Selection and Characterization of Single Chain Antibody Fragments Specific for Hsp90 as a Potential Cancer Targeting Molecule.

Edyta Petters,Aleksandra Sokolowska-Wedzina,Jacek Otlewski

doi:10.3390/ijms160819920

Edyta Petters, Aleksandra Sokolowska-Wedzina + Show 1 more

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https://doi.org/10.3390/ijms160819920

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Abstract

Heat shock proteins play an essential role in facilitating malignant transformation and they have been recognized as important factors in human cancers. One of the key elements of the molecular chaperones machinery is Hsp90 and it has recently become a target for anticancer therapeutic approaches. The potential and importance of Hsp90-directed agents becomes apparent when one realizes that disruption of Hsp90 function may influence over 200 oncogenic client proteins. Here, we described the selection and characterization of Hsp90-specific antibody fragments from commercially available Tomlinson I and J phage display libraries. The affinities of Hsp90-binding scFv variants were measured using SPR method. Then, based on the best clone selected, we performed the affinity maturation procedure and obtained valuable Hsp90-specific clones. The selected binders were expressed and applied for immunostaining, ELISA and SPR analysis using model cancer cell lines. All performed experiments confirmed the ability of selected antibodies to interact with the Hsp90. Therefore, the presented Hsp90-specific scFv, might be a starting point for the development of a novel antibody-based strategy targeting cancer.

Highlights

Heat shock protein 90 (Hsp90) is an evolutionary conserved protein that accounts for 1%–2% of total cellular proteins and is essential for cell viability
Hsp90 protein is commonly overexpressed in a wide variety of human cancers, where it helps cells to tolerate imbalanced signaling caused by oncoproteins, supporting the malignant transformation of tumor cells [4,6]
Phage particles displaying scFv proteins were rescued from E. coli TG1 and used for panning against the antigen

Summary

Introduction

Heat shock protein 90 (Hsp90) is an evolutionary conserved protein that accounts for 1%–2% of total cellular proteins and is essential for cell viability. Hsp is ATP-dependent molecular chaperone that assists client proteins in proper folding [1]. There are over 200 protein substrates of Hsp90 [2], many of which are the key factors in cancer development and progression, including tyrosine kinases (e.g., Src), serine-threonine kinases (e.g., Raf-1, AKT), cell cycle kinases (e.g., Wee, POLO-1) [3,4], transcription factors (such as HIF-1), steroid receptors (e.g., estrogen, androgen, progesterone or glucocorticoid) and non-steroid receptors (e.g., HER2), as well as mutated forms of p53 [4,5]. Hsp is one of the key players in breast carcinogenesis. It was shown that single nucleotide polymorphism within Hsp90α gene (Gln488His) is associated with a higher risk of breast cancer [7]

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