ALBERT

Description: Background: PNH is a rare, acquired blood disorder. A somatic mutation in the PIGA gene of one or more hematopoietic stem cells generates a clone of abnormal erythrocytes (RBCs) that lack alternative pathway (AP) regulatory complement proteins CD55 and CD59. This leads to uncontrolled complement activation on affected RBCs and intravascular hemolysis (IVH). Standard of care is C5 inhibition to prevent membrane attack complex (MAC) formation. Despite preventing MAC-mediated IVH, many patients experience extravascular hemolysis (EVH). Despite C5 inhibition, ~ 70% of patients remain anemic and 〉1/3 were transfused 〉 1 time in the prior 12 months. Factor D (FD), a serine protease, catalyzes complement factor B cleavage, allowing formation of AP C3 convertase. By inhibiting FD, oral danicopan blocks C3 convertase formation, the control point for AP activation and amplification of all pathways. This leads to inhibition of C3 cleavage, C3 fragment deposition, terminal pathway activation and MAC formation. Thus, danicopan can control both IVH and EVH therefore, making FD a promising target. Aim: Demonstrate that danicopan is a potential treatment for PNH patients with an inadequate response to C5 inhibition. Methods: Data are presented for this Ph 2, dose-finding, proof of concept trial of danicopan in patients with an inadequate response to eculizumab who were transfusion dependent. Additional criteria in Table 1. In addition to the current eculizumab regimen, danicopan starting doses were 100-150 mg TID, with dose escalation up to 200 mg TID, based on clinical and biochemical response, at protocol defined time points (Figure 1). The primary endpoint was change in Hgb at Treatment Week (TW) 24. Secondary efficacy parameters included transfusion needs, effect on lactate dehydrogenase (LDH), and an exploratory an endpoint of Functional Assessment of Chronic Illness Therapy (FACIT) FATIGUE scores. General safety, tolerability, and PK/PD of danicopan were measured. After TW 24, patients entered a long-term extension. Results: Twelve patients received at least one dose of danicopan. One patient discontinued after 2 doses, due to a serious adverse event of worsening of an underlying condition (pulmonary hypertension/edema), considered unlikely related to danicopan. This patient is excluded from this analysis. Eleven patients continue to receive treatment. Dose titrations are shown in Table 2. Benefits were observed in multiple laboratory markers of PNH, shown in Table 3. Hgb improved in all patients, with a mean Hgb gain of 2.6 g/dL at 24 Weeks of treatment (n=4). Meaningful improvement in FACIT Fatigue scores were reported, with a mean increase of 8 points relative to the baseline on eculizumab. A 3-point change is clinically meaningful on this scale. Transfusion needs dramatically reduced, with one patient receiving one transfusion during the trial, as compared to 34 transfusions (58 units) in 10 patients, in the 6 months prior to screening. One patient (who does not accept blood products due to religious objections and also has hereditary elliptocytosis) had a baseline Hgb=5 g/dL with a 3.5 g/dL improvement at TW24 and significant improvement in fatigue. C3 fragment deposition was inhibited; reticulocytes, total/ direct bilirubin and LDH returned to normal range. Baseline C5 inhibition (classical pathway activity) was essentially inhibited (mean=1 [range 60-144]). Three of 11 patients received 〉 900 mg eculizumab, making PK a less likely reason for inadequate response. Danicopan has been well tolerated; 96% of treatment emergent adverse events (TEAEs) were mild to moderate in severity and no discontinuations due to TEAEs. Two patients had Grade 3 TEAEs which resolved and continued in the trial. Conclusion: Proof of concept is established with danicopan, an oral, small molecule FD inhibitor in the treatment of PNH in combination with eculizumab. Meaningful improvements in Hgb, transfusion needs, FACIT-FATIGUE and other parameters of interest were achieved. These clinically significant improvements demonstrate that further benefit can be achieved by blocking the alternative pathway at FD with danicopan, in combination with C5 inhibition. This benefit is likely due to the prevention of C3-mediated EVH, in addition to control of IVH. Danicopan targets an unmet need in PNH and will be further evaluated in a pivotal trial in combination with standard of care C5 inhibition. Disclosures Kulasekararaj: Achillion: Consultancy; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Alexion: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Ra Pharma: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Akari: Honoraria, Membership on an entity's Board of Directors or advisory committees; Alnylam: Membership on an entity's Board of Directors or advisory committees. Risitano:Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amyndas: Consultancy; Achillion: Research Funding; Amyndas: Consultancy; Apellis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Samsung: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees; Alexion: Honoraria, Research Funding, Speakers Bureau; Alexion: Honoraria, Research Funding, Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Alnylam: Research Funding; Achillion: Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Ra Pharma: Research Funding; Biocryst: Membership on an entity's Board of Directors or advisory committees; Alnylam: Research Funding; Roche: Membership on an entity's Board of Directors or advisory committees; Biocryst: Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; Ra Pharma: Research Funding; Samsung: Membership on an entity's Board of Directors or advisory committees; Apellis: Honoraria, Membership on an entity's Board of Directors or advisory committees. Maciejewski:Novartis: Consultancy; Alexion: Consultancy. Notaro:Alexion: Membership on an entity's Board of Directors or advisory committees, Other: letture fees. Browett:Janssen: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck: Membership on an entity's Board of Directors or advisory committees; Amgen: Research Funding; Beigene: Research Funding; AbbVie: Membership on an entity's Board of Directors or advisory committees; Achillion: Research Funding. Lee:Alexion: Consultancy, Honoraria, Research Funding; Achillion: Research Funding. Huang:Achillion: Employment, Equity Ownership. Geffner:Achillion: Employment, Equity Ownership. Brodsky:Alexion: Membership on an entity's Board of Directors or advisory committees, Other: Grant funding; Achillion: Research Funding.

Description: Idiopathic aplastic anemia (IAA) is an acquired bone marrow disease probably caused by an auto-immune attack against hematopoietic stem cells (HSCs), which leads to bone marrow failure. Many abnormalities have been observed in the T cell compartment, but the putative auto-antigen(s) remain elusive. A large body of evidence links paroxysmal nocturnal hemoglobinuria (PNH) to IAA, supporting the notion that autoimmunity is a key pathogenic mechanism in both diseases. In PNH, auto-reactive T cells may be the 'noxious agent' capable of killing GPI positive HSCs while sparing GPI negative HSCs. Recently, CD1d restricted, GPI-specific T-cells have been demonstrated in PNH patients. Here, we investigate whether CD1d restricted, GPI-specific T cells are also present in IAA patients. When peripheral blood mononuclear cells (PBMNCs) from 14 newly diagnosed IAA patients [12 of whom had a small percentage (between 0.003% and 5%) of GPI-negative granulocytes] were co-cultured with antigen presenting cells (APCs) expressing CD1d and competent for the synthesis of GPI, we detected GPI specific T cells (CD8+CD1d/GPI dimer+ T cells) in 10 out of 14 patients (71%) at a significantly higher abundance than in co-culture experiments performed with PBMNCs from healthy controls (Fig. 1). In fact, the frequency of CD8+CD1d/GPI dimer+ T cells was below the cutoff value of 0.35% in all the 15 healthy controls but only in 4 out 14 IAA patients (Fisher test, P

Description: Background: In PNH, a somatic mutation in the PIGA gene of one or a few hematopoietic stem cells generates a clone of abnormal erythrocytes which lack two key alternative pathway (AP) regulatory proteins, CD55 and CD59, leading to uncontrolled complement activation on affected erythrocytes (RBCs) and membrane attack complex (MAC)-mediated lysis. Factor D (FD), a serine protease, catalyzes the cleavage of complement factor B into Ba and Bb, which allows for the formation of the AP C3 convertase. By inhibiting FD, danicopan, an oral small molecule FD inhibitor, blocks C3 convertase formation, the control point for AP activation as well as the amplification of all pathways. This leads to inhibition of C3 cleavage, C3 fragment deposition, terminal pathway activation and MAC formation. Therefore, FD is a promising target in diseases of excess activation of the AP, such as PNH. Aims: Correlation between complement activity biomarkers and clinical/laboratory efficacy of danicopan, administered as monotherapy, in the treatment of PNH. Given that danicopan is potential first in class AP inhibitor for the treatment of PNH, it is important to investigate changes and relationships among relevant biomarkers of the AP to better understand the mechanism of action of danicopan in vivo. Methods: Danicopan starting doses ranged from 100-150 mg Q8H, with subsequent dose escalation based on clinical and biochemical response to doses as high as 200 mg Q8H. Danicopan plasma concentrations were determined by liquid chromatography/tandem mass spectrometry (LC/MS-MS). Danicopan pharmacodynamics (PD) were determined by measuring serum AP activity (AP Wieslab). Plasma Bb concentration, serum FD concentration, serum complement C3 and C4 concentrations, and serum classical pathway (CP) activity were also measured. C3 fragment deposition on RBCs was measured by flow cytometry with FITC conjugated anti- human C3d antibody. Results: Clinical results were previously presented, Table 1. Pharmacokinetic (PK) and PD analysis of danicopan concentration and AP activity measured ex vivo showed a clear correlation: ≤10% AP activity was seen for 6 hours following dosing with some recovery in AP activity prior to the next danicopan dose, Figure 1. Mean baseline Bb levels in this untreated PNH population were elevated relative to the reference range, demonstrating ongoing AP activity. Bb levels decreased into normal range with danicopan, demonstrating decreased in vivo C3 convertase formation, Figure 2. A strong positive correlation existed between Bb concentration and LDH level, demonstrating Bb as a reliable biomarker of AP activation in vivo during therapeutic complement inhibition in PNH. Strong correlations were also observed between danicopan concentration with Bb concentration (positive), AP activity (negative) and LDH level (positive), validating the role of danicopan in the changes of these endpoints. As expected, serum FD concentration was unchanged throughout treatment as was CP activity, which was measured with an in vitro assay designed to exclude any AP contribution. Notably, in contrast with C5 inhibition, C3 fragment deposition was not observed (95% inhibition of AP at steady state. Disclosures Risitano: Apellis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Samsung: Membership on an entity's Board of Directors or advisory committees; Achillion: Research Funding; Alexion: Honoraria, Research Funding, Speakers Bureau; Ra Pharma: Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Apellis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees; Biocryst: Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; Ra Pharma: Research Funding; Alexion: Honoraria, Research Funding, Speakers Bureau; Biocryst: Membership on an entity's Board of Directors or advisory committees; Achillion: Research Funding; Amyndas: Consultancy; Alnylam: Research Funding; Amyndas: Consultancy; Alnylam: Research Funding; Roche: Membership on an entity's Board of Directors or advisory committees; Samsung: Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees. Lee:Achillion: Research Funding; Alexion: Consultancy, Honoraria, Research Funding. Notaro:Alexion: Membership on an entity's Board of Directors or advisory committees, Other: letture fees. Brodsky:Alexion: Membership on an entity's Board of Directors or advisory committees, Other: Grant funding; Achillion: Research Funding. Kulasekararaj:Akari: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Achillion: Consultancy; Alexion: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Ra Pharma: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Alnylam: Membership on an entity's Board of Directors or advisory committees. Maciejewski:Alexion: Consultancy; Novartis: Consultancy. Huang:Achillion: Employment, Equity Ownership. Geffner:Achillion: Employment, Equity Ownership. Browett:AbbVie: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Merck: Membership on an entity's Board of Directors or advisory committees; Achillion: Research Funding; Amgen: Research Funding; Roche: Membership on an entity's Board of Directors or advisory committees, Research Funding; Beigene: Research Funding.

Description: C5-blockade with eculizumab prevents complement-mediated intravascular hemolysis in PNH patients and its clinical consequences. However, a distinct population of PNH red blood cells bound with C3 fragments appears in almost all treated patients. This C3 binding results in extravascular hemolysis that in some patients reduces the clinical benefit from eculizumab. In each PNH patients on eculizumab there are always two distinct populations of PNH red blood cells, one with (C3+) and one without (C3-) C3 binding. This phenomenon is somehow paradoxical since the glycosylphosphatidylinositol (GPI)-linked complement regulators, CD55 and CD59, are uniformly deficient on the surface of PNH red cells. To investigate this phenomenon, we have modeled in vitro the C3 binding in the context of C5 blockade by incubating red blood cells from PNH patients with AB0-matched sera from patients on eculizumab. Complement alternative pathway has been activated by mild acidification (in presence of Mg/EGTA to prevent the activation of complement classical pathway) and C3 binding has been assessed by flow cytometry at serial time points. In these experimental conditions a fraction of PNH red blood cells, similar to what happens in vivo, become promptly C3+ and its size increases with the time: from 9.4±2.7% after 5 minutes to 21.2±9.5% after 24 hours. The membrane defects of PNH cells suggested that the deficiency of CD55, which regulates the formation and accelerates the dissociation of C3 convertases, should be responsible for C3 binding to PNH red blood cells in presence of eculizumab (Parker CJ. Hematology Am Soc Hematol Educ Program. 2011;2011:21-29). In order to verify experimentally this hypothesis we have inactivated CD55 or CD59 on normal red blood cells by using blocking monoclonal antibodies (moAb - listed in the figure legend), and we have tested them in vitro upon activation of complement alternative pathway by mild acidification in presence or absence of C5 blockade. We found that CD55 inactivation on normal red blood cells results neither in hemolysis (without C5 blockade) nor in any C3 binding (with C5 blockade). As expected without C5 blockade CD59-inactivated normal red blood cells undergo hemolysis but, surprisingly, we found that in presence of C5 blockade they become bound with C3 fragments (Figure 1), just as it occurs in vivo in PNH patients on eculizumab. The simultaneous inactivation of both CD55 and CD59 further increased the level of C3 binding. Thus, at variance with the starting hypothesis, the deficiency of CD59, not that of CD55, plays the major role in C3 binding to PNH red cells of patients on eculizumab. Therapeutic C5 blockade in PNH patients has unmasked a novel function of CD59: in addition to prevent MAC formation, it plays a central role also in the regulation of C3 activation on cell surface through molecular mechanisms not elucidated yet. It remains to be established the physiological role, if any, of this novel function of CD59 and whether it play a role in determining the pleomorphic clinical features of the congenital CD59 deficiency. Finally, these findings may lead to investigate innovative approaches to reduce C3 binding and extravascular hemolysis in PNH patients on eculizumab and, in a broader context, to modulate complement activity. Figure 1 Figure 1. Disclosures Risitano: Novartis: Research Funding; Alexion Pharmaceuticals: Other: lecture fees, Research Funding; Rapharma: Research Funding; Alnylam: Research Funding.

Description: Introduction Eculizumab (Ecu) is the standard treatment for paroxysmal nocturnal hemoglobinuria (PNH), as it results in sustained control of intravascular hemolysis and prevention of thrombosis, and significant improvement of long-term survival. However, the hematological benefits remain heterogeneous among patients, and most response analyses are restricted to transfusion independence. In the era of new anti-complement therapies, we found appropriate to investigate in detail the quality of hematological response of Ecu-treated PNH patients. Methods We have recently proposed (Risitano et al, Frontiers in Immunology 2019) a classification of hematological response to anti-complement therapies based on hemoglobin (Hb) levels and laboratory markers of hemolysis. Six distinct categories were determined (Table 1): i. complete response (no transfusion requirement with normal stable Hb and no evidence of hemolysis); ii. major response (no transfusion requirement with normal Hb but evidence of residual intravascular or extravascular hemolysis); iii. good response (no transfusion requirement with persistent chronic mild anemia); iv. partial response (persistent chronic moderate anemia and/or with occasional red blood cell transfusions); v. minor response (3-6 red blood cell transfusions every 6 months); vi. no response (〉6 blood cell transfusions every 6 months). Here we have retrospectively exploited this classification to assess hematological response in a large cohort of 93 PNH patients evaluated in six international reference PNH centers (Paris, Naples, London, Florence, São Paulo, and Ribeirão Preto). Results Forty-two patients were male and 51 were female; the median age at diagnosis (treatment starting) was 36 years (range, 12 to 74 years). All the patients have started Ecu treatment for hemolytic PNH; 57% of patients had classic PNH, whereas 30% and 13% had aplastic anemia (AA)/PNH or intermediate PNH (Peffault de Latour et al, Blood 2006), respectively. Two-thirds of patients had been receiving red blood cell (RBC) transfusion before starting Ecu; the mean treatment duration was 97 months (range 6-179). The majority of patients (87%) received Ecu at the standard schedule of 900 mg every 2 weeks; due to chronic, residual, intravascular hemolysis 13% were treated with higher doses (1200 mg) or with shorter administration interval (10-12 days). Hematological responses were evaluated early during the treatment course (at 6 and 12 months), and then later during the last 6 months of treatment (Figure 1). After 6 (n=80) and 12 months (n=79) of Ecu, respectively, rates of hematological response were as follows: complete response 9% and 10%, major response 4% and 6%, good response 34% and 47%, partial response 34% and 28%, minor response 14% and 6% and no response 6% and 3%. In 83 patients treated for more than 18 months late response rates also were evaluated; most patients responded and the rates of hematological response during the last 6 months of treatment were as follows: complete response, 11%; major response, 6%; good response, 41%; partial response, 29%; minor response, 7% and no response, 6%. Breakthrough episodes were recorded in 21% of patients (equally split between pharmacokinetic and pharmacodynamic ones); thromboembolic events and AA were seen in 2% and 3% of patients, respectively. These data suggest that, albeit individual and unpredictable, the extent of the hematological benefit of Ecu in most PNH patient can be determined quite early in the treatment course, after a minimum of 12 months. Conclusion This is the first attempt to classify hematological response to Ecu based on Hb levels and residual hemolysis. The proposed classification allows the identification of distinct patient subgroups with different medical needs, as well as of meaningful clinical goals for future anti-complement therapies. In our cohort, about 60% of patients are well controlled with Ecu, even if less than 20% of patients reach normal Hb values; about 40% of patients remain anemic, and this represents a goal for future therapies in PNH. This classification is helpful to characterize more precisely the clinical response, which should be assessed after 1 year of treatment. Even if it should be validated in independent, larger cohorts, this classification represents a useful tool to design future trials, and to compare the clinical results of the novel anti-complement agents in development for PNH. Disclosures Notaro: Alexion: Membership on an entity's Board of Directors or advisory committees, Other: letture fees. Scheinberg:Pfizer,: Speakers Bureau; Celgene: Speakers Bureau; Roche: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Alexion: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Kulasekararaj:Alexion: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene Corporation: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Achilleon: Consultancy; Ra Pharma: Honoraria, Membership on an entity's Board of Directors or advisory committees; Akari Therapeutics: Consultancy. Risitano:Biocryst: Membership on an entity's Board of Directors or advisory committees; Samsung: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees; Ra Pharma: Research Funding; Samsung: Membership on an entity's Board of Directors or advisory committees; Achillion: Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Apellis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Alexion: Honoraria, Research Funding, Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Ra Pharma: Research Funding; Alnylam: Research Funding; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; Alnylam: Research Funding; Amyndas: Consultancy; Achillion: Research Funding; Amyndas: Consultancy; Biocryst: Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; Apellis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Alexion: Honoraria, Research Funding, Speakers Bureau; Roche: Membership on an entity's Board of Directors or advisory committees. Peffault de Latour:Pfizer: Consultancy, Honoraria, Research Funding; Alexion: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Amgen: Research Funding.