Modification of Different IgG1 Antibodies via Glutamine and Lysine using Bacterial and Human Tissue Transglutaminase

Abstract

The modification of proteins by chemical methods is well-established, however usually difficult to control. In this paper, we describe the posttranslational modification of different IgGs via the Lys or Gln side chains catalyzed by bacterial and human tissue transglutaminase (BTGase and TG2). For proof of concept, different IgG1s (commercial bovine IgG1, and L1CAM targeting chCE7 and chCE7 aglycosylated) were enzymatically functionalization with different fluorescent TGase substrates based on the CY3 analogue Dy547. The optimal reaction conditions were determined in order to assess the two enzymes. The efficiency of the enzymatic method was also compared with a standard chemical method employing a reactive NHS ester of Dy547. Three new TGase substrates were synthesized for this study including Lys-substrate 1 useful for BTGase and TG2 and two Gln-substrates tailor-made for BTGase (substrate 2) and TG2 (substrate 3). Of the two TGases tested, BTGase incorporated Lys-substrate 1 more efficiently than TG2. On the other hand, both enzymes reacted equally efficiently with the corresponding Gln-substrates 2 and 3. Reproducible labeling could be achieved in a broad concentration "window" of the substrates (up to 400 microM) without the risk of overlabeling of chCE7 or chCE7 aglycosylated. The biological activities of the functionalized antibodies were unaltered as shown by in vitro antigen affinity measurements and cell internalization experiments using confocal laser scanning microscopy. A maximum label-to-protein ratio of approximately 1 was achieved with chCE7 aglycosylated and substrate 1 using BTGase. It is important to recognize that the enzymatic activity of TGases enables the stable functionalization of proteins via the side chains of Gln, which is not possible by any chemical method available today. In addition, we could prove that the enzymatic modification of all antibodies occurred selectively at the heavy chain whereas the chemical method led to labeling of both the heavy and the light chains.

Transglutaminase‐mediated methods for site‐selective modification of human growth hormone

“…This is not possible to the same degree in the reversed reaction where protein lysines are modified because of the much lower molar solubility of proteins. Although this type of reaction has been reported previously, 23,24 it was an initial concern that the hydrolysis sidereaction would be predominant. This appeared not to be the case.…”

Site-Specifically Labeled Immunoconjugates for Molecular Imaging—Part 2: Peptide Tags and Unnatural Amino Acids

“…Most notably, Schibli and coworkers at ETH Zurich have exploited TGases for chemoenzymatic antibody conjugations [18]. Working primarily with microbial transglutaminase (mTGase), the researchers have found that while antibodies possess many glutamine residues, only aglycosylated or deglycosylated antibodies can be functionalized appreciably using mTGase [19, 20]. The deglycosylation of the antibody at the N297 position increases the flexibility of the peptide backbone, exposing Q295 for reaction with the enzyme.…”

“…4; Fig. 5a) [20]. In subsequent investigations, the laboratory used mTGase to modify variants of chCE7 and rituximab that had been enzymatically deglycosylated with PNGaseF (chCE7degl and RTXdegl).…”

Antibody Conjugates: From Heterogeneous Populations to Defined Reagents

“…The first approaches to enzymatically conjugate biotin and fluorescent dyes to antibodies yielded heterogeneous antibody conjugates with regard to the PAR while the site of conjugation could not be determined [134,135]. It was not until Jeger et al discovered a single glutamine in the human IgG heavy chain domain (Q295), which is next to the antibody glycosylation site (N297), to be the sole site of modification for MTGase-mediated bioconjugation.…”