1. ¹Dipartimento di Patologia Sperimentale, Università di Bologna, Bologna, Italy
2. ²Servizio di Immunoematologia e Trasfusionale, Policlinico 'S Orsola', Bologna, Italy
3. ³Istituto di Ematologia e Oncologia medica 'L & A Seràgnoli', Università di Bologna, Bologna, Italy

Correspondence: Professor A Bolognesi, Dipartimento di Patologia Sperimentale, Università di Bologna, via San Giacomo 14, 40126 Bologna, Italy. Fax: +39 0512094746; E-mail: andrea.bolognesi@unibo.it

Received 4 August 2003; Accepted 12 March 2004; Published online 22 April 2004.

Abstract

Immunotoxins are chimeric proteins consisting of a toxin coupled to an antibody. To date, several clinical trials have been conducted, and some are still ongoing, to evaluate their anti-tumor efficacy. In this view, we chemically constructed an anti-CD20 immunotoxin with the mAb Rituximab and the type 1 ribosome-inactivating protein (RIP) saporin-S6, designed for B cells non-Hodgkin's lymphoma (NHL) therapy. This immunotoxin showed a specific cytotoxicity for the CD20+ cell lines Raji and D430B, evidenced by inhibition of protein synthesis, evaluation of apoptosis and clonogenic assay. Upon conjugation, saporin-S6 increased its toxicity on target cells by at least 2 logs, with IC₅₀ values of 0.1–0.3 nM. The percentage of AnnexinV+ cells was over 95% in both cell lines treated with 10 nM immunotoxin. A complete elimination of Raji clones was reached with the 10 nM immunotoxin, whereas a mixture of free RIP and mAb gave about 90% of clonogenic growth. Rituximab/saporin-S6, at 10 nM concentration, also induced apoptosis in 80% of lymphoma cells from NHL patients. Moreover, sensitivity of Raji to Rituximab/saporin-S6 was augmented when cells were coincubated with Fludarabine. The synergistic toxic effect of the two drugs led to a total elimination of the neoplastic population.