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

Hadjabdelhafid-Parisien, Adem; Bitsch, Sebastian; Palacios, Arturo Macarrón; Deweid, Lukas; Kolmar, Harald; Pelletier, Joelle N.

doi:10.1039/d2ra05630e

Cited by 5 publications

(4 citation statements)

References 34 publications

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“…[22] Therefore, site-specific and tag-free modifications are required to avoid the time-consuming process of installing tags on proteins. [15][16][17] LplA is a unique enzyme with versatile substrate recognition site (s). [23] This enzyme has the potential to be used for modification of specific lysine (Lys) residues incorporating a specific sequence consisting of 13 amino acid residues, termed "LplA acceptor peptide (LAP) tag".…”

Section: Introductionmentioning

confidence: 99%

“…In addition, cell culture conditions are difficult to optimize, posing potential challenges for scalability and reproducibility [22] . Therefore, site‐specific and tag‐free modifications are required to avoid the time‐consuming process of installing tags on proteins [15–17] …”

Section: Introductionmentioning

confidence: 99%

“…A wide variety of enzyme conjugations have been reported in peer-reviewed literature as these catalytic modifications are favored owing to their mild reaction conditions. [12,13] Some of these enzymatic approaches such as microbial transglutaminases (MTG), [14][15][16][17] sortase A, [18] glycan remodeling, [19] protein farnesyltransferase (PFTase), [20] and formyl glycine-generating enzymes (FGE) [21] resulted in the ADCs being utilized in clinical trials; however, most of these enzymatic modifications required the use of tags. Protein engineering requires time-consuming optimization to determine a suitable insertion site to produce efficacious/stable ADCs, complicating the process development towards ADC manufacturing.…”

Section: Introductionmentioning

confidence: 99%

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Tag‐Free Antibody Modification Mediated by Lipoic Acid Ligase A: Application to Antibody‐Drug Conjugates Production

et al. 2023

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Enzymatic modifications of proteins are of great interest in the pharmaceutical industry as it enables the production of homogeneous protein conjugates such as antibody‐drug conjugates, which are considered to be promising for cancer medicine. Several biotechnology companies have established proprietary conjugation technologies using specific enzymes. Most enzymatic modifications require the use of tags and enzyme‐recognition moieties. Therefore, site‐specific and “tag‐free” modifications are required to avoid the time‐consuming process of installing tags on proteins. Lipoic acid ligase A (LplA), is a recently reported enzyme having the potential to be used for the site‐specific modification of native antibodies without requiring a tag moiety. In this manuscript, investigations focused on the optimization of this enzymatic modification applied to antibody‐drug conjugates (ADCs) production. First, an analysis of the pH dependency was performed. Next, the effects of excess adenosine mono‐ and di‐phosphate were also examined to show that these by‐products from LplA modification did not have a significant impact on inhibiting the reaction efficiency. Furthermore, ADC syntheses by LplA‐mediated tag‐free modification followed by click reaction were achieved. To the best of our knowledge, this is the first example of actual ADC synthesis produced by LplA modification of tag‐free antibodies. A stability assessment of the resulting ADCs indicated that this enzymatic approach is a promising tool for installing biorthogonal groups to native antibodies for the next generation of ADC manufacturing.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Tag‐Free Antibody Modification Mediated by Lipoic Acid Ligase A: Application to Antibody‐Drug Conjugates Production

et al. 2023

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“…11 However, IgG antibodies possess numerous Gln and Lys residues, and these amino acids on the antibody surface exhibit low enzymatic reactivity. Therefore, the pretreatment of IgG via enzymatic deglycosylation 12,13 or genetic mutagenesis 14 is necessary for the site-specific modification of native antibodies. These pre-treatment procedures may potentially adversely affect the inherent functionality and the production yield of the antibodies.…”

mentioning

confidence: 99%

Transglutaminase-mediated proximity labeling of a specific Lys residue in a native IgG antibody

Nishioka,

Iida,

Minamihata

et al. 2024

Chem. Commun.

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Analytical studies on the conjugation site specificity of trastuzumab modified by Escherichia coli lipoate ligase A: multiple-enzyme digestion approach for peptide mapping

Shikida,

Yamazaki,

Takahashi

et al. 2023

Anal Bioanal Chem

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

Abstract: Substitution I253Q on a glycosylated IgG1 antibody allows microbial transglutaminase-mediated conjugation of a fluorophore or a clickable auristatin. The resulting antibody–drug conjugate showed excellent cell toxicity and no FcRn-mediated recycling.

Cited by 5 publications

References 34 publications

Tag‐Free Antibody Modification Mediated by Lipoic Acid Ligase A: Application to Antibody‐Drug Conjugates Production

Tag‐Free Antibody Modification Mediated by Lipoic Acid Ligase A: Application to Antibody‐Drug Conjugates Production

Transglutaminase-mediated proximity labeling of a specific Lys residue in a native IgG antibody

Analytical studies on the conjugation site specificity of trastuzumab modified by Escherichia coli lipoate ligase A: multiple-enzyme digestion approach for peptide mapping

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