Understanding and improving the mechanisms of action of anti-GD2 monoclonal antibody therapy in neuroblastoma

Abstract

High-risk neuroblastoma (HR-NBL) is an aggressive paediatric cancer that develops in the peripheral nervous system, and shows very poor outcome despite intensive multi-agent treatment regimens. In recent years, treatment with anti-GD2 monoclonal antibody (mAb) immunotherapy has been found to improve the survival of HR-NBL patients, however more than 50% of these patients will still eventually die from their disease. The anti-cancer function of GD2 targeting mAbs is considered to depend on multiple mechanisms of action including, Fc-dependent immune effector functions and Fc-independent functions such as the blockade of GD2 signalling. This leads to a repertoire of anti-tumoral activity such as inhibition of growth, proliferation and cell adhesion. Fc-dependent mechanisms are also associated to on-target off-tumour antibody related neuropathic pain and neurotoxicity, which is difficult to treat even with opioid analgesia, and can be dose-limiting in some cases. Complement activation triggered by antibody binding to GD2 expressed on healthy neurons is thought to activate this pain response. Suppressing complement activation by K322A point mutation however failed to completely alleviate the toxicities of anti-GD2 mAb therapy in phase I trial, indicating that other mechanisms may be involved. The dominant mechanisms underlying the toxicities and therapeutic efficacy of anti-GD2 mAb are unknown. The aim of the current study was to explore the contribution of Fc-dependent and -independent mechanisms in the anti-tumoral functions and neurotoxicity of anti-GD2 mAbs in vitro and in vivo, using a panel of Fc modified antibodies. The Fc variants were altered to confer differential FcγR and C1q binding affinities, and in turn varying levels of immune effector functions. In this study, we demonstrate that reducing complement activation can prevent damage to human neurons, derived from induced pluripotent stem cells, with Fc mutations DLE and PG-LALA which suppress C1q binding, but not with the K322A mutation. The K322A modification was found to reduce the Fc stability of human IgG1 which led to highly variable complement activities. Incubating human neurons with the K322A variant in the presence of human serum led to widespread myelin and axon disintegration, and significant neuron lysis which may explain the pain responses seen in patients. In contrast, human IgG1DLE and PG-LALA variants were found to be relatively more stable and significantly less neurotoxic when compared to the K322A and wildtype variant. Next, we used a GD2 overexpressing immunocontent murine model (CT26-GD2 BALB/c model) to study the mechanisms responsible for the therapeutic efficacy of anti-GD2 mAbs. We found that the mouse IgG2aPG-LALA, which does not bind FcγRs and C1q, led to comparable levels of therapeutic efficacy as wildtype antibody supporting the direct contribution of GD2 blockade. We also demonstrated that FcγR engagement may be involved in the initiation of the adverse responses triggered by anti-GD2 mAbs. Compared to the mouse IgG2a wildtype antibody, the PG-LALA and IgG1 variants, which have reduced affinities for FcγRs, showed significantly decreased adverse reactions. Preliminary experiments highlighted that knocking out the Fc receptor gamma chain in mice significantly suppressed adverse responses to anti-GD2 mAb mouse IgG2a wildtype injection, while this was not observed in FcγRI or FcγRIV deficient mice. In addition to this, we also detected significant amounts of antibody immune complexes in the serum of treated mice and the presence of anti-GD2 mAbs on neuronal tissue harvested from mice experiencing toxicities following anti-GD2 mAb injection. These findings collectively indicate the contribution of GD2 recognition and FcγR dependent mechanisms in the adverse responses of anti-GD2 mAb. Overall, the findings show that the human IgG1K322A variant has highly variable complement activities, and therefore is potentially unreliable for use as treatment in HR-NBL patients. We provide first evidence to show that FcγR interactions significantly contribute to the adverse responses of anti-GD2 antibody, and that blocking GD2 signalling has a direct role in the therapeutic efficacy of anti-GD2 mAb. Diminishing the affinity that anti-GD2 mAb has for FcγRs alleviated adverse responses without compromising its therapeutic efficacy. These findings will hopefully inform the development of novel clinical anti-GD2 antibodies with increased anti-tumoral efficacy and reduced treatment-related toxicities.

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Identifiers

ORCID for Sathurthika Kulanthaivadivel: orcid.org/0009-0008-1019-6621

ORCID for Juliet Gray: orcid.org/0000-0002-5652-4722

ORCID for Stephen Beers: orcid.org/0000-0002-3765-3342

ORCID for Jane Willoughby: orcid.org/0000-0002-6326-4519

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