Statistical Approaches for Establishing Appropriate Immunogenicity Assay Cut Points: Impact of Sample Distribution, Sample Size, and Outlier Removal.

The statistical assessments needed to establish anti-drug antibody (ADA) assay cut points (CPs) can be challenging for bioanalytical scientists. Poorly established CPs that are too high could potentially miss treatment emergent ADA or, when set too low, result in detection of responses that may have no clinical relevance. We evaluated 16 validation CP datasets generated with ADA assays at Regeneron's bioanalytical laboratory and compared results obtained from different CP calculation tools. We systematically evaluated the impact of various factors on CP determination including biological and analytical variability, number of samples for capturing biological variability, outlier removal methods, and the use of parametric vs. non-parametric CP determination. In every study, biological factors were the major component of assay response variability, far outweighing the contribution from analytical variability. Non-parametric CP estimations resulted in screening positivity in drug-naïve samples closer to the targeted rate (5%) and were less impacted by skewness. Outlier removal using the boxplot method with an interquartile range (IQR) factor of 3.0 resulted in screening positivity close to the 5% targeted rate when applied to entire drug-naïve dataset. In silico analysis of CPs calculated using different sample sizes showed that using larger numbers of individuals resulted in CP estimates closer to the CP of the entire population, indicating a larger sample size (~ 150) for CP determination better represents the diversity of the study population. Finally, simpler CP calculations, such as the boxplot method performed in Excel, resulted in CPs similar to those determined using complex methods, such as random-effects ANOVA.

Mitigating target interference in bridging immunogenicity assay with target-blocking reagents and mild basic pH.

Background: Soluble drug target in clinical study samples generated false positive results in anti-drug antibody (ADA) bridging assays due to target-mediated bridging. Results: The combination of two target-blocking reagents and mild basic assay pH resulted in high tolerance to recombinant target protein and reduced levels of positivity in clinical study samples with pharmacokinetic profiles that did not indicate significant ADA response. Testing with low-affinity ADA positive serum from immunized rabbits and known ADA positive samples from nonclinical studies in rats confirmed the assay's ability to detect ADA positive samples and the minimal impact of basic pH and target-blocking reagents on ADA detection. Conclusion: These strategies provide alternatives for mitigating target interference when standard target-blocking antibodies alone are ineffective.

Bridging immunogenicity assays for IgG4 therapeutics: mitigating interference from Fc-Fc interactions.

AIM: A bridging immunogenicity assay for a human IgG4 mAb therapeutic was transferred to an automation system to increase throughput. However, background signal increased five- to six-fold during the 6- to 8-h run. RESULTS: Noncovalent Fc contacts formed between labeled IgG4 drugs in reagent solutions stored during the automation run. This generated substantial background signal, reducing assay sensitivity by approximately sixfold. Fc interactions also significantly impacted the confirmation assay. Fc contacts formed between labeled and unlabeled drug, significantly increasing signal inhibition (∼7-70%) in the 6-h run. CONCLUSION: Storing labeled antibody solutions separately and combining them immediately before adding to samples reduced interference from Fc interactions. Preincubation time for reagent solutions should be strictly controlled for anti-drug antibody assays with IgG4 drugs to avoid false-positive results.

Hematopoietic cells and osteoblasts are derived from a common marrow progenitor after bone marrow transplantation.

Bone and bone marrow are closely aligned physiologic compartments, suggesting that these tissues may represent a single functional unit with a common bone marrow progenitor that gives rise to both osteoblasts and hematopoietic cells. Although reports of multilineage engraftment by a single marrow-derived stem cell support this idea, more recent evidence has challenged claims of stem cell transdifferentiation and therefore the existence of a multipotent hematopoietic/osteogenic progenitor cell. Using a repopulation assay in mice, we show here that gene-marked, transplantable marrow cells from the plastic-nonadherent population can generate both functional osteoblasts/osteocytes and hematopoietic cells. Fluorescent in situ hybridization for the X and Y chromosomes and karyotype analysis of cultured osteoblasts confirmed the donor origin of these cells and excluded their generation by a fusion process. Molecular analysis demonstrated a common retroviral integration site in clonogenic hematopoietic cells and osteoprogenitors from each of seven animals studied, establishing a shared clonal origin for these ostensibly independent cell types. Our findings indicate that the bone marrow contains a primitive cell able to generate both the hematopoietic and osteocytic lineages. Its isolation and characterization may suggest novel treatments for genetic bone diseases and bone injuries.