Radiopharmaceuticals Division, Bhabha Atomic Research Centre, Mumbai, Maharashtra, India.
Homi Bhabha National Institute, Mumbai, Maharashtra, India.
J Cancer Res Ther. 2020 Oct-Dec;16(6):1426-1434. doi: 10.4103/jcrt.JCRT_20_19.
Trastuzumab is a Food and Drug Administration-approved humanized monoclonal antibody which targets the extracellular domain of human epidermal growth factor receptor 2 (HER2) receptor overexpressed on HER2-positive breast cancer cells. The combination of Lutetium-177 ( Lu) (t= 6.7 days, E497 keV (78.6%) and trastuzumab makes it a suitable targeting agent for radioimmunotherapy. In preclinical and clinical studies, Lu-Trastuzumab has proven to be effective for the treatment of HER2-positive malignancies such as breast and ovarian cancer.
In this study, we report the mechanism of action of Lu-CHX-A"-diethylenetriaminepentaacetic acid (DTPA)-trastuzumab at the cellular and molecular level by performing various in vitro assays in HER2-positive MDA-MB-453 breast cancer cells.
Trastuzumab was conjugated to the bifunctional chelating agent (BFCA) para-isothiocyanatobenzyl-DTPA and radiolabeled with Lu. In vitro cell binding studies were carried out in MDA-MB-453 cells to confirm the specificity of the complex toward the receptor. Cellular toxicity, cell cycle, and cell death analysis were also performed for exploring the potential of the radioimmunoconjugate at cellular and molecular level.
In vitro cell binding studies showed a maximum binding of 10.7 ± 0.1% which reduced to 2.9 ± 0.1% on coincubation with unlabeled antibody. Our study revealed that the cellular toxicity was dose dependent, and mode of cell death was predominantly by apoptosis. The radioimmunoconjugate retarded the cell in the S phase of cell cycle with two-fold increase in G2/M arrest which justifies the enhanced apoptosis at higher doses.
The study revealed that the formulation can execute a dose-dependent cellular toxicity through induction of apoptosis.
曲妥珠单抗是一种已获美国食品药品监督管理局批准的人源化单克隆抗体，靶向 HER2 阳性乳腺癌细胞上过表达的人表皮生长因子受体 2（HER2）受体的细胞外结构域。镥-177（Lu）（t=6.7 天，E497keV（78.6%）与曲妥珠单抗的结合使它成为放射免疫治疗的理想靶向剂。在临床前和临床研究中，Lu-曲妥珠单抗已被证明对治疗 HER2 阳性恶性肿瘤（如乳腺癌和卵巢癌）有效。
本研究通过在 HER2 阳性 MDA-MB-453 乳腺癌细胞中进行各种体外检测，从细胞和分子水平报告 Lu-CHX-A"-二乙基三胺五乙酸（DTPA）-曲妥珠单抗的作用机制。
曲妥珠单抗与双功能螯合剂（BFCA）对异硫氰酸苯甲基-DTPA 缀合，并与 Lu 标记。在 MDA-MB-453 细胞中进行细胞结合研究，以证实该复合物对受体的特异性。还进行了细胞毒性、细胞周期和细胞死亡分析，以探讨放射性免疫偶联物在细胞和分子水平上的潜力。
体外细胞结合研究表明，最大结合率为 10.7±0.1%，而与未标记抗体共同孵育时降至 2.9±0.1%。我们的研究表明，细胞毒性呈剂量依赖性，且细胞死亡方式主要为凋亡。放射性免疫偶联物使细胞周期停滞在 S 期，G2/M 期阻滞增加一倍，这证明了在较高剂量下凋亡增强。
该研究表明，该制剂可以通过诱导凋亡来执行剂量依赖性细胞毒性。
