Foundations of Pretargeted Radioimmunotherapy

描述(申请人提供)：将电离辐射的细胞杀伤量输送到肿瘤是抗体导向放射免疫疗法(RIT)的目标。然而，在实践中，对健康骨髓和肾脏的附带损害限制了最大辐射剂量。前靶向RIT(PRIT)旨在通过分离肿瘤靶向和放射性核素传递的药代动力学来克服这一限制。第一代PRIT利用链霉亲和素/生物素相互作用捕获放射性核素，并在动物肿瘤异种移植模型中展示了良好的前景。然而，链霉亲和素的免疫原性、肾脏定位和内源性生物素的问题本质上限制了这种方法。这项建议的观点是，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制剂，并且必须对肿瘤渗透的药代动力学进行严格的工程分析。该项目汇集了生物工程和放射肿瘤学的教师，共同开发必要的试剂和剂量策略，使PRIT发挥最大的效果。通过定向进化的蛋白质工程将被应用于：1)构建有效地不可逆地捕获放射性金属螯合物(与Y、镓或铋的DOTA)的人抗体片段；以及2)开发通过酸性隔间的内吞运输循环而持久结合的抗CEA抗体片段。抗体片段在微转移瘤中的扩散、结合和代谢过程将通过单个LS174T人结肠腺癌细胞和球形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，以确定极限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LS174T裸鼠异种移植瘤的定量生物分布研究来验证。总而言之，这些研究将为优化PRIT奠定坚实的基础。在该提案的第二次也是最后一次允许的修订中，我们取消了辐射剂量测定，并基于LS174T细胞快速摄取抗体的直接证据，将抗体片段的内吞运输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提供了计划中的努力。

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.