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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 affinity maturation for scFv47 clone. (A) Monoclonal ELISA for scFv clones selected after the second round of panning against Hsp90α. Of all clones, 35 were selected for further analysis by SPR on Biacore® 3000. The best clone scFvA4 is highlighted with orange; (B,C) Binding curves for scFvA4 clone against Hsp90α and Hsp90β, respectively. Varied concentrations of purified protein were injected on two CM5 sensor chips immobilized with Hsp90 isoforms. The BIAevaluation software was used for calculations of kinetic constants; (D) The amino acids selected at CDR1, CDR2 and CDR3 of parental and affinity matured clones.
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ijms-16-19920-f002: The results of affinity maturation for scFv47 clone. (A) Monoclonal ELISA for scFv clones selected after the second round of panning against Hsp90α. Of all clones, 35 were selected for further analysis by SPR on Biacore® 3000. The best clone scFvA4 is highlighted with orange; (B,C) Binding curves for scFvA4 clone against Hsp90α and Hsp90β, respectively. Varied concentrations of purified protein were injected on two CM5 sensor chips immobilized with Hsp90 isoforms. The BIAevaluation software was used for calculations of kinetic constants; (D) The amino acids selected at CDR1, CDR2 and CDR3 of parental and affinity matured clones.

Mentions: The panning procedure was modified to favor selection of scFv variants with improved koff values. Therefore, we run so-called “off-rate selection”, wherein for the second round of panning we used five molar excess of soluble Hsp90α. This strategy enabled the enrichment of slower dissociating clones and led to the isolation of 35 positive variants (Figure 2A). The best clone selected, scFvA4, exhibited KD of 185 nM (Figure 2B). Although the improvement in KD was not significant (two-fold), the scFvA4 showed over sixfold lower koff in comparison with parental scFv47. Moreover, we observed 10-fold weaker binding affinity of scFvA4 to Hsp90β (Figure 2C). Such result could be beneficial for future therapeutic applications, as cancer cells express and constitutively secrete primarily the isoform α of Hsp90 [38,39]. In addition, we compared our selected antibody fragments with commercially available anti-Hsp90 antibody using SPR method. We injected the same concentration of antibodies/scFvs on Hsp90α immobilized on sensor chip. We observed similar binding profiles of scFvA4 and commercial antibody (Figure S1).


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 affinity maturation for scFv47 clone. (A) Monoclonal ELISA for scFv clones selected after the second round of panning against Hsp90α. Of all clones, 35 were selected for further analysis by SPR on Biacore® 3000. The best clone scFvA4 is highlighted with orange; (B,C) Binding curves for scFvA4 clone against Hsp90α and Hsp90β, respectively. Varied concentrations of purified protein were injected on two CM5 sensor chips immobilized with Hsp90 isoforms. The BIAevaluation software was used for calculations of kinetic constants; (D) The amino acids selected at CDR1, CDR2 and CDR3 of parental and affinity matured clones.
© Copyright Policy
Related In: Results  -  Collection

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

ijms-16-19920-f002: The results of affinity maturation for scFv47 clone. (A) Monoclonal ELISA for scFv clones selected after the second round of panning against Hsp90α. Of all clones, 35 were selected for further analysis by SPR on Biacore® 3000. The best clone scFvA4 is highlighted with orange; (B,C) Binding curves for scFvA4 clone against Hsp90α and Hsp90β, respectively. Varied concentrations of purified protein were injected on two CM5 sensor chips immobilized with Hsp90 isoforms. The BIAevaluation software was used for calculations of kinetic constants; (D) The amino acids selected at CDR1, CDR2 and CDR3 of parental and affinity matured clones.
Mentions: The panning procedure was modified to favor selection of scFv variants with improved koff values. Therefore, we run so-called “off-rate selection”, wherein for the second round of panning we used five molar excess of soluble Hsp90α. This strategy enabled the enrichment of slower dissociating clones and led to the isolation of 35 positive variants (Figure 2A). The best clone selected, scFvA4, exhibited KD of 185 nM (Figure 2B). Although the improvement in KD was not significant (two-fold), the scFvA4 showed over sixfold lower koff in comparison with parental scFv47. Moreover, we observed 10-fold weaker binding affinity of scFvA4 to Hsp90β (Figure 2C). Such result could be beneficial for future therapeutic applications, as cancer cells express and constitutively secrete primarily the isoform α of Hsp90 [38,39]. In addition, we compared our selected antibody fragments with commercially available anti-Hsp90 antibody using SPR method. We injected the same concentration of antibodies/scFvs on Hsp90α immobilized on sensor chip. We observed similar binding profiles of scFvA4 and commercial antibody (Figure S1).

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