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. 2014 May 8;123(19):3016-26.
doi: 10.1182/blood-2013-10-533398. Epub 2014 Mar 20.

CD16xCD33 Bispecific Killer Cell Engager (BiKE) Activates NK Cells Against Primary MDS and MDSC CD33+ Targets

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Free PMC article

CD16xCD33 Bispecific Killer Cell Engager (BiKE) Activates NK Cells Against Primary MDS and MDSC CD33+ Targets

Michelle K Gleason et al. Blood. .
Free PMC article

Abstract

Myelodysplastic syndromes (MDS) are stem cell disorders that can progress to acute myeloid leukemia. Although hematopoietic cell transplantation can be curative, additional therapies are needed for a disease that disproportionally afflicts the elderly. We tested the ability of a CD16xCD33 BiKE to induce natural killer (NK) cell function in 67 MDS patients. Compared with age-matched normal controls, CD7(+) lymphocytes, NK cells, and CD16 expression were markedly decreased in MDS patients. Despite this, reverse antibody-dependent cell-mediated cytotoxicity assays showed potent degranulation and cytokine production when resting MDS-NK cells were triggered with an agonistic CD16 monoclonal antibody. Blood and marrow MDS-NK cells treated with bispecific killer cell engager (BiKE) significantly enhanced degranulation and tumor necrosis factor-α and interferon-γ production against HL-60 and endogenous CD33(+) MDS targets. MDS patients had a significantly increased proportion of immunosuppressive CD33(+) myeloid-derived suppressor cells (MDSCs) that negatively correlated with MDS lymphocyte populations and CD16 loss on NK cells. Treatment with the CD16xCD33 BiKE successfully reversed MDSC immunosuppression of NK cells and induced MDSC target cell lysis. Lastly, the BiKE induced optimal MDS-NK cell function irrespective of disease stage. Our data suggest that the CD16xCD33 BiKE functions against both CD33(+) MDS and MDSC targets and may be therapeutically beneficial for MDS patients.

Figures

Figure 1
Figure 1
Aberrant NK cell frequency and NK cell CD16 expression in MDS PBMCs. (A-C) PBMCs from normal donors and MDS patients were stained with anti-CD56, anti-CD3, and anti-CD16 mAbs. (A-B) Gating strategy for the evaluation of the lymphocyte populations in normal donors (A) and MDS patients (B). Plots are representative from 1 normal donor (#12 of 20) and 1 higher-risk MDS patient (#49 of 67), and gate frequency indicates population percent normalized to the all-cell fraction based on the forward scatter (FSC)/side scatter (SSC) gate excluding debris. (C) Percent of CD56/CD3+ T cells and CD56+/CD3 NK cells (normalized to all-cell fraction based on the FSC/SSC gate excluding debris) and (D) percent of CD16+ NK cells (calculated as the percent of CD56+/CD3 NK cells) and mean fluorescence intensity of CD16 expression on NK cells were determined by fluorescence-activated cell sorter (FACS) analysis (****P < .0001). ECD, energy-coupled dye.
Figure 2
Figure 2
NK cell activation induced through CD16 crosslinking is intact in MDS-NK cells. (A-C) Resting PBMCs from normal donors and MDS patients were coated with 10 μg/mL of the indicated mAbs and cocultured with P815 targets, and an R-ADCC assay was performed. NK cell CD107a expression (A) and intracellular TNF-α (B) and IFN-γ (C) production were evaluated by FACS analysis (*P < .05, ***P < .001, ****P < .0001). mIgG, mouse immunoglobulin G.
Figure 3
Figure 3
CD16xCD33 BiKE enhances MDS-NK cell function against CD33+ targets. Resting PBMCs from normal donors and MDS patients were coated with 10 μg/mL of the CD16xCD33 BiKE or scFv CD16 control and cocultured with (A-C) or without (D-F) CD33+ HL-60 targets. NK cell CD107a expression and intracellular TNF-α and IFN-γ production were evaluated by FACS analysis (*P < .05, **P < .01, ***P < .001, ****P < .0001).
Figure 4
Figure 4
MDS patients have a significant increase in MDSCs that negatively correlates with lymphocyte populations. (A) PBMCs from normal donors and MDS patients were stained with anti-CD45, anti-CD33, anti-CD11b, anti-CD14, and anti-HLA-DR mAbs and the percent of MDSCs (phenotypically defined as CD33+/CD11b+/CD14/HLA-DRlo/−) of the all-cell fraction was evaluated (****P < .0001). (B-D) Based on the all-cell fraction, the correlation between percent MDSCs and percent CD7+ lymphocytes, percent T cells, percent NKT cells, and percent NK cells among all MDS patients (B), lower-risk MDS patients (C), and higher-risk MDS patients (D) is shown. Correlation coefficients (r) and statistical significance are indicated in the figure.
Figure 5
Figure 5
CD16xCD33 BiKE enhances NK cell cytotoxicity and cytokine production against MDSC targets. (A) Carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled NK cells were cultured for 5 days in basal medium or medium supplemented with 10 ng/mL IL-15 in the presence or absence (E:T ratio = 1:2) of cytokine-derived MDSCs from normal PBMCs, and proliferation was evaluated via FACS analysis. Histogram plots represent 1 of 6 normal donors. (B) PBMCs from normal donors were coated with 10 μg/mL of CD16xCD33 BiKE or scFv CD16 control and cocultured with cytokine-derived MDSC or HL-60 targets, and NK cell-mediated cytotoxicity was evaluated via a 51Cr-release assay (****P < .0001; n = 4). (C-E) PBMCs from normal donors were coated with 10 μg/mL of CD16xCD33 BiKE or scFv CD16 control and cocultured with cytokine-derived MDSC or HL-60 targets, and NK cell CD107a expression and intracellular TNF-α and IFN-γ production were evaluated via FACS analysis (**P < .01, ****P < .0001; n = 6). (F-H) Mononuclear cells from paired PB and BM samples were isolated from normal donors (n = 4) and MDS patients (n = 10), coated with 10 μg/mL of CD16xCD33 BiKE or scFv CD16 control, and cocultured with cytokine-derived MDSC, and NK cell (F) CD107a expression and intracellular (G) TNF-α and (H) IFN-γ production were evaluated via FACS analysis (*P < .05, **P < .01, ***P < .01, ****P < .0001).
Figure 6
Figure 6
CD16xCD33 BiKE consistently enhances NK cell function at all disease stages. The percent of CD56+/CD3 MDS-NK cells (A), total CD33+ cells (B), and MDS-MDSC (C) of the all-cell fraction was stratified according to their respective FAB classification and IPSS karyotype groups. (D-E) CD16xCD33 BiKE-induced MDS-NK cell functions (degranulation [CD107a], intracellular TNF-α and IFN-γ production) in the absence of HL-60 targets (D) and in the presence of HL-60 targets (E) were stratified according to their respective FAB classification and IPSS karyotype groups. The y-axes of graphs in panels D and E represent the percent of NK cells positive for each function listed in the graph legend.

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