Total antibody quantification for MMAE-conjugated antibody-drug conjugates: impact of assay format and reagents.

Antibody-drug conjugates (ADCs) are target-specific anticancer agents consisting of cytotoxic drugs covalently linked to a monoclonal antibody. The number of ADCs in the clinic is growing, and therefore thorough characterization of the quantitative assays used to measure ADC concentrations in support of pharmacokinetic, efficacy, and safety studies is of increasing importance. Cytotoxic drugs such as the tubulin polymerization inhibiting auristatin, monomethyl auristatin E, have been conjugated to antibodies via cleavable linkers (MC-vc-PAB) through internal cysteines.

Micro-volume wall-less immunoassays using patterned planar plates.

Miniaturization of immunoassays has numerous potential advantages over traditional ELISAs. Here we present a novel approach using patterned planar plates (PPPs). These 'wall-less' plates consist of a 16 × 24 array of 2 mm diameter hydrophilic regions surrounded by a hydrophobic polytetrafluoroethylene (PTFE) coating.

Challenges in developing bioanalytical assays for characterization of antibody-drug conjugates.

With more than 34 targets being investigated and nearly 20 clinical trials at various phases of development, antibody-drug conjugates (ADCs) hold a lot of promise for improving oncological malignancy therapy. This therapeutic strategy designed to specifically or preferentially deliver a cytotoxic agent to tumor cells through conjugation to a monoclonal antibody is not new. Although this approach is relatively simple conceptually, the history of ADCs clearly attests to the high degree of complexity in their development.

Evaluation of heterophilic antibody blocking agents in reducing false positive interference in immunoassays for IL-17AA, IL-17FF, and IL-17AF.

IL-17AA, IL-17FF, and IL-17AF are proinflammatory cytokines that have been implicated in the pathogenesis of autoimmune diseases such as rheumatoid arthritis (RA). In order to measure the levels of these cytokines in synovial fluid and serum samples from RA patients, immunoassays specific for IL-17AA, FF, and AF were developed. Although these assays could tolerate up to 50% pooled normal human serum, false positive reactivity was problematic in patient samples suggesting interference from heterophilic antibodies.

Targeting HER2-positive breast cancer with trastuzumab-DM1, an antibody-cytotoxic drug conjugate.

HER2 is a validated target in breast cancer therapy. Two drugs are currently approved for HER2-positive breast cancer: trastuzumab (Herceptin), introduced in 1998, and lapatinib (Tykerb), in 2007. Despite these advances, some patients progress through therapy and succumb to their disease.

Anti-CD22-MCC-DM1 and MC-MMAF conjugates: impact of assay format on pharmacokinetic parameters determination.

CD22 represents a promising target for antibody-drug conjugate therapy in the context of B cell malignancies since it rapidly internalizes, importing specifically bound antibodies with it. To determine the pharmacokinetic parameters of anti-CD22-MCC-DM1 and MC-MMAF conjugates, various approaches to quantifying total and conjugated antibody were investigated. Although the total antibody assay formats gave similar results for both conjugates, the mouse pharmacokinetic profile for the anti-CD22-MCC-DM1 and MC-MMAF appeared significantly different depending on the conjugated antibody assay format.

Species-dependent serum interference in a sandwich ELISA for Apo2L/TRAIL.

To support pre-clinical studies of Apo2L/TRAIL in rodents and non-human primates, a sandwich ELISA was developed using two mouse monoclonal anti-Apo2L/TRAIL antibodies. Mouse, rat, cynomolgus monkey, and chimpanzee serum at concentrations of > or =1% were found to interfere with accurate quantitation of Apo2L/TRAIL. Moreover, the characteristics of the serum interference for each species were different.

The pharmacokinetics of an albumin-binding Fab (AB.Fab) can be modulated as a function of affinity for albumin.

An AB.Fab (albumin-binding Fab) consists of a Fab and a phage-derived albumin-binding peptide. This molecule is capable of binding both antigen and albumin simultaneously.

Substrate capacity considerations in developing kinase assays.

In developing a screening assay for a serine/threonine kinase, we evaluated various formats of an in-plate enzyme-linked immunosorbent assay (ELISA), as well as solution-phase kinase assays using either ELISA or AlphaScreen detection. Substrate was available both as a biotinylated 15-residue peptide and as a 25-residue peptide containing the same sequence expressed as a glutathione S-transferase fusion protein. When increasing concentrations of either of these substrates were coated directly onto ELISA plates, the rates of the kinase reactions progressively increased.

Nucleic acid capture assay, a new method for direct quantitation of nucleic acids.

Technologies allowing direct detection of specific RNA/DNA sequences occasionally serve as an alternative to amplification methods for gene expression studies. In these direct methods the hybridization of probes takes place in complex mixtures, thus specificity and sensitivity still limit the use of current technologies. To address these challenges, we developed a new technique called the nucleic acid capture assay, involving a direct multi-capture system.

Development of a frozen cell array as a high-throughput approach for cell-based analysis.

Recent advances in molecular biology, human genetics, and functional genomics tremendously increase the number of molecular targets available for potential therapeutic and diagnostic use. To complement DNA array data, cost-efficient high-throughput technologies providing reliable information at the protein level need to be developed. Here we describe the generation of a frozen cell array that required the use of single cell suspensions and could serve various applications such as the analysis of specific antibody or ligand binding to a large panel of different cell types.