Enzyme-mediated Modification and Conjugation

Creative Biolabs has extensive expertise in antibody-drug conjugate (ADC) design and production. Antibody enzymatic modification is one of our featured services that enables the chemical compatibility of antibodies to meet several unique conjugation approaches. Three sophisticated technological strategies are employed to create specific conjugation sites on antibodies to serve your ADC development projects.

Enzymes that react with a particular amino acid in a specific peptide sequence are utilized in our platform to achieve site-specific modification of antibodies. Payload drugs are then attached to the antibodies via those modified amino acid side chains using proper conjugation chemistries. Comparing to conjugation using endogenous sites (antibody Lys or Cys residues), enzyme-mediated conjugation allows precise control over the drug to antibody ratio (DAR)/stoichiometry, yields well-defined conjugation site distributions, and subsequently, homogeneous conjugates. Our technological strategies include:

SortA-mediated conjugation

This strategy is based on the Sortase A (SortA)-mediated peptide coupling. As a bacteria transpeptidase, SortA from Staphylococcus aureus recognizes a C-terminal pentapeptide with the sequence LPXTG (where X can be any amino acid) and catalyzes the replacement of the terminal Gly with the conjugation partner containing a N-terminal Gly residue. By utilizing recombinant protein expression system, scientists at Creative Biolabs install the LPXTG consensus sequence for SortA modification into antibody C terminus without compromising its bioactivity. The modified antibodies can then be used to conjugate with glycine-functionalized cargo molecules to produce various ADCs. Alternatively, incorporation of the pentapeptide into a drug-linker complex while modify the antibody N terminus to a Gly residue to enable this type of conjugation is also feasible for ADC development.

SortA-mediate conjugation: by replacing the Gly residue at the C terminus after Thr with the conjugation partner equipped with a N terminal Gly residue (Bioconjugate Chem, 2015).

TGs-mediated conjugation

This strategy works by using transglutaminases (TGs) to generate ADCs. Generally, microbial transglutaminases catalyze amide bond formation between glutamine (Gln, Q) side chains and small molecules containing a primary amine. To this end, TGs can be used to conjugate small molecules onto antibodies Gln residues, for instance Q295 on IgG, to form ADCs with a potential DAR of 2. The removal or size reduction of N297 glycans is prerequisite to reduce spacial hindrance for TG activity. Alternatively, a mutation that converts N297 to Q also facilitates the TG-mediated conjugation and results in ADCs with a potential DAR of up to 4.

One special TG, the transglutaminase from Streptoverticillium mobaraense, has an atypical catalytic site comparing to other TGs. S. mobaraense TG recognizes a “glutamine (Q) tag”, usually in the sequence of LLQG. Taking advantage of this specificity, the LLQG quapeptide tag can be introduced into various locations on the heavy or light chains of an antibody to achieve site-selective attachment of payloads.

Transglutaminase (TG) conjugation of primary amine-containing small molecules onto antibodies via endogens Q295 sites or glutamine (Q) tag (S. mobaraense TG, Bioconjugate Chem, 2015)

FGE-mediated conjugation 

This strategy employs a formylglycine generating enzyme (FGE) to generate specific conjugation sites for ADC production. FGE recognizes a tag comprising a six-residue LCxPxR (where X is usually serine, threonine, alanine or glycine) peptide and oxidizes the cysteine to formylglycine, thereby creating a bio-orthogonal aldehyde handle for drug attachment by means of Hydrazino-iso-Pictet-Spengler (HIPS) ligation. Like the Q-tag for S. mobaraense TG, this LCxPxR peptide tag can also be engineered into desired locations on antibodies to achieve site-specific conjugation of the payloads.

FGE- mediate conjugation that converts the Cys residue on LCxPxR into formylglycine for site-specific conjugation (Bioconjugate Chem, 2015).

Advantages of enzyme-mediated modification and conjugation strategies:

• Recognition sequence can be resided on either the antibody or the conjugation partner (especially in the case of SortA-mediated conjugations)
• The recognition sequence incorporation is easy to achieve
• Suitable for modifications on all regions (Fab, scFv, Fc) of an antibody and on whole antibodies
• Incorporated conjugation sites have no significant influence on antigen binding affinity, specificity, and antibody stability
• Generating site-specific, homogeneous Antibody−Drug Conjugates with homogenous drug-to-antibody ratios (DARs)

With years of experience in antibody engineering, Creative Biolabs provides comprehensive antibody modification and conjugation strategies to help our clients with various ADC development projects, please contact us for more information and a detailed quote.

References:

1. Agarwal, P.; et al. Site-specific antibody−drug conjugates: the nexus of bioorthogonal chemistry, protein engineering, and drug development. Bioconjugate Chem. 2015, 26: 176−192.
2. Dennler, P.; et al. Antibody conjugates: from heterogeneous populations to defined reagents. Antibodies. 2015, 4: 197-224.