Markus Anliker

Incomplete inhibition by eculizumab: mechanistic evidence for residual C5 activity during strong complement activation

Eculizumab inhibits the terminal, lytic pathway of complement by blocking the activation of the complement protein C5 and shows remarkable clinical benefits in certain complement-mediated diseases. However, several reports suggest that activation of C5 is not always completely suppressed in patients even under excess of eculizumab over C5, indicating that residual C5 activity may derogate the drugs therapeutic benefit under certain conditions. By using eculizumab and the tick-derived C5 inhibitor coversin, we determined conditions ex vivo in which C5 inhibition is incomplete. The degree of such residual lytic activity depended on the strength of the complement activator and the resulting surface density of the complement activation product C3b, which autoamplifies via the alternative pathway (AP) amplification loop. We show that at high C3b densities required for binding and activation of C5, both inhibitors reduce but do not abolish this interaction. The decrease of C5 binding to C3b clusters in the presence of C5 inhibitors correlated with the levels of residual hemolysis. However, by employing different C5 inhibitors simultaneously, residual hemolytic activity could be abolished. The importance of AP-produced C3b clusters for C5 activation in the presence of eculizumab was corroborated by the finding that residual hemolysis after forceful activation of the classical pathway could be reduced by blocking the AP. By providing insights into C5 activation and inhibition, our study delivers the rationale for the clinically observed phenomenon of residual terminal pathway activity under eculizumab treatment with important implications for anti-C5 therapy in general.

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

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.

A new blood group antigen is defined by anti-CD59, detected in a CD59-deficient patient

CD59 is a cell surface glycoprotein of approximately 20u2009kDa limiting the lytic activity of the terminal complement complex C5b‐9. Although CD59 is known as a red blood cell (RBC) antigen defined by monoclonal antibodies, it so far has not been identified as a blood group antigen, since the description of a human alloantibody was missing. In this study we show the presence of an anti‐CD59 in a patient affected by a homozygous CD59 deficiency.

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

The serum proteins factor H (FH), consisting of 20 complement control protein modules (CCPs), and its splice product FH-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 an 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 a paroxysmal nocturnal hemoglobinuria 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.

Complement activation by human red blood cell antibodies: hemolytic potential of antibodies and efficacy of complement inhibitors assessed by a sensitive flow cytometric assay: RBC ANTIBODIES AND COMPLEMENT

Therapeutic intervention strategies in complement‐mediated hemolytic diseases are still inappropriate, and lethal events cannot be reliably prevented. As an in vitro model of intravascular hemolysis, a sensitive flow cytometric assay was designed using red blood cells (RBCs) of patients with paroxysmal nocturnal hemoglobinuria (PNH) as target cells. Complement activation by human allo‐ and autoantibodies directed against RBC antigens and the effect of different complement inhibitors were studied.

The Monoclonal Anti-CD157 Antibody Clone SY11B5, Used for High Sensitivity Detection of PNH Clones on WBCs, Fails to Detect a Common Polymorphic Variant Encoded by BST-1 : ANTI-CD157 ANTIBODIES FOR PNH ANALYSIS

CD157, encoded by BST‐1, has been described as a useful flow cytometric marker for the analysis of paroxysmal nocturnal hemoglobinuria (PNH) as it is a glycosylphosphatidylinositol (GPI)‐linked molecule highly expressed on normal monocytes and neutrophils. We and others observed isolated CD157 signal dropouts during intended PNH analysis. We hypothesize that these negative populations occur due to an antibody failure. To investigate the reason for this finding, we compared two different anti‐CD157 antibody clones for PNH analysis.

Hämatologische Labordiagnostik im interdisziplinären hämatologischen Kompetenzzentrum (IHK) der hämatologisch onkologischen Klinik V der Universitätsklinik Innsbruck und des Zentrallabors der Universitätsklinik Innsbruck/Laboratory examination of hematologic diseases in the Interdiscipline Hematologic Competence Center (IHK) of Innsbruck of the Clinic for Hemato-Oncology V of the University Hospital Innsbruck and the Central Laboratory of the University Hospital Innsbruck

Zusammenfassung Die hämatologische Labordiagnostik hat in den letzten 30 Jahren große Fortschritte gemacht. Sie basiert heute im Wesentlichen auf vier Säulen: Zytomorphologie, Immunphänotypisierung, Zytogenetik und Molekulargenetik. Weiterhin unerlässlich steht am Anfang jeder Abklärung die Zytomorphologie meist gefolgt von einer Immunphänotypisierung. Beide Untersuchungen erfordern vom Laborteam hohe Expertise und oft Informationsaustausch mit den klinischen Zuweisern. Trotz hochspezialisierter Mitarbeiter ist es für kleine Speziallaboratorien eine große Herausforderung den Anforderungen an Qualitätssicherung und Wirtschaftlichkeit zu genügen. Eine Lösung kann die Zusammenarbeit zwischen der Labormedizin einerseits (meist in einem Zentrallabor angesiedelt) und den meist in der Hämatologie angesiedelten kleineren Speziallaboren sein, wie sie in Innsbruck seit 2008 modellhaft im interdisziplinären hämatologischen Kompetenzzentrum (IHK) praktiziert wird. Auf diese Weise wurde in Innsbruck eine interdisziplinäre Einrichtung geschaffen, welche die gesamte hämatologische Laborkompetenz der Klinik aus Zytomorphologie und Durchflusszytometrie zentral bündelt. Neben der Abklärung bekannter hämatologischer Erkrankungen bietet das IHK auch optimale Vorraussetzungen zur gezielten, zeitnahen Erstdiagnostik hämatologischer Erkrankungen (z.B. Zufallsbefunde) aber auch zum Screening von Blutproben eines großen Klinikums, die aufgrund von morphologischen oder numerischen Auffälligkeiten einer weiteren diagnostischen Abklärung bedürfen. Für die weitere Entwicklung des IHK ist eine zusätzliche Einbindung der zytogenetischen bzw. molekularbiologischen Diagnostik unerlässlich. Die interdisziplinäre Zusammenarbeit von Labormedizinern, Hämatologen, Zytogenetikern und Molekularbiologen fördert den Wissenstransfer und die Expertise aller beteiligten Mitarbeiter und steigert das Leistungspotential und Qualitätsniveau dieser Laborbereiche. Weitere Vorteile bieten sich im Bereich wichtiger Laborentwicklungsprozesse (z.B. Laborakkreditierung, Einführung neuer teurer Ausrüstungsgegenstände, wie etwa 10-Farben-Flowzytometer, moderner Labor-EDV oder Bildarchivierungssysteme), wobei durch interdiszplinäre Zusammenarbeit und gemeinsame Nutzung hohe Synergieeffekte zu erwarten sind. Die Etablierung interdisziplinärer Kompetenzzentren für hämatologische Diagnostik stellt daher einen Meilenstein in der modernen Labordiagnostik dar. Abstract The laboratory examination of hematologic diseases has made great progress in the last 30 years and is now based on a four-fold strategy: cytomorphology, flow cytometry, cytogenetics, and molecular genetics. Cytomorphology is still the crucial first step, and then it usually followed by flow cytometric immunophenotyping and of relevant cell population. Both investigations require a highly expert laboratory team and, often, an exchange of information among the clinicians. It is a challenge for small specialized laboratories to reach quality standards and economic efficiency nowadays, even with highly specialized personnel. One solution is the collaboration among institutes of laboratory medicine (usually a central laboratory), smaller laboratories specialized in hematology, and usually residents in departments of hematologic diseases, as is the practice in the Interdiscipline Hematologic Competence Center (IHK) of Innsbruck since 2008. Thus, in Innsbruck, this competence center concentrates the whole expertise in the hematology laboratory of the hospital based on cytomorphology and flow cytometry. Besides the clarification of already identified hematologic diseases, the IHK has optimal conditions not only for the specific and prompt first-time diagnosis of hematologic diseases (e.g., additional diagnostic findings) but also for the screening of blood samples, from tertiary care hospitals, that require further diagnostic workup for specific disease clarification based on numeric and morphologic abnormalities. For the further progression of the IHK, the integration of cytogenetic and molecular genetic diagnostics is essential. The interdisciplinary collaboration of specialists in laboratory medicine, hematology, cytogenetics and molecular genetics improves the knowledge tranfer and expertise of all members and enhances the efficiency and quality level of such a collaborating laboratory division. Further advantages can be achieved in important laboratory development processes (e.g., laboratory accreditation, implementation of expensive laboratory equipment, such as ten-color flow cytometers, introduction of modern laboratory information systems, or image-archiving systems), with a high potential of synergetic effects stemming from the collaboration. The establishment of interdisciplinary competence centers for hematologic diagnostics is therefore a milestone in modern laboratory diagnostics.

Laboratory examination of hematologic diseases at the interdisciplinary hematologic competence center (IHK) of Innsbruck of the clinic for hemato-oncology V of the university hospital Innsbruck and the central laboratory of the university hospital Innsbruck (ZIMCL)1)

Abstract The laboratory examination of hematologic diseases has made great progress in the last 30 years and is now based on a fourfold strategy: cytomorphology, flow cytometry, cytogenetics, and molecular genetics. Cytomorphology is still the crucial first step, and then it usually followed by flow cytometric immunophenotyping of relevant cell population. Both investigations require a highly expert laboratory team and, often, an exchange of information among the clinicians. It is a challenge for small specialized laboratories to reach quality standards and economic efficiency nowadays, even with highly specialized personnel. One solution is the collaboration among institutes of clinical pathology (usually a central laboratory), smaller laboratories specialized in hematology, and usually residents in departments of hematologic diseases, as is the practice in the Interdiscipline Hematologic Competence Center (IHK) of Innsbruck since 2008. Thus, in Innsbruck, this competence center concentrates the whole expertise in the hematology laboratory of the hospital based on cytomorphology and flow cytometry. Besides the clarification of already identified hematologic diseases, the IHK has optimal conditions not only for the specific and prompt first-time diagnosis of hematologic diseases (e.g., additional diagnostic findings) but also for the screening of blood samples, from tertiary care hospitals, that require further diagnostic workup for specific disease clarification based on numeric and morphologic abnormalities. For the further progression of the IHK, the integration of cytogenetic and molecular genetic diagnostics is essential. The interdisciplinary collaboration of clinical pathologists, hematology clinicians, and cytogenetic and molecular genetics experts improves the knowledge transfer and expertise of all members and enhances the efficiency and quality level of such a collaborating laboratory division. Further advantages can be achieved in important laboratory development processes (e.g., laboratory accreditation, implementation of expensive laboratory equipment, such as ten-color flow cytometers, introduction of modern laboratory information systems, or image-archiving systems), with a high potential of synergetic effects stemming from the collaboration. The establishment of interdisciplinary competence centers for hematologic diagnostics is therefore a milestone in modern laboratory diagnostics.