Photodisruption of the Structurally Conserved Cys-Cys-Trp Triads Leads to Reduction-Resistant Scrambled Intrachain Disulfides in an IgG1 Monoclonal Antibody.

Photostability conditions as prescribed by ICH guidelines induced highly reduction-resistant scrambled disulfides that contribute to the population of apparent nonreducible aggregates in an IgG1 mAb. Photoinduced cross-linked species were isolated under reducing conditions using an organic phase size exclusion chromatography (OP-SEC) method, followed by O18-labeling tryptic mapping to identify cross-linked peptides. Disulfide scrambling was observed within the IgG1 structurally conserved-intrachain cysteine-cysteine-tryptophan triads (Cys-Cys-Trp), and correlated with Trp-to-kynurenine (Kyn) photodegradation within these triads. We hypothesize that intrachain disulfides protect the proximal Trp within the Cys-Cys-Trp triads from photodegradation by enabling dissipation of Trp-absorbed UV energy via electron transfer to the disulfide bond. Finally, we propose three distinct mechanisms of photochemical degradation of monoclonal antibodies mediated by Trp residues.

Antitherapeutic antibody-mediated hepatotoxicity of recombinant human Apo2L/TRAIL in the cynomolgus monkey.

Apo2L/TRAIL is a member of the tumor necrosis factor superfamily and an important inducer of apoptosis. Recombinant human (rhu) Apo2L/TRAIL has been attractive as a potential cancer therapeutic because many types of tumor cells are sensitive to its apoptosis-inducing effects. Nonclinical toxicology studies were conducted to evaluate the safety of rhuApo2L/TRAIL for possible use in humans. The cynomolgus monkey was chosen for this safety assessment based on high protein sequence homology between human and cynomolgus Apo2L/TRAIL and comparable expression of their receptors. Although hepatotoxicity was observed in repeat-dose monkey studies with rhuApo2L/TRAIL, all animals that displayed hepatotoxicity had developed antitherapeutic antibodies (ATAs). The cynomolgus ATAs augmented the cytotoxicity of rhuApo2L/TRAIL but not of its cynomolgus counterpart. Of note, human and cynomolgus Apo2L/TRAIL differ by four amino acids, three of which are surface-exposed. In vivo studies comparing human and cynomolgus Apo2L/TRAIL supported the conclusion that these distinct amino acids served as epitopes for cross-species ATAs, capable of crosslinking rhuApo2L/TRAIL and thus triggering hepatocyte apoptosis. We describe a hapten-independent mechanism of immune-mediated, drug-related hepatotoxicity - in this case - associated with the administration of a human recombinant protein in monkeys. The elucidation of this mechanism enabled successful transition of rhuApo2L/TRAIL into human clinical trials.