Development of a therapeutic anti-HtrA1 antibody and the identification of DKK3 as a pharmacodynamic biomarker in geographic atrophy.

Genetic polymorphisms in the region of the trimeric serine hydrolase high-temperature requirement 1 (HTRA1) are associated with increased risk of age-related macular degeneration (AMD) and disease progression, but the precise biological function of HtrA1 in the eye and its contribution to disease etiologies remain undefined. In this study, we have developed an HtrA1-blocking Fab fragment to test the therapeutic hypothesis that HtrA1 protease activity is involved in the progression of AMD. Next, we generated an activity-based small-molecule probe (ABP) to track target engagement in vivo. In addition, we used N-terminomic proteomic profiling in preclinical models to elucidate the in vivo repertoire of HtrA1-specific substrates, and identified substrates that can serve as robust pharmacodynamic biomarkers of HtrA1 activity. One of these HtrA1 substrates, Dickkopf-related protein 3 (DKK3), was successfully used as a biomarker to demonstrate the inhibition of HtrA1 activity in patients with AMD who were treated with the HtrA1-blocking Fab fragment. This pharmacodynamic biomarker provides important information on HtrA1 activity and pharmacological inhibition within the ocular compartment.

Prevention of Fatal C3 Glomerulopathy by Recombinant Complement Receptor of the Ig Superfamily.

Background C3 glomerulopathy (C3G) is a life-threatening kidney disease caused by dysregulation of the alternative pathway of complement (AP) activation. No approved specific therapy is available for C3G, although an anti-C5 mAb has been used off-label in some patients with C3G, with mixed results. Thus, there is an unmet medical need to develop other inhibitors of complement for C3G.Methods We used a murine model of lethal C3G to test the potential efficacy of an Fc fusion protein of complement receptor of the Ig superfamily (CRIg-Fc) in the treatment of C3G. CRIg-Fc binds C3b and inhibits C3 and C5 convertases of the AP. Mice with mutations in the factor H and properdin genes (FHm/mP-/-) develop early-onset C3G, with AP consumption, high proteinuria, and lethal crescentic GN.Results Treatment of FHm/mP-/- mice with CRIg-Fc, but not a control IgG, inhibited AP activation and diminished the consumption of plasma C3, factor B, and C5. CRIg-Fc-treated FHm/mP-/- mice also had significantly improved survival and reduced proteinuria, hematuria, BUN, glomerular C3 fragment, C9 and fibrin deposition, and GN pathology scores.Conclusions Therapeutics developed on the basis of the mechanism of action of soluble CRIg may be effective for the treatment of C3G and should be explored clinically.

The trimeric serine protease HtrA1 forms a cage-like inhibition complex with an anti-HtrA1 antibody.

High temperature requirement A1 (HtrA1) is a trypsin-fold serine protease implicated in the progression of age-related macular degeneration (AMD). Our interest in an antibody therapy to neutralize HtrA1 faces the complication that the target adopts a trimeric arrangement, with three active sites in close proximity. In the present study, we describe antibody 94, obtained from a human antibody phage display library, which forms a distinct macromolecular complex with HtrA1 and inhibits the enzymatic activity of recombinant and native HtrA1 forms. Using biochemical methods and negative-staining EM we were able to elucidate the molecular composition of the IgG94 and Fab94 complexes and the associated inhibition mechanism. The 246-kDa complex between the HtrA1 catalytic domain trimer (HtrA1_Cat) and Fab94 had a propeller-like organization with one Fab bound peripherally to each protomer. Low-resolution EM structures and epitope mapping indicated that the antibody binds to the surface-exposed loops B and C of the catalytic domain, suggesting an allosteric inhibition mechanism. The HtrA1_Cat-IgG94 complex (636 kDa) is a cage-like structure with three centrally located IgG94 molecules co-ordinating two HtrA1_Cat trimers and the six active sites pointing into the cavity of the cage. In both complexes, all antigen-recognition regions (paratopes) are found to bind one HtrA1 protomer and all protomers are bound by a paratope, consistent with the complete inhibition of enzyme activity. Therefore, in addition to its potential therapeutic usefulness, antibody 94 establishes a new paradigm of multimeric serine protease inhibition.

Inhibiting alternative pathway complement activation by targeting the factor D exosite.

By virtue of its amplifying property, the alternative complement pathway has been implicated in a number of inflammatory diseases and constitutes an attractive therapeutic target. An anti-factor D Fab fragment (AFD) was generated to inhibit the alternative complement pathway in advanced dry age-related macular degeneration. AFD potently prevented factor D (FD)-mediated proteolytic activation of its macromolecular substrate C3bB, but not proteolysis of a small synthetic substrate, indicating that AFD did not block access of the substrate to the catalytic site. The crystal structures of AFD in complex with human and cynomolgus FD (at 2.4 and 2.3 Å, respectively) revealed the molecular details of the inhibitory mechanism. The structures show that the AFD-binding site includes surface loops of FD that form part of the FD exosite. Thus, AFD inhibits FD proteolytic function by interfering with macromolecular substrate access rather than by inhibiting FD catalysis, providing the molecular basis of AFD-mediated inhibition of a rate-limiting step in the alternative complement pathway.

A novel inhibitor of the alternative pathway of complement reverses inflammation and bone destruction in experimental arthritis.

Complement is an important component of the innate and adaptive immune response, yet complement split products generated through activation of each of the three complement pathways (classical, alternative, and lectin) can cause inflammation and tissue destruction. Previous studies have shown that complement activation through the alternative, but not classical, pathway is required to initiate antibody-induced arthritis in mice, but it is unclear if the alternative pathway (AP) plays a role in established disease. Previously, we have shown that human complement receptor of the immunoglobulin superfamily (CRIg) is a selective inhibitor of the AP of complement. Here, we present the crystal structure of murine CRIg and, using mutants, provide evidence that the structural requirements for inhibition of the AP are conserved in human and mouse. A soluble form of CRIg reversed inflammation and bone loss in two experimental models of arthritis by inhibiting the AP of complement in the joint. Our data indicate that the AP of complement is not only required for disease induction, but also disease progression. The extracellular domain of CRIg thus provides a novel tool to study the effects of inhibiting the AP of complement in established disease and constitutes a promising therapeutic with selectivity for a single complement pathway.

Structure of C3b in complex with CRIg gives insights into regulation of complement activation.

The complement system is a key part of the innate immune system, and is required for clearance of pathogens from the bloodstream. After exposure to pathogens, the third component of the complement system, C3, is cleaved to C3b which, after recruitment of factor B, initiates formation of the alternative pathway convertases. CRIg, a complement receptor expressed on macrophages, binds to C3b and iC3b mediating phagocytosis of the particles, but it is unknown how CRIg selectively recognizes proteolytic C3-fragments and whether binding of CRIg to C3b inhibits convertase activation. Here we present the crystal structure of C3b in complex with CRIg and, using CRIg mutants, provide evidence that CRIg acts as an inhibitor of the alternative pathway of complement. The structure shows that activation of C3 induces major structural rearrangements, including a dramatic movement (>80 A) of the thioester-bond-containing domain through which C3b attaches to pathogen surfaces. We show that CRIg is not only a phagocytic receptor, but also a potent inhibitor of the alternative pathway convertases. The structure provides insights into the complex macromolecular structural rearrangements that occur during complement activation and inhibition. Moreover, our structure-function studies relating the structural basis of complement activation and the means by which CRIg inhibits the convertases provide important clues to the development of therapeutics that target complement.

Complement receptor of the Ig superfamily enhances complement-mediated phagocytosis in a subpopulation of tissue resident macrophages.

An important function of the complement cascade is to coat self and foreign particles with C3-proteins that serve as ligands for phagocytic receptors. Although tissue resident macrophages play an important role in complement-mediated clearance, the receptors coordinating this process have not been well characterized. In the present study, we identified a subpopulation of resident peritoneal macrophages characterized by high expression of complement receptor of the Ig superfamily (CRIg), a recently discovered complement C3 receptor. Macrophages expressing CRIg showed significantly increased binding and subsequent internalization of complement-opsonized particles compared with CRIg negative macrophages. CRIg internalized monovalent ligands and was able to bind complement-opsonized targets in the absence of Ca(2+) and Mg(2+), which differs from the beta(2)-integrin CR3 that requires divalent cations and polyvalent ligands for activation of the receptor. Although CRIg dominated in immediate binding of complement-coated particles, CRIg and CR3 contributed independently to subsequent particle phagocytosis. CRIg thus identifies a subset of tissue resident macrophages capable of increased phagocytosis of complement C3-coated particles, a function critical for immune clearance.

Integration of eQTL and a Single-Cell Atlas in the Human Eye Identifies Causal Genes for Age-Related Macular Degeneration.

Age-related macular degeneration (AMD) is a leading cause of vision loss. To better understand disease pathogenesis and identify causal genes in GWAS loci for AMD risk, we present a comprehensive database of human retina and retinal pigment epithelium (RPE). Our database comprises macular and non-macular RNA sequencing (RNA-seq) profiles from 129 donors, a genome-wide expression quantitative trait loci (eQTL) dataset that includes macula-specific retina and RPE/choroid, and single-nucleus RNA-seq (NucSeq) from human retina and RPE with subtype resolution from more than 100,000 cells. Using NucSeq, we find enriched expression of AMD candidate genes in RPE cells. We identify 15 putative causal genes for AMD on the basis of co-localization of genetic association signals for AMD risk and eye eQTL, including the genes TSPAN10 and TRPM1. These results demonstrate the value of our human eye database for elucidating genetic pathways and potential therapeutic targets for ocular diseases.

Publisher Correction: Classical and alternative complement activation on photoreceptor outer segments drives monocyte-dependent retinal atrophy.

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Classical and alternative complement activation on photoreceptor outer segments drives monocyte-dependent retinal atrophy.

Geographic atrophy (GA), the advanced form of dry age-related macular degeneration (AMD), is characterized by progressive loss of retinal pigment epithelium cells and photoreceptors in the setting of characteristic extracellular deposits and remains a serious unmet medical need. While genetic predisposition to AMD is dominated by polymorphisms in complement genes, it remains unclear how complement activation contributes to retinal atrophy. Here we demonstrate that complement is activated on photoreceptor outer segments (POS) in the retina peripheral to atrophic lesions associated with GA. When exposed to human serum following outer blood-retinal barrier breakdown, POS act as potent activators of the classical and alternative complement pathway. In mouse models of retinal degeneration, classical and alternative pathway complement activation on photoreceptors contributed to the loss of photoreceptor function. This was dependent on C5a-mediated recruitment of peripheral blood monocytes but independent of resident microglia. Genetic or pharmacologic inhibition of both classical and alternative complement C3 and C5 convertases was required to reduce progressive degeneration of photoreceptor rods and cones. Our study implicates systemic classical and alternative complement proteins and peripheral blood monocytes as critical effectors of localized retinal degeneration with potential relevance for the contribution of complement activation to GA.

IL-33 amplifies an innate immune response in the degenerating retina.

Age-related macular degeneration (AMD), a leading cause of vision impairment in the ageing population, is characterized by irreversible loss of retinal pigment epithelial (RPE) cells and photoreceptors and can be associated with choroidal neovascularization. Mononuclear phagocytes are often present in AMD lesions, but the processes that direct myeloid cell recruitment remain unclear. Here, we identify IL-33 as a key regulator of inflammation and photoreceptor degeneration after retina stress or injury. IL-33(+) Müller cells were more abundant and IL-33 cytokine was elevated in advanced AMD cases compared with age-matched controls with no AMD. In rodents, retina stress resulted in release of bioactive IL-33 that in turn increased inflammatory chemokine and cytokine expression in activated Müller cells. Deletion of ST2, the IL-33 receptor α chain, or treatment with a soluble IL-33 decoy receptor significantly reduced release of inflammatory mediators from Müller cells, inhibited accumulation of mononuclear phagocytes in the outer retina, and protected photoreceptor rods and cones after a retina insult. This study demonstrates a central role for IL-33 in regulating mononuclear phagocyte recruitment to the photoreceptor layer and positions IL-33 signaling as a potential therapeutic target in macular degenerative diseases.

Interleukin-13 gene therapy reduces inflammation, vascularization, and bony destruction in rat adjuvant-induced arthritis.

Rheumatoid arthritis (RA) is a chronic autoimmune inflammatory disease characterized by synovial pannus formation, leukocyte infiltration, and angiogenesis. Adenoviral production of interleukin-13 (IL-13) reduces levels of proinflammatory mediators in an explant model of RA synovial tissue in vitro. To assess this approach in an animal model of arthritis, we compared intra-articular injections of an adenovirus producing rat IL-13 (AxCArIL-13), a control virus, and rat ankles receiving phosphate-buffered saline (PBS) in rat adjuvant-induced arthritis (AIA). We demonstrate that IL-13 levels are normally low in ankles throughout the course of rat AIA. We show that administration of AxCArIL-13 before arthritis onset significantly reduces ankle circumference, paw volume, bony destruction, the number of polymorphonuclear cells (PMNs), the quantity of blood vessels, and levels of monocyte chemoattractant protein (MCP)-1 in ankles. When administered as a treatment to inflamed ankles, AxCArIL-13 decreases articular index scores, paw volumes, bony destruction, vascularization, tumor necrosis factor-alpha (TNF-alpha) levels, and the quantity of monocytes, lymphocytes, and PMNs. Thus, increasing IL-13 levels significantly ameliorates the course of rat AIA, suggesting that similar strategies for the treatment of human RA are worthy of further study.

CRIg mediates early Kupffer cell responses to adenovirus.

Whereas adenoviral vectors are known to activate the complement cascade, leading to fixation of C3 proteins to the viral capsid, the consequences of this activation for viral clearance from the circulation are not known. Liver KCs, the macrophage population responsible for early uptake and elimination of many blood-borne pathogens, express CRIg, a complement receptor for C3 proteins. Here, we find that CRIg is important for the early elimination of C3-coated adenoviral vectors from the sinusoidal bloodstream by KCs. We further demonstrate that by acting as a critical receptor for adenovirus phagocytosis, CRIg plays an important role in regulating virus-induced KC death and depletion of these cells from the liver sinusoidal lumen. Our study thus identifies a critical pathway regulating KC function and survival in response to systemic viral infection.

Activation of the alternative complement pathway in vitreous is controlled by genetics in age-related macular degeneration.

PURPOSE: To determine if the progression of age-related macular degeneration (AMD) is associated with complement activation in the eye. METHODS: Immunohistochemistry and ELISAs were used to determine the distribution, concentration, and activation of the alternative pathway complement proteases factor B (FB) and factor D (FD) and the central complement protein C3 in genotyped human postmortem donor eyes graded as having no or minimal drusen (category 1; controls), large drusen (category 3), and large drusen with advanced AMD (category 4). RESULTS: C3, FB, and FD were present in vitreous and Bruch's membrane choroid (BM/C) interface of the macula of eyes in all tested AMD severity categories (n = 100). C3, FB, and FD were predominantly located to the choroidal vasculature and Bruch's membrane and, together with the serum proteins transferrin and albumin, elevated in BM/C extracts of category 4 eyes (n = 23) compared with category 1 eyes (n = 24). A significant increase in FB activation was found only in vitreous of category 4 eyes (n = 23) compared with category 1 eyes (n = 25). Genetic variants of complement factor H (CFH), C3, C2, and FB associated with increased risk of AMD were correlated with alternative pathway complement activation in vitreous, but not with complement proteins in BM/C protein extracts. CONCLUSIONS: Increased activation of the alternative complement pathway in vitreous was controlled by disease stage and genetic variation in the complement pathway, supporting a role for complement activation in AMD disease pathogenesis.

Negative regulation of autoimmune demyelination by the inhibitory receptor CLM-1.

Multiple sclerosis and its preclinical model, experimental autoimmune encephalomyelitis, are marked by perivascular inflammation and demyelination. Myeloid cells, derived from circulating progenitors, are a prominent component of the inflammatory infiltrate and are believed to directly contribute to demyelination and axonal damage. How the cytotoxic activity of these myeloid cells is regulated is poorly understood. We identify CMRF-35-like molecule-1 (CLM-1) as a negative regulator of autoimmune demyelination. CLM-1 is expressed on inflammatory myeloid cells present in demyelinating areas of the spinal cord after immunization of mice with MOG35-55 (myelin oligodendrocyte glycoprotein) peptide. Absence of CLM-1 resulted in significantly increased nitric oxide and proinflammatory cytokine production, along with increased demyelination and worsened clinical scores, whereas T cell responses in the periphery or in the spinal cord remained unaffected. This study thus identifies CLM-1 as a negative regulator of myeloid effector cells in autoimmune demyelination.

Structural and functional analysis of a C3b-specific antibody that selectively inhibits the alternative pathway of complement.

Amplification of the complement cascade through the alternative pathway can lead to excessive inflammation. Targeting C3b, a component central to the alternative pathway of complement, provides a powerful approach to inhibit complement-mediated immune responses and tissue injury. In the present study, phage display technology was employed to generate an antibody that selectively recognizes C3b but not the non-activated molecule C3. The crystal structure of C3b in complex with a Fab fragment of this antibody (S77) illustrates the structural basis for this selectivity. Cleavage of C3 to C3b results in a plethora of structural changes within C3, including the rearrangement of macroglobulin domain 6 enabling binding of S77 to the adjacent macroglobulin domain 7 domain. S77 blocks binding of factor B to C3b inhibiting the first step in the formation of the alternative pathway C3 convertase. In addition, S77 inhibits C5 binding to C3b. This results in significantly reduced formations of anaphylatoxins and membrane-attack complexes. This study for the first time demonstrates the structural basis for complement inhibition by a C3b-selective antibody and provides insights into the molecular mechanisms of alternative pathway complement activation.

CRIg: a macrophage complement receptor required for phagocytosis of circulating pathogens.

The complement system serves an important role in clearance of pathogens, immune complexes, and apoptotic cells present in the circulation. Complement fragments deposited on the particle surface serve as targets for complement receptors present on phagocytic cells. Although Kupffer cells, the liver resident macrophages, play a dominant role in clearing particles in circulation, complement receptors involved in this process have yet to be identified. Here we report the identification and characterization of a Complement Receptor of the Immunoglobulin superfamily, CRIg, that binds complement fragments C3b and iC3b. CRIg expression on Kupffer cells is required for efficient binding and phagocytosis of complement C3-opsonized particles. In turn, Kupffer cells from CRIg-deficient mice are unable to efficiently clear C3-opsonized pathogens in the circulation, resulting in increased infection and mortality of the host. CRIg therefore represents a dominant component of the phagocytic system responsible for rapid clearance of C3-opsonized particles from the circulation.

A novel function for a glucose analog of blood group H antigen as a mediator of leukocyte-endothelial adhesion via intracellular adhesion molecule 1.

The 4A11 antigen is a unique cytokine-inducible antigen up-regulated on rheumatoid arthritis synovial endothelium compared with normal endothelium. In soluble form, this antigen, Lewisy-6/H-5-2 (Ley/H), or its glucose analog, 2-fucosyllactose (H-2g), mediates angiogenesis. The Ley/H antigen is structurally related to the soluble E-selectin ligand, sialyl Lewisx, and is selectively expressed in skin, lymphoid organs, thymus, and synovium, suggesting that it may be important in leukocyte homing or adhesion. In the present study, we used H-2g as a functional substitute to demonstrate a novel property for Ley/H antigen in inducing leukocyte-endothelial adhesion. H-2g significantly enhanced the expression of human dermal microvascular endothelial cells (HMVECs) intercellular adhesion molecule-1 (ICAM-1), but not vascular cell adhesion molecule-1, E-selectin, and P-selectin. Immunoprecipitation and Western blotting showed glycolipids Ley-6, H-5-2, or the glucose analog H-2g quickly activated human microvascular endothelial cell line-1 (HMEC-1) Janus kinase 2 (JAK2) and that the JAK2 inhibitor, AG-490, completely inhibited HMVEC ICAM-1 expression and HL-60 adhesion to HMEC-1s. Use of a JAK/signal transducer and activator of transcription (STAT) profiling system confirmed that H-2g selectively activated STAT3 but not STAT1 and STAT2. AG-490 inhibited H-2g-induced Erk1/2 and PI3K-Akt activation, suggesting that JAK2 is upstream of the Erk1/2 and PI3K-Akt pathways. Furthermore, the JAK2 inhibitor AG-490, the Erk1/2 inhibitor PD98059, or the phosphatidylinositol 3-kinase inhibitor LY294002 or antisense oligodeoxynucleotides directed against JAK2, Erk1/2, or phosphatidylinositol 3-kinase blocked H-2g-induced HMVEC ICAM-1 expression and HL-60 adhesion to HMEC-1s. Hence, H-2g signals through JAK2 and its downstream signal transducers STAT3, Erk1/2, and phosphatidylinositol 3-kinase result in ICAM-1 expression and cell adhesion. Potential treatment strategies through the inhibition of JAK-dependent pathways to target H-2g signals may provide a useful approach in inflammation-driven diseases like rheumatoid arthritis.