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Reactive oxygen species (ROS) generated by xanthine oxidoreductase (XOR) were toxic to B lymphoma-derived Raji cells (positive for 8A monoclonal antibody, mAb). The sensitivity of these malignant cells to the hypoxanthine/XOR system was higher than that observed in peripheral human lymphocytes. The understanding of the mechanisms of cytotoxicity induced by XOR-produced ROS is essential in view of a possible clinical application. Cell death mostly had the feature of apoptosis and post-apoptotic necrosis and depended on the activity of XOR. Catalase, but not superoxide dismutase, protected cells from the toxicity of XOR, thus indicating that cell damage depended on the production of hydrogen peroxide. The toxicity of ROS was selectively targeted to malignant Raji cells by antibody-XOR conjugation, either directly, with an 8A-XOR conjugate, or indirectly, with an 8A mAb plus an anti-mouse IgG-XOR. Both direct and indirect immunotoxins induced apoptotic death to target cells in a dose-dependent manner. These conjugates showed no aspecific cytotoxicity in conditions very similar to the ex vivo treatment of cell suspension for bone marrow transplantation. Moreover, the prevalence of apoptotic death over necrosis may reduce the in vivo inflammatory response and its local and systemic consequences, thus becoming relevant in the construction of immunotoxins with therapeutic potential.

The anti-CD30 immunotoxin (IT) Ber-H2/saporin is effective in patients with refractory Hodgkin's disease. However, responses are short and partial, one of the main reasons being the inability to repeat IT doses because of formation of human antibodies against the murine antibody and/or the toxin. To overcome this problem, we constructed two new anti-CD30 ITs by covalently linking the mouse monoclonal antibody Ber-H2 to the type 1 ribosome-inactivating proteins (RIPs) momordin (MOM) and pokeweed antiviral protein from seeds (PAP-S), which do not cross-react with each other or with saporin. Both ITs inhibited protein synthesis by Hodgkin's disease and anaplastic large-cell lymphoma (ALCL)-derived CD30+ target cell lines with a very high efficiency (IC50 ranging from5 x 10(-13) M to 2.75 x 10(-11) M, as RIP). In a SCID mouse model of xenografted CD30+ human ALCL, a 3d treatment with non-toxin doses of Ber-H2/MOM (50%LD50), started 24 h after transplantation, prevented tumour development in about 40% of the animals and significantly delayed tumour growth rate in the others. Main toxicity signs in mice and rabbits were dose-related increase of serum transaminases (AST and ALT) and creatine phosphokinase (CPK). LD50 (as RIP) in Swiss mice was 7 mg/kg for Ber-H2/MOM and 0.45 mg/kg for Ber-H2/PAP-S. Sequential administration of two anti-CD30 ITs (Ber-H2/MOM and Ber-H2/saporin) was well tolerated and did not result in formation of antibodies cross-reacting and with the two plant toxins. The results presented in this paper suggest that in the future, sequential administration of anti-CD30 humanized antibodies linked to antigenically distinct type 1 RIPs (saporin, MOM, PAP-S) should be feasible.

Human CD34+ hematopoietic cells were purified using the avidin-biotin immunoabsorption technique. The selected population showed 78.6 +/- 3% CD34+ cells and the overall recovery of CD34+ cells, CFU-GM and BFU-E from the starting population was 34 +/- 5%, 71 +/- 4% and 67 +/- 2%, respectively. Hematopoietic progenitor cell purification also resulted in 3 log of normal T cell depletion from the bone marrow (BM) by immunofluorescence analysis. Moreover, when unseparated BM cells were mixed with the CD34- lymphoma cell lines D430B and Raji, the removal of greater than 3 log of tumor cells from the enriched CD34+ cell fraction was demonstrated. To increase the neoplastic cell purging, several immunotoxins (IT) containing the ribosome-inactivating protein (RIP) saporin and directed toward the lymphoid-associated antigens CD30 and CD2 were prepared. Our experiments showed only a minimal toxicity of the immunoconjugates on colony-forming cells (CFC) derived from purified CD34+ cells. Conversely, all the ITs were very effective in inhibiting protein synthesis and growth of normal and neoplastic lymphoid cells. Further experiments demonstrated that the sequential combination of CD34+ cells purification and IT treatment resulted in 5 or more log of tumor cell purging with no additional loss of BM progenitor cells.