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

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.