Destruction of cancer cells by therapeutic antibodies occurs, at least in part, through antibody-dependent cellular cytotoxicity (ADCC), and this can be mediated by various Fc-receptor-expressing immune cells, including neutrophils. However, the mechanism(s) by which neutrophils kill antibody-opsonized cancer cells has not been established. Here, we demonstrate that neutrophils can exert a mode of destruction of cancer cells, which involves antibody-mediated trogocytosis by neutrophils. Intimately associated with this is an active mechanical disruption of the cancer cell plasma membrane, leading to a lytic (i.e., necrotic) type of cancer cell death. Furthermore, this mode of destruction of antibody-opsonized cancer cells by neutrophils is potentiated by CD47-SIRPα checkpoint blockade. Collectively, these findings show that neutrophil ADCC toward cancer cells occurs by a mechanism of cytotoxicity called trogoptosis, which can be further improved by targeting CD47-SIRPα interactions.

†, # These authors contributed equally Ethical Compliance Animal experiments were in compliance with all relevant ethical regulations approved by the IVD committee (Utrecht, the Netherlands). Blood samples from healthy donors was collected after informed consent. The use of human blood samples was in compliance with all relevant ethical regulations approved by the Sanquin Ethics Advisory Council of Sanquin Blood Supply (Amsterdam, the Netherlands). Reporting summary. Further information on experimental design is available in the Nature Research Reporting Summary linked to this article. Data availability All sequencing datasets have been deposited in the NCBI Sequence Read Archive under accession number SRP144590. In addition, all processed screen results are accessible in an interactive database (https://phenosaurus.nki.nl/). All data presented in this manuscript are available from the corresponding authors upon reasonable request Author contributions M.E.W.L. conceived the project, designed and performed experiments, interpreted data and co-wrote the manuscript. M.R., A.F. an T.R.B. designed, performed and interpreted the haploid genetic screens. M.T. and J.N. designed, performed and interpreted biochemical data. J.H.M.J., A.M.B. and J.H.W.L. designed, performed and interpreted anti-Her2 in vitro and in vivo data, and J.H.W.L. co-wrote the manuscript. K.F., H.L.M. and T.K.v.d.B. designed, performed and interpreted in vitro data with human effector cells. S.v.d.S. supported and performed flow cytometry analyses. R.G.-E. and N.A.M.B. designed, performed and interpreted in vitro studies with human T cells. J.H.v.d.B. and J.B.A.G.H. supervised analyses of T cell reactivity. K.A.M. performed and interpreted experiments. M.V. designed experiments and provided reagents. F.A.S. and T.N.S. conceived the project, designed experiments, interpreted data and co-wrote the manuscript.

The function of the low-affinity IgG-receptor FcγRIIIb (CD16b), which is uniquely and abundantly expressed on human granulocytes, is not clear. Unlike the other Fcγ receptors (FcγR), it is a glycophosphatidyl inositol (GPI) -anchored molecule and does not have intracellular signaling motifs. Nevertheless, FcγRIIIb can cooperate with other FcγR to promote phagocytosis of antibody-opsonized microbes by human neutrophils. Here we have investigated the role of FcγRIIIb during antibody-dependent cellular cytotoxicity (ADCC) by neutrophils toward solid cancer cells coated with either trastuzumab (anti-HER2) or cetuximab (anti-EGFR). Inhibiting FcγRIIIb using CD16-F(ab')2 blocking antibodies resulted in substantially enhanced ADCC. ADCC was completely dependent on FcγRIIa (CD32a) and the enhanced ADCC seen after FcγRIIIb blockade therefore suggested that FcγRIIIb was competing with FcγRIIa for IgG on the opsonized target cells. Interestingly, the function of neutrophil FcγRIIIb as a decoy receptor was further supported by using neutrophils from individuals with different gene copy numbers of FCGR3B causing different levels of surface FcγRIIIb expression. Individuals with one copy of FCGR3B showed higher levels of ADCC compared to those with two or more copies. Finally, we show that therapeutic antibodies intended to improve FcγRIIIa (CD16a)-dependent natural killer (NK) cell ADCC due to the lack of fucosylation on the N-linked glycan at position N297 of the IgG1 heavy chain Fc-region, show decreased ADCC as compared to regularly fucosylated antibodies. Together, these data confirm FcγRIIIb as a negative regulator of neutrophil ADCC toward tumor cells and a potential target for enhancing tumor cell destruction by neutrophils.