High-dimensional profiling reveals predominant depletion of NK cell effector phenotypes in relapsed/refractory multiple myeloma patients treated with anti-CD38 therapy

Abstract Background: Anti-CD38 monoclonal antibodies (mAbs) have become a standard part of multiple myeloma (MM) therapy. Their activity is mostly mediated by NK cells through antibody-dependent cellular cytotoxicity (ADCC). Although the association of increased CD38+ NK cells with worse outcomes was demonstrated, the underlying mechanisms of resistance to anti-CD38 mAbs remain poorly understood. Aims: To identify immune effector cell subsets predictive of treatment efficacy and driving resistance to anti-CD38 mAbs. Methods: Bone marrow (BM) samples (N=111) of relapsed/refractory MM (RRMM) treated with anti-CD38-based regimens (CD38-IMID [N=64]; CD38-PI [N=33]; CD38-mono [N=14] were assessed at baseline (BASE; N=110) and progression (PD; N=23), using conventional EuroFlow 8-color MM panel, followed by high-dimensional spectral cytometryesigned for deep profiling of lymphoid subsets and their activation/exhaustion status. Subset proportions were expressed as percentage of lymphocytes, unless stated otherwise. Results: Patients were treated predominantly with daratumumab over isatuximab (92%, 82%, and 100% of CD38-IMID, CD38-PI, and CD38-mono regimens). Treatment groups differed by line of therapy (p <0.001), with CD38-IMID mainly used in line 2 (56%), CD38-PI in lines 2-3 (55%, 30%), and CD38-mono in line ≥4 (86%). Median progression-free survival (PFS) for the CD38-IMID group was 37 months, compared to CD38-PI (16 months; p = 0.012) and CD38-mono (3 months; p <0.001). First, RRMM BM (N=110) collected at BASE were assessed by the EuroFlow MM panel. Lymphocyte pool consisted of median 13.9% B (CD19+CD56-), 19.7% NK (CD19-CD56+), and 61.6% T cell (CD19-CD56-) lineage. 79.7% NK cells were CD38+, while only 29.2% T cells were CD38+. Elevated total NK cells were observed in the PD group (p=0.07). This difference was driven by increased CD38+ NK cell proportion (p <0.028). Lasso-penalized multivariable Cox regression adjusted for therapy group and all immune subsets identified higher percentages of CD38+ NK cells and mature B cells (CD19+CD38low/−CD81low) as significant predictors of PFS at BASE. Increased CD38+ NK cells were associated with worse PFS (HR 1.65, p=0.003), while higher levels of mature B cells correlated with improved PFS (HR 0.79, p=0.002). These findings remained significant also in uniform CD38-IMID group (p <0.007). To better characterize the immune landscape driving these associations, detailed subset distribution was explored using spectral cytometry at BASE (N=14). CD16-, CD16+CD57-, CD16+CD57+ cells represented median 1.68%, 4.26%, and 6.17% of lymphocytes. Comparing CD38+/- NK compartments, CD16+CD57− subset was significantly enriched in the CD38+ NK pool (p=0.006). Gating on 8 other key NK markers revealed that KLRB1+ (p=0.063) cells were also enriched in the CD38+ pool, whereas NKG2C+ cells were more frequent in the CD38- pool (p <0.001). To further investigate the impact of anti-CD38 mAbs on immune cells, paired BASE and PD samples were analyzed using conventional (N=23) and spectral (N=14) panels. Basic exploratory analysis using both methods showed that proportion of total T cells increased (p <0.017), while total B cells (p <0.012) and total NK cells (p=0.023) dropped at PD. Furthermore, both CD38+ NK and CD38+ T cells decreased (p <0.004). Finally, spectral cytometry was utilized in both BASE and PD to identify NK subsets most affected by anti-CD38 therapy. As expected, most reduced NK cells in lymphocytes were CD38+ (median drop, log2-fold change: 6.84%, 1.72; p=0.008), followed by CD16+CD57+ (5.85%; 1.31; p=0.023), KIR2DL+ (5.11%; 1.52; p=0.008), KLRB1+ (4.58%, 1.46; p=0.008), and CD16+CD57- cells (2.42%, 1.04; p=0.023). Conclusion: This study provides in-depth analysis of immune cells in anti-CD38 mAb-treated patients using conventional and spectral cytometry. Higher BASE proportion of CD38+ NK cells was associated with worse PFS, while higher levels of mature B cells predicted better outcomes. Post-therapy, CD38+ NK and CD38+ T cells, total NK cells, and total B cells decreased, while total T cells increased. Importantly, deeper profiling revealed CD16+CD57- and KLRB1+ cells among those enriched in the CD38+ NK pool at BASE. Post-treatment, CD16+CD57+, KIR2DL+, and KLRB1+ NK cells showed the greatest reduction. These results indicate a complex interplay of fratricide, resulting in preferential depletion of mature effector NK cell phenotypes by anti-CD38 therapy.