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Prolonged activation of vascular endothelial growth factor receptor-2 (VEGFR-2) due to mis-regulation of the VEGF pathway induces aberrant blood vessel expansion, which supports growth and survival of solid tumors. Therapeutic interventions that inhibit the VEGFR-2 pathway have therefore become a mainstay of cancer treatment. Non-clinical studies have recently revealed that blockade of angiogenesis can modulate the tumor microenvironment and enhance the efficacy of concurrent immune therapies. Ramucirumab is an FDA-approved anti-angiogenic antibody that inhibits VEGFR-2 and is currently being evaluated in clinical studies in combination with anti-programmed cell death (PD-1) axis checkpoint inhibitors (pembrolizumab, durvalumab, or sintilimab) across several cancer types. The purpose of this study is to establish a mechanistic basis for the enhanced activity observed in the combined blockade of VEGFR-2 and PD-1-axis pathways. Pre-clinical studies were conducted in murine tumor models known to be responsive to anti-PD-1 axis therapy, using monoclonal antibodies that block mouse VEGFR-2 and programmed death-ligand 1 (PD-L1). Combination therapy resulted in enhanced anti-tumor activity compared to anti-PD-L1 monotherapy. VEGFR-2 blockade at early timepoints post-anti-PD-L1 therapy resulted in a dose-dependent and transient enhanced infiltration of T cells, and establishment of immunological memory. VEGFR-2 blockade at later timepoints resulted in enhancement of anti-PD-L1-driven immune cell infiltration. VEGFR-2 and PD-L1 monotherapies induced both unique and overlapping patterns of immune gene expression, and combination therapy resulted in an enhanced immune activation signature. Collectively, these results provide new and actionable insights into the mechanisms by which concurrent VEGFR-2 and PD-L1 antibody therapy leads to enhanced anti-tumor efficacy.

Background Modulation of the PD-1/PD-L1 axis through antagonist antibodies that block either receptor or ligand has been shown to reinvigorate the function of tumor-specific T cells and unleash potent anti-tumor immunity, leading to durable objective responses in a subset of patients across multiple tumor types. Results Here we describe the discovery and preclinical characterization of LY3300054, a fully human IgG1λ monoclonal antibody that binds to human PD-L1 with high affinity and inhibits interactions of PD-L1 with its two cognate receptors PD-1 and CD80. The functional activity of LY3300054 on primary human T cells is evaluated using a series of in vitro T cell functional assays and in vivo models using human-immune reconstituted mice. LY3300054 is shown to induce primary T cell activation in vitro, increase T cell activation in combination with anti-CTLA4 antibody, and to potently enhance anti-tumor alloreactivity in several xenograft mouse tumor models with reconstituted human immune cells. High-content molecular analysis of tumor and peripheral tissues from animals treated with LY3300054 reveals distinct adaptive immune activation signatures, and also previously not described modulation of innate immune pathways. Conclusions LY3300054 is currently being evaluated in phase I clinical trials for oncology indications.

Purpose: Combination strategies leveraging chemotherapeutic agents and immunotherapy have held the promise as a method to improve benefit for patients with cancer. However, most chemotherapies have detrimental effects on immune homeostasis and differ in their ability to induce immunogenic cell death (ICD). The approval of pemetrexed and carboplatin with anti-PD-1 (pembrolizumab) for treatment of non–small cell lung cancer represents the first approved chemotherapy and immunotherapy combination. Although the clinical data suggest a positive interaction between pemetrexed-based chemotherapy and immunotherapy, the underlying mechanism remains unknown. Experimental Design: Mouse tumor models (MC38, Colon26) and high-content biomarker studies (flow cytometry, Quantigene Plex, and nCounter gene expression analysis) were deployed to obtain insights into the mechanistic rationale behind the efficacy observed with pemetrexed/anti-PD-L1 combination. ICD in tumor cell lines was assessed by calreticulin and HMGB-1 immunoassays, and metabolic function of primary T cells was evaluated by Seahorse analysis. Results: Pemetrexed treatment alone increased T-cell activation in mouse tumors in vivo, robustly induced ICD in mouse tumor cells and exerted T-cell–intrinsic effects exemplified by augmented mitochondrial function and enhanced T-cell activation in vitro. Increased antitumor efficacy and pronounced inflamed/immune activation were observed when pemetrexed was combined with anti-PD-L1. Conclusions: Pemetrexed augments systemic intratumor immune responses through tumor intrinsic mechanisms including immunogenic cell death, T-cell–intrinsic mechanisms enhancing mitochondrial biogenesis leading to increased T-cell infiltration/activation along with modulation of innate immune pathways, which are significantly enhanced in combination with PD-1 pathway blockade. See related commentary by Buque et al., p. 6890

e18541 Background: Despite considerable advancements in the standard therapies including surgery, chemotherapy and radiotherapy, the clinical outcome for head and neck squamous cell carcinoma (HNSCC) remains poor due to tumor recurrence and metastasis. Molecular-targeted therapies have evolved as novel and promising treatment for HNSCC patients. Herein, we identified a CHK1 inhibitor, prexasertib, as a therapeutic target that enhanced the infiltration of innate and adaptive immune markers in the mice tumor immune microenvironment (TIME) and subsequently sensitizes the tumors. Methods: For this study we used syngeneic mouse model of HNSCC developed using mouse tonsil epithelial cells transformed with HRAS expression and PTPN13 knockdown to represent tobacco-induced HNSCC (MTE-Ras). In vitro drug activity of prexasertib was determined using immunoblotting, flow cytometry, cell proliferation and colony formation assays. The effect of prexasertib on TIME was quantified by Quantigene Plex panel and multiplex immunohistochemistry (mIHC). Results: We found that in vitro treatment with prexasertib increased the amount of DNA damage in the cancer cells and eventually lead to their death. Similarly, our in vivo data showed that treatment with prexasertib resulted in significant tumor regression and increased mice survival. At the molecular level, prexasertib treatment resulted in an upregulation of transcripts associated with T-cell activation, cytokines, chemokines and macrophages indicating an upregulation of inflammatory gene signature. This was further supported by our findings from mIHC staining of mice tumors showing increased infiltration of natural killer cells, natural killer T cells and dendritic cells, following prexasertib treatment. Interestingly, we also saw an increase in the Tregs after prexasertib treatment, which is indicative of an immunosuppressive environment, and we speculate this could be as a result of negative feedback following immune activation by prexasertib treatment. Conclusions: Our results uncover a previously unidentified role of prexasertib in regulating the innate and adaptive immune response in HNSCC, and therefore further corroborate CHK1 as a promising therapeutic target in HNSCC.

Background TGFβ signaling plays a pleotropic role in tumor biology, promoting tumor proliferation, invasion and metastasis, and escape from immune surveillance. Inhibiting TGFβ’s immune suppressive effects has become of particular interest as a way to increase the benefit of cancer immunotherapy. Here we utilized preclinical models to explore the impact of the clinical stage TGFβ pathway inhibitor, galunisertib, on anti-tumor immunity at clinically relevant doses. Results In vitro treatment with galunisertib reversed TGFβ and regulatory T cell mediated suppression of human T cell proliferation. In vivo treatment of mice with established 4T1-LP tumors resulted in strong dose-dependent anti-tumor activity with close to 100% inhibition of tumor growth and complete regressions upon cessation of treatment in 50% of animals. This effect was CD8+ T cell dependent, and led to increased T cell numbers in treated tumors. Mice with durable regressions rejected tumor rechallenge, demonstrating the establishment of immunological memory. Consequently, mice that rejected immunogenic 4T1-LP tumors were able to resist rechallenge with poorly immunogenic 4 T1 parental cells, suggesting the development of a secondary immune response via antigen spreading as a consequence of effective tumor targeting. Combination of galunisertib with PD-L1 blockade resulted in improved tumor growth inhibition and complete regressions in colon carcinoma models, demonstrating the potential synergy when cotargeting TGFβ and PD-1/PD-L1 pathways. Combination therapy was associated with enhanced anti-tumor immune related gene expression profile that was accelerated compared to anti-PD-L1 monotherapy. Conclusions Together these data highlight the ability of galunisertib to modulate T cell immunity and the therapeutic potential of combining galunisertib with current PD-1/L1 immunotherapy.

Background Modulation of the PD-1/PD-L1 axis through antagonist antibodies that block either receptor or ligand has been shown to reinvigorate the function of tumor-specific T cells and unleash potent anti-tumor immunity, leading to durable objective responses in a subset of patients across multiple tumor types. Results Here we describe the discovery and preclinical characterization of LY3300054, a fully human IgG1λ monoclonal antibody that binds to human PD-L1 with high affinity and inhibits interactions of PD-L1 with its two cognate receptors PD-1 and CD80. The functional activity of LY3300054 on primary human T cells is evaluated using a series of in vitro T cell functional assays and in vivo models using human-immune reconstituted mice. LY3300054 is shown to induce primary T cell activation in vitro, increase T cell activation in combination with anti-CTLA4 antibody, and to potently enhance anti-tumor alloreactivity in several xenograft mouse tumor models with reconstituted human immune cells. High-content molecular analysis of tumor and peripheral tissues from animals treated with LY3300054 reveals distinct adaptive immune activation signatures, and also previously not described modulation of innate immune pathways. Conclusions LY3300054 is currently being evaluated in phase I clinical trials for oncology indications. Electronic supplementary material The online version of this article (10.1186/s40425-018-0329-7) contains supplementary material, which is available to authorized users.

Combination strategies leveraging chemotherapy and immunotherapy have held the promise as a method to improve benefit to cancer patients. However, most chemotherapies have detrimental effects on immune homeostasis and do not induce immunogenic cell death. The positive phase III trial of pemetrexed/carboplatin with the PD-1 antibody pembrolizumab in NSCLC (Keynote-189) lead to the first chemotherapy/immunotherapy combination ever approved. While this suggests a positive interaction between pemetrexed-based chemotherapy and PD-1 therapy, the underlying mechanism is unknown. Therefore, it is important to understand the role of pemetrexed in modulating antitumor immune response to assure application of this therapy to the appropriate patients. To characterize the effects of pemetrexed on intra-tumor immune response, murine tumor models which were sensitive to pemetrexed and known to be sensitive to PD-L1, were treated with pemetrexed with or without carboplatin, or anti-mouse PD-L1. In MC38 tumors, pemetrexed monotherapy demonstrated a trend towards an increased frequency of intra-tumor leukocytes that was accompanied by immune-related gene expression changes indicative of enhanced T cell infiltration and/or activation. Gene expression induced by pemetrexed was largely unaffected by carboplatin. Treatment of both MC38 and Colon26 tumor cells in vitro with pemetrexed induced release of HMGB1, indicative of immunogenic cell death. Although proliferation of primary human T cells was slightly reduced by pemetrexed, at clinically relevant concentrations, treatment lead to an enhanced T cell activation phenotype, including upregulation of multiple interferon gamma-induced genes, and increased mitochondrial respiration. This correlated with improved antigen specific in vitro cytotoxic activity of OT-1 TCR transgenic CD8 T cells when treated with pemetrexed during priming with OVA peptide. Treatment with pemetrexed and PD-L1 demonstrated a combination benefit compared to either monotherapy in both tumor models. Pathway Analysis of gene expression data revealed that improved antigen presentation, enhanced T cell and cytokine signaling and an engagement of CD4+ T cell-mediated immunity during the combination. This correlated with upregulation of MHC-I & II on monocytes, macrophages and tumor cells, suggesting increased immune priming. Accordingly, treatment with S1P1R antagonist (FTY720, preventing T cell LN egress) after initiation of therapy resulted in a loss of combination efficacy. This data suggests that pemetrexed promotes intra-tumor T cell-mediated immune response through immunogenic tumor cell death and increased activation and metabolic fitness of T cells. The combination of these effects results in enhanced T cell function leading to an improved anti-tumor efficacy in combination with PD-L1 antibody Citation Format: David A. Schaer, Nelusha Amaladas, Zhao Hai Lu, Erik Rasmussen, Andreas Sonyi, Darin Chin, Andrew Capen, Yanxia Li, Catalina Meyer, Bonita Jones, Xiaodong Hong, Shuang Luo, Carmine Carpenito, Kenneth Roth, Alexander Nikolayev, Bo Tan, Manisha Brahmachary, Krishna Chodavarapu, Frank Charles Dorsey, Jason Manro, Thompson Doman, Greg Donoho, David Surguladze, Gerald Hall, Sandaruwan Geeganage, Michael Kalos, Ruslan Novosiadly. The folate pathway inhibitor pemetrexed pleiotropically enhances effects of cancer immunotherapy via immunogenic tumor cell death and T cell-intrinsic mechanisms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3945.

O1 IL-15 primes an mTOR-regulated gene-expression program to prolong anti-tumor capacity of human natural killer cells #### Andreas Lundqvist1, Vincent van Hoef1, Xiaonan Zhang1, Erik Wennerberg2, Julie Lorent1, Kristina Witt1, Laia Masvidal Sanz1, Shuo Liang1, Shannon Murray3, Ola Larsson1

Background: Recently, pemetrexed and carboplatin in combination with PD–1 antibody (pembrolizumab) demonstrated markedly improved clinical outcome in NSCLC patients (KEYNOTE–021G trial) suggesting a positive interaction between pemetrexed–based chemotherapy and immunotherapy. However, the role of pemetrexed in modulating antitumor immune response is largely unknown. The objective of this study was to characterize the effects of pemetrexed on intra–tumor immune response in monotherapy and combination with carboplatin or PD–1 pathway blockade in preclinical models. Methods: Mice bearing syngeneic MC38 or Colon26 tumors were treated with pemetrexed with or without carboplatin or anti–mouse PD–L1 antibody (178G7). Immune cell subsets and immune–related changes in mouse tumor tissue and T cells were assessed by flow cytometry and gene expression analysis (Quantigene Plex and nanoString nCounter assays). Effects of pemetrexed on immunogenic cell death in tumor cells and mitochondrial respiration in T cells were evaluated by HMGB1 ELISA and Agilent Seahorse XF analysis, respectively. Results: In MC38 tumors, pemetrexed monotherapy demonstrated a trend towards an increased frequency of intratumoral leukocytes and total and cycling (Ki67+) T cells accompanied by immune–related gene expression changes indicative of enhanced antigen presentation, T cell infiltration and/or activation and down–modulation of the myeloid cell compartment. Immune gene expression signature induced by pemetrexed was largely unaffected by carboplatin. In both MC38 and Colon26 tumor cells, in vitro treatment with pemetrexed induced a robust release of HMGB1 indicative of immunogenic cell death. Proliferation of primary human T cells stimulated with CD3/CD28 was inhibited by pemetrexed in a dose–dependent manner. At clinically relevant concentrations pemetrexed also enhanced T cell activation phenotype exemplified by an increased frequency of CD137+, GITR+ and PD–L1+ T cells as well as upregulation of multiple interferon gamma–induced genes, and increased mitochondrial respiration. In both MC38 and Colon26 models, treatment with pemetrexed and 178G7 demonstrated a combination benefit compared to either monotherapy, and nCounter profiling of Colon26 tumors followed by Ingenuity Pathway Analysis revealed that improved antigen presentation, enhanced T cell and cytokine signaling and an engagement of CD4+ T cell–mediated immunity might underlie this combination effect. Conclusions: Pemetrexed promotes intra–tumor T cell–mediated immune response through immunogenic tumor cell death and increased activation and metabolic fitness of T cells leading to an enhanced anti–tumor efficacy in combination with PD–L1 antibody. Citation Format: Ruslan Novosiadly, David Schaer, Nelusha Amaladas, Erik Rasmussen, Zhao Hai Lu, Andreas Sonyi, Carmine Carpenito, Yanxia Li, Shuang Luo, Andrew Capen, Catalina Meyer, Xiaodong Huang, Jason Manro, Gregory Donoho, Thompson Doman, Gerald Hall, Sandaruwan Geeganage, Michael Kalos. Pemetrexed enhances anti-tumor efficacy of PD1 pathway blockade by promoting intra tumor immune response via immunogenic tumor cell death and T cell intrinsic mechanisms [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 4549.

Necitumumab (EGFR inhibitor) in combination with chemotherapy provides a modest, yet a significant improvement in overall survival over chemotherapy alone in patients with advanced squamous non-small cell lung carcinoma (NSCLC). However, combination therapies targeting EGFR and PD-1 pathway blockers may represent a better way to extend clinical benefit to more cancer patients given that PD-(L)1 antibodies have emerged as a standard of care in NSCLC. Antibodies targeting EGFR have the potential to promote an inflamed tumor microenvironment through engagement of Fc-gamma receptors (FcγR) on innate immune cells resulting in an improved antigen presentation and T cell priming. Therefore, the present study was initiated to understand the combinatorial effect of immune checkpoint (PD-(L)1) inhibitors with necitumumab. Preclinical modeling of EGFR/PD-(L)1 mAb combination in mice is challenging due to the lack of cross-reactivity of necitumumab with mouse EGFR. Syngeneic mouse tumor models widely used to study effects of immunomodulatory agents express low or no EGFR. To overcome this limitation, we used two immunocompetent model systems to study the combination effect of EGFR mAb with PD-1 (RMP1-14) or PD-L1 (178G7) mAbs: 1) genetically engineered mouse model of lung adenocarcinoma (TD model) driven by mutant forms of human EGFR (exon 19 deletion and T790M mutation) and 2) CT26 syngeneic mouse tumor model with ectopic expression of human EGFR (CT26-hEGFR). To engage mouse immune cells more efficiently, a murinized version of necitumumab was generated through antibody engineering, with human EGFR binding Fabs and a mouse Fc backbone. Intratumor immune response was evaluated by immunohistochemistry and a custom-made immune profiling Quantigene Plex (QGP) gene expression panel. In both models, targeting EGFR and PD-1 pathway resulted in the combinatorial antitumor efficacy exemplified by decreased tumor burden compared to the monotherapy groups. QGP analysis of CT26-hEGFR tumor tissue revealed that the combination treatment enhanced intra-tumor immune response exemplified by an upregulation of immune-related genes indicative of T cell infiltration (Cd3e, Cd4, Cd8b1), T cell activation (Ifng, Cd274, Pdcd1lg2, Icos, Tnfrsf4, Tnfrsf18, Cd69, Ido1, Havcr2, Lag3), myeloid cell infiltration (Cd86, Timd4, Vista, Cd68, Mpo, Nos2). Histopathological analysis confirmed an increase in T cell infiltration indicating an improved immune response in the combination therapy group. Taken together, these results provide a rationale for further evaluation of EGFR and PD-(L)1 mAbs in clinical setting. Citation Format: Veena Kandaswamy, Amelie Forest, Marianne Deroose, David A. Schaer, Ting Chen, Shengwu Liu, David Surguladze, Yung-mae Yao, Thompson Doman, Gerald Hall, Kwok-Kin Wong, Michael Kalos, Ruslan D. Novosiadly. Combination of EGFR antibody with PD-1 pathway inhibitors improves anti-tumor efficacy and enhances intra-tumor immune response in preclinical mouse tumor models [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 3632.

Modulating the PD-L1/PD-1 axis in man through function blocking antibodies can release potent anti-tumor immunity, leading to durable objective responses across multiple tumor types. Here we describe the discovery and preclinical characterization of LY3300054, a fully human IgG1λ antibody capable of binding human PD-L1 with high affinity and inhibiting binding to its two cognate receptors, PD-1 and CD80. LY3300054 is an antagonist monoclonal antibody recognizing human PD-L1 with high affinity (KD 0.08 nM), selected and derived from a scFv phage library. LY3300054 was engineered and expressed as an IgG1-Fc null monoclonal antibody to ablate immune effector function. Multiple in vitro assays, including mixed leukocyte reaction (MLR) and tetanus toxoid recall assay were utilized to demonstrate LY3300054 potent functional activity in enhancing the activation of primary human T cells in culture. The biological activity of LY3300054 on T cells was also shown to be enhanced by co-administration of anti-CTLA4 mAb (ipilimumab) in MLR. Further, we evaluated LY3300054 activity in vivo using xenograft mouse tumor models reconstituted with human immune cells. LY3300054 demonstrated anti-tumor activity in both NCI-H292 xenografts co-implanted with human PBMCs and HCC827 xenograft model upon infusion of human T cells. LY3300054 in vivo activity was also tested in HCC827- and Ov79-bearing immunodeficient NSG/NOG mice reconstituted with human hematopoietic stem cells. In this setting, LY3300054 displayed robust anti-tumor and immunomodulatory effects exemplified by T cell inflamed phenotype in the tumor and peripheral tissues. High-content molecular profiling identified distinct gene expression changes indicative of T cell activation in all models tested. A mutational strategy based on integrating the PD-1/PD-L1 structure data with the orthologous sequence data of PD-L1 has identified a residue on PD-L1, which is part of the PD-L1/PD-1 interface, that is critical for the species specificity of LY3300054. This study demonstrates that LY3300054 is novel anti-PD-L1 monoclonal antibody, capable of potently enhancing human T cell function both in vitro and in vivo, and provides previously not described insights into the effects of PD-L1 blockade on the intra- and extra-tumoral immune response. LY3300054 is currently under clinical evaluation in monotherapy and combination with other therapeutic modalities in multiple tumor types (NCT02791334; NCT03099109; NCT02791334; NCT02791334) Citation Format: Carmine Carpenito, Yiwen Li, George X. Wang, Maria S. Malabunga, Jaafar N. Haidar, Amelie Forest, Mary Y. Murphy, Gerald E. Hall, Cindy Wang, Leyi Shen, Andreas Sonyi, Darin Chin, Anthony L. Pennello, Ivan V. Inigo, David Surguladze, Yung-mae Yao, Douglas Burtrum, Ruslan D. Novosiadly, Kris Persaud, Dale L. Ludwig, Michael D. Kalos. Preclinical characterization of the anti-PD-L1 monoclonal antibody LY3300054 [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 2730.

First- , second- , and third-generation EGFR tyrosine kinase inhibitors (TKIs) demonstrate profound tumor responses in EGFR mutation-positive NSCLC. Nevertheless, most patients invariably develop acquired resistance. The objective of the present study was to evaluate the effects of dual EGFR blockade using a ligand-blocking antibody (necitumumab) and second- or third-generation TKIs (afatinib or osimertinib) in preclinical NSCLC models with EGFR mutations. Afatinib and osimertinib (AZD9291), second- and third-generation EGFR TKIs, respectively, are irreversible covalent inhibitors of the EGFR tyrosine kinase, with the latter specifically targeting a mutant form of the receptor (EGFR-T790M). We sought to investigate whether combination of necitumumab (LY3012211) with afatinib and osimertinib would provide any benefit over the monotherapies in preclinical mouse models of lung cancer. Using PC-9 and NCI-H1975 xenograft models of NSCLC that harbor EGFR mutations, we observed that dual EGFR blockade of ligand binding and receptor tyrosine kinase activity improved the antitumor efficacy and, more importantly, resulted in durable tumor remissions compared to either single-agent therapy. Additionally, we explored two dosing schedules of necitumumab/osimertinib combination treatment (concurrent versus phased). Concurrent administration of both agents for four weeks was significantly more efficacious compared to the phased schedule (e.g., administration of osimertinib for one week followed by necitumumab and osimertinib combination for additional three weeks). To provide mechanistic insights into the combinatorial activity observed, we employed high-content gene expression analysis using nCounter Pan-Cancer Pathways assay. Pathway analysis of the combination and single-agent treatment groups identified multiple shared and treatment-specific pathway alterations. Genes implicated in FGFR, PI3K pathways and extracellular remodeling appeared to be upregulated by combination and monotherapies and suggested potential common mechanisms of acquired resistance to EGFR inhibitors, whereas compromised DNA repair mechanisms were identified in PC-9 and NCI-H1975 tumors upon treatment with both combinations. In conclusion, these data demonstrate that combination of necitumumab with second- or third-generation EGFR TKIs results in an improved antitumor efficacy compared to the respective monotherapies; a more profound inhibition of EGFR pathway and compromised DNA repair mechanisms may underlie this effect. A phase I clinical trial of necitumumab and osimertinib in EGFR mutation-positive stage IV or recurrent NSCLC who have progressed on a previous EGFR TKI is currently ongoing (NCT02496663). Citation Format: Amelie Forest, Erik R. Rasmussen, Thompson N. Doman, Michael Amatulli, Rajiv Bassi, Gerald E. Hall, Jason R. Manro, Manisha Brahmachary, David Surguladze, Yung-mae M. Yao, Michael D. Kalos, Ruslan D. Novosiadly. Combination of necitumumab with second- or third-generation EGFR tyrosine kinase inhibitors downregulates DNA repair mechanisms and induces durable tumor remissions in xenograft NSCLC models with EGFR mutations [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A160.

Necitumumab (Nmab) in combination with chemotherapy has demonstrated overall survival benefit in patients with advanced or metastatic squamous non-small cell lung cancer (SqNSCLC). The PI3K/ mTOR pathway shows aberrant activity in multiple tumor types and is implicated in the development of intrinsic and acquired resistance to anti-EGFR therapy. LY3023414, a potent dual PI3K/mTOR inhibitor, is currently undergoing clinical evaluation. The objective of this study was to evaluate the combination effects of Nmab and LY3023414 in multiple tumor models. Nmab and/or LY3023414 were evaluated using Cancer Cell Sensitivity Profiling (CCSP) using a panel of ~500 genetically and histologically diverse human tumor cell lines. The combination treatment showed greater inhibition of tumor cell growth as compared to single-agent treatments in a large fraction of the cell lines tested. The combination effect was further assessed in 21 patient-derived xenograft (PDX) models across multiple tumor types with and without genetic alterations in the PI3K pathway. Compared to both monotherapies, the combination treatment demonstrated greater efficacy in 16 of 21 models, and reached statistical significance in 6 models. Nmab -/+ LY3023414 was also evaluated in a panel of 40 SqNSCLC PDX models using a single-mouse trial format. Combination therapy exhibited significant benefit over either monotherapy as exemplified by disease control (61% for combination vs 17.5% and 34.3% for Nmab and LY3023414, respectively) and overall response rate (22% for combination vs 10% and 5.7% for Nmab and LY3023414, respectively). Median time to tumor doubling or event-free survival (mEFS) was also in favor of the combination therapy (mEFS = 38.9 days for combination vs 14.2 and 32.4 days for Nmab and LY3023414, respectively). To gain mechanistic insights into the combinatorial effect observed, we employed a high-content gene/protein expression assay (nCounter Vantage 3D RNA:Protein Solid Tumor Signaling Pathways Panel) in a SqNSCLC PDX model harboring PIK3CA mutation that was highly sensitive to the combination therapy. nCounter Vantage 3D analysis confirmed inhibition of the PI3K/mTOR pathway in LY3023414-treated tumors. A more robust effect on signaling events downstream of mTOR was observed in the combination group. Furthermore, pathway analysis of the gene expression data suggested alterations in cell cycle/apoptosis, DNA repair, Notch, TGFb, and Wnt signaling in the combination group. Collectively, these data indicate that the Nmab/LY3023414 combination provides a benefit over both monotherapies. A more profound blockade of the PI3K/ mTOR pathway accompanied by inhibition of tumor cell cycle, survival, and DNA repair mechanisms may underlie this effect. Citation Format: Maxine A. Melchior, Xuemei Guo, Gregory P. Donoho, Philip W. Iversen, Thompson N. Doman, Manisha Brahmachary, Xueqian Gong, Gerald E. Hall, Jason R. Manro, Yung-mae M. Yao, Sean Buchanan, Tod Smeal, Michael D. Kalos, Ruslan D. Novosiadly. Antitumor efficacy of EGFR antibody necitumumab and dual PI3K/mTOR inhibitor (LY3023414) in preclinical tumor models [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B108.

The combination of tumor targeted therapeutics with PD-L1 checkpoint blockade is being explored as a method to increase the clinical benefits of immunotherapy, and expand response to additional cancer types. Merestinib (Mer) is a kinase inhibitor targeting several oncokinases1 (including MET, MST1R, AXL, MERTK, and MKNK1/2) that can potentially modulate immune function, angiogenesis, as well as target the tumor 1-5. To determine the combinatorial potential with immunotherapy, the effects of Mer were evaluated in vitro on human T cells, PBMCs and murine tumor lines CT26 colon carcinoma (harbors KRASmt G12D expresses low Met/no p-Met/high Axl/p-Axl) and B16F10 melanoma (expressing high Met/pMet/peIF4E). Additionally, the anti-tumor effect of Mer was tested in vivo on established CT26 and B16F10 tumors compared to MET specific TKIs (savolitinib, PF4217903) alone or in combination with PD-L1 antibody (Ab) blockade. In vitro, Mer showed no significant effects on either T cells or PBMCs, but was able to inhibit downstream signaling in both CT26 and B16F10 showing activity on murine tumor cell lines. In vivo, daily Mer monotherapy (6, 12 or 24 mg/kg) showed significant anti-tumor effect at all doses in both CT26 and B16F10, that was not seen with either savolitinib or PF4217903. Concurrent combination of Mer (12 mg/kg) and anti-PD-L1 Ab (0.5 mg qw) in CT26 was found to have anti-tumor activity that was synergistic as compared to each single agent alone. While the effect of Mer monotherapy was lost when treatment ended, tumors continued to regress in the combination group even upon cessation of therapy. The combination was well tolerated and resulted in 90% complete responders compared to 30% with anti-PD-L1 Ab alone, 35 days after completing dosing. To test the ability to generate immunologic memory, complete responders were re-challenged with CT26 cells on the contralateral side. All mice in the combination group resisted re-challenge, showing that Mer/PD-L1 Ab combination was triggering immunologic memory. Although there was no significant change in intra-tumor immune cell populations between groups, combination therapy showed an enhanced and unique intra-tumor immune activation/inflammation gene expression signature compared to PD-L1 Ab monotherapy. The enhanced immune activation of the combination therapy, leading to synergistic anti-tumor efficacy, demonstrates that merestinib has the potential to augment immunotherapy while targeting the tumor directly. This preclinical data provides the rationale for the clinical investigation of merestinib in combination with checkpoint therapies targeting the PD-L1/PD1 axis (NCT02791334). 1 - Yan et al. Invest New Drugs 2013;31:833-44 2 - Balan et al. J Biol Chem 2015;290:8110-20 3 - Eyob et al. Cancer Discov 2013;3:751-60 4 - Lemke G. CSH Persp Biol 2013;5:a009076 5 - Piccirillo et al. Nat Immunol 2014;15:503-11 Citation Format: Sau-Chi Betty Yan, Victoria L. Peek, Jennifer R. Stephens, Um L. Um, Amaladas Nelusha, Colleen A. Burns, Kelly M. Credille, Thompson N. Doman, Scott W. Eastman, Beverly L. Falcon, Gerald E. Hall, Philip W. Iversen, Bruce W. Konicek, Jason R. Manro, Any T. Pappas, Julie A. Stewart, Michael B. Topper, Swee-Seong Wong, Michael Kalos, Ruslan D. Novosiadly, Richard A. Walgren, David Schaer. Combination of an oncokinase inhibitor merestinib with anti-PD-L1 results in enhanced immune mediated antitumor activity in CT26 murine tumor model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5590. doi:10.1158/1538-7445.AM2017-5590

Matrix attachment regions (MARs) can be operationally defined as DNA fragments that bind to the nuclear matrix. We have created a library of randomly obtained MARs from tobacco (Nicotiana tobacum) by cloning DNA fragments that co-isolate with nuclear matrixes prepared by a method involving lithium diiodosalicylate. The interactions of several of the cloned MARs with nuclear matrixes were tested by an in vitro binding assay in which genomic DNA was used as competitor. Based on this assay, the MARs were classified as strong, medium, and weak binders. Examples of each of the binding classes were further studied by in vitro binding using self- and cross-competition. Estimates of dissociation constants for several MARs ranged from 6 to 11 nM and correlated inversely with binding strength. The number of binding sites per matrix for several MARs ranged from 4 x 10(5) to 9 x 10(5) and correlated directly with binding strength. We conclude that binding strength, as we have measured it, is a function of both numbers of binding sites and affinity for the sites. The tobacco MARs were sequenced and analyzed for overall AT content, for distribution of AT-rich regions, and for the abundance of several MAR-related motifs. Previously identified MAR motifs correlate to various degrees with binding strength. Notably, the Drosophila topoisomerase II motif does not correlate with binding strength of the tobacco MARs. A newly identified motif, the "90%AT Box," correlates better with binding strength than any of the previously identified motifs we investigated.

TGFb signaling is known to play a central role in tumor biology, via inducing and/or enhancing tumor cell growth and differentiation, modulating the extracellular matrix in the stroma, inducing epithelial to mesenchymal transition, modulating angiogenesis, and inhibiting immune surveillance and anti-tumor immunity. Galunisertib is a pharmacological inhibitor of the TGFb pathway which acts by inhibiting signaling though TGFbRI. Galunisertib is currently being evaluated clinically in several Phase I and II studies; as a monotherapy, galunisertib has shown antitumor activity against a variety of tumors, including durable and long-term responses in patients with glioma. To explore the impact of Galunisertib monotherapy on anti-tumor T cell immunity, we utilized murine tumor models. Treatment of mice with well-established 4T1-LP (poorly immunogenic 4T1 breast tumor engineered to express luciferase) implanted in the mammary fat pad resulted in strong dose-dependent anti-tumor activity with nearly 100% inhibition of tumor growth across doses during the dosing period, with complete tumor responses upon cessation of treatment in ~50% of animals at the highest dose tested; depletion studies demonstrated that regression of 4T1-LP was dependent on the presence of CD8+ T cells. Rechallenge of treated, tumor free mice resulted in complete rejection of 4T1-LP tumor cells but no rejection of EMT6-LM2 tumor cells, demonstrating the establishment of a durable response and immunological memory. Treatment of mice bearing established parental 4T1 tumors in the mammary fat pad resulted in no significant inhibition of tumor growth, indicating that the presence of a foreign antigen (i.e. LP), potentially enhanced the ability to regress the 4T1-LP derivative. Animals that rejected the immunogenic 4T1-LP tumors were able to also reject 4T1 parental cells upon rechallenge, suggesting the development of a secondary immune response via antigen spreading as a consequence of effective tumor targeting. In the CT26 murine colon carcinoma model, treatment of established tumors with galunisertib or anti-PD-L1 as monotherapies resulted in tumor growth inhibition compared to control of 75% and 86%, respectively (T/C values of 25% and 14%); complete responders were observed in about 20% of treated animals in both monotherapy groups. Combination of galunisertib with anti-PD-L1 resulted in an enhanced tumor growth inhibition of 98% (T/C value of ~2%), and a complete response rate of ~50%, suggesting at least additive activity with potential for synergy when targeting the TGFb and PD-1 pathways. Taken together, these data demonstrate the potential for galunisertib treatment to enhance the development of anti- tumor T cell immunity, which can be enhanced by combinations with immune check point inhibitors. Citation Format: David Schaer, Yanxia Li, Stephen Castaneda, Ivan Inigo, David Surguladze, Xiaohong Xu, Desiree Nugent, Mary Murphy, Gerald Hall, Karim Benhadji, Susan Guba, Yiwen Li, Michael Kalos, Kyla Driscoll. Targeting the TGFb pathway with galunisertib, a TGFbRI SMI, promotes antitumor immunity leading to durable, complete responses, as monotherapy and in combination with checkpoint inhibition. [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 A091.

The β-phaseolin promoter yields high levels of spatially and temporally regulated expression in transgenic plants. It is shown that elements within 5′ and 3′ regions flanking the coding sequence bind strongly and specifically to matrix preparations from tobacco nuclei. Both of these matrix attachment regions (MARs) are A/T-rich and contain topoisomerase II, Abox, T-box and MAR motifs frequently found in MARs in Drosophila and other organisms. The β-phaseolin 5′ MAR is located on a 1047 bp fragment with a core element of 673 bp (positions −1096 to −424), that includes a 55 bp region (−682 to −628) previously shown to function as an enhancer. A 1205 bp fragment (positions +1879 to +3083) 3′ of the phaseolin coding region also showed strong and specific binding to nuclear matrix preparations and contained consensus MAR motifs. A β-phaseolin promoter-GUS fusion construct (p1470/phas) that included the MAR elements exhibited higher expression levels and lower plant-to-plant variability in stably transformed tobacco plants than did a similar construct lacking MAR sequences. Seeds of plants containing two intact copies of the insert had higher expression levels than did those of plants containing a single copy. These findings, which are the first to report a plant gene being flanked both 5′ and 3′ by MARs, are in accordance with previous observations of increased DNase I sensitivity associated with the onset of phaseolin transcription in bean and with the consistently high and position-independent expression observed in transgenic plants from the β-phaseolin promoter. The results suggest that the native β-phaseolin gene is located on a small chromatin loop that functions as an active and independent domain.

The yeast ARS-1 element contains a scaffold attachment region (SAR) that we have previously shown can bind to plant nuclear scaffolds in vitro. To test effects on expression, constructs in which a chimeric beta-glucuronidase (GUS) gene was flanked by this element were delivered into tobacco suspension cells by microprojectile bombardment. In stably transformed cell lines, GUS activity averaged 12-fold higher (24-fold on a gene copy basis) for a construct containing two flanking SARs than for a control construct lacking SARs. Expression levels were not proportional to gene copy number, as would have been predicted if the element simply reduced position effect variation. Instead, the element appeared to reduce an inhibitory effect on expression in certain transformants containing multiple gene copies. The effect on expression appears to require chromosomal integration, because SAR constructs were only twofold more active than the controls in transient assays.

Many of the questions we have about how biological systems work are ultimately questions about the regulation of gene expression. For this reason, the control of transcription is fundamental and has received well-deserved attention. From a simplistic point of view, transcription can be though of as being regulated at two levels. The first level (coarse control) involves access of RNA polymerase and transacting factors to the specific DNA sequences with which they interact. Access is a function of chromatin structure. In chromatin fibers of both condensed metaphase chromosomes and interphase chromatin, DNA is highly compacted and essentially inaccessible to RNA polymerase and trans-acting factors. In order to make the DNA accessible, chromatin fibers must in some way take on a more open, less compact structure. Once an open (transcriptionally poised) chromatin structure is obtained, further regulation of transcription involving availability and interactions of transacting factors (fine control), come into play. For reviews see Paranjape et al. (1994) and Reeves (1984).

The control of gene expression in eukaryotic organisms is generally considered to be multi-leveled, and one of the levels is thought to involve the effect of the structure of chromatin fibers on the access of transcription factors to DNA [ 1 ]. It has become increasingly apparent in recent years that the organization of chromatin fibers within the nucleus also has a role to play in the control of gene expression. Evidence has accumulated that chromatin fibers are organized through association with a proteinaceous network, variously referred to as the nuclear scaffold or nuclear matrix, which runs throughout the nucleus [2]. Chromatin fibers are believed to associate with the scaffold at specific, AT-rich DNA sequences that have been termed SARs (for scaffold associated regions) or MARs (for matrix associated regions).2 The chromatin fibers between the attachment sites would then form ‘loop domains’ along the chromatin fibers. Some of the evidence supporting this view comes from electron micrographs of histone depleted chromosomes in which DNA segments can be seen to form large loops (on the order of 50 to 100 kb) attached at their bases to a proteinaceous scaffold [3]. In at least some cases, these SAR-bounded physical loop domains coincide with transcriptionally active chromatin domains as assayed by DNase I sensitivity (reviewed by Zlatanova and Van Holde [4]. This has led to the idea that SARs form the boundaries of topologically isolated chromatin domains that function as units of gene regulation [5, 6].

DNA in the nuclei of eukaryotic organisms undergoes a hierarchy of folding to be packaged into interphase and metaphase chromosomes. The first level of packaging is the 11-nm nucleosome fiber, which is further coiled into a 30-nm fiber. Evidence from fungal and animal systems reveals the existence of higher order packaging consisting of loops of the 30-nm fibers attached to a proteinaceous nuclear scaffold by an interaction between the scaffold and specific DNA sequences called scaffold-attachment regions (SARs). Support for the ubiquitous nature of such higher order packaging of DNA is presented here by our work with plants. We have isolated scaffolds from tobacco nuclei using buffers containing lithium diiodosalicylate to remove histones and then using restriction enzymes to remove the DNA not closely associated with the scaffold. We have used Southern hybridization to show that the DNA remaining bound to the scaffolds after nuclease digestion includes SARs flanking three root-specific tobacco genes. This assay for SARs is termed the endogenous assay because it identifies genomic sequences as SARs by their endogenous association with the scaffold. Another assay, the exogenous assay, depends upon the ability of scaffolds to specifically bind exogenously added DNA fragments containing SARs. The tobacco scaffolds specifically bind a well-characterized yeast SAR, and cloned DNA fragments derived from the 3'-flanking regions of the root-specific genes are confirmed to contain SARs by this exogenous assay.

We have previously shown that yeast scaffold attachment regions (SARs) flanking a chimeric beta-glucuronidase (GUS) reporter gene increased per-copy expression levels by 24-fold in tobacco suspension cell lines stably transformed by microprojectile bombardment. In this study, we examined the effect of a DNA fragment originally identified in a tobacco genomic clone by its activity in an in vitro binding assay. The tobacco SAR has much greater scaffold binding affinity than does the yeast SAR, and tobacco cell lines stably transformed with constructs containing the tobacco SAR accumulated greater than fivefold more GUS enzyme activity than did lines transformed with the yeast SAR construct. Relative to the control construct, flanking the GUS gene with plant SARs increased overall expression per transgene copy by almost 140-fold. In transient expression assays, the same construct increased expression only approximately threefold relative to a control without SARs, indicating that the full SAR effect requires integration into chromosomal DNA. GUS activity in individual stable transformants was not simply proportional to transgene copy number, and the SAR effect was maximal in cell lines with fewer than approximately 10 transgene copies per tobacco genome. Lines with significantly higher copy numbers showed greatly greatly reduced expression relative to the low-copy-number lines. Our results indicate that strong SARs flanking a transgene greatly increases expression without eliminating variation between transformants. We propose that SARs dramatically reduce the severity or likelihood of homology-dependent gene silencing in cells with small numbers of transgenes but do not prevent silencing of transgenes present in many copies.

Within the Aşvan Project we are concerned with the examination of the presentday architecture of the region, and utilizing this as a basic framework of reference, to investigate any areas of relevance, implication and relationship it may have to the stated objectives of the project.As much of our work to date has been of a preliminary and exploratory nature, with our collected data still at a very basic and fragmented level, we have had to limit this paper to a very general synopsis of the various aspects of our work within the project.Our paper consists of four main sections:1. Outline of conceptual and methodological approach.2. General appraisal of architecture within the region.3. Preliminary appraisal of Aşvan village.4. Summary of archaeological relevance.The project objectives are concerned with the investigation of Man's activities, with particular reference to subsistence economy systems within the Aşvan region.