE-1d coded another protein (GAGED2, isoform b, 69 amino acids). The first half of XAGE-1b and 1d protein (1-32 amino acids) is same sequence. On the other hand, XAGE-1c transcript codes some peptides but not a protein. Previously, immunogenic analyses of XAGE-1b have been performed because of its dominant expression, however, few analysis of XAGE-1d was performed. In this study, we investigated the immunogenicity of XAGE-1d in patients with non-small cell lung cancer (NSCLC). MATERIALS AND METHODS: Tumor tissue samples for extracting mRNA were obtained from NSCLC patients treated surgical resection at Nagasaki University Hospital during the period from 2005 to 2010. Sera and PBMCs were obtained from NSCLC patients who visited Kawasaki Medical School Hospital during the period from 2005 to 2010. Splising variants of XAGE-1 gene were analyzed by quantitative real-time PCR with specific TaqMan probe-primer sets. Antibody responses to XAGE-1d were analyzed by ELISA using synthesized XAGE-1b protein. In antibody-positive patients, CD4+ T-cell responses against XAGE-1d were examined by IFN-γ ELISA. Epitope-peptides recognized by XAGE-1d specific T-cells were determined by ELISA using XAGE-1d-overlapping peptides (OLPs). RESULTS: In quantitative real-time PCR, expression of XAGE-1d mRNA was observed in 16/38 specimens (42.1%) that were also positive with XAGE-1b mRNA. The correlation coefficiency of the expression was 0.8047. Protein expression of XAGE-1d was observed in cytoplasm, being different from that of XAGE-1b in the nucleus in lung cancer cell lines by immunohistochemical staining. Antibody against XAGE-1d was detected in lung cancer patients who were also positive with XAGE-1b antibody. By a limiting dilution, a CD4+ T-cell clone was obtained from PBMCs in a seropositive patient. HLA class II restricted recognition of XAGE-1d was confirmed using various allogeneic EBV-B cells as APC. 14-mer minimal epitope peptide was determined. CONCLUSION: The findings indicate that XAGE-1d co-expresses with XAGE-1b and is immunogenic in NSCLC patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 518. doi:1538-7445.AM2012-518

BACKGROUND: XAGE-1b is a cancer/testis antigen identified in lung cancer using autologous patient serum by SEREX. The expression of XAGE-1b is observed frequently in lung adenocarcinoma and rarely in some other tumors. Recently, we showed spontaneous T-cell response against XAGE-1b in XAGE-1b-antibody positive non-small cell lung cancer (NSCLC) patients. In this study, we analyzed the frequency of XAGE-1b-reactive CD4 and CD8 T-cells in NSCLC patients showing high, intermediate and low antibody responses. MATERIALS AND METHODS: Sera and PBMCs were obtained from NSCLC patients who visited Kawasaki Medical School Hospital between 2009 and 2011. Antibody response was determined for XAGE-1b protein by ELISA using O.D. values at 1:900 serum dilution and classified as high ≥ 3.0, 3.0 > intermediate ≥ 1.0 and 1.0 > low. CD4 and CD8 T-cell responses against XAGE-1b were examined by IFN-γ ELISA using 12- or 16-mer XAGE-1b-overlapping peptides (OLPs) spanning the entire XAGE-1b protein in antibody-positive patients. Cytotoxicity was analyzed determining GAPDH release by aCellaTox kit. RESULTS: The number of XAGE-1b-antibody positives was 29 (9.0%) of 323 NSCLC patients. Within those, the patients showing high, intermediate and low antibody response was 6, 11 and 12, respectively. The frequency of XAGE-1b-reactive CD4 T-cells in patients showing high, intermediate and low antibody response was 5.5 ±2.1 x 10-5, 1.5 ±0.7 x 10-5 and < 1.1 x 10-5, respectively. The frequency of XAGE-1b reactive CD8 T-cells was 6.9 ±1.6 x 10-6, 4.6 ±1.6 x 10-6 and < 1.1 x 10-6, respectively. The ratio of the CD8 T-cells recognizing XAGE-1b-expressing tumor to the XAGE-1b-peptide reactive CD8 T-cells in patients showing high and intermediate antibody responses was 60.0% and 14.3%, respectively. CONCLUSION: Our findings indicate correlation of antibody response to CD4 and CD8 T-cell responses against XAGE-1b in NSCLC patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 508. doi:1538-7445.AM2012-508

Background: Analysis of cytokine and chemokine production by tumor cell lines including 5 lung cancers, a malignant mesothelioma and a malignant melanoma recently established in our laboratory showed rather high production of IL-8 in all tumors and IL-6 in one lung cancer, the malignant mesothelioma and the malignant melanoma. We investigated migration of PBMCs to these tumor cells using Transwell plates and showed enrichment of Foxp3+ CD4 Tregs in migrated T-cells to both IL-6- and IL-8-producing tumors. Marked induction of CXCR1 expression on Foxp3+ CD4 Tregs by IL-6 followed by IL-8-mediated migration appeared to be the mechanisms for enriched migration. Frequent production of IL-8 by the tumors and Treg migration to those tumors through induction of IL-8 receptor expression by IL-6 is one of the mechanisms for tumor escape. Purpose: In this study, we investigated cytokine and chemokine production by tumor cell lines including 5 lung cancers, a malignant mesothelioma and a malignant melanoma recently established in our laboratory. We observed IL-8 production in all tumors and IL-6 production in one lung cancer, a malignant mesothelioma and a malignant melanoma. We investigated migration of Foxp3+ CD4 Tregs in PBMCs to those tumor cells using Transwell plates. Experimental Design: Cytokine and chemokine production was analyzed in tumor cell lines including 5 lung cancers, a malignant mesothelioma and a malignant melanoma recently established in our laboratory. IL-8 production was detected in all tumors and IL-6 production in 3 of them. We then investigated the role of IL-6 and IL-8 on migration of Foxp3+ CD4 Tregs to those tumor cells using Transwell plates. Induction of IL-8 receptor on Foxp3+ CD4 T-cells by IL-6 was investigated by flow cytometry and its role on their migration was examined by anti-IL-8R or anti-IL-6R blocking. Furthermore, to investigate relationships between the effect of IL-6 and STAT3 signaling in the induction of IL-8R expression, we examined whether IL-8R expression would inhibit by blocking of STAT3 signaling. Results: We showed enrichment of Foxp3+ CD4 Tregs in migrated T-cells to both IL-6 and IL-8-producing tumors. Marked induction of CXCR1 (IL-8RA) expression was observed on Foxp3+ CD4 Tregs by the treatment with IL-6. Migration of IL-6-treated Foxp3+ CD4 T-cells to the tumors was blocked by the addition of anti-CXCR1 or anti-IL-6R. Furthermore, high level of IL-6 was detected in pleural effusion in most of lung cancer patients.Conclusions: Frequent production of IL-8 by the tumors and Treg migration to those tumors through induction of IL-8 receptor expression by IL-6 is one of the mechanisms for tumor escape. The findings indicate that the blocking of Treg migration by inhibiting IL-6-mediated IL-8 receptor expression could be a new strategy for tumor immunotherapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3653. doi:10.1158/1538-7445.AM2011-3653

BACKGROUND: Several cancer/testis(CT) antigens such as NY-ESO-1 antigen, etc., have been shown to elicit immune responses in cancer patients spontaneously. Because of their restricted expression in normal tissues and high immunogenicity, CT antigens have been thought to be attractive targets for cancer vaccine. We previously described XAGE-1b (GAGED2a), one of the CT-associated antigens, was predominantly expressed in lung adenocarcinoma by immunohistochemistry using XAGE-1b mAb (clone USO 9-13) and could elicit both humoral and cellular immune response in patients with non-small cell lung cancer (NSCLC). In this study, we established CD4 and CD8 T cell clones from PBMC in XAGE-1b antibody-positive patients and analyzed the antigen recognition. MATERIALS AND METHODS: Sera and PBMCs were obtained from NSCLC patients who visited Kawasaki Medical School Hospital between 2005 and 2010. Antibody response to XAGE-1b was analyzed by ELISA using synthesized XAGE-1b protein. CD4 and CD8 T cell responses against XAGE-1b were examined by IFN-γ ELISA and/or capture assay using 17 16- or 17-mer XAGE-1b-overlapping peptides (OLPs) spanning the entire XAGE-1b protein by FACS in antibody-positive patients. Cytotoxicity was analyzed by bioluminescence assay (GAPDH). RESULTS: Spontaneous CD4 and CD8 T cell responses were also detected in most of the antibody positive patients. Occurrence of antibody response, and CD4 and CD8 T cell responses in XAGE-1b (GAGED2a) antibody positive patients indicated strong immunogenicity of XAGE-1b (GAGED2a) antigen in NSCLC patients. Furthermore, We identified the minimal epitope peptide bound to HLA-DR4.1 and DP5 recognized by CD4 and those bound to HLA-A2, B61 and Cw1 recognized by CD8 T cell clones. CD4 and CD8 T cell clones recognized naturally processed XAGE-1b (GAGED2a) antigen on APC (antigen-presenting cell) and the CD8 T cell clone was stained with the peptide/MHCI tetramer and showed cytotoxicity against XAGE-1b (GAGED2a)-expressing, appropriate HLA class I expressing tumor cell line. CONCLUSION: Our findings indicate validity of XAGE-1b (GAGED2a) for cancer vaccine. Now we plan to global clinical trial of cancer vaccine against XAGE-1b in NSCLC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2689. doi:10.1158/1538-7445.AM2011-2689

Background: Several mechanisms by which tumors evade the host immune response have been elucidated. A tumor modulates its antigenicity by immunoediting during development of an overt tumor, and frequently loses MHC and/or tumor antigen expression. Recently, it has been shown that regulatory T-cells were closely involved in immune impairment against tumors. Naturally occurring Foxp3+ CD25 high CD4 Tregs were observed in local tumor sites in some ovarian cancer patients and infiltration of those Tregs in the tumor correlated well with poor patient prognosis. Moreover, recently, an increase in the number of Tregs in the peripheral blood has been shown in advanced cancer patients and immune down-regulation systemically occurring in those patients is one cause of the ineffectiveness of immunization in clinical trials using cancer vaccines. The evidence suggests that it is necessary to regulate Tregs function and their accumulation to tumor local site for induction of effective immune response against tumors. Purpose: In this study, we investigated cytokine and chemokine production by tumor cell lines including 5 lung cancers, a malignant mesothelioma and a malignant melanoma recently established in our laboratory. We observed IL-8 production in all tumors and IL-6 production in one lung cancer, a malignant mesothelioma and a malignant melanoma. We investigated migration of Foxp3+ CD4 Tregs in PBMCs to those tumor cells using Transwell plates. Experimental Design: Cytokine and chemokine production was analyzed in tumor cell lines including 5 lung cancers, a malignant mesothelioma and a malignant melanoma recently established in our laboratory. IL-8 production was detected in all tumors and IL-6 production in 3 of them. We then investigated the role of IL-6 and IL-8 on migration of Foxp3+ CD4 Tregs to those tumor cells using Transwell plates. Induction of IL-8 receptor on Foxp3+ CD4 T-cells by IL-6 was investigated by flow cytometry and its role on their migration was examined by anti-IL-8R blocking. Results: We showed enrichment of Foxp3+ CD4 Tregs in migrated T-cells to both IL-6 and IL-8-producing tumors. Marked induction of CXCR1 (IL-8RA) expression was observed on Foxp3+ CD4 Tregs by the treatment with IL-6. Migration of IL-6-treated Foxp3+ CD4 T-cells to the tumors was blocked by the addition of anti-CXCR1. In fact, high level of IL-6 was detected in pleural effusion in most of lung cancer patients. Conclusions: Frequent production of IL-8 by the tumors and Treg migration to those tumors through induction of IL-8 receptor expression by IL-6 is one of the mechanisms for tumor escape. The findings indicate that the blocking of Treg migration by inhibiting IL-6-mediated IL-8 receptor expression could be a new strategy for tumor immunotherapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1915.

BACKGROUND: Cancer/testis (CT) antigen is expressed only in the testis in normal adult tissues and in various types of cancers. There is no expression of MHC antigen in the testis. Therefore, CT antigen is thought to be a promising target for cancer immunotherapy. The XAGE-1 gene has been identified as a CT-like antigen and consists of several isoforms. We recently reported that XAGE-1b was predominantly expressed in lung adenocarcinoma by immunohistochemistry using XAGE-1b mAb (clone USO 9-13). In this study, we investigated humoral and cellular immune responses against XAGE-1b in patients with non-small cell lung cancer (NSCLC). MATERIALS AND METHODS: Sera and PBMCs were obtained from NSCLC patients who visited Kawasaki Medical School Hospital between 2005 and 2009. Antibody response to XAGE-1b was analyzed by ELISA using synthesized XAGE-1b protein. CD4 and CD8 T-cell responses against XAGE-1b were examined by IFN-γ ELISA and/or secretion assay using 17 16- or 17-mer XAGE-1b-overlapping peptides (OLPs) spanning the entire XAGE-1b protein in antibody-positive patients. Epitope peptides recognized by antibody and T-cells were determined by ELISA using XAGE-1b OLPs. RESULTS: Antibody positive frequencies of total 200 NSCLC patients and 69 stage IIIB/IV lung adenocarcinoma patients were 10.0% (20/200) and 18.8% (13/69), respectively. The peptide regions frequently recognized by the antibody were XAGE1b peptides 25-40, 29-44 and 33-48. CD4 and CD8 T-cell responses against XAGE-1b were observed in 14 (87.5%) of 16 patients and 6 (66.7%) of 9 patients, respectively, in the antibody-positive patients. No response was observed in 5 antibody-negative patients and 5 healthy donors. The XAGE-1b peptide regions dominantly recognized by CD4 and CD8 T-cells were XAGE-1b peptides 13-28 and 33-48 for CD4 and XAGE-1b peptide 29-44 for CD8. Furthermore, the minimal epitope peptides bound to HLA-DR4.1 and DP5 recognized by CD4 T-cell clones and those bound to HLA-A2 and Cw1 recognized by CD8 T-cell clones were determined. CONCLUSION: Our findings indicate that CT antigen XAGE-1b is highly immunogenic in NSCLC patients inducing antibody, and CD4 and CD8 T-cell responses. Thus, XAGE-1b is a promising target antigen for immunotherapy against lung adenocarcinoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2394.