In vitro evaluation of 213Bi-rituximab versus external gamma irradiation for the treatment of B-CLL patients: relative biological efficacy with respect to apoptosis induction and chromosomal damage

Abstract

External source radiotherapy and beta radioimmunotherapy (RIT) are effective treatments for lymphoid malignancies. The development of RIT with alpha emitters is attractive because of the high linear energy transfer (LET) and short path length, allowing higher tumour cell kill and lower toxicity to healthy tissues. We assessed the relative biological efficacy (RBE) of alpha RIT (in vitro) compared to external gamma irradiation with respect to induction of apoptosis in B chronic lymphocytic leukaemia (B-CLL) and induction of chromosomal damage in healthy donor B and T lymphocytes. The latter was measured by a micronucleus assay. 213Bi was eluted from a 225Ac generator and conjugated to CD20 antibody (rituximab) with CHX-A"-DTPA as a chelator. B-CLL cells from five patients were cultured for 24 h in RPMI/10% FCS while exposed to 213Bi conjugated to CD20 antibody or after external 60Co gamma irradiation. Binding assays were performed in samples of all patients to calculate the total absorbed dose. Apoptosis was scored by flow cytometric analyses of the cells stained with annexin V-FITC and 7-AAD. Apoptosis was expressed as % excess over spontaneous apoptosis in control. Full dose range experiments demonstrated 213Bi-conjugated CD20 antibody to be more effective than equivalent doses of external gamma irradiation, but showed that similar plateau values were reached at 10 Gy. The RBE for induction of apoptosis in B-CLL was 2 between 1.5 and 7 Gy. The micronucleus yield in lymphocytes of healthy volunteers was measured to assess the late toxicity caused by induction of chromosomal instability. While gamma radiation induced a steady increase in micronucleus yields in B and T cells, the damage induced by 213Bi was more dramatic, with RBE ranging from 5 to 2 between 0.1 Gy and 2 Gy respectively. In contrast to gamma irradiation, 213Bi inhibited mitogen-stimulated mitosis almost completely at 2 Gy. In conclusion, high-LET targeted alpha particle exposure killed B-CLL cells more effectively than did external gamma irradiation at a low dose (RBE=2), while a plateau was reached at a high dose. Long-term toxicity on healthy B and T lymphocytes was systematically higher for the alpha emitter (RBE=5 to 2).

mFISH analysis of chromosome aberrations in workers occupationally exposed to mixed radiation

“…Hence, the findings demonstrate that translocations are a biological marker responding to the external gamma-ray exposure when concerning mixed radiation exposures. This may be correlated with a higher RBE and a higher long-term toxicity by alpha emitters (Vandenbulcke et al 2003) leading to depletion of lethally hit lymphocytes with time.…”

Feasibility of the radioastatination of a monoclonal antibody with astatine‐211 purified by wet extraction

“…4 This makes them highly cytotoxic with a relative biologic effectiveness 5-20 times that of b À -particles. 5,6 Another advantage of a-particles, compared with b À -particles, is that they exhibit a low dependence on dose rate and oxygen enhancement effects. 7 Astatine-211 is the heaviest halogen and it is one of the most promising a-emitters for medical applications.…”

213Bi-induced death of HSC45-M2 gastric cancer cells is characterized by G2 arrest and up-regulation of genes known to prevent apoptosis but induce necrosis and mitotic catastrophe

“…Insight into these mechanisms could lead to improved strategies for radioimmunotherapy with a-emitters. Incubation of human prostate cancer cells and lymphocytes from leukemia patients with 213 Bi-immunoconjugates has been reported to activate apoptotic pathways (11,12), whereas treatment of gastric cancer cells expressing d9-E-cadherin with 213 Bi-d9MAb triggered nonapoptotic pathways (10).…”

“…BrdUrd was removed completely by repeated washings with BrdUrd-free culture medium. At defined time points after start of irradiation (4,6,12, and 24 h), cells were detached with 1 mmol/L EDTA in PBS and fixed in 80% ethanol. For preparation of nuclei, cells were treated with RNase A (Sigma) and subsequently incubated with pepsin (Merck; 0.7 FIP-U/mg; 5% in 0.05 N HCl) for 10 min at 37jC.…”