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Integrin binding human antibody constant domains—Probing the C-terminal structural loops for grafting the RGD motif

Paper ID Volume ID Publish Year Pages File Format Full-Text
23719 43467 2011 10 PDF Available
Title
Integrin binding human antibody constant domains—Probing the C-terminal structural loops for grafting the RGD motif
Abstract

Recently, it has been demonstrated that loops of the crystallizable fragment of IgG1 (IgG1-Fc) can be engineered to form antigen-binding sites. In this work C-terminal structural loops in the CH3 domains of homodimeric IgG1-Fc have been functionalized to form integrin-binding sites in order to probe the effect of engineering on structural integrity and thermal stability of IgG1-Fc as well as on binding to the ligands Protein A, CD16 and FcRn, respectively. The peptide sequence GCRGDCL – a disulfide-bridged cyclic heptapeptide that confers binding to human αvβ3 integrin was introduced into AB, CD and/or EF loops and single and double mutants were heterologously expressed in Pichia pastoris. Integrin binding of engineered IgG-Fc was tested using both binding to coated αvβ3 integrin in ELISA or to αvβ3-expressing K562 cells in FACS analysis. Additionally, blocking of αvβ3-mediated cell adhesion to vitronectin was investigated. The data presented in this report demonstrate that bioactive integrin-binding peptide(s) can be grafted on the C-terminal loops of IgG-Fc without impairing binding to effector molecules. Observed differences between the investigated variants in structural stability and integrin binding are discussed with respect to the known structure of IgG-Fc and its structural loops.

► We grafted the RGD-motif on the structural loops of the CH3 domains of IgG1-Fc. ► Loop engineered IgG1-Fc mutants bound to avb3 integrin. ► Structural integrity and thermal stability of loop engineered mutants were analyzed. ► Binding of loop engineered IgG1-Fc-variants to FcRn, CD16 and Protein A was analyzed.

Keywords
mAb, monoclonal antibody; IgG1-Fc, crystallizable fragment of immunoglobulin G class 1; CDR, complementarity determining region; Fcab, antigen binding crystallizable domain; wt, wild-type; ADCC, antibody-dependent cellular cytotoxicity; CDC, complement-de
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Integrin binding human antibody constant domains—Probing the C-terminal structural loops for grafting the RGD motif
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 155, Issue 2, 10 September 2011, Pages 193–202
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us