Incorporation of a repeated polypeptide sequence in therapeutic antibodies as a universal masking procedure: A case study of T cell-engaging bispecific antibodies.

Prodrug design is a promising approach for reducing the off-target effects of therapeutic antibodies, particularly bispecific antibodies (bsAbs) that recruit T cells for activation; this design uses masking sequences that inhibit antibody binding until they reach the tumor microenvironment, where they are removed. In this study, we propose PAS, a polypeptide sequence composed of repeated Pro, Ala, and Ser residues, as a universal masking sequence. PAS has no specificity, but can inhibit antibody binding through steric hindrance caused by its large fluid dynamic radius and disordered structure; additionally, its length can be adjusted. We fused PAS to the N-terminus of an anti-CD3 single-chain variable fragment (scFv) and a bsAb, that targets both the epidermal growth factor receptor and CD3, via a recognition sequence cleaved by cancer-related proteases. PAS integration inhibited anti-CD3 scFv binding with higher efficacy than the epitope sequence, and the extent of inhibition was proportional to the length of the PAS sequence. For masked bsAbs, T cell-binding ability, cancer growth inhibition effects, and T cell activation effects were also reduced depending on the length of PAS and were fully restored upon removing PAS sequences using protease. The masking procedure using PAS was successfully applied to another scFv. The provision to adjust the masking effects of PAS by tuning its length, makes PAS fusion a valuable tool for the universal design of prodrug antibodies.

Pediatric Respiratory Virus Infections During the COVID-19 Pandemic in a Region Without Active Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Circulation.

Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections were not prevalent in Yonago and its vicinity during autumn 2020, and the relative frequencies of pathogen-induced respiratory infections during this period are unclear. Methods: We collected 109 nasopharyngeal swabs from 93 pediatric patients who visited Tottori University Hospital between October 1, 2020, and March 31, 2021. These samples were comprehensively tested for 18 pathogens with the FilmArray® respiratory panel test (v2.1) using nested real-time polymerase chain reaction, and the frequency of pathogens detected per month was calculated. In addition, we compared the duration of fever and the blood test results of patients infected with each pathogen or multiple pathogens. Results: Of the 109 samples, 42 were obtained from female patients and 67 from male patients (median age, 3 years; range, 0-15 years). Overall, 62 patients (56.9%) had a fever ≥ 38 °C at the time of examination, and the median duration of fever ≥ 38 °C was 2 days (1-12). During the study period, the highest number of samples (22) were collected in November 2020. Among samples that tested positive, the most common pathogens were rhino/enteroviruses (52 samples; 76.5%), followed by adenoviruses (7 samples; 10.3%), coronavirus NL63 (6 samples; 8.8%), coronavirus OC43, parainfluenza virus type 1, and parainfluenza virus type 2 (1 sample each; 1.5% each). The duration of fever was significantly longer in adenovirus-infected patients than in patients infected with other viruses (P < 0.05). Hemoglobin and sodium levels were also significantly lower among the adenovirus-infected patients. However, these variations were mostly within the normal range. No clinically meaningful differences were found between rhino/enterovirus-infected and non-rhino/enterovirus-infected cases, between coronavirus NL63-infected and non-coronavirus NL63-infected cases, and between cases with multiple- and single-pathogen infections. Conclusion: Rhino/enteroviruses were the most common viruses causing respiratory tract infections in areas without endemic SARS-CoV-2.

Evaluation of intercellular cross-linking abilities correlated with cytotoxicities of bispecific antibodies with domain rearrangements using AFM force-sensing.

Bispecific antibodies (bsAbs) are a promising engineered antibody format; thus, technologies for the fabrication and evaluation of functional bsAbs are attracting increasing attention. Here, based on atomic force microscopy (AFM) force-sensing integrated with a metal cup-attached AFM chip (cup-chip) to ensure efficient capture of a target cell on a cantilever, we established a novel method for measuring cross-linking ability that is correlated with the cytotoxicities of bsAbs targeting two cells. We previously reported that domain rearrangements of bsAbs affected their cytotoxicities; however, no differences in cross-linking ability for soluble antigens were observed by surface plasmon resonance. We predicted that there would be differences in molecular configurations to avoid steric hindrance in the cross-linking of the two whole target cells. A picked-up T cell lymphoma cell on the cantilever using a cup-chip was moved to approach a cancer cell adhered to a dish, and force-curve measurements were performed. The resulting forces mediated by the cross-linking of bsAbs with different domain orders were well-correlated with their cytotoxicities. The AFM force-sensing method established herein may reflect steric hindrance of intercellular cross-linking, and thus has the potential to evaluate the net function of bsAbs and contribute to the generation of functional bsAbs.