Investigating the agonistic requirements of anti-human 4-1BB monoclonal antibodies for cancer immunotherapy

Abstract

Antibody immunotherapy has emerged as a promising therapeutic approach for cancer, a key factor to its success being the ability to activate cytotoxic T cell responses against tumours. The immune co-stimulatory receptor 4-1BB (CD137) represents an attractive target for monoclonal antibody (mAb)-based cancer immunotherapy as it promotes the anti-tumour potential of cytotoxic T cells. Despite this, the approval of 4-1BB mAbs for clinical use has been hampered by difficulties balancing activity and toxicity due to a lack of understanding of the molecular characteristics which contribute to efficacy. Through the functional and molecular characterisation of a panel of 33 anti-human (h)4-1BB mAb, including two in prior clinical development, this work aimed to identify optimal h4-1BB agonists, and to investigate which properties determine activity.
To investigate the molecular properties of these mAbs, the epitope on 4-1BB bound by each mAb and their binding kinetics were assessed by flow cytometry and surface plasmon resonance. Their ability to induce signalling downstream of 4-1BB was assessed by flow cytometry using a combination of reporter cell lines and in vitro T cell proliferation assays. From the panel, mAbs with a range of agonistic activities were identified. Of most interest we identified a strong agonist, 6H05, which also displayed different binding properties and lower affinity compared to the established clinical candidate urelumab. The agonistic activity of this mAb was further evaluated in vivo in transgenic mice expressing human 4-1BB. 6H05 triggered potent dose-dependent antigen-specific expansion of CD8+ T cells in an immunisation model, across several different isotype formats. Importantly, it induced complete tumour regressions in a subcutaneous MC38 tumour model both as monotherapy and in combination with anti-PD-1 mAb, resulting in prolonged survival compared to urelumab. The potential in vivo toxicity of 6H05 was also compared to that of urelumab in transgenic h4-1BB mice, and results revealed different levels of immune infiltration into the spleen and liver after treatment with each mAb.
Overall, this thesis reveals a novel anti-h4-1BB mAb with strong agonistic activity in vitro and in vivo and with a different binding profile to existing clinical candidates. Moreover, the results presented here highlight different properties, including binding affinity, which influence the ability of anti-h4-1BB mAbs to elicit receptor agonism. Finally, this work provides insight which could be utilised to rationally design h4-1BB mAbs to achieve desired levels of activity, with the aim of improving clinical outcomes.

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Identifiers

ORCID for Alba Isabel Gonzalez Garcia: orcid.org/0000-0002-6329-2798

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

ORCID for Kirstie Cleary: orcid.org/0000-0001-6200-4945

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

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