Across the animal kingdom, multivalency discriminates antibodies from all other immunoglobulin superfamily members. The evolutionary forces conserving multivalency above other structural hallmarks... Show moreAcross the animal kingdom, multivalency discriminates antibodies from all other immunoglobulin superfamily members. The evolutionary forces conserving multivalency above other structural hallmarks of antibodies remain, however, incompletely defined.Here, we engineer monovalent either Fc-competent or -deficient antibody formats to investigate mechanisms of protection of neutralizing antibodies (nAbs) and non-neutralizing antibodies (nnAbs) in virus-infected mice. Antibody bivalency enables the tethering of virions to the infected cell surface, inhibits the release of virions in cell culture, and suppresses viral loads in vivo independently of Fc gamma receptor (Fc gamma R) interactions. In return, monovalent antibody formats either do not inhibit virion release and fail to protect in vivo or their protective efficacy is largely Fc gamma R dependent. Protection in mice correlates with virus-release-inhibiting activity of nAb and nnAb rather than with their neutralizing capacity.These observations provide mechanistic insights into the evolutionary conservation of antibody bivalency and help refining correlates of nnAb protection for vaccine development. Show less
Legrain, S.; Su, D.; Gaignage, M.; Breukel, C.; Claassens, J.; Brouwers, C.; ... ; Coutelier, J.P. 2021
Infection with viruses, such as the lactate dehydrogenase-elevating virus (LDV), is known to trigger the onset of autoimmune anemia through the enhancement of the phagocytosis of autoantibody... Show moreInfection with viruses, such as the lactate dehydrogenase-elevating virus (LDV), is known to trigger the onset of autoimmune anemia through the enhancement of the phagocytosis of autoantibody-opsonized erythrocytes by activated macrophages. Type I interferon receptor-deficient mice show enhanced anemia, which suggests a protective effect of these cytokines, partly through the control of type II interferon production. The development of anemia requires the expression of Fc gamma receptors (Fc gamma R) I, III, and IV. Whereas LDV infection decreases Fc gamma R III expression, it enhances Fc gamma R I and IV expression in wild-type animals. The LDV-associated increase in the expression of Fc gamma R I and IV is largely reduced in type I interferon receptor-deficient mice, through both type II interferon-dependent and -independent mechanisms. Thus, the regulation of the expression of Fc gamma R I and IV, but not III, by interferons may partly explain the exacerbating effect of LDV infection on anemia that results from the enhanced phagocytosis of IgG autoantibody-opsonized erythrocytes. Show less
Braster, R.; Bogels, M.; Benonisson, H.; Wuhrer, M.; Plomp, R.; Bentlage, A.E.H.; ... ; Vidarsson, G. 2021
Simple SummaryCancer treatments are increasingly based on therapeutic antibodies to clear tumors. While in vivo mouse models are useful to predict effectiveness of human antibodies it is not... Show moreSimple SummaryCancer treatments are increasingly based on therapeutic antibodies to clear tumors. While in vivo mouse models are useful to predict effectiveness of human antibodies it is not completely clear how useful these models are to test antibodies engineered with enhanced effector functions designed for humans. One of the changes considered for many new antibody-based drugs is the removal of fucose (resulting in afucosylated IgG) which enhances IgG-Fc receptor (Fc gamma R) mediated effector functions in humans through Fc gamma RIIIa. Here we show that afucosylated human IgG1 also have enhanced effector functions against peritoneal metastasis of melanoma cells in mice through the evolutionary related mouse Fc gamma RIV. This shows that afucosylated human IgG is functionally recognized across species and shows that mouse tumor models can be used to assess the therapeutic potential of afucosylated IgG1.Promising strategies for maximizing IgG effector functions rely on the introduction of natural and non-immunogenic modifications. The Fc domain of IgG antibodies contains an N-linked oligosaccharide at position 297. Human IgG antibodies lacking the core fucose in this glycan have enhanced binding to human (Fc gamma R) IIIa/b, resulting in enhanced antibody dependent cell cytotoxicity and phagocytosis through these receptors. However, it is not yet clear if glycan-enhancing modifications of human IgG translate into more effective treatment in mouse models. We generated humanized hIgG1-TA99 antibodies with and without core-fucose. C57Bl/6 mice that were injected intraperitoneally with B16F10-gp75 mouse melanoma developed significantly less metastasis outgrowth after treatment with afucosylated hIgG1-TA99 compared to mice treated with wildtype hhIgG1-TA99. Afucosylated human IgG1 showed stronger interaction with the murine Fc gamma RIV, the mouse orthologue of human Fc gamma RIIIa, indicating that this glycan change is functionally conserved between the species. In agreement with this, no significant differences were observed in tumor outgrowth in Fc gamma RIV-/- mice treated with human hIgG1-TA99 with or without the core fucose. These results confirm the potential of using afucosylated therapeutic IgG to increase their efficacy. Moreover, we show that afucosylated human IgG1 antibodies act across species, supporting that mouse models can be suitable to test afucosylated antibodies. Show less
Yu, X.J.; Chan, H.T.C.; Fisher, H.; Penfold, C.A.; Kim, J.; Inzhelevskaya, T.; ... ; Cragg, M.S. 2020
Anti-CD40 monoclonal antibodies (mAbs) comprise agonists and antagonists, which display promising therapeutic activities in cancer and autoimmunity, respectively. We previously showed that epitope... Show moreAnti-CD40 monoclonal antibodies (mAbs) comprise agonists and antagonists, which display promising therapeutic activities in cancer and autoimmunity, respectively. We previously showed that epitope and isotype interact to deliver optimal agonistic anti-CD40 mAbs. The impact of Fc engineering on antagonists, however, remains largely unexplored. Here, we show that clinically relevant antagonists used for treating autoimmune conditions can be converted into potent FcyR-independent agonists with remarkable antitumor activity by isotype switching to hIgG2. One antagonist is converted to a super-agonist with greater potency than previously reported highly agonistic anti-CD40 mAbs. Such conversion is dependent on the unique disulfide bonding properties of the hIgG2 hinge. This investigation highlights the transformative capacity of the hIgG2 isotype for converting antagonists to agonists to treat cancer. Show less
Koelink, P.J.; Bloemendaal, F.M.; Li, B.F.; Westera, L.; Vogels, E.W.M.; Roest, M. van; ... ; Brink, G.R. van den 2020
Objective Macrophage interleukin (IL)-10 signalling plays a critical role in the maintenance of a regulatory phenotype that prevents the development of IBD. We have previously found that anti... Show moreObjective Macrophage interleukin (IL)-10 signalling plays a critical role in the maintenance of a regulatory phenotype that prevents the development of IBD. We have previously found that anti-tumour necrosis factor (TNF) monoclonal antibodies act through Fc gamma-receptor (Fc gamma R) signalling to promote repolarisation of proinflammatory intestinal macrophages to a CD206+ regulatory phenotype. The role of IL-10 in anti-TNF-induced macrophage repolarisation has not been examined.Design We used human peripheral blood monocytes and mouse bone marrow-derived macrophages to study IL-10 production and CD206+ regulatory macrophage differentiation. To determine whether the efficacy of anti-TNF was dependent on IL-10 signalling in vivo and in which cell type, we used the CD4+CD45Rb(high) T-cell transfer model in combination with several genetic mouse models.Results Anti-TNF therapy increased macrophage IL-10 production in an Fc gamma R-dependent manner, which caused differentiation of macrophages to a more regulatory CD206+ phenotype in vitro. Pharmacological blockade of IL-10 signalling prevented the induction of these CD206+ regulatory macrophages and diminished the therapeutic efficacy of anti-TNF therapy in the CD4+CD45Rb(high) T-cell transfer model of IBD. Using cell type-specific IL-10 receptor mutant mice, we found that IL-10 signalling in macrophages but not T cells was critical for the induction of CD206+ regulatory macrophages and therapeutic response to anti-TNF.Conclusion The therapeutic efficacy of anti-TNF in resolving intestinal inflammation is critically dependent on IL-10 signalling in macrophages. Show less
The use of Trastuzumab (Herceptin), a monoclonal antibody (mAb) targeting HER2/neu, results in an increased median survival in Her2(+) breast cancer patients. The tumour mutational burden and the... Show moreThe use of Trastuzumab (Herceptin), a monoclonal antibody (mAb) targeting HER2/neu, results in an increased median survival in Her2(+) breast cancer patients. The tumour mutational burden and the presence of tumour infiltrating lymphocytes (TILs) clearly correlate with response to trastuzumab. Here, we investigated if the immunogenicity of the transplantable rat-neu(+) tumour cell line (TUBO) derived from a BALB/c-NeuT primary tumour is associated with the response to anti-neu mAb therapy. We compared the TUBO tumour outgrowth and tumour infiltrating T cells in isogenic (BALB/c-NeuT) and non-isogenic (WT BALB/c) recipient mice. Furthermore, therapeutic efficacy of anti-neu mAb and the contribution of T cells were examined in both mouse strains. The outgrowth of untreated tumours was significantly better in BALB/c-NeuT than WT BALB/c mice. Moreover, tumour infiltrating T cells were more abundantly present in WT BALB/c than BALB/c-NeuT mice, showing that the TUBO tumour was more immunogenic in WT BALB/c mice. In TUBO tumour bearing WT BALB/c mice, anti-neu mAb therapy resulted in an increase of tumour infiltrating T cells and long-term survival. When T cells were depleted, this strong anti-tumour effect was reduced to an outgrowth delay. In contrast, in TUBO tumour bearing BALB/c-NeuT mice, treatment with anti-neu mAb resulted only in tumour outgrowth delay, both in the presence and absence of T cells. We concluded that in immunogenic tumours the response to anti-neu mAb therapy is enhanced by additional T cell involvement compared to the response to anti-neu mAb in non-immunogenic tumours. Show less
Infection of C57BL/6 mice with lymphocytic choriomeningitis virus (LCMV) strain Armstrong (Arm) induces an acute infection with rapid virus clearance by CD8(+) T cells independently of CD4(+) T... Show moreInfection of C57BL/6 mice with lymphocytic choriomeningitis virus (LCMV) strain Armstrong (Arm) induces an acute infection with rapid virus clearance by CD8(+) T cells independently of CD4(+) T cell help. Residual viral antigen may, however, persist for a prolonged time. Here, we demonstrate that mice that had been transiently depleted of CD4(+) T cells during acute LCMV Arm infection generated high levels of virus-specific IgG antibodies (Ab) after viral clearance. Robust induction of LCMV-specific IgG after transient CD4(+) T cell depletion was dependent on Fc gamma receptors but not on the complement receptors CD21/CD35. In contrast to the potent production of LCMV-specific IgG, the generation of LCMV-specific isotype-switched memory B cells after transient CD4(+) T cell depletion was considerably reduced. Moreover, mice depleted of CD4(+) T cells during acute infection were strongly impaired in generating a secondary LCMV-specific B cell response upon LCMV rechallenge. In conclusion, our data indicate that LCMV antigen depots after viral clearance were capable of inducing high levels of virus-specific IgG. They failed, however, to induce robust virus-specific B cell memory revealing a previously unappreciated dichotomy of specific Ab production and memory cell formation after priming with residual antigen. Show less
Benonisson, H.; Altintas, I.; Sluijter, M.; Verploegen, S.; Labrijn, A.F.; Schuurhuis, D.H.; ... ; Hall, T. van 2019
Dendritic cells (DCs) express Fc receptors (FcRs) for the binding immune complexes (ICs) consisting of IgG and antigens (Ags). IC-FcR interactions have been demonstrated to enhance activation and... Show moreDendritic cells (DCs) express Fc receptors (FcRs) for the binding immune complexes (ICs) consisting of IgG and antigens (Ags). IC-FcR interactions have been demonstrated to enhance activation and antigen-presenting functions of DCs. Utilizing Friend virus (FV), an oncogenic mouse retrovirus, we investigated the effect of IgG-opsonization of retroviral particles on the infection of DCs and the subsequent presentation of viral antigens by DCs to virus-specific CD8 T cells. We found that opsonization by virus-specific non-neutralizing IgG abrogated DC infection and as a consequence significantly reduced the capacity of DCs to activate virus-specific CD8 T cells. Effects of IgG-opsonization were mediated by the high-affinity FcR type I, CD64, expressed on DCs. Our results suggest that different opsonization patterns on the retroviral surface modulate infection and antigen-presenting functions of DCs, whereby, in contrast to complement, IgG reduces the capacity of DCs to activate cytotoxic T cell (CTL) responses. Show less
Immunomodulatory antibodies blocking interactions of coinhibitory receptors to their ligands such as CTLA-4, PD1 and PD-L1 on immune cells have shown impressive therapeutic efficacy in clinical... Show moreImmunomodulatory antibodies blocking interactions of coinhibitory receptors to their ligands such as CTLA-4, PD1 and PD-L1 on immune cells have shown impressive therapeutic efficacy in clinical studies. The therapeutic effect of these antibodies is mainly mediated by reactivating antitumor T cell immune responses. Detailed analysis of anti-CTLA4 antibody therapy revealed that an optimal therapeutic efficacy also requires binding to Fc receptors for IgG, Fc gamma R, mediating depletion of intratumoral regulatory T cells. Here, we investigated the role of Fc binding in anti-PD-L1 antibody therapy in the MC38 C57BL/6 and CT26 BALB/c colon adenocarcinoma tumor models. In the MC38 tumor model, all IgG subclasses anti-PD-L1 showed similar therapeutic efficacy when compared to each other in either wild-type mice or in mice deficient for all Fc gamma R. In contrast, in the CT26 tumor model, anti-PD-L1 mIgG2a, the IgG subclass with the highest affinity for activating Fc gamma R, showed stronger therapeutic efficacy than other IgG subclasses. This was associated with a reduction of a myeloid cell subset with high expression of PD-L1 in the tumor microenvironment. This subclass preference for mIgG2a was lost in C57BL/6 x BALB/c F1 mice, indicating that the genetic background of the host may determine the additional clinical benefit of the high affinity antibody subclasses. Based on these data, we conclude that Fc gamma R are not crucial for anti-PD-L1 antibody therapy but might play a role in some tumor models. Show less
