Abstract
Natural killer (NK) cells have the capacity to target tumors and are ideal candidates for immunotherapy. Viral vectors have been used to genetically modify in vitro expanded NK cells to express chimeric antigen receptors (CARs), which confer cytotoxicity against tumors. However, use of viral transduction methods raises the safety concern of viral integration into the NK cell genome. In this study, we used trogocytosis as a non-viral method to modify NK cells for immunotherapy. A K562 cell line expressing high levels of anti-CD19 CARs was generated as a donor cell to transfer the anti-CD19 CARs onto NK cells via trogocytosis. Anti-CD19 CAR expression was observed in expanded NK cells after these cells were co-cultured for one hour with freeze/thaw-treated donor cells expressing anti-CD19 CARs. Immunofluorescence analysis confirmed the localization of the anti-CD19 CARs on the NK cell surface. Acquisition of anti-CD19 CARs via trogocytosis enhanced NK cell-mediated cytotoxicity against the B-cell acute lymphoblastic leukemia (B-ALL) cell lines and primary B-ALL cells derived from patients. To our knowledge, this is the first report that describes the increased cytotoxicity of NK cells following the acquisition of CARs via trogocytosis. This novel strategy could be a potential valuable therapeutic approach for the treatment of B-cell tumors.
Highlights
Natural killer (NK) cells have the ability to recognize and eliminate tumor cells, making them ideal candidates for tumor immunotherapy [1,2]
After 7 days of expansion, the peripheral blood mononuclear cells (PBMCs) co-cultured with irradiated K562-mbIL15-41BBL cells produced 95.8% CD56+CD3- NK cells (Figure 1A, left upper panel), whereas K562-mbIL15-41BBL cells subjected to one freeze/thaw cycle yielded 81.8% CD56+CD3- cells (Figure 1A, right upper panel)
By day 7 in cultures, approximately 85% (n = 3; Figure 1B, NK IR) of the NK cells expanded from PBMCs co-cultured with irradiated K562-mb15-41BBL cells were CD56-positive, and this number increased to 95% following CD3 depletion
Summary
Natural killer (NK) cells have the ability to recognize and eliminate tumor cells, making them ideal candidates for tumor immunotherapy [1,2]. We have previously genetically modified in vitro expanded NK cells to express DAP10 and the chimeric NKG2D receptor containing the CD3f signal domain, which altered the balance between the activating and inhibitory signals of NK cells and enhanced the cytotoxicity against NKG2D ligand-bearing tumors [3]. Expression of anti-CD19 chimeric antigen receptors (CARs) containing 41BB and CD3f signal domains on NK cells enhanced the activating signals originating from CD19 antigen engagement, leading to cytotoxicity against B-cell leukemia [4]. Trogocytosis is a process in which membrane patches are exchanged between target and immune cells [5,6,7]. The chemokine receptor CCR7 has been shown to be transferred from donor cells onto the surface of NK cells via trogocytosis, and this transfer stimulated NK cell migration, leading to enhanced lymph node homing [10,11]. T cells captured NKG2D and NKp46 ligands on tumor cells through trogocytosis and promoted NK cell activity [12]
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