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

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Fort Lauderdale, Fla. – November 1, 2011 – ImmunoSite Technologies (IST), LLC, a leading provider of immune monitoring services, has entered into a strategic agreement with Goodwin Biotechnology, Inc. (GBI), a full service Contract Manufacturing Organization focused on process development and GMP manufacturing of cell culture derived biopharmaceuticals, based in Plantation, Fla.

In compliance with cGLP quality standards, IST specializes in confirming the validity of processes in assay solutions to evaluate the safety, immunogenicity and efficacy of vaccines and biologics. Conversely, GBI offers a full range of mammalian cell culture, cell culture and bioconjugation development and manufacturing services. GBI is a cGMP contract manufacturing organization (CMO) and has established robust quality systems to support  the manufacturing of materials that can be injected into humans. The partnership provides expanded capabilities for both companies allowing each to outsource appropriate projects across all phases of clinical trials, offering a more streamlined process of developing pharmaceuticals.

“When it comes to developing pharmaceuticals, streamlining processes saves time, resources, and, ultimately, money,” said Wade Barton, Ph.D., president of IST. “Our goal is to help our clients bring pharmaceuticals to market with the highest quality control as quickly as possible. Our partnership with GBI has garnered a trusted name in the industry benefiting both of our client bases.”

“IST’s capabilities in developing and performing cell-based and cytotoxicity assays as well as bioanalytical testing such as flow cytometry was an incredible advantage over others in this field,” said David Cunningham, Director of Business Development for GBI. “GBI and IST can now offer more of a ‘one-stop shop’, which is extremely valuable if you’ve ever had the experience of dealing with several different vendors.”

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

For more information on IST, please visit http://immunositetechnologies.com/; follow on Twitter at http://twitter.com/ImmunoSite; or Facebook at http://www.facebook.com/immunosite. For more information on GBI, visit http://www.goodwinbio.com/.

About ImmunoSite Technologies

Formed in 2009 as a spin-off of Beckman Coulter, Inc., ImmunoSite Technologies, LLC (IST) offers a full range of contract research (CRO) for immune monitoring, particle testing, and process automation services for biotechnology, pharmaceutical, manufacturing and academic organizations around the world. Fully GLP compliant and based in Fort Lauderdale, Fla., IST’s team has 110 years of rigorous product development experience, has been published over 300 peer-reviewed scientific publications, has authored over 30 U.S. and international patents, and has developed and successfully commercialized over 200 diagnostic (IVD) product reagents, kits and instrument systems. IST is distinguished by its 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 immune monitoring and cell analysis R&D experience qualifies IST scientists to accelerate vaccine, biologic and drug discovery, and to comply with complex and demanding international scientific and governmental regulations.

About Goodwin Biotechnology, Inc.

Goodwin Biotechnology is a fully integrated cGMP contract manufacturer of monoclonal antibodies, recombinant proteins and vaccines. GBI has the expertise and experience in cell line development, process development and GMP manufacturing of recombinant proteins and antibodies, as well as conjugated therapeutic proteins (e.g., antibodies conjugated to linkers for radioimmune therapy and diagnostics, other antibodies, proteins, chemotoxins, or plant toxins) by leveraging our proprietary conjugation technology. By working with GBI, our clients can enhance the value of their product candidates with clear development and manufacturing strategies and a road map to meet product requirements from the milligram, gram and kilogram range as the product candidates move along the clinical approval pathway. With nearly 20 years of experience as an independent contract manufacturer, GBI has worked with companies of all sizes from small university spin-offs to major research institutes, government agencies and large, established biopharmaceutical companies.

IST Press Contact:

Marketing Matters

Kyle E. Glass, Public Relations/Marketing Manager

Ph: 502-409-5953

Email: kyle@marketingmatters.net

Web: http://www.marketingmatters.net

GBI Press Contact:

Goodwin Biotechnology

Dave Cunningham, Director of Business Development

Ph: 954-327-9639

Email: DCunningham@goodwinbio.com

Web: http://www.goodwinbio.com

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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.

Press Contact:

Marketing Matters

Kyle E. Glass, Public Relations/Marketing Manager

Ph: 502-409-5953

Email: kyle@marketingmatters.net

Web: http://www.marketingmatters.net

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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.

What Do You Seek In A Contract Research Partnership?

In a time of tight budgets and careful examination of the cost/benefit of partnering with a contract research service provider, ImmunoSite Technologies, LLC (IST) offers a rare combination of experience, knowledge, responsiveness, and trustworthiness.  IST knows what clients need to be successful, and how to provide the highest quality in service.  Clients can depend on ImmunoSite Technologies to be the ideal partner that helps accelerate their success without encumbering their resources.

Cost efficiency – As R&D costs escalate, you need a CRO partner that fits well with your business from a cost/value point-of-view, as well as a shared values point-of-view.  IST understands this and is ready to work with you to craft a partnership that works for you from a time, resource, and cost perspective, all the while delivering the results you need.  IST demonstrates value in the services they provide, by getting the work done quickly and on budget without sacrificing quality.

Knowledge and experience – Backed by years in pharmaceutical, biotech, and diagnostics product development, IST knows the challenges of developing the right assay to get the accuracy, precision and reproducibility needed to succeed.

Compatibility and communication – Essential to a great partnership, is a high level of compatibility and great communications.  IST’s professionals understand this.  You need a team that understands your company and your goals, your particular research objectives and challenges.  You need professionals that will work with you to plan, implement and work through all challenges along the way.  The team at IST has partnered with organizations from around the globe so they know the value of accurate communications and a shared vision to bring creative solutions to fruition, while fostering long-term and productive relationships.

Quality – Outsourcing your assay automation project to a qualified partner like IST is a key strategy for ensuring quality.  In a business where service, precision, accuracy, and timeliness are the key deliverables, IST stands apart by putting the “service” back into the service provider partnership.  Quality is central to their work and the unique value they deliver.  The IST quality system is in compliance with GxP guidelines and international safety and regulatory requirements.

Specialization in assay automation – This is IST’s niche and they provide the unique service that validates the automated assay performance with the client’s samples before the system ever leaves the ImmunoSite Technologies facility.  This offers great peace of mind to their clients who are investing significantly in automation to enhance their productivity, but don’t have the time or people to dedicate to implementing a new automated assay project.  IST has designed and developed automated systems for handling biological specimens from live cells to protein to nucleic acids.  Automation doesn’t have to fill a room to meet your needs.  Engineers at IST are cognizant of space and budget requirements when designing the best system for a client so the solution can be a simple benchtop or hood-sized system.

Explore a Partnership With ImmunoSite Technologies

If great service from a dependable, trustworthy, and experienced partner is what you are seeking for your assay automation project, then contact IST now. The technical experts and management team at ImmunoSite Technologies will meet with you to explore the fit between your company, your project needs and IST capabilities.  When it is a great fit, everybody knows.  IST assay automation services

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.