Quintarelli et al. developed a feeder-cell-free NK cell manufacturing approach amenable to lentivirus transfection with CAR constructs to produce CD19 CAR-NK cells with high efficiency, maintaining a diverse differentiation pattern and low expression of PD-1. In vitro, CAR-NK cells were cytotoxic against multiple CD19-expressing leukemia lines, including primary ALL blasts. Expression of key NK activating receptors was not required for CAR-NK activity but was maintained, potentially mitigating antigen escape via CD19 loss. In vivo, CAR-NK cells demonstrated equivalent efficacy to CAR T cells against DAUDI tumors but without cytokine-mediated fatal toxicity.
We developed an innovative and efficient, feeder-free culture method to genetically modify and expand peripheral blood-derived NK cells with high proliferative capacity, while preserving the responsiveness of their native activating receptors. Activated peripheral blood NK cells were efficiently transduced by a retroviral vector, carrying a second-generation CAR targeting CD19. CAR expression was demonstrated across the different NK-cell subsets. CAR.CD19-NK cells display higher antileukemic activity toward CD19(+) cell lines and primary blasts obtained from patients with B-cell precursor ALL compared with unmodified NK cells. In vivo animal model data showed that the antileukemia activity of CAR.CD19-NK cell is superimposable to that of CAR-T cells, with a lower xenograft toxicity profile. These data support the feasibility of generating feeder-free expanded, genetically modified peripheral blood NK cells for effective "off-the-shelf" immuno-gene-therapy, while their innate alloreactivity can be safely harnessed to potentiate allogeneic cell therapy.
Author Info: (1) Department of Onco-Haematology and Cell and Gene Therapy, Bambino Gesu Children's Hospital, IRCCS, 00146, Rome, Italy. Department of Clinical Medicine and Surgery, Federico II
Author Info: (1) Department of Onco-Haematology and Cell and Gene Therapy, Bambino Gesu Children's Hospital, IRCCS, 00146, Rome, Italy. Department of Clinical Medicine and Surgery, Federico II University of Naples, 81100, Naples, Italy. (2) Department of Experimental Medicine (DIMES), University of Genoa, 16132, Genoa, Italy. Centre of Excellence for Biomedical Research (CEBR), University of Genoa, 16132, Genoa, Italy. (3) Department of Onco-Haematology and Cell and Gene Therapy, Bambino Gesu Children's Hospital, IRCCS, 00146, Rome, Italy. (4) Department of Experimental Medicine (DIMES), University of Genoa, 16132, Genoa, Italy. (5) Istituto G Gaslini, Laboratorio di Immunologia Clinica e Sperimentale, Dipartimento di Ricerca e Diagnostica, 16147, Genoa, Italy. (6) Department of Onco-Haematology and Cell and Gene Therapy, Bambino Gesu Children's Hospital, IRCCS, 00146, Rome, Italy. (7) Department of Onco-Haematology and Cell and Gene Therapy, Bambino Gesu Children's Hospital, IRCCS, 00146, Rome, Italy. (8) Department of Onco-Haematology and Cell and Gene Therapy, Bambino Gesu Children's Hospital, IRCCS, 00146, Rome, Italy. (9) Department of Onco-Haematology and Cell and Gene Therapy, Bambino Gesu Children's Hospital, IRCCS, 00146, Rome, Italy. (10) Department of Onco-Haematology and Cell and Gene Therapy, Bambino Gesu Children's Hospital, IRCCS, 00146, Rome, Italy. (11) Department of Onco-Haematology and Cell and Gene Therapy, Bambino Gesu Children's Hospital, IRCCS, 00146, Rome, Italy. (12) Department of Onco-Haematology and Cell and Gene Therapy, Bambino Gesu Children's Hospital, IRCCS, 00146, Rome, Italy. (13) Department of Onco-Haematology and Cell and Gene Therapy, Bambino Gesu Children's Hospital, IRCCS, 00146, Rome, Italy. (14) Department of Onco-Haematology and Cell and Gene Therapy, Bambino Gesu Children's Hospital, IRCCS, 00146, Rome, Italy. (15) Department of Onco-Haematology and Cell and Gene Therapy, Bambino Gesu Children's Hospital, IRCCS, 00146, Rome, Italy. (16) Department of Onco-Haematology and Cell and Gene Therapy, Bambino Gesu Children's Hospital, IRCCS, 00146, Rome, Italy. (17) Department of Onco-Haematology and Cell and Gene Therapy, Bambino Gesu Children's Hospital, IRCCS, 00146, Rome, Italy. (18) Department of Onco-Haematology and Cell and Gene Therapy, Bambino Gesu Children's Hospital, IRCCS, 00146, Rome, Italy. (19) Department of Onco-Haematology and Cell and Gene Therapy, Bambino Gesu Children's Hospital, IRCCS, 00146, Rome, Italy. (20) Department of Onco-Haematology and Cell and Gene Therapy, Bambino Gesu Children's Hospital, IRCCS, 00146, Rome, Italy. (21) Sandhill Therapeutics, Dallas, TX, 75231, USA. (22) Department of Onco-Haematology and Cell and Gene Therapy, Bambino Gesu Children's Hospital, IRCCS, 00146, Rome, Italy. biagio.deangelis@opbg.net. (23) Department of Immunology, Bambino Gesu Children's Hospital, IRCCS, 00146, Rome, Italy. (24) Department of Onco-Haematology and Cell and Gene Therapy, Bambino Gesu Children's Hospital, IRCCS, 00146, Rome, Italy. franco.locatelli@opbg.net. Department of Pediatrics, Sapienza University of Rome, Rome, Italy. franco.locatelli@opbg.net.
