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1.
Blood ; 125(1): 102-10, 2015 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-25395427

RESUMO

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.


Assuntos
Anticorpos Monoclonais Humanizados/química , Monócitos/citologia , Receptores de IgG/metabolismo , Regulação para Cima , Animais , Linfócitos B/citologia , Antígenos CD28/metabolismo , Células CHO , Proliferação de Células , Técnicas de Cocultura , Cricetinae , Cricetulus , Citocinas/metabolismo , Humanos , Leucócitos Mononucleares/citologia , Linfócitos T/citologia , Linfócitos T/imunologia , Transfecção
2.
Cancer Immunol Immunother ; 63(6): 559-69, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24658837

RESUMO

Neuroblastoma, a childhood tumour of neuroectodermal origin, accounts for 15 % of paediatric cancer deaths, which is often metastatic at diagnosis and despite aggressive therapies, it has poor long-term prognosis with high risk of recurrence. Monoclonal antibody (mAb) therapy targeting GD2, a disialoganglioside expressed on neuroblastoma, has shown promise in recent trials with natural killer cell (NK)-mediated antibody-dependent cellular cytotoxicity (ADCC) thought to be central to efficacy, although other immune effectors may be important. To further enhance therapy, immunomonitoring of patients is essential to elucidate the in vivo mechanisms of action and provides surrogate end points of efficacy for future clinical trials. Our aim was to establish a 'real-time' ex vivo wholeblood (WB) immunomonitoring strategy to perform within the logistical constraints such as limited sample volumes, anticoagulant effects, sample stability and shipping time. A fluorescent dye release assay measuring target cell lysis was coupled with flow cytometry to monitor specific effector response. Significant target cell lysis with anti-GD2 antibody (p < 0.05) was abrogated following NK depletion. NK up-regulation of CD107a and CD69 positively correlated with target cell lysis (r > 0.6). The ADCC activity of WB correlated with peripheral blood mononuclear cells (r > 0.95), although WB showed overall greater target cell lysis attributed to the combination of NK-mediated ADCC, CD16+ granulocyte degranulation and complement- dependent cytotoxicity. Response was maintained in heparinised samples stored for 24 h at room temperature, but not 4 °C. Critically, the assay showed good reproducibility (mean % CV < 6.4) and was successfully applied to primary neuroblastoma samples. As such, WB provides a resourceful analysis of multiple mechanisms for efficient end point monitoring to correlate immune modulation with clinical outcome.


Assuntos
Citotoxicidade Celular Dependente de Anticorpos/imunologia , Gangliosídeos/metabolismo , Leucócitos Mononucleares/imunologia , Neuroblastoma/imunologia , Adulto , Citometria de Fluxo , Humanos , Leucócitos Mononucleares/metabolismo , Neuroblastoma/sangue , Neuroblastoma/patologia , Células Tumorais Cultivadas
3.
Cytometry A ; 77(4): 328-37, 2010 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-20140969

RESUMO

Traditionally, flow cytometry analysis of dendritic cells (DC) has followed a negative selection procedure, often limiting the characterization of individual DC subsets to enumeration. We demonstrate the development, evaluation, and clinical application of a novel 6 color/8 parameter flow cytometry panel to allow enumeration and monitoring of activation status of circulating human myeloid (MDC1) and plasmacytoid (PDC) dendritic cells in human whole blood. Enumeration showed a trend of greater numbers of MDC1s and PDCs being collected for fresh whole blood than frozen PBMCs, with this difference being statistically significant (P = 0.04) for unstimulated PDC enumeration. Intra-assay variation had a coefficient of variation <10% and interassay results between operators showed good correlation (r > 0.95). Our results on fresh whole blood showed a significant up regulation of CD83 on both MDC1 and PDC at 4 h post Toll-like ligand stimulus and this activity was comparable in frozen PBMC samples. Comparison for the late activation marker CCR7 showed a significant difference (P <0.05) in expression between fresh and frozen samples, precluding its use for batch analysis of frozen samples. In addition, the level of activation is dependent on the anticoagulant used for sample collection. For CD83 expression at 4 h both EDTA and lithium heparin samples are comparable for MDC1 and PDC populations. Whereas for CCR7 expression, lithium heparin is preferable as EDTA increases the background expression in PDC, preventing further functional assessments. We demonstrate the importance of establishing the kinetic profile of activation marker expression and the importance of evaluating sample collection tubes and sample type before application of novel cytometry panels to a clinical study. We have shown that this DC enumeration flow cytometry panel is a robust analysis system that allows the flexibility of including activation markers.


Assuntos
Células Sanguíneas/citologia , Células Dendríticas/citologia , Citometria de Fluxo/métodos , Células Mieloides/citologia , Bioensaio , Células Sanguíneas/efeitos dos fármacos , Células Dendríticas/efeitos dos fármacos , Ácido Edético/farmacologia , Heparina/farmacologia , Humanos , Lítio/farmacologia , Células Mieloides/efeitos dos fármacos , Fenótipo
4.
Exp Lung Res ; 36(1): 1-11, 2010 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-20128677

RESUMO

Environmental particulates impact first on airway epithelium, whereas circulating infiltrating cells are recruited through the underlying endothelium. An effective cellular immune response requires coordination between endothelium and epithelium. The authors have developed a bilayer culture model consisting of human bronchial epithelial derived cells (16HBE 14o-) and human umbilical vein endothelial cells (HUVECs) cultured as confluent layers on either side of a porous membrane. Confocal microscopy with epithelial and endothelial-specific antibodies showed segregated cell layers. By scanning and transmission electron microscopy, both cell types are polarized and tight junctions formed at the apical interface between cells. Epithelial cells grown in a bilayer showed significantly increased transepithelial resistance (TER) of 2260 +/- 64 Omega.cm(2) compared to epithelial or endothelial monolayers alone (1400 +/- 70 or 80 +/- 12 Omega.cm(2), respectively). This reflected decreased permeability and was unrelated to cell density or height. Increased TER coincided with increased occludin mRNA and protein in the epithelial cell layer as determined by polymerase chain reaction (PCR) and immunoblotting. Conditioned medium showed that decreased permeability was mediated by soluble endothelial-derived factor(s). This model reflects the in vivo relationship of human airway endothelial cells and epithelial cells. Altered tight junction permeability in cocultures indicates that these cells can work together as an active part of the mucosal barrier.


Assuntos
Comunicação Celular/fisiologia , Células Endoteliais/metabolismo , Células Epiteliais/metabolismo , Mucosa Respiratória/citologia , Junções Íntimas/metabolismo , Brônquios/citologia , Polaridade Celular , Técnicas de Cocultura , Endotélio Vascular/citologia , Células Epiteliais/química , Humanos , Proteínas de Membrana/análise , Proteínas de Membrana/genética , Microscopia Eletrônica , Ocludina , Permeabilidade , RNA Mensageiro/análise
5.
Cancer Immunol Immunother ; 58(5): 789-800, 2009 May.
Artigo em Inglês | MEDLINE | ID: mdl-19066888

RESUMO

Clinical trials are governed by an increasingly stringent regulatory framework, which applies to all levels of trial conduct. Study critical immunological endpoints, which define success or failure in early phase clinical immunological trials, require formal pre-trial validation. In this case study, we describe the assay validation process, during which the sensitivity, and precision of immunological endpoint assays were defined. The purpose was the evaluation of two multicentre phase I/II clinical trials from our unit in Southampton, UK, which assess the effects of DNA fusion vaccines on immune responses in HLA-A2+ patients with carcinoembryonic antigen (CEA)-expressing malignancies and prostate cancer. Validated immunomonitoring is being performed using ELISA and IFNgamma ELISPOTs to assess humoral and cellular responses to the vaccines over time. The validated primary endpoint assay, a peptide-specific CD8+ IFNgamma ELISPOT, was tested in a pre-trial study and found to be suitable for the detection of low frequency naturally occurring CEA- and prostate-derived tumour-antigen-specific T cells in patients with CEA-expressing malignancies and prostate cancer.


Assuntos
Anticorpos Antineoplásicos/sangue , Antígenos de Neoplasias/imunologia , Vacinas Anticâncer/imunologia , Antígeno Carcinoembrionário/imunologia , Ensaios Clínicos Fase II como Assunto/normas , Ensaio de Imunoadsorção Enzimática , Antígeno HLA-A2/imunologia , Interferon gama/sangue , Estudos Multicêntricos como Assunto/normas , Neoplasias/terapia , Fragmentos de Peptídeos/imunologia , Antígeno Prostático Específico/imunologia , Toxina Tetânica/imunologia , Vacinas de DNA/imunologia , Adenocarcinoma/imunologia , Adenocarcinoma/terapia , Animais , Anticorpos Antineoplásicos/biossíntese , Anticorpos Antineoplásicos/imunologia , Seguimentos , Humanos , Imunidade Celular , Masculino , Camundongos , Neoplasias/imunologia , Fragmentos de Peptídeos/genética , Antígeno Prostático Específico/genética , Neoplasias da Próstata/imunologia , Neoplasias da Próstata/terapia , Proteínas Recombinantes de Fusão/imunologia , Linfócitos T/metabolismo , Toxina Tetânica/genética , Reino Unido
6.
J Immunol Methods ; 340(1): 55-64, 2009 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-18983846

RESUMO

Biomarker research has rapidly expanded over recent years aided by the progressive development of research tools, in particular the different multiplex technologies allowing simultaneous measurement of multiple analytes. It is foreseeable that such technology will have an integral role in clinical studies for establishing biomarker profiles of disease status, but validation of the tools is essential to confirm the reliability of their application. More comparable studies between multiplex platforms are required to enable users to determine which of these are best for a particular clinical study, as different platforms will have varying levels of performance for the validation parameters. Comparison of two multiplex platforms, the Luminex and the Mesoscale Discovery, has been performed to determine their performance for the validation parameters of sensitivity, precision and accuracy for the cytokines IL-2, IL-4, IL-8, IL-10, IL-12, IFNgamma and TNFalpha. When measuring high concentrations both platforms show good accuracy (within +/-25% recovery) with all cytokines except IL-12 for the MSD. At low concentrations, +/-25% recovery was seen with all cytokines except IL-2 and IL-8 for the Luminex and IL-2 and IL-12 for the MSD. Although quantitative differences are found, relative differences are comparable, and consequently both platforms have been shown to be suitable for analyzing trends in multiple cytokine profiles, with the Luminex having better precision and the Mesoscale Discovery having greater sensitivity.


Assuntos
Citocinas/análise , Medições Luminescentes/normas , Kit de Reagentes para Diagnóstico/normas , Humanos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
7.
Bioanalysis ; 8(20): 2169-76, 2016 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-27628669

RESUMO

We focus here on how the interface in academia has adapted in their approach to assessing the PDs of biological agents to better understand mechanisms at an early stage. This understanding enables drugs to be modified early and to be reassessed before progressing to late stage trials. We discuss how these efforts are now being bolstered by a network of consortia involving industry, academia and regulatory bodies, to bring together resources, knowledge and a harmonization in bioanalytical techniques. We highlight how the regulatory guidance still lags behind the rapid advancement in biologicals and associated analytical techniques, especially in immunotherapies and immunological bioassays. Despite this, new collaborative groups are working together to deliver robust and accurate results essential for identifying the most promising drugs to progress from early phase academic research to late phase industry based trials. We show how the relationship between academia and not-for-profit organizations with large pharma and emerging biotech companies has shifted toward a more collaborative effort in bringing new therapies to the forefront.


Assuntos
Academias e Institutos , Indústria Farmacêutica , Preparações Farmacêuticas/metabolismo , Pesquisa , Transferência de Tecnologia , Biomarcadores/análise , Comportamento Cooperativo , Avaliação Pré-Clínica de Medicamentos , Preparações Farmacêuticas/análise
8.
Clin Cancer Res ; 21(6): 1321-8, 2015 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-25589626

RESUMO

PURPOSE: This phase I study aimed to establish the biologic effects and MTD of the agonistic IgG1 chimeric anti-CD40 antibody ChiLob7/4 in patients (pts) with a range of CD40-expressing solid tumors and diffuse large B-cell lymphoma, resistant to conventional therapy. Potential mechanisms of action for agonistic anti-CD40 include direct cytotoxic effects on tumor cells and conditioning of antigen-presenting cells. EXPERIMENTAL DESIGN: ChiLob7/4 was given by IV infusion weekly for 4 doses at a range from 0.5 to 240 mg/dose. Validated ELISAs were used to quantify ChiLob7/4 in serum and test for anti-chimeric MAb (HACA) responses. Pharmacodynamic assessments included quantitation of T-cell, natural killer-cell, and B-cell numbers and activation in blood by flow cytometry and a panel of cytokines in plasma by Luminex technology. Planned dose escalation was in cohorts of 3 patients until MTD or biologic effect, defined as reduction of peripheral blood CD19(+) B cells to 10% or less of baseline. RESULTS: Twenty-nine courses of treatment were given to 28 subjects. The MTD was 200 mg × 4, with dose-limiting toxicity of liver transaminase elevations at 240 mg. At 200 mg (range between 2.1 mg/kg and 3.3 mg/kg based on patient body weight), the trough level pretreatment was above 25 µg/mL. Grade 1-2 infusion reactions were seen above the dose of 16 mg, but could be prevented with single-dose corticosteroid premedication. HACA responses were seen after doses between 1.6 mg and 50 mg, but not above this. There were dose-dependent falls in blood B-cell numbers accompanied by reduced expression of CD21, and transient reductions in NK cell numbers with increased CD54 expression from 50 mg upward. MIP-1ß and IL12 plasma concentrations rose after doses above 16 mg. Fifteen of 29 treatments were accompanied by disease stabilization for a median 6 months, the longest for 37 months. CONCLUSIONS: ChiLob7/4 can activate B and NK cells at doses that can be administered safely, and should be tested in combination with other antibodies and chemotherapy agents.


Assuntos
Linfócitos B/imunologia , Antígenos CD40/antagonistas & inibidores , Imunoglobulina G/uso terapêutico , Células Matadoras Naturais/imunologia , Ativação Linfocitária/efeitos dos fármacos , Linfócitos T/imunologia , Adulto , Idoso , Anticorpos Monoclonais/uso terapêutico , Células Apresentadoras de Antígenos/imunologia , Antineoplásicos/uso terapêutico , Antígenos CD40/biossíntese , Antígenos CD40/imunologia , Quimiocina CCL4/sangue , Feminino , Humanos , Imunoglobulina G/efeitos adversos , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Molécula 1 de Adesão Intercelular/biossíntese , Subunidade p35 da Interleucina-12/sangue , Ativação Linfocitária/imunologia , Contagem de Linfócitos , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Linfoma Difuso de Grandes Células B/patologia , Masculino , Dose Máxima Tolerável , Pessoa de Meia-Idade , Receptores de Complemento 3d/biossíntese , Transaminases/metabolismo
9.
Bioanalysis ; 1(2): 285-91, 2009 May.
Artigo em Inglês | MEDLINE | ID: mdl-21083167

RESUMO

The current decade has seen an evolution in biomarker research, with a breakthrough from traditional single analyte studies to simultaneous multiple analyte technologies, aided by the progressive development of research tools and the discovery of many novel biomarkers. It is foreseeable that the application of such technologies will have an integral role in clinical studies for establishing biomarker profiles of disease status and prognosis. However, the transfer of such complex procedures to a regulated environment presents many obstacles. Here, we discuss some of these applied technologies and the validation approaches we have taken as an academic unit to prove their suitability and appropriateness for clinical application. We discuss the advantages and limitations for such end point assays in early Phase clinical trials.


Assuntos
Biomarcadores/análise , Pesquisa Biomédica/métodos , Técnicas de Química Analítica , Aprovação de Drogas , Pesquisa Translacional Biomédica , Ensaios Clínicos como Assunto , Humanos , Ciência de Laboratório Médico , Prognóstico , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
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