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Abstract 1524 CSL362, a novel humanized, affinity matured monoclonal antibody (mAB), is currently under development at CSL Limited. CSL362 neutralizes Interleukin-3 (IL-3) by binding to the human IL-3 receptor alpha chain (IL-3Ra/CD123). Furthermore, CSL362 has been engineered to bind to FcγRIIIa receptor (CD16) with high affinity and engages with effector cells of the innate immune system to elicit antibody-dependent cellular cytotoxicity (ADCC) against target cells expressing IL-3Ra such as AML blasts and leukemic stem cells. A non-clinical program was conducted to investigate the general safety and toxicity profile as well as the pharmacokinetic and pharmacodynamic properties of CSL362 in non-human primates. Cynomolgus monkeys (n=6 per dose cohort) were administered weekly intravenous infusions of CSL362 at doses of either 0, 3, 10, 30 or 100 mg/kg for 5 weeks with a 6 week recovery period. The potential of CSL362 to induce hypersensitivity or allergic reactions, its impact on safety pharmacology variables as well as local tolerance were assessed. Furthermore, the possible adverse effects of CSL362 on the proliferation, differentiation and maturation of bone marrow derived hematopoetic progenitors were explored. The potential for immunotoxicity of CSL362 was assessed by means of peripheral blood immunophenotyping, T cell-dependent antibody response measurements after keyhole limpet hemocyanin-immunization, measurements of natural killer cell (NK), macrophage and granulocyte function, and quantification of serum cytokine release. The ability to ablate cells expressing IL-3Ra was also determined by evaluating the numbers of basophils, plasmacytoid dendritic cells (pDCs) and monocytes in the peripheral blood. Plasma concentrations of CSL362 were determined to evaluate the pharmacokinetic characteristics. In parallel, the specific immune response against CSL362 was monitored throughout the 11 week study duration. Plasma level determination of CSL362 revealed adequate exposure and a PK profile similar to other humanized mABs. Specific anti-CSL362 antibody formation was observed in 18 % of the study animals. Treatment with CSL362 did not result in any anaphylactic or allergic reaction and had no adverse effect on systemic toxicity parameters or safety pharmacology variables measured. As predicted from in vitro and ex vivo studies, CSL362 produced a marked, dose-dependent and sustained depletion of peripheral blood basophils and pDCs which was associated with a transient decrease in NK cell numbers. No adverse effects of CSL362 on peripheral blood monocytes or pluripotent progenitor cells in the bone marrow was observed, furthermore the function of normal peripheral blood cells expressing IL-3Ra remained unaffected by CSL362. Intriguingly, a clear dose-dependent trend to NK cell recovery was observed following the final dose at week 5 during the subsequent recovery period which preceded the reappearance of basophils and pDC's. Together with the outcome of a tissue-cross reactivity study this proof of specific pharmacodynamic activity confirmed the Cynomolgus monkey as a relevant species. Overall, treatment with CSL362 was very well tolerated and 100 mg/kg, the highest tested dose, was designated the no-observed-adverse-effect-level. In conclusion, the results from this non-clinical safety program demonstrate an excellent tolerance and favorable safety profile of CSL362. This study supports the further clinical development of this mAB in the remission maintenance of patients with CD123-positive acute myeloid leukemia. Disclosures: Herzog: CSL Behring GmbH: Employment. Busfield:CSL Ltd.: Employment. Biondo:CSL Ltd.: Employment. Vairo:CSL Ltd.: Employment. DeWitte:CSL Behring: Employment. Pragst:CSL Behring GmbH: Employment. Dickneite:CSL Behring GmbH: Employment. Nash:CSL Ltd.: Employment. Zollner:CSL Behring GmbH: Employment.

Abstract 1117 The recombinant fusion protein linking the human coagulation factor IX to human albumin, rIX-FP, developed by CSL Behring GmbH, is currently undergoing investigation in clinical phase II/III trials (PROLONG - 9FP) for prophylaxis and on-demand treatment of bleeding in haemophilia B patients. The present study has been conducted to evaluate the biodistribution of rIX-FP in comparison to the marketed recombinant factor IX product BeneFIX®. Therefore, [3H]-rIX-FP or [3H]-BeneFIX®, labeled at lysine or terminal NH2 residues using the N-Succinimidyl [2,3,-3H] propionate (NSP) method, were administered intravenously to male Sprague Dawley (SD) rats at a single dose leading to a radioactive dose of 400 μCi/kg. Using whole-body autoradiography (QWBA), tissue radioactivity was determined at 0.25, 1, 3, 8, 24, 72, 120 and 240 hours following [3H]-rIX-FP, and at 0.25, 1, 3 and 24 hours following [3H]-BeneFIX® administration. In addition to full body sections, the hind limbs were separately subjected to QWBA to obtain more detailed information on the product distribution within the bone marrow, articular capsule and synovial region of the knee joints. In parallel, plasma, urine and faeces were collected at pre-dose and at several sampling points throughout the 24 and 240 hour study period, respectively, to calculate excretion balance and assess physiological elimination pathways. The radioactivity associated with the purified [3H]-labelled protein was determined by quantitative radiochemical analysis (QRA) and high performance liquid chromatography (HPLC). Biological activity of [3H]-labelled rIX-FP and BeneFIX® was confirmed using a chromogenic assay for factor IX activity. The radioactivity associated with plasma, urine and faeces samples was determined by QRA. HPLC-mass spectrometry (MS) techniques were employed to further characterize individual components identified following profiling of plasma and urine samples by size exclusion chromatography (SEC). Overall, the tissue distribution of [3H]-rIX-FP and [3H]-BeneFIX® was comparable, both penetrating predominantly into well perfused tissues and organs. Both products are also rapidly present in synovial and mineralized regions of knee joint sections and seem to mostly localize to the zone of calcified cartilage within the growth plate regions of long bones. For both, the longest retention time was observed in the bone marrow and endosteum of long bones. However, whereas [3H]-rIX-FP was detectable over 120 hours after administration, [3H]-BeneFIX® signal could only be detected until 24 hours post dosing. This is also reflected in the pharmacokinetic parameters determined based on the QRA of plasma and urine samples which suggested a terminal half life of 20.4 and 6.1 hours for [3H]-rIX-FP and [3H]-BeneFIX®, respectively, following correction for total radioactivity attributable to intact product. For both proteins, the major route of elimination was urinary. In case of [3H]-rIX-FP 73% of radioactivity was recovered in urine at 240 hours, the latest sampling point investigated. Less than 5% of radioactivity was eliminated in faeces and approximately 20% of radioactivity was present in tissues after 240 hours. Plasma profiling showed that up to 8 hours, 100% of the radioactivity could be assigned to unchanged [3H]-rIX-FP. From 24 to 240 hours, increasing levels of low molecular weight components (LMW) could be observed in plasma. Intriguingly, no high molecular protein components like [3H]-rIX-FP or albumin were detected in urine. Only LMW [3H]-components were found to be renally excreted. Such LMW components could be either be free [3H]-Lysine or bigger peptide fragments derived from [3H]-rIX-FP, which occur as a result of physiological protein catabolism. An exact identification of such renally excreted fragments is currently underway using HPLC-MS. Overall, the observed data are consistent with the hypothesis, that recycling via FcRn receptor is likely to be the physiological retention route for [3H]-rIX-FP. Consequently, this study shows that rIX-FP exhibits equal biodistribution compared to other marketed recombinant factor IX products such as BeneFIX®, but clearly distinguishes itself from BeneFIX® by its extended plasma half-life allowing a reduction in dosing frequency leading to increased therapeutic convenience and compliance. Disclosures: Herzog: CSL Behring GmbH: Employment. Harris:Quotient Bioresearch Metabolic Chemistry: Commercial Research Organisation Other. McEwen:Quotient Bioresearch Metabolic Chemistry: Commercial Research Organisation Other. Pragst:CSL Behring GmbH: Employment. Dickneite:CSl Behring GmbH: Employment. Schulte:CSL Behring GmbH: Employment. Zollner:CSL Behring GmbH: Employment.

Many procedures in cardiac surgery require extracorporeal circulation (EC). During EC, blood is pumped from a venous line to an oxygenator, cooled and infused back via an arterial line. This procedure is known to compromise the coagulation system, especially under prolonged hypothermia. The aim of the present study was to investigate the effects of EC and hypothermia on the coagulation system in a porcine model. The efficacy of a substitution therapy with a prothrombin complex concentrate (PCC) regarding normalization of coagulation and decrease in hemorrhage was evaluated.

Severe traumatic or intraoperative blood loss necessitates massive transfusion. This blood loss and the dilution of coagulation factors result in insufficient haemostasis. Treatments to enhance the haemostatic capacity need to be evaluated. An acquired bleeding model due to a dilutional coagulopathy was developed in the rabbit to estimate the contribution of PCC (Beriplex P/N; CSL Behring) on haemorrhage.

Introduction The preclinical efficacy and safety of rVIII-SingleChain (CSL627), a novel recombinant single-chain factor VIII, was assessed in a series of animal studies. Materials and Methods In the tail-clip bleeding model, hemophilia A mice were injected with escalating doses (1–150 IU/kg) of rVIII-SingleChain, B-domain deleted (BDD) rFVIII (ReFacto AF ® ), or full-length rFVIII products (Advate ® , Helixate ® ). Total blood loss and the percentage of animals in which hemostasis occurred were assessed in this observer-blinded, randomized study. In a second non-randomized study in hemophilia A mice, thromboelastographic analysis, thrombin generation, and activated partial thromboplastin time assays were performed. General safety and toxicity were assessed in three animal species, including determination of the prothrombotic potential of rVIII-SingleChain in a rabbit venous thrombosis model. Results Under acute bleeding conditions, the effect of rVIII-SingleChain on total blood loss and hemostasis was indistinguishable from BDD and full-length rFVIII. rVIII-SingleChain and full-length rFVIII (both 20 IU/kg) corrected thromboelastographic parameters, activated partial thromboplastin time, and thrombin generation to a similar degree in hemophilia A mice. In a thrombosis model, the effect of rVIII-SingleChain on thrombus incidence was non-significant and comparable to BDD rFVIII at doses up to 500 IU/kg. Treatment with rVIII-SingleChain did not cause anaphylactic reaction or local intolerance in safety and toxicity studies, and demonstrated an excellent overall safety profile. Conclusions rVIII-SingleChain showed convincing hemostatic efficacy and excellent tolerability in animal studies, warranting continued investigation in human Phase I/III trials (AFFINITY).

Introduction A novel fusion protein linking coagulation factor VIIa with albumin (rVIIa-FP) is currently undergoing clinical investigations. Objective This study was conducted to examine the biodistribution of rVIIa-FP in comparison to recombinant factor VIIa (rFVIIa). Materials and Methods [ 3 H]-rVIIa-FP (10 mg kg − 1 ) or [ 3 H]-rFVIIa (1.6 mg kg − 1 ) were administered intravenously to rats, followed by quantitative whole-body and knee joint autoradiography for 24 ([ 3 H]-rFVIIa) or 240 ([ 3 H]-rVIIa-FP) hours post-dose. Pharmacokinetic and excretion balance analyses were performed. Results In contrast to [ 3 H]-albumin, the tissue distributions of [ 3 H]-rVIIa-FP and [ 3 H]-rFVIIa were similar. Within the knee, both were rapidly present within synovial and mineralized regions. Importantly, rVIIa-FP- and albumin-derived radioactivity were detectable up to 72–120 hours, whereas [ 3 H]-rFVIIa signals were already close to detection limits at 24 hours. The longest rVIIa-FP retention times were observed in bone marrow and endosteum, in which the retention times were up to 5 times longer for rVIIa-FP compared with rFVIIa. Up to 8 hours post-dose, 100% of radioactivity was assigned to unchanged [ 3 H]-rVIIa-FP. Elimination of both proteins occurred primarily via the urine. Conclusions The data suggest that the FVIIa moiety is directing rVIIa-FP’s tissue distribution while the albumin moiety is responsible for the prolonged tissue retention. Importantly, rVIIa-FP is highly concentrated and retained over a long period in the growth plate of the knee joint − a vulnerable site in haemophilia patients. Overall, these improved tissue distribution characteristics of rVIIa-FP may enhance compliance and allow a more convenient dosing frequency.

The efflux of cellular cholesterol mediated by apoA-I and ATP-binding cassette transporter A1 (ABCA1) is thought to underlie many of the cardiovascular benefits of HDL. Several groups have manufactured apoA-I preparations with the aim of making an infused HDL therapy for the acute treatment of