Trisaccharide linker, linker-payload comprising trisaccharide linker, and glycan chain-remodeled antibody-drug conjugate, and preparation methods therefor and uses thereof

Abstract

The present invention relates to a linker-payload compound comprising a trisaccharide group, wherein the trisaccharide group is linked to the remainder of the compound by means of an amide bond. The present invention further relates to an antibody-drug conjugate comprising the linker-payload compound, wherein a glycan chain in an antibody is remodeled using the trisaccharide group in the linker-payload compound. The present invention further relates to preparation methods and uses of the described substances.

Description

Trisaccharide linker, linker-load and sugar chain remodeled antibody-drug conjugate comprising the trisaccharide linker, preparation method and use thereof Technical Field

[0001] The present invention relates to a trisaccharide linker. It also relates to a linker-load compound comprising a trisaccharide group, wherein the trisaccharide group is linked to the remainder of the compound via an amide bond. It also relates to an antibody-drug conjugate comprising the linker-load compound, wherein the trisaccharide group in the linker-load compound is used to reshape the sugar chains in the antibody. The present invention also relates to methods for preparing and using the aforementioned substances. Background Art

[0002] Antibody-drug conjugates (ADCs) are a novel class of drugs that link antibodies to physiologically active effector molecules. They leverage the targeting properties of antibodies and the high physiological activity of effector molecules to achieve targeted delivery of effector molecules, reducing off-target toxicity caused by drug distribution to non-target organs and tissues. They also further improve the limited efficacy of traditional antibody-based drugs against solid tumors. Currently, more than ten ADCs containing small molecule toxins have been approved for marketing worldwide, providing improved survival benefits for cancer patients (Dumontet C, Reichert JM, Senter PD, et al. Antibody–drug conjugates come of age in oncology. Nat. Rev. Drug. Discov. 2023, 22, 641). Take breast cancer treatment as an example. Traditional treatments for breast cancer, such as surgery, chemotherapy, radiotherapy, and endocrine therapy, are associated with permanent damage, significant side effects, and limited prognostic benefits. The advent of targeted monoclonal antibody therapies, such as trastuzumab, has revolutionized breast cancer treatment and, in general, cancer overall. They have significantly reduced breast cancer mortality and benefited millions of patients. However, trastuzumab has its limitations. It is only effective in the approximately 20% of breast cancer patients with HER2-positive disease, while the much larger proportion of patients with HER2-low-expressing disease struggles to benefit from trastuzumab. Enhertu, an antibody-drug conjugate based on trastuzumab and the topoisomerase I inhibitor DXd, has revolutionized targeted therapy. Enhertu has achieved an overall response rate of 60.3% in patients with HER2-positive breast cancer who have developed resistance after receiving at least two prior HER2-targeted therapies. Furthermore, for patients with metastatic breast cancer with low HER2 expression who have no available targeted therapy, Enhertu has reduced the risk of death or tumor progression by 50% compared to standard chemotherapy. In short, antibody-drug conjugates fully utilize the advantages of antibodies and effector molecules through the organic connection of both, improving the targeting, safety and efficacy of treatment, and bringing about changes in cancer treatment.

[0003] Most of the antibody-drug conjugates currently on the market or in clinical development are based on traditional conjugation methods such as lysine or cysteine ​​conjugation on antibodies, which often leads to random conjugation sites and high product heterogeneity. In addition, due to the limitations of the conjugation method, the antibody backbone may be affected by unstable linkers or interchain disulfide bonds after the reduction step. Therefore, traditional conjugation methods face severe challenges in process stability, quality control, antibody-drug conjugate drug stability, metabolic consistency and safety. In order to overcome the above problems, in recent years, the industry has developed a new generation of site-specific conjugation technologies such as site-specific conjugation based on the introduction of specific amino acids through antibody engineering, site-specific conjugation based on transpeptidase catalysis, affinity-mediated site-specific conjugation and enzymatic antibody sugar chain site-specific conjugation, which to a certain extent solve the limitations of traditional conjugation heterogeneity. However, these technologies still have certain limitations. For example, some coupling technologies require additional antibody modification or engineering transformation, the screening and synthesis of affinity ligands are relatively complex, the coupling conditions are not mild and green enough, the process flow is complicated, and the applicability to substrates is limited (Walsh SJ, Bargh JD, Dannheim FM, et al. Site-selective modification strategies in antibody–drug conjugates. Chem. Soc. Rev. 2021, 50, 1305).

[0004] Enzymatic antibody glycan site-specific conjugation technology uses glycosidases or endoglycosidases to modify antibody glycans and introduce effector molecules, without the need for additional antibody engineering. However, previous studies have often not fully resolved issues such as the complex conjugation process (which requires the participation of multiple enzymes and multi-step reactions) and the large substrate limitations (Manabe S, Yamaguchi Y, Antibody Glycoengineering and Homogeneous Antibody-Drug Conjugate Preparation. Chem. Rec. 2021, 21, 1). In 2022, Shi W. et al. reported a one-step antibody sugar chain site-specific coupling scheme based on the 6'-aldehyde-N-acetyllactosamine disaccharide derivative structure catalyzed by the endoglycosidase Endo S2. This method does not require antibody engineering modification. The same enzyme is used to catalyze the glycosyl cleavage reaction of the N-297 sugar group in the antibody Fc region and continue to catalyze the transglycosylation reaction of the disaccharide linker-effector molecule, completing the site-specific modification of the antibody in one pot to obtain an antibody-drug conjugate (Shi W., Li W., Zhang J., et.al. One-step synthesis of site-specific antibody edrug conjugates by reprograming IgG glycoengineering with LacNAc-based substrates, Acta Pharmaceutica Sinica B 2022, 12, 2417.). Similarly, Zhang X. et al. reported in the same year a one-step antibody sugar chain site-specific coupling scheme catalyzed by endoglycosidase Endo S2 and based on a disaccharide derivative structure functionalized with 6'-alkylamine (Zhang X., Ou C., Liu H., et.al. Synthesis and Evaluation of Three Azide-Modified disaccharide Oxazolines as Enzyme Substrates for Single-Step Fc Glycan Mediated Antibody-Drug Conjugation, Bioconjugate Chem. 2022, 33, 1179.). Summary of the Invention

[0005] The present invention provides a novel trisaccharide linker, and methods for preparing and using the same. Also provided are linker-load compounds comprising a trisaccharide group, wherein the trisaccharide group is linked to the remainder of the compound via an amide bond. Specifically, the present invention provides a linker-load compound having the formula (I): D—L—(P) t

[0006] Formula (I)

[0007] in

[0008] P is the load;

[0009] L is a linker, and L is directly connected to D via its terminal -NH-, wherein when L is an unbranched linker, it is connected to 1 P, and t is 1, and when L is a branched linker, each branch can be connected to 1 P, and t is an integer greater than 1 (e.g., 2, 3, 4, 5, 6, 7, 8, 9 or 10);

[0010] D is a trisaccharide unit structure, which contains a first hexose unit or a derivative thereof, a second hexose unit or a derivative thereof, and a third hexose unit or a derivative thereof;

[0011] The 6-OH group of the first hexose unit or its derivative is oxidized to -C(O)- and connected to the terminal -NH- group of L;

[0012] The first hexose unit or its derivative portion is connected to the second hexose unit or its derivative portion via an α-(1→6) glycosidic bond;

[0013] The second hexose unit or its derivative is connected to the third hexose unit or its derivative via a β-(1→4) glycosidic bond;

[0014] The third hexose unit or its derivative has the following structure:

[0015] (β-DN-acetylglucosamine moiety) or (β-D-glucose oxazoline moiety).

[0016] In one embodiment, L can be cleaved from P chemically (eg, hydrolytically) or biologically (eg, enzymatically) to release P.

[0017] The present invention also relates to an antibody-drug conjugate comprising the linker-load compound, wherein the trisaccharide groups in the linker-load compound are used to reshape the sugar chains in the antibody. Specifically, the present invention provides an antibody-drug conjugate having formula (II) based on site-directed attachment of the N-glycosylation site in the Fc region of the antibody:

[0018] in

[0019] P is the load;

[0020] R is hydrogen or α-L-fucosyl;

[0021] q is 1 or 2;

[0022] Ab is an antibody or antigen-binding fragment;

[0023] The first hexose unit or its derivative portion is linked to the second hexose unit or its derivative portion via an α-(1→6) glycosidic bond;

[0024] The second six-carbon sugar unit or its derivative is connected to the β-DN-acetylglucosamine portion via a β-(1→4) glycosidic bond;

[0025] The 6-OH of the first hexose unit or its derivative is oxidized to -C(O)-;

[0026] L is a linker, and L is directly connected to the -C(O)- in the first hexose unit or its derivative portion via the terminal -NH- therein; wherein when L is an unbranched linker, it is connected to 1 P, and t is 1, and when L is a branched linker, each branch can be connected to 1 P, and t is an integer greater than 1 (e.g., 2, 3, 4, 5, 6, 7, 8, 9 or 10).

[0027] In one embodiment, the first six-carbon sugar unit or its derivative moiety is selected from glucosyl, mannosyl, galactosyl, fructosyl, gulosyl, idosyl or derivatives thereof.

[0028] In one embodiment, the second six-carbon sugar unit or its derivative moiety is selected from glucosyl, mannosyl, galactosyl, fructosyl or derivatives thereof.

[0029] In one embodiment, the -NHC(O)CH2- group in formula (II) that is attached to Ab is derived from asparagine at position 297 of the antibody Fc region.

[0030] The present invention also relates to a preparation method and application of the substance. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figures 1 and 2 show the results of hydrophobic interaction high performance liquid chromatography (HIC-HPLC) and size exclusion chromatography (SEC-HPLC) analysis of the antibody-drug conjugate ADC-1, respectively.

[0032] FIG3 and FIG4 respectively show the hydrophobic interaction high performance liquid chromatography (HIC-HPLC) and size exclusion chromatography (SEC-HPLC) analysis results of the antibody-drug conjugate ADC-2.

[0033] FIG5 and FIG6 respectively show the hydrophobic interaction high performance liquid chromatography (HIC-HPLC) and size exclusion chromatography (SEC-HPLC) analysis results of the antibody-drug conjugate ADC-3.

[0034] FIG7 and FIG8 respectively show the hydrophobic interaction high performance liquid chromatography (HIC-HPLC) and size exclusion chromatography (SEC-HPLC) analysis results of the antibody-drug conjugate ADC-4.

[0035] Figures 9-11 show the in vitro activities of antibody-drug conjugates ADC-1 to ADC-4 on SKBR-3, NCI-N87, and MDA-MB-468 cell lines, respectively.

[0036] Figures 12-14 show the in vitro activities of antibody-drug conjugates ADC-5 to ADC-8 on SKBR-3, NCI-N87, and MDA-MB-468 cell lines, respectively.

[0037] Figures 15-17 show the in vitro activities of antibody-drug conjugates ADC-9 to ADC-11 on SKBR-3, NCI-N87, and MDA-MB-468 cell lines, respectively.

[0038] Figures 18-20 show the in vitro activities of antibody-drug conjugates ADC-12 to ADC-14 on SKBR-3, NCI-N87, and MDA-MB-468 cell lines, respectively.

[0039] FIG21 shows the affinity detection of ADC-5 to ADC-8 for cell surface HER2.

[0040] FIG22 shows the in vivo efficacy evaluation of ADC-6 to ADC-8.

[0041] FIG23 shows the ADCC effects of ADC-5 to ADC-8. DETAILED DESCRIPTION

[0042] General Definition

[0043] Unless otherwise defined below, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art. The technology used herein refers to the technology generally understood in the art, including variations and equivalent substitutions that are obvious to those skilled in the art. Although it is believed that the following terms are readily understood by those skilled in the art, the following definitions are set forth to better illustrate the present invention. When a trade name appears herein, it refers to the corresponding commercial product or its active ingredient. All patents, published patent applications and publications cited herein are incorporated herein by reference.

[0044] When a certain amount, concentration or other numerical value or parameter is set forth in the form of a range, a preferred range or a preferred upper limit or a preferred lower limit, it 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 the range is clearly stated. Unless otherwise stated, the numerical ranges listed herein are intended to include all integers and fractions (decimals) within the endpoints and ranges of the range. For example, the expression "i is an integer from 1 to 20" means that i is any integer from 1 to 20, for example, i 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 such as j, g, k, etc. should also be understood in a similar manner.

[0045] Unless the context clearly dictates otherwise, singular forms such as "a", "an", and "the" include plural forms. The expression "one or more" or "at least one" may mean 1, 2, 3, 4, 5, 6, 7, 8, 9 or more.

[0046] The terms "about" and "approximately" when used with a numerical variable generally mean that the value of the variable and all values ​​of the variable are within the range of experimental error (e.g., within a 95% confidence interval about the mean) or within ±10% or more of the stated value.

[0047] The term "optionally" means that the subsequently described event may or may not occur, and that the description includes instances where said event or circumstance occurs or does not occur.

[0048] The expressions "comprising," "including," "containing," and "having" are open ended and do not exclude additional unrecited elements, steps, or ingredients. The expression "consisting of excludes any elements, steps, or ingredients not specified. The expression "consisting essentially of means that the scope is limited to the specified elements, steps, or ingredients, as well as the optional presence of elements, steps, or ingredients that do not materially affect the basic and novel characteristics of the claimed subject matter. It should be understood that the expression "comprising" encompasses the expressions "consisting essentially of" and "consisting of."

[0049] The term "targeting molecule" refers to a molecule that has an affinity for a specific target (e.g., a receptor, a cell surface protein, a cytokine, a tumor-specific antigen, etc.). Targeting molecules can deliver a payload to a specific site in the body through targeted delivery. Targeting molecules can recognize one or more targets. A specific target is defined by the target it recognizes. For example, a receptor-targeting targeting molecule can deliver a cytotoxin to a site containing a large number of receptors. Examples of targeting molecules include, but are not limited to, antibodies, binding proteins for a given antigen, antibody mimics, scaffold proteins with affinity for a given target, ligands, etc. Targets recognized by targeted molecules include but are not limited to CD19, CD22, CD25, CD30 / TNFRSF8, CD33, CD37, CD44v6, CD56, CD70, CD71, CD74, CD79b, CD117 / KIT, CD123, CD138, CD142, CD174, CD227 / MUC1, CD352, CLDN18.2, DLL3, ErbB2 / HER2, ErbB3 / HER3, CN33, GPNMB, ENP P3, Nectin-4, EGFRvⅢ, SLC44A4 / AGS-5, CEACAM5, PSMA, TIM1, LY6E, LIV1, Nectin4, SLITRK6, HGFR / cMet, SLAMF7 / CS1, EGFR, BCMA, AXL, NaPi2B, GCC, STEAP1, MUC16, Mesothelin, ETBR, EphA2, 5T4, FOLR1, LAMP1, Cadherin 6, FGFR2, FGFR3, CA6, CanAg, Integrin αV, TDGF1, Ephrin A4, TROP2, PTK7, NOTCH3, C4.4A, FLT3, B7H3 / 4, TF (Tissue Factor) and ROR1 / 2, as well as combinations of the above targets. The antibodies of the present invention include, but are not limited to, antibodies based on the above targets, antibody fragments, and bispecific antibodies and multispecific antibodies based on a combination of the above targets.

[0050] As used herein, the term "antibody" is used in a broad manner, and its definition covers conventional antibodies, recombinant antibodies / genetically engineered antibodies, particularly including complete monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (such as bispecific antibodies) and antibody fragments, as long as they have the desired biological activity. The antibody can be any subtype (such as IgG, IgE, IgM, IgD and IgA) or subclass, and can be derived from any suitable species. In some embodiments, the antibody is human or mouse. The antibody can also be a fully human antibody, a humanized antibody or a chimeric antibody prepared by a recombinant method.

[0051] As used herein, a monoclonal antibody refers to an antibody obtained from a substantially homogeneous antibody population, i.e., the individual antibodies comprising the population are identical except for a few possible naturally occurring mutations. Monoclonal antibodies are highly specific for a single antigenic site. The term "monoclonal" refers to the characteristic of an antibody as being derived from a substantially homogeneous antibody population and should not be construed as requiring some specific method for producing the antibody.

[0052] An intact antibody or full-length antibody essentially comprises an antigen-binding variable region and a light chain constant region (C L ) and heavy chain constant region (C H ), which may include C H 1, C H 2. C H 3 and C H 4, depending on the subtype of the antibody. The antigen-binding variable region (also called fragment variable region, Fv fragment) usually contains the light chain variable region (V L ) and heavy chain variable region (V H The constant region can be a native sequence constant region (e.g., a human native sequence constant region) or an amino acid sequence variant thereof. The variable region recognizes and interacts with the target antigen. The constant region can be recognized and interacted with by the immune system.

[0053] Antibody fragments may comprise a portion of an intact antibody, preferably its antigen binding region or variable region. Examples of antibody fragments include Fab, Fab', F(ab')2, H and C H 1 domain, Fd fragment, Fv fragment, single domain antibody (dAb) fragment and isolated complementary determining region (CDR). Fab fragment is an antibody fragment obtained by papain digestion of full-length immunoglobulin, or a fragment with the same structure produced by, for example, recombinant expression. Fab fragment contains light chain (including V L and C L ) and another chain, wherein the other chain comprises the variable region of the heavy chain (V H ) and a constant region of the heavy chain (C H1). F(ab')2 fragments are antibody fragments obtained by digesting immunoglobulins with pepsin at pH 4.0-4.5, or fragments with the same structure produced by, for example, recombinant expression. F(ab')2 fragments essentially contain two Fab fragments, wherein each heavy chain portion contains several additional amino acids, including cysteine ​​that forms a disulfide bond connecting the two fragments. Fab' fragments are fragments that contain half of a F(ab')2 fragment (a heavy chain and a light chain). The antibody fragment may contain multiple chains linked together, for example, by disulfide bonds and / or by peptide linkers. Examples of antibody fragments also include single-chain Fv (scFv), Fv, dsFv, diabody, Fd and Fd' fragments, as well as other fragments, including modified fragments. Antibody fragments typically contain at least or about 50 amino acids, typically at least or about 200 amino acids. Antigen-binding fragments may include any antibody fragment that, when inserted into an antibody framework (for example, by replacing the corresponding region), can obtain an antibody that immunospecifically binds to an antigen.

[0054] In particular, the antibody-drug conjugates of the present application are site-specifically conjugated based on any site containing natural N-glycosylation modifications of the antibody Fc region. Molecules containing sugar chains in the antibody Fc region (including but not limited to antibodies / bispecific antibodies / Fc fusion proteins / single-chain antibodies / nanoantibodies, etc.) can be prepared in a one-step process using the trisaccharide-containing linker-load of the present application. Therefore, the antibodies of the present application are not particularly limited, and their Fc regions can contain sugar chains, and can be natural antibodies. Drugs are not limited to small molecule therapeutic drugs, as long as they have therapeutic, diagnostic, and preventive effects. Including but not limited to oligonucleotides (such as ASOs, siRNAs, etc.), polypeptides, cytokines, etc.

[0055] In addition, the antibodies of the present invention can also be prepared using techniques well known in the art, such as the following techniques or combinations thereof: recombinant technology / genetic engineering technology, phage display technology, synthetic technology, or other techniques known in the art. For example, genetically engineered recombinant antibodies can be expressed in a suitable culture system (e.g., E. coli or mammalian cells). The genetic engineering can refer to, for example, the introduction of a ligase-specific recognition sequence at its terminus.

[0056] As used herein, the term "targeting molecule-drug conjugate" is referred to as a "conjugate." Examples of conjugates include, but are not limited to, antibody-drug conjugates.

[0057] Small molecule compounds refer to molecules of a size comparable to organic molecules commonly used in pharmaceuticals. The term does not encompass biological macromolecules (e.g., proteins, nucleic acids, etc.), but does encompass low molecular weight peptides or derivatives thereof, such as dipeptides, tripeptides, tetrapeptides, pentapeptides, etc. Typically, small molecule compounds have a molecular weight of, for example, about 100 to about 2000 Da, about 200 to about 1000 Da, about 200 to about 900 Da, about 200 to about 800 Da, about 200 to about 700 Da, about 200 to about 600 Da, or about 200 to about 500 Da.

[0058] Cytotoxins refer to substances that inhibit or prevent the expression activity, cell function, and / or cause cell damage. Currently, cytotoxins commonly used in antibody-drug conjugates are more toxic than chemotherapy drugs. Examples of cytotoxins include, but are not limited to, drugs that target the following targets: microtubule cytoskeleton, DNA, RNA, kinesin-mediated protein transport, and regulation of apoptosis. Drugs that target the microtubule cytoskeleton can, for example, be microtubule stabilizers or tubulin polymerization inhibitors. Examples of microtubule stabilizers include, but are not limited to, taxanes. Examples of tubulin polymerization inhibitors include, but are not limited to, maytansinoids, auristatins, vinblastines, colchicines, and dolastatins. Drugs that target DNA can, for example, be drugs that directly destroy DNA structure or topoisomerase inhibitors. Examples of drugs that directly destroy DNA structure include, but are not limited to, DNA double strand breakers, DNA alkylating agents, and DNA intercalators. DNA double-strand disruptors can be, for example, enediyne antibiotics, including but not limited to danemycin, esperamicin, neocarzinostatin, uncialamycin, etc. DNA alkylating agents can be, for example, DNA bis-alkylators (i.e., DNA cross-linkers) or DNA mono-alkylators. Examples of DNA alkylating agents include but are not limited to pyrrolo[2,1-c][1,4]benzodiazepines. (PBD) dimer, 1-(chloromethyl)-2,3-dihydro-1H-benzo[e]indole (CBI) dimer, CBI-PBD heterodimer, dihydroindole benzodiazepine (IGN) dimers, duocarmycin-like compounds, etc. Examples of topoisomerase inhibitors include, but are not limited to, exatecan and its derivatives (e.g., DX8951f, DXd-(1) and DXd-(2), the structures of which are described below), camptothecins, and anthracyclines. Drugs targeting RNA may, for example, be drugs that inhibit splicing, examples of which include, but are not limited to, pladienolide. Drugs targeting kinesin-mediated protein transport may, for example, be mitotic kinesin inhibitors, including, but not limited to, kinesin spindle (KSP) inhibitors.

[0059] Small molecule compounds may also include, for example, small molecule drugs with various mechanisms of action, including various traditional small molecule drugs, photoacoustic dynamic therapy drugs, photothermal therapy drugs, etc., such as chemotherapeutic drugs, small molecule targeted drugs, immune agonists, etc., such as traditional cytotoxic drugs such as cisplatin, paclitaxel, 5-fluorouracil, cyclophosphamide, and bendamustine; small molecule targeted drugs such as imatinib mesylate, gefitinib, and anlotinib; immune agonists such as STING agonists, TLR agonists, etc.). The cargo of the present invention may also be a nucleic acid and nucleic acid analog, a tracer molecule, including a fluorescent molecule, biotin, a fluorophore, a chromophore, a spin resonance probe, and a radiolabel, or a short peptide, polypeptide, peptidomimetic, or protein.

[0060] "Spacer" refers to a structure located between different structural modules that can spatially separate the structural modules. The definition of a spacer does not limit whether it has a certain function or whether it can be cut or degraded in vivo. Examples of spacers include, but are not limited to, amino acid and non-amino acid structures, wherein 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 serves as a spacer, and its examples include, but are not limited to, a single amino acid, a sequence containing multiple amino acids, for example, a sequence containing two amino acids, such as GA, or for example, GGGGS (SEQ ID No. 3), GGGGSGGGGS (SEQ ID No. 4), GGGGSGGGGSGGGGS (SEQ ID No. 5), etc. A self-immolative spacer (such as self-immolative spacer Sp1) is a covalent component that causes two chemical bonds to be cleaved sequentially after activation of the protective moiety in the precursor: the protective moiety (such as the cleavable sequence) is removed after activation, triggering a cascade of decomposition reactions, resulting in the release of smaller molecules in a sequential order. Examples of self-immolative spacers include, but are not limited to, PABC (p-aminobenzyloxycarbonyl), acetals, heteroacetals, and combinations thereof.

[0061] As used herein, the term "amino acid" includes "natural amino acids" and "unnatural amino acids."

[0062] The term "natural amino acids" refers to amino acids, which are protein-building amino acids, including the common twenty amino acids (alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine), as well as the less common selenocysteine ​​and pyrrolysine.

[0063] As used herein, the term "unnatural amino acid" refers to an amino acid that is not a protein-forming amino acid. Specifically, the term refers to an amino acid that is not a natural amino acid as defined above.

[0064] The term "alkyl" refers to a straight or branched chain saturated aliphatic hydrocarbon group consisting of carbon atoms and hydrogen atoms, which is connected to the rest of the molecule by a single bond. Alkyl groups can have 1 to 20 carbon atoms and refer to "C1-C 20 "alkyl", such as C1-C4 alkyl, C1-C3 alkyl, C1-C2 alkyl, C3 alkyl, C4 alkyl, C3-C6 alkyl. Non-limiting examples of alkyl include, but are not limited to, methyl, ethyl, propyl, butyl, pentyl, hexyl, isopropyl, isobutyl, sec-butyl, tert-butyl, isopentyl, 2-methylbutyl, 1-methylbutyl, 1-ethylpropyl, 1,2-dimethylpropyl, neopentyl, 1,1-dimethylpropyl, 4-methylpentyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 2-ethylbutyl, 1-ethylbutyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1 -dimethylbutyl, 2,3-dimethylbutyl, 1,3-dimethylbutyl or 1,2-dimethylbutyl, or isomers thereof. A divalent free radical refers to a group obtained by removing a hydrogen atom from a carbon atom with free valence electrons of the corresponding monovalent free radical. A divalent free radical has two attachment sites to the rest of the molecule. For example, "alkylene" or "alkylene group" refers to a saturated straight or branched divalent hydrocarbon group. Examples of "alkylene" include, but are not limited to, methylene (-CH2-), ethylene (-C2H4-), propylene (-C3H6-), butylene (-C4H8-), pentylene (-C5H 10 -), hexamethylene (-C6H 12 -), 1-methylethylene (-CH(CH3)CH2-), 2-methylethylene (-CH2CH(CH3)-), methylpropylene or ethylpropylene, etc.

[0065] The term "cycloalkyl" refers to a cyclic saturated aliphatic group consisting of carbon atoms and hydrogen atoms, which is connected to the rest of the molecule by a single bond. A cycloalkyl group can have 3 to 10 carbon atoms, i.e., "C3-C 10 "Cycloalkyl" refers to a divalent cycloalkyl radical, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl or cyclodecyl. "Cycloalkylene" refers to a divalent cycloalkyl radical.

[0066] The term "heterocyclyl" refers to a cycloalkyl group in which one or more carbon atoms are replaced by a heteroatom selected from nitrogen, oxygen, and sulfur, such as azepine, oxa-, or thiirane, azepine, oxa-, or thietanyl, pyrrolidinyl, pyrazolidinyl, imidazolidinyl, tetrahydrofuranyl, tetrahydrothiophenyl, piperidinyl, piperazinyl, tetrahydropyranyl, or tetrahydrothiopyranyl. "Heterocyclylene" refers to a divalent cycloalkyl group.

[0067] When "substituted" is mentioned herein, unless otherwise indicated, the relevant substituent is selected from alkyl, halogen, amino, monoalkylamino, dialkylamino, nitro, cyano, formyl, alkylcarbonyl, carboxyl, alkyloxycarbonyl, alkylcarbonyloxy, aminocarbonyl, monoalkylaminocarbonyl, dialkylaminocarbonyl, formylamino, alkylcarbonylamino, formyl(monoalkyl)amino or alkylcarbonyl(monoalkyl)amino.

[0068] As used herein, when a group is combined with another group, the connection between the groups can be linear or branched, provided that a chemically stable structure is formed. The structure formed by such a combination can be connected to other parts of the molecule through any suitable atom in the structure, preferably through a specified chemical bond. For example, when two or more groups selected from -CR 1 R 2 -、C 1-10 Alkylene, C4-10 cycloalkylene, C 4-10 The heterocyclic group and the divalent group of -(CO)- are combined to form a combination, and two or more divalent groups can form a linear connection with each other, such as -CR 1 R 2 -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 R2'-(CO)-、-CR 1 R 2 -CR ’R 2’ -CR 1 ”R 2 ”-(CO)-, etc. The resulting bivalent structure can be further linked to other parts of the molecule.

[0069] When multiple identical letters representing chemical groups appear in the same chemical formula, they are independently selected and not necessarily the same. For example, multiple M in formula I-2 are independently selected from LKa-L 2 ―L 1 ―B―P; and multiple L 2 For example, if two a or i appear in the same structural formula at the same time, they are also independently selected and not necessarily the same.

[0070] As used herein, the expressions "antibody-drug conjugate" and "antibody-drug conjugate" have the same meaning.

[0071] Linker-load compound

[0072] In a first aspect, the present invention provides a linker-load compound having the formula (I): D—L—(P) t

[0073] Formula (I)

[0074] in

[0075] P is the load;

[0076] L is a linker, and L is directly connected to D via its terminal -NH-, wherein when L is an unbranched linker, it is connected to 1 P, and t is 1, and when L is a branched linker, each branch can be connected to 1 P, and t is an integer greater than 1 (e.g., 2, 3, 4, 5, 6, 7, 8, 9 or 10);

[0077] D is a trisaccharide unit structure, which contains a first hexose unit or a derivative thereof, a second hexose unit or a derivative thereof, and a third hexose unit or a derivative thereof;

[0078] The 6-OH group of the first hexose unit or its derivative is oxidized to -C(O)- and connected to the terminal -NH- of L;

[0079] The first hexose unit or its derivative portion is linked to the second hexose unit or its derivative portion via an α-(1→6) glycosidic bond;

[0080] The second hexose unit or its derivative portion is linked to the third hexose unit or its derivative portion via a β-(1→4) glycosidic bond;

[0081] The third hexose unit or its derivative has the following structure:

[0082] or

[0083] In one embodiment, formula (I) has the following structure:

[0084] Wherein, R1-R 12 Selected from -OH, or H;

[0085] Preferably, the two substituents on the same carbon atom are different;

[0086] More preferably, the two substituents on the same carbon atom are different; and R3 is H, and R4 is -OH;

[0087] More preferably, the two substituents on the same carbon atom are different; and R3 is H, R4 is -OH; R9 is H, R 10 is -OH;

[0088] More preferably, the two substituents on the same carbon atom are different; and R3 is H, R4 is -OH; R9 is H, R 10 is -OH; R8 is H, R7 is -OH; R 12 H, R 11 is -OH;

[0089] The definitions of L and P refer to those of formula (I).

[0090] In one embodiment, the structure of formula (III) is as shown in formula (III-1):

[0091] Wherein, R1, R2, R5, and R6 are selected from -OH or H; and the two substituents on the same carbon atom are different.

[0092] In one embodiment, the first hexose unit or its derivative portion is selected from glucosyl, mannosyl, galactosyl, fructosyl, gulosyl, idosyl or their derivatives; wherein the 6-hydroxyl group of the first hexose unit or its derivative portion is oxidized to a -C(O)- form and connected to the terminal -NH- of the L.

[0093] In one embodiment, the first six-carbon sugar unit or its derivative is selected from in, represents the site of connection to L; * represents the site of connection to the second hexose unit or its derivative.

[0094] In one embodiment, the second hexose unit or its derivative portion is selected from glucosyl, mannosyl, galactosyl, fructosyl or their derivatives; wherein the first hexose unit or its derivative portion is linked to the second hexose unit or its derivative portion via an α-(1→6) glycosidic bond; and the second hexose unit or its derivative portion is linked to the third hexose unit or its derivative portion via a β-(1→4) glycosidic bond.

[0095] In one embodiment, the second six-carbon sugar unit or its derivative moiety is selected from

[0096] in, represents the site of attachment to the third hexose unit or its derivative; * represents the site of attachment to the first hexose unit or its derivative.

[0097] In one embodiment, the derivatives in the hexose unit or its derivative part are independently selected from uronic acid or monosaccharide derivatives in which the hydroxyl group is replaced by an acylamino group (e.g., an alkanoylamino group, such as formylamino, acetylamino, propionylamino, etc., especially acetylamino).

[0098] In one embodiment, the trisaccharide unit structure has the following structure:

[0099] In one embodiment, -L-(P) t -L 2 -L 1 -BP, that is, formula (I): D——L2——L1——B——P

[0100] Formula (I-1)

[0101] in

[0102] B is independently absent, or is 1) below, or is 2) below, or is a combination of 1) and 2) below: 1) a self-immolative spacer Sp1; 2) a divalent group, or a combination of two or more divalent groups, wherein the divalent group is selected from: -CR 1 R 2 -、C 1-10 Alkylene, C 4-10 Cycloalkylene, C 4-10 heterocyclylene and -(CO)-;

[0103] L 1 independently absent; or a non-cleavable sequence; or a cleavable sequence comprising an enzymatically cleavable amino acid sequence comprising 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids;

[0104] L 2 Independently absent; or the following 1); or the following 2); or the following 1) and 2) combination:

[0105] 1)-NH-C 2-20 Alkylene, wherein one or more -CH2- structures in the alkylene are optionally replaced by: -CR 3 R 4 -, -O-, -(CO)-, -S-, -S(=O)2-, -NR 5 -、-N ⊕ R 6 R 7 -、C 4-10 Cycloalkylene, C 4-10 Heterocyclylene, phenylene, wherein cycloalkylene, heterocyclylene and phenylene are each independently unsubstituted or selected from halogen, -C 1- 10 Alkyl, -C 1-10 Haloalkyl, -C 1-10 Alkylene-NH-R 8 and -C 1-10 Alkylene-OR 9 is substituted with at least one substituent;

[0106] 2) Amino acid residue sequence, i.e. -*(AA) n **-, n is an integer from 1 to 100, AA is independently an amino acid residue at each occurrence, * represents the N-terminus of the corresponding amino acid, ** represents the C-terminus of the corresponding amino acid, and -(C2H4-O) is optionally present between the amino group and the α-carbon of an amino acid m -(CH2) p -, wherein m is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; p is 0, 1, 2 or 3, and the * end forms an amide bond with the carbonyl group in the trisaccharide structure;

[0107] Among them, B, L 1 and L 2 Not existing at the same time;

[0108] R 1 、R 2 、R 3 、R 4 、R 5 、R 6 、R 7 、R 8 、R 9 are each independently selected from hydrogen, halogen, substituted or unsubstituted -C 1-10 Alkyl, C 4-10 Cycloalkylene; or R1 and R 2 and together with the carbon atoms to which they are attached form a 3-6 membered cycloalkylene group, and / or R 3 and R 4 and together with the carbon atoms to which they are attached form a 3-6 membered cycloalkylene group;

[0109] P is with B part, or L 1 Part, or L 2 partially connected loads;

[0110] D is as defined herein.

[0111] In another embodiment, -L-(P) t for That is, formula (I) is:

[0112] in,

[0113] Ld2 and each Ld1 are independently a bond; or selected from -NH-C 1-20 Alkylene-(CO)-, -NH-(PEG) i -(CO)-; or each independently unsubstituted or -CO-(PEG) on the side chain j -R 11 Substituted natural amino acids or oligomeric natural amino acids having a degree of polymerization of 2-10 (i.e., 2, 3, 4, 5, 6, 7, 8, 9 or 10);

[0114] -(PEG) i -and-(PEG) j - Each is a PEG fragment comprising a specified number of consecutive -(O-C2H4)- structural units or consecutive -(C2H4-O)- structural units, optionally with C appended at one end 1-10 alkylene;

[0115] M is hydrogen or LKa-L 2 ―L 1 ―B―P;

[0116] Q is NH2 or L 2 ―L 1 ―B―P;

[0117] Provided that the following is not included: M is hydrogen and at the same time Q is NH2;

[0118] Each LKa is independently selected

[0119] opSu is or a mixture thereof; wherein * represents and L 2 The connecting part;

[0120] Each L 2 Independently absent; or the following 1); or the following 2); or the following 1) and 2) combination:

[0121] 1)-NH-C 2-20 Alkylene, wherein one or more -CH2- structures in the alkylene are optionally replaced by: -CR 3 R 4 -, -O-, -(CO)-, -S-, -S(=O)2-, -NR 5 -、-N ⊕ R 6 R 7 -、C 4-10 Cycloalkylene, C 4-10 Heterocyclylene, phenylene, wherein cycloalkylene, heterocyclylene and phenylene are each independently unsubstituted or selected from halogen, -C 1- 10 Alkyl, -C 1-10 Haloalkyl, -C 1-10 Alkylene-NH-R 8 and -C 1-10 Alkylene-OR 9 substituted with at least one substituent; preferably, -NH-(C2H4O)2-(C2H4)CO-;

[0122] 2) Amino acid residue sequence, i.e. -*(AA) n **-, n is an integer from 1 to 100, AA is independently an amino acid residue at each occurrence, * represents the N-terminus of the corresponding amino acid, ** represents the C-terminus of the corresponding amino acid, and -(C2H4-O) is optionally present between the amino group and the α-carbon of an amino acid m -(CH2) p -, wherein m is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; p is 0, 1, 2 or 3, and the * end forms an amide bond with the carbonyl group in the trisaccharide structure; preferably, -Gly-Gly-Gly-;

[0123] L 1 Independently absent; or a non-cleavable sequence, for example, coupling the load to the antibody via a thioether bond; or a cleavable sequence comprising an amino acid sequence cleavable by an enzyme, the amino acid sequence cleavable by the enzyme comprising 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids;

[0124] Each B is independently absent, or is 1) below, or is 2) below, or is a combination of 1) and 2) below: 1) a self-immolative spacer Sp1; 2) a divalent group, or a combination of two or more divalent groups, wherein the divalent group is selected from: -CR1 R 2 -、C 1-10 Alkylene, C 4-10 Cycloalkylene, C 4-10 heterocyclylene and -(CO)-;

[0125] Among them, B, L 1 and L 2 Not existing at the same time;

[0126] P is with B part, or L 1 Part, or L 2 partially connected loads;

[0127] R 1 、R 2 、R 3 、R 4 、R 5 、R 6 、R 7 、R 8 、R 9 are each independently selected from hydrogen, halogen, substituted or unsubstituted -C 1-10 Alkyl, C 4-10 Cycloalkylene; or R 1 and R 2 and together with the carbon atoms to which they are attached form a 3-6 membered cycloalkylene group, and / or R 3 and R 4 and together with the carbon atoms to which they are attached form a 3-6 membered cycloalkylene group;

[0128] R 11 It is C 1-10 alkyl;

[0129] d is 0, 1, 2, 3, 4, 5, or 6;

[0130] Each i is independently an integer from 1 to 100 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10), preferably 1 to 20; preferably, each i is independently an integer from 1 to 12; more preferably, 2 to 8; especially 4;

[0131] Each j is independently an integer from 1 to 100 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10), preferably 1 to 20; preferably, each j is independently an integer from 1 to 12; more preferably, from 8 to 12; especially, 8, 9, 12, or 13.

[0132] In one embodiment, B, L 1 and L 2 At least one of them is not "not present".

[0133] In one embodiment, L 2is the amino acid residue sequence, i.e. -*(AA) n **-, n is an integer from 1 to 100, AA is independently an amino acid residue at each occurrence, * represents the N-terminus of the corresponding amino acid, ** represents the C-terminus of the corresponding amino acid, and -(C2H4-O) is optionally present between the amino group and the α-carbon of an amino acid m -(CH2) p -, wherein m is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; p is 0, 1, 2 or 3, and the * end forms an amide bond with the carbonyl group in the trisaccharide structure. In one embodiment, AA is independently 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 at each occurrence. In one embodiment, n is an integer from 1 to 50, preferably an integer from 1 to 30, preferably an integer from 1 to 20, preferably an integer from 1 to 10, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10.

[0134] In one embodiment, L 1 A cleavable sequence comprising an amino acid sequence cleavable by an enzyme, wherein the amino acid sequence cleavable by the enzyme comprises 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 -Val-Ala-, -Gly-Gly-Phe-Gly-, -Phe-Lys-, -Val-Cit-, -Val-Lys-, -Gly-Phe-Leu-Gly-, -Ala-Leu-Ala-Leu-, -Ala-Ala-Ala- and combinations thereof; a preferred 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 -Val-Ala-, -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 It means -Val-Cit-.

[0135] In one embodiment, Sp1 is selected from PABC (p-aminobenzyloxycarbonyl), acetal, heteroacetal and a combination thereof; preferably, Sp1 is acetal, heteroacetal or PABC; further preferably, the heteroacetal is selected from N,O-heteroacetal; more preferably, Sp1 is -O-CH2-U- or -NH-CH2-U-, wherein -O- or -NH- is linked to an amino acid sequence that can be cleaved by the enzyme, U is absent, or is CH2, O, S or NH, preferably O or S; further preferably, Sp1 is PABC.

[0136] In one embodiment, B is absent. In one embodiment, B is 1), 2), or a combination of 1) and 2): 1) a self-immolative spacer Sp1; 2) a divalent group, or a combination of two or more divalent groups, wherein the divalent group is selected from: -CR 1 R 2 -、C 1-10 In one embodiment, B is connected to the support through an amide bond, 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)-; wherein g is 1, 2, 3, 4, 5 or 6; U is absent or is CH2, O, S or NH, preferably O or S.

[0137] In one embodiment, -L 1 -B- stands for -Val-Cit-PABC-.

[0138] In one embodiment, -L 1 -B- stands for -Gly-Gly-Phe-Gly-.

[0139] In one embodiment, -L 2 -L 1 -B- denotes -Gly-Gly-Gly-Val-Cit-PABC-.

[0140] In one embodiment, -L 2 -L 1 -B- represents -HN-(C2H4-O) m -(CH2) p -Gly-Gly-Phe-Gly-.

[0141] In one embodiment, Ld2 and each Ld1 are independently selected from a bond, or

[0142] Each i, j and k is independently selected from an integer from 1-100.

[0143] In one embodiment, each i, j, and k is independently selected from an integer from 1 to 20. In one embodiment, each i, j, and k is independently selected from an integer from 1 to 12.

[0144] In one embodiment, each i is independently selected from an integer from 2 to 8; particularly 4.

[0145] In one embodiment, each j is independently selected from an integer from 8 to 12; particularly 8 or 12.

[0146] In one embodiment, each k is independently selected from an integer of 1-7; in particular 1 or 3 or 5.

[0147] In one embodiment, Ld2 and each Ld1 are independently selected from a bond; or a C with an amino group and a carbonyl group at both ends. 1- 20 Alkylene, or a PEG fragment of a certain length with an amino group and a carbonyl group at both ends (represented as -(PEG) i -), or one or more natural amino acids, each of which is independently unsubstituted or substituted with a PEG fragment of a certain length (represented as -CO-(PEG) j -)replace.

[0148] In one embodiment, -(PEG) i -Contains -(O-C2H4) i -or-(C2H4-O) i -, and optionally appended with C at one end 1-10 Alkylene; -(PEG) j -Contains -(O-C2H4) j -or-(C2H4-O) j -, and optionally appended with C at one end 1- 10 In a very specific embodiment, -(PEG) i - contains -C2H4-(O-C2H4) i -or-(C2H4-O) i -C2H4-.

[0149] In one embodiment, the load can be selected from the group consisting of small molecule compounds (e.g., small molecule drugs with various mechanisms of action, including various traditional small molecule drugs, photoacoustic dynamic therapy drugs, photothermal therapy drugs, etc., such as chemotherapy drugs, small molecule targeted drugs, immune agonists, etc., such as traditional cytotoxic drugs, such as cisplatin, paclitaxel, 5-fluorouracil, cyclophosphamide and bendamustine, etc.; small molecule targeted drugs, such as imatinib mesylate, gefitinib and anlotinib, etc.; 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 radioactive labels, etc.), short peptides, polypeptides, peptidomimetics and proteins. In one embodiment, the load is selected from the group consisting of small molecule compounds and nucleic acid molecules. In a preferred embodiment, the load is selected from small molecule compounds. In a more preferred embodiment, the load is selected from the group consisting of cytotoxins and fragments thereof.

[0150] In one embodiment, the cargo is a cytotoxin or a fragment thereof, optionally derivatized to attach to the L portion of formula (I), or the B portion, L portion of the compound of formula (I-1) or formula (I-2). 2 or L 1 part.

[0151] In one embodiment, the cytotoxin is selected from the group consisting of drugs that target the microtubule cytoskeleton. In a preferred embodiment, the cytotoxin is selected from the group consisting of taxanes, maytansinoids, auristatins, epothilones, combretastatin A-4 phosphate, combretastatin A-4 and its derivatives, indole-sulfonamides, vinca alkaloids such as vinblastine, vincristine, vindesine, vinorelbine, vinflunine, vinglycinate, anhy-drovinblastine, dolastatin 10 and its analogs, halichondrin B, indole-3-oxyacetamides, podophyllotoxins, 7-diethylamino-3-(2'-benzoxazolyl)-coumarin (DBC), discodermolide, and laulimalide. In another embodiment, the cytotoxin is selected from the group consisting of DNA topoisomerase inhibitors such as camptothecins and their derivatives, mitoxantrone, and mitoguanidine. In a preferred embodiment, the cytotoxin is selected from the group consisting of nitrogen mustards such as chlorambucil, naphthyl nitrogen mustard, cholophosphamide, estramustine, ifosfamide, nitrogen mustard, nitrogen oxide hydrochloride, melphalan, new nitrogen mustard, methionine, phenylephrine, prednimustine, trofosfamide, and uramustine. In another preferred embodiment, the cytotoxin is selected from the group consisting of nitrosoureas such as carmustine, flubenzuron, formoterol, lomustine, nimustine, and ranimustine. In one embodiment, the cytotoxin is selected from the group consisting of aziridines. In a preferred embodiment, the cytotoxin is selected from the group consisting of benzodopa, carboquinone, metodepa and uredepa. In one embodiment, the cytotoxin is selected from the group consisting of antitumor antibiotics. In a preferred embodiment, the cytotoxin is selected from the group consisting of enediyne antibiotics. In a more preferred embodiment, the cytotoxin is selected from the group consisting of dynemicin, esperamicin, neocarzinostatin, aclarubicin.In another preferred embodiment, the cytotoxin is selected from the group consisting of actinomycin, anthramycin, bleomycins, actinomycin C, carabinin, carminomycin, carminomycin, actinomycin D, daunorubicin, detorubicin, doxorubicin, epirubicin, esorubicin, idarubicin, mexicomycin, mitomycins, noramycin, olivomycin, peplomycin, porfibrinocin, puromycin, ferroxorubicin, rhodorubicin, streptozocin, streptozocin, zoloft, and daunorubicin. In yet another preferred embodiment, the cytotoxin is selected from the group consisting of trichothecenes. In a more preferred embodiment, the cytotoxin is selected from the group consisting of T-2 toxin, verracurin A, baculozolin A, and anguidine. In one embodiment, the cytotoxin is selected from the group consisting of antitumor amino acid derivatives. In a preferred embodiment, the cytotoxin is selected from the group consisting of ubenimex, azaserine, and 6-diazo-5-oxo-L-norleucine. In another embodiment, the cytotoxin is selected from the group consisting of folic acid analogs. In a preferred embodiment, the cytotoxin is selected from the group consisting of folate, methotrexate, pteropterin, trimetrexate, and edatrexate. In one embodiment, the cytotoxin is selected from the group consisting of purine analogs. In a preferred embodiment, the cytotoxin is selected from the group consisting of fludarabine, 6-mercaptopurine, thiabendine, and thioguanine. In yet another embodiment, the cytotoxin is selected from the group consisting of pyrimidine analogs. In a preferred embodiment, the cytotoxin is selected from the group consisting of ancitabine, gemcitabine, enocitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, and floxuridine. In one embodiment, the cytotoxin is selected from the group consisting of androgens. In a preferred embodiment, the cytotoxin is selected from the group consisting of captestosterone, drostanolone propionate, cyclothiocarbamate, melastosane, and testolactone. In another embodiment, the cytotoxin is selected from the group consisting of anti-adrenal drugs. In a preferred embodiment, the cytotoxin is selected from the group consisting of aminoglutethimide, mitotane, and trilostane. In one embodiment, the cytotoxin is selected from the group consisting of antiandrogens. In a preferred embodiment, the cytotoxin is selected from the group consisting of flutamide, nilutamide, bicalutamide, leuprolide acetate, and goserelin. In yet another embodiment, the cytotoxin is selected from the group consisting of protein kinase inhibitors and proteasome inhibitors.In another embodiment, the cytotoxin is selected from the group consisting of vinca alkaloids, colchicines, taxanes, auristatins, maytansinoids, calicheamicin, doxonubicin, duocarmucin, SN-38, cryptophycin analogs, deruxtecan, duocarmazine, calicheamicin, centanamycin, dolastansine, pyrrolobenzodiazepine (PBD), and exatecan. In one embodiment, the cytotoxin is selected from the group consisting of vinca alkaloids, colchicines, taxanes, auristatins, and maytansinoids.

[0152] In one embodiment, the cytotoxin is exatecan or a derivative thereof, such as DX8951f and the like.

[0153] In one embodiment, the cytotoxin is a maytansinoid, such as DM1. It should be noted that when a cytotoxin comprising a sulfhydryl moiety is used, the sulfhydryl moiety can react with a maleimide moiety to form a sulfosuccinimide, such as a maytansinoid, such as DM1, and the cytotoxin can be directly linked via the sulfosuccinimide. In this case, it will be understood that in some embodiments, the cargo and the sulfhydryl moiety together constitute the cytotoxin, and thus in this case, the cargo represents the remainder of the cytotoxin molecule excluding the sulfhydryl moiety.

[0154] In one embodiment, the cytotoxin is an auristatin, such as MMAE (monomethyl auristatin E), MMAF (monomethyl auristatin F), MMAD (monomethyl auristatin D), etc. In one embodiment, the cytotoxin is MMAE. The synthesis and structure of auristatin compounds are described in US20060229253, the entire disclosure of which is incorporated herein by reference.

[0155] The carrier contains an active group that can react with the active group in the compound of formula (I) and thus covalently conjugate the carrier to the compound of formula (I). Compounds without active groups require appropriate derivatization to obtain the carrier.

[0156] In one embodiment, the cytotoxin is a compound of formula (i)

[0157] Where g is any integer from 1 to 6;

[0158] In one embodiment, g is any integer from 1 to 3, preferably 1.

[0159] In one embodiment, the cytotoxin is selected from the following compounds 1-16; wherein the wavy bond represents the attachment site to the compound of formula (I).

[0160] In one embodiment, the linker-load compound having formula (I) is:

[0161] For example,

[0162] In another aspect, the present invention provides a linker-load compound obtained by a method comprising the steps of:

[0163] (i) oxidizing the 6-primary alcohol of the terminal first hexose unit in a trisaccharide containing a terminal first hexose unit and a terminal N-acetylglucosamine (GlcNAc) unit to a carboxyl group to obtain an intermediate compound (a) having a carboxyl group, wherein a second hexose unit or a derivative thereof is present between the terminal first hexose unit and the terminal N-acetylglucosamine (GlcNAc) unit;

[0164] (ii) reacting the carboxyl group in the intermediate compound (a) obtained in step (i) with the reactive group in the linker-carrier compound (b) having a reactive group at the end to obtain the linker-carrier compound.

[0165] In one embodiment of this other aspect,

[0166] The first six-carbon sugar unit is selected from glucosyl, mannosyl, galactosyl, fructosyl, gulosyl or idosyl; and / or

[0167] The second six-carbon sugar unit is selected from glucosyl, mannosyl, galactosyl or fructosyl; and / or

[0168] The first hexose unit or its derivative in the trisaccharide structure is connected to the second hexose unit or its derivative via an α-(1→6) glycosidic bond; and / or

[0169] The second hexose unit or its derivative in the trisaccharide is linked to the β-DN-acetylglucosamine portion or the β-D-glucose oxazoline portion via a β-(1→4) glycosidic bond; and / or

[0170] The derivatives are independently selected from derivatives in which the hydroxyl groups of the monosaccharide are replaced by acylamino groups (eg alkanoylamino groups, such as formylamino, acetylamino, propionylamino, etc., especially acetylamino groups).

[0171] In one embodiment of this other aspect,

[0172] The first six-carbon sugar unit is selected from mannosyl, glucosyl or galactosyl; and / or

[0173] The second six-carbon sugar unit is selected from mannosyl, glucosyl or galactosyl.

[0174] In one embodiment of this other aspect, the trisaccharide in step (i) has the following structure:

[0175] In one embodiment of this other aspect, the trisaccharide in step (i) has the following structure:

[0176] In one embodiment of this other aspect, the trisaccharide in step (i) has the following structure:

[0177] In one embodiment of this other aspect, the trisaccharide in step (i) has the following structure:

[0178] In one embodiment of this other aspect, the trisaccharide in step (i) has the following structure:

[0179] In one embodiment of this other aspect, the trisaccharide in step (i) has the following structure:

[0180] In one embodiment of this other aspect, the trisaccharide in step (i) has the following structure:

[0181] In one embodiment of this other aspect, the trisaccharide in step (i) has the following structure:

[0182] In one embodiment of this other aspect, the trisaccharide in step (i) has the following structure:

[0183] In one embodiment of this other aspect, the reactive group of the linker-carrier compound (b) with a reactive group at the end in step (ii) is an amino group.

[0184] In one embodiment of this other aspect, the method may further include the step of converting the intermediate compound (a) having a carboxyl group into an acyl halide, and further reacting it with a linker-carrier compound (b) having a reactive group at the end to obtain the linker-carrier compound.

[0185] In one embodiment of this further aspect, the linker-load compound is a linker-load compound as defined in the first aspect or various embodiments thereof.

[0186] Preparation method of linker-load compound

[0187] In a second aspect, the present invention provides methods of preparing linker-load compounds having formula (I), wherein unless otherwise indicated, the various variables are as defined in the first aspect or various embodiments thereof.

[0188] In one embodiment, the method comprises reacting D with L'-(P) t An amide formation reaction is performed to form D——L——(P) t The step of preparing the method of claim 1, wherein L' is the same as L defined in the first aspect or various embodiments thereof, except that the -NH- in L connected to D is H2N- in L'.

[0189] In one embodiment, when D in the prepared linker-load compound of formula (I) is When the method can further include Condensation In one embodiment, the condensation is carried out in the presence of water, a base and 2-chloro-1,3-dimethylimidazolinium chloride (DMC, cas: 37091-73-9). In one embodiment, the base is an inorganic base or an organic base. In one embodiment, the inorganic base is potassium carbonate, potassium phosphate, etc. In one embodiment, the organic base is an amine, such as a tertiary amine, such as triethylamine (Et3N).

[0190] In one embodiment, the amide formation reaction is carried out in the presence of an organic solvent, an organic base and a condensation reagent. In one embodiment, the organic solvent is selected from N,N-dimethylformamide (DMF) and N,N-dimethylacetamide (DMA). In one embodiment, the organic base is selected from diisopropylethylamine (DIPEA) and N-methylmorpholine (NMM). In one embodiment, the condensation reagent is selected from 2-(7-benzotriazole oxide)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU), 2-(1H-benzotriazol L-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU), 1H-benzotriazol-1-yloxytripyrrolidino hexafluorophosphate (PyBOP), N,N'-diisopropylcarbodiimide (DIC) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCI).

[0191] In one embodiment, D-OH is

[0192] In one embodiment, Depend on Preparation. In one embodiment, the preparation is carried out under conditions where the substrate is debenzylated. In one embodiment, the deprotection is carried out in the presence of hydrogen and a palladium catalyst. In one embodiment, the palladium catalyst is selected from palladium carbon and palladium hydroxide on carbon. In one embodiment, the palladium catalyst is palladium on carbon.

[0193] In one embodiment, Depend on Preparation. In one embodiment, the preparation is carried out under alkaline conditions, and then the reaction system is adjusted to acidic conditions for purification. In one embodiment, the alkaline conditions are a reaction system with a pH of about 11-12, and the acidic conditions are a reaction system with a pH of about 6-7.

[0194] In one embodiment, Depend on Preparation. In one embodiment, the preparation is carried out in the presence of thioacetic acid (AcSH) and an organic solvent. In one embodiment, the organic solvent is selected from chloroform and pyridine or a mixture thereof.

[0195] In one embodiment, Depend on and Prepared by forming an α-(1→6) glycosidic bond. In one embodiment, the preparation is carried out under glycosidic bond forming conditions. In one embodiment, the glycosidic bond forming conditions include using N-iodosuccinimide and silver trifluoromethanesulfonate in an anhydrous environment.

[0196] In one embodiment, Depend on Preparation. In one embodiment, the preparation comprises the use of dibutylboron trifluoromethanesulfonate and borane-tetrahydrofuran.

[0197] In one embodiment, Depend on Preparation. In one embodiment, the preparation comprises the use of acetic anhydride.

[0198] In one embodiment, Depend on Preparation. In one embodiment, the preparation comprises the use of iodomethane.

[0199] In one embodiment, Depend on Preparation. In one embodiment, the preparation comprises the use of 2,2,6,6-tetramethylpiperidinyloxide (TEMPO) and iodophenyldiacetic acid.

[0200] In one embodiment, Depend on Preparation. In one embodiment, the preparation is carried out in the presence of an acid. In one embodiment, the acid is p-toluenesulfonic acid.

[0201] In one embodiment, Depend on Preparation. In one embodiment, the preparation comprises using a catalyst and acetic anhydride. In one embodiment, the catalyst is 4-dimethylaminopyridine (DMAP).

[0202] Antibody-drug conjugates

[0203] In a third aspect, the present invention provides an antibody-drug conjugate having formula (II) based on site-directed attachment of the N-glycosylation site in the Fc region of an antibody:

[0204] in

[0205] P is the load;

[0206] R is hydrogen or α-L-fucosyl;

[0207] q is 1 or 2;

[0208] Ab is an antibody or antigen-binding fragment (for example, -NHC(O)CH2- linked to Ab in formula (II) is derived from asparagine at position 297 of the antibody Fc region);

[0209] The first hexose unit or its derivative portion is linked to the second hexose unit or its derivative portion via an α-(1→6) glycosidic bond;

[0210] The second six-carbon sugar unit or its derivative is connected to the β-DN-acetylglucosamine portion via a β-(1→4) glycosidic bond;

[0211] The 6-OH of the first hexose unit or its derivative is oxidized to -C(O)-;

[0212] L is a linker, and L is directly connected to the -C(O)- in the first hexose unit or its derivative portion via the terminal -NH- therein; wherein when L is an unbranched linker, it is connected to 1 P, and t is 1, and when L is a branched linker, each branch can be connected to 1 P, and t is an integer greater than 1 (e.g., 2, 3, 4, 5, 6, 7, 8, 9 or 10).

[0213] In one embodiment, the first six-carbon sugar unit or its derivative moiety is selected from glucosyl, mannosyl, galactosyl, fructosyl, gulosyl, idosyl or derivatives thereof.

[0214] In one embodiment, the first six-carbon sugar unit or its derivative is selected from in, represents the site of connection to L; * represents the site of connection to the second hexose unit or its derivative.

[0215] In one embodiment, the second six-carbon sugar unit or its derivative is selected from glucosyl, mannosyl, galactosyl, fructosyl or their derivatives. In one embodiment, the second six-carbon sugar unit or its derivative is selected from

[0216] in, indicates the site of attachment to the β-DN-acetylglucosamine moiety; * indicates the site of attachment to the first six-carbon sugar unit or its derivative.

[0217] In one embodiment, the derivatives in the hexose unit or its derivative part are independently selected from uronic acid or monosaccharide derivatives in which the hydroxyl group is replaced by an acylamino group (e.g., an alkanoylamino group, such as formylamino, acetylamino, propionylamino, etc., especially acetylamino).

[0218] In one embodiment, the -NHC(O)CH2- group in formula (II) that is attached to Ab is derived from asparagine at position 297 of the Fc region of an antibody.

[0219] In some embodiments, Ab is a monoclonal antibody. In some embodiments, Ab is an anti-human HER2 antibody or its antigen-binding fragment. Examples of anti-human HER2 antibodies include but are not limited to Pertuzumab and Trastuzumab. In one embodiment, Ab is Trastuzumab.

[0220] In one embodiment, formula (II) is formula (II-1):

[0221] In one embodiment, formula (II) is formula (II-2):

[0222] In one embodiment, formula (II) is formula (II-3):

[0223] In one embodiment, formula (II) is formula (II-4):

[0224] In one embodiment, formula (II) is formula (II-5):

[0225] In one embodiment, formula (II) is formula (II-6):

[0226] In one embodiment, formula (II) is formula (II-7):

[0227] In one embodiment, formula (II) is formula (II-8):

[0228] In one embodiment, formula (II) is formula (II-9):

[0229] in

[0230] R is hydrogen or α-L-fucosyl;

[0231] q is 1 or 2;

[0232] Ab is an antibody or antigen-binding fragment (for example, -NHC(O)CH2- linked to Ab in formula (II-1) is derived from asparagine at position 297 of the antibody Fc region);

[0233] The remaining variables are as defined in the first aspect or various embodiments thereof.

[0234] In a preferred embodiment, Ab is an anti-ErbB2 / HER2 antibody (e.g., trastuzumab);

[0235] (P) t -L- is:

[0236] In one embodiment, R in formula (II) is α-L-fucosyl;

[0237] q is 2;

[0238] Ab is trastuzumab;

[0239] (P) t -L- is

[0240] For example,

[0241] The -NHC(O)CH2- group in formula (II) that is connected to Ab is derived from asparagine at position 297 in the Fc region of the antibody.

[0242] Method for preparing antibody-drug conjugates

[0243] In a fourth aspect, the present invention provides a method for preparing an antibody-drug conjugate having formula (II), comprising conjugating a linker-load compound having formula (I) to an antibody Ab, wherein unless otherwise indicated, each variable is as defined in the third aspect or its various embodiments.

[0244] In one embodiment, the method comprises the steps of:

[0245] a) Under the catalysis of glycosidase or its mutant, the antibody Ab is cleaved from its N-glycan chain to obtain an antibody whose Fc region N-glycosylation site is modified with N-acetylglucosamine or fucosyl-α-1,6-N-acetylglucosamine;

[0246] b) coupling the modified antibody obtained in step a) with the linker-load compound described above under the catalysis of glycosidase or a mutant thereof;

[0247] The glycosidase or its mutant used in step a) and step b) may be the same or different.

[0248] In one embodiment, the glycosidase or its mutant used in steps a) and b) is a fucosylate, an N-acetylglucosamine endohydrolase, or a mutant thereof. In one embodiment, the N-acetylglucosamine endohydrolase comprises at least one selected from the group consisting of 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); preferably, the endoglycosidase is Endo H, Endo D, Endo F2, Endo F3, Endo M, Endo CC1, Endo CC2, Endo Om, Endo S, and Endo S2. In one embodiment, the enzyme is Endo S2.

[0249] In one embodiment, steps a) and b) are performed by one-pot enzymatic catalysis.

[0250] In one embodiment, steps a) and b) are performed by one-pot enzymatic catalysis, and the enzyme is Endo S2.

[0251] trisaccharide linker

[0252] In one embodiment, a trisaccharide structure of the following structural formula (IV) is disclosed:

[0253] Wherein, in formula (IV), R1-R 12 Selected from -OH, or H;

[0254] Preferably, the two substituents on the same carbon atom are different;

[0255] More preferably, the two substituents on the same carbon atom are different; and R3 is H, and R4 is -OH;

[0256] More preferably, the two substituents on the same carbon atom are different; and R3 is H, R4 is -OH; R9 is H, R 10 is -OH;

[0257] More preferably, the two substituents on the same carbon atom are different; and R3 is H, R4 is -OH; R9 is H, R 10 is -OH; R8 is H, R7 is -OH; R 12 H, R 11 is -OH;

[0258] The definitions of L and P refer to those of formula (I).

[0259] In one embodiment, the structure of formula (IV) is shown in formula (IV-1):

[0260] Wherein, R1, R2, R5, and R6 are selected from -OH or H; and the two substituents on the same carbon atom are different.

[0261] In one embodiment, the trisaccharide structure of formula (IV) or (IV-1) is combined with an amino group-containing fragment to form an amide bond to link the drug fragment, or is connected to a linker with a bioorthogonal functional group in the form of an amide bond, and then reacted with a drug fragment having the aforementioned bioorthogonal functional group to form an antibody drug conjugate.

[0262] Pharmaceutical compositions and pharmaceutical preparations

[0263] Another object of the present invention is to provide a pharmaceutical composition comprising a preventive or therapeutically effective amount of the antibody-drug conjugate of the present invention and at least one pharmaceutically acceptable carrier.

[0264] The pharmaceutical composition of the present invention can be administered in any manner as long as it achieves the effect of preventing, alleviating, preventing or treating the symptoms of humans or animals. For example, various suitable dosage forms can be prepared according to the route of administration, particularly injections such as lyophilized powder injections, injections or sterile injection powders.

[0265] The term "pharmaceutically acceptable" means that it does not produce undue toxicity, irritation or allergic reaction when in contact with patient tissues within the scope of normal medical judgment, has a reasonable ratio of advantages to disadvantages, and is effective for the intended use.

[0266] The term "pharmaceutically acceptable carrier" refers to any carrier material that is pharmaceutically acceptable and does not interfere with the biological activity and performance of the antibody-drug conjugate. Examples of aqueous carriers include, but are not limited to, buffered saline. Pharmaceutically acceptable carriers also include substances that allow the composition to approach physiological conditions, such as pH adjusters and buffers, toxicity modifiers, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, and the like.

[0267] In one embodiment, the drug / antibody ratio (DAR) of the pharmaceutical composition of the present invention is an integer or non-integer of 1-20, such as about 1 to about 10, about 1 to about 8, about 1 to about 6, about 1 to about 4, about 1 to about 3, about 1 to about 2.5, or about 1 to about 2. In a specific embodiment, the DAR of the antibody-drug conjugate of the present invention is about 2, about 4, about 6, or about 8. In another specific embodiment, the DAR of the antibody-drug conjugate of the present invention is about 2. In yet another specific embodiment, the DAR of the antibody-drug conjugate of the present invention is about 1.65.

[0268] Treatment methods and uses

[0269] The antibody-drug conjugates of the present invention can be used to treat tumors and / or autoimmune diseases. Tumors sensitive to antibody-drug conjugate therapy include those characterized by specific tumor-associated antigens or cell surface receptors. These tumor cells can be recognized by the targeting molecules in the antibody-drug conjugate and, in turn, killed by the cargo / cytotoxin in the antibody-drug conjugate.

[0270] Therefore, in another aspect, the present invention also provides use of the antibody-drug conjugate of the present invention or the pharmaceutical composition of the present invention in the preparation of a medicament for treating a disease, disorder or condition selected from a tumor or an autoimmune disease.

[0271] In another aspect, the present invention provides an antibody-drug conjugate or a pharmaceutical composition of the present invention for use in treating tumors or autoimmune diseases.

[0272] In a further aspect, the present invention provides a method for treating tumors or autoimmune diseases, comprising administering an effective amount of the antibody-drug conjugate of the present invention or the pharmaceutical composition of the present invention to an individual in need thereof.

[0273] In one embodiment, the antibody-drug conjugate provided herein, formed by linking an anti-human HER2 antibody to a small molecule cytotoxin, can specifically bind to HER2 on the surface of tumor cells, selectively killing HER2-expressing tumor cells. In another embodiment, the present invention provides the use of the antibody-drug conjugate or pharmaceutical composition of the present invention in the preparation of a medicament for treating a disease, disorder, or condition selected from HER2-positive tumors. In a more preferred embodiment, the disease, disorder, or condition is selected from the group consisting of breast cancer, gastric cancer, lung cancer, ovarian cancer, and urothelial carcinoma.

[0274] The dosage of the antibody-drug conjugate administered to a subject can be adjusted to a considerable extent. The dosage can vary depending on the specific route of administration and the needs of the subject and can be subject to the judgment of a healthcare professional. Beneficial effects

[0275] The present invention expands the scope of antibody-drug conjugates obtained by sugar chain remodeling technology due to the use of a novel trisaccharide structure that forms an amide bond. In the present invention, a unique trisaccharide carboxylic acid substrate (e.g., ) can be well connected with amine-containing compounds through a mild and compatible amide bond formation method to achieve one-step connection, simplifying and accelerating the synthesis of linker-load compounds without the generation of adverse by-products, while enriching the source of substrates. Therefore, under predictable circumstances, all compounds with NH2 (such as L'-(P) t Compounds of the structure wherein P and t are as defined herein, except that -NH- in L connected to D is H2N- in L', and L' is the same as L as defined herein) can be formed via the above trisaccharide structure by a simple amide reaction to form a linker-loaded substance D——L——(P) comprising a trisaccharide linker. t The present invention further demonstrates that the linker-load can be efficiently and site-specifically coupled to an antibody via a one-pot enzyme-catalyzed coupling method, forming a highly uniform antibody-drug conjugate product.

[0276] The present invention utilizes modular combinatorial chemistry to efficiently synthesize a series of linker-loads based on trisaccharide structures, and verifies that it can achieve efficient coupling of different payloads. Due to the introduction of trisaccharides, its water solubility has increased to a certain extent, which has a meaningful improvement on the stability of antibody-drug conjugate products. The above linker-loads based on trisaccharide structures are a further supplement and improvement to the one-step sugar chain site-specific coupling technology based on disaccharides, especially enriching the source of substrates, improving the connection mode between disaccharides and effector molecules, enriching the sugar chain pattern of the resulting antibody-drug conjugates, improving the water solubility of antibody-drug conjugates, avoiding the limitations of the prior art that coupling still requires a certain amount of organic solvent to promote dissolution, and further improving the coupling efficiency of some substrates.

[0277] At the same time, the present invention further developed a series of trisaccharide molecular libraries with novel structures and differentiated characteristics, and based on this molecular library, constructed a group of antibody-drug conjugate molecular libraries containing structurally differentiated glycoforms in the antibody Fc region. These molecular libraries exhibit unique antibody-dependent cell-mediated cytotoxicity (ADCC) activity, which is significantly different from existing ADC technologies based on disaccharide linkers. The use of disaccharide linkers in traditional technologies often leads to the loss of ADCC function in antibody-drug conjugates such as ADCs. However, the technology we developed, through the introduction of trisaccharide linkers, enables new molecular libraries to meet the diverse needs of drug molecule design.

[0278] Furthermore, compared to disaccharide linkers, trisaccharide molecules possess greater hydrophilicity and are more compatible with hydrophobic payload molecules. This property not only enhances the water solubility of ADC molecules but also broadens the scope of ADC design. Therefore, by employing structurally diverse trisaccharide linkers, ADC molecules with differentiated Fc glycoforms and diverse physicochemical properties can be readily constructed, thereby better meeting the diverse needs of ADC drug development.

[0279] Example

[0280] The solution of the present invention is further described in detail below with reference to specific embodiments.

[0281] It should be noted that the following examples are merely examples for clearly illustrating the technical solutions of the present invention, and are not intended to limit the present invention. For those skilled in the art, other variations or modifications may be made based on the description of the present invention. It is not necessary and is not possible to exhaustively enumerate all embodiments herein, and the obvious variations or modifications derived therefrom are still within the scope of protection of the present invention. Unless otherwise indicated, the instruments, equipment, and reagents used herein are all commercially available.

[0282] 1. Preparation of trisaccharide linkers

[0283] Example 1: Preparation of Compound 1-1 Containing the First Hexose Unit or Its Derivatives

[0284] The following steps were used to prepare compound 1-1, whose structure is as follows:

[0285] (1) Preparation of compound 1-1b

[0286] Compound 1-1a (500 mg, 1.39 mmol, CAS No. 159407-19-9), dichloromethane (7.5 mL), and triethylamine were added sequentially at room temperature to a 25 mL single-necked flask and dissolved. The reaction system was then brought to 0°C, and 4-dimethylaminopyridine and acetic anhydride were added. The flask was sealed with a retractable stopper and allowed to naturally return to room temperature with stirring for approximately 6 hours. After TLC confirmed the completion of the reaction of the starting materials, the reaction was quenched by the addition of saturated sodium bicarbonate solution (20 mL) and extracted with dichloromethane (20 mL x 3). The resulting organic phase was washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by column chromatography (eluent: EtOAc / PE = 1 / 100-1 / 10) to afford compound 1-1b (554 mg, 90% yield, colorless, viscous oil). 1 H NMR (400MHz, chloroform-d) δ7.35-7.54(m,10H),5.65(dd,1H,J=1.4,3.4Hz),5.64(s,1H),5.48(s,1H),5.46(dd,1H,J=7.0,10.4Hz),5. 52(td,1H,J=4.8,9.8Hz), 4.30(dd,1H,J=4.9,10.4Hz), 4.17(t,1H,J=9.8Hz), 3.91(t,1H,J=10.3Hz), 2.21(s,3H), 2.09(s,3H). C 23 H 24 7S + [M+Na] + MS (ESI): calculated value: 467.11, found value: 467.22.

[0287] (2) Preparation of compound 1-1c

[0288] At room temperature, compound 1-1b (554 mg, 1.25 mmol), methanol (3 mL), tetrahydrofuran (6 mL), and p-toluenesulfonic acid were added to a 25 mL single-necked flask in sequence and dissolved. The flask was sealed with a retractable stopper and stirred at room temperature for approximately 3 hours. After TLC monitoring indicated that the starting material had reacted completely, saturated sodium bicarbonate solution (20 mL) was added to quench the reaction, and the reaction system was extracted with ethyl acetate (20 mL x 3). The resulting organic phase was washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by column chromatography (eluent: EtOAc / PE = 1 / 100-1 / 1) to obtain compound 1-1c (345 mg, 78% yield, colorless viscous oil). 1 H NMR (400 MHz, chloroform-d) δ 7.30-7.49 (m, 5H), 5.50 (dd, 1H, J = 1.6, 3.4 Hz), 5.44 (d, 1H, J = 1.5 Hz), 5.18 (dd, 1H, J = 3.3, 9.9 Hz), 4.26-4.22 (m, 1H), 4.09 (t, 1H, J = 9.8 Hz), 3.91 (s, 1H), 3.90 (s, 1H), 2.11 (s, 3H), 2.10 (s, 3H). 16 H 20 7S + [M+Na] + MS (ESI): calculated value: 379.08, found value: 379.15.

[0289] (3) Preparation of compound 1-1e

[0290] At 0°C, compound 1-1c (345 mg, 0.97 mmol), tert-butyl alcohol, dichloromethane, water, 2,2,6,6-tetramethylpiperidinyl oxide (TEMPO), and iodophenyl diacetic acid were added to a 25 mL single-necked bottle in sequence and dissolved. The mixture was allowed to naturally return to room temperature and stirred open for about 12 hours. After TLC monitoring showed that the reaction of the starting material was complete, a saturated sodium thiosulfate solution (15 mL) was added to quench the reaction, and the reaction system was extracted with dichloromethane (15 mL × 3). The resulting organic phase was washed with saturated brine (30 mL), then dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by column chromatography (eluent: EtOAc / PE = 1 / 100-1 / 1) to obtain compound 1-1d (201 mg, yield 56%, colorless viscous oily liquid). 16 H 18 O8S - [MH] - MS (ESI): calculated value: 369.06, found value: 369.10.

[0291] At room temperature, compound 1-1d (201 mg, 0.54 mmol), DMF (2 mL), cesium carbonate, and iodomethane were added to a 25 mL single-necked flask in sequence and dissolved. The mixture was stirred at room temperature for approximately 2 hours. After TLC monitoring indicated that the starting material had reacted completely, water (15 mL) was added to quench the reaction, and the reaction system was extracted with ethyl acetate (15 mL × 3). The resulting organic phase was washed with water (30 mL × 3) and saturated brine (30 mL), then dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by column chromatography (eluent: EtOAc / PE = 1 / 100-1 / 2) to obtain compound 1-1e (111 mg, 53% yield, as a white solid). 1 H NMR (400 MHz, chloroform-d) δ 7.30-7.52 (m, 5H), 5.66 (d, 1H, J = 3.4 Hz), 5.35 (t, 1H, J = 8.3 Hz), 5.31 (t, 1H, J = 3.4 Hz), 4.74 (d, 1H, J = 8.2 Hz), 4.11 (dd, 1H, J = 3.4, 8.5 Hz), 3.77 (s, 3H), 2.16 (s, 3H), 2.14 (s, 3H). 17 H 20 8SNa + [M+Na] + MS (ESI): calculated value: 407.08, found value: 407.16.

[0292] (4) Preparation of Compound 1-1

[0293] At room temperature, compound 1-1e (182 mg, 0.47 mmol), dichloromethane (4 mL), triethylamine, and acetic anhydride were added sequentially to a 25 mL single-necked flask and dissolved. The flask was sealed with a retractable stopper and stirred at room temperature for approximately 12 hours. After TLC monitoring indicated that the starting material had reacted completely, a saturated sodium thiosulfate solution (10 mL) was added to quench the reaction, and the reaction system was extracted with dichloromethane (10 mL x 3). The resulting organic phase was washed with saturated brine (30 mL), dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by column chromatography (eluent: EtOAc / PE = 1 / 100-1 / 4) to obtain compound 1-1 (119 mg, 59% yield, colorless viscous oil). 1H NMR (400 MHz, chloroform-d) δ 7.33-7.57 (m, 5H), 5.63 (d, 1H, J = 3.8 Hz), 5.50 (t, 1H, J = 8.2 Hz), 5.46 (t, 1H, J = 3.4 Hz), 5.37 (dd, 1H, J = 3.2, 8.5 Hz), 4.83 (d, 1H, J = 7.9 Hz), 3.81 (s, 3H), 2.17 (s, 3H), 2.13 (s, 3H), 2.07 (s, 3H). 19 H 22 9SNa + [M+Na] + MS (ESI): calculated value: 449.09, found value: 449.17.

[0294] Example 2: Preparation of Compound 1-2 Containing the First Hexose Unit or Its Derivatives

[0295] According to the method of the literature (Beilstein Journal of Organic Chemistry, 2015, 11, 155–161), compound 1-2a (20 g, 51.2 mmol, CAS number: 604-68-2) was synthesized through 4 steps to finally obtain compound 1-2 (7.1 g, total yield 30%, colorless oily liquid). 1 H NMR(400MHz,DMSO-d)δ7.36–7.18(m,9H),5.64(s,1H),5.37(t,1H,J=9.0Hz),5.23(d,1H,J=10.0Hz),4.8 3(t,1H,J=8.9Hz),4.27(dd,1H,J=3.8,9.5Hz),3.84–3.73(m,3H),2.30(s,3H),2.04(s,3H),1.97(s,3H). C 24 H 26 7S + [M+Na] + MS (ESI): calculated value: 481.13, found value: 481.16.

[0296] Example 3: Preparation of Compounds 1-3 Containing the First Hexose Unit or Its Derivatives

[0297] According to the method of the literature (Synlett, 2009, 4, 603–606), compound 1-3a (15 g, 52.3 mmol, CAS number: 28244-98-6) was synthesized in 2 steps to finally give compound 1-3 (11.5 g, total yield 48%, colorless oily liquid).1 HNMR(400MHz,DMSO-d)δ7.40–7.09(m,9H),5.55(s,1H),5.18–5.15(m,1H),5.05–4.98(m,2H), 4.36(d,1H,J=3.4Hz),4.10–4.01(m,2H),3.84(s,1H),2.28(s,3H),1.98(s,3H),1.92(s,3H). C 24 H 26 7S + [M+Na] + MS (ESI): calculated value: 481.13, found value: 481.18.

[0298] Example 4: Preparation of Compounds 1-4 Containing the First Hexose Unit or Its Derivatives

[0299] At room temperature, compound 1-1 (6 g, 14.1 mmol), acetone (30 mL) and N-bromosuccinimide were added to a 250 mL single-necked flask in sequence and stirred at room temperature for about 1 hour. After TLC monitoring indicated that the reaction of the starting material was complete, a saturated sodium thiosulfate solution (10 mL) was added to quench the reaction, and the excess acetone was removed by concentration under reduced pressure. The reaction system was extracted with ethyl acetate (50 mL × 3). The resulting organic phase was washed with saturated brine (150 mL), then dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by column chromatography (eluent: EtOAc / PE = 1 / 10-1 / 2) to obtain compound 1-4a (3.8 g, yield 80%, colorless oily liquid). 13 H 22 NO 10 + [M+NH4] + MS (ESI): calculated value: 352.12, found value: 352.18.

[0300] Compound 1-4a (3.8 g, 11.3 mmol), anhydrous dichloromethane (50 mL), and trichloroacetonitrile were added to a 250 mL single-necked flask at 0°C and stirred for half an hour. 1,8-diazabicyclo[5.4.0]undec-7-ene was then added dropwise to the reaction mixture. After returning to room temperature, the mixture was stirred for 2 hours, concentrated under reduced pressure, and purified by column chromatography (eluent: EtOAc / PE = 1 / 10-1 / 1) to obtain compound 1-4 (2.5 g, total yield 3.4%, colorless oily liquid). 1H NMR (400MHz, DMSO-d) δ8.83 (s, 1H), 6.40 (d, 1H, J = 4.0Hz), 5.50–5.31 (m, 3H) ,4.51(d,1H,J=8.0Hz),3.86(s,3H),2.09(s,3H),2.05(s,3H),2.02(s,3H). C 15 H 18 Cl3NO 10 + [M+H] + MS (ESI): calculated value: 478.01, found value: 478.05.

[0301] Example 5: Preparation of disaccharide substrate compound 2-1

[0302] The following steps were used to prepare compound 2-1, the structure of which is as follows:

[0303] Preparation of compound 2-1

[0304] Compound 2-1a (500 mg, 0.55 mmol), anhydrous dichloromethane (10 mL), dibutylboron trifluoromethanesulfonate, and borane-tetrahydrofuran were added sequentially to a 25 mL Schlenk reaction flask at 0°C under nitrogen and dissolved. The mixture was stirred at 0°C for approximately 4 hours. After TLC confirmed the completion of the reaction of the starting materials, water was slowly added to the reaction system at 0°C to quench the reaction until bubbling ceased. The reaction system was then extracted with ethyl acetate (20 mL x 3). The resulting organic phase was washed sequentially with saturated sodium bicarbonate solution (50 mL), saturated ammonium chloride solution (50 mL), 1 M sodium hydroxide solution (50 mL), and saturated brine (50 mL), then dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by column chromatography (eluent: EtOAc / PE = 1 / 100-1 / 5) to obtain compound 2-1 (335 mg, 67% yield, colorless viscous oil). 1H NMR (400MHz, chloroform-d) δ7.30-7.45 (m, 30H), 5.10 (d, 1H, J = 11.8Hz), 4.97 (d, 1H, J = 12.0Hz), 4.90 (d, 1H, J = 10.9Hz), 4.88 (d, 1H, J = 11.8Hz), 4.83 (d, 1H, J = 11.8Hz), 4.73 (d, 1H, J = 12.0Hz), 4.69 (d, 1H, J = 10.8Hz), 4.67 (d, 1H, J = 12.1Hz), 4.61 (d, 1H, J = 10.9Hz ),4.55(s,2H),4.50(d,1H,J=11.6Hz),4.48(s,1H),4.35(d,1H,J=8.1Hz),3.95(t,1H,J=9.3Hz),3.83(t,1H,J=9.5Hz),3.76( d,1H,J=2.9Hz),3.72(dd,1H,J=2.3,11.2Hz),3.64–3.68(m,2H),3.51(t,1H,J=9.9Hz),3.34–3.40(m,4H),3.13–3.17(m,1H). C 54 H 57 N3O 10 Na + [M+Na] + MS (ESI): calculated value: 930.39, found value: 930.55.

[0305] Example 6: Preparation of disaccharide substrate compound 2-2

[0306] The following steps were used to prepare compound 2-2, the structure of which is as follows:

[0307] (1) Synthesis of compound 2-2b

[0308] To a 250 mL three-necked flask were added compound 2-2a (5 g, 10.5 mmol, CAS number: 342640-42-0), compound 1-2 (equivalent to 0.1-5 equivalents of 2-2a), 4A molecular sieves, and dichloromethane (100 mL) at room temperature. The mixture was stirred at room temperature for approximately 3 hours. The reaction system was cooled to -60°C, and N-iodosuccinimide and trifluoromethanesulfonic acid were added, followed by stirring at this temperature for 2 hours. After TLC confirmed the completion of the reaction of the starting materials, triethylamine was added to quench the reaction. The mixture was concentrated under reduced pressure and purified by column chromatography (eluent: EtOAc / PE = 1 / 10-1 / 5) to afford compound 2-2b (6.6 g, 82% yield, as a colorless, viscous oil). 1H NMR(400MHz,DMSO-d)δ7.43–7.29(m,20H),5.60(s,1H),5.22–5.18(m,1H),4.89 (d,1H,J=10.8Hz),4.84–4.81(m,3H),4.66(d,1H,J=12.3Hz),4.63(d,1H,J=12.0 Hz),4.62(d,1H,J=11.0Hz),4.58–4.54(m,2H),4.06–4.02(m,1H),3.84–3.75(m ,3H),3.68(dd,1H,J=4.5,11.3Hz),3.58–3.43(m,5H),1.98(s,3H),1.97(s,3H). C 44 H 51 N4O 12 + [M+NH4] + MS (ESI): calculated value: 827.35, found value: 827.27.

[0309] (2) Synthesis of Compound 2-2

[0310] To a 250 mL Schlenk reaction flask at 0°C under nitrogen was added compound 2-2b (6.6 g, 8.15 mmol), borane tetrahydrofuran complex, and dibutylboron trifluoromethanesulfonate. After dissolution, the mixture was stirred at 0°C for approximately 40 minutes. After TLC confirmed the complete reaction of the starting materials, triethylamine (6 mL) and methanol (6 mL) were added to the reaction system. The mixture and methanol were then concentrated under reduced pressure three times. Purification by column chromatography (eluent: EtOAc / PE = 1 / 5-1 / 2) afforded compound 2-2 (2.4 g, 36% yield, colorless, viscous oil). 1 H NMR (400MHz, DMSO-d) δ7.47–7.25(m,20H),5.09(t,1H,J=9.4Hz),4.97(d,1H,J=11.1Hz),4.82(d,1H,J=12.0Hz),4.75(d,1H,J=8.0Hz),4.70–4.60(m ,6H),4.55–4.49(m,3H),3.85(t,1H,J=8.9Hz),3.76–3.63(m,4H),3.57–3. 52(m,2H),3.50–3.46(m,2H),3.34–3.30(m,1H),1.92(s,3H),1.90(s,3H). C 44 H 49 N3O 12 Na +[M+Na] + MS (ESI): calculated value: 834.32, found value: 834.34.

[0311] Example 7: Preparation of disaccharide substrate compound 2-3

[0312] The following steps were used to prepare compound 2-3, the structure of which is as follows:

[0313] (1) Preparation of compound 2-3a

[0314] To a 250 mL three-necked flask were added compound 2-2a (5 g, 10.5 mmol, CAS number: 342640-42-0), compound 1-3 (equivalent to 0.1-5 equivalents of 2-2a), 4A molecular sieves, and dichloromethane (100 mL) at room temperature. The mixture was stirred at room temperature for approximately 3 hours. The reaction system was cooled to -60°C, and N-iodosuccinimide and trifluoromethanesulfonic acid were added, followed by stirring at this temperature for 2 hours. After completion of the reaction of the starting materials as monitored by TLC, triethylamine was added to quench the reaction. The mixture was concentrated under reduced pressure and purified by column chromatography (eluent: EtOAc / PE = 1 / 10-1 / 5) to afford compound 2-3a (6.6 g, 82% yield, as a colorless, viscous oil). 1 H NMR(400MHz, DMSO-d)δ7.41–7.17(m,20H),5.58(s,1H),5.07(d,1H,J=11.0Hz),5.03–4.95(m,2H),4.7 8(d,1H,J=12.1Hz),4.72(d,1H,J=7.7Hz),4.62(d,1H,J=12.1Hz),4.60(d,1H,J=10.9Hz),4.72(d,1H, J=12.1Hz),4.51(d,1H,J=7.7Hz),4.50(d,1H,J=12.2Hz),4.34(d,1H,J=3.4Hz),4.04–3.94(m,2H),3. 79(d,1H,J=9.5Hz),3.75–3.64(m,2H),3.55–3.49(m,2H),3.46–3.43(m,2H),1.96(s,3H),1.93(s,3H). C 44 H 51 N4O 12 + [M+NH4] + MS (ESI): calculated value: 827.35, found value: 827.31.

[0315] (2) Preparation of Compound 2-3

[0316] To a 250 mL Schlenk reaction flask at 0°C under nitrogen was added compound 2-3a (6.6 g, 8.15 mmol), borane tetrahydrofuran complex, and dibutylboron trifluoromethanesulfonate. After dissolution, the mixture was stirred at 0°C for approximately 40 minutes. After TLC confirmed the complete reaction of the starting materials, triethylamine (6 mL) and methanol (6 mL) were added to the reaction system. The mixture and methanol were then concentrated under reduced pressure three times. Purification by column chromatography (eluent: EtOAc / PE = 1 / 5-1 / 2) afforded compound 2-3 (2.4 g, 36% yield, colorless, viscous oil). 1 H NMR(400MHz,DMSO-d)δ7.39–7.20(m,20H),5.06(dd,1H,J=7.9,10.4Hz),4.93(d,1H ,J=10.5Hz),4.89(d,1H,J=10.2Hz),4.88(d,1H,J=10.4Hz),4.81(d,1H,J=12.1Hz) ,4.67–4.58(m,5H),4.54–4.51(m,3H),3.99(d,1H,J=3.2Hz),3.81(t,1H,J=9.2Hz) ,3.75–3.65(m,2H),3.54–3.47(m,4H),3.44–3.39(m,2H),1.98(s,3H),1.96(s,3H). C 44 H 53 N4O 12 + [M+NH4] + MS (ESI): calculated value: 829.37, found value: 829.34.

[0317] Example 8: Preparation of trisaccharide substrate compound 3-1

[0318] The following steps were used to prepare compound 3-1, whose structure is as follows:

[0319] (1) Preparation of compound 3-1a

[0320] Under oil pump vacuum conditions, a 25 mL Schlenk reaction flask was baked with a heat gun for 5 minutes. After cooling, activated molecular sieves were added to the system and baked for another 5 minutes. The nitrogen atmosphere was replaced by vacuum three times. Under nitrogen protection, a certain amount of compound 1-1 (0.1 to 10 equivalents relative to compound 2-1) and compound 2-1 (1.0 equivalent, 90.6 mg, 0.10 mmol) dissolved in anhydrous dichloromethane (6 mL) were added to the system. The reaction system was placed at 0°C and stirred for a period of time. Subsequently, a certain amount of N-iodosuccinimide and silver trifluoromethanesulfonate were added to the system. The system was allowed to naturally return to room temperature and stirred until the reaction was complete. Saturated sodium bicarbonate solution (10 mL) was added to quench the reaction, and the reaction system was extracted with ethyl acetate (10 mL × 3). The obtained organic phase was washed with saturated sodium thiosulfate solution (30 mL) and saturated brine (30 mL), then dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by column chromatography (eluent: EtOAc / PE = 1 / 100-1 / 9) to obtain compound 3-1a (64 mg, yield 52%, colorless viscous oily liquid). 1 H NMR (400MHz, chloroform-d) δ7.25-7.43(m,30H),5.36–5.38(m,2H),5.20(t,1H,J=2.4Hz),5.02(d,1H,J=11.2Hz),4.99(d,1H,J=2.1Hz),4.97 (d,2H,J=11.5Hz),4.85(s,2H),4.76(d,1H,J=9.5Hz),4.73(d,1H,J=10.1Hz),4.68(d,1H,J=12.2Hz),4.61(d,1H,J=11.1Hz),4.52(d ,1H,J=12.2Hz),4.47(d,1H,J=9.5Hz),4.46(s,1H),4.45(d,1H,J=10.0Hz),4.36(d,1H,J=8.3Hz),4.33–4.36(m,1H),4.04(t,1H,J=9 .3Hz),3.77(d,1H,J=2.8Hz),3.58–3.72(m,7H),3.54(d,1H,J=1.7,9.8Hz),3.29-3.47(m,5H),2.11(s,3H),2.05(s,3H),1.99(s,3H). C 67 H 73 N3O 19 Na + [M+Na] + MS (ESI): calculated value: 1246.47, found value: 1246.61.

[0321] (2) Preparation of compound 3-1b

[0322] At room temperature, compound 3-1a (150 mg, 0.12 mmol), chloroform, pyridine, and AcSH were added to a 10 mL Schlenk reaction flask and dissolved. The flask was sealed with a stopper and stirred at 60°C until the reaction was essentially complete as determined by HPLC, which took approximately 18 hours. After concentration under reduced pressure to remove most of the solvent, ethyl acetate (20 mL) was added to the flask. The flask was washed sequentially with saturated sodium bicarbonate solution (20 mL), 1 M hydrochloric acid (20 mL x 4), saturated sodium bicarbonate solution (20 mL), and saturated brine (20 mL). The organic phase was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by column chromatography (eluent: EtOAc / PE = 1 / 10-1 / 1) to afford compound 3-1b (72 mg, 47% yield, as a white solid). 1 H NMR (400 MHz, chloroform-d) δ7.15–7.32 (m, 30H), 5.25–5.27 (m, 2H), 5.16 (t, 1H, J = 2.4 Hz), 4.79–4.88 (m, 4H), 4.74 (s, 2H), 4.73 (d, 1H, J = 11.4 Hz), 4.55 (d, 1H, J = 11.4 Hz), 4.48–4.51 (m, 4H), 4.41 (d, 1H, J = 1 1.8Hz),4.38(d,1H,J=11.8Hz),4.35(d,1H,J=11.5Hz),4.24–4.28(m,1H),3.90–3.92(m,2H),3.59 –3.75(m,9H),3.57(s,3H),3.24–3.31(m,2H),1.97(s,3H),1.89(s,3H),1.85(s,3H),1.67(s,3H). C 69 H 77 NO 20 Na + [M+Na] + MS (ESI): calculated value 1262.49, found value 1262.67.

[0323] (3) Preparation of compound 3-1c

[0324] At room temperature, compound 3-1b (72 mg, 0.06 mmol), methanol (2 mL), and sodium methoxide were added to a 25 mL single-necked bottle in sequence. The pH of the reaction system was adjusted to 11-12 and stirred at room temperature for 4 hours until the reaction was detected by HPLC. The pH of the reaction system was adjusted to 6-7 and the reaction system was extracted with ethyl acetate (10 mL × 3). The resulting organic phase was washed with water (30 mL) and saturated brine (30 mL), then dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The reaction solution was purified by semi-preparative HPLC to obtain compound 3-1c (42 mg, yield 66%, white solid). 1 H NMR (400 MHz, chloroform-d) δ 7.21-7.41 (m, 30H), 4.94 (d, 1H, J = 11.0 Hz), 4.73-4.91 (m, 9H), 4.65 (d, 1H, J = 12.0 Hz), 4.58 (d, 1H, J = 10.4 Hz), 4.55 (d, 1H, J = 11.4 Hz), 4.51 (s, 1H), 4.50 (s ,1H),4.46(s,1H),4.45(d,1H,J=11.9Hz),4.24(d,1H,J=9.6Hz),3.66-4.03(m,11H),3 .59-3.62(m,1H),3.39(dd,1H,J=2.8,9.2Hz),3.34(dd,1H,J=4.8,9.9Hz),1.66(s,3H). C 62 H 70 NO 17 + [M+H] + MS (ESI): calculated value 1100.49, found value 1100.62.

[0325] (4) Preparation of Compound 3-1

[0326] At room temperature, compound 3-1c (14 mg, 0.01 mmol), tetrahydrofuran, methanol, and a certain amount of palladium / activated carbon catalyst were added to a 25 mL single-necked flask in sequence. The mixture was stirred under a hydrogen atmosphere until the starting material disappeared as detected by LC-MS. The reaction system was filtered, concentrated under reduced pressure, and pumped dry to obtain compound 3-1 (5 mg, 70% yield, as a white solid). 20 H 34 NO 17 + [M+H] + MS (ESI): calculated value 560.18, found value 560.30.

[0327] Example 9: Preparation of trisaccharide substrate compound 3-2

[0328] The following steps were used to prepare compound 3-2, the structure of which is as follows:

[0329] (1) Preparation of compound 3-2a

[0330] At room temperature, under nitrogen, compound 1-4 (0.1-10 equivalents relative to compound 2-2), compound 2-2 (1.2 g, 1.48 mmol), and toluene (100 mL) were added sequentially to a 250 mL three-necked flask. After dissolution, scandium trifluoromethanesulfonate was added to the system, and the mixture was heated to 60°C and stirred for approximately 2 hours. After TLC monitoring of the reaction of the starting materials, water (100 mL) was added to quench the reaction, and the mixture was extracted with dichloromethane (100 mL × 2). The organic phase was collected, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by column chromatography (eluent: EtOAc / PE = 1 / 50-1 / 5) to obtain compound 3-2a (0.9 g, 55% yield, colorless viscous oily liquid). 1 H NMR (400MHz, DMSO-d) δ7.36–7.21 (m, 20H), 5.17–5.06 (m, 4H), 4.96 (d, 1H, J = 2.2Hz), 4.81 (d, 1H, J = 12. 0Hz),4.76(d,1H,J=12.1Hz),4.71–4.65(m,3H),4.61(d,1H,J=12.3Hz),4.57(d,1H,J=12.1Hz),4.55( d,1H,J=11.3Hz),4.50–4.45(m,2H),4.28(d,1H,J=9.0Hz),3.80(t,1H,J=9.0Hz),3.70–3.57(m,6H),3 .53–3.50(m,5H),3.42–3.38(m,2H),1.97(s,3H),1.93(s,3H),1.88(s,3H),1.88(s,3H),1.87(s,3H). C 57 H 69 N4O 21 + [M+NH4] + MS (ESI): calculated value: 1145.45, found value: 1145.41.

[0331] (2) Preparation of compound 3-2b

[0332] Compound 3-2a (900 mg, 0.798 mmol), chloroform, pyridine, and AcSH were added sequentially to a 25 mL Schlenk reaction flask at room temperature and dissolved. The system was stirred at room temperature for 18 hours. After completion of the reaction of the starting materials as monitored by TLC, the mixture was concentrated under reduced pressure and purified by semi-preparative HPLC (mobile phase: H2O / MeCN = 70%) to afford compound 3-2b (550 mg, 60% yield, as a white solid). 1 H NMR (400MHz, DMSO-d) δ7.81 (d, 1H, J = 8.9Hz), 7.38–7.24 (m, 20H), 5.18–5.1 0(m,4H),4.96(d,1H,J=2.4Hz),4.78–4.68(m,4H),4.62–4.48(m,6H),4.28 (d,1H,J=8.3Hz),3.81(t,1H,J=9.0Hz),3.75–3.65(m,6H),3.60–3.51(m,6 H),3.47–3.43(m,1H),1.98(s,3H),1.97(s,3H),1.92(s,9H),1.73(s,3H). C 59 H 70 NO 22 + [M+H] + MS (ESI): calculated value: 1144.44, found value: 1144.46.

[0333] (3) Preparation of compound 3-2c

[0334] Compound 3-2b (550 mg, 0.481 mmol) and methanol (10 mL) were added to a 25 mL single-necked vial at room temperature. After dissolution, 1 M sodium hydroxide solution (1 mL) was slowly added over 10 minutes. The mixture was stirred at room temperature for 2 hours until the reaction was complete as determined by HPLC. Acetic acid (1 mL) was added to the reaction system to quench the reaction. The mixture was concentrated under reduced pressure to afford crude compound 3-2c (300 mg, 68% yield, colorless oil). 1H NMR(400MHz,DMSO-d)δ7.99(d,1H,J=8.8Hz),7.37–7.23(m,20H),5.43–5.39(m,1H),4.90 (d,1H,J=10.7Hz),4.84(d,1H,J=11.4Hz),4.76(d,1H,J=11.4Hz),4.74(s,1H),4.67–4.6 6(m,1H),4.60–4.49(m,7H),4.42(d,1H,J=7.8Hz),3.90–3.80(m,3H),3.72–3.61(m,3H), 3.58–3.49(m,6H),3.37–3.35(m,2H),3.15–3.14(m,1H),3.08–3.04(m,1H),1.77(s,3H). C 48 H 58 NO 17 + [M+H] + MS (ESI): calculated value: 920.37, found value: 920.24.

[0335] (4) Preparation of Compound 3-2

[0336] At room temperature, compound 3-2c (150 mg, 0.163 mmol), ethanol (10 mL), and water (5 mL) were added sequentially to a 25 mL single-necked flask. Pd / C was then added and stirred under a hydrogen atmosphere for 21 hours until the complete disappearance of the starting material was detected by LC-MS. The reaction system was filtered, concentrated under reduced pressure, dissolved in water, and lyophilized to obtain compound 3-2 (72 mg, 79% yield, as a white solid). 20 H 34 NO 17 + [M+H] + MS (ESI): calculated value 560.18, found value 560.15.

[0337] Example 10: Preparation of trisaccharide substrate compound 3-3

[0338] The following steps were used to prepare compound 3-3, the structure of which is as follows:

[0339] (1) Preparation of compound 3-3a

[0340] At room temperature, under nitrogen, compound 1-4 (0.1-10 equivalents relative to compound 2-3), compound 2-3 (1.2 g, 1.48 mmol), and toluene (100 mL) were added sequentially to a 250 mL three-necked flask. After dissolution, scandium trifluoromethanesulfonate was added to the system, and the mixture was heated to 60°C and stirred for approximately 2 hours. After TLC monitoring of the reaction of the starting materials, water (100 mL) was added to quench the reaction, and the mixture was extracted with dichloromethane (100 mL × 2). The organic phase was collected, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by column chromatography (eluent: EtOAc / PE = 1 / 50-1 / 5) to obtain compound 3-3a (0.9 g, 55% yield, colorless viscous oily liquid). 1 H NMR (400MHz, DMSO-d) δ7.39–7.19(m,20H),5.17–5.15(m,2H),5.09–5.05(m,2H),5.00(dd,1H,J=3.0Hz,10.4Hz),4.87(d,1H,J=10.7Hz),4. 81(d,1H,J=12.0Hz),4.77(d,1H,J=2.4Hz),4.69(d,1H,J=11.6Hz),4.65(d,1H,J=12.2Hz),4.64(d,1H,J=10.9Hz),4.63(d,1H,J=3.3Hz),4 .60–4.49(m,4H),4.34–4.32(m,1H),3.93(d,1H,J=3.6Hz),3.83(t,1H,J=9.0Hz),3.78(t,1H,J=6.6Hz),3.74–3.71(m,1H),3.65(dd,1H,J= 4.3Hz,11.2Hz),3.60–3.58(m,4H),3.56–3.51(m,2H),3.45–3.41(m,2H),2.10(s,3H),2.02(s,3H),2.01(s,3H),1.95(s,3H),1.94(s,3H). C 57 H 69 N4O 21 + [M+NH4] + MS (ESI): calculated value: 1145.45, found value: 1145.33.

[0341] (2) Preparation of compound 3-3b

[0342] Compound 3-3a (900 mg, 0.798 mmol), chloroform, pyridine, and AcSH were added sequentially to a 25 mL Schlenk reaction flask at room temperature and dissolved. The system was stirred at room temperature for 18 hours. After completion of the reaction of the starting materials as monitored by TLC, the mixture was concentrated under reduced pressure and purified by column chromatography (eluent: EtOAc / PE = 1 / 4 - 2 / 3) to afford compound 3-3b (550 mg, 60% yield, as a white solid). 1 H NMR(400MHz, DMSO-d)δ7.90(d,1H,J=9.0Hz),7.41–7.18(m,20H),5.16–5.14(m,2H),5.06–5.03(m ,2H),4.78–4.71(m,3H),4.68–4.63(m,3H),4.56–4.51(m,2H),4.49–4.42(m,2H),4.30(t,1H,J=4. 2Hz),3.92(d,1H,J=2.8Hz),3.80–3.74(m,2H),3.72–3.67(m,3H),3.61(s,3H),3.56–3.53(m,2H) ,3.50–3.40(m,4H),2.10(s,3H),2.03(s,3H),2.01(s,3H),1.97(s,3H),1.94(s,3H),1.80(s,3H). C 59 H 70 NO 22 + [M+H] + MS (ESI): calculated value: 1144.44, found value: 1144.42.

[0343] (3) Preparation of compound 3-3c

[0344] Compound 3-3b (550 mg, 0.481 mmol) and methanol (10 mL) were added to a 25 mL single-necked vial at room temperature. After dissolution, 1 M sodium hydroxide solution (1 mL) was slowly added over 10 minutes. The mixture was stirred at room temperature for 2 hours until the reaction was complete as detected by HPLC. Acetic acid (1 mL) was added to the reaction system to quench the reaction. The mixture was concentrated under reduced pressure to obtain crude compound 3-3c (300 mg, 68% yield, colorless oil). 1H NMR (400MHz, DMSO-d) δ8.00 (d, 1H, J = 8.8Hz), 7.36–7.17 (m, 20H), 5.17–5.14 (m, 2H), 4. 92(d,1H,J=11.4Hz), 4.83(d,1H,J=10.9Hz), 4.76(d,1H,J=12.5Hz), 4.71(d,1H,J=4.3 Hz),4.64–4.60(m,2H),4.57–4.44(m,6H),4.35–4.33(m,1H),4.87–4.86(m,2H),3.80( t,1H,J=9.1Hz),3.68–3.56(m,4H),3.52–3.40(m,9H),3.37–3.36(m,1H),1.81(s,3H). C 48 H 58 NO 17 + [M+H] + MS (ESI): calculated value: 920.37, found value: 920.34.

[0345] (4) Preparation of Compound 3-3

[0346] At room temperature, compound 3-3c (150 mg, 0.163 mmol), ethanol (10 mL), and water (5 mL) were added sequentially to a 25 mL single-necked flask. Pd / C was then added and stirred under a hydrogen atmosphere for 21 hours until the complete disappearance of the starting material was detected by LC-MS. The reaction system was filtered, concentrated under reduced pressure, dissolved in water, and lyophilized to obtain compound 3-3 (73 mg, 80% yield, as a white solid). 20 H 34 NO 17 + [M+H] + MS (ESI): calculated value 560.18, found value 560.17.

[0347] 2. Preparation of Linker-Payload (LP) Compounds

[0348] Example 11: Preparation of Linker-Loader Compound 4-1

[0349] The structure of the linker-load compound 4-1 is as follows:

[0350] (1) Preparation of compound 4-1b

[0351] At room temperature, a certain amount of compound 3-1 (0.1-10 equivalents), compound 4-1a (1.0 equivalent, 10.0 mg, 0.0077 mmol, CAS number: 2684216-48-4), DMF (1.3 mL), and a certain amount of DIPEA and HATU were added to a 4 mL centrifuge tube in sequence. The resulting reaction solution was stirred at room temperature until the reaction was complete as detected by HPLC. The reaction solution was purified by semi-preparative HPLC to obtain compound 4-1b (yield 82%) as a white solid. 84 H 136 N 14 O 31 2+ [M+2H] 2+ / 2 MS (ESI): calculated value 918.47, found value 918.69.

[0352] (2) Preparation of compound 4-1

[0353] At room temperature, compound 4-1b (2.5 mg, 0.0014 mmol), H2O, a certain amount of Et3N, and DMC (2-chloro-1,3-dimethylimidazolinium chloride, CAS No. 37091-73-9) were added sequentially to a 4 mL centrifuge tube. The resulting reaction solution was stirred at room temperature until the reaction was complete as detected by HPLC. The reaction solution was purified by semi-preparative HPLC to obtain compound 4-1 (2 mg, 81% yield, white solid). 84 H 134 N 14 O 30 2+ [M+2H] 2+ / 2 MS (ESI): calculated value 909.47, found value 909.97.

[0354] Example 12: Preparation of Linker-Loader Compound 4-2

[0355] The structure of the linker-load compound 4-2 is as follows:

[0356] (1) Preparation of compound 4-2a

[0357] At room temperature, compound 3-2 (0.1-10 equivalents relative to compound 4-1a), compound 4-1a (19.4 mg, 0.015 mmol, CAS No.: 2684216-48-4), DMF (500 μL), and a certain amount of DIPEA and HATU were added to a 4 mL centrifuge tube in sequence. The resulting reaction solution was stirred at room temperature until the reaction was complete as detected by HPLC. The reaction solution was purified by semi-preparative HPLC to obtain compound 4-2a, which was directly used in the next step (white solid). 84 H 136 N 14 O 31 2+ [M+2H] 2+ / 2 MS (ESI): calculated value 918.47, found value 918.92.

[0358] (2) Preparation of compound 4-2

[0359] Compound 4-2a (27.5 mg, 0.015 mmol) and Tris buffer (pH = 9.0, 2 mL) were added to a 15 mL centrifuge tube at 0°C. After dissolution, a certain amount of triethylamine and 2-chloro-1,3-dimethylimidazoline chloride were added. The resulting reaction solution was stirred at 0°C until the reaction was complete as detected by HPLC. The reaction solution was purified by semi-preparative HPLC to obtain compound 4-2 (15.5 mg, two-step yield 57%, white solid). 84 H 134 N 14 O 30 2+ [M+2H] 2+ / 2 MS (ESI): calculated value 909.47, found value 909.73.

[0360] Example 13: Preparation of Linker-Loader Compound 4-3

[0361] The structure of the linker-load compound 4-3 is as follows:

[0362] (1) Preparation of compound 4-3a

[0363] At room temperature, compound 3-3 (0.1-10 equivalents relative to compound 4-1a), compound 4-1a (19.8 mg, 0.015 mmol, CAS No.: 2684216-48-4), DMF (500 μL), and a certain amount of DIPEA and HATU were added to a 4 mL centrifuge tube in sequence. The resulting reaction solution was stirred at room temperature until the reaction was complete as detected by HPLC. The reaction solution was purified by semi-preparative HPLC to obtain compound 4-3a, which was directly used in the next step (white solid). 84 H 136 N 14 O 31 2+ [M+2H] 2+ / 2 MS (ESI): calculated value 918.47, found value 918.94.

[0364] (2) Preparation of compound 4-3

[0365] Compound 4-3a (28.0 mg, 0.015 mmol) and Tris buffer (pH = 9.0, 2 mL) were added to a 15 mL centrifuge tube at 0°C. After dissolution, a certain amount of triethylamine and 2-chloro-1,3-dimethylimidazoline chloride were added. The resulting reaction solution was stirred at 0°C until the reaction was complete as detected by HPLC. The reaction solution was purified by semi-preparative HPLC to obtain compound 4-3 (14.4 mg, two-step yield 52%, white solid). 84 H 134 N 14 O 30 2+ [M+2H] 2+ / 2 MS (ESI): calculated value 909.47, found value 909.71.

[0366] Example 14: Preparation of Linker-Loader Compound 4-4

[0367] The structure of the linker-load compound 4-4 is as follows:

[0368] For the synthesis of compound 4-4, please refer to Example 6 of WO2023232144A1.

[0369] Example 15: Preparation of Linker-Loader Compound 4-5

[0370] The structure of linker-load compound 4-5 is as follows:

[0371] (1) Preparation of compound 4-5-1

[0372] 4-5-1 was synthesized using peptide solid phase synthesis, and the synthesis steps were similar to the synthesis of LP-6-2 in patent (WO2023232144A1). The crude product after cleavage was prepared by Prep-HPLC. After freeze-drying, 4-5-1 was obtained. 76 H 125 N9O 31 2+ [M+2H] 2+ / 2 MS (ESI): calculated value 829.92, found value 830.56.

[0373] (2) Preparation of compound 4-5-2

[0374] Step A: Preparation of compound 4-5-2b

[0375] Weigh 4-5-1 (21.4 g, 12.9 mmol) and 4-5-2a (0.1-10 equivalents relative to 4-5-1) into a round-bottom flask and dissolve in DMF (450 mL). After dissolution, cool to 0-5°C in an ice bath. Stir for 5 minutes, then add HATU to dissolve. Finally, add 2,4,6-collidine, stir at 0-5°C for 1 hour, then return to room temperature and stir for 1 hour. After checking that the reaction is complete, prepare the reaction by prep-HPLC. After lyophilization, 4-5-2b (31.5 g, 81.6% yield) was obtained as a white solid. 144 H 223 N 24 O 49 3+ [M+3NH4] 3+ / 3 MS (ESI): calculated value 1024.19, actual value 1024.72.

[0376] Step B and C: Preparation of compound 4-5-2

[0377] 4-5-2b (38.5 g, 12.7 mmol) was dissolved in DMF (400 mL) in a reaction flask. A certain amount of Pd / C was then added, and the gas was replaced with hydrogen three times. The reaction was allowed to proceed at room temperature for 2 h. Samples were collected and tested by HPLC. After the reaction was complete, the Pd / C was filtered off with diatomaceous earth and the temperature was cooled to 0-5°C in an ice bath. 4-5-2d (0.1-10 equivalents relative to 4-5-2b) and PyBop were added. After stirring to dissolve, DIEA was added dropwise. The reaction was stirred at room temperature for 2 h. Samples were collected and tested by HPLC. After the reaction was complete, DEA was added. The reaction was stirred at room temperature for 30 min. After sampling and testing, the pH was adjusted to 5-6 and the product was prepared by Prep-HPLC. 4-5-2 was obtained after lyophilization (27.4 g, 61.6% yield, as a pink solid). 161H 232 Cl2F2N 29 O 53 3+ [M+H+2NH4] 3+ / 3 MS (ESI): calculated value 1175.86, found value 1176.45.

[0378] For the synthesis of compound 4-5-2a, please refer to patent WO2023232144A1.

[0379] For the synthesis of compound 4-5-2d, please refer to patent CN202211428194.6.

[0380] (3) Preparation of compound 4-5a

[0381] At room temperature, compound 3-1 (0.1-10 equivalents relative to 4-5-2), compound 4-5-2 (33 mg, 0.010 mmol), DMF (500 μL), and a certain amount of DIPEA and HATU were added to a 4 mL centrifuge tube in sequence. The resulting reaction solution was stirred at room temperature until the reaction was complete as detected by HPLC. The reaction solution was purified by semi-preparative HPLC to obtain compound 4-5a (22.4 mg, 59% yield, white solid). 181 H 260 Cl2F2N 29 O 69 3+ [M+2H+NH4] 3+ / 3 MS (ESI): calculated value 1350.57, found value 1351.75.

[0382] (4) Preparation of Compound 4-5

[0383] Compound 4-5a (22.4 mg, 0.006 mmol) and Tris buffer (pH = 9.0, 2 mL) were added to a 15 mL centrifuge tube at 0°C. After dissolution, a certain amount of triethylamine and 2-chloro-1,3-dimethylimidazoline chloride were added. The resulting reaction solution was stirred at 0°C until the reaction was complete as detected by HPLC. The reaction solution was purified by semi-preparative HPLC to obtain compound 4-5 (20.1 mg, 90% yield, white solid). 181 H 261 Cl2F2N 30 O 68 3+ [M+H+2NH4] 3+ / 3 MS (ESI): calculated value 1350.24, found value 1352.14.

[0384] Example 16: Preparation of Linker-Loader Compounds 4-6

[0385] The structures of linker-load compounds 4-6 are as follows:

[0386] (1) Preparation of compound 4-6a

[0387] At room temperature, compound 3-2 (0.1-10 equivalents relative to 4-5-2), compound 4-5-2 (32.5 mg, 0.009 mmol), DMF (500 μL), and a certain amount of DIPEA and HATU were added to a 4 mL centrifuge tube in sequence. The resulting reaction solution was stirred at room temperature until the reaction was complete as detected by HPLC. The reaction solution was purified by semi-preparative HPLC to obtain compound 4-6a (25.6 mg, 68% yield, white solid). 181 H 263 Cl2F2N 30 O 69 3+ [M+H+2NH4] 3+ / 3 MS (ESI): calculated value 1356.24, found value 1357.06.

[0388] (2) Preparation of Compound 4-6

[0389] Compound 4-6a (25.6 mg, 0.006 mmol) and Tris buffer (pH = 9.0, 2 mL) were added to a 15 mL centrifuge tube at 0°C. After dissolution, a certain amount of triethylamine and 2-chloro-1,3-dimethylimidazoline chloride were added. The resulting reaction solution was stirred at 0°C until the reaction was complete as detected by HPLC. The reaction solution was purified by semi-preparative HPLC to obtain compound 4-6 (21.7 mg, 85% yield, white solid). 181 H 261 Cl2F2N 30 O 68 3+ [M+H+2NH4] 3+ / 3 MS (ESI): calculated value 1350.24, found value 1351.43.

[0390] Example 17: Preparation of Linker-Loader Compounds 4-7

[0391] The structures of linker-load compounds 4-7 are as follows:

[0392] (1) Preparation of compound 4-7a

[0393] At room temperature, compound 3-3 (0.1-10 equivalents relative to 4-5-2), compound 4-5-2 (31.7 mg, 0.009 mmol), DMF (500 μL), and a certain amount of DIPEA and HATU were added to a 4 mL centrifuge tube in sequence. The resulting reaction solution was stirred at room temperature until the reaction was complete as detected by HPLC. The reaction solution was purified by semi-preparative HPLC to obtain compound 4-7a (29.2 mg, 80% yield, white solid). 181 H 266 Cl2F2N 31 O 69 3+ [M+3NH4] 3+ / 3 MS (ESI): calculated value 1361.92, found value 1362.87.

[0394] (2) Preparation of Compound 4-7

[0395] Compound 4-7a (29.2 mg, 0.007 mmol) and Tris buffer (pH = 9.0, 2 mL) were added to a 15 mL centrifuge tube at 0°C. After dissolution, a certain amount of triethylamine and 2-chloro-1,3-dimethylimidazoline chloride were added. The resulting reaction solution was stirred at 0°C until the reaction was complete as detected by HPLC. The reaction solution was purified by semi-preparative HPLC to obtain compound 4-7 (15.2 mg, 52% yield, white solid). 181 H 261 Cl2F2N 30 O 68 3+ [M+H+2NH4] 3+ / 3 MS (ESI): calculated value 1350.24, found value 1351.67.

[0396] Example 18: Preparation of Linker-Loader Compounds 4-8

[0397] The structures of linker-load compounds 4-8 are as follows:

[0398] The linker cargo 4-8 was synthesized using similar steps to those used for the synthesis of 4-5.

[0399] Example 19: Preparation of Linker-Loader Compounds 4-9

[0400] The structures of linker-load compounds 4-9 are as follows:

[0401] (1) Preparation of compound 4-9-1

[0402] Step A: Preparation of compound 4-9-1c

[0403] At room temperature, 4-9-1a (1 g, 1.662 mmol, CAS No.: 159858-22-7) was weighed and placed in a 250 mL round-bottom flask. DMA (50 mL) was added and, after dissolution, DBU was added and stirred for 2.5 h. HPLC monitoring showed that the reaction of the raw materials was complete. PPTS was added to the reaction system and stirred for 10 min. EDCI, 4-9-1b (0.1-10 equivalents relative to 4-9-1a, CAS No.: 557756-85-1), and HOBT were then added to the reaction solution in sequence. The mixture was stirred for 12 h until the reaction was complete as detected by HPLC. The reaction solution was purified by semi-preparative HPLC to obtain compound 4-9-1c (1.3 g, 92% yield, white solid). 44 H 61 N6O 11 + [M+H] + MS (ESI): calculated value 849.44, found value 849.33.

[0404] Step B: Preparation of compound 4-9-1d

[0405] At 0°C, 4-9-1c (1.3 g, 1.531 mmol) was placed in a 100 mL round-bottom flask and DMF (25 mL) was added. After dissolving, p-(dinitrobenzene) carbonate and DIEA were added. After stirring at 0°C for 0.5 h, the ice-water bath was removed and the mixture was stirred at room temperature for 12 h. TLC monitoring showed that the reaction of the raw materials was complete. Water (200 mL) was added to the reaction system and extracted with EA (150 mL × 3). The organic phase was collected, washed with saturated sodium chloride solution (500 mL), dried over anhydrous sodium sulfate, filtered, concentrated, slurried with diethyl ether, filtered, and the solid was pumped dry with an oil pump to obtain compound 4-9-1d (1.43 g, 92% yield, white solid). 51 H 64 N7O 15 + [M+H] + MS (ESI): calculated value 1014.45, found value 1014.30.

[0406] Step C: Preparation of compound 4-9-1

[0407] At room temperature, 4-9-1d (1.43 g, 1.417 mmol) was placed in a 100 mL round-bottom flask, and DMF (25 mL) was added. After dissolution, HOBT and DIEA were added. After stirring for 5 minutes, 4-9-1e (0.1-10 equivalents relative to 4-9-1d, CAS number: 474645-27-7) was added and stirred at room temperature for 12 hours until the reaction was complete as detected by HPLC. Diethylamine (2.5 mL) was added to the reaction solution and stirred for 0.5 hours until the reaction was complete as detected by HPLC. The reaction solution was purified by semi-preparative HPLC to obtain compound 4-9-1 (856.6 mg, 44% yield, white solid). 69 H 117 N 11 O 17 2+ [M+2H] 2+ / 2 MS (ESI): calculated value 685.93, found value 686.20.

[0408] (2) Preparation of compound 4-9-2

[0409] At room temperature, 4-5-1 (873 mg, 0.282 mmol) and 4-9-1 (0.1-10 equivalents relative to 4-5-1) were placed in a 25 mL round-bottom flask, DMF (8 mL) was added, and after cooling to 0°C in an ice bath, DIEA was added. After stirring for 5 minutes, HATU was added and stirred at room temperature for 2 hours until the reaction was complete as detected by HPLC. Diethylamine (800 μL) was added to the reaction solution and stirred for 0.5 hours until the reaction was complete as detected by HPLC. The reaction solution was purified by semi-preparative HPLC to obtain compound 4-9-2 (1.2 g, yield 88.8%, white solid). 199 H 346 N 32 O 61 4+ [M+3H+NH4] 4+ / 4 MS (ESI): calculated value 1040.13, found value 1040.89.

[0410] (2) Preparation of compound 4-9a

[0411] At room temperature, compound 3-2 (0.1-10 equivalents relative to 4-9-2), compound 4-9-2 (29.6 mg, 0.007 mmol), DMF (500 μL), and a certain amount of DIPEA and HATU were added to a 4 mL centrifuge tube in sequence. The resulting reaction solution was stirred at room temperature until the reaction was complete as detected by HPLC. The reaction solution was purified by semi-preparative HPLC to obtain compound 4-9a (white solid), which was directly used in the next step.219 H 376 N 33 O 77 3+ [M+2H+NH4] 3+ / 3 MS (ESI): calculated value 1566.88, found value 1567.40.

[0412] (3) Preparation of Compound 4-9

[0413] Compound 4-9a (33 mg, 0.007 mmol) and Tris buffer (pH = 9.0, 2 mL) were added to a 15 mL centrifuge tube at 0°C. After dissolution, a certain amount of triethylamine and 2-chloro-1,3-dimethylimidazoline chloride were added. The resulting reaction solution was stirred at 0°C until the reaction was complete as detected by HPLC. The reaction solution was purified by semi-preparative HPLC to obtain compound 4-9 (15.3 mg, two-step yield 46%, white solid). 219 H 374 N 33 O 76 3+ [M+2H+NH4] 3+ / 3 MS (ESI): calculated value 1560.88, found value 1561.60.

[0414] Example 20: Preparation of Linker-Loader Compound 4-10

[0415] The structure of linker-load compound 4-10 is as follows:

[0416] (1) Preparation of compound 4-10a

[0417] At room temperature, compound 3-3 (0.1-10 equivalents relative to 4-9-2), compound 4-9-2 (29.6 mg, 0.007 mmol), DMF (500 μL), and a certain amount of DIPEA and HATU were added to a 4 mL centrifuge tube in sequence. The resulting reaction solution was stirred at room temperature until the reaction was complete as detected by HPLC. The reaction solution was purified by semi-preparative HPLC to obtain compound 4-10a (white solid), which was directly used in the next step. 219 H 376 N 33 O 77 3+ [M+2H+NH4] 3+ / 3 MS (ESI): calculated value 1566.88, actual value 1567.56.

[0418] (2) Preparation of Compound 4-10

[0419] Compound 4-10a (33 mg, 0.007 mmol) and Tris buffer (pH = 9.0, 2 mL) were added to a 15 mL centrifuge tube at 0°C. After dissolution, a certain amount of triethylamine and 2-chloro-1,3-dimethylimidazoline chloride were added. The resulting reaction solution was stirred at 0°C until the reaction was complete as detected by HPLC. The reaction solution was purified by semi-preparative HPLC to obtain compound 4-10 (21.2 mg, two-step yield 64%), as a white solid. 219 H 374 N 33 O 76 3+ [M+2H+NH4] 3+ / 3 MS (ESI): calculated value 1560.88, actual value 1561.44.

[0420] Example 21: Preparation of Linker-Loader Compound 4-11

[0421] The structure of linker-load compound 4-11 is as follows:

[0422] The linker cargo 4-11 was synthesized using similar steps to those used for the synthesis of 4-9.

[0423] Example 22: Preparation of Linker-Loader Compound 4-12

[0424] The structure of linker-load compound 4-12 is as follows:

[0425] (1) Preparation of compound 4-12-1

[0426] 4-12a was synthesized using peptide solid phase synthesis method, the steps are as follows:

[0427] Step 1: Swelling of the resin

[0428] 19.2 g of 2-CTC-Resin (Sub=1.04 mmol / g) was weighed into a synthesis tube and swelled with 190 mL of DCM for 30 min.

[0429] Step 2: Coupling of Fmoc-Phe-OH

[0430] Weigh Fmoc-Phe-OH (1-10 equivalents) and dissolve it in 190 mL of DCM. Add DIEA and add the mixture to a synthesis tube. Bubble nitrogen for 2 hours. Then, add 50 mL of MeOH:DIEA (5:1) to cap the reaction for 30 minutes. Drain the solvent and wash once with DCM and then three times with DMF.

[0431] Step 3: Coupling of Fmoc-Gly-Gly-OH

[0432] Add 190 mL of deprotection reagent and react for 10 ± 10 min. Wash with DMF 4 times. Kaiser assay shows black. Wash with DMF 4 times.

[0433] Weigh Fmoc-Gly-Gly-OH (1-10 equivalents) and dissolve ethyl 2-oximecyanoacetate in 190 mL of DMF. Add DIC and activate for 3 minutes. Pour the mixture into a reaction tube and purge with nitrogen for 2 hours. Kaiser assay indicates colorless. Wash with DMF three times.

[0434] Step 4: Coupling of Fmoc-PEG2-CH2CH2COOH

[0435] Add 190 mL of deprotection reagent and react for 10 ± 10 min. Wash with DMF 4 times. Kaiser assay shows black. Wash with DMF 4 times.

[0436] Weigh Fmoc-PEG2-CH2CH2COOH (1-10 equivalents) and dissolve ethyl 2-oximecyanoacetate in 190 mL of DMF. Add DIC and activate for 3 minutes. Pour the mixture into a reaction tube and purge with nitrogen for 2 hours. Kaiser assay indicates colorless. Wash twice with DMF and three times with DCM, then dry overnight.

[0437] Step 5: Resin cracking

[0438] Prepare 370 mL of TFA:H2O=95:5 solution, add the resin to the solution, stir and react for 2 hours, then filter, concentrate the solvent, prepare by Prep-HPLC, and freeze-dry to obtain 4-12-1a (12 g, yield 91%, white solid). 35 H 41 N4O9 + [M+H] + MS (ESI): Calculated 661.29, found 661.22.

[0439] Step A: Preparation of compound 4-12-1

[0440] 4-12-1a (12 g, 18.2 mmol) and Gly-OBn·HCl were dissolved in DMF (120 mL). HATU was added after dissolution, followed by dropwise addition of DIEA. The mixture was stirred at room temperature for 3 h, and samples were collected for HPLC analysis. After the reaction of the raw materials was complete, DEA was added and stirred for 30 min, and samples were collected for HPLC analysis. After the reaction of the raw materials was complete, prep-HPLC preparation was performed, and lyophilization was performed to obtain 4-12-1 (8.9 g, yield 84%, white solid). 29 H 40 N5O8 + [M+H] + MS (ESI): Calculated 586.29, found 586.02.

[0441] (2) Preparation of compound 4-12-2

[0442] Step A: Preparation of compound 4-12-2a

[0443] At room temperature, 4-5-1 (3.5 mg, 2.111 mmol) and 4-12-1 (0.1-10 equivalents relative to 4-5-1) were placed in a 100 mL round-bottom flask, and DMF (30 mL) was added. After cooling to 0°C in an ice bath, DIEA was added. After stirring for 5 minutes, HATU was added and stirred at room temperature for 1 hour until the reaction was complete as detected by HPLC. The reaction solution was purified by semi-preparative HPLC to obtain compound 4-12-2a (4.1 g, yield 69.5%, white solid). 134 H 205 N 21 O 45 2+ [M+2NH4] 2+ / 2 MS (ESI): calculated value 1414.22, found value 1415.20.

[0444] Step B: Preparation of compound 4-12-2b

[0445] At room temperature, 4-12-2a (957 mg, 0.343 mmol) was placed in a 50 mL round-bottom flask, DMF (10 mL) was added, and Pd / C was added. After replacing the hydrogen atmosphere, the mixture was stirred at room temperature for 1.5 h until the reaction was complete as detected by HPLC. The reaction solution was filtered and purified by semi-preparative HPLC to obtain compound 4-12-2b (668 mg, 74.4% yield, white solid). 120 H 190 N 20 O 45 2+ [M+H+NH4] 2+ / 2 MS (ESI): calculated value 1315.66, found value 1316.51.

[0446] Step C: Preparation of compound 4-12-2d

[0447] At room temperature, 4-12-2b (250 mg, 0.096 mmol) and 4-12-2c (equivalent to 0.1-10 equivalents of 4-12-12b, CAS number: 253128-41-5) were placed in a 25 mL round-bottom flask. DMF (5 mL) was added, followed by DIEA. After stirring for 5 minutes, HATU was added and the mixture was stirred at room temperature for 1 hour until the reaction was complete as detected by HPLC. The reaction solution was purified by semi-preparative HPLC to obtain compound 4-12-2d (311.8 mg, 80.7% yield, as a white solid). 200 H 302 N 21 O 65 3+ [M+3H] 3+ / 3 MS (ESI): calculated value 1346.03, found value 1346.95.

[0448] Step D: Preparation of compound 4-12-2

[0449] At room temperature, 4-12-2d (311.8 mg, 0.077 mmol) was placed in a 25 mL round-bottom flask, DMF (4 mL) was added, and diethylamine was added. The mixture was stirred at room temperature for 20 minutes until the reaction was complete as detected by HPLC. The reaction solution was filtered and purified by semi-preparative HPLC to obtain compound 4-12-2 (255.8 mg, 72.2% yield, white solid). 185 H 291 N 21 O 63 2+ [M+2H] 2+ / 2 MS (ESI): calculated value 1907.52 actual value 1908.72.

[0450] (3) Preparation of compound 4-12a

[0451] At room temperature, compound 3-2 (0.1-10 equivalents relative to 4-12-2), compound 4-12-2 (27 mg, 0.007 mmol), DMF (500 μL), and a certain amount of DIPEA and HATU were added to a 4 mL centrifuge tube in sequence. The resulting reaction solution was stirred at room temperature until the reaction was complete as detected by HPLC. The reaction solution was purified by semi-preparative HPLC to obtain compound 4-12a (21.2 mg, 70% yield, white solid). 205 H329 N 24 O 79 3+ [M+H+2NH4] 3+ / 3 MS (ESI): calculated value 1463.75, found value 1464.61.

[0452] (4) Preparation of Compound 4-12

[0453] Compound 4-12a (21.2 mg, 0.005 mmol) and Tris buffer (pH = 9.0, 2 mL) were added to a 15 mL centrifuge tube at 0°C. After dissolution, a certain amount of triethylamine and 2-chloro-1,3-dimethylimidazoline chloride were added. The resulting reaction solution was stirred at 0°C until the reaction was complete as detected by HPLC. The reaction solution was purified by semi-preparative HPLC to obtain compound 4-12 (17.87 mg, 85% yield, white solid). 205 H 327 N 24 O 78 3+ [M+H+2NH4] 3+ / 3 MS (ESI): calculated value 1457.75, found value 1458.33.

[0454] Example 23: Preparation of Linker-Loader Compound 4-13

[0455] The structure of linker-load compound 4-13 is as follows:

[0456] (1) Preparation of compound 4-13a

[0457] At room temperature, compound 3-3 (equivalent to 0.1-10 equivalents of 4-12-2), compound 4-12-2 (27 mg, 0.007 mmol), DMF (500 μL), and a certain amount of DIPEA and HATU were added to a 4 mL centrifuge tube in sequence. The resulting reaction solution was stirred at room temperature until the reaction was complete as detected by HPLC. The reaction solution was purified by semi-preparative HPLC to obtain compound 4-13a (26.2 mg, 84% yield, white solid). 205 H 329 N 24 O 79 3+ [M+H+2NH4] 3+ / 3 MS (ESI): calculated value 1463.75, found value 1464.41.

[0458] (2) Preparation of Compound 4-13

[0459] Compound 4-13a (26.2 mg, 0.006 mmol) and Tris buffer (pH = 9.0, 2 mL) were added to a 15 mL centrifuge tube at 0°C. After dissolution, a certain amount of triethylamine and 2-chloro-1,3-dimethylimidazoline chloride were added. The resulting reaction solution was stirred at 0°C until the reaction was complete as detected by HPLC. The reaction solution was purified by semi-preparative HPLC to obtain compound 4-13 (20.01 mg, 77% yield, white solid). 205 H 327 N 24 O 78 3+ [M+H+2NH4] 3+ / 3 MS (ESI): calculated value 1457.75, found value 1458.28.

[0460] Example 24: Preparation of Linker-Loader Compound 4-14

[0461] The structure of linker-load compound 4-14 is as follows:

[0462] The linker cargo 4-14 was synthesized using similar steps to those used for the synthesis of 4-12.

[0463] Example 25: Preparation of Linker-Loader Compound 4-15

[0464] The structure of linker-load compound 4-15 is as follows:

[0465] (1) Preparation of compound 4-15-1

[0466] Step A: Preparation of compound 4-15-1c

[0467] At room temperature, 4-15-1a (0.1-10 equivalents relative to 4-15-1b) and 4-15-1b (1 g, 4.582 mmol) were placed in a 100 mL round-bottom flask. DMF (25 mL) was added and the mixture was cooled to 0°C in an ice bath. DIEA was then added and stirred for 5 minutes before HATU was added. The mixture was stirred at room temperature for 1 hour until the reaction was complete as determined by HPLC. Water (150 mL) was added to the reaction solution and washed with ethyl acetate (150 mL x 3). The organic phase was collected and washed with saturated sodium chloride solution (450 mL), dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by column chromatography (eluent: MeOH / DCM = 1 / 50-1 / 15) to afford compound 4-15-1c (white solid), which was directly used in the next step. 29 H41 N8O8 + [M+NH4] + MS (ESI): calculated value 629.30, found value 629.25.

[0468] Step B: Preparation of compound 4-15-1

[0469] At room temperature, 4-15-1c was placed in a 50 mL round-bottom flask, and DMF (15 mL) was added, followed by diethylamine. The mixture was stirred at room temperature for 20 minutes until the reaction was complete as detected by HPLC. The reaction solution was filtered and purified by semi-preparative HPLC to obtain compound 4-15-1 (1.32 g, yield 74.2%), as a white solid. 14 H 28 N7O6 + [M+H] + MS (ESI): calculated value 390.21, found value 390.25.

[0470] (2) Preparation of compound 4-15a

[0471] At room temperature, compound 3-2 (equivalent to 0.1-10 equivalents of 4-15-1), compound 4-15-1 (30 mg, 0.077 mmol), DMF (1 mL), and a certain amount of DIPEA and HATU were added to a 4 mL centrifuge tube in sequence. The resulting reaction solution was stirred at room temperature until the reaction was complete as detected by HPLC. The reaction solution was purified by semi-preparative HPLC to obtain compound 4-15a (57.4 mg, 80% yield, white solid). 34 H 62 N9O 22 + [M+NH4] + MS (ESI): Calculated value 948.40, found value 948.45.

[0472] (3) Preparation of Compound 4-15

[0473] Compound 4-15a (57.4 mg, 0.062 mmol) and Tris buffer (pH = 9.0, 2 mL) were added to a 15 mL centrifuge tube at 0°C. After dissolution, a certain amount of triethylamine and 2-chloro-1,3-dimethylimidazoline chloride were added. The resulting reaction solution was stirred at 0°C until the reaction was complete as detected by HPLC. The reaction solution was purified by semi-preparative HPLC to obtain compound 4-15 (41.1 mg, 73% yield, white solid). 34 H 57 N8O 21+ [M+H] + MS (ESI): calculated value 913.36, found value 913.38.

[0474] 3. Selection or Preparation of Antibodies

[0475] Antibody selection

[0476] The macromolecular portion of the antibody-drug conjugate prepared using the substrate and conjugation method of the present invention can be any molecule containing a carbohydrate chain in the Fc region of an antibody, including but not limited to antibodies / diabodies / Fc fusion proteins / single-chain antibodies, etc. For example, trastuzumab can be selected, which is commercially available.

[0477] In addition, antibodies with engineered modifications may also be selected. The production, purification, and identification of the anti-human ErbB2 / HER2 antibody mAb-1 were described in Example 1 of patent CN 106856656 B, the entire disclosure of which is incorporated herein by reference.

[0478] ADC Preparation and Characterization

[0479] Example 26: Preparation and Characterization of ADC-1

[0480] 26.1 Preparation of Antibody-Drug Conjugate ADC-1

[0481] In the following, the sugar chain at the Fc end of the antibody mAb-1 is remodeled by endoglycosidase, and the linker-load is specifically coupled to the antibody to form the corresponding ADC drug.

[0482] 1) Treatment of mAb-1: The antibody was treated by ultrafiltration, dialysis, or desalting column, and its storage buffer was replaced with 50 mM Tris-HCl (pH 5-8) and 150 mM NaCl.

[0483] 2) Preparation of ADC-1: ADC-1 was prepared by coupling the antibody mAb-1 with the linker-load compound 4-1 using the endoglycosidase Endo S2. In 1× endoglycosidase buffer, the antibody mAb-1 and compound 4-1 were thoroughly mixed at an appropriate molar ratio (1:1 to 1:100), and then the endoglycosidase Endo S2 was added and mixed thoroughly. While mixing, the coupling reaction was carried out at 4-40°C for 0.5-20 hours. After completion of the reaction, purification was performed. The purified ADC-1 was stored in 1× PBS (pH 7.4) at 4°C or -80°C.

[0484] The characterization test methods and results of the antibody-drug conjugate ADC-1 are described in 26.2-26.3 below.

[0485] 26.2 Hydrophobic Interaction High-Performance Liquid Chromatography (HIC-HPLC) Analysis of Antibody-Drug Conjugate ADC-1

[0486] A Proteomix HIC Butyl-NP5 4.6 x 35 mm 5 μm Non-Porous column (Manufacturer: Saifen, PN: 431NP5-4603) was used. Mobile phase A consisted of 1.5 M ammonium sulfate and 20 mM phosphate buffer (pH 7.0) and mobile phase B consisted of a 7:3 ratio (v / v) of 20 mM phosphate buffer (pH 7.0) and isopropanol. The flow rate was 0.8 mL / min, and the gradient increased from 10% to 70% phase B over 8 minutes. The detection wavelength was 280 nm to measure the DAR distribution of the ADC drug ADC-1.

[0487] The test results are shown in Figure 1. The unconjugated cytotoxic antibody is less than 5%; the conjugated product is mainly DAR2. Overall, the DAR value of the ADC drug is about 1.88 (under better conjugation conditions).

[0488] 26.3 Detection and Analysis of ADC-1 by Size Exclusion Liquid Chromatography (SEC-HPLC)

[0489] A TSKgel G3000SWXL 7.8mm ID x 30cm, 5μm column (Tosoh, PN: 0008541) was used as the mobile phase, with a ratio of 2xPBS to acetonitrile (9:1 v / v). The flow rate was 1.0 mL / min and the isocratic run was performed at room temperature for 15 minutes. The detection wavelength was 280 nm to analyze the high-molecular-weight aggregation of the ADC.

[0490] The test results are shown in FIG2 . The high molecular weight aggregates in the ADC drug are less than 6%, and the ADC sample (8.03 min) mainly exists in the monomer form. The damage of the coupling reaction to the antibody is almost negligible.

[0491] Example 27: Preparation and Characterization of ADC-2

[0492] 27.1 Preparation of Antibody-Drug Conjugate ADC-2

[0493] In the following, the sugar chain at the Fc end of the antibody mAb-1 is remodeled by endoglycosidase, and the linker-load is specifically coupled to the antibody to form the corresponding ADC drug.

[0494] 1) Treatment of mAb-1: The antibody was treated by ultrafiltration, dialysis, or desalting column, and its storage buffer was replaced with 50 mM Tris-HCl (pH 5-8) and 150 mM NaCl.

[0495] 2) Preparation of ADC-2: ADC-2 was prepared by catalyzing the coupling reaction between the antibody mAb-1 and the linker-load compound 4-2 using the endoglycosidase Endo S2. In 1× endoglycosidase buffer, the antibody mAb-1 and compound 4-2 were thoroughly mixed at an appropriate molar ratio (1:1 to 1:100), and then the endoglycosidase Endo S2 was added and mixed thoroughly. While mixing, the coupling reaction was carried out at 4-40°C for 0.5-20 hours. After completion of the reaction, purification was performed. The purified ADC-2 was stored in 1× PBS (pH 7.4) at 4°C or -80°C.

[0496] The characterization test methods and results of the antibody-drug conjugate ADC-2 are described in 27.2-27.3 below.

[0497] 27.2 Hydrophobic Interaction High-Performance Liquid Chromatography (HIC-HPLC) Analysis of Antibody-Drug Conjugate ADC-2

[0498] A Proteomix HIC Butyl-NP5 4.6*100mm 5um Non-Porous column (manufacturer: Saifen, PN: 431NP5-4610) was used; 1.5M ammonium sulfate + 20mM phosphate buffer, pH 7.0 was used as mobile phase A; 20mM phosphate buffer, pH 7.0: isopropanol = 8:2 (v / v) was used as mobile phase B; the flow rate was 0.8mL / min; a gradient method was used: phase B increased from 10% to 100% within 8 minutes; and 280nm was selected as the detection wavelength to detect the DAR distribution of ADC drugs.

[0499] The test results are shown in Figure 3. The unconjugated cytotoxic antibody is less than 1%; the conjugated product is mainly DAR2, and the overall DAR value of the ADC drug is about 1.90.

[0500] 27.3 Detection and Analysis of ADC-2 by Size Exclusion Liquid Chromatography (SEC-HPLC)

[0501] A BioCore SEC-300 5μm, 7.8*300mm column (PN: B213-050030-07830S) was used; the mobile phase was a 9:1 (v / v) mixture of 2*PBS:acetonitrile; the flow rate was 1.0mL / min; the run time was 15min; and the detection wavelength was 280nm to analyze and detect the high molecular weight aggregation of ADC drugs.

[0502] The test results are shown in FIG4 . The high molecular weight aggregates in the ADC drug are less than 2%, and the ADC sample (8.54 min) mainly exists in the monomer form. The damage of the coupling reaction to the antibody is almost negligible.

[0503] Example 28: Preparation and Characterization of ADC-3

[0504] The preparation and characterization were performed according to the method described in Example 27, except that the linker-payload used was 4-3. The characterization data of the antibody-drug conjugate ADC-3 are described below.

[0505] ADC-3 was analyzed by hydrophobic interaction high performance liquid chromatography (HIC-HPLC). The results are shown in FIG5 . The unconjugated cytotoxic antibody was less than 1%; the conjugated product was mainly DAR2. Overall, the DAR value of the ADC drug was about 1.90.

[0506] ADC-3 was analyzed by high-performance liquid phase size exclusion chromatography (SEC-HPLC). The test results are shown in Figure 6. The high molecular weight aggregates in the ADC drug were less than 2%. The ADC sample (8.53 min) existed mainly in monomeric form, and the damage to the antibody caused by the coupling reaction was almost negligible.

[0507] Example 29: Preparation and Characterization of ADC-4

[0508] The preparation and characterization were performed according to the method described in Example 27, except that the linker-payload used was 4-4. The characterization data of the antibody-drug conjugate ADC-4 are described below.

[0509] ADC-4 was analyzed by hydrophobic interaction high performance liquid chromatography (HIC-HPLC). The results are shown in FIG7 . The unconjugated cytotoxic antibody was less than 1%; the conjugated product was mainly DAR2. Overall, the DAR value of the ADC drug was about 1.95.

[0510] ADC-4 was analyzed by high-performance liquid-phase size-exclusion chromatography (SEC-HPLC). The results are shown in Figure 8 . High-molecular-weight aggregates in the ADC drug were less than 2%, and the ADC sample (8.53 min) existed primarily in monomeric form. Damage to the antibody caused by the coupling reaction was almost negligible.

[0511] Example 30: Preparation and Characterization of ADC-5

[0512] The preparation and characterization were performed according to the method described in Example 27, except that the linker-payload used was 4-5. The characterization data of the antibody-drug conjugate ADC-5 are described below.

[0513] Hydrophobic interaction high performance liquid chromatography (HIC-HPLC) analysis of ADC-5 showed that the unconjugated cytotoxic antibody was less than 1%; the conjugated product was mainly DAR4, and the overall DAR value of the ADC drug was about 3.82.

[0514] High-performance liquid-phase size exclusion chromatography (SEC-HPLC) analysis of ADC-5 showed that the high-molecular-weight aggregates in the ADC drug were less than 3%, and the ADC sample (8.55 min) mainly existed in the monomer form. The damage to the antibody caused by the coupling reaction was almost negligible.

[0515] Example 31: Preparation and Characterization of ADC-6

[0516] The preparation and characterization were performed according to the method described in Example 27, except that the linker-payload used was 4-6. The characterization data of the antibody-drug conjugate ADC-6 are described below.

[0517] Hydrophobic interaction high performance liquid chromatography (HIC-HPLC) analysis of ADC-6 showed that the unconjugated cytotoxic antibody was less than 1%; the conjugated product was mainly DAR4, and the overall DAR value of the ADC drug was about 3.91.

[0518] High-performance liquid-phase size-exclusion chromatography (SEC-HPLC) analysis of ADC-6 showed that the high-molecular-weight aggregates in the ADC drug were less than 3%, and the ADC sample (8.54 min) mainly existed in the monomer form. The damage to the antibody caused by the coupling reaction was almost negligible.

[0519] Example 32: Preparation and Characterization of ADC-7

[0520] The preparation and characterization were performed according to the method described in Example 27, except that the linker-payload used was 4-7. The characterization data of the antibody-drug conjugate ADC-7 are described below.

[0521] Hydrophobic interaction high performance liquid chromatography (HIC-HPLC) analysis of ADC-7 showed that the unconjugated cytotoxic antibody was less than 1%; the conjugated product was mainly DAR4, and the overall DAR value of the ADC drug was about 3.98.

[0522] High-performance liquid phase size exclusion chromatography (SEC-HPLC) analysis of ADC-7 showed that the high molecular weight aggregates in the ADC drug were less than 3%, and the ADC sample (8.53 min) mainly existed in the monomer form. The damage to the antibody caused by the coupling reaction was almost negligible.

[0523] Example 33: Preparation and Characterization of ADC-8

[0524] The preparation and characterization were performed according to the method described in Example 27, except that the linker-payload used was 4-8. The characterization data of the antibody-drug conjugate ADC-8 are described below.

[0525] Hydrophobic interaction high performance liquid chromatography (HIC-HPLC) analysis of ADC-8 showed that the unconjugated cytotoxic antibody was less than 1%; the conjugated product was mainly DAR4, and the overall DAR value of the ADC drug was about 3.87.

[0526] High-performance liquid-phase size-exclusion chromatography (SEC-HPLC) analysis of ADC-8 showed that the high-molecular-weight aggregates in the ADC drug were less than 3%, and the ADC sample (8.52 min) mainly existed in the monomer form. The damage to the antibody caused by the coupling reaction was almost negligible.

[0527] Example 34: Preparation and Characterization of ADC-9

[0528] The preparation and characterization were performed according to the method described in Example 27, except that the linker-payload used was 4-9. The characterization data of the antibody-drug conjugate ADC-9 are described below.

[0529] Hydrophobic interaction high performance liquid chromatography (HIC-HPLC) analysis of ADC-9 showed that the unconjugated cytotoxic antibody was less than 1%; the conjugated product was mainly DAR4, and the overall DAR value of the ADC drug was about 3.76.

[0530] High-performance liquid-phase size-exclusion chromatography (SEC-HPLC) analysis of ADC-9 showed that the high-molecular-weight aggregates in the ADC drug were less than 2%, and the ADC sample (8.53 min) mainly existed in monomeric form. The damage to the antibody caused by the coupling reaction was almost negligible.

[0531] Example 35: Preparation and Characterization of ADC-10

[0532] The preparation and characterization were performed according to the method described in Example 27, except that the linker-payload used was 4-10. The characterization data of the antibody-drug conjugate ADC-10 are described below.

[0533] Hydrophobic interaction high performance liquid chromatography (HIC-HPLC) analysis of ADC-10 showed that the unconjugated cytotoxic antibody was less than 2%; the conjugated product was mainly DAR4, and the overall DAR value of the ADC drug was about 3.72.

[0534] High-performance liquid-phase size exclusion chromatography (SEC-HPLC) analysis of ADC-10 showed that the high-molecular-weight aggregates in the ADC drug were less than 3%, and the ADC sample (8.51 min) mainly existed in the monomer form. The damage to the antibody caused by the coupling reaction was almost negligible.

[0535] Example 36: Preparation and Characterization of ADC-11

[0536] The preparation and characterization were performed according to the method described in Example 27, except that the linker-payload used was 4-11. The characterization data of the antibody-drug conjugate ADC-11 are described below.

[0537] Hydrophobic interaction high performance liquid chromatography (HIC-HPLC) analysis of ADC-11 showed that the unconjugated cytotoxic antibody was less than 1%; the conjugated product was mainly DAR4, and the overall DAR value of the ADC drug was about 3.86.

[0538] High-performance liquid-phase size exclusion chromatography (SEC-HPLC) analysis of ADC-11 showed that the high-molecular-weight aggregates in the ADC drug were less than 2%, and the ADC sample (8.52 min) mainly existed in monomeric form. The damage to the antibody caused by the coupling reaction was almost negligible.

[0539] Example 37: Preparation and Characterization of ADC-12

[0540] The preparation and characterization were performed according to the method described in Example 27, except that the linker-payload used was 4-12. The characterization data of the antibody-drug conjugate ADC-12 are described below.

[0541] Hydrophobic interaction high performance liquid chromatography (HIC-HPLC) analysis of ADC-12 showed that the unconjugated cytotoxic antibody was less than 1%; the conjugated product was mainly DAR4, and the overall DAR value of the ADC drug was about 3.64.

[0542] High-performance liquid-phase size exclusion chromatography (SEC-HPLC) analysis of ADC-12 showed that the high-molecular-weight aggregates in the ADC drug were less than 2%, and the ADC sample (8.56 min) mainly existed in monomeric form. The damage to the antibody caused by the coupling reaction was almost negligible.

[0543] Example 38: Preparation and Characterization of ADC-13

[0544] The preparation and characterization were performed according to the method described in Example 27, except that the linker-payload used was 4-13. The characterization data of the antibody-drug conjugate ADC-13 are described below.

[0545] Hydrophobic interaction high performance liquid chromatography (HIC-HPLC) analysis of ADC-13 showed that the unconjugated cytotoxic antibody was less than 1%; the conjugated product was mainly DAR4, and the overall DAR value of the ADC drug was about 3.74.

[0546] High-performance liquid-phase size-exclusion chromatography (SEC-HPLC) analysis of ADC-13 showed that the high-molecular-weight aggregates in the ADC drug were less than 2%, and the ADC sample (8.55 min) mainly existed in monomeric form. The damage to the antibody caused by the coupling reaction was almost negligible.

[0547] Example 39: Preparation and Characterization of ADC-14

[0548] The preparation and characterization were performed according to the method described in Example 27, except that the linker-payload used was 4-14. The characterization data of the antibody-drug conjugate ADC-14 are described below.

[0549] Hydrophobic interaction high performance liquid chromatography (HIC-HPLC) analysis of ADC-14 showed that the unconjugated cytotoxic antibody was less than 1%; the conjugated product was mainly DAR4, and the overall DAR value of the ADC drug was about 3.84.

[0550] High-performance liquid chromatography-exclusion chromatography (SEC-HPLC) analysis of ADC-14 showed that the high molecular weight aggregates in the ADC drug were less than 2%, and the ADC sample (8.56 min) mainly existed in the monomer form. The damage to the antibody caused by the coupling reaction was almost negligible.

[0551] Example 40: Preparation and Characterization of ADC-15

[0552] The preparation and characterization were performed according to the method described in Example 27, except that the linker-payload used was 4-15. The characterization data of the antibody-drug conjugate ADC-15 are described below.

[0553] Hydrophobic interaction high performance liquid chromatography (HIC-HPLC) analysis of ADC-15 showed that the unconjugated cytotoxic antibody was less than 1%; the conjugated product was mainly DAR2, and the overall DAR value of the ADC drug was about 1.92.

[0554] High-performance liquid-phase size exclusion chromatography (SEC-HPLC) analysis of ADC-15 showed that the high-molecular-weight aggregates in the ADC drug were less than 2%, and the ADC sample (8.53 min) mainly existed in the monomeric form. The damage to the antibody caused by the coupling reaction was almost negligible.

[0555] 5. In vitro activity testing of ADC

[0556] Example 41 In vitro activity test of ADC-1, ADC-2, ADC-3 and ADC-4 (SKBR-3, NCI-N87, MDA-MB-468)

[0557] The effects of ADC-1, ADC-2, ADC-3, and ADC-4 on the proliferation of tumor cells with different HER2 expression levels were tested using the following method:

[0558] 1) HER2-positive human breast cancer cells SKBR-3, HER2-positive human gastric cancer cells NCI-N87, and HER2-negative human breast cancer cells MDA-MB-468 were seeded into 96-well cell plates at 100 μL per well (containing 1,000-10,000 cells) and cultured overnight in a cell culture incubator at 37°C, 5% CO2, 95% air, and 100% humidity.

[0559] 2) ADC-1, ADC-2, ADC-3, and ADC-4 were added to HER2-positive and HER2-negative cells cultured overnight at varying concentrations (40, 10, 2.5, 0.625, 0.156, 0.039, 0.00977, 0.00244, 0.00061, and 0.00015). A no-drug group and a 5 μM Puromycin group were also set up as 100% and 0% cell viability controls, respectively. After drug administration, cells were placed in a cell culture incubator and incubated at 37°C for 72-120 hours.

[0560] 3) Remove the cell plate from the cell culture incubator and equilibrate to room temperature. Add 100 μL of CellTiter Glo reagent to each well, shake on a shaker for 2 minutes, and then let it stand at room temperature in the dark for 10 minutes. Measure the luminescence (RLU) using a Cytation 3 microplate reader.

[0561] 4) The results of the inhibitory effects of different drugs on tumor cell proliferation are shown in Table 1 and Figures 9-11. ADC-1, ADC-2, ADC-3, and ADC-4 showed significant inhibitory effects on HER2-positive cells. Furthermore, there was no significant difference in efficacy between the disaccharide ADC-4 and the trisaccharides ADC-1, ADC-2, and ADC-3 at the same DAR. ADC-1, ADC-2, ADC-3, and ADC-4 had no inhibitory effect on HER2-negative cells.

[0562] Table 1. Inhibitory effects of different drugs on tumor cell proliferation (IC 50 ,nM)

[0563] Example 42 In vitro activity testing of ADC-5, ADC-6, ADC-7 and ADC-8 (SKBR-3, NCI-N87, MDA-MB-468)

[0564] The effects of ADC-5, ADC-6, ADC-7, and ADC-8 on the proliferation of tumor cells with different HER2 expression levels were tested using the following method:

[0565] Referring to the method described in Example 41, the antibody-drug conjugates ADC-5, ADC-6, ADC-7, and ADC-8 were tested for their proliferative effects on tumor cells at varying HER2 expression levels. The results of the inhibitory effects of the different drugs on tumor cell proliferation are shown in Table 2 and Figures 12-14. ADC-5, ADC-6, ADC-7, and ADC-8 showed significant inhibitory effects on HER2-positive cells. There was no significant difference in efficacy between the disaccharide ADC-8 and the trisaccharides ADC-5, ADC-6, and ADC-7 at the same DAR. ADC-5, ADC-6, ADC-7, and ADC-8 had no inhibitory effect on HER2-negative cells.

[0566] Table 2. Inhibitory effects of different drugs on tumor cell proliferation (IC 50 ,nM)

[0567] Example 43 In vitro activity test of ADC-9, ADC-10 and ADC-11 (SKBR-3, NCI-N87, MDA-MB-468)

[0568] The effects of ADC-9, ADC-10, and ADC-11 on the proliferation of tumor cells with different HER2 expression levels were tested using the following method:

[0569] Referring to the method described in Example 41, the antibody-drug conjugates ADC-9, ADC-10, and ADC-11 were tested for their proliferative effects on tumor cells at varying HER2 expression levels. The results of the inhibitory effects of the different drugs on tumor cell proliferation are shown in Table 3 and Figures 15-17. ADC-9, ADC-10, and ADC-11 showed significant inhibitory effects on HER2-positive cells. There was no significant difference in efficacy between the disaccharide ADC-11 and the trisaccharides ADC-9 and ADC-10 at the same DAR. ADC-9, ADC-10, and ADC-11 had no inhibitory effect on HER2-negative cells.

[0570] Table 3. Inhibitory effects of different drugs on tumor cell proliferation (IC 50 ,nM)

[0571] Example 44 In vitro activity test of ADC-12, ADC-13 and ADC-14 (SKBR-3, NCI-N87, MDA-MB-468)

[0572] The effects of ADC-12, ADC-13, and ADC-14 on the proliferation of tumor cells with different HER2 expression levels were tested as follows:

[0573] Referring to the method described in Example 41, the antibody-drug conjugates ADC-12, ADC-13, and ADC-14 were tested for their proliferative effects on tumor cells with varying HER2 expression levels. The results of the inhibitory effects of the different drugs on tumor cell proliferation are shown in Table 4 and Figures 18-20. ADC-12, ADC-13, and ADC-14 showed significant inhibitory effects on HER2-positive cells. There was no significant difference in efficacy between the disaccharide ADC-14 and the trisaccharides ADC-12 and ADC-13 at the same DAR. ADC-12, ADC-13, and ADC-14 had no inhibitory effect on HER2-negative cells.

[0574] Table 4. Inhibitory effects of different drugs on tumor cell proliferation (IC 50 ,nM)

[0575] Example 45 Detection of affinity of ADC-5, ADC-6, ADC-7 and ADC-8 for cell surface HER2 (NCI-N87)

[0576] The antigen binding rates of ADC-5, ADC-6, ADC-7, and ADC-8 in the NCI-N87 cell environment were tested as follows:

[0577] 1. Cell Sample Preparation

[0578] 1) Cell Harvesting: Detach adherent cells from the culture flask using 0.25% trypsin in a cell digestion buffer. Place the cell suspension into a 15 mL centrifuge tube. Centrifuge the cell suspension at 1000 rpm for 5 minutes. Remove the supernatant and resuspend the cells in an appropriate amount of FACS buffer. Determine cell viability using trypan blue staining.

[0579] 2) Cell dilution: Take an appropriate amount of cell suspension and add FACS buffer to prepare 10mL 1-2×10 6 Cells / mL of cell suspension.

[0580] 2. Cell-antibody and ADC drug binding

[0581] 1) Plate the cells using a V-bottom plate. Invert several times before plating to mix the cells thoroughly. Add 100 μL of cell solution to each well. Add 100 μL of FACS buffer to the cell-only control group and 100 μL of antibody solution to the other experimental groups. Immediately incubate on ice for 60 minutes.

[0582] 2) Wash unbound antibody or ADC: Pellet cells by centrifugation at 2000 rpm for 3 min at 4°C and discard the supernatant. Resuspend cells in 200 μL / well of ice-cold FACS buffer. Pellet cells by centrifugation again at 2000 rpm for 3 min at 4°C and discard the supernatant.

[0583] 3) Secondary antibody binding: Use 500-fold diluted Goat anti-Human IgG (H+L), Superclonal TM Recombinant Secondary Antibody, Alexa Fluor TM Resuspend cells in 100 μL / well of Plus 647 and incubate on ice for 60 minutes in the dark. Repeat step 2 to wash away unbound antibody. Resuspend cells in 100 μL / well of buffer and store at 4°C in the dark until ready for flow cytometry.

[0584] 3. Cytometer Detection

[0585] 1) Set appropriate acquisition parameters

[0586] 2) Cell sample injection

[0587] 3) Analyze using FACSDiva software

[0588] The binding results of different drugs to tumor cells are shown in Table 5 and Figure 21. The binding affinity of the disaccharide ADC-8, the trisaccharides ADC-5, ADC-6, and ADC-7, and the monoclonal antibody OL1302 on NCI-N87 cells was comparable, and the binding behavior was concentration-dependent. The negative control antibody Isotype showed no significant specific binding to NCI-N87 cells.

[0589] Table 5. Binding affinity of different drugs to NCI-N87 cells (EC 50 ,nM)

[0590] 6. In vivo activity testing of ADCs

[0591] Example 46 In vivo efficacy evaluation of ADC drugs ADC-6, 7, and 8

[0592] 1) Collect NCI-N87 gastric cancer cells in the logarithmic growth phase and adjust the cell density to 10×10 6 cells / mL, and 0.2 mL of the prepared NCI-N87 cell suspension was subcutaneously inoculated into the right scapula of each Balb / c nude mouse.

[0593] 2) Measure the tumor diameter with a vernier caliper and calculate the diameter using the formula V = 0.5 × a × b 2 The tumor volume was calculated (where a is the longest diameter of the tumor and b is the shortest diameter of the tumor). On day 6 after cell inoculation, the average tumor volume reached 180 mm 3The animals were randomly divided into a vehicle control group, a 5 mg / kg ADC-6 group, a 5 mg / kg ADC-7 group, and a 5 mg / kg ADC-8 group, with 5 animals in each group. The animals were injected via the tail vein. The tumor volume of each group of animals was measured twice a week after administration, and the tumor volume of animals on day 21 was compared between groups. The T / C (%) value and TGI (%) value were calculated based on the tumor volume. The calculation formula is as follows: T / C% = T RTV / C RTV ×100%(T RTV :RTV in treatment group; C RTV : RTV of vehicle control group). Relative tumor volume (RTV) was calculated based on the results of tumor measurement. The calculation formula was RTV = Vt / V0, where V0 was the average tumor volume measured at the time of grouping (i.e., D0), Vt was the average tumor volume at a certain measurement, and T RTV with C RTV Data were collected on the same day. Calculation of TGI (%): TGI (%) = [1 - (average tumor volume of a treatment group at the end of drug administration - average tumor volume of the treatment group at the start of drug administration) / (average tumor volume of the vehicle control group at the end of treatment - average tumor volume of the vehicle control group at the start of treatment)] × 100%.

[0594] 3) On day 21 after administration, the results of the inhibitory effects of different drugs on xenograft tumors in mice are shown in Table 6 and Figure 22. The average tumor volume of the vehicle control group was 450 mm 3 The average tumor volumes of the 5mg / kg ADC-6, 5mg / kg ADC-7, and 5mg / kg ADC-8 groups were 12mm 3 , 10mm 3 and 12mm 3 It can be seen that all three ADC drugs have significant and superior tumor inhibition effects, and there is no significant difference in efficacy among ADC-6, ADC-7 and ADC-8.

[0595] Table 6. Inhibitory effects of ADC-6, ADC-7, and ADC-8 on NCI-N87 mouse xenograft tumors

[0596] a. Mean ± SEM;

[0597] bT / C%=T RTV / C RTV x 100%(T RTV :RTV in treatment group; C RTV: RTV of vehicle control group). Relative tumor volume (RTV) was calculated based on the results of tumor measurement. The calculation formula is RTV=V 21 / V0, where V0 is the average tumor volume measured at the time of group administration (i.e., D0), V 21 T is the average tumor volume on day 21 after administration. RTV with C RTV Get data from the same day;

[0598] c. TGI (%) = [1-(T 21 -T0) / (V 21 -V0)]×100) calculation;

[0599] d. Statistical analysis was performed using One-way ANOVA.

[0600] 7. ADCC activity test of ADC

[0601] Example 47 Evaluation of ADCC activity of ADC drugs ADC-5, 6, 7, and 8

[0602] Since the antibody-dependent cell-mediated cytotoxicity (ADCC) function is closely related to the affinity of the antibody for the FcγR series of receptors and is directly affected by the glycoform structure at position N-297 of the antibody Fc region. Through enzyme-catalyzed glycosyl coupling reactions, oligosaccharide linkers can be introduced to reshape the glycosyl structure of the antibody Fc region, thereby regulating the ADCC function of the generated ADC molecule. Given the differences between the trisaccharide structure and the existing disaccharide structure, this example aims to test the difference in ADCC activity between the ADC obtained using the trisaccharide linker of the present invention and the activity of the disaccharide linker.

[0603] In the experiment, HER2-positive SK-BR-3 cells were used as target cells and primary human NK cells were used as effector cells. The two cells were co-cultured in a ratio of 1:5 (SK-BR-3:NK). After the effector cells and target cells were incubated with the test article for 4 hours, the ADCC effect of the test drug was reflected by detecting the release of LDH.

[0604] The experimental results show that ADCs with trisaccharide linkers have stronger ADCC activity than those with disaccharide linkers, and different glycoforms have different degrees of influence on ADCC activity. The specific results are shown in Figure 23.

[0605] Sequence Listing

Claims

1. A linker-carrier compound having the structure of formula (I): D——L——(P) t Formula (I) in P is the load; L is a linker, and L is directly connected to D via the terminal -NH- therein, wherein when L is an unbranched linker, it is connected to 1 P, and t is 1, and when L is a branched linker, each branch can be connected to 1 P, and t is an integer greater than 1 (e.g., 2, 3, 4, 5, 6, 7, 8, 9 or 10); D is a trisaccharide unit structure, which contains a first hexose unit or a derivative thereof, a second hexose unit or a derivative thereof, and a third hexose unit or a derivative thereof; The 6-OH of the first hexose unit or its derivative is oxidized to -C(O)- and connected to the terminal -NH- of L; The first hexose unit or its derivative part is connected to the second hexose unit or its derivative part via an α-(1→6) glycosidic bond; The second hexose unit or its derivative part is connected to the third hexose unit or its derivative part via a β-(1→4) glycosidic bond; The third hexose unit or its derivative part has the following structure: or 2. The linker-load compound of claim 1, wherein The first hexose unit or its derivative part is selected from glucosyl, mannosyl, galactosyl, fructosyl, gulosyl, idosyl or their derivatives.

3. The linker-load compound of claim 2, wherein: The first hexose unit or its derivative part is selected from in, represents the site of connection to L; * represents the site of connection to the second hexose unit or its derivative.

4. The linker-load compound according to claim 1, in, The second six-carbon sugar unit or its derivative part is selected from glucosyl, mannosyl, galactosyl, fructosyl or their derivatives.

5. The linker-load compound of claim 4, wherein: The second six-carbon sugar unit or its derivative part is selected from in, indicates the site of connection to the third hexose unit or its derivative; * indicates the site of connection to the first hexose unit or its derivative.

6. The linker-load compound of claim 1, wherein The trisaccharide unit structure has the following structure:

7. The linker-load compound of claim 1, wherein -L-(P) t For -L 2 -L 1 -BP, that is, formula (I) is: D——L 2 ——L 1 ——B——P Formula (I-1) in B is independently absent, or is the following 1), or is the following 2), or is a combination of the following 1) and 2): 1) a self-removing spacer Sp1; 2) a divalent group, or a combination of two or more divalent groups, wherein 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 independently absent; or a non-cleavable sequence; or a cleavable sequence comprising an amino acid sequence cleavable by an enzyme, wherein the amino acid sequence cleavable by the enzyme comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids; L 2 Independently absent; or the following 1); or the following 2); or the following 1) and 2) combination: 1) -NH-C 2-20 Alkylene, wherein one or more -CH2- structures in the alkylene are optionally replaced by the following groups: -CR 3 R 4 -, -O-, -(CO)-, -S-, -S(=O)2-, -NR 5 -、-N ⊕ R 6 R 7 -、C 4-10 Cycloalkylene, C 4-10 cycloalkylene, heterocyclylene, phenylene, wherein cycloalkylene, heterocyclylene and phenylene are each independently unsubstituted or selected from halogen, -C 1- 10 Alkyl, -C 1-10 Haloalkyl, -C 1-10 Alkylene-NH-R 8 and -C 1-10 Alkylene-OR 9 is substituted with at least one substituent; 2) Amino acid residue sequence, i.e. -*(AA) n **-, n is an integer from 1 to 100, AA is independently an amino acid residue at each occurrence, * represents the N-terminus of the corresponding amino acid, ** represents the C-terminus of the corresponding amino acid, and -(C2H4-O) is optionally present between the amino group and the α-carbon of an amino acid m -(CH2) p -, wherein m is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; p is 0, 1, 2 or 3, and the * end forms an amide bond with the carbonyl group in the trisaccharide structure; Among them, B, L 1 and L 2 Not existing at the same time; R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 are each independently selected from hydrogen, halogen, substituted or unsubstituted -C 1-10 Alkyl, C 4-10 Cycloalkylene; or R 1 and R 2 and together with the carbon atoms to which they are attached form a 3-6 membered cycloalkylene group, and / or R 3 and R 4 and together with the carbon atoms to which they are attached form a 3-6 membered cycloalkylene group; P is with B part, or L 1 Part, or L 2 Partially connected loads; D is as defined in claim 1.

8. The linker-carrier compound of claim 1, wherein -L-(P) t for That is, formula (I) is: in, Ld2 and each Ld1 are independently a bond; or selected from -NH-C 1-20 Alkylene-(CO)-, -NH-(PEG) i -(CO)-; or each independently unsubstituted or -CO-(PEG) on the side chain j -R 11 Substituted natural amino acids or oligomeric natural amino acids having a degree of polymerization of 2-10 (i.e. 2, 3, 4, 5, 6, 7, 8, 9 or 10); -(PEG) i -and-(PEG) j - Each is a PEG fragment comprising a specified number of consecutive -(O-C2H4)- structural units or consecutive -(C2H4-O)- structural units, optionally with C appended at one end 1-10 Alkylene; M is hydrogen or LKa-L 2 ―L 1 ―B―P; Q is NH2 or L 2 ―L 1 ―B―P; The conditions do not include the following: M is hydrogen and at the same time Q is NH2; Each LKa is independently selected from opSu or a mixture thereof; wherein * represents and L 2 The connecting part; Among them, D and L 2 , L 1 , B as defined in claim 7; R 11 It is C 1-10 alkyl; d is 0, 1, 2, 3, 4, 5, or 6; Each i is independently an integer from 1 to 100 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10); Each j is independently an integer from 1 to 100 (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10).

9. The linker-load compound according to claim 7 or 8, wherein L 2 is the amino acid residue sequence, i.e. -*(AA) n **-, n is an integer from 1 to 100; AA is independently an amino acid residue at each occurrence, * represents the N-terminus of the corresponding amino acid, ** represents the C-terminus of the corresponding amino acid, and -(C2H4-O) is optionally present between the amino group and the α-carbon of an amino acid m -(CH2) p -, wherein m is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; p is 0, 1, 2 or 3, and the * end forms an amide bond with the carbonyl group in the trisaccharide structure.

10. The linker-carrier compound of claim 9, wherein AA is independently 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 at each occurrence.

11. The linker-carrier compound according to claim 7 or 8, wherein n is an integer of 1-50, preferably an integer of 1-30, preferably an integer of 1-20, preferably an integer of 1-10, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10.

12. The linker-carrier compound according to claim 7 or 8, wherein L 1 is a cleavable sequence comprising an amino acid sequence cleavable by an enzyme, wherein the amino acid sequence cleavable by the enzyme comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids; preferably, L 1 It is any one of Val, Cit, Phe, Lys, Gly, Ala, Leu, and Asn, or any combination thereof; more preferably, it is -Val-Ala-, -Gly-Gly-Phe-Gly-, -Phe-Lys-, -Val-Cit-, -Val-Lys-, -Gly-Phe-Leu-Gly-, -Ala-Leu-Ala-Leu-, -Ala-Ala-Ala-, and combinations thereof; further preferably, it is -Val-Cit-, -Gly-Gly-Phe-Gly-.

13. The linker-carrier compound according to claim 7 or 8, wherein B is absent, or is selected from: or -NH-CH2-U-; or -NH-CH2-U-(CH2) g -(CO)-, wherein g is 1, 2, 3, 4, 5 or 6; U is absent or is CH2, O, S or NH, preferably O or S.

14. The linker-load compound according to claim 7 or 8, wherein -L 1 -B- represents -Val-Cit-PABC-; or -Gly-Gly-Phe-Gly-; or -Gly-Gly-Phe-Gly-NH-CH2-U-, wherein U is absent, or is O, S or NH, preferably O or S.

15. The linker-carrier compound according to claim 7 or 8, wherein -L 2 - is the amino acid residue sequence, i.e. -*(AA) n **-, n is an integer from 1 to 10, AA is independently an amino acid residue at each occurrence, * represents the N-terminus of the corresponding amino acid, ** represents the C-terminus of the corresponding amino acid, and -(C2H4-O) is optionally present between the amino group and the α-carbon of an amino acid m -(CH2) p -, wherein m is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; p is 0, 1, 2 or 3, and the * end forms an amide bond with the carbonyl group in the trisaccharide structure, preferably, -L 2 - is -Gly-Gly-Gly-; or -L 2 -NH-C 2-20 Alkylene, wherein one or more -CH2- structures in the alkylene are optionally replaced by the following groups: -O-, -(CO)-.

16. The linker-carrier compound according to any one of claims 1 to 15, wherein the carrier P is selected from the group consisting of small molecule compounds, nucleic acids and nucleic acid analogs, tracer molecules, short peptides, polypeptides, peptidomimetics and proteins.

17. The linker-carrier compound of claim 7, selected from: Each a is independently 0, 1, 2, 3, 4, or 5.

18. The linker-carrier compound of claim 8, selected from: Each a is independently 0, 1, 2, 3, 4, or 5; Each b is independently selected from an integer of 1-30, preferably an integer of 1-15; more preferably 6, 7, 8, 9, 10, 11, 12.

19. An antibody-drug conjugate based on site-specific attachment of N-glycosylation sites in the Fc region of an antibody, having a structure of formula (II): in P is the load; R is hydrogen or α-L-fucosyl; q is 1 or 2; Ab is an antibody or antigen-binding fragment; The first hexose unit or its derivative part is as defined in claim 2 or 3; and / or The second hexose unit or its derivative part is as defined in claim 4 or 5; and / or The derivatives are independently selected from derivatives in which the hydroxyl groups of uronic acid or monosaccharide are replaced by acylamino groups; and / or The first hexose unit or its derivative part is connected to the second hexose unit or its derivative part via an α-(1→6) glycosidic bond; The remaining monosaccharide moieties are linked by β-(1→4) glycosidic bonds; -L-(P) t As defined in any one of claims 1 to 18.

20. The antibody-drug conjugate according to claim 19, wherein formula (II) is selected from formula (II-1), (II-2), (II-3), (II-4), (II-5), (II-6), (II-7), (II-8) or (II-9): in R is hydrogen or α-L-fucosyl; q is 1 or 2; Ab is an antibody or antigen-binding fragment; -L-(P) t As defined in any one of claims 1 to 19.

21. The antibody-drug conjugate according to claim 20, wherein: Ab is an anti-HER2 antibody.

22. A pharmaceutical composition comprising the antibody-drug conjugate according to any one of claims 20-21.

23. Use of the antibody-drug conjugate according to any one of claims 20 to 21 in the preparation of a medicament for treating or preventing tumors or autoimmune diseases.

24. A method for treating or preventing tumors or autoimmune diseases, comprising administering a therapeutically effective amount of the antibody-drug conjugate according to any one of claims 20-21 to a subject in need thereof.

25. A trisaccharide linker having the following formula (IV): in, In formula (IV), R1-R 12 is selected from -OH, or H; Preferably, the two substituents on the same carbon atom are different; More preferably, the two substituents on the same carbon atom are different; and R3 is H, and R4 is -OH; More preferably, the two substituents on the same carbon atom are different; and R3 is H, R4 is -OH; R9 is H, R 10 is -OH; More preferably, the two substituents on the same carbon atom are different; and R3 is H, R4 is -OH; R9 is H, R 10 is -OH; R8 is H, R7 is -OH; R 12 For H, R 11 It is -OH.

26. The trisaccharide linker according to claim 25, wherein the specific structure is as follows:

27. Use of the trisaccharide linker according to the structure shown in claim 25 or 26 in the preparation of conjugated drugs.

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