Characterization of a Powerful High Affinity Antagonist That Inhibits Biological Activities of Human Interleukin-13

Ôshima, Yasuo; Puri, Raj K.

doi:10.1074/jbc.m010159200

Cited by 26 publications

(18 citation statements)

References 56 publications

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“…29 The marked specificity of these agents makes them attractive potential drugs. The availability of large quantities of characterized yeast expressed Epo (R103A) will allow further studies to determine if this agent will have clinical utility as member of this new class of drugs.…”

Section: Discussionmentioning

confidence: 99%

Purification and characterization of the yeast-expressed erythropoietin mutant Epo (R103A), a specific inhibitor of human primary hematopoietic cell erythropoiesis

Burns

Arcasoy

et al. 2002

Blood

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A drug that specifically inhibits erythropoiesis would be clinically useful. The erythropoietin (Epo) mutant Epo (R103A) could potentially be used for this purpose. Epo (R103A) has a single amino acid substitution of alanine for arginine at position 103. Because of this mutation, Epo (R103A) is only able to bind to one of the 2 subunits of the erythropoietin receptor (EpoR) homodimer and is thus a competitive inhibitor of Epo activity. To produce large quantities of Epo (R103A) to test in animal models of thalassemia and sickle cell disease, we expressed and purified recombinant Epo (R103A) from the yeast Pichia pastoris. IntroductionBlood transfusion is commonly used in the treatment hemoglobinopathies. One of the goals of blood transfusion in these diseases is the suppression of abnormal endogenous erythropoiesis. For instance, thalassemia major is associated with ineffective erythropoiesis and massive bone marrow expansion. This bone marrow expansion has numerous adverse effects, including hypersplenism, bone abnormalities, growth retardation, increased susceptibility to infection, and endocrine abnormalities. 1 Transfusion therapy regimens can dramatically ameliorate these problems.Patients with sickle cell disease may have a relative erythrocytosis such that oxygen delivery begins to decrease at hematocrits as low as 30% to 35%. 2 This decreased oxygen delivery is a consequence of the abnormal rheology of sickle red blood cells. 3 The resulting hyperviscosity is associated with serious clinical conditions such as acute chest syndrome and acute multiorgan damage syndrome. 4,5 These complications require treatment with exchange blood transfusions.In both thalassemia and sickle cell disease chronic transfusion therapy can have important complications including iron overload, viral hepatitis, and alloimmunization. Drug therapy that specifically and safely inhibits erythropoiesis would be desirable for these patients.A potential drug for specific inhibition of erythropoiesis is the erythropoietin (Epo) mutant Epo (R103A). Epo (R103A) has a mutation of alanine for arginine at amino acid position 103. Epo (R103A) was discovered during mutational studies to define important amino acid residues required for Epo activity. 6 Epo (R103A) completely lacks the ability to support mitogenesis in cells expressing the erythropoietin receptor (EpoR). 6,7 Biochemical and crystallographic studies have shown that Epo has nonidentical binding sites for each of the 2 identical EpoR subunits in the preformed EpoR homodimer. 8,9 Epo binding site 1 interacts with the first EpoR subunit with nanomolar affinity. 10 Epo binding site 2 interacts with the second subunit of the EpoR homodimer with micromolar affinity. 10 The resulting subnanomolar affinity of Epo for the EpoR dimer is the consequence of the 2 binding interactions. The interaction of Epo with the receptor homodimer results in a conformational shift of the subunits such that cytoplasmic signal transduction is activated. Arginine 103 of Epo is the primary residue involved in...

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Section: Discussionmentioning

confidence: 99%

Purification and characterization of the yeast-expressed erythropoietin mutant Epo (R103A), a specific inhibitor of human primary hematopoietic cell erythropoiesis

Burns

Arcasoy

et al. 2002

Blood

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“…Anti-IL-4 mAb was tested by ELISA. IL-13K13K was tested using the IL-13-dependent cell line TF-1 (44). The efficiency of anti-IL-10 receptor and anti-TGF-␤ mAbs was confirmed by their ability to induced colitis after in vivo treatment.…”

Section: Abs and Reagentsmentioning

confidence: 99%

“…(3 g/ mouse) twice daily for 7 days. IL13E13K, a powerful IL-13 antagonist, in which the glutamic acid at position 13 is replaced by a lysine, was expressed and purified as previously described (44). The efficiency of each cytokine-blocking reagent was confirmed by in vitro or in vivo assays.…”

Section: Abs and Reagentsmentioning

confidence: 99%

Inhibition of T Cell Differentiation into Effectors by NKT Cells Requires Cell Contacts

Novák

Beaudoin

Griseri

et al. 2005

The Journal of Immunology

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NKT cells are potent regulatory T cells that prevent the development of several autoimmune diseases. Analysis of NKT cell regulatory function in the NOD mouse has revealed that NKT cells inhibit the development of type 1 diabetes by impairing the differentiation of anti-islet T cells into Th1 effector cells. In the present study, we have performed in vitro and in vivo experiments to determine the respective role of cytokines and cell contacts in the blockade of T cell differentiation by NKT cells. These experiments reveal that cytokines such as IL-4, IL-10, IL-13, and TGF-β, that have been involved in other functions of NKT cells, play only a minor role if any in the blockade of T cell differentiation by NKT cells. Diabetes is still prevented by NKT cells in the absence of functional IL-4, IL-10, IL-13, and TGF-β. In contrast, we show for the first time that cell contacts are crucial for the immunoregulatory function of NKT cells.

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“…We have also generated a mutant of human IL-13, in which a glutamic acid (E) residue at position 13 was substituted by a lysine (K) residue (17). This mutant (termed IL-13E13K) was found to act as a powerful antagonist of IL-13 in that it inhibited biological activities of IL-13 (17).…”

Section: Introductionmentioning

confidence: 99%

Analysis of Antitumor Activity of an Interleukin-13 (IL-13) Receptor-Targeted Cytotoxin Composed of IL-13 Antagonist and Pseudomonas Exotoxin

Kioi

Kawakami

Puri

2004

Clinical Cancer Research

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We have shown previously that a chimeric fusion protein composed of human interleukin-13 (IL-13) and Pseudomonas exotoxin (PE), termed IL-13 cytotoxin (IL13-PE38), is specifically cytotoxic to various cancer cell lines and primary cell cultures derived from a variety of solid cancers. In addition, we have shown that IL-13 mutant IL-13E13K, in which glutamic acid (E) residue at position 13 of IL-13 molecule was substituted by a lysine (K), is a powerful antagonist of IL-13 and binds to IL-13 receptor with a higher affinity compared with wild-type IL-13. In this study, we have generated an IL-13 cytotoxin IL13E13K-PE38, in which IL-13 antagonist is fused to PE to determine whether this molecule has improved cytotoxicity to tumor cells compared with wild type (wt)IL13-PE38. Highly purified IL13E13K-PE38 was tested in various tumor cell lines including seven glioblastoma multiforme cell lines to compare its binding to the cells, in vitro cytotoxicity, in vivo antitumor activity, and safety in mouse model with wtIL13-PE38. IL13E13K-PE38 bound to U251MG and IL-13R␣2 chaintransfected tumor cell lines with 3 to 10 times higher affinity compared with wtIL13-PE38. However, IL13E13K-PE38 did not show higher cytotoxicity compared with wtIL13-PE38 in glioblastoma multiforme or any other cell lines tested. The antitumor activity of IL13E13K-PE38, when administered intraperitoneally to nude mice bearing U251 tumors, was also similar to wtIL13-PE38. Some improvement in antitumor activity was observed when lower doses of IL13E13K-PE38 were injected intratumorally in subcutaneous tumors. These results indicate that in general, IL13E13K-PE38 mediates similar cytotoxicity and antitumor activity to wtIL13-PE38 despite its improved binding affinity to IL-13 receptors.

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Characterization of a Powerful High Affinity Antagonist That Inhibits Biological Activities of Human Interleukin-13

Cited by 26 publications

References 56 publications

Purification and characterization of the yeast-expressed erythropoietin mutant Epo (R103A), a specific inhibitor of human primary hematopoietic cell erythropoiesis

Purification and characterization of the yeast-expressed erythropoietin mutant Epo (R103A), a specific inhibitor of human primary hematopoietic cell erythropoiesis

Inhibition of T Cell Differentiation into Effectors by NKT Cells Requires Cell Contacts

Analysis of Antitumor Activity of an Interleukin-13 (IL-13) Receptor-Targeted Cytotoxin Composed of IL-13 Antagonist and Pseudomonas Exotoxin

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