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

Abstract The common β chain (βc) of the granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), and IL-5 receptors is the major signaling subunit of these receptors coupling ligand binding to multiple biological activities. It is thought that these multiple functions arise as a consequence of the recruitment of specific signaling molecules to tyrosine-phosphorylated residues in the cytoplasmic domain of βc. However, the contribution of serine phosphorylation in βc to the recruitment of signaling molecules is not known. We show here the identification of a phosphoserine motif in the cytoplasmic domain of βc that interacts with the adaptor protein 14-3-3ζ. Coimmunoprecipitation and pull-down experiments with a glutathione S-transferase (GST):14-3-3ζ fusion protein showed that 14-3-3 directly associates with βc but not the GM-CSF receptor  chain. C-terminal truncation mutants of βcfurther showed that a region between amino acids 544 and 626 in βc was required for its association with 14-3-3ζ. This region contains the sequence 582HSRSLP587, which closely resembles the RSXSXP (where S is phosphorylated) consensus 14-3-3 binding site identified in a number of signaling molecules, including Raf-1. Significantly, substitution of582HSRSLP587 for EFAAAA completely abolished interaction of βc with GST–14-3-3ζ. Furthermore, the interaction of βc with GST–14-3-3 was greatly reduced in the presence of a peptide containing the 14-3-3 binding site, but only when 585Ser was phosphorylated. Direct binding experiments showed that the peptide containing phosphorylated 585Ser bound 14-3-3ζ with an affinity of 150 nmol/L. To study the regulation of 585S phosphorylation in vivo, we raised antibodies that specifically recognized 585Ser-phosphorylated βc. Using these antibodies, we showed that GM-CSF stimulation strongly upregulated 585Ser phosphorylation in M1 myeloid leukemic cells. The proximity of the SHC-binding site (577Tyr) to the 14-3-3–binding site (582HSRSLP587) and their conservation between mouse, rat, and human βc but not in other cytokine receptors suggest that they form a distinct motif that may subserve specialized functions associated with the GM-CSF, IL-3, and IL-5 receptors.

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Identification of a 14-3-3 Binding Sequence in the Common β Chain of the Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF), Interleukin-3 (IL-3), and IL-5 Receptors That Is Serine-Phosphorylated by GM-CSF

Blood ◽

10.1182/blood.v94.6.1933.418k10_1933_1942 ◽

1999 ◽

Vol 94 (6) ◽

pp. 1933-1942 ◽

Cited By ~ 1

Author(s):

F.C. Stomski ◽

M. Dottore ◽

W. Winnall ◽

M.A. Guthridge ◽

J. Woodcock ◽

...

Keyword(s):

Binding Site ◽

Signaling Molecules ◽

Colony Stimulating Factor ◽

Interleukin 3 ◽

Gm Csf ◽

Macrophage Colony Stimulating Factor ◽

The Common ◽

Macrophage Colony ◽

Β Chain ◽

Stimulating Factor

The common β chain (βc) of the granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), and IL-5 receptors is the major signaling subunit of these receptors coupling ligand binding to multiple biological activities. It is thought that these multiple functions arise as a consequence of the recruitment of specific signaling molecules to tyrosine-phosphorylated residues in the cytoplasmic domain of βc. However, the contribution of serine phosphorylation in βc to the recruitment of signaling molecules is not known. We show here the identification of a phosphoserine motif in the cytoplasmic domain of βc that interacts with the adaptor protein 14-3-3ζ. Coimmunoprecipitation and pull-down experiments with a glutathione S-transferase (GST):14-3-3ζ fusion protein showed that 14-3-3 directly associates with βc but not the GM-CSF receptor  chain. C-terminal truncation mutants of βcfurther showed that a region between amino acids 544 and 626 in βc was required for its association with 14-3-3ζ. This region contains the sequence 582HSRSLP587, which closely resembles the RSXSXP (where S is phosphorylated) consensus 14-3-3 binding site identified in a number of signaling molecules, including Raf-1. Significantly, substitution of582HSRSLP587 for EFAAAA completely abolished interaction of βc with GST–14-3-3ζ. Furthermore, the interaction of βc with GST–14-3-3 was greatly reduced in the presence of a peptide containing the 14-3-3 binding site, but only when 585Ser was phosphorylated. Direct binding experiments showed that the peptide containing phosphorylated 585Ser bound 14-3-3ζ with an affinity of 150 nmol/L. To study the regulation of 585S phosphorylation in vivo, we raised antibodies that specifically recognized 585Ser-phosphorylated βc. Using these antibodies, we showed that GM-CSF stimulation strongly upregulated 585Ser phosphorylation in M1 myeloid leukemic cells. The proximity of the SHC-binding site (577Tyr) to the 14-3-3–binding site (582HSRSLP587) and their conservation between mouse, rat, and human βc but not in other cytokine receptors suggest that they form a distinct motif that may subserve specialized functions associated with the GM-CSF, IL-3, and IL-5 receptors.

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Association of RACK1 and PKCβ with the common β-chain of the IL-5/IL-3/GM-CSF receptor

Oncogene ◽

10.1038/sj.onc.1202896 ◽

1999 ◽

Vol 18 (36) ◽

pp. 5126-5130 ◽

Cited By ~ 68

Author(s):

Niels Geijsen ◽

Marcel Spaargaren ◽

Jan AM Raaijmakers ◽

Jan-Willem J Lammers ◽

Leo Koenderman ◽

...

Keyword(s):

Gm Csf ◽

The Common ◽

Β Chain

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Erythropoietin: multiple targets, actions, and modifying influences for biological and clinical consideration

Journal of Experimental Medicine ◽

10.1084/jem.20122760 ◽

2013 ◽

Vol 210 (2) ◽

pp. 205-208 ◽

Cited By ~ 78

Author(s):

Hal E. Broxmeyer

Keyword(s):

Cell Types ◽

Clinical Consideration ◽

Cell Type ◽

Erythroid Progenitors ◽

Chain Component ◽

Gm Csf ◽

Cell Tissue ◽

Wide Range ◽

The Common ◽

Β Chain

Erythropoietin (EPO), a humoral regulator of erythropoiesis and replacement therapy for selected red blood cell disorders in EPO-deficient patients, has been implicated in a wide range of activities on diverse cell, tissue, and organ types. EPO signals via two receptors, one comprising EPO receptor (EPOR) homodimers and the other a heterodimer of EPOR and CD131—the common β chain component of the GM-CSF, interleukin (IL)-3, and IL-5 receptors. Ligation of EPORs triggers various signaling pathways, including the JAK2–STAT5 and MAPK–NF-κB pathways, depending both on the receptor and the target cell type. A new study in this issue reveals a novel EPO-triggered pathway involving a Spi2A serpin–lysosome–cathepsin cascade that is initiated through the homodimeric EPOR complex and is required for the survival of erythroid progenitors. A full understanding of EPO’s effects on various cell types and their potential clinical relevance requires more work on the signaling events initiated through both EPORs, the effects of other cytokines and growth factors that modulate EPO’s actions, and a comparison of the effects of full-length versus truncated forms of EPO.

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A multitargeted antisense therapy directed at CCR3 and the common β-chain of IL-3/IL-5/GM-CSF

New Drugs and Targets for Asthma and COPD - Progress in Respiratory Research ◽

10.1159/000320834 ◽

2010 ◽

pp. 297-302

Author(s):

Luc Paquet ◽

Paolo Renzi ◽

Nicolay Ferrari ◽

Mark Parry-Billings

Keyword(s):

Antisense Therapy ◽

Gm Csf ◽

The Common ◽

Β Chain

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Inhibition of IL-13: A New Pathway for Atopic Dermatitis

Journal of Cutaneous Medicine and Surgery ◽

10.1177/1203475420982553 ◽

2020 ◽

pp. 120347542098255

Author(s):

Kayadri Ratnarajah ◽

Michelle Le ◽

Anastasiya Muntyanu ◽

Steve Mathieu ◽

Simon Nigen ◽

...

Keyword(s):

Monoclonal Antibody ◽

Atopic Dermatitis ◽

Severity Index ◽

Severe Atopic Dermatitis ◽

Treatment Alternative ◽

Control Groups ◽

Rapid Improvement ◽

Common Receptor ◽

The Common ◽

And Control

Dupilumab, a monoclonal antibody against the common receptor of interleukin (IL)-4 and IL-13, was the first biologic therapy approved in Canada for treatment of moderate-to-severe atopic dermatitis (AD). While it is considered safe and effective, dupilumab is not universally effective and 8%-38% of patients develop conjunctivitis, while some patients develop head and neck dermatitis. Thus, new therapeutic options are warranted. While both IL-4 and IL-13 play important roles in the pathogenesis of AD, it has been recently demonstrated that IL-13 is the primary upregulated cytokine in AD skin biopsy samples. A placebo-controlled phase 2b clinical trial evaluating the efficacy and safety of lebrikizumab, an IL-13 inhibitor, in AD demonstrated that, at 16 weeks, Eczema Area and Severity Index (EASI) 75 and Investigator’s Global Assessment (IGA) 0/1 were achieved by 60.6% and 44.6% of patients taking lebrikizumab at its highest dose (vs 24.3% and 15.3% of patients taking placebo, respectively). Moreover, treatment with lebrikizumab was associated with rapid improvement of pruritus and low rates of conjunctivitis (1.4%-3.8%). Another IL-13 monoclonal antibody, tralokinumab, was evaluated for safety and efficacy in moderate-to-severe AD. By week 12, among adults receiving 300 mg tralokinumab, 42.5% achieved EASI-75 and 26.7% achieved IGA 0/1 score (vs 15.5% and 11.8% in the placebo group, respectively). Both lebrikizumab and tralokinumab demonstrated acceptable safety profiles in AD (and non-AD) trials with adverse events often being comparable between treatment and control groups. Thus, IL-13 inhibitors may provide a safe and effective treatment alternative for patients with moderate-to-severe AD.

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