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. Figure 1 Figure 1. Disclosures Wong: 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.

Atypical Hemolytic Uremic Syndrome after ChAdOx1 nCoV-19 Vaccination in a Patient with Homozygous CFHR3/CFHR1 Gene Deletion

Hemolytic uremic syndrome (HUS) is a thrombotic microangiopathy (TMA) affecting the kidneys. Compared with typical HUS due to an infection from shiga toxin-producing <i>Escherichia coli</i>, atypical HUS involves a genetic or acquired dysregulation of the complement alternative pathway. In the presence of a mutation in a complement gene, a second trigger is often necessary for the development of the disease. We report a case of a 54-year-old female, with a past medical history of pulmonary tuberculosis, who was admitted to the emergency service with general malaise and reduction in urine output, 5 days after vaccination with ChAdOx1 nCoV-19. Laboratory results revealed microangiopathic hemolytic anemia, thrombocytopenia, and acute kidney injury. Given the clinical picture of TMA, plasma exchange (PEX) was immediately started, along with hemodialysis. Complementary laboratory workup for TMA excluded thrombotic thrombocytopenic purpura and secondary causes. Complement study revealed normal levels of factors H, B, and I, normal activity of the alternate pathway, and absence of anti-factor H antibodies. Genetic study of complement did not show pathogenic variants in the 12 genes analyzed, but revealed a deletion in gene CFHR3/CFHR1 in homozygosity. Our patient completed 10 sessions of PEX, followed by eculizumab, with both clinical and laboratorial improvement. Actually, given the short time lapse between vaccination with ChAdOx1 nCoV-19 and the clinical manifestations, we believe that vaccine was the trigger for the presentation of aHUS in this particular case.

Properdin Deficiency Impairs Phagocytosis and Enhances Injury at Kidney Repair Phase Post Ischemia–Reperfusion

Properdin, a positive regulator of complement alternative pathway, participates in renal ischemia–reperfusion (IR) injury and also acts as a pattern-recognition molecule affecting apoptotic T-cell clearance. However, the role of properdin in tubular epithelial cells (TECs) at the repair phase post IR injury is not well defined. This study revealed that properdin knockout (PKO) mice exhibited greater injury in renal function and histology than wild-type (WT) mice post 72-h IR, with more apoptotic cells and macrophages in tubular lumina, increased active caspase-3 and HMGB1, but better histological structure at 24 h. Raised erythropoietin receptor by IR was furthered by PKO and positively correlated with injury and repair markers. Properdin in WT kidneys was also upregulated by IR, while H2O2-increased properdin in TECs was reduced by its small-interfering RNA (siRNA), with raised HMGB1 and apoptosis. Moreover, the phagocytic ability of WT TECs, analyzed by pHrodo Escherichia coli bioparticles, was promoted by H2O2 but inhibited by PKO. These results were confirmed by counting phagocytosed H2O2-induced apoptotic TECs by in situ end labeling fragmented DNAs but not affected by additional serum with/without properdin. Taken together, PKO results in impaired phagocytosis at the repair phase post renal IR injury. Properdin locally produced by TECs plays crucial roles in optimizing damaged cells and regulating phagocytic ability of TECs to effectively clear apoptotic cells and reduce inflammation.

Genome-wide association studies for production, respiratory disease, and immune-related traits in Landrace pigs

Identification of a quantitative trait locus (QTL) related to a chronic respiratory disease such as Mycoplasmal pneumonia of swine (MPS) and immune-related traits is important for the genetic improvement of disease resistance in pigs. The objective of this study was to detect a novel QTL for a total of 22 production, respiratory disease, and immune-related traits in Landrace pigs. A total of 874 Landrace purebred pigs, which were selected based on MPS resistance, were genotyped using the Illumina PorcineSNP60 BeadChip. We performed single nucleotide polymorphism (SNP)-based and haplotype-based genome-wide association studies (GWAS) to detect a novel QTL and to evaluate the possibility of a pleiotropic QTL for these traits. SNP-based GWAS detected a total of six significant regions in backfat thickness, ratio of granular leucocytes to lymphatic cells, plasma concentration of cortisol at different ages, and complement alternative pathway activity in serum. The significant region detected by haplotype-based GWAS was overlapped across the region detected by SNP-based GWAS. Most of these detected QTL regions were novel regions with some candidate genes located in them. With regard to a pleiotropic QTL among traits, only three of these detected QTL regions overlapped among traits, and many detected regions independently affected the traits.

Quantitative alterations in complement alternative pathway and related genetic analysis in severe phenotype preeclampsia

Background: Preeclampsia and HELLP (hemolysis, elevated liver enzymes, low platelets) syndrome share many clinical and biologic features with thrombotic microangiopathy syndromes caused by complement abnormalities. Our hypothesis was that similar functional and genetic alterations in the complement alternative pathway (CAP) are present in these disorders of pregnancy. Methods: Quantitative analysis of proteins involved in CAP using ELISA and nephelometry was conducted on prospectively collected blood samples from cases of severe phenotype preeclampsia (defined as delivery <34 weeks due to preeclampsia), HELLP syndrome or eclampsia, and matched normotensive controls (n=25 in each arm) between 2011 and 2016. Sequencing was performed to interrogate 14 genes encoding CAP components. Results: Both groups were similar in age, gravidity, parity, marital status and race; the study group had a higher BMI (mean ± SD; 32±8 vs 25±4 kg/m2; p=0.002) and earlier gestational age at delivery (32.5±3.6 vs 40.3±1 weeks; p<0.001). Serological studies demonstrated elevated Bb subunit (median [range]; 1.2 [0.5-4.3] vs 0.6 [0.5-1] mcg/mL; p<0.001), complement C5 concentration (28 [18-33] vs 24 [15-34] mg/dL; p=0.03) and sMAC (371 [167-761] vs 184 [112-249] ng/mL; p<0.001) concentrations in patients with preeclampsia. Two-thirds had at least 1 non-synonymous sequence variant in CAP genes. Conclusion: Patients with severe phenotype preeclampsia manifest functional alterations in CAP activation. Genetic variants in the CAP genes were detected in several patients, a larger population study is necessary to fully evaluate genetic risk. Genetic screening and complement-targeted treatment may be useful in risk stratification and novel therapeutic approaches.

Generation and Characterization of Mouse Models of C3 Glomerulonephritis With CFI D288G and P467S Mutations

C3 glomerulopathy (C3GP) is a disease entity caused by abnormality of the complement alternative pathway (AP) and characterized by C3 deposition in glomeruli. Many variations or mutations of complement factors are believed to underlie the susceptibility to C3GP, but there is a lack of experimental evidence. We have recently reported a patient with C3 glomerulonephritis (C3GN) and compound heterozygosity of two novel variations in the complement factor (CFI). Here, we generated a mouse model to mimic the CFI variations for studying pathogenicity of CFI variations in C3GN development. We used the CRISPR/Cas9 system to make mutant mouse lines that carried D288G and P467S mutations in CFI, respectively, and crossed them to generate mice with compound heterozygosity of CFI D288G and P467S. The mice were all normal in either SPF (specific pathogen free) or regular environment. When treated with lipopolysaccharides (LPS), a bacterial endotoxin that mimics infection and sepsis, the mice developed albuminuria, kidney function impairment, and C3 glomerular deposition at levels comparable with the wild-type mice. The mice with other genotypes concerning CFI D288G and P467S were also tested in parallel. Unexpectedly, we found that the D288G homozygotes all developed severe mesangial deposition of C3 in the LPS model, indicating that CFI D288G variation was involved in the C3 deposition, a key feature of C3GN. The mouse lines generated in the present study can be used to further study the role of CFI variations in C3GN development; in addition, they may be used to screen and test infections and environmental factors capable of triggering C3GN.