HER2-CAR-NK cells exhibit enhanced cytotoxic activity towards HER2-positive tumors

Targeting of adoptive immune cells is among modern approaches to therapy of solid tumors. Expression of an epidermal growth factor receptor HER2 is a common finding (20% of breast tumors) and is associated with a negative outcome. In this regard, obtaining HER2-specific effector cells carrying a chimeric CAR receptor is a relevant task. NK cells have a wide spectrum of activating receptors capable of recognizing tumor-associated markers and do not initiate a graft-versus-host reaction. The goal of this study was to obtain effector CAR-NK cells capable of eliminating the HER2-positive tumor targets. The NK cells were obtained by negative magnetic separation from peripheral mononuclear cells isolated from volunteers’ blood using density gradient centrifugation. Activated NK cells were modified by retroviral transduction. Preliminarily transfected Phoenix Ampho cells were used to accumulate retroviral particles carrying HER2- CAR construct. The DARPin 9-29-HER2 molecule with affinity for the HER2 distal domain I was used as the antigen-recognizing domain. The proportion of transduced NK cells was measured by means of GFP reporter protein expression. The surface emergence of HER2-CAR receptors was detected by expression of the c-Myc extracellular domain. All modified GFP⁺NK cells were capable of expressing HER2-CAR receptors on the cell membrane. Functional activity of HER2-CAR NK cells was measured using flow cytometry, by degranulation intensity and IFNγ production in the presence of HER2-positive target cells BT-474. To assess lytic activity of HER2-CAR NK cells, the cultures of HER2-CAR-expressing GFP⁺NK cells and unmodified GFP^-NK cells were obtained by cell sorting. Lysis of BT-474 targets was measured by calcein release upon incubation with HER2-CAR and GFP^- effectors. HER2-CAR NK cells were characterized by higher levels of degranulation and IFNγ production compared to GFP^-NK cells. Moreover, HER2-CAR-NK cells had higher lytic activity towards BT-474. Thus, by means of genetic modification based on primary NK cells, we obtained highly effective HER2-CAR-NK agents capable of HER2-positive target recognition and possessing cytotoxic and cytokine-producing potential in presence of tumor cells. Expanding the variety of cellular effectors aimed at treating HER2-positive breast tumors will increase the potential of personalized tumor therapy in the future.