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Development of tumor-specific, cytosol-penetrating antibody with improved endosome-escape efficacy
- Author(s)
- 김지선
- Advisor
- 김용성
- Department
- 일반대학원 분자과학기술학과
- Publisher
- The Graduate School, Ajou University
- Publication Year
- 2020-02
- Language
- eng
- Alternative Abstract
- Therapeutic antibodies are the most widely researched because of high specificity, high affinity and long serum half-life. However, since antibodies can’t directly penetrate cell membranes as a result of their large molecular size and hydrophilicity, they can only target cell surface expressed receptors and secreted proteins. My group previously reported full-length human IgG format antibodies, called cytotransmabs, which penetrate into the cytoplasm of live cells. To further improve the practical applicability of cytoplasm-penetrating antibodies, I attempted to improve the cytoplasmic delivery of cytotransmab. In chapter 2, to enhance the endosome-escape efficacy of cytotransmab, I first investigated the endosome-escape mechanism of cytotransmab and defined the endosome-escape motif and engineered w/ increasing the interaction of lipid and endosome-escape motif. The local conformational changes of the endosome-escape motif of cytotransmab in response to the acidified pH of endosome were crucial for the membrane pore formation, which allows the cytotransmab to escape into the cytoplasm. Ultimately, I was able to developed the cytotransmab w/ 3-fold improved endosome-escape efficacy. In chapter 3, I successfully generated the VH domain which has ability to localize in cytoplasm by grafting the endosome-escape motif into VH-CDR3 and the VL domain which specifically binds to EpCAM receptor instead of HSPG receptor. Combined the VH and VL domain, I developed a tumor-specific cytoplasm-penetrating cytotransmab w/ improved endosome-escape efficacy. In chapter 4, I demonstrate that immunotoxin, consist of targeting moiety and toxin molecule, can exhibit high cytotoxicity and low side-effect because of tumor-specific delivery of toxins. Because toxin molecules derived from bacteria/plant cause immunogenicity, I generated fully humanized immunotoxin using cytotransmab and hpRNase, which exhibited cytotoxicity in EpCAM-positive human colorectal cancer cells, w/o noticeable cytotoxicity in EpCAM-negative cancer cells. Consequently, cytotransmab technology holds the applicability to develop human IgG format antibodies which can deliver of exogenous cytotoxic molecules, using tumor-specific cytotransmab w/ improved endosome-escape efficacy. Therefore, engineering an antibodies to have efficient endosome-escape ability can overcome limitation of antibodies and provide an opportunity to design an antibodies for antibodies-based agents which carry a wide applicability of bioactive cargos into the cytoplasm.
- Fulltext
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- Graduate School of Ajou University > Department of Molecular Science and Technology > 4. Theses(Ph.D)
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