Validation of high-sensitivity assays to quantitate cerebrospinal fluid and serum ß-galactosidase activity in patients with GM1-gangliosidosis.

GM1-gangliosidosis (GM1) is a lysosomal storage disorder caused by mutations in the galactosidase beta 1 gene (GLB1) that leads to reduced ß-galactosidase (ß-gal) activity. This enzyme deficiency results in neuronal degeneration, developmental delay, and early death. A sensitive assay for the measurement of ß-gal enzyme activity is required for the development of disease-modifying therapies. We have optimized fluorometric assays for quantitative analysis of ß-gal activity in human cerebrospinal fluid (CSF) and serum for the development of a GLB1 gene replacement therapy. Assay analytical performance was characterized by assessing sensitivity, precision, accuracy, parallelism, specificity, and sample stability. Sensitivity of the CSF and serum ß-gal activity assays were 0.05 and 0.20 nmol/mL/3 h, respectively. Assay precision represented by inter-assay percent coefficient of variation of the human CSF and serum was <15% and <20%, respectively. The effect of pre-analytical factors on ß-gal activity was examined, and rapid processing and freezing of samples post-collection was critical to preserve enzyme activity. These assays enabled measurement of CSF and serum ß-gal activities in both healthy individuals and patients with GM1-gangliosidosis. This CSF ß-gal activity assay is the first of its kind with sufficient sensitivity to quantitatively measure ß-gal enzyme activity in CSF samples from GM1 patients.

Best Practices for Development and Validation of Enzymatic Activity Assays to Support Drug Development for Inborn Errors of Metabolism and Biomarker Assessment.

Aberrant or dysfunctional cellular enzymes are responsible for a wide range of diseases including cancer, neurodegenerative conditions, and metabolic disorders. Deficiencies in enzyme level or biofunction may lead to intracellular accumulation of substrate to toxic levels and interfere with overall cellular function, ultimately leading to cell damage, disease, and death. Marketed therapeutic interventions for inherited monogenic enzyme deficiency disorders include enzyme replacement therapy and small molecule chaperones. Novel approaches of in vivo gene therapy and ex vivo cell therapy are under clinical evaluation and provide promising opportunities to expand the number of available disease-modifying treatments. To support the development of these different therapeutics, assays to quantify the functional activity of protein enzymes have gained importance in the diagnosis of disease, assessment of pharmacokinetics and pharmacodynamic response, and evaluation of drug efficacy. In this review, we discuss the technical aspects of enzyme activity assays in the bioanalytical context, including assay design and format as well as the unique challenges and considerations associated with assay development, validation, and life cycle management.

A flow cytometric assay for HLA-DR expression on monocytes validated as a biomarker for enrollment in sepsis clinical trials.

PURPOSE: Decreased expression of HLA-DR on monocytes (mHLA-DR) is a reliable indicator of immunosuppression in patients with sepsis and is correlated with increased risk of secondary infection and mortality. A flow cytometry-based laboratory developed test for the measurement of mHLA-DR in whole blood was validated for clinical trial enrollment, which is considered medical decision-making, for patients with severe sepsis or septic shock. METHODS: The BD Quantibrite™ anti-HLA-DR/anti-monocyte reagent measures antibodies bound per cell of HLA-DR on CD14+ monocytes. The mHLA-DR assay was planned to support inclusion/exclusion of patients for a clinical trial and was validated according to New York State Department of Health (NYSDOH) requirements for a new non-malignant leukocyte immunophenotyping assay. RESULTS: Normal, healthy donor and sepsis patient samples were stable up to 72 h post-collection in Cyto-Chex BCT phlebotomy tubes. Pre-determined acceptance criteria were met for precision parameters (average %CV ≤ 20%) and global laboratory-to-laboratory comparisons (average %Δ ≤ 20%). The approaches taken to evaluate and report accuracy, analytical specificity and sensitivity, reportable range, reference interval, and the proposed multi-level quality control were accepted by NYSDOH. CONCLUSIONS: In this study, the validation strategy necessary when the intended use of assay results changes from exploratory to medical decision making (patient enrollment), which successfully resulted in regulatory approval, is described.

EKLF/KLF1 is ubiquitinated in vivo and its stability is regulated by activation domain sequences through the 26S proteasome.

Erythroid Krüppel-like factor (EKLF/KLF1) is an erythroid specific, C(2)H(2) zinc finger transcription factor that is essential for the proper chromatin structure and expression of the adult beta-globin gene. Herein, we determine that 26S proteasome inhibitors lead to an accumulation of EKLF protein in murine erythroleukemia (MEL) cells. In addition, EKLF half-life in both MEL cells (<3h) and fetal liver cells (between 6 and 9h) is stabilized in the presence of these inhibitors. EKLF is ubiquitinated in vivo, however its modification does not rely on a particular internal lysine. Finally, EKLF contains two PEST sequences within its N-terminus that have no effect on the ability of EKLF to be ubiquitinated but contribute to its destabilization.

Validation of a flow cytometry-based assay to assess C5aR receptor occupancy on neutrophils and monocytes for use in drug development.

The C5a/C5a receptor (C5aR) pathway, a key component in the proinflammatory immune response, is an attractive therapeutic target since its dysregulation is implicated in a variety of autoimmune and inflammatory disorders. The objective of the present study was to validate a receptor occupancy (RO) assay for a human anti-C5aR monoclonal antibody drug candidate, NNC0215-0384 (NN0384). This flow cytometry-based assay measures the percentage (%), median fluorescence intensity (MFI), and molecules of equivalent soluble fluorochrome (MESF) of NN0384 binding to its target cells, neutrophils and monocytes, in whole blood from normal healthy donors and rheumatoid arthritis (RA) patients with clinically active disease. The validation parameters assessed included postcollection and postprocessing sample stability, intra- and interassay precision, an analyst-to-analyst comparison, a comparison of normal healthy donor and RA patient sample postcollection stability, and a laboratory-to-laboratory comparison and assay transfer. The cumulative results indicate that the assay was reproducible, met the clearly defined acceptance criteria for the validation parameters tested, and was transferable to another laboratory. In conclusion, this RO assay is suitable for use to accrue pharmacodynamic biomarker data in a multicenter, global clinical trial.

Non-random subcellular distribution of variant EKLF in erythroid cells.

EKLF protein plays a prominent role during erythroid development as a nuclear transcription factor. Not surprisingly, exogenous EKLF quickly localizes to the nucleus. However, using two different assays we have unexpectedly found that a substantial proportion of endogenous EKLF resides in the cytoplasm at steady state in all erythroid cells examined. While EKLF localization does not appear to change during either erythroid development or terminal differentiation, we find that the protein displays subtle yet distinct biochemical and functional differences depending on which subcellular compartment it is isolated from, with PEST sequences possibly playing a role in these differences. Localization is unaffected by inhibition of CRM1 activity and the two populations are not differentiated by stability. Heterokaryon assays demonstrate that EKLF is able to shuttle out of the nucleus although its nuclear re-entry is rapid. These studies suggest there is an unexplored role for EKLF in the cytoplasm that is separate from its well-characterized nuclear function.

IRX-2, a novel in vivo immunotherapeutic, induces maturation and activation of human dendritic cells in vitro.

IRX-2 is a uniform, well-defined set of natural cytokines currently in Phase II clinical trials for squamous cell carcinoma of the head and neck (HNSCC). In preliminary clinical studies of HNSCC patients, IRX-2 therapy has shown promising results, increasing overall survival of patients from 32% to 61% at 48 months. Although it is known that specific cytokines in IRX-2 enhance T cell activity [e.g., interleukin-2 (IL-2), interferon-gamma, IL-1beta], we chose to investigate the influence of IRX-2 on monocyte-derived dendritic cells (Mo-DCs) isolated from human peripheral blood in an effort to further understand the clinical findings. We show here that IRX-2 treatment of human monocyte-derived DC resulted in morphologic, phenotypic, and functional changes consistent with the development of mature activated DC. Specifically, IRX-2-treated DC increased expression of CD83 and CCR7, markers for DC maturation and migration, respectively, and increased the expression of HLA-DR, CD54, and the costimulatory molecules CD86 and CD40, which are critical mediators of T cell activation. Functional changes in DC induced by IRX-2 included a reduced endocytic capacity, increased ability to stimulate T cells and increased IL-12 cytokine production. These results provide a plausible mechanistic explanation for the in vivo clinical activity of IRX-2 and an additional rationale for the use of IRX-2-based immunotherapy in patients.

Krüppel-like zinc fingers bind to nuclear import proteins and are required for efficient nuclear localization of erythroid Krüppel-like factor.

Erythroid Krüppel-like Factor (EKLF/KLF-1) is an erythroid-specific transcription factor that contains three C(2)H(2) zinc fingers and is required for correct chromatin structure and expression of the beta-globin locus. However, regions within the EKLF protein that serve as signals for its nuclear localization and the proteins that may enable it to become localized are unknown. Two approaches were used to address these issues. First, green fluorescent protein or pyruvate kinase was fused to EKLF domains, and localization was monitored and quantitated by confocal microscopy. Two necessary and sufficient nuclear localization signals (NLSs) were identified: one (NLS1) adjacent to the zinc finger DNA binding domain within a highly basic stretch of amino acids (275-296), and another more efficient signal (NLS2) within the zinc finger domain itself (amino acids 293-376). Interestingly, each zinc finger contributes to the overall effectiveness of NLS2 and requires an intact finger structure. Second, each NLS was tested in vitro for binding to importin proteins. Surprisingly, both EKLF NLSs, but principally the zinc finger domain, bind importin alpha and importin beta. These findings demonstrate that two nuclear localization signals target EKLF to the nucleus and suggest this transport relies primarily on a novel zinc finger/importin protein interaction.