Enzymatic conjugation of polypeptides
Inventors
Bregeon, Delphine • Dennler, Patrick • Belmant, Christian • Gauthier, Laurent • Romagne, François • Fischer, Eliane • Schibli, Roger
Assignees
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Publication Number
US-10434180-B2
Publication Date
2019-10-08
Expiration Date
Abstract
The present application 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 a method for conjugating a hydrophobic compound to an antibody by providing an antibody having at least one acceptor glutamine residue, wherein the residue at position 297 according to Kabat numbering is not glycosylated. The method uses a transglutaminase (TGase) reaction with a linker comprising a primary amine and a reactive group (R) to obtain an antibody comprising an acceptor glutamine linked to the reactive group (R) via the linker.
The reaction mixture for the TGase step is free of organic solvent or contains less than 10% (v/v) organic solvent. A subsequent coupling step reacts the antibody from the TGase step with a compound comprising a moiety of interest (Z) in the presence of at least 5% (v/v) organic solvent, where Z is a hydrophobic compound selected from auristatins, dolastatins, duocarmycins, maytansanoids, and pyrrolobenzodiazepine.
The reactive group (R') is capable of reacting with reactive group (R) so that the acceptor glutamine is linked to moiety of interest (Z) via the linker. R and R' are each bioorthogonal-reaction compatible reactive groups selected from azide and a strained alkene.
Claims Coverage
The consolidated claim coverage centers on one independent claim: a two-step TGase-mediated installation of a non-glycosylated acceptor glutamine at Kabat position 297, followed by solvent-conditioned coupling to a hydrophobic moiety of interest (Z) using a bioorthogonal reactive-group pair. The inventive features are the acceptor glutamine positioning, the primary-amine linker with reactive group R, the solvent constraints for both steps, and the azide/strained alkene pairing for R and R'.
TGase-mediated acceptor glutamine at Kabat position 297 not glycosylated
Providing an antibody having at least one acceptor glutamine residue, wherein the residue at position 297 of the antibody according to Kabat numbering is not glycosylated.
Linker with primary amine and reactive group R under limited organic solvent
Reacting the antibody with a linker comprising a primary amine and a reactive group (R), in the presence of transglutaminase (TGase), under conditions sufficient to obtain an antibody comprising an acceptor glutamine linked to a reactive group (R) via the linker, wherein the reaction mixture is free of organic solvent or contains less than 10% (v/v) organic solvent.
Solvent-conditioned bioorthogonal coupling to hydrophobic moiety Z
Reacting, in the presence of at least 5% (v/v) organic solvent, an antibody comprising an acceptor glutamine linked to a reactive group (R) with a compound comprising a moiety of interest (Z), wherein Z is a hydrophobic compound selected from auristatins, dolastatins, duocarmycins, maytansanoids, and pyrrolobenzodiazepine, and a reactive group (R') capable of reacting with reactive group R, under conditions sufficient to obtain an antibody comprising an acceptor glutamine linked to moiety of interest (Z) via the linker.
Bioorthogonal reactive group pairing selected from azide and strained alkene
Selecting R and R' as bioorthogonal-reaction compatible reactive groups selected from an azide and a strained alkene.
Overall, the claims cover TGase-mediated installation of an acceptor glutamine-linked reactive group at Kabat position 297, followed by bioorthogonal coupling to a hydrophobic moiety of interest (Z) under defined organic solvent conditions using azide and strained-alkene reactive groups.
Stated Advantages
Enables conjugation of a hydrophobic compound to an antibody by linking an acceptor glutamine to the hydrophobic moiety of interest (Z).
Provides a solvent-conditioned process in which the TGase coupling uses a reaction mixture free of organic solvent or containing less than 10% (v/v) organic solvent, while the subsequent coupling is carried out with at least 5% (v/v) organic solvent.
Uses bioorthogonal-reaction compatible reactive groups selected from azide and a strained alkene for the R/R' coupling step.
Documented Applications
Conjugation of hydrophobic cytotoxic payloads (Z), including auristatins, dolastatins, duocarmycins, maytansanoids, and pyrrolobenzodiazepines, to an antibody.
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