Improved Peptide-based Tumor Targeting Agents Using Phage Display

描述（由申请人提供）：虽然在早期阶段可以治疗，但乳腺癌、肺癌和卵巢癌的转移形式通常是致命的。癌症存活率的提高将源于一种更直接的方法，即靶向肿瘤上过度表达的抗原。尽管在基于抗体的放射性药物发现中正在利用有限数量的肿瘤生物标志物，但仍然迫切需要确定新的靶向载体和生物标志物。基于肽的探针促进癌症的检测和治疗正在从组合化学和噬菌体（噬菌体）展示方法迅速发展。许多放射性标记肽和小蛋白在体内显示出良好的肿瘤靶向倾向，然而，由于它们几乎普遍的高肾吸收和保留，它们转化为临床有用的药物的速度很慢。虽然观察到的摄取和保留机制尚不清楚，但人们认为带正电荷的肽结合带负电荷的肾细胞导致高剂量的肾辐射。目前阻止这种保留的技术，包括赖氨酸或利尿剂的使用，效果甚微，而且有有害的副作用。拟议研究的总体目标是采用创新的功能选择噬菌体展示方法来改善肿瘤靶向放射性标记肽的成像和潜在的治疗效果。我们假设，体内噬菌体展示可以用来识别噬菌体和相应的肽，这些肽在尿液中迅速排出，而不是保留在肾脏中或通过网状内皮系统被清除。这些肽序列将被附加到肿瘤靶向肽中，并检查其减少肾潴留的能力。噬菌体展示和亲和成熟研究也将在肿瘤靶向微文库中进行，以产生具有改善肿瘤结合和减少体内肾潴留的肽。我们选择了从噬菌体展示中发现的ErbB-2受体靶向肽KCCYSL作为模型系统。先前的研究表明，放射性标记的KCCYSL结合Erbb-2受体和体内成像的乳腺癌，尽管观察到高放射性标记的肾脏摄取和保留。这种特征明确的肽将成为这些研究的重点，因为它最初是在噬菌体的背景下被发现的，我们知道当它在噬菌体上表达或化学合成时，肽保持其肿瘤靶向特性。此外，ErbB-2的过表达和二聚化激活了促进乳腺、肺和卵巢肿瘤发生的几种信号通路，使其成为开发新的癌症成像和治疗剂的一个有吸引力的靶点。本研究的目的是：1)开发新的策略来选择非荷瘤小鼠的噬菌体展示文库，以鉴定快速排泄到尿液中的噬菌体和相应的肽；2)在乳腺荷瘤小鼠体内亲和力成熟15个氨基酸噬菌体文库，显示ErbB-2最小结合基序（CCYSL），以鉴定高肿瘤摄取和低肾保留的肽；3)检测111in放射性标记的ErbB-2靶向肽构建物的单光子发射计算机断层成像效果。这项工作与NCI的使命和患者护理高度相关，因为所发现的肽将为开发新的耐受性良好的靶向药物奠定基础，用于检测和治疗乳腺癌、卵巢癌和肺癌。公共卫生相关性：用于靶向癌症检测和治疗的肽和小分子放射性药物的发展受到肾脏几乎普遍摄取和保留肽和小分子的阻碍。我们的工作是相关的，使用高度创新的噬菌体展示技术将发现新的分子，通过选择噬菌体和相应的展示肽来减少肾中放射性标记肽的摄取，这些肽绕过网状内皮系统的清除并迅速从尿液中排出。这些发现的分子是否能够减少肾脏对放射性标记肽的摄取，这些肽与乳腺、卵巢和肺部肿瘤上过度表达的ErbB-2受体结合，这些能力将在活体动物中进行研究。

DESCRIPTION (provided by applicant): While treatable in their earliest stages, metastatic forms of breast, lung, and ovarian cancer are often lethal. Improved cancer survival will result from a more directed approach in which antigens overexpressed on tumors are targeted. Even though a limited number of tumor biomarkers are being exploited in antibody-based radiopharmaceutical drug discovery, there remains a critical need to identify new targeting vehicles and biomarkers. Peptide-based probes to facilitate cancer detection and therapy are rapidly evolving from combinatorial chemistry and bacteriophage (phage) display approaches. Many radiolabeled peptides and small proteins have shown good tumor-targeting propensity in vivo, however their translation into clinically useful agents has been slowed by their almost universal high renal uptake and retention. While the mechanisms for the observed uptake and retention are not clear, it is believed that positively charged peptides bind negatively charged kidney cells resulting in high doses of kidney radiation. Current techniques to block this retention, including lysine or diuretic administration are marginally effective and have harmful side effects. The overall goal of the proposed research is to employ innovative functional selection phage display approaches to improve the imaging and, potentially, therapeutic efficacy of tumor-targeting radiolabeled peptides. We hypothesize that in vivo phage display can be exploited to identify phage and corresponding peptides that are rapidly excreted in the urine and not retained in the kidneys or cleared through the reticuloendothelial system. These peptide sequences will be appended to tumor-targeting peptides and examined for their abilities to reduce renal retention. Phage display and affinity maturation studies will also be performed with a tumor-targeting micro-library, to generate peptides with improved tumor binding and reduced kidney retention in vivo. We have chosen as a model system the ErbB-2 receptor (ErbB-2)-targeting peptide, KCCYSL, discovered from phage display. Previous studies demonstrated that radiolabeled versions of KCCYSL bound the Erbb-2 receptor and imaged breast carcinomas in vivo, although high radiolabel kidney uptake and retention was observed. This well-characterized peptide will be the focus of these studies because it was originally identified in the context of phage and we know that the peptide retains its tumor targeting properties when it is expressed on phage or chemically synthesized. Furthermore, ErbB-2 overexpression and dimerization activates several signaling pathways that promote breast, lung, and ovarian tumorigenesis- making it an attractive target for development of new cancer imaging and therapeutic agents. The objectives of this proposed research are to: 1) develop novel strategies to select phage display libraries in non-tumor bearing mice in order to identify phage and corresponding peptides that are rapidly excreted into the urine; 2) in vivo affinity maturate 15 amino acid phage libraries displaying the ErbB-2 minimum binding motif, CCYSL, in breast tumor bearing mice to identify peptides with high tumor uptake and low kidney retention, and 3) examine the single photon emission computed tomography imaging efficacy of 111In-radiolabeled ErbB-2-targeting peptide constructs. This work is highly relevant to the mission of NCI and patient care in that the discovered peptides will form the foundation for new, well tolerated targeting agents for the detection and potential treatment of breast, ovary, and lung cancer. PUBLIC HEALTH RELEVANCE: The development of peptide and small molecule-based radiopharmaceutical for targeted cancer detection and therapy is hindered by the almost universal uptake and retention of peptides and small molecules in the kidneys. Our work is relevant in that new molecules will be discovered using highly innovative phage display techniques to reduce radiolabeled peptide uptake in the kidney by selecting phage and corresponding displayed peptides that bypass clearance through the reticuloendothelial system and are rapidly excreted in the urine. The ability of the discovered molecules to reduce kidney uptake of radiolabeled peptides that bind the ErbB-2 receptor overexpressed on breast, ovarian, and lung tumors will be investigated in living animals.