PO.ET01.01 · 实验与分子治疗
针对人胰腺细胞上过表达的整合素alpha6beta4的新型单克隆抗体的开发与表征
Development and characterisation of a novel monoclonal antibody against overexpressed integrin alpha6beta4 on human pancreatic cells
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摘要 Abstract
中文摘要
尽管在癌症患者的诊断和治疗方面取得了重大进展，胰腺癌仍然是最具侵袭性和致死性的癌症类型之一。迫切需要发现更多的治疗靶点并开发更有效的疗法。单克隆抗体（mAb）技术是发现过表达细胞表面肿瘤抗原的优秀工具，所产生的抗体可能具有重要的诊断和治疗价值。在此，我们报告了一种新型抗体KU44.71的产生与表征，该抗体使用杂交瘤技术针对人导管胰腺腺癌肝转移细胞系CFPAC-1而制备。经筛选后，选出了针对胰腺癌细胞表面过表达抗原的mAb KU44.71，并通过亲和层析进行纯化。进一步通过ELISA、流式细胞术、细胞增殖检测、内化研究、免疫沉淀和质谱、蛋白质印迹以及免疫组化对该mAb进行了表征。我们发现KU44.71识别整合素alpha6beta4的胞外结构域，该整合素在源自不同来源（包括原发性胰腺癌、肝转移、腹水和淋巴结转移）的胰腺癌细胞系中以不同程度过表达。用裸抗体治疗不影响表达alpha6beta4的胰腺癌细胞的生长。相反，暴露于抗alpha6beta4的KU44.71诱导了胰腺癌细胞中整合素alpha6beta4的内化。此外，发现KU44.71可有效通过蛋白质印迹和免疫组化检测alpha6beta4的表达。我们的结果表明，mAb KU44.71是研究alpha6beta4在人类癌症复杂生物学中作用的有用工具，也可用于研究整合素alpha6beta4在胰腺癌患者中的相对表达、预后意义和预测价值。有必要开展进一步研究，以阐明这种新型mAb（包括其人源化或药物偶联型）在alpha6beta4过表达的胰腺癌及其他类型癌症患者中的治疗潜力。
查看英文原文 English abstract
Despite major advances in the diagnosis and treatment of patients with cancer, pancreatic cancer remains one of the most aggressive and fatal cancer types. There is an urgent need for the discovery of additional therapeutic targets and the development of more effective therapeutics. Monoclonal antibody (mAb) technology is an excellent tool for the discovery of overexpressed cell surface tumour antigens, and antibodies generated may have significant diagnostic and therapeutic value. Here, we report the generation and characterisation of a novel antibody KU44.71 against the human ductal pancreatic adenocarcinoma metastasised to the liver cell line CFPAC-1, using hybridoma technology. Following screening, mAb KU44.71 which was directed against overexpressed cell surface antigens on pancreatic cancer cells was selected and purified by affinity chromatography. Further characterisation of the mAb was performed by ELISA, flow cytometry, cell proliferation assay, internalisation studies, immunoprecipitation and mass spectrometry, western blotting, and immunohistochemistry. We discovered that KU44.71 recognises the external domain of integrin alpha6beta4 that is overexpressed by varying amounts in pancreatic cancer cell lines, derived from different sources including primary pancreatic cancer, liver metastasis, ascites and lymph node metastasis. Treatment with the naked antibody did not affect growth of alpha6beta4 expressing pancreatic cancer cells. In contrast, exposure to anti-alpha6beta4 KU44.71 induced internalisation of integrin alpha6beta4 in pancreatic cancer cells. Furthermore, KU44.71 was found to be effective in detecting alpha6beta4 expression by western blot and immunohistochemistry. Our results suggest that mAb KU44.71 is an useful tool for studying the role of alpha6beta43 in the complex biology of human cancer, and for investigating the relative expression, prognostic significance, and predictive value of integrin alpha6beta43 in patients with pancreatic cancer. Further studies are warranted to elucidate the therapeutic potential of this novel mAb including its humanised or drug conjugated versions in patients with alpha6beta43 overexpressing pancreatic and other type of cancer.
利益披露 Disclosure
G. Arias, None..
A. G. Dalgleish, None..
I. Bagwan, None..
S. Khelwatty, None..
S. Mudan, None..
T. Walker, None..
H. Modjtahedi, None.