S-Space College of Medicine/School of Medicine (의과대학/대학원) Dept. of Biomedical Sciences (대학원 의과학과) Theses (Ph.D. / Sc.D._의과학과)
(The) versatility of framework regions of chicken VH and VL to mutations
닭 항체 가변영역 프레임워크 돌연변이의 다양성
- 의과대학 의과학과
- Issue Date
- 서울대학교 대학원
- Monoclonal antibody; Framework regions; Randomization; mutation; Phage display; site directed conjugation; artificial cysteine and Next-generation sequencing
- 학위논문 (박사)-- 서울대학교 대학원 : 의과대학 의과학과 의과학전공, 2016. 2. 정준호.
- Introduction: Diversification of chicken immunoglobulin occur by somatic recombination and hypermutation during immune responses. Different to mutations in framework regions, mutations in Complemenatarity determining regions may be positively selected by antigen, leading to enhanced binding but not in framework regions.
Aim: To select replaceable amino acids from the randomized chicken antibody framework residues and replacing part of them with cysteine for PEGlytion.
Reactivity of anti-PSA monoclonal antibody selected from chicken antibody library and each of its framework regions randomized to antigen was measured by enzyme-linked immunosorbent assay. In addition, we conjugated Polyethylene glycol to cysteine replaced scFvs. Lastly, we applied next-generation sequencing analysis to select rare and diverse scFvs by using phagemid DNA which were prepared after bio-panning.
Following the results, most residues could be replaced to other residues while retaining high affinity to antigen. Although framework region could be replaced with various amino acids, the somatic mutation in framework may be lost during clonal expansion because of negative mutations. In addition, three VL (L5, L6 and L7) and two VH (H13 and H16) cysteine mutants as scFv-Ckappa fusion proteins showed that PEG-conjugation to the sulfhydryl group of the artificial cysteine was achievable in all the five mutants. Furthermore, we selected various scFvs by NGS analysis.
Therefore, chicken antibody could be useful to select diverse antibodies with antigen binding activity and the replacement of framework of chicken antibody may be useful for an approach to chicken antibody humanization, affinity maturation and drug conjugation.