A Traceless Site-Specific Conjugation on Native Antibodies Enables Efficient One-Step Payload Assembly

Antibody-drug conjugates (ADCs) are composed of monoclonal antibodies targeting specific antigens and small-molecule cytotoxic drugs linked by linkers, which have both the powerful killing effect of traditional small-molecule chemotherapy and the tumor targeting of antibody drugs. Up to date, 14 antibody-drug conjugates (ADCs) have been approved for the treatment of various cancers, and approximately 100 ADC candidates are in clinical trials worldwide.

Over recent years, studies have found that the site-specific ADC drugs formed by quantitatively coupling small-molecule drugs to specific sites of antibodies have a better therapeutic index, and have gradually become the focus of research and development in the field of ADC drugs. However, due to the complex amino acid composition of antibodies, site-specific conjugation of small-molecule drugs by chemical methods has always been challenging. Among them, ligand-directed site-specific coupling technology is a potential method, but the difficult release of redundant ligands or the complex release process limits the application of this technology in site-specific ADC drug development.

Recently, Huang group from Shanghai Institute of Materia Medica Chinese Academy of Sciences developed a novel ligand-directed acylation reagent based on a thioester structure, which can automatically release redundant ligand structures while site-directed modification of lysines at specific sites of antibodies, and realize “one-step”, “no trace” preparation of site-directed antibody drug conjugates for the first time by chemical methods, providing a novel preparation for the development of targeted ADC drugs.

The research team applied this technology to realize various forms of functional molecular modification, such as the “one-step” site-specific azation, biotinylation and various small molecule drugs, all of which were specifically coupled to two molecular compounds. This traceless method has demonstrated excellent efficiency in one-step IgG conjugation with various cargoes including azide (Fig.2), biotin (Fig.3), toxins (Fig.4), etc. Analysis methods such as high-resolution mass spectrometry and mass spectrometry proteomics have proved that the small molecule modification occurs at the targeted site in the Fc region of the antibody, with extremely high site selectivity.

Meanwhile, this technology is also applicable to IgG1 antibodies such as rituximab, pertuzumab, bevacizumab, IgG2 antibodies such as panitumumab, and IgG4 antibodies such as nivolumab, and shows good substrate universality.

Subsequently, the team prepared four “one-step” site-directed ADC compounds with different drug linkers based on trastuzumab, and three “two-step” site-directed ADC compounds based on bioorthogonal reactions, and carried out the preliminary pharmaceutical evaluation. All ADC compounds exhibited good structural homogeneity (DAR=2), strong in vitro tumor cell inhibitory activity (<0.1 nM) and very low cytotoxicity (≥1000 nM). At the same time, compared with the positive ADC compounds prepared by random coupling (DAR≈3.3), the multiple site-directed ADC compounds obtained by this technique have lower drug loadings (DAR=2), but show stronger tumor in vivo inhibitory activity.

In conclusion, the research team has realized the “one-step” and “no trace” site-specific quantitative modification of lysine at specific sites of natural antibodies by designing novel acylation reagents and optimizing the structure of ligands. For the first time, this technology realizes the efficient and uniform preparation of site-directed ADC compounds by chemical means without the need for antibody engineering and bioorthogonal reactions, and is compatible with diverse substrate structures and antibody types, which exhibits broad potential applications in ligand-directed protein modifications.

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