항종양 면역반응을 유도하는 나노물질 하이브리드 유전자전달체와 수지상세포 융합치료의 전신투여 효율성 평가

Abstract

수지상 세포(Dendritic cell)는 T세포에 항원제시를 수행 하는 대표적인 항원제시세포로서, 낮은 독성과 종양 특이적 면역반응을 유도하므로 암 치료 연구에 널리 쓰이고 있다. 수지상 세포가 가지는 여러 가지 이점에도 불구하고 임상실험에서 낮은 효과가 나타나고 있는데, 이는 종양 내 면역 억제 환경에 의해 수지상 세포의 효과가 감소되기 때문이다. 본 실험실 이전 연구에서 종양 내 면역 억제 환경 극복과 수지상 세포의 치료효능을 증가시키기 위해 인터류킨(interleukin, IL)-12와 과립구 마크로파지 콜로니 자극인자(granulocyte-macrophage colony-stimulating factor, GM-CSF)를 동시 발현하는 종양 특이적 살상 아데노바이러스(oncolytic adenovirus)를 수지상 세포와 병용투여 함으로써 향상된 항종양 효과를 이미 확인하였다. 본 연구에서는 아데노바이러스의 치료 효험(therapeutic efficacy)과 수지상 세포의 보조제(adjuvant)로써 아데노바이러스의 효험을 증가시키기 위해 아르지닌이 접합된 생분해성 중합체(arginine-grafted bioreducible polymer)에 페길화된 파클리탁셀(PEGylated PTX)이 연결된(conjugated) APP 중합체(ABP-PEG3.5k-Paclitaxel, APP polymer)와 사이토카인 발현 아데노바이러스를 복합체화 하여 나노물질 하이브리드 유전자전달체로 이용 하였으며, 수지상 세포와 병용 투여 하였다. 아데노바이러스와 APP 복합체는 종양세포 내에서 아데노바이러스 대비 증가된 사이토카인 발현을 보였으며, 이는 종양 내 면역억제 환경을 극복하게 하였다. 더욱이, APP 중합체에 연결 되어 있는 파클리탁셀이 세포 내 이입 이후 종양세포의 세포자살(apoptosis)을 유도하여 종양 관련 항원(tumor-associated antigen)을 수지상 세포에 제공함으로써 수지상 세포의 분화 및 2차 면역기관으로의 이동을 증가 시켰다. 향상된 수지상 세포의 기능은 종양 내로의 CD4+, CD8+ T세포의 침투(infiltration)와 축적(accumulation)을 증가 시키며, 항종양 면역반응을 유도하였다. 결론적으로 사이토카인 동시발현 종양 특이적 살상 아데노바이러스와 APP 복합체는 아데노바이러스에 비해 보다 증가된 전달효율 및 사이토카인 발현을 통해 종양 내 면역 억제 환경 극복을 용이하게 할 뿐만 아니라, 수지상 세포의 효험을 증가시킴으로 써 보다 증가된 항종양 효과를 유도하게 된다. 이는 기존 아데노바이러스의 치료 효험(therapeutic efficacy)과 수지상 세포 보조제 효험(adjuvant efficacy)을 동시에 증가시킬 수 있는 새로운 방법이라고 사료된다.|Dendritic cell (DC)-based vaccines have emerged as a promising strategy in cancer immunotherapy due to low toxicity and immune stimulatory attributes. Despite these promising attributes of DC-based vaccines, therapeutic efficacy of DCs is insufficient as a monotherapy due to highly immunosuppressive tumor microenvironment. Previously we have shown that interleukin (IL)-12 and granulocyte-macrophage colony-stimulating factor (GM-CSF) co-expressing oncolytic adenovirus (ΔB7/IL-12/GM-CSF) in combination with DCs can induce potent antitumor immune response through enhanced infiltration and activation of immune effector cells in tumor tissue. To enhance the therapeutic potential of oncolytic adenovirus (Ad), we have previously explored complexation of oncolytic Ad with cationic polymers to shield oncolytic Ad from the host environment. In our present study, we have investigated paclitaxel-conjugated polymeric micelle consisting of PEG and arginine-grafted bioreducible polymer (ABP), ABP-PEG3.5k-Paclitaxel (APP) as a nanohybrid gene carrier for oncolytic Ad to enhance the cellular internalization, viral replication, and transgene expression of oncolytic Ad (ΔB7/IL-12/GM-CSF). Ad/APP complex mediated tumor-specific expression of therapeutic cytokines (IL-12 and GM-CSF) ameliorated immunosuppressive tumor microenvironment, thus enhancing DC vaccination efficacy. APP-coated oncolytic Ad in combination with DC enhanced intratumoral expression of antitumor cytokines, resulting in restoration of antitumor immune function in immunosuppressive tumor microenvironment. Furthermore, APP with potent chemotherapeutic attributes induced tumor cell apoptosis, which generates tumor-associated antigens (TAAs) for DC uptake, leading to enhanced stimulation of DC differentiation and migration to lymphoid organs. Importantly, enhanced DC function positively correlated with improved infiltration and accumulation of CD4+ and CD8+ T cells in tumor tissues, ultimately leading to induction of potent antitumor immune response and antitumor effect. Taken together, these results demonstrate that Ad/APP complex in combination with DC can be a promising candidate for cancer immunotherapy.; Dendritic cell (DC)-based vaccines have emerged as a promising strategy in cancer immunotherapy due to low toxicity and immune stimulatory attributes. Despite these promising attributes of DC-based vaccines, therapeutic efficacy of DCs is insufficient as a monotherapy due to highly immunosuppressive tumor microenvironment. Previously we have shown that interleukin (IL)-12 and granulocyte-macrophage colony-stimulating factor (GM-CSF) co-expressing oncolytic adenovirus (ΔB7/IL-12/GM-CSF) in combination with DCs can induce potent antitumor immune response through enhanced infiltration and activation of immune effector cells in tumor tissue. To enhance the therapeutic potential of oncolytic adenovirus (Ad), we have previously explored complexation of oncolytic Ad with cationic polymers to shield oncolytic Ad from the host environment. In our present study, we have investigated paclitaxel-conjugated polymeric micelle consisting of PEG and arginine-grafted bioreducible polymer (ABP), ABP-PEG3.5k-Paclitaxel (APP) as a nanohybrid gene carrier for oncolytic Ad to enhance the cellular internalization, viral replication, and transgene expression of oncolytic Ad (ΔB7/IL-12/GM-CSF). Ad/APP complex mediated tumor-specific expression of therapeutic cytokines (IL-12 and GM-CSF) ameliorated immunosuppressive tumor microenvironment, thus enhancing DC vaccination efficacy. APP-coated oncolytic Ad in combination with DC enhanced intratumoral expression of antitumor cytokines, resulting in restoration of antitumor immune function in immunosuppressive tumor microenvironment. Furthermore, APP with potent chemotherapeutic attributes induced tumor cell apoptosis, which generates tumor-associated antigens (TAAs) for DC uptake, leading to enhanced stimulation of DC differentiation and migration to lymphoid organs. Importantly, enhanced DC function positively correlated with improved infiltration and accumulation of CD4+ and CD8+ T cells in tumor tissues, ultimately leading to induction of potent antitumor immune response and antitumor effect. Taken together, these results demonstrate that Ad/APP complex in combination with DC can be a promising candidate for cancer immunotherapy.