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

描述（由申请人提供）：本提案的目标是开发一种新的治疗系统来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69000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。该治疗系统在体外和体内已被证明可诱导b细胞凋亡。为实现项目目标，我们将对NHL的治疗系统进行优化。该治疗系统利用一对带相反电荷的多肽，形成一个反平行的卷曲线圈。螺旋状肽（CC1/CC2）通过生物识别形成物理交联。生物识别导致b细胞上CD20受体的交联。cd20特异性Fab‘片段附着在一个卷曲的卷曲肽（Fab’- CC1）上，互补肽的多个拷贝附着在N-（2-羟丙基）甲基丙烯酰胺（HPMA）共聚物（P- cc2； P是聚合物主链）上。我们假设通过我们先前设计的共轭结构修饰增加CD20的交联将导致细胞凋亡增加。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含有L-和/或d -氨基酸残基的不同结构和卷曲卷曲肽的共轭物。这些缀合物将是高度可调的，并且由于肽之间的特定相互作用，可以自组装成高度有序的结构。将评估共轭生物识别，并确定增强生物识别的设计参数。优化的结构特征包括：HPMA主链的分子量、肽接枝的间距、肽与聚合物之间的间隔长度/Fab'。这些研究将提供有价值的见解，具体的设计特点，提高自组装线圈系统一般。利用超分辨率光学成像技术评估细胞表面共轭物的自组装和CD20的交联。超分辨率成像可以量化CD20的聚类程度。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引起的整体效应联系起来。我们将评估CD20交联与诱导细胞凋亡的关系。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。优化后的偶联物将在体内进行测试，结果将与CD20聚类结果相关联。这种新的治疗系统将提高NHL患者的应答率，也可能应用于其他b细胞相关疾病，如多发性硬化症和类风湿性关节炎。通过对细胞表面卷曲肽的生物识别来诱导细胞信号传导是一种令人兴奋的治疗疾病的策略。此外，使用超分辨率成像来评估自组装和蛋白质交联是评估治疗方法的一种新方法。

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.