Bi-Specific Killer Cell Engagers (BiKEs) Signaling Through CD16 and Targeting CD33 (CD16 × CD33), Triggers Natural Killer (NK) Cell Cytotoxic and Cytokine Production Against Refractory Acute Myeloid Leukemia (AML)
Authors: Wiernik Andres , Foley Bree , Zhang Bin , Verneris Michael R. , Warlick Erica , Ross Julie A. , Weiner Louis M. , Weisdorf Daniel J. , Vallera Daniel A , Miller Jeffrey S. 
Published: October 2019

Abstract
Abstract 256
AML accounts for a large number of annual deaths due to leukemia worldwide. NK cells are effectors of the innate immune system that mediate the graft versus leukemia (GVL) effect in patients with AML and other hematologic malignancies. The safety and success of using haploidentical NK cell infusions to treat patients with AML has been previously demonstrated, but this therapeutic approach has limitations of potency and lacks specificity for leukemic targets. NK cell antibody-dependent cell-mediated cytotoxicity (ADCC) usually occurs trough the binding of the activating receptor FcγRIIIA (also known as CD16) to the Fc portion of antibodies. CD16 is expressed on most CD56dim NK cells and induces NK cell activation, leading to interferon (IFN-γ) and tumor necrosis factor (TNF-α) secretion. CD16 shedding occurs upon NK cell activation, an effect that we have shown is mediated by a specific metalloproteinase called a disintegrin and metalloproteinase-17 (ADAM-17). We hypothesized that a BiKE antibody molecule, developed specifically to signal through CD16 and targeting the myeloid differentiation antigen CD33 (i.e., CD16 × CD33 BiKE) would enhance NK cell function against AML. In addition, we predicted that selective inhibition of ADAM-17 activity would prevent CD16 shedding and enhance the activity of the CD16 × CD33 BiKE. Bone marrow (BM) samples from 10 patients with primary refractory AML were obtained from our leukemia tissue bank and used as targets. CD33 was expressed on 8 of the 10 BM samples. Purified NK cells from healthy donors were isolated and incubated overnight in the absence of cytokines. NK cells and AML BM samples were treated with 1 ug/mL of bscFv CD16 × CD33 BiKE, scFv anti-CD16 (negative control) or DTCD33 × CD33 (anti-CD33 plus anti-CD33 spliced to truncated diphtheria toxin - negative control). NK cells under the above conditions were all treated with or without 5 uM of an ADAM17 inhibitor (Incyte). After 4 hours in culture, intracellular CD107a degranulation assay and intracellular TNF-α and IFN-γ assays were performed. A MTS survival assay was also performed on the target cells. In 8 of 10 AML samples, NK cells significantly degranulated and secreted cytokines (TNF-α and IFN-γ) only when treated with the CD16 × CD33 BiKE reagent. The MTS survival assay confirmed significant AML target cell death in the presence of the CD16 × CD33 BiKE. Combined treatment with the CD16 × CD33 BiKE and the ADAM17 inhibitor led to a significant increase in cytokine TNF-α and IFN-γ secretion by NK cells when compared to treatment with CD16 × CD33 BiKE alone. Phenotypic analysis of the NK cells after treatment with the ADAM17 inhibitor revealed a significant decrease in CD16 shedding as predicted. Of note, no NK cell activity was triggered by the 2 AML BM samples that lacked CD33 expression arguing in favor of the specificity of this molecule for CD33 positive AML. We then analyzed the ability of NK cells to kill multiple targets in the presence of the CD16 × CD33 BiKE over time. NK cells and HL60 cells (CD33 positive targets) were treated with 1 ug/mL of bscFv CD16 × CD33 BiKE and incubated overnight in the presence or absence of the ADAM17 inhibitor. On the following day, a second target exposure with chromium (Cr-) labeled HL60 cells was added to the culture in order to visualize the effect of NK cells on second targets. After 4 hours, intracellular CD107a, TNF-α, IFN-γ and standard Cr- release assays were performed. In the presence of the CD16 × CD33 BiKE, NK cells showed significantly more degranulation killing and secreted more cytokines in response to secondary targets. Cytokine secretion was also enhanced by the addition of the ADAM17 inhibitor to the BiKE. Collectively, these findings support the ability of a CD16 × CD33 BiKE to trigger NK cell activation through direct signaling of CD16, inducing secretion of cytokines, lytic granules and causing target cell death in resistant AML BM samples and HL60 targets. BiKE enhanced AML killing occurs over a wide range of CD33 target cell density as long as some expression is present. In addition, targeting ADAM17 prevents activation induced CD16 shedding and enhances NK cell cytokine production when combine with therapeutic antibodies. NK cell directed therapy by these compounds could specifically enhance the anti-leukemic potency and efficacy of NK cell adoptive therapy against myeloid disorders.

Disclosures:
Miller: Celgene: Membership on an entity's Board of Directors or advisory committees; Coronado Bioscience: Membership on an entity's Board of Directors or advisory committees.