Jay Duffner

M402, a novel heparan sulfate mimetic, targets multiple pathways implicated in tumor progression and metastasis.

Heparan sulfate proteoglycans (HSPGs) play a key role in shaping the tumor microenvironment by presenting growth factors, cytokines, and other soluble factors that are critical for host cell recruitment and activation, as well as promoting tumor progression, metastasis, and survival. M402 is a rationally engineered, non-cytotoxic heparan sulfate (HS) mimetic, designed to inhibit multiple factors implicated in tumor-host cell interactions, including VEGF, FGF2, SDF-1α, P-selectin, and heparanase. A single s.c. dose of M402 effectively inhibited seeding of B16F10 murine melanoma cells to the lung in an experimental metastasis model. Fluorescent-labeled M402 demonstrated selective accumulation in the primary tumor. Immunohistological analyses of the primary tumor revealed a decrease in microvessel density in M402 treated animals, suggesting anti-angiogenesis to be one of the mechanisms involved in-vivo. M402 treatment also normalized circulating levels of myeloid derived suppressor cells in tumor bearing mice. Chronic administration of M402, alone or in combination with cisplatin or docetaxel, inhibited spontaneous metastasis and prolonged survival in an orthotopic 4T1 murine mammary carcinoma model. These data demonstrate that modulating HSPG biology represents a novel approach to target multiple factors involved in tumor progression and metastasis.

Adjuvanted influenza-H1N1 vaccination reveals lymphoid signatures of age-dependent early responses and of clinical adverse events

Adjuvanted vaccines afford invaluable protection against disease, and the molecular and cellular changes they induce offer direct insight into human immunobiology. Here we show that within 24 h of receiving adjuvanted swine flu vaccine, healthy individuals made expansive, complex molecular and cellular responses that included overt lymphoid as well as myeloid contributions. Unexpectedly, this early response was subtly but significantly different in people older than ∼35 years. Wide-ranging adverse clinical events can seriously confound vaccine adoption, but whether there are immunological correlates of these is unknown. Here we identify a molecular signature of adverse events that was commonly associated with an existing B cell phenotype. Thus immunophenotypic variation among healthy humans may be manifest in complex pathophysiological responses.

Bioactivity screening of partially desulfated low-molecular-weight heparins: A structure/activity relationship study

A series of size-defined low-molecular-weight heparins were generated by regioselective chemical modifications and profiled for their in vitro and in vivo activities. The compounds displayed reduced anti-coagulant activity, demonstrated varying affinities toward angiogenic growth factors (fibroblast growth factor-2, vascular endothelial growth factor and stromal cell-derived factor-1α), inhibited the P-selectin/P-selectin glycoprotein ligand-1 interaction and, notably, exhibited anti-tumor efficacy in a murine melanoma experimental metastasis model. Our results demonstrate that modulating specific sequences, especially the N-domains (-NS or -NH(2) or -NHCOCH(3)) in these polysaccharide sequences, has a major impact on the participation in a diverse range of biological activities. These results also suggest that the 6-O-sulfates, but not the 2-O-sulfates, critically affect the binding of a desulfated derivative to certain angiogenic proteins as well as its ability to inhibit P-selectin-mediated B16F10 melanoma metastases. Furthermore, N-desulfation followed by N-acetylation regenerates the affinity/inhibition properties to different extents in all the compounds tested in the in vitro assays. This systematic study lays a conceptual foundation for detailed structure function elucidation and will facilitate the rational design of targeted heparan sulfate proteoglycan-based anti-metastatic therapeutic candidates.

Characterization of heparin–protein interaction by saturation transfer difference (STD) NMR

The binding affinity and specificity of heparin to proteins is widely recognized to be sulfation-pattern dependent. However, for the majority of heparin-binding proteins (HBPs), it still remains unclear what moieties are involved in the specific binding interaction. Here, we report our study using saturation transfer difference (STD) nuclear magnetic resonance (NMR) to map out the interactions of synthetic heparin oligosaccharides with HBPs, such as basic fibroblast growth factor (FGF2) and fibroblast growth factor 10 (FGF10), to provide insight into the critical epitopes of heparin ligands involved. The irradiation frequency of STD NMR was carefully chosen to excite the methylene protons so that enhanced sensitivity was obtained for the heparin–protein complex. We believe this approach opens up additional application avenues to further investigate heparin–protein interactions.

Equivalent Gene Expression Profiles between Glatopa™ and Copaxone®

Glatopa™ is a generic glatiramer acetate recently approved for the treatment of patients with relapsing forms of multiple sclerosis. Gene expression profiling was performed as a means to evaluate equivalence of Glatopa and Copaxone®. Microarray analysis containing 39,429 unique probes across the entire genome was performed in murine glatiramer acetate—responsive Th2-polarized T cells, a test system highly relevant to the biology of glatiramer acetate. A closely related but nonequivalent glatiramoid molecule was used as a control to establish assay sensitivity. Multiple probe-level (Student’s t-test) and sample-level (principal component analysis, multidimensional scaling, and hierarchical clustering) statistical analyses were utilized to look for differences in gene expression induced by the test articles. The analyses were conducted across all genes measured, as well as across a subset of genes that were shown to be modulated by Copaxone. The following observations were made across multiple statistical analyses: the expression of numerous genes was significantly changed by treatment with Copaxone when compared against media-only control; gene expression profiles induced by Copaxone and Glatopa were not significantly different; and gene expression profiles induced by Copaxone and the nonequivalent glatiramoid were significantly different, underscoring the sensitivity of the test system and the multiple analysis methods. Comparative analysis was also performed on sets of transcripts relevant to T-cell biology and antigen presentation, among others that are known to be modulated by glatiramer acetate. No statistically significant differences were observed between Copaxone and Glatopa in the expression levels (magnitude and direction) of these glatiramer acetate-regulated genes. In conclusion, multiple methods consistently supported equivalent gene expression profiles between Copaxone and Glatopa.

Demonstration of Biological and Immunological Equivalence of a Generic Glatiramer Acetate

Background: In April 2015, the US Food and Drug Administration approved the first generic glatiramer acetate, Glatopa® (M356), as fully substitutable for Copaxone® 20 mg/mL for relapsing forms of multiple sclerosis (MS). This approval was accomplished through an Abbreviated New Drug Applica-tion that demonstrated equivalence to Copaxone. Method: This article will provide an overview of the methods used to establish the biological and immu-nological equivalence of the two glatiramer acetate products, including methods evaluating antigen-presenting cell (APC) biology, T-cell biology, and other immunomodulatory effects. Results: In vitro and in vivo experiments from multiple redundant orthogonal assays within four biologi-cal processes (aggregate biology, APC biology, T-cell biology, and B-cell biology) modulated by glati-ramer acetate in MS established the biological and immunological equivalence of Glatopa and Copaxone and are described. The following were observed when comparing Glatopa and Copaxone in these exper-iments: equivalent delays in symptom onset and reductions in “disease” intensity in experimental autoim-mune encephalomyelitis; equivalent dose-dependent increases in Glatopa- and Copaxone-induced mono-kine-induced interferon-gamma release from THP-1 cells; a shift to a T helper 2 phenotype resulting in the secretion of interleukin (IL)-4 and downregulation of IL-17 release; no differences in immunogenicity and the presence of equivalent “immunofingerprints” between both versions of glatiramer acetate; and no stimulation of histamine release with either glatiramer acetate in basophilic leukemia 2H3 cell lines. Conclusion: In summary, this comprehensive approach across different biological and immunological pathways modulated by glatiramer acetate consistently supported the biological and immunological equiv-alence of Glatopa and Copaxone.

Abstract LB-43: M402, a novel heparin sulphate mimetic, synergizes with gemcitabine to improve survival and reduce metastasis and epithelial-to-mesenchymal transition (EMT) in a genetically engineered mouse model for pancreatic cancer

Introduction: Heparan sulphate proteoglycans (HSPGs) play a central role in tumor progression and metastasis by presenting and modulating growth factors, cytokines, and other soluble factors. A novel heparin sulphate mimetic (M402), engineered from heparin to have low anti-coagulant activity, has shown promising anti-tumor efficacy in several pre-clinical tumor models. This study was designed to probe the efficacy and mechanism of action of M402 in a genetically engineered mouse (GEM) model for pancreatic cancer. Methods: Mice that spontaneously develop pancreatic cancer (LSL-KRASG12D/+; Trp53 LSL-R172H/flox; pdx-CRE) were treated with twice weekly i.p. doses of saline or gemcitabine (50 mg/kg) starting at Day 30, or with saline or M402 (40 mg/kg/day) administered by a subcutaneous osmotic minipump from Day 30-90, or with a combination of gemcitabine plus M402. Results: Treatment with M402 alone did not prolong survival and gemcitabine alone showed only a modest improvement in survival; however the combination of M402 and gemcitabine significantly improved survival. Moreover, mice treated with the combination of M402 and gemcitabine showed a substantially lower incidence of metastasis. RT-qPCR analysis revealed that M402 treated mice had significantly lower levels of TGF-alpha mRNA than the saline control group, which is corroborated by a corresponding decrease in tumor cell proliferation. Immunohistochemical analysis revealed that M402 treated mice developed reduced areas of epithelial-to-mesenchymal transition (EMT), as defined by negative staining for E-Cadherin, strongly positive staining for vimentin and positive nuclear staining for SNAIL. As there is a direct link between EGFR activation and the nuclear localisation of SNAIL we propose that M402 affects gemcitabine sensitivity and metastasis formation by reducing the expression of TGF-alpha. Discussion: M402 increased the anti-tumor efficacy of gemcitabine in a GEM model for pancreatic cancer resulting in increased survival and, interestingly, decreased incidence of metastasis. One potential mechanism is that the observed reduction in EMT may be due to the reduced expression of TGF-alpha, a cognate ligand for EGFR. These data suggest that the EGFR pathway is active in KRAS mutant tumors. Overall, these results provide a rationale for investigating the clinical use of M402 in combination with gemcitabine in the treatment of human pancreatic cancer. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-43.

Abstract 302: Characterization of effects of M402 on EMT in pancreatic ductal adenocarcinoma.

Background: Pancreatic ductal adenocarcinoma (PDA) is an almost uniformly lethal disease. The current treatment for resectable disease consists of surgery and adjuvant gemcitabine therapy. However, for more than 80 percent of the patients with PDA surgery is not an option. For these patients palliative chemotherapy using gemcitabine, or most recently FOLFIRINOX, is considered standard of care. Even with palliative chemotherapy the median overall survival for patients with metastatic PDA is dismal and improved therapeutic strategies are a clear unmet medical need. Heparins may be of interest for the treatment of PDA. They play an important role in tumor progression and metastasis by binding at heparin binding domains, thereby preventing growth factor gradients created along proteoglycan heparan sulfates and thus disrupting signaling. We used a novel heparan sulfate mimetic, M402, which binds to multiple growth factors, adhesion molecules, and chemokines. We previously showed that M402 decreases both epithelial-to-mesynchymal transition and chemotherapy resistance in PDA. Here we further evaluated the underlying mechanisms of M402 in EMT. Results: We studied the effects of M402 on PDA using a genetically engineered mouse model (GEMM) for PDA (LSL-KRAS G12D/+ ; Trp53 LSL-R172H/flox ; pdx-CRE), which recapitulates human PDA. Combination therapy of M402 with gemcitabine significantly prolonged the average survival of the mice when compared to mice treated with gemcitabine monotherapy (87 days versus 78 days) and significantly reduced metastasis and local invasion into the small intestine. These data suggest an effect of M402 on invasiveness and EMT. Gemcitabine treatment increased EMT as determined by staining for E-cadherin and Fsp1 double positive cells. In contrast, M402 treatment resulted in a decrease in E-cadherin and Fsp1 double positive cells in pancreatic tumors. We next sought to determine whether M402 affects cancer cells or stromal cells. In vitro experiments show that cell lines derived from our model respond to M402 by decreasing their invasive behavior in 3D culture and scratch assays. Moreover, we found that M402 augments the gemcitabine effect in vitro. These data suggest that M402 affects both EMT and gemcitabine response. Further identification by whole transcriptome microarray analysis of treated tumors hints towards a role of M402 in affecting multiple signaling pathways involved in the regulation of EMT. These data will undoubtedly lead to better insight into the mechanism of action of M402 and will increase our understanding of the pathways PDA cells use to evade the effects of chemotherapy. M402 is currently being investigated in a phase 1/2 M402 gemcitabine combination study to assess if these findings can be translated into a clinical benefit in pancreatic cancer patients. Conclusion: These data suggest that M402 reduces acquired chemotherapy resistance in a GEMM for PDA by decreasing EMT. Citation Format: Ilse Oosterom, Birgit C. Schultes, Chia Lin Chu, Zoya Galcheva-Gargova, Elma Kurtagic, He Zhou, Jay Duffner, Emile E. Voest, David A. Tuveson, Martijn Lolkema. Characterization of effects of M402 on EMT in pancreatic ductal adenocarcinoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 302. doi:10.1158/1538-7445.AM2013-302

Role of M402, a novel heparan sulfate mimetic, in pancreatic cancer cell invasion and metastasis: Inhibition of the Sonic Hedgehog pathway and heparanase activity.

25 Background: M402 is a novel heparin sulfate mimetic that binds to multiples growth factors, adhesion molecules, and cytokines to inhibit tumor angiogenesis, progression, and metastasis in nonclinical studies. We investigated if M402 could modulate tumor-stroma interactions in pancreatic cancer by inhibiting the Sonic Hedgehog (Shh) pathway as well as inhibit the activity of the extracellular matrix degrading enzyme, heparanase. METHODS Surface plasmon resonance (SPR) was used for analysis of M402 binding to Shh in vitro. A cell based Gli-1 reporter assay was implemented to assess the effect of M402 on Shh signaling. Immunohistochemistry and RT-qPCR were utilized to investigate M402s effect on Shh activity in an orthotopic Capan-2 model in nude mice. The effect of M402 on heparanase activity in vitro and on Capan-2 tumor samples isolated from treated and untreated mice was measured using an HTRF-FRET assay. RESULTS There was specific binding of M402 to Shh in vitro. Additionally, Shh signaling was inhibited in the presence of M402. Immunohistochemistry and RT-qPCR of Capan-2 tumor samples from animals treated with M402 also demonstrated reduction of Shh signaling via Gli, its targeted transcription factor. The degree of inhibition of heparanase activity, as measured in the HTRF assay, was affected by the size, structure, and sulfation pattern of the different heparin sulfate mimetics evaluated. M402 was the most potent inhibitor of heparanse activity in vitro from all compounds tested. In addition, treatment with M402 inhibited heparanase activity in the pancreatic tumor lysates in a dose-dependent manner. CONCLUSIONS M402 was shown in nonclinical studies to modulate tumor-stroma interactions involved in the metastatic, invasive, and desmoplastic pathways by simultaneously inhibiting two distinct pathways: Shh signaling and the activity of heparanase. M402 regulates a variety of polysaccharide-based binding proteins, which provides a rationale for the clinical investigation of M402 in a range of cancers.

Abstract 1524: M402, a novel heparan sulfate mimetic, inhibits pancreatic tumor growth and desmoplasia potentially via sonic hedgehog signaling in an orthotopic mouse model

One of the hallmarks of pancreatic ductal adenocarcinoma (PDAC) is extensive desmoplasia in the surrounding tumor microenvironment that prevents delivery of chemotherapeutics (such as gemcitabine). Dysregulation of sonic hedgehog (SHH, a heparin-binding morphogen), has been implicated in pancreatic cancer tumorigenesis. We have rationally designed a heparan sulfate mimetic, M402, that has previously been shown in animal studies to reduce metastatic seeding through disruption of several key heparin-binding growth factors (including FGF2, VEGF, SDF-1α and P-Selectin). We hypothesized that M402, alone or in combination with gemcitabine, could potentially modulate tumor-stroma interactions in an orthotopic pancreatic cancer model (Capan-2 cell line) which secretes SHH and displays a desmoplastic response in vivo. Capan-2 human adenocarcinoma cells (∼1x10 6 cells) were injected into the pancreata of immunodeficient mice (Nu/Nu CD-1). M402 (10-40 mg/kg/day) or saline treatment commenced on Day 4 or 32. Gemcitabine (30-60 mg/kg, twice weekly, IP) treatment commenced between Weeks 3-6. At the termination of each study (Week 8), gross anatomical observations were made of the primary pancreatic tumor and metastatic lesions in the surrounding tissues including the spleen, intestines and liver. Gemcitabine was increasingly less effective when started at later time points, but still reduced the primary tumor weight by 60-70% (at 30-45 mg/kg) when treatment was started at week 5. While the addition of M402 to gemcitabine showed some additive effect on primary tumor burden, metastasis, invasion, and surrounding fibrotic lesions appeared particularly impacted by the combination treatment. M402 was also effective as monotherapy showing a dose-dependent reduction in primary tumor weight and fibrotic lesions. Immunohistochemical and qPCR analyses showed reduced fibrosis and SHH signaling with M402 and gemcitabine combination treatment. These results demonstrate that M402 can modulate tumor-stroma interactions involved in the desmoplastic response in a murine model of pancreatic cancer and provide a rationale for the clinical investigation of M402 as a potential anti-desmoplastic agent in pancreatic cancer therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1524. doi:1538-7445.AM2012-1524