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

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?