研發抗PD-L1抗體片段嵌合微脂體藥物運用於肺癌標靶藥物傳輸

Abstract

肺癌為全球目前最常見的癌症，根據2018年最新統計數據結果，肺癌是致死率最高的癌症，且五年存活率居於最末，可見如手術、化療、免疫療法等單一療法，雖有一定療效但有所不足，於是對於肺癌具高效並精準的聯合療法(combination therapy)成為現今研發的重點。PD1/PD-L1為當今聞名的免疫檢查點之一，在肺癌與其他多數癌症中，癌細胞藉由表現PD-L1得以躲避免疫系統的監視。近年臨床數據指出，透過阻斷PD-1/PD-L1的免疫療法，病患的生存率得以有效提升，因此針對該免疫檢查點開發創新的治療方式，可望克服肺癌病患高死亡率與低五年存活率的瓶頸。Doxorubicin及Vinorelbine為當今常見之抗癌化療藥物，而以微脂體形式(liposomal drug)的投藥方式能夠明顯降低該兩種藥物的生物毒性與其他不良的副作用，同時微脂體更具有生物可分解性，延長藥物於血液循環的時間，進而提升藥物於腫瘤組織中之濃度等優勢。本實驗室先前已利用噬菌體顯現法(phage display)與親合力成熟技術(affinity maturation)篩選出一株能專一辨認PD-L1的單株抗體(anti-PD-L1 antibody)；於本篇研究，我們產出該抗體的抗原結合片段(Fab)命名為PL15.2 Fab，驗證其對於PD-L1擁有良好的鍵結能力，並可經內噬作用進入細胞當中。我們進一步將PL15.2 Fab接合於微脂體藥物之外膜，使得微脂體藥物擁有專一辨識癌細胞的能力，同時證實這些帶有PL15.2 Fab的微脂體藥物可被肺癌細胞內化，並找出微脂體藥物最佳的抗體片段嵌合率。另外，在人類肺癌細胞移植的免疫缺陷小鼠中，進行微脂體藥物的治療，發現接上PL15.2 Fab的微脂體藥物確實能夠抑制腫瘤的生長，且帶有PL15.2 Fab的微脂體比單純只有微脂體或藥物呈現更好的療效。綜合上述之實驗結果，PL15.2 Fab能夠專一地辨認PD-L1，可作為帶有抗肺癌藥物之微脂體膜外的標靶，於生物體中辨認高度表達PD-L1的肺癌細胞，使得微脂體藥物更有效地投遞至肺癌細胞，不僅建立一個新穎而精確的藥物投遞系統，進而增進藥物的療效。

Lung cancer is the most common cancer globally. According to the latest statistics in 2018, malignant lung cancer has the highest mortality rate as well as the lowest five-year overall survival rate among different cancers. This phenomenon indicates that monotherapy such as surgery, chemotherapy, and immunotherapy is somewhat insufficient for the treatment of lung cancer. Therefore, it is urgent to develop combination therapy with better therapeutic efficacy for the treatment of lung cancer. PD-1/PD-L1 is one of the well-known immune checkpoints. In lung cancer and most other tumors, cancer cells could escape from immune surveillance by expressing PD-L1. Recently, some clinical data indicate that the survival rate of patients suffering from cancers can be improved by blocking PD-1/PD-L1 interaction. Therefore, the development of innovative therapeutic methods for PD-1/PD-L1 immune checkpoint may overcome the bottleneck of high mortality and low survival rate of lung cancer patients. Liposomes are biodegradable molecules, which can prolong the circulation time of anti-cancer drugs in blood, and thereby increase the concentration of drugs in tumor tissues. Drugs in the liposomal form can also significantly reduce their biotoxicity and avoid other harmful side effects. Doxorubicin and vinorelbine are commonly used as small molecular drugs for the treatment of cancer in chemotherapy. We previously produced a monoclonal anti-PD-L1 antibody via phage display technology and affinity maturation, namely PL15.2, which has been validated to exhibit high binding affinity to PD-L1. In this study, the PL15.2 Fab, which was further conjugated onto the drug-containing liposomes to give liposomal drugs the ability to recognize lung cancer cells specifically, subsequently internalize into the cells. These targeting liposomes, which combine the advantages of chemotherapy and immunotherapy, can be properly applied to the drug delivery system as a highly efficient therapeutic method. In human lung cancer xenograft model, the PL15.2 Fab-conjugated liposomal drugs significantly decreased the tumors growth , and showed better therapeutic efficacy than liposomal drugs or free drugs. Based on the results mentioned above, we demonstrated that PL15.2 Fab, which specifically recognizing PD-L1-expressing cancer cells, could be inserted to the outside of the liposome to facilitate the delivery of the liposome to the target lung cancer cells. Furthermore, the PL15.2 Fab-conjugated liposomal drugs can accumulate precisely in tumor cells in vivo. Thus, the PL15.2 Fab has great potential as a targeted drug delivery system in the treatment of lung cancer.