Elotuzumab directly enhances NK cell cytotoxicity against myeloma via CS1 ligation: evidence for augmented NK cell function complementing ADCC

Abstract

Elotuzumab is a monoclonal antibody in development for multiple myeloma (MM) that targets CS1, a cell surface glycoprotein expressed on MM cells. In preclinical models, elotuzumab exerts anti-MM efficacy via natural killer (NK)-cell-mediated antibody-dependent cellular cytotoxicity (ADCC). CS1 is also expressed at lower levels on NK cells where it acts as an activating receptor. We hypothesized that elotuzumab may have additional mechanisms of action via ligation of CS1 on NK cells that complement ADCC activity. Herein, we show that elotuzumab appears to induce activation of NK cells by binding to NK cell CS1 which promotes cytotoxicity against CS1(+) MM cells but not against autologous CS1(+) NK cells. Elotuzumab may also promote CS1-CS1 interactions between NK cells and CS1(+) target cells to enhance cytotoxicity in a manner independent of ADCC. NK cell activation appears dependent on differential expression of the signaling intermediary EAT-2 which is present in NK cells but absent in primary, human MM cells. Taken together, these data suggest elotuzumab may enhance NK cell function directly and confer anti-MM efficacy by means beyond ADCC alone.

Fig. 1

Elotuzumab activates NK cells a Elotuzumab, b elo-G2M3, and c elo-F(ab’)₂ enhance healthy donor NK cell and d patient-derived NK cell activation in the absence of MM targets as measured by CD69 expression. e Elotuzumab increased NK cell IFN-γ production 2.5–3.4-fold (all pair-wise p < 0.05) against CS1(+) L363 MM cells (results represent n = 3 independent experiments)

Fig. 2

Elotuzumab augments tumor-directed NK cell killing by ADCC and by direct NK cell activation a Isotype-treated NK cells produced an average of GrB 50 spots/well (±4 SEM) against isotype-treated MM targets. Against G2M3-treated MM tumor cells, as expected, no enhancement of killing was observed (GrB mean 55 ± 2, p = n/s compared to isotype-treated targets); however, against elotuzumab-treated MM targets, isotype-treated NK cells produced 127 ± 6 GrB spots/well, confirming ADCC as a mechanism of killing (“*”, p = 0.001). Elo-G2M3-treated NK cells showed evidence of enhanced cytotoxicity against isotype-treated MM tumor cells (“**”, mean GrB = 84 ± 3, p < 0.05) as did elotuzumab-treated NK cells (“***”, mean GrB = 117 ± 7, p < 0.05). b These results were validated at higher E:T ratios where elo-G2M3 treatment of NK cells enhanced killing of elotuzumab-treated MM targets, suggesting complementary effects of CS1 ligation on NK cells and MM cells by elotuzumab. c Utilizing patient-derived NK cells as effectors, and purified, CD138(+) autologous MM cells as targets, pre-treatment of either effectors or targets or both with elotuzumab enhanced killing as compared to isotype control (“*” each condition p < 0.05 pair-wise versus control, n = 3 independent experiments). d In whole marrow aspirates, elotuzumab enhanced NK cell degranulation measured by CD107a expression (p = 0.04, n = 3 independent experiments). e Pre-treatment of NK cells with elotuzumab for 72 h enhanced killing of CS1(−) K562 targets at multiple E:T ratios (p < 0.05 for all “*” comparisons indicated). f Elotuzumab did not enhance CS1(+) NK cell cytotoxicity against autologous CS1(+) NK cells, perhaps due in part to (g) upregulation of MHC class I molecules on NK cells in response to elotuzumab, MFI: p = 0.02, MRFI: p = 0.01

Fig. 3

Elotuzumab facilitates NK cell killing by modulating CS1–CS1 interaction a The CS1(+), C16(−) NK cell line NK92 shows increased cytotoxicity against CS1(+) Ba/F3 cells as compared to parental Ba/F3 or Ba/F3 expressing murine CS1. b Elotuzumab or elo-F(ab’)₂ enhanced NK92 killing of CS1(+)-Ba/F3 targets as compared to relevant isotype or non-competing controls. c Increasing concentrations of elotuzumab enhanced NK92 killing of CS1(+)-Ba/F3 cells as compared to a non-competing, anti-CS1 control

Fig. 4

Elotuzumab recruits EAT-2 to CS1 in NK cells and induces downstream signal transduction a Elotuzumab facilitates association of CS1 with EAT-2. Lysates from NK cells incubated in either isotype or elotuzumab were immunoprecipitated with an EAT-2 antibody and immunoblotting was performed with an anti-CS1 antibody. Results show that elotuzumab increased co-localization of CS1 with EAT-2 (EAT-2 immunoblot shown as loading control, results representative of n = 2 independent experiments in independent donors. The complementary experiment was attempted using the CS1 antibody to immunoprecipitate and EAT-2 to blot, but this was not successful as the CS1 antibody was not able to function in this manner.) b Elotuzumab also appeared to enhance ERK phosphorylation in NK cells (left panel) but not in CS1(+) MM cell lines. c Neither EAT-2 protein nor transcript was observed in freshly isolated, primary CD138(+), human MM cells (center bar) as compared to peripheral blood mononuclear cells (left bar) or CD138(−) marrow elements (right bar)