An indium-111-labelled membrane-targeted peptide for cell tracking with radionuclide imaging.

Cell labelling agents that enable longitudinal in vivo tracking of administered cells will support the clinical development of cell-based therapies. Radionuclide imaging with gamma and positron-emitting radioisotopes can provide quantitative and longitudinal mapping of cells in vivo. To make this widely accessible and adaptable to a range of cell types, new, versatile and simple methods for directly radiolabelling cells are required. We have developed [111In]In-DTPA-CTP, the first example of a radiolabelled peptide that binds to the extracellular membrane of cells, for tracking cell distribution in vivo using Single Photon Emission Computed Tomography (SPECT). [111In]In-DTPA-CTP consists of (i) myristoyl groups for insertion into the phospholipid bilayer, (ii) positively charged lysine residues for electrostatic association with negatively charged phospholipid groups at the cell surface and (iii) a diethylenetriamine pentaacetate derivative that coordinates the γ-emitting radiometal, [111In]In3+. [111In]In-DTPA-CTP binds to 5T33 murine myeloma cells, enabling qualitative SPECT tracking of myeloma cells' accumulation in lungs immediately after intravenous administration. This is the first report of a radiolabelled cell-membrane binding peptide for use in cell tracking.

Regression of Atherosclerosis in ApoE-/- Mice Via Modulation of Monocyte Recruitment and Phenotype, Induced by Weekly Dosing of a Novel "Cytotopic" Anti-Thrombin Without Prolonged Anticoagulation.

Background Anticoagulants induce atherosclerosis regression in animal models but exploiting this clinically is limited by bleeding events. Here we test a novel thrombin inhibitor, PTL060, comprising hirulog covalently linked to a synthetic myristoyl electrostatic switch to tether to cell membranes. Methods and Results ApoE-/- mice were fed chow or high-fat diets, before transplantation of congenic aortic segments or injection of PTL060, parental hirulog, control saline, or labeled CD11b positive cells. Aortic transplants from transgenic mice expressing anticoagulants on endothelium did not develop atherosclerosis. A single intravenous injection of PTL060, but not hirulog inhibited atheroma development by >50% compared with controls when assessed 4 weeks later. Mice had prolonged bleeding times for only one seventh of the time that PTL060 was biologically active. Repeated weekly injections of PTL060 but not hirulog caused regression of atheroma. We dissected 2 contributory mechanisms. First, the majority of CCR2+ (C-C chemokine receptor type 2+) monocytes recruited into plaques expressed CCR7 (C-C chemokine receptor type 7), ABCA1 (ATP-binding cassette transporter - 1), and interleukin-10 in PTL060 mice, a phenotype seen in <20% of CCR2+ recruits in controls. Second, after several doses, there was a significant reduction in monocyte recruits, the majority of which were CCR2-negative with a similar regression-associated phenotype. Regression equivalent to that induced by intravenous PTL060 was induced by adoptive transfer of CD11b+ cells pre-coated with PTL060. Conclusions Covalent linkage of a myristoyl electrostatic switch onto hirulog in PTL060 uncouples the pharmacodynamic effects on hemostasis and atherosclerosis, such that plaque regression, mediated predominantly via effects on monocytes, is accompanied by only transient anticoagulation.

Protein-inspired antibiotics active against vancomycin- and daptomycin-resistant bacteria.

The public health threat posed by a looming 'post-antibiotic' era necessitates new approaches to antibiotic discovery. Drug development has typically avoided exploitation of membrane-binding properties, in contrast to nature's control of biological pathways via modulation of membrane-associated proteins and membrane lipid composition. Here, we describe the rejuvenation of the glycopeptide antibiotic vancomycin via selective targeting of bacterial membranes. Peptide libraries based on positively charged electrostatic effector sequences are ligated to N-terminal lipophilic membrane-insertive elements and then conjugated to vancomycin. These modified lipoglycopeptides, the 'vancapticins', possess enhanced membrane affinity and activity against methicillin-resistant Staphylococcus aureus (MRSA) and other Gram-positive bacteria, and retain activity against glycopeptide-resistant strains. Optimised antibiotics show in vivo efficacy in multiple models of bacterial infection. This membrane-targeting strategy has potential to 'revitalise' antibiotics that have lost effectiveness against recalcitrant bacteria, or enhance the activity of other intravenous-administered drugs that target membrane-associated receptors.

Disentangling the roles of cholesterol and CD59 in intermedilysin pore formation.

The plasma membrane provides an essential barrier, shielding a cell from the pressures of its external environment. Pore-forming proteins, deployed by both hosts and pathogens alike, breach this barrier to lyse target cells. Intermedilysin is a cholesterol-dependent cytolysin that requires the human immune receptor CD59, in addition to cholesterol, to form giant ß-barrel pores in host membranes. Here we integrate biochemical assays with electron microscopy and atomic force microscopy to distinguish the roles of these two receptors in mediating structural transitions of pore formation. CD59 is required for the specific coordination of intermedilysin (ILY) monomers and for triggering collapse of an oligomeric prepore. Movement of Domain 2 with respect to Domain 3 of ILY is essential for forming a late prepore intermediate that releases CD59, while the role of cholesterol may be limited to insertion of the transmembrane segments. Together these data define a structural timeline for ILY pore formation and suggest a mechanism that is relevant to understanding other pore-forming toxins that also require CD59.

C1 esterase inhibitor reduces lower extremity ischemia/reperfusion injury and associated lung damage.

BACKGROUND: Ischemia/reperfusion injury of lower extremities and associated lung damage may result from thrombotic occlusion, embolism, trauma, or surgical intervention with prolonged ischemia and subsequent restoration of blood flow. This clinical entity is characterized by high morbidity and mortality. Deprivation of blood supply leads to molecular and structural changes in the affected tissue. Upon reperfusion inflammatory cascades are activated causing tissue injury. We therefore tested preoperative treatment for prevention of reperfusion injury by using C1 esterase inhibitor (C1 INH). METHODS AND FINDINGS: Wistar rats systemically pretreated with C1 INH (n = 6), APT070 (a membrane-targeted myristoylated peptidyl construct derived from human complement receptor 1, n = 4), vehicle (n = 7), or NaCl (n = 8) were subjected to 3h hind limb ischemia and 24h reperfusion. The femoral artery was clamped and a tourniquet placed under maintenance of a venous return. C1 INH treated rats showed significantly less edema in muscle (P<0.001) and lung and improved muscle viability (P<0.001) compared to controls and APT070. C1 INH prevented up-regulation of bradykinin receptor b1 (P<0.05) and VE-cadherin (P<0.01), reduced apoptosis (P<0.001) and fibrin deposition (P<0.01) and decreased plasma levels of pro-inflammatory cytokines, whereas deposition of complement components was not significantly reduced in the reperfused muscle. CONCLUSIONS: C1 INH reduced edema formation locally in reperfused muscle as well as in lung, and improved muscle viability. C1 INH did not primarily act via inhibition of the complement system, but via the kinin and coagulation cascade. APT070 did not show beneficial effects in this model, despite potent inhibition of complement activation. Taken together, C1 INH might be a promising therapy to reduce peripheral ischemia/reperfusion injury and distant lung damage in complex and prolonged surgical interventions requiring tourniquet application.

Structural basis for recognition of the pore-forming toxin intermedilysin by human complement receptor CD59.

Pore-forming proteins containing the structurally conserved membrane attack complex/perforin fold play an important role in immunity and host-pathogen interactions. Intermedilysin (ILY) is an archetypal member of a cholesterol-dependent cytolysin subclass that hijacks the complement receptor CD59 to make cytotoxic pores in human cells. ILY directly competes for the membrane attack complex binding site on CD59, rendering cells susceptible to complement lysis. To understand how these bacterial pores form in lipid bilayers and the role CD59 plays in complement regulation, we determined the crystal structure of human CD59 bound to ILY. Here, we show the ILY-CD59 complex at 3.5 Å resolution and identify two interfaces mediating this host-pathogen interaction. An ILY-derived peptide based on the binding site inhibits pore formation in a CD59-containing liposome model system. These data provide insight into how CD59 coordinates ILY monomers, nucleating an early prepore state, and suggest a potential mechanism of inhibition for the complement terminal pathway.

Local production and activation of complement up-regulates the allostimulatory function of dendritic cells through C3a-C3aR interaction.

Donor cell expression of C3 enhances the alloimmune response and is associated with the fate of transplantation. To clarify the mechanism for enhancement of the immune response, we have explored the role of C3a receptor (C3aR)-ligand interaction on murine bone marrow dendritic cells (DCs). We show that DCs either lacked receptor for C3a (a C3 cleavage product) or were treated with C3aR antagonist, elicited defective T-cell priming against alloantigen expressed on the DCs. This was associated with reduced surface expression of major histocompatibility complex (MHC) and costimulatory molecules on the DCs, and with defective priming in skin allograft rejection. In addition, DCs lacking factor B were unable to generate potent T-cell responses against donor antigen, whereas lack of C4 had no detectable effect, suggesting a role for the alternative pathway contributing to allostimulation. Furthermore, therapeutic complement regulator can down-regulate DC allostimulatory function. These findings suggest that the capacity of DCs for allostimulation depends on their ability to express, activate, and detect relevant complement components leading to C3aR signaling. This mechanism, in addition to underpinning the cell-autonomous action of donor C3 on allostimulation, has implications for a wider range of immune responses in self-restricted T-cell priming.

High-resolution structures of bacterially expressed soluble human CD59.

CD59 is a membrane-bound glycoprotein that protects host cells from lysis by inhibiting the terminal pathway of complement, preventing the formation and insertion of the membrane attack complex (MAC). Crystals of bacterially expressed and nonglycosylated recombinant soluble human CD59 have been obtained from three crystallization conditions, each of which gave rise to a distinct crystal form. Each crystal form led to a crystal structure at high resolution (1.15, 1.35 and 1.8 A). In one of these structures the electron-density map shows an as yet unidentified small molecule in the predicted C8/C9-binding site. The presence/absence of this ligand is linked to alternate conformations of the amino acids implicated in C8/C9 binding.

Therapeutic strategy with a membrane-localizing complement regulator to increase the number of usable donor organs after prolonged cold storage.

A shortage of donor organs and increasing dependence on marginal grafts with prolonged ischemic times have meant that new methods are needed to prevent postischemic damage. Herein is reported a new strategy aimed to protect donor kidney from complement-mediated postischemic damage and therefore increase the number of successful transplants. Rat donor kidneys were perfused with a membrane-localizing complement regulator derived from human complement receptor type 1 (APT070) and then subjected to prolonged periods of cold storage (at 4 degrees C). A relationship was found between the duration of cold ischemia and the extent of complement-mediated tubule damage and loss of graft function. After 16 h of cold storage, APT070-treated kidneys that were transplanted into syngeneic recipients showed a significant increase in the number of surviving grafts, compared with control-treated grafts (63.6 versus 26.3%). Surviving grafts also displayed less acute tubular injury and better preservation of renal function. These results not only enhance the understanding of the mechanism by which prolonged cold ischemia reduces immediate graft survival but also provide essential information about the effectiveness of membrane-localizing complement regulator with prolonged cold storage. This could lead to more effective strategies for improving the use of severely ischemic donor organs.

Expression of glycosylphosphatidylinositol-anchored CD59 on target cells enhances human NK cell-mediated cytotoxicity.

NK cell-mediated cytotoxicity of target cells is the result of a balance between the activating and inhibitory signals provided by their respective ligand-receptor interactions. In our current study, we have investigated the significance of CD59 on human target cells in modulating this process. A range of CD59 site-specific Abs were used in NK cytotoxicity blocking studies against the CD59-expressing K562 target cell line. Significantly reduced cytotoxicity was observed in the presence of Abs previously shown to lack blocking capacity for C-mediated lysis. We investigated the consequences for alternative membrane attachment modalities, namely bis-myristoylated-peptidyl (BiMP) and GPI anchoring, on CD59-negative U937 cells. Expression of GPI-anchored CD59 either via transfection or incorporation rendered U937 targets more susceptible to NK cytotoxicity, whereas incorporation of CD59 via a BiMP anchor to similar levels did not alter susceptibility to NK cytotoxicity. Localization of both BiMP- and GPI-anchored CD59 proteins was shown to be within the lipid raft microdomain. A role for the GPI anchor and independence from glycosylation status was confirmed by expression of transmembrane-anchored CD59 or unglycosylated CD59 and by testing in NK cytotoxicity assays. To investigate mechanisms, we compared the signaling capacity of the various forms of expressed and incorporated CD59 following Ab cross-linking in calcium flux assays. GPI-anchored CD59, with or without glycosylation, mediated activation events, whereas CD59 forms lacking the GPI anchor did not. The data show that the increased susceptibility of target cells expressing CD59 to NK cytotoxicity requires GPI anchor-mediating signaling events, likely mediated by interactions between GPI-anchored CD59 on targets and NK receptors.

Protection of erythrocytes from human complement-mediated lysis by membrane-targeted recombinant soluble CD59: a new approach to PNH therapy.

Paroxysmal nocturnal hemoglobinuria (PNH) results from the expansion of a hematopoietic clone that is deficient in glycosylphosphatidylinositol-anchored molecules. PNH is characterized by chronic hemolysis with acute exacerbations due to the uncontrolled activity of complement on PNH cells, which lack the inhibitor of homologous complement, CD59. Symptoms include severe fatigue, hemoglobinuria, esophageal spasm, erectile dysfunction, and thrombosis. We report the use of a novel synthetically modified recombinant human CD59, rhCD59-P, a soluble protein that attaches to cell membranes. In vitro treatment of PNH erythrocytes with rhCD59-P resulted in levels of CD59 equivalent to normal erythrocytes and effectively protected erythrocytes from complement-mediated hemolysis. The administration of rhCD59-P to CD1 mice resulted in levels of CD59 on erythrocytes, which protected them from complement-mediated lysis. Thus, rhCD59-P corrects the CD59 deficiency in vitro and can bind to erythrocytes in an in vivo murine model, protecting the cells from the activity of human complement, and represents a potential therapeutic strategy in PNH.

Efficient production of complement (C3d)3 fusion proteins using the baculovirus expression vector system.

Proteins fused to activated complement (C) fragments elicit enhanced immunogenicity. This "natural adjuvant" effect may have important implications when considering novel vaccination approaches. Here we describe both the construction of a novel fusion protein, consisting of a well characterized test antigen fused to multiple copies of the activated complement component (C3d)3, as well as an efficient method for its expression and production in insect cells. Using the inherent biological advantages of the baculovirus expression system, as well as applying specific infection and harvesting modifications, we have optimized the efficiency of protein production. Our modifications allow purification of fusion proteins directly from cell supernatant in a single anion exchange chromatographic step. This alleviates the requirement for the inclusion of protein affinity tags. The integrity of the purified recombinant protein was evaluated by SDS PAGE analysis, reactivity with antibodies, as well as in vivo by administration as an immunogen.

Complement inhibition abrogates nerve terminal injury in Miller Fisher syndrome.

A large body of clinical and experimental data indicate that complement activation is an important mechanism for neuronal and glial injury in Guillain-Barré syndromes. Inhibition of complement activation therefore might be expected to limit the progression of the disease. Using in vitro and in vivo models of the Guillain-Barré syndrome variant, Miller Fisher syndrome, we have shown previously that anti-GQ1b ganglioside antibodies target the presynaptic motor nerve terminal axon and surrounding perisynaptic Schwann cells, thereby mediating destructive injury through deposition of membrane attack complex. Here, we have used this model to investigate the effects of a novel therapeutic inhibitor of complement activation, APT070 (Mirococept), both in vitro and in vivo. In these models, APT070 completely prevents membrane attack complex formation, and thereby has a major neuroprotective effect at the nerve terminal, as assessed by immunohistology of perisynaptic Schwann cell and axonal integrity. These data provide a rationale for considering clinical trials of APT070 in Guillain-Barré syndrome, its variant forms, and other complement dependent neuromuscular disorders.

An atomic resolution model for assembly, architecture, and function of the Dr adhesins.

Pathogenic bacteria possess adhesion protein complexes that play essential roles in targeting host cells and in propagating infection. Although each family of adhesion proteins is generally associated with a specific human disease, the Dr family from Escherichia coli is a notable exception, as its members are associated with both diarrheal and urinary tract infections. These proteins are reported to form both fimbrial and afimbrial structures at the bacterial cell surface and target a common host cell receptor, the decay-accelerating factor (DAF or CD55). Using the newly solved three-dimensional structure of AfaE, we have constructed a robust atomic resolution model that reveals the structural basis for assembly by donor strand complementation and for the architecture of capped surface fibers.

Biological activity, membrane-targeting modification, and crystallization of soluble human decay accelerating factor expressed in E. coli.

Decay-accelerating factor (DAF, CD55) is a glycophosphatidyl inositol-anchored glycoprotein that regulates the activity of C3 and C5 convertases. In addition to understanding the mechanism of complement inhibition by DAF through structural studies, there is also an interest in the possible therapeutic potential of the molecule. In this report we describe the cloning, expression in Escherichia coli, isolation and membrane-targeting modification of the four short consensus repeat domains of soluble human DAF with an additional C-terminal cysteine residue to permit site-specific modification. The purified refolded recombinant protein was active against both classical and alternative pathway assays of complement activation and had similar biological activity to soluble human DAF expressed in Pichia pastoris. Modification with a membrane-localizing peptide restored cell binding and gave a large increase in antihemolytic potency. These data suggested that the recombinant DAF was correctly folded and suitable for structural studies as well as being the basis for a DAF-derived therapeutic. Crystals of the E. coli-derived protein were obtained and diffracted to 2.2 A, thus permitting the first detailed X-ray crystallography studies on a functionally active human complement regulator protein with direct therapeutic potential.

Generation of a recombinant, membrane-targeted form of the complement regulator CD59: activity in vitro and in vivo.

Inappropriate activation of complement contributes to pathology in diverse inflammatory diseases. Soluble recombinant forms of the natural cell membrane regulators of complement are effective in animal models and some human diseases. However, their use is limited for reasons related to cost, short half lives, and propensity to cause unwanted systemic effects. Some of these limitations may be overcome by use of bacterial expression systems, specific targeting moieties, and judicious choice of regulator. Here we describe the application of these strategies to the generation of a membrane-targeted form of CD59. A recombinant soluble form of rat CD59, comprising the first 71 residues of the mature protein and missing the membrane-anchoring signal, was expressed in bacteria, purified, and refolded in a fully active form. The protein was coupled through its carboxyl terminus to a short, synthetic address tag that confers membrane binding activity. Attachment of the membrane address tag markedly increased complement-inhibitory activity assessed in vitro in hemolysis assays. Intra-articular administration of the tagged agent markedly suppressed disease in a model of rheumatoid arthritis in Lewis rats. This novel type of agent, termed sCD59-APT542, offers for the first time the prospect of efficient and specific inhibition of membrane attack complex activity in vivo.

Nontransgenic hyperexpression of a complement regulator in donor kidney modulates transplant ischemia/reperfusion damage, acute rejection, and chronic nephropathy.

Complement activation during ischemia and reperfusion contributes to the development of tissue injury with severe negative impact on outcomes in transplantation. To counter the effect of complement, we present a strategy to deliver a novel complement regulator stabilized on cell surfaces within donor organs. The membrane-bound complement regulator is able to inhibit complement activation when the donor organ is revascularized and exposed to host-circulating complement. Application of this construct to donor kidneys protected transplanted tissues from ischemia/reperfusion injury and reduced the deposition of activated complement and histological signs of damage under conditions in which a nontargeted control construct was ineffective. Treatment of donor organs in this way improved graft performance in the short and long term. An analysis of the immune response in allograft recipients showed that reducing graft damage at the time of transplantation through complement regulation also modulated the alloresponse. Additionally, the results of perfusion studies with human kidneys demonstrated the feasibility of targeting endothelial and epithelial surfaces with this construct, to allow investigation in clinical transplantation.

Stimulation and inhibition of fibril formation by a peptide in the presence of different concentrations of SDS.

Sodium dodecyl sulphate (SDS), a detergent that mimics some characteristics of biological membranes, has been found to affect significantly fibril formation by a peptide from human complement receptor 1. In aqueous solution the peptide is unfolded but slowly aggregates to form fibrils. In sub-micellar concentrations of SDS the peptide is initially alpha-helical but converts rapidly to a beta-sheet structure and large quantities of fibrils form. In SDS above the critical micellar concentration the peptide adopts a stable alpha-helical structure and no fibrils are observed. These findings demonstrate the sensitivity of fibril formation to solution conditions and suggest a possible role for membrane components in amyloid fibril formation in living systems.

Bacterial expression and membrane targeting of the rat complement regulator Crry: a new model anticomplement therapeutic.

Inappropriate or unregulated activation of complement can contribute to pathology in inflammatory diseases. Previous studies have shown that soluble recombinant regulators of complement are effective in animal models and some human diseases. However, limitations include cost, rapid clearance, and unwanted systemic effects. To avoid some of these problems, bacterial expression of regulators has been optimized and methods for the addition of a membrane-targeting moiety to the complement regulator developed. When administered directly to sites of inflammation, membrane-targeted human regulators are retained and inhibit complement-activation locally. To test the efficacy of membrane-targeted complement regulators in vivo, we have undertaken the expression and membrane targeting of the rat-complement regulator Crry. A soluble recombinant form of Crry, containing only the first four short consensus repeats, was expressed in a mammalian expression system and shown to be functional as a fluid phase regulator. To generate the quantities required for testing in vivo, Crry was expressed in bacteria and refolded successfully. Refolded protein had full-complement regulatory activity in vitro. Attachment of a membrane address tag conferred membrane-binding capacity and greatly increased complement regulatory function in vitro. This novel anticomplement agent can now be applied to rat models of arthritis and other inflammatory diseases.