Abstract Interleukin-6 (IL-6) is a key pleiotropic cytokine implicated in kidney injury; however, the therapeutic potential of IL-6 blockade against acute ischemia–reperfusion injury (IRI) remains uncertain, with previous murine studies yielding conflicting results. This study investigated the protective effects of the high-affinity chimeric anti-IL-6 monoclonal antibody, cALD518-P18, in a non-transplant mouse model of warm renal IRI (22 min ischemia, 24 h reperfusion). Male C57BL/6 mice received a single 40 mg/kg dose of cALD518-P18 or an isotype control, administered either intraperitoneally 30 min before ischemia or intravenously immediately after ischemia. At 24 h, control mice exhibited significant kidney dysfunction (serum creatinine, urea), injury, and inflammation, consistent with elevated pro-inflammatory IL-6 trans-signalling (soluble IL-6R (sIL-6R) and tubular STAT3 phosphorylation (pSTAT3)). Treatment with cALD518-P18, in both regimens, significantly protected the kidneys from IRI, demonstrating improved renal function and reduced tissue damage. The antibody attenuated inflammation, endothelial activation and pSTAT3 signalling, significantly downregulating gene expression of the acute tubular stress marker NGAL. These findings confirm the pathogenic role of IL-6 and highlight its early blockade with cALD518-P18 as a promising therapeutic strategy to improve outcomes in acute kidney injury and transplantation.

Objectives We previously reported that CCL17 gene-deficient mice are protected from developing pain-like behaviour and exhibit less disease in destabilization of medial meniscus (DMM)-induced OA, as well as in high-fat diet (HFD)-exacerbated DMM-induced OA. Here, we explored if therapeutic neutralization of CCL17, using increasing doses of a neutralizing monoclonal antibody (mAb), would lead to a dose-dependent benefit in these two models. Design DMM-induced OA was initiated in male mice either fed with a control diet (7% fat) or 8 weeks of a 60% HFD, followed by therapeutic intraperitoneal administration (i.e. when pain is evident) of an anti-CCL17 mAb (B293, 25mg/kg, 5mg/kg or 1mg/kg) or isotype control (BM4; 25mg/kg). Pain-like behaviour and arthritis were assessed by relative static weight distribution and histology, respectively. The effects of B293 (25mg/kg) on HFD-induced metabolic changes, namely oral glucose tolerance test, insulin tolerance test and liver triglyceride levels, were examined. Results Therapeutic administration of B293 results in a dramatic amelioration of DMM-induced OA pain-like behaviour and the inhibition of disease progression, compared to BM4 (isotype control) treatment. A similar therapeutic effect was observed in HFD-exacerbated OA pain-like behaviour and disease. B293 treatment did not alter the measured HFD-induced metabolic changes. Conclusions Based on the data presented, CCL17 could be a therapeutic target in OA patients with joint injury alone or with obesity.

Background: Complement activation is a key driver of inflammation following lung transplantation and contributes to the development of primary graft dysfunction (PGD). Excessive complement activity leads to endothelial damage, increased vascular permeability, and immune cell infiltration, worsening lung injury post-transplant. Given this here we assessed the therapeutic efficacy of CSL040, a novel soluble CR1-based complement therapeutic, as a means to reduce the severity of PGD, thereby improving lung transplant outcomes. Methods: To assess CSL040 therapeutic efficacy, we utilize two rodent models (mouse and rat) of lung transplantation. For clinical relevance we utilized a brain-dead donor (BD) allogeneic lung transplant model. Donor lungs were procured from BD donors (Fischer and Balb/c), cold stored for 18 h, prior to orthotopic transplantation into allogeneic recipients (Wistar and C57Bl/6). CSL040 was administered immediately post-transplant (0 h) and at 24 h post transplantation, at a dose of 30 mg/kg. Grafts were harvested at 48 h post-transplant and assessed using the American Thoracic Society recommendations. ATS guidelines were devised to ensure consistency in preclinical lung research and to improve translational potential to human disease. Specifically, three domains were assessed: lung histopathology (e.g., alveolar damage, inflammatory cell infiltration, edema), gas exchange (e.g., arterial blood gases), and inflammation (MPO). Additionally, the impact on local complement activation was assessed by immunohistochemistry for C3d. Results: CSL040 was similarly effective in both rat and mouse lung transplant models. CSL040 provided effective protection from PGD as determined by significantly reduced histological evidence of injury, improved lung function as measured by PaO2, and reduced inflammation, as shown by reduced immune cell infiltration into the transplanted lung (MPO+ cells). Analysis of complement deposition showed that CSL040 treatment was associated with a significant reduction in C3d, in both species. Conclusion: Taken together here we demonstrate, using two rodent lung transplant models that incorporate the clinically relevant BD donor injury, that CSL040 significantly improved transplant outcomes. These findings highlight CSL040 as a promising therapeutic strategy for enhancing lung transplant success and warrant further investigation in clinical settings.

Objectives: We have previously identified a granulocyte macrophage-colony stimulating factor (GM-CSF)/C-C motif ligand 17 (CCL17) pathway in monocytes/macrophages, in which GM-CSF regulates the formation of CCL17, and it is important for an experimental osteoarthritis (OA) model. We explore here additional OA models, including in the presence of obesity, such as a requirement for this pathway.Design: The roles of GM-CSF, CCL17, CCR4, and CCL22 in various experimental OA models, including those incorporating obesity (eight-week high-fat diet), were investigated using gene-deficient male mice. Painlike behavior and arthritis were assessed by relative static weight distribution and histology, respectively. Cell populations (flow cytometry) and cytokine messenger RNA (mRNA) expression (qPCR) in knee infrapatellar fat pad were analyzed. Human OA sera were collected for circulating CCL17 levels (ELISA) and OA knee synovial tissue for gene expression (qPCR).Results: We present evidence that: i) GM-CSF, CCL17, and CCR4, but not CCL22, are required for the development of pain-like behavior and optimal disease in three experimental OA models, as well as for exacerbated OA development due to obesity, ii) obesity alone leads to spontaneous knee joint damage in a GM-CSF- and CCL17-dependent manner, and iii) in knee OA patients, early indications are that BMI correlates with a lower Oxford Knee Score (r = -0.458 and p = 0.0096), with elevated circulating CCL17 levels (r = 0.2108 and p = 0.0153) and with elevated GM-CSF and CCL17 gene expression in OA synovial tissue.Conclusions: The above findings indicate that GM-CSF, CCL17, and CCR4 are involved in obesity-associated OA development, broadening their potential as targets for possible treatments for OA. (c) 2023 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

OBJECTIVES:We have previously identified a granulocyte macrophage-colony stimulating factor (GM-CSF)/C-C motif ligand 17 (CCL17) pathway in monocytes/macrophages, in which GM-CSF regulates the formation of CCL17, and it is important for an experimental osteoarthritis (OA) model. We explore here additional OA models, including in the presence of obesity, such as a requirement for this pathway. DESIGN:The roles of GM-CSF, CCL17, CCR4, and CCL22 in various experimental OA models, including those incorporating obesity (eight-week high-fat diet), were investigated using gene-deficient male mice. Pain-like behavior and arthritis were assessed by relative static weight distribution and histology, respectively. Cell populations (flow cytometry) and cytokine messenger RNA (mRNA) expression (qPCR) in knee infrapatellar fat pad were analyzed. Human OA sera were collected for circulating CCL17 levels (ELISA) and OA knee synovial tissue for gene expression (qPCR). RESULTS:We present evidence that: i) GM-CSF, CCL17, and CCR4, but not CCL22, are required for the development of pain-like behavior and optimal disease in three experimental OA models, as well as for exacerbated OA development due to obesity, ii) obesity alone leads to spontaneous knee joint damage in a GM-CSF- and CCL17-dependent manner, and iii) in knee OA patients, early indications are that BMI correlates with a lower Oxford Knee Score (r = -0.458 and p = 0.0096), with elevated circulating CCL17 levels (r = 0.2108 and p = 0.0153) and with elevated GM-CSF and CCL17 gene expression in OA synovial tissue. CONCLUSIONS:The above findings indicate that GM-CSF, CCL17, and CCR4 are involved in obesity-associated OA development, broadening their potential as targets for possible treatments for OA.

Background 3F7 is a monoclonal antibody targeting the enzymatic pocket of activated factor XII (FXIIa), thereby inhibiting its catalytic activity. Given the emerging role of FXIIa in promoting thromboinflammation, along with its apparent redundancy for hemostasis, the selective inhibition of FXIIa represents a novel and highly attractive approach targeting pathogenic processes that cause thromboinflammation-driven cardiovascular diseases. Methods The effects of FXIIa inhibition were investigated using three distinct mouse models of cardiovascular disease—angiotensin II-induced abdominal aortic aneurysm (AAA), an ApoE−/− model of atherosclerosis, and a tandem stenosis model of atherosclerotic plaque instability. 3F7 or its isotype control, BM4, was administered to mice (10 mg/kg) on alternate days for 4 to 8 weeks, depending on the experimental model. Mice were examined for the development and size of AAAs, or the burden and instability of atherosclerosis and associated markers of inflammation. Results Inhibition of FXIIa resulted in a reduced incidence of larger AAAs, with less acute aortic ruptures and an associated fibro-protective phenotype. FXIIa inhibition also decreased stable atherosclerotic plaque burden and achieved plaque stabilization associated with increased deposition of fibrous structures, a >2-fold thicker fibrous cap, increased cap-to-core ratio, and reduction in localized and systemic inflammatory markers. Conclusion Inhibition of FXIIa attenuates disease severity across three mouse models of thromboinflammation-driven cardiovascular diseases. Specifically, the FXIIa-inhibiting monoclonal antibody 3F7 reduces AAA severity, inhibits the development of atherosclerosis, and stabilizes vulnerable plaques. Ultimately, clinical trials in patients with cardiovascular diseases such as AAA and atherosclerosis are warranted to demonstrate the therapeutic potential of FXIIa inhibition.

Factor XII (FXII) is a serine protease involved in multiple cascades, including the kallikrein-kinin system. It may play a role in diseases in which the downstream cascades are dysregulated, such as hereditary angioedema. Garadacimab (CSL312) is a first-in-class, fully human, monoclonal antibody targeting activated FXII (FXIIa). We describe how translational pharmacokinetic (PK) and pharmacodynamic (PD) modeling enabled dose selection for the phase I, first-in-human trial of garadacimab. The PK/PD data used for modeling were derived from preclinical PK/PD and safety studies. Garadacimab plasma concentrations rose with increasing dose, and clear dose-related PD effects were observed (e.g., a mechanism-based prolongation of activated partial thromboplastin time). The PK/PD profile from cynomolgus monkeys was used to generate minimal physiologically-based pharmacokinetic (mPBPK) models with target-mediated drug disposition (TMDD) for data prediction in cynomolgus monkeys. These models were later adapted for prediction of human data to establish dose selection. Based on the final mPBPK model with TMDD and assuming a weight of 70 kg for an adult human, a minimal inhibition (<10%) of FXIIa with a starting dose of 0.1 mg/kg garadacimab and a near maximal inhibition (>95%) at 10 mg/kg garadacimab were predicted. The phase I study is complete, and data on exposure profiles and inhibition of FXIIa-mediated kallikrein activity observed in the trial support and validate these simulations. This emphasizes the utility and relevance of translational modeling and simulation in drug development.

Hereditary angioedema (HAE) is a rare genetic disorder with 4 known disease types that are all clinically characterized by local swellings of the skin (ie, edema of the extremities, genitals, and face), abdominal pain attacks, and, occasionally, life-threatening attacks of laryngeal edema.1Bork K. Pasteurized C1 inhibitor concentrate in hereditary angioedema: pharmacology, safety, efficacy and future directions.Expert Rev Clin Immunol. 2008; 4: 13-20Crossref PubMed Scopus (37) Google Scholar In the event of an HAE attack, therapeutic options include the intravenous administration of C1-inhibitor (C1-INH) concentrate,1Bork K. Pasteurized C1 inhibitor concentrate in hereditary angioedema: pharmacology, safety, efficacy and future directions.Expert Rev Clin Immunol. 2008; 4: 13-20Crossref PubMed Scopus (37) Google Scholar, 2Gompels M.M. Lock R.J. Abinun M. Bethune C.A. Davies G. Grattan C. et al.C1 inhibitor deficiency: consensus document.Clin Exp Immunol. 2005; 139: 379-394Crossref PubMed Scopus (379) Google Scholar kallikrein inhibitors (eg, Ecallantide), and a bradykinin (BK) receptor antagonist (eg, Icatibant). Despite a number of acute treatment options for HAE attacks, the prophylactic treatment of HAE remains an area of unmet medical need. Current prophylactic treatments include intravenously administered C1-INH (CINRYZE), and the recently approved subcutaneous C1-INH product HAEGARDA. Factor XII (FXII) is the principal initiator of the plasma contact system and autoactivates to FXIIa upon contact with a negatively charged surface. Activation of FXII leads to the production of the proinflammatory mediator BK through the kallikrein-kinin system (KKS). The binding of BK to BK type 2 receptors activates various intracellular signaling pathways that dilate vessels, induce chemotaxis of neutrophils, and increase vascular permeability and fluid efflux.3Björkqvist J. Jämsä A. Renné T. Plasma kallikrein: the bradykinin-producing enzyme.Thromb Haemost. 2013; 110: 399-407Crossref PubMed Scopus (106) Google Scholar BK production is increased during HAE attacks and is thought to be the primary mediator of swelling in HAE.4Nussberger J. Cugno M. Amstutz C. Cicardi M. Pellacani A. Agostoni A. Plasma bradykinin in angio-oedema.Lancet. 1998; 351: 1693-1697Abstract Full Text Full Text PDF PubMed Scopus (639) Google Scholar, 5Nussberger J. Cugno M. Cicardi M. Agostoni A. Local bradykinin generation in hereditary angioedema.J Allergy Clin Immunol. 1999; 104: 1321-1322Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar Like BK, plasma levels of FXIIa have also been shown to increase in patients during HAE attacks.6Csuka D. Veszeli N. Imreh É. Zotter Z. Skopál J. Prohászka Z. et al.Comprehensive study into the activation of the plasma enzyme systems during attacks of hereditary angioedema due to C1-inhibitor deficiency.Orphanet J Rare Dis. 2015; 10: 132Crossref PubMed Scopus (33) Google Scholar, 7Cugno M. Cicardi M. Coppola R. Agostoni A. Activation of factor XII and cleavage of high molecular weight kininogen during acute attacks in hereditary and acquired C1-inhibitor deficiencies.Immunopharmacology. 1996; 33: 361-364Crossref PubMed Scopus (62) Google Scholar However, although FXII is key to the initiation of the plasma contact system, it has not been explored as a target for the treatment of HAE. We previously reported the generation of a fully human recombinant antibody (3F7) that specifically and potently inhibited FXIIa and prevented contact-mediated thrombosis without impairing hemostasis in mouse and rabbit models.8Larsson M. Rayzman V. Nolte M.W. Nickel K.F. Björkqvist J. Jämsä A. et al.A factor XIIa inhibitory antibody provides thromboprotection in extracorporeal circulation without increasing bleeding risk.Sci Transl Med. 2014; 6: 222ra17Crossref PubMed Scopus (232) Google Scholar In this study, we investigate the potential for 3F7, and its affinity matured variant CSL312, to modulate KKS-mediated BK production and subsequent edema formation in a number of in vitro assays and in vivo models. We first examined the effect of 3F7 on mast cell heparin-initiated activation of the KKS using a murine passive cutaneous anaphylaxis model.9Oschatz C. Maas C. Lecher B. Jansen T. Björkqvist J. Tradler T. et al.Mast cells increase vascular permeability by heparin-initiated bradykinin formation in vivo.Immunity. 2011; 34: 258-268Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar 3F7 administered intraperitoneally 4 hours before antigen challenge dose-dependently inhibited ear skin edema, with approximately 90% inhibition observed at 25 mg/kg of 3F7 (Fig 1, A and B). Benchmarking studies were performed to compare the efficacy of 3F7 with the HAE drugs Icatibant (a BK receptor 2 antagonist) and Ecallantide (a plasma kallikrein inhibitor). When administered 10 minutes before antigen challenge, Icatibant and Ecallantide treatment inhibited edema by approximately 40% and 65%, respectively, compared with approximately 90% inhibition with 3F7 (Fig 1, C). Although 3F7 maintained marked inhibition of the passive cutaneous anaphylaxis response when administered 4 hours before antigen injection, the other inhibitors administered at the same time frame displayed reduced efficacy (Fig 1, D), likely because of faster serum clearance. These results highlight the potential of anti-FXIIa antibodies to provide prolonged protection from edema, with significance for prophylactic management of HAE. The effect of 3F7 was also investigated in a C1-INH−/− murine model of angiotensin-converting enzyme inhibitor–induced angioedema. Captopril treatment increases vascular permeability, which can be quantitated by extraction of coadministered Evans blue dye from the colons of treated mice, and was greater in C1-INH−/− than in their wild-type littermates (Fig 1, E). 3F7 administered before captopril treatment ablated vascular permeability increase at doses of 2.5 mg/kg or greater (Fig 1, F). To enhance the potency of 3F7, affinity maturation was undertaken and a lead antibody clone (CSL312) was selected for further development. Compared with the parental 3F7 antibody,8Larsson M. Rayzman V. Nolte M.W. Nickel K.F. Björkqvist J. Jämsä A. et al.A factor XIIa inhibitory antibody provides thromboprotection in extracorporeal circulation without increasing bleeding risk.Sci Transl Med. 2014; 6: 222ra17Crossref PubMed Scopus (232) Google Scholar CSL312 displays a 44-fold increase in affinity to activated βFXIIa (KD = 140 pM) and improved potency in an activated partial thromboplastin time (aPTT) assay with human plasma (see Fig E1 in this article's Online Repository at www.jacionline.org). Although displaying picomolar affinity to activated FXII, CSL312 binding to zymogen FXII is a weaker interaction with rapid dissociation (see Fig E2 in this article's Online Repository at www.jacionline.org). CSL312 specificity for human FXII was tested against a panel of relevant human serine proteases (FVIIa, FIXa, FXa, FXIa, kallikrein, tissue plasminogen activator, activated protein C, and urokinase plasminogen activator) using in vitro chromogenic activity assays. At enzyme to antibody ratios of up to 1:500, no IC50 concentration was reached for any protease tested (data not shown). Prothrombin time was unaltered at any CSL312 concentration tested (data not shown). CSL312 potently inhibits FXIIa chromogenic activity following dextran sulfate (DXS) activation of diluted plasma from normal, HAE types I/II, HAE with normal C1-INH (nC1-INH-HAE), and acquired angioedema with C1-INH deficiency (C1-INH-AAE) donors (see Fig E3 in this article's Online Repository at www.jacionline.org), with maximal FXIIa inhibition observed at 10 μg/mL for all plasma samples tested. A small but statistically significant increase in FXII levels was observed in patients with C1-INH-HAE type I and nC1-INH-HAE when compared with normal donors. An LC-MS/MS assay was developed to measure the concentration of BK in undiluted plasma. Using DXS-activated pooled healthy human plasma, CSL312 completely inhibited BK generation at concentrations of more than 133 nM (Fig 2, A). When compared against clinically approved HAE therapeutics (C1-INH and Ecallantide), CSL312 displayed the most potent inhibition (Fig 2, A). This assay was also used to investigate the efficacy of CSL312 with 48 HAE plasma samples and 4 cynomolgus plasma samples (Fig 2, B). Here, it can be seen that all HAE and cynomolgus samples were completely protected against DXS-mediated activation, with most HAE samples requiring less than 200 nM (30 μg/mL) of CSL312 for complete inhibition and no observed difference in the efficacy of CSL312 between HAE types. Cynomolgus plasma samples require a slightly higher CSL312 concentration (267 nM) for complete protection. The pharmacokinetic and pharmacodynamic profile of CSL312 following intravenous infusion to cynomolgus monkeys was investigated. The pharmacokinetics of CSL312 in cynomologus monkeys were typical for an mAb (Fig 2, C). The beta phase constituted approximately 40% of total clearance, and the terminal half-life was approximately 9.1 days, with neither parameter showing any significant nonlinearity over the dose range (3, 9, 27 mg/kg), as determined by ANOVA. CSL312 administration was well tolerated and a dose-dependent and prolonged increase in aPTT was observed in animals treated with CSL312 (Fig 2, D). There was no increase in prothrombin time at any doses tested (data not shown). In this study, we show that specific antibody-mediated inhibition of FXII protease activity effectively reduced edema formation in animal models and prevented BK formation following contact activation of normal, HAE (C1-INH-HAE type I/II and HAE with normal C1-INH), and acquired angioedema plasma samples. CSL312 displayed a long plasma half-life in cynomolgous monkeys of 9.1 days and prolonged inhibition of FXIIa activity following a single dose over the course of a 36-day study. Coupled with the ability to formulate at high concentrations (>100 mg/mL), it is well suited to subcutaneous prophylactic application in HAE with potential dosing regimens of once every 2 weeks, or less frequently. We are currently undertaking a phase I clinical study with CSL312 in normal human volunteers and aim to investigate the efficacy of CSL312 in patients with HAE in the near future. 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To date, the major target of biologic therapeutics in systemic lupus erythematosus (SLE) has been the B cell, which produces pathogenic autoantibodies. Recently, targeting type I IFN, which is elaborated by plasmacytoid dendritic cells (pDCs) in response to endosomal TLR7 and TLR9 stimulation by SLE immune complexes, has shown promising results. pDCs express high levels of the IL-3Rα chain (CD123), suggesting an alternative potential targeting strategy. We have developed an anti-CD123 monoclonal antibody, CSL362, and show here that it affects key cell types and cytokines that contribute to SLE. CSL362 potently depletes pDCs via antibody-dependent cell-mediated cytotoxicity, markedly reducing TLR7, TLR9, and SLE serum-induced IFN-α production and IFN-α-upregulated gene expression. The antibody also inhibits TLR7- and TLR9-induced plasmablast expansion by reducing IFN-α and IL-6 production. These effects are more pronounced than with IFN-α blockade alone, possibly because pDC depletion reduces production of other IFN subtypes, such as type III, as well as non-IFN proinflammatory cytokines, such as IL-6. In addition, CSL362 depletes basophils and inhibits IL-3 signaling. These effects were confirmed in cells derived from a heterogeneous population of SLE donors, various IFN-dependent autoimmune diseases, and healthy controls. We also demonstrate in vivo activity of CSL362 following its s.c. administration to cynomolgus monkeys. This spectrum of effects provides a preclinical rationale for the therapeutic evaluation of CSL362 in SLE.

Hereditary angioedema (HAE) is characterized by recurrent and potentially life-threatening bradykinin-mediated edema. There is an unmet medical need for a safe and long-lasting prophylactic treatment for HAE. We have generated a potent and specific FXIIa blocking antibody and demonstrated that targeting FXII represents a promising prophylactic therapy for HAE. A human phage display antibody library was used to generate a fully human recombinant anti-FXIIa antibody. Enzyme assays were performed to determine its specific and potent inhibition of FXIIa. The in vivo efficacy was investigated in various murine edema models. The anti-FXIIa mAb 3F7 is a potent and highly specific inhibitor of the proteolytic activity of FXIIa. 3F7 binds to rabbit, mouse and human activated FXII. Administration of 3F7 abrogated skin edema induced by contact activation triggered by mast-cell released heparin in mice. 3F7 was also shown to abolish bradykinin-mediated increase of vascular permeability induced by the angiotensin-converting enzyme (ACE) inhibitor captopril in C1-inhibitor deficient mice. Comparison of 3F7 with current HAE therapeutics in these murine edema models revealed that 3F7 has potent and prolonged efficacy. CSL312, a variant of 3F7 with improved affinity and potency, effectively inhibited dextran sulphate-triggered FXII contact activation and bradykinin formation in plasma of healthy donors and HAE patients. The preclinical data provide strong evidence that CSL312, a potent and specific inhibitor of FXIIa, represents an effective prophylactic treatment option for HAE.

Despite the remarkable efficacy of tyrosine kinase inhibitors (TKIs) in eliminating differentiated chronic myeloid leukemia (CML) cells, recent evidence suggests that leukemic stem and progenitor cells (LSPCs) persist long term, which may be partly attributable to cytokine-mediated resistance. We evaluated the expression of the interleukin 3 (IL-3) receptor α subunit (CD123), an established marker of acute myeloid leukemia stem cells, on CML LSPCs and the potential of targeting those cells with the humanized anti-CD123 monoclonal antibody CSL362. Compared with normal donors, CD123 expression was higher in CD34(+)/CD38(-) cells of both chronic phase and blast crisis CML patients, with levels increasing upon disease progression. CSL362 effectively targeted CML LSPCs by selective antibody-dependent cell-mediated cytotoxicity (ADCC)-facilitated lysis of CD123(+) cells and reduced leukemic engraftment in mice. Importantly, not only were healthy donor allogeneic natural killer (NK) cells able to mount an effective CSL362-mediated ADCC response, but so were CML patients' autologous NK cells. In addition, CSL362 also neutralized IL-3-mediated rescue of TKI-induced cell death. Notably, combination of TKI- and CSL362-induced ADCC caused even greater reduction of CML progenitors and further augmented their preferential elimination over normal hematopoietic stem and progenitor cells. Thus, our data support the further evaluation of CSL362 therapy in CML.

Introduction: Early molecular response (EMR, BCR-ABL (IS) ≤ 10% at 3 months) is a strong predictor of outcome in imatinib-treated chronic phase chronic myeloid leukemia (CP-CML) patients, but for patients who transform early 3 months may be too late for effective therapeutic intervention. Thus, alternative approaches are required to identify poor responders at the time of diagnosis. The aim of this study was to identify plasma biomarkers at diagnosis that will predict for subsequent EMR failure, early transformation or the development of BCR-ABL1 kinase domain mutations. Cytokine profiling has proven valuable in identifying prognostic factors in myelofibrosis and myelodysplastic syndromes; however, similar comprehensive studies are lacking to date in CML.

Abstract 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 Abstract 3598 The interleukin-3 receptor alpha chain (IL-3Rα/CD123) is expressed in a variety of hematological malignancies including AML, MDS, B-ALL, Hodgkin's lymphoma, hairy cell leukemia, systemic mastocytosis, plasmacytoid dendritic cell leukemia and CML. In AML, the majority of AML blasts express CD123 and this receptor is selectively over expressed on CD34+CD38− leukemic stem cells (LSC) compared to normal hematopoietic stem cells. This difference may provide a biological advantage to the leukemic cells given the survival and proliferation-promoting activities of IL-3, whilst at the same time providing an opportunity to target these malignant cells selectively. We have shown previously that 7G3, a mouse monoclonal antibody (mAb) which blocks IL-3 binding to CD123, is capable of eliminating human LSC in a mouse model of human AML by a combination of mechanisms, including engagement of the innate immune system via Fc-dependent mechanisms (Jin et al., 2009 Cell Stem Cell, 5:31). We have subsequently humanised and affinity-matured this antibody and, in addition, have engineered the Fc-domain to optimise potential cytotoxicity against AML cells. The resultant antibody, CSL362, retains the ability to neutralise IL-3 and has enhanced affinity for the FcγRIIIa (CD16) on NK cells. In vitro studies have demonstrated that the increased affinity for CD16 correlates with greater antibody-dependent cell-mediated cytotoxicity (ADCC) against CD123 expressing cell lines compared to CSL360, a non Fc-engineered anti-CD123 mAb. The improved activity was evident as both an increased maximal level of target cell lysis and as a shift in the EC50 of the antibody to lower concentrations. Importantly, both primary AML blasts and CD34+CD38−CD123+LSC were susceptible to CSL362-induced ADCC and this was seen even in samples that were resistant to ADCC by a non Fc-engineered anti-CD123 mAb. In an AML xenograft mouse model, where treatment with the antibody was initiated 4 weeks after engraftment of leukemia cells, CSL362 was more effective in reducing leukemic growth than the non Fc-engineered anti-CD123 mAb. The evaluation of neutrophils, monocytes, macrophages and NK cells in ADCC assays has revealed that the major effector cell responsible for CSL362-mediated cytotoxicity in human peripheral blood is the NK cell. In clinical samples we have been able to demonstrate autologous depletion ex vivo of target AML blasts (collected at diagnosis and cryopreserved) following incubation with CSL362 and peripheral blood mononuclear cells (taken from the same patient at first remission), indicating that NK cell number and function is sufficiently preserved in such patients for CSL362-directed killing of leukemic target cells. The pre-clinical data generated thus far strongly support the clinical development of CSL362 for the treatment of AML in patients with adequate NK cell function. A Phase 1 study of CSL362 in patients with CD123 positive AML in remission is underway (Clinical Trials.gov identifier: NCT01632852). Disclosures: Busfield: CSL Limited: Employment. Biondo:CSL Limited: Employment. Wong:CSL Limited: Employment. Ramshaw:CSL Limited: Research Funding. Lee:CSL Limited: Research Funding. Martin:CSL Limited: Employment. Ghosh:CSL Limited: Employment. Braley:CSL Limited: Employment. Tomasetig:CSL Limited: Employment. Panousis:CSL Limited: Employment. Vairo:CSL Limited: Employment. Roberts:CSL Limited: Research Funding. DeWitte:CSL Behring: Employment. Lock:CSL Limited: Consultancy, Research Funding. Lopez:CSL Limited: Consultancy, Research Funding. Nash:CSL Limited: Employment.

RATIONALE:Sublingual allergen-specific immunotherapy is gaining popularity for treatment of allergic diseases, but underlying immunological mechanisms are unresolved.OBJECTIVES:To perform detailed immunological investigation of sublingual house dust mite (HDM) immunotherapy.METHODS:A 12-month randomized double-blind placebo-controlled study of sublingual HDM immunotherapy in 30 HDM-allergic subjects was performed, with 1-year open extension in 9 patients on active treatment. HDM-stimulated blood mononuclear cells were analyzed for proliferation, cytokines, and regulatory T cells (Tregs) by flow cytometry and ELISA. Effects of blocking transforming growth factor (TGF)-beta and IL-10 on proliferation were determined. Treg suppressor function and allergen-specific antibody levels were measured. Clinical efficacy was assessed by symptom, medication, and Juniper quality-of-life scores.MEASUREMENTS AND MAIN RESULTS:Allergen-induced CD4(+) T-cell division and IL-5 production were significantly decreased after 6- and 12-months' active treatment but not placebo. sTGF-betaRII blocked immunotherapy-induced suppression of allergen-specific T-cell proliferation, maximal at 6 months. Decreased allergen-specific CD4(+) T-cell proliferation and increased IL-10 secretion and serum Der p 2-specific IgG(4) were maximal at 24 months' active treatment. Treg (CD4(+)CD25(+)CD127(lo)/Foxp3(+)) function was demonstrated by suppression of allergen-specific effector T-cell (CD4(+)CD25(-)CD127(hi)) proliferation and cytokine production. Clinical efficacy of immunotherapy was supported by significantly decreased rhinitis symptom score, total asthma score, and Juniper quality-of-life score.CONCLUSIONS:This study establishes the novel finding that TGF-beta mediates the immunological suppression seen early in clinically effective sublingual HDM immunotherapy in addition to an increase in Tregs with suppressor function. Clinical trial registered with www.clinicaltrials.gov (NCT00250263).

CD123 (IL-3Rα) is a phenotypic marker of putative leukemic stem cells (LSC) in AML (Jordan, Leukemia 2000; 14:1777). We and others have found that CD34+38− cells from AML patients (pts) express high levels of CD123, in contrast to absence of expression on CD34+38− cells in normal individuals. Binding of CD123 by monoclonal antibody (mAb) 7G3 inhibits IL-3-dependent signalling and proliferation in vitro. In a NOD-SCID xenograft model 7G3 inhibits human AML engraftment, but not normal human hematopoiesis (Lock ASH 2007; Abs161). CSL360, a recombinant chimeric IgG1 mAb derived from 7G3, binds the same epitope. CSL360 concentrations ≥ 0.1μg/mL in vitro inhibited 90% AML cell growth in the presence of supraphysiological IL-3 levels. Preclinical toxicology studies with doses up to 100 mg/kg weekly × 4 in cynomolgus monkeys showed no CSL360-related effects in clinical signs, hematology, chemistry, urinalysis, gross pathology or histopathology. A Phase 1 study of safety, pharmacokinetics (PK) and bioactivity of CSL360 in relapsed, refractory or high risk AML began in March 2007. Pts receive 12 weekly iv infusions if not withdrawn early due to treatment-related toxicity or disease progression. Additional treatments may be given to pts who achieve a response. Bone marrow aspirates/trephine samples are obtained at screening, after dose 3 and before doses 5 and 11. More than 180 infusions have been administered to 26 AML pts (21 M, 5 F; 17 de novo, 8 MDS-related, 1 treatment-related AML) in 5 dose level cohorts: 0.1, 0.3, 1.0, 3.0 and 10 mg/kg. There is no intra-patient dose escalation. PK parameters over the dose range, estimated in 19 pts over 7 days after doses 1 and 4, were linear with dose-proportional increases in the AUC and Cmax; dose 1 Cmax ranged from 0.62 – 287.33 μg/mL and dose 4 Cmax from 1.02 – 178.22 μg/mL. CSL360 mean plasma half-life (dose 1, 83 ± 33 h; dose 4, 117 ± 59 h) appears to be independent of dose and treatment number. Dose 1 systemic clearance (0.21 ± 0.16 L/h) and volume of distribution (0.39 ± 0.22 L/kg) were relatively low, consistent with this size IgG. In all pre-treatment samples anti-CSL360 antibody titers were negative, determined by enzyme immunoassay. Anti-CSL360 antibodies were detected post-treatment in 8/12 pts; these antibodies have not been fully quantified or characterised. CSL360 has been well tolerated; a MTD has not been defined. Seven pts received all 12 doses, 13 pts were withdrawn due to progressive disease or investigator's decision, 3 pts were withdrawn in association with infections, 2 pts withdrew consent, and 1 pt is ongoing. Three serious adverse events have been considered possibly related or related to CSL360: 1 invasive fungal infection (Gr 5), and 2 infusion reactions (Gr 2; hospitalised). Other adverse events are consistent with expectations for the disease population. Of 8 pts in the 3 mg/kg and 10 mg/kg cohorts who are evaluable for response after ≥ 4 doses, 1 complete response (CR) has been observed. A 22 yr old male, de novo FAB M1 cytogenetically normal AML, who had relapsed post-2 allogeneic SCT, achieved a morphologic leukemia free state after 3 doses (3.0 mg/kg) and CR after 12 doses, sustained for > 9 weeks. The pt received 17 doses before withdrawal to treat co-morbidities. Flow cytometry studies with anti-CD123 antibodies demonstrated dose-dependent CSL360 coating of both AML blasts and LSC. Saturation of target antigen on marrow and blood cells was observed 1 day after dosing at 0.3mg/kg, associated with decreased expression of CD123 detected by an antibody to a different epitope. At higher dose levels saturation of CD123 was maintained 7 days post dosing, associated with ongoing reduction in surface CD123 expression. In a representative sample, plasma from a pt treated at 10 mg/kg specifically inhibited IL-3-induced proliferation of AML blasts ex vivo, indicating sufficient circulating concentration of CSL360 to inhibit IL-3 mediated effects in vivo. Effects of CSL360 on proliferation and apoptosis of AML cells in treated patients are being investigated. These preliminary results show anti-CD123 mAb therapy with CSL360 is safe and tolerable; biological effects have been demonstrated; a sustained CR was achieved in 1 advanced, refractory AML pt. The study is continuing, with 20 evaluable patients to be accrued and treated at 10 mg/kg weekly; at this dose level the PK and correlative assays predict that complete blockade of IL-3 signalling through CD123 can be achieved in vivo.

Autoimmune diseases remain a significant health problem in our society, despite the best efforts to understand and treat these conditions. Current clinical treatments are aimed at alleviating the consequences of these diseases, with limited prospects for cure. Our studies with the experimental model of autoimmune gastritis have led us to explore potential curative strategies that can reverse the autoimmune condition. Using mouse models, we have shown that expression of the known gastric autoantigen in the thymus results in immunological tolerance and resistance to the induction of autoimmune gastritis. Also, induced tolerance in donor mice can be transferred to syngeneic recipient mice by bone marrow cells. Strategies based on these observations could lead to reversal of established disease. Transfer of ensuing knowledge to the cure of serious human autoimmune diseases is our ultimate goal.

Autoimmune diseases remain a significant health problem in our society, despite the best efforts to understand and treat these conditions. Current clinical treatments are aimed at alleviating the consequences of these diseases, with limited prospects for cure. Our studies with the experimental model of autoimmune gastritis have led us to explore potential curative strategies that can reverse the autoimmune condition. Using mouse models, we have shown that expression of the known gastric autoantigen in the thymus results in immunological tolerance and resistance to the induction of autoimmune gastritis. Also, induced tolerance in donor mice can be transferred to syngeneic recipient mice by bone marrow cells. Strategies based on these observations could lead to reversal of established disease. Transfer of ensuing knowledge to the cure of serious human autoimmune diseases is our ultimate goal.

Pernicious anaemia is the commonest cause of vitamin B 12 deficiency in man affecting similar to2% of the population. Autoimmune gastritis is the underlying cause of this disease. The gastritis results in loss of parietal cells that secrete intrinsic factor required for vitamin B 12 absorption in the small intestine. The target antigen of these parietal cell antibodies is the gastric H/K ATPase responsible for secretion of HCl. To examine the role of the gastric H/K ATPase in the pathogenesis of autoimmune gastritis, we adopted and established mouse models of autoimmune gastritis. We showed that the gastritis is initiated by the 0 subunit of the ATPase because transgenic expression of this subunit in the thymus of gastritis-susceptible BALB/c or BALB/cCrS1c mice renders the mice resistant to development of autoimmune gastritis. We showed that immunological tolerance to the beta subunit was established by expression of this subunit in the thymus because thymocytes from transgenic mice failed to transfer gastritis to naive recipients. The mouse gastritis is mediated entirely by TH1-type CD4 T cells. We showed that early lesions of gastritis are characterised by influx of CD4 T cells that secrete a mix of TH1 and TH2 cytokines with the exception of IL-4. A single injection of antibodies to interferon gamma prevented the development of gastritis. An unresolved question is the nature of the trigger that initiates gastritis in genetically-susceptible mice. Microbial organisms may initiate gastritis by acting as molecular mimics or through by-stander activation following the generation of pro-inflammatory cytokines. We investigated the role of the pro-inflammatory cytokine GM-CSF in the initiation of gastritis by the transgenic production of this cytokine in the stomach. We selected this cytokine because we have previously shown that it is generated in gastritic lesions and because GM-CSF is a potent activator of antigen-presenting cells that links innate to adaptive immunity. We showed that transgenic expression of GM-CSF in the stomach was alone sufficient to initiate gastritis in genetically-susceptible mice, Our studies support the role of pro-inflammatory by-stander activation in the initiation of gastritis.