The epidermal growth factor receptor (EGFR) is a member of the erbB family of receptors and is overexpressed in many tumor types. A repebody is a newly designed nonantibody protein scaffold for tumor targeting that contains leucine-rich repeat modules. In this study, 3 ⁶⁴Cu-labeled anti-EGFR repebodies with different chelators were synthesized, and their biologic characteristics were assessed in cultured cells and tumor-bearing mice. Methods: Repebodies were synthesized with the chelators 2-(p-isothiocyanatobenzyl)-1,4,7-triazacyclononane-N,N′,N,″-triacetic acid trihydrochloride ([p-SCN-Bn]-NOTA), 2,2′,2″-(10-(2-(2,5-dioxopyrrolidin-1-yloxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl) triacetic acid (DOTA-N-hydroxysuccinimide ester), or 1-(p-isothiocyanatobenzyl)diethylenetriamine pentaacetic acid trihydrochloride ([p-SCN-Bn]-DTPA) in 1.0 M NaHCO₃ buffer (pH 9.2) for 24 h. Purified NOTA-, DOTA-, and DTPA-conjugated repebody were radiolabeled with ⁶⁴Cu in 0.1 M NH₄OAc buffer (pH 5.5). To compare the EGFR-binding affinities of the repebodies, cellular uptake studies were performed with the human non–small cell lung cancer cell line H1650 (high expression of EGFR) and the human colon adenocarcinoma cell line SW620 (low expression of EGFR). Biodistribution and small-animal PET imaging studies were performed using H1650 tumor–bearing mice. Results: Radiochemical yields of the ⁶⁴Cu-labeled repebodies were approximately 70%–80%. Cellular uptake of the NOTA-, DOTA-, and DTPA-repebodies was over 4-fold higher in H1650 cells than in SW620 cells at 1 h. The 3 repebodies had accumulated specifically in H1650 tumor–bearing nude mice by 1 h after intravenous injection and were retained for over 24 h, as measured by the percentage injected dose per gram of tissue (%ID/g). Tumor uptake of all repebodies increased from 1 to 6 h (at 1 h, 6.28, 8.46, and 6.91 %ID/g for NOTA-, DOTA-, and DTPA-repebody, respectively; at 6 h, 9.4, 8.28, and 10.1 %ID/g, respectively). H1650 tumors were clearly visible after injection of each repebody, with high tumor-to-background ratios (at 1 h, 3.43, 4.89, and 2.38 for NOTA-, DOTA-, and DTPA-repebody, respectively; at 6 h, 3.05, 4.36, and 2.08; at 24 h, 3.81, 4.58, and 2.86). Conclusion: The 3 ⁶⁴Cu-repebody complexes demonstrated specific and rapid uptake in EGFR-expressing tumors within 1 h and may have potential as novel EGFR imaging agents for PET.

表皮生长因子受体（EGFR）是erbB受体家族的成员，在许多肿瘤类型中均过表达。repebody是一种新设计的用于肿瘤靶向的非抗体蛋白支架，其中包含富含亮氨酸的重复模块。在这项研究中，合成了3 ^64个具有不同螯合剂的Cu标记的抗EGFR重复序列，并在培养的细胞和荷瘤小鼠中评估了它们的生物学特性。方法：用螯合剂2-（对异硫氰酸根合苄基）-1,4,7-三氮杂环壬烷-N，N'，N，''-三乙酸三盐酸盐（[ p-SCN-Bn] -NOTA），2,2'，2''-（10-（2-（2,5-二氧杂吡咯烷-1-基氧基）-2-氧代乙基）-1,4,7,10-四氮杂环十二烷- 1.0 M NaHCO ₃缓冲液（pH ）中的1,4,7-三基）三乙酸（DOTA- N-羟基琥珀酰亚胺酯）或1-（对-异硫氰酸根合苄基）二亚乙基三胺五乙酸三盐酸盐（[ p -SCN-Bn] -DTPA）9.2）24小时。用⁶⁴ Cu在0.1 M NH _4中对纯化的NOTA-，DOTA-和DTPA共轭重铸抗体进行放射性标记。OAc缓冲液（pH 5.5）。为了比较重复序列的EGFR结合亲和力，对人非小细胞肺癌细胞株H1650（EGFR高表达）和人结肠腺癌细胞系SW620（EGFR低表达）进行了细胞摄取研究。使用荷瘤小鼠H1650进行了生物分布和小动物PET成像研究。结果：^64种的放射化学收率铜标记的重链约占70％–80％。1小时时，H1650细胞中的NOTA，DOTA和DTPA重复序列的细胞摄取比SW620细胞高4倍以上。根据每克组织的注射剂量百分比（％ID / g）测得，静脉注射后1 h时，这3个重排在含H1650荷瘤的裸鼠中特别蓄积，并保留了24 h以上。所有重复序列的肿瘤吸收从1小时增加到6小时（NOTA-，DOTA-和DTPA-重复体分别在1 h，6.28、8.46和6.91％ID / g处；在6 h，9.4、8.28和10.1处）。分别为％ID / g）。注射每个重复体后，H1650肿瘤清晰可见，肿瘤与背景的比率很高（NOTA，DOTA和DTPA重复体分别在1 h，3.43、4.89和2.38； 6 h，3.05， 4.36和2.08；在24小时，3.81、4.58和2.86时）。结论： 3 ⁶⁴ Cu-repebody复合物在1小时内显示了EGFR表达肿瘤的特异性和快速摄取，可能作为PET的新型EGFR成像剂具有潜力。