As Immunologists, we are encouraged by the fact that the FDA approved the biotech drug known chemically as ipilimumab, even though it only worked in a small segment of patients studied, and on average they lived just four months longer than patients given older medications. Ipilimumab is part of a group of targeted cancer medicines that harness the body’s immune system to fight off cancer, rather than attacking the disease with outside chemicals like chemotherapy. The drug works by blocking a molecule linked to immune cell response called CTLA-4, and maintaining activity of white blood cells. When the molecule is blocked, the cells maintain an “activated phenotype” and are able to fight off cancer.

The fact that Yervoy (ipilimumab) proved to prolong life for some patients is a good start in finding more effective ways to treat melanoma, which is often unresponsive to therapy.  While some will argue the value of the cost and time involved for this result, experts in immune monitoring and surveillance see promising research progress that may someday soon lead to more significant and meaningful treatments for those suffering from metastatic melanoma.

Two-tiered strategy may improve outcome for treating melanoma

Why is it an important milestone for treating melanoma? “If a single biologic, CTLA4, enhances the outcome, now researchers have the capability of adding in therapeutic strategies that are scientifically relevant to hopefully further improve the outcome”, says Sybil D’Costa, Ph.D., VP of R&D at ImmunoSite Technologies. 

For example, D’Costa suggests a two-tiered strategy that would be immunologically relevant and scientifically sound: possibly a “tickle” of melanoma specific T cells with DCs pulsed with melanoma peptides, and then the CTLA4 therapy to lengthen the timeline of the activation. This would also limit the immune-related side effects by targeting the immune response to the cancer.

In that instance the immune monitoring panels would be:

1. An evaluation of Dendritic cell function pre and post autologous cancer presenting Dendritic cell pulse

2. A determination of downstream effects of the DC pulse by evaluating surface activation and rafting of cancer specific CD4 and CD8 pre and post DC pulse and CTLA4 treatment

3. An evaluation of antigen specific functionality in helper and cytotoxic T cells pre and post CTLA4 treatment

4. An evaluation of shift in T regulatory cell activity

5. An evaluation of Tumor infiltrating lymphocytes in comparison to antigen (cancer) specific circulating lymphocytes

Such combinatorial strategies that are scientifically relevant are already being discussed and evaluated in the literature: 2011 Apr 5. CTLA-4 blockade increases antigen-specific CD8(+) T cells in prevaccinated patients with melanoma: three cases. http://www.ncbi.nlm.nih.gov/pubmed/21465316.

Contract Research Organizations (CROs) like ImmunoSite Technologies (IST) are very involved with pre-clinical and clinical research to assist drug and biologic companies in developing immune monitoring assays to assess the performance of drugs such as this.  While the initial results may be marginal, the progress towards a better drug treatment is clear.  It is significant that the FDA has approved the drug Yervoy for late-stage or metastatic melanoma. It should encourage more drug research approaches such as the two-tiered strategy outlined here.

Progress leads to more questions

Do you agree that the drug Yervoy (ipilimumab) has proven to be a “significant milestone” in treating melanoma?

What are your thoughts on the two-tiered strategy outlined in this post?  How would you amend it?

What are your thoughts on the potential for future cancer medicines that harness the body’s immune system to fight off cancer?  What research would you like to see conducted?

Update:  Two-tiered Strategy Proves Successful at Dana Farber Cancer Institute

In an April 27th announcement (http://www.dana-farber.org/abo/news/press/2011/new-technique-extends-cancer-fighting-cells-potency-in-melanoma-patients.html), the Dana Farber Cancer Institute talks about successful use of a two-tiered or combinational strategy for immune modulation in cancer.  This small study supports the two-tiered strategy suggested in this post.  In the Dana Farber Cancer Institute study, the positive results, five of five patients seemed to respond, indicate that such a combinational strategy would in fact help make a drug like Ipilimumab a scientifically relevant drug for immune modulation in cancer.

Until the investment is made to identify these surrogates, the marketplace will be condemned to relive the current paradigm of high failure rates, very high overall costs associated with vaccine and drug development, few therapies reaching patients with needs, and very slow changes in prevention, morbidity and mortality rates.  Identification of a few surrogate markers in this field would have a profound effect on all of the above and would be a wise investment.

As the search continues for surrogates to determine vaccine efficacy and therapeutic response,   the utility of cell mediated immunity (CMI) assays in the assessment of immune response or immunogenicity is increasing significantly.

Once critical assay reproducibility and robustness of data has been established, what needs to be done in relevant clinical trial settings to identify immune markers that can be used as surrogates of efficacy?

ELISPOT, ICS assays or both to determine CMI?

There are several advantages to using Enzyme-linked Immunosorbent Spot (ELISPOT) assays to interrogate cell functionality.  ELISPOT assays do require fewer cells compared to intracellular cytokine staining (ICS) assays and there have been studies that have shown comparability of fresh to frozen PBMCs in ELISPOT validation studies. Both these parameters are critical for the logistics of running clinical trials. ELISPOT is also an easier assay to run compared to ICS (ELISA with cells), however, there continues to be issues with precision in both assays being run manually with acceptable CVs being as high as 70%.  (Ref: Clin Vaccine Immunol. 2009 February; 16(2): 147–155 Concordant Proficiency in Measurement of T-Cell Immunity in Human Immunodeficiency Virus Vaccine Clinical Trials by Peripheral Blood Mononuclear Cell and Enzyme-Linked Immunospot Assays in Laboratories from Three Continents (CVs of 30% or less but some as high as 70% for positivity 50spots/10 6 cells), and J Immunol Methods. 2011 Jan 5;363(2):143-57. Quality assurance of intracellular cytokine staining assays: analysis of multiple rounds of proficiency testing. (CVs of less than 35% for 0.2% and higher positivity).

A key disadvantage of ELISPOT is the inability for polyspectral immunophenotyping using lineage-specific markers to identify the responding cell populations. Given the publications in the past five years on the association of long-term non-progression in HIV positive individuals with higher polyfunctionality of the cellular response, it is anticipated that these are the kinds of studies that will need to be conducted in large scale clinical trials to determine if vaccine and therapeutic strategies elicit similar responses. (Ref: HIV nonprogressors preferentially maintain highly functional HIV-specific CD8 T cells; Blood 2006;107:4781-4789)

Recent publications in the field have identified biomarkers of tolerance in transplant patients that have required a holistic systems biology approach (combining gene expression-secreted protein profiling, polyfunctional flow cytometry evaluations as well as ELISPOT evaluations). (Ref: J Clin Invest. 2010 Jun 1;120(6):1848-61, Development of a cross-platform biomarker signature to detect renal transplant tolerance in humans)

Can CMI assays be performed in a more cost conscious manner?

Both ELISPOT and polyspectral flow cytometry (PSFC) assays are more complex than most of the assays used in current clinical trial settings.  First, both assay formats are configured to assess function and not just presence of cell types.  And, although we would like to identify a single marker as a surrogate, many studies now predict that there will be a mosaic signature, whether it is a cellular or gene expression assay.  In some initial studies with clinical trial organizations, ImmunoSite Technologies (IST) used up to 60 different five-color combinations as screening tools to dissect the immune response and to find the candidate markers of choice.

Once the candidate marker cocktails have been identified and validated, the cost associated with testing can be significantly reduced.  Plus, the automation of these assays has not only reduced associated labor costs, but has improved reproducibility and has significantly reduced the cost of retesting of samples.  All of these progressive steps have reduced sample testing costs while at the same time improved assay results.

Can the paradigm be changed?

Given critical assay reproducibility and robustness of data, would not a holistic approach be best to identify immune markers that can be used as surrogates of efficacy in relevant clinical trial settings?  And given that the right screening can identify the immune response and find candidate surrogate markers of efficacy, should not investment be made to identify these markers?

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.

Tuesday, March 29, 2011 – Please join in with the topical discussions about the newest trends in clinical drug development, and learn strategies to avoid costly mistakes and improve efficiency.  The focus of this BioFlorida SE Chapter-sponsored event is on advancing biomedical products through the clinical development process, and is being led by Planning Committee Chair Mike Keller of McDermott Will & Emery.  A nationally recognized panel of speakers will be providing an overview of clinical drug development for the novice, as well as providing high level insights for the experienced life sciences professional.

ImmunoSite Technologies, LLC will be participating at this important event.  Contact two of the IST owners attending, Dr. Carlos Aparicio and Julie Wilkinson, to arrange one-on-one discussions about the criticality of immune monitoring and immune surveillance during drug development, or just meet them there for impromptu discussions on other thought-provoking topics.

Tuesday, March 29, 2011

5:00 p.m.calculate tax refund 2011estimate tax refund 2011 – 8:30 p.m.

Lois Pope LIFE Center
Univ. of Miami Miller School of Medicine
1095 NW 14th Terrace
7th Floor Apex Auditorium
Miami, FL 33136

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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.

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.