Delivery of immune checkpoint inhibitor protein using mesoporous polydopamine nanoparticles for cancer immunotherapy = 메조다공성 폴리도파민 나노입자를 이용한 면역관문억제 단백질의 전달 및 항암면역치료 응용

국문 초록 (Abstract)

면역관문억제제(Immune checkpoint inhibitors, ICIs)는 다양한 암에 대해 임상 종양학을 성공적으로 혁신하였습니다. 그러나 낮은 반응률과 심각한 부작용으로 인해 그 광범위한 활용에는 한계가 있으며, 이러한 문제를 해결하기
위해 안전하고 효과적인 접근법이 필요합니다. 본 연구에서는 anti-programmed cell death-1 (αPD-1) 항체 단백질을 나노엔지니어링하여 종양 내 직접적인 국소 투여 후 치료 효과를 증진시키는 전략을 보고합니다. 구체적으로, 우리는 생체적합성 메조다공성 폴리도파민 나노입자(mesoporous polydopamine nanoparticles, MPNs)를 이용하여 αPD-1 나노제형을 제작하였으며, 이는 단백질에 대한 잠재적 반응성을 통해 αPD-1 의 표면 기능화를 용이하고 효율적으로 가능하게 합니다. 해당 나노제형은 αPD-1 의 PD-1 수용체에 대한 길항 활성을 증가시켜 T 세포에서 PD-1 면역관문 신호를 효과적으로 차단하여 이들의 활성화와 효과기능을 회복시켰습니다. 국소 종양 내 주입을 통해 투여된 나노제형은 αPD-1 의 종양 내 잔류를 향상시키고, 국소 종양에 대한 강력한 항종양 효능과 장기적인 종양 재발 억제를 유도하였습니다. 우리의 결과는 나노ICI 치료를 통한 국소 종양 내 강력한 면역관문 신호 차단이 효과적으로 항종양 면역을 조율하여 국소 및 전신 암 치료를 가능하게 함을 보여줍니다. 결론적으로, 본 연구는 국소화된 종양 치료를 통해 항체 기반 ICI 요법의 효능과 안전성을 향상시킬 수 있는 생체재료 기반 나노엔지니어링 접근법의 잠재력을 확인하였습니다.

다국어 초록 (Multilingual Abstract)

Immune checkpoint inhibitors (ICIs) have successfully transformed clinical oncology against various cancers. However, their widespread utility is limited by low response rates and severe adverse events; thus, a safe and effective approach is required to address these issues. Here, we report the nanoengineering of an anti- programmed cell death-1 antibody (αPD-1) to boost the therapeutic effects following direct local administration into tumors. Specifically, we prepared an αPD-1 nanoformulation using biocompatible mesoporous polydopamine nanoparticles (MPNs) that allow facile and efficient surface functionalization of αPD-1 via latent reactivity to proteins. The nanoformulation increased the antagonistic activity of αPD- 1 against PD-1 receptors by enhancing their avidity interactions, effectively blocking PD-1 immune checkpoint signaling in T cells to restore their activation and effector function. The nanoformulation administered via local intratumoral injection enhanced tumor retention of αPD-1 and elicited strong antitumor efficacy against local tumors and long-term tumor recurrence. Our results indicate that robust immune checkpoint signaling blockade in the local tumors using nano-ICI treatment can effectively orchestrate antitumor immunity for local and systemic cancer treatment. Overall, this study underscores the potential of a biomaterial-based nanoengineering approach for improving the efficacy and safety of antibody-based ICI therapy with localized tumor treatment. Keywords: Polydopamine, Nanoparticles, Immune checkpoint inhibitor, anti-PD-1 antibody, Nano-immunotherapy

목차 (Table of Contents)

• ◼ CONTENTSi
• ◼ LIST OF TABLES iv
• ◼ LIST OF FIGURES v
• ◼ LIST OF ABBREVIATIONS vii
• ◼ ABSTRACTix
• ◼ INTRODUCTION. 1
• 1. Cancer and immunotherapy 2
• 2. Types of cancer immunotherapy treatments 4
• 3. Role of immune check points in cancer immunotherapy 7
• 4. Over the time breakthroughs in checkpoint therapy 10
• 5. Limitataions of ICI therapy 12
• 6. Nanoengineering in cancer immunotherapy 13
• 7. Polydopamine based therapies in cancer treatment 15
• ◼ PURPOSE OF THE STUDY 18
• ◼ MATERIALS AND METHODS 20
• 1. Materials 21
• 2. Synthesis of MPNs and PMPNs and αPD-1@PMPNs 22
• 3. Nanoparticle characterization 23
• 4. Hemolysis assay 23
• 5. Release of αPD-1 23
• 6. Fluorophore conjugation to αPD-1 24
• 7. Isolation of OT-1 splenocytes 24
• 8. In vitro studies 25
• 9. In vivo studies 26
• 10. Gene expression analysis 27
• 11. Western blot analysis 27
• 12. Statistical analysis 28
• ◼ RESULTS 29
• 1. Synthesis and characterization of MPNs, PMPNs, and αPD-1@PMPNs 30
• 2. PEGylation of MPNs to form PMPNs and evaluation of blood compatibility 32
• 3. αPD-1 loading onto PMPNs and release profile 34
• 4. PD-1 expression and binding of αPD-1@PMPNs in EL4 T cells 38
• 5. PD-1/PD-L1 coexpression and cellular uptake of αPD-1@PMPNs in EL4 T
• cells 40
• 6. Blockade of PD-1 signaling and cytokine secretion in EL4 T cells 42
• 7. PD-1/PD-L1 coexpression and cellular uptake of αPD-1@PMPNs in OT-1
• splenocytes 44
• 8. Functional blockade of PD-1 signaling and cytokine production in OT-1
• splenocytes 46
• 9. Local tumor retention of PMPNs and αPD-1@PMPNs in CT26 tumors 48
• 10. Antitumor efficacy and survival benefit of αPD-1@PMPNs in CT26
• tumors 50
• 11. Effects of αPD-1@PMPNs on CD8⁺ T cells and proinflammatory cytokines in
• local CT26 tumors. 53
• 12. αPD-1@PMPNs modulate innate immune cells in local tumors 55
• 13. αPD-1@PMPNs enhance immune activation systemically in tumor
• rechallenge and lymph nodes 58
• 14. αPD-1@PMPNs enhance immune activation systemically in spleen 60
• 15. Assessment of systemic toxicity to normal organs 62
• ◼ DISCUSSION 64
• ◼ CONCLUSION 70
• ◼ REFERENCES 72
• ◼ APPENDIX 90
• ◼ ABSTRACT (초록) 92
• ◼ ACKNOWLEDGMENTS 94