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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.

IST quite literally helped “write the book” on cell analysis testing.  They were asked to contribute a chapter of a book titled Validation of Cell-Based Assays in the GLP Setting: A Practical Guide, by authors/editors Uma Prabhakar and Marian Kelley (Publisher John Wiley and Sons, 2008, ISBN 0470028769, 9780470028766).  Chapter 8 of this book entitled “Intracellular cytokine detection by flow cytometry” was authored by IST scientists Julie G. Wilkinson, Carlos L. Aparicio, and Wade E. Bolton.

Readers gain an understanding of the details and the high level considerations of assay qualification for difficult cell-based assays.  The level of optimization described for cell-based assays lends itself to biomarker assay automation which can be used for biomarker qualification and validation studies to the satisfaction of the FDA, EMEA, and other regulatory agencies.   Cell-based assay platforms covered are flow cytometry, intracellular cytokine ICS, immunophenotyping, elispot, IHC, cylex, neutralization bioassays, and endpoint assays.