CSL311, a novel, potent, therapeutic monoclonal antibody for the treatment of diseases mediated by the common beta chain of the IL-3, GM-CSF and IL-5 receptors

The β common-signaling cytokines interleukin (IL)-3, granulocyte-macrophage colony stimulating factor (GM-CSF) and IL-5 stimulate pro-inflammatory activities of haematopoietic cells via a receptor complex incorporating cytokine-specific α and shared β common (βc, CD131) receptor. Evidence from animal models and recent clinical trials demonstrate that these cytokines are critical mediators of the pathogenesis of inflammatory airway disease such as asthma. However, no therapeutic agents, other than steroids, that specifically and effectively target inflammation mediated by all 3 of these cytokines exist. We employed phage display technology to identify and optimize a novel, human monoclonal antibody (CSL311) that binds to a unique epitope that is specific to the cytokine-binding site of the human βc receptor. The binding epitope of CSL311 on the βc receptor was defined by X-ray crystallography and site-directed mutagenesis. CSL311 has picomolar binding affinity for the human βc receptor, and at therapeutic concentrations is a highly potent antagonist of the combined activities of IL-3, GM-CSF and IL-5 on primary eosinophil survival in vitro. Importantly, CSL311 inhibited the survival of inflammatory cells present in induced sputum from human allergic asthmatic subjects undergoing allergen bronchoprovocation. Due to its high potency and ability to simultaneously suppress the activity of all 3 β common cytokines, CSL311 may provide a new strategy for the treatment of chronic inflammatory diseases where the human βc receptor is central to pathogenesis. The coordinates for the βc/CSL311 Fab complex structure have been deposited with the RCSB Protein Data Bank (PDB 5DWU).Con Panousis, Urmi Dhagat, Kirsten M. Edwards, Veronika Rayzman, Matthew P. Hardy, Hal Braley, Gail M. Gauvreau, Timothy R. Hercus, Steven Smith, Roma Sehmi, Laura McMillan, Mara Dottore, Barbara J. McClure, Louis J. Fabri, Gino Vairo, Angel F Lopez, Michael W. Parker, Andrew D. Nash, Nicholas J. Wilson, Michael J. Wilson and Catherine M. Owczare

Distinct Assemblies of Heterodimeric Cytokine Receptors Govern Stemness Programs in Leukemia

Published first May 16, 2023Leukemia stem cells (LSC) possess distinct self-renewal and arrested differentiation properties that are responsible for disease emergence, therapy failure, and recurrence in acute myeloid leukemia (AML). Despite AML displaying extensive biological and clinical heterogeneity, LSC with high interleukin-3 receptor (IL3R) levels are a constant yet puzzling feature, as this receptor lacks tyrosine kinase activity. Here, we show that the heterodimeric IL3Rα/βc receptor assembles into hexamers and dodecamers through a unique interface in the 3D structure, where high IL3Rα/βc ratios bias hexamer formation. Importantly, receptor stoichiometry is clinically relevant as it varies across the individual cells in the AML hierarchy, in which high IL3Rα/βc ratios in LSCs drive hexamer-mediated stemness programs and poor patient survival, while low ratios mediate differentiation. Our study establishes a new paradigm in which alternative cytokine receptor stoichiometries differentially regulate cell fate, a signaling mechanism that may be generalizable to other transformed cellular hierarchies and of potential therapeutic significance.Winnie L. Kan, Urmi Dhagat, Kerstin B. Kaufmann, Timothy R. Hercus, Tracy L. Nero, Andy G.X. Zeng, John Toubia, Emma F. Barry, Sophie E. Broughton, Guillermo A. Gomez, Brooks A. Benard, Mara Dottore, Karen S. Cheung Tung Shing, Héléna Boutzen, Saumya E. Samaraweera, Kaylene J. Simpson, Liqing Jin, Gregory J. Goodall, C. Glenn Begley, Daniel Thomas, Paul G. Ekert, Denis Tvorogov, Richard J. D, Andrea, John E. Dick, Michael W. Parker, and Angel F. Lope

Translating the biology of B common receptor-engaging cytokines into clinical medicine

The family of cytokines that comprises IL-3, IL-5, and GM-CSF was discovered over 30 years ago, and their biological activities and resulting impact in clinical medicine has continued to expand ever since. Originally identified as bone marrow growth factors capable of acting on hemopoietic progenitor cells to induce their proliferation and differentiation into mature blood cells, these cytokines are also recognized as key mediators of inflammation and the pathobiology of diverse immunologic diseases. This increased understanding of the functional repertoire of IL-3, IL-5, and GM-CSF has led to an explosion of interest in modulating their functions for clinical management. Key to the successful clinical translation of this knowledge is the recognition that these cytokines act by engaging distinct dimeric receptors and that they share a common signaling subunit called β-common or βc. The structural determination of how IL-3, IL-5, and GM-CSF interact with their receptors and linking this to their differential biological functions on effector cells has unveiled new paradigms of cell signaling. This knowledge has paved the way for novel mAbs and other molecules as selective or pan inhibitors for use in different clinical settings.Harshita Pant, Timothy R. Hercus, Damon J. Tumes, Kwok Ho Yip, Michael W. Parker, Catherine M. Owczarek, Angel F. Lopez, and David P. Husto

Messing with beta c: a unique receptor with many goals

Our understanding of the biological role of the βc family of cytokines has evolved enormously since their initial identification as bone marrow colony stimulating factors in the 1960's. It has become abundantly clear over the intervening decades that this family of cytokines has truly astonishing pleiotropic capacity, capable of regulating not only hematopoiesis but also many other normal and pathological processes such as development, inflammation, allergy and cancer. As noted in the current pandemic, βc cytokines contribute to the cytokine storm seen in acutely ill COVID-19 patients. Ongoing studies to discover how these cytokines activate their receptor are revealing insights into the fundamental mechanisms that give rise to cytokine pleiotropy and are providing tantalizing glimpses of how discrete signaling pathways may be dissected for activation with novel ligands for therapeutic benefit.Winnie L. Kan, Karen S. Cheung Tung Shing, Tracy L. Nero, Timothy R. Hercus, Denis Tvorogov, Michael W. Parker, Angel F. Lope

Accumulation of JAK activation loop phosphorylation is linked to type I JAK inhibitor withdrawal syndrome in myelofibrosis

Treatment of patients with myelofibrosis with the type I JAK (Janus kinase) inhibitor ruxolitinib paradoxically induces JAK2 activation loop phosphorylation and is associated with a life-threatening cytokine-rebound syndrome if rapidly withdrawn. We developed a time-dependent assay to mimic ruxolitinib withdrawal in primary JAK2V617F and CALR mutant myelofibrosis patient samples and observed notable activation of spontaneous STAT signaling in JAK2V617F samples after drug washout. Accumulation of ruxolitinib-induced JAK2 phosphorylation was dose dependent and correlated with rebound signaling and the presence of a JAK2V617F mutation. Ruxolitinib prevented dephosphorylation of a cryptic site involving Tyr1007/1008 in JAK2 blocking ubiquitination and degradation. In contrast, a type II JAK inhibitor, CHZ868, did not induce JAK2 phosphorylation, was not associated with withdrawal signaling, and was superior in the eradication of flow-purified JAK2V617F mutant CD34+ progenitors after drug washout. Type I inhibitor-induced loop phosphorylation may act as a pathogenic signaling node released upon drug withdrawal, especially in JAK2V617F patients.Denis Tvorogov, Daniel Thomas, Nicholas P.D. Liau, Mara Dottore, Emma F. Barry, Maya Lathi, Winnie L. Kan, Timothy R. Hercus, Frank Stomski, Timothy P. Hughes, Vinay Tergaonkar, Michael W. Parker, David M. Ross, Ravindra Majeti, Jeffrey J. Babon, Angel F. Lope

Targeting the human βc Receptor inhibits contact dermatitis in a transgenic mouse model

Allergic contact dermatitis (ACD) is a prevalent and poorly controlled inflammatory disease caused by skin infiltration of T cells and granulocytes. The beta common (bc) cytokines GM-CSF, IL-3, and IL-5 are powerful regulators of granulocyte function that signal through their common receptor subunit bc, a property that has made bc an attractive target to simultaneously inhibit these cytokines. However, the species specificity of bc has precluded testing of inhibitors of human bc in mouse models. To overcome this problem, we developed a human bc receptor transgenic mouse strain with a hematopoietic cell‒specific expression of human bc instead of mouse bc. Human bc receptor transgenic cells responded to mouse GM-CSF and IL-5 but not to IL-3 in vitro and developed tissue pathology and cellular inflammation comparable with those in wild-type mice in a model of ACD. Similarly, Il3e/e mice developed ACD pathology comparable with that of wild-type mice. Importantly, the blocking antiehuman bc antibody CSL311 strongly suppressed ear pinna thickening and histopathological changes typical of ACD and reduced accumulation of neutrophils, mast cells, and eosinophils in the skin. These results show that GM-CSF and IL-5 but not IL-3 are major mediators of ACD and define the human bc receptor transgenic mouse as a unique platform to test the inhibitors of bc in vivo.Kwok Ho Yip ... Barbara J. McClure ... Angel F. Lopez ... Harshita Pant ... Hayley S. Ramshaw ... et. a