Makoto Nakakido

Accurate identification of epitope residues is essential for developing biopharmaceuticals and understanding the mechanisms of immune recognition. However, experimental approaches for residue‐level epitope mapping remain time‐consuming and labor‐intensive, while accurate computational prediction of protein–protein interfaces remains challenging. Here, we present MAXTIA, a high‐throughput kinetic screening platform that integrates cell‐free protein synthesis with high‐throughput surface plasmon resonance and demonstrate its application to alanine scanning‐based functional epitope mapping. This workflow enables the rapid preparation and kinetic characterization of up to 384 protein variants, allowing the identification of functional epitope residues within 3 days while simultaneously providing binding affinity and kinetic parameters ( K _D , k _on , and k _off ). We applied MAXTIA to map the epitope of the single‐domain antibody (VHH) N1 against the pentraxin domain of neuronal pentraxin‐2 (NP2 PTX). Alanine substitutions that cause substantial affinity losses clustered within a localized region on the AlphaFold3‐predicted NP2 PTX structure, defining a functional epitope site. These residues closely matched the interface observed in the NP2 PTX–VHH N1 crystal structure, validating the accuracy of MAXTIA. Beyond epitope identification, MAXTIA provides a simple and versatile platform for the quantitative analysis of protein–protein interactions, including high‐throughput screening of antibody variants for affinity optimization. This approach should accelerate biopharmaceutical development and facilitate mechanistic studies of molecular recognition.

Single domain antibodies (sdAbs) can be generated from variable regions of heavy‐chain antibodies, which lack light chain and CH1 region. They have attracted attention due to their small size and molecular characteristics. Hydrophilic hallmark amino acids at framework region 2 (FR2) are key residues involved in the solubility of sdAbs. Nevertheless, previous studies reported that several sdAbs with human VH‐like hydrophobic hallmark residues were soluble in a monomeric state and suggested that solubility also depends on the amino acid sequences in the complementarity‐determining region. In this study, we obtained two sdAbs (sdAb A and B) with VH‐like hallmark residues and low solubility from an alpaca immune library. We introduced VHH‐like mutations (V37Y, G44E, L45R, W47L) into the hallmark residues in FR2 of both sdAb A and B. We were able to prepare sdAb A as a monomer without an additive in the buffer, but sdAb B was polydispersed when arginine was not added to the buffer. We also predicted the hydrophobicity of the sdAb B surface by spatial aggregation propensity calculations and identified W99 as the residue responsible for its low solubility. Subsequently, we obtained the sdAb B mutant as a monomer by introducing the W99A mutation. We characterized the engineered sdAbs using structural, physicochemical, and biophysical analyses and found that the solubility‐improved sdAbs retained their functionality. Our findings can be applied to improving the solubility of sdAbs even in the absence of structural information.

Group A Streptococcus (GAS) are gram-positive bacteria that cause various symptoms. The treatment of GAS infections currently relies on antibiotics, but new treatment options are needed due to the spread of antibiotic resistance. To develop novel treatment methods that circumvent the generation of antibiotic resistance, we used virtual screening followed by several biophysical-based screening methods to identify antibacterial compounds that target SPs0871, which is a maltodextrin-binding protein that is involved in carbohydrate catabolism in GAS. We narrowed down the list of compounds in the library via multi-step screening and finally isolated a compound that bacteriostatically inhibited the growth of GAS. Together with our previous study showing that an anti-SPs0871 variable heavy domain of heavy chain antibody, which completely blocked ligand binding, did not suppress bacterial growth, our results provide guidelines for designing an antistreptococcal therapeutic.

This study reports the potent antiviral ability of proton-type zeolites without the aid of any metal cations. Antiviral activities of zeolites with different topologies and chemical compositions are investigated using M13 phage and influenza virus. Proton-type zeolites exhibit excellent antiviral activity, equivalent to 99% inactivated. Antiviral tests using a centricon device suggest that direct contact of viruses on the external surface of zeolites is required to inactivate the viruses. This discovery is of importance in an interdisciplinary research field covering both zeolite science and virological science and shed light on the possibility of the development of low-cost and environmentally friendly antiviral zeolites.

Protein aggregation and liquid‒liquid phase separation (LLPS), as key physicochemical processes, orchestrate protein behavior and function, and engineering a protein surface charge offers a robust approach to modulate protein‒protein interactions and, consequently, aggregation and phase separation. Among protein surface engineering methods, supercharging leads to a drastic increase in the protein net charge by replacing surface residues with charged amino acid residues. Previous studies have reported that some physicochemical properties of proteins are improved by supercharging, and changing the surface charge is considered to affect intermolecular interactions. In this study, we designed a new supercharged antigen-binding fragment (Fab) antibody mutant and investigated its aggregation behavior. Upon examination of the physicochemical properties of the designed supercharged antibody, the thermal stability, structure, and ligand binding affinity of the antibody were retained despite having the same charge pairing of both the antibody and the antigen. Furthermore, we revealed that the antibody exhibited reversible ligand- and salt concentration-dependent aggregation. Our study demonstrated how supercharging can potentially modulate protein aggregation and LLPS. It is expected that this approach can be extended to other proteins, through which its applicability in various biological and biotechnological fields can be explored.

Single‐domain V_HH antibodies are promising therapeutic and diagnostic tools. The third complementarity‐determining region (CDR3) is usually the most critical region for antigen recognition by V_HH antibodies. When CDR3 adopts a short and extended β‐hairpin conformation, framework region 2 (FR2) often interacts directly with the antigen. However, the importance of these interactions in antigen recognition remains unclear. In this research, we investigated the role of FR2 residues in V_HH antibodies with β‐hairpin CDR3s. We found that several FR2 residues, particularly at positions 35 and 37, are critical for high‐affinity antigen binding. Notably, a trade‐off was observed: introducing a charged residue at position 35 enhanced binding affinity but reduced thermal stability. These findings provide insights into optimizing FR2 in single‐domain antibodies to improve their functionality for diagnostic and therapeutic applications.

Due to the high affinity and specificity of antibodies toward antigens, various antibody-based applications have been developed. Recently, variable antigen-binding domains of heavy-chain antibodies (VHH) have become an attractive alternative to conventional fragment antibodies due to their unique molecular characteristics. As an antibody-generating strategy, synthetic VHH libraries (including humanized VHH libraries) have been developed using distinct strategies to constrain the diversity of amino acid sequences. In this study, we designed and constructed several novel synthetic humanized VHH libraries based on biophysical analyses conducted using the complementarity determining region-grafting method and comprehensive sequence analyses of VHHs deposited in the protein data bank. We obtained VHHs from the libraries, and hit clones exhibited considerable thermal stability. We also found that VHHs from distinct libraries tended to have different epitopes. Based on our results, we propose a strategy for generating humanized VHHs with distinct epitopes toward various antigens by utilizing our library combinations.