Archive | 2010
In a FOCUS workshop at Heidelberg Technology Park Dr. Bernd Liedert of Merck Serono (Darmstadt, Germany) provided his views on a risk-based strategy for the assessment of immunogenicity directed against therapeutic proteins.
He started out with the fundamental 2004 citation of the FDA’s Dr. Amy Rosenberg that “All proteins are potentially immunogenic”. Immunogenicity results from two mechanisms, a reaction to neo-antigens and/or the breakdown of immune tolerance, whereby the latter is very much dependent on aggregate formation of the antigen. Aggregate formation allows direct B lymphocyte activation through cross-linking of surface-bound IgM / IgD containing B-cell receptors without the need for antigen-specific help by T lymphocytes.
Dr. Liedert put the topic into perspective by summarizing the potential consequences of anti-drug antibodies (ADAs): Although formation of these ADAs has no clinical consequences in most cases, a sometimes even dramatic impact on safety and/or efficacy cannot be ruled out (Fig. 1).
In the first major part of his presentation Dr. Liedert reviewed the current regulatory framework in Europe (EMA; Fig 2) and the US, whereby in the US a series of four white papers provided basic advices, which were recently compiled in a first FDA Guidance on assay development (Fig. 3). Dr. Liedert emphasized that analysis of immunogenicity is one essential pillar in the assessment of biosimilars and therefore part of corresponding regulations.
In the second part Dr. Liedert addressed the tiered concept of “screening” and “confirmatory” assays for immunogenicity testing and outlined some technical aspects and challenges of current anti-drug antibody assay formats (Figs. 4 and 5).
A major issue in the development process of a given therapeutic protein is assessment of its immunogenicity risk. To this end, Dr. Liedert provided his view on the evaluation of primary and secondary immunogenicity risk factors, which describe and rate clinical consequences and probability of anti-drug immune responses (Fig. 6 and 7). This evaluation then leads to allocation of the drug into a low / medium or high risk category.
The classification helps to define a risk-adapted immunogenicity testing strategy tailored to the specific compound (Fig. 8). Extent and frequency of testing has to be defined as well as degree of assay validation. Concomitant analysis of pharmacokinetics and monitoring of appropriate safety/efficacy marker might support association of positive findings with potential clinical consequences.
In most cases it is acceptable to analyze samples, collected for immunogenicity testing, after the end of a clinical trial. For therapeutic proteins, where immunogenicity-induced depletion of an unique endogenous counterpart is a serious concern, testing may be performed in real time and NAb-positivity may be defined as withdrawal criterion. In this case it should be warranted that the applied assays are capable to measure ADAs in the presence of drug (especially for protein-drugs with a long half-life).
Taken together, Dr. Liedert provided very valuable information derived from his hands-on experience in immunogenicity testing with a clear focus on decision making in specific development projects. He concluded by stating that “any immunogenicity strategy will always have to be adapted individually for each project” and that sponsors should not hesitate to seek Scientific Advice at the Health Authorities.
Disclaimer: The views expressed in this summary are the personal views of Dr. Bernd Liedert and may not be understood or quoted as being made on behalf of, or reflecting the position of Merck Serono.
In the recent FOCUS workshop at Heidelberg Technology Park Dr. Isabel Buettel of the Paul-Ehrlich-Institute provided information on the perspective of European Regulators on immunogenicity testing for therapeutic proteins.
She addressed the role of the Paul-Ehrlich-Institute and the CHMP guideline on “Immunogenicty assessment of biotechnology derived therapeutic proteins” and the Concept Paper on “Immunogenicity assessment of Monoclonal antibodies intended for in vivo clinical use”. Biotechnological products are more than the drug substance, since e.g. small changes in the production process can have a high impact on the resulting product, in particular on its immunogenicity.
In other words: for biotechnological products “The process is the product” and the complete production process needs to be considered when designing an immunogenicity testing strategy.
Dr. Buettel also provided advice on factors influencing the immunogenicity of biotechnology products and the risk factors to be considered when developing an immunogenicity testing strategy (Fig. 1), the technical key principles of an assay strategy (Fig. 2), and the key principles of immunogenicity testing in clinical trials (Fig. 3) and in pharmacovigilance. For the latter, immunogenicity aspects should always be included into the risk management plan.
Dr. Buettel stressed that the EMEA sees biotechnology derived products as individuals and “that standard algorithms cannot be used for immunogenicity testing of a given compound”.
Dr. Buettel concluded with an overview on a recent workshop held at the Paul-Ehrlich-Institute on immunogencity testing (“Taking immunogenicity assessment of therapeutic proteins to the next level”), the summary of which will be published soon.
Inhibitors of Tumor Necrosis Factor-alpha (TNF) have improved the therapy of chronic inflammatory diseases. One such inhibitor molecule is etanercept, a competitive inhibitor for TNF.
Zaba and colleagues explored the pharmacodynamics of etanercept in psoriatic patients. By using gene expression profiling they compared lesional and non-lesional skin biopsies from responders and non-responders under etanercept therapy.
Their comparison revealed a rapid down-regulation of IL-1ß and IL-8 in both groups. However, IL-17-related pathway genes were down-regulated to baseline levels, in responders only.
This was taken to conclude that in psoriasis “… ultimate Th17 suppression is necessary for response to TNF-inhibiting drugs.” In other words, the IL-17 producing T-Helper (Th17) cell appears as the critical target in the etanercept therapy of psoriasis vulgaris.
FOCUS Immunology is prepared to provide state of the art analyses for Th17-related PD studies, under GLP conditions.
If you are interested in learning more about FOCUS Immunology’s experience and offers or if you want to discuss your specific experimental needs, please feel free to contact Dr. Eddy Bruyns, Head of FOCUS Immunology Laboratory via e-mail eddy.bruyns@focus-cdd.com or via telephone +49 6221 64935124.
Source
Zaba et al.
Effective treatment of psoriasis with etanercept is linked to suppression of IL-17 signaling, not immediate response TNF genes
J Allergy Clin Immunol 2009;124:1022-1030
On Wednesday, September 22, 2010, from 13:00 until 17:00 h, a workshop will be held at Technologiepark Heidelberg GmbH, Conference Room Europa, Im Neuenheimer Feld 582, 69120 Heidelberg on:
Immunogenicity of Biologicals
Identification and handling of immunogenicity – strategies and practical approaches
The detailed programme can be found here:
Participants are welcome!
T lymphocytes mature into CD4- or CD8-expressing single positive cells in the thymus from where they become “peripheral T lymphocytes” which populate the secondary lymphoid organs. Upon interaction with antigen-presenting cells, peripheral T lymphocytes become activated, proliferate and expand to differentiate into effector and memory cells.
Until some years ago two functionally different CD4+ T helper (Th) cell types were known, i.e. “Th1 cells” which are induced by the action of IFN-gamma and IL-12 to protect against specific intracellular pathogens, and “Th2 cells” which are induced by IL-4 to encounter parasitic infections and also to participate in allergic reactions.
Additional CD4+ Th subtypes have been described meanwhile.
First the “Th17 effector T cell”, which produces IL-17 and IL-6 and which is generated by the action of IL-23. Th17 cells function to clear a range of pathogens distinct from Th1 and under pathological conditions they are involved in autoimmune responses. The latter has sparked general interest for these cells as targets for immuno-modulatory and anti-inflammatory therapies.
“Treg cells” (CD4+, CD25+, FoxP3+) represent another variant of CD4+ effector T-cells with a pivotal role in the regulation of immune responses.
FOCUS Immunology provides state-of-the-art scientific support and services to study these cell types under GLP conditions. If you are interested in learning more about FOCUS Immunology’s experience and offers or if you want to discuss your specific experimental needs, please feel free to contact:
Dr. Eddy Bruyns, Head of FOCUS Immunology Laboratory via e-mail eddy.bruyns@focus-cdd.com or via telephone +49 6221 64935124.
Bettelli et al Nat Immunol 2007 8:345-350
Shevach Immunol Rev 2006 212: 60-73
Murphy Nat Rev Immunol 2002 2: 933-944
In a workshop hosted by the European Medicine Agency the possible role of in vitro cytokine release assays for prediction of unwanted side effects of novel (immuno-) therapeutics has been discussed. With respect to the latter the “cytokine release syndrome” and “cytokine storm” are severe and sometimes fatal adverse clinico-pathological events for which predictive assay systems are urgently needed.
In the workshop the central role of so-called “multiple cytokine secreting cells” (also termed “multiple cytokine producing cells”) in the pharmaco-dynamics of immuno-modulatory compounds and associated adverse events was pointed out.
As the name implies, these cells are characterized by the production of several cytokines in any single cell and thus contrast cells in which only one or two cytokines may be detected. In other words: “Multiple Cytokine Secretion” / “Multiple Cytokine Production” is a functional characteristic of a given cell and pretty much every T cell may become such a multiple cytokine secreting cell upon activation. Multiple cytokine secretion is a temporary state, only as long as the cell is being activated.
Physiological stimulation of T cells to multiple cytokine secretion is required for host defense and tissue remodeling. However, aberrant or un-intentional stimulation under pathological conditions or by (over-dosing of) immuno-modulatory compounds may result in severe pathologies, such as the above-mentioned “Cytokine Release Syndrome“.
The analysis of multiple cytokine secreting cells requires sophisticated multi-parameter assays, like multiplex immunological assays for measuring cytokine profiles in cellular secretions under various stimulation conditions. Ultimately, it requires multi-color intracellular cytokine staining by flow cytometry, which allows multiple cytokine determinations at the single level.
FOCUS Immunology is experienced in providing state-of-the-science analyses for multiple cytokine secreting cells under GLP conditions. If you are interested in learning more about FOCUS Immunology’s experience and offers or if you want to discuss your specific experimental needs, please feel free to contact
Dr. Eddy Bruyns, Head of FOCUS Immunology Laboratory via e-mail eddy.bruyns@focus-cdd.com or via telephone +49 6221 64935124.
EMEA workshop: “In vitro cytokine release assays to predict Cytokine Release Syndrome: the current-state-of-the science”
In his presentation Dr. Greb, president and CEO of Focus Clinical Drug Development GmbH (Neuss, Germany), summarizes pertinent issues of early phase development of biologicals.
He points out that careful analysis of the molecular situation is required and that the relevant target structures and species need to be chosen in preclinical evaluation of the compounds activity.
You may wish to download Dr. Greb’s presentation for your personal use. To order a complimentary copy of Dr. Greb’s presentationon specific challenges in human pharmacology trials with biologicals enter your email address below and press the submit button:
A detailed complimentary copy of Dr. Greb’s presentation 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.
Zhang and colleagues explored the immunomodulatory activity of the cholesterol-lowering agent simvastatin. Their pharmaco-dynamic studies indicated that the immuno-modulatory activity of statins may involve two basic mechanisms. First, it may involve an increase in SOCS-3 and SOCS -7, i.e. members of the molecular family of “Suppressors of Cytokine Secretion”. And second, it may involve the inhibition of Interleukin-17 (IL-17), a central pro-inflammatory mediator.
The members of the SOCS family of molecules are important negative feedback regulators in adaptive and innate immune responses, while IL-17 producing CD4+ cells, the so-called “Th17 cells”, play a central role in the development of autoimmune diseases.
From their findings the authors conclude as follows: “Based on the described immunomodulatory mechanisms, good safety profile and oral biovailabalility, statins represent a promising therapeutic approach for multiple sclerosis and other chronic inflammatory diseases.”
Along the lines of this study by Zhang and colleagues, FOCUS Immunology is prepared to provide state-of-the-art services for pharmacodynamic (PD) analyses of immuno-modulatory compounds under GLP conditions.
If you are interested in learning more or in discussing your specific experimental needs, please feel free to contact Dr. Eddy Bruyns, Head of FOCUS Immunology Laboratory via e-mail eddy.bruyns@focus-cdd.com or via telephone +49 6221 64935124.
Source
Zhang et al.
Simvastatin Inhibits IL-17 Secretion by Targeting Multiple IL-17-Regulatory Cytokines and by Inhibiting the Expression of IL-17 Transcription Factor RORC in CD4+ Lymphocytes
The Journal of Immunology 2008; 180; 6988-6996
Hyperactivation of the immune system can lead to tissue destruction and auto-immunity. Therefore, the amplitude and duration of immune responses after antigenic and cytokine signaling are regulated in a feedback manner.
So-called “Suppressors of Cytokine Signaling (SOCS)” are important molecular players in this negative feedback regulation. 7 members of the SOCS molecular family have been described so-far. The SOCS family members 1 and 3 are of particular importance, since they are involved in adaptive and innate immune responses.
SOCS-1 and -3 are induced by LPS stimulation via the Toll-like Receptor-4 (TLR-4). While SOCS-1 directly targets the downstream signaling molecules of TLR4, SOCS-3 regulates the secondary effects of many LPS-induced cytokines. E.g. IL-1R signalling pathways are negatively regulated by SOCS-3.
In adaptive immune responses SOCS-1 and -3 play a role in the regulation of T cell activation, proliferation and diferentiation.
Recently, SOCS molecules were found to be central elements in the pharmacodynamics of the immuno-modulatory compound simvastatin and probiotic lactobacilli.
FOCUS Immunology is prepared to provide state-of-the-art services for pharmacodynamic (PD) studies for immunomodulatory compounds under GLP conditions. This includes studies on the regulation of intra- and extracellular mediators of inflammationand immunity.
If you are interested in learning more about FOCUS Immunology’s experience and offers or if you want to discuss your specific experimental needs, please feel free to contact Dr. Eddy Bruyns, Head of FOCUS Immunology Laboratory via e-mail eddy.bruyns@focus-cdd.com or via telephone +49 6221 64935124.
Source
Dimitriou et al.
Putting out the fire: coordinated suppression of the innate and adaptive immune systems by SOCS1 and SOCS3 proteins
Immunological reviews 2008, Vol. 224: 265-283
More information on SOCS1: Wikipedia on SOCS1
More information on SOCS3: Wikipedia on SOCS3
Suppressors of Cytokine Signaling (SOCS) are a group of proteins that play an important role in attenuating immune responses. These molecules may thus be important players in the pharmacodynamics (PD) of immuno-modulatory therapies.
In a recent publication Lee and colleagues explored whether probiotics induced anti-inflammatory properties through induction of SOCS.
Helicobacter pylori (H. pylori) or lipopolysaccharides derived thereof were found to increase the expression of pro-inflammatory cytokines in a human gastric cell line. Pre-treatment of the cells with probiotic bacteria of the Lactobacillus group reduced the H.Pylori-induced expression of these cytokines.
When the underlying molecular mechanisms were explored in more detail, the administration of probiotics was found to increase the expression of SOCS-2 and SOCS-3.
The authors conclude that the “Anti-inflammatory signals of SOCS … might be a key anti-inflammatory mechanism of probiotics …”
FOCUS Immunology is prepared to provide state-of-the-art servcies for pharmacodynamic (PD) studies for immunomodulatory compounds under GLP conditions.
If you are interested in learning more about FOCUS Immunology’s experience and offers or if you want to discuss your specific experimental needs, please feel free to contact Dr. Eddy Bruyns, Head of FOCUS Immunology Laboratory via e-mail eddy.bruyns@focus-cdd.com or via telephone +49 6221 64935124.
Source
Jeong Sang Lee et al.
Anti-inflammatory actions of probiotics through activating suppressor of cytokine signaling (SOCS) expression and signaling in Helicobacter pylori infection: A novel mechanism
Journal of Gastroenterology and Hepatology 25 (2010) 194-202
Tuesday, December 14, 2010
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