Abstract
BackgroundThe natural killer cell line, NK-92MI, is cytotoxic against various types of cancer. The aim of this study was to develop chimeric antigen receptor-modified (CAR) NK-92MI cells targeting carcinoembryonic antigen-expressing (CEA) tumours and increase killing efficacy by pharmacologically modifying CEA-expression.ResultWe generated anti-CEA-CAR NK-92MI cells by retroviral vector transduction. This genetically-modified cell line recognised and lysed high CEA-expressing tumour cell lines (LS174T) at 47.54 ± 12.60% and moderate CEA-expressing tumour cell lines (WiDr) at 31.14 ± 16.92% at a 5:1 effector: target (E/T) ratio. The cell line did not lyse low CEA-expressing tumour cells (HCT116) as they did their parental cells (NK-92MI cells). The histone deacetylase-inhibitor (HDAC) sodium butyrate (NaB) and the methylation-inhibitor 5-azacytidine (5-AZA), as epigenetic modifiers, induced CEA-expression in HCT116 and WiDr cells. Although the IC50 of 5 fluorouracil (5-FU) increased, both cell lines showed collateral sensitivity to anti-CEA-CAR NK-92MI cells. The cytolytic function of anti-CEA-CAR NK-92MI cells was increased from 22.99 ± 2.04% of lysis background to 69.20 ± 11.92% after NaB treatment, and 69.70 ± 9.93% after 5-AZA treatment, at a 10:1 E/T ratio in HCT116 cells. The WiDr cells showed similar trend, from 22.99 ± 4.01% of lysis background to 70.69 ± 10.19% after NaB treatment, and 59.44 ± 10.92% after 5-AZA treatment, at a 10:1 E/T ratio.ConclusionsThis data indicates that the effector-ability of anti-CEA-CAR NK-92MI increased in a CEA-dependent manner. The combination of epigenetic-modifiers like HDAC-inhibitors, methylation-inhibitors, and adoptive-transfer of ex vivo-expanded allogeneic-NK cells may be clinically applicable to patients with in 5-FU resistant condition.
Highlights
The natural killer cell line, natural killer cells (NK)-92MI, is cytotoxic against various types of cancer
This data indicates that the effector-ability of anti-carcinoembryonic antigenexpressing (CEA)-chimeric antigen receptor-modified (CAR) NK-92MI increased in a CEA-dependent manner
Expression of anti-CEA-CAR in NK-92MI cells To construct the anti-CEA specific CAR, the cDNAs of variable heavy-chain (VH) and light-chain (VL) domains of the humanised-monoclonal-anti-CEA antibody, the human influenza hemagglutinin (HA)-tag sequence, the CD8α hinge region, and the transmembrane and intracellular domains of CD3ζ were assembled stepwise into a pGEM-1 plasmid (Promega, Madison, WI, USA)
Summary
The natural killer cell line, NK-92MI, is cytotoxic against various types of cancer. The aim of this study was to develop chimeric antigen receptor-modified (CAR) NK-92MI cells targeting carcinoembryonic antigenexpressing (CEA) tumours and increase killing efficacy by pharmacologically modifying CEA-expression. NK cells do not require antigen representation for target recognition They show tumour cytotoxicity in a major histocompatibility complex-unrestricted (MHC-unrestricted) manner [1, 2]. Genetically-modified effector cells with a chimeric antigen receptor (CAR) were chosen with the intention of enhancing their reactivity against antigen-expressing tumour cells [15,16,17]. In recent years several CAR-modified NK-92 and NK-92MI cells have been developed They were highly cytotoxic both in vitro and in vivo [22,23,24,25,26,27]. We proposed that CEA-targeted adoptive immunotherapy is a good example of collateral sensitivity to 5-FU-resistant CEA-expression in tumours
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