Phage Display for Improved Peptide-based Tumor Targeting and Imaging Agents

描述(由申请人提供)： 癌症是美国和美国退伍军人的第二大死因。2009年，美国退伍军人中超过28%的癌症诊断是前列腺癌、乳腺癌和卵巢癌。然而，这些癌症在疾病的初期通常是无症状的。前列腺癌是男性退伍军人的主要健康问题，据估计，超过25%的人已经患有前列腺癌。与性别相关的乳腺癌和卵巢癌也令人担忧，特别是因为每15名退伍军人中就有一名是女性。为了更好地满足我们女退伍军人日益增长的医疗需求，已经建立了退伍军人中心。虽然这些癌症的筛查和检测方法已经存在，但它们都有局限性。可以预见，使用专门针对癌症和转移瘤的抗原的分子将导致改进癌症检测和治疗方式。放射性标记抗体和多肽目前正在被探索作为癌症诊断显像剂。然而，作为体内肿瘤显像剂，大分子抗体和小肽可能并不理想。只有少数放射性标记抗体和多肽具有作为肿瘤显像剂所需的高亲和力、特异性、稳定性和肿瘤摄取。因此，寻找新的癌症靶向载体和癌症相关抗原是该领域的中心目标。毫无疑问，如果退伍军人管理局要应对我们过去、现在和未来军事人员的医疗挑战，那么开发可以安全和非侵入性地用于检测和治疗癌症的特定肿瘤靶向分子是一个重要的优先事项。由于噬菌体(噬菌体)展示方法的实施，新的基于多肽的分子探针正在迅速演变，以促进癌症成像。虽然放射性标记多肽在体外表现出良好的肿瘤靶向性，但它们进入临床的速度因肿瘤保留不佳以及几乎普遍存在的高肾摄取和保留而变得缓慢。我们利用噬菌体展示技术获得了针对肿瘤相关ErbB-2受体Galectin-3(GAL-3)及其碳水化合物配体Thomsen-Friedenreich(TF)抗原的多肽，这些多肽与癌细胞的黏附和信号转导有关。我们推测，结合ErbB-2、GAL-3和Tf的多肽支架一旦被放射性标记，将形成新型单光子发射计算机断层扫描(SPECT)和正电子发射计算机断层扫描(PET)显像剂的基础，用于前列腺、乳腺和卵巢肿瘤。我们将扩大噬菌体展示的应用，利用体内创新的功能选择方法来提高肿瘤靶向放射性标记多肽的成像和潜在的治疗效果。放射性标记的结合物可用作诊断放射性药物，用于检测原发癌和转移癌以及治疗反应的指示物。从长远来看，这些多肽可能在体内发挥作用，通过阻断参与肿瘤发生的信号通路来减少肿瘤的生长和转移。将采用不同的分子生物学、蛋白质工程、放射化学和光学成像方法。本研究的目的是：1)根据ErbB-2和Gal-3的靶向序列在小鼠体内筛选噬菌体展示文库，以鉴定具有高肿瘤摄取率和低肾脏滞留率的噬菌体和相应的多肽；2)将优化的多肽基序设计成稳定的小分子半胱氨酸约束支架，以提高体内稳定性、亲和力和快速排泄； 3)比较111铟-放射性标记ErbB-2-、Gal-3-和Tf-靶向线性多肽和工程多肽支架在至少一个合适的肿瘤模型(如乳腺、前列腺或卵巢)中的体内SPECT成像效率；以及4)开发64个铜标记多肽对应物，用于敏感的PET成像。这项工作对退伍军人管理局的任务意义重大，因为这里发现的多肽可能会转化为我们退伍军人的前列腺癌、乳腺癌和卵巢癌的新诊断和治疗方法。 公共卫生相关性： 前列腺癌是美国退伍军人中最常被诊断出的癌症之一。退伍军人管理局最近关注的是与女性相关的癌症，包括乳腺癌和卵巢癌。由于近10%的女性退伍军人人口，与女性癌症相关的研究和保健现在是退伍军人管理局的优先事项。如果在转移发生之前及早发现，这些癌症是可以治疗的。不幸的是，标准的筛查测试要么不具特异性，要么难以管理，要么昂贵。因此，需要更简便和更具体的手段来检测这些癌症。我们的研究与退伍军人管理局的使命相关，因为我们正在开发基于多肽支架的创新肿瘤靶向试剂，用于前列腺癌、乳腺癌和卵巢癌的早期放射成像检测。这些药物将针对这些癌症上过度表达的肿瘤相关抗原ErbB-2和Galectin-3/Thomsen-Friedenreich。这种放射显像剂专门针对癌细胞上的分子，在患者中具有很好的耐受性，并且不会充满有害的副作用。

DESCRIPTION (provided by applicant): Cancer is the second leading cause of death in the United States and US Veterans. Over 28% of cancer diagnoses in US Veterans in 2009 were for prostate, breast, and ovarian carcinoma. However, these cancers are often asymptomatic in beginning stages of the disease. Prostate cancer is a major health problem in male Veterans and it has been estimated that over 25% already have prostate cancer. Gender-associated breast and ovarian cancers are also of concern, especially since one out of every 15 Veterans is female. A VA Center for Women has been established in order to better address the rising medical needs of our female Veterans. While screening and detection methods exist for these cancers, they all have limitations. It can be envisioned that the use of molecules that specifically target antigens on carcinomas and metastases will lead to improved cancer detection and treatment modalities. Radiolabeled antibodies and peptides are currently being explored as diagnostic cancer imaging agents. However, high molecular weight antibodies as well as small peptides may not be ideal as cancer imaging agents in vivo. Only a small number of radiolabeled antibodies and peptides possess the requisite high affinity, specificity, stability, and tumor uptake to serve as cancer imaging agents. Thus, finding new cancer targeting vehicles and cancer-associated antigens is a central goal of the field. Development of specific tumor-targeting molecules that can be used safely and non- invasively to detect and treat cancer is undeniably an important priority for the Veterans Administration if it is to meet the health care challenges of our past, current, and future military personnel. New peptide-based molecular probes to facilitate cancer imaging are rapidly evolving due to implementation of bacteriophage (phage) display approaches. While radiolabeled peptides have shown good tumor-targeting propensity in vitro, their translation into the clinic has been slowed by sub-optimal tumor retention and almost universal high renal uptake and retention. We have used phage display to obtain peptides that target the tumor-associated ErbB-2 receptor, galectin-3 (gal-3) and its carbohydrate ligand Thomsen- Friedenreich (TF) antigen, which are involved in cancer cell adhesion and signaling. We hypothesize that peptides that bind ErbB-2, gal-3, and TF grafted into constrained loops of polypeptide scaffolds once radiolabeled, will form the foundation for novel single photon emission computed tomography (SPECT) and positron emission computed tomography (PET) imaging agents for prostate, breast, and ovarian tumors. We will expand the applications of phage display to employ innovative functional selection approaches in vivo to improve the imaging and, potentially, therapeutic efficacy of the tumor-targeting radiolabeled peptides. The radiolabeled conjugates may serve as diagnostic radiopharmaceuticals for the detection of primary and metastatic cancer and indicators of response to therapy. In the long term, the peptides may function in vivo to reduce tumor growth and metastasis by blocking signaling pathways involved in tumorigenesis. Diverse molecular biology, protein engineering, radiochemistry, and optical imaging approaches will be employed. The objectives of this proposed research are to: 1) select phage display libraries based on the ErbB-2 and gal-3-targeting sequences in mice in order to identify phage and corresponding peptides with high tumor uptake and low kidney retention; 2) engineer the optimized peptide motifs into stable small molecular weight cysteine-constrained scaffolds for enhanced in vivo stability, affinity, and rapid excretion; 3) compare the in vivo SPECT imaging efficacy of 111Indium-radiolabeled ErbB-2-, gal-3-, and TF- targeting linear peptides and engineered peptide scaffolds in at least one appropriate tumor model (i.e. breast, prostate or ovary); and 4) develop 64Copper-labeled peptide counterparts for sensitive PET imaging. This work is significant and relevant to the VA mission because the peptides discovered here may translate into novel prostate, breast, and ovarian cancer diagnostics and therapeutics for our veterans. PUBLIC HEALTH RELEVANCE: Prostate cancer is one of the most frequently diagnosed cancers among United States Veteran males. Of recent concern to the VA are female-related cancers including breast and ovarian cancer. Research and health care related to female cancers is now a VA priority due to the almost 10% female Veteran population. These cancers are treatable if detected early, before metastasis occurs. Unfortunately, standard screening tests are either not specific, difficult to administer, or expensive. Thus, facile and more specific means to detect these, cancers are needed. Our research is relevant to the Veterans Administration Mission in that we are developing innovative tumor-targeting agents based on peptide scaffolds for early radioimaging detection of prostate, breast, and ovarian cancer. The agents will target the tumor-associated antigens, ErbB-2 and galectin- 3/Thomsen-Friedenreich that are overexpressed on these cancers. Being specifically targeted to molecules on cancer cells, the radioimaging agents will be well tolerated in patients and not fraught with harmful side effects.