血管内皮生长因子受体KDR的靶向制剂的制备

目的：研究从噬菌体肽库中筛选到的与血管内皮生长因子受体Ⅱ(KDR)有特异结合活性的小肽，做为KDR靶向药物的先导物质的应用。方法：从噬菌体肽库中筛选能特异结合KDR的噬菌体克隆，挑选结合力最强的克隆测序并化学合成小肽P5，梯度ELISA、阻断实验和竞争结合实验测定小肽在体外与KDR的结合活性。将P5与生物素(NHS-d-Biotin)、BSA化学偶联，ELISA法和细胞免疫组化实验检测偶联物与KDR的结合活性。结果：化学合成的小肽P5在体外能特异结合KDR，Kd=168．6nmol／L，约是KDR与配体血管内皮生长因子(VEGF165)亲和力的1／30，P5能阻断VEGF165与KDR的结合活性，但不能竞争VEGF165与KDR的结合活性。将P5作为导向物质化学合成的P5-BSA-Biotin，在体外同样具有与KDR和细胞表面KDR分子结合的特性。结论：化学合成的小肽P5有望作为先导分子，在以KDR为靶点的肿瘤靶向治疗中得到应用。

Synthesis and Characterization of Chemical Conjugate Targeting KDR

Objective: One strategy for improving the selectively and toxicity profile of antitumor agents is to design drug carrier systems. Thus a reagent targeting KDR expressed on tumor vasculature was prepared by a peptide binding to KDR specifically which screened from C7 peptide library coupled covalently to NHS-d-Biotin and BSA. Methods: A high affinity peptide specific for KDR was screened by phage display. ELISA, Gradient-ELISA, Competitive-ELISA and Blocking-ELISA were used to detect whether synthesized peptide bind to KDR. To explore whether peptide could be used to deliver agent to target site, synthesized peptide was chemically conjugated with large molecule-BSA and NHS-d-Biotin easily detected by avidin. The binding activity to KDR of chemical compound was determined by Cell-ELISA and Cell-immunohistology. Results: Synthesized P5 peptide having a dissociation constant (Kd) of about 168.6 nM with KDR was approximately 3 fold lower than Kd of VEGF with KDR. P5 blocked VEGF-KDR interaction while did not competes effectively with VEGF for binding KDR. The chemical conjugate, P5-BSA-Biotin, binds specifically to soluble KDR as well as KDR expressed on human endothelial cells. Conclusion: The peptide P5 that home to KDR expressed on tumor vasculature may also be useful in targeting therapies specifically to tumors.