Translating the biology of β 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.

Evaluation of Phage Display Biopanning Strategies for the Selection of Anti-Cell Surface Receptor Antibodies.

Monoclonal antibodies (mAbs) are one of the most successful and versatile protein-based pharmaceutical products used to treat multiple pathological conditions. The remarkable specificity of mAbs and their affinity for biological targets has led to the implementation of mAbs in the therapeutic regime of oncogenic, chronic inflammatory, cardiovascular, and infectious diseases. Thus, the discovery of novel mAbs with defined functional activities is of crucial importance to expand our ability to address current and future clinical challenges.

Targeting the human β receptor inhibits inflammatory myeloid cells and lung injury caused by acute cigarette smoke exposure.

Background And Objective: Chronic obstructive pulmonary disease (COPD) is a devastating disease commonly caused by cigarette smoke (CS) exposure that drives tissue injury by persistently recruiting myeloid cells into the lungs. A significant portion of COPD patients also present with overlapping asthma pathology including eosinophilic inflammation. The β cytokine family includes granulocyte monocyte-colony-stimulating factor, IL-5 and IL-3 that signal through their common receptor subunit β to promote the expansion and survival of multiple myeloid cells including monocytes/macrophages, neutrophils and eosinophils.

Sialylation-dependent pharmacokinetics and differential complement pathway inhibition are hallmarks of CR1 activity in vivo.

Human Complement Receptor 1 (HuCR1) is a potent membrane-bound regulator of complement both in vitro and in vivo, acting via interaction with its ligands C3b and C4b. Soluble versions of HuCR1 have been described such as TP10, the recombinant full-length extracellular domain, and more recently CSL040, a truncated version lacking the C-terminal long homologous repeat domain D (LHR-D). However, the role of N-linked glycosylation in determining its pharmacokinetic (PK) and pharmacodynamic (PD) properties is only partly understood.

Blocking the human common beta subunit of the GM-CSF, IL-5 and IL-3 receptors markedly reduces hyperinflammation in ARDS models.

Acute respiratory distress syndrome (ARDS) is triggered by various aetiological factors such as trauma, sepsis and respiratory viruses including SARS-CoV-2 and influenza A virus. Immune profiling of severe COVID-19 patients has identified a complex pattern of cytokines including granulocyte macrophage-colony stimulating factor (GM-CSF) and interleukin (IL)-5, which are significant mediators of viral-induced hyperinflammation. This strong response has prompted the development of therapies that block GM-CSF and other cytokines individually to limit inflammation related pathology.

Targeting the Human β 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 (β) cytokines GM-CSF, IL-3, and IL-5 are powerful regulators of granulocyte function that signal through their common receptor subunit β, a property that has made β an attractive target to simultaneously inhibit these cytokines. However, the species specificity of β has precluded testing of inhibitors of human β in mouse models.

Modular Platform for the Development of Recombinant Hemoglobin Scavenger Biotherapeutics.

Cell-free hemoglobin (Hb) is a driver of disease progression in conditions with intravascular or localized hemolysis. Genetic and acquired anemias or emergency medical conditions such as aneurysmal subarachnoid hemorrhage involve tissue Hb exposure. Haptoglobin (Hp) captures Hb in an irreversible protein complex and prevents its pathophysiological contributions to vascular nitric oxide depletion and tissue oxidation.

Glycoproteomic measurement of site-specific polysialylation.

Polysialylation is the enzymatic addition of a highly negatively charged sialic acid polymer to the non-reducing termini of glycans. Polysialylation plays an important role in development, and is involved in neurological diseases, neural tissue regeneration, and cancer. Polysialic acid (PSA) is also a biodegradable and non-immunogenic conjugate to therapeutic drugs to improve their pharmacokinetics.

Mining the Plasma Cell Transcriptome for Novel Cell Surface Proteins.

Antibody Secreting Cells (ASCs) are a fundamental component of humoral immunity, however, deregulated or excessive antibody production contributes to the pathology of autoimmune diseases, while transformation of ASCs results in the malignancy Multiple Myeloma (MM). Despite substantial recent improvements in treating these conditions, there is as yet no widely used ASC-specific therapeutic approach, highlighting a critical need to identify novel methods of targeting normal and malignant ASCs. Surface molecules specifically expressed by the target cell population represent ideal candidates for a monoclonal antibody-based therapy.

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.

An Optimized Hepatitis C Virus E2 Glycoprotein Core Adopts a Functional Homodimer That Efficiently Blocks Virus Entry.

The hepatitis C virus (HCV) envelope glycoprotein E2 is the major target of broadly neutralizing antibodies and is the focus of efforts in the rational design of a universal B cell vaccine against HCV. The E2 glycoprotein exhibits a high degree of amino acid variability which localizes to three discrete regions: hypervariable region 1 (HVR1), hypervariable region 2 (HVR2), and the intergenotypic variable region (igVR). All three variable regions contribute to immune evasion and/or isolate-specific structural variations, both important considerations for vaccine design.

CSL311, a novel, potent, therapeutic monoclonal antibody for the treatment of diseases mediated by the common β 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.

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.

Identification of potent antagonist antibodies against mouse IL-13Rα1 using novel bioassays.

Interleukin-13 (IL-13) is a cytokine implicated in airway diseases such as asthma and idiopathic pulmonary fibrosis. IL-13 signals through a heterodimeric receptor complex consisting of IL-13Rα1 and IL-4Rα, known as the type II IL-4R. IL-4 also signals through this receptor and as such many of the biological effects of IL-13 and IL-4 are similar.

Soluble IFN receptor potentiates in vivo type I IFN signaling and exacerbates TLR4-mediated septic shock.

Circulating levels of a soluble type I IFNR are elevated in diseases, such as chronic inflammation, infections, and cancer, but whether it functions as an antagonist, agonist, or transporter is unknown. In this study, we elucidate the in vivo importance of the soluble type I IFNAR, soluble (s)IFNAR2a, which is generated by alternative splicing of the Ifnar2 gene. A transgenic mouse model was established to mimic the 10-15-fold elevated expression of sIFNAR2a observed in some human diseases.

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.

Acute delivery of EphA4-Fc improves functional recovery after contusive spinal cord injury in rats.

Blocking the action of inhibitory molecules at sites of central nervous system injury has been proposed as a strategy to promote axonal regeneration and functional recovery. We have previously shown that genetic deletion or competitive antagonism of EphA4 receptor activity promotes axonal regeneration and functional recovery in a mouse model of lateral hemisection spinal cord injury. Here we have assessed the effect of blocking EphA4 activation using the competitive antagonist EphA4-Fc in a rat model of thoracic contusive spinal cord injury.

EphA4 receptor tyrosine kinase is a modulator of onset and disease severity of experimental autoimmune encephalomyelitis (EAE).

The EphA4 receptor tyrosine kinase is a major regulator of axonal growth and astrocyte reactivity and is a possible inflammatory mediator. Given that multiple sclerosis (MS) is primarily an inflammatory demyelinating disease and in mouse models of MS, such as experimental autoimmune encephalomyelitis (EAE), axonal degeneration and reactive gliosis are prominent clinical features, we hypothesised that endogenous EphA4 could play a role in modulating EAE. EAE was induced in EphA4 knockout and wildtype mice using MOG peptide immunisation and clinical severity and histological features of the disease were then compared in lumbar spinal cord sections.

Characterization of the type I interferon locus and identification of novel genes.

The human type I interferon (IFN) genes are clustered on human chromosome 9p21 and the mouse genes are located in the region of conserved synteny on mouse chromosome 4. We have identified two novel mouse Ifna genes (Ifna12, Ifna13) and Ifnl2 (IFN-like 2, a homologue of Limitin/IFN-like 1). Another type I IFN gene was designated Ifne1.

Identification and characterization of polymorphisms at the HAS alpha1-acid glycoprotein (ORM*) gene locus in Caucasians.

Human alpha(1)-acid glycoprotein (AGP) or orosomucoid (ORM) is a major acute phase protein that is thought to play a crucial role in maintaining homeostasis. Human AGP is the product of a cluster of at least two adjacent genes located on HSA chromosome 9. Using a range of restriction endonucleases we have investigated DNA variation at the locus encoding the AGP genes in a group of healthy Caucasians.

Characterization and transcriptional analysis of the mouse Chromosome 16 cytokine receptor gene cluster.

The class II cytokine receptor (CIICR) genes Il10r2 and Ifnar1 are localized on mouse Chr 16 in a cluster that also contains the CIICR genes Ifnar2 and Ifngr2. The structure of the Il10r2 gene was deduced and consisted of 7 exons and 6 introns arrayed in an organization similar to its human ortholog. We also present a revised Il10r2 cDNA sequence with a total of 100 bp of additional nucleotide sequence in the 5' and 3' untranslated regions, and report the first extensive profiles of Il10r2 and Ifnar1 mRNA developmental stage and adult tissue expression.

Multiple regions within the promoter of the murine Ifnar-2 gene confer basal and inducible expression.

The (murine) type I interferon (IFN) receptor, muIfnar-2, is expressed ubiquitously, and exists as both transmembrane and soluble forms. In the present study we show that the gene encoding muIfnar-2 spans approx. 33 kb on mouse chromosome 16, and consists of nine exons and eight introns.