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

Petters E, Sokolowska-Wedzina A, Otlewski J - Int J Mol Sci (2015)

Bottom Line: 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.All performed experiments confirmed the ability of selected antibodies to interact with the Hsp90.

View Article: PubMed Central - PubMed

Affiliation: Faculty of Biotechnology, Department of Protein Engineering, University of Wroclaw, Joliot-Curie 14a, 50-383 Wroclaw, Poland. edyta_petters@op.pl.

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.

No MeSH data available.


Related in: MedlinePlus

The immunofluorescence detection of intracellular Hsp90 in fixed and permeabilized. (A) MDA MB 453 cell line; and (B) MDA MB 231 cell line. The intracellular Hsp90 was stained green either by scFv47-FITC, scFvA4-FITC or anti-Hsp90/secondary goat anti-mouse Alexa 488-conjugated antibodies. Nuclei were stained with DAPI (blue) and the cytoskeletal actin filament network was visualized with Alexa 594 phalloidin (red). Original magnification ×20, for all panels.
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ijms-16-19920-f003: The immunofluorescence detection of intracellular Hsp90 in fixed and permeabilized. (A) MDA MB 453 cell line; and (B) MDA MB 231 cell line. The intracellular Hsp90 was stained green either by scFv47-FITC, scFvA4-FITC or anti-Hsp90/secondary goat anti-mouse Alexa 488-conjugated antibodies. Nuclei were stained with DAPI (blue) and the cytoskeletal actin filament network was visualized with Alexa 594 phalloidin (red). Original magnification ×20, for all panels.

Mentions: The immunofluorescence experiments were preformed to test the ability of selected scFvs to specifically recognize Hsp90α in the cell model. We used two human breast cancer cell lines, MDA MB 435 and MDA MB 231, as Hsp90-expressing examples [19,40,41]. To visualize the scFv47 and scFvA4 binding to Hsp90, we conjugated those proteins to fluorescein isothiocyanate (FITC). As a positive control, commercially available mouse antibody specific for Hsp90 was used. Additionally, the nuclei were stained with DAPI and the cytoskeletal actin filament network was visualized with Alexa 594 phalloidin (Figure 3).


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

Petters E, Sokolowska-Wedzina A, Otlewski J - Int J Mol Sci (2015)

The immunofluorescence detection of intracellular Hsp90 in fixed and permeabilized. (A) MDA MB 453 cell line; and (B) MDA MB 231 cell line. The intracellular Hsp90 was stained green either by scFv47-FITC, scFvA4-FITC or anti-Hsp90/secondary goat anti-mouse Alexa 488-conjugated antibodies. Nuclei were stained with DAPI (blue) and the cytoskeletal actin filament network was visualized with Alexa 594 phalloidin (red). Original magnification ×20, for all panels.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4581332&req=5

ijms-16-19920-f003: The immunofluorescence detection of intracellular Hsp90 in fixed and permeabilized. (A) MDA MB 453 cell line; and (B) MDA MB 231 cell line. The intracellular Hsp90 was stained green either by scFv47-FITC, scFvA4-FITC or anti-Hsp90/secondary goat anti-mouse Alexa 488-conjugated antibodies. Nuclei were stained with DAPI (blue) and the cytoskeletal actin filament network was visualized with Alexa 594 phalloidin (red). Original magnification ×20, for all panels.
Mentions: The immunofluorescence experiments were preformed to test the ability of selected scFvs to specifically recognize Hsp90α in the cell model. We used two human breast cancer cell lines, MDA MB 435 and MDA MB 231, as Hsp90-expressing examples [19,40,41]. To visualize the scFv47 and scFvA4 binding to Hsp90, we conjugated those proteins to fluorescein isothiocyanate (FITC). As a positive control, commercially available mouse antibody specific for Hsp90 was used. Additionally, the nuclei were stained with DAPI and the cytoskeletal actin filament network was visualized with Alexa 594 phalloidin (Figure 3).

Bottom Line: 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.All performed experiments confirmed the ability of selected antibodies to interact with the Hsp90.

View Article: PubMed Central - PubMed

Affiliation: Faculty of Biotechnology, Department of Protein Engineering, University of Wroclaw, Joliot-Curie 14a, 50-383 Wroclaw, Poland. edyta_petters@op.pl.

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.

No MeSH data available.


Related in: MedlinePlus