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Table S1 and Table S2- Patients' characteristics. Table S3 - Cell lines. Table S4 - Antibodies used. Table S5 - Expression of PD-L1 and Galectin-9 in lung cancer. Table S6 - Expression of XAGE1 in lung cancer. Table S7 - Cut-off values for each T-cell parameter. Table S8 - Standardized coefficients in discriminant functions. Table S9 - Univariate and multivariate analysis.

PDF file 90K, Supplementary Figure Legends

Antibody response and CD4 and CD8 T-cell responses during treatment.

Representative results of analyses of CD4 (A) and CD8 (B) T cells expressing T cell activation and inhibitory molecules in PBMCs from an XAGE1 (GAGED2a) antibody-positive patient, KLU456 by FACS.

Patient characteristics

Immune response

Clinical course of 3 NSCLC patients evaluated as SD at 12 weeks after treatment.

The changes in the specific populations in PBMCs, lymphocytes and CD4 T-cells.

OS of the advanced adenocarcinoma patients with EGFRwt or EGFRmt tumors (A), and with XAGE1 (GAGED2a) antigen-positive EGFRwt or EGFRmt tumors and antibody, and XAGE1 (GAGED2a) antigen-negative EGFRmt tumors (B).

PDF file 79K, GAG-HERV-K ch22q11.23 features. Endogenous retrovirus K from chromosome 22 is located on the q11.23 region, nearby the BCR, ZDHHC8P, IGLL1 and RGL4 genes. HERV-K ch22q11.23 has GAG-PRO, POL and ENV genes flanked by LTR sequences. The GAG gene has an ORF encoding a 715aa protein that contains P10, P24 and Zinc finger domains. {delta}: deletions; I: early stop codons; --> primer binding regions; ABS: antibody binding site for monoclonal antibody TI-35 (see Supplementary Fig. S3C)

Supplementary Table 1A. Patient characteristics (n=145). Supplementary Table 1B. EGFR mutation types (n=44). Supplementary Table 2A. Univariate Cox regression analysis. Supplementary Table 2B. Multivariate Cox regression analysis.

Purpose: The cancer/testis antigen XAGE1 (GAGED2a) is expressed in approximately 40% of advanced lung adenocarcinomas. We investigated the clinical relevance of the XAGE1 (GAGED2a) immune responses in patients with advanced lung adenocarcinoma.Experimental Design: The XAGE1 (GAGED2a) antigen expression and EGFR mutation were determined with tumor tissues. The XAGE1 (GAGED2a) antibody and T-cell immune responses, as well as immune cell phenotypes, were analyzed with blood samples. Patients with EGFR wild-type (EGFRwt) tumors were treated with conventional platinum-based doublet chemotherapy and patients with EGFR-mutated (EGFRmt) tumors were treated with EGFR-TKI and conventional chemotherapy. The overall survival (OS) rates of the antibody-positive and -negative patients were investigated.Results: The results showed that the OS of antibody-positive patients was prolonged significantly compared with that of antibody-negative patients with either XAGE1 (GAGED2a) antigen-positive EGFRwt (31.5 vs. 15.6 months, P = 0.05) or EGFRmt (34.7 vs. 11.1 months, P = 0.001) tumors. Multivariate analysis showed that the presence of the XAGE1 (GAGED2a) antibody was a strong predictor for prolonged OS in patients with XAGE1 (GAGED2a) antigen-positive tumors and in patients with either EGFRwt or EGFRmt tumors. On the other hand, XAGE1 (GAGED2a) antigen expression was a worse predictor in patients with EGFRmt tumors. Phenotypic and functional analyses of T cells indicated immune activation in the antibody-positive patients.Conclusions: The findings suggest that production of the XAGE1 (GAGED2a) antibody predicts good prognosis for patients with lung adenocarcinoma as an immune biomarker and the protective effect of this naturally occurring immune response supports the concept of immunotherapy. Clin Cancer Res; 20(19); 5052–63. ©2014 AACR.

OS of the advanced lung adenocarcinoma patients with different EGFR mutation types.

Supplementary Figure 1. Pharmacokinetic (PK) analysis after KW-0761 infusions in solid cancer patients. Supplementary Figure 2. Clinical course of 3 NSCLC patients evaluated as SD at 12 weeks after treatment. Supplementary Figure 3. The changes in the specific populations in PBMCs, lymphocytes and CD4 T-cells. Supplementary Figure 4. Antibody response and CD4 and CD8 T-cell responses during treatment.

Pharmacokinetic (PK) analysis after KW-0761 infusions in solid cancer patients.

PDF file 204K, Alignment of GAG proteins derived from human endogenous retroviruses and respective epitopes recognized by prostate cancer patient sera. A, Detail alignment from several GAG-HERV-K family members and the TI-35 binding site (red) highlighting the amino-acid differences present in GAG-HERV-K ch22q11.23 (blue). B, Western-blot analysis of immunoprecipitation of GAG-HERV-K from VCaP and LNCaP prostate cancer cell lines. WFP prostate cancer patient serum (WFP) was incubated with VCaP or LNCaP protein extract for 2 hours at 4{degrees}C. Protein-antibody complexes were pulled down using anti-human IgG sepharose beads. The pull-down pellet was submitted to SDS-PAGE and Western-blot analysis. Reactivity to GAG-HERV-K protein was developed by incubating the membrane with TI-35 monoclonal antibody. A pool of healthy donor sera was used as control (Con). C, Summary of ELISA results from epitope mapping using 20mers peptides overlapping by 10aa (71 peptides tested in total from JPT Innovative Peptide Solution, Berlin, Germany) spanning GAG-HERV-K and tested individually for recognition by monoclonal antibody TI-35 and different prostate cancer patient sera seropositive for GAG-HERV-K protein. The square symbol represents a positive reaction to the corresponding peptide (labeled by aminoacid start and end position). A pool of healthy donor sera was used as control (Con pool). Plates were coated with 5 uM peptide and the reaction was performed as previously described in methods

Autoantibody response

Purpose: Human endogenous retroviruses (HERV) encode 8% of the human genome. While HERVs may play a role in autoimmune and neoplastic disease, no mechanistic association has yet been established. We studied the expression and immunogenicity of a HERV-K GAG protein encoded on chromosome 22q11.23 in relation to the clinical course of prostate cancer.Experimental Design:In vitro expression of GAG-HERV-K was analyzed in panels of normal and malignant tissues, microarrays, and cell lines, and effects of demethylation and androgen stimulation were evaluated. Patient sera were analyzed for seroreactivity to GAG-HERV-K and other self-antigens by ELISA and seromics (protein array profiling).Results: GAG-HERV-K expression was most frequent in prostate tissues and regulated both by demethylation of the promoter region and by androgen stimulation. Serum screening revealed that antibodies to GAG-HERV-K are found in a subset of patients with prostate cancer (33 of 483, 6.8%) but rarely in male healthy donors (1 of 55, 1.8%). Autoantibodies to GAG-HERV-K occurred more frequently in patients with advanced prostate cancer (29 of 191 in stage III–IV, 21.0%) than in early prostate cancer (4 of 292 in stages I–II, 1.4%). Presence of GAG-HERV-K serum antibody was correlated with worse survival of patients with prostate cancer, with a trend for faster biochemical recurrence in patients with antibodies to GAG-HERV-K.Conclusions: Preferential expression of GAG-HERV-K ch22q11.23 in prostate cancer tissue and increased frequency of autoantibodies observed in patients with advanced prostate cancer make this protein one of the first bona fide retroviral cancer antigens in humans, with potential as a biomarker for progression and biochemical recurrence rate of prostate cancer. Clin Cancer Res; 19(22); 6112–25. ©2013 AACR.

PDF file 63K, Correlation between antibodies against GAG-HERV-K and group risk or Gleason score in cancer patients. Prostate cancer patients were split (A) in 3 groups according to Gleason score and classified from group risk 1 to 3 and compared to healthy donors (HD), or (B) in 3 groups according to Gleason score as indicated. Antibodies anti-GAG-HERV-K were detected and analyzed as in Fig. 4. Asterisk denotes significantly higher frequency of seropositive patients (titer > 100) in risk group 3 and Gleason {greater than or equal to} 8 (Chi-square test, p

ICS of CD4 and CD8 T cells reactive against XAGE1 (GAGED2a).

PDF file 341K, qPCR gene expression analysis of chromosome 22q11.23 region in cancer tissues. Total RNA from cancer tissues was submitted to DNAse treatment and reverse transcriptase reaction for cDNA synthesis. The resulting cDNA was used on qPCR to detect the expression of GAG-HERV-K and the neighbor genes BCR, IGGL1, RGL4 and ZDHHC8P. The results are expressed as relative expression to GAG-HERV-K levels in normal prostate. GAG-HERV-K expression is relative to GAPDH. BCR, IGGL1, RGL4 and ZDHHC8P expression is relative to TFRC

Supplementary Figure Legend from Prolongation of Overall Survival in Advanced Lung Adenocarcinoma Patients with the XAGE1 (GAGED2a) Antibody

Purpose: FoxP3+ Tregs inhibit immune responses against tumors. KW-0761 is a humanized anti-human CCR4 monoclonal antibody (mAb) that has antibody-dependent cellular cytotoxicity activity. Depletion of CCR4-expressing FoxP3+ CD4 Tregs by KW-0761 infusion was investigated in solid cancer patients.Experimental Design: We conducted a phase Ia clinical trial of KW-0761 infusion in 7 lung and 3 esophageal cancer patients. Toxicity, clinical efficacy, changes in lymphocyte subpopulations, including Tregs, and induction of immune responses were analyzed.Results: The results showed that KW-0761 infusion in a dose range between 0.1 mg/kg and 1.0 mg/kg was safe and well tolerated. No dose-limiting toxicity was observed. Four of 10 patients showed stable disease during treatment and were long survivors. The monitoring of FoxP3+ Tregs in the peripheral blood mononuclear cells during treatment indicated efficient depletion of those cells, even at the lowest dose of 0.1 mg/kg used. The reduction in Th 1 CD4 T cells and CD8 T cells was limited, whereas a significant reduction was observed with Th 2 and Th 17 CD4 T cells. Immune responses to cancer/testis (CT) antigens and an autoantibody response to thyroid peroxidase were observed in some patients.Conclusions: The findings showed Tregs depletion and the possible occurrence of an immune response following KW-0761 infusion. Combined use of KW-0761 to deplete FoxP3+ Tregs with other immunotherapies, such as cancer vaccines or checkpoint inhibitors, is a promising approach to augment immune responses. Clin Cancer Res; 21(19); 4327–36. ©2015 AACR.

Correlation of OS with XAGE1 (GAGED2a) antibody titer in patients with XAGE1 (GAGED2a) antigen-positive tumors.

Developing Treg cell-targeted therapies is an important aspect in cancer immunotherapy. Here, we investigate anti-CCR4 mAb (mogamulizumab) with solid cancer patients in phase 1b clinical trial based on the predominant detection of CCR4-expressing eTreg cells in tumors. While eTreg cells in peripheral blood were significantly decreased after mogamulizumab administration in all patients, clinical responses were hardly detected. Comprehensive immune-monitoring revealed that concomitant reduction of central-memory CD8+ T cells with CCR4 expression, that reportedly play an important role in antitumor activity. This reduction was subsided in long survivors in whom central-memory CD8+ T cells possessed lower CCR4 expression and/or NK cells exhibited an exhausted phenotype. Thus, excess doses of mogamulizumab harboring enhanced antibody-dependent cellular cytotoxicity could unexpectedly deplete central-memory CD8+ T cells with CCR4 expression. We therefore need to carefully determine the optimal dose of mogamulizumab for successful clinical application as cancer immunotherapy to avoid unexpected depletion of effector components.

Regulatory T (Treg) cells are important negative regulators of immune homeostasis, but in cancers they tone down the anti-tumor immune response. They are distinguished by high expression levels of the chemokine receptor CCR4, hence their targeting by the anti-CCR4 monoclonal antibody mogamulizumab holds therapeutic promise. Here we show that despite a significant reduction in peripheral effector Treg cells, clinical responses are minimal in a cohort of patients with advanced CCR4-negative solid cancer in a phase Ib study (NCT01929486). Comprehensive immune-monitoring reveals that the abundance of CCR4-expressing central memory CD8+ T cells that are known to play roles in the antitumor immune response is reduced. In long survivors, characterised by lower CCR4 expression in their central memory CD8+ T cells possessed and/or NK cells with an exhausted phenotype, cell numbers are eventually maintained. Our study thus shows that mogamulizumab doses that are currently administered to patients in clinical studies may not differentiate between targeting effector Treg cells and central memory CD8+ T cells, and dosage refinement might be necessary to avoid depletion of effector components during immune therapy., Elimination of regulatory T cells via the anti-CCR4 monoclonal antibody, mogamulizumab, is expected to augment anti-tumour immune response. Authors show here that although regulatory T cell targeting is successful, clinical improvement remains minimal in patients with solid tumours due to concomitant and unintended depletion of central memory CD8+ T cells.

The cancer-testis antigens (CTA) are a large family of tumor-associated antigens expressed by a variety of cancer cells and primitive germ cells of the adult testis and placenta. These tumor-restricted expressing patterns suggest that CTA would be ideal targets for tumor-specific immunotherapy. XAGE-1 is a CTA that was originally identified by computer-based screening, and four transcription variants, XAGE-1a, -1b, -1c and -1d, have been characterized to date. Although the presence of XAGE-1 transcripts has been reported in various cancers, the expression of XAGE-1b in melanoma has not been fully characterized. In this study, we performed immunohistochemical staining of XAGE-1b together with NY-ESO-1, a well-known CTA, in 113 melanoma samples obtained from 84 patients and evaluated their expression in tumor cells. The effects of expression on tumor progression and patient prognosis were analyzed. Both XAGE-1b and NY-ESO-1 were expressed at high levels in lymph node metastasis and skin metastasis samples compared with the primary site (P < 0.01 in XAGE-1b and P < 0.05 in NY-ESO-1). In a subgroup analysis of 22 patients with stage III lymph node metastasis, overall survival was significantly higher in the XAGE-1b and NY-ESO-1 double-negative group than in the other groups (P < 0.05). These results suggest that lack of XAGE-1b and NY-ESO-1 expression could have a positive influence on clinical outcome in patients with melanoma.

Humoral immune responses against tumor-associated antigens (TAAs) or cancer/testis antigens (CTAs) aberrantly expressed in tumor cells are frequently observed in cancer patients. Recent clinical studies have elucidated that anticancer immune responses with increased levels of anti-TAA/CTA antibodies improve cancer survival rates. Thus, these antibody levels are promising biomarkers for diagnosing the efficiency of cancer immunotherapy. Full-length antigens are favored for detecting anti-TAA/CTA antibodies because candidate antigen proteins contain multiple epitopes throughout their structures. In this study, we developed a methodology to prepare purified water-soluble and full-length antigens by using cysteine sulfhydryl group cationization (S-cationization) chemistry. S-Cationized antigens can be prepared from bacterial inclusion bodies, and they exhibit improved protein solubility but preserved antigenicity. Anti-TAA/CTA antibodies detected in cancer patients appeared to recognize linear epitopes, as well as conformational epitopes, and because the frequency of cysteine side-residues on the epitope-paratope interface was low, any adverse effects of S-cationization were virtually negligible for antibody binding. Furthermore, S-cationized antigen-immobilized Luminex beads could be successfully used in highly sensitive quantitative-multiplexed assays. Indeed, patients with a more broadly induced serum anti-TAA/CTA antibody level showed improved progression-free survival after immunotherapy. The comprehensive anti-TAA/CTA assay system, which uses S-cationized full-length and water-soluble recombinant antigens, may be a useful diagnostic tool for assessing the efficiency of cancer immunotherapy.

Metformin, a prescribed drug for type 2 diabetes, has been reported to have anti-cancer effects; however, the underlying mechanism is poorly understood. Here we show that this mechanism may be immune-mediated. Metformin enabled normal but not T-cell-deficient SCID mice to reject solid tumors. In addition, it increased the number of CD8(+) tumor-infiltrating lymphocytes (TILs) and protected them from apoptosis and exhaustion characterized by decreased production of IL-2, TNFα, and IFNγ. CD8(+) TILs capable of producing multiple cytokines were mainly PD-1(-)Tim-3(+), an effector memory subset responsible for tumor rejection. Combined use of metformin and cancer vaccine improved CD8(+) TIL multifunctionality. The adoptive transfer of antigen-specific CD8(+) T cells treated with metformin concentrations as low as 10 μM showed efficient migration into tumors while maintaining multifunctionality in a manner sensitive to the AMP-activated protein kinase (AMPK) inhibitor compound C. Therefore, a direct effect of metformin on CD8(+) T cells is critical for protection against the inevitable functional exhaustion in the tumor microenvironment.

We conducted a clinical trial of a cancer vaccine using NY-ESO-1 protein with polyinosinic-polycytidylic acid-poly-L-lysine carboxymethylcellulose (poly-ICLC) and/or OK-432 against solid tumors. A total of 15 patients were sequentially enrolled in 4 cohorts. Patients in cohort 1 received NY-ESO-1 protein; cohort 2a received NY-ESO-1 protein+OK-432; cohort 2b received NY-ESO-1 protein+poly-ICLC; cohort 3 received NY-ESO-1 protein+OK-432+poly-ICLC with Montanide ISA-51. The endpoints of this trial were safety, NY-ESO-1 immune responses, and clinical response. Vaccine-related adverse events observed were fever and injection-site reaction (grade 1). Two patients showed stable disease after vaccination. NY-ESO-1 antibodies were observed in 4 patients at the baseline (sero-positive) and augmented in all patients after vaccination. Eleven patients showed a conversion of negative antibody responses at baseline to positive after vaccination (seroconversion). The seroconversions were observed in all 11 sero-negative patients by the fourth immunization; in particular, it was observed by the second immunization in patients with poly-ICLC, and these induced antibody responses were stronger than those in patients immunized without poly-ICLC. The number of NY-ESO-1-specific interferon (IFN)γ-producing T cells was increased in patients immunized with poly-ICLC and/or OK-432, and furthermore, the increase of IFNγ-producing CD8 T cells in patients immunized with poly-ICLC was significantly higher than that in patients without poly-ICLC. Nonspecific activations of T-cell or antigen presenting cells were not observed. Our present study showed that poly-ICLC is a promising adjuvant for cancer vaccines.

Regulatory T (Treg) and type 1 regulatory T (Tr1) cells facilitate hepatitis C virus (HCV) recurrence after orthotopic liver transplantation (OLT). However, their frequencies and effects on HCV-specific immune responses have not been well investigated.We determined Treg and Tr1 frequencies in OLT patients with hepatitis C and assessed their associations with HCV-specific T cell responses. These patients comprised the following groups: an early post-transplantation group (n=14); an OLT-chronic active hepatitis C group (n=14) with active hepatitis C (alanine aminotransferase ofupper limit of normal/positive for HCV-RNA); an OLT-persistently normal alanine aminotransferase group (n=12) without active hepatitis C (not interferon/positive for HCV-RNA); and an OLT-sustained viral response group (n=6) with sustained viral responses using interferon treatment (negative for HCV-RNA). The frequencies of HCV-specific CD4+ T cells that secreted interferon-γ were determined by enzyme-linked immunosorbent spot assay (except for the OLT early group).Treg and Tr1 frequencies were low during the early post-transplantation period. OLT patients with sustained viral responses had lower Treg frequencies than those with chronic hepatitis C, whereas Tr1 frequencies were significantly reduced in OLT patients with persistently normal alanine aminotransferase levels compared to those with chronic hepatitis C (p0.05). Treg frequencies positively correlated with HCV NS3 antigen-specific interferon-γ responses, which corresponded to HCV clearance.Increased Treg frequencies and reduced HCV-NS3 antigen-specific responses recovered after viral eradication in post-OLT chronic hepatitis C patients. Reduced Tr1 frequencies were associated with hepatitis activity control, which may facilitate controlling chronic hepatitis C in patients after OLT.

Summary GM (γ marker) allotypes, genetic variants of immunoglobulin γ chains, have been reported to be associated strongly with susceptibility to lung cancer, but the mechanism(s) underlying this association is not known. One mechanism could involve their contribution to humoral immunity to lung tumour-associated antigens. In this study, we aimed to determine whether particular GM and KM (κ marker) allotypes were associated with antibody responsiveness to XAGE-1b, a highly immunogenic lung tumour-associated cancer-testis antigen. Sera from 89 patients with non-small cell lung cancer (NSCLC) were allotyped for eight GM and two KM determinants and characterized for antibodies to a synthetic XAGE-1b protein. The distribution of various GM phenotypes was significantly different between XAGE-1b antibody-positive and -negative patients (P = 0·023), as well as in the subgroup of XAGE-1b antigen-positive advanced NSCLC (P = 0·007). None of the patients with the GM 1,17 21 phenotype was positive for the XAGE-1b antibody. In patients with antigen-positive advanced disease, the prevalence of GM 1,2,17 21 was significantly higher in the antibody-positive group than in those who lacked the XAGE-1b antibody (P = 0·026). This phenotype also interacted with a particular KM phenotype: subjects with GM 1,2,17 21 and KM 3,3 phenotypes were almost four times (odds ratio = 3·8) as likely to be positive for the XAGE-1b antibody as the subjects who lacked these phenotypes. This is the first report presenting evidence for the involvement of immunoglobulin allotypes in immunity to a cancer-testis antigen, which has important implications for XAGE-1b-based immunotherapeutic interventions in lung adenocarcinoma.

We conducted a clinical trial of an NY-ESO-1 cancer vaccine using 4 synthetic overlapping long peptides (OLP; peptides #1, 79-108; #2, 100-129; #3, 121-150; and #4, 142-173) that include a highly immunogenic region of the NY-ESO-1 molecule. Nine patients were immunized with 0.25 mg each of three 30-mer and a 32-mer long NY-ESO-1 OLP mixed with 0.2 KE Picibanil OK-432 and 1.25 mL Montanide ISA-51. The primary endpoints of this study were safety and NY-ESO-1 immune responses. Five to 18 injections of the NY-ESO-1 OLP vaccine were well tolerated. Vaccine-related adverse events observed were fever and injection site reaction (grade 1 and 2). Two patients showed stable disease after vaccination. An NY-ESO-1-specific humoral immune response was observed in all patients and an antibody against peptide #3 (121-150) was detected firstly and strongly after vaccination. NY-ESO-1 CD4 and CD8 T-cell responses were elicited in these patients and their epitopes were identified. Using a multifunctional cytokine assay, the number of single or double cytokine-producing cells was increased in NY-ESO-1-specific CD4 and CD8 T cells after vaccination. Multiple cytokine-producing cells were observed in PD-1 (-) and PD-1 (+) CD4 T cells. In conclusion, our study indicated that the NY-ESO-1 OLP vaccine mixed with Picibanil OK-432 and Montanide ISA-51 was well tolerated and elicited NY-ESO-1-specific humoral and CD4 and CD8 T-cell responses in immunized patients.

We established CD4 T-cell clones, Mz-1B7, and Ue-21, which recognized the NY-ESO-1 121-138 peptide from peripheral blood mononuclear cells (PBMCs) of an esophageal cancer patient, E-2, immunized with an NY-ESO-1 protein and determined the NY-ESO-1 minimal epitopes. Minimal peptides recognized by Mz-1B7 and Ue-21 were NY-ESO-1 125-134 and 124-134, respectively, both in restriction to DRB1*08:03. Using a longer peptide, 122-135, and five other related peptides, including either of the minimal epitopes recognized by the CD4 T-cell clones, we investigated the free peptide/DR recognition on autologous EBV-B cells as APC and peptide/DR tetramer binding. The results showed a discrepancy between them. The tetramers with several peptides recognized by either Mz-1B7 or the Ue-21 CD4 T-cell clone did not bind to the respective clone. On the other hand, unexpected binding of the tetramer with the peptide not recognized by CD4 T-cells was observed. The clone Mz-1B7 did not recognize the free peptide 122-135 on APC, but the peptide 122-135/DRB1*08:03 tetramer bound to the TCR on those cells. The failure of tetramer production and the unexpected tetramer binding could be due to a subtly modified structure of the peptide/DR tetramer from the structure of the free peptide/DR molecule. We also demonstrated that the NY-ESO-1 123-135/DRB1*08:03 tetramer detected ex vivo CD4 T-cell responses in PBMCs from patients after NY-ESO-1 vaccination in immunomonitoring.

Purpose: Human endogenous retroviruses (HERV) encode 8% of the human genome. While HERVs may play a role in autoimmune and neoplastic disease, no mechanistic association has yet been established. We studied the expression and immunogenicity of a HERV-K GAG protein encoded on chromosome 22q11.23 in relation to the clinical course of prostate cancer. Experimental Design: In vitro expression of GAG-HERV-K was analyzed in panels of normal and malignant tissues, microarrays, and cell lines, and effects of demethylation and androgen stimulation were evaluated. Patient sera were analyzed for seroreactivity to GAG-HERV-K and other self-antigens by ELISA and seromics (protein array profiling). Results: GAG-HERV-K expression was most frequent in prostate tissues and regulated both by demethylation of the promoter region and by androgen stimulation. Serum screening revealed that antibodies to GAG-HERV-K are found in a subset of patients with prostate cancer (33 of 483, 6.8%) but rarely in male healthy donors (1 of 55, 1.8%). Autoantibodies to GAG-HERV-K occurred more frequently in patients with advanced prostate cancer (29 of 191 in stage III–IV, 21.0%) than in early prostate cancer (4 of 292 in stages I–II, 1.4%). Presence of GAG-HERV-K serum antibody was correlated with worse survival of patients with prostate cancer, with a trend for faster biochemical recurrence in patients with antibodies to GAG-HERV-K. Conclusions: Preferential expression of GAG-HERV-K ch22q11.23 in prostate cancer tissue and increased frequency of autoantibodies observed in patients with advanced prostate cancer make this protein one of the first bona fide retroviral cancer antigens in humans, with potential as a biomarker for progression and biochemical recurrence rate of prostate cancer. Clin Cancer Res; 19(22); 6112–25. ©2013 AACR.

Aim: Accumulating evidence has indicated that hyperthermia using magnetite nanoparticles induces antitumor immunity. This study investigated the diversity of T-cell receptors (TCRs) in tumor-infiltrating lymphocytes after hyperthermia using magnetite nanoparticles.Materials & methods: Functionalized magnetite nanoparticles, N-propionyl-4-S-cysteaminylphenol (NPrCAP)/magnetite, were synthesized by conjugating the melanogenesis substrate NPrCAP with magnetite nanoparticles. NPrCAP/magnetite nanoparticles were injected into B16 melanomas in C57BL/6 mice, which were subjected to an alternating magnetic field for hyperthermia treatment. Results: Enlargement of the tumor-draining lymph nodes was observed after hyperthermia. The TCR repertoire was restricted in tumor-infiltrating lymphocytes, and expansion of Vβ11+ T cells was preferentially found. DNA sequences of the third complementaritydetermining regions revealed the presence of clonally expanded T cells. Conclusion: These results indicate that the T-cell response in B16 melanomas after hyperthermia is dominated by T cells directed toward a limited number of epitopes and that epitope-specific T cells frequently use a restricted TCR repertoire. Original submitted 14 May 2012; Revised submitted 30 July 2012; Published online 15 October 2012

The sensitivity and specificity of a new rapid Mycoplasma pneumoniae antigen immunochromatography (IC) test, DK-MP-001, were determined using particle agglutination (PA) antibody response and loop-mediated isothermal amplification (LAMP) gene detection as the gold standard. Of 165 patients, 59 were diagnosed with M. pneumoniae infection based on a ≥fourfold rise of serum PA antibody during the course of the illness. Of the first visit swabs, 60 were positive for M. pneumoniae on LAMP, and 49 were positive for M. pneumoniae antigen on IC test. Compared with PA antibody and LAMP, the sensitivity/specificity of the IC test were 81.4% (48/59) and 99.1% (105/106); and 81.7% (49/60) and 100% (105/105), respectively. IC test detected antigen in pharyngeal swabs more sensitively than in nasal swabs for the same subjects (P

Background A combination of hepatitis B immunoglobulin and nucleos(t)ide analogues is the current standard of care for controlling hepatitis B recurrence after orthotopic liver transplantation (OLT). However, frequent immunoglobulin treatment is expensive and inconvenient. This study investigated the efficacy of hepatitis B virus (HBV) vaccination in preventing the recurrence of hepatitis B after living donor OLT. Methods Twenty-seven patients who had undergone living donor OLT participated in the study; five had acute HBV infected liver failure (ALF-OLT) and 22 had HBV related liver cirrhosis (LC-OLT). Hepatitis B surface antigen (HBsAg)-containing vaccine was administered to them for at least 1 year after transplantation and continued once monthly for up to 36 months post-OLT. Patients who had anti-HBs antibody titers above 100 mIU/mL for a minimum of 6 months without immunoglobulin administration were defined as good responders; the others were defined as poor responders. Interferon-γ enzyme-linked immunospot assays against HBs and HBc antigens were used to assay cellular immune responses. Results All five of the ALF-OLT patients had good responses after a median of four (range 2.5–5) vaccinations. Nine of the 22 LC-OLT patients had good responses after a median of 19 (range 11.5–30) vaccinations. Among the LC-OLT group, those with livers donated by relatively higher-aged, marital and high-titer anti-HBs antibody donors were good responders. LC-OLT patients classed as good responders showed interferon-γ responses comparable to those of the ALF-OLT patients. Conclusions The ALF-OLT and LC-OLT patients who received livers from relatively higher-aged, marital, high-titer anti-HBs antibody donors were the best candidates for HBV vaccine administration. Boosting donors before transplantation may facilitate later vaccine response of the recipients.

Background: NY-ESO-1 antibodies are specifically observed in patients with NY-ESO-1-expressing tumours. We analysed whether the NY-ESO-1 humoral immune response is a useful tumour marker of gastric cancer. Methods: Sera from 363 gastric cancer patients were screened by enzyme-linked immunosorbent assay (ELISA) to detect NY-ESO-1 antibodies. Serial serum samples were obtained from 25 NY-ESO-1 antibody-positive patients, including 16 patients with curative resection and 9 patients who received chemotherapy alone. Results: NY-ESO-1 antibodies were detected in 3.4% of stage I, 4.4% of stage II, 25.3% of stage III, and 20.0% of stage IV patients. The frequency of antibody positivity increased with disease progression. When the NY-ESO-1 antibody was used in combination with carcinoembryonic antigen and CA19-9 to detect gastric cancer, information gains of 11.2% in stages III and IV, and 5.8% in all patients were observed. The NY-ESO-1 immune response levels of the patients without recurrence fell below the cutoff level after surgery. Two of the patients with recurrence displayed incomplete decreases. The nine patients who received chemotherapy alone continued to display NY-ESO-1 immune responses. Conclusion: When combined with conventional tumour markers, the NY-ESO-1 humoral immune response could be a useful tumour marker for detecting advanced gastric cancer and inferring the post-treatment tumour load in seropositive patients.

The clearance of solid low-level radioactive laboratory waste (LLRW) after decay-in-storage (DIS) obtained from a research institute and thoroughly separated using the separation and classification protocols presented in this study was evaluated. METHOD: The radioisotope (RI) content of incinerated LLRW from the specified RI research group (group A); the RI content of LLRW obtained in fiscal year 2000, which contained radionuclides with half-lives of less than 164 d (LLRW2); and the RI content of the LLRW reported in group A's disposal records were compared. The LLRW2 and LLRW of group A were incinerated after 2 y of decay-in-storage and immediately after storage, respectively. RESULTS: The highest ratio of the RI of incinerated LLRW to the value in the disposal records was 2.52 for ⁵¹Cr. The radioactivities of radionuclides in both the LLRW2 and LLRW for ³⁵S, ⁴⁵Ca, ⁵¹Cr, ¹²⁵I, ³²P, ³³P, and ⁹⁹mTc and the incinerated ash after 1 y later of decay-in-storage were below the clearance level defined by the RS-G-1.7 of the International Basic Safety Standard without contamination by ³H and ¹⁴C. These remains contained very small amounts of some long-half-life radionuclides of natural origin after 7 y of decay-in-storage. CONCLUSION: This LLRW separation protocol was effective for the separation of ³H and ¹⁴C. LLRW2 after 2 years of DIS and its incinerated ash after one year later of DIS were below the clearance level for radioactivity and radioactivity concentration.

Metastatic melanoma is resistant to conventional therapies. N-propionyl-4-S-cysteaminylphenol (NPrCAP), an N-protected sulfur-amine analog of tyrosine, is a good substrate for tyrosinase and is selectively incorporated into melanoma cells, causing cytotoxicity in vitro and in vivo. We have recently shown that intratumoral injections of NPrCAP suppress not only the growth of primary B16F1 melanoma tumors but also of secondary, re-challenged tumors. The participation of CD8(+) T cells has been suggested for the NPrCAP-mediated anti-B16 melanoma immunity. In this study, the molecular mechanism of the NPrCAP cytotoxicity and immunogenicity was examined. The phenol NPrCAP was shown to be activated by mushroom tyrosinase to the ortho-quinone N-propionyl-4-S-cysteaminyl-1,2-benzoquinone (NPrCAQ), and the structure was confirmed by reducing it to the corresponding catechol. NPrCAQ reacted rapidly with biologically relevant sulfhydryl compounds such as cysteine, glutathione and bovine serum albumin. The NPrCAQ-thiol adduct formation was proven with a model thiol N-acetylcysteine by spectroscopic methods. The production and release of NPrCAQ-protein adducts was verified in B16F1 melanoma cells in vitro and in B16F1 melanoma-bearing mice in vivo through the detection of 5-S-cysteaminyl-3-S-cysteinylcatechol after acid hydrolysis of the protein fraction. These results suggest that the phenol NPrCAP, acting as a prohapten, can be activated in melanoma cells by tyrosinase to the quinone-hapten NPrCAQ, which binds to melanosomal proteins through their cysteine residues to form possible neo-antigens, thus triggering the immunological response. NPrCAP thus represents a potential new approach to immunotherapy against metastatic melanoma.

Background N -propionyl-4- S -cysteaminylphenol (NPr-4- S -CAP) is selectively incorporated into melanoma cells and degrades them. However, it remains unclear whether NPr-4- S -CAP can induce cell death associated with the induction of host immune responses and tumor suppression in vivo. Objective To examine the molecular mechanism of NPr-4- S -CAP-mediated cytotoxicity toward melanoma cells and to test whether NPr-4- S -CAP can suppress transplanted primary and secondary B16F1 melanomas. Methods Cytotoxicity and apoptosis of melanoma cells were assessed by cell counting, flow cytometry, and detection of reactive oxygen species (ROS) and apoptotic molecules. NPr-4- S -CAP-associated host immunity was studied using a B16F1 mouse melanoma model through the application of CD4- and CD8-specific antibodies and tetramer assay. Results NPr-4- S -CAP suppressed growth of pigmented melanoma cells associated with an increase of intracellular ROS, activation of caspase 3 and DNA fragmentation, suggesting that NPr-4- S -CAP mediated ROS production, eliciting apoptosis of melanoma cells. Growth of transplanted B16F1 melanomas was inhibited after the consecutive intratumoral injections of NPr-4- S -CAP, and the tumor growth after rechallenge of B16F1 was significantly suppressed in the treated mice. This suppression occurred when the treated mice were given the anti-CD4 antibody, but not the anti-CD8 antibody. Tetramer assay demonstrated increased TYRP-2-specific CD8 + T cells in the lymph node and spleen cells prepared from NPr-4- S -CAP-treated B16F1-bearing mice. Conclusions These suggest that NPr-4- S -CAP induces apoptosis in melanoma cells through ROS production and generates CD8 + cell immunity resulting in the suppression of rechallenged B16F1 melanoma.

Esophageal cancer is a highly aggressive disease, and improved modalities for its treatment are needed. We performed chemoimmunotherapy involving the intratumoral administration of 111In-labeled dendritic cells (DC) in combination with preoperative chemotherapy in 5 esophageal cancer patients. Mature DC were generated and traced by scintigraphy after their administration. No adverse events that were directly related to the intratumoral DC administration were observed. Delayed-type hypersensitivity skin tests against keyhole limpet hemocyanin, which was added to the culture medium, detected a positive response in 3 patients, and keyhole limpet hemocyanin antibody production was observed in 4 patients, suggesting that intratumorally administered DC migrate to the lymph nodes, where they function as antigen-presenting cells. However, scintigraphic images obtained after the DC administration demonstrated that the DC remained at the esophageal tumor injection sites in all cases, and no DC accumulation was observed elsewhere. The accumulation of CD83+ cells in the primary tumor was also observed in 2 out of 4 patients in an immunohistochemical analysis using surgically resected specimens. Although the induction of tumor-specific immune responses during chemoimmunotherapy was also analyzed in enzyme-linked immunosorbent assay against 28 tumor antigens, none of the antibodies against the antigens displayed enhanced titers. No changes of NY-ESO-1-specific cellular immune response was observed in a patient who displayed NY-ESO-1 antibody production before the DC administration. These results suggest that the intratumoral administration of 111In-labeled mature DC during chemotherapy does not lead to detectable DC migration from the primary tumor to the draining lymph nodes, and therefore, might not achieve an optimal clinical response.