Robert MacLeod

Knockdown of endogenous circulating C1 inhibitor induces neurovascular impairment, neuroinflammation and cognitive decline

Plasma proteins and activated immune cells are known contributors of vascular brain disorders. However, the mechanisms and routes involved are still unclear. In order to understand the cross-talk between plasma proteins and the brain, we knocked down circulating C1 inhibitor (C1INH) in wild-type (WT) mice using antisense-oligonucleotide (ASO) technique and examined the brain. C1INH is a plasma protein inhibitor of vascular inflammation induced by activation of the kallikrein-kinin system (KKS) and the complement system. This knockdown induced the activation of the KKS but spared the activation of the classical complement system. Activation of the KKS induced an upregulation of the bradykinin pathway in the periphery and the brain, resulting in hypotension. Blood-brain barrier (BBB) permeability, plasma protein extravasations, activated glial cells and elevated levels of IL-1beta, IL-6, TNF-alpha, and iNOS were detected in brains of C1INH ASO treated mice. Infiltrating innate immune cells were evident, entering the brain through the lateral ventricle walls and the neurovascular units. The mice showed normal motor functions, however, cognition was impaired. Altogether, our results highlight the important role of regulated plasma-C1INH as a gatekeeper of the neurovascular system. Thus, manipulation of C1INH in neurovascular disorders might be therapeutically beneficial.Activation of the innate immune system, mediated by the complement and the contact systems, induces inflammation. The inhibition of these pathways by C1 inhibitor (C1INH) has been shown to decrease pro-inflammatory response and mediate vascular permeability in the periphery. However, the role of peripheral C1INH has not been associated with brain function. Using an antisense oligonucleotide (ASO) to deplete circulating liver-derived C1INH in wildtype (WT) mice, we induced the activation of the kallikrein-kinin system (KKS), which produced bradykinin in the plasma, resulting in hypotension. Interestingly, the complement system was quiescent. Depletion of liver-derived C1INH increased blood-brain barrier (BBB) permeability, increased expression of bradykinin 2 receptor, activated resident glial cells to secrete pro-inflammatory mediators such as Il-1beta, Il-6, TNF-alpha and iNOS, and induced cognitive decline. The results of this study emphasize the important role of circulating C1INH in mediating brain function through the activation of the KKS. Thus, manipulation of C1INH in neuro-vascular disorders might be therapeutically beneficial.

Knock-down of endogenous circulating C1 inhibitor induces neurovascular impairment and neuroinflammation

Plasma proteins and activated immune cells are known contributors of vascular brain disorders. However, the mechanisms and routes involved are still unclear. In order to understand the cross-talk between plasma proteins and the brain, we knocked down circulating C1 inhibitor (C1INH) in wild-type (WT) mice using antisense-oligonucleotide (ASO) technique and examined the brain. C1INH is a plasma protein inhibitor of vascular inflammation induced by activation of the kallikrein-kinin system (KKS) and the complement system. This knockdown induced the activation of the KKS but spared the activation of the classical complement system. Activation of the KKS induced an upregulation of the bradykinin pathway in the periphery and the brain, resulting in hypotension. Blood-brain barrier (BBB) permeability, plasma protein extravasations, activated glial cells and elevated levels of IL-1beta, IL-6, TNF-alpha, and iNOS were detected in brains of C1INH ASO treated mice. Infiltrating innate immune cells were evident, entering the brain through the lateral ventricle walls and the neurovascular units. The mice showed normal motor functions, however, cognition was impaired. Altogether, our results highlight the important role of regulated plasma-C1INH as a gatekeeper of the neurovascular system. Thus, manipulation of C1INH in neurovascular disorders might be therapeutically beneficial.Activation of the innate immune system, mediated by the complement and the contact systems, induces inflammation. The inhibition of these pathways by C1 inhibitor (C1INH) has been shown to decrease pro-inflammatory response and mediate vascular permeability in the periphery. However, the role of peripheral C1INH has not been associated with brain function. Using an antisense oligonucleotide (ASO) to deplete circulating liver-derived C1INH in wildtype (WT) mice, we induced the activation of the kallikrein-kinin system (KKS), which produced bradykinin in the plasma, resulting in hypotension. Interestingly, the complement system was quiescent. Depletion of liver-derived C1INH increased blood-brain barrier (BBB) permeability, increased expression of bradykinin 2 receptor, activated resident glial cells to secrete pro-inflammatory mediators such as Il-1beta, Il-6, TNF-alpha and iNOS, and induced cognitive decline. The results of this study emphasize the important role of circulating C1INH in mediating brain function through the activation of the KKS. Thus, manipulation of C1INH in neuro-vascular disorders might be therapeutically beneficial.

Knockdown of Liver-Derived C1 Inhibitor Results in Brain Pathology in mice

Plasma proteins and activated immune cells are known contributors of vascular brain disorders. However, the mechanisms and routes involved are still unclear. In order to understand the cross-talk between plasma proteins and the brain, we knocked down circulating C1 inhibitor (C1INH) in wild-type (WT) mice using antisense-oligonucleotide (ASO) technique and examined the brain. C1INH is a plasma protein inhibitor of vascular inflammation induced by activation of the kallikrein-kinin system (KKS) and the complement system. This knockdown induced the activation of the KKS but spared the activation of the classical complement system. Activation of the KKS induced an upregulation of the bradykinin pathway in the periphery and the brain, resulting in hypotension. Blood-brain barrier (BBB) permeability, plasma protein extravasations, activated glial cells and elevated levels of IL-1beta, IL-6, TNF-alpha, and iNOS were detected in brains of C1INH ASO treated mice. Infiltrating innate immune cells were evident, entering the brain through the lateral ventricle walls and the neurovascular units. The mice showed normal motor functions, however, cognition was impaired. Altogether, our results highlight the important role of regulated plasma-C1INH as a gatekeeper of the neurovascular system. Thus, manipulation of C1INH in neurovascular disorders might be therapeutically beneficial.Activation of the innate immune system, mediated by the complement and the contact systems, induces inflammation. The inhibition of these pathways by C1 inhibitor (C1INH) has been shown to decrease pro-inflammatory response and mediate vascular permeability in the periphery. However, the role of peripheral C1INH has not been associated with brain function. Using an antisense oligonucleotide (ASO) to deplete circulating liver-derived C1INH in wildtype (WT) mice, we induced the activation of the kallikrein-kinin system (KKS), which produced bradykinin in the plasma, resulting in hypotension. Interestingly, the complement system was quiescent. Depletion of liver-derived C1INH increased blood-brain barrier (BBB) permeability, increased expression of bradykinin 2 receptor, activated resident glial cells to secrete pro-inflammatory mediators such as Il-1beta, Il-6, TNF-alpha and iNOS, and induced cognitive decline. The results of this study emphasize the important role of circulating C1INH in mediating brain function through the activation of the KKS. Thus, manipulation of C1INH in neuro-vascular disorders might be therapeutically beneficial.

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, PAnnAZD9150 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.nnIHC 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.nnClinical 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.nnPreclinical 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.nnThese 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.nnCitation 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 C136: Exploring the functional contribution of STAT3 activity in tumor and tumor-associated stromal cells with species-selective generation 2.5 antisense oligonucleotides in preclinical cancer models.

The transcription factor STAT3 is a point of convergence for multiple signaling pathways and is activated in both tumor and non-tumor stromal cells in the tumor microenvironment. STAT3 activation mediates the expression of genes involved in many aspect of tumorigenesis. Thus, STAT3 has been hotly pursued as a target for cancer. Although inhibitors of STAT3 upstream signaling pathways have shown anti-tumor activity in preclinical animal models, the relative contributions of STAT3 in tumor cells versus tumor-associated stromal cells to the observed antitumor effects remains unclear. To address this question we have developed potent next generation (Generation 2.5) antisense oligonucleotides (ASOs) that are selective for either mouse or human STAT3 and we haveassessed their specificity, potency, and antitumor activities in several preclinical cancer models. The safety of targeting STAT3 with ASOs was first confirmed in both rodents and non-human primates. STAT3 ASOs were very well tolerated at doses that resulted in complete abrogation of STAT3 expression in the liver of treated animals, Next, we evaluated the relative contribution of STAT3 in tumor cells and tumor-associated stromal cells in a human ovarian cancer xenograft model (SKOV3), using both mouse and human specific STAT3 ASOs. Mouse and human STAT3 ASOs selectively reduced their respective target STAT3 mRNA levels in SKOV3 tumors. However, when we evaluated the level of IL-6, a key cytokine in ovarian cancer and known to be regulated by STAT3, it was significantly reduced only by the inhibition of the STAT3 in the tumor associated-stromal cells (mouse STAT3). Moreover, the antitumor activity of the mouse STAT3 was superior to that observed with inhibition of STAT3 in the tumor cells themselves, suggesting that the tumor promoting effects of STAT3 in this model is mediated through STAT3 activation in tumor-associated stromal cells. To evaluate the effects of STAT3 inhibition in a mouse model of cancer we employed the APC min model in which mice develop intestinal neoplasia at a young age. Treatment of APC min mice with mouse STAT3 ASOs led to a >90% inhibition of STAT3 mRNA levels in both the small intestine and colon. In addition, STAT3 ASO treatment resulted in a significant decrease in the number of polyps in the intestine. Finally, we evaluated the effects of human-specific STAT3 ASO in NSCLC s.c. xenograft models using either PC9 or primary human tumor explants. Human STAT3 ASO treatment resulted in >50% inhibition in STAT3 mRNA levels in the tumor cells and resulted in significant tumor growth inhibition (∼50% and 90% >, respectively), suggesting that relative contributions of mouse stromal cells to tumor growth may vary in different tumor types. Taken together, these results demonstrate that STAT3 ASO can selectively downregulate STAT3 in both tumor and stromal compartments, affect anti-tumor activity in vivo and that stromal derived STAT3 activity is important contributor to tumor growth and survival. 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 C136.

Abstract LB-302: Inhibition of STAT3 following systemic delivery of STAT3 antisense oligonucleotide results in target inhibition in tumor and tumor-associated stromal cells and dramatic reduction in circulating pro-tumorigenic cytokines

Constitutive activation of STAT3 has been documented in a broad range of neoplastic tissues. STAT3 is a transcription factor that regulates the expression of numerous genes involved in the proliferation, survival, and metastasis of tumor cells. In addition, STAT3 contributes to tumorigenesis by modulating immune responses mediated by tumor stromal cells in the tumor microenvironment. The immuno-suppressive, pro-tumorigenic effects of STAT3 are regulated by induction or inhibition of several key cytokines. However STAT3 remains a difficult target to drug with conventional approaches. Here, we have studied the effects of selective downregulation of STAT3 in various cancer settings by optimized antisense oligonucleotides (ASOs). We assessed the effects of STAT3 downregulation on the expression of cytokines and the progression of cancer. Initially, the safety of inhibiting STAT3 was demonstrated in tolerability studies, where no notable toxicities were observed despite near complete abrogation of STAT3 expression in the liver following systemic delivery of ASO in both rodents and in non-human primate. ASO treatment led to STAT3 inhibition in tumor cells and several non-tumor stromal cell populations of the tumor. In addition, dramatic reduction in key pro-tumorigenic cytokines including IL-6 and IL-1b was observed in C26 colon cancer-bearing animals, which correlated with the decrease of STAT3 level in tumor and stromal cells. This result suggests that STAT3 might not only transmit the IL-6 signal from upstream pathway, but regulates IL-6 level possibly in an auto-feedback manner. Downregulation of STAT3 also led to the inhibition of IL-6-induced C-reactive protein (CRP) expression, which was recently reported to play a key role in the progression of multiple myeloma (MM). Moreover, inhibition of STAT3 resulted in a significant delay in tumor growth in STAT3-dependent MDA-MB-231 breast cancer and U251 glioblastoma xenograft models following systematic ASO treatment. The results from this study collectively suggest that STAT3 expression can be specifically modulated by ASOs in vivo tumor models and that in addition to effects in tumor cells STAT3 ASOs regulate the balance of tumorigenic cytokines in the tumor microenvironment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-302. doi:10.1158/1538-7445.AM2011-LB-302