Inhibition of MICA and MICB Shedding Enhances Cytokine-Induced Memory-like NK Cell-Mediated Activity Against Multiple Myeloma

Abstract

Introduction Natural Killer (NK) cells are important mediators of cytotoxic immune responses against MM, and the efficacy of existing MM therapies relies substantially on NK cell function. However, MM cells develop mechanisms to escape NK cell recognition by loss of ligands for activating receptors and by inducing functional defects. To overcome reduced NK cell functionality, adoptive transfer of cytokine-induced memory-like (CIML) NK cells are of particular interest, as these cells acquire memory-like features resulting in enhanced activity upon restimulation, greater IFN-γ response, and greater anti-tumor cytotoxicity. The success of adoptive transfer of CIML NK cells in MM is dependent on the ability of NK cells to recognize malignant plasma cells (PCs). NKG2D, one of the major activating receptors on NK cells, is upregulated on CIML NK cells. NKG2D plays an important role in the immune recognition of malignant PCs through interaction with its ligands MICA and MICB. However, MM cells evade this recognition by proteolytic shedding of MICA/B from the cell surface, leading to NKG2D internalization. Rationally designed antibodies like the 7C6 monoclonal antibody (mAb) targeting the proteolytic MICA α3 domain stabilize cell surface MICA/B expression and can enhance NK cell recognition and susceptibility to NK cell-mediated cytotoxicity. Furthermore, it has been shown that Bortezomib, an FDA-approved proteasome inhibitor with potent anti-MM activity, augments MICA expression in MM. We hypothesized that use of the 7C6 mAb would allow simultaneous blockade of MICA/B shedding and ADCC by CIML NK cells resulting in enhanced targeting of MM cells, which would further synergize with Bortezomib treatment. Results Flowcytometric analysis of surface MICA/B levels on 10 human myeloma cell lines (HMCLs) demonstrated heterogeneous protein expression. MICA/B transcript levels for corresponding HMCLs were queried from the CCLE database. There was no correlation between transcript and protein expression, suggesting substantial post-translational modifications, such as proteolytic cleavage, determining surface expression levels of MICA/B in MM. To determine if proteolytic cleavage occurs in MM, we treated HMCL with the 7C6 mAb, targeting the proteolytic cleavage site. Flowcytometric analysis of MM.1S revealed that 7C6 was able to upregulate basal expression of MICA/B in a dose-dependent manner as well as inhibit MICA/B shedding. These results suggest proteolytic cleavage of MICA/B occurs in MM and that this process can be inhibited by a MICA/B stabilizing antibody. Previously, it has been shown that MICA/B levels can be upregulated in MM by Bortezomib. As heterogeneity in MICA/B expression levels was seen among HMCLs, we hypothesized that the use of 7C6 would synergize with Bortezomib. Indeed, the addition of Bortezomib to 7C6 substantially increased MICA/B surface expression. To assess the functional consequences of MICA/B stabilization, we examined the effects on NK cell activation in co-culture assays of NK cells with MM.1S pretreated with 7C6 or IC mAb. We observed enhanced NK cell function as assessed by increased CD107a degranulation, IFN-γ and TNF-α production in co-culture assays of conventional NK cells with MM.1S pre-treated with 7C6 vs IC. Next, we measured the activity of CIML NK cells to myeloma targets. CIML NK cells exhibited enhanced CD107a in response to MM targets when compared to control NK cells from the same donors (Figure 1A). Furthermore, CIML NK cells demonstrated significantly higher cytotoxicity against 7C6 pretreated MM.1S, which was dependent on increased MICA/B and NKG2D levels. CIML NK cells in NSG mice treated with 7C6-hIgG1 mAb were significantly more effective in controlling early MM tumor cell outgrowth compared to CIML NK cells in mice treated with isotype control antibodies (Figure 1B). Conclusion Here we demonstrate that inhibition of MICA/B shedding with a novel antibody 7C6 enhances CIML NK cell-mediated function against MM in vitro and in a xenograft mouse model. Preliminary data demonstrate that this treatment strategy could synergize with current treatment regimens including Bortezomib. Our data support the hypothesis that combination strategies to enhance NK cell activation can be effective to enhance NK cell-mediated immune surveillance in MM. This combined approach highlights their potential use as novel immunotherapy in MM. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal