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The ScFV Interdomain Linker: A Protein Engineering Hotspot for Introducing Novel Functions into and Tuning the Biophysical Properties of ScFV Antibody Fragments

Ryan-Simkins, Michael Alfred
http://rave.ohiolink.edu/etdc/view?acc_num=osu1660945890529193

Abstract Details

2022, Doctor of Philosophy, Ohio State University, Biochemistry Program, Ohio State.
Antibodies are important tools in scientific research and medicine due to their ability to bind a wide variety of targets with high specificity. The minimal binding fragment of human antibodies, the single-chain FV (scFV) fragment, is engineered by genetically fusing the two binding domains of an antibody together and spacing them with an artificial linker. The length of the linker has been demonstrated to change a host of biophysical characteristics such as binding affinity, stability, and oligomeric state. However, the effect that the linker composition has on these properties is not well understood as previous comprehensive linker studies have been primarily completed with limited variations in linker composition. In chapter 2 we constructed and purified scFV constructs with varying lengths and four different compositions, (EAAAK)n, (DDAKK)n, Pn, An, and compared them to the commonly used (GGGGS)n linker. Constructs were characterized by differential scanning fluorimetry, gel filtration, and protease assays to determine thermal stability, oligomeric state, and proteolytic stability, respectively. We show that the majority oligomeric state when the interdomain linker is 15 amino acids or fewer is dimer. This suggests that despite the popular use of 15 amino acid long linkers in scFV construction, the linker should be at least 20 amino acids in length when monomeric scFVs are desired. Additionally, scFV dimers with higher stability and homogeneity can be generated with linkers that have higher conformational rigidity. Overall, our results contribute to greater understanding of how the design of the linker composition and length impacts the of the biophysical properties of scFVs. In chapter 3 we explore the incorporation of functional peptides into the scFV interdomain linker. We hypothesized that the artificial interdomain linker region would be a favorable place to insert a functional cell penetrating peptide (CPP) with minimal negative effects on the biophysical properties of the overall scFV. We created four such scFV.CPP constructs using our model scFV 3E8, which binds to the glycoprotein TAG-72 commonly found in adenocarcinoma. Here we demonstrate that these 3E8 CPP constructs have thermal stability, binding affinity, and oligomeric state distributions comparable to scFVs generated using the popular Gly/Ser linkers ((GGGGS)4/(G4S)4). We evaluated the cellular internalization using flow cytometry and live-cell confocal microcopy. Our results show an increase in internalization from all four 3E8 CPP constructs and a 3E8.205C when compared to 3E8.(GGGGS)4. Overall, we establish the scFV interdomain linker as a promising, modular site for engineering cell penetrating sequences into scFV antibody fragments, and that the 205C linker has modest cell-penetrating properties. Chapter four contains a collection of work in progress on projects such as flipped Caspase-3, Cys-free 3E8, and 3E8-GFP genetic fusions. The primary the purpose of this chapter is to provide a helpful starting point for those who come after me in the Magliery lab. Overall, this work demonstrates the compositional tolerance of the scFV interdomain linker, its effect on the overall biophysical properties of the scFV and establishes the linker as a protein engineering hotspot for regulating the biophysical properties of the scFV or for adding additional functionality with functional peptides.
Tom Magliery (Advisor)
Mike Tweedle (Committee Member)
Ross Dalbey (Committee Member)
Mark Foster (Committee Member)
170 p.
Biochemistry
antibody engineering, protein engineering, linker length, linker composition, CPP, cysteine replacement

Recommended Citations

Citations

  • Ryan-Simkins, M. A. (2022). The ScFV Interdomain Linker: A Protein Engineering Hotspot for Introducing Novel Functions into and Tuning the Biophysical Properties of ScFV Antibody Fragments [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1660945890529193

    APA Style (7th edition)

  • Ryan-Simkins, Michael. The ScFV Interdomain Linker: A Protein Engineering Hotspot for Introducing Novel Functions into and Tuning the Biophysical Properties of ScFV Antibody Fragments. 2022. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1660945890529193.

    MLA Style (8th edition)

  • Ryan-Simkins, Michael. "The ScFV Interdomain Linker: A Protein Engineering Hotspot for Introducing Novel Functions into and Tuning the Biophysical Properties of ScFV Antibody Fragments." Doctoral dissertation, Ohio State University, 2022. http://rave.ohiolink.edu/etdc/view?acc_num=osu1660945890529193

    Chicago Manual of Style (17th edition)

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This open access ETD is published by The Ohio State University and OhioLINK.
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