Structure-based Development of Humanized Anti-Human Transferrin Receptor Antibody with Enhanced BBB Penetration = BBB 투과도가 향상된 인간화 트랜스페린 수용체 특이적 항체의 구조 기반 개발

다국어 초록 (Multilingual Abstract)

Receptor-mediated transcytosis (RMT) is one of the notable systems in the research field of drug delivery for neurological diseases, such as Alzheimer’s disease, Parkinson’s disease, since it allows selective drug delivery across the blood-brain barrier (BBB). The BBB tightly regulates the flux of molecules into the brain parenchyma by forming the tight junction between microvascular endothelial cells. Therefore, the main point of the study is to selectively increase the permeability of BBB and deliver the drug into the brain. Among the receptors, transferrin receptor (TfR), highly expressed on the brain endothelial cells, plays a pivotal role in transporting iron into the brain. Using this role of TfR, anti-TfR antibody, which non-competitively binds to TfR, has been considered as a promising tool for delivering the therapeutic agents. Nevertheless, the permeability of anti-TfR antibody is still low and there is no therapeutic antibody that has been approved for clinical application. Especially, few structure-based engineering of anti-TfR antibody for enhancing the BBB permeability has been reported. Modification of interaction between TfR and anti-TfR antibody through antibody engineering based on structural analysis can have a significant impact on biological functions. Also, humanization of non-human derived antibody is required essentially for clinical use. In this study, I designed and produced humanized anti-human TfR antibody with preserved. Also, I determined the X-ray crystal structure of humanized anti-human TfR antibody Fab fragments. X-ray diffraction data were collected at 1.63 Å resolution. Based on the solved crystal structure, mutagenesis of various amino acids was performed on the binding domain of humanized anti-human TfR antibody. As a result, enhanced penetration of specific mutant antibody was identified using in vitro BBB transcytosis assay. The humanized anti-human TfR with improved BBB permeability demonstrated in this study will enable efficient delivery of therapeutic substances into the brain.

목차 (Table of Contents)

• 1. Introduction 8
• 2. Materials and methods 13
• 2.1 Design of humanized anti-human TfR antibody 13
• 2.2 Antibody expression and purification 14
• 2.3 Human Transferrin Receptor ELISA 15
• 2.4 In vitro BBB Transcytosis Assay 16
• 2.5 Quantification of penetrated antibody by ELISA 18
• 2.6 Crystallization 19
• 2.7 X-ray diffraction and data collection 20
• 3. Results 21
• 3.1 Humanization of anti-human TfR antibody 21
• 3.2 Comparison of binding activity with human TfR by ELISA 23
• 3.3 In vitro BBB Transcytosis assay 26
• 3.4 Purification of humanized anti-human TfR Fab fragments for crystallization 29
• 3.5 Crystallization 30
• 3.6 X-ray data collection 31
• 3.7 Overall structure of humanized anti-human TfR Fab fragment 34
• 3.8 Preparation of antibodies for in vitro BBB assay 38
• 3.9 Optimization of BBB permeability 39
• 4. Discussion 41
• 5. References 44