ABSTRACT
Background: NK cells are being extensively studied as a cell therapy for cancer. Their effector functions are induced by the recognition of ligands on tumor cells and by various cytokines. IL-15 is broadly used to stimulate endogenous and adoptively transferred NK cells in cancer patients. These stimuli activate the membrane protease ADAM17, which then cleaves assorted receptors on the surface of NK cells as a negative feedback loop to limit their activation and function. We have shown that ADAM17 inhibition can enhance IL-15-mediated NK cell proliferation in vitro and in vivo . In this study, we investigated the underlying mechanism of this process. Methods: PBMCs or enriched NK cells from human peripheral blood, either unlabeled or labeled with a cell proliferation dye, were cultured for up to 7 days in the presence of rhIL-15 +/- an ADAM17 function-blocking antibody. Different versions of the antibody were generated; Medi-1 (IgG1), Medi-4 (IgG4), Medi-PGLALA, Medi-F(ab') 2 , and TAB16 (anti-ADAM17 and anti-CD16 bispecific) to modulate CD16A engagement on NK cells. Flow cytometry was used to assess NK cell proliferation and phenotypic markers, immunoblotting to examine CD16A signaling, and IncuCyte-based live cell imaging to measure NK cell anti-tumor activity. Results: The ADAM17 function-blocking mAb Medi-1 markedly increased initial NK cell activation by IL-15. Using different engineered versions of the antibody revealed that the activating Fcγ receptor CD16A, a well-described ADAM17 substrate, was critical for enhancing IL-15 stimulation. Hence, Medi-1 bound to ADAM17 on NK cells can be engaged by CD16A and block its shedding, inducing and prolonging its signaling. This process did not promote evident NK cell fratricide, phagocytosis, or dysfunction. Synergistic activity by Medi-1 and IL-15 enhanced the upregulation of CD137 on CD16A + NK cells and augmented their proliferation in the presence of PBMC accessory cells. Conclusions: Our data reveal for the first time that CD16A and CD137 underpin Medi-1 enhancement of IL-15-driven NK cell activation and proliferation, respectively. The use of Medi-1 represents a novel strategy to enhance IL-15-driven NK cell proliferation, and it may be of therapeutic importance by increasing the anti-tumor activity of NK cells in cancer patients. What is already known on this topic: NK cell therapies are being broadly investigated to treat cancer. NK cell stimulation by IL-15 prolongs their survival in cancer patients. Various stimuli including IL-15 activate ADAM17 in NK cells, a membrane protease that regulates the cell surface density of various receptors as a negative feedback mechanism. What this study adds: Treating NK cells with the ADAM17 function-blocking mAb Medi-1 markedly enhanced their activation and proliferation. Our study reveals that the Fc and Fab regions of Medi-1 function synergistically with IL-15 in NK cell activation. Medi-1 treatment augments the upregulation of CD137 by NK cells, which enhances their proliferation in the presence of PBMC accessory cells. How this study might affect research practice or policy: Our study is of translational importance as Medi-1 treatment in combination with IL-15 could potentially augment the proliferation and function of endogenous or adoptively transferred NK cells in cancer patients.
ABSTRACT
Natural killer (NK) cell-mediated cancer immunotherapy has grown significantly over the past two decades. More recently, multi-specific engagers have been developed as cancer therapeutics to effectively arm endogenous NK cells to more potently induce specific cytolytic responses against tumor targets. This review explores the bi- and tri-specific NK/tumor engagers that are emerging as a new generation of immunotherapeutics. These molecules vary in configuration, but they typically have small molecular weights and domains that engage specific tumor antigens and NK cell-activating receptors such as CD16, NKp30, NKp46, and NKG2D. They have demonstrated compelling potential in boosting NK cell cytotoxicity against specific tumor targets. This highly adaptable off-the-shelf platform, which in some formats also integrates cytokines, is poised to revolutionize targeted NK cell immunotherapy, either as a monotherapy or in combination with other effective anti-cancer therapies.
Subject(s)
Killer Cells, Natural , Neoplasms , Cytokines , Humans , Immunotherapy , Neoplasms/therapyABSTRACT
Adrenergic receptor (AR) expression has been demonstrated at several sites of primary and metastatic tumour growth and may influence proliferation, survival, metastasis and angiogenesis. AR antagonists like propranolol and carvedilol inhibit proliferation, induce apoptosis and synergize with chemotherapy agents in some cancers. Radiation resistance is mediated in many cells by upregulation of pro-survival pathways, which may be influenced by ARs. Studies evaluating AR antagonists combined with radiation are limited. The purpose of this study was to determine the effect of propranolol and carvedilol on viability and radiosensitivity in sarcoma cell lines. The hypothesis was that propranolol and carvedilol would increase radiosensitivity in four primary bone sarcoma cell lines. Single agent propranolol or carvedilol inhibited cell viability in all cell lines in a concentration-dependent manner. The mean inhibitory concentrations (IC50 ) for carvedilol were approximately 4-fold lower than propranolol and may be clinically relevant in vivo. Immunoblot analysis confirmed AR expression in both human and canine sarcoma cell lines; however, there was no correlation between baseline AR protein expression and radiosensitivity. Short duration treatment with carvedilol and propranolol did not significantly affect clonogenic survival. Prolonged exposure to propranolol and carvedilol significantly decreased the surviving fraction of canine osteosarcoma cells after 3Gy radiation. Based on our results and possible in vivo activity in dogs, further studies investigating the effects of carvedilol on sarcoma are warranted.
