Paroxysmal Nocturnal Hemoglobinuria Patients Research Articles

Post Hoc Analysis of the Effect of Pegcetacoplan Treatment of Patients with Paroxysmal Nocturnal Hemoglobinuria and Baseline Hemoglobin Levels Greater Than 10 Grams per Deciliter

Post Hoc Analysis of the Effect of Pegcetacoplan Treatment of Patients with Paroxysmal Nocturnal Hemoglobinuria and Baseline Hemoglobin Levels Greater Than 10 Grams per Deciliter

Changes in Hemoglobin Measures Observed in PNH Patients Treated with Both C5 Inhibitors Ravulizumab and Eculizumab: Real-World Evidence from a US-Based EMR Network

Changes in Hemoglobin Measures Observed in PNH Patients Treated with Both C5 Inhibitors Ravulizumab and Eculizumab: Real-World Evidence from a US-Based EMR Network

Sars-Cov-2 Vaccination in Patients with Paroxysmal Nocturnal Hemoglobinuria: An Italian Multicenter Survey

SARS-CoV-2 infection and vaccination have raised concerns in paroxysmal nocturnal hemoglobinuria (PNH). In fact, PNH patients carry an increased infectious risk secondary to complement inhibition treatment or associated bone marrow failure (BMF), and may therefore benefit from preventive strategies such as vaccinations. On the contrary, vaccines can be numbered among inflammatory complement amplifiers (e.g., infections, traumas, surgery), potentially triggering a disease exacerbation. In PNH patients on complement inhibitors, this phenomenon has been defined pharmacodynamic breakthrough hemolysis (BTH). Based on isolated reports of BTH following SARS-CoV-2 vaccines, we conducted a survey among 5 Italian reference centers to evaluate complications and BTH occurrence in PNH patients who completed the SARS-CoV-2 vaccination schedule from January, 2 2021 until the time of writing. Adverse events, hematologic and hemolytic parameters were recorded within 7-10 days before and after each dose of vaccine. A total of 67 patients (females/males 43/24, median age 47.6 years, range 21-90.5) were eligible for the analysis. According to the International PNH Interest Group classification, 45 patients suffered from hemolytic PNH, 20 from PNH in the context of BMF syndromes (aplastic anemia or myelodysplastic syndrome), and 2 from subclinical PNH. Fifty-five subjects (82%) were on regular complement inhibition therapy, i.e., eculizumab (N=35), ravulizumab (N=13), subcutaneous anti-C5 (N=3), anti-factor B (N=2) and ravulizumab + anti-factor D combination (N=2). Vaccines (Comirnaty/Pfizer-BioNTech N=53, mRNA-1273/Moderna N=12, and ChAdOx1 nCov-19/AstraZeneca N=2) were complessively well-tolerated, with 3 non-hematologic adverse events after the first dose (2 fever and 1 exercise-induced tachycardia, grade 1 according to CTCAE v5.0) and 2 after the second one (fever, accompanied by vomit in one patient, grade 1). During the observation period, 3 BTH and 1 hemolytic exacerbation were recorded (5.9% of patients), as detailed in Table 1. The most severe episode occurred in a young woman (Patient 3) on subcutaneous ravulizumab who experienced a hemoglobin (Hb) drop >2 g/dL, marked clinical signs of intravascular hemolysis and lactate dehydrogenase (LDH) increase >1.5 x upper limit of normal (ULN) from baseline, which is considered a clinical BTH according to the criteria proposed by the Severe Aplastic Anemia Working Party of the European group for Bone Marrow Transplantation. The patient required hospitalization for additional treatment with recombinant erythropoietin and anti-thombotic/bacterial prophylaxis. The second more severe BTH was registered in a male patient (Patient 1) on oral anti-factor B who experienced a Hb drop >2 g/dL without an overt hemolytic flare, and required hospitalization for intravenous antibiotic therapy (concomitant urinary tract infection). The remaining two patients experienced a subclinical BTH (Patient 2) and a hemolytic flare (Patient 4, not on complement inhibition). On the whole, a median delta variation from usual values of Hb and LDH of -25% (range -26+3%) and +80% (+18+105%) were observed, respectively. Of note, 3 episodes occurred after the second dose of vaccine, generally within 24-48 hours. Anti-complement drugs were not modified/discontinued in any of the 3 patients on regular treatment. Patients not experiencing BTH (94.1%) showed stable hematologic parameters after the first dose (Hb/LDH median delta variations from baseline -1%/+1%, range -14+12%/-32+40%) and the second dose of vaccine (Hb/LDH median delta variations from baseline +1%/0%, range -18+47%/-76+41%). Of note, 4 patients with a previous SARS-CoV-2 infection completed the vaccination without any complication/PNH exacerbation.In conclusion, this survey shows that BTH/hemolytic flares following SARS-CoV-2 vaccines are observed in about 6% of PNH patients, may be clinically relevant but manageable, and should not discourage vaccination. BTH has been registered mostly few days after the second dose of vaccine, suggesting a “booster” effect favoring a higher inflammatory response. Watchful clinical and laboratory monitoring is advised, in order to promptly recognize severe hemolytic flares in both treated and naïve patients. [Display omitted] DisclosuresFattizzo: Novartis: Speakers Bureau; Kira: Speakers Bureau; Alexion: Speakers Bureau; Annexon: Consultancy; Momenta: Honoraria, Speakers Bureau; Apellis: Speakers Bureau; Amgen: Honoraria, Speakers Bureau. Bianchi: Agios pharmaceutics: Consultancy, Membership on an entity's Board of Directors or advisory committees. Sica: Jazz Pharma: Consultancy; Alexion: Consultancy. Barcellini: Novartis: Other: Invited speaker, Research Funding; Agios: Membership on an entity's Board of Directors or advisory committees; Alexion Pharmaceuticals, Inc: Membership on an entity's Board of Directors or advisory committees, Other: Invited speaker, Research Funding; Bioverativ: Membership on an entity's Board of Directors or advisory committees; Incyte: Membership on an entity's Board of Directors or advisory committees.

SUZ12 Participates in PNH Cloning Proliferation By Regulating Histone H3K27me3 Methylation Level

Paroxysmal nocturnal hemoglobinuria (PNH) is a disease of hematopoietic stem cell membrane defects due to acquired PIG-Amutation. Our previous study found some secondary gene mutations in PNH patients by WES. However, it is not clear exactly which mutations are associated with the disease. So, 97 target genes were selected as a target gene panel and tested in 23 PNH patients by DNA sequencing of specific target regions. We found that all PNH patients had other gene mutations except PIG-Amutations, including TTN, NCOR2, CPS1, MUC4, SUZ12, LFNG, CELSR2, JAK2, SETBP1 and KMT2D (Figure1A). Through harmful analysis, KEGG enrichment, GO enrichment analysis and protein interaction analysis, we screened out the secondary mutant gene SUZ12 that may be involved in the cloning proliferation of PNH. We detected the mRNA and protein expression levels of SUZ12 and H3K27me3 methylation in PNH patients and health volunteers, the results showed that the mRNA and protein expression levels of SUZ12 and H3K27me3 methylation in peripheral blood CD59 -cells of PNH patients were higher than those in CD59 + cells of PNH patients and healthy controls (Figure1B). The relative expression level of SUZ12 in peripheral blood CD59 -cells of PNH patients was correlated with (r=0.4162, p=0.0385), CD59 -erythrocyte ratio (r=0.4636, p=0.0196), CD59 -monocyte ratio (r=0.4052, p=0.0495), Flaer -monocyte ratio (r=0.6769, p=0.0004) and Flaer -granulocytic ratio (r=0.6146, p=0.0018), indicating that SUZ12 may be involved in abnormal PNH cloning and proliferation by regulating H3K27me3. To verify the role of SUZ12 in the proliferation of PNH cloning, we used CRISPR/Cas9 to knockdown PIG-A expression in THP-1 cells to construct A PNH cell model, the expression level of PIG-A protein in the cell model was significantly decreased, and the proportion of CD59 - cells accounted was stable at 95%. Then lentivirus transfection was used to knockdown the expression of SUZ12 in PNH cell model. The results showed when the SUZ12 expression was knockdown, the methylation level of histone H3K27me3 was decreased, the cell proliferation activity was decreased, apoptosis was increased, and the cell cycle was arrested at G0/G1 phase. The proportion of CD59 + cells increased gradually from 3 weeks after transfection, and significantly increased at 4 weeks after transfection, while no changes were observed in the empty virus group and control group (Figure1C). Four weeks after lentivirus transfection, the expression of PIG-A protein recovered in SUZ12 knockdown group compared with empty virus group and control group (Figure1D). In conclusion, SUZ12 mutation leads to the overexpression of SUZ12, which can affect cell proliferation, apoptosis and cell cycle by regulating the methylation level of histone H3K27me3, thereby promoting the proliferation of PNH abnormal cloning and participating in the pathogenesis of PNH. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Immunodynamics of Paroxysmal Nocturnal Hemoglobinuria

In the context of immune-mediated attack in aplastic anemia (AA), PIGA mutations produce a clonally-restricted escape phenotype in an attempt to restore hematopoiesis. Along with PIGA lesions, HLA class I/II mutations may represent an alternative way to render cytotoxic T cell recognition less effective. Microdeletions of 6p21, uniparental disomy [UPD] of 6p and mutations of HLA genes have been previously identified in AA patients. One could stipulate that paroxysmal nocturnal hemoglobinuria (PNH) may be an alternative mode of clonal evolution as opposed to HLA mutation acquisition or instead be a redundant pathway of immune escape co-existing with such lesions. Here, we took advantage of a large cohort (n=92) of patients with hemolytic PNH (granulocytes clone size >20% and LDH>x2.5 ULN) to elucidate the immunodynamics of PIGA/HLA clone(s) (Fig1A).We first used a deep targeted sequencing panel covering HLA classical loci and applied an in-house newly developed pipeline on 62 patients with PNH. Overall, 35% of cases had HLA alterations, which were found at a similar rate in patients studied at onset (33%) and during the course of the disease (32%). These alterations were classified as allelic losses in 15% of cases while majority of mutations were missense (57%) followed by 5‘ or 3‘ UTR (20%), nonsense (15%) and frameshifts (8%) with a median variant allelic frequency (VAF) of 5% (range, 2-95). Of note, the canonical exon 1 HLA B14:02 mutation (c.19C>T, p.R7X) 1,2was found in 2 patients who both had de novo PNH with a granulocyte clone size of 60% at disease onset (Fig1B).To clarify whether the PNH phenotype conveys an absolute resistance from immune attack, one should determine whether in these patients HLA mutations arise indeed within non-PNH cells. Consequently, we also performed HLA sequencing on 9 patients whose samples (n=18) were previously sorted by flow cytometry to enrich for CD59+ and CD59- fractions. HLA mutations were identified in both CD59+ and CD59- fractions at similar rates (44% each) and VAF (5% vs 3%, p=0.3) while no “canonical” exon 1 mutation was found.When looking at associations with disease-specific characteristics, we did not identify any significant differences in terms of burden of HLA events and disease ontogeny (primary vs secondary to AA cases) as well as baseline demographics characteristics (gender, age at presentation), dominant erythrocytes type (II vs III), and thrombotic events. No correlations with specific types of PIGA mutations nor their burden were found in HLA-mutated vs non-mutated groups as also confirmed by the absence of relationship with PNH granulocyte clone size (62% vs 72%, p=0.5). Of note, in HLA mutants the VAFs of HLA lesions were significantly lower than those of PIGA (5% vs 18%, p<0.001) underlying their possible less powerful role in driving immune escape within the context of AA overshooting reactions. To validate these results, we have designed a highly quantitative flow cytometric method for detection of HLA expression (A, B, C for class I; DQ, DR, DP for class II) on CD59+ vs CD59- fractions of CD34+ and CD15+ cells obtained from PNH patients. Preliminary results in 7 patients showed a general downregulation of HLA expression within PNH clones (delta HLA MFI of CD59+ vs CD59- fractions), consistent with our ability to detect HLA mutations also in PNH cells (Fig1C). This preferential down-modulation on PNH cells suggests a possible selection mechanism operating agonistically and in a non-mutually exclusive fashion with the PNH-intrinsic privilege. Moreover, these findings may also represent a byproduct of antigen processing machinery impairment due to proteasomal accumulation of proteins, which would have been GPI-linked and exported to the membrane.In sum, our study indicates that PNH phenotype conveyed by PIGA mutations operates independently from HLA mutations as means of immune escape. In addition, based on these results we stipulate that HLA down-modulation via various mechanisms is operative in the context of the PNH phenotype and the immune privilege of PIGA clones is not absolutely protective. Patients with PNH may have elements of immune suppression. In fact, their responses in the context of reduced-intensity transplant do eliminate hosts PNH clones following transplantation. 3 [Display omitted] DisclosuresPatel: Alexion: Consultancy, Other: educational talks, Speakers Bureau; Apellis: Consultancy, Other: educational talks, Speakers Bureau. Maciejewski: Bristol Myers Squibb/Celgene: Consultancy; Novartis: Consultancy; Alexion: Consultancy; Regeneron: Consultancy.

Categorized Hematologic Response to Pegcetacoplan and Correlations with Quality of Life in Patients with Paroxysmal Nocturnal Hemoglobinuria: Post Hoc Analysis of Data from Phase 1b, Phase 2a, and Phase 3 Trials

Background: Paroxysmal nocturnal hemoglobinuria (PNH) is a complement-mediated, hematologic disease characterized by hemolysis, anemia, and fatigue that impairs quality of life. Pegcetacoplan (PEG), a C3-inhibitor recently approved by the Food and Drug Administration for treatment of PNH, controls intravascular hemolysis (IVH) and prevents extravascular hemolysis (EVH). The PADDOCK and PALOMINO trials demonstrated PEG's improvement of key hematologic and clinical parameters, including hemoglobin (Hb) level and Functional Assessment of Chronic Illness Therapy (FACIT)-Fatigue score. The PEGASUS trial demonstrated that PEG was more effective than eculizumab (ECU) in improving these parameters in PNH patients with suboptimal response to prior ECU treatment.Aims: This post hoc analysis categorized hematologic response to PEG in 3 trials: PADDOCK (phase 1b, open-label, two-cohort [NCT02588833]), PALOMINO (phase 2a, open-label, single-cohort [NCT03593200]), and PEGASUS (phase 3, randomized, open-label, active-comparator controlled [NCT03500549]).Methods: PADDOCK and PALOMINO included adult complement inhibitor-naïve patients, while PEGASUS included adult patients with Hb <10.5 g/dL despite ≥3 months of ECU treatment. PADDOCK was a multiple ascending dose pilot study of 2 cohorts: cohort 1 received a suboptimal PEG dose (180 mg/day subcutaneously [SQ]) for 28 days; cohort 2 received a PEG dose of 270-360 mg/day SQ for up to one year. Patients could be enrolled in both cohorts. PALOMINO patients received a PEG dose of 270-360 mg/day SQ for up to one year. PEGASUS comprised a 4-week run-in period where patients received both ECU (continued dosing regimen) and PEG (1080 mg SQ twice-weekly), 16-week randomized controlled period (PEG or ECU monotherapy), and 32-week open-label period (PEG monotherapy) including a 4-week run-in period for the ECU-to-PEG group.Hematologic response category was assessed at Days 113/337 for patients in the combined PADDOCK/PALOMINO trials (N=24) via manual categorization by two independent, blinded observers. Categorization was assessed at Weeks 16/48 for PEGASUS patients (N=80) using SAS 9.4M5. Statistical significance for hematologic response categories within arms at Week 16 vs. 48, and between arms at Week 48, was evaluated using Wilcoxon-Mann-Whitney and Chi-square testing. FACIT-Fatigue scores for PEGASUS patients, summarized using descriptive statistics, were also correlated to hematologic response category.Response categories were defined per the following criteria (Risitano A, et al., Front Immunol, 2019;10:1157): complete- no transfusions required, stable Hb (normal range), no evidence of hemolysis (lactate dehydrogenase [LDH]≤1.5×upper limit of normal [ULN] U/L, absolute reticulocyte count [ARC]≤150,000/µL); major- no transfusion, normal Hb, but with evidence of hemolysis (LDH>1.5×ULN U/L and/or ARC>150,000/µL); good- no transfusions, but with chronic mild anemia or evidence of hemolysis; partial- chronic moderate anemia and/or occasional transfusions (<3 units/6 months); minor- regular transfusions required (3-6 units/6 months); no response- regular and frequent transfusions required (>6 units/6 months).PADDOCK/PALOMINO patients missing data at an assessment timepoint, or PEGASUS patients missing data within 6 weeks prior to an assessment timepoint, were not evaluated.Results: Most PADDOCK/PALOMINO patients achieved at least a good hematologic response at Days 113 and 337 (Table 1). At Week 48, most patients in both PEGASUS arms achieved a good/major/complete hematologic response (inter-arm p=0.4390), with a significant increase in the percent of ECU-to-PEG patients achieving at least a good response after switching to PEG at Week 16 (Week 16 vs. 48: intra-arm p<0.0001; Table 1). In PEGASUS, patients with better response categories had higher FACIT-Fatigue scores when compared to the rest of the cohort (Week 16: p<0.001, Week 48: p=0.028; Table 2).Conclusions: In post hoc analyses of PADDOCK, PALOMINO, and PEGASUS trial data, a substantial proportion of patients achieved and maintained good, major, or complete hematologic responses to PEG, suggesting that PEG can lead to sustained improvements in hematologic parameters. PEGASUS data also demonstrated correlation of improved hematologic response category with clinically meaningful improvements in quality of life, as measured by FACIT-Fatigue. [Display omitted] DisclosuresRisitano: Jazz: Other: Lecture fees, Speakers Bureau; Achillion: Membership on an entity's Board of Directors or advisory committees, Other: Lecture fees; Biocryst: Membership on an entity's Board of Directors or advisory committees; RA Pharma: Research Funding; Amyndas: Consultancy; Samsung: Membership on an entity's Board of Directors or advisory committees; Alexion: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Lecture fees, Research Funding, Speakers Bureau; Alnylam: Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Other: Lecture fees, Research Funding, Speakers Bureau; F. Hoffmann-La Roche Ltd.: Membership on an entity's Board of Directors or advisory committees; Pfizer: Other: Lecture fees, Speakers Bureau; Apellis Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Other: Lecture fees, Speakers Bureau. Wong: Roche: Consultancy, Honoraria, Research Funding, Speakers Bureau; Apellis Pharmaceuticals: Research Funding, Speakers Bureau; Alexion: Consultancy, Honoraria, Research Funding, Speakers Bureau. Al-Adhami: Apellis Pharmaceuticals: Current Employment. Chen: Apellis Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Peffault De Latour: Swedish Orphan Biovitrum AB: Consultancy, Honoraria; Apellis Pharmaceuticals: Consultancy, Honoraria; Alexion Pharmaceuticals: Consultancy, Honoraria, Research Funding; Amgen: Research Funding; Pfizer: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding.

Evaluation of the Long-Term Safety and Efficacy of Pegcetacoplan Treatment for Paroxysmal Nocturnal Hemoglobinuria Patients: An Extension Study

Background: Paroxysmal nocturnal hemoglobinuria (PNH) is a rare, potentially life-threatening disease characterized by complement mediated hemolysis and thrombosis. Current C5-inhibitors, such as eculizumab and ravulizumab, are able to partially treat these symptoms by controlling intravascular but not extravascular hemolysis, manifesting as persistent anemia in up to 72% of treated patients and transfusion dependence in up to 36% of patients.Pegcetacoplan (PEG) is a Food and Drug Administration (FDA)-approved C3-inhibitor that controls both intravascular and extravascular hemolysis (IVH and EVH). In the PEGASUS phase 3, multi- center, active-comparator controlled, randomized trial (NCT03500549), improvements in hemoglobin levels from baseline to Week 16 demonstrated the superiority of PEG over eculizumab; 85% of the patients receiving PEG achieved transfusion independence when compared with the 15% of patients on eculizumab. The extension study will evaluate the long-term safety and efficacy of PEG in patients with PNH.Acute hemolytic (AH) events occurred during the PEG clinical development program. As part of the long-term extension study, we aim to better understand how to manage acute hemolytic events through dose adjustments in patients treated with PEGAims: Determine the long-term safety and efficacy of PEG for PNH treatment, as well as evaluate a treatment regimen to address acute hemolytic (AH) events in these patients.Study Design: The PEG extension study (NCT03531255) is an active, multi-center, open-label, non-randomized trial with an estimated enrollment of up to160 adult PNH patients (≥18 years), who have completed prior PEG trials - phase 1b PADDOCK (NCT02588833) and PHAROAH (NCT02264639), phase 2a PALOMINO (NCT03593200), and the phase 3 PEGASUS (NCT03500549) and PRINCE (NCT04085601) trials. The main study will determine long-term safety and efficacy of PEG dosing on complement-mediated hemolytic activity, hemoglobin levels, and transfusion independence in PNH patients. For enrolled subjects experiencing AH episodes, a substudy to evaluate safety and efficacy of intensive SC or IV dosing of PEG acute treatments will be offered. Dose adjustments will be offered for AH subjects declining to enter the substudy, but continuing to participate in the main study.Inclusion criteria for the management of AH substudy specify that lactate dehydrogenase (LDH) levels in patients must be >2 times the upper limit of normal, accompanied by new or worsening clinical signs of hemolysis (hemoglobinuria, fatigue, decreased hemoglobin, etc.). Notable exclusion criteria include subjects who have withdrawn from a previous PEG trial, co-morbidities and infections that can enhance risk profiles for adverse events or confound study interpretation, and complement deficiency.The safety population consisting of patients that will receive at least one dose of the drug will be used for all efficacy analyses. Descriptive statistics will be used in documenting changes from baseline, along with qualitative descriptors (improved, maintained, or worsened).Intervention: Patients in the long-term extension study receive subcutaneous (SC) administration of 1080 mg PEG twice weekly, every 3 days, or 3 times weekly. For AH events substudy, patients with steady-state PEG dose of 1080 mg SC twice weekly will receive a loading dose of 1080 mg IV once, or 1080 mg SC every 24 hours for 3 doses, followed by increase in maintenance dose to 1080 mg SC every 3 days; for those receiving 1080 mg SC every 3 days, identical loading dose will be followed by subsequent maintenance dose of 1080 mg SC 3 times weekly.Primary and Secondary Outcome Measures: The study will monitor safety outcomes such as occurrence and severity of adverse events within a 2-year timeframe from baseline. Additional biomarkers include recurrent acute hemolysis, thromboembolism, laboratory and electrocardiogram (ECG) parameters. Efficacy endpoint measurements over a 48-week timeframe include mean change from baseline levels of hemoglobin, LDH, and indirect bilirubin, absolute reticulocyte counts, RBC transfusions, and functional assessment of fatigue scale scores.Results: Trial results are anticipated in 2022, upon study completion. DisclosuresHoffman: Apellis Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Machaidze: Apellis Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Yeh: Apellis Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Weitz: Alexion: Consultancy, Honoraria, Speakers Bureau; Apellis Pharmaceuticals: Consultancy, Honoraria; Biocryst: Consultancy, Honoraria; Novartis Corporation: Consultancy, Honoraria; Sanofi Genxyme: Consultancy, Honoraria. OffLabel Disclosure:Pegcetacoplan is a Food and Drug Administration approved C3-inhibitor that controls both intravascular and extravascular hemolysis. As part of the long-term extension study, we aim to better understand how to manage acute hemolytic events through dose adjustments in patients treated with pegcetacoplan.

12-Month Analysis of a Phase 2 Study of Iptacopan (LNP023) Monotherapy for Paroxysmal Nocturnal Hemoglobinuria

Background: Paroxysmal nocturnal hemoglobinuria (PNH) is a rare, acquired hematological disorder caused by somatic mutations in the phosphatidylinositol glycan A (PIGA) gene in hematopoietic stem cells, resulting in complement alternative pathway (AP)-mediated severe hemolysis, life-threatening thrombosis, and impaired bone marrow function. The current standard-of-care for PNH consists of anti-C5 blockade with either eculizumab or ravulizumab. While both monoclonal antibodies effectively control intravascular hemolysis (IVH), reduce thrombosis and improve long-term survival, a significant proportion of patients remains anemic and continues to require transfusions, largely due to persistent extravascular hemolysis (EVH). Conversely, pegcetacoplan, a recently FDA-approved anti-C3 inhibitor, prevents both IVH and EVH and showed superiority to eculizumab in improving hemoglobin (Hb) levels in PNH. Nevertheless, the need for effective oral treatment options in PNH remains unmet. Iptacopan (LNP023) is a new, oral, selective and potent first-in-class inhibitor of factor B, a key component of the AP. Recent phase 2 data showed that iptacopan effectively controls both IVH and EVH and leads to rapid, transfusion-free improvements in Hb levels in the majority of PNH patients.Methods: CLNP023X2204 (NCT03896152) is a multicenter, randomized, open-label phase 2 study in adult PNH patients with active hemolysis and no complement inhibitor treatment within 3 months prior to study entry. Patients are randomized to receive twice daily (BID) iptacopan in one of two dose-sequences, either 25 mg for 4 weeks followed by 100 mg for up to 2 years (Cohort 1) or 50 mg followed by 200 mg (Cohort 2). The key objectives are to assess the effect of iptacopan on markers of IVH/EVH (incl. lactate dehydrogenase (LDH)) and on Hb levels, as well as safety. 12-month interim results are summarized below.Results: The study has completed enrolment. A total of 13 patients (mean age 38.2 years, 7 female) were randomized to either Cohort 1 (n=7) or 2 (n=6). Mean (SD) lab values at baseline were LDH 2097.8 (911.2) U/L, reticulocytes 203.3 (82.9) x10E9/L, bilirubin 32.4 (10.1) µmol/L, and Hb 85.8 (13.2) g/L, and most patients required red blood cell transfusions in the prior 12 months (median 3.0, range 0-19). At the time of the interim analysis, 11 patients had been treated with iptacopan for at least 52 weeks, with a maximum treatment duration of 81 weeks; 2 patients discontinued treatment early, one after 2 days due to a non-serious adverse event of headache (hence not evaluable for the primary endpoint), the other after 13 weeks due to physician decision. Amongst the 12 evaluable patients, all of whom were anti-C5 naive, all reached the primary endpoint of lowering LDH by at least 60% within the first 12 weeks. The LDH response was rapid and durable, with all patients treated with ≥50 mg BID reaching this threshold after only one week of treatment and all ongoing patients except one maintaining the threshold up until the data cutoff, i.e., for at least 52 weeks. Equivalent improvements were also observed for other markers of IVH and EVH. Similarly, Hb levels improved significantly and durably in most patients, and all except one of the ongoing patients have remained transfusion-free since the start of iptacopan treatment. Moreover, no thromboembolic events occurred during the study, and the FACIT fatigue score improved significantly in most patients. Iptacopan monotherapy was safe, with no severe or serious adverse reported up to the data cutoff.Conclusion: Iptacopan is a new, well tolerated oral complement AP inhibitor that blocks both IVH and EVH in adult PNH patients with hemolytic PNH. 12-month results from this non-pivotal study demonstrate that iptacopan monotherapy leads to rapid and durable improvements in various hemolytic markers and meaningful and sustained clinical benefit as seen in improvements in Hb levels, transfusion requirement and FACIT fatigue score. These results suggest that proximal inhibition of the complement cascade parallels and further improves the hematological benefit seen with anti-C5 therapies, paving the way for the phase 3 evaluation of iptacopan as potentially new oral first-line therapy for patients with PNH. [Display omitted] DisclosuresWong: Astellas Pharma, INc.: Research Funding. Li: Novartis: Current Employment, Current equity holder in publicly-traded company. Rozenberg: Novartis: Current Employment, Current equity holder in publicly-traded company. Nidamarthy: Novartis: Current Employment, Current equity holder in publicly-traded company. Chawla: Novartis: Current Employment, Current equity holder in publicly-traded company. Junge: Novartis: Current Employment, Current equity holder in publicly-traded company. OffLabel Disclosure:Iptacopan (LNP023) is a new, oral, selective and potent first-in-class inhibitor of factor B, a key component of the complement alternative pathway. It is currently being investigated in paroxysmal nocturnal hemoglobinuria (PNH) as well as in several renal indications.

COVID19 infection in a patient undergoing treatment for Paroxysmal Nocturnal Hemoglobinuria (PNH) with Ravulizumab

BackgroundIn the recent COVID19 pandemic, patients with hematological disorders were considered at high risk for severe disease. Limited data is available regarding the course of COVID19 infection in this subgroup.Case PresentationWe describe a case of a 32-year-old man with paroxysmal nocturnal hemoglobinuria (PNH) undergoing treatment with ravulizumab (Ultomiris) who presented with COVID19 infection. He experienced only mild symptoms and had a rapid recovery from COVID19 infection.ConclusionThis case may demonstrate the beneficial effects of ravulizumab on complement mediated inflammatory damage linked with COVID19 infection especially in PNH patients.

Allogeneic Hematopoietic Stem Cell Transplantation for Paroxysmal Nocturnal Hemoglobinuria

To analyze the clinical efficacy and safety of allogeneic hematopoietic stem cell transplantation (allo-HSCT) for paroxysmal nocturnal hemoglobinuria (PNH), and preliminarily explore the role of an improved post-transplantation cyclophosphamide (PTCy) based conditioning regimen in PNH patients receiving transplantation. Clinical related data of PNH sufferers receiving allo-HSCT in Department of Hematology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology were collected, and hematopoietic reconstitution, chimerism, PNH cloning, graft-versus-host disease (GVHD), infection, and survival were analyzed. Totally five PNH patients receiving allo-HSCT were enrolled, including 1 case with classic PNH, 3 cases with aplastic anemia-PNH syndrome, 1 case with myelodysplastic syndrome, three of them (case 1-3) received the improved PTCy based conditioning regimen before HSCT. All sufferers engrafted successfully within 28 days, the median time of neutrophil and platelet engraftment was 11 days and 12 days, respectively, no patient occurred acute or chronic GVHD, after a median follow-up of 16 months, all recipients survived and completely eliminated PNH cloning. Allo-HSCT can completely clear PNH cloning and restore hematopoietic function with controllable complications, and the improved PTCy based conditioning regimen is proved to be effective in PNH transplantation.

MDS-134: Efficacy and Safety at 48 Weeks of Pegcetacoplan in Adult Paroxysmal Nocturnal Hemoglobinuria Patients with Suboptimal Response to Prior Eculizumab Treatment

Context: Pegcetacoplan (PEG) is a C3 complement inhibitor recently approved by the FDA for the treatment of paroxysmal nocturnal hemoglobinuria (PNH). PEG was superior to eculizumab (ECU; C5-inhibitor) in improving hemoglobin levels at week 16 during the phase 3 PEGASUS trial (NCT03500549) in patients with suboptimal response to prior ECU treatment. Objective: We report on efficacy and safety of PEG and results from a post hoc time-aligned analysis based on start of PEG dosing. Design: Eighty PNH patients (≥18 years, hemoglobin levels Results: PEG-to-PEG patients achieved sustained improvements in hemoglobin levels at week 16 through the OLP (week 48 mean hemoglobin level: 11.3 g/dL; CFB: 2.5 g/dL). ECU-to-PEG patients displayed improved hemoglobin levels during the OLP (week 48 mean hemoglobin level: 11.6 g/dL; CFB: 2.9 g/dL). Timepoint alignment demonstrated no significant difference (p=0.64) between improvements in hemoglobin levels at 28 and 48 weeks among PEG-to-PEG and ECU-to-PEG groups, respectively. Thirty percent of patients reported a serious AE, 6% possibly PEG-related. Common AEs for PEG-treated patients were injection site reactions (36%), hemolysis (24%), and diarrhea (21%). Twelve patients (15%) discontinued PEG due to AEs: 3 in RCP, 8 in OLP, 1 during follow-up, including one death due to COVID-19, unrelated to PEG. Conclusions: PEG-treated patients experienced sustained improvements in hemoglobin levels at week 48, and the safety profile of PEG was consistent with previously reported data. The treatment effect of PEG on hemoglobin levels over time was similar between PEG-to-PEG and ECU-to-PEG groups. Thus, PEG represents a new effective therapeutic option for PNH patients.

MDS-138: Management of Acute Hemolytic Events in Paroxysmal Nocturnal Hemoglobinuria Patients Taking Pegcetacoplan

Context: Acute hemolytic events occurred in completed paroxysmal nocturnal hemoglobinuria (PNH) studies during the pegcetacoplan clinical development program. As part of the long-term extension study, we aim to better understand how to manage acute hemolytic events through dose adjustments in patients treated with the C3 inhibitor pegcetacoplan, recently approved by the FDA. Objective: Establish the long-term safety and efficacy of pegcetacoplan dose adjustments in patients with PNH. Design: This is a multicenter, open-label, nonrandomized extension study (NCT03531255) in PNH patients treated with pegcetacoplan who have previously completed a pegcetacoplan study. Study participants who experience an acute hemolytic event will be offered the opportunity to receive either intensive subcutaneous (SC) or intravenous (IV) pegcetacoplan as an acute treatment. Patients must have lactate dehydrogenase (LDH) >2 times the upper limit of normal and the presence of at least one new or worsening sign or symptom of hemolysis (e.g., decrease in hemoglobin, hemoglobinuria, fatigue), warranting acute intervention in the opinion of the investigator. Interventions: In patients with a steady-state pegcetacoplan dose of 1080 mg SC twice weekly, a loading dose of 1080 mg IV once or 1080 mg SC every 24 hours for 3 doses, followed by an increase in the maintenance dose to 1080 mg SC every 3 days would be used. In patients at steady-state on a pegcetacoplan dose of 1080 mg SC every 3 days, a loading dose of 1080 mg IV once or 1080 mg SC every 24 hours for 3 doses, followed by an increase in the maintenance dose to 1080 mg SC 3 times weekly would be used. Main Outcome Measures: Main outcome measures include incidence and severity of treatment-emergent adverse events, thromboembolic events, hemoglobin level, LDH level, and red blood cell transfusions. Results: Results will be presented upon study completion, estimated in 2022.

Poster: MDS-138: Management of Acute Hemolytic Events in Paroxysmal Nocturnal Hemoglobinuria Patients Taking Pegcetacoplan

Poster: MDS-138: Management of Acute Hemolytic Events in Paroxysmal Nocturnal Hemoglobinuria Patients Taking Pegcetacoplan

CT-121: Phase 3 Study of the Efficacy and Safety of Iptacopan (LNP023), an Oral Factor B Inhibitor, in Adult Patients with Paroxysmal Nocturnal Hemoglobinuria (PNH) Naïve to Complement Inhibitor Therapy

Context Standard of care in PNH is based on complement C5 inhibition with anti-C5 monoclonal antibodies (MAbs), which are generally effective at treating intravascular hemolysis (IVH), but unmet needs remain. Hematological responses to anti-C5 antibody treatment can be heterogeneous, and a substantial number of patients experience residual anemia associated with breakthrough hemolysis (BTH). Iptacopan, a first-in-class, oral, small-molecule, selective, and reversible complement factor B inhibitor, inhibits both IVH and extravascular hemolysis. In an ongoing Ph2 study (NCT03439839), iptacopan has demonstrated improvements in hematological response and biomarkers of disease activity in PNH adult patients who had active hemolysis, despite eculizumab treatment. Objective APPOINT-PNH study (NCT04820530) will assess iptacopan efficacy and safety in PNH adult patients naive to complement inhibitors, including anti-C5 MAbs. Design The study comprises ≤8-wk screening; 24-wk, single-arm, open-label core treatment; and 24-wk, open-label treatment extension. On completion of week 24, patients who benefit from iptacopan treatment can enter extension. Eligible adult patients (N≈40) have confirmed PNH diagnosis (RBCs and WBCs [granulocyte/monocyte] clone size ≥10%); anemia (mean Hb 1.5× ULN). Key exclusion criteria: prior treatment with complement inhibitor; known or suspected hereditary complement deficiency; history of HSCT; laboratory evidence of bone marrow failure; active, systemic infection ≤14 days preceding study treatment; and history of recurrent, invasive infections caused by encapsulated microorganisms. Primary objective will assess hematologic response, defined as achieving sustained increase in Hb ≥2g/dL (in absence of RBC transfusions). Primary endpoint will evaluate proportion of patients achieving an increase from baseline in Hb ≥2g/dL between D126–D168 (in absence of RBC transfusions between D14–D168). Secondary endpoints include proportion of patients reaching Hb ≥12g/dL; absence of RBC transfusions; changes from baseline in Hb; changes in FACIT-Fatigue scores; changes from baseline in reticulocytes; % change from baseline in LDH; occurrence of BTH; and occurrence of major adverse vascular events (including thrombosis). Additional analyses include safety assessments; evolution of PNH clone size and C3d deposition on RBCs; and other PROs. Conclusions Ph3 studies to assess iptacopan efficacy in a wider population of PNH patients, including those naive to anti-C5 MAbs treatment, are needed. Study sponsored by Novartis Pharmaceuticals Corporation.

Poster: MDS-134: Efficacy and Safety at 48 Weeks of Pegcetacoplan in Adult Paroxysmal Nocturnal Hemoglobinuria Patients with Suboptimal Response to Prior Eculizumab Treatment

Poster: MDS-134: Efficacy and Safety at 48 Weeks of Pegcetacoplan in Adult Paroxysmal Nocturnal Hemoglobinuria Patients with Suboptimal Response to Prior Eculizumab Treatment

The Clinical Characteristics and Prognosis of Patients with Acute Myeloid Leukemia Combine Paroxysmal Nocturnal Hemoglobinuria

To investigate the clinical characteristics and prognosis of patients with acute myeloid leukemia(AML) combined with paroxysmal nocturnal hemoglobinuria(PNH). The clinical data of 13 AML combined with PNH patients treated in our hospital from January 2017 to May 2019 were collected and retrospective analyzed. The complete remission(CR) rate for induction chemotherapy was analyzed. The level of PNH+ cell before and after chemotherapy were tested by Paired t test. Kaplan-Meier method and multi-factorial Cox regression model were used to analyze the influencing factors of prognosis. Among the 13 patients, 11 (84.6%) cases were CR after first induction chemotherapy. The median overall survival(OS) time was 17 months(0-30 months), the median progression-free survival(PFS) time was 16 months(2-26 months). There were no significant difference in the number of PNH+ cell before and after chemotherapy (P＞0.05). Multivariate Cox regression analysis showed that age，sex，the level of hemoglobin, platelet were not related to the OS of the patients(P＞0.05), the level of WBC, LDH and risk stratification at first diagnosed were related to the OS of the patients（P＜0.05）. Kaplan-Meier survival analysis showed that the OS rate of AML combined with PNH patients with leukocyte lower than 10×109/L at first diagnosed was better than that of the patients with leukocyte higher than 10×109/L (P=0.0261). The OS rate of patients with low or standard risk was better than the patients with high risk group(P=0.0010). The patients of AML combined with PNH have higher CR rate after the first induction chemotherapy. The level of WBC and LDH at first diagnosed are the factors that affecting the OS of the patients. The OS of patients with WBC lower than 10×109/L, at first diagnosed low and medium risk are better than the other patients.

Centennial development of paroxysmal nocturnal hemoglobinuria research in Peking Union Medical College Hospital

<p indent=0mm>Paroxysmal nocturnal hemoglobinuria (PNH) is a rare acquired clonal hematopoietic stem cell (HSC) disease. A somatic phosphatidylinositol glycan-A (PIGA) gene mutation in HSCs translates into abnormal biosynthesis of glycosyl phosphatidylinositol (GPI) anchors on the cell surface. The absence of GPI-anchored complements regulators leads to complement-driven blood cell lysis. Hemolysis and chronic anemia are the most common clinical manifestations, while bone marrow failure is a rare but life-threatening complication. Thrombosis is the leading cause of mortality. Transfusion and supportive care have been introduced as symptomatic treatments since the early decades of the last century. Eculizumab, a monoclonal antibody that blocks terminal complement at C5, is a breakthrough medicine that changed the standard treatment methods of PNH. However, in cases where eculizumab is not accessible, allogeneic HSC transplantation could be an alternative strategy for refractory and aplastic patients. The Peking Union Medical College Hospital (PUMCH) is approaching its centennial anniversary in 2021. The first case of PNH in China was reported by physicians at PUMCH in the 1950s. During the past century, generations of hematologists established the largest cohort of PNH patients in China while continuously developing novel laboratory tests for this disease. They collaborated with investigators of basic science on the pathogenetic mechanisms of bone marrow failure and thrombosis. Although great historical changes have impacted academic progress, the spirit of perseverance, commitment, and innovation in older generations is passed on to the next generations. In this paper, we summarize the clinical evolution of PNH treatment along with basic science at PUMCH, which in turn facilitates great progress in PNH in the era of the 21st century in China.

Complement Blockade With a C1 Esterase Inhibitor In Paroxysmal Nocturnal Hemoglobinuria

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare, clonal, hematopoietic stem cell disorder that manifests with a complement-mediated hemolytic anemia, bone marrow failure and a propensity for thrombosis. PNH confers deficiency of GPI anchored proteins that protect erythrocytes at different stages of the complement cascade. CD55 regulates the formation and stability of the C3 and C5 convertases, whereas CD59 blocks the formation of the membrane attack complex (MAC). These patients experience both intra- and extravascular hemolysis due to complement activation. Chronic treatment with eculizumab results in sustained control of intravascular hemolysis leading to transfusion independence for many. Eculizumab compensates for the CD59 deficiency on PNH erythrocytes; however, it does not compensate for the CD55 deficiency. PNH patients receiving eculizumab accumulate C3 on their erythrocytes; this leads to opsonization and extravascular hemolysis. There remains an unmet clinical need for a complement inhibitor with activity early in the complement cascade, which would not only reduce C3 accumulation on PNH erythrocytes, but also inhibit C3 activation by the alternative pathway (APC).C1 esterase inhibitor (C1 INH) is an endogenous human plasma protein that has broad inhibitory activity in the complement pathway. C1 INH inhibits the classical pathway of the complement system by binding C1r and C1s and inhibits the mannose-binding lectin-associated serine proteases in the lectin pathway. Thus, C1 INH is an ideal therapeutic for diseases of complement pathways. Nanofiltered plasma derived C1 INH (Cinryze®; ViroPharma) is FDA approved for routine prophylaxis against angioedema attacks in patients with hereditary angioedema (HAE), a disease characterized by constitutional deficiency or dysfunction of endogenous C1 INH. We hypothesized that this plasma-derived C1 INH would inhibit C3 deposition and the APC on PNH erythrocytes in patients treated with eculizumab.Peripheral blood of 3 healthy donors and 5 PNH patients (all with PNH type III erythrocyte proportion >5%) treated with eculizumab were obtained with written informed consent through a protocol approved by the local institutional review board. Clinical parameters for persistent extravascular hemolysis were noted at the time of patient sampling. Commercial vials of C1 INH were used for C1 inhibition assays ex vivo. Hemolysis experiments with acidified serum were performed in erythrocytes from PNH patients and healthy donors. We were able to show in the PNH patients that C1 inhibition is concentration dependent with increasing inhibition by serial dilutions of C1 INH up to 12 U/mL. The amount of hemolysis correlates with the percentage of CD55 deficient erythrocytes, but is attenuated by C1 INH in all patients. Flow cytometry was used to analyze deposition of C3 activation fragments on intact and lysed PNH erythrocytes. At time 0, small amounts of C3 deposition were observed on the PNH erythrocytes, but not the normal erythrocytes. The amount of C3 deposition was increased after incubation in acidified serum; however, co-incubation of C1 INH in the acidified serum markedly attenuated the amount of C3 deposition on the CD55 deficient erythrocytes. This demonstrated that C1 INH blocks APC-medicated deposition of C3 on the PNH erythrocytes in PNH patients.In this study, we show that the commercially available plasma derived C1-INH (Cinryze®) prevents lysis induced by the APC in PNH erythrocytes. The results presented here show that plasma-derived C1 INH can inhibit both APC- mediated hemolysis and C3 deposition. Importantly, C1-INH was able to block the accumulation of C3 degradation products on CD55 deficient erythrocytes from PNH patients who were being treated with eculizumab. This suggests a role for inhibition of earlier phases of the complement cascade than currently inhibited by eculizumab. A clinical trial to explore this hypothesis in vivo with patients who are suboptimal responders to eculizumab could be considered. Disclosures:Off Label Use: A nanofiltered plasma derived C1 INH (Cinryze®; ViroPharma) is FDA approved for routine prophylaxis against angioedema attacks in adolescent and adult patients with hereditary angioedema. Uknis:ViroPharma, Incorporated: Employment. Saye:ViroPharma, Incorporated: Employment. Brodsky:Alnylam Pharmaceuticals : Consultancy; Prolong Pharmaceuticals : Consultancy; ViroPharma, Incorporated: Research Funding; Alexion Pharmaceuticals : Membership on an entity's Board of Directors or advisory committees.

Whole-Exome Sequencing of Paroxysmal Nocturnal Hemoglobinuria

OBJECTIVE: In order to elucidate the possible mechanism on the proliferation of paroxysmal nocturnal hemoglobinuria (PNH) clone, and investigate the significance of gene mutation in PNH patients with thrombosis and provide a theoretical basis for related molecules markers and potential therapeutic targets in PNH, we performed the whole genome exon sequencing in PNH patients.METHODS: The peripheral blood and nail specimens of 7 PNH and 6 AA-PNH patients were obtained and we have used V2's NimbleGen capture probe to capturethe whole exon group (WES), and performed whole genome sequencing of exon in patients, meanwhile, processing and analysis of sequencing data were obtained and compared with the 1000 genome project database, to obtain high frequency variation genes and further gene enrichment pathway analysis in these patients. Using a combination of KEGG pathway analysis and GO pathway enrichment analysis methods to analyze the experimental data.RESULTS: We detected somatic PIGA mutations in the PNH fractions of 8 of 13 cases analyzed by WES. And we identified a total of 4 shear site, 4 non-synonymous, 4 truncation, and 6 frame-shift mutations in cases analyzed by WES. All mutations were grade one. We performed KEGG pathway enrichment analysis and GO gene annotation of 5717 mutated genes screened, the union as a target gene set, there were 25 target genes, SUZ12, CUX1, MLL2, MUC4, CTBP1, AXIN2, MLL, SDHA, CARD11, MLH1, RB1, SMO and NKX2-1 mutations were recurrent in PNH patients; FGFR3, ERCC5, MPL, FLT3, TSC2, POLE, TET2, MLL3, MSH6, FAT1, CIC and ZFHX3 mutations were recurrent in AA-PNH patients. Among them, SUZ12, FLT3, CUX1, MUC4, TET2, and RB1 gene are involved in the regulation of cell proliferation and differentiation, promote the growth, metastasis and invasion of tumor cells. In addition, the pathways enriched by mutant genes included pathways in cancer, central carbon metabolism in cancer signaling pathway and cell cycle signal pathways. GO analysis showed that the coding protein CDP has the functions of cell proliferation regulation, metabolism regulation, gene expression regulation, cell invasiveness, cell motility, cell apoptosis, extracellular matrix compositions, and so on. Furthermore, we analyzed the gene mutations in patients with thrombosis, which includes several genes with the higher deleterious mutation in protein structure, such as BMPR2, ZFPM2, F5, F8, F12, F13A1, MCL1, MMRN1, NBEAL2, NOS1and PC. The major functions of mutation sites were non synonymous single nucleotide variants and synonymous single nucleotide variants. The pathways enriched by mutant genes included the Notch signaling pathway, Ribosome biogenesis in eukaryotes signaling pathway, Wnt signaling pathway and Arachidonic acid metabolism signaling pathways, which may be related to the pathogenesis of PNH associated thrombosis.CONCLUSION: We identified additional somatic mutations that resulted in a complex hierarchical clonal architecture, similar to that observed in myeloid neoplasms and solid tumors. In addition to PIG-A mutations, mutations were found in genes known to be involved in myeloid neoplasm pathogenesis and solid tumors, including SUZ12, FLT3, CUX1, MUC4, TET2, and RB1. Our data indicate that in addition to PIG-A mutations, accessory genetic events are frequent in PNH, suggesting a stepwise clonal evolution derived from a singular stem cell clone. Meanwhile, several genes with higher deleterious mutation in PNH patients with thrombosis were found: F5, F8, F12, F13A1, THBD, ITGA2 and BMPR2, which enriched the Notch signaling pathway, Ribosome biogenesis in eukaryotes signaling pathway, Wnt signaling pathway and Arachidonic acid metabolism signaling pathways. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

In Vitro Combination Studies of ACH-4471 with Eculizumab to Assess a Potential “Switch” Treatment Approach for Paroxysmal Nocturnal Hemoglobinuria

Introduction: ACH-4471 is an oral complement factor D (fD) inhibitor that has demonstrated clinical efficacy in an ongoing phase 2 clinical study for naïve patients suffering from paroxysmal nocturnal hemoglobinuria (PNH). PNH is a rare clonal blood disorder in which erythrocytes lacking the GPI-anchored complement regulators CD55 and CD59 are susceptible to destruction by the terminal complement complex (also known as membrane attack complex, MAC) following the normal low-level activation of the complement alternative pathway (AP) in the fluid phase. The therapeutic agent eculizumab, a monoclonal antibody directed against the terminal complement component C5, blocks MAC assembly on erythrocyte membranes and so prevents most intravascular hemolytic destruction. Eculizumab treatment however leads to increased deposition of complement C3 fragments on PNH membranes, which can result in extravascular phagocytic elimination of opsonized erythrocytes (A. M. Risitano et al, Blood, 2009, 113:4094) and incomplete inhibition of intravascular hemolysis (M. J. Harder et al, Blood, 2017, 129:970), and consequently to the continued anemia and transfusion dependence observed in a significant subset of patients. As an AP inhibitor, in contrast to eculizumab, ACH-4471 prevents both MAC assembly and C3 fragment deposition on PNH cell surfaces and therefore is expected to address the medical needs of these suboptimal responders to eculizumab. To assess this notion directly and also to support a future switch treatment approach, a further clinical study has been planned to evaluate ACH-4471 co-administration in PNH patients who respond suboptimally to eculizumab. In the present study, we conducted in vitro combination experiments to explore the potential pharmacological consequences when ACH-4471 is added on to eculizumab or another anti-C5 monoclonal antibody, using functional AP assays that include the hemolysis of erythrocytes from a PNH patient.Methods: Concentration series of ACH-4471 were assessed in pairwise combinations with eculizumab and a second monoclonal antibody inhibitor of complement C5. Functional assays for human serum AP activity included a single-plate ELISA-based functional assay (Wieslab) and the hemolysis of red blood cells from a PNH patient under AP-restricted conditions but at neutral pH. Analyses of interactions were performed using a three-dimensional surface graphing method and, where applicable, a median-effect plot method.Results: We assessed pairwise interactions using two assays for AP function. In the AP Wieslab assay, interactions between ACH-4471 and an anti-C5 monoclonal antibody ranged from additive to moderately synergistic. Next, to better approximate the physiological setting, we assessed inhibitor interactions against the AP-mediated hemolysis of red blood cells obtained from a PNH patient. ACH-4471 in this assay showed strongly synergistic interactions when tested with eculizumab. In light of a recent report on incomplete inhibition by eculizumab following strong complement activation, we propose a possible mechanism for synergy between ACH-4471 and eculizumab wherein ACH-4471 reduces the density of deposited C3b clusters, to which C5 binding is thereby more readily inhibited by eculizumab.Conclusions: ACH-4471 showed strong synergy with eculizumab. These results suggest the potential clinical utility of ACH-4471 as a PNH therapeutic agent that may be used in suboptimal eculizumab responders in support of a “switch” treatment strategy. DisclosuresPatel:Achillion: Employment. Thanassi:Achillion: Employment. Yang:Achillion: Employment. Brodsky:Alexion Pharmaceuticals, Inc.: Membership on an entity's Board of Directors or advisory committees, Other: Grant Funding. Huang:Achillion: Employment. Podos:Achillion: Employment.