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Development of cytosol-penetrating antibody platform technology for inhibition of intracellular protein-protein interaction in living cells
- Alternative Title
- Development of cytosol-penetrating antibody platform technology for inhibition of intracellular protein-protein interaction in living cells
- Author(s)
- 최동기
- Alternative Author(s)
- Dong-ki Choi
- Advisor
- 김용성
- Department
- 일반대학원 분자과학기술학과
- Publisher
- The Graduate School, Ajou University
- Publication Year
- 2016-08
- Language
- eng
- Keyword
- antibody; cell penetrating; Ras; affinity maturation
- Alternative Abstract
- Protein–protein interactions (PPIs) are central to most biological processes and therefore represent a large and important class of targets for human therapeutics. Targeting the interfaces between intracellular proteins has huge therapeutic potential, but discovering antagonistic drugs that disrupt PPIs is an enormous challenge. Antibodies are appropriate for antagonizing PPIs with high specificity and affinity for targeted proteins and have large binding interfaces, but they cannot penetrate into the cytosol of living cells because of their large size and hydrophilicity. Many attempts have been made to directly deliver antibodies into the cytosol. Although this research has had some success in delivering antibodies into living cells, many issues, including nucleus localization, loss of antibody stability, and difficulties with systemic administration, remain unresolved. Here I report an antibody technology platform using full-length IgG for penetrating the cytosol and targeting intracellular PPIs. In chapter 2, I report on a cytosol-penetrating antibody called cytotransmab, in the form of a full-length IgG that is created by incorporating a cytosol-penetrating light chain variable domain (VL) into light chains (LCs) and then co-expressing the LCs with the heavy chains (HCs). Cytotransmab was internalized into living cells through a physiological endocytic pathway and then reached the cytosol regions. In chapter 3, I report the application of cytotransmab to directly develop a Ras·guanosine triphosphate (GTP)-specific cytotransmab, called Ras·GTP iMab (internalizing and interfering monoclonal antibody). Ras·GTP iMab was generated by incorporating a Ras·GTP-specific heavy chain variable domain (VH) into the HC of cytotransmab, which has a VL that is able to penetrate into the cytosol of living cells. Ras·GTP iMab efficiently blocked the interaction between Ras·GTP and effector molecules, thus inhibiting downstream cell growth and survival signaling pathways such as Raf-MEK-Erk1/2 and PI3K/Akt. In addition, tumor-expressed integrin-targeted Ras·GTP iMab significantly impaired in vivo tumor growth in human xenograft tumor-bearing mice. In chapter 4, to improve the therapeutic potency of Ras·GTP iMab, I performed affinity maturation using complementarity determining region (CDR)-focused randomization as predicted by computational homology modeling and molecular docking. Isolated variants had a ~5-fold increased affinity against active Ras. My conclusion was that the cytosol-penetrating antibody technology platform has the potential to deliver antibodies into the cytosol of living cells and to directly target the “undruggable” intracellular PPIs. Therefore, this technology platform should be widely applicable to the study of diverse biologic questions, making it attractive for clinical applications.
- Fulltext
- Appears in Collections:
- Graduate School of Ajou University > Department of Molecular Science and Technology > 4. Theses(Ph.D)
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