A novel soluble complement receptor 1 fragment with enhanced therapeutic potential
2021; Elsevier BV; Volume: 296; Linguagem: Inglês
10.1074/jbc.ra120.016127
ISSN1083-351X
AutoresSandra Wymann, Yun Dai, Anup G. Nair, Helen Cao, Glenn A. Powers, Anna Schnell, Genevieve Martin-Roussety, David Tai Leong, Jason Simmonds, Kim G. Lieu, Mitchell J. de Souza, Marcel Mischnik, Shirley M. Taylor, Saw Yen Ow, M. Spycher, R.E. Butcher, Martin J. Pearse, Adrian W. Zuercher, Adriana Baz Morelli, Con Panousis, Michael J. Wilson, Tony Rowe, Matthew P. Hardy,
Tópico(s)Immune Cell Function and Interaction
ResumoHuman complement receptor 1 (HuCR1) is a pivotal regulator of complement activity, acting on all three complement pathways as a membrane-bound receptor of C3b/C4b, C3/C5 convertase decay accelerator, and cofactor for factor I-mediated cleavage of C3b and C4b. In this study, we sought to identify a minimal soluble fragment of HuCR1, which retains the complement regulatory activity of the wildtype protein. To this end, we generated recombinant, soluble, and truncated versions of HuCR1 and compared their ability to inhibit complement activation in vitro using multiple assays. A soluble form of HuCR1, truncated at amino acid 1392 and designated CSL040, was found to be a more potent inhibitor than all other truncation variants tested. CSL040 retained its affinity to both C3b and C4b as well as its cleavage and decay acceleration activity and was found to be stable under a range of buffer conditions. Pharmacokinetic studies in mice demonstrated that the level of sialylation is a major determinant of CSL040 clearance in vivo. CSL040 also showed an improved pharmacokinetic profile compared with the full extracellular domain of HuCR1. The in vivo effects of CSL040 on acute complement-mediated kidney damage were tested in an attenuated passive antiglomerular basement membrane antibody-induced glomerulonephritis model. In this model, CSL040 at 20 and 60 mg/kg significantly attenuated kidney damage at 24 h, with significant reductions in cellular infiltrates and urine albumin, consistent with protection from kidney damage. CSL040 thus represents a potential therapeutic candidate for the treatment of complement-mediated disorders. Human complement receptor 1 (HuCR1) is a pivotal regulator of complement activity, acting on all three complement pathways as a membrane-bound receptor of C3b/C4b, C3/C5 convertase decay accelerator, and cofactor for factor I-mediated cleavage of C3b and C4b. In this study, we sought to identify a minimal soluble fragment of HuCR1, which retains the complement regulatory activity of the wildtype protein. To this end, we generated recombinant, soluble, and truncated versions of HuCR1 and compared their ability to inhibit complement activation in vitro using multiple assays. A soluble form of HuCR1, truncated at amino acid 1392 and designated CSL040, was found to be a more potent inhibitor than all other truncation variants tested. CSL040 retained its affinity to both C3b and C4b as well as its cleavage and decay acceleration activity and was found to be stable under a range of buffer conditions. Pharmacokinetic studies in mice demonstrated that the level of sialylation is a major determinant of CSL040 clearance in vivo. CSL040 also showed an improved pharmacokinetic profile compared with the full extracellular domain of HuCR1. The in vivo effects of CSL040 on acute complement-mediated kidney damage were tested in an attenuated passive antiglomerular basement membrane antibody-induced glomerulonephritis model. In this model, CSL040 at 20 and 60 mg/kg significantly attenuated kidney damage at 24 h, with significant reductions in cellular infiltrates and urine albumin, consistent with protection from kidney damage. CSL040 thus represents a potential therapeutic candidate for the treatment of complement-mediated disorders. The complement system, comprising more than 30 proteins in plasma and on cell surfaces, is a key element of the innate immune system and a primary mechanism of host defense against pathogens. It also functions as an important link to the adaptive immune and coagulation systems (1Ricklin D. Hajishengallis G. Yang K. Lambris J.D. Complement: a key system for immune surveillance and homeostasis.Nat. Immunol. 2010; 11: 785-797Crossref PubMed Scopus (2295) Google Scholar, 2Kolev M. Le Friec G. Kemper C. Complement--tapping into new sites and effector systems.Nat. Rev. Immunol. 2014; 14: 811-820Crossref PubMed Scopus (200) Google Scholar, 3Amara U. Rittirsch D. Flierl M. Bruckner U. Klos A. Gebhard F. Lambris J.D. Huber-Lang M. Interaction between the coagulation and complement system.Adv. Exp. Med. Biol. 2008; 632: 71-79PubMed Google Scholar). Targeting of cells and immune complexes by complement results in phagocytosis via C4b/C3b-mediated opsonization, direct cell lysis through the formation of the pore-forming membrane attack complex, and in inflammation mediated primarily through the anaphylatoxins C3a and C5a. The complement cascade is initiated via the classical pathway (CP), lectin pathway, and alternative pathway (AP) by a variety of activators ranging from immune complexes, neoepitopes, endotoxin, certain carbohydrates, and in the case of the AP, spontaneously by what is referred to as a tick-over mechanism (4Lachmann P.J. The amplification loop of the complement pathways.Adv. Immunol. 2009; 104: 115-149Crossref PubMed Scopus (152) Google Scholar) involving hydrolysis of C3. These pathways converge at the level of C3, leading to cellular activation, phagocytosis, or lysis (1Ricklin D. Hajishengallis G. Yang K. Lambris J.D. Complement: a key system for immune surveillance and homeostasis.Nat. Immunol. 2010; 11: 785-797Crossref PubMed Scopus (2295) Google Scholar). Regulatory proteins, including complement receptor 1 (CR1) (5Liu D. Niu Z.X. The structure, genetic polymorphisms, expression and biological functions of complement receptor type 1 (CR1/CD35).Immunopharmacol. Immunotoxicol. 2009; 31: 524-535Crossref PubMed Scopus (55) Google Scholar), factor H (6Ferreira V.P. Pangburn M.K. Cortes C. Complement control protein factor H: the good, the bad, and the inadequate.Mol. Immunol. 2010; 47: 2187-2197Crossref PubMed Scopus (249) Google Scholar), factor I (7Lachmann P.J. The story of complement factor I.Immunobiology. 2019; 224: 511-517Crossref PubMed Scopus (13) Google Scholar), CD46 (8Yamamoto H. Fara A.F. Dasgupta P. Kemper C. CD46: the 'multitasker' of complement proteins.Int. J. Biochem. Cell Biol. 2013; 45: 2808-2820Crossref PubMed Scopus (62) Google Scholar), CD55 (9Dho S.H. Lim J.C. Kim L.K. Beyond the role of CD55 as a complement component.Immune Netw. 2018; 18e11Crossref PubMed Scopus (28) Google Scholar), and CD59 (10Davies A. Simmons D.L. Hale G. Harrison R.A. Tighe H. Lachmann P.J. Waldmann H. 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. 1989; 170: 637-654Crossref PubMed Scopus (525) Google Scholar), have evolved to tightly regulate the complement cascade and prevent unintended damage to host cells or tissues. In addition to its role in pathogen clearance, complement is intimately involved in the pathogenesis of a number of human disease states, such as post-transplant graft rejection and ischemia reperfusion injury (11Salvadori M. Rosso G. Bertoni E. Complement involvement in kidney diseases: from physiopathology to therapeutical targeting.World J. Nephrol. 2015; 4: 169-184Crossref PubMed Google Scholar, 12Sacks S.H. Zhou W. The role of complement in the early immune response to transplantation.Nat. Rev. Immunol. 2012; 12: 431-442Crossref PubMed Scopus (148) Google Scholar, 13Powell J.T. Tsapepas D.S. Martin S.T. Hardy M.A. Ratner L.E. Managing renal transplant ischemia reperfusion injury: novel therapies in the pipeline.Clin. Transplant. 2013; 27: 484-491Crossref PubMed Scopus (30) Google Scholar, 14Chan R.K. Ibrahim S.I. Verna N. Carroll M. Moore Jr., F.D. Hechtman H.B. Ischaemia-reperfusion is an event triggered by immune complexes and complement.Br. J. Surg. 2003; 90: 1470-1478Crossref PubMed Scopus (76) Google Scholar, 15Asgari E. Zhou W. Sacks S. Complement in organ transplantation.Curr. Opin. Organ Transplant. 2010; 15: 486-491Crossref PubMed Scopus (47) Google Scholar), atypical hemolytic uremic syndrome (16Noris M. Remuzzi G. Glomerular diseases dependent on complement activation, including atypical hemolytic uremic syndrome, membranoproliferative glomerulonephritis, and C3 glomerulopathy: core curriculum 2015.Am. J. Kidney Dis. 2015; 66: 359-375Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar), glomerulonephritis (GN) (17Thurman J.M. Many drugs for many targets: novel treatments for complement-mediated glomerular disease.Nephrol. Dial. Transplant. 2017; 32: i57-i64Crossref PubMed Scopus (12) Google Scholar, 18Berger S.P. Daha M.R. Complement in glomerular injury.Semin. Immunopathol. 2007; 29: 375-384Crossref PubMed Scopus (38) Google Scholar), paroxysmal nocturnal hemoglobinuria (19Parker C.J. Paroxysmal nocturnal hemoglobinuria: an historical overview.Hematol. Am. Soc. Hematol. Educ. Program. 2008; : 93-103Crossref PubMed Scopus (16) Google Scholar), myasthenia gravis (20Tuzun E. Huda R. Christadoss P. Complement and cytokine based therapeutic strategies in Myasthenia gravis.J. Autoimmun. 2011; 37: 136-143Crossref PubMed Scopus (56) Google Scholar), systemic lupus erythematosus (21Macedo A.C. Isaac L. Systemic Iupus erythematosus and deficiencies of early components of the complement classical pathway.Front. Immunol. 2016; 7: 55Crossref PubMed Scopus (131) Google Scholar), macular degeneration (22Charbel Issa P. Chong N.V. Scholl H.P. The significance of the complement system for the pathogenesis of age-related macular degeneration - current evidence and translation into clinical application.Graefes Arch. Clin. Exp. Ophthalmol. 2011; 249: 163-174Crossref PubMed Scopus (73) Google Scholar), and septic shock (23Charchaflieh J. Wei J. Labaze G. Hou Y.J. Babarsh B. Stutz H. Lee H. Worah S. Zhang M. The role of complement system in septic shock.Clin. Dev. Immunol. 2012; 2012: 407324Crossref PubMed Scopus (51) Google Scholar). These diseases and/or injuries can occur not only when complement is hyperactivated but also when expression of fluid-phase or membrane-associated complement regulation is aberrant (1Ricklin D. Hajishengallis G. Yang K. Lambris J.D. Complement: a key system for immune surveillance and homeostasis.Nat. Immunol. 2010; 11: 785-797Crossref PubMed Scopus (2295) Google Scholar, 24Ricklin D. Lambris J.D. Complement in immune and inflammatory disorders: pathophysiological mechanisms.J. Immunol. 2013; 190: 3831-3838Crossref PubMed Scopus (322) Google Scholar, 25McGeer P.L. Lee M. McGeer E.G. A review of human diseases caused or exacerbated by aberrant complement activation.Neurobiol. Aging. 2017; 52: 12-22Crossref PubMed Scopus (28) Google Scholar). Mutations in genes that encode complement proteins are also associated with disease (26Degn S.E. Jensenius J.C. Thiel S. Disease-causing mutations in genes of the complement system.Am. J. Hum. Genet. 2011; 88: 689-705Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). Examples of this are the association of the factor H gene mutations with atypical hemolytic uremic syndrome (27Servais A. Noel L.H. Dragon-Durey M.A. Gubler M.C. Remy P. Buob D. Cordonnier C. Makdassi R. Jaber W. Boulanger E. Lesavre P. Fremeaux-Bacchi V. Heterogeneous pattern of renal disease associated with homozygous factor H deficiency.Hum. Pathol. 2011; 42: 1305-1311Crossref PubMed Scopus (34) Google Scholar), C1q gene mutations with a systemic lupus erythematosus–like syndrome (28Lipsker D. Hauptmann G. Cutaneous manifestations of complement deficiencies.Lupus. 2010; 19: 1096-1106Crossref PubMed Scopus (34) Google Scholar), and C9 gene mutations with meningococcal infection (29Khajoee V. Ihara K. Kira R. Takemoto M. Torisu H. Sakai Y. Guanjun J. Hee P.M. Tokunaga K. Hara T. Founder effect of the C9 R95X mutation in orientals.Hum. Genet. 2003; 112: 244-248Crossref PubMed Scopus (16) Google Scholar). Human complement receptor 1 (HuCR1), also known as CD35, is type I membrane protein that forms part of a family of complement receptors that include CR2, CR3, and CR4 (5Liu D. Niu Z.X. The structure, genetic polymorphisms, expression and biological functions of complement receptor type 1 (CR1/CD35).Immunopharmacol. Immunotoxicol. 2009; 31: 524-535Crossref PubMed Scopus (55) Google Scholar). It is expressed on erythrocytes, eosinophils, monocytes, macrophages, neutrophils, B and some T cells, mast cells, glomerular podocytes, and follicular dendritic cells (30Ahearn J.M. Fearon D.T. Structure and function of the complement receptors, CR1 (CD35) and CR2 (CD21).Adv. Immunol. 1989; 46: 183-219Crossref PubMed Scopus (370) Google Scholar). The extracellular domain of HuCR1 is 1971 amino acids long and composed of 30 short consensus repeat (SCR) domains, each of approximately 60 amino acids and of variable homology to each other (31Krych-Goldberg M. Atkinson J.P. Structure-function relationships of complement receptor type 1.Immunol. Rev. 2001; 180: 112-122Crossref PubMed Scopus (201) Google Scholar, 32Weis J.H. Morton C.C. Bruns G.A. Weis J.J. Klickstein L.B. Wong W.W. Fearon D.T. A complement receptor locus: genes encoding C3b/C4b receptor and C3d/Epstein-Barr virus receptor map to 1q32.J. Immunol. 1987; 138: 312-315PubMed Google Scholar). SCR domains 1 to 28 of HuCR1 are further arranged into four long homologous repeat (LHR) domains, each consisting of seven SCR domains (31Krych-Goldberg M. Atkinson J.P. Structure-function relationships of complement receptor type 1.Immunol. Rev. 2001; 180: 112-122Crossref PubMed Scopus (201) Google Scholar, 33Klickstein L.B. Wong W.W. Smith J.A. Weis J.H. Wilson J.G. Fearon D.T. Human C3b/C4b receptor (CR1). Demonstration of long homologous repeating domains that are composed of the short consensus repeats characteristics of C3/C4 binding proteins.J. Exp. Med. 1987; 165: 1095-1112Crossref PubMed Scopus (191) Google Scholar). Very low levels (approximately 50 ng/ml) of soluble HuCR1 occur naturally in plasma, likely produced as a consequence of proteolytic shedding (34Karthikeyan G. Baalasubramanian S. Seth S. Das N. Low levels of plasma soluble complement receptor type 1 in patients receiving thrombolytic therapy for acute myocardial infarction.J. Thromb. Thrombolysis. 2007; 23: 115-120Crossref PubMed Scopus (3) Google Scholar). HuCR1 has multiple functions. It acts as the receptor for both C3b and C4b, and to a lesser extent, iC3b and C3dg (35Cooper N.R. Immune adherence by the fourth component of complement.Science. 1969; 165: 396-398Crossref PubMed Scopus (88) Google Scholar, 36Fearon D.T. Identification of the membrane glycoprotein that is the C3b receptor of the human erythrocyte, polymorphonuclear leukocyte, B lymphocyte, and monocyte.J. Exp. Med. 1980; 152: 20-30Crossref PubMed Scopus (438) Google Scholar, 37Ross G.D. Lambris J.D. Identification of a C3bi-specific membrane complement receptor that is expressed on lymphocytes, monocytes, neutrophils, and erythrocytes.J. Exp. Med. 1982; 155: 96-110Crossref PubMed Scopus (120) Google Scholar). It also acts as a cofactor for factor I-mediated cleavage of C3b to iC3b, and for further cleavage of iC3b to C3dg and C3c (38Medof M.E. Iida K. Mold C. Nussenzweig V. Unique role of the complement receptor CR1 in the degradation of C3b associated with immune complexes.J. Exp. Med. 1982; 156: 1739-1754Crossref PubMed Scopus (222) Google Scholar, 39Ross G.D. Lambris J.D. Cain J.A. Newman S.L. Generation of three different fragments of bound C3 with purified factor I or serum. I. Requirements for factor H vs CR1 cofactor activity.J. Immunol. 1982; 129: 2051-2060PubMed Google Scholar). A similar cofactor function for C4b cleavage has also been observed (40Iida K. Nussenzweig V. Complement receptor is an inhibitor of the complement cascade.J. Exp. Med. 1981; 153: 1138-1150Crossref PubMed Scopus (188) Google Scholar). The third main function of HuCR1 is to regulate complement by accelerating convertase decay via binding to C3b or C4b and displacing C2a or Bb in the C3 or C5 convertases (36Fearon D.T. Identification of the membrane glycoprotein that is the C3b receptor of the human erythrocyte, polymorphonuclear leukocyte, B lymphocyte, and monocyte.J. Exp. Med. 1980; 152: 20-30Crossref PubMed Scopus (438) Google Scholar, 40Iida K. Nussenzweig V. Complement receptor is an inhibitor of the complement cascade.J. Exp. Med. 1981; 153: 1138-1150Crossref PubMed Scopus (188) Google Scholar). Additional functions, such as clearance of immune complexes (41Birmingham D.J. Hebert L.A. CR1 and CR1-like: the primate immune adherence receptors.Immunol. Rev. 2001; 180: 100-111Crossref PubMed Scopus (86) Google Scholar) and enhancement of phagocytosis (42Dustin M.L. Complement receptors in myeloid cell adhesion and phagocytosis.Microbiol. Spectr. 2016; 4Crossref PubMed Scopus (17) Google Scholar), have also been described. HuCR1 inhibits complement activity in multiple species, including mouse (43Pemberton M. Anderson G. Vetvicka V. Justus D.E. Ross G.D. Microvascular effects of complement blockade with soluble recombinant CR1 on ischemia/reperfusion injury of skeletal muscle.J. Immunol. 1993; 150: 5104-5113PubMed Google Scholar, 44Yazdanbakhsh K. Kang S. Tamasauskas D. Sung D. Scaradavou A. Complement receptor 1 inhibitors for prevention of immune-mediated red cell destruction: potential use in transfusion therapy.Blood. 2003; 101: 5046-5052Crossref PubMed Scopus (33) Google Scholar, 45Huang J. Kim L.J. Mealey R. Marsh Jr., H.C. Zhang Y. Tenner A.J. Connolly Jr., E.S. Pinsky D.J. Neuronal protection in stroke by an sLex-glycosylated complement inhibitory protein.Science. 1999; 285: 595-599Crossref PubMed Scopus (297) Google Scholar), rat (46Couser W.G. Johnson R.J. Young B.A. Yeh C.G. Toth C.A. Rudolph A.R. The effects of soluble recombinant complement receptor 1 on complement-mediated experimental glomerulonephritis.J. Am. Soc. Nephrol. 1995; 5: 1888-1894Crossref PubMed Google Scholar, 47Goodfellow R.M. Williams A.S. Levin J.L. Williams B.D. Morgan B.P. Soluble complement receptor one (sCR1) inhibits the development and progression of rat collagen-induced arthritis.Clin. Exp. Immunol. 2000; 119: 210-216Crossref PubMed Scopus (56) Google Scholar, 48Mulligan M.S. Yeh C.G. Rudolph A.R. Ward P.A. Protective effects of soluble CR1 in complement- and neutrophil-mediated tissue injury.J. Immunol. 1992; 148: 1479-1485PubMed Google Scholar, 49Pratt J.R. Hibbs M.J. Laver A.J. Smith R.A. Sacks S.H. Effects of complement inhibition with soluble complement receptor-1 on vascular injury and inflammation during renal allograft rejection in the rat.Am. J. Pathol. 1996; 149: 2055-2066PubMed Google Scholar, 50Weisman H.F. Bartow T. Leppo M.K. Marsh Jr., H.C. Carson G.R. Concino M.F. Boyle M.P. Roux K.H. Weisfeldt M.L. Fearon D.T. Soluble human complement receptor type 1: in vivo inhibitor of complement suppressing post-ischemic myocardial inflammation and necrosis.Science. 1990; 249: 146-151Crossref PubMed Scopus (863) Google Scholar), rabbit (51Heller A. Kunz M. Samakas A. Haase M. Kirschfink M. Koch T. The complement regulators C1 inhibitor and soluble complement receptor 1 attenuate acute lung injury in rabbits.Shock. 2000; 13: 285-290Crossref PubMed Scopus (19) Google Scholar, 52Homeister J.W. Satoh P.S. Kilgore K.S. Lucchesi B.R. Soluble complement receptor type 1 prevents human complement-mediated damage of the rabbit isolated heart.J. Immunol. 1993; 150: 1055-1064PubMed Google Scholar), and pig (53Pruitt S.K. Kirk A.D. Bollinger R.R. Marsh Jr., H.C. Collins B.H. Levin J.L. Mault J.R. Heinle J.S. Ibrahim S. Rudolph A.R. et al.The effect of soluble complement receptor type 1 on hyperacute rejection of porcine xenografts.Transplantation. 1994; 57: 363-370Crossref PubMed Scopus (232) Google Scholar, 54Schmid R.A. Zollinger A. Singer T. Hillinger S. Leon-Wyss J.R. Schob O.M. Hogasen K. Zund G. Patterson G.A. Weder W. Effect of soluble complement receptor type 1 on reperfusion edema and neutrophil migration after lung allotransplantation in swine.J. Thorac. Cardiovasc. Surg. 1998; 116: 90-97Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). A recombinant, soluble, and full extracellular domain form of HuCR1 comprising of all 28 SCR domains and known as TP-10 has previously been developed for therapeutic use (55Li J.S. Jaggers J. Anderson P.A. The use of TP10, soluble complement receptor 1, in cardiopulmonary bypass.Expert Rev. Cardiovasc. Ther. 2006; 4: 649-654Crossref PubMed Scopus (31) Google Scholar). Phase I open-label ascending dose study (56Zimmerman J.L. Dellinger R.P. Straube R.C. Levin J.L. Phase I trial of the recombinant soluble complement receptor 1 in acute lung injury and acute respiratory distress syndrome.Crit. Care Med. 2000; 28: 3149-3154Crossref PubMed Scopus (69) Google Scholar) showed that TP-10 had a mean half-life of 69 h, with doses greater than 1 mg/kg inhibiting complement activity. Two clinical trials examining the effect of TP-10–mediated complement inhibition in patients during cardiopulmonary bypass demonstrated a gender-specific effect, with only males showing clinically significant improvement (57Lazar H.L. Bokesch P.M. van Lenta F. Fitzgerald C. Emmett C. Marsh Jr., H.C. Ryan U. Obe TP10 Cardiac Surgery Study GroupSoluble human complement receptor 1 limits ischemic damage in cardiac surgery patients at high risk requiring cardiopulmonary bypass.Circulation. 2004; 110: II274-II279Crossref PubMed Scopus (78) Google Scholar, 58Lazar H.L. Keilani T. Fitzgerald C.A. Shapira O.M. Hunter C.T. Shemin R.J. Marsh Jr., H.C. Ryan U.S. TP10 Cardiac Surgery Study GroupBeneficial effects of complement inhibition with soluble complement receptor 1 (TP10) during cardiac surgery: is there a gender difference?.Circulation. 2007; 116: I83-I88Crossref PubMed Scopus (23) Google Scholar). However, a study testing TP-10 at 10 mg/kg in 59 lung transplant recipients (59Keshavjee S. Davis R.D. Zamora M.R. de Perrot M. Patterson G.A. A randomized, placebo-controlled trial of complement inhibition in ischemia-reperfusion injury after lung transplantation in human beings.J. Thorac. Cardiovasc. Surg. 2005; 129: 423-428Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar) has shown clinical efficacy, with 50% of patients in the TP-10 group extubated at 24 h compared with 19% in the placebo group. In addition, the mean duration of mechanical ventilation was reduced to 10.6 days in TP-10–treated patients compared with 21.5 days in standard-of-care patients (59Keshavjee S. Davis R.D. Zamora M.R. de Perrot M. Patterson G.A. A randomized, placebo-controlled trial of complement inhibition in ischemia-reperfusion injury after lung transplantation in human beings.J. Thorac. Cardiovasc. Surg. 2005; 129: 423-428Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). This clinical trial data show that not only that a soluble CR1 has potential value as a therapeutic but also the choice of indication is important. In this report, we assessed the role of individual and combined HuCR1 LHR domains by comparing their complement inhibitory activity as soluble proteins in both ELISA-based and red blood cell hemolytic assays. From these in vitro studies, we identified and characterized CSL040, a truncated soluble variant of HuCR1 lacking the LHR-D domain exhibiting greater in vitro potency and an extended in vivo half-life compared with full-length HuCR1. We demonstrated the importance of sialylation to reduce in vivo clearance rate of CSL040 as well as its ability to attenuate damage in vivo in a mouse model of immune complex–mediated kidney injury. We sought to identify potential therapeutic candidates based on HuCR1 by determining the relative contributions of the LHR domains of soluble HuCR1 to its overall potency in vitro. A series of recombinant soluble N-terminal and C-terminal truncation mutants were generated that lacked 1, 2, or 3 of its four LHR domains (Fig. 1) and were shown to be monomeric by SDS–PAGE (Fig. S1). These proteins were purified, and their potencies compared with the full-length extracellular domain HuCR1(1971) in vitro. Using the Wieslab complement system kits for all three complement pathways or red blood cell hemolytic assays specific for the CP and AP (Table 1), we showed that purified single LHR domain variants of HuCR1 had greatly reduced complement inhibitory activity in vitro compared with HuCR1(1971). Indeed, recombinant HuCR1(1393–1971) had no detectable activity (Table 1) in any of the assays employed. Mutants with three contiguous LHR domains (LHR-AB, LHR-BC, and LHR-CD) exhibited increased inhibitory activity compared with singly expressed LHR domains, with HuCR1(939) generally being more potent than HuCR1(490–1392) and HuCR1(940–1971) (Table 1). HuCR1(490–1971) had lower activity than soluble full-length HuCR1(1971), particularly as measured in the hemolytic assays; although for some assays, this was not determined to be statistically significant (Table 1). Surprisingly, HuCR1(1392) containing LHR-ABC was a significantly more potent inhibitor than full-length HuCR1(1971), of two of three pathways in the Wieslab assay, and both hemolytic assays (Table 1). The potency improvements for HuCR1(1392) over HuCR1(1971) were found to be approximately twofold for both the CP and lectin pathway–specific assays and approximately threefold for both Wieslab and hemolytic AP assays (Table 1). Removal of the histidine (His)-tag from HuCR1(1392) had no effect on its potency in vitro (Fig. S2). HuCR1(1392) was designated as CSL040 and is described as such in the remainder of the article.Table 1Potency of human soluble CR1 truncation variants in complement pathway–specific Wieslab and red blood cell hemolytic assaysHuCR1 truncationsWieslab IC50 (nM) ± SEMHemolytic IC50 (nM) ± SEMConstructLHR domainsClassicalLectinAlternativeClassicalAlternativeHuCR1(1971)ABCD1.28 ± 0.261.05 ± 0.170.53 ± 0.291.21 ± 0.212.71 ± 0.94HuCR1(1392)/CSL040ABC0.53 ± 0.33ap < 0.05.0.47 ± 0.16ap < 0.05.0.17 ± 0.100.42 ± 0.10bp < 0.005.0.90 ± 0.54ap < 0.05.HuCR1(490–1971)BCD4.18 ± 2.493.12 ± 0.59bp < 0.005.0.29 ± 0.1732.96 ± 15.34ap < 0.05.76.85 ± 49.15HuCR1(939)AB2.59 ± 1.301.87 ± 0.641.31 ± 1.824.61 ± 1.85ap < 0.05.13.15 ± 2.18bp < 0.005.HuCR1(490–1392)BC10.06 ± 2.55bp < 0.005.5.80 ± 0.65cp < 0.0005.0.70 ± 0.3610.08 ± 5.52ap < 0.05.12.48 ± 4.30ap < 0.05.HuCR1(940–1971)CD98.57 ± 71.68120.74 ± 49.29ap < 0.05.1.26 ± 1.17677.80 ± 278.35ap < 0.05.95.51 ± 21.06bp < 0.005.HuCR1(489)A36.91 ± 7.25bp < 0.005.22.35 ± 14.361.10 ± 1.4368.38 ± 9.52cp < 0.0005.49.87 ± 24.76ap < 0.05.HuCR1(490–939)BNo activityNo activity8.18 ± 9.63959.37 ± 243.92bp < 0.005.63.38 ± 31.03ap < 0.05.HuCR1(940–1392)CNo activity1879.7 ± 1545.30.79 ± 0.61733.10 ± 203.47bp < 0.005.66.36 ± 18.10bp < 0.005.HuCR1(1393–1971)DNo activityNo activityNo activityNo activityNo activityThe IC50 values listed are the mean ± SD of three independent experiments. Statistically significant differences for individual HuCR1 fragment IC50 values compared with parental HuCR1(1971) for each pathway assay were calculated by one-way ANOVA.CR1, complement receptor 1; HuCR1, human complement receptor 1; LHR, long homologous repeat.a p < 0.05.b p < 0.005.c p < 0.0005. Open table in a new tab The IC50 values listed are the mean ± SD of three independent experiments. Statistically significant differences for individual HuCR1 fragment IC50 values compared with parental HuCR1(1971) for each pathway assay were calculated by one-way ANOVA. CR1, complement receptor 1; HuCR1, human complement receptor 1; LHR, long homologous repeat. As HuCR1 is a receptor for C3b and C4b (31Krych-Goldberg M. Atkinson J.P. Structure-function relationships of complement receptor type 1.Immunol. Rev. 2001; 180: 112-122Crossref PubMed Scopus (201) Google Scholar), we examined the affinity of CSL040 for these ligands by surface plasmon resonance (SPR). Both the C3b and C4b used were commercially sourced plasma-derived proteins that under nonreducing conditions appeared predominantly monomeric (Fig. S3). However, a noticeable dimeric fraction was also observed for both C3b and C4b. As shown in Figure 2A and Table 2, the affinity of CSL040 to human C3b was determined by SPR to be 264.6 ± 14.4 nM, whereas the control HuCR1(1971) affinity was measured at 385.7 ± 17.1 nM (Fig. 2B and Table 2). This difference was small but statistically significant (p = 0.0007). Binding of human C4b to CSL040 (Fig. 2C) and HuCR1(1971) (Fig. 2D) was also observed, but no affinity measurements could be determined as the interaction was biphasic and could not be fitted to a 1:1 model (Table 2). However, there were no qualitative difference in the binding of CSL040 to human C4b compared with HuCR1(1971). These data demonstrate that removal of the LHR-D domain has no or minimal effect on the binding of CSL040 to C3b or C4b and suggests that the improved potency of CSL040 is likely not because of increased ligand-binding affinity.Table 2Kinetic rate constants and affinity of plasma-derived human C3b and C4b binding to CSL040 or HuCR1(1971)ReceptorLigandka (1/Ms)kd (1/s)KD (nM)CSL040C3b2.48 × 1046.54 × 10−3264.6 ± 14.3HuCR1(1971)1.96 × 1047.54 × 10−3385.7 ± 17.1CSL040/HuCR1(1971)C4bBiphasic (nonfitted)KD indicated as mean ± SD (N = 3); p = 0.0007; unpaired t test. Values were calculated from sensorgram data fit to a 1:1 binding model for C3b.HuCR1, human complement receptor 1; ka, association rate constant; kd, dissociation rate constant; KD, equilibrium dissociation constant. Open table in a new tab KD indicated as mean ± SD (N = 3); p = 0.0007; unpaired t test. Values were calculated from sensorgram data fit to a 1:1 binding model for C3b. HuCR1, human complement receptor 1; ka, association rate constant; kd, dissociation rate constant; KD, equilibrium dissociation constant. We next sought to examine the complement inhibitory properties of CSL040 in greater detail, using decay acceleration assays to compare the ability of CSL040 and HuCR1(1971) to modify CP-specific C3 or C5 convertases. As shown in Figure 3A, CSL040 retained its increased potency compared with H
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