New Insights on Complement Inhibitor CD59 in Mouse Laser-Induced Choroidal Neovascularization: Mislocalization After Injury and Targeted Delivery for Protein Replacement.

PURPOSE: The membrane attack complex (MAC) in choriocapillaris (CC) and retinal pigment epithelium (RPE) increase with age and disease (age-related macular degeneration). MAC assembly can be inhibited by CD59, a membrane-bound regulator. Here we further investigated the role of CD59 in murine choroidal neovascularization (CNV), a model involving both CC and RPE, and tested whether CR2-CD59, a soluble targeted form of CD59, provides protection. METHODS: Laser-induced CNV was generated in wild type and CD59a-deficient mice (CD59-/-). CNV size was measured by optical coherence tomography, and CR2-CD59 was injected intraperitoneally. Endogenous CD59 localization and MAC deposition were identified by immunohistochemistry and quantified by confocal microscopy. Cell-type-specific responses to MAC were examined in retinal pigment epithelial cells (ARPE-19) and microvascular endothelial cells (HMEC-1). RESULTS: CD59 levels were severely reduced and protein was mislocalized in the RPE surrounding the lesion. CNV lesion size and subretinal fluid accumulation were exacerbated in CD59-/- when compared with those in WT mice, and an increase in MAC deposition was noted. In contrast, CR2-CD59 significantly reduced both structural features of CNV severity. In vitro, MAC inhibition in ARPE-19 cells prevented barrier function loss and accelerated wound healing and cell adhesion, whereas in HMEC-1 cells, CR2-CD59 decelerated wound healing and cell adhesion. CONCLUSION: These data further support the importance of CD59 in controlling ocular injury responses and indicate that pharmacological inhibition of the MAC with CR2-CD59 may be a viable therapeutic approach for reducing complement-mediated ocular pathology.

Dissecting the complement pathway in hepatic injury and regeneration with a novel protective strategy.

Liver resection is commonly performed under ischemic conditions, resulting in two types of insult to the remnant liver: ischemia reperfusion injury (IRI) and loss of liver mass. Complement inhibition is recognized as a potential therapeutic modality for IRI, but early complement activation products are also essential for liver regeneration. We describe a novel site-targeted murine complement inhibitor, CR2-CD59, which specifically inhibits the terminal membrane attack complex (MAC), and we use this protein to investigate the complement-dependent balance between liver injury and regeneration in a clinical setting of pharmacological inhibition. CR2-CD59 did not impact in vivo generation of C3 and C5 activation products but was as effective as the C3 activation inhibitor CR2-Crry at ameliorating hepatic IRI, indicating that the MAC is the principle mediator of hepatic IRI. Furthermore, unlike C3 or C5 inhibition, CR2-CD59 was not only protective but significantly enhanced hepatocyte proliferation after partial hepatectomy, including when combined with ischemia and reperfusion. Remarkably, CR2-CD59 also enhanced regeneration after 90% hepatectomy and improved long-term survival from 0 to 70%. CR2-CD59 functioned by increasing hepatic TNF and IL-6 levels with associated STAT3 and Akt activation, and by preventing mitochondrial depolarization and allowing recovery of ATP stores.

A new treatment for neurogenic inflammation caused by EV71 with CR2-targeted complement inhibitor.

BACKGROUND: Enterovirus 71 (EV71), one of the most important neurotropic EVs, has caused death and long-term neurological sequelae in hundreds of thousands of young children in the Asia-Pacific region in the past decade. The neurological diseases are attributed to infection by EV71 inducing an extensive peripheral and central nervous system (CNS) inflammatory response with abnormal cytokine production and lymphocyte depletion induced by EV71 infection. In the absence of specific antiviral agents or vaccines, an effective immunosuppressive strategy would be valuable to alleviate the severity of the local inflammation induced by EV71 infection. PRESENTATION OF THE HYPOTHESIS: The complement system plays a pivotal role in the inflammatory response. Inappropriate or excessive activation of the complement system results in a severe inflammatory reaction or numerous pathological injuries. Previous studies have revealed that EV71 infection can induce complement activation and an inflammatory response of the CNS. CR2-targeted complement inhibition has been proved to be a potential therapeutic strategy for many diseases, such as influenza virus-induced lung tissue injury, postischemic cerebral injury and spinal cord injury. In this paper, a mouse model is proposed to test whether a recombinant fusion protein consisting of CR2 and a region of Crry (CR2-Crry) is able to specifically inhibit the local complement activation induced by EV71 infection, and to observe whether this treatment strategy can alleviate or even cure the neurogenic inflammation. TESTING THE HYPOTHESIS: CR2-Crry is expressed in CHO cells, and its biological activity is determined by complement inhibition assays. 7-day-old ICR mice are inoculated intracranially with EV71 to duplicate the neurological symptoms. The mice are then divided into two groups, in one of which the mice are treated with CR2-Crry targeted complement inhibitor, and in the other with phosphate-buffered saline. A group of mice deficient in complement C3, the breakdown products of which bind to CR2, are also infected with EV71 virus. The potential bioavailability and efficacy of the targeted complement inhibitor are evaluated by histology, immunofluorescence staining and radiolabeling. IMPLICATIONS OF THE HYPOTHESIS: CR2-Crry-mediated targeting complement inhibition will alleviate the local inflammation and provide an effective treatment for the severe neurological diseases associated with EV71 infection.

A targeted complement-dependent strategy to improve the outcome of mAb therapy, and characterization in a murine model of metastatic cancer.

Complement inhibitors expressed on tumor cells provide an evasion mechanism against mAb therapy and may modulate the development of an acquired antitumor immune response. Here we investigate a strategy to amplify mAb-targeted complement activation on a tumor cell, independent of a requirement to target and block complement inhibitor expression or function, which is difficult to achieve in vivo. We constructed a murine fusion protein, CR2Fc, and demonstrated that the protein targets to C3 activation products deposited on a tumor cell by a specific mAb, and amplifies mAb-dependent complement activation and tumor cell lysis in vitro. In syngeneic models of metastatic lymphoma (EL4) and melanoma (B16), CR2Fc significantly enhanced the outcome of mAb therapy. Subsequent studies using the EL4 model with various genetically modified mice and macrophage-depleted mice revealed that CR2Fc enhanced the therapeutic effect of mAb therapy via both macrophage-dependent FcγR-mediated antibody-dependent cellular cytotoxicity, and by direct complement-mediated lysis. Complement activation products can also modulate adaptive immunity, but we found no evidence that either mAb or CR2Fc treatment had any effect on an antitumor humoral or cellular immune response. CR2Fc represents a potential adjuvant treatment to increase the effectiveness of mAb therapy of cancer.

Design and development of TT30, a novel C3d-targeted C3/C5 convertase inhibitor for treatment of human complement alternative pathway-mediated diseases.

To selectively modulate human complement alternative pathway (CAP) activity implicated in a wide range of acute and chronic inflammatory conditions and to provide local cell surface and tissue-based inhibition of complement-induced damage, we developed TT30, a novel therapeutic fusion protein linking the human complement receptor type 2 (CR2/CD21) C3 fragment (C3frag = iC3b, C3dg, C3d)-binding domain with the CAP inhibitory domain of human factor H (fH). TT30 efficiently blocks ex vivo CAP-dependent C3frag accumulation on activated surfaces, membrane attack complex (MAC) formation and hemolysis of RBCs in a CR2-dependent manner, and with a ∼ 150-fold potency gain over fH, without interference of C3 activation or MAC formation through the classic and lectin pathways. TT30 protects RBCs from hemolysis and remains bound and detectable for at least 24 hours. TT30 selectively inhibits CAP in cynomolgus monkeys and is bioavailable after subcutaneous injection. Using a unique combination of targeting and effector domains, TT30 controls cell surface CAP activation and has substantial potential utility for the treatment of human CAP-mediated diseases.

Targeted inhibition of the complement alternative pathway with complement receptor 2 and factor H attenuates collagen antibody-induced arthritis in mice.

The alternative pathway (AP) of complement is required for the induction of collagen Ab-induced arthritis (CAIA) in mice. The objective of this study was to examine the effect of a recombinant AP inhibitor containing complement receptor 2 and factor H (CR2-fH) on CAIA in mice. CR2 binds to tissue-fixed activation fragments of C3, and the linked fH is a potent local inhibitor of the AP. CAIA was induced in C57BL/6 mice by i.p. injections of 4 mAb to type II collagen (CII) on day 0 and LPS on day 3. PBS or CR2-fH (250 or 500 microg) were injected i.p. 15 min after the mAb to CII on day 0 and 15 min after LPS on day 3; the mice were sacrificed on day 10. The disease activity score (DAS) was decreased significantly (p < 0.001) in both groups receiving CR2-fH compared with the PBS. Histology scores for inflammation, pannus, bone damage, and cartilage damage decreased in parallel with the DAS. C3 deposition in the synovium and cartilage was significantly reduced (p < 0.0001) in the mice treated with CR2-fH. In vitro studies with immune complexes containing type II collagen and mAb to CII showed that CR2-fH specifically inhibited the AP with minimal effect on the classical pathway (CP) and no effect on the lectin pathway (LP). The relative potency of CR2-fH in vitro was superior to mAbs to factor B and C5. Thus, CR2-fH specifically targets and inhibits the AP of complement in vitro and is effective in CAIA in vivo.

Complement-dependent P-selectin expression and injury following ischemic stroke.

The mechanisms that contribute to inflammatory damage following ischemic stroke are poorly characterized, but studies indicate a role for both complement and P-selectin. In this study, we show that compared with wild-type mice, C3-deficient mice showed significant improvement in survival, neurological deficit, and infarct size at 24 h after middle cerebral artery occlusion and reperfusion. Furthermore, P-selectin protein expression was undetectable in the cerebral microvasculature of C3-deficient mice following reperfusion, and there was reduced neutrophil influx, reduced microthrombus formation, and increased blood flow postreperfusion in C3-deficient mice. We further investigated the use of a novel complement inhibitory protein in a therapeutic paradigm. Complement receptor 2 (CR2)-Crry inhibits complement activation at the C3 stage and targets to sites of complement activation. Treatment of normal mice with CR2-Crry at 30 min postreperfusion resulted in a similar level of protection to that seen in C3-deficient mice in all of the above-measured parameters. The data demonstrate an important role for complement in cerebrovascular thrombosis, inflammation, and injury following ischemic stroke. P-selectin expression in the cerebrovasculature, which is also implicated in cerebral ischemia and reperfusion injury, was shown to be distal to and dependent on complement activation. Data also show that a CR2-targeted approach of complement inhibition provides appropriate bioavailability in cerebral injury to enable complement inhibition at a dose that does not significantly affect systemic levels of serum complement activity, a potential benefit for stroke patients where immunosuppression would be undesirable due to significantly increased susceptibility to lung infection.

Complement plays an important role in spinal cord injury and represents a therapeutic target for improving recovery following trauma.

Initiation of an inflammatory cascade following traumatic spinal cord injury (SCI) is thought to cause secondary injury and to adversely impact functional recovery, although the mechanisms involved are not well defined. We report on the dynamics of complement activation and deposition in the mouse spinal cord following traumatic injury, the role of complement in the development of SCI, and the characterization of a novel targeted complement inhibitor. Following traumatic injury, mice deficient in C3 had a significantly improved locomotor score when compared with wild-type controls, and analysis of their spinal cords revealed significantly more tissue sparing, with significantly less necrosis, demyelination, and neutrophil infiltration. Wild-type mice were also treated with CR2-Crry, a novel inhibitor of complement activation that targets to sites of C3 deposition. A single intravenous injection of CR2-Crry 1 hour after traumatic injury improved functional outcome and pathology to an extent similar to that seen in C3-deficient animals. CR2-Crry specifically targeted to the injured spinal cord in a distribution pattern corresponding to that seen for deposited C3. As immunosuppression is undesirable in patients following SCI, targeted CR2-Crry may provide appropriate bioavailability to treat SCI at a dose that does not significantly affect systemic levels of serum complement activity.

Protection against influenza virus infection by intranasal administration of C3d-fused hemagglutinin.

For the induction of mucosal immune responses by intranasal vaccination, cholera toxin B subunits (CTB) and Escherichia coli heat-labile toxin (LT) are often administered as mucosal adjuvants in order to enhance immune responses to mucosally co-administered bystander antigens. However, these toxin also are the causative agents of diarrhea. There is a demand for the establishment of an effective and safer adjuvant or vaccine that elicits mucosal immunity, but does not require the use of CTB or LT adjuvants. In order to induce protective mucosal immune responses in the nasal area against influenza virus infection, we have examined the recombinant protein composed of the complement component, C3d, which is fused to the secreted form of hemagglutinin (sHA-mC3d3) in the influenza-BALB/c mouse model. The fusion protein sHA-mC3d3, the secretory form of hemagglutinin, and the transmembrane form of HA (tmHA) from the influenza virus were intranasally administered to the mice with or without CTB containing a trace amount of holotoxin (CTB*) as an adjuvant. After intranasal administration of these proteins with CTB*, all mice produced nasal IgA and serum IgG antibodies (Abs) against the viral HA. In addition, viral infection was completely inhibited in these mice. In contrast, in the absence of the adjuvant, only sHA-mC3d3-induced locally secreted IgA and serum IgG Abs and provided complete protection against the influenza virus challenge. Thus, C3d fused to the influenza HA antigen is an effective and safe tool for mucosal vaccination.