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R.M., K.U., M.I., J.F.P., Y.Y. residues was generated. A high-throughput SPR analysis was performed against human and mouse PD-1, showing that the mutation in the specific region enhanced the affinity for mouse PD-1. Furthermore, deep mutational scanning of the region revealed two mutants with > 100-fold increased affinity for mouse PD-1, demonstrating the potential efficacy of antibody design using data-driven approach. Subject terms:High-throughput screening, Antibody therapy, Protein design, Surface plasmon resonance, High-throughput screening == Introduction == The number of developing or approved antibody drugs is increasing yearly, and binding affinity to the target antigen is one Fucoxanthin of the most important properties. The affinity is defined as the dissociation equilibrium constant (KD). Typical antibody drugs haveKDvalues on the order of pMnM. Some antibodies obtained from animal immunizations or synthetic libraries may have relatively weak affinities in the tens of nanomolar to micromolar. Humanization of antibodies derived from animals often results in lower affinities than those of the original antibodies1. In cases where high-affinity antibodies cannot be obtained, the affinity itself is sufficient, but the dissociation rate needs to be improved, or other high-affinity antibodies are obtained but do not bind to the appropriate epitope and do not Angiotensin Acetate have the desired biological activity, an approach is taken to optimize the sequence of the obtained antibody to improve its affinity. The conventional approach is to randomize certain residues and obtain higher-affinity clones using antibody display technology2, such as phage and yeast displays. Recently, several studies have reported that the affinity and specificity of antibodies can be improved using machine learning by evaluating the antigen binding of many antibodies using mammalian cell display or yeast display36. Other studies have designed antibodies based on changes in the composition of sequences in the phage pool before and after panning, using next-generation sequencing technology710. Although these display methods are suitable for analyzing information on clones that can bind to the target antigen from a large library, they do not provide direct information on theKDof the clones. Other approaches for designing higher-affinity antibodies are based on computer-aided design11. These approaches introduce mutations in silico to antibody residues at the interaction interface based on the antigenantibody complex structure. Candidate mutant residues with improved affinities were identified by comparing the interaction energy scores before and after mutation. Although several successful examples have been reported1218, these approaches require experimental evaluation of many clones because the predictive performance is not usually high. The bottleneck here is that only tens of clones can be evaluated because of the low throughput of the experiment; therefore, the display method is sometimes combined Fucoxanthin to screen antibodies with high binding ability. Surface plasmon resonance (SPR) is a popular interaction analysis method19,20. In this method, one of the two molecules whose interaction is to be evaluated is immobilized on a sensor chip, and a solution containing the other molecule is injected. The interaction is observed using real-time detection of the mass change on the sensor chip. In SPR, the association rate constant,kon, and the dissociation rate constant,koff, can be obtained, from whichKDcan be calculated. From a drug discovery perspective,koffis believed to be related to drug efficacy21,22, Fucoxanthin although some argue thatkonis also important for drug discovery23,24. In either case, a kinetic argument is essential for drug discovery25. Therefore, SPR is often used to characterize drug candidates. The Biacore series are the most popular SPR instruments and market leaders because of their excellent sensitivity and wide options for sensor chips and applications. However, Biacore 8 K, which is currently the highest throughput system among the Biacore series, can only measure a maximum of eight interactions, and its microfluidic channels are prone to clogging, which prevents the use of crude samples and makes it unsuitable for high-throughput measurements. Recently, an SPR instrument capable of simultaneously measuring 384 interactions was launched by Carterra Inc. This instrument, LSA, can immobilize purified antibodies26,27and antibodies in crude solutions, such as hybridoma culture medium28, and allows high-throughput evaluation of many clones simultaneously. However, using this instrument for analyses that require prior cloning, such as mutational analysis,.