Preparation method and application of a glycosyl site-directed conjugated antibody drug conjugate

Abstract

The application belongs to the technical field of biological medicine, and discloses a preparation method and application of an antibody drug conjugate with site-specific glycosyl coupling. The application discloses a recombinant cell containing a nucleic acid molecule encoding trastuzumab, and the FUT8 gene and the B4GALT1 gene are deleted. The application provides a recombinant cell which can efficiently and stably prepare trastuzumab with a uniform A2 glycosylation type. Based on the trastuzumab, 2, 4 or 6 azido groups can be introduced at specific sites of the antibody by using alpha 1, 6-fucosyltransferase and / or beta 1, 4-galactosyltransferase, and a toxin molecule is further connected, so that an antibody conjugate with uniformity and DAR value = 2, DAR value = 4 or DAR value = 6 is prepared.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a method for preparing a glycosylated site-conjugated antibody-drug conjugate and its application. Background Technology

[0002] Antibody-drug conjugates (ADCs) are a novel biotherapy approach that covalently links potent cytotoxins to antibodies and has been extensively studied. To date, 15 ADCs have been approved for marketing by the U.S. Food and Drug Administration (FDA). These approved ADCs primarily work by randomly conjugating several toxin molecules to lysine or cysteine ​​residues of antibodies to form an effective payload. However, the randomness of conjugation leads to heterogeneity in site and drug-antibody ratios (DARs). Over the past decade, site-specific conjugation has proven to be a viable strategy for improving the therapeutic index of ADCs, such as through the introduction of non-natural amino acids into the THIOMAB technology (Junutula, J., Raab, H., Clark, S.). et al. Site-specific conjugation of a cytotoxic drug to an antibody improves thetherapeutic index. Nat BiotechnolHomogeneous ADCs with a DAR value of 2 were obtained (see references 26, 925–932 (2008). https: / / doi.org / 10.1038 / nbt.1480), and enzymatic ligation using sorting enzymes or transglutaminases (Anami Y, Tsuchikama K. Transglutaminase-mediated conjugations[M] / / Antibody-Drug Conjugates: Methods and Protocols. New York, NY: Springer US, 2019: 71-82.). To avoid introducing extra cysteine ​​or non-natural amino acids, the conserved N-glycosylation site on Asn297 in the antibody Fc fragment has gained attention as a payload loading site. This N-glycosylation site is far from the complementarity-determining region (CDR), resulting in less impact of drug loading on antigen binding. Several strategies have been developed to achieve conjugation at the N-glycosylation site. Most of these are two-step reactions: (1) a glycoengineered antibody anchors a bioorthogonal tag (such as an azide) to an N-glycosylation site; and (2) a subsequent click chemistry conjugates the payload. Hsu's group recently reported on conjugating the payload to bisected GlcNAc (Hsu YP, Nourzaie O, Tocher AE, et al. Site-specific antibody conjugation using modified bisected N-glycans: method development and potential toward tunable effector function[J]. Bioconjugate Chemistry, 2023,34(9): 1633-1644.). Their strategy involved cleaving the glycan chain into the FA2 glycoform using galactosidase, then further converting it to FA2B-Az via Gn-TⅢ-catalyzed transglycosylation, and finally conjugating the payload to bisected GlcNAc via click chemistry.Furthermore, researchers have provided a similar payload-linking strategy by introducing branched fucose onto bisected or truncated bisected sugar chains (Yang Y, Song Z, Tian T, et al. Trimming crystallizable fragment (Fc) Glycans enables the direct enzymatic transfer of biomacromolecules to antibodies as therapeutics[J]. Angewandte Chemie, 2023,135(36): e202308174.). In another similar approach, heterogeneous N-glycans are removed via Endo-S and Alfc, and the resulting GlcNAc-modified IgG is reacted with UDP-Gal in the presence of GalT to obtain the LacNAc glycoform, which is further coupled to the GDP-Fuc-linker-payload via α1,3-FucT-2HR. These studies provide three novel forms of glycosylation site-specific ADCs by linking the payload to bisected GlcNAc or branched Fuc.

[0003] The process of modifying glycan chains on antibodies is often complex, involving multiple deglycosylation and transglycosylation steps catalyzed by different glycosidases and glycosyltransferases. To address this issue, scientists have been working to achieve one-step glycosylation engineering of natural IgG. ENGases have been widely used in one-step glycosylation engineered antibodies, with the preferred glycan substrate being the pentasaccharide Man3GlcNAc2. Wang's research group found that wild-type Endo-S2 exhibits significant activity when transferring site-selectively modified azide compounds, biotin, or fluorescently labeled functionalized disaccharides ManGlcNAc onto antibodies (Zhang X, Ou C, Liu H, et al. General and robust chemoenzymatic method for glycan-mediated site-specific labeling and conjugation of antibodies: facilesynthesis of homogeneous antibody–drug conjugates[J]. ACS chemical biology,2021, 16(11): 2502-2514.). Because Endo-S2 has extremely strong activity in hydrolyzing heterogeneous N-glycan chains on IgGs, natural IgGs can be "one-step" modified into ManGlcNAc glycoforms and coupled with toxin molecules using Endo-S2 and ManGlcNAc substrates. One-step synthesis of site-specific antibody–drug conjugates by reprogramming IgG glycoengineering with LacNAc-based substrates[J]. Acta Pharmaceutica Sinica B, 2022, 12(5): 2417-2428. These methods share several problems: ① The antibody glycoforms produced by engineered cells are heterogeneous, requiring the introduction of enzymatic reactions to obtain homogeneous reaction substrates, which complicates production due to the numerous steps involved; ② The glycoforms after enzymatic digestion can only support one specific reaction, limiting the possibility of conjugating different amounts of drugs (DARs) to antibodies and thus restricting the application of gsADCs. Currently, a novel conjugation approach is needed to achieve antibody substrate homogenization and the synthesis of multiple DAR values. Summary of the Invention

[0004] This invention aims to solve at least one of the technical problems existing in the prior art. Through gene editing technology, CHO cells capable of producing homogeneous A2 glycoform antibodies are obtained. Using recombinant α1,6-fucosyltransferase (FUT8) or β1,4-galactosyltransferase (GALT1), two or four azide groups are introduced at specific sites of the antibody A2 glycoform, and further linked with a toxin molecule, thereby preparing a homogeneous antibody conjugate with a DAR value of 2 or 4. By combining recombinant FUT8 and GALT, six azide groups are introduced at specific sites of the antibody A2 glycoform, and further linked with a toxin molecule, thereby preparing a homogeneous antibody conjugate with a DAR value of 6.

[0005] The first aspect of the present invention is to provide a recombinant cell.

[0006] A second aspect of the present invention is to provide a trastuzumab.

[0007] A third aspect of the present invention is to provide an antibody-drug conjugate.

[0008] The fourth aspect of this invention is to provide a method for preparing the antibody-drug conjugate of the third aspect of this invention.

[0009] The fifth aspect of this invention aims to provide the use of the recombinant cells of the first aspect of this invention, the trastuzumab of the second aspect of this invention, or the antibody-drug conjugate of the third aspect of this invention in the preparation of antitumor drugs.

[0010] To achieve the above objectives, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention provides a recombinant cell containing a nucleic acid molecule encoding trastuzumab, but lacking the FUT8 gene and the B4GALT1 gene.

[0011] In some embodiments of the present invention, the cells include CHO cells.

[0012] In some embodiments of the present invention, the nucleic acid molecule encoding trastuzumab includes a nucleic acid molecule encoding the trastuzumab heavy chain and a nucleic acid molecule encoding the trastuzumab light chain.

[0013] In some embodiments of the present invention, the amino acid sequence of the trastuzumab heavy chain is shown in SEQ ID NO:14, and the amino acid sequence of the trastuzumab light chain is shown in SEQ ID NO:13.

[0014] In some embodiments of the present invention, the nucleotide sequence of the FUT8 gene is shown in SEQ ID NO:1.

[0015] In some embodiments of the present invention, the nucleotide sequence of the B4GALT1 gene is shown in SEQ ID NO:2.

[0016] In some embodiments of the present invention, the method for constructing the recombinant cells includes the following steps: (1) sgRNAs were designed targeting the exon regions of the FUT8 and B4GALT1 genes in CHO cells, and FUT8 knockout vector plasmids and B4GALT1 knockout vector plasmids were constructed using CRISPR / Cas9. (2) Transfect CHO cells sequentially with FUT8 knockout vector plasmid and B4GALT1 knockout vector plasmid to obtain engineered cells with FUT8 and B4GALT1 genes knocked out. (3) The plasmid expressing trastuzumab was introduced into engineered cells with FUT8 and B4GALT1 genes knocked out to obtain recombinant cells.

[0017] In some preferred embodiments of the present invention, the nucleotide sequence of the sgRNA is as shown in SEQ ID NO:3-4 (for the sixth exon of the cellular FUT8 gene) and SEQ ID NO:5-6 (for the first exon of the B4GALT1 gene).

[0018] In some preferred embodiments of the present invention, the construction process of the FUT8 knockout vector plasmid is as follows: the sgRNA targeting FUT8 is digested and ligated with the vector plasmid HP180 to obtain the ligation product, which is then transformed into DH5α competent cells to obtain the FUT8 knockout vector plasmid.

[0019] In some preferred embodiments of the present invention, the construction process of the B4GALT1 knockout vector plasmid is as follows: the sgRNA targeting B4GALT1 is digested and ligated with the vector plasmid HP180 to obtain the ligation product, which is then transformed into DH5α competent cells to obtain the B4GALT1 knockout vector plasmid.

[0020] In some preferred embodiments of the present invention, the reaction system of the enzyme digestion and ligation is as shown in Table 3, and the reaction conditions are 35-38℃ for 2-4 h.

[0021] In some embodiments of the present invention, the transfection of CHO cells includes the use of Fectopro transfection reagent.

[0022] In some embodiments of the present invention, the plasmid expressing trastuzumab contains the coding genes for the trastuzumab light chain and the coding genes for the trastuzumab heavy chain.

[0023] In some preferred embodiments of the present invention, the introduction includes electroporation.

[0024] In some preferred embodiments of the present invention, the electroporation conditions are 1600-1650 V, 8-12 ms, and 1-5 Pulzes.

[0025] The recombinant cells provided by this invention can stably produce homogeneous A2 glycoform trastuzumab.

[0026] A second aspect of the present invention provides a trastuzumab obtained by culturing the recombinant cells of the first aspect of the present invention.

[0027] In some embodiments of the present invention, the trastuzumab is an A2 glycoform monoclonal antibody.

[0028] In some embodiments of the present invention, the N-glycans at the conserved glycosylation sites in the Fc region of the trastuzumab lack fucose and galactose.

[0029] In some embodiments of the present invention, the structure of the A2 glycoform monoclonal antibody is shown below: , In the above structure, the Y-shaped structure represents trastuzumab. It represents acetamide glucose. It refers to mannose.

[0030] In some embodiments of the present invention, the amino acid sequence of the trastuzumab heavy chain is shown in SEQ ID NO:14, and the amino acid sequence of the trastuzumab light chain is shown in SEQ ID NO:13.

[0031] A third aspect of the present invention provides an antibody-drug conjugate, the antibody-drug conjugate comprising: Antibodies with N-glycosylation sites; Connect segment 1 and / or connect segment 2; drug; The drug is coupled to the N-glycosylation site of the antibody via a linker fragment; The linker fragment 1 includes a linker, a reactive functional group, and a sugar structure, wherein the sugar structure is linked to the N-glycosylation site of the antibody, and the linker links the reactive functional group and the sugar structure. The connecting segment 2 includes a linker and a reactive functional group, wherein the linker is connected to a drug.

[0032] In some embodiments of the present invention, the antibody includes IgG having a conserved N-glycosylation site in the Fc region.

[0033] In some embodiments of the present invention, the IgG includes IgG1, IgG2 or IgG4.

[0034] In some embodiments of the present invention, the antibody is a monoclonal antibody, a polyclonal antibody, a bifunctional antibody, a trifunctional antibody, a nanobody fused with an Fc domain, a therapeutic antibody or a functional antibody from different species.

[0035] In some embodiments of the present invention, the targets of the antibody include HER2, Claudin18.2, EGFR, c-Met, NECTIN4, CD276, HER3, CD3, FOLR1, BCMA, CD20, DLL3, MUC1, PD-L1, ROR1, TF, CD19, CD22, CD30, CD70, CD79B, FGFs, MSLN, NT5E, TNFα, CD147, CD24, CD38, CD47, CDH3, CDK4, CDK6, CEACAM5, CLDN6, CTLA4, DDR1, DR5, FAPα, FGFR3, GPRC5D, GR, HLA-DR, ICAM1, IL2R, MELTF, ROR2, TPBG, VTCN1, Z1P6, CD33, CD25, RSV, VEGF, RANKL, VEGFR2, CTLA-4, CD52, CD319, PD- 1. CD274, IgE, IL-6, IL-12, IL-2, C5, IL-17A, CD25, SLAMF7, F10, factorIXa, HAb18G, PCSK9, BLyS, 1L23, C4β7, IL-4R-α, HAE, FGF23 or IL6R.

[0036] In some embodiments of the present invention, the antibody is selected from trastuzumab, inetuzumab, zineb, pertuzumab, sacituzumab, datopotamab, zotuzumab, cetuximab, panitumumab, nimotuzumab, nexituzumab, elevantuzumab, telisotuzumab, enfortumab, vobramitamab, mirzotamab, deparetuzumab, morotumab, caputuzumab, belintoomeb, and others. Mirvetuximab, Belantuzumab, Atoxizumab, Rituximab, Lovatozumab, Talatuzumab, Clivatuzumab, Sontuzumab, Gatipotuzumab, Vofatamab, Atezolizumab, Averumab, Duvalumab, Duvalimumab, Atezolizumab, Zilovertamab, Tisotumab, Denintuzumab, Inotuzumab, Moxetumomab, Brentuximab, Vorsetuzumab, Polatuzumab, Iladatuzumab, Burosumab, Bemattouzumab, Efruxifermin, Anetumab, Amatuximab, MEDI9447, Adalimumab, Infliximab, Golimumab, Ziralimumab, ATG-031, Daraerumumab, Daraetumumab, Letaplimab, Lezolimumab, Lefalimumab, Tusamitamab, Labetuzumab, IMAB027, Tremelimumab, Zeflimumab, Botensilimab, Numulimab, Ipilimumab, Iprimumab, Tigatuzumab, Lexatumumab, Vofatamab, Talquetamab, Bersanlimab, Enlimo mab, Inolimomab, Ozuriftamab, Ladiratuzumab, Gemtuzumab, Camidanlumab, Nirsevimab, Felvizumab, Bevacizumab, Ramosinumab, Ranibizumab, Denosumab, Denizumab, Trimelimumab, Zeflimumab, Zeflimumab, Alemumab, Pallizumab, Nivolumab, Pembrolizumab, Nivolumab, Pembrolizumab, Tripeptide Rimab, Sintilimab, Camrelizumab, Lesabelimab, Omalizumab, Tocilizumab, Ustenolimab, Baliximab, Ikuzumab, Brolinizumab, Ikizumab, Secukinizumab, Daliximab, Elotuzumab, Emexizumab, Iometuzumab, Ivolucrata, Emexizumab, Belimumab, Alicizumab, Gusekizumab, Vedolizumab, Duprelimin, Lanalimumab, Broshulimab, Sattelizumab.

[0037] In some embodiments of the present invention, the antibody is trastuzumab.

[0038] In some embodiments of the present invention, the antibody is trastuzumab, which is the second aspect of the present invention.

[0039] In some embodiments of the present invention, the drug is selected from MMAE, MMAF, Dxd, Dx8951, SN38, DM1, DM4, PBD, PBD dimer, eribulin, docamycin, muscarinic acid, vincristine, vinorelbine, VP-16, camptothecin, paclitaxel, docetaxel, epothilone A, epothilone B, olaparib, nocodazole, colchicine, estradiol, simadastin, ilexeloselin, or derivatives of the above drugs.

[0040] In some embodiments of the present invention, the drug is Dxd, with the structural formula [insert structural formula here]. .

[0041] In some embodiments of the present invention, the linker includes a PEG chain, a carbon chain, or a pyrolytic linker.

[0042] In some embodiments of the present invention, the connector is PEG4.

[0043] In some embodiments of the present invention, the sugar structure is selected from monosaccharides, disaccharides, oligosaccharides, or branched sugar structures.

[0044] In some embodiments of the present invention, the reactive functional group is selected from azide group, tetrazine group, TCO group, alkynyl structure, aldehyde structure, carbonyl structure, hydroxylamine structure, isothiocyanate, amino, carboxyl, mercapto, alkenyl, maleimide, and hydrazone structure.

[0045] In some embodiments of the present invention, the DAR value of the antibody-drug conjugate is 1-6, such as 2, 4 or 6.

[0046] The antibody-drug conjugate has the structures shown in Formula I, Formula II, and Formula III as follows:

[0047] Formula I

[0048] Formula II

[0049] Formula III In the above structure, the Y-shaped structure represents trastuzumab. It represents N-acetylgalactosamine. It represents acetamide glucose. It means mannose. It represents fucose. Indicates reactive functional groups, Indicates connector, Indicates medicine.

[0050] In some embodiments of the present invention, the antibody-drug conjugate has the structure shown in formulas a, b, and c:

[0051] Formula a

[0052] Formula b

[0053] Formula c.

[0054] A fourth aspect of the present invention provides a method for preparing the antibody-drug conjugate of the third aspect of the present invention. For formula a, the preparation method includes mixing the antibody, α1,6-fucosyltransferase and sugar structure-reactive functional group complex 1 into a buffer solution, reacting to obtain a glycan-mediated site-directed antibody-reactive functional group conjugate with antibody glycosylation site modified reactive functional group; adding a toxic drug-reactive functional group complex, reacting again to obtain an antibody-drug conjugate; For formula b, the preparation method includes mixing the antibody, β1,4-galactosyltransferase and glycostructure-reactive functional group complex 2 into a buffer solution, reacting to obtain a glycan-mediated site-directed antibody-reactive functional group conjugate with antibody glycosylation site modified reactive functional group; adding a toxic drug-reactive functional group complex, reacting again to obtain an antibody-drug conjugate; For formula c, the preparation method includes mixing the antibody, α1,6-fucosyltransferase, and sugar structure-reactive functional group complex 1 in a buffer solution, performing a first reaction to obtain glycan site-directed antibody-reactive functional group conjugate 1 with antibody glycosylation site modified reactive functional group; adding β1,4-galactosyltransferase and sugar structure-reactive functional group complex 2, performing a second reaction to obtain glycan site-directed antibody-reactive functional group conjugate 2 with antibody glycosylation site modified reactive functional group; adding a toxic drug-reactive functional group complex, performing a third reaction to obtain the antibody-drug conjugate.

[0055] In some embodiments of the present invention, the sugar structure-reactive functional group complex 1 is GDP-Fuc-AM-AZ, with the following structural formula: .

[0056] In some embodiments of the present invention, the sugar structure-reactive functional group complex 2 is UDP-GalNAz, with the following structural formula: .

[0057] In some embodiments of the present invention, the toxic drug-reactive functional group complex is Propargyl-PEG4-GGFG-DXd, with the following structural formula:

[0058] In some embodiments of the present invention, the amino acid sequence of the α1,6-fucosyltransferase is shown in SEQ ID NO:15.

[0059] In some embodiments of the present invention, the amino acid sequence of the α1,6-fucosyltransferase is shown in SEQ ID NO:18.

[0060] In some embodiments of the present invention, during the preparation of formula a, the reaction conditions are incubation at 35-38°C for 20-26 hours.

[0061] In some embodiments of the present invention, during the preparation of formula a, the condition for the second reaction is incubation at room temperature for 3-5 h.

[0062] In some embodiments of the present invention, during the preparation of formula a, the mass ratio of the antibody, α1,6-fucosyltransferase and sugar structure-reactive functional group complex 1 is (14-20):(1-5):1; further, (15-18):(1-4):1; and even further, (16-17):(2-3):1.

[0063] In some embodiments of the present invention, the buffer solution used in the preparation of formula a includes PBS.

[0064] In some embodiments of the present invention, during the preparation of formula a, the toxic drug-reactive functional group complex solution further includes aminoguanidine, THPTA (trihydroxypropyltriazine), CuSO4 and sodium ascorbate.

[0065] In some embodiments of the present invention, during the preparation of formula a, the mass ratio of the antibody to the toxic drug-reactive functional group complex is (150-165):1; further, (155-165):1; and even further, (158-160):1.

[0066] In some embodiments of the present invention, during the preparation of formula b, the reaction conditions are incubation at 35-38°C for 34-38 h.

[0067] In some embodiments of the present invention, during the preparation of formula b, the condition for the second reaction is incubation at room temperature for 3-5 h.

[0068] In some embodiments of the present invention, during the preparation of formula b, the mass ratio of the antibody, β1,4-galactosyltransferase and sugar structure-reactive functional group complex 2 is (35-45):1:(3-8); further, it is (36-40):1:(4-7); and even further, it is (39-40):1:(5-6).

[0069] In some embodiments of the present invention, during the preparation of formula b, the buffer solution is TBS containing MnCl2. The final concentration of MnCl2 in the buffer solution is 4-6 mM.

[0070] In some embodiments of the present invention, during the preparation of formula b, the toxic drug-reactive functional group complex solution further includes aminoguanidine, THPTA (trihydroxypropyltriazine), CuSO4 and sodium ascorbate.

[0071] In some embodiments of the present invention, during the preparation of formula b, the mass ratio of the antibody to the toxic drug-reactive functional group complex is (45-60):1; further, (50-60):1; and even further, (55-56):1.

[0072] In some embodiments of the present invention, during the preparation of formula c, the conditions for the first reaction are incubation at 35-38°C for 20-26 h.

[0073] In some embodiments of the present invention, during the preparation of formula c, the conditions for the second reaction are incubation at 35-38°C for 34-38 h.

[0074] In some embodiments of the present invention, during the preparation of formula c, the second reaction is carried out under the condition of incubation at room temperature for 3-5 h.

[0075] In some embodiments of the present invention, during the preparation of formula c, the mass ratio of the antibody, α1,6-fucosyltransferase and sugar structure-reactive functional group complex 1 is (14-20):(1-5):1; further, (15-18):(1-4):1; and even further, (16-17):(2-3):1.

[0076] In some embodiments of the present invention, during the preparation of formula c, the mass ratio of the antibody, β1,4-galactosyltransferase and sugar structure-reactive functional group complex 2 is (50-60):1:(5-10); further, it is (50-58):1:(6-8); and even further, it is (54-55):1:(7-8).

[0077] In some embodiments of the present invention, the buffer solution used in the preparation of formula c is PBS.

[0078] In some embodiments of the present invention, during the preparation of formula c, the toxic drug-reactive functional group complex solution further includes aminoguanidine, THPTA (trihydroxypropyltriazine), CuSO4 and sodium ascorbate.

[0079] In some embodiments of the present invention, during the preparation of formula c, the mass ratio of the antibody to the toxic drug-reactive functional group complex is (50-65):1; further, (55-60):1; and even further, (58-59):1.

[0080] In some embodiments of the present invention, the preparation method further includes a purification step, wherein the purification includes a purification step using Protein A magnetic beads.

[0081] A fifth aspect of the present invention provides the use of the recombinant cells of the first aspect of the present invention, the tocilizumab of the second aspect of the present invention, or the antibody-drug conjugate of the third aspect of the present invention in the preparation of antitumor drugs.

[0082] In some embodiments of the present invention, the tumor includes at least one of gastric adenocarcinoma, esophageal adenocarcinoma, colorectal cancer, bile duct cancer, gallbladder cancer, gastric cancer, lung cancer, cholangiocarcinoma, bladder cancer, esophageal cancer, melanoma, ovarian cancer, liver cancer, prostate cancer, pancreatic cancer, small bowel cancer, head and neck cancer, uterine cancer, cervical cancer, brain cancer, and breast cancer.

[0083] The beneficial effects of this invention are: This invention provides a recombinant cell capable of efficiently and stably producing homogeneous A2 glycoform trastuzumab. Based on this trastuzumab, using α1,6-fucosyltransferase and / or β1,4-galactosyltransferase, two, four, or six azide groups can be introduced at specific sites on the antibody, and further linked to a toxin molecule, thereby preparing homogeneous antibody conjugates with DAR values ​​of 2, 4, or 6.

[0084] This invention provides a method for preparing antibody-drug conjugates (ADCs). This method allows for efficient glycosylation conjugation of antibody substrates produced from engineered cells in vitro using FUT8 and GALT, ultimately yielding site-specific, homogeneous ADCs with controllable DAR values ​​(2, 4, 6) via a Click reaction. This effectively addresses the instability in production and clinical efficacy caused by the heterogeneity of ADC drugs. This method also has broader applications; by changing different toxic small molecules and varying the amount of conjugated drugs, ADCs can achieve more ideal pharmacodynamic and pharmacokinetic parameters, playing a positive role in the further exploration and development of ADCs. It overcomes the limitations of current glycosylation conjugation schemes in controlling and optimizing DAR values, providing a novel approach for optimizing DAR values ​​in the development of different ADCs. Attached Figure Description

[0085] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein: Figure 1 The diagram shows the structural schematic of trastuzumab with FUT8 and B4GALT1 knocked out (A) and the construction process of antibody-drug conjugates with different DAR values ​​(B). In the figures, Representative: trastuzumab; Represents fucose, Represents N-acetylgalactosamine; Represents PEG chains, carbon chains, and pyrolytic linkers; Selected from azide group, tetrazine group, TCO group, alkynyl structure, aldehyde structure, carbonyl structure, hydroxylamine structure, isothiocyanate, amino, carboxyl, mercapto, alkenyl, maleimide, and hydrazone structure; This refers to toxic drugs.

[0086] Figure 2 This is a plasmid map of the vector plasmid HP180.

[0087] Figure 3 FOR CHO FUT8 - / - Cellular FUT8 active site gene sequencing results (A) and CHO FUT8 - / - / B4GALT1 - / - Sequencing results of the B4GALT1 active site gene in cells (B).

[0088] Figure 4 FOR CHO FUT8 - / - / B4GALT1 - / - Results of Fc glycoform analysis in transient cell transformation.

[0089] Figure 5 FOR CHO FUT8 - / - / B4GALT1 - / - Results of transient cell Fc mass spectrometry analysis.

[0090] Figure 6 SDS-PAGE and mass spectrometry analysis of the FUT8 and B4GALT1 double knockout trastuzumab prepared in Example 1.

[0091] Figure 7 The results of SDS-PAGE analysis of recombinant FUT8 enzyme are shown.

[0092] Figure 8 The results of SDS-PAGE analysis of recombinant GALT (Y289L) enzyme.

[0093] Figure 9 This is a schematic diagram of the preparation process of Tras-F-2DXD, an antibody-drug conjugate with a DAR of 2.

[0094] Figure 10 This is a schematic diagram of the structure of Tras-F-2DXD, an antibody-drug conjugate with a DAR of 2.

[0095] Figure 11 This is the heavy chain mass spectrum of Tras-F-2DXD, an antibody-drug conjugate with a DAR of 2.

[0096] Figure 12 The results of SDS-PAGE analysis of the antibody-drug conjugates Tras-F-2DXD, Tras-G-4DXD, and Tras-Fg-6DXD are shown.

[0097] Figure 13 This is a schematic diagram of the preparation process of Tras-G-4DXD, an antibody-drug conjugate with a DAR of 4.

[0098] Figure 14 This is a schematic diagram of the structure of Tras-G-4DXD, an antibody-drug conjugate with a DAR of 4.

[0099] Figure 15 This is the heavy chain mass spectrum of Tras-G-4DXD, an antibody-drug conjugate with a DAR of 4.

[0100] Figure 16 This is a schematic diagram of the preparation process of Tras-Fg-6DXD, an antibody-drug conjugate with a DAR of 6.

[0101] Figure 17 This is a schematic diagram of the structure of Tras-Fg-6DXD, an antibody-drug conjugate with a DAR of 6.

[0102] Figure 18 The image shows the heavy chain mass spectrum of Tras-Fg-6DXD, an antibody-drug conjugate with a DAR of 6.

[0103] Figure 19 The values ​​represent ADC cell binding and average fluorescence intensity.

[0104] Figure 20 For NCI-N87 (HER) 2 + ELISA of cells yielded saturation binding curves.

[0105] Figure 21 The anti-cytotoxic effects of various antibody-drug conjugates on Calu3 cells were studied, with TF-DXD showing the highest IC50 value. 50 The IC of TG-DXD is 4.62 nM. 50 The IC of TFG-DXD is 1.32 nM. 50 It is 0.31 nM.

[0106] Figure 22 The anti-cytotoxic effects of various antibody-drug conjugates on NCI-N87 cells were studied, with TF-DXD showing the highest IC50 value. 50 The IC of TG-DXD is 4.02 nM. 50 The IC of TFG-DXD is 1.72 nM. 50 It is 0.35 nM.

[0107] Figure 23The anti-cytotoxic effects of various antibody-drug conjugates on SK-BR-3 cells were studied, with TF-DXD showing the highest IC50 value. 50 The IC of TG-DXD is 5.48 nM. 50 The IC of TFG-DXD is 0.77 nM. 50 It is 0.41 nM.

[0108] Figure 24 The cytotoxic effects of various antibody-drug conjugates on MCF-7 cells. Detailed Implementation

[0109] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0110] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0111] The objective of this invention is to first obtain a natural antibody with a uniform A2 glycoform, then use an in vitro recombinase-catalyzed coupling reaction to couple azide-modified fucose or galactose to the glycan chain, and finally couple an alkyne-modified toxin molecule to the glycan chain via a "Click" reaction, thereby achieving site-specific and uniform DAR ADC synthesis. Figure 1 The specific steps include: (1) sgRNAs containing gene sequences targeting α1,6-fucosyltransferase (FUT8) and β1,4-galactosyltransferase (B4GALT1) were constructed for gene knockout experiments based on the CRISPR-Cas9 system. Single-clonal cell lines successfully knocking out FUT8 and B4GALT1 (named CHOFUT8) were screened by liposome transfection, flow cytometry sorting, and genome sequencing. - / - / B4GALT1 - / - ); (2) The plasmid encoding the monoclonal antibody Trastuzumab was stably transfected into the obtained CHO FUT8 using electroporation transfection technology. - / - / B4GALT1 - / - From cell lines, monoclonal strains with high expression levels are screened out; antibodies are produced and purified. Figure 1 (A) (3) In vitro, azide-modified fucose or galactose is coupled to the antibody glycan chain using recombinant enzymes FUT8 or GALT (Y289L) or simultaneously using FUT8 and GALT1. Then, the toxin molecule DXD (Propargyl-PEG4-GGFG-DXd) with alkynyl modification is coupled to the glycan chain through a "Click" reaction. Figure 1 (Middle B) (4) The obtained ADC molecules were characterized for molecular weight, uniformity, and aggregation stability, and their activity was evaluated. Structural characterization mainly included SDS-PAGE and LC-MS, while activity characterization mainly included HER2 binding assay (ELISA / FlowCytometry), cytotoxicity assay (CCK8), and in vivo toxicity assay.

[0112] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0113] Example 1: Construction of the natural antibody Trastuzumab with a homogeneous A2 glycoform 1. Construction of FUT8 and B4GALT1 knockout cell lines (1) Constructing gene knockout plasmids based on CRISPR Cas9 Synthesize the sixth exon of the FUT8 gene (Gene ID: 100751648) (nucleotide sequence: gtgaagtgaaggacaaaaatgttcaagtggtcgagctccccattgtagacagcctccatcctcgtcctccttacttacccttggctgtaccagaagaccttgcagatcgactcctgagagtccatggtgatcctgcagtgtggtgggtatcccagtttgtcaaatacttgatccgtccacaaccttggctggaaagggaaatagaagaaaccaccaagaagcttggcttcaaacatccagttattgg, SEQ ID NO:1) and the B4GALT1 gene (Gene ID: The sgRNA sequence of the first exon of the FUT8 gene (SEQ ID NO:2) is F: GATCCGTCCACAACCTTGGC (SEQ ID NO:3), R: gccaaggttgtggacggatc (SEQ ID NO:3). The sgRNA sequence designed to target the sixth exon of the FUT8 gene is F: GATCCGTCCACAACCTTGGC (SEQ ID NO:3), R: gccaaggttgtggacggatc (SEQ ID NO:3). NO:4); The sgRNA sequence designed targeting the first exon of the B4GALT1 gene is F: GGGCGGTCGTTATTCCCCCA (SEQ ID NO:5), R: TGGGGGAATAACGACCGCCC (SEQ ID NO:6)). The synthesized gRNA was annealed, digested and ligated, amplified, and sequenced to construct the vector plasmid HP180 (the plasmid contains the sgRNA scaffold, the Cas9 protein-coding gene, and the green fluorescent protein-coding gene; the plasmid map is shown below). Figure 2As shown, this plasmid is the same as the HP180_px330_GFP plasmid in the literature "HUOMengfei, MENG Fanming, WANG Sutian, et al. Construction and functional validation of CRISPR / Cas9 vector targeting pig Y chromosome cutting[J]. Journal of South China Agricultural University, 2023, 44(2): 187-196. DOI:10.7671 / j.issn.1001-411X.202201022", respectively, to obtain CRISPR-Cas9 knockout plasmids targeting exons of FUT8 and B4GALT1 genes. The annealing reaction system and procedure are shown in Tables 1 and 2. The enzyme digestion and ligation reaction system is shown in Table 3, and the reaction conditions are 37℃ for 2-4 h. DH5α competent cells were thawed on ice, the enzyme ligation product was gently added, and the cells were incubated on ice for 30 min. After heat shock in a 42℃ water bath for 60 s, add 0.5 mL of LB medium and incubate at 37℃ with shaking at 200 rpm for 45 min. Spread 100 μL of bacterial culture evenly onto LB agar plates containing the appropriate antibiotic (carbenicillin, 100 μg / mL) and incubate upside down at 37℃ for 12–16 h. Pick a single colony and inoculate it into 5 mL of LB medium containing antibiotic, incubate at 37℃ with shaking for 6–8 h, and then send it to a sequencing company.

[0114] Table 1 Annealing reaction system

[0115] Table 2 Annealing Reaction Procedure

[0116] Table 3 Enzyme digestion and ligation reaction system

[0117] (2) Gene knockout plasmid transfection of CHO cells First, CHO cells in the logarithmic growth phase were... 6 Inoculate at a density of 2 mL / well in 6-well plates (2 × 10⁶ m² / mL). 6 In cells), the specific procedure is as follows: trypan blue staining is used to count the density of CHO cells, and 2 × 10⁻⁶ cells are taken. 6Cells were added to a new 1.5 mL EP tube, centrifuged at low speed (200 g, 3 min), and the supernatant was discarded. After washing the cells with 500 μL PBS, the cells were resuspended in 1.8 mL of culture medium (GibcoOptiPRO™ SFM + 2 v / v% Glutamax (Gibco, catalog number 35050061), preheated) and transferred to 6-well plates for culture (37℃ shaker, 5% CO2). Transfection was performed after 24 h, as follows: 2 μg of the gene knockout plasmid constructed in step (1) was added to 200 μL of preheated OPTI-SFM medium, and the diluted plasmid was added to 4 μL of Fectopro transfection reagent (Sartorius, catalog number 101000014) and mixed. The mixture was then incubated at room temperature for 10 min. The mixed culture medium was then evenly added to the seeded 6-well plates of cells and incubated for 48 h (37℃ shaker, 5% CO2).

[0118] (3) Monoclonal screening Transfected cells were transferred to 15 mL centrifuge tubes, centrifuged at low speed (200 g, 3 min), and the supernatant was discarded. Cells were washed once with 500 μL PBS, and then resuspended in 500 μL PBS. The resuspended cells were filtered through a cell strainer into flow cytometry tubes. Single positive cells with moderate green fluorescence were collected using a flow cytometer and placed into each well of a 96-well plate. Each well contained 200 μL of CHO Fusion serum-free media (Sigma, catalog number 14365C) ​​+ 20 v / v% FetalBovine Serum + 2 v / v% Glutamax + 1 v / v% pen / strep medium. The collected monoclonal cells were cultured in a cell culture incubator for approximately 14 days. Clumps of cells were broken up, and two-thirds of the cells were transferred to a 96-well PCR plate, while the remaining one-third was cultured further. DNA templates were extracted from cells transferred to a 96-well PCR plate as follows: 100 μL of alkaline lysis buffer (25 mM NaOH, 0.2 mM EDTA) was added to each well, and the cells were incubated at 98°C for 45 min for lysis. Then, an equal volume of acid (40 mM Tris-HCl) was added for neutralization to obtain the DNA template from the monoclonal cells. PCR amplification of the target gene fragment (covering the gRNA target site) was performed using the primer pairs in Table 4, followed by Sanger sequencing. The two allele knockout monoclonal cells, CHO FUT8, were used for further analysis. - / - CHO B4GALT1 - / - (genotype such as) Figure 3 (As shown) to carry out expanded culture and preservation.

[0119] Table 4 Primers for PCR amplification of target gene fragments

[0120] (4) Verification of glycoform in gene knockout monoclonal cell lines The expression plasmid containing the antibody Fc fragment (IGHG1, Gene ID: 3500) (named pCGS3-Fc, the plasmid sequence contains the EF-1α promoter, the glutamine synthase encoding gene, which has been disclosed in Chinese invention patent CN116478948A) was used. Fc fragment expression was performed using Fectopro transfection reagent (same procedure as step (2)) to transfect wild-type CHO cell lines, FUT8 knockout CHO cell lines, and B4GALT1 knockout CHO cell lines. The antibody Fc fragment was purified using Protein A+G affinity chromatography resin. The specific procedure was as follows: After high-speed centrifugation of the cell culture medium, the supernatant was collected and filtered through a 0.22 μm filter membrane to obtain the mother liquor. Affinity chromatography was performed using a Beyotime Protein A+G Agarose column. The equilibration buffer was PBS solution. The column was washed and equilibrated with 10 column volumes of PBS. The mother liquor was then loaded onto the column using a constant flow pump. After the mother liquor passed through the column, the column was washed with 10 column volumes of 1 M NaCl to remove unbound and non-specifically bound proteins. After washing, the bound antibody was eluted with 100 mM Glycine-HCl (pH 2.7), and the protein concentration was calculated by measuring the absorbance of the eluent at 280 nm. The eluent was neutralized with Tris-HCl (pH 9.0) (30 μL of neutralizing solution added to 1 mL of eluent), followed by solution displacement and concentration of the protein using a 10 kD ultrafiltration tube. Finally, the protein was stored in PBS.

[0121] The purified Fc fragment was replaced with 50 mM TEAB for glycoform analysis. The specific procedure was as follows: PNGase F glycosidase (Wuhan HanHai New Enzyme Biotechnology Co., Ltd., catalog number HH6901) was added to the Fc solution to digest the Fc fragment (1 μL of enzyme per 60 μg protein, overnight digestion at 37℃) to release N-glycans. The protein and glycans were then separated and purified using a C8-C18 membrane to obtain the N-glycans. The glycans were labeled with procainamide, and further purified using an NH2 column to obtain procainamide-labeled N-glycans. The purified N-glycans were then separated and detected using UPLC with a HILIC column and a fluorescence detector.

[0122] The specific experimental steps are as follows: a) Add the purified Fc fragment (80 μg) elution buffer to a 0.5 mL ultrafiltration tube with a molecular weight cutoff of 10 KD, centrifuge at 14000 g for 7 min, discard the effluent, add 0.5 mL of 50 mM TEAB buffer, centrifuge at 14000 g for 7 min, and discard the effluent.

[0123] b) Repeat the addition of 50 mM TEAB buffer and centrifuge 5 times to concentrate the Fc fragment in 50-100 μL of 50 mM TEAB buffer.

[0124] c) PNGase F glycosidase cleavage to release glycans: Add 1 μL of PNGase F glycosidase to the concentrated Fc fragment, incubate overnight at 37°C, then add 0.5 μL of PNGase F glycosidase and continue incubation at 50°C for 1 h to allow full release of glycans.

[0125] d) Stage-Tip desalting columns made using C18 and C8 membranes were used to separate proteins from glycans, obtaining a glycan solution. The solvent was then removed using a centrifugal concentrator to obtain evaporated glycans. The specific procedure is as follows: Stage-Tip desalting columns were made by sequentially fabricating C18, C8, C18, and C8-C18 membranes. Column rinsing: 200 μL of acetonitrile was added, and the column was centrifuged at 3000 g for 3 min. The effluent was discarded, and the rinsing was repeated twice. Second washing: 200 μL of 85% acetonitrile was added, and the column was centrifuged at 3000 g for 3 min. The effluent was discarded, and the washing was repeated twice. Column equilibration: 200 μL of 0.1% FA was added, and the column was centrifuged at 3000 g for 3 min. The effluent was discarded, and the washing was repeated twice. Sample loading: Transfer the enzyme-digested solution obtained in step c) to the column, centrifuge at 3000 g for 3 min, collect the eluent, repeat the loading once, collect the eluent and store it. Elution: Add 200 μL of 0.1% FA, centrifuge at 3000 g for 3 min, collect the eluent, repeat the elution once and collect the eluent again. Transfer the two collected eluents to the same tube, evaporate the solvent in a centrifugal concentrator to obtain the glycans.

[0126] e) Add 10 μL of a mixture of 0.4 M procainamide and 1 M 2-methylpyridine-N-methylborane to the sugar chain and react at 65 °C for 1 h to label the sugar chain with procainamide.

[0127] f) Purification of procainamide-labeled glycans using a Sep-Pak NH2 amino solid-phase extraction column. The specific procedure is as follows: Fix the Sep-Pak NH2 amino solid-phase extraction column onto a rack, add 1 mL of acetonitrile to rinse the column, repeating 5 times. Add 1 mL of 85% acetonitrile to equilibrate the column, repeating 2 times. Loading: Load the procainamide-labeled glycan solution from the previous step onto the column. Washing: Add 1 mL of 85% acetonitrile to equilibrate the column, repeating 2 times to wash away as many other impurities as possible. Elution: Add 100 μL of 100 mM ammonium acetate solution containing 5% acetonitrile to elute the glycans, collect the eluent, repeating 3 times to collect a total of 300 μL of eluent. Remove the solvent by centrifugation and rotary evaporation to obtain procainamide-labeled glycans.

[0128] g) Add 20 μL of ultrapure distilled water to reconstitute the sugar chain, and centrifuge at 21000 g for 20 min at 4℃.

[0129] h) Pipette 10 μL of the supernatant solution of the sugar chain into a 1.5 mL centrifuge tube, add 20 μL of HPLC-grade acetonitrile and 10 μL of LDM to prepare the injection solution, and inject it into UPLC to characterize the sugar form. The UPLC chromatographic conditions are shown in Table 5.

[0130] Table 5 UPLC Chromatographic Conditions

[0131] like Figure 4 As shown, the N-glycoforms of the CHO-WT cell line mainly include G0F, G1F, and G2F; CHO FUT8 - / - The elution time of the N-glycan glycan chain in the CHO-WT cell line was 2 minutes earlier than that in the CHO-WT cell line, indicating that the loss of fucose led to a decrease in the hydrophilicity of the glycan chain, thus confirming that the CHO-WT cell line... - / - The FUT8 gene is lost in the monoclonal cell line.

[0132] The purified Fc fragment was replaced with mass spectrometry-grade water and then analyzed by mass spectrometry: the protein molecules were determined using liquid chromatography-mass spectrometry (LC-MS) by Thermo Scientific Orbitrap Exploratory. TM A 240 mass spectrometer was used, equipped with an RP column (MAbPaCTM RP, 4 μm, 2.1 × 50 mm). Figure 5 As shown, CHO FUT8 - / - The overall mass of Fc protein expressed in the CHO FUT8 cell line was reduced by approximately 292.4 Da compared to the CHO WT cell line. Theoretically, the reduction of two fucose glycosides at both ends of the Fc glycosides would lead to a mass decrease of 292.32 Da, indicating that the CHO FUT8...- / - The FUT8 gene in the cell line is not functioning.

[0133] (5) Obtain FUT8 / CIGALT1 double gene knockout cell lines Transfect the HP180 plasmid containing B4GALT1-sgRNA into the FUT8 knockout monoclonal CHO cell line (construction process is described in step (1)). Repeat steps (2)-(3) above to obtain the FUT8 and B4GALT1 double gene knockout monoclonal cell line CHO FUT8. - / - / B4GALT1 - / - (genotype such as) Figure 3 As shown), and UPLC glycoform analysis (detection process same as step (4)) revealed CHO FUT8 - / - / B4GALT1 - / - N-glycans in cell lines ( Figure 4 The peak shape completely disappeared after 10 minutes, indicating that the sugar chain no longer contains galactose, confirming CHO FUT8. - / - / B4GALT1 - / - The B4GALT1 gene in the monoclonal cell line is loss of function. Mass spectrometry results are shown below. Figure 5 As shown, CHO FUT8 - / - / B4GALT1 - / - All galactose-containing peaks in the Fc protein of the cell line disappeared, confirming the loss of function of FUT8 and B4GALT1.

[0134] 2.CHO FUT8 - / - / B4GALT1 - / - Screening of stable trastuzumab monoclonal cell lines (1) A plasmid expressing trastuzumab (pcGS-Trastuzumab) was constructed. This plasmid contains elements such as the trastuzumab light chain protein encoding gene, the trastuzumab heavy chain protein encoding gene, the glutamine synthase encoding gene, and the EF-1α promoter. The expression sequence of trastuzumab was optimized for CHO cell expression based on DrugBank (ID DB00072). The specific construction process is as follows: 1) Trastuzumab was searched on the DrugBank website to determine the amino acid sequence of the IgG antibody. The gene sequence was synthesized and optimized by Beijing Qingke Biotechnology Co., Ltd., resulting in plasmids pUC57-Trastuzumab-LC-Crigr and pUC57-Trastuzumab-HC-Crigr.

[0135] 2) Based on the synthesized gene sequence, primers Trastuzumab-HC-F (5'-aaaacaagcttgccaccatgg-3', SEQ ID NO:11) and Trastuzumab-HC-R (5'-aaaagaattccctcacttgccgggag-3', SEQ ID NO:12) were designed and synthesized.

[0136] 3) Prepare the reaction system according to Table 6. Double digest the vector plasmid pCGS3-Fc-Fc-GS (i.e., pCGS3-Fc) and the plasmid pUC57-Trastuzumab-LC-Crigr containing the trastuzumab light chain using NotI-HF and XhoI restriction endonucleases (37℃, 4h). Use a 0.8% agarose gel to determine the target band. Purify and recover the enzyme products using a DNA gel recovery kit to obtain the digested linear vector plasmid and antibody light chain fragment. Prepare the enzyme ligation reaction system according to Table 7. Ligate the digested linearized vector and trastuzumab light chain using T4 DNA ligase.

[0137] Table 6. Double enzyme digestion reaction system

[0138] Table 7 Enzyme-linked reaction system

[0139] 4) After transformation, plating of bacterial culture, overnight culture at 37°C, selection and amplification of single clones, and sequencing of bacterial culture, the above enzyme reaction solution was successfully used to construct an IgG antibody LC fragment expression plasmid, named pCGS-Trastuzumab-LC-Crigr.

[0140] 5) Using pUC57-Trastuzumab-HC-Crigr as a template, the PCR reaction system was prepared according to Table 8 for amplification. The target band was determined using a 0.8% agarose gel, and the enzyme product was purified and recovered using a DNA gel extraction kit. Table 8 PCR Reaction System

[0141] 6) Prepare the reaction system according to Table 9. Perform a double digestion reaction using HindIII and EcoRI on the PCR product (i.e., the antibody HC fragment) of the vector plasmid pCGS-Trastuzumab-LC-Crigr with the HC fragment extracted using primers Trastuzumab-HC-F and Trastuzumab-HC-R. Run the gel on a 0.8% agarose gel to determine the target band, and purify and recover the enzyme product using a DNA gel recovery kit. This yields the linearized vector and the trastuzumab heavy chain fragment.

[0142] Table 9. Double enzyme digestion reaction system

[0143] 7) Prepare the enzyme ligation reaction system according to Table 10, and use T4 DNA ligase to ligate the linearized vector and trastuzumab heavy chain fragment after enzyme digestion.

[0144] Table 10 Enzyme-linked reaction system

[0145] After transformation, bacterial culture plasmid plasmid plasmid was plated, cultured overnight at 37 °C, single clone amplification, and bacterial culture sequencing, the above enzyme reaction solution was successfully constructed and named pcGS-Trastuzumab.

[0146] The amino acid sequence encoding the Trastuzumab light chain (LC) in the plasmid pcGS-Trastuzumab is: DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:13).

[0147] The amino acid sequence encoding the Trastuzumab heavy chain (HC) in this plasmid is: EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSN TKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:14).

[0148] (2) The pcGS-Trastuzumab plasmid was introduced into CHO FUT8 using an Invitrogen Neon MPK3000 electroporation system. - / - / B4GALT1 - / - In the cells, the specific operation is as follows: First, 4×10 6 One cell line was seeded into a 6-well plate containing 2 mL of complete culture medium (95% EX-CELL® CD CHO Fusion (Sigma, catalog number 14365C) ​​+ 4 v / v% Glutamax + 1 v / v% P / S) and cultured for 24 h before transfection. 3 × 10⁶ cells were then centrifuged in centrifuge tubes. 6After mixing each cell with 6-9 μg of plasmid, electroporation was performed once at 1620 V, 10 ms, and 3 Pulzes. The cells were then transferred to 6-well plates containing 2 mL of complete culture medium (95% EX-CELL® CD CHO Fusion (Sigma, catalog number 14365C) ​​+ 4 v / v% Glutamax + 1 v / v% P / S) and incubated at 37°C in a 5% CO2 humidified incubator. After 48 h, cells were seeded at 2000 cells / well in 96-well plates for glutamine-free culture to screen for stable cell pools. After 10-14 days, the supernatant from the surviving cell pools was used for ELISA to screen for trastuzumab-overexpressing cell pools. The specific ELISA procedure was as follows: 10 μL of the supernatant from the surviving cell pools was added to a 96-well plate, followed by 40 μL of Coating Buffer (50 mM sodium bicarbonate solution) and overnight incubation at 4°C to coat the protein. The next day, the plate was washed three times with 0.05% PBST buffer (200 μL / well / wash) and the solution was removed. The plate was then sealed by incubation in 3% BSA-buffered PBS (200 μL / well) at room temperature for 1 h, followed by washing as described above. Secondary antibody solution (Goat Anti-Human IgG H& (HRP) (MCE, catalog number HY-P83662)) (1:5000 dilution, 50 μL / well) was added and incubated at room temperature for 1 h, followed by washing as described above. TMB chromogenic solution (50 μL / well) was added and incubated at room temperature for 30 min, followed by termination of the reaction with 0.5 M sulfuric acid solution (50 μL / well). The absorbance at 450 nm was compared in each well of the 96-well plate to select cell lines with high expression levels. The selected high-expression cell lines were then subjected to monoclonal screening as described above, repeating the ELISA steps until monoclonal cell lines with high expression levels were selected.

[0149] After antibody purification, SDS-PAGE was used to detect the expression of the protein's light and heavy chains. Disulfide bonds between the antibodies were reduced using DTT. LC-MS analysis revealed that the heavy chain molecular weight was 50446.2 Da and the light chain molecular weight was 23436.27 Da. Figure 6 The light and heavy chain quality is consistent with that of currently commercialized trastuzumab, proving that the obtained antibody is trastuzumab, that is, a trastuzumab monoclonal antibody with a uniform A2 glycoform is obtained.

[0150] Example 2: Plasmid construction, expression, and purification of recombinant human FUT8 and GALT enzymes 1. Expression and purification of recombinant human FUT8 enzyme After PCR, the gene encoding the human FUT8 protein fragment (NCBI Gene ID: 2530, 32-575) was ligated into the expression vector pSecTag2 A (which contains a T7 promoter and a 6×His tag) to obtain the plasmid pSec-hsFUT8, which was used to express the human FUT8 enzyme with the His6 tag in eukaryotic cells.

[0151] The amino acid sequence encoding the human FUT8 protein fragment (32-575) in plasmid pSec-hsFUT8 is: (SEQ ID NO:15).

[0152] Specific steps for constructing plasmid pSec-hsFUT8: Purchase FUT8 expression plasmid pCMV-FUT8(huaman)-6XHis-Neo (catalog number P50735) from Wuhan Miaoling Biotechnology Co., Ltd., synthesize primer fragments FUT8-F1 (5'-AAAAGGATCCtgataatgaccatcctgatcactctagcc-3', SEQ ID NO:16) and FUT8-R1 (5'-AAAACTCGAGCtttctcagcctcaggatatgtgg-3', SEQ ID NO:17), and perform PCR amplification according to the reaction system in Table 11 and the reaction procedure in Table 12 to obtain the target FUT8 fragment. Prepare the enzyme digestion reaction system according to Table 13, and perform FUT8 enzyme digestion at 37℃ for 4 hours to obtain the FUT8 fragment. Prepare the pSec-Tag2 digestion reaction system according to Table 14, and digest the pSec-Tag2 vector to obtain Linearized pSec-Tag2. The digestion conditions are 37℃ for 4 h. Prepare the enzyme ligation reaction system according to Table 15, and ligate the Linearized pSec-Tag2 with the FUT8 fragment. The reaction conditions are 37℃ for 2 h. Thaw DH5α competent cells on ice, gently add the enzyme ligation product, and incubate on ice for 30 min. After heat shock in a 42℃ water bath for 60 s, add 0.5 mL of LB medium and incubate at 37℃ with shaking at 200 rpm for 45 min. Spread 100 μL of bacterial culture evenly on LB agar plates containing the corresponding antibiotic (carbenicillin, 100 μg / mL) and incubate upside down at 37℃ for 12-16 h. Pick a single colony and inoculate it into 5 mL of LB medium containing antibiotic, incubate at 37℃ with shaking for 6-8 h, and then send it to the sequencing company.

[0153] Table 11 PCR reaction system

[0154] Table 12 PCR reaction procedure

[0155] Table 13 Enzyme digestion reaction system

[0156] Table 14 Enzymatic digestion system of pSec-Tag2

[0157] Table 15 Enzyme-linked reaction system

[0158] The pSec-hsFUT8 plasmid was transfected into 293F cells using PEI reagent. After transient expression for 96 h, the supernatant was collected, and the recombinant FUT8 enzyme was obtained by separation and purification using an NTA-Ni column. The SDS-PAGE analysis results are shown below. Figure 7 As shown.

[0159] 2. Expression and purification of recombinant GALT enzyme The sequence of the recombinant GALT enzyme is referenced in the literature (Ramakrishnan B, Qasba P K. In vitro folding of β-1,4-galactosyltransferase and polypeptide-α-N-acetylgalactosaminyltransferase from the inclusion bodies[M] / / Glycosyltransferases: Methods and Protocols. Totowa, NJ: Humana Press, 2013:321-333.), where the tyrosine residue at position 289 is mutated to leucine (amino acid sequence shown in SEQ ID NO:18). Protein expression and purification procedures were performed in accordance with the reference "Thompson JW, Griffin ME, Hsieh-Wilson L C. Methods for the detection, study, and dynamic profiling of O-GlcNAc glycosylation[M] / / Methods in enzymology. Academic Press, 2018, 598: 101-135." The purified protein was confirmed using SDS-PAGE gel chromatography and Coomassie Brilliant Blue staining. The SDS-PAGE analysis results of the recombinant GALT enzyme (denoted as recombinant GALT(Y289L) enzyme, amino acid sequence as shown in SEQ ID NO:19) are as follows: Figure 8 As shown.

[0160] MKFREPLLGGSAAMPGASLQRACRLLVAVCALHLGVTLVYYLAGRDLRRLPQLVGVHPPLQGSSHGAAAIGQPSGELLRRGVAPPPPLQNSSKPRSRAPSNLDAYSHPGPGPGSNLTSAPVPSTTTRSLTACPEESPLLVGPMLIEFNIPVDLKLVEQQNPKVKLGGRYTPMDCISPHKVAIIIPFRNRQEHLKYWLYYLH PILQRQQLDYGIYVINQAGESMFNRAKLLNVGFKEALKDYDYNCFVFSDVDLIPMNDHNTYRCFSQPRHISVAMDKFGFSLPYVQLFGGVSALSKQQFLSI NGFPNNYWGWGGEDDDIYNRLAFRGMSVSRPNAVIGKCRMIRHSRDKKNEPNPQRFDRIAHTKETMLSDGLNSLTYMVLEVQRYPLYTKITVDIGTPS (SEQ ID NO:18); RDLRRLPQLVGVHPPLQGSSHGAAAIGQPSGELRLRGVAPPPPLQNSSKPRSRAPSNLDAYSHPGPGPGPGSNLTSAPVPSTTTRSLTACPEESPLLVGPMLIEFNIPVDLKLVEQQNPKVKLGGRYTPMDCISPHKVAIIIPFRNRQEHLKYWLYYLHPILQRQQLDYGIYVINQAGESM FNRAKLLNVGFKEALKDYDYNCFVFSDVDLIPMNDHNTYRCFSQPRHISVAMDKFGFSLPYVQLFGGVSALSKQQFLSINGFPNNYWGWGGEDDDIYNRLAFRGMSVSRPNAVIGKCRMIRHSRDKKNEPNPQRFDRIAHTKETMLSDGLNSLTYMVLEVQRYPLYTKITVDIGTPS (SEQ ID NO:19).

[0161] Example 3 Preparation of antibody conjugate Tras-F-2DXD This embodiment provides a method for preparing the antibody-drug conjugate Tras-F-2DXD with a DAR of 2 (flow diagram shown below). Figure 9 ), including the following steps: (1) Fucose reaction In a 50 μL reaction system, 350 μg of A2 glycoform trastuzumab (prepared in Example 1), 57.6 μg of recombinant FUT8 enzyme (prepared in Example 2), and 20.9 μg of GDP-Fuc-AM-AZ (molecular weight 877.2 Da, disclosed in "Yang Y, Song Z, Tian T, et al. Trimming crystallizable fragment (Fc) Glycansenables the direct enzymatic transfer of biomacromolecules to antibodies astherapeutics[J]. Angewandte Chemie, 2023, 135(36): e202308174.") were mixed evenly in PBS solution, incubated at 37°C for 24 h, and the unreacted GDP-Fuc-AM-AZ was removed by ultrafiltration (Millipore 30 kDa) to obtain the reaction product.

[0162] The structure of GDP-Fuc-AM-AZ is:

[0163] (2) Click reaction 2.2 μg of Propargyl-PEG4-GGFG-DXd (molecular weight 1083.12 Da, CAS number 2762518-94-3), aminoguanidine (final concentration 5 mM), THPTA (final concentration 5 mM), CuSO4 (final concentration 1 mM), and sodium ascorbate (final concentration 15 mM) were added to the above reaction product. After incubation at room temperature for 4 h, the product was purified using Protein A magnetic beads. Finally, the product was transferred to an HBS buffer system (10 mM histidine, 10% trehalose, 0.02% Tween 20, pH 5.5) to obtain the antibody conjugate with a DAR of 2, denoted as Tras-F-2DXD or TF-DXD (structural schematic diagram shown in figure). Figure 10 ).

[0164] The structural formula of Propargyl-PEG4-GGFG-DXd is:

[0165] The antibody-drug conjugate Tras-F-2DXD prepared above was analyzed using LC-MS as follows: DTT was added to the above product (i.e., antibody-drug conjugate Tras-F-2DXD) to a final concentration of 100 mM, and the mixture was incubated at 37°C for 1 h to reduce the disulfide bonds within the antibody. Subsequently, the protein was replaced with mass-grade pure water, and LC-MS (Orbitrap Exploris) mass spectrometry was performed. TM 240) Analysis.

[0166] The results are as follows Figure 11 As shown, the actual measured molecular weight of the heavy chain of the antibody-drug conjugate Tras-F-2DXD was 51964.00 Da, which is close to the theoretically calculated molecular weight (51963.89 Da), resulting in a DAR of 2 for Tras-F-2DXD. 6 μg of Tras-F-2DXD was subjected to SDS-PAGE gel chromatography and Coomassie Brilliant Blue staining. The results are as follows... Figure 12 As shown.

[0167] Example 4 Preparation of antibody conjugate Tras-G-4DXD This embodiment provides a method for preparing the antibody-drug conjugate Tras-G-4DXD with a DAR of 4 (flow diagram shown below). Figure 13 ), including the following steps: (1) Galactose conversion reaction In a 50 μL reaction system, 250 μg of A2 glycoform trastuzumab (prepared in Example 1), 6.4 μg of recombinant GALT (Y289L) enzyme (prepared in Example 2), 35.7 μg of UDP-GalNAz (molecular weight 648.37 Da, CAS: 608514-41-6), and MnCl2 (final concentration 5 mM) were mixed evenly in TBS solution and incubated at 37°C for 36 h. Unreacted UDP-GalNAz was removed by ultrafiltration (Millipore 30 kDa) to obtain the reaction product.

[0168] The structure of UDP-GalNAz is:

[0169] (2) Click reaction Add 4.5 μg Propargyl-PEG4-GGFG-DXd (molecular weight 1083.12 Da), aminoguanidine (final concentration 5 mM), THPTA (final concentration 5 mM), CuSO4 (final concentration 1 mM), and sodium ascorbate (final concentration 15 mM) to the above reaction product. After incubation at room temperature for 4 h, purify using Protein A magnetic beads. Finally, replace the product with an HBS buffer system (10 mM histidine, 10% trehalose, 0.02% Tween 20, pH 5.5) to obtain the antibody conjugate, denoted as Tras-G-4DXD or TG-DXD (structural schematic diagram shown). Figure 14 (As shown).

[0170] The antibody-drug conjugate Tras-G-4DXD prepared above was analyzed using LC-MS as follows: DTT was added to the above product (i.e., antibody-drug conjugate Tras-G-4DXD) to a final concentration of 100 mM, and the mixture was incubated at 37°C for 1 h to reduce the disulfide bonds within the antibody. Subsequently, the protein was replaced with mass-grade pure water, and LC-MS (Orbitrap Exploris) mass spectrometry was performed. TM 240) Analysis.

[0171] The results are as follows Figure 15 As shown, the actual measured molecular weight of the heavy chain of the antibody-drug conjugate Tras-G-4DXD was 53101.52 Da, which is close to the theoretically calculated molecular weight (53100.97 Da), resulting in a DAR of 4 for Tras-G-4DXD. 6 μg of Tras-G-4DXD was subjected to SDS-PAGE gel chromatography and Coomassie Brilliant Blue staining. The results are as follows... Figure 12 As shown.

[0172] Example 5 This embodiment provides a method for preparing the antibody-drug conjugate Tras-FG-6DXD with a DAR of 6 (flow diagram shown below). Figure 16 ), including the following steps: (1) Fucose reaction In a 50 μL reaction system, 350 μg of A2 glycoform trastuzumab (prepared in Example 1), 57.6 μg of recombinant FUT8 enzyme (prepared in Example 2), and 20.9 μg of GDP-Fuc-AM-AZ (molecular weight 877.2 Da) were mixed evenly in PBS solution and incubated at 37°C for 24 h. Unreacted GDP-Fuc-AM-AZ was removed by ultrafiltration (Millipore 30 kDa), and the solution was replaced with TBS solution to obtain the reaction product.

[0173] (2) Galactose conversion reaction In step (1), 6.4 μg of recombinant GALT (Y289L) enzyme (prepared in Example 2), 49.4 μg of UDP-GalNAz (molecular weight 648.37 Da), and 5 mM of MnCl2 were added to the reaction product and mixed thoroughly. The mixture was incubated at 37°C for 36 h and then ultrafiltered (Millipore 30 kDa) to remove unreacted UDP-GalNAz, thus obtaining the reaction product.

[0174] (3) Click reaction Add 6 μg Propargyl-PEG4-GGFG-DXd (molecular weight 1083.12 Da), aminoguanidine (5 mM), THPTA (5 mM), CuSO4 (1 mM), and sodium ascorbate (15 mM) to the reaction product in step (2). After incubation at room temperature for 4 h, purify using Protein A magnetic beads. Finally, replace the product with an HBS buffer system (10 mM histidine, 10% trehalose, 0.02% Tween 20, pH 5.5) to obtain the antibody conjugate, denoted as Tras-FG-6DXD or TFG-DXD (structural schematic diagram shown in Figure 1). Figure 17 ).

[0175] The antibody-drug conjugate Tras-FG-6DXD prepared above was analyzed using LC-MS as follows: DTT was added to the Click reaction product (i.e., antibody-drug conjugate Tras-FG-6DXD) to a final concentration of 100 mM, and incubated at 37°C for 1 h to reduce the disulfide bonds within the antibody. Subsequently, the protein was replaced with mass-grade pure water, and LC-MS (Orbitrap Exploratory Spectrometry) was performed. TM 240) Analysis.

[0176] The results are as follows Figure 18 As shown, the actual measured molecular weight of the heavy chain of the antibody-drug conjugate Tras-FG-6DXD was 54617.45 Da, which is close to the theoretically calculated molecular weight (53618.04 Da), resulting in a DAR of 6 for Tras-FG-6DXD. 6 μg of Tras-FG-6DXD was subjected to SDS-PAGE gel chromatography and Coomassie Brilliant Blue staining. The results are as follows. Figure 12 As shown. From Figure 12 It can be seen that after the reaction, the heavy chains of ADCs with more conjugated drugs show a significant upward shift in the gel image, which proves the increase in DAR value.

[0177] Example 6 Activity test of antibody conjugate This embodiment performs cell binding analysis, cell surface antigen-based ELISA detection, and cytotoxicity evaluation on the antibody-drug conjugates prepared in Examples 3-5, as detailed below: 1. Cell binding analysis Collect SK-BR-3 cells (human breast adenocarcinoma cells) (1×10⁻⁶ cells per group) 6 (100 cells) were collected and resuspended in cold PBS, washed once with cold PBS, centrifuged, and then resuspended in 500 μL of staining buffer (DPBS, pH 7.4, 2% FBS) so that each 500 μL buffer contained 1 × 10⁻⁶ cells. 6 Cells were randomly divided into five groups: Ctrl1: trastuzumab was added, but FITC-conjugated Goat Anti-Human IgG H&L (MCE, catalog number HY-P83649) was not added; Ctrl2: trastuzumab was not added, but FITC-conjugated Goat Anti-Human IgG H&L was added; Tras: trastuzumab and FITC-conjugated Goat Anti-Human IgG H&L were added; TF-DXD: Tras-F-2DXD and FITC-conjugated Goat Anti-Human IgG H&L were added; TG-DXD: Tras-G-4DXD and FITC-conjugated Goat Anti-Human IgG H&L were added; TFG-DXD: Tras-FG-6DXD and FITC-conjugated Goat Anti-Human IgG H&L were added. According to the group assignments, 5 μg of the test antibody or antibody-conjugate was added to 500 μL of cell suspension and incubated at 4°C for 30 min. After washing with cold PBS and centrifuging, the cells were resuspended in 500 μL of staining buffer. Then, 1 μL of the secondary antibody FITC-conjugated Goat Anti-Human IgG H&L was added to the cell suspension, and the cells were incubated again at 4°C for 30 min. After washing with PBS and centrifuging, the cells were resuspended in 500 μL of staining buffer. Antibody binding analysis was performed on the treated cells using a CytoFLEX S flow cytometer.

[0178] like Figure 19As shown, groups Ctrl1 and Ctrl2 showed extremely low fluorescence intensity because no primary antibody (trastuzumab or antibody-drug conjugate) or secondary antibody was added, indicating that false positives were unlikely in this experiment. The fluorescence intensity of groups Tras, TF-DXD, TG-DXD, and TFG-DXD was significantly higher than that of the negative control group. Furthermore, the fluorescence intensity of groups TF-DXD, TG-DXD, and TFG-DXD was similar to that of group Tras, indicating that the ability to recognize and bind antigens after antibody conjugation did not significantly change, and the drug molecules did not affect antibody binding to target cells.

[0179] 2. ELISA detection based on cell surface antigens NCI-N87 cells (human gastric cancer cells) were used at a rate of 1×10⁻⁶. 4 The cells were seeded into 96-well plates and incubated statically for 24 h. They were fixed with paraformaldehyde for 10 min, blocked with 0.2% BSA in PBS for 1 h, and then incubated overnight at 4°C with gradient concentrations (0.0001, 0.001, 0.01, 0.1, 1, 10, 100 nM) of antibody or antibody-drug conjugate. The next day, after washing three times with PBST, the cells were incubated with Goat Anti-Human IgG H&L (HRP) (MCE, catalog number HY-P83662, dilution 1:5000) at room temperature for 1 h. After washing again, TMB chromogenic buffer was added, and the cells were incubated in the dark for 30 min. The reaction was terminated by adding 0.5 M H2SO4, and the absorbance at 450 nm was recorded using a BioTek Synergy microplate reader.

[0180] The results are as follows Figure 20 As shown, the Kd values ​​for each group were obtained through fitting. The Kd value of trastuzumab against NCI-N87 cells was 0.5856 nM, the Kd value of Tras-F-2DXD against NCI-N87 cells was 0.5383 nM, the Kd value of Tras-G-4DXD against NCI-N87 cells was 0.5369 nM, and the Kd value of Tras-Fg-6DXD against NCI-N87 cells was 0.5192 nM. It can be seen that there is no significant change in the Kd values ​​among the groups, indicating that the drugs do not affect antigen-antibody binding.

[0181] 3. Cytotoxicity evaluation SK-BR-3, NCI-N87, Calu3 (human lung adenocarcinoma cells), and MCF-7 (human breast cancer cells) were selected for drug toxicity evaluation. The first three cell types were HER2-positive, while MCF-7 was HER2-negative. Cell viability was detected using the CCK8 assay. The specific procedures were as follows: 100 μL of PBS was added to the outermost ring of a 96-well plate, and 4000 cells (100 μL of complete culture medium) were added to each of the remaining wells. After incubation at 37°C in a CO2 incubator for 24 h, the culture medium was removed from the wells. 100 μL of a 10-fold serially diluted test antibody or antibody-drug conjugate was added to each well, starting at a final concentration of 68 nM, for a total of 8 concentrations, with 5 replicates for each concentration. Five wells in each 96-well plate were seeded with cells only, and five wells were filled with culture medium only. 100 μL of complete culture medium was added to each well as a control and a blank control group. The plates were then incubated at 37°C in a CO2 incubator for 96 h. After replacing the old medium with 100 μL of fresh medium, add 10 μL of Cell Counting Kit 8 (CCK-8) to each well and incubate at 37°C for 3 h. Record the absorbance at 450 nm using a BioTek Synergy microplate reader. Calculate the IC50 using a Graph Pad Prism 9. 50 Value. The complete culture medium mentioned above is 89% DMEM (Gibco, 11965092) + 10 v / v% FBS (Gibco, A5256701) + 1 v / v% P / S.

[0182] The results are as follows Figures 21-24 All three antibody-drug conjugates (ADCs) exhibited good in vitro killing activity against HER2-positive cells, with the killing ability further increasing with increasing DAR values. However, the ADCs did not kill HER2-negative MCF-7 cells, indicating that the ADCs all possess good targeting and stability, and will not damage normal cells that do not express HER2, resulting in lower off-target effects during use.

[0183] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments, and various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. A recombinant cell, characterized in that, The recombinant cells contain nucleic acid molecules encoding trastuzumab, but lack the FUT8 and B4GALT1 genes.

2. The recombinant cell according to claim 1, characterized in that, The cells include CHO cells; And / or, the nucleic acid molecule encoding trastuzumab includes a nucleic acid molecule encoding the trastuzumab heavy chain and a nucleic acid molecule encoding the trastuzumab light chain; And / or, the nucleotide sequence of the FUT8 gene is shown in SEQ ID NO:1; And / or, the nucleotide sequence of the B4GALT1 gene is shown in SEQ ID NO:2; Preferably, the amino acid sequence of the trastuzumab heavy chain is shown in SEQ ID NO:14; and the amino acid sequence of the trastuzumab light chain is shown in SEQ ID NO:

13.

3. A trastuzumab, obtained by culturing the recombinant cells as described in claim 1 or 2; Preferably, the trastuzumab is an A2 glycoform monoclonal antibody; Preferably, the N-glycans at the conserved glycosylation sites in the Fc region of the trastuzumab lack fucose and galactose; Preferably, the structure of the A2 glycoform monoclonal antibody is shown below: ， in, In the above structure, the Y-shaped structure represents trastuzumab. It represents acetamide glucose. It refers to mannose.

4. An antibody-drug conjugate, characterized in that, The antibody-drug conjugate includes: Antibodies with N-glycosylation sites; Connect segment 1 and / or connect segment 2; drug; The drug is coupled to the N-glycosylation site of the antibody via a linker fragment; The linker fragment 1 includes a linker, a reactive functional group, and a sugar structure, wherein the sugar structure is linked to the N-glycosylation site of the antibody, and the linker links the reactive functional group and the sugar structure. The connecting segment 2 includes a linker and a reactive functional group, wherein the linker is connected to a drug. Preferably, the antibody comprises IgG with a conserved N-glycosylation site in the Fc region; Preferably, the IgG includes IgG1, IgG2, or IgG4; Preferably, the antibody is a monoclonal antibody, a polyclonal antibody, a bifunctional antibody, a trifunctional antibody, a nanobody fused with an Fc domain, a therapeutic antibody or a functional antibody from different species. Preferably, the targets of the antibody include HER2, Claudin18.2, EGFR, c-Met, NECTIN4, CD276, HER3, CD3, FOLR1, BCMA, CD20, DLL3, MUC1, PD-L1, ROR1, TF, CD19, CD22, CD30, CD70, CD79B, FGFs, MSLN, NT5E, TNFα, CD147, CD24, CD38, CD47, CDH3, CDK4, CDK6, CEACAM5, CLDN6, CTLA4, DDR1, DR5, FAPα, and FGFR.

3. GPRC5D, GR, HLA-DR, ICAM1, IL2R, MELTF, ROR2, TPBG, VTCN1, Z1P6, CD33, CD25, RSV, VEGF, RANKL, VEGFR2, CTLA-4, CD52, CD319, PD-1, CD274, IgE, IL-6, IL-12, IL-2, C5, IL-17A, CD25, SLAMF7, F10, factorIXa, HAb18G, PCSK9, BLyS, 1L23, C4β7, IL-4R-α, HAE, FGF23 or IL6R; Preferably, the antibody is selected from trastuzumab, inetuzumab, zineb, pertuzumab, sacituzumab, datopotamab, zotuzumab, cetuximab, panitumumab, nimotuzumab, nexituzumab, ervantuzumab, telisotuzumab, enfortumab, vobramitamab, mirzotamab, deparetuzumab, morotumab, caputuzumab, belintoumab, and bonatuzumab. Mirvetuximab, Belantuzumab, Atoxizumab, Rituximab, Lovatozumab, Talatuzumab, Clivatuzumab, Sontuzumab, Gatipotuzumab, Vofatamab, Atezolizumab, Averumab, Duvalumab, Duvalimumab, Atezolizumab, Zilovertamab, Tisotumab, Denintuzumab, Inotuzumab, Moxetumomab, Brentuximab, Vorsetuzumab, Polatuzumab, Iladatuzumab, Burosumab, Bemattouzumab, Efruxifermin, Anetumab, Amatuximab, MEDI9447, Adalimumab, Infliximab, Golimumab, Ziralimumab, ATG-031, Daraerumumab, Daraetumumab, Letaplimab, Lezolimumab, Lefalimumab, Tusamitamab, Labetuzumab, IMAB027, Tremelimumab, Zeflimumab, Botensilimab, Numulimab, Ipilimumab, Iprimumab, Tigatuzumab, Lexatumumab, Vofatamab, Talquetamab, Bersanlimab, Enlimo mab, Inolimomab, Ozuriftamab, Ladiratuzumab, Gemtuzumab, Camidanlumab, Nirsevimab, Felvizumab, Bevacizumab, Ramosinumab, Ranibizumab, Denosumab, Denizumab, Trimelimumab, Zeflimumab, Zeflimumab, Alemumab, Pallizumab, Nivolumab, Pembrolizumab, Nivolumab, Pembrolizumab, Tripeptide Rimab, Sintilimab, Camrelizumab, Lesabelimab, Omalizumab, Tocilizumab, Ustekinumab, Baliximab, Ikuzumab, Brolinumab, Ikizumab, Secukinumab, Daliximab, Elotuzumab, Emexizumab, Iometuzumab, Ivolucrata, Emexizumab, Belimumab, Alicizumab, Gusegezumab, Vedolimumab, Dupreliminumab, Lanalimumab, Broshuzumab, Sattelizumab; Preferably, the antibody is trastuzumab; Preferably, the antibody is the trastuzumab described in claim 3.

5. The antibody-drug conjugate according to claim 4, characterized in that, The drug is selected from MMAE, MMAF, Dxd, Dx8951, SN38, DM1, DM4, PBD, PBD dimer, eribulin, docamycin, muscarinic acid, vincristine, vinorelbine, VP-16, camptothecin, paclitaxel, docetaxel, epothilone A, epothilone B, olaparib, nocodazole, colchicine, estradiol, simadalide, ilexeloseluxol, or derivatives of the above drugs; And / or, the linker includes a PEG chain, a carbon chain, or a pyrolytic linker; And / or, the sugar structure is selected from monosaccharides, disaccharides, oligosaccharides, or branched sugar structures; And / or, the reactive functional group is selected from azide group, tetrazine group, TCO group, alkynyl structure, aldehyde structure, carbonyl structure, hydroxylamine structure, isothiocyanate, amino, carboxyl, mercapto, alkenyl, maleimide, and hydrazone structure.

6. The antibody-drug conjugate according to claim 4 or 5, characterized in that, The DAR value of the antibody-drug conjugate is 1-6.

7. The antibody-drug conjugate according to claim 4 or 5, characterized in that, The antibody-drug conjugate has the structures shown in Formula I, Formula II, and Formula III as follows: Formula I Formula II Formula III In the above structure, the Y-shaped structure represents an antibody. It represents N-acetylgalactosamine. It represents acetamide glucose. It means mannose. It represents fucose. Indicates reactive functional groups, Indicates connector, Indicates medicine.

8. The antibody-drug conjugate according to claim 7, characterized in that, The antibody-drug conjugate has the structures shown in formulas a, b, and c below: Formula a Formula b Formula c.

9. The method for preparing the antibody-drug conjugate according to claim 8, characterized in that, For formula a, the preparation method includes mixing the antibody, α1,6-fucosyltransferase and sugar structure-reactive functional group complex 1 into a buffer solution, reacting to obtain a glycan-mediated site-directed antibody-reactive functional group conjugate with antibody glycosylation site modified reactive functional group; adding a toxic drug-reactive functional group complex solution, reacting again to obtain an antibody-drug conjugate; For formula b, the preparation method includes mixing the antibody, β1,4-galactosyltransferase and glycostructure-reactive functional group complex 2 into a buffer solution, reacting to obtain a glycan-mediated antibody-reactive functional group conjugate with antibody glycosylation site modified reactive functional group; A solution of a toxic drug-reactive functional group complex was added, and the reaction was repeated to obtain an antibody-drug conjugate. For formula c, the preparation method includes mixing an antibody, α1,6-fucosyltransferase, and sugar structure-reactive functional group complex 1 in a buffer solution, performing a first reaction to obtain glycan site-directed antibody-reactive functional group conjugate 1 with antibody glycosylation site modified reactive functional group; adding β1,4-galactosyltransferase and sugar structure-reactive functional group complex 2, performing a second reaction to obtain glycan site-directed antibody-reactive functional group conjugate 2 with antibody glycosylation site modified reactive functional group; adding a toxic drug-reactive functional group complex solution, performing a third reaction to obtain the antibody-drug conjugate; Preferably, the sugar structure-reactive functional group complex 1 is GDP-Fuc-AM-AZ, with the following structural formula: ； Preferably, the sugar structure-reactive functional group complex 2 is UDP-GalNAz, with the following structural formula: ； Preferably, the toxic drug-reactive functional group complex is Propargyl-PEG4-GGFG-DXd, with the following structural formula: 。 10. The use of the recombinant cells of claim 1 or 2, the totuzumab of claim 3, or the antibody-drug conjugate of any one of claims 4-8 in the preparation of antitumor drugs; Preferably, the tumor includes at least one of gastric adenocarcinoma, esophageal adenocarcinoma, colorectal cancer, bile duct cancer, gallbladder cancer, gastric cancer, lung cancer, cholangiocarcinoma, bladder cancer, esophageal cancer, melanoma, ovarian cancer, liver cancer, prostate cancer, pancreatic cancer, small intestine cancer, head and neck cancer, uterine cancer, cervical cancer, brain cancer, and breast cancer.

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