Endonuclease S2 mutant, preparation method thereof and application of endonuclease S2 mutant in antibody glycosylation modification

Abstract

The invention relates to an endonuclease S2 mutant, a preparation method of the endonuclease S2 mutant and application of the endonuclease S2 mutant in antibody glycosylation transformation. The glycosidase S2 mutant comprises 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.

Description

Technical Field

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

[0002] The Fc glycan of IgG antibody 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). It and Endo S, Endo F1, Endo F2 and Endo F3 of the same family can hydrolyze and deglycosylate a wide range of glycoforms of glycoproteins, especially IgG, with significant Fc deglycosylation activity. Related literature reports the molecular basis of the broad-spectrum N-glycan specificity and processing of endoglycosidase S2 (Endo-S2) for therapeutic IgG monoclonal antibodies, discloses the crystal structure of S2 and explains the hydrolytic activity of endoglycosidase.

[0004] According to previous reports, a series of single amino acid mutants obtained by amino acid mutations 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 inhibition of hydrolysis activity. CN108026518A discloses a series of Endo S mutant enzymes that exhibit reduced hydrolysis activity for the activity of 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 hydrolysis activity. These mutant enzymes with inhibited hydrolysis activity play a key role in the protein / antibody glycoengineering process. This finding also further illustrates that Endo S and Endo-S2 have a high degree of structural consistency, and each domain plays the same or similar role in the catalytic reaction of the enzyme. Based on literature reports, Endo-S and Endo-S2 show differences in activity in the recognition of different glycoform substrates, while previous reports have focused on the exploration of hydrolysis 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, develops 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. Summary of the invention

[0006] 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 a hydrolysis activity equivalent to that of the wild-type Endo-S2 and a more stable transglycosylation activity than the wild-type Endo-S2, and can therefore be effectively used for the conjugation of antibody drugs.

[0007] Another technical purpose of the present invention is to provide a method for preparing the above-mentioned endoglycosidase S2 mutant.

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

[0009] Another technical object of the present invention is to provide a recombinant vector comprising the above-mentioned polynucleotide.

[0010] Another technical purpose of the present invention is to provide a host cell transformed with the above-mentioned recombinant vector.

[0011] Another technical purpose of the present invention is to provide the use of the above-mentioned endoglycosidase S2 mutant in antibody glycosylation modification.

[0012] Another technical purpose of the present invention is to provide a method for glycosylation modification of antibodies by using the above-mentioned endoglycosidase S2 mutant.

[0013] In one aspect, the present invention provides a glycosidase S2 mutant, which comprises 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.

[0014] On the other hand, the present invention provides a method for preparing the above-mentioned endoglycosidase S2 mutant, the method comprising:

[0015] 1) connecting 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;

[0016] 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

[0017] 3) obtaining the endoglycosidase S2 mutant by expressing the target gene in a recombinant cell,

[0018] Preferably, the method further comprises step 4): isolating and purifying the endoglycosidase S2 mutant expressed in step 3).

[0019] In yet 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.

[0020] In yet another aspect, the present invention provides a recombinant vector comprising the above polynucleotide.

[0021] 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, and preferably, the host cell is selected from Pichia pastoris cells, Escherichia coli cells, Saccharomyces cerevisiae cells, insect ovary cells, and Bacillus subtilis cells.

[0022] In another aspect, the present invention provides the use of the above-mentioned endoglycosidase S2 mutant in antibody glycosylation modification.

[0023] The glycosylation modification includes hydrolysis of sugar groups in antibodies, transglycosylation of antibodies, or both.

[0024] 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.

[0025] Wherein, the disaccharide-small molecule drug conjugate is represented by the following formula III:

[0026]

[0027] 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,

[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]

[0035] 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:

[0036]

[0037] In Formula II, represents a monosaccharide selected from galactose, mannose and glucose, wherein the C5 position is substituted by Y,

[0038] Wherein, Y is selected from the following structures:

[0039]

[0040] 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;

[0041] m represents an integer from 1 to 6, for example, m is 1, 2, 3, 4, 5, 6;

[0042] 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;

[0043] L1 represents a dipeptide linker, such as -Val-Cit-, -Val-Ala-;

[0044] PAB represents p-aminophenylethanol unit;

[0045] D represents a small molecule drug unit, such as MMAE and MMAF;

[0046] Indicates the attachment position to the monosaccharide.

[0047] 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.

[0048] In a specific embodiment, the antibody is selected from the group consisting of antibodies having a glycosylation site.

[0049] In a specific embodiment, the antibody is an antibody having an N-glycosylation site.

[0050] In a specific embodiment, the antibody is a biantennary antibody having an N-glycosylation site.

[0051] In a specific embodiment, the antibody is an IgG with a conserved N-glycosylation site at N297 in the Fc region.

[0052] 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 different species.

[0053] Specifically, the antibody is a human antibody, a mouse antibody or a chimeric antibody;

[0054] 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, and 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;

[0055] In a specific embodiment, the antibody comprises trastuzumab, rituximab, pertuzumab, panitumumab, toripalimab, toripalimab, nivolumab.

[0056] In a specific embodiment, the method comprises:

[0057] 1) A method for hydrolyzing sugar groups in an antibody, wherein the antibody is incubated with the above-mentioned endoglycosidase S2 mutant; and / or

[0058] 2) using a sugar substrate, subjecting the antibody to glycosylation modification under the transglycosylation action of the endoglycosidase S2 mutant,

[0059] in,

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

[0061]

[0062] in, It means a monosaccharide selected from galactose, mannose and glucose, and the connecting bond between disaccharides is a 1,4 glycosidic bond;

[0063] or,

[0064] The sugar substrate is a disaccharide derivative represented by the following formula II:

[0065]

[0066] 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:

[0067]

[0068] 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;

[0069] m represents an integer from 1 to 6, for example, m is 1, 2, 3, 4, 5, 6;

[0070] 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;

[0071] L1 represents a dipeptide linker, such as -Val-Cit-, -Val-Ala-;

[0072] PAB represents p-aminophenylethanol unit;

[0073] D represents a small molecule drug unit, such as MMAE and MMAF;

[0074] represents the attachment position to the monosaccharide,

[0075] or,

[0076] The sugar substrate is a disaccharide-small molecule drug conjugate represented by the following formula III:

[0077]

[0078] In Formula III, represents a monosaccharide selected from galactose, mannose and glucose, wherein the C5 position is substituted by Y',

[0079] Y' is

[0080] Wherein, L1 represents a dipeptide linker, for example, -Val-Cit-, -Val-Ala-;

[0081] PAB represents p-aminophenylethanol unit;

[0082] D represents a small molecule drug unit, such as MMAE and MMAF;

[0083] represents the attachment position to the monosaccharide,

[0084] Specifically, the sugar substrate is selected from:

[0085]

[0086] or,

[0087] The sugar substrate is a sugar substrate in the ring-opened form of the oxazoline ring in the above formula I, II or III;

[0088] 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.

[0089] Beneficial Effects

[0090] The endoglycosidase S2 mutants S2M3 and V3 obtained by fusing different structural domains of Endo-S and Endo-S2 in the present invention have hydrolysis activity equivalent to that of wild-type Endo-S2.

[0091] The endoglycosidase S2 mutants S2M3 and V3 obtained by the modification of the present invention can be used for antibody glycosylation modification, and have good transglycosylation activity for various glycoforms such as LacNAc-ox, Man-GlcNAc-ox, Glc-GlcNAc-ox and these glycoform derivatives, among which S2M3 has a more stable transglycosylation activity than Endo-S2.

[0092] The endoglycosidase S2 mutants S2M3 and V3 obtained by transformation of the present invention can be effectively used for antibody-drug coupling, and thus can be used for preparing ADC. BRIEF DESCRIPTION OF THE DRAWINGS

[0093] Figure 1 The results of Endo-S2, S2M1, S2M3, S2M4 and V3 hydrolyzing the sugar chain at the N-glycosylation site of trastuzumab are shown. Figure A shows the hydrolysis of the sugar chain at the N-glycosylation site of the antibody after each enzyme and trastuzumab were co-incubated for 1 hour, and Figure B shows the hydrolysis of the sugar chain at the N-glycosylation site of the antibody after each enzyme and trastuzumab were co-incubated for 4 hours.

[0094] Figure 2The results of the transglycosylation activity test of endoglycosidase using Acylhydrazone-LacNAc-ox as the sugar substrate are shown. A total of four mutant enzymes were tested.

[0095] Figure 3 The RP test results (A) and LC-MS (B) test results of the antibody drug conjugate (ADC) prepared in Example 4 are shown.

[0096] Figure 4 The sugar transglycosylation activities of S2M1, S2M4 and V3 after increasing the sugar substrate equivalent are shown.

[0097] Figure 5 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 are shown.

[0098] Figure 6 The figure shows the effect of the amount of Endo-S2 and S2M3 enzymes on the transglycosylation activity of the Az-LacNAc-ox antibody.

[0099] Figure 7 The effect of the amount of Endo-S2 and S2M3 enzymes on the transglycosylation activity of the diAz-LacNAc-ox antibody is shown.

[0100] Figure 8 The results of the activity analysis of diAz-LacNAc-ox antibody transglycosylation under different enzyme amounts of Endo-S2 and S2M3 and reaction time are shown. DETAILED DESCRIPTION

[0101] The technical solution of the present application is 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.

[0102] the term

[0103] In the present application, "endoglycosidase S2 mutant", "novel endoglycosidase" and "mutant enzyme" can be used interchangeably.

[0104] In this application, the monosaccharide symbols of antibody glycosylation sites represent the following meanings: is galactose, ■ is N-acetylglucosamine, For mannose, For fucose.

[0105] In the present 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:

[0106]

[0107] In the present application, the drug-linker NH2O-VC-PAB-MMAE was obtained by customization.

[0108] Az-LacNAc-ox see G3 of CN114949236A.

[0109] diAz-LacNAc-ox see G14 of CN114949236A.

[0110] Az-PEG3-LacNAc-ox is commercially available.

[0111] Az-PEG6-LacNAc-ox is commercially available.

[0112] Az-PEG 12 -LacNAc-ox is commercially available.

[0113] Az-PEG 24 -LacNAc-ox is commercially available.

[0114] Az-PEG3-Man-GlcNAc-ox is commercially available.

[0115] Az-PEG3-Glc-GlcNAc-ox is commercially available.

[0116] Acylhydrazone-LacNAc-ox is commercially available.

[0117] NH2O-VC-PAB-MMAE is compound D1 in CN114949236A.

[0118] General Procedure 1: Expression and purification of endoglycosidase

[0119] 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 30 minutes. Heat shock in a 42℃ water bath for 90sec, then transfer to ice and let it stand for 3-5min. Add 500μL of LB medium without antibiotics, mix well, and shake at 37℃, 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 medium. Wait until OD 600 When it reaches 0.6-0.8, 50 μL of bacterial solution is transferred to a sequencing company for Sanger sequencing.

[0120] 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 1μL of plasmid (10ng / μL) and transform it into 50μL of E.coli BL21 (DE3) competent cells, and let it stand in an ice bath for 30 minutes. Heat shock in a 42℃ water bath for 90sec, and then transfer to ice and let it stand for 3-5min. Add 500μL of LB medium without antibiotics, mix well, and shake at 37℃, 220rpm for 1-2h. Then take 200-300μL of bacterial solution and spread it on the LB plate containing ampicillin antibiotics, invert it in a constant temperature incubator, and culture it at 37℃ overnight. The next day, pick a single clone and expand it in 3-5mL LB medium. When OD 600 When the pH reaches 0.6-0.8, add IPTG to the bacterial solution to a final concentration of 0.5 mM, and place the bacterial solution at 20°C and 200 rpm for overnight expression. After the expression is completed, centrifuge at 4500 rpm for 20 minutes, discard the supernatant and collect the bacteria. Resuspend the bacteria in 30 mL of phosphate buffer (0.01 M, pH 7.4), ultrasonically disrupt, and centrifuge at 12000 rpm for 15 minutes to collect the supernatant for protein purification.

[0121] Use nickel ion affinity chromatography to purify the overexpressed endoglycosidase. Use 5 column volumes of phosphate buffer (0.01M, pH7.4) to balance the column bed, then load the supernatant after the above-mentioned sterilization centrifugation into the chromatography column, repeat 3 times to allow the target protein to bind to the column filler. Then use 10 times the column volume of 50mM, 100mM and 250mM imidazole to elute, and collect the eluate in separate tubes.

[0122] 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).

[0123] The protein concentration was determined by UV280 and the molecular weight was determined by LC-MS.

[0124] General operation 2: endoglycosidase specific hydrolysis antibody N-glycan activity test

[0125] Trastuzumab (5 mg / mL) was added to phosphate buffer (pH 7.0), followed by endoglycosidase (100 ng / mg), incubated at 30°C for 1 hour and 4 hours, and then sampled for LC-MS analysis.

[0126] General operation 3: Endoglycosidase to achieve antibody glycosylation modification

[0127] The antibody (5 mg / mL) and oxazoline (0.3-2 mM) (also referred to as "ox" herein) were added to a phosphate buffer (pH 6.5-7.0), and after the solution pH was controlled at 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 and identified by SDS-PAGE or LC-MS.

[0128] The following operations of this application are all completed based on the above general operations 1-3.

[0129] Example 1: Transformation of endoglycosidase mutants

[0130] Based on the wild-type Endo-S2 and Endo S, their different domains were combined and fused to obtain four mutants, which were named V3, S2M1, S2M3, and S2M4, as shown in Table 1 below.

[0131] Table 1 Structural information of novel endoglycosidases

[0132] Endoglycosidase / mutant name sequence Endo-S2 SEQ ID NO: 1 Endo-S SEQ ID NO: 2 S2M3 SEQ ID NO: 3 S2M4 SEQ ID NO: 4 V3 SEQ ID NO: 5 S2M1 SEQ ID NO: 6

[0133] Example 2: Expression and purification of the novel endoglycosidase designed in Example 1

[0134] The novel endoglycosidases V3, S2M1, S2M3, and S2M4 were obtained by expression and purification using the general operation 1.

[0135] Example 3: Antibody glycosylation hydrolysis activity test of novel endoglycosidase

[0136] The antibody glycosylation hydrolysis activity of the novel endoglycosidases V3, S2M1, S2M3, and S2M4 was tested using general procedure 2. The results are shown in Figure 1 The results showed that the modified mutant enzymes can hydrolyze the sugar chains of wild-type antibodies, S2M3 has better hydrolysis activity than Endo-S2, and the hydrolysis activities of S2M1, S2M4 and V3 are comparable to Endo-S2.

[0137] Example 4: Novel endoglycosidase realizes the transglycosylation of Acylhydrazone-LacNAc-ox antibody and two-step preparation of sugar chain-directed ADC compound

[0138]

[0139] Step 1: Add Acylhydrazone-LacNAc-ox (0.5mM) to phosphate buffer (pH 6.5), add wild-type Herceptin (5mg / mL), endoglycosidase (40μg / mg, S2M1, S2M3, S2M4, V3) to the above reaction system, and incubate at 30℃ for 2 hours. Mass spectrometry results show that S2M3 has the best transglycosylation activity for Acylhydrazone-LacNAc-ox, and the ratio of coupling two sugar substrates is extremely high; the transglycosylation activity of V3 is weaker than that of S2M3, and S2M1 and S2M4 have poor transglycosylation activity.

[0140] (Purification): Use AKTA pure 150 (chromatographic column: Merck VL 11 250; filler: Mabselect SureXL 2mL) to remove excess Endo-S2 and sugar substrate in the sample.

[0141] Step 2: Add 50 mM NaOAc buffer (pH 4.6), DMSO (10% V / V), and then 10 mg / mL H2NO-VC-PAB-MMAE drug linker intermediate solution to the sugar substrate-modified antibody solution obtained in step 1. Incubate at 30° C. for 2 hours.

[0142] Figure 2 The results of the transglycosylation activity test of endoglycosidase using Acylhydrazone-LacNAc-ox as the sugar substrate are shown. A total of four mutant enzymes were tested. Figure 2 It can be seen that S2M3 has the best transglycosylation activity for Acylhydrazone-LacNAc-ox, and the ratio of coupling two sugar substrates is extremely high; the transglycosylation activity of V3 is weaker than that of S2M3, and S2M1 and S2M4 have poor transglycosylation activity.

[0143] The ADC compound prepared above was subjected to RP detection and LC-MS detection: the test results are shown in Table 2 and the attached Figure 3 .

[0144] Table 2:

[0145]

[0146] Example 5: Sugar transfection activity of S2M1, S2M4 and V3 under increased sugar substrate equivalent

[0147] The same operation as in Example 4 was performed, except that the sugar substrate equivalent was increased to 60 relative to the wild-type Herceptin, and the sugar conversion effect was measured. Figure 4 As shown in the figure, after increasing the sugar substrate equivalent, the three enzymes S2M1, S2M4 and V3 all have certain transfer activity, among which S2M4 is better than V3, and V3 is better than S2M1. It can be seen that S2M4 and V3 containing the CBM region of Endo-S2 have better sugar transfer activity.

[0148] Example 6: One-step preparation of sugar chain-directed ADC compounds using a novel endoglycosidase

[0149]

[0150] MMAE-PAB-VC-NH-LacNAc-ox (0.5 mM) was added to phosphate buffer (pH 6.5), and wild-type Herceptin (5 mg / mL) and endoglycosidase (80 μg / mg, S2M1, S2M3, S2M4, V3, respectively) were added to the above reaction system in sequence, and incubated at 30°C for 2 hours. Figure 5 shown.

[0151] S2M3 has the best transglycosylation activity, and S2M1, S2M4 and V3 all have certain transglycosylation activity, among which S2M4 is better than V3, and V3 is better than S2M1. It can be seen that S2M3, S2M4 and V3 containing the CBM region of Endo-S2 have better transglycosylation activity.

[0152] Example 7: Endo-S2 and S2M3 enzymes for the transglycosylation activity of antibodies on Az-LacNAc-ox substrates

[0153] Az-LacNAc-ox (0.5 mM, structural formula as follows) was added to phosphate buffer (pH 6.5), and trastuzumab (5 mg / mL) and endoglycosidase (Endo-S2 or S2M3, 0.02-2.4 mg / mL) were added to the above reaction system in sequence. The mixture was incubated at 30°C for 2 hours and then analyzed by LC-MS.

[0154]

[0155] The specific design is shown in Table 3:

[0156] Table 3 Effect of Endo-S2 and S2M3 enzyme amounts on the antibody transglycosylation activity of Az-LacNAc-ox substrate

[0157]

[0158] As shown in Table 3, the antibody transglycosylation activity was tested when the amount of Endo-S2 or S2M3 enzyme was 4, 20, 40, 80, 160, 240, 320 and 480 μg / mg (enzyme amount / antibody amount), wherein the concentration of trastuzumab was 5 mg / mL, the concentration of the sugar substrate Az-LacNAc-ox was 0.5 mM, the reaction was carried out in a 50 mM phosphate, pH 6.5 buffer system, and LC-MS analysis was performed after reacting at 30°C for 2 hours. Figure 6 shown.

[0159] The results showed that under low enzyme dosage conditions (about <50μg / mg), both wild-type Endo-S2 and mutant enzyme S2M3 had a positive effect on DAR values. Under higher enzyme dosage conditions, Endo-S2 showed a downward trend in DAR value with increasing enzyme dosage, while S2M3 showed a longer plateau period and was able to maintain a high DAR value in a wide range (about 100-500μg / mg), indicating that the mutant enzyme S2M3 has a more stable transglycosylation activity than Endo-S2.

[0160] Example 8: Endo-S2 and S2M3 enzyme activity test on antibody transglycosylation activity of diAz-LacNAc-ox substrate

[0161] Add diAz-LacNAc-ox (0.5 mM, as shown in the structural formula) to phosphate-HCl buffer (pH 6.5), and add trastuzumab (5 mg / mL) and endoglycosidase (0.02 -2.4 mg / mL) to the above reaction system in sequence. Incubate at 30°C, and take samples at different time points for LC-MS analysis and identification.

[0162]

[0163] The specific design is shown in Table 4:

[0164] Table 4 Endo-S2 and S2M3 enzyme amounts for antibody transglycosylation activity of diAz-LacNAc-ox substrate reaction conditions

[0165]

[0166] As shown in Table 4, the antibody transglycosylation activity was tested when the amount of Endo-S2 or S2M3 enzyme was 4, 20, 40, 80, 160, 240, 320 and 480 μg / mg (enzyme amount / antibody amount), wherein the concentration of trastuzumab was 5 mg / mL, the concentration of the sugar substrate diAz-LacNAc-ox 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 FIG. Figure 7 shown.

[0167] The results showed that as the reaction time increased, at various enzyme dosages, the mutant enzyme S2M3 showed lower and slower hydrolysis activity than Endo-S2, which was more conducive to process production.

[0168] Experimental results:

[0169] Based on the results of Examples 5-8, compared with Endo-S2, the mutant enzyme S2M3 shows significant superiority in the antibody transglycosylation reaction of different sugar substrates. The reaction curve of the mutant S2M3 has obvious enzymatic reaction characteristics, its plateau remains stable, and the high DAR value of the product is continuously maintained within a wide range of enzyme amounts. This shows that after the mutant enzyme S2M3 hydrolyzes the antibody Fc sugar chain, it transfers the Az-LacNAc-ox sugar substrate to the antibody with efficient transglycosylation activity, and does not have hydrolysis activity on the sugar-engineered antibody after the transglycosylation. This efficient and stable transglycosylation activity enables the mutant enzyme to have the durability characteristics of enzyme amount in the preparation process of antibody conjugates. On the one hand, the longer plateau weakens the effect of enzyme amount on the product DAR value, and on the other hand, the reduction in substrate hydrolysis activity highlights the enhancement of the transfer activity of the mutant enzyme. In summary, the mutant enzyme has a more superior practical application value in the process.

[0170] Example 9: Antibody transglycosylation activity test on diAz-LacNAc-ox substrate with the same enzyme amount Endo-S2 and S2M3 and different reaction times

[0171] The operation was the same as in Example 6. For details, see Table 5 below, which examined the effect of reaction time on the antibody transglycosylation activity of the diAz-LacNAc-ox substrate under the same enzyme amounts of Endo-S2 and S2M3.

[0172] Table 5 Endo-S2 and S2M3 enzyme amounts for antibody transglycosylation activity of diAz-LacNAc-ox substrate reaction conditions

[0173]

[0174] The results are as follows Figure 8 As shown, from Figure 8It can be seen that 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.

[0175] In the following, the general procedure 3 was used to prepare non-natural glycoengineered antibodies using different antibodies and different sugar substrates as raw materials.

[0176] Example 10: Synthesis of Ab-1

[0177] The non-natural glycoengineered antibody Ab-1 was obtained by the compound Az-LacNAc-ox and the wild-type antibody Rituxan (Human IgG1) through the general procedure 3 (S2M3 enzyme). The measured value after HRMS deconvolution was 145780.

[0178] Example 11: Synthesis of Ab-2

[0179] The non-natural glycoengineered antibody Ab-2 was obtained by combining the compound Az-LacNAc-ox and the wild-type antibody Pertuzumab (Human IgG1) through general procedure 3 (S2M3 enzyme). The measured value after HRMS deconvolution was 146791.

[0180] Example 12: Synthesis of Ab-3

[0181] The non-natural glycoengineered antibody Ab-3 was obtained by combining the compound Az-LacNAc-ox and the wild-type antibody panitumumab (Human IgG2) through general procedure 3 (S2M3 enzyme). The measured value after HRMS deconvolution was 145631.

[0182] Example 13: Synthesis of Ab-4

[0183] The non-natural glycoengineered antibody Ab-4 was obtained by the compound Az-LacNAc-ox and the wild-type antibody toripalimab (Human IgG4) through the general procedure 3 (S2M3 enzyme). The measured value after HRMS deconvolution was 148616.

[0184] Example 14: Synthesis of Ab-5

[0185] The non-natural glycoengineered antibody Ab-5 was obtained by combining the compound Az-LacNAc-ox and the wild-type antibody nivolumab (Human IgG4) through general procedure 3 (S2M3 enzyme). The measured value after HRMS deconvolution was 144929.

[0186] Example 15: Synthesis of Ab-6

[0187] The non-natural glycoengineered antibody Ab-6 was obtained by the compound Az-LacNAc-ox and mouse monoclonal antibody-1 through general operation 3 (S2M3 enzyme). The measured value after HRMS deconvolution was 146927.

[0188] Example 16: Synthesis of Ab-7

[0189] The non-natural glycoengineered antibody Ab-7 was obtained by the compound Az-LacNAc-ox and mouse monoclonal antibody-2 through general operation 3 (S2M3 enzyme). The measured value after HRMS deconvolution was 147312.

[0190] Example 17: Synthesis of Ab-8

[0191] The non-natural glycoengineered antibody Ab-8 was obtained by the compound Az-LacNAc-ox and the mouse monoclonal antibody-3 through the general procedure 3 (S2M3 enzyme). The measured value after HRMS deconvolution was 146550.

[0192] Example 18: Synthesis of Ab-9

[0193] The non-natural glycoengineered antibody Ab-9 is obtained by combining the compound Man-GlcNAc-ox and trastuzumab through general procedure 3 (S2M3 enzyme). The measured value after HRMS deconvolution is 146590.

[0194] Example 19: Synthesis of Ab-10

[0195] The non-natural glycoengineered antibody Ab-10 is obtained by combining the compound Glc-GlcNAc-ox and trastuzumab through general procedure 3 (S2M3 enzyme). The measured value after HRMS deconvolution is 146591.

[0196] Example 20: Synthesis of Ab-11

[0197] The non-natural glycoengineered antibody Ab-11 is obtained by combining the compound Az-PEG3-Man-GlcNAc-ox and trastuzumab through general procedure 3 (S2M3 enzyme). The measured value after HRMS deconvolution is 146904.

[0198] Example 21: Synthesis of Ab-12

[0199] The non-natural glycoengineered antibody Ab-12 is obtained by combining the compound Az-PEG3-Glc-GlcNAc-ox and trastuzumab through general procedure 3 (S2M3 enzyme). The measured value after HRMS deconvolution is 146904.

[0200] Example 22: Synthesis of Ab-13

[0201] The non-natural glycoengineered antibody Ab-13 was obtained by combining the compound Az-PEG3-LacNAc-ox and trastuzumab through general procedure 3 (S2M3 enzyme). The measured value after HRMS deconvolution was 146996.

[0202] Example 23: Synthesis of Ab-14

[0203] The non-natural glycoengineered antibody Ab-14 was obtained by combining the compound Az-PEG6-LacNAc-ox and trastuzumab through general procedure 3 (S2M3 enzyme). The measured value after HRMS deconvolution was 147260.

[0204] Example 24: Synthesis of Ab-15

[0205] The non-natural glycoengineered antibody Ab-15 is the compound Az-PEG 12 -LacNAc-ox and trastuzumab were obtained by general procedure 3 (S2M3 enzyme). Found 147784 after HRMS deconvolution.

[0206] Example 25: Synthesis of Ab-16

[0207] The non-natural glycoengineered antibody Ab-16 is the compound Az-PEG 24 -LacNAc-ox and trastuzumab were obtained by general procedure 3 (S2M3 enzyme). HRMS deconvolution found 148840.

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 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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