癌症是人類的主要死因之一，預計又將因人口增加、人口老齡化和與增加癌症易感性相關的生活方式選擇等因素而不減反增。根據世界衛生組織（WHO）的預測，到2040年，全球癌症發生率將增加到約3000萬例，死亡率增加到1600萬例。目前，癌症在全世界人口的第二大死因，對全球公共衛生構成重大挑戰。
目前癌症治療包括化學治療、手術切除、放射治療和激素治療以及癌症免疫治療等。癌症免疫治療相關的藥物包括了抗體、小分子、細胞因子、腫瘤疫苗和胜肽，用以激活免疫系統的特定成員或抑制癌細胞規避免疫反應的信號。其中細胞因子白細胞介素-2（IL-2）在癌症免疫治療中顯示出重要作用。IL-2是由活化T細胞合成的一種強效生長因子，能夠刺激各種免疫細胞群體的活化，IL-2也成為美國食品和藥物管理局批准的第一種用於癌症治療的免疫治療藥物。但IL-2治療在臨床上因為高劑量的全身性施打以及高頻率的給藥導致了高毒性和副作用。為了解決這樣的限制，研究團隊從兩個層面來改善IL-2療法，一個是將全身性的靜脈注射改成局部的瘤內注射，另一個則是將IL-2進行修飾。
根據其他研究團隊先前的研究，他們將抗癌肽與IL-2接合在一起形成抗癌肽-IL-2融合蛋白。在我們實驗室先前的研究中，我們實驗室開發出了多種具有抗癌能力的抗癌胜肽，其中W5K11展現出了對多個癌細胞廣效的抗癌能力同時對正常細胞有著良好的選擇性。因此在這篇研究當中，我們將W5K11與IL-2接合在一起形成W5K11-IL-2，並利用大腸桿菌蛋白質表現系統表達W5K11-IL-2以及IL-2。根據實驗結果，在W5K11-IL-2中，W5K11端有著跟W5K11相似的抗癌能力，同時IL-2端也有著跟IL-2本身相近的刺激、活化免疫細胞的能力。這樣的結果表明了W5K11-IL-2是一個相當有潛力治療多個癌症的新型癌症免疫治療藥物。

Cancer is one of the leading causes of death worldwide, and its prevalence is expected to rise due to factors such as population growth, aging demographics, and lifestyle choices associated with increased cancer susceptibility. According to the World Health Organization (WHO), by 2040, global cancer incidence is projected to increase to approximately 30 million cases, with mortality rates rising to 16 million cases. Currently, cancer ranks as the second leading cause of death globally, posing a significant public health challenge.
Current cancer treatments include surgical excision, radiotherapy, chemotherapy, hormone therapy, and cancer immunotherapy. Cancer immunotherapy involves various pharmacological agents, including antibodies, small molecules, cytokines, tumor vaccines, and peptides, aimed at activating specific components of the immune system or inhibiting signals that suppress the immune response against cancer cells. Among these, the cytokine interleukin-2 (IL-2) has demonstrated significant efficacy in cancer immunotherapy. IL-2, synthesized by activated T cells, stimulates activation across various immune cell populations and has been the first immunotherapeutic agent approved by the US Food and Drug Administration for cancer treatment. The clinical utility of IL-2 therapy has been constrained by its elevated systemic dosing and frequent administration, resulting in pronounced toxicity and adverse reactions. In response to these constraints, the research team has enhanced IL-2 therapy through dual avenues: transitioning from systemic intravenous delivery to localized intratumoral injection, and undertaking IL-2 modification.
In this study, we incorporated insights from prior research conducted by other teams and amalgamated anticancer peptides with IL-2 to generate anticancer peptide-IL-2 fusion proteins. In preceding investigations within our laboratory, we synthesized a range of anticancer peptides exhibiting robust anticancer efficacy. Notably, W5K11 displayed broad-spectrum anticancer activity against multiple cancer cell types, while demonstrating favorable selectivity towards normal cells.In this study, we fused W5K11 with IL-2 to form W5K11-IL-2 and expressed it using the Escherichia coli protein expression system, alongside IL-2 alone. Experimental results showed that W5K11-IL-2 retained similar anticancer activity to W5K11 and exhibited comparable stimulatory and activation capabilities to IL-2. These findings suggest that W5K11-IL-2 holds promise as a novel cancer immunotherapy agent for treating multiple cancers.

摘要 1
Abstract 3
Abbreviations 5
Chapter 1 Introduction 8
1.1 Cancer trends 8
1.2 Immunotherapy of cancer treatment 8
1.3 Limitations of IL-2 therapy 9
1.4 Optimizations in IL-2 therapy 10
1.5 Anti-cancer peptides 11
1.6 W5K series antimicrobial peptides and W5K11 12
1.7 Aim of this study 13
Chapter 2 Materials and Methods 14
2.1 Reagents, chemical, and cell lines 14
2.2 Construction of the IL-2 and W5K11-IL-2 14
2.3 Expression of IL-2 and W5K11-IL-2 16
2.4 Purification of IL-2 and W5K11-IL-2 and refolding 16
2.5 Further purification of IL-2 and W5K11-IL-2 by RP-HPLC 18
2.6 SDS-PAGE and Western-Blot analysis 19
2.7 IL-2 and W5K11-IL-2 concentration determination 20
2.8 IL-2 enzyme-linked immunosorbent assay (ELISA) 21
2.9 Proliferation assay of CTLL-2 T cell 21
2.10 Cancer cell inhibition assay 22
2.11 Statistical analyses 23
Chapter 3 Results 24
3.1 Construction of the IL-2&W5K11-IL-2 plasmid 24
3.2 Expression and purification of the IL-2&rW5K11-IL-2 24
3.3 Characterization of the IL-2&W5K11-IL-2 25
3.4 Purification of the IL-2&W5K11-IL-2 by RP-HPLC 26
3.5 Binding ability of the IL-2&W5K11-IL-2 with anti-IL-2 antibody 27
3.6 The biological activity of IL-2 of the IL-2&W5K11-IL-2 27
3.7 In vitro cell cytotoxicity of the IL-2&W5K11-IL-2 28
Chapter 4 Discussion 29
Chapter 5 Conclusion 31
Chapter 6 Figuers & Tables 32
Figure 1. The functions of IL-2 in cancer immunotherapy 32
Figure 2. The therapeutic mechanisms of cationic ACPs in cancer cells 33
Figure 3. Gene map of the recombinant plasmid pET-26b(+)-IL-2 34
Figure 4. Gene map of the recombinant plasmid pET-26b(+)-W5K11-IL-2 35
Figure 5. SDS gel page analysis of the IL-2 36
Figure 6. SDS gel page analysis of the W5K11-IL-2 37
Figure 7. Western blot analysis of the IL-2 & W5K11-IL-2 38
Figure 8. Reversed-phase HPLC purification of the IL-2 39
Figure 9. Reversed-phase HPLC purification of the W5K11-IL-2 40
Figure 10. MALDI-TOF mass analysis of the purified IL-2 41
Figure 11. MALDI-TOF mass analysis of the purified W5K11-IL-2 42
Figure 12. Direct ELISA binding assay of the IL-2 standard, purified IL-2, and purified W5K11-IL-2 43
Figure 13. The biological activity of IL-2 of the IL-2 standard, purified IL-2, and purified W5K11-IL-2 44
Figure 14. Anticancer activities of W5K11, purified W5K11-IL2, and purified IL-2 45
Table 1. IC50 (μM) of W5K11, purified W5K11-IL2, and purified IL-2 46
Chapter 7 References 47

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