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American Macular Degeneration Foundation
American Neurological Association
Complement Pathways in Disease
Tuesday, April 25, 2017
The New York Academy of Sciences, 7 World Trade Center, 250 Greenwich St Fl 40, New York, USA
The complement system is the first line of defense against infection, and is thus a critical step in maintaining biological health in response to invading pathogens. However, uncontrolled activation of complement – occurring commonly as a sequela to a number of diseases including kidney transplant, immune-complex mediated glomerulonephritis (lupus nephritis, membranous nephropathy), myasthenia gravis, neuromyelitis optica, multiple sclerosis, Alzheimer’s disease, and autoimmune uveitis – can lead to host cell injury and ultimately end organ damage, creating a significant medical burden in a large population of already compromised individuals.
Although the therapeutic role for complement blockade is not clear, it is an active area of research. This symposium will focus on novel insights into the complement pathway in a number of clinical disorders and offer potential therapeutic implications.
Registration Pricing
| Member | $60 |
| Member (Student / Postdoc / Fellow) | $25 |
| Nonmember (Academia) | $105 |
| Nonmember (Corporate) | $160 |
| Nonmember (Non-profit) | $105 |
| Nonmember (Student / Postdoc / Fellow) | $70 |
Organizers
Keynote Speaker
Speakers
Agenda
* Presentation times are subject to change.
Tuesday, April 25, 2017 | |
8:30 AM | Breakfast and Registration |
9:00 AM | Introduction and Welcome Remarks |
9:15 AM | Keynote Lecture |
Session 1 | |
10:00 AM | The Role of Complement in Experimental Renal Diseases |
10:35 AM | Networking Coffee Break |
11:05 AM | Utilizing Complement Evasion Strategies to Design Complement-Based Antibacterial Immuno-therapeutics: Lessons from the Pathogenic Neisseriae |
11:40 AM | Data Evaluation of Anti-human Complement Component 5 (C5) Antibody in Humanized C5 Mice |
11:55 AM | Complement and Microglia Mediate Sensory-Motor Synaptic Loss in Spinal Muscular Atrophy |
12:10 PM | Networking Lunch and Poster Viewing |
Session 2 | |
1:40 PM | The Role of the Complement Alternative Pathway in C3 Glomerulopathy (C3G) |
2:15 PM | New Insights into Complement and Transplantation |
2:50 PM | Networking Coffee Break |
3:20 PM | Complement-Activation and Age-related Macular Degeneration: Mechanisms, Treatments, and Diagnostics |
3:55 PM | How Complement Eliminates Synapses in Health and Disease |
4:30 PM | Closing Remarks and F1000 Poster Prize Presentation |
4:40 PM | Networking Reception |
5:40 PM | Adjourn |
Organizers
Kishor Devalaraja, PhD, DVM
Regeneron Pharmaceuticals
Dr. Kishor Devalaraja-Narashimha obtained Doctor of Veterinary Medicine degree from India before moving to the USA to pursue research as his career. He joined University of Nebraska Medical Center, Omaha, Nebraska, to pursue PhD. Kishor's thesis research focus was to elucidate molecular mechanisms of programmed cell death in acute kidney injury. He has identified PARP1 and cyclophilin-D as key molecules contributing to programmed necrotic cell death of proximal tubular cells following acute kidney injury. He also uncovered novel biological role of PARP1 and cyclophilin D in obesity. His work has been published in several peer-reviewed journals. After completing his doctoral degree, Kishor joined Regeneron as a postdoctoral scientist in the Cardiovascular and Renal therapeutic focus area, where he established preclinical research work to support target discovery for a number of kidney diseases. He currently works as a senior staff scientist in the company where he leads a team focusing on therapeutic target research and discovery, and drug development for both acute and chronic kidney diseases.
Scott MacDonnell, PhD
Regeneron Pharmaceuticals
Dr. Scott MacDonnell obtained his undergraduate and master's degrees in exercise physiology from the University of Delaware and completed his doctoral work in cardiovascular physiology at Temple University in Philadelphia, PA. He completed a post-doctoral fellowship at Temple University Medical School in the lab of Dr. Steve Houser. His fellowship research focused on identifying mechanisms responsible for the pathogenesis of heart failure. Specifically, his work examined the role of CaMKII in altered contractility, myocytes apoptosis, and transcriptional regulation associated with heart failure progression. This work has been published in Circulation Research and recognized as a best manuscript by the editorial board in 2010. Additionally, Scott was recognized by the International Society for Heart Research and awarded the young investigator of the year in 2008. Scott worked for 8-years as a principal scientist at Boehringer Ingelheim within the department of CardioMetabolic Disease Research where his research focused on identifying novel therapeutic treatment options for chronic kidney disease, heart failure, and fibrosis. Scott currently works at Regeneron Pharmaceuticals in the department of Cardiovascular and Renal Disease where he leads an in vitro group supporting both screening and functional assay development for heart failure, systemic hypertension, pulmonary fibrosis and hypertension, and both acute and chronic renal indications.
Lori Morton, PhD
Regeneron Pharmaceuticals
George Zavoico, PhD
JonesTrading Institutional Services
George B. Zavoico, PhD, is a Senior Equity Analyst, Healthcare, at JonesTrading Institutional Services, a leading equity trading firm with a focus on block trading and a growing capital markets business. He has over 11 years of experience as a life sciences analyst writing research on publicly traded equities. His principal focus is on biotechnology, biopharmaceutical, specialty pharmaceutical, and molecular diagnostics companies. He received The Financial Times / Starmine Award two years in a row for being among the top-ranked earnings estimators in the biotechnology sector. In 2009, Zavoico was hired as the first equity analyst at MLV & Co., a New York-based boutique investment bank and institutional broker-dealer at the time, where he helped establish its Healthcare research team. He returned to MLV in mid-2014 after serving for a brief period as a Senior Equity Analyst at H.C. Wainwright & Co. in early 2014, and then joined JonesTrading in early 2015.
Previously, Zavoico was an equity research analyst in the healthcare sector at Westport Capital Markets and Cantor Fitzgerald. Prior to working as an analyst, Zavoico established his own consulting company serving the biotech and pharmaceutical industries, providing competitive intelligence and marketing research, due diligence services and guidance in regulatory affairs. Zavoico began his career as a senior research scientist at Bristol-Myers Squibb Co., moving on to management positions at Alexion Pharmaceuticals Inc. and T Cell Sciences Inc. (now Celldex Therapeutics Inc.). Zavoico has a bachelor's degree in biology from St. Lawrence University and a PhD in physiology from the University of Virginia. He held post-doctoral fellowships at the University of Connecticut School of Medicine and Harvard Medical School / Brigham & Women's Hospital. He has published more than 30 papers in peer-reviewed journals and has coauthored four book chapters.
Sonya Dougal, PhD
The New York Academy of Sciences
Caitlin McOmish, PhD
The New York Academy of Sciences
Keynote Speaker
V. Michael Holers, MD
University of Colorado School of Medicine
Dr. Holers received his MD degree from Washington University School of Medicine in St. Louis. Following clinical training in medicine and rheumatology, he joined the laboratory of Dr. John Atkinson at Washington University, and in 1986 he became a faculty member there. In 1993 Dr. Holers moved to the University of Colorado as the first Smyth Professor of Rheumatology. Following promotion to Professor of Medicine and Immunology, in 2000 he became Head of the Division of Rheumatology and in 2008 the Scoville Professor. The focus of Dr. Holers' complement research program has been on the molecular genetics of complement C3 receptors and membrane regulatory proteins, the in vivo roles of these proteins, and the development of complement therapeutics. The Holers' laboratory has recently focused on defining the unexpectedly important role of the lectin pathway and the alternative pathway amplification loop, as well as novel innate immune tissue-recognition mechanisms, in the generation of complement-dependent tissue injury in vivo. These mechanisms form the basis for new therapeutic and diagnostic approaches to the treatment of complement-dependent diseases. In recognition of his scientific advances and academic roles, Dr. Holers was elected to membership in the ASCI in 1993 and the AAP in 2000.
Speakers
David Apelian, MD, PhD, MBA
Achillion Pharmaceuticals
Dr. Apelian brings more than 18 years of industry clinical development experience from Bristol-Myers Squibb (BMS), Schering Plough and GlobeImmune where he focused on hepatology, immuno-oncology, and infectious diseases. Dr. Apelian was Clinical Director in the Infectious Diseases Group at BMS, serving as medical co-lead for the clinical development and NDA submission of entecavir for chronic hepatitis B viral infection. Prior to BMS, Dr. Apelian served as Clinical Director in the Department of Hepatology/Gastroenterology at Schering Plough, coordinating a supplemental NDA filing for interferon alpha-2b and ribavirin for the treatment of pediatric patients with chronic hepatitis C viral infection. Most recently, Dr. Apelian served as Senior Vice President of Research and Development and Chief Medical Officer at GlobeImmune, where he was responsible for clinical development, regulatory affairs, clinical immunology, development of companion diagnostics, as well as target discovery and preclinical research. Dr. Apelian currently serves as Executive Vice President and Chief Medical Officer of Achillion Pharmaceuticals where his responsibilities include the clinical development of complement alternative pathway inhibitors that target factor D for the treatment of rare debilitating diseases such as paroxysmal nocturnal hemoglobinuria and C3 glomerulonepropathy. Dr. Apelian completed his residency training in Pediatrics at New York Hospital, Cornell Medical Center. He received his MD from the University of Medicine and Dentistry of New Jersey, and his PhD in Biochemistry and BA from Rutgers University. He also holds an MBA from Quinnipiac University.
Peter S. Heeger, MD
Icahn School of Medicine at Mount Sinai
Peter S. Heeger, MD is a graduate of the University of Pennsylvania School of Medicine (1984). He completed medical residency training along with a Chief Medical Resident year at Temple University Hospital in Philadelphia (1984-1988) and then clinical and research Nephrology training at the University of Pennsylvania (1988–93). After developing a research interest in transplantation in Cleveland OH (Case Western and the Cleveland Clinic), Dr. Heeger moved to the Icahn School of Medicine at Mount Sinai (NY) where he is currently the Irene and Arthur Fishberg Professor of Medicine and Director of the Translational Transplant Research Center. In addition to being a dedicated educator (currently directs the Immunology course at Icahn School of Medicine at Mount Sinai), Dr. Heeger has been continuously funded through the National Institutes of Health for 21 years for studies in transplantation immunobiology. He currently leads an NIH consortium, Clinical Trials in Organ Transplantation, which is conducting multiple international trials to assess the utility of noninvasive biomarkers to predict outcomes and guide treatment strategies in transplant recipients. Dr. Heeger's basic science research interests are in mechanisms of allograft injury and tolerance, including studies on interactions between the complement system and T lymphocytes. Dr. Heeger is the author of more than 180 publications. He served as the Chair and a member of the TTT NIH study section for grant reviews, is an associate editor and reviewer for multiple immunology, transplant and nephrology journals (including J Clin Invest and Am J Transplantation), and was the 2011 recipient of the Established Investigator Award from the American Society of Transplantation.
Ying Hu, PhD
Regeneron Pharmaceuticals, Inc.
Ying Hu is a staff scientist in Ophthalmology at Regeneron Pharmaceutical Inc. Ying earned his medical degree in Nankai University in China. He was awarded his PhD with Distinction in Neuroscience from the University of Western Australia. In 2007 he moved to the USA for his postdoctoral training in Dr. Jeffery Goldberg's lab at Bascom Palmer Eye Institute. His research has been focused on neuroprotection and axonal regeneration of retinal ganglion cells. Recently, he also works on translating animal research into medicines in the disease such as glaucoma, and dry age-related macular degeneration.
Richard J. Quigg, Jr, MD
University at Buffalo, The State University of New York
Dr. Quigg graduated summa cum laude from the Boston University Six-Year Medical Program. Following clinical training in medicine and nephrology, he trained in the research laboratory of Dr. David Salant at Boston University. In 1994, Dr. Quigg moved to the University of Chicago. In 1999 he became Chief of the Section of Nephrology. He left the University of Chicago in 2013 as Emeritus Professor of Medicine to become the inaugural Arthur Morris Professor of Medicine and Chief of the Division of Nephrology at the State University of New York at Buffalo. He is also Professor of Biomedical Informatics and Board Certified in Clinical Informatics. Dr. Quigg was elected to membership in the ASCI in 2003 and is an alumnus inductee of the Alpha Omega Alpha National Honor Medical Society. The focus of Dr. Quigg's complement research program has been on the role of complement C3 and C5 receptors and membrane regulatory proteins in experimental and clinical renal diseases.
Peter A. Rice, MD
University of Massachusetts Medical School
Dr. Rice is Professor of Medicine at the University of Massachusetts Medical School, His research involves studies of human immunology of Neisseria gonorrhoeae infection. In particular, his current research focuses on immune mechanisms, antibodies in particular, that may confer protection against gonococcal infection. Dr. Rice also examines the role of complement (C) in gonococcal (and meningococcal) infection, particularly the role of complement regulators such as human factor H, an alternative pathway of complement regulator and human C4b-binding protein (C4BP). All strains of N. gonorrhoeae bind human factor H and many bind human C4BP. These complement regulators, factor H and C4BP, (negatively) influence complement mediated functions of antibodies and can subvert potentially protective antibody functions such as those induced by vaccine candidates—in Dr. Rice's case, gonococcal vaccine candidates, which Dr. Rice's laboratory has also developed and tested in human complement regulator transgenic mice. In addition to vaccine development, Dr. Rice is also developing and testing antibody therapy directed against antimicrobial resistant (AMR) N. gonorrhoeae that will serve as adjunctive therapy for the treatment of AMR gonococcal disease.
Bärbel Rohrer, PhD
Medical University of South Carolina
Dr. Bärbel (Barb) Rohrer is Professor and Endowed Chair in the Department of Ophthalmology at MUSC and is an academic and innovative leader in diseases of the retina. Her lab is investigating mechanism of photoreceptor degeneration and neuroprotection, focusing on two areas: targeting complement activation in models of age-related macular degeneration and improving mitochondrial metabolism as a means to promote life-span in neurons. She holds eleven U.S. and international patents, with an additional 24 applications pending. Her IP provided the foundation for three start-up companies, one of which she co-founded. Dr. Rohrer has published more than 80 manuscripts; PI'd or Co-PI'd 40+ grants; and mentored 50+ trainees. In addition to the numerous intramural committees on which Dr. Rohrer serves, she is a Foundation Fighting Blindness Scientific Advisory Board member, member of multiple professional societies, including the Association for Research in Vision and Ophthalmology and the Society for Neuroscience, and elected member of the National Academy of Inventors.
Beth Stevens, PhD
Harvard Medical School, Boston Children's Hospital
Beth Stevens is an associate professor at Harvard Medical School in the FM Kirby Neurobiology Research Center at Boston Children's Hospital, and an institute member of the Broad Institute. Her research seeks to understand the mechanisms that regulate the development and elimination of synapses by focusing on how microglia and immune-related molecules mediate this process. Beth received her PhD in Neuroscience in 2003 at the University of Maryland, College Park. She performed her dissertation research at the National Institutes of Health (NICHD) in the area of neuron-glia interactions. In her postdoctoral work with Ben Barres at Stanford University, she discovered that the classical complement cascade, part of the innate immune system, helps to mediate developmental CNS synapse elimination. Their findings have raised many questions about how the complement cascade normally works to eliminate synapses and especially whether it becomes abnormally reactivated in brain diseases such as AD that impair synaptic connectivity. In 2008, Dr. Stevens established her independent laboratory in the FM Kirby Neurobiology Center at Children's Hospital where she is currently using a combination of molecular, physiological and high resolution imaging techniques to dissect the mechanisms by which microglial cells and immune-related molecules (ie.complement, cytokines) regulate synapse function during health and disease. She is investigating the mechanisms that drive synapse loss and dysfunction in AD, Huntington's disease, as well as neurodevelopmental disorders, such as autism and schizophrenia. Beth is a recipient of several young investigator awards, including: Ellison Medical Foundation New Scholar in Aging, John Merck Scholar (2011), Presidential Early Career Award for Scientists and Engineers (PECASE), and a 2015 MacArthur Fellow Award.
Aleksandra Vukojicic, MSc
Center for Motor Neuron Biology and Disease, Columbia University
Aleksandra Vukojicic is a PhD candidate in Dr. George Mentis laboratory at Columbia University. Her doctoral studies are focused on the mechanisms involved in the loss and dysfunction of synapses contacting motor neurons early in the time course in the neurodegenerative disease of Spinal Muscular Atrophy.
Abstracts
Keynote: Harnessing Innate Immune Recognition of Injured Tissues for Therapeutic and Diagnostic Purposes
V. Michael Holers, MD, University of Colorado School of Medicine, Denver
The complement system plays a central role in the pro-inflammatory mechanisms that drive the pathogenesis of many human diseases. Two important therapeutic goals have been to develop drugs that minimize systemic inhibition of this pathway by their targeting to sites of complement activation as well as a means to monitor levels of in vivo complement activation in these specific tissues. Our studies have identified specific injury-associated neo-epitopes that develop locally and are recognized by a group of target-restricted pathogenic natural antibodies. In both ischemic and non-ischemic tissues, unique monoclonal antibodies (mAbs) from this natural antibody subset can initiate complement-mediated injury. In a novel therapeutic strategy, the ScFv from one pathogenic mAb has been linked to the murine complement C3/C5 convertase inhibitor Crry and been shown to durably target to injured tissues in wild type mice and protect from the development of complement-dependent injury. In parallel, as complement C3d fragments, derived from the circulation or from intracellular C3, are fixed to host cells and tissues during the immune response, and thus can serve as biomarkers of ongoing inflammation, we have developed a method to perform quantitative in vivo live animal imaging of C3d fixation to complement-targeted tissue sites. In aggregate, these studies illustrate a new therapeutic targeting and related biomarker monitoring strategy which takes advantage of new insights into the mechanisms of "danger" recognition and subsequent local complement fixation by natural antibodies in order to modulate specific targets in the pathway in a safer, more precise and highly informed manner.
Coauthors: Stephen Tomlinson1, Catherine A. Foss2, Liudmila Kulik3, Carl Atkinson1, Bärbel Rohrer1, Nirmal K. Banda3, Martin G. Pomper2, and Joshua M. Thurman3.
1. Medical University of South Carolina, Charleston.
2. Johns Hopkins University School of Medicine, Baltimore.
3. University of Colorado School of Medicine, Denver.
The Role of the Complement Alternative Pathway in C3 Glomerulopathy (C3G)
David Apelian, MD, PhD, MBA, Achillion Pharmaceuticals
The complement system, a complex network of proteins functioning in a tightly regulated cascade, is a critical component of the human immune and defense system. It consists of three inter-related pathways: lectin, classical and alternative (AP). The AP is different from the other two in that its activation does not require interaction with a pathogen or other activating molecule. Rather, it is constitutively active at low levels due to continuous and spontaneous hydrolysis of C3 (C3 tick-over). Furthermore, it amplifies classical and lectin activity via the "amplification loop."
The term C3G was introduced to categorize kidney lesions with the common feature of predominant C3 glomerular deposition in the setting of excessive AP activity. Prior classifications (e.g. membranoproliferative glomerulonephritis Types I, II and III) did not differentiate based on the underlying pathophysiology (1). Animal models have firmly established that AP dysregulation leads to C3G. Furthermore, the majority of genetic and acquired factors predisposing to clinical C3G are known to drive AP dysregulation (1, 2).
Given the pathophysiology of C3G as a disease of excessive AP activity, treatment of the disease with an AP complement inhibitor warrants further development.
References:
1. Kidney International (2013) 84, 1079–1089
2. J Am Soc Nephrol (2015) Vol 26, No. 12, 2917–2929
Utilizing Complement Evasion Strategies to Design Complement-Based Antibacterial Immuno-therapeutics: Lessons from the Pathogenic Neisseriae
Peter A. Rice, MD, Division of Infectious Diseases and Immunology, University of Massachusetts Medical School
Novel therapies are urgently needed to combat the global threat of multidrug-resistant pathogens. Complement forms an important arm of innate defenses against infections. In physiological conditions, complement activation is tightly controlled by soluble and membrane-associated complement inhibitors, but must be selectively activated on invading pathogens to facilitate microbial clearance. Many pathogens, including Neisseria gonorrhoeae and N. meningitidis, express glycans, including N-acetylneuraminic acid (Neu5Ac), that mimic host structures to evade host immunity. Neu5Ac is a negatively charged 9-cabon sugar that inhibits complement, in part by enhancing binding of the complement inhibitor factor H (FH) through C-terminal domains (19 and 20) on FH. Other microbes also bind FH, in most instances through FH domains 6 and 7 or 18-20. Here we describe two strategies to target complement activation on Neisseriae. First, microbial binding domains of FH were fused to IgG Fc to create FH18-20/Fc (binds gonococci) and FH6,7/Fc (binds meningococci). A point mutation in FH domain 19 eliminated hemolysis caused by unmodified FH18-20, but retained binding to gonococci. FH18-20/Fc and FH6,7/Fc mediated complement-dependent killing in vitro and showed efficacy in animal models of gonorrhea and meningococcal bacteremia, respectively. The second strategy utilized CMP-nonulosonate (CMP-NulO) analogs of sialic acid that were incorporated into LOS and prevented complement inhibition by physiologic CMP-Neu5Ac that resulted in attenuated gonococcal infection in mice. While studies to establish the safety of these agents are needed, enhancing complement activation on microbes may represent a promising strategy to treat antimicrobial resistant organisms.
Coauthors: Sanjay Ram, Jutamas Shaughnessy, Rosane B. DeOliveira, Lisa A. Lewis, and Sunita Gulati; Division of Infectious Diseases and Immunology, University of Massachusetts Medical School.
Complement-Activation and Age-related Macular Degeneration: Mechanisms, Treatments, and Diagnostics
Bärbel Rohrer, PhD, Medical University of South Carolina, Charleston
Age-related macular degeneration (AMD) is the leading cause of blindness in the U.S. Polymorphisms in complement components are associated with increased AMD risk. It has been hypothesized that an overactive complement system is involved in AMD pathology, resulting from multifactorial injury by the combination of anaphylatoxin- (C3a and C5a) and membrane attack complex-signaling. Our goal is to understand mechanisms, pathways and target-cells of complement in AMD, and to develop rational complement therapeutics and diagnostics. Mouse models that exhibit features of AMD are used; laser-induced choroidal neovascularization (CNV) and long-term smoke-induced ocular pathology (SIOP). In these models, we showed that the alternative pathway (AP) of complement is required for controlling angiogenesis, for triggering RPE damage, and for Bruch's membrane thickening and vision loss. C3d deposition was demonstrated in lesions in vivo using fluorescently-labeled antibodies. We developed a strategy for "addressable" inhibitors that are targeted to sites of complement activation, by generating complement-receptor-2 (CR2)-based inhibitors that recognize the complement activation product C3d. The AP-inhibitor CR2-factor-H (CR2-fH) slowed CNV progression and reversed SIOP. Also, comparing effects of CR2-fH with a C3aR antagonist or C3aR/C5aR deficiency on CNV regression, revealed that anaphylatoxins not only promote CNV, but also play a role in its repair. Overall, this suggests that CR2-fH provides sufficient complement inhibition to provide protection during injury, while allowing for lectin and/or classical pathway generation of anaphylatoxins at levels sufficient for promoting recovery. This duality of complement in injury and repair needs to be considered when designing a complement inhibitory strategy for AMD.
Coauthors: Beth Coughlin1, Nathaniel Parsons1, Alex Woodell1, Balasubramaniam Annamalai1, Bryan Jones2, Joshua Thurman3, Carl Atkinson1, Stephen Tomlinson1, and V. Michael Holers3.
1. Medical University of South Carolina, Charleston.
2. University of Utah, Salt Lake City.
3. University of Colorado, Denver.
An Expanded Role for Complement in Transplantation
Peter S. Heeger, MD, Icahn School of Medicine at Mount Sinai
In addition to functioning as an effector arm of alloantibody initiated transplant injury, we previously demonstrated that immune cell-derived complement drives effector T cell expansion and inhibits regulatory T cells, together resulting in rejection of solid organ allografts and graft vs. host disease (GVHD). Building upon this work, we tested the effects of C5a/C5aR1 ligations on development and function of follicular helper T cells (TFH) and antibody dependent chronic GVHD. Using a semi-allogeneic model of TFH and germinal center (GC) B cell differentiation we demonstrate that absent host C3, absent donor cell C5aR1, and pharmacological C5aR1 blockade each abrogate alloimmune generation/expansion of TFH, GC B cells and autoantibody formation (p<0.05 for each). In a second multi-organ chronic GVHD model, B10.BR mice conditioned with cyclophosphamide/irradiation and transplanted with B6 bone marrow and T cells develop TFH-dependent bronchiolitis obliterans syndrome and chronic GVHD over 8 weeks, manifested by increased pulmonary resistance/elastance with decreased compliance. C5aR1 antagonist (C5aR1-A) administration beginning on d28 fully prevented the clinical disease (p<0.05) and also abrogated differentiation of TFH and GC B cells in this cGVHD system (p<0.05). Mechanistic studies showed that in vitro, IL-6-dependent, T cell differentiation into IL-21-producers is diminished in the absence of C5aR1. C5aR1-initiated signals on T cells, transmitted through PI3-Kgamma and AKT amplify mTOR-dependent phosphorylation of pS6 kinase and IL-21 production. Together, our findings link complement and C5a/C5aR1 ligations to IL-6-dependent TFH and GC B cell formation and provide a foundation for testing effects of C5aR1 blockade on cGVHD in humans.
Evaluation of Anti-human Complement Component 5 (C5) Antibody in Humanized C5 Mice
Ying Hu, PhD, Regeneron Pharmaceuticals, Inc.
Complement has been implicated in ocular inflammatory and retinal degenerative diseases. The present study was undertaken to evaluate pharmacokinetic/pharmacodynamic (PK/PD) properties of a fully human anti-C5 monoclonal antibody (mAb) in humanized C5 mice (C5hu/hu). Lead anti-human C5 mAb was generated in VelocImmune® mice and selected based on activity in classical (CP) and alternative pathway (AP) hemolysis bioassays in vitro. C5hu/hu mice were generated using VelociGene technology and used to assess PK/PD in vivo / ex vivo. Each mouse received a single subcutaneous injection of anti-C5 or isotype control mAb. Mouse serum was collected pre-dose and through day 60. PD was analyzed by ex vivo CP assay. Excess human C3 (80ug/ml) was added ex vivo prior to hemolysis assay. Human C5 concentrations in serum were evaluated by LC-MS-MRM (liquid chromatography-mass spectrometry-multiple reaction monitoring), and anti-C5 mAb concentrations were analyzed by LC-MS-MRM and immunoassay. Anti-C5 mAb blocked CP and AP hemolysis in a dose-dependent manner, with IC50 values of 18.3 nM and 27.4 nM, respectively. A single 15 mg/kg SC dose of anti-C5 mAb significantly blocked CP hemolytic activity ex vivo for 35 days post-treatment. Anti-hemolytic activity was correlated with a C5/ anti-C5 mAb molar ratio &tl;2 as measured by LC-MS-MRM and lack of C5 accumulation in serum. Our findings demonstrate that C5hu/hu mice, ex vivo hemolytic assays, and LC-MS-MRM can be used to elucidate PK/PD relationships of anti-C5 mAbs in vivo.
Coauthors: Adrianna Latuszek, Yashu Liu, Randi Foster, Irena Lovric, Ming Yuan, Henry Chen, Pamela Krueger, Tammy Huang, William Poueymirou, Brian Zambrowicz, Jingtai Cao, Carmelo Romano, and William Olson; Regeneron Pharmaceuticals, Inc.
How Complement Eliminates Synapses in Development and Disease
Beth Stevens, PhD, Boston Children's Hospital
One of the major unsolved mysteries in neuroscience is how synapses are eliminated in the healthy and diseased brain. During development, neural circuitry undergoes a remodeling process in which excess synapses are eliminated and the remaining synapses are strengthened. This pruning process is required for precise brain wiring; however the mechanisms that drive the elimination of specific synapses in the brain remain unclear. Emerging evidence implicate microglia and the classical complement cascade in developmental synaptic pruning. Our recent studies support a model in which 'weaker' or less active synapses in the developing brain are targeted by complement proteins (C1q, C3) and then eliminated by phagocytic microglia that express receptors for complement and other immune molecules. These findings raise the question of how microglia differentiate the synapses or axons to prune from those to leave intact. Microglia-mediated synaptic refinement appears to depend on a careful balance of "eat me" (ie. complement) and a group of novel immune-related protective signals. An early hallmark of many neurodegenerative diseases (NDDs) is a progressive, region-specific degeneration of synapses; however, molecular mechanisms that drive synapse loss remains elusive. Our recent work suggest that aberrant activation of some of these normal immune-related pruning pathways mediate early synapse loss in neurodegenerative diseases (NDDs), including Alzheimer's Disease (AD) and Huntington's disease (HD). Thus, understanding how these immune mechanisms drive developmental pruning may provide novel insight into how to protect synapses in NDDS and other disorders of synaptic dysfunction, including autism and schizophrenia.
Complement and Microglia Mediate Sensory-Motor Synaptic Loss in Spinal Muscular Atrophy
Aleksandra Vukojicic, MSc, Columbia University
Spinal muscular atrophy (SMA) is a neurodegenerative disease caused by reduced levels of the ubiquitously expressed SMN protein. The hallmarks of SMA are loss of motor neurons (MN) and abnormal postural reflexes. We have shown that reduction of select synapses and sensory-motor circuit dysfunction precedes MN loss. The mechanisms leading to this selective synapse loss remain unknown. Here we investigated whether complement-dependent pathways are activated and cause synapse elimination in SMA. Immunohistochemical assays in a mouse model of SMA revealed that C1q, the initiating protein of the classical complement cascade, associates abnormally with excitatory—but not inhibitory—synapses on MNs. We show further that both C1q and C3, a downstream complement protein, are tagging proprioceptive synapses on vulnerable MNs. Furthermore we show that synaptic elimination is mediated by phagocytic activity of reactive microglia. Additionally, we find that microglia is the major source of C1q in SMA. Finally, we asked whether in vivo immunotherapy against C1q, rescues synapses destined to be eliminated and whether prevention of early synaptic loss alleviates the severe SMA phenotype. Strikingly, behavioral and morphological analysis revealed significant rescue of synapses, improved righting time and lifespan. Importantly, physiological assays demonstrated that synapses rescued from elimination are functional, providing further evidence that SMA is a disease of motor circuits. Collectively, our findings suggest that aberrant activation of classical complement pathway and microglial phagocytic activity mediate synaptic loss in a mouse model of SMA and identify blockade of C1q as a novel therapeutic target.
Support: This work was supported by grants from Department of Defense W81XWH-11-1-0689 (GZM) and NIH-NINDS: R01NS078375 (GZM), R21 NS084185 (GZM).
Coauthors: Nicolas Delestree1,2, Emily V. Fletcher1,2, Sethu Sankaranarayanan3, Ted Yednock3, Ben A. Barres3,4, and George Z. Mentis1,2.
1. Center for Motor Neuron Biology and Disease, Columbia University.
2. Depts. of Pathology & Cell Biology and Neurology, Columbia University.
3. Annexon Biosciences, 280 Utah Avenue Suite 110, South San Francisco.
4. Dept. of Neurobiology, Stanford University School of Medicine.