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Impact of target antigen properties on antibody effector mechanisms

Impact of target antigen properties on antibody effector mechanisms
Impact of target antigen properties on antibody effector mechanisms
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.
University of Southampton
Cleary, Kirstie
16e11432-9855-4b5b-8c3f-be86f0ef51f0
Cleary, Kirstie
16e11432-9855-4b5b-8c3f-be86f0ef51f0
Elliott, Timothy
16670fa8-c2f9-477a-91df-7c9e5b453e0e
Collins, Jane
be0e66f1-3036-47fa-9d7e-914c48710ba4

Cleary, Kirstie (2015) Impact of target antigen properties on antibody effector mechanisms. University of Southampton, Doctoral Thesis, 210pp.

Record type: Thesis (Doctoral)

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.

Text
KCleary Thesis 2016 Final - Version of Record
Available under License University of Southampton Thesis Licence.
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Published date: September 2015

Identifiers

Local EPrints ID: 434168
URI: http://eprints.soton.ac.uk/id/eprint/434168
PURE UUID: f8c7abe0-3caa-4eeb-b861-5cb7aa720072
ORCID for Kirstie Cleary: ORCID iD orcid.org/0000-0001-6200-4945
ORCID for Timothy Elliott: ORCID iD orcid.org/0000-0003-1097-0222

Catalogue record

Date deposited: 13 Sep 2019 16:30
Last modified: 14 Sep 2019 00:37

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Contributors

Author: Kirstie Cleary ORCID iD
Thesis advisor: Timothy Elliott ORCID iD
Thesis advisor: Jane Collins

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