Ferdousi Chowdhury

Validation and comparison of two multiplex technologies, Luminex and Mesoscale Discovery, for human cytokine profiling.

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.

Interactions between endothelial cells and epithelial cells in a combined cell model of airway mucosa: effects on tight junction permeability.

ABSTRACT 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 Ω..cm2 compared to epithelial or endothelial monolayers alone (1400 ± 70 or 80 ± 12 Ω..cm2, 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.

Clinical and Biological Effects of an Agonist Anti-CD40 Antibody: A Cancer Research UK Phase I Study

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-1β 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. Clin Cancer Res; 21(6); 1321–8. ©2015 AACR.

PD-L1 and CD8+PD1+ lymphocytes exist as targets in the pediatric tumor microenvironment for immunomodulatory therapy

Recent monoclonal antibody trials targeting the PD1/PD-L1 immune-checkpoint pathway have shown remarkable success in treating adult malignancies, with PD-L1-expressing tumors showing the most objective response. However, little is known as to whether pediatric cancers have also adopted this immune evasion mechanism. We evaluated 115 pediatric tumors (taken at diagnosis) for PD-L1 expression and the presence of CD8+ tumor-infiltrating lymphocytes (TILs). Tumors with >5% PD-L1 membrane staining were scored positive. The presence of CD8+ TILs expressing PD-1 was assessed using dual-labeling immunohistochemistry. Data were evaluated against clinical demographics. The proportion of PD-L1+ tumors was 86% for alveolar rhabdomyosarcoma (12/14), 72% for high-risk neuroblastoma (31/43), 57% for Ewings sarcoma (8/14), 50% for embryonal rhabdomyosarcoma (8/16) and 47% for osteosarcoma (7/15). Increased proportions of CD8+ TILs significantly correlated with PD-1 expression. When grouped by cancer type, those with the highest proportion of PD-L1 positivity showed poorest survival. PD-L1+ patients with a particularly high frequency of CD8+ TILs (but not those with low numbers CD8+ TILs) had significantly better survival compared to PD-L1 negative patients. This study reveals the presence of an active PD-L1 pathway in a high proportion of pediatric cancers, as demonstrated by strong PD-L1 positivity and the presence of PD-1 expressing CD8+ TILs. In addition, patients with high proportions of CD8+ TILs showed better survival, suggesting that bolstering CD8+ T-cell responses through PD-1/PD-L1 blockade would be a viable treatment strategy, providing support for expediting these targeted immunotherapies in children.

Fit for purpose? A case study: validation of immunological endpoint assays for the detection of cellular and humoral responses to anti-tumour DNA fusion vaccines

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 IFNγ ELISPOTs to assess humoral and cellular responses to the vaccines over time. The validated primary endpoint assay, a peptide-specific CD8+ IFNγ 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.

Ex Vivo Assays of Dendritic Cell Activation and Cytokine Profiles as Predictors of In Vivo Effects in an Anti-Human CD40 Monoclonal Antibody ChiLob 7/4 Phase I Trial

Chowdhury and colleagues use whole blood samples in ex vivo assays to show that ChiLob 7/4 induces a pattern of DC activation and cytokine secretion matching the in vivo responses, providing a strategy to predict dosages and cytokine release syndromes. Immunostimulatory antibodies entering the clinic create challenge in terms of not only pharmacodynamics for monitoring anticipated mechanisms but also predetermining cytotoxicity. We show the use of ex vivo whole-blood samples to predict the activation requirements, cytokine signature, and adverse events of an anti-human-CD40 chimeric IgG1 antibody, ChiLob 7/4. Assessments were initially undertaken on human myeloid (mDC1) and plasmacytoid (pDC) dendritic cells, in which an absolute need for cross-linking was shown through the upregulation of activation markers CD83 and CCR7. Subsequent cytokine secretion evaluations of ex vivo whole blood showed the cross-linked antibody-induced increases in MIP1β, interleukin (IL)-8, IL-12, TNFα, and IL-6. This cytokine signature compared favorably with the Toll-like receptor (TLR) ligand lipopolysaccharide (LPS), in which levels of TNFα and IL-6 were significantly higher, suggesting a less intense proinflammatory response and possible modified cytokine release syndrome when used in human trials. Following first-in-human use of this agent within a dose escalation study, in vivo evaluations of dendritic cell activation and secreted cytokines closely matched the predetermined immunomonitoring endpoints. Patients showed a comparable pattern of MIP1β, IL-8, and IL-12 secretion, but no TNFα and IL-6 were identified. Mild symptoms relating to a cytokine release syndrome were seen at an equivalent dosage to that observed for dendritic cell activation and cytokine release. In summary, ChiLob 7/4 induces a distinctive pattern of dendritic cell activation and cytokine secretion in ex vivo assays that can be predictive of in vivo responses. Such preclinical approaches to monoclonal antibody evaluation may inform both the starting dosages and the anticipated cytokine release events that could occur, providing a valuable adjunct for future first-in-human assessments of immunostimulatory antibodies. Cancer Immunol Res; 2(3); 229–40. ©2013 AACR.

Enumeration and Phenotypic Assessment of Human Plasmacytoid and Myeloid Dendritic Cells in Whole Blood

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.

From research to regulated: challenges in transferring methods

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.

Abstract LB-142: A trial of agonistic anti-CD40 antibody: Biological effects in a Cancer Research UK phase I study.

Introduction: This phase I study aimed to establish the biological effects and maximum tolerated dose (MTD) of the agonistic IgG1 chimeric anti-CD40 antibody, Chi Lob 7/4, in patients (pts) with a range of CD40 expressing solid tumors and diffuse large B-cell non-Hodgkin lymphoma (DLBL), resistant to conventional therapy. Potential mechanisms of action for agonistic anti-CD40 include direct cytotoxic effects on tumor cells, recruitment of immune effectors and conditioning of antigen-presenting cells Methodology: Chi Lob 7/4 was given by IV infusion weekly for 4 doses at a range from 0.5 to 240mg/dose. Validated ELISA9s were used to quantify Chi Lob 7/4 in serum and test for anti-chimeric MAb (HACA) responses. Pharmacodynamic assessments included quantitation of T-, NK-, and B-cell numbers and activation in blood by flow cytometry, a panel of cytokines in plasma by Luminex® technology and phenotypic assessment of human plasmacytoid and myeloid dendritic cells (DC) by a novel 6 colour flow cytometry panel Planned dose escalation was in cohorts of 3 pts until MTD or biological effect, defined as reduction of peripheral blood CD19+ B-cells to 10% or less of baseline. Results: Twenty-eight pts with CD40+ tumors were treated. 11 had mesothelioma, 3 melanoma, 2 oesophageal, 2 colorectal, 2 head and neck, 2 cervical, 2 pancreatic, 2 DLBL and one each lung and thymic carcinoma. One pt with mesothelioma was re-treated with 240mg at an interval of more than 3 years, having experienced prolonged disease stabilisation after initial treatment at the 1.6mg level. Doses of up to 160mg x 4 were well-tolerated, but two pts treated with 240mg experienced grade 3 elevation of hepatic transaminase levels after 1 dose, indicating dose-limiting toxicity. The dose was reduced to 200mg x 4, and 6 pts have been treated at this level, with 1 experiencing grade 3 elevation of liver enzymes after 2 doses but 5 completing 4 doses without difficulties. Infusion reactions occurred at 16mg doses, largely preventable by corticosteroid premedication. Chi Lob 7/4 levels were measurable at doses of 50mg and above. At 200mg, Cmax on day 1 was 47-60mcg/ml, with terminal half life > 7 days and levels > 30 mcg/ml detectable at 1 week. HACA responses were common at low doses (5-50mg) but absent at higher doses (160-240mg), coincident with dose-dependent partial depletion of peripheral blood CD19+ B-cells. Transient falls in blood NK-cells occurred after the first dose of Chi Lob 7/4 at 16mg or greater, with elevated MIP-1beta maximal at 3-6 hours. Dose-dependent elevations in IL-12 occurred above 160mg, rising throughout the period of treatment. Activation of NK cells and monocytes was detected by elevated CD54 expression at doses of 160mg or above, but there was no consistent change in T-cell markers. Response assessment at week 8 showed stable disease after 15 courses, and progression after 14. The median duration of disease stabilization was 6 months, maximum 37. The pt re-treated for progressive mesothelioma after 3 years remains with stable disease at 17 months after a single 240mg dose. Conclusion: This antibody elicits B-cell depletion, and there was evidence of consistent NK and macrophage activation. The dose-limiting toxicity was liver enzyme rise, with the MTD being 200mg/dose (range between 2.1 - 3.3 mg/kg based on patient body weight). Disease stabilization in some cases suggests that further testing is indicated in CD40-expressing malignancies. Citation Format: Peter W. Johnson, Ruth Challis, Ferdousi Chowdhury, Claude Chan, Anna Smith, Neil Steven, Ceri Edwards, Margaret Ashton-Key, Elisabeth Hodges, Alison Tutt, Christian Ottensmeier, Anthony Williams, Martin Glennie. A trial of agonistic anti-CD40 antibody: Biological effects in a Cancer Research UK phase I study. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-142. doi:10.1158/1538-7445.AM2013-LB-142

Transfer from research/academia to clinical/regulated

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.