RESUMO
Anti-CD20 antibodies such as rituximab are broadly used to treat B-cell malignancies. These antibodies can induce various effector functions, including immune cell-mediated antibody-dependent cellular cytotoxicity (ADCC). Neutrophils can induce ADCC toward solid cancer cells by trogoptosis, a cytotoxic mechanism known to be dependent on trogocytosis. However, neutrophils seem to be incapable of killing rituximab-opsonized B-cell lymphoma cells. Nevertheless, neutrophils do trogocytose rituximab-opsonized B-cell lymphoma cells, but this only reduces CD20 surface expression and is thought to render tumor cells therapeutically resistant to further rituximab-dependent destruction. Here, we demonstrate that resistance of B-cell lymphoma cells toward neutrophil killing can be overcome by a combination of CD47-SIRPα checkpoint blockade and sodium stibogluconate (SSG), an anti-leishmaniasis drug and documented inhibitor of the tyrosine phosphatase SHP-1. SSG enhanced neutrophil-mediated ADCC of solid tumor cells but enabled trogoptotic killing of B-cell lymphoma cells by turning trogocytosis from a mechanism that contributes to resistance into a cytotoxic anti-cancer mechanism. Tumor cell killing in the presence of SSG required both antibody opsonization of the target cells and disruption of CD47-SIRPα interactions. These results provide a more detailed understanding of the role of neutrophil trogocytosis in antibody-mediated destruction of B cells and clues on how to further optimize antibody therapy of B-cell malignancies.
Assuntos
Antígeno CD47 , Neutrófilos , Citotoxicidade Celular Dependente de Anticorpos , Gluconato de Antimônio e Sódio , Antígeno CD47/metabolismo , Neutrófilos/metabolismo , Rituximab/farmacologia , Rituximab/uso terapêuticoRESUMO
Antibody-mediated blood disorders ensue after auto- or alloimmunization against blood cell antigens, resulting in cytopenia. Although the mechanisms of cell destruction are the same as in immunotherapies targeting tumor cells, many factors are still unknown. Antibody titers, for example, often do not strictly correlate with clinical outcome. Previously, we found C-reactive protein (CRP) levels to be elevated in thrombocytopenic patients, correlating with thrombocyte counts, and bleeding severity. Functionally, CRP amplified antibody-mediated phagocytosis of thrombocytes by phagocytes. To investigate whether CRP is a general enhancer of IgG-mediated target cell destruction, we extensively studied the effect of CRP on in vitro IgG-Fc receptor (FcγR)-mediated cell destruction: through respiratory burst, phagocytosis, and cellular cytotoxicity by a variety of effector cells. We now demonstrate that CRP also enhances IgG-mediated effector functions toward opsonized erythrocytes, in particular by activated neutrophils. We performed a first-of-a-kind profiling of CRP binding to all human FcγRs and IgA-Fc receptor I (FcαRI) using a surface plasmon resonance array. CRP bound these receptors with relative affinities of FcγRIa = FcγRIIa/b = FcγRIIIa > FcγRIIIb = FcαRI. Furthermore, FcγR blocking (in particular FcγRIa) abrogated CRP's ability to amplify IgG-mediated neutrophil effector functions toward opsonized erythrocytes. Finally, we observed that CRP also amplified killing of breast-cancer tumor cell line SKBR3 by neutrophils through anti-Her2 (trastuzumab). Altogether, we provide for the first time evidence for the involvement of specific CRP-FcγR interactions in the exacerbation of in vitro IgG-mediated cellular destruction; a trait that should be further evaluated as potential therapeutic target e.g., for tumor eradication.
Assuntos
Proteína C-Reativa/metabolismo , Imunoglobulina G/imunologia , Receptores Fc/metabolismo , Receptores de IgG/metabolismo , Adulto , Animais , Células Cultivadas , Citofagocitose/imunologia , Citotoxicidade Imunológica , Eritrócitos/imunologia , Feminino , Humanos , Imunoglobulina G/metabolismo , Masculino , Camundongos , Pessoa de Meia-Idade , Modelos Biológicos , Neutrófilos/imunologia , Neutrófilos/metabolismo , Explosão Respiratória/imunologia , Adulto JovemRESUMO
Therapeutic monoclonal antibodies (mAb), directed toward either tumor antigens or inhibitory checkpoints on immune cells, are effective in cancer therapy. Increasing evidence suggests that the therapeutic efficacy of these tumor antigen-targeting mAbs is mediated-at least partially-by myeloid effector cells, which are controlled by the innate immune-checkpoint interaction between CD47 and SIRPα. We and others have previously demonstrated that inhibiting CD47-SIRPα interactions can substantially potentiate antibody-dependent cellular phagocytosis and cytotoxicity of tumor cells by IgG antibodies both in vivo and in vitro IgA antibodies are superior in killing cancer cells by neutrophils compared with IgG antibodies with the same variable regions, but the impact of CD47-SIRPα on IgA-mediated killing has not been investigated. Here, we show that checkpoint inhibition of CD47-SIRPα interactions further enhances destruction of IgA antibody-opsonized cancer cells by human neutrophils. This was shown for multiple tumor types and IgA antibodies against different antigens, i.e., HER2/neu and EGFR. Consequently, combining IgA antibodies against HER2/neu or EGFR with SIRPα inhibition proved to be effective in eradicating cancer cells in vivo In a syngeneic in vivo model, the eradication of cancer cells was predominantly mediated by granulocytes, which were actively recruited to the tumor site by SIRPα blockade. We conclude that IgA-mediated tumor cell destruction can be further enhanced by CD47-SIRPα checkpoint inhibition. These findings provide a basis for targeting CD47-SIRPα interactions in combination with IgA therapeutic antibodies to improve their potential clinical efficacy in tumor patients.
Assuntos
Antineoplásicos Imunológicos/farmacologia , Neoplasias da Mama/imunologia , Neoplasias da Mama/terapia , Antígeno CD47/antagonistas & inibidores , Imunoglobulina A/imunologia , Neutrófilos/imunologia , Receptores Imunológicos/antagonistas & inibidores , Animais , Anticorpos Monoclonais/farmacologia , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Antígenos de Diferenciação/imunologia , Neoplasias da Mama/patologia , Antígeno CD47/imunologia , Linhagem Celular Tumoral , Receptores ErbB/antagonistas & inibidores , Feminino , Humanos , Imunoterapia/métodos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos NOD , Camundongos SCID , Fagocitose/efeitos dos fármacos , Fagocitose/imunologia , Receptor ErbB-2/antagonistas & inibidores , Receptores Imunológicos/imunologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
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.
Assuntos
Citotoxicidade Celular Dependente de Anticorpos , Neutrófilos/imunologia , Animais , Anticorpos Monoclonais/uso terapêutico , Antígeno CD11b/metabolismo , Antígenos CD18/metabolismo , Antígeno CD47/metabolismo , Linhagem Celular Tumoral , Feminino , Humanos , Masculino , Melanoma Experimental/metabolismo , Melanoma Experimental/patologia , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias/tratamento farmacológico , Neoplasias/imunologia , Neoplasias/patologia , Receptores de IgG/metabolismo , Receptores Imunológicos/genética , Receptores Imunológicos/metabolismo , Transplante HomólogoRESUMO
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.
Assuntos
Citotoxicidade Celular Dependente de Anticorpos/imunologia , Imunoglobulina G/metabolismo , Neoplasias/tratamento farmacológico , Neutrófilos/imunologia , Receptores de IgG/metabolismo , Citotoxicidade Celular Dependente de Anticorpos/efeitos dos fármacos , Linhagem Celular Tumoral , Cetuximab/metabolismo , Cetuximab/farmacologia , Cetuximab/uso terapêutico , Variações do Número de Cópias de DNA , Proteínas Ligadas por GPI/antagonistas & inibidores , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/imunologia , Proteínas Ligadas por GPI/metabolismo , Humanos , Imunoglobulina G/farmacologia , Imunoglobulina G/uso terapêutico , Células Matadoras Naturais/imunologia , Neoplasias/imunologia , Neoplasias/patologia , Neutrófilos/metabolismo , Receptores de IgG/antagonistas & inibidores , Receptores de IgG/genética , Receptores de IgG/imunologia , Trastuzumab/metabolismo , Trastuzumab/farmacologia , Trastuzumab/uso terapêuticoRESUMO
The efficacy of cancer therapeutic antibodies varies considerably among patients. Anti-cancer antibodies act through different mechanisms, including antibody-dependent cellular cytotoxicity (ADCC) triggered via Fcγ receptors (FcγR). This phagocyte ADCC can be promoted by interference with CD47-SIRPα interactions, but the magnitude of this enhancement also varies among individuals. Both FcγR and SIRPα display considerable genetic variation, and we investigated whether this explains some of the variability in ADCC. Because of linkage disequilibrium between FcγR variants the interpretation of previous reports suggesting a potential link between FcγR polymorphisms and ADCC has been troublesome. We performed an integrated genetic analysis that enables stratification. ADCC by activated human neutrophils towards Trastuzumab-coated breast cancer cells was predominantly dependent on FcγRIIa. Neutrophils from individuals with the FcγRIIa-131H polymorphic variant displayed significantly higher killing capacity relative to those with FcγRIIa-131R. Furthermore, ADCC was consistently enhanced by targeting CD47-SIRPα interactions, and there were no significant functional differences between the two most prevalent SIRPα polymorphic variants. Thus, neutrophil ADCC capacity is directly related to the FcγRIIa polymorphism, and targeting CD47-SIRPα interactions enhances ADCC independently of FcγR and SIRPα genotype, thereby further suggesting that CD47-SIRPα interference might be a generic strategy for potentiating the efficacy of antibody therapy in cancer.
Assuntos
Citotoxicidade Celular Dependente de Anticorpos/genética , Antígenos de Diferenciação/genética , Neoplasias da Mama/genética , Genótipo , Imunoterapia/métodos , Neutrófilos/fisiologia , Receptores de IgG/genética , Receptores Imunológicos/genética , Antígenos de Diferenciação/metabolismo , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Neoplasias da Mama/imunologia , Antígeno CD47/metabolismo , Linhagem Celular Tumoral , Feminino , Predisposição Genética para Doença , Humanos , Desequilíbrio de Ligação , Polimorfismo Genético , Receptor ErbB-2/imunologia , Receptores de IgG/metabolismo , Receptores Imunológicos/metabolismo , Trastuzumab/farmacologia , Trastuzumab/uso terapêutico , Resultado do TratamentoRESUMO
Abs of the IgG isotype are glycosylated in their Fc domain at a conserved asparagine at position 297. Removal of the core fucose of this glycan greatly increases the affinity for FcγRIII, resulting in enhanced FcγRIII-mediated effector functions. Normal plasma IgG contains â¼94% fucosylated Abs, but alloantibodies against, for example, Rhesus D (RhD) and platelet Ags frequently have reduced fucosylation that enhances their pathogenicity. The increased FcγRIII-mediated effector functions have been put to use in various afucosylated therapeutic Abs in anticancer treatment. To test the functional consequences of Ab fucosylation, we produced V-gene-matched recombinant anti-RhD IgG Abs of the four different subclasses (IgG1-4) with and without core fucose (i.e., 20% fucose remaining). Binding to all human FcγR types and their functional isoforms was assessed with surface plasmon resonance. All hypofucosylated anti-RhD IgGs of all IgG subclasses indeed showed enhanced binding affinity for isolated FcγRIII isoforms, without affecting binding affinity to other FcγRs. In contrast, when testing hypofucosylated anti-RhD Abs with FcγRIIIa-expressing NK cells, a 12- and 7-fold increased erythrocyte lysis was observed with the IgG1 and IgG3, respectively, but no increase with IgG2 and IgG4 anti-RhD Abs. Notably, none of the hypofucosylated IgGs enhanced effector function of macrophages, which, in contrast to NK cells, express a complex set of FcγRs, including FcγRIIIa. Our data suggest that the beneficial effects of afucosylated biologicals for clinical use can be particularly anticipated when there is a substantial involvement of FcγRIIIa-expressing cells, such as NK cells.
Assuntos
Fucose/química , Imunoglobulina G/química , Imunoglobulina G/imunologia , Receptores de IgG/imunologia , Citotoxicidade Celular Dependente de Anticorpos , Fucose/imunologia , Fucose/metabolismo , Proteínas Ligadas por GPI/química , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/imunologia , Glicosilação , Humanos , Fragmentos Fc das Imunoglobulinas/química , Fragmentos Fc das Imunoglobulinas/imunologia , Imunoglobulina G/isolamento & purificação , Imunoglobulina G/metabolismo , Células Matadoras Naturais/imunologia , Macrófagos/imunologia , Ligação Proteica , Receptores de IgG/química , Receptores de IgG/genética , Sistema do Grupo Sanguíneo Rh-Hr/imunologia , Ressonância de Plasmônio de SuperfícieRESUMO
Neutrophils play an important role in cancer. This does not only relate to the well-established prognostic value of the presence of neutrophils, either in the blood or in tumor tissue, in the context of cancer progression or for the monitoring of therapy, but also to their active role in the progression of cancer. In the current review, we describe what is known in general about the role of neutrophils in cancer. What is emerging is a complex, rather heterogeneous picture with both pro- and anti-tumorigenic roles, which apparently differs with cancer type and disease stage. Furthermore, we will discuss the well-known role of neutrophils as myeloid-derived suppressor cells (MDSC), and also on the role of neutrophils as important effector cells during antibody therapy in cancer. It is clear that neutrophils contribute substantially to cancer progression in multiple ways, and this includes both direct effects on the cancer cells and indirect effect on the tumor microenvironment. While in many cases neutrophils have been shown to promote tumor progression, for instance by acting as MDSC, there are also protective effects, particularly when antibody immunotherapy is performed. A better understanding of the role of neutrophils is likely to provide opportunities for immunomodulation and for improving the treatment of cancer patients.