Conjugation method involving transglutaminase at the FC region containing trimmed N-glycans

Abstract

Described herein are methods for conjugating moieties of interest, such as pharma- ceutical active molecules, to targeting molecules, such as antibodies, using a transglutaminase-based process.

Description

[Technical field]

[0001] The present disclosure relates to methods for conjugating a moiety of interest, such as a pharma- ceutical active molecule, to a targeting molecule, such as an antibody. [Background technology]

[0002] Various methods are known for the attachment of pharma- ceutical active substances to antibodies and related molecules. For example, drug linkers have been attached to natural or engineered cysteine ​​residues in immunoglobulins using maleimide groups. Other approaches are based on using an N-linked glycosylation site, N297, which in some approaches is remodeled to trim back the glycan to a core GlcNAc moiety, which is then reacted with a UDP-sugar in the presence of GalT enzyme, which further contains a reactive azide moiety that allows for the subsequent addition of a drug linker using click chemistry. Another approach has been to use endogenous or engineered glutamine residues together with microbial transglutaminase (mTGase). Summary of the Invention

[0003] Previous approaches to using mTGase to conjugate endogenous glutamine residues at position Q295 (Kabat EU numbering) in the Fc region of immunoglobulins are based on using PNGaseF to remove all of the N-linked glycan at the adjacent position N297 to avoid interference with the TGase reaction. This leaves a negatively charged aspartic acid residue at D297, and therefore the modified approach involves mutating D297 to remove the negative charge (and also the need to remove the N-glycan). We have now found that using a different enzymatic treatment of the N-glycan based on EndoS to leave the core GlcNAc moiety intact allows a very efficient mTGase reaction at Q295. Furthermore, the core GlcNAc moiety at N297 is then available for a second conjugation, and we have surprisingly found that this is very efficient, even though a drug linker is already present at the adjacent Q295.

[0004] Thus, in a first aspect, the disclosure relates to a method for conjugating a moiety of interest to an Fc-containing polypeptide (e.g., an antibody), the method comprising: (i) providing an Fc-containing polypeptide having an N-linked glycosylation at a site in the Fc region of the polypeptide adjacent to a site that contains a glutamine residue; (ii) treating the polypeptide with an enzyme, e.g., EndoS, to trim back N-linked glycans at sites of N-linked glycosylation to optionally fucosylated, core N-acetylglucosamine (GlcNAc) moieties; (iii)(1) reacting the polypeptide obtained in step (ii) with a moiety of interest that includes an acceptor moiety that is a substrate for TGase in the presence of transglutaminase (TGase), such that the moiety of interest is attached to the polypeptide via a glutamine residue.

[0005] In a second, related aspect, the disclosure also relates to a method for conjugating a moiety of interest to an Fc-containing polypeptide (e.g., an antibody), the method comprising: (i) providing an Fc-containing polypeptide having an N-linked glycosylation at a site in the Fc region of the polypeptide adjacent to a site that contains a glutamine residue; (ii) treating the polypeptide with an enzyme, e.g., EndoS, to trim back N-linked glycans at sites of N-linked glycosylation to optionally fucosylated, core N-acetylglucosamine (GlcNAc) moieties; (iii)(2) reacting, in the presence of transglutaminase (TGase), the polypeptide resulting from step (ii) with an acceptor moiety that is a substrate for TGase, the acceptor moiety further comprising a reactive group (R) that allows for subsequent attachment of a moiety of interest via reaction between the reactive group (R) and a complementary reactive group (R') attached to the moiety of interest, such that the acceptor moiety is attached to the polypeptide via a glutamine residue; (iv) reacting the polypeptide conjugate formed in step (iii)(2) with a moiety of interest attached to R' under conditions such that R reacts with R' to form R-R'.

[0006] In one embodiment of the above aspect, the acceptor moiety is selected from amine, aminooxy, hydrazide, hydrazino, and aryl derivatives thereof.

[0007] In one embodiment, the acceptor moiety is of the formula Ac-Sp-R, where Ac is selected from amine, aminooxy, hydrazide, hydrazino, and aryl derivatives thereof, Sp is absent or a spacer moiety, and R is a reactive group, e.g., azide.

[0008] In one embodiment, the N-linked glycosylation site is at position 297, or an equivalent position, according to the Kabat numbering system, and / or the site containing a glutamine residue is at position 295, or an equivalent position, according to the Kabat numbering system.

[0009] In one embodiment, the moiety of interest comprises a drug linker.

[0010] The present disclosure also relates to a second aspect, wherein in the method of the first aspect above, the second moiety of interest is conjugated to an Fc-containing polypeptide, the method comprising the steps of: (v) reacting the polypeptide resulting from step (iii)(1), step (iii)(2), or step (iv) with a sugar derivative conjugated to a second moiety of interest in the presence of a galactosyltransferase (GalT), such that the sugar derivative is conjugated to the polypeptide via a core N-acetylglucosamine (GlcNAc) moiety.

[0011] In a related embodiment, the second moiety of interest is conjugated to an Fc-containing polypeptide and the method comprises the steps of: (v) reacting, in the presence of a galactosyltransferase (GalT), the polypeptide resulting from step (iii))(1), step (iii)(2), or step (iv) with a sugar derivative attached to R that allows for subsequent attachment of a moiety of interest via reaction between the reactive group (R) and a complementary reactive group (R') attached to the moiety of interest, such that the sugar derivative is attached to the polypeptide via a core N-acetylglucosamine (GlcNAc) moiety; (vi) reacting the polypeptide conjugate formed in step (v) with a moiety of interest attached to R' under conditions such that R reacts with R' to form R-R'.

[0012] The present disclosure relates in a third aspect to an Fc-containing polypeptide conjugate, comprising: (i) an asparagine residue present in an Fc region of a polypeptide having an optionally fucosylated, GlcNAc N-glycan core that lacks any additional endogenous glycan residues; and (ii) (Q)-NH-LD, or a pharma- ceutically acceptable salt or solvate thereof, wherein Q is a glutamine residue present in an Fc region of a polypeptide, L is a linker, optionally comprising a conditionally cleavable moiety, and D is a drug moiety; The asparagine residue is adjacent to the glutamine residue.

[0013] In one embodiment, in the Fc-containing polypeptide conjugate, L comprises L1-R-R'-L2, where L1 is absent or a spacer moiety, R-R' is formed by reaction of a reactive group R with a complementary reactive group R', and L2 is a linker or spacer moiety to which D is attached and optionally includes a conditionally cleavable moiety.

[0014] The present disclosure relates in a fourth aspect to an Fc-containing polypeptide conjugate, comprising: (i) (Q)-NH-LA-D1, or a pharma- ceutically acceptable salt or solvate thereof, wherein Q is a glutamine residue present in an Fc region of a polypeptide, LA is a linker, and D is a drug moiety; (ii) (N)-GS-R2-R2'-LB-D2, or a pharma- ceutically acceptable salt or solvate thereof, wherein N is an asparagine residue present in an Fc region of a polypeptide, G is a GlcNAc N-glycan core, optionally fucosylated, S is a sugar derivative, R2-R2' is formed by reaction of reactive group R2 with a complementary reactive group R2', LB is a linker, optionally comprising a conditionally cleavable moiety, and D1 and D2 are each independently a drug moiety; The asparagine residue is adjacent to the glutamine residue.

[0015] In one embodiment, in the Fc-containing polypeptide conjugate, LA comprises LA1-R1-R1'-LA2, where LA1 is absent or a spacer moiety, R1-R1' is formed by reaction of reactive group R1 with a complementary reactive group R1', and LA2 is a linker or spacer moiety to which D1 is attached and optionally includes a conditionally cleavable moiety.

[0016] In an embodiment of the third or fourth aspect, the Fc-containing polypeptide conjugate is an IgG, such as an IgG that specifically binds to a tumor antigen.

[0017] In an embodiment of the third or fourth aspect, the drug is a cytotoxin.

[0018] In an embodiment of the third or fourth aspect, the asparagine residue is at position 297, or an equivalent position, according to the Kabat numbering system, and / or the glutamine residue is at position 295, or an equivalent position, according to the Kabat numbering system.

[0019] The present disclosure also relates to an Fc-containing polypeptide conjugate obtained or obtainable by the process of the first or second aspect described above.

[0020] In a fifth aspect, the present disclosure relates to a pharmaceutical composition comprising an Fc-containing polypeptide conjugate described herein together with one or more pharma- ceutically acceptable carriers or diluents, such as a buffer, sugar, and / or detergent. [Brief description of the drawings]

[0021] [Figure 1] FIG. 1 shows MTGase-mediated conjugation of amine-PEG(3u)-azide to antibodies deglycosylated using PNGase F (top) or glycans trimmed using EndoS (bottom). [Diagram 2]Figure 1 shows dual conjugation to an antibody functionalized with amine-PEG(4u)-MTZ via MTGase-mediated conjugation and GalNAz via glycan remodeling. The process was modeled using TCO-Cy5 and DBCO-AF488, conjugated with MTZ and azide functional groups, respectively. [Figure 3A] FIG. 1 shows reduced PLRP of antibody X and antibody X dual-labeled with TCO-Cy5 and DBCO-AF488. [Figure 3B] FIG. 13 shows reduced PLRP of antibody X dual-labeled with TCO-Cy5 and DBCO-AF488 with absorbance maxima at 280 nm (protein), 488 nm (AF488), and 651 nm (Cy5) overlay. [Figure 4A] FIG. 1 shows double- and single-labeled non-reduced PLRP of antibody X labeled with TCO-Cy5 and DBCO-AF488. [Figure 4B] FIG. 1 shows UV / Vis spectra of doubly and singly labeled antibody X. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0022] antibody Antibodies for use in the processes described herein include an N-glycosylated asparagine residue, such as wild-type N297, or an equivalent position in the heavy chain of an immunoglobulin. N-glycosylation sites can also be engineered into antibodies based on, for example, the consensus sequence Asn-Xaa-Ser / Thr, where Xaa is any amino acid except proline (see Reslan et al., 2020, Int. J. Biol. Macromol 158:189-196; Cruz et al., 2021 Pharmaceutics 13(11):1747; Bano-Polo et al., 2011, Protein Sci. 20(1):179-186).

[0023] The N-glycosylated asparagine residue is flanked by glutamine residues that are substrates for microbial transglutaminase. For example, an immunoglobulin with an N297 glycosylation site typically has a glutamine residue at position 295 (i.e., two residues from the N297 residue). By "flanked" it is meant that the glutamine residue is within two, three, or four amino acids on either side (i.e., toward the N-terminus or C-terminus) of the N-glycosylated asparagine residue. In one embodiment, the glutamine residue is at least two amino acids, e.g., + / - two amino acids, from the N-glycosylated asparagine residue.

[0024] The glutamine may be naturally occurring / wild-type, such as the glutamine residue at position 295 in an immunoglobulin molecule, or its equivalent, or may be engineered into the antibody amino acid sequence.

[0025] The term antibody includes full-length immunoglobulins, e.g., IgG, and fragments thereof that contain an antigen-binding site. In one embodiment, an antibody comprises an Fc domain or a fragment thereof that contains a CH2 domain.

[0026] In one embodiment, the N-glycosylation site and the adjacent glutamine residue are present in the constant region of the antibody, e.g., the Fc domain or the CH2 domain.

[0027] Antibodies for use in and resulting from the processes described herein may bind to any antigen of interest. In one embodiment, the antigen is present on the surface of a cell, such as a cell in a human or animal body. Such a cell / antigen may be associated with a disease or disorder, e.g., a proliferative disease. Expression of the antigen may be upregulated in the cell of interest. In one embodiment, the antigen is a tumor antigen. In another embodiment, the antigen is a cell surface molecule found on immune cells, such as regulatory T cells. Particular cell surface proteins / tumor antigens include CD19, CD22, CD25, AXL, KAAG1, DLK-1, and PSMA.

[0028] Antibody Remodeling In one aspect of the invention, the antibody is treated to trim back the N-glycan, such as the N-glycan at N297, to a core glycan, such as a core N-acetylglucosamine (GlcNAc) moiety, optionally fucosylated. This can be done by incubating the antibody with an enzyme capable of removing most of the glycan. Previously, N-glycan removal has been performed using PNGaseF. However, this results in the removal of all glycan residues and converts asparagine to a negatively charged aspartic acid residue. Instead, the process of the present disclosure uses a suitable enzyme to trim back the N-linked glycan at the site of N-linked glycosylation to a core N-acetylglucosamine (GlcNAc) moiety, optionally fucosylated.

[0029] Depending on the nature of the glycan, a suitable endoglycosidase can be selected. The endoglycosidase is preferably selected from the group consisting of Endo S, Endo A, EfEndo18A, Endo F (e.g., F1, F2, or F3), Endo M, Endo D, and Endo H enzymes, and / or combinations thereof, the selection depending on the nature of the glycan. In a further preferred embodiment, the endoglycosidase is Endo S, Endo S49, Endo F, or a combination thereof. A combined endoglycosidase is disclosed in US10,858,41, which comprises two different Endo S enzymes as a fusion protein. The advantage of using a combination is that a larger number of glycoforms can be recognized and trimmed in a single reaction.

[0030] EndoS is an endoglycosidase isolated from the human pathogen Streptococcus pyogenes. Different forms of EndoS, such as EndoS (Collin et al., 2001. EMBO J. 20:3046-3055) and EndoS2 (Sjogren et al., 2013. Biochem J. 455:107-118), have been shown to hydrolyze the N-linked glycans of human immunoglobulin G with different glycoform selectivities (Sjogen et al., 2015. Glycobiology 25(10):1053-1063). EndoS and EndoS2 are available from Genovis AB (IgGZERO™, GlycINATOR™).

[0031] The antibody of interest is incubated with one or more enzymes under suitable conditions to remove all or a substantial portion of the N-glycans, leaving behind a core N-acetylglucosamine (GlcNAc) moiety. Suitable methods are described in the Examples and in Sjogen et al., 2015, supra.

[0032] An antibody glycan having a core N-acetylglucosamine is defined herein as an antibody that comprises an optionally fucosylated glycan that is attached to the antibody via the C1 of GlcNAc (core-GlcNAc).

[0033] Addition of the desired moiety using TGase Moieties of interest for conjugation to an Fc-containing polypeptide of interest include pharma- ceutically active substances / drugs, diagnostic reagents, and detectable labels. In one embodiment, the active moiety is covalently attached to a linker or spacer construct.

[0034] In the first step of the process, conjugation is performed at the glutamine residue of interest adjacent to the N-glycosylation site using transglutaminase.

[0035] Microbial transglutaminase (mTG) catalyzes the transamidation reaction between specific surface-exposed glutamine (Gln) and lysine (Lys) residues on protein substrates, linking them via an isopeptide bond. The catalytic mechanism of mTG involves nucleophilic attack of the target acyl donor Gln by the active site cysteine ​​of the enzyme forming a thioester intermediate. Subsequent nucleophilic attack of the thioester by an amine substrate, which functions as an acyl acceptor, leads to the formation of the transamidation product.

[0036] The most commonly used mTGase is a 38 kDa enzyme derived from Streptomyces mobaraensis. Another transglutaminase found in bacteria is derived from Bacillus subtilisin (see US 5,731,183). Various variants of mTGase have been developed, such as those disclosed in WO2017 / 059158 and WO2017 / 059160 (Merck), which have amino acid substitutions selected from the group consisting of (A) E300A, (B) I240A and P241A, (C) E249Q, and (D) E300A and Y302A.

[0037] Thus, the remodeled antibody can be conjugated to a moiety of interest using a transglutaminase (such as mTGase), where the moiety of interest contains / is bound to an acyl acceptor that forms a substrate for mTGase, and the glutamine residue of interest in the remodeled antibody provides an acyl donor for the transamidation reaction. The acyl acceptor commonly used in this reaction is a primary amine. However, recent studies have shown that other acyl acceptors containing a terminal -NH group can be used, i.e., alpha effect nucleophiles such as hydrazines, hydrazides, and alkoxyamines (Chio et al., 2020. Angew Chem Int Ed Engl 59(33):13814-13820). Those skilled in the art will understand that the term "alpha effect" refers to the increased nucleophilicity of an atom due to the presence of an adjacent (alpha) atom with a lone pair of electrons, in this context, the increased nucleophilicity of the nitrogen atom of the terminal -NH of the acyl acceptor. In one embodiment, the acyl acceptor is selected from an amine, such as a primary amine; hydrazine; hydrazide; alkoxyamine or aryloxyamine. In a further embodiment, the amine, hydrazine, hydrazide, or oxyamine is an aryl derivative, in particular an optionally substituted phenyl or an optionally substituted benzyl derivative, such as an optionally substituted phenylhydrazine. In one embodiment, the acyl acceptor is a hydrazine or hydrazide. In one embodiment, the acyl acceptor is a hydrazide or an optionally substituted arylhydrazine derivative. In a further embodiment, the acyl acceptor is an optionally substituted arylhydrazine derivative or an optionally substituted arylhydrazide derivative. In yet a further embodiment, the acyl acceptor is an optionally substituted phenylhydrazine or an optionally substituted phenylhydrazide derivative.

[0038] In one embodiment, the acyl acceptor is [ka] especially [ka] The invention includes an optionally substituted moiety selected from: *Indicates attachment point.

[0039] Alternatively, the acyl acceptor is [ka] especially [ka] The invention includes an optionally substituted moiety selected from:

[0040] In a further embodiment, the acyl acceptor is [ka] especially [ka] The invention includes an optionally substituted moiety selected from:

[0041] In a further alternative embodiment, the acyl acceptor is [ka] especially [ka] Includes.

[0042] In one embodiment, the aryl derivatives can be multiply substituted such that the acyl acceptor is branched with multiple points of attachment.

[0043] In one embodiment, the acyl acceptor is [ka] especially [ka] The invention includes an optionally substituted moiety selected from: *Indicates attachment point.

[0044] Alternatively, the acyl acceptor is [ka] especially [ka] The invention includes an optionally substituted moiety selected from:

[0045] Specific examples include the following: [Table 1-1] [Table 1-2]

[0046] In one embodiment, the acyl acceptor is selected from Examples 1-12, in particular Examples 3, 4, 7, 8, 11, and 12.

[0047] The azide groups in Examples 1-12 can be replaced with other reactive groups described below for the two-step reaction, such as BCN and DBCO. The azide groups can also be replaced with other linker moieties.

[0048] As used herein, the term "amine" refers to a derivative of ammonia in which one or more hydrogen atoms are replaced by a substituent, such as an aliphatic (e.g., alkyl or heteroalkyl) or aromatic (e.g., aryl or heteroaryl) group. A primary amine has only one of the three hydrogen atoms in ammonia replaced by a substituent.

[0049] As used herein, the term "hydrazine" refers to a derivative of hydrazine (H2N-NH2) in which one of the hydrogen atoms has been replaced by a substituent such as an aliphatic (e.g., alkyl or heteroalkyl) or aromatic (e.g., aryl or heteroaryl) group.

[0050] As used herein, the term "hydrazide" refers to a derivative of inorganic hydrazine (H2N-NH2) in which one of the hydrogen atoms has been replaced by a substituent such as an acyl, sulfonyl, or phosphoryl group, especially an acyl group.

[0051] As used herein, the term "alkyl" refers to a fully saturated branched or unbranched hydrocarbon moiety having up to 20 carbon atoms. Unless otherwise provided, alkyl refers to a hydrocarbon moiety having 1-16 carbon atoms, 1-10 carbon atoms, 1-7 carbon atoms, or 1-4 carbon atoms. Representative examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl, n-decyl, and the like.

[0052] As used herein, the term "alkylene" refers to a divalent alkyl group as defined herein above having 1 to 20 carbon atoms. It includes 1 to 20 carbon atoms, and unless otherwise provided, alkylene refers to a moiety having 1 to 16 carbon atoms, 1 to 10 carbon atoms, 1 to 7 carbon atoms, or 1 to 4 carbon atoms. Representative examples of alkylene include, but are not limited to, methylene, ethylene, n-propylene, iso-propylene, n-butylene, sec-butylene, iso-butylene, tert-butylene, n-pentylene, isopentylene, neopentylene, n-hexylene, 3-methylhexylene, 2,2-dimethylpentylene, 2,3-dimethylpentylene, n-heptylene, n-octylene, n-nonylene, n-decylene, and the like.

[0053] As used herein, the term "alkynyl" refers to a branched or unbranched hydrocarbon moiety containing one triple bond having up to 20 carbon atoms. Unless otherwise provided, alkynyl refers to a hydrocarbon moiety having 2-16 carbon atoms, 2-10 carbon atoms, 2-7 carbon atoms, or 2-4 carbon atoms. Representative examples of alkyl include, but are not limited to, ethynyl, propynyl, butynyl, pentynyl, hexynyl, heptynyl, octynyl, nonynyl, decynyl, and the like.

[0054] The term "heteroalkyl" is given its ordinary meaning in the art and refers to an alkyl group, as described herein, in which one or more carbon atoms is replaced by a heteroatom (eg, oxygen, nitrogen, sulfur).

[0055] The term "aryl" refers to an aromatic hydrocarbon group having 6 to 20 carbon atoms in the ring portion. Typically, the aryl is a monocyclic, bicyclic, or tricyclic aryl having 6 to 20 carbon atoms.

[0056] Furthermore, as used herein, the term "aryl" refers to an aromatic substituent which may be a single aromatic ring or multiple aromatic rings fused together.

[0057] Non-limiting examples include phenyl, naphthyl, or tetrahydronaphthyl, each of which can be optionally substituted with 1 to 4 substituents, such as alkyl, trifluoromethyl, cycloalkyl, halogen, hydroxy, alkoxy, acyl, alkyl-C(O)—O—, aryl-O—, heteroaryl-O—, amino, thiol, alkyl-S—, aryl-S—, nitro, cyano, carboxy, alkyl-OC(O)—, carbamoyl, alkyl-S(O)—, sulfonyl, sulfonamido, phenyl, and heterocyclyl.

[0058] As used herein, the term "alkoxy" refers to alkyl-O-, where alkyl is defined herein above. Representative examples of alkoxy include, but are not limited to, methoxy, ethoxy, propoxy, 2-propoxy, butoxy, tert-butoxy, pentyloxy, hexyloxy, cyclopropyloxy-, cyclohexyloxy-, and the like. Typically, an alkoxy group has about 1-7 carbons, more preferably about 1-4 carbons.

[0059] As used herein, the term "aryloxy" refers to both an --O-aryl and an --O-heteroaryl group, where aryl and heteroaryl are defined herein.

[0060] As used herein, the term "heteroaryl" refers to a 5-14 membered monocyclic or bicyclic or tricyclic aromatic ring system having 1-8 heteroatoms selected from N, O, or S. Typically, a heteroaryl is a 5-10 membered ring system (e.g., a 5-7 membered monocyclic or an 8-10 membered bicyclic) or a 5-7 membered ring system. Exemplary heteroaryl groups include 2- or 3-thienyl, 2- or 3-furyl, 2- or 3-pyrrolyl, 2-, 4-, or 5-imidazolyl, 3-, 4-, or 5-pyrazolyl, 2-, 4-, or 5-thiazolyl, 3-, 4-, or 5-isothiazolyl, 2-, 4-, or 5-oxazolyl, 3-, 4-, or 5-isoxazolyl, 3- or 5-1,2,4-triazolyl, 4- or 5-1,2,3-triazolyl, tetrazolyl, 2-, 3-, or 4-pyridyl, 3- or 4-pyridazinyl, 3-, 4-, or 5-pyrazinyl, 2-pyrazinyl, and 2-, 4-, or 5-pyrimidinyl.

[0061] The term "heteroaryl" also refers to groups in which a heteroaromatic ring is fused to one or more aryl, alicyclic, or heterocyclyl rings, and the radical or point of attachment is on the heteroaromatic ring. Non-limiting examples include 1-, 2-, 3-, 5-, 6-, 7-, or 8-indolizinyl, 1-, 3-, 4-, 5-, 6-, or 7-isoindolyl, 2-, 3-, 4-, 5-, 6-, or 7-indolyl, 2-, 3-, 4-, 5-, 6-, or 7-indazolyl, 2-, 4-, 5-, 6-, 7-, or 8-purinyl, 1-, 2-, 3-, 4-, 6-, 7-, 8-, or 9-quinolizinyl, 2-, 3-, 4-, 5-, 6-, 7-, or 8-quinolyl, 1 ... 8-Isoquinolyl, 1-, 4-, 5-, 6-, 7-, or 8-phthalazinyl, 2-, 3-, 4-, 5-, or 6-naphthyridinyl, 2-, 3-, 5-, 6-, 7-, or 8-quinazolinyl, 3-, 4-, 5-, 6-, 7-, or 8-cinnolinyl, 2-, 4-, 6-, or 7-pteridinyl, 1-, 2-, 3-, 4-, 5-, 6-, 7-, or 8-4aHcarbazolyl, 1-, 2-, 3-, 4-, 5-, 6-, 7-, or 8-carbazolyl, 1-, 3-, 4-, 5-, 6-, 7-, or 8-carbazolyl, 1-, 3-, 4-, 5-, 6-, 7-, 8-, or 9-caproyl. 1-, 2-, 3-, 4-, 6-, 7-, 8-, 9-, or 10-phenanthridinyl; 1-, 2-, 3-, 4-, 5-, 6-, 7-, 8-, or 9-acridinyl; 1-, 2-, 4-, 5-, 6-, 7-, 8-, or 9-perimidinyl; 2-, 3-, 4-, 5-, 6-, 8-, 9-, or 10-phenathrolinyl; 1-, 2-, 3-, 4-, 6-, 7-, 8-, or 9-phenazinyl; 1-, 2-, 3-, 4-, 6-, 7-, 8-, 9-, or 10-phenothiazinyl; -, 4-, 6-, 7-, 8-, 9-, or 10-phenoxazinyl; 2-, 3-, 4-, 5-, 6-, or 1-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, or 10-benzisoquinolinyl; 2-, 3-, 4-, or thieno[2,3-b]furanyl; 2-, 3-, 5-, 6-, 7-, 8-, 9-, 10-, or 11-7H-pyrazino[2,3-c]carbazolyl; 2-, 3-, 5-, 6-, or 7-2H-furo[3,2-b]pyranyl; 2-, 3-, 4-, 5-, 7-, or 8-5H-pyrido[2,3-d]-o-oxazinyl, 1-, 3-, or 5-1H-pyrazolo[4,3-d]-oxazolyl, 2-, 4-, or 54H-imidazo[4,5-d]thiazolyl, 3-, 5-, or 8-pyrazino[2,3-d]pyridazinyl, 2-, 3-, 5-, or 6-imidazo[2,1-b]thiazolyl, 1-, 3-, 6-, 7-, 8-, or 9-furo[3,4-c]cinnolinyl, 1-, 2-, 3-, 4-, 5-, 6-, 8-, 9-, 10, or 11-4H-pyrido[2,3-c]carbazolyl, 2-, 3-, 6-, or 7-imidazo[ 1,2-b][1,2,4]triazinyl, 7-benzo[b]thienyl, 2-, 4-, 5-, 6-, or 7-benzoxazolyl, 2-, 4-, 5-, 6-, or 7-benzimidazolyl, 2-, 4-, 4-, 5-, 6-, or 7-benzothiazolyl, 1-, 2-, 4-, 5-, 6-, 7-, 8-, or 9-benzoxapinyl, 2-, 4-, 5-, 6-, 7-, or 8-benzoxazinyl, 1-, 2-, 3-, 5-, 6-, 7-, 8-, 9-, 10-, or 11-1H-pyrrolo[1,2-b][2]benzazapinyl. Exemplary fused heteroaryl groups include, but are not limited to, 2-, 3-, 4-, 5-, 6-, 7-, or 8-quinolinyl, 1-, 3-, 4-, 5-, 6-, 7-, or 8-isoquinolinyl, 2-, 3-, 4-, 5-, 6-, or 7-indolyl, 2-, 3-, 4-, 5-, 6-, or 7-benzo[b]thienyl, 2-, 4-, 5-, 6-, or 7-benzoxazolyl, 2-, 4-, 5-, 6-, or 7-benzimidazolyl, and 2-, 4-, 5-, 6-, or 7-benzothiazolyl.

[0062] The heteroaryl group may be substituted with 1 to 5 substituents independently selected from the group consisting of: (a) alkyl, (b) hydroxy (or protected hydroxy), (c)Haro, (d) oxo, i.e., =O; (e) amino, alkylamino, or dialkylamino, (f) alkoxy, (g) cycloalkyl, (h) carboxyl, (i) heterocyclooxy (heterocyclooxy denotes a heterocyclic group attached through an oxygen bridge); (j) alkyl-OC(O)-, (k) mercapto, (l) nitro, (m) Cyano, (n) sulfamoyl or sulfonamide, (o) aryl, (p) alkyl-C(O)—O—, (q) aryl-C(O)—O—, (r) aryl-S-, (s) aryloxy, (t) alkyl-S-, (u) formyl, i.e., HC(O)-, (v) carbamoyl, (w) aryl-alkyl-, and (x) Aryl substituted with alkyl, cycloalkyl, alkoxy, hydroxy, amino, alkyl-C(O)—NH—, alkylamino, dialkylamino, or halogen.

[0063] As used herein, the term "acyl" refers to a substituted carbonyl group in which a carbon atom is replaced by a substituent such as an aliphatic (e.g., alkyl or heteroalkyl) or aromatic (e.g., aryl or heteroaryl) group.

[0064] As used herein, the term "halogen" or "halo" refers to fluoro, chloro, bromo, and iodo.

[0065] As used herein, unless otherwise specified, the term "optionally substituted" refers to a group that is unsubstituted or substituted with one or more, typically 1, 2, 3 or 4, suitable non-hydrogen substituents, each of which independently represents: (a) alkyl, (b) hydroxy (or protected hydroxy), (c)Haro, (d) oxo, i.e., =O; (e) amino, alkylamino, or dialkylamino, (f) alkoxy, (g) cycloalkyl, (h) carboxyl, (i) heterocyclooxy (heterocyclooxy denotes a heterocyclic group attached through an oxygen bridge); (j) alkyl-OC(O)-, (k) mercapto, (l) nitro, (m) Cyano, (n) sulfamoyl or sulfonamide, (o) aryl, (p) alkyl-C(O)—O—, (q) aryl-C(O)—O—, (r) aryl-S-, (s) aryloxy, (t) alkyl-S-, (u) formyl, i.e., HC(O)-, (v) carbamoyl, (w) aryl-alkyl-, and (x) is selected from the group consisting of alkyl, cycloalkyl, alkoxy, hydroxy, amino, alkyl-C(O)—NH—, alkylamino, dialkylamino, or aryl substituted with halogen.

[0066] The acyl acceptor may comprise / be bound to a spacer moiety, such as polyethylene glycol, e.g., polyethylene glycol having 1 to 50 ethylene glycol units, e.g., 1 to 30 ethylene glycol units. Suitable spacer moieties may include, but are not limited to, substituted or unsubstituted alkylene or heteroalkylene chains, where the length of the carbon chain (n) is, for example, an integer selected from 2 to 20.

[0067] The acyl acceptor may also include / be bound to other components of the drug linker construct. A wide variety of linker technologies for binding drugs, such as cytotoxins, to cell-binding agents are available in the art. Linkers can incorporate a variety of different moieties to aid in antibody-drug conjugate stability and determine drug release characteristics. For example, linkers can include cleavable moieties, such as those cleavable by cathepsin B (e.g., valine-alanine or valine-citrulline). Another strategy is to use pH-sensitive linkers, where the lower pH of endosomes and lysosomes partitions the hydrolysis of acid-labile groups in the linker, such as hydrazones, carbonates, silyl ethers, or silanols (e.g., -Si(Me2)-O-Si(Me2)-see WO2016 / 183359). Alternative linkers may be non-cleavable, which can avoid or reduce off-target effects and improve plasma stability in the circulation.

[0068] In another embodiment, the linker may comprise a spacer moiety, such as, for example, polyethylene glycol, and / or a substituted or unsubstituted alkylene or heteroalkylene chain, attached to an acyl acceptor or otherwise, where the length of the carbon chain (n) is an integer selected from 2 to 20. The spacer moiety may also be a hydrophilic spacer, such as -NH-SO2-NH-, to increase the overall hydrophilicity of the resulting conjugate (taking into account that many drugs of interest may be highly hydrophobic).

[0069] The linker may also include a self-immolative group attached to the drug moiety so that the drug can be released without leaving any linker group behind. Examples of such groups are p-aminobenzyl alcohol (PABA) and p-aminobenzyloxycarbonyl (PABC). Self-immolation is the spontaneous and irreversible fragmentation of a multicomponent compound into small molecules through a cascade of cyclization or elimination reactions. In this context, a self-immolative group is a moiety that undergoes self-immolation and acts as a linker to temporarily connect a cleavable protecting group (represented as a "trigger") to the drug moiety. The self-immolative group maintains the space between the trigger and the drug moiety, enhancing the entropic driving force that promotes complete and traceless deprotection.

[0070] Linkers include both linear and branched configurations. Branched linkers allow multiple active agents to be attached via one conjugation point on the antibody. Various branched linkers are described in WO2016 / 183359. Branched linkers include branched PEG linkers such as branched PEG azide and branched PEG maleimide.

[0071] The drug moiety (D) can be any suitable pharmacoactive agent that is desired to be delivered to a target cell. Examples include cytotoxins such as taxanes, anthracyclines, camptothecins, epothilones, mitomycins, combretastatins, vinca alkaloids, nitrogen mustards, maytansinoids, calicheamicins, duocarmycins, tubulysins, amanitins / amatoxins, dolastatins, auristatins, enediynes, pyrrolobenzodiazepines, ecteinascidins, and ethylenimines. Other examples include immune stimulatory molecules such as TLR agonists (e.g., TLR7, TLR8, and TLR9 agonists) and STING agonists. In one embodiment where there are two or more drug moieties, at least one drug moiety has a first mode of action and at least one drug moiety has a second, different, mode of action. For example, at least one is an immune stimulatory molecule and at least one is a cytotoxin.

[0072] The drug linker can be attached to the antibody in one or more steps. For example, in a one-step process, a drug linker bearing an acyl acceptor moiety is reacted with an antibody of interest bearing mTGase.

[0073] In one embodiment, the resulting molecule comprises the following formula: (Q)-NH-LD, where Q is a glutamine residue present in an Fc region of the polypeptide, NH is from the acyl acceptor, L is a linker, and D is a drug moiety.

[0074] In certain embodiments, taking into account the relevant chemical nature of the acyl acceptor, the resulting molecule may comprise any one of the following formulas: [Table 2]

[0075] (Ar) represents an optionally substituted aryl group, including, but not limited to, optionally substituted phenyl, optionally substituted benzyl, and optionally substituted benzoyl.

[0076] In one embodiment, taking into account the relevant chemical properties of the acyl acceptor, the resulting molecule is [ka] especially [ka] The invention includes an optionally substituted moiety selected from: *Indicates point of attachment to linker.

[0077] Alternatively, the acyl acceptor is [ka] especially [ka] The invention includes an optionally substituted moiety selected from:

[0078] In a further embodiment, the acyl acceptor is [ka] especially [ka] The invention includes an optionally substituted moiety selected from:

[0079] In a further alternative embodiment, the acyl acceptor is [ka] especially [ka] The compound includes a moiety selected from

[0080] In one embodiment, the aryl derivatives can be multiply substituted such that the acyl acceptor is branched with multiple points of attachment.

[0081] In certain embodiments, taking into account the relevant chemical nature of the acyl acceptor, the resulting molecule may comprise any one of the following formulas: [Table 3] wherein y is 2, 3, 4 or 5, particularly 2, 3 or 4, especially 2 or 3, most particularly 2.

[0082] L and D can be the same or different at each occurrence.

[0083] Alternatively, it may be convenient to use a two-step procedure in which the acyl acceptor moiety contains a reactive group (R), which allows the remainder of the moiety of interest, such as a drug linker, to be added in a second step upon further reaction with the reactive group. In such embodiments, the remainder of the moiety of interest contains a complementary reactive group R' that can react with R.

[0084] For example, in one embodiment, R or R' is a moiety that includes a bioorthogonal reaction compatible reactive group, such as an unprotected or protected thiol, epoxide, maleimide, haloacetamide, o-phosphene aromatic ester, azide, fulminate, sulfonate ester, alkyne, cyanide, amino-thiol, carbonyl, aldehyde, generally any group capable of oxime and hydrazine formation, 1,2,4,5-tetrazine, norbornene, other dyed or otherwise electronically activated alkenes, substituted or unsubstituted cycloalkynes, generally any reactive group that forms 1,3- or 1,5-disubstituted triazoles via a bioorthogonal cycloaddition reaction, any diene or strained alkene dienophile that can react via an inverse electron demand Diels-Alder (IEDDA) reaction, protected or unprotected amine, carboxylic acid, aldehyde, oxamine.

[0085] R or R' can be selected to undergo, for example, a thio-maleimide (or haloacetamide) addition, a Staudinger ligation, a Huisgen 1,3-cycloaddition (click reaction), or a Diels-Alder cycloaddition with the complementary reactive group R' on the remainder attached to a moiety of interest, such as a drug linker.

[0086] In one embodiment, the reactive group is a haloacetamide (e.g., bromo-acetamide, iodo-acetamide, chloro-acetamide). Such reactive groups will be more stable in vivo (and in serum) compared to maleimide groups. In one embodiment, the reactive group is a reagent capable of undergoing a "click" reaction. For example, 1,3-dipole functional compounds can react with alkynes in a cyclization reaction to form heterocyclic compounds, preferably in the substantial absence of an added catalyst (e.g., Cu(I)). A variety of compounds having at least one 1,3-dipole group attached thereto (having a three-atom π-electron system containing four electrons delocalized on three atoms) can be used to react with the alkynes disclosed herein. Exemplary 1,3-dipole groups include, but are not limited to, azides, nitrile oxides, nitrones, azoxy groups, and acyldiazo groups.

[0087] Examples include o-phosphene aromatic esters, azides, fulminates, alkynes (including any strained cycloalkynes), cyanides, anthracene, 1,2,4,5-tetrazine, or norbornene (or other strained cycloalkenes).

[0088] In one embodiment, R or R' is a group having a terminal alkyne or azide, such moieties are described, for example, in US Pat. No. 7,763,736.

[0089] In one embodiment, R or R' is a substituted or unsubstituted cycloalkyne, such as a substituted or unsubstituted heterocyclic strained alkyne. For example, R or R' can be a DBCO (dibenzylcyclooctyl) group or a BCN (bicyclononyne) group.

[0090] Alkynes such as those described herein above can be reacted with at least one 1,3-dipole functional compound in a cyclization reaction, preferably in the substantial absence of an added catalyst (e.g., Cu(I)), to form a heterocyclic compound. A wide variety of compounds having at least one 1,3-dipole group attached thereto (having a three-atom π-electron system containing four electrons delocalized on three atoms) can be used to react with the alkynes disclosed herein. Exemplary 1,3-dipole groups include, but are not limited to, azides, nitrile oxides, nitrones, azoxy groups, and acyldiazo groups.

[0091] Thus, in one embodiment, after the first step of the process, the resulting molecule has the following formula: (Q)-NH-L1-R, where Q is a glutamine residue present in the Fc region of the polypeptide, NH is from an acyl acceptor, L1 is an optional linker or spacer moiety, and R is a reactive group as described above. This can then be reacted with a drug comprising R', for example, R'-L2-D, where L2 is an optional linker or spacer moiety, and D is a drug. Thus, in this embodiment with respect to the resulting product, -L1-R-R'-L2- collectively form a linker, L, as described above. The resulting molecule thus has the following formula: (Q)-NH-L1-R-R'-L2-D, where Q is a glutamine residue present in the Fc region of the polypeptide, NH is derived from an acyl acceptor, L1 and L2 are independently absent, a linker, or a spacer moiety (L1-R-R'-L2 collectively forming the linker, L, as described above), and preferably at least one of L1 and L2 is not absent, and D is a drug moiety.

[0092] As regards the single step process described above, since different acyl acceptors can be used, certain embodiments include (Q)-NH-L1-R-R'-L2-D, (Q)-NH-Ar-L1-R-R'-L2-D, (Q)-NH-O-L1-R-R'-L2-D, (Q)-NH-O-Ar-L1-R-R'-L2-D, (Q)-NH-NHC(O)-L1-R-R'-L2-D, (Q)-NH-NHC(O)-Ar-L1-R-R'-L2-D, (Q)-NH-NH-L1-R-R'-L2-D, Includes (Q) -NH-NH-Ar-L1-R-R'-L2-D.

[0093] (Ar) represents an optionally substituted aryl group, including, but not limited to, optionally substituted phenyl, optionally substituted benzyl, and optionally substituted benzoyl.

[0094] In certain embodiments, taking into account the relevant chemical nature of the acyl acceptor, the resulting molecule may comprise any one of the following formulas: [Table 4] wherein y is 2, 3, 4 or 5, particularly 2, 3 or 4, especially 2 or 3, most particularly 2.

[0095] L1, R, R', and D can be the same or different in each occurrence.

[0096] With reference to the above examples of R and R', R-R' can be, for example, an addition product of a thio-maleimide (or haloacetamide) addition, such as an NS-disubstituted-3-thio-pyrrolidine-2,5-diones; a Staudinger ligation, such as an N,3- or N,4-substituted-5-diphenylphosphinoxide-benzoic acid amide; a Huisgen 1,3-cycloaddition (click reaction), such as an NS-disubstituted-3-thio-pyrrolidine-2,5-diones, a 1,4-disubstituted-1,2,3-triazole, a 3,5-disubstituted-isoxazole, or a 3,5-disubstituted-tetrazole; a Diels-Alder cycloaddition adduct, such as an O or N 2,4-cycloaddition products between O- or N-substituted-5-norbornene-2-carboxylic acid esters or amides, N-substituted-5-norbornene-2,3-dicarboxylic acid imides, O- or N-substituted-7-oxonorbornene-5-carboxylic acid esters or amides, or N-substituted-7-oxonorbornene-5,6-dicarboxylic acid imides, and 9-substituted anthracenes or 3-substituted 1,2,4,5-tetrazines; inverse electron demand Diels-Alder cycloaddition adducts, for example, 2,4-cycloaddition products between substituted cyclopropenyls and 3-substituted 1,2,4,5-tetrazines; or any high yield selective amidation or imidization reaction.

[0097] Examples of R-R' are: [ka] where (*) indicates the site of attachment of L1 and L2 or D. R-R' can be in either orientation with respect to their attachment to L1 and L2 or D.

[0098] Conjugation of a second drug linker at the adjacent remodeled N-glycosylation site The N-glycosylation site, treated with EndoS to trim back to the core GlcNAc, can be used for a second conjugation.

[0099] In one embodiment, the process involves the use of enzymatic addition of a sugar derivative linked to a reactive group R (see previous section for examples of R), such as azide, to the core GlcNAc moiety. Galactosyltransferases are a family of enzymes that catalyze the addition of galactose residues from an activated sugar nucleotide donor UDP-Gal to an acceptor in an α1-3-, α1-4-, β1-3-, or β1-4-linkage. In one particular embodiment, the enzyme is β-1-4-galactosyltransferase (GalT), such as a mutant version with a Y298L mutation (Ramakrishnan and Qasba, 2002, The Journal of Biological Chemistry 277(23):20833-20839). Other GalT mutants of interest include GalT Y289N, GalT Y289I, GalT Y289F, GalT Y289M, GalT Y289V, GalT Y289G, GalT Y289I, and GalT Y289A.

[0100] The general term "sugar" is used herein to refer to monosaccharides, such as glucose (Glc), galactose (Gal), mannose (Man), and fucose (Fuc). The term "sugar derivative" is used herein to refer to a monosaccharide, i.e., a derivative of a monosaccharide that includes a substituent and / or functional group.

[0101] In one embodiment, the sugar derivative together with the reactive group R is a sugar derivative Su(A)xP derived from a sugar or sugar derivative Su containing x functional groups A, where P is a nucleoside monophosphate or diphosphate (P is part of the sugar derivative substrate for the enzymatic reaction, but is consumed during the reaction, leaving the sugar derivative Su(A)x attached to the core GlcNAc residue). The sugar derivative Su(A)x contains one or more functional groups A. When Su(A)x contains two or more functional groups A, each functional group A is independently selected, i.e., one Su(A)x may contain a different functional group A. In one embodiment, A is independently selected from the group consisting of an azide group, a keto group, and an alkynyl group, where x is 1, 2, 3, or 4, and P is a nucleoside mono- or diphosphate, such as selected from the group consisting of uridine diphosphate (UDP), guanosine diphosphate (GDP), and cytidine diphosphate (CDP). In a further embodiment, the sugar derivative Su(A)x is preferably derived from the group consisting of galactose (Gal), mannose (Man), N-acetylglucosamine (GlcNAc), glucose (Glc), N-acetylgalactosamine (GalNAc), glucuronic acid (Gcu), fucose (Fuc) and N-acetylneuraminic acid (sialic acid), in particular GlcNAc, Glc, Gal and GalNAc. In yet a further embodiment, Su(A)x is derived from Gal or GalNAc, in particular Su(A)x is derived from GalNAc.

[0102] In one embodiment, one or more functional groups A in Su(A)x are present on C2 and / or C6 of the sugar or sugar derivative Su.

[0103] When A is an azido group, A is typically attached at C2, C3, C4, or C6. As described above, one or more azido substituents in Su(A)x may be attached to C2, C3, C4, or C6 of the sugar or sugar derivative S, instead of a hydroxyl (OH) group, or, in the case of 6-azidofucose (6-AzFuc), instead of a hydrogen atom. Alternatively or in addition, the N-acetyl substituent of the amino sugar derivative may be replaced by an azidoacetyl substituent. In one embodiment, Su(A)x is 2-azidoacetamidogalactose (GalNAz), 6-azido-6-deoxygalactose (6-AzGal), 6-azido-6-deoxy-2-acetamidogalactose (6-AzGalNAc), 4-azido-4-deoxy-2-acetamidogalactose (4-AzGalNAc), 6-azido-6-deoxy-2-azidoacetamidogalactose (6-AzGalNAz), 2-azidoacetamidoglucose (GlcNAz), 6-azido -6-deoxyglucose (6-AzGlc), 6-azido-6-deoxy-2-acetamido glucose (6-AzGlcNAc), 4-azido-4-deoxy-2-acetamido glucose (4-AzGlcNAc), and 6-azido-6-deoxy-2-azidoacetamido glucose (6-AzGlcNAz), in particular from the group consisting of GalNAz, 6-AzGal, 6-AzGalNAc, 4-AzGalNAc, GlcNAz, and 6-AzGlcNAc.

[0104] When A is a keto group, typically A is attached to C2 instead of the OH group of Su. Alternatively, A may be attached to the N atom of an amino sugar derivative, preferably a 2-amino sugar derivative. The sugar derivative is then -NC(O)R 3 Contains the substituents. 3 is preferably an optionally substituted C2 to C4 alkyl group.

[0105] In one embodiment, R 3is an ethyl group. In a further embodiment, Su(A) is selected from the group consisting of 2-deoxy-(2-oxopropyl)galactose (2-ketoGal), 2-N-propionylgalactosamine (2-N-propionylGalNAc), 2-N-(4-oxopentanoyl)galactosamine (2-N-LevGal), and 2-N-butyrylgalactosamine (2-N-butyrylGalNAc), more preferably 2-ketoGalNAc and 2-N-propionylGalNAc.

[0106] When A is an alkynyl group, particularly a terminal alkynyl group or a (hetero)cycloalkynyl group, the alkynyl group is typically present on a 2-amino sugar derivative. An example of Su(A)x where A is an alkynyl group is 2-(but-3-yonic acid amide)-2-deoxy-galactose.

[0107] In certain embodiments, Su(A)xP is selected from the group consisting of GalNAz-UDP6-AzGal-UDP, 6-AzGalNAc-UDP, 4-AzGalNAz-UDP, 6-AzGalNAz-UDP, 6-AzGlc-UDP, 6-AzGlcNAz-UDP, 2-ketoGal-UDP, 2-N-propionyl-GalNAc-UDP, and 2-(but-3-yonic acid amide)-2-deoxy-galactose-UDP. Most particularly, S(A)xP is GalNAz-UDP, 4-AzGalNAc-UDP, or 6-AzGalNAc-UDP.

[0108] Several compounds of formula Su(A)xP, in which a nucleoside monophosphate or a nucleoside diphosphate P is linked to a sugar derivative Su(A)x, are known in the art. For example, Wang et al, Chem. Eur. J. 2010, 16, 13343-13345, Piller et al, ACS Chem. Biol. 2012, 7, 753, Piller et al, Bioorg. Med. Chem. Lett. 2005, 15, 5459-5462, WO 2009 / 102820, WO2014 / 065661, WO2015 / 057063, WO2015 / 057065, and WO2021 / 015622, all of which are incorporated herein by reference, disclose several compounds Su(A)xP and their synthesis.

[0109] Suitable conditions for the reaction of a core GlcNAc moiety with a sugar derivative containing a reactive group in the presence of GalT are described, for example, in EP 2,911,699, WO 2016 / 022027, and WO 2016 / 170186, all of which are incorporated herein by reference.

[0110] In one embodiment, where the process involves using enzymatic addition of a sugar derivative linked to a reactive group R to the core GlcNAc moiety, where the sugar derivative together with the reactive group R is the sugar derivative Su(A)xP as defined above, the resulting Fc-containing polypeptide, e.g., an antibody, comprises one or more of the following: (N)-GSR, where N is an asparagine residue present in the Fc region of the polypeptide, G is the GlcNAc N-glycan core, optionally fucosylated, S is a sugar derivative, R is a reactive group, and SR is the sugar derivative Su(A)x as defined above. The skilled artisan will understand that in this context, the nucleoside monophosphate or diphosphate P is not retained in the resulting Fc-containing polypeptide, such as an antibody, that contains the reactive group.

[0111] The resulting Fc-containing polypeptide, such as an antibody, containing a reactive group, such as an azide group, can then be conjugated to a second moiety of interest that contains an R' reactive group, such as a drug linker (e.g., as described above for the two-step process for the first drug linker).

[0112] In one embodiment, the resulting Fc-containing polypeptide conjugate, e.g., an antibody conjugate, comprises one or more of the following: (N)-GSR-R'-LD, or a pharma- ceutically 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-R' is the reaction product between reactive group R and complementary reactive group R', L is a linker, and D is a drug moiety.

[0113] To distinguish between the linker attached during the first conjugation and the linker attached during the second conjugation, the former may be referred to as LA and the latter as LB.

[0114] In one embodiment, SR is Su(A)x as defined above.

[0115] R-R', L (LA and LB), and D are as defined above for the first step of the process, where the glutamine residue is the conjugation site. For the first conjugation, D can be referred to as D1, and for the second conjugation, D can be referred to as D2. D1 and D2 can be the same or different in the two conjugations, i.e., the same to increase the drug-to-antibody ratio (DAR) for a particular drug, or different to allow simultaneous targeting of two different drugs to the same cell, such as drugs with different modes of action (e.g., a tubulin inhibitor and a DNA binder). In a preferred embodiment, the R-R' for the second conjugation is typically different from the R-R' for the first conjugation to avoid any cross-reactivity, i.e., for the first conjugation at the glutamine residue, R-R' is R1-R1' and for the second conjugation at the asparagine-GlcNAc, R-R' is R2-R2', where R1 does not react with R2' and R2 does not react with R1'.

[0116] Thus, in combination with the first process for the addition of a moiety of interest at the glutamine residue, in one embodiment the Fc-containing polypeptide conjugate, e.g. an antibody conjugate, is (i) (Q)-NH-LA-D1, or a pharma- ceutically acceptable salt or solvate thereof, where Q is a glutamine residue present in an Fc region of a polypeptide, LA is a linker, and D is a drug moiety; and (ii) (N)-GSR-R'-LB-D2, or a pharma- ceutically acceptable salt or solvate thereof, wherein N is an asparagine residue present in an Fc region of a polypeptide, G is a GlcNAc N-glycan core, optionally fucosylated, S is a sugar derivative, R-R' is the reaction product between reactive group R and complementary reactive group R', LB is a linker, and D is a drug moiety; The asparagine residue is adjacent to the glutamine residue.

[0117] Regarding (i), all acyl acceptor variants described above in relation to the TGase reaction are included.

[0118] When two sites are used, one based on using TGase at the glutamine residue and the other based on a GalT-mediated reaction at the asparagine-GlcNAc site, it is not necessary to conjugate the complete first moiety of interest construct prior to the GalT-mediated reaction. For example, TGase can be used to conjugate the acyl acceptor reactive moiety (R), and then a sugar derivative and a second reactive R group can be attached to the asparagine-GlcNAc N-glycan core using a GalT-mediated reaction. If the R and R' groups for the two sites are chosen to be different and not cross-reactive, the first and second moieties of interest can be added simultaneously, or sequentially if preferred, in a "one-pot" reaction.

[0119] Thus, the following intermediate can be produced, which is an Fc-containing polypeptide, such as an antibody: (i) (Q)-NH-L1-R1, where Q is a glutamine residue present in an Fc region of a polypeptide, L1 is absent or a linker or spacer moiety, such as PEG, and R1 is a reactive group; and (ii) (N)-GS-R2, where N is an asparagine residue present in an Fc region of a polypeptide, G is an optionally fucosylated GlcNAc N-glycan core, S is a sugar derivative, and R2 is a reactive group; The asparagine residue is adjacent to the glutamine residue.

[0120] This intermediate can then be reacted with (i) R1'-L2-D1 and (ii) R2'-L2-D2, where R1' and R2' are complementary reactive groups that react with R1 and R2, respectively, specifically, i.e., R1 does not react with R2' and R2 does not react with R1'. As an example, R1 can be tetrazine, R1' can be trans-cyclooctene (TCO), R2 can be azide, and R2' is dibenzocyclooctyne (DBCO).

[0121] This intermediate can be reacted with (i) R1'-L2-D1 and (ii) R2'-L2-D2, either simultaneously or sequentially, in either order.

[0122] "Pharmaceutically acceptable salt" is intended to mean a free acid or base salt of a compound of the present invention that is non-toxic, biologically acceptable, or otherwise biologically suitable for administration to a subject. See generally, GS Paulekuhn, et al., "Trends in Active Pharmaceutical Ingredient Salt Selection based on Analysis of the Orange Book Database", J.Med.Chem., 2007, 50:6665-72; SM Berge, et al., "Pharmaceutical Salts", J Pharm Sci., 1977, 66:1-19; and Handbook of Pharmaceutical Salts, Properties, Selection, and Use, Stahl and Wermuth, Eds., Wiley-VCH and VHCA, Zurich, 2002.

[0123] Examples of pharma- ceutically acceptable salts are those that are pharmacologically effective and suitable for contact with the tissues of a subject without undue toxicity, irritation, or allergic response. The compounds of the present invention may possess sufficiently acidic groups, sufficiently basic groups, or both types of functional groups, and thus may react with a number of inorganic or organic bases, and inorganic and organic acids, to form pharma- ceutically acceptable salts.

[0124] Pharmaceutically acceptable acid addition salts include those of inorganic and organic acids, such as acetate, aspartate, benzoate, besylate, bromide / hydrobromide, bicarbonate / carbonate, bisulfate / sulfate, camphorsulfonate, chloride / hydrochloride, chlortheophyllonate, citrate, ethanedisulfonate, fumarate, gluceptate, gluconate, glucuronate, hippurate, hydroiodide / iodide, isethionate, lactate, lactobionate, lauryl sulfate, phosphate, phosphate, phosphate-1, phosphate-2 ... It can be formed in the salts of goate, maleate, malonate, mandelate, mesylate, methylsulfate, naphthoate, napsylate, nicotinate, nitrate, octadecanoate, oleate, oxalate, palmitate, pamoate, phosphate / hydrogen phosphate / dihydrogen phosphate, polygalacturonate, propionate, stearate, succinate, sulfosalicylate, tartrate, tosylate, trifluoroacetate, and trifluoromethylsulfonate.

[0125] Inorganic acids from which salts can be derived include, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, etc. Organic acids from which salts can be derived include, for example, acetic acid, propionic acid, glycolic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, toluenesulfonic acid, trifluoromethylsulfonic acid, sulfosalicylic acid, etc. Pharmaceutically acceptable base addition salts can be formed with inorganic and organic bases.

[0126] Inorganic bases from which salts may be derived include, for example, ammonium salts and metals from columns I-XII of the periodic table, in certain embodiments, salts are derived from sodium, potassium, ammonium, calcium, magnesium, iron, silver, zinc, and copper, with particularly suitable salts including the ammonium, potassium, sodium, calcium, and magnesium salts.

[0127] Organic bases from which salts can be derived include, for example, primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, basic ion exchange resins, etc. Particular organic amines include isopropylamine, benzathine, cholate, diethanolamine, diethylamine, lysine, meglumine, piperazine, and tromethamine.

[0128] Examples of pharma- ceutically acceptable salts include, inter alia, sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate, monohydrogen-phosphate, dihydrogen-phosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caproate, heptanoate, propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, butyrate-1,4-dioate, hexaphosphate ... Examples of suitable salts include syn-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, sulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, gamma-hydroxybutyrate, glycolate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, and mandelate salts.

[0129] The compound of the present invention or its pharma-ceutically acceptable salt can be obtained as a solvate.Solvates include those formed from the interaction or complexation of the compound of the present invention with one or more solvents, either in solution or in solid or crystalline form.In some embodiments, the solvent is water, and then the solvate is a hydrate.

[0130] Pharmaceutical Compositions Conjugates disclosed herein that contain one or more pharma- ceutically active substances / drugs will typically be subjected to one or more purification steps, for example, to remove unused reactants and reaction by-products. Such purification techniques include one or more of affinity purification and size-exclusion purification. The resulting purified conjugates can then be combined with one or more pharma- ceutically acceptable carriers or diluents. These typically include one or more of a buffer to maintain pH at a desired level, such as pH 5 to pH 7, a detergent, such as polysorbate, and, particularly in the case of lyophilized formulations, a lyoprotectant, such as a sugar, e.g., sucrose.

[0131] The pharmaceutical compositions may be used to treat a disease or disorder in an animal or human subject, such as a proliferative disorder, e.g., cancer.

[0132] Specific embodiments [1] A method for conjugating a moiety of interest to an Fc-containing polypeptide (e.g., an antibody), comprising: (i) providing an Fc-containing polypeptide having an N-linked glycosylation at a site in the Fc region of the polypeptide adjacent to a site that contains a glutamine residue; (ii) treating the polypeptide with an enzyme to trim back N-linked glycans at sites of N-linked glycosylation to optionally fucosylated, core N-acetylglucosamine (GlcNAc) moieties; (iii)(1) reacting the polypeptide obtained in step (ii) with a moiety of interest that comprises an acceptor moiety that is a substrate for TGase in the presence of transglutaminase (TGase), such that the moiety of interest is attached to the polypeptide via a glutamine residue.

[0133] [2]. A method for conjugating a moiety of interest to an Fc-containing polypeptide (e.g., an antibody), comprising: (i) providing an Fc-containing polypeptide having an N-linked glycosylation at a site in the Fc region of the polypeptide adjacent to a site that contains a glutamine residue; (ii) treating the polypeptide with an enzyme to trim back N-linked glycans at sites of N-linked glycosylation to optionally fucosylated, core N-acetylglucosamine (GlcNAc) moieties; (iii)(2) reacting, in the presence of transglutaminase (TGase), the polypeptide resulting from step (ii) with an acceptor moiety that is a substrate for TGase, the acceptor moiety further comprising a reactive group (R) that allows for subsequent attachment of a moiety of interest via reaction between the reactive group (R) and a complementary reactive group (R') attached to the moiety of interest, such that the acceptor moiety is attached to the polypeptide via a glutamine residue; (iv) reacting the polypeptide conjugate formed in step (iii)(2) with a moiety of interest attached to R' under conditions such that R reacts with R' to form R-R'.

[0134] The method according to [1] or [2], wherein the acceptor moiety is selected from amine, aminooxy, hydrazide, hydrazino, and aryl derivatives thereof.[3].

[0135] [4]. The method according to [2] or [3], wherein the acceptor moiety is of the formula Ac-Sp-R, where Ac is selected from amine, aminooxy, hydrazide, hydrazino, and aryl derivatives thereof, Sp is absent or a spacer moiety, and R is a reactive group, such as azide.

[0136] [5]. The method according to any one of the preceding embodiments, wherein the enzyme is EndoS.

[0137] [6] The method according to any one of the preceding embodiments, wherein the N-linked glycosylation site is at position 297, or an equivalent position, according to the Kabat numbering system, and / or the site containing a glutamine residue is at position 295, or an equivalent position, according to the Kabat numbering system.

[0138] [7]. The method according to any one of the preceding embodiments, wherein the moiety of interest comprises a drug linker.

[0139] [8]. The second moiety of interest is conjugated to an Fc-containing polypeptide, and the method comprises the steps of: The method according to any one of the preceding embodiments, further comprising the step of: (v) reacting the polypeptide obtained in step (iii)(1), step (iii)(2), or step (iv) with a sugar derivative conjugated to a second moiety of interest in the presence of a galactosyltransferase (GalT), such that the sugar derivative is conjugated to the polypeptide via a core N-acetylglucosamine (GlcNAc) moiety.

[0140] [9]. The second moiety of interest is conjugated to an Fc-containing polypeptide, and the method comprises the steps of: (v) reacting, in the presence of a galactosyltransferase (GalT), the polypeptide resulting from step (iii))(1), step (iii)(2), or step (iv) with a sugar derivative attached to R that allows for subsequent attachment of a moiety of interest via reaction between the reactive group (R) and a complementary reactive group (R') attached to the moiety of interest, such that the sugar derivative is attached to the polypeptide via a core N-acetylglucosamine (GlcNAc) moiety; (vi) reacting the polypeptide conjugate formed in step (v) with a moiety of interest bound to R' under conditions such that R reacts with R' to form R-R'.

[0141]

[10] . (i) an asparagine residue present in an Fc region of a polypeptide having an optionally fucosylated, GlcNAc N-glycan core that lacks any additional endogenous glycan residues; and (ii) (Q)-NH-LD, or a pharma- ceutically acceptable salt or solvate thereof, wherein Q is a glutamine residue present in an Fc region of a polypeptide, L is a linker, optionally comprising a conditionally cleavable moiety, and D is a drug moiety; An Fc-containing polypeptide conjugate, wherein an asparagine residue is adjacent to a glutamine residue.

[0142]

[11] . L comprises L1-R-R'-L2, An Fc-containing polypeptide conjugate according to

[10] , wherein L1 is absent or a spacer moiety, R-R' is formed by reaction of a reactive group R with a complementary reactive group R', and L2 is a linker or spacer moiety to which D is attached and optionally comprising a conditionally cleavable moiety.

[0143]

[12] . (i) (Q)-NH-LA-D1, or a pharma- ceutically acceptable salt or solvate thereof, wherein Q is a glutamine residue present in an Fc region of a polypeptide, LA is a linker, and D is a drug moiety; (ii) (N)-GS-R2-R2'-LB-D2, or a pharma- ceutically acceptable salt or solvate thereof, wherein N is an asparagine residue present in an Fc region of a polypeptide, G is a GlcNAc N-glycan core, optionally fucosylated, S is a sugar derivative, R2-R2' is formed by reaction of reactive group R2 with a complementary reactive group R2', LB is a linker, optionally comprising a conditionally cleavable moiety, and D1 and D2 are each independently a drug moiety; An Fc-containing polypeptide conjugate, wherein an asparagine residue is adjacent to a glutamine residue.

[0144]

[13] LA comprises LA1-R1-R1'-LA2; An Fc-containing polypeptide conjugate according to

[12] , wherein LA1 is absent or a spacer moiety, R1-R1' is formed by reaction of reactive group R1 with a complementary reactive group R1', and LA2 is a linker or spacer moiety to which D1 is attached, optionally comprising a conditionally cleavable moiety.

[0145]

[14] . An Fc-containing polypeptide conjugate according to any one of

[10] to

[15] , wherein D is a cytotoxin or an immunostimulatory molecule.

[0146]

[15] . An Fc-containing polypeptide conjugate according to any one of

[10] to

[14] , wherein an asparagine residue is at position 297, or an equivalent position, according to the Kabat numbering system, and / or a glutamine residue is at position 295, or an equivalent position, according to the Kabat numbering system.

[0147] Certain aspects and embodiments of the present disclosure are described in more detail below with reference to the following examples, which are illustrative only and non-limiting.

[0148] For an example, see the diagram below. [Figure 1] - MTGase-mediated conjugation of amine-PEG(3u)-azide to antibodies deglycosylated using PNGase F (top) or glycans trimmed using EndoS (bottom). [Figure 2] Dual conjugation to an antibody functionalized with amine-PEG(4u)-MTZ via MTGase-mediated conjugation and GalNAz via glycan remodeling. The process was modeled using TCO-Cy5 and DBCO-AF488, conjugated with MTZ and azide functional groups, respectively. [Figure 3] - (A) Reduced PLRP of antibody X and antibody X dual-labeled with TCO-Cy5 and DBCO-AF488, (B) Reduced PLRP of antibody X dual-labeled with TCO-Cy5 and DBCO-AF488 with absorbance maxima at 280 nm (protein), 488 nm (AF488), and 651 nm (Cy5) overlay. [Figure 4] - (A) Doubly and singly labeled non-reduced PLRP of antibody X labeled with TCO-Cy5 and DBCO-AF488, (B) UV / Vis spectra of doubly and singly labeled antibody X. EXAMPLES

[0149] Example 1 - EndoS vs PNGase F before MTGase treatment The transamidation reaction between primary amine donors and the side chains of glutamine residues can be catalyzed by microbial transglutaminase. This biochemistry has been exploited for the conjugation of cytotoxic drugs to antibodies. IgG has a conserved site for transglutaminase-mediated conjugation at Q295. To ensure sufficient surface accessibility to Q295, antibodies are typically deglycosylated at the proximal N297 residue using PNGase F prior to MTGase-mediated conjugation. We have developed a new process in which the glycan at N297 is trimmed to the core GlcNAc[Fuc] using EndoS prior to MTGase conjugation of the azide-based acyl acceptor, amine-PEG(3u)-azide.

[0150] Materials and Methods PNGase deglycosylation of antibody X Antibody X (2.5 mg) was deglycosylated using PNGase F (379 kU) at an antibody concentration of 1.33 mg / mL and the digest was incubated for 18 h at 37° C. Deglycosylated antibody X was purified by Protein A chromatography followed by buffer exchange into PBS and concentration by UF / DF.

[0151] EndoS glycan trimming of antibody X Antibody X (2.5 mg) glycans were trimmed using EndoS (12.5 μg) at an antibody concentration of 1.0 mg / mL and the digest was incubated for 18 h at 37° C. Trimmed Antibody X was purified by Protein A chromatography followed by buffer exchange into PBS and concentration by UF / DF.

[0152] MTGase-mediated conjugation of linkers 1-4, 7, 8, and 11 Solutions of linkers 1-4, 7, 8, and 11 (Table 1) were prepared in PBS. Deglycosylated / trimmed antibody X samples (0.1 mg, 2 mg / mL) were then functionalized with linkers 1-4, 7, 8, and 11 (200 equivalents) via MTGase-mediated conjugation using 6.7 U of enzyme per mg of antibody. The resulting reactions were incubated at 37 °C for 18 h. The functionalized antibody X samples were purified by Protein A chromatography followed by buffer exchange into PBS and concentration via UF / DF. Conjugation efficiency for the purified samples was determined by LC-MS (Table 2). [Table 5]

[0153] result [Table 6] These results indicate that the process based on the EndoS process works well.

[0154] Example 2 - Conjugation of DBCO-PEG (5 kDa) and DBCO-Cy5 to an azide-functionalized antibody X sample To model the conjugation process, the functionalized antibody X sample from Example 1 was conjugated with DBCO-PEG (5 kDa) and DBCO-Cy5. Briefly, the functionalized antibody X sample (50 μg) was incubated with either DBCO-PEG (5 kDa) and DBCO-Cy5 (10 equivalents) at an antibody concentration of 0.5 mg / mL. The resulting reaction mixture was incubated at 22° C. for 19 hours. To assess the conjugation efficiency, the DBCO-PEG (5 kDa) conjugation was directly analyzed by SDS-PAGE. The DBCO-Cy5 conjugate was buffer exchanged into PBS via gel filtration and then assessed by LC-MS.

[0155] PEGylation of the heavy chain of antibody X was observed by reducing SDS-PAGE with a degree of PEGylation of 1.3 and 1.5 determined for PNGase F and EndoS, respectively. Cy5 conjugation was monitored by MS: the PNGase F process was complete at the DBCO-Cy5 conjugation step with 74% efficiency (DoL=1.5), whereas the EndoS process was efficient with 94% incorporation of DBCO-Cy5 observed (DoL=1.9).

[0156] Thus, the EndoS process was observed to be over 25% more efficient than the PNGase F process.

[0157] Example 3 - Dual labeling at Q295 / N297 with MTGase and galactosyltransferase The single conjugation approach in Examples 1 and 2 was combined with a second conjugation at the EndoS trimming site at N297.

[0158] Materials and Methods [ka] [ka] [ka]

[0159] EndoS glycan trimming and MTGase-mediated conjugation of amine-PEG(4u)-MTZ of antibody X Antibody X glycans were trimmed at 1 mg scale using EndoS according to Example 1. Trimmed antibody X (0.65 mg) was then functionalized with amine-PEG(4u)-MTZ (80 equiv.) via MTGase-mediated conjugation using 6 U / mL enzyme at an antibody concentration of 0.8 mg / mL. The reaction was incubated overnight at 37° C. Purification of the conjugate was performed by Protein A chromatography followed by buffer exchange into PBS and concentration via UF / DF.

[0160] Glycan remodeling for monofunctional antibody X Monofunctionalized antibody X (0.5 mg) was remodeled via GalT(Y289L) transfer of UDP-GalNAz using the SiteClick™ conjugation kit according to the manufacturer's instructions. Remodeling was performed overnight at 30° C. Purification of bifunctionalized antibody X was performed by UF / DF, buffer exchange into TBS, pH 7.

[0161] Conjugation of TCO-Cy5 and / or DBCO-AF488 to dual-functionalized antibody X Solutions of TCO-Cy5 and DBCO-AF488 in DMSO were prepared at a concentration of 10 mM. Bifunctional antibody X (0.1 mg) was labeled with TCO-Cy5 (10 eq.) and DBCO-AF488 (10 eq.) at an antibody concentration of 4 mg / mL. A control reaction in which only one fluorophore was added was also performed. The conjugation reaction was incubated overnight at 22° C. Purification of the conjugate was performed by Protein A chromatography followed by buffer exchange into PBS and concentration via UF / DF. The purified conjugate was analyzed by PLRP and LC-MS.

[0162] result Conjugation was confirmed by a shift in retention time of the heavy chain of the conjugate compared to the naked antibody using RP-UHPLC (Vanquish™). Absorbance at 651 nm (Cy5) and 488 nm (AF488) for the heavy chain confirmed conjugation with both fluorophores (Figure 3).

[0163] PLRP of the dual-labeled conjugates and single-labeled samples using UV / Vis spectroscopy clearly shows that the dual-labeled conjugates contain both fluorophores, while the single-labeled conjugates show only a single absorbance maximum for each fluorophore (Figure 4).

[0164] MS analysis showed that the incorporation level of amine-PEG(4u)-methyltetrazine was 90%. Glycan remodeling with GalNAz led to a complete reaction, and conjugation of TCO-Cy5 and DBCO-AF488 to their respective sites also led to complete conversion.

[0165] conclusion We have shown that using a combination of two conjugation techniques, we can efficiently use both the Q295 site and the N297 site in the same antibody to dual-label antibodies. This dual-labeling method can be used to create near-homogeneous antibody drug conjugates (ADCs) with two different cytotoxins attached to one antibody. As many systemic cancer treatments use drug combinations, the two mechanisms of action may improve treatment outcomes and reduce drug resistance. Homogeneity of the ADC has been shown to increase efficacy and safety, as suboptimal conjugate species deliver too little payload (low DAR variants) or potentially have suboptimal pharmacokinetics (high DAR variants). ********

[0166] Several publications are cited above in order to more fully describe and disclose the disclosure and the state of the art to which the invention herein may pertain. The entirety of each of the references mentioned in this disclosure is incorporated herein by reference.

Claims

1. 1. A method for conjugating a moiety of interest to an Fc-containing polypeptide (e.g., an antibody), comprising: (i) providing an Fc-containing polypeptide having an N-linked glycosylation at a site in the Fc region of said polypeptide adjacent to a site containing a glutamine residue; (ii) treating the polypeptide with an enzyme to trim back N-linked glycans at the sites of N-linked glycosylation to optionally fucosylated, core N-acetylglucosamine (GlcNAc) moieties; (iii) (1) reacting the polypeptide obtained in step (ii) with a moiety of interest comprising an acceptor moiety that is a substrate for transglutaminase (TGase) in the presence of said TGase, such that said moiety of interest is attached to said polypeptide via said glutamine residue.

2. 1. A method for conjugating a moiety of interest to an Fc-containing polypeptide (e.g., an antibody), comprising: (i) providing an Fc-containing polypeptide having an N-linked glycosylation at a site in the Fc region of said polypeptide adjacent to a site containing a glutamine residue; (ii) treating the polypeptide with an enzyme to trim back N-linked glycans at the sites of N-linked glycosylation to optionally fucosylated, core N-acetylglucosamine (GlcNAc) moieties; (iii)(2) reacting, in the presence of transglutaminase (TGase), the polypeptide obtained in step (ii) with an acceptor moiety that is a substrate for said TGase, said acceptor moiety further comprising a reactive group (R) that allows for subsequent attachment of said moiety of interest via a reaction between said reactive group (R) and a complementary reactive group (R') attached to said moiety of interest, such that said acceptor moiety is attached to said polypeptide via said glutamine residue; (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. 3. The method of claim 1 or 2, wherein the acceptor moiety is selected from amine, aminooxy, hydrazide, hydrazino, and aryl derivatives thereof.

4. 3. The method of claim 1 or 2, wherein the acceptor moiety is of the formula Ac-Sp-R, where Ac is selected from amine, aminooxy, hydrazide, hydrazino, and aryl derivatives thereof, Sp is absent or a spacer moiety, and R is a reactive group, e.g., azide.

5. The method of claim 1 or 2, wherein the enzyme is EndoS.

6. 3. The method of claim 1 or 2, wherein the site of N-linked glycosylation is at position 297, or an equivalent position, according to the Kabat numbering system, and / or the site containing a glutamine residue is at position 295, or an equivalent position, according to the Kabat numbering system.

7. The method of claim 1 or 2, wherein the moiety of interest comprises a drug linker.

8. A second moiety of interest is conjugated to said Fc-containing polypeptide, the method comprising the steps of:

3. The method of claim 1 or 2, further comprising: (v) reacting the polypeptide obtained in step (iii)(1), step (iii)(2), or step (iv) with a sugar derivative attached to the second moiety of interest in the presence of a galactosyltransferase (GalT), such that the sugar derivative is attached to the polypeptide via the core N-acetylglucosamine (GlcNAc) moiety.

9. A second moiety of interest is conjugated to said Fc-containing polypeptide, the method comprising the steps of: (v) reacting, in the presence of a galactosyltransferase (GalT), the polypeptide obtained in step (iii))(1), step (iii)(2), or step (iv) with a sugar derivative attached to R that allows for subsequent attachment of the moiety of interest via reaction between the reactive group (R) and a complementary reactive group (R') attached to the moiety of interest, such that the sugar derivative is attached to the polypeptide via the core N-acetylglucosamine (GlcNAc) moiety; (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. (i) an asparagine residue present in an Fc region of a polypeptide having an optionally fucosylated, GlcNAc N-glycan core that lacks any additional endogenous glycan residues; and (ii) (Q)-NH-L-D, or a pharma- ceutically acceptable salt or solvate thereof, wherein Q is a glutamine residue present in an Fc region of a polypeptide, L is a linker, optionally comprising a conditionally cleavable moiety, and D is a drug moiety; The Fc-containing polypeptide conjugate, wherein said asparagine residue is adjacent to said glutamine residue.

11. L, L １ -R-R'-L ２ Including, In the ceremony, L １ is absent or a spacer moiety, R-R' is formed by reaction of a reactive group R with a complementary reactive group R', and L ２ 11. The Fc-containing polypeptide conjugate of claim 10, wherein is a linker or spacer moiety to which D is attached, optionally comprising a conditionally cleavable moiety.

12. (i) (Q)-NH-LA-D1, or a pharma- ceutically acceptable salt or solvate thereof, wherein Q is a glutamine residue present in the Fc region of a polypeptide, LA is a linker, and D is a drug moiety; (ii) (N)-G-S-R ２ -R ２ or a pharma- ceutically acceptable salt or solvate thereof, wherein N is an asparagine residue present in an Fc region of a polypeptide, G is a GlcNAc N-glycan core, optionally fucosylated, S is a sugar derivative, R ２ -R ２ ' is a reactive group R ２ and a complementary reactive group R ２ ', where LB is a linker, optionally including a conditionally cleavable moiety, and D1 and D2 are each independently a drug moiety; The Fc-containing polypeptide conjugate, wherein said asparagine residue is adjacent to said glutamine residue.

13. LA, LA １ -R １ -R １ '-LA ２ Including, During the ceremony, L.A. １ is absent or a spacer moiety, R １ -R １ ' is a reactive group R １ and a complementary reactive group R １ ', which is formed by reaction with LA ２ is a linker or spacer moiety to which D1 is attached, optionally comprising a conditionally cleavable moiety.

14. 14. The Fc-containing polypeptide conjugate of any one of claims 10 to 13, wherein D is a cytotoxin or an immunostimulatory molecule.

15. 14. The Fc-containing polypeptide conjugate according to any one of claims 10 to 13, wherein said asparagine residue is at position 297, or an equivalent position, according to the Kabat numbering system, and / or said glutamine residue is at position 295, or an equivalent position, according to the Kabat numbering system.

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