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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

Interaction of scFv47 with eHsp90: (A) Western blot analysis of concentrated serum-free media derived from MDA MB 231 and MDA MB 453 cell cultures using specific anti-Hsp90 antibodies. Cell lysates containing cytoplasmic Hsp90 and recombinant Hsp90α served as positive controls; (B) The representative ELISA using scFv47 to detect eHsp90 in concentrated culture media derived from MDA MB 231, MDA MB 453 and BJ cells. The plate was coated with scFv47 and cell medium was applied. Detection of bound Hsp90 was performed using commercially available mouse anti-Hsp90 followed by anti-mouse-HRP antibody. Absorbance value from the controls with no antigen was subtracted from the obtained results, which were subsequently normalized as a fraction of absorbance from the wells where 10 µg/mL of recombinant Hsp90α was loaded (positive control); (C) The SPR sensograms showing the binding of Hsp90α to immobilized scFv47. After 38 hours of culturing MDA MB 231 and MDA MB 453 cells (in serum-free DMEM) media were collected, concentrated 100 times, buffer exchanged and injected on a sensor chip coated with ca. 1500 RU scFv47. Fifty micrograms per milliliters of the recombinant Hsp90α was injected as a positive control.
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ijms-16-19920-f004: Interaction of scFv47 with eHsp90: (A) Western blot analysis of concentrated serum-free media derived from MDA MB 231 and MDA MB 453 cell cultures using specific anti-Hsp90 antibodies. Cell lysates containing cytoplasmic Hsp90 and recombinant Hsp90α served as positive controls; (B) The representative ELISA using scFv47 to detect eHsp90 in concentrated culture media derived from MDA MB 231, MDA MB 453 and BJ cells. The plate was coated with scFv47 and cell medium was applied. Detection of bound Hsp90 was performed using commercially available mouse anti-Hsp90 followed by anti-mouse-HRP antibody. Absorbance value from the controls with no antigen was subtracted from the obtained results, which were subsequently normalized as a fraction of absorbance from the wells where 10 µg/mL of recombinant Hsp90α was loaded (positive control); (C) The SPR sensograms showing the binding of Hsp90α to immobilized scFv47. After 38 hours of culturing MDA MB 231 and MDA MB 453 cells (in serum-free DMEM) media were collected, concentrated 100 times, buffer exchanged and injected on a sensor chip coated with ca. 1500 RU scFv47. Fifty micrograms per milliliters of the recombinant Hsp90α was injected as a positive control.

Mentions: Based on previous studies that reported the secretion of Hsp90 by human breast cancer cells MDA MB 231 [42] and MDA MB 453 [21,41], lysates of MDA MB 231 and MDA MB 453 cells as well as concentrated supernatants derived from the serum-free cell medium upon 38 h of culture were analyzed by Western blotting using commercially available anti-Hsp90 antibody. As shown in Figure 4A, Hsp90 was present not only in lysates (cytoplasmic Hsp90) but also in the media (extracellular Hsp90), indicating that both breast cancer cell lines tested secreted Hsp90. The absence of tubulin in the medium fraction (Figure 4A) confirmed that there was no contamination with the intracellular components during the experimental procedure.


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)

Interaction of scFv47 with eHsp90: (A) Western blot analysis of concentrated serum-free media derived from MDA MB 231 and MDA MB 453 cell cultures using specific anti-Hsp90 antibodies. Cell lysates containing cytoplasmic Hsp90 and recombinant Hsp90α served as positive controls; (B) The representative ELISA using scFv47 to detect eHsp90 in concentrated culture media derived from MDA MB 231, MDA MB 453 and BJ cells. The plate was coated with scFv47 and cell medium was applied. Detection of bound Hsp90 was performed using commercially available mouse anti-Hsp90 followed by anti-mouse-HRP antibody. Absorbance value from the controls with no antigen was subtracted from the obtained results, which were subsequently normalized as a fraction of absorbance from the wells where 10 µg/mL of recombinant Hsp90α was loaded (positive control); (C) The SPR sensograms showing the binding of Hsp90α to immobilized scFv47. After 38 hours of culturing MDA MB 231 and MDA MB 453 cells (in serum-free DMEM) media were collected, concentrated 100 times, buffer exchanged and injected on a sensor chip coated with ca. 1500 RU scFv47. Fifty micrograms per milliliters of the recombinant Hsp90α was injected as a positive control.
© Copyright Policy
Related In: Results  -  Collection

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

ijms-16-19920-f004: Interaction of scFv47 with eHsp90: (A) Western blot analysis of concentrated serum-free media derived from MDA MB 231 and MDA MB 453 cell cultures using specific anti-Hsp90 antibodies. Cell lysates containing cytoplasmic Hsp90 and recombinant Hsp90α served as positive controls; (B) The representative ELISA using scFv47 to detect eHsp90 in concentrated culture media derived from MDA MB 231, MDA MB 453 and BJ cells. The plate was coated with scFv47 and cell medium was applied. Detection of bound Hsp90 was performed using commercially available mouse anti-Hsp90 followed by anti-mouse-HRP antibody. Absorbance value from the controls with no antigen was subtracted from the obtained results, which were subsequently normalized as a fraction of absorbance from the wells where 10 µg/mL of recombinant Hsp90α was loaded (positive control); (C) The SPR sensograms showing the binding of Hsp90α to immobilized scFv47. After 38 hours of culturing MDA MB 231 and MDA MB 453 cells (in serum-free DMEM) media were collected, concentrated 100 times, buffer exchanged and injected on a sensor chip coated with ca. 1500 RU scFv47. Fifty micrograms per milliliters of the recombinant Hsp90α was injected as a positive control.
Mentions: Based on previous studies that reported the secretion of Hsp90 by human breast cancer cells MDA MB 231 [42] and MDA MB 453 [21,41], lysates of MDA MB 231 and MDA MB 453 cells as well as concentrated supernatants derived from the serum-free cell medium upon 38 h of culture were analyzed by Western blotting using commercially available anti-Hsp90 antibody. As shown in Figure 4A, Hsp90 was present not only in lysates (cytoplasmic Hsp90) but also in the media (extracellular Hsp90), indicating that both breast cancer cell lines tested secreted Hsp90. The absence of tubulin in the medium fraction (Figure 4A) confirmed that there was no contamination with the intracellular components during the experimental procedure.

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