کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
2184593 1095892 2013 23 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
Combinatorial Design of an Anticalin Directed against the Extra-Domain B for the Specific Targeting of Oncofetal Fibronectin
موضوعات مرتبط
علوم زیستی و بیوفناوری بیوشیمی، ژنتیک و زیست شناسی مولکولی بیولوژی سلول
پیش نمایش صفحه اول مقاله
Combinatorial Design of an Anticalin Directed against the Extra-Domain B for the Specific Targeting of Oncofetal Fibronectin
چکیده انگلیسی

The oncofetal isoform of the extracellular matrix protein fibronectin (Fn), which carries the extra-domain B (ED-B) and is exclusively expressed in neovasculature, has gained interest for tumor diagnosis and therapy using engineered antibody fragments. We have employed the human lipocalin 2 (Lcn2) as a small and robust non-immunoglobulin scaffold to select ED-B-specific Anticalins from a new advanced random library using bacterial phage display and ELISA screening against appropriately engineered Fn fragments. As a result, we have isolated and biochemically characterized four different Anticalins that all show low nanomolar affinities for ED-B, right in the range between the monomeric and dimeric forms of the single-chain variable antibody fragment L19 that has been widely applied in this area before. All Anticalins can be readily expressed in Escherichia coli as soluble and strictly monomeric proteins, and they show specific staining of ED-B-positive tumor cells in immunofluorescence microscopy while BIAcore affinity analyses indicate recognition of distinct ED-B epitopes. The crystal structure for one Anticalin, N7A, in complex with the Fn7B8 fragment, was solved at 2.6 Å resolution and reveals binding to the gfcc′ sheet and cc′ loop on ED-B. This is the second example of a protein-specific Lcn2-based Anticalin, which illustrates the remarkable plasticity of the calyx-like ligand pocket of lipocalins with their four structurally hypervariable loops supported by a highly conserved β-barrel. The ED-B-specific Anticalins resulting from this study should provide useful reagents in research and biomedical drug development, both for in vivo imaging and for directed cancer therapy.

Graphical AbstractFigure optionsDownload high-quality image (82 K)Download as PowerPoint slideHighlights
► The ED-B of Fn is relevant for tumor diagnosis and therapy.
► Anticalins were selected from a novel random library based on human Lcn2.
► These Anticalins specifically recognize the ED-B in the context of its flanking domains.
► The stably monomeric Anticalins have high affinities and stain ED-B-positive cells.
► X-ray analysis of the Anticalin N7A bound to Fn7B8 reveals new structural insights.

ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Journal of Molecular Biology - Volume 425, Issue 4, 22 February 2013, Pages 780–802
نویسندگان
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