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Development of cell-penetrating antibody targeting intracellular activated Ras proteins to block the interaction with effector proteins
- Alternative Title
- Development of cell-penetrating antibody targeting intracellular activated Ras proteins to block the interaction with effector proteins
- Author(s)
- 신승민
- Alternative Author(s)
- Seung-Min Shin
- Advisor
- 김용성
- Department
- 일반대학원 분자과학기술학과
- Publisher
- The Graduate School, Ajou University
- Publication Year
- 2017-08
- Language
- eng
- Alternative Abstract
- Antibody is very powerful therapeutic reagents targeting protein-protein interactions (PPIs) associated with disease and cancer. However, their target proteins are limited to cell surface receptors and some secreted proteins, because of their hydrophilicity and large size. Impairment of these interactions is more efficient with antibodies than with small molecules because of the large and hydrophilic interaction surfaces of antibodies. Although some approaches have succeeded in delivering antibodies into the cytosol of live cells, many problems, including difficulty in systemic administration, nonspecific cytotoxicity, and loss of stability, remain unresolved. In chapter 2, I report the development of cytotransmabs, full-length human IgG format cell-penetrating antibodies, which penetrate the cytosol of live cells. When VL of IgG format antibodies was replaced with a humanized cell-penetrating VL domain, all cytotransmabs were efficiently internalized and localized in the cytosol. Cytotransmabs that escaped from early endosomes were degraded by the proteasome pathway but not by the lysosome pathway. Furthermore, a human lysyl-tRNA synthetase (KRS)-targeting cytotransmab, called KT4, bound to cytosolic KRS proteins when it was treated to live cells, showing that cytotransmabs can target proteins in the cytosol. In chapter 3, I successfully developed RT11, full-length human IgG format cell-penetrating antibody, which selectively bound to activated Ras proteins. RT11 penetrated into the cytosolic region of live cells and interfered with interactions of effector proteins, thereby suppressing downstream signaling and in vitro growth of oncogenic Ras-mutated cancer cells. Moreover, systemic administration of integrin ανβ3- and ανβ5-binding peptide fused RT11, exhibited anti-tumor activity in oncogenic Ras-mutated tumor xenograft mouse models but not in Ras wild type tumors. In chapter 4, I demonstrated that TRIM21 can degrade a full-length human IgG format cell-penetrating antibody without pathogen infection and affect the anti-tumor efficacy of a Ras·GTP-targeting iMab. I generated an Fc variant of the Ras·GTP-targeting iMab by substituting N434 with aspartic acid to reduce the interaction with TRIM21 and the rate of degradation in the cytosol. Compared with the Ras·GTP-targeting iMab with wild-type Fc, Fc variants exhibited enhanced anti-tumor efficacy in oncogenic Ras-mutated human colorectal and lung cancer cells, without noticeable cytotoxicity in Ras wild-type cancer cells. My conclusion is that the iMab platform technology has the potential of generating human IgG format antibodies for the inhibition of intracellular PPIs associated with disease, using systemic administration such as conventional therapeutic antibody regimens. Therefore, the iMab can extend the antibody target space to proteins in cytosolic region from the current limit of only extracellular and secreted proteins.
- Fulltext
- Appears in Collections:
- Graduate School of Ajou University > Department of Molecular Science and Technology > 4. Theses(Ph.D)
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