title: Obinutuzumab-mediated high-affinity ligation of FcγRIIIA/CD16 primes NK cells for IFNγ production.
abstract: Natural killer (NK) cell-mediated antibody-dependent cellular cytotoxicity (ADCC), based on the recognition of IgG-opsonized targets by the low-affinity receptor for IgG FcγRIIIA/CD16, represents one of the main mechanisms by which therapeutic antibodies (mAbs) mediate their antitumor effects. Besides ADCC, CD16 ligation also results in cytokine production, in particular, NK-derived IFNγ is endowed with a well-recognized role in the shaping of adaptive immune responses. Obinutuzumab is a glycoengineered anti-CD20 mAb with a modified crystallizable fragment (Fc) domain designed to increase the affinity for CD16 and consequently the killing of mAb-opsonized targets. However, the impact of CD16 ligation in optimized affinity conditions on NK functional program is not completely understood. Herein, we demonstrate that the interaction of NK cells with obinutuzumab-opsonized cells results in enhanced IFNγ production as compared with parental non-glycoengineered mAb or the reference molecule rituximab. We observed that affinity ligation conditions strictly correlate with the ability to induce CD16 down-modulation and lysosomal targeting of receptor-associated signaling elements. Indeed, a preferential degradation of FcεRIγ chain and Syk kinase was observed upon obinutuzumab stimulation independently from CD16-V158F polymorphism. Although the downregulation of FcεRIγ/Syk module leads to the impairment of cytotoxic function induced by NKp46 and NKp30 receptors, obinutuzumab-experienced cells exhibit an increased ability to produce IFNγ in response to different stimuli. These data highlight a relationship between CD16 aggregation conditions and the ability to promote a degradative pathway of CD16-coupled signaling elements associated to the shift of NK functional program.
authors.affiliation: Department of Experimental Medicine, Laboratorio Pasteur Italia Fondazione Cenci Bolognetti, Sapienza University , Rome, Italy.
authors.forename: Cristina
authors.initials: C
authors.lastname: Capuano
authors.affiliation: Department of Molecular Medicine, Laboratorio Pasteur Italia Fondazione Cenci Bolognetti, Sapienza University , Rome, Italy.
authors.forename: Chiara
authors.initials: C
authors.lastname: Pighi
authors.affiliation: Department of Molecular Medicine, Laboratorio Pasteur Italia Fondazione Cenci Bolognetti, Sapienza University , Rome, Italy.
authors.forename: Rosa
authors.initials: R
authors.lastname: Molfetta
authors.affiliation: Department of Molecular Medicine, Laboratorio Pasteur Italia Fondazione Cenci Bolognetti, Sapienza University , Rome, Italy.
authors.forename: Rossella
authors.initials: R
authors.lastname: Paolini
authors.affiliation: Department of Molecular Medicine, Laboratorio Pasteur Italia Fondazione Cenci Bolognetti, Sapienza University , Rome, Italy.
authors.forename: Simone
authors.initials: S
authors.lastname: Battella
authors.affiliation: Department of Experimental Medicine, Laboratorio Pasteur Italia Fondazione Cenci Bolognetti, Sapienza University , Rome, Italy.
authors.forename: Gabriella
authors.initials: G
authors.lastname: Palmieri
authors.affiliation: Department of Molecular Medicine, Laboratorio Pasteur Italia Fondazione Cenci Bolognetti, Sapienza University , Rome, Italy.
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Giannini
authors.affiliation: Department of Molecular Medicine, Laboratorio Pasteur Italia Fondazione Cenci Bolognetti, Sapienza University , Rome, Italy.
authors.forename: Francesca
authors.initials: F
authors.lastname: Belardinilli
authors.affiliation: Department of Molecular Medicine, Laboratorio Pasteur Italia Fondazione Cenci Bolognetti, Sapienza University, Rome, Italy; IRCCS Neuromed, Pozzilli, Italy.
authors.forename: Angela
authors.initials: A
authors.lastname: Santoni
authors.affiliation: Department of Experimental Medicine, Laboratorio Pasteur Italia Fondazione Cenci Bolognetti, Sapienza University , Rome, Italy.
authors.forename: Ricciarda
authors.initials: R
authors.lastname: Galandrini
keywords: ADCC; Fc glycoengineering; FcγRIIIA/CD16; IFNγ; NK cells; obinutuzumab