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-10675359-B2
Publication Date
2020-06-09
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 human or humanized antibodies that include one or more 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 variable or constant region. The solvent-exposed glutamine residues are present in a light chain variable domain (VL) CDR at a Kabat position selected from 27, 55, or a combination thereof, and the acceptor glutamine residue is used to conjugate one or more moieties-of-interest (Z) through a linker containing an NH-(C)n-moiety.
The conjugation linker is defined by a substituted or unsubstituted alkyl or heteroalkyl chain as the (C)n part, where n is an integer selected from 2 to 20 and any carbon in the chain may be substituted with alkoxy, hydroxyl, alkylcarbonyloxy, alkyl-S—, thiol, alkyl-C(O)S—, amine, alkylamine, amide, or alkylamide. Z is a reactive moiety or a moiety that improves pharmacokinetic properties, a therapeutic moiety, or a diagnostic moiety.
The disclosure also grounds the conjugation design in acceptor glutamine/Q locations on antibody heavy chains, including Q295/N297Q and N297S variants, and describes quantitative control over conjugation stoichiometry and distribution of moieties-of-interest on the antibody, including mean Z:antibody ratio and the proportion of antibodies with more than two Z. The document further provides payload/linker context including a valine-citrulline conditionally-cleavable dipeptide, a PAB self-eliminating spacer, an MMAF payload, and epothilone derivatives as Z.
Claims Coverage
The provided independent claim coverage centers on a conjugatable human or humanized antibody with engineered solvent-exposed glutamine residues and acceptor glutamine positions, and on the NH-(C)n linker used to attach moieties-of-interest (Z). The main inventive features are the specific glutamine placement in the light chain CDR, the acceptor glutamine conjugation in the constant region or fused sequence, the defined linker structure, the permitted Z classes, and the specified heavy and light chain variable region sequences.
Solvent-exposed glutamine residues in a light chain variable domain CDR at Kabat positions 27 and/or 55
A human or humanized antibody comprising one or more solvent-exposed glutamine residues in a variable region, where the solvent-exposed glutamines are present in a light chain variable domain (VL) CDR at a Kabat position selected from 27, 55, or a combination thereof.
Acceptor glutamine in the constant region or fused sequence for conjugation
The antibody further comprises at least one acceptor glutamine residue in its constant region or in a sequence fused to a variable or constant region, wherein the antibody is conjugated via the acceptor glutamine residue to one or more moieties-of-interest (Z).
NH-(C)n linker with substituted or unsubstituted alkyl or heteroalkyl chain and n from 2 to 20
Conjugation to Z is through a linker comprising a NH-(C)n-moiety, where (C)n is a substituted or unsubstituted alkyl or heteroalkyl chain optionally substituted on any carbon as defined, and n is an integer selected from among the range of 2 to 20.
Z as reactive, pharmacokinetic, therapeutic, or diagnostic moiety
Z is a reactive moiety or a moiety that improves the pharmacokinetic properties, a therapeutic moiety, or a diagnostic moiety.
Specific heavy and light chain variable region sequences
The antibody comprises heavy chain variable region sequence selected from SEQ ID NOs: 3-239 and light chain variable region sequence selected from SEQ ID NOs: 240-299.
Across the independent claim, the inventive concept is the engineered placement of solvent-exposed glutamines in the light chain CDR at Kabat position 27 and/or 55, paired with acceptor glutamine conjugation in the constant region or a fused sequence, linked via an NH-(C)n structure that connects to moieties-of-interest (Z) defined as reactive, pharmacokinetic-improving, therapeutic, or diagnostic. The independent claim also restricts the antibody to specified heavy and light chain variable region sequence sets.
Stated Advantages
Provides quantitative control over conjugation stoichiometry and distribution of moieties-of-interest on the antibody, including mean Z:antibody ratio and the proportion of antibodies with more than two Z.
BTG-mediated quantitative coupling may be limited when payloads are large, hydrophobic, or charged.
Spacer length can affect coupling, for example C6 versus C2 DOTA.
Documented Applications
Z includes a therapeutic moiety or a diagnostic moiety.
Epothilone derivatives can serve as Z.
Named cytotoxic moieties such as auristatins and epothilones are included as payload examples.
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