通过调整药物结合物的分子大小、价数和药代动力学特征，可以改善其治疗窗口，特别是通过影响肿瘤渗透、肾脏清除和短暂的全身暴露。在这方面，小分子肿瘤靶向配体备受关注。本研究中，我们展示了小型Nanofitin替代支架（7 kDa）作为选择性肿瘤靶向模块用于生成药物结合物的好处，重点关注针对上皮生长因子受体（EGFR）的Nanofitins B10和D8。由于其小尺寸和单价格式，这两种Nanofitins在BALB/c裸鼠的EGFR阳性A431异种移植瘤中经静脉注射后表现出快速而深入的肿瘤渗透，免疫组织化学结果显示针对肿瘤细胞的靶向效率分别为67.9%±14.1和98.9%±0.7。与单甲基艾霉素E毒素的结合提供了均匀的Nanofitin-药物结合物，在EGFR阳性的BALB/c裸鼠A431细胞中进行体内评估，在治愈性异种移植模型中，使用生物发光方式监测2个月内肿瘤体积和生物发光水平的变化结果显示，D8基础构建物的静脉注射在体内显示出重要的抗肿瘤效果。

Adjusting the molecular size, the valency and the pharmacokinetics of drug conjugates are as many leverages to improve their therapeutic window, notably by affecting tumor penetration, renal clearance and short systemic exposure. In that regard, small tumor-targeting ligands are gaining attention. In this study, we demonstrate the benefits of the small Nanofitin alternative scaffolds (7 kDa) as selective tumor-targeting modules for the generation of drug conjugates, focusing on Nanofitins B10 and D8 directed against the Epithelial Growth Factor Receptor (EGFR). Owing to their small size and monovalent format, the two Nanofitins displayed a fast and deep tumor penetration in EGFR-positive A431 xenografts in BALB/c nude mice after intravenous administration, yielding to a targeting of respectively 67.9%±14.1 and 98.9%±0.7 of the tumor cells as demonstrated by immunohistochemistry. Conjugation with the monomethyl auristatin E toxin provided homogeneous Nanofitin-drug conjugates, with an overall yield of ≥97%, for in vivo assessment in a curative xenograft model using bioluminescent, EGFR-positive, A431 cells in BALB/c nude mice. Internalization was found critical for efficient release of the toxin. Hence, the intravenous administration of the D8-based construct showed significant anti-tumor effect in vivo as determined by monitoring tumor volumes and bioluminescence levels over 2 months.