Messing with βc: 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.

The mechanism of GM-CSF inhibition by human GM-CSF auto-antibodies suggests novel therapeutic opportunities.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a hematopoietic growth factor that can stimulate a variety of cells, but its overexpression leads to excessive production and activation of granulocytes and macrophages with many pathogenic effects. This cytokine is a therapeutic target in inflammatory diseases, and several anti-GM-CSF antibodies have advanced to Phase 2 clinical trials in patients with such diseases, e.g.

NLS-Cholic Acid Conjugation to IL-5Rα-Specific Antibody Improves Cellular Accumulation and In Vivo Tumor-Targeting Properties in a Bladder Cancer Model.

Receptor-mediated internalization followed by trafficking and degradation of antibody-conjugates (ACs) via the endosomal-lysosomal pathway is the major mechanism for delivering molecular payloads inside target tumor cells. Although a mainstay for delivering payloads with clinically approved ACs in cancer treatment and imaging, tumor cells are often able to decrease intracellular payload concentrations and thereby reduce the effectiveness of the desired application. Thus, increasing payload intracellular accumulation has become a focus of attention for designing next-generation ACs.

A dual role for the N-terminal domain of the IL-3 receptor in cell signalling.

The interleukin-3 (IL-3) receptor is a cell-surface heterodimer that links the haemopoietic, vascular and immune systems and is overexpressed in acute and chronic myeloid leukaemia progenitor cells. It belongs to the type I cytokine receptor family in which the α-subunits consist of two fibronectin III-like domains that bind cytokine, and a third, evolutionarily unrelated and topologically conserved, N-terminal domain (NTD) with unknown function. Here we show by crystallography that, while the NTD of IL3Rα is highly mobile in the presence of IL-3, it becomes surprisingly rigid in the presence of IL-3 K116W.

Targeting IL-5Rα with antibody-conjugates reveals a strategy for imaging and therapy for invasive bladder cancer.

Despite the high interest and concern due to an increasing incidence and death rate, patients who develop muscle invasive bladder cancer (MIBC) have few options available. However, the past decade has produced many candidate bladder tumor-specific markers but further development of these markers is still needed for creating effective targeted medications to solve this urgent need. Interleukin-5 receptor α-subunit (IL-5Rα) has recently been reported to be involved in MIBC progression.

Role of the β Common (βc) Family of Cytokines in Health and Disease.

The β common ([βc]/CD131) family of cytokines comprises granulocyte macrophage colony-stimulating factor (GM-CSF), interleukin (IL)-3, and IL-5, all of which use βc as their key signaling receptor subunit. This is a prototypic signaling subunit-sharing cytokine family that has unveiled many biological paradigms and structural principles applicable to the IL-2, IL-4, and IL-6 receptor families, all of which also share one or more signaling subunits. Originally identified for their functions in the hematopoietic system, the βc cytokines are now known to be truly pleiotropic, impacting on multiple cell types, organs, and biological systems, and thereby controlling the balance between health and disease.

Conformational Changes in the GM-CSF Receptor Suggest a Molecular Mechanism for Affinity Conversion and Receptor Signaling.

The GM-CSF, IL-3, and IL-5 receptors constitute the βc family, playing important roles in inflammation, autoimmunity, and cancer. Typical of heterodimeric type I cytokine receptors, signaling requires recruitment of the shared subunit to the initial cytokine:α subunit binary complex through an affinity conversion mechanism. This critical process is poorly understood due to the paucity of crystal structures of both binary and ternary receptor complexes for the same cytokine.

Ywhaz/14-3-3ζ Deletion Improves Glucose Tolerance Through a GLP-1-Dependent Mechanism.

Multiple signaling pathways mediate the actions of metabolic hormones to control glucose homeostasis, but the proteins that coordinate such networks are poorly understood. We previously identified the molecular scaffold protein, 14-3-3ζ, as a critical regulator of in vitro β-cell survival and adipogenesis, but its metabolic roles in glucose homeostasis have not been studied in depth. Herein, we report that Ywhaz gene knockout mice (14-3-3ζKO) exhibited elevated fasting insulin levels while maintaining normal β-cell responsiveness to glucose when compared with wild-type littermate controls.

The Interferon-induced Transmembrane Proteins, IFITM1, IFITM2, and IFITM3 Inhibit Hepatitis C Virus Entry.

The interferon-induced transmembrane (IFITM) family of proteins have recently been identified as important host effector molecules of the type I interferon response against viruses. IFITM1 has been identified as a potent antiviral effector against hepatitis C virus (HCV), whereas the related family members IFITM2 and IFITM3 have been described to have antiviral effects against a broad range of RNA viruses. Here, we demonstrate that IFITM2 and IFITM3 play an integral role in the interferon response against HCV and act at the level of late entry stages of HCV infection.

14-3-3ζ coordinates adipogenesis of visceral fat.

The proteins that coordinate complex adipogenic transcriptional networks are poorly understood. 14-3-3ζ is a molecular adaptor protein that regulates insulin signalling and transcription factor networks. Here we report that 14-3-3ζ-knockout mice are strikingly lean from birth with specific reductions in visceral fat depots.

Efficacy of an Fc-modified anti-CD123 antibody (CSL362) combined with chemotherapy in xenograft models of acute myelogenous leukemia in immunodeficient mice.

The prognosis of older patients with acute myelogenous leukemia is generally poor. The interleukin-3 receptor α-chain (CD123) is highly expressed on the surface of acute leukemia cells compared with normal hematopoietic stem cells. CSL362 is a fully humanized, CD123-neutralizing monoclonal antibody containing a modified Fc structure, which enhances human natural killer cell antibody-dependent cell-mediated cytotoxicity.

The βc receptor family - Structural insights and their functional implications.

Granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3) and IL-5 are members of a small family of cytokines that share a beta receptor subunit (βc). These cytokines regulate the growth, differentiation, migration and effector function activities of many hematopoietic cells in bone marrow, blood and sites of inflammation. Excessive or aberrant signaling can result in chronic inflammatory conditions and myeloid leukemias.

Unexpected mechanisms of action for a cytokine receptor-blocking antibody.

CSL362 is a humanized interleukin-3 (IL-3)-neutralizing monoclonal antibody with enhanced effector function that binds the α subunit of the IL-3 receptor (IL3Rα). The crystal structure of an IL3Rα:CSL362 complex shows that IL3Rα adopts "open" and "closed" conformations. CSL362 blocks IL-3 function through both IL3Rα conformations but via distinct and unexpected mechanisms.

Dual mechanism of interleukin-3 receptor blockade by an anti-cancer antibody.

Interleukin-3 (IL-3) is an activated T cell product that bridges innate and adaptive immunity and contributes to several immunopathologies. Here, we report the crystal structure of the IL-3 receptor α chain (IL3Rα) in complex with the anti-leukemia antibody CSL362 that reveals the N-terminal domain (NTD), a domain also present in the granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-5, and IL-13 receptors, adopting unique "open" and classical "closed" conformations. Although extensive mutational analyses of the NTD epitope of CSL362 show minor overlap with the IL-3 binding site, CSL362 only inhibits IL-3 binding to the closed conformation, indicating alternative mechanisms for blocking IL-3 signaling.

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).

Crystallization and preliminary X-ray diffraction analysis of the interleukin-3 alpha receptor bound to the Fab fragment of antibody CSL362.

Interleukin-3 (IL-3) is a member of the beta common family of cytokines that regulate multiple functions of myeloid cells. The IL-3 receptor-specific alpha subunit (IL3Rα) is overexpressed on stem cells/progenitor cells of patients with acute myeloid leukaemia, where elevated receptor expression correlates clinically with a reduced patient survival rate. The monoclonal antibody (MAb) CSL362 is a humanized MAb derived from the murine MAb 7G3, originally identified for its ability to specifically recognize the human IL-3 receptor and for blocking the signalling of IL-3 in myeloid and endothelial cells.

ER stress does not cause upregulation and activation of caspase-2 to initiate apoptosis.

A recent report claimed that endoplasmic reticulum (ER) stress activates the ER trans-membrane receptor IRE1α, leading to increased caspase-2 levels via degradation of microRNAs, and consequently induction of apoptosis. This observation casts caspase-2 into a central role in the apoptosis triggered by ER stress. We have used multiple cell types from caspase-2-deficient mice to test this hypothesis but failed to find significant impact of loss of caspase-2 on ER-stress-induced apoptosis.

Signalling by the βc family of cytokines.

The GM-CSF, IL-3 and IL-5 family of cytokines, also known as the βc family due to their receptors sharing the signalling subunit βc, regulates multiple biological processes such as native and adaptive immunity, inflammation, normal and malignant hemopoieis, and autoimmunity. Australian scientists played a major role in the discovery and biological characterisation of the βc cytokines and their recent work is revealing unique features of cytokine receptor assembly and signalling. Furthermore, specific antibodies have been generated to modulate their function.

Molecular basis of cytokine receptor activation.

Cytokines are secreted soluble peptides that precisely regulate multiple cellular functions. Amongst these the GM-CSF/IL-3/IL-5 family of cytokines controls whether hematopoietic cells will survive or apoptose, proliferate, differentiate, migrate, or perform effector functions such as phagocytosis or reactive oxygen species release. Their potent and pleiotropic activities are mediated through binding to high affinity membrane receptors at surprisingly low numbers per cell.