RESUMO
The assembly of tissue-damaging membrane attack complexes (MACs; C5b-9) is a major mechanism by which excessive complement activation causes diseases. We previously developed a mouse anti-human C6 monoclonal antibody (mAb) 1C9 that selectively inhibits the assembly of MACs in human and non-human primates. In this project, we found that 1C9 also cross-reacted with rat and guinea pig C6, and determined its binding domains on C6 using different truncated C6 proteins. We then humanized the anti-C6 mAb by molecular modeling and complementarity-determining region grafting. After screening a library of 276 humanized variants with different combinations of humanized light and heavy chains in biophysical assays, we identified clone 3713 with the best developability profile, and an increased affinity against C6 when compared with the parental 1C9 mAb. This humanized 3713 mAb inhibited human, monkey, and rat complement-mediated hemolysis in vitro, and more importantly, it significantly reduced complement-mediated hemolysis in vivo in rats. These results demonstrated the successful humanization of the anti-C6 mAb and suggested that the humanized 3713 mAb could be further developed as a new therapeutic that selectively targets MAC for certain complement-mediated pathological conditions.
Assuntos
Anticorpos Monoclonais , Complemento C6 , Hemólise , Animais , Humanos , Ratos , Cobaias , Camundongos , Hemólise/efeitos dos fármacos , Hemólise/imunologia , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Complemento C6/imunologia , Anticorpos Monoclonais Humanizados/imunologia , Anticorpos Monoclonais Humanizados/farmacologia , Ativação do Complemento/imunologia , Ativação do Complemento/efeitos dos fármacos , Complexo de Ataque à Membrana do Sistema Complemento/imunologia , Reações Cruzadas/imunologiaRESUMO
Anti-D immunoglobulin (Anti-D Ig) prophylaxis prevents haemolytic disease of the fetus and newborn. Monoclonal IgG anti-Ds (mAb-Ds) would enable unlimited supplies but have differed in efficacy in FcγRIIIa-mediated ADCC assays and clinical trials. Structural variations of the oligosaccharide chains of mAb-Ds are hypothesised to be responsible. Quantitative data on 12 Fc-glycosylation features of 23 mAb-Ds (12 clones, 5 produced from multiple cell lines) and one blood donor-derived anti-D Ig were obtained by HPLC and mass spectrometry using 3 methods. Glycosylation of mAb-Ds from human B-lymphoblastoid cell lines (B) was similar to anti-D Ig although fucosylation varied, affecting ADCC activity. In vivo, two B mAb-Ds with 77-81% fucosylation cleared red cells and prevented D-immunisation but less effectively than anti-D Ig. High fucosylation (>89%) of mouse-human heterohybridoma (HH) and Chinese hamster ovary (CHO) mAb-Ds blocked ADCC and clearance. Rat YB2/0 mAb-Ds with <50% fucosylation mediated more efficient ADCC and clearance than anti-D Ig. Galactosylation of B mAb-Ds was 57-83% but 15-58% for rodent mAb-Ds. HH mAb-Ds had non-human sugars. These data reveal high galactosylation like anti-D Ig (>60%) together with lower fucosylation (<60%) as safe features of mAb-Ds for mediating rapid red cell clearance at low doses, to enable effective, inexpensive prophylaxis.
Assuntos
Anticorpos Monoclonais/imunologia , Eritroblastose Fetal/terapia , Imunoglobulina G/imunologia , Imunoglobulina rho(D)/imunologia , Animais , Anticorpos Monoclonais/metabolismo , Anticorpos Monoclonais/uso terapêutico , Linhagem Celular , Cricetulus , Fucose/metabolismo , Galactose/metabolismo , Glicosilação , Humanos , Hibridomas/imunologia , Imunoglobulina G/metabolismo , Camundongos , Ácido N-Acetilneuramínico/metabolismo , Ratos , Imunoglobulina rho(D)/metabolismo , Imunoglobulina rho(D)/uso terapêutico , Resultado do TratamentoRESUMO
Neonatal Fc-receptor (FcRn), the major histocompatibility complex (MHC) class I-like Fc-receptor, transports immunoglobuline G (IgG) across cell layers, extending IgG half-life in circulation and providing newborns with humoral immunity. IgG1 and IgG2 have similar half-lives, yet IgG2 displays lower foetal than maternal concentration at term, despite all known FcRn binding residues being preserved between IgG1 and IgG2. We investigated FcRn mediated transcytosis of VH-matched IgG1 and IgG2 and mutated variants thereof lacking Fc-gamma receptor (FcγR) binding in human cells expressing FcRn. We observed that FcγR binding was not required for transport and that FcRn transported less IgG2 than IgG1. Transport of IgG1 with a shortened lower hinge (ΔGly236, absent in germline IgG2), was reduced to levels equivalent to IgG2. Conversely, transport of IgG2 + Gly236 was increased to IgG1 levels. Gly236 is not a contact residue between IgG and FcRn, suggesting that its absence leads to an altered conformation of IgG, possibly due to a less flexible Fab, positioned closer to the Fc portion. This may sterically hinder FcRn binding and transport. We conclude that the lack of Gly236 is sufficient to explain the reduced FcRn-mediated IgG2 transcytosis and accounts for the low maternal/fetal IgG2 ratio at term.
Assuntos
Glicina/genética , Antígenos de Histocompatibilidade Classe I/metabolismo , Imunidade Materno-Adquirida , Imunoglobulina G/metabolismo , Receptores Fc/metabolismo , Transcitose , Sítios de Ligação/genética , Linhagem Celular Tumoral , Feminino , Sangue Fetal , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Imunoglobulina G/genética , Imunoglobulina G/imunologia , Recém-Nascido , Leucócitos , Troca Materno-Fetal , Mutação , Circulação Placentária , Gravidez , Cultura Primária de Células , Receptores Fc/imunologiaRESUMO
Modification of immunoglobulin G (IgG) 1 proteins in cancer treatment is a rapidly growing field of research. Antibody-drug conjugates (ADCs) exploit the targeted nature of this immunotherapy by conjugating highly potent drugs to antibodies, allowing for effective transport of cargo(s) to cancerous cells. Of the many bioconjugation strategies now available for the formation of highly homogeneous ADCs, disulfide modification is considered an effective, low-cost, and widely accepted method for modifying IgG1s for improved clinical benefit. However, little is known about how disulfide modification impacts clinically relevant fragment crystallizable (Fc) region interactions. Although often overlooked as a secondary ADC function, Fc interactions could prove key in the rational design of cancer cell-targeting ADCs through consideration of potent mechanisms such as antibody-dependent cellular cytotoxicity (ADCC). This work explores different IgG1 disulfide modification techniques and the effect they have on quantifiable secondary IgG1 Fc interactions (e.g., CD16a and FcRn). The solvent accessible disulfide residues of trastuzumab, a clinically relevant IgG1, were modified to provide a range of bioconjugates with differing amounts of interchain covalent linkages. It was found that by natively rebridging the IgG1 model, all tested Fc functionalities were not significantly affected. Additionally, in non Fc-specific biophysical experiments (e.g., thermal stability/aggregation), the natively rebridged species provided an exceptional profile, showing no significant change from the tested native antibody. Conjugates with significant disruption of the covalent connectivity of IgG1 chains resulted in a suboptimal Fc profile (CD16a kinetics or ADCC activity), in addition to substandard non Fc-specific attributes (thermal stability). These results advocate native disulfide rebridging as an excellent synthetic strategy for forming homogeneous IgG1 bioconjugates, with no reported negative impact on biophysical profile relative to the native antibody.
Assuntos
Dissulfetos/química , Fragmentos Fc das Imunoglobulinas/química , Imunoglobulina G/química , Citotoxicidade Celular Dependente de Anticorpos , Fenômenos Biofísicos , Humanos , Imunoconjugados/administração & dosagem , Imunoconjugados/química , Receptores de IgG/química , Trastuzumab/químicaRESUMO
We have previously generated human IgG1 antibodies that were engineered for reduced binding to the classical Fcγ receptors (FcγRI-III) and C1q, thereby eliminating their destructive effector functions (constant region G1Δnab). In their potential use as blocking agents, favorable binding to the neonatal Fc receptor (FcRn) is important to preserve the long half-life typical of IgG. An ability to cross the placenta, which is also mediated, at least in part, by FcRn is desirable in some indications, such as feto-maternal alloimmune disorders. Here, we show that G1Δnab mutants retain pH-dependent binding to human FcRn but that the amino acid alterations reduce the affinity of the IgG1:FcRn interaction by 2.0-fold and 1.6-fold for the two antibodies investigated. The transport of the modified G1Δnab mutants across monolayers of human cell lines expressing FcRn was approximately 75% of the wild-type, except that no difference was observed with human umbilical vein endothelial cells. G1Δnab mutation also reduced transport in an ex vivo placenta model. In conclusion, we demonstrate that, although the G1Δnab mutations are away from the FcRn-binding site, they have long-distance effects, modulating FcRn binding and transcellular transport. Our findings have implications for the design of therapeutic human IgG with tailored effector functions.
Assuntos
Antígenos de Histocompatibilidade Classe I/metabolismo , Imunoglobulina G/metabolismo , Placenta/metabolismo , Receptores Fc/metabolismo , Células Cultivadas , Feminino , Antígenos de Histocompatibilidade Classe I/química , Antígenos de Histocompatibilidade Classe I/genética , Células Endoteliais da Veia Umbilical Humana , Humanos , Imunoglobulina G/química , Imunoglobulina G/genética , Cinética , Troca Materno-Fetal/fisiologia , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Mutação , Gravidez , Ligação Proteica , Receptores Fc/química , Receptores Fc/genética , Receptores de IgG/metabolismoRESUMO
Invasive non-typhoidal Salmonella are a common cause of invasive disease in immuno-compromised individuals and in children. Multi-drug resistance poses challenges to disease control, with a critical need for effective vaccines. Flagellin is an attractive vaccine candidate due to surface exposure and high epitope copy number, but its potential as a target for opsonophacytic antibodies is unclear. We examined the effect of targeting flagella with different classes of IgG on the interaction between Salmonella Typhimurium and a human phagocyte-like cell line, THP-1. We tagged the FliC flagellar protein with a foreign CD52 mimotope (TSSPSAD) and bacteria were opsonized with a panel of humanised CD52 antibodies with the same antigen-binding V-region, but different constant regions. We found that IgG binding to flagella increases bacterial phagocytosis and reduces viable intracellular bacterial numbers. Opsonisation with IgG3, followed by IgG1, IgG4, and IgG2, resulted in the highest level of bacterial uptake and in the highest reduction in the intracellular load of viable bacteria. Taken together, our data provide proof-of-principle evidence that targeting flagella with antibodies can increase the antibacterial function of host cells, with IgG3 being the most potent subclass. These data will assist the rational design of urgently needed, optimised vaccines against iNTS disease.
RESUMO
Aberrant angiogenesis is implicated in diseases affecting nearly 10% of the world's population. The most widely used anti-angiogenic drug is bevacizumab, a humanized IgG1 monoclonal antibody that targets human VEGFA. Although bevacizumab does not recognize mouse Vegfa, it inhibits angiogenesis in mice. Here we show bevacizumab suppressed angiogenesis in three mouse models not via Vegfa blockade but rather Fc-mediated signaling through FcγRI (CD64) and c-Cbl, impairing macrophage migration. Other approved humanized or human IgG1 antibodies without mouse targets (adalimumab, alemtuzumab, ofatumumab, omalizumab, palivizumab and tocilizumab), mouse IgG2a, and overexpression of human IgG1-Fc or mouse IgG2a-Fc, also inhibited angiogenesis in wild-type and FcγR humanized mice. This anti-angiogenic effect was abolished by Fcgr1 ablation or knockdown, Fc cleavage, IgG-Fc inhibition, disruption of Fc-FcγR interaction, or elimination of FcRγ-initated signaling. Furthermore, bevacizumab's Fc region potentiated its anti-angiogenic activity in humanized VEGFA mice. Finally, mice deficient in FcγRI exhibited increased developmental and pathological angiogenesis. These findings reveal an unexpected anti-angiogenic function for FcγRI and a potentially concerning off-target effect of hIgG1 therapies.
RESUMO
We previously produced a recombinant version of the human anti-RhD antibody Fog-1 in the rat myeloma cell line, YB2/0. When human, autologous RhD-positive red blood cells (RBC) were sensitised with this IgG1 antibody and re-injected, they were cleared much more rapidly from the circulation than had been seen earlier with the original human-mouse heterohybridoma-produced Fog-1. Since the IgG have the same amino acid sequence, this disparity is likely to be due to alternative glycosylation that results from the rat and mouse cell lines. By comparing the in vitro properties of YB2/0-produced Fog-1 IgG1 and the same antibody produced in the mouse myeloma cell line NS0, we now have a unique opportunity to pinpoint the cause of the difference in ability to clear RBC in vivo. Using transfected cell lines that express single human FcγR, we showed that IgG1 made in YB2/0 and NS0 cell lines bound equally well to receptors of the FcγRI and FcγRII classes but that the YB2/0 antibody was superior in FcγRIII binding. When measuring complexed IgG binding, the difference was 45-fold for FcγRIIIa 158F, 20-fold for FcγRIIIa 158V and approximately 40-fold for FcγRIIIb. The dissimilarity was greater at 100-fold in monomeric IgG binding assays with FcγRIIIa. When used to sensitise RBC, the YB2/0 IgG1 generated 100-fold greater human NK cell antibody-dependent cell-mediated cytotoxicity and had a 103-fold advantage over the NS0 antibody in activating NK cells, as detected by CD54 levels. In assays of monocyte activation and macrophage adherence/phagocytosis, where FcγRI plays major roles, RBC sensitised with the two antibodies produced much more similar results. Thus, the alternative glycosylation profiles of the Fog-1 antibodies affect only FcγRIII binding and FcγRIII-mediated functions. Relating this to the in vivo studies confirms the importance of FcγRIII in RBC clearance.
Assuntos
Anticorpos/imunologia , Eritrócitos/metabolismo , Imunoglobulina G/imunologia , Receptores de IgG/metabolismo , Animais , Linhagem Celular , Eritrócitos/imunologia , Humanos , Camundongos , RatosRESUMO
G1Δnab is a mutant human IgG1 constant region with a lower ability to interact with FcγR than the natural IgG constant regions. Radiolabelled RBCs and platelets sensitised with specific G1Δnab Abs were cleared more slowly from human circulation than IgG1-sensitised counterparts. However, non-destructive splenic retention of G1Δnab-coated RBCs required investigation and plasma radioactivities now suggest this also occurred for platelets sensitised with an IgG1/G1Δnab mixture. In vitro assays with human cells showed that G1Δnab-sensitised RBCs did not cause FcγRI-mediated monocyte activation, FcγRIIIa-mediated antibody-dependent cell-mediated cytotoxicity (ADCC) or macrophage phagocytosis although they did adhere to macrophages. Thus, FcγRII was implicated in the adhesion despite the Δnab mutation reducing the already low-affinity binding to this receptor class. Additional contacts via P-selectin enhance the interaction of sensitised platelets with monocytes and this system provided evidence of FcγRII-dependent activation by G1Δnab. These results emphasise the physiological relevance of low-affinity interactions: It appears that FcγRII interactions of G1Δnab allowed splenic retention of G1Δnab-coated RBCs with inhibitory FcγRIIb binding preventing RBC destruction and that FcγRIIb engagement by G1Δnab on IgG1/G1Δnab-sensitised platelets overcame activation by IgG1. Considering therapeutic blocking Abs, G1Δnab offers lower FcγR binding and a greater bias towards inhibition than IgG2 and IgG4 constant regions.
Assuntos
Plaquetas/imunologia , Plaquetas/metabolismo , Eritrócitos/imunologia , Eritrócitos/metabolismo , Imunoglobulina G/imunologia , Receptores de IgG/metabolismo , Antígenos de Plaquetas Humanas/imunologia , Sobrevivência Celular/imunologia , Sobrevivência Celular/efeitos da radiação , Humanos , Imunoglobulina G/metabolismo , Integrina beta3 , Monócitos/imunologia , Proteínas Nucleares/imunologia , Ligação Proteica , Sistema do Grupo Sanguíneo Rh-Hr/imunologia , Fatores de Transcrição/imunologiaRESUMO
BACKGROUND: Allergen recognition by dendritic cells (DCs) is a key event in the allergic cascade leading to production of IgE antibodies. C-type lectins, such as the mannose receptor and DC-SIGN, were recently shown to play an important role in the uptake of the house dust mite glycoallergen Der p 1 by DCs. In addition to mannose receptor (MR) and DC-SIGN the high and low affinity IgE receptors, namely FcεRI and FcεRII (CD23), respectively, have been shown to be involved in allergen uptake and presentation by DCs. OBJECTIVES: This study aims at understanding the extent to which IgE- and IgG-facilitated Der p 1 uptake by DCs influence T cell polarisation and in particular potential bias in favour of Th2. We have addressed this issue by using two chimaeric monoclonal antibodies produced in our laboratory and directed against a previously defined epitope on Der p 1, namely human IgE 2C7 and IgG1 2C7. RESULTS: Flow cytometry was used to establish the expression patterns of IgE (FcεRI and FcεRII) and IgG (FcγRI) receptors in relation to MR on DCs. The impact of FcεRI, FcεRII, FcγRI and mannose receptor mediated allergen uptake on Th1/Th2 cell differentiation was investigated using DC/T cell co-culture experiments. Myeloid DCs showed high levels of FcεRI and FcγRI expression, but low levels of CD23 and MR, and this has therefore enabled us to assess the role of IgE and IgG-facilitated allergen presentation in T cell polarisation with minimal interference by CD23 and MR. Our data demonstrate that DCs that have taken up Der p 1 via surface IgE support a Th2 response. However, no such effect was demonstrable via surface IgG. CONCLUSIONS: IgE bound to its high affinity receptor plays an important role in Der p 1 uptake and processing by peripheral blood DCs and in Th2 polarisation of T cells.
Assuntos
Alérgenos/imunologia , Apresentação de Antígeno/imunologia , Células Dendríticas/imunologia , Imunoglobulina E/imunologia , Alérgenos/metabolismo , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Antígenos de Dermatophagoides/imunologia , Antígenos de Dermatophagoides/metabolismo , Proteínas de Artrópodes/imunologia , Proteínas de Artrópodes/metabolismo , Células Cultivadas , Cisteína Endopeptidases/imunologia , Cisteína Endopeptidases/metabolismo , Citocinas/imunologia , Citocinas/metabolismo , Células Dendríticas/metabolismo , Citometria de Fluxo , Humanos , Imunoglobulina E/metabolismo , Imunoglobulina G/imunologia , Imunoglobulina G/metabolismo , Lectinas Tipo C/imunologia , Lectinas Tipo C/metabolismo , Ativação Linfocitária/imunologia , Mananas/imunologia , Mananas/farmacologia , Receptor de Manose , Lectinas de Ligação a Manose/imunologia , Lectinas de Ligação a Manose/metabolismo , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/imunologia , Receptores de Superfície Celular/imunologia , Receptores de Superfície Celular/metabolismo , Receptores de IgE/imunologia , Receptores de IgE/metabolismo , Receptores de IgG/imunologia , Receptores de IgG/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Células Th1/imunologia , Células Th1/metabolismo , Células Th2/imunologia , Células Th2/metabolismo , Fatores de TempoRESUMO
UNLABELLED: Cryptococcus neoformans is one of the most common causes of fungal disease in HIV-infected persons, but not all of those who are infected develop cryptococcal disease (CD). Although CD4(+) T cell deficiency is a risk factor for HIV-associated CD, polymorphisms of phagocytic Fc gamma receptors (FCGRs) have been linked to CD risk in HIV-uninfected persons. To investigate associations between FCGR2A 131 H/R and FCGR3A 158 F/V polymorphisms and CD risk in HIV-infected persons, we performed PCR-based genotyping on banked samples from 164 men enrolled in the Multicenter AIDS Cohort Study (MACS): 55 who were HIV infected and developed CD and a matched control group of 54 who were HIV infected and 55 who were HIV uninfected. Using additive and allelic statistical models for analysis, the high-affinity FCGR3A 158V allele was significantly associated with CD status after adjusting for race/ethnicity (odds ratio [OR], 2.1; P = 0.005), as was the FCGR3A 158 VV homozygous genotype after adjusting for race/ethnicity, rate of CD4(+) T cell decline, and nadir CD4(+) T cell count (OR, 21; P = 0.005). No associations between CD and FCGR2A 131 H/R polymorphism were identified. In binding studies, human IgG (hIgG)-C. neoformans complexes exhibited more binding to CHO-K1 cells expressing FCGR3A 158V than to those expressing FCGR3A 158F, and in cytotoxicity assays, natural killer (NK) cells expressing FCGR3A 158V induced more C. neoformans-infected monocyte cytotoxicity than those expressing FCGR3A 158F. Together, these results show an association between the FCGR3A 158V allele and risk for HIV-associated CD and suggest that this polymorphism could promote C. neoformans pathogenesis via increased binding of C. neoformans immune complexes, resulting in increased phagocyte cargo and/or immune activation. IMPORTANCE: HIV-associated CD4(+) T cell deficiency is a sine qua non for HIV-associated cryptococcal disease (CD), but not all patients with CD4(+) T cell deficiency develop CD despite serological evidence of previous infection. At present, there are no biomarkers that predict HIV-associated CD risk. The goal of our study was to understand whether Fc gamma receptor (FCGR) polymorphisms that have been shown to portend CD risk in HIV-uninfected people are associated with CD risk in HIV-infected people. Such biomarkers could identify those who would benefit most from targeted prophylaxis and/or earlier treatment, particularly in sub-Saharan Africa, where there are nearly a million cases of HIV-associated CD annually. A biomarker of risk could also identify potential candidates for immunization, should there be a vaccine for Cryptococcus neoformans.
Assuntos
Infecções Oportunistas Relacionadas com a AIDS/genética , Criptococose/genética , Polimorfismo Genético , Receptores de IgG/genética , Infecções Oportunistas Relacionadas com a AIDS/imunologia , Infecções Oportunistas Relacionadas com a AIDS/microbiologia , Adulto , Citotoxicidade Celular Dependente de Anticorpos , Contagem de Linfócito CD4 , Estudos de Coortes , Criptococose/imunologia , Criptococose/microbiologia , Cryptococcus neoformans , Genótipo , Humanos , Células Matadoras Naturais/imunologia , Masculino , Pessoa de Meia-Idade , Mutação de Sentido Incorreto , Receptores de IgG/imunologia , Fatores de RiscoRESUMO
Fetomaternal alloimmune thrombocytopenia, caused by the maternal generation of antibodies against fetal human platelet antigen-1a (HPA-1a), can result in intracranial hemorrhage and intrauterine death. We have developed a therapeutic human recombinant high-affinity HPA-1a antibody (B2G1Δnab) that competes for binding to the HPA-1a epitope but carries a modified constant region that does not bind to Fcγ receptors. In vitro studies with a range of clinical anti-HPA-1a sera have shown that B2G1Δnab blocks monocyte chemiluminescence by >75%. In this first-in-man study, we demonstrate that HPA-1a1b autologous platelets (matching fetal phenotype) sensitized with B2G1Δnab have the same intravascular survival as unsensitized platelets (190 hours), while platelets sensitized with a destructive immunoglobulin G1 version of the antibody (B2G1) are cleared from the circulation in 2 hours. Mimicking the situation in fetuses receiving B2G1Δnab as therapy, we show that platelets sensitized with a combination of B2G1 (representing destructive HPA-1a antibody) and B2G1Δnab survive 3 times as long in circulation compared with platelets sensitized with B2G1 alone. This confirms the therapeutic potential of B2G1Δnab. The efficient clearance of platelets sensitized with B2G1 also opens up the opportunity to carry out studies of prophylaxis to prevent alloimmunization in HPA-1a-negative mothers.
Assuntos
Anticorpos/uso terapêutico , Proteínas Recombinantes/uso terapêutico , Trombocitopenia Neonatal Aloimune/tratamento farmacológico , Antígenos de Plaquetas Humanas/imunologia , Plaquetas/imunologia , Vasos Sanguíneos/patologia , Sobrevivência Celular/imunologia , Feminino , Citometria de Fluxo , Humanos , Imunoglobulina G/sangue , Integrina beta3 , Masculino , Proteínas Mutantes/imunologia , Software , Trombocitopenia Neonatal Aloimune/sangue , Trombocitopenia Neonatal Aloimune/imunologiaRESUMO
Several classes and multiple subclasses of immunoglobulins are produced towards protein and polysaccharide antigens in response to Salmonella infection and play a key role in protection against systemic disease. The targeting of Salmonella to Fc receptors (FcR) on phagocytes is a key step in the antibody-mediated antibacterial functions of host cells. We wished to compare the relative efficiency of different human IgG subclasses, which targeted the Salmonella enterica OmpA surface protein in modulating the interaction of bacteria with human phagocytes. To this end, we developed a novel system by tagging OmpA with a foreign CD52 mimotope (TSSPSAD) and opsonizing the bacteria with a panel of humanized CD52 antibodies that share the same antigen-binding V-region, but have constant regions of different subclasses. Our data revealed that opsonization with all the IgG subclasses increases Salmonella uptake by human phagocytes. IgG3 resulted in the highest level of bacterial uptake and the highest average bacterial load per infected cell, which was closely followed by IgG1, then IgG4 and lastly IgG2. Phagocytosis mediated by IgG1, IgG3 and IgG4 had a higher dependency on FcγRI than FcγRIIA, whereas IgG2-mediated phagocytosis required FcγRIIA more than FcγRI. The results show that IgG binding to OmpA increases the uptake of Salmonella by human phagocytic cells and that the efficiency of this process depends both on the subclass of the IgG and the type of FcR that is available for antibody binding.
Assuntos
Imunoglobulina G/imunologia , Isotipos de Imunoglobulinas/imunologia , Monócitos/imunologia , Fagócitos , Receptores de IgG/imunologia , Infecções por Salmonella/imunologia , Salmonella typhimurium/imunologia , Imunofluorescência , Humanos , Monócitos/microbiologiaRESUMO
The efficacy of a therapeutic IgG molecule may be as dependent on the optimisation of the constant region to suit its intended indication as on the selection of its variable regions. A crucial effector function to be maximised or minimised is antibody-dependent cell-mediated cytotoxicity by natural killer cells. Traditional assays of ADCC activity suffer from considerable inter-donor and intra-donor variability, which makes the measurement of antibody binding to human FcgammaRIIIa, the key receptor for ADCC, an attractive alternative method of assessment. Here, we describe the development of cell lines and assays for this purpose. The transmembrane receptor, FcgammaRIIIa, requires co-expression with signal transducing subunits to prevent its degradation, unlike the homologous receptor FcgammaRIIIb that is expressed as a GPI-anchored molecule. Therefore, to simplify the production of cell lines as reliable assay components, we expressed FcgammaRIIIa as a GPI-anchored molecule. Separate, stable CHO cell lines that express either the 158F or the higher-affinity 158V allotype of FcgammaRIIIa were isolated using fluorescence-activated cell sorting. The identities of the expressed receptors were confirmed using a panel of monoclonal antibodies that distinguish between subclasses and allotypes of FcgammaRIII and the cell lines were shown to have slightly higher levels of receptor than FcgammaRIII-positive peripheral blood mononuclear cells. Because the affinity of FcgammaRIIIa for IgG is intermediate amongst the receptors that bind IgG, we were able to use these cell lines to develop flow cytometric assays to measure the binding of both complexed and monomeric immunoglobulin. Thus, by choosing the appropriate method, weakly- or strongly-binding IgG can be efficiently compared. We have quantified the difference in the binding of wildtype IgG1 and IgG3 molecules to the two functional allotypes of the receptor and report that the FcgammaRIIIa-158V-antibody interaction is 3- to 4-fold stronger that the interaction with FcgammaRIIIa-158F. Overall, these robust assays should be valuable for batch-testing clinical material as well as providing tools for improving the design of therapeutic IgG.
Assuntos
Anticorpos Monoclonais/metabolismo , Bioensaio , Imunoglobulina G/metabolismo , Receptores de IgG/metabolismo , Sequência de Aminoácidos , Animais , Citotoxicidade Celular Dependente de Anticorpos , Sequência de Bases , Células CHO , Separação Celular , Cricetinae , Cricetulus , Citometria de Fluxo , Proteínas Ligadas por GPI , Glicosilfosfatidilinositóis/metabolismo , Humanos , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Dados de Sequência Molecular , Ligação Proteica , Receptores de IgG/genética , TransfecçãoRESUMO
Fetomaternal alloimmune thrombocytopenia (FMAIT) is caused by maternal generation of antibodies specific for paternal platelet antigens and can lead to fetal intracranial hemorrhage. A SNP in the gene encoding integrin beta3 causes a clinically important maternal-paternal antigenic difference; Leu33 generates the human platelet antigen 1a (HPA-1a), whereas Pro33 generates HPA-1b. As a potential treatment to prevent fetal intracranial hemorrhage in HPA-1a alloimmunized pregnancies, we generated an antibody that blocks the binding of maternal HPA-1a-specific antibodies to fetal HPA-1a1b platelets by combining a high-affinity human HPA-1a-specific scFv (B2) with an IgG1 constant region modified to minimize Fcgamma receptor-dependent platelet destruction (G1Deltanab). B2G1Deltanab saturated HPA-1a+ platelets and substantially inhibited binding of clinical HPA-1a-specific sera to HPA-1a+ platelets. The response of monocytes to B2G1Deltanab-sensitized platelets was substantially less than their response to unmodified B2G1, as measured by chemiluminescence. In addition, B2G1Deltanab inhibited chemiluminescence induced by B2G1 and HPA-1a-specific sera. In a chimeric mouse model, B2G1 and polyclonal Ig preparations from clinical HPA-1a-specific sera reduced circulating HPA-1a+ platelets, concomitant with transient thrombocytopenia. As the Deltanab constant region is uninformative in mice, F(ab')2 B2G1 was used as a proof of principle blocking antibody and prevented the in vivo platelet destruction seen with B2G1 and polyclonal HPA-1a-specific antibodies. These results provide rationale for human clinical studies.
Assuntos
Anticorpos/imunologia , Antígenos de Plaquetas Humanas/imunologia , Trombocitopenia Neonatal Aloimune/terapia , Anticorpos/metabolismo , Antígenos de Plaquetas Humanas/genética , Plaquetas/imunologia , Plaquetas/metabolismo , Feminino , Humanos , Imunoglobulina G/química , Imunoglobulina G/genética , Região Variável de Imunoglobulina/imunologia , Região Variável de Imunoglobulina/metabolismo , Recém-Nascido , Integrina beta3 , Modelos Moleculares , Mutação , Contagem de Plaquetas , Gravidez , Ligação Proteica , Receptores de IgG/imunologia , Receptores de IgG/metabolismo , Proteínas Recombinantes/imunologia , Trombocitopenia Neonatal Aloimune/etiologiaRESUMO
Alloimmune feto-maternal destruction of blood cells is thought to be mediated by binding of alloantibodies to Fc receptors on effector cells. Blocking the antigen using inert antibodies might prolong cell survival. We have performed a "proof of principle" study in volunteers to measure the intravascular survival of autologous red cells coated with human recombinant IgG antibody containing a novel constant region, G1Deltanab, devoid of in vitro cytotoxic activity. RhD-positive red blood cells (RBCs), labeled with chromium-51 or technetium-99m, were separately coated to equal levels with wild-type IgG1 or G1Deltanab anti-D antibody (Fog-1). After re-injection, there was complete, irreversible clearance of IgG1-coated RBCs by 200 minutes, concomitant with appearance of radiolabel in plasma. Gamma camera imaging revealed accumulation in spleen and, at higher coating levels, in liver. In contrast, clearance of G1Deltanab-coated cells was slower, incomplete, and transient, with whole blood counts falling to 7% to 38% injected dose by about 200 minutes before increasing to 12% to 67% thereafter. There was no appearance of plasma radiolabel and no hepatic accumulation. These findings suggest that G1Deltanab-coated RBCs were not hemolysed but temporarily sequestered in the spleen and that our approach merits investigation in larger studies.
Assuntos
Eritrócitos/citologia , Eritrócitos/imunologia , Imunoglobulina G/imunologia , Isoanticorpos/genética , Anticorpos Monoclonais/imunologia , Formação de Anticorpos , Contagem de Células Sanguíneas , Sobrevivência Celular , Contagem de Eritrócitos , Engenharia Genética , Humanos , Isoanticorpos/imunologia , Imunoglobulina rho(D)RESUMO
Human vascular adhesion protein-1 (VAP-1) is a homodimeric 170-kDa sialoglycoprotein that is expressed on the surface of endothelial cells and functions as a semicarbazide-sensitive amine oxidase and as an adhesion molecule. Blockade of VAP-1 has been shown to reduce leukocyte adhesion and transmigration in in vivo and in vitro models, suggesting that VAP-1 is a potential target for anti-inflammatory therapy. In this study we have constructed mouse-human chimeric antibodies by genetic engineering in order to circumvent the potential problems involved in using murine antibodies in man. Our chimeric anti-VAP-1 antibodies, which were designed to lack Fc-dependent effector functions, bound specifically to cell surface-expressed recombinant human VAP-1 and recognized VAP-1 in different cell types in tonsil. Furthermore, the chimeric antibodies prevented leukocyte adhesion and transmigration in vitro and in vivo. Hence, these chimeric antibodies have the potential to be used as a new anti-inflammatory therapy.
Assuntos
Amina Oxidase (contendo Cobre)/antagonistas & inibidores , Amina Oxidase (contendo Cobre)/fisiologia , Anticorpos Bloqueadores/uso terapêutico , Moléculas de Adesão Celular/antagonistas & inibidores , Moléculas de Adesão Celular/fisiologia , Inibição de Migração Celular , Mediadores da Inflamação/antagonistas & inibidores , Mediadores da Inflamação/fisiologia , Amina Oxidase (contendo Cobre)/genética , Amina Oxidase (contendo Cobre)/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Bloqueadores/genética , Sítios de Ligação de Anticorpos , Adesão Celular/imunologia , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/imunologia , Movimento Celular/imunologia , Endotélio Vascular/fisiologia , Citometria de Fluxo , Humanos , Mediadores da Inflamação/imunologia , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , RatosRESUMO
Varicella-Zoster virus (VZV) immune globulin (VZIG) derived from pooled human serum is currently used in immunotherapy of VZV-associated complications of chickenpox and shingles. We developed a mouse-human chimeric antibody against a VZV glycoprotein E (gE) epitope as a safer replacement for VZIG. Variable (V) heavy- and V kappa light-chain exons, derived from an anti-VZV gE antibody secreting mouse hybridoma cell line, were cloned into expression vectors containing an immunoglobulin promoter and enhancer, and human IgG1 or kappa constant (C) region genes. The expression vectors were cotransfected into mouse myeloma cell line (NSO), generating transformants that secreted chimeric human-mouse IgGs. The chimeric and the parent mouse antibody were indistinguishable in their antigen binding specificity. VZV gE chimeric antibody may prove to be a prophylactic antibody that could provide significant advantages over VZIG in having defined specificity, lessened possibility of contamination with viral pathogens, and consistent availability.
Assuntos
Anticorpos Monoclonais/genética , Herpesvirus Humano 3/metabolismo , Proteínas do Envelope Viral/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Sequência de Bases , Ensaio de Imunoadsorção Enzimática , Vetores Genéticos , Humanos , Hibridomas , Camundongos , Dados de Sequência Molecular , Mapeamento por Restrição , Análise de Sequência de DNARESUMO
Recombinant HPA-1a antibodies with Fc, mutated to remove destructive effector functions, have been developed as a potential therapy for fetomaternal alloimmune thrombocytopenia (FMAIT), via blockade of binding of human HPA-1a polyclonal antibodies to fetal HPA-1a1b platelets. We have assessed the effect of the IgG1 HPA-1a antibody B2G1 and two mutated derivatives in various functional assays in resting and agonist-stimulated platelets of the three HPA-1 genotypes. With HPA-1a1b platelets (fetal genotype), the antibodies did not activate signalling or CD62P expression in resting platelets, did not change in vitro bleeding time (IVBT), and did not inhibit platelet adhesion to collagen in flowing blood. Adhesion of HPA-1a1b platelets to fibrinogen was reduced by 20%, and aggregation induced by ADP by 50%, but collagen-related peptide (CRP-XL)-induced aggregation was normal. With HPA-1a1a platelets, aggregation to both ADP and CRP-XL was inhibited, with total blockade of adhesion to fibrinogen and of IVBT responses. Interestingly, a monovalent antibody fragment with identical specificity had no inhibitory effect on aggregation. In static adhesion assays using human alphaIIbbeta3 or alphaVbeta3 transfectants of HPA-1a (Leu(33)) phenotype, attachment to fibrinogen of the latter but not of the former was completely blocked by the HPA-1a antibodies. These observations are best explained by antibody-mediated blockade of the RGD binding site on beta3 by a mechanism of steric hindrance. As the effect on platelet function is modest with HPA-1a1b (fetal type) platelets, the mutated HPA-1a antibodies described here could be developed further for FMAIT therapy.
Assuntos
Anticorpos/farmacologia , Antígenos de Plaquetas Humanas/imunologia , Ativação Plaquetária/imunologia , Polimorfismo de Nucleotídeo Único/imunologia , Anticorpos/genética , Anticorpos/imunologia , Colágeno/metabolismo , Feminino , Fibrinogênio/metabolismo , Humanos , Imunoglobulina G/genética , Imunoglobulina G/farmacologia , Integrina alfaVbeta3/metabolismo , Integrina beta3/imunologia , Leucina , Testes de Função Plaquetária , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Gravidez , Engenharia de Proteínas , Proteínas RecombinantesRESUMO
We are investigating the interactions of recombinant human IgG antibodies with Fc receptors to enable selection of a constant region giving minimal depletion of antigen-bearing cells. Eight variant constant regions were made by substituting motifs between human IgG subclasses in the lower hinge region and/or a specially close loop of the CH2 domain. Mutations in the lower hinge region were shown to eliminate FcgammaRI binding and monocyte activation [Eur. J. Immunol. 29 (1999) 2613]. Here, we detail interactions with FcgammaRIIa of the 131R and 131H allotypes and FcgammaRIIb. Lower hinge mutations caused large reductions in binding whereas modification of residues 327, 330 and 331 had less dramatic effects. However, like the wildtype IgG subclass binding hierarchies, the effect of the mutations varied between different receptors. We identified IgG1 variants which react with the activating receptor, FcgammaRIIa, at least 10-fold less efficiently than wildtype IgG1 but whose binding to the inhibitory receptor, FcgammaRIIb, is only four-fold reduced. Manipulation of interactions with FcgammaRIIb separately from those with activating receptors provides potential for designing antibodies with novel and effective combinations of attributes. In addition, insight is gained into the evolution of functional differences in human IgG subclasses.