Abstract
Natural killer (NK) cells are highly specialized effectors of the innate immune system that hold promise for adoptive cancer immunotherapy. Their cell killing activity is primarily mediated by the pro-apoptotic serine protease granzyme B (GrB), which enters targets cells with the help of the pore-forming protein perforin. We investigated expression of a chimeric GrB fusion protein in NK cells as a means to augment their antitumoral activity. For selective targeting to tumor cells, we fused the epidermal growth factor receptor (EGFR) peptide ligand transforming growth factor α (TGFα) to human pre-pro-GrB. Established human NKL natural killer cells transduced with a lentiviral vector expressed this GrB-TGFα (GrB-T) molecule in amounts comparable to endogenous wildtype GrB. Activation of the genetically modified NK cells by cognate target cells resulted in the release of GrB-T together with endogenous granzymes and perforin, which augmented the effector cells' natural cytotoxicity against NK-sensitive tumor cells. Likewise, GrB-T was released into the extracellular space upon induction of degranulation with PMA and ionomycin. Secreted GrB-T fusion protein displayed specific binding to EGFR-overexpressing tumor cells, enzymatic activity, and selective target cell killing in the presence of an endosomolytic activity. Our data demonstrate that ectopic expression of a targeted GrB fusion protein in NK cells is feasible and can enhance antitumoral activity of the effector cells.
Highlights
Natural killer (NK) cells are highly specialized effectors of the innate immune system
Expression of the granzyme B-transforming growth factor a (TGFa) fusion protein GrB-T in NK cells cDNA encoding human pre-pro-GrB was fused via a flexible (Gly4Ser)4-His6 linker to a sequence encoding the epidermal growth factor receptor (EGFR)-specific peptide ligand TGFa followed by C-terminal Myc and hexahistidine tags in the lentiviral transfer vector pSIEW, that encodes enhanced green fluorescent protein (EGFP) as a marker (Fig. 1A)
After generation of lentiviral particles and transduction of human NKL cells, EGFP-expressing NKL/GrB-T cells were enriched by two rounds of flow cytometric cell sorting
Summary
Natural killer (NK) cells are highly specialized effectors of the innate immune system. They play an important role in the defense against viral infection and the elimination of neoplastic cells [1]. GrB is expressed as an inactive precursor protein This pre-pro-GrB carries an Nterminal signal peptide, directing packaging of the protein into secretory granules, followed by the activation dipeptide Gly-Glu. This pre-pro-GrB carries an Nterminal signal peptide, directing packaging of the protein into secretory granules, followed by the activation dipeptide Gly-Glu Removal of this peptide by the cysteine protease cathepsin C generates the enzymatically active form of GrB [7], which is stored together with other granzymes and perforin in the dense core of lytic granules. Upon release from cytotoxic lymphocytes, GrB enters target cells in cooperation with perforin, and rapidly induces apoptosis via caspase-dependent and caspase-independent mechanisms [8]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
