Anti-drug Antibody Sample Testing and Reporting Harmonization.

A clear scientific and operational need exists for harmonized bioanalytical immunogenicity study reporting to facilitate communication of immunogenicity findings and expedient review by industry and health authorities. To address these key bioanalytical reporting gaps and provide a report structure for documenting immunogenicity results, this cross-industry group was formed to establish harmonized recommendations and a develop a submission template to facilitate agency filings. Provided here are recommendations for reporting clinical anti-drug antibody (ADA) assay results using ligand-binding assay technologies. This publication describes the essential bioanalytical report (BAR) elements such as the method, critical reagents and equipment, study samples, results, and data analysis, and provides a template for a suggested structure for the ADA BAR. This publication focuses on the content and presentation of the bioanalytical ADA sample analysis report. The interpretation of immunogenicity data, including the evaluation of the impact of ADA on safety, exposure, and efficacy, is out of scope of this publication.

Soluble amyloid precursor protein α and ß in CSF in Alzheimer's disease.

OBJECTIVE: Cerebral accumulation of amyloid ß (Aß) is a pathological hallmark of Alzheimer's disease (AD). Proteolytic processing of amyloid precursor protein (APP) by α- or ß-secretase results in two soluble metabolites, sAPPα and sAPPß, respectively. However, previous data have shown that both α- and ß-secretase have multiple cleavage sites. The aim of this study was to characterize the C-termini of sAPPα and sAPPß in cerebrospinal fluid (CSF) by mass spectrometry (MS) and to evaluate whether different combinations of these fragments better separate between AD patients and controls by comparing two different sAPP immunoassays. METHODS: Using immunoprecipitation and high resolution MS, the APP species present in CSF were investigated. CSF levels of sAPPα and sAPPß from patients with AD (n=43) and from non-demented controls (n=44) were measured using AlphaLISA and MSD immunoassays that employ different antibodies for C-terminal recognition of sAPPα. RESULTS: Four different C-terminal forms of sAPP were identified, sAPPß-M671, sAPPß-Y681, sAPPα-Q686, and sAPPα-K687 (APP770 numbering). Neither immunoassay for the sAPP species could separate the two patient groups. The correlation (R(2)) between the two immunoassays was 0.41 for sAPPα and 0.45 for sAPPß. CONCLUSION: Using high resolution MS, we show here for the first time that sAPPα in CSF ends at Q686 and K687. The findings also support the conclusion from several previous studies that sAPPα and sAPPß levels are unaltered in AD.

Aß1-15/16 as a potential diagnostic marker in neurodegenerative diseases.

Cerebrospinal fluid (CSF) biomarkers for Alzheimer's disease (AD) reflect brain biochemistry. Using combined immunoprecipitation and mass spectrometry, we have shown that amyloid beta 1-15 (Aß1-15) is produced by concerted ß- and α-secretase cleavage of amyloid precursor protein (APP) and that the relative levels of Aß1-16 in AD compared to controls are increased. Furthermore, drug-induced γ-secretase inhibition enhances the relative levels of Aß1-15 and Aß1-16. Here, we investigate a novel immunoassay for Aß1-15/16 in a broad range of neurodegenerative conditions. The CSF level of Aß1-15/16 was measured by the bead-based amplified luminescent proximity homogeneous assay (Alpha technology). Concentrations of Aß1-15/16 were analyzed in subjects with Parkinson disease (PD; n = 90), PD with dementia (PDD) (n = 32), dementia with Lewy bodies (DLB) (n = 68), AD (n = 48), progressive supranuclear palsy (PSP) (n = 45), multiple system atrophy (MSA) (n = 46), and corticobasal degeneration (CBD) (n = 12). The detecting antibody is specific to the C-terminal epitope of Aß15. We found that a carboxypeptidase (CPB) present in fetal bovine serum (FBS), a component of the buffers used, degrades Aß1-16 to Aß1-15, which is then detected by the Aß1-15/16 assay. Significantly, lower levels of Aß1-15/16 were detected in PD, PDD, PSP, and MSA compared to other neurodegenerative diseases and controls. Using the specific Aß1-15/16 assay, a reliable quantification of Aß1-15 or Aß1-15/16 in CSF samples is obtained. We found reduced levels of Aß1-15 in parkinsonian disease groups. The molecular mechanism behind this reduction is at present unknown.

Catalytic specificity of human protein tyrosine kinases revealed by Peptide substrate profiling.

Out of the 90 human protein tyrosine kinases, 81 were assayed with short peptides derived from well-characterized [CDK1(Tyr15), IRS1(Tyr983), and JAK1(Tyr1023)] or generic [polyGlu:Tyr(4:1) and poly-Glu:Ala:Tyr(1:1:1)] substrates. As expected, the CDK1 peptide is a substrate for all Src family kinases. On the other hand, some of the activities are novel and lead to a better understanding of the function of certain kinases. Specifically, the CDK1 peptide is a substrate for many of the Eph family members. Interestingly, profiling of nearly all the human protein tyrosine kinases revealed a distinct pattern of selectivity towards the CDK1 and IRS1 peptides.

Single-well monitoring of protein-protein interaction and phosphorylation-dephosphorylation events.

We combined oxygen channeling assays with two distinct chemiluminescent beads to detect simultaneously protein phosphorylation and interaction events that are usually monitored separately. This novel method was tested in the ERK1/2 MAP kinase pathway. It was first used to directly monitor dissociation of MAP kinase ERK2 from MEK1 upon phosphorylation and to evaluate MAP kinase phosphatase (MKP) selectivity and mechanism of action. In addition, MEK1 and ERK2 were probed with an ATP competitor and an allosteric MEK1 inhibitor, which generated distinct phosphorylation-interaction patterns. Simultaneous monitoring of protein-protein interactions and substrate phosphorylation can provide significant mechanistic insight into enzyme activity and small molecule action.