RESUMEN
The anti-CD28 superagonist antibody TGN1412 caused life-threatening cytokine release syndrome (CRS) in healthy volunteers, which had not been predicted by preclinical testing. T cells in fresh peripheral blood mononuclear cells (PBMCs) do not respond to soluble TGN1412 but do respond following high-density (HD) preculture. We show for the first time that this response is dependent on crystallizable fragment gamma receptor IIb (FcγRIIb) expression on monocytes. This was unexpected because, unlike B cells, circulating monocytes express little or no FcγRIIb. However, FcγRIIb expression is logarithmically increased on monocytes during HD preculture, and this upregulation is necessary and sufficient to explain TGN1412 potency after HD preculture. B-cell FcγRIIb expression is unchanged by HD preculture, but B cells can support TGN1412-mediated T-cell proliferation when added at a frequency higher than that in PBMCs. Although low-density (LD) precultured PBMCs do not respond to TGN1412, T cells from LD preculture are fully responsive when cocultured with FcγRIIb-expressing monocytes from HD preculture, which shows that they are fully able to respond to TGN1412-mediated activation. Our novel findings demonstrate that cross-linking by FcγRIIb is critical for the superagonist activity of TGN1412 after HD preculture, and this may contribute to CRS in humans because of the close association of FcγRIIb-bearing cells with T cells in lymphoid tissues.
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Anticuerpos Monoclonales Humanizados/química , Monocitos/citología , Receptores de IgG/metabolismo , Regulación hacia Arriba , Animales , Linfocitos B/citología , Antígenos CD28/metabolismo , Células CHO , Proliferación Celular , Técnicas de Cocultivo , Cricetinae , Cricetulus , Citocinas/metabolismo , Humanos , Leucocitos Mononucleares/citología , Linfocitos T/citología , Linfocitos T/inmunología , TransfecciónRESUMEN
FcγRs are key regulators of the immune response, capable of binding to the Fc portion of IgG Abs and manipulating the behavior of numerous cell types. Through a variety of receptors, isoforms, and cellular expression patterns, they are able to fine-tune and direct appropriate responses. Furthermore, they are key determinants of mAb immunotherapy, with mAb isotype and FcγR interaction governing therapeutic efficacy. Critical to understanding the biology of this complex family of receptors are reagents that are robust and highly specific for each receptor. In this study, we describe the development and characterization of mAb panels specific for both mouse and human FcγR for use in flow cytometry, immunofluorescence, and immunocytochemistry. We highlight key differences in expression between the two species and also patterns of expression that will likely impact on immunotherapeutic efficacy and translation of therapeutic agents from mouse to clinic.
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Anticuerpos Monoclonales/inmunología , Inmunoglobulina G/inmunología , Receptores de IgG/biosíntesis , Receptores de IgG/inmunología , Animales , Médula Ósea/inmunología , Células CHO , Línea Celular , Cricetinae , Cricetulus , Citometría de Flujo , Células HEK293 , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Tonsila Palatina/inmunología , Isoformas de Proteínas/genética , Isoformas de Proteínas/inmunología , Ratas , Ratas Wistar , Bazo/inmunologíaRESUMEN
Hematopoietic cells constitutively express CD31/PECAM1, a signaling adhesion receptor associated with controlling responses to inflammatory stimuli. Although expressed on CD4(+) T cells, its function on these cells is unclear. To address this, we have used a model of systemic Salmonella infection that induces high levels of T cell activation and depends on CD4(+) T cells for resolution. Infection of CD31-deficient (CD31KO) mice demonstrates that these mice fail to control infection effectively. During infection, CD31KO mice have diminished numbers of total CD4(+) T cells and IFN-γ-secreting Th1 cells. This is despite a higher proportion of CD31KO CD4(+) T cells exhibiting an activated phenotype and an undiminished capacity to prime normally and polarize to Th1. Reduced numbers of T cells reflected the increased propensity of naive and activated CD31KO T cells to undergo apoptosis postinfection compared with wild-type T cells. Using adoptive transfer experiments, we show that loss of CD31 on CD4(+) T cells alone is sufficient to account for the defective CD31KO T cell accumulation. These data are consistent with CD31 helping to control T cell activation, because in its absence, T cells have a greater propensity to become activated, resulting in increased susceptibility to become apoptotic. The impact of CD31 loss on T cell homeostasis becomes most pronounced during severe, inflammatory, and immunological stresses such as those caused by systemic Salmonella infection. This identifies a novel role for CD31 in regulating CD4 T cell homeostasis.
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Apoptosis/inmunología , Activación de Linfocitos/inmunología , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/inmunología , Infecciones por Salmonella/inmunología , Salmonella/inmunología , Células TH1/inmunología , Traslado Adoptivo , Animales , Apoptosis/genética , Supervivencia Celular/genética , Supervivencia Celular/inmunología , Inflamación/genética , Inflamación/inmunología , Inflamación/microbiología , Activación de Linfocitos/genética , Ratones , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/genética , Salmonella/genética , Infecciones por Salmonella/genéticaRESUMEN
Clonotypic αß T cell responses to cargoes presented by major histocompatibility complex (MHC), MR1, or CD1 proteins underpin adaptive immunity. Those responses are mostly mediated by complementarity-determining region 3 motifs created by quasi-random T cell receptor (TCR) gene rearrangements, with diversity being highest for TCRγδ. Nonetheless, TCRγδ also displays nonclonotypic innate responsiveness following engagement of germline-encoded Vγ-specific residues by butyrophilin (BTN) or BTN-like (BTNL) proteins that uniquely mediate γδ T cell subset selection. We now report that nonclonotypic TCR engagement likewise induces distinct phenotypes in TCRαß+ cells. Specifically, antibodies to germline-encoded human TCRVß motifs consistently activated naïve or memory T cells toward core states distinct from those induced by anti-CD3 or superantigens and from others commonly reported. Those states combined selective proliferation and effector function with activation-induced inhibitory receptors and memory differentiation. Thus, nonclonotypic TCRVß targeting broadens our perspectives on human T cell response modes and might offer ways to induce clinically beneficial phenotypes in defined T cell subsets.
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Receptores de Antígenos de Linfocitos T alfa-beta , Receptores de Antígenos de Linfocitos T gamma-delta , Humanos , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Subgrupos de Linfocitos T , Butirofilinas/genética , Butirofilinas/metabolismo , Fenotipo , InmunoterapiaRESUMEN
BACKGROUND: Hypoxia is a hallmark of the tumor microenvironment (TME) and in addition to altering metabolism in cancer cells, it transforms tumor-associated stromal cells. Within the tumor stromal cell compartment, tumor-associated macrophages (TAMs) provide potent pro-tumoral support. However, TAMs can also be harnessed to destroy tumor cells by monoclonal antibody (mAb) immunotherapy, through antibody dependent cellular phagocytosis (ADCP). This is mediated via antibody-binding activating Fc gamma receptors (FcγR) and impaired by the single inhibitory FcγR, FcγRIIb. METHODS: We applied a multi-OMIC approach coupled with in vitro functional assays and murine tumor models to assess the effects of hypoxia inducible factor (HIF) activation on mAb mediated depletion of human and murine cancer cells. For mechanistic assessments, siRNA-mediated gene silencing, Western blotting and chromatin immune precipitation were utilized to assess the impact of identified regulators on FCGR2B gene transcription. RESULTS: We report that TAMs are FcγRIIbbright relative to healthy tissue counterparts and under hypoxic conditions, mononuclear phagocytes markedly upregulate FcγRIIb. This enhanced FcγRIIb expression is transcriptionally driven through HIFs and Activator protein 1 (AP-1). Importantly, this phenotype reduces the ability of macrophages to eliminate anti-CD20 monoclonal antibody (mAb) opsonized human chronic lymphocytic leukemia cells in vitro and EL4 lymphoma cells in vivo in human FcγRIIb+/+ transgenic mice. Furthermore, post-HIF activation, mAb mediated blockade of FcγRIIb can partially restore phagocytic function in human monocytes. CONCLUSION: Our findings provide a detailed molecular and cellular basis for hypoxia driven resistance to antitumor mAb immunotherapy, unveiling a hitherto unexplored aspect of the TME. These findings provide a mechanistic rationale for the modulation of FcγRIIb expression or its blockade as a promising strategy to enhance approved and novel mAb immunotherapies.
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Leucemia Linfocítica Crónica de Células B , Receptores de IgG , Animales , Anticuerpos Monoclonales/farmacología , Humanos , Hipoxia/metabolismo , Inmunoterapia , Leucemia Linfocítica Crónica de Células B/metabolismo , Macrófagos/metabolismo , Ratones , Receptores de IgG/genética , Receptores de IgG/metabolismo , Microambiente TumoralRESUMEN
Among the diverse tumor resident immune cell types, tumor-associated macrophages (TAMs) are often the most abundant, possess an anti-inflammatory phenotype, orchestrate tumor immune evasion and are frequently associated with poor prognosis. However, TAMs can also be harnessed to destroy antibody-opsonized tumor cells through the process of antibody-dependent cellular phagocytosis (ADCP). Clinically important tumor-targeting monoclonal antibodies (mAb) such as Rituximab, Herceptin and Cetuximab, function, at least in part, by inducing macrophages to eliminate tumor cells via ADCP. For IgG mAb, this is mediated by antibody-binding activating Fc gamma receptors (FcγR), with resultant phagocytic activity impacted by the level of co-engagement with the single inhibitory FcγRIIb. Approaches to enhance ADCP in the tumor microenvironment include the repolarization of TAMs to proinflammatory phenotypes or the direct augmentation of ADCP by targeting so-called 'phagocytosis checkpoints'. Here we review the most promising new strategies targeting the cell surface molecules present on TAMs, which include the inhibition of 'don't eat me signals' or targeting immunostimulatory pathways with agonistic mAb and small molecules to augment tumor-targeting mAb immunotherapies and overcome therapeutic resistance.
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BACKGROUND: The major sites for fast synaptic inhibition in the central nervous system (CNS) are ion channels activated by γ-aminobutyric acid (GABA). These receptors are referred as GABA(A) receptors (GABA(A)R). Recent evidence indicates a role of GABA(A)R in modulating the immune response. This work aimed to discern the role of GABA and GABA(A)Rs in human and mouse T cell activity. METHODS: Mouse splenocytes or human peripheral blood mononuclear cells (PBMCs) were activated with anti-CD3 antibodies and the proliferation of both CD8+ and CD4+ T cells assessed through flow cytometry. Subsequently, the effects on T cell proliferation of either GABA(A)R modulation by diazepam that is also capable of activating mitochondrial based translocator protein (TSPO), alprazolam and allopregnanolone or inhibition by bicucculine methiodide (BMI) and (1,2,5,6-Tetrahydropyridin-4-yl)methylphosphinic acid (TPMPA) were assessed. RESULTS: Positive modulation of GABA(A)Rs either by benzodiazepines or the neurosteroid allopregnanolone inhibits both mouse and human T cell proliferation. GABAergic inhibition of T cell proliferation by benzodiazepines could be rescued by GABA(A)R blocking. Our data suggest that benzodiazepines influence T cell proliferation through both TSPO and GABA(A)Rs activation. CONCLUSIONS: We conclude that activation of GABA(A)Rs provides immunosuppression by inhibiting T cell proliferation.
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Benzodiazepinas/farmacología , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD8-positivos/metabolismo , Proliferación Celular/efectos de los fármacos , Pregnanolona/farmacología , Receptores de GABA-A/metabolismo , Animales , Humanos , Ratones , Receptores de GABA/metabolismoRESUMEN
BACKGROUND: Previous data suggests that anti-OX40 mAb can elicit anti-tumor effects in mice through deletion of Tregs. However, OX40 also has powerful costimulatory effects on T cells which could evoke therapeutic responses. Human trials with anti-OX40 antibodies have shown that these entities are well tolerated but to date have delivered disappointing clinical responses, indicating that the rules for the optimal use of anti-human OX40 (hOX40) antibodies is not yet fully understood. Changes to timing and dosages may lead to improved outcomes; however, here we focus on addressing the role of agonism versus depleting activity in determining therapeutic outcomes. We investigated a novel panel of anti-hOX40 mAb to understand how these reagents and mechanisms may be optimized for therapeutic benefit. METHODS: This study examines the binding activity and in vitro activity of a panel of anti-hOX40 antibodies. They were further evaluated in several in vivo models to address how isotype and epitope determine mechanism of action and efficacy of anti-hOX40 mAb. RESULTS: Binding analysis revealed the antibodies to be high affinity, with epitopes spanning all four cysteine-rich domains of the OX40 extracellular domain. In vivo analysis showed that their activities relate directly to two key properties: (1) isotype-with mIgG1 mAb evoking receptor agonism and CD8+ T-cell expansion and mIgG2a mAb evoking deletion of Treg and (2) epitope-with membrane-proximal mAb delivering more powerful agonism. Intriguingly, both isotypes acted therapeutically in tumor models by engaging these different mechanisms. CONCLUSION: These findings highlight the significant impact of isotype and epitope on the modulation of anti-hOX40 mAb therapy, and indicate that CD8+ T-cell expansion or Treg depletion might be preferred according to the composition of different tumors. As many of the current clinical trials using OX40 antibodies are now using combination therapies, this understanding of how to manipulate therapeutic activity will be vital in directing new combinations that are more likely to improve efficacy and clinical outcomes.
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Isotipos de Inmunoglobulinas/inmunología , Inmunoterapia/métodos , Receptores OX40/inmunología , Animales , Femenino , Humanos , RatonesRESUMEN
Monoclonal antibody (mAb) immunotherapy has transformed the treatment of allergy, autoimmunity, and cancer. The interaction of mAb with Fc gamma receptors (FcγR) is often critical for efficacy. The genes encoding the low-affinity FcγR have single nucleotide polymorphisms (SNPs) and copy number variation that can impact IgG Fc:FcγR interactions. Leukocyte-based in vitro assays remain one of the industry standards for determining mAb efficacy and predicting adverse responses in patients. Here we addressed the impact of FcγR genetics on immune cell responses in these assays and investigated the importance of assay format. FcγR genotyping of 271 healthy donors was performed using a Multiplex Ligation-Dependent Probe Amplification assay. Freeze-thawed/pre-cultured peripheral blood mononuclear cells (PBMCs) and whole blood samples from donors were stimulated with reagents spanning different mAb functional classes to evaluate the association of FcγR genotypes with T-cell proliferation and cytokine release. Using freeze-thawed/pre-cultured PBMCs, agonistic T-cell-targeting mAb induced T-cell proliferation and the highest levels of cytokine release, with lower but measurable responses from mAb which directly require FcγR-mediated cellular effects for function. Effects were consistent for individual donors over time, however, no significant associations with FcγR genotypes were observed using this assay format. In contrast, significantly elevated IFN-γ release was associated with the FCGR2A-131H/H genotype compared to FCGR2A-131R/R in whole blood stimulated with Campath (p ≤ 0.01) and IgG1 Fc hexamer (p ≤ 0.05). Donors homozygous for both the high affinity FCGR2A-131H and FCGR3A-158V alleles mounted stronger IFN-γ responses to Campath (p ≤ 0.05) and IgG1 Fc Hexamer (p ≤ 0.05) compared to donors homozygous for the low affinity alleles. Analysis revealed significant reductions in the proportion of CD14hi monocytes, CD56dim NK cells (p ≤ 0.05) and FcγRIIIa expression (p ≤ 0.05), in donor-matched freeze-thawed PBMC compared to whole blood samples, likely explaining the difference in association between FcγR genotype and mAb-mediated cytokine release in the different assay formats. These findings highlight the significant impact of FCGR2A and FCGR3A SNPs on mAb function and the importance of using fresh whole blood assays when evaluating their association with mAb-mediated cytokine release in vitro. This knowledge can better inform on the utility of in vitro assays for the prediction of mAb therapy outcome in patients.
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Anticuerpos Monoclonales/inmunología , Afinidad de Anticuerpos/genética , Síndrome de Liberación de Citoquinas/genética , Técnicas Inmunológicas , Polimorfismo de Nucleótido Simple , Receptores de IgG/genética , Anticuerpos Monoclonales/farmacología , Citocinas/biosíntesis , Genotipo , Humanos , Leucocitos Mononucleares/inmunología , Receptores de IgG/inmunologíaRESUMEN
Tumors routinely attract and co-opt macrophages to promote their growth, angiogenesis, and metastasis. Macrophages are also the key effector cell for mAb therapies. Here we report that the tumor microenvironment creates an immunosuppressive signature on tumor-associated macrophages (TAM), which favors expression of inhibitory rather than activating Fcγ receptors (FcγR), thereby limiting the efficacy of mAb immunotherapy. We assessed a panel of TLR and STING agonists (a) for their ability to reprogram macrophages to a state optimal for mAb immunotherapy. Both STINGa and TLRa induced cytokine release, modulated FcγR expression, and augmented mAb-mediated tumor cell phagocytosis in vitro However, only STINGa reversed the suppressive FcγR profile in vivo, providing strong adjuvant effects to anti-CD20 mAb in murine models of lymphoma. Potent adjuvants like STINGa, which can improve FcγR activatory:inhibitory (A:I) ratios on TAM, are appealing candidates to reprogram TAM and curb tumor-mediated immunosuppression, thereby empowering mAb efficacy. Cancer Res; 77(13); 3619-31. ©2017 AACR.
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Anticuerpos Monoclonales/farmacología , Resistencia a Antineoplásicos/inmunología , Inmunización Pasiva/métodos , Linfoma/inmunología , Linfoma/terapia , Proteínas de la Membrana/agonistas , Proteínas de la Membrana/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Femenino , Humanos , Linfoma/metabolismo , Ratones , Ratones Endogámicos BALB C , Receptores de IgG/inmunología , Microambiente Tumoral/inmunología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Monoclonal antibodies (mAbs) can destroy tumors by recruiting effectors such as myeloid cells, or targeting immunomodulatory receptors to promote cytotoxic T cell responses. Here, we examined the therapeutic potential of combining a direct tumor-targeting mAb, anti-CD20, with an extended panel of immunomodulatory mAbs. Only the anti-CD27/CD20 combination provided cures. This was apparent in multiple lymphoma models, including huCD27 transgenic mice using the anti-huCD27, varlilumab. Detailed mechanistic analysis using single-cell RNA sequencing demonstrated that anti-CD27 stimulated CD8+ T and natural killer cells to release myeloid chemo-attractants and interferon gamma, to elicit myeloid infiltration and macrophage activation. This study demonstrates the therapeutic advantage of using an immunomodulatory mAb to regulate lymphoid cells, which then recruit and activate myeloid cells for enhanced killing of mAb-opsonized tumors.
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Anticuerpos Monoclonales/farmacología , Antineoplásicos/farmacología , Linfoma/inmunología , Miembro 7 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/agonistas , Animales , Anticuerpos Monoclonales Humanizados , Humanos , Inmunoterapia/métodos , Activación de Linfocitos/efectos de los fármacos , Activación de Linfocitos/inmunología , Activación de Macrófagos/efectos de los fármacos , Activación de Macrófagos/inmunología , Ratones , Ratones TransgénicosRESUMEN
Helicobacter pylori infections are usually established in early childhood and continuously stimulate immunity, including T-helper 1 (Th1), Th17, and regulatory T-cell (Treg) responses, throughout life. Although known to be the major cause of peptic ulcer disease and gastric cancer, disease occurs in a minority of those who are infected. Recently, there has been much interest in beneficial effects arising from infection with this pathogen. Published data robustly show that the infection is protective against asthma in mouse models. Epidemiological studies show that H. pylori is inversely associated with human allergy and asthma, but there is a paucity of mechanistic data to explain this. Since Th1 and Treg responses are reported to protect against allergic responses, we investigated if there were links between the human systemic Th1 and Treg response to H. pylori and allergen-specific IgE levels. The human cytokine and T-cell responses were examined using peripheral blood mononuclear cells (PBMCs) from 49 infected and 58 uninfected adult patients. Concentrations of total and allergen-specific plasma IgE were determined by ELISA and ImmunoCAP assays. These responses were analyzed according to major virulence factor genotypes of the patients' colonizing H. pylori strains. An in vitro assay was employed, using PBMCs from infected and uninfected donors, to determine the role of Treg cytokines in the suppression of IgE. Significantly higher frequencies of IL-10-secreting CD4(+)CD25(hi) Tregs, but not H. pylori-specific Th1 cells, were present in the peripheral blood of infected patients. Total and allergen-specific IgE concentrations were lower when there was a strong Treg response, and blocking IL-10 in vitro dramatically restored IgE responses. IgE concentrations were also significantly lower when patients were infected with CagA(+) strains or those expressing the more active i1 form of VacA. The systemic IL-10(+) Treg response is therefore likely to play a role in H. pylori-mediated protection against allergy in humans.
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Recent research has demonstrated that infection with the bacterial pathogen Helicobacter pylori is less common amongst patients with multiple sclerosis (MS), an inflammatory demyelinating disease of the central nervous system (CNS). We aimed to compare the prevalence of H. pylori amongst MS patients and healthy controls, and also investigated the impact of this infection on an animal model for MS, experimental autoimmune encephalomyelitis (EAE). The H. pylori status of 71 MS patients and 42 healthy controls was determined by serology. Groups of C57BL/6 mice were infected with H. pylori, or given diluent alone as a placebo, prior to inducing EAE. Clinical scores were assessed for all mice, and spleens and spinal cord tissue were harvested. CD4(+) T cell subsets were quantified by flow cytometry, and T cell proliferation assays were performed. In MS patients the seroprevalence of H. pylori was half that of healthy controls (p = 0.018). Over three independent experiments, prior H. pylori infection had a moderate effect in reducing the severity of EAE (p = 0.012). In line with this, the antigen-specific T cell proliferative responses of infected animals were significantly reduced (p = 0.001), and there was a fourfold reduction in the number of CD4(+) cells in the CNS. CD4(+) populations in both the CNS and the spleens of infected mice also contained greatly reduced proportions of IFNγ(+), IL-17(+), T-bet(+), and RORγt(+) cells, but the proportions of Foxp3(+) cells were equivalent. There were no differences in the frequency of splenic CD4(+)cells expressing markers of apoptosis between infected and uninfected animals. H. pylori was less prevalent amongst MS patients. In mice, the infection exerted some protection against EAE, inhibiting both Th1 and Th17 responses. This could not be explained by the presence of increased numbers of Foxp3(+) regulatory T cells, or T cell apoptosis. This is the first direct experimental evidence showing that H. pylori may provide protection against inflammatory demyelination in the CNS.
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Cancer immunotherapy has been revolutionised by the use monoclonal antibodies (mAb) that function through their interaction with Fc gamma receptors (FcγRs). The low-affinity FcγR genes are highly homologous, map to a complex locus at 1p23 and harbour single nucleotide polymorphisms (SNPs) and copy number variation (CNV) that can impact on receptor function and response to therapeutic mAbs. This complexity can hinder accurate characterisation of the locus. We therefore evaluated and optimised a suite of assays for the genomic analysis of the FcγR locus amenable to peripheral blood mononuclear cells and formalin-fixed paraffin-embedded (FFPE) material that can be employed in a high-throughput manner. Assessment of TaqMan genotyping for FCGR2A-131H/R, FCGR3A-158F/V and FCGR2B-232I/T SNPs demonstrated the need for additional methods to discriminate genotypes for the FCGR3A-158F/V and FCGR2B-232I/T SNPs due to sequence homology and CNV in the region. A multiplex ligation-dependent probe amplification assay provided high quality SNP and CNV data in PBMC cases, but there was greater data variability in FFPE material in a manner that was predicted by the BIOMED-2 multiplex PCR protocol. In conclusion, we have evaluated a suite of assays for the genomic analysis of the FcγR locus that are scalable for application in large clinical trials of mAb therapy. These assays will ultimately help establish the importance of FcγR genetics in predicting response to antibody therapeutics.
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Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Receptores de IgG/genética , Anticuerpos Monoclonales/uso terapéutico , ADN/genética , ADN/aislamiento & purificación , Variaciones en el Número de Copia de ADN , Humanos , Leucocitos Mononucleares/inmunología , Reacción en Cadena de la Polimerasa Multiplex , Polimorfismo de Nucleótido Simple , Análisis de Secuencia de ADN/métodosRESUMEN
Therapeutic antibodies have transformed cancer therapy, unlocking mechanisms of action by engaging the immune system. Unfortunately, cures rarely occur and patients display intrinsic or acquired resistance. Here, we demonstrate the therapeutic potential of targeting human (h) FcγRIIB (CD32B), a receptor implicated in immune cell desensitization and tumor cell resistance. FcγRIIB-blocking antibodies prevented internalization of the CD20-specific antibody rituximab, thereby maximizing cell surface accessibility and immune effector cell mediated antitumor activity. In hFcγRIIB-transgenic (Tg) mice, FcγRIIB-blocking antibodies effectively deleted target cells in combination with rituximab, and other therapeutic antibodies, from resistance-prone stromal compartments. Similar efficacy was seen in primary human tumor xenografts, including with cells from patients with relapsed/refractory disease. These data support the further development of hFcγRIIB antibodies for clinical assessment.