本實驗室已成功地篩選專一性結合腸癌之新穎標的胜肽pHCT74，相關研究成果已發表於頂尖轉譯醫學期刊Science Translational Medicine，受到審查委員高度肯定，並認為對於癌症治療是一重大突破。期刊出版後也被重要的生技期刊Biocentury專訪，獲得高度的評價，認為未來的發展極具市場價值。此標的微脂體明顯抑制腫瘤的生長及大幅提升其存活率。在標靶抗癌藥物傳輸系統研究過程中，我們發現此新一代標靶微脂體比原來的微脂體藥物更能有效地將抗癌藥物艾黴素(doxorubicin)以及溫諾平(vinorelbine)共同傳輸至腫瘤部位，並在腫瘤部位累積更高劑量的藥物。故此標靶藥物傳輸系統明顯對腫瘤更具療效且能大幅降低副作用，也改善了目前微脂體藥物的缺點。

此外，我們也已成功鑑定出pHCT74胜肽分子靶標蛋白是α-enolase (ENO1)。ENO1的表現與許多癌症的存活率降低和預後不良顯著相關。然而，ENO1在癌症發生中的功能仍然是未知的。我們發現ENO1的高量表達存在於高轉移性肺癌細胞系中，並且與肺癌患者的較差存活率有相關性。在細胞及動物實驗中，觀察到ENO1可以增加癌細胞增殖和侵襲力能力。ENO1除了可以降低上皮細胞標誌蛋白E-cadherin的表現量之外，還可以增強間質細胞標誌蛋白N-cadherin和vimentin的表現量，以及上皮細胞間質轉化調節因子SLUG的蛋白質表現量。此外，ENO1可通過上調Wnt共受體LRP5/6和降低GSK-3β活性來激活Wnt訊號傳遞。在機轉方面，ENO1與肝細胞生長因子受體 (HGFR) 相互作用並藉由增加HGFR和Wnt共同受體 LRP5/6 的磷酸化，激活HGFR和Wnt訊號。這些訊號軸的激活透過 Src-PI3K-AKT 訊號傳遞和β-連環蛋白破壞複合物的失活降低了 GSK-3β 的活性，最終上調了 SLUG 和 β-連環蛋白(如下圖)。我們進一步研發出抗ENO1的治療性單株抗體，chENO1-22，此抗體可以降低癌細胞的增殖和侵襲能力。chENO1-22 透過抑制ENO1介導的 GSK3β 失活以促進 SLUG 蛋白泛素化和降解來減弱癌細胞侵襲。此外，chENO1-22 在動物模型中預防肺腫瘤轉移並延長實驗動物的壽命 (如下圖)。總結，這些發現闡明了 ENO1 在肺癌轉移中扮演的功能及分子機制，並證明針對 ENO1 的新型抗體用以治療肺癌的潛力。

我們進一步將chENO1-22進行人源化，以降低免疫原性並提升臨床應用潛力，開發出人源化單株抗體huENO1-22-1，並藉由噬菌體顯示技術自人類抗體庫中成功篩選出人類(全人源)抗ENO1抗體株hENO1-14。隨後，我們對hENO1-14進行親和力成熟工程，產生親和力更佳的hENO1-14-AF3。經體外與小鼠實驗證明，hENO1-14-AF3能有效抑制人類肺癌細胞的增殖與轉移，並顯著延長存活時間。整體治療過程中未觀察到明顯的毒性或副作用，顯示其具備良好的安全性與臨床應用潛力。此外，經Epitope mapping分析，我們發現hENO1-14及與hENO1-14-AF3對ENO1有全新結合表位，與目前進行臨床試驗之競爭抗體所標靶的區域明顯不同，這項特性有助提升專利佈局的優勢與商業化潛力。

另一方面，我們發現ENO1的過度表現不僅促進腫瘤侵襲，亦能誘導肺癌細胞獲得幹細胞樣特性，顯著增加幹細胞關鍵基因如Oct4、Sox2與c-Myc的表達，這一過程主要由Wnt/β-catenin訊號途徑所驅動。當以hENO1-14或hENO1-14-AF3處理肺癌細胞時，可有效抑制上述基因的表達與癌幹細胞性狀的形成。我們推測這兩株抗體之作用機轉（MOA）除可阻斷HGFR與Wnt訊號軸外，亦可能下調Yamanaka因子，藉此抑制肺癌幹細胞，進而減緩腫瘤轉移與惡化。近期，我們接續改良hENO1-14-AF3，研製出兼具結合人類ENO1與交叉辨識小鼠ENO1能力的新抗體hENO1-14-AF5，現正進行臨床前毒理學試驗，以全面評估其安全性與後續發展潛力。

（105年度TBF學術講座、中研院生醫轉譯研究中心　吳漢忠特聘研究員）

ENO1 promotes lung cancer metastasis via HGFR and WNT signaling-driven epithelial-mesenchymal transition

We have successfully concocted a targeted drug by conjugating these peptides to liposomes. This novel targeted liposomal doxorubicin and vinorelbine combination could accurately deliver chemotherapeutics to tumors, resulting in a much higher dose of drugs being accumulated at the tumor site. This significantly increased the tumor inhibition abilities of these two types of chemotherapeutic drugs and effectively eliminated cancer without inducing side effects for 120 days, with no trace of recurrence. These significant discoveries were published in the top ranking journal, Science Translational Medicine. The results were highly regarded as an important breakthrough in cancer therapy. Biocentury, the leading source of biotech/pharmaceutical industry newsletter, dedicated an entire article in their Innovations section introducing our research results, with special highlight on its effect at curing cancer.

 We have successfully identified the molecular target of the pHCT74 peptide to be α-enolase (ENO1). ENO1 expression is significantly correlated with reduced survival and poor prognosis in many cancer types, including lung cancer. However, the function of ENO1 in carcinogenesis remains elusive. In one study, we found that high expression of ENO1 is present in metastatic lung cancer cell lines and malignant tumors and is associated with poor overall survival of lung cancer patients. Knockdown of ENO1 decreases cancer cell proliferation and invasiveness, whereas overexpression of ENO1 enhances these processes. Moreover, ENO1 expression promotes tumor growth in orthotopic models and enhances lung tumor metastasis in tail-vein injection models. These effects are mediated by upregulation of mesenchymal markers N-cadherin and vimentin and the epithelial-mesenchymal transition (EMT) regulator SLUG, along with concurrent downregulation of E-cadherin. Mechanistically, ENO1 interacts with hepatocyte growth factor receptor (HGFR) and activates HGFR and Wnt signaling via increased phosphorylation of HGFR and the Wnt co-receptor LRP5/6. Activation of these signaling axes decreases GSK-3β activity via Src-PI3K-AKT signaling and attenuates inactivation of the β-catenin destruction complex to ultimately upregulate SLUG and β-catenin. Additionally, we have generated a chimeric anti-ENO1 monoclonal antibody (chENO1-22) that can decrease cancer cell proliferation and invasion. chENO1-22 attenuates cancer cell invasion by inhibiting ENO1-mediated GSK3β inactivation to promote SLUG protein ubiquitination and degradation. Moreover, chENO1-22 prevents lung tumor metastasis and prolongs survival in animal models (Figure). Taken together, these findings illuminate the molecular mechanisms underlying the function of ENO1 in lung cancer metastasis and supports the therapeutic potential of a novel antibody targeting ENO1 for treating lung cancer.

Furthermore, we humanized chENO1-22 to reduce its immunogenicity and improve its therapeutic usefulness, resulting in the development of the humanized Ab huENO1-22-1. Using phage display screening with human naïve antibody libraries, we successfully identified hENO1-14, a fully human antibody targeting ENO1. Subsequently, affinity maturation of hENO1-14 yielded a higher-affinity version, hENO1-14-AF3. Both in vitro and in vivo studies, including mice models bearing human lung cancer, demonstrated that hENO1-14-AF3 effectively inhibited tumor cell growth and metastasis while significantly prolonged survival. No apparent toxicity or adverse effects were observed during treatment, highlighting the antibody’s favorable safety profile and therapeutic potential. Epitope mapping indicated that hENO1-14 and hENO1-14-AF3, recognize a new epitope on ENO1 that is distinct from the epitope targeted by a competing anti-ENO1 antibody currently under clinical trials. This unique binding specificity not only improves the antibody's potential efficacy, but also provides competitive advantages in regard to intellectual property and future commercialization opportunity.

The mechanism of action (MOA) of anti-ENO1 antibodies involves the suppression of ENO1 overexpression-induced acquisition of cancer stem cell-like properties in lung cancer cells. This is achieved partly through inhibition of the Wnt/β-catenin signaling pathway and downregulation of critical stemness-associated genes such as Oct4, Sox2, and c-Myc. Treatment with hENO1-14 or hENO1-14-AF3 significantly decreased the expression of these genes and impaired the formation of spheroid sphere enriched with lung cancer stem cells. Collectively, the MOA of these antibodies likely includes disruption of the HGFR-Wnt signaling axis and suppression of Yamanaka factors, resulting in reduced cancer stemness, diminished metastatic capability, and improved therapeutic efficacy. To further advance preclinical development, we engineered hENO1-14-AF5, a derivative of hENO1-14-AF3, that maintains high affinity for human ENO1 while acquiring cross-reactivity with murine ENO1, an important characteristic for preclinical evaluation in mouse models. hENO1-14-AF5 is currently conducting preclinical toxicity studies to ensure its safety and support future clinical translation and technology transfer.

(2016 TBF Chair in Biotechnology, Dr. Han-Chung Wu)