Multifunctional polymeric carriers for the intracellular delivery of protein cancer therapeutics

用于细胞内递送蛋白质癌症治疗药物的多功能聚合物载体

• 批准号: 8832442
• 负责人: Hanna Beth Kern
• 金额: $ 3.72万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-04-01 至 2019-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8832442
• 关键词: Adverse effects; Affinity; Animals; Antibodies; Apoptosis; Apoptotic; B-Cell Lymphomas; BCL-2 Protein; BCL2L11 gene; BIM Bcl-2-binding protein; Binding; Biocompatible; Biodistribution; Biological Assay; Biotin; Blood; Blood Circulation; Bortezomib; Cancer cell line; Caspase; Cell Culture Techniques; Cell Line; Cell Separation; Cell Survival; Cell membrane; Cells; Cellular biology; Clinical; Cyclophosphamide; Cytoplasm; Cytosol; Deposition; Development; Disulfide Linkage; Disulfides; Dose; Drug Delivery Systems; Drug Formulations; Drug Kinetics; Endosomes; Engineering; Erythrocytes; Family; Fred Hutchinson Cancer Research Center; Generations; Hemolysis; High Pressure Liquid Chromatography; Human; Human Herpesvirus 4; Human herpesvirus 4 BHRF1 protein; Label; Malignant Neoplasms; Measures; Membrane; Micelles; Microscopy; Molecular Sieve Chromatography; Mus; NMR Spectroscopy; Oncogenic; Peptides; Pharmaceutical Preparations; Polymers; Positioning Attribute; Probability; Protein Binding; Proteins; Radiation; Resistance; Solubility; Solutions; Specificity; Staining method; Stains; Streptavidin; Technology; Testing; Therapeutic; Time; Toxic effect; Translations; Treatment Efficacy; Viral; Xenograft Model; annexin A5; aqueous; biomaterial compatibility; cancer cell; cancer therapy; caspase-3; chemotherapeutic agent; chemotherapy; clinical application; design; di-block copolymer; fluorophore; in vivo; in vivo imaging; innovation; light scattering; meetings; novel; overexpression; polymerization; pro-apoptotic protein; public health relevance; research study; success; synergism; therapeutic protein; tumor; tumor growth; tumorigenesis; uptake

 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.

 描述（由应用提供）：对于癌症的治疗，蛋白质治疗比常规化疗和放射线具有重要优势，例如高目标特异性和广泛的靶标曲目。不幸的是，蛋白质药物的临床应用受到一组常见的药物递送挑战的阻碍。蛋白质在血液中降解，沉积在肿瘤中，无法越过细胞膜和进入细胞内靶标。该提案的目的是开发一种促进蛋白质药物的生物相容性多功能聚合物递送平台（1）循环稳定性，（2）肿瘤靶向和（3）细胞内递送。所提出的聚合物设计的尖端特征是pH依赖性的膜 - 止动活性，它使蛋白质可以逃脱酸性内体并访问细胞胞质。该模块化药物递送系统还结合了强大的肿瘤特异性抗体和可还原二硫基团，以促进细胞质中蛋白质结合并释放。该提案将开发两种密切相关的促凋亡蛋白，即在UW的贝克实验室中设计的肽BIM和蛋白质Bindi，以拮抗具有无与伦比的结合亲和力（<0.1 NM）和特异性的致癌性Epstein-Barr病毒（EBV）蛋白BHRF1，BHRF1。有效的治疗分娩或BINDI将在A中进行验证 EBV阳性B细胞淋巴瘤的鼠色谱模型。此外，将评估与化学治疗剂环磷酰胺（CY）和硼替佐米的潜在协同作用。为了实现这些目标，已经定义了三个具体目标。在AIM 1中，将采用可逆添加碎片（RAFT）聚合来合成二嵌段共聚物胶束载体进行抗体靶向的细胞内蛋白质递送。使用动态光散射（DLS）和pH响应性膜 - 降解活性，使用良好的红细胞溶血测定法对胶束尺寸进行优化。在AIM 2中，将优化抗体聚合物 - 聚合物 - 蛋​​白结合物，以用于细胞内BIM/BINDI递送和凋亡活性 癌细胞癌细胞系。 In Aim 3, the conjugates will be optimized in a murine xenographic 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.最后，将测试优化的抗体 - 聚合物 - 蛋​​白质结合物，以抑制肿瘤生长和延长动物生存。该项目的完成将证明pH响应性聚合物的创新家族用于蛋白质癌症治疗的临床实用性。此外，它将结合Baker Lab的设计师蛋白，Stayton Lab的药物输送系统，Hockenbery Lab的细胞生物学和体内成像专业知识，以及新闻实验室在Fred Hutchinson癌症研究中心（FHCRC）的临床开发能力，以对迅速和广泛的临床构成技术和人类的影响和人类的影响和人类的影响和人类的影响和人类构成和人类的影响。