Nanobiotechnology for the Treatment of Mantle Cell Lymphoma

描述(申请人提供)：套细胞淋巴瘤(MCL)是一种以多种癌基因调节失调为特征的B细胞恶性肿瘤。有证据表明，维甲酸及其衍生物全反式维甲酸(ATRA)是一种有用的药物，可以增强细胞的凋亡或抗增殖作用，因此建议在培养的MCL细胞中进行靶向传递ATRA的策略。利用一种新型的递送载体，其中ATRA被溶解在纳米级的蛋白质稳定的脂质颗粒中，被称为纳米盘(ND)，蛋白质工程方法将ATRA-ND靶向存在于B淋巴细胞表面的CD20抗原。这将通过构建单链抗体(ScFv)载脂蛋白(Apo)融合蛋白来实现。推测1-CD20scFv7apoA-I融合蛋白能够在保持亲本ScFv抗原识别特性的同时形成ND。重组融合蛋白将在大肠杆菌中表达、分离和鉴定。嵌合体与脂质结合和诱导ND形成的能力将被确定，而融合蛋白的1-CD20单链抗体部分的抗原识别特性将通过Western印迹和流式细胞术来评估。并用野生型apoA-I制备，作为这些研究的对照。在第二个目的中，将评估1-CD20 scFv7apoA-I ATRA-ND对培养的MCL细胞的影响。在MCL细胞培养模型中，假设含有靶向ND的维甲酸将显示出更强的诱导细胞凋亡的作用。不同的MCL细胞株将用于评估CD20靶向scFv7apoA-I ATRA-ND暴露后的靶向效率、浓度有效性和细胞活力。将培养的细胞暴露在对照ATRA-ND和1-CD20 scFv7apoA-I ATRA-ND中，然后测量细胞存活率、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ND进行研究，这些ND含有相关的合成和天然维甲酸。这些研究结果将通过展示细胞/组织特异性靶向来扩大ND介导的药物传递的潜力，为在MCL动物模型中进行靶向ND的研究提供一个框架。公共卫生相关性：尽管在淋巴瘤的广泛类别中取得了进展，但套细胞淋巴瘤(MCL)仍然是一种治疗不善的疾病，中位生存期约为3至4年。新的治疗方案提高了完全缓解率，但对改变总体存活率几乎没有什么作用。本文提出的研究将评估靶向药物有效载荷输送到培养的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，建立靶向纳米盘作为平台，可用于其他形式的癌症，潜在地减轻与这种疾病相关的公共负担。

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.