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Publication Number
US-10071169-B2
Publication Date
2018-09-11
Expiration Date
Abstract
One embodiment relates to methods for the enzymatic 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, TGase-mediated stoichiometric functionalization of antibodies for drug conjugates in which antibodies comprise an acceptor glutamine residue. The antibody acceptor glutamine residues are reacted with a linking reagent comprising a primary amine and a moiety of interest in the presence of bacterial transglutaminase (BTG) to obtain functionalized antibodies. The disclosure further specifies antibody-conjugate compositions suitable for pharmaceutical compositions, including acceptor glutamine residues naturally present in an Fc region and at residue 295 by Kabat numbering.
The invention controls TGase-mediated deamidation of acceptor glutamine residues by reducing BTG/TGase activity relative to the antibody and by using a BTG amount determined by a hydroxamate activity assay. The resulting conjugation is performed while maintaining deamidation at a controlled level of no more than 10% of deamidated acceptor glutamine residue, with dependent refinements including no more than 5% and no more than 2%.
Linker architectures are described using primary-amine lysine-based linking reagents and general linker formulas, including cleavable or non-cleavable spacer systems with optional val-cit motifs, di-, tri-, or oligopeptide spacer elements, and moiety-of-interest selection. Documented outcomes include higher drug:antibody ratios under controlled deamidation limits, with examples associated with 2 acceptor glutamines and 4 acceptor glutamines.
Claims Coverage
The patent includes independent claims directed to BTG-mediated linking of antibody acceptor glutamine residues using a primary-amine linking reagent, while controlling BTG amount by a hydroxamate activity assay and limiting deamidation to no more than 10%. The independent coverage is presented as both a method for producing the conjugated antibody composition and a vessel/system comprising the defined components in defined amounts.
BTG-controlled enzymatic linking of acceptor glutamine using a primary-amine linking reagent
A method for producing an antibody conjugated to a moiety of interest, comprising providing a composition comprising a plurality of antibodies each comprising an acceptor glutamine residue and reacting said antibody with a linking reagent comprising a primary amine and a moiety of interest in the presence of bacterial transglutaminase (BTG) providing between 0.004 and 0.03 enzyme units (U), as determined by a hydroxamate activity assay, per nanomole (nmole) of acceptor glutamine to obtain a composition comprising antibodies with acceptor glutamine linked to the linking reagent.
Deamidation-limited acceptor glutamine composition
The method yields a composition wherein no more than 10% of the antibodies in the composition comprise a deamidated acceptor glutamine residue.
Primary-amine moiety-of-interest selection for conjugation
The moiety of interest is selected from the group consisting of a hydrophobic organic compound, an organic compound having an electrically negative charge, an organic compound having a molecular weight of at least 400 g/mol, and any combination thereof.
Vessel/system with acceptor glutamine antibodies, linking reagent, and BTG within hydroxamate-assayed units
A vessel comprising a plurality of antibodies each comprising an acceptor glutamine residue, a linking reagent comprising a primary amine and a moiety of interest selected from the group consisting of a hydrophobic organic compound, an organic compound having an electrically negative charge, an organic compound having a molecular weight of at least 400 g/mol, and any combination thereof, and a bacterial transglutaminase (BTG) in the amount of between 0.004 and 0.03 enzyme units (U), as determined by a hydroxamate activity assay, per nanomole (nmole) of acceptor glutamine.
Deamidation-limited vessel composition
The vessel includes antibodies wherein no more than 10% of the antibodies comprise a deamidated acceptor glutamine residue.
The independent claims define BTG-controlled, hydroxamate-assay-determined enzymatic linking of antibody acceptor glutamine residues to a primary-amine linking reagent carrying defined classes of moiety of interest, together with an explicit limit on deamidated acceptor glutamine residues (no more than 10%). The second independent claim recasts the same component set and constraints as a vessel/system comprising antibodies, linking reagent, and BTG within the defined activity range.
Stated Advantages
Increases conjugation while maintaining low deamidation (no more than 10% deamidated acceptor glutamine residue, optionally lower deamidation limits such as ≤5% and ≤2%).
Achieves higher drug:antibody ratios under the deamidation limits.
Improved coupling, with increased drug-antibody ratio (DAR) under low activity BTG conditions while limiting deamidation of acceptor glutamine.
Conjugation avoids BTG-driven deamidation that rapidly reduces available acceptor glutamine when BTG is incubated without linker, supporting effective linking under controlled BTG dosing.
Documented Applications
Production and provision of functionalized antibody compositions for drug conjugates using TGase-mediated stoichiometric functionalization of antibodies.
Producing an antibody conjugate by reacting antibodies with acceptor glutamine residues with a linking reagent comprising a primary amine in the presence of bacterial transglutaminase (BTG) under hydroxamate-amount and deamidation limits.
Using a vessel or system comprising antibodies with acceptor glutamine residues, a primary-amine linking reagent with a moiety of interest from the defined classes, and BTG dosed by hydroxamate activity to generate the specified conjugation composition.
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