Development of an interleukin-2 displaying M1 macrophage extracellular vesicle platform for multi-functional cancer immunotherapy = 다기능 암 면역치료를 위한 인터루킨-2 표면 디스플레이된 M1 대식세포유래 세포밖소포체 플랫폼 개발

다국어 초록 (Multilingual Abstract)

Cancer immunotherapy has transformed the paradigm of cancer treatment. however, its therapeutic efficacy remains limited by the immunosuppressive tumor microenvironment (TME), which is characterized by a predominance of M2 macrophages and abundant inactivated T cells. In this study, a multi-functional cancer immunotherapy platform was developed by displaying interleukin-2 (IL-2) on the surface of M1 macrophage-derived extracellular vesicles (M1EV) to simultaneously regulate innate and adaptive immune responses within the TME. In addition, M1EV engineered to present an optimized level of surface-displayed IL-2 were generated to further improve the overall efficiency of the platform.
M1EV were first purified to high purity using ultrafiltration and size-exclusion chromatography and were subsequently engineered via metabolic glycoengineering to display IL-2 (M1EV_IL2). Physicochemical analyses confirmed that IL-2 surface display did not alter vesicle size, polydispersity, zeta potential, or morphology, and that representative EV markers were preserved. The engineered M1EV_IL2 retained the intrinsic function of M1EV in inducing M2 to M1 macrophage repolarization, while surface displayed IL-2 promoted T cell activation. These findings demonstrate that M1EV_IL2 constitutes a multi-functional cancer immunotherapeutic platform capable of successfully stimulating both innate and adaptive immune responses in vitro and ex vivo.
Subsequently, to achieve more efficient IL-2 surface display and enhance overall performance, a DSPE-conjugated lipid insertion strategy (M1EV@IL2) was employed, enabling a simpler, faster, and higher-yield approach to EV surface engineering. Compared with the metabolic glycoengineering approach, this method enabled a greater amount of IL-2 to be displayed on the vesicle surface while reducing production time and cost. Moreover, M1EV@IL2 was found to contribute to tumor growth suppression as an additional therapeutic effect. In vivo biodistribution studies further showed that M1EV@IL2 rapidly entered the circulation and was able to circulate systemically within 24 hours. Furthermore, evaluation using an in vivo tumor model demonstrated that M1EV@IL2 treatment effectively suppressed tumor growth, suggesting that the M1EV+IL-2 platform retains antitumor efficacy.
Collectively, these results suggest that an immunotherapeutic platform based on IL-2 displaying M1EV is expected to exert strong synergistic effects within the TME. Furthermore, DSPE-conjugated lipid insertion allows faster and higher-level IL-2 display than metabolic glycoengineering, thereby shortening production time and reducing IL-2 consumption, and thus improving the overall platform. These findings indicate that IL-2 expressing M1EV, particularly M1EV@IL2, may provide a scalable platform capable of enhancing both innate and adaptive antitumor immunity, leading to tumor suppression and elimination through regulation of the TME. This approach may complement existing immunotherapies and contribute to the development of combination strategies that more effectively overcome TME‑associated immune resistance.

국문 초록 (Abstract)

암 면역요법은 암 치료의 패러다임을 변화시켰습니다. 그러나 M2 대식세포가 우세하고 불활성화된 T 세포가 풍부한 면역억제성 종양 미세환경(TME)으로 인해 치료 효능이 제한적입니다. 본 연구에서는 M1 대식세포 유래 세포밖소포체(M1EV) 표면에 인터루킨-2(IL-2)를 디스플레이하여 TME 내에서 선천적 면역 반응과 적응 면역 반응을 동시에 조절하는 다기능 암 면역치료 플랫폼을 개발했습니다.
먼저, M1EV는 한외여과 및 크기 배제 크로마토그래피를 사용하여 고순도로 정제한 후, 대사 당공학을 통해 IL-2를 발현하도록 조작했습니다(M1EV_IL2). 물리화학적 분석 결과, IL-2 표면 발현이 소포 크기, 다분산성, 제타 전위 또는 형태를 변화시키지 않았으며, 대표적인 EV 마커가 보존됨을 확인했습니다. 조작된 M1EV_IL2는 M2 대식세포에서 M1 대식세포로의 재분극을 유도하는 M1EV의 고유한 기능을 유지하는 반면, 표면에 디스플레이된 IL-2는 T 세포 활성화를 촉진했습니다. 이러한 결과는 M1EV_IL2가 in vitro 및 ex vivo에서 선천 면역 반응과 적응 면역 반응을 모두 자극할 수 있는 다기능성 암 면역치료 플랫폼임을 보여줍니다.
이후, 더욱 효율적인 IL-2 표면 디스플레이를 달성하고 전반적인 성능을 향상시키기 위해 DSPE-접합 지질 삽입 전략(M1EV@IL2)을 사용하여 보다 간단하고 빠르며 높은 수율의 표면 엔지니어링 방법을 개발했습니다. 대사 당공학 접근법과 비교하여, 이 방법은 생산 시간과 비용을 절감하면서 M1EV 표면에 더 많은 양의 IL-2를 디스플레이할 수 있었습니다. 또한, M1EV@IL2의 추가적인 치료 효과로서 종양 성장 억제에 기여하는 것을 확인하였습니다. 생체 내 생체 분포 연구 결과, M1EV@IL2는 빠르게 혈액 순환계로 유입되어 24시간 이내에 전신 순환을 나타내는 것으로 확인되었으며 생체 내 종양 모델을 이용한 평가에서 M1EV@IL2가 종양 성장을 효과적으로 억제하는 것으로 나타나, M1EV+IL-2 플랫폼이 항종양 효능을 할 수 있음을 시사합니다.
이러한 결과는 IL-2를 디스플레이하는 M1EV를 기반 면역 치료 플랫폼이 TME 내에서 강력한 시너지 효과를 발휘할 것으로 예상됨을 시사합니다. 더욱이, DSPE-접합 지질 삽입은 대사 당공학보다 더 빠르고 더 높은 수준의 IL-2 디스플레이를 가능하게 하여 생산 시간을 단축하고 IL-2 소비를 줄임으로써 전반적인 플랫폼을 개선합니다. 이러한 결과는 M1EV+IL-2 플랫폼이 선천 및 적응 면역 반응을 모두 향상시키고 종양 억제 기능을 통해 TME를 효과적으로 조절할 수 있는 플랫폼을 제공할 수 있음을 시사합니다. 이러한 접근법은 병용 요법 개발에 기여할 수 있으며, 기존 면역치료의 한계를 보완하고 TME을 효과적으로 조절할 수 있는 새로운 치료 전략으로 활용될 수 있을 것으로 기대됩니다.

목차 (Table of Contents)

• Abstact i
• Table of Contents ii
• List of Tables iii
• List of Figures iv
• Chapter 1. Introduction 1
• 1.1. Cancer: a major global health challenge 1
• 1.2. Cancer immunotherapy: a transformative modality in modern cancer treatment 1
• 1.3. Tumor microenvironment: barrier to effective can er therapy 3
• 1.4. Interleukin-2: immunological significance and therapeutic limitation 6
• 1.5. Extracellular vesicles: Biological functions and immunomodulatory potential 6
• 1.6. Extracellular vesicles engineering: strategies and applications for functional enhancement 9
• 1.6.1. Metabolic glycoengineering 10
• 1.6.2. DSPE- conjugated lipid modification 10
• 1.6.3. Current extracellular vesicle engineering approaches and remaining challenges for cancer immunotherapy 12
• 1.7. M1EV-IL-2 conjugates: a multi-functional platform for cancer immunotherapy 12
• Chapter 2. Materials and Methods 15
• 2.1. Cell culture 15
• 2.1.1. Culture of cell lines 15
• 2.1.2.Culture of naïve CD8+ T cells from mouse spleens 15
• 2.2. Animal 16
• 2.3. Differentiation of M1 macrophages for M1EV production 16
• 2.4. Isolation of M1EV 17
• 2.5. Construction of multi-functional immunotherapeutic M1EV displaying surface IL-2 17
• 2.5.1. Generation of IL-2-displaying M1EV via metabolic glycoengineering (M1EV_IL2). 17
• 2.5.2. Generation of IL-2-displaying M1EV via DSPE-conjugated lipid insertion (M1EV@IL2) 19
• 2.6. Investigation of macrophage repolarization from M2 to M1 20
• 2.7. Investigation of CD8⁺ T cell activation and proliferation 20
• 2.8. Fluorescence imaging for analysis of EV uptake 21
• 2.9. Investigation of Tumor growth suppression 21
• 2.10. Investigation of in vivo biodistribution 21
• 2.11. Mouse tumor model 22
• 2.12. Hematoxylin and eosin staining 23
• 2.13. EV characterization 23
• 2.14. Western blot analysis 23
• 2.15. ELISA 24
• 2.16. qRT-PCR analysis 24
• 2.17. WST-1 assay 25
• 2.18. Statistical analysis 25
• Chapter 3. Results and Discussion 27
• 3.1. Differentiation of monocytes to M1 macrophages 27
• 3.2. Isolation and purification of M1EV 29
• 3.3. Development of IL‑2 surface displayed M1EV: a multi-functional cancer immunotherapeutic generated via metabolic glycoengineering31
• 3.3.1. Metabolic glycoengineering of M1 macrophages for the generation of IL-2-displaying M1EV 31
• 3.3.2. Isolation and purification of M1EV and M1EV_IL2 34
• 3.3.3. Physicochemical characterization of M1EV andM1EV_IL2 36
• 3.3.4. M2 to M1 macrophage repolarization induced by M1EV and M1EV_IL2 39
• 3.3.5. T cell activation by M1EV_IL2 41
• 3.3.6. Challenges in the M1EV_IL2 platform and rationale for optimization 45
• 3.4. Advancement of IL‑2 surface displayed M1EV: a multi-functional cancer immunotherapeutic generated via DSPE-conjugated lipid engineering 46
• 3.4.1. Optimization of DSPE-IL2 conjugation and isolati n of M1EV@IL2 46
• 3.4.2. Comparative Advantages of M1EV@IL2 Over M1EV_IL2 . 49
• 3.4.3. Physicochemical characterization of M1EV and M1EV_IL2 51
• 3.4.4. M2 to M1 macrophage repolarization induced by M1EV and M1EV@IL2 53
• 3.4.5. Tumor-suppressive effects of M1EV and M1EV@IL2 55
• 3.4.6. In vivo biodistribution of M1EV and M1EV@IL2 57
• 3.4.7. In vivo tumor growth evaluation in mouse tumor model 59
• 3.4.8. In vivo EV biocompatibility evaluation in mouse tumor model 61
• Chapter 4. Conclusion 63
• Reference 65
• 국문초록 78