Coupling complement regulators to immunoglobulin domains ...

Clin Exp Immunol 2002; 129:198–207

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Coupling complement regulators to immunoglobulin domains generates effective anti-complement reagents with extended half-life in vivo C. L. HARRIS*, A. S. WILLIAMS†, S. M. LINTON† & B. P. MORGAN* Departments of *Medical Biochemistry and †Rheumatology, University of Wales College of Medicine, Cardiff, UK

(Accepted for publication 13 May 2002)

SUMMARY Complement activation and subsequent generation of inflammatory molecules and membrane attack complex contributes to the pathology of a number of inflammatory and degenerative diseases, including arthritis, glomerulonephritis and demyelination. Agents that specifically inhibit complement activation might prove beneficial in the treatment of these diseases. Soluble recombinant forms of the naturally occurring membrane complement regulatory proteins (CRP) have been exploited for this purpose. We have undertaken to design better therapeutics based on CRP. Here we describe the generation of soluble, recombinant CRP comprising rat decay accelerating factor (DAF) or rat CD59 expressed as Fc fusion proteins, antibody-like molecules comprising two CRP moieties in place of the antibody Fab arms (CRP-Ig). Reagents bearing DAF on each arm (DAF-Ig), CD59 on each arm (CD59Ig) and a hybrid reagent containing both DAF and CD59 were generated. All three reagents inhibited C activation in vitro. Compared with soluble CRP lacking Fc domains, activity was reduced, but was fully restored by enzymatic release of the regulator from the Ig moiety, implicating steric constraints in reducing functional activity. In vivo studies showed that DAF-Ig, when compared to soluble DAF, had a much extended half-life in the circulation in rats and concomitantly caused a sustained reduction in plasma complement activity. When given intra-articularly to rats in a model of arthritis, DAF-Ig significantly reduced severity of disease. The data demonstrate the potential of CRP-Ig as reagents for sustained therapy of inflammatory disorders, including arthritis, but emphasize the need for careful design of fusion proteins to retain function. Keywords complement

decay accelerating factor

INTRODUCTION The complement (C) system forms a powerful arm of the innate immune system. Targeting of a cell by C results in phagocytosis through opsonization with C3b, cell damage or death through formation of the cytolytic macromolecular membrane attack complex (MAC) and inflammation due to production of the inflammatory mediators (C5a, C3a and MAC). To protect from ‘inappropriate’ targeting by this potentially harmful defence mechanism, self-cells express on their membranes an armoury of complement regulatory proteins (CRP) which rapidly and efficiently inactivate ‘accidental’ foci of C activation [1,2]. CRP function either by inactivating the enzymes formed during C

Correspondence: Claire Harris, Department of Medical Biochemistry, University of Wales College of Medicine, Heath Park, Cardiff, CF14 4XN, UK E-mail: [email protected]

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activation which are responsible for cleavage of C3 and C5, the C3 and C5 convertases, respectively, or by interfering with MAC formation. In the human, the CRP membrane cofactor protein (MCP; CD46), decay accelerating factor (DAF; CD55) and complement receptor 1 (CR1; CD35) inactivate the convertases by either accelerating the natural decay of the enzymes (DAF and CR1) or by acting as cofactor for the serine protease factor I which irreversibly cleaves and inactivates C3b and C4b (MCP and CR1). A fourth regulator, CD59, acts by binding C8 in the forming MAC and inhibiting C9 polymerization and MAC formation. DAF and CD59 are linked to the plasma membrane through a GPI anchor, whereas MCP and CR1 are transmembrane proteins. Rodents have an additional regulator of the convertase enzymes, termed Crry [3]. This transmembrane regulator has both decay and cofactor activities and contains the same building blocks, short concensus repeats (SCR), found in MCP, DAF and CR1 [4]. SCR are globular protein domains consisting of approximately 60 amino acids, many of which are highly © 2002 Blackwell Science

CRP-Ig as therapeutic reagents conserved, and are linked end to end to form the flexible, elongated structures characteristic of most activation pathway regulators. The functional activity of SCR-containing regulators resides in these domains. The entire extracellular domain of the most common isoform of CR1 contains 30 of these repeat units, forming three C3b/C4b binding sites, whereas MCP and DAF each contain four SCR. CD59 is structurally unrelated and functionally distinct from these activation pathway regulators. In normal circumstances, these control mechanisms are sufficient to protect cells from damage by homologous C. However, evidence of C activation is abundant in inflammatory diseases including rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), lupus nephritis and multiple sclerosis. In RA, soluble products of C activation are present in the synovial fluid of affected joints and complement deposits are evident on synovial tissue [5–7]. Affected joints are full of leucocytes (neutrophils and T cells) attracted to the site by a gradient of C5a and other chemoattractants. Whilst C itself is not always the primary cause of these diverse diseases, it acts to sustain the pro-inflammatory cycle and perpetuate tissue damage. The involvement of C in the perpetuation and exacerbation of these disorders is indisputable and has driven the search for therapeutic reagents capable of inhibiting the C cascade. Two reagents are currently in clinical trials for treatment of acute inflammatory disorders such as adult respiratory distress syndrome (ARDS), or ischaemia-reperfusion injury. The first reagent, a single chain Fv (scFv), binds C5 and prevents its enzymatic cleavage [8]. The second reagent is a soluble, recombinant form of CR1 (sCR1), this reagent inhibits formation of the amplification enzymes responsible for C5a production [9]. We and others have used human sCR1 to treat animal models of arthritis. Administration of sCR1 intravenously (iv) in rat collagen-induced arthritis (CIA) inhibited disease development when given as prophylactic treatment and significantly reduced disease severity in established disease [10]. However, sCR1 is rapidly cleared and in order to maintain an anti-C effect it had to be administered twice daily at a dose of 15 mg/kg. Further, the duration of treatment was restricted to 5 days due to the development of an anti-human CR1 antibody response. Soluble recombinant forms of other human and rodent regulators have been generated and tested in complement-mediated inflammatory conditions such as the Arthus reaction and rejection in xenotransplantation. Again, therapeutic effects are evident, but their use is limited by short circulating half-lives; reported b phase half-lives in rodents vary from 90 min to 8 h [11]. The rapid clearance of these ‘first-generation’ CRP-based anti-C reagents necessitates continual transfusion to maintain an anti-C effect, and limits their use to treatment of acute conditions. Here we have attempted to address some of these limitations by generating CRP-containing reagents that, by virtue of their attachment to an antibody Fc domain, are anticipated to have extended half-lives in vivo. We have chosen to use rat CRP in these reagents to facilitate their testing in rats [12,13]. CRP-Ig, including rat DAF-Ig, rat CD59-Ig and a hybrid CRP-Ig containing both DAF and CD59 have been generated and tested in vitro. We have further analysed the functional properties in vivo of rat DAF-Ig, and have contrasted its long half-life with the rapid clearance of soluble DAF lacking an Fc domain. The results demonstrate that CRP-Ig have potential as therapeutic reagents, but highlight the requirement for careful molecular design to ensure maximum functional activity and therapeutic benefit.

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MATERIALS AND METHODS Materials Chemicals and reagents were from Fisher Scientific (Loughborough, UK) or Sigma (Poole, UK) unless otherwise stated below. All tissue culture reagents and plastics were from Life Technologies (Paisley, UK). pDR2DEF1a was a gift from Dr I. Anegon (INSERM U437, Nantes, France) [14], Signal pIgplus and pIgplus were from R & D Systems (Abingdon, UK). Sheep erythrocytes in Alsever’s solution were from TCS Microbiology (Claydon, UK), guinea pig erythrocytes and rat serum were from the local animal facility. Rabbit anti-sheep erythrocyte antibody (Amboceptor) was from Boehring Diagnostics GmbH (Marburg, Germany), goat anti-mouse IgG-HRPO was purchased from BioRad Ltd (Hemel Hempstead, UK) and goat anti-human FcHRPO was from Sigma. Monoclonal antibodies recognizing rat DAF (RDIII-7, RDII-24) and rat CD59 (6D1) were raised in this laboratory [15]. Soluble, recombinant human C receptor 1 (sCR1) was a gift from T Cell Sciences Inc (Needham, MA), pure human IgG1 and papain were from Sigma. Prosep A was from Bioprocessing Ltd (Consett, UK). PBS is 8·1 mM Na2PO4, 1·5 mM KH2PO4, 137 mM NaCl, 2·7 mM KCl, pH 7·4 (Oxoid Ltd, Basingstoke, UK). C fixation diluent (CFD; Oxoid Ltd) is 2·8 mM barbituric acid, 145·5 mM NaCl, 0·8 mM MgCl2, 0·3 mM CaCl2, 0·9 mM sodium barbital pH 7·2. GVB is CFD, 0·1% (w/v) gelatin. Preparation of recombinant proteins DAF-Ig and CD59-Ig. DNA encoding the four SCR of rat DAF (C-terminal residue as in the published sequences of the mature protein: Arg252 [13]) was cloned into the expression vector SigpIg (R & D Systems) and that encoding the signal peptide and entire extracellular domain of CD59, omitting the GPI anchor signal sequence (C-terminal residue as in the published sequences of the mature protein: Lys76 [12]), was cloned into the vector pIgPlus (R & D Systems). Cloning procedures were as described previously [16]. Vent DNA proof-reading polymerase was used in the PCR reactions and sequencing confirmed that no errors had been introduced by PCR. In both cases DNA encoding the regulator was cloned upstream of and in frame with DNA encoding the hinge and Fc domains of human IgG1. In order to achieve high levels of expression, DNA encoding the signal peptide, regulator and Fc domains was then subcloned using PCR into the high expression vector pDR2DEF1a. CHO cells were transfected using lipofectamine (Life Technologies) according to the manufacturer’s instructions and stable lines were established by selection with 400 mg/ml Hygromycin B (Life Technologies). Supernatant was collected and passed over a Prosep A column (Bioprocessing Ltd, Consett, UK) to purify the fusion protein. The column was washed with PBS and with 0·1 M citrate buffer pH 5·0 to remove contaminating bovine Ig and the fusion protein was eluted with 0·1 M Glycine/HCl pH 2·5. Eluted protein was neutralized with Tris, concentrated by ultrafiltration and dialysed into PBS. A control SCR-containing fusion protein, comprising SCRs from pig DAF, was also prepared in an identical manner, this fusion protein had no C-regulatory function. A second CD59-containing fusion protein was also prepared in which an 11 amino acid spacer comprising the amino acids (SerGly-Gly-Gly-Gly)2-Ser was inserted between CD59 and the antibody hinge using two stage PCR. Briefly, DNA encoding CD59 and the Fc domains was reamplified in two separate reactions using new primers that incorporated the sequence of the

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spacer domain at the 3¢ end of CD59 and at the 5¢ end of the Ig hinge. The two PCR products were mixed together and allowed to anneal at complementary DNA sequences encoding the spacer domain. Following PCR using outside primers, the product was ligated into the expression vector pDR2DEF1a. Cells were transfected and the second CD59-Ig protein was purified as described above. Protein concentrations were determined using the Pierce Comassie assay (Perbio Science UK Ltd, Tattenhall, UK) with bovine serum albumin as a standard. Soluble rat DAF. DNA encoding the signal peptide and four SCRs of rat DAF (C-terminal residue Lys254) was cloned directly into the expression vector pDR2DEF1a. CHO cells were transfected as described above. sDAF was purified from supernatant by affinity chromatography on a monoclonal anti-DAF (RDII-24) column [15]. Protein was eluted using 50 mM diethylamine pH 11 and immediately lyophilized. The dried protein was solubilized in phosphate buffer with 1 M NaCl and was applied to a Superose 12 gel filtration column (Amersham-Pharmacia Biotech AB, Uppsala, Sweden). Proteins were eluted with PBS and fractions containing DAF were identified. The pure DAF was concentrated by ultrafiltration. Soluble CD59 containing the entire extracellular portion (omitting the GPI anchor) was also produced in transfected CHO cells and was a gift from Dr E Lafferty (UWCM and idENTIGENcyf, Cardiff, UK). Cleavage of protein using papain. In some experiments soluble DAF or CD59 was released by cleaving the Fc domain from the fusion protein using papain. Fusion protein was dialysed into 0·1 M phosphate buffer pH 7, 2 mM EDTA. Lyophilized papain was dissolved in the same buffer supplemented with 10 mM cysteine and incubated at 37°C for 30 min to activate. The enzyme was then passed over a PD10 buffer exchange column (Amersham-Pharmacia Biotech AB) equilibrated in phosphate/EDTA to remove cysteine and added immediately to the fusion protein at 4% (w : w). The incubation was allowed to proceed at 37°C until the Fc domain had been digested (generally overnight) and was quenched using 0·1 mg/ml iodoacetamide. Undigested fusion protein and the free Fc domain were removed from the incubation by adsorption on solid phase protein A (Bioprocessing Ltd). Proteins were dialysed into CFD or PBS and protein concentration was determined by Coomassie assay.

SDS-PAGE and Western blot analysis Tissue culture supernatants or purified proteins were subjected to SDS-PAGE and separated proteins were stained with Commassie Blue R-250 or with silver [17]. For Western blot analysis, proteins were transferred to nitrocellulose membrane (Schleicher & Schuell, London, UK), the membrane was blocked with 5% (w : v) non-fat milk and incubated with primary antibody overnight. The membrane was incubated with in-house monoclonal antibodies 6D1 or RDIII-7 to detect rat CD59 or DAF, respectively, or with a goat anti-human Fc-HRPO conjugate in order to detect Ig fusion proteins. Membranes probed with 6D1 or RDIII-7 were washed with PBS/0·1% Tween 20 and then incubated with goat anti-mouse Ig HRPO-conjugate in PBS/5% milk for one hour. Membranes were washed three times in PBS/Tween and three times in PBS; bands were visualized using ECL (Perbio Science) and autoradiographic film. Haemolysis assays DAF function. In order to assess function of DAF, antibody coated sheep erythrocytes (E; 2% (v : v)) were prepared by

incubating cells in PBS for 30 min with 1/500 dilution of rabbit anti-sheep E (Amboceptor). Sensitized E (EA) were washed three times in GVB and resuspended to 2%. In order to determine a concentration of rat serum giving partial lysis (50–80%), EA were incubated for 30 min at 37°C with different dilutions of serum. Following pelleting of cells by centrifugation, lysis was quantified by adding an aliquot of supernatant (50 ml) to water (100 ml) and measuring absorbance at 415 nm. Control samples were prepared by adding buffer only (0%) or 0·1% TritonX100 (100%) to EA instead of serum.% lysis was calculated as follows: %lysis = 100 ¥ ( A 415 sample - A 415 0% control) ( A 415 100% control - A 415 0% control). To test function of the recombinant inhibitors, EA were incubated with the predetermined dilution of rat serum giving 50–80% lysis and different dilutions of the test protein. Following incubation at 37°C, percentage lysis was determined as described above. CH50 determination. Plasma CH50 was determined using a modification of the haemolysis assay described above. EA were prepared and incubated with different dilutions of pretreatment plasma or with plasma from rats administered DAF-Ig. Percentage lysis was determined at each serum dilution and amount of serum required to give 50% haemolysis (CH50) was determined according to the method of Kabat and Mayer [18]. CD59 function. Guinea pig E (GPE) were washed and resuspended in GVB at 2% (v : v). These were incubated for 30 min at 37°C with an equal volume of 25% (v : v) normal human serum from which C8 had been depleted by passage over a monoclonal anti-C8 affinity column. The resulting cells (GPEC5b-7) were washed and resuspended at 2% in PBS/10 mM EDTA. The amount of rat serum giving 50–80% lysis was determined by incubating GPE-C5b-7 for 30 min at 37°C with dilutions of rat serum in PBS/EDTA. In order to assess function of soluble CD59, GPE-C5b-7 were incubated in EDTA with dilutions of the test reagent and the predetermined concentration of rat serum. 0% and 100% controls were included and percentage lysis was determined as described above.

In vivo function of DAF-Ig and clearance studies DAF-Ig in PBS was injected intravenously into five 150 g PVG rats at a dose of 10 mg/kg. At various timepoints following administration of the reagent, a sample of blood was removed from the tail vein. Plasma CH50 was analysed by haemolysis assay and concentration of DAF-Ig was determined by ELISA as follows. ELISA plates (Titertek Immuno Assay-Plate, ICN Biomedicals Inc., Basingstoke, UK) were coated with RDII-24 anti-rat DAF (1 mg/ml) in carbonate buffer pH 9·6. The plate was blocked with 5% (w : v) non-fat milk in PBS and then incubated with dilutions of the test plasma. Standards were included on the plate. Captured DAF-Ig was detected using goat anti-human Fc-HRPO (1/1000 dilution) and the assay was developed using OPD substrate (DAKO Ltd (High Wycombe, UK). Concentration of DAFIg in plasma was determined from the standard curve. In separate experiments, half life of DAF-Ig was compared to that of sDAF. In this case, the proteins were labelled with 125I using Iodogen (Sigma) according to the method of Fraker and Speck [19]. Wistar rats (5 in each group, average weight 270 g) were injected through the tail vein with approximately 12 ¥ 106 cpm radiolabelled protein. At specific timepoints, 20 ml blood was removed via the


CRP-Ig as therapeutic reagents tail vein and added to 0·5 ml 0·2 M EDTA. Foetal calf serum (0·5 ml) was added and total cpm determined using a Wallac Multigamma Gamma Counter. Protein bound 125I was determined by adding 1 ml 20% TCA (trichloroacetic acid), precipitated protein was pelleted in a centrifuge and counts remaining in supernatant (free iodine) were determined. Protein bound 125I was calculated (total cpm – free cpm).

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Prophylactic treatment of antigen induced arthritis (AIA) AIA was induced in male Lewis rats as described previously [20]. Briefly, methylated BSA (mBSA) was introduced into the right knee of rats preimmunized with mBSA; 0·45 mg DAF-Ig or the same volume of saline (control animals) was included with the antigen (5 rats per group). Disease progression was monitored by comparing swelling of the right knee to that of the left as described [20].

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RESULTS Generation of CRP-Ig and soluble rat DAF CRP-Ig are antibody-like molecules in which the Fab arms of antibody have been replaced with CRP moieties. Expression vectors were constructed that contained DNA encoding either the extracellular domain of rat CD59 or the four amino terminal SCR of rat DAF upstream of DNA encoding human IgG1 hinge, CH2 and CH3 domains. In the case of CD59-Ig, a second form was generated in which a short polypeptide spacer domain, consisting of two Ser-Gly-Gly-Gly-Gly repeat units, was inserted between CD59 and the antibody hinge. CHO cells were transfected with plasmids containing CD59-Ig, DAF-Ig, or with both plasmids together. Stable cell lines were generated and supernatant harvested. Western blot analysis of culture supernatant demonstrated the presence of CD59-Ig or DAF-Ig in supernatants of cells transfected with one plasmid alone (Fig. 1, lanes 1 and 5). Cotransfection of cells with both plasmids resulted in production of three species of CRP-Ig: DAF-Ig, CD59-Ig and a hybrid molecule consisting of one moiety each of CD59 and DAF attached to Ig (Fig. 1, lanes 2–4). Lane 1 in all panels represents transfection with 2 mg CD59-Ig plasmid and lane 5 represents transfection with 2 mg DAF-Ig plasmid. Lanes 2–4 represent cotransfection with 2 mg total DNA with the ratio of DAF-Ig: CD59-Ig plasmid being 2 : 1, 1 : 1 and 1 : 2, respectively. Supernatant was harvested from transfected cells three days following transfection and analysed by Western blot (Fig. 1). In Fig. 1a, probed with a polyclonal anti-human Fc antibody, CD59-Ig was detected in lane 1, DAF-Ig in lane 5 and all three species of fusion protein in lanes 2–4, the hybrid molecule having an intermediate mobility. Expression of CD59-Ig in supernatant was less efficient than DAF-Ig (comparison of lanes 1 and 5), explaining the apparent decrease in overall yield of CRP-Ig with increased input of CD59-Ig in the transfection. The preferential formation of the hybrid molecule over CD59-Ig can also be explained by the greater efficiency of expression of DAF-Ig in the cotransfected cells (lanes 2–4). In Fig. 1b, probed with a monoclonal anti-CD59 antibody, although CD59-Ig was strongly detected (lane 1), and the amount of CD59Ig lower band increased with increasing amount of CD59-Ig plasmid used in the tranfection (lanes 2–4), the DAF/CD59-Ig hybrid molecule, clearly visible in (a), was detected only weakly (lanes 2–4). In Fig. 1c, probed with monoclonal anti-rat DAF, both the DAF-Ig and the hybrid molecule were detected and the relative band intensity was comparable to that in (a). Weak detection

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62 Fig. 1. Western blot analysis of supernatant from transfected cells. Supernatant from cells transfected with plasmid encoding CD59-spacer-Ig (lane 1), DAF-Ig (lane 5) or both plasmids together (lanes 2–4) was subjected to SDS-PAGE on a 7·5% nonreducing gel and Western blot analysis. Lanes 2–4 represent cotransfection with 2 mg total DNA with the ratio of DAFIg: CD59-Ig plasmid being 2 : 1, 1 : 1 and 1 : 2, respectively. Blots were probed with HRPO-conjugated polyclonal anti-human Fc (a), monoclonal anti-rat CD59 (b) or monoclonal anti-rat DAF (c). Monoclonal antibodies were detected using HRPO conjugated secondary antibody and bands were visualized using ECL.

of the CD59 moiety in the hybrid protein is thus likely due to steric hindrance masking the anti-CD59 Mab epitope. DAF-Ig and CD59-Ig were purified from CHO supernatant by protein A affinity chromatography. A soluble form of rat DAF, comprising the four SCR domains (sDAF), was generated in the same expression vector. Soluble DAF was purified from supernatant on a monoclonal anti-DAF affinity column and gel filtration. Purified proteins were analysed by SDS-PAGE (Fig. 2). The apparent molecular masses were: CD59-Ig, 77 kD; CD59-spacerIg, 78·5 kD; DAF-Ig, 122 kD; sDAF, 32 kD with a dimer band at 64 kD. These molecular masses were confirmed by MALDI-TOF mass spectrometry (Bruker, Coventry, UK; data not shown).

In vitro functional analysis of CRP-Ig The capacities of DAF-Ig and sDAF to inhibit the classical pathway of C were compared using a haemolysis assay. Comparison with sCR1 was also made. Soluble DAF and sCR1 were


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Fig. 2. Purification of CRP-Ig and sDAF. DAF-Ig, CD59-Ig and CD59-spacer-Ig were purified from culture supernatant by protein A affinity chromatography. Purified proteins were subjected to SDS-PAGE on a (a) 7·5% or (b) 10% nonreducing gel and stained with Coomassie Blue. Human IgG1 was run for comparison. (c) Soluble, recombinant rat DAF was purified from culture supernatant by affinity chromatography on a monoclonal anti-rat DAF column followed by gel filtration. The pure protein was subjected to nonreducing (NR) or reducing (R) SDS-PAGE on a 12·5% gel. Proteins were visualized by silver staining.

equally potent at inhibiting C lysis whereas DAF-Ig showed an approximate 24-fold decrease in ability to inhibit lysis when compared to sDAF on a weight-for weight basis (Fig. 3a; IH50 DAF-Ig = 1·32 mg/ml, IH50 sDAF = 0·055 mg/ml). Approximately half the mass of DAF-Ig is contributed by the Fc domain, accounting for at most a two-fold decrease in activity. The residual loss of activity is likely a consequence of tethering the CRP into the fusion protein. In order to confirm that the reduced activity of DAF-Ig was due to the presence of the Fc domains, soluble DAF was released from DAF-Ig using papain. The sDAF produced this way had identical activity to sDAF secreted from CHO cells (Fig. 3b). The ability of CD59-Ig and CD59-spacer-Ig to inhibit C was tested using haemolysis assays specific for the terminal pathway. Soluble CD59 (sCD59) secreted from CHO cells was an effective inhibitor of the terminal pathway, however, when expressed as a fusion protein with human IgG1, its activity, while still detectable, was dramatically reduced (IH50 of 29 mg/ml compared to 0·24 mg/ml for sCD59) (Fig. 4). Even allowing for the fact that the Fc domain comprises 65% by mass of CD59-Ig, this represents an approximately 37fold reduction in activity. The presence of the 11 amino-acid spacer region enhanced regulatory function of CD59-Ig by 2–3-fold (Fig. 4a). CD59 released from the fusion protein had comparable activity to soluble, recombinant CD59 secreted from CHO cells (Fig. 4b).

Clearance of DAF-Ig and sDAF In order to assess the effect of the Fc domain on in vivo clearance of CRP-Ig in rats, DAF-Ig and sDAF were radiolabelled with 125I. Animals were administered a single intravenous (iv) dose of either reagent and samples of blood were removed at intervals. Protein was precipitated using TCA and protein bound counts were measured in a gamma counter. sDAF was cleared extremely rapidly with a half-life of 20 min, DAF-Ig on the other hand had a much prolonged half-life (33 h), illustrating the anticipated effect on half-life of the Fc domains (Fig. 5). Effect of DAF-Ig on plasma haemolytic activity To further assess the effect of the DAF-Ig in vivo, the protein was administered iv to normal rats in a single dose of 10 mg/kg. Although the inhibitory activity of DAF-Ig was lower than sDAF, it nevertheless had sufficient residual activity, even at this low dose, to cause measurable effects on plasma haemolytic activity. Blood was removed at various time-points postinjection, the plasma concentration of the DAF-Ig was measured by ELISA and plasma CH50 was assessed (Fig. 6). At 1 h following administration of the reagent, average plasma concentration was 140 mg/ml. Plasma concentration then fell slowly, reaching 28% of the 1 h level at the 48 h timepoint (Fig. 6a). Plasma CH50 was reduced to approximately 60% of that prior to treatment at 1 h and this was maintained to 24 h (Fig. 6b). At the 48 h


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Effect of DAF-Ig on AIA The therapeutic effect of DAF-Ig was tested in a rat arthritis model, antigen induced arthritis (AIA). Immune animals were injected intra-articularly with methylated-BSA and either saline or DAF-Ig. Disease progression was monitored over the course of a week by measuring joint swelling. Rat DAF-Ig caused a significant reduction in swelling (Fig. 7) and disease severity (clinical score; not shown) compared to control animals from day 2 through to day 7.

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Fig. 3. In vitro complement regulatory function of DAF-Ig. (a) Sensitized erythrocytes were incubated in GVB with rat serum and different concentrations of sCR1 (), rat DAF-Ig (), sDAF () or a nonregulatory Ig fusion protein (). Haemolysis was assessed by release of haemoglobin to the supernatant and percent lysis was determined. Results represent the mean value ± SD of three determinations. (b) Inhibition of lysis by sDAF () is compared to that achieved with soluble DAF released from DAF-Ig using papain () and a nonregulatory Ig fusion protein ().

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DISCUSSION The use of recombinant, soluble CRP as anti-complement therapeutic reagents was pioneered over 10 years ago when a soluble recombinant form of CR1 was generated and shown to reduce the extent of myocardial infarction in a rat model of ischaemiareperfusion injury [9]. Since then sCR1 has been used successfully in many animal models of disease, particularly ischaemiareperfusion injuries of various organs, but also in experimental demyelination, experimental myasthenia gravis, glomerulonephritis and others [21–23]. sCR1 has also reached the clinic and is in trials for treatment of the adult respiratory distress syndrome and cardiopulmonary bypass [24,25]. Soluble, recombinant


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forms of the other naturally occurring membrane CRP have now been generated and tested. For example, soluble recombinant forms of human DAF and MCP have been effective in reducing immune complex-mediated inflammation in the reverse passive Arthus reaction [26–28]. Strategies to increase the efficacy of these reagents have included the production of reagents containing two different CRP moieties. CAB-2 (C activation blocker-2) contains the four SCRs of human MCP joined in immediate proximity to the SCRs and ST region of human DAF [11,29]. CAB2 has both decay and cofactor activities, has greater inhibitory activity than soluble DAF or MCP alone and is effective in the reverse passive Arthus reaction and models of cardiac xenotransplantation. Soluble forms of CD59 have been generated and tested in vitro [30] but to the best of our knowledge have not been used to inhibit C in vivo. The ability to inhibit only the terminal C pathway in vivo is an attractive proposition, as physical damage and proinflammatory effects caused by MAC would be averted whilst physiological roles played by the C system, such as protection from infection and immune complex solubilization, would be sustained. The reagents described above have several disadvantages as therapeutics. All must be given systemically and, with the exception of sCR1 (200 kD), soluble recombinant CRP are small, ranging in size from 12 kD (soluble CD59 [30]) to 40–50 kD (DAF, MCP; Crry [26,27,31]). These reagents, soluble CD59 in particular, are cleared rapidly through the kidney in vivo. This rapid clearance is a major drawback which makes them unsuitable for long-term therapy or treatment of chronic inflammatory disorders and has restricted therapy, even with sCR1, to acute inflammatory conditions. We set out to generate soluble forms of CRP with enhanced therapeutic efficiency by combining the regulatory domains of CRP with Fc domains of antibody. By fusing the active moiety to antibody Fc, circulating half-life in vivo should be increased dramatically, a concept illustrated by an abundant literature [32–34]. Several biological reagents based on Ig-fusion proteins have already had success in the clinic; for

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example, Etanercept (Enbrel, Immunex, Seattle), a genetically engineered fusion protein consisting of the recombinant human TNF-receptor p75 fused to the Fc domain of human IgG1, has been enormously successful in the treatment of rheumatoid arthritis [35]. With one exception, this strategy has not previously been exploited for CRP-based therapeutics. Recently, a soluble form of murine Crry fused to murine IgG1 hinge, CH2 and CH3 domains (Crry-Ig) was generated and shown to have a long half life in vivo (40 h) and to be efficacious in murine antibodymediated glomerulonephritis and ischaemia/reperfusion-induced intestinal mucosal injury [36,37]. No comparisons with Crry minus the Fc domain were made in these studies. Apart from the possibility of extending half-life in vivo, generation of CRP-Ig reagents enables the creation of hybrid bifunctional molecules containing multiple CRP with complementary activities. Previous attempts by others to combine two activities into one reagent


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2 3 4 5 6 Time post disease induction (days)

7

Fig. 7. Therapeutic effect of DAF-Ig on AIA. Methylated BSA was introduced into the right knee of immune rats. DAF-Ig () or saline (, control) was administered to the joint at the same time. Swelling of the joint was measured daily and compared to that of the left knee. Results represent the mean of five animals ± SEM (* P < 0·01; ** P < 0·001).

have involved the generation of linear, inflexible molecules where the CRP are fused end to end [11]. Linkage of the CRP through the antibody hinge is likely to allow the CRP moieties to move and work independently as is seen with the Fab arms in native antibody. Further, hybrid molecules containing a CRP on one ‘arm’ and a targeting moiety on the other could be created, to focus the C regulatory activity at a specific site. Strategies that target sCR1 to cell membranes, such as the addition of the sLex carbohydrate moiety, a ligand for E- and P-selectins on activated endothelia, have been used successfully to increase its therapeutic efficiency [38]. APT070, a truncated form of CR1 with a carboxy-terminal membrane-targeting tail, termed an ‘addressin’ is far more effective at C inhibition that its untailed counterpart [39,40]. Targeting strategies have also been used with other CRP. Localization of soluble, recombinant CD59 and DAF to membranes by fusing these proteins to the carboxy terminus of a targeting antibody increased their C-inhibitory effect [41,42]. Finally, a chimeric molecule combining rat CD59 with Crry had greater ability to regulate MAC formation than soluble CD59 alone, possibly due to localization of CD59 to the target membrane through binding of Crry to the opsonin, C3b [43]. We here describe the generation and functional characterization of Ig fusion proteins which possess regulatory activities of CD59 or DAF. In addition, we demonstrate the feasibility of combining these activities into one molecule (Fig. 1). Functional analysis of CRP with or without the Fc domains showed that attachment to the Fc markedly reduced the C inhibitory capacity of the CRP: 12-fold for DAF and almost 40-fold for CD59 (Figs 3 and 4). This finding was unexpected and raised the possibility that steric hindrance in the CRP–Ig inhibited interaction of the CRP with its target. In the CRP-Ig the active site of the regulatory domains cannot efficiently access and bind the large multimolecular substrate, the C3/C5 convertase and the MAC for DAF and CD59, respectively. This hypothesis was supported by the observation that inclusion of a short spacer region between CD59 and the Fc partially restored C regulatory activity and confirmed by showing

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that enzymatic removal of the Fc domains restored native function to both CD59 and DAF. Despite the reduced activity compared to sDAF, DAF-Ig nevertheless retained considerable anti-C activity in vitro and was therefore chosen for testing in vivo to assess the effects of the Fc on plasma half life and ability to inhibit plasma C activity. First, radiolabelled sDAF and DAF-Ig were administered intravenously and clearance rates were compared. Within 1 h of administration, sDAF levels were less than 20% of the peak at 3 min whereas DAF-Ig levels were more than 80% of those at peak. Even at 48 h, DAF-Ig was still detectable in the plasma, demonstrating the enhancement of half life as a consequence of fusion to Fc domains (Fig. 5). In a separate experiment, DAF-Ig was given iv to rats as a single dose of 10 mg/kg. Plasma levels of DAF-Ig were near 50% of peak at 24 h and plasma haemolytic activity was reduced by 40% up to 24 h postdose (Fig. 6). We have previously shown that a dose of sCR1 of 20 mg/kg was necessary to reduce plasma haemolytic activity to approximately 35% at 1 h following administration of the reagent [20]. Given the reduced activity of DAF-Ig compared to sCR1 and the smaller dose of reagent used, it is unsurprising that plasma C activity was not reduced further in our experiments. Based on experiments with sCR1 in rats, a decrease in plasma C activity of 40% is likely to be sufficient for a therapeutic effect in chronic inflammatory conditions [20]. To test for therapeutic efficacy we chose a model in which the agent could be administered locally and in which a therapeutic effect of sCR1 had already been demonstrated. A single dose of DAF-Ig given intra-articularly had a marked inhibitory effect on disease progression in rat antigen arthritis, lasting for the full 7 day course of the experiment (Fig. 7). The data presented here demonstrate that CRP-Ig fusion proteins have considerable potential as therapeutic reagents in the clinic. The reduced C regulatory activity is compensated by the much increased half life in vivo and the agents are effective in inflammatory disease models. Nevertheless, it should be possible to increase the anti-C activity in the CRP-Ig by careful molecular engineering to enable the active sites in the CRP to bind their target ligands efficiently. Attainment of optimum efficiency may simply require the incorporation of protein spacer domains between the CRP and antibody hinge, shown here to be partly effective for CD59-Ig. Judicious choice of the antibody isotype used as Fc donor may also be relevant. Increased flexibility at the hinge region may reduce steric hindrance and IgG3, which has the most flexible upper hinge of all human IgG, could be substituted for human IgG1 used in these studies [44]. Other modifications to reduce effector functions in the Fc might also be useful and the targeting strategies discussed above could be incorporated into the design of the CRP-Ig fusion proteins. Etanercept, the TNFreceptor p75-Fc fusion protein used in treatment of rheumatoid arthritis, is given twice weekly by subcutaneous injection [35]. Similar treatment protocols might be envisaged for CRP-Ig reagents, obviating the need for continual infusion or multiple iv injections. These new reagents therefore offer the real prospect of C regulatory therapeutics that can be used for long-term treatment of chronic diseases.

ACKNOWLEDGEMENTS This work was supported by The Wellcome Trust (Senior Research Fellowship to BPM, (Grant 016668/Z) and The Arthritis Research Campaign (SML and ASW). We thank Nick Amos and Yvonne McGrath for invaluable advice and discussion.


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