Membrane-targeted complement inhibitors

Undesirable complement activation contributes to the pathology of many human diseases by damaging tissue and promoting inflammation. Because complement-mediated damage is caused by the deposition of complement components on the cell surface, several strategies have been devised to target complement regulator proteins to cell membranes. These strategies have resulted in engineered proteins that have improved potency in vitro and enhanced therapeutic benefit in animal models of disease. One membrane-targeted complement inhibitor has now entered clinical development and this class of second-generation agents may provide effective therapies for the treatment of a variety of disease states.     

Two complement inhibitors in mouse sera

Two inhibitors of the guinea pig complement components C1 and C3 are demonstrated in normal DBA and anti C57 DBA isoimmune sera. The specificity of the inhibitors is proved by their complete absorption with EAC1 and EAC1423 cells, respectively. Both inhibitors are found in the 7S fraction of normal DBA mouse serum, which correlates well with the data of the C1 and C3 inactivator of human, rabbit, and guinea pig sera.     

Targeted inhibition of complement using complement receptor 2-conjugated inhibitors attenuates EAE

Multiple sclerosis (MS) is the most common autoimmune demyelinating disease, affecting millions of individuals worldwide. In the last two decades, many therapeutic options for the treatment of MS have become available, however they are limited in terms of effectiveness and some remain plagued by safety issues. The currently available treatment options target relapsing remitting forms of MS and are not effective against the more progressive forms of the disease. These limitations highlight a significant unmet treatment need for MS. In experimental autoimmune encephalomyelitis (EAE) studies from our laboratory, we have previously shown, using a number of complement mutant and transgenic mice, that inhibition of the alternative complement pathway and the C3 convertase confers significant protection from disease. We report here that targeted inhibition of complement activation using complement receptor 2 (CR2)-conjugated inhibitors significantly attenuates EAE. Administration of CR2-Crry (blocks all complement pathways at C3 activation) and CR2-fH (specifically blocks the alternative pathway) just prior to and during the onset of EAE blocks progression of both acute and chronic disease. These data indicate that inhibition of complement may offer an effective therapeutic approach to treating both acute and chronic forms of demyelinating disease through blocking the alternative pathway or complement convertases.     

Therapeutic uses of recombinant complement protein inhibitors

Conclusion In conclusion, it is apparent that researchers are poised at the threshold of developing inhibitors of complement activation from the molecules in the RCA family. By creating soluble forms of these protective proteins for in vivo administration, or by making transgenic animals expressing these proteins or their derivatives, it may be possible to inhibit complement-mediated pathology stemming from autoimmune disease, reperfusion injuries, and physical trauma. This technology combined with current attempts to protect allografts from cellular rejection with monoclonal antibodies against members of the integrin family of adhesion molecules [52] makes it possible that the excessive mortality due to the severe shortage of human donor organs could be overcome by the use of xenografts.     

Low-molecular weight inhibitors of the alternative complement pathway

Dysregulation of the alternative complement pathway predisposes individuals to a number of diseases. It can either be evoked by genetic alterations in or by stabilizing antibodies to important pathway components and typically leads to severe diseases such as paroxysmal nocturnal hemoglobinuria, atypical hemolytic uremic syndrome, C3 glomerulopathy, and age-related macular degeneration. In addition, the alternative pathway may also be involved in many other diseases where its amplifying function for all complement pathways might play a role. To identify specific alternative pathway inhibitors that qualify as therapeutics for these diseases, drug discovery efforts have focused on the two central proteases of the pathway, factor B and factor D. Although drug discovery has been challenging for a number of reasons, potent and selective low-molecular weight (LMW) oral inhibitors have now been discovered for both proteases and several molecules are in clinical development for multiple complement-mediated diseases. While the clinical development of these inhibitors initially focuses on diseases with systemic and/or peripheral tissue complement activation, the availability of LMW inhibitors may also open up the prospect of inhibiting complement in the central nervous system where its activation may also play an important role in several neurodegenerative diseases.     

Injury site-specific targeting of complement inhibitors for treating stroke

Cumulative evidence indicates a role for the complement system in both pathology and recovery after ischemic stroke. Here, we review the current understanding of the dual role of complement in poststroke injury and recovery, and discuss the challenges of anti-complement therapies. Most complement directed therapeutics currently under investigation or development systemically inhibit the complement system, but since complement is important for immune surveillance and is involved in various homeostatic activities, there are potential risks associated with systemic inhibition. Depending on the target within the complement pathway, other concerns are high concentrations of inhibitor required, low efficacy and poor bioavailability. To overcome these limitations, approaches to target complement inhibitors to specific sites have been investigated. Here, we discuss targeting strategies, with a focus on strategies developed in our lab, to specifically localize complement inhibition to sites of tissue injury and complement activation, and in particular to the postischemic brain. We discuss various injury site-specific targeted complement inhibitors as potential therapeutic agents for the treatment of ischemic stroke treatment, as well as their use as investigative tools for probing complement-dependent pathophysiological processes.     

Extensive complement activation in hereditary porcine membranoproliferative glomerulonephritis type II (porcine dense deposit disease).

Massive glomerular deposits of C3 and the terminal C5b-9 complement complex (TCC), but no immune complex deposits were detected by immunofluorescence in porcine membranoproliferative glomerulonephritis type II. TCC deposits were always observed with concomitant deposits of vitronectin (S-protein) in membranoproliferative glomerulonephritis, in contrast to a piglet with mesangial glomerulopathy where TCC was present without vitronectin co-deposition. Enzyme immunoassays revealed extensive systemic complement activation in 1-week-old affected piglets, observed by low plasma C3 (about 5% of normal) and high plasma TCC (about 10 x normal). Affected piglets revealed some plasma complement activation already at birth, 3 to 4 weeks before recognizable clinical disease. It is concluded that porcine membranoproliferative glomerulonephritis represents a nonimmune complex-mediated glomerulonephritis caused by unrestricted systemic complement activation with C3 consumption, TCC formation, and glomerular trapping of complement activation products. A pathogenetic mechanism of a defective or missing complement regulation protein is suggested.     

猪睾丸Sertoli细胞抵御人血清中天然抗体介导的补体杀伤作用

目的 探讨新生猪睾丸Sertoli细胞抵御人血清中灭然抗体介导的补体杀伤作用及其主要机制.方法 分离培养10～15口龄的湖北白猪睾丸Sertoli细胞;以猪血管内皮细胞(PED)为对照,采用流式细胞仪(FACS)及免疫荧光检测并比较两种细胞天然抗原α-Gal的表达;FACS枪测并比较两种细胞分别与正常人血清(NHS)中天然抗体IgM、IgG的结合情况;乳酸脱氢酶(LDH)检测20%NHS对Serto-li细胞及PED细胞的杀伤;MTT法检测两种细胞在20%NHS中培养的活性;细胞免疫荧光检测补体C3c、C4d在Sertoli细胞、PED细胞的沉积;细胞免疫组化及免疫荧光检测补体C5b-9在两种细胞上的沉积.结果 猪睾丸Sertoli细胞表达ot-Gal,但明显弱于PED细胞,猪睾丸Sertoli细胞能与天然抗体(主要是IgM)结合;20%NHS对Sertoli细胞、PED细胞的杀伤率分别为24.38%±0.50%、53.13%±14.53%,P<0.01;两种细胞在20%NttS中的活性分别为98.73%±18.84%、52.43%±8.08%,P<0.01;免疫荧光及组化可见PED细胞上有补体C3c、C4d、C5b-9的沉积;但Sertoli细胞仅有C3c、C4d沉积,而未见C5b-9沉积.结论 猪睾丸Sertoli细胞可以显著抵御人血清巾天然抗体介导的杀伤作用,其机制可能与Sertoli细胞低表达α-Gal并抑制补体攻膜复合物形成有关.     

人补体对猪血管内皮细胞的溶细胞效应研究

选择猪血管内皮细胞为靶，人血清为天然抗体和补体源，用四唑盐法（ＭＴＴ）行补体依赖的细胞毒反应（ＣＤＣ），建立了体外超急性排斥模型。人血清能溶解５８±５％的猪血管内皮细胞。Ｃ１ｑ缺乏的人血清（阻断经典途径）仅溶解３７±７％猪血管内皮细胞（Ｐ＜０．０１）。Ｂ因子缺乏的人血清（阻断旁路途径）仅溶４２±１０％的猪血管内皮细胞（Ｐ＜０．０１）。同种猪血清及加热灭活补体的人血清不溶猪血管内皮细胞。将经典途径及旁路途径缺陷的人血清等体积混合，其细胞毒作用恢复正常。同样，Ｃ１ｑ缺乏的人血清和Ｂ因子缺乏的人血清分别补加Ｃ１ｑ和Ｂ因子后，血清细胞毒作用亦恢复正常。因此，补体经典和旁路两条途径可能均参与体内超急性排斥反应。提示抑制猪／人之间的超急性排斥应考虑补体旁路途径激活的问题。     

Lack of complement inhibitors in the outer intracranial artery aneurysm wall associates with complement terminal pathway activation

Inflammation and activation of the complement system predispose to intracranial artery aneurysm (IA) rupture. Because disturbances in complement regulation may lead to increased susceptibility to complement activation and inflammation, we looked for evidence for dysregulation of the complement system in 26 unruptured and 26 ruptured IAs resected intraoperatively. Immunohistochemical and immunofluorescence results of parallel IA sections showed that deposition of the complement activation end-product C5b-9 was lacking from the luminal part of the IA wall that contained complement inhibitors factor H, C4b binding protein, and protectin as well as glycosaminoglycans. In contrast, the outer, less cellular part of the IA wall lacked protectin and had enabled full complement activation and C5b-9 formation. Decay accelerating factor and membrane cofactor protein had less evident roles in complement regulation. The Factor H Y402H variant, studied in 97 IA patients, was seen as often in aneurysm patients with or without aneurysm rupture as in the control population. The regulatory capacity of the complement system thus appears disturbed in the outer part of the IA wall, allowing full proinflammatory complement activation to occur before aneurysm rupture. Insufficient complement control might be due to matrix remodeling and cell loss by mechanical hemodynamics and/or inflammatory stress. Apparently, disturbed complement regulation leads to an increased susceptibility to complement activation, inflammation, and tissue damage in the IA wall.     

Strategies for targeting complement inhibitors in ischaemia/reperfusion injury

A transplanted organ suffers inherently from an ischaemic insult and subsequent reperfusion injury. The severity of such early events is thought to influence the success of the transplant procedure, not only in the immediate post-transplant period, but also to predispose the graft to both acute and chronic rejection. In this paper, we review the influence of the complement system upon ischaemia,reperfusion injury. The recognition of the involvement of complement has led to novel strategies to try to modulate ischaemia/reperfusion injury, some of which we have summarized. Finally, we note our own strategy to target complement inhibition in ischaemic tissues.     

Lipopolysaccharides as complement inhibitors by complex formation with the purified third complement component (C3)

Lipopolysaccharides (LPS) from different bacteria in smooth or rough form (Y. enterocolitica, Y. pseudotuberculosis, E. coli, S. typhimurium, S. marcescens) strongly inhibited hemolytic C3 in incubation mixtures with purified C3. LPS from a core deficient mutant was still reactive, whereas lipid A no longer affected C3 activity. The physical state of LPS was critical for its effect on C3. Strand-like LPS structures formed by Ca++-induced aggregation of solubilized LPS, as shown by electron microscopy, demonstrated the highest reactivity with C3. Inhibition of hemolytic C3 was found to be due to complex formation between LPS and C3 by a hydrophobic reaction. The binding capacity of 1 microgram LPS-R and LPS-S was as high as 125 ng C3 and 56 ng C3, respectively. The C3b fragment required different reaction conditions for maximal binding. The strong binding capacity of LPS for the complement component C3 raises the possibility that LPS act as inhibitors of complement by interruption of the reaction cascade at local infectious sites with gram-negative bacteria.     

Membrane-attack complexes and membrane complement inhibitors on leukocyte surface during combined exposure to meningococcal lipopolysaccharide and complement

Leukocytes from donors and subjects with terminal complement component deficiency were incubated for 30 min at 37°C in autologous serum with or without meningococcal lipopolysaccharide (LPS), followed by treatment with monoclonal antibodies to membrane-attack complexes (MAC) and complement inhibitors and cytofluorometry. After incubation in normal serum, about 97% granulocytes and 65% lymphocytes expressed CD55 and CD59. MAC were detected on only 5% of both types of cells. After incubation with the serum and meningococcal LPS, up to 40% granulocytes expressed MAC, expression of CD55 was decreased, and of CD59 virtually did not change. MAC were not detected in cells incubated in the serum with terminal complement component deficiency. LPS-dependent MAC-mediated hyperactivation of granulocytes may play an unfavorable role in meningococcal infection Translated fromBuulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 127, No. 4, pp. 433–438, April, 1999     

Effects of two types of cobra venom factor on porcine complement activation and pulmonary artery pressure

Autologous porcine plasma that has been incubated with cuprophan haemodialysis membranes causes pulmonary hypertension and peripheral leucopenia following reinfusion into swine. These effects appear to be mediated by biologically active fragments of C3 and C5 that are generated as a consequence of ex vivo activation of complement. Putatively, C5a induces the leucopenia; however, the specific contributions of products of C3 and C5 activation to the pulmonary vasoconstriction have not been elucidated. In the present study, the effects of in vivo infusion of two different types of cobra venom factor (CVF) on peripheral leucocyte count and pulmonary artery pressure in the swine are reported. The CVF from Naja n. naja (CVF(TN)) was shown to activate both porcine C3 and C5, whereas the CVF from Naja h. haje (CVF(NH)) activated only C3. Both types of CVF produced pulmonary hypertension. Significant peripheral leucopenia, however, was observed only with CVF(TN). These results suggest that activation products of C3 contribute to the pulmonary hypertension but not to the peripheral leucopenia observed during haemodialysis using dialysis membranes that activate complement.     

Mechanism of complement activation in the hyperacute rejection of porcine organs transplanted into primate recipients.

The authors investigated the importance of natural antibody and complement in the pathogenesis of hyperacute xenograft rejection using in vivo and in vitro pig to primate models. Studies were carried out in rhesus monkeys transplanted with a pig heart or kidney in which hyperacute rejection was observed within a few hours. The rejected organs showed deposits of IgM, C3, C4, C5, and C9 neoantigen along small blood vessels, but few deposits of factors B and P. Removal of anti-endothelial cell "natural" antibodies by plasmapheresis, immunoabsorption, and immunosuppression techniques resulted in marked prolongation of the survival of a subsequently transplanted heart, even when complement levels were within the normal range. Thus, complement, in the absence of natural antibodies, did not initiate hyperacute rejection in this species combination. The requirements for complement activation in human serum to cause cytotoxicity of porcine endothelial cells were then evaluated. Cytotoxicity was abrogated by depleting human serum of IgM, C2, or C5, but not of factor B. Restoration of the effect of serum on endothelial cells was achieved by reconstitution of the respective depleted sera with purified IgM or with the corresponding complement proteins, indicating that IgM and the classical, but not the alternative, pathway of complement, were involved. Identical conclusions were drawn from experiments to ascertain the requirements for complement activation in human serum to mediate binding of iC3b to porcine endothelial cells. The authors conclude that in a pig to primate xenograft complement does not directly initiate injury to the graft but rather requires activation by bound xenoreactive natural antibodies; IgM antibodies directed against endothelial cells activate the classical complement pathway, which then contributes to endothelial cell activation and subsequent events characteristic of hyperacute rejection.