Membrane-targeted complement inhibitors

Molecular Immunology 38 (2001) 249– 255 www.elsevier.com/locate/molimm

Review

Membrane-targeted complement inhibitors Geoffrey P. Smith *, Richard A.G. Smith * Adprotech, Chesterford Research Park, Little Chesterford, Saffron Walden, Essex, CB10 1XL, UK

Abstract 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. © 2001 Elsevier Science Ltd. All rights reserved. Keywords: Cell membrane; Human disease; Cytolytic pore

1. Introduction The power of the complement system in controlling infection needs to be regulated to prevent the destruction of host tissue. Complement activation is associated with numerous human diseases such as rheumatoid arthritis (Perrin et al., 1977), multiple sclerosis (MS, Compston et al., 1989; Gasque et al., 2000) and stroke (Lindsberg et al., 1996). In these diseases, many of the complement regulators that normally protect the membrane surface from complement damage are downregulated or may provide insufficient protection from excessive complement activation. Host tissue is directly destroyed by complement in diseases such as rheumatoid arthritis and MS through the deposition of a cytolytic pore, termed the membrane attack complex (MAC). Complement also exacerbates the inflammaAbbre6iations: MAC, membrane attack complex; GPI, glycosylphosphatidylinositol; (s)CR1, (soluble) complement receptor 1; DAF, decay accelerating factor; MCP, membrane cofactor protein; CHO, Chinese hamster ovary; Dansyl, 5-dimethylaminonaphthalene1-sulphonyl; sLex, Sialyl Lewisx; SCR, short consensus repeat; MSWP1, myristoylated switch peptide 1; MARCKS, myristoylated alanine-rich C kinase substrate; CVF, Cobra venom factor. * Corresponding author. Tel.: +44-870-444-6144; fax: +44-870444-7244. E-mail address: [email protected] (G.P. Smith).

tory process through the generation of two anaphylatoxins, C3a and C5a, that bind to specific receptors on neutrophils and macrophages. Four cell surface proteins act as regulatory molecules and protect the cells on which they are situated from complement attack (reviewed in Morgan and Harris, 1999). Three of these proteins Complement Receptor 1 (CR1, CD35), Decay-accelerating factor (DAF, CD55) and Membrane cofactor protein (MCP, CD46) inhibit the activity of highly specific multi-subunit proteases (convertases) that cleave fluid phase serum proteins C3 and C5. Convertase activity results in the production of anaphylatoxins and generates two labile complement fragments C3b and C5b that bind to nearby activating surfaces through a highly reactive thioester group that is exposed in these proteins. The deposition of C3b on the cell surface seeds further convertase activity on the membrane, whilst membrane-bound C5b initiates the terminal pathway of complement activation that generates the MAC. The formation of the terminal complement pathway is controlled by a fourth membrane-bound complement regulator, CD59. This protein binds C8 in the forming MAC and blocks the recruitment of multiple copies of C9 that are required to form the cytolytic pore. Human CR1 is an integral membrane glycoprotein of Mr 160 –250 kDa that plays a key role in immune

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complex clearance and complement inhibition at the cell surface (Fearon, 1980). CR1 has an extracellular region that, in the major allotypes, comprises 30 repeating short consensus repeat (SCR) domains, a transmembrane region and a small cytoplasmic region (Ahearn and Fearon, 1989). CR1 binds the activated products C3b and C4b (Ahearn and Fearon, 1989), and has also been recently identified as a receptor for C1q (Klickstein et al., 1997). CR1 regulates complement activation by accelerating the decay of C3 and C5 convertases of both the classical and alternative pathways. CR1 is also a cofactor for the serum protease factor I which degrades C3b and C4b to complement fragments that play no further part in the convertase activity. A soluble form of CR1 (sCR1, TP10) has been expressed in Chinese hamster ovary (CHO) cells (Weisman et al., 1990) and is a potent inhibitor of complement activation in a number of disease states (reviewed in Morgan and Harris, 1999). Both classical and alternative pathways of complement activation are inhibited by sCR1, and the protein retains the key regulatory activities of CR1 (Weisman et al., 1990; Yeh et al., 1991). Two other complement regulators, MCP and DAF possess independently the two major functions of CR1, factor I cofactor activity and decay acceleration (reviewed in Morgan and Harris, 1999). The functional similarity of these proteins to CR1 is reflected in their similar structures, both MCP and DAF being composed of four SCR domains. DAF is a 70-kDa glycosylated protein that is tethered to the membrane by a glycophosphatidylinositol (GPI) anchor (Davitz et al., 1986; Medof et al., 1986). By contrast, MCP is an integral membrane glycoprotein, and has a Mr of 39 kDa (Lublin et al., 1988). In contrast to the other membrane-bound complement regulators, CD59 is a single domain protein whose structure is based on the Ly-6 rather than the SCR fold (Davies et al., 1989). The protein has a Mr of Table 1 Strategies for membrane targeting Targeting ligand

Mechanism of membrane binding

Complement inhibitors

sLex carbohydrate

Specific binding sites for Eand P-selectin receptors expressed on the cell surface Cooperative binding that involves membrane insertion and charge interaction

sCR1sLex (TP20)

MSWP1 peptide

Antibody fragments

Specific binding to cells modified by the dansyl hapten

SCR1-3 –MSWP1 (APT070) CD59-MSWP1 DAF-MSWP1 CH1-CD59 H-CD59 CH3-CD59

: 12 kDa and is tethered to the membrane surface by a GPI anchor (Davies et al., 1989). The protein is highly glycoslyated although the function of this modification is unclear because the removal of the carbohydrate does not appear to impair the potency of the protein to inhibit MAC formation (reviewed in Morgan and Harris, 1999). There are a growing number of complement inhibitors that are under development for the treatment of complement-mediated tissue injury and inflammatory disease (reviewed in Sahu and Lambris, 2000). Because complement proteins are deposited at cell surfaces, and the natural complement regulators CR1, MCP, DAF and CD59 are bound to the cell membrane, several groups have investigated strategies to target complement inhibitors to the cell surface in an attempt to enhance their activity. We review the mechanisms behind these various membrane-targeting strategies (Table 1) and discuss the advantages of membrane-targeting for the production of effective complement inhibitors for clinical use.

2. SCR1sLex (TP20) TP20 is a derivative of sCR1 that is targeted to the endothelium (Mulligan et al., 1999). TP20 is different to sCR1 in the composition of N-linked carbohydrate moieties that are added to the protein during expression (Rittershaus et al., 1999). Although both sCR1 and TP20 are produced in CHO cells, TP20 is expressed in a CHO cell line that has been selected for the incorporation of high levels of the sialylated and fucosylated tetrasaccharide sialyl Lewisx (sLex; Fig. 1(A)) (Rittershaus et al., 1999). sCR1 has 25 potential sites for N-glycosylation; : 16 of these sites in TP20 are modified by carbohydrate, of which just over half incorporate sLex (Fig. 1(B)) (Rittershaus et al., 1999). During inflammation, leukocytes are recruited from the bloodstream to the inflamed tissue through their interaction with various adhesion molecules (Springer, 1990). This process allows leukocytes to accumulate at local sites of inflammation and is an essential process for the body’s elimination of infectious agents. Upregulated and/or overexpressed cell adhesion molecules can be found in several diseases where inflammation and immune cells are involved (e.g. arthritis, asthma, ischaemia-reperfusion injury, transplant rejection and stroke) (Bevilacqua et al., 1994; Menger and Vollmar, 1996). Two adhesion molecules that are upregulated during the early inflammatory response are E-selectin (CD62E) and P-selectin (CD62P) (Springer, 1990), both of which contain binding sites for sLex (Lasky, 1992). Selectin blockade is a possible therapeutic way to reduce inflammation, and sLex has been demonstrated to reduce tissue damage in animal models of acute lung

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Fig. 1. Representation of selected membrane-targeting ligands and membrane-targeted complement inhibitors. (A) Structure of sLex. (B) Diagram of TP20 in which individual SCR domains are depicted by circles and sLex moieties by arrows. (C) Structure of MSWP1. (D) Diagram of APT070 in which numbered circles indicate SCR domains of CR1, and the membrane-targing peptide MSWP1 is represented by the ‘ + ’ characters and wavy line. (E) Diagram of chimeras between CD59 (boxes) and various dansyl-specific antibody fragments. Ovals represent individual antibody light and heavy (black) chain domains.

injury (Mulligan et al., 1993a,b) and myocardial reperfusion (Buerke et al., 1994). By decorating sCR1 with multiple copies of sLex, TP20 targets the cell membrane at sites of vascular injury (Table 1), with the potential of inhibiting complement activation and blocking selectin-mediated leukocyte adhesion. The activity of TP20 has been investigated both in vitro and in vivo. In vitro studies have shown that TP20 binds to cells that express E-selectin on the cell surface (Rittershaus et al., 1999). TP20 is also a potent complement inhibitor of cell lysis by both the classical and alternative pathways (Rittershaus et al., 1999). The protein inhibits the binding of neutrophils to activated endothelial cells in culture with an IC50 value that reflects the multivalent display of sLex on the protein (Rittershaus et al., 1999). In vivo, in a model of lung injury induced by Cobra venom factor (CVF) (Mulli-

gan et al., 1999), the administration of TP20 showed a significantly greater degree of protection than sCR1. Furthermore, rats treated with TP20 showed reduced neutrophil accumulation compared to sCR1. Immunostaining of rat lung after CVF treatment demonstrated accumulation of TP20 in the tissue; by contrast, no inhibitor was detected in rats treated with sCR1. These data suggested that the additional protective effects of TP20 were due to its targeting to vascular tissue. Similar increases in potency over sCR1 were demonstrated for TP20 in a mouse stroke model (Huang et al., 1999). In this study, mice were subjected to cerebral ischaemia for 45 min, followed by reperfusion for 24 h. TP20 reduced the infarct volume to a greater extent than sCR1, and also showed reduced neutrophil and platelet accumulation and increased cerebral blood flow. Furthermore, mice treated with TP20 showed reduced neu-

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rological deficit compared to mice treated with sCR1. TP20 has also been evaluated in two other preclinical studies: a rat lung allograft model (Stammberger et al., 2000), and a rat myocardial ischaemia model (Zacharowski et al., 1999).

3. SCR1-3 –MSWP1 (APT070) Studies on the structure and function of CR1 revealed that there was a discrete binding site for C4b located in the N-terminal SCRs, and further binding sites for C3b in SCRs 8– 10 and 15– 17 (Klickstein et al., 1988; Krych et al., 1991). The location of these important regulatory sites in fragments of CR1 prompted an evaluation of such CR1 fragments as possible therapeutic compounds. To date, the most advanced studies have been conducted with the first three SCRs (SCR1-3) at the N-terminus of CR1. SCR1-3 of CR1 was expressed in Escherichia coli and refolded from inclusion bodies (Dodd et al., 1995). Contrary to earlier proposals, this protein fragment contained the inhibitory functions for both the classical and alternative pathways of complement activation and was also a cofactor for factor I-mediated cleavage of both C3b and C4b (Mossakowska et al., 1999). However, the biological activity of SCR1-3 was significantly lower than that of full-length sCR1, due to the multiple binding sites for C3b and/or C4b that exist within the native 30 domain CR1. Because the C3b/C4b-containing targets of SCR1-3 are located on the membrane surface, it was postulated that the potency of this complement inhibitor may be enhanced by membrane targeting. The membranetargeting approach that was devised was inspired by studies of the membrane association processes regulated by myristoylation (McLaughlin and Aderem, 1995). The attachment of many intracellular proteins (e.g. myristoylated alanine-rich C kinase substrate (MARCKS), p21ras variants, Src) to the inner leaflet of the plasma membrane occurs through a two-site interaction: firstly, the insertion into the lipid bilayer of a lipophilic acyl chain, such as myristate (C14); and secondly, the association of a stretch of basic amino acids in the protein with the negatively charged headgroups of phosphatidylserine which is sequestered in this leaflet of the membrane (reviewed in McLaughlin and Aderem, 1995; Murray et al., 1997). Interspersed within the basic region of MARCKS are serine residues that can be phosphorylated. This modification changes the net charge of the attachment domain to neutral and causes the detachment of the protein from the membrane. This process has been termed the myristoyl-electrostatic switch (McLaughlin and Aderem, 1995). The apparent lack of constraints on the spatial relationship between the membrane-insertive and basic attachment

domain suggested that the essential features of the two-site membrane attachment mechanism of MARCKS could be mimicked in a minimized peptide structure (Table 1). Furthermore, by incorporating an activated disulphide group into the peptide during the synthesis, these membrane-targeting peptides could be coupled to proteins via standard thiol-interchange chemistry. The structure of a typical membrane-targeting peptide, MSWP1, is shown in Fig. 1(C). The structure of the membrane-targeted derivative of SCR1-3 of CR1 (APT070) is depicted in Fig. 1(D). The synthetic gene that encoded SCR1-3 protein was modified to introduce a cysteine at the C-terminus of the protein. This protein, SCR1-3cys, was successfully expressed in E. coli, and when refolded, had identical biological activity to the parent compound, SCR1-3 (Smith et al., 1998a and manuscript in preparation). Following selective reduction of SCR1-3cys, the membrane-targeting peptide MSWP1 was coupled to the protein by disulphide exchange to produce APT070 (Smith et al., 1998a,b). APT070 was soluble in common biological buffers in the absence of detergents. The biological activity of APT070 was enhanced over 100-fold compared to its parent protein SCR1-3 in the classical pathway haemolytic assay. By contrast, the activity of the parent protein was not increased by modification by a myristoyl group alone, nor by the extension of SCR1-3 by a sequence corresponding to the basic membrane association domain. These data demonstrated that the complement inhibitory activity of SCR1-3 was increased by the post-translational attachment of a synthetic membrane-targeting peptide that contained both a membrane-insertive and membrane-associative elements. Fluorescence microscopy has shown that APT070 binds to a wide variety of cell types (Dodd et al., 2000). APT070 is a potent complement inhibitor in vivo. Compared to the unmodified SCR1-3 protein, APT070 demonstrated a dramatic increase in the protection of rats from vascular shock in a model of anti-basement membrane disease (Smith et al., 1998b). APT070 was also an effective agent in the prevention of acute kidney rejection resulting from ischaemia-reperfusion damage in the rat (Dong et al., 1999), and was also effective in reducing the swelling in the antigen-induced rat model of arthritis (Linton et al., 2000). In both the kidney transplantation model and arthritis model, APT070 could be visualised binding to the membranes of the target tissues in the protected animals. These studies have provided proof-of-principle data that demonstrate that combining complement inhibition and membrane targeting is an effective therapeutic strategy. APT070 has been manufactured to cGMP for human trials (Dodd et al., 2000).

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4. CD59-MSWP1 and DAF-MSWP1 In their native states both CD59 and DAF are tethered to the membrane via GPI-anchors (Davitz et al., 1986; Medof et al., 1986; Davies et al., 1989). Although GPI-anchored CD59 and DAF can be extracted from biological membranes (Medof et al., 1984; Sugita et al., 1988), this process yields too little protein to be considered viable for the manufacture of a therapeutic protein. In addition, GPI-anchored proteins are only soluble in aqueous solution in the presence of detergents and localise poorly on biological membranes due to serum absorption (Moran et al., 1992; Sugita et al., 1994). Soluble forms of both CD59 and DAF have been produced by recombinant means (Moran et al., 1992; Kieffer et al., 1994; Sugita et al., 1994) and exhibit the expected complement inhibitory functions in vitro. However, the specific activity of these anchorless proteins is significantly lower than for the native proteins in their GPI-anchored forms. Despite these problems, an anchorless derivative of DAF was effective in the reversed passive Arthus reaction in the rat (Moran et al., 1992). However, it could be anticipated that the potency of these soluble inhibitors would be increased by appropriate membrane-targeting. The membrane-targeting strategy that was devised for SCR1-3 of CR1 has also been applied to CD59 and DAF. In preliminary experiments (Smith et al., 2000), soluble forms of CD59 were derivatized with 2-iminothiolane (Traut’s reagent) to introduce free thiol groups randomly into the protein. This modified form of CD59 was then reacted with the MSWP1 membrane-targeting peptide. Although the modification procedure was a random process (2-iminothiolane modifies free amine groups), reaction conditions could be found that resulted in the derivatization of CD59 by only a single MSWP1 peptide. This product, CD59-MSWP1, was soluble in aqueous buffers in the absence of detergent. Flow cytometry demonstrated that CD59-MSWP1 bound to cells. When the activity of CD59-MSWP1 was examined in the reactive lysis haemolytic assay, the compound was equal in potency to native (GPI-anchored) CD59 and approximately 100-fold more potent than soluble (anchorless) CD59. These data demonstrated that the in vitro specific activity of CD59 could be improved by membranetargeting. To our knowledge, no membrane-targeted version of CD59 has been tested in vivo at least in a non-transgenic setting. However, efforts under underway in our laboratory to produce a derivative of CD59 that can be modified by membrane-targeted peptides in a site-specific manner suitable for use as a therapeutic compound. The production of a membrane-targeted version of DAF has been achieved in a similar manner to the

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process developed for APT070 (White et al., manuscript in preparation). A recombinant gene that encoded the four SCRs of DAF was modified to introduce a unique cysteine at the C-terminus of the encoded polypeptide. The resulting construct was expressed in E. coli and refolded. After selective reduction of the C-terminal thiol, the protein was converted to a membrane-targeted form by reaction with MSWP1. The resulting compound, DAF-MSWP1, possessed complement inhibitory activity in both classical and alternative pathway assays. In these in vitro haemolytic assays, the specific activity of DAFMSWP1 was identical to APT070. Quantities of DAF-MSWP1 have been produced that permit assessment of the efficacy of this compound in rodent-based animal models of disease. Furthermore, a scaleable manufacturing process has been devised that would permit the production of this protein to cGMP.

5. CD59-IgG The specific targeting of proteins to different cell types can be achieved by construction of fusion proteins that incorporate a cell-specific antibody (or antibody fragment). An appropriate target for such a strategy for complement inhibition would be the cell surface of a specific organ or tissue undergoing an inflammatory process. To produce cell-targeted derivatives of CD59, Zhang et al. (1999) produced several constructs that encoded CD59 fused to antibody fragments that bound the hapten 5-dimethylaminonaphthalene-1sulphonyl (dansyl). In this study, CD59 was fused to the antibody combining site after either the CH1 domain (CH1-CD59), after the hinge (H-CD59), or after the CH3 Ig region (CH3-CD59; Fig. 1(E)). The recombinant proteins were expressed in mammalian cells to produce refolded chimeras that specifically recognised dansyl. Flow cytometry showed that these proteins bound to CHO cells that had been labelled with dansyl on the cell surface; by contrast, these proteins did not bind to wild type CHO cells. The chimeric proteins also protected dansyl-labelled CHO cells from complement-mediated lysis. However, these proteins differed in their specific activities. The most potent compounds, the CH1 and hinge chimeras, positioned CD59 close to the membrane surface; by comparison, CD59 fused to the full length antibody had significantly lower activity. These data suggested that in addition to being membrane-targeted, CD59 must also be located at the correct distance form the cell membrane to be an effective complement inhibitor. Similar constraints on the distance of CD59 from the membrane surface have been inferred from other studies (Fodor et al., 1995).

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6. Conclusions Membrane targeting has been demonstrated to be an effective strategy to increase both the potency and the effective selectivity of recombinant versions of complement regulatory proteins. Different research groups have devised alternative approaches to achieve this success: modification by the glycoprotein sLex; modification by short, synthetic membrane-targeting peptides; and fusion to cell-specific antibody fragments. Two of these membrane targeted complement inhibitor proteins, TP20 and APT070, have been shown to be extremely effective in animal models of disease. The benefits of targeting complement inhibitors to the membrane outweigh the additional process implications and costs involved in their production and it is likely that these proteins will become the drugs of choice for limiting the injury associated with complement-mediated disease. Furthermore, for some complement regulators, such as CD59, the true effectiveness of these compounds will only be assessed by their delivery in membrane-targeted forms. Finally, membrane-targeting that allows stable high-affinity attachment to membranes (as the myristoyl-switch approach apparently does) will permit new therapeutic modalities using local delivery and ex-vivo cell or organ modification.

Acknowledgements We thank the research team at Adprotech for their contributions to the membrane-targeted complement inhibitor programme and numerous collaborators for their advice and support of that programme especially: B.P. Morgan, P.J. Lachmann, A. Davies, S.H. Sacks, J.R. Pratt and D.T. Fearon.

References Ahearn, J.M., Fearon, D.T., 1989. Structure and function of the complement receptors, CR1 (CD35) and CR2 (CD21). Adv. Immunol. 46, 183 – 219. Bevilacqua, M.P., Nelson, R.M., Mannori, G., Cecconi, O., 1994. Endothelial-leukocyte adhesion molecules in human disease. Annu. Rev. Med. 45, 361 –378. Buerke, M., Weyrich, A.S., Zheng, Z., Gaeta, F.C., Forrest, M.J., Lefer, A.M., 1994. Sialyl Lewisx-containing oligosaccharide attenuates myocardial reperfusion injury in cats. J. Clin. Invest. 93, 1140 – 1148. Compston, D.A., Morgan, B.P., Campbell, A.K., Wilkins, P., Cole, G., Thomas, N.D., Jasani, B., 1989. Immunocytochemical localization of the terminal complement complex in multiple sclerosis. Neuropathol. Appl. Neurobiol. 15, 307 –316. Davies, A., Simmons, D.L., Hale, G., Harrison, R.A., Tighe, H., Lachmann, P.J., Waldmann, H., 1989. CD59, an LY-6-like protein expressed in human lymphoid cells, regulates the action of the complement membrane attack complex on homologous cells. J. Exp. Med. 170, 637 –654.

Davitz, M.A., Low, M.G., Nussenzweig, V., 1986. Release of decayaccelerating factor (DAF) from the cell membrane by phosphatidylinositol-specific phospholipase C (PIPLC). Selective modification of a complement regulatory protein. J. Exp. Med. 163, 1150 – 1161. Dodd, I., Mossakowska, D.E., Camilleri, P., Haran, M., Hensley, P., Lawlor, E.J., McBay, D.L., Pindar, W., Smith, R.A., 1995. Overexpression in Escherichia coli, folding, purification, and characterization of the first three short consensus repeat modules of human complement receptor type 1. Protein Exp. Purif. 6, 727 – 736. Dodd, I., Oldroyd, R., Powell, S., Affleck, L., bamber, L., Gallagher, S., Rowling, P., Ragnauth, C., Smith, G., Pratt, J.R., Sacks, S.H., Linton, S.M., Morgan, B.P., Smith, R., 2000. Development of a membrane-targeted complement inhibitor for clinical use. Immunopharmacology 49, 63. Dong, J., Pratt, J.R., Smith, R.A., Dodd, I., Sacks, S.H., 1999. Strategies for targeting complement inhibitors in ischaemia/reperfusion injury. Mol. Immunol. 36, 957 – 963. Fearon, D.T., 1980. Identification of the membrane glycoprotein that is the C3b receptor of the human erythrocyte, polymorphonuclear leukocyte, B lymphocyte, and monocyte. J. Exp. Med. 152, 20 – 30. Fodor, W.L., Rollins, S.A., Guilmette, E.R., Setter, E., Squinto, S.P., 1995. A novel bifunctional chimeric complement inhibitor that regulates C3 convertase and formation of the membrane attack complex. J. Immunol. 155, 4135 – 4138. Gasque, P., Dean, Y.D., McGreal, E.P., VanBeek, J., Morgan, B.P., 2000. Complement components of the innate immune system in health and disease in the CNS. Immunopharmacology 49, 171 – 186. Huang, J., Kim, L.J., Mealey, R., Marsh, H.C. Jr., Zhang, Y., Tenner, A.J., Connolly, E.S Jr., Pinsky, D.J., 1999. Neuronal protection in stroke by an sLex-glycosylated complement inhibitory protein. Science 285, 595 – 599. Kieffer, B., Driscoll, P.C., Campbell, I.D., Willis, A.C., van der Merwe, P.A., Davis, S.J., 1994. Three-dimensional solution structure of the extracellular region of the complement regulatory protein CD59, a new cell-surface protein domain related to snake venom neurotoxins. Biochemistry 33, 4471 – 4482. Klickstein, L.B., Barbashov, S.F., Liu, T., Jack, R.M., NicholsonWeller, A., 1997. Complement receptor type 1 (CR1, CD35) is a receptor for C1q. Immunity 7, 345 – 355. Klickstein, L.B., Bartow, T.J., Miletic, V., Rabson, L.D., Smith, J.A., Fearon, D.T., 1988. Identification of distinct C3b and C4b recognition sites in the human C3b/C4b receptor (CR1, CD35) by deletion mutagenesis. J. Exp. Med. 168, 1699 – 1717. Krych, M., Hourcade, D., Atkinson, J.P., 1991. Sites within the complement C3b/C4b receptor important for the specificity of ligand binding. Proc. Natl. Acad. Sci. USA 88, 4353 – 4357. Lasky, L.A., 1992. Selectins: interpreters of cell-specific carbohydrate information during inflammation. Science 258, 964 – 969. Linton, S.M., Williams, A.S., Dodd, I., Smith, R., Williams, B.D., Morgan, B.P., 2000. Therapeutic efficacy of a novel membranetargeted complement regulator in antigen-induced arthritis in the rat. Arthritis Rheum 43, 2590 – 2597. Lindsberg, P.J., Ohman, J., Lehto, T., Karjalainen-Lindsberg, M.L., Paetau, A., Wuorimaa, T., Carpen, O., Kaste, M., Meri, S., 1996. Complement activation in the central nervous system following blood– brain barrier damage in man. Ann. Neurol. 40, 587 –596. Lublin, D.M., Liszewski, M.K., Post, T.W., Arce, M.A., Le Beau, M.M., Rebentisch, M.B., Lemons, L.S., Seya, T., Atkinson, J.P., 1988. Molecular cloning and chromosomal localization of human membrane cofactor protein (MCP). Evidence for inclusion in the multigene family of complement-regulatory proteins. J. Exp. Med. 168, 181 – 194. McLaughlin, S., Aderem, A., 1995. The myristoyl-electrostatic switch: a modulator of reversible protein – membrane interactions. Trends Biochem. Sci. 20, 272 – 276.

G.P. Smith, R.A.G. Smith / Molecular Immunology 38 (2001) 249–255 Medof, M.E., Kinoshita, T., Nussenzweig, V., 1984. Inhibition of complement activation on the surface of cells after incorporation of decay-accelerating factor (DAF) into their membranes. J. Exp. Med. 160, 1558 – 1578. Medof, M.E., Walter, E.I., Roberts, W.L., Haas, R., Rosenberry, T.L., 1986. Decay accelerating factor of complement is anchored to cells by a C-terminal glycolipid. Biochemistry 25, 6740 – 6747. Menger, M.D., Vollmar, B., 1996. Adhesion molecules as determinants of disease: from molecular biology to surgical research. Br. J. Surg. 83, 588 – 601. Moran, P., Beasley, H., Gorrell, A., Martin, E., Gribling, P., Fuchs, H., Gillett, N., Burton, L.E., Caras, I.W., 1992. Human recombinant soluble decay accelerating factor inhibits complement activation in vitro and in vivo. J. Immunol. 149, 1736 –1743. Morgan, B.P., Harris, C.L., 1999. Complement Regulatory Proteins. Academic Press, London. Mossakowska, D., Dodd, I., Pindar, W., Smith, R.A., 1999. Structure-activity relationships within the N-terminal short consensus repeats (SCR) of human CR1 (C3b/C4b receptor, CD35): SCR 3 plays a critical role in inhibition of the classical and alternative pathways of complement activation. Eur. J. Immunol. 29, 1955 – 1965. Mulligan, M.S., Lowe, J.B., Larsen, R.D., Paulson, J., Zheng, Z.L., DeFrees, S., Maemura, K., Fukuda, M., Ward, P.A., 1993a. Protective effects of sialylated oligosaccharides in immune complex-induced acute lung injury. J. Exp. Med. 178, 623 –631. Mulligan, M.S., Paulson, J.C., De Frees, S., Zheng, Z.L., Lowe, J.B., Ward, P.A., 1993b. Protective effects of oligosaccharides in P-selectin-dependent lung injury. Nature 364, 149 –151. Mulligan, M.S., Warner, R.L., Rittershaus, C.W., Thomas, L.J., Ryan, U.S., Foreman, K.E., Crouch, L.D., Till, G.O., Ward, P.A., 1999. Endothelial targeting and enhanced antiinflammatory effects of complement inhibitors possessing sialyl Lewisx moieties. J. Immunol. 162, 4952 –4959. Murray, D., Ben-Tal, N., Honig, B., McLaughlin, S., 1997. Electrostatic interaction of myristoylated proteins with membranes: simple physics, complicated biology. Structure 5, 985 –989. Perrin, L.H., Nydegger, U.E., Zubler, R.H., Lambert, P.H., Miescher, P.A., 1977. Correlation between levels of breakdown products of C3, C4 and properdin factor B in synorial fluids from patients with rhemated arthritis. Arthritis Rheum. 20, 647 – 652. Rittershaus, C.W., Thomas, L.J., Miller, D.P., Picard, M.D., Geoghegan-Barek, K.M., Scesney, S.M., Henry, L.D., Sen, A.C., Bertino, A.M., Hannig, G., Adari, H., Mealey, R.A., Gosselin, M.L., Couto, M., Hayman, E.G., Levin, J.L., Reinhold, V.N., Marsh, H.C. Jr., 1999. Recombinant glycoproteins that inhibit complement activation and also bind the selectin adhesion molecules. J. Biol. Chem. 274, 11237 –11244.

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Sahu, A., Lambris, J., 2000. Complement inhibitors: a resurgent concept in anti-inflammatory therapeutics. Immunopharmacology 49, 133 – 148. Smith, G., Dodd, I., Davies, A., Morgan, B.P., Lachmann, P.J., Smith, R., 2000. Derivatization of soluble human CD59 with a myristoylated peptide creates a potent membrane-bound inhibitor of complement-mediated lysis. Immunopharmacology 49, 53. Smith, R.A.G., Dodd, I., Mossakowska, D.E.I., 1998a. Conjugates of soluble peptidic compounds with membrane-binding agents. International Patent Publication No. WO 98/02454. Smith, R., Dodd, I., Rowling, P., Cox, V., Mossakowska, D., Oldroyd, R., Lachmann, P., 1998b. Cell surface engineering using a complement regulatory molecule modified with a synthetic myristoyl-electrostatic switch derivative. Mol. Immunol. 35, 400. Springer, T.A., 1990. Adhesion receptors of the immune system. Nature 346, 425 – 434. Stammberger, U., Hamacher, J., Hillinger, S., Schmid, R.A., 2000. sCR1sLe ameliorates ischemia/reperfusion injury in experimental lung transplantation. J. Thorac. Cardiovasc. Surg. 120, 1078 – 1084. Sugita, Y., Ito, K., Shiozuka, K., Suzuki, H., Gushima, H., Tomita, M., Masuho, Y., 1994. Recombinant soluble CD59 inhibits reactive haemolysis with complement. Immunology 82, 34 – 41. Sugita, Y., Nakano, Y., Tomita, M., 1988. Isolation from human erythrocytes of a new membrane protein, which inhibits the formation of complement transmembrane channels. J. Biochem. (Tokyo) 104, 633 – 637. Weisman, H.F., Bartow, T., Leppo, M.K., Marsh, H.C. Jr., Carson, G.R., Concino, M.F., Boyle, M.P., Roux, K.H., Weisfeldt, M.L., Fearon, D.T., 1990. Soluble human complement receptor type 1: in vivo inhibitor of complement suppressing post-ischemic myocardial inflammation and necrosis. Science 249, 146 – 151. Yeh, C.G., Marsh, H.C. Jr., Carson, G.R., Berman, L., Concino, M.F., Scesney, S.M., Kuestner, R.E., Skibbens, R., Donahue, K.A., Ip, S.H., 1991. Recombinant soluble human complement receptor type 1 inhibits inflammation in the reversed passive arthus reaction in rats. J. Immunol. 146, 250 – 256. Zacharowski, K., Otto, M., Hafner, G., Marsh, H.C. Jr., Thiemermann, C., 1999. Reduction of myocardial infarct size with sCR1sLe(x), an alternatively glycosylated form of human soluble complement receptor type 1 (sCR1), possessing sialyl Lewis x. Br. J. Pharmacol. 128, 945 – 952. Zhang, H.F., Yu, J., Bajwa, E., Morrison, S.L., Tomlinson, S., 1999. Targeting of functional antibody-CD59 fusion proteins to a cell surface. J. Clin. Invest. 103, 55 – 61.