Converting Conventional Antibodies into ScFv and ScFv-Fc Fusion Fragments to Improve Expression and Function
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CitationCreesy, Alicia. 2021. Converting Conventional Antibodies into ScFv and ScFv-Fc Fusion Fragments to Improve Expression and Function. Master's thesis, Harvard University Division of Continuing Education.
AbstractAntibodies are used in many diagnostic, therapeutic, and research applications around the world. Traditionally, conventional full-length antibodies have been used for these applications, however, with the development of antibody engineering the use of antibody fragments has become more appealing.
Conventional antibodies contain an Fc region which can be beneficial as well as detrimental. In regard to therapeutics, the Fc region can elicit a heightened immune response which can be damaging to a human’s health. The large size of conventional antibodies also does not allow the antibody to easily penetrate tissues and tumors. This can be challenging when it comes to drug development. The presence of the Fc region in conventional antibodies can also contribute to non-specific binding in research applications (Johnson, 2013).
To combat these issues, antibody fragments can be made to reduce immune cell activation and antibody effector functions. Antibody fragments often have a smaller size, which allows for better nuclear and tissue penetration. They can also be linked to each other to create multimeric forms or to other molecules to better target specific cells or tumors (Chames, Van Regenmortel, Weiss, & Baty, 2009). These features may be beneficial for therapeutic purposes as well as research assays. Lastly, antibody fragments can sometimes be more rapidly and less costly manufactured than conventional antibodies. This is due to the ability to be expressed in different less expensive systems (Jørgensen et al., 2014)..
This study aimed to compare different antibody formats to see how expression and functional levels were affected. Conventional full-length antibodies were selected that were considered high value and low expressing from Cell Signaling Technologies™ mouse monoclonal antibody catalog. As well as the commercially available trastuzumab as a control antibody. ScFv and scFv-Fc fusion antibodies were constructed in either the variable heavy (VH) followed by variable light (VL) or variable light (VL) followed by variable heavy (VH) orientation. Newly constructed antibodies were expressed using the mammalian ExpiCHO™ system. Antibodies were then purified and quantified before being subjected to western blot and flow cytometry analysis.
It was determined that full-length antibodies had higher expression levels in the ExpiCHO™ system compared to the previously made antibodies expressed in the HEK293 systems. It was also determined that the VL-VH orientation seemed to be preferred over the VH-VL orientation based on expression levels. However, orientation preference seemed to be target specific. Furthermore, all antibody formats behaved functionally similar in western blot and flow cytometry analysis. This indicated that removing portions of the constant region did not affect functionality. Results from this study laid the groundwork for future experiments using the ExpiCHO™ system as well as different antibody formats to improve expression and functional levels of other high value targets.
Citable link to this pagehttps://nrs.harvard.edu/URN-3:HUL.INSTREPOS:37367509
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