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: 9058421
• 负责人: Hanna Beth Kern
• 金额: $ 3.76万
• 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/9058421
• 关键词: Adverse effects; Affinity; Animals; Antibodies; Apoptosis; Apoptotic; B-Cell Lymphomas; BCL-2 Protein; BCL2L11 gene; BIM Bcl-2-binding protein; Binding; Binding Proteins; Biocompatible; Biodistribution; Biological Assay; Biotin; Blood; Blood Circulation; Bortezomib; CASP3 gene; 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 Kinetics; Endosomes; Engineering; Erythrocytes; Family; Formulation; Fred Hutchinson Cancer Research Center; Generations; Hemolysis; High Pressure Liquid Chromatography; Human; Human Herpesvirus 4; Label; Malignant Neoplasms; Measures; Membrane; Micelles; Microscopy; Molecular Sieve Chromatography; Mus; NMR Spectroscopy; Oncogenic; Peptides; Pharmaceutical Preparations; Polymers; Positioning Attribute; Probability; Protein Engineering; Proteins; Radiation; Resistance; Solubility; Specificity; Staining method; Stains; Streptavidin; Technology; Testing; Therapeutic; Time; Toxic effect; Translations; Treatment Efficacy; Viral; Viral Proteins; Xenograft Model; annexin A5; aqueous; biomaterial compatibility; cancer cell; cancer therapy; 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（由申请人提供）：对于癌症的治疗，蛋白质治疗剂提供了优于常规化疗和放疗的重要优势，例如高靶特异性和宽靶谱。不幸的是，蛋白质药物的临床应用受到一系列常见的药物递送挑战的阻碍。蛋白质在血液中降解，在肿瘤中存款较差，并且不能穿过细胞膜并到达细胞内靶点。本提案的目的是开发用于蛋白质药物的生物相容性多功能聚合物递送平台，其促进（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一个尖端特征是pH依赖性膜去稳定活性，其允许蛋白质逃离酸性内体并进入细胞胞质溶胶。这种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的二硫键基团，以促进蛋白质缀合和在细胞质中释放。该提案将开发两种密切相关的促凋亡蛋白，肽BIM和蛋白质BINDI在UW的Baker实验室中工程化，以拮抗致癌EB病毒（EBV）蛋白BHRF1，具有无与伦比的结合亲和力（<0.1 nM）和特异性。有效的治疗输送或BINDI将在一个 EB病毒阳性B细胞淋巴瘤的小鼠异种移植模型。此外，将评价与化疗剂环磷酰胺（CY）和硼替佐米的潜在协同作用。为实现这些目标，确定了三个具体目标。目的1：利用可逆加成断裂（RAFT）聚合法合成二嵌段共聚物胶束载体，用于抗体靶向细胞内蛋白质的递送。将使用动态光散射（DLS）和pH响应性膜去稳定活性（使用成熟的红细胞溶血试验）对载体的胶束大小进行优化。在目标2中，抗体-聚合物-蛋白质缀合物将针对细胞内BIM/BINDI递送和细胞凋亡活性进行优化。 癌细胞癌细胞系。在目的3中，将在B细胞淋巴瘤的鼠异种移植模型中优化缀合物，以用于（1）多剂量毒性实验中的低毒性，（2）使用荧光标记的蛋白质的药代动力学/生物分布研究中的肿瘤靶向，和（3）使用生物发光半胱天冬酶底物的肿瘤内凋亡活性。最后，将测试优化的抗体-聚合物-蛋白质缀合物对肿瘤生长的抑制和延长的动物存活。该项目的完成将证明一个创新的pH响应聚合物家族用于蛋白质癌症治疗的临床实用性。此外，它将结合联合收割机贝克实验室的设计师蛋白质，Stayton实验室的药物输送系统，Hockenbery实验室的细胞生物学和体内成像专业知识，以及Fred哈钦森癌症研究中心（FHCRC）的Press实验室的临床开发能力，以定位一种创新和广泛适用的技术，用于快速临床转化和人类影响。