Mark Biondo

Targeting of acute myeloid leukemia in vitro and in vivo with an anti-CD123 mAb engineered for optimal ADCC.

Acute myeloid leukemia (AML) is a biologically heterogeneous group of related diseases in urgent need of better therapeutic options. Despite this heterogeneity, overexpression of the interleukin (IL)-3 receptor α-chain (IL-3 Rα/CD123) on both the blast and leukemic stem cell (LSC) populations is a common occurrence, a finding that has generated wide interest in devising new therapeutic approaches that target CD123 in AML patients. We report here the development of CSL362, a monoclonal antibody to CD123 that has been humanized, affinity-matured and Fc-engineered for increased affinity for human CD16 (FcγRIIIa). In vitro studies demonstrated that CSL362 potently induces antibody-dependent cell-mediated cytotoxicity of both AML blasts and CD34+CD38−CD123+ LSC by NK cells. Importantly, CSL362 was highly effective in vivo reducing leukemic cell growth in AML xenograft mouse models and potently depleting plasmacytoid dendritic cells and basophils in cynomolgus monkeys. Significantly, we demonstrated CSL362-dependent autologous depletion of AML blasts ex vivo, indicating that CSL362 enables the efficient killing of AML cells by the patient’s own NK cells. These studies offer a new therapeutic option for AML patients with adequate NK-cell function and warrant the clinical development of CSL362 for the treatment of AML.

A cytotoxic anti-IL-3Rα antibody targets key cells and cytokines implicated in systemic lupus erythematosus

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.

THU0311 IL3R-Alpha as A Novel Therapeutic Target in Systemic Lupus Erythematosus

Background Despite currently available therapies, a significant morbidity, and mortality remains in SLE. Major therapeutic targets have been the auto-antibody producing B cell, and more recently, type I IFN, which is produced by pDCs activated via TLR7 and TLR9 stimulation by SLE immune complexes. Alternate ways of reducing type I IFN, such as by modulating pDCs, are now emerging (1, 2). We have developed a novel mAb targeting the IL-3Rα, CSL362, which both depletes IL-3Rα expressing cells, and neutralizes signaling through IL-3. Objectives Our objective was to explore the potential utility of CSL362 as a therapeutic in SLE, via its effects on multiple pathogenic cell types and cytokines. Methods In vitro studies were undertaken in a heterogenous population of SLE donors, and healthy and autoimmune disease controls, and major findings confirmed in vivo in cynomolgus macaques. The effect of CSL362 on peripheral blood mononuclear cell types was evaluated by flow cytometry, on cytokine production by ELISA or Luminex assays, and on gene expression by qRT-PCR. Results CSL362 potently and specifically depleted CD123hi pDCs and basophils (average depletion 86.07%±2.07 (SEM), p<0.05, n=72 for pDCs, and 59.39%±3.57, p<0.05, n=71 for basophils). This occurred mainly through antibody-dependent cell-mediated cytotoxicity, although neutralization of IL-3 alone with the Fab fragment of CSL362 resulted in pDC, but not basophil, depletion at higher doses. Through pDC depletion, TLR7-, TLR9- and SLE serum- stimulated IFNα production, and IFN-inducible gene expression was markedly reduced (average reduction in TLR9-stimulated IFNα production 99.36%±0.39, p<0.05, n=29). Additionally, TLR7- and TLR9-induced plasmablast expansion was inhibited by CSL362. This depended upon depletion of pDCs by CSL362, which decreased not only IFNα, but IL-6, production that was required for plasmablast expansion. Importantly, we found that IFN-inducible gene expression and plasmablast expansion were more effectively inhibited by CSL362 than by IFNα blocking mAbs. This may be because pDC depletion by CSL362 reduced production of cytokines other than IFNα, such as IL-6, and interferon types other than type I. Indeed, CSL362 reduced production of type III IFN, a cytokine which has been postulated as a cause of ongoing disease activity despite type I IFN blockade (3). Administration of a single subcutaneous dose of CSL362 to cynomolgus macaques confirmed pDC and basophil depletion, correlating with a decrease in IFN-inducible gene expression, that lasted several weeks post dose, with no major adverse events observed. Conclusions The unique ability of CSL362 to affect the major pathogenic targets of type I IFN and the B cell, and newer targets such as the basophil (4), IL-3 (5) and type III IFN, presents a strong rationale for its therapeutic evaluation in SLE. References Pellerin A et al, EMBO Mol Med, 2015;7:464–476 Zhan Y et al, Arthritis Rheumatol, 2015;67:797–808 Amezcua-Guerra LM et al, Rheumatology (Oxford), 2015;54:203–205 Charles N et al, Nat Med, 2010;16:701–707 Renner K et al, Kidney Int, 2015;88:1088–1098 Disclosure of Interest S. Oon Grant/research support from: CSL Limited, H. Huynh Employee of: CSL Limited, T. Y. Tai Employee of: CSL Limited, M. Ng Employee of: CSL Limited, K. Monaghan Shareholder of: CSL Limited, Employee of: CSL Limited, M. Biondo Employee of: CSL Limited, G. Vairo Shareholder of: CSL Limited, Employee of: CSL Limited, E. Maraskovsky Shareholder of: CSL Limited, Employee of: CSL Limited, A. Nash Shareholder of: CSL Limited, Employee of: CSL Limited, N. Wilson Shareholder of: CSL Limited, Employee of: CSL Limited, I. Wicks Grant/research support from: CSL Limited