溶瘤病毒(Oncolytic viruses)因其固有的腫瘤選擇性和細胞殺傷能力，作為創新的癌症治療手段已引起了廣泛關注。雖然美國食品藥物管理局(U.S. Food and Drug Administration)已批准病毒療法作為一種腫瘤內免疫療法，但單獨使用溶瘤病毒由於體內分佈(In vivo biodistribution)不佳，導致臨床效果僅屬普通。這一局限性促使人們對「組合策略」產生興趣，其靈感來自抗體-藥物複合體(Antibody-Drug Conjugate)等標靶療法，即在遞送腫瘤特異性細胞毒性的同時，將化療劑量降至最低。延伸此一概念，病毒-藥物複合體(Virus-Drug Conjugates, VDCs)具有提升治療效果的潛力。例如，將轉導增強劑(Transduction-enhancing agent)連接到對目標細胞具有天然特異性的病毒上，從而促進它們的共同遞送，並可能增加治療的整體影響。雖然Doxorubicin (Dox)通常作為化療藥物，透過造成DNA鏈斷裂來抑制細胞增生，但使用極低細胞毒性劑量的Dox卻能反常地放大病毒轉導的速率與效率。

活化T細胞上的程序性細胞死亡蛋白-1 (Programmed cell death protein 1, PD-1)與其配體程序性死亡-配體 1 (Programmed death-ligand 1, PD-L1)之間的交互作用，形成了一個調節T細胞活性的關鍵抑制性檢查點。許多癌症利用此路徑過度表達PD-L1，使其能逃避免疫偵測並抑制抗腫瘤免疫反應。因此，鎖定PD-L1/PD-1已成為癌症免疫療法的重點。抑制癌症相關基因的一個潛力方法是RNA干擾(RNA interference, RNAi)，即利用小干擾RNA (small-interfering RNAs, siRNAs)來抑制基因表現。

雖然siRNAs在癌症治療中展現潛力，但如何有效遞送仍是一大挑戰。值得注意的是，利用DNA編碼的短髮夾RNA (Short hairpin RNAs, shRNAs)是產生小RNA觸發因子的有效方法。重組腺相關病毒(Recombinant adeno-associated virus, AAV)載體約20 nm，是治療多種人類疾病的基因遞送平台。AAV雖已獲授權臨床使用，但仍受限於生物分佈不佳的問題。利用AAV作為shRNAs的遞送載體，為siRNA療法提供了一個有前景的替代方案。

綜合上述，我們開發了一種治療策略，將編碼PD-L1 shRNA的AAV血清型第二型(AAV2)與Dox結合(AAV2-Dox)。與一般合併療法不同，這確保了藥物與病毒利用相同的受體介導內吞路徑。此方法增強了癌細胞中PD-L1 shRNA的表現，導致其表面PD-L1被抑制(Knockdown)。PD-L1水平的降低重新活化了T細胞，使其能啟動腫瘤特異性的免疫反應。此外，腫瘤微環境(Tumor microenvironment)獨特的物理化學特徵如缺氧和微酸性pH (6.7 – 7.0)不僅促進癌細胞進展，也為增強病毒療法的特異性與功效提供了關鍵標靶目標。為了建立響應腫瘤微環境的pH遞送系統，我們利用pH敏感的苯甲酸亞胺(Benzoic imine)連接體，策略性地將具隱形特性的聚乙二醇(Polyethene glycol, PEG)連接到AAV2-Dox (圖一)。此連接體作為一種環境感測器：苯甲酸亞胺的動態共價鍵在健康組織典型的中性pH條件下保持穩定，使PEG能在系統循環中屏蔽VDC。然而，該連接體在實體腫瘤常見的酸性環境中是不穩定的。暴露於較低pH值會觸發連接體的水解斷裂，隨後使PEG屏蔽脫落。這種腫瘤微環境特異性的活化，使AAV2-Dox複合體精準地在目標位置「去屏蔽」，促進癌細胞的感染及其治療成分的局部釋放。這種整合式、pH活化的VDC方法可經工程設計以克服系統性遞送的限制，並在改善癌症病毒療法的標靶上極具潛力。

（110年TBF吳火獅醫學獎、台大藥理學科暨藥理學研究所　曾士傑副教授）

pH-Responsive Virus-Drug Conjugates for Targeted PD-L1 Silencing and Enhanced Cancer Immunotherapy

The inherent tumor selectivity and cell-killing capabilities of oncolytic viruses have generated considerable interest in their development as innovative cancer treatments. While the U.S. Food and Drug Administration (FDA) have approved virotherapy as an intratumoral immunotherapy, oncolytic viruses alone have yielded only modest clinical benefits due to poor biodistribution. This limitation has spurred interest in combination strategies, drawing inspiration from targeted therapies like antibody-drug conjugates (ADCs), which deliver tumor-specific cytotoxicity while minimizing chemotherapy dosage. Extending this concept, virus-drug conjugates (VDCs) could potentially boost therapeutic effectiveness. For example, by linking a transduction-enhancing agent to viruses with a natural specificity for target cells, thereby facilitating their co-delivery and potentially increasing the overall impact of the treatment. While doxorubicin (Dox) typically acts as a chemotherapeutic by causing DNA strand breaks to inhibit cell proliferation, using a minimally cytotoxic Dox dosage can paradoxically amplify the rate and efficiency of viral transduction.

The interaction between programmed cell death protein 1 (PD-1) on activated T cells and its ligand, programmed death-ligand 1 (PD-L1), forms a crucial inhibitory checkpoint that regulates T-cell activity. Many cancers exploit this pathway by overexpressing PD-L1, allowing them to evade immune detection and suppress anti-tumor immune responses. Targeting PD-L1/PD-1 signaling has become a major focus in cancer immunotherapy. One promising approach for silencing cancer-related genes is RNA interference (RNAi), where small-interfering RNAs (siRNAs) are used to suppress gene expression. While siRNAs have shown potential in cancer therapy, delivering them effectively remains a challenge. Synthetic siRNA duplexes and plasmid-mediated siRNAs suffer from limitations such as short half-life, low transfection efficiency, and cytotoxicity associated with the delivery process. These barriers have hindered their clinical application. Notably, an efficient method for generating small RNA triggers involves using DNA-encoded short hairpin RNAs (shRNAs).

Recombinant adeno-associated virus (AAV) vectors, measuring approximately 20 nm, are a leading nanoplatform for gene delivery in the treatment of various human diseases. AAV products have gained authorization for clinical use, but they continue to struggle with poor biodistribution. Leveraging AAVs as delivery vectors for shRNAs offers a promising alternative to siRNA-based therapies. Taken together, we developed a therapeutic strategy conjugating AAV serotype 2 (AAV2) encoding PD-L1 shRNA with Dox treatment (AAV2-Dox). Unlike combination therapies, this ensured that the drug and virus utilize the same receptor-mediated endocytosis pathway. This approach enhanced the expression of PD-L1 shRNA in Dox-treated cancer cells, resulting in the knockdown of PD-L1 on their surface. The reduction in PD-L1 levels reactivated T cells, allowing them to initiate a tumor-specific immune response. Additionally, the distinct physicochemical characteristics of the tumor microenvironment—such as hypoxia and mildly acidic pH (6.7–7.0)—not only promote cancer progression but also offer key targets for enhancing the specificity and efficacy of virotherapy. To create a pH delivery system responsive to the tumor microenvironment, we attached polyethene glycol (PEG) with stealth properties to the AAV2-Dox conjugate strategically utilizing a pH-sensitive benzoic imine linker (Figure 1). This linker acted as an environmental sensor: the benzoic imine's dynamic covalent bond is stable under neutral pH conditions typical of healthy tissues, allowing the PEG layer to shield the VDC systemically. However, the linker is labile in the acidic environment often found in solid tumors. Exposure to this lower pH triggers hydrolytic cleavage of the linker and subsequent detachment of the PEG shield. This tumor microenvironment-specific activation unmasks the AAV2-Dox conjugate precisely at the target site, promoting enhanced uptake by cancer cells and localized release of its therapeutic components. Furthermore, AAV transduction involves viral entry via endocytosis, subsequent movement to the nucleus, capsid uncoating in the nucleolus, and final release of the viral genome to express the transgene. Concurrently, the transduction-enhancing agent Dox with minimal cytotoxicity has been released from the AAV2-Dox conjugate within the nucleus. This integrated, pH-activated VDC approach could be engineered to overcome limitations of systemic delivery and holds significant promise for improving the targeted efficacy of cancer virotherapy.

(2021 TBF Wo Ho-Su Medical Award, Associate Professor S.-Ja Tseng)