Abstract
Immunotoxins are chimeric molecules, which combine antibody specificity to recognize and bind with high-affinity tumor-associated antigens (TAA) with the potency of the enzymatic activity of a toxin, in order to induce the death of target cells. Current immunotoxins present some limitations for cancer therapy, driving the need to develop new prototypes with optimized properties. Herein we describe the production, purification and characterization of two new immunotoxins based on the gene fusion of the anti-carcinoembryonic antigen (CEA) single-chain variable fragment (scFv) antibody MFE23 to α-sarcin, a potent fungal ribotoxin. One construct corresponds to a conventional monomeric single-chain immunotoxin design (IMTXCEAαS), while the other one takes advantage of the trimerbody technology and exhibits a novel trimeric format (IMTXTRICEAαS) with enhanced properties compared with their monomeric counterparts, including size, functional affinity and biodistribution, which endow them with an improved tumor targeting capacity. Our results show the highly specific cytotoxic activity of both immunotoxins in vitro, which was enhanced in the trimeric format compared to the monomeric version. Moreover, the trimeric immunotoxin also exhibited superior antitumor activity in vivo in mice bearing human colorectal cancer xenografts. Therefore, trimeric immunotoxins represent a further step in the development of next-generation therapeutic immunotoxins.
Highlights
Www.nature.com/scientificreports cell surface expressed tumor-associated antigens (TAA), the complex internalization effectiveness and the speed of the toxin release pathway and its own intrinsic potency and specificity
Whereas the first immunotoxins were constructed using chemical conjugation based on full-length mAbs, currently most of them are designed as recombinant fusion proteins based on antibody fragments, linked by a flexible peptide, a disulfide bridge or both, with reduced size and improved tumor penetration, to achieve greater specificity and antitumor efficacy[11,20,21,22,23,24,25]
This TIEXVIII domain and the antibody are directly joined by linkers of different lengths, which provide the necessary flexibility for the intended use[29,32]
Summary
Www.nature.com/scientificreports cell surface expressed TAA, the complex internalization effectiveness and the speed of the toxin release pathway and its own intrinsic potency and specificity. The antibody engineering field has driven the development of a myriad of new formats with improved properties, which potentially can be included within immunotoxin constructs[27] This new generation of antibody formats overcomes some of the scFv limitations, mainly lower tumor retention due to its monovalence, and fast blood clearance given its size under the glomerular filtration threshold (70 kDa)[28]. Trimerbodies are trivalent antibodies formed by fusing a collagen XVIII-derived homotrimerization (TIEXVIII) domain to the C-terminal edge of a scFv or sdAb5,27,31 This TIEXVIII domain and the antibody are directly joined by linkers of different lengths, which provide the necessary flexibility for the intended use[29,32]. Its specific ribonucleolytic activity against one single bond, strategically located at the large rRNA, causes ribosome inactivation and, thereby, protein biosynthesis inhibition and apoptosis[47,48,49]
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