結合單株抗體與奈米明膠之藥物載體於肺癌治療之探討

Abstract

近年來肺癌成為大多數已開發國家的主要疾病之一，也位居國人癌症死亡之冠。儘管醫學已有相當進展，但是肺癌的長期存活率低，且傳統化療之副作用多，因此為了進一步改善治療效果，發展低毒性的治療，目前主要的研究方向是標靶治療，而藥物作用的標靶包括了癌細胞具有的特殊抗原、特殊生長因子受體、腫瘤血管新生相關因子、訊息傳遞路徑中的各類分子。其中，表皮生長因子受體 (epidermal growth factor receptor, EGFR)是本實驗的主要研究目標，它在癌症細胞的增殖扮演重要的角色。大部分上皮性腫瘤均會表現或過度表現EGFR；尤其是在非小細胞肺癌上，EGFR的過度表現高達40 %~80 %。 本研究分為兩部分，第一部分是比較可標的癌細胞表面EGFR之導向配位體(ligand)之間的親和性(affinity)，比較的方式是利用原子力顯微鏡(atomic force microscopy, AFM)來偵測受體和配位體之間的作用力，以及使用表面電漿共振系統(surface plasmon resonance, SPR)來獲得結合係數或分離係數等動力學參數。而比較的對象是EGFR的天然配位體-表皮生長因子(EGF)和單株抗體(anti-EGFR antibody，mAb LA1)及由噬菌體表現庫篩選出的胜肽GE11。由SPR得到的結果顯示，EGF，mAb LA1以及胜肽GE11的平衡解離常數分別為1.77× 10-7、2.07× 10-9、4.59× 10-4 M-1，表示mAb LA1和EGFR之間的親合性最強，EGF是其次，胜肽GE11的親合性則最弱。在AFM的部分，EGF-EGFR以及mAb LA1-EGFR的作用力(unbinding force)皆為150-210 pN (loading rate range: 450-5000 pN/s)，而胜肽GE11的作用力僅有上述的三分之一，50-60 pN (loading rate range: 150-7500 pN/s)，顯示胜肽GE11的作用力依然最弱。 從第一部分的結果得知，mAb LA1與EGFR的親和性是最強的，此外，也不會造成嚴重的癌細胞增生問題(cell proliferation)，因此在本實驗的第二部分，我們選用mAb LA1結合明膠奈米粒子，來製備具標的EGFR功能的藥物載體。首先，我們以two-step desolvation method製備出顆粒大小約為230 nm、表面電位約為-3 mV的明膠奈米顆粒(GPs)，其表面經由改質後與卵白素(Neutravidin)鍵結，再結合以生物素修飾之單株抗體(GP-Av-bmAb)，以達到標的效果。經過多層修飾後的明膠奈米顆粒並無顯著差異，其顆粒大小為240 nm、表面電位約為- 4 mV，在細胞實驗中，也證明對於正常人類肺細胞沒有顯著的毒性影響。以EGFR 過度表現之癌細胞做為標的效果測試，在共軛焦顯微鏡的觀察下，可發現人類肺腺癌細胞，A549，以GP-Av-bmAb 顆粒培養有較多的綠色螢光分佈，因此確認於細胞階段可有效標的EGFR過度表現細胞。

Lung cancer is the most common disease in developed countries, also in the predominant place of cancer-related death worldwide. Although medical technology is more progressive than before, the long-term survival rate of lung cancer patients is still far from satisfactory. In order to improve treatment effectively, the most widely investigated field is targeted therapy. Epidermal growth factor receptor (ErbB1, EGFR) is a trans-membrane protein which is overexpressed in a variety of human lung cancer cells, since it can be considered as a rational target for drug delivery. This thesis is divided into two parts. The first part is comparison between binding affinities of EGFR and its related ligands which are including EGF, anti-EGFR antibody (mAb LA1) and peptide GE11. Surface plasmon resonance (SPR) was used to get some kinetics information such as rate constants and equilibrium constants. Atomic force microscopy (AFM) was performed to detect unbinding forces between receptor and ligands. The results from SPR experiments revealed each equilibrium dissociation constants of EGF, mAb LA1, and peptide GE11 are 1.77 × 10-7,2.07 × 10-9,4.59 × 10-4 M-1, respectively. Among these three ligands, mAb LA1-EGFR showed the strongest binding affinity, peptide GE11 showed the weakest affinity. In AFM experiments, the unbinding forces of EGF-EGFR and mAb LA1-EGFR are both 150-210 pN under loading rate ranging from 450 to 5000 pN/s. However, unbinding force of peptide GE11 was smaller than them by 3-fold, approximately 50-60 pN (loading rate range: 150-7500 pN/s). It indicated that the unbinding force of peptide GE11 was still the smallest. Since mAb LA1 showed the strongest binding affinity to EGFR and not caused cell proliferation problems, we can use it as the efficient targeting moiety for our drug delivery system in lung cancer treatment. In the second part, gelatin nanoparticles (GPs) were prepared firstly by two-step desolvation method. And size of particles was about 230 nm, net surface charge was about -3 mV. After modification, surfaces of gelatin nanoparticles were covalently bound with NeutravidinFITC, which can then linked with biotinylated mAb LA1 due to the non-covalent interactions of avidin and biotin. The EGFR-seeking nanoparticles (GP-Av-bmAb) had no significant difference between original gelatin nanoparticles and showed 240 nm in diameter, -4 mV in net surface charge. By the in vitro test, GP-Av-bmAb performed no apparently cytotoxicity effect in human normal lung cells (HFL1). The cell uptake of GP-Av-bmAb to the EGFR-overexpressed lung adenocarcinoma cells, A549 cells, was performed by confocal microscopy. The results indicated that GP-Av-bmAb possessed efficient targeting ability to A549 cells and can be considered as a good drug carrier in lung cancer therapy.