Development of On-Target Hexamerisation for Effective Antibody Immunotherapy

Abstract

Monoclonal antibodies represent one of the fastest growing sectors of human therapeutics used in the treatment of cancer and autoimmune diseases. The majority of monoclonal antibodies approved for use in cancer elicit a therapeutic response via Fc-mediated effector functions. Antibodies have been shown to have natural Fc:Fc interactions that help to mediate these effector functions. Specifically, when the Fc-molecules are arranged hexamerically at the target cell surface this leads to the optimal activation of complement-dependent cytotoxicity.
Herein is described a novel antibody engineering approach based around the tailpiece of the naturally multimeric IgM. Fusion of the C-terminal ‘tailpiece’ sequence of IgM promoted stable hexamerisation of hIgG1 in solution. Mutation of the penultimate cysteine residue of the tailpiece to serine (C575S) ablated covalent hexamer formation. However, hIgG1 C575S tailpiece mutants retained the potential to self-associate into reversible, non-covalent hexamers after concentration in solution. These hIgG1 IgM tailpiece C575S fusion antibodies could be expressed and purified with standard hIgG protocols, without any loss of yield or additional quality control processes, whereas the native hIgG1 tailpiece required additional purification steps to isolate a single species and delivered lower yields.
First, constructs were assessed in the context of CD20 specificity. Anti-CD20 hIgG tailpiece hexamers demonstrated enhanced C1q affinity and complement-dependent cytotoxicity in vitro, but exhibited reduced depletion of target cells in whole blood assays, lacked in vivo efficacy, and demonstrated a significantly faster clearance rate than wild-type hIgG1. The hIgG1 IgM tailpiece C575S also had an enhanced ability to recruit C1q and elicit complement-dependent cytotoxicity, in the absence of enhanced C1q affinity. Additionally, there was no compromise to FcγR-mediated effector functions, with whole blood depletion comparable to wild-type hIgG1. In vivo efficacy, safety, and half-life were also comparable to wild-type hIgG1, with enhanced depletion in the lymph node for some anti-CD20 antibodies. Hence, the hIgG1 IgM tailpiece C575S represents a more suitable in vivo format.
The same technology was also applied to anti-CD40 monoclonal antibodies, which rely on effective receptor clustering for agonistic activity, and so were proposed to benefit from hexamerisation. In B cell stimulation assays in vitro, hexameric hIgG1, hIgG2, and hIgG4 demonstrated potent CD40 agonism, however, only the hIgG4 tailpiece C575S showed enhanced agonism compared to the inactive hIgG4 wild-type. In contrast, the hIgG1 tailpiece C575S did not exhibit more agonism and all hIgG2 formats were equivalently active. Further analysis of the hIgG4 anti-CD40 antibodies in vivo showed the native tailpiece hexamer was inactive, whereas the tailpiece C575S promoted CD40 agonism and humoral responses, in an FcγR-independent manner.
These results are in-line with the proposed activity of IgG on-target hexamer formation. Therefore, the IgG IgM tailpiece C575S represents a novel format for promoting on-target antibody hexamerisation for enhanced complement-dependent cytotoxicity or agonistic activities

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

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