Antibody optimization encompasses several strategies which improve the safety and efficacy of antibodies, which is extremely important for therapeutic use. Before entering clinical trials, therapeutic antibody candidates typically undergo several phases of research and development, which include antibody discovery and screening based on antigen binding, lead selection based on biological function, and antibody optimization. Safety methods include antibody humanization and deimmunization, and efficacy methods include affinity maturation and Fc effector function engineering.
Safety
Antibody humanization is a method to reduce the immunogenicity of antibodies from non-human species. It is often used to develop monoclonal antibodies for human administration by modifying protein sequences to increase similarity to antibody variants produced naturally in humans. Typically, this is done by grafting antibody complementarity-determining regions (CDRs) from the non-human antibody onto a human variable region framework, depending on if a human residue would affect binding affinity. If the non-human residue is maintained, this is called ‘back mutation’.1
Deimmunization and tolerization are processes which can be used when fully humanized mAbs are still displaying immunogenicity due to epitope sequences in the antibody. Human T-cell epitopes may activate helper T-cells, causing the sustained production of antibodies and neutralization of the therapeutic effect. Deimmunization allows for the identification and removal of these epitopes using unspecific shielding approaches or site-directed mutagenesis, through either experimental or computational approaches.2,3
Efficacy
Affinity maturation refers to the process of improving antibody affinity and binding interactions to target antigens. This is performed in the lab in vitro by random mutagenesis, targeted mutagenesis, chain shuffling or in silico approaches, with subsequent selection. This directed evolution process is similar to the somatic hypermutation that naturally occurs in mammalian B cells in vivo.4
Fc effector function improvement is useful for therapeutic effectiveness as the antibody’s Fc region mediates effector functions such as antibody dependent cell-mediated cytotoxicity (ADCC), antibody induced complement dependent cytotoxicity (CDC), and antibody dependent cell-mediated phagocytosis (ADCP), which all lead to phagocytosis or cell death. Approaches to improve the affinity of Fc regions include glycosyl modifications, computational designing, and high-throughput screening.1
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Wang, B., Kankanamalage, S. G., Dong, J., & Liu, Y. (2021). Optimization of therapeutic antibodies. Antibody Therapeutics, 4(1), 45-54. https://doi.org/10.1093/abt/tbab003
Jones, T.D., Crompton, L.J., Carr, F.J., Baker, M.P. (2009). Deimmunization of Monoclonal Antibodies. In: Dimitrov, A. (eds) Therapeutic Antibodies. Methods in Molecular Biology™, vol 525. Humana Press. https://doi.org/10.1007/978-1-59745-554-1_21
Zinsli, L. V., Stierlin, N., Loessner, M. J., & Schmelcher, M. (2021). Deimmunization of protein therapeutics – Recent advances in experimental and computational epitope prediction and deletion. Computational and Structural Biotechnology Journal, 19, 315-329. https://doi.org/10.1016/j.csbj.2020.12.024
Denice T.Y. Chan, Maria A.T. Groves; Affinity maturation: highlights in the application of in vitro strategies for the directed evolution of antibodies. Emerg Top Life Sci 12 November 2021; 5 (5): 601–608. doi: https://doi.org/10.1042/ETLS20200331;
The therapeutic efficacy of antibodies is closely related to their ability to recognize and bind specific epitopes on target antigens. Epitopes, or antigenic determinants, are a group of amino acids or other chemical groups that are part of a molecule to which an antibody attaches itself. Epitope characterization can help reveal the mechanism of antibody binding and apply intellectual property (patent) protection for novel antibodies, in addition to designing antibodies with high specificity and minimal cross-reactivity.
Understanding the differences between antibody specificity and selectivity is essential for designing and interpreting antibody-based assays in research for experimental accuracy and data interpretation. Antibody specificity refers to an antibody's ability to recognize and bind to a particular epitope—a unique part of an antigen that elicits an immune response.
Antibody-based assays are essential tools in biomedical research, providing the means to detect, quantify, and visualize specific proteins or antigens within complex biological samples. These assays' efficacy hinges on the antibodies' precise properties. While affinity, avidity, specificity, and selectivity are fundamental to antibody performance, the ultimate impact of these properties is heavily influenced by the experimental context in which the antibody is employed.
Biologics, particularly antibodies, have become indispensable in biomedical research and therapeutic development. Research-use-only (RUO) biologics play a pivotal role in preclinical studies, providing researchers with the necessary tools to explore antibody functions and therapeutic potential in vivo.