Update on the rational use of tositumomab and iodine-131 tositumomab radioimmunotherapy for the treatment of non-Hodgkin’s lymphoma

Michael J Burdick, Roger M MacklisDepartment of Radiation Oncology, Taussig Cancer Center and Cleveland Clinic Lerner College of Medicine, Cleveland, OH, USAAbstract: Targeted radioimmunotherapy in non-Hodgkin’s B-cell lymphoma (NHL) offers an efficacious therapy and minimal toxicity compared to conventional chemotherapy. Iodine 131 tositumomab (131I-TST) is a murine monoclonal antibody against the CD20 cell surface protein and is directly covalently conjugated to 131I, a radioactive β and γ emitter. While initially approved for use in relapsed, refractory, or transformed low grade B-cell NHL, investigational uses with promising results include autologous stem cell transplant, intermediate grade NHL, and the frontline management of indolent NHL. This review summarizes the 131I-TST literature on mechanism of action, treatment indications, treatment delivery, efficacy, investigational uses, and future prospects.Keywords: tositumomab, radioimmunotherapy, non-Hodgkin’s lymphoma, Bexxa

Surface levels of CD20 determine anti-CD20 antibodies mediated cell death in vitro.

The sensitivity of human Burkitt's lymphoma cells to rituximab (Rtx) and tositumomab (Tst) was assessed on cells expressing different levels of CD20 on surface. Cells that harbor low CD20 levels may resists against therapeutics response to CD20-specific antibodies. We postulated that, radiation-induced modulation of CD20 surface levels may play a crucial and central role in determining the relative efficacy of rituximab and tositumomab in treating Burkitt's lymphoma disease. Here, we examined the γ-radiation-induced CD20 expression in the Burkitt lymphoma cell line 'Daudi' and the relation of differential levels of CD20 with anti-CD20 mAbs mediated cell death.In this study we examined kinetics of CD20 expression following sub lethal doses ofγ-radiation to Daudi cells and thereafter anti-CD20 mAbs (rituximab and tositumomab) were added in cell suspensions. The correlation of kinetics of CD20 expression and cells treated with anti-CD20 mAbs/or corresponding isotype Abs with special reference to changes in mitochondrial membrane potential and reactive oxygen species generation was also examined. Further, we also investigated the efficacy of anti-CD20 mAbs and possible induction of cell death in relation to levels of CD20 cell surface expression.This report provides evidence that CD20 expression can be induced by exposure of cells to γ-radiation. In addition, these findings demonstrated that the efficacy of anti-CD20 mAbs is dependent on the surface levels of CD20. Based on these findings, we hypothesized (i) irradiation just prior to immunotherapy may provide new treatment options even in aggressive B cell tumors, which are resistant to current therapies in vivo (ii) The efficacy of induction of apoptosis varies with type of monoclonal antibodies in vitro

Synthesis of hemoglobin F in adult simian erythroid progenitor-derived colonies.

The simian hematopoietic system is known to respond to anemic stress with the production of erythrocytes containing large amounts of fetal hemoglobin. To determine the regulatory mechanism responsible for hemoglobin F (HbF) production in stress erythropoiesis, adult simian bone marrow cells were cultured in plasma clots in the presence or absence of erythropoietin and burst-promoting activities, and the HbF content of progenitor-derived colonies was determined by radioimmunoligand assay. Three classes of erythroid progenitors were detected: BFU-E, CFU-E, and a very mature cohort of dense highly erythropoietin-responsive cells (HERC). These classes varied in inverse proportion to their maturity with respect to their potential for HbF accumulation in the colonies to which they give rise. Both erythropoietin and burst-promoting activity stimulated HbF production, particularly in colonies derived from immature progenitors. For example, under conditions of high erythropoietin stimulation, BFU-E colonies contained 13.7-37.7% HbF, CFU-E colonies contained 2.8-13.5% HbF, and HERC colonies 0-1% HbF. These results suggest that under nonstress conditions simian erythrocytes are derived almost entirely from HERC progenitors and proerythroblasts in which gamma chain synthesis is suppressed. During stress erythropoiesis, augmented HbF accumulation could be explained by the rapid entrance into the marrow of proerythroblasts directly derived from immature progenitors. Gamma chain production in these proerythroblasts is additionally regulated by the changes in environmental erythropoietin and burst-promoting activities

The changes in ROS and MMP (ΔΨm).

<p>(A, B and C) To measure changes ROS levels, fluorescence of CM-DCF was acquired at Ex λ of 488±10 nm and Em λ 517–527 nm and the results are expressed as the mean fluorescence ±SD of three independent experiments and statistical analysis was performed using ONE way ANOVA. Significant values are represented as; ***<i>p</i><0.001 for Rtx <i>vs</i> 0.5Gy+Rtx or 1.5Gy+Rtx, ***<i>p</i><0.001 for Tst <i>vs</i> 0.5Gy+Tst or 1.5Gy+Tst, ##<i>p</i><0.01 for sham irradiated control <i>vs</i> 0.5Gy, ##<i>p</i><0.001 for sham irradiated control <i>vs</i> 1.5Gy. Corresponding Isotype controls antibodies were used to measure changes in ROS levels. (D and E) The changes in ΔΨm were expressed from the mean fluorescence ±SD of three independent experiments and statistical analysis was performed using ONE way ANOVA. Significant values represents as; #p<0.05 for control <i>vs</i> 1.5Gy, *p<0.05 for control <i>vs</i> Rtx or 0.5Gy+RTX or 0.5Gy+Tst or 1.5Gy+Tst, **p<0.01 for control <i>vs</i> 1.5Gy+Rtx. Corresponding Isotypic controls were used to measure changes in ΔΨm levels [Human IgG1 (for Rtx) and Mouse IGG2a (Tst)].</p

Cross-linking or homotypic adhesions (aggregations) and cell death.

<p>(A–B) The binding of Fab regions of Anti-CD20 mAbs (Rtx and Tst) on CD20 and Fc region binding to FcγRIIB1 thereby induction of cross-linking or homotypic adhesions (aggregations) as well as extra cross-linking induced by corresponding secondary antibodies were observed microscopically (20x). Isotype control antibodies were taken separately to measure non-specific cross-linking. (C, D and E) Anti-CD20 mAbs induced cell death was measured using PI uptake by flowcytometrically. PI is membrane impermeable, generally excluded from viable cells and therefore, commonly used for identifying alterations in biological membrane and thereby death of cells in a population. The fold induction cell death was measured from mean fluorescence ±SD and statistical analysis was performed using ONE way ANOVA. Significant values represented as; ***<i>p</i><0.001 for Rtx <i>vs</i> 0.5Gy+Rtx and 1.5Gy+Rtx. ***<i>p</i><0.001 for Tst <i>vs</i> 1.5Gy+Tst, **<i>p</i><0.01 Tst <i>vs</i> 0.5Gy+Tst. Further, ###<i>p</i><0.001 for sham irradiated control <i>vs</i> 1.5Gy, and $$$<i>p</i><0.001 for sham irradiated control <i>vs</i> Tst. Isotype control antibodies were taken separately to measure non-specific induction of cell death. (F) Induction of cell death by extra cross-linking using secondary antibodies: Cells expressing different levels of CD20 and treated with monoclonal anti-CD20 antibodies or isotypes (separately) were further incubated with corresponding secondary antibodies. PI uptake was also measured flowcytometrically to measure extra crosslinking induced cell death. (G) Cell cycle analysis: For cell cycle analysis, binding of PI with DNA were used as a marker of DNA content which depicts phase of cells in cell cycle.</p

The kinetics of changes in CD20 expression on Daudi cells following exposure to 0.5 Gy γ-radiation.

<p>(A and B) Total CD20 levels: The changes in CD20 expression were measured using anti-CD20 antibodies. Results are expressed as ratio of levels of CD20 (CD20/β-Actin) with respect to 0 hr control. (C) CD20 expression on cell surface: The levels of CD20 at cell surface levels were calculated using quantiBRITE beads and expressed as numbers of CD20 molecules/cell. The index histogram is showing levels of CD20 at 20 hr (0.5Gy) with respect to 0 hr. Statistical analysis was done using Student's t-test (***<i>p</i><0.001).</p

Anti-CD20 mAbs-induced programed cell death.

<p>Schematic diagram illustrating the kinetics of changes in CD20 expression followed 0.5Gy γ-radiation and sequence of events in the proposed possible cell death pathway evoked by type I anti-CD20 mAbs (Rtx) and type I anti-CD20 mAbs (Tst). Anti-CD20 mAbs ligation results in cross-linking and homotypic adhesions followed by generation of ROS and create genotoxic stress which ultimately culminates in apoptotic and non-apoptotic cell death. *SB =  Strand Break.</p