發展針對人類程序性死亡配體-1的奈米抗體

Abstract

目前對於獲得抗藥性帶有突變表皮生長因子受體非小細胞肺癌，酪氨酸激酶抑製劑治療的療效不彰。因此，在臨床上迫切需要開發新的癌症治療方法。程序性死亡蛋白-1 (programmed death protein 1, PD-1)/程序性死亡配體-1 (programmed death-ligand 1, PD-L1) 靶向免疫檢查點抑制劑為癌症治療提供了新希望。PD-L1是一種在癌細胞表面表達的膜結合配體。PD-L1與其受體PD-1結合，在T細胞上表達訊息，進而抑制 T 細胞活性。腫瘤細胞會透過PD-L1/PD-1抑制T細胞，干擾抗癌免疫來進行免疫逃避。 PD-L1在臨床上可作為一種生物標誌物，預測多種腫瘤類型中病患對 PD-1/PD-L1 阻斷治療的反應。然而，PD-L1 胞外結構域的重度醣基化影響了 FDA 批准的 PD-L1 抗體在免疫組織化學檢測的準確性。對 PD-L1表現量的錯誤評估，導致對 PD-1/PD-L1 阻斷治療的治療反應的預測不佳。此外，PD-L1 也存在於細胞外囊泡表面，稱為外泌體 PD-L1 (exosomal PD-L1)。越來越多的證據發現外泌體 PD-L1 會影響全身 T 細胞所參與的免疫反應，促進腫瘤發展。然而，外泌體 PD-L1 的調控機制尚不完全清楚，追蹤PD-L1運輸的工具也尚待發展建立。因此，我們計劃開發對針對醣基化人類PD-L1的奈米抗體，使臨床樣本的檢測能更精準，並且應用於研究PD-L1。與傳統的IgG單株抗體相比，奈米抗體是一種小型的單變異區抗體。除了體積小外，奈米抗體具有更好的水溶性，在極端環境中也相對穩定以及對蛋白酶具有較好的抵抗性等優點。在這項研究中，我們從奈米抗體的合成庫中篩選出了辨認PD-L1的奈米抗體，並獲得了兩株奈米抗體 (Nb 4-9 和 Nb 15H)。另外，我們製造了重組PD-L1 Nb 4-9-Fc融合蛋白 (Nb 4-9-Fc)，在未來將測試其應用於免疫組織化學染色測定法 (IHC) 的可能性。我們將進一步測試開發的PD-L1奈米抗體是否可作為改善PD-L1檢測和探討癌細胞中PD-L1運輸機制的工具。

The acquired resistance of non-small cell lung cancer (NSCLC) harboring mutant epidermal growth factor receptor (EGFR) limits the treatment efficacy of tyrosine kinase inhibitors (TKIs). Therefore, it is urgent to develop new therapies for NSCLC. Programmed death protein 1 (PD-1)/programmed death-ligand 1 (PD-L1)-targeted immune checkpoint inhibitors provide new hope for cancer treatment. PD-L1 is a membrane-bound ligand expressed on the surface of cancer cells. PD-L1 is associated with PD-1 expressed on T cells to suppress T cell activity. Tumor cells adapt the PD-1/PD-L1 pathway to evade the immune system by inhibiting T cell-mediated anti-cancer immunity. Therefore, PD-L1 can be used as a biomarker to predict the patient response to PD-1/PD-L1 blockade treatment in multiple tumor types. However, the heavy glycosylation of the PD-L1 extracellular domain hinders immunohistochemistry detection using FDA-approved PD-L1 antibodies. Inaccurate assessment of PD-L1 levels in clinical biopsies leads to the poor prediction of therapeutic response of PD-1/PD-L1 blockade treatment. In addition, PD-L1 also exists on the surface of extracellular vesicles (EVs), known as exosomal PD-L1. Growing evidence showed that exosomal PD-L1 influences systemic T cell-mediated immunity to promote tumor progression. However, the regulatory mechanisms of exosomal PD-L1 are not fully understood, and the PD-L1 tracking tool is unavailable. Therefore, we plan to develop anti-glycosylated human PD-L1 nanobodies for better detecting abilities in clinical diagnosis and research application. Nanobody is a small-size single variable domain antibody compared to conventional monoclonal IgG antibodies. In addition to the small size, nanobodies have better solubility and stability in extreme environments and resistance to proteases. In this study, we screened PD-L1 nanobodies from the synthetic nanobody library and got two candidates (Nb 4-9 and Nb 15H). We further fused PD-L1 Nb 4-9 with a fragment crystallizable (Fc) region for immunohistochemical (IHC) assay purposes. We will test whether the anti-human PD-L1 Nb 4-9 and 15H can be used to improve the detection of PD-L1 in clinical samples and study the PD-L1 trafficking in cancer cells.