Department of Medicinal Chemistry, University of Minnesota, Minneapolis, Minnesota 55455, United States.
Mol Pharm. 2012 Nov 5;9(11):3218-27. doi: 10.1021/mp300303k. Epub 2012 Oct 16.
Chemically self-assembled antibody nanorings (CSANs) displaying multiple copies of single-chain variable fragments can be prepared from dihydrofolate reductase (DHFR) fusion proteins and bis-methotrexate (bisMTX). We have designed and synthesized a bisMTX chemical dimerizer (bisMTX-NH(2)) that contains a third linker arm that can be conjugated to fluorophores, radiolabels, and drugs. Monovalent, divalent, and higher-order AntiCD3 CSANs were assembled with a fluorescein isothiocyanate (FITC)-labeled bis-methotrexate ligand (bisMTX-FITC) and found to undergo rapid internalization and trafficking by HPB-MLT, a CD3+ T-leukemia cell line, to the early and late endosome and lysosome. Because the fluorescence of bisMTX-FITC when incorporated into CSANs was found to be significantly greater than that of the free ligand, the stability of the endocytosed AntiCD3 CSANs could be monitored. The internalized CSANs were found to be stable for several hours, while treatment with the nontoxic DHFR inhibitor trimethoprim resulted in a rapid loss (>80%) of cellular fluorescence within minutes, consistent with efficient intracellular disassembly of the nanorings. Over longer time periods (24 h), cellular fluorescence decreased by 75-90%, regardless of whether cells had been treated with DMSO or trimethoprim. Although bisMTX is a potent inhibitor of DHFR, it was found to be nontoxic (GI(50) > 20 μM) to HPB-MLT cells. In contrast, AntiCD3 CSANs prepared with bisMTX were found to be at least 13-fold more cytotoxic (GI(50) = 0.5-1.5 μM) than bisMTX at 72 h. Consistent with our findings from CSAN stability studies, no increase in cytotoxicity was observed upon treatment with trimethoprim. Taken together, our results suggest that cell receptor targeting CSANs prepared with trifunctional bisMTX could be used as potential tissue selective drug carriers.
化学自组装抗体纳米环（CSANs）可以由二氢叶酸还原酶（DHFR）融合蛋白和双甲氨蝶呤（bisMTX）制备，展示多个单链可变片段。我们设计并合成了一种双甲氨蝶呤化学二聚体（bisMTX-NH(2)），它包含第三个连接臂，可以与荧光团、放射性标记物和药物结合。单价、二价和更高阶的 AntiCD3 CSANs 与荧光素异硫氰酸酯（FITC）标记的双甲氨蝶呤配体（bisMTX-FITC）组装，并发现它们被 CD3+T 白血病细胞系 HPB-MLT 快速内化和运输到早期和晚期内体和溶酶体。由于发现当将 bisMTX-FITC 掺入 CSANs 时，其荧光显著大于游离配体的荧光，因此可以监测内吞作用的 AntiCD3 CSANs 的稳定性。发现内化的 CSANs 在数小时内稳定，而用非毒性 DHFR 抑制剂三甲氧苄二氨嘧啶处理会导致细胞内荧光在数分钟内迅速丧失（>80%），这与纳米环的有效细胞内解组装一致。在较长的时间段内（24 小时），无论细胞是否用 DMSO 或三甲氧苄二氨嘧啶处理，细胞荧光都会降低 75-90%。尽管 bisMTX 是 DHFR 的有效抑制剂，但它对 HPB-MLT 细胞无毒（GI(50)>20 μM）。相比之下，我们发现用 bisMTX 制备的 AntiCD3 CSANs 在 72 小时时的细胞毒性至少比 bisMTX 高 13 倍（GI(50)=0.5-1.5 μM）。与我们从 CSAN 稳定性研究中得出的结果一致，在用三甲氧苄二氨嘧啶处理时，未观察到细胞毒性增加。综上所述，我们的结果表明，用三功能双甲氨蝶呤制备的细胞受体靶向 CSANs 可用作潜在的组织选择性药物载体。
