Construction and Characterization of Adenovirus Vectors Encoding Aspartate-β-Hydroxylase to Preliminary Application in Immunotherapy of Hepatocellular Carcinoma.

Yujiao Zhou,Xiaolei Xu,Limin Chen,Yuanling Zhang,Guo Shi,Jingjie Fu,Chengmin Li,Aizhong Zeng,Feifei Liu,Xue Luo

doi:10.1155/2018/9832467

Abstract

Dendritic cells (DCs) harboring tumor-associated antigen are supposed to be a potential immunotherapy for hepatocellular carcinoma (HCC). Aspartate-β-hydroxylase (AAH), an overexpressed tumor-associated cell surface protein, is considered as a promising biomarker and therapeutic target for HCC. In this study, we constructed adenovirus vector encoding AAH gene by gateway recombinant cloning technology and preliminarily explored the antitumor effects of DC vaccines harboring AAH. Firstly, the total AAH mRNA was extracted from human HCC tissues; the cDNA was amplified by RT-PCR, verified, and sequenced after TA cloning. Gateway technology was used and the obtained 18T-AAH was used as a substrate, to yield the final expression vector Ad-AAH-IRES2-EGFP. Secondly, bone marrow-derived DCs were infected by Ad-AAH-IRES2-EGFP to yield AAH-DC vaccines. Matured DCs were demonstrated by increased expression of CD11c, CD80, and MHC-II costimulatory molecules. A dramatically cell-killing effect of T lymphocytes coculturing with AAH-DCs on HepG2 HCC cell line was demonstrated by CCK-8 and FCM assays in vitro. More importantly, in an animal experiment, the lysis effect of cytotoxic T lymphocytes (CTLs) on HepG2 cells in the AAH-DC group was stronger than that in the control groups. In conclusion, the gateway recombinant cloning technology is a powerful method of constructing adenovirus vector, and the product Ad-AAH-IRES2-EGFP may present as a potential candidate for DC-based immunotherapy of HCC.

Highlights

Hepatocellular carcinoma (HCC) is a common malignant tumor, and despite curable strategies such as resection or liver transplantation, patients with advanced HCC continue to present a poor outcome [1, 2]
The DNA fragment of the AAH gene was amplified by reverse transcription polymerase chain reaction (RT-PCR)
The titer of Ad-AAHIRES2-EGFP was detected as 5.0 × 109 ifu/ml according to the immunoassay aforementioned

Summary

Introduction

Hepatocellular carcinoma (HCC) is a common malignant tumor, and despite curable strategies such as resection or liver transplantation, patients with advanced HCC continue to present a poor outcome [1, 2]. Sorafenib has been shown to improve survival of advanced HCC patients, it failed to show any improvement in outcomes for the treatment of HCC in randomized studies. The clinical trials of new drugs including lenvatinib, regorafenib, and pembrolizumab have shown promise; more convictive clinical evidences are still needed [3]. AAH has been found highly expressed in a variety of human malignancies including HCC, cholangiocellular carcinoma, and breast, pancreatic, and non-small lung cancer [4,5,6,7]; it is rarely expressed in normal tissues and lowly expressed in tumor-adjacent tissues [4]. Previous researches have demonstrated that overexpression of AAH strikingly increases

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