Impact of target antigen properties on antibody effector mechanisms

Abstract

Monoclonal antibody (mAb) immunotherapy has proven effective in the treatment of haematological malignancies. Antibodies towards unique epitopes within the same antigen can engage different effector mechanisms to facilitate cell clearance. Understanding what drives the engagement of these mechanisms is important for the development of new therapeutics. This thesis investigated the role of the antigen, in particular the epitope bound by a mAb in defining the effector mechanisms engaged. Specifically, the role of distance between the epitope and the target cell membrane in relation to the engagement of the effector mechanisms; complement dependent cytotoxicity (CDC), antibody dependent cellular phagocytosis (ADCP) and antibody dependent cellular cytotoxicity (ADCC) were assessed. A panel of model antigens were generated; incorporating the same epitope for a clinically relevant mAb attached to the N-terminus of various CD137 constructs. Extracellular domains of CD137 were removed or added in order to change the distance of the clinically relevant epitope from the cell membrane. These constructs were transfected into CHO-S and A20 cells and tested in vitro to assess the engagement of the three aforementioned effector mechanisms. It was found that the engagement of CDC and ADCC was diminished when targeting the largest (8-domain) construct (therefore most distal from the membrane), whilst ADCP was impaired with the smallest (membrane-flush) construct and required the presence of at least one extracellular domain for activity. However, ADCP engagement was restored when the membrane-flush epitope was tethered to the membrane via a GPI anchor rather than a transmembrane peptide domain. These findings were confirmed using two separate epitopes targeted by rituximab and CAMPATH-1H antibodies. Finally, the therapeutic response when targeting either the membrane-flush or 8-domain constructs was investigated in vivo. Mice who received A20 cells expressing the membraneflush construct exhibited clearance of tumour in the spleen following antibody therapy, whilst those that received tumours expressing the 8-domain construct did not respond to therapy. Together the work in this thesis demonstrates how the effector mechanisms engaged by a mAb can be altered dependent on the position of the epitope in relation to the cell membrane.<br/