Margaret Veldman-Jones

Dual IGF-I/II-neutralizing antibody MEDI-573 potently inhibits IGF signaling and tumor growth.

Insulin-like growth factors (IGF), IGF-I and IGF-II, are small polypeptides involved in regulating cell proliferation, survival, differentiation, and transformation. IGF activities are mediated through binding and activation of IGF-1R or insulin receptor isoform A (IR-A). The role of the IGF-1R pathway in promoting tumor growth and survival is well documented. Overexpression of IGF-II and IR-A is reported in multiple types of cancer and is proposed as a potential mechanism for cancer cells to develop resistance to IGF-1R-targeting therapy. MEDI-573 is a fully human antibody that neutralizes both IGF-I and IGF-II and inhibits IGF signaling through both the IGF-1R and IR-A pathways. Here, we show that MEDI-573 blocks the binding of IGF-I and IGF-II to IGF-1R or IR-A, leading to the inhibition of IGF-induced signaling pathways and cell proliferation. MEDI-573 significantly inhibited the in vivo growth of IGF-I- or IGF-II-driven tumors. Pharmacodynamic analysis demonstrated inhibition of IGF-1R phosphorylation in tumors in mice dosed with MEDI-573, indicating that the antitumor activity is mediated via inhibition of IGF-1R signaling pathways. Finally, MEDI-573 significantly decreased (18)F-fluorodeoxyglucose ((18)F-FDG) uptake in IGF-driven tumor models, highlighting the potential utility of (18)F-FDG-PET as a noninvasive pharmacodynamic readout for evaluating the use of MEDI-573 in the clinic. Taken together, these results demonstrate that the inhibition of IGF-I and IGF-II ligands by MEDI-573 results in potent antitumor activity and offers an effective approach to selectively target both the IGF-1R and IR-A signaling pathways.

Evaluating Robustness and Sensitivity of the NanoString Technologies nCounter Platform to Enable Multiplexed Gene Expression Analysis of Clinical Samples.

Analysis of clinical trial specimens such as formalin-fixed paraffin-embedded (FFPE) tissue for molecular mechanisms of disease progression or drug response is often challenging and limited to a few markers at a time. This has led to the increasing importance of highly multiplexed assays that enable profiling of many biomarkers within a single assay. Methods for gene expression analysis have undergone major advances in biomedical research, but obtaining a robust dataset from low-quality RNA samples, such as those isolated from FFPE tissue, remains a challenge. Here, we provide a detailed evaluation of the NanoString Technologies nCounter platform, which provides a direct digital readout of up to 800 mRNA targets simultaneously. We tested this system by examining a broad set of human clinical tissues for a range of technical variables, including sensitivity and limit of detection to varying RNA quantity and quality, reagent performance over time, variability between instruments, the impact of the number of fields of view sampled, and differences between probe sequence locations and overlapping genes across CodeSets. This study demonstrates that Nanostring offers several key advantages, including sensitivity, reproducibility, technical robustness, and utility for clinical application.

MEDI0639: a novel therapeutic antibody targeting Dll4 modulates endothelial cell function and angiogenesis in vivo

The Notch signaling pathway has been implicated in cell fate determination and differentiation in many tissues. Accumulating evidence points toward a pivotal role in blood vessel formation, and the importance of the Delta-like ligand (Dll) 4-Notch1 ligand–receptor interaction has been shown in both physiological and tumor angiogenesis. Disruption of this interaction leads to a reduction in tumor growth as a result of an increase in nonfunctional vasculature leading to poor perfusion of the tumor. MEDI0639 is an investigational human therapeutic antibody that targets Dll4 to inhibit the interaction between Dll4 and Notch1. The antibody cross-reacts to cynomolgus monkey but not mouse species orthologues. In vitro MEDI0639 inhibits the binding of Notch1 to Dll4, interacting via a novel epitope that has not been previously described. Binding to this epitope translates into MEDI0639 reversing Notch1-mediated suppression of human umbilical vein endothelial cell growth in vitro. MEDI0639 administration resulted in stimulation of tubule formation in a three-dimensional (3D) endothelial cell outgrowth assay, a phenotype driven by disruption of the Dll4-Notch signaling axis. In contrast, in a two-dimensional endothelial cell–fibroblast coculture model, MEDI0639 is a potent inhibitor of tubule formation. In vivo, MEDI0639 shows activity in a human endothelial cell angiogenesis assay promoting human vessel formation and reducing the number of vessels with smooth muscle actin-positive mural cells coverage. Collectively, the data show that MEDI0639 is a potent modulator of Dll4-Notch signaling pathway. Mol Cancer Ther; 11(8); 1650–60. ©2012 AACR.

Reproducible, Quantitative, and Flexible Molecular Subtyping of Clinical DLBCL Samples Using the NanoString nCounter System

Purpose: Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous disease with distinct molecular subtypes. The most established subtyping approach, the “Cell of Origin” (COO) algorithm, categorizes DLBCL into activated B-cell (ABC) and germinal center B-cell (GCB)-like subgroups through gene expression profiling. Recently developed immunohistochemical (IHC) techniques and other established methodologies can deliver discordant results and have various technical limitations. We evaluated the NanoString nCounter gene expression system to address issues with current platforms. Experimental Design: We devised a scoring system using 145 genes from published datasets to categorize DLBCL samples. After cell line validation, clinical tissue segmentation was tested using commercially available diagnostic DLBCL samples. Finally, we profiled biopsies from patients with relapsed/refractory DLBCL enrolled in the fostamatinib phase IIb clinical trial using three independent RNA expression platforms: NanoString, Affymetrix, and qNPA. Results: Diagnostic samples showed a typical spread of subtypes with consistent gene expression profiles across matched fresh, frozen, and formalin-fixed paraffin-embedded tissues. Results from biopsy samples across platforms were remarkably consistent, in contrast to published IHC data. Interestingly, COO segmentation of longitudinal fostamatinib biopsies taken at initial diagnosis and then again at primary relapse showed 88% concordance (15/17), suggesting that COO designation remains stable over the course of disease progression. Conclusions: DLBCL segmentation of patient tumor samples is possible using a number of expression platforms. However, we found that NanoString offers the most flexibility and fewest limitations in regards to robust clinical tissue subtype characterization. These subtype distinctions should help guide disease prognosis and treatment options within DLBCL clinical practice. Clin Cancer Res; 21(10); 2367–78. ©2014 AACR. See related commentary by Rimsza, p. 2204

In vitro pharmacological profiling of R406 identifies molecular targets underlying the clinical effects of fostamatinib.

Off‐target pharmacology may contribute to both adverse and beneficial effects of a new drug. In vitro pharmacological profiling is often applied early in drug discovery; there are fewer reports addressing the relevance of broad profiles to clinical adverse effects. Here, we have characterized the pharmacological profile of the active metabolite of fostamatinib, R406, linking an understanding of drug selectivity to the increase in blood pressure observed in clinical studies. R406 was profiled in a broad range of in vitro assays to generate a comprehensive pharmacological profile and key targets were further investigated using functional and cellular assay systems. A combination of traditional literature searches and text‐mining approaches established potential mechanistic links between the profile of R406 and clinical side effects. R406 was selective outside the kinase domain, with only antagonist activity at the adenosine A3 receptor in the range relevant to clinical effects. R406 was less selective in the kinase domain, having activity at many protein kinases at therapeutically relevant concentrations when tested in multiple in vitro systems. Systematic literature analyses identified KDR as the probable target underlying the blood pressure increase observed in patients. While the in vitro pharmacological profile of R406 suggests a lack of selectivity among kinases, a combination of classical searching and text‐mining approaches rationalized the complex profile establishing linkage between off‐target pharmacology and clinically observed effects. These results demonstrate the utility of in vitro pharmacological profiling for a compound in late‐stage clinical development.

The MEK inhibitor selumetinib complements CTLA-4 blockade by reprogramming the tumor immune microenvironment

Background T-cell checkpoint blockade and MEK inhibitor combinations are under clinical investigation. Despite progress elucidating the immuno-modulatory effects of MEK inhibitors as standalone therapies, the impact of MEK inhibition on the activity of T-cell checkpoint inhibitors remains incompletely understood. Here we sought to characterize the combined effects of MEK inhibition and anti-CTLA-4 mAb (anti-CTLA-4) therapy, examining effects on both T-cells and tumor microenvironment (TME). Methods In mice, the effects of MEK inhibition, via selumetinib, and anti-CTLA-4 on immune responses to keyhole limpet haemocyanin (KLH) immunization were monitored using ex vivo functional assays with splenocytes. In a KRAS-mutant CT26 mouse colorectal cancer model, the impact on the tumor microenvironment (TME) and the spleen were evaluated by flow cytometry. The TME was further examined by gene expression and immunohistochemical analyses. The combination and sequencing of selumetinib and anti-CTLA-4 were also evaluated in efficacy studies using the CT26 mouse syngeneic model. Results Anti-CTLA-4 enhanced the generation of KLH specific immunity following KLH immunization in vivo; selumetinib was found to reduce, but did not prevent, this enhancement of immune response by anti-CTLA-4 in vivo. In the CT26 mouse model, anti-CTLA-4 treatment led to higher expression levels of the immunosuppressive mediators, Cox-2 and Arg1 in the TME. Combination of anti-CTLA-4 with selumetinib negated this up-regulation of Cox-2 and Arg1, reduced the frequency of CD11+ Ly6G+ myeloid cells, and led to the accumulation of differentiating monocytes at the Ly6C+ MHC+ intermediate state in the tumor. We also report that MEK inhibition had limited impact on anti-CTLA-4-mediated increases in T-cell infiltration and T-cell activation in CT26 tumors. Finally, we show that pre-treatment, but not concurrent treatment, with selumetinib enhanced the anti-tumor activity of anti-CTLA-4 in the CT26 model. Conclusion These data provide evidence that MEK inhibition can lead to changes in myeloid cells and immunosuppressive factors in the tumor, thus potentially conditioning the TME to facilitate improved response to anti-CTLA-4 treatment. In summary, the use of MEK inhibitors to alter the TME as an approach to enhance the activities of immune checkpoint inhibitors warrants further investigation in clinical trials. Electronic supplementary material The online version of this article (doi:10.1186/s40425-017-0268-8) contains supplementary material, which is available to authorized users.BackgroundT-cell checkpoint blockade and MEK inhibitor combinations are under clinical investigation. Despite progress elucidating the immuno-modulatory effects of MEK inhibitors as standalone therapies, the impact of MEK inhibition on the activity of T-cell checkpoint inhibitors remains incompletely understood. Here we sought to characterize the combined effects of MEK inhibition and anti-CTLA-4 mAb (anti-CTLA-4) therapy, examining effects on both T-cells and tumor microenvironment (TME).MethodsIn mice, the effects of MEK inhibition, via selumetinib, and anti-CTLA-4 on immune responses to keyhole limpet haemocyanin (KLH) immunization were monitored using ex vivo functional assays with splenocytes. In a KRAS-mutant CT26 mouse colorectal cancer model, the impact on the tumor microenvironment (TME) and the spleen were evaluated by flow cytometry. The TME was further examined by gene expression and immunohistochemical analyses. The combination and sequencing of selumetinib and anti-CTLA-4 were also evaluated in efficacy studies using the CT26 mouse syngeneic model.ResultsAnti-CTLA-4 enhanced the generation of KLH specific immunity following KLH immunization in vivo; selumetinib was found to reduce, but did not prevent, this enhancement of immune response by anti-CTLA-4 in vivo. In the CT26 mouse model, anti-CTLA-4 treatment led to higher expression levels of the immunosuppressive mediators, Cox-2 and Arg1 in the TME. Combination of anti-CTLA-4 with selumetinib negated this up-regulation of Cox-2 and Arg1, reduced the frequency of CD11+ Ly6G+ myeloid cells, and led to the accumulation of differentiating monocytes at the Ly6C+ MHC+ intermediate state in the tumor. We also report that MEK inhibition had limited impact on anti-CTLA-4-mediated increases in T-cell infiltration and T-cell activation in CT26 tumors. Finally, we show that pre-treatment, but not concurrent treatment, with selumetinib enhanced the anti-tumor activity of anti-CTLA-4 in the CT26 model.ConclusionThese data provide evidence that MEK inhibition can lead to changes in myeloid cells and immunosuppressive factors in the tumor, thus potentially conditioning the TME to facilitate improved response to anti-CTLA-4 treatment. In summary, the use of MEK inhibitors to alter the TME as an approach to enhance the activities of immune checkpoint inhibitors warrants further investigation in clinical trials.

Identification of Pharmacodynamic Transcript Biomarkers in Response to FGFR Inhibition by AZD4547

The challenge of developing effective pharmacodynamic biomarkers for preclinical and clinical testing of FGFR signaling inhibition is significant. Assays that rely on the measurement of phospho-protein epitopes can be limited by the availability of effective antibody detection reagents. Transcript profiling enables accurate quantification of many biomarkers and provides a broader representation of pathway modulation. To identify dynamic transcript biomarkers of FGFR signaling inhibition by AZD4547, a potent inhibitor of FGF receptors 1, 2, and 3, a gene expression profiling study was performed in FGFR2-amplified, drug-sensitive tumor cell lines. Consistent with known signaling pathways activated by FGFR, we identified transcript biomarkers downstream of the RAS-MAPK and PI3K/AKT pathways. Using different tumor cell lines in vitro and xenografts in vivo, we confirmed that some of these transcript biomarkers (DUSP6, ETV5, YPEL2) were modulated downstream of oncogenic FGFR1, 2, 3, whereas others showed selective modulation only by FGFR2 signaling (EGR1). These transcripts showed consistent time-dependent modulation, corresponding to the plasma exposure of AZD4547 and inhibition of phosphorylation of the downstream signaling molecules FRS2 or ERK. Combination of FGFR and AKT inhibition in an FGFR2-mutated endometrial cancer xenograft model enhanced modulation of transcript biomarkers from the PI3K/AKT pathway and tumor growth inhibition. These biomarkers were detected on the clinically validated nanoString platform. Taken together, these data identified novel dynamic transcript biomarkers of FGFR inhibition that were validated in a number of in vivo models, and which are more robustly modulated by FGFR inhibition than some conventional downstream signaling protein biomarkers. Mol Cancer Ther; 15(11); 2802–13. ©2016 AACR.

Abstract A94: STAT3 antisense treatment decreases M2 macrophage infiltration and enhances the activity of checkpoint inhibitors in preclinical tumor models

AZD9150 is a gen2.5 antisense oligonucleotide (ASO) targeting STAT3. Gen2.5 ASOs exhibit enhanced drug-like properties compared to previous generations of antisense therapeutics, including increased stability and resistance to nucleases, a marked decrease in proinflammatory effects, and enhanced potency. The immune suppressive effects of STAT3 signaling are well established (Kortylewski et al.; Nat. Med. 2005 and Curr. Opin. Immunol. 2008). Preclinical experiments were carried out to determine the potential for combination of a STAT3 ASO with checkpoint inhibitor therapy, and to explore the mechanism of the antitumor activity of STAT3 ASOs observed in mouse models. The antitumor activity of STAT3 ASO treatment, as a single agent and in combination with checkpoint inhibitors, was evaluated in syngeneic mouse models, including CT-26. ASO uptake and STAT3 knockdown were evaluated by immunohistochemistry (IHC), and the pharmacodynamic effects of STAT3 ASO treatment in mice were evaluated by IHC, flow cytometry and immune gene expression profiling. In contrast to many tumor cell lines, CT26 cells take up ASOs poorly, including gen2.5 ASOs, thus as expected the tumor-associated activity of STAT3 ASOs in CT26 tumors in vivo was primarily in stromal and immune compartments (including circulating PBMCs), rather than directly in tumor cells. This was observed in several murine tumor models, as well as in clinical samples. Expression analysis of CT-26 tumor lysates taken from STAT3 ASO-treated (50 mg/kg QDx5/wk) mice using the Nanostring mouse immunology panel (561 immune related genes) identified CD163 as the gene most consistently modulated after STAT3 ASO treatment, with an average knockdown of 85% relative to control treated tumors, across 3 independent experiments. This result was confirmed by immunohistochemistry, which showed a marked reduction in the number of tumor infiltrating CD163+ cells after STAT3 ASO treatment. CD163 is a marker of M2 macrophages, and the presence of M2 macrophages in a tumor can create an immune suppressive environment which has the potential to reduce the effectiveness of checkpoint inhibitor therapy. Consistent with this hypothesis, the addition of STAT3 ASO treatment enhanced the response to a checkpoint inhibitor antibody targeting PD-L1 (clone 10F.9G2). When treatment began 2 days after tumor implant, single agent STAT3 ASO and PD-L1 antibody treatments resulted in response rates (regression or no tumor growth during the course of the experiment) of 0% and 14% respectively, while the rate was 50% with the combination. The combination remained active when treatments were delayed until established tumors were present (140 mm3 average tumor volume), with single agent and combination regression rates of 0%, and 20% respectively. These results suggest the addition of AZD9150 treatment as a promising approach to enhance the response to checkpoint inhibitor therapy Citation Format: Richard D. Woessner, Patricia McCoon, Shaun Grosskurth, Paul Lyne, Kirsten Bell, Mike Collins, Nanhua Deng, Rachel DuPont, Michele Johnstone, Youngsoo Kim, Deborah Lawson, Robert MacLeod, Lourdes Pablo, Corinne Reimer, Vasu Sah, Margaret Veldman-Jones. STAT3 antisense treatment decreases M2 macrophage infiltration and enhances the activity of checkpoint inhibitors in preclinical tumor models. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A94.

Abstract CT239: Clinical and preclinical evidence of an immune modulating role for the STAT3-targeting ASO AZD9150 and potential to enhance clinical responses to anti-PDL1 therapy

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA AZD9150 is a therapeutic Generation 2.5 antisense oligonucleotide (ASO) targeting STAT3 that has completed two phase I clinical studies, in patients with HCC and DLBCL, with durable clinical responses seen in both trials. Biomarker studies using patient samples and related preclinical experiments were performed to investigate the mechanism of action of AZD9150. Patients were treated with three loading doses of AZD9150 in the first week followed by weekly dosing, at doses ranging from 1.0 to 3.0 mg/kg. In the DLBCL study, paired tumor biopsies were collected pre-treatment and on-treatment to evaluate drug uptake and target knockdown by immunohistochemistry (IHC). In the HCC study, blood samples were collected at baseline and at multiple time points on-treatment to evaluate target knockdown and gene expression changes. IHC staining of DLBCL patients’ tumor biopsies (at 2 & 3 mg/kg) demonstrated that the drug distributes to the tumor, with strongest uptake in stromal cells, including endothelium, fibroblasts, and immune cells. Pronounced decreases (absence of staining on-treatment) in STAT3 were observed in the endothelium of several samples. More limited STAT3 modulation was observed in tumor cells. Flow cytometry analysis of HCC patients’ blood samples revealed an average decrease in STAT3 protein staining of 49% across all peripheral leukocyte populations in the 1 mg/kg cohort. Clinical pharmacodynamics and mechanism of action were explored further by conducting a gene expression study with the Nanostring nCounter Human Immunology Panel v2 to evaluate STAT3 RNA knockdown and 593 additional immune genes in peripheral leukocytes collected from HCC patients. Statistically significant decreases of >30% in STAT3 expression were observed in 14/32 patients by the fourth week of treatment. These STAT3 changes are accompanied by +/- 40% changes in expression by additional genes associated with decreased myeloid trafficking and function, increased antigen presentation, and increased CD8 effector cell function. These data provide evidence that AZD9150 treatment may remove or reprogram immunosuppressive elements employed by tumors, leading to therapeutic benefit. Preclinical studies were carried out to investigate immune cell changes within tumors and the benefit of combining STAT3 ASO with PDL1 blockade. Monotherapy STAT3 ASO treatment resulted in CT26 tumor growth inhibition (80%) when tested in immune competent Balb/c but not immune-deficient NSG mice, and was associated with two-fold increases in CD45+ and CD8+ cell infiltrate into tumors. Mice treated with STAT3 ASO and anti-PD-L1 blocking antibody resulted in a 50% response rate for the combination treatment, vs. only 14% with anti-PD-L1 Ab alone. These data suggest that the effects of STAT3 ASO are mechanistically complementary to immune checkpoint inhibitors and that the combination with AZD9150 could broaden clinical responses to these important therapies. This hypothesis will be tested in upcoming clinical trials with AZD9150 and MEDI4736. Citation Format: Patricia E. Mccoon, Rich Woessner, Shaun Grosskurth, Chris Womack, Mason Yamashita, Gene Hung, Robert MacLeod, Kirsten Bell, Mike Collins, Rachel DuPont, Vivian Jacobs, Michele Johnstone, Margaret Veldman-Jones, Paul Lyne. Clinical and preclinical evidence of an immune modulating role for the STAT3-targeting ASO AZD9150 and potential to enhance clinical responses to anti-PDL1 therapy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr CT239. doi:10.1158/1538-7445.AM2015-CT239

Abstract B238: Novel imidazo[1,2-a]pyridines as inhibitors of the IGF-1R tyrosine kinase.

The Insulin-like Growth Factor-1 Receptor (IGF-1R) is a receptor tyrosine kinase which plays an important role in cancer by promoting proliferation and survival through activation of the Ras/Raf/MAPK and PI3K/Akt signaling pathways. Deregulation of the IGF-1R axis has been reported in a variety of solid cancers and occurs by overexpression of either the receptor or its ligands (IGF-1 and IGF-2) or by down-regulation of IGF binding proteins (in particular IGFBP3). Therapeutic approaches to inhibit IGF-1R signaling include antibodies targeting the receptor and small molecule inhibitors of the tyrosine kinase. The latter strategy offers a potential advantage by simultaneously inhibiting the closely related Insulin Receptor A (IR-A) tyrosine kinase. Indeed, IR-A can drive tumor growth in some cancer cell lines and has been suggested has a potential escape mechanism for IGF-1R inhibition. Following a cell based high throughput screening, we have identified a series of imidazo[1,2-a]pyridines inhibiting the IGF-1R kinase. Although hit compounds carried a significant activity against CDK2, modifications of the aniline part has led to more potent and selective inhibitors. We selected 1-[4-[4-[(5-chloro-4-imidazo[1,2-a]pyridin-3-yl-pyrimidin-2-yl)amino]-3-methoxy-phenyl]piperazin-1-yl]ethanone (5) as a lead compound having good pharmacokinetics in pre-clinical species. Compound (5) displays a dual inhibition of IGF-1R and the insulin receptor tyrosine kinase as measured in biochemical assays as well as in cellular auto-phosphorylation assays. Structure-activity relationship around (5) is described for IGF-1R inhibition as well as for the hERG ion channel. Compound (5) has been assessed in an in vivo efficacy model based on the NIH 3T3 fibroblast cell line transfected to express human IGF-1R and IGF1 ligand. In contrast to the non-transfected parent line, this cell line rapidly grows as a sub-cutaneous xenograft in nude mouse, thus providing a suitable model to measure anti-IGF-1R pharmacology by tumor growth inhibition. The high expression of IGF-1R also enables the generation of good quality PD data using receptor phosphorylation as an endpoint. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B238.