Development of viral T cell epitope-fused cytotransmab cancer vaccine that instructs pre-existing virus-specific cytotoxic T cells to eliminate tumors
- 주제(키워드) cancer thearpy , immunotherapy , virus-specific T cell , antibody , cell-penetrating antibody
- 발행기관 아주대학교
- 지도교수 김용성
- 발행년도 2020
- 학위수여년월 2020. 8
- 학위명 석사
- 학과 및 전공 일반대학원 분자과학기술학과
- 실제URI http://www.dcollection.net/handler/ajou/000000030311
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
The cancer vaccine activates tumor-specific T cells by providing immunogenicity in tumors with low immunogenicity. Conferring to immunogenicity to cancer using personalized neoantigens has limitation in application to large number of patients because of the inter-/intra-tumor heterogeneity. To overcome these limitations, I am intended to convert cancer cells into targets of virus-specific T cell by delivering virus-derived antigens commonly infected to many people. To this end, viral antigen containing T cell epitope was fused to tumor-specific cytosol penetrating cytotransmab to deliver antigen to cytosol of tumor cells. I selected the cytomegalovirus (CMV) that infected with a population of 60-80% worldwide, and the 495-503 sequence of the CMV pp65 antigen is the highly immunogenic epitope which binds to HLA-A*02:01 subtype of MHC-I. To efficiently produce peptide/MHC-I complex on the cell surface, I constructed various format: only T cell epitope, N-terminally extended, C-terminally extended and both extended fragments-fused cytotransmabs. Because N-terminally-extended fragment of the T cell epitope fused cytotransmab showed cell surface MHC-I binding, both terminally-extended fragment formed peptide/MHC-I only through intracellular processing in cytosol without no surface MHC-I binding ability. I showed that CMV pp65-specific CD8 T cells isolated from healthy donors efficiently kill HLA-A*02:01 positive cancer cells, after the extracellular treatment with CMV pp65-derived T cell epitope fragment-fused cytotransmab. Lastly, since the CMV pp65-derived T cell epitope fragment-fused cytotransmab located in the cytosol is degraded by proteasome to generate T cell epitope precursor, I confirmed that the activity of CMV pp65-specific CD8 T cells is reduced through proteasome inhibitor treatment. These data demonstrate the viral antigen-derived T cell epitope-fused cytotransmab can be used to treat cancer with reduced immunogenicity as a new cancer vaccine platform. In addition, by fusing other viral antigen which infected in many people besides CMV to cytotransmab will be provide the possibility of expanding the technology.
more목차
1. Introduction 1
2. Material and methods 13
2.1 Cell lines 13
2.2 Peptides 13
2.3 Flow cytometry 13
2.4 Construction of antibody expression plasmids 14
2.5 Expression and purification of antibodies 15
2.6 PBMC preparations 15
2.7 Expansion of CMV pp65-specific CD8+ T cells 16
2.8 Phenotype analysis of CMV pp65-specific CD8+ T cells from PBMCs 16
2.9 Cytotoxicity assay 17
2.10 Intracellular IFN-γ staining 17
2.11 Statistical analysis 18
3. Results 19
3.1 Generation of viral antigen-derived T cell epitope-fused cytotransmab 19
3.2 T cell receptor-like antibody recognize the peptide/MHC-I complex on the surface of tumor cells 22
3.3 Comparison of generated peptide/MHC-I complex between control constructs 26
3.4 Applicability of antigen-derived T cell epitope fragment-fused cytotransmab 30
3.5 Frequency and phenotype of CMV pp65-specific CD8 T cells in healthy donor-derived PBMCs 32
3.6 CMV pp65-specific CD8 T cells efficiently kill CMV pp65-derived T cell epitope fragment-fused cytotransmab-treated tumor cells 39
3.7 Validation of efficiency of tumor cell lysis according to CMV pp65-derived T cell epitope fragment-fused length and treatment time 42
3.8 IFN-γ cytokine production induced by CMV pp65-derived T cell epitope fragment-fused cytotransmab 45
4. Discussion 47
5. References 50
