Interdomain disulfide bonds of rabbit kappa light chain allotypes influence mouse-rabbit chimeric antibody performance

Yang-Nim Park; Douglas W Houston

doi:10.1101/2025.09.17.676883

Chimeric monoclonal antibodies have emerged as powerful tools to extend the capabilities of traditional monoclonal antibodies. These antibodies are made by replacing the variable region of a monoclonal antibody with the variable region of another antibody, typically from a different species, enabling use in a wider range of applications. Although theoretically compatible, interspecies differences in antibody structure can complicate chimeric antibody design and performance. In this study, we evaluated the impact of rabbit light chain allotype on the expression and function of chimeric antibodies containing mouse light chain variable regions. We found that constructs using the rabbit kappa 1 b4 (K1-b4) allotype frequently exhibited poor recombinant expression and, in some cases, lost antigen recognition. Structural analysis implicated disruption of an intrachain, interdomain disulfide bond as a contributing factor. Restoration of key residues predicted to re-establish this bond partially rescued both expression and activity. Additionally, chimeric antibodies incorporating the rabbit kappa 1 b9 (K1-b9) allotype, which contains a disulfide bond not disrupted by mouse variable region sequences, consistently maintained robust production and antigen-binding activity across multiple applications, including immunoblotting, immunoprecipitation, and immunostaining, for several test antibodies. Our findings underscore the importance of light chain scaffold selection in recombinant antibody engineering and provide practical guidance for optimizing chimeric antibody design to preserve both expression and function.Chimeric monoclonal antibodies have emerged as powerful tools to extend the capabilities of traditional monoclonal antibodies. These antibodies are made by replacing the variable region of a monoclonal antibody with the variable region of another antibody, typically from a different species, enabling use in a wider range of applications. Although theoretically compatible, interspecies differences in antibody structure can complicate chimeric antibody design and performance. In this study, we evaluated the impact of rabbit light chain allotype on the expression and function of chimeric antibodies containing mouse light chain variable regions. We found that constructs using the rabbit kappa 1 b4 (K1-b4) allotype frequently exhibited poor recombinant expression and, in some cases, lost antigen recognition. Structural analysis implicated disruption of an intrachain, interdomain disulfide bond as a contributing factor. Restoration of key residues predicted to re-establish this bond partially rescued both expression and activity. Additionally, chimeric antibodies incorporating the rabbit kappa 1 b9 (K1-b9) allotype, which contains a disulfide bond not disrupted by mouse variable region sequences, consistently maintained robust production and antigen-binding activity across multiple applications, including immunoblotting, immunoprecipitation, and immunostaining, for several test antibodies. Our findings underscore the importance of light chain scaffold selection in recombinant antibody engineering and provide practical guidance for optimizing chimeric antibody design to preserve both expression and function.

Interdomain disulfide bonds of rabbit kappa light chain allotypes influence mouse-rabbit chimeric antibody performance

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