To demonstrate that radio-immunotherapy i s more effective than the combination of the cold antibody with an equal absorbed dose of gamma radiation we compared gamma irradiation of B-CLL cells in vitro with an alpha-emitter conjugated to an membranous (CD20) and an internalized antibody (CD19). In a previous study alpha-RIT with 213 Bi-Rituximab was indeed shown to induce apoptosis in vitro in CLL cells more effectively than gamma plus cold antibody (RBE=2.2) in patients who were sensitive to chlorambucil, but not significantly in those who had become resistant to alkylator or fludarabine chemotherapy. To measure if the response initiated by alpha-RIT can be amplified or restored by radiosensitizers or agents that cripple antiapoptotic defenses induced or present in tumor cells, we combined this with fludarabine and/or corticosteroids .
213-Bi was eluted from an 225-Ac generator and used conjugated to anti-CD20or anti-CD19 with CHX-A''-DTPA as a chelator. B-CLL cells from 32 patients were cultured for 24 hours in CRPMI/10%FCS while exposed to the following treatments : (1) 213-Bi conjugated anti-CD20(2Gy) (2) 213-Bi conjugated anti-CD19(2Gy) (3) external 60-Co gamma-irradiation (2Gy), (4) Fludarabine 5 mM, (5) methylprednisolone 10 ยตM, (6) 213Bi antiCD19 and 20 plus prednisolone and/or fludarabine (7) gamma radiation plus prednisolone and/or fludarabine. Apoptosis was scored by flowcytometry staining with AnnexinV-FITC and 7-AAD. The apoptosis score was expressed as % excess over spontaneous apoptosis in control culture medium. 213 Bi-Rituximab alone induced significantly more apoptosis than external gamma radiation (24.5+14.8 % vs 17+11.5%, p <0.001) 213 Bi-antiCD-19 was superior to 213 Bi-Rituximab with borderline statistical significance (32.7+17.8%, p=0.048, paired t). Treatment with prednisolone ga ve 22.6+13.2% and combination with 213BiantiCD20, CD19 or gamma increased this to 46+13.9, 51.6+16.9 and 40.1+14.9% respectively. Fludarabine alone resulted in 13.4+ 12.7 and in combination with 213BiantiCD20, CD19 or gamma this increased to 39.9+15.8, 46.6+19 and 30.7+18.1%. When combining fludarabine and prednisolone apoptosis was induced in 31.1+15.9 % of cells and addition of 213BiantiCD20, CD19 or gamma increased this to 51.6+16.9, 55.9+17.2 and 44.9+16.7% respectively.
In conclusion, both fludarabine and corticosteroids were at least additive in inducing apoptosis when combined with gamma radiation or alpha-radioimmunotherapy of B-CLL in vitro. S99 ABSTRACTS