ALBERT

Description: Abstract 731 Paroxysmal Nocturnal Hemoglobinuria (PNH) is an acquired disorder of hemopoietic stem cells (HSCs). Affected individuals experience intravascular hemolysis and have a predisposition to thromboembolism, renal impairment and pulmonary hypertension. These symptoms vary in severity, but in general, the higher the proportion of PNH blood cells produced, the more severe the symptoms. The molecular pathogenesis in PNH is known to relate to a single gene mutation in the X-linked phosphatidylinositol glycan class A gene (PIG-A) which causes a complete or partial deficiency of glycophosphatidylinositol (GPI) anchored proteins leading to the symptoms of the disease. The recent development of eculizumab therapy in PNH has had a dramatic impact in reducing both morbidity and mortality in the disease but PNH remains incurable. A sub-optimal response to eculizumab, certainly when assessed by transfusion requirements, is often due to the underlying bone marrow failure that is considered to be universally present in PNH. The factors that lead from the development of a mutant clone to clonal expansion and symptomatic disease are poorly understood but are the key to improving responses and potentially to move towards a cure. Bone marrow failure appears to provide the environment necessary for expansion of PNH clones and small PNH clones are often detected in aplastic anemia and less commonly in myelodysplasia. There is no evidence that PNH HSCs have an intrinsic proliferative advantage compared to normal HSCs and an immune-mediated extrinsic suppression of normal hematopoiesis with a selective advantage for the PNH cells over residual normal stem cells is likely to explain the preferential development of PNH clones concurrent with bone marrow failure. To gain a better understanding of clonal expansion in PNH we have developed an in vitro PNH bone marrow culture model using a stromal cell line which allows PNH stem cells to be maintained in long term cultures and their capacity to form progenitor cells in myeloid colony forming assays to be assessed. We have evaluated bone marrow from 11 patients with PNH (median age 47 years, median granulocyte clone size 95.3%) and 10 normal controls (median age 42 years) within these long term bone marrow culture experiments. Unmanipulated bone marrow mononuclear cells (MNCs), CD34 selected cells and MNCs with their T-cell component depleted were used in this model. This in vitro model provides the environment for PNH stem cells to be maintained for up to eight weeks and, unlike previous studies, produce progenitor cells. When the patient's MNC's are used to seed the culture system there is poor growth of the culture which is only maintained for a median of 2 weeks. However if CD34 selected cells are used then the cultures are maintained for up to 8 weeks (similar to the normal controls) suggesting that there is a component within the MNC's that is responsible for suppressing the marrow culture which is removed by CD34 select. We next selectively removed the T-cells from the PNH MNC's and demonstrated that the marrow cultures now survived to the extent of the controls (see Figure). This demonstrates that the immune suppression in PNH resides in the T-cells. The progenitor cells produced in both the CD34 selected or the T-cell depleted MNCs over the course of the long term culture experiments show an increase in the proportion of normal progenitors the longer the cultures are maintained. This supports the hypothesis that PNH stem cells have no intrinsic proliferative advantage over normal HSCs and that T-cells are the critical cell suppressing the normal hematopoiesis in PNH. We are now examining the specific cell type that causes the myelosuppression in PNH and which will facilitate a targeted approach to the treatment of bone marrow failure in PNH and related disorders. In conclusion we have developed an in vitro PNH bone marrow culture model that allows the immune insult in PNH to be evaluated. Furthermore, this work confirms the important role that T-cells play in the etiology of PNH and provides a model with which to define the exact mechanism of suppression of hematopoiesis in PNH (and aplastic anemia). This information is essential to develop targeted therapies for the marrow failure seen in PNH and aplastic anemia. Disclosures: Kelly: Alexion Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Richards:Alexion Pharmaceuticals: Honoraria, Speakers Bureau. Arnold:Alexion Pharmaceuticals: Honoraria. Hill:Alexion Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Hillmen:Alexion Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

Description: In past 22 years, we have identified using flow cytometry 705 patients with detectable PNH (GPI deficient) populations of granulocytes, monocytes and red cells in the peripheral blood in samples sent for diagnosis. We undertook an analysis of presenting clinical features, blood count data and PNH clone sizes in order to better understand the natural history and provide a more objective classification of disease. Based on serial flow cytometry measurements of PNH clone sizes, we also studied disease stability, frequency of recovery and progression with an aim to guiding future management of individual patients. Clinical classification of patients at presentation was as follows; aplastic anemia (58%), hemolytic anemia (36.1%); myelodysplasia (2.5%); thrombosis (2.4%); hemolysis & thrombosis (0.6%), myeloproliferative neoplasm (0.3%); Fanconi anemia (0.1%). Median age at presentation was 45 years (observed range 0.5 – 90 years) and the Male:Female ratio was 1.05. Descriptive statistical analysis of presenting blood count data revealed novel gender related features not previously described in PNH. At presentation, pancytopenia was found in 61% of male and 47% of female patients; a normal blood count was present in only 0.3% of males and 4% of females. A combined low red blood cell count (RBC) and white cell count (WBC) was the most frequent bicytopenia affecting 19% males and 22% females. Leucopenia as a sole abnormality did not occur in males and was present in

Description: Abstract 639 PNH is an acquired clonal hemolytic anemia associated with severe symptoms, life-threatening thrombosis, pulmonary hypertension (PH), chronic renal impairment (CRI) and bone marrow failure resulting in reduced quality of life (QoL) and survival (median: 10 – 15 years). Eculizumab is a monoclonal humanized antibody that inhibits terminal complement activity and thereby prevents intravascular hemolysis, reduces transfusions, improves QoL and protects against PH, CRI and thromboses. Eculizumab was approved for PNH in 2007 as a result of studies with short follow-up and with no information on it's impact on survival. We present data on 79 patients treated with eculizumab from the Leeds PNH Center between May 2002 – July 2010 including 34 patients from the clinical trials and a further 45 treated since, funded in England by the National Commissioning Group (NCG) or locally in Scotland and Wales. The NCG indications for treatment include transfusion-dependent hemolysis (4 or more transfusions in 12 months) or significant PNH-related complications (i.e. thrombosis or renal failure) regardless of transfusion history. Three patients did not fulfil these criteria but were treated for profound symptoms as agreed with the NCG. Forty men and 39 women were treated for a mean of 39 months (1-98). Median age at diagnosis was 37yo (12-79) and at initiation of eculizumab was 46yo (14-84). Median granulocyte clone size at the start of eculizumab was 96.38% (41.78-99.98). Patient survival on eculizumab was compared with age and sex matched controls obtained using data from the UK Office of National Statistics. We previously published that there was a significantly worse survival for PNH patients compared to matched controls with ~ 50% of patients dying as a result of PNH (Hillmen et al., NEJM 1995). In the current series there was no difference in mortality between patients on eculizumab and the normal population (P=0.46; Fig 1). Patients over 70yo had worse survival (P=0.0042) with none of the 45 patients under the age of 50yo dying. Three patients died: a 55yo man from metastatic caecal carcinoma diagnosed prior to eculizumab, a 76yo woman who died from pneumionia following a long history of recurrent bronchopneumonia prior to eculizumab and a 79yo man with a preceding history of ischaemic heart disease who died from cardiac failure. Three patients have had a clonal evolution of their PNH, 1 to acute myeloid leukemia and 2 to myelodysplasia. Two patients have stopped eculizumab: - 1 due to pre-existing aplastic anemia and 1 with spontaneous remission of his PNH. Seventy four patients remain on eculizumab to date.Figure 1Kaplan-Meier survival plots depicting PNH patients on eculizumab compared to age and sex matched controlsFigure 1. Kaplan-Meier survival plots depicting PNH patients on eculizumab compared to age and sex matched controls There were 34 thrombotic episodes in 21/79 patients (27%) prior to eculizumab and only 2 thromboses since. Importantly primary prophylaxis with warfarin has now been stopped in 21 patients (mean duration of 8.4 months (range: 1–25) and total of 〉 14 years) with no thromboses occurring. Seven patients had thrombosis within 12 months prior to starting eculizumab and they had no further thromboses on therapy. Therefore eculizumab is effective in preventing new, or evolution of pre-existing thrombosis, and appears to remove the need for primary prophylactic anticoagulation. We have not systematically stopped the anticoagulation of patients with a previous history of thrombosis. Patients had a mean of 19.9 units transfused (0-156) in the 12 months before eculizumab. Of the 64 patients on eculizumab for at least a year, 43 (67%) have been transfusion independent for 〉12 months. Twenty one patients still needing transfusions had a significant (P=0.028) reduction in their mean requirement of 24.6 (4-52) units in the 12 months before eculizumab compared to a mean of 14.6 (2-50) units in the last 12 months. Eculizumab is well tolerated long-term (〉 8 years of therapy) with continued improvement in PNH associated symptoms, reduction in transfusion requirements and a higher proportion of transfusion independent patients than previously seen. There is a significant reduction in the development of thromboembolism on eculizumab therapy and importantly we have been able to stop primary prophylaxis with warfarin in 21 patients without any thrombotic complications. We demonstrate for the first time that eculizumab has a major impact on survival in PNH so that survival is comparable to an age- and sex-matched control population. Disclosures: Kelly: Alexion Pharmaceuticals: Honoraria. Hill:Alexion Pharmaceuticals: Honoraria, Speakers Bureau. Richards:Alexion Pharmaceuticals: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Arnold:Alexion Pharmaceuticals: Honoraria. Hillmen:Alexion Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Description: Abstract 3478 Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal disorder of hematopoietic stem cells characterised by deficiency of cell membrane glycosylphosphatidylinositol-anchored proteins, rendering the cells susceptible to complement attack. Budd-Chiari syndrome (BCS) describes obstruction of the hepatic venous outflow tract. Complications such as ascites and varices often occur at the time of acute thrombosis. Conventional management includes anticoagulation, transjugular intrahepatic portosystemic shunt (TIPSS) placement, peripheral/local thrombolysis and orthotopic liver transplantation (OLT). The morbidity and mortality of PNH patients with BCS is particularly high: a new thrombotic event or thrombosis extension in anticoagulated patients is reported in 27%; a significant proportion of TIPSS fail due to recurrent thromboses; historically, mortality rates of 67% have been reported; hematopoietic stem cell transplantation and OLT have unacceptably high transplant-related mortalities. Eculizumab prevents cleavage of C5 and reduces risk of thrombosis in PNH. The role of this monoclonal antibody in the management of BCS with PNH has yet to be evaluated. Nineteen PNH patients (12 female, 7 male) with a BCS diagnosis who were subsequently given eculizumab, were identified from the Leeds PNH Service. Patients were diagnosed with PNH between 1991–2012 (median follow up from PNH diagnosis 7.5 yrs, range 2 mths – 21 yrs; from BCS diagnosis 3.5 yrs). Median age at PNH diagnosis was 30 yrs (range 19–87) and at BCS 32 yrs (range 19–87). The mean granulocyte clone size at PNH diagnosis (n=14) was 86.3% (range 59.7–99%) and at BCS (n=12) was 93.1% (range 80.9–99.6%). BCS was the presenting feature of PNH in 9 (47.4%). Six were diagnosed as part of diagnostic work-up during the acute BCS. In 3, a diagnosis of PNH was made 1, 6 and 26 months post BCS. Ten (52.6%) were already known PNH at the time of their BCS. None were receiving eculizumab. Median time from PNH to BCS was 30 mths, range 3–132. Seven were anticoagulated (5 primary prophylaxis) prior to BCS. Eight PNH patients (Group 1) developed BCS before the availability of eculizumab and remained alive long enough to receive therapy. Six in Group 1 (75%) developed BCS complications prior to commencing eculizumab including development of varices (n=1), progression of thrombus despite full anticoagulation (n=1), ascites necessitating TIPSS (n=2), hepatomegaly (n=3) and splenomegaly (n=3). Two did not endure BCS complications but suffered cerebral vein thrombotic events on anticoagulation. The median time to first dose of eculizumab therapy in Group 1 was 3 yrs (range 2 mths – 11 yrs). All patients remain on eculizumab (median follow-up 6.9 yrs, range 2.8 – 7.1); there have been no further thrombotic events in this cohort. Group 2 consists of 11 PNH patients in whom BCS was diagnosed during the availability of eculizumab. Four received this less than 14 days from onset of BCS without any subsequent complications. The remaining 7 started eculizumab 2 or more weeks from probable onset of BCS. Complications included ascites necessitating TIPSS and/or gastric/esophageal varices. TIPSS procedures were effective in all patients once on eculizumab with no occlusions. Importantly, 3 of the 19 patients have stopped secondary anticoagulation after starting eculizumab (due to thrombocytopenia, hemorrhage or perceived bleeding risk due to varices). There have been no further thrombotic episodes in these patients. This case series is the first to document 19 patients, managed with eculizumab with both PNH and BCS. Rapid intervention with eculizumab in addition to anticoagulation when safe to administer, appears to prevent the development of BCS associated complications. If complications are established at diagnosis, eculizumab can prevent progression and reoccurrence; it also facilitates successful use of TIPSS. We can conclude: a) primary prophylaxis is not sufficient to prevent BCS in patients with PNH b) prompt treatment with eculizumab may negate long-term complications of BCS and allows effective and safe use of TIPSS c) in selected cases, eculizumab therapy may facilitate the withdrawal of anticoagulation without increasing subsequent risk of thrombosis and d) survival for this cohort is currently 100%. The management of BCS in PNH with immediate commencement of eculizumab and, where appropriate, use of TIPSS, is associated with improved outcomes. Disclosures: Kelly: Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria. Richards:Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria. Arnold:Alexion Pharmaceuticals, Inc: Honoraria. Hillmen:Alexion Pharmaceuticals, Inc: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Hill:Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria.

Description: Abstract 4368 Paroxysmal nocturnal hemoglobinuria (PNH) is a rare, acquired bone marrow disorder characterised by intravascular hemolysis with resultant anemia often leading to transfusion dependence, severe disabling symptoms and, frequently, life threatening thrombosis. Historically the management of PNH was largely supportive, relatively ineffective and resulted in frequent visits to hospital, admissions, an inability to function normally including loss of employment or other daily activities. PNH is a chronic condition and in most patients persists for the remainder of the patient’s life. Eculizumab was first used for PNH in 2002 and has been licensed since 2007. Eculizumab has been reported to improve all symptoms due to hemolysis in PNH as well as preventing the common complications, such as thrombosis and renal failure and normalising survival. However eculizumab has to be given as an intravenous infusion every 2 weeks indefinitely. In the UK PNH is managed in a shared care model between local hematologists and the National PNH Service from two Centres based in St James’s University Hospital, Leeds and Kings College Hospital, London. Here we report the management of patients treated with eculizumab within the PNH National Service. A total of 130 patients have been treated with eculizumab since May 2002 with 120 currently receiving therapy. 5 patients have died and none were directly related to PNH or eculizumab. 99 patients requiring transfusions prior to eculizumab have been on treatment for at least a year and 65 (66%) of these have not required transfusions for at least the last 12 months. The rarity of PNH means that patients frequently have to travel long distances for review and treatment. This leads to major issues both in terms of time commitment and expense. In order to allow patients to lead as normal lives as possible we have developed a service model in which Specialist PNH Clinics are performed regionally by the PNH Centre and in which patients receive eculizumab every 2 weeks in their homes delivered by a homecare nursing team. In the UK, the PNH Service and Healthcare at Home Ltd (www.hah.co.uk) have been working in partnership for over 7 years during the clinical trials of eculizumab and since its license in 2007. The PNH Service manages the prescription and delivery of eculizumab including an education program for the homecare nurses. This innovative home infusion programme ensures the safe administration of eculizumab outside of the hospital environment, leading to enhanced treatment-associated convenience for patients and their families. Each year the home infusion program has grown, now over 3000 infusions are given annually including whilst patients are on holiday, visiting family, at University or in the workplace. A recent patient survey has been conducted from the 2 PNH Centres to assess the patients’ experience of their PNH diagnosis and treatment. 122 patients responded with 70 of these patients receiving treatment with eculizumab and all currently on the home infusion programme. 63 of 68 patients reported the homecare service as excellent or very good compared to 1 reporting it as poor and 66 of the 68 patients preferred to have their treatment at home compared to hospital. The patients main concerns before starting treatment were reduced life expectancy and the requirement for blood transfusions along with fatigue. With eculizumab treatment and the convenience of homecare 30 patients reported being able to return to work. The homecare service is supported by contact between the clinic appointments, 56 of 67 patients having contact with their PNH Specialist Centre by phone or email in addition to the care of the patient’s local hematology team that over 90% of the patients continue to see. The impact of PNH on patients lives before eculizumab treatment was rated and improved from a median of 3 out of 10 (0 = no quality of life; 10 = normal) prior to eculizumab to a median of 8 out of 10 on treatment. In summary, a novel model of provision of care in PNH with Outreach Specialist Clinics, a 24 hour on call service and homecare delivery of eculizumab permits the normalisation of patients’ lives and overcomes most of the hurdles associated with prolonged regular intravenous therapy. This allows patients to benefit fully from eculizumab including reduction in transfusions, the prevention of serious complications, normalisation of quality of life and where appropriate a return to work. Disclosures: Arnold: Alexion Pharmaceuticals: Honoraria. Kelly:Alexion Pharmaceuticals: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Speakers Bureau. Hill:Alexion Pharmaceuticals: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Speakers Bureau. Richards:Alexion Pharmaceuticals: Honoraria, Speakers Bureau. Elebute:Alexion Pharmaceuticals: Honoraria, Membership on an entity’s Board of Directors or advisory committees. Hillmen:Alexion Pharmaceuticals: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding, Speakers Bureau.

Description: Abstract 3472 Paroxysmal Nocturnal Hemoglobinuria (PNH) is an acquired clonal stem cell disorder arising on the background of bone marrow failure and resulting in hemolytic anemia, thrombosis, pulmonary hypertension (PHT) and chronic kidney disease (CKD) through uncontrolled complement activation. Approximately half of patients treated with supportive therapies alone die as a result of their PNH. Eculizumab blocks C5 and thereby terminal complement activation. Complications are therefore prevented with reduction in intravascular hemolysis, transfusion requirements, thromboses, pulmonary pressures and renal impairment all associated with significantly improved quality of life. The UK has a nationally commissioned PNH service led by 2 Centres, Leeds and King's. Between May 2002 - April 2012, a total of 153 patients (76 male; 77 female) were treated with eculizumab for PNH in the UK for a mean of 42 mths (0.4 – 119). Patients are treated if they have 1) transfusion-dependent hemolysis or 2) independent of transfusions, either i) thrombosis, ii) CKD, iii) PHT, iv) pregnancy or v) LDH〉1.5× upper limit normal with anemia and symptoms including fatigue, dysphagia, dyspnea or abdominal pain due to PNH. Median age at PNH diagnosis was 34 yrs (range 12–80) and at initiation of eculizumab was 42 yrs (range 14–84). There were 28 thrombotic episodes in 15/65 (23%) patients anticoagulated prior to commencing eculizumab therapy. Nine patients have died - 3 due to progression of their underlying bone marrow failure to MDS/AML, one died immediately after BMT and the remaining 5 deaths are not directly related to PNH. Seven patients discontinued eculizumab - one had predominant aplastic anemia (AA), 2 had spontaneous remissions of the PNH clone, 3 commenced for a pregnancy discontinuing after and one who had a successful transplant for very severe AA. One hundred and thirty-seven patients remain on eculizumab. There were 36 thrombotic episodes in 22 patients in the 12 months prior to eculizumab and 3 thromboses in 3 patients in the most recent 12 months on therapy (1 Budd-Chiari during complement blockade breakthrough caused by infection, 1 CVA during reversal of coumadin overanticoagulation, 1 TIA/lacunar infarct thought to be due to diabetic small vessel disease) (P

Description: Introduction Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired bone marrow disorder characterised by intravascular hemolysis and hemoglobinuria, potentially life-threatening thrombosis and an association with aplastic anemia. Most of the clinical features and complications of PNH are due to the unopposed activity of complement due to the absence of CD59 and CD55, two key regulators of complement. Eculizumab prevents the cleavage of C5 complement thereby preventing terminal complement activity and protecting PNH cells from lysis. The inhibition of C5 preserves the early part of complement pathway and leads to the build up of C3 on the PNH red cells, perhaps in part due to their lack of CD55. The majority of PNH patients receiving eculizumab have evidence of extravascular haemolysis that can be clinically significant, including with anemia, hyperbilirubinemia and in some a continued requirement for transfusions. This extravascular hemolysis in thought to be due to the C3 loading of PNH red cells. Methods We report the C3-loading of the PNH red cells from 119 patients treated with eculizumab and correlate this with hemoglobin, LDH, bilirubin, reticulocytes and transfusions. We have studied genetic polymorphisms that affect both C3 and FCγR. We have genotyped 46 eculizumab patients for a functional mutation in the C3 gene (rs2230199). The two alleles of this gene can be distinguished by the presence or absence of a HindIII restriction site that distinguishes the electophoretically slow (arg80) from the electrophoretically fast (gly80) allotype. The fast (C3F) allotype allele of this snp is associated with a range of disorders including age-related macular degeneration, IgA nephropathy, systemic vasculitis and partial lipodystrophy. APL-1 is a small cyclic peptide that binds to and inhibits the activation of complement C3. APL-2 is a large conjugate of APL-1 with enhanced bioactivity and a long systemic half-life. APL-1 and APL-2 molecules as well as other complement inhibitors were studied for lysis of red cells and C3 loading in vitro in a modified Ham test in which flow cytometry was used to identify non-lysed cells. Results Out of the 119 Eculizumab treated patients, 55 (46.2%) required at least one transfusion on treatment. 110 patients had C3 detectable by flow cytometry on their PNH red cells (mean of 19.8%; range:

Description: Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal hematopoietic disorder with increased mortality and morbidity resulting from intravascular hemolysis. Eculizumab, a monoclonal antibody against the complement protein 5, stops the intravascular hemolysis in PNH. We evaluated 79 consecutive patients treated with eculizumab in Leeds between May 2002 and July 2010. The survival of patients treated with eculizumab was not different from age- and sex-matched normal controls (P = .46) but was significantly better than 30 similar patients managed before eculizumab (P = .030). Three patients on eculizumab, all over 50 years old, died of causes unrelated to PNH. Twenty-one patients (27%) had a thrombosis before starting eculizumab (5.6 events per 100 patient-years) compared with 2 thromboses on eculizumab (0.8 events per 100 patient-years; P 〈 .001). Twenty-one patients with no previous thrombosis discontinued warfarin on eculizumab with no thrombotic sequelae. Forty of 61 (66%) patients on eculizumab for more than 12 months achieved transfusion independence. The 12-month mean transfusion requirement reduced from 19.3 units before eculizumab to 5.0 units in the most recent 12 months on eculizumab (P 〈 .001). Eculizumab dramatically alters the natural course of PNH, reducing symptoms and disease complications as well as improving survival to a similar level to that of the general population.

Description: Coversin is a clinical stage recombinant protein molecule (16.7kDa) derived from a salivary molecule from the Ornithodros moubata tick where it assists the parasite to feed without provoking a host immunological response. It prevents activation of complement C5 and binds leukotriene B4 (LTB4) with high affinity. It is not an antibody and can therefore be manufactured in prokaryotic cells such as E. coli leading to potential savings in manufacturing costs. As its mode of action on C5 is similar to that of the monoclonal antibody eculizumab (Soliris®), it is considered to be a promising alternative therapeutic approach to thrombotic and hemolytic diseases mediated by complement activation including atypical hemolytic uremic syndrome (aHUS), catastrophic anti-phospholipid syndrome (APS) and paroxysmal nocturnal hemoglobinuria (PNH). In order to test this hypothesis, experiments were undertaken to determine whether Coversin could inhibit the hemolysis of PNH red cells in vitro using a modified Ham test combined with visualisation of the results by flow cytometric measurement of CD59 expression. Initial experiments showed maximum inhibition of hemolysis compared to control occurred at a concentration of approximately 10mcg/mL. Further experiments using PNH red blood cells also demonstrated the effectiveness of Coversin in blocking in vitro hemolysis of PNH red cells that had both type III (complete GPI-deficiency) and type II (partial GPI deficiency) red cells and also the deposition and accumulation of C3d on both types of PNH red cells. In a comparative experiment Coversin 10mcg/mL was found to be as effective as eculizumab 50mcg/mL, a molar equivalent dose. As Coversin is a non-humanised xenologous protein a mouse study was performed in order to assess immunogenicity and its potential to induce the formation of neutralising antibodies. Daily repeat subcutaneous doses of Coversin (0.57mg/kg) or vehicle were administered to 5 groups of 6 BALB/c mice and blood samples taken after 7, 14, and 28 days administration with an additional group where samples were taken 14 days following cessation of 28 days dosing. Immunoblots showed that by Day 14 33% of mice had developed low titre IgG antibodies to Coversin. By 28 days this had risen to 75% of mice receiving the active compound. Using CH50 lytic assays the antibodies were shown to be non-neutralising and no animals exhibited clinical signs of allergy or injection site reactions. The group of 6 mice that received vehicle showed no evidence of antibodies at 28 days. A heterozygous C5 mutation leading to a p.Arg885His polymorphism and interfering with the binding of eculizumab was recently reported in Japanese PNH patients and this was associated with a poor response to therapy. This was subsequently found to affect 3.5% of the general Japanese and Han Chinese population. A similar polymorphism has now been shown in a 4 year old Caucasian child with persistent thrombocytopenia following a stem cell transplant for chronic granulomatous disease. Prolonged eculizumab treatment was ineffective leading to discovery of the mutation. Serum from the patient was spiked with ascending concentrations of Coversin and CH50 activity was measured by ELISA. This produced a dose response curve corresponding to that found in serum from normal subjects. Coversin, which binds to a slightly different site on the C5 molecule, seems therefore to be unaffected by the polymorphism and to achieve superior inhibition of C5 activation compared to eculizumab. A Phase I, single ascending dose clinical trial was performed in 24 healthy volunteers. In the top dose cohort total blockade of complement C5 was achieved following subcutaneous injection at the predicted therapeutic loading dose of 0.57mg/kg. Activity remained below 50% for 48 hours suggesting that dosing once a day is feasible at steady state (see figure). The drug was well tolerated and no drug-related side effects were seen. Phase II clinical trials in PNH and other thrombotic micro-angiopathies (TMAs) are planned. Coversin appears to be a promising alternative to eculizumab for patients with PNH and other TMAs including those with polymorphisms which interfere with optimal binding of eculizumab to complement C5. Figure 1 Figure 1. Disclosures Weston-Davies: Volution Immuno Pharmaceuticals (VIP) SA: Consultancy. Nunn:Volution Immuno Pharm: Consultancy. Prudo:Volution Immuno Pharmaceuticals (VIP) SA: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.