Domain interactions in the Fab fragment: A comparative evaluation of the single-chain Fv and Fab format engineered with variable domains of different stability
Open accessAbstract
Recombinant antibody fragments, most notably Fab and scFv, have become important tools in research, diagnostics and therapy. Since different recombinant antibody formats exist, it is crucial to understand the difference in their respective biophysical properties. We assessed the potential stability benefits of changing the scFv into the Fab format, the influence of the variable domains on the stability of the Fab fragment, and the influence of the interchain disulfide bond in the Fab fragment. To analyze domain interactions, the Fab fragment was broken down into its individual domains, several two-domain assemblies and one three-domain assembly. The equilibrium denaturation properties of these constructs were then compared to those of the Fab fragment. It was found that mutual stabilization occurred across the VH/VL and the CH1/CL interface, whereas the direct interaction between the V) and the CL domain had no influence on the stability of either domain. This observation can be explained by the different interfaces used for interaction. In contrast, the whole CH1CL and VHVL unit showed significant mutual stabilization, indicating a high degree of cooperation between the VH/VL and CH1/CL interface. The interchain disulfide bond in the Fab fragment plays an essential role in this stabilization. In addition to the effects of domain association on the thermodynamic (equilibrium) stability, Fab fragments differ from scFv fragments of similar equilibrium stability by having a very…
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Fab fragments are usually biochemically more stable than scFv counterparts due to mutual stabilization between VH/VL and CH1/CL interfaces.
“Usually, Fab fragments are biochemically more stable than scFv counterparts due to the mutual stabilization that occurs between the VH/VL and CH1/CL interfaces [37].”
From Evolution of Escherichia coli Expression System in Producing Antibody Recombinant Fragments (2020) · cites this paper as [37]
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Evolution of Escherichia coli Expression System in Producing Antibody Recombinant Fragments
Sandomenico, A., Sivaccumar, J. P., & Ruvo, M. (2020) · International Journal of Molecular Sciences · doi:10.3390/ijms21176324 · Cited by 134 · see citing works
“Usually, Fab fragments are biochemically more stable than scFv counterparts due to the mutual stabilization that occurs between the VH/VL and CH1/CL interfaces [37].”
BackgroundRezaie, F., Davami, F., Mansouri, K., Agha Amiri, S., Fazel, R., Mahdian, R., Davoudi, N., Enayati, S., Azizi, M., & Khalaj, V. (2017) · Journal of Applied Microbiology · doi:10.1111/jam.13483 · Cited by 15 · see citing works
- Authors
- Röthlisberger, D., Honegger, A., & Plückthun, A.
- Affiliations
- University of Zurich (CH)
- Corresponding author
- Plückthun, Andreas
- Venue
- Journal of Molecular Biology
- Publisher
- Elsevier BV
- Published
- 2005-04
- Volume
- 347
- Issue
- 4
- Pages
- 773-789
- ISSN
- 0022-2836 (ISSN-L)
- OpenAlex
- W2137663279
- Cited by
- 289
- References
- 0
- Citations by year
- 2026: 8 · 2025: 11 · 2024: 6 · 2023: 13 · 2022: 14 · 2021: 16 · 2020: 14 · 2019: 14 · 2018: 15 · 2017: 19 · 2016: 14 · 2015: 23 · 2014: 12 · 2013: 14 · 2012: 16
- FWCI
- 9.08
- Type
- journal article
- Language
- en
- Open access
- green
- doi.org
- Locations
- Journal of Molecular Biology (publishedVersion)
- PubMed (publishedVersion)
- Zurich Open Repository and Archive (University of Zurich) (acceptedVersion)
- Zurich Open Repository and Archive (University of Zurich) (submittedVersion)
- Zurich Open Repository and Archive (University of Zurich)
- Funders
- Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung
- Topics
- Health Sciences › Medicine › Radiology, Nuclear Medicine and Imaging › Monoclonal and Polyclonal Antibodies Research
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein purification and stability
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Structure and Dynamics
- Keywords
- Fab fragment, scFv, antibody fragments, domain interactions, interchain disulfide bonds, equilibrium stability, kinetic stability, unfolding rate, variable domains, constant domains, protein stability engineering
- MeSH terms
- Disulfides, Immunoglobulin Fab Fragments, Immunoglobulin Heavy Chains, Kinetics, Models, Molecular, Protein Binding, Protein Denaturation, Thermodynamics, Urea, Protein Engineering, Protein Structure, Tertiary, Protein Folding, Guanidine, Protein Structure, Quaternary
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