Endoglycosidase s2 mutant, preparation method therefor and use thereof in antibody glycosylation modification

Abstract

An endoglycosidase S2 mutant, a preparation method therefor and the use thereof in antibody glycosylation modification. The endoglycosidase S2 mutant comprises a carbohydrate-binding module CBM defined by M677-D843 in SEQ ID NO: 1, and the amino acid sequence of the endoglycosidase S2 mutant comprises SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5.

Description

Endoglycosidase S2 mutant, preparation method thereof and application thereof in antibody glycosylation modification Technical Field

[0001] The present application belongs to the field of glycoengineered or enzyme-engineered antibodies, and specifically relates to a glycosidase S2 mutant with antibody transglycosylation function, a preparation method thereof, and its application in antibody glycosylation modification. Background Art

[0002] The Fc glycan of IgG antibodies is mainly a core-fucosylated biantennary complex disaccharide structure. After co-incubation with Endo-S2 under certain conditions, the glycosylation at the N297 site of the antibody will be hydrolyzed and deglycosylated, thereby exposing a GlcNAc disaccharide structure with core fucose.

[0003] Endo-S2 is an endoglycosidase belonging to glycoside hydrolase family 18 (GH18). Like its family members, Endo S, Endo F1, Endo F2, and Endo F3, Endo-S2 can hydrolyze and deglycosylate a wide range of glycoforms on glycoproteins, with particular activity against IgG Fc deglycosylation. Related literature reports the molecular basis for the broad-spectrum N-glycan specificity and processing of therapeutic IgG monoclonal antibodies by Endo-S2, the crystal structure of S2, and the description of its hydrolytic activity.

[0004] According to previous reports, a series of single amino acid mutants obtained by mutating amino acids at key sites of glycoside hydrolases, such as the D233 mutant (based on Endo S) and the D184 mutant (based on Endo-S2) (see CN109071630A), have obvious hydrolysis activity inhibition. CN108026518A discloses a series of Endo S mutant enzymes that exhibit reduced hydrolytic activity for binding to (N297-linked sugar chains of Asn at position 297 in IgG, and CN110234341A discloses a series of Endo-S2 mutant enzymes with improved transglycosylation activity and reduced hydrolytic activity. These mutant enzymes with inhibited hydrolytic activity play a key role in the protein / antibody glycoengineering process. This discovery further demonstrates that Endo S and Endo-S2 have a high degree of structural consistency, and that each domain plays the same or similar role in the enzyme's catalytic reaction. Based on literature reports, Endo-S and Endo-S2 exhibit differences in activity in recognizing different glycoform substrates, while previous reports have focused on exploring hydrolytic activity against IgG substrates.

[0005] In the glycosylation modification of antibodies, enzymes play a special and important role. In antibody-drug conjugates, enzyme site-specific conjugation technology, as a third-generation technology, has developed a class of enzymes with transglycosylation activity, or both hydrolysis and transglycosylation activity, which has good application prospects in the fields of antibodies and antibody-drug conjugates.

[0006] Summary of the Invention

[0007] One of the technical objectives of the present invention is to provide a glycosidase S2 mutant that fuses different structural domains of Endo-S and Endo-S2, which has hydrolytic activity comparable to that of wild-type Endo-S2 and more stable transglycosylation activity compared to wild-type Endo-S2. Therefore, it can be effectively used for antibody-drug conjugation.

[0008] Another technical purpose of the present invention is to provide a method for preparing the above-mentioned endoglycosidase S2 mutant.

[0009] Another technical purpose of the present invention is to provide a polynucleotide encoding the above-mentioned endoglycosidase S2 mutant.

[0010] Another technical object of the present invention is to provide a recombinant vector comprising the above-mentioned polynucleotide.

[0011] Another technical purpose of the present invention is to provide a host cell transformed with the above-mentioned recombinant vector.

[0012] Another technical purpose of the present invention is to provide the use of the above-mentioned endoglycosidase S2 mutant in antibody glycosylation modification.

[0013] Another technical object of the present invention is to provide a method for glycosylation modification of antibodies by using the above-mentioned endoglycosidase S2 mutant.

[0014] In one aspect, the present invention provides a glycosidase S2 mutant comprising a carbohydrate binding module CBM defined by M677-D843 in SEQ ID NO: 1, and the amino acid sequence of the glycosidase S2 mutant comprises SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5.

[0015] On the other hand, the present invention provides a method for preparing the above-mentioned endoglycosidase S2 mutant, the method comprising:

[0016] 1) ligating the target gene encoding the endoglycosidase S2 mutant according to claim 1 or 2 to an expression vector (e.g., pBSYA1S1Z plasmid, pPICZα plasmid, pPIC9K plasmid, pPIC3K plasmid, pPICZA plasmid, pPIC3.5K plasmid, pET22b plasmid, pET28a plasmid, pET30a plasmid, pET32a plasmid, pGEX-6p-1 plasmid, pTrcHis plasmid, pLysS plasmid, pLysE plasmid, pBAD plasmid, pCS plasmid, pGEX-4T-1 plasmid, pET SUMO plasmid, pUC19 plasmid, pBR322 plasmid, pYES2 plasmid, pBV plasmid, pHT01 plasmid and pHT254 plasmid) to construct a recombinant vector;

[0017] 2) transforming the recombinant vector into a host cell (e.g., Pichia pastoris cells, Escherichia coli cells, Saccharomyces cerevisiae cells, insect ovary cells, Bacillus subtilis cells) to obtain a recombinant cell, and

[0018] 3) obtaining the endoglycosidase S2 mutant by expressing the target gene in a recombinant cell,

[0019] Preferably, the method further comprises step 4): isolating and purifying the endoglycosidase S2 mutant expressed in step 3).

[0020] In another aspect, the present invention provides a polynucleotide encoding the above-mentioned endoglycosidase S2 mutant, preferably, its sequence comprises SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9.

[0021] In another aspect, the present invention provides a recombinant vector comprising the above polynucleotide.

[0022] In another aspect, the present invention provides a host cell transformed with the above-mentioned recombinant vector, wherein the host cell is a prokaryotic cell or a eukaryotic cell. Preferably, the host cell is selected from Pichia pastoris cells, Escherichia coli cells, Saccharomyces cerevisiae cells, insect ovary cells, and Bacillus subtilis cells.

[0023] In another aspect, the present invention provides the use of the above-mentioned endoglycosidase S2 mutant in antibody glycosylation modification.

[0024] The glycosylation modification includes hydrolysis of sugar groups in antibodies, transglycosylation of antibodies, or both.

[0025] For example, the glycosylation modification includes preparing an antibody drug conjugate by a one-step method using an antibody and a disaccharide-small molecule drug conjugate.

[0026] Wherein, the disaccharide-small molecule drug conjugate is represented by the following formula III:

[0027] In Formula III, represents a monosaccharide selected from galactose, mannose and glucose, wherein the C5 position is substituted by Y', and the linkage between disaccharides is a 1,4 glycosidic bond,

[0028] Y' is

[0029] Wherein, L1 represents a dipeptide linker, for example, -Val-Cit-, -Val-Ala-;

[0030] PAB represents p-aminophenylethanol unit;

[0031] D represents a small molecule drug unit, for example, MMAE, MMAF or maytansine;

[0032] represents the attachment position to the monosaccharide,

[0033] Preferably, the disaccharide-small molecule drug conjugate has the following structure:

[0034] Alternatively, the glycosylation engineering comprises preparing an antibody drug conjugate by a two-step method using an antibody and a sugar substrate of the following formula II:

[0035] In Formula II, represents a monosaccharide selected from galactose, mannose and glucose, wherein the C5 position is substituted by Y,

[0036] Wherein, Y is selected from the following structures:

[0037] wherein R1 represents a C1-C7 alkyl group, preferably a C2-C6 alkyl group, preferably a C3-C5 alkyl group, preferably a C3-C4 alkyl group, such as methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl;

[0038] m represents an integer from 1 to 6, for example, m is 1, 2, 3, 4, 5, or 6;

[0039] n represents an integer from 1 to 24, for example, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24;

[0040] L1 represents a dipeptide linker, for example, -Val-Cit-, -Val-Ala-;

[0041] PAB represents p-aminophenylethanol unit;

[0042] D represents a small molecule drug unit, such as MMAE and MMAF;

[0043] Indicates the position of attachment to the monosaccharide.

[0044] In another aspect, the present invention provides a method for glycosylation modification of an antibody, the method comprising using the above-mentioned endoglycosidase S2 mutant in the glycosylation modification process.

[0045] In a specific embodiment, the antibody is selected from the group consisting of antibodies having glycosylation sites.

[0046] In a specific embodiment, the antibody is an antibody having N-glycosylation sites.

[0047] In a specific embodiment, the antibody is a biantennary antibody having N-glycosylation sites.

[0048] In a specific embodiment, the antibody is an IgG with a conserved N-glycosylation site at N297 of the Fc region.

[0049] In a specific embodiment, the antibody is a monoclonal antibody, a polyclonal antibody, a bifunctional antibody, a trifunctional antibody, a nanobody fused with an Fc domain, a therapeutic antibody or a functional antibody derived from a different species.

[0050] Specifically, the antibody is a human antibody, a murine antibody or a chimeric antibody;

[0051] Specifically, the antibody is IgG1, IgG2, or IgG4; in a specific embodiment, the targets of the antibody include HER2, Claudin 18.2, EGFR, c-Met, NECTIN4, CD276, HER3, CD3, FOLR1, BCMA, CD20, DLL3, MUC1, PD-L1, ROR1, TF, CD19, CD22, CD30, CD70, CD79B, FGFs, MSLN, NT5E, TNFα, CD147, CD24, CD38, CD47, CDH3, CDK4, CDK6, CEACAM5, CLDN6, CTLA4, DDR1, DR5, FAPα, FGFR3, GPRC5D, GR, HLA-DR, ICAM1, IL2R, MELTF, ROR2, TPBG (5T4), VTCN1, ZIP6, CD33, CD25, RSV, VEGF, RANKL, VEGFR2, CTLA-4, CD52, CD319, PD-1, CD274, IgE, IL-6, IL-12, IL-2, C5, IL-17A, CD25, SLAMF7, F10, factor IXa, HAb18G, PCSK9, BLyS, IL23, α4β7, IL-4R-α, HAE, FGF23 and IL6R; preferably, the targets of the antibody include HER2, CD20, EGFR, PD-1;

[0052] In a specific embodiment, the antibody comprises trastuzumab, rituximab, pertuzumab, panitumumab, toripalimab, toripalimab, or nivolumab.

[0053] In a specific embodiment, the method comprises:

[0054] 1) A method for hydrolyzing glycosides in an antibody, wherein the antibody is incubated with the above-mentioned endoglycosidase S2 mutant; and / or

[0055] 2) using a sugar substrate, performing glycosylation modification on the antibody under the transglycosylation action of the endoglycosidase S2 mutant,

[0056] in,

[0057] The sugar substrate is a disaccharide represented by the following formula I:

[0058] in, It represents a monosaccharide selected from galactose, mannose and glucose, and the linkage between disaccharides is a 1,4 glycosidic bond;

[0059] or,

[0060] The sugar substrate is a disaccharide derivative represented by the following formula II:

[0061] In Formula II, represents a monosaccharide selected from galactose, mannose and glucose, wherein the C5 position is substituted by Y, wherein Y is selected from the following structures:

[0062] wherein R1 represents a C1-C7 alkyl group, preferably a C2-C6 alkyl group, preferably a C3-C5 alkyl group, preferably a C3-C4 alkyl group, such as methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl;

[0063] m represents an integer from 1 to 6, for example, m is 1, 2, 3, 4, 5, or 6;

[0064] n represents an integer from 1 to 24, for example, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24;

[0065] L1 represents a dipeptide linker, for example, -Val-Cit-, -Val-Ala-;

[0066] PAB represents p-aminophenylethanol unit;

[0067] D represents a small molecule drug unit, such as MMAE and MMAF;

[0068] represents the attachment position to the monosaccharide,

[0069] or,

[0070] The sugar substrate is a disaccharide-small molecule drug conjugate represented by the following formula III:

[0071] In Formula III, represents a monosaccharide selected from galactose, mannose and glucose, wherein the C5 position is substituted by Y',

[0072] Y' is

[0073] Wherein, L1 represents a dipeptide linker, for example, -Val-Cit-, -Val-Ala-;

[0074] PAB represents p-aminophenylethanol unit;

[0075] D represents a small molecule drug unit, such as MMAE and MMAF;

[0076] represents the attachment position to the monosaccharide,

[0077] Specifically, the sugar substrate is selected from:

[0078] or,

[0079] The sugar substrate is a sugar substrate in the ring-opened form of the oxazoline ring in the above formula I, II or III;

[0080] Preferably, the method further comprises reacting the glycosylated antibody modified with the sugar substrate represented by Formula II with a drug-linker to prepare an antibody-drug conjugate. Preferably, the drug-linker is a hydroxylamine-containing functional group, such as NH2O-VC-PAB-MMAE. Beneficial effects

[0081] The endoglycosidase S2 mutants S2M3 and V3 obtained by fusing different structural domains of Endo-S and Endo-S2 have hydrolysis activities comparable to those of wild-type Endo-S2.

[0082] The modified endoglycosidase S2 mutants S2M3 and V3 obtained by the present invention can be used for antibody glycosylation modification and have good transglycosylation activity on various glycoforms such as LacNAc-ox, Man-GlcNAc-ox, Glc-GlcNAc-ox and their derivatives. Among them, S2M3 has more stable transglycosylation activity than Endo-S2.

[0083] The modified endoglycosidase S2 mutants S2M3 and V3 obtained by the present invention can be effectively used for antibody-drug coupling, and thus can be used to prepare ADC. BRIEF DESCRIPTION OF THE DRAWINGS

[0084] Figure 1 shows the hydrolysis of trastuzumab N-glycosylation site glycans by Endo-S2, S2M1, S2M3, S2M4, and V3. Panel A shows the hydrolysis of antibody N-glycosylation site glycans after 1 hour of incubation with each enzyme, and Panel B shows the hydrolysis of antibody N-glycosylation site glycans after 4 hours of incubation with each enzyme.

[0085] FIG2 shows the results of the transglycosidation activity test of endoglycosidase using Acylhydrazone-LacNAc-ox as the sugar substrate. A total of four mutant enzymes were tested.

[0086] FIG3 shows the RP detection results (A) and LC-MS (B) of the antibody drug conjugate (ADC) prepared in Example 4.

[0087] FIG4 shows the transglycosylation activities of S2M1, S2M4, and V3 after increasing the sugar substrate equivalent.

[0088] FIG5 shows the results of the transglycosylation activity test of the mutant enzymes (S2M1, S2M3, S2M4, V3) using MMAE-PAB-VC-NH-LacNAc-ox as the sugar substrate in a one-step method.

[0089] FIG6 shows the effect of the amount of Endo-S2 and S2M3 enzymes on the transglycosylation activity of the Az-LacNAc-ox antibody.

[0090] FIG7 shows the effect of the amount of Endo-S2 and S2M3 enzymes on the transglycosylation activity of the diAz-LacNAc-ox antibody.

[0091] FIG8 shows the results of the analysis of the effect of reaction time on the transglycosylation activity of diAz-LacNAc-ox antibody under different enzyme amounts of Endo-S2 and S2M3. DETAILED DESCRIPTION

[0092] The technical solutions of the present application are described in detail below through specific embodiments so that those skilled in the art can better understand the present application. However, these embodiments are not intended to limit the scope of the present application.

[0093] the term

[0094] In this application, "endoglycosidase S2 mutant", "novel endoglycosidase", and "mutant enzyme" can be used interchangeably.

[0095] In this application, the monosaccharide symbols of the antibody glycosylation sites represent the following meanings: is galactose, ■ is N-acetylglucosamine, For mannose, Fucose.

[0096] In this application, the transglycosylation activity of the novel endoglycosidase was tested, and the following sugar substrates Az-LacNAc-ox, diAz-LacNAc-ox, Az-PEG3-Man-GlcNAc-ox, Az-PEG3-Glc-GlcNAc-ox, Acylhydrazone-LacNAc-ox, and MMAE-PAB-VC-NH-LacNAc-ox were obtained by customization:

[0097] In this application, the drug-linker NH2O-VC-PAB-MMAE was obtained by customization.

[0098] Az-LacNAc-ox refers to G3 of CN114949236A.

[0099] diAz-LacNAc-ox refers to G14 of CN114949236A.

[0100] Az-PEG3-LacNAc-ox is commercially available.

[0101] Az-PEG6-LacNAc-ox is commercially available.

[0102] Az-PEG 12 -LacNAc-ox is commercially available.

[0103] Az-PEG 24 -LacNAc-ox is commercially available.

[0104] Az-PEG3-Man-GlcNAc-ox is commercially available.

[0105] Az-PEG3-Glc-GlcNAc-ox is commercially available.

[0106] Acylhydrazone-LacNAc-ox is commercially available.

[0107] NH2O-VC-PAB-MMAE is compound D1 in CN114949236A.

[0108] General Procedure 1: Expression and Purification of Endoglycosidase

[0109] Connect the target gene (SEQ ID NO:7 / SEQ ID NO:8 / SEQ ID NO:9 / SEQ ID NO:10) to the pET22b(+) vector to construct a plasmid. Transform the plasmid into E.coli DH5α competent cells for cloning. Take 1μL of the constructed plasmid (10ng / μL) and transform it into 50μL of E.coli DH5α competent cells and let it stand in an ice bath for 30min. Heat shock in a 42℃ water bath for 90sec, then transfer to ice and let it stand for 3-5min. Add 500μL of LB culture medium without antibiotics, mix well and shake at 37℃ and 220rpm for 1-2h. Then take 200-300μL of bacterial solution and spread it on an LB plate containing ampicillin antibiotics, invert it in a constant temperature incubator, and culture it overnight at 37℃. The next day, pick a single clone and expand it in 3-5mL LB culture medium. Wait until OD 600 When the concentration reaches 0.6-0.8, 50 μL of bacterial solution is transferred to a sequencing company for Sanger sequencing.

[0110] After the sequencing results are confirmed to be correct, the plasmid is transformed into E.coli BL21 (DE3) competent cells, and IPTG is used to induce the expression of endoglycosidase. Take 1 μL of plasmid (10 ng / μL) and transform it into 50 μL of E.coli BL21 (DE3) competent cells, and let it stand in an ice bath for 30 minutes. Heat shock in a 42°C water bath for 90 seconds, then transfer to ice and let it stand for 3 to 5 minutes. Add 500 μL of LB culture medium without antibiotics, mix well, and shake at 37°C and 220 rpm for 1 to 2 hours. Then take 200 to 300 μL of bacterial solution and spread it on the LB plate containing ampicillin antibiotics, invert it in a constant temperature incubator, and culture it overnight at 37°C. The next day, pick a single clone and expand it in 3 to 5 mL of LB culture medium. Wait until OD 600 When the pH reaches 0.6-0.8, add IPTG to the bacterial solution to a final concentration of 0.5 mM. Incubate the solution at 20°C, 200 rpm, and allow overnight expression. After expression is complete, centrifuge at 4500 rpm for 20 minutes, discard the supernatant, and collect the cells. Resuspend the cells in 30 mL of phosphate buffer (0.01 M, pH 7.4), disrupt with ultrasound, and centrifuge at 12000 rpm for 15 minutes. Collect the supernatant for protein purification.

[0111] Purify the overexpressed endoglycosidase using nickel affinity chromatography. Equilibrate the column with five column volumes of phosphate buffer (0.01M, pH 7.4). Then, load the supernatant from the above-mentioned centrifugation onto the column. Repeat three times to allow the target protein to bind to the column packing. Elute with 10 column volumes of 50mM, 100mM, and 250mM imidazole, respectively. Collect the eluates in separate tubes.

[0112] The eluate was subjected to polyacrylamide gel electrophoresis to determine the location of the sample, concentrated by ultrafiltration, and the buffer was replaced with phosphate buffer (0.01 M, pH 7.4).

[0113] The protein concentration was determined by UV280, and the molecular weight was determined by LC-MS.

[0114] General operation 2: endoglycosidase specific hydrolysis antibody N-glycan activity test

[0115] Trastuzumab (5 mg / mL) was added to phosphate buffer (pH 7.0), followed by the addition of endoglycosidase (100 ng / mg). The mixture was incubated at 30°C for 1 hour and 4 hours, and samples were collected for LC-MS analysis.

[0116] General Operation 3: Antibody Glycosylation Modification Using Endoglycosidase

[0117] The antibody (5 mg / mL) and oxazoline (0.3-2 mM) (herein also referred to as "ox") were added to a phosphate buffer (pH 6.5-7.0). After adjusting the pH of the solution to 6.5-7.0, endoglycosidase (0.1-0.8 mg / mL) was added. The mixture was incubated at 30°C for 2 hours and then analyzed by SDS-PAGE or LC-MS.

[0118] The following operations of this application are all completed based on the above general operations 1-3.

[0119] Example 1: Transformation of endoglycosidase mutants

[0120] Based on the wild-type Endo-S2 and Endo S, their different domains were combined and fused to obtain four mutants, named V3, S2M1, S2M3, and S2M4, as shown in Table 1 below.

[0121] Table 1 Structural information of novel endoglycosidases

[0122] Example 2: Expression and purification of the novel endoglycosidase designed in Example 1

[0123] The novel endoglycosidases V3, S2M1, S2M3, and S2M4 were expressed and purified using the general procedure 1.

[0124] Example 3: Antibody glycosylation hydrolysis activity test of the new endoglycosidase

[0125] The antibody glycosylation hydrolysis activities of the novel endoglycosidases V3, S2M1, S2M3, and S2M4 were tested using General Procedure 2. The results are shown in Figure 1. The results show that all of the modified mutant enzymes can hydrolyze wild-type antibody glycans. S2M3 has superior hydrolysis activity compared to Endo-S2, while the hydrolysis activities of S2M1, S2M4, and V3 are comparable to Endo-S2.

[0126] Example 4: Novel endoglycosidase achieves transglycosylation of Acylhydrazone-LacNAc-ox antibody and two-step preparation of sugar chain-directed ADC compound

[0127] Step 1: Acylhydrazone-LacNAc-ox (0.5 mM) was added to phosphate buffer (pH 6.5). Wild-type Herceptin (5 mg / mL) and endoglycosidases (40 μg / mg, S2M1, S2M3, S2M4, and V3, respectively) were added to the reaction system in sequence, and the mixture was incubated at 30°C for 2 hours. Mass spectrometry results showed that S2M3 had the best transglycosylation activity for Acylhydrazone-LacNAc-ox, with a very high ratio of coupling to two sugar substrates. V3's transglycosylation activity was weaker than that of S2M3, while S2M1 and S2M4 had poorer transglycosylation activity.

[0128] (Purification): Use AKTA pure 150 (chromatographic column: Merck VL 11 250; filler: Mabselect Sure XL 2 mL) to remove excess Endo-S2 and sugar substrate in the sample.

[0129] Step 2: Add 50 mM NaOAc buffer (pH 4.6) and DMSO (10% V / V) to the sugar substrate-modified antibody solution obtained in step 1, followed by 10 mg / mL H2NO-VC-PAB-MMAE drug-linker intermediate solution, and incubate at 30°C for 2 hours.

[0130] Figure 2 shows the results of transglycosylation activity testing of four endoglycosidase mutants using Acylhydrazone-LacNAc-ox as the sugar substrate. Figure 2 shows that S2M3 exhibits the best transglycosylation activity toward Acylhydrazone-LacNAc-ox, with a very high ratio of coupling to both sugar substrates. V3 exhibits weaker transglycosylation activity than S2M3, while S2M1 and S2M4 exhibit relatively poor transglycosylation activity.

[0131] The ADC compound prepared above was subjected to RP detection and LC-MS detection: the detection results are shown in Table 2 below and Figure 3.

[0132] Table 2:

[0133] Example 5: Transglycosylation activity of S2M1, S2M4, and V3 under increased sugar substrate equivalents

[0134] The same procedures as in Example 4 were followed, except that the sugar substrate equivalent was increased to 60 relative to wild-type Herceptin, and the transglycosylation effect was measured. The results are shown in Figure 4. As can be seen, after increasing the sugar substrate equivalent, all three enzymes, S2M1, S2M4, and V3, exhibited a certain degree of transglycosylation activity, with S2M4 outperforming V3, which in turn outperformed S2M1. This indicates that S2M4 and V3, which contain the CBM region of Endo-S2, exhibited superior transglycosylation activity.

[0135] Example 6: One-step preparation of sugar chain-directed ADC compounds using a novel endoglycosidase

[0136] MMAE-PAB-VC-NH-LacNAc-ox (0.5 mM) was added to phosphate buffer (pH 6.5). Wild-type Herceptin (5 mg / mL) and endoglycosidases (80 μg / mg, S2M1, S2M3, S2M4, and V3, respectively) were added to the reaction system, followed by incubation at 30°C for 2 hours. The results are shown in Figure 5.

[0137] S2M3 has the best transglycosylation activity, while S2M1, S2M4, and V3 all have some transglycosylation activity. S2M4 is superior to V3, which in turn is superior to S2M1. This suggests that S2M3, S2M4, and V3, which contain the CBM region of Endo-S2, have superior transglycosylation activity.

[0138] Example 7: Endo-S2 and S2M3 enzymes for the transglycosylation activity of antibodies on Az-LacNAc-ox substrates

[0139] Az-LacNAc-ox (0.5 mM, structural formula shown below) was added to phosphate buffer (pH 6.5). Trastuzumab (5 mg / mL) and endoglycosidase (Endo-S2 or S2M3, 0.02-2.4 mg / mL) were then added to the reaction system. The mixture was incubated at 30°C for 2 hours, and LC-MS analysis was performed.

[0140] The specific design is shown in Table 3:

[0141] Table 3 Reaction conditions of Endo-S2 and S2M3 enzyme amounts for the transglycosylation activity of antibodies on Az-LacNAc-ox substrate

[0142] As shown in Table 3, the antibody transglycosylation activity was tested at Endo-S2 or S2M3 enzyme amounts of 4, 20, 40, 80, 160, 240, 320, and 480 μg / mg (enzyme amount / antibody amount), wherein the trastuzumab concentration was 5 mg / mL, the sugar substrate Az-LacNAc-ox concentration was 0.5 mM, and the reaction was carried out in a 50 mM phosphate, pH 6.5 buffer system. LC-MS analysis was performed after reacting at 30°C for 2 hours, as shown in Figure 6.

[0143] The results showed that at low enzyme dosages (approximately <50 μg / mg), both wild-type Endo-S2 and mutant enzyme S2M3 positively promoted DAR values. At higher enzyme dosages, Endo-S2 showed a decreasing DAR value with increasing enzyme dosage, while S2M3 exhibited a longer plateau and maintained high DAR values ​​over a wide range (approximately 100-500 μg / mg), indicating that mutant enzyme S2M3 possesses more stable transglycosylation activity than Endo-S2.

[0144] Example 8: Endo-S2 and S2M3 enzymes for diAz-LacNAc-ox substrate antibody transglycosylation activity test

[0145] diAz-LacNAc-ox (0.5 mM, structural formula shown) was added to phosphate-HCl buffer (pH 6.5). Trastuzumab (5 mg / mL) and endoglycosidase (0.02-2.4 mg / mL) were then added to the reaction system. The mixture was incubated at 30°C, and samples were collected at different time points for LC-MS analysis.

[0146] The specific design is shown in Table 4:

[0147] Table 4 Reaction conditions of Endo-S2 and S2M3 enzyme amounts for antibody transglycosylation activity on diAz-LacNAc-ox substrate

[0148] As shown in Table 4, the antibody transglycosylation activity was tested at Endo-S2 or S2M3 enzyme amounts of 4, 20, 40, 80, 160, 240, 320, and 480 μg / mg (enzyme amount / antibody amount), wherein the trastuzumab concentration was 5 mg / mL, the sugar substrate diAz-LacNAc-ox concentration was 0.5 mM, the reaction was carried out in a 50 mM phosphate, pH 6.5 buffer system, the reaction was carried out at 30°C, and samples were taken at different time points for LC-MS analysis, as shown in Figure 7.

[0149] The results showed that as the reaction time increased, at various enzyme dosages, the mutant enzyme S2M3 exhibited lower and slower hydrolysis activity than Endo-S2, making it more conducive to process production.

[0150] Experimental results:

[0151] Based on the results of Examples 5-8, the mutant enzyme S2M3 demonstrated significant superiority over Endo-S2 in antibody transglycosylation reactions on various sugar substrates. The reaction curve of mutant S2M3 exhibited distinct enzymatic reaction characteristics, with a stable plateau phase and consistently high product DAR values ​​across a wide range of enzyme dosages. This demonstrates that after hydrolyzing the antibody Fc sugar chain, the mutant enzyme S2M3, through its efficient transglycosylation activity, transfers the Az-LacNAc-ox sugar substrate to the antibody, but lacks hydrolysis activity on the transglycosylated glycoengineered antibody. This efficient and stable transglycosylation activity provides the mutant enzyme with robust enzyme dosage in the preparation of antibody conjugates. While the longer plateau phase mitigates the effect of enzyme dosage on product DAR values, the reduced substrate hydrolysis activity further highlights the enhanced transfer activity of the mutant enzyme. In summary, this mutant enzyme possesses superior practical application value in the process.

[0152] Example 9: Antibody transglycosylation activity test on diAz-LacNAc-ox substrate with the same enzyme dosage of Endo-S2 and S2M3 and different reaction times

[0153] The operation was the same as in Example 6. For details, see Table 5 below. The effect of reaction time on the antibody transglycosylation activity of the diAz-LacNAc-ox substrate was investigated under the same enzyme amounts of Endo-S2 and S2M3.

[0154] Table 5 Reaction conditions of Endo-S2 and S2M3 enzyme amounts for antibody transglycosylation activity on diAz-LacNAc-ox substrate

[0155] The results are shown in FIG8 . As can be seen from FIG8 , as the reaction time increases, at various enzyme amounts, compared with Endo-S2, the mutant enzyme S2M3 shows lower and slower hydrolysis activity, which is more conducive to process production.

[0156] In the following, general procedure 3 was used to prepare non-natural glycoengineered antibodies using different antibodies and different sugar substrates as raw materials.

[0157] Example 10: Synthesis of Ab-1

[0158] The non-natural glycoengineered antibody Ab-1 was obtained by combining the compound Az-LacNAc-ox with the wild-type antibody Rituxan (Human IgG1) using General Procedure 3 (S2M3 enzyme). The measured value after HRMS deconvolution was 145780.

[0159] Example 11: Synthesis of Ab-2

[0160] The non-natural glycoengineered antibody Ab-2 was obtained by combining the compound Az-LacNAc-ox with the wild-type antibody pertuzumab (Human IgG1) using General Procedure 3 (S2M3 enzyme). The observed value after HRMS deconvolution was 146791.

[0161] Example 12: Synthesis of Ab-3

[0162] The unnatural glycoengineered antibody Ab-3 was obtained by combining the compound Az-LacNAc-ox with the wild-type antibody panitumumab (Human IgG2) using General Procedure 3 (S2M3 enzyme). The observed value after HRMS deconvolution was 145631.

[0163] Example 13: Synthesis of Ab-4

[0164] The non-natural glycoengineered antibody Ab-4 was obtained by combining the compound Az-LacNAc-ox with the wild-type antibody toripalimab (Human IgG4) using General Procedure 3 (S2M3 enzyme). The observed value after HRMS deconvolution was 148616.

[0165] Example 14: Synthesis of Ab-5

[0166] The unnatural glycoengineered antibody Ab-5 was synthesized from the compound Az-LacNAc-ox and the wild-type antibody nivolumab (Human IgG4) using General Procedure 3 (S2M3 enzyme). The observed value after HRMS deconvolution was 144929.

[0167] Example 15: Synthesis of Ab-6

[0168] The non-natural glycoengineered antibody Ab-6 was obtained by reacting the compound Az-LacNAc-ox with murine monoclonal antibody-1 via general procedure 3 (S2M3 enzyme). The HRMS deconvolution result was 146927.

[0169] Example 16: Synthesis of Ab-7

[0170] The non-natural glycoengineered antibody Ab-7 was obtained by reacting the compound Az-LacNAc-ox with murine monoclonal antibody-2 via general procedure 3 (S2M3 enzyme). The HRMS deconvolution result was 147312.

[0171] Example 17: Synthesis of Ab-8

[0172] The non-natural glycoengineered antibody Ab-8 was obtained by combining the compound Az-LacNAc-ox and murine monoclonal antibody-3 using general procedure 3 (S2M3 enzyme). The observed value after HRMS deconvolution was 146550.

[0173] Example 18: Synthesis of Ab-9

[0174] The unnatural glycoengineered antibody Ab-9 was obtained by combining the compound Man-GlcNAc-ox and trastuzumab via general procedure 3 (S2M3 enzyme). The observed value after HRMS deconvolution was 146590.

[0175] Example 19: Synthesis of Ab-10

[0176] The unnatural glycoengineered antibody Ab-10 was obtained by combining the compound Glc-GlcNAc-ox and trastuzumab via general procedure 3 (S2M3 enzyme). The observed value after HRMS deconvolution was 146591.

[0177] Example 20: Synthesis of Ab-11

[0178] The non-natural glycoengineered antibody Ab-11 was prepared by combining the compound Az-PEG3-Man-GlcNAc-ox and trastuzumab via general procedure 3 (S2M3 enzyme). The observed value after HRMS deconvolution was 146904.

[0179] Example 21: Synthesis of Ab-12

[0180] The non-natural glycoengineered antibody Ab-12 was prepared by combining the compound Az-PEG3-Glc-GlcNAc-ox and trastuzumab via general procedure 3 (S2M3 enzyme). The observed value after HRMS deconvolution was 146904.

[0181] Example 22: Synthesis of Ab-13

[0182] The non-natural glycoengineered antibody Ab-13 was prepared by combining the compound Az-PEG3-LacNAc-ox and trastuzumab via general procedure 3 (S2M3 enzyme). The observed value after HRMS deconvolution was 146996.

[0183] Example 23: Synthesis of Ab-14

[0184] The non-natural glycoengineered antibody Ab-14 was prepared by combining the compound Az-PEG6-LacNAc-ox and trastuzumab via general procedure 3 (S2M3 enzyme). The observed value after HRMS deconvolution was 147260.

[0185] Example 24: Synthesis of Ab-15

[0186] The non-natural glycoengineered antibody Ab-15 is the compound Az-PEG 12 -LacNAc-ox and trastuzumab were obtained using general protocol 3 (S2M3 enzyme). HRMS deconvolution yielded a value of 147,784.

[0187] Example 25: Synthesis of Ab-16

[0188] The non-natural glycoengineered antibody Ab-16 is the compound Az-PEG 24 -LacNAc-ox and trastuzumab were obtained using general protocol 3 (S2M3 enzyme). HRMS deconvolution yielded a value of 148,840.

Claims

1. A glycosidase S2 mutant comprising a carbohydrate binding module CBM defined by M677-D843 in SEQ ID NO: 1, and the glycosidase S2 mutant amino acid sequence comprises SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO:

5.

2. The method for preparing the endoglycosidase S2 mutant according to claim 1, comprising: 1) connecting the target gene encoding the endoglycosidase S2 mutant of claim 1 to an expression vector (e.g., pBSYA1S1Z plasmid, pPICZα plasmid, pPIC9K plasmid, pPIC3K plasmid, pPICZA plasmid, pPIC3.5K plasmid, pET22b plasmid, pET28a plasmid, pET30a plasmid, pET32a plasmid, pGEX-6p-1 plasmid, pTrcHis plasmid, pLysS plasmid, pLysE plasmid, pBAD plasmid, pCS plasmid, pGEX-4T-1 plasmid, pET SUMO plasmid, pUC19 plasmid, pBR322 plasmid, pYES2 plasmid, pBV plasmid, pHT01 plasmid and pHT254 plasmid) to construct a recombinant vector; 2) transforming the recombinant vector into a host cell (eg, Pichia pastoris cells, Escherichia coli cells, Saccharomyces cerevisiae cells, insect ovary cells, Bacillus subtilis cells) to obtain a recombinant cell, and 3) obtaining the endoglycosidase S2 mutant by expressing the target gene in a recombinant cell, Preferably, the method further comprises step 4): isolating and purifying the endoglycosidase S2 mutant expressed in step 3).

3. A polynucleotide encoding the endoglycosidase S2 mutant according to claim 1, preferably, its sequence comprises SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO:

9. A recombinant vector comprising the polynucleotide according to claim 3.

5. A host cell transformed with the recombinant vector according to claim 4, wherein the host cell is a prokaryotic cell or a eukaryotic cell, and preferably, the host cell is selected from the group consisting of Pichia pastoris cells, Escherichia coli cells, Saccharomyces cerevisiae cells, insect ovary cells and Bacillus subtilis cells.

6. Use of the endoglycosidase S2 mutant according to claim 1 in antibody glycosylation modification, in, The glycosylation modification includes hydrolysis of sugar groups in antibodies, transglycosylation of antibodies, and both. For example, the glycosylation modification includes preparing an antibody drug conjugate by a one-step method using an antibody and a disaccharide-small molecule drug conjugate. Wherein, the disaccharide-small molecule drug conjugate is represented by the following formula III: In Formula III, represents a monosaccharide selected from galactose, mannose and glucose, wherein the C5 position is substituted by Y', and the connecting bond between disaccharides is a 1,4 glycosidic bond, Y' is Wherein, L1 represents a dipeptide linker, for example, -Val-Cit-, -Val-Ala-; PAB represents p-aminophenylethanol unit; D represents a small molecule drug unit, for example, MMAE, MMAF or maytansine; represents the attachment position to the monosaccharide, Alternatively, the glycosylation modification comprises preparing an antibody drug conjugate by a two-step method using an antibody and a sugar substrate of the following formula II: In Formula II, represents a monosaccharide selected from galactose, mannose and glucose, wherein the C5 position is substituted by Y, and the connecting bond between the disaccharides is a 1,4 glycosidic bond, Wherein, Y is selected from the following structures: wherein R1 represents a C1-C7 alkyl group, preferably a C2-C6 alkyl group, preferably a C3-C5 alkyl group, preferably a C3-C4 alkyl group, such as methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl; m represents an integer from 1 to 6, for example, m is 1, 2, 3, 4, 5, 6; n represents an integer of 1-24, for example, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24; L1 represents a dipeptide linker, such as -Val-Cit-, -Val-Ala-; PAB represents p-aminophenylethanol unit; D represents a small molecule drug unit, such as MMAE and MMAF; Indicates the attachment position to the monosaccharide.

7. A method for glycosylation modification of an antibody, the method comprising using the endoglycosidase S2 mutant according to claim 1 in the glycosylation modification process.

8. The use according to claim 6 or the method according to claim 7, wherein The antibody is selected from the group consisting of antibodies having glycosylation sites, Preferably, the antibody is an antibody having an N-glycosylation site, More preferably, the antibody is a biantennary antibody having an N-glycosylation site, Most preferably, the antibody is an IgG with a conserved N-glycosylation site at N297 in the Fc region, Specifically, the antibody is a monoclonal antibody, a polyclonal antibody, a bifunctional antibody, a trifunctional antibody, a nanobody fused with an Fc domain, a therapeutic antibody or a functional antibody derived from different species, Specifically, the antibody is a human antibody, a mouse antibody or a chimeric antibody; Specifically, the antibody is IgG1, IgG2, or IgG4; Specifically, the targets of the antibodies include HER2, Claudin 18.2, EGFR, c-Met, NECTIN4, CD276, HER3, CD3, FOLR1, BCMA, CD20, DLL3, MUC1, PD-L1, ROR1, TF, CD19, CD22, CD30, CD70, CD79B, FGFs, MSLN, NT5E, TNFα, CD147, CD24, CD38, CD47, CDH3, CDK4, CDK6, CEACAM5, CLDN6, CTLA4, DDR1, D R5, FAPα, FGFR3, GPRC5D, GR, HLA-DR, ICAM1, IL2R, MELTF, ROR2, TPBG (5T4), VTCN1, ZIP6, CD33, CD25, RSV, VEGF, RANKL, VEGFR2, CTLA-4, CD52, CD319, PD-1, CD274, IgE, IL-6, IL-12, IL-2, C5, IL-17A, CD25, SLAMF7, F10, factor IXa, HAb18G, PCSK9, BLyS, IL23, α4β7, IL-4R-α, HAE, FGF23 and IL6R; preferably, the targets of the antibody include HER2, CD20, EGFR, PD-1; Specifically, the antibodies include trastuzumab, rituximab, pertuzumab, panitumumab, toripalimab, toripalimab, and nivolumab.

9. The method according to claim 7, wherein: The method comprises: 1) hydrolyzing sugar groups in the antibody, wherein the antibody is incubated with the endoglycosidase S2 mutant; and / or 2) using a sugar substrate, and subjecting the antibody to glycosylation modification under the transglycosylation action of the endoglycosidase S2 mutant, in, The sugar substrate is a disaccharide represented by the following formula I: in, It means a monosaccharide selected from galactose, mannose and glucose, and the connecting bond between disaccharides is a 1,4 glycosidic bond; or, The sugar substrate is a disaccharide derivative represented by the following formula II: In Formula II, represents a monosaccharide selected from galactose, mannose and glucose, wherein the C5 position is substituted by Y, Wherein, Y is selected from the following structures: wherein R1 represents a C1-C7 alkyl group, preferably a C2-C6 alkyl group, preferably a C3-C5 alkyl group, preferably a C3-C4 alkyl group, such as methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl; m represents an integer from 1 to 6, for example, m is 1, 2, 3, 4, 5, 6; n represents an integer of 1-24, for example, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24; L1 represents a dipeptide linker, such as -Val-Cit-, -Val-Ala-; PAB represents p-aminophenylethanol unit; D represents a small molecule drug unit, such as MMAE and MMAF; represents the attachment position to the monosaccharide, or, The sugar substrate is a disaccharide-small molecule drug conjugate represented by the following formula III: In Formula III, represents a monosaccharide selected from galactose, mannose and glucose, wherein the C5 position is substituted by Y', Y' is Wherein, L1 represents a dipeptide linker, for example, -Val-Cit-, -Val-Ala-; PAB represents p-aminophenylethanol unit; D represents a small molecule drug unit, such as MMAE and MMAF; represents the attachment position to the monosaccharide, Specifically, the sugar substrate is selected from: or, The sugar substrate is a sugar substrate in the ring-opened form of the oxazoline ring in the above formula I, II or III; Preferably, the method further comprises reacting the glycosylated antibody modified with the sugar substrate represented by formula II with a drug-linker to prepare an antibody-drug conjugate. Preferably, the drug-linker is a functional group containing hydroxylamine, such as NH2O-VC-PAB-MMAE.

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