Mandler R, Wu C, Sausville E A, Roettinger A J, Newman D J, Ho D K, King C R, Yang D, Lippman M E, Landolfi N F, Dadachova E, Brechbiel M W, Waldmann T A
Metabolism Branch, Division of Clinical Sciences, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.
J Natl Cancer Inst. 2000 Oct 4;92(19):1573-81. doi: 10.1093/jnci/92.19.1573.
HER2 is a membrane receptor whose overexpression is strongly associated with poor prognosis in breast carcinomas. Inhibition of HER2 activity can reduce tumor growth, which led to the development of Herceptin, an anti-HER2 monoclonal antibody (MAb) that is already in clinical use. However, the objective response rate to Herceptin monotherapy is quite low. HER2 activity can also be inhibited by the highly cytotoxic antibiotic geldanamycin (GA). However, GA is not used clinically because of its adverse toxicity. Our purpose was to enhance the inhibitory activity of anti-HER2 MAb by coupling it to GA.
We synthesized 17-(3-aminopropylamino)GA (17-APA-GA) and conjugated it to the anti-HER2 MAb e21, to form e21 : GA. The noninternalizing anti-HER2 MAb AE1 was used as a control. Internalization assays and western blot analyses were used to determine whether the anti-HER2 MAbs and their immunoconjugates were internalized into HER2-expressing cells and reduced HER2 levels. All statistical tests were two-sided.
The immunoconjugate e21 : GA inhibited the proliferation of HER2-overexpressing cell lines better than unconjugated e21 (concentration required for 50% inhibition = 40 versus 1650 microg/mL, respectively). At 15 microg/mL, e21 : GA reduced HER2 levels by 86% within 16 hours, whereas unconjugated e21, 17-APA-GA, or AE1 : GA reduced HER2 levels by only 20%. These effects were not caused by release of 17-APA-GA from the immunoconjugate because immunoconjugates containing [(3)H]GA were stable in serum at 37 degrees C. Furthermore, e21 : GA did not significantly inhibit proliferation of the adult T-cell leukemia cell line HuT102, which is HER2 negative yet highly sensitive to GA.
Our findings suggest that conjugating GA to internalizing MAbs enhances the inhibitory effect of the MAbs. This approach might also be applied in cellular targeting via growth factors and may be of clinical interest.
HER2是一种膜受体，其过表达与乳腺癌的不良预后密切相关。抑制HER2活性可减少肿瘤生长，这促使了赫赛汀的研发，赫赛汀是一种已用于临床的抗HER2单克隆抗体（MAb）。然而，赫赛汀单药治疗的客观缓解率相当低。高细胞毒性抗生素格尔德霉素（GA）也可抑制HER2活性。然而，由于其不良毒性，GA未用于临床。我们的目的是通过将抗HER2 MAb与GA偶联来增强其抑制活性。
我们合成了17 -（3 -氨丙基氨基）GA（17 - APA - GA），并将其与抗HER2 MAb e21偶联，形成e21:GA。非内化抗HER2 MAb AE1用作对照。采用内化试验和蛋白质印迹分析来确定抗HER2 MAbs及其免疫缀合物是否被内化到HER2表达细胞中并降低HER2水平。所有统计检验均为双侧检验。
免疫缀合物e21:GA比未偶联的e21更能有效抑制HER2过表达细胞系的增殖（50%抑制所需浓度分别为40和1650μg/mL）。在15μg/mL时，e21:GA在16小时内使HER2水平降低了86%，而未偶联的e21、17 - APA - GA或AE1:GA仅使HER2水平降低了20%。这些效应并非由免疫缀合物中17 - APA - GA的释放引起，因为含有[³H]GA的免疫缀合物在37℃血清中是稳定的。此外，e21:GA并未显著抑制成人T细胞白血病细胞系HuT102的增殖，该细胞系HER2阴性但对GA高度敏感。
我们的研究结果表明，将GA与内化MAb偶联可增强MAb的抑制作用。这种方法也可能应用于通过生长因子进行细胞靶向，可能具有临床意义。
