Rituximab Blocks Binding of Radiolabeled Anti-CD20 Antibodies (Ab) but Not Radiolabeled Anti-CD45-Ab.
Authors: Gopal Ajay K. , Press Oliver W. , Wilbur Shani M. , Maloney David G. , Pagel John M. 
Published: October 2019

Abstract
The use of rituximab for treatment of B-cell non-Hodgkin lymphoma (B-NHL) is associated with a long in vivo persistence (months), which is enhanced by protracted maintenance regimens. At the time of progression, the radioimmunoconjates131I-Tositumomab and 90Y-Ibritumomab Tiuxetan are often employed to induce remissions in patients with relapsed B-NHL. Despite this common clinical scenario, little is known about the potential impact of high levels of circulating anti-CD20 antibody (Ab) on targeting of a second radiolabeled anti-CD20-Ab. Furthermore, alternate strategies to overcome potential blocking of the radiolabeled Ab have not been identified. In order to address this critical question we evaluated the impact of rituximab on both CD20 and CD45 targeting of B-NHL. First, we demonstrated that CD45 and CD20 were expressed in comparable densities on B-NHL lines (FL-18, Granta, Ramos). Next, blocking by rituximab was assessed by co-incubation of B-NHL lines with increasing concentrations of rituximab (0–2000μg/ml), followed by secondary incubation with either fluoresceinated tositumomab or fluoresceinated BC8 (anti-CD45). Evaluation by flow cytometry revealed that rituximab concentrations as low as 5μg/mL reduced tositumomab binding to CD20 by 86% (p=.00027), with complete blocking occurring at concentrations as low as 50μg/mL (p=0.00017). In contrast, rituximab had no effect on CD45 ligation (p=0.89). We then showed that serum derived from patients treated with rituximab (> 4 weeks prior to blood draw) had rituximab concentrations ranging from 14.6–82.2μg/ml and could fully block the binding of tositumomab to rituximab-naïve, patient-derived B-NHL cells at concentrations as low as 7.3μg/mL. Rituximab-containing serum exhibited no impact on binding of anti-CD45 Ab. Experiments assessing the biodistribution of radiolabeled Ab in human B-NHL xenografts in athymic mice demonstrated that the estimated radiation dose that would be delivered to tumor sites (% injected dose/g tumor) with 131I-labeled Ab was comparable with Tositumomab or BC8 in a rituximab-free system (p=0.54). In contrast, mice pre-treated with rituximab exhibited a 55% reduction in the radiation dose that would be delivered to tumor xenografts using 131I-labeled Tositumomab (p=0.00026), but no effect was observed on the estimated radiation dose that would be delivered by radioiodinated BC8 (p=.66). Therapy experiments confirmed the predictions of the biodistribution studies and demonstrated similar tumor control for 131I-Tositumomab and 131I-BC8 in the absence of rituximab (p=.21), but inferior tumor control for rituximab pre-treated mice receiving 131I-Tositumomab as compared to rituximab + 131I-BC8 (p=.00016). Likewise, survival was inferior for mice treated with rituximab followed by 131I-Tositumomab compared to mice treated with 131I-Tositumomab alone (p=.05), whereas the survival of mice treated with 131I-BC8 were not impacted by rituximab pre-treatment (p=.36). Our data indicate that even low concentrations of long half-lived Ab such as rituximab, can impair the binding of a secondary monoclonal Ab targeting the same antigen. These results identify major implications for the current clinical use of CD20-directed lymphoma therapy and uncover potential limitations for the development of novel therapeutic antibodies that compete for identical antigens.