Development of a Coiled-Coil therapeutic for Non-Hodgkin's Lymphoma

描述(申请人提供)：这项提案的目标是开发一种新的治疗系统，以改善非霍奇金淋巴瘤(NHL)的治疗。自1980年以来，NHL病例增加了一倍，2013年估计有6.9万例新诊断。B细胞淋巴瘤占NHL病例的85%，因此，新的抗癌治疗依赖于B细胞表面CD20受体的交联来诱导细胞凋亡。该治疗系统已被证明在体外和体内都能诱导B细胞的凋亡。为实现项目目标，我们将优化治疗NHL的治疗系统。该治疗系统利用一对相反电荷的多肽，形成一个反平行的螺旋线圈。卷曲多肽(CC1/CC2)通过生物识别形成物理交联物。生物识别导致B细胞上CD20受体的交联性。CD20特异的Fab‘片段连接到卷曲多肽之一(Fab’-CC1)，多个拷贝的互补多肽连接到N-(2-羟丙基)甲基丙烯酰胺(HPMA)共聚物(P-CC2；P是聚合物主干)。我们假设，通过对先前设计的共轭结构的修改来增加CD20的交联度将导致更多的细胞凋亡。为了验证我们的假设并实现我们的目标，我们建议设计和合成不同结构的偶联物和含有L和/或D-氨基酸残基的卷曲多肽。这些偶联物将是高度可调的，并可以由于多肽之间的特定相互作用而自组装成高度有序的结构。将对共轭生物识别进行评估，并确定增强生物识别的设计参数。需要优化的结构特征包括：HPMA主链的相对分子质量、多肽接枝的间距以及多肽与聚合物/Fab‘之间的间隔物长度。这些研究将提供有价值的洞察具体的设计特征，以改善卷绕系统的总体自组装。将使用超分辨率光学成像技术评估CD20在细胞表面的自组装和交联性。超分辨率成像可以用来量化CD20的聚集程度。生物材料系统的超分辨率成像将更清楚地将生物机制与生物材料诱导的整体效应联系起来。我们将评估CD20的交联性与诱导细胞凋亡之间的关系。此外，生物材料的作用机制可以通过识别涉及的其他蛋白质或细胞结构来确定。优化的结合物随后将在体内进行测试，结果将与CD20聚类结果相关联。这一新的治疗系统将提高NHL患者的应答率，也可能适用于其他与B细胞相关的疾病，如多发性硬化症和类风湿性关节炎。通过在细胞表面识别卷曲多肽来诱导细胞信号传递是治疗疾病的一种令人兴奋的策略。此外，使用超分辨率成像来评估自组装和蛋白质交联是一种新的治疗评估方法。

DESCRIPTION (provided by applicant): The goal of this proposal is to develop a new therapeutic system to improve the treatment of non-Hodgkin's lymphoma (NHL). Cases of NHL have doubled since 1980 and there are an estimated 69,000 new diagnoses in 2013. B-cell lymphomas constitute 85% of NHL cases; therefore, the new anti-cancer treatment relies on the cross-linking of CD20 receptors on the B-cell surface to induce apoptosis. The therapeutic system has already been shown to induce apoptosis of B-cells in vitro and in vivo. To achieve the goal of the project, we will optimize the therapeutic system for the treatment of NHL. The therapeutic system utilizes a pair of oppositely charged peptides that form an antiparallel coiled coil. The coiled coil peptides (CC1/CC2) form physical crosslinks through biorecognition. Biorecognition leads to crosslinking of CD20 receptors on B-cells. A CD20-specific Fab' fragment is attached to one of the coiled coil peptides (Fab'- CC1), and multiple copies of the complementary peptide are attached to a N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer (P-CC2; P is the polymer backbone). We hypothesize that increasing the crosslinking of CD20 through conjugate structure modifications of our previous design will result in increased apoptosis. To test our hypothesis and accomplish our goals, we propose to design and synthesize conjugates of varying architecture and coiled coil peptides containing L- and/or D-amino acid residues. These conjugates will be highly tunable, and can self-assemble into highly ordered structures due to specific interactions between peptides. The conjugate biorecognition will be evaluated and design parameters will be identified that enhance biorecognition. Structural features to optimize include: Mw of HPMA backbone, spacing of peptide grafts, and spacer length between peptides and polymer/Fab'. These studies will provide valuable insight into specific design features that improve self-assembly of coiled coil systems in general. Self-assembly of the conjugates at the cell surface and the crosslinking of CD20 will be evaluated using super-resolution optical imaging techniques. Super-resolution imaging can be used to quantify the degree of CD20 clustering. Super-resolution imaging of biomaterial systems will more clearly link biological mechanisms to the overall effect induced by the biomaterial. The relationship between the crosslinking of CD20 and induction of apoptosis will be evaluated. Furthermore, the mechanism of action of a biomaterial can be determined by identifying other proteins or cellular structures involved. Optimized conjugates will then be tested in vivo and the results will be correlated with CD20 clustering results. This new therapeutic system will improve response rates in patients with NHL, and also may be applied to other B-cell related diseases such as multiple sclerosis and rheumatoid arthritis. The biorecognition of coiled-coil peptides at the cell surface to induce cellular signaling is an exciting strategy in treating diseases. Furthermore, using super-resolution imaging to evaluate self-assembly and protein crosslinking is a novel approach in evaluating therapeutics.