Universität Zürich, Biochemisches Institut, Winterthurerstrasse, 8057 Zürich, Switzerland.
Cancer Res. 2010 Feb 15;70(4):1595-605. doi: 10.1158/0008-5472.CAN-09-2724. Epub 2010 Feb 2.
Slow-clearing, tumor-targeting proteins such as monoclonal antibodies typically exhibit high tumor accumulation but low tissue contrast, whereas intermediate-sized proteins such as scFvs show faster clearance but only moderate tumor accumulation. For both, tumor targeting does not seem to improve further above an optimal affinity. We show here that with very small high-affinity proteins such as designed ankyrin repeat proteins (DARPins), these limits can be overcome. We have systematically investigated the influence of molecular mass and affinity on tumor accumulation with DARPins with specificity for HER2 in SK-OV-3.ip nude mouse xenografts. DARPins with a mass of 14.5 kDa and affinities between 270 nmol/L and 90 pmol/L showed a strong correlation of tumor accumulation with affinity to HER2, with the highest affinity DARPin reaching 8% ID/g after 24 hours and 6.5% ID/g after 48 hours (tumor-to-blood ratio >60). Tumor autoradiographs showed good penetration throughout the tumor mass. Genetic fusion of two DARPins (30 kDa) resulted in significantly lower tumor accumulation, similar to values observed for scFvs, whereas valency had no influence on accumulation. PEGylation of the DARPins increased the circulation half-life, leading to higher tumor accumulation (13.4% ID/g after 24 hours) but lower tumor-to-blood ratios. Affinity was less important for tumor uptake of the PEGylated constructs. We conclude that two regimes exist for delivering high levels of drug to a tumor: small proteins with very high affinity, such as unmodified DARPins, and large proteins with extended half-life, such as PEGylated DARPins, in which the importance of affinity is less pronounced.
清除缓慢、靶向肿瘤的蛋白质，如单克隆抗体，通常表现出高肿瘤积累，但组织对比度低，而中等大小的蛋白质，如 scFv，则表现出更快的清除速度，但只有适度的肿瘤积累。对于两者，肿瘤靶向似乎不会在最佳亲和力之上进一步提高。我们在这里表明，对于非常小的高亲和力蛋白质，如设计的锚蛋白重复蛋白（DARPins），可以克服这些限制。我们系统地研究了分子质量和亲和力对 SK-OV-3.ip 裸鼠异种移植中 HER2 特异性 DARPins 肿瘤积累的影响。质量为 14.5 kDa、亲和力在 270 nmol/L 至 90 pmol/L 之间的 DARPins 与对 HER2 的亲和力呈强相关性，亲和力最高的 DARPin 在 24 小时后达到 8% ID/g，48 小时后达到 6.5% ID/g（肿瘤与血液比值>60）。肿瘤放射自显影显示良好的穿透性，整个肿瘤质量均有较高的积累。两个 DARPins（30 kDa）的遗传融合导致肿瘤积累显著降低，与 scFv 观察到的值相似，而价数对积累没有影响。DARPins 的 PEG 化增加了循环半衰期，导致更高的肿瘤积累（24 小时后达到 13.4% ID/g），但肿瘤与血液的比值降低。亲和力对 PEG 化构建体的肿瘤摄取的重要性较低。我们得出结论，存在两种向肿瘤输送高剂量药物的机制：具有非常高亲和力的小蛋白质，如未修饰的 DARPins，以及半衰期延长的大蛋白质，如 PEG 化的 DARPins，其中亲和力的重要性不那么明显。
