Abstract 1630: FceR1g negative NK-cells (g-NK) enhance antibody-dependent cellular cytotoxicity and in vivo efficacy of therapeutic monoclonal antibodies against hematologic malignanices
Authors: Bigley Austin Basil , Agha Nadia H. , Spade Shanae , Dipierro Gaetano , Martell Ronald , Hann Byron C. , Shah Nina , Wiita Arun P. 
Published: September 2020

Abstract
Monoclonal antibodies (mAbs) are a central component of hematologic malignancy therapy; however, not all patients initially respond to these agents and resistance is a major clinical issue. A major mechanism of action for these mAbs in vivo is NK cell antibody-dependent cellular cytotoxicity (ADCC). Recent studies led to the discovery of a novel subset of human NK cells that lack expression of FceR1g (“g-NK cells”) and have a multi-fold increase in ADCC activity after CD16 crosslinking. Here, we validate the potency of g-NKs as an off-the shelf cellular immunotherapy to enhance the efficacy of therapeutic mAbs.
g-NK cells were enriched and expanded using a proprietary method from CMV-positive blood donors selected for high levels of circulating g-NKs. For ADCC cytotoxicity assays, expanded NK cells were sorted by flow cytometry into populations of “conventional” NK cells (cNK), adaptive (NKG2C+) NK cells, and g-NK cells. After sorting, NK-cells were co-cultured 4 hr at 37° C with Raji or MM.1S target cells (1e4 cells) at varying NK-cell:target cell ratios. For in vivo persistence studies a single dose of 1e7 g-NK or cNK cells was injected into NOD scid gamma (NSG) mice. For in vivo efficacy studies with rituximab (RIT), NSG mice were inoculated with 5e5 luciferase-labeled Raji lymphoma cells and dosed weekly with 15e6 g-NK cells +/-200 ug RIT or vehicle. For in vivo efficacy studies with daratumumab (DARA) and elotuzumab (ELO), NSG mice were inoculated with 1e6 luciferase-labeled MM.1S cells and dosed every 6d with expanded g-NKs (20e6), resting cNKs (3e5, equivalent to physiological levels of NK cells/kg in humans, and +/- 10 ug DARA or 10 ug ELO. In all studies, mice received IL-15 intraperitoneally every 3 days.
Our expansion method was able to expand g-NKs from selected CMV-seropositive donors ~800-fold (n=8). By flow cytometry we found an increase in the percentage of g-NK from 28% of NK cells at input to 82% post-expansion. In vitro ADCC assays demonstrated that g-NKs enhanced cytotoxicity of RIT to Raji cells (70% at 1:1 E:T) when compared to both NKG2C+ NKs (29%) and cNKs (27%) (p&lt;0.001). Notably, we saw similar ADCC of g-NK cells whether used fresh or thawed after viable freezing (70% vs. 63%, p=0.71). In addition, g-NK cells enhanced cytotoxicity of DARA and ELO to MM.1S cells (58% and 54% at 1:1 E:T) when compared to cNK (14% and 12% at 1:1 E:T) (p&lt;0.001). We confirmed in NSG mice that g-NK persistence was markedly improved (&gt;90%) over cNKs in blood at multiple time points (p&lt;0.001), and spleen at Day 22 (p&lt;0.001). Efficacy studies using Raji cells showed the combination of g-NK and RIT led to a &gt;90% reduction in tumor burden versus RIT alone (p&lt;0.001). Evaluation of expanded g-NKs in a disseminated orthotopic xenograft model of multiple myeloma found a dramatic reduction (&gt;70%) in tumor burden with DARA+g-NK vs. DARA alone or DARA+cNK (p&lt;0.001). Similar results were found with ELO.
Here we demonstrate that adoptive transfer of expanded g-NK cells markedly enhances anti-tumor ADCC of therapeutic mAbs in several preclinical models. Importantly, because adoptive transfer of NK-cells in humans does not result in severe graft-versus-host disease, we propose that this long-lived, highly potent NK-cell therapy could be administered in an “off-the-shelf” manner to supercharge mAb efficacy.
Citation Format: Austin Basil Bigley, Nadia H. Agha, Shanae Spade, Gaetano Dipierro, Ronald Martell, Byron C. Hann, Nina Shah, Arun P. Wiita. FceR1g negative NK-cells (g-NK) enhance antibody-dependent cellular cytotoxicity and in vivo efficacy of therapeutic monoclonal antibodies against hematologic malignanices [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1630.