Contextual Cellular Analysis Powers Pharmaceutical and Clinical Research

Cell-based analysis of potential therapeutic targets and vaccines provides researchers with the high level data required to make informed decisions about the function and efficacy of their compounds. Having assays designed so that the appropriate cellular targets can be located and functionally monitored, and so that the assay can be standardized and, where necessary, automated, is a very complex and time-consuming process.

Combining a large portfolio of reagents and the ability to stabilize, standardize and customize assays, with a varied and sophisticated instrument line, Beckman Coulter, Inc. is uniquely positioned to provide high quality data and biological insight to pharma customers. Furthermore, the company offers a corps of experienced MDs and PhDs who work directly with clients to develop assays that will fully elucidate their compounds and targets.

Beckman Coulter scientists work with pharmaceutical clients to identify their assay needs, and to help configure those assays, with a primary focus on designing tests that truly monitor the therapeutic efficacy of compounds and the stratification of patients in clinical trials. In the drug discovery realm, Beckman Coulter scientists are able to develop customized assays and provide the sophisticated instrumentation necessary for high throughput contextual cellular analysis.

Once assays are identified, they are standardized so that every test site across an organization, at all locations, arrives at the same answers. When appropriate, these assays can be fully automated. This means that once the sample is placed on the instrument, it is prepared and stained, cells are washed and then sent for analysis. In this manner, complete cellular analysis can be achieved through high-level, multi-color flow cytometry.

Cell-based screening provides a level of context that monitoring target-compound interaction does not. This is significant because testing targets in isolation puts them at a remove from the signaling pathways that can negate hoped for therapeutic benefits. Compounds characterized by cell-based screening are more fully understood, and can more readily move into pre-clinical screening and clinical trials.

To provide contextual cellular analysis, functional assays look at targets within a cell. To do this, the cell must be permeabilized. The appropriate combination of reagents and fluorochromes must be chosen and conjugated. Using processes developed by Beckman Coulter scientists in collaboration with customers, the cell-signaling pathway can be looked at reproducibly. Immune response functions such as intracellular cytokine analysis can be characterized. Regulatory T-cells, for example, can be studied, to better understand immune function, immune competence or immune suppression. The decision to move forward with a compound can be made with confidence, or the compound can fail earlier and new candidates can move into testing.

Finding interrelated needles in the haystack of a cell
Drug discovery companies often have huge numbers of compounds that they believe might have therapeutic potential. For example, consider a pharmaceutical research team that wants to screen a million compounds – 50,000 a day – against a target related to inflammatory response. The team wants to shut down the inflammation process by targeting one specific node within the cell-signaling pathway. The objective is to find the pathways that are associated with immune response, to identify reagents that will look at those pathways and to see if they are turned on or turned off.

Beckman Coulter has the resources to develop a cocktail of reagents that the researcher can use to determine which pathways are turned on or off, and if the inflammatory response has been down-regulated. These tests can often be used in further characterizations and cell studies. There are times when the same assay can by used for analysis with an animal model. The researcher can draw some of the animal’s blood, look at the white cells and determine whether or not the pathway was still being shut down, or were there other factors within the animal that reversed it, or that degraded the drug.

Beckman Coulter is uniquely qualified to provide assays for clinical trials as well. In Phase I trials, these assays can measure response to compounds in a human system, check toxicology, adsorption and other related factors. White cells can be isolated from the patient’s blood and be examined for response to the compound.

Patient stratification testing requires another level of assay. For example, with a target that is presenting multi-drug resistance at the genetic or proteomic level, the pharmaceutical researcher can work with Beckman Coulter scientists to develop an assay to prescreen patients and determine which individuals may be eligible to participate in the trial, and who may not. Moving on through Phase I and Phase II trials, dosages may be changed or multiple sets of patients may be followed.

Cell-based assays allow the study of the proteomics and the functioning of the cell. Knowing if a specific gene that indicates for a disease is present or not is of course significant. Knowing if the gene is expressed and if the proteins are functioning properly provides more detailed and more useful information. To do this, it is imperative that proper antibodies be developed, so that the  right receptors are identified.

Beckman Coulter consultants first determine a client’s understanding of their compound. The next steps are to determine what type of assay would monitor that target, the kind of reagents that are needed. After determining what to study – T-cell related response, or B-cell or NK-cell related response, the various pathways and the major nodes – a recommendation is put forth as to what to study to look at the efficacy of the drug. A variety of reagents are used to make an assay that will target the appropriate nodes. Fluorochromes are used that will not interfere or that do not interact with intracellular components. Next the assay is tested. Genetically altered cells are used to make sure that the correct target is being monitored. Pathways are shut down and up-regulated. Once the assay is confirmed on the target, the screening process can begin. To facilitate screening, Beckman Coulter offers a variety of automation workstations. An integration group within the company can bring together all types of instruments and robotics to scale the process to client-throughput requirements. Automation provides reproducibility and standardization of procedures wherever testing may take place.

Testing vaccines
A new vaccine must be efficacious. It also must not interfere with any of the other vaccines a patient may be receiving. Current vaccines can be composed of as many as 23 products, because of the number and variety of epitopes on infectious agents. An enormous amount of testing is required, and it is complicated by the fact that every major country has a different immunization schedule.

New vaccines require more than just antibody response – they require a cellular-level response in which targeted cells are located and killed in an environment in which a lot of antigen receptor signaling and secondary signals are being secreted. Beckman Coulter personnel have the experience to provide standardized and reproducible tests for these vaccines.

Some very sophisticated biochemical products are available for the study of epitopes, and to identify those that are most likely to elicit an immune response. Epitope discovery systems identify the most immunogenic regions of a peptide or protein, by mimicking peptide binding in vivo, thus simulating an immune response. After the target epitopes are known, reagents called tetramers are used to determine if antigen specific T-cells are being generated. In testing a new vaccine, this ability to combine efficiency and specificity to the design process can be highly advantageous and save considerable time. To wait for therapeutic efficacy to be determined by patient response could take years: with an infection such as HIV, it could take ten years or more. Cellular analysis of vaccine function can determine, in as little as three months, specifically which epitopes are being targeted. Measuring cellular response to specific antigens or epitopes on the surface of a cell will confirm whether T-cells were armed and activated. Conversely, it can also confirm that T-cells were not armed, that there is a problem in the design or presentation of the vaccine.

A sophisticated line of reagents
Beckman Coulter offers a broad line of unique reagents for the assembly of cellular analysis assays, and the instruments and software to collect and analyze the data. A prime example of the level of scientific acumen they bring to bear is the iTopia™ Epitope Discovery System, which, by mimicking in vivo peptide-binding reactions, may help researchers advance important candidates more quickly through the development process. It does so not only by facilitating peptide identification and ranking based on binding characterization, but also through its ability to assist the selection of individual populations most likely to respond. This may improve the productivity of clinical trials and can form the basis for an immune monitoring system to follow individuals.

Once specific epitopes of interest have been identified and qualified, tetramers can be used for further testing and monitoring purposes. Tetramer technology has revolutionized T-cell immunology by its ability to directly quantitate antigen-specific T-cells. T-cells are the primary cells involved in cell-mediated immune responses. Cell-mediated immune responses are central to the control of initial infection, inflammation, disease development and therapy monitoring.

Beckman Coulter offers two types of Tetramers, Class I and Class II. iTag™ MHC Tetramers are complexes of four MHC (Major Histocompatibility Complex) molecules that are associated with a specific peptide and bound to a fluorochrome. Class I Tetramers bind to a distinct set of T cell receptors (TCRs) on a subset of CD8+ T-cells, and Class II Tetramers bind to a distinct population of CD4+ T-cells. Patented iTag MHC tetramers reduce non-specific binding to allow unique detection and enumeration of Ag specific T-cells.

The Class I Tetramers, (human and rhesus macaque) have been mutated to minimize binding of the MHC molecule to CD8 cell surface receptor. These reagents show diminished CD8-mediated binding to the general CD8 positive lymphocyte population, but retain MHC peptide-specific binding to T-cell regulators, facilitating accurate discrimination of rare, specific T cells (less than 1% of CD8+).

By mixing Tetramers with PBMCs or whole blood and using flow cytometry as a detection system, a count of all CD4+ or CD8+ T-cells that are specific for one peptide and its matched MHC allele is provided, regardless of functionality. These reagents allow measurement of the cellular response directed toward single-peptide specificity and meet the regulatory stability and quality requirements needed for clinical trials

Multicolor analysis is an important tool used in the identification and investigation of cellular subpopulations. Many common immunophenotyping tests involve analyzing multiple parameters. The additional parameters needed to perform these tests require the use of an increasing number of fluorochromes. Beckman Coulter scientists have developed Tandem dyes to expand the dye portfolio to meet these needs.

The quality of a Tandem dye can be measured by the intensity of its acceptor fluorescence and crossover value (ratio of peak emissions for the donor and acceptor dyes). The processes in place at Beckman Coulter have been validated for lot-to-lot reproducibility within tight target specifications, a key requirement for the use of antibody conjugates of these dyes in longitudinal or multi-site studies. Comparative studies are available that demonstrate the quality of tandem dye conjugates for both fluorescence intensity and energy-transfer efficiency

Instrumentation
Developed for the research and biopharmaceutical markets with input from a number of investigators, the multi-color, multi-laser Gallios™* flow cytometry system couples sensitivity, resolution and dynamic range with high-speed data collection. Unprecedented detection capabilities and easy-to-use software combine with new dyes – some with proprietary tandem dye technology – to facilitate superior performance of multi-color flow cytometry assays.
An innovative forward-scatter detector provides up to three measurements of cell size and visualization of particles down to 0.4 μm in diameter. A side-scatter detector incorporates an independently focused, high-performance photodiode with electronic attenuation. Six fluorescence detectors provide simultaneous acquisition of up to six fluorescence signals. Up to four fluorescence detectors can be added to the system, enabling concurrent reading of up to 10 colors. Simultaneous measurements of integral, peak and width are available for all parameters, including scatter and fluorescence signals. A selection of up to 62 parameters can be processed per analysis, at acquisition rates of 25,000 events-per-second, with high yield.
Flow cytometry is a core technology and provides a comprehensive and sensitive way to understand the immune response. According to PubMed, the number of papers published annually containing both flow and cytometry has gone from less than 100 in 1980 to more than 7000 in 2007.

Summary
As scientists move from locating and identifying genes to integrated global studies, cellular analysis offers researchers new perspectives on biological information. This view helps researchers validate targets and biomarkers and eliminate process bottlenecks.

By delivering easy access to a full spectrum of interrelated and synergistic tools and automation, Beckman Coulter cellular analysis products help accelerate the identification of new signal transduction pathways, and provide a better understanding of what is happening when there is a reaction to a bacteria or a virus and which part of that cell is eliciting strong reaction.

For drug research, the company supplies systems that accelerate analyses of hundreds of potential therapeutic compounds; systems to analyze drug toxicity; solutions that evaluate drug response; and systems to assess targets and monitor the effects of target candidates.

Offering the broadest range of cellular analysis systems in the world, Beckman Coulter provides a variety of reagents, kits, sample preparation systems, instruments and software to meet the diverse needs of laboratories in applications from basic research to drug discovery to clinical diagnostics.

*For research use only. Not for use in diagnostic procedures.

Contacts:
Celeste Speier, Beckman Coulter, E: cmspeier@beckman.com | T: 714.993.8955
Steve Ernst, Veris Communications, E: veriscomm@gmail.com | T: 973.702.7249

Captions
Figure 1. Initial MHC Binding Assay. Initial screening enables elimination of non-binders while generating data for ranking of binding candidates.
Figure 2. Tetramers Enhance Specificity. Proprietary mutation at α3 domain reduces non-specific binding while the MHC-peptide-TCR interaction is preserved.
Figure 3. The Gallios Flow Cytometer. This 10 color/3 laser research benchtop analyzer couples extraordinary sensitivity, resolution and dynamic range with high-speed data collection.