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International Clinical Trials

Flow Cytometry and Clinical Trials

Carla G Hill and Thomas W McCloskey of ICON Central Laboratories explain how flow cytometry, monoclonal antibody therapy and cell-based biomarkers can be utilised in clinical trials

The past decade has witnessed a remarkable increase in large molecule, antibody-based therapies – a trend which is predicted to continue. Monoclonal antibody drugs have unique laboratory testing requirements that typically include cellbased assays. Flow cytometry is a powerful cellular analysis technology which has proven valuable in both research and clinical trial environments to further drug development. Innovative flow cytometric methods can yield valuable clinical information from cell-based biomarkers in drug development and in clinical trials for safety and efficacy.

FLOW CYTOMETRY

Flow cytometry is a laser-based, cellular analysis technology. A flow cytometer is an instrument in which cells, labelled with fluorescent reagents, travel in a fluid stream past a laser beam with the resulting fluorescence measured by sensitive optical detectors called photomultiplier tubes (1). These instruments are capable of performing quantitative measurements on cells at very rapid rates, sometimes over 10,000 cells per second. In addition, modern digital flow cytometry instruments are capable of measuring multiple fluorescence wavelengths simultaneously – eight wavelengths for instruments typically used in the clinical setting, and 14 or more for instruments designed for research laboratories (2). Flow cytometry offers a very unique type of measurement technology because although measurements are performed on large numbers of cells – up to a million in some circumstances – each measurement is performed on each individual cell. For example, a digital instrument used in a clinical trial laboratory may measure forward scatter, side scatter, and eight fluorescence channels on a single cell in approximately 10 μsec. The advantage of this type of measurement is that the data obtained is not an average of all cells; rather, each cell is measured individually, allowing subpopulations to be distinguished from one another.

The most common sample type analysed in flow cytometry is peripheral blood. Whole blood is drawn by venipuncture into vacutainer tubes containing anticoagulant; typically ethylenediaminetetraacetic acid (EDTA), sodium heparin, or acid citrate dextrose (ACD). Cells tend to be less stable in EDTA, so either sodium heparin or ACD are most commonly used for flow cytometry testing where sample analysis is expected to be delayed. Recently, Cyto-Chex® Blood Collection Tubes (BCT) have been utilised to provide extended stability for particular phenotypic markers. If absolute cell counts of specific subpopulations are required, an additional EDTA tube may be drawn for a complete blood count on a haematology analyser, providing absolute numbers of subpopulations by a dual platform method. Alternatively, by using relative bead counting approaches, absolute cell counts may be determined on a single platform flow cytometric basis. Lymphocytes are often the object of analysis, although for particular biologics, monocytes or neutrophils may be assessed. Red blood cells may be lysed, or white blood cells may be isolated via ficoll gradient centrifugation, buffy coat procedure, or the use of Cell Preparation Tubes (CPT)TM, prior to analysis.

Fluorescent reagents are used to label cells prior to flow cytometric analysis. These are most often monoclonal antibody reagents which bind to a specific protein on the surface or in the cytoplasm of the cells. These monoclonal antibody reagents are specialised molecules, labelled with fluorochromes which, when excited by the correct wavelength of light, emit their own particular wavelength. The laser provides the excitation wavelength, while the photomultiplier tube in combination with appropriate optical filters captures the emission wavelength. Other types of reagents include dyes which stain a specific cellular constituent such as nucleic acid.

Bead-based assays are another growing area of flow cytometry testing. These methods use fluorescent beads, which may be distinguished from each other by light scatter or fluorescence intensity parameters, to measure proteins contained in a soluble matrix. For example, one bead may be coated with anti-IL-2 antibody while another is coated with anti-γIFN antibody. The beads are incubated with serum collected from the subject and then a second step indicator antibody is added. The beads are processed on the cytometer and the resulting fluorescence output of each bead correlates with the amount of specific cytokine present in the serum.

MONOCLONAL ANTIBODY THERAPIES

Most drugs developed during the 20th century were small molecule drugs. With the introduction of biologics as therapies, the entire pharmaceutical landscape changed.

Biologics are large molecules produced in living cell systems. Monoclonal antibodies are the most common biologic; other examples are cytokines and hormones. Small molecule drugs can be synthesised chemically and during clinical trials for small molecule drugs, typical laboratory tests performed are chemistry, haematology and urinalysis. Monoclonal antibodies are produced by immunisation followed by isolation of a single antibody producing cell which is then grown into a clone. The supernatant is harvested and immunoglobulin purified. For development of large molecule drugs, however, in addition to basic safety testing, it is also necessary to monitor cellular constituents of the subjects receiving the drug. Thus, flow cytometry is taking on an increasingly important role in the development of new drugs.

The increasing role of monoclonal antibodies as a component of a therapeutic regimen is particularly apparent in oncology. While chemotherapy and radiation have served as cancer treatments for decades, they are cytotoxic and non-specific, often resulting in severe adverse side effects. Monoclonal antibody-based treatments serve to target specific cellular proteins and thus offer greater efficacy with fewer side effects (3). For example, Alemtuzumab specifically binds to CD52, Cetuximab specifically binds EGFR, and Rituximab specifically binds CD20 (4). The result is that these drugs can bind to and specifically target the cellular subpopulations which express these molecules. In addition, a toxin may be conjugated to the monoclonal antibody as this approach allows a cytotoxic blow to be delivered to specific target cells. Other monoclonal antibody therapies are designed to modulate the host immune response and thus ameliorate disease. This approach to drug target selection is much more selective than chemotherapy or radiation treatments.

Monoclonal antibodies are the most rapidly expanding segment of new human medicines as they represent an extremely appealing approach to treatment of disease as humanised or fully human monoclonal antibodies with low immunogenicity, enhanced antigen binding, and reduced cellular toxicity provide better clinical efficacy (5). A review of the industry research pipeline and sales data indicates a paradigm shift in industrial research and development from pharmaceuticals to biologics. The higher clinical success rate and the overcoming of technical hurdles in large scale manufacturing have attracted funds and resources towards research and development in the monoclonal antibody arena. Hundreds of monoclonal antibodies are in clinical trials for treatment of various diseases including cancers, immune disorders and infections (6). Therapeutic and business successes reflect the major advances in antibody engineering, which have resulted in the generation of safe, specific, high affinity and non-immunogenic antibodies during the last three decades. Antibody-based therapeutics are currently enjoying unprecedented success, growth in research and revenues, and recognition of their potential; many large pharmaceutical companies have recently made acquisitions in order to obtain antibody-based therapies under development.

CELL-BASED BIOMARKERS

Biomarkers have characteristics that are measured and evaluated objectively as indicators of normal biological or pathogenic processes, or of pharmacological responses to a therapeutic intervention (7). Blood pressure and cholesterol are examples of well accepted biomarkers that have long played an important role in diagnosing disease and monitoring the efficacy of therapy. With the emergence of biologic therapy and new technology platforms such as DNA sequencing and high-throughput screening over the past few decades, the drug development paradigm has changed dramatically, requiring new approaches to streamline the process. Biomarker research has exploded, and new biomarkers are continually being identified. Biomarkers have become a hot topic for exchange of information between researchers and clinicians in drug development. Anti-TNFα is one success story where open communication and new developments in cytokine blockades and cellular interactions of the inflammatory process has resulted in bringing improved treatment for millions of patients who suffer with autoimmune diseases such as rheumatoid arthritis, Crohn’s disease and psoriasis (8). Biomarkers are now routinely incorporated across all phases of drug discovery and development with hopes to speed the process of bringing safe and effective therapies to market (9).

Immunoassays and mass spectrometry have historically been used for pharmacodynamic biomarker evaluation of small molecule compound treatments. As monoclonal drugs enter into clinical trials, new biomarker assay platforms will be required to evaluate in vivo cellular effects of antibody-based therapeutics. Flow cytometric analysis is emerging as a leading biomarker technology, with its capability for measuring multiple parameters at the single cell level without the need for physical cell separation. Customised compound-specific applications are being developed and implemented to improve clinical management and patient stratification. Cellular assays measuring receptor occupancy and activation status are becoming increasingly popular in clinical trials. Other assays measuring cell cycle and intracellular cytokines are being explored as clinical tools for therapeutic efficacy (10,11). Flow cytometry is also being applied in early phases of drug discovery to screen compounds for their effect on cancer stem cells (12).

The use of novel cellular biomarkers in clinical trials may result in significant cost reductions for pharmaceutical companies. Early identification of an informative biomarker can facilitate implementation in Phase 1 clinical trials. Early biomarker information can be used to support pharmacokinetic dose response, mechanism of action, or differentiate a compound over its competition to support fast track submission to regulatory agencies for expedited marketing. Likewise, toxic compounds and those not showing improvement over current therapy can be halted, thus minimising losses (13). Measurement of multiple biomarkers from a single cellular sample may also reduce clinical costs. The expanded data set from patients receiving therapy may be used to identify a subset of patients that are more likely respond to therapy or less likely to experience an adverse effect (14).

CONCLUSION

The future of medicine will likely include a combination of small and large molecule compound therapy. Cellular biomarkers will be necessary in defining disease pathology, toxicological assessment and efficacy. Flow cytometry assays, where multiple measurements can be obtained from single cells, are valuable in clinical development to identify key cellular interactions for the identification of successful therapies (15-18). Autoimmune disease, oncology and immunodeficiency syndromes are currently realising an increased demand for customised flow cytometry assays to support biological development, bringing safe and effective therapy to patients in need.

References
  1. Givan AL, Flow cytometry: an introduction, Methods Mol Biol 699: pp1-29, 2011
  2. Chattopadhyay PK, Hogerkorp CM and Roederer M, A chromatic explosion: the development and future of multiparameter flow cytometry, Immunology 125: pp441-449, 2008
  3. Bono JS and Ashworth A, Translating cancer research into targeted therapeutics, Nature 467: pp542-549, 2010
  4. Gerber DE, Targeted therapies: a new generation of cancer treatments, Am Fam Physician 77: pp311-319, 2008
  5. Maggon K, Monoclonal antibody ‘gold rush’, Curr Med Chem 14: pp1,978-1,987, 2007
  6. Dimitrov DS and Marks JD, Therapeutic antibodies: current state and future trends – is a paradigm change coming soon? Methods Mol Biol 525: pp1-27, 2009
  7. Sawyers CL, The cancer biomarker problem, Nature 452: pp548-552, 2008
  8. Feldmann M, Translating molecular insights in autoimmunity into effective therapy, Ann Rev Immunol 27: pp1-17, 2009
  9. Biomarker Definitions Working Group, Biomarkers and Surrogate endpoints: preferred definitions and conceptual framework, Clin Pharmacol Ther 69: pp89-95, 2001
  10. Estevam J, Danaee H, Liu R, Ecsedy J, Trepicchio WL and Wyant T, Validation of a flow cytometry based G(2)M delay cell cycle assay for use in evaluating the pharmacodynamic response to Aurora A inhibition, J Immunol Methods 5,363(2): pp135-142, January 2011
  11. Jaimes MC, Maecker HT, Yan M, Maino VC, Hanley MB, Greer A, Darden JM and D’Souza MP, Quality assurance of intracellular cytokine staining assays: analysis of multiple rounds of proficiency testing, J Immunol Methods 5,363(2): pp143-157, January 2011
  12. Gupta V, Zhang QJ and Liu YY, Evaluation of anticancer agents using flow cytometry analysis of cancer stem cells, Methods Mol Biol 716: pp179-191, 2011
  13. Litwin V and O’Gorman, Flow cytometry-based biomarkers in translational medicine and drug development, J Immunol Methods, doi:10.1016/j.jim.2010.09.035, 2010
  14. Vesterqvist O and Reddy M, Introduction to Biomarkers, In Litwin V, Marder P, eds, Flow Cytometry in Drug Discovery and Development, Wiley-Blackwell, John Wiley & Sons, Inc, pp55-70, 2010
  15. Hill C, Wu D, Ferbas J, Litwin V and Reddy M, Regulatory compliance and method validation, In Litwin V and Marder P, (eds), Flow Cytometry in Drug Discovery and Development, Wiley-Blackwell, John Wiley & Sons, Inc, 2010
  16. Wu D, Patti-Diaz L and Hill C, Development and validation of flow cytometry methods for pharmacodynamic clinical biomarkers, Bioanalysis 2: pp1,617-1,626, 2010
  17. O’Hara DM, Xu Y, Liang Z, Reddy MP, Wu DY and Litwin V, Recommendations for the validation of flow cytometric testing during drug development: assays, J Immunol Methods 363: pp120-134, 2011
  18. Green CL, Brown L, Stewart JJ, Xu Y, Litwin V and Mc Closkey TW, Recommendations for the validation of flow cytometric testing during drug development: instrumentation, J Immunol Methods 363: pp104-119, 2011


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Carla G Hill specialises in transitioning flow cytometry biomarker assays into clinical trials. She serves on the MetroFlow NY/NJ Flow Cytometry Steering Committee and the Medical Laboratory Technology Advisory Commission at Mercer County Community College in New Jersey. She has an MSc in Biology from Georgian Court University in New Jersey, a BSc in Microbiology from Pennsylvania State University, and a Medical Technology ASCP certification. Email: carla.hill@iconplc.com

Thomas W McCloskey specialises in the validation of complex novel cell-based assays for use in clinical trials. He obtained his BSc in Biology from Hofstra University in New York and his PhD in Immunology from Rutgers University in New Jersey, and has authored more than 40 manuscripts. In 1998, Thomas won the Presidential Award of Excellence, which recognised him as the Top Young Investigator in Flow Cytometry in a worldwide competition. Email: thomas.mccloskey@iconplc.com

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