Integrated analysis of phase 1a and 1b randomized controlled trials; Treg-targeted cancer immunotherapy with the humanized anti-CCR4 antibody, KW-0761, for advanced solid tumors.

INTRODUCTION: Regulatory T cells (Tregs) have attracted attention as a novel therapeutic target to augment the clinical efficacy of immunotherapy. We conducted phase Ia and Ib trials to examine the safety and efficacy of the anti-CCR4 antibody, KW-0761 (mogamulizumab), which may eliminate effector Tregs (eTregs). We herein overviewed the results of these trials, presented cases with a durable clinical response, and investigated factors associated with the clinical effects of KW-0761. METHODS: Forty-nine patients with CCR4-negative solid cancers were enrolled in the phase Ia and Ib trials on KW-0761. An integral analysis of safety, clinical responses, prognosis, blood laboratory data, and cancer testis antigen-specific immune responses was performed. RESULTS: Grade 3-4 treatment-related adverse events were reported in 21 (42.9%) out of 49 patients, all of which were manageable. A partial response and stable disease were observed in 1 and 9 patients, respectively. A durable clinical response was noted in 2 esophageal and 2 lung cancer patients. eTreg depletion in peripheral blood was confirmed in most patients, and eTreg depletion was sustained during the KW-0761 treatment. High lymphocyte levels at baseline and 2 weeks after the initiation of KW-0761 were associated with a favorable clinical outcome. CONCLUSIONS: A durable clinical response was noted in some patients, and high lymphocyte levels before treatment initiation may be a biomarker for the efficacy of KW-0761. The synergistic effect of KW-0761 for depleting Tregs and other immunotherapies is expected in the future.

Immune checkpoint therapy and response biomarkers in non-small-cell lung cancer: Serum NY-ESO-1 and XAGE1 antibody as predictive and monitoring markers.

Immune checkpoint inhibitors (ICI) are key drugs in systemic therapy for advanced non-small-cell lung cancer (NSCLC) and have recently been incorporated into neoadjuvant and adjuvant settings for surgical resection. Currently, ICI combinations with cytotoxic agents are frequently used in clinical practice, although several ICI clinical trials have failed to produce long-term clinical benefits. Unfortunately, clinical benefit is moderate and limited considering physical and financial burden. Therefore, selecting appropriate patients and regimens for ICI therapy is important, and biomarkers are necessary for their selection. Tumor PD-L1 expression is universally used as a biomarker; however, PD-L1 assays show low analytical validity and reproducibility due to the visual-scoring system by pathologists. Recent tumor immunology studies explore that neoantigens derived from somatic mutations and the collaboration between T and B cells efficiently elicit antitumor responses. This suggests that high tumor mutational burden and T-cell infiltration are predictive biomarkers. However, B cells producing antibody (Ab) remain poorly understood and analyzed as biomarkers. We found that NY-ESO-1 and XAGE1 of cancer-testis antigen frequently elicit spontaneous humoral and cellular immune responses in NSCLC. Serum Ab against these antigens were detected in approximately 25% of NSCLC patients and predicted ICI monotherapy responses. In addition, the Ab levels were decreased with tumor shrinkage after ICI therapy. Thus, NY-ESO-1 and XAGE1 Ab are potentially biomarkers predicting and monitoring response to ICI therapy. For clinical applications, a fully-automated assay system measuring the Ab was developed. Here, we review current ICI therapy, tumor immunology, and biomarkers in NSCLC, and discuss the applicability of the serum biomarkers NY-ESO-1 and XAGE1 Ab.

Predictive value of serum high-mobility group box 1 levels for checkpoint inhibitor pneumonitis.

BACKGROUND AND OBJECTIVE: Checkpoint inhibitor pneumonitis (CIP), caused by the anti-programmed cell death-1 (PD-1)/programmed cell death ligand-1 (PD-L1) antibody, can be a fatal adverse event in cancer patients. However, no predictive biomarkers for CIP have been identified. Because high-mobility group box 1 (HMGB1) can aggravate lung injury and potentially increase the immune response, it was investigated as a predictive blood marker. METHODS: Blood samples, prospectively stored before anti-PD-1/PD-L1 monotherapy between December 2015 and October 2020, were obtained at two university hospitals from 87 and 43 non-small cell lung cancer (NSCLC) patients (discovery and validation cohorts, respectively). We retrospectively evaluated the association of serum HMGB1 levels with the incidence of CIP developed within 3 months of initiating anti-PD-1/PD-L1 therapy. RESULTS: CIP was observed in 9 (10.3%) and 6 (14.0%) patients in the discovery and validation cohorts, respectively. In each cohort, serum HMGB1 levels were significantly and reproducibly higher in patients with CIP. In the discovery cohort, an HMGB1 cut-off level of 11.24 ng/ml was identified by receiver operating characteristic analysis. CIP incidence in the HMGB1high subgroup was significantly higher than that in the HMGB1low subgroup in the discovery (41.2% vs. 2.9%) and validation cohorts (36.4% vs. 6.3%). In an exploratory pooled analysis, three patients died of grade 5 CIP; a 19.29 ng/ml HMGB1 cut-off level detected grade 5 CIP with 100% sensitivity and 96.85% specificity. CONCLUSION: Our results suggest that HMGB1 may be a potential blood marker to predict the development and severity of CIP in NSCLC patients.

Inhibition of pancreatic cancer-cell growth and metastasis in vivo by a pyrazole compound characterized as a cell-migration inhibitor by an in vitro chemotaxis assay.

Pancreatic cancer is recalcitrant to treatment as it is highly metastatic and rapidly progressive. While observing the behavior of human pancreatic BxPC-3 cells using an optical assay device called TAXIScan, we found that several synthetic pyrazole and pyrimidine derivatives inhibited cell migration. One such compound, 14-100, inhibited metastasis of fluorescence-labeled BxPC-3 cells, which were transplanted into the pancreas of nude mice as a subcutaneously grown cancer fragment. Surprisingly, despite its low cytotoxicity, the compound also showed an inhibitory effect on cancer cell proliferation in vivo, suggesting that the compound alters cancer cell characteristics needed to grow in situ. Single-cell RNA-sequencing revealed changes in gene expression associated with metastasis, angiogenesis, inflammation, and epithelial-mesenchymal transition. These data suggest that the compound 14-100 could be a good drug candidate against pancreatic cancer.

Phase Ib study on the humanized anti-CCR4 antibody, KW-0761, in advanced solid tumors.

Tregs infiltrate tumors and inhibit antitumor immunity. KW-0761 (Mogamulizumab) is a humanized anti-CCR4 monoclonal antibody that could eliminate activated Tregs with high immunosuppressive activity that express CCR4. In this phase Ib trial, KW-0761 was used as a cancer immunotherapeutic reagent to deplete Tregs in patients with advanced or recurrent solid CCR4-negative tumors. Thirty-nine patients with solid cancer were treated with KW-0761 at a dose of 0.1 or 1.0 mg/kg. The safety, clinical responses, and effects of Treg depletion were analyzed. Any grade and grade 3-4 treatment-related adverse events (AEs) were observed in 36 (92%) and 14 (36%) out of 39 patients, respectively. All treatment-related AEs were manageable. One and 5 patients achieved a partial response and stable disease, respectively, during treatment and were long survivors. The efficient depletion of Treg in peripheral blood was confirmed in both cohorts. Therefore, the administration of KW-0761 was safe, resulting in the depletion of Tregs in peripheral blood and potential immune responses in patients with solid cancer. The combined use of KW-0761 to deplete Tregs and other immunotherapies is a promising approach to augment immune responses.

Tumor PD-L1 and VEGF Expression, and CD8 T Cell Infiltration Predict Clinical Response to Immune Checkpoint Inhibitors in Non-small Cell Lung Cancer.

BACKGROUND/AIM: We evaluated the efficacy of "the tumor immune microenvironment (TIME) classification" for predicting clinical response to immune checkpoint inhibitors (ICIs) in patients with non-small cell lung cancer (NSCLC). In addition, we aimed to evaluate the "modified TIME classification", which adds the vascular endothelial growth factor (VEGF) status to TIME. MATERIALS AND METHODS: Programmed cell death receptor ligand-1 (PD-L1), CD8 T cell tumor-infiltrating lymphocytes (CD8+TILs) count and VEGF expression analyses were performed using immuno - histochemistry in 44 patients who had undergone ICI monotherapy. RESULTS: Regarding TIME classification, type-I (PD-L1 high and CD8+TILs high) had a significantly higher response than the other types. Using the modified TIME classification, type-IA (PD-L1 high, CD8+TILs high, and VEGF low) had a significantly higher response than the other types. CONCLUSION: The modified TIME classification, which adds tumor VEGF expression to "the TIME classification", could be useful in predicting clinical response to ICI monotherapy.

A novel automated immunoassay for serum NY-ESO-1 and XAGE1 antibodies in combinatory prediction of response to anti-programmed cell death-1 therapy in non-small-cell lung cancer.

BACKGROUND: Anti-programmed cell death-1 (PD-1) antibodies (Abs) are key drugs in non-small-cell lung cancer (NSCLC) treatment; however, clinical benefits with anti-PD-1 monotherapy are limited. We reported that serum Abs against cancer-testis antigens NY-ESO-1 and XAGE1 predicted clinical benefits. We aimed to develop a fully automated immunoassay system measuring NY-ESO-1/XAGE1 Abs. METHODS: Sera from 30 NSCLC patients before anti-PD-1 monotherapy were reacted with recombinant NY-ESO-1 protein- or synthetic XAGE1 peptide-coated magnetic beads. ALP-conjugated Ab and chemiluminescent substrate were added and luminescence measured. These procedures were automated using high sensitivity chemiluminescent enzyme immunoassay (HISCL™). NY-ESO-1/XAGE1 Ab stability was tested under various conditions. Response prediction accuracy was evaluated using area under receiver operating curve (AUROC). RESULTS: HISCL detected specific serum NY-ESO-1/XAGE1 Abs, which levels in ELISA and HISCL were highly correlated. The Ab levels in HISCL were stable at four temperatures, five freeze/thaw cycles, and long-term storage; the levels were not interfered by common blood components. The Ab levels in 15 NSCLC responders to anti-PD-1 monotherapy were significantly higher than those in non-responders and healthy donors. The AUROC was the highest (0.91; 95% CI, 0.78-1.0) in combinatory prediction with NY-ESO-1/XAGE1 Abs. CONCLUSION: Our immunoassay system is useful to predict clinical benefits with NSCLC immune-checkpoint therapy.

Generalized herpes zoster and cutaneous metastasis during chemotherapy for non-small cell lung cancer: A case report.

Although herpes zoster is known to occur in some patients with lung cancer, generalized (disseminated) herpes zoster is an uncommon form whereby hematogenous dissemination of the virus occurs and leads to the development of widespread cutaneous lesions. Similarly, skin is an uncommon site of metastasis in patients with lung cancer. Here, we report a clinical case of a 53-year-old male patient who developed generalized herpes zoster during chemotherapy for non-small cell lung cancer (squamous cell carcinoma) and subsequently developed cutaneous metastasis of lung cancer after generalized herpes zoster was cured by treatment with intravenous aciclovir. The coincidence of these two conditions, generalized herpes zoster and cutaneous metastasis, in the patient during lung cancer treatment might be associated with an impaired or dysregulated immune system partly due to repeated chemotherapy, indicating a poor prognosis. Close observation and accurate diagnosis of changes in the skin of patients with lung cancer are important when evaluating their immune status and considering their therapy and prognosis.

Successful treatment of gastric cancer after complete response of lung cancer by nivolumab: a case report.

BACKGROUND: Nivolumab is effective for gastric cancer and lung cancer, but complete response is rare. We experienced a case of synchronous gastric cancer and lung cancer who was treated by nivolumab and laparoscopic gastrectomy. CASE PRESENTATION: A 63-year-old male consulted our institution and was found to have gastric cancer cT1(SM)N0M0 Stage IA and lung cancer cT2N2M1(PUL) Stage IV. He received eight chemotherapy treatments plus radiation, but the lung disease remained progressive. Finally, he received nivolumab therapy and complete response of both cancers was obtained. The gastric cancer recurred, but was successfully treated by laparoscopic gastrectomy. The resected specimen revealed three lesions, each being pT1aN0M0 Stage IA. The primary gastric cancer seemed to have completely vanished without scarring. CONCLUSIONS: This was thought to be a rare case of gastric cancer recurrence after complete response of gastric cancer and lung cancer to nivolumab.

Detachment and recovery of an X-ray opaque tip of a disposable guide sheath kit: A rare complication of endobronchial ultrasound-guided transbronchial biopsy.

Endobronchial ultrasound with a guide sheath (EBUS-GS)-guided transbronchial biopsy offers advantages and is frequently used for the diagnosis of peripheral pulmonary lesions. A previously healthy 75-year-old man was hospitalized to undergo bronchoscopy for the diagnosis of a mass with a diameter of about 40 mm in right S3 area. The mass was detected during a regular medical check-up, and lung cancer was suspected. EBUS-GS guided transbronchial biopsy was performed through the right B3. Following the bronchoscopic procedure and removal of the GS, we observed that an X-ray opaque tip attached to the point of the GS was missing. We examined the lung field through X-ray fluoroscopy and found that the detached opaque tip was located in the right middle lung field. We re-inserted the bronchoscope, and successfully recovered it using transbronchial biopsy forceps. The rate of complications in EBUS-GS is low, and the complication presented in this report is rare. Physicians should exercise caution when performing this procedure and carefully check the condition of the kit to reduce the risk of such complications.

Thoracoscopic Findings in IgG4-related Pleuritis.

A 46-year-old Japanese man was admitted to our hospital with a 1-year history of dyspnea and persistent right-dominant bilateral pleural effusions. Chest and abdominal computed tomography (CT) revealed no notable findings apart from the bilateral pleural effusions. 2-deoxy-2-[18F]-fluoro-D-glucose (FDG) positron emission tomography-CT showed no accumulation of FDG in the thorax and abdomen. Thoracoscopy revealed numerous small (approximately 2-3 mm in size), blister-like nodules on the left parietal pleura extending from the lower third of the chest wall to the diaphragm. A pathological examination revealed lymphocyte and plasma cell infiltrates with increasing numbers of IgG4-positive plasma cells in the fibrotic pleura, indicating IgG4-related pleuritis.

Serum Antibody Against NY-ESO-1 and XAGE1 Antigens Potentially Predicts Clinical Responses to Anti-Programmed Cell Death-1 Therapy in NSCLC.

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 antibody-positive 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 CD8+ 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.

NY-ESO-1 Protein Cancer Vaccine With Poly-ICLC and OK-432: Rapid and Strong Induction of NY-ESO-1-specific Immune Responses by Poly-ICLC.

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.

Basic and clinical evaluation of anti-CCR4 mAb in cancer immunotherapy.

CCR4 is one of the receptors for chemokines and is expressed on Th2, Th17, skin-homing T cells and regulatory T cells (Tregs). Activated Tregs highly express CCR4 and are induced into tumor environment by M2 macrophages-producing CCL22. Tregs show strong suppres- sive functions in cancer immunity, resulting in tumor progression. On the other hand, moga- mulizumab is a humanized anti-human CCR4 monoclonal antibody with a defucosylated Fe region which markedly enhances ADCC activity, and the antibody has been shown to effi- ciently deplete CCR4-positive cells. Therefore, a phase Ia clinical study of mogamulizumab was conducted in the treatment of solid cancers. Mogamulizumab almost depeleted activated Tregs in peripheral blood, and then re-activated CTL function with low grade of adverse events. However, no clinical responses were observed in ten patients. A phase lb study is now on-going, and further clinical studies may be needed in combination with other anti- tumor drugs.

Survival of Lung Adenocarcinoma Patients Predicted from Expression of PD-L1, Galectin-9, and XAGE1 (GAGED2a) on Tumor Cells and Tumor-Infiltrating T Cells.

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.

Phase Ia Study of FoxP3+ CD4 Treg Depletion by Infusion of a Humanized Anti-CCR4 Antibody, KW-0761, in Cancer Patients.

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.

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 CD127 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.

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.

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-1ß in CD14⁺ monocytes and monocytic cell line THP-1 cells and activated the NLRP3-inflammasome, resulting in production of mature IL-1ß. Use of ASC and NLRP3-deficient THP-1 cell lines established involvement of the NLRP3 inflammasome in an IL-1ß secretion in treatment with NPCMD. Inhibition of IL-1ß 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.