An expanded genetic code facilitates antibody chemical conjugation involving the lambda light chain

• Published in: Biochemical and biophysical research communications 2021-03, Vol.546, p.35-39
• Main Authors: Kato, Akifumi, Ohtake, Kazumasa, Tanaka, Yoshitaka, Yokoyama, Shigeyuki, Sakamoto, Kensaku, Shiraishi, Yasuhisa
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Antibodies, Bispecific - chemistry; Antibodies, Bispecific - genetics; Antibodies, Bispecific - immunology; Bispecific antibody; Codon reassignment; Genetic Code; Humans; Immunoconjugates - chemistry; Immunoconjugates - genetics; Immunoconjugates - immunology; Immunoglobulin Fab Fragments - chemistry; Immunoglobulin Fab Fragments - genetics; Immunoglobulin Fab Fragments - immunology; Immunoglobulin Isotypes - chemistry; Immunoglobulin Isotypes - genetics; Immunoglobulin Isotypes - immunology; Immunoglobulin kappa-Chains - chemistry; Immunoglobulin kappa-Chains - genetics; Immunoglobulin kappa-Chains - immunology; Immunoglobulin lambda-Chains - chemistry; Immunoglobulin lambda-Chains - genetics; Immunoglobulin lambda-Chains - immunology; Lambda light chain; Lysine - chemistry; Lysine - genetics; Models, Molecular; Non-natural amino acid; Protein Multimerization; Receptor, ErbB-2 - immunology; Receptor, IGF Type 1 - immunology; Site-specific conjugation
• ISSN: 0006-291X; 1090-2104
• DOI: 10.1016/j.bbrc.2021.02.005

Most of the currently approved therapeutic antibodies are of the immunoglobulin gamma (IgG) κ isotype, leaving a vast opportunity for the use of IgGλ in medical treatments. The incorporation of designer amino acids into antibodies enables efficient and precise manufacturing of antibody chemical conjugates. Useful conjugation sites have been explored in the constant domain of the human κ-light chain (LCκ), which is no more than 38% identical to its LCλ counterpart in amino acid sequence. In the present study, we used an expanded genetic code for site-specifically incorporating Nε-(o-azidobenzyloxycarbonyl)-l-lysine (o-Az-Z-Lys) into the antigen-binding fragment (Fab) of an IgGλ, cixutumumab. Ten sites in the LCλ constant domain were found to support efficient chemical conjugation exploiting the bio-orthogonal azido chemistry. Most of the identified positions are located in regions that differ between the two light chain isotypes, thus being specific to the λ isotype. Finally, o-Az-Z-Lys was incorporated into the Fab fragments of cixutumumab and trastuzumab to chemically combine them; the resulting bispecific Fab-dimers showed a strong antagonistic activity against a cancer cell line. The present results expand the utility of the chemical conjugation method to the whole spectrum of humanized antibodies, including the λ isotype. •A designer amino acid was site-specifically introduced into the human λ light chain.•The bio-orthogonal azido chemistry was used for site-specific chemical conjugation.•Multiple positions useful for the conjugation were identified in the λ light chain.•Bispecific Fab dimers made of the κ and λ isotypes showed antagonistic activity.