Site‐Specific and Stoichiometric Modification of Antibodies by Bacterial Transglutaminase

The therapeutic efficacy of antibodies can be substantially enhanced by conjugation of cytotoxic compounds such as chemotherapeutics and particle-emitting radionuclides. Intuitively one would assume that the therapeutic index would improve as the number of cytotoxic entities conjugated to the antibody increases. However, recent studies on auristatin–antibody conjugates in mice have demonstrated that a drug/antibody molar ratio of 4:1 results in optimal efficacy and in vivo tolerability. Unfortunately, conventional chemical strategies for protein modification are difficult to control and give rise to heterogeneous populations of immunoconjugates with variable stoichiometries, each of which has its own in vivo characteristics. The introduction of artificial, bio-orthogonal groups for site-specific and stoichiometric protein modification offers a potential solution to this problem. Such strategies are en vogue but are often laborious and still risk product heterogeneity. Transglutaminases (TGs, E.C. 2.3.2.13) catalyze acyltransfer reactions between the g-carboxamide group of glutamine (a side chain, which is otherwise chemically inert under physiological conditions) and the primary e-amino group of lysine, to form catabolically stable isopeptide bonds (Figure 1a). Most TGs are promiscuous with respect to the lysine substrate and accept even simple 5-aminopentyl groups as lysine surrogates. The criteria for a glutamine residue to be recognized by the enzyme, however, are muchmore stringent: it should be both located in a flexible region of the protein and flanked by specific amino acids. Given this inherent selectivity, we hypothesized that TG would be an alternative for the site-specific and stoichiometric functionalization of antibodies. For this study we used bacterial transglutaminase (BTG) because it is robust, inexpensive, and easy to handle. Our group is interested in radioimmunoconjugates for diagnostic and therapeutic applications, where low off-target accumulation of radioactivity is crucial. Earlier studies performed with radiolabeled monoclonal antibodies (mAbs) demonstrated that high numbers of metal chelators adversely affect the biological behavior of radioimmunoconjugates. Therefore, we tested the features of BTG for the preparation of immunoconjugates that are functionalized with different metal chelators and radiolabeled with different diagnostic and therapeutic radionuclides. Deferoxamine (DF, 1), an antidote for metal poisoning, has recently been identified as a suitable chelator for radionuclides such as Ga and Zr. During the course of our studies we recognized that without further derivatization deferoxamine is already a potent BTG substrate. Furthermore, the metal chelating system 4-(1,4,8,11tetraazacyclotetradec-1-yl)methyl benzoic acid (CPTA, 2) was derivatized with a 1,5-diaminopentane (cadaverine) spacer (Figure 1b; see the Supporting Information for details on the synthesis). To probe the scope of the new strategy we investigated other (model) substrates, which are of potential Figure 1. a) TG-mediated modification of Gln (Q) with a substrate containing lysine or a lysine surrogate. b) Substrates used in this study.