아주대학교

목차

1. Introduction 1
1.1 Antibody 1
1.2 Antibody therapeutics 4
1.3 Antibody-drug conjugates (ADCs) 5
1.4 Methods for antibody conjugation 6
1.4.1 Traditional conjugation methods 6
1.4.2 Site-specific conjugation using enzymatic modification of antibody 8
1.4.3 Site-specific conjugation using unnatural amino acid (UAA) 10
1.4.4 PEptide-DIrected Photocrosslinking (PEDIP) 11
1.5 Bacterial display 13
1.6 Fluorescence activated cell sorting (FACS) 14
2. Materials and Methods 15
2.1 FcIII mutant libraries construction fused to CPX 15
2.2 Bacterial strains and growth conditions 18
2.3 Screening and sorting peptide libraries based on FACS 18
2.4 DNA amplification of individual E. coli clones and further rounds of selection 19
2.5 Compare selected mutants (M3,B1) with FcIII V10Bpa 21
2.6 Preparation of Trastuzumab-B1-MMAE conjugate via PEDIP and click reaction 21
2.7 In vitro cell cytotoxicity test for human breast cancer cell line with ADCs 22
3. Results 23
3.1 Bacterial display of FcIII peptide 23
3.2 Directed evolution for FcIII variants 26
3.3 Characterization with B1 peptide 38
3.4 Production and evaluation of ADCs 41
4. Conclusion 43
5. Reference 44