Nanobiotechnology for the Treatment of Mantle Cell Lymphoma

描述（由申请人提供）：套细胞淋巴瘤（MCL）是一种 B 细胞恶性肿瘤，其特征是各种癌基因的失调。基于视黄酸及其衍生物、全反式视黄酸 (ATRA) 是增强细胞凋亡或抗增殖作用的有用药物的证据，建议寻求一种将 ATRA 靶向递送至培养中的 MCL 细胞的策略。使用一种新的递送载体，其中ATRA溶解在纳米级、蛋白质稳定的脂质颗粒中，称为纳米盘(ND)，蛋白质工程方法将用于将ATRA-ND靶向存在于B淋巴细胞表面上的CD20抗原。这将通过构建单链可变抗体（scFv）载脂蛋白（apo）融合蛋白来实现。假设 1-CD20 scFv7apoA-I 融合蛋白将能够形成 ND，同时保留亲本 scFv 的抗原识别特性。重组融合蛋白将在大肠杆菌中表达、分离和表征。将确定嵌合体与脂质结合并诱导ND形成的能力，同时将通过蛋白质印迹和流式细胞术评估融合蛋白的1-CD20 scFv部分的抗原识别特性。用野生型 apoA-I 制备的 ND 将作为这些研究的对照。第二个目标是评估 1-CD20 scFv7apoA-I ATRA-ND 对培养的 MCL 细胞的影响。据推测，含有类视黄醇的靶向 ND 将在 MCL 细胞培养模型中表现出增强的细胞凋亡诱导作用。不同的 MCL 细胞系将​​用于旨在评估暴露于 CD20 靶向 scFv7apoA-I ATRA-ND 后的靶向效率、浓度有效性和细胞活力的研究。培养的细胞将暴露于对照 ATRA-ND 和 1-CD20 scFv7apoA-I ATRA-ND，然后测量细胞活力、细胞凋亡和自噬。将进行剂量反应和时间过程研究以确定最佳条件。还将对含有相关合成和天然类视黄醇的 ND 进行研究。这些研究的结果将通过证明细胞/组织特异性靶向来扩大 ND 介导的药物递送的潜力，为 MCL 动物模型中靶向 ND 的体内研究提供框架。 公共卫生相关性：尽管在大类淋巴瘤方面取得了进展，但套细胞淋巴瘤 (MCL) 仍然是一种治疗效果不佳的疾病，中位生存时间约为 3 至 4 年。新的治疗方案提高了完全缓解率，但对改变总体生存率几乎没有作用。本文提出的研究将评估靶向药物有效负载递送至培养的 MCL 细胞的有效性。这些研究将建立一种具有广泛适用性的新方法，将靶向纳米盘建立为可用于其他形式癌症的平台，有可能减轻与这种疾病相关的公众负担。

DESCRIPTION (provided by applicant): Mantle cell lymphoma (MCL) is a B-cell malignancy that is characterized by dysregulation of various oncogenes. Building on evidence that retinoic acid and its derivative, all trans retinoic acid (ATRA), are useful agents that potentiate apoptosis or anti-proliferative effects, it is proposed to pursue a strategy of targeted delivery of ATRA to MCL cells in culture. Using a novel delivery vehicle wherein ATRA is solubilized in nanoscale, protein stabilized lipid particles, termed nanodisks (ND), protein engineering methods will be employed to target ATRA-ND to the CD20 antigen present on the surface of B lymphocytes. This will be achieved by construction of a single chain variable antibody (scFv) apolipoprotein (apo) fusion protein. It is hypothesized that 1-CD20 scFv7apoA-I fusion protein will be capable of forming ND while retaining the antigen recognition properties of the parent scFv. Recombinant fusion protein will be expressed in E. coli, isolated and characterized. The ability of the chimera to associate with lipid and induce formation of ND will be determined while the antigen recognition properties of the 1-CD20 scFv portion of the fusion protein will be evaluated by Western blot and flow cytometry. ND prepared with wild type apoA-I will serve as control for these studies. In a second aim the effect of 1-CD20 scFv7apoA-I ATRA-ND on MCL cells in culture will be assessed. It is hypothesized that retinoid containing, targeted ND, will display enhanced induction of apoptosis in cell culture models of MCL. Different MCL cell lines will be employed in studies designed to evaluate targeting efficiency, concentration effectiveness and cell viability following exposure to CD20 targeted scFv7apoA-I ATRA-ND. Cultured cells will be exposed to control ATRA-ND and 1-CD20 scFv7apoA-I ATRA-ND followed by measurements of cell viability, apoptosis and autophagy. Dose- response and time course studies will be conducted to define optimal conditions. Studies will also be performed with ND harboring related synthetic and natural retinoids. The results of these studies will expand the potential of ND mediated drug delivery by demonstrating cell / tissue specific targeting, providing a framework for in vivo studies of targeted ND in animal models of MCL. PUBLIC HEALTH RELEVANCE: Despite progress made in the broad category of lymphomas, mantle cell lymphoma (MCL) remains a poorly treated disease with median survival time of approximately 3 to 4 years. New therapy regimens have increased the complete remission rate but they have done little to change overall survival. Research proposed herein will evaluate the effectiveness of targeted drug payload delivery to cultured MCL cells. These studies will establish a novel approach with broad applicability, establishing targeted nanodisks as a platform that can be used with other forms of cancer, potentially decreasing the public burden associated with this disease.