非载药纳米免疫脂质体治疗淋巴瘤的机制研究

Represented with rituximab, antibodies have been playing a significant role in clinical lymphoma treatment. Development of immunoliposomal drug delivery systems has become one of the most popular areas in antibody-based research to conquer cancer including lymphoma. It is a technique to immobilize antibodies onto the surface of modified liposomes so as to realize targeting delivery and release the encapsulated drugs by utilizing the specificity of antibodies. However, rare studies have considered immunoliposome without drug as a new antibody formulation and explore its mechanism as well as efficiency. In our previous research work about lymphoma, we found that immunoliposomes without drug showed equivalent or improved efficiency compared to the corresponding free antibody both in vitro and in vivo, with an unknown reason. This project aims at exploring and comparing the mechanism alteration of rituximab due to the effect of immunoliposomal formulation. From both overall and individual level, the cellular impact caused by rituximab-immunoliposomes will be fully studied. Specifically, those well-known mechanisms of free rituximab in lymphoma treatment, including antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC) and biomarkers in direct killing cytotoxicity pathways will be evaluated and compared after treatment with rituximab-immunoliposomes. Additionally, lymphoma engraftment mouse model will be used to characterize tissue distribution, pharmacokinetics and efficacy of rituximab-immunoliposomes. Results of this study should be helpful and informative to evaluate and provide the basis for feasibility of immunoliposomes in clinical lymphoma application as a new antibody drug formulation. The mechanism study of rituximab-immunoliposomes will also contribute to future antibody-based immunoliposomal discovery and application in cancer diseases not limited to lymphoma.

以利妥昔单抗为代表的抗体药物在淋巴瘤临床治疗中发挥着重要作用。国内外利用抗体药物治疗淋巴瘤等肿瘤的研究的热点之一，是利用抗体的特异性修饰脂质体表面形成免疫脂质体药物传递系统，从而达到靶向传递释放药物的目的；但几乎没有考虑将免疫脂质体视作一种新型抗体药物剂型，在不包埋其他药物的情况下，对其作用机理和效果进行研究。前期研究已观察到免疫脂质体在体内和体外都比游离抗体产生更好效果，但原因不明。本项目将从细胞整体和个体水平对利妥昔单抗免疫脂质体与游离单抗的作用机制区别进行研究，侧重考察免疫脂质体对已知的利妥昔单抗作用机制如抗体依赖细胞毒效应（ADCC）、补体依赖细胞毒效应（CDC）和直接作用通路中生物标记物的变化影响，并使用淋巴瘤小鼠模型考察利妥昔单抗免疫脂质体的组织分布和药理药效。期望通过研究，能为免疫脂质体作为抗体新剂型在临床应用的可行性提供基础依据，并有助于改善现有抗体药物治疗淋巴瘤的局限性。

以利妥昔单抗为代表的抗体药物在淋巴瘤临床治疗中发挥着重要作用。本课题在基于前期利用抗体的特异性修饰脂质体表面形成免疫脂质体的基础上，将该剂型作为利妥昔单抗的一种新的给药剂型，在不包埋药物的情况下，对其治疗淋巴瘤的效果和作用机制进行研究。通过对包括Raji细胞在内的多种细胞活性检测，发现利妥昔单抗免疫脂质体对Raji细胞能够产生最好的效果，并确定了后续实验的药物浓度和处理时间。在体内使用Raji尾静脉接种免疫缺陷（SCID）小鼠后，对利妥昔单抗免疫脂质体和游离抗体在模拟治疗淋巴瘤的效果上进行体内验证，结果与体外实验结果相符，表明利妥昔单抗免疫脂质体具有更好的治疗淋巴瘤的效果，能显著延长小鼠生存率。在考察和比较了游离药物的三种主要作用机制后，发现利妥昔单抗免疫脂质体并非通过抗体以来细胞毒效应（ADCC）或补体依赖细胞毒效应（CDC）引起恶性B细胞凋亡，但却具有增强抗体交联效应的作用。通过在Raji细胞模型在小鼠体内对利妥昔单抗的血药浓度和组织分布进行检测，发现利妥昔单抗免疫脂质体能够更有效地分布于骨髓和脾脏等淋巴瘤的靶器官，在药物分布上具有更好的特性。且由于脂质体本身能够延长药物在体内长循环的原因，利妥昔单抗免疫脂质体在小鼠体内能够在一定程度上维持血药浓度。根据以上结果，进一步研究利妥昔单抗免疫脂质体提高治疗效果可能存在的机制，研究结果表明，利妥昔单抗免疫脂质体可通过交联效应增加钙离子内流和调节线粒体信号通路。本项目的研究结果将为后续利妥昔单抗免疫脂质体作为抗体新剂型在临床应用的可行性提供参考，并对包括免疫脂质体在内的利妥昔单抗药物，乃至以抗体为基础的淋巴瘤药物治疗的作用机制提供新的基础依据，也为改善现有抗体药物治疗淋巴瘤的局限开拓思路。