Identification of CD21-binding peptides with phage display and investigation of binding properties of HPMA copolymer-peptide conjugates
Identification of CD21-binding peptides with phage display and investigation of binding properties of HPMA copolymer-peptide conjugates
复制标题
DOI：
10.1021/bc0503162
复制
发表时间：
2006-03-01
影响因子：
4.7
通讯作者：
Kopecek, J
中科院分区：
文献类型：
--
作者：
Ding, H;Prodinger, WM;Kopecek, J
随着组合肽技术如噬菌体展示的出现，用肽靶向癌症变得很有希望。在严格筛选条件下使用噬菌体展示，我们选择了五种特异性识别CD 21受体（恶性B细胞淋巴瘤的细胞表面标志物）的不同肽。排除了两个高度疏水的序列（RLAYWCFSGLFLLVC和PVAAVSFVPYLVKTY）。用荧光猝灭分析其它三种选定肽（RMWPSSTVNLSAGRR、PNLDFSPTCSFRFGC和GRVPSMFGGHFFFSR）对CD 21的结合亲和力。它们的解离常数被确定为在微摩尔范围内。基于噬菌体ELISA、竞争性噬菌体ELISA和荧光猝灭的结果，发现所选的三种肽的结合位点位于CD 21（SCRI-4）的前四个短共有重复序列内。将多肽RMWPSSTVNLSAGRR（P1）与N-（2-羟丙基）甲基丙烯酰胺（HPMA）共聚物（一种潜在的化疗药物载体）结合，研究了HPMA共聚物-P1结合物的表面结合性质。观察到HPMA共聚物-P1缀合物与表面结合受体之间的特异性相互作用。HPMA共聚物-P1缀合物的结合与表面（MaxiSorp板）结合受体的量直接相关，并且缀合物的结合可以通过施加超过缀合物中肽浓度3-4个数量级的过量游离肽来抑制。聚合物结合肽的结合增强归因于HPMA共聚物-P1缀合物与表面结合的CD 21受体之间的多价相互作用。
Cancer targeting with peptides has become promising with the emergence of combinatorial peptide techniques such as phage display. Using phage display under stringent screening conditions, we selected five distinct peptides that specifically recognized the CD21 receptor, a cell surface marker of malignant B cell lymphoma. Two highly hydrophobic sequences were excluded (RLAYWCFSGLFLLVC and PVAAVSFVPYLVKTY). The binding affinity toward CD21 of the other three selected peptides (RMWPSSTVNLSAGRR, PNLDFSPTCSFRFGC, and GRVPSMFGGHFFFSR) was analyzed with fluorescence quenching. Their dissociation constants were determined to be within the micromolar range. On the basis of the results of phage ELISA, competitive phage ELISA, and fluorescence quenching, the binding sites of the three selected peptides were found to reside within the first four short consensus repeats of CD21 (SCRI-4). The peptide RMWPSSTVNLSAGRR (P1) was bound to the N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer, a potential drug carrier for chemotherapeutic agents, and the surface binding properties of HPMA copolymer-P1 conjugates were investigated. Specific interactions were observed between HPMA copolymer-P1 conjugates and surface-bound receptor. Binding of HPMA copolymer-P1 conjugates was directly related to the amount of surface (MaxiSorp plate) bound receptor, and the binding of the conjugates could be inhibited by the application of a 3-4 orders-of-magnitude excess of free peptide over the peptide concentration in conjugates. The enhanced binding of polymer-bound peptide was ascribed to multivalent interactions between the HPMA copolymer-P1 conjugate and the surface-bound CD21 receptor.