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Understanding the role for Fc gamma receptors in response to anti-CD20 monoclonal antibody therapy

Understanding the role for Fc gamma receptors in response to anti-CD20 monoclonal antibody therapy
Understanding the role for Fc gamma receptors in response to anti-CD20 monoclonal antibody therapy
Research into cancer therapeutics has utilized the antigen specificity of antibodies to provide targeted cancer treatments. One notable therapeutic is rituximab, a chimeric human IgG1 antibody that has specificity to the CD20 protein on the surface of B cells. This antibody is currently used in the treatment of leukaemia and lymphoma. However, due to reasons not fully understood patients often relapse and become resistant to therapy. Therefore an increasingly important area of research is to uncover the key mechanisms of action of anti-CD20 antibodies.

It has been demonstrated that antibody Fc: FcγR interactions are critical foreffective anti-CD20 antibody dependent cellular cytotoxicity (ADCC). During this project the role of Fc: FcγR interactions and their ability to mediate anti-CD20 mediated B cell depletion was explored. We assessed two different types of anti-CD20 antibody: type I, rituximab has been shown to have high levels of antibody:antigen internalisation following CD20 engagement. This process has been termed antigenic modulation. In contrast, type II, obinutuzumab do not display a high level of antigenic modulation. We assessed the ability of rituximab and obinutuzumab to deplete normal and malignant B cells. Additionally, macrophages and NK cells have both been implicated as key FcγRexpressing effector cells. Therefore, we assessed rituximab and obinutuzumab using in vitro macrophage or NK cell mediated ADCC assays and looked at the impact antigenic modulation had on effector cell activity. In vivo analysis revealed that obinutuzumab give more sustained B cell depletion compared to rituximab. Furthermore, we demonstrate that this sustained depletion is due to a lack of antigenic modulation preventing NK cell and macrophage mediated ADCC. In contrast rituximab undergoes significant internalisation preventing both NK cell and macrophage mediated ADCC.

In addition, it has been well documented that the tumour microenvironment will affect the phenotype of the surrounding stroma, particularly macrophages. Established tumours are typically associated with low levels of apoptosis. Therefore, we investigated the impact dysregulated apoptosis had on the ability of macrophages to orchestrate ADCC. Using apoptosis resistant Vav Bcl-2 mice, as well as other apoptosis defective strains, we demonstrate that there is a decrease in the activatory to inhibitory FcγR ratio on effector cell subsets.This was largely due to an increase in inhibitory FcγRIIb. Subsequent analysisrevealed that an increase in FcγRIIb may be due to the autoimmune-likephenotype of the Vav Bcl-2 mouse.

This knowledge will be beneficial to future consideration of antibody selection and provides further avenues of research into antibody and FcγR interactions.
University of Southampton
Tipton, Thomas R.W.
a0a94b14-7d09-4411-b988-84c9d39eb0d6
Tipton, Thomas R.W.
a0a94b14-7d09-4411-b988-84c9d39eb0d6
Cragg, Mark
ec97f80e-f3c8-49b7-a960-20dff648b78c

Tipton, Thomas R.W. (2015) Understanding the role for Fc gamma receptors in response to anti-CD20 monoclonal antibody therapy. University of Southampton, Doctoral Thesis, 271pp.

Record type: Thesis (Doctoral)

Abstract

Research into cancer therapeutics has utilized the antigen specificity of antibodies to provide targeted cancer treatments. One notable therapeutic is rituximab, a chimeric human IgG1 antibody that has specificity to the CD20 protein on the surface of B cells. This antibody is currently used in the treatment of leukaemia and lymphoma. However, due to reasons not fully understood patients often relapse and become resistant to therapy. Therefore an increasingly important area of research is to uncover the key mechanisms of action of anti-CD20 antibodies.

It has been demonstrated that antibody Fc: FcγR interactions are critical foreffective anti-CD20 antibody dependent cellular cytotoxicity (ADCC). During this project the role of Fc: FcγR interactions and their ability to mediate anti-CD20 mediated B cell depletion was explored. We assessed two different types of anti-CD20 antibody: type I, rituximab has been shown to have high levels of antibody:antigen internalisation following CD20 engagement. This process has been termed antigenic modulation. In contrast, type II, obinutuzumab do not display a high level of antigenic modulation. We assessed the ability of rituximab and obinutuzumab to deplete normal and malignant B cells. Additionally, macrophages and NK cells have both been implicated as key FcγRexpressing effector cells. Therefore, we assessed rituximab and obinutuzumab using in vitro macrophage or NK cell mediated ADCC assays and looked at the impact antigenic modulation had on effector cell activity. In vivo analysis revealed that obinutuzumab give more sustained B cell depletion compared to rituximab. Furthermore, we demonstrate that this sustained depletion is due to a lack of antigenic modulation preventing NK cell and macrophage mediated ADCC. In contrast rituximab undergoes significant internalisation preventing both NK cell and macrophage mediated ADCC.

In addition, it has been well documented that the tumour microenvironment will affect the phenotype of the surrounding stroma, particularly macrophages. Established tumours are typically associated with low levels of apoptosis. Therefore, we investigated the impact dysregulated apoptosis had on the ability of macrophages to orchestrate ADCC. Using apoptosis resistant Vav Bcl-2 mice, as well as other apoptosis defective strains, we demonstrate that there is a decrease in the activatory to inhibitory FcγR ratio on effector cell subsets.This was largely due to an increase in inhibitory FcγRIIb. Subsequent analysisrevealed that an increase in FcγRIIb may be due to the autoimmune-likephenotype of the Vav Bcl-2 mouse.

This knowledge will be beneficial to future consideration of antibody selection and provides further avenues of research into antibody and FcγR interactions.

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Tom Tipton Thesis - Version of Record
Available under License University of Southampton Thesis Licence.
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Published date: September 2015

Identifiers

Local EPrints ID: 415837
URI: http://eprints.soton.ac.uk/id/eprint/415837
PURE UUID: 5e33b51f-8332-4795-aec2-64aece0bfc7b
ORCID for Mark Cragg: ORCID iD orcid.org/0000-0003-2077-089X

Catalogue record

Date deposited: 24 Nov 2017 17:30
Last modified: 14 Mar 2019 01:49

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Contributors

Author: Thomas R.W. Tipton
Thesis advisor: Mark Cragg ORCID iD

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