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A method to conjugate full-length IgG antibody with protein and its application to immunotoxin
| DC Field | Value | Language |
|---|---|---|
| dc.contributor.advisor | 유태현 | - |
| dc.contributor.author | 이유미 | - |
| dc.date.accessioned | 2022-11-29T02:31:57Z | - |
| dc.date.available | 2022-11-29T02:31:57Z | - |
| dc.date.issued | 2018-02 | - |
| dc.identifier.other | 27246 | - |
| dc.identifier.uri | https://dspace.ajou.ac.kr/handle/2018.oak/19436 | - |
| dc.description | 학위논문(석사)--아주대학교 일반대학원 :분자과학기술학과,2018. 2 | - |
| dc.description.tableofcontents | 1. Introduction 1.1. Antibody 1 1.1.1 Monoclonal Antibody therapeutics 2 1.1.2 Antibody-drug conjugates 4 1.1.3 THIOMAB technology 5 1.1.4 Immunotoxin 6 1.2. Pseudomonas Exotoxin A based immunotoxin 8 1.2.1 Pseudomonas Exotoxin A 8 1.2.2 PE based immunotoxin Mechanism of Action 11 1.2.3 Development of Pseudomonas Exotoxin A construct 12 1.3. Incorporation of unnatural amino acids into proteins 14 1.3.1 Unnatural amino acid 14 1.3.2 Method for incorporation of unnatural amino acids into proteins 15 1.4. Project aims 16 2. Materials and Methods 2.1. Cell lines 17 2.2. Construction of THIOMAB 17 2.3. Production and purification of Herceptin N425C construct 20 2.4. Construction of plasmids for expressing deimmunized Pseudomonas Exotoxin A (PE24) 20 2.5. Expression and purification of modified deimmunized Pseudomonas Exotoxin A (PE24) 21 2.6. Conjugation of Herceptin-HC-N425C and modified PE24 22 2.7. Purification of Herceptin-PE24 conjugates 22 2.8. Evaluation of ErbB2 binding affinity by ELISA 24 2.9. ADP-ribosylation assay 24 2.10. Photo screens of Herceptin-PE24 conjugates 25 2.11. In vitro cell viability on a variety of breast cancer cell lines 25 2.12. Protein synthesis inhibition assay 25 3. Results 3.1. Screening of introduced cysteine positions in Herceptin 27 3.2. Expression and purification of PE24 30 3.3. Conjugation of HC-N425C and PE24 32 3.4. Antigen binding affinity of the Herceptin-PE24 conjugate 35 3.5. ADP-ribosylation of EF2 by Herceptin-PE24 37 3.6. Cytotoxicity of Herceptin-PE24 for breast cancer cell lines 38 4. Discussion 42 5. References 44 6. Abstract in Korean 51 | - |
| dc.language.iso | eng | - |
| dc.publisher | The Graduate School, Ajou University | - |
| dc.rights | 아주대학교 논문은 저작권에 의해 보호받습니다. | - |
| dc.title | A method to conjugate full-length IgG antibody with protein and its application to immunotoxin | - |
| dc.title.alternative | A method to conjugate full-length IgG antibody with protein and its application to immunotoxin | - |
| dc.type | Thesis | - |
| dc.contributor.affiliation | 아주대학교 일반대학원 | - |
| dc.contributor.alternativeName | Yumi Lee | - |
| dc.contributor.department | 일반대학원 분자과학기술학과 | - |
| dc.date.awarded | 2018. 2 | - |
| dc.description.degree | Master | - |
| dc.identifier.localId | 800899 | - |
| dc.identifier.url | http://dcoll.ajou.ac.kr:9080/dcollection/jsp/common/DcLoOrgPer.jsp?sItemId=000000027246 | - |
| dc.subject.keyword | antibody-protein conjugate | - |
| dc.subject.keyword | immunotoxin | - |
| dc.subject.keyword | pseudomonas exotoxin a | - |
| dc.description.alternativeAbstract | In the past decade, immunotoxins have been developed for targeted cancer therapy. These chimeric proteins consisting of a targeting moiety of monoclonal antibody or ligand binding to cancer cell surface receptor and potent proteins such as bacterial and plant-derived toxins have demonstrated a powerful ability to kill cancer cells. Chemical conjugation or fusion protein methods have been explored as means to obtain immunotoxins. However, most chemical conjugation methods result in heterogeneous products, and the fused forms of immunotoxin have been reported only for antibody fragments rather than full-length ones. Here, we report a site-specifically conjugated immunotoxin composed of Herceptin, targeting the HER2/neu receptor, and PE24 derived from Pseudomonas exotoxin A. the conjugation was performed by THIOMAB technology and method to incorporate unnatural amino acids into protein. The THIOMAB technology allows products to avoid heterogeneity through an incorporation of engineered cystine to heavy chain or light chain of antibody, thus that enable the site-specific conjugation. The incorporation of unnatural amino acids into protein also used to facilitate site-specific conjugation via bi-functional linker. using this strategy, Herceptin and PE24 were conjugated site-specifically. The Herceptin-PE24 conjugate was does not affect their biochemical characterization such as antigen binding of antibody and catalytic activity of PE24. Also, its cytotoxicity was demonstrated for the HER2/neu-positive cells such as BT-474, SK-BR-3 and ZR-75-1 breast cancer cell lines, and showed comparable previous fused immunotoxin. We expect that the conjugation approach described in this study can be applied to prepare various antibody-protein conjugates. | - |
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- Graduate School of Ajou University > Department of Molecular Science and Technology > 3. Theses(Master)
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