Singh Sunanda, Murillo Genoveva, Chen Dong, Parihar Ashutosh S, Mehta Rajendra G
Singh Biotechnology and Tampa Bay Technology Incubator, University of South Florida, Tampa Bay, FL, USA.
IIT Research Institute, Chicago, IL, USA.
Breast Cancer (Auckl). 2018 Jan 3;12:1178223417750858. doi: 10.1177/1178223417750858. eCollection 2018.
The serendipitous discovery of heavy-chain antibodies devoid of light chains in camelids and the subsequent development of VHHs (variable region of camelid heavy chain) have provided a very important tool for research and possibly for therapeutics. In this study, we synthesized single-domain 15-kDa antibody SBT-100 (anti-STAT3 B VHH13) against human STAT3 (signal transducer and activator of transcription) that binds selectively to STAT3 and suppresses the function of phosphorylated STAT3 (p-STAT3).
Single-chain VHH nanobodies were generated by immunizing camelid with humanized STAT3. Commercially available breast cancer cell lines including MDA-MB-231, MDA-MB-468, MDA-MB-453, MCF-7, and BT474 were used. Cell proliferation was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The association of anti-STAT3 B VHH13 with STAT3 and p-STAT3 was determined by immunoprecipitation and Western blot analyses. The efficacy of SBT-100 on the growth of MDA-MB-231 xenografts in vivo was determined using athymic mice. for cell proliferation was determined using analysis of variance. If a significant difference ( < .05) was observed, then Tukey-Kramer multiple comparison test was conducted.
SBT-100 suppressed cell proliferation of triple-negative breast cancer cells ( < .01) as well as provided significant inhibition of tumor growth ( < .05) in a xenograft model without any toxicity. Results are presented to show that anti-STAT3 B VHH13 selectively binds to STAT3 suggesting that the effects were mediated by inhibiting STAT3.
A very large number of human malignancies and benign diseases have constitutive STAT3 activation. Therefore, the results described here suggest that anti-STAT3 B VHH13 can be developed for therapeutic intervention for cancer cells expressing STAT3 or p-STAT3.
在骆驼科动物中偶然发现无轻链的重链抗体，随后开发出骆驼科动物重链可变区（VHHs），这为研究乃至治疗提供了非常重要的工具。在本研究中，我们合成了针对人信号转导与转录激活因子3（STAT3）的单域15 kDa抗体SBT-100（抗STAT3 B VHH13），它能选择性结合STAT3并抑制磷酸化STAT3（p-STAT3）的功能。
用人源化STAT3免疫骆驼科动物以产生单链VHH纳米抗体。使用包括MDA-MB-231、MDA-MB-468、MDA-MB-453、MCF-7和BT474在内的市售乳腺癌细胞系。通过3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴盐法测定细胞增殖。通过免疫沉淀和蛋白质印迹分析确定抗STAT3 B VHH13与STAT3和p-STAT3的结合。使用无胸腺小鼠确定SBT-100对体内MDA-MB-231异种移植瘤生长的疗效。使用方差分析确定细胞增殖情况。如果观察到显著差异（<0.05），则进行Tukey-Kramer多重比较检验。
SBT-100抑制三阴性乳腺癌细胞的增殖（<0.01），并且在异种移植模型中显著抑制肿瘤生长（<0.05），且无任何毒性。结果表明抗STAT3 B VHH13选择性结合STAT3，提示其作用是通过抑制STAT3介导的。
大量人类恶性肿瘤和良性疾病存在组成型STAT3激活。因此，本文所述结果表明，抗STAT3 B VHH13可开发用于对表达STAT3或p-STAT3的癌细胞进行治疗干预。
