Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2023 Jan-Dec;15(1):2149057.
doi: 10.1080/19420862.2022.2149057.

A glyco-engineering approach for site-specific conjugation to Fab glycans

Maria L Jaramillo  1 , Traian Sulea  1 , Yves Durocher  1 , Mauro Acchione  1 , Melissa J Schur  2 , Anna Robotham  2 , John F Kelly  2 , Marie-France Goneau  2 , Alma Robert  1 , Yuneivy Cepero-Donates  1 , Michel Gilbert  2
Affiliations

A glyco-engineering approach for site-specific conjugation to Fab glycans

Maria L Jaramillo et al. MAbs. 2023 Jan-Dec.

Abstract

Effective processes for synthesizing antibody-drug conjugates (ADCs) require: 1) site-specific incorporation of the payload to avoid interference with binding to the target epitope, 2) optimal drug/antibody ratio to achieve sufficient potency while avoiding aggregation or solubility problems, and 3) a homogeneous product to facilitate approval by regulatory agencies. In conventional ADCs, the drug molecules are chemically attached randomly to antibody surface residues (typically Lys or Cys), which can interfere with epitope binding and targeting, and lead to overall product heterogeneity, long-term colloidal instability and unfavorable pharmacokinetics. Here, we present a more controlled process for generating ADCs where drug is specifically conjugated to only Fab N-linked glycans in a narrow ratio range through functionalized sialic acids. Using a bacterial sialytransferase, we incorporated N-azidoacetylneuraminic acid (Neu5NAz) into the Fab glycan of cetuximab. Since only about 20% of human IgG1 have a Fab glycan, we extended the application of this approach by using molecular modeling to introduce N-glycosylation sites in the Fab constant region of other therapeutic monoclonal antibodies. We used trastuzumab as a model for the incorporation of Neu5NAz in the novel Fab glycans that we designed. ADCs were generated by clicking the incorporated Neu5NAz with monomethyl auristatin E (MMAE) attached to a self-immolative linker terminated with dibenzocyclooctyne (DBCO). Through this process, we obtained cetuximab-MMAE and trastuzumab-MMAE with drug/antibody ratios in the range of 1.3 to 2.5. We confirmed that these ADCs still bind their targets efficiently and are as potent in cytotoxicity assays as control ADCs obtained by standard conjugation protocols. The site-directed conjugation to Fab glycans has the additional benefit of avoiding potential interference with effector functions that depend on Fc glycan structure.

Keywords: Actinobacillus; Fab glycan; antibody; cetuximab; drug conjugate; sialyltransferase; trastuzumab.

PubMed Disclaimer

Conflict of interest statement

No potential conflict of interest was reported by the author(s).

Figures

Strategy used to target specifically the Fab glycans of an antibody for site-specific conjugation of a label or a drug.
Figure 1.
Overview of the conjugation to antibody glycans using sialyltransferases with different specificities. The mAbs are produced in CHO cells and a sialidase is used to remove the sialic acids (Neu5Ac) while the B4GalT1 galactosyltransferase is used to increase the presence of terminal β-1,4-linked galactose residues. Neu5NAz is transferred to the Fc and Fab glycans using ST6Gal1 or specifically to the Fab glycan using a bacterial sialyltransferase (AST-03). A label or a drug can then be covalently attached to the functionalized sialic acid (Neu5NAz). For simplicity, the N-glycans are depicted on one heavy chain monomer only.
Effect of MMAE-conjugated cetuximab ADCs on the viability of U87 glioblastoma cells that overexpress EGFR. Cetuximab antibodies conjugated to their glycan residues were as potent as antibodies labeled on random Cys residues.
Figure 2.
Growth inhibitory effect of glyco-engineered cetuximab conjugated with MMAE in U87 glioblastoma cancer cells engineered to overexpress EGFR. Potency was determined according to concentration of antibody (Panel a) or MMAE drug (Panel b).
Locations of de novo glycosylation sites highlighted on a typical Fab crystal structure of high resolution. The heavy chain is in black rendering and the light chain is in white rendering.
Figure 3.
The locations of the de novo glycosylation sites are highlighted on a typical Fab crystal structure of high resolution (PDB code 3MXW at 1.8 Å resolution, heavy chain in black, light chain in white rendering). Shown distances are in Ångstrom units.
Effect of MMAE-conjugated trastuzumab ADCs based on the viability of HER2-expressing SKOV3 cells. Growth inhibition potencies were similar when comparing the glyco-engineered versions with the trastuzumab ADC conjugated on random Cys residues.
Figure 4.
Growth inhibitory effect of glyco-engineered wild-type trastuzumab and designed single mutants conjugated with MMAE in HER2-expressing SKOV3 cells. Potency was determined according to molar concentration of antibody (Panel a) or MMAE (Panel b) component.
See this image and copyright information in PMC

References

    1. Ducry L, Stump B.. Antibody-drug conjugates: linking cytotoxic payloads to monoclonal antibodies. Bioconjugate Chem. 2010;21(1):5–15. doi:10.1021/bc9002019. - DOI - PubMed
    1. Panowski S, Bhakta S, Raab H, Polakis P, Junutula JR. Site-specific antibody drug conjugates for cancer therapy. MAbs. 2014;6(1):34–45. doi:10.4161/mabs.27022. - DOI - PMC - PubMed
    1. Junutula JR, Raab H, Clark S, Bhakta S, Leipold DD, Weir S, Chen Y, Simpson M, Tsai SP, Dennis MS, et al. Site-specific conjugation of a cytotoxic drug to an antibody improves the therapeutic index. Nat Biotechnol. 2008;26:925–32. doi:10.1038/nbt.1480. - DOI - PubMed
    1. Axup JY, Bajjuri KM, Ritland M, Hutchins BM, Kim CH, Kazane SA, Halder R, Forsyth JS, Santidrian AF, Stafin K, et al. Synthesis of site-specific antibody-drug conjugates using unnatural amino acids. Proc Natl Acad Sci U S A. 2012;109(40):16101–06. doi:10.1073/pnas.1211023109. - DOI - PMC - PubMed
    1. Italia JS, Addy PS, Erickson SB, Peeler JC, Weerapana E, Chatterjee A. Mutually orthogonal nonsense-suppression systems and conjugation chemistries for precise protein labeling at up to three distinct sites. J Am Chem Soc. 2019;141(15):6204–12. doi:10.1021/jacs.8b12954. - DOI - PMC - PubMed

Publication types

LinkOut - more resources