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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 results of phage display selection against Hsp90α. (A) Monoclonal ELISA for 64 scFv clones selected after the third round of panning against Hsp90α. Bacterial supernatants containing soluble antibody fragments were applied onto the target protein immobilized directly on the plastic surface of 96-well Nunc MaxiSorp® plate. The binding clones were detected with the use of monoclonal mouse antibody 9E10. Of all clones, 51 showing the highest absorption at 450 nm were selected for further analysis by SPR on Biacore® 3000. The best clone scFv47 is highlighted with orange; (B,C) SPR response curves for measurements of scFv47 affinity for Hsp90. Pure scFv47 in five different concentrations was injected on Hsp90 isoform α (A) or β (B) immobilized on CM5 sensor chip. The calculations of kinetic constants were done with the use of BIAevaluation 4.1 software.
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ijms-16-19920-f001: The results of phage display selection against Hsp90α. (A) Monoclonal ELISA for 64 scFv clones selected after the third round of panning against Hsp90α. Bacterial supernatants containing soluble antibody fragments were applied onto the target protein immobilized directly on the plastic surface of 96-well Nunc MaxiSorp® plate. The binding clones were detected with the use of monoclonal mouse antibody 9E10. Of all clones, 51 showing the highest absorption at 450 nm were selected for further analysis by SPR on Biacore® 3000. The best clone scFv47 is highlighted with orange; (B,C) SPR response curves for measurements of scFv47 affinity for Hsp90. Pure scFv47 in five different concentrations was injected on Hsp90 isoform α (A) or β (B) immobilized on CM5 sensor chip. The calculations of kinetic constants were done with the use of BIAevaluation 4.1 software.

Mentions: After the third round of selection, we conducted monoclonal ELISA and we screened 64 individual scFv clones for binding to the target molecule. The assay showed that most of the investigated proteins exhibited some preference for Hsp90 (Figure 1A). Among them, 51 demonstrated the highest absorption signal and were employed for preliminary surface plasmon resonance (SPR) screening. The selected scFv fragments contained in bacterial supernatants were verified for binding to the Hsp90α immobilized on the CM5 sensor chip. Overall, 25 of them showed promising binding profile and were subsequently sequenced. The analysis of the sequencing results revealed no sequence identity among all clones examined, although there were some evident preferences for particular amino acid at given positions. For example, T or S was highly favored at the position 50 in HCDR2 and there were clear preferences for T, S and Y at the positions 95/96, 97 and 98 of HCDR3, respectively (data not shown). The amino acid preferences were more explicit for randomized positions in Tomlinson I library (DVT randomization scheme) than for Tomlinson J where NNK randomization was applied. Next, all 25 clones were overexpressed in bacteria, purified on Ni-NTA resin and subjected to the affinity measurements on Biacore® 3000. The estimated KD values of tested scFv fragments were mostly in the micromolar range and the highest affinity for Hsp90α was observed for scFv47 clone and was equal to 387 nM (Figure 1B).


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 results of phage display selection against Hsp90α. (A) Monoclonal ELISA for 64 scFv clones selected after the third round of panning against Hsp90α. Bacterial supernatants containing soluble antibody fragments were applied onto the target protein immobilized directly on the plastic surface of 96-well Nunc MaxiSorp® plate. The binding clones were detected with the use of monoclonal mouse antibody 9E10. Of all clones, 51 showing the highest absorption at 450 nm were selected for further analysis by SPR on Biacore® 3000. The best clone scFv47 is highlighted with orange; (B,C) SPR response curves for measurements of scFv47 affinity for Hsp90. Pure scFv47 in five different concentrations was injected on Hsp90 isoform α (A) or β (B) immobilized on CM5 sensor chip. The calculations of kinetic constants were done with the use of BIAevaluation 4.1 software.
© Copyright Policy
Related In: Results  -  Collection

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

ijms-16-19920-f001: The results of phage display selection against Hsp90α. (A) Monoclonal ELISA for 64 scFv clones selected after the third round of panning against Hsp90α. Bacterial supernatants containing soluble antibody fragments were applied onto the target protein immobilized directly on the plastic surface of 96-well Nunc MaxiSorp® plate. The binding clones were detected with the use of monoclonal mouse antibody 9E10. Of all clones, 51 showing the highest absorption at 450 nm were selected for further analysis by SPR on Biacore® 3000. The best clone scFv47 is highlighted with orange; (B,C) SPR response curves for measurements of scFv47 affinity for Hsp90. Pure scFv47 in five different concentrations was injected on Hsp90 isoform α (A) or β (B) immobilized on CM5 sensor chip. The calculations of kinetic constants were done with the use of BIAevaluation 4.1 software.
Mentions: After the third round of selection, we conducted monoclonal ELISA and we screened 64 individual scFv clones for binding to the target molecule. The assay showed that most of the investigated proteins exhibited some preference for Hsp90 (Figure 1A). Among them, 51 demonstrated the highest absorption signal and were employed for preliminary surface plasmon resonance (SPR) screening. The selected scFv fragments contained in bacterial supernatants were verified for binding to the Hsp90α immobilized on the CM5 sensor chip. Overall, 25 of them showed promising binding profile and were subsequently sequenced. The analysis of the sequencing results revealed no sequence identity among all clones examined, although there were some evident preferences for particular amino acid at given positions. For example, T or S was highly favored at the position 50 in HCDR2 and there were clear preferences for T, S and Y at the positions 95/96, 97 and 98 of HCDR3, respectively (data not shown). The amino acid preferences were more explicit for randomized positions in Tomlinson I library (DVT randomization scheme) than for Tomlinson J where NNK randomization was applied. Next, all 25 clones were overexpressed in bacteria, purified on Ni-NTA resin and subjected to the affinity measurements on Biacore® 3000. The estimated KD values of tested scFv fragments were mostly in the micromolar range and the highest affinity for Hsp90α was observed for scFv47 clone and was equal to 387 nM (Figure 1B).

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