Ruban Mangadu

SD-208, a Novel Transforming Growth Factor β Receptor I Kinase Inhibitor, Inhibits Growth and Invasiveness and Enhances Immunogenicity of Murine and Human Glioma Cells In vitro and In vivo

The cytokine transforming growth factor (TGF)-β, by virtue of its immunosuppressive and promigratory properties, has become a major target for the experimental treatment of human malignant gliomas. Here we characterize the effects of a novel TGF-β receptor (TGF-βR) I kinase inhibitor, SD-208, on the growth and immunogenicity of murine SMA-560 and human LN-308 glioma cells in vitro and the growth of and immune response to intracranial SMA-560 gliomas in syngeneic VM/Dk mice in vivo. SD-208 inhibits the growth inhibition of TGF-β–sensitive CCL64 cells mediated by recombinant TGF-β1 or TGF-β2 or of TGF-β–containing glioma cell supernatant at an EC50 of 0.1 μmol/L. SD-208 blocks autocrine and paracrine TGF-β signaling in glioma cells as detected by the phosphorylation of Smad2 or TGF-β reporter assays and strongly inhibits constitutive and TGF-β–evoked migration and invasion, but not viability or proliferation. Peripheral blood lymphocytes or purified T cells, cocultured with TGF-β–releasing LN-308 glioma cells in the presence of SD-208, exhibit enhanced lytic activity against LN-308 targets. The release of interferon γ and tumor necrosis factor α by these immune effector cells is enhanced by SD-208, whereas the release of interleukin 10 is reduced. SD-208 restores the lytic activity of polyclonal natural killer cells against glioma cells in the presence of recombinant TGF-β or of TGF-β–containing glioma cell supernatant. The oral bioavailability of SD-208 was verified by demonstrating the inhibition of TGF-β–induced Smad phosphorylation in spleen and brain. Systemic SD-208 treatment initiated 3 days after the implantation of SMA-560 cells into the brains of syngeneic VM/Dk mice prolongs their median survival from 18.6 to 25.1 days. Histologic analysis revealed no difference in blood vessel formation, proliferation, or apoptosis. However, animals responding to SD-208 showed an increased tumor infiltration by natural killer cells, CD8 T cells, and macrophages. These data define TGF-β receptor I kinase inhibitors such as SD-208 as promising novel agents for the treatment of human malignant glioma and other conditions associated with pathological TGF-β activity.

A Selective p38α Mitogen-Activated Protein Kinase Inhibitor Reverses Cartilage and Bone Destruction in Mice with Collagen-Induced Arthritis

Destruction of cartilage and bone is a poorly managed hallmark of human rheumatoid arthritis (RA). p38 Mitogen-activated protein kinase (MAPK) has been shown to regulate key proinflammatory pathways in RA, including tumor necrosis factor α, interleukin (IL)-1β, and cyclooxygenase-2, as well as the process of osteoclast differentiation. Therefore, we evaluated whether a p38α MAPK inhibitor, indole-5-carboxamide (SD-282), could modulate cartilage and bone destruction in a mouse model of RA induced with bovine type II collagen [collagen-induced arthritis (CIA)]. In mice with early disease, SD-282 treatment significantly improved clinical severity scores, reduced bone and cartilage loss, and reduced mRNA levels of proinflammatory genes in paw tissue, including IL-1β, IL-6, and cyclooxygenase-2. Notably, SD-282 treatment of mice with advanced disease resulted in significant improvement in clinical severity scoring and paw swelling, a reversal in bone and cartilage destruction as assessed by histology, bone volume fraction and thickness, and three-dimensional image analysis. These changes were accompanied by reduced osteoclast number and lowered levels of serum cartilage oligomeric matrix protein, a marker of cartilage breakdown. Thus, in a model of experimental arthritis associated with significant osteolysis, p38α MAPK inhibition not only attenuates disease progression but also reverses cartilage and bone destruction in mice with advanced CIA disease.

p38 MAPK inhibition reduces diabetes-induced impairment of wound healing

In healthy tissue, a wound initiates an inflammatory response characterized by the presence of a hematoma, infiltration of inflammatory cells into the wound and, eventually, wound healing. In pathological conditions like diabetes mellitus, wound healing is impaired by the presence of chronic nonresolving inflammation. p38 mitogen-activated protein kinase (MAPK) inhibitors have demonstrated anti-inflammatory effects, primarily by inhibiting the expression of inflammatory cytokines and regulating cellular traffic into wounds. The db/db mouse model of type 2 diabetes was used to characterize the time course of expression of activated p38 during impaired wound healing. The p38α-selective inhibitor, SCIO-469, was applied topically and effects on p38 activation and on wound healing were evaluated. A topical dressing used clinically, Promogran™, was used as a comparator. In this study, we established that p38 is phosphorylated on Days 1 to 7 post-wounding in db/db mice. Further, we demonstrated that SCIO-469, at a dose of 10 μg/wound, had a positive effect on wound contraction, granulation tissue formation, and re-epithelialization, and also increased wound maturity during healing. These effects were similar to or greater than those observed with Promogran™. These results suggest a novel approach to prophylactic and therapeutic management of chronic wounds associated with diabetes or other conditions in which healing is impaired.

Selective p38α mitogen-activated protein kinase inhibitor attenuates lung inflammation and fibrosis in IL-13 transgenic mouse model of asthma

p38 Mitogen-activated protein kinase (MAPK) plays a critical role in the activation of inflammatory cells. We investigated the anti-inflammatory effects of a p38α-selective MAPK inhibitor (SD-282) in a mouse transgenic (CC10:IL-13) asthma model. The CC-10-driven over-expression of IL-13 in the mouse lung/airway has been shown to result in a remarkable phenotype recatitulating many features of asthma and characterized by eosinophilic and mononuclear inflammation, with airway epithelial cell hypertrophy, mucus cell metaplasia, the hyperproduction of neutral and acidic mucus, the deposition of Charcot–Leyden-like crystal, and airway sub-epitheilial fibrosis. Here we show how activated p38 MAPK can be observed in the lungs at the onset of asthma ie, around 8 weeks of age in both female and male mice. We also show that administration of a p38α MAPK selective inhibitor, SD-282 at 30 or 90 mg/kg, twice a day for a period of four weeks beginning at the onset of asthma, significantly reduced the inflammation (p < 0.001); hyperplasia of airway epithelium (p < 0.05); goblet cell metaplasia and mucus hypersecretion (p < 0.001) and reduced lung remodeling and fibrosis (p < 0.01), alleviating the severity of lung damage as measured by a composite score (p < 0.05). Furthermore, SD-282 significantly reduced activated p38 MAPK in the lymphocytes and epithelial cells (p < 0.001). Simultaneously, identical studies were conducted with an anti-fibrotic TGFβR1 kinase inhibitor (SD-208) which demonstrated anti-fibrotic but not anti-inflammatory properties. These findings suggest that the p38α-selective MAPK inhibitor may have dual therapeutic potential in attenuating both the inflammatory component and the fibrotic component of asthma and other Th2-polarized inflammatory lung diseases.

Preventive and Therapeutic Potential of p38α-Selective Mitogen-Activated Protein Kinase Inhibitor in Nonobese Diabetic Mice with Type 1 Diabetes

Mitogen-activated protein kinases (MAPKs) and heat shock proteins (HSPs) are ubiquitous proteins that function within T cells in both normal and stress-related pathophysiological states, including type 1 diabetes. The nonobese diabetic (NOD) mouse spontaneously develops T cell-mediated autoimmune pancreatic beta cell destruction that is similar to type 1 diabetes in humans. Because p38 MAPKs have been shown to modulate T cell function, we studied the effects of a p38α MAPK-selective inhibitor, indole-5-carboxamide (SD-169), on the development and progression of type 1 diabetes in the NOD mouse. In preventive treatment studies, SD-169 significantly reduced p38 and HSP60 expression in T cells of the pancreatic beta islets. Following treatment, the incidence of diabetes as determined by blood glucose levels was significantly lower, and immuno-histochemistry of pancreatic beta islet tissue demonstrated significant reduction in CD5+ T cell infiltration in the SD-169 treatment group as compared with untreated NOD mice. In therapeutic studies using mildly and moderately hyperglycemic NOD mice, SD-169 treatment lowered blood glucose and improved glucose homeostasis. Furthermore, following cessation of SD-169 treatment, NOD mice showed significant arrest of diabetes. In conclusion, we report that this p38α-selective inhibitor prevents the development and progression of diabetes in NOD mice by inhibiting T cell infiltration and activation, thereby preserving beta cell mass via inhibition of the p38 MAPK signaling pathway. These results have bearing on current prophylactic and therapeutic protocols using p38α-selective inhibitors in the prediabetic period for children at high risk of type 1 diabetes, in the honeymoon period, and for adults with latent autoimmune diabetes.

Characterization of MK-4166, a clinical agonistic antibody that targets human GITR and inhibits the generation and suppressive effects of T regulatory cells

GITR is a T-cell costimulatory receptor that enhances cellular and humoral immunity. The agonist anti-mouse GITR antibody DTA-1 has demonstrated efficacy in murine models of cancer primarily by attenuation of Treg-mediated immune suppression, but the translatability to human GITR biology has not been fully explored. Here, we report the potential utility of MK-4166, a humanized GITR mAb selected to bind to an epitope analogous to the DTA-1 epitope, which enhances the proliferation of both naïve and tumor-infiltrating T lymphocytes (TIL). We also investigated the role of GITR agonism in human antitumor immune responses and report here the preclinical characterization and toxicity assessment of MK-4166, which is currently being evaluated in a phase I clinical study. Expression of human GITR was comparable with that of mouse GITR in tumor-infiltrating Tregs despite being drastically lower in other human TILs and in many human peripheral blood populations. MK-4166 decreased induction and suppressive effects of Tregsin vitro In human TIL cultures, MK-4166 induced phosphorylation of NFκB and increased expression of dual specificity phosphatase 6 (DUSP6), indicating that MK-4166 activated downstream NFκB and Erk signaling pathways. Furthermore, MK-4166 downregulated FOXP3 mRNA in human tumor infiltrating Tregs, suggesting that, in addition to enhancing the activation of TILs, MK-4166 may attenuate the Treg-mediated suppressive tumor microenvironment. Cancer Res; 77(16); 4378-88. ©2017 AACR.

In vivo anti-tumor efficacy of afucosylated anti-CS1 monoclonal antibody produced in glycoengineered Pichia pastoris.

Monoclonal antibody (mAb) therapy has been successfully used for the treatment of B-cell lymphomas and is currently extended for the treatment of multiple myeloma (MM). New developments in MM therapeutics have achieved significant survival gains in patients but the disease still remains incurable. Elotuzumab (HuLuc63), an anti-CS1 monoclonal IgG1 antibody, is believed to induce anti-tumor activity and MM cytotoxicity through antibody dependent cellular cytotoxicity (ADCC) and inhibition of MM cell adhesion to bone marrow stromal cells (BMSCs). Modulations of the Fc glycan composition at the N297 site by selective mutations or afucosylation have been explored as strategies to develop bio-better therapeutics with enhanced ADCC activity. Afucosylated therapeutic antibodies with enhanced ADCC activity have been reported to possess greater efficacy in tumor growth inhibition at lower doses when compared to fucosylated therapeutic antibodies. The N-linked glycosylation pathway in Pichia pastoris has been engineered to produce human-like N-linked glycosylation with uniform afucosylated complex type glycans. The purpose of this study was to compare afucosylated anti-CS1 mAb expressed in glycoengineered Pichia pastoris with fucosylated anti-CS1 mAb expressed in mammalian HEK293 cells through in vitro ADCC and in vivo tumor inhibition models. Our results indicate that Fc glycosylation is critical for in vivo efficacy and afucosylated anti-CS1 mAb expressed in glycoengineered Pichia pastoris shows a better in vivo efficacy in tumor regression when compared to fucosylated anti-CS1 mAb expressed in HEK293 cells. Glycoengineered Pichia pastoris could provide an alternative platform for generating homogeneous afucosylated recombinant antibodies where Fc mediated immune effector function is important for efficacy.

Abstract 269: Evaluation of the antitumor activity of anti-PD-1 immunotherapy as a single agent and in combination with approved agents in preclinical tumor models

Pembrolizumab (MK-3475), a humanized monoclonal IgG4 antibody against programmed death receptor 1 (PD-1), is currently being studied in clinical trials across more than 30 types of cancers. To further support the clinical development of pembrolizumab and to aid the mechanistic understanding of anti-PD-1 immunotherapy, we generated a surrogate PD-1-blocking antibody (muDX400). We have used muDX400 to determine the antitumor activity, pharmacokinetics, and pharmacodynamics of PD-1 inhibition in multiple preclinical syngeneic tumor model systems. Response to muDX400 treatment in several syngeneic tumor models was broadly classified into 3 categories: highly responsive (ie, complete and durable tumor regressions were observed), partially responsive (ie, tumor growth inhibition was observed), and intrinsically resistant to therapy. Gene and protein expression signatures revealed that the more responsive models expressed higher levels of both PD-1 ligand (PD-L1) and tumor-infiltrating lymphocytes compared with nonresponsive models. To further evaluate mechanisms that could potentially enhance the antitumor activity of anti-PD-1 in these tumor models, muDX400 was combined with a number of different chemotherapies, targeted therapies, and other immunotherapies. Because immune suppression is a common side effect associated with many standard-of-care therapies, we evaluated the potential abrogation of muDX400-mediated antitumor activity when combined with approved therapies by scheduling the dosing regimen to examine concurrent and sequential administration of these agents. In the models in which enhanced antitumor activity was evident, we evaluated the immune landscape of blood, tumors, and draining lymph nodes by immuno-phenotyping and molecular profiling. These data provide preclinical support to expand the clinical development of pembrolizumab into additional cancer types as both a single agent and in combination with other approved anticancer therapies. Additional studies with muDX400 are ongoing to further elucidate the mechanism of action of PD-1 blockade and to better understand the antitumor responses observed in clinical trials of pembrolizumab. Citation Format: Elaine M. Pinheiro, Ruban Mangadu, Uyen T. Phan, Mingmei Cai, Yanhong Ma, Heather A. Hirsch, Terrill K. McClanahan, Raymond J. Moniz, Ali-Samer Al-Assaad, Samik Basu, Yaolin Wang, Venkataraman Sriram, Joseph H. Phillips, Brian J. Long. Evaluation of the antitumor activity of anti-PD-1 immunotherapy as a single agent and in combination with approved agents in preclinical tumor models. [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 269. doi:10.1158/1538-7445.AM2015-269

Abstract B114: Evaluation of the antitumor activity and molecular characterization of mouse syngeneic tumor models in response to anti-PD-1 treatment as a single agent and in combination with approved agents

Pembrolizumab (MK-3475), a humanized monoclonal IgG4 antibody against programmed death receptor 1 (PD-1), is currently being studied in clinical trials across more than 30 types of cancers. To further support the clinical development of pembrolizumab and to aid in the mechanistic understanding of anti–PD-1 immunotherapy, we generated a surrogate PD-1–blocking antibody (muDX400). We have used muDX400 to determine the antitumor activity, pharmacokinetics, and pharmacodynamics of PD-1 inhibition in multiple preclinical syngeneic tumor model systems. Response to muDX400 treatment in several syngeneic tumor models was broadly classified into 3 categories: highly responsive (ie, complete and durable tumor regressions were observed), partially responsive (ie, tumor growth inhibition was observed), and intrinsically resistant to therapy. Using a multifaceted approach, tumors from these models were extensively characterized at the molecular and cellular level by gene expression profiling, whole exome sequencing, fluorescence-activated cell sorting, and immunohistochemistry to help elucidate mechanisms of action and biology associated with response and resistance to anti-PD1 treatment. Analyses included but were not limited to the evaluation of mutational burden, immune cell activation and migration, interferon signaling, antigen presentation (major histocompatibility [MHC] class I and II), and expression of novel targets.To further evaluate mechanisms that could potentially enhance the antitumor activity of anti–PD-1 in these tumor models, muDX400 was combined with a number of different chemotherapies, targeted therapies, and other immunotherapies. In the models in which enhanced antitumor activity was evident, we evaluated the immune landscape of blood, tumors, and draining lymph nodes by molecular profiling. These data provide preclinical support to expand the clinical development of pembrolizumab into additional cancer types as both a single agent and in combination with other approved anticancer therapies. Additional studies with muDX400 are ongoing to further elucidate the mechanism of action of PD-1 blockade and to better understand the antitumor responses observed in clinical trials of pembrolizumab. Citation Format: Heather Hirsch, Ruban Mangadu, Mingmei Cai, Yanhong Ma, Uyen Phan, Yaolin Wang, Venkataraman Sriram, Joseph H. Phillips, Terri McClanahan, Brian Long, Elaine M. Pinheiro. Evaluation of the antitumor activity and molecular characterization of mouse syngeneic tumor models in response to anti-PD-1 treatment as a single agent and in combination with approved agents. [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 B114.

Abstract 543: Preclinical combination strategies to enhance the efficacy of the anti-PD-1 antibody pembrolizumab

Pembrolizumab (MK-3475), a humanized monoclonal IgG4 antibody against programmed death receptor 1 (PD-1), has shown activity in multiple types of cancer. Although pembrolizumab is showing a dramatic impact in patients that respond, a large fraction of patients do not respond to single agent therapy. Combination approaches may be the key to improving response rates in these patients. One of the challenges is how best to tailor combination strategies to provide maximal benefit to patients. How we prioritize new combinations will require an understanding of which mechanism is best for which tumor. To elucidate mechanisms that may create an immunogeneic tumor microenvironment and enhance the antitumor activity of PD-1 blockade, we have tested combination strategies with muDX400, a murine anti-PD-1 antibody. The murine preclinical syngeneic tumor models selected for these studies had varied tumor microenvironment compositions and displayed a range of responses to therapy with single agent anti-PD-1. Here we show enhanced anti-tumor activity when PD-1 blockade is combined with conventional chemotherapies, small molecule therapies and additional immunotherapy approaches that target multiple steps in the immune-oncology pathway. Pre and post treatment tumors from these models were extensively characterized by gene expression profiling, whole exome sequencing, flow cytometry and immunohistochemistry analysis. Molecular and cellular correlates of response to anti-PD-1 as a single agent and in combination with these agents were evaluated. These data support potential combination strategies that could either increase efficacy or expand the patient population in which pembrolizumab will demonstrate benefit. Citation Format: Elaine M. Pinheiro, Marlene Hinton, Ruban Mangadu, Mingmei Cai, Uyen Phan, Michael Nebozhyn, Heather Hirsch, Andrey Loboda, Sarah Javaid, Yaolin Wang, Venkataraman Sriram, Joseph H. Phillips, Terri McClanahan, Brian Long. Preclinical combination strategies to enhance the efficacy of the anti-PD-1 antibody pembrolizumab. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 543.