Background Intravitreal injection (IVI) of antibody biologics is a key treatment approach in ophthalmology. Pharmaceutical compounding and storage of prefilled syringes for IVI must take place without impairing the structure and function of the biologics. This study investigated the effect of withdrawing and storing the therapeutic antibody faricimab (Vabysmo, Roche, Basel, Switzerland) in the Zero Residual silicone oil-free, 0.2-mL syringe (SJJ Solutions, The Hague, the Netherlands). Methods To assess the effect of syringe withdrawal on faricimab, we compared samples from syringes prepared at day 0 with samples taken directly from faricimab vials. To assess the effect of syringe storage on faricimab, we kept prefilled syringes in the dark at 4 o C for 7, 14, or 37 days and compared samples from these syringes with day 0. We measured protein concentration (with spectrophotometry), stability and integrity (with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), size-exclusion chromatography (SEC), and melting temperature (Tm)), as well as binding of faricimab to its cognate antigens: vascular endothelial growth factor A (VEGF-A) and angiopoietin-2 (Ang-2) (with enzyme-linked immunosorbent assay (ELISA)). Results Faricimab migrated in line with its expected molecular mass under both reducing and non-reducing conditions for all time points when analyzed with SDS-PAGE, without any sign of degradation products or aggregation. The SEC elution profiles were identical for all time points. There were slight variations in Tm for different time points compared to day 0 but without consistent relationship with storage time. ELISA did not detect differences in VEGF-A or Ang-2 binding between time points, and faricimab did not bind the neonatal Fc receptor. Conclusions Withdrawal and storage of faricimab in syringes for up to day 37 did not impair the structure and bi-specific binding properties of the therapeutic antibody.

Immunocompromised patients often fail to raise protective vaccine-induced immunity against the global emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants. Although monoclonal antibodies have been authorized for clinical use, most have lost their ability to potently neutralize the evolving Omicron subvariants. Thus, there is an urgent need for treatment strategies that can provide protection against these and emerging SARS-CoV-2 variants to prevent the development of severe coronavirus disease 2019. Here, we report on the design and characterization of a long-acting viral entry-blocking angiotensin-converting enzyme 2 (ACE2) dimeric fusion molecule. Specifically, a soluble truncated human dimeric ACE2 variant, engineered for improved binding to the receptor-binding domain of SARS-CoV-2, was fused with human albumin tailored for favorable engagement of the neonatal fragment crystallizable receptor (FcRn), which resulted in enhanced plasma half-life and allowed for needle-free transmucosal delivery upon nasal administration in human FcRn-expressing transgenic mice. Importantly, the dimeric ACE2-fused albumin demonstrated potent neutralization of SARS-CoV-2 immune escape variants.

Depletion of the Anaphase-Promoting Complex/Cyclosome (APC/C) activator Cdc20 arrests cells in metaphase with high levels of the mitotic cyclin (Cyclin B) and the Separase inhibitor Securin. In mammalian cells this arrest has been exploited for the treatment of cancer with drugs that engage the spindle assembly checkpoint and, recently, with chemical inhibitors of the APC/C. While most cells arrested in mitosis for prolonged periods undergo apoptosis, others skip cytokinesis and enter G1 with unsegregated chromosomes. This process, known as mitotic slippage, generates aneuploidy and increases genomic instability in the cancer cell. Here, we analyze the behavior of fission yeast cells arrested in mitosis through the transcriptional silencing of the Cdc20 homolog slp1. While depletion of slp1 readily halts cells in metaphase, this arrest is only transient and a majority of cells eventually undergo cytokinesis and show steady mitotic dephosphorylation. Notably, this occurs in the absence of Cyclin B (Cdc13) degradation. We investigate the involvement of phosphatase activity in these events and demonstrate that PP2A-B55Pab1 is required to prevent septation and, during the arrest, its CDK-mediated inhibition facilitates the induction of cytokinesis. In contrast, deletion of PP2A-B56Par1 completely abrogates septation. We show that this effect is partly due to this mutant entering mitosis with reduced CDK activity. Interestingly, both PP2A-B55Pab1 and PP2A-B56Par1, as well as Clp1 (the homolog of the budding yeast mitotic phosphatase Cdc14) are required for the dephosphorylation of mitotic substrates during the escape. Finally, we show that the mitotic transcriptional wave controlled by the RFX transcription factor Sak1 facilitates the induction of cytokinesis and also requires the activity of PP2A-B56Par1 in a mechanism independent of CDK.

Abstract While the established route for vaccines against the pandemic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is intramuscular, it may be preferable to deliver vaccines intranasally to secure mucosal protection at the site of infection. This will limit the spread of the virus, ease administration and likely improve vaccine acceptance. Here, we report on a subunit vaccine platform, where the antigen is genetically fused to engineered human albumin. Upon intranasal delivery the subunit vaccines target the neonatal Fc receptor (FcRn) and induce both local and systemic antigen-specific antibody responses at magnitudes higher than after intramuscular delivery. We provide evidence that such needle-free vaccination induces production of antibodies with neutralizing capacity against SARS-CoV-2 or influenza A. Thus, the vaccine platform is particularly well suited for design of subunit vaccines against these and other infectious respiratory diseases.

Extracellular cues regulate cell fate, and this is mainly achieved through the engagement of specific transcriptional programs. The TORC1 and TORC2 complexes mediate the integration of nutritional cues to cellular behavior, but their interplay is poorly understood. Here, we use fission yeast to investigate how phosphatase activity participates in this interplay during the switch from proliferation to sexual differentiation. We find that loss of PP2A-B55Pab1 enhances the expression of differentiation-specific genes and leads to premature conjugation. pab1 deletion brings about a transcriptional profile similar to TORC1 inactivation, and deletion of pab1 overcomes the repression of differentiation genes in cells overexpressing TORC1. Importantly, we show that this effect is mediated by an increased TORC2-AKT (Gad8) signaling. Under nutrient-rich conditions, PP2A-B55Pab1 dephosphorylates Gad8 Ser546, repressing its activity. Conversely, TORC1 inactivation upon starvation leads to the inactivation of PP2A-B55Pab1 through the Greatwall-Endosulfin pathway. This results in the activation of Gad8 and the commitment to differentiation. Thus, PP2A-B55Pab1 enables a crosstalk between the two TOR complexes that controls cell-fate decisions in response to nutrient availability.