Milazzo Ferdinando Maria, Vesci Loredana, Anastasi Anna Maria, Chiapparino Caterina, Rosi Antonio, Giannini Giuseppe, Taddei Maurizio, Cini Elena, Faltoni Valentina, Petricci Elena, Battistuzzi Gianfranco, Salvini Laura, Carollo Valeria, De Santis Rita
Biotechnology R&D, Alfasigma SpA, Rome, Italy.
Dipartimento di Biotecnologia, Chimica e Farmacia, Università degli Studi di Siena, Siena, Italy.
Front Oncol. 2020 Jan 23;9:1534. doi: 10.3389/fonc.2019.01534. eCollection 2019.
Targeted therapy using monoclonal antibodies conjugated to toxins is gaining space in the treatment of cancer. Here, we report the anti-tumor effect of a new antibody drug conjugate (ADC) delivering a HDAC inhibitor to ErbB2+ solid tumors. Trastuzumab was partially reduced with tris [2-carboxyethyl] phosphine (TCEP) and conjugated to ST7464AA1, the active form of the prodrug HDAC inhibitor ST7612AA1, through a maleimide-thiol linker to obtain the Antibody Drug Conjugate (ADC) ST8176AA1. The average drug/antibody ratio (DAR) was 4.5 as measured by hydrophobic interaction chromatography (HIC). Binding of ST8176AA1 to ErbB2 receptor and internalization in tumor cells were investigated by enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance (SPR), cytofluorimetry, and High Content Screening (HCS) Imaging. The biological activity of the ADC was evaluated and by measuring cell proliferation/cell cycle, apoptosis/DNA damage, tubulin, and histone acetylation and modulation of Epithelial/Mesenchymal Transition (EMT) markers. Receptor binding and internalization of ST8176AA1 were confirmed to be similar to trastuzumab. Higher anti-tumor activity of ST8176AA1 compared to trastuzumab was observed in tumor cell lines. Such higher activity correlated with increased acetylation of histones and alfa-tubulin as a consequence of HDAC inhibitor-mediated epigenetic modulation that also induced increased expression of ErbB2 and estrogen receptor in triple negative breast cancer cells. Consistently with data, ST8176AA1 exhibited higher tumor growth inhibition than trastuzumab in xenograft models of ovary and colon carcinoma and in two patient-derived xenograft (PDX) models of pancreatic carcinoma. Immunohistochemistry analysis of tumor masses showed lower expression of the proliferation marker Ki67 and higher expression of cleaved caspase-3 in mice treated with the ADC compared to those treated with trastuzumab and results correlated with increased acetylation of both histones and tubulin. Collectively, present data indicate that ADC ST8176AA1 can target epigenetic modulation to ErbB2+ tumors. Interestingly, the amount of HDACi estimated to be delivered at the ST8176AA1 effective dose would correspond to ~1/1,000 of ST7612AA1 effective dose. Therefore, ST8176AA1 is an attractive new therapeutic candidate because it exhibits increased anti-tumor potency compared to trastuzumab by exerting epigenetic modulation at a much safer dose compared to standard HDACi-based therapeutic protocols.
使用与毒素偶联的单克隆抗体进行靶向治疗在癌症治疗中越来越受关注。在此，我们报告了一种新型抗体药物偶联物（ADC）对ErbB2阳性实体瘤的抗肿瘤作用，该偶联物可将组蛋白去乙酰化酶（HDAC）抑制剂递送至肿瘤部位。曲妥珠单抗用三[2-羧乙基]膦（TCEP）部分还原，并通过马来酰亚胺-硫醇连接子与前药HDAC抑制剂ST7612AA1的活性形式ST7464AA1偶联，从而获得抗体药物偶联物（ADC）ST8176AA1。通过疏水相互作用色谱法（HIC）测得平均药物/抗体比（DAR）为4.5。通过酶联免疫吸附测定（ELISA）、表面等离子体共振（SPR）、细胞荧光测定法和高内涵筛选（HCS）成像研究了ST8176AA1与ErbB2受体的结合以及在肿瘤细胞中的内化情况。通过测量细胞增殖/细胞周期、凋亡/DNA损伤、微管蛋白、组蛋白乙酰化以及上皮/间质转化（EMT）标志物的调节来评估ADC的生物学活性。证实ST8176AA1的受体结合和内化情况与曲妥珠单抗相似。在肿瘤细胞系中观察到ST8176AA1比曲妥珠单抗具有更高的抗肿瘤活性。这种更高的活性与组蛋白和α-微管蛋白乙酰化增加相关，这是HDAC抑制剂介导的表观遗传调节的结果，该调节还诱导了三阴性乳腺癌细胞中ErbB2和雌激素受体表达增加。与这些数据一致，在卵巢癌和结肠癌的异种移植模型以及两种胰腺癌患者来源的异种移植（PDX）模型中显示，ST8176AA1比曲妥珠单抗表现出更高的肿瘤生长抑制作用。对肿瘤块的免疫组织化学分析显示，与用曲妥珠单抗治疗的小鼠相比，用ADC治疗的小鼠中增殖标志物Ki67的表达较低，而裂解的半胱天冬酶-3的表达较高，结果与组蛋白和微管蛋白的乙酰化增加相关。总体而言，目前的数据表明ADC ST8176AA1可以靶向对ErbB2阳性肿瘤进行表观遗传调节。有趣的是，估计在ST8176AA1有效剂量下递送的HDACi量约相当于ST7612AA1有效剂量的1/1000。因此，ST8176AA1是一种有吸引力的新型治疗候选物，因为与曲妥珠单抗相比，它通过以比基于标准HDACi的治疗方案更安全的剂量发挥表观遗传调节作用，从而表现出更高的抗肿瘤效力。
