Complement Pathways in Disease

Agenda

* Presentation times are subject to change.

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 (T_FH) and antibody dependent chronic GVHD. Using a semi-allogeneic model of T_FH 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 T_FH, 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 T_FH-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 T_FH 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 T_FH 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 (C5^hu/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. C5^hu/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 C5^hu/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 Delestree^1,2, Emily V. Fletcher^1,2, Sethu Sankaranarayanan³, Ted Yednock³, Ben A. Barres^3,4, and George Z. Mentis^1,2.
 
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.