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.

IntroductionAntibodies have been studied extensively for their use in immunotherapy of cancer. [1][2][3][4] It is evident that receptors for the Fc portion of immunoglobulins (FcRs) on myeloid cells are critical in triggering antitumor cytotoxicity in vivo. 5,6 Antibody-dependent cellular cytotoxicity (ADCC), considered crucial for antibody-mediated tumor cell degradation, can be mediated by polymorphonuclear leukocytes (PMNs), monocytes/macrophages, eosinophils, and natural killer (NK) cells. 7,8 These effector cells use different cytotoxic mechanisms, depending on their activation state and the nature of the target. 7,9-12 PMNs, representing the most populous type of white blood cell, exhibit fast recruitment activity in vivo. Potent and very rapid (within 30 minutes) PMN cytotoxicity toward various tumor targets has been documented. 10,[13][14][15] Two classes of immunoglobulin (Ig)G receptors (Fc␥RIIa, CD32, and Fc␥RIIIb, CD16) and one class of IgA receptor (Fc␣RI, CD89) have been identified on human PMNs, whereas Fc␥RI (CD64) expression is inducible on PMNs on stimulation with granulocyte colony-stimulating factor. 16 PMNs can trigger ADCC by engagement of Fc␥RI, Fc␥RIIa, and Fc␣RI. Fc␣RI, however, has been observed to be the most effective FcR for PMN-mediated tumor cell killing. 14,17 Mac-1 (CR3, CD11b/CD18) is a member of the ␤ 2 integrin family, which includes Mac-1, LFA-1 (CD11a/CD18), and gp150/95 (CR4, CD11c/CD18). 18,19 These receptors, sharing a common ␤-chain (CD18), can bind multiple ligands and can regulate various leukocyte functions. Mac-1 represents the predominant ␤ 2 integrin on PMNs and is furthermore expressed on monocytes/macrophages and NK cells. Several PMN functions are regulated by Mac-1, including adhesion, migration, chemotaxis, phagocytosis, respiratory burst activity, and degranulation. 18 On activation, Mac-1 is able to initiate signaling by its linkage to the actin cytoskeleton and associated signaling proteins. 20,21 A number of studies described Mac-1 cooperation with different receptors on PMNs, indicating Mac-1 to be a signaling partner for other receptors. 22 These include FMLP receptors, LPS/LBP receptors (CD14), urokinase plasminogen activator receptor (CD87), and Fc receptors. [22][23][24][25][26] Mac-1 was found to trigger Ab-dependent phagocytosis by Fc␥RIIIb in fibroblasts transfected with both Mac-1 and Fc␥RIIIb, whereas cells expressing only Mac-1 or Fc␥RIIIb were unable to ingest Ab-opsonized particles. 27 Mac-1 cooperation with Fc␥RIIIb in the generation of PMN respiratory burst has been described as well. 28 Furthermore, Mac-1 restored IgG-dependent phagocytosis of transfectants with Fc␥RIIa tail-minus mutants. 29 Importantly, PMNs from patients with leukocyte adhesion deficiency, who lack CD18, were shown to be severely impaired in mediating phagocytosis and ADCC. 18,[30][31][32] Although the relative contribution of individual ␤ 2 integrins remains to be determined, various studies point to an important role of Mac-1 in FcR-mediated cytotoxicity. Targets st...

Development of human therapeutic Abs has led to reduced immunogenicity and optimal interactions with the human immune system in patients. Humanization had as a consequence that efficacy studies performed in mouse models, which represent a crucial step in preclinical development, are more difficult to interpret because of gaps in our knowledge of the activation of murine effector cells by human IgG (hIgG) remain. We therefore developed full sets of human and mouse isotype variants of human Abs targeting epidermal growth factor receptor and CD20 to explore the crosstalk with mouse FcγRs (mFcγRs) and murine effector cells. Analysis of mFcγR binding demonstrated that hIgG1 and hIgG3 bound to all four mFcγRs, with hIgG3 having the highest affinity. hIgG1 nevertheless was more potent than hIgG3 in inducing Ab-dependent cellular cytotoxicity (ADCC) and Ab-dependent cellular phagocytosis with mouse NK cells, mouse polymorphonuclear leukocytes, and mouse macrophages. hIgG4 bound to all mFcγRs except mFcγRIV and showed comparable interactions with murine effector cells to hIgG3. hIgG4 is thus active in the murine immune system, in contrast with its inert phenotype in the human system. hIgG2 bound to mFcγRIIb and mFcγRIII, and induced potent ADCC with mouse NK cells and mouse polymorphonuclear leukocytes. hIgG2 induced weak ADCC and, remarkably, was unable to induce Ab-dependent cellular phagocytosis with mouse macrophages. Finally, the isotypes were studied in s.c. and i.v. tumor xenograft models, which confirmed hIgG1 to be the most potent human isotype in mouse models. These data enhance our understanding of the crosstalk between hIgGs and murine effector cells, permitting a better interpretation of human Ab efficacy studies in mouse models.

Emerging evidence suggests that FcγR-mediated cross-linking of tumor-bound mAbs may induce signaling in tumor cells that contributes to their therapeutic activity. In this study, we show that daratumumab (DARA), a therapeutic human CD38 mAb with a broad-spectrum killing activity, is able to induce programmed cell death (PCD) of CD38+ multiple myeloma tumor cell lines when cross-linked in vitro by secondary Abs or via an FcγR. By comparing DARA efficacy in a syngeneic in vivo tumor model using FcRγ-chain knockout or NOTAM mice carrying a signaling-inactive FcRγ-chain, we found that the inhibitory FcγRIIb as well as activating FcγRs induce DARA cross-linking–mediated PCD. In conclusion, our in vitro and in vivo data show that FcγR-mediated cross-linking of DARA induces PCD of CD38-expressing multiple myeloma tumor cells, which potentially contributes to the depth of response observed in DARA-treated patients and the drug’s multifaceted mechanisms of action.

Key Points• Phagocytosis of CLL targets by neutrophils is a novel mechanism of action of the glycoengineered anti-CD20 antibody obinutuzumab.• This mechanism takes place in physiological conditions and requires CD16B and CD32A.Obinutuzumab (GA101) is a glycoengineered type 2 CD20 antibody with enhanced CD16A-binding and natural killer-mediated cytotoxicity. CD16B is highly homologous to CD16A and a major FcgR on human polymorphonuclear neutrophils (PMNs). We show here that glycoengineered obinutuzumab or rituximab bound CD16B with approximately sevenfold higher affinity, compared with nonglycoengineered wild-type parental antibodies. Furthermore, glycoengineered obinutuzumab activated PMNs, either purified or in chronic lymphoblastic leukemia whole blood, more efficiently than wild-type rituximab. Activation resulted in a 50% increase in CD11b expression and 70% down-modulation of CD62L on neutrophils and in release of tumor necrosis factor alpha, IL-6, and IL-8. Activation was not accompanied by generation of reactive oxygen species or antibody-dependent cellular cytotoxicity activity, but led to up to 47% phagocytosis of glycoengineered anti-CD20 opsonized chronic lymphoblastic leukemia targets by purified PMNs. Significant phagocytosis was observed in whole blood, but only in the presence of glycoengineered antibodies, and was followed by up to 50% PMN death. Finally we show, using anti-CD16B and anti-CD32A Fab and F(ab') 2 fragments, that both of these receptors are involved in PMN activation, phagocytosis, and cell death induced by glycoengineered antibodies. We conclude that phagocytosis by PMNs is an additional mechanism of action of obinutuzumab mediated through its higher binding affinity for CD16B. (Blood. 2013;122(20):3482-3491) IntroductionThe chimeric unmodified, wild-type CD20 IgG1 monoclonal antibody rituximab (MabThera and Rituxan) has shown significant therapeutic activity in B-non-Hodgkin lymphoma (B-NHL) and chronic lymphocytic leukemia (CLL).1,2 Rituximab is thought to act largely through immune-mediated mechanisms: complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC), and/or antibody-dependent phagocytosis (ADCP) by macrophages/monocytes.3,4 CD20 IgG1 antibodies can differ in their functional properties according to their binding mode to CD20. They are classified as type 1 when they show high CDC such as rituximab or as type 2 when they show high homotypic adhesion and direct cell death, respectively. 5,6 Improved versions of rituximab have been developed with the scope of enhancing response rates and reducing relapse or resistance. 7,8 One modification applied for CD20 and other therapeutic antibodies is termed "glycoengineering" and results in the decreased fucosylation of the carbohydrate attached to the Asn-297 glycosylation site of the Fc portion of the antibody. [9][10][11][12][13][14][15] Obinutuzumab (GA101) is a glycoengineered CD20 antibody derived from the murine Bly-1 antibody and is currently in pivotal clinical trials for the treatment of...

†, # 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 success of passive immunization suggests that antibody-based therapies will be effective at controlling malaria. We describe the development of fully human antibodies specific for Plasmodium falciparum by antibody repertoire cloning from phage display libraries generated from immune Gambian adults. Although these novel reagents bind with strong affinity to malaria parasites, it remains unclear if in vitro assays are predictive of functional immunity in humans, due to the lack of suitable animal models permissive for P. falciparum. A potentially useful solution described herein allows the antimalarial efficacy of human antibodies to be determined using rodent malaria parasites transgenic for P. falciparum antigens in mice also transgenic for human Fc-receptors. These human IgG1s cured animals of an otherwise lethal malaria infection, and protection was crucially dependent on human FcγRI. This important finding documents the capacity of FcγRI to mediate potent antimalaria immunity and supports the development of FcγRI-directed therapy for human malaria.

This review aims to provide an in depth overview of the current knowledge of the effects of bovine immunoglobulins on the human immune system. The stability and functional effects of orally ingested bovine immunoglobulins in milk products are described and potential mechanisms of action are discussed. Orally ingested bovine IgG (bovine IgG) can be recovered from feces, ranging from very low levels up to 50% of the ingested IgG that has passed through the gastrointestinal tract. In infants the recovered levels are higher than in adults most likely due to differences in stomach and intestinal conditions such as pH. This indicates that bovine IgG can be functionally active throughout the gastrointestinal tract. Indeed, a large number of studies in infants and adults have shown that bovine IgG (or colostrum as a rich source thereof) can prevent gastrointestinal tract infections, upper respiratory tract infections, and LPS-induced inflammation. These studies vary considerably in target group, design, source of bovine IgG, dosage, and endpoints measured making it hard to draw general conclusions on effectiveness of bovine immunoglobulin rich preparations. Typical sources of bovine IgG used in human studies are serum-derived IgG, colostrum, colostrum-derived IgG, or milk-derived immunoglobulins. In addition, many studies have used IgG from vaccinated cows, but studies using IgG from nonimmunized animals have also been reported to be effective. Mechanistically, bovine IgG binds to many human pathogens and allergens, can neutralize experimental infection of human cells, and limits gastrointestinal inflammation. Furthermore, bovine IgG binds to human Fc receptors which, enhances phagocytosis, killing of bacteria and antigen presentation and bovine IgG supports gastrointestinal barrier function in in vitro models. These mechanisms are becoming more and more established and explain why bovine IgG can have immunological effects in vivo. The inclusion of oral bovine immunoglobulins in specialized dairy products and infant nutrition may therefore be a promising approach to support immune function in vulnerable groups such as infants, children, elderly and immunocompromised patients.