ICCS Portland Plenary Session V: Translational Medicine.
Chair: Virginia Litwin, PhD.

The ICCS Portland Translational Medicine plenary session gave a good overview of the types of flow cytometric assays used in translational medicine.  Although several of the techniques described resemble those currently used in clinical laboratories, others are less well established.  However, all procedures require rigorous validation before they can be implemented in clinical trials.  Virginia Litwin commenced the session by reminding the audience that flow cytometry is used in all stages of drug development, including drug discovery, toxicity studies (in vitro and ex vivo), as a biomarker, in clinical trials to monitor response to treatment, and in companion diagnostics.  Virginia announced that PharmaFlow, an official part of the AAPS BioTec Section Action Program Committee, recently published two articles in the field: the January issue of the Journal of Immunological Methods dedicated to flow cytometry-based biomarkers in translational medicine and drug development, edited by Virginia Litwin and Maurice O'Gorman, and a book entitled Flow Cytometry in Drug Discovery and Development, edited by Virginia Litwin and Philip Marder. 

The session translational medicine session included four presentations that illustrate the application of a variety of flow cytometric techniques to translational medicine.

Of Mice, Men, and Monkeys: Flow Cytometry-Based Biomarkers at Each Stage of rhuMAb Beta7 Drug Development. 
Marna Williams, Ph.D. Genentech, Inc. and F. Hoffmann-La Roche Ltd.

Abstract: Pharmacodynamic biomarkers provide information central in understanding biological effects and pharmacology of drugs, as well as in guiding program decision-making.  This presentation focused on the evaluation of pharmacologic effects of rhuMAb Beta7, a humanized antibody to the integrin Beta7 that is being clinically investigated for the treatment of inflammatory bowel disease. Flow-based biomarkers were utilized to evaluate pharmacodynamic (PD) effects of rhuMAb Beta7 at each stage of drug development to date (preclinical, nonclinical, and clinical).  Flow-based biomarkers provide information critical in informing important aspects of the program—e.g., confirming target binding, demonstrating biological effects of target binding, and providing pharmacodynamic and pharmacokinetic (PD/PK) correlations for exploratory dosing regimens.

Relevant published literature: E. G. Stefanich, D. M. Danilenko, H. Wang, R. Erickson, T. Gelzleichter, H. Chiu, S. Ivelja, S. Jeet, S. Gadkari, O. Hwang, F. Fuh, K. Howell, M. Balazs, K. Refino, S. Fong, S. Iyer, and M. Williams (2011). A Humanized Monoclonal Antibody Targeting the Beta7 Integrin Selectively Blocks Intestinal Homing of T Lymphocytes, British Journal of Pharmacology 162 (8): 1855-1870.

The first presentation, by Marna Williams, illustrated the use of flow-based biomarkers by describing studies evaluating the pharmacologic effects of a humanized antibody to integrin Beta7 (rhuMAb Beta7) in the treatment of inflammatory bowel disease.  She discussed how biomarkers provide information central to the understanding of biological effects and pharmacology of drugs, as well as for guiding program decision-making.  In the study of rhuMAb Beta7, flow-based biomarkers were utilized to evaluate pharmacodynamic (PD) effects at each stage of drug development: preclinical, nonclinical, and clinical.  Initial studies were performed using murine models of autoimmune disease and demonstrated block of lymphocyte homing to the colon using a mouse colitis model and no effect on homing of leukocytes to the brain using an EAE model [Stefanich EG et al.].  Two types of flow cytometric assay were performed using antibodies with differing specificity: an occupancy assay where binding of the fluorochrome labelled detection antibody was inhibited by bound drug and a tracking assay utilizing an anti-Beta7 non-competing fluorochrome labelled antibody that binds even when drug is bound.  Study of peripheral blood T-cells demonstrated occupancy of CD8 positive and CD4 positive T-cells with correlating down regulation of Beta7 receptors on the respective T-cell subsets. 
Further studies were performed using a similar assay in non-human primates to assess the effect on  circulating mucosal-homing versus peripheral-homing T-cells.  In cynomolgus monkeys, rhuMAb Beta7 induced a moderate increase in peripheral blood lymphocytes that was largely due to a marked increase in CD45RA-Beta7high CD4+ and CD45RA-Beta7high CD8+ T-cells i.e. subsets similar to the memory/effector T-cells in humans and mice that preferentially home to mucosal LN and tissues [Stefanich EG et al].  This increase correlated with occupation of Beta7 receptors by the drug. Furthermore, there was a consistent relationship between serum drug concentrations required to maintain drug occupancy.  Finally studies were performed in humans to evaluate safety, tolerability, pharmokinetic (PK) and pharmacodynamic (PD) profiles.   A peripheral blood PD/PK model was built and data from preclinical, nonclinical and clinical studies was utilized to project doses for phase I and predict efficacious dose ranges for phase II. 
In summary, theses studies demonstrated how flow-based biomarkers can provide information at all stages of drug development, including confirming target binding, demonstrating the biological effects of target binding, and providing pharmacodynamic and pharmacokinetic (PD/PK) correlations for exploratory dosing regimens.

Fit-for-purpose validation of a flow-based assay to evaluate pharmacodynamic response of Aurora A inhibition in PBMCs. 
Jose Estevan.  Millennium Pharmaceutical, a Takeda Oncology Company.

Abstract: Pharmacodynamic assays are important aspects for understanding molecularly targeted anticancer agents to investigate the relationship between drug concentration and drug “effect” or biological activity. As new drug entities are developed that affect DNA cell cycle, a pharmacodynamic assay which measures cell cycle perturbation would be a valuable clinical trial tool.  Here we report the validation of a flow cytometry based cell cycle G2/M delay assay for use in evaluating the effect of investigational drug Alisertib (MLN8237), a small molecule inhibitor of a mitotic kinase Aurora A, for clinical trial use. The assay method was validated by examining assay robustness, repeatability, reproducibility, and determining a cutoff for a true drug effect based on biostatistical modeling.

Jose Estevan started the second presentation by mentioning how the development of new drug entities affecting the cell cycle has led to the need for pharmacodynamic assays which measure cell cycle perturbation.  Pharmacodynamic assays are important for understanding molecularly targeted anticancer agents and are used to investigate the relationship between drug concentration and drug “effect” or biological activity.  Jose then went on to describe the development and validation of a flow cytometry based cell cycle, G2/M delay, assay for use in evaluating the effect of the investigational drug Alisertib (MLN8237).  This drug is a small molecule inhibitor of the mitotic kinase Aurora A resulting in mitotic defects.  The assay that was developed uses DNA content staining to determine accumulation of cells in G2/M and >G2/M i.e. aneuploid fractions.  Peripheral blood mononuclear cells are isolated by Ficoll separation, stimulated ex vivo, and DNA content measured by flow cytometry using propidium iodide (PI) staining.  It was necessary to develop a no-wash technique so that the cells would remain exposed to drug.  Jose described the steps involved in method development.  First the protocol was optimized using normal peripheral blood spiked with drug to address the following variables: collection method, matrix effect, PHA concentration and time, permeabilization buffer, assay stability, drug spike/recovery and drug loss with cell culture, wash versus no-wash, optimal drug concentration, and analysis method including the need for exclusion of aggregates.  In order to further characterize the variability of staining, the assay was out-sourced to a clinical research organization to evaluate the following: intra-assay variability, inter- and intra-donor reproducibility, and assay range, and during this transition the DNA staining was converted from PI to DRAQ5.  These studies revealed more assay variability than expected and therefore, biostatistical modeling analysis was performed to establish an appropriate cut-off to reliably identify drug effect.  Subsequently, the validated assay has been incorporated into three clinical trials evaluating Alisertib (MLN8237) therapy for hematologic malignancy including non-Hodgkin lymphoma, myelodysplastic syndrome and acute myeloid leukemia.  Additional details of the validation process are available in the following publication: Estevam J, et al., Validation of a flow cytometry based G2M delay cell cycle assay for use in evaluating the pharmacodynamic response to Aurora A inhibition.  Journal of Immunological Methods 363;135-142:2011.  This work illustrates detailed validation studies that resemble those required for clinical laboratory test development.

Phosphoflow: from Lead Optimization through Preclinical Development.
Becky Penhallow, Bristol-Myers Squibb Oncology.

In the third presentation, Becky Penhallow described development of an assay to evaluate potential new drugs for the treatment of myeloproliferative neoplasms.  The goal was to develop an assay using pertinent biology to better understand the compounds in vitro activities using human whole blood with minimal manipulation.  The assay that was developed utilizes cytokine receptor signaling to evaluate the JAK-Stat signalling pathway.  Becky initially chose to evaluate a single chain receptor using thrombopoietin stimulation of platelets in human whole blood, identified by surface CD61 staining and low forward scatter, and measuring intracllular pStat 5.  In subsequent studies she utilized a 6-color flow cytometric assay to evaluate different JAK family members by gating on monocytes and lymphocytes and measuring various intracellular Stat phosphoproteins after stimulation with series of cytokines.  The assay was used to assess different inhibitors for selectivity for JAK family kinases and potency.  This preliminary work was followed by utilization of a mouse model for PK/PD studies.  In these studies, in addition to measurement of serum compound levels, whole blood was stimulated ex vivo and Stat5 phosphorylation was measured using phosphoflow methods.  For these mouse studies in vitro IC50s correlated well with ex-vivo EC50.  Further studies were then performed using rat, dog and cynomolgus monkey, and more recently, two diseased blood samples. 
Becky concluded that phosphoflow analysis is a powerful method to discriminate compound activity and measure selectivity.  Furthermore, it is a useful tool for determining compound potency in blood, monitoring efficacy, and for PK/PD studies in multiple species.  Finally, Becky mentioned that it is possible that this methodology may provide baseline study data on diseased blood that could be utilized to track treatment effects in the clinic.

Flow Cytometry “A Powerful Tool During Drug Development”
Shabnam Tangri, Ph.D., Genoptix (A Novartis Company).

Abstract:  Biomarkers are increasingly being utilized as a part of drug development.  Flow cytometry is a powerful tool that has as is widely utilized for biomarker discovery and supporting drug development in various stages of clinical trials. In early phase clinical trials, flow cytometry has been utilized as a tool for assessing proof of principle/mechanism, dose/schedule selection and in safety monitoring. In late stage development, flow cytometry has been used to facilitate appropriate patient selection serve as early surrogate end points of efficacy. This discussion will focus on showing case studies that demonstrate how flow cytometry helped address several key questions during the drug development phase.

In the final presentation, Shabnam Tangri used several examples to illustrate the power of flow cytometry in clinical drug development.  She described how biomarkers can be used in the decision tree at each stage of study, Phase 0 – IV, to facilitate advancement of drugs through development.  Shabham described flow cytometric assays to measure receptor saturation for the selection of optimal dose, rare cell analysis and phosphoflow for PD effect and mechanism of action (MOA), target expression analysis, apoptosis assays to determine MOA, and assay of prognosis/predicative/efficacy markers.

Fiona Craig, MD
University of Pittsburgh Medical Center, Pittsburgh, PA