In silico Humanization of Human Tumor Necrosis Factor-A-Specific Immunoglobulin-Domain Antibody (I-DAb): A Case Study

The evolving landscape of antibody therapeutics has enabled the discovery and development of next-gen antibodies i.e., camelid nanobodies or heavy chain-only Immunoglobulin Domain Antibodies (I-DAbs), which have demonstrated high therapeutic potential. To mitigate the risk of immunogenicity in humans, it is frequently necessary to humanize therapeutic antibodies obtained from animal sources. Generally, humanization results in the loss of binding affinities that are restored by introducing back-mutations within the humanized sequence. Here, an exemplified process of humanization and optimization of back-mutations is reported for an anti-human Tumor Necrosis Factor-α (hTNF-α) I-DAb via computational methods. This case study describes the computational humanization of a camelid anti-hTNF-α I-DAb, utilizing an enhanced Back-Mutation (BM) protocol recently published by Sulea 2022. To ease this process, molecular docking using HADDOCK v2.4 and a humanness evaluation tool, T20 analyzer, were additionally incorporated.