Abstract
Elotuzumab (formerly known as HuLuc63) is a humanized monoclonal antibody targeting CS1 (CD319, SLAMF7), a protein which is highly and uniformly expressed on myeloma cells. CS1 is also expressed at lower levels on selected lymphocyte subsets in humans, including natural killer (NK) cells and a subset of CD8+ T cells. Elotuzumab mediates killing of multiple myeloma cell lines (OPM-2, L363) via antibody-dependent cellular cytotoxicity in vitro and has significant anti-tumor activity in mouse xenograft models. Elotuzumab is currently being investigated in Phase I safety studies in relapsed multiple myeloma (MM). To further characterize the effects of elotuzumab on different immune cell subsets, we evaluated its effects in human whole blood cultures in vitro. In resting human whole blood, >95% of the CD56dim (CD3−CD56+CD16++) NK population expressed CS1. In contrast, only 50–75% of CD56bright NK cells expressed CS1. The expression level of CS1, as measured by mean fluorescence intensity, was also lower in CD56bright than in CD56dim NK cells. In 24-hr whole blood cultures, elotuzumab selectively activated CD56dim NK cells, as evidenced by up-regulation of CD69, CD11b, CD54, and down-modulation of CD16 expression, while having little or no effect on CD56bright NK cells. Monocytes in the same whole blood culture were also activated as determined by up-regulation of CS1, HLA-DR, and CD54. Monocyte activation was significantly correlated with CD56dim NK cell activation. In contrast, elotuzumab induced little to no activation of either CD4+ or CD8+ T cell subsets. In whole blood cultures, elotuzumab induced low levels of IFN-γ and only negligible levels of TNF-α. However, elotuzumab induced robust levels of the chemokines IP-10 and MCP-1 which correlated significantly with the level of CD56dim NK cell activation. The induction of chemokines was both dose and Fc-dependent, as F(ab′)2 fragments of elotuzumab failed to induce either NK cell activation or release of chemokines. Interestingly, when elotuzumab was added to purified NK cell cultures, no increase in the levels of IP-10 and MCP-1 was observed. Addition of neutralizating anti-IFN-g mAb to elotuzumab-treated whole blood cultures had little effect on NK cell activation but partially reduced monocyte activation and IP-10 release. Finally, addition of anti-CD18 mAb abrogated the elotuzumab-mediated activation of NK cells and monocytes suggesting cell-cell contact plays an important role in elotuzumab-induced activation of CD56dim NK cells and monocytes. In conclusion, the result from this study demonstrated that elotuzumab may activate NK cells via CS1 and CD16 signaling, resulting in the release of low levels of IFN-g and subsequently monocyte-dependant chemokine production including IP-10 and MCP-1. When dosed in a phase I clinical trial, elotuzumab induced a transient elevation of the chemokines IP-10 and MCP-1 in the serum of MM patients, indicating that the whole blood culture system enables the identification of appropriate pharmacodynamic markers.
Development, validation and applications of the monocyte activation test for pyrogens based on human whole blood