High-Throughput and Format-Agnostic Mispairing Assay for Multispecific Antibodies Using Intact Mass Spectrometry.

Multispecific antibodies have gained significant importance in a broad indication space due to their ability to engage multiple epitopes simultaneously and to thereby overcome therapeutic barriers. With growing therapeutic potential, however, the molecular complexity increases, thus intensifying the demand for innovative protein engineering and analytical strategies. A major challenge for multispecific antibodies is the correct assembly of light and heavy chains. Engineering strategies exist to stabilize the correct pairing, but typically individual engineering campaigns are required to arrive at the anticipated format. Mass spectrometry has proven to be a versatile tool to identify mispaired species. However, due to manual data analysis procedures, mass spectrometry is limited to lower throughputs. To keep pace with increasing sample numbers, we developed a high-throughput-capable mispairing workflow based on intact mass spectrometry with automated data analysis, peak detection, and relative quantification using Genedata Expressionist. This workflow is capable of detecting mispaired species of ∼1000 multispecific antibodies in three weeks and thus is applicable to complex screening campaigns. As a proof of concept, the assay was applied to engineering a trispecific antibody. Strikingly, the new setup has not only proved successful in mispairing analysis but has also revealed its potential to automatically annotate other product-related impurities. Furthermore, we could confirm the assay to be format-agnostic, as shown by analyzing several different multispecific formats in one run. With these comprehensive capabilities, the new automated intact mass workflow can be applied as a universal tool to detect and annotate peaks in a format-agnostic approach and in high-throughput, thus enabling complex discovery campaigns.

A multivalent antibody assembled from different building blocks using tag/catcher systems: a case study.

The field of therapeutic antibodies and, especially bi- or multispecific antibodies, is growing rapidly. Especially for treating cancers, multispecific antibodies are very promising, as there are multiple pathways involved and multispecific antibodies offer the possibility to interfere at two or more sites. Besides being used as therapeutic, multispecific antibodies can be helpful tools in basic research. However, the design and choice of the most appropriate multispecific antibody format are far from trivial. The generation of multispecific antibodies starts with the generation of antibodies directed against the desired targets and then combining the different antigen-binding sites in one molecule. This is a time-consuming and laborious approach since the most suitable geometry cannot be predicted. The SpyTag technology is based on a split-protein system, where a small peptide of said protein, the SpyTag, can bind to the remaining protein, the SpyCatcher. An irreversible isopeptide bond between the SpyTag and the SpyCatcher is formed. A related Tag-Catcher system is the SnoopTag-SnoopCatcher. These systems offer the opportunity to separately produce proteins fused to the tag-peptides and to the catcher-domains and assemble them in vitro. Our goal was to design and produce different antibody fragments, Fab domains and Fc-containing domains, with different tags and/or catchers as building blocks for the assembly of different multivalent antibodies. We have shown that large multivalent antibodies consisting of up to seven building blocks can be prepared. Binding experiments demonstrated that all binding sites in such a large molecule retained their accessibility to their corresponding antigens.

The role of proximal circumstances and child behaviour in toddlers' risk for minor unintentional injuries.

BACKGROUND: Much of the research on child injury risk has focused on trait-like factors (eg, hyperactivity, child gender) that influence injury risk rather than state-like factors (eg, environmental circumstances, child behaviour). Additional research is needed to better identify the proximal risk factors for children's risk for unintentional injury. OBJECTIVES: The present study examined the antecedents to minor unintentional injury events and whether unusual circumstances and child behaviour predicted injury risk. METHODS: The study used archival data that were collected via biweekly in-person interviews with 170 mothers of toddlers (15-36 months) for 6 months. A case crossover design was used to predict children's risk for injury from proximal risk factors. RESULTS: Children were at a higher risk for injury when circumstances were unusual and when they were engaging in an unusual behaviour. When a child was engaging in an unusual behaviour, higher levels of maternal supervision predicted lower injury risk. Children were more likely to be injured in a new environment, in an environment with animals or other people, in an environment with hazards or when engaging in a new activity or in a familiar activity performed in an unfamiliar way. CONCLUSIONS: The results indicate that toddlers may be at a greater risk for minor unintentional injury when environmental circumstances are outside of the norm or when a child is engaging in unusual behaviours. The findings also indicate that higher levels of caregiver supervision may be especially beneficial when children are engaging in new or unfamiliar activities.

Toddlers' unintentional injuries: the role of maternal-reported paternal and maternal supervision.

UNLABELLED: Research indicates that mothers' supervision protects children from injuries. However, little research has examined the role of fathers' supervision in children's injuries. OBJECTIVES: This study compared the role of maternal and paternal supervision in children's injury risk and severity using maternal reports. METHODS: Mothers (n = 170) of toddlers were interviewed for 6 months about their children's unintentional injuries. RESULTS: Children were more likely to engage in high activity levels and were at higher risk for injury when being cared for by their fathers. Although higher supervision predicted lower injury risk for both mothers and fathers, fathers' close supervision (as reported by mothers) was a stronger predictor of injury risk than mothers' supervision. CONCLUSION: Children's higher levels of activities may have accounted for their higher risk of injury when in their fathers' care. These findings indicate the need to include fathers in injury prevention efforts.

Genome-wide pooling approach identifies SPATA5 as a new susceptibility locus for alopecia areata.

Alopecia areata (AA) is a common hair loss disorder, which is thought to be a tissue-specific autoimmune disease. Previous research has identified a few AA susceptibility genes, most of which are implicated in autoimmunity. To identify new genetic variants and further elucidate the genetic basis of AA, we performed a genome-wide association study using the strategy of pooled DNA genotyping (729 cases, 656 controls). The strongest association was for variants in the HLA region, which confirms the validity of the pooling strategy. The selected top 61 single-nucleotide polymorphisms (SNPs) were analyzed in an independent replication sample (454 cases, 1364 controls). Only one SNP outside of the HLA region (rs304650) showed significant association. This SNP was then analyzed in a second independent replication sample (537 cases, 657 controls). The finding was not replicated on a significant level, but showed the same tendency. A combined analysis of the two replication samples was then performed, and the SNP rs304650 showed significant association with P=3.43 × 10(-4) (OR=1.24 (1.10-1.39)). This SNP maps to an intronic region of the SPATA5 (spermatogenesis-associated protein 5) gene on chromosome 4. The results therefore suggest the SPATA5 locus is a new susceptibility locus for AA.

Maternal self-efficacy and associated parenting cognitions among mothers of children with autism.

Feelings of competency in the parental role, termed parenting self-efficacy, have been associated with well-being and positive parenting outcomes. Given the unique stresses inherent in raising a child with autism, parents may find it challenging to maintain a positive sense of well-being and self-efficacy. Study aims were to investigate associations between maternal self-efficacy and parenting cognitions among mothers of children with autism. Mothers (n = 170) completed questionnaires on paper or via the Internet. In a hierarchical linear regression, depression, parenting stress, agency, and guilt each accounted for unique variance in maternal self-efficacy when controlling for time since diagnosis and the presence of a second child with a disability. Autism knowledge was not associated with parenting self-efficacy. Self-efficacy appears to be associated with well-being, agency, and feelings of guilt among mothers of children with autism. Parent- and family-based interventions designed to support parental well-being and focusing on parenting cognitions may enhance parenting self-efficacy.