Novel endo-β-N-acetylglucosaminidase

Abstract

The present invention provides: an endo-β-N-acetylglucosaminidase (Endo-Si) cloned from a bacterial strain belonging to Streptococcus iniae, and a mutant enzyme thereof; a gene encoding said enzyme; a recombinant plasmid; a transformant transformed by said plasmid, and a use thereof; and a method for producing a sugar chain-remodeling antibody or the like using said enzyme.　This polypeptide has an amino acid sequence set forth in amino acid numbers 34-928 in SEQ ID NO: 2, or an amino acid sequence including mutations into one or more amino acid moieties selected from the group consisting of 241st (D241), 190th (T190), 311st (Q311), and 360th (E360) in the amino acid sequence, and exhibits hydrolysis activity and / or sugar chain-transferring activity.

Description

[Technical Field]

[0001] The present invention relates to endo-β-N-acetylglucosaminidase (Endo-Si), a gene encoding the enzyme, a recombinant plasmid, a transformant transformed with the plasmid and its use, and a method for producing a glycan-remodeling antibody or the like using the enzyme. [Background technology]

[0002] Antibodies are glycoprotein molecules that have an N-linked glycan (N297-linked glycan) attached to the side chain of Asn at position 297 located in the Fc region of the heavy chain molecule. Antibodies are important molecules in basic research and medical fields, and research and development of antibodies as therapeutic antibodies has been actively progressing, and the various effects of glycans are being clarified (Non-Patent Document 1). Currently, the majority of therapeutic antibodies are IgG class molecules, and such antibodies are generally produced using cultured animal cells, such as CHO cells or NS0 cells. The N297-linked glycan of antibodies produced in these animal cells is a biantennary complex-type glycan, but is heterogeneous in terms of core fucose, terminal sialic and galactosyl groups, and bisecting GlcNAc (Non-Patent Document 2). It has been shown that the N297-linked glycans of antibodies significantly affect effector activities, including antibody ADCC (Antibody-Dependent Cell-Mediated Cytotoxicity) and CDC (Complement-Dependent Cytotoxicity) (Non-Patent Documents 3 and 4), and it has also been suggested that they may affect the serum half-life of antibodies (Non-Patent Document 5). Furthermore, it has been shown that antibodies in which the non-reducing terminus of the N297-linked glycans is 2,6-sialylated are the major active ingredients in intravenous immunoglobulin (IVIG) (Non-Patent Document 6). Furthermore, in therapeutic molecules, including IgG and Fc fragments, heterogeneity of the N297-linked glycans is thought to significantly affect the properties and quality of the active ingredient, and the possibility cannot be denied that even trace amounts of heterogeneously glycosylated molecules may significantly alter the properties of the final product.

[0003] Given this situation, technologies for homogenizing glycans are being developed for the production of glycoprotein molecules, including therapeutic antibodies and antibody Fc regions. Enzymatic transglycosylation is a known method for homogenizing glycans attached to glycoproteins (Non-Patent Documents 7-9). This is a multistep process consisting of cleavage of glycans (hydrolysis) and condensation of other glycans (transglycosylation) in an in vitro environment. For the purpose of N-glycan conversion in particular, a group of enzymes known as endo-β-N-acetylglucosaminidases (ENGases) are used. The required properties of these enzymes are: 1) the ability to hydrolyze complex glycans as a substrate specificity, and 2) the ability to transglycosylate specific structures. Known methods for glycosyltransferase reactions include a method in which a single ENGase is used to transfer a sugar chain whose reducing end has been oxazolined to a GlcNAc (N-acetylglucosamine) acceptor (Non-Patent Documents 7-8), and a one-pot method in which two types of ENGase are used to directly transfer a sugar chain to a GlcNAc acceptor (Non-Patent Document 9, Patent Document 1). ENGase has been isolated from various biological species, and wild-type or mutant enzymes are used depending on the type of sugar chain used as a substrate.

[0004] As ENGase, Endo-A (enzyme derived from Arthrobacter protophormiae) (Non-patent document 10), Endo-D (enzyme derived from Streptococcus pneumoniae) (Non-patent document 11), Endo-M (enzyme derived from Mucor hiemalis) (Non-patent document 12), Endo-H (Non-patent document 13), Endo-F2 (Flavobacterium Meningosepticum-derived enzyme), Endo-F3 (Flavobacterium meningosepticum-derived enzyme) (Non-patent Document 14), Endo-E (Enterococcus faecalis-derived enzyme) (Non-patent Document 15), Endo-S (Streptococcus pygenes-derived enzyme) (Non-Patent Document 16), Endo-Tsp1006 (derived from bacteria of the genus Tannerella), Endo-Tsp1263 (derived from bacteria of the genus Tannerella), Endo-Bno1263 (derived from bacteria of the genus Bacteroides), Endo-Tsp1457 (derived from bacteria of the genus Tannerella), Endo-Bac1008 (derived from bacteria of the genus Muribaculum), Endo-Tsp1603 (derived from bacteria of the genus Tannerella), Endo-Tsp1263 (derived from bacteria of the genus Tannerella) (Patent Document 2), endo-β-N-acetyltransferases derived from bacteria of the genus Sphingobacterium Endo-β-N-acetylglucosaminidase (ORF1152), endo-β-N-acetylglucosaminidase derived from Sphingobacterium bacteria (ORF1188), endo-β-N-acetylglucosaminidase derived from Sphingobacterium bacteria (ORF3046), endo-β-N-acetylglucosaminidase derived from Sphingobacterium bacteria (ORF3750), endo-β-N-acetylglucosaminidase derived from Cordyceps fungi, endo-β-N-acetylglucosaminidase derived from Beauveria fungi (Non-Patent Document 17), Endo-CC1 (enzyme derived from Coprinopsis cinerea), Endo-CC2 (enzyme derived from Coprinopsis cinerea) (Non-Patent Document 18), Endo-Om (enzyme derived from Ogataea minuta) (Non-Patent Document 19), Endo-CE (enzyme derived from CaenorhabditisEndo-Rp (enzyme derived from Rhizomucor pusillus) (Patent Document 3), Endo-S2 or Endo-S49 (Non-Patent Document 24), and the like are known.

[0005] Among these, EndoS (Non-Patent Document 25), EndoS2 (Non-Patent Document 26), and Endo-F3 (Non-Patent Document 27) are known as enzymes that have been confirmed to have both hydrolytic activity and transglycosylation activity using complex N297-linked glycans containing the core fucose of antibodies as substrates.

[0006] It is known that the hydrolysis activity of the mutant EndoS enzyme, EndoS D233Q, is suppressed to some extent, and that this mutant enzyme selectively undergoes transglycosylation under conditions in which a large amount of intermediates in which the reducing terminal of the sugar chain is oxazolinated is present in the reaction system (Patent Document 4, Non-Patent Document 8).

[0007] It is also known that introducing additional mutations into EndoS D233Q increases the transglycosylation activity or decreases the hydrolysis activity compared to the EndoS D233Q enzyme (Patent Document 5).

[0008] The EndoS2 mutant enzyme (D184Q) is known to exhibit increased transglycosylation activity and decreased hydrolysis activity compared to the wild-type EndoS2 enzyme (Patent Documents 6 to 8, Non-Patent Document 28).

[0009] The Endo-F3 mutant enzymes (D165A and D165Q) are known to have reduced hydrolytic activity and enhanced transglycosylation activity of sugar oxazolines compared to wild-type Endo-F3 enzymes. Furthermore, the Endo-F3 mutant enzymes can use bi- and tri-antennary oxazolines as substrates for transglycosylation reactions (Non-Patent Document 27).

[0010] Known ENGases used in the one-pot method for directly transferring sugar chains to GlcNAc acceptors include a combination of two enzymes: an EndoS / EndoS mutant enzyme and an Endo-M / Endo-M mutant enzyme or an Endo-CC / Endo-CC mutant enzyme (Patent Document 1, Non-Patent Document 9).

[0011] Streptococcus iniae (Non-Patent Document 29) is known as a fish pathogen, causing significant damage to farmed flounder and red sea bream in Japan (Non-Patent Document 30). For this reason, M Back Inie (Matsuken Pharmaceutical Co., Ltd.) is sold as an inactivated vaccine for flounder β-hemolytic streptococcosis. Streptococcus strains, which are pathogenic bacteria that have a serious impact on humans and the agriculture, forestry, and fisheries industries, have been extensively classified using genome analysis (Non-Patent Document 31). The presence of an ENGase sequence in S. iniae was known, but its enzyme activity had not yet been investigated (Non-Patent Document 24). [Prior art documents] [Patent documents]

[0012] [Patent Document 1] WO2018 / 003983 [Patent Document 2] JP2020-022440 [Patent Document 3] WO2018 / 101451 [Patent Document 4] WO2013 / 120066 [Patent Document 5] WO2017 / 010559 [Patent Document 6] WO2017 / 124084

Patent document 7

Patent document 8

Non-licensed literature

[0013] [Non-licensed document 1] Arnold JN, et al., Annu Rev Immunol. 2007, 25, 21-50 [Non-licensed document 2] Jefferis R, Biotechnol Prog. 2005, 21, 11-16 [Non-licensed document 3] Nimmerjahn F, et al., Nat Rev Immunol. 2008, 8, 34-47

Non-licensed Document 4

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Non-licensed Document 7

Non-licensed literature 9

Non-licensed literature 10

Non-licensed Document 11

Non-licensed Document 12

Non-licensed Document 13

Non-licensed Document 14

Non-licensed Document 15

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Non-licensed Document 19

Non-licensed Document 20

Non-licensed Document 21

Non-Patent Document 22

Non-Patent Document 23

Non-Patent Document 24

Non-Patent Document 25

Non-Patent Document 26

Non-Patent Document 27

Non-Patent Document 28

Non-Patent Document 29

Non-Patent Document 30

Non-Patent Document 31

Summary of the Invention

[0014] An object of the present invention is to provide a novel endo-β-N-acetylglucosaminidase having hydrolytic activity and / or transglycosylation activity on the N297-linked sugar chain of a glycoprotein. [Means for solving the problem]

[0015] As a result of extensive research aimed at solving the above-mentioned problems, the present inventors discovered that endo-β-N-acetylglucosaminidase (Endo-Si), cloned from a strain belonging to Streptococcus iniae, has hydrolytic activity against N297-linked glycans, and that by introducing a mutation into Endo-Si, the hydrolytic activity can be further suppressed and a certain level of transglycosylation activity can be maintained, thereby completing the present invention.

[0016] The present invention provides the following inventions. [1] A polypeptide having an amino acid sequence set forth in amino acid numbers 34 to 928 of SEQ ID NO: 2, or an amino acid sequence containing a mutation at one or more amino acid positions selected from the group consisting of amino acids 241 (D241), 190 (T190), 311 (Q311), and 360 (E360) in the amino acid sequence, and exhibiting glycosylation activity and / or transglycosylation activity. [2] The polypeptide of [1], characterized in that the mutations are present at 1 to 3 amino acid sites selected from the group consisting of amino acids D241, T190, Q311, and E360. [3] The polypeptide of [1] or [2], which has one or more mutations selected from the group consisting of the following (A) to (D): (A) In the amino acid sequence of SEQ ID NO: 2, the 241st amino acid (D241) is mutated to glutamine (D241Q), methionine (D241M), or alanine (D241A); (B) In the amino acid sequence of SEQ ID NO: 2, the 190th amino acid (T190) is mutated to glutamine (T190Q); (C) In the amino acid sequence of SEQ ID NO: 2, the 311th amino acid (Q311) is mutated to leucine (Q311L), and (D) In ​​the amino acid sequence of SEQ ID NO: 2, the 360th amino acid (E360) is mutated to glutamine (E360Q), alanine (E360A), asparagine (E360N), or aspartic acid (E360D). [4] One or more mutations selected from the group consisting of the following (A) to (D): a polypeptide according to any one of [1] to [3], (A) D241Q or D241M, (B) T190Q, (C)Q311L, and (D)E360Q. [5] (A) to (C) below Either a polypeptide according to any one of [1] to [4], comprising the amino acid sequence set forth in (A) an amino acid sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11; (B) an amino acid sequence having at least 90% homology or identity to an amino acid sequence other than the 241st, 190th, 311th, or 360th amino acids in each of the sequences of (A); or (C) An amino acid sequence in which one or more amino acids have been deleted, substituted, and / or added in the amino acid sequence other than the amino acid at position 241, 190, 311, or 360 in the sequence of (A). [6] Hydrolytic activity and / or activity against N-linked glycans or A polypeptide according to any one of [1] to [5], which exhibits transglycosylation activity. [7] The polypeptide of [6], wherein the N-linked glycan is an N-linked glycan in a glycoprotein. [8] The polypeptide of [6] or [7], wherein the glycoprotein is an antibody or a molecule containing the Fc region of an antibody (Fc region-containing molecule). [9] A polypeptide according to any one of [6] to [8], wherein the N-linked glycan is an N-linked glycan that binds to Asn at position 297 of the antibody (N297-linked glycan).

[10] The polypeptide of [9], wherein the non-reducing end of the N297-linked glycan is a complex glycan which may be chemically modified.

[11] The polypeptide of [9] or

[10] , wherein the N297-linked glycan is an N297-linked glycan in which fucose may be added to the core GlcNAc.

[12] A polynucleotide encoding any one of the polypeptides [1] to

[11] .

[13] An expression vector comprising the polynucleotide of

[12] .

[14] Host cells transformed with the expression vector of

[13] .

[15] A method for producing any one of the polypeptides [1] to

[11] , comprising the steps of culturing the host cell of

[14] and collecting the target polypeptide from the culture obtained in said step.

[16] A polypeptide obtained by the production method of

[15] .

[17] A method for producing an antibody or an Fc region-containing molecule thereof, characterized by reacting an acceptor molecule which is an antibody having a core GlcNAc to which fucose may be attached as an N297-linked glycan or an Fc region-containing molecule thereof with a glycan donor molecule containing GlcNAc whose reducing end is activated in the presence of any of the polypeptides [1] to

[11] .

[18] The method of producing

[17] , in which the GlcNAc having an activated reducing end is an oxazolinated GlcNAc.

[19] The method of producing

[17] or

[18] , wherein the sugar chain donor molecule is a complex sugar chain whose non-reducing end may be chemically modified.

[20] The method of any one of

[17] to

[19] , wherein the sugar chain donor molecule is SG(10)-Ox, MSG1(9)-Ox, MSG2(9)-Ox, or a mixture of MSG1(9)-Ox and MSG2(9)-Ox, each of which may have a chemically modified non-reducing end.

[21] The method according to any one of

[17] to

[20] , wherein the sugar chain donor molecule is [N3-PEG(3)]2-SG(10)-Ox, [N3-PEG(3)]-MSG1(9)-Ox, [N3-PEG(3)]-MSG2(9)-Ox, or a mixture of [N3-PEG(3)]-MSG1(9)-Ox and [N3-PEG(3)]-MSG2(9)-Ox.

[22] The method of

[21] further comprising a step of reacting the azide group (N3-) with a molecule having an alkyne structure.

[23] Molecules with alkyne structures are used in a wide range of applications, including chemotherapeutic agents, molecular targeted drugs, immune activators, toxins, antibacterial agents, antiviral agents, diagnostic agents, proteins, peptides, amino acids, nucleic acid molecules,

[22] A method for producing a compound selected from nucleic acids, antigens, lipids, liposomes, vitamins, and hormones.

[24] The method of

[23] , wherein the chemotherapeutic agent is selected from camptothecin, pyrrolobenzodiazepine, doxorubicin, auristatin, taxane, or a derivative thereof.

[25] The manufacturing method of

[23] , wherein the immune activator is selected from STING agonists, TLR agonists, A2AR antagonists, IDO inhibitors, antagonists of the CTLA-4, LAG-3 and PD-1 pathways, checkpoint inhibitors, vascular endothelial growth factor (VEGF) receptor inhibitors, smoothen inhibitors, alkylating agents, antimetabolites, retinoids, anti-cancer vaccines, and adjuvants.

[26] The method according to any one of

[23] to

[25] , wherein the molecule having an alkyne structure is selected from the group consisting of (A) to (E). (A) N-[4-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]glycylglycyl-L-valyl-N-{4-[({[(11'S,11a'S)-11'-hydroxy-7'-methoxy-8'-[(5-{[(11aS)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,10,11,11 a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl]oxy}pentyl)oxy]-5'-oxo-11',11a'-dihydro-1'H-spiro[cyclopropane-1,2'-pyrrolo[2,1-c][1,4]benzodiazepin]-10'(5'H)-yl]carbonyl}oxy)methyl]phenyl}-L-alaninamide, (B) N-[4-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]glycylglycyl-L-valyl-N-[4-({[(11'S,11'aS)-11'-hydroxy-7'-methoxy-8'-(3-{[(11aS)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,10,11,11 a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl]oxy}propoxy)-5'-oxo-11',11'a-dihydro-1'H,3'H-spiro[cyclopropane-1,2'-pyrrolo[2,1-c][1,4]benzodiazepine]-10'(5'H)-carbonyl]oxy}methyl)phenyl]-L-alaninamide, (C) N-[4-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]glycylglycyl-L-valyl-N-{4-[({[(11'S,11a'S)-11'-hydroxy-7'-methoxy-8'-[(5-{[(11a'S)-7'-methoxy-5'-oxo-5',11a'-dihydro-1'H-spiro[cyclo cyclopropane-1,2'-pyrrolo[2,1-c][1,4]benzodiazepine]-8'-yl]oxy}pentyl)oxy]-5'-oxo-11',11a'-dihydro-1'H-spiro[cyclopropane-1,2'-pyrrolo[2,1-c][1,4]benzodiazepine]-10'(5'H)-yl]carbonyl}oxy)methyl]phenyl}-L-alaninamide, (D) N-[4-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]glycylglycyl-L-valyl-N-{4-[({[(11'S,11a'S)-11'-hydroxy-7'-methoxy-8'-[(5-{[(11a'S)-7'-methoxy-5'-oxo-5',10',11',11a'-tetrahydro-1'H-spiro [cyclopropane-1,2'-pyrrolo[2,1-c][1,4]benzodiazepine]-8'-yl]oxy}pentyl)oxy]-5'-oxo-11',11a'-dihydro-1'H-spiro[cyclopropane-1,2'-pyrrolo[2,1-c][1,4]benzodiazepine]-10'(5'H)-yl]carbonyl}oxy)methyl]phenyl}-L-alaninamide, and (E) (Bis(N,N-diethylethanaminium) N-[4-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]glycylglycyl-L-phenylalanyl-N-[(2-{9-[(5R,7R,8R,12aR,14R,15R,15aR,16R)-15-fluoro-16-hydroxy-2,10-dioxo-2,10-disulfide-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5-Furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-1-yl}ethoxy)methyl]glycinamide.

[27] The method for producing any one of

[17] to

[26] , wherein the acceptor molecule is an antibody or an Fc region-containing molecule having an N297-linked glycan consisting of a core GlcNAc optionally having fucose attached thereto.

[28] In the presence of endo-β-N-acetylglucosaminidase (enzyme A (also referred to as Enzyme A)) which uses the complex glycan of any of the polypeptides [1] to

[11] and the glycan donor molecule whose reducing end is not activated as a substrate, but does not use the N297-linked glycan as a substrate, A method for producing an antibody or an Fc region-containing molecule, characterized by reacting an acceptor molecule that is an antibody having a core GlcNAc that may have fucose attached as the N297-linked glycan or an Fc region-containing molecule thereof with a glycan donor molecule that contains a GlcNAc whose reducing end is not activated.

[29] A method for producing a saccharide-donor molecule comprising reacting a polypeptide according to any one of [1] to

[11] , an enzyme A, an acceptor molecule, and a sugar chain donor molecule in the same reaction solution. 28 ] manufacturing method.

[30] The method of producing

[28] or

[29] , wherein the glycan donor molecule is a complex glycan whose non-reducing end may be chemically modified.

[31] A method according to any one of

[28] to

[30] , wherein the glycan donor molecule is SGP, (SG-)Asn, (MSG1-)Asn, (MSG2-)Asn, or a mixture of (MSG1-)Asn and (MSG2-)Asn, each of which may have a chemically modified non-reducing end.

[32] The method of any one of

[28] to

[31] , wherein the sugar chain donor molecule is ([N3-PEG(3)]2-SG-)Asn-PEG(3)-N3, ([N3-PEG(3)]-MSG1-)Asn-PEG(3)-N3, ([N3-PEG(3)]-MSG2-)Asn-PEG(3)-N3, or a mixture of ([N3-PEG(3)]-MSG1-)Asn-PEG(3)-N3 and ([N3-PEG(3)]-MSG2-)Asn-PEG(3)-N3.

[33] The method of

[32] further comprising a step of reacting the azide group (N3-) with a molecule having an alkyne structure.

[34] The method of

[33] , wherein the molecule having an alkyne structure is selected from chemotherapeutic agents, molecular targeted drugs, immune activators, toxins, antibacterial agents, antiviral agents, diagnostic agents, proteins, peptides, amino acids, nucleic acids, antigens, lipids, liposomes, vitamins, and hormones.

[35] The method of

[34] , wherein the chemotherapeutic agent is selected from camptothecin, pyrrolobenzodiazepine, doxorubicin, auristatin, taxane, or a derivative thereof.

[36] The manufacturing method of

[34] , wherein the immune activator is selected from STING agonists, TLR agonists, A2AR antagonists, IDO inhibitors, antagonists of the CTLA-4, LAG-3 and PD-1 pathways, checkpoint inhibitors, vascular endothelial growth factor (VEGF) receptor inhibitors, smoothen inhibitors, alkylating agents, antimetabolites, retinoids, anti-cancer vaccines, and adjuvants.

[37] The method for producing any one of

[34] to

[36] , wherein the molecule having an alkyne structure is selected from the group consisting of (A) to (E). (A) N-[4-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]glycylglycyl-L-valyl-N-{4-[({[(11'S,11a'S)-11'-hydroxy-7'-methoxy-8'-[(5-{[(11aS)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,10,11,11 a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl]oxy}pentyl)oxy]-5'-oxo-11',11a'-dihydro-1'H-spiro[cyclopropane-1,2'-pyrrolo[2,1-c][1,4]benzodiazepin]-10'(5'H)-yl]carbonyl}oxy)methyl]phenyl}-L-alaninamide, (B) N-[4-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]glycylglycyl-L-valyl-N-[4-({[(11'S,11'aS)-11'-hydroxy-7'-methoxy-8'-(3-{[(11aS)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,10,11,11 a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl]oxy}propoxy)-5'-oxo-11',11'a-dihydro-1'H,3'H-spiro[cyclopropane-1,2'-pyrrolo[2,1-c][1,4]benzodiazepine]-10'(5'H)-carbonyl]oxy}methyl)phenyl]-L-alaninamide, (C) N-[4-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]glycylglycyl-L-valyl-N-{4-[({[(11'S,11a'S)-11'-hydroxy-7'-methoxy-8'-[(5-{[(11a'S)-7'-methoxy-5'-oxo-5',11a'-dihydro-1'H-spiro[cyclo cyclopropane-1,2'-pyrrolo[2,1-c][1,4]benzodiazepine]-8'-yl]oxy}pentyl)oxy]-5'-oxo-11',11a'-dihydro-1'H-spiro[cyclopropane-1,2'-pyrrolo[2,1-c][1,4]benzodiazepine]-10'(5'H)-yl]carbonyl}oxy)methyl]phenyl}-L-alaninamide, (D) N-[4-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]glycylglycyl-L-valyl-N-{4-[({[(11'S,11a'S)-11'-hydroxy-7'-methoxy-8'-[(5-{[(11a'S)-7'-methoxy-5'-oxo-5',10',11',11a'-tetrahydro-1'H-spiro [cyclopropane-1,2'-pyrrolo[2,1-c][1,4]benzodiazepine]-8'-yl]oxy}pentyl)oxy]-5'-oxo-11',11a'-dihydro-1'H-spiro[cyclopropane-1,2'-pyrrolo[2,1-c][1,4]benzodiazepine]-10'(5'H)-yl]carbonyl}oxy)methyl]phenyl}-L-alaninamide, and (E) (Bis(N,N-diethylethanaminium) N-[4-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]glycylglycyl-L-phenylalanyl-N-[(2-{9-[(5R,7R,8R,12aR,14R,15R,15aR,16R)-15-fluoro-16-hydroxy-2,10-dioxo-2,10-disulfide-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ 5 ,10λ 5 -Furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-1-yl}ethoxy)methyl]glycinamide.

[38] The method for producing any one of

[28] to

[37] , wherein the acceptor molecule is an antibody having an N297-linked glycan consisting of a core GlcNAc optionally containing fucose, or an Fc region-containing molecule.

[39] The method of any one of

[28] to

[38] , wherein Enzyme A is an enzyme having glycosylation activity from SGP to an acceptor having GlcNAc.

[40] The method of any one of

[28] to

[39] , wherein enzyme A is Endo-M, Endo-Rp, Endo-Om, Endo-CC, or a mutant enzyme thereof with reduced hydrolytic activity.

[41] Mutant enzymes with reduced hydrolytic activity were identified as Endo-Rp N172Q, Endo-Rp N172H, Endo-Rp N172A, Endo-Rp N172C, Endo-Rp N172D, Endo-Rp N172E, Endo-Rp N172G, Endo-Rp N172I, Endo-Rp N172L, Endo-Rp N172M, Endo-Rp N172P, Endo-Rp N172S, Endo-Rp N172T, Endo-Rp N172V, Endo-Rp W278F / S216V, Endo-Rp W278F / N246D, Endo-Rp W278F / D276N, Endo-Rp W278F / A310D, and Endo-Rp

[40] The method for producing Endo-Rp W278F / N172D / F307Y, Endo-Rp W278F / N172D / F307H, Endo-Rp W278F / N172D / A310D, Endo-Rp W214F / F307Y / L306I, Endo-M N175Q, Endo-CC N180H, and Endo-Om N194Q.

[42] An antibody or Fc region-containing molecule obtained by any of the production methods described in

[17] to

[41] .

[43] A method for producing an antibody or Fc region-containing molecule having only a core GlcNAc, optionally with fucose attached, characterized by reacting an antibody or Fc region-containing molecule with a polypeptide having an amino acid sequence set forth in amino acid numbers 34 to 928 of SEQ ID NO: 2.

[44] An antibody or Fc region-containing molecule having only core GlcNAc, obtained by the production method of

[43] .

[0017] This specification includes the disclosure of Japanese Patent Application No. 2020-147745, from which the present application claims priority. [Effects of the Invention]

[0018] The Endo-Si enzyme of the present invention has excellent hydrolytic activity and can act on N-linked glycans containing the N297 bond of glycoproteins, efficiently cleaving the β1,4-glycosidic bond between GlcNAc in the core chitobiose structure present in the glycan. The released glycans can be used as samples for glycoprotein glycan structural analysis and as raw materials for glycan derivatives. When glycoproteins are used as substrates, the glycoproteins whose glycans have been hydrolyzed can be used as acceptor molecules for glycan remodeling.

[0019] Furthermore, compared with wild-type Endo-Si, Endo-Si mutant enzymes with mutations introduced into them have reduced hydrolytic activity and enhanced transglycosylation activity, making it possible to efficiently obtain antibodies or glycosylated molecules (including Fc region-containing molecules) with uniform glycosylation by glycoremodeling, with high purity. Therefore, it is also possible to reduce the molecular weight of the glycosyl donor used in glycoremodeling, leading to reduced production costs for glycoremodeled antibodies or glycosylated molecules. [Brief explanation of the drawings]

[0020] [Figure 1] The structural formula of [N3-PEG(3)]-MSG1(9)-Ox is shown. [Figure 2] The structural formula of SGP is shown below. [Figure 3] 1 is a graph showing the time course of the hydrolysis activity of Endo-Si (◯) and Endo-S (X) against Trastuzumab (mAb1). The X axis represents the time elapsed after the start of the reaction, and the Y axis represents the rate of glycosylation. [Figure 4] FIG. 1 is a schematic diagram of the hydrolysis reaction of the N297-linked glycan of an antibody using Endo-Si or an Endo-Si mutant enzyme. [Figure 5] FIG. 1 is a schematic diagram of the glycosylation reaction using an oxazoline sugar donor and Endo-Si or an Endo-Si mutant enzyme. [Figure 6] FIG. 1 shows the sequence of SEQ ID NO: 1 (Endo-Si base sequence). [Figure 7] FIG. 1 shows the sequence of SEQ ID NO: 2 (Endo-Si amino acid sequence). [Figure 8] FIG. 1 shows the sequence of SEQ ID NO: 3 (Endo-Si amino acid sequence D241Q). [Figure 9] FIG. 1 shows the sequence of SEQ ID NO: 4 (Endo-Si amino acid sequence D241Q / Q311L). [Figure 10] FIG. 1 shows the sequence of SEQ ID NO: 5 (Endo-Si amino acid sequence D241Q / E360Q). [Figure 11] FIG. 1 shows the sequence of SEQ ID NO: 6 (Endo-Si amino acid sequence D241M). [Figure 12] FIG. 1 shows the sequence of SEQ ID NO: 7 (Endo-Si amino acid sequence D241M / Q311L). [Figure 13] FIG. 1 shows the sequence of SEQ ID NO: 8 (Endo-Si amino acid sequence D241M / E360Q). [Figure 14] FIG. 1 shows the sequence of SEQ ID NO: 9 (Endo-Si amino acid sequence T190Q / D241Q). [Figure 15] FIG. 1 shows the sequence of SEQ ID NO: 10 (Endo-Si amino acid sequence T190Q). [Figure 16] FIG. 1 shows the sequence of SEQ ID NO: 11 (Endo-Si amino acid sequence T190Q / D241M). [Figure 17] Schematic diagram of the glycosylation reaction using SGP as a donor, Endo-Si mutant enzymes, and enzyme A. [Figure 18] FIG. 1 is a diagram showing the relationship between the reaction temperature of Endo-Si and EndoS and the rate of glycosylation. [Figure 19] FIG. 1 shows the relationship between the reaction pH of Endo-Si and EndoS and the rate of glycosylation. [Figure 20] FIG. 1 shows a comparison of the hydrolytic activity of Endo-Si, EndoS, and PNGaseF against various antibodies. [Figure 21]Schematic diagram of the glycosylation reaction using ([N3-PEG(3)]-MSG1-)Asn-PEG(3)-N3 as a donor, Endo-Si mutant enzymes, and enzyme A. [Figure 22] Schematic diagram of the glycosylation reaction using ([N3-PEG(3)]2-SG-)Asn-PEG(3)-N3 as a donor, Endo-Si mutant enzymes, and enzyme A. DETAILED DESCRIPTION OF THE INVENTION

[0021] The present invention will be described in detail below.

[0022] Herein, the notation of amino acids contained in a molecule follows the convention in this field, and when indicating a mutation site, it is represented by the single-letter code of the wild-type amino acid (or nucleic acid) and its number (for example, Asp at position 241 is represented as "D241"). Note that, herein, a mutation at an amino acid site refers to the substitution, deletion, insertion, or addition of an amino acid, and preferably refers to the substitution of an amino acid. Furthermore, a mutation is represented by the single-letter code of the wild-type amino acid (or nucleic acid), its number, and the single-letter code of the amino acid (or nucleic acid) after the mutation (for example, a mutation in which Asp at position 241 is replaced with Gln is represented as "D241Q"). Furthermore, specific mutant enzymes having mutations are represented by the molecular name and the mutation (for example, a mutant enzyme in which Asp at position 241 of Endo-Si is replaced with Gln is referred to as "Endo-Si D241Q"). When multiple mutations are present, the mutations are separated by a " / " (for example, a mutant enzyme in Endo-Si D241Q that has an additional mutation in which Gln at position 241 is replaced with Leu is referred to as "Endo-Si D241Q / Q311L").

[0023] In the present invention, the term "N297-linked glycan" refers to an N-linked glycan bound to the side chain of Asn at position 297 of the IgG heavy chain. When IgG is fragmented, even glycans bound to the corresponding Asn in peptide fragments containing the Asn are included in the N297-linked glycan. Generally, the N297-linked glycan in IgG produced in animals or the like has a basic structure consisting of the structure of formula (I) or (II) below, and its non-reducing end may be further chemically modified, for example, by the addition of galactose (Gal) or sialic acid (Sia).

[0024] [ka]

[0025] [ka] Many of the N297-linked glycans of IgG produced by cells have diverse glycan structures, including those with additional glycans attached to the reducing terminal GlcNAc (core GlcNAc), non-reducing terminals, and branched sugars. The core GlcNAc may be modified at position 6 with fucose (Fuc) linked to it in an α1,6-linked structure ((Fucα1,6)GlcNAc). In the case of the branched sugar Man, a triantennary glycan may be formed by further attaching a glycan containing GlcNAc to position 5. The non-reducing terminal GlcNAc may also be further attached with a glycan containing galactose or sialic acid.

[0026] In the present invention, sialyl glycan (hereinafter referred to as "SG") has a basic structure consisting of the following structural formula and sequence formula:

[0027] A representative example of SG is the sugar chain contained in sialyl glycopeptide (hereinafter referred to as "SGP") contained in the yolk of chicken eggs.

[0028] [ka]

[0029] [ka] (In the formula, "-(N / Q)" indicates an N-glycosidic bond with the side chain of Asn or Gln.) Disialooctasaccharide (Tokyo Chemical Industry Co., Ltd.) consisting only of a sugar chain in which one GlcNAc is missing from the reducing end of the sugar chain portion of SG (hereinafter referred to as "SG(10)") is commercially available. In this specification, the sugar chain structure in which sialic acid is missing from the non-reducing end of only one of the β-mannose (β-Man) branches of SG(10) is referred to as MSG(9), and one having sialic acid only on the 1-3 sugar chain of the branched chain will be referred to as MSG1(9), and one having sialic acid only on the 1-6 sugar chain of the branched chain will be referred to as MSG2(9) (Patent Document 1, WO2019 / 065964).

[0030] In the present invention, a "glycan donor molecule" refers to a glycan-containing molecule having an activated GlcNAc at the reducing end of the glycan, preferably an oxazolinated GlcNAc, and molecules with a variety of glycan structures can be used. Activation refers to a state in which the reactivity of the anomeric sugar position is enhanced, including oxazolination or halogenation. Examples of glycan donor molecules include [N3-PEG(3)]-MSG1(9)-Ox (Figure 1), SG(9)-Ox (oxazoline), MSG1(9)-Ox, MSG2(9)-Ox, or a mixture of MSG1(9)-Ox and MSG2(9)-Ox.

[0031] Another embodiment of the glycan donor molecule is a glycan-containing molecule having an inactivated GlcNAc at the reducing end of the glycan, preferably SGP (Figure 2), (SG-)Asn, (MSG1-)Asn, (MSG2-)Asn, or a mixture of (MSG1-)Asn and (MSG2-)Asn.

[0032] In the present invention, unless otherwise specified, a partial structure in which an amino acid is linked to a sugar chain at its side chain will be represented by placing the side chain portion in parentheses, for example, as "(SG-)Asn."

[0033] The sugar chain donor molecule may be chemically modified, and includes, for example, SGP with a chemically modified non-reducing end, (SG-)Asn, (MSG1-)Asn, (MSG2-)Asn, a mixture of (MSG1-)Asn and (MSG2-)Asn, SG(10)-Ox, MSG1(9)-Ox, MSG2(9)-Ox, or a mixture of MSG1(9)-Ox and MSG2(9)-Ox. Preferably, ([N3-PEG(3)]2-SG(10))-Ox, [N3-PEG(3)]-MSG1(9)-Ox, [N3-PEG(3)]-MSG2(9)-Ox, or a mixture of [N3-PEG(3)]-MSG1(9)-Ox and [N3-PEG(3)]-MSG2(9)-Ox, or ([N3-PEG(3)]2-SG-)Asn-PEG(3)-N3, ( Examples include [N3-PEG(3)]-MSG1-)Asn-PEG(3)-N3, ([N3-PEG(3)]-MSG2-)Asn-PEG(3)-N3, or a mixture of ([N3-PEG(3)]-MSG1-)Asn-PEG(3)-N3 and ([N3-PEG(3)]-MSG2-)Asn-PEG(3)-N3 (Patent Document 1, WO2019 / 065964).

[0034] When used for glycan remodeling for drug discovery, it is preferable to use glycan donors with human-type or human-compatible glycans, which pose few problems when applied to humans. Such glycans are known to be non-antigenic in the human body, and N-linked glycans include high-mannose, hybrid, and complex types. These three types share a common basic structure. High-mannose glycans have a mannose-rich structure in which multiple mannoses are consecutively attached to two branched chains (1-3 chain and 1-6 chain) branched from a mannose (β-mannose) located near the reducing end. Hybrid glycans have a structure in which one of the two branched chains (1-3 chain and 1-6 chain) branched from a mannose (β-mannose) located near the reducing end contains GlcNAc. Complex glycans have a structure in which GlcNAc is attached to two branched chains (1-3 chain and 1-6 chain) branching from the mannose (β-mannose) located near the reducing end, and have a variety of structures including the presence or absence of galactose, the presence or absence of sialic acid, and the bond and positional isomerism of these. Complex glycans are known to be biantennary, triantennary, and tetraantennary.

[0035] Examples of high mannose, hybrid and complex structures are shown below.

[0036] [ka] Types of human N-linked glycans In the present invention, an "acceptor molecule" refers to a molecule containing a sugar structure with GlcNAc at the non-reducing end. When the acceptor molecule is reacted with a sugar chain donor molecule in the presence of Endo-Si or its mutant enzyme, the oxazoline ring or an active intermediate (Non-Patent Document 9) of the sugar chain donor molecule reacts with position 4 of the GlcNAc at the non-reducing end, forming a chitobiose structure.

[0037] A typical acceptor molecule is an IgG or its Fc fragment derived from a monoclonal antibody, which has an N297-linked glycan consisting only of a core GlcNAc, optionally linked to core Fuc. The core GlcNAc may or may not be linked to core Fuc, depending on the antibody from which it is derived and the method for producing it. Acceptor molecules can be derived from a variety of monoclonal antibodies, glycan-containing molecules, or Fc region-containing molecules (e.g., Fc, CLCH, which combines CH consisting only of the constant region obtained by deleting the variable region from the heavy chain with CL consisting only of the constant region from the light chain), but preferred examples include (Fucα1,6)-GlcNAc-IgG (e.g., (Fucα1,6)GlcNAc-mAb1 in Figure 4), (Fucα1,6)-GlcNAc-Fc, and (Fucα1,6)-GlcNAc-CLCH (Patent Document 1).

[0038] In the present invention, "Endo-Si" refers to a type of endo-β-N-acetylglucosaminidase (ENGase) derived from Streptococcus iniae, and its nucleotide sequence is shown in SEQ ID NO: 1, and its amino acid sequence is shown in SEQ ID NO: 2. Endo-Si is an enzyme (EC 3.2.1.96, GH18) consisting of an amino acid sequence in which the 241st amino acid in the amino acid sequence of amino acid numbers 34 to 928 of SEQ ID NO: 2 (amino acids 1 to 33 represent a signal sequence. SignalP-5.0, a tool provided by CBS, was used to predict the signal sequence), is Asp. Endo-Si specifically recognizes N-linked glycans (e.g., N297-linked glycans) and possesses both hydrolysis activity and transglycosylation activity.

[0039] The hydrolytic activity of Endo-Si specifically hydrolyzes the β1,4 glycosidic bond contained in the core chitobiose of the N-linked glycan having the basic structure described above (unless otherwise specified, the term "hydrolytic activity" refers to this activity in this specification. A reaction diagram is shown in Figure 4).

[0040] The transglycosylation activity of Endo-Si is the activity of glycosidically linking the reducing end of the above-mentioned glycan donor molecule (a glycan-containing molecule having a reducing-end activated GlcNAc or a reducing-end inactivated GlcNAc) to an acceptor molecule containing an Fc moiety with only a core GlcNAc (which may or may not have a core fucose attached) at N297 (hereinafter referred to as "transglycosylation activity"; a reaction scheme is shown in Figure 5 or Figure 17).

[0041] The substrate specificity of Endo-Si for various antibodies is as follows: It exhibits glycosylation activity for all four IgG subclasses, but not for IgA or IgE. Furthermore, its substrate specificity for various N-linked glycans is as follows: It exhibits glycosylation activity for both high-mannose glycans and complex-type biantennary glycans, but has higher specificity for complex-type biantennary glycans than for high-mannose glycans, with the highest hydrolysis activity for G0 glycans. Furthermore, it exhibits hydrolysis activity for sialyl glycans and fucosylated glycans, but does not exhibit hydrolysis activity for complex-type triantennary glycans.

[0042] The G0 sugar chain is a biantennary complex sugar chain in which the non-reducing end is GlcNAc bound to two branched chains (1-3 chain, 1-6 chain) and does not contain any galactose residues.

[0043] The enzyme of the present invention is not limited to the enzymes having the specific sequences obtained in the examples, as long as it has the above-mentioned properties, and may be an enzyme isolated from nature, or an enzyme artificially produced or modified based on the sequence information of the enzyme of the present invention. When isolated from nature, the biological species from which it is isolated is not particularly limited, but is preferably a bacterium, more preferably a bacterium of the genus Streptococcus, and even more preferably a bacterium belonging to Streptococcus iniae.

[0044] Based on sequence comparison with EndoS, whose crystal structure has been analyzed (B. Trastoy et al., PNAS (2014) Vol. 111, No. 18, pp. 6714-6719), the active domain and carbohydrate-binding module (CBM) of Endo-Si are presumed to be the regions of amino acids 106 to 447 and 762 to 897 of SEQ ID NO: 2, respectively. These two regions are considered to be important sites for hydrolytic activity and / or transglycosylation activity and for antibody interaction. Therefore, examples of the enzyme of the present invention include polypeptides that contain the amino acid sequence of amino acids 106 to 447 and / or 762 to 897 of SEQ ID NO: 2, preferably the amino acid sequence of amino acids 106 to 897 of SEQ ID NO: 2, more preferably the amino acid sequence of amino acids 106 to 928 of SEQ ID NO: 2, and even more preferably the amino acid sequence of amino acids 34 to 928 of SEQ ID NO: 2, and that exhibit hydrolytic activity and / or transglycosylation activity.

[0045] <Mutant enzymes of the present invention> The present invention provides an Endo-Si mutant enzyme, which has an amino acid sequence comprising mutations at one or more amino acid positions selected from the group consisting of amino acids 241 (D241), 190 (T190), 311 (Q311), and 360 (E360) in the amino acid sequence set forth in amino acid numbers 34 to 928 of SEQ ID NO: 2, and which exhibits glycosylation activity and / or transglycosylation activity. Preferably, as shown in Example 6, the present invention provides an Endo-Si mutant enzyme, which contains a region required for transglycosylation activity in the amino acid sequence set forth in amino acid numbers 34 to 928 of SEQ ID NO: 2, and which is characterized by reduced hydrolysis activity and improved transglycosylation activity against the N297-linked glycan of IgG compared to Endo-Si WT (hereinafter, a wild-type strain without a mutation in the amino acid sequence is referred to as "WT").

[0046] The amino acid substitutions / mutations of the present invention are substitutions / mutations that exhibit the above-mentioned characteristics, preferably substitutions / mutations of at least one or more amino acid sites selected from T190, D241, Q311, and E360 of SEQ ID NO: 2, more preferably D241Q, D241M, D241A, T190Q, Q311L, E360Q, E360A, E360N, or E360D, even more preferably D241Q, D241M, T190Q, Q311L, and E360Q, and most preferably D241Q, D241Q / Q311L, D241Q / E360Q, D241M, D241M / Q311L, D241M / E360Q, T190Q / D241Q, T190Q, and T190Q / D241M. As long as the above properties are exhibited, the polypeptide may contain further substitutions / mutations in addition to the substitutions / mutations at T190, D241, Q311, and E360 in SEQ ID NO: 2.

[0047] Endo-β-N-acetylglucosaminidase possesses both hydrolytic and transglycosylation activities (hereinafter, the combination of both activities is referred to as "having the enzyme activity"). Therefore, enzymes with strong hydrolytic activity may hydrolyze the glycan transferred to the core GlcNAc of an acceptor molecule (an antibody having a core GlcNAc as the N297-linked glycan or a molecule containing its Fc domain) as a substrate, failing to properly obtain the desired transglycosylation product. Therefore, mutant enzymes with improved transglycosylation activity are useful in the synthesis of glycosylation remodeling antibodies or glycosylated compounds. The mutant enzymes of the present invention are characterized by both reduced hydrolytic activity and enhanced transglycosylation activity compared to Endo-Si WT.

[0048] The transglycosylation activity of the mutant enzyme can be evaluated by the method of Example 6 or Example 7 described below.

[0049] Among the enzymatic activities possessed by the mutant enzymes of the present invention, the transglycosylation activity exceeds that of Endo-Si WT. That is, under conditions of pH 7 to 8 (e.g., pH 7.5) and the presence of 5 to 10 equivalents (e.g., 8 equivalents) of a sugar chain donor (e.g., a sugar chain containing GlcNAc with an activated reducing end (e.g., oxazolinated GlcNAc)) relative to the acceptor molecule, the transglycosylation rate exceeds that of Endo-Si WT at any time point from 1 to 24 or 48 hours after the start of the reaction. Preferably, the transglycosylation rate exceeds 50% by 24 or 48 hours after the start of the reaction, more preferably, the transglycosylation rate exceeds 60% by 24 hours after the start of the reaction, even more preferably, the transglycosylation rate exceeds 80% by 24 hours after the start of the reaction, and even more preferably, the transglycosylation rate exceeds 95% by 24 hours after the start of the reaction.

[0050] Another aspect of the transglycosylation activity of the mutant enzymes of the present invention is that they exceed the transglycosylation activity of Endo-Si WT. That is, the transglycosylation rate at any time point between 1 and 48 hours after the start of the reaction under conditions of pH 7 to 8 (e.g., pH 7.5) and the presence of a glycan donor containing GlcNac at an inactivated reducing end in an amount of 10 to 100 equivalents (e.g., 50 equivalents) of the acceptor molecule is equal to or greater than that of Endo-Si WT. Preferably, the transglycosylation rate exceeds 50% within 24 or 48 hours after the start of the reaction, more preferably, the transglycosylation rate exceeds 60% within 48 hours after the start of the reaction, even more preferably, the transglycosylation rate exceeds 80% within 48 hours after the start of the reaction, and even more preferably, the transglycosylation rate exceeds 90% within 48 hours after the start of the reaction.

[0051] The mutant enzyme of the present invention does not need to be the full-length sequence, as long as it has mutations at one or more (preferably 1 to 3, more preferably 1 or 2) amino acid sites selected from the group consisting of D241, T190, Q311, and E360 in the amino acid sequence of amino acid numbers 34 to 928 of SEQ ID NO: 2, and retains a region important for the transglycosylation activity of Endo-Si. Domain analysis of EndoS has revealed that the catalytic domain (amino acid numbers 106 to 447 of SEQ ID NO: 2) and / or CBM (amino acid numbers 762 to 897 of SEQ ID NO: 2) are important, and any mutant enzyme containing these can be used as the mutant enzyme of the present invention.

[0052] The mutant enzymes of the present invention are polypeptides containing the mutations described above in "Amino acid substitutions / mutations of the present invention," and specifically include polypeptides containing an amino acid sequence selected from the group consisting of SEQ ID NO: 3 (Endo-Si D241Q), SEQ ID NO: 4 (Endo-Si D241Q / Q311L), SEQ ID NO: 5 (Endo-Si D241Q / E360Q), SEQ ID NO: 6 (Endo-Si D241M), SEQ ID NO: 7 (Endo-Si D241M / Q311L), SEQ ID NO: 8 (Endo-Si D241M / E360Q), SEQ ID NO: 9 (Endo-Si T190Q / D241Q), SEQ ID NO: 10 (Endo-Si T190Q), and SEQ ID NO: 11 (Endo-Si T190Q / D241M).

[0053] The amino acid sequence of the mutant enzyme of the present invention may contain one to several amino acid substitutions, deletions, insertions, and / or additions at positions other than the essential mutations (D241, T190, Q311, or E360) to the extent that the activity of the enzyme is not affected. While any position may be selected as the site for such amino acid mutations as long as the activity of the enzyme is not affected, a site other than amino acid positions 241, 190, 311, or 360 is preferred, a site other than the catalytic domain (amino acid positions 106 to 447 of SEQ ID NO: 2) and the CBM (amino acid positions 762 to 897 of SEQ ID NO: 2) is more preferred, and a site within the region of amino acid positions 34 to 105 or amino acid positions 898 to 928 of SEQ ID NO: 2 is even more preferred.

[0054] In the present invention, several means 30 or 20 or less, preferably 10 or less, more preferably 5 or less, and most preferably 4, 3, 2 or 1.

[0055] In the present invention, the amino acid to be substituted by mutation is not particularly limited as long as the resulting mutant enzyme has the enzymatic activity of the present invention. Various amino acids can be used, including naturally occurring amino acids, artificially synthesized amino acids, and modified amino acids thereof. However, naturally occurring amino acids are preferred, naturally occurring L-amino acids are more preferred, and essential amino acids are even more preferred.

[0056] In the amino acid sequence of the mutant enzyme of the present invention, the amino acid sequence has at least 80% or more, preferably 85% or more, more preferably 90% or more, and even more preferably 95%, 96%, 97%, 98% or 99% or more homology or identity to the amino acid sequence other than the amino acid of the essential mutation (D241, T190, Q311 or E360), to the extent that it does not affect the activity of the enzyme.

[0057] The identity or homology between two amino acid sequences is 、B The Blast algorithm version 2.2.2 can be determined using the default parameters of Blast algorithm version 2.2.2 (Altschul, SF, et al., Nucleic Acids Res. 1997, 25, 3389-3402). The Blast algorithm can also be accessed, for example, on the Internet at http: / / blast.ncbi.nlm.nih.gov / .

[0058] <Gene, host cells, enzyme production method> The present invention further provides recombinant genes encoding the Endo-Si (SEQ ID NO: 1) or Endo-Si mutant enzymes described above, gene constructs such as plasmids and expression vectors containing the recombinant genes, host cells transformed with the gene constructs, and methods for producing the enzymes of the present invention, including a step of recovering the Endo-Si or Endo-Si mutant enzymes of the present invention from a culture of the host cells. These recombinant genes, gene constructs, host cells, etc. can be prepared using known genetic engineering techniques based on the amino acid sequence of the mutant enzymes of the present invention. The nucleotide sequence of Endo-Si for E. coli is shown in SEQ ID NO: 16.

[0059] Host cells (cells typically used for protein production, such as animal cells, plant cells, Escherichia coli, and yeast, can be appropriately selected) transformed by introducing a gene encoding the enzyme of the present invention are cultured under conditions appropriate for the cell type, and the enzyme of the present invention can be recovered from the culture. Enzymes can be recovered by appropriately combining conventional purification techniques, taking advantage of the physical properties of the enzyme. For easier recovery, a gene construct can be designed so that the enzyme is expressed in a form linked to a tag peptide, such as a His tag or GST tag, and recovery can be performed by utilizing the affinity of the tag peptide. The tag peptide may be removed after purification, but if this does not affect enzymatic activity, the enzyme with the tag peptide still attached may be used in reactions such as glycan remodeling. The enzymes of the present invention include enzymes having an amino acid sequence linked to such a tag peptide.

[0060] <Glycan remodeling> The present invention provides a method for remodeling the glycans of glycoproteins using the Endo-Si or Endo-Si mutant enzyme of the present invention, a glycoprotein having a glycan with a substantially uniform structure produced by the glycan remodeling, and a method for producing a glycoprotein having a glycan with a substantially uniform structure by the glycan remodeling.

[0061] One embodiment of the present invention provides a method for glycan remodeling of N-linked glycans containing an N297 bond in an antibody or an Fc region-containing molecule thereof using the Endo-Si or Endo-Si mutant enzyme of the present invention, and a glycoprotein, preferably an antibody or an Fc region-containing molecule, having an N-linked glycan containing an N297 bond with a substantially uniform structure produced by the glycan remodeling. The present invention also provides a method for producing a glycoprotein, preferably an antibody or an Fc region-containing molecule, having an N-linked glycan containing an N297 bond with a substantially uniform structure by the glycan remodeling. The antibody is preferably an IgG. IgG will be described below. In the present invention, a glycoprotein refers to a protein present in animal and plant tissues, the cell membrane or cell wall of eukaryotic microorganisms, etc., to which at least one O-linked glycan or one N-linked glycan is bound within the amino acid sequence of the protein, and may be naturally derived or synthetic. Examples of glycoproteins include monoclonal antibody IgG or IgG Fc fragments, and Fc region-containing molecules such as CLCH consisting only of the constant region (Patent Document 1, WO2018 / 003983).

[0062] "Glycosylation remodeling" refers to a method for producing an IgG or its Fc region-containing molecule in which the N297-linked glycan of a specific glycoprotein, for example, an Fc region-containing molecule such as a monoclonal antibody IgG or an Fc fragment of IgG, or CLCH consisting only of the constant region, is removed leaving a core GlcNAc (which may have core fucose attached) to prepare an acceptor molecule, and then, using the glycosylation activity of the Endo-Si mutant enzyme of the present invention, a glycosylation derived from a glycan donor is transferred to the core GlcNAc of the acceptor molecule, thereby producing an IgG or its Fc region-containing molecule in which the N297-linked glycan has a uniform glycosylation structure derived from the glycan donor.

[0063] The IgG or Fc region-containing molecule used for glycosylation remodeling is preferably derived from an IgG heavy chain consisting of the same amino acid sequence and produced in a form having an N297-linked glycan. There are no particular limitations on the production method, and IgG produced by commonly known monoclonal antibody production methods, IgG CLCH, or Fc fragments obtained by enzymatic treatment thereof can be used. Furthermore, a mixture of samples obtained by different production methods or different lots of such IgG or Fc fragments may also be used.

[0064] Acceptor molecules for use in glycan remodeling can be prepared by treating the aforementioned IgG or Fc region-containing molecules with ENGase, which specifically hydrolyzes the 1,4-glycosidic bond (GlcNAcβ1-4GlcNAc) between GlcNAcs in the core chitobiose structure of the N297-linked glycan. Various ENGases, including Endo-Si WT, Endo-A, Endo-D, Endo-E, Endo-F3, Endo-H, Endo-S, and Endo-S2, can be used.

[0065] Glycan donor molecules with various glycan structures can be used for glycan remodeling. However, when the goal is to use the remodeled antibody as an antibody drug, it is preferable to use a glycan donor with a human-type glycan or a human-compatible glycan structure that is similar to or identical to the glycan structure possessed by humans.

[0066] Representative examples of such glycan donor molecules include those in which the core GlcNAc has been removed and the second GlcNAc from the reducing end has been activated in the basic N-linked glycan structure described above, such as SG(10)-Ox, ([N3-PEG(3)]2-SG(10))-Ox, [N3-PEG(3)]-MSG1(9)-Ox, [N3-PEG(3)]-MSG2(9)-Ox, and mixtures of [N3-PEG(3)]-MSG1(9)-Ox and [N3-PEG(3)]-MSG2(9)-Ox (WO2019 / 065964).

[0067] Other embodiments of sugar chain donor molecules include molecules in which the second GlcNAc from the reducing end is not activated, such as ([N3-PEG(3)]2-SG-)Asn-PEG(3)-N3, ([N3-PEG(3)]-MSG1-)Asn-PEG(3)-N3, ([N3-PEG(3)]-MSG2-)Asn-PEG(3)-N3, or a mixture of ([N3-PEG(3)]-MSG1-)Asn-PEG(3)-N3 and ([N3-PEG(3)]-MSG2-)Asn-PEG(3)-N3 (Patent Document 1, WO2019 / 065964).

[0068] In this process, the glycan of the donor molecule is conjugated to the core GlcNAc residue of the glycan-cleaved IgG or Fc domain-containing molecule, by coexisting an endo-β-N-acetylglucosaminidase (enzyme A) that uses the complex glycan of the glycan donor molecule as a substrate but not the N297-linked glycan, with the Endo-Si or Endo-Si mutant enzyme of the present invention, preferably the Endo-Si mutant enzyme. When Enzyme A used here has the activity of transferring the glycan from the glycan donor SGP to the GlcNAc-bearing acceptor, Enzyme A exhibits high transglycosylation efficiency in a one-pot method. Specifically, Enzyme A can be selected from endo-β-N-acetylglucosaminidases that use the complex glycan of the glycan donor molecule with an inactivated reducing end as a substrate but not the N297-linked glycan, based on their transglycosylation activity toward the GlcNAc-bearing acceptor. Endo-β-N-acetylglucosaminidases that do not use N297-linked glycans as substrates include Endo-M, Endo-Rp, Endo-Om, Endo-CC, and mutant enzymes thereof with reduced hydrolytic activity. Examples of mutant enzymes with reduced hydrolytic activity include known Endo-Rp N172Q and Endo-Rp N172H (Patent Document 3), Endo-M N175Q (Umekawa M. et al., J. Biol. Chem. 2010, 285, 511-521), Endo-CC N180H, and Endo-Om N194Q (Chiba Y., Kagaku to Seibutsu 2015, 53, 236-244). Preferred examples include Endo-Rp N172Q, Endo-Rp N172H, Endo-Rp N172A, Endo-Rp N172C, Endo-Rp N172D, Endo-Rp N172E, Endo-Rp N172G, Endo-Rp N172I, Endo-Rp N172L, Endo-Rp N172M, Endo-Rp N172P, Endo-Rp N172S, Endo-Rp N172T, and Endo-Rp N172V.Examples of mutant enzymes with two amino acid substitutions include Endo-Rp W278F / S216V, Endo-Rp W278F / N246D, Endo-Rp W278F / D276N, and Endo-Rp W278F / A310D. Examples of mutant enzymes with three amino acid substitutions include Endo-Rp W278F / N172D / F307Y, Endo-Rp W278F / N172D / F307H, Endo-Rp W278F / N172D / A310D, and Endo-Rp W214F / F307Y / L306I.

[0069] The amino acid sequences of the mutant enzymes of Endo-Rp are shown in SEQ ID NOs: 17 to 43, the amino acid sequence of Endo-M in SEQ ID NO: 44, the amino acid sequence of Endo-Om in SEQ ID NO: 45, and the amino acid sequence of Endo-CC in SEQ ID NO: 46.

[0070] Examples of combinations of the Endo-Si mutant enzymes of the present invention and Enzyme A include the combinations shown in Tables 1 and 2.

[0071] [Table 1] TIFF0007798773000007.tif24993TIFF0007798773000008.tif25192TIFF0007798773000009.tif25092TIFF0007798773 000010.tif25092TIFF0007798773000011.tif25090TIFF0007798773000012.tif24992TIFF0007798773000013.tif14493

[0072] [Table 2] TIFF0007798773000015.tif2882 From the viewpoint of producing glycoproteins having sugar chains with a substantially uniform structure by sugar chain remodeling, preferred combinations of the Endo-Si mutant enzyme of the present invention and Enzyme A include those shown in Table 3.

[0073] [Table 3]

[0074] In the presence of the polypeptide of the present invention exhibiting transglycosylation activity and endo-β-N-acetylglucosaminidase (enzyme A) which uses as a substrate the complex glycan of a sugar chain donor molecule with an inactivated reducing end but not the N297-linked glycan, an acceptor molecule which is an antibody having a core GlcNAc to which fucose may be attached as the N297-linked glycan or a molecule containing the Fc region thereof, is transglycosylated with an acceptor molecule having an inactivated reducing end. GlcNAc The saccharide can be reacted with a glycan donor molecule containing the following (Figure 17).

[0075] The reaction conditions for the hydrolysis reaction to prepare acceptor molecules in glycosylation remodeling can be selected appropriately based on known conditions for other enzymes. They can also be selected taking into consideration factors such as enzyme activity, antibody properties, recovery rate in the purification process, and processing time. The reaction is carried out in a buffer solution, which can be appropriately selected from buffers commonly used in conventional enzyme reactions, such as citrate buffer (pH 3.5-5.5), acetate buffer (pH 4.5-6.0), phosphate buffer (pH 6.0-7.5), MOPS-NaOH buffer (pH 6.5-8.0), and Tris-HCl buffer (pH 7.0-9.0). Phosphate buffer (pH 6.0-7.5) or Tris-HCl buffer (pH 7.0-9.0) is preferred. Additives that do not inhibit the enzymatic reaction may be added to the reaction solution to stabilize the enzyme, but they are not required.

[0076] The reaction temperature can be appropriately selected from the range of 4°C to 50°C, but is preferably 15°C to 45°C, more preferably 18°C ​​to 40°C, and even more preferably 20°C to 35°C.

[0077] The reaction pH for the hydrolysis reaction of Endo-Si can be appropriately selected between pH 5.8 and 9.5, but is preferably between pH 6.2 and pH 8.0, and more preferably between pH 6.5 and pH 7.5.

[0078] The reaction time can be selected appropriately between 10 minutes and 96 hours, but is preferably 0.5 to 80 hours, more preferably 1 to 60 hours, more preferably 8 to 48 hours, and even more preferably 12 to 24 hours. The completion of the reaction can be determined by periodically sampling a small amount of the reaction solution and monitoring the progress of hydrolysis. Generally, the progress of the glycosylation reaction can be monitored by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), a fully automated electrophoresis system, or liquid chromatography-mass spectrometry (LC-MS). In this patent, a commercially available antibody or a glycosylation-remodeling antibody was fragmented into heavy and light chains, and then the change in retention time of only the heavy chain with the N297-linked glycosylation chain was confirmed using a fully automated electrophoresis system.

[0079] In glycan remodeling, the reaction conditions for the glycosyltransfer reaction using GlcNAc with an activated reducing end as the glycan donor (e.g., oxazolinated GlcNAc) or non-activated GlcNAc as the glycan donor can be appropriately selected based on the conditions known for other enzymes (Patent Document 1, WO2019 / 065964, etc.).

[0080] The reaction is carried out in a buffer solution, preferably one that does not promote the decomposition of the sugar chain donor, GlcNAc, with or without activated reducing ends. This can be appropriately selected from phosphate buffer (pH 6.0-7.5), MOPS-NaOH buffer (pH 6.5-8.0), Tris-HCl buffer (pH 7.0-9.0), etc. Tris-HCl buffer (pH 7.0-9.0) is preferred. To stabilize the enzyme, an additive that does not inhibit the enzymatic reaction may be added to the reaction solution, but it is not necessary to add it.

[0081] The reaction temperature can be appropriately selected from the range of 4°C to 50°C, but is preferably 15°C to 45°C, more preferably 20°C to 40°C, and even more preferably 25°C to 40°C.

[0082] The reaction pH for the hydrolysis reaction of Endo-Si can be appropriately selected between pH 5.8 and 9.5, but is preferably between pH 6.2 and pH 8.0, and more preferably between pH 6.5 and pH 7.5.

[0083] The reaction time can be selected appropriately between 10 minutes and 96 hours, preferably between 0.5 and 80 hours, more preferably between 2 and 70 hours, more preferably between 12 and 60 hours, more preferably between 16 and 48 hours, and even more preferably between 16 and 28 hours. The completion of the reaction can be determined by periodically sampling a small amount of the reaction mixture and monitoring the progress of the transglycosylation reaction. Generally, the progress of the transglycosylation reaction can be monitored by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), a fully automated electrophoresis system, or liquid chromatography-mass spectrometry (LC-MS). In this patent, a commercially available antibody or a glycosylation-remodeling antibody was fragmented into heavy and light chains, and then the change in retention time of only the heavy chain with the N297-linked glycan attached was confirmed using a fully automated electrophoresis system.

[0084] Glycosylation remodeling can also be performed in a one-pot manner by directly transferring the glycan of a glycan donor molecule to an acceptor molecule, an antibody or its Fc domain-containing molecule, which has a core GlcNAc optionally fucose-linked as the N297-linked glycan, using both ENGase that retains the activity of specifically hydrolyzing the 1,4-glycosidic bond (GlcNAcβ1-4GlcNAc) between GlcNAcs in the core chitobiose structure of the N297-linked glycan and the above-mentioned enzyme A. The ENGase that retains the activity of specifically hydrolyzing the 1,4-glycosidic bond (GlcNAcβ1-4GlcNAc) between GlcNAcs in the core chitobiose structure of the N297-linked glycan is preferably the above-mentioned mutant enzyme of Endo-Si, and the enzyme A is preferably the above-mentioned mutant enzyme of Endo-Rp with reduced hydrolysis activity. In the one-pot method, examples of glycan donor molecules include SGP with chemically modified non-reducing ends, (SG-)Asn, (MSG1-)Asn, (MSG2-)Asn, a mixture of (MSG1-)Asn and (MSG2-)Asn, SG(10)-Ox, MSG1(9)-Ox, MSG2(9)-Ox, or a mixture of MSG1(9)-Ox and MSG2(9)-Ox. Preferably, ([N3-PEG(3)]2-SG(10))-Ox, [N3-PEG(3)]-MSG1(9)-Ox, [N3-PEG(3)]-MSG2(9)-Ox, or a mixture of [N3-PEG(3)]-MSG1(9)-Ox and [N3-PEG(3)]-MSG2(9)-Ox, or ([N3-PEG(3)]2-SG-)Asn-PEG(3)-N3, ([ ([N3-PEG(3)]-MSG1-)Asn-PEG(3)-N3, ([N3-PEG(3)]-MSG2-)Asn-PEG(3)-N3, or a mixture of ([N3-PEG(3)]-MSG1-)Asn-PEG(3)-N3 and ([N3-PEG(3)]-MSG2-)Asn-PEG(3)-N3 (Patent Document 1, WO2019 / 065964), etc. can be used.

[0085] Glycoproteins (antibodies or Fc region-containing molecules) produced by the glycochain remodeling method can be further modified chemically or biochemically. For example, the azide group (N3-) can be reacted with an alkyne structure such as a (hetero)cycloalkynyl group (e.g., dibenzocyclooctyne (DBCO)) to form a 1,2,3-triazole ring (SPAAC (strain-promoted alkyne azide cycloaddition: Agard NJ, et al., J Am Chem Soc. 2004, 126, 46, 15046-15047)).Therefore, the glycan remodeling antibody obtained using the donor molecule having the above-mentioned azide group (N3-) can be used to synthesize a molecule having a (hetero)cycloalkynyl group and having a desired activity (a pharmaceutically active compound (e.g., a chemotherapeutic agent, a molecular targeted drug, an immune activator (e.g., a STING agonist (WO2020 / 050406, WO2014 / 099824, WO2014 / 179335, WO2014 / 189805, WO2014 / 189 806, WO2015 / 074145, WO2015 / 185565, WO2016 / 096714, WO2016 / 012305, WO2016 / 145102, WO2017 / 027646, WO2017 / 0 27645, WO2017 / 075477, WO2017 / 093933, WO2017 / 100305, WO2017 / 123669, WO2017 / 161349, WO2017 / 175147, WO2017 / 175156, WO2018 / 009466, WO2018 / 045204, WO2018 / 060323, WO2018 / 067423, WO2018 / 065360, WO2014 / 093936, WO2018 / 009648, WO2018 / 100558), TLR agonists, A2AR antagonists, IDO inhibitors, antagonists of the CTLA-4, LAG-3 and PD-1 pathways, checkpoint inhibitors, vascular Further modified antibodies having desired activity (e.g., antibody-drug conjugates) can be obtained by reacting the antibodies with endothelial growth factor (VEGF) receptor inhibitors, smoothen inhibitors, alkylating agents, antimetabolites, retinoids, and anti-cancer vaccines, adjuvants, lipids, liposomes, toxins, antibacterial agents, antiviral agents, diagnostic agents, proteins, peptides, amino acids, nucleic acids, antigens, vitamins, hormones, etc.).As chemotherapeutic agents or toxins, camptothecin (e.g., WO2014 / 057687), pyrrolobenzodiazepines (e.g., WO2013 / 173496, WO2014 / 130879, WO2017 / 004330, WO2017 / 004025, WO2017 / 020972, WO2016 / 036804, WO201 5 / 095124, WO2015 / 052322, WO2015 / 052534, WO2016 / 011519, WO2015 / 052321, WO2015 / 031693, WO2011 / 130613, WO2019 / 065964), doxorubicin, auristatin, taxane or a derivative thereof.

[0086] Examples of donor molecules having the azide group (N3-) include drug linkers described in WO2020 / 050406 and WO2019 / 065964. For example, N-[4-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]glycylglycyl-L-valyl-N-{4-[({[(11'S,11a'S)-11'-hydroxy-7'-methoxy-8'-[(5-{[(11aS)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,10,11,1 1a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl]oxy}pentyl)oxy]-5'-oxo-11',11a'-dihydro-1'H-spiro[cyclopropane-1,2'-pyrrolo[2,1-c][1,4]benzodiazepin]-10'(5'H)-yl]carbonyl}oxy)methyl]phenyl}-L-alaninamide, N-[4-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]glycylglycyl-L-valyl-N-[4-({[(11'S,11'aS)-11'-hydroxy-7'-methoxy-8'-(3-{[(11aS)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl]oxy}propoxy]-5'-oxo-11',11'a-dihydro-1'H,3'H-spiro[cyclo Propane-1,2'-pyrrolo[2,1-c][1,4]benzodiazepine]-10'(5'H)-carbonyl]oxy}methyl)phenyl]-L-alaninamide, N-[4-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]glycylglycyl-L-valyl-N-{4-[({[(11'S,11a'S)-11'-hydroxy-7'-methoxy-8'-[(5-{[(11a'S)-7'-methoxy-5'-oxo-5',11a'-dihydro-1'H-spiro[cyclopropane-1,2'-pi N-[4-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]glycylglycyl-L-valyl-N-{4-[({[(11'S,11a'S)-11'-hydro hydroxy-7'-methoxy-8'-[(5-{[(11a'S)-7'-methoxy-5'-oxo-5',10',11',11a'-tetrahydro-1'H-spiro[cyclopropane-1,2'-pyrrolo[2,1-c][1,4]benzodiazepine]-8'-yl]oxy}pentyl)oxy]-5'-oxo-11',11a'-dihydro-1'H-spiro[cyclopropane-1,2'-pyrrolo[2,1-c][1,4]benzodiazepine]-10'(5'H)-yl]carbonyl}oxy)methyl]phenyl}-L-alaninamide, (bis(N,N-diethylethanaminium) N-[4-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]glycylglycyl-L-phenylalanyl-N-[(2-{9-[(5R,7R,8R,12aR,14R,15R,15aR,16R)-15-fluoro-16-hydroxy-2,10-dioxo-2,10-disulfide-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ, 5 ,10λ 5 -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-1-yl}ethoxy)methyl]glycinamide. [Example]

[0087] The present invention will be specifically described below using examples. The examples are merely examples of embodiments of the present invention, and the present invention is not limited thereto.

[0088] The protein concentrations described herein were quantified using an ultra-microspectrophotometer NanoDrop1000 (Thermo Fisher Scientific) or NanoDrop2000 (Thermo Fisher Scientific).

[0089] In the examples, [N3-PEG(3)]-MSG1(9)-Ox refers to the compound in Figure 1. SGP refers to the compound in Figure 2. mAb1 refers to commercially available Trastuzumab (purchased from Chugai Pharmaceutical). (Fucα1,6)GlcNAc-mAb1 refers to a glycosylated form of Trastuzumab. mAb2 refers to an antibody prepared by the method described in Example 136 of WO2019065964. The amino acid sequences of the light and heavy chains of mAb2 are shown in SEQ ID NOs: 12 and 13. (Fucα1,6)GlcNAc-mAb2 refers to a glycosylated form of (Fucα1,6)GlcNAc-anti-CLDN6 antibody (H1L1) prepared by the method described in Example 61, Step 1 of WO2019 / 065964.

[0090] The progress of glycosylation and transglycosylation was confirmed using protein gel electrophoresis (Patent Document 4, Non-Patent Document 8). A fully automated protein electrophoresis system was used, including LabChip GX II (PerkinElmer) and Protein Express LabChip and Protein Express Reagent Kit (PerkinElmer).

[0091] Example 1: Obtaining endo-β-N-acetylglucosaminidase from S. iniae The gene sequence of endo-β-N-acetylglucosaminidase was obtained from S. iniae strain SIO1002 by the following method.

[0092] First, genomic DNA was isolated from formalin-inactivated S. iniae SIO1002 cells (Japanese strain, purchased from Kyoritsu Seiyaku Co., Ltd.). One mL of 0.1% (w / v) formalin solution was centrifuged (6,000 rpm, 10 min, 4°C), and the precipitate was washed with 1 mL of sterile water. The mixture was centrifuged again under the same conditions, and the precipitate was suspended in 200 μL of InstaGene DNA Purification Matrix (Bio-Rad). This suspension was heat-treated at 56°C for 30 minutes and then at 99°C for 8 minutes, and then centrifuged (12,000 rpm, 10 min, 4°C). The resulting supernatant was used as the DNA extract.

[0093] The DNA extract was used as a template to amplify the gene encoding endo-β-N-acetylglucosaminidase using Primer 1 (SEQ ID NO: 14) and Primer 2 (SEQ ID NO: 15) and PrimeSTAR Max DNA Polymerase (Takara Bio). The gene is 2787 bases long (including the termination codon) (SEQ ID NO: 1) and encodes a protein with a molecular weight of 104,644 and 928 amino acid residues (SEQ ID NO: 2). This protein was named Endo-Si.

[0094] Example 2: Expression of Endo-Si using E. coli A nucleic acid sequence (SEQ ID NO: 16) optimized for heterologous expression in E. coli was designed based on the endo-β-N-acetylglucosaminidase gene obtained in Example 1, with a 6xHis tag added to the C-terminus. An artificially synthesized gene was prepared at Eurofins Genomics. This was cloned into the pET24b(+) vector and transformed into E. coli BL21(DE3). The transformed bacterial solution was inoculated into 2 mL of LB medium (1% (w / v) Tryptone, 0.5% (w / v) Yeast extract, 0.5% (w / v) NaCl, 50 μg / mL Kanamycin) in a 12 mL conical tube and cultured overnight at 37°C with shaking (600 rpm, O / N). 1.2 mL of this preculture was inoculated into 100 mL of TB medium (1.2% (w / v) Tryptone, 2.4% (w / v) Yeast extract, 0.94% (w / v) KHPO, 0.22% (w / v) KHPO, 50 μg / mL Kanamycin, 0.01% (w / v) Antifoam 204, 2 mM MgSO) in a 500 mL baffled flask, and shaking culture was initiated at 37°C (210 rpm). After 1.5 hours of culture at 37°C, the incubator temperature was lowered to 16°C and culture was continued for 1 hour. After confirming that the culture temperature had dropped to 16°C, IPTG was added to a final concentration of 0.2 mM, and culture was continued for 24 hours. After the culture was completed, the bacteria were harvested by centrifugation.

[0095] The collected cells were suspended in 5 mL of binding buffer (50 mM HEPES (pH 8.0), 0.5 M NaCl, 20 mM imidazole, 5% (w / v) glycerol), and then ultrasonically disrupted and centrifuged. The supernatant was purified using Ni Sepharose 6 Fast Flow (GE Healthcare). The yield (A 280 The concentration (converted into an extinction coefficient) was 8.43 mg / 100 mL broth.

[0096] Example 3: Hydrolytic activity of Endo-Si against antibody sugar chains The hydrolytic activity of the enzyme obtained in Example 2 was measured by the following method, using EndoS as a control.

[0097] 60 mg of mAb1 was dissolved in 5 mL of sterile water, and the solution was concentrated with Vivaspin 20 (30,000 MWCO, PES, manufactured by Sartorius) while replacing the solution with 50 mM Tris-HCl buffer (pH 7.5).

[0098] A reaction mixture (total volume 50 μL) containing 0.1 mg of this mAb1 and 10 ng of enzyme was prepared and incubated at 37°C. Samples were taken from the reaction mixture after 0.5, 1, and 2 hours and analyzed using the automated protein electrophoresis system described above. The resulting chromatogram confirmed the presence of separate peaks representing the unreacted material and hydrolysate. The glycosylation rate was calculated from the peak area ratio between the unreacted material and hydrolysate using the following formula:

[0099] Glycosylation rate (%) = [[(Fucα1,6)GlcNAc-mAb1-derived H chain peak area] / {[mAb1-derived H chain peak area] + [(Fucα1,6)GlcNAc-mAb1-derived H chain peak area]}] × 100 The change in reaction yield over time is shown in Figure 3. The glycan hydrolysis rate after 0.5 hours was 57.4% for Endo-Si and 41.8% for EndoS, indicating that Endo-Si exhibited stronger hydrolytic activity than EndoS.

[0100] Example 4: Examination of Endo-Si reaction conditions The reaction temperature and pH of Endo-Si were investigated. (4-1) Evaluation of Endo-Si reaction temperature The glycosylation rate for Endo-Si and EndoS at each temperature was measured as follows: 50 μL of 50 mM Tris-HCl buffer (pH 7.5) containing 0.1 mg mAb1 and 10 ng Endo-Si WT or EndoS WT was prepared and incubated at 27, 29, 31, 35, 37, 40, 43, 46, 48, and 50°C. After 0.5 hours of reaction, the reaction mixture was sampled, and the glycosylation rate was calculated as described above.

[0101] The results are shown in Figure 18. Endo-Si exhibits good hydrolysis at an optimum temperature (temperature at which the enzyme functions well) in the hydrolysis reaction in the range of 25°C to 45°C, more preferably in the range of 30°C to 42°C, and particularly in the range of 35°C to 39°C, which is close to 37°C. It was also confirmed that the hydrolysis activity at each temperature was higher than that of EndoS.

[0102] (4-2) Evaluation of Endo-Si reaction pH The glycosylation rate for Endo-Si and EndoS at each pH was measured as follows: 50 μL of 50 mM citrate-sodium phosphate buffer (pH 5.0 or 5.5), sodium phosphate buffer (pH 6.0, 6.5, or 7.0), or Tris-HCl buffer (pH 7.5, 8.0, 8.5, or 9.0) containing 0.1 mg mAb1 and 10 ng Endo-Si WT or EndoS WT was prepared and incubated at 37°C. The reaction mixture was sampled after 0.5 hours of reaction, and the glycosylation rate was calculated as described above.

[0103] The results are shown in Figure 19. Endo-Si exhibited optimal pH (the pH at which the enzyme functions well) in the hydrolysis reaction in the range of pH 6.3 to 9.0, more preferably in the range of pH 6.7 to 8.8, and exhibited particularly good hydrolysis at pH 7.2 to 8.0 near pH 7.5. It was confirmed that Endo-Si had higher activity than EndoS at pH conditions near pH 6.7 or higher.

[0104] <Example 5> Substrate specificity of Endo-Si (5-1) Evaluation of substrate specificity of Endo-Si for various antibodies The hydrolytic activity of Endo-Si, EndoS, and PNGase F toward various antibodies was measured as follows. 50 μL of 50 mM Tris-HCl buffer (pH 7.5) containing 10 μg of each substrate and 1 μg of Endo-Si WT was prepared and incubated at 37°C. Human IgG1-4, IgA, and IgE (all from Sigma-Aldrich) were used as substrates. 1 μg of EndoS WT and 500 U of PNGase F PRIME (from N-zyme Scientifics) were used as controls. After 2 hours, the reaction mixture was sampled and analyzed using the automated protein electrophoresis system described above.

[0105] The results are shown in Figure 20. When the sugar chains are hydrolyzed by the addition of enzyme, the protein band shifts to a lower molecular weight compared to when no enzyme is added. The experiment showed that Endo-Si WT exhibited activity against all four IgG subclasses. However, it did not exhibit hydrolytic activity against IgA or IgE. EndoS WT, used as a control, was also confirmed to exhibit similar substrate specificity.

[0106] (5-2) Evaluation of substrate specificity of Endo-Si for various sugar chains The substrate specificity of Endo-Si for various glycans was measured as follows. 10 μL of 50 mM Tris-HCl buffer (pH 7.5) containing 5 pmol of each 2-AB-labeled glycan (Agilent Technologies) and 20 μg of Endo-Si WT was prepared. The mixture was incubated at 37°C for 24 hours, and then the reaction was stopped by heating at 95°C for 5 minutes. The reaction solution was analyzed by HPLC under the following conditions.

[0107] [HPLC analysis conditions] HPLC equipment: 1200 Infinity LC (Agilent Technologies) Column: ACQUITY UPLC Glycan Amide 130Å, 1.7 μm, 2.1 x 150 mm (Waters) Column temperature: 40℃ Detector: Fluorescence detector RF-20Axs (Shimadzu Corporation) Mobile phase A: H2O+0.1% HCOOH Mobile phase B: acetonitrile + 0.1% HCOOH Gradient (mobile phase B%): 90% (0 min), 40% (25 min) Flow rate: 0.2 mL / min

[0108] Enzyme activity was calculated from the peak area ratio of the substrate and its hydrolysate, GlcNAc-2AB. Table 4 shows the relative activities for various glycans, with activity for G0 glycans taken as 100%. Endo-Si exhibited activity for both high-mannose and complex-type biantennary glycans, but was more specific for complex-type biantennary glycans than for high-mannose glycans, with the highest activity for G0 glycans. It also exhibited activity for sialyl glycans and fucosylated glycans, but not for complex-type triantennary glycans. [Table 4] Hydrolysis activity of Endo-Si on various sugar chains

[0109] [Table 4]

[0110] <Example 6> Modification of Endo-Si and measurement of metastatic activity Preparation of (6-1)[N3-PEG(3)]-MSG1(9)-Ox [N3-PEG(3)]-MSG1(9)-Ox, which will be used as a sugar chain donor in the following examples, was produced by the method described in Example 56 of WO2019 / 065964.

[0111] [ka]

[0112] (6-2) Modification of Endo-Si and confirmation of transglycosylation activity Mutations were introduced to obtain Endo-Si mutant enzymes with high transglycosylation activity. Based on the three-dimensional structure of EndoS (PDB ID: 4NUY), various mutant enzymes shown in Table 1 were designed and their transglycosylation activity against the antibody was measured.

[0113] Transglycosylation activity was evaluated as follows. 45 μL of 50 mM Tris-HCl buffer (pH 7.5) containing 0.5 mg of (Fucα1,6)GlcNAc-mAb2, 50.4 μg (8 eq.) of the glycosylated oxazoline derivative [N3-PEG(3)]-MSG1(9)-Ox, and 1.25 μg of enzyme was prepared and incubated at 28°C. Reaction mixture samples were taken at 1, 2, 4, 6, and 24 hours and analyzed using the automated protein electrophoresis system described above. The resulting chromatograms showed distinct peaks representing the unreacted product and the transglycosylated product mAb2-(MSG1-N3)2. The transglycosylation rate was calculated from the peak area ratio of the unreacted product to the transglycosylated product using the following formula:

[0114] Transglycosylation rate (%) = [[peak area of ​​H chain derived from mAb2-(MSG1-N3)2] / {[peak area of ​​H chain derived from (Fucα1,6)GlcNAc-mAb2] + [peak area of ​​H chain derived from mAb2-(MSG1-N3)2]}] × 100 Similarly, the glycosylation rate for each Endo-Si mutant enzyme at each reaction time was calculated (Table 5). [Table 5] Time course of glycosylation rates for Endo-Si WT and each mutant enzyme using an oxazoline-based glycosyl donor

[0115] [Table 5] Except for Endo-Si WT, all of the Endo-Si mutant enzymes showed high transglycosylation activity.

[0116] <Example 7> Measurement of glycosylation activity using SGP as a sugar chain donor Transglycosylation activity was evaluated as follows. 17 μL of 50 mM sodium phosphate buffer (pH 7.5) containing 0.5 mg of (Fucα1,6)GlcNAc-mAb2, 0.485 mg (50 eq.) of SGP, 2.5 mU of Endo-M N175Q (Tokyo Chemical Industry Co., Ltd.), and 10 μg of each Endo-Si mutant enzyme was prepared and incubated at 23°C. Reaction mixture samples were taken at 2, 4, 6, 24, and 48 hours and analyzed using the automated protein electrophoresis system described above. The resulting chromatograms showed distinct peaks representing the unreacted product and the transglycosylated product mAb2-(SG)2. The transglycosylation rate was calculated from the peak area ratio of the unreacted product to the transglycosylated product using the following formula:

[0117] Transglycosylation rate (%) = [[peak area of ​​H chain derived from mAb2-(SG)2] / {[peak area of ​​H chain derived from (Fucα1,6)GlcNAc-mAb2] + [peak area of ​​H chain derived from mAb2-(SG)2]}] × 100 Similarly, the glycosylation rate for each Endo-Si mutant enzyme at each reaction time was calculated (Table 6). [Table 6] Time course of glycosylation rates for Endo-Si WT and each mutant enzyme using SGP as a glycan donor

[0118] [Table 6] Except for Endo-Si WT, all of the Endo-Si mutant enzymes showed high transglycosylation activity.

[0119] <Example 8> Examination of one-pot method in combination with Endo-Rp Endo-Rp, which is known to transfer glycosylation from the glycan donor SGP to a GlcNAc derivative, was used as a representative example of enzyme A to examine the properties of enzyme A that can be combined with Endo-Si.

[0120] The glycosyltransferase activity of antibodies was evaluated using a one-pot method as follows. 17 μL of 50 mM sodium phosphate buffer (pH 7.5) containing 0.5 mg of (Fucα1,6)GlcNAc-mAb2, 0.485 mg (50 eq.) of SGP, 2.5 mU of Endo-M N175Q (Tokyo Chemical Industry Co., Ltd.) or 8 μg of each Endo-Rp mutant enzyme, and 5 μg of Endo-Si D241Q was prepared and incubated at 28°C. Reaction mixture samples were taken at 2, 4, 6, 24, and 48 hours and analyzed using the automated protein electrophoresis system described above. The glycosyltransferase rate was calculated from the resulting chromatogram using the formula described in Example 5.

[0121] The results are shown in Table 7. Endo-Rp mutant enzymes with low activity in transferring SGP to GlcNAc derivatives, such as the N172F, N172K, N172L, N172R, N172W, and N172Y mutant enzymes that showed no transfer activity at all in Patent Document 3, also showed low transfer efficiency in the one-pot method.

[0122] From the above, the transglycosylation activity of enzyme A itself is correlated with its ability to activate glycan raw materials. Therefore, it is possible to identify appropriate enzymes A capable of activating glycan raw materials using the transfer activity from SGP to GlcNAc derivatives as an indicator. In general, Endo-M, Endo-Om, and Endo-CC are thought to have similar reactivity to Endo-Rp, so it is possible to identify enzymes A effective for the one-pot method by replacing Endo-Rp with Endo-M, Endo-Om, or Endo-CC. Furthermore, this method can also be applied to Endo-S and Endo-S2, which have the same transglycosylation activity toward antibodies as Endo-Si.

[0123] Thus, the scope of application of the above-mentioned identification method is not limited to the combination of Endo-Si and Endo-Rp, and it is believed that any enzyme having properties similar to those of the respective enzymes can be used. [Table 7] Time course of glycosylation activity of various mutant enzymes on antibodies using the one-pot method

[0124] [Table 7]

[0125] <Example 9> Examination of the one-pot method using chemically modified sugar chain derivatives as donors As a donor of sugar chain derivatives other than the natural sugar chain SGP, derivatives in which the non-reducing and reducing terminal amino acids were azide-modified were prepared, and the one-pot transglycosylation reaction was examined. (9-1) Preparation of ([N3-PEG(3)]-MSG1-)Asn-PEG(3)-N3 ([N3-PEG(3)]-MSG1-)Asn-PEG(3)-N3, which will be used as a sugar chain donor in the following examples, was produced by the method described in step 3 of Example 154 of WO2019065964.

[0126] [ka]

[0127] (9-2) Preparation of ([N3-PEG(3)]2-SG-)Asn-PEG(3)-N3 ([N3-PEG(3)]2-SG-)Asn-PEG(3)-N3, which will be used as a sugar chain donor in the following examples, was produced by the method described in Example 1-12, Step 1-12A of WO2018003983.

[0128] [ka]

[0129] (9-3) Evaluation of glycosylation activity Transglycosylation activity was evaluated as follows: 0.5 mg of (Fucα1,6)GlcNAc-mAb2, 0.485 mg (50 eq.) of SGP, 0.415 mg (50 eq.) of ([N3-PEG(3)]-MSG1-)Asn-PEG(3)-N3, or 0.495 mg (50 eq.) of ([N3-PEG(3)]2-SG-)Asn-PEG(3)-N3, 8 μg of Endo-Rp N172H, and 10 μg of Endo-Si D241M / Q311L in 17 μL of 50 mM sodium phosphate buffer (pH 7.5) were prepared and incubated at 28°C. Reaction mixtures were sampled at 2, 4, 6, 24, and 48 hours and analyzed using the automated protein electrophoresis system described above. When SGP was used as the donor, the transglycosylation reaction product was mAb2-(SG)2 as described in Example 7. When ([N3-PEG(3)]-MSG1-)Asn-PEG(3)-N3 was used as the donor, mAb2-(MSG1-N3)2 (Figure 21). When ([N3-PEG(3)]2-SG-)Asn-PEG(3)-N3 was used as the donor, mAb2-[SG-(N3)2]2 (Figure 22, referred to as mAb2-(SG-N3)2 in the formula) was generated. The resulting chromatogram confirmed the unreacted product and each transglycosylation product as separate peaks. The transglycosylation rate when SGP was used as the donor was calculated using the formula described in Example 5. The transglycosylation rate when ([N3-PEG(3)]-MSG1-)Asn-PEG(3)-N3 was used as the donor was calculated using the formula described in Example 4-2. The transglycosylation rate when ([N3-PEG(3)]2-SG-)Asn-PEG(3)-N3 was used as the donor was calculated using the following formula. Transglycosylation rate (%) = [[peak area of ​​H chain derived from mAb2-(SG-N3)2] / {[peak area of ​​H chain derived from (Fucα1,6)GlcNAc-mAb2] + [peak area of ​​H chain derived from mAb2-(SG-N3)2]}] × 100 The results are shown in Table 8. It was confirmed that the one-pot transfer reaction proceeded not only when natural glycan structures but also when chemically modified glycans were used as donors. [Table 8] Time course of glycosylation activity of various glycan donors to antibodies using the one-pot method

[0130] [Table 8] [Industrial Applicability]

[0131] Antibodies or sugar chain-containing molecules having uniform sugar chains obtained using the Endo-Si enzyme of the present invention can be obtained efficiently or with high purity, and the antibodies, etc. can be used as pharmaceuticals. [Sequence List Free Text]

[0132] SEQ ID NO: 1: Endo-Si base sequence SEQ ID NO: 2: Endo-Si amino acid sequence SEQ ID NO: 3: Endo-Si amino acid sequence D241Q SEQ ID NO: 4: Endo-Si amino acid sequence D241Q / Q311L SEQ ID NO: 5: Endo-Si amino acid sequence D241Q / E360Q SEQ ID NO: 6: Endo-Si amino acid sequence D241M SEQ ID NO: 7: Endo-Si amino acid sequence D241M / Q311L SEQ ID NO: 8: Endo-Si amino acid sequence D241M / E360Q SEQ ID NO: 9: Endo-Si amino acid sequence T190Q / D241Q SEQ ID NO: 10: Endo-Si amino acid sequence T190Q SEQ ID NO: 11: Endo-Si amino acid sequence T190Q / D241M SEQ ID NO: 12: mAb2 light chain amino acid sequence SEQ ID NO: 13: mAb2 heavy chain amino acid sequence SEQ ID NO: 14: Primer 1 SEQ ID NO: 15: Primer 2 SEQ ID NO: 16: Endo-Si E. coli sequence SEQ ID NO: 17: Endo-Rp amino acid sequence N172Q SEQ ID NO: 18: Endo-Rp amino acid sequence N172H SEQ ID NO: 19: Endo-Rp amino acid sequence N172A SEQ ID NO: 20: Endo-Rp amino acid sequence N172C SEQ ID NO: 21: Endo-Rp amino acid sequence N172D SEQ ID NO: 22: Endo-Rp amino acid sequence N172E SEQ ID NO: 23: Endo-Rp amino acid sequence N172F SEQ ID NO: 24: Endo-Rp amino acid sequence N172G SEQ ID NO: 25: Endo-Rp amino acid sequence N172I SEQ ID NO: 26: Endo-Rp amino acid sequence N172K SEQ ID NO: 27: Endo-Rp amino acid sequence N172L SEQ ID NO: 28: Endo-Rp amino acid sequence N172M SEQ ID NO: 29: Endo-Rp amino acid sequence N172P SEQ ID NO: 30: Endo-Rp amino acid sequence N172R SEQ ID NO: 31: Endo-Rp amino acid sequence N172S SEQ ID NO: 32: Endo-Rp amino acid sequence N172T SEQ ID NO: 33: Endo-Rp amino acid sequence N172V SEQ ID NO: 34: Endo-Rp amino acid sequence N172W SEQ ID NO: 35: Endo-Rp amino acid sequence N172Y SEQ ID NO: 36: Endo-Rp amino acid sequence W278F / S216V SEQ ID NO: 37: Endo-Rp amino acid sequence W278F / N246D SEQ ID NO: 38: Endo-Rp amino acid sequence W278F / D276N SEQ ID NO: 39: Endo-Rp amino acid sequence W278F / A310D SEQ ID NO: 40: Endo-Rp amino acid sequence W278F / N172D / F307Y SEQ ID NO: 41: Endo-Rp amino acid sequence W278F / N172D / F307H SEQ ID NO: 42: Endo-Rp amino acid sequence W278F / N172D / A310D SEQ ID NO: 43: Endo-Rp amino acid sequence W278F / F307Y / L306I SEQ ID NO: 44: Endo-M amino acid sequence SEQ ID NO: 45: Endo-Om amino acid sequence SEQ ID NO: 46: Endo-CC amino acid sequence

[0133] All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety.

Claims

1. A polypeptide having an amino acid sequence including the amino acid sequence set forth in amino acid numbers 34 to 928 of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO:

11.

2. A polypeptide described in claim 1, comprising the amino acid sequence described in SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO:

11.

3. The polypeptide according to claim 1 or 2, which exhibits hydrolytic activity and / or transglycosylation activity against N-linked sugar chains.

4. The polypeptide according to claim 3 , wherein the N-linked sugar chain is an N-linked sugar chain in a glycoprotein.

5. The polypeptide according to claim 4 , wherein the glycoprotein is an antibody or a molecule containing the Fc region of an antibody (Fc region-containing molecule).

6. The polypeptide according to any one of claims 3 to 5, wherein the N-linked sugar chain is an N-linked sugar chain that is bound to Asn at position 297 of the antibody (N297-linked sugar chain).

7. The polypeptide according to claim 6, wherein the N297-linked sugar chain is a complex-type sugar chain whose non-reducing end may be chemically modified.

8. The polypeptide according to claim 6 or 7, wherein the N297-linked glycan is an N297-linked glycan in which fucose may be added to the core GlcNAc.

9. A polynucleotide encoding the polypeptide of any one of claims 1 to 8.

10. An expression vector comprising the polynucleotide of claim 9.

11. A host cell transformed with the expression vector of claim 10.

12. A method for producing a polypeptide described in any one of claims 1 to 8, characterized in that it comprises a step of culturing the host cell described in claim 11, and a step of collecting the target polypeptide from the culture obtained in said step.

13. A method for producing an antibody or an Fc region-containing molecule thereof, comprising reacting an acceptor molecule that is an antibody having a core GlcNAc optionally having fucose attached as an N297-linked sugar chain or an Fc region-containing molecule thereof with a sugar chain donor molecule containing GlcNAc whose reducing end has been activated in the presence of the polypeptide according to any one of claims 1 to 8.

14. The method according to claim 13, wherein the GlcNAc having an activated reducing end is an oxazolinated GlcNAc.

15. The method according to claim 13 or 14, wherein the sugar chain donor molecule is a complex sugar chain whose non-reducing end may be chemically modified.

16. The method according to any one of claims 13 to 15, wherein the sugar chain donor molecule is SG(10)-Ox, MSG1(9)-Ox, MSG2(9)-Ox, or a mixture of MSG1(9)-Ox and MSG2(9)-Ox, each of which may have a chemically modified non-reducing end.

17. The sugar chain donor molecule is [N ３ -PEG(3)] ２ -SG(10)-Ox, [N ３ -PEG(3)]-MSG1(9)-Ox, [N ３ -PEG(3)]-MSG2(9)-Ox, or [N ３ -PEG(3)]-MSG1(9)-Ox and [N ３ The method according to any one of claims 13 to 16, wherein the mixture is a mixture of [-PEG(3)]-MSG2(9)-Ox.

18. Furthermore, an azide group (N ３ 18. The method according to claim 17, comprising a step of reacting the hydroxyl group with a molecule having an alkyne structure.

19. The method according to claim 18, wherein the molecule having an alkyne structure is selected from a chemotherapeutic agent, a molecular targeted drug, an immunoactivator, a toxin, an antibacterial agent, an antiviral agent, a diagnostic agent, a protein, a peptide, an amino acid, a nucleic acid molecule, a nucleic acid, an antigen, a lipid, a liposome, a vitamin, and a hormone.

20. 20. The method of claim 19, wherein the chemotherapeutic agent is selected from camptothecin, pyrrolobenzodiazepine, doxorubicin, auristatin, taxane, or a derivative thereof.

21. 20. The method of claim 19, wherein the immune activator is selected from a STING agonist, a TLR agonist, an A2AR antagonist, an IDO inhibitor, an antagonist of the CTLA-4, LAG-3, and PD-1 pathways, a checkpoint inhibitor, a vascular endothelial growth factor (VEGF) receptor inhibitor, a smoothen inhibitor, an alkylating agent, an antimetabolite, a retinoid, an anticancer vaccine, and an adjuvant.

22. The method according to any one of claims 19 to 21, wherein the molecule having an alkyne structure is selected from the group consisting of (A) to (E). (A) N-[4-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]glycylglycyl-L-valyl-N-{4-[({[(11'S,11a'S)-11'-hydroxy-7'-methoxy-8'-[(5-{[(11aS)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,10,11,11 a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl]oxy}pentyl)oxy]-5'-oxo-11',11a'-dihydro-1'H-spiro[cyclopropane-1,2'-pyrrolo[2,1-c][1,4]benzodiazepin]-10'(5'H)-yl]carbonyl}oxy)methyl]phenyl}-L-alaninamide, (B) N-[4-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]glycylglycyl-L-valyl-N-[4-({[(11'S,11'aS)-11'-hydroxy-7'-methoxy-8'-(3-{[(11aS)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,10,11,11 a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl]oxy}propoxy)-5'-oxo-11',11'a-dihydro-1'H,3'H-spiro[cyclopropane-1,2'-pyrrolo[2,1-c][1,4]benzodiazepine]-10'(5'H)-carbonyl]oxy}methyl)phenyl]-L-alaninamide, (C) N-[4-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]glycylglycyl-L-valyl-N-{4-[({[(11'S,11a'S)-11'-hydroxy-7'-methoxy-8'-[(5-{[(11a'S)-7'-methoxy-5'-oxo-5',11a'-dihydro-1'H-spiro[cyclo[ cyclopropane-1,2'-pyrrolo[2,1-c][1,4]benzodiazepine]-8'-yl]oxy}pentyl)oxy]-5'-oxo-11',11a'-dihydro-1'H-spiro[cyclopropane-1,2'-pyrrolo[2,1-c][1,4]benzodiazepine]-10'(5'H)-yl]carbonyl}oxy)methyl]phenyl}-L-alaninamide, (D) N-[4-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]glycylglycyl-L-valyl-N-{4-[({[(11'S,11a'S)-11'-hydroxy-7'-methoxy-8'-[(5-{[(11a'S)-7'-methoxy-5'-oxo-5',10',11',11a'-tetrahydro-1'H-spiro [cyclopropane-1,2'-pyrrolo[2,1-c][1,4]benzodiazepine]-8'-yl]oxy}pentyl)oxy]-5'-oxo-11',11a'-dihydro-1'H-spiro[cyclopropane-1,2'-pyrrolo[2,1-c][1,4]benzodiazepine]-10'(5'H)-yl]carbonyl}oxy)methyl]phenyl}-L-alaninamide, and (E) bis(N,N-diethylethanaminium) N-[4-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]glycylglycyl-L-phenylalanyl-N-[(2-{9-[(5R,7R,8R,12aR,14R,15R,15aR,16R)-15-fluoro-16-hydroxy-2,10-dioxo-2,10-disulfido-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ ５ , 10λ ５ -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-1-yl}ethoxy)methyl]glycinamide.

23. The production method according to any one of claims 13 to 22, wherein the acceptor molecule is an antibody or an Fc region-containing molecule having an N297-linked sugar chain consisting of core GlcNAc optionally having fucose attached thereto.

24. In the presence of endo-β-N-acetylglucosaminidase (enzyme A) which uses as substrates the complex type sugar chain of the polypeptide according to any one of claims 1 to 8 and a sugar chain donor molecule whose reducing end is not activated, but does not use as a substrate the N297-linked sugar chain, A method for producing an antibody or an Fc region-containing molecule, comprising reacting an acceptor molecule that is an antibody having a core GlcNAc to which fucose may be attached as an N297-linked glycan or an Fc region-containing molecule thereof with a glycan donor molecule that contains a GlcNAc whose reducing end is not activated.

25. The manufacturing method described in claim 24, characterized in that the polypeptide described in any one of claims 1 to 8, enzyme A, acceptor molecule and glycan donor molecule are reacted in the same reaction solution.

26. The method according to claim 24 or 25, wherein the sugar chain donor molecule is a complex sugar chain whose non-reducing end may be chemically modified.

27. The method according to any one of claims 24 to 26, wherein the sugar chain donor molecule is SGP, (SG-)Asn, (MSG1-)Asn, (MSG2-)Asn, or a mixture of (MSG1-)Asn and (MSG2-)Asn, each of which may have a chemically modified non-reducing end.

28. The sugar chain donor molecule is ([N ３ -PEG(3)] ２ -SG-)Asn-PEG(3)-N ３ , ([N ３ -PEG(3)]-MSG1-)Asn-PEG(3)-N ３ , ([N ３ -PEG(3)]-MSG2-)Asn-PEG(3)-N ３ , or ([N ３ -PEG(3)]-MSG1-)Asn-PEG(3)-N ３ and ([N ３ -PEG(3)]-MSG2-)Asn-PEG(3)-N ３ The method according to any one of claims 24 to 27, wherein the mixture is a mixture of

29. Furthermore, an azide group (N ３ 29. The method according to claim 28, comprising a step of reacting the hydroxyl group with a molecule having an alkyne structure.

30. The method according to claim 29, wherein the molecule having an alkyne structure is selected from the group consisting of chemotherapeutic agents, molecular targeted drugs, immunoactivators, toxins, antibacterial agents, antiviral agents, diagnostic agents, proteins, peptides, amino acids, nucleic acids, antigens, lipids, liposomes, vitamins, and hormones.

31. 31. The method of claim 30, wherein the chemotherapeutic agent is selected from camptothecin, pyrrolobenzodiazepine, doxorubicin, auristatin, taxane, or a derivative thereof.

32. The method of claim 30, wherein the immune activator is selected from a STING agonist, a TLR agonist, an A2AR antagonist, an IDO inhibitor, an antagonist of the CTLA-4, LAG-3, and PD-1 pathways, a checkpoint inhibitor, a vascular endothelial growth factor (VEGF) receptor inhibitor, a smoothen inhibitor, an alkylating agent, an antimetabolite, a retinoid, an anticancer vaccine, and an adjuvant.

33. The production method according to any one of claims 30 to 32, wherein the molecule having an alkyne structure is selected from the group consisting of (A) to (E). (A) N-[4-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]glycylglycyl-L-valyl-N-{4-[({[(11'S,11a'S)-11'-hydroxy-7'-methoxy-8'-[(5-{[(11aS)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,10,11,11 a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl]oxy}pentyl)oxy]-5'-oxo-11',11a'-dihydro-1'H-spiro[cyclopropane-1,2'-pyrrolo[2,1-c][1,4]benzodiazepin]-10'(5'H)-yl]carbonyl}oxy)methyl]phenyl}-L-alaninamide, (B) N-[4-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]glycylglycyl-L-valyl-N-[4-({[(11'S,11'aS)-11'-hydroxy-7'-methoxy-8'-(3-{[(11aS)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,10,11,11 a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl]oxy}propoxy)-5'-oxo-11',11'a-dihydro-1'H,3'H-spiro[cyclopropane-1,2'-pyrrolo[2,1-c][1,4]benzodiazepine]-10'(5'H)-carbonyl]oxy}methyl)phenyl]-L-alaninamide, (C) N-[4-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]glycylglycyl-L-valyl-N-{4-[({[(11'S,11a'S)-11'-hydroxy-7'-methoxy-8'-[(5-{[(11a'S)-7'-methoxy-5'-oxo-5',11a'-dihydro-1'H-spiro[cyclo[ cyclopropane-1,2'-pyrrolo[2,1-c][1,4]benzodiazepine]-8'-yl]oxy}pentyl)oxy]-5'-oxo-11',11a'-dihydro-1'H-spiro[cyclopropane-1,2'-pyrrolo[2,1-c][1,4]benzodiazepine]-10'(5'H)-yl]carbonyl}oxy)methyl]phenyl}-L-alaninamide, (D) N-[4-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]glycylglycyl-L-valyl-N-{4-[({[(11'S,11a'S)-11'-hydroxy-7'-methoxy-8'-[(5-{[(11a'S)-7'-methoxy-5'-oxo-5',10',11',11a'-tetrahydro-1'H-spiro [cyclopropane-1,2'-pyrrolo[2,1-c][1,4]benzodiazepine]-8'-yl]oxy}pentyl)oxy]-5'-oxo-11',11a'-dihydro-1'H-spiro[cyclopropane-1,2'-pyrrolo[2,1-c][1,4]benzodiazepine]-10'(5'H)-yl]carbonyl}oxy)methyl]phenyl}-L-alaninamide, and (E) bis(N,N-diethylethanaminium) N-[4-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]glycylglycyl-L-phenylalanyl-N-[(2-{9-[(5R,7R,8R,12aR,14R,15R,15aR,16R)-15-fluoro-16-hydroxy-2,10-dioxo-2,10-disulfido-14-(6,7,8,9-tetrahydro-2H-2,3,5,6-tetraazabenzo[cd]azulen-2-yl)octahydro-2H,10H,12H-5,8-methano-2λ ５ , 10λ ５ -furo[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-1-yl}ethoxy)methyl]glycinamide.

34. The production method according to any one of claims 24 to 33, wherein the acceptor molecule is an antibody or an Fc region-containing molecule having an N297-linked sugar chain consisting of core GlcNAc optionally having fucose attached thereto.

35. The production method according to any one of claims 24 to 34, wherein the enzyme A is an enzyme having an activity of transferring a glycosyl chain from SGP to an acceptor having GlcNAc.

36. The production method according to any one of claims 24 to 35, wherein the enzyme A is Endo-M, Endo-Rp, Endo-Om, Endo-CC, or a mutant enzyme thereof in which the hydrolysis activity thereof has been reduced.

37. The mutant enzymes with reduced hydrolytic activity are Endo-Rp N172Q, Endo-Rp N172H, Endo-Rp N172A, Endo-Rp N172C, Endo-Rp N172D, Endo-Rp N172E, Endo-Rp N172G, Endo-Rp N172I, Endo-Rp N172L, Endo-Rp N172M, Endo-Rp N172P, Endo-Rp N172S, Endo-Rp N172T, Endo-Rp N172V, Endo-Rp W278F / S216V, and Endo-Rp N172C.

37. The method of claim 36, wherein the endothelial cell line is selected from the group consisting of Endo-Rp W278F / N246D, Endo-Rp W278F / D276N, Endo-Rp W278F / A310D, Endo-Rp W278F / N172D / F307Y, Endo-Rp W278F / N172D / F307H, Endo-Rp W278F / N172D / A310D, Endo-Rp W214F / F307Y / L306I, Endo-M N175Q, Endo-CC N180H, and Endo-Om N194Q.

38. A method for producing an antibody or an Fc region-containing molecule having only core GlcNAc optionally with fucose attached, comprising reacting the antibody or the Fc region-containing molecule with the polypeptide of any one of claims 1 to 8.

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