Investigating the Mechanisms of Antibody-induced Cell Death and Radioimmunotherapy in B-cell Malignancies

Abstract

Monoclonal antibodies (mAbs) have revolutionised the treatment of B-cell malignancies improving overall survival. Despite this success many patients relapse with treatment-resistant disease warranting the development of novel mAb-based therapies with improved therapeutic efficacy. Central to this development is an enhanced understanding of the cytotoxic mechanisms of mAb. Although Fc-dependent mechanisms of mAb-mediated tumour clearance have been extensively studied, the ability of mAbs to directly induce programmed cell death (PCD) on antigen ligation remains largely under-investigated. The aim of this thesis was to dissect the cellular mechanisms underlying mAb-induced PCD in B-cell malignancies, as well as to assess its contribution to the therapeutic efficacy of radioisotope-conjugated anti-CD20 mAbs (radioimmunotherapy), which demonstrated impressive clinical responses in certain B-cell lymphoma subtypes.We first demonstrate that the ability of mAbs to evoke direct PCD correlates with the induction of intercellular homotypic adhesion (HA). Using the type II anti-CD20 mAb tositumomab and anti-HLA DR mAb we reveal that HA and the ensuing cell death were dependent on the peripheral relocalisation of actin and plasma membrane cholesterol. Cell death was independent of apoptosis and autophagy, but required lysosomes that permeabilise and release cathepsins into the cytoplasm and surrounding environment. This lysosomal cell death was potently elicited by the next generation humanised type II anti-CD20 mAb GA101, in contrast to the type I anti-CD20 mAbs rituximab and ofatumumab. Given the significant role of reactive oxygen species (ROS) in non-apoptotic cell death, their contribution to the non-apoptotic lysosomal cell death evoked by mAb was next investigated. The production of ROS by mAb was critical for cell death and appears to occur downstream of HA and lysosomal permeabilisation. MAb-induced ROS were produced by the specialised ROS-generating enzyme complex NADPH oxidase, with the NOX2 isoform contributing to mAb-induced cell death.Finally, an animal model was developed to help assess the contribution of mAb-induced non-apoptotic cell death, which increases when combined with radiation therapy (RT), to the therapeutic efficacy of anti-CD20-based radioimmunotherapy. Preliminary results in xenograft and syngeneic lymphoma models suggest that the induction of additive cell death with anti-CD20 mAb in combination with RT in vitro correlates with enhanced tumour clearance by anti-CD20 mAb and RT in vivo. In summary this thesis describes a novel non-apoptotic cell death pathway directly triggered by mAb. The contribution of this mode of cell death to the therapeutic efficacy of mAb-based therapies should be further investigated to establish whether it can be exploited to improve clinical outcomes and help treat patients refractory to conventional chemotherapy and immunotherapy.

Date of Award	31 Dec 2011
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Timothy Illidge (Supervisor) & Jamie Honeychurch (Supervisor)