The antibody with cytotoxic molecule conjugated (antibody-drug complex, ADC) can critically make damages on the solid tumors which are hardly damaged only by normal antibody functions. For chemical conjugation on proteins, the... [ view full abstract ]
The antibody with cytotoxic molecule conjugated (antibody-drug complex, ADC) can critically make damages on the solid tumors which are hardly damaged only by normal antibody functions. For chemical conjugation on proteins, the use of the thiol group in cysteine residue is a simple approach for site-specific conjugation; however, in the case of antibody, the activation of thiol groups lead to instabilities of antibody structures, because the disulfide linkages in each domain of antibody are critical for the structural stability of antibody. Recently, recombinant small antibodies constructed only from antigen-binding domain are studies, because they can penetrate deep into the tumor mass. However, their structures are seriously dependent on the formation of the disulfide linkages. In this study, we applied the amino group in lysine residue for the site-specific chemical conjugation of organic molecule on recombinant small antibody and we tried to identify the prior conjugated lysine residues by mass spectrometry and extract rules characters the small antibody’s lysine residues conjugated organic molecule (Fig. 1).
In this study, biotin molecules are applied instead of drug and small antibody is the bivalent antibody fragment (diabody). NHS-bound biotin molecules were mixed with recombinant small antibody fragments at 5 times moles in the solution at several pH values. We analyzed the small antibody-biotin conjugate with the object of chemical reaction rate and bound positions by HABA assay and LC/ESI-MS/MS.
The chemical reaction rate at pH6.5, 7.4 and 8.5 became gradually higher. We identified the modified lysine residues and analyzed the modified rate of each lysine residue from mass spectrometry intensity (Fig.2). The most high modified lysine residue (VL107) was bound at 46.87% where is C-terminal of small antibody. We investigated homo diabody Crystal structures on Protein Data Bank (PDB): consequently we found the same orientation homo diabody 5GRW. The Kabat numbering residues of 5GRW same as modified lysine residues have a highest side chain solvent accessibility and average isotropic displacement in the other residues.
In conclusion, we succeeded that identified the prior modified lysine residues in small antibody and characterized the modified lysine rule from the same orientation homo diabody.
