Recognition tags for TGase-mediated conjugation
Inventors
Fischer, Eliane • Romagne, François • Dennler, Patrick
Assignees
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Publication Number
US-10036010-B2
Publication Date
2018-07-31
Expiration Date
Abstract
The present application relates to methods for the functionalization of antibodies using transglutaminase, in particular antibodies lacking Fc regions. Also disclosed herein are peptide tags for transglutaminase, linking reagents, functionalized antibodies, multi-specific antibodies, pharmaceutical compositions, and method of treating disease and/or conditions.
Core Innovation
The invention relates to antibody-drug conjugate linker chemistry using an antibody lacking an Fc domain, including scFv, affibody, V_H domain, V_L domain, V-NAR domain and V_HH domain. The antibody comprises a TGase recognition tag containing a functionalized acceptor glutamine residue (Q) of Formula IV, with defined structural components including C, X, L, V and Y.
Conjugation uses complementary reactive group pairs R and R', wherein RR' is an addition product formed by a Huisgen 1,3-cycloaddition reaction and one of R and R' is an azide and the other is a strained cycloalkene. The defined moiety M links the TGase-tagged antibody to a moiety-of-interest Z through the RR' system.
The tag architecture further defines V as absent, a non-cleavable moiety, or a conditionally-cleavable moiety, and Y as absent, a bond, or a spacer system comprised of one or more spacers. The moiety-of-interest Z includes pharmacokinetic-improving moieties, therapeutic moieties, diagnostic moieties, and other drug-like payload categories.
Claims Coverage
The consolidated content provides one independent claim centered on an Fc-lacking antibody bearing a TGase recognition tag with a functionalized acceptor glutamine (Q) of Formula IV. The inventive features focus on the defined tag structure and attachment of a moiety-of-interest Z through an RR' addition product using an azide and a strained cycloalkene.
Fc-lacking antibody with TGase recognition tag of Formula IV
An antibody lacking an Fc domain selected from scFv, affibody, V_H domain, V_L domain, V-NAR domain and V_HH domain, comprising a TGase recognition tag having a functionalized acceptor glutamine residue (Q) of Formula IV with defined structural elements C, X, L, V and Y.
Huisgen 1,3-cycloaddition via azide and strained cycloalkene addition product RR'
The antibody comprises a moiety M including an RR' addition product between a reactive moiety R and a complementary reactive moiety R', where one of R and R' is an azide and the other is a strained cycloalkene.
Conditionally-cleavable or non-cleavable moiety V and defined spacer system Y
The TGase recognition tag framework includes V as absent, a non-cleavable moiety, or a conditionally-cleavable moiety, and Y as absent, a bond, or a spacer system comprised of one or more spacers.
Moiety-of-interest Z attached through defined tag components
Z is a moiety-of-interest attached within the defined M structure, including a moiety that improves pharmacokinetic properties, a therapeutic moiety, or a diagnostic moiety.
Overall claim coverage centers on an Fc-lacking antibody format that includes a TGase recognition tag defined by Formula IV around an acceptor glutamine Q, coupled to attachment logic using an azide/strained cycloalkene Huisgen 1,3-cycloaddition addition product (RR'). The coverage further includes controlled cleavability and spacer-system flexibility via V and Y, and supports attachment of a broadly defined moiety-of-interest Z.
Stated Advantages
The coupling is controlled without direct lysine-to-glutamine isopeptide formation between antibodies that can cause polymerization.
The moiety-of-interest can improve pharmacokinetic properties, or be a therapeutic moiety or a diagnostic moiety.
The platform supports formation of multimeric multispecific antibodies.
Documented Applications
Multimeric polypeptide assembly through TGase-mediated conjugation of the acceptor glutamine residue.
Functionalization and analytical characterization using LC-MS, SDS-PAGE and Western blot with streptavidin-HRP.
Evaluation of conjugates by binding, affinity, cytotoxicity and pharmacokinetics using standard assays.
MTGase-mediated coupling to scFv, affibody and nanobody using a myc-tag.
Synthesis of TGase substrates/linker molecules with analytical characterization.
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