Bevacizumab Plus Atezolizumab After Progression on Atezolizumab Monotherapy in Pretreated Patients With NSCLC: An Open-Label, Two-Stage, Phase 2 Trial.

INTRODUCTION: Vascular endothelial growth factor promotes an immunosuppressive tumor microenvironment that can be reverted by an antiangiogenic therapy. This two-stage, phase 2 study aimed to determine the treatment efficacy of adding bevacizumab to atezolizumab in patients with metastatic NSCLC whose disease had progressed after atezolizumab monotherapy. METHODS: Immune checkpoint inhibitor-naive patients with NSCLC, without EGFR or ALK alterations, whose disease progressed after at least one line of platinum-based chemotherapy were eligible. The patients received atezolizumab 1200 mg once every 3 weeks until radiographic progression (stage I). Then, bevacizumab 15 mg/kg was combined with atezolizumab 1200 mg once every 3 weeks (stage II). The primary end point was the disease control rate (DCR) confined to stage II. RESULTS: A total of 42 and 24 patients were enrolled in stages I and II, respectively. Most patients had negative programmed death ligand-1 expression (71.4%) and received one or two lines of therapy (95.2%). In stage I, patients achieved a DCR of 35.7% (95% confidence interval [CI]: 21.6-52.0). In stage II, three (12.5%) and 18 (75.0%) of 24 patients had partial response and stable disease, respectively, leading to a DCR of 87.5% (95% CI: 67.6-97.3). For 24 patients enrolled in stage II, the median progression-free survival was 5.6 (95% CI: 4.1-7.1) months and the overall survival was 14.0 (95% CI: 10.7-17.4) months. Treatment-related adverse events occurred in 25% of the patients in stage II, but all were of grade 1 or 2. CONCLUSIONS: Combination of bevacizumab plus atezolizumab for patients with metastatic NSCLC whose disease had progressed after atezolizumab monotherapy was found to have a promising antitumor activity with good tolerability.

Therapeutic efficacy of cancer vaccine adjuvanted with nanoemulsion loaded with TLR7/8 agonist in lung cancer model.

Although immune checkpoint inhibitors have significantly improved clinical outcomes in various malignant cancers, only a small proportion of patients reap benefits, likely due to the low number of T cells and high number of immunosuppressive cells in the tumor microenvironment (TME) of patients with advanced disease. We developed a cancer vaccine adjuvanted with nanoemulsion (NE) loaded with TLR7/8 agonist (R848) and analyzed its therapeutic effect alone or in combination with immune checkpoint inhibitors, on antitumor immune responses and the reprogramming of suppressive immune cells in the TME. NE (R848) demonstrated robust local and systemic antitumor immune responses in both subcutaneous and orthotopic mouse lung cancer models, inducing tumor-specific T cell activation and mitigating T cell exhaustion. Combination with anti-PD-1 antibodies showed synergistic effects with respect to therapeutic efficacy and survival rate. Thus, NE (R848)-based cancer vaccines could prevent tumor recurrence and prolong survival by activating antitumor immunity and reprogramming immunosuppression.

PD-1 blockade-unresponsive human tumor-infiltrating CD8+ T cells are marked by loss of CD28 expression and rescued by IL-15.

Blockade of programmed death-1 (PD-1) reinvigorates exhausted CD8+ T cells, resulting in tumor regression in cancer patients. Recently, reinvigoration of exhausted CD8+ T cells following PD-1 blockade was shown to be CD28-dependent in mouse models. Herein, we examined the role of CD28 in anti-PD-1 antibody-induced human T cell reinvigoration using tumor-infiltrating CD8+ T cells (CD8+ TILs) obtained from non-small-cell lung cancer patients. Single-cell analysis demonstrated a distinct expression pattern of CD28 between mouse and human CD8+ TILs. Furthermore, we found that human CD28+CD8+ but not CD28-CD8+ TILs responded to PD-1 blockade irrespective of B7/CD28 blockade, indicating that CD28 costimulation in human CD8+ TILs is dispensable for PD-1 blockade-induced reinvigoration and that loss of CD28 expression serves as a marker of anti-PD-1 antibody-unresponsive CD8+ TILs. Transcriptionally and phenotypically, PD-1 blockade-unresponsive human CD28-PD-1+CD8+ TILs exhibited characteristics of terminally exhausted CD8+ T cells with low TCF1 expression. Notably, CD28-PD-1+CD8+ TILs had preserved machinery to respond to IL-15, and IL-15 treatment enhanced the proliferation of CD28-PD-1+CD8+ TILs as well as CD28+PD-1+CD8+ TILs. Taken together, these results show that loss of CD28 expression is a marker of PD-1 blockade-unresponsive human CD8+ TILs with a TCF1- signature and provide mechanistic insights into combining IL-15 with anti-PD-1 antibodies.

Regulatory (FoxP3+) T cells and TGF-ß predict the response to anti-PD-1 immunotherapy in patients with non-small cell lung cancer.

Antitumor immune responses induced by immune checkpoint inhibitors anti-PD-1 or anti-PD-L1 have been used as therapeutic strategies in advanced non-small cell lung cancer (NSCLC) patients over the last decade. Favorable antitumor activity to immune checkpoint inhibitors is correlated with high PD-L1 expression, increased tumor-infiltrating lymphocytes, and decreased suppressive immune cells including Treg cells, myeloid-derived suppressor cells, or tumor-associated macrophages in various cancer types. In this study, we investigated the potential correlation between clinical outcomes and peripheral blood immune cell profiles, specifically focused on FoxP3+ Treg cells, collected at baseline and one week after anti-PD-1 therapy in two independent cohorts of patients with NSCLC: a discovery cohort of 83 patients and a validation cohort of 49 patients. High frequencies of circulating Treg cells one week after anti-PD-1 therapy were correlated with a high response rate, longer progression-free survival, and overall survival. Furthermore, high levels of TGF-ß and Treg cells were associated with favorable clinical outcomes. Our results suggest that higher levels of FoxP3+ Treg cells and TGF-ß can predict a favorable response to anti-PD-1 immunotherapy in patients with advanced NSCLC.

MDSC subtypes and CD39 expression on CD8+ T cells predict the efficacy of anti-PD-1 immunotherapy in patients with advanced NSCLC.

The major suppressive immune cells in tumor sites are myeloid derived suppressor cells (MDSCs), tumor-associated macrophages (TAMs), and Treg cells, and the major roles of these suppressive immune cells include hindering T-cell activities and supporting tumor progression and survival. In this study, we analyzed the pattern of circulating MDSC subtypes in patients with non-small cell lung cancer (NSCLC) whether those suppressive immune cells hinder T-cell activities leading to poor clinical outcomes. First, we verified PMN-MDSCs, monocytic-MDSCs (M-MDSCs), and Treg cells increased according to the stages of NSCLC, and MDSCs effectively suppressed T-cell activities and induced T-cell exhaustion. The analysis of NSCLC patients treated with anti-PD-1 immunotherapy demonstrated that low PMN-MDSCs, M-MDSCs, and CD39+ CD8+ T cells as an individual and all together were associated with longer progression free survival and overall survival, suggesting PMN-MDSCs, M-MDSCs, and CD39+ CD8+ T cells frequencies in peripheral blood might be useful as potential predictive and prognostic biomarkers.

Immune-related adverse events are clustered into distinct subtypes by T-cell profiling before and early after anti-PD-1 treatment.

Although anti-programmed death-1 (PD-1) treatment has shown remarkable anti-tumor efficacy, immune-related adverse events (irAEs) develop with heterogeneous clinical manifestations. However, the immunological understanding of irAEs is currently limited. In the present study, we analyzed peripheral blood T cells obtained from cancer patients who received anti-PD-1 treatment to determine the immunological characteristics of irAEs. This study included 31 patients with refractory thymic epithelial tumor (TET) who were enrolled in a phase II trial of pembrolizumab (NCT02607631) and 60 patients with metastatic non-small cell lung cancer (NSCLC) who received pembrolizumab or nivolumab. T-cell profiling was performed by multicolor flow cytometry using peripheral blood obtained before treatment and 7 days after the first dose of anti-PD-1 antibodies. irAEs developed in 21 TET patients and 24 NSCLC patients. Severe (≥ grade 3) irAEs occurred in 7 TET patients (22.6%) and 6 NSCLC patients (10.0%). Patients with severe irAEs exhibited a significantly lower fold increase in the frequency of effector regulatory T (eTreg) cells after anti-PD-1 treatment, a higher ratio of T helper-17 (Th17) and T helper-1 cells at baseline, and a higher percentage of Ki-67+ cells among PD-1+CD8+ T cells posttreatment. In clustering analysis using the T-cell parameters, patients with irAEs were grouped into four distinct subtypes: Th17-related, TNF-related, CD8-related Treg-compensated, and CD8-related Treg-uncompensated. The T-cell parameters showed a predictive value for the development of each subtype of severe irAEs. In conclusion, severe irAEs after anti-PD-1 treatment were clustered into four immunological subtypes, and potential biomarkers for early prediction of severe irAEs were proposed.

Immunological Characteristics of Hyperprogressive Disease in Patients with Non-small Cell Lung Cancer Treated with Anti-PD-1/PD-L1 Abs.

Hyperprogressive disease (HPD) is a distinct pattern of progression characterized by acceleration of tumor growth after treatment with anti-PD-1/PD-L1 Abs. However, the immunological characteristics have not been fully elucidated in patients with HPD. We prospectively recruited patients with metastatic non-small cell lung cancer treated with anti-PD-1/PD-L1 Abs between April 2015 and April 2018, and collected peripheral blood before treatment and 7-days post-treatment. HPD was defined as ≥2-fold increase in both tumor growth kinetics and tumor growth rate between pre-treatment and post-treatment. Peripheral blood mononuclear cells were analyzed by multi-color flow cytometry to phenotype the immune cells. Of 115 patients, 19 (16.5%) developed HPD, 52 experienced durable clinical benefit (DCB; partial response or stable disease ≥6 months), and 44 experienced non-hyperprogressive progression (NHPD). Patients with HPD had significantly lower progression-free survival (p<0.001) and overall survival (p<0.001). When peripheral blood immune cells were examined, the pre-treatment frequency of CD39+ cells among CD8+ T cells was significantly higher in patients with HPD compared to those with NHPD, although it showed borderline significance to predict HPD. Other parameters regarding regulatory T cells or myeloid derived suppressor cells did not significantly differ among patient groups. Our findings suggest high pre-treatment frequency of CD39+CD8+ T cells might be a characteristic of HPD. Further investigations in a larger cohort are needed to confirm our results and better delineate the immune landscape of HPD.

The First-week Proliferative Response of Peripheral Blood PD-1+CD8+ T Cells Predicts the Response to Anti-PD-1 Therapy in Solid Tumors.

PURPOSE: To investigate blood-based dynamic biomarkers that predict responses to anti-programmed cell death protein 1 (PD-1) therapy in solid tumors. EXPERIMENTAL DESIGN: Preplanned biomarker analysis was performed as part of a phase II clinical trial (NCT02607631) in patients with metastatic or refractory thymic epithelial tumors (TETs; n = 31) who received pembrolizumab. The biomarker was further tested in an independent cohort of prospectively recruited patients with metastatic non-small cell lung cancer (NSCLC) who received pembrolizumab or nivolumab (NSCLC cohort 1; n = 33) and validated in an independent cohort of patients with NSCLC (NSCLC cohort 2; n = 46). Peripheral blood samples were obtained immediately before treatment (D0) and 7 days after the first dose (D7) and analyzed using multi-color flow cytometry. RESULTS: A higher fold-change in the percentage of Ki-67+ cells among PD-1+CD8+ T cells 7 days after the first dose (Ki-67D7/D0) significantly predicted durable clinical benefit (DCB; P < 0.001) and prolonged progression-free survival (PFS; P = 0.027) in patients with TETs. Ki-67D7/D0 ≥ 2.8 was also associated with better DCB, PFS, and overall survival (OS) in NSCLC cohort 1 (all P < 0.05). Ki-67D7/D0 was subsequently validated in NSCLC cohort 2, and Ki-67D7/D0 ≥ 2.8 significantly predicted better DCB (P = 0.001), PFS (P = 0.002), and OS (P = 0.037). Ki-67D7/D0 had a low correlation with tumor PD-L1 expression and combining both factors did not improve the predictive power of Ki-67D7/D0. CONCLUSIONS: The proliferative response of peripheral blood PD-1+CD8+ T cells, measured as the fold-change in the percentage of Ki-67+ cells 7 days after treatment (Ki-67D7/D0), may be a useful surrogate biomarker for predicting the response and prognosis to anti-PD-1 therapy in solid tumors.

Mutational status of TP53 defines the efficacy of Wee1 inhibitor AZD1775 in KRAS-mutant non-small cell lung cancer.

KRAS is frequently mutated in non-small cell lung cancer (NSCLC). However, direct targeting of KRAS has proven to be challenging, and inhibition of KRAS effectors has resulted in limited clinical efficacy. Wee1 kinase is an important regulator of the G2 checkpoint and is overexpressed in various cancers. Inhibition of Wee1 exerts anticancer effects as a monotherapy or in combination with DNA-damaging agents when cancer cells harbor TP53 mutations. However, its role in KRAS-mutant NSCLC, especially as a single agent, has not been explored. Here, we investigate the anticancer potential of Wee1 inhibitor AZD1775 as a monotherapy and uncover a possible cellular context underlying sensitivity to AZD1775. Our data show that treatment with AZD1775 significantly inhibited cell survival, growth, and proliferation of TP53-mutant (TP53MUT) compared to TP53 wild-type (TP53WT) in KRAS-mutant (KRASMUT) NSCLC cells. In KRASMUT/TP53MUT cells, AZD1775 treatment led to DNA damage, a decrease of survival signaling, and cell death by apoptosis. Interestingly, cell death through apoptosis was found to be heavily dependent on specific cellular genetic context, rather than inhibition of Wee1 kinase activity alone. In addition, AZD1775 treatment was well tolerated and displayed single-agent efficacy in a mouse xenograft model. This study provides rationale for inhibiting Wee1 using AZD1775 as a potential anticancer therapy against the TP53MUT subgroup of KRASMUT NSCLC.

AZD9291 overcomes T790 M-mediated resistance through degradation of EGFR(L858R/T790M) in non-small cell lung cancer cells.

The discovery of activating mutations of epidermal growth factor receptor (EGFR) has resulted in the development of more effective treatments for non-small cell lung cancer (NSCLC). Although first-generation EGFR tyrosine kinase inhibitors (EGFR TKIs) provide significant clinical benefit, acquired resistance often occurs, most commonly (>50 %) via a T790 M resistance mutation. Although AZD9291 is selective for both T790 M and activating EGFR mutations over wild-type EGFR, it is highly active when T790 M is present, especially EGFR(L858R/T790M), and modestly active when T790 M is absent. The aim of this study was to elucidate the underlying mechanism of the high sensitivity of NSCLC cells harboring EGFR(L858R/T790M) to AZD9291. In H1975 cells harboring EGFR(L858R/T790M), AZD9291 potently inhibited cellular growth and EGFR signaling pathways together with depletion of mutant EGFR protein. AZD9291-induced depletion of EGFR(L858R/T790M) protein was abrogated through inhibition of the proteasome with MG132. However, AZD9291 had no effect on protein levels of EGFR(WT) and EGFR(L858R). In addition, AZD9291 induced apoptosis and caused expression changes in cell cycle-related genes. Moreover, oral administration of AZD9291 as a single agent induced tumor regression in vivo in a H1975 tumor xenograft model and reduced EGFR(L858R/T790M) protein levels in xenograft tumors. Taken together, our results provide a potential mechanism for the sensitivity of EGFR(L858R/T790M) cells to AZD9291 and suggest that AZD9291 may be effective in cases of T790 M-positive EGFR resistance.

The CDK4/6 inhibitor LY2835219 has potent activity in combination with mTOR inhibitor in head and neck squamous cell carcinoma.

Deletion of CDKN2A (p16) or amplification of CCND1 (cyclin D1) occurs commonly in head and neck squamous cell carcinoma (HNSCC) and induces sustained cyclin-dependent kinase (CDK) 4/6 activation. Here, we report the antiproliferative activity of LY2835219, a selective CDK4/6 inhibitor through inhibition of CDK4/6-dependent Ser780 phosphorylation in retinoblastoma (RB) and induction of cell cycle arrest in HNSCC cells. In addition, we demonstrated the antitumor effects of HNSCC xenografts to LY2835219 in vivo. Given the limited effect in HNSCC as a single-agent treatment with LY2835219, a combinational strategy is required to enhance antitumor activity. At the molecular level, we found that LY2835219 inhibited activation of AKT and ERK, but not mTOR. The combination of LY2835219 with mTOR inhibitor was found to be more effective than either drug alone in vitro and in vivo. Taken together, our findings suggest that a combinational treatment with LY2835219 and mTOR inhibitor is a promising therapeutic approach for HNSCC.

A Phase Ib/II Study of Afatinib in Combination with Nimotuzumab in Non-Small Cell Lung Cancer Patients with Acquired Resistance to Gefitinib or Erlotinib.

PURPOSE: In this phase Ib/II study, we aimed to assess the safety and efficacy of afatinib plus nimotuzumab (N) in advanced non-small cell lung cancer (NSCLC) patients with acquired resistance to gefitinib or erlotinib. EXPERIMENTAL DESIGN: In phase Ib stage, patients received afatinib (40 mg or 30 mg once daily) plus nimotuzumab (100 mg or 200 mg once weekly) for 28-day cycles to determine the recommended phase II dose (RPIID). The safety and efficacy of RPIID dose was evaluated in phase II stage. RESULTS: In total, 50 patients were enrolled (13 to phase Ib and 37 to phase II). In the first dose-finding cohort (afatinib 40 mg plus nimotuzumab 100 mg), one patient experienced dose-limiting toxicity (DLT) of grade 3 diarrhea and in the subsequent cohort (afatinib 40 mg plus nimotuzumab 200 mg), two DLTs (grade 3 diarrhea and grade 3 neutropenia) occurred in 2 of 6 patients. Accordingly, RPIID was determined as afatinib 40 mg plus nimotuzumab 100 mg. In 44 patients treated with RPIID, 7 (16%) patients had grade 3 toxicities; skin rash (7%), diarrhea (5%), acne (2%), and fatigue (2%). The overall response rate was 23% and the median duration of response was 4.3 months (range, 0.7-16.2 months). The median progression-free survival and overall survival were 4.0 months [95% confidence interval (CI), 2.3-5.7 months] and 11.7 months (95% CI, 9.4-14.0 months), respectively. CONCLUSIONS: Combination treatment of afatinib and nimotuzumab demonstrated an acceptable safety profile and encouraging antitumor activity in advanced NSCLC patients with acquired resistance to gefitinib or erlotinib. Larger phase III trial is warranted to confirm its efficacy and safety. Clin Cancer Res; 22(9); 2139-45. ©2015 AACR.

Susceptibility of CD24(+) ovarian cancer cells to anti-cancer drugs and natural killer cells.

Natural killer cells are lymphocytes of the innate immune system that play a key role in the direct elimination of transformed or virus-infected cells. Recently, it has been reported that NK cells can attack cancer cells with stem cell-like properties. In this study, we isolated ovarian cancer cell lines CAOV3 and TOV21G with and without CD24, which has been reported as an ovarian cancer stem cell marker, and compared their drug resistance and susceptibility to NK cell lysis. The isolated CD24(+) CAOV3 and TOV21G cells were more resistant to cisplatin and doxorubicin anti-cancer drugs. Also, CD24(+) CAOV3 and TOV21G cells were more susceptible to NK cell lysis compared with CD24(-) cells. In order to identify reasons for the differing NK cell susceptibility, we examined NK cell-killing mechanisms against CD24(+) cancer cell lines by analyzing NKG2D ligands, MHC class I molecules, and natural cytotoxic receptor ligands expression on target cells. Consistently, CD24(+) CAOV3 and TOV21G cells showed up-regulated NKG2D ligands and down-regulated MHC class I molecule expression. These findings show that CD24(+) ovarian cancer cell lines are more resistant to antitumor drugs but are more susceptible to NK cell lysis; thus, NK cell immunotherapy might be useful in eliminating ovarian cancer stem cells and preventing tumor recurrence and metastasis.