Chemoenzymatic Conjugation of Toxic Payloads to the Globally Conserved N-Glycan of Native mAbs Provides Homogeneous and Highly Efficacious Antibody-Drug Conjugates.

A robust, generally applicable, nongenetic technology is presented to convert monoclonal antibodies into stable and homogeneous ADCs. Starting from a native (nonengineered) mAb, a chemoenzymatic protocol allows for the highly controlled attachment of any given payload to the N-glycan residing at asparagine-297, based on a two-stage process: first, enzymatic remodeling (trimming and tagging with azide), followed by ligation of the payload based on copper-free click chemistry. The technology, termed GlycoConnect, is applicable to any IgG isotype irrespective of glycosylation profile. Application to trastuzumab and maytansine, both components of the marketed ADC Kadcyla, demonstrate a favorable in vitro and in vivo efficacy for GlycoConnect ADC. Moreover, the superiority of the native glycan as attachment site was demonstrated by in vivo comparison to a range of trastuzumab-based glycosylation mutants. A side-by-side comparison of the copper-free click probes bicyclononyne (BCN) and a dibenzoannulated cyclooctyne (DBCO) showed a surprising difference in conjugation efficiency in favor of BCN, which could be even further enhanced by introduction of electron-withdrawing fluoride substitutions onto the azide. The resulting mAb-conjugates were in all cases found to be highly stable, which in combination with the demonstrated efficacy warrants ADCs with a superior therapeutic index.

[1]  C. Bertozzi,et al.  Site-Specific Antibody–Drug Conjugates: The Nexus of Bioorthogonal Chemistry, Protein Engineering, and Drug Development , 2014, Bioconjugate chemistry.

[2]  D. Goldenberg,et al.  Carbohydrates engineered at antibody constant domains can be used for site-specific conjugation of drugs and chelates. , 1998, Journal of immunological methods.

[3]  Y. Karamanos.  Endo-N-Acetyl-β-D-Glucosaminidases and Peptide-N4-(N-acetyl-β-D-Glucosaminyl) Asparagine Amidases: More Than Just Tools , 2013 .

[4]  Bruce Tidor,et al.  Aglycosylated immunoglobulin G1 variants productively engage activating Fc receptors , 2008, Proceedings of the National Academy of Sciences.

[5]  P. Friedl,et al.  Readily Accessible Bicyclononynes for Bioorthogonal Labeling and Three-Dimensional Imaging of Living Cells , 2010, Angewandte Chemie.

[6]  Suzanne F. Jones,et al.  Phase I study of trastuzumab-DM1, an HER2 antibody-drug conjugate, given every 3 weeks to patients with HER2-positive metastatic breast cancer. , 2010, Journal of clinical oncology : official journal of the American Society of Clinical Oncology.

[7]  P. Senter,et al.  Self-hydrolyzing maleimides improve the stability and pharmacological properties of antibody-drug conjugates , 2014, Nature Biotechnology.

[8]  R. Linhardt,et al.  Chemoenzymatic synthesis of uridine diphosphate-GlcNAc and uridine diphosphate-GalNAc analogs for the preparation of unnatural glycosaminoglycans. , 2012, The Journal of organic chemistry.

[9]  Alain Van Dorsselaer,et al.  Characterization of therapeutic antibodies and related products. , 2013, Analytical chemistry.

[10]  P. Bondarenko,et al.  High-mannose glycans on the Fc region of therapeutic IgG antibodies increase serum clearance in humans. , 2011, Glycobiology.

[11]  C. Bertozzi,et al.  Probing glycosyltransferase activities with the Staudinger ligation. , 2004, Journal of the American Chemical Society.

[12]  M. Beeram,et al.  A phase 1 study of weekly dosing of trastuzumab emtansine (T‐DM1) in patients with advanced human epidermal growth factor 2–positive breast cancer , 2012, Cancer.

[13]  Mark Tatham,et al.  The General Approach , 2006 .

[14]  Yang Feng,et al.  Site-specific antibody-drug conjugation through an engineered glycotransferase and a chemically reactive sugar , 2014, mAbs.

[15]  B. Gorovits,et al.  Proposed mechanism of off-target toxicity for antibody–drug conjugates driven by mannose receptor uptake , 2013, Cancer Immunology, Immunotherapy.

[16]  B. G. Davis,et al.  An endoglycosidase with alternative glycan specificity allows broadened glycoprotein remodelling. , 2012, Journal of the American Chemical Society.

[17]  V. Nizet,et al.  EndoS2 is a unique and conserved enzyme of serotype M49 group A Streptococcus that hydrolyses N-linked glycans on IgG and α1-acid glycoprotein , 2013, The Biochemical journal.

[18]  B. Ramakrishnan,et al.  Structure-based design of beta 1,4-galactosyltransferase I (beta 4Gal-T1) with equally efficient N-acetylgalactosaminyltransferase activity: point mutation broadens beta 4Gal-T1 donor specificity. , 2002, The Journal of biological chemistry.

[19]  Natalia Gomez,et al.  Effect of temperature, pH, dissolved oxygen, and hydrolysate on the formation of triple light chain antibodies in cell culture , 2010, Biotechnology progress.

[20]  S. Jung,et al.  Aglycosylated IgG variants expressed in bacteria that selectively bind FcγRI potentiate tumor cell killing by monocyte-dendritic cells , 2009, Proceedings of the National Academy of Sciences.

[21]  A. Borrmann,et al.  Highly accelerated inverse electron-demand cycloaddition of electron-deficient azides with aliphatic cyclooctynes , 2014, Nature Communications.

[22]  R. Chari,et al.  Antibody-drug conjugates: an emerging concept in cancer therapy. , 2014, Angewandte Chemie.

[23]  Peter G Schultz,et al.  A general approach to site-specific antibody drug conjugates , 2014, Proceedings of the National Academy of Sciences.

[24]  G. Mathé,et al.  Effet sur la leucémie 1210 de la souris d'un combinaison par diazotation d'A-méthoptèrine et de gamma-globulines de hamsters porteurs de cette leucèmie par hétérogreffe. , 1958 .

[25]  Jennifer A. Prescher,et al.  A strain-promoted [3 + 2] azide-alkyne cycloaddition for covalent modification of biomolecules in living systems. , 2004, Journal of the American Chemical Society.

[26]  Paul Polakis,et al.  Site-specific conjugation of a cytotoxic drug to an antibody improves the therapeutic index , 2008, Nature Biotechnology.

[27]  Linda C Hsieh-Wilson,et al.  A chemoenzymatic approach toward the rapid and sensitive detection of O-GlcNAc posttranslational modifications. , 2003, Journal of the American Chemical Society.

[28]  Xiuru Li,et al.  Preparation of well-defined antibody-drug conjugates through glycan remodeling and strain-promoted azide-alkyne cycloadditions. , 2014, Angewandte Chemie.

[29]  Jason S. Lewis,et al.  Enzyme-mediated methodology for the site-specific radiolabeling of antibodies based on catalyst-free click chemistry. , 2013, Bioconjugate chemistry.

[30]  Louise Robinson,et al.  Long-term stabilization of maleimide-thiol conjugates. , 2015, Bioconjugate chemistry.

[31]  B. Ramakrishnan,et al.  Site specific conjugation of fluoroprobes to the remodeled Fc N-glycans of monoclonal antibodies using mutant glycosyltransferases: application for cell surface antigen detection. , 2009, Bioconjugate chemistry.

[32]  M. Debets,et al.  Bioconjugation with strained alkenes and alkynes. , 2011, Accounts of chemical research.

[33]  M. Dorywalska,et al.  Location matters: site of conjugation modulates stability and pharmacokinetics of antibody drug conjugates. , 2013, Chemistry & biology.

[34]  Armin Ruf,et al.  Unique carbohydrate–carbohydrate interactions are required for high affinity binding between FcγRIII and antibodies lacking core fucose , 2011, Proceedings of the National Academy of Sciences.

[35]  Aza-dibenzocyclooctynes for fast and efficient enzyme PEGylation via copper-free (3+2) cycloaddition. , 2010, Chemical communications.

[36]  A. Olsén,et al.  EndoS, a novel secreted protein from Streptococcus pyogenes with endoglycosidase activity on human IgG , 2001, The EMBO journal.

[37]  B. Ramakrishnan,et al.  The N-terminal stem region of bovine and human beta1,4-galactosyltransferase I increases the in vitro folding efficiency of their catalytic domain from inclusion bodies. , 2003, Protein expression and purification.

[38]  M. Sliwkowski,et al.  Conjugation site modulates the in vivo stability and therapeutic activity of antibody-drug conjugates , 2012, Nature Biotechnology.

[39]  G. Baisch,et al.  Convenient chemoenzymatic synthesis of beta-purine-diphosphate sugars (GDP-fucose-analogues). , 1997, Bioorganic & medicinal chemistry.

[40]  M. Dorywalska,et al.  Effect of attachment site on stability of cleavable antibody drug conjugates. , 2015, Bioconjugate chemistry.

[41]  Janice M. Reichert.  Note of appreciation to reviewers , 2017, mAbs.

[42]  Frank Loganzo,et al.  Mild method for succinimide hydrolysis on ADCs: impact on ADC potency, stability, exposure, and efficacy. , 2014, Bioconjugate chemistry.

[43]  C. J. Murray,et al.  Production of site-specific antibody-drug conjugates using optimized non-natural amino acids in a cell-free expression system. , 2014, Bioconjugate chemistry.