現今，針對免疫檢查點（immune checkpoint）所開發的抑制劑儼然成為促進癌症免疫治療最重要的進展，近期研究更指出抑制調控巨噬細胞吞噬作用的訊號調節蛋白 α—整聯素相關蛋白 47（signal-regulatory protein α-CD47, SIRPα-CD47）傳遞路徑能夠有效增進巨噬細胞吞噬腫瘤細胞的能力進而達到清除腫瘤細胞的目的。儘管科學家已於多種癌症模式中證實抑制 SIRPα-CD47 路徑能有效改善種腫瘤免疫抑制的微環境，但因缺乏直接的免疫反應活化導致此策略僅能夠些微的抑制腫瘤生長，其有限的治療效果促使我們思索可改善的方法。有研究曾指出，透過促進干擾素基因刺激蛋白（stimulator of interferon gene, STING）路徑，可有效引發先天免疫細胞活化並活化第一型干擾素的製造。於此，我們致力於開發標靶腫瘤巨噬細胞的奈米載體並搭載 SIRPα 小分子干擾核糖核酸與 STING 路徑配體。此奈米載體可有效遞送此兩種免疫調節劑，並於載體表面修飾能夠專一標靶腫瘤巨噬細胞的胜肽以減少引起全身性免疫反應的風險，並透過抑制 SIRPα 達到改善腫瘤免疫抑制微環境並同時以STING 路徑配體刺激先天免疫細胞活化以達到抑制腫瘤生長與癌症進程。

Inhibition of immune checkpoint blockade has emerged as one of the most important advances in cancer immunotherapy. Recent study indicated that the inhibitory of signal-regulatory protein (SIRP) α-CD47 pathway, a phagocytosis checkpoint in macrophage, can promote the ability of macrophage to phagocytose and eliminate tumor cells. Although numerous attempts have been made to introducing SIRPα-CD47 blockades for reversing immunosuppressive tumor microenvironment, these strategies only showed a moderate anti-cancer effect regarding the disability of immune cell activation. STING agonist, cGAMP, had been reported to enhance anti-tumor immunity through the innate immune cell activation and the further type I IFN production. Hence, we hypothesize to co-administrate SIRPα siRNA as SIRPα-CD47 blockades and cGAMP to synergistically activate anti-tumor immunity and suppress cancer progression. However, nonspecific immune response arising from systemic administration, poor intracellular delivery ability, and short half-life of these immune modulators hindered the application of this approach in cancer treatment. Herein, we developed a tumor-associated macrophage (TAM)-targeted lipid-based nanoparticles (NPs) to specifically deliver SIRPα siRNA and cGAMP to TAMs. Our result indicated that with the TAM-targeted peptide, which enhanced the in vitro and in vivo uptake by TAM, SIRPα siRNA-cGAMP NPs elicit strong STING activation and M2 repolarization in macrophages. SIRPα siRNA-cGAMP NPs treatment not only successfully reversing the immunosuppressive TME, but activating the cytotoxic T lymphocyte and inhibit the tumor growth in orthotopic pancreatic ductal adenocarcinoma (PDAC).

中文摘要 i
Abstract ii
致謝 iii
Table of Contents iv
Table of Charts vi
Table of Tables viii
Abbreviation ix
Chapter 1 Motivation and Aims 1
1.1 Motivation 2
1.2 Aims 3
Chapter 2 Literature Review 6
2.1 Cancer and Immune System 7
2.1.1 Immune system 7
2.1.2 Macrophages 8
2.1.3 T cells 10
2.1.4 Natural killer cells 11
2.2 Cancer Immunotherapy 12
2.2.1 Tumor-associated macrophage as a therapeutic target 12
2.2.2 Signal regulatory protein-α-CD47 immune checkpoint 13
2.2.3 STING-based therapy 15
2.2.4 PD-1/PD-L1 immune checkpoint 17
2.3 Advantages of Utilizing NPs for Advanced Cancer Immunotherapy 18
Chapter 3 Materials and Methods 20
3.1 Materials 21
3.2 Animals 22
3.3 Cell lines 23
3.4 Isolation of bone marrow derived macrophages 23
3.5 Phage display 24
3.6 ELISA assay 25
3.7 Preparation of cGAMP-siRNA nanoparticle 26
3.8 Characterization of nanoparticle 27
3.9 In vitro cellular uptake 28
3.10 In vivo cellular uptake 28
3.11 Reverse transcription quantitative real time PCR 29
3.13 Western blot analysis 32
3.14 Treatment study 32
3.15 Flow cytometry analysis and cell sorting 32
3.16 Immunostaining 34
3.17 Statistics 34
Chapter 4 Results 35
4.1 Production and characterization of macrophage-targeted immunotherapeutic agent delivery nanocarriers 36
4.2 In vitro efficacy of macrophage-specific peptides modified-nanoparticles 38
4.3 In vivo anti-tumor efficacy of SIRPα siRNA-cGAMP MT nanoparticles in orthotopic PDAC model 45
4.4 SIRPα siRNA-cGAMP MT nanoparticles sensitize PDAC to immunotherapy 53
4.5 Isolation of macrophage-specific phage ex vivo and in vitro 55
4.6 Verification and characterization of selected phage peptide sequences 57
Chapter 5 Conclusion 58
Chapter 6 Discussion and prospect 60
6.1 Discussion and Prospect 61
Chapter 7 Reference 64

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