Enzymatic antibody modification by bacterial transglutaminase

Area of Science:

• Biochemistry
• Protein Engineering
• Immunology

Background:

• Enzymatic protein modification offers superior site control compared to chemical methods.
• Homogeneous antibody conjugates possess enhanced properties for research and therapeutic applications.
• Bacterial transglutaminase (BTGase) enables specific protein conjugation.

Purpose of the Study:

• To provide a protocol for BTGase-mediated conjugation of substrates to IgG1 antibodies.
• To demonstrate the generation of homogeneous antibody conjugates with defined substrate-to-antibody ratios.
• To establish a method for analyzing the uniformity of antibody conjugates.

Main Methods:

• Enzymatic removal of N-linked glycans from IgG1 followed by BTGase-mediated conjugation.
• Generation of a mutant aglycosylated IgG1 variant with an additional glutamine via site-directed mutagenesis.
• ESI-TOF mass spectrometry for analyzing conjugate uniformity.

Main Results:

• Stable bond formation between glutamine 295 of IgG1 heavy chain and cadaverine-derivatized substrates.
• Achieved a defined substrate/antibody ratio of 2:1 with glycan removal, or 4:1 with a mutant IgG1.
• Demonstrated the generation of homogeneous antibody conjugates.

Conclusions:

• The presented BTGase-mediated approach facilitates the facile generation of homogeneous IgG1 antibody conjugates.
• This method allows for precise control over conjugation site and stoichiometry.
• The protocol is applicable to various IgG1 antibodies and diverse cadaverine-derivatized substrates for research and therapeutic development.