title: High in Vitro Anti-Tumor Efficacy of Dimeric Rituximab/Saporin-S6 Immunotoxin.
abstract: The anti-CD20 mAb Rituximab has revolutionized lymphoma therapy, in spite of a number of unresponsive or relapsing patients. Immunotoxins, consisting of toxins coupled to antibodies, are being investigated for their potential ability to augment Rituximab efficacy. Here, we compare the anti-tumor effect of high- and low-molecular-weight Rituximab/saporin-S6 immunotoxins, named HMW-IT and LMW-IT, respectively. Saporin-S6 is a potent and stable plant enzyme belonging to ribosome-inactivating proteins that causes protein synthesis arrest and consequent cell death. Saporin-S6 was conjugated to Rituximab through an artificial disulfide bond. The inhibitory activity of HMW-IT and LMW-IT was evaluated on cell-free protein synthesis and in two CD20⁺ lymphoma cell lines, Raji and D430B. Two different conjugates were separated on the basis of their molecular weight and further characterized. Both HMW-IT (dimeric) and LMW-IT (monomeric) maintained a high level of enzymatic activity in a cell-free system. HMW-IT, thanks to a higher toxin payload and more efficient antigen capping, showed stronger in vitro anti-tumor efficacy than LMW-IT against lymphoma cells. Dimeric HMW-IT can be used for lymphoma therapy at least for ex vivo treatments. The possibility of using HMW-IT augments the yield in immunotoxin preparation and allows the targeting of antigens with low internalization rates.
authors.affiliation: Department of Experimental, Diagnostic and Specialty Medicine-DIMES, Alma Mater Studiorum, University of Bologna, Via San Giacomo 14, 40126 Bologna, Italy. massimo.bortolotti2@unibo.it.
authors.forename: Massimo
authors.initials: M
authors.lastname: Bortolotti
authors.affiliation: Department of Experimental, Diagnostic and Specialty Medicine-DIMES, Alma Mater Studiorum, University of Bologna, Via San Giacomo 14, 40126 Bologna, Italy. andrea.bolognesi@unibo.it.
authors.forename: Andrea
authors.initials: A
authors.lastname: Bolognesi
authors.affiliation: Department of Experimental, Diagnostic and Specialty Medicine-DIMES, Alma Mater Studiorum, University of Bologna, Via San Giacomo 14, 40126 Bologna, Italy. mariagiulia.battelli@unibo.it.
authors.forename: Maria Giulia
authors.initials: MG
authors.lastname: Battelli
authors.affiliation: Department of Experimental, Diagnostic and Specialty Medicine-DIMES, Alma Mater Studiorum, University of Bologna, Via San Giacomo 14, 40126 Bologna, Italy. letizia.polito@unibo.it.
authors.forename: Letizia
authors.initials: L
authors.lastname: Polito
mesh_terms: D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology*; D045744:Cell Line, Tumor; D006801:Humans; D000922:Immunotoxins / Q000494:pharmacology*; D014176:Protein Biosynthesis / Q000187:drug effects; D054789:Ribosome Inactivating Proteins, Type 1 / Q000494:pharmacology*; D000069283:Rituximab / Q000494:pharmacology*; D000076985:Saporins
chemical_list: D000972:Antineoplastic Agents, Phytogenic; D000922:Immunotoxins; D054789:Ribosome Inactivating Proteins, Type 1; D000069283:Rituximab; D000076985:Saporins
keywords: B-cell lymphomas; Rituximab; Saporin-S6; immunotherapy; immunotoxin; ribosome-inactivating protein