Neutralization of Biological Activity and Inhibition of Receptor Binding by Antibodies Against Human Thrombopoietin

Tahara, Tomoyuki; Kuwaki, Tomoaki; Matsumoto, Atsushi; Morita, Hirohisa; Watarai, Hiroshi; Inagaki, Yoshimasa; Ohashi, Hideya; Ogami, Kinya; Miyazaki, Hiroshi; Kato, Takashi

doi:10.1002/stem.160054

Cited by 17 publications

(18 citation statements)

References 33 publications

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“…FN1 and FC1, which are mouse monoclonal antibodies (IgG 1 ) with neutralizing activity to FGF23, were affinity‐purified from the conditioned media of the hybridoma cells using Protein G Sepharose (GE Healthcare) and reconstituted in PBS. As an isotype‐matched control antibody, we used anti‐human thrombopoietin (TPO) mouse monoclonal antibody (IgG 1 ) without cross‐reactivity to murine TPO (25) . FN1 structurally recognizes the N‐terminal portion of FGF23, whereas FC1 recognizes single region comprised of several residues in the C‐terminal domain of FGF23.…”

Section: Methodsmentioning

confidence: 99%

Therapeutic Effects of Anti-FGF23 Antibodies in Hypophosphatemic Rickets/Osteomalacia

Aono

Yamazaki

Yasutake

et al. 2009

Journal of Bone and Mineral Research

236

177

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X-linked hypophosphatemia (XLH), characterized by renal phosphate wasting, is the most common cause of vitamin D-resistant rickets. It has been postulated that some phosphaturic factor plays a causative role in XLH and its murine homolog, the Hyp mouse. Fibroblast growth factor 23 (FGF23) is a physiological phosphaturic factor; its circulatory level is known to be high in most patients with XLH and Hyp mice, suggesting its pathophysiological role in this disease. To test this hypothesis, we treated Hyp mice with anti-FGF23 antibodies to inhibit endogenous FGF23 action. A single injection of the antibodies corrected the hypophosphatemia and inappropriately normal serum 1,25-dihydroxyvitamin D. These effects were accompanied by increased expressions of type IIa sodium-phosphate cotransporter and 25-hydroxyvitamin-D1a-hydroxylase and a suppressed expression of 24-hydroxylase in the kidney. Repeated injections during the growth period ameliorated the rachitic bone phenotypes typically observed in Hyp mice, such as impaired longitudinal elongation, defective mineralization, and abnormal cartilage development. Thus, these results indicate that excess actions of FGF23 underlie hypophosphatemic rickets in Hyp mice and suggest a novel therapeutic potential of the FGF23 antibodies for XLH.

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Section: Methodsmentioning

confidence: 99%

Therapeutic Effects of Anti-FGF23 Antibodies in Hypophosphatemic Rickets/Osteomalacia

Aono

Yamazaki

Yasutake

et al. 2009

Journal of Bone and Mineral Research

236

177

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“…The hTPO gene (26) containing the N-terminal region from residues 1 to 163 (hTPO 163 ) was expressed in Escherichia coli and prepared as reported (27). The TN1-neutralizing IgG (subclass IgG1) was raised against recombinant hTPO (28) and prepared by hybridoma cultivation followed by affinity chromatography with a protein G agarose column (Amersham Pharmacia Biosciences). The TN1-IgG showed half-maximal binding at 0.7 nM against 3.3 pmol of hTPO in ELISAs.…”

Section: Methodsmentioning

confidence: 99%

“…The arrangement of the molecules in the asymmetric unit was determined with AMORE (28). The cell dimensions and crystal packing of the P2 1 and C2 crystal forms are in fact nearly identical.…”

Section: Methodsmentioning

confidence: 99%

Structure of the receptor-binding domain of human thrombopoietin determined by complexation with a neutralizing antibody fragment

Feese

Tamada

Kato

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

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The cytokine thrombopoietin (TPO), the ligand for the hematopoietic receptor c-Mpl, acts as a primary regulator of megakaryocytopoiesis and platelet production. We have determined the crystal structure of the receptor-binding domain of human TPO (hTPO 163) to a 2.5-Å resolution by complexation with a neutralizing Fab fragment. The backbone structure of hTPO 163 (1) predicted the existence of a potent, lineage-specific soluble factor, which they called thrombopoietin (TPO), that stimulates megakaryocytopoiesis and platelet production. It was not until 1994 that unequivocal evidence for the existence of this elusive molecule was provided by the nearly simultaneous isolation and cloning of TPO by five independent research groups (2-6). This cytokine has proven to be a primary factor in megakaryocytopoiesis from megakaryocyte colony formation to platelet production and the differentiation and proliferation of progenitor cells of multiple hematopoietic lineages (7). As such, TPO is being investigated for its potential to treat thrombocytopenia resulting from AIDS and chemotherapy and radiation treatments for cancer and leukemia and for the in vivo and ex vivo expansion of hematopoietic stem cells for bone marrow transplantation.Human TPO (hTPO) is a heavily glycosylated protein with two distinct regions. The 153-residue N-terminal region is homologous to human erythropoietin (EPO) with which it shares 23% sequence identity and is sufficient for receptor binding and signal transduction (2,3,8). The 179-residue C-terminal region has a large number of proline and glycine residues and six N-linked glycosylation sites. Its function is not known, although recent work indicates a role in secretion and protection from proteolysis (9, 10).The TPO receptor c-Mpl was first identified as an oncogene of the murine myeloproliferative leukemia virus (11, 12) that was able to immortalize hematopoietic progenitor cells and was later cloned from human and mouse (13,14). c-Mpl is expressed in some pluripotent hematopoietic stem cells (15) and in the megakaryocyte lineage from progenitor cells to platelets (16). It is a class I cytokine receptor of the hematopoietic superfamily of receptors and signals by the JAK͞STAT, Ras, and mitogenactivated protein kinase pathways (17-21). Class I hematopoietic receptors bind to their cytokine ligands by Ϸ200-aa Ig-like extracellular domains called cytokine receptor homology (CRH) or hematopoietic receptor domains that contain a distinctive WSXWS sequence motif (13).Cytokines possess two distinct interaction sites that bind with differing affinities [high affinity (nanomolar range) and low affinity (micromolar range)] to the same cytokinerecognition surface of the CRH domain. Crystal structures of human EPO and human growth hormone (hGH) in complex with the extracellular CRH domains of their receptors (22, 23) have shown the cytokine-CRH interaction in detail. However, unlike EPO receptor (EPOR) and hGH receptor (hGHR), which have only one CRH domain, c-Mpl belongs to a subset of hematopoietic ...

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“…Previously, the crystal structure of hTPO in complex with an antigen‐binding fragment (Fab) derived from the hTPO‐neutralizing murine monoclonal antibody TN1 was described; however, the crystal structure of free hTPO has yet to be reported. The difficulties associated with the crystallographic analysis of free hTPO may relate to its physicochemical properties; possibly, hTPO is highly‐flexible and structurally stabilized by complexation with TN1.…”

Section: Introductionmentioning

confidence: 99%

An insight into the thermodynamic characteristics of human thrombopoietin complexation with TN1 antibody

et al. 2016

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Human thrombopoietin (hTPO) primarily stimulates megakaryocytopoiesis and platelet production and is neutralized by the mouse TN1 antibody. The thermodynamic characteristics of TN1 antibody-hTPO complexation were analyzed by isothermal titration calorimetry (ITC) using an antigen-binding fragment (Fab) derived from the TN1 antibody (TN1-Fab). To clarify the mechanism by which hTPO is recognized by TN1-Fab the conformation of free TN1-Fab was determined to a resolution of 2.0 Å using X-ray crystallography and compared with the hTPO-bound form of TN1-Fab determined by a previous study. This structural comparison revealed that the conformation of TN1-Fab does not substantially change after hTPO binding and a set of 15 water molecules is released from the antigen-binding site (paratope) of TN1-Fab upon hTPO complexation. Interestingly, the heat capacity change (DCp) measured by ITC (21.52 6 0.05 kJ mol 21 K 21) differed significantly from calculations based upon the X-ray structure data of the hTPO-bound and unbound forms of TN1-Fab (21.02~0.25 kJ mol 21 K 21) suggesting that hTPO undergoes an induced-fit conformational change combined with significant desolvation upon TN1-Fab binding. The results shed light on the structural biology associated with neutralizing antibody recognition.

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Neutralization of Biological Activity and Inhibition of Receptor Binding by Antibodies Against Human Thrombopoietin

Cited by 17 publications

References 33 publications

Therapeutic Effects of Anti-FGF23 Antibodies in Hypophosphatemic Rickets/Osteomalacia

Therapeutic Effects of Anti-FGF23 Antibodies in Hypophosphatemic Rickets/Osteomalacia

Structure of the receptor-binding domain of human thrombopoietin determined by complexation with a neutralizing antibody fragment

An insight into the thermodynamic characteristics of human thrombopoietin complexation with TN1 antibody

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