Improved Peptide-based Tumor Targeting Agents Using Phage Display

描述(申请人提供)：虽然在早期阶段是可以治疗的，但转移性乳腺癌、肺癌和卵巢癌往往是致命的。通过一种更直接的方法来提高癌症存活率，在这种方法中，肿瘤上过度表达的抗原是有针对性的。尽管在基于抗体的放射性药物发现中使用的肿瘤生物标记物数量有限，但仍然迫切需要识别新的靶向载体和生物标记物。有助于癌症检测和治疗的基于多肽的探针正在从组合化学和噬菌体(噬菌体)展示方法迅速演变。许多放射性标记的多肽和小蛋白在体内表现出良好的肿瘤靶向性，但由于它们几乎普遍存在于肾脏的高摄取和滞留，它们转化为临床有用的药物的速度慢了下来。虽然观察到的摄取和滞留的机制尚不清楚，但人们认为带正电的多肽与带负电的肾细胞结合会导致高剂量的肾脏辐射。目前阻止这种滞留的技术，包括赖氨酸或利尿剂给药，都是略微有效的，而且有有害的副作用。拟议研究的总体目标是使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多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被用来识别在尿液中快速排泄的噬菌体和相应的肽，而不是滞留在肾脏中或通过网状内皮系统清除的噬菌体和相应的肽。这些多肽序列将被附加到肿瘤靶向多肽上，并检查它们减少肾脏滞留的能力。还将利用肿瘤靶向微库进行噬菌体展示和亲和力成熟研究，以产生具有更好的肿瘤结合和减少体内肾脏滞留的多肽。我们选择了从噬菌体展示中发现的ErbB-2受体(ErbB-2)靶向肽KCCYSL作为模型系统。以前的研究表明，KCCYSL的放射性标记版本与ErbB-2受体结合，并在体内成像乳腺癌，尽管观察到高放射性标记肾脏摄取和滞留。这个特性良好的多肽将是这些研究的重点，因为它最初是在噬菌体的背景下发现的，而且我们知道，当它在噬菌体上表达或化学合成时，它仍然具有肿瘤靶向性。此外，ErbB-2的过度表达和二聚化激活了几个促进乳腺癌、肺癌和卵巢肿瘤发生的信号通路-使其成为开发新的癌症成像和治疗药物的有吸引力的靶点。本项拟议研究的目的是：1)开发新的策略来选择非荷瘤小鼠的噬菌体展示文库，以确定快速排泄到尿液中的噬菌体及相应的多肽；2)在体内亲和成熟15个氨基酸的噬菌体文库，展示ErbB-2的最小结合基序CCYSL，以确定具有高肿瘤摄取和低肾脏滞留的多肽；以及3)检测111 In标记的ErbB-2靶向多肽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，因为所发现的多肽将为新的、耐受性良好的靶向药物奠定基础，用于检测和潜在治疗乳腺癌、卵巢癌和肺癌。与公共卫生相关：基于多肽和小分子的放射性药物用于靶向癌症检测和治疗的发展受到几乎普遍的多肽和小分子在肾脏中的摄取和滞留的阻碍。我们的工作是相关的，因为将使用高度创新的噬菌体展示技术发现新的分子，通过选择绕过网状内皮系统清除并在尿液中快速排泄的噬菌体和相应的展示肽来减少肾脏对放射性标记的肽的摄取。将在活体动物身上研究发现的分子减少肾摄取放射性标记多肽的能力，这些多肽结合在乳腺、卵巢和肺肿瘤上过度表达的ErbB-2受体。

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.