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.
Highlights
CD20 (B1) is a membrane protein highly expressed by mature B lymphocytes
This cluster determinant represents an excellent target for monoclonal antibody-based immunotherapy because of several favorable properties: (i) it is expressed on approximately 90% of B-cell non-Hodgkin’s lymphomas (NHLs); (ii) it is not expressed on B cell precursors nor on other tissues; (iii) it is widely expressed on the cell membrane; and (iv) it is not normally shed from the cell [1]
The optimal derivatization condition for Rituximab, to obtain a dimeric immunotoxin, was reached with 0.5 mM 2-iminothiolane, which yielded 3.66 thiol groups inserted per molecule
Summary
CD20 (B1) is a membrane protein highly expressed by mature B lymphocytes. This cluster determinant represents an excellent target for monoclonal antibody (mAb)-based immunotherapy because of several favorable properties: (i) it is expressed on approximately 90% of B-cell non-Hodgkin’s lymphomas (NHLs); (ii) it is not expressed on B cell precursors nor on other tissues; (iii) it is widely expressed on the cell membrane; and (iv) it is not normally shed from the cell [1]. Rituximab is an anti-CD20 mouse-human chimeric mAb that has proven to be effective for the treatment of CD20+. This mAb can activate different cell death mechanisms, primarily complement-dependent cytotoxicity (CDC), and antibody-dependent cell cytotoxicity (ADCC) and, to a lesser extent, apoptosis. Rituximab is not effective for all patients, and another problem arises from an acquired resistance to Rituximab that has been reported for some patients [5]
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