Abstract
One mechanism of action for clinical efficacy by therapeutic antibodies is the promotion of immune-related functions, such as cytokine secretion and cytotoxicity, driven by FcγRIIIa (CD16) expressed on natural killer (NK) cells. These observations have led to research focusing on methods to increase Fc receptor-mediated events, which include removal of a fucose moiety found on the Fc portion of the antibody. Further studies have elucidated the mechanistic changes in signaling, cellular processes, and cytotoxic characteristics that increase ADCC activity with afucosylated antibodies. Additionally, other studies have shown the potential benefits of these antibodies in combination with small molecule inhibitors. These experiments demonstrated the molecular and cellular mechanisms underlying the benefits of using afucosylated antibodies in combination settings. Many of these observations were based on an artificial in vitro activation assay in which the FcγRIIIa on human NK cells was activated by therapeutic antibodies. This assay provided the flexibility to study downstream effector NK cell functions, such as cytokine production and degranulation. In addition, this assay has been used to interrogate signaling pathways and identify molecules that can be modulated or used as biomarkers. Finally, other therapeutic molecules (i.e., small molecule inhibitors) have been added to the system to provide insights into the combination of these therapeutics with therapeutic antibodies, which is essential in the current clinical space. This manuscript aims to provide a technical foundation for performing this artificial human NK cell activation assay. The protocol demonstrates key steps for cell activation as well as potential downstream applications that range from functional readouts to more mechanistic observations.
Highlights
Over the last few decades, there has been tremendous focus on developing targeted cancer therapies using antibodies
It is essential that natural killer (NK) cell purity is high because the Fc portion of antibodies can bind the FcγRIIIa expressed on other cell types, such as monocytes
To ensure events were driven by the FcγRIIIa, western blots for phospho-AKT, phospho-PRAS40, and phospho-ERK1/2 were performed, in which NK cells were activated for 1–5 min
Summary
Over the last few decades, there has been tremendous focus on developing targeted cancer therapies using antibodies. Combined with the fact that there are numerous research programs developing therapeutic antibodies to utilize FcγRIIIa-driven mechanisms to target cancer cells, it is imperative to develop in vitro assays that examine the molecular and cellular aspects promoted by these antibodies. The artificial in vitro activation assay used to define the signaling and cellular characteristics described above has been designed to study FcγRIIIa-driven events in NK cells mediated by antibodies in the absence of target cells. Depending on the length of stimulation, researchers can assess signaling, cellular processes, cytotoxic characteristics, and effector functions[8,9] This assay provides flexibility in studying these events when antibodies are combined with other molecules[9].
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