Design and In Vitro Evaluation of a Cytotoxic Conjugate Based on the Anti-HER2 Affibody Fused to the Fc Fragment of IgG1
Abstract
:1. Introduction
2. Results
2.1. Expression and Purification of the ZHER2:2891-Fc from Mammalian Cells
2.2. Specificity of the ZHER2:2891-Fc Fusion Protein
2.3. The ZHER2:2891-Fc-MMAE Conjugate
2.4. In Vitro Efficacy of the ZHER2:2891-Fc-MMAE
2.5. Stability of the ZHER2:2891-Fc-MMAE Conjugate in Serum
3. Discussion
4. Materials and Methods
4.1. Construction of the ZHER2:2891-Fc Expression Plasmid
4.2. Protein Expression
4.3. Protein Purification
4.4. Mass Spectrometry
4.5. Western Blotting
4.6. The ZHER2:2891-Fc-MMAE Conjugate Preparation
4.7. ZHER2:2891-Fc Labeling with FITC
4.8. Immunostaining
4.9. Microscopy
4.10. Cell Lines
4.11. Cytotoxicity Assays
4.12. Monitoring Stability of the ZHER2:2891-Fc-MMAE Conjugate in Mouse and Human Sera
Acknowledgments
Author Contributions
Conflicts of Interest
References
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Cytotoxic Agent | SK-BR-3 (10) * | MDA-MB-453 (8) * | T-47-D (5) * | MDA-MB-231 (2) * | |
---|---|---|---|---|---|
IC50 (nM) | free MMAE [19] | 1.76 ± 0.26 (n = 3) | 1.27 ± 0.46 (n = 3) | 3.25 ± 1.06 (n =3) | 5.21 ± 1.46 (n = 3) |
ZHER2:2891-MMAE [19] | 5.16 ± 1.09 (n = 4) | 24.83 ± 5.62 (n =3) | 135.55 ± 22.7 (n = 4) | 161.53 ± 49.9 (n = 3) | |
ZHER2:2891-Fc-MMAE | 0.13 ± 0.01 (n = 7) | 1.87 ± 0.27 (n = 4) | 45.67 ± 7.43 (n = 3) | 98.22 ± 30.9 (n = 7) |
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Sochaj-Gregorczyk, A.M.; Ludzia, P.; Kozdrowska, E.; Jakimowicz, P.; Sokolowska-Wedzina, A.; Otlewski, J. Design and In Vitro Evaluation of a Cytotoxic Conjugate Based on the Anti-HER2 Affibody Fused to the Fc Fragment of IgG1. Int. J. Mol. Sci. 2017, 18, 1688. https://doi.org/10.3390/ijms18081688
Sochaj-Gregorczyk AM, Ludzia P, Kozdrowska E, Jakimowicz P, Sokolowska-Wedzina A, Otlewski J. Design and In Vitro Evaluation of a Cytotoxic Conjugate Based on the Anti-HER2 Affibody Fused to the Fc Fragment of IgG1. International Journal of Molecular Sciences. 2017; 18(8):1688. https://doi.org/10.3390/ijms18081688
Chicago/Turabian StyleSochaj-Gregorczyk, Alicja M., Patryk Ludzia, Emilia Kozdrowska, Piotr Jakimowicz, Aleksandra Sokolowska-Wedzina, and Jacek Otlewski. 2017. "Design and In Vitro Evaluation of a Cytotoxic Conjugate Based on the Anti-HER2 Affibody Fused to the Fc Fragment of IgG1" International Journal of Molecular Sciences 18, no. 8: 1688. https://doi.org/10.3390/ijms18081688