Coiled-coil Based Drug-Free Macromolecular Therapeutics

描述（由申请人提供）：非霍奇金淋巴瘤（NHL）是一种常见的癌症，2008年在美国估计有66,000例确诊病例，自1980年以来发病率翻了一番。尽管在FDA批准Rituxan后，nhl的治疗方法得到了极大的改善，但至少三分之一的患者仍发生对当前治疗无反应的屈光性恶性肿瘤，这表明需要改进治疗策略。CD20是B细胞NHL最可靠的生物标志物和治疗靶点之一，它是一种非内化抗原，当与互补抗体结合时仍留在细胞表面。cd20结合抗体与二抗交联可导致这些细胞凋亡。该项目的总体目标是产生新的、无药物的大分子治疗方法，以改善非霍奇金淋巴瘤的治疗。提出了一种基于N-（2-羟丙基）甲基丙烯酰胺（HPMA）接枝共聚物介导的B细胞表面螺旋状异源二聚体形成的细胞凋亡诱导新体系。设计的基本原理是缺乏低分子量药物和CD20在b细胞表面交联导致细胞凋亡的事实。该体系由一对互补的卷曲肽构成反平行异二聚体；抗cd20抗体1F5的Fab'片段；和HPMA共聚物。一个肽与Fab片段结合，另一个肽与polyHPMA多次嫁接结合。先前设计了一对带相反电荷的五七肽（CCE和CCK），它们形成具有高度生物识别的反平行盘绕线圈异二聚体。据推测，CCK和CCE肽的独特生物识别可能是设计一类新型大分子治疗药物的基础。事实上，CD20+ Raji B细胞暴露于Fab'-CCE后，通过抗原-抗体片段生物识别，细胞表面出现了多个拷贝的CCE肽。将CCE修饰的细胞进一步暴露于接枝多个CCK拷贝的HPMA共聚物中，导致细胞表面形成CCE-CCK线圈状异源二聚体。这第二次生物识别事件诱导CD20受体交联并引发Raji B细胞凋亡。系统将在初步数据的基础上进行优化。将设计和评估含有L-和/或d-氨基酸残基的较短的盘状线圈形成序列。通过五种细胞类型和三种细胞凋亡实验，将评估HPMA接枝共聚物介导的细胞表面线圈形成与伴随的受体交联和细胞凋亡诱导。重要的是，无药物大分子疗法的功效和生物相容性将在动物模型中进行评估。将评估人体分布、剂量和给药频率对治疗效果的影响，以选择主要化合物和最佳治疗方式。最后，这一新概念将发展成为一种新的治疗实体——无药大分子治疗学。

DESCRIPTION (provided by applicant): Non-Hodgkin's lymphoma (NHL) is a prevalent cancer with an estimated 66,000 cases diagnosed in the United States in 2008, with the incidence doubling since 1980. Although treatments for NHLs greatly improved following the FDA approval of Rituxan, refractive malignancies still occur that are nonresponsive to current therapies in at least a third of all patients, indicating that improved treatment strategies are needed. One of the most reliable biomarkers and therapeutic targets for B cell NHL is CD20, which is a non- internalizing antigen that remains on the cell surface when bound to a complementary antibody. The crosslinking of CD20-bound antibodies with a secondary antibody results in apoptosis of these cells. The overall goal of this project is to generate new, drug-free macromolecular therapeutics for improved treatment of NHL. A new apoptosis induction system is proposed which is based on N-(2- hydroxypropyl)methacrylamide (HPMA) graft copolymer mediated formation of coiled-coil heterodimers at B- cell surface. The rationale of the design is the absence of low molecular weight drugs and the fact that crosslinking of CD20 at B-cell surface results in apoptosis. The system is composed of a pair of complementary coiled-coil peptides forming antiparallel heterodimers; Fab' fragment of the 1F5 anti-CD20 antibody; and HPMA copolymer. One peptide is conjugated to the Fab' fragment, the other is conjugated in multiple grafts to polyHPMA. A pair of oppositely charged pentaheptad peptides (CCE and CCK) that formed antiparallel coiled-coil heterodimers with a high degree of biorecognition was previously designed. It was hypothesized that the unique biorecognition of CCK and CCE peptides could be a basis for the design of a novel class of macromolecular therapeutics. Indeed, the exposure of CD20+ Raji B cells to Fab'-CCE resulted in the decoration of the cell surface with multiple copies of the CCE peptide via antigen-antibody fragment biorecognition. Further exposure of the CCE decorated cells to HPMA copolymer grafted with multiple copies of CCK resulted in the formation of CCE-CCK coiled-coil heterodimers on the cell surface. This second biorecognition event induced crosslinking of CD20 receptors and triggered apoptosis of Raji B cells. The system will be optimized based on the preliminary data. Shorter coiled-coil forming sequences containing L- and/or D-amino acid residues will be designed and evaluated. HPMA graft copolymer mediated coiled-coil formation at cell surface with concomitant receptor crosslinking and apoptosis induction will be assessed in vitro using five cell types and three apoptosis assays. Importantly, the efficacy and biocompatibility of drug-free macromolecular therapeutics will be evaluated in an animal model. Body distribution, impact of the dose and frequency of administration on the efficacy of treatment will be evaluated with the aim to select leading compound(s) and optimal treatment modalities. Finally, this new concept will be developed into a new therapeutic entity, drug-free macromolecular therapeutics.