This study evaluated the effectiveness of the Reverse Transcription-droplet digital PCR (RT-ddPCR) method in measuring T-cell-mediated immunity by quantifying IFN-γ mRNA expression. The results demonstrated that peak IFN-γ expression occurred approximately 4 h after stimulation of whole blood and peripheral blood mononuclear cells (PBMCs) by stimulants. The fold activation of IFN-γ mRNA expression in 100 µL of blood challenged with CEF peptides was lower than that observed in PBMCs.
View Article and Find Full Text PDFPre-existing anti-AAV antibodies can be detected using ligand binding-based assay formats. One such format is the MSD-based bridging assay, which uses sulfo-tag-labeled AAV vectors as detection reagents. However, no method has been developed to accurately measure the degree of sulfo-tag labeling on AAV vectors.
View Article and Find Full Text PDFPre-existing adeno-associated viruses (AAV) neutralizing antibodies (NAb) can prevent AAV vectors from transducing target tissues. The immune responses can include binding/total antibodies (TAb) and neutralizing antibodies (NAb). This study is aimed at comparing total antibody assay (TAb) and cell-based NAb assay against AAV8 to help inform the best assay format for patient exclusion criteria.
View Article and Find Full Text PDFPre-existing neutralizing antibodies (NAb) to adeno-associated virus (AAV) may diminish the efficacy of AAV-based therapies depending on the titer. To support gene therapy studies in pigs, the seroprevalence of NAb to AAV1, 2, 5, 6, 8, and 9 serotypes were assessed in the sera of 3 different strains of pigs consisting of 60 Norsvin Topigs-20 strain, 22 Gottingen minipigs, and 40 Yucatan minipigs. Cell-based NAb assays were developed for various AAV serotypes.
View Article and Find Full Text PDFPurpose: The purpose of this work was to investigate the role of the lymphatic system in the pharmacokinetics of etanercept, a fusion protein.
Methods: Etanercept 1 mg/kg was administered intravenously (IV) and subcutaneously (SC) to thoracic lymph duct-cannulated and sham-operated control rats. Blood and lymph samples were obtained for up to 6 days.
The growing fraction (GF) of tumor has been reported as one of the predictive markers of the efficacy of chemotherapeutics. Therefore, a semi-mechanistic model has been developed that describes tumor growth on the basis of cell cycle, allowing the incorporation of the GF of a tumor in pharmacokinetic/pharmacodynamic (PK/PD) modeling. Efficacy data of anti-glypican 3 (GPC3) antibody drug conjugate (ADC) in a hepatocellular carcinoma (HCC) patient derived xenograft (PDX) model was used for evaluation of this proposed model.
View Article and Find Full Text PDFAcoustic liquid handlers deliver small volumes (nL-µL) of multiple fluid types with accuracy and dynamic viscosity profiling. They are widely used in the pharmaceutical industry with applications extending from high-throughput screening in compound management to gene expression sequencing, genomic and epigenetic assays, and cell-based assays. The capability of the Echo to transfer small volumes of multiple types of fluids could benefit bioanalysis assays by minimization of sample volume and by simplifying dilution procedures by direct dilution.
View Article and Find Full Text PDFCo-inhibitory immune receptors can contribute to T cell dysfunction in patients with cancer. Blocking antibodies against cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and programmed cell death 1 (PD-1) partially reverse this effect and are becoming standard of care in an increasing number of malignancies. However, many of the other axes by which tumours become inhospitable to T cells are not fully understood.
View Article and Find Full Text PDFLigand-binding assay (LBA) performance depends on quality reagents. Strategic reagent screening and characterization is critical to LBA development, optimization and validation. Application of advanced technologies expedites the reagent screening and assay development process.
View Article and Find Full Text PDFMyostatin, also known as growth differentiation factor 8 (GDF-8), is a protein acting as a negative regulator in skeletal muscle growth. Inhibition of myostatin by therapeutic agents provides opportunities for current unmet medical needs. In order to better understand drug engagement to aid the drug development, we have developed a hybrid LC-MS/MS method which can differentially measure myostatin and another protein from the same GDF family, GDF-11.
View Article and Find Full Text PDFBackground: Isomerization of aspartic acid and deamidation of asparagine are two common amino acid modifications that are of particular concern if located within the complementarity-determining region of therapeutic antibodies. Questions arise as to the extent of modification occurring in circulation due to potential exposure of the therapeutic antibody to different pH regimes.
Results: To enable evaluation of site-specific isomerization and deamidation of human mAbs in vivo, immunoprecipitation (IP) has been combined with LC-MS providing selective enrichment, separation and detection of naive and modified forms of tryptic peptides comprising complementarity-determining region sequences.
Background: Antibody-drug conjugates (ADCs) are complex drug constructs with multiple species in the heterogeneous mixture that contribute to their efficacy and toxicity. The bioanalysis of ADCs involves multiple assays and analytical platforms.
Methods: A series of ligand binding and LC-MS/MS (LB-LC-MS/MS) hybrid assays, through different combinations of anti-idiotype (anti-Id), anti-payload, or generic capture reagents, and cathepsin-B or trypsin enzyme digestion, were developed and evaluated for the analysis of conjugated-payload as well as for species traditionally measured by ligand-binding assays, total-antibody and conjugated-antibody.
LC-MS/MS has been investigated to quantify protein therapeutics in biological matrices. The protein therapeutics is digested by an enzyme to generate surrogate peptide(s) before LC-MS/MS analysis. One challenge is isolating protein therapeutics in the presence of large number of endogenous proteins in biological matrices.
View Article and Find Full Text PDFBackground: Therapeutic protein discovery study highlights the need for the development of quantitative bioanalytical methods for determining the levels of both the therapeutic protein and the target protein, as well.
Results: For the quantitation of BMS-986089, both accuracy (99-103%) and precision (2.4-12%) were obtained for the analysis of the surrogate peptide (ITYGGNSPVQEFTVPGR), in addition to the accuracy (100-108%) and precision (0.
J Chromatogr B Analyt Technol Biomed Life Sci
October 2015
Antibody drug conjugates (ADCs) are complex molecules composed of two pharmacologically distinct components, the cytotoxic payload and the antibody. The measurement of the payload molecules that are attached to the antibody in vivo is important for the evaluation of the safety and efficacy of ADCs, and can also provide distinct information compared to the antibody-related analytes. However, analyzing the antibody-conjugated payload is challenging and in some cases may not be feasible.
View Article and Find Full Text PDFBackground: The bioanalytical strategy for antibody-drug conjugates (ADC) includes numerous measurements integrally designed to provide comprehensive characterization of PK, PD and immunogenicity. This manuscript describes the utilization of reagents specifically tailored to an ADC with a microtubule polymerization inhibitor payload and cathepsin B cleavable linker.
Methods: The PK strategy includes the evaluation of physiological levels of total antibody, active ADC, total ADC, antibody-conjugated payload and unconjugated payload.
Background: FGF21-AdPKE is a fusion protein and functionally inactivated in vivo by cleavage around the C-terminus. It is important to quantify the intact active protein in serum.
Results & Discussion: Taking advantage of a uniquely acid-labile aspartyl-prolyl amide bond, we developed an acid hydrolysis procedure based on heating FGF21-AdPKE in dilute formic acid to generate a surrogate peptide encompassing the last 17 amino acids at the C-terminus.
J Allergy Clin Immunol
November 2011
Background: IL-5 plays a central role in the development and maintenance of eosinophilia (EO) and eosinophil activation in a wide variety of eosinophilic disorders. Although IL-5, IL-3, and GM-CSF can modulate the expression of IL-5 receptor α (IL-5Rα) on eosinophils in vitro, little is known about soluble and surface IL-5Rα levels in vivo.
Objective: To assess soluble and surface IL-5Rα levels in patients with EO and/or mastocytosis.
Background: Peripheral blood eosinophilia and lung mucosal eosinophil infiltration are hallmarks of bronchial asthma. IL-5 is a critical cytokine for eosinophil maturation, survival, and mobilization. Attempts to target eosinophils for the treatment of asthma by means of IL-5 neutralization have only resulted in partial removal of airway eosinophils, and this warrants the development of more effective interventions to further explore the role of eosinophils in the clinical expression of asthma.
View Article and Find Full Text PDFRationale: Chronic obstructive pulmonary disease (COPD) is characterized by airway inflammation and remodeling. High-mobility group box 1 (HMGB1), a nuclear protein that is released during inflammation and repair, interacts with proinflammatory cytokines and with the receptor for advanced glycation end products (RAGE), which is highly expressed in the lung.
Objectives: To determine whether HMGB1 is augmented in COPD and is associated with IL-1beta and RAGE.
Chronic obstructive pulmonary disease (COPD) is characterized by chronic airway inflammation and emphysematous alveolar destruction. In this study, we have investigated whether chitotriosidase (ChTRase) and acidic mammalian chitinase, two chitinases with chitinolytic activity, are selectively augmented in COPD and contribute to its pathogenesis. We found that smokers with COPD, but not asthmatics, had higher chitinolytic activity and increased levels of ChTRase in bronchoalveolar lavage, more ChTRase-positive cells in bronchial biopsies, and an elevated proportion of alveolar macrophages expressing ChTRase than smokers without COPD or never-smokers.
View Article and Find Full Text PDFMouse breast regression protein 39 (BRP-39; Chi3l1) and its human homologue YKL-40 are chitinase-like proteins that lack chitinase activity. Although YKL-40 is expressed in exaggerated quantities and correlates with disease activity in asthma and many other disorders, the biological properties of BRP-39/YKL-40 have only been rudimentarily defined. We describe the generation and characterization of BRP-39(-/-) mice, YKL-40 transgenic mice, and mice that lack BRP-39 and produce YKL-40 only in their pulmonary epithelium.
View Article and Find Full Text PDFAlthough respiratory syncytial virus (RSV) infection is the most important cause of bronchiolitis in infants, the pathogenesis of RSV disease is poorly described. We studied histopathologic changes in a panel of lung tissue specimens obtained from infants with fatal cases of primary RSV infection. In these tissues, airway occlusion with accumulations of infected, apoptotic cellular debris and serum protein was consistently observed.
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