The Impact of Conjugation Mode and Site on Tubulysin Antibody‐Drug‐Conjugate Efficacy and Stability

Antibody-drug conjugates (ADCs) represent a prominent class of biotherapeutics engineered to selectively deliver cytotoxic payloads to tumors, thereby facilitating targeted cell killing. While first-generation ADCs, created by conjugating payloads to surface-accessible lysine or hinge-cysteine residues, have achieved clinical success, several site-specific ADCs with defined drug-to-antibody ratios are currently under clinical investigation. Herein, the efficacy, stability, and pharmacokinetics of ADCs generated by attaching the drug linker to surface-exposed lysine residues, hinge-cysteine residues, and the C'E loop in the CH2 domain (mediated by bacterial transglutaminase) using a tubulysin payload are compared. In N87 xenograft mice, the order of efficacy is C'E loop > hinge-cysteine > lysine-conjugated ADCs. Among the three ADCs evaluated, the site-specific ADC demonstrates superior in vivo stability (minimal payload-linker deconjugation and limited payload metabolism/deacetylation) and favorable pharmacokinetics (longer half-life, low clearance, high exposure). In contrast, the lysine-conjugated ADC exhibits the least stability and poorest pharmacokinetics, which directly correlate with its efficacy. Further investigation into cysteine-engineered site-specific ADCs with payloads conjugated at various sites confirms that both the conjugation chemistry and the site of conjugation significantly influence the in vivo stability and pharmacokinetics of site-specific ADCs.