Some monoclonal antibodies undergo liquid–liquid phase separation owing to self-attractive associations involving electrostatic and other soft interactions, thereby rendering monoclonal antibodies unsuitable as therapeutics. To mitigate the phase separation, formulation optimization is often performed. However, this is sometimes unsuccessful because of the limited time for the development of therapeutic antibodies. Thus, protein mutations with appropriate design are required. In this report, we describe a case study involving the design of mutants of negatively charged surface residues to reduce liquid–liquid phase separation propensity. Physicochemical analysis of the resulting mutants demonstrated the mutual correlation between the sign of second virial coefficient B 2 , the Fab dipole moment, and the reduction of liquid–liquid phase separation propensity. Moreover, both the magnitude and direction of the dipole moment appeared to be essential for liquid–liquid phase separation propensity, where electrostatic interaction was the dominant mechanism. These findings could contribute to a better design of mutants with reduced liquid–liquid phase separation propensity and improved drug-like biophysical properties.

ORAI1 constitutes the pore‐forming subunit of the calcium release‐activated calcium (CRAC) channel, which is responsible for store‐operated calcium entry into lymphocytes. It is known that ORAI1 is essential for the activation of T cells and mast cells and is considered to be a potent therapeutic target for autoimmune and allergic diseases. Here, we obtained a new humanized antibody, DS‐2741a, that inhibits ORAI1 function. DS‐2741a bound to human‐ORAI1 with high affinity and without cross‐reactivity to rodent Orai1. DS‐2741a demonstrated suppression of CRAC‐mediated human and mouse T‐cell activation and mast cell degranulation in human ORAI1 knock‐in mice. Furthermore, DS‐2741a ameliorated house dust mite antigen‐induced dermatitis in the human ORAI1 knock‐in mouse. Taken together, DS‐2741a inhibited T‐cell and mast cell functions, thus improving skin inflammation in animal models of atopic dermatitis and reinforcing the need for investigation of DS‐2741a for the treatment of allergic diseases in a clinical setting.

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