Abstract 2304: Expanded and engineered NK cells upregulate expression of activation and survival genes associated with increased cytotoxicity and persistence
Authors: Tohme Mira , Lazetic Sasha , Jamboretz Kate , Buren Luxuan , Guo Chao , Trager James 
Published: August 2019

Background: Natural Killer (NK) cells expanded using irradiated K562 cells engineered to express membrane-bound IL-15 and 4-1BBL (K562-mb15-4-1BBL) have been employed in the clinic [1]. Expansion upregulated activating NK cell receptors and increased NK cytotoxicity against tumor cells. Expanded NK cells can be engineered to express chimeric receptors to further improve their cytotoxicity. Chimeric receptors commonly incorporate an antigen binding domain, a costimulatory domain (i.e. 41BB, CD28) and a CD3ζ signaling domain. Receptor structure can bias engineered cells to chronic activation and exhaustion [2,3], and can regulate metabolic pathways that influence cell phenotype and function. Characterization of NK cells expressing activating NK receptor (aNKr)-expressing NK cells may enable assessment of their clinical potency. We therefore evaluated the phenotypic characteristics of expanded and engineered NK cells and investigated signatures of activation or exhaustion upon antigen stimulation.

Material and Methods: NK cells from peripheral blood mononuclear cells were expanded on irradiated K562-mbIL15-41BBL and transduced with NKG2D based chimeras (NKG2D.aNKr) and mbIL15. The expression of relevant receptors on expanded and transduced NK cells was assessed by flow cytometry. To evaluate the impact of tonic aNKr signaling versus antigen-driven stimulation on NK activation, we analyzed gene expression after stimulation with either isotype or anti-NKG2D coated beads using a Nanostring immune function panel.

Results: Ex vivo expansion generates potent, activated NK cells with high expression of NKG2D, Nkp30, Nkp44, Nkp46, CD69, CD25, and DNAM-1 activation markers and decreased expression of KLRG1 and CD158b inhibitory receptors. Expanded NK cells also upregulated TIGIT and TIM-3 expression, markers associated with functional exhaustion. However, expanded NK cells retained enhanced tumor cell cytotoxicity and cytokine production.

NKG2D.aNKr construct expression induced tonic signaling in engineered NK cells characterized by the upregulation of genes involved in cell survival (Bcl-2), cell activation (CD25) and cell mediated cytotoxicity (TRAIL).

NKG2D engagement further enhanced expression of anti-apoptotic genes, and genes encoding cytokines and chemokines required to control cell cytotoxicity (INF-γ, TNF-α) and recruitment to tumor sites (XCL1, CCL3, CCL4). Lastly, NKG2D stimulation of engineered cells led to a significant increase in the expression of TNF family members (41BB, Ox40L, Ox40, TRAIL). Increased 41BB, Ox40L and TRAIL expression was confirmed by flow cytometry and correlated with enhanced

cytotoxicity. Our data suggest that, upon stimulation, NKG2D.aNKr cells are highly activated with greater longevity. Moreover, our data allow the identification of biomarker candidates that reflect NK.aNKr potency.

Citation Format: Mira Tohme, Sasha Lazetic, Kate Jamboretz, Luxuan Buren, Chao Guo, James Trager. Expanded and engineered NK cells upregulate expression of activation and survival genes associated with increased cytotoxicity and persistence [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2304.