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. 2013 Jul 15;19(14):3844-55.
doi: 10.1158/1078-0432.CCR-13-0505. Epub 2013 May 20.

Targeting Natural Killer Cells to Acute Myeloid Leukemia in Vitro With a CD16 X 33 Bispecific Killer Cell Engager and ADAM17 Inhibition

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Free PMC article

Targeting Natural Killer Cells to Acute Myeloid Leukemia in Vitro With a CD16 X 33 Bispecific Killer Cell Engager and ADAM17 Inhibition

Andres Wiernik et al. Clin Cancer Res. .
Free PMC article

Abstract

Purpose: The graft versus leukemia effect by natural killer (NK) cells prevents relapse following hematopoietic stem cell transplantation. We determined whether a novel bispecific killer cell engager (BiKE) signaling through CD16 and targeting CD33 could activate NK cells at high potency against acute myelogenous leukemia (AML) targets.

Experimental design: We investigated the ability of our fully humanized CD16 × CD33 (CD16 × 33) BiKE to trigger in vitro NK cell activation against HL60 (CD33(+)), RAJI (CD33(-)), and primary AML targets (de novo and refractory) to determine whether treatment with CD16 × 33 BiKE in combination with an ADAM17 inhibitor could prevent CD16 shedding (a novel inhibitory mechanism induced by NK cell activation) and overcome inhibition of class I MHC recognizing inhibitory receptors.

Results: NK cell cytotoxicity and cytokine release were specifically triggered by the CD16 × 33 BiKE when cells were cultured with HL60 targets, CD33(+) de novo and refractory AML targets. Combination treatment with CD16 × 33 BiKE and ADAM17 inhibitor resulted in inhibition of CD16 shedding in NK cells, and enhanced NK cell activation. Treatment of NK cells from double umbilical cord blood transplant (UCBT) recipients with the CD16 × 33 BiKE resulted in activation, especially in those recipients with cytomegalovirus reactivation.

Conclusion: CD16 × 33 BiKE can overcome self-inhibitory signals and effectively elicit NK cell effector activity against AML. These in vitro studies highlight the potential of CD16 × 33 BiKE ± ADAM17 inhibition to enhance NK cell activation and specificity against CD33(+) AML, which optimally could be applied in patients with relapsed AML or for adjuvant antileukemic therapy posttransplantation.

Conflict of interest statement

COI Statement: There are no conflicts of interest to on behalf of the co-authors.

Figures

Fig 1
Fig 1. CD16x33 BiKE enhances NK cell function against CD33+ targets in a dose-dependent manner
Purified NK cells from healthy donors (n=4) were cultured with HL60 (CD33+) target cells (E:T ratio of 2:1) and treated with the indicated concentrations of CD16x33 BiKE. (A) After 4 hours in culture, intracellular CD107a degranulation, TNF-α, and IFN-γ were measured by flow cytometry, and (B) cytotoxicity was measured in a 4-hour chromium release assay. (C) NK cell function (intracellular CD107a degranulation, TNF-α and IFN-γ measured by flow cytometry) and (D) cytotoxicity were determined at the indicated E:T ratios (n=2) with or without 5 ug/mL of CD16 x CD33 BiKE. (E) Specificity of the CD16 x CD33 BiKE was tested by culturing NK cells with HL60 (CD33+) and Raji targets (CD33) for 4 hours (E:T ratio of 2:1) with or without 5 ug/mL of CD16 x CD33 BiKE (n=4). (*P<0.05; **P<0.01, ***P<0.001).
Fig 2
Fig 2. CD16x33 BiKE activates NK cells against CD33+ HL60 targets in the presence of CD33+ monocytes
(A) Monocytes (CD33+) and PBMC containing NK cells (PBMC) [NK cells + PBMC (NK)] [NK:PBMC; ratio of 2:1] were tested against HL60 cells at the indicated effector-to-HL60 ratios after treatment with or without 5 ug/mL of the CD16x33 BiKE. Cytotoxicity against HL60 targets was determined by 4-hour chromium release assay. (B) Purified NK cells from healthy donors were cultured with autologous Monocytes (CD33+) and with HL60 (CD33+) target cells (E:T ratio of 2:1), and PBMC (containing NK cells and monocytes) were cultured with HL60 targets (E:T ratio of 2:1) for 4 hours with or without 5 ug/mL of CD16x33 BiKE. Flow cytometry was used to determine intracellular CD107a degranulation, TNF-α, and IFN-γ. (C) The same conditions were used to determine CD16 and KIR expression on NK cells after 4 hours of activation. (n=3 for all experiments, *P<0.05; **P<0.01, ***P<0.001).
Fig 3
Fig 3. CD16x33 BiKE triggers NK cell degranulation and cytokine production against de-novo CD33+ AML targets, an effect that is enhanced by ADAM17 inhibition
Purified NK cells isolated from healthy donors (n=6) were cultured with de-novo CD33+ AML cells (E:T ratio of 2:1). Cells were treated with or without 5 ug/mL of CD16x33 BiKE and with or without 2uM of an ADAM17 inhibitor (INCB003619). (A) Representative flow cytometry analysis of CD56+ NK cells after 4 hours in culture. Aggregate data of (B) intracellular CD107a degranulation, TNF-α, and IFN-γ for the indicated conditions and (C) CD16 expression (baseline CD16 expression of NK cells shown in the dotted line) (n=6, *P<0.05; **P<0.01, ***P<0.001).
Fig 4
Fig 4. MHC class I blockade in AML targets triggers NK cell activation, effect enhanced with CD16x33 BiKE activation
AML target cells were pre-incubated with 20 ug/mL of a pan-HLA blocking antibody (HP-1F7) for 30 mins prior to culturing with NK cells. Purified NK cells isolated from healthy donors (n=5) were cultured with de-novo CD33+ AML cells (E:T ratio of 2:1). Cells were treated with or without 5 ug/mL of CD16x33 BiKE as indicated and cultured for 4 hours. Aggregate data of NK cell intracellular CD107a degranulation, TNF-α, and IFN-γ for the indicated conditions were determined by flow cytometry. (n=5, *P<0.05; **P<0.01, ***P<0.001).
Fig 5
Fig 5. CD16x33 BiKE triggers NK cell degranulation and cytokine production against relapse/refractory CD33+ AML targets
(A) CD33 surface expression measured by flow cytometry analysis of bone marrow samples from 10 patients with relapse/refractory AML. Percent expression and median fluorescence intensity (MFI) are shown for each sample. (B and C) Purified NK cells isolated from two healthy donors were cultured with each refractory AML sample (E:T ratio of 2:1) (n=20 separate assays). Cells were treated with or without 5 ug/mL of CD16x33 BiKE. After 4 hours in culture, intracellular CD107a degranulation, TNF-α, and IFN-γ were determined by flow cytometry for (B) CD33+ AML targets (n=16 from 8 CD33+ AML targets derived from 2 donors each) and (C) CD33 AML targets (n=4 from 2 CD33 AML targets derived from 2 donors each). (*P<0.05; **P<0.01, ***P<0.001).
Fig 6
Fig 6. CD16x33 BiKE-stimulated NK cells after SCT exhibit time- and CMV reactivation-dependent activity against primary CD33+ AML targets
PBMCs were collected from double umbilical cord blood (UCB) recipients without CMV reactivation (UCB CMV NEG)(n=6) and from recipients with CMV reactivation (UCB CMV POS)(n=6) who had reactivated CMV in the first one hundred days post-transplant. PBMC samples were collected at 3 months and 1 year post-transplant and compared to PBMCs obtained from healthy donors (n=7). (A) CD16 expression on NK cells was determined by flow cytometry (baseline CD16 expression of NK cells from healthy donors is shown in dotted line) at the indicated time points post-transplant. (B) PBMCs were treated with or without 5 ug/mL of CD16 x CD33 BiKE. After 4 hours in culture, levels of NK cell intracellular CD107a degranulation, TNF-α, and IFN-γ were determined by flow cytometry and compared to PBMC from healthy donors (*P<0.05; **P<0.01, ***P<0.001).

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