Bispecific antibodies (BsAbs) that redirect cytotoxic T lymphocytes (CTLs) via CD3 engagement represent a promising approach in cancer immunotherapy. Here, we explore the development of VNAR-based BsAbs for improved tumor targeting. Shark-derived VNARs possess unique properties such as small size, high stability, and antigen specificity, making them ideal candidates for T cell engagement. We immunized banded houndsharks (Triakis scyllium) with the recombinant human CD3 epsilon domain to isolate VNARs that specifically recognize CD3. The isolated anti-CD3 VNARs showed moderate affinity for recombinant human CD3 and bound to CD3+ cell lines. These results suggest the potential of these VNARs for the construction of VNAR-based BsAbs as T cell engagers, providing a novel avenue for cancer immunotherapy.
The neonatal Fc receptor (FcRn) extends the serum half-life of immunoglobulins G (IgG) through a pH-dependent interaction that protects and recycles IgG during intracellular trafficking. To understand the molecular basis of the prolonged half-life observed with the Fc variant KU-1, this study employs X-ray crystallography and in silico modeling to elucidate the mode of interaction (MOI) between FcRn and KU-1. Deciphering this interaction has the potential to significantly impact the development of KU-1-based biopharmaceuticals, paving the way for enhanced therapeutic efficacy and safety profiles.
CD27, a costimulatory molecule of the TNF receptor superfamily expressed on T lymphocytes, plays a critical role in regulating T cell survival, differentiation, and effector function. Upon binding its ligand, CD70, CD27 signaling enhances T cell proliferation and differentiation into effector and memory T cells. This agonistic activity positions CD27 as a promising target for immunomodulatory cancer therapy. This study investigates the development of therapeutic agents targeting the CD27 for cancer immunotherapy. We employed a recombinant human CD27 extracellular domain to immunize an alpaca (Vicugna pacos), generating antigen-specific single-domain antibodies (nanobodies). Through biopanning using the immunized phage display library, we identified the anti-CD27 cpNb4 clone exhibiting high binding affinity for recombinant human CD27 and specific binding to cell-surface CD27 expressed on CHO-K1 cells. These findings establish cpNb4 as a promising candidate for further investigation, potentially paving the way for its integration into combination immunomodulatory cancer therapy regimens.
Cellular senescence is a permanent cell proliferation arrest compromising cell regeneration and tissue repair process which gradually leads to age-related disorders. The driving factor is considered to be senescence-associated secretory phenotype(SASP) a diverse pro-inflammatory secretory factors exerted to nearby cells resulting in normal cell aging. Targeted therapies with complex drug that can selectively modulate these cells offer promising avenues for anti-aging interventions. In this study, we developed extracellular vesicles(EVs) with specific ligands as a novel drug carrier system aimed to selectively target senescent cells. By attaching ligands on the surface of EVs, we enhanced their affinity for senescent cells over normal cells and the targeting efficiency was assessed using fluorescence-activated cell sorting(FACs). The results demonstrated a significantly higher uptake of modified EVs in senescent cells which suggest that this selective delivery can effectively serve as a precision drug carrier. Future work will focus on loading therapeutic agents that exhibit senolytic or senomorphic activities to these ligand-modified EVs aiming to selectively attenuate cellular senescence.
The accumulation of aggregates of the microtubule-binding protein Tau represents a pathological hallmark in Alzheimer’s disease (AD). While Tau is primarily recognized for its interaction with microtubules, recent findings suggest the presence of Tau clusters near the plasma membrane, potentially serving as binding partners for Axonal Initial Segment (AIS)-related membrane proteins and synaptic proteins. Additionally, during AD, pathogenic tau is known to traverse the membrane via cell-to-cell transport. Furthermore, recently our group identified lipidation as a process enabling Tau’s interaction with the membrane. However, despite tau’s hydrophilic nature, the precise mechanism through which Tau dynamics might fulfill a novel physiological function by facilitating its interaction with hydrophobic lipid membranes remains elusive. In this study, we performed single-molecule imaging with total internal reflection fluorescence microscopy (TIRF) to observe tau dynamics near the plasma membrane of differentiated PC12 cells. Indeed, expression of Tau mutant constructs with inhibited lipidation in PC12 cells resulted in increased mobility of Tau near the plasma membrane. Moreover, treatment with an inhibitor targeting lipidation produced similar effects as observed with Tau mutants, suggesting that lipidation-mediated membrane interaction slows Tau mobility. In primary hippocampal neurons, we observed colocalization of Tau with lipidation-related proteins, and the Proximity Ligation Assay (PLA) confirmed their presence within 40nm proximity. This study introduces a novel post-translational modification mechanism enabling Tau interaction with the membrane. It show that Tau exhibits distinctive dynamic characteristics in close proximity to the plasma membrane, where its interaction with membrane-associated proteins could potentially serve as a potent mechanism for spatially guiding tau towards native membrane-mediated functions.
The trans-activating CRISPR RNA (tracrRNA) is fundamental to the CRISPR/Cas9 system, forming guide RNA with crRNA. Despite its known importance in crRNA maturation and Cas9 RNP-mediated DNA cleavage, the exact function of tracrRNA scaffolds remains unclear. In this investigation, we generated five tracrRNA variants by removing specific scaffolds, including Stem loops 1, 2, and 3, and the Linker. Using a new single-molecule assay, we directly observed target binding and cleavage processes guided by Cas9 RNP. Our findings underscore the vital role of the Linker in initiating R-loops and highlight the significance of Stem loop 2 in identifying PAM-distal mismatches within target DNA. Furthermore, we explored cleavage efficiency by adding tracrRNA segments, indicating that maintaining the integrity of Stem loops 2 and 3 is crucial for potent Cas9 activity. We believe that these results deepen our understanding of Cas9 functionality and offer insights into its detailed mechanism from target binding to cleavage.
Tau, known primarily as a microtubule-binding protein, is also found in the nucleus where it binds to DNA. Recent investigations have focused on its role in stabilizing DNA and chromosomes, but the biophysical understanding of its molecular mechanisms, particularly regarding tau’s phase separation properties, remains limited. In this study, we used in vitro single-molecule assays to show that tau interacts with DNA to form co-condensates, significantly altering the mechanical properties of DNA. Our findings indicate that tau can wet the DNA strand in low-salt conditions, effectively condensing and stiffening the DNA. At high concentrations, tau also forms droplet-shaped condensates on DNA, similar to its interaction with microtubules. Notably, these condensates are mobile and may act as nucleation sites for microtubule growth. This study reveals previously unknown effects of Tau-DNA condensation and suggests that these interactions could influence microtubule dynamics during mitosis.
Extracellular vesicles (EVs) are released from cells and can be taken up by other cells to mediate communication among distant cells. The process of vesicle uptake is initiated by the docking of the vesicles onto membrane proteins on cells, but a generalizable technique for quantitatively observing these vesicle–protein interactions (VPIs) is lacking. Here, we develop a technique that measures VPIs between single vesicles and cell-surface proteins using total internal reflection fluorescence microscopy. We first describe a simple procedure that can effectively label vesicles without complex purification. Subsequently, we quantify the interaction between the labeled vesicles and target proteins either attached to a surface or embedded in a lipid bilayer. By employing cell-derived vesicles (CDVs) and intercellular adhesion molecule-1 (ICAM-1) as a model system, we determine the binding affinity of vesicles toward the ICAM-1 depending on cell types of vesicle origin. Moreover, controlling the surface density of proteins also reveals robust support from a tetraspanin protein CD9, with a critical dependence on molecular proximity. We expect that VPI imaging will be a useful tool to dissect the molecular mechanisms of vesicle uptake and to guide the development of therapeutic vesicles.
Pharmaceutical and biological researchers consistently explore questions related to protein structures and mutations to better understand virus evolution. In thermodynamics, protein structures are predicted through computational simulations, such as molecular dynamic simulation, which calculates free energy, intermediate states, mutation effects, and protein-protein interactions. However, this novel method has limitations in deciphering complex protein structures. To bridge this gap in protein understanding, machine learning and deep learning are applied to study virus evolution. Notably, escape mutations of SARS-CoV-2 have been predicted using natural language processing techniques, which interpret amino acid sequences in terms of semantic change (antigenic variant) and grammatical quality (viability/fitness). Surprisingly, training models using only amino acid sequences was sufficient to predict escape mutations without additional information on protein structure and function.
Despite the potential of natural language models to suggest possible escape mutations, there is a need to enhance the accuracy of these predictions to minimize the selection of unnecessary candidates. In this study, we evaluated and refined a novel language model by incorporating nucleotide substitutions to improve prediction accuracy. Biologically, amino acid sequences are determined by nucleotide compositions, and most mutations occur at the DNA or RNA level. Although deep learning models might indirectly learn this information from amino acid sequences, integrating direct nucleotide data into the model has resulted in more precise estimations with higher accuracy. This approach has enabled us not only to reduce unnecessary candidates for escape mutations and but also to enhance prediction of characteristic and dominant mutations.
Cellular senescence is a permanent cell proliferation arrest compromising cell regeneration and tissue repair process which gradually leads to age-related disorders. The driving factor is considered to be senescence-associated secretory phenotype(SASP) a diverse pro-inflammatory secretory factors exerted to nearby cells resulting in normal cell aging. Targeted therapies with complex drug that can selectively modulate these cells offer promising avenues for anti-aging interventions. In this study, we developed extracellular vesicles(EVs) with specific ligands as a novel drug carrier system aimed to selectively target senescent cells. By attaching ligands on the surface of EVs, we enhanced their affinity for senescent cells over normal cells and the targeting efficiency was assessed using fluorescence-activated cell sorting(FACs). The results demonstrated a significantly higher uptake of modified EVs in senescent cells which suggest that this selective delivery can effectively serve as a precision drug carrier. Future work will focus on loading therapeutic agents that exhibit senolytic or senomorphic activities to these ligand-modified EVs aiming to selectively attenuate cellular senescence.