Site-specific antibody-drug conjugates: the nexus of bioorthogonal chemistry, protein engineering, and drug development

Abstract

Antibody-drug conjugates (ADCs) combine the specificity of antibodies with the potency of small molecules to create targeted drugs. Despite the simplicity of this concept, generation of clinically successful ADCs has been very difficult. Over the past several decades, scientists have learned a great deal about the constraints on antibodies, linkers, and drugs as they relate to successful construction of ADCs. Once these components are in hand, most ADCs are prepared by nonspecific modification of antibody lysine or cysteine residues with drug-linker reagents, which results in heterogeneous product mixtures that cannot be further purified. With advances in the fields of bioorthogonal chemistry and protein engineering, there is growing interest in producing ADCs by site-specific conjugation to the antibody, yielding more homogeneous products that have demonstrated benefits over their heterogeneous counterparts in vivo. Here, we chronicle the development of a multitude of site-specific conjugation strategies for assembly of ADCs and provide a comprehensive account of key advances and their roots in the fields of bioorthogonal chemistry and protein engineering.

Figure 1

Potential sites of modification and theoretical product distributions for lysine-conjugated and site-specifically conjugated antibodies. (A) All lysine residues of a human IgG1 are highlighted in red, indicating potential sites of conjugation with activated esters. The number of regioisomers is calculated based on 40 reactive lysine residues. (B) A site-specifically modifiable antibody with one conjugation site on each heavy chain highlighted in red; a fully conjugated antibody has a DAR of 2. PDB ID: 1IGY.

Figure 2

Methods for site-specific ADC production based on cysteine conjugation. (A) Reduction and alkylation of all interchain cysteine disulfides. (B) Reduction and alkylation of interchain cysteine residues on antibodies containing several cysteine-to-serine mutations. (C) Reduction and bridging alkylation of interchain cysteine residues with bis-sulfone linkers. (D) Reduction and bridging alkylation of interchain cysteine residues with propargyldibromomaleimide, followed by Cu-click ligation. (E) Reduction of cysteine residues followed by reoxidation of interchain disulfides and selective alkylation to produce THIOMABs. (F) Reductive and nucleophilic deblocking of N-terminal cysteine residues on a diabody followed by thiazolidine ligation. Abbreviations: DTT, dithiothreitol; TCEP, tris(carboxyethyl)phosphine; THPTA, tris(3-hydroxypropyltriazolylmethyl)amine.

Figure 3

Methods for chemical conjugation on the N-glycan of IgG. (A) Schematic structure of glycans found at N297 of recombinantly expressed IgG. Dashed lines indicate partial occupancy. (B) Chemical structure of a fully elaborated complex-type N-glycan. (C) Periodate oxidation of fucose followed by hydrazone condensation. (D) Enzymatic transfer of galactose and sialic acid followed by periodate oxidation and oxime condensation. (E) Enzymatic transfer of galactose and 9-azidosialic acid followed by Cu-free click reaction. (F) Enzymatic removal of terminal galactose followed by enzymatic transfer of GalNAz and Cu-free click reaction. (G) Metabolic incorporation of 6-thiofucose followed by maleimide conjugation. Abbreviations: GalT, galactosyltransferase; SiaT, sialyltransferase; 9-N₃Sia, 9-azidosialic acid; GalNAz, N-azidoacetylgalactosamine.

Figure 4

Conjugation methods based on UAA incorporation. (A) Incorporation of p-acetylphenylalanine followed by oxime condensation. (B) Simultaneous incorporation of p-acetylphenylalanine and an azido-lysine derivative followed by oxime condensation and Cu-free click chemistry to attach a fluorophore (green star). (C) Cell-free incorporation of p-azidomethylphenylalanine followed by Cu-free click chemistry. (D) Incorporation of selenocysteine followed by mild reduction and alkylation.

Figure 5

Conjugation methods based on enzymatic modification of peptide tags. (A) Glycosidase treatment for access to Q295 followed by transglutaminase-mediated conjugation of amine-functionalized small molecules. (B) Transglutaminase-mediated conjugation in an N297Q mutant at sites Q295 and Q297. (C) Glycosidase treatment followed by transglutaminase-mediated conjugation of an azido-PEG-amine and Cu-free click chemistry. (D) Transglutaminase-mediated conjugation of amine-functionalized drugs to an engineered LLQGA site or (E) several engineered LLQGA sites simultaneously. (F) Sortase-mediated conjugation of a glycine-functionalized chelator near the C-terminus of an scFv. (G) Formylglycine generating enzyme mediated conversion of cysteine to formylglycine followed by HIPS ligation. Abbreviations: PNGase, peptide N-glycosidase; FGly, formylglycine; HIPS, hydrazino-Pictet–Spengler.