Enzymatic conjugation of antibodies
Inventors
Dennler, Patrick • Bregeon, Delphine • Gauthier, Laurent • Romagné, François • Belmant, Christian • Fischer, Eliane • Schibli, Roger
Assignees
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Publication Number
US-9764038-B2
Publication Date
2017-09-19
Expiration Date
Abstract
The present application relates to methods for the functionalization of immunoglobulins, in particular with drugs. Also disclosed herein are linking reagents, functionalized antibodies, pharmaceutical compositions, and method of treating disease and/or conditions.
Core Innovation
The invention relates to enzymatic, site-specific antibody functionalization using transglutaminase to attach a lysine-based linker to a human or humanized antibody. The antibody contains solvent exposed glutamine residues in a variable region and at least one acceptor glutamine residue in a constant region or in a sequence fused to a constant region or fused to a variable region, enabling formation of an isopeptide bond between the lysine-based linker and the acceptor glutamine residue.
The solvent exposed glutamine residues are present in a light chain variable domain CDR at Kabat positions selected from 27, 55, or a combination thereof. The method focuses conjugation to acceptor glutamine residues in defined regions and excludes acceptor sites in CDRs, while also covering antibody fragments such as Fab, Fv, scFv, and nanobody/dAb.
The invention additionally describes lysine-based linker architectures intended to provide controlled drug-antibody ratios and homogeneous stoichiometry. Linker structures include a lysine-like moiety and a reactive group such as azide, alkyne, norbornene, strained or electronically activated alkenes, substituted or unsubstituted cycloalkynes, or other strained or electronically activated alkenes, and engineered glutamine acceptor sites include constant-region glutamine acceptors and glycosylation elimination substitutions such as N297X.
Claims Coverage
The independent claim set covers a method of attaching a lysine-based linker to a human or humanized antibody using transglutaminase, with acceptor glutamine residues positioned for site-specific covalent conjugation. The consolidated claim coverage includes two inventive features: selection of solvent-exposed VL CDR glutamine residues and TGase-mediated attachment to acceptor glutamine residues in constant or fused regions.
TGase-mediated covalent attachment of a lysine-based linker to an acceptor glutamine in antibody constant region
Reacting a human or humanized antibody containing an acceptor glutamine residue in a constant region or in a sequence fused to a constant region or fused to a variable region with a lysine-based linker in the presence of transglutaminase under conditions permitting the lysine-based linker to be covalently linked to the acceptor glutamine residue.
Solvent-exposed VL CDR glutamine selection at Kabat 27 and 55
Providing a human or humanized antibody in which one or more solvent exposed glutamine residues are present in a light chain variable domain CDR at a Kabat position selected from 27, 55, or a combination thereof, in connection with the method of attaching the lysine-based linker to the acceptor glutamine residue.
The inventive concept is site-specific covalent conjugation by transglutaminase that attaches a lysine-based linker to acceptor glutamine residues in antibody constant or fused regions. The claim coverage is anchored by selection of solvent-exposed VL CDR glutamine residues at Kabat positions 27 and/or 55 and by covalent linkage to the acceptor glutamine residue.
Stated Advantages
Allows covalent, site-specific attachment of a lysine-based linker to an antibody acceptor glutamine residue using transglutaminase.
Enables attachment at defined acceptor glutamine locations in constant regions or constant-/variable-fused sequences rather than unspecific antibody sites.
Provides controlled drug-antibody ratios and homogeneous stoichiometry.
Spacer and linker tuning affects coupling outcomes, with a 6-carbon spacer improving coupling compared with a C2-DOTA spacer.
Removal of negative charge at the +2 position via N297S substitutions enables quantitative coupling for fluorescent, TAMRA, and MMAF or large hydrophobic cytotoxic-linker constructs.
The method solves the problem of enabling covalent attachment of a lysine-based linker to antibody glutamine residues using a transglutaminase.
Documented Applications
Preparation of antibody conjugates carrying a moiety of interest, including drug-antibody conjugates with enumerated cytotoxic and therapeutic agent classes.
Conjugation of moieties of interest to acceptor glutamine residues on human or humanized antibodies, including use with antibody fragments such as Fab, Fv, scFv, and nanobody/dAb.
Antibody conjugates using the described lysine-based linker system.
Therapeutic and diagnostic and cytotoxic classes, including hydrophobic drugs, taxanes, camptothecins, epothilones, auristatins, radioisotopes, therapeutic proteins, toxins, and fragments.
Experimental coupling of lysine-based linkers or payloads to the heavy chain of chADC1.
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