Antibody Engineering

Vinodh B. Kurella, Reddy Gali, Kurella, Vinodh B., Gali, Reddy

Antibody humanization process converts any nonhuman antibody sequence into humanized antibodies. This can be achieved using different methods of antibody design and engineering. This chapter will primarily focus on antibody design using a homology model followed by framework shuffling of murine to human germline sequence for humanization. Historically, mouse antibodies have been humanized using sequence-based approaches, in which all the murine frameworks are replaced with most homologous human germline sequence or related scaffold. Most often this humanized antibody design, when tested, has a significantly reduced binding or no binding to the cognate antigen. This is due to noncompatibility of mouse CDRs being supported by non-native human framework scaffold. This mismatch between mouse, human structural fold, and elimination of key conformational residues often leads to antibody humanization failures. Recently, there has been advent of homology modelor structure-guided antibody humanization. Instead of humanization based on linear sequence, this approach takes into account the tertiary structure and fold of the mouse antibody. A mouse homology model of the fragment variable is created, and based on sequence alignment with human germline, residues that are different in mouse are replaced with humanized sequence in the model. Energy minimization is applied to this humanized model that also delineates residues which might have steric clashes due to change in the overall tertiary conformation of the humanized antibody. Therefore, a homology model-guided with rational mutations, and reintroduction of key conformational residues from mouse antibody not only eliminates steric clashes but might also restore function in relation to binding affinity to its antigen.