O-linked glucosylation of a therapeutic recombinant humanised monoclonal antibody produced in CHO cells.

An unpredictable modification of a therapeutic recombinant humanised monoclonal antibody (rh-mAbX) produced using CHO cells was found. LC/MS analysis of rh-mAbX indicated the presence of heterogeneity in the light chain with a corresponding mass shift of 162 Da compared to the theoretical mass. To characterise the heterogeneity, that is, the attached moiety, several analyses were performed. Peptide mapping of rh-mAbX indicated that the attached moiety was located in the amino acid sequence from Leu20 to Lys45, which is a part of the variable region of the light chain. The peptide was efficiently purified in two-steps by RP-HPLC by utilising two different types of RP columns. N-terminal sequencing and LC/MS/MS analysis of the peptide suggested that Ser29 of the light chain was the modification site, and that the attached moiety was a single O-linked hexose. HPAEC-PAD analysis following ß-elimination indicated the presence of an O-linked glucose in the modified peptide. Monosaccharide composition analysis after acid hydrolysis supported this result. The content of antibodies containing this species was determined to be approximately 10% by Lys-C peptide mapping detected at 280 nm. Thus, this study demonstrated the formation of a unique O-linked glucosylation posttranslational modification in a recombinant humanised monoclonal antibody produced in CHO cells.

Antibody recycling by engineered pH-dependent antigen binding improves the duration of antigen neutralization.

For many antibodies, each antigen-binding site binds to only one antigen molecule during the antibody's lifetime in plasma. To increase the number of cycles of antigen binding and lysosomal degradation, we engineered tocilizumab (Actemra), an antibody against the IL-6 receptor (IL-6R), to rapidly dissociate from IL-6R within the acidic environment of the endosome (pH 6.0) while maintaining its binding affinity to IL-6R in plasma (pH 7.4). Studies using normal mice and mice expressing human IL-6R suggested that this pH-dependent IL-6R dissociation within the acidic environment of the endosome resulted in lysosomal degradation of the previously bound IL-6R while releasing the free antibody back to the plasma to bind another IL-6R molecule. In cynomolgus monkeys, an antibody with pH-dependent antigen binding, but not an affinity-matured variant, significantly improved the pharmacokinetics and duration of C-reactive protein inhibition. Engineering pH dependency into the interactions of therapeutic antibodies with their targets may enable them to be delivered less frequently or at lower doses.

VH/VL interface engineering to promote selective expression and inhibit conformational isomerization of thrombopoietin receptor agonist single-chain diabody.

Thrombopoietin receptor agonist humanized VB22B single-chain diabody (hVB22B (scFv)(2)) was found to be expressed as a mixture of two conformational isomers, a single-chain diabody form and a bivalent scFv form, which had different V(H)/V(L) (variable region of the heavy chain/light chain) association patterns. The single-chain diabody form showed significantly higher biological activity than the bivalent scFv form and, when incubated at elevated temperatures, exhibited novel isomerization to the inactive bivalent scFv form. Therefore, therapeutic development of hVB22B (scFv)(2) would require separation of the purified single-chain diabody form from the mixture of the two conformational isomers and also inhibition of isomerization into an inactive bivalent scFv form during storage. Novel V(H)/V(L) interface engineering in hVB22 (scFv)(2), in which hydrogen bonding between H39 and L38 was substituted with electrostatic interaction to enhance the desired V(H)/V(L) association and inhibit the undesired V(H)/V(L) association, enabled selective expression of the desired conformational isomer without any reduction in biological activity or thermal stability. Moreover, V(H)/V(L) interface-engineered hVB22 (scFv)(2) was completely resistant to isomerization. Because the hydrogen bonding interaction between H39 and L38 and the surrounding residues are highly conserved in human antibody sequences, V(H)/V(L) interface engineering could be generally applied to various (scFv)(2) molecules for selective expression and inhibition of the isomerization of conformational isomers.