- 學位論文
人類化雙功能抗體(mPEG×CD20)非共價鍵修飾 於甲氧基聚乙二醇奈米藥物來提升 B 細胞淋巴癌的選擇性和腫瘤療效
Non-covalent Modification of mPEGylated Nanodrugs with Anti-PEG Bispecific Antibody (mPEG×CD20) to Enhance Tumor specific Targeting and Therapeutic Efficacy in B cell Lymphoma
摘要
修飾甲氧基聚乙二醇之微脂體(mPEGylated Nanoparticles； mPEG-NPs)目前常被應用於癌症奈米藥物，例如 Lipo-Dox，微脂體表 面修飾親水性長鏈的 PEG，提升藥物之生物相容性以提高半衰期，但 在血液中缺乏腫瘤專一性，對一般細胞造成毒性、病患免疫力下降， 並且藥物無法主動內化作用進入細胞，無法提升治療效果。修飾抗體 於 mPEG-NPs 上被報導可提升藥物腫瘤專一性及藥物內化作用。目 前常使用之化學修飾抗體的方式受限於抗體修飾的方向性及穩定性， 可能使得抗體的功能降低。我們創新簡單的修飾方法，設計出人類化 雙功能抗體(mPEG×CD20)，一端能結合mPEG-NPs上的mPEG分子； 一端能辨認 B 細胞淋巴癌上過度表達的 CD20 分子，改善較簡易的修 飾方式，亦能達到增強 mPEG-NPs 對腫瘤的專一性及增加藥物內化 作用的優點。我們確認 mPEG×CD20 具備雙邊功能，以一步製成(Onestep mixing)之非共價鍵方式修飾於 Lipo-Dox，使得 Lipo-Dox 轉化為 具抗 CD20 的標靶微脂體藥物(αCD20/Lipo-Dox)，確立 αCD20/LipoDox 的最佳修飾比例(mPEG:bsAb = 40:2)後，證實 αCD20/Lipo-Dox 具 備藥物腫瘤專一性、只靶向 CD20+之 B 細胞淋巴癌細胞(Raji)，內化 能力也隨時間增加而提升、相較於 Lipo-Dox 之細胞毒性也提升 15.2 倍。此外，更進一步證實 αCD20/Lipo-DiD 在活體中能夠專一性累積 在小鼠皮下的 B 細胞淋巴癌腫瘤區，相較於無腫瘤專一性之奈米造 影劑提升了 300 倍的訊號。在小鼠腫瘤模式中，顯著的提升抗腫瘤的 活性、延長小鼠約 90%的存活率。因此，mPEG×CD20 以一步製成的 非共價鍵修飾與 mPEGylated nanodrugs 結合，不但提升藥物對腫瘤的 專一性、增強藥物對腫瘤細胞內化的能力及細胞毒性，亦提升活體的 存活率。期望能提升現今 B 細胞淋巴癌的治療療效，未來亦能置換不 同辨認癌細胞端的抗體或修飾 mPEG 的藥物，便能運用在其他治療 上，使用更廣泛
並列摘要
Methoxy PEGylated nanoparticles (mPEG-NPs) are increasingly used for cancer therapy, such as Lipo-Dox. Because hydrophilic long-chain mPEG could be used to modify surface of liposomal nanodrugs to protect drug molecules from prolonging the half-life in circulation. However, mPEG-NPs are still non-specific to tumor cells, and result in toxicity to normal cells, leading to limited drug uptake by cells and impaired immunity of patients. It couldn’t improve the therapeutic effect. Recently, the antibody-modified mPEG-NPs (immunoliposome) could enhance the specificity and internalization of drugs. But the chemical modification is affected by directionality and stability of the antibody modification, which results in the decline of antibody function and stability of the drug. Here we described a simple modified method that could enhance the specificity and internalization of mPEG-NPs. That is humanized bispecific antibody (mPEG×CD20) which can simultaneously bind to both mPEG molecules t mPEG-NPs and CD20 molecules overexpressed by the lymphoma cells. Improving the modification method could enhance the specificity of mPEG-NPs and increase the internalization of drugs. We confirmed that the bifuctional mPEG×CD20 could be non-covalent modified of Lipo-Dox with mPEG×CD20 transformed the Lipo-Dox into CD20 targeted liposomes (αCD20/Lipo-Dox). After establishing the optimally modified ratio of αCD20/Lipo-Dox (mPEG: bsAb = 40:2), we confirmed that both anti-CD20 and anti-PEG have binding activities to their corresponding antigens. αCD20/Lipo-Dox could induce receptor-mediated internalization in Raji B cell lymphoma cells, and it also increased 15.2-fold cytotoxicity compared to Lipo-Dox. Moreover, αCD20/Lipo-Dox could specifically target to cancer cells and significantly have higher anti-tumor activity against Raji tumors rather than Lipo-Dox in Raji-bearing mice. In the therapeutic model, the survival rate of the mouse was prolonged by about 90%. Therefore, the non-covalent modification of Lipo-Dox with iv mPEG×CD20 is a simple method for tumor specificity, increasing drug internalization, enhancing the anti-cancer efficacy and improve survival rate against CD20 overexpressing B cell lymphoma cancer. We expected that it can significantly improve the therapeutic efficacy of B cell lymphoma, and in the future, it can replace different antibodies that recognize cancer cells or drugs that modify mPEG, and can be used in other treatments and used more widely.