Multifunctional polymeric carriers for the intracellular delivery of protein cancer therapeutics

 描述(申请人提供)：对于癌症的治疗，蛋白质疗法比传统的化疗和放射疗法具有重要的优势，如高靶标特异性和广泛的靶标谱系。遗憾的是，蛋白质类药物的临床应用受阻于S一套常见的给药挑战。蛋白质在血液中降解，在肿瘤中沉积不良，无法穿过细胞膜和接触细胞内靶点。这项建议的目标是开发一种生物相容的多功能蛋白质药物给药平台，以促进(1)循环稳定，(2)肿瘤靶向和(3)细胞内给药。建议的聚合物设计的一个尖端特征是依赖于pH的膜不稳定活性，它允许蛋白质逃脱酸性内体并进入细胞胞浆。这种模块化的药物输送系统还结合了强大的肿瘤特异性抗体和可还原的二硫基，以促进蛋白质结合和在细胞质中的释放。该提案将开发两种密切相关的促凋亡蛋白，BIM肽和Bindi蛋白，由威斯康星大学贝克实验室设计，以对抗致癌的EB病毒(EBV)蛋白BHRF1，具有无与伦比的结合亲和力(&lt；0.1 NM)和特异性。有效的治疗交付或BINDI将在 EB病毒阳性B细胞淋巴瘤小鼠移植模型的建立。此外，还将评估与化疗药物环磷酰胺(CY)和硼替佐米的潜在协同作用。为了实现这些目标，已经确定了三个具体目标。在目标1中，将采用可逆加成裂解(RAFT)聚合来合成用于抗体靶向胞内蛋白输送的两嵌段共聚物胶束载体。载体将使用动态光散射(DLS)优化胶束大小，并使用成熟的红细胞溶血测试来优化pH响应膜的不稳定活性。在目标2中，抗体-聚合物-蛋白质结合物将优化细胞内BIM/BINDI递送和细胞内凋亡活性 癌细胞系癌细胞系。在目标3中，这些结合物将在B细胞淋巴瘤小鼠移植模型中进行优化，以(1)在多剂量毒性实验中的低毒性，(2)在药代动力学/生物分布研究中使用荧光标记蛋白的肿瘤靶向性，以及(3)使用生物发光caspase底物的肿瘤内的凋亡活性。最后，优化的抗体-聚合物-蛋白质结合物将用于抑制肿瘤生长和延长动物生存时间的测试。该项目的完成将展示一种创新的pH响应型聚合物家族在提供蛋白质癌症疗法方面的临床实用价值。此外，它还将结合贝克实验室的设计蛋白质、Stayton实验室的药物输送系统、霍根贝里实验室的细胞生物学和活体成像专业知识，以及Press实验室在弗雷德·哈钦森癌症研究中心(FHCRC)的临床开发能力，以定位一种创新且广泛适用的技术，用于快速临床翻译和人类影响。

 DESCRIPTION (provided by applicant): For the treatment of cancer, protein therapeutics offer important advantages over conventional chemotherapy and radiation, such as high target specificity and a wide target repertoire. Unfortunately, the clinical application of protein drugs s hindered by a common set of drug delivery challenges. Proteins are degraded in the blood, deposit poorly in tumors, and are unable to cross cell membranes and access intracellular targets. It is the objective of this proposal to develop a biocompatible multifunctional polymeric delivery platform for protein drugs that facilitates (1) circulation stability, (2) tumor targeting and (3) intracellular delivery. A cutting-edge feature of the proposed polymer design is pH-dependent membrane-destabilizing activity, which allows proteins to escape acidic endosomes and access the cell cytosol. This modular drug delivery system also incorporates powerful tumor-specific antibodies and reducible disulfide groups to facilitate protein conjugation and release in the cell cytoplasm. The proposal will develop two closely related pro-apoptotic proteins, the peptide BIM and the protein BINDI engineered in the Baker lab at UW to antagonize an oncogenic Epstein-Barr virus (EBV) protein, BHRF1, with unmatched binding affinity (< 0.1 nM) and specificity. Effective therapeutic delivery or BINDI will be validated in a murine xenograft model of EBV-positive B-cell lymphoma. Furthermore, potential synergism with the chemotherapeutic agents cyclophosphamide (CY) and bortezomib will be evaluated. To achieve these objectives, three Specific Aims have been defined. In Aim 1, reversible addition fragmentation (RAFT) polymerization will be employed to synthesize diblock copolymer micelle carriers for antibody-targeted intracellular protein delivery. The carriers will be optimized for micelle size using dynamic light scattering (DLS) and pH-responsive membrane-destabilizing activity using a well-established red blood cell hemolysis assay. In Aim 2, antibody-polymer-protein conjugates will be optimized for intracellular BIM/BINDI delivery and apoptotic activity in cancer cell cancer cell lines. In Aim 3, the conjugates will be optimized in a murine xenograft model of B-cell lymphoma for (1) low toxicity in a multidose toxicity experiment, (2) tumor targeting in a pharmacokinetic/biodistribution study using fluorescently labeled protein, and (3) intratumoral apoptotic activity using a bioluminescent caspase substrate. Lastly, the optimized antibody- polymer-protein conjugate will be tested for inhibition of tumor growth and prolonged animal survival. Completion of this project will demonstrate the clinical utility of an innovative family of pH-responsive polymers for the delivery of protein cancer therapeutics. Furthermore, it will combine the Baker lab's designer proteins, the Stayton lab's drug delivery systems, the Hockenbery lab's cellular biology and in vivo imaging expertise, and the Press lab's clinical development capabilities at the Fred Hutchinson Cancer Research Center (FHCRC), in order to position an innovative and widely applicable technology for rapid clinical translation and human impact.