Selective tumor inhibition by tumor-homing angiogenesis-suppressing nanofibers

 描述（由申请人提供）：血管生成是癌症进展中的重要过程。在缺乏血管支持的情况下，癌细胞会坏死甚至凋亡。因此，抗血管生成疗法成为治疗癌症的有希望的策略。血管生成素（Angiogenin，Ang）是一种主要由癌细胞分泌的用于血管生成的蛋白质。阻断其与内皮细胞表面Ang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抑制剂已经被开发出来，甚至被FDA批准用于阻断血管紧张素及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，并且可能导致严重的副作用，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血管紧张素抑制剂，以改善癌症抗血管生成治疗。本申请的目的是（i）通过主要外壳噬菌体展示技术鉴定可以选择性结合并干扰Ang的常规功能的Ang结合肽;和（ii）通过在同一噬菌体的顶端双展示我们最近发现的乳腺肿瘤归巢肽和在侧壁上双展示所鉴定的Ang结合肽来构建双功能重组噬菌体。在其表面上展示肿瘤归巢肽和Ang结合肽的所得噬菌体抗体预期首先归巢到乳腺肿瘤，然后结合肿瘤内的Ang以抑制肿瘤血管生成。我们的假设是：（1）Ang-binding peptides可以从主要的外壳展示噬菌体文库中鉴定，以及（2）双重展示Ang-binding和肿瘤-归巢肽的双功能肿瘤归巢/Ang-binding噬菌体纳米纤维可以作为智能血管生成抑制剂选择性地归巢并保留在乳腺肿瘤内，并结合Ang以阻断肿瘤血管生成并随后抑制肿瘤生长。我们将进行两个目标来验证我们的假设：目标1：通过生物淘选从主要的噬菌体展示库中筛选出Ang结合肽。我们将利用噬菌体展示技术对市售Ang进行生物淘选，并从主要的外套展示噬菌体库中选择能够特异性靶向并结合Ang的Ang结合肽。所选肽的Ang结合特异性将通过噬菌体捕获ELISA、肽抑制测定和噬菌体-Ang相互作用测定来验证。目的2：构建具有导向/结合Ang-binding双重功能的乳腺癌噬菌体，并评价其选择性抑制肿瘤生长的作用。我们将利用我们建立的双展示技术在单个噬菌体上展示乳腺肿瘤归巢肽和Ang结合肽，并评估其体外抗肿瘤活性。我们将噬菌体注射到乳腺肿瘤模型中，以验证噬菌体可以归巢肿瘤，与肿瘤细胞分泌的Ang结合，抑制血管生成，抑制肿瘤生长。该项目将导致肿瘤归巢Ang-binding纳米纤维，可以作为选择性抑制癌症血管生成和进展而不会引起副作用的纳米药物。

 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.