Adaptation of an ELISA assay for detection of IgG responses against therapeutic monoclonal antibodies in a mouse immunization model.

Biotherapeutic monoclonal antibodies (mAb) play important roles in clinical medicine but their potential to elicit immune responses in patients remains a major issue. In a study designed to investigate the effect of aggregation on immunogenic responses, mice were immunized with two monoclonal antibodies (mAb1 and mAb2). Serum levels of total IgG, IgG, and IgG were measured by ELISA.

The clonal structure and dynamics of the human T cell response to an organic chemical hapten.

Diphenylcyclopropenone (DPC) is an organic chemical hapten which induces allergic contact dermatitis and is used in the treatment of warts, melanoma, and alopecia areata. This therapeutic setting therefore provided an opportunity to study T cell receptor (TCR) repertoire changes in response to hapten sensitization in humans. Repeated exposure to DPC induced highly dynamic transient expansions of a polyclonal diverse T cell population.

Human neutrophil antigens: Nature, clinical significance and detection.

Granulocytes are an essential part of both the innate and adaptive immune systems. Human neutrophil antigens (HNAs) are a family of epitopes that are located on glycoproteins that are mostly expressed on human granulocytes. Antibodies that recognize these epitopes have been associated with neutropenia, transfusion complications, haematopoietic stem cell transplant nonengraftment and renal transplant rejection.

The impact of the analytical performance specifications of calcium and albumin on adjusted calcium.

Aim: The generation of accurate, comparable results from traceable measurement procedures is a primary goal in harmonization efforts. In this study, the analytical performance of routine methods for calcium and albumin measurement is assessed to define the impact of the analytical bias of calcium and albumin on adjusted calcium equation performance and on reference intervals.

Method: In collaboration with the Wales External Quality Assurance Scheme, six months' worth of anonymized data that cover a concentration range of clinical interest were collected.

Albumin and calcium reference interval using healthy individuals and a data-mining approach.

Background: Harmonization of reference intervals for analytes that have a sound calibration and metrological traceability is a widely recommended practice. The UK Pathology Harmony has recently harmonized reference intervals for calcium and albumin. In this study, we have determined the reference intervals for calcium and albumin on the UK's most commonly used analytical platforms.

The effect of different analytical platforms and methods on the performance of population-specific adjusted calcium equation.

Background: A recent attempt to improve the diagnostic value of adjusted calcium addressed a primary care-specific adjusted calcium equation, but validated the new equation for Roche Cobas, BCG and NM-BAPTA methods only. In this study, we aim to validate a population-specific equation for other methods and platforms.

Method: We collected retrospective patient data-sets from 15 hospital laboratories using a range of commercially available analytical platforms and methods for calcium and albumin measurements.

Prospective study comparing the outcome of a population-specific adjusted calcium equation to ionized calcium.

Background: Calcium circulates bound to albumin and changes in albumin concentration will therefore affect total calcium measurements. In order to mitigate this, correction factors are frequently used. The most widely used correction equation was described by Payne and colleagues in 1973.

The impact of thioredoxin reduction of allosteric disulfide bonds on the therapeutic potential of monoclonal antibodies.

Therapeutic mAbs are used to manage a wide range of cancers and autoimmune disorders. However, mAb-based treatments are not always successful, highlighting the need for a better understanding of the factors influencing mAb efficacy. Increased levels of oxidative stress associated with several diseases are counteracted by the activities of various oxidoreductase enzymes, such as thioredoxin (Trx), which also reduces allosteric disulfide bonds in proteins, including mAbs.

Proteomic and Bioinformatic Analyses for the Identification of Proteins With Low Allergenic Potential for Hazard Assessment.

Use of botanicals and natural substances in consumer products has increased in recent years. Such extracts can contain protein that may theoretically represent a potential risk of IgE-mediated allergy. No method has yet been generally accepted or validated for assessment of the allergenic potential of proteins.

T lymphocyte phenotype of contact-allergic patients: experience with nickel and p-phenylenediamine.

Background: There is considerable interest in understanding the immunological variables that have the greatest influence on the effectiveness of sensitization by contact allergens, particularly in the context of developing new paradigms for risk assessment of novel compounds.

Objectives: To examine the relationship between patch test score for three different contact allergens and the characteristics of T cell responses.

Methods: A total of 192 patients with confirmed nickel, p-phenylenediamine (PPD) or methylisothiazolinone (MI) allergy were recruited from the Contact Dermatitis Investigation Unit at Salford Royal Hospital.

Impact of a Heat Shock Protein Impurity on the Immunogenicity of Biotherapeutic Monoclonal Antibodies.

Purpose: Anti-drug antibodies can impair the efficacy of therapeutic proteins and, in some circumstances, induce adverse health effects. Immunogenicity can be promoted by aggregation; here we examined the ability of recombinant mouse heat shock protein 70 (rmHSP70) - a common host cell impurity - to modulate the immune responses to aggregates of two therapeutic mAbs in mice.

Methods: Heat and shaking stress methods were used to generate aggregates in the sub-micron size range from two human mAbs, and immunogenicity assessed by intraperitoneal exposure in BALB/c mice.

Identification of B cell epitopes enhanced by protein unfolding and aggregation.

Aggregation of therapeutic proteins is a key factor in the generation of unwanted immunogenicity, and can result in reduced serum half-life, neutralization of function and adverse health effects. There is currently little information regarding how aggregates interact with B-cell receptors or cognate antibodies at the protein sequence level, or whether non-native, aggregate-induced epitopes predominate in these interactions. Using an antibody fragment (single chain antibody variable fragment; scFv) that forms aggregates readily at low temperature, anti-scFv IgG antibody responses were generated by intraperitoneal injection of BALB/c strain mice with monomer or aggregate preparations.

Impaired Langerhans cell migration in psoriasis is due to an altered keratinocyte phenotype induced by interleukin-17.

Background: Psoriasis is a common skin condition driven by increased expression of interleukin (IL)-17. Langerhans cells (LCs) are epidermal dendritic cells that regulate cutaneous immune responses. Within the uninvolved skin of patients with psoriasis, LCs display impaired migration from the epidermis.

Lower levels of interleukin-1β gene expression are associated with impaired Langerhans' cell migration in aged human skin.

Langerhans' cells (LC) play pivotal roles in skin immune responses, linking innate and adaptive immunity. In aged skin there are fewer LC and migration is impaired compared with young skin. These changes may contribute to declining skin immunity in the elderly, including increased skin infections and skin cancer.

Investigating Liquid-Liquid Phase Separation of a Monoclonal Antibody Using Solution-State NMR Spectroscopy: Effect of Arg·Glu and Arg·HCl.

Liquid-liquid phase separation (LLPS) of monoclonal antibody (mAb) formulations involves spontaneous separation into dense (protein-rich) and diluted (protein-lean) phases and should be avoided in the final drug product. Understanding the factors leading to LLPS and ways to predict and prevent it would therefore be highly beneficial. Here we describe the link between LLPS behavior of an IgG1 mAb (mAb5), its solubility, and parameters extracted using H NMR spectroscopy, for various formulations.

Surrogate CD16-expressing effector cell lines for determining the bioactivity of therapeutic monoclonal antibodies.

Traditional antibody dependent cellular cytotoxicity (ADCC) assays use donor derived natural killer (NK) or peripheral blood mononuclear cells, but donor genetic variability and the technically challenging nature of the assay means that alternative in vitro assay formats are required. We explored the utility of two reporter gene cell lines, the J2 and J9, as surrogate effector cells for ADCC assays. Both express the ADCC relevant Fcγ receptor CD16, crosslinking of which leads to firefly luciferase expression.

The T Cell Response to the Contact Sensitizer Paraphenylenediamine Is Characterized by a Polyclonal Diverse Repertoire of Antigen-Specific Receptors.

Paraphenylenediamine (PPD) is a common component of hair dyes and black henna tattoos and can cause skin sensitization and allergic contact dermatitis (ACD). The cutaneous inflammatory reaction associated with ACD is driven by both CD4+ and CD8+ T cells. However, the characteristics of such responses with respect to clonal breadth and magnitude are poorly defined.

Influence of Escherichia coli chaperone DnaK on protein immunogenicity.

The production of anti-drug antibodies can impact significantly upon the safety and efficacy of biotherapeutics. It is known that various factors, including aggregation and the presence of process-related impurities, can modify and augment the immunogenic potential of proteins. The purpose of the investigations reported here was to characterize in mice the influence of aggregation and host cell protein impurities on the immunogenicity of a humanized single-chain antibody variable fragment (scFv), and mouse albumin.

Editor's Highlight: Subvisible Aggregates of Immunogenic Proteins Promote a Th1-Type Response.

Protein aggregation is associated with enhanced immunogenicity of biotherapeutics. As a result, regulatory guidelines recommend screening for aggregation during bioprocessing. However, the mechanisms underlying the enhanced immunogenicity of aggregates are poorly understood.

T lymphocyte dynamics in methylisothiazolinone-allergic patients.

Background: Methylisothiazolinone (MI), a preservative that is commonly used in personal care products, is now recognized as an important contact allergen in both cosmetic and occupational settings.

Objectives: To analyse T lymphocyte responses to MI, in order to provide important information regarding the relationship between the nature of such responses and skin sensitization potency.

Methods: Proliferative responses to free MI and to an MI-human serum albumin (HSA) conjugate were measured according to [(3) H]thymidine incorporation (n = 56 donors; patch test scores of + in 20, ++ in 29, and +++ in 7).

The effects of arginine glutamate, a promising excipient for protein formulation, on cell viability: Comparisons with NaCl.

The effects of an equimolar mixture of l-arginine and l-glutamate (Arg·Glu) on cell viability and cellular stress using in vitro cell culture systems are examined with reference to NaCl, in the context of monoclonal antibody formulation. Cells relevant to subcutaneous administration were selected: the human monocyte cell line THP-1, grown as a single cell suspension, and adherent human primary fibroblasts. For THP-1 cells, the mechanism of cell death caused by relatively high salt concentrations was investigated and effects on cell activation/stress assessed as a function of changes in membrane marker and cytokine (interleukin-8) expression.

Evaluation of 5-methylcytosine and 5-hydroxymethylcytosine as potential biomarkers for characterisation of chemical allergens.

Epigenetic regulation of gene expression plays a pivotal role in the orchestration of immune responses. Chemical allergens form two categories: skin sensitizing chemicals associated with allergic contact dermatitis, and chemicals that cause sensitization of the respiratory tract and occupational asthma. In mice these are characterized by different T helper (Th) cell responses.