Expression and function of plant-derived recombinant multiple monoclonal antibodies for the recognition of human colorectal cancer cells

Abstract

Large-scale production of therapeutic proteins in transgenic plants has several advantages over other expression systems such as mammalian cell lines. Monoclonal antibodies (mAbs) recognize several specific epitopes highly expressed on the surface of cancer cells and can enhance the anticancer efficacy of antibody-mediated targeted immunotherapy. We describe the expressions of multiple mAbs in a single F₁ tobacco line obtained through cross-pollination between a transgenic line expressing anticancer mAb^P CO17-1A (mAb^P CO) and another expressing anticancer mAb^P BR55 (mAb^P BR), which recognize GA733 and Lewis-Y antigens, respectively, which are found on human colorectal cancer cells. The presence of heavy chains (HCs) and light chains (LCs) in mAbs and their protein sizes were confirmed by immunoblotting analysis. N-glycan structure analysis showed similar glycan structure profiles between individually expressed mAbs (mAb^P CO and mAb^P BR) and multiple mAbs (mAb^P CO × BR). The interaction of GA733 with mAb^P CO was tested using surface plasmon resonance, and the results showed that mAb^P CO × BR had lower binding affinity to the antigen compared to individual mAb CO. Boyden chamber assay indicated that mAb^M CO, mAb^P CO, and mAb^P CO × BR had similar inhibitory properties against migration of colon cancer cell. The antibody-dependent cell-mediated cyctotoxicity assay showed a significant synergistic effect of mAb^P CO × BR on colorectal cancer cell. These results suggested that the F₁ plants produced both mAb^P CO and mAb^P BR, but the HCs and LCs of mAb^P CO and mAb^P BR were randomly assembled in a single plant, resulting in chimeric HC and LC assemblies.