High-avidity multimeric cancer imaging agent

描述(由申请人提供) 肿瘤靶向抗体可以为肿瘤特异性显像剂的产生提供一个通用的起点。特别是，通过蛋白质工程，可以生产出针对体内肿瘤靶向、血液清除以及正常组织分布和清除模式进行优化的重组抗体片段。在这些片段与发射正电子的放射性核素(如铜-和1-124)结合并进行放射性标记后，工程抗体片段可用于使用小动物成像手段(如微PET)在小鼠肿瘤异种移植模型中进行灵敏、高分辨率的成像。我们建议利用一种新的蛋白质多聚体技术来开发高亲和力的多聚体肿瘤显像剂。作为一个模型系统，针对癌胚抗原(CEA)的单链抗体将被重组以产生二聚体和四聚体。1)在细菌表达和纯化后，将在体外测定生化和结合特性。2)将重组抗体标记为放射性碘或与螯合部分(DOTA)偶联，用于放射性金属标记，用于结合和生物分布研究。蛋白质还将被放射性标记1-124和铜-，用于微型正电子发射计算机断层扫描成像研究。此外，还将开发一套C端Cys残基修饰的高亲和力多聚体抗CEA抗体片段，用于位点特异性连接和放射性标记。3)将在CEA阳性(LS1741)和CEA阴性(C6)异种移植的裸鼠中评价抗体制剂的性能，在活体小鼠中进行生物分布研究和系列microPET成像研究。4)对新型抗体的肿瘤靶向性、生物分布和成像潜力进行了评价。二聚体和四聚体将被比较，以确定由四个结合结构域赋予的高亲和力是否提高了整体在体内的表现。这些新的片段还将与现有的抗体形式进行比较。通过这项工作获得的知识将加速癌症特异性显像剂的开发，这些显像剂来自各种针对肿瘤相关抗原的抗体，用于单光子或PET成像应用。

DESCRIPTION (provided by applicant) Cancer-targeting antibodies can provide a versatile starting point for generation of tumor-specific imaging agents. In particular, through protein engineering, recombinant antibody fragments can be produced that are optimized for in vivo tumor targeting, blood clearance, and normal tissue distribution and clearance patterns. Following conjugation and radiolabeling of such fragments with positron-emitting radionuclides such as Cu-64 and 1-124, engineered antibody fragments can be used for sensitive, high-resolution imaging in murine tumor xenograft models using small-animal imaging modalities such as microPET (positron emission tomography). We propose to develop high-avidity multimeric cancer imaging agents utlizing a novel protein multimerization technology. As a model system, single chain antibodies specific for carcinoembryonic antigen (CEA) will be reformatted to generate dimers and tetramers. 1) Following bacterial expression and purification biochemical and binding properties will be determined in vitro. 2) Recombinant antibodies will be labeled with radioiodine or conjugated with a chelating moiety (DOTA) for radiometal labeling for binding and biodistribution studies. Proteins will also be radiolabeled with 1-124 and Cu-64 for microPET imaging studies. In addition, a set of high-avidity multimeric anti-CEA antibody fragments modified with C-terminal Cys residues will be developed for site-specific conjugation and radiolabeling. 3) Performance of the antibody agents will be evaluated in athymic mice bearing CEA-positive (LS1741) and CEA-negative (C6) xenografts, in biodistribution studies and by serial microPET imaging studies in living mice. 4) The tumor targeting biodistribution, and imaging potential of the novel antibody constructs will be evaluated. The dimer and tetramer will be compared to determine whether the high avidity imparted by four binding domains enhances overall in vivo performance. The novel fragments will also be evaluated against existing antibody formats. Knowledge gained through this work will accelerate the development of cancer-specific imaging agents derived from any of a variety of antibodies specific for tumor-associated antigens, for single-photon or PET imaging applications.