RESUMO
Immunogenicity assessment of Adeno-Associated Virus (AAV) vectors is a critical part of gene therapy drug development. Whether the assays are used for inclusion/exclusion criteria or to monitor the safety and efficacy of the gene therapy, they are critical bioanalytical assessments. While total anti-AAV assays are perceived as easier to develop and implement than neutralizing anti-AAV assays, the gene therapy field is still nascent, and it is not yet clear which of the assays should be implemented at what stage of drug development. Recently AAVrh.10 has gained interest for use in gene therapies targeting cardiac, neurological, and other diseases due to its enhanced transduction efficiency. There is limited information on anti-AAVrh.10 antibodies and their clinical impact; thus, the information presented herein documents the validation of both a total antibody assay (TAb) and a neutralizing antibody (NAb) assay for anti-AAVrh.10 antibodies. In this manuscript, the validation was performed in accordance with the 2019 FDA immunogenicity guidance with additional evaluations to comply with CLIA where applicable. The AAVrh.10 TAb and NAb assays were compared in terms of sensitivity, drug tolerance, and precision, along with a concordance analysis using the same individual serum samples. This comparison gave insight into the utility of each format as a screening assay for inclusion into clinical studies.
Assuntos
Anticorpos Neutralizantes , Dependovirus , Anticorpos Neutralizantes/genética , Dependovirus/genética , Sorogrupo , Bioensaio , Terapia Genética , Vetores GenéticosRESUMO
The appropriate regulation of neutrophil activation is critical for maintaining host defense and limiting inflammation. Polymorphonuclear neutrophils (PMNs) express a number of cytoplasmic tyrosine kinases that regulate signaling pathways leading to activation. One of the most highly expressed, but least studied, kinases in PMNs is proline rich kinase 2 (Pyk2). By analogy to the related focal adhesion kinase, Pyk2 has been implicated in regulating PMN adhesion and migration; however, its physiologic function has yet to be described. Using pyk2(-/-) mice, we found that this kinase was required for integrin-mediated degranulation responses, but was not involved in adhesion-induced cell spreading or activation of superoxide production. Pyk2-deficient PMNs also manifested reduced migration on fibrinogen-coated surfaces. The absence of Pyk2 resulted in a severe reduction in paxillin and Vav phosphorylation following integrin ligation, which likely accounts for the poor degranulation and cell migration. Pyk2(-/-) mice were unable to efficiently clear infection with Staphylococcus aureus in a skin abscess model, owing in part to the poor release of granule contents at the site of infection. However, Pyk2-deficient PMNs responded normally to soluble agonists, demonstrating that this kinase functions mainly in the integrin pathway. These data demonstrate the unrealized physiologic role of this kinase in regulating the adhesion-mediated release of PMN granule contents.
Assuntos
Degranulação Celular/imunologia , Quinase 2 de Adesão Focal/fisiologia , Ativação de Neutrófilo/imunologia , Neutrófilos/imunologia , Infecções Estafilocócicas/enzimologia , Infecções Estafilocócicas/imunologia , Abscesso/enzimologia , Abscesso/imunologia , Abscesso/microbiologia , Animais , Células da Medula Óssea/imunologia , Células da Medula Óssea/microbiologia , Células da Medula Óssea/patologia , Degranulação Celular/genética , Movimento Celular/genética , Movimento Celular/imunologia , Modelos Animais de Doenças , Quinase 2 de Adesão Focal/deficiência , Quinase 2 de Adesão Focal/genética , Imunidade Inata/genética , Integrinas/fisiologia , Macrófagos/enzimologia , Macrófagos/imunologia , Macrófagos/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ativação de Neutrófilo/genética , Neutrófilos/enzimologia , Neutrófilos/patologia , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Pele/imunologia , Pele/microbiologia , Pele/patologia , Infecções Estafilocócicas/prevenção & controle , Staphylococcus aureus/imunologiaRESUMO
Major histocompatibility complex (MHC)-Associated Peptide Proteomics (MAPPs) is an ex vivo method used to assess the immunogenicity risk of biotherapeutics. MAPPs can identify potential T-cell epitopes within the biotherapeutic molecule. Using adalimumab treated human monocyte derived dendritic cells (DCs) and a pan anti-HLA-DR antibody (Ab), we systematically automated and optimized biotin/streptavidin (SA)-capture antibody coupling, lysate incubation with capture antibody, as well as the washing and elution steps of a MAPPs method using functionalized magnetic beads and a KingFisher Magnetic Particle processor. Automation of these steps, combined with capturing using biotinylated-Ab/SA magnetic beads rather than covalently bound antibody, improved reproducibility as measured by minimal inter-and intra-day variability, as well as minimal analyst-to-analyst variability. The semi-automated MAPPs workflow improved sensitivity, allowing for a lower number of cells per analysis. The method was assessed using five different biotherapeutics with varying immunogenicity rates ranging from 0.1 to 48% ADA incidence in the clinic. Biotherapeutics with ≥10%immunogenicity incidence consistently presented more peptides (1.8-28 fold) and clusters (10-21 fold) compared to those with <10% immunogenicity incidence. Our semi-automated MAPPs method provided two main advantages over a manual workflow- the robustness and reproducibility affords confidence in the epitopes identified from as few as 5 to 10 donors and the method workflow can be readily adapted to incorporate different capture Abs in addition to anti-HLA-DR. The incorporation of semi-automated MAPPs with biotinylated-Ab/SA bead-based capture in immunogenicity screening strategies allows the generation of more consistent and reliable data, helping to improve immunogenicity prediction capabilities in drug development. MHC associated peptide proteomics (MAPPs), Immunogenicity risk assessment, in vitro/ex vivo, biotherapeutics, Major Histocompatibility Complex Class II (MHC II), LC-MS, Immunoaffinity Capture, streptavidin magnetic beads.
Assuntos
Proteômica , Espectrometria de Massas em Tandem , Humanos , Estreptavidina , Reprodutibilidade dos Testes , Peptídeos/metabolismo , Anticorpos , Epitopos de Linfócito T , Desenvolvimento de MedicamentosRESUMO
Evolving immunogenicity assay performance expectations and a lack of harmonized neutralizing antibody validation testing and reporting tools have resulted in significant time spent by health authorities and sponsors on resolving filing queries. A team of experts within the American Association of Pharmaceutical Scientists' Therapeutic Product Immunogenicity Community across industry and the Food and Drug Administration addressed challenges unique to cell-based and non-cell-based neutralizing antibody assays. Harmonization of validation expectations and data reporting will facilitate filings to health authorities and are described in this manuscript. This team provides validation testing and reporting strategies and tools for the following assessments: (1) format selection; (2) cut point; (3) assay acceptance criteria; (4) control precision; (5) sensitivity including positive control selection and performance tracking; (6) negative control selection; (7) selectivity/specificity including matrix interference, hemolysis, lipemia, bilirubin, concomitant medications, and structurally similar analytes; (8) drug tolerance; (9) target tolerance; (10) sample stability; and (11) assay robustness.
Assuntos
Anticorpos Neutralizantes , Preparações Farmacêuticas , Tolerância a MedicamentosRESUMO
Antibody-based therapeutics are powerful tools to treat disease. While their mechanism of action (MOA) always involves binding to a specific target via the antibody-binding fragment (Fab) region of the antibody, the induction of immune-mediated effector functions through the fragment crystallizable (Fc) region is a vital aspect of antibody therapeutics targeting tumor cells. Cross-linking of the Fc gamma receptors (FcγRs) via cell-bound antibodies activate immune effector cells, leading to antibody-dependent cellular cytotoxicity via natural killer (NK) cells. Linking of FcγRs on macrophages triggers the process of antibody-dependent cellular phagocytosis (ADCP), where antibody-opsonized target cells are internalized in phagosomes and degraded through the process of phagosome maturation and acidification. ADCP activity can be challenging to measure accurately due to the difficulty in differentiating target cells that are bound to a macrophage versus those that are internalized within phagosomes. In this chapter, we describe a protocol that measures ADCP activity by labeling target cells with a pH-sensitive dye that fluoresces brightly in mature phagosomes. The ADCP activity of therapeutics is then measured via flow cytometry. This assay is capable of detecting glycosylation differences arising from manufacturing processes and is suitable for evaluation of ADCP activity of monoclonal antibodies (mAb) to support in vitro biological characterization of drug candidates and lead candidate selection for desirable effector functions.
Assuntos
Fagocitose , Anticorpos Monoclonais , Citotoxicidade Celular Dependente de Anticorpos , Macrófagos , Fagossomos , Receptores de IgGRESUMO
The treatment of diseases with biologic agents can result in the formation of antidrug antibodies (ADA). Although drivers for ADA formation are unknown, a role for antigen presentation is likely, and variation in human leukocyte antigen (HLA) genes has been shown to be associated with occurrence of ADA for several biologics. Here, we performed an HLA-wide association study in 1982 patients treated with the anti-PD-L1 antibody atezolizumab across eight clinical trials. On average, 29.8% of patients were ADA-positive (N = 591, range of 13.5%-38.4% per study) and 14.6% of patients were positive for ADA that were neutralizing in vitro (neutralizing antibodies [NAb], N = 278, range of 6.4%-21.9% per study). In a meta-analysis of logistic regression coefficients, we found statistically significant associations between HLA class II alleles and ADA status. The top-associated alleles were HLA-DRB1*01:01 in a comparison of ADA-positive versus ADA-negative patients (p = 3.4 × 10-5 , odds ratio [OR] 1.96, 95% confidence interval [95% CI] 1.64-2.28) and HLA-DQA1*01:01 when comparing NAb-positive with ADA-negative patients (p = 2.8 × 10-7 , OR 2.31, 95% CI 1.98-2.66). Both alleles occur together on a common HLA haplotype, and analyses considering only NAb-negative, ADA-positive patients did not yield significant results, suggesting that the genetic association is mainly driven by NAb status. In conclusion, our study showed that HLA class II genotype is associated with the risk of developing ADA, and specifically NAb, in patients treated with atezolizumab, but the effect estimates suggest that immunogenetic factors are not sufficient as clinically meaningful predictors.
Assuntos
Anticorpos Monoclonais Humanizados , Anticorpos Neutralizantes , Cadeias HLA-DRB1 , Neoplasias , Alelos , Anticorpos Monoclonais Humanizados/uso terapêutico , Anticorpos Neutralizantes/imunologia , Cadeias HLA-DRB1/genética , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/genéticaRESUMO
Adoptive cell therapies (ACTs) have shown transformative efficacy in oncology with five US Food and Drug Administration (FDA) approvals for chimeric antigen receptor (CAR) T-cell therapies in hematological malignancies, and promising activity for T cell receptor T-cell therapies in both liquid and solid tumors. Clinical pharmacology can play a pivotal role in optimizing ACTs, aided by modeling and simulation toolboxes and deep understanding of the underlying biological and immunological processes. Close collaboration and multilevel data integration across functions, including chemistry, manufacturing, and control, biomarkers, bioanalytical, and clinical science and safety teams will be critical to ACT development. As ACT is comprised of alive, polyfunctional, and heterogeneous immune cells, its overall physicochemical and pharmacological property is vastly different from other platforms/modalities, such as small molecule and protein therapeutics. In this review, we first describe the unique kinetics of T cells and the appropriate bioanalytical strategies to characterize cellular kinetics. We then assess the distinct aspects of clinical pharmacology for ACTs in comparison to traditional small molecule and protein therapeutics. Additionally, we provide a review for the five FDA-approved CAR T-cell therapies and summarize their properties, cellular kinetic characteristics, dose-exposure-response relationship, and potential baseline factors/variables in product, patient, and regimen that may affect the safety and efficacy. Finally, we probe into existing empirical and mechanistic quantitative techniques to understand how various modeling and simulation approaches can support clinical pharmacology strategy and propose key considerations to be incorporated and explored in future models.
Assuntos
Neoplasias , Farmacologia Clínica , Receptores de Antígenos Quiméricos , Humanos , Imunoterapia Adotiva/efeitos adversos , Imunoterapia Adotiva/métodos , Receptores de Antígenos de Linfócitos T , Linfócitos TRESUMO
Aim: Immunogenicity risk assessment assays are powerful tools that assess the relative immunogenicity of potential biotherapeutics. We detail here the development of a novel assay that measures the degree of antibody internalization by antigen-presenting cells as a predictor of immunogenicity. Results & methodology: The assay uses the fluorescence signal from the antibody bound to the outside of the cell as well as inside the cell to determine internalization. To calculate the amount of internalized antibody, the fluorescent signal from the outside was subtracted from the fluorescent signal from the inside, which is referred to as the internalization index. Conclusion: This assay format demonstrated that antibody-based biotherapeutics with higher clinical immunogenicity internalized to a higher degree than therapeutic antibodies with lower clinical immunogenicity.
Assuntos
Anticorpos , Células Dendríticas , Medição de RiscoRESUMO
T cell-specific adaptor protein (TSAd) is a T lineage-restricted signaling adaptor molecule that is thought to participate in the assembly of intracellular signaling complexes in T cells. Previous studies of TSAd-deficient mice have revealed a role for TSAd in the induction of T cell interleukin 2 secretion and proliferation. We now show that TSAd-deficient mice are susceptible to lupus-like autoimmune disease. On the nonautoimmune-prone C57BL/6 genetic background, TSAd deficiency results in hypergammaglobulinemia that affects all immunoglobulin (Ig)G subclasses. Older C57BL/6 TSAd-deficient mice (1 yr of age) accumulate large numbers of activated T and B cells in spleen, produce autoantibodies against a variety of self-targets including single stranded (ss) and double stranded (ds) DNA, and, in addition, develop glomerulonephritis. We further show that immunization of younger C57BL/6 TSAd-deficient mice (at age 2 mo) with pristane, a recognized nonspecific inflammatory trigger of lupus, results in more severe glomerulonephritis compared with C57BL/6 controls and the production of high titer ss and ds DNA antibodies of the IgG subclass that are not normally produced by C57BL/6 mice in this model. The development of autoimmunity in TSAd-deficient mice is associated with defective T cell death in vivo. These findings illustrate the role of TSAd as a critical regulator of T cell death whose absence promotes systemic autoimmunity.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas de Transporte/imunologia , Hipergamaglobulinemia/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Linfócitos T/imunologia , Animais , Doenças Autoimunes/imunologia , Doenças Autoimunes/patologia , Linfócitos T CD4-Positivos/imunologia , Proteínas de Transporte/genética , Morte Celular , Modelos Animais de Doenças , Hipergamaglobulinemia/genética , Imunoglobulina G/sangue , Imunoglobulina G/classificação , Imunoglobulina M/sangue , Interleucina-2/metabolismo , Lúpus Eritematoso Sistêmico/patologia , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transdução de Sinais/imunologiaRESUMO
T cell-specific adapter (TSAd) protein and adapter protein in lymphocytes of unknown function (ALX) are two related Src homology 2 (SH2) domain-containing signaling adapter molecules that have both been shown to regulate TCR signal transduction in T cells. TSAd is required for normal TCR-induced synthesis of IL-2 and other cytokines in T cells and acts at least in part by promoting activation of the LCK protein tyrosine kinase at the outset of the TCR signaling cascade. By contrast, ALX functions as a negative-regulator of TCR-induced IL-2 synthesis through as yet undetermined mechanisms. In this study, we report a novel T cell-expressed adapter protein named SH2D4A that contains an SH2 domain that is highly homologous to the TSAd protein and ALX SH2 domains and that shares other structural features with these adapters. To examine the function of SH2D4A in T cells we produced SH2D4A-deficient mice by homologous recombination in embryonic stem cells. T cell development, homeostasis, proliferation, and function were all found to be normal in these mice. Furthermore, knockdown of SH2D4A expression in human T cells did not impact upon their function. We conclude that in contrast to TSAd and ALX proteins, SH2D4A is dispensable for TCR signal transduction in T cells.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/deficiência , Proteínas Adaptadoras de Transdução de Sinal/genética , Sequência de Aminoácidos , Animais , Linhagem Celular , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/deficiência , Listeriose/genética , Listeriose/imunologia , Listeriose/metabolismo , Proteínas de Membrana/química , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , RNA Interferente Pequeno/genética , Alinhamento de SequênciaRESUMO
In macrophages, enzymes that synthesize or hydrolyze phosphatidylinositol (3,4,5)-trisphosphate [PI(3,4,5)P(3)] regulate Fcgamma receptor-mediated phagocytosis. Inhibition of phosphatidylinositol 3-kinase (PI3K) or overexpression of the lipid phosphatases phosphatase and tensin homologue (PTEN) and Src homology 2 domain-containing inositol phosphatase (SHIP-1), which hydrolyze PI(3,4,5)P(3) to phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 3,4-bisphosphate [PI(3,4)P(2)], respectively, inhibit phagocytosis in macrophages. To examine how these enzymes regulate phagosome formation, the distributions of yellow fluorescent protein (YFP) chimeras of enzymes and pleckstrin homology (PH) domains specific for their substrates and products were analyzed quantitatively. PTEN-YFP did not localize to phagosomes, suggesting that PTEN regulates phagocytosis globally within the macrophage. SHIP1-YFP and p85-YFP were recruited to forming phagosomes. SHIP1-YFP sequestered to the leading edge and dissociated from phagocytic cups earlier than did p85-cyan fluorescent protein, indicating that SHIP-1 inhibitory activities are restricted to the early stages of phagocytosis. PH domain chimeras indicated that early during phagocytosis, PI(3,4,5)P(3) was slightly more abundant than PI(3,4)P(2) at the leading edge of the forming cup. These results support a model in which phagosomal PI3K generates PI(3,4,5)P(3) necessary for later stages of phagocytosis, PTEN determines whether those late stages can occur, and SHIP-1 regulates when and where they occur by transiently suppressing PI(3,4,5)P(3)-dependent activities necessary for completion of phagocytosis.
Assuntos
PTEN Fosfo-Hidrolase/metabolismo , Fagossomos/enzimologia , Fosfatidilinositol 3-Quinases/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Animais , Linhagem Celular , Humanos , Inositol Polifosfato 5-Fosfatases , Camundongos , Microscopia de Fluorescência , Modelos Biológicos , Fagocitose , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases , Fosfatidilinositóis/metabolismo , Estrutura Terciária de Proteína , Transporte Proteico , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Antibody-dependent cellular cytotoxicity (ADCC) is an important mechanism of action (MOA) of monoclonal antibody (mAb) therapeutics. Target cells opsonized with therapeutic antibody bind and activate FcγR-bearing immune effector cells, resulting in target cell lysis. A key step in mAb drug development is the characterization of ADCC activity for its potential to inform mAb efficacy and safety. A number of in vitro assays are commonly used to assess ADCC. Most are endpoint assays that measure a surrogate marker of cell lysis. Newer imaging technologies allow direct measurement of ADCC-mediated cell lysis over time. In this study, we detail the development and characterization of a kinetic ADCC assay applicable to multiple target and effector cell types. This kinetic assay shows comparable sensitivity to an endpoint fluorescence release ADCC assay, while offering the advantages of a simpler set up and shorter assay time. Our results demonstrate that kinetic ADCC activity is a valid alternative assay format for measuring in vitro ADCC of mAbs.
Assuntos
Anticorpos Monoclonais Humanizados/farmacologia , Citotoxicidade Celular Dependente de Anticorpos/efeitos dos fármacos , Antineoplásicos Imunológicos/farmacologia , Citometria de Fluxo , Fluoresceínas/metabolismo , Corantes Fluorescentes/metabolismo , Linfoma de Células B/tratamento farmacológico , Rituximab/farmacologia , Linhagem Celular Tumoral , Humanos , Cinética , Linfoma de Células B/imunologia , Linfoma de Células B/metabolismo , Linfoma de Células B/patologia , Reprodutibilidade dos TestesRESUMO
Antibody-based therapeutics are powerful tools to treat disease. While their mechanism of action (MOA) always involves binding to a specific target via the Fab region of the antibody, the induction of effector functions through the Fc region of the antibody is equally important for antibody therapeutics designed to deplete tumor cells. By binding of the Fc region to Fc gamma receptors (FcγRs) on the surface of immune cells or complement factors, antibody therapeutics exert effector functions such as antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), both of which induce target cell death and aid in the efficacy of treatment. Another major Fc effector function is antibody-dependent cellular phagocytosis (ADCP). ADCP is the mechanism by which antibody-opsonized target cells activate the FcγRs on the surface of macrophages to induce phagocytosis, resulting in internalization and degradation of the target cell through phagosome acidification. ADCP has been implicated as a major MOA of several biologics, but this activity is difficult to measure in in vitro. Most assays measure the association of target cells and macrophages; however, co-localization can represent cell attachment rather than internalization. Here, we describe the development of a novel method to accurately measure ADCP activity. By labeling target cells with a pH sensitive dye that only fluoresces in mature phagosomes, the ADCP activity of antibody therapeutics can be accurately quantitated via flow cytometry.
Assuntos
Anticorpos Monoclonais Humanizados/farmacologia , Antineoplásicos Imunológicos/farmacologia , Citotoxicidade Imunológica/efeitos dos fármacos , Citometria de Fluxo , Corantes Fluorescentes/metabolismo , Macrófagos/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Fagocitose/efeitos dos fármacos , Fagossomos/efeitos dos fármacos , Rituximab/farmacologia , Anticorpos Monoclonais Humanizados/metabolismo , Linhagem Celular Tumoral , Glicosilação , Humanos , Concentração de Íons de Hidrogênio , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/patologia , Neoplasias/imunologia , Neoplasias/metabolismo , Neoplasias/patologia , Fagossomos/imunologia , Fagossomos/metabolismo , Fagossomos/patologia , Receptores de IgG/metabolismo , Rituximab/metabolismoRESUMO
The neonatal Fc receptor (FcRn) binds to the Fc domain of IgG in a pH-dependent manner, guides the intracellular movement of the bound antibodies and protects them from lysosomal degradation. Proper characterization of Fc-FcRn interactions is fundamental to successful design, development, and production of Fc-containing therapeutic proteins because of the potential impact of such interactions on their in vivo pharmacokinetic behaviors. Here, we describe the development and characterization of a cell-based, label-free FcRn-mediated transcytosis assay that provides a functional readout to reflect the totality of Fc-FcRn interactions, including pH-dependent association and dissociation, as well as the intracellular trafficking of Fc-containing molecules in complex with FcRn. Our study demonstrates that this transcytosis assay can be used to evaluate FcRn binding of therapeutic antibodies and Fc-fusion proteins, including wild-type and engineered Fc variants with varying FcRn binding affinities, as well as oxidized and aggregated antibody samples. These results support the utility of an FcRn-dependent transcytosis assay for evaluation of both Fc-FcRn interactions and the structural integrity of Fc-containing therapeutic proteins pertinent to their pharmacokinetic behavior in vivo.
Assuntos
Anticorpos/análise , Bioensaio/métodos , Antígenos de Histocompatibilidade Classe I/imunologia , Fragmentos Fc das Imunoglobulinas/análise , Receptores Fc/imunologia , Proteínas Recombinantes de Fusão/análise , Transcitose/imunologia , Animais , Anticorpos/imunologia , Anticorpos/uso terapêutico , Cães , Antígenos de Histocompatibilidade Classe I/genética , Humanos , Fragmentos Fc das Imunoglobulinas/imunologia , Fragmentos Fc das Imunoglobulinas/uso terapêutico , Células Madin Darby de Rim Canino , Receptores Fc/genética , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/uso terapêuticoRESUMO
In vitro antibody-dependent cell-mediated cytotoxicity (ADCC) assays are routinely performed to support the research and development of therapeutic antibodies. In ADCC assays, target cells bound by the antibodies are lysed by activated effector cells following interactions between the Fc region of the bound antibody and Fcγ receptors on effector cells. Target cell lysis is typically measured by quantification of released endogenous enzymes, e.g., lactate dehydrogenase, or measurement of released exogenous labels, e.g., 51Cr, europium or calcein. ADCC assays based on the detection of exogenous labels released from lysed target cells generally show higher sensitivity and require shorter incubation times. However, target cells are usually labeled immediately prior to assay, which inadvertently introduces additional assay variations due to differences in target cell conditions and labeling/handling processes. In this report, we describe the use of thaw-and-use pre-labeled target cells for ADCC assays. Thaw-and-use target cells in our experiments were pre-labeled with the fluorescent dye calcein AM, cryopreserved in single-use aliquots and used directly in assays after thawing. Upon thaw, the pre-labeled cells displayed viability and label retention comparable to freshly labeled cells, responded to ADCC mediated by both peripheral blood mononuclear cells and engineered natural killer cells, performed stably for at least 3 years and provided favorable precision and accuracy to ADCC assays. Implementation of thaw-and-use pre-labeled target cells in ADCC assays can help to alleviate both cell culture and dye labeling derived variability, increase the flexibility of assay scheduling and improve assay consistency and robustness.
Assuntos
Testes Imunológicos de Citotoxicidade/métodos , Fluoresceínas , Coloração e Rotulagem/métodos , Anticorpos Monoclonais , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Criopreservação/métodos , Testes Imunológicos de Citotoxicidade/normas , Humanos , Células Matadoras Naturais/imunologia , Leucócitos Mononucleares/imunologia , Manejo de Espécimes/métodosRESUMO
This corrects the article DOI: 10.1038/ncomms14234.
RESUMO
Influenza B virus (IBV) causes annual influenza epidemics around the world. Here we use an in vivo plasmablast enrichment technique to isolate a human monoclonal antibody, 46B8 that neutralizes all IBVs tested in vitro and protects mice against lethal challenge of all IBVs tested when administered 72 h post infection. 46B8 demonstrates a superior therapeutic benefit over Tamiflu and has an additive antiviral effect in combination with Tamiflu. 46B8 binds to a conserved epitope in the vestigial esterase domain of hemagglutinin (HA) and blocks HA-mediated membrane fusion. After passage of the B/Brisbane/60/2008 virus in the presence of 46B8, we isolated three resistant clones, all harbouring the same mutation (Ser301Phe) in HA that abolishes 46B8 binding to HA at low pH. Interestingly, 46B8 is still able to protect mice against lethal challenge of the mutant viruses, possibly owing to its ability to mediate antibody-dependent cellular cytotoxicity (ADCC).
Assuntos
Anticorpos Monoclonais/uso terapêutico , Anticorpos Neutralizantes/uso terapêutico , Imunoglobulina G/uso terapêutico , Vírus da Influenza B , Infecções por Orthomyxoviridae/terapia , Animais , Anticorpos Neutralizantes/imunologia , Epitopos , Hemaglutininas , Humanos , Concentração de Íons de Hidrogênio , Imunoglobulina G/imunologia , Camundongos , Modelos Moleculares , Infecções por Orthomyxoviridae/virologia , Oseltamivir , Conformação ProteicaRESUMO
We report that particles of ß-glucan, one of the surface components of yeasts, are powerful inducers of neutrophil extracellular trap (NET) formation in human neutrophils. ß-Glucan triggered a prolonged phosphorylation of Src family kinases and Syk that were suppressed by the Src family inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3, 4-d] pyrimidine (PP2) and a novel Syk inhibitor, PRT-060318, respectively. PP2 and PRT-060318 also inhibited ß-glucan-induced NET formation and reactive oxygen species (ROS) generation, suggesting that both responses are triggered by a Src/Syk-regulated signaling pathway. Given that the NADPH oxidase inhibitor diphenyleneiodonium chloride (DPI) markedly inhibited NET formation, our findings suggest that ROS are required for the full-blown formation of NETs in response to ß-glucan particles. Contrary to ß-glucan, ROS generation triggered by phorbol myristate acetate (PMA) was unaffected by PP2 and PRT-060318, but these compounds, as well as DPI, suppressed Src/Syk phosphorylation triggered by PMA. Whereas PP2 had no effect on PMA-induced NET formation, PRT-060318 had a significant, albeit partial, inhibitory effect, thus suggesting that ROS induce NET formation in part via activation of Syk. These findings were substantiated by the evidence that neutrophils from mice with the conditional deletion of Syk were defective in formation of NETs in response to ß-glucan.
Assuntos
Armadilhas Extracelulares/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Neutrófilos/imunologia , Proteínas Tirosina Quinases/imunologia , Transdução de Sinais/imunologia , Quinases da Família src/imunologia , Animais , Carcinógenos/farmacologia , Cicloexilaminas/farmacologia , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/genética , Ativação Enzimática/imunologia , Armadilhas Extracelulares/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/genética , Camundongos , Camundongos Knockout , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/genética , Pirimidinas/farmacologia , Espécies Reativas de Oxigênio/imunologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Quinase Syk , Acetato de Tetradecanoilforbol/farmacologia , Quinases da Família src/antagonistas & inibidores , Quinases da Família src/genéticaRESUMO
Candida albicans is a dimorphic yeast that enters macrophages (Mphi) via the beta-glucan receptor dectin-1. Phagocytosis of C. albicans is characterized by actin polymerization, Syk kinase activation and rapid acquisition of phagolysosomal markers. In mice, C. albicans are able to resist the harsh environment of the phagosome and form pseudohyphae inside the phagolysosomal compartment, eventually extending from the Mphi. In this study, we investigated these unique C. albicans phagosomes and found that actin localized dynamically around the phagosomes, before disintegrating. Membrane phosphoinositides, PI(4,5)P(2), PI(3,4,5)P(3), PI(3,4)P(2), and PI(3)P also localized to the phagosomes. Localization was not related to actin polymerization, and inhibitor studies showed that polymerization of actin on the C. albicans phagosome was independent of PI3K. The ability of mature C. albicans phagosomes to stimulate actin polymerization could facilitate the escape of the growing yeast from the Mphi.