Immobilized endoglycosidase fusion protein and its use

Abstract

The present invention relates to a method for preparing an antibody-drug conjugate, wherein the antibody-drug conjugate is site-specifically conjugated based on the N-glycosylation site of the antibody Fc region. The method includes: (1) providing a donor containing an oxazoline oligosaccharide, an antibody containing a GlcNAc motif, and an immobilized endoglycosidase having glycosyltransferase activity; and (2) covalently bonding the activated donor containing the oxazoline oligosaccharide to the antibody containing the GlcNAc motif by the catalysis of the endoglycosidase, thereby realizing one-step conjugation. The present invention further relates to an endoglycosidase fusion protein containing a covalently bonded endoglycosidase, Halo tag and / or His tag. The present invention further relates to an immobilized endoglycosidase fusion protein obtained by immobilizing the endoglycosidase fusion protein on a support, a prepacked column filled with the immobilized fusion protein, and the use of the immobilized endoglycosidase fusion protein in the purification of an antibody conjugate.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and specifically to a method for preparing a drug conjugate or an antibody-drug conjugate with a glycoside transfer activity by an immobilized endoglycosidase fusion protein, wherein the antibody-drug conjugate is a one-step site-specific conjugation based on the N-glycosylation site of the antibody Fc region. The present invention also relates to an endoglycosidase fusion protein comprising a covalently linked endoglycosidase, Halo tag and / or His tag, and an immobilized endoglycosidase fusion protein obtained by immobilizing the endoglycosidase fusion protein on a support.

Background Art

[0002] The demand for high-quality conjugates, especially bioconjugates for bioscience research, diagnosis or treatment, is increasing rapidly. However, part of the reason why simply producing bioconjugates with high quality and stability is not satisfactory is that it is difficult to meet the high-quality standards of bioconjugates due to the complexity of biomolecules and the difficulty of amplification.

[0003] An antibody-drug conjugate (ADC) is a novel targeted drug that falls within the category of bioconjugates. It is a combination of a highly active small molecule drug and a monoclonal antibody by chemical bonding, which combines the high activity of the small molecule drug and the targeting properties of the antibody-drug, effectively eliminates the strong toxic side effects of the small molecule toxin on the human body, and can eliminate the limitation that the effect of antibody therapy on solid tumors is limited. Therefore, it has great potential as an anti-tumor drug.

[0004] With the development and increasing maturity of antibody-drug conjugates, antibody conjugation technology has experienced several generations of updates. Generally, it has gone through three generations of technological development, including random conjugation, site-specific conjugation based on antibody genetic engineering, and site-specific conjugation technology that does not rely on antibody genetic engineering (Walsh SJ, Bargh JD, Dannheim FM et al., Site-selective modification strategies in antibody-drug conjugates, Chem. Soc. Rev. 2021, 50, 1305-1353). The first-generation conjugation technology mainly employs random conjugation based on lysine or cysteine residues. Generally, the conjugation sites are random, the drug / antibody ratio (DAR) is non-uniform, and a highly heterogeneous mixture is obtained, which has problems in terms of process stability, quality control, drug stability, metabolic consistency, safety, etc. The second-generation conjugation technology usually requires introducing specific amino acids or inserting polypeptide fragments with specific sequences into the antibody through engineering mutations, and based on this, realizing site-specific conjugation of the antibody through site-specific chemical conjugation or enzymatic conjugation. The third-generation conjugation technology does not require engineering modification of the antibody. It focuses on selecting specific amino acid sites and performing site-specific conjugation under specific conditions or proximity effects. Specifically, there are modifications of inter-chain disulfide bonds, chemoselective modifications, proximity effect-based modifications, and glycosyl modifications. Overall, when compared comprehensively, glycosyl modification does not require engineering modification of the antibody, does not change the core backbone structure of the antibody, does not affect the binding ability of the antibody to the antigen-binding domain, does not require fine adjustment of chemical reaction conditions, and has advantages such as preferable conjugation reproducibility and uniformity, making it a research hotspot in antibody conjugation technology.At position 297 of the antibody Fc region, there is a highly conserved glycosylation (N-297 glycan), and site-specific binding in antibodies of different molecules can be achieved by glycan remodeling at this site (Wang LX, Tong X, Li C. Glycoengineering of Antibodies for Modulating Functions, Annu Rev Biochem, 2019, 88, 433-459). The site-specific conjugation technology based on antibody Fc glycosylation modification is mainly as follows.

[0005] 1) Based on chemical modification by glycosyl groups, sodium periodate is used to oxidize core fucose (Zuberbuhler K, Casi G, Bernardes GJ et al., Fucose-specific conjugation of hydrazide derivatives to a vascular-targeting monoclonal antibody in IgG format, Chem Commun, 2012, 48, 7100-7102) or o-diol in the sialic acid at the glycosyl terminus (Zhou Q, Stefano JE, Manning C et al., Site-specific antibody-drug conjugation through glycoengineering, Bioconjugate Chem, 2014, 25, 510-520) to obtain the corresponding aldehyde. The aldehyde carbonyl group can be used for the preparation of ADC by conjugation with small molecule toxin fragments. Such a method has certain substrate limitations because due to the diversity of the N-297 glycosyl structure, not all monoclonal antibody glycosyls contain core fucose or sialic acid reaction sites.

[0006] 2) Based on modification by enzyme catalysts, deglycosylation-transglycosylation tandem catalysts of tool enzymes such as endoglycosidase and glycosyltransferase are used to achieve glycan remodeling to introduce bioorthogonal reactive groups, and further through subsequent chemical reactions, the preparation by site-specific conjugation of ADCs is realized (Wang LX, Tong X, Li C, Glycoengineering of antibodies for modulating functions, Annu Rev Biochem, 2019, 88, 433-459, and Zeng Y, Tang F, Shi W et al., Recent advances in synthetic glycoengineering for biological applications, Current Opinion in Biotechnol, 2022, 74, 247-255).

[0007] In 2012, Wang Laixi et al. reported a site-specific conjugation technology based on the tandem catalysis of endoglycosidase Endo S and its mutants for antibody glycan remodeling, and synthesized glycan-remodeled antibodies with a single glycoform structure containing azide modification (Huang W, Giddens J, Fan SQ et al., Chemoenzymatic glycoengineering of intact IgG antibodies for gain of functions, J. Am. Chem. Soc., 2012, 134, 12308). Based on this, Wang Laixi, Huang Wei et al. developed a large variety of ADC site-specific conjugation synthesis technologies by a three-step process of deglycosylation-transglycosylation-click chemistry based on the tandem catalysis of tool enzymes such as endoglycosidase Endo S or Endo S2 and their mutants (Zeng Y, Tang F, Shi W et al., Recent advances in synthetic glycoengineering for biological applications, Current Opinion in Biotechnol, 2022, 1074, 247-255). In 2021, Wang Laixi et al. reported Endo S2-catalyzed site-specific conjugation by one-pot antibody glycan remodeling, which introduced a bioorthogonal azide functional group into the antibody and obtained ADC molecules through one click reaction step (Zhang X, Ou C, Liu H et al., General and robust chemoenzymatic method for glycan-mediated site-specific labeling and conjugation of antibodies: facile synthesis of homogeneous antibody-drug conjugates, ACS Chem. Biol., 2021, 16, 11, 2502-2514).

[0008] Another type of tool enzyme commonly used in site-specific conjugation technology by glycan remodeling is β-1,4-galactosyltransferase (β-1,4-Gal-T1) and its variant (β-1,4-Gal-T1 Y289L). This enzyme transfers galactose (Gal) to the non-reducing end of N-acetylglucosamine (Glc-NAc) of glycoproteins using uridine diphosphate galactose (Gal-UDP) as a donor. Based on this strategy, in 2009, Qasba et al. (Boeggeman E, Ramakrishnan B, Pasek M et al., Site specific conjugation of fluoroprobes to the remodeled Fc N-glycans of monoclonal antibodies using mutant glycosyltransferases: application for cell surface antigen detection, Bioconjugate Chem., 2009, 20, 6, 1228-1236) first reported an antibody site-specific conjugation technology using β-1,4-Gal-T1 and β-1,4-Gal-T1 Y289L as tool enzymes and C2-keto-Gal-UDP or N-azidoacetylgalactosamine-UDP (GalNAz-UDP) as donors, and prepared keto-carbonyl or azide-modified antibodies. Subsequently, in 2014, they first reported a method for synthesizing ADC molecules by a three-step process of deglycosylation-transglycosylation-bioorthogonal reaction based on β-1,4-Gal-T1 and its variant catalysis (Zhu Z, Ramakrishnan B, Li J et al., Site-specific antibody-drug conjugation through an engineered glycotransferase and a chemically reactive sugar, mAbs, 2014, 6, 1190-1200).Based on the above incorporation, Synaffix combined and utilized the tandem catalysis of endoglycosidase Endo S and β-1,4-Gal-T1 Y289L tool enzyme, and developed an ADC three-step synthesis method of deglycosylation-transglycosylation-bioorthogonal reaction (Van Geel R, Wijdeven MA, Heesbeen R et al., Chemoenzymatic conjugation of toxic payloads to the globally conserved N-glycan of native mAbs provides homogeneous and highly efficacious antibody-drug conjugates, Bioconjugate Chem., 2015, 26, 2233-2242). In addition, this literature also reported a four-step synthesis method of ADC of deglycosylation-transglycosylation-transglycosylation-bioorthogonal reaction (Li X, Fang T, Boons GJ, Preparation of well-defined antibody-drug conjugates through glycan remodeling and strain-promoted azide-alkyne cycloadditions, Angew. Chem. Int. Ed., 2014, 53, 7179-7182), and a total of three types of tool enzymes, namely β-1,4-Gal-T1, β-1,4-Gal-T1 Y289L and sialyltransferase, were used in this method.

[0009] All of the above conjugation technologies based on glycan remodeling utilize multi-step reactions by liquid-phase enzyme catalysis, which have certain limitations. For example, a high proportion of endoglycosidase catalyst equivalents is required for liquid-phase enzyme catalysis, and the requirements for enzyme purity are high (the process is complex and the preparation cost is high). A large amount of enzyme-related impurities (such as host proteins and nucleic acids) are easily introduced into the reaction system, and it is difficult to remove impurities downstream of the subsequent drug conjugate.

[0010] Currently, there are only a few publicly reported sugar chain conjugation reaction modes based on immobilized enzyme catalysts. For example, in 2018, Wang Laixi et al. reported realizing a continuous flow tandem catalyst of deglycosylation-transglycosylation based on the tandem column conjugation of covalently immobilized Endo S2 enzyme and covalently immobilized Endo S2 D184M enzyme to obtain a glycosyl-modified sugar chain homogeneous antibody (Li T, Li C, Quan DN, et al., Site-specific immobilization of endoglycosidases for streamlined chemoenzymatic glycan remodeling of antibodies, Carbohydr. Res. 2018, 458 - 459, 77 - 84). In 2021, Wu Zongyi et al. reported obtaining a glycosyl-modified sugar chain homogeneous antibody by the tandem catalysis of liquid-phase suspension immobilized enzymes Endo S2 and Endo S2 T138Q (Chuang H, Huang C, Hung T, et al., Development of biotinylated and magnetic bead-immobilized enzymes for efficient glyco-engineering and isolation of antibodies, Bioorg. Chem. 2021, 112, 104863). Currently, the one-step preparation of ADC drugs by sugar chain remodeling based on immobilized enzyme catalysts has not been reported yet. In short, currently, the problem of the need to operate antibodies with other site-specific conjugation technologies by conventional sugar chain remodeling reactions has been solved, but there are still very large limitations. For example, the conjugation steps are too long, the operation is complicated, at least two-step enzyme reactions and one-step chemical reactions are required, that is, ADC can be obtained only after at least three-step reactions and three complete purifications.On the one hand, most of the conventional glycan remodeling is based on liquid-phase enzymatic reactions, which have problems such as difficulty in scaling up, difficulty in separating and removing glycosidases, and difficulty in industrialization. According to literature reports, even if trace amounts of tool enzymes remain in ADC products, the ADC products may be deglycosylated and decomposed, causing the shedding of toxin molecules (Li T, Li C, Quan DN et al., Site-specific immobilization of endoglycosidases for streamlined chemoenzymatic glycan remodeling of antibodies, Carbohydrate Research 2018, 458 - 459, 77 - 84), which may induce serious toxic reactions, pose major challenges to drug research and development and production, and also pose significant potential risks to the safety of ADC drugs. The present invention aims to solve these problems.

Summary of the Invention

[0011] Antibody-drug conjugates can be conjugated based on site-specific conjugation technology by glycan remodeling. There is highly conserved glycosylation at asparagine 297 in the Fc region of antibodies, and site-specific conjugation of different molecules on the antibody can be achieved by glycan remodeling at this site. Proteins containing the Fc region (such as Fc fusion proteins) can also achieve site-specific conjugation of different molecules based on asparagine 297 in the Fc region of antibodies, where asparagine at position 297 in the protein containing the Fc region is localized according to the amino acid sequence of the antibody.

[0012] This application remodels the Fc-terminal glycan of an antibody with an immobilized endoglycosidase having glycosyltransferase activity, and conjugates a linker-payload efficiently, specifically to a protein or antibody containing Fc by a suspension liquid-phase catalysis or column continuous flow catalysis process, obtaining a highly homogeneous antibody-drug conjugate in one step.

[0013] In one aspect, the present invention is a method for preparing a drug conjugate, wherein the drug conjugate is site-specifically conjugated based on the Fc region N-glycosylation site, and the method comprises: (1) providing a donor containing an oxazoline oligosaccharide, a protein containing an Fc region, and an immobilized endoglycosidase having glycosyltransferase activity, wherein the Fc contains a GlcNAc motif; (2) covalently bonding the donor containing the oxazoline oligosaccharide to the protein containing the Fc motif by the catalytic action of the endoglycosidase. A method is provided that includes these steps.

[0014] In some embodiments, the protein containing the Fc region is an antibody or an Fc fusion protein. In one aspect, the present invention is a method for preparing an antibody-drug conjugate, wherein the antibody-drug conjugate is site-specifically conjugated based on the antibody Fc region N-glycosylation site, and the method comprises: (1) providing a donor containing an oxazoline oligosaccharide, an antibody containing a GlcNAc motif, and an immobilized endoglycosidase having glycosyltransferase activity; and (2) covalently bonding the activated donor containing the oxazoline oligosaccharide to the antibody containing the GlcNAc motif by the catalytic action of the endoglycosidase. A method is provided that includes these steps.

[0015] In some embodiments, the donor containing the oxazoline oligosaccharide further comprises a payload. In some embodiments, the payload is selected from the group consisting of small molecule compounds, agonists, nucleic acids, nucleic acid analogs, fluorescent molecules, radionuclides, and immunomodulatory proteins (such as interleukins). In some embodiments, the payload is a small molecule compound (e.g., various small molecule drugs with various mechanisms of action including various conventional small molecule drugs, photoacoustic therapy drugs, photothermal therapy drugs, etc., such as small molecule drugs for chemotherapy, small molecule target drugs, immune agonists, etc., such as conventional cytotoxic drugs like cisplatin, paclitaxel, 5-fluorouracil, cyclophosphamide, and bendamustine, small molecule target drugs like imatinib mesylate, gefitinib, and anlotinib, immune agonists like STING agonists, TLR agonists, etc.), nucleic acids and nucleic acid analogs, tracer molecules (including fluorescent molecules, biotin, fluorophores, chromophores, spin resonance probes, and radiolabels, etc.), short-chain peptides, polypeptides, peptidomimetics, and proteins.

[0016] In some embodiments, the oxazoline oligosaccharide is one or more selected from the group consisting of disaccharide oxazoline, trisaccharide oxazoline, tetrasaccharide oxazoline, pentasaccharide oxazoline, hexasaccharide oxazoline, heptasaccharide oxazoline, octasaccharide oxazoline, nonasaccharide oxazoline, decasaccharide oxazoline, and undecasaccharide oxazoline. In some embodiments, the oxazoline oligosaccharide has the structure of a first hexosyl or its derivative - (a second hexosyl or its derivative) f -β-D-glucopyranosyl oxazoline, where f is 0, 1, 2, 3, 4, 5, or 6, and the structure of β-D-glucopyranosyl oxazoline is as follows.

Chemical formula

[0017] In some embodiments, the first hexosyl or its derivative is selected from the group consisting of glucosyl, mannosyl, galactosyl, fructosyl, glucosyl, idosyl or their derivatives, and / or the carbon at its 6-position is in the form of -C(O)-, and / or, the second hexosyl or its derivative is independently selected from the group consisting of glucosyl, mannosyl, galactosyl, fructosyl or their derivatives for each occurrence, and / or each monosaccharide moiety in the oligosaccharide structure is linked via a β-(1→4) glycosidic bond, and / or, the derivatives (i.e., the first hexosyl derivative and the second hexosyl derivative) are independently selected from derivatives in which the uronic acid or the hydroxyl group of the monosaccharide is substituted by acylamino.

[0018] In some embodiments, the oxazoline oligosaccharide, has the structure of a first hexosyl or its derivative-β-D-glucopyranosyl oxazoline, and the first hexosyl or its derivative is mannitol (mannosyl) or its derivative.

[0019] In some embodiments, the oxazoline oligosaccharide, has the structure of a first hexosyl or its derivative-β-D-glucopyranosyl oxazoline, and the first hexosyl or its derivative is galactosyl or its derivative.

[0020] In some embodiments, the oxazoline oligosaccharide, is a first hexosyl or its derivative-β-D-glucopyranosyl oxazoline, having a structure in which the first hexosyl or its derivative is glucosyl or its derivative, or, is a first hexosyl or its derivative-β-D-glucopyranosyl oxazoline, having a structure in which the first hexosyl or its derivative is fructosyl or its derivative, or, is a first hexosyl or its derivative-β-D-glucopyranosyl oxazoline, having a structure in which the first hexosyl or its derivative is glucosyl or its derivative, or, It is a 1-hexosyl or its derivative-β-D-glucopyranosyloxazoline, and has a structure in which the 1-hexosyl or its derivative is idosyl or its derivative.

[0021] In some embodiments, the structure of the oxazoline oligosaccharide is as follows.

Chemical formula

[0022] In some embodiments, the structure of the oxazoline oligosaccharide is as follows.

Chemical formula

[0023] In one aspect, the present invention is a method for preparing a drug conjugate, wherein the antibody-drug conjugate is site-specifically conjugated based on the Fc region N-glycosylation site, and the method comprises: (1) providing a donor containing an oxazoline oligosaccharide, a protein containing an Fc region, and an immobilized endoglycosidase having glycosyltransferase activity, wherein the Fc region contains a GlcNAc motif, the step that the donor containing an oxazoline oligosaccharide is a linker-payload compound of formula (I),

Chemical formula

Chemical formula

[0024] In some embodiments, -L-(P)t is as shown in the following formula I-1 or formula I-2.

[0025] In some embodiments, the first hexosyl or its derivative is selected from glucosyl, mannosyl, galactosyl, fructosyl, glucosyl, idosyl or their derivatives, and / or the second hexosyl or its derivative is independently selected from glucosyl, mannosyl, galactosyl, fructosyl or their derivatives for each occurrence, and / or each monosaccharide moiety in the oligosaccharide structure is bonded via a β-(1→4) glycosidic bond, and / or the first hexosyl derivative and the second hexosyl derivative are independently selected from derivatives in which the uronic acid or the hydroxyl group of the monosaccharide is substituted by acylamino. In some embodiments, the protein containing the Fc region is an antibody or an Fc fusion protein.

[0026] In one aspect, the present invention is a method for preparing an antibody-drug conjugate, wherein the antibody-drug conjugate is site-specifically conjugated based on the N-glycosylation site of the antibody Fc region, and the method comprises (1) Providing a donor containing an oxazoline oligosaccharide, an antibody containing a GlcNAc motif, and an immobilized endoglycosidase having glycosyltransferase activity, wherein the donor containing the oxazoline oligosaccharide is a linker-payload compound of formula (I); [Chemical formula] (2) Covalently bonding the donor containing the oxazoline oligosaccharide to the antibody containing the GlcNAc motif by the catalysis of the endoglycosidase; The structure of the antibody-drug conjugate is as shown in formula (II-1), (II-2), (II-3), (II-4) or (II-5), [Chemical formula] wherein P is a payload, D-C(O)-L- is a linker D-C(O)- is a disaccharide structure, L is a linker, and L is directly bonded to the carbonyl in D-C(O)- through -NH- therein. When L is an unbranched linker, it is bonded to one P and t = 1; when L is a branched linker, each branch can be bonded to one P and t is an integer greater than 1; R is hydrogen or α-L-fucosyl, q is 1 or 2, and Ab is an antibody or an antigen-binding fragment thereof.

[0027] In one aspect, the present invention provides a method for preparing an antibody-drug conjugate, wherein the antibody-drug conjugate is site-specifically conjugated based on the N-glycosylation site of the antibody Fc region, and the method comprises: (1) Providing a donor containing an oxazoline oligosaccharide, an antibody containing a GlcNAc motif, and an immobilized endoglycosidase having glycosyltransferase activity, wherein A step in which a donor containing an oxazoline oligosaccharide is a linker-payload compound of formula (I),

Chemical formula

Chemical formula

Chemical formula

[0028] In some embodiments, -L-(P) in formula (I) t is -L 2 -L 1 -B-P, in which case formula (I) is as follows,

Chemical formula

Chemical formula

[0029] In one aspect, the present invention is a method for preparing an antibody-drug conjugate, wherein the antibody-drug conjugate is site-specifically conjugated based on the N-glycosylation site of the antibody Fc region, and the method comprises providing a linker-payload compound of formula (I), an antibody, and an immobilized endoglycosidase having glycosyltransferase activity;

Chemical formula

[0030] In some embodiments, -L-(P) in formula (I) t is -L 2 -L 1 -B-P, in which case formula (I) is as follows,

Chemical formula

Chem.

[0031] In some embodiments, -L-(P)t is

Chemical formula

Chemical formula

Chem.

Chem.

Chem.

[0032] In some embodiments, L 2 is an amino acid residue sequence of -*(AA) n- wherein n is an integer from 1 to 100, AA is an amino acid residue independently for each occurrence, * represents the N-terminus of the corresponding amino acid, ** represents the C-terminus of the corresponding amino acid, and optionally -(C2H4-O) is between the amino and α-carbon of one amino acid m -(CH2) p - is present, m is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, p is 0, 1, 2 or 3, and the *-end and the carbonyl in the disaccharide structure form an amide bond. In some embodiments, AA is independently for each occurrence any one of Phe, Lys, Gly, Ala, Leu, Asn, Val, Ile, Pro, Trp, Ser, Tyr, Cys, Met, Asp, Gln, Glu, Thr, Arg, His or any combination thereof.

[0033] In some embodiments, n is an integer from 1 to 100. In some embodiments, n is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 20, 22, 25, 28, 32, 34, 40, 50, 52, 60, 70, 86, 90, 100, or a value (or endpoint value) between any two values. In some embodiments, n is about 1 to 50. In some embodiments, n is about 1 to 30. In some embodiments, n is about 1 to 20. In some embodiments, n is about 1 to 10.

[0034] In some embodiments, L 1 is a cleavable sequence containing an amino acid sequence cleavable by an enzyme, and the amino acid sequence cleavable by the enzyme contains 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids. In some embodiments, L 1 is any one of Val, Cit, Phe, Lys, Gly, Ala, Leu, Asn or any combination thereof, preferably -Gly-Gly-Phe-Gly-, -Phe-Lys-, -Val-Cit-, -Val-Lys-, -Gly-Phe-Leu-Gly-, -Ala-Leu-Ala-Leu-, -Ala-Ala-Ala- and combinations thereof. In some embodiments, L1 is -Val-Cit-.

[0035] In some embodiments, B is

Chemical formula

[0036] In some embodiments, -L 1 -B- represents -Val-Cit-PABC-. In some embodiments, -L 2 -L 1 -B- represents -Gly-Gly-Gly-Val-Cit-PABC-.

[0037] In some embodiments, the payload P is selected from the group consisting of small molecule compounds (e.g., small molecule drugs of various mechanisms of action including various conventional small molecule drugs, photoacoustic dynamic therapy drugs, photothermal therapy drugs, etc., such as small molecule drugs for chemotherapy, small molecule target drugs, immune agonists, etc., such as conventional cytotoxic drugs such as cisplatin, paclitaxel, 5-fluorouracil, cyclophosphamide and bendamustine, small molecule target drugs such as imatinib mesylate, gefitinib and anlotinib, immune agonists such as STING agonists, TLR agonists, etc.), nucleic acids and nucleic acid analogs, tracer molecules (including fluorescent molecules, biotin, fluorophores, chromophores, spin resonance probes and radioisotopes, etc.), short chain peptides, polypeptides, peptidomimetics and proteins. In some embodiments, the payload P is a cytotoxin or a fragment thereof and is optionally derivatized for attachment to the L moiety in the compound of formula (I).

[0038] In some embodiments, the cytotoxin is a taxane, a maytansinoid, an auristatin, an epothilone, combretastatin A-4 phosphate, combretastatin A-4 and its derivatives, an indole sulfonamide, vinblastine, vincristine, vindesine, vinorelbine, vinflunine, vinglycinate, anhydrovinblastine and other vinblastines, dolastatin 10 and its analogs, halichondrin B, eribulin, indole-3-oxoacetamides, podophyllotoxins, 7-diethylamino-3-(2'-benzoxazolyl)-coumarin (DBC), discodermolide, laulimalide, camptothecins and their derivatives, mitoxantrone, mitoguazone, nitrogen mustards, nitrosoureas, aziridines, benzodopa, carboquone, meturedepa, uredepa, dynemicin, esperamicin, neocarzinostatin, aclacinomycin, actinomycin, anthramycin, bleomycin, actinomycin C, carvisine, calminomycin, sarcomycin, calminomycin, actinomycin D, daunorubicin, detorubicin, doxorubicin, epirubicin, elsorubicin, idarubicin, marcellomycin, mitomycins, nogalamycin, olivomycin, peplomycin, porfiromycin, puromycin, keramycin, rhodrubicin, streptozotocin, streptomycin, dinostatin, zorubicin, trichothecenes, T-2 toxin, verracurin AA), lolidine A, anguidine, ubenimex, azaserine, 6-diazo-5-oxo-L-norleucine, denopterin, methotrexate, pteropterin, trimethoprim, edatrexate, fludarabine, 6-purinethiol, thiampurine, thioguanine, ancitabine, gemcitabine, enocitabine, azacitidine, 6-azauridine, carmofur, cytarabine, didoxyridine, doxifluridine, floxuridine, calusterone, drostanolone propionate, epithioestanol, mepitiostane, testolactone, aminoglutethimide, mitotane, trilostane, flutamide, nilutamide, bicalutamide, leuprorelin acetate, protein kinase inhibitors and proteasome inhibitors, and / or, The cytotoxic agent is selected from the group consisting of vinca alkaloids, colchicine alkaloids, taxanes, auristatins, maytansinoids, calicheamicin, doxorubicin, duocarmycin, SN-38, cryptophycin analogs, deruxtecan, duocarmazine, calicheamicin, centanamycin, dolastansine, pyrrolobenzodiazepine and exatecan and its derivatives, and / or, The cytotoxic agent is selected from auristatin, especially MMAE, MMAF or MMAD, and / or, The cytotoxic agent is selected from exatecan and its derivatives such as DX8951f, and / or, The cytotoxic agent is selected from DXd-(1) and DXd-(2), preferably DXd-(1).

[0039] In some embodiments, the linker-payload compound is as shown in formula (I-2).

Chem.

Chem.

[0040] In some embodiments, the antibody is selected from the group consisting of an anti-CD19 antibody, an anti-CD20 antibody, an anti-CD22 antibody, an anti-CD25 antibody, an anti-CD30 / TNFRSF8 antibody, an anti-CD33 antibody, an anti-CD37 antibody, an anti-CD44v6 antibody, an anti-CD56 antibody, an anti-CD70 antibody, an anti-CD71 antibody, an anti-CD74 antibody, an anti-CD79b antibody, an anti-CD117 / KITk antibody, an anti-CD123 antibody, an anti-CD138 antibody, an anti-CD142 antibody, an anti-CD174 antibody, an anti-CD227 / MUC1 antibody, an anti-CD352 antibody, an anti-CLDN18.2 antibody, an anti-DLL3 antibody, an anti-ErbB2 / HER2 antibody, an anti-CN33 antibody, an anti-GPNMB antibody, an anti-ENPP3 antibody, an anti-Nectin-4 antibody, an anti-EGFRvIII antibody, an anti-SLC44A4 / AGS-5 antibody, an anti-CEACAM5 antibody, an anti-PSMA antibody, an anti-TIM1 antibody, an anti-LY6E antibody, an anti-LIV1 antibody, an anti-Nectin4 antibody, an anti-SLITRK6 antibody, an anti-HGFR / cMet antibody, an anti-SLAMF7 / CS1 antibody, an anti-EGFR antibody, an anti-BCMA antibody, an anti-AXL antibody, an anti-NaPi2B antibody, an anti-GCC antibody, an anti-STEAP1 antibody, an anti-MUC16 antibody, an anti-Mesothelin antibody, an anti-ETBR antibody, an anti-EphA2 antibody, an anti-5T4 antibody, an anti-FOLR1 antibody, an anti-LAMP1 antibody, an anti-Cadherin6 antibody, an anti-FGFR2 antibody, an anti-FGFR3 antibody, an anti-CA6 antibody, an anti-CanAg antibody, an anti-integrin αV antibody, an anti-TDGF1 antibody, an anti-Ephrin A4 antibody, an anti-TROP2 antibody, an anti-PTK7 antibody, an anti-NOTCH3 antibody, an anti-C4.4A antibody, an anti-FLT3 antibody, an anti-B7H3 / 4 antibody, an anti-TF (Tissue Factor) antibody, an anti-ROR1 / 2 / antibody, preferably an anti-CD19 antibody, an anti-ErbB2 / HER2 antibody, an anti-CLDN18.2 antibody, an anti-Nectin-4 antibody, an anti-FGFR3 antibody, an anti-Trop2 antibody.

[0041] In some embodiments, the endoglycosidase having the glycosyltransferase activity is an N-acetylglucosamine endohydrolase. In some embodiments, the N-acetylglucosamine endohydrolase comprises at least one selected from Endo S (Streptococcus pyogenes endoglycosidase-S), Endo F3 (Elizabethkingia miricola endoglycosidase-F3), Endo S2 (Endoglycosidase-S2, Streptococcus pyogenes endoglycosidase-S2), Endo Sd (Endoglycosidase-Sd, Streptococcus pyogenes endoglycosidase-Sd), and Endo CC (Endoglycosidase-CC, Streptococcus pyogenes endoglycosidase-CC), or the N-acetylglucosamine endohydrolase comprises at least one selected from Endo H, Endo D, Endo F2, Endo F3, Endo M, Endo CC1, Endo CC2, Endo Om, Endo S, and Endo S2.

[0042] In some embodiments, the endoglycosidase having the glycosyltransferase activity has a Halo tag covalently attached thereto, and the endoglycosidase fusion protein is immobilized on a support containing a haloalkyl linker by the Halo tag. The Halo tag is a dehalogenase or a variant or truncated functional active portion thereof. In some embodiments, the Halo tag is attached to one end of the endoglycosidase, and a His tag (the His tag is a histidine polypeptide, such as His4, His5, His6, His8, His 10 、His 12 、or His 14is attached. In some embodiments, a Halo tag is attached to the amino terminus of the endoglycosidase, and a His tag is attached to the carboxyl terminus of the endoglycosidase, i.e., it becomes Halo-endoglycosidase-His (where His here refers to the His tag, His4, His5, His6, His8, His 10 His 12 or His 14 is. The same applies hereinafter). In some embodiments, a Halo tag is attached to the amino terminus of the endoglycosidase, and a His tag is attached to the carboxyl terminus of the endoglycosidase, and the endoglycosidase is Endo-S2, i.e., it becomes Halo-Endo S2-His.

[0043] In some embodiments, the support contains the chloroalkyl linker such that the endoglycosidase fusion protein is immobilized on the support by the covalent interaction between the chloroalkyl linker and the Halo tag. In some embodiments, the chloroalkyl linker is generated by a chloroalkyl substrate having the structure of formula (III),

Chemical formula

[0044] In some embodiments, the support has the structure of formula (IV),

Chemical formula

Chemical formula

[0045] In one aspect, the present invention provides an endoglycosidase fusion protein comprising a covalently attached endoglycosidase and a Halo tag, wherein the Halo tag is a dehalogenase or a variant or truncated functional portion thereof. In some embodiments, the endoglycosidase fusion protein consists of a covalently attached endoglycosidase and Halo.

[0046] In some embodiments, the fusion protein has a Halo tag covalently attached to one end of the endoglycosidase and a His tag covalently attached to the other end of the endoglycosidase. In some embodiments, the Halo tag is covalently attached to the amino terminus of the endoglycosidase and the His tag is covalently attached to the carboxyl terminus of the endoglycosidase.

[0047] In some embodiments, the endoglycosidase is at least one selected from Endo S (Streptococcus pyogenes endoglycosidase - S), Endo F3 (Elizabethkingia miricola endoglycosidase - F3), Endo S2 (Endoglycosidase - S2, Streptococcus pyogenes endoglycosidase - S2), Endo Sd (Endoglycosidase - Sd, Streptococcus pyogenes endoglycosidase - Sd) and Endo CC (Endoglycosidase - CC, Streptococcus pyogenes endoglycosidase - CC), or the endoglycosidase is at least one selected from Endo H, Endo D, Endo F2, Endo F3, Endo M, Endo CC1, Endo CC2, Endo Om, Endo S and Endo S2.

[0048] In one embodiment, the endoglycosidase is endo - β - N - acetylglucosaminidase. In one embodiment, the endoglycosidase is selected from the group consisting of Endo H, Endo D, Endo F2, Endo F3, Endo M, Endo CC1, Endo CC2, Endo Om, Endo S and Endo S2.

[0049] In some embodiments, the His - tag is a plurality of consecutive histidine residues. In some embodiments, the His - tag is 3 histidines, 4 histidines, 5 histidines, 6 histidines, 7 histidines, 8 histidines, 9 histidines or 10 histidines. In some embodiments, the His - tag is His6. In one embodiment, the His - tag is His8. In one embodiment, the His - tag is His 10 is.

[0050] In one embodiment, the endoglycosidase fusion protein is the amino acid sequence shown in SEQ ID NO: 1, or has at least 80% identity, at least 85% identity, at least 90% identity with SEQ ID NO: 1, or has one or more conservative amino acid substitutions with SEQ ID NO: 1.

[0051] In some embodiments, the endoglycosidase fusion protein comprises or consists of the amino acid sequence shown in SEQ ID NO: 1.

[0052] In some embodiments, the endoglycosidase fusion protein comprises the amino acid sequence shown at positions 1 to 1150 in SEQ ID NO: 1, or has at least 90% identity compared to the amino acids shown at positions 1 to 1150 in SEQ ID NO: 1, or has one or more conservative amino acid substitutions compared to the amino acids shown at positions 1 to 1150 in SEQ ID NO: 1. In some embodiments, the endoglycosidase fusion protein comprises or consists of the amino acid sequence shown at positions 1 to 1150 in SEQ ID NO: 1.

[0053] In some embodiments, the pI of the endoglycosidase fusion protein is about 4 to 7. In some embodiments, the pI of the endoglycosidase fusion protein is about 4, 4.1, 4.3, 4.5, 4.7, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, or a value between any two of these values (including the endpoint values).

[0054] In one aspect, the present invention is a method for preparing the endoglycosidase fusion protein of the present invention, comprising: (a) providing a nucleic acid sequence of an endoglycosidase; and (b) binding one end of the nucleic acid sequence of the endoglycosidase to the nucleic acid sequence of a Halo tag. A method is provided in which the obtained nucleic acid sequence is cloned into a suitable vector, and then the vector is transformed into a suitable host cell to express the endoglycosidase fusion protein of the present invention within the host cell.

[0055] In some embodiments, a method for preparing the endoglycosidase fusion protein of the present invention comprises: (a) providing a nucleic acid sequence of an endoglycosidase; (b) binding one end of the nucleic acid sequence of the endoglycosidase to the nucleic acid sequence of a Halo tag; and (c) binding the other end of the nucleic acid sequence of the endoglycosidase to the nucleic acid sequence of a His tag.

[0056] In some embodiments, the nucleic acid sequence of the Halo tag is bound to the amino terminus of the endoglycosidase sequence. In some embodiments, the nucleic acid sequence of the Halo tag is bound to the amino terminus of the nucleic acid sequence of the endoglycosidase, and the nucleic acid sequence of the His tag is bound to the carboxyl terminus of the nucleic acid sequence of the endoglycosidase.

[0057] In one aspect, the present invention provides an immobilized endoglycosidase fusion protein comprising the endoglycosidase fusion protein of the present invention immobilized on a support.

[0058] In some embodiments, the support comprises a haloalkyl linker such that the endoglycosidase fusion protein is immobilized on the support by a covalent interaction between the haloalkyl linker and the Halo tag.

[0059] In some embodiments, the support comprises a chloroalkyl linker such that the endoglycosidase fusion protein is immobilized on the support by a covalent interaction between the chloroalkyl linker and the Halo tag. In some embodiments, the chloroalkyl linker is generated by a chloroalkyl substrate having the structure of formula (III),

Chemical formula

[0060] In some embodiments, the support has the structure of formula (IV),

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0061] In another aspect, the present invention provides a prepacked column filled with an immobilized endoglycosidase fusion protein.

[0062] In another aspect, the present invention further provides the use of the above immobilized endoglycosidase fusion protein and / or the above prepacked column in the preparation and / or purification of a drug conjugate or an antibody conjugate of the above prepacked column. In some embodiments, the above immobilized endoglycosidase fusion protein and / or the above prepacked column can be easily industrially scaled up in the production (e.g., purification) of a drug conjugate or an antibody conjugate.

[0063] The one-step conjugation system using the immobilized endoglycosidase catalyst of the present application has the following advantages. 1) In the case of a liquid-phase enzyme catalyst, a large amount of endoglycosidase needs to be introduced, and a large amount of enzyme-related impurities (e.g., host proteins and nucleic acids) are introduced into the reaction system. Therefore, the requirement for enzyme purity is high (the process is complex and the preparation cost is high), and it is difficult to remove impurities downstream of the subsequent drug conjugate. In contrast, the immobilization of the enzyme does not require high purity of the starting endoglycosidase (the enzyme preparation process is simple and the cost is low), the impurities introduced into the reaction system are very few, the impurity removal process downstream of the drug conjugate is greatly simplified, and the immobilization of the enzyme is useful for separation and purification, and the tool enzyme is more easily removed from the product, reducing the possibility of sugar chain and small molecule dropout of the product due to the residue of the tool enzyme, so the toxicity caused by potential product decomposition can be reduced. 2) The immobilization of the enzyme is useful for the repeated use of the enzyme. 3) By the intelligent continuous flow conjugation technology, the procedures of reaction and purification are greatly simplified, the efficiency of drug discovery is greatly improved, and the process robustness in the drug production process is guaranteed. 4) The one-step conjugation of the antibody and the linker-payload is realized by the endoglycosidase immobilized on the column. 5) The PI of the endoglycosidase fusion protein of the present invention is about 5.5, and the PI of the ADC conjugate is 8-9. By adopting AEX and / or CEX, it is easy to remove the endoglycosidase fusion protein from the ADC product in the later stage. 6) The conjugation column catalyst of the endoglycosidase fusion protein of the present invention can achieve excellent conjugation efficiency, easy linear scale-up, and easy removal of residual enzymes, making it more suitable for the industrial production of ADCs.

Brief Description of the Drawings

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Mode for Carrying Out the Invention

[0065] 1. Definitions Unless otherwise defined herein, all technical and scientific terms used in the present invention have the same meaning as commonly understood by one of ordinary skill in the art. Also, terms and experimental methods related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology are terms and common methods widely used in the art. The trade names described herein refer to the corresponding products or their active ingredients. All patents, published patent applications, and publications cited herein are incorporated herein by reference. Further, to better understand the present invention, definitions and explanations of related terms are provided as follows.

[0066] As used herein, the expressions "at least one" or "one or more" or "one kind" or "plural kinds" refer to one, two, three, four, five, six, seven, eight, nine, one hundred, two hundred, three hundred, four hundred, five hundred, six hundred, seven hundred, eight hundred, nine hundred, or more. As used herein, unless specifically indicated to the contrary, "one" and "one kind" should be understood as "at least one" or "at least one kind".

[0067] The fact that a particular quantity, concentration, or other value or parameter is described in the form of a range, a preferred range, or a preferred upper limit or a preferred lower limit should be understood to be equivalent to specifically disclosing any range formed by combining any upper limit or preferred value with any lower limit or preferred value, regardless of whether it is explicitly stated. Unless otherwise specified, the numerical ranges recited herein are intended to include the endpoints of the range and all integers and fractions (decimals) within the range, as well as the values between any two values. For example, the expression "u is an integer from 1 to 20" should be understood to mean that u is any integer from 1 to 20. For example, u can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. Other similar expressions should be understood in the same way.

[0068] The terms "about" and "approximately", when used in connection with a numerical variable, such as a concentration, isoelectric point (pI), pH, temperature, or a particular range, generally mean that the value of the variable and all values of the variable are within the range of experimental error (e.g., within the 95% confidence interval of the mean value) or within ±10% of the specified value, or within a wider range.

[0069] The term "optional" or "optionally" means that the event described thereafter may occur but does not necessarily occur, and the expression includes the case where the event or situation occurs or does not occur.

[0070] Expressions such as "comprising", "including", "containing", and "having" are open-ended and do not exclude additional unenumerated elements, steps, or components. The expression "consisting of" excludes any elements, steps, or components not expressly indicated. The expression "consisting essentially of" means that the scope is limited to the specified elements, steps, or components, and any elements, steps, or components that are optionally present and do not substantially affect the important and novel features of the claimed subject matter. The expression "comprising" should be understood to encompass the terms "consisting essentially of" and "consisting of".

[0071] As used herein, the definition of "biomolecule" includes proteins, nucleic acids, lipids, carbohydrates, small nucleotides, amino acids, and derivatives thereof.

[0072] As used herein, "nucleic acid" or "polynucleotide" refers to a polymer formed by linking at least two nucleotides or nucleotide derivatives by a phosphodiester bond, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).

[0073] As used herein, a "vector" is a vehicle that transfers an exogenous nucleic acid that amplifies or is expressed within a host cell into the host cell. As used herein, the definition of "vector" includes plasmids (e.g., linearized plasmids), viral vectors, cosmids, phage vectors, phagemids, artificial chromosomes (e.g., yeast artificial chromosomes and mammalian artificial chromosomes), and the like. As used herein, for a vector to be capable of being expressed and / or replicated within a host cell means that the vector can express an RNA polynucleotide or polypeptide within the host cell and / or can generate multiple copies of the vector. For a vector to be "capable of being expressed" or "capable of being replicated", the vector may contain a nucleic acid sequence or element operably linked to a promoter or replicon. As used herein, "operably linked" with respect to a nucleic acid sequence or element means that these nucleic acid sequences are functionally related to each other. For example, a promoter may be operably linked to a nucleic acid sequence encoding a polypeptide, whereby the promoter regulates or mediates the transcription of the nucleic acid. Those skilled in the art can select and use a suitable vector according to a specific purpose.

[0074] An Fc fusion protein is a recombinant protein obtained by fusing a bioactive protein or polypeptide with the hinge region or Fc fragment of IgG. Examples of the bioactive protein or polypeptide include ligands, cytokines, receptors, antigens, and cyclic polypeptides that bind to cell surface antigens. The Fc fusion protein confers more antibody properties, such as an extended plasma half-life and the exertion of functional effects specific to the Fc fragment, on the fused protein.

[0075] The GlcNAc motif refers to a sugar chain in which two conserved N-glycosylation sites are present at asparagine (Asn) at position 297 in the Fc region of an antibody, and the sugar chain contains N-acetylglucosamine-β-(1,4)-N-acetylglucosamine covalently bound to Asn. Glycosylation of the Fc region is a complex post-translational modification process that forms sugar chains of various lengths, compositions, and structures through the involvement of various enzymes, and the effects of sugar chains on the biological activity, conformation, stability, solubility, pharmacokinetics, etc. of proteins are different. In some embodiments, the Fc glycosylated sugar molecule has a complex double antenna core structure consisting of two pentose molecules, mannose and N-acetylglucosamine. Different glycoforms contain, in addition to the core structure, different numbers of sugar molecules, such as fucose, mannose, N-acetylglucosamine, galactose, bisecting N-acetylglucosamine, and sialic acid, and heterogeneity often occurs due to galactosylation and allylation of the terminal sugar. Depending on the quantity of terminal galactose, three different subtypes (G0, G1, and G2) are divided, and each subtype contains various forms of core fucose or fucose without a core and bisecting N-acetylglucosamine, that is, there are a total of 16 neutral complex structures.

[0076] As used herein, "peptide", "polypeptide", or "protein" refers to two or more amino acids that are covalently bonded. Unless specifically described otherwise, these terms can be used interchangeably.

[0077] An "amino acid" is an organic compound containing both an amino group and a carboxyl group, such as, for example, an α-amino acid, and can be encoded directly or in the form of a precursor by nucleic acids. A single amino acid is encoded by a nucleic acid consisting of three nucleotides (so-called codon or triplet). Each amino acid is encoded by at least one codon. The fact that the same amino acid is encoded by different codons is called "degeneracy of the genetic code". Amino acids include natural amino acids and non-natural amino acids. Natural amino acids include alanine (three-letter code: Ala, one-letter code: A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine (Cys, C), glutamine (Gln, Q), glutamic acid (Glu, E), glycine (Gly, G), histidine (His, H), isoleucine (Ile, I), leucine (Leu, L), lysine (Lys, K), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y) and valine (Val, V).

[0078] As used herein, "sequence identity" has the meaning recognized in the art, and the percentage of sequence identity between two polypeptides may be calculated by comparing the two sequences with publicly available algorithms such as the Basic Local Alignment Search Tool (BLAST) and the Fast Adaptive Shrinkage / Threshold Algorithm (FASTA) (see, for example, Computational Molecular Biology, Lesk, A.M. ed., Oxford University Press, New York, 1988, Biocomputing: Informatics and Genome Projects, Smith, D.W. ed., Academic Press, New York, 1993, Computer Analysis of Sequence Data, Part I, Griffin, A.M. and Griffin, H.G. eds., Humana Press, New Jersey, 1994). There are various methods for measuring identity between two polypeptides, but the term "identity" is well known to those of skill in the art (Carrillo, H. & Lipman, D., SIAM J Applied Math 48:1073 (1988)).

[0079] As used herein, the term "variant" refers to a protein (or nucleic acid) having one or more residue (or nucleotide) substitutions, deletions, or insertions as compared to a reference protein (or nucleic acid). The reference protein can be a naturally occurring protein isolated from a natural source (i.e., a wild-type protein) or an engineered protein. As used herein, the function or activity of a variant such as an EndoS variant, an EndoS2 variant, or a HaloTag variant is substantially the same as or equivalent to or greater than the function or activity of the reference EndoS, EndoS2, or HaloTag, respectively.

[0080] As used herein, the term "endoglycosidase" refers to an enzyme that catalyzes the hydrolysis of internal glycosidic linkages of oligosaccharide chains and polysaccharides and can be used to cleave polysaccharides from glycoproteins. In some embodiments, the "endoglycosidase" of the present application can be used to hydrolyze the β-(1→4) glycosidic bond between two N-acetylglucosamines at the N-glycosylation site of the antibody Fc region. "Glycosyltransferase activity" refers to the binding of a catalytically activated sugar to different receptor molecules such as proteins, nucleic acids, oligosaccharides, lipids, and small molecules. In the present application, a donor containing an oxazoline oligosaccharide can bind to the GlcNAc of the antibody Fc region.

[0081] As used herein, the term "HaloTag" refers to a haloalkane dehalogenase or variant thereof that removes a halogen from a haloalkyl substrate (e.g., a reagent containing a haloalkyl moiety -(CH2) 2-30 -X, where X is a halogen, such as F, Cl, Br, I, especially Cl or Br) and forms a covalent bond with the remaining portion of the substrate. Mutant haloalkane dehalogenases are described, for example, in WO2006 / 093529 and WO2008 / 054821, the relevant content of which is incorporated herein by reference. Mutant haloalkane dehalogenases that can be used in the present invention include, but are not limited to, variants of Xanthobacter dehalogenase (e.g., Xanthobacter autotrophicus dehalogenase (DhIA)) or Rhodococcus dehalogenase (e.g., Rhodococcus rhodochrous dehalogenase (DhaA)). For example, as described in WO2008 / 054821, those containing one or more substitutions at the residues of the catalytic triad, those with His272 substituted with Phe / Ala / Gly / Gln / Asn, those with Asp106 substituted with Cys, or those due to other substitutions. The prerequisite is that the mutant haloalkane dehalogenase can form a covalent bond with the haloalkyl substrate.

[0082] The "His tag" is a polypeptide consisting of histidine, such as His-His (His2), His-His-His (His3), His-His-His-His (His4), His-His-His-His-His (His5), His-His-His-His-His-His (His6), His-His-His-His-His-His-His-His-His-His (His 10 ) etc.

[0083] As used herein, the term "fusion protein" refers to a protein product in which two or more genes obtained by DNA recombination technology are co-expressed. For example, by deleting the stop codon of the coding gene of the first protein and then ligating the second protein gene having a stop codon, co-expression of the two genes can be achieved. The endoglycosidase fusion protein may further contain one or more additional elements such as, for example, an additional polypeptide or a tag such as a Halo tag and / or a His tag. In some embodiments, the endoglycosidase fusion protein substantially retains the desired properties.

[0084] As used herein, the term "resin" refers to an organic polymer that has a range in which it softens or melts when heated, tends to flow when subjected to an external force during softening, and is solid, semi-solid or liquid at room temperature. "Halo resin" refers to a new compound formed by substituting at least one functional group in the resin with a halo.

[0085] As used herein, the term "conjugation" refers to a covalent bond of at least two moieties (e.g., at least two molecules or at least two termini of the same molecule).

[0086] As used herein, a "conjugate" can be prepared by a covalent bond of at least two moieties (e.g., at least two molecules or at least two termini / side chains of the same molecule).

[0087] As used herein, the term "antibody-drug conjugate" refers to a conjugate that includes an antibody or antibody fragment covalently linked to a payload, where the payload is a small molecule compound, agonist, nucleic acid, nucleic acid analog, or fluorescent molecule.

[0088] As used herein, the term "drug conjugate" refers to a conjugate that includes a protein containing an Fc region covalently linked to a payload, where the payload is a small molecule compound, agonist, nucleic acid, nucleic acid analog, or fluorescent molecule.

[0089] The term "antibody" includes a wide variety of biochemically distinguishable polypeptides. One of ordinary skill in the art will understand that the heavy chain categories include gamma, mu, alpha, delta, or epsilon (γ, μ, α, δ, ε), and several subunits (e.g., γ1-γ4). The "type" of antibody is determined by the nature of the chain to be IgG, IgM, IgA, IgG, or IgE, respectively. Immunoglobulin subunits (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgG5, etc., are well characterized and the functional specificities conferred are also known. All types of immunoglobulins are included within the scope of protection disclosed in the present invention. In some embodiments, the immunoglobulin molecule is of the IgG type. IgG typically includes two identical light chain polypeptides with a molecular weight of about 23,000 daltons and two identical heavy chain polypeptides with a molecular weight of about 53,000-70,000. These four chains are joined in a "Y" configuration by disulfide bonds, where the light chains extend from the "Y" ports through the variable regions and surround the heavy chains.

[0090] Light chains can be divided into kappa (κ) or lambda (λ). Each heavy chain can bind to a κ or λ light chain. Generally, when immunoglobulins are produced by hybridomas, B cells, or genetically engineered host cells, their light and heavy chains are covalently linked, and the "tail" portions of the two heavy chains are linked by either covalent disulfide bonds or non-covalent bonds. In the heavy chain, the amino acid sequence extends from the N-terminus at the branched end of the Y configuration to the C-terminus at the bottom of each chain. The immunoglobulin κ light chain variable region is Vκ, and the immunoglobulin λ light chain variable region is Vλ.

[0091] Both light and heavy chains are divided into regions that are structurally and functionally homologous. The terms "constant" and "variable" are used according to function. The variable regions of the light chain (VL) and heavy chain (VH) determine antigen recognition and specificity. The constant regions of the light and heavy chains confer important biological properties such as secretion, transplacental transfer, Fc receptor binding, complement binding, etc. Conventionally, the numbers of the constant regions increase as they move farther from the antigen-binding site or the amino terminus of the antibody. The N-terminal portion is the variable region, and the C-terminal portion is the constant region. The CH3 and CL domains contain the carboxyl termini of the heavy and light chains, respectively.

[0092] In a naturally occurring antibody, assuming that the antibody is in a three-dimensional arrangement in a water-containing environment, the six "complementary determining regions" or "CDRs" present in each antigen-binding domain are short discontinuous amino acid sequences that form the antigen-binding domain and specifically bind to the antigen. The remaining other amino acids, called the "framework" regions in the antigen-binding domain, have little intermolecular variation. Most of the framework regions adopt a β-sheet conformation, and the CDRs either form a loop structure that binds to it or, in some cases, form part of the β-sheet structure. Thus, the framework regions localize the CDRs in the correct orientation by forming a scaffold through intermolecular non-covalent binding interactions. The antigen-binding domain with CDRs at specific positions forms a surface complementary to the epitope on the antigen, and this complementary surface promotes the non-covalent binding of the antibody to its antigen epitope. For a given heavy or light chain variable region, those skilled in the art can identify the amino acids including the CDRs and framework regions by methods known in the art (see Kabat, E. et al., U.S. Department of Health and Human Services, Sequences of Proteins of Immunological Interest, (1983) and Chothia and Lesk, J. Mol. Biol., 196:901-917 (1987)).

[0093] CDRs defined according to Kabat and Chothia include overlapping or subsets of amino acid residues when compared to each other. Nevertheless, it is within the scope of the present invention to apply any definition for referring to the CDRs of an antibody or its variants. The exact numbering of residues including a particular CDR varies depending on the sequence and size of the CDR. One of ordinary skill in the art can usually determine which particular residues are included in the CDRs according to the amino acid sequence of the variable region of the antibody. Kabat et al. have also defined a numbering system applicable to the variable region sequences of any antibody. One of ordinary skill in the art can apply the "Kabat numbering" system to any variable region sequence without relying on other experimental data than the sequence itself. "Kabat numbering" refers to the numbering system proposed by Kabat et al. in "Sequence of Proteins of Immunological Interest" (1983) by the U.S. Dept. of Health and Human Services. Antibodies may adopt the EU numbering system.

[0094] The light chain constant region includes a partial amino acid sequence derived from an antibody light chain. Preferably, the light chain constant region (CL) includes at least one of the constant κ domain or the constant λ domain. "Light chain-heavy chain pair" refers to a set of a light chain and a heavy chain that can form a dimer by a disulfide bond between the CL domain of the light chain and the CH1 domain of the heavy chain.

[0095] The "Fc region" is the tail region of an antibody and interacts with some proteins of cell surface receptors and the complement system. By this property, antibodies are enabled to activate the immune system. In IgG, IgA, and IgD antibody isotypes, the Fc region consists of two identical protein fragments and is derived from the second and third constant regions of the two heavy chains of the antibody. In IgM and IgE antibody isotypes, the Fc region includes three heavy chain constant domains (CH2-4). The Fc region of IgG has a highly conserved N-glycosylation site. Glycosylation of the Fc fragment is necessary for Fc receptor-mediated activity, and the influence of glycoforms on the pharmacological properties of therapeutic antibodies varies.

[0096] Antibodies can be prepared by conventional recombinant DNA techniques. Cell lines that produce antibodies can be selected, constructed, and cultured by techniques known to those skilled in the art. These techniques are described in various laboratory manuals and major publications. Technical references suitable for use in the present invention are described below. For example, Current Protocols in Immunology, edited by Coligan et al., Green Publishing Associates and Wiley-Interscience, John Wiley and Sons, New York (1991), and Recombinant DNA Technology for Production of Protein Therapeutics in Cultured Mammalian Cells, D.L. Hacker, F.M. Wurm, in Reference Module in Life Sciences, 2017. All of their contents, including supplementary content, are hereby incorporated by reference into this specification.

[0097] In some embodiments, according to the conventional method, DNA encoding an antibody is designed and synthesized according to the antibody amino acid sequences described herein, placed in an expression vector, and then host cells are transfected, and the transfected host cells are cultured in a medium to produce monoclonal antibodies. In some embodiments, the expression antibody vector contains at least one promoter element, an antibody coding sequence, a transcription termination signal, and a polyA tail. Other elements include enhancers, Kozak sequences, and donor and acceptor sites for RNA splicing on both sides of the inserted sequence. Efficient transcription may be achieved by the early and late promoters of SV40, long terminal repeat sequences from retroviruses such as RSV, HTLV1, HIVI, and the early promoter of cytomegalovirus, or other cellular promoters such as the actin promoter may be used. Suitable expression vectors may include pIRES1neo, pRetro-Off, pRetro-On, PLXSN, or Plncx, pcDNA3.1(+ / -), pcDNA / Zeo(+ / -), pcDNA3.1 / Hygro(+ / -), PSVL, PMSG, pRSVcat, pSV2dhfr, pBC12MI, and pCS2, etc. Commonly used mammalian cells include 293 cells, Cos1 cells, Cos7 cells, CV1 cells, mouse L cells, and CHO cells, etc.

[0098] In some embodiments, the inserted gene fragment needs to contain a screening marker, and common screening markers include dihydrofolate reductase screening gene, glutamine synthetase screening gene, neomycin resistance screening gene, hygromycin resistance screening gene. By these, cells that have successfully transfected are screened and separated. The constructed plasmid is transfected into host cells that do not contain the above genes, and the cells that have successfully transfected grow in large numbers by culturing in a selective medium and produce the desired target protein.

[0099] As used herein, the term "isoelectric point (pI)" refers to the pH (hydrogen ion concentration index) value of an aqueous solution when a molecule (such as a protein) has no net surface charge, and is expressed in pH units. The pI of a protein can be experimentally measured using methods well known in the art, such as imaging capillary isoelectric focusing (iCIEF) and capillary isoelectric focusing (CIEF). Different biomolecules (such as proteins, nucleic acids, polysaccharides, etc.) with different pIs may carry different charges at a given pH value, thereby enabling their separation by methods such as ion exchange chromatography or isoelectric focusing.

[0100] As used herein, a molecule having an "alkaline pI" refers to a molecule whose pI is higher than 7.0. As used herein, a molecule having an "acidic pI" refers to a molecule whose pI is lower than 7.0.

[0101] As used herein, "ion exchange chromatography (IEX)" refers to a technique for separating biomolecules based on differences in the net surface charge of biomolecules and differences in affinity for an ion exchanger (also called a medium, resin, or stationary phase). For example, in anion exchange chromatography, a protein with a pI lower than the buffer pH has a negative net surface charge and binds to a positively charged anion exchanger. However, another protein with a pI higher than the buffer pH has a positive net surface charge and does not bind to the positively charged anion exchanger, and thus passes through the medium with the buffer.

[0102] As used herein, the term "support" refers to a water-insoluble substance separable from the reaction mixture in solid or semi-solid form, such as a surface, gel, polymer, matrix, particle, resin, bead, or membrane.

[0103] "Spacer" refers to a structure that is located between different structural modules and can spatially separate the structural modules. The definition of a spacer is not limited by whether it has a specific function or can be cleaved or degraded in vivo. Examples of spacers include, but are not limited to, amino acids and non-amino acid structures. Among them, the non-amino acid structure can be, but is not limited to, an amino acid derivative or analog. "Spacer sequence" refers to an amino acid sequence that acts as a spacer, and examples thereof include a single amino acid, a sequence containing multiple amino acids, such as a sequence containing two amino acids such as GA, or sequences containing, but not limited to, GGGGS, GGGGSGGGGS, GGGGSGGGGSGGGGS, etc. A self-cleaving spacer (such as self-cleaving spacer Sp1) is a covalent assembly that sequentially cleaves two chemical bonds after activation of the protecting moiety of the precursor. Upon stimulation, the protecting moiety (such as a cleavable sequence) is removed, triggering a cascade of degradation reactions, resulting in the sequential release of smaller molecules. Examples of self-cleaving spacers include, but are not limited to, PABC (p-aminobenzyloxycarbonyl), acetal, heteroacetal, and combinations thereof.

[0104] The term "alkyl group" means a straight-chain or branched saturated aliphatic hydrocarbon group consisting of carbon atoms and hydrogen atoms, and the saturated aliphatic hydrocarbon group is bonded to the rest of the molecule by a single bond. The alkyl group may contain 1 to 20 carbon atoms, i.e., "C1-C" 20It can be an "alkyl group", for example, a C1-C4 alkyl group, a C1-C3 alkyl group, a C1-C2 alkyl group, a C3 alkyl group, a C4 alkyl group, a C3-C6 alkyl group. Non-limiting examples of the alkyl group include methyl group, ethyl group, propyl group, butyl group, pentyl group, hexyl group, isopropyl group, isobutyl group, sec-butyl group, tert-butyl group, isopentyl group, 2-methylbutyl group, 1-methylbutyl group, 1-ethylpropyl group, 1,2-dimethylpropyl group, neopentyl group, 1,1-dimethylpropyl group, 4-methylpentyl group, 3-methylpentyl group, 2-methylpentyl group, 1-methylpentyl group, 2-ethylbutyl group, 1-ethylbutyl group, 3,3-dimethylbutyl group, 2,2-dimethylbutyl group, 1,1-dimethylbutyl group, 2,3-dimethylbutyl group, 1,3-dimethylbutyl group, or 1,2-dimethylbutyl group, or their isomers, but not limited thereto. The divalent radical means a group obtained by removing one hydrogen atom from the carbon atom having a free valence electron of the corresponding monovalent radical. The divalent radical has two bonding sites bonded to the rest of the molecule. For example, the "alkylene group" or "alkylidene group" refers to a linear or branched saturated divalent hydrocarbon group. Examples of the "alkylene group" include methylene (-CH2-), ethylene (-C2H4-), propylene (-C3H6-), butylene (-C4H8-), pentylene (-C5H 10 -), hexylene (-C6H 12 -), 1-methylethylene (-CH(CH3)CH2-), 2-methylethylene (-CH2CH(CH3)-), methylpropylene group, ethylpropylene group, etc., but not limited thereto.

[0105] The term "cycloalkyl" refers to a cyclic saturated aliphatic group composed of carbon atoms and hydrogen atoms, and is bonded to the rest of the molecule by a single bond. Cycloalkyl can have 3 to 10 carbon atoms, that is, "C3-C 10It may be "cycloalkyl", for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl or cyclodecyl. "Cycloalkylene" refers to divalent cycloalkyl.

[0106] The term "heterocyclyl" refers to one or more carbon atoms in the above cycloalkyl being replaced by heteroatoms selected from nitrogen, oxygen and sulfur, for example, aziridine, oxirane or thiirane, azetidine, oxetane or thietane, pyrrolidinyl, pyrazolidinyl, imidazolidinyl, tetrahydrofuryl, tetrahydrothienyl, piperidinyl, piperazinyl, tetrahydropyranyl, or tetrahydrothiopyranyl. "Heterocyclylene" refers to divalent cycloalkyl.

[0107] When "substituted" is referred to in this specification, unless otherwise specified, the relevant substituents are selected from alkyl, halogen, amino, monoalkylamino, dialkylamino, nitro, cyano, formyl, alkylcarbonyl, carboxy, alkyloxycarbonyl, alkylcarbonyloxy, aminocarbonyl, monoalkylaminocarbonyl, dialkylaminocarbonyl, formylamino, alkylcarbonylamino, formyl(monoalkyl)amino or alkylcarbonyl(monoalkyl)amino.

[0108] As used in this specification, when a group is combined with another group, as long as a chemically stable structure is formed, the groups may be bonded linearly or branched. The structure formed by such a combination can be bonded to other parts of the molecule via any suitable atom in the structure, preferably via the specified chemical bond. For example, -CR 1 R 2 -, C 1-10 alkylene, C 4-10 cycloalkylene, C 4-10 When two or more divalent groups selected from heterocyclylene and -(CO)- are bonded to form a combination, the two or more divalent groups are, for example, -CR 1 R2 -C 1-10 alkylene-(CO)-, -CR 1 R 2 -C 4-10 cycloalkylene-(CO)-, -CR 1 R 2 -C 4-10 cycloalkylene-C 1-10 alkylene-(CO)-, -CR 1 R 2 -CR 1 R 2’ -(CO)-, -CR 1 R 2 -CR 1’ R 2’ -CR 1’’ R 2’’ They may be linearly linked to each other, such as -(CO)- and the like. The resulting divalent structure may be further linked to other parts of the molecule.

[0109] 2. Linker-Payload Compounds In one aspect, the present invention provides a linker-payload compound having the formula (I), wherein

Chemical formula

[0110] In some embodiments, -L-(P) t is

Chemical formula

Chem.

[0111] In one embodiment, B, L 1 and L 2At least one of them is not "non-existent".

[0112] In one embodiment, L 2 is -NH-(CH2) a -(CH2)2(CO)-, where a is an integer of 0, 1, 2, 3, 4 or 5, and

Chemical formula

[0113] In one embodiment, L 2 is the amino acid residue sequence -*(AA) n **-, where n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, AA is an amino acid residue independently for each occurrence, * represents the N-terminus of the corresponding amino acid, ** represents the C-terminus of the corresponding amino acid, and optionally -(C2H4-O) is between the amino and α-carbon of one amino acid m -(CH2) p - exists, m is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, p is 0, 1, 2 or 3, and the * end and the carbonyl in the disaccharide structure form an amide bond. In one embodiment, AA is independently for each occurrence any one of Phe, Lys, Gly, Ala, Leu, Asn, Val, Ile, Pro, Trp, Ser, Tyr, Cys, Met, Asp, Gln, Glu, Thr, Arg, His or any combination thereof. In one embodiment, n is an integer from 2 to 100, preferably an integer from 2 to 50, preferably an integer from 2 to 30, preferably an integer from 2 to 20, preferably an integer from 2 to 10, preferably 2, 3, 4, 5, 6, 7, 8, 9.

[0114] In one embodiment, L 1contains a cleavable sequence of an amino acid sequence cleavable by an enzyme, and the amino acid sequence cleavable by the enzyme contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids. In one embodiment, the amino acid sequence cleavable by the enzyme is selected from -Gly-Gly-Phe-Gly-, -Phe-Lys-, -Val-Cit-, -Val-Lys-, -Gly-Phe-Leu-Gly-, -Ala-Leu-Ala-Leu-, -Ala-Ala-Ala- and combinations thereof. Preferably, the amino acid sequence cleavable by the enzyme is -Gly-Gly-Phe-Gly-. In one embodiment, L 1 is any one of Val, Cit, Phe, Lys, Gly, Ala, Leu, Asn or any combination thereof, preferably -Gly-Gly-Phe-Gly-, -Phe-Lys-, -Val-Cit-, -Val-Lys-, -Gly-Phe-Leu-Gly-, -Ala-Leu-Ala-Leu-, -Ala-Ala-Ala- and combinations thereof. In one embodiment, L 1 represents -Val-Cit-.

[0115] In one embodiment, Sp1 is selected from PABC (p-aminobenzyloxycarbonyl), acetal, heteroacetal and combinations thereof, preferably Sp1 is acetal, heteroacetal or PABC, more preferably the heteroacetal is selected from N,O-heteroacetals, and even more preferably Sp1 is -O-CH2-U- or -NH-CH2-U-, where -O- or -NH- is linked to the amino acid sequence cleavable by the enzyme and U is absent or CH2, O, S or NH, preferably O or S.

[0116] In one embodiment, B is absent or is -NH-CH2-U- or -NH-CH2-U-(CH2) g-(CO)-, where g is 1, 2, 3, 4, 5 or 6, and U is absent or is CH2, O, S or NH, preferably O or S. In one embodiment, B is absent. In one embodiment, B is either 1) a self-cleaving spacer Sp1, 2) a single divalent group, or a combination of two or more divalent groups, where the divalent group is selected from -CR 1 R 2 -, C 1-10 alkylene and -(CO)-. In one embodiment, B is -NH-CH2-U- or -NH-CH2-U-(CH2) g -(CO)-, where U is absent or is CH2, O, S or NH, preferably O or S. In one embodiment, B is attached to the payload via an amide bond or an ester bond or an ether bond. In one embodiment, B is selected from (-PABC-), -NH-CH2-U- or -NH-CH2-U-(CH2) g -(CO)-, where g is 1, 2, 3, 4, 5 or 6, and U is absent or is CH2, O, S or NH, preferably O or S.

[0117] In one embodiment, -L 1 -B- represents -Val-Cit-PABC-.

[0118] In one embodiment, -L 2 -L 1 -B- represents -Gly-Gly-Gly-Val-Cit-PABC-.

[0119] In one embodiment, Ld2 and each Ld1 are independently a bond or

Chemical formula

[0120] In one embodiment, each i, j, and k is independently selected from integers from 1 to 20. In one embodiment, each i, j, and k is independently selected from integers from 1 to 12.

[0121] In one embodiment, each i is independently selected from integers from 2 to 8, and in one embodiment, i is about 4.

[0122] In one embodiment, each j is independently selected from integers from 8 to 12, and in one embodiment, j is about 8 or 12.

[0123] In one embodiment, each k is independently selected from integers from 1 to 7, and in one embodiment, k is about 1 or 3 or 5.

[0124] In one embodiment, Ld2 and each Ld1 are independently selected from a bond, or a C alkylene having an amino and a carbonyl at each end, or a PEG fragment of a certain length having an amino and a carbonyl at each end (-(PEG)- as shown), or one or more natural amino acids, where the natural amino acids are each independently unsubstituted or substituted by a PEG fragment of a certain length (-(PEG)-CO- as shown). 1-20 In one embodiment, -(PEG)- is selected from -(O-C2H4)- or -(C2H4-O)-, optionally with a C alkylene added to one end, and -(PEG)- is selected from -(O-C2H4)- or -(C2H4-O)-, optionally with a C alkylene added to one end. In one embodiment, -(PEG)- contains -C2H4-(O-C2H4)- or -(C2H4-O)-C2H4-. i - and is optionally substituted at one end with a C alkylene, and -(PEG)- contains -C2H4-(O-C2H4)- or -(C2H4-O)-C2H4-. j - and is optionally substituted at one end with a C alkylene, and -(PEG)- contains -C2H4-(O-C2H4)- or -(C2H4-O)-C2H4-.

[0125] In one embodiment, -(PEG)- i - is selected from -(O-C2H4)- i - or -(C2H4-O)- i - and optionally has a C alkylene added to one end, and -(PEG)- 1-10 - is selected from -(O-C2H4)- j - or -(C2H4-O)- j - and optionally has a C alkylene added to one end, and -(PEG)- j - is selected from -(O-C2H4)- 1-10 - or -(C2H4-O)- i - and optionally has a C alkylene added to one end, and -(PEG)- i - contains -C2H4-(O-C2H4)- i - or -(C2H4-O)-C2H4-.

[0126] 3. Immobilization of Endoglycosidase The support is in the form of a solid or semi-solid made of any material. Non-limiting examples of the support include resins (e.g., agarose resin, silicone resin, polymethyl methacrylate resin, epoxy resin or cellulose resin), gels (e.g., alginic acid hydrogel), beads / microspheres / particles (e.g., polystyrene beads, magnetic particles), plates, wells, tubes, films, membranes, matrices and glass (e.g., glass slides), but are not limited thereto.

[0127] In some embodiments, the support is a resin. In some embodiments, the support is selected from the group consisting of agarose resin, silicone resin, polymethyl methacrylate resin and cellulose resin. In some embodiments, the support is a highly cross-linked agarose resin.

[0128] Methods for enzyme immobilization include adsorption, covalent or non-covalent bonding, incorporation, encapsulation and cross-linking. It is desirable that the maximum enzyme activity of endoglycosidase is maintained after immobilization and that the free endoglycosidase is present in a minimum amount in the conjugate product after the conjugation reaction. In some embodiments, the surface of the support is modified to include one or more functional groups so that the endoglycosidase fusion protein can be covalently immobilized on the support.

[0129] In some embodiments, the support includes one or more chemically active functional groups, and the chemically active functional groups can form a covalent bond with a reactive group (such as amine, thiol, and carboxylate, etc.) of the endoglycosidase fusion protein or a reactive group in a haloalkyl substrate, or the support includes one or more binding partners of the corresponding binding tag / affinity label, and the binding tag / affinity label is included in the endoglycosidase fusion protein. The corresponding relationship between the chemically active functional group and the reactive group, or the corresponding relationship between the binding tag / affinity label and the binding partner is known in the art.

[0130] In some embodiments, the support comprises a chemically active functional group capable of forming a covalent bond with a reactive group (such as amine, thiol, carboxylate, etc.) on the endoglycosidase fusion protein or a reactive group in the haloalkyl substrate. In some embodiments, the functional group contained in the support is selected from the group consisting of cyanate ester, isothiocyanate, isocyanate, carbodiimide, N-hydroxysuccinimide (NHS) ester, amine, carbonate, epoxide, maleimide, haloacetyl, aziridine, ethyl chloroformate, and aliphatic aldehyde.

[0131] In some embodiments, the support is an epoxy-activated resin, CNBr (cyanogen bromide)-activated resin or NHS-activated resin. In some embodiments, the support is an epoxy-activated resin. In some embodiments, the support is an epoxy-activated agarose resin. In some embodiments, the support is a highly cross-linked epoxy-activated agarose resin. In some embodiments, before reacting with the haloalkyl substrate, the epoxy-activated resin is pretreated to introduce amino groups. In some embodiments, the pretreatment of the epoxy-activated resin is carried out using ammonia. In some embodiments, by the pretreatment of the epoxy-activated resin, amino groups are introduced into the oxirane ring, and hydroxyl groups are obtained by ring-opening of the oxirane ring. Such hydroxyl groups are optionally end-capped in subsequent steps for preparing the support. In some embodiments, by the pretreatment of the epoxy-activated resin, amino groups are introduced into the oxirane ring, and hydroxyl groups are obtained by ring-opening of the oxirane ring, and such hydroxyl groups are esterified with an esterification reagent (such as an acetylation reagent such as Ac2O) in subsequent steps for preparing the support. The epoxy-activated resin pretreated in this way is within the scope of the "epoxy-activated resin" defined above. In some embodiments, the resin is an agarose resin (such as a highly cross-linked agarose resin) or a polymethyl methacrylate resin.

[0132] In some other embodiments, the support comprises one or more binding partners of the corresponding binding tag / affinity label, and the binding tag / affinity label is included in the endoglycosidase fusion protein, such as an additional tag or an affinity label. The corresponding relationship between the reactive groups or between the binding tag / affinity label and the binding partner is known in the art. Examples of the binding tag / affinity label and the corresponding binding partner include His tag and Ni 2+ , biotin / SPB tag / Strep tag / Strep tag II and streptavidin / avidin / neutravidin, GST tag and glutathione, Fc tag and protein A, calmodulin tag and Ca 2+ , MBP and amylose, S tag and ribonuclease S-protein, SNAP tag and benzylguanine (BG) derivative, and CLIP tag and benzylcytosine (BC) derivative, but are not limited thereto.

[0133] In some embodiments, the support is functionalized by including a haloalkyl linker to form a covalent interaction with the Halo tag. The haloalkyl linker can be introduced into the support by covalently bonding one or more functional groups contained in the support and one or more reactive groups in the haloalkyl substrate, and the resulting support is also called a haloalkyl linker-modified support. The haloalkyl linker-modified support is within the scope of the "support" defined above. Examples of the haloalkyl substrate include, but are not limited to, those described in, for example, US20060024808A1 and WO2006093529. For the haloalkyl substrate and the method for preparing such a support, reference may be made to the descriptions in, for example, US Pat. Nos. 7,429,472, 7,888,086 and 8,202,700, and Japanese Patent No. 4748685, the relevant contents of which are incorporated herein by reference.

[0134] The haloalkyl substrate may include a haloalkyl moiety containing a primary halo or a secondary halo. In some embodiments, the haloalkyl substrate contains a primary halo. The halo in the haloalkyl moiety is selected from F, Cl, Br, and I. In some embodiments, the halo in the haloalkyl moiety is selected from Cl and Br. In some embodiments, the haloalkyl substrate has the structure of formula (V) below, [Chemical formula] wherein, F1 and F2 are each independently a moiety containing a reactive group and can form a covalent bond with a chemically active functional group contained in the support, H1 and H2 are each independently selected from haloC 2-30 alkyl, Lh is a chemical bond or C 3-200 alkylene, wherein one or more (-CH2-) structures in the alkylene are optionally substituted by -O-, -NH-, -(CO)-, -NH(CO)-, and -(CO)NH-, Lh is optionally substituted with one, two, or three substituents, and the substituents are selected from -O-C 1-10 alkyl, -NH-C 1-10 alkyl, -(CO)-C 1-10 alkyl, -NH(CO)-C 1-10 alkyl, and -(CO)NH-C 1-10 alkyl, Provided that o and e are different, o is 0 or 1, and e is 0 or 1, r is an integer from 1 to 100, s is an integer from 1 to 100.

[0135] In some embodiments, r is an integer from 1 to 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, s is an integer from 1 to 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, the reactive group in F1 or F2 is selected from amino, amine, thiol, and active ester. In some embodiments, the active ester contains one or more carboxylic acid radicals (e.g., in a monoester of carbonic acid of a suitable alcohol or phenol such as an electron-deficient phenol like 4-nitrophenol, or, e.g., in an NHS ester or a sulfo-NHS ester), or one or more sulfonic acid radicals (e.g., in a methanesulfonic acid active ester such as MsO-). In a specific embodiment, F1 or F2 is

Chemical formula

[0136] In some embodiments, H1 and H2 are independently selected from halo C 2-20 alkyl. In some embodiments, H1 and H2 are independently selected from halo C 2-10 alkyl, particularly halo C6 alkyl. In some specific embodiments, the alkyl in H1 or H2 is a straight-chain alkyl. In some embodiments, H1 or H2 is (CH2) 2-30 -X. In some embodiments, H1 or H2 is (CH2) 2-20 -X. In some embodiments, H1 or H2 is (CH2) 2-10 -X, particularly (CH2)6-X, where X is a halogen selected from F, Cl, Br, and I.

[0137] In some embodiments, the support is HaloLink (trademark) resin (Promega).

[0138] In some embodiments, the support is a resin that may contain a haloalkyl linker, and the haloalkyl linker is -(CH2)2-30 -including the structure of -X, where X is a halogen selected from F, Cl, Br, and I. In a specific embodiment, the support is a haloallyl linker-modified resin. In some embodiments, the support is an agarose resin or a polymethyl methacrylate resin. In some embodiments, the support is a highly cross-linked agarose resin.

[0139] In some embodiments, o is 1, e is 0, r is 1, s is 1, F1 is

Chemical formula

Chemical formula

Chemical formula

[0140] In a specific embodiment, u is 3, v is 2, w is 5, and the chloroalkyl substrate has the structure of the following formula (III-1).

Chemical formula

[0141] In some embodiments, the support is a chloroalkyl linker-modified support, having the structure of formula (IV),

Chemical formula

Chemical formula

[0142] In some embodiments, the chloroalkyl linker-modified support represented by formula (IV) is

Chemical formula

[0143] In some embodiments, the chloroalkyl linker-modified support represented by formula (IV) is prepared from a pretreated epoxy-activated resin, and the epoxy-activated resin is prepared by introducing an amino group onto the oxirane ring of the epoxy-activated resin. Hydroxyl is obtained by ring-opening of the oxirane ring in the pretreatment process, and the hydroxyl is optionally esterified with Ac2O in a subsequent procedure for preparing the support. The support represented by formula (IV) has the structure of formula (IV-1),

Chemical formula

Chemical formula

Chemical formula

Chemical Structure

[0144] In some embodiments, the immobilized endoglycosidase fusion protein has the following structure,

Chemical Structure

Chemical Structure

[0145] 4. Conjugation equipment The conjugation equipment disclosed in patent application WO2022170676A or CN114480115A can be used for site-specific conjugation of the N-glycosylation site of the antibody Fc region and the payload, specifically used in the method for preparing the conjugate or antibody-drug conjugate of the present invention. The conjugation equipment includes a flow reactor and a fluid transport unit. The flow reactor is filled with immobilized endoglycosidase. The fluid transport unit is in fluid communication with the inlet of the flow reactor and pumps a donor containing oxazoline oligosaccharide and an antibody (or a protein containing the Fc region) containing a GlcNAc motif into the flow reactor.

[0146] In some embodiments, the conjugation equipment has an inlet and an outlet, and is filled with a medium (such as a matrix of chromatography beads, fibers or films, etc.), and a flow reactor in which endoglycosidase is immobilized on the medium, and is in fluid communication with the inlet of the flow reactor, and is configured to supply at least one reaction fluid to the flow reactor according to different stages of the conjugation process. The at least one reaction fluid includes a fluid transport unit containing a donor containing oxazoline oligosaccharide and an antibody (or a protein containing the Fc region) containing a GlcNAc motif, and is in fluid communication with the outlet of the flow reactor, and is configured to control the recovery of the fluid flowing out from the outlet of the flow reactor according to different stages of the conjugation process. A fluid recovery unit. While at least one reaction fluid continuously flows through the flow reactor, the donor containing oxazoline oligosaccharide and the antibody (or the protein containing the Fc region) containing the GlcNAc motif undergo a conjugation reaction under the catalysis of endoglycosidase to generate an antibody conjugate or a drug conjugate.

[0147] In some embodiments, the endoglycosidase is oriented and immobilized on the medium and filled in the flow reactor, so that when the reaction fluid flows through the flow reactor, the two reaction components of the conjugate to be generated contained in the reaction fluid are stably and continuously conjugated.

[0148] Compared with chemical conjugation, the conjugation equipment of the present invention significantly reduces the process steps, greatly reduces the complexity of the process, and is very suitable for saving high manufacturing costs. In addition, by using a flow reactor, linear scale-up and continuous flow production of the conjugation process can be realized, meeting the industrial demand for higher yields, shortening the unit conjugation time, and reducing the occupied space at the manufacturing site. By producing bioconjugates with the conjugation equipment, site-specific conjugation between the payload-linker and the antibody (or protein containing the Fc region) can be realized, improving the uniformity, thus expanding the therapeutic window. Furthermore, the conjugation process can be integrated into the production process flow of biomolecules such as monoclonal antibodies. For example, conjugation can be performed at either the production stage of the monoclonal antibody intermediate or the monoclonal antibody bulk solution. Therefore, the process is highly flexible and consistent.

[0149] In some embodiments, at least one reaction fluid includes a first reaction fluid and a second reaction fluid, the first reaction fluid includes a donor containing oxazoline oligosaccharide or an antibody (or protein containing the Fc region) containing a GlcNAc motif, and the second reaction fluid includes an antibody (or protein containing the Fc region) containing a GlcNAc motif or a donor containing oxazoline oligosaccharide.

[0150] In some embodiments, the conjugation process includes equilibration before reaction, conjugation reaction, recovery after reaction, and rinsing after recovery in this order. Further, the fluid transport unit is configured to continuously supply a buffer solution to the flow reactor during equilibration before reaction, recovery after reaction, and rinsing after recovery, and to simultaneously and continuously supply a donor containing oxazoline oligosaccharide and an antibody containing a GlcNAc motif (or a protein containing an Fc region) to the flow reactor during the conjugation reaction.

[0151] In some embodiments, the buffer solution, the first reaction fluid, and the second reaction fluid are stored in a first container, a second container, and a third container, respectively. The fluid transport unit includes a first transport pump and a second transport pump. The first container and the second container are connected to the first transport pump via a first container outlet pipe and a second container outlet pipe, respectively. The third container is connected to the second transport pump via a third container outlet pipe. The first transport pump and the second transport pump are connected to an inlet main pipe via a first inlet branch pipe and a second inlet branch pipe, respectively. The inlet main pipe is connected to the inlet of the flow reactor. Further, during equilibration before reaction, recovery after reaction, and rinsing after recovery, the first transport pump pumps the buffer solution in the first container into the inlet main pipe. During the conjugation reaction, the first transport pump pumps the first reaction fluid in the second container into the inlet main pipe, and the second transport pump pumps the second reaction fluid in the third container into the inlet main pipe.

[0152] In some embodiments, the fluid transport unit further includes a first valve, a second valve, a third valve, and a fourth valve. The first valve, the second valve, and the third valve are provided on the first container outlet pipe, the second container outlet pipe, and the third container outlet pipe, respectively, for controlling the flow of fluid in the first container outlet pipe, the second container outlet pipe, and the third container outlet pipe, respectively. The fourth valve is provided on the first inlet branch pipe for controlling the flow of fluid in the first inlet branch pipe.

[0153] In some embodiments, during pre - reaction equilibration, post - reaction recovery, and post - recovery rinse, the first valve and the fourth valve are open, and the second valve and the third valve are closed. During the conjugation reaction, the first valve is closed, and the second valve, the third valve, and the fourth valve are open.

[0154] In some embodiments, the first container outlet pipe, the second container outlet pipe, the third container outlet pipe, the first inlet branch pipe, the second inlet branch pipe, and the inlet main pipe are disposable or non - disposable and are each manufactured from one of stainless steel, titanium, and silicone. The first container, the second container, and the third container are each selected from a disposable liquid storage bag, a disposable liquid storage bottle, a stainless steel container, and a disposable or non - disposable glass or plastic container.

[0155] In some embodiments, the fluid recovery unit is further configured to collect the fluid flowing out from the outlet of the flow reactor into the fourth container during pre - reaction equilibration and post - recovery rinse, and to collect the fluid flowing out from the outlet of the flow reactor into the fifth container during the conjugation reaction and post - reaction recovery.

[0156] In some embodiments, the fourth container and the fifth container are each connected to an outlet main pipe connected to the outlet of the flow reactor via a fourth container inlet pipe and a fifth container inlet pipe, respectively. The fluid recovery unit includes a fifth valve and a sixth valve provided on the fourth container inlet pipe and the fifth container inlet pipe, respectively, for controlling the flow of fluid in the fourth container inlet pipe and the fifth container inlet pipe.

[0157] In some embodiments, during pre - reaction equilibration and post - recovery rinse, the fifth valve is open and the sixth valve is closed. During the conjugation reaction and post - reaction recovery, the fifth valve is closed and the sixth valve is open.

[0158] In some embodiments, the fourth container inlet pipe and the fifth container inlet pipe are disposable or non-disposable and are each manufactured from one of stainless steel, titanium, and silicone. The fourth container and the fifth container are each one selected from a disposable liquid storage bag, a disposable liquid storage bottle, a stainless steel container, and a disposable or non-disposable glass or plastic container.

[0159] In some embodiments, the conjugation facility further includes a temperature control unit configured to control the temperatures of the fluid flowing into the inlet of the flow reactor and the fluid flowing out of the outlet of the flow reactor during the conjugation process.

[0160] In some embodiments, the temperature control unit includes a heating module provided at the inlet of the flow reactor for heating the fluid flowing into the inlet, and a cooling module provided at the outlet of the flow reactor for cooling the fluid flowing out of the outlet.

[0161] In some embodiments, the conjugation facility is in fluid communication with the outlet of the flow reactor, samples a sample fluid from the fluid flowing out of the outlet of the flow reactor according to a predetermined sampling time, and further includes a sampling detection unit that indicates whether the conjugate meets a predetermined standard based on the detection result obtained by detecting the conjugate in the sample fluid.

[0162] In some embodiments, the sampling detection unit includes a sampling pump, a first switching valve, an elution pump, at least one analytical column, and a detector. The sampling pump is connected to the outlet of the flow reactor through a sampling tube, a sample loop is provided on the first switching valve, and the first switching valve switches between a first state and a second state according to a predetermined sampling time. When the first switching valve is in the first state, the sampling pump is in fluid communication with the sample loop, and samples fluid from the fluid flowing out of the outlet of the flow reactor through the sampling tube and pumps it into the sample loop. When the first switching valve is in the second state, the elution pump, the sample loop, at least one analytical column, and the detector are in fluid communication through a detection tube. The elution pump pumps the eluent into the detection tube, and the eluent flows through the sample loop so that the sample fluid in the sample loop flows through one of the at least one analytical column and then flows into the detector in a coordinated manner.

[0163] In some embodiments, there are two analytical columns, and the sampling detection unit further includes a second switching valve and a cleaning pump. The second switching valve switches between two states. When the second switching valve is in any state, the sample loop and the detector are in fluid communication with one of the two analytical columns, and the eluent is coordinated so that the sample fluid in the sample loop flows into the one analytical column. The cleaning pump is in fluid communication with the other of the two analytical columns and pumps a buffer solution into the other analytical column for equilibration.

[0164] In some embodiments, the first switching valve is a six-way valve, the second switching valve is a ten-way valve, and the elution pump is a quaternary pump.

[0165] In some embodiments, the conjugation facility further includes a recycling unit provided between the inlet and the outlet of the flow reactor. When the detection result indicates that the conjugate does not meet a predetermined standard, the fluid recovery unit is configured to stop recovering the fluid flowing out of the outlet of the flow reactor, and the recycling unit is configured to control the fluid flowing out of the outlet of the flow reactor to newly flow into the inlet for performing the conjugation reaction again in the flow reactor.

[0166] In some embodiments, the recycling unit includes a seventh valve provided on the recovery pipe. The recovery pipe is connected between the inlet and the outlet of the flow reactor, and a recovery container is provided on the recovery pipe. When the detection result indicates that the conjugate does not meet a predetermined standard, the seventh valve is open, and the fluid flowing out of the outlet of the flow reactor flows through the recovery pipe and the recovery container and then flows into the inlet.

[0167] In some embodiments, the flow reactor is a conjugation column (i.e., a prepacked column).

[0168] In some embodiments, the conjugation facility further includes at least one selected from the group consisting of a pressure sensing module, a flow rate detection module, a pH measurement module, a conductivity measurement module, and a UV detection module. In some embodiments, the modules may be provided at the inlet and / or the outlet respectively.

[0169] The patent application WO2022170676A or CN114480115A is incorporated herein by reference in its entirety.

[0170] The present invention will be further described below by way of examples, but the present invention is not limited to the scope of the described examples. In the following examples, the experimental methods for which specific conditions are not explicitly stated are selected according to conventional methods and conditions, or the product manuals.

[0171] Apparatus, materials and reagents Unless otherwise specified, all the apparatus and reagents used in the examples are commercially available. The reagents can be used directly without further purification.

[0172] Ni Sepharose 6 FF: A metal chelate affinity chromatography medium, also called immobilized metal ion affinity chromatography. Its principle is to separate proteins based on the fact that specific amino acids on the protein surface, such as histidine, tryptophan, cysteine, etc., can specifically interact with metal ions (Cu 2+ , Zn 2+ , Ni 2+ , Co 2+ , Fe 3+ ). These interactions include binding by coordination bonds, electrostatic adsorption, and binding by covalent bonds. Among them, binding by coordination bonds is the main one, and among which the 6-histidine tag (His-Tag) is the most widely used.

[0173] Q Sepharose FF / Capto S impact: The separation of molecules by ion exchange is based on the difference in surface electrostatic charges. Proteins are composed of a number of different amino acids containing weak acid groups and weak base groups, and the surface electrostatic charge gradually changes with the change of the pH value of the surrounding environment. That is, protein molecules are amphoteric molecules. In the ion exchange chromatography separation process, the binding and elution of specific molecules are realized by controlling the reversible interaction with the ion exchange packing with the opposite charge of the charged molecule, and the separation effect is achieved. Proteins in an environment with the same pH value as the isoelectric point have a surface electrostatic charge of zero and do not interact with the charged packing. When the pH value of the environment is higher than its isoelectric point, the protein binds to the positively charged packing, that is, the anion exchanger. When the pH value of the environment is lower than its isoelectric point, the protein binds to the negatively charged packing, that is, the cation exchanger.

[0174] SDS-PGAE: Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). SDS is an anionic detergent. As a denaturing agent and co-solvent, it can break intramolecular and intermolecular hydrogen bonds, eliminate intramolecular hydrophobic interactions to unfold molecules, and disrupt the secondary and tertiary structures of protein molecules. SDS binds to denatured proteins to make them negatively charged. The amount of SDS bound to denatured proteins is proportional to the molecular weight. In a saturated state, 1 gram of protein can bind 1.4 g of SDS. The negative charge carried far exceeds the original charge amount of the protein, eliminating the charge differences and structural differences between different molecules. Therefore, the migration rate of SDS-protein complexes in acrylamide gel electrophoresis is only related to the size of the protein. Strong reducing agents (such as β-mercaptoethanol, dithiothreitol) can open intramolecular and intermolecular disulfide bonds and separate proteins more effectively.

[0175] Example 1 Preparation of Endoglycosidase Fusion Protein 1.1 Amino acid sequence of Halo-EndoS2-His (Halo is underlined, His Tag is italicized, and GGGGSGGGGS is the linker sequence) MAEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSYVWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIHDWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLIIDQNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVEEYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLIGSEIARWLSTLEISGGGGGSGGGGSMDKHLLVKRTLGCVCAATLMGAALATHHDSLNTVKAEEKTVQTGKTDQQVGAKLVQEIREGKRGPLYAGYFRTWHDRASTGIDGKQQHPENTMAEVPKEVDILFVFHDHTASDSPFWSELKDSYVHKLHQQGTALVQTIGVNELNGRTGLSKDYPDTPEGNKALAAAIVKAFVTDRGVDGLDIDIEHEFTNKRTPEEDARALNVFKEIAQLIGKNGSDKSKLLIMDTTLSVENNPIFKGIAEDLDYLLRQYYGSQGGEAEVDTINSDWNQYQNYIDASQFMIGFSFFEESASKGNLWFDVNEYDPNNPEKGKDIEGTRAKKYAEWQPSTGGLKAGIFSYAIDRDGVAHVPSTYKNRTSTNLQRHEVDNISHTDYTVSRKLKTLMTEDKRYDVIDQKDIPDPALREQIIQQVGQYKGDLERYNKTLVLTGDKIQNLKGLEKLSKLQKLELRQLSNVKEITPELLPESMKKDAELVMVGMTGLEKLNLSGLNRQTLDGIDVNSITHLTSFDISHNSLDLSEKSEDRKLLMTLMEQVSNHQKITVKNTAFENQKPKGYYPQTYDTKEGHYDVDNAEHDILTDFVFGTVTKRNTFIGDEEAFAIYKEGAVDGRQYVSKDYTYEAFRKDYKGYKVHLTASNLGETVTSKVTATTDETYLVDVSDGEKVVHHMKLNIGSGAIMMENLAKGAKVIGTSGDFEQAKKIFDGEKSDRFFTWGQTNWIAFDLGEINLAKEWRLFNAETNTEIKTDSSLNVAKGRLQILKDTTIDLEKMDIKNRKEYLSNDENWTDVAQMDDAKAIFNSKLSNVLSRYWRFCVDGGASSYYPQYTELQILGQRLSNDVANTLKDHHHHHHHHHH(SEQ ID NO: 1) 1.2 Halo-Endo S2-His Clone The nucleic acid sequence encoding Halo-Endo S2-His was synthesized by standard gene synthesis methods and inserted into a pET expression vector. The Halo-Endo S2-His expression plasmid was transformed into Escherichia coli BL21(DE3). The corresponding antibiotic was added to LB (Luria-Bertani) medium, and the cells were cultured at 37 °C until OD600 = 0.5 - 1.0, and the cell bodies were preserved.

[0176] 1.3 Purification of Halo-Endo S2-His Tag (Reactor) The glycerol stock was taken out, inoculated into LB liquid medium containing the corresponding antibiotic, and cultured with shaking at 100 - 300 rpm and 37 °C for 2 - 8 h. When OD600 reached 0.5 - 1.0, it was inoculated into a 10 L reactor, cultured for 5 - 10 h, then IPTG with a final concentration of 0.2 mM was added, and induced expression was carried out overnight at 16 °C. Centrifugation was performed at 2 - 8 °C and a rotational speed of 3000 - 5000 rpm for 10 - 30 min to collect the bacterial precipitate, the equilibrium buffer was added to resuspend the cells, and the cells were disrupted under high pressure. Centrifugation was performed at 2 - 8 °C and 5000 - 10,000 rpm for 10 - 60 min to separate the supernatant and the precipitate. Ni column affinity purification was performed. The Ni chromatography column was connected to the protein purification system, first rinsed thoroughly with purified water and the equilibrium buffer, and the supernatant was filled at a low flow rate. It was washed with the equilibrium buffer and 80 mM imidazole buffer respectively. Elution was carried out with 500 mM imidazole buffer. SDS-PAGE detection and analysis were performed.

[0177] As shown in Figure 1, Halo-Endo S2-His adhered to the Ni magnetic beads, there was no target sample in the flow-through, and Halo-Endo S2-His was eluted under 500 mM imidazole conditions. According to this result, Halo-Endo S2-His can be cultured with scale-up by the reactor, and linear production scale-up can be realized by the Ni chromatography column.

[0178] Example 2 Preparation of Immobilized Halo-Endo S2-His Preparation Procedure 2.1 Preparation of Chloroalkyl-Linker Modified Resin (Chloro Resin) For the preparation method of the chlorine resin, reference may be made to, for example, U.S. Pat. Nos. 7,429,472, 7,888,086, and 8,202,700, the entire contents of which are incorporated herein by reference. The resins for preparing the chlorine resin are shown in Table 1. [Table 1]

[0179] Method (1) Pretreatment For NHS-activated resin (Bestchrom) and CNBr-activated resin (Bestchrom), Filter the resin with isopropanol, wash the filter cake once with DMF (N,N-dimethylformamide), and then drain. Transfer the filter cake to a flask with DMF and stir. Next, add ethylenediamine to the mixture and stir for 10 to 15 hours. Filter and wash the filter cake with DMF. Then drain. For epoxy-activated resin, Filter the resin with isopropanol, wash the filter cake once with H2O, and then drain. Transfer the filter cake to a flask and stir. Next, add 25% - 28% concentrated aqueous ammonia to the mixture, slowly heat the reaction system to 40 - 50 °C, and react with stirring at 40 - 50 °C. Cool the reaction system to 20 - 30 °C and filter the mixture. Wash the filter cake with H2O until the filtrate pH reaches about 7 - 8. Then wash the filter cake with DMF and drain.

[0180] (2) Transfer the filter cake from step (1) to a flask and stir. Next, add DMF and triethylamine containing the chloroalkyl substrate of formula (III-1) to the reaction system in sequence. Stir and react. Subsequently, filter the reaction system, wash the filter cake with DMF, and finally drain. [Chemical Formula]

[0181] (3) The filter cake from step (2) was transferred to a flask. The flask was stirred while open. Next, Ac2O and triethylamine were sequentially added to the mixture. The mixture was stirred for reaction. Subsequently, the mixture was filtered and the filter cake was washed with DMF. Then the filter cake was washed with H2O and drained. Finally, the mixture was transferred to a container with 20% ethanol and stored.

[0182] Result: A chlorine resin having the structure of formula (IV-1) was obtained.

Chemical formula

Chemical formula

[0183] Halo-Endo S2-His and chlorine resin were mixed at room temperature and incubated for 10 min to 24 h. The washing step was repeated three times with a buffer of 20 mM Tris-HCl, 150 mM NaCl, pH 6.0 to 10.0. The activity of the immobilized endoglycosidase fusion protein (where Halo-Endo S2-His and chlorine resin are covalently bonded) was detected. After passing the detection, it was washed with 20 mM Tris-HCl, 150 mM NaCl, and finally stored at 2 - 8 °C.

[0184] 2.2 Immobilized enzyme loading 1) Weighed 250 μl of various chlorine resins to be measured, added an excess of glycosidase, placed it on a rotary mixer, and immobilized it at room temperature for 2 h. 2) During the immobilization of Halo-Endo S2-His, the supernatant of the immobilized enzyme was taken at 15 min, 30 min, 1 h, and 2 h. Each time, it was centrifuged at 3000 g for 3 min at room temperature, the supernatant was aspirated, and the concentration of the enzyme in the supernatant was detected with a Nanodrop ultraviolet spectrophotometer. 3) Calculate the endoglycosidase concentration at each time point, and subtract the concentration at each time point from the starting concentration of the enzyme to calculate the amount of immobilized enzyme at the timing time, and plot the load change curve of the Halo-Endo S2-His medium immobilization process.

[0185] Detect the change in the protein trend in the supernatant by SDS-PAGE. As shown in Figure 2, the results indicate that as time progresses, Halo-Endo S2-His in the supernatant decreases and is specifically immobilized on the chlorine resin.

[0186] Example 3 Preparation of Linker-Payload 3.1 Preparation of Disaccharide Substrate Preparation of disaccharide matrix compound 1 Compound 1 was prepared in the following steps, and its structure is as follows.

Chemical formula

[0187] (1) Preparation of Compound 1c

Chemical formula

[0188] Under nitrogen protection, at room temperature, 1-(phenylsulfinyl)piperidine (BSP, 1.37 g, 6.56 mmol) and 2,4,6-tri-tert-butylpyrimidine (TTBP, 2.94 g, 11.94 mmol) were added to the solution of the dried compound 1a above, and the mixture was stirred for another 20 minutes. The reaction flask was placed in a dry ice / ethyl acetate bath and cooled to -65 °C. Then, trifluoromethanesulfonic anhydride (1.2 mL, 7.16 mmol) was added to the reaction system. After 2 minutes, a dichloromethane solution of the pre-dried compound 1b was added to the reaction system, and the resulting reaction mixture was stirred at -65 °C until the reaction was detected to be complete by TLC (developing solvent: EtOAc / PE = 1 / 8) (about 3 h). A saturated sodium bicarbonate solution was added to the reaction system to quench the reaction, and the mixture was extracted and separated with dichloromethane (150 mL × 3). The combined organic phases were washed successively with water and saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, wet-packed, and isolated by column chromatography (eluent: EtOAc / PE = 1 / 12 - 1 / 10) to obtain compound 1c (2.72 g, yield 75.4%, colorless viscous oily liquid). 1 H NMR (400 MHz, chloroform-d) δ 7.55 (dd, J = 7.6, 2.1 Hz, 2H), 7.51 - 7.28 (m, 28H), 5.60 (s, 1H), 5.14 (d, J = 10.4 Hz, 1H), 5.00 (d, J = 12.0 Hz, 1H), 4.94 (d, J = 11.8 Hz, 1H), 4.90 - 4.80 (m, 2H), 4.76 (d, J = 12.0 Hz, 1H), 4.74 - 4.63 (m, 3H), 4.57 (s, 1H, H 1’ )、4.48 (d, J = 12.1 Hz, 1H)、4.37 (d, J = 8.1 Hz, 1H, H 1 )、4.22 - 4.10 (m, 2H)、4.05 (t, J = 9.3 Hz, 1H)、3.80 (d, J = 3.1 Hz, 1H)、3.72 (dd, J = 11.2, 2.2 Hz, 1H)、3.67 - 3.52 (m, 3H)、3.49 (dd, J = 9.8, 3.1 Hz, 1H)、3.41 (t, J = 9.3 Hz, 1H)、3.35 (dt, J = 9.8, 2.9 Hz, 1H)、3.16 (td, J = 9.7, 4.8 Hz, 1H). C 54 H 56 N3O 10+ [M+H] + The MS (ESI) m / z of [[M+H]] is 906.4 (calculated) and 906.7 (measured).

[0189] (2) Preparation of Compound 1d [Chemical Structure] At room temperature, compound 1c (228 mg, 0.252 mmol), chloroform (1.2 mL), pyridine (1 mL), and AcSH (1.2 mL) were successively added to a 25 mL Schlenk reaction flask and dissolved. The flask was sealed with a sleeve stopper septum, and the reaction system was stirred at 60 °C until the reaction was detected to be basically complete by HPLC (about 18 hours). After concentration under reduced pressure to remove most of the solvent, ethyl acetate (50 mL) was added to the reaction system, and it was washed successively with saturated sodium bicarbonate solution (30 mL), 1 M hydrochloric acid (10 mL × 4), and saturated sodium bicarbonate solution (30 mL). The organic phase was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and then purified by column chromatography (eluent: EtOAc / PE = 1 / 10 - 1 / 1) to obtain compound 1d (200 mg, yield 86%, white solid). 1 H NMR (400 MHz, chloroform-d) δ 7.57 - 7.49 (m, 2H), 7.48 - 7.39 (m, 5H), 7.39 - 7.24 (m, 23H), 5.80 (d, J = 8.1 Hz, 1H), 5.59 (s, 1H), 5.00 (d, J = 6.7 Hz, 1H), 4.97 - 4.87 (m, 3H), 4.87 - 4.75 (m, 2H), 4.69 - 4.59 (m, 4H), 4.57 (s, 1H), 4.45 (d, J = 12.0 Hz, 1H), 4.19 - 4.05 (m, 3H), 3.96 (t, J = 7.3 Hz, 1H), 3.85 - 3.76 (m, 2H), 3.73 - 3.59 (m, 4H), 3.49 (dd, J = 9.8, 3.1 Hz, 1H), 3.18 (td, J = 9.7, 4.8 Hz, 1H), 1.77 (d, J = 1.2 Hz, 3H). C 56 H 60 NO 11 + [M+H] + The MS (ESI) m / z of [[M+H]] is 922.4 (calculated) and 922.4 (measured).

[0190] (3) Preparation of Compound 1e

Chem.

[0191] (4) Preparation of Compound 1f

Chem.

[0192] (5) Preparation of Compound 1

Chemical Structure

[0193] Preparation of disaccharide matrix compound 2 Compound 2 was prepared in the following steps and its structure is as follows.

Chemical formula

[0194] (1) Synthesis of compound 2-1

Chemical formula

[0195] Step B: Synthesis of compound 2-1c ​To a single-necked flask were sequentially added 2-1b crude product (1 equiv), acetonitrile, camphorsulfonic acid (0.1 equiv), and benzaldehyde dimethyl acetal (4 equiv), and the reaction system was stirred overnight at room temperature. The progress of the reaction was monitored by TLC. After the reaction was completed (about 24 h), saturated sodium bicarbonate solution was added to the reaction system to quench the reaction, and the mixture was extracted and separated with ethyl acetate. The combined organic phases were washed with water, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, wet-packed, and purified by silica gel column chromatography (eluent: PE / EtOAc = 5:1) to obtain 2-1c (white solid, 83% yield). 1 H NMR (400 MHz, chloroform-d) δ 7.51 - 7.49 (m, 2H, Ar-H), 7.45 (d, J = 8.0 Hz, 2H, Ar-H), 7.42 - 7.37 (m, 5H, Ar-H), 7.36 - 7.31 (m, 3H, Ar-H), 7.15 (d, J = 8.0 Hz, 2H, Ar-H), 5.56 (s, 1H, PhCH), 4.97 (d, J = 11.6 Hz, 1H, PhCH2), 4.81 (d, J = 11.6 Hz, 1H, PhCH2), 4.58 (d, J = 9.6 Hz, 1H), 4.40 (dd, J = 10.2, 4.8 Hz, 1H), 3.80 (dd, J = 10.0, 10.4 Hz, 1H), 3.70 (dd, J = 9.2, 9.2 Hz, 1H), 3.65 (dd, J = 9.2, 9.2 Hz, 1H), 3.53 - 3.48 (m, 2H), 2.64 (brs, 1H, -OH), 2.36 (s, 3H). C 27 H 29 O5S + [M+H] + The MS (ESI) m / z of [M+H] was calculated to be 465.2 and the measured value was 465.3. 1 The 1H NMR data was consistent with that reported in the literature. Refer to compound 2b1 in Nature 2007, 446, 896.

[0196] Step C: Synthesis of compound 2-1d 2-1c (1 equivalent), dichloromethane, and triethylamine (5 equivalents) were sequentially added to a one-necked flask. The reaction system was cooled to 0 °C, acetic anhydride (2 equivalents) was added, and the mixture was stirred for 10 min. Then, the reaction system was warmed to room temperature and stirred. The progress of the reaction was monitored by TLC. After the reaction was completed (about 6 h), saturated sodium bicarbonate solution was added to the reaction system to quench the reaction. The mixture was extracted and separated with dichloromethane. The combined organic phases were washed with water, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, wet-packed, and purified by column chromatography (eluent: PE / EtOAc = 5:1) to obtain 2-1d (white solid, 89% yield). 1 H NMR (400 MHz, chloroform-d) δ 7.51 - 7.48 (m, 2H, Ar-H), 7.43 - 7.36 (m, 5H, Ar-H), 7.34~7.25 (m, 5H, Ar-H), 7.13 (d, J = 8.0 Hz, 2H, Ar-H), 5.58 (s, 1H, PhCH), 5.00 (dd, J = 8.0, 8.0 Hz, 1H, H-2), 4.87 (d, J = 12.0 Hz, 1H, PhCH2), 4.67 (d, J = 12.0 Hz, 1H, PhCH2), 4.64 (d, J = 10.4 Hz, 1H, H-1), 4.39 (dd, J = 10.8 Hz, J = 5.2 Hz, 1H), 3.81 (t, J = 10.4 Hz, 1H), 3.78 - 3.70 (m, 2H), 3.48 (ddd, J = 9.6, 5.4, 10.0 Hz, 1H), 2.35 (s, 3H, Ar-CH3), 2.05 (s, 3H, OAc). C 29 H 31 O6S + [M+H] + The MS (ESI) m / z of 1 was calculated to be 507.2 and the measured value was 507.4. The H NMR data was consistent with that reported in the literature. Refer to compound 3b1 in Nature 2007, 446, 896.

[0197] Step D: Synthesis of compound 2-1e In a 100 mL two-necked flask under a nitrogen atmosphere, compound 2-1d (1 equivalent), dichloromethane, and borane tetrahydrofuran (10 equivalents, 1 M in THF) were added in sequence. The reaction system was cooled to 0 °C, and dibutylboron trifluoromethanesulfonate (1.4 equivalents, 1.0 M in DCM) was added. The reaction was maintained at 0 °C until TLC indicated completion of the reaction (about 5 hours). Then, at 0 °C, a triethylamine solution was added to the reaction system to quench the dibutylboron trifluoromethanesulfonate in the reaction system. Further, methanol quenching borane tetrahydrofuran was slowly added dropwise. After the reaction system stopped generating a large amount of bubbles, water was added to sufficiently quench it. Then, extraction and liquid separation were performed with ethyl acetate. The combined organic phase was washed with water, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and wet-packed. A crude product 2-1e (white solid) was obtained by high-speed column chromatography (eluent: PE / EtOAc = 4:1). The structure of the product was confirmed to be correct by LCMS. C 29 H 33 O6S + [M+H] + The MS (ESI) m / z of, calculated value 509.2, measured value 509.3. The crude product was directly used in the next reaction without further purification.

[0198] Step E / F: Synthesis of compound 2-1f Step E: In a single-necked flask, 2-1e (1 equivalent), iodobenzene diacetate (3 equivalents), TEMPO (0.5 equivalent), and tert-butanol / dichloromethane / water (volume ratio 4:4:1) were added in sequence. The obtained mixed reaction system was stirred at room temperature, and the progress of the reaction was monitored by TLC (developing solvent: PE / EtOAc = 2 / 1, containing 1 v / v% acetic acid). After the reaction was completed, saturated sodium thiosulfate was added to the reaction system to quench the reaction. Extraction and liquid separation were performed with dichloromethane. The combined organic phase was washed with water, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and the obtained carboxylic acid intermediate was directly used in the next reaction without further purification.

[0199] Step F: Dissolve the carboxylic acid intermediate obtained in the previous step in DMF, add iodomethane (3 eq) and potassium carbonate (5 eq), stir at room temperature, monitor the progress of the reaction by TLC (developing solvent: PE / EtOAc = 5 / 1), after the reaction is complete (about 2 h), add water to the reaction system, extract and separate with ethyl acetate, wash the organic phase with water, wash with saturated brine, dry over anhydrous sodium sulfate, filter, concentrate, and purify by column chromatography (eluent: PE / EtOAc = 7:1) to obtain compound 2-1f (white solid, total yield of 3 steps 59%). 1 H NMR (400 MHz, chloroform-d) δ, 7.42 - 7.26 (m, 12H, Ar-H), 7.16 (d, J = 7.6 Hz, 2H, Ar-H), 5.02 (dd, J = 8.4, 8.0 Hz, 1H), 4.83 (d, J = 11.6 Hz, 1H, PhCH2), 4.79 (d, J = 11.2 Hz, 1H, PhCH2), 4.70 (d, J = 11.6 Hz, 1H), 4.64 (d, J = 11.2 Hz, 1H), 4.62 (d, J = 10.0 Hz, 1H), 3.97 (d, J = 10.0 Hz, 1H), 3.90 (dd, J = 8.4, 8.0 Hz, 1H), 3.79 (s, 3H, OMe), 3.72 (dd, J = 8.8, 8.8 Hz, 1H), 2.38 (s, 3H, Ar-CH3), 2.04 (s, 3H, OAc). C 30 H 33 O7S + [M+H] + The MS (ESI) m / z of [M+H] is calculated to be 537.2 and the measured value is 537.4.

[0200] Step G: Synthesis of compound 2-1g 2-1f (1.0 equivalent) and acetone were successively added to a 50 mL single-necked flask, and the reaction system was cooled to 0 °C. N-Bromobutanimide (1.4 equivalents) was added, and the mixture was stirred at 0 °C for about 1 hour. TLC (developing solvent: PE / EtOAc = 4 / 1) indicated the completion of the reaction. A saturated sodium thiosulfate solution was added to the reaction system to quench the reaction, and acetone was removed by rotary evaporation under reduced pressure. Water was added, and the mixture was separated and extracted with ethyl acetate. The organic phases were combined, washed with water, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography (eluent: PE / EtOAc = 6:1) to obtain compound 2-1g (white solid, yield 90%). C 23 H 27 O8 + [M+H] + The MS (ESI) m / z of

[0201] Step H: Synthesis of Compound 2-1 Under a nitrogen atmosphere, 2-1g (1 equivalent), 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU, 0.1 equivalent), and dichloromethane were successively added to a 50 mL single-necked flask. The reaction system was cooled to 0 °C and stirred uniformly. Then, trichloroacetonitrile (4 equivalents) was added. The ice-water bath was removed, and the temperature was allowed to rise to room temperature naturally and stirred. The reaction was monitored by TLC (developing solvent: PE / EtOAc = 2 / 1). After the reaction was completed (about 2 hours), the solvent was removed by rotary evaporation under reduced pressure, wet-packed, and purified by column chromatography (eluent: PE / EtOAc = 4:1) to obtain compound 2-1 (pale yellow viscous oily liquid, yield 87%). C 23 H 25 O7 + [M-Cl3CC(NH)O - + The MS (ESI) m / z of

[0202] (2) Synthesis of Compound 2-2

Chemical Structure

[0203] At room temperature, Compound 2-2a (1 equivalent), methanol, and sodium methoxide (0.1 equivalent, 5 M in MeOH) were added to a one-necked flask, stirred at room temperature, and the reaction was monitored by TLC (developing solvent: PE / EtOAc = 2 / 1). After the reaction was completed (about 1 hour), dilute hydrochloric acid (1 M) was added to neutralize the solution to pH = 7. After concentrating the reaction solution, toluene was added, and rotary evaporation was carried out under reduced pressure to remove the residual water in the reaction system by the azeotropic effect, and a light brown oily crude product 2-2b was obtained, which was directly used in the next reaction without purification.

[0204] Step B: Synthesis of Compound 2-2c At room temperature, p-toluenesulfonic acid monohydrate (0.2 equivalent) and anhydrous acetonitrile were successively added to the crude product 2-2b (1 equivalent) from the previous step and stirred uniformly. The nitrogen atmosphere of the reaction system was replaced, benzaldehyde dimethyl acetal (5 equivalents) was added, and the resulting reaction solution was stirred at room temperature overnight until TLC (developing solvent: PE / EtOAc = 5 / 1) indicated that the reaction was completed. Then, a saturated sodium carbonate solution was added to the reaction system to quench the reaction, and extraction and liquid separation were carried out with dichloromethane. The combined organic phases were washed with water, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, wet-packed, and purified by silica gel column chromatography (eluent: PE / EtOAc = 5 / 1) to obtain Compound 2-2c (white solid, total yield of 2 steps 85%). 11H NMR (400 MHz, chloroform-d) δ 7.63 - 7.54 (m, 2H), 7.52 - 7.39 (m, 5H), 7.25 - 7.14 (m, 2H), 5.60 (s, 1H), 5.53 (d, J = 5.4 Hz, 1H), 4.52 - 4.39 (m, 1H), 4.28 (dd, J = 10.4, 4.9 Hz, 1H), 4.07 (t, J = 9.5 Hz, 1H), 3.92 (dd, J = 10.0, 5.6 Hz, 1H), 3.79 (t, J = 10.3 Hz, 1H), 3.60 (t, J = 9.3 Hz, 1H), 3.08 (brs, 1H), 2.40 (s, 3H). C 20 H 22 N3O4S + [M + H] + The MS (ESI) m / z of [M + H], calculated value 400.1, measured value 399.9. 1 The 1H NMR data is consistent with that reported in the literature. Refer to compound 47 in Angew. Chem. Int. Ed. 2021, 60, 12413.

[0205] Step C: Synthesis of compound 2 - 2d Under a nitrogen atmosphere, compound 2 - 2c (1 equiv), anhydrous tetrahydrofuran (reaction concentration 0.2 M) were sequentially added to a dry two-necked flask, and the mixture was placed in an ice bath and cooled to 0 °C. Sodium hydride (1.2 equiv, 60% content, dispersed in mineral oil) was added. After the addition was complete, the ice bath was removed and the temperature was raised to room temperature and stirred. After 1 hour, tetrabutylammonium iodide (0.1 equiv) and benzyl bromide (1.5 equiv) were added. The reaction system obtained was stirred at room temperature until the reaction was shown to be complete by TLC (developing solvent: PE / EtOAc = 8 / 1) (about 6 hours). After the reaction was completed, water was added dropwise to quench it, and the mixture was extracted and separated with ethyl acetate. The combined organic phases were washed successively with water and saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by silica gel column chromatography (eluent: PE / EtOAc = 5 / 1) to obtain compound 2 - 2d (white solid, yield 97%). 11H NMR (400 MHz, chloroform-d) δ 7.63 - 7.55 (m, 2H), 7.53 - 7.33 (m, 10H), 7.20 (d, J = 8.4 Hz, 2H), 5.67 (s, 1H), 5.56 (d, J = 4.6 Hz, 1H), 5.05 (d, J = 10.9 Hz, 1H), 4.90 (d, J = 10.9 Hz, 1H), 4.59 - 4.47 (m, 1H), 4.31 (dd, J = 10.4, 4.9 Hz, 1H), 4.10 - 3.97 (m, 2H), 3.90 - 3.77 (m, 2H), 2.41 (s, 3H). C 27 H 28 N3O4S + [M + H] + The MS (ESI) m / z of [[M + H]] is calculated to be 490.2 and the measured value is 490.5. 1 The 1H NMR data is consistent with that reported in the literature. Refer to compound 20 in Bioorg. Med. Chem. 2011, 19, 30.

[0206] Step D: Synthesis of Compound 2 - 2e Compound 2 - 2d (1 equiv), tetrahydrofuran / methanol (v / v = 1:1, reaction concentration 0.5 M), and p - toluenesulfonic acid monohydrate (0.2 equiv) were added to a single - necked flask and stirred at room temperature overnight. The progress of the reaction was monitored by TLC (developing solvent: PE / EtOAc = 8 / 1). After the reaction was completed (about 12 h), saturated sodium bicarbonate solution was added to the reaction system to quench the reaction. The mixture was extracted and separated with ethyl acetate. The combined organic phases were washed with water, then with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, wet - packed, and purified by silica gel column chromatography (eluent: EtOAc / PE = 1 / 1) to obtain compound 2 - 2e. 1 1H NMR (400 MHz, chloroform-d) δ 7.41 - 7.34 (m, 7H), 7.14 (d, J = 8.0 Hz, 2H), 5.49 (d, J = 5.2 Hz, 1H), 5.01 (d, J = 11.2 Hz, 1H), 4.77 (d, J = 11.2 Hz, 1H), 4.24 - 4.20 (m, 1H), 3.89 - 3.85 (m, 1H), 3.79 - 3.77 (m, 2H), 3.69 - 3.65 (m, 2H), 2.42 (brs, 1H), 2.34 (s, 3H). C20 H 27 N4O4S + [M+NH4] + The MS (ESI) m / z of [[M+NH4]] is calculated as 419.2 and the measured value is 419.2.

[0207] Step E: Synthesis of Compound 2-2 To a single-neck flask were sequentially added Compound 2-2e (1 equivalent), dichloromethane (reaction concentration 0.5 M), imidazole (2 equivalents), and tert-butylchlorodiphenylsilane (1.5 equivalents). The mixture was stirred at room temperature, and the progress of the reaction was monitored by TLC (developing solvent: EtOAc / PE = 1 / 12). After the reaction was completed (about 6 hours), a saturated ammonium chloride solution was added to the reaction system to quench the reaction. Then, extraction and liquid separation were performed with dichloromethane. The combined organic phases were washed with water, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, wet-filled, and purified by silica gel column chromatography (eluent: PE / EtOAc / DCM = 15 / 1 / 1) to obtain Compound 2-2 (a yellow viscous oily liquid, yield 66%). 1 H NMR (400 MHz, chloroform-d) δ 7.73 - 7.68 (m, 4H), 7.48 - 7.35 (m, 13H), 7.06 (d, J = 7.6 Hz, 1H), 5.49 (d, J = 5.2 Hz, 1H), 4.94 (ABq, J = 10.8 Hz, 2H), 4.32 - 4.27 (m, 1H), 3.96 - 3.90 (m, 2H), 3.87 (dd, J = 4.8, 5.2 Hz, 1H), 3.82 (dt, J = 2.8, 8.8 Hz, 1H), 3.74 - 3.69 (m, 1H), 2.70 (d, J = 2.8 Hz, OH), 2.33 (s, 3H), 1.09 (s, 9H). C 36 H 42 N3O4SSi + [M+H] + The MS (ESI) m / z of [[M+H]] is calculated as 640.3 and the measured value is 640.2.

[0208] (3) Synthesis of Compound 2

Chemical Structure

[0209] Step B: Synthesis of compound 2b Add compound 2a (1 equivalent), mercaptoacetic acid (CAS: 68-11-1) / pyridine / trichloromethane (v / v / v = 1:1:1) to the reaction flask, heat the reaction system to 60 °C and stir, monitor the progress of the reaction by TLC. After the reaction is completed (about 12 h), concentrate under reduced pressure to remove most of the solvent, add an appropriate amount of ethyl acetate, wash with saturated sodium bicarbonate solution, extract and separate the layers. Then wash the organic phase with 1 M hydrochloric acid solution, wash again with saturated sodium bicarbonate solution after separation, dry the obtained organic phase over anhydrous sodium sulfate, filter, concentrate, and purify by column chromatography to obtain compound 2b (white solid, 71% yield). 1 H NMR (400 MHz, chloroform-d) δ 7.70 (d, J = 6.8 Hz, 2H), 7.66 (d, J = 7.2 Hz, 2H), 7.44 - 7.28 (m, 20H), 7.26 - 7.21 (m, 3H), 7.00 (d, J = 7.9 Hz, 2H), 5.68 (d, J = 4.8 Hz, 1H), 5.36 - 5.30 (m, 1H), 5.09 (dd, J = 9.5, 8.1 Hz, 1H), 4.92 (d, J = 12.4 Hz, 1H), 4.85 (d, J = 11.5 Hz, 1H), 4.75 - 4.66 (m, 3H), 4.63 - 4.57 (m, 2H), 4.29 (ddd, J = 9.3, 7.7, 4.8 Hz, 1H), 4.10 (t, J = 7.8 Hz, 1H), 4.01 (dd, J = 11.5, 3.2 Hz, 1H), 3.94 - 3.89 (m, 2H), 3.86 - 3.84 (m, 1H), 3.80 (dd, J = 11.5, 2.5 Hz, 1H), 3.67 (s, 3H), 3.60 - 3.51 (m, 2H), 2.28 (s, 3H), 1.85 (s, 3H), 1.82 (s, 3H), 1.07 (s, 9H). C 61 H 70 NO 12 SSi + [M+H] + The MS (ESI) m / z of [M+H] is calculated to be 1068.4, and the measured value is 1068.4.

[0210] Step C: Synthesis of compound 2c Compound 2b (1 eq) and tetrahydrofuran were added to a reaction flask, and then TBAF (2 eq, 1 M in THF) was added to the reaction system. The mixture was stirred at room temperature, and the progress of the reaction was monitored by TLC. After the reaction was completed (about 10 h), a saturated ammonium chloride solution was added to the reaction system to quench the reaction. The mixture was further extracted and separated with ethyl acetate. The combined organic phases were washed with water and saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, wet-filled, and preliminarily purified by silica gel column chromatography to obtain compound 2c (white solid, crude yield 56%). C 45 H 52 NO 12 S + [M+H] + The MS (ESI) m / z of was 830.3 calculated and 830.6 found.

[0211] Step D: Synthesis of compound 2d Compound 2c was dissolved in THF / MeOH (v / v = 3:1), and an aqueous sodium hydroxide solution (1 M) was slowly added to adjust the pH of the reaction system to 12. The resulting reaction solution was stirred at room temperature, and the progress of the reaction was monitored by HPLC. After the reaction was sufficient (about 12 h), 1 M hydrochloric acid was added to the reaction system to adjust the pH to 7 - 8, and the mixture was concentrated under reduced pressure to remove most of the organic solvents. The resulting crude product was purified by HPLC to obtain compound 2d (white solid, 82% yield). 11H NMR (400 MHz, chloroform-d) δ 7.43 - 7.27 (m, 17H), 7.07 (d, J = 7.9 Hz, 2H), 5.54 (d, J = 5.0 Hz, 1H), 5.33 (d, J = 8.0 Hz, 1H), 4.93 (d, J = 12.1 Hz, 1H), 4.86 (d, J = 11.3 Hz, 1H), 4.82 - 4.77 (m, 3H), 4.74 (d, J = 6.8 Hz, 1H), 4.69 (d, J = 12.2 Hz, 1H), 4.36 (ddd, J = 10.6, 7.9, 5.0 Hz, 1H), 4.25 - 4.22 (m, 1H), 4.13 - 4.02 (m, 3H), 3.88 (t, J = 8.6 Hz, 1H), 3.81 (dd, J = 12.7, 2.2 Hz, 1H), 3.74 (dd, J = 10.7, 8.5 Hz, 1H), 3.68 - 3.59 (m, 2H), 3.40 (brs, 1H), 2.30 (s, 3H), 1.77 (s, 3H). 13 13C NMR (100 MHz, chloroform-d) δ 170.73, 169.33, 138.20, 138.06, 137.81, 137.55, 132.37, 129.99, 129.44, 128.84, 128.76, 128.58, 128.42, 128.30, 127.90, 103.24, 88.40, 83.43, 78.89, 77.85, 77.35, 77.24, 77.03, 76.92, 76.72, 75.17, 74.93, 74.57, 74.02, 73.93, 72.95, 60.66, 52.95, 23.08, 21.06 (In the aromatic region, three carbon signals overlapped and no peak appeared). C 42 H 46 NO 11 S - [M - H] - The MS (ESI) m / z of [M - H], calculated value 772.3, measured value 772.2.

[0212] Step E: Synthesis of Compound 2e Compound 2d was weighed and dissolved in acetone. The solution was stirred in an ice-water bath for 5 min to cool down. NBS was weighed and added to the reaction solution, and the reaction was carried out in an ice-water bath for about 1 h. A sample was taken and detected by HPLC to confirm the completion of the reaction. After quenching with a saturated sodium thiosulfate solution, the solution was concentrated to remove acetone, prepared by preparative HPLC, and freeze-dried to obtain 2e (white solid, yield 45%). C 35 H 40 NO 12 - [M-H] - The MS (ESI) m / z of [M-H] was calculated to be 666.3 and the measured value was 666.4.

[0213] Step F: Synthesis of Compound 2 At room temperature, compound 2e, tetrahydrofuran, methanol, and palladium-carbon catalyst were sequentially added to a 50 mL single-necked flask, and the reaction system was stirred under a hydrogen atmosphere until it was detected by TLC that all the raw materials had disappeared. The mixture was filtered, concentrated under reduced pressure, and dried with an oil pump to obtain compound 2 (white solid, yield 95%). C 14 H 22 NO 12 - [M-H + - The MS (ESI) m / z of [M-H] was calculated to be 396.1 and the measured value was 396.1.

[0214] Preparation of disaccharide matrix compound 3 Compound 3 was prepared in the following steps, and its structure is as follows.

Chemical formula

[0215] The preparation process of compound 3 may refer to the same method as the synthesis of compound 2, and the specific route is as follows.

Chemical formula

[0216] The final product, compound 3, was verified by mass spectrometry. C 14 H​22 NO 12 - [M-H + - The MS(ESI) m / z of [M-H] is 396.1 (calculated) and 396.0 (measured).

[0217] Preparation of disaccharide matrix compound 4 Compound 4 was prepared in the following steps and its structure is as follows.

Chemical formula

[0218] The preparation process of Compound 4 may refer to the same method as the synthesis of Compound 2, and the specific route is as follows.

Chemical formula

[0219] The final product, Compound 4, was verified by mass spectrometry. C 14 H 22 NO 12 - [M-H + - The MS(ESI) m / z of [M-H] is 396.1 (calculated) and 396.1 (measured).

[0220] Preparation of disaccharide matrix compound 5 Compound 5 was prepared in the following steps and its structure is as follows.

Chemical formula

[0221] The preparation process of Compound 5 may refer to the same method as the synthesis of Compound 2, and the specific route is as follows.

Chemical formula

[0222] The final product, Compound 5, was verified by mass spectrometry. C​​14 H 22 NO 12 - [M-H + - The MS(ESI) m / z of [[M-H]] is 396.1 (calculated) and 396.0 (measured).

[0223] Example 3.2 Preparation of Linker-Payload (abbreviated as LP) Preparation of LP-1 The structure of linker-payload 1 (LP-1) is as follows.

Chemical Structure

[0224] (1) Preparation of Compound LP-1b

Chemical Structure

[0225] (2) Preparation of Compound LP-1

Chemical Structure

[0226] Preparation of LP-2 The structure of linker payload 2 (LP-2) is as follows.

Chemical Structure

[0227] The preparation process is as follows.

Chemical Structure

[0228] 20.1 Step A: Preparation of compound LP-2a At room temperature, to a 10 mL single-necked flask were sequentially added compound 2 (0.5 - 5.0 equivalents), compound LP-1a (1.0 equivalent, GGG-VC-PAB-MMAE, CAS number: 2684216-48-4, commercially available), DMF, DIPEA (1 - 10 equivalents), and HATU (0.5 - 10 equivalents). The resulting reaction solution was stirred at room temperature until it was detected by HPLC that the reaction was completed. The reaction solution was purified by semi-preparative HPLC to obtain compound LP-2a (white solid, yield 84%). C 78 H 126 N 14 O 26 2+ [M+2H] 2+The MS(ESI) m / z, calculated value 837.4, measured value 837.8.

[0229] 20.2 Step B: Preparation of Compound LP-2 At room temperature, to a 10 mL single-neck flask were sequentially added Compound LP-2a (1 equivalent), H2O, Et3N (1 - 100 equivalents), and DMC (2-chloro-1,3-dimethylimidazolidinium chloride, CAS: 37091-73-9, 1 - 100 equivalents). The resulting reaction solution was stirred at 0 °C and monitored by HPLC until the reaction was complete. The reaction solution was purified by semi-preparative HPLC to obtain Compound LP-2 (yield 87%, white solid). C 78 H 124 N 14 O 25 2+ [M + 2H] 2+ The MS(ESI) m / z, calculated value 828.4, measured value 828.7.

[0230] Preparation of LP-3, LP-4, LP-5 The same steps as those for the preparation of LP-2 were adopted to prepare linker payloads LP-3, LP-4, and LP-5 with the following structures.

Chemical Structure

[0231] Preparation of LP-6

Chemical Structure

[0232] (1) Synthesis of LP-6-1

Chemical Structure

[0233] Step A: Synthesis of Intermediate LP-6-1b Compound LP-6-1a (1.0 equiv) and DMF (dissolution concentration 1 g / mL) were added to a reaction flask, stirred and dissolved under a nitrogen atmosphere, cooled to 0 - 5 °C, and then DIEA (3 equiv) was added dropwise. The resulting reaction system was stirred at 5 °C for 10 min, and then benzyl bromide (1.3 equiv) was added dropwise. After the addition was complete, the reaction system was allowed to warm to room temperature naturally and stirred for 16 h. The reaction solution was slowly poured into ice water, methyl tert-butyl ether was added, stirred, and then allowed to stand for liquid separation. The aqueous phase was extracted 4 times with methyl tert-butyl ether. The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated to obtain a yellow oily crude product, wet-filled, and purified by silica gel column chromatography (eluent: PE / EA = 6:1) to obtain the product LP-6-1b (pale yellow oil, quantitative yield).

[0234] Step B: Synthesis of Intermediate LP-6-1d Under nitrogen protection, intermediate LP-6-1b (2.0 equiv), compound LP-6-1c (1.0 equiv) and THF (dissolution concentration 10 g / mL) were added to a reaction flask, stirred and dissolved, TsOH (0.1 equiv) was added to the reaction, and the reaction system was reacted at room temperature for 4 h. The reaction solution was slowly poured into ice water, extracted 3 times with ethyl acetate, and the combined organic phases were washed successively with saturated aqueous sodium bicarbonate solution, water and saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated to obtain a crude product, which was purified by silica gel column chromatography (eluent: PE / EA = 1:1) to obtain the product LP-6-1d (white solid, yield 40%).

[0235] Step C: Synthesis of Intermediate LP-6-1e Under nitrogen protection, compound LP-6-1d and N,N-dimethylacetamide (DMAc, dissolution concentration 10 g / mL) were added to a reaction flask, stirred and dissolved, the reaction system was cooled to 14 - 18 °C, DBU (0.5 equiv) was added dropwise, and the reaction was carried out at this temperature until TLC indicated that the reaction was complete (about 1.5 h) to obtain intermediate LP-6-1e, which was used directly in the next reaction without purification.

[0236] Step D: Synthesis of Intermediate LP-6-1g Cool the reaction solution from the previous step to 0 - 5 °C. Sequentially add pyridinium 4-toluenesulfonate (PPTS, 0.5 equivalent), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCI, 1.0 equivalent), 1-hydroxybenzotriazole (HOBT, 1.0 equivalent), and LP-6-1f (0.85 equivalent). React the reaction system at 0 - 10 °C until LCMS indicates the completion of the reaction (about 4 h). Add the reaction solution to ice water, add 2-methyltetrahydrofuran for extraction once, extract the aqueous phase with 2-methyltetrahydrofuran twice. Combine the organic phases and wash them successively with 0.5 M hydrochloric acid, saturated aqueous NaHCO3 solution, water, and saturated brine. Dry over anhydrous sodium sulfate, filter, concentrate, and purify by silica gel column chromatography (eluent: DCM / MeOH) to obtain the product LP-6-1g (white solid, yield 78%).

[0237] Step E: Synthesis of Intermediate LP-6-1h Under nitrogen protection, add LP-6-1g and DMAc (dissolution concentration 10 g / mL) to a reaction flask, stir to dissolve. Cool the temperature to 14 - 18 °C, dropwise add DBU (0.5 equivalent), stir and react at this temperature for 1.5 h. Monitor the progress of the reaction by TLC. After the reaction is complete, obtain intermediate LP-6-1h and directly use it in the next reaction without purification.

[0238] Step F: Synthesis of Intermediate LP-6-1j Cool the reaction solution of LP-6-1h in the previous step to 0-5 °C, add PPTS (0.5 equivalent), EDCI (1 equivalent), HOBT (1 equivalent) and compound 3i (0.85 equivalent), react at 0-10 °C for 3-4 h, monitor the progress of the reaction by LCMS. After the reaction is completed, add the reaction solution to ice water, add 2-methyltetrahydrofuran for extraction once, extract the aqueous phase with 2-methyltetrahydrofuran twice more, combine the organic phases, wash successively with 0.5 M hydrochloric acid, saturated NaHCO3 aqueous solution, water, saturated brine, dry over anhydrous sodium sulfate, filter, concentrate, dry-mix, and purify by column chromatography (eluent: DCM / MeOH) to obtain LP-6-1j (white solid, yield 50%).

[0239] Step G: Synthesis of Compound LP-6-1 Under nitrogen protection, dissolve the intermediate LP-6-1j in DCM (at a concentration of 15 g / mL), add DBU (0.5 equivalent) dropwise at 20 °C, and maintain the temperature and stir the reaction until HPLC indicates that the reaction is complete. Then add DCM to the reaction system to dilute the reaction solution, directly perform wet packing, and purify by column chromatography (eluent: DCM / MeOH) to obtain compound LP-6-1 (white solid, yield 82%). C 34 H 47 N6O 10 + [M+H] + The MS (ESI) m / z of [M+H] is calculated to be 699.4 and the measured value is 699.6.

[0240] (2) Synthesis of LP-6-2

Chemical Structure

[0241] Step 1: Preparation of NH2-Asp(OtBu)-Rink Amide Resin 400 g of Rink amide resin was placed into a reaction kettle, immersed in 2400 mL of DCM for 0.5 h to fully swell the resin, and then drained. 2400 mL of cap removal reagent was added for washing, and then drained. 2400 mL of cap removal reagent was added, nitrogen was blown in, and the mixture was stirred and reacted at 25 ± 1 °C for 0.5 h, and then drained. It was washed twice with 2400 mL of DMF, 2400 mL of absolute ethanol, 2400 mL of DCM, and 2400 mL of DMF in sequence, and then drained. The Kaiser test showed blue color.

[0242] 88.87 g of Fmoc-Asp(OtBu)-OH and 29.19 g of HOBT were dissolved in 2000 mL of DMF solution and 80 mL of DIC, left in an ice bath at -10 °C for 0.5 h, and then slowly added to the reaction kettle for reaction. Nitrogen was blown in at room temperature, and the mixture was stirred and reacted for 2 h, and then drained. It was washed twice with 2400 mL of DMF, 2400 mL of absolute ethanol, 2400 mL of DCM, and 2400 mL of DMF in sequence, and then drained. The Kaiser test showed a light blue color. 2400 mL of DCM was added, and further 60 mL of capping reagent was added. Nitrogen was blown in, and the mixture was stirred and reacted at 25 ± 1 °C for 1 h, and then drained. It was washed twice with 2400 mL of DMF, 2400 mL of absolute ethanol, 2400 mL of DCM, and 2400 mL of DMF in sequence, and then drained. The Kaiser test showed colorless.

[0243] 2400 mL of cap removal reagent was added for washing, and then drained. 2400 mL of cap removal reagent was added, nitrogen was blown in, and the mixture was stirred and reacted at 25 ± 1 °C for 0.5 h, and then drained. It was washed twice with 2400 mL of DMF, 2400 mL of absolute ethanol, 2400 mL of DCM, and 2400 mL of DMF in sequence, and then drained. The Kaiser test showed blue color.

[0244] Step 2: Preparation of NH2-PEG4-Asp(OtBu)-Rink amide resin 131.64 g of Fmoc-PEG4-OH and 48.64 g of HOBT were dissolved in 2000 mL of DMF solution and 80.0 mL of DIC, and after standing in an ice bath at -10 °C for 0.5 h, it was slowly added to the reaction kettle for reaction. Nitrogen was blown in at room temperature, and the mixture was stirred and reacted for 2 - 4 h, and then drained. It was washed twice with 2400 mL of DMF, 2400 mL of absolute ethanol, 2400 mL of DCM, and 2400 mL of DMF in sequence, and then drained. The Kaiser test showed it was colorless.

[0245] 2400 mL of cap removal reagent was added for washing and then drained. Another 2400 mL of cap removal reagent was added, nitrogen was blown in, and the mixture was stirred and reacted at 25 ± 1 °C for 0.5 h, and then drained. It was washed twice with 2400 mL of DMF, 2400 mL of absolute ethanol, 2400 mL of DCM, and 2400 mL of DMF in sequence, and then drained. The Kaiser test showed it was blue.

[0246] Step 3: Preparation of NH2-Asp(OtBu)-PEG4-Asp(OtBu)-Rink amide resin 222.18 g of Fmoc-Asp(OtBu)-OH and 72.96 g of HOBT were dissolved in 2000 mL of DMF solution and 80 mL of DIC, and after standing in an ice bath at -10 °C for 0.5 h, it was slowly added to the reaction kettle for reaction. Nitrogen was blown in at room temperature, and the mixture was stirred and reacted for 2 - 4 h, and then drained. It was washed twice with 2400 mL of DMF, 2400 mL of absolute ethanol, 2400 mL of DCM, and 2400 mL of DMF in sequence, and then drained. The Kaiser test showed it was colorless.

[0247] 2400 of cap removal reagent was added for washing and then drained. Another 2400 mL of cap removal reagent was added, nitrogen was blown in, and the mixture was stirred and reacted at 25 ± 1 °C for 0.5 h, and then drained. It was washed twice with 2400 mL of DMF, 2400 mL of absolute ethanol, 2400 mL of DCM, and 2400 mL of DMF in sequence, and then drained. The Kaiser test showed it was blue.

[0248] Step 4: Preparation of Dde-Lys(NH2)-Asp(OtBu)-PEG4-Asp(OtBu)-Rink amide resin 191.75 g of Dde-Lys(Fmoc)-OH and 48.64 g of HOBT were dissolved in 2000 mL of DMF solution and 80.0 mL of DIC, left in an ice bath at -10 °C for 0.5 h, then slowly added to the reaction kettle for reaction, nitrogen was blown in at room temperature, stirred and reacted for 2 - 4 h, and drained. It was washed twice with 2400 mL of DMF, 2400 mL of absolute ethanol, 2400 mL of DCM, and 2400 mL of DMF in sequence and drained. It was colorless in the Kaiser test.

[0249] 2400 ± 100 mL of cap removal reagent was added for washing and drained. Another 2400 mL of cap removal reagent was added, nitrogen was blown in, stirred and reacted at 25 ± 1 °C for 0.5 h, and drained. It was washed twice with 2400 mL of DMF, 2400 mL of absolute ethanol, 2400 mL of DCM, and 2400 mL of DMF in sequence and drained. It showed blue in the Kaiser test.

[0250] Step 5: Preparation of Dde-Lys(mPEG12)-Asp(OtBu)-PEG4-Asp(OtBu)-Rink amide resin 170.84 g of m-PEG12-CH2CH2COOH and 48.64 g of HOBT were dissolved in 2000 mL of DMF solution and 80.0 mL of DIC, left in an ice bath at -10 °C for 0.5 h, then slowly added to the reaction kettle for reaction, nitrogen was blown in at room temperature, stirred and reacted for 2 - 4 h, and drained. It was washed twice with 2400 mL of DMF, 2400 mL of absolute ethanol, 2400 mL of DCM, and 2400 mL of DMF in sequence and drained. It was colorless in the Kaiser test.

[0251] Step 6: Preparation of NH2-Lys(PEG12)-Asp(OtBu)-PEG4-Asp(OtBu)-Rink amide resin 2400 mL of dde-free reagent was added, nitrogen was bubbled in, and the mixture was stirred at 25 ± 1 °C for 10 min. After draining, this operation was repeated three times. Then, it was washed twice with 2400 mL of DMF, 2400 mL of absolute ethanol, 2400 mL of DCM, and 2400 mL of DMF in sequence and drained. The Kaiser test showed a blue color.

[0252] Step 7: Preparation of Fmoc-Gly-Gly-Gly-Lys(PEG12)-Asp(OtBu)-PEG4-Asp(OtBu)-Rink amide resin 111.08 g of Fmoc-Gly-Gly-Gly-OH and 48.64 g of HOBT were dissolved in 2000 mL of DMF solution and 80.0 mL of DIC. After standing in an ice bath at -10 °C for 0.5 h, it was slowly added to the reaction kettle for reaction. Nitrogen was bubbled in at room temperature, and the mixture was stirred at 2 - 4 h and drained. It was washed twice with 2400 mL of DMF, 2400 mL of absolute ethanol, 2400 mL of DCM, and 2400 mL of DMF in sequence and drained. The Kaiser test showed a colorless result. The resin peptide was washed three times with 2400 mL of absolute ethanol and drained in preparation for cleavage.

[0253] Step 8: Preparation of LP-6-2 10000 mL of cleavage reagent (TFA:TIS:H2O = 95:2.5:2.5) was added to a 10 L reaction kettle and cooled to -10 ± 2 °C. After drying, the weighed resin was added, and then the temperature was raised. Nitrogen was passed through at 20 ± 5 °C and stirred for 2 h. It was filtered, and the resin was washed once with 100 mL of TFA. The filtrate was combined with the washing solution.

[0254] 40 L of pre-cooled (-10 °C or lower) cold ether was added, stirred for 10 min, then centrifuged and precipitated. After centrifugation and precipitation, the supernatant was discarded. The precipitate was mixed with cold ether and shaken uniformly, then centrifuged and precipitated again (this step was repeated three times, with the usage amount being 10 L, 10 L, and 10 L each time. For each centrifugation, the rotation speed was 3600 revolutions, the centrifugation time was 5 min, and the inner chamber temperature of the centrifuge was -5 °C).

[0255] Recovery of the precipitate gave the crude product LP-6-2, which was purified by preparative HPLC and lyophilized to give LP-6-2. C 74 H 121 N9O 31 2+ [M+2H] 2+ MS (ESI) m / z for [M+2H], calcd 815.9, found 816.3.

[0256] (3) Synthesis of LP-6-3

Chemical formula

[0257] Step A: Synthesis of intermediate LP-6-3a Compound LP-6-1 (2.2 equiv) and compound LP-6-2 (1.0 equiv) were added to a reaction flask, dissolved in DMF, and then DIPEA (5.0 equiv) was added and stirred uniformly. Subsequently, HATU (2.5 equiv) was added to the reaction system, and the reaction was carried out at room temperature and monitored by HPLC until the reaction was complete (about 2 h). The reaction system was directly prepared by preparative HPLC, and the preparation solution was lyophilized to obtain compound LP-6-3a (white solid, yield 52%). C 143 H 212 O 48 N 21 3+ [M+3H] 3+ MS (ESI) m / z for [M+3H], calcd 997.2, found 875.9 (some functional groups are lost after ionization).

[0258] Step B: Synthesis of intermediate LP-6-3b Compound LP-6-3a was dissolved in purified water, a certain amount of palladium hydroxide (Pd(OH)2 on 10 wt% carbon) was added, the reaction system was replaced with hydrogen three times, and stirred at room temperature for 1.5 h. During this period, the progress of the reaction was monitored, and the reaction was stopped immediately after the raw material was exhausted to prevent the increase of the de-Fmoc product. The reaction solution was filtered and prepared by preparative HPLC to obtain compound LP-6-3b (white solid, yield 76%). C 128 H 200 O 48 N 213+ [M + 3H] 3+ The MS(ESI) m / z of [[M+3H]] is 937.1 (calculated) and 875.9 (measured) (some functional groups are lost after ionization).

[0259] Step C: Synthesis of Intermediate LP-6-3d Compound LP-6-3b (1.0 equivalent) and compound LP-6-3c (2.2 equivalents) were dissolved in DMF, DIPEA (5.0 equivalents) was added and stirred uniformly, then HATU (2.5 equivalents) was added, and the reaction was carried out at room temperature. The reaction was monitored by HPLC until completion (about 16 h). The reaction system was directly prepared by preparative HPLC, and after freeze-drying the preparation solution, compound LP-6-3d (yellow solid, yield 66%) was obtained. C 174 H 232 O 55 Cl2F2N 27 3+ [M + 3H] 3+ The MS(ESI) m / z of [[M+3H]] is 1229.2 (calculated) and 1229.3 (measured).

[0260] Step D: Synthesis of Compound LP-6-3 Compound LP-6-3d was dissolved in DMF, diethylamine was added, and the reaction was carried out at room temperature. The reaction was monitored by HPLC until completion (about 0.5 h). After the reaction was completed, the pH was adjusted to neutral, and then it was prepared by preparative HPLC. After freeze-drying, compound LP-6-3 (yellow solid, yield 73%) was obtained. C 159 H 222 O 53 Cl2F2N 27 3+ [M + 3H] 3+ The MS(ESI) m / z of [[M+3H]] is 1155.2 (calculated) and 1155.3 (measured).

[0261] Here, for the synthesis of compound LP-6-3c, please refer to Patent CN202211428194.6

[0262] (4) Synthesis of LP-6

Chemical Structure

[0263] Synthesis of LP-6 Compound LP-6a was dissolved in purified water, and the temperature was lowered by stirring in an ice-water bath. Then Et3N (1 - 100 equivalents) and DMC (2-chloro-1,3-dimethylimidazolidinium chloride, CAS: 37091-73-9, 1 - 100 equivalents) were added. The progress of the reaction was monitored by HPLC. After the reaction was completed, it was purified by preparative HPLC, and the prepared solution was freeze-dried to obtain compound LP-6 (light yellow solid, yield 74%). C 173 H 241 O 63 Cl2F2N 28 3+ [M + 3H] 3+ The MS (ESI) m / z of is calculated to be 1275.5 and the measured value is 1275.9.

[0264] Preparation of LP-7 The structure of LP-7 is as follows.

Chemical formula

[0265] The synthesis route is as follows.

Chemical formula

[0266] Step A: Synthesis of Compound LP-7b Compound LP-7a was synthesized by the method of solid-phase synthesis of polypeptides. For the same method as LP-6-2, please refer to it.

[0267] Compound LP-7a (1.0 equivalent) and compound LP-6-1 (3.6 equivalents) were added to the reaction flask, dissolved in DMF, and then DIPEA (6.0 equivalents) was added and stirred uniformly. After that, HATU (3.6 equivalents) was added to the reaction system, and the reaction was carried out at room temperature. The reaction was monitored by HPLC until completion (about 2 h). The reaction system was directly prepared by preparative HPLC, and the prepared solution was lyophilized to obtain compound LP-7b (white solid, yield 56%). C 191 H 280 O 66 N 29 3+ [M+3H] 3+ The MS (ESI) m / z of , calculated value 1345.3, measured value 1345.8.

[0268] Step B: Synthesis of Intermediate LP-7c Compound LP-7b was dissolved in purified water, a certain amount of palladium hydroxide (Pd(OH)2 on 10 wt% activated carbon) was added, the reaction system was replaced with hydrogen three times, stirred at room temperature for 1.5 h, and the progress of the reaction was monitored during this period. The reaction solution was filtered and directly prepared by preparative HPLC to obtain compound LP-7c (white solid, yield 80%). C 170 H 262 O 66 N 29 3+ [M+3H] 3+ The MS (ESI) m / z of , calculated value 1255.3, measured value 1255.9.

[0269] Step C: Synthesis of Intermediate LP-7d Compound LP-7c (1.0 equiv) and compound LP-6-3 (1 - 10 equiv) were dissolved in DMF, DIPEA (1 - 20 equiv) was added and stirred uniformly, then HATU (1 - 10 equiv) was added, and the reaction was carried out at room temperature. The reaction was monitored by HPLC until completion (about 2 h), and the reaction product was used directly in the next reaction. C 239 H 313 O 75 Cl3F3N 38 3+ [M+3H] 3+ The MS (ESI) m / z of is calculated to be 1692.4 and the measured value is 1692.9.

[0270] Step D: Synthesis of compound LP-7e Compound LP-7d was dissolved in DMF, diethylamine was added, and the reaction was carried out at room temperature. The reaction was monitored by HPLC until completion (about 0.5 h). After the reaction was completed, the pH was adjusted to neutral, and then it was prepared by preparative HPLC. After lyophilization, compound LP-7e (yellow solid, yield 73%) was obtained. C 224 H 303 O 73 Cl3F3N 38 3+ [M+3H] 3+ The MS (ESI) m / z of is calculated to be 1618.3 and the measured value is 1618.5.

[0271] Step E: Synthesis of compound LP-7f Compound 1 (0.5 - 5.0 equiv) and compound LP-7e (1.0 equiv) were dissolved in DMF, DIPEA (1 - 10 equiv) was added and stirred uniformly at 0 °C, HATU (0.5 - 10 equiv) was added, and the reaction system was stirred at 0 °C. The progress of the reaction was monitored by HPLC until completion (about 2 h). The reaction system was directly prepared by preparative HPLC, and after lyophilizing the preparation solution, compound LP-7f (yellow solid, yield 65%) was obtained. C 238 H 325 O 84 Cl3F3N 39 4+ [M+4H] 4+ The MS (ESI) m / z of is calculated to be 1308.8 and the measured value is 1309.0.

[0272] 15.6 Step F: Synthesis of Compound LP-7 Compound LP-7f was dissolved in purified water and stirred in an ice-water bath to lower the temperature. Then, Et3N (1 - 100 equivalents) and DMC (2-chloro-1,3-dimethylimidazolidinium chloride, CAS: 37091-73-9, 1 - 100 equivalents) were added. The progress of the reaction was monitored by HPLC. After the reaction was completed, it was purified by preparative HPLC. The preparation solution was lyophilized to obtain Compound LP-7 (pale yellow solid, yield 50%).

[0273] Example 4 Preparation of Anti-Her2 Antibody Anti-human ErbB2 / Her2 antibody T-LCCT L The production, purification and identification of -HC (mAb-1) and anti-human TROP2 antibody mAb-2 may refer to Example 1 of Patent CN106856656B, which is hereby incorporated by reference in its entirety.

[0274] Example 5 Suspension Conjugation Solid-phase Preparation of ADC-1 Based on the conjugation reaction of Halo-Endo S2-His catalytic antibody and linker-payload, an ADC drug was prepared. In an endonuclease buffer, the antibody and linker-payload were thoroughly mixed at an appropriate molar ratio (1:1 to 1:100), and the Halo-Endo S2-His medium was added and mixed uniformly. The conjugation reaction in the uniformly mixed state was carried out at 4 - 40 °C for 0.5 - 20 hours. After the reaction was completed, centrifugation was performed, and the solid-phase conjugation reaction mixture was taken out. Unreacted drug intermediates were removed by purification, ultrafiltration or dialysis. The purified ADC1 was stored in 1×PBS pH7.4 at 4 °C or -80 °C.

[0275] 1) SDS-PAGE Detection and Analysis After the conjugation reaction, the purity and conjugation efficiency of ADC-1 can be detected by SDS-PAGE method. The SDS-PAGE detection result of ADC1 is as shown in Figure 3. The conjugation reaction occurred site-specifically in the heavy chain of the antibody (the heavy chain conjugated with the cytotoxic agent had a significantly changed molecular weight compared to the heavy chain not conjugated with the cytotoxic agent). No heavy chain not conjugated with the cytotoxic agent was detected in the conjugation product, and the conjugation efficiency was 95% or more.

[0276] 2) HIC-HPLC detection and analysis HIC-HPLC: Proteomix HIC Butyl-NP5 4.6*100mm 5μm Non-Porous column, column temperature 30°C, mobile phase A: 1.5M ammonium sulfate + 20mM phosphate buffer salt, pH 7.0, mobile phase B: 20mM phosphate buffer salt: isopropanol = 7:3 (v / v), flow rate 0.8 mL / min, gradient method: increase the B phase from 10% to 100% within 8 minutes, select 280nm as the detection wavelength, and detect the DAR distribution of the ADC1 drug.

[0277] The detection results are as shown in Figure 4 and Table 2. The antibody not conjugated to compound LP-1 is <1%, the conjugation product is mainly DAR2, and the DAR value of the ADC-1 drug is 1.93.

Table 2

[0278] 3) Mass spectrometry High-precision molecular weight mass spectrometry (ESI-MS) of ADC-1 When ADC-1 was analyzed and detected by high-precision molecular weight mass spectrometry, its apparent molecular weight was 150539.27, and the theoretical molecular weight was 150538.78, which was in line with expectations, and it was confirmed that one cytotoxic molecule was conjugated to each Fc end of the heavy chain.

[0279] Example 6 Column conjugation Antibody and small molecule toxin (LP-1) reaction solutions were prepared respectively, with the antibody reaction solution concentration being 1 - 100 mg / ml and the small molecule reaction solution concentration being 0.1 - 50 mg / ml. The treated immobilized enzyme (Halo-Endo S2-His) medium was packed into a column, and conduction heating was carried out to 10 - 40 °C by air or water bath, and preheated for >30 min in advance. The antibody reaction solution and the small molecule reaction solution were uniformly mixed at a certain ratio, passed through a conjugation column for reaction, and the effluent was the ADC drug, and the residence time in the conjugation column was 5 min to 24 h. Samples of the conjugation reaction solution were subjected to SDS-PAGE, HIC-HPLC and RP-HPLC detections.

[0280] 1) SDS-PAGE detection and analysis After the conjugation reaction is completed, the purity and conjugation efficiency of ADC-1 can be detected by SDS-PAGE method. According to the SDS-PAGE detection results of ADC-1, the conjugation reaction occurs site-specifically in the heavy chain of the antibody, and no heavy chain that is not conjugated to the cytotoxin is detected in the conjugation product, and the conjugation efficiency is 95% or more.

[0281] 2) HIC-HPLC detection and analysis According to the detection results, the DAR of ADC-1 after the conjugation reaction is 1.93, and the conjugation efficiency is 96.5%.

[0282] The efficiencies of column conjugation and suspension conjugation can both reach 95% or more, and the results of column conjugation are used to support the linear production scale-up of ADC drugs.

[0283] The specific operation procedure of column conjugation is as follows. An appropriate tube (e.g., a silicon tube of a peristaltic pump) was attached to an appropriate pump head (e.g., a peristaltic pump head (Chongqing Jieheng, BT-600CA / DG4(10))), and the tube was equilibrated. Then the outlet end of the tube was connected to the inlet end of the conjugation column, and another tube was connected to the lower end of the conjugation column. The connected conjugation column was placed in a temperature control device (e.g., a hybridizer (UVP / HB-1000 Hybridizer)) with the temperature set at 10 - 40 °C, and the outlet end of the lower end tube of the conjugation column was placed outside the temperature control device. The inlet end of the tube was placed in the conjugation reaction solution, the pump speed was activated to carry out the conjugation reaction, the conjugation reaction solution was collected, and after the filling of the conjugation reaction solution was completed, the conjugation column was rinsed with the conjugation equilibrium buffer and collected with the same recovery volume. The effluent was the ADC drug. After the collection was completed, the concentration of the collected solution was detected with an ultraviolet spectrophotometer, and the unreacted drug intermediate was removed by purification, ultrafiltration or dialysis. The purified ADC was stored at 4 °C or -80 °C in 1×PBS pH7.4. The conjugation reaction solution sample was subjected to SDS-PAGE, HIC-HPLC or RP-HPLC detection.

[0284] The solid-phase conjugation column reaction can achieve linear scale-up. By adjusting the corresponding process (such as pump flow rate) according to the size of the conjugation column under the condition of ensuring that the main process parameters (such as retention time) are the same, the conjugation column can be linearly scaled up to a volume of several hundred milliliters (500 mL), several liters (3 L) or more. The glycosidase conjugation column catalyst and offline DAR detection were achieved at the laboratory scale, and linear scale-up of drug conjugates and online DAR value detection were realized using larger-volume conjugation columns at higher flow rates. The conjugation equipment in patent application WO2022170676A can be applied to the glycosylation process of the present invention. This example verified the feasibility of applying conjugation equipment of different scales to the glycosylation conjugation platform.

[0285] Example 7 Separation of Halo-Endo S2-His and ADC When the environmental pH value is higher than its isoelectric point, the protein binds to a positively charged packing, i.e., an anion exchanger. When the environmental pH value is lower than its isoelectric point, the protein binds to a negatively charged packing, i.e., a cation exchanger. The isoelectric point of Halo-Endo S2-His is 5.5, and under the conditions of pH 6.0 to pH 8.0, the surface of the Halo-Endo S2-His protein is negatively charged. The Halo-Endo S2-His protein enters a passing mode with a negatively charged packing, i.e., a cation exchanger, and the Halo-Endo S2-His protein binds to a positively charged packing, i.e., an anion exchanger. The isoelectric point of ADC is generally 8 to 9, and under the conditions of pH 6.0 to pH 8.0, the surface of the ADC protein is positively charged. The ADC binds to a negatively charged packing, i.e., a cation exchanger, and the ADC enters a passing mode with a positively charged packing, i.e., an anion exchanger. There is a significant difference of more than 2 between the isoelectric points of Halo-Endo S2-His and ADC. By selecting pH 6.0 to pH 8.0 / buffer, ADC and Halo-Endo S2-His can be effectively separated by ion exchange chromatography. That is, when Halo-Endo S2-His catalyzes ADC site-specifically, even if a small amount of the Halo-Endo S2-His protein immobilized on the column drops into the ADC product, Halo-Endo S2-His can be effectively removed by subsequent anion and cation chromatography.

[0286] 1) Anion exchange chromatography (AEX) Select Q Sepharose FF packing to fill the column, equilibrate the chromatography column with 20 mM Tris-HCl pH 7.5, and load the sample. After loading the sample, continue to equilibrate to the baseline with 20 mM Tris-HCl pH 7.5, and finally elute the sample with 20 mM Tris-HCl, 1 M NaCl pH 7.5, and perform CIP (cleaning) with 1 M NaOH. Purify ADC and Halo-Endo S2-His respectively by the same method and packing, mix ADC-1 and Halo-Endo S2-His and then purify.

[0287] The results are as shown in Figure 5A. The ADC-1 sample is subjected to the flow-through mode of the Q FF chromatography column under the condition of pH 7.5.

[0288] The Halo-Endo S2-His sample is subjected to the binding mode of the Q FF chromatography column under the condition of pH 7.5 and eluted with 20 mM Tris-HCl, 1 M NaCl pH 7.5. As shown in Figure 5B, the mixed-loading sample of ADC-1 and Halo-Endo S2-His can be effectively separated after being subjected to the Q FF chromatography column, with ADC-1 flowing through and Halo-Endo S2-His binding.

[0289] 2) Cation Exchange Chromatography (CEX) Capto S impact cation packing was selected to pack the column, and the chromatography column was equilibrated with 20 mM citric acid / citric sodium pH 6.2, and then the sample was loaded. After the sample loading was completed, it was continuously equilibrated to the baseline with 20 mM citric acid / citric sodium, pH 6.2, and finally the sample was eluted with 20 mM citric acid / citric sodium, 160 mM NaCl pH 6.2. The chromatography column was regenerated with 20 mM citric acid / citric sodium, 1 M NaCl pH 6.2, and the CIP of the chromatography column was performed with 1 M NaOH. The purification of ADC and Halo-Endo S2-His was carried out respectively with the same method and packing, and then they were mixed and purified.

[0290] The results are as shown in Figure 6A. The ADC-1 sample is subjected to the binding mode of the Capto S impact cation chromatography column under the condition of pH 6.2.

[0291] The Halo-Endo S2-His sample is subjected to the passing mode of a Capto S impact cation chromatography column under the condition of pH 7.5. As shown in Figure 6B, the mixed filling sample of ADC-1 and Halo-Endo S2-His can be effectively separated after being subjected to the Capto S impact cation chromatography column, such that ADC-1 binds and Halo-Endo S2-His passes through.

[0292] Example 8 Detection of the affinity of ADC-1 for cell surface ErbB2 / Her2 Take Her2 ECD, prepare it to be 1 μg / ml with CBS coating buffer (0.1 M carbonate buffer, pH 9.6), coat it at 100 μl / well at 25 °C for 60 min. After the coating is completed, wash the plate 3 times with PBST, block it with blocking buffer (5% skim milk powder) (300 μl / well), and incubate it at 25 °C and 200 rpm for 60 min. After the blocking is completed, wash the plate 3 times with PBST, and add antibody T-LCCT L -HC and ADC-1 (100 μl / well), leave it at 25 °C, and incubate it at 200 rpm for 60 min. After the incubation with the sample is completed, wash the plate 3 times with PBST, add anti-human IgG Fc-HRP at 100 μl / well, and incubate it at 25 °C and 200 rpm for 60 min. After the 60-min incubation is completed, develop the color with TMB (100 μl / well) for 5 min, then stop the reaction with stop buffer, and read it at OD450.

[0293] The results of the Elisa test show that, as shown in Figure 7, there is no significant difference in the antigen affinity between the T-LCCT L -HC unbound monoclonal antibody and ADC-1 for Her2 ECD.

[0294] Example 9 Influence of ADC-1 on tumor cell proliferation at different expression levels of ErbB2 / Her2 1) Inoculate 100 μl per well (containing 1000 - 10000 cells) of ErbB2 / Her2-positive human breast cancer cells BT-474, ErbB2 / Her2-positive human gastric cancer cells NCI-N87, and ErbB2 / Her2-negative human liver cancer cells HepG2 into a 96-well plate and culture overnight in a cell incubator at 37 °C, 5% CO2, and 100% humidity.

[0295] 2) Add ADC-1 or antibody T-LCCT L -HC at different concentrations (10, 3.3, 1.1, 0.37, 0.12, 0.041, 0.014, 0.0046, 0.0015, 0.00051 nM) or MMAE (monomethyl auristatin E) at different concentrations (30, 10, 3.3, 1.1, 0.37, 0.12, 0.041, 0.014, 0.0046, 0.0015 nM) to the ErbB2 / Her2-positive cells cultured overnight. Add ADC-1 or antibody T-LCCTL-HC at different concentrations (100, 10, 3.3, 1.1, 0.37, 0.12, 0.041, 0.014, 0.0046, 0.0015 nM) or MMAE at different concentrations (30, 10, 3.3, 1.1, 0.37, 0.12, 0.041, 0.014, 0.0046, 0.0015 nM) to the ErbB2 / Her2-negative cells cultured overnight. Add puromycin at a concentration of 50 μM to the control group. Continue to incubate at 37 °C for 72 - 120 h.

[0296] 3) Remove the cell plate from the 37 °C cell incubator and equilibrate it at room temperature for about 30 minutes. Add 100 μl of CellTiter Glo reagent per well, shake it on a shaker for 2 min, then let it stand in the dark at room temperature for 10 min, and measure the relative luminescence units (RLU) with an MD M4 microplate reader.

[0297] 4) The results of the inhibitory effects of different drugs on tumor cell proliferation are as shown in Table 3 and Figures 8 - 10. Both ADC1 and the MMAE small molecule toxin have obvious growth inhibitory effects on ErbB2 / Her2-positive cells, and the antibody T-LCCT L-HC monoclonal antibody has a slight growth inhibitory effect on ErbB2 / Her2 positive cells, and ADC-1 is T-LCCT L -HC monoclonal antibody and MMAE small molecule toxin were significantly exceeded. ADC-1 and T-LCCT L -HC monoclonal antibody has no inhibitory effect on ErbB2 / Her2 negative cells.

Table 3

[0298] Example 10 In vivo activity test of ADC-1 (NCI-N87 CDX mouse model) The effect of ADC-1 on tumor growth in ErbB2 / HER2 NCI-N87 CDX mouse model was tested by the following method 1) Cell culture: NCI-N87 human gastric cancer tumor cells (ATCC, Manassas, VA, cat# CRL-5822) in the logarithmic growth phase were collected, and the cell density was adjusted with matrix gel buffer (PBS:Matrigel = 1:1). 0.2 mL of the prepared NCI-N87 cell suspension was subcutaneously injected into the right scapula of 7-9 week-old SPF-grade female BALB / c nude mice at a cell seeding size of 10×10 6 / mouse.

[0299] 2) Tumor diameter was measured with calipers, and tumor volume (where a is the longest diameter of the tumor and b is the shortest diameter of the tumor) was calculated using the formula V = 0.5a×b 2 Five days after cell seeding, when the average tumor volume range reached 100 - 300 mm 3 the animals were randomly divided into a solvent control group and an ADC-1 3 mg / kg group, with 5 animals in each group. The animals were injected via the tail vein, and the control group was administered an equal volume of solvent (Vehicle). The day of grouping and administration was defined as Day0. The tumor volume of the animals in each group was measured twice a week within 35 days after administration, and the tumor volume of the animals on day 35 was compared between groups. The T / C value and TGI value were calculated based on the tumor volume. The calculation formula is T / C% = T RTV / C RTV ×100% (T RTV : RTV of the treatment group, C RTV: It was the vehicle control group (RTV). Based on the results of tumor measurement, the relative tumor volume (RTV) was calculated, and the calculation formula is RTV = Vt / V0, where V0 is the average tumor volume measured at the time of grouping, Vt is the average tumor volume at a certain measurement, and T RTV and C RTV were obtained from the data of the same day. Calculation of TGI(%): TGI(%) = [1 - (average tumor volume at the end of treatment in a certain treatment group - average tumor volume at the start of administration in the same treatment group) / (average tumor volume at the end of treatment in the vehicle control group - average tumor volume at the start of administration in the vehicle control group)] × 100%.

[0300] 3) The data between groups were statistically analyzed by independent samples t-test, and all analyses were performed using SPSS 17.0. P < 0.05 indicates that the difference is statistically significant.

[0301] 4) Table 4 and Figure 11 show that ADC-1 can significantly inhibit tumor growth in the NCI-N87 CDX mouse model compared with the vehicle control group.

Table 4

[0302] Example 11 Preparation and Characterization of ADC-2 ADC-2 was prepared with reference to the above preparation method. It differed from ADC-1 in that the linker-payload was LP-6, and the characterization data of the antibody-drug conjugate ADC-2 are as follows.

[0303] SDS-PAGE Detection and Analysis of Antibody-Drug Conjugate ADC-2: After the conjugation reaction was completed, the purity and conjugation efficiency of ADC-2 were detected by SDS-PAGE. According to the SDS-PAGE detection results of ADC-2, the conjugation reaction occurred site-specifically in the heavy chain of the antibody, and the ADC-2 heavy chain conjugated to Linker-payload had a significantly changed molecular weight compared with the monoclonal antibody heavy chain with cleaved sugar chains, indicating that the Linker-payload was successfully conjugated to the monoclonal antibody heavy chain site-specifically. Basically, no antibody not conjugated to Linker-payload was observed in the conjugation product, the conjugation efficiency was over 95%, and the purity of the conjugation product met the expectations.

[0304] Regarding the HIC-HPLC detection and analysis of ADC-2, as shown in Figure 12 in the detection results, the antibody not conjugated to the cytotoxic was <5%, the conjugation product mainly had a DAR of 4, and overall the DAR value of the ADC-2 drug was about 3.8.

[0305] Plasma Stability Test of ADC-2 Test Design Healthy human pooled plasma (5 males and 5 females, mixed in equal volume) was collected, ADC-2 was added to reach a specific final concentration, divided into 4 portions, 450 μL per portion, incubated in a 37 °C incubator, the sampling times were 0 h, 24 h, 48 h, and 96 h, and stored in a refrigerator at -60 to -90 °C after collection to detect the free payload dropout rate and DAR change rate.

[0306] LC-MS Detection of Small Molecule Toxin (the small molecule toxin of LP-6 according to this example is represented by payload) Collect 40 μL of each sample (excluding Double Blank), add a certain amount of internal standard precipitant (1 ng / mL DXd), shake for at least 10 min. Collect 40 μL of blank matrix from Double Blank, add precipitant without internal standard of 120 μL, shake for at least 10 min, centrifuge at 3600 g and 4 °C for 15 min, aspirate 60 μL of supernatant into the sample injection vial, add a certain amount of 0.1% FA ultrapure water, shake for at least 3 min, and perform HPLC detection.

[0307] DAR detection by hybrid LC-MS method Weigh 1 g of CNBr-activated agarose (Sigma, Cat# C9142) into a 50 mL centrifuge tube, add 50 mL of pre-cooled 1 mM HCl, place it on a rotary mixer and incubate for at least 30 min. The incubation conditions are 4 °C and 10 RPM. Centrifuge for a certain time to remove 1 mM HCl. Wash the sol once with 5 - 10 volumes of deionized water, and then wash it three times with 0.1 M NaHCO3. Replace an appropriate amount of HER2 ECD with a 30 kD ultrafiltration tube (Buffer is 0.1 M NaHCO3). Then mix the replaced HER2 ECD with the packing, place it on a rotary mixer, and incubate at 25 °C and 10 RPM for 2 h ± 10 min to perform conjugation. Then add 5 mL of 0.1 M NaHCO3 buffer, centrifuge for a certain time, and wash three times to remove unconjugated proteins. Add a certain amount of glycine (pH 8.0) to the packing, let it stand at 2 - 8 °C for 16 h to block, and block the unreacted chemical groups on the packing. First, wash the packing with 5 mL of 0.1 M NaHCO3, centrifuge for a certain time, and then wash the packing with a certain amount of acetate buffer, wash for a certain time, and repeat the washing cycle multiple times. Put 0.1 mL of each sample into a 1.5 mL EP tube, add 0.1 mL of the packing conjugated with HER2 ECD, incubate at 25 °C and 10 RPM for 2 h, wash three times with 1 mL of PBST, and centrifuge for a certain time. Elute and centrifuge to take the supernatant, measure the concentration, and perform LC-MS detection.

[0308] Result analysis When ADC-2 was incubated in healthy human pooled plasma for 0, 24, 48, and 96 h, the DAR change rates were 100.00%, 106.7%, 104.2%, and 106.5%, respectively. The detachment ratios of the small molecule toxin were only 0.006%, 0.179%, 0.373%, and 1.07%, respectively. According to the above results, after ADC-2 was incubated in healthy human pooled plasma at 37 °C for 96 h, the amount of toxin detachment was extremely low, the DAR was stable, and the low toxin detachment indicated that the toxic side effects caused by free toxin clinically were significantly reduced. The high DAR stability indicated that the antibody molecule could target the tumor site and transport more toxin molecules there, improving the drug efficacy. The synergistic effect of both could significantly expand the therapeutic window of ADC-2.

[0309] Example 13 In vitro activity test of ADC-2 (SK-BR-3, HCC1954, MDA-MB-468) The effect of ADC-2 on tumor cell proliferation at different expression levels of ErbB2 / HER2 was tested by the following method Referring to the method described in Example 9, the inhibitory effect of the antibody-drug conjugate ADC-2 on cancer cell proliferation at various ErbB2 / HER2 expression levels was tested. For example, ErbB2 / HER2-positive human tumor cells such as SK-BR-3 and HCC1954, and MDA-MB-468 ErbB2 / HER2-negative human tumor cells were selected. The results of the inhibitory effect of different drugs on tumor cell proliferation are as shown in Table 5 and Figures 13 to 15. Among them, both ADC-2 and the small molecule toxin had an obvious inhibitory effect on ErbB2 / HER2-positive cells. The antibody mAb-1 monoclonal antibody had a certain inhibitory effect on ErbB2 / HER2-positive cells, and ADC-2 was significantly higher than mAb-1. ADC-2 and the mAb-1 monoclonal antibody had no inhibitory effect on ErbB2 / HER2-negative cells, showing good targeting performance.

Table 5

[0310] Example 14 Preparation and Characterization of ADC-3 Referring to the above preparation method, the antibody-drug conjugate ADC-3 was prepared, which differed from ADC-1 in that the antibody used was an anti-TROP2 antibody, namely mAb-2, and the linker-payload used was LP-6. The characterization data of the antibody-drug conjugate ADC-3 are as follows.

[0311] According to the SEC-HPLC of ADC-3, the high molecular weight polymer is <5%, the ADC sample mainly exists in the form of monomers, and the damage to the antibody by the conjugation reaction is very slight.

[0312] High-Resolution Mass Spectrometry (ESI-MS) DAR Value Analysis of Antibody-Drug Conjugate ADC-3 The molecular weight of ADC-3 was analyzed by a high-resolution mass spectrometer, and the deconvoluted mass spectrum is shown in Figure 16. Based on the measured molecular weight information, by comparing with the theoretical molecular weight, each major molecular weight variant was assigned, and the DAR value analysis was performed according to the mass spectrometry abundance of each major molecular weight variant. The average DAR value was calculated to be 3.93.

[0313] Example 15 In Vitro Activity Test of ADC-3 (BxPC-3, FaDu, HepG2) Referring to the same operation as the method described in Example 9, the inhibitory effect of the antibody-drug conjugate ADC-3 on cancer cell proliferation at different TROP2 expression levels with different concentration gradients was tested. Human tumor cells such as BxPC-3, FaDu, and HepG2 were selected. The results of the inhibitory effect of different drugs on tumor cell proliferation are shown in Table 6 and Figures 17-19. Among them, both ADC-3 and the small molecule toxin have an obvious inhibitory effect on TROP2-positive cells, while the TROP2 antibody, namely mAb-2, has no obvious inhibitory effect on TROP2-positive cells, and ADC-3 significantly exceeds the monoclonal antibody and the small molecule toxin. Neither ADC-3 nor the mAb-2 monoclonal antibody has an inhibitory effect on TROP2-negative cells, indicating good targeting performance.

Table 6

[0314] In vivo activity test of Example 16 ADC-3 (NCI-N87 CDX mouse model) Referring to the method similar to that described in Example 10, the effect of ADC-3 on inhibiting tumor growth in the NCI-N87 CDX mouse model was evaluated. The tumor growth curve and body weight change curve after administration are as shown in Figures 20 - 21. ADC-3 showed good tumor growth inhibitory effect and high safety, and no toxicity related to body weight loss was observed in the experimental mice.

[0315] Preparation and characterization of Example 17 ADC-4 Referring to the above preparation method, antibody-drug conjugate ADC-4 was prepared. It is different from ADC-1 in that the antibody used is anti-TROP2 antibody, namely mAb-2. The characterization data of antibody-drug conjugate ADC-4 are as follows.

[0316] The HIC-HPLC detection analysis of ADC-4 shows that the detection results are as shown in Figure 22. The antibody not conjugated to the cytotoxin is <5%, the conjugation product is mainly DAR2, and the overall DAR value of ADC-4 drug is 1.89. According to the SEC-HPLC detection results of ADC-4, the high molecular weight polymer in the ADC drug is <5%, and the ADC sample mainly exists in the form of monomers.

[0317] Preparation and characterization of Example 18 ADC-5 Referring to the above preparation method, antibody-drug conjugate ADC-5 was prepared. It is different from ADC-1 in that the antibody is anti-TROP2 antibody mAb-2 and the linker-payload is LP-2. The characterization data of antibody-drug conjugate ADC-5 are as follows.

[0318] HIC-HPLC detection analysis of ADC-5. The detection results are as shown in Figure 23. The antibody not conjugated to the cytotoxin is <5%. The conjugation product is mainly DAR2. Overall, the DAR value of the ADC-5 drug is 1.87. According to the SEC-HPLC detection of ADC-5, the high molecular weight polymer in the ADC drug is <5%, and the ADC sample mainly exists in the form of monomers.

[0319] Example 19 In vitro activity comparison of ADC-4 and ADC-5 (BxPC-3, FaDu, HepG2) Referring to the method described in Example 9, the inhibitory effects of the antibody-drug conjugates ADC-4 and ADC-5 on cancer cell proliferation at various TROP2 expression levels were tested. TROP2-positive human tumor cells such as BxPC-3 (human pancreatic cancer cells) and FaDu (human pharyngeal cancer cells), and TROP2-negative tumor cells such as HepG2 (human liver cancer cells) were selected. The results of the inhibitory effects of different drugs on tumor cell proliferation are as shown in Table 7 and Figures 24-26. Among them, ADC-4, ADC-5, and the MMAE small molecule toxin all have obvious inhibitory effects on the proliferation of positive cells, and there is no significant difference in the activities of ADC-4 and ADC-5. The monoclonal antibody has no obvious inhibitory effect on TROP2-positive cells. ADC-4, ADC-5, and the monoclonal antibody have no inhibitory effect on antigen-negative cells, showing good targeting performance.

Table 7

[0320] Example 20 Preparation and characterization of ADC-6 An antibody-drug conjugate was prepared with reference to the above preparation method. It differs from ADC-1 in that the antibody used is mAb-3 (Trastuzumab) and the linker-payload is LP-6. The characterization data of the antibody-drug conjugate ADC-6 are as follows. For the HIC-HPLC detection analysis of ADC-6, the detection results are as shown in Figure 27. The antibody not conjugated to the cytotoxin is <5%, the conjugation product is mainly DAR4, and overall the DAR value of the ADC-6 drug is 3.93. According to the SEC-HPLC detection of ADC-6, the high molecular weight polymer in the ADC drug is <5%, and the ADC sample mainly exists in the form of monomers.

[0321] Example 21 In vitro Activity Test of ADC-6 (SK-BR-3, NCI-N87) With reference to the method described in Example 9, the inhibitory effect of the antibody-drug conjugate ADC-6 on cancer cell proliferation at various HER2 expression levels was tested. Two groups of HER2 cell lines, SK-BR-3 and NCI-N87, were selected, and the difference in activity between ADC-2 (containing the Sortase recognition sequence) and ADC-6 (not containing the Sortase recognition sequence) was compared. It was found that regardless of the presence or absence of the specific recognition short peptide sequence of the Srt A enzyme (Sortase recognition sequence), there was no difference in the cell killing activity of the target ADC for the above two cell lines (see Figures 28 - 29).

[0322] The sequences according to this application are as follows. SEQ ID NO: 1 (Halo-Endo S2-His): MAEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSYVWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIHDWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLIIDQNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVEEYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLIGSEIARWLSTLEISGGGGGSGGGGSMDKHLLVKRTLGCVCAATLMGAALATHHDSLNTVKAEEKTVQTGKTDQQVGAKLVQEIREGKRGPLYAGYFRTWHDRASTGIDGKQQHPENTMAEVPKEVDILFVFHDHTASDSPFWSELKDSYVHKLHQQGTALVQTIGVNELNGRTGLSKDYPDTPEGNKALAAAIVKAFVTDRGVDGLDIDIEHEFTNKRTPEEDARALNVFKEIAQLIGKNGSDKSKLLIMDTTLSVENNPIFKGIAEDLDYLLRQYYGSQGGEAEVDTINSDWNQYQNYIDASQFMIGFSFFEESASKGNLWFDVNEYDPNNPEKGKDIEGTRAKKYAEWQPSTGGLKAGIFSYAIDRDGVAHVPSTYKNRTSTNLQRHEVDNISHTDYTVSRKLKTLMTEDKRYDVIDQKDIPDPALREQIIQQVGQYKGDLERYNKTLVLTGDKIQNLKGLEKLSKLQKLELRQLSNVKEITPELLPESMKKDAELVMVGMTGLEKLNLSGLNRQTLDGIDVNSITHLTSFDISHNSLDLSEKSEDRKLLMTLMEQVSNHQKITVKNTAFENQKPKGYYPQTYDTKEGHYDVDNAEHDILTDFVFGTVTKRNTFIGDEEAFAIYKEGAVDGRQYVSKDYTYEAFRKDYKGYKVHLTASNLGETVTSKVTATTDETYLVDVSDGEKVVHHMKLNIGSGAIMMENLAKGAKVIGTSGDFEQAKKIFDGEKSDRFFTWGQTNWIAFDLGEINLAKEWRLFNAETNTEIKTDSSLNVAKGRLQILKDTTIDLEKMDIKNRKEYLSNDENWTDVAQMDDAKAIFNSKLSNVLSRYWRFCVDGGASSYYPQYTELQILGQRLSNDVANTLKDHHHHHHHHHH Sequence number 2 (mAb-1 light chain): DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGALPETGG Sequence number 3 (mAb-1 heavy chain): EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Sequence number 4 (mAb-2 light chain): DIQLTQSPSSLSASVGDRVSITCKASQDVSIAVAWYQQKPGKAPKLLIYSASYRYTGVPDRFSGSGSGTDFTLTISSLQPEDFAVYYCQQHYITPLTFGAGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGALPETGG Accession number 5 (mAb-2 heavy chain): QVQLQQSGSELKKPGASVKVSCKASGYTFTNYGMNWVKQAPGQGLKWMGWINTYTGEPTYTDDFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFCARGGFGSSYWYFDVWGQGSLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Accession number 6 (mAb-3 light chain): DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Accession number 7 (mAb-3 heavy chain): EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0323] The applicant explains the detailed method of the present invention by the above embodiments in the present invention, but the present invention is not limited to the above detailed method, that is, the applicant claims that the present invention can be implemented without the above detailed method. Those skilled in the art should understand that any improvement to the present invention, the equivalent substitution of each raw material of the product of the present invention, the addition of auxiliary components, the selection of specific methods, etc. are all included within the protection scope and disclosure scope of the present invention.

Claims

1. A method for preparing a drug conjugate, wherein the drug conjugate is site-specifically conjugated based on an N-glycosylation site in the Fc region, and the method is (1) A step of providing a donor containing an oxazoline oligosaccharide, a protein containing an Fc region, and an immobilized endoglycosidase having glycosidic transfer activity, wherein the Fc contains a GlcNAc motif, (2) A method comprising the step of covalently bonding the donor containing the oxazoline oligosaccharide to the protein containing the Fc motif by catalytic action of the endoglycosidase.

2. The method according to claim 1, characterized in that the protein containing the Fc region is an antibody or an Fc fusion protein.

3. The donor containing the oxazoline oligosaccharide further comprises a payload, The payload is selected from the group consisting of small molecule compounds, agonists, nucleic acids, nucleic acid analogs, fluorescent molecules, radionuclides, and immunomodulatory proteins, or The method according to claim 1, wherein the payload is selected from the group consisting of small molecule compounds (for example, small molecule drugs with various mechanisms of action, including various conventional small molecule drugs, photoacoustic therapy drugs, photothermal therapy drugs, etc., for example, chemotherapeutic drugs, small molecule targeted drugs, immune agonists, etc., for example, conventional cytotoxic drugs such as cisplatin, paclitaxel, 5-fluorouracil, cyclophosphamide and bendamustine, for example, small molecule targeted drugs such as imatinib mesylate, gefitinib and anlotinib, for example, immune agonists such as STING agonists and TLR agonists), nucleic acids and nucleic acid analogs, tracer molecules (including fluorescent molecules, biotin, fluorophores, chromophores, spin resonance probes and radiolabeling, etc.), short-chain peptides, polypeptides, peptide mimes and proteins.

4. The oxazoline oligosaccharide is one or more selected from the group consisting of disaccharide oxazoline, trisaccharide oxazoline, tetrasaccharide oxazoline, pentasaccharide oxazoline, hexasaccharide oxazoline, heptasaccharide oxazoline, octasaccharide oxazoline, nonusaccharide oxazoline, decasaccharide oxazoline, and decacsaccharide oxazoline. Characterized by being a number, the oxazoline oligosaccharide is first hexosyl or its derivative - (second hexosyl or its derivative) f The method according to claim 1, characterized in that it has the structure of -β-D-glucopyranosyloxazoline, f is 0, 1, 2, 3, 4, 5 or 6, and the structure of β-D-glucopyranosyloxazoline is as follows. 【Chemistry 1】

5. The first hexosyl or its derivative is selected from glucosyl, mannosyl, galactosyl, fructosyl, grosyl, idosyl or its derivatives, and / or The carbon at position 6 is in the form of -C(O)-, and / or The second hexosyl or its derivative is independently selected from glucosyl, mannosyl, galactosyl, fructosyl or its derivatives for each occurrence, and / or Each monosaccharide portion in the oligosaccharide structure is linked via a β-(1→4) glycosidic bond, and / or The method according to claim 4, characterized in that the first hexosyl derivative and the second hexosyl derivative are independently selected from derivatives in which the hydroxyl group of a uronic acid or monosaccharide is substituted with an acylamino group.

6. The oxazoline oligosaccharide has the structure of first hexosyl or its derivative-β-D-glucopyranosyloxazoline, and the first hexosyl or its derivative is mannosyl or its derivative, or The oxazoline oligosaccharide has the structure of 1-hexosyl or its derivative--β-D-glucopyranosyloxazoline, and the 1-hexosyl or its derivative is galactosyl or its derivative, or The oxazoline oligosaccharide has the structure of 1-hexosyl or its derivative--β-D-glucopyranosyloxazoline, and the 1-hexosyl or its derivative is glucosyl or its derivative, or The oxazoline oligosaccharide has the structure of 1-hexosyl or its derivative--β-D-glucopyranosyloxazoline, and the 1-hexosyl or its derivative is fructosyl or its derivative, or The oxazoline oligosaccharide has the structure of 1-hexosyl or its derivative--β-D-glucopyranosyloxazoline, and the 1-hexosyl or its derivative is grosyl or its derivative, or The method according to claim 4, characterized in that the oxazoline oligosaccharide has the structure of first hexosyl or its derivative-β-D-glucopyranosyloxazoline, and the first hexosyl or its derivative is idosyl or its derivative.

7. The method according to claim 4, characterized in that the structure of the oxazoline oligosaccharide is as follows. 【Chemistry 2】

8. A method for preparing a drug conjugate, wherein the antibody-drug conjugate is site-specifically conjugated based on an N-glycosylation site in the Fc region, and the method is (1) A step of providing a donor containing an oxazoline oligosaccharide, a protein containing an Fc region, and an immobilized endoglycosidase having glycosidic transfer activity, wherein the Fc region contains a GlcNAc motif, The step of the donor containing oxazoline oligosaccharide being a linker-payload compound of formula (I), 【Transformation 3】 (2) The step of covalently attaching the donor containing the oxazoline oligosaccharide to the protein containing the Fc region by catalytic action of the endoglycosidase, wherein the structure of the drug conjugate is as shown in formula (II), 【Chemistry 4】 In the formula, P is the payload, D-C(O)-L- is a linker, The carbon at position 6 of the first hexosyl or its derivative moiety is in the form of -C(O)-, and f is 0, 1, 2, 3, 4, 5, or 6. L is a linker, and L directly bonds to the carbonyl in D-C(O)- via -NH- within it, and if L is a non-branched linker, it bonds to one P and t is 1, and if L is a branched linker, each branch can bond to one P and t is an integer greater than 1, R is hydrogen or α-L-fucosyl. q is either 1 or 2. A method characterized in that the protein is a protein containing an Fc region.

9. The first hexosyl or its derivative is selected from glucosyl, mannosyl, galactosyl, fructosyl, grosyl, idosyl or its derivatives, and / or The second hexosyl or its derivative is independently selected from glucosyl, mannosyl, galactosyl, fructosyl or its derivatives for each occurrence, and / or Each monosaccharide portion in the oligosaccharide structure is linked via a β-(1→4) glycosidic bond, and / or The method according to claim 8, characterized in that the first hexosyl derivative and the second hexosyl derivative are independently selected from derivatives in which the hydroxyl group of a uronic acid or monosaccharide is substituted with an acylamino group.

10. The method according to claim 8, characterized in that the protein containing the Fc region is an antibody or an Fc fusion protein.

11. A method for preparing an antibody-drug conjugate, wherein the antibody-drug conjugate is an antibody The Fc region is conjugated in a site-specific manner based on the N-glycosylation site, and the method described above is (1) A step of providing a donor containing oxazoline oligosaccharide, an antibody containing a GlcNAc motif, and an immobilized endoglycosidase having glycosidic transfer activity, The step of the donor containing oxazoline oligosaccharide being a linker-payload compound of formula (I), 【Transformation 5】 (2) The step of covalently binding the donor containing the oxazoline oligosaccharide to the antibody containing the GlcNAc motif by catalytic action of the endoglycosidase, The structure of the antibody-drug conjugate is as shown in formulas (II-1), (II-2), (II-3), (II-4), or (II-5), 【Transformation 6】 【Transformation 7】 【Transformation 8】 【Chemistry 9】 【Chemistry 10】 In the formula, P is the payload, D-C(O)-L- is a linker, D-C(O)- is a disaccharide structure, L is a linker, and L directly bonds to the carbonyl in D-C(O)- via -NH- within it, and if L is a non-branched linker, it bonds to one P and t is 1, and if L is a branched linker, each branch can bond to one P and t is an integer greater than 1, R is hydrogen or α-L-fucosyl. q is either 1 or 2. A method characterized in that Ab is an antibody or an antigen-binding fragment thereof.

12. A method for preparing an antibody-drug conjugate, wherein the antibody-drug conjugate is site-specifically conjugated based on an N-glycosylation site of the antibody Fc region, and the method is (1) A step of providing a donor containing oxazoline oligosaccharide, an antibody containing a GlcNAc motif, and an immobilized endoglycosidase having glycosidic transfer activity, The step of the donor containing oxazoline oligosaccharide being a linker-payload compound of formula (I), 【Chemistry 11】 (2) The step of covalently binding the donor containing the oxazoline oligosaccharide to the antibody containing the GlcNAc motif by catalytic action of the endoglycosidase, The structure of the antibody-drug conjugate is as shown in formula (II), 【Chemistry 12】 In the formula, P is the payload, D-C(O)-L- is a linker, D-C(O)- is a disaccharide structure. 【Chemistry 12-1】 And, L is a linker, and L is directly bonded to the carbonyl in D-C(O)- via -NH- within it, and if L is a non-branched linker, it is bonded to one P and t is 1, and if L is a branched linker, each branch can be bonded to one P and t is an integer greater than 1, R is hydrogen or α-L-fucosyl. q is either 1 or 2. A method characterized in that Ab is an antibody or an antigen-binding fragment thereof.

13. -L-(P) t Ha-L 2 -L 1 -B-P, and therefore equation (I) is as follows: 【Chemistry 13】 During the ceremony, B does not exist independently or is any one or a combination of 1) a self-cleaving spacer Sp1 and 2) one divalent group or a combination of two or more divalent groups, and the divalent group is selected from -CR 1 R 2 -, C 1-10 alkylene, C 4-10 cycloalkylene, C 4-10 heterocyclylene and -(CO)-, L 1 It is either a sequence that does not exist independently, or a sequence that cannot be cleaved, or a cleavable sequence that includes an amino acid sequence that can be cleaved by an enzyme, wherein the amino acid sequence that can be cleaved by an enzyme contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids. L 2 They do not exist independently, or, 1) One or more -CH groups in the alkylene 2 - Arbitrary structure - CR 3 R 4 -, -O-, -(CO)-, -S-, -S(=O) 2 -, -NR 5 - 【Chemistry 13-1】 C 4-10 Cycloalkylene, C 4-10 Substitution by groups such as heterocyclylene and phenylene Furthermore, cycloalkylene, heterocyclene, and phenylene are each independently unsubstituted, or they are halogens, -C 1-10 Alkyl, -C 1-10 Haloalkyl, -C 1-10 Alkilen-NHR 8 and -C 1-10 Alkilen-OR 9 -NH-C substituted with at least one substituent selected from 2-20 Alkilen and, 2) n is an integer from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 to 100, AA is an amino acid residue each time it appears independently, * represents the N-end of the corresponding amino acid, ** represents the C-end of the corresponding amino acid, and there is optionally -(C) between the amino and α-carbon of one amino acid. 2 H 4 -O) m - (CH 2 ) p - is an amino acid residue sequence in which -* (AA) exists, m is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, p is 0, 1, 2, or 3, and the * end and the carbonyl in the disaccharide structure form an amide bond. n **-and, It is one of the above or a combination thereof. R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 Each is independently hydrogen, halogen, substituted or unsubstituted -C 1-10 Alkyl, C 4-10 Selected from cycloalkylenes, or R 1 and R 2 The carbon atoms to which they are bonded together form a 3-6 membered ring cycloalkylene, and / or R 3 and R 4 The carbon atoms to which they are bonded together form a 3- to 6-membered ring cycloalkylene. P is part B, or L 1 Part, or L 2 The method according to claim 8, characterized in that it is a payload that is coupled to a portion.

14. -L-(P)t is, 【Chemistry 14】 Therefore, equation (I) is, 【Chemistry 15】 And, During the ceremony, Ld2 and each Ld1 are independently bonded, or -NH-C 1-20 Alkylene-(CO)-,-NH-(PEG) i -(CO)- is selected, or the side chains are independently unsubstituted or -CO-(PEG) j -R 11 A natural amino acid substituted by or a natural amino acid oligomer with a degree of polymerization of 2 to 10 (i.e., 2, 3, 4, 5, 6, 7, 8, 9, or 10), R 11 is C 1-10 It is alkyl, d is 0, 1, 2, 3, 4, 5, or 6. - (PEG) i - and - (PEG) j Each of the hyphens represents a predetermined number of consecutive hyphens (O-C). 2 H 4 ) - Structural unit or continuous - (C 2 H 4 -O)- A structural unit, with C at either end. 1-10 This is a PEG fragment with an alkylene attached, where each i is an independent integer between 1 and 100, and each j is an independent integer between 1 and 100. M is hydrogen or LKa-L 2 ―L 1 ―B-P, Q is NH 2 or L 2 ―L 1 ―B-P, However, M is hydrogen and Q is NH 2 Cases where this is the case are excluded, Each LKa is independent 【Chemistry 16】 Selected from, opSu is 【Chemistry 17】 or a mixture thereof, where * is L 2 This represents the part that is joined to, B either does not exist independently, or it is either 1) a self-cutting spacer Sp1, or 2) one divalent group, or a combination of two or more divalent groups, or a combination thereof, wherein the divalent group is -CR 1 R 2 -, C 1-10 Alkylene, C 4-10 Cycloalkylene, C 4-10 Selected from heterocyclylene and -(CO)-, L 1 It is either a sequence that does not exist independently, or a sequence that cannot be cleaved, or a cleavable sequence that includes an amino acid sequence that can be cleaved by an enzyme, wherein the amino acid sequence that can be cleaved by an enzyme contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids. L 2 They do not exist independently, or, 1) One or more -CH groups in the alkylene 2 - Arbitrary structure - CR 3 R 4 -, -O-, -(CO)-, -S-, -S(=O) 2 -, -NR 5 - 【Chemistry 17-1】 C 4-10 Cycloalkylene, C 4-10 Substituted with groups such as heterocyclylene and phenylene, with cycloalkylene, heterocyclylene, and phenylene each independently being unsubstituted, or with halogens, -C 1-10 Alkyl, -C 1-10 Haloalkyl, -C 1-10 Alkilen-NHR 8 and -C 1-10 Alkilen-OR 9 -NH-C substituted with at least one substituent selected from 2-20 Alkilen and, 2) n is an integer of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 to 100; AA is an amino acid residue independent for each occurrence; * represents the N-terminus of the corresponding amino acid; ** represents the C-terminus of the corresponding amino acid; randomly - (C[[ERR]] 2 H 4 -O) m -(CH 2 ) p - exists; m is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; p is 0, 1, 2 or 3; it is an amino acid residue sequence in which the *-terminus and the carbonyl in the disaccharide structure form an amide bond -*(AA) n **- and It should be noted that there seems to be an error in the original text at the end of line where "-(C[[ERR]]" is incomplete. This might affect the accuracy of the overall translation. Please check and correct the original text if possible for a more precise translation. It is one of the above or a combination thereof. R 1 、R 2 、R 3 、R 4 、R 5 、R 6 、R 7 、R 8 、R 9 are each independently hydrogen, halogen, substituted or unsubstituted -C 1-10 alkyl, C 4-10 cycloalkylene, or R 1 and R 2 and the carbon atom to which they are attached together form a 3- to 6-membered cycloalkylene ring, and / or R 3 and R 4 and the carbon atom to which they are attached together form a 3- to 6-membered cycloalkylene ring, P is part B, or L 1 Part, or L 2 The method according to claim 8, wherein the payload is coupled to the portion.

15. L 2 This is an amino acid residue sequence - * (AA) n ** is an integer from 1 to 100, AA is an independent amino acid residue each time it appears, * represents the N-end of the corresponding amino acid, ** represents the C-end of the corresponding amino acid, and there is an arbitrary -(C) between the amino and α-carbon of one amino acid. 2 H 4 -O) m - (CH 2 ) p The method according to claim 13, characterized in that a - exists, m is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, p is 0, 1, 2 or 3, and the * end and the carbonyl in the disaccharide structure form an amide bond.

16. The endoglycosidase having glycosidic transfer activity is characterized by being N-acetylglucosamine endohylase, The N-acetylglucosamine endohylase comprises at least one selected from Endo S (Streptococcus pyogenes endoglycosidase-S), Endo F3 (Elizabethkingia miricola endoglycosidase-F3), Endo S2 (Endoglycosidase-S2, Streptococcus pyogenes endoglycosidase-S2), Endo Sd (Endoglycosidase-Sd, Streptococcus pyogenes endoglycosidase-Sd), and Endo CC (Endoglycosidase-CC, Streptococcus pyogenes endoglycosidase-CC), or The method according to claim 1, characterized in that the N-acetylglucosamine endohlase comprises at least one selected from Endo H, Endo D, Endo F2, Endo F3, Endo M, Endo CC1, Endo CC2, Endo Om, Endo S, and Endo S2.

17. The method according to claim 16, characterized in that an endoglycosidase having glycosidic transfer activity is covalently bonded to a Halo tag, is immobilized on a support containing a haloalkyl linker by the Halo tag, and the Halo tag is a dehalogenase or a variant thereof or a shortened functional active portion.

18. An endoglycosidase fusion protein comprising a covalently bound endoglycosidase and a Halo tag, wherein the Halo tag is a dehalogenase or a variant thereof or a shortened functional active portion.

19. The present invention is characterized in that a Halo tag is covalently bonded to the amino end of the endoglycosidase, and a His tag is covalently bonded to the carboxyl end of the endoglycosidase. The aforementioned endoglycosidases include Endo S (Streptococcus pyogenes endoglycosidase-S) and Endo F3 (Elizabethkingia myricola). At least one selected from (milicola) endoglycosidase-F3), Endo S2 (Endoglycosidase-S2, Streptococcus pyogenes endoglycosidase-S2), Endo Sd (Endoglycosidase-Sd, Streptococcus pyogenes endoglycosidase-Sd), and Endo CC (Endoglycosidase-CC, Streptococcus pyogenes endoglycosidase-CC), or The endoglycosidase fusion protein according to claim 18, characterized in that the endoglycosidase is at least one selected from Endo H, Endo D, Endo F2, Endo F3, Endo M, Endo CC1, Endo CC2, Endo Om, Endo S, and Endo S2.

20. The endoglycosidase fusion protein according to claim 18, characterized in that the endoglycosidase fusion protein contains the amino acid sequence shown in SEQ ID NO: 1, or has at least 80% identity with SEQ ID NO: 1, at least 90% identity with SEQ ID NO: 1, or has one or more conserved amino acid substitutions with SEQ ID NO:

1.

21. An immobilized endoglycosidase fusion protein, characterized by comprising the endoglycosidase fusion protein described in claim 18, immobilized on a support.

22. The immobilized endoglycosidase fusion protein according to claim 21, wherein the support comprises a haloalkyl linker such that the endoglycosidase fusion protein is immobilized on the support by a covalent interaction between the haloalkyl linker and the Halo tag, and preferably the haloalkyl linker is a chloroalkyl linker.

23. A pre-packed column characterized by being filled with the immobilized endoglycosidase fusion protein described in claim 21.

24. Use of the immobilized endoglycosidase fusion protein according to claim 21 or 22 and / or the pre-packed column according to claim 23 in the preparation and / or purification of drug conjugates or antibody conjugates.

25. Use in an implementation of the method according to any one of claims 1 to 17 of a conjugation apparatus comprising a flow reactor filled with immobilized endoglycosidase and a fluid transport unit, wherein the fluid transport unit is in fluid communication with the inlet of the flow reactor, and a donor containing oxazoline oligosaccharide and an antibody containing a GlcNAc motif are transported to the flow reactor.

26. The aforementioned conjugation equipment is A flow reactor having an inlet and an outlet, filled with a medium, and in which endoglycosidase is immobilized, A fluid transport unit is fluidically connected to the inlet of the flow reactor and configured to continuously supply at least one reaction fluid to the flow reactor according to different stages of the conjugation process, wherein the at least one reaction fluid comprises a donor containing an oxazoline oligosaccharide and an antibody containing a GlcNAc motif or a protein containing an Fc region. The use according to claim 25, characterized in that it includes a fluid recovery unit that is in fluid communication with the outlet of the flow reactor and is configured to control the recovery of fluid that has flowed out of the outlet of the flow reactor according to different stages of the conjugation process.

27. The at least one reaction fluid comprises a first reaction fluid and a second reaction fluid, the buffer, the first reaction fluid and the second reaction fluid are stored in a first container, a second container and a third container, respectively, the fluid transport unit comprises a first transport pump and a second transport pump, the first container and the second container are connected to the first transport pump via a first container outlet pipe and a second container outlet pipe, respectively, the third container is connected to the second transport pump via a third container outlet pipe, the first transport pump and the second transport pump are connected to an inlet main pipe via a first inlet branch pipe and a second inlet branch pipe, respectively, the inlet main pipe is connected to the inlet of a flow reactor, The use according to claim 26, characterized in that, during equilibration before the reaction, recovery after the reaction, and rinsing after recovery, the first transport pump pumps the buffer solution in the first container into the inlet main pipe, and during the conjugation reaction, the first transport pump pumps the first reaction fluid in the second container into the inlet main pipe, and the second transport pump pumps the second reaction fluid in the third container into the inlet main pipe.

28. The conjugation equipment is fluidically connected to the outlet of the flow reactor, and a sample fluid is taken from the fluid flowing out of the outlet of the flow reactor according to a predetermined sampling time, and the conjugate in the sample fluid is detected to obtain the detection result. The use according to claim 26, further comprising a sampling detection unit that indicates whether or not the conjugate meets a predetermined criterion.

29. The conjugation equipment further includes a recycling unit located between the inlet and outlet of the flow reactor, wherein if the detection results indicate that the conjugate does not meet predetermined criteria, the fluid recovery unit is configured to stop recovering the fluid that has flowed out of the outlet of the flow reactor, and / or the recycling unit is configured to control the fluid that has flowed out of the outlet of the flow reactor to flow back into the inlet of the flow reactor in order to perform the conjugation reaction again within the flow reactor, and / or The use according to claim 28, wherein the sampling detection unit further comprises a sampling pump, a first switching valve, an elution pump, at least one analytical column, and a detector, wherein the sampling pump is connected to the outlet of the flow reactor via a sampling tube, a sample loop is provided in the first switching valve, the first switching valve switches between a first state and a second state according to a predetermined sampling time, when the first switching valve is in the first state, the sampling pump is in fluidic communication with the sample loop and collects a sample fluid from the fluid flowing out of the outlet of the flow reactor via the sampling tube and pumps it into the sample loop, and when the first switching valve is in the second state, the elution pump, sample loop, at least one analytical column, and detector are in fluidic communication via a detection tube, the elution pump pumps the eluate into the detection tube, and the eluate flows through the sample loop, thereby coordinating so that the sample fluid in the sample loop flows through one of the at least one analytical columns before flowing into the detector.

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