The contribution of the Fc RIIB immunoreceptor tyrosine-based inhibitory motif (ITIM) to the modulation of antibody immunotherapy

Abstract

Monoclonal antibodies (mAb) are an increasingly important therapeutic tool in the treatment of cancers and autoimmunity. Many mAbs elicit their efficacy through engagement of activatory Fc gamma receptors (Fc R), resulting in target cell depletion by immune effector cells. These activatory Fc Rs are negatively influenced by the single inhibitory Fc R, Fc RIIB. Fc RIIB has been shown to reduce the efficacy of mAbs through impairment of activatory Fc R function and upregulation of Fc RIIB has been identified as a resistance mechanism of mAb therapeutics in cancer. Fc RIIB contains an intracellular immunoreceptor-tyrosine-based inhibition motif (ITIM), which delivers inhibitory signalling. Whether the ITIM is required for all Fc RIIB-mediated inhibitory activities of therapeutic mAbs is not yet clear. To address this issue, we developed and characterised a novel transgenic mouse model (NoTIM), in which the endogenous inhibitory mouse (m)Fc RII is replaced with a non-signalling ITIM mutant of human (h)Fc RIIB. Cells from mice expressing the NoTIM receptor were no longer able to elicit phosphorylation of the Fc RIIB ITIM, mediate efficient internalisation of immune complexes or prevent anti-IgM-mediated calcium flux, confirming the expected phenotype. To understand how Fc RIIB inhibitory signalling impacted mAb-mediated depletion of target cells, NoTIM mice were treated with anti-mouse CD20 mAbs. The extent of depletion was compared to mice either lacking mFc RII or where mFc RII was replaced with a signalling competent hFc RIIB (hFc RIIB Tg). B cell depletion was reduced in both the NoTIM and hFc RIIB Tg mice compared to those lacking mFc RII. Subsequent experiments revealed this was not due to accelerated mAb internalisation nor enhanced clearance from the serum. Using a series of adoptive transfer models we determined that the NoTIM hFc RIIB was mediating inhibition, by competing with activatory Fc Rs on the surface of myeloid cells for the Fc of the direct targeting mAb. These findings were then assessed with respect to T regulatory cell and malignant murine B cell depletion. It was found that in both cases, depletion was negatively impacted by the expression of Fc RIIB at themyeloid surface rather than by its ability to signal. These data indicate that signalling through the ITIM is not critical for the ability of hFc RIIB to prevent mAb-mediated depletion of target cells, questioning current paradigms for its mechanism of action.

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

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