Department of Nuclear Medicine, Ruijin Hospital, Shanghai Jiaotong University, Shanghai, China.
Department of Radiology, Molecular Imaging Program at Stanford (MIPS), Cancer Center, Bio-X Program, Stanford University, Stanford, California.
J Nucl Med. 2010 Feb;51(2):251-258. doi: 10.2967/jnumed.109.069831.
Recently, a truncated form of the agouti-related protein (AgRP), a 4-kDa cystine-knot peptide of human origin, was used as a scaffold to engineer mutants that bound to alpha(v)beta(3) integrin with high affinity and specificity. In this study, we evaluated the potential of engineered integrin-binding AgRP peptides for use as cancer imaging agents in living subjects.
Engineered AgRP peptides were prepared by solid-phase peptide synthesis and were folded in vitro and purified by reversed-phase high-performance liquid chromatography. Competition assays were used to measure the relative binding affinities of engineered AgRP peptides for integrin receptors expressed on the surface of U87MG glioblastoma cells. The highest-affinity mutant, AgRP clone 7C, was site-specifically conjugated with 1,4,7,10-tetra-azacyclododecane-N,N',N''N'''-tetraacetic acid (DOTA). The resulting bioconjugate, DOTA-AgRP-7C, was radiolabeled with (64)Cu for biodistribution analysis and small-animal PET studies in mice bearing U87MG tumor xenografts. In addition to serum stability, the in vivo metabolic stability of (64)Cu-DOTA-AgRP-7C was assessed after injection and probe recovery from mouse kidney, liver, tumor, and urine.
AgRP-7C and DOTA-AgRP-7C bound with high affinity to integrin receptors expressed on U87MG cells (half maximal inhibitory concentration values, 20 +/- 4 and 14 +/- 2 nM, respectively). DOTA-AgRP-7C was labeled with (64)Cu with high radiochemical purity (>99%). In biodistribution and small-animal PET studies, (64)Cu-DOTA-AgRP-7C displayed rapid blood clearance, good tumor uptake and retention (2.70 +/- 0.93 percentage injected dose per gram [%ID/g] and 2.37 +/- 1.04 %ID/g at 2 and 24 h, respectively), and high tumor-to-background tissue ratios. The integrin-binding specificity of (64)Cu-DOTA-AgRP-7C was confirmed in vitro and in vivo by showing that a large molar excess of the unlabeled peptidomimetic c(RGDyK) could block probe binding and tumor uptake. Serum stability and in vivo metabolite assays demonstrated that engineered AgRP peptides are sufficiently stable for in vivo molecular imaging applications.
A radiolabeled version of the engineered AgRP peptide 7C showed promise as a PET agent for tumors that express the alpha(v)beta(3) integrin. Collectively, these results validate AgRP-based cystine-knot peptides for use in vivo as molecular imaging agents and provide support for the general use of AgRP as a scaffold to develop targeting peptides, and hence diagnostics, against other tumor receptors.
最近，使用一种截短形式的刺鼠相关蛋白（AgRP）作为支架来设计突变体，该突变体与 αvβ3 整合素具有高亲和力和特异性结合。在本研究中，我们评估了工程化整合素结合 AgRP 肽作为活体研究中癌症成像剂的潜力。
通过固相肽合成制备工程化的 AgRP 肽，并在体外折叠和通过反相高效液相色谱法进行纯化。竞争测定用于测量工程化 AgRP 肽对 U87MG 神经胶质瘤细胞表面表达的整合素受体的相对结合亲和力。最高亲和力的突变体 AgRP 克隆 7C 被特异性地与 1,4,7,10-四氮杂环十二烷-N,N',N'',N'''-四乙酸（DOTA）缀合。所得的生物缀合物 DOTA-AgRP-7C 用（64）Cu 标记用于生物分布分析和携带 U87MG 肿瘤异种移植物的小鼠的小动物 PET 研究。除了血清稳定性之外，还评估了（64）Cu-DOTA-AgRP-7C 在注射后和从小鼠肾脏、肝脏、肿瘤和尿液中回收探针后的体内代谢稳定性。
AgRP-7C 和 DOTA-AgRP-7C 与 U87MG 细胞上表达的整合素受体具有高亲和力（半最大抑制浓度值分别为 20 ± 4 和 14 ± 2 nM）。DOTA-AgRP-7C 用（64）Cu 标记，具有高放射化学纯度（> 99%）。在生物分布和小动物 PET 研究中，（64）Cu-DOTA-AgRP-7C 表现出快速的血液清除，良好的肿瘤摄取和保留（2 小时和 24 小时分别为 2.70 ± 0.93%注入剂量/克[ID/g]和 2.37 ± 1.04%ID/g），并且肿瘤与背景组织的比值很高。（64）Cu-DOTA-AgRP-7C 的整合素结合特异性在体外和体内均得到证实，表明大量未标记的肽模拟物 c（RGDyK）可以阻断探针结合和肿瘤摄取。血清稳定性和体内代谢物测定表明，工程化的 AgRP 肽在体内分子成像应用中足够稳定。
工程化 AgRP 肽 7C 的放射性标记版本有望成为表达 αvβ3 整合素的肿瘤的 PET 试剂。总之，这些结果验证了基于 AgRP 的半胱氨酸结肽在体内用作分子成像剂的用途，并为 AgRP 作为支架开发针对其他肿瘤受体的靶向肽以及诊断试剂提供了支持。
