A glyco-engineering approach for site-specific conjugation to Fab glycans
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)...
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Taylor & Francis Group
2023-12-01
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| Series: | mAbs |
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| Online Access: | https://www.tandfonline.com/doi/10.1080/19420862.2022.2149057 |
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| author | Maria L. Jaramillo Traian Sulea Yves Durocher Mauro Acchione Melissa J. Schur Anna Robotham John F. Kelly Marie-France Goneau Alma Robert Yuneivy Cepero-Donates Michel Gilbert |
| author_facet | Maria L. Jaramillo Traian Sulea Yves Durocher Mauro Acchione Melissa J. Schur Anna Robotham John F. Kelly Marie-France Goneau Alma Robert Yuneivy Cepero-Donates Michel Gilbert |
| author_sort | Maria L. Jaramillo |
| collection | DOAJ |
| description | 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. |
| format | Article |
| id | doaj-art-d087d6bfdce74a5586de95d8d3bafdd9 |
| institution | DOAJ |
| issn | 1942-0862 1942-0870 |
| language | English |
| publishDate | 2023-12-01 |
| publisher | Taylor & Francis Group |
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| series | mAbs |
| spelling | doaj-art-d087d6bfdce74a5586de95d8d3bafdd92025-08-20T02:50:17ZengTaylor & Francis GroupmAbs1942-08621942-08702023-12-0115110.1080/19420862.2022.2149057A glyco-engineering approach for site-specific conjugation to Fab glycansMaria L. Jaramillo0Traian Sulea1Yves Durocher2Mauro Acchione3Melissa J. Schur4Anna Robotham5John F. Kelly6Marie-France Goneau7Alma Robert8Yuneivy Cepero-Donates9Michel Gilbert10Human Health Therapeutics Research Centre, National Research Council Canada, 6100 Royalmount Avenue, H4P 2R2, Montreal, Qc, CanadaHuman Health Therapeutics Research Centre, National Research Council Canada, 6100 Royalmount Avenue, H4P 2R2, Montreal, Qc, CanadaHuman Health Therapeutics Research Centre, National Research Council Canada, 6100 Royalmount Avenue, H4P 2R2, Montreal, Qc, CanadaHuman Health Therapeutics Research Centre, National Research Council Canada, 6100 Royalmount Avenue, H4P 2R2, Montreal, Qc, CanadaHuman Health Therapeutics Research Centre, National Research Council Canada, 100 Sussex Drive, K1A 0R6, Ottawa, ON, CanadaHuman Health Therapeutics Research Centre, National Research Council Canada, 100 Sussex Drive, K1A 0R6, Ottawa, ON, CanadaHuman Health Therapeutics Research Centre, National Research Council Canada, 100 Sussex Drive, K1A 0R6, Ottawa, ON, CanadaHuman Health Therapeutics Research Centre, National Research Council Canada, 100 Sussex Drive, K1A 0R6, Ottawa, ON, CanadaHuman Health Therapeutics Research Centre, National Research Council Canada, 6100 Royalmount Avenue, H4P 2R2, Montreal, Qc, CanadaHuman Health Therapeutics Research Centre, National Research Council Canada, 6100 Royalmount Avenue, H4P 2R2, Montreal, Qc, CanadaHuman Health Therapeutics Research Centre, National Research Council Canada, 100 Sussex Drive, K1A 0R6, Ottawa, ON, CanadaEffective 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.https://www.tandfonline.com/doi/10.1080/19420862.2022.2149057Actinobacillusantibodycetuximabdrug conjugateFab glycansialyltransferase |
| spellingShingle | Maria L. Jaramillo Traian Sulea Yves Durocher Mauro Acchione Melissa J. Schur Anna Robotham John F. Kelly Marie-France Goneau Alma Robert Yuneivy Cepero-Donates Michel Gilbert A glyco-engineering approach for site-specific conjugation to Fab glycans mAbs Actinobacillus antibody cetuximab drug conjugate Fab glycan sialyltransferase |
| title | A glyco-engineering approach for site-specific conjugation to Fab glycans |
| title_full | A glyco-engineering approach for site-specific conjugation to Fab glycans |
| title_fullStr | A glyco-engineering approach for site-specific conjugation to Fab glycans |
| title_full_unstemmed | A glyco-engineering approach for site-specific conjugation to Fab glycans |
| title_short | A glyco-engineering approach for site-specific conjugation to Fab glycans |
| title_sort | glyco engineering approach for site specific conjugation to fab glycans |
| topic | Actinobacillus antibody cetuximab drug conjugate Fab glycan sialyltransferase |
| url | https://www.tandfonline.com/doi/10.1080/19420862.2022.2149057 |
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