Monitoring of adalimumab and antibodies-to-adalimumab levels in patient serum by the homogeneous mobility shift assay.

This report describes the analytical validation and application of the homogeneous mobility shift assay (HMSA) method for the measurement of adalimumab and human antibodies-to-adalimumab (ATA) in serum samples from patients who have lost response to adalimumab treatment. Validation of the ATA- and the adalimumab-HMSA revealed a lower limit of detection to be 0.026 U/mL for ATA and 0.018 µg/mL for adalimumab in serum samples. Intra-assay and inter-assay precision determination yielded a coefficient of variation of less than 15%, and the accuracy of both assays was within 20%. Adalimumab drug tolerance in the ATA-HMSA was up to 20 µg/mL in the test serum. Serum samples from 100 drug-naïve healthy subjects were tested to set-up the cut point of 0.55 U/mL for ATA and 0.68 µg/mL for adalimumab. Analysis of 100 serum samples from patients who were losing response to adalimumab showed that 26% had an adalimumab level below the cut point, of these 68% were ATA positive. Overall, 44% of the patients (44/100) were positive for ATA. This study presents evidence that drug and anti-drug antibody levels are important determinants of patient response to therapy.

Development and validation of a homogeneous mobility shift assay for the measurement of infliximab and antibodies-to-infliximab levels in patient serum.

Antibody-based drugs such as infliximab (IFX) are effective for the treatment of inflammatory bowel disease (IBD) and other immune-mediated disorders. The development of antibodies against these drugs may result in unfavorable consequences, including the loss of drug efficacy, hypersensitivity reactions, and other adverse events. Therefore, accurate monitoring of serum drug and anti-drug antibody levels should be an important part of therapy for patients being treated with an antibody-based drug. Current methods for the assessment of anti-drug antibodies and drug levels, involving various bridging ELISA and radioimmunoassay techniques, are limited by their sensitivity, interference, and/or complexity. To overcome these limitations, we have developed a non-radiolabeled homogeneous mobility shift assay (HMSA) to measure the antibodies-to-infliximab (ATI) and IFX levels in serum samples. Full method validation was performed on both the ATI- and IFX-HMSA, and the clinical sample test results were also compared with those obtained from a bridging ELISA method to evaluate the difference in performance between the two assays. Validation of the ATI-HMSA revealed a lower limit of quantitation of 0.012 µg/mL in serum. The linear range of quantitation was 0.029-0.54 µg/mL. The intra- and inter-assay precision was less than 20% of coefficient of variation (CV), and the accuracy (% error) of the assay was less than 20%. In serum samples, ATI as low as 0.036 µg/mL can be measured, even in the presence of 60 µg/mL of IFX in the serum. Sera from 100 healthy subjects were tested to determine the cut point of the assay. ATI-positive samples that had been previously analyzed by using a bridging ELISA from 100 patients were also measured by the new method. There was a high correlation between the two methods for ATI levels (p<0.001). Significantly, the new method identified five false-positive samples from the bridging ELISA method. Validation of the mobility shift IFX assay also showed high assay sensitivity, precision and accuracy. The HMSA method may also be applied to other protein-based drugs to accurately detect serum drug and anti-drug antibody levels.

Identification of novel pathways involved in the pathogenesis of human adamantinomatous craniopharyngioma.

Activating mutations in the gene encoding ß-catenin have been identified in the paediatric form of human craniopharyngioma (adamantinomatous craniopharyngioma, ACP), a histologically benign but aggressive pituitary tumour accounting for up to 10% of paediatric intracranial tumours. Recently, we generated an ACP mouse model and revealed that, as in human ACP, nucleocytoplasmic accumulation of ß-catenin (ß-cat(nc)) and over-activation of the Wnt/ß-catenin pathway occurs only in a very small proportion of cells, which form clusters. Here, combining mouse genetics, fluorescence labelling and flow-sorting techniques, we have isolated these cells from tumorigenic mouse pituitaries and shown that the ß-cat(nc) cells are enriched for colony-forming cells when cultured in stem cell-promoting media, and have longer telomeres, indicating shared properties with normal pituitary progenitors/stem cells (PSCs). Global gene profiling analysis has revealed that these ß-cat(nc) cells express high levels of secreted mitogenic signals, such as members of the SHH, BMP and FGF family, in addition to several chemokines and their receptors, suggesting an important autocrine/paracrine role of these cells in the pathogenesis of ACP and a reciprocal communication with their environment. Finally, we highlight the clinical relevance of these findings by showing that these pathways are also up-regulated in the ß-cat(nc) cell clusters identified in human ACP. As well as providing further support to the concept that pituitary stem cells may play an important role in the oncogenesis of human ACP, our data reveal novel disease biomarkers and potential pharmacological targets for the treatment of these devastating childhood tumours.