Applications of Yeast Surface Display Technology for Development of Therapeutic Proteins and Peptides
Applications of Yeast Surface Display Technology for Development of Therapeutic Proteins and Peptides
- 주제(키워드) 헝채 , 항체공학 , 항체 라이브러리 , Humanization , Yeast Display , 뉴로필린 , 펩타이드
- 발행기관 아주대학교
- 지도교수 김용성
- 발행년도 2018
- 학위수여년월 2018. 8
- 학위명 박사
- 학과 및 전공 일반대학원 분자과학기술학과
- 실제URI http://www.dcollection.net/handler/ajou/000000027926
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
The yeast surface display technology have evolved into a versatile tool for research and industrial applications. The works presented in this thesis aimed at the application of yeast surface display technology for developing therapeutic proteins and peptides and engineering their affinity, specificity and stability. I applied the yeast surface display technology to construction of fully synthetic human Fab libraries with large diversity for rapid isolation of human antibody candidates against given targets, humanization of phosphorylated epitope specific non-human antibody and development of novel neuropilin-1 binding peptides. Firstly, I report a large synthetic human Fab yeast surface-displayed library generated by stepwise optimization of yeast mating conditions. Yeast surface-displayed antibody libraries provide an efficient and quantitative screening resource for given antigens, but suffer from typically modest library sizes owing to low yeast transformation efficiency. Yeast mating is an attractive method for overcoming the limit of yeast transformation to construct a large, combinatorial antibody library, but the optimal conditions have not been reported. Yeast mating conditions were optimized in the order of cell density, media pH, and cell growth phase, yielding a mating efficiency of ~58% between the two haploid cells carrying HC and LC libraries. I constructed two combinatorial Fab libraries with VH-CDR3 of 9 or 11 residues in length with colony diversities of more than 109 by one round of yeast mating between the two haploid HC and LC libraries, with modest diversity sizes of ~107. The synthetic human Fab yeast-displayed libraries exhibited relative amino acid compositions in each position of the six CDRs that were very similar to those of the designed repertoires, suggesting that they are extremely functional in isolating human Fab antibodies with improved biophysical properties and reduced immunogenicity for given antigens. Secondly, I report humanization of a chicken pT231 antibody specific to a tau protein-derived peptide carrying the phosphorylated threonine at 231 residue (pT231-peptide) as a model for better understanding of the phosphoepitope recognition mechanism. There have been no humanization reports of phosphospecific non-human antibodies until now. By using yeast surface-displayed combinatorial library with permutations of 11 FR residues potentially affecting CDR loop conformations, I efficiently identified critical 5 FR residues, the back mutation of which with the corresponding chicken residues completely recovered the pT231-peptide binding affinity and specificity of humanized antibody, while simple grafting six CDRs of the chicken antibody into a homologous human framework (FR) template resulted in complete loss of the pT231-peptide binding. Importantly back mutation of FR 76 residue of VH (H76) (Asn to Ser) was critical in preserving the pT231-binding motif conformation via allosteric regulation of ArgH71, which closely interacts with ThrH52 and SerH52a residues on VH-CDR2 to induce the unique phosphate-binding bowl-like conformation. My humanization approach of CDR grafting plus permutations of FR residues by combinatorial library screening can be applied for non-human antibodies containing unique binding motif on CDRs specific to post-translationally modified epitopes. The successfully humanized antibodies in this study could be used for diagnostic and/or therapeutic purposes associated with the pT231 tau protein, and also served as a valuable template for generating pThr-peptide focused human antibody library to bypass animal immunization. Lastly, I report immunoglobulin Fc-fused NRP1-specific peptides deviating from CendR. VEGF and Sema3 family ligands specifically bind to the arginine-binding pocket in the b1 domain of NRP1 through a C-terminal (R/K)XX(R/K) amino acid sequence motif, where X stands for any amino acid and these interactions trigger NRP1-mediated vascular permeability. In fact, all known proteins and peptides binding to the VEGF-binding region in NRP1-b1 domain share the basic sequence motif at the free C-terminus of these ligands and peptides and this distinct motif must be exposed, with a stringent requirement for the Arg (or rarely Lys) residue at their C-terminus; this prerequisite for binding to NRP1 is known as the “C-end rule” (CendR). Moreover, previous studies have reported that lose their binding activity and biological function upon substitution of their C-terminal Arg or Lys residue with another amino acid. However, the development of non-CendR peptides is essential for the future practical applications because the C-terminal Arg and Lys residues of Fc-fused CendR peptides may be removed by carboxypeptidases in cell culture or blood circulation after systemic administration, as shown with antibody. Here, I screened a yeast surface-displayed Fc-fused non-CendR peptide library against NRP1 and isolated Fc-V12, wherein V12 peptide comprising 12 amino acids has a PPRV sequence at its C-terminal end. Although Fc-V12 lacked the CendR motif, it showed selective binding to the VEGF-binding site of NRP1 and triggered cellular internalization of NRP1, resulting in enhanced extravasation into tumor tissues and tumor tissue penetration of the Fc-fused peptide along with the co-injected chemical drug in tumor-bearing mice. Through a saturation mutagenesis study, I identified that the Val residue at the C-terminus of Fc-V12 is essential for NRP1 binding. I further improved NRP1 affinity of Fc-V12 (KD = ~761 nM) through directed evolution of the upstream sequence of PPRV to obtain Fc-V12-33 (KD = ~17.4 nM), which exhibited enhanced NRP1-mediated vascular permeability as compared with Fc-V12. My results provide a new avenue for the development of optimized NRP1-targeting peptides without the restriction of CendR peptides.
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ABSTRACT OF DISSERTATION I
TABLE OF CONTENTS III
LIST OF FIGURES VI
LIST OF TABLES VIII
CHAPTER 1. General introduction 1
CHAPTER 2. Construction of a large synthetic human Fab yeast-displayed library by an optimized yeast mating 6
2.1 Abstract 6
2.2 Introduction 7
2.3 Materials and Methods 9
2.3.1 Yeast strains and media 9
2.3.2 Construction of HC and LC expression vectors 9
2.3.3 Preparation of synthetic VH and VL gene libraries 10
2.3.4 Transformation of HC and LC library into haploid strains 10
2.3.5 Fab library construction on diploid cells by yeast mating 11
2.3.6 Yeast colony PCR for sequencing VH and VL 12
2.3.7 Fab library analysis 12
2.3.8 Statistical analysis 13
2.4 Results and Discussion 15
2.4.1 Construction of HC and LC expression vectors for yeast surface display of Fab 15
2.4.2 Design of fully human synthetic VH and VL library with six diversified CDRs 17
2.4.3 Construction of HC and LC libraries on haploid yeasts 22
2.4.4 Optimization of yeast mating conditions 27
2.4.5 Generation of synthetic Fab libraries by the optimized yeast mating conditions 30
2.5 Conclusion 34
CHAPTER 3. Humanization of a phosphothreonine-peptide specific chicken antibody by combinatorial library optimization of the phosphoepitope binding motif 35
3.1 Abstract 35
3.2 Introduction 36
3.3 Materials and Methods 38
3.3.1 Materials 38
3.3.2 Construction of VH and VL gene libraries with FR substitutions on yeast haploid cells 38
3.3.3 Combinatorial Fab library construction on diploid cells by yeast mating 38
3.3.4 Screening of the Fab library 39
3.3.5 Expression and purification of humanized IgG antibody 39
3.3.6 Binding analysis by ELISA 39
3.3.7 Structure modeling 40
3.4 Results and Discussion 41
3.4.1 Design of humanization for phosphospecific chicken antibody 41
3.4.2 Construction and screening of a combinatorial Fab library 44
3.4.3 Expression of humanized antibodies in IgG format and determination of their antigen-binding activity 48
3.4.4 Structural analysis of the pThr-binding motif on VH-CDR2 of humanized antibody 50
3.5 Conclusion 52
CHAPTER 4. An immunoglobulin Fc-fused peptide without C-terminal Arg or Lys residue augments neuropilin-1–dependent tumor vascular permeability 53
4.1 Abstract 53
4.2 Introduction 54
4.3 Materials and Methods 56
4.3.1 Generation and screening of Fc-fused non-CendR peptide library displayed on the surface of yeast 56
4.3.2 Construction and purification of Fc-fused peptides 57
4.3.3 Expression and purification of recombinant NRP proteins 57
4.3.4 Binding analysis by enzyme-linked immunosorbent assay (ELISA) 58
4.3.5 Surface plasmon resonance (SPR) analysis 59
4.3.6 Endothelial permeability assay 59
4.3.7 Immunofluorescence microscopy of cells 60
4.3.8 Xenograft tumor models 60
4.3.9 Immunofluorescence microscopy of tumor tissues 61
4.3.10 Statistical analysis 61
4.4 Results and Discussion 62
4.4.1 Design and construction of Fc-fused non-CendR peptide library 62
4.4.2 Isolation and characterization of NRP1-specific Fc-V12 65
4.4.3 Fc-V12 homes to tumor vessels and penetrates into tumor tissues 67
4.4.4 Essential elements of Fc-V12 for binding to NRP1 70
4.4.5 Affinity-matured Fc-V12 derivative shows more potent vascular permeability 72
4.5 Conclusion 79
CONCLUSION 80
REFERENCES 82
ABSTRACT IN KOREAN 94
PUBLICATIONS AND PATENTS 97
