Generation of soluble interleukin-1 receptor from an immunoadhesin by specific cleavage

Beck, Joanne T.; Marsters, Scot A.; Harris, Reed J.; Carter, Paul; Ashkenazi, Avi; Chamow, Steven M.

doi:10.1016/0161-5890(94)90052-3

Cited by 17 publications

(10 citation statements)

References 30 publications

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“…Attempts to express the MISIIR ECD protein from transiently transfected mammalian cells failed to yield the desired molecule. Accordingly, we decided to produce MISIIR ECD as a fusion protein with human IgG 1 Fc domain which has been widely employed to aid in the expression and secretion of a wide variety of poorly expressing proteins (51,52). Although it is easier to pan libraries using a pure antigen as a target, selections can be successfully done against fusion proteins if care is taken to rule out the targeting of the carrier protein or unique sequences created at the fusion junction.…”

Section: Discussionmentioning

confidence: 99%

Development of engineered antibodies specific for the Müllerian inhibiting substance type II receptor: a promising candidate for targeted therapy of ovarian cancer

Yuan

Simmons

Robinson

et al. 2006

Molecular Cancer Therapeutics

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The Müllerian inhibiting substance type II receptor (MISIIR) is involved in Müllerian duct regression as part of the development of the male reproductive system. In adult females, MISIIR is present on ovarian surface epithelium and is frequently expressed on human epithelial ovarian cancer cells. Müllerian inhibiting substance has been found to be capable of inhibiting the growth of primary human ovarian cancer cells derived from ascites and ovarian cancer cell lines. This suggested to us that MISIIR could be an attractive target for antibody-based tumor targeting and growth inhibition strategies. Here, we describe the production of recombinant human MISIIR extracellular domain-human immunoglobulin Fc domain fusion proteins and their use as targets for the selection of MISIIR-specific human single-chain variable fragments (scFv) molecules from a human nonimmune scFv phage display library. The binding kinetics of the resulting anti-MISIIR scFv clones were characterized and two were employed as the basis for the construction of bivalent scFv:Fc antibody-based molecules. Both bound specifically to human ovarian carcinoma cells in flow cytometry assays and cross-reacted with mouse MISIIR. These results indicate that antibody-based constructs may provide a highly specific means of targeting MISIIR on human ovarian carcinoma cells for the purpose of diagnosing and treating this disease.

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Section: Discussionmentioning

confidence: 99%

Development of engineered antibodies specific for the Müllerian inhibiting substance type II receptor: a promising candidate for targeted therapy of ovarian cancer

Yuan

Simmons

Robinson

et al. 2006

Molecular Cancer Therapeutics

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“…We expressed each immunoadhesin by transient transfection of human 293 cells and purified it by protein A affinity chromatography. We then cleaved at the junction by immobilized Genenase I and isolated the ECD by removing the accompanying Fc fragment with protein A-agarose (24). Using this procedure, we obtained >95% pure ECD preparations, as judged by SDS/PAGE and silver staining (data not shown).…”

mentioning

confidence: 99%

“…We generated cDNA constructs encoding immunoglobulin fusion proteins (immunoadhesins) based on the ECD of human (h) or murine (m) IFN--yR a or , chain, as described (21,22). By site-directed mutagenesis, we inserted at the ECD-immunoglobulin junction a sequence encoding the hexapeptide AAHYTL, which is cleaved specifically by a mutant subtilisin BPN' called Genenase I (23), as described (24). We expressed each immunoadhesin by transient transfection of human 293 cells and purified it by protein A affinity chromatography.…”

mentioning

confidence: 99%

Interferon gamma signals via a high-affinity multisubunit receptor complex that contains two types of polypeptide chain.

Marsters¹,

Pennica²,

Bach³

et al. 1995

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

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Signaling by interferon y (IFN--y) requires two structurally related cell surface proteins: a ligand-binding polypeptide, known as the IFN-,y receptor (IFN-,yR), and an accessory factor. However, it is not known whether IFN--y forms a ternary complex with the IFN--yR and accessory factor to initiate signaling. Here we demonstrate complex formation between IFN-y and the two proteins, both in solution and at the cell surface. We observe complexes containing ligand, two molecules of IFN--yR ( IFN--y have been elucidated (2-4). IFN--y stimulates the tyrosine kinases Jakl and Jak2, leading to tyrosine phosphorylation of the IFN--y receptor (IFN--yR). Statl, a latent cytoplasmic transcription factor, binds to the phosphorylated IFN--yR, undergoes tyrosine phosphorylation, and forms homodimers that translocate to the nucleus to initiate transcription of IFN--y-inducible genes.The cell surface events that initiate IFN-,y signaling remain unclear. A protein known widely as the IFN-,yR binds IFN-y in a species-specific manner in human or murine species (1, 5).The IFN--yR belongs to the class 2 cytokine receptor family (6), which includes tissue factor (7), the two known chains of the IFN-a/13 receptor (8, 9), the interleukin 10 receptor (10), and the receptor-like molecule CRF2-4 (11). IFN--y signaling requires the IFN-,yR as well as an additional protein known as the IFN--yR accessory factor (1, 12). Functional complementation experiments suggest that a species-specific interaction between the IFN--yR extracellular domain (ECD) and the accessory factor is required for signaling (13-15). However, direct biochemical evidence for this deduced interaction is lacking, and its nature is undefined. For example, while the IFN-,yR is sufficient for ligand binding, it is not known whether the accessory factor is involved in this function as well. In addition, while the homodimeric IFN--y molecule (16) and IFN-,yR form a complex with a stoichiometry of 1:2 (17, 18), it is not known whether a ternary complex that contains the The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. accessory factor as well mediates signal transduction. Recently, human and murine accessory factor cDNAs have been isolated (19,20). The accessory factor is a type 1 transmembrane protein that resembles the class 2 cytokine receptor family (6). In this study, we use IFN--yR and accessory factor cDNAs to investigate whether their encoded proteins interact and to examine whether signaling can be blocked by preventing such interaction. Our results provide the basis for defining the subunit structure of the IFN--yR signaling complex and suggest an approach to inhibiting specific functions of IFN by blocking the interaction of receptor subunits.MATERIALS AND METHODS a-and 1-Chain ECDs. We generated cDNA constructs encoding immunoglobulin fusion proteins (immunoadhesins) based on the ECD of human ...

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“…To facilitate preparation of monomeric forms of KDR, a Genenase 1 protease cleavage site (18) was engineered at the junction of the last KDR IgG domain and the CH2 domain (19). The cleaved KDR was shown to be monomeric based on its mobility in nonreducing SDS-PAGE and gel filtration.…”

Section: The Igg-like Domains 2-3 In Kdr Are Sufficient For Highmentioning

confidence: 99%

Requirements for Binding and Signaling of the Kinase Domain Receptor for Vascular Endothelial Growth Factor

Fuh¹,

Li²,

Crowley³

et al. 1998

Journal of Biological Chemistry

241

213

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Vascular endothelial growth factor (VEGF) is a dimeric hormone that controls much of vascular development through binding and activation of its kinase domain receptor (KDR). We produced analogs of VEGF that show it has two receptor-binding sites which are located near the poles of the dimer and straddle the interface between subunits. Deletion experiments in KDR indicate that of the seven IgG-like domains in the extracellular domain, only domains 2-3 are needed for tight binding of VEGF. Monomeric forms of the extracellular domain of KDR bind ϳ100 times weaker than dimeric forms showing a strong avidity component for binding of VEGF to predimerized forms of the receptor. Based upon these structure-function studies and a mechanism in which receptor dimerization is critical for signaling, we constructed a receptor antagonist in the form of a heterodimer of VEGF that contained one functional and one non-functional site. These studies establish a functional foundation for the design of VEGF analogs, mimics, and antagonists.

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Generation of soluble interleukin-1 receptor from an immunoadhesin by specific cleavage

Cited by 17 publications

References 30 publications

Development of engineered antibodies specific for the Müllerian inhibiting substance type II receptor: a promising candidate for targeted therapy of ovarian cancer

Development of engineered antibodies specific for the Müllerian inhibiting substance type II receptor: a promising candidate for targeted therapy of ovarian cancer

Interferon gamma signals via a high-affinity multisubunit receptor complex that contains two types of polypeptide chain.

Requirements for Binding and Signaling of the Kinase Domain Receptor for Vascular Endothelial Growth Factor

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