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ABP 798 is a biosimilar to Rituxan® (rituximab reference product [RP]). Non-clinical assessments relevant to the primary and secondary mechanisms of action (MOA) contribute to the totality of the evidence (TOE) in supporting biosimilarity and are critical in providing scientific evidence for extrapolation of indications. Similarity of ABP 798 with rituximab RP was investigated across a range of biological activities which have potential impact on pharmacokinetics and clinical efficacy with non-clinical assessments relevant to MOA such as CD20 internalization, trogocytosis, binding to primary human natural killer (NK) cells as well as the ability to induce antibody-dependent cellular phagocytosis (ADCP) in peripheral blood mononuclear cells. Additionally, in vitro synergy of ABP 798 or RP with chemotherapeutic agents, in vivo xenograft studies in mice, and toxicological assessments in cynomolgus monkeys (including B cell depletion and toxicokinetics) were also conducted. Results from these non-clinical assessments contribute to the TOE supporting the biosimilarity between ABP 798 and rituximab RP across a range of primary and secondary MOAs and support justification for extrapolation to all indications of use for ABP 798 for which the RP is approved.

Subcutaneous (SubQ) injection is a common administration route for biotherapeutics. However, limited tools are available for understanding the dynamic relationships between drug products and resident cells following injection. Advances in tissue engineering have enabled the production of in vitro skin models that recapitulate the morphological structure and functional activity of human skin. Here we explore the use of a commercially available skin model to investigate potential immune activation in response to subcutaneously injected biotherapeutics. Exposure to high levels of a mixture of process-related impurities (that are known potent immune system activators) induced a robust immune response from the skin model, as indicated by enhanced metabolic activity and increased secretion of 19 cytokines and chemokines. The skin model also responded to aggregated antibodies (generated by extreme mechanical stirring and pH-jump stress, which resulted in orders of magnitude higher particle numbers than that found in products), as shown by the secretion of several signature cytokines (GM-CSF, RANTES, and MCP-1). However, the magnitude of the responses to the aggregates were significantly lower than the response to the impurities. These results highlight the promising utility of in vitro skin models for investigating the potential immune response to process-related impurities and biotherapeutic attributes in a subcutaneous environment. The use of skin models for assessing drug safety may provide new insights to help guide drug product and process development, and potentially mitigate the risk of injection site reactions and systemic immunogenic responses that may compromise the safety and efficacy of subcutaneously administered drugs.

Antibody therapeutics with poor solubility in the subcutaneous matrix may carry unintended risks when administered to patients. The objective of this work was to estimate the risk of antibodies that precipitate in vitro at neutral pH by determining the impact of poor solubility on distribution of the drug from the injection site as well as immunogenicity in vivo. Using fluorescence imaging in a mouse model, we show that one such precipitation-prone antibody is retained at the injection site in the subcutaneous space longer than a control antibody. In addition, we demonstrate that retention at the injection site through aggregation is concentration-dependent and leads to macrophage association and germinal center localization. Although there was delayed disposition of the aggregated antibody to draining lymph nodes, no overall impact on the immune response in lymph nodes, systemic exposure of the antibody, or enhancement of the anti-drug antibody response was evident. Unexpectedly, retention of the precipitated antibody in the subcutaneous space delayed the onset of the immune response and led to an immune suppressive response. Thus, we conclude that precipitation due to poor solubility of high doses of antibody formulations delivered subcutaneously may not be of special concern in terms of exposure or immunogenicity.

Purpose The in vitro and in vivo pharmacologic assessment of ABP 980 similarity to its reference product is intended to compare the activity of ABP 980 and trastuzumab and support the overall conclusion of similarity based on a comprehensive analytical and functional evaluation. Methods This work complements the primary assessment of functional similarity with additional in vitro assays, binding studies, and non-clinical studies including human epidermal growth factor receptor-2 (HER2) kinetic binding, HER2 signaling, HER2 internalization, synergy with docetaxel chemotherapy, FcγR kinetic binding, primary natural killer and monocyte cell binding, antibody-dependent cellular phagocytosis activity, in vivo xenograft studies, and toxicokinetic parameters. Results The results contribute to the totality of evidence with respect to functional similarity and support that ABP 980 is similar to trastuzumab in all primary and secondary mechanisms of action. Conclusions These results also support the scientific justification of extrapolation to all approved indications of trastuzumab given the established functional similarity of the two products and the same mechanisms of action across all conditions of use.

Receptor occupancy measurements demonstrate the binding of a biotherapeutic agent to its extra-cellular target and represent an integral component of the pharmacodynamic (PD) portfolio utilized to advance the development and commercialization of a therapeutic agent. Coupled with traditional pharmacokinetic (PK) assessments derived from serum drug concentration, receptor occupancy data can be used to model PK/PD relationships and validate dose selection decisions throughout the drug development lifecycle. Receptor occupancy assays can be even more challenging to develop than other flow cytometric methods (e.g. surface immunophenotyping). In addition to typical considerations regarding stability of the cell type of interest, stability of the target-bound therapeutic agent and stability of the target receptor must be taken into account. Reagent selection is also challenging as reagents need to be evaluated for the potential to compete with the therapeutic agent and bind with comparable affinity. This article provides technical guidance for the development and validation of cytometry-based receptor occupancy assays.

The measurement of the binding of a biotherapeutic to its cellular target, receptor occupancy (RO), is increasingly important in development of biologically-based therapeutic agents. Receptor occupancy (RO) assays by flow cytometry describe the qualitative and/or quantitative assessment of the binding of a therapeutic agent to its cell surface target. Such RO assays can be as simple as measuring the number of cell surface receptors bound by an antireceptor therapeutic agent or can be designed to address more complicated scenarios such as internalization or shedding events once a receptor engages the administered therapeutic agent. Data generated from RO assays can also be used to model whether given doses of an experimental therapeutic agent and their administration schedules lead to predicted levels of receptor occupancy and whether the receptor is modulated (up or down) on cells engaged by the therapeutic agent. There are a variety of approaches that can be used when undertaking RO assays and with the ability to measure distinct subsets in heterogeneous populations, flow cytometry is ideally suited to RO measurements. This article highlights the importance of RO assays on the flow cytometric platform in the development of biotherapeutic agents.

The aim of the study was to characterize performance of a complementary set of assays to measure antigen-specific immune responses in subjects immunized with a neoantigen. Healthy volunteers (HV) (n = 8) and patients with systemic lupus erythematosus (SLE) (n = 6) were immunized with keyhole limpet haemocyanin (KLH) on days 1 and 29. Serum antibodies were detected using a flow cytometric bead array (CBA) that multiplexed the KLH response alongside pre-existing anti-tetanus antibodies. Peripheral blood mononuclear cells were studied by B cell ELISPOT. These assays were built upon precedent assay development in cynomolgus monkeys, which pointed towards their utility in humans. Primary anti-KLH IgG responses rose to a mean of 65-93-fold above baseline for HV and SLE patients, respectively, and secondary responses rose to a mean of 260-170-fold above baseline. High levels of anti-tetanus IgG were detected in pre-immunization samples and their levels did not change over the course of study. Anti-KLH IgG1-4 subclasses were characterized by a predominant IgG1 response, with no significant differences in subclass magnitude or distribution between HV and SLE subjects. Anti-KLH IgM levels were detectable, although the overall response was lower. IgM was not detected in two SLE subjects whodid generate an IgG response. All subjects responded to KLH by B cell ELISPOT, with no significant differences observed between HV and SLE subjects. The CBA and B cell ELISPOT assays reliably measured anti-KLH B cell responses, supporting use of this approach and these assays to assess the pharmacodynamic and potential safety impact of marketed/investigational immune-therapeutics.

Objectives To evaluate the safety, tolerability, pharmacokinetics (PK) and pharmacodynamics (PD) of single-dose and multiple-dose administration of AMG 557, a human anti-inducible T cell co-stimulator ligand (ICOSL) monoclonal antibody, in subjects with systemic lupus erythematosus (SLE). Methods Patients with mild, stable SLE (n=112) were enrolled in two clinical trials to evaluate the effects of single (1.8–210 mg subcutaneous or 18 mg intravenous) and multiple (6 –210 mg subcutaneous every other week (Q2W)×7) doses of AMG 557. Subjects received two 1 mg intradermal injections 28 days apart of keyhole limpet haemocyanin (KLH), a neoantigen, to assess PD effects of AMG 557. Safety, PK, target occupancy, anti-KLH antibody responses, lymphocyte subset analyses and SLE-associated biomarkers and clinical outcomes were assessed. Results AMG 557 demonstrated an acceptable safety profile. The PK properties were consistent with an antibody directed against a cell surface target, with non-linear PK observed at lower concentrations and linear PK at higher concentrations. Target occupancy by AMG 557 was dose dependent and reversible, and maximal occupancy was achieved in the setting of this trial. Anti-AMG 557 antibodies were observed, but none were neutralising and without impact on drug levels. A significant reduction in the anti-KLH IgG response was observed with AMG 557 administration without discernible changes in the anti-KLH IgM response or on the overall IgG levels. No discernible changes were seen in lymphocyte subsets or in SLE-related biomarkers and clinical measures. Conclusions The selective reduction in anti-KLH IgG demonstrates a PD effect of AMG 557 in subjects with SLE consistent with the biology of the ICOS pathway and supports further studies of AMG 557 as a potential therapeutic for autoimmune diseases. Trial registration numbers NCT02391259 and NCT00774943.

Mast cells (MCs) have important functional roles in leukocyte recruitment, pain, and wound healing, and increased tissue resident MC function has been associated with several fibrotic diseases. Consequently, the study of MCs in situ can be a direct approach to studying the pharmacodynamic impact of MC-directed therapeutics in tissues. Here we describe an automated laser scanning cytometry assay that was used to characterize the kinetics of MC accumulation in healing skin wounds and to study the effect of inhibiting CD117 (cKit) signaling. The number of tryptase-positive MCs approximately doubled 14 days after cutaneous injury in nonhuman primates. Treatment of animals with anti-CD117 or imatinib mesylate (Gleevec®) reduced MC accumulation at the edge of healing wounds in mice and nonhuman primates, respectively. In translating this MC assay to become a biomarker for human studies, no differences in dermal MC numbers were evident between genders, ages or body mass index from 20 healthy donors. These data suggest that skin is a practical and useful tissue for tracking pharmacodynamic effects of MC-directed therapies. © 2008 International Society for Advancement of Cytometry

Flow Cytometry has become a mainstay technique for measuring fluorescent and physical attributes of single cells in a suspended mixture. These data are reduced during analysis using a manual or semiautomated process of gating. Despite the need to gate data for traditional analyses, it is well recognized that analyst-to-analyst variability can impact the dataset. Moreover, cells of interest can be inadvertently excluded from the gate, and relationships between collected variables may go unappreciated because they were not included in the original analysis plan. A multivariate non-gating technique was developed and implemented that accomplished the same goal as traditional gating while eliminating many weaknesses. The procedure was validated against traditional gating for analysis of circulating B cells in normal donors (n = 20) and persons with Systemic Lupus Erythematosus (n = 42). The method recapitulated relationships in the dataset while providing for an automated and objective assessment of the data. Flow cytometry analyses are amenable to automated analytical techniques that are not predicated on discrete operator-generated gates. Such alternative approaches can remove subjectivity in data analysis, improve efficiency and may ultimately enable construction of large bioinformatics data systems for more sophisticated approaches to hypothesis testing. © 2008 International Society for Advancement of Cytometry

Immunogenicity profiles of recombinant therapeutic proteins are important to understand because antibodies raised against these molecules may have important clinical sequelae. The purpose of the present study was to demonstrate that a flow cytometric bead array could be used to detect clinically relevant antibodies with specificity to such therapeutics. We chose to evaluate well-characterized specimens from persons treated with epoetin alfa that developed antibody-mediated pure red blood cell aplasia as a means to demonstrate the utility of this platform. Our data show that this assay is capable of detecting anti-epoetin alfa antibodies with a relative antibody concentration of 50 ng/ml, where 25 of 25 sera spiked with antibodies at this concentration scored positive. Moreover, the assay was designed to include positive and negative control beads for each specimen that is processed to ensure the specificity of the signal when detected. Measurement of interassay precision supports quantitative estimates of relative antibody concentrations in the range of 313 to 5,000 ng/ml, where the percent coefficient of variation did not exceed 20%. With respect to clinical specimens, antibodies with specificity for epoetin alfa could be easily detected in a set of specimens from persons with pure red blood cell aplasia that had prior exposure to the EPREX brand of recombinant epoetin alfa. Further development and validation of this approach may facilitate successful widespread application of the method for detection of anti-epoetin alfa antibodies, as well as antibodies directed against other recombinant therapeutic proteins.

Blisibimod is a potent B cell-activating factor (BAFF) antagonist that binds to both cell membrane-expressed and soluble BAFF. The goal of these first-in-human studies was to characterize the safety, tolerability, and pharmacokinetic and pharmacodynamic profiles of blisibimod in subjects with systemic lupus erythematosus (SLE). SLE subjects with mild disease that was stable/inactive at baseline received either a single dose of blisibimod (0.1, 0.3, 1, or 3 mg/kg subcutaneous [SC] or 1, 3, or 6 mg/kg intravenous [IV]) or placebo (phase 1a; N = 54), or four weekly doses of blisibimod (0.3, 1, or 3 mg/kg SC or 6 mg/kg IV) or placebo (phase 1b; N = 63). Safety and tolerability measures were collected, and B cell subset measurements and pharmacokinetic analyses were performed. All subjects (93 % female; mean age 43.7 years) carried the diagnosis of SLE for ≥ 1 year. Single- and multiple-dose treatment with blisibimod produced a decrease in the number of naive B cells (24–76 %) and a transient relative increase in the memory B cell compartment, with the greatest effect on IgD-CD27+; there were no notable changes in T cells or natural killer cells. With time, memory B cells reverted to baseline, leading to a calculated 30 % reduction in total B cells by approximately 160 days after the first dose. In both the single- and multiple-dosing SC cohorts, the pharmacokinetic profile indicated slow absorption, dose-proportional exposure from 0.3 through 3.0 mg/kg SC and 1 through 6 mg/kg IV, linear pharmacokinetics across the dose range of 1.0–6.0 mg/kg, and accumulation ratios ranging from 2.21 to 2.76. The relative increase in memory B cells was not associated with safety signals, and the incidence of adverse events, anti-blisibimod antibodies, and clinical laboratory abnormalities were comparable between blisibimod- and placebo-treated subjects. Blisibimod changed the constituency of the B cell pool and single and multiple doses of blisibimod exhibited approximate dose-proportional pharmacokinetics across the dose range 1.0–6.0 mg/kg. The safety and tolerability profile of blisibimod in SLE was comparable with that of placebo. These findings support further studies of blisibimod in SLE and other B cell-mediated diseases. Clinicaltrials.gov NCT02443506 . Registered 11 May 2015. NCT02411136 Registered 7 April 2015.

The measurement of the binding of a biotherapeutic to its cellular target, receptor occupancy (RO), is increasingly important in development of biologically-based therapeutic agents. Receptor occupancy (RO) assays by flow cytometry describe the qualitative and/or quantitative assessment of the binding of a therapeutic agent to its cell surface target. Such RO assays can be as simple as measuring the number of cell surface receptors bound by an antireceptor therapeutic agent or can be designed to address more complicated scenarios such as internalization or shedding events once a receptor engages the administered therapeutic agent. Data generated from RO assays can also be used to model whether given doses of an experimental therapeutic agent and their administration schedules lead to predicted levels of receptor occupancy and whether the receptor is modulated (up or down) on cells engaged by the therapeutic agent. There are a variety of approaches that can be used when undertaking RO assays and with the ability to measure distinct subsets in heterogeneous populations, flow cytometry is ideally suited to RO measurements. This article highlights the importance of RO assays on the flow cytometric platform in the development of biotherapeutic agents.

The host immune factors that determine susceptibility to HIV-1 infection are poorly understood. We compared multiple immunologic parameters in three groups of HIV-1-seronegative men: 14 highly exposed (HR10), 7 previously reported possibly to have sustained transient infection (PTI), and a control group of 14 low risk blood bank donors (BB). Virus-specific cellular immune assays were performed for CD4(+) T helper cell responses, CD8(+) cytotoxic T lymphocyte activity, CD8(+) cell chemokine release, and CD8(+) cell-derived antiviral soluble factor activity. General immune parameters evaluated included CCR5 genotype and phenotype, interferon alpha production by PBMCs, leukocyte subset analysis, and detailed T lymphocyte phenotyping. Comparisons revealed no detectable group-specific differences in measures of virus-specific immunity. However, the HR10 group differed from the BB group in several general immune parameters, having higher absolute monocyte counts, higher absolute CD8(+) T cell counts and percentages, lower naive and higher terminal effector CD8(+) cells, and lower levels of CD28(+)CD8(+) cells. These changes were not associated with seropositivity for other chronic viral infections. The PTI men appeared to have normal levels of monocytes and slightly elevated levels of CD8(+) T cells (also with increased effector and decreased naive cells). Although we cannot entirely exclude the contribution of other chronic viral infections, these findings suggest that long-lived systemic cellular antiviral immunity as detected by our assays is not a common mechanism for resistance to infection, and that resistance may be multifactorial. General immune parameters reflected by CD8(+) T cell levels and activation, and monocyte concentrations may affect the risk of infection with HIV-1, and/or serve as markers of exposure.

Background: Combined analysis of DNA content and immunofluorescence on single cells by flow cytometry provides information on the proliferative response of subpopulations to stimuli in mixed cell preparations; however, in low-viability cell preparations, dead cells interfere with accurate flow cytometric data analysis because of nonspecific binding of antibodies and altered DNA-staining profiles. Light scatter differences between nonviable and viable cells are unreliable, particularly after the cell permeabilization step that is necessary for DNA staining. We developed a method for identification of nonviable cells by fluorescence in cell preparations that are stained simultaneously for cell surface or intracellular immunofluorescence and DNA content. Materials and Methods: Nonviable cells that have lost membrane integrity are identified by uptake of 7-amino-actinomycin D (7-AAD). Transfer of 7-AAD from stained nonviable cells to unstained viable cells after permeabilization is prevented by blocking DNA binding with nonfluorescent actinomycin D (AD). Pyronin Y(G) (PY) is used for DNA staining because the orange spectral emission of PY can be separated from the green fluorescein isothiocyanate (FITC) emission and the red emission of 7-AAD, respectively. Results: Application of the method to the analysis of the T-cell leukemia cell line Molt-4f and of cultured human peripheral blood mononuclear cells is presented. In both cell preparations, 7-AAD staining permitted reliable dead cell exclusion. Live, 7-AAD–negative Molt-4f cells showed higher expression levels of cell surface CD4 and of intracellular CD3, showed a higher proportion of cells in the G1 phase of the cell cycle, and showed a lower coefficient of variation of the G1 peak compared with data obtained from all the cells in the preparation. Live, CD8+ lymphocytes from OKT3-stimulated cultures of human peripheral blood mononuclear cells showed a specific proliferative response as measured by DNA content analysis. Conclusions: The results show that cells stained with FITC–labeled antibodies can be analyzed by single-laser flow cytometry for DNA content combined with dead cell discrimination. Furthermore, they emphasize the need for exclusion of dead cells from the analysis of cell preparations with low viability to obtain reliable data on immunofluorescence and cell-cycle distributions. Cytometry 35:64–74, 1999. © 1999 Wiley-Liss, Inc.

The relationship between host and virus was examined during the initial stages of human immunodeficiency virus type 1 (HIV) infection in a volunteer from the Multicenter AIDS Cohort Study (MACS). The individual was asymptomatic and unaware of his infection during an initial donation of blood and inguinal lymphoid tissue. Proviral DNA, however, was present in cells from both sources, HIV RNA was detected in the plasma, and CD4+ cell levels were reduced by approximately 50% compared with previous donations in the MACS. In a second blood donation 12 days later, plasma HIV RNA increased 200-fold in tandem with viral isolates with an increased growth phenotype in vitro. HIV burden was ultimately suppressed upon seroconversion and the emergence of HIV-specific CD8+ cytotoxic T lymphocytes. These observations provide further evidence that the potential benefits of early treatment may be maximized during the early stages of infection, when viral fitness may be low but is unopposed by immune responses.

The aim of the study was to characterize performance of a complementary set of assays to measure antigen-specific immune responses in subjects immunized with a neoantigen. Healthy volunteers (HV) (n = 8) and patients with systemic lupus erythematosus (SLE) (n = 6) were immunized with keyhole limpet haemocyanin (KLH) on days 1 and 29. Serum antibodies were detected using a flow cytometric bead array (CBA) that multiplexed the KLH response alongside pre-existing anti-tetanus antibodies. Peripheral blood mononuclear cells were studied by B cell ELISPOT. These assays were built upon precedent assay development in cynomolgus monkeys, which pointed towards their utility in humans. Primary anti-KLH IgG responses rose to a mean of 65–93-fold above baseline for HV and SLE patients, respectively, and secondary responses rose to a mean of 260-170-fold above baseline. High levels of anti-tetanus IgG were detected in pre-immunization samples and their levels did not change over the course of study. Anti-KLH IgG1-4 subclasses were characterized by a predominant IgG1 response, with no significant differences in subclass magnitude or distribution between HV and SLE subjects. Anti-KLH IgM levels were detectable, although the overall response was lower. IgM was not detected in two SLE subjects whodid generate an IgG response. All subjects responded to KLH by B cell ELISPOT, with no significant differences observed between HV and SLE subjects. The CBA and B cell ELISPOT assays reliably measured anti-KLH B cell responses, supporting use of this approach and these assays to assess the pharmacodynamic and potential safety impact of marketed/investigational immune-therapeutics.

Multiphoton-induced second-harmonic generation (SHG) has developed into a very powerful approach for in depth visualization of some biological structures with high specificity. In this unit, we describe the basic principles of three-dimensional SHG microscopy. In addition, we illustrate how SHG imaging can be utilized to assess collagen fibrils in biological tissues. Some technical considerations are also addressed. Curr. Protoc. Cytom. 61:6.33.1-6.33.11. © 2012 by John Wiley & Sons, Inc. Keywords: multiphoton microscopy; second-harmonic generation; laser scanning microscopy; collagen; extracellular matrix; articular cartilage

Flow Cytometers are key devices used to monitor the composition of cells in the blood in the setting of a variety of disease states. Recent advances have produced a range of instruments that range from simple desktop-type devices to multi-laser platforms that allow for high complexity measurements. This variety of instrumentation makes the technology suitable for different budgets, expertise levels and intended uses (research versus diagnostic) with a set of reagents that can effectively be used on any platform so long as the laser line can excite the given fluorochrome and the optics are set up to discriminate emission from the excitation wavelength. Traditional medical applications for flow cytometers include evaluation of CD4 T cell depletion and associated immunophenotypic changes in HIV-infected persons as well as characterization of aberrant cell types used to diagnose hematologic malignancies. More recently, investigators have not only extended immunophenotyping campaigns to other disease settings, but have also taken advantage of fluorescent probes that provide insight into cellular function. For example, it can be inferred that a cell that expresses CD107 on its surface has likely participated in the delivery of cytotoxic granules to a target cell (Michael R.Betts, 2004). Likewise, amine-reactive dyes can be used to track cell division, probes that fluoresce only after enzymatic cleavage can report on caspase activities in apoptosis experiments and intracellular phosphorylation can be measured with specific antibodies and cell permeabilization buffers (phosphoflow) (Maxwell et al., 2009; Krutzik and Nolan, 2006; Wu et al., 2010). The elegance of the flow cytometry platform relies on its simplicity in as much as any combination of fluorescently-conjugated probes can be used to address contemporary hypotheses in cell biology and immunology. It is therefore not surprising that investigators have introduced flow cytometric measurements in biomarker campaigns to study a variety of activities of an immunomodulatory therapeutics), including effects of proximal signaling events as influenced by agonist or antagonistic drugs, or cell immunophenotype as a representative distal pharmacodynamic marker in treated persons.

Subsets of activated CD8+ lymphocytes defined by membrane expression of the activation antigens HLA-DR and CD38 were counted by three-color flow cytometry in homosexual men who subsequently became seropositive for human immunodeficiency virus type 1 (HIV). Profound CD8+ cell activation was seen in all subjects at seroconversion and 6 and 12 months later. The HLA-DR+ CD38+ CD8+ cell population, which has potent direct HIV cytotoxic T cell activity, was markedly elevated at seroconversion in all subjects. In some men, these levels remained elevated throughout the first year of infection. During the next 5 years, these men had stable CD4+ cell levels, whereas the others did not. Long-term survivors (seropositive for 9 years, > 800 CD4+ cells/mm3) also had elevated levels of this subset, despite few other activated CD8+ cells. Thus, selective elevation of HLA-DR+ CD38- CD8+ cells was a marker of subsequent stable HIV disease.

Quantitation of activated caspases in xenograft models by laser scanning cytometry has demonstrated mechanism-specific biological activity of Anti-Trail Receptor immunoglobulin therapies in situ. These preclinical data confirmed that caspase activation is an early event that precedes tumor regression. To apply this platform for clinical monitoring of caspase activation using fine needle aspirate (FNA) biopsies, additional assay feasibility and validation experiments need be addressed. Furthermore, important instrument parameters should be considered including the maintenance and operation of the cytometer in a controlled state to ensure aspects like data traceability, reliability, and integrity. In the present chapter we describe a method to evaluate caspase activation in Colo205 cells and fine needle aspirate tumors by slide-based, laser scanning cytometry. This approach can be applied to cell cultures, preclinical and clinical fine needle aspirate material.

Quantitation of activated caspases in xenograft models by laser scanning cytometry has demonstrated mechanism-specific biological activity of Anti-Trail Receptor immunoglobulin therapies in situ. These preclinical data confirmed that caspase activation is an early event that precedes tumor regression. To apply this platform for clinical monitoring of caspase activation using fine needle aspirate (FNA) biopsies, additional assay feasibility and validation experiments need be addressed. Furthermore, important instrument parameters should be considered including the maintenance and operation of the cytometer in a controlled state to ensure aspects like data traceability, reliability, and integrity. In the present chapter we describe a method to evaluate caspase activation in Colo205 cells and fine needle aspirate tumors by slide-based, laser scanning cytometry. This approach can be applied to cell cultures, preclinical and clinical fine needle aspirate material.

Complexities in sample handling, instrument setup and data analysis are barriers to the effective use of flow cytometry to monitor immunological parameters in clinical trials. The novel use of a central laboratory may help mitigate these issues.

Background: 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. Methods: Five reagents lyophilized from ethanol or CHAPS buffer stock solution of phorbol 12-myristate 13-acetate (PMA) and ionomycin were benchmarked against standard DMSO liquid formulation for their stimulation equivalency. The median fluorescence intensity of phosphorylated ribosomal protein S6 in lymphocytes was assessed on a BD FACSCalibur™. Results: We demonstrate here that tubes pre-loaded with lyophilized versions of the liquid reagents can provide equivalent stimulation in healthy volunteer specimens. Conclusions: The value of this approach is that it safeguards against omission or erroneous addition of bulk liquid formulations of PMA and ionomycin to the reaction vessel (i.e., plate or tube) and also lends itself to extended stability/shelf-life of these reagents. On the basis of this initial success, we plan to expand our evaluation of lyophilized reagents so that they can be incorporated into our clinical biomarker campaigns as appropriate. © 2009 Clinical Cytometry Society

Objective: Characterization of peripheral leukocytes is an important aspect of monitoring the effect of immunotherapeutic interventions in systemic lupus erythematosus (SLE). We analyzed cell surface markers commonly used to assess patients with SLE, focusing on the effect of holding blood prior to processing/analysis and the relative reliability of the measurements that were conducted. Methods: Healthy volunteers (HV; n = 20) and patients with SLE (n = 42) were studied. Whole blood was collected for flow cytometric analysis on days 1, 8, 15, 105, 195, 285, and 375 and held overnight for analysis. A subset of samples was additionally analyzed on the day of collection. Results: Variability arising from overnight storage of whole blood was found to be within 20% for most lymphocyte subsets. There was greater between rather than within subject variability over a 1-year period. As anticipated, the data showed higher CD38 and lower CD19 densities on B cells from patients with SLE compared to HV. Although a higher percentage of cells with markers of plasmablasts/cells were observed in the blood of patients with SLE relative to HV, these measurements were found to be among the least reliable (i.e., most variable). Conclusions: This study provides technical perspectives for those conducting immunophenotypic analyses of B-cells in patients with SLE. We envision that our data, which addresses sample stability issues and presents a method to describe the relative reliability of one measure over another, holds value for clinical assessments of B-cells in SLE and the evaluation of investigational agents designed to modify the B-cell compartment. © 2009 Clinical Cytometry Society

Production of droplets and microdroplets (aerosols) is part of the normal operation of a cell sorter. These aerosols may contain toxic, carcinogenic, or teratogenic fluorophores or known or unknown pathogens from viable biological specimens. Most newer models of commercially available instruments incorporate features designed to reduce the production of aerosols and prevent their release into the room. This unit presents two protocols for assessment of aerosol containment on jet-in-air flow sorters. In both procedures, lytic T4 bacteriophage is run through the instrument at high concentrations to tag aerosol droplets. The instrument is tested in normal operating mode and in simulated failure mode. Aerosols are detected by plaque formation on susceptible E. coli lawns. With the continuing increase in the sorting of viable human cells, it is vital for cytometrists to be aware of the potential dangers.

A surface plasmon resonance (SPR)-based biosensor immunoassay was developed and validated using the Biacore 3000 instrument to detect, semi-quantitate, and characterize serum antibodies against darbepoetin alfa (Aranesp) and epoetin alfa (EPOGEN). In this sensitive, dual-flow cell assay, epoetin alfa and darbepoetin alfa are covalently immobilized onto consecutive flow cells of a carboxymethyl dextran-coated sensor chip. Diluted human serum samples are injected sequentially over both surfaces. The binding of serum antibodies to the immobilized proteins are detected and recorded in real time based on the principles of SPR. Furthermore, antibody binding is confirmed with a secondary anti-human immunoglobulin antibody. Positive samples are further characterized to determine the relative concentration of the antibodies using an affinity-purified, rabbit anti-epoetin alfa antibody as a reference control. The assay can detect 80ng/ml and 100ng/ml of antibody to epoetin alfa and darbepoetin alfa, respectively. The dynamic range of the assay is from 0.078microg/ml to 10microg/ml using a rabbit antibody with demonstrated accuracy and intra- and inter-assay precision. Approximately 80 serum samples can be analyzed on each sensor chip while maintaining a stable baseline and consistent immunological reactivity. The analysis of serum samples from subjects administered with epoetin alfa or darbepoetin alfa provided evidence that the assay can detect varying concentrations of antibodies of different off rates, isotypes, and IgG subclasses.

Since 1984, the Multicenter AIDS Cohort Study (MACS) has utilized four flow cytometry laboratories to measure T-lymphocyte subset levels semiannually in a large cohort of homosexual men. This report summarizes the steps taken in the MACS laboratories to attain comparability of lymphocyte subset determinations across the centers and over time. Identical flow cytometers, monoclonal antibodies, and analytic procedures have been used, and over a period of time, the procedure for sample preparation was also standardized. Interlaboratory proficiency testing utilizing identical specimens analyzed in the four laboratories was performed to evaluate the comparability of the data among the laboratories. Our results verify that such testing can identify technical bias in flow cytometric evaluations performed at different laboratories. Temporal laboratory consistency in flow cytometric measurements was evaluated using data from each site's HIV-seronegative homosexual reference group. Both sequential 95% confidence intervals (mean ± 2 × SEM) and the within-person standard deviations of the immune measurements were considered. Significant variation in CD3, CD4, and CD8 lymphocyte subset percentages over time in the seronegative reference population was observed. Our observations indicate that the lymphocyte subset values of this seronegative group should be used to adjust those obtained on the seropositive study participants during a particular time period, thereby allowing improved discrimination of the effects of HIV on particular time period, thereby allowing improved discrimination of the effects of HIV on T cells in infected individuals. The data presented are of use for designing epidemiologic and intervention studies in HIV-1-infected individuals, especially for calculating sample sizes. The methods we have used to assess the quality of data in the MACS have general application to quality control programs in flow cytometry laboratories. In particular, comparison of sequential confidence intervals and within-person standard deviations for lymphocyte subset determinations on control populations are essential to a comprehensive proficiency testing program because they permit assessment of consistency within a laboratory over time.

Background: From May 1998 to November 2000, 13 European patients developed antibody-mediated pure red cell aplasia during treatment with recombinant human erythropoietin (rHuEPO), reinforcing the need for analytical testing for antibodies against erythropoietic agents. Specimens from 8 patients were provided for further antibody testing and characterization. Methods: We evaluated 4 analytical methods with these sera: radioimmune precipitation (RIP), enzyme-linked immunosorbent assay (ELISA), biosensor immunoassay, and a bioassay for identification of neutralizing antibodies. Results: The RIP, biosensor immunoassay, and biological assay performed equally to detect and quantify anti-rHuEPO antibodies. The ELISA failed to detect antibodies in 2 patient samples. The biosensor immunoassay could determine antibody isotypes, subclasses, and dissociation rates and the bioassay could determine whether these antibodies were able to neutralize the biological effect of the drug; the other assays could not make this determination. Conclusion: We recommend the use of the biosensor immunoassay followed by a bioassay to best identify and characterize anti-rHuEPO antibodies. The biosensor immunoassay allows for identification of all classes and subclasses of immunoglobulins and is a preferred method for the identification of lower-affinity antibodies. The bioassay is needed to determine if the antibodies identified have the capacity to neutralize a biological effect of the drug in a cell-based system.

Inactivation of viral particles is the basis for several vaccines currently in use. Initial attempts to use simian immunodeficiency virus to model a killed human immunodeficiency virus type 1 (HIV-1) vaccine were unsuccessful, and limited subsequent effort has been directed toward a systematic study of the requirements for a protective killed HIV-1 vaccine. Recent insights into HIV-1 virion and glycoprotein structure and neutralization epitopes led us to revisit whether inactivated HIV-1 particles could serve as the basis for an HIV-1 vaccine. Our results indicate that relatively simple processes involving thermal and chemical inactivation can inactivate HIV-1 by at least 7 logs. For some HIV-1 strains, significant amounts of envelope glycoproteins are retained in high-molecular-weight fractions. Importantly, we demonstrate retention of each of three conformation-dependent neutralization epitopes. Moreover, reactivity of monoclonal antibodies directed toward these epitopes is increased following treatment, suggesting greater exposure of the epitopes. In contrast, treatment of free envelope under the same conditions leads only to decreased antibody recognition. These inactivated virions can also be presented by human dendritic cells to direct a cell-mediated immune response in vitro. These data indicate that a systematic study of HIV-1 inactivation, gp120 retention, and epitope reactivity with conformation-specific neutralizing antibodies can provide important insights for the development of an effective killed HIV-1 vaccine.

RANTES (regulated on activation, normal T expressed and secreted), macrophage inflammatory protein (MIP)-1alpha, and MIP-1beta are human immunodeficiency virus (HIV) suppressor factors by virtue of their ability to compete with HIV for access to cell surface R5. Their ability to block HIV infection in vitro is unequivocal; however, their role as HIV suppressor factors in vivo is not firmly established. We therefore conducted a study to test the hypothesis that production of these factors in vitro was a correlate of decreased virus burden in vivo. Moreover, we asked whether higher beta chemokine production could be demonstrated with cells from people who are R5D32 heterozygotes, compared with people who are R5 wild-type homozygotes. Our data support the thesis that RANTES, MIP-1alpha, and MIP-1beta production is associated with decreased in vivo virus load. Moreover, enhanced production of these factors may be explained in part by the genetic background of the host.

The lack of clinical progression in some individuals despite prolonged human immunodeficiency virus type 1 (HIV-1) infection may result from infection with less-pathogenic viral strains. To address this question, we examined the HIV-1 envelope protein from a donor with a low viral burden, stable CD4+ T-lymphocyte counts, and little evidence of CD8+ T-cell expansion, activation, or immune activity. To avoid potential changes in envelope function resulting from selection in vitro, envelope clones were constructed by using viral RNA isolated from uncultured peripheral blood mononuclear cells (PBMC). The data showed that recombinant viruses containing envelope sequences derived from RNA isolated from patient PBMC replicated poorly in primary CD4+ T cells but demonstrated efficient growth in macrophages. The unusual phenotype of these viruses could not be explained solely by differential utilization of coreceptors since the chimeric viruses, as well as an uncloned isolate obtained from the same visit date, can utilize CCR5. In addition, the donor’s own cells appeared resistant to infection with chimeric viruses containing autologous envelope sequences. Genotype analysis revealed that the donor was heterozygous for the previously described 32-bp deletion in CCR5 which may be linked with prolonged survival in HIV-1-infected individuals. These data suggest that the changes in envelope sequences confer properties of viral attenuation, which together with the CCR5 +/Δ32 genotype could account for the long-term survival of this patient.

Persons infected with human immunodeficiency virus (HIV) for > 8 years were studied to delineate virologic and immunologic attributes of long-term survival. Whereas those with 300-700 CD4 + cells/μL often had circulating cytotoxic T lymphocytes (CTL) against HIV antigens, those with >1000 CD4 + cells/μL did not. The subjects with >1000 CD4 + cells/μL had low virus burden, low levels of Gag-specific CTL precursors, and minimal CD8 + cell activation. Overall, elevated levels of CD8 + cells, CD38 antigen expression on CD8 + cells, and anti-HIV functions were correlated with increased virus burden, provirus load, and HIV plasma RNA levels. A factor that suppressed HIV replication was spontaneously secreted from CD8 + cells of most subjects but not from those with high CD4 + cell counts. CD8 + cell activities, therefore, may reflect chronic viral stimulation of the immune system. Long-term survivors with high levels of CD4 + cells maintained control of viral replication but lacked the CD8 + cell activities.

Biohazardous aerosols generated during cell sorting have been of increased concern recently because of interest in sorting specimens containing human immunodeficiency virus type 1 (HIV-1). Current flow cytometers have features designed to contain such aerosols within the sorting chamber, but the efficacy of these features has not been established. Therefore, we tested aerosol containment by two ELITE flow cytometers (Coulter Cytometry, Inc., Hialeah, FL) during sorting of specimens containing high titers of bacteriophage. Agar plates confluent with susceptible Escherichia coli were used to detect infectious units released from the sorting chamber. Under recommended operating conditions very few infectious units were released from the sorting chambers. Release increased when the center stream was not optimally collected in a vacuum-exhausted tube or the chamber door was not completely closed. Failure of the negative pressure and high efficiency particle air (HEPA) filtration features had less of an effect. The data indicate that these standard safety features provide a rational expectation of safety for the flow cytometry operator. © 1995 Wiley-Liss, Inc.

We previously reported that human immunodeficiency virus type 1 (HIV-1), herpes simplex virus (HSV), and Sendai virus induce higher levels of alpha interferon (IFN-alpha) in blood dendritic cells than in monocytes of healthy donors. In the present study, the levels of IFN-alpha induced by T-cell tropic (IIIb and RF) and monocytotropic (BaL) strains of HIV-1 and by HSV were significantly decreased in peripheral blood mononuclear cells (PBMCs) derived from subjects with asymptomatic and symptomatic HIV-1 infection. In contrast, Sendai virus, a paramyxovirus that induces proportionally more IFN-alpha in monocytes and B cells than do either HIV-1 or HSV, stimulated normal levels of IFN-alpha in PBMCs from the HIV-1-infected men. The IFN-alpha produced by PBMCs from the HIV-1-seropositive subjects was partially acid labile, whereas the IFN-alpha produced by PBMCs from the HIV-1-seronegative donors was acid stable. We hypothesize that there is a selective defect in IFN-alpha production by peripheral blood dendritic cells, whereas the host retains the IFN-alpha-producing capacity of monocytes and B lymphocytes. The loss of IFN-alpha production in response to HIV-1, herpesviruses, and possibly other pathogens could contribute to the progression of HIV-1 infection and to the development of AIDS.

A basic immunophenotyping panel that employed dual-color combinations of fluorescein isothiocyanate (FITC) and phycoerythrin (PE) conjugated monoclonal antibodies (mAb; FITC-CD45/PE-CD14, FITC-IgG1/PE-IgG2, FITC-CD3/PE-CD8, FITC-CD3/PE-CD4, FITC-CD3/PE-CD16+PE-CD56, and PE-CD19) was utilized in a quality assurance program to determine whether the 4 laboratories participating in a multicenter AIDS study obtained similar lymphocyte subset percentage values for T cells, B cells, NK cells, and CD4+ and CD8+ T cells. Over a 1½ year period, 78 shared peripheral blood specimens Were prepared and analyzed in each laboratory. The CD45brightCD14− percentage for each specimen was used to correct that individual's lymphocyte subset values. Interlaboratory coefficients of variation (CV) for the human immunodeficiency virus type I (HIV) seronegative (n = 38) and HIV-seropositive (n = 40) specimens using this panel were

T-regulatory cells (Treg) are critically responsible for controlling unwanted immunological activity. Therefore, potential perturbation of Tregs can be important to consider when studying therapeutic intervention of inflammatory disorders with novel agents. Moreover, reliable assays are needed to allow detection of changes resulting from treatment. In the clinical setting intracellular staining for antigens such as Foxp3 can be unwieldy. Therefore, we chose to identify Treg using cell surface markers. Recent reports indicate that using a CD25hi and CD127dim phenotype on CD4+ T-cells allows identification of an enriched fraction of Treg using surface staining. On average, however, only 85 % of these cells are reported to be Foxp3+ Treg. We evaluated use of CCR4 together with CD25 and CD127 to refine detection of Treg. Foxp3 co-staining was performed as well to characterize the proportion of bonafide Treg. More than 90 % of cells in the CD25hi, CD127dim, CCR4+ CD4+ T-cell gate were found to co-express Foxp3. We then validated a 5-color method incorporating CCR4 as a marker for T-reg enumeration. 26 healthy volunteers and 13 asthmatic subjects were evaluated. Pre-analytical variables and assay performance characteristics will be discussed. We conclude that using CCR4 imparts additional specificity for phenotypic identification of Treg. Implementation is planned for clinical studies where therapeutic intervention has the potential to impact Treg.