Distribution and determinants of circulating complement factor H concentration determined by a high-throughput immunonephelometric assay

Sofat, Reecha; Mangione, Palma; Gallimore, J. Ruth; Hakobyan, Svetlana; Hughes, Timothy R.; Shah, Tina; Goodship, Tim; D’Aiuto, F; Langenberg, Claudia; Wareham, Nick; Morgan, B. Paul; Pepys, Mark B.; Hingorani, Aroon D.

doi:10.1016/j.jim.2013.01.009

Cited by 32 publications

(23 citation statements)

References 30 publications

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“…LPS coated to micro-titer plates was exposed to serum spiked (separately) with various amounts of either the natural regulator FH or one of the engineered inhibitors. Of note, serum already contains FH (~300 to 500 μg/ml (Hakobyan et al, 2008; Sofat et al, 2013) equating to ~2–3 μM), so the effective FH concentration in this assay, with a final serum proportion of 25%, is about 0.6 μM higher than the amount of externally added FH. Addition of purified FH to a final concentration of 2 μM completely abolished AP-activation by LPS, which was measured by detecting (or failing to detect) the C3-activation products C3b and iC3b (Figure 2A).…”

Section: Resultsmentioning

confidence: 99%

Selectivity of C3-opsonin targeted complement inhibitors: A distinct advantage in the protection of erythrocytes from paroxysmal nocturnal hemoglobinuria patients

et al. 2016

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Paroxysmal nocturnal hemoglobinuria (PNH) is characterized by complement-mediated cell lysis due to deficiency of GPI-anchored complement regulators. Blockage of the lytic pathway by eculizumab is the only available therapy for PNH patients and shows remarkable benefits, but regularly yields PNH erythrocytes opsonized with fragments of complement protein C3, rendering such erythrocytes prone to extravascular hemolysis. This effect is associated with insufficient responsiveness seen in a subgroup of PNH patients. Novel C3-opsonin targeted complement inhibitors act earlier in the cascade, at the level of activated C3 and are engineered from parts of the natural complement regulator Factor H (FH) or complement receptor 2 (CR2). This inhibitor class comprises three variants of “miniFH” and the clinically developed “FH-CR2” fusion-protein (TT30). We show that the approach of FH-CR2 to target C3-opsonins was more efficient in preventing complement activation induced by foreign surfaces, whereas the miniFH variants were substantially more active in controlling complement on PNH erythrocytes. Subtle differences were noted in the ability of each version of miniFH to protect human PNH cells. Importantly, miniFH and FH-CR2 interfered only minimally with complement-mediated serum killing of bacteria when compared to untargeted inhibition of all complement pathways by eculizumab. Thus, the molecular design of each C3-opsonin targeted complement inhibitor determines its potency in respect to the nature of the activator/surface providing potential functionality in PNH.

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Section: Resultsmentioning

confidence: 99%

Selectivity of C3-opsonin targeted complement inhibitors: A distinct advantage in the protection of erythrocytes from paroxysmal nocturnal hemoglobinuria patients

et al. 2016

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“…How the FH splice product FHL‐1, consisting of only the first seven N‐terminal FH CCPs and a unique stretch of four amino acids, contributes to AP regulation was largely unaddressed. FHL‐1 is found at approximately 1 μM in serum, which corresponds to 30–50% of the FH concentration in the blood . Initial functional characterization of FHL‐1, in an isolated system in which FHL‐1 was allowed to exclusively probe DAA on sheep erythrocytes, indicated that FHL‐1 exhibits only approximately 1% of the activity of FH in this system .…”

Section: Novel Insights In Fh and Fhl‐1 Biologymentioning

confidence: 99%

“…Although possible, this approach required a doubling of the concentration of FH to achieve full protection . Given the rather high plasma concentration of FH (2–3 μM; 0.3–0.5 mg/ml), the protein amounts needed for supplementation treatment would be very high. Engineered, targeted FH‐based inhibitors that exceed the regulatory activity of FH could potentially alleviate this problem.…”

Section: Engineered Fh‐based Regulatorsmentioning

confidence: 99%

Protection of host cells by complement regulators

Schmidt

Lambris

Ricklin

2016

Immunological Reviews

182

174

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Summary The complement cascade is an ancient immune-surveillance system that not only provides protection from pathogen invasion but has also evolved to participate in physiological processes to maintain tissue homeostasis. The alternative pathway (AP) of complement activation is the evolutionarily oldest part of this innate immune cascade. It is unique in that it is continuously activated at a low level and arbitrarily probes foreign, modified-self, and also unaltered self-structures. This indiscriminate activation necessitates the presence of preformed regulators on autologous surfaces to spare self-cells from the undirected nature of AP activation. Although the other two canonical complement activation routes, the classical and lectin pathways, initiate the cascade more specifically through pattern recognition, their activity still needs to be tightly controlled to avoid excessive reactivity. It is the perpetual duty of complement regulators to protect the self from damage inflicted by inadequate complement activation. Here, we review the role of complement regulators as preformed mediators of defense, explain their common and specialized functions, and discuss selected cases in which alterations in complement regulators lead to disease. Finally, rational engineering approaches using natural complement inhibitors as potential therapeutics are highlighted.

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“…FH is composed of 20 CCPs, whereas FHL-1 consists of FH CCPs 1–7 and an additional four C-terminal residues. FH occurs in the blood at concentrations of ~2–3 µM (6–8), while FHL-1 is less abundant (~1 µM) (8). Both regulators specifically adhere, via a polyanion-binding site in CCP 7 and another in CCP 20 in the case of FH (9, 10), to glycosaminoglycans (GAGs) and sialic acids on host surfaces.…”

Section: Introductionmentioning

confidence: 99%

Comparative Analysis of Novel Complement-Targeted Inhibitors, MiniFH, and the Natural Regulators Factor H and Factor H–like Protein 1 Reveal Functional Determinants of Complement Regulation

Harder

Höchsmann

Simmet

et al. 2016

The Journal of Immunology

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The serum proteins Factor H (FH), consisting of 20 complement control protein modules (CCPs), and its splice product Factor H-like protein 1 (FHL-1; consisting of CCPs 1–7) are major regulators of the alternative pathway (AP) of complement activation. The engineered version of FH, miniFH, contains only the N- and C-terminal portions of FH linked by an optimized peptide and shows ~10-fold higher ex vivo potency. We explored the hypothesis that regulatory potency is enhanced by unmasking of a ligand-binding site in the C-terminal CCPs (19–20) that is cryptic in full-length native FH. Therefore we produced a FH variant lacking the central domains 10–15 (FHΔ10–15). To explore how avidity affects regulatory strength, we generated a duplicated version of miniFH, termed midiFH. We compared activities of FHΔ10–15 and midiFH to miniFH, FH and FHL-1. Relative to FH, FHΔ10–15 exhibited an altered binding profile toward C3 activation products and a 5-fold-enhanced complement regulation on PNH patient’s erythrocytes. Contrary to dogma, FHL-1 and FH exhibited equal regulatory activity, suggesting that the role of FHL-1 in AP regulation has been underestimated. Unexpectedly, a substantially increased avidity for complement opsonins, as seen in midiFH, did not potentiate the inhibitory potential on host cells. In conclusion, comparisons of engineered and native FH-based regulators have identified features that determine high AP regulatory activity on host cells. Unrestricted availability of FH CCPs 19–20 and an optimal spatial orientation between the N- and C-terminal FH regions are key.

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Distribution and determinants of circulating complement factor H concentration determined by a high-throughput immunonephelometric assay

Cited by 32 publications

References 30 publications

Selectivity of C3-opsonin targeted complement inhibitors: A distinct advantage in the protection of erythrocytes from paroxysmal nocturnal hemoglobinuria patients

Selectivity of C3-opsonin targeted complement inhibitors: A distinct advantage in the protection of erythrocytes from paroxysmal nocturnal hemoglobinuria patients

Protection of host cells by complement regulators

Comparative Analysis of Novel Complement-Targeted Inhibitors, MiniFH, and the Natural Regulators Factor H and Factor H–like Protein 1 Reveal Functional Determinants of Complement Regulation

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