Transglutaminase-Based Chemo-Enzymatic Conjugation Approach Yields Homogeneous Antibody–Drug Conjugates

Abstract

Most chemical techniques used to produce antibody−drug conjugates (ADCs) result in a heterogeneous mixture of species with variable drug-to-antibody ratios (DAR) which will potentially display different pharmacokinetics, stability, and safety profiles. Here we investigated two strategies to obtain homogeneous ADCs based on site-specific modification of deglycosylated antibodies by microbial transglutaminase (MTGase), which forms isopeptidic bonds between Gln and Lys residues. We have previously shown that MTGase solely recognizes Gln295 within the heavy chain of IgGs as a substrate and can therefore be exploited to generate ADCs with an exact DAR of 2. The first strategy included the direct, onestep attachment of the antimitotic toxin monomethyl auristatin E (MMAE) to the antibody via different spacer entities with a primary amine functionality that is recognized as a substrate by MTGase. The second strategy was a chemo-enzymatic, two-step approach whereby a reactive spacer entity comprising a bio-orthogonal thiol or azide function was attached to the antibody by MTGase and subsequently reacted with a suitable MMAE-derivative. To this aim, we investigated two different chemical approaches, namely, thiol-maleimide and strain-promoted azide−alkyne cycloaddition (SPAAC). Direct enzymatic attachment of MMAE-spacer derivatives at an 80 molar excess of drug yielded heterogeneous ADCs with a DAR of between 1.0 to 1.6. In contrast to this, the chemo-enzymatic approach only required a 2.5 molar excess of toxin to yield homogeneous ADCs with a DAR of 2.0 in the case of SPAAC and 1.8 for the thiol-maleimide approach. As a proof-of-concept, trastuzumab (Herceptin) was armed with the MMAE via the chemo-enzymatic approach using SPAAC and tested in vitro. Trastuzumab-MMAE efficiently killed BT-474 and SK-BR-3 cells with an IC 50 of 89.0 pM and 21.7 pM, respectively. Thus, the chemo-enzymatic approach using MTGase is an elegant strategy to form ADCs with a defined DAR of 2. Furthermore, the approach is directly applicable to a broad variety of antibodies as it does not require prior genetic modifications of the antibody sequence.

Antibody Drug Conjugates for Cancer Therapy

“…Site-dependent differences in pharmacokinetics, particularly in rat, were again noted. Dennler et al (2014) demonstrated an additional method involving mTG catalyzed crosslinking, but lacking the requirement for any engineering of the antibody cDNA sequence. The approach takes advantage of conserved heavy chain residue glutamine-295, which is the only site recognized by mTG in deglycosylated IgG.…”

Antibody Drug Conjugates: Design and Selection of Linker, Payload and Conjugation Chemistry

“…Schibli and co-workers reported, however, that deglycosylating IgGs at N297 exposed a glutamine residue at the 295 position to enzymatic ligation to create ADCs with a DAR of 2 (31). Further, by producing a N297 to Q297 mutant IgG1, they were able to introduce two viable sites for enzymatic labeling to create ADCs with a DAR of 4 (31,32).…”

Antibody-Drug Conjugates in Head and Neck Cancer

“…6B). A transglutaminase derived from Streptomyces mobaraensis catalyzes transpeptidation where a primary amine-containing linker is covalently attached to the primary amide side chain of a specific glutamine (Q295) within deglycosylated antibodies, resulting in ADCs with a defined DAR of 2 (one conjugation site per heavy chain) (Jeger et al, 2010;Dennler et al, 2014). An N297Q mutation prior to this conjugation provides two more reaction sites (DAR = 4).…”