암세포는 혈관, 면역세포, 신호전달 분자 및 세포외 기질 (extracellular matrix, ECM) 등으로 구성된 복잡한 종양미세환경 (tumor microenvironment, TME)에 둘러싸여 존재한다. 종양미세환경에서 암세포는 면역관문 (immune checkpoint) 단백질, 면역 억제성 효소, 신호전달 물질 등을 과발현시켜 종양미세환경을 생존에 유리하도록 변화시킴으로써 체내 ...
암세포는 혈관, 면역세포, 신호전달 분자 및 세포외 기질 (extracellular matrix, ECM) 등으로 구성된 복잡한 종양미세환경 (tumor microenvironment, TME)에 둘러싸여 존재한다. 종양미세환경에서 암세포는 면역관문 (immune checkpoint) 단백질, 면역 억제성 효소, 신호전달 물질 등을 과발현시켜 종양미세환경을 생존에 유리하도록 변화시킴으로써 체내 면역반응을 회피한다. 이를 극복하고, 종양 주변의 면역시스템을 재활성화시켜 항종양 면역반응을 유도하기위해 면역관문억제제 (immune checkpoint blockade, ICB)와 같은 다양한 면역항암제가 개발되었다. 그러나, 일부 암 환자 및 제한된 암 종에서만 효능을 보이거나 내성이 발생하는 등 한계점이 여전히 존재하고있다. 최근 몇 가지 화학항암제가 면역세포를 활성화 시켜 종양 내 면역세포의 침투를 유도할 수 있음이 밝혀졌다. 따라서 기존 면역항암제에 낮은 반응률을 보이는 종양에서 항종양 면역 반응을 증가시키고 복잡한 종양미세환경의 다중 면역억제 활성을 역전시키기위한 전략으로 면역원성 화학항암제와 면역항암제의 병용치료가 제시되었다. 체내 조직으로 다중 약물을 공동전달하기 위해 리포솜 (liposome)과 같은 약물전달체 (drug carrier)의 적용은 약물의 안정성을 증가시킴으로써 치료효과를 높일 수 있다. 그러나 종양에 약물을 전달하기 위하여 현재까지 개발된 다양한 약물전달체 기술은 대부분 표적화 분자 (targeting molecule)없이 EPR(Enhanced Permeation and Retention) 효과에만 의존한다. 이는 정상조직으로의 비특이적인 약물 전달에 의한 부작용 우려뿐만 아니라, 다양한 유형의 세포가 존재하는 종양부위에서 암 세포에 대한 전달 특이성 부족으로 인해 치료 효과가 경감되는 단점이 있다. 따라서, 본 연구에서는 암세포를 표적으로 하는 압타머 (aptamer)가 수식된 리포솜을 이용해 종양 특이적 화학면역요법을 위한 표적지향형 다중 약물 전달시스템을 개발하는 것을 목표로 한다. 첫 번째 장에서는, 리포솜을 이용한 약물전달시스템의 개요와 핵산 (nucleic acid)으로 이루어진 표적화 분자, 압타머 (aptamer) 를 이용한 표적지향형 약물 전달시스템에 대해 설명한다. 두 번째 장에서는, 면역원성세포사멸 유도제 (immunogenic cell death inducer)독소루비신 (doxorubicin, DOX)과 IDO1 (Indoleamine 2,3-dioxygenase) siRNA를 공동전달하기 위해 유방암세포의 막 항원 (이하 CD44와 PD-L1)에 특이적으로 결합하는 압타머를 부착시킨 리포솜을 개발했다 (이하 Aptm[DOX/IDO1]). Aptm[DOX/IDO1]은 표적 유방암 모델에서 증가된 종양 내 약물 축적 효과와 종양 성장 및 2차 전이에 대한 우수한 억제 효능을 나타냈다. 이는 독소루비신에 의해 유도되는 ‘면역원성 세포사멸’과 IDO1 siRNA에 의한 면역억제성 단백질 ‘IDO1’의 발현 억제를 통해 종양 미세환경이 면역 작용에 유리한 환경으로 변화되어 항종양 면역반응 증가함에 따른 것임을 확인했다. 세 번째 장에서는, 히스톤 탈아세틸화효소 억제제(Histone deacetylase inhibitor, HDACi)와 PD-L1 (Programmed Death-Ligand 1) siRNA를 전달하기 위해 폐암 세포의 막 항원 PD-L1에 특이적으로 결합하는 압타머를 부착시킨 리포솜과 리포플렉스 (lipoplex)를 개발했다 (이하 aptamosome[SAHA], aptaplex[siPD-L1]). Aptamosome[SAHA]과 aptaplex[siPD-L1]은 폐암 마우스 모델에서 증가된 약물 전달 효율과 높은 종양 성장 및 2차 전이 억제 효과를 나타냈다. 이는 종양조직에서 SAHA에 의한 아포토시스 (apoptosis)와 면역유도성 케모카인 (chemokine) 분비와 PD-L1 siRNA에 의한 면역관문 단백질 ‘PD-L1’ 발현 저해가 촉진됨으로써 종양 내 세포 사멸과 항암 면역반응을 지속적으로 유도하여 항종양 효과를 나타냄을 입증했다. 본 연구의 압타머 접합 리포솜을 기반으로 한 표적지향형 약물전달체는 종양 특이적 다중 약물 전달을 위한 다목적 플랫폼으로서 이용될 수 있을 것이라 기대한다.
암세포는 혈관, 면역세포, 신호전달 분자 및 세포외 기질 (extracellular matrix, ECM) 등으로 구성된 복잡한 종양미세환경 (tumor microenvironment, TME)에 둘러싸여 존재한다. 종양미세환경에서 암세포는 면역관문 (immune checkpoint) 단백질, 면역 억제성 효소, 신호전달 물질 등을 과발현시켜 종양미세환경을 생존에 유리하도록 변화시킴으로써 체내 면역반응을 회피한다. 이를 극복하고, 종양 주변의 면역시스템을 재활성화시켜 항종양 면역반응을 유도하기위해 면역관문억제제 (immune checkpoint blockade, ICB)와 같은 다양한 면역항암제가 개발되었다. 그러나, 일부 암 환자 및 제한된 암 종에서만 효능을 보이거나 내성이 발생하는 등 한계점이 여전히 존재하고있다. 최근 몇 가지 화학항암제가 면역세포를 활성화 시켜 종양 내 면역세포의 침투를 유도할 수 있음이 밝혀졌다. 따라서 기존 면역항암제에 낮은 반응률을 보이는 종양에서 항종양 면역 반응을 증가시키고 복잡한 종양미세환경의 다중 면역억제 활성을 역전시키기위한 전략으로 면역원성 화학항암제와 면역항암제의 병용치료가 제시되었다. 체내 조직으로 다중 약물을 공동전달하기 위해 리포솜 (liposome)과 같은 약물전달체 (drug carrier)의 적용은 약물의 안정성을 증가시킴으로써 치료효과를 높일 수 있다. 그러나 종양에 약물을 전달하기 위하여 현재까지 개발된 다양한 약물전달체 기술은 대부분 표적화 분자 (targeting molecule)없이 EPR(Enhanced Permeation and Retention) 효과에만 의존한다. 이는 정상조직으로의 비특이적인 약물 전달에 의한 부작용 우려뿐만 아니라, 다양한 유형의 세포가 존재하는 종양부위에서 암 세포에 대한 전달 특이성 부족으로 인해 치료 효과가 경감되는 단점이 있다. 따라서, 본 연구에서는 암세포를 표적으로 하는 압타머 (aptamer)가 수식된 리포솜을 이용해 종양 특이적 화학면역요법을 위한 표적지향형 다중 약물 전달시스템을 개발하는 것을 목표로 한다. 첫 번째 장에서는, 리포솜을 이용한 약물전달시스템의 개요와 핵산 (nucleic acid)으로 이루어진 표적화 분자, 압타머 (aptamer) 를 이용한 표적지향형 약물 전달시스템에 대해 설명한다. 두 번째 장에서는, 면역원성세포사멸 유도제 (immunogenic cell death inducer)독소루비신 (doxorubicin, DOX)과 IDO1 (Indoleamine 2,3-dioxygenase) siRNA를 공동전달하기 위해 유방암세포의 막 항원 (이하 CD44와 PD-L1)에 특이적으로 결합하는 압타머를 부착시킨 리포솜을 개발했다 (이하 Aptm[DOX/IDO1]). Aptm[DOX/IDO1]은 표적 유방암 모델에서 증가된 종양 내 약물 축적 효과와 종양 성장 및 2차 전이에 대한 우수한 억제 효능을 나타냈다. 이는 독소루비신에 의해 유도되는 ‘면역원성 세포사멸’과 IDO1 siRNA에 의한 면역억제성 단백질 ‘IDO1’의 발현 억제를 통해 종양 미세환경이 면역 작용에 유리한 환경으로 변화되어 항종양 면역반응 증가함에 따른 것임을 확인했다. 세 번째 장에서는, 히스톤 탈아세틸화효소 억제제(Histone deacetylase inhibitor, HDACi)와 PD-L1 (Programmed Death-Ligand 1) siRNA를 전달하기 위해 폐암 세포의 막 항원 PD-L1에 특이적으로 결합하는 압타머를 부착시킨 리포솜과 리포플렉스 (lipoplex)를 개발했다 (이하 aptamosome[SAHA], aptaplex[siPD-L1]). Aptamosome[SAHA]과 aptaplex[siPD-L1]은 폐암 마우스 모델에서 증가된 약물 전달 효율과 높은 종양 성장 및 2차 전이 억제 효과를 나타냈다. 이는 종양조직에서 SAHA에 의한 아포토시스 (apoptosis)와 면역유도성 케모카인 (chemokine) 분비와 PD-L1 siRNA에 의한 면역관문 단백질 ‘PD-L1’ 발현 저해가 촉진됨으로써 종양 내 세포 사멸과 항암 면역반응을 지속적으로 유도하여 항종양 효과를 나타냄을 입증했다. 본 연구의 압타머 접합 리포솜을 기반으로 한 표적지향형 약물전달체는 종양 특이적 다중 약물 전달을 위한 다목적 플랫폼으로서 이용될 수 있을 것이라 기대한다.
Cancer cells have various complex immune evasion mechanisms to resist the immune response by reprogramming the tumor microenvironment (TME), such as overexpressing immune checkpoint proteins, immunosuppressive enzymes, and signaling molecules. Thus, chemoimmunotherapy combining immunogenic chemother...
Cancer cells have various complex immune evasion mechanisms to resist the immune response by reprogramming the tumor microenvironment (TME), such as overexpressing immune checkpoint proteins, immunosuppressive enzymes, and signaling molecules. Thus, chemoimmunotherapy combining immunogenic chemotherapeutic can evoke anticancer immune response and immunotherapeutic drugs can inhibit immunosuppressive activities has emerged as a reliable strategy for improving cancer treatment. The application of nanocarriers, such as liposomal formulation, can improve the in vivo pharmacokinetic (PK) behavior of anticancer drugs through increasing the drug stability. To date, numerous nanocarrier platforms have been developed to deliver the multiple drugs, which mainly depends on the enhanced permeation and retention (EPR). These nanocarriers are insufficient to accomplish tissue selectivity in tumor sites harboring various cell types, such as cancer cells and immune cells. Therefore, to achieve the tumor-specific immune response, targeted delivery platform is highly recommended for systematic delivery of the anticancer drugs into tumors. Throughout my doctorate studies, I developed a targeted drug delivery systems based on nano-sized liposomes functionalized with cancer cell-targeting DNA aptamers for chemoimmunotherapy. In the first chapter, there is an overview regarding drug delivery systems using various liposomes and drug delivery vehicles with nucleic acid aptamer for cancer-targeting. In the second chapter, shown is the development of a nano-sized liposome conjugated with anti-CD44 and anti-PD-L1(programmed death-ligand 1) DNA aptamers, which target breast cancer cells and inhibit PD-1/PD-L1 interaction between cancer cells and T-cells. To reverse immunosuppressive TME and reactivate immune response, cancer-targeting nano-liposomes were prepared to contain immunogenic cell death inducer (Doxorubicin, DOX) and IDO1 (indoleamine 2,3-dioxygenase-1) siRNA, namely Aptm[DOX/IDO1]. The Aptm[DOX/IDO1] specifically delivered the loaded DOX and IDO1 siRNA into target breast cancer cells through aptamer-mediated endocytosis. Cancer-targeted DOX/IDO1 siRNA delivery enhanced immunogenic cell death (ICD) and suppressed IDO1 expression with significantly high toxicity in cancer cells. I demonstrated that Aptm[DOX/IDO1] could achieve synergistic antitumor effects by facilitating ICD response and simultaneous reversal of the immunosuppressive TME with IDO1 knockdown in the subcutaneous breast cancer model mice, thus reducing tumor size. These antitumor effects were exerted with intratumoral infiltration of CD8+ cytotoxic T lymphocyte as well as attenuation of regulatory T-cell recruitment in the tumor sites. I further proved that our Aptm[DOX/IDO1] strategy significantly reduced tumor metastasis in tumor-xenograft mice through a synergistic combination of cancer cell-targeted ICD induction and reversal of the IDO1-mediated immunosuppressive TME. In the third chapter, another version of DNA aptamer-conjugated nano-liposomes are developed for targeted delivery of histone deacetylase (HDAC) inhibitor (suberoylanilide hydroxamic acid, SAHA) and PD-L1 siRNA to tumors, in which two liposomal carriers were conjugated with anti-PD-L1 DNA aptamer for tumor-specific delivery of each liposome. Although SAHA, which is a first HDAC inhibitor approved by the FDA, exhibited great therapeutic efficacy in epigenetic anticancer therapy, repeated treatment caused acute relapse of tumor development in clinical trials, which is likely attributed by upregulation of PD-L1 expression. Thus, I hypothesized that PD-L1 suppression in combination with SAHA would provide synergistically enhanced anticancer efficacy through reactivating of immune response in TME. For combinatorial treatment of synergistic drugs, SAHA was encapsulated in neutral liposomes (liposome[SAHA]) and negatively charged PD-L1 siRNA was complexed with the cationic liposomes (lipoplex[siPD-L1]). The liposome[SAHA] and lipoplex[siPD-L1] were subsequently functionalized with antagonistic anti-PD-L1 aptamers on the surface for targeting the cancer cells and inhibiting PD-1/PD-L1 interaction (namely, aptamosome[SAHA] and aptaplex[siPD-L1], respectively). I evaluated the aptamer-mediated specific binding of these anti-PD-L1 aptamer-conjugated liposomal formulations in PD-L1 expressing cancer cells. I then investigated the anticancer efficacy of combinational treatment with aptamosome[SAHA] and aptaplex[siPD-L1] in target cancer cells through immune-activating chemokine secretion, PD-L1 suppression, and cytotoxicity. Furthermore, I demonstrated that tumor-specific accumulation of aptamosome[SAHA] and aptaplex[siPD-L1] in tumor-bearing mice results in effective suppression of tumor growth in mice. Taken together, I developed the liposome-based targeted drug delivery systems functionalized with cancer-targeting DNA aptamers for chemoimmunotherapy, which induced immune-favorable TME through tumor-specific immune modulation. The immunogenic chemotherapeutics and/or nucleic acid drugs were loaded to liposomal formulations with different methods depending on physical properties of drugs. In summary, I firstly developed tumor-specific anti-PD-L1 DNA aptamer conjugated two liposomal carriers for combinatorial treatment of epigenetic drug SAHA and PD-L1 siRNA. Furthermore, I developed a targeted co-delivery system harboring ICD inducer DOX and IDO1 siRNA based on cationic liposomes conjugated with tumor-specific anti-CD44 and anti-PD-L1 aptamers. These nano-sized liposomal formulations conjugated with DNA aptamers targeting the cancer cell surface marker proteins can be applied as versatile nanocarrier platform for targeted co-delivery of various drug types, such as hydrophobic, hydrophilic, and nucleic acid drugs.
Cancer cells have various complex immune evasion mechanisms to resist the immune response by reprogramming the tumor microenvironment (TME), such as overexpressing immune checkpoint proteins, immunosuppressive enzymes, and signaling molecules. Thus, chemoimmunotherapy combining immunogenic chemotherapeutic can evoke anticancer immune response and immunotherapeutic drugs can inhibit immunosuppressive activities has emerged as a reliable strategy for improving cancer treatment. The application of nanocarriers, such as liposomal formulation, can improve the in vivo pharmacokinetic (PK) behavior of anticancer drugs through increasing the drug stability. To date, numerous nanocarrier platforms have been developed to deliver the multiple drugs, which mainly depends on the enhanced permeation and retention (EPR). These nanocarriers are insufficient to accomplish tissue selectivity in tumor sites harboring various cell types, such as cancer cells and immune cells. Therefore, to achieve the tumor-specific immune response, targeted delivery platform is highly recommended for systematic delivery of the anticancer drugs into tumors. Throughout my doctorate studies, I developed a targeted drug delivery systems based on nano-sized liposomes functionalized with cancer cell-targeting DNA aptamers for chemoimmunotherapy. In the first chapter, there is an overview regarding drug delivery systems using various liposomes and drug delivery vehicles with nucleic acid aptamer for cancer-targeting. In the second chapter, shown is the development of a nano-sized liposome conjugated with anti-CD44 and anti-PD-L1(programmed death-ligand 1) DNA aptamers, which target breast cancer cells and inhibit PD-1/PD-L1 interaction between cancer cells and T-cells. To reverse immunosuppressive TME and reactivate immune response, cancer-targeting nano-liposomes were prepared to contain immunogenic cell death inducer (Doxorubicin, DOX) and IDO1 (indoleamine 2,3-dioxygenase-1) siRNA, namely Aptm[DOX/IDO1]. The Aptm[DOX/IDO1] specifically delivered the loaded DOX and IDO1 siRNA into target breast cancer cells through aptamer-mediated endocytosis. Cancer-targeted DOX/IDO1 siRNA delivery enhanced immunogenic cell death (ICD) and suppressed IDO1 expression with significantly high toxicity in cancer cells. I demonstrated that Aptm[DOX/IDO1] could achieve synergistic antitumor effects by facilitating ICD response and simultaneous reversal of the immunosuppressive TME with IDO1 knockdown in the subcutaneous breast cancer model mice, thus reducing tumor size. These antitumor effects were exerted with intratumoral infiltration of CD8+ cytotoxic T lymphocyte as well as attenuation of regulatory T-cell recruitment in the tumor sites. I further proved that our Aptm[DOX/IDO1] strategy significantly reduced tumor metastasis in tumor-xenograft mice through a synergistic combination of cancer cell-targeted ICD induction and reversal of the IDO1-mediated immunosuppressive TME. In the third chapter, another version of DNA aptamer-conjugated nano-liposomes are developed for targeted delivery of histone deacetylase (HDAC) inhibitor (suberoylanilide hydroxamic acid, SAHA) and PD-L1 siRNA to tumors, in which two liposomal carriers were conjugated with anti-PD-L1 DNA aptamer for tumor-specific delivery of each liposome. Although SAHA, which is a first HDAC inhibitor approved by the FDA, exhibited great therapeutic efficacy in epigenetic anticancer therapy, repeated treatment caused acute relapse of tumor development in clinical trials, which is likely attributed by upregulation of PD-L1 expression. Thus, I hypothesized that PD-L1 suppression in combination with SAHA would provide synergistically enhanced anticancer efficacy through reactivating of immune response in TME. For combinatorial treatment of synergistic drugs, SAHA was encapsulated in neutral liposomes (liposome[SAHA]) and negatively charged PD-L1 siRNA was complexed with the cationic liposomes (lipoplex[siPD-L1]). The liposome[SAHA] and lipoplex[siPD-L1] were subsequently functionalized with antagonistic anti-PD-L1 aptamers on the surface for targeting the cancer cells and inhibiting PD-1/PD-L1 interaction (namely, aptamosome[SAHA] and aptaplex[siPD-L1], respectively). I evaluated the aptamer-mediated specific binding of these anti-PD-L1 aptamer-conjugated liposomal formulations in PD-L1 expressing cancer cells. I then investigated the anticancer efficacy of combinational treatment with aptamosome[SAHA] and aptaplex[siPD-L1] in target cancer cells through immune-activating chemokine secretion, PD-L1 suppression, and cytotoxicity. Furthermore, I demonstrated that tumor-specific accumulation of aptamosome[SAHA] and aptaplex[siPD-L1] in tumor-bearing mice results in effective suppression of tumor growth in mice. Taken together, I developed the liposome-based targeted drug delivery systems functionalized with cancer-targeting DNA aptamers for chemoimmunotherapy, which induced immune-favorable TME through tumor-specific immune modulation. The immunogenic chemotherapeutics and/or nucleic acid drugs were loaded to liposomal formulations with different methods depending on physical properties of drugs. In summary, I firstly developed tumor-specific anti-PD-L1 DNA aptamer conjugated two liposomal carriers for combinatorial treatment of epigenetic drug SAHA and PD-L1 siRNA. Furthermore, I developed a targeted co-delivery system harboring ICD inducer DOX and IDO1 siRNA based on cationic liposomes conjugated with tumor-specific anti-CD44 and anti-PD-L1 aptamers. These nano-sized liposomal formulations conjugated with DNA aptamers targeting the cancer cell surface marker proteins can be applied as versatile nanocarrier platform for targeted co-delivery of various drug types, such as hydrophobic, hydrophilic, and nucleic acid drugs.
주제어
#Liposome Aptamer Cancer-targeting Targeted drug delivery siRNA delivery Immunogenic cell death Tumor microenvironment Immunotherapy Combination therapy
학위논문 정보
저자
김민희
학위수여기관
건국대학교 대학원
학위구분
국내박사
학과
생명공학과
지도교수
김동은
발행연도
2023
총페이지
139
키워드
Liposome Aptamer Cancer-targeting Targeted drug delivery siRNA delivery Immunogenic cell death Tumor microenvironment Immunotherapy Combination therapy
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