Archive | 2009
Dr. Eddy Bruyns, FOCUS Immunology, has provided a white paper on FOCUS Immunologies‘ pre-analytical services. He addresses aspects of sample collection, processing and shipment in clinical trials.
He points out procedures that allow cell preparation and transportation for functional analysis in international multi-center trials as well as procedures for collection, transportation and central testing of whole blood sample for flow cytometry.
You may download the white paper here.
Dr. Eddy Bruyns and Dr. Matthias Baumann of FOCUS CDD have summarized information on biomarkers of innate immune system functions. They provide latest information on assay systems for innate immune system parameters (Toll-like receptors [TLR], NOD-like receptors) and the implementation of such assays as PD biomarkers in exploratory clinical studies.
Finally case studies and a compilation of pertinent literature is provided.
To order a complimentary copy of the FOCUS expert report on biomarkers of innate immune system functions enter your email address below and press the submit button:
A detailed complimentary copy of FOCUS’ expert report can be ordered by entering your email address below and pressing the submit button:
“FOCUS Immunology Laboratories Heidelberg” offer testing of biomarkers for immune cell functions tailored to the specific requirements of a given clinical trial.
For further information please contact Dr. Eddy Bruyns at eddy.bruyns(at)focus-cdd.com.
Our basic test program for innate immunity testing quantifies the extent to which ligands specific for the nine known human Toll-like receptors (TLR) stimulate peripheral blood mononuclear cells (PBMC) isolated from individuals. The degree of stimulation is quantified via the amount of Tumor Necrosis Factor-alpha (TNF-alpha) secreted into the culture supernatants.
An example for a typical experiment is given here (click on the image to enlarge view):
PBMC were isolated from the blood of two human volunteers and cultured in the presence of distinct ligands for toll-like receptors (TLR-1 to -9). After a given interval of time the culture supernatants were harvested and tested for the amount of secreted TNF-alpha by enzyme-linked immunosorbent assay (ELISA).
The ligands and the corresponding TLRs are shown on the X-axis. The Y-axis provides the read-out parameter, i.e. amount of TNF-alpha in the culture supernatants. The left-most sample in both diagrams represents TNF-alpha secretion the unstimulated control culture.
Strongest responses were found for the TLR-4 ligand lipopolysaccharide (LPS) and the TLR 8 ligand single-stranded RNA (ssRNA40). The weak-responding TLR 3, 7 and 9 stimulations are again highlighted in the right hand diagram with a differently-scaled Y-axis in order to demonstrate the above-background response.
Please note, that this basic ex vivo assay design can be adapted to the specific requirements of a given project – be it for immune screening purposes in clinical trials or be it for mode-of-action or PD measurements in compound development.
The aim of FOCUS Immunology’s “Immune Screening” testing package is to find out whether novel or known compounds affect basic immune functions; this may alert early on in development to potential side effects in clinical studies and on the other hand it may pave the way towards definition of new indications.
To this end FOCUS Immunology provides a selection of assays comprised in the “Immune Screening Package”. This package is purposefully set to address the “major” aspects of immune cell activation but this package may at any time be complemented by more refined assays from the the “Specific Immune System Package”, the “Innate Immune System Package”, or the “Biologicals (of higher risk) Package”.
Specifically, the following assays are offered to screen for possible immuno-modulatory effects of known or to be developed compounds:
Cell proliferation: Upon stimulation, cell proliferation is determined either in bulk cultures by metabolic assays or by measuring DNA synthesis (BrdU incorporation). Alternatively, cell proliferation can be determined on the single cell level by using a fluorescence-based assay (CFSE assay).
Cytokine release: Upon stimulation cytokine production can be measured using either of three assays: (a) ELISA, to determine the total amount of cytokine(s) secreted into the cell culture medium (link to List), (b) EliSpot, to determine the frequency of cells secreting (a) particular cytokine(s) and (c) Intracellular Cytokine Staining (ICS), to measure multiple cytokines and correlate their expression on the single cell level with lineage markers.
Expression of activation markers: Immune cells are not only characterized by cell-type-specific markers such as CD3 for T cells, CD19 or CD20 for B cells or CD14 for monocytes, but also by a continuously growing panel of CD markers to distinguish subpopulations. Examples are CD4 and CD8 for helper and cytotoxic T cells and CD4 + CD25 for regulatory T cells. Additionally, cells of the lymphoid lineage acquire specific activation markers upon stimulation, which are not expressed on resting cells. Examples for activation markers of T cells are CD69 and CD86.
The combination of lineage and activation markers in one assay (“multi-color flow cytometry”) allows analyzing the functional states of immune cells on the single cell level. This provides refined information on the effects test-compounds on the functionality of immune cell subpopulations.
FOCUS Immunology operates a dual-laser FACSCalibur from BD Biosciences to analyze up to four different markers in parallel allowing a detailed analysis of cell surface and/or intracellular markers.
Cytokines are important mediators of immune cell functionality and may strongly influence the outcome of an immune response. Accordingly, it is important to know whether a compound alters the production and secretion of cytokines partaking in an immune response.
FOCUS Immunology has established ex vivo assays in order to detect effects of compounds on cytokine secretion. To this effect, whole blood cultures or purified blood cells are suboptimally stimulated in order to activate both lympoid and myeloid cells in the presence or absence of the test-compound.
Suboptimal stimulation is used in order to test the compounds in the linear range of the dose-response-curve of the stimulant and which allows the detection of compound-mediated effects.
At termination of the culture, the following cytokines are determined from the supernatant by ELISA: IL-6 and TNF-alpha as prototype pro-inflammatory cytokines, IL-8 as mediator of chemotaxis, IL-2 as a central regulator of immune cell functions, IFN-gamma for its involvement in anti-viral immune responses and IL-10 as the major immunosuppressive cytokine.
FOCUS Immunology’s cytokine package was designed to keep the assay concise and to answer many questions with a reasonable use of resources.
It is of course possible to add or leave out any cytokine for your specific project.
We have experience in analyzing a great many more than the above listed cytokines and are also willing to establish any assay for which reagents are available or if the need is dire enough to develop our own reagents.
The innate or non-specific immune system provides immediate defence reactions against invading pathogens. Granulocytes, monocytes/macrophages, NK-cells, mast cells and dendritic cells are important cellular constituents of the innate immune system.
Cells of the innate immune system do not possess antigen-specific receptors but instead recognize conserved molecules expressed by large numbers of microorganisms. These conserved molecules are referred to as pathogen-associated molecular patterns or PAMPS and the corresponding receptors on cells of the innate immune system are called pattern-recognition receptors or PRRs.
Toll-like receptors (TLR) are an example for PRRs. TLRs recognize different types of molecules expressed by bacteria, viruses, fungi and protozoa. Extensive research has allowed correlating TLRs with the molecules they recognize: e.g. the endotoxin LPS, which is widely expressed by gram negative bacteria, is recognized by TLR 4, while unmethylated CpG-DNA sequences, which distinguishes bacterial DNA from mammalian DNA, is recognized by TLR-9.
FOCUS Immunology has established a complete range of assays to functionally analyse the triggering of TLRs and the subsequent activation of the respective cell.
Ligands for the 9 well characterized human TLRs (TLR1 – TLR9) are used to stimulate Peripheral Blood Mononuclear Cells (PBMC) in vitro and analyze the secretion of the cytokine TNF-alpha.
This assay allows analyzing the effect any compound, whether NCE or NBE, may have on TLR-mediated stimulation of cells of the innate immune system.
See also:
Expert Report – you can obtain your free expert report here.
The advent of biologicals has brought new challenges to drug development. Proteins with immunomodulatory activity such as monoclonal antibodies directed against major switches of the immune system or proteins such as receptor antagonists or agonists may have dramatic effects on immune cell function.
The recent past has shown just how dramatic these effects may be for the well-being of – in this case – volunteers in a clinical study.
The ensuing investigation of this incident resulted in committee recommendations among which the use of human ex vivo / in vitro cultures had a prominent place. This is based on the rationale that biologicals are very often species-specific and thus traditional animal models are in all likelihood not adequate to investigate effects on the immune system.
FOCUS Immunology offers a range of assays to assess the effects of biologicals on different aspects of immune cell activity, which allow a comprehensive analysis of possible immunomodulatory activities.
Specifically, the following assays are provided by FOCUS Immunology : (i) cytokine release, (ii) cell proliferation, (iii) expression of activation markers, (iv) oxidative burst reaction, (v) chemotaxis, (vi) NK cell activity, (vii) cytotoxicity (CDC, ADCC), (viii) NO production, (ix) histamine release, (x) changes in the frequency of defined cell populations.
It may be advisable to test not only resting cells but also cells after suboptimal stimulation in order to account for activation processes which may not suffice by themselves to activate resting cells but may strongly augment activation processes occurring independently.
It also may be advisable to test different means of presenting the tested biological to the cells, from soluble to different types of surface-bound presentation.
FOCUS Immunology has developed methods for on-site isolation of immune cells from peripheral blood, their transportation to Heidelberg, and subsequent functional testing under conditions of international multicenter clinical trials.
In a recent project patients were recruited at medical centers in several countries. At these sites FOCUS implemented its robust and simple protocol for on-site cell preparation. Then FOCUS organised the controlled transportation of the cells to the central testing laboratory in Heidelberg, Germany.
Incoming samples were QC-ed, and cytokine production as well as proliferation was determined in cultures of the isolated immune cells together with various defined stimuli of immune functions.
It took FOCUS only 6 weeks to implement standardized sample generation, sample logistics and testing.
The studies led to further understanding of the compound’s mode-of-action.
FOCUS Immunology offers immune monitoring tailored to the needs of given clinical trial protocols. For further information please contact immunology@focus-cdd.com or contact directly FOCUS’ Head of the Immunology Laboratory Dr. Eddy Bruyns at eddy.bruyns@focus-cdd.com, Tel.: +49 (0) 6221 649 35 124.
Biologicals used as therapeutics may induce unwanted immune responses in treated patients. Thus, the EMEA has recently issued a guideline for immunogenicity assessment. Immunogenicity testing should be part of clinical efficacy and safety studies.
Key elements of the EMEA recommendations in brief: Immunogenicity should be evaluated in all study participants and not only in symptomatic individuals. Bioassays for neutralizing antibodies need to be designed in a product-dependent manner. Clear criteria have to be defined to distinguish positive from negative findings and to confirm positive results.
FOCUS Immunology supports its clients to integrate these recommendations into their repsective clinical trial protocols. FOCUS’ services comprise assay development and validation for preclinical and clinical studies, testing under GLP conditions, implementation of confirmatory assays, development of bioassays for testing of neutralising antibodies and sample logistics in international multicenter trials.
If you have questions or want to discuss immunogenicity testing in your specific development project, please do not hesitate to contact our Head of the Immunology Laboratory Dr. Eddy Bruyns via email (eddy.bruyns@focus-cdd.com) or via telephone (+49 6221 649 35 124).
The current EMEA guideline on immunogenicity testing can be found here.
The overall intent of exploratory clinical studies is to provide information that allows assessing the clinical feasibility of new drug candidates early in the development process. Exploratory clinical trials therefore aim at early evaluation in humans of pharmacokinetic and and phamacodynamic profiles of novel compounds.
The doses applied in such studies are too low to be therapeutic or even to allow any meaningful PD assessment by conventional methods. Therefore, sensitive and specific (“surrogate”) PD markers – “biomarkers” – are required.
In an expert report, Dr. Eddy Bruyns, Dr. Matthias Baumann and Dr. Maikel Raghoebar of FOCUS have compiled information on the use of biomarkers in exploratory clinical studies and they provide a summary of the pertinent regulatory background.
They point out that PD biomarkers are feasible in many therapeutic areas, yet they are especially useful in the development of drugs for anti-inflammation and immuno-modulation. This is exemplified by several case studies.
To order a complimentary copy of the FOCUS expert report on the use of biomarkers in exploratory clinical studies enter your email address below and press the submit button:
A detailed complimentary copy of FOCUS’ expert report can be ordered by entering your email address below and pressing the submit button:
“FOCUS Immunology Laboratories Heidelberg” offer testing of biomarkers for immune cell functions tailored to the specific requirements of a given clinical trial.
For further information please contact Dr. Eddy Bruyns at eddy.bruyns@focus-cdd.com.
Wednesday, October 21, 2009
0 Comments