The interleukin (IL)-23/T helper (Th)17 axis plays a critical role in autoimmune diseases, and there is an increasing number of biologic therapies that target IL-23 and IL-17. The transcription factor retinoic acid receptor-related orphan nuclear receptor γt (RORγt) is important for the activation and differentiation of Th17 cells and thus is an attractive pharmacologic target for the treatment of Th17-mediated diseases. A novel series of pyrazinone RORγ antagonists was discovered through hybridization of two distinct screening hits and scaffold hopping. The series offers attractive potency and selectivity in combination with favorable druglike properties, such as metabolic stability and aqueous solubility. Lead optimization identified a clinical candidate, compound (S)-11 (BI 730357), for the treatment of autoimmune diseases.

Dysregulated IL-23/IL-17 responses have been linked to psoriatic arthritis and other forms of spondyloarthritides (SpA). RORγt, the key Thelper17 (Th17) cell transcriptional regulator, is also expressed by subsets of innate-like T cells, including invariant natural killer T (iNKT) and γδ-T cells, but their contribution to SpA is still unclear. Here we describe the presence of particular RORγt+T-betloPLZF- iNKT and γδ-hi T cell subsets in healthy peripheral blood. RORγt+ iNKT and γδ-hi T cells show IL-23 mediated Th17-like immune responses and were clearly enriched within inflamed joints of SpA patients where they act as major IL-17 secretors. SpA derived iNKT and γδ-T cells showed unique and Th17-skewed phenotype and gene expression profiles. Strikingly, RORγt inhibition blocked γδ17 and iNKT17 cell function while selectively sparing IL-22+ subsets. Overall, our findings highlight a unique diversity of human RORγt+ T cells and underscore the potential of RORγt antagonism to modulate aberrant type 17 responses.

Exploring various cyclization strategies, using a submicromolar pyrazole HTS screening hit 6 as a starting point, a novel indazole based CCR1 antagonist core was discovered. This report presents the design and SAR of CCR1 indazole and azaindazole antagonists leading to the identification of three development compounds, including 19e that was advanced to early clinical trials.

Background and Aims: Despite the negative results of blocking IL-17 in Crohn's disease (CD) patients, selective modulation of Th17-dependent responses warrants further study. Inhibition of retinoic acid-related orphan receptor gamma (RORγt), the master regulator of the Th17 signature, is currently being explored in inflammatory diseases. Our aim was to determine the effect of a novel oral RORγt antagonist (BI119) in human CD and on an experimental model of intestinal inflammation. Methods: 51 CD patients and 11 healthy subjects were included. The effects of BI119 were tested on microbial-stimulated peripheral blood mononuclear cells (PBMCs), intestinal crypts and biopsies from CD patients. The ability of BI119 to prevent colitis in vivo was assessed in the CD4+CD45RBhigh T cell transfer model. Results: In bacterial antigen-stimulated PBMCs from CD patients, BI119 inhibits Th17-related genes and proteins, while upregulating Treg and preserving Th1 and Th2 signatures. Intestinal crypts cultured with supernatants from BI119-treated commensal-specific CD4+ T cells showed decreased expression of CXCL1, CXCL8 and CCL20. BI119 significantly reduced IL17 and IL26 transcription in colonic and ileal CD biopsies and did not affect IL22. BI119 has a more profound effect in ileal CD with additional significant downregulation of IL23R, CSF2, CXCL1, CXCL8, and S100A8, and upregulation of DEFA5. BI119 significantly prevented development of clinical, macroscopic and molecular markers of colitis in the T-cell transfer model. Conclusions: BI119 modulated CD-relevant Th17 signatures, including downregulation of IL23R while preserving mucosa-associated IL-22 responses, and abrogated experimental colitis. Our results provide support to the use of RORγt antagonists as a novel therapy to CD treatment.

Background: Ventana Medical Systems, Inc. is developing in collaboration with Genentech, Inc., an immunohistochemical (IHC) companion diagnostic to aid in selecting patients with metastatic castration-resistant prostate cancer (mCRPC) that may benefit from ipatasertib, an Akt inhibitor therapy. The phosphatase and tensin (PTEN) protein plays an important role in controlling cell survival and cell cycle progression as a negative regulator of the phosphoinositide 3-kinase/AKT pathway. Methods: PTEN (SP218) rabbit monoclonal primary antibody was optimized for use with the VENTANA OptiView DAB IHC Detection Kit on the automated BenchMark ULTRA platform (PTEN (SP218) RxDx Assay). The PTEN (SP218) RxDx Assay was developed for use in formalin-fixed, paraffin-embedded tissue samples of mCRPC in a series of studies addressing sensitivity, specificity, robustness and precision. The scoring algorithm was defined using statistical analysis of clinical outcome data analysis, and the PTEN (SP218) RxDx Assay staining pattern and prevalence of PTEN loss of expression in a set of tissue samples. Inter-reader precision was established by 3 pathologists evaluating 90 mCRPC samples across the range of PTEN expression levels. Results: The Ventana PTEN (SP218) RxDx Assay met all pre-defined acceptance criteria. mCRPC tissue samples are designated with PTEN-Loss status when ≥ 50% of viable malignant cells have no specific cytoplasmic staining with PTEN (SP218) in the presence of acceptable internal controls. Inter-reader precision in determining PTEN status resulted in agreement rates greater than 97%. Conclusion: These results highlight the robustness and reproducibility of the Ventana PTEN (SP218) RxDx Assay. In the clinical outcome analysis, patients identified to have PTEN-loss status demonstrated clinically meaningful improvements when treated with ipatasertib. The clinical utility of the PTEN (SP218) RxDx assay will be further validated in additional patients in subsequent ipatasertib studies, including a Phase 3 study (IPATential150) beginning in 2017. Citation Format: Crystal Stephens, Erica Harnish, Rebecca Bowermaster, Azita Djalilvand, Dustin Smith, Doris Kim, Steven Gendreau, Edmundo Del Valle. Development of a companion diagnostic assay for the detection of phosphatase and tensin (PTEN) protein loss and treatment with ipatasertib in metastatic castration-resistant prostatic cancer (mCRPC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4531.

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.

Cancer immunotherapies, such as atezolizumab, are proving to be a valuable therapeutic strategy across indications, including non–small cell lung cancer (NSCLC) and urothelial cancer (UC). Here, we describe a diagnostic assay that measures programmed-death ligand 1 (PD-L1) expression, via immunohistochemistry, to identify patients who will derive the most benefit from treatment with atezolizumab, a humanized monoclonal anti-PD-L1 antibody. We describe the performance of the VENTANA PD-L1 (SP142) Assay in terms of specificity, sensitivity, and the ability to stain both tumor cells (TC) and tumor-infiltrating immune cells (IC), in NSCLC and UC tissues. The reader precision, repeatability and intermediate precision, interlaboratory reproducibility, and the effectiveness of pathologist training on the assessment of PD-L1 staining on both TC and IC were evaluated. We detail the analytical validation of the VENTANA PD-L1 (SP142) Assay for PD-L1 expression in NSCLC and UC tissues and show that the assay reliably evaluated staining on both TC and IC across multiple expression levels/clinical cut-offs. The reader precision showed high overall agreement when compared with consensus scores. In addition, pathologists met the predefined training criteria (≥85.0% overall percent agreement) for the assessment of PD-L1 expression in NSCLC and UC tissues with an average overall percent agreement ≥95.0%. The assay evaluates PD-L1 staining on both cell types and is robust and precise. In addition, it can help to identify those patients who may benefit the most from treatment with atezolizumab, although treatment benefit has been demonstrated in an all-comer NSCLC and UC patient population.

Lupus nephritis (LN) patients currently lack highly effective and safe treatment options. Bruton’s tyrosine kinase (BTK) is a tyrosine kinase which is important for B cell and macrophage function. Based on preliminary studies in an inducible model of nephritis that highlighted the importance of BTK dependent macrophage effector function in the pathogenesis of disease, we extended our studies to explore the effect of BTK inhibition on remission induction of established, proliferative nephritis. Nephritis was induced in female 129 sv/J mice (10 weeks of age) via the passive transfer of nephrotoxic serum (NTS). Once a mouse developed severe nephritis (>300 mg/dl proteinuria for two consecutive days, or a single measurement of >2000 mg/dl), treatment begun via daily oral gavage with 3 mg/kg of BI-BTK-1, a novel, highly selective and potent BTK inhibitor. Each treated mouse was matched to a control mouse that received daily gavage of the vehicle alone. Within two days of beginning treatment, mice treated with BI-BTK-1 had a significant reversal of the proteinuria compared to vehicle control treated mice (p<0.05). Terminal urine revealed a significantly reduced A:C ratios (p <0.001). Additionally, terminal serum revealed normalized BUN levels. Histological assessment revealed significant structural protection in the treated compared to control treated mice, including both glomerular (p<0.001) and tubular compartments (p < 0.001). Kidney RT-PCR and RNA-seq analysis are currently in progress. Therapeutic treatment with BI-BTK-1 reversed severe, established nephritis induced by pathogenic antibodies. These exciting results indicate the novel therapeutic potential of BTK inhibition for treating LN patients with established disease.

Methods: A mechanism-based pharmacokinetic-target occupancy (PK-TO) model of a novel, highly selective, irreversible BTK inhibitor was developed based on non-clinical and clinical findings reported in the literature or generated in house. The model was trained with published clinical PK and TO data sets for ibrutinib and CC-292, and further validated with pre-clinical in-vivo observations for in-house compounds including a highly selective development candidate, BI-BTK-1.

Lupus nephritis (LN) is a potentially dangerous end organ pathology that affects upwards of 60% of lupus patients. Bruton's tyrosine kinase (BTK) is important for B cell development, Fc receptor signaling, and macrophage polarization. In this study, we investigated the effects of a novel, highly selective and potent BTK inhibitor, BI-BTK-1, in an inducible model of LN in which mice receive nephrotoxic serum (NTS) containing anti-glomerular antibodies. Mice were treated once daily with vehicle alone or BI-BTK-1, either prophylactically or therapeutically. When compared with control treated mice, NTS-challenged mice treated prophylactically with BI-BTK-1 exhibited significantly attenuated kidney disease, which was dose dependent. BI-BTK-1 treatment resulted in decreased infiltrating IBA-1+ cells, as well as C3 deposition within the kidney. RT-PCR on whole kidney RNA and serum profiling indicated that BTK inhibition significantly decreased levels of LN-relevant inflammatory cytokines and chemokines. Renal RNA expression profiling by RNA-seq revealed that BI-BTK-1 dramatically modulated pathways related to inflammation and glomerular injury. Importantly, when administered therapeutically, BI-BTK-1 reversed established proteinuria and improved renal histopathology. Our results highlight the important role for BTK in the pathogenesis of immune complex-mediated nephritis, and BTK inhibition as a promising therapeutic target for LN.

11590 Background: Expression of PD-L1 in tumor cells (TC) and infiltrating immune cells (IC) allows TCs to escape from host’s antitumor immunity by inducing tolerance among tumor-specific T cells. Treatment with the anti-PD-L1 agent atezolizumab has been shown to result in significantly improved outcomes in patients with advanced urothelial bladder cancer (UBC) and non-small cell lung cancer (NSCLC). Higher levels of PD-L1-expression have been associated with a greater efficacy. An ideal assessment of TCs and ICs in patients with anti-PD-L1 treatment requires simultaneous immunohistochemistry (IHC) detection of multiple biomarkers. Methods: A fully automated 5-Plex fluorescence IHC assay was developed on the BenchMark ULTRA automated slide stainer using rabbit monoclonal antibodies PD-L1 (SP142), CD3, CD8, CD68 and FoxP3 by tyramide signal amplification. The stained slides were scanned with Zeiss AXIO Z1, and analyzed with in-house digital image analysis tools. Results: A cohort of archived NSCLC and UBC specimens were evaluated for PD-L1 positive staining in tumor areas or on tumor infiltrating immune cells. The workflow consisted of spectral unmixing to determine marker colocalization, and followed by detection of individual cells within the tumor region and classifying them into tumor or one of the immune phenotypes through a supervised machine learning algorithm trained on expert-provided examples. The spatial relationship of immune and PD-L1-positive tumor cells was quantitatively explained using descriptive statistics of the distances of immune cells to the closest PD-L1 positive tumor and PD-L1 positive immune cells, and density of different immune cell phenotypes within the tumor and peri-tumoral regions. Hot and cold spots of immune cell makeup were also identified in the heat maps of immune cells within the tumor region. Conclusions: The fully automated 5-Plex IHC assay together with its respective digital analysis could serve as a tool for further characterizing tumors and their microenvironment and provide a better understanding of which patients may benefit from immune-therapies.

Abstract 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 <1% overlap, each benefiting from atezo. In POPLAR, ORR in TC3 and IC3 subgroups was 40% and 30%, respectively, vs 14.6% in all pts treated with atezo. IC3 tumors had a high frequency of immune infiltrates, including CD8+ T cells, localized in the intra-epithelium, epithelial/stroma interface and stroma. These tumors also exhibited high expression of genes associated with effector T cells, consistent with PD-L1 regulated by an adaptive IFNγ-driven mechanism. However, high infiltration of CD8+ T cells within the tumor at baseline was not associated with response to atezo (P=.39), suggesting that the mechanism of response is not exclusively due to adaptive antitumor T-cell immunity. In contrast, TC3 tumors had distinct histopathologic characteristics, with a dense desmoplastic and sclerotic TME and low intratumoral CD8 infiltrate. PD-L1 on TC appeared to be regulated by intrinsic tumor mechanisms, including promoter methylation. TC0 and IC0 tumors (lowest/no PD-L1 expression; ≈30% of NSCLC) showed little/no evidence of immune infiltration or activation, consistent with immunologic ignorance. 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 Cancers may escape immune surveillance and eradication through the expression of programmed death-ligand 1 (PD-L1) on tumor cells and in the tumor microenvironment. PD-L1 expression has been reported in various cell populations within the tumor, and its expression associated with prognosis for various tumors. Further, clinical studies have shown that this pathway is an important target for immunotherapy and PD-L1 expression on tumor cells and in the tumor microenvironment has been associated with enhanced response. Understanding PD-L1's complex biological function not only on tumor cells but also within the tumor microenvironment requires simultaneous interrogation of multiple biomarkers, ranging from cancer immunology checkpoint markers, tumor infiltrating immune cell markers, and tumor specific markers, etc. Multiplex immunohistochemistry (IHC) allows simultaneous detection of multiple markers to explore the potential cellular composition of immune/stromal/cancer cells in tumor microenvironment. Development of a multiplex IHC assay remains challenging due to antibody species similarity and cross reactivity, stability of fluorophores through multiple rounds of processing, balancing high and low signals and measurement of weakly expressed markers. We present here the development of a fully automated multiplex IHC assay (PD-L1, CD3, CD8, CD68 and FoxP3) using rabbit primary antibodies with a heat deactivation process between each antigen staining cycles on the BenchMark ULTRA automated slide stainer. As part of the technology validation, we compared the 5-plex IHC to the respective single-plex chromogenic IHC assays. Using the automated 5-plex fluorescent IHC assay, we tested a cohort of non-small cell lung (NSCLC) and bladder cancer tissue specimens and characterized PD-L1 and immune marker expression in both tumor and infiltrate immune cells. To provide an objective and reliable readout of the assay, image analysis tools are being developed for automated identification and quantification of the labelled biomarkers and their co-expression on a cell-by-cell basis. This automated multiplex PD-L1 5-Plex IHC assay could be utilized as a tool for further characterizing tumors and its microenvironment and gain a better understanding of which patients may benefit from immune-therapies. Citation Format: Wenjun Zhang, Antony Hubbard, Adriana Racolta, Nick Cummins, Mehrnoush Khojasteh, Liping Zhang, Karl Garsha, Joerg Bredno, Dustin Harshman, Srabani Bhaumik, Tobin Jones, Marcin Kowanetz, Sanjeev Mariathasan, Ian McCaffery, Dustin Smith, J Andrew Williams, Lidija Pestic-Dragovich, Larry Morrison, Lei Tang. An automated 5-plex fluorescent immunohistochemistry enabled characterization of PD-L1 expression and tumor infiltrating immune cells in lung and bladder cancer specimens. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5117.

IMPORTANCE Dysregulation of the mesenchymal-epithelial transition (MET) signaling pathway is associated with poor prognosis in gastroesophageal adenocarcinoma (GEC). We report results of METGastric, a phase 3 trial of the MET inhibitor onartuzumab plus standard first-line chemotherapy for human epidermal growth factor receptor 2 (HER2)-negative, MET-positive, advanced GEC.OBJECTIVE To determine whether the addition of onartuzumab to first-line fluorouracil, leucovorin, and oxaliplatin (mFOLFOX6) improves efficacy compared with mFOLFOX6 plus placebo in HER2-negative, MET-positive GEC.DESIGN, SETTING, AND PARTICIPANTS Randomized, double-blind, multicenter trial conducted from November 2012 to March 2014. Patients were 18 years or older with an adenocarcinoma of the stomach or gastroesophageal junction with metastatic disease not amenable for curative therapy. Tumor samples were centrally tested for MET expression using Ventana anti-Total c-MET (SP44) rabbit monoclonal antibody, HER2 status, and Lauren histologic subtype. MET-positive tumors were defined as at least 50% of tumor cells showing weak, moderate, and/or strong staining intensity (MET 1+/2+/3+, respectively) by immunohistochemistry.INTERVENTIONS Patients with HER2-negative, MET-positive GEC were enrolled and randomized 1: 1 to receivem FOLFOX6 with or without onartuzumab (10mg/kg).MAIN OUTCOMES AND MEASURES Co-primary end points: overall survival in the intent-to-treat (ITT) population and in patients with MET 2+/3+ GEC. Secondary end points: progression-free survival (PFS), overall response rate (ORR), and safety.RESULTS Enrollment was stopped early due to sponsor decision, which was agreed with an independent data monitoring committee. At the data cutoff (April 25, 2014) there were 562 patients in the ITT population (n = 283 placebo plus mFOLFOX6 [median age, 58 y; 65% male]; n = 279 onartuzumab plus mFOLFOX6 [median age, 60 y; 67% male]); 109 (38.5%) and 105 (37.6%) of the ITT population were MET 2+/3+, respectively. Addition of onartuzumab to mFOLFOX6 did not significantly improve OS, PFS, or ORR vs placebo plus mFOLFOX6 in the ITT (OS hazard ratio [HR], 0.82; 95% CI, 0.59-1.15; P =.24; PFS HR, 0.90; 95% CI, 0.71-1.16; P =.43; ORR, 46.1% vs 40.6%) or MET 2+/3+ populations (OS HR, 0.64; 95% CI, 0.40-1.03; P =.06; PFS HR, 0.79; 95% CI, 0.54-1.15; P =.22; ORR, 53.8% vs 44.6%). Safety was as expected for onartuzumab.CONCLUSIONS AND RELEVANCE Addition of onartuzumab to first-line mFOLFOX6 did not significantly improve clinical benefits in the ITT or MET 2+/3+ populations.

Abstract Background: Understanding of immune tolerance mechanisms of cancer has prompted the development of cancer immunotherapies such as atezolizumab (anti-PD-L1, MPDL3280A). Robust, durable responses have been observed, leading to Breakthrough status designation by the FDA for atezolizumab for previously treated NSCLC and bladder cancer (UBC) patients. Roche/Ventana have developed a companion diagnostic (CDx) for atezoliziumab using a robust immunohistochemistry (IHC) assay and antibody clone (SP142), optimized to detect PD-L1 expression in both tumor cells (TC) and tumor-infiltrating immune cells (IC). Here we describe the characteristics of the SP142 assay, PD-L1 expression patterns observed by immunohistochemistry in TC and IC compartments, and their association with clinical efficacy for NSCLC and UBC patients. Methods: The anti-human PD-L1 rabbit monoclonal antibody SP142 was optimized for detection of both TC and IC expression of PD-L1 with the Ventana OptiView DAB IHC Detection Kit on the automated BenchMark ULTRA platform. The VENTANA PD-L1 (SP142) CDx assay was validated for use in formalin-fixed, paraffin-embedded samples of NSCLC and UBC in a series of studies addressing sensitivity, specificity, robustness, and precision. Formalin fixed, paraffin embedded tumor specimens were obtained from patients prescreened and/or enrolled into Genentech Ph I study PCD4989g, and PD-L1 expression was measured using the SP142 assay and PCR gene expression assays. Results: The SP142 assay met pre-defined acceptance criteria for TC and IC assessment of PD-L1 expression in NSCLC and UBC tumors, including >90% inter-reader concordance between pathologist readers. Rapid and durable responses were observed in the Ph I study (PCD4989g), and correlated with PD-L1 expression patterns observed by immunohistochemistry in TC and IC. In the same Ph I study (PCD4989g), PD-L1 expression as measured by PCR did not demonstrate the same predictive value as observed for IHC. Conclusions: The PD-L1 IHC (SP142) CDx is a robust assay to measure PD-L1 expression in both IC and TC. Further, the high reproducibility of results between pathologists shows that the scoring algorithms in UBC and NSCLC are precise, reproducible, and practical in the diagnostic setting. The results indicate that favorable atezolizumab efficacy for UBC is strongly associated with higher IC levels as assessed by the sensitive and specific PD-L1 IHC (SP142) CDx assay. In NSCLC, favorable atezolizumab efficacy is associated with tumors expressing either high TC or high IC levels by the PD-L1 IHC (SP142) CDx assay. These data support the predictive value of tumor PD-L1 levels by IHC for NSCLC and UBC patients receiving atezoliziumab. Citation Format: Zachary S. Boyd, Dustin Smith, Brian Baker, Bharathi Vennapusa, Hartmut Koeppen, Marcin Kowanetz, Sanjeev Mariathasan, Jean-Marie Bruey, Howard Mackey, Gregg Fine, Simonetta Mocci, Priti Hegde, J. Andrew Williams, Ian McCaffery. Development of a PD-L1 companion diagnostic IHC assay (SP142) for atezolizumab. [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 B001.

Poor solubility and cationic amphiphilic drug-likeness were liabilities identified for a lead series of S1P3-sparing, S1P1 agonists originally developed from a high-throughput screening campaign. This work describes the subsequent optimization of these leads by balancing potency, selectivity, solubility and overall molecular charge. Focused SAR studies revealed favorable structural modifications that, when combined, produced compounds with overall balanced profiles. The low brain exposure observed in rat suggests that these compounds would be best suited for the potential treatment of peripheral autoimmune disorders.

16 Background: False-negative HER2 testing in GEC may deny patients the benefit of HER2-targeted therapy. The aim of this study is to estimate the false-negative rate for HER2 testing in GEC and to evaluate predictive factors that might be used to select patients for re-testing. Methods: Two clinical trials conducted in patients with HER2-negative GEC were conducted by Genentech (NCT01662869 and NCT01590719). Information on local HER2 results was collected and testing was repeated centrally by immunohistochemistry (IHC; Ventana, 4B5 antibody) and FISH. HER2 positivity was determined using published criteria (Mod Pathol 2012;25:637). Results: Of 1,189 patients screened, 738 GECs were HER2 negative by local testing and had central results available. Twenty-nine patients (3.9%, 95% confidence interval [CI] 2.5–5.3) were locally HER2 negative, but centrally HER2 positive. The cases showed a wide geographic distribution, a mixture of biopsies (n=19) and resections (n=10), and a mixture of gastric cancer (n=20) and GEC (n=9). All false-negative cases were centrally confirmed to be Lauren’s intestinal (n=25) or mixed (n=4) morphology; amongst intestinal/mixed GEC, the false-negative rate was 6.4% (95% CI 4.1–8.6). Detailed local testing results were available for 25 cases, of which 21 cases showed local IHC scores of 0 or 1+ but were centrally IHC 2+/FISH positive (n=17) or IHC 3+ (n=4). The remaining 4 cases were locally HER2 IHC 2+, FISH negative, but centrally IHC 2+, FISH positive. Conclusions: A clinically significant false-negative rate of 6.4% was observed for HER2 testing in GEC (intestinal/mixed type). Several measures might improve the accuracy of HER2 testing, including running FISH and IHC on all cases, carefully evaluating internal positive controls, improving the education of pathologists, and retesting cases with mixed or intestinal morphology. Clinical trial information: NCT01590719 and NCT01662869.