Production of recombinant protein ltb-ctla4 by transient plant-based expression using geminiviral vector

Cancer is one of the leading global causes of death. In 2018, WHO estimated that 9.6 million patients died from cancer, with 18.1 million new cases emerged. In recent years, immunotherapy is considered as a treatment improving cancer-related immune function by manipulating the immune checkpoints in the immune system. Cytotoxic-T-lymphocyte antigen 4 (CTLA-4) is a immune checkpoint transmembrane protein. The expression of CTLA-4 on the surface of activated T-cell could downregulate the production and proliferation of cytotoxic T-lymphocyte cells (CTLs). CTLs are the host-defended immune cells that could retard the growth of cancer cells by recognizing the tumor-associated antigens expressed on the membrane of cancer cells, then eliminate them before spreading and evading throughout the body. The overexpression of CTLA-4 founded in cancer has a suppressive effect on cytotoxic T-lymphocyte cells, encouraging the tumor cell's growth. Recently, the monoclonal antibodies targeting CTLA-4 have successfully proved to be an active agent in immunotherapy. However, the large amount and multiple dosing had been a major hindrance. In this study, we developed heat-labile enterotoxin B subunit (LTB) fused CTLA-4 or LTB-CTLA4 as a cancer vaccine, to reduce the disadvantage in term of quantity, price, and frequency of dosing. LTB-CTLA4 was produced in Nicotiana benthamiana plant as a bio-factory, a promising platform for the plant-derived biopharmaceutical production which has abilities to produce high value but cost-effective vaccines. The highest LTB-CTLA4 level reached 1.29 µg per gram plant fresh weight and induced BALB/c mice to produce specific IgG against CTLA-4. LTB-CTLA4 has a potential as a candidate agent for cancer vaccine either to prevent the development of cancer or to treat cancer in the future.