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.

Figure S1 - OS of lung adenocarcinoma patients with TILs. Figure S2 - Discriminant scores of each lung adenocarcinoma patient calculated by the discriminant function. Figure S3 - OS of lung adenocarcinoma patients belonging to predicted clusters with the study cohort and validation cohort. Figure S4 - Correlation of PD-L1 and Galectin-9 expression and T-cell infiltration in the tumor - Figure S5 - RT-PCR analysis of XAGE1 mRNA. Figure S6 - Effect of IFNgamma on PD-L1 and Galectin-9 expression in cell lines.

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.

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.

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

Representative results of analyses on Th1, Th2, Th17 and TFH CD4 T cells, and resting and activated CD4 Tregs, and M- and PMN-MDSCs in PBMCs from an XAGE1 (GAGED2a) antibody-positive patient, KLU213 or from an XAGE1 (GAGED2a) antibody-negative patient, KLU320 by FACS.

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

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.

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

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).

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

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

Introduction: Programmed cell death-1 (PD-1) inhibitors effectively treat NSCLC and prolong survival. Robust biomarkers for predicting clinical benefits of good response and long survival with anti-PD-1 therapy have yet to be identified; therefore, predictive biomarkers are needed to select patients with benefits. Methods: We conducted a prospective study to explore whether serum antibody against NY-ESO-1 and/or XAGE1 cancer-testis antigens predicted primarily good clinical response and secondarily long survival with anti-PD-1 therapy for NSCLC. The serum antibody was detected by enzyme-linked immunosorbent assay, and tumor immune microenvironment and mutation burden were analyzed by immunohistochemistry and next-generation sequencing. Results: In the discovery cohort (n = 13), six antibodypositive NSCLC cases responded to anti-PD-1 therapy (two complete and four partial responses), whereas seven antibody-negative NSCLC cases did not. Antibody positivity was associated with good response and survival, regardless of tumor programmed death ligand 1 (PD-L1) expression, mutation burden, and CD8thorn T-cell infiltration. In the validation cohort (n = 75), 17 antibody-positive NSCLC cases responded well to anti-PD-1 therapy as compared with 58 negative NSCLC cases (objective response rate 65% versus 19%, p = 0.0006) and showed significantly prolonged progression-free survival and overall survival. Antibody titers highly correlated with tumor reduction rates. In the multivariate analysis, response biomarkers were tumor programmed death ligand 1 expression and antibody positivity, and only antibody positivity was a significantly better predictive biomarker of progression-free survival (hazard ratio = 0.4, p = 0.01) and overall survival (hazard ratio = 0.2, p = 0.004). Conclusions: Our results suggest that NY-ESO-1 and/or XAGE1 serum antibodies are useful biomarkers for predicting clinical benefits in anti-PD-1 therapy for NSCLC and probably for other cancers. (C) 2019 International Association for the Study of Lung Cancer. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

162 Background: Cancer-testis antigens (CTAs) are expressed predominantly in the testis and various types of cancer. Some of CTAs are highly immunogenic and attractive targets for cancer immunotherapy. We identified XAGE1 as a dominant CTA in lung adenocarcinoma (LAD). In this study, we examined immune responses to XAGE1 and the clinical significance in LAD patients. Methods: Expression of XAGE1 and immune checkpoint molecules in LAD tissues was determined by immunohistochemistry. The XAGE1 antibody and T cell immune responses were analyzed in blood samples. Then, overall survival (OS) of the XAGE1 antigen-positive and -negative, and XAGE1 antibody-positive and -negative patients was analyzed. Results: The XAGE1 antigen was expressed in approximately 40% of LAD, and the expression reflected shortened OS of pStage I-IIIA LAD in two japanese cohorts. In addition, expression profiles of XAGE1 and immune checkpoint molecules of PD-L1 and galectin-9 on tumor cells efficiently predicted OS of pStage I-IIIA LAD patients. The XAGE1 antibody response was observed in 6% (9/155) of our pStage I-IIIA, and 20% (34/167) of cStage IIIB-IV LAD, respectively, showing a higher antibody response rate in more advanced stages. In the antibody-positive patients, CD4 and CD8 T cell responses were frequently elicited, and phenotypic and functional analyses of T cells indicated immune activation. Furthermore, the OS of antibody-positive patients significantly prolonged as compared with that of antibody-negative patients with either XAGE1 antigen-positive EGFRwt (31.5 vs 15.6 months, P = 0.05) or EGFRmt (34.7 vs 11.1 months, P = 0.001) LAD. Multivariate analysis showed that the XAGE1 antigen expression was a worse predictor in EGFRmt LAD (HR: 5.23). On the other hand, the presence of the XAGE1 antibody was a strong predictor for prolonged OS in XAGE1 antigen positive LAD (HR: 0.18) and in either EGFRwt or EGFRmt LAD. Conclusions: The XAGE1 antigen induced strong immune responses in LAD patients with more advanced stages, and the production of XAGE1 antibody in these patients showed good prognosis. Our results indicate that the XAGE1 immunity is probably a good prognostic biomarker in LAD and a promising target for immunotherapy of LAD.

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 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.

Patients with advanced lung cancer have poor survival, although they have received multidisciplinary therapy. Therefore, the novel effective therapy is needed. In various malignancies, tumor cells escape the host immune defenses, in which regulatory T cells (Tregs) play an important role. Tregs, maintaining self-tolerance and homeostasis in the immune system, suppress antitumor immune responses in cancer patients. Thus, Tregs are crucial in controlling antitumor immune responses. Several clinical studies show that a number of Tregs at tumor site was correlated with poor prognosis and Tregs suppress the antigen-specific T-cell induction in immunotherapy.

Cancer/testis (CT) antigen is a class of antigens that express predominantly in the testes and various tumor types. Some CT antigens have been shown to be highly immunogenic and are considered to be attractive targets for cancer immunotherapy. We identified XAGE1 as a dominant CT antigen in lung adenocarcinoma (LAD). In this study, we investigated the correlation of clinical response and XAGE1 immunity in LAD.

Abstract The immune status of tumors varies, and this may affect the overall survival (OS) of patients. We examined tumors from 120 patients with lung adenocarcinomas with a tissue microarray for T-cell infiltration and the expression of PD-L1 and Galectin-9 (both ligands for inhibitory receptors on T cells), and cancer/testis (CT) antigen XAGE1 (GAGED2a; a tumor antigen often found on lung tumors) expression, to determine their relevance to OS. Patients defined as pStage I–IIIA could be grouped, based on the expression profiles of PD-L1, Galectin-9, and XAGE1, into cluster A, who had prolonged survival, and cluster B, who had shorter survival. The difference in survival of the clusters was confirmed separately for pStage I and pStage II–IIIA patients. Cluster A patients who also had CD4 and CD8 T-cell infiltration showed even better survival, as expected. The findings were confirmed by examining an independent validation cohort of 68 pStage I lung adenocarcinoma patients. Our data showed that PD-L1 expression was a positive indicator, whereas Galectin-9 and XAGE1 expression was negative. In vitro analyses suggested that PD-L1 expression was upregulated by IFNγ secreted from activated T cells in the tumor and Galectin-9 expression was counteracting those T cells. Thus, use of these immune markers enables the creation of a discriminant function with which to classify tumors and predict survival. Cancer Immunol Res; 4(12); 1049–60. ©2016 AACR.