腫瘤微環境 (tumor microenvironment，TME) 已被視為癌症進展 (progression) 與腫瘤轉移的關鍵構成因素。TME 主要由腫瘤細胞與非腫瘤細胞組成，非腫瘤細胞包含纖維母細胞、內皮細胞以及免疫細胞如巨噬細胞。在 TME 中，被稱作 TAMs (tumor-associated macrophages) 的 M2 巨噬細胞深受 TME 影響，且 TAMs 與腫瘤的生長與侵入 (invasion) 有關。固態腫瘤中 TAMs 可構成超出 50% 的腫瘤滲入細胞。TAMs 的增加和病人較差的預後與較差的存活率有關。Macrophage CSF-1R (macrophage colony-stimulating factor-1 receptor) 高度表現於 M2 巨噬細胞，CSF-1R 會被其配體 CSF-1 活化進而刺激細胞的增殖 (proliferation)。過度表現 (overexpression) 的 CSF-1R 與 M2巨噬細胞的增殖、分化 (differentiation)、移動 (migration) 以及生存能力有關。且滲入腫瘤的 TAMs 的增加 (accumulation) 與聚集 (recruitment) 皆由巨噬細胞的 CSF-1R 的訊息傳遞路徑所調控──自 CSF-1 的結合為起點，最終促成腫瘤生長，故 CSF-1R 相關訊息傳遞路徑可視為抗腫瘤處置的有效切入點。本研究利用融合瘤細胞技術產出對 CSF-1R 具專一性結合能力且對 CSF-1 誘導的 CSF-1R 訊息傳遞路徑具中和 (neutralize) 能力的單株抗體，並且測試了三株單株抗體 (命名為 35H、210H、13F) 的能力，其中包括對細胞表面的 CSF-1R 的結合與對 CSF-1R 的雙體化 (dimerization) 導致的自體磷酸化 (autophosphorylation) 的抑制，也測試了本研究中的單株抗體 (35H、210H) 對 CSF-1R 的結合常數 (binding constants)，得到單株抗體具有中等結合能力 (~10-8 µM) 的結果，將來可進一步利用 affinity maturation 進行結合能力的改良。綜上所述，本研究製作出了三種對 CSF-1R 訊息傳遞路徑具有功能性抑制的單株抗體。為了使本研究的單株抗體具更廣泛的應用，優化抗體的結合能力並進行antibody engineering 進行 humanization 是未來可進一步發展的工作。

Tumor microenvironment (TME) has been recognized as a key contributor for cancerous disease progression and tumor metastasis. TME consists of tumor cells and the non-tumor cells including stromal fibroblasts, endothelial cells, and immune cells such as macrophages. In TME, polarized M2 macrophages called tumor-associated macrophages (TAMs) are educated by TME and associated with tumor progression and invasion. In solid tumors, TAMs have been shown to contribute more than 50% of the tumor-infiltrated cells. The increase of TAMs is associated with a poor prognosis and lower survival rates. Macrophage colony-stimulating factor-1 receptor (CSF-1R) activated by its ligand CSF-1 to stimulate cell proliferation is highly expressed on M2 macrophages. Overexpression of CSF-1R associates with the proliferation, differentiation, migration and survival of M2 macrophages. In addition, infiltration and recruitment of TAMs into the tumors are mediated by the CSF-1R signaling activation initiated by CSF-1 binding, and consequently facilitate tumor growth. Therefore, targeting CSF-1R-associated signaling provides a promising target for the anticancer treatments. By applying hybridoma technology, we produced monoclonal antibodies that specifically bind to CSF-1R and neutralize CSF-1R signaling induced by CSF-1, respectively. We have extensively characterized these hybridoma clones (named 35H, 210H, and 13F) for their activities of cell-surface binding and inhibition of autophosphorylation the receptors resulted from dimerization of CSF-1R. We also estimated the binding constants of anti-CSF-1R monoclonal antibodies with moderate levels of binding affinities (~10-8 µM), suggesting a need for affinity maturation. In summary, we have identified three functional inhibitory monoclonal antibodies against CSF-1R signaling in vitro. Further investigation including improvement of their binding affinities and antibody engineerings such as humanization for wider application are warranted.

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