ALBERT

Description: Cold agglutinin disease is a difficult-to-treat autoimmune hemolytic anemia in which immunoglobulin M antibodies bind to erythrocytes and fix complement, resulting in predominantly extravascular hemolysis. This trial tested the hypothesis that the anti-C1s antibody sutimlimab would ameliorate hemolytic anemia. Ten patients with cold agglutinin disease participated in the phase 1b component of a first-in-human trial. Patients received a test dose of 10-mg/kg sutimlimab followed by a full dose of 60 mg/kg 1 to 4 days later and 3 additional weekly doses of 60 mg/kg. All infusions were well tolerated without premedication. No drug-related serious adverse events were observed. Seven of 10 patients with cold agglutinin disease responded with a hemoglobin increase 〉2 g/dL. Sutimlimab rapidly increased hemoglobin levels by a median of 1.6 g/dL within the first week, and by a median of 3.9 g/dL (interquartile range, 1.3-4.5 g/dL; 95% confidence interval, 2.1-4.5) within 6 weeks (P = .005). Sutimlimab rapidly abrogated extravascular hemolysis, normalizing bilirubin levels within 24 hours in most patients and normalizing haptoglobin levels in 4 patients within 1 week. Hemolytic anemia recurred when drug levels were cleared from the circulation 3 to 4 weeks after the last dose of sutimlimab. Reexposure to sutimlimab in a named patient program recapitulated the control of hemolytic anemia. All 6 previously transfused patients became transfusion-free during treatment. Sutimlimab was safe, well tolerated, and rapidly stopped C1s complement–mediated hemolysis in patients with cold agglutinin disease, significantly increasing hemoglobin levels and precluding the need for transfusions. This trial was registered at www.clinicaltrials.gov as #NCT02502903.

Description: Background: The investigational anti von Willebrand Factor (vWF) aptamer ARC1779 effectively inhibits vWF activity in blood samples of controls and of patients suffering from thrombotic thrombocytopenic purpura (TTP) (Jilma et al, Blood2007;110:279a, Gilbert et al. Circulation2007;116:2678–2686). Methods: A 39 year old comatose male patient with acute (TTP) was treated with daily plasma exchange. Further, the patient received rituximab (375mg/m2 first treatment on day 8, and weekly thereafter for 8 weeks) and was splenectomized on day 18. Due to the refractory nature of his TTP, the patient received a concomitant intravenous infusion of ARC1779 at a rate of 2 μg/kg/min beginning on day 30. Results: ARC1779 increased the platelet count slightly from 7 to a maximum of 30/nL; during this period septicaemia and DIC may have blunted the rise in platelet counts. However, platelet counts dropped to 5/nL by 16h after cessation of infusion (day 34). The infusion of ARC1779 was re-started on day 37, and platelet counts increased from 9 to 45/nL. (Figure) Due to a temporary lack of drug, the dose of ARC1779 was stopped at 78h, and platelet counts fell to 12/nL by 12 h after interruption of the infusion. (circle in the Figure) When ARC1779 was re-started, platelet counts increased to a maximum of 97/nL and the patient’s neurologic status improved to near normal under therapy with ARC1779 over the next week. Conclusions: ARC1779 was well-tolerated, and caused a reproducible rise in platelet counts, which alleviated severe thrombocytopenia, in an otherwise refractory TTP case. This effect was reproducible under serial “re-challenge”. Together with the observed improvement in neurologic function, the data provide clinical proof-of-concept and suggest that ARC1779 treatment might improve the organ dysfunction which typically occurs in acute TTP. These data provide a rational basis for ongoing and planned phase II trials of ARC1779. Figure Figure

Description: Autoantibody mediated classical complement pathway (CP) activation has been hypothesized to drive hemolytic anemia in cold agglutinin disease (CAD) patients. Red blood cell (RBC) destruction is believed to occur as a result of C3 opsonin mediated extravascular hemolysis in the liver. We recently reported that TNT009, a humanized monoclonal antibody targeting the CP specific serine protease C1s, rapidly restores hemoglobin levels in severely anemic CAD patients. Here we describe the pharmacodynamic changes in the complement profile of patients to provide a mechanistic understanding of the hematological responses and therapeutic benefit observed following TNT009 treatment. In a Phase 1b trial, we enrolled 5 female CAD patients with severe anemia, one of whom had a lymphoplasmacytic lymphoma with 〉70% bone marrow infiltration and no measurable CP activity prior to dosing. This patient did not respond to TNT009 while on study and will be omitted from subsequent analyses. Patients were given an initial 10 mg/kg test dose of TNT009 on Day 1, followed by four weekly doses of 60 mg/kg on Day 2 or Day 5. Patients were followed for 4 weeks following the last dose (washout). Plasma and serum samples were collected throughout the study to measure TNT009 concentrations and to monitor serological markers of anemia and hemolysis. Additionally, futhan-containing plasma samples were collected to assess the levels of CP specific components including C1s, C1s-C1INH, and C1q by ELISA. RBCs were collected to monitor cell surface complement deposition (C3 fragments) via flow cytometry. Finally, C4 levels and an ELISA-based readout of CP activity were examined as measures of the pharmacodynamic effect of TNT009. Baseline levels of circulating C4 were either low or undetectable in CAD patients. Accordingly, serum CP activity was reduced compared to normal human serum samples. Following the first 60 mg/kg TNT009 dose, CP activity was immediately and completely inhibited within 15 minutes of dosing in all patients and remained inhibited for 3 weeks after the last dose. During this period of inhibition, C4 levels rose from a median circulating concentration of

Description: Background: ARC1779 is an aptamer which blocks the binding of the vWF A1 domain to platelet GPIb receptors. In TTP there is an excess of ultra-large multimers of vWF which are especially avid for binding GPIb and give rise to disseminated platelet thrombi which are fibrin-poor and vWF-rich in composition. ARC1779 is being evaluated for use as front-line therapy of acute TTP in conjunction with plasma exchange. ARC1779 has already been demonstrated in healthy volunteers to inhibit vWF activity and vWF-dependent platelet function. ARC1779 has no anticoagulant effect and does not inhibit other pathways of platelet activation. ARC1779 is expected to normalize platelet dysfunction and prevent the thrombotic end-organ complications of TTP based upon the mechanism of action defined for ARC1779 and the mechanism of thrombosis defined for TTP. Methods: We first assessed vWF activity (vWF:RiCO) and platelet function in blood samples taken from TTP patients and age-matched, healthy controls. We then studied the ex vivo dose response curves for ARC1779 on vWF activity (free A1 domain sites) and on platelet function assessed by the Platelet Function Analyzer (PFA-100®), cone and plate analyzer (IMPACT®), and agonist-induced impedence platelet aggregometry (Multiplate®) of TTP patients (N=10, 2 in acute phase and 8 in remission) and healthy age-matched controls (N=23). Results: vWF:RiCO activity (p=0.002) and vWF-dependent platelet plug formation (p=0.001) were increased in TTP patients relative to healthy controls, but agonist-induced platelet aggregation (ADP, arachidonic acid, collagen, TRAP) was not. ARC1779 fully blocked platelet plug formation as measured by PFA-100® with an IC100 of ∼ 1 mcg/mL with citrate anticoagulation, and ∼ 3–4 mcg/mL with hirudin anticoagulation in both TTP patients and in healthy controls. ARC1779 fully blocked shear-dependent platelet adhesion measured by the IMPACT® analyzer with an IC100 of ∼ 1 mcg/mL with citrate anticoagulation in both TTP patients and in healthy controls. ARC1779 fully blocked vWF activity (free A1 domain sites) with an IC90 of ∼ 6 mcg/mL in TTP patients and ∼ 2 mcg/mL in young controls (p

Description: Cold agglutinin disease (CAD) is an autoimmune hemolytic anemia (AIHA) characterized by the presence of autoantibodies (cold agglutinins) that bind red blood cells (RBCs) and activate the classical complement pathway (CP). We have previously shown in vitro that in contrast to C5 inhibition, inhibition of the CP specific protease C1s prevents complement opsonin deposition on cold agglutinin-sensitized RBCs and protects them from phagocytosis, underscoring the necessity to block upstream CP activity (Shi et al., Blood, 2014). Based on the strong scientific rationale and nonclinical data, a Phase 1 clinical trial for TNT009, a monoclonal antibody (mAb) inhibitor of C1s, has commenced at the Medical University of Vienna, Austria. Phase 1a consists of healthy volunteer cohorts in single- and multiple-ascending dose protocols for which interim study results will be presented. In the integrated protocol design of Phase 1b, TNT009 will be dosed in patients with diseases in which pathological CP activity has been implicated, including CAD, warm AIHA and additional non-hematologic indications. In anticipation of the clinical trial, we initiated a screening campaign in Vienna to find prospective CAD patients with serological markers of anemia and hemolysis. To date, plasma and serum samples have been collected from 15 CAD patients. Serum samples from 10 patients induce robust complement activation (C3b/iC3b deposition and/or hemolysis) on AET-treated human RBCs incubated in the patient's own complement-containing serum. In contrast to isotype control (IC), 100 mcg/mL of TNT003 (mouse parental mAb of TNT009), showed near complete inhibition of patient serum mediated C3b/iC3b deposition (90 ± 4%, n = 10; p〈 1 x 10-5) and hemolysis (93 ± 5 %, n = 9; p〈 1 x 10-5) (Fig. 1). To further support the rationale of C1s inhibition in CAD, we asked whether serological signs of anemia and hemolysis were associated with evidence of increased in vivo CP activity in patient samples. We first examined how well experimental laboratory results agreed with standard clinical readouts. We found good concordance between patient sample induced C3 deposition on RBCs (FACS) and clinical C3 DAT scores (p〈 .05). Furthermore, IgM staining on RBCs incubated in patient samples (FACS) correlated well with cold agglutinin titers determined in the clinic (p 〈 .001). Next, we observed that the extent of in vitro hemolysis correlated with C3d DAT scores (p〈 .05), LDH levels (p〈 .05), and bilirubin levels (p= .05). The agreement between the results from our in vitro patient sample-induced hemolysis assay with serologicalmarkers of complement activity, hemolysis and anemia used in the clinic suggest that our in vitro paradigm serves as a good model for in vivo complement activity in CAD patients. We then measured plasma C4 levels and CP activity in CAD serum samples (Wieslab Classical Pathway ELISA). We found that plasma C4 positively correlated with hemoglobin levels (p = .05). Additionally, we found an inverse correlation between serum CP activity and reticulocyte count (p 〈 .05) and bilirubin levels (p = .05). These data demonstrate that in vivo consumption of the CP and its components (low CP activity, low C4) is associated with markers of anemia and hemolysis (low hemoglobin, high reticulocyte counts, high bilirubin). Finally, an emerging literature calls attention to an increased thromboembolic risk in AIHA, similar to that seen in patients with other hemolytic anemias such as paroxysmal nocturnal hemoglobinuria. We therefore measured D-dimer levels and found it significantly elevated in CAD patient plasma compared to healthy controls (p 〈 .0001). Preliminary analyses show an inverse correlation of C4 and D-dimer in patient plasma (p 〈 .05) suggesting that in vivo CP activity may contribute to the elevated thromboembolic risk in patients (Fig. 2). On-going analyses for other markers of thrombosis, in addition to other experimental approaches to assess the hypercoagulable state in these patients will seek to corroborate this finding. The successful identification of CAD patients with altered complement and hematological profiles provides a unique opportunity to assess proof-of-concept early in the clinical development of TNT009. Figure 1. TNT009 Parental mAb (TNT003) Inhibits CAD Serum Mediated Complement Activation on AET-Treated Human RBCs Figure 1. TNT009 Parental mAb (TNT003) Inhibits CAD Serum Mediated Complement Activation on AET-Treated Human RBCs Figure 2. Elevated D-dimer Correlates with Lower C4 Levels in CAD Patient Plasma Figure 2. Elevated D-dimer Correlates with Lower C4 Levels in CAD Patient Plasma Disclosures Jaeger: Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; True North Therapeutics, Inc.: Research Funding; Hoffmann La Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Rose:True North Therapeutics, Inc.: Employment, Equity Ownership. Singh:True North Therapeutics, Inc.: Employment, Equity Ownership. Jilma:True North Therapeutics, Inc.: Consultancy, Research Funding. Gilbert:True North Therapeutics, Inc.: Employment, Equity Ownership. Panicker:True North Therapeutics, Inc.: Employment, Equity Ownership, Patents & Royalties.

Description: Background: We recently presented that the humanized anti-complement C1s monoclonal antibody, TNT009, rapidly stoppedhemolysisand correctedanemiain patients with severe CAD in an ongoing Phase 1b clinical trial. After completion of washout from protocol treatment, these patients suffered relapse of CAD with a drop inhemoglobinto pre-dose levels. We now report on the clinical experience of their extended re-treatment under the Named Patient provision of the Austrian Drug Act, which provided the occasion: 1) to explore lower dose regimens and alternative dosing schedules; 2) to try to recapitulate the initial success of complete remission under protocol treatment; 3) to test the durability of response with a longer term maintenance treatment with TNT009. Methods: In order to test a lower dose regimen of TNT009, the Phase 1b trial dosing of 60 mg/kg IV once weekly for both loading and maintenance was reduced to 45 mg/kg IV once weekly (4x) followed by maintenance with 45 mg/kg every other week. A simplified dosing regimen was then tried, using a single priming dose of 60 mg/kg on Day 0 followed by 60 mg/kg every other week (biweekly) thereafter beginning on Day 7. This TNT009 biweekly maintenance with 60 mg/kg was continued indefinitely to test the durability of the effect, to monitor for safety, and to serially assess their hematologic response to TNT009. Results:All infusions were well tolerated without premedication and without relevant adverse effects. TNT009 re-exposure immediately decreased CH50 activity and increased complement C4 levels, confirming the rapid onset ofpharmacodynamiceffect. The concordant and sustained effect of TNT009 on hematologic parameters related tohemolysisandanemiasis illustrated in the composite Figure 1 for the first patient treated, C1001. This patient has undergone three treatment episodes with TNT009: weekly dosing at 60 mg/kg under the Phase 1b protocol (left); weekly → biweekly 45 mg/kg (center); 60 mg/kg on Day 0 and biweekly thereafter starting on Day 7 (right). Note that the CH50 serves as apharmacodynamicmarker for the effective inhibition of the classical complement pathway by TNT009. Each new episode of treatment with TNT009 induced remission ofhemolyticanemia, and relapse occurred following washout (60 mg/kg weekly x 4) orpharmacodynamicbreakthrough (45 mg/kg maintenance regimen). Although it appears that the reduced dose maintenance regimen of 45 mg/kg biweekly does not suffice to maintain full C1s inhibition, there is no evidence oftachyphylaxisas restoration of full dose treatment at 60 mg/kg biweekly was able to restore remission again. A similar pattern of results was observed in the other patients offered Named Patient treatment with TNT009, but with two instructive exceptions. Patient C1004 had unsuspected erythropoietin deficiency and did not achieve complete normalization ofhemoglobinuntil concomitant treatment with erythropoietin was begun. Patient C1003 did not have CAD but Cold Agglutinin Syndrome (CAS) secondary to an extensivelymphoplasmacyticlymphoma (LPL). Initially she was treated with TNT009 monotherapy and did not respond. TNT009 was then stopped and her LPL was treated withibrutinib, resulting in a gradual amelioration of heranemiauntil anintercurrentinfection triggered relapse of herhemolyticanemia. At that time re-treatment with TNT009 (whileibrutinibwas continued) quickly normalized her hematologic parameters. Eventually TNT009 was able to increasehemoglobinlevels to 〉12 g/dLin all 5 of 5 treated patients. Conclusion:TNT009 was previously shown to induce rapid correction ofhemolyticanemiain severe CAD patients in a Phase 1b trial. It has now been shown that subsequent washout of TNT009 leads to relapse in all patients, and that re-induction of remission by TNT009 is possible in all. Long-term maintenance of remission can be achieved by dosing TNT009 at 60 mg/kg biweekly, the dose regimen to be tested in larger-scale clinical trials of CAD. Figure Figure. Disclosures Gilbert: Truenorth Therapeutics, Inc.: Employment, Equity Ownership. Panicker:Truenorth Therapeutics, Inc.: Employment. Parry:Truenorth Therapeutics, Inc.: Employment, Equity Ownership. Jaeger:Janssen: Consultancy, Honoraria, Other: Travel, Accommodations, Expenses.

Description: Background: Thrombotic thrombocytopenic purpura (TTP) has a high morbidity and mortality rate despite current standard therapy comprising plasma exchange (PEX). Aim of this prospective clinical trial was to test the safety and efficacy of the anti von Willebrand Factor aptamer ARC1779 in patients with relapsing TTP, and to find possible proof of concept. Methods: Three patients with relapsing TTP (with detectable anti-ADAMTS13 autoantibodies) received bolus primed continuous intravenous infusions of ARC1779 (0.002 mg/kg/min) on top of standard PEX therapy until remission of TTP was induced, or for 14 days whatever came first. ARC1779 concentrations were quantified by a highperformance liquid chromatography/ultraviolet assay, the inhibitory effect of ARC1779 on vWF activity was evaluated with an ELISA kit (READDS vWF Activity ELISA Test Kit, Corgenix, Inc, Westminster, Colo), and platelet function was assessed with the platelet function analyzer (PFA-100). Results: ARC1779 was well tolerated without any evidence for bleeding in these patients. Median ARC1779 concentrations of approximately 10μg/mL in plasma were reached under steady state, and inhibited the collagen binding site of the vWF A1 domain by 〉95%. While plasma exchange (PEX) removed some 50% of the drug from the blood, ARC1779 concentrations could be restored by subsequent mini boluses. Platelet counts in all patients normalized with concurrent ARC1779 and PEX: 2 days after initiation of ARC1779 in two patients, and after 6 days in the third. In the third, the time course of response (see Figure) provides support for the concept that ARC1779 can restore platelet counts, and thus interferes with the disease process. While PEX was stopped when platelet counts reached 125/nL, 4 more days of therapy with ARC1779 increased platelet counts to 260/nL. Discontinuation of the ARC1779 infusion was associated with an immediate drop in platelet counts, so that PEX had to be restarted after 2 days (Figure). Conclusion: These data indicate that ARC1779 effectively inhibits VWF and thereby increases platelet counts in TTP patients. Addition of ARC1779 to PEX may have the potential to accelerate recovery from organ dysfunction in TTP and thereby decrease morbidity/mortality. Figure Figure

Description: Introduction: Von Willebrand Disease (VWD) Type 2B is characterized by increased affinity of VWF for the platelet glycoprotein receptor Ib (GPIb). Consequently, infusion of desmopressin, a standard of treatment for other forms of VWD, causes transient thrombocytopenia in patients with VWD-2B. The aim of the study was to test whether the novel anti-VWF aptamer ARC1779, which inhibits the interaction of VWF with GPIb, could abrogate the transient desmopressin-induced thrombocytopenia in VWD-2B. Patients and Methods: This was part of a clinical trial using a double-blind, randomized, placebo-controlled, crossover design. A patient with VWD 2B received desmopressin (0.4 mcg/kg) alone, or desmopressin after pre-treatment with ARC1779 targeted to a plasma concentration of 10 mcg/mL. Multiple blood samples were obtained over 24 hours to measure von Willebrand Factor Antigen, Ristocetin Cofactor (RiCO), FVIII activity and multimers. Results: Desmopressin alone (open circles) increased VWF:Ag by 38% (not shown), and VWF:RiCo 3.4-fold within 30 min, and profoundly decreased platelet counts by 87%. In striking contrast, ARC1779 (solid triangles) completely prevented the desmopressin induced fall in platelet counts (Figure), although the increase in vWF:Ag and vWF:RiCO was greater (2.7-fold and 12-fold, respectively). Conclusions: This randomized, double-blind, placebo-controlled experiment shows that ARC1779 effectively prevents consumption of VWF and platelets in response to desmopressin in VWD Type 2B. It provides in vivo proof of concept that ARC1779 is a potent inhibitor of the VWF A1 domain interaction with GPIb. Figure Figure