將促進腫瘤生長之細胞激素轉化為毒殺腫瘤之特洛伊木馬: 研發以腫瘤壞死因子-α作為媒介之細胞內癌症藥物運輸抗體載體

Abstract

近年來，抗體藥物複合體在癌症的治療上得到十分顯著的效果。然而，鑑定出得以使抗體藥物複合體順利地被內化進入腫瘤細胞之抗原往往成為抗體藥物複合體研發的主要瓶頸之一。腫瘤壞死因子-α，做為一個參與包括發炎反應、細胞生長、細胞凋亡，許多不同生理現象的細胞激素，已經被證明在腫瘤細胞與腫瘤微環境中之骨髓性細胞裡過量表現，並藉由自分泌與旁分泌的方式結合至腫瘤細胞上之受體，進而促進腫瘤細胞的存活、生長、與轉移。明確來說，當結合上其受體，腫瘤壞死因子-α會引發配體與受體複合體的內化，進而將整個複合體帶入細胞內，從而活化下游訊息傳遞，此現象暗示了以腫瘤壞死因子-α作為媒介，針對腫瘤細胞進行帶有選擇性的細胞內藥物運輸之可能性。進一步的研究也證明利用針對腫瘤壞死因子-α之中和性抗體，得以在臨床前動物實驗當中增強免疫抑制劑療法的治療效果，顯示出針對腫瘤壞死因子-α路徑作為抑制標的，在癌症治療當中的潛力。因此，在此篇研究當中我們提出假設，利用一株抗腫瘤壞死因子-α的內化性抗體，同時結合抗體藥物複合體的技術，得以將抗體做為一個細胞內藥物運輸的載體，在癌症治療中針對腫瘤組織發揮具有專一性的毒殺效果。利用噬菌體展現與酵母菌展現技術，我們成功分離出得以結合抗腫瘤壞死因子-α的高親和力抗體，y#2。進一步的細胞實驗確認了抗體的非中和特性，及其在結合腫瘤壞死因子-α與其受體後被黑色素瘤細胞內化的現象。在後續的腫瘤細胞毒殺實驗當中，接有抗癌藥物DM1與免疫毒素Pseudomonas exotoxin A的y#2抗體藥物複合體得以對黑色素瘤細胞產生對腫瘤壞死因子-α具專一性的抑制效果。然而特別的是，我們所分離出之抗體y#2並無法在有腫瘤壞死因子-α之情況下結合至其他腫瘤細胞，甚至是表現有全長腫瘤壞死因子-α受體的細胞。此現象促使我們將已初步對腫瘤壞死因子-α篩選過之酵母菌展現抗體庫，利用更貼近實際配體受體結合之生理環境的方法再次分選。初步結果顯示有多株抗體具有以高親和力結合上與受體作用之腫瘤壞死因子-α的潛力，後續則尚需要透過進一步細胞與動物實驗確認抗體的功能性與療效。綜合以上結果，我們證明了以抗腫瘤壞死因子-α的內化性抗體藥物複合體做為癌症治療的可能性，同時提出了利用抗體載體在細胞內藥物運輸上一個創新的治療策略。

Antibody-drug conjugate-based therapy has achieved a great success in recent years. However, the difficulties in the identification of competent tumor-specific neoantigens that license the intracellular uptake of the antibody-drug conjugate into tumor cells has become a major issue. TNF-α, a pleiotropic cytokine that participates in the regulations of inflammation, cellular apoptosis, and proliferation, has been defined to be highly-expressed and essential for the tumoral survival, proliferation, and metastasis in various types of tumor cells. Through the binding to the receptor, TNF-α induces the internalization of the ligand-receptor complex into the target cells, which provides a potential TNF-α-mediated intracellular drug delivery approach for the selective cytotoxicity against tumors. Besides, previous studies that incorporated TNF-α-neutralizing antibodies into the combinational therapy had shown improved immune checkpoint blockade-dependent efficacy, suggesting the anti-tumor effects of inhibiting TNF-α signaling axis in cancer therapy. Thus, in this study, we aimed to design an anti-TNF-α antibody vehicle that selectively and intracellularly delivers the cytotoxic payload into tumor cells. The high-affinity antibody, y#2, was successfully isolated by phage and yeast surface display. Cell-based assays revealed the noncompetitive and internalization property of the antibody. DM1- and Pseudomonas exotoxin A-conjugated antibody induced TNF-α-dependent cytotoxicity against melanoma cells in vitro. However, cytometric analysis demonstrated the impaired binding of y#2 against other types of tumor cells and, surprisingly, the full-length mTNFR1 expressing cells, which prompted us to employ a more physiologically relevant selection method, in terms of the ligand-receptor interaction, on the pre-enriched antibody yeast library. In preliminary results, multiple potential high-affinity binders against TNF-α-TNFR1 complex were isolated. Further in vitro and in vivo assays are still required to characterize the functional property and the anti-tumor efficacy of the selected antibodies. Based on these results, we concluded that tumoral TNF-α may present a novel and viable target for the therapeutic intracellular drug delivery in cancers and provided an innovative concept on the utilization of antibody vehicles.