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

基本信息

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
项目类别：
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

项目摘要

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，蛋白质疗法提供了优于化疗和放疗的重要优势，例如高靶点特异性和广泛的常规靶点库。不幸的是，蛋白质药物的临床应用受到一组常见的障碍的阻碍。药物递送挑战。蛋白质在血液中降解，在肿瘤中沉积不良，并且无法穿过细胞膜并进入细胞内靶标。该提案的目的是开发一种生物相容性多功能聚合物递送平台，用于蛋白质药物。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前沿特征是pH依赖性膜不稳定活性，它允许蛋白质逃离酸性内体并进入细胞质。该模块化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团，以促进细胞质中蛋白质的结合和释放。该提案将开发两种密切相关的促凋亡蛋白，即肽 BIM 和蛋白质。 BINDI 在威斯康星大学贝克实验室进行设计，可拮抗致癌的 Epstein-Barr 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，有效的治疗递送或 BINDI 将在一项研究中得到验证。此外，将评估化疗药物环磷酰胺 (CY) 和硼替佐米的潜在协同作用，在目标 1 中定义了三个具体目标： RAFT）聚合将用于合成二嵌段共聚物胶束载体，用于抗体靶向细胞内蛋白质递送。使用动态光散射 (DLS) 确定胶束大小，并使用成熟的红细胞溶血测定确定 pH 响应膜不稳定活性。在目标 2 中，将针对细胞内 BIM/BINDI 递送和细胞凋亡对抗体-聚合物-蛋白质缀合物进行优化。活动于在目标 3 中，将在 B 细胞淋巴瘤的小鼠异种移植模型中优化缀合物，以实现 (1) 多剂量毒性实验中的低毒性，(2) 使用荧光的药代动力学/生物分布研究中的肿瘤靶向。标记的蛋白质，以及（3）使用生物发光半胱天冬酶底物的瘤内细胞凋亡活性最后，将测试优化的抗体-聚合物-蛋白质缀合物对肿瘤生长和延长动物的抑制作用。该项目的完成将展示创新的 pH 响应聚合物系列在蛋白质癌症治疗中的临床应用，此外，它将结合 Baker 实验室的设计蛋白、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 Hutchinson 癌症研究中心 (FHCRC) 新闻实验室的临床开发能力，以便定位一种创新且广泛适用的技术，用于快速临床转化和人类影响。