Utility of lyophilized PMA and ionomycin to stimulate lymphocytes in whole blood for immunological assays

Belouski, S. S.*, Wilkinson, J.^, Thomas, J.*, Kelley, K.*, Wang, S.-W.*, Suggs, S.* and Ferbas, J.* (2010), . Cytometry, 78B: 59–64.

doi: 10.1002/cyto.b.20492

The need to implement robust biomarkers in clinical trials has never been greater, and such efforts can be easily compromised by reagent instability or simple human error during assay set-up. Many biotechnology and pharmaceutical companies are introducing efforts to conduct biomarker studies under more rigorous settings, and the use of plates or tubes pre-loaded with stimulation or staining reagents could be of value for studies that involve flow cytometry.

Author Information:

* Amgen Inc., Department of Medical Sciences

^ Beckman Coulter Inc., Custom BioPharma Group, Miami, Florida (author Julie Wilkinson current address is ImmunoSite Technologies, Fort Lauderdale, FL)

Email: John Ferbas (jferbas@amgen.com)

*One Amgen Center Drive, Mailstop 30E-3-C, Thousand Oaks 91320-1799, CA

Dextramers are superior reagents for the detection of antigen-specific T cells, having the ability to interact simultaneously with multiple receptors on a single T cell with unsurpassed avidity. The increased avidity of Dextramers compared to conventional MHC multimer reagents enhances resolution and signal-to-noise ratio providing a more accurate assessment of the T cell response, and clearly identifying responses previous generation technologies might miss.

In compliance with cGLP quality standards, IST specializes in incorporating antigen-specific responses in assay solutions to evaluate the safety and efficacy of vaccines and biologics.  IST offers expertise in confirming the validity of processes involved in such studies. Together with Immudex’s Dextramer™ technology, IST now offers enhanced capabilities to validate the effectiveness of potential vaccines and biologics, and thereby significantly improve the efficiency and cost-effectiveness of drug development and production.

Through the partnership, IST can refer clients that need to gauge T cell response to Immudex, and Immudex can refer clients that have other immune monitoring needs to IST. IST immune monitoring services include custom assay design, optimization, automation and validation; cell mediated immunity; functional immune monitoring and much more. This alliance adds to the capabilities of both companies while streamlining processes – providing for faster, more accurate results for customers. By accurately gauging the effect of an immunotherapy, researchers can reduce a study’s timeline by weeks or even months, and more importantly, can avoid wrong and costly decisions based on weak and inaccurate data. This can prove invaluable during all phases of clinical trials, driving significant cost savings and productivity.

“If you’ve ever been on a conference call with eight separate vendors trying to figure out if a pharmaceutical is working, then you’ll understand why this partnership is beneficial to our industry,” said Wade Bolton, Ph.D., President of IST. “Immudex offers a technology that is superior in gauging T cell responses and we’re honored to call them partners.”

“IST’s standardization, validation and automation of custom assays are the best in the industry,” said Stephen Haley, Ph.D., Vice President and Director of U.S. Operations for Immudex. “This partnership greatly increases our offering capabilities and is poised to be a great asset to our current and future clients.”

The alliance is not an exclusive partnership. IST still has the ability to use other technologies when deemed more appropriate or to meet a client’s request.

For more information on ImmunoSite Technologies, please visit http://immunositetechnologies.com/; follow IST on Twitter at http://twitter.com/ImmunoSite; or follow on Facebook at http://www.facebook.com/pages/ImmunoSite-Technologies-LLC/167604859950599. For more information on Immudex, visit http://www.immudex.com/.

About ImmunoSite Technologies

Based in Ft. Lauderdale, FL, ImmunoSite Technologies, LLC (IST) offers a full range of contract research (CRO) immune monitoring services to leading biotechnology, pharmaceutical, and academic organizations around the world to provide products and services that span all stages of drug discovery and development. IST is rapidly building a worldwide reputation for services related to qualifying, standardizing, and when appropriate, automating assays associated with cell-mediated immunity. By using qualified reagents, controls, standards and processes, IST-developed functional assays perform within tight specifications and yield reproducible results, helping pharmaceutical and biotechnology companies to accelerate drug discovery, document clinical relevance and reduce costs. The IST team has been distinguished by their ongoing partnerships with best-in-class clinical trial organizations such as: the Immune Tolerance Network, the Immune Tolerance Institute, and the Imperial College of London-managed CD4 Initiative. This extensive cell analysis R&D experience qualifies IST scientists to comply with complex and demanding international scientific and governmental regulations.

About Immudex

Based in Copenhagen, Denmark with North American operations based in Fairfax, Virginia, Immudex is the sole proprietor of the MHC Dextramer technology. Immudex develops and commercializes products for the quantitation, characterization, and generation of antigen-specific T-cell responses for life science research, in vitro diagnostics and vaccine development. Immudex has a number of Research Use Only (RUO) products on the market, two products under development for in vitro diagnostic use, as well as a vaccine candidate in development for one of the most deadly of human diseases.

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Kyle E. Glass, Public Relations/Marketing Manager

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Email: kyle@marketingmatters.net

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Can this be done with any level of confidence in the outcome? Can these assays be performed routinely?

Questions like these are being asked more and more often.

In these days of tight budgets, companies are understandably careful in examining the costs/benefits of sponsoring cell mediated immunity (CMI) assays to interrogate correlates of immunity. Certainly the costs are high, but the benefits can be very significant and far reaching.

In fact, the utility of CMI assays in the assessment of immune response or immunogenicity is increasing significantly as we search for biomarker surrogates to determine vaccine efficacy or therapeutic response.  But though the utility is well supported by science and theory, there have to date been no definitive human clinical trials where CMI assays on their own have correlated with clinical outcome.

This was the main topic of a special Cell Mediated Immunity session conducted as part of the Phacillitate Vaccine Forum in Washington DC on January 25^th, 2011. 

The session was attended by a group of senior executives representing the pharmaceutical, biotechnology and CRO industries, and was moderated by Wade Bolton, Ph.D., CEO and President of ImmunoSite Technologies.

The Phacillitate meeting participants agreed that, while there are plenty of theoretical and logical immunological arguments, and there are ample animal and non-human primate  studies in the literature that demonstrate surrogacy to varying degree, there are no human clinical trials that indicate conclusively that cell mediated immunity assays can provide surrogate biomarkers.

So the next logical question the CMI group addressed was, “Why should I have these assays performed?  It may raise more questions than answers.”  The discussions to address this question were scientific, philosophical and financial.  Currently, there are no validated cellular surrogates of clinical outcome being used routinely in clinical trials and the time and money spent on vaccines and therapies that eventually fail are staggering…in the hundreds of millions of dollars.

 At the same time, there are some immunological truths documented in numerous publications which validate that T-helper cellular response shifts, T-regulatory responses and T-cytotoxic responses are important for an effective cellular immune response dependent on the disease state.  If that is a true statement, then a qualitative and quantitative assessment of intracellular cytokines for indicating T helper responses of a particular type would be a harbinger of clinical outcome.  This would be true for vaccines as well as all therapies that are being administered to either up-regulate or down-regulate immune response.  This would include transplantation, autoimmunity (diabetes, allergy, asthma, MS, Lupus), AIDS and oncology.  Additionally, this would be relevant for all trials evaluating the cellular immunogenicity of small and large molecule biologics.

The Search For Surrogacy: Promise For Vaccine Development & Immunogenicity

With Cell Mediated Immunity functional assays come man challenges, choices and issues; from assay selection and assay design to timing and quality control.  These are ‘functional’ assays, and as such, require stringent assay conditions and tight quality control, from selection of reagents and permeabilizers, to critical timing of assay steps and incubations.

In the search for surrogacy, Dr. Bolton’s team at ImmunoSite Technologies (IST) has spent years developing, standardizing, validating and fully automating CMI functional assays for the determination of intracellular cytokine levels and cytotoxic activities using polyspectral flow cytometry (PSFC) to monitor functional cellular response, as well as assays to determine proliferation, activation, apoptosis, cell signaling, regulatory responses, and antigen specific response.  Using PSFC, they can not only determine cell activity, but also dissect the phenotypic signature to better characterize the immune response.

 From an immunological standpoint, these determinations should be useful in vaccine development and in most therapies that are being administered to regulate immune response.  In addition, these assays are critical to understanding the immunogenicity of both small and large molecule biologics.  But in order to be of value in demonstrating surrogacy, the data collected in the clinical trial setting would need to be highly specific and reproducible.  The challenges in doing so are significant.

“In our laboratories, it became obvious in the very beginning of CMI assay development that every step of the assay had to be evaluated and validated with appropriate controls and, when available, clinical samples,” explains IST’s Bolton. “To address the need for critical timing and incubation issues, automation became very desirable, even necessary, in order to get tight statistics in the data.”  In fact, for a number of years now IST scientists have worked with clinical trial organizations, such as the Immune Tolerance Network and the Immune Tolerance Institute, to standardize assays like these with very promising results.

Most of the Phacillitate CMI session participants believed that CMI assays such as these will become the surrogates for immune response, and are eagerly waiting for the definitive studies to show clinical correlation.  The industry wants standardized assays to interrogate surrogate correlates of immunity.  There is a widespread need for high-throughput, validated, and automated assays to measure immunogenicity.

There is much work to be done, and an investment to be made, but the possibilities of enhancing the success of immune based therapies are great.

What are your thoughts?

So, what do you think about the practicality of running validated and standardized cellular assays to interrogate surrogate correlates of immunity in clinical trial settings?  Can this be done with any level of confidence in the outcome? Can these assays be performed routinely?  We would like to know your thoughts on this topic.

Topics included: Do you want standardized assays to interrogate surrogate correlates of immunity? Do you need high-throughput, validated, and automated assays to measure immunogenicity?

Summary of group discussion can be found in the Immune Monitoring Update Blog.