Tag-free, specific conjugation of glycosylated IgG1 antibodies using microbial transglutaminase.

We present an efficient approach for tag-free, site-specific conjugation of a fully glycosylated antibody using microbial transglutaminase (mTG). We created variants of trastuzumab where a single surface-exposed residue of the human crystallizable fragment had been substituted to glutamine, with the objective of enabling site-specific mTG-mediated conjugation with primary amine payloads. MTG reactivity was determined by conjugation to an amino fluorophore, demonstrating effective tag-free conjugation at the newly introduced I253Q site. The conjugation of one payload per antibody heavy chain was confirmed by mass spectrometry. We further demonstrated two-step mTG/click chemistry-based conjugation of I253Q trastuzumab with monomethyl auristatin E. Cytotoxicity and specificity of the resulting antibody-drug conjugate were indistinguishable from trastuzumab conjugated by another method although binding to the neonatal Fc receptor was impaired. The resulting fully glycosylated ADC is unique in that it results from minimal modification of the antibody sequence and offers potential for application to cellular imaging, fluorescence microscopy, western blotting or ELISA.

Glutamine-walking: Creating reactive substrates for transglutaminase-mediated protein labeling.

Chemically modified proteins are increasingly being tested and approved as therapeutic products. Batch-to-batch homogeneity is crucial to ensure safety and quality of therapeutic products. Highly selective protein modification may be achieved using enzymatic routes. Microbial transglutaminase (mTG) is a robust, easy to use and well-established enzyme that is used at a very large scale in the food industry such that its efficacy and its safety for human consumption are well established. In the context of therapeutic protein modification, mTG should crosslink one or more glutamines on the target protein with an aminated moiety such as a solubilizer, a tracer or a cytotoxic moiety. mTG has the advantage of being unreactive toward the majority of surface-exposed glutamines on most proteins, reducing sample heterogeneity. The caveat is that there may be no reactive glutamine on the target protein, or else a reactive glutamine may be found in a location where its modification compromises function of the target protein. Here we describe the glutamine-walk (Gln-walk), a straightforward method to create a glutamine-substrate site that is reactive to mTG in a target protein. Iterative substitution of single amino acids to a glutamine is followed by facile identification of reactivity with mTG, where covalent labeling of the target with an aminated fluorophore allows visualization of the most reactive modified targets. The approach is empirical; knowledge of the target protein structure and functional regions facilitates application of the method.