ALBERT

Description: A fusion protein genetically linking recombinant human coagulation FIX with recombinant human albumin (rIX-FP) has been developed with an improved PK profile, improving hemophilia B treatment by allowing less frequent dosing than required with standard plasma-derived (pd) and recombinant (r) FIX products. The PROLONG-9FP clinical program has evaluated the use of rIX-FP for prophylaxis and on-demand treatment of bleeding in patients with severe hemophilia B. The clinical program is comprised of 5 clinical studies, including four completed studies (a Phase I pharmacokinetic (PK) study, a Phase II study, and two Phase III studies in adults and children), and an ongoing extension study, which includes previously untreated patients. Over 100 subjects from 42 hemophilia treatment centers in 12 countries have participated in the PROLONG-9FP clinical program. Here, we report on the long-term safety and efficacy of rIX-FP in 15 subjects who have continuously participated in 3 clinical studies (Phase II, Phase III and the on-going Phase III extension studies) over a period of 4 years. Subjects began either weekly prophylaxis treatment or on-demand treatment with rIX-FP in the Phase II 2004 study, and continued that treatment regimen in the Phase III 3001 study. The Phase III global study evaluated safety and efficacy of rIX-FP for prophylaxis treatment (PT) of every 7-, 10- and 14-days and on-demand treatment (ODT) of bleeding episodes. Subjects in the on-demand arm received only ODT for 6 months and then switched to 7-day PT. Subjects in the prophylaxis arm received 7-day PT for 6 months, and eligible subjects switched to 10- or 14-day PT interval. Upon completion of the 3001 study, subjects entered the on-going extension study 3003 and either continued the treatment regimen or switched to a longer prophylaxis interval of 10-, 14- or 21-days. Within subject comparisons of the annualized spontaneous bleeding rates (AsBR), total ABR over time and other efficacy parameters between regimens will be presented. Long-term use of rIX-FP is safe and well-tolerated. No subjects developed inhibitors to FIX or antibodies to rIX-FP during the 4 year treatment period, with a mean of 180 exposure days (EDs) for PT subjects and 125 EDs for ODT subjects. ABR decreased over time with rIX-FP prophylaxis, and longer treatment intervals were possible with no increase in consumption. Disclosures Lubetsky: CSL Behring: Consultancy. Martinowitz:CSL Behring: Honoraria, Speakers Bureau. Voigt:CSL Behring: Employment. Wolko:CSL Behring: Employment. Jacobs:CSL Behring: Employment. Santagostino:Biotest: Speakers Bureau; Kedrion: Speakers Bureau; Roche: Speakers Bureau; Octapharma: Speakers Bureau; Novo Nordisk: Speakers Bureau; Baxter/Baxalta: Speakers Bureau; Bayer: Speakers Bureau; Biogen/Sobi: Speakers Bureau; Pfizer: Research Funding, Speakers Bureau; CSL Behring: Speakers Bureau.

Description: Introduction Under the auspices of the EC-Horizon 2020 project "TREGeneration", we are conducting five clinical phase I/II trials investigating the safety and efficacy of donor regulatory T cell (Treg) infusion in patients with chronic Graft-versus-Host Disease (cGvHD), a serious complication following hematopoietic stem cell transplantation (HSCT). Workpackage 4 evaluates T cell receptor (TCR) repertoire changes by high-throughput sequencing following the infusion of donor Tregs. Methods For repertoire analysis, four different T cell subsets (Treg, conventional CD4 T cells - Tcon, total CD4 and CD8 T cells) are FACS sorted from the infused product and from the cGvHD patients' peripheral blood before the infusion and at 10 time-points up to 12 months post infusion (Fig. 1A). To monitor TCR repertoire changes over time, we developed a novel quantitative "DeepImmune" technique for TCRβ amplification and sequencing. We amplify all TCRβ chain family transcripts from total RNA using a normalized primer set including molecular identifiers for unbiased and quantitative determination of TCRβ copy numbers. We have normalized the concentrations of our TCRβ primers using synthetic TCRβ gene sequences, thus minimizing amplification biases during RT-PCR to ≤3-fold differences among gene families. The inclusion of molecular identifiers during the RT-PCR step allows us to computationally remove additional biases introduced during the secondary PCR step for Illumina library preparation. Thus, our method results in ultra-deep characterization of immune receptor repertoires for sensitive detection of differences among samples and sample cohorts. Depending on T cell numbers in the samples, we routinely identify 1,000 - 150,000 clonotypes per sample. Results As first quality control for repertoire analysis, we cluster the top 200 TCRβ clonotypes from the different sequenced T cell subsets for each patient, resulting in heatmaps showing prominent repertoire differences among the analyzed T cell subsets (Fig. 1B; infused patient example shown). Next, we track the fate of infused donor Treg cells in the time course samples of the patients after Treg infusion, allowing us to quantify the number and frequency of TCRβ clonotypes that persist or expand in the patients after the infusion. We report that TCRβ clonotypes specific to the infused Treg cells (which were not present in the patients before Treg infusion) can be detected in the patients' blood after the infusion. Infusion-specific Treg clonotypes can reach up to 10-15% of the Treg repertoire of the patients and usually decline over time (Fig. 1C; boxplot shows clonotype overlap between infusion-specific Treg clonotypes and patient Treg clonotypes from five patients of the Lisbon cohort over 12 months). In the Tcon compartment of the patients, infusion-specific Treg clonotypes typically reach only 1-2% of the Tcon repertoire of the patients and also decline over time (Fig. 1D), similarly to what is observed in the CD8 T cell compartment (data not shown). This suggests that infused Tregs are not reverting to Tcons, an important control and promising result. We apply the same type of analysis to track clonotypes that were shared between the infused product and the patient prior to infusion, in addition to those clonotypes that were unique to the patient before infusion and not present in the infused product. Conclusion By applying our robust and sensitive TCRβ sequencing platform to samples from cGvHD patients infused with donor Treg, we are able to detect unique clonotypes of the infused product up to 1 year post-infusion. It remains to be be determined if there is any correlation between the level of detection of the infused clonotypes and the clinical responses of cGVHD to donor Treg therapy. Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.

Description: A recombinant fusion protein linking coagulation factor IX (FIX) with human albumin (rIX-FP) has been developed to facilitate hemophilia B treatment by less frequent FIX dosing. This first-in-human dose-escalation trial in 25 previously treated subjects with hemophilia B (FIX ≤ 2 IU/dL) examined the safety and pharmacokinetics of 25, 50, and 75 IU/kg rIX-FP. Patients in the 50-IU/kg cohort underwent a comparative pharmacokinetics assessment with their previous FIX product (plasma-derived or recombinant). No allergic reactions or inhibitors were observed. Four mild, possibly treatment-related adverse events were reported. In the 50-IU/kg cohort (13 subjects), the mean half-life of rIX-FP was 92 hours, more than 5 times longer than the subjects' previous FIX product. After 25 or 50 IU/kg rIX-FP administration, the baseline-corrected mean FIX activity remained elevated at day 7 (7.4 IU/dL and 13.4 IU/dL, respectively) and day 14 (2.5 IU/dL and 5.5 IU/dL, respectively). The incremental recovery of rIX-FP was higher than both recombinant and plasma-derived FIX (1.4 vs 0.95 and 1.1 IU/dL per IU/kg, respectively). These results demonstrated both the safety and improved pharmacokinetics of rIX-FP, thus indicating this new product with extended half-life as possibly able to control and prevent bleeding with less frequent injection. The trial was registered at www.clinicaltrials.gov as no. NCT01233440.

Description: Abstract 1121 Factor IX (FIX) replacement therapy is the standard of care for patients with haemophilia B. FIX products currently available have a relatively short half-life, requiring 2–3 times a week intravenous prophylactic treatment to achieve a significant bleeding reduction. A recombinant FIX albumin fusion protein (rIX-FP) was generated by genetic fusion of human recombinant albumin to rFIX to extend the half life of FIX. A Phase I/II open-label, multicenter, clinical study of rIX-FP has been completed in previously treated patients with severe hemophilia B (FIX ≤ 2%) as part of the PROLONG - 9FP clinical developmental program. The objectives of the study were to evaluate the prevention of bleeding episodes during once weekly prophylaxis and to assess the hemostatic efficacy of rIX-FP for the treatment of bleeding, in addition to safety and pharmacokinetic (PK) assessments. The study consisted of a 10 to 14 day PK evaluation period, and a 3 to 12 month safety and efficacy evaluation period, during which subjects received either on-demand or prophylaxis treatment. Subjects receiving weekly prophylactic treatment were initially treated with a dose based upon the subject's PK profile, bleeding phenotype, and physical activity level. The dose could be adjusted based on clinical outcome, while maintaining a 7 day treatment interval. Subjects receiving on-demand treatment were treated with a dose based upon the subject's PK profile and WFH recommendations for treatment of bleeding episodes. Seventeen study subjects from hemophilia treatment centers in Israel and Bulgaria participated in the study, 13 of whom received weekly prophylaxis treatment and 4 of whom received on-demand treatment. Following a single infusion of 25 IU/kg rIX-FP (n=13), the PK parameters (t1/2 = 94 hrs, AUC0-inf= 3414 hr*IU/dL) were comparable to those previously reported from the Phase I study (Blood prepublished Aug 2, 2012). In addition, rIX-FP maintained a baseline-corrected mean trough level of 3.8% and 2.7% at Day 7 and Day 14, respectively, after 25 IU/kg rIX-FP administration. There were no AEs considered as possibly related to rIX-FP. There were no allergic reactions, inhibitors to FIX or antibodies to rIX-FP reported. All 10 prophylaxis subjects who were previously receiving routine prophylaxis with FIX were maintained successfully on weekly treatment with rIX-FP for the entire study. Furthermore, three prophylaxis subjects who were previously treated on-demand were maintained successfully on weekly prophylaxis treatment with rIX-FP with at least 85% reduction in the annualized spontaneous and total bleeding rate compared to the annualized bleeding rate prior to study entry. All of the bleeding events were treated successfully, including approximately 90% of the events with a single infusion of rIX-FP. The mean weekly product consumption of rIX-FP (IUs) was reduced compared to the consumption of the previous FIX product (IUs). No subject was withdrawn from the study due to safety concerns or lack of hemostatic efficacy. This Phase I/II study has demonstrated the clinical efficacy of rIX-FP for once weekly routine prophylaxis to prevent spontaneous bleeding episodes and treatment of bleeding episodes, in addition to the excellent safety characteristics and improved PK profile. A detailed analysis of the efficacy of weekly routine prophylaxis and treatment of bleeding episodes, improved PK and safety properties of rIX-FP will be presented. Disclosures: Martinowitz: CSL Behring: Honoraria, Investigator for CSL clinical study of rIX-FP Other. Lubetsky:CSL Behring: Investigator for CSL clinical trial of rIX-FP Other. Santagostino:CSL Behring: Honoraria, Investigator for CSL Behring clinical trial of rIX-FP Other, Research Funding, Speakers Bureau. Jotov:CSL Behring: sub-investigator for CSL clinical trial of rIX-FP Other. Barazani-Brutman:CSL Behring: study coordinator for CSL Behring clinical trial of rIX-FP Other. Voigt:CSL Behring: Employment. Moises:CSL Behring: Employment. Jacobs:CSL Behring: Employment. Lissitchkov:CSL Behring: Investigator for CSL Behgring clinical trial of rIX-FP Other.

Description: Background The standard of care for patients with severe hemophilia B is replacement treatment with Factor IX (FIX) 2-3 times a week. A fusion protein genetically linking recombinant human coagulation FIX with recombinant human albumin (rIX-FP) was developed with the aim to extend the half-life of FIX. In the completed Phase I pharmacokinetic study, the mean half-life of rIX-FP was found to be over 5 times longer than the subjects’ previous FIX. Thus, rIX-FP has potential to prevent bleedings for longer periods, allowing reduction in the frequency of injections compared to standard FIX and to reduce the number of injections required to treat a single bleed. Aims This was a Phase I/II open-label, multicenter study of rIX-FP in previously treated patients 12-65 years of age with severe hemophilia B (FIX ≤ 2%). The study evaluated the safety and efficacy of rIX-FP, including prevention of bleeding episodes during weekly prophylaxis of rIX-FP. Methods After completion of a 14-day rIX-FP pharmacokinetic assessment, 13 subjects in the prophylaxis arm received weekly prophylaxis of rIX-FP for approximately 11 months, and 4 subjects in the on-demand arm received rIX-FP upon occurrence of bleeding events. The treatment doses were initially selected based upon the pharmacokinetic profile of rIX-FP and subject’s bleeding phenotype, and doses could be adjusted at the Investigator’s discretion. Results Seventeen subjects were enrolled from hemophilia treatment centers in Israel and Bulgaria; the mean age was 26 years (range 13 to 46 years). Following a single injection of 25 IU/kg rIX-FP (n=13), the mean FIX activity level was 3.75% and 2.67% above baseline at Day 7 and Day 14, respectively, and the mean half-life of rIX-FP was 95 hours (comparable to the previously reported Phase I data). Over the 11 month treatment period, rIX-FP demonstrated a good safety profile with a total of over 700 EDs. The treatment was well tolerated and no FIX inhibitor formation was observed. There was no AE considered to be related to treatment with rIX-FP. No subject was withdrawn from the study due to safety concerns or lack of hemostatic efficacy. All 13 prophylaxis subjects were successfully maintained on a weekly routine regimen of rIX-FP for the entire duration of the study, with annualized spontaneous bleeding rates of 1.255 and 1.134 (mean and median respectively). Furthermore, three prophylaxis subjects who received only on-demand treatment prior to study entry had greater than 80% reduction in the annualized bleeding rate compared to their annualized bleeding rate prior to study entry. All bleeding events were treated successfully with ≤ 2 injections of rIX-FP, with approximately 90% of bleeds treated with a single injection of rIX-FP. The mean weekly consumption of rIX-FP was reduced markedly compared to the subjects’ weekly consumption of the previous FIX product. Conclusion This Phase I/II study demonstrated the clinical efficacy of rIX-FP for once weekly routine prophylaxis to prevent spontaneous bleeding episodes and for the treatment of bleeding episodes. In addition, rIX-FP showed an excellent safety and an improved PK profile over currently marketed factor IX products. Disclosures: Lubetsky: CSL Behring: Investigator for CSL clinical trial of rIX-FP Other. Lissitchkov:CSL Behring: Investigator for CSL Behring clinical trial of rIX-FP Other. Santagostino:CSL Behring: Honoraria, Investigator for CSL Behring clinical trial of rIX-FP Other, Research Funding, Speakers Bureau. Jotov:CSL Behring: sub-investigator for CSL Begring trial of rIX-FP Other. Barazani-Brutman:CSL Behring: study coordinator for CSL Behring rIX-FP trials Other. Voigt:CSL Behring: Employment. Moises:CSL Behring: Employment. Jacobs:CSL Behring: Employment. Martinowitz:CSL Behring: Honoraria, Investigator for CSL rIX-FP trials Other, Speakers Bureau.

Description: Key Points rIX-FP maintains mean trough of 20 and 12 IU/dL FIX activity with 40 IU/kg weekly and 75 IU/kg every 2 weeks prophylaxis, respectively. Weekly and 14-day prophylaxis regimens with rIX-FP were well tolerated and provided low bleeding rates and target joint improvement.

Description: A fusion protein genetically linking recombinant human coagulation FIX with recombinant human albumin (rIX-FP) has been developed with an improved PK profile, improving hemophilia B treatment by allowing less frequent dosing and FIX consumption for perioperative management. A surgical sub-study was included in 3 Phase III studies, including an on-going Phase IIIb extension study in subjects with hemophilia B, as part of the global PROLONG-9FP clinical program. This report will focus on the use of rIX-FP for perioperative prophylaxis. Efficacy was evaluated by Investigator assessment (4-point scale), predicted and estimated actual blood loss, and consumption of rIX-FP during the 14-day perioperative period. Safety, including inhibitors to FIX, was assessed. The pre-operative dose of rIX-FP was based on individual incremental recovery, targeting 80-100% FIX activity. FIX activity was monitored at local laboratories to determine repeat doses during the surgical period. Five major orthopedic surgeries have been assessed to date. Hemostatic response was rated by investigators as excellent or good for all surgeries. A single dose of rIX-FP prior to surgery was sufficient to maintain hemostasis during surgery. Estimated actual blood loss was consistent with predicted blood loss in patients without hemophilia B. The total number of rIX-FP injections ranged from 6-7 over the 14 day post-operative period. The median rIX-FP consumption prior to surgery, Days 1-3, Days 1-7 and Days 8-14 post surgery were 84, 33, 81 and 107 IU/kg, respectively. There were no inhibitors to FIX or antibodies to rIX-FP during the study. The extension study is still ongoing, and additional orthopedic surgeries data may be presented. These results support the efficacy and safety of rIX-FP for perioperative management in patients with hemophilia B, with prolonged dosing intervals and lower FIX consumption than conventional therapies. Disclosures Negrier: CSL Behring: Speakers Bureau. Wolko:CSL Behring: Employment. Voigt:CSL Behring: Employment. Jacobs:CSL Behring: Employment. Santagostino:CSL Behring: Speakers Bureau; Pfizer: Research Funding, Speakers Bureau; Baxter/Baxalta: Speakers Bureau; Biotest: Speakers Bureau; Kedrion: Speakers Bureau; Roche: Speakers Bureau; Octapharma: Speakers Bureau; Biogen/Sobi: Speakers Bureau; Novo Nordisk: Speakers Bureau; Bayer: Speakers Bureau.

Description: A fusion protein genetically linking recombinant human coagulation FIX with recombinant human albumin (rIX-FP) has been developed with an improved PK profile, thus improving hemophilia B treatment by allowing less frequent dosing. Two Phase 3 studies (CSL654-3001 and CSL654-3002) were completed. CSL654-3001 study evaluated safety and efficacy of rIX-FP for prophylaxis treatment (PT) of every 7-, 10- and 14-day and on-demand (ODT) of bleeding episodes in 63 previously treated patients (PTP), 12-61 years of age with hemophilia B (FIX ≤ 2%). Subjects in the on-demand arm received only ODT for 6 months and then switched to every 7-day PT. Subjects in the prophylaxis arm received every 7-day PT, and eligible subjects switched to every 10- or 14-day PT for approximately 12-18 months. Annualized spontaneous bleeding rates (AsBR) were compared between ODT and PT periods (in on-demand arm), and between 7-day PT and 10- or 14-day PT (in prophylaxis arm). CSL654-3002 study evaluated safety and efficacy of rIX-FP for weekly prophylaxis regimen in 27 previously treated patients younger than 12 years with hemophilia B (FIX ≤ 2%) for approximately 12 months. Annualized spontaneous bleeding rates (AsBR) were calculated. The median annualized spontaneous bleeding rate were all 0.00 for all treatment interval (7-day, 10-day or 14-day) and in both studies age groups (1-11 years and 12-65 years) during the two completed phase 3 studies. Seventy-six subjects from both studies continued their prophylaxis regimen in the on-going extension study. In addition, subjects (including children), switched to longer treatment intervals of 10-day, 3 times per month or 14-day or lowered their weekly prophylaxis dose. Nine subjects switched to 21-day treatment interval with 100 IU/kg rIX-FP. As of 28 July 2015, at least 50 subjects (PTP) had achieved 100 EDs without developing an inhibitor to FIX or antibodies to rIX-FP. The long term safety and efficacy of rIX-FP will be presented. This presentation includes the new information regarding the change to longer than 7-day treatment regimens in the extension study, among those subjects (1-61 years of age) that previously participated in the lead in studies. Conclusion: The Prolong - 9FP clinical programdemonstrated the clinical efficacy of rIX-FP for routine prophylaxis every 7-, 10- and 14-day treatment intervals. Routine prophylaxis once every 21 days may be effective in preventing bleeding episodes in a selected patient population. In addition, rIX-FP demonstrated favorable long-term safety and tolerability. Disclosures Santagostino: Novo Nordisk: Speakers Bureau; Bayer: Speakers Bureau; CSL Behring: Speakers Bureau; Baxter/Baxalta: Speakers Bureau; Pfizer: Research Funding, Speakers Bureau; Biogen/Sobi: Speakers Bureau; Biotest: Speakers Bureau; Kedrion: Speakers Bureau; Octapharma: Speakers Bureau; Roche: Speakers Bureau. Voigt:CSL Behring: Employment. Wolko:CSL Behring: Employment. Cole:CSL Behring: Employment. Li:CSL Behring: Employment. Jacobs:CSL Behring: Employment.