The role of Fc gamma receptors in the activity of therapeutic monoclonal antibodies

Abstract

Fc gamma receptors (FcγRs) are the major family of receptors responsible for interacting with immunoglobulin G (IgG). They are known to be required for the anti-tumour activity of direct targeting mAbs through expression on NK cells and macrophages. Furthermore, recent work has suggested that cross-linking via FcγRs is required for the activity of agonistic, immune modulatory mAb. This thesis sought to investigate the requirement for these receptors for different aspects of mAb activity; from T cell activation to tumour depletion, using a combination of in vitro and in vivo systems.

A panel of CHO-K1 cells were generated and transfected to express the polymorphic variants of human FcγRs. These were characterised for their ability to bind IgG before being used as feeder cells in T cell proliferation assays. The assays found that cross-linking of the anti-CD28 mAb, TGN1412 by FcγRIIb (CD32b) or FcγRIIa (CD32a) but not FcγRIIIa (CD16a) transfected cells induced T cell proliferation. Furthermore, this was accompanied by the release of pro-inflammatory cytokines including TNF-α, IFN-γ and IL-2. With the importance of cross-linking via CD32b demonstrated, experiments probed the mechanism of expression using Ramos and Raji cells. These experiments investigated the gene and promoter sequences as well as the effect of epigenetic inhibitors, however further investigation is required.

Next a novel mouse model was developed to investigate the efficacy of anti-human (h)CD20 mAbs. The type II mAb obinutuzumab was found to give prolonged tumour clearance compared to the type I rituximab in a fully syngeneic model with the target antigen expressed on malignant and normal B cells. The use of immune compromised mice confirmed that activatory FcγRs were required for efficient anti-CD20 mediated tumour clearance. Further experiments demonstrated that anti-CD20 mAbs could be combined with the PI3Kδ inhibitor, GS9820, to prolong tumour clearance. These experiments unexpectedly revealed that hIgG1 had an abnormally short half-life in NOD SCID mice, causing the basis for this to be examined as NOD SCID mice are widely used for immunotherapy experiments, particularly patient derived xenografts. Further investigations found that half-life could be restored through genetic deletion of mouse CD32 or by reconstitution with IgG. The polymorphic variant of CD32 found in NOD SCID mice had a higher affinity for hIgG1 which could explain the short half-life.

Overall the results presented here demonstrate the multiple roles played by FcγRs in the many aspects of mAb immunotherapy, from effector cell activation to cross-linking and altering mAb half-life. This knowledge will help guide the next generation of therapeutic mAbs.

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Identifiers

ORCID for Mark Cragg: orcid.org/0000-0003-2077-089X

ORCID for Ali Roghanian: orcid.org/0000-0003-1316-4218

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