Edward E. Kadel

The antileukemia activity of a human anti-CD40 antagonist antibody, HCD122, on human chronic lymphocytic leukemia cells

B-cell chronic lymphocytic leukemia (B-CLL) is a lymphoproliferative disorder characterized by the surface expression of CD20, CD5 antigens, as well as the receptor CD40. Activation of CD40 by its ligand (CD40L) induces proliferation and rescues the cells from spontaneous and chemotherapy-induced apoptosis. CD40 activation also induces secretion of cytokines, such as IL-6, IL-10, TNF-alpha, IL-8, and GM-CSF, which are involved in tumor cell survival, migration, and interaction with cells in the tumor microenvironment. Here we demonstrate that in primary B-CLL tumor cells, the novel antagonist anti-CD40 monoclonal antibody, HCD122, inhibits CD40L-induced activation of signaling pathways, proliferation and survival, and secretion of cytokines. Furthermore, HCD122 is also a potent mediator of antibody-dependent cellular cytotoxicity (ADCC), lysing B-CLL cells more efficiently than rituximab in vitro, despite a significantly higher number of cell surface CD20 binding sites compared with CD40. Unlike rituximab, however, HCD122 (formerly CHIR-12.12) does not internalize upon binding to the cells. Our data suggest that HCD122 may inhibit B-CLL growth by blocking CD40 signaling and by ADCC-mediated cell lysis.

TGFβ attenuates tumour response to PD-L1 blockade by contributing to exclusion of T cells

Therapeutic antibodies that block the programmed death-1 (PD-1)–programmed death-ligand 1 (PD-L1) pathway can induce robust and durable responses in patients with various cancers, including metastatic urothelial cancer. However, these responses only occur in a subset of patients. Elucidating the determinants of response and resistance is key to improving outcomes and developing new treatment strategies. Here we examined tumours from a large cohort of patients with metastatic urothelial cancer who were treated with an anti-PD-L1 agent (atezolizumab) and identified major determinants of clinical outcome. Response to treatment was associated with CD8+ T-effector cell phenotype and, to an even greater extent, high neoantigen or tumour mutation burden. Lack of response was associated with a signature of transforming growth factor β (TGFβ) signalling in fibroblasts. This occurred particularly in patients with tumours, which showed exclusion of CD8+ T cells from the tumour parenchyma that were instead found in the fibroblast- and collagen-rich peritumoural stroma; a common phenotype among patients with metastatic urothelial cancer. Using a mouse model that recapitulates this immune-excluded phenotype, we found that therapeutic co-administration of TGFβ-blocking and anti-PD-L1 antibodies reduced TGFβ signalling in stromal cells, facilitated T-cell penetration into the centre of tumours, and provoked vigorous anti-tumour immunity and tumour regression. Integration of these three independent biological features provides the best basis for understanding patient outcome in this setting and suggests that TGFβ shapes the tumour microenvironment to restrain anti-tumour immunity by restricting T-cell infiltration.

Atezolizumab versus chemotherapy in patients with platinum-treated locally advanced or metastatic urothelial carcinoma (IMvigor211): a multicentre, open-label, phase 3 randomised controlled trial

BACKGROUND Few options exist for patients with locally advanced or metastatic urothelial carcinoma after progression with platinum-based chemotherapy. We aimed to assess the safety and efficacy of atezolizumab (anti-programmed death-ligand 1 [PD-L1]) versus chemotherapy in this patient population. METHODS We conducted this multicentre, open-label, phase 3 randomised controlled trial (IMvigor211) at 217 academic medical centres and community oncology practices mainly in Europe, North America, and the Asia-Pacific region. Patients (aged ≥18 years) with metastatic urothelial carcinoma who had progressed after platinum-based chemotherapy were randomly assigned (1:1), via an interactive voice and web response system with a permuted block design (block size of four), to receive atezolizumab 1200 mg or chemotherapy (physicians choice: vinflunine 320 mg/m2, paclitaxel 175 mg/m2, or 75 mg/m2 docetaxel) intravenously every 3 weeks. Randomisation was stratified by PD-L1 expression (expression on <1% [IC0] or 1% to <5% [IC1] of tumour-infiltrating immune cells vs ≥5% of tumour-infiltrating immune cells [IC2/3]), chemotherapy type (vinflunine vs taxanes), liver metastases (yes vs no), and number of prognostic factors (none vs one, two, or three). Patients and investigators were aware of group allocation. Patients, investigators, and the sponsor were masked to PD-L1 expression status. The primary endpoint of overall survival was tested hierarchically in prespecified populations: IC2/3, followed by IC1/2/3, followed by the intention-to-treat population. This study, which is ongoing but not recruiting participants, is registered with ClinicalTrials.gov, number NCT02302807. FINDINGS Between Jan 13, 2015, and Feb 15, 2016, we randomly assigned 931 patients from 198 sites to receive atezolizumab (n=467) or chemotherapy (n=464). In the IC2/3 population (n=234), overall survival did not differ significantly between patients in the atezolizumab group and those in the chemotherapy group (median 11·1 months [95% CI 8·6-15·5; n=116] vs 10·6 months [8·4-12·2; n=118]; stratified hazard ratio [HR] 0·87, 95% CI 0·63-1·21; p=0·41), thus precluding further formal statistical analysis. Confirmed objective response rates were similar between treatment groups in the IC2/3 population: 26 (23%) of 113 evaluable patients had an objective response in the atezolizumab group compared with 25 (22%) of 116 patients in the chemotherapy group. Duration of response was numerically longer in the atezolizumab group than in the chemotherapy group (median 15·9 months [95% CI 10·4 to not estimable] vs 8·3 months [5·6-13·2]; HR 0·57, 95% CI 0·26-1·26). In the intention-to-treat population, patients receiving atezolizumab had fewer grade 3-4 treatment-related adverse events than did those receiving chemotherapy (91 [20%] of 459 vs 189 [43%] of 443 patients), and fewer adverse events leading to treatment discontinuation (34 [7%] vs 78 [18%] patients). INTERPRETATION Atezolizumab was not associated with significantly longer overall survival than chemotherapy in patients with platinum-refractory metastatic urothelial carcinoma overexpressing PD-L1 (IC2/3). However, the safety profile for atezolizumab was favourable compared with chemotherapy, Exploratory analysis of the intention-to-treat population showed well-tolerated, durable responses in line with previous phase 2 data for atezolizumab in this setting. FUNDING F Hoffmann-La Roche, Genentech.

Learning and confirming with preclinical studies: modeling and simulation in the discovery of GDC-0917, an inhibitor of apoptosis proteins antagonist.

The application of modeling and simulation techniques is increasingly common in the preclinical stages of the drug development process. GDC-0917 [(S)-1-((S)-2-cyclohexyl-2-((S)-2-(methylamino)propanamido)acetyl)-N-(2-(oxazol-2-yl)-4-phenylthiazol-5-yl)pyrrolidine-2-carboxamide] is a potent second-generation antagonist of inhibitor of apoptosis (IAP) proteins that is being developed for the treatment of various cancers. GDC-0917 has low to moderate clearance in the mouse (12.0 ml/min/kg), rat (27.0 ml/min/kg), and dog (15.3 ml/min/kg), and high clearance in the monkey (67.6 ml/min/kg). Accordingly, oral bioavailability was lowest in monkeys compared with other species. Based on our experience with a prototype molecule with similar structure, in vitro–in vivo extrapolation was used to predict a moderate clearance (11.5 ml/min/kg) in humans. The predicted human volume of distribution was estimated using simple allometry at 6.69 l/kg. Translational pharmacokinetic-pharmacodynamic (PK-PD) analysis using results from MDA-MB-231-X1.1 breast cancer xenograft studies and predicted human pharmacokinetics suggests that ED50 and ED90 targets can be achieved in humans using acceptable doses (72 mg and 660 mg, respectively) and under an acceptable time frame. The relationship between GDC-0917 concentrations and pharmacodynamic response (cIAP1 degradation) was characterized using an in vitro peripheral blood mononuclear cell immunoassay. Simulations of human GDC-0917 plasma concentration-time profile and cIAP1 degradation at the 5-mg starting dose in the phase 1 clinical trial agreed well with observations. This work shows the importance of leveraging information from prototype molecules and illustrates how modeling and simulation can be used to add value to preclinical studies in the early stages of the drug development process.

Transcriptional-Based Screens for Pathway-Specific, High-Throughput Target Discovery in Endothelial Cells

With the sequence of the human genome at hand, target discovery strategies are needed that can rapidly identify novel gene products involved in human disease pathways. In this article, the authors describe a cell-based, high-throughput assay that can identify gene products capable of modulating the vascular endothelial growth factor (VEGF) and tumor necrosis factor • (TNFa) signaling pathways in human endothelial cells. The assay uses real-time PCRtechnology tomeasure downstreamreporter mRNA transcripts induced upon cytokine stimulation in a 96-well plate format and has been adapted for use with recombinant adenoviruses. The authors specifically demonstratemodulation of cytokine-driven reporter transcripts using drug inhibitors and through adenoviral-mediated expression of known signaling intermediates of the respective pathways. In addition, they have used an arrayed library of 350 recombinant adenoviruses to screen for novel modulators of the VEGF and TNF• pathways. The high-throughput screening capacity and sensitivity of this system make it a useful tool for new drug target identification.

Immune biomarkers associated with clinical benefit from atezolizumab (MPDL3280a; anti-PD-L1) in advanced urothelial bladder cancer (UBC)

Meeting abstracts Atezolizumab (anti-PD-L1) has demonstrated robust clinical activity in UBC [[1][1]]. Elevated PD-L1 expression on tumor-infiltrating immune cells (IC) is associated with increased clinical efficacy; however, the contribution of other immune biomarkers is unknown. In this study, we

Abstract 4978: Lung cancers regulate the immune suppressor PD-L1 by multiple mechanisms, altering its role in tumor survival.

The immune system has multiple mechanisms by which it can eliminate cancer and yet, tumors are able to adapt resistance to host immune surveillance and continue to survive and grow. One critical resistance mechanism involves Programmed cell death 1 ligand 1 (PD-L1, CD274, B7-H1), the predominant ligand for PD-1, an inhibitory receptor expressed on T cells following activation. PD-L1 also binds to B7.1 (CD80), inhibiting its ability to provide an immune stimulatory signal. PD-L1 is expressed broadly on multiple peripheral blood mononuclear cell subtypes, placenta, and numerous cancers, including NSCLC. Tumor-specific T cells infiltrate tumors and recognize tumor cells, releasing Interferon-gamma (IFNγ), initiating signaling of the Janus kinase/Signal Transducer and Activator of Transcription (JAK/STAT) pathway in the tumor cells. IFNγ induces and/or greatly enhances the expression of PD-L1 in the tumor, among other cell types, allowing the tumor to become resistant to the host T cell response. Blockade of PD-L1 binding to PD-1 and B7.1 can reinvigorate the host immune response against the tumor and overcome tumor adaptive resistance. The regulation of PD-L1 expression is complex and likely involves multiple types of pre- and post-translational events. We show that basal PD-L1 expression levels can vary greatly in cancer cells, as can PD-L1 induction by IFNγ. Here we describe the different categories of PD-L1 basal expression and IFNγ–dependent regulation across multiple lung cell lines and human tumor samples. Reverse Phase Protein Array and RNA microarray data show that the JAK/STAT canonical pathways are still intact among all of these distinct categories, so alternative mechanisms of expression regulation must be active in these cell lines and tumor samples. We provide evidence that a combination of mechanisms regulate both the basal and stimulated expression levels of PD-L1 across these distinct categories. This data regarding PD-L1 expression regulation provides valuable information to better understand the PD-1/PD-L1 pathway as a therapeutic target. Citation Format: Edward (Ward) E. Kadel, Kimberly Walter, Rupal Desai, Juliet Carbon, Marigold Boe, David Shames, Marcin Kowanetz. Lung cancers regulate the immune suppressor PD-L1 by multiple mechanisms, altering its role in tumor survival. [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 4978. doi:10.1158/1538-7445.AM2013-4978

Differential regulation of PD-L1 expression by immune and tumor cells in NSCLC and the response to treatment with atezolizumab (anti–PD-L1)

Significance Programmed death-ligand 1 (PD-L1) expression on tumor cells and tumor-infiltrating immune cells is regulated by distinct mechanisms and has nonredundant roles in regulating anticancer immunity, and PD-L1 on both cell types is important for predicting best response to atezolizumab in non-small cell lung cancer. Programmed death-ligand 1 (PD-L1) expression on tumor cells (TCs) by immunohistochemistry is rapidly gaining importance as a diagnostic for the selection or stratification of patients with non-small cell lung cancer (NSCLC) most likely to respond to single-agent checkpoint inhibitors. However, at least two distinct patterns of PD-L1 expression have been observed with potential biological and clinical relevance in NSCLC: expression on TC or on tumor-infiltrating immune cells (ICs). We investigated the molecular and cellular characteristics associated with PD-L1 expression in these distinct cell compartments in 4,549 cases of NSCLC. PD-L1 expression on IC was more prevalent and likely reflected IFN-γ–induced adaptive regulation accompanied by increased tumor-infiltrating lymphocytes and effector T cells. High PD-L1 expression on TC, however, reflected an epigenetic dysregulation of the PD-L1 gene and was associated with a distinct histology described by poor immune infiltration, sclerotic/desmoplastic stroma, and mesenchymal molecular features. Importantly, durable clinical responses to atezolizumab (anti–PD-L1) were observed in patients with tumors expressing high PD-L1 levels on either TC alone [40% objective response rate (ORR)] or IC alone (22% ORR). Thus, PD-L1 expression on TC or IC can independently attenuate anticancer immunity and emphasizes the functional importance of IC in regulating the antitumor T cell response.

Abstract A017: PD-L1 as a predictive biomarker for atezolizumab (MPDL3280A; anti-PDL1) in non-small cell lung cancer (NSCLC)

Background: Programmed death ligand-1 (PD-L1), a ligand for PD-1 and B7.1, is broadly expressed on tumor cells (TC) and tumor-infiltrating immune cells (IC) in many human cancers. PD-L1 expression on either TC or IC can negatively regulate antitumor T-cell function within the tumor microenvironment (TME). Consistent with this, the ORR, PFS and OS benefit of atezolizumab (atezo) across PhI and PhII studies appeared to correlate with increasing baseline PD-L1 expression levels on TC and/or IC. Therefore, we explored the biologic reasons for PD-L1 expression on TC and IC, the association with response to atezo and the intrapatient heterogeneity of PD-L1 expression in NSCLC. Methods: Tumor specimens were obtained from patients (pts) prescreened and/or enrolled in NSCLC atezo trials (PhI PCD4989g, PhII POPLAR and FIR [n=1360]) and from pts treated at MSKCC (n=39). Samples included 14 synchronous and 106 metachronous pairs collected in FIR or at MSKCC. Using the SP142 IHC assay, which has been optimized to detect PD-L1 on both TC and IC, PD-L1 expression was scored at 4 levels (TC0-3 and IC0-3) based on increasing expression. A subset of samples was further characterized by histopathologic review and gene expression by RNAseq. CD8 expression (clone C8/144B) was assessed in the tumor center, invasive margin and periphery by IHC. Results: PD-L1 was expressed on IC only, on TC only or on both TC and IC within the TME. Tumors with the highest (TC3 or IC3), moderate/high (TC2/3 or IC2/3) and any (TC1/2/3 or IC1/2/3) PD-L1 expression represented ≈15%, ≈38% and ≈70% of NSCLC, respectively. PD-L1 expression was similar across all paired synchronous and metachronous tissues. At the TC3 or IC3 cutoff, PD-L1 status remained unchanged in 86% of paired synchronous specimens and in 78% of metachronous pairs. Analysis of PD-L1 expression patterns revealed the existence of exclusive TC and IC subpopulations at each PD-L1 expression level, unique to NSCLC and not seen in other cancers, e.g. UBC. Strikingly, TC3 and IC3 tumors represented 2 distinct populations, with Conclusions: These data demonstrated that NSCLC has unique PD-L1 expression patterns. High expression of PD-L1 on TC and/or IC in NSCLC confers sensitivity to atezo, despite exhibiting distinct immunologic profiles. These results further our understanding of how atezo promotes responses in tumors expressing PD-L1 on TC and/or IC and emphasizes the need to assess PD-L1 on both TC and IC in NSCLC. In addition, intrapatient heterogeneity in PD-L1 expression was relatively low in both synchronous and metachronous tissues, indicating that various types of tumor samples (e.g. primary or metastatic, fresh or archival) can be reliably used to assess PD-L1 expression with the SP142 assay. Citation Format: Marcin Kowanetz, Hartmut Koeppen, Wei Zou, Sanjeev Mariathasan, Matthew Hellmann, Mark Kockx, Colombe Chappey, Edward Kadel, Dustin Smith, Natasha Miley, Vincent Leveque, Roel Funke, Alan Sandler, Ian McCaffery, Lukas Amler, Daniel Chen, Priti Hegde. PD-L1 as a predictive biomarker for atezolizumab (MPDL3280A; anti-PDL1) in non-small cell lung cancer (NSCLC). [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A017.

Abstract 4409: A quantitative MSD assay to measure c-IAP1 degradation in PBMC, cell lines and tumor cells as a pharmacodynamic biomarker following antagonism of IAP family members

The Inhibitor of Apoptosis (IAP) protein family members are widely expressed in many cancers, providing a mechanism to protect these cancer cells from apoptosis. The IAP proteins block activation of the canonical and non-canonical NF-kB pathway, the subsequent expression of TNFα and its initiation of the extrinsic apoptosis pathway. IAP proteins also directly bind and inhibit caspases activated during the induction of apoptosis. Using MDA-MB-231 cells as a model system to assess the effect of candidate IAP antagonists on markers of extrinsic and intrinsic pathway inhibition, we observe that the antagonism of the major IAP family members results in apoptosis in vitro and inhibits their tumor growth in nude mice in vivo. These effects are coincident with changes in the cellular levels of IAP family members such as c-IAP1, detected by Western analysis. These data are consistent with previously described autoubiquitination and subsequent proteasomal degradation of c-IAP1 following IAP antagonism. We have developed a high-throughput, quantifiable and sensitive assay to measure c-IAP1 levels in multiple cell lines, xenografts and in PBMCs using the MesoScale Discovery (MSD) Platform. The c-IAP1 MSD assay has a lower limit of quantification of 1.6 ng/ml of cell lysate, has precision of Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4409. doi:10.1158/1538-7445.AM2011-4409