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Understanding the molecular mechanisms underpinning the biological activity of antibodies against the inhibitory CD32b receptor

Understanding the molecular mechanisms underpinning the biological activity of antibodies against the inhibitory CD32b receptor
Understanding the molecular mechanisms underpinning the biological activity of antibodies against the inhibitory CD32b receptor
Fc gamma receptor (FcγR) IIB (CD32b) is the only inhibitory FcγR in humans and regulates the action of Immunoreceptor Tyrosine-based Activation Motif (ITAM) containing receptors such as the B cell receptor and activatory FcR. The inhibitory actions of CD32b are detrimental in a cancer immunotherapy setting, yet have the potential to regulate aberrant immune responses in autoimmune conditions, making CD32b an attractive target for immunotherapy.

A panel of human anti-human CD32b monoclonal antibodies (mAb) were recently developed, capable of blocking IgG immune complex binding to the receptor. Although predicted to bind within a similar region of the receptor, opposing biological responses were observed with these mAb, with some activating the receptor (agonists) and others blocking CD32b phosphorylation and consequent activation (antagonists). This project aimed to identify the molecular mechanisms that determined the agonist or antagonist activity of these anti-CD32b mAb.

Microscopy studies revealed that agonist but not antagonist antibodies induced clustering of CD32b, which was necessary for CD32b activation. The ability of the anti-CD32b mAb to cluster CD32b was therefore thought to drive the opposing biological responses observed and was proposed to derive from the subtle differences in epitopes engaged by the agonist and antagonist mAb.

The crystal structure of an antagonist complex was not available, therefore small angle X-ray scattering studies were conducted to explore the basis behind the opposing biological responses of the anti-CD32b mAb. In solution, both agonist and antagonist F(ab) were observed to adopt similar conformations. In contrast, when F(ab) were in complex with the extracellular domain of CD32b, antagonist F(ab) appeared to form more elongated complexes with CD32b, showing a greater radius of gyration (Rg) and maximal dimension (Dmax), compared to agonist F(ab):CD32b complexes.

Current crystal structures of the agonist 6G08 F(ab):CD32b complex showed poor agreement to the solution data, therefore molecular dynamics simulations were used to determine atomic structures of agonist 6G08 and an antagonist F(ab), 6G11, that agreed with SAXS data for the proteins in solution. The simulations confirmed previous observations from SAXS studies, that these proteins adopted similar conformations in solution. These methods in VI addition to metadynamics were further applied to the crystal structure of the 6G08 F(ab):CD32b complex and a homology model of the antagonist 6G11 F(ab):CD32b complex.

The structures identified from these simulations revealed a difference in binding orientation between the agonist and antagonist F(ab), with the 6G11 F(ab) binding in an orientation which resulted in a more linear complex with CD32b in comparison to the 6G08 F(ab). When predicting the interactions of an IgG anti-CD32b mAb at the cell surface, the difference in binding orientation between the 6G08 and 6G11 F(ab) were proposed to result in the CD32b receptors coming in to close proximity when bound to 6G08 IgG, whereas 6G11 IgG was predicted to hold the receptors further apart in the cell membrane.

Taken together, a model for determining the biological activity of the anti-CD32b was generated. The model proposes that binding orientation of the F(ab) determines the ability of the anti-CD32b to cluster CD32b in the cell membrane, and that the lower affinity of agonist mAb facilitates clustering of CD32b. In contrast, the extended binding geometry and higher affinity of the antagonist F(ab) results in receptors being held apart in the membrane with less potential for clustering. Further studies will continue to test this model in order to design increasingly effective agonist or antagonist antibodies for the treatment of autoimmunity or cancer, respectively.
University of Southampton
Sutton, Emma, Jill
3d0922bd-d83b-4d82-b2de-3346b82217c4
Sutton, Emma, Jill
3d0922bd-d83b-4d82-b2de-3346b82217c4
Cragg, Mark
ec97f80e-f3c8-49b7-a960-20dff648b78c
Tews, Ivo
9117fc5e-d01c-4f8d-a734-5b14d3eee8dd

Sutton, Emma, Jill (2018) Understanding the molecular mechanisms underpinning the biological activity of antibodies against the inhibitory CD32b receptor. University of Southampton, Doctoral Thesis, 303pp.

Record type: Thesis (Doctoral)

Abstract

Fc gamma receptor (FcγR) IIB (CD32b) is the only inhibitory FcγR in humans and regulates the action of Immunoreceptor Tyrosine-based Activation Motif (ITAM) containing receptors such as the B cell receptor and activatory FcR. The inhibitory actions of CD32b are detrimental in a cancer immunotherapy setting, yet have the potential to regulate aberrant immune responses in autoimmune conditions, making CD32b an attractive target for immunotherapy.

A panel of human anti-human CD32b monoclonal antibodies (mAb) were recently developed, capable of blocking IgG immune complex binding to the receptor. Although predicted to bind within a similar region of the receptor, opposing biological responses were observed with these mAb, with some activating the receptor (agonists) and others blocking CD32b phosphorylation and consequent activation (antagonists). This project aimed to identify the molecular mechanisms that determined the agonist or antagonist activity of these anti-CD32b mAb.

Microscopy studies revealed that agonist but not antagonist antibodies induced clustering of CD32b, which was necessary for CD32b activation. The ability of the anti-CD32b mAb to cluster CD32b was therefore thought to drive the opposing biological responses observed and was proposed to derive from the subtle differences in epitopes engaged by the agonist and antagonist mAb.

The crystal structure of an antagonist complex was not available, therefore small angle X-ray scattering studies were conducted to explore the basis behind the opposing biological responses of the anti-CD32b mAb. In solution, both agonist and antagonist F(ab) were observed to adopt similar conformations. In contrast, when F(ab) were in complex with the extracellular domain of CD32b, antagonist F(ab) appeared to form more elongated complexes with CD32b, showing a greater radius of gyration (Rg) and maximal dimension (Dmax), compared to agonist F(ab):CD32b complexes.

Current crystal structures of the agonist 6G08 F(ab):CD32b complex showed poor agreement to the solution data, therefore molecular dynamics simulations were used to determine atomic structures of agonist 6G08 and an antagonist F(ab), 6G11, that agreed with SAXS data for the proteins in solution. The simulations confirmed previous observations from SAXS studies, that these proteins adopted similar conformations in solution. These methods in VI addition to metadynamics were further applied to the crystal structure of the 6G08 F(ab):CD32b complex and a homology model of the antagonist 6G11 F(ab):CD32b complex.

The structures identified from these simulations revealed a difference in binding orientation between the agonist and antagonist F(ab), with the 6G11 F(ab) binding in an orientation which resulted in a more linear complex with CD32b in comparison to the 6G08 F(ab). When predicting the interactions of an IgG anti-CD32b mAb at the cell surface, the difference in binding orientation between the 6G08 and 6G11 F(ab) were proposed to result in the CD32b receptors coming in to close proximity when bound to 6G08 IgG, whereas 6G11 IgG was predicted to hold the receptors further apart in the cell membrane.

Taken together, a model for determining the biological activity of the anti-CD32b was generated. The model proposes that binding orientation of the F(ab) determines the ability of the anti-CD32b to cluster CD32b in the cell membrane, and that the lower affinity of agonist mAb facilitates clustering of CD32b. In contrast, the extended binding geometry and higher affinity of the antagonist F(ab) results in receptors being held apart in the membrane with less potential for clustering. Further studies will continue to test this model in order to design increasingly effective agonist or antagonist antibodies for the treatment of autoimmunity or cancer, respectively.

Text
Emma_Sutton_PhD_Thesis_FINAL - Version of Record
Restricted to Repository staff only until 28 May 2021.
Available under License University of Southampton Thesis Licence.

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Published date: October 2018

Identifiers

Local EPrints ID: 436544
URI: http://eprints.soton.ac.uk/id/eprint/436544
PURE UUID: 0b498d11-0470-41a4-bba3-dcd04d5de225
ORCID for Mark Cragg: ORCID iD orcid.org/0000-0003-2077-089X
ORCID for Ivo Tews: ORCID iD orcid.org/0000-0002-4704-1139

Catalogue record

Date deposited: 12 Dec 2019 17:30
Last modified: 13 Dec 2019 01:38

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Contributors

Author: Emma, Jill Sutton
Thesis advisor: Mark Cragg ORCID iD
Thesis advisor: Ivo Tews ORCID iD

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