HER2-CAR-NK CELLS EXHIBIT ENHANCED CYTOTOXIC ACTIVITY TOWARDS HER2-POSITIVE TUMORS

Abstract

Targeting by adoptive immune cells is one of the modern approaches to the solid tumors therapy. Expression of the epidermal growth factor receptor HER2 is common for 20% of breast tumors and is associated with a negative outcome. In this regard, obtaining HER2-specific cellular effectors carrying a chimeric CAR receptor appears to be a relevant task. NK cells have a wide arsenal 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 CAR-NK cell effectors capable of HER2-positive tumor targets directed elimination. 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 upon the GFP reporter protein expression, and the surface emergence of HER2-CAR receptors was detected upon expression of the c-Myc extracellular domain. All modified GFP⁺ NK cells were capable of expressing HER2-CAR receptors on the cell membrane. HER2-CAR NK cells’ functional activity was measured via flow cytometry by degranulation intensity and IFNγ production in the presence of HER2-positive target cells BT-474. To assess the lytic activity of HER2-CAR NK cells, 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. Also, HER2-CAR-NK cells have 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 tumor cells targeted recognition and realizing cytotoxic and cytokine-producing potential in their presence. Expanding the arsenal of cellular effectors aimed at treating HER2-positive breast tumors will increase the potential of personalized tumor therapy in the future.