Foundations of Pretargeted Radioimmunotherapy

预定位放射免疫治疗的基础

• 批准号: 6976911
• 负责人: Karl Dane Wittrup
• 金额: $ 28.15万
• 依托单位: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-08-01 至 2009-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6976911
• 关键词: antibody specificity; athymic mouse; cell line; directed evolution; fluorescence microscopy; gene targeting; immunoconjugates; immunologic substance development /preparation; mathematical model; neoplasm /cancer radioimmunotherapy; neoplasm /cancer transplantation; pharmacokinetics; protein engineering; protein transport; proteomics; xenotransplantation

DESCRIPTION (provided by applicant): Delivery of cell-killing doses of ionizing radiation to tumors is the objective of antibody-directed radioimmunotherapy (RIT). In practice, however, collateral damage to healthy bone marrow and kidneys limits the maximum delivered radiation dose. Pretargeted RIT (PRIT) aims to overcome this limitation by separating the pharmacokinetics of tumor targeting and radionuclide delivery. First-generation PRIT utilizes the streptavidin/biotin interaction for radionuclide capture, and exhibits promise in animal tumor xenograft models. However, problems with streptavidin immunogenicity, kidney localization, and endogenous biotin intrinsically limit this approach. The perspective of this proposal is that the principle of PRIT is sound, but that to reach its full potential the protein targeting agents must be optimized, and the pharmacokinetics of tumor penetration must be subjected to rigorous engineering analysis. This project brings together faculty from Biological Engineering and Radiation Oncology to collaboratively develop the essential reagents and dosing strategies to enable PRIT to be maximally effective. Protein engineering by directed evolution will be applied to: 1) construct human antibody fragments that capture radiometal chelates (DOTA with yttrium, gallium, or bismuth) effectively irreversibly; and 2) to develop anti-CEA antibody fragments that bind persistently through cycles of endocytic trafficking through acidic compartments. The processes of diffusion, binding, and metabolism of antibody fragments in micrometastases will be characterized by quantitative fluorescence microscopy of single LS174T human colon adenocarcinoma tumor cells and spheroid cultures, and analyzed within a mathematical modeling framework to determine the limiting kinetic processes and predict necessary concentrations and times for saturation binding. These predictions will be tested with quantitative biodistribution studies in LS174T xenografts in nude mice. Taken together, these studies will establish a firm foundation from which to optimize PRIT. In this second and final allowed revision of the proposal, we have eliminated radiation dosimetry and focused on endocytic trafficking of antibody fragments as a critical process limiting the permeation and retention of bispecific antibodies, based on direct evidence for rapid antibody uptake by LS174T cells. This hypothesis informs the planned efforts in protein engineering, cell culture, and in vivo biodistribution studies.

描述（由申请方提供）：向肿瘤递送细胞杀伤剂量的电离辐射是抗体导向放射免疫治疗（RIT）的目的。然而，在实践中，对健康骨髓和肾脏的附带损害限制了最大的辐射剂量。预靶向RIT（PRIT）旨在通过分离肿瘤靶向和放射性核素递送的药代动力学来克服这一限制。第一代PRIT利用链霉亲和素/生物素相互作用进行放射性核素捕获，并在动物肿瘤异种移植模型中表现出希望。然而，链霉亲和素免疫原性、肾脏定位和内源性生物素的问题本质上限制了这种方法。该提案的观点是，PRIT的原理是合理的，但为了充分发挥其潜力，必须优化蛋白质靶向剂，并且肿瘤渗透的药代动力学必须进行严格的工程分析。该项目汇集了来自生物工程和放射肿瘤学的教师，共同开发基本试剂和给药策略，使PRIT能够最大限度地发挥作用。通过定向进化的蛋白质工程将用于：1）构建有效地不可逆地捕获放射性金属螯合物（具有钇、镓或铋的DOTA）的人抗体片段;和2）开发通过酸性区室的内吞运输循环持续结合的抗CEA抗体片段。微转移中抗体片段的扩散、结合和代谢过程将通过单个LS 174 T人结肠腺癌肿瘤细胞和球状体培养物的定量荧光显微镜来表征，并在数学建模框架内进行分析，以确定限制性动力学过程并预测饱和结合所需的浓度和时间。这些预测将通过裸鼠中LS 174 T异种移植物的定量生物分布研究进行检验。这些研究将为优化PRIT奠定坚实的基础。在该提案的第二次和最终允许的修订中，我们基于LS 174 T细胞快速抗体摄取的直接证据，取消了辐射剂量测定，并将重点放在抗体片段的内吞运输上，作为限制双特异性抗体渗透和保留的关键过程。这一假设为蛋白质工程、细胞培养和体内生物分布研究的计划工作提供了信息。