CONJUGATION METHOD INVOLVING A TRANSGLUTAMINASE AT THE FC REGION COMPRISING A TRIMMED N-GLYCAN
Described herein are methods for conjugating moieties of interest, such as pharmaceutically active molecules, to targeting molecules such as antibodies using transglutaminase-based processes.
1 . A method for conjugating a moiety of interest to an Fc-containing polypeptide (e.g. an antibody), which method comprises:
(i) providing an Fc-containing polypeptide with N-linked glycosylation at a site present in the Fc region of the polypeptide, adjacent a site comprising a glutamine residue;
(ii) treating said polypeptide with an enzyme to trim back the N-linked glycans at the site of N-linked glycosylation to an optionally fucosylated, core N-acetylglucosamine (GlcNAc) moiety; and
(iii)(1) reacting, in the presence of a transglutaminase (TGase), the polypeptide obtained in step (ii) with a moiety of interest comprising an acceptor moiety which is a substrate for the TGase, so that the moiety of interest is attached to the polypeptide via the glutamine residue.
2 . A method for conjugating a moiety of interest to an Fc-containing polypeptide (e.g. an antibody), which method comprises:
(i) providing an Fc-containing polypeptide with N-linked glycosylation at a site present in the Fc region of the polypeptide, adjacent a site comprising a glutamine residue;
(ii) treating said polypeptide with an enzyme to trim back the N-linked glycans at the site of N-linked glycosylation to an optionally fucosylated, core N-acetylglucosamine (GlcNAc) moiety; and
(iii)(2) reacting, in the presence of a transglutaminase (TGase), the polypeptide obtained in step (ii) with an acceptor moiety which is a substrate for the TGase and which acceptor moiety further comprises a reactive group (R) which enables the subsequent attachment of the moiety of interest via a reaction between R and a complementary reactive group (R′) attached to the moiety of interest, such that the acceptor moiety is attached to the polypeptide via the glutamine residue; and
(iv) reacting the polypeptide conjugate formed in step (iii)(2) with the moiety of interest attached to R′ under conditions such that R reacts with R′ to form R—R′.
3 . A method according to claim 1 or claim 2 wherein the acceptor moiety is selected from an amine, an aminooxy, a hydrazido, a hydrazino and aryl derivatives thereof.
4 . A method according to claim 2 or claim 3 wherein the acceptor moiety is of formula Ac-Sp-R where Ac is selected from an amine, an aminooxy, a hydrazido, a hydrazino and aryl derivatives thereof, Sp is absent or a spacer moiety; and R is a reactive group, e.g. an azide.
5 . A method according to any one of the preceding claims wherein the enzyme is EndoS.
6 . A method according to any one of the preceding claims wherein the N-linked glycosylation site is at position 297 according to the Kabat numbering system, or an equivalent position and/or the site comprising a glutamine residue is at position 295 according to the Kabat numbering system, or an equivalent position.
7 . A method according to any one of the preceding claims wherein the moiety of interest comprises a drug-linker.
8 . A method according to any one of the preceding claims wherein a second moiety of interest is conjugated to the Fc-containing polypeptide, which method further comprises the following step:
(v) reacting, in the presence of a galactosyl transferase (GalT), the polypeptide obtained in step (iii)(1), step (iii)(2) or step (iv) with a sugar derivative attached to the second moiety of interest, so that the sugar derivative is attached to the polypeptide via the core N-acetylglucosamine (GlcNAc) moiety.
9 . A method according to any one of claims 1 to 7 wherein a second moiety of interest is conjugated to the Fc-containing polypeptide, which method further comprises the following steps:
(v) reacting, in the presence of a galactosyl transferase (GalT), the polypeptide obtained in step (iii))(1), step (iii)(2) or step (iv) with a sugar derivative attached to a reactive group (R) which enables the subsequent attachment of the moiety of interest via a reaction between R and a complementary reactive group (R′) attached to the moiety of interest, so that the sugar derivative is attached to the polypeptide via the core N-acetylglucosamine (GlcNAc) moiety; and
(vi) reacting the polypeptide conjugate formed in step (v) with the moiety of interest attached to R′ under conditions such that R reacts with R′ to form R—R′.
10 . An Fc-containing polypeptide conjugate, which comprises:
(i) an asparagine residue present in the Fc region of the polypeptide with an optionally fucosylated, GlcNAc N-glycan core lacking any additional endogenous glycan residues; and
(ii) (Q)-NH-L-D, or a pharmaceutically acceptable salt or solvate thereof; wherein Q is a glutamine residue present in the Fc region of the polypeptide; L is a linker, which optionally comprises a conditionally-cleavable moiety; and D is a drug moiety;
wherein the asparagine residue is adjacent the glutamine residue.
11 . An Fc-containing polypeptide conjugate according to claim 10 , wherein L comprises L 1 -R—R′-L 2 ;
wherein L 1 is absent or a spacer moiety, R—R′ is formed by the reaction of a reactive group R and a complementary reactive group R′; and L 2 is a linker or spacer moiety to which D is attached and which optionally comprises a conditionally-cleavable moiety.
12 . An Fc-containing polypeptide conjugate, which comprises:
(i) (Q)-NH-LA-D1, or a pharmaceutically acceptable salt or solvate thereof; wherein Q is a glutamine residue present in the Fc region of the polypeptide; LA is a linker; and D is a drug moiety;
(ii) (N)-G-S—R 2 —R 2 ′-LB-D2, or a pharmaceutically acceptable salt or solvate thereof; where N is an asparagine residue present in the Fc region of the polypeptide; G is an optionally fucosylated, GlcNAc N-glycan core; S is a sugar derivative; R 2 —R 2 ′ is formed by the reaction of a reactive group R 2 and a complementary reactive group R 2 ′; LB is a linker, which optionally comprises a conditionally-cleavable moiety; and D1 and D2 are each independently a drug moiety; and
wherein the asparagine residue is adjacent the glutamine residue.
13 . An Fc-containing polypeptide conjugate according to claim 12 , wherein LA comprises LA 1 -R 1 —R 1 ′-LA 2 ;
wherein LA 1 is absent or a spacer moiety, R 1 —R 1 ′ is formed by the reaction of a reactive group R 1 and a complementary reactive group R 1 ′; and LA 2 is a linker or spacer moiety to which D1 is attached and which optionally comprise a conditionally-cleavable moiety.
14 . An Fc-containing polypeptide conjugate according to any one of claims 10 to 15 wherein D is a cytotoxin or immunostimulatory molecule.
15 . An Fc-containing polypeptide conjugate according to any one of claims 10 to 14 wherein the asparagine residue is at position 297 according to the Kabat numbering system, or an equivalent position and/or the glutamine residue is at position 295 according to the Kabat numbering system, or an equivalent position.