Generation of Anti-complement “Prodrugs”
2003; Elsevier BV; Volume: 278; Issue: 38 Linguagem: Inglês
10.1074/jbc.m306351200
ISSN1083-351X
AutoresClaire L. Harris, Clare Hughes, Anwen S. Williams, Ian Goodfellow, David J. Evans, Bruce Caterson, B. Paul Morgan,
Tópico(s)Biochemical and Structural Characterization
ResumoExpression of biologically active molecules as fusion proteins with antibody Fc can substantially extend the plasma half-life of the active agent but may also influence function. We have previously generated a number of fusion proteins comprising a complement regulator coupled to Fc and shown that the hybrid molecule has a long plasma half-life and retains biological activity. However, several of the fusion proteins generated had substantially reduced biological activity when compared with the native regulator or regulator released from the Fc following papain cleavage. We have taken advantage of this finding to engineer a prodrug with low complement regulatory activity that is cleaved at sites of inflammation to release active regulator. Two model prodrugs, comprising, respectively, the four short consensus repeats of human decay accelerating factor (CD55) linked to IgG4 Fc and the three NH2-terminal short consensus repeats of human decay accelerating factor linked to IgG2 Fc have been developed. In each, specific cleavage sites for matrix metalloproteinases and/or aggrecanases have been incorporated between the complement regulator and the Fc. These prodrugs have markedly decreased complement inhibitory activity when compared with the parent regulator in vitro. Exposure of the prodrugs to the relevant enzymes, either purified, or in supernatants of cytokine-stimulated chondrocytes or in synovial fluid, efficiently cleaved the prodrug, releasing active regulator. Such agents, having negligible systemic effects but active at sites of inflammation, represent a paradigm for the next generation of anti-C therapeutics. Expression of biologically active molecules as fusion proteins with antibody Fc can substantially extend the plasma half-life of the active agent but may also influence function. We have previously generated a number of fusion proteins comprising a complement regulator coupled to Fc and shown that the hybrid molecule has a long plasma half-life and retains biological activity. However, several of the fusion proteins generated had substantially reduced biological activity when compared with the native regulator or regulator released from the Fc following papain cleavage. We have taken advantage of this finding to engineer a prodrug with low complement regulatory activity that is cleaved at sites of inflammation to release active regulator. Two model prodrugs, comprising, respectively, the four short consensus repeats of human decay accelerating factor (CD55) linked to IgG4 Fc and the three NH2-terminal short consensus repeats of human decay accelerating factor linked to IgG2 Fc have been developed. In each, specific cleavage sites for matrix metalloproteinases and/or aggrecanases have been incorporated between the complement regulator and the Fc. These prodrugs have markedly decreased complement inhibitory activity when compared with the parent regulator in vitro. Exposure of the prodrugs to the relevant enzymes, either purified, or in supernatants of cytokine-stimulated chondrocytes or in synovial fluid, efficiently cleaved the prodrug, releasing active regulator. Such agents, having negligible systemic effects but active at sites of inflammation, represent a paradigm for the next generation of anti-C therapeutics. Complement (C) 1The abbreviations used are: C, complement; DAF, decay accelerating factor; CReg, complement regulatory protein; SCR, short consensus repeat; MMP, matrix metalloproteinases; IGD, interglobular domain of aggrecan; ADAMTS, a disintegrin-like and metalloproteinase with thrombospondin motifs; SF, synovial fluid; AIA, antigen induced arthritis; RA, rheumatoid arthritis; MAC, membrane attack complex; CR1, complement receptor 1; IL, interleukin; TNF-α, tumor necrosis factor-α; PBS, phosphate-buffered saline; sDAF, soluble decay accelerating factor; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; HRP, horseradish peroxidase. is a core component of the immune system, performing vital roles in immune surveillance. Its activation results in production of proinflammatory mediators (such as C3a and C5a) and formation of the cytolytic membrane attack complex (MAC). Host cells are protected from damage by proteins present on cell membranes, complement regulatory proteins (CReg). These proteins include complement receptor 1 (CR1; CD35), membrane cofactor protein (CD46), and decay accelerating factor (DAF; CD55), which act early in the C cascade to inactivate the amplification enzymes, and CD59, which acts late in the cascade and prevents MAC formation (1Morgan B.P. Meri S. Springer Semin. Immunopathol. 1994; 15: 369-396Crossref PubMed Scopus (236) Google Scholar, 2Morgan B.P. Harris C.L. Complement Regulatory Proteins. Academic Press, London1999Google Scholar). The genes encoding the regulators of the activation cascades are linked on chromosome 1 and the proteins are structurally related, comprising a number of protein domains known as short consensus repeats (SCRs). These domains consist of ∼60 amino acids, many of which are highly conserved, and are linked end-to-end in the CReg to give flexible, elongated structures (3Reid K.B. Day A.J. Immunol. Today. 1989; 10: 177-180Abstract Full Text PDF PubMed Scopus (268) Google Scholar, 4Hourcade D. Holers V.M. Atkinson J.P. Adv. Immunol. 1989; 45: 381-416Crossref PubMed Scopus (380) Google Scholar). DAF and membrane cofactor protein comprise four SCR domains, whereas the most common isoform of CR1 has 30. Under normal circumstances the CReg are sufficient to protect cells from damage by homologous C. However, the active products that enable complement to perform its physiological roles can inappropriately target self-tissues; C-mediated inflammation and tissue destruction is an important drive to pathology in diseases such as rheumatoid arthritis (RA), systemic lupus erythematosus, glomerulonephritis, multiple sclerosis, ischemia/reperfusion injury, and transplantation, where it acts to sustain the "vicious cycle" of inflammation and tissue damage. In RA, soluble products of C activation are present in the synovial fluid of affected joints and complement deposits are evident on synovial tissue (5Morgan B.P. Daniels R.H. Williams B.D. Clin. Exp. Immunol. 1988; 73: 473-478PubMed Google Scholar, 6Brodeur J.P. Ruddy S. Schwartz L.B. Moxley G. Arthritis Rheum. 1991; 34: 1531-1537Crossref PubMed Scopus (109) Google Scholar, 7Oleesky D.A. Daniels R.H. Williams B.D. Amos N. Morgan B.P. Clin. Exp. Immunol. 1991; 84: 250-255PubMed Google Scholar). In addition, affected joints are full of leukocytes (neutrophils and T cells) attracted to the site by a gradient of C5a and other chemoattractants. Although C itself is not always the primary cause of disease in these inflammatory diseases, it acts to sustain the proinflammatory cycle and perpetuate tissue damage. The involvement of C in the perpetuation and exacerbation of these disorders has driven the search for therapeutic reagents capable of inhibiting dysregulated C activation. Drugs mimicking the action of CReg have been effective at controlling pathological activation of complement in vivo in many animal models of disease (8Goodfellow R.M. Williams A.S. Levin J.L. Williams B.D. Morgan B.P. Clin. Exp. Immunol. 1997; 110: 45-52Crossref PubMed Scopus (44) Google Scholar, 9Goodfellow R.M. Williams A.S. Levin J.L. Williams B.D. Morgan B.P. Clin. Exp. Immunol. 2000; 119: 210-216Crossref PubMed Scopus (58) Google Scholar). These reagents are frequently recombinant forms of membrane CReg that have been engineered without their membrane-anchoring domains as soluble proteins (10Harris C.L. Fraser D.A. Morgan B.P. Biochem. Soc. Trans. 2002; 30: 1019-1026Crossref PubMed Scopus (21) Google Scholar). Some of these reagents, such as soluble CR1 (sCR1), have progressed to trials in humans and have been beneficial in the treatment of acute inflammation, such as in adult respiratory distress syndrome (11Rioux P. Curr. Opin. Investig. Drugs. 2001; 2: 364-371PubMed Google Scholar). However, there are drawbacks associated with anti-C therapy, including rapid clearance of reagents in vivo, which can be within minutes of administration, and the side effects of long term systemic inhibition of the C system. Individuals genetically deficient in C components are prone to recurrent bacterial infections and immune complex disease, provoking caution in the use of anti-C agents. Such agents might be suitable for short term therapy, for example, in myocardial infarction or adult respiratory distress syndrome, but cannot be used for chronic treatment. To circumvent the short half-life of CReg-based therapeutic reagents, we and others have previously fused CReg to antibody Fc domains, generating "antibody-like" molecules that have a much enhanced half-life in vivo and are effective in animal models of C-mediated disease (12Quigg R.J. Kozono Y. Berthiaume D. Lim A. Salant D.J. Weinfeld A. Griffin P. Kremmer E. Holers V.M. J. Immunol. 1998; 160: 4553-4560PubMed Google Scholar, 13Harris C.L. Williams A.S. Linton S.M. Morgan B.P. Clin. Exp. Immunol. 2002; 129: 198-207Crossref PubMed Scopus (47) Google Scholar). We demonstrated that fusion of DAF to antibody domains increased the half-life in rats from 20 min to 33 h. However, functional analysis of two reagents, rat DAF-Ig and rat CD59-Ig, indicated that the CReg function was substantially impaired in the fusion protein. Comparison of activities (moles of CReg) indicated that DAF-Ig had a 10-fold reduction in activity, whereas CD59-Ig had a 35-fold reduction in activity (13Harris C.L. Williams A.S. Linton S.M. Morgan B.P. Clin. Exp. Immunol. 2002; 129: 198-207Crossref PubMed Scopus (47) Google Scholar). Insertion of "spacing" domains between the CReg and the antibody hinge partially restored function, which was totally restored if the Fc domains were removed with papain. These data implicated steric constraints in the reduction in function. We have taken advantage of this phenomenon to generate anti-C prodrugs by deliberately fusing functional domains of human CReg to inflexible antibody hinge regions. Insertion of a short enzyme site between the CReg, illustrated here with DAF, and the Ig hinge generates a reagent that can be targeted by specific enzymes to release active DAF from the fusion protein in the relevant tissue. Our model prodrugs contain short regions from aggrecan, the major proteoglycan in cartilage that is degraded by aggrecanases and MMPs in arthritis (14Little C.B. Hughes C.E. Curtis C.L. Janusz M.J. Bohne R. Wang-Weigand S. Taiwo Y.O. Mitchell P.G. Otterness I.G. Flannery C.R. Caterson B. Matrix Biol. 2002; 21: 271-288Crossref PubMed Scopus (113) Google Scholar). Aggrecan comprises two globular domains at the amino terminus (G1, G2) separated by a polypeptide stretch of ∼150 amino acids, termed the interglobular domain (IGD), followed by a long glycosaminoglycan attachment region that separates G2 from a third globular domain at the COOH terminus, G3. Extensive proteolytic degradation of aggrecan is associated with cartilage destruction in arthritic diseases; two major sites of catabolism have been identified within the IGD of aggrecan. The major site of cleavage appears to be hydrolysis of the peptide bond Glu373-Ala374 (human sequence enumeration (15Doege K.J. Sasaki M. Kimura T. Yamada Y. J. Biol. Chem. 1991; 266: 894-902Abstract Full Text PDF PubMed Google Scholar)) generated by the activity of the aggrecanases (members of a disintegrin-like and metalloproteinase with thrombospondin motifs (ADAMTS) family, namely ADAMTS-4, -5, and -1) (16Abbaszade I. Liu R.Q. Yang F. Rosenfeld S.A. Ross O.H. Link J.R. Ellis D.M. Tortorella M.D. Pratta M.A. Hollis J.M. Wynn R. Duke J.L. George H.J. Hillman Jr., M.C. Murphy K. Wiswall B.H. Copeland R.A. Decicco C.P. Bruckner R. Nagase H. Itoh Y. Newton R.C. Magolda R.L. Trzaskos J.M. Hollis G.F. Arner E.C. Burn T.C. J. Biol. Chem. 1999; 274: 23443-23450Abstract Full Text Full Text PDF PubMed Scopus (447) Google Scholar, 17Tortorella M.D. Burn T.C. Pratta M.A. Abbaszade I. Hollis J.M. Liu R. Rosenfeld S.A. Copeland R.A. Decicco C.P. Wynn R. Rockwell A. Yang F. Duke J.L. Solomon K. George H. Bruckner R. Nagase H. Itoh Y. Ellis D.M. Ross H. Wiswall B.H. Murphy K. Hillman Jr., M.C. Hollis G.F. Arner E.C. Science. 1999; 284: 1664-1666Crossref PubMed Scopus (625) Google Scholar), whereas a secondary cleavage is generated by hydrolysis of the Asn341-Phe342 bond by a number of MMPs and recently reported by ADAMTS-4. Various other minor sites of cleavage within the IGD have also been characterized (18Buttner F.H. Hughes C.E. Margerie D. Lichte A. Tschesche H. Caterson B. Bartnik E. Biochem. J. 1998; 333: 159-165Crossref PubMed Scopus (58) Google Scholar, 19Tortorella M.D. Pratta M. Liu R.Q. Austin J. Ross O.H. Abbaszade I. Burn T. Arner E. J. Biol. Chem. 2000; 275: 18566-18573Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar). Cleavage of aggrecan is enhanced in inflammatory joint disease resulting in cartilage damage, indeed soluble products of aggrecan catabolism generated by aggrecanases and MMPs can be found in synovial fluid and cartilage extracts of patients with arthritis (14Little C.B. Hughes C.E. Curtis C.L. Janusz M.J. Bohne R. Wang-Weigand S. Taiwo Y.O. Mitchell P.G. Otterness I.G. Flannery C.R. Caterson B. Matrix Biol. 2002; 21: 271-288Crossref PubMed Scopus (113) Google Scholar, 20Lohmander L.S. Neame P.J. Sandy J.D. Arthritis Rheum. 1993; 36: 1214-1222Crossref PubMed Scopus (382) Google Scholar, 21Ishiguro N. Ito T. Oguchi T. Kojima T. Iwata H. Ionescu M. Poole A.R. Arthritis Rheum. 2001; 44: 2503-2511Crossref PubMed Scopus (74) Google Scholar, 22Matsuno H. Yudoh K. Watanabe Y. Nakazawa F. Aono H. Kimura T. J. Rheumatol. 2001; 28: 22-28PubMed Google Scholar). In vitro culture systems have also demonstrated that a variety of inflammatory cytokines, such as IL-1α and TNF-α stimulate cleavage at these same sites (23Fosang A.J. Tyler J.A. Hardingham T.E. Matrix. 1991; 11: 17-24Crossref PubMed Scopus (91) Google Scholar, 24Hughes C.E. Caterson B. Fosang A.J. Roughley P.J. Mort J.S. Biochem. J. 1995; 305: 799-804Crossref PubMed Scopus (197) Google Scholar, 25Little C.B. Flannery C.R. Hughes C.E. Mort J.S. Roughley P.J. Dent C. Caterson B. Biochem. J. 1999; 344: 61-68Crossref PubMed Scopus (171) Google Scholar, 26Caterson B. Flannery C.R. Hughes C.E. Little C.B. Matrix Biol. 2000; 19: 333-344Crossref PubMed Scopus (248) Google Scholar). The enzyme cleavage sites, and the minimal sequence lengths for recognition by target enzymes, have been previously documented (27Niedzwiecki L. Teahan J. Harrison R.K. Stein R.L. Biochemistry. 1992; 31: 12618-12623Crossref PubMed Scopus (74) Google Scholar, 28Nagase H. Fields G.B. Biopolymers. 1996; 40: 399-416Crossref PubMed Google Scholar, 29Horber C. Buttner F.H. Kern C. Schmiedeknecht G. Bartnik E. Matrix Biol. 2000; 19: 533-543Crossref PubMed Scopus (19) Google Scholar, 30Mercuri F.A. Maciewicz R.A. Tart J. Last K. Fosang A.J. J. Biol. Chem. 2000; 275: 33038-33045Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). The prodrugs described here combine three important characteristics. First, the reagent has enhanced circulating half-life because of the antibody Fc domains; second, it is designed to have little or no systemic activity; and third, it is a targeted reagent, having potential to unleash powerful anti-C activity at sites that express an abundance of the target enzyme. Chemicals and reagents were from Fisher (Loughborough, United Kingdom) or Sigma unless otherwise stated. All tissue culture reagents and plastics were from Invitrogen. pDR2ΔEF1α was a gift from Dr. I. Anegon (INSERM U437, Nantes, France) and has been described previously (31Charreau B. Cassard A. Tesson L. Le Mauff B. Navenot J.M. Blanchard D. Lublin D. Soulillou J.P. Anegon I. Transplantation. 1994; 58: 1222-1229PubMed Google Scholar). Restriction enzymes were from Amersham Biosciences, T4 DNA ligase from Promega (Southampton, UK), dNTPs from Bioline (London, UK), and Vent DNA polymerase from New England Biolabs Ltd. (Hitchin, UK). Primers and other molecular biology reagents were from Invitrogen. Chinese hamster ovary cells were obtained from the European Collection of Animal Cell Cultures (ECACC, Salisbury, UK). All tissue culture reagents and plastics were from Invitrogen (Paisley, UK). Chinese hamster ovary cells were cultured in Ham's F-12, 50 units of penicillin/streptomycin, 1 μg/ml amphotericin B, 2 mm glutamine, 1 mm sodium pyruvate, 4% fetal calf serum. Goat anti-human Fc-HRP was from Sigma, goat anti-mouse Ig-HRP was from Bio-Rad. Neoepitope antibodies BC-3 recognizing the new NH2 terminus ARGSV... generated by aggrecanases, BC-4 recognizing the new COOH terminus... DIPEN, and BC-14 recognizing the new NH2 terminus FFGVG... generated by MMPs and aggrecanases, and anti-human CD55 (HD1A) were produced in-house (24Hughes C.E. Caterson B. Fosang A.J. Roughley P.J. Mort J.S. Biochem. J. 1995; 305: 799-804Crossref PubMed Scopus (197) Google Scholar, 26Caterson B. Flannery C.R. Hughes C.E. Little C.B. Matrix Biol. 2000; 19: 333-344Crossref PubMed Scopus (248) Google Scholar, 33Caterson B. Hughes C.E. Roughley P. Mort J.S. Acta Orthop. Scand. Suppl. 1995; 266: 121-124Crossref PubMed Scopus (43) Google Scholar, 34Harris C.L. Lublin D.M. Morgan B.P. J. Immunol. Methods. 2002; 268: 245-258Crossref PubMed Scopus (25) Google Scholar). Human serum was obtained by venepuncture from healthy volunteers. MMP3 (recombinant, catalytic domain) and MMP8 (purified from granulocytes) were purchased from Calbiochem (Nottingham, UK). Recombinant, human IL-1α and TNF-α were from Totam Biologicals, Cambridge, UK; retinoic acid was from Sigma. Recombinant ADAMTS-4 was a gift from Wyeth Genetic Institute, Boston, MA. 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 (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 C fixation diluent, 0.1% (w/v) gelatin. FACS buffer is PBS, 0.1% (w/v) sodium azide, 1% bovine serum albumin, pH 7.4. MMP digest buffer is 50 mm Tris, pH 7.5, 100 mm NaCl, 10 mm CaCl2·2H2O. Construction of Ig Fusion Protein Expression Vectors—Total RNA was prepared from human peripheral blood mononuclear cells using Ultraspec total RNA isolation reagent (Biotecx Laboratories Inc., Houston, TX) and was reverse transcribed according to standard protocols using Superscript reverse transcriptase (Invitrogen) and oligo(dT) (CCAGTGAGCAGAGTGACGAGGACTGGAGCTCAAGCT17). DNA encoding the hinge, CH2, and CH3 regions of human IgG2 or IgG4 was amplified by PCR with primers (see below) that incorporated restriction sites (indicated in bold) enabling direct ligation into the expression vector pDR2ΔEF1α. DNA encoding either three or four SCR of human DAF was amplified using plasmid template and primers that incorporated restriction sites (indicated in bold) enabling direct ligation into pDR2ΔEF1α immediately upstream of and in-frame with DNA encoding the Ig domains. Sequencing confirmed that no errors had been introduced by PCR. Primers for IgG and DAF were as follows: IgG2 sense, 5′-CACGGATCCGAGCGCAAATGTTGTGTCG; IgG4 sense, 5′-GTTGGATCCAAATATGGTCCCCCATG for DAF-IgG4 and AACGGATCCGTGGACAAGAGAGTT for DAF4-IGD75-IgG4 prodrug; IgG2 and IgG4 antisense, 5′-CGAGATATCGGGGAGCGGGGGCTTGC; DAF sense, GCTTCTAGACTAACCCGGCGCGCCATGACC; DAF4-IgG4 antisense, TAGGGATCCTCCTCTGCATTCAGGTGGTGG; DAF4-IGD75-IgG4 prodrug antisense, TGGGGATCCCTTGGAAGTTAGAGATTTTC; three SCR forms of DAF antisense TGCGGATCCATAAATTTCTCTGCACTCTGG. Incorporation of Enzyme Sites—To incorporate the short enzyme site derived from the aggrecan IGD (Fig. 2) into the DAF (3 SCRs)-IgG2 construct, complementary DNA oligomers were used that encoded the polypeptide flanked on both sides by DNA forming the restriction site for BamHI (sense primer, 5′-GCAGGATCCGGAGAAGACTTTGTAGATATTCCAGAAAATTTCTTCGGCGTCGGTGGAGAGGAGGACGGATCCACG, encoding amino acids GEDFVDIPENFFGVGGEED). Oligomers were annealed, digested with BamHI, and ligated into the vector encoding DAF3-IgG2 at the BamHI site (located at 3′ end of DAF, 5′ end of IgG2). To generate the larger construct, DAF4-IGD75-IgG4, DNA encoding 75 amino acids of aggrecan IGD sequence (sequence, GYTGEDFVDIPENF342FGVGGEEDITVQTVTWPDMELPLPRNITEG EA374RGSVILTVKPIFEVSPSPLEPEEPFTFAP) was amplified by PCR from plasmid template, primers encoded the BamHI restriction site at either end of the construct enabling ligation into the expression vector between DAF and the Ig hinge. The major aggrecanase and MMP cleavage sites are underlined. In all cases sequencing confirmed orientation and fidelity of DNA sequence. Cell Transfection and Purification of Fusion Proteins—Chinese hamster ovary cells were transfected with expression vectors using LipofectAMINE (Invitrogen) according to the manufacturer's instructions and stable lines were established by selection with 400 μg/ml hygromycin B (Invitrogen). Tissue culture 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 most 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 dialyzed into PBS. In some cases reagents were applied to a Superose 12 size exclusion column to exchange buffer to MMP digest buffer and to remove minor contaminants such as bovine Ig. Protein concentration was determined using BCA protein assay reagent (Pierce & Warriner, Chester, UK) according to manufacturer's instructions and using bovine serum albumin as a standard. Purity was assessed by SDS-PAGE and either Coomassie or silver stain (35Morrissey J.H. Anal. Biochem. 1980; 117: 307-310Crossref Scopus (2942) Google Scholar). Soluble DAF without Fc—Soluble forms of DAF comprising either the amino-terminal three or all four SCR domains from human DAF have been described previously (36Powell R.M. Ward T. Evans D.J. Almond J.W. J. Virol. 1997; 71: 9306-9312Crossref PubMed Google Scholar, 37Daniels G.L. Green C.A. Powell R.M. Ward T. Transfusion. 1998; 38: 332-336Crossref PubMed Scopus (25) Google Scholar, 38Lea S.M. Powell R.M. McKee T. Evans D.J. Brown D. Stuart D.I. van der Merwe P.A. J. Biol. Chem. 1998; 273: 30443-30447Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). In brief, DAF was expressed as a soluble protein in Pichia pastoris with a carboxyl-terminal oligohistidine tag to enable purification on nickel-nitrilotriacetic acid columns. The soluble forms of DAF produced had NMR spectra typical of SCR-containing proteins and bound a large range of monoclonal CD55 antibodies (37Daniels G.L. Green C.A. Powell R.M. Ward T. Transfusion. 1998; 38: 332-336Crossref PubMed Scopus (25) Google Scholar). Sheep E (TCS Microbiology, Claydon, UK) were sensitized by incubating 1 volume of 4% E (v/v) with 1 volume of 1/250 rabbit anti-sheep E (Amboceptor; Behring Diagnostics) for 20 min at 37 °C. Sensitized cells were washed twice in GVB and resuspended to 2%. A 50-μl aliquot was incubated with 50 μl of normal human serum and 50 μl of a dilution of DAF-Ig or control protein. All dilutions were made in GVB and serum was previously titered to yield between 50 and 70% lysis in the absence of inhibitor. Cells were incubated at 37 °C for 30 min, pelletted, and 75 μl of supernatant was removed for measurement of absorbance at 415 nm (hemoglobin released). Control incubations included cells incubated in buffer only (0%) or in 0.03% Nonidet P-40 (100%). Percent lysis was calculated as follows: percent lysis = 100 × (A 415 test sample - A 415 0% control)/(A415 100% control - A 415 0% control). A non-regulatory fusion protein was used as a negative control protein and inhibitory activity of the test protein was calculated: percent inhibition = 100 × (% lysis with negative control protein - % lysis test sample)/% lysis with negative control protein (13Harris C.L. Williams A.S. Linton S.M. Morgan B.P. Clin. Exp. Immunol. 2002; 129: 198-207Crossref PubMed Scopus (47) Google Scholar). The IH50 represents the concentration of test protein at which 50% inhibition was obtained. Where reagents had been incubated in MMP digest buffer and activity subsequently assessed by hemolysis assay, an equivalent volume of this buffer was added to all other incubations. Proteins were separated by SDS-PAGE using the Hoeffer Mighty Small Gel system (Hoeffer Scientific Instruments, Newcastle-under-Lyme, UK). Gels were electroblotted onto nitrocellulose that was then blocked for 30 min at room temperature in PBS, 5% nonfat dried milk and incubated overnight with primary antibody (BC-4, BC-3, and BC-14, all at 1/100 dilution tissue culture supernatant; HD1A anti-DAF at 10 μg/ml) or polyclonal goat anti-human Fc-HRP (1/1000). Blots incubated with mouse monoclonal antibodies were then washed three times in PBS, 0.1% Tween 20 for 5 min each, followed by incubation with HRP-conjugated goat anti-mouse Ig at a dilution of 1:1000 in PBS, 5% milk for 1 h. Blots were then washed three times in PBS, 0.1% Tween 20 for 5 min each, followed by PBS, and were developed using a chemiluminescent substrate (SuperSignal, Pierce & Warriner) and Kodak x-ray film. Where samples were analyzed following culture with chondrocytes, an alkaline phosphatase-conjugated secondary antibody was used to detect primary antibody and blots were developed as described previously (39Hughes C.E. Buttner F.H. Eidenmuller B. Caterson B. Bartnik E. J. Biol. Chem. 1997; 272: 20269-20274Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 40Hughes C.E. Little C.B. Buttner F.H. Bartnik E. Caterson B. J. Biol. Chem. 1998; 273: 30576-30582Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). Reagents that were subjected to enzymatic cleavage by MMPs or aggrecanase were first buffer-exchanged into digest buffer on a Superose 12 column. Purified enzymes (MMP3, MMP8, or ADAMTS-4) were added to the final concentrations stated in the text and reagents were incubated at 37 °C for the specified time. To analyze cleavage by native enzyme released from cytokine-stimulated chondrocytes, porcine chondrocytes were isolated from metacarpophangeal joints as described previously (39Hughes C.E. Buttner F.H. Eidenmuller B. Caterson B. Bartnik E. J. Biol. Chem. 1997; 272: 20269-20274Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 40Hughes C.E. Little C.B. Buttner F.H. Bartnik E. Caterson B. J. Biol. Chem. 1998; 273: 30576-30582Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar), embedded in agarose, and cultured with prodrug and various cytokines as stated under "Results." After 4 days, medium was removed, dialyzed against water, and lyophilized. Samples were reconstituted in SDS-PAGE sample loading buffer and proteins were separated on a 10% reducing SDS-PAGE gel. Proteins were transferred to nitrocellulose and immunoblotted with neoepitope antibodies as described above. To analyze ex vivo cleavage by native enzyme, synovial fluid (SF) was collected aseptically from the knees of patients with rheumatoid arthritis. All patients gave written consent and had symptomatic knee effusions needing aspiration for clinical relief. These human SFs were preadsorbed on Prosep A prior to addition of the prodrug to deplete human IgG already present in the joint fluid. SF was also collected from knee joints of rats with antigen-induced arthritis (AIA), the disease was induced as described elsewhere (8Goodfellow R.M. Williams A.S. Levin J.L. Williams B.D. Morgan B.P. Clin. Exp. Immunol. 1997; 110: 45-52Crossref PubMed Scopus (44) Google Scholar) and fluid was aspirated from the knee joint 9 days following disease induction. The prodrug was added to the fluid at 0.33 (human SF) or 0.2 mg/ml (rat SF) and incubated for 16 h at 37 °C. Fluid was incubated with 50 μl of Prosep A (solid phase) to bind human Fc fragments, the pellet was washed once with PBS, and bound protein was eluted with HCl, pH 1.5. The eluate was lyophilized, reconstituted in SDS-PAGE loading buffer, and analyzed by Western blotting as described above, blots were probed with neoepitope antibodies reacting with the Fc portion of the cleaved molecule. In vivo cleavage was analyzed in the joints of rats with AIA 9 days following disease induction, 200 μg of the DAF4-IGD75-IgG4 prodrug or an equivalent molar concentration of a reagent lacking an enzyme site (DAF3-IgG2) was injected into the knee joint. Four hours later, fluid was aspirated, human Fc-containing fragments were harvested and analyzed as described above. In Vitro Functional Analysis of Human DAF-Ig—We have previously demonstrated that fusion of rat DAF or CD59 to human IgG1 Fc domains reduces their C-inhibitory function (13Harris C.L. Williams A.S. Linton S.M. Morgan B.P. Clin. Exp. Immunol. 2002; 129: 198-207Crossref PubMed Scopus (47) Google Scholar). In that study, comparison of rat DAF-Ig to soluble DAF (no Fc) that had been generated in the same cell line demonstrated an approximate 10-fold increase in function when compared on a moles of DAF basis and that of CD59-Ig to soluble CD59 a 35-fold increase in regulatory function. To generate the most effective prodrug based on human CReg, we first determined which fusion protein best abrogated function of human DAF. We assessed the effect on function of both antibody isotype and the length of the DAF molecule incorporated into the fusion protein. The two most "inflexible" human antibody isotypes are documented as IgG4 and IgG2 (42Dangl J.L. Wensel T.G. Morrison S.L. Stryer L. Herzenberg L.A. Oi V.T. EMBO J. 1988; 7: 1989-1994Crossref PubMed Scopus (255) Google Scholar), this
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