Selective tumor inhibition by tumor-homing angiogenesis-suppressing nanofibers

 描述(申请人提供)：血管生成是癌症进展过程中的一个重要过程。在没有血管支持的情况下，癌细胞会变得坏死甚至凋亡。因此，抗血管生成治疗成为治疗癌症的一种很有前途的策略。血管生成素(Angigenin，Ang)是一种主要由癌细胞分泌的血管生成蛋白。阻断其与血管内皮细胞表面血管紧张素受体的相互作用可使其功能丧失 诱导癌症血管生成，从而抑制癌症的进展。虽然一些血管紧张素转换酶抑制剂已经被FDA开发并批准用于阻断血管紧张素转换酶与其受体之间的相互作用来治疗癌症，但这些抑制剂缺乏肿瘤归巢能力，可能会导致严重的副作用，如出血和高血压。为了解决这个问题，我们建议开发一种人类安全的基于噬菌体的肿瘤归巢Ang抑制剂，以改进癌症抗血管生成治疗。本申请的目的是(I)通过主要的衣壳噬菌体展示技术，鉴定能够选择性结合和干扰Ang的正常功能的Ang结合肽；以及(Ii)通过在同一纳米纤维的顶端双重展示我们最近发现的乳腺肿瘤归巢肽，以及在同一纳米纤维的侧壁双重展示我们最近发现的Ang结合肽，构建双功能重组噬菌体纳米纤维。由此产生的噬菌体纳米纤维表面既有肿瘤归巢又有Ang结合肽，有望首先与乳腺肿瘤结合，然后与肿瘤内的Ang结合，抑制肿瘤血管生成。我们的假设是：(1)Ang结合肽可以从主要的外壳展示噬菌体文库中鉴定出来，(2)双展示Ang结合和肿瘤归巢的双功能噬菌体纳米纤维可以作为一种智能的血管生成抑制因子选择性地定位和保留在乳腺肿瘤中，并与Ang结合来阻断肿瘤的血管生成，从而抑制肿瘤的生长。我们将实现两个目标来验证我们的假设：目标1：通过对Ang进行生物扫描，从主要的外衣展示噬菌体文库中鉴定Ang结合肽。我们将利用噬菌体展示技术对商业购买的Ang进行生物扫描，并从主要的外衣展示噬菌体文库中选择能够与Ang特异靶向和结合的Ang结合肽。通过噬菌体捕获酶联免疫吸附试验、多肽抑制试验和噬菌体-血管紧张素相互作用试验来验证所选择的多肽的血管紧张素结合特异性。目的：构建双功能乳腺肿瘤归巢/血管紧张素结合噬菌体，并评价其选择性抑制肿瘤生长的作用。我们将使用我们建立的双展示技术在单噬菌体纳米纤维上展示乳腺肿瘤归巢肽和血管紧张素结合肽，并评价其体外抗肿瘤活性。然后我们将该噬菌体注射到乳腺肿瘤模型中，以验证该噬菌体可以与肿瘤细胞结合，与肿瘤细胞分泌的Ang结合，从而抑制血管生成，抑制肿瘤生长。该项目将导致肿瘤归巢和血管结合纳米纤维，可以作为纳米药物，选择性地抑制癌症血管生成和进展，而不会引起副作用。

 DESCRIPTION (provided by applicant): Angiogenesis is an important process in the progression of cancer. In the absence of vascular support, cancer cells will become necrotic or even apoptotic. Therefore, antiangiogenic therapy becomes a promising strategy for treating cancers. Angiogenin (Ang) is a protein predominantly secreted by cancer cells for angiogenesis. Blocking its interaction with Ang receptors on endothelial cell surface can disable its function of inducing cancer angiogenesis and thus inhibit the progression of cancers. Although some Ang inhibitors have been developed and even approved by the FDA for blocking the interactions between Ang and its receptors to treat cancer, these inhibitors lack the tumor-homing capability and may lead to serious side effects, such as bleeding and hypertension. To address this problem, we propose to develop a human-safe phage-based tumor-homing Ang inhibitor for improved cancer antiangiogenic therapy. The objectives of this Application are (i) to identify Ang- binding peptides that can selectively bind to and interfere with the regular functions of Ang by major coat phage display technique; and (ii) to construct a dual-functional recombinant phage nanofiber by double-displaying our recently discovered breast tumor-homing peptide at the tip and the identified Ang-binding peptide on the side- walls of the same nanofiber. The resultant phage nanofiber displaying both tumor-homing and Ang-binding peptides on its surface is expected to first home to breast tumors and then bind to the Ang within the tumors to inhibit the tumor angiogenesis. Our hypotheses are that (1) Ang-binding peptides can be identified from a major coat-displayed phage library, and (2) the dual-functional tumor-homing/Ang-binding phage nanofibers double- displaying Ang-binding and tumor-homing peptides can serve as a smart angiogenic inhibitor to selectively home to and retain within breast tumors, and bind Ang to block the tumor angiogenesis and subsequently inhibit the tumor growth. We will carry out two aims to test our hypothesis: Aim 1: Identify Ang-binding peptides from a major coat-displayed phage library by biopanning against Ang. We will use phage display technique to conduct biopanning against commercially purchased Ang and select Ang-binding peptides that can specifically target and bind to Ang from the major coat-displayed phage library. The Ang-binding specificity of the selected peptides will be verified by phage capture ELISA, peptide inhibition assay, and phage-Ang interaction assay. Aim 2: Construct dual functional breast tumor-homing/Ang-binding phage and evaluate its use in selectively inhibiting tumor growth. We will use our established double display technique to display both the breast tumor-homing and Ang- binding peptides on the single phage nanofiber and evaluate its antitumor activity in vitro. We will then inject the phage into the breast tumor models to verify that the phage can home to tumor, bind to Ang secreted by tumor cells to suppress angiogenesis and inhibit tumor growth. This project will lead to tumor-homing Ang-binding nanofibers that can serve as nanomedicines for selectively inhibiting cancer angiogenesis and progression without causing the side effects.