Novel Mechanisms of Non-Apoptotic Cell Death Evoked by Type II Anti-CD20 (Tositumomab) and HLA-DR Monoclonal Antibodies
Authors: Illidge Tim , Ivanov Andrei , Beers Stephen A , Walshe Claire , Chan Claude , Alduaij Waleed , Glennie Martin J. , Cragg Mark S 
Published: October 2019

Abstract
Monoclonal antibodies (mAb) directed to a range of cell surface antigens on malignant lymphoid cells are increasingly a key component of the treatment of a whole range of haematological malignancies and remain the focus of intense ongoing clinical investigation. The anti-CD20 mAb rituximab has over the last decade substantially increased clinical responses leading to a marked improvement in outcome for many patients with B cell malignancies. The pursuit of mAb to improve on the efficacy of rituximab is currently undergoing intensive research, with several new candidate mAb now undergoing clinical evaluation. These newer generation anti-CD20 mAb have largely focused on improvements in increasing the affinity of the Fc-FcgR interactions and the recruitment of immune effector cells as these are thought to underlie much of the mAb therapeutic effect. However the immune effector functions of mAb does not explain why certain mAb specificities are more potent than others in inducing tumour cell death. Previously, we have demonstrated that Type II anti-CD20 mAb (Tositumomab) were able to evoke direct cell death via a non-apoptotic mode of death that appeared to be linked with the induction of homotypic adhesion. In this study we have considerably extended these observations using both in vitro cell lines and primary CLL cells. Using time-lapse microscopy and the FRAP technique, we revealed that de novo F-actin synthesis within cell contact area is critical for both homotypic adhesion and cell death induced by both Type II anti-CD20 mAb (Tositumomab) and HLA DR Class II mAb (L243). We have demonstrated that the mode of cell death engaged is rapid, non-apoptotic and non-autophagic as detected by inability to inhibit this form of cell death with pan-caspase inhibitor QVD, overexpressed Bcl2 as well as with siRNA against key autophagy regulators Beclin 1 and ATG-12. Scanning and transmission electon microscopy studies revealed the cytoplasmic nature of cell death involving lysosomes which swell and then disperse their contents into the cytoplasm. The increase in the lysosomal compartment followed by lysosome membrane permeabilisation and subsequent cell death has also been confirmed by flow cytometry and fluorescence microscopy, using lysosome specific probe lysotracker. Furthermore, this cell death has been shown to be dependent on active V-type ATPase but not on chymotrypsinlike or trypsin-like serine proteases. Using time lapse microscopy, we show here for the first time that malignant B cells, undergoing homotypic adhesion, actively communicate via ~ 5 nm wide temporary inter-cytoplasmic bridges. The formation of these channels is accompanied by the exchange of plasma membrane components. Moreover, the extent of plasma membrane swapping correlates with the extent of cell death induced by both anti-CD20 and anti-HLA DR antibodies. This data provide new insights into the potential mechanisms underlying mAb-induced cell death and may guide the rational design of more effective mAb therapy in B cell malignancies.