C1q acts in the tumour microenvironment as a cancer-promoting factor independently of complement activation

Complement C1q is the activator of the classical pathway. However, it is now recognized that C1q can exert functions unrelated to complement activation. Here we show that C1q, but not C4, is expressed in the stroma and vascular endothelium of several human malignant tumours. Compared with wild-type (WT) or C3- or C5-deficient mice, C1q-deficient (C1qa(-/-)) mice bearing a syngeneic B16 melanoma exhibit a slower tumour growth and prolonged survival. This effect is not attributable to differences in the tumour-infiltrating immune cells. Tumours developing in WT mice display early deposition of C1q, higher vascular density and an increase in the number of lung metastases compared with C1qa(-/-) mice. Bone marrow (BM) chimeras between C1qa(-/-) and WT mice identify non-BM-derived cells as the main local source of C1q that can promote cancer cell adhesion, migration and proliferation. Together these findings support a role for locally synthesized C1q in promoting tumour growth

New insight into antiphospholipid syndrome: antibodies to \u3b22glycoprotein I-domain 5 fail to induce thrombi in rats

Clinical studies have reported different diagnostic/predictive values of antibodies to domain 1 or 4/5 of \u3b22glycoproteinI in terms of risk of thrombosis and pregnancy complications in patients with antiphospholipid syndrome. To obtain direct evidence for the pathogenic role of anti-domain 1 or anti-domain 4/5 antibodies, we analysed the in vivo pro-coagulant effect of two groups of 5 serum IgG each reacting selectively with domain 1 or domain 5 in LPS-treated rats. Antibody-induced thrombus formation in mesenteric vessels was followed by intravital microscopy and vascular deposition of \u3b22glycoproteinI, human IgG and C3 was analyzed by immunofluorescence. Five serum IgG with undetectable anti-\u3b22glycoproteinI antibodies served as controls. All the anti-domain 1 positive IgG exhibited potent pro-coagulant activity while the anti-domain 5 positive and the negative control IgG failed to promote blood clot and vessels occlusion. A stronger granular deposit of IgG/C3 was found on the mesenteric endothelium of rats treated with anti-domain 1 antibodies, as opposed to a mild linear IgG staining and absence of C3 observed in rats receiving anti-domain 5 antibodies. Purified anti-domain 5 IgG, unlike anti-domain 1 IgG, did not recognize cardiolipin-bound \u3b22glycoprotein I while able to interact with fluid-phase \u3b22glycoproteinI. These findings may explain the failure of anti-domain 5 antibodies to exhibit in vivo thrombogenic effect and the interaction of these antibodies with circulating \u3b22glycoproteinI suggest their potential competitive role with the pro-coagulant activity of anti-domain 1 antibodies. These data aim at better defining really at risk patients for more appropriate treatments to avoid recurrences and disability

Bispecific antibodies targeting tumor-associated antigens and neutralizing complement regulators increase the efficacy of antibody-based immunotherapy in mice.

The efficacy of antibody-based immunotherapy is due to the activation of apoptosis, the engagement of antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity (CDC). We developed a novel strategy to enhance CDC using bispecific antibodies (bsAbs) that neutralize the C-regulators CD55 and CD59 to enhance C-mediated functions. Two bsAbs (MB20/55 and MB20/59) were designed to recognize CD20 on one side. The other side neutralizes CD55 or CD59. Analysis of CDC revealed that bsAbs could kill 4 to 25 times more cells than anti-CD20 recombinant antibody in cell lines or cells isolated from patients with chronic lymphocytic leukemia. The pharmacokinetics of the bsAbs was evaluated in a human-SCID model of Burkitt lymphoma. The distribution profile of bsAbs mimics the data obtained by studying the pharmacokinetics of anti-CD20 antibodies, showing a peak in the tumor mass 3-4 days after injection. The treatment with bsAbs completely prevented the development of human/SCID lymphoma. The tumor growth was blocked by the activation of the C cascade and by the recruitment of macrophages, PMN and NK cells. This strategy can easily be applied to the other anti-tumor C-fixing antibodies currently used in the clinic or tested in preclinical studies using the same vector with the appropriate modifications

Selective therapeutic control of C5a and the terminal complement complex by anti-C5 single-chain Fv in an experimental model of antigen-induced arthritis in rats.

To determine the role of the terminal complement complex (TCC) in the development of experimental antigen-induced arthritis (AIA) and the therapeutic effects of human anti-C5 single-chain Fv (scFv).Two different anti-C5 scFv, one that inhibits both release of C5a and assembly of the TCC (TS-A 12/22) and another that selectively blocks formation of the TCC (TS-A 8), were injected at the onset of AIA. The effects of these scFv on disease severity were evaluated for up to 21 days and compared with the effects of injection of an unrelated scFv. AIA was also established in C6-deficient and C6-sufficient PVG rats to obtain further information on the role of the TCC in this model.TS-A 12/22 and TS-A 8 proved to be equally effective in reducing joint swelling, cell counts and tumor necrosis factor alpha levels in synovial lavage fluids, and the degree of histomorphologic changes compared with the effects of the unrelated scFv. TS-A 12/22 and TS-A 8 prevented the deposition of C9 but not that of C3, confirming the ability of the 2 scFv to neutralize C5. Administration of the 2 anti-C5 scFv after AIA onset also reduced disease severity. In C6-deficient rats with AIA, disease activity was reduced markedly compared with that in C6-sufficient rats.These 2 human anti-C5 scFv could represent potential therapeutic reagents to be used in patients with rheumatoid arthritis. In addition, the finding that TS-A 8 was as effective as TS-A 12/22 in reducing disease severity suggests that the TCC is mainly responsible for the joint inflammation and damage observed in AIA

Osteoprotegerin increases leukocyte adhesion to endothelial cells both in vitro and in vivo.

Recombinant osteoprotegerin (OPG) promoted the adhesion of both primary polymorphonuclear neutrophils (PMNs) and leukemic HL60 cells to endothelial cells. Leukocyte/endothelial cell adhesion was promoted by short (peak at 1 hour) preincubation of either endothelial cells or PMNs with OPG, and the peak of proadhesive activity was observed in the same range of OPG concentrations detected in the sera of patients affected by cardiovascular diseases. Although the cognate high-affinity ligands for OPG, membrane receptor activator of nuclear factor-kappaB ligand (RANKL) and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), were detected at significant levels on both PMNs and HL60 cells, they were not expressed on the surface of endothelial cells. However, preincubation of OPG with heparin abrogated its proadhesive activity, whereas pretreatment of endothelial cells with chondroitinase plus heparinases significantly decreased the proadhesive activity of OPG. Taken together, these findings suggest the involvement of both the ligand binding and the N-terminal heparin-binding domains of OPG in mediating its pro-adhesive activity. The relevance of these in vitro findings was underscored by in vivo experiments, in which the topical administration of recombinant OPG increased leukocyte rolling and adhesion to rat mesenteric postcapillary venules. Our data suggest that a pathological increase of OPG serum levels might play an important role in promoting leukocyte/endothelial cell adhesion

Osteoprotegerin increases leukocyte adhesion to endothelial cells both in vitro and in vivo

Recombinant osteoprotegerin (OPG) promoted the adhesion of both primary polymorphonuclear neutrophils (PMNs) and leukemic HL60 cells to endothelial cells. Leukocyte/endothelial cell adhesion was promoted by short (peak at 1 hour) preincubation of either endothelial cells or PMNs with OPG, and the peak of proadhesive activity was observed in the same range of OPG concentrations detected in the sera of patients affected by cardiovascular diseases. Although the cognate high-affinity ligands for OPG, membrane receptor activator of nuclear factor-kappaB ligand (RANKL) and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), were detected at significant levels on both PMNs and HL60 cells, they were not expressed on the surface of endothelial cells. However, preincubation of OPG with heparin abrogated its proadhesive activity, whereas pretreatment of endothelial cells with chondroitinase plus heparinases significantly decreased the proadhesive activity of OPG. Taken together, these findings suggest the involvement of both the ligand binding and the N-terminal heparin-binding domains of OPG in mediating its pro-adhesive activity. The relevance of these in vitro findings was underscored by in vivo experiments, in which the topical administration of recombinant OPG increased leukocyte rolling and adhesion to rat mesenteric postcapillary venules. Our data suggest that a pathological increase of OPG serum levels might play an important role in promoting leukocyte/endothelial cell adhesion

Prevention of arthritis by locally synthesized recombinant antibody neutralizing complement component C5

Treatment of patients suffering from chronic diseases such as rheumatoid arthritis with recombinant antibodies is time consuming and fairly expensive and can be associated with side effects due to generalized depletion of the target molecule. We have addressed these issues by developing an alternative approach consisting of the intraarticular injection of a DNA vector encoding for the anti-C5 neutralizing recombinant miniantibody MB12/22. This method allows local production of the antibody in sufficient amount to be effective in preventing joint inflammation in a rat model of antigen-induced arthritis. Injection of the DNA vector in a right knee of normal rats resulted in the production of the minibody detected in the synovial washes by western blot with a strong signal peaking at 3 days after administration. DNA encoding for the minibody was shown for 14 days in the synovial tissue and was undetectable in the controlateral knee and in other organs. The preventive effect of this approach was evaluated in rats receiving a single injection of the vector 3 days before the induction of antigen-induced arthritis and analyzed 3 days later. The treated rats exhibited a lower increase in swelling, associated with a lower number of PMN in the articular washes and reduced deposition of C9 in synovial tissue compared to control rats. These results suggest that treating the inflamed joints with a vector that induces a local production of a neutralizing anti-C5 antibody may represent a useful strategy to inhibit in situ complement activation and to treat patients with monoarthritis. Moreover, this approach may be adopted as a novel therapeutic strategy to prevent monoarthritis as an alternative to local treatment with antibodies commonly used in this form of arthritis, with the advantages of the lower cost and the longer persistence of antibody production

Posttransplant ischemia-reperfusion injury in transplanted heart is prevented bya minibody to the fifth component of complement.

BACKGROUND: Complement activation has been implicated in the development of posttransplant ischemia-reperfusion (I/R) which is responsible for the delayed function of 20% to 30% of grafts. C5a and the terminal complement complex (TCC) are the complement activation products mainly involved in tissue injury caused by I/R. METHODS: To control activation of the terminal step of the complement activation pathways, we used a neutralizing minibody to C5 containing a human single-chain fragment variable (scFv) linked to the hinge region, CH2, and CH3 domains of rat IgG1. RESULTS: The minibody acts on C5 inhibiting the release of C5a and the assembly of TCC and depletes circulating C5 in Sprague-Dawley rats with a therapeutic activity of 4 hr. Administration of the minibody to rats 30 min before heart allotransplantation prevented tissue deposition of TCC, apoptosis, and necrosis of the graft and increase in the levels of serum creatine phosphokinase and tumor necrosis factor-alpha observed in control transplanted rats. CONCLUSIONS: These data suggest that an anti-C5 therapy is effective in preventing graft injury caused by I/R. A minibody containing the human scFv linked to the hinge region and the CH2 and CH3 domains of human IgG1 is ready for use in clinical transplantation

\u201dFluvastatin treatment inhibits leucocyte adhesion and extravasation in models of complement-mediated acute inflamation\u201dF.Fischetti, R. Carretta, G. Borotto, P. Durigutto, R. Bulla, P.L. Meroni, F. Tedesco.

Abstract Complement activation plays a relevant role in the development of tissue damage under inflammatory conditions, and clinical and experimental observations emphasize its contribution to inflammatory vasculitides. Statins have recently been shown to reduce cardiovascular morbidity independently of plasma cholesterol lowering and in vitro studies support a direct anti-inflammatory action of these drugs. The aim of this study was to verify the in vivo effect of fluvastatin on complement-mediated acute peritoneal inflammation. The effect of oral treatment with fluvastatin was investigated in normo-cholesterolaemic rats that received intraperitoneal injection of either yeast-activated rat serum (Y-act RS) or lipopolysaccharide to induce peritoneal inflammation monitored by the number of PMN recruited in peritoneal fluid washes. In addition, vascular adherence and extravasation of leucocytes were evaluated by direct videomicroscopy examination on mesentery postcapillary venules topically exposed to Y-act RS. The number of PMN in the peritoneal washes of rats treated with fluvastatin was 38% lower than that of untreated animals (P < 0.05) 12 h after LPS injection, and was even lower (56%) in rats treated with Y-act RS already 8 h after injection (P < 0.02). Firm adhesion to endothelium and extravasation of leucocytes evaluated under direct videomicroscopy observation were significantly inhibited in fluvastatin treated rats (77% and 72%, respectively; P < 0.01), 120 min after treatment with Y-act RS. Our results demonstrate that fluvastatin inhibits in vivo complement-dependent acute peritoneal inflammation and suggest a role for statins in preventing the inflammatory flares usually associated with complement activation in chronic diseases, such as SLE or rheumatoid arthritis