Akiko Uenaka

Increase in Activated Treg in TIL in Lung Cancer and In Vitro Depletion of Treg by ADCC Using an Antihuman CCR4 mAb (KM2760)

Introduction: Tregs infiltrate tumors and inhibit immune responses against them. Methods: We investigated subpopulations of Foxp3+ CD4 T cells previously defined by Miyara et al. (Immunity 30, 899–911, 2009) in peripheral blood mononuclear cells (PBMCs) and tumor infiltrating lymphocytes (TILs) in lung cancer. We also showed that Tregs in healthy donors that express CCR4 could be efficiently eliminated in vitro by cotreatment with antihuman (h) CCR4 mAb (KM2760) and NK cells. Results: In lung cancer, the number of activated/effector Tregs and non-Tregs, but not resting/naive Tregs, was increased in TILs compared with the number of those cells in PBMCs. The non-Treg population contained Th2 and Th17. CCR4 expression on activated/effector Tregs and non-Tregs in TILs was down-regulated compared with that on those cells in PBMCs. Chemokinetic migration of CD25+ CD4 T cells containing the Treg population sorted from the PBMCs of healthy donors to CCL22/MDC was abrogated by pretreatment with anti-hCCR4 mAb (KM2760). The inhibitory activity of CD25+ CD127dim/− CD4 Tregs on the proliferative response of CD4 and CD8 T cells stimulated with anti-CD3/CD28 coated beads was abrogated by adding an anti-hCCR4 mAb (KM2760) and CD56+ NK cells to the culture. Conclusions: The findings suggested the CCR4 on activated/effector Tregs and non-Tregs was functionally involved in the chemokinetic migration and accumulation of those cells to the tumor site. In vitro findings of efficient elimination of Tregs may give the basis for implementation of a clinical trial to investigate Treg depletion by administration of an anti-hCCR4 mAb to solid cancer patients.

Induction of CD8 T-cell responses restricted to multiple HLA class I alleles in a cancer patient by immunization with a 20-mer NY-ESO-1f (NY-ESO-1 91-110) peptide.

Immunogenicity of a long 20‐mer NY‐ESO‐1f peptide vaccine was evaluated in a lung cancer patient TK‐f01, immunized with the peptide with Picibanil OK‐432 and Montanide ISA‐51. We showed that internalization of the peptide was necessary to present CD8 T‐cell epitopes on APC, contrasting with the direct presentation of the short epitope. CD8 T‐cell responses restricted to all five HLA class I alleles were induced in the patient after the peptide vaccination. Clonal analysis showed that B*35:01 and B*52:01‐restricted CD8 T‐cell responses were the two dominant responses. The minimal epitopes recognized by A*24:02, B*35:01, B*52:01 and C*12:02‐restricted CD8 T‐cell clones were defined and peptide/HLA tetramers were produced. NY‐ESO‐1 91‐101 on A*24:02, NY‐ESO‐1 92‐102 on B*35:01, NY‐ESO‐1 96‐104 on B*52:01 and NY‐ESO‐1 96‐104 on C*12:02 were new epitopes first defined in this study. Identification of the A*24:02 epitope is highly relevant for studying the Japanese population because of its high expression frequency (60%). High affinity CD8 T‐cells recognizing tumor cells naturally expressing the epitopes and matched HLA were induced at a significant level. The findings suggest the usefulness of a long 20‐mer NY‐ESO‐1f peptide harboring multiple CD8 T‐cell epitopes as an NY‐ESO‐1 vaccine. Characterization of CD8 T‐cell responses in immunomonitoring using peptide/HLA tetramers revealed that multiple CD8 T‐cell responses comprised the dominant response.

Prolongation of overall survival in advanced lung adenocarcinoma patients with the XAGE1 (GAGED2a) antibody.

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.

Detection and Tracking of NY-ESO-1-Specific CD8+ T Cells by High-Throughput T Cell Receptor β (TCRB) Gene Rearrangements Sequencing in a Peptide-Vaccinated Patient

Comprehensive immunological evaluation is crucial for monitoring patients undergoing antigen-specific cancer immunotherapy. The identification and quantification of T cell responses is most important for the further development of such therapies. Using well-characterized clinical samples from a high responder patient (TK-f01) in an NY-ESO-1f peptide vaccine study, we performed high-throughput T cell receptor β-chain (TCRB) gene next generation sequencing (NGS) to monitor the frequency of NY-ESO-1-specific CD8+ T cells. We compared these results with those of conventional immunological assays, such as IFN-γ capture, tetramer binding and limiting dilution clonality assays. We sequenced human TCRB complementarity-determining region 3 (CDR3) rearrangements of two NY-ESO-1f-specific CD8+ T cell clones, 6-8L and 2F6, as well as PBMCs over the course of peptide vaccination. Clone 6-8L possessed the TCRB CDR3 gene TCRBV11-03*01 and BJ02-01*01 with amino acid sequence CASSLRGNEQFF, whereas 2F6 possessed TCRBV05-08*01 and BJ02-04*01 (CASSLVGTNIQYF). Using these two sequences as models, we evaluated the frequency of NY-ESO-1-specific CD8+ T cells in PBMCs ex vivo. The 6-8L CDR3 sequence was the second most frequent in PBMC and was present at high frequency (0.7133%) even prior to vaccination, and sustained over the course of vaccination. Despite a marked expansion of NY-ESO-1-specific CD8+ T cells detected from the first through 6th vaccination by tetramer staining and IFN-γ capture assays, as evaluated by CDR3 sequencing the frequency did not increase with increasing rounds of peptide vaccination. By clonal analysis using 12 day in vitro stimulation, the frequency of B*52:01-restricted NY-ESO-1f peptide-specific CD8+ T cells in PBMCs was estimated as only 0.0023%, far below the 0.7133% by NGS sequencing. Thus, assays requiring in vitro stimulation might be underestimating the frequency of clones with lower proliferation potential. High-throughput TCRB sequencing using NGS can potentially better estimate the actual frequency of antigen-specific T cells and thus provide more accurate patient monitoring.

Abstract 4732: Depletion of Tregs from PBMCs by treatment with defucosylated anti-CCR4 mAb.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC 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

Sensitive Multiplexed Quantitative Analysis of Autoantibodies to Cancer Antigens with Chemically S-Cationized Full-Length and Water-Soluble Denatured Proteins

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.

Production of NY-ESO-1 peptide/DRB1*08:03 tetramers and ex vivo detection of CD4 T-cell responses in vaccinated cancer 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.

TLR4 and NLRP3 inflammasome activation in monocytes by N-propionyl cysteaminylphenol-maleimide-dextran (NPCMD).

BACKGROUND N-propionyl cysteaminylphenol-maleimide-dextran (NPCMD) is a toxic tyrosinase substrate developed to treat melanoma. OBJECTIVE We investigated the effect of NPCMD on innate immune responses in monocytes. METHODS CD14⁺ monocytes and a monocytic cell line, THP-1, were stimulated with NPCMD in vitro. Cytokines in the culture supernatants were determined by ELISA and flow cytometry. RESULTS NPCMD stimulated CD14⁺ monocytes and THP-1 cells to secrete TNFα, IL-6 and IL-8, but not IL-10 or IL-12. TNFα secretion from THP-1 cells stimulated with NPCMD was inhibited by addition of an anti-TLR4 mAb in culture. Moreover, NPCMD stimulated production of pro-IL-1β in CD14⁺ monocytes and monocytic cell line THP-1 cells and activated the NLRP3-inflammasome, resulting in production of mature IL-1β. Use of ASC and NLRP3-deficient THP-1 cell lines established involvement of the NLRP3 inflammasome in an IL-1β secretion in treatment with NPCMD. Inhibition of IL-1β secretion by an endocytosis inhibitor, cytochalasin B, and a lysosomal enzyme cathepsin B inhibitor, CA-074 Me, suggested the involvement of lysosomal rupture and leakage of cathepsin B into the cytosol in NLRP3 activation by NPCMD. CONCLUSION The immunopotentiating effect of NPCMD mediated by TLR4 and NLRP3 inflammasome activation could be useful for eliciting effective adaptive immune responses against melanoma and other tumors.

Abstract 495: Immune response to XAGE-1b (GAGED2a) in NSCLC patients and its clinical relevance.

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 − 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 − 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

Abstract 508: Correlation of antibody and T-cell responses against XAGE-1b in NSCLC patients

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL 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