Structural characterization of the maytansinoid–monoclonal antibody immunoconjugate, huN901–DM1, by mass spectrometry

Abstract

Immunoconjugates are being explored as novel cancer therapies with the promise of target-specific drug delivery. The immunoconjugate, huN901-DM1, composed of the humanized monoclonal IgG 1 antibody, huN901, and the maytansinoid drug, DM1, is being tested in clinical trials to treat small cell lung carcinoma (SCLC). huN901-DM1 contains an average of three to four DM1 drug molecules per huN901 antibody molecule. The drug molecules are linked to huN901 through random modification of huN901 at e-amino groups of lysine residues, thus yielding a heterogeneous population of conjugate species. We studied the drug distribution profile of huN901-DM1 by electrospray time-of-flight mass spectrometry (ESI-TOFMS), which showed that one to six DM1 drug molecules were attached to an antibody molecule. Both light and heavy chains contained linked drugs. The conjugation sites in both chains were determined by peptide mapping using trypsin and Asp-N protease digestion. Trypsin digestion identified modified lysine residues, since these residues were no longer susceptible to enzymatic cleavage after conjugation with the drug. With respect to Asp-N digestion, modified peptides were identified by observing a mass increase corresponding to the modification. The two digestion methods provided consistent results, leading to the identification of 20 modified lysine residues in both light and heavy chains. Each lysine residue was only partially modified. No conjugation sites were found in complementarity determining regions (CDRs). Using structural models of human IgG 1 , it was found that modified lysine residues were on the surface in areas of structural flexibility and had large solvent accessibility.

Synthesis of site-specific antibody-drug conjugates using unnatural amino acids

“…There is limited stoichiometric control because of the large number of disulfide bonds and lysine residues in antibody molecules, which leads to a typical distribution of zero to eight toxins per antibody (16,17). In addition, coupling occurs at many different sites leading to a heterogeneous mixture of ADCs, the components of which likely have distinct affinities, stabilities, pharmacokinetics, efficacies, and safety profiles.…”

Antibody–Drug Conjugates for Tumor Targeting—Novel Conjugation Chemistries and the Promise of non-IgG Binding Proteins

“…Thus, the 40-86 lysine residues on the antibody surface are modified to a variable degree, resulting in a sample with potentially 4.5 million different molecules in terms of regioisomerism and drug load 45,46 . If thiol-maleimide coupling is used instead, cysteine conjugation occurs after mild reduction of the four inter-chain disulfide bonds in the hinge region of an IgG1.…”

A modern paradigm for treating HER2-positive advanced breast cancer: Dual HER2 blockade and antibody-drug conjugates

“…DM1 (derivative of maytansine 1) является ингибитором полимеризации микротрубочек, близким по механизму действия к винкаалкалоидам, но примерно в 100 раз более активным [30]. В исследовани-ях in vitro на клеточных линиях различных опухолей DM1 продемонстрировал очень высокую цитотоксичность, в 25-270 раз превосходящую таковую у паклитаксела [31].…”