Human Complement Receptor Research Articles

The Molecular Mechanisms of Complement Receptor 1-It Is Complicated.

Human complement receptor 1 (CR1) is a membrane-bound regulator of complement that has been the subject of recent attempts to generate soluble therapeutic compounds comprising different fragments of its extracellular domain. This review will focus on the extracellular domain of CR1 and detail how its highly duplicated domains work both separately and together to mediate binding to its main ligands C3b and C4b, and to inhibit the classical, lectin, and alternative pathways of the complement cascade via the mechanisms of decay acceleration activity (DAA) and co-factor activity (CFA). Understanding the molecular basis of CR1 activity is made more complicated by the presence not only of multiple ligand binding domains within CR1 but also the fact that C3b and C4b can interact with CR1 as both monomers, dimers, and heterodimers. Evidence for the interaction of CR1 with additional ligands such as C1q will also be reviewed. Finally, we will bring the mechanistic understanding of CR1 activity together to provide an explanation for the differential complement pathway inhibition recently observed with CSL040, a soluble CR1-based therapeutic candidate in pre-clinical development.

Inhibition of acute complement responses towards bolus-injected nanoparticles using targeted short-circulating regulatory proteins.

Effective inhibition of the complement system is needed to prevent the accelerated clearance of nanomaterials by complement cascade and inflammatory responses. Here we show that a fusion construct consisting of human complement receptor 2 (CR2) (which recognizes nanosurface-deposited complement 3 (C3)) and complement receptor 1 (CR1) (which blocks C3 convertases) inhibits complement activation with picomolar to low nanomolar efficacy on many types of nanomaterial. We demonstrate that only a small percentage of nanoparticles are randomly opsonized with C3 both in vitro and in vivo, and CR2-CR1 immediately homes in on this subpopulation. Despite rapid in vivo clearance, the co-injection of CR2-CR1 in rats, or its mouse orthologue CR2-Crry in mice, with superparamagnetic iron oxide nanoparticles nearly completely blocks complement opsonization and unwanted granulocyte/monocyte uptake. Furthermore, the inhibitor completely prevents lethargy caused by bolus-injected nanoparticles, without inducing long-lasting complement suppression. These findings suggest the potential of the targeted complement regulators for clinical evaluation.

139 Antibodies targeting human complement receptor C3aR

139 Antibodies targeting human complement receptor C3aR

Soluble Complement Receptor 1 Therapeutics

Human Complement Receptor 1 (CR1/CD35) is a potent negative regulator of the complement system. Its mechanism of action is through interaction with the complement activation fragments, C3b and C4b to mediate decay acceleration of the C3 and C5 convertase complexes as well as cleavage of both ligands into inactive fragments via cofactor activity. The result is inhibition of the classical, lectin, and alternative complement pathways. This article will focus on recombinant soluble forms of CR1 that have been generated as potential therapeutics for complement-mediated disorders. Specifically, we will review and contrast the in vitro and in vivo properties of: sCR1 (BRL55730/TP10/CDX-1135), the soluble full-length extracellular domain of human CR1; sCR1-sLex (TP20), a glyco-engineered version of sCR1 additionally targeted to activated endothelium; APT070 (Mirococept), a CR1 fragment conjugated to a myristoylated peptide to enhance tissue targeting; and CSL040, a soluble truncated version of the CR1 extracellular domain which exhibits altered potency and pharmacokinetic properties as compared to the parental molecule. The data obtained from studies on the effects of these CR1-based molecules in animal models of disease and their therapeutic applications will also be discussed.

Sialylation-dependent pharmacokinetics and differential complement pathway inhibition are hallmarks of CR1 activity in vivo.

Human Complement Receptor 1 (HuCR1) is a potent membrane-bound regulator of complement both in vitro and in vivo, acting via interaction with its ligands C3b and C4b. Soluble versions of HuCR1 have been described such as TP10, the recombinant full-length extracellular domain, and more recently CSL040, a truncated version lacking the C-terminal long homologous repeat domain D (LHR-D). However, the role of N-linked glycosylation in determining its pharmacokinetic (PK) and pharmacodynamic (PD) properties is only partly understood. We demonstrated a relationship between the asialo-N-glycan levels of CSL040 and its PK/PD properties in rats and non-human primates (NHPs), using recombinant CSL040 preparations with varying asialo-N-glycan levels. The clearance mechanism likely involves the asialoglycoprotein receptor (ASGR), as clearance of CSL040 with a high proportion of asialo-N-glycans was attenuated in vivo by co-administration of rats with asialofetuin, which saturates the ASGR. Biodistribution studies also showed CSL040 localization to the liver following systemic administration. Our studies uncovered differential PD effects by CSL040 on complement pathways, with extended inhibition in both rats and NHPs of the alternative pathway compared with the classical and lectin pathways that were not correlated with its PK profile. Further studies showed that this effect was dose dependent and observed with both CSL040 and the full-length extracellular domain of HuCR1. Taken together, our data suggests that sialylation optimization is an important consideration for developing HuCR1-based therapeutic candidates such as CSL040 with improved PK properties and shows that CSL040 has superior PK/PD responses compared with full-length soluble HuCR1.

A potent truncated form of human soluble CR1 is protective in a mouse model of renal ischemia\u2013reperfusion injury

The complement system is a potent mediator of ischemia–reperfusion injury (IRI), which detrimentally affects the function and survival of transplanted kidneys. Human complement receptor 1 (HuCR1) is an integral membrane protein that inhibits complement activation by blocking the convertases that activate C3 and C5. We have previously reported that CSL040, a truncated form of recombinant soluble HuCR1 (sHuCR1), has enhanced complement inhibitory activity and improved pharmacokinetic properties compared to the parent molecule. Here, we compared the capacity of CSL040 and full-length sHuCR1 to suppress complement-mediated organ damage in a mouse model of warm renal IRI. Mice were treated with two doses of CSL040 or sHuCR1, given 1 h prior to 22 min unilateral renal ischemia and again 3 h later. 24 h after reperfusion, mice treated with CSL040 were protected against warm renal IRI in a dose-dependent manner, with the highest dose of 60 mg/kg significantly reducing renal dysfunction, tubular injury, complement activation, endothelial damage, and leukocyte infiltration. In contrast, treatment with sHuCR1 at a molar equivalent dose to 60 mg/kg CSL040 did not confer significant protection. Our results identify CSL040 as a promising therapeutic candidate to attenuate renal IRI and demonstrate its superior efficacy over full-length sHuCR1 in vivo.

A novel soluble complement receptor 1 fragment with enhanced therapeutic potential

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.

Ex vivo delivery of Mirococept: A dose-finding study in pig kidney after showing a low dose is insufficient to reduce delayed graft function in human kidney.

The complement system plays a pivotal role in the pathogenesis of ischemia-reperfusion injury in solid organ transplantation. Mirococept is a potent membrane-localizing complement inhibitor that can be administered ex vivo to the donor kidney prior to transplantation. To evaluate the efficacy of Mirococept in reducing delayed graft function (DGF) in deceased donor renal transplantation, we undertook the efficacy of mirococept (APT070) for preventing ischaemia-reperfusion injury in the kidney allograft (EMPIRIKAL) trial (ISRCTN49958194). A dose range of 5-25mg would be tested, starting with 10mg in cohort 1. No significant difference between Mirococept at 10mg and control was detected; hence the study was stopped to enable a further dose saturation study in a porcine kidney model. The optimal dose of Mirococept in pig kidney was 80mg. This dose did not induce any additional histological damage compared to controls or after a subsequent 3hours of normothermic machine perfusion. The amount of unbound Mirococept postperfusion was found to be within the systemic dose range considered safe in the Phase I trial. The ex vivo administration of Mirococept is a safe and feasible approach to treat DGF in deceased donor kidney transplantation. The porcine kidney study identified an optimal dose of 80mg (equivalent to 120mg in human kidney) that provides a basis for further clinical development.

A novel mouse model expressing human forms for complement receptors CR1 and CR2

BackgroundThe complement cascade is increasingly implicated in development of a variety of diseases with strong immune contributions such as Alzheimer’s disease and Systemic Lupus Erythematosus. Mouse models have been used to determine function of central components of the complement cascade such as C1q and C3. However, species differences in their gene structures mean that mice do not adequately replicate human complement regulators, including CR1 and CR2. Genetic variation in CR1 and CR2 have been implicated in modifying disease states but the mechanisms are not known.ResultsTo decipher the roles of human CR1 and CR2 in health and disease, we engineered C57BL/6J (B6) mice to replace endogenous murine Cr2 with human complement receptors, CR1 and CR2 (B6.CR2CR1). CR1 has an array of allotypes in human populations and using traditional recombination methods (Flp-frt and Cre-loxP) two of the most common alleles (referred to here as CR1long and CR1short) can be replicated within this mouse model, along with a CR1 knockout allele (CR1KO). Transcriptional profiling of spleens and brains identified genes and pathways differentially expressed between mice homozygous for either CR1long, CR1short or CR1KO. Gene set enrichment analysis predicts hematopoietic cell number and cell infiltration are modulated by CR1long, but not CR1short or CR1KO.ConclusionThe B6.CR2CR1 mouse model provides a novel tool for determining the relationship between human-relevant CR1 alleles and disease.

Complement Receptor 2 Based Immunoassay Measuring Activation of the Complement System at C3-Level in Plasma Samples From Mice and Humans.

We aimed at establishing a sensitive and robust assay for estimation of systemic complement activation at complement component C3 level in mouse and human plasma samples. In order to capture the activation products iC3b and C3dg in a specific and physiological relevant manner we utilized a construct consisting of the iC3b/C3dg-binding site of human complement receptor 2 (CR2) attached to an Fc-part of mouse IgG. This construct binds C3dg and iC3b from both mice and humans. We purified the CR2-IgG construct from mouse B myeloma cell line supernatants, J558L-CR2-IgG, by protein G affinity chromatography. The CR2-IgG construct was used for capturing C3 fragments in microtiter wells and an anti-mouse or an anti-human-C3 antibody was used for detection of bound C3 fragments. Initially we tested the specificity of the assays with the use of purified C3 fragments. Further, with the use of the CR2-based assay, we measured an up to three-fold higher signal in activated mouse serum as compared to non-activated mouse serum, whereas activated serum from a C3 knock-out mouse gave no signal. We tested in vivo generated samples from a mouse experiment; complement activation was induced by injecting cobra venom factor or heat aggregated IgG into C57bl6 mice, followed by withdrawal of EDTA blood samples at different time points and measurement of iC3b/C3dg. We observed a clear time-dependent distinction in signals between samples with expected high and low complement activation. Furthermore, with the use of the assay for human C3 fragments, we observed that patients with systemic lupus erythematosus (SLE) (n = 144) had significantly higher iC3b/C3dg levels as compared to healthy individuals (n = 144) (p < 0.0001). We present two functional immunoassays, that are able to measure systemic levels of the C3-activation products iC3b and C3dg in mice and humans. To our knowledge, these are the first assays for complement activation that use a physiological relevant capture construct such as CR2. These assays will be a relevant tool when investigating mouse models and human diseases involving the complement system.

Size-Selective Phagocytic Clearance of Fibrillar α-Synuclein through Conformational Activation of Complement Receptor 4.

Aggregation of α-synuclein (αSN) is an important histological feature of Parkinson disease. Recent studies showed that the release of misfolded αSN from human and rodent neurons is relevant to the progression and spread of αSN pathology. Little is known, however, about the mechanisms responsible for clearance of extracellular αSN. This study found that human complement receptor (CR) 4 selectively bound fibrillar αSN, but not monomeric species. αSN is an abundant protein in the CNS, which potentially could overwhelm clearance of cytotoxic αSN species. The selectivity of CR4 toward binding fibrillar αSN consequently adds an important αSN receptor function for maintenance of brain homeostasis. Based on the recently solved structures of αSN fibrils and the known ligand preference of CR4, we hypothesize that the parallel monomer stacking in fibrillar αSN creates a known danger-associated molecular pattern of stretches of anionic side chains strongly bound by CR4. Conformational change in the receptor regulated tightly clearance of fibrillar αSN by human monocytes. The induced change coupled concomitantly with phagolysosome formation. Data mining of the brain transcriptome in Parkinson disease patients supported CR4 as an active αSN clearance mechanism in this disease. Our results associate an important part of the innate immune system, namely complement receptors, with the central molecular mechanisms of CNS protein aggregation in neurodegenerative disorders.

Partial ligand-receptor engagement yields functional bias at the human complement receptor, C5aR1

The human complement component, C5a, binds two different seven-transmembrane receptors termed C5aR1 and C5aR2. C5aR1 is a prototypical G-protein-coupled receptor that couples to the Gαi subfamily of heterotrimeric G-proteins and β-arrestins (βarrs) following C5a stimulation. Peptide fragments derived from the C terminus of C5a can still interact with the receptor, albeit with lower affinity, and can act as agonists or antagonists. However, whether such fragments might display ligand bias at C5aR1 remains unexplored. Here, we compare C5a and a modified C-terminal fragment of C5a, C5apep, in terms of G-protein coupling, βarr recruitment, endocytosis, and extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase activation at the human C5aR1. We discover that C5apep acts as a full agonist for Gαi coupling as measured by cAMP response and extracellular signal-regulated kinase 1/2 phosphorylation, but it displays partial agonism for βarr recruitment and receptor endocytosis. Interestingly, C5apep exhibits full-agonist efficacy with respect to inhibiting lipopolysaccharide-induced interleukin-6 secretion in human macrophages, but its ability to induce human neutrophil migration is substantially lower compared with C5a, although both these responses are sensitive to pertussis toxin treatment. Taken together, our data reveal that compared with C5a, C5apep exerts partial efficacy for βarr recruitment, receptor trafficking, and neutrophil migration. Our findings therefore uncover functional bias at C5aR1 and also provide a framework that can potentially be extended to chemokine receptors, which also typically interact with chemokines through a biphasic mechanism.

Protein-engineered molecules carrying GAD65 epitopes and targeting CD35 selectively down-modulate disease-associated human B lymphocytes.

Type 1 diabetes mellitus is an autoimmune metabolic disorder characterized by chronic hyperglycemia, the presence of autoreactive T and B cells and autoantibodies against self-antigens. A membrane-bound enzyme on the pancreatic beta-cells, glutamic acid decarboxylase 65 (GAD65), is one of the main autoantigens in type 1 diabetes. Autoantibodies against GAD65 are potentially involved in beta-cell destruction and decline of pancreatic functions. The human complement receptor type 1 (CD35) on B and T lymphocytes has a suppressive activity on these cells. We hypothesized that it may be possible to eliminate GAD65-specific B cells from type 1 diabetes patients by using chimeric molecules, containing an anti-CD35 antibody, coupled to peptides resembling GAD65 B/T epitopes. These molecules are expected to selectively bind the anti-GAD65 specific B cells by the co-cross-linking of the immunoglobulin receptor and CD35 and to deliver a suppressive signal. Two synthetic peptides derived from GAD65 protein (GAD65 epitopes) and anti-CD35 monoclonal antibody were used for the construction of two chimeras. The immunomodulatory activity of the engineered antibodies was tested in vitro using peripheral blood mononuclear cells (PBMCs) from type 1 diabetes patients. A reduction in the number of anti-GAD65 IgG antibody-secreting plasma cells and increased percentage of apoptotic B lymphocytes was observed after treatment of these PBMCs with the engineered antibodies. The constructed chimeric molecules are able to selectively modulate the activity of GAD65-specific B lymphocytes and the production of anti-GAD65 IgG autoantibodies by co-cross-linking of the inhibitory CD35 and the B cell antigen receptor (BCR). This treatment presents a possible way to alter the autoimmune nature of these cells.

The human complement receptor type 2 (CR2)/CR1 fusion protein TT32, a novel targeted inhibitor of the classical and alternative pathway C3 convertases, prevents arthritis in active immunization and passive transfer mouse models

Complement activation in human diseases is characterized by the local covalent deposition of the long-lived C3 fragments iC3b/C3dg/C3d. Previously, TT30, a complement alternative pathway (AP)-selective inhibitor, was designed as a fusion protein linking the first four short consensus repeats (SCRs) of human complement receptor type 2 (CR2) with the first five SCRs of human factor H (fH). TT30 acts by utilizing CR2 SCR1–4 to bind the initially formed iC3b/C3dg/C3d fragments and delivering surface-targeted inhibition of AP C3 and C5 convertases through fH SCR 1-5. In order to combine classical (CP) and lectin (LP) pathway inhibitory abilities employing CR2-mediated targeting, TT32 was developed. TT32 is a CR2-CR1 fusion protein using the first ten SCRs of CR1, chosen because they contain both C3 and C5 convertase inhibitory activity through utilization of decay-acceleration and cofactor activity for both AP and CP. In Wieslab assays, TT32 showed potent inhibition of the CP and AP with IC50 of 11 and 46 nM, respectively. The TT32 inhibitory activity is partially blocked with a molar excess of a competing anti-CR2 mAb, thus demonstrating the importance of the CR2 targeting. TT32 was studied in the type II (CII) collagen-induced arthritis (CIA), an active immunization model, and the CII antibody-induced arthritis (CAIA) passive transfer model. In CIA, injection of 2.0 mg TT32 at day 21 and 28 post disease induction, but not untargeted CR1 alone, resulted in a 51.5% decrease in clinical disease activity (CDA). In CAIA, treatment with TT32 resulted in a 47.4% decrease in CDA. Therefore, a complement inhibitor that targets both the AP and CP/LP C3/C5 convertases was shown to limit complement-mediated tissue damage and inflammation in disease models in which all three complement activation pathways are implicated.

Human complement receptor type 1 (CR1) protein levels and genetic variants in chronic Chagas Disease

Complement is an essential element in both innate and acquired immunity contributing to the immunopathogenesis of many disorders, including Chagas Disease (CD). Human complement receptor 1 (CR1) plays a role in the clearance of complement opsonized molecules and may facilitate the entry of pathogens into host cells. Distinct CR1 exon 29 variants have been found associated with CR1 expression levels, increased susceptibility and pathophysiology of several diseases. In this study, CR1 plasma levels were assessed by ELISA and CR1 variants in exon 29 by sequencing in a Brazilian cohort of 232 chronic CD patients and 104 healthy controls. CR1 levels were significantly decreased in CD patients compared to controls (p < 0.0001). The CR1 rs1704660G, rs17047661G and rs6691117G variants were significantly associated with CD and in high linkage disequilibrium. The CR1*AGAGTG haplotype was associated with T. cruzi infection (p = 0.035, OR 3.99, CI 1.1-14.15) whereas CR1*AGGGTG was related to the risk of chagasic cardiomyopathy (p = 0.028, OR 12.15, CI 1.13-113). This is the first study that provides insights on the role of CR1 in development and clinical presentation of chronic CD.

A novel C3d-containing oligomeric vaccine provides insight into the viability of testing human C3d-based vaccines in mice

The use of C3d, the final degradation product of complement protein C3, as a “natural” adjuvant has been widely examined since the initial documentation of its immunogenicity-enhancing properties as a consequence of binding to complement receptor 2. Subsequently it was demonstrated that these effects are most evident when oligomeric, rather than when monomeric forms of C3d, are linked to various test protein antigens. In this study, we examined the feasibility of enhancing the adjuvant properties of human C3d further by utilizing C4b-binding protein (C4BP) to provide an oligomeric arrayed scaffold fused to the model antigen, tetanus toxin C fragment (TTCF).High molecular weight, C3d-containing oligomeric vaccines were successfully expressed, purified from mammalian cells and used to immunize groups of mice. Surprisingly, anti-TTCF antibody responses measured in these mice were poor. Subsequently we established by in vitro and in vivo analysis that, in the presence of mouse C3, human C3d does not interact with either mouse or even human complement receptor 2.These data confirm the requirement to develop murine versions of C3d based adjuvant compounds to test in mice or that mice would need to be developed that express both human C3 and human CR2 to allow the testing of human C3d based adjuvants in mouse in any capacity.

Geographical distribution of complement receptor type 1 variants and their associated disease risk.

BackgroundPathogens exert selective pressure which may lead to substantial changes in host immune responses. The human complement receptor type 1 (CR1) is an innate immune recognition glycoprotein that regulates the activation of the complement pathway and removes opsonized immune complexes. CR1 genetic variants in exon 29 have been associated with expression levels, C1q or C3b binding and increased susceptibility to several infectious diseases. Five distinct CR1 nucleotide substitutions determine the Knops blood group phenotypes, namely Kna/b, McCa/b, Sl1/Sl2, Sl4/Sl5 and KCAM+/-.MethodsCR1 variants were genotyped by direct sequencing in a cohort of 441 healthy individuals from Brazil, Vietnam, India, Republic of Congo and Ghana.ResultsThe distribution of the CR1 alleles, genotypes and haplotypes differed significantly among geographical settings (p≤0.001). CR1 variants rs17047660A/G (McCa/b) and rs17047661A/G (Sl1/Sl2) were exclusively observed to be polymorphic in African populations compared to the groups from Asia and South-America, strongly suggesting that these two SNPs may be subjected to selection. This is further substantiated by a high linkage disequilibrium between the two variants in the Congolese and Ghanaian populations. A total of nine CR1 haplotypes were observed. The CR1*AGAATA haplotype was found more frequently among the Brazilian and Vietnamese study groups; the CR1*AGAATG haplotype was frequent in the Indian and Vietnamese populations, while the CR1*AGAGTG haplotype was frequent among Congolese and Ghanaian individuals.ConclusionThe African populations included in this study might have a selective advantage conferred to immune genes involved in pathogen recognition and signaling, possibly contributing to disease susceptibility or resistance.

Peripheral complement interactions with amyloid β peptide: Erythrocyte clearance mechanisms

IntroductionAlthough amyloid β peptide (Aβ) is cleared from the brain to cerebrospinal fluid and the peripheral circulation, mechanisms for its removal from blood remain unresolved. Primates have uniquely evolved a highly effective peripheral clearance mechanism for pathogens, immune adherence, in which erythrocyte complement receptor 1 (CR1) plays a major role. MethodsMultidisciplinary methods were used to demonstrate immune adherence capture of Aβ by erythrocytes and its deficiency in Alzheimer's disease (AD). ResultsAβ was shown to be subject to immune adherence at every step in the pathway. Aβ dose-dependently activated serum complement. Complement-opsonized Aβ was captured by erythrocytes via CR1. Erythrocytes, Aβ, and hepatic Kupffer cells were colocalized in the human liver. Significant deficits in erythrocyte Aβ levels were found in AD and mild cognitive impairment patients. DiscussionCR1 polymorphisms elevate AD risk, and >80% of human CR1 is vested in erythrocytes to subserve immune adherence. The present results suggest that this pathway is pathophysiologically relevant in AD.

The Structure-Function Relationships of Complement Receptor Type 2 (CR2; CD21).

Human complement receptor type 2 (CR2; CD21) is a surface-associated glycoprotein which binds to a variety of endogenous ligands, including the complement component C3 fragments iC3b, C3dg and C3d, the low-affinity IgE receptor CD23, and the type I cytokine, interferon-alpha. CR2 links the innate complement-mediated immune response to pathogens and foreign antigens with the adaptive immune response by binding to C3d that is covalently attached to targets, and which results in a cell signalling phenomenon that lowers the threshold for B cell activation. Variations or deletions of the CR2 gene in humans, or the Cr2 gene in mice associate with a variety of autoimmune and inflammatory conditions. A number of infectious agents including Epstein-Barr virus (EBV), Human Immunodeficiency Virus (HIV) and prions also bind to CR2 either directly or indirectly by means of C3d-targeted immune complexes. In this review we discuss the interactions that CR2 undertakes with its best characterized ligands C3d, CD23 and the EBV gp350/220 envelope protein. To date only a single physiologically relevant complex of CR2 with one of its ligands, C3d, has been elucidated. By contrast, the interactions with CD23 and EBV gp350/220, while being important from physiologic and disease-associated standpoints, respectively, are only incompletely understood. A detailed knowledge of the structure-function relationships that CR2 undergoes with its ligands is necessary to understand the implications of using recombinant CR2 in therapeutic or imaging agents, or alternatively targeting CR2 to down-regulate the antibody mediated immune response in cases of autoimmunity.

Safety and Pharmacokinetics of the Complement Inhibitor TT30 in a Phase I Trial for Untreated PNH Patients

▪TT30 is a novel, recombinant human fusion protein, linking the C3d-binding domain of human complement receptor 2 (CR2) with the complement regulatory domain of human factor H (fH). TT30 is designed to inhibit the complement alternative pathway (CAP) at sites of local activation; it has been shown to be effective in preventing hemolysis and C3b-mediated opsonization in paroxysmal nocturnal hemoglobinuria (PNH) in vitro. This first in human study, TT30-PNH-002, was a multi-center open-label, single-dose, dose-escalation study of two formulations of TT30, ALXN1102 (10mg/ml) and ALXN1103 (50mg/ml). The study was conducted in patients with a diagnosis of PNH, defined as a PNH clone size of ≥10% or a PNH clone size between 5% and 10% with evidence of intravascular hemolysis documented within the previous 12 months; patients were not receiving concurrent treatment or treatment with a complement inhibitor within 60 days prior to screening. The primary endpoint of the study was assessment of the safety and tolerability of a single dose of TT30 when administered by IV infusion or separately by SC injection; secondary endpoints included pharmacokinetics (PK), pharmacodynamics (PD), and immunogenicity. The ALXN1102 formulation was administered by IV infusion to the 0.1 (n=1), 0.3 (n=1), and 1 mg/kg (n=3) dose level cohorts. The ALXN1103 formulation was administered by IV infusion to the 3 mg/kg (n=1) dose level cohort and by SC injection to the 1 (n=2) and 3 mg/kg (n=2) dose level cohorts. Patients were dosed one at a time, and each patient was evaluated before the next patient could be dosed. All 10 patients completed the study as planned; across all groups, 28 mild and 5 moderate treatment-emergent adverse event (TEAEs) were experienced. Treatment with both formulations of TT30 was considered safe, with no apparent dose-related safety risks. One patient who received ALXN1102 at 1.0 mg/kg IV experienced transient hypocalcemia associated with transient asymptomatic prolongation of the QT interval, both of which spontaneously resolved by 24 hours without intervention and may have been related to citrate in the formulation. Subsequent patients supplemented with oral calcium at the start of the infusion did not experience these events, nor were citrate-related toxicities seen in patients who received ALXN1103, which contains a lower amount of citrate. The secondary endpoints of the study were to characterize the PK of a single dose of TT30, to assess CAP, complement classical pathway (CCP) inhibition, PD effects on markers of intravascular hemolysis such as lactate dehydrogenase (LDH), and to characterize the immunogenicity of TT30. Serum TT30 total and peak (AUCs and Cmax) exposures increased with increase in IV or SC dose. Serum CAP activity declined in a dose-dependent manner following dosing in all IV dose cohorts, and approached zero % of baseline value (complete CAP inhibition) in the 1 mg/kg and 3 mg/kg IV dosing cohorts, when circulating levels of TT30 were in the range of approximately 5-10 ug/ml. CAP activity returned to baseline by 24 hours, when circulating TT30 was below 1 ug/ml, and appeared to remain stable thereafter. Serum CCP activity remained near baseline in all patients at all times. LDH levels dropped by 20% in the 1 mg/kg and 3 mg/kg IV dose cohorts and rose over time after CAP activity returned to baseline. Similarly, there was a decline in serum CAP activity and LDH levels in the 1 mg/kg and 3 mg/kg SC dose cohorts that returned to pre-treatment baseline over time, by 24 hours. No immunogenicity was observed in any patient, following either IV or SC dosing of a single dose of TT30. In conclusion, both TT30 formulations were safe and well tolerated; preliminary PK and PD data support the concept that treatment by targeted complement inhibitors like TT30 result in pharmacologically relevant CAP inhibition in PNH, as demonstrated by LDH decrease. Nevertheless, such therapeutic inhibition is short-lasting, and further strategies are needed to sustain pharmacological levels of TT30 for chronic treatment in PNH. DisclosuresRisitano:Alnylam: Consultancy, Research Funding; Novartis: Research Funding; Pfizer: Consultancy; Alexion Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Rapharma: Consultancy, Research Funding. Off Label Use: The presentation will include discussion of a clinical trial with a non-approved medication. Storek:Alexion Pharmaceuticals, Inc.: Employment, Other: stock owner. Sahelijo:Alexion Pharmaceuticals: Employment, Equity Ownership. Doyle:Alexion Pharmaceuticals, Inc.: Employment, Other: stock owner. Dai:Alexion Pharmaceuticals, Inc.: Employment. Weitz:Alexion Pharmaceuticals, Inc.: Consultancy, Speakers Bureau. Elebute:Alexion Pharmaceuticals, Inc.: Speakers Bureau. O'Connell:Celgene: Speakers Bureau; Incyte Corporation: Membership on an entity's Board of Directors or advisory committees; Portola: Membership on an entity's Board of Directors or advisory committees. Kulasekararaj:Alexion: Consultancy. Lundberg:Alexion Pharmaceuticals, Inc.: Employment.