Oligosaccharide linker, linker-payload containing the oligosaccharide linker, and antibody-drug conjugate with remodeled sugar chains, method for producing the same, and use thereof

Abstract

The present invention relates to oligosaccharide groups, particularly linker-payload compounds containing disaccharide groups, wherein the oligosaccharide groups are linked to other parts of the compound via amide bonds. The present invention further relates to an antibody-drug conjugate (ADC) containing the linker-payload compound, and uses the oligosaccharide group in the linker-payload compound to remodel the sugar chain of the antibody. The present invention further relates to methods for producing and using the above-described substances.

Description

Technical Field

[0001] The present invention relates to oligosaccharide (especially disaccharide) linkers. Further, it relates to linker-payload compounds containing oligosaccharide groups, especially disaccharide groups, wherein the oligosaccharide groups are linked to other parts of the compound via amide bonds. The present invention further relates to antibody-drug conjugates (ADCs) containing the linker-payload compounds, wherein the oligosaccharide groups in the linker-payload compounds remodel the sugar chains in the antibodies. The present invention further relates to methods for manufacturing and uses of the above-described substances.

Background Art

[0002] Cancer is one of the main causes of death in humans. Every year, approximately one-sixth of the deaths worldwide are related to cancer. In 2017, there were 24.5 million new cancer cases and 9.6 million cancer deaths globally. The main treatments for cancer are surgery, radiotherapy, and drug therapy. With the development and application of these therapies, the survival situation of cancer patients has been significantly improved. The drug therapy for cancer has gone through three generations of development: chemotherapy, targeted therapy, and immunotherapy. Chemotherapy occupies an important position in cancer treatment, but it has relatively large non-therapeutic toxic side effects. While killing cancer cells, it also kills a large number of normal cells. Targeted therapy reduces the severe toxic side effects in traditional chemotherapy to a certain extent. This therapy mainly uses small molecule targeted drugs (currently mainly tyrosine kinase inhibitors) or monoclonal antibodies to target specific genes or proteins (i.e., target sites) involved in the growth and survival of tumor cells, and kills tumor cells and tissues. Antibody-drug conjugate (ADC) is a new type of targeted drug, which is a new drug molecule formed by linking a small molecule drug with high activity to a monoclonal antibody by chemical bonding. ADC drugs have both the high activity of small molecule drugs and the high specificity and targeting of antibody drugs. They can reduce the non-therapeutic toxic side effects of small molecule toxins on important tissues and organs such as the liver, kidney, nerves, and heart to a certain extent, and can eliminate the limitation that the therapeutic effect of antibody therapy on solid tumors is limited. Therefore, it has now become one of the hot directions in the research and development of anti-tumor drugs.

[0003] The development of ADC drugs involves systems engineering that carefully controls four elements: monoclonal antibodies, bioactive small molecules, linkers, and conjugation methods. Among these, the conjugation method has a significant impact on drug-related properties such as drug efficacy stability, metabolic consistency, and quality control (Non-Patent Document 1). Currently, commercially available and clinical-stage ADC drugs mainly employ random conjugation via lysine or cysteine residues, often resulting in a highly heterogeneous mixture with random conjugation sites and a non-uniform drug / antibody ratio (DAR). This easily leads to problems in aspects such as process stability, quality control, drug stability, metabolic consistency, and safety.

[0004] To address the above-mentioned problems caused by random conjugation, since 2008, the academic and industrial communities have intensively studied the development and application of site-specific conjugation strategies and achieved some encouraging progress. These site-specific conjugation methods can be broadly classified into three types: conjugation by introducing specific amino acids through gene-manipulated mutations, conjugation facilitated by enzymes inserted with polypeptides, and site-specific conjugation techniques that promote glycan remodeling by enzymes. The exploration and application of these site-specific conjugation techniques in ADC drug development have successfully solved many problems caused by random conjugation. However, in the first two types of the above-mentioned site-specific conjugation techniques, usually, it is necessary to genetically manipulate or modify the antibody, lacking versatility in manufacturing ADCs for different target antibodies. For each new antibody, a lot of repetitive and cumbersome cell engineering is required, significantly reducing the efficiency of new drug development. On the other hand, site-specific conjugation by remodeling the Fc glycan of the antibody does not require antibody modification and cell engineering, greatly reducing the development difficulty and workload, and can become a general-purpose platform technology for site-specific conjugation of antibodies.

[0005] At position 297 of the antibody Fc region, there is a highly conserved glycosylation (N-297Glycan), and site-specific binding of different molecules on the antibody can be achieved by glycan remodeling at this site (Non-Patent Document 2). Site-specific binding technology by glycosylation modification of the antibody Fc region is roughly divided into the following two types.

[0006] 1) In the chemical method, sodium periodate is used to oxidize core fucose (Non-Patent Document 3) or the o-diol of sialic acid at the glycan terminus (Non-Patent Document 4) to obtain the corresponding aldehyde, and the aldehyde carbonyl group can be used for the production of ADC by binding to a low-molecular-weight toxin fragment. The limitation of such a method is that the structures of the glycans at position N-297 are diverse, and not all monoclonal antibody glycans contain reactive sites, and the substrate limitation is relatively large.

[0007] 2) In the enzyme-catalyzed method, glycan remodeling is achieved by a catalytic relay of deglycosylation-transglycosylation using tool enzymes such as endoglycosidase and glycosyltransferase, and a bioorthogonal reaction group is introduced. Furthermore, the production of site-specific binding ADC can be realized by subsequent chemical reactions (Non-Patent Document 2 and Non-Patent Document 5).

[0008] In 2012, Wang Laixi et al. reported a site-specific binding technology by glycan remodeling of antibodies using endoglycosidase Endo S and its mutants as catalysts (Non-Patent Document 6). This technology utilizes the selective endo-deglycosylation of wild-type Endo S on the β-1,4-glycosidic bond between GlcNAc-GlcNAc in the oligosaccharide structure at position N-297 and the transglycosylation of the mutant enzyme Endo S D233Q to synthesize glycan-remodeled antibodies with a single glycan structure and modified with azide groups. Based on this work, Wang Laixi, Huang Wei et al. developed a major category of ADC site-specific binding technologies based on a three-step method of deglycosylation-transglycosylation-click chemical reaction using tool enzymes such as endoglycosidase Endo S, or Endo S2, and its mutants as catalysts (Non-Patent Document 5). In 2021, Wang Laixi et al. reported site-specific binding of antibody glycan remodeling by catalytic action of one-pot Endo S2, introduced a bioorthogonal azide functional group into the antibody, and further performed click chemical reaction to obtain ADC molecules (Non-Patent Document 7). Currently, all of the above-mentioned technologies are in the basic research stage, and no reports have been made yet on the clinical development of ADC drugs based on the corresponding technologies.

[0009] Another commonly used tool enzyme in site-specific binding technology for glycan remodeling is β-1,4-galactosyltransferase (β-1,4-Gal-T1) and its variant (β-1,4-Gal-T1 Y289L). This enzyme uses uridine diphosphate galactose (Gal-UDP) as a donor to transfer the galactosyl group Gal to the non-reducing end of N-acetylglucosamine (Glc-NAc) of glycoproteins. In 2009, Qasba et al. (Non-Patent Document 8) first reported an antibody site-specific binding technology using β-1,4-Gal-T1 and β-1,4-Gal-T1 Y289L as tool enzymes with C2-keto-Gal-UDP or N-azidoacetylgalactosamine-UDP (GalNAz-UDP) as donors, and produced antibodies modified with a ketone carbonyl group or an azide group. Based on this, in 2014, they reported an ADC molecule synthesis method based on a three-step method of deglycosylation-transglycosylation-bioorthogonal reaction using β-1,4-Gal-T1 and its variant as catalysts for the first time. It should be noted here that the corresponding ADC molecule retains a fairly high affinity for the FcγRIIIa and FcγRI receptors, retains a certain degree of antibody-dependent cell cytotoxicity (ADCC) effect, and the molecule showed potential killing power in the Her2-positive JIMT-1 breast cancer cell line (Non-Patent Document 9). Based on the above work, Synaffix developed a three-step synthesis method for ADC by combining the use of the endoglycosidase Endo S and the tool enzyme β-1,4-Gal-T1 Y289L (Non-Patent Document 10). In addition, the literature further reports a four-step synthesis method for ADC of deglycosylation-transglycosylation-transglycosylation-bioorthogonal reaction (Non-Patent Document 11). In this method, a total of three types of tool enzymes, namely β-1,4-Gal-T1, β-1,4-Gal-T1 Y289L, and sialyltransferase, were used.

[0010] The above-mentioned sugar chain remodeling reaction avoids the problem of the need for genetic manipulation of antibodies in other site-specific binding techniques, but still has very significant limitations. On the one hand, the binding process is very long and the operation is complicated. At least two enzymatic catalytic reactions plus one chemical reaction, that is, at least three reactions and three complete purifications are required to obtain an ADC. On the other hand, even if a trace amount of the tool enzyme remains in the product, the ADC product may be deglycosylated and decomposed, and the toxin molecule may fall off, causing a serious toxic reaction, posing a major problem in drug research and development and production, and also bringing a major risk to the safety of ADC drugs. More importantly, in the above-mentioned sugar chain remodeling reaction, the method of linking the sugar group and the toxin molecule is still limited, and it may be necessary to adjust the linking method according to the demand by additional reactions, and these additional reactions may also bring many adverse by-products. The object of the present invention is to solve these problems.

Prior Art Documents

Non-Patent Documents

[0011]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Non-Patent Document 6

Non-Patent Document 7

Non-Patent Document 8

Non-Patent Document 9

Non-Patent Document 10

Non-Patent Document 11

Summary of the Invention

Means for Solving the Problems

[0012] The present invention provides a novel oligosaccharide (especially disaccharide) linker, and a method for producing and using the same. Further, a linker-payload compound containing an oligosaccharide group is provided, wherein the oligosaccharide group is linked to other parts of the compound via an amide bond. Specifically, the present invention provides a linker-payload compound having the formula (I):

Chemical formula

Chemical formula

Chemical formula

[0013] The present invention further relates to an antibody-drug conjugate (ADC) comprising the linker-payload compound, wherein the oligosaccharide group in the linker-payload compound is used to remodel the sugar chain of the antibody. Specifically, the present invention provides an antibody-drug conjugate having the formula (II) by site-specific binding to the N-glycosylation site of the antibody Fc region:

Chemical formula

[0014] The present invention further relates to a method for manufacturing and using the above-described substances.

[0015] The present invention expands the scope of ADCs obtained by glycan remodeling technology by using a novel oligosaccharide structure that forms an amide bond. In a contemplated scenario, compounds having NH2 (e.g., L’-(P) t a compound having the structure, where P and t in the formula are as defined in the text, and except that the -NH- linked to D-C(O)- in L is H2N- in L’, L’ is the same as L defined in the text) are all, via the above oligosaccharide structure, by a simple amide reaction, a linker-payload

Chemical formula

Chemical formula

Chemical formula

Brief Description of the Drawings

[0016]

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Modes for Carrying Out the Invention

[0017] General Definitions Unless otherwise defined hereinafter, all technical and scientific terms used in this text shall have the same meaning as commonly understood by those skilled in the art. The technologies used in this text are those generally understood in the art and include obvious modifications and equivalent alternatives for those skilled in the art. The following terms are considered to be easily understood by those skilled in the art, but the definitions are explained below for better description of the present invention. When a product name is described in this text, it means the corresponding product or its active ingredient. All patents, published patent applications and publications cited in this text are hereby incorporated herein by reference.

[0018] When describing an amount, concentration, or other numerical value or parameter in the form of a range, a preferred range, or a preferred upper limit, or a preferred lower limit, it should be understood that any range formed by combining any upper limit or preferred value with any lower limit or preferred value is equivalent to the specifically disclosed one, regardless of whether the range is specified or not. Unless otherwise explained, the numerical ranges given in this text shall include the endpoints of the range and all integers and fractions (decimals) within 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 for j, g, k, etc., should be understood in a similar manner.

[0019] Unless otherwise clearly specified in the foregoing or following text, singular forms such as "one kind (piece)" and "the kind (piece)" also include the plural form. Expressions such as "one kind (piece) or a plurality of kinds (pieces)", or "at least one kind (piece)" represent 1, 2, 3, 4, 5, 6, 7, 8, 9, or more.

[0020] When the terms "about" and "approximately" are used with a numerical variable, generally, both the value of the variable and all values of the variable are within the range of experimental error (for example, within the 95% confidence interval of the average value), or within a wider range of ±10% or more of the specified value.

[0021] The term "optional" means that such a situation may occur, but does not necessarily occur, and includes descriptions whether the situation or circumstances occur or not.

[0022] Expressions such as "comprising", "including", "containing" and "having" are open languages and do not exclude other unmentioned elements, steps or components. The expression "consisting of ···" does not include undefined elements, steps or components. The expression "consisting essentially of ···" means that its scope is limited to the defined elements, steps, or components, and elements, steps, or components that do not substantially affect the basis and novel features of the theme for which protection is sought and may exist. The expression "comprising" should be understood to include expressions such as "consisting essentially of ···" and "consisting of ···".

[0023] The term "targeted molecule" refers to a molecule that has an affinity for a specific target (e.g., receptor, cell surface protein, cytokine, tumor-specific antigen, etc.). The targeted molecule can deliver a payload to a specific site in vivo by targeted delivery. The targeted molecule can identify one or more targets. The specific target site is defined by the target identified by the targeted molecule. For example, a targeted molecule that targets a receptor can deliver a cytotoxin to a site containing many receptors. Examples of targeted molecules include, but are not limited to, antibodies, binding proteins for specific antigens, antibody mimetics, scaffold proteins having an affinity for a specific target, ligands, etc. Targets identified 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, CN33, GPNMB, ENPP3, Nectin-4, EGFRv III, SLC44A4 / AGS-5, CEACAM5, PSMA, TIM1, LY6E, LIV1, Nectin 4, 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.

[0024] HER2 refers to human epidermal growth factor receptor-2 and belongs to the epidermal growth factor (EGFR) receptor tyrosine kinase family. In the present application, the terms ErbB2 and HER2 have the same meaning and can be used interchangeably with each other.

[0025] TROP2 is a transmembrane glycoprotein encoded by the Tacstd2 gene. TROP2 is a type of intracellular calcium signal sensor and is overexpressed in multiple types of tumors.

[0026] CLDN18.2 (Claudin-18 isoform 2) is a member of the human Claudin family. CLDN18.2 is a pan-cancer target that is expressed in primary and metastatic lesions of multiple human cancer types.

[0027] As used herein, the term "antibody" is used in a broad sense, and its definition includes general antibodies, recombinant antibodies / antibodies genetically engineered, and in particular, complete monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, provided they have the required biological activity. Antibodies may be of any subtype (e.g., IgG, IgE, IgM, IgD, and IgA), or subclass, and may be derived from any suitable species. In some embodiments, the antibodies are of human or murine origin. Antibodies may be fully human antibodies, humanized antibodies, or chimeric antibodies produced by recombinant methods.

[0028] As used herein, a monoclonal antibody is an antibody obtained from a basically homogeneous antibody population, i.e., with the exception of a few possible natural mutations, the individual antibodies constituting the population are identical. Monoclonal antibodies are highly specific for a single antigenic site. The term "monoclonal" means that the characteristics of the antibody are derived from a basically homogeneous antibody population and should not be construed as requiring the antibody to be produced by certain specific methods.

[0029] A complete antibody or full-length antibody basically comprises an antigen-binding variable region, a light-chain constant region (CL), and a heavy-chain constant region (CH), and may include CH1, CH2, CH3, and CH4, depending on the antibody subtype. The antigen-binding variable region (also called the fragment variable region, Fv fragment) usually comprises a light-chain variable region (VL) and a heavy-chain variable region (VH). The constant region may be a constant region having a native sequence (e.g., a constant region having a human native sequence) or a variant of the amino acid sequence. The variable region identifies and interacts with the target antigen. The constant region can be recognized by and interact with the immune system.

[0030] An antibody fragment can comprise a portion of a full antibody, preferably its antigen-binding or variable region. Examples of antibody fragments include Fab, Fab’, F(ab’)2, an Fd fragment composed of a VH and a CH1 domain, an Fv fragment, a single-domain antibody (dAb) fragment, and an isolated complementarity-determining region (CDR). A Fab fragment is an antibody fragment obtained by digesting a full-length immunoglobulin with papain or a fragment having the same structure produced, for example, by recombinant expression. A Fab fragment contains a light chain (including VL and CL) and another chain, and the other chain contains the variable region of the heavy chain (VH) and one constant region of the heavy chain (CH1). An F(ab’)2 fragment is an antibody fragment obtained by digesting an immunoglobulin with pepsin at pH 4.0 - 4.5 or a fragment having the same structure produced, for example, by recombinant expression. An F(ab’)2 fragment basically contains two Fab fragments, where each heavy-chain portion contains some extra amino acids and a cysteine that forms a disulfide bond connecting the two fragments. A Fab’ fragment is a fragment containing half of an F(ab’)2 fragment (one heavy chain and one light chain). The antibody fragment may include, for example, multiple chains linked by disulfide bonds and / or peptide linker termini. Examples of antibody fragments further include single-chain Fv (scFv), Fv, dsFv, bispecific antibodies, Fd and Fd’ fragments, and other fragments including modified fragments. Antibody fragments usually contain at least 50 or about 50 amino acids and usually contain at least 200 or about 200 amino acids. An antigen-binding fragment may include any antibody fragment that gives an immunospecific binding antigen when inserted into an antibody framework (e.g., by substitution of the corresponding region).

[0031] In particular, the antibody-drug conjugate of the present application performs site-specific binding based on any site containing the natural N-glycosylation modification of the antibody FC region, and any molecule containing the antibody FC region containing a sugar chain (including, but not limited to, antibodies / bispecific antibodies / FC fusion proteins / single-chain antibodies / nanobodies, etc.) was produced by a one-step method using the linker-payload containing the oligosaccharide of the present application. Therefore, the antibody of the present application is not particularly limited, and as long as its FC region contains a sugar chain, it may be a natural antibody.

[0032] It should be noted that the antibodies of the present invention can also be produced using techniques well known in the art, such as recombinant techniques / gene engineering techniques, phage display techniques, synthetic techniques, or other techniques known in the art, or combinations thereof. For example, recombinant antibodies by gene engineering can be expressed in an appropriate culture system (e.g., Escherichia coli (E.Coli), or mammalian cells). The gene engineering mentioned above is, for example, introducing a ligase-specific recognition sequence at its end.

[0033] As used herein, the term "targeted molecule-drug conjugate" is referred to as "conjugate". Examples of conjugates include, but are not limited to, antibody-drug conjugates.

[0034] A small molecule compound is a molecule whose size corresponds to an organic molecule used in ordinary drugs. The term does not include biopolymers (e.g., proteins, nucleic acids, etc.), but includes low molecular weight peptides or their derivatives, such as dipeptides, tripeptides, tetrapeptides, pentapeptides, etc. Usually, the molecular weight of a small molecule compound may be, 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, about 200 to about 500 Da.

[0035] A cytotoxin is a substance that inhibits or prevents the expression activity and cell function of cells and / or causes cell destruction. Currently, the cytotoxin commonly used in ADCs is more toxic than chemotherapeutic drugs. Examples of cytotoxins are drugs that target targets including, but not limited to, the microtubule cytoskeleton, DNA, RNA, protein transport via kinesin, and regulation of apoptosis. Drugs that target the microtubule cytoskeleton may be, for example, microtubule stabilizers or tubulin polymerization inhibitors. Examples of microtubule stabilizers include, but are not limited to, the taxane series. Examples of tubulin polymerization inhibitors include, but are not limited to, maytansinoid series, auristatin series, vinblastine series, colchicine series, and aplisiatoxin series. Drugs that target DNA may be, for example, drugs that directly disrupt the DNA structure or topoisomerase inhibitors. Examples of drugs that directly disrupt the DNA structure include, but are not limited to, DNA double strand breakers, DNA alkylating agents, and DNA intercalators. DNA double strand breakers may be, for example, enediyne antibiotics and include, but are not limited to, dynemicin, esperamicin, neocarzinostatin, uncialamycin, etc. DNA alkylating agents may be, for example, DNA bis-alkylating agents (i.e., DNA-cross linkers) or DNA mono-alkylating agents. Examples of DNA alkylating agents include, but are not limited to, pyrrolo[2,1-c][1,4]benzodiazepine (PBD) dimers, 1-(chloromethyl)-2,3-dihydro-1H-benzo[e]indole (CBI) dimers, CBI-PBD heterodimers, indoline 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 as follows), camptothecin-based, and anthracycline-based. Drugs targeting RNA may be, for example, drugs that inhibit cleavage, and examples thereof include, but are not limited to, pladienolide. Drugs targeting protein transport via kinesin may be, for example, mitotic kinesin inhibitors, including but not limited to kinesin spindle protein (KSP) inhibitors.

[0036] As used herein, the term "spacer" is a structure that is located between different structural modules and spatially separates the structural modules. The definition of a spacer does not limit the presence or absence of a certain function, nor does it limit whether it is cleaved or degraded in vivo. Examples of spacers include, but are not limited to, amino acids and non-amino acid structures, and the non-amino acid structures may be, but are not limited to, amino acid derivatives or analogs. A "Spacer sequence" is an amino acid sequence as a spacer, and examples thereof include a single amino acid, a sequence containing a plurality of amino acids, for example, a sequence containing two amino acids such as GA, or, for example, GGGGS, GGGGSGGGGS, GGGGSGGGGSGGGGS, etc., but are not limited thereto. A self-destructive spacer (e.g., self-destructive spacer Sp1) is a covalent component that causes two chemical bonds to break successively after the protecting moiety in the precursor is activated: specifically, after the protecting moiety (e.g., a cleavable sequence) is activated, it is removed, causing a cascade of degradation reactions to sequentially release relatively low molecular weight substances. Examples of self-destructive spacers include, but are not limited to, PABC (p-aminobenzyloxycarbonyl group), acetal, heteroacetal, and combinations thereof.

[0037] As used herein, the term "amino acid" includes "natural amino acids" and "non-natural amino acids".

[0038] The term "natural amino acid" refers to an amino acid that is a constituent amino acid of a protein and includes the 20 commonly found 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 commonly found selenocysteine and pyrrolysine.

[0039] As used herein, the term "unnatural amino acid" refers to an amino acid that is not a constituent amino acid of a protein. Specifically, the term means an amino acid that is not a natural amino acid as defined above.

[0040] The term "alkyl group" refers to a straight-chain or branched-chain saturated aliphatic hydrocarbon group composed of carbon atoms and hydrogen atoms and is linked to the other part of the molecule via a single bond. The alkyl group may have 1 to 20 carbon atoms and is designated as "C1 - C 20"Alkyl group", for example, means a C1-C4 alkyl group, a C1-C3 alkyl group, a C1-C2 alkyl group, a C3 alkyl group, a C4 alkyl group, or a C3-C6 alkyl group. Non-limiting examples of alkyl groups include methyl group, ethyl group, propyl group, butyl group, pentyl group, hexyl group, isopropyl group, isobutyl group, s-butyl group, t-butyl group, isopentyl group, 2-methylbutyl group, 1-methylbutyl group, 1-ethylpropyl group, 1,2-dimethylpropyl group, neopentyl group, 1,1-dimethylpropyl group, 4-methylpentyl group, 3-methylpentyl group, 2-methylpentyl group, 1-methylpentyl group, 2-ethylbutyl group, 1-ethylbutyl group, 3,3-dimethylbutyl group, 2,2-dimethylbutyl group, 1,1-dimethylbutyl group, 2,3-dimethylbutyl group, 1,3-dimethylbutyl group, or 1,2-dimethylbutyl group, or their isomers, but are not limited thereto. A divalent free radical is a group obtained by removing one hydrogen atom from a carbon atom having a free valence electron of the corresponding monovalent free radical. A divalent free radical has two linking sites for linking to other parts of the molecule. For example, "alkylene group" or "alkylidene group" is a saturated straight-chain or branched-chain divalent hydrocarbon group. Examples of "alkylene group" include, for example, methylene group (-CH2-), ethylene group (-C2H4-), propylene group (-C3H6-), butylene group (-C4H8-), pentylene group (-C5H 10 -), hexylene group (-C6H 12 -), 1-methylethylene group (-CH(CH3)CH2-), 2-methylethylene group (-CH2CH(CH3)-), methylpropylene group, or ethylpropylene group, etc., but are not limited thereto.

[0041] The term "cycloalkyl group" is a cyclic saturated aliphatic group composed of carbon atoms and hydrogen atoms, and is linked to other parts of the molecule through a single bond. The cycloalkyl group may have 3 to 10 carbon atoms, that is, "C3-C 10is a "cycloalkyl group", for example, a cyclopropyl group, a cyclobutyl group, a cyclopentyl group, a cyclohexyl group, a cycloheptyl group, a cyclooctyl group, a cyclononyl group, or a cyclodecyl group. A "cycloalkylene group" is a divalent cycloalkyl group.

[0042] The term "heterocyclyl group" refers to a group in which one or more carbon atoms in the above cycloalkyl group are replaced by heteroatoms selected from nitrogen, oxygen, and sulfur, for example, an aza-, oxa- or thiocyclopropyl group, an aza-, oxa- or thiocyclobutyl group, a pyrrolidinyl group, a pyrazolidinyl group, an imidazolidinyl group, a tetrahydrofuranyl group, a tetrahydrothiophenyl group, a piperidyl group, a piperazinyl group, a tetrahydropyranyl group, or a tetrahydrothiopyranyl group. A "heterocyclylene group" is a divalent cycloalkyl group.

[0043] When "substituted" is mentioned in the present text, unless otherwise specified, the relevant substituents are selected from an alkyl group, a halogen, an amino group, a monoalkylamino group, a dialkylamino group, a nitro group, a cyano group, a formyl group, an alkylcarbonyl group, a carboxyl group, an alkyloxycarbonyl group, an alkylcarbonyloxy group, an aminocarbonyl group, a monoalkylaminocarbonyl group, a dialkylaminocarbonyl group, a formylamino group, an alkylcarbonylamino group, a formyl(monoalkyl)amino group, or an alkylcarbonyl(monoalkyl)amino group.

[0044] As used herein, when a group is combined with another group, on the premise of forming a chemically stable structure, the linkage between the groups may be linear or branched. The structure formed by such a combination may be linked to other parts of the molecule through any suitable atom in the structure, preferably through a predetermined chemical bond. For example, -CR 1 R 2 、C 1~10 alkylene group, C 4~10 cycloalkylene group, C 4~10Two or more divalent groups selected from a heterocyclylene group and -(CO)- are combined to form a combination, and the two or more divalent groups are linearly combined, for example, -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 R 2’ -(CO)-, -CR 1 R 2 -CR 1’ R 2’ -CR 1” R 2” -(CO)-, etc. may be formed. The obtained divalent structure may be further linked to other parts of the molecule.

[0045] When there are multiple of the same alphabet representing chemical groups in one chemical structural formula, they are each independently selected and not necessarily the same. For example, multiple Ms in Formula I-2 are each independently selected from LKa-L 2 -L 1 -B-P; and multiple Ls 2 etc. are also independent of each other and not necessarily the same.

[0046] As used herein, the expressions "antibody conjugate drug" and "antibody-drug conjugate" have the same meaning.

[0047] Linker-payload compound In a first aspect, the present invention provides a linker-payload compound having formula (I):

Chemical formula

Chemical formula

Chemical formula

[0048] In one embodiment, -L-(P) t is -L 2 -L 1 -B-P, that is, formula (I) is

Chemical formula

Chemical formula

[0049] In another embodiment, -L-(P) t is

Chemical formula

Chemical formula

[0050] Each LKa is each independently

Chemical formula

Chemical formula

[0051] In one embodiment, B, L 1 and L 2 at least one of them is not "absent".

[0052] In one embodiment, L 2 is -NH-(CH2) a -(CH2)2(CO)- (a is an integer of 0, 1, 2, 3, 4, or 5);

Chemical formula

[0053] In one embodiment, L 2 is an amino acid residue sequence, i.e., -*(AA) n **-, where n is an integer from 1 to 100, AA is independently an amino acid residue each time it appears, * represents the N-terminus of the corresponding amino acid, ** represents the C-terminus of the corresponding amino acid, and between the amino group of one amino acid and the α-carbon, there is -(C2H4-O) m -(CH2) p- may be present, where 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 oligosaccharide structure. In one embodiment, each time AA appears independently, it is any one of Phe, Lys, Gly, Ala, Leu, Asn, Val, Ile, Pro, Trp, Ser, Tyr, Cys, Met, Asp, Gln, Glu, Thr, Arg, His, or any combination thereof. In one embodiment, n is an integer from 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.

[0054] In one embodiment, L 1 contains a cleavable sequence of an amino acid sequence cleavable by an enzyme, and the amino acid sequence cleavable by the enzyme contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids. In one embodiment, the amino acid sequence cleavable by the enzyme is selected from -Gly-Gly-Phe-Gly-, -Phe-Lys-, -Val-Cit-, -Val-Lys-, -Gly-Phe-Leu-Gly-, -Ala-Leu-Ala-Leu-, -Ala-Ala-Ala-, and combinations thereof; preferably, the amino acid sequence cleavable by the enzyme is -Gly-Gly-Phe-Gly-. In one embodiment, L 1 is any one of Val, Cit, Phe, Lys, Gly, Ala, Leu, Asn, or any combination thereof, preferably -Gly-Gly-Phe-Gly-, -Phe-Lys-, -Val-Cit-, -Val-Lys-, -Gly-Phe-Leu-Gly-, -Ala-Leu-Ala-Leu-, -Ala-Ala-Ala-, and combinations thereof. In one embodiment, L 1 represents -Val-Cit-.

[0055] In one embodiment, Sp1 is selected from PABC (p-aminobenzyloxycarbonyl group), ethyl acetal, heteroacetal, and combinations thereof; preferably, Sp1 is an acetal, heteroacetal, or PABC; more preferably, the heteroacetal is selected from N,O-heteroacetals; even more preferably, Sp1 is -O-CH2-U- or -NH-CH2-U-, wherein -O- or -NH- is linked to an enzymatically cleavable amino acid sequence, and U is absent or is CH2, O, S, or NH, preferably O or S.

[0056] In one embodiment, B is absent or is -NH-CH2-U- or -NH-CH2-U-(CH2) g -(CO)-, where g is 1, 2, 3, 4, 5, or 6, and U is absent or is CH2, O, S, or NH, preferably O or S. In one embodiment, B is absent. In one embodiment, B is one of the following 1), 2), or a combination of 1) and 2): 1) a self-destructive spacer Sp1; 2) one divalent group, or a combination of two or more divalent groups, said divalent group being selected from -CR 1 R 2 -, C 1~10 an alkylene group, and -(CO)-. In one embodiment, B is -NH-CH2-U- or -NH-CH2-U-(CH2) g -(CO)-, where U is absent or is CH2, O, S, or NH, preferably O or S. In one embodiment, B is linked to the payload via an amide bond, or an ester bond, or an ether bond. In one embodiment, B is

Chemical formula

[0057] In one embodiment, -L 1 -B- represents -Val-Cit-PABC- or -Gly-Gly-Phe-Gly-.

[0058] In one embodiment, -L 2 -L 1 -B- represents -Gly-Gly-Gly-Val-Cit-PABC- or -HN-(C2H4-O) m -(CH2) p -Gly-Gly-Phe-Gly-.

[0059] In one embodiment, Ld2 and each Ld1 are independently a bond or

Chemical formula

[0060] In one embodiment, each i, j, and k is independently selected from integers from 1 to 20. In one embodiment, each i, j, and k is independently selected from integers from 1 to 12.

[0061] In one embodiment, each i is independently selected from integers from 2 to 8; in particular, 4.

[0062] In one embodiment, each j is independently selected from integers from 8 to 12; in particular, 8 or 12.

[0063] In one embodiment, each k is independently selected from integers from 1 to 7; in particular, 1, or 3, or 5.

[0064] In one embodiment, Ld2 and each Ld1 are independently a bond; or a C alkylene group having an amino group and a carbonyl group at each of both ends, or a PEG fragment of a certain length having an amino group and a carbonyl group at each of both ends (represented by -(PEG)-), or one or more selected from natural amino acids, and the natural amino acids are each independently unsubstituted or substituted with a PEG fragment of a certain length (represented by -CO-(PEG)-). 1~20 An alkylene group, or a PEG fragment of a certain length having an amino group and a carbonyl group at each of both ends (represented by -(PEG)-), or one or more selected from natural amino acids, and the natural amino acids are each independently unsubstituted or substituted with a PEG fragment of a certain length (represented by -CO-(PEG)-). i -). j -).

[0065] In one embodiment, -(PEG)- i - is -(O-C2H4)- i - or -(C2H4-O)- i - and may have a C alkylene group added to one end; -(PEG)- 1~10 - is -(O-C2H4)- j - or -(C2H4-O)- j - and may have a C alkylene group added to one end. In a very specific embodiment, -(PEG)- j - is -C2H4-(O-C2H4)- 1~10 - or -(C2H4-O)- i -C2H4-. i - or -(C2H4-O)- i -C2H4-.

[0066] In one embodiment, the payload may be selected from the group consisting of small molecule compounds (e.g., small molecule drugs of each mechanism of action including each conventional small molecule drug, photodynamic / sonodynamic therapy drug, photothermal therapy drug, etc., such as chemotherapeutic drugs, small molecule targeted drugs, immune agonists, etc., such as conventional 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 and TLR agonists), nucleic acids and nucleic acid analogs, probe molecules (including fluorescent molecules, biotin, fluorophores, chromophores, spin resonance probes, and radiolabels, etc.), oligopeptides, polypeptides, peptidomimetics, and proteins. In one embodiment, the payload is selected from the group consisting of small molecule compounds and nucleic acid molecules. In a preferred embodiment, the payload is selected from small molecule compounds. In a more preferred embodiment, the payload is selected from the group consisting of cytotoxins and fragments thereof.

[0067] In one embodiment, the payload is a cytotoxin or a fragment thereof, and is linked to the L moiety in formula (I), or the B moiety in the compound of formula (I-1) or formula (I-2), L 2 or L 1 has any derivatization for linking to the moiety.

[0068] 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 a taxane, maytansinoid, auristatin, epothilone, combretastatin A-4 phosphate, combretastatin A-4 and its derivatives, indole-sulfonamide, vinblastine such as vinblastine series, vincristine, vindesine, vinorelbine, vinflunine, vinglycinate, anhydrovinblastine, dolastatin 10 and its analogs, halichondrin B, indole-3-oxoacetamide, podophyllotoxin, 7-diethylamino-3-(2'-benzoxazolyl)-coumarin (DBC), discodermolide, laulimalide. In another embodiment, the cytotoxin is selected from the group consisting of DNA topoisomerase inhibitors, such as camptothecin and its derivatives, mitoxantrone, mitoguazone. In a preferred embodiment, the cytotoxin is a nitrogen mustard, such as chlorambucil, chloronaphazine, cholophosphamide, estramustine, ifosfamide, nitrogen mustard, nitrobicin hydrochloride, melphalan, novembicin, phenamet, phenesterine, prednimustine, trophosphamide, uramustine. In yet another preferred embodiment, the cytotoxin is a nitrosourea, such as carmustine, flubenzuron, formoterol, lomustine, nimustine, 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, carbocon, metsredepa, 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 enjicin antibiotics. In a more preferred embodiment, the cytotoxin is selected from the group consisting of dynemicin, esperamicin, neocarzinostatin, and aclacinomycin. In another preferred embodiment, the cytotoxin is selected from the group consisting of actinomycin, anthramycin, bleomycin series, actinomycin C, carabicin, calminomycin, sarcomycin, calminomycin, actinomycin D, daunorubicin, detorubicin, doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin series, nogalamycin, olivomycin, peplomycin, porfiromycin, promycin, keramycin, rhodrubicin, streptozocin, streptozocin, dinostatin, and zorubicin. In yet another preferred embodiment, the cytotoxin is selected from the group consisting of trichothecene series. In a more preferred embodiment, the cytotoxin is selected from the group consisting of T-2 toxin, verracurin A, loridine 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 denopterin, methotrexate, pteropterin, trimethoprim, 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, thiampurine, 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, didoxuridine, doxifluridine, 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 calusterone, drostanolone propionate, epithioestanol, mepitiostane, testolactone. In another embodiment, the cytotoxin is selected from the group consisting of anti-adrenergics. In a preferred embodiment, the cytotoxin is selected from the group consisting of aminoglutethimide, mitotane, trilostane. In one embodiment, the cytotoxin is selected from the group consisting of anti-androgens. In a preferred embodiment, the cytotoxin is selected from the group consisting of flutamide, nilutamide, bicalutamide, leuprorelin 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 vinblastine-based, colchicine-based, taxane-based, auristatin-based, maytansinoid-based, calicheamicin, doxonubicin, duocarmucin, SN-38, cryptophycin analog, deruxtecan, duocarmazine, calicheamicin, centanamycin, dolastansine, pyrrolobenzodiazepine, and exatecan. In one embodiment, the cytotoxin is selected from the group consisting of vinblastine-based, colchicine-based, taxane-based, auristatin-based, and maytansinoid-based.

[0069] In one embodiment, the cytotoxin is exatecan or a derivative thereof, such as DX8951f and the like.

[0070] In one embodiment, the cytotoxin is a maytansinoid, such as DM1 and the like. It should be noted that when using a cytotoxin containing a thiol group moiety, the thiol group moiety can react with a maleimide moiety to form succinimide. For example, a cytotoxin such as a maytansinoid such as DM1 can be directly linked via succinimide. In such cases, in some embodiments, since the payload and the thiol group moiety together constitute the cytotoxin, in such cases, the payload can be understood to represent the other part of the cytotoxin molecule excluding the thiol group moiety.

[0071] In one embodiment, the cytotoxin is an auristatin, such as MMAE (monomethyl auristatin E), MMAF (monomethyl auristatin F), MMAD (monomethyl auristatin D), and the like. The synthesis and structure of auristatin compounds are described in US20060229253, the entire disclosure of which is incorporated herein by reference.

[0072] The payload contains an active group that can react with the active group in the compound of formula (I), thereby covalently bonding the payload and the compound of formula (I). A compound that does not contain an active group cannot obtain a payload unless appropriately derivatized.

[0073] In one embodiment, the cytotoxin is a compound of the following formula (i).

Chemical formula

[0074] In one embodiment, the cytotoxin is selected from the following Compounds 1-16, and the wavy bond represents the linking site linked to the compound of formula (I).

[0075]

Chemical formula

[0076] In some embodiments, the payload is selected from DX8951f (Compound 9), DXd-(1) (Compound 10), and DXd-(2) (Compound 14), Compound 15, preferably DX8951f or DXd-(1), and more preferably DXd-(1).

[0077] In one embodiment, the first hexosyl group or its derivative moiety is selected from a glucosyl group, a mannosyl group, a galactosyl group, a fructosyl group, a glucosyl group, an idosyl group, or a derivative thereof, and the carbon at its 6-position is in the form of -C(O)-.

[0078] In one embodiment, each time the second hexosyl group or its derivative moiety appears, it is independently selected from a glucosyl group, a mannosyl group, a galactosyl group, a fructosyl group, or a derivative thereof.

[0079] In one embodiment, each monosaccharide moiety in the oligosaccharide structure is linked via a β-(1→4) glycosidic bond.

[0080] In one embodiment, the derivatives are independently selected from derivatives in which the uronic acid or the hydroxyl group of the monosaccharide is replaced by an acylamino group (e.g., an alkanoylamino group, e.g., a formylamino group, an acetylamino group, a propionylamino group, etc., especially an acetylamino group).

[0081] In one embodiment, the first hexosyl group or its derivative moiety is

Chemical formula

Chemical formula

[0082] In one embodiment, f is 0.

[0083] In one embodiment, D-C(O)- is a disaccharide structure

Chemical formula

[0084] In one embodiment, D-C(O)- is a disaccharide structure

Chemical formula

[0085] In one embodiment, the linker-payload compound having formula (I) is:

Chemical formula

Chemical formula

[0086] In another aspect, the present invention provides a linker-payload compound obtainable by a method comprising the following steps.

[0087] (i) Oxidizing the primary alcohol at the 6-position of the terminal first hexosyl unit in an oligosaccharide containing a terminal first hexosyl unit and a terminal N-acetylglucosamine (GlcNAc) unit to a carboxyl group to obtain an intermediate compound (a) having a carboxyl group, wherein between the terminal first hexosyl unit and the terminal N-acetylglucosamine (GlcNAc) unit, there may be 1, 2, 3, 4, 5, or 6 second hexosyl units or derivative moieties thereof; (ii) Reacting the carboxyl group in the intermediate compound (a) obtained in step (i) with the reactive group of a linker-terminal payload compound (b) having a reactive group at the terminal to obtain the linker-payload compound.

[0088] In one embodiment of this other aspect, the first hexosyl unit is selected from a glucosyl group, a mannosyl group, a galactosyl group, or a fructosyl group; and / or, the second hexosyl unit is independently selected, each time it appears, from a glucosyl group, a mannosyl group, a galactosyl group, or a fructosyl group, and / or, each monosaccharide moiety in the oligosaccharide is linked via a β-(1→4) glycosidic bond; and / or, the derivative is independently selected from derivatives in which the hydroxyl group of the monosaccharide is replaced with an acylamino group (e.g., an alkanoylamino group, e.g., a formylamino group, an acetylamino group, a propionylamino group, etc., in particular, an acetylamino group).

[0089] In one embodiment of this other aspect, the first hexosyl unit is a mannosyl group or a glucosyl group; and / or, the second hexosyl unit or its derivative moiety is absent.

[0090] In one embodiment of this other aspect, the oligosaccharide in step (i) has the following structure:

Chemical formula

[0091] In one embodiment of this other aspect, the oligosaccharide in step (i) has the following structure:

Chemical formula

[0092] In one embodiment of this other aspect, the reactive group of the linker-terminal-payload compound (b) having a reactive group at the terminal in step (ii) is an amino group.

[0093] In one embodiment of this other aspect, the method may further include converting an intermediate compound (a) having a carboxyl group into an acid halide and further reacting it with a linker-terminal payload compound (b) having a reactive group at the terminal to obtain the linker-payload compound.

[0094] In one embodiment of this other aspect, the linker-payload compound is the linker-payload compound defined in the first aspect or each of its embodiments.

[0095] Method for producing linker-payload compound In a second aspect, the present invention provides a method for producing a linker-payload compound having the formula (I), and unless otherwise specified, each variable is as defined in the first aspect or each of its embodiments.

[0096] In one embodiment, the method is D-C(O)-OH and L'-(P) t by an amide formation reaction to

Chemical formula

[0097] In one embodiment, D-C(O)- in the linker-payload compound of formula (I) to be produced is

Chemical formula

Chemical formula

Chemical formula

[0098] In one embodiment, the amidation reaction is carried out in the presence of an organic solvent, an organic base and a condensing 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 condensing reagent is selected from HATU, HBTU, TBTU and PyBOP.

[0099] In one embodiment, D-C(O)-OH is

Chemical formula

[0100] In one embodiment,

Chemical formula

Chemical formula

[0101] In one embodiment, [Chemical formula] is produced from [Chemical formula] In one embodiment, the production is carried out in the presence of an oxidizing agent and an optional oxidation catalyst. In one embodiment, the oxidizing agent is iodosobenzene diacetate. In one embodiment, the oxidation catalyst is 2,2,6,6 - tetramethyl - 1 - piperidinyloxy.

[0102] In one embodiment, [Chemical formula] is produced from [Chemical formula] In one embodiment, the production is carried out in the presence of an acid. In one embodiment, the acid is p - toluenesulfonic acid.

[0103] In one embodiment, [Chemical formula] is produced from [Chemical formula] In one embodiment, the production 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 of both.

[0104] In one embodiment, [Chemical formula] is produced from [Chemical formula] It is produced by forming a β-(1→4) glycosidic bond. In one embodiment, the production is carried out under glycosidic bond formation conditions. In one embodiment, the glycosidic bond formation conditions include using trifluoromethanesulfonic anhydride in an anhydrous situation.

[0105] The present invention further provides the following compounds:

Chemical formula

[0106] Antibody-drug conjugate In a third aspect, the present invention provides an antibody-drug conjugate having formula (II) by site-specific binding to the N-glycosylation site of the antibody Fc region:

Chemical formula

[0107] In one embodiment, the first hexosyl group or its derivative moiety is selected from a glucosyl group, a mannosyl group, a galactosyl group, a fructosyl group, a glucosyl group, an idosyl group, or a derivative thereof, and the carbon at the 6-position thereof has the form of -C(O)-.

[0108] In one embodiment, each time the second hexosyl group or its derivative moiety appears, it is independently selected from a glucosyl group, a mannosyl group, a galactosyl group, a fructosyl group, or a derivative thereof.

[0109] In one embodiment, each monosaccharide moiety is linked via a β-(1→4) glycosidic bond.

[0110] In one embodiment, the derivatives are independently selected from derivatives in which the uronic acid or the hydroxyl group of the monosaccharide is replaced with an acylamino group (e.g., an alkanoylamino group, e.g., a formylamino group, an acetylamino group, a propionylamino group, etc., particularly an acetylamino group).

[0111] In one embodiment, the first hexosyl group or its derivative moiety is

Chemical formula

[0112] In one embodiment, f is 0.

[0113] In one embodiment, -NHC(O)CH2- in formula (II) is derived from asparagine at position 297 of the antibody Fc region.

[0114] In one embodiment, the target identified by Ab is 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, CN33, GPNMB, ENPP3, Nectin-4, EGFRvIII, SLC44A4 / AGS-5, CEACAM5, PSMA, TIM1, LY6E, LIV1, Nectin 4, 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, including but not limited to these.

[0115] In some embodiments, Ab is a monoclonal antibody. In some embodiments, Ab is an anti-human HER2 antibody, or an antigen-binding fragment thereof. Examples of anti-human HER2 antibodies include, but are not limited to, Pertuzumab and Trastuzumab. In one embodiment, Ab is Trastuzumab.

[0116] In one embodiment, formula (II) is formula (II-1):

Chemical formula

[0117] In one embodiment, formula (II) is formula (II-2):

Chemical formula

[0118] In one embodiment, formula (II) is formula (II-3): [Chemical formula]

[0119] In one embodiment, formula (II) is formula (II-4): [Chemical formula]

[0120] In one embodiment, formula (II) is formula (II-5): [Chemical formula] Wherein R is hydrogen or an α-L-fucosyl group; q is 1 or 2; Ab is an antibody or an antigen-binding fragment (for example, -NHC(O)CH2- in formula (II-1) is derived from asparagine at position 297 of the Fc region of the antibody); The other variables are as defined in the first aspect or each of its embodiments.

[0121] In one embodiment, formula (II) is selected from formula (II-1), formula (II-2), formula (II-3), formula (II-4), formula (II-5): [Chemical formula] Wherein R is hydrogen or an α-L-fucosyl group; q is 1 or 2, for example, q is 2; Ab is an anti-CD19 antibody, anti-CD20 antibody, anti-CD22 antibody, anti-CD25 antibody, anti-CD30 / TNFRSF8 antibody, anti-CD33 antibody, anti-CD37 antibody, anti-CD44v6 antibody, anti-CD56 antibody, anti-CD70 antibody, anti-CD71 antibody, anti-CD74 antibody, anti-CD79b antibody, anti-CD117 / KITk antibody, anti-CD123 antibody, anti-CD138 antibody, anti-CD142 antibody, anti-CD174 antibody, anti-CD227 / MUC1 antibody, anti-CD352 antibody, anti-CLDN18.2 antibody, anti-DLL3 antibody, anti-ErbB2 / HER2 antibody, anti-CN33 antibody, anti-GPNMB antibody, anti-ENPP3 antibody, anti-nectin-4 antibody, anti-EGFRvIII antibody, anti-SLC44A4 / AGS-5 antibody, anti-CEACAM5 antibody, anti-PSMA antibody, anti-TIM1 antibody, anti-LY6E antibody, anti-LIV1 antibody, anti-nectin-4 antibody, anti-SLITRK6 antibody, anti-HGFR / cMet antibody, anti-SLAMF7 / CS1 antibody, anti-EGFR antibody, anti-BCMA antibody, anti-AXL antibody, anti-NaPi2B antibody, anti-GCC antibody, anti-STEAP1 antibody, anti-MUC16 antibody, anti-mesothelin antibody, anti-ETBR antibody, anti-EphA2 antibody, anti-5T4 antibody, anti-FOLR1 antibody, anti-LAMP1 antibody, anti-cadherin-6 antibody, anti-FGFR2 antibody, anti-FGFR3 antibody, anti-CA6 antibody, anti-CanAg antibody, anti-integrin αV antibody, anti-TDGF1 antibody, anti-Ephrin A4 antibody, anti-TROP2 antibody, anti-PTK7 antibody, anti-NOTCH3 antibody, anti-C4.4A antibody, anti-FLT3 antibody, anti-B7H3 / 4 antibody, anti-TF (tissue factor) antibody, anti-ROR1 / 2 antibody; preferably, it is an anti-CD19 antibody, anti-ErbB2 / HER2 antibody, anti-CLDN18.2 antibody, anti-nectin-4 antibody, anti-FGFR3 antibody, anti-Trop2 antibody; more preferably, it is an anti-ErbB2 / HER2 antibody, anti-Trop2 antibody; particularly preferably, it is an anti-ErbB2 / HER2 antibody (e.g., trastuzumab);

[0122] (P) t -L- is [Chemical formula] selected from

[0123] In one embodiment, R in formula (II) is an α-L-fucosyl group; q is 2; Ab is trastuzumab;

[0124] (P) t -L- is

Chemical formula

Chemical formula

[0125] Method for producing an antibody-drug conjugate In a fourth aspect, the present invention provides a method for producing an antibody-drug conjugate having formula (II), comprising binding a linker-payload compound having formula (I) to an antibody Ab, and unless otherwise specified, each variable is as defined in the third aspect or its respective embodiments.

[0126] In one embodiment, the method comprises the following steps.

[0127] a) By the catalytic action of a glycosidase or its variant, the antibody Ab excises the N-glycan moiety thereof, and an antibody is obtained in which the N-glycosylation site in its Fc region is modified with N-acetylglucosamine or fucosyl-α-1,6-N-acetylglucosamine.

[0128] b) By the catalytic action of a glycosidase or its variant, the modified antibody obtained in step a) is bound to the linker-payload compound.

[0129] Wherein, the glycosidase or its variant used in step a) and step b) may be the same or different.

[0130] In one embodiment, the glycosidase or its variant used in steps a) and b) is a fucosidase, an N-acetylglucosaminidase, or a variant thereof. In one embodiment, the N-acetylglucosaminidase comprises at least one selected from Endo-S (Streptococcus pyogenes endoglycosidase-S), Endo-F3 (Elizabethkingia miricola endoglycosidase-F3), Endo-S2 (Endoglycosidase-S2, Streptococcus pyogenes endoglycosidase-S2), Endo-Sd (Endoglycosidase-Sd, Streptococcus pyogenes endoglycosidase-Sd), and Endo-CC (Endoglycosidase-CC, Streptococcus pyogenes endoglycosidase-CC); 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.

[0131] In one embodiment, steps a) and b) are carried out by one-pot enzymatic catalysis.

[0132] In one embodiment, steps a) and b) are carried out by one-pot enzymatic catalysis, and the enzyme is Endo S2.

[0133] Pharmaceutical compositions and pharmaceutical preparations Another object of the present invention is to provide a pharmaceutical composition comprising a prophylactically or therapeutically effective dose of the complex of the present invention and at least one pharmaceutically acceptable carrier.

[0134] The pharmaceutical composition of the present invention may be administered by any method as long as it has the effect of preventing, alleviating, preventing or treating human or animal symptoms. For example, it may be manufactured into various appropriate dosage forms according to the administration route, particularly, injection agents, such as freeze-dried powder injection agents · injection agents, or sterile injection agents · powders.

[0135] The term "pharmaceutically acceptable" means that when contacting with the tissues of a patient within the scope of normal medical judgment, it does not cause toxicity, irritation or allergic reactions that should not occur, has a reasonable benefit-risk ratio, and is effective for the expected use.

[0136] The term "pharmaceutically acceptable carrier" refers to a carrier material that is pharmaceutically acceptable and does not interfere with the biological activity and performance of the complex. Examples of aqueous carriers include, but are not limited to, buffered saline. Pharmaceutically acceptable carriers further include carrier substances that bring the composition closer to physiological conditions, such as pH adjusters and buffers, toxicity adjusters, etc., and include sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc.

[0137] In one embodiment, the drug / antibody ratio (DAR) of the pharmaceutical composition of the present invention is an integer or non-integer from 1 to 20, for example, 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, about 1 to about 2. In a particular embodiment, the DAR of the complex of the present invention is about 2, about 4, about 6, or about 8.

[0138] Therapeutic methods and uses The antibody-drug conjugate of the present invention can be used for the treatment of tumors and / or autoimmune diseases. Tumors sensitive to treatment with the antibody-drug conjugate include tumors characterized by specific tumor-related antigens or cell surface receptors, and these tumor cells are identified by the targeting molecule in the antibody-drug conjugate and can be killed by the payload / cytotoxin in the antibody-drug conjugate.

[0139] Accordingly, in another aspect, the present invention further provides the use of the antibody-drug conjugate of the present invention or the pharmaceutical composition of the present invention for manufacturing a therapeutic agent for a disease, illness or medical condition, wherein the disease, illness or medical condition is selected from a tumor or an autoimmune disease.

[0140] In another aspect, the present invention provides the antibody-drug conjugate of the present invention or the pharmaceutical composition of the present invention for treating a tumor or an autoimmune disease.

[0141] In a further aspect, the present invention provides a method for treating a tumor or an autoimmune disease, which comprises administering to an individual in need thereof an effective amount of the antibody-drug conjugate of the present invention or the pharmaceutical composition of the present invention.

[0142] In one embodiment, the antibody-drug conjugate formed by conjugating an anti-human HER2 or Trop2 antibody according to the present invention with a small molecule cytotoxin specifically binds to HER2 or Trop2 on the surface of tumor cells and selectively kills tumor cells expressing HER2. In another embodiment, the present invention provides the use of the antibody-drug conjugate of the present invention or the pharmaceutical composition of the present invention for manufacturing a therapeutic agent for a disease, illness or medical condition, wherein the disease, illness or medical condition is selected from tumors positive for HER2 or Trop2. In a more preferred embodiment, the disease, illness or medical condition is selected from the group consisting of breast cancer, gastric cancer, lung cancer, ovarian cancer, urothelial cancer, and the like.

[0143] The dose of the antibody-drug conjugate administered to a subject can be adjusted within a wide range. The dose can be changed according to the specific administration route and the needs of the subject, and can also be determined by medical and healthcare professionals.

[0144] Examples I. Preparation of disaccharide linker

[0145] Example 1 Preparation of disaccharide substrate compound 1 Compound 1 was prepared using the following procedure, and its structure is as follows: [Chemistry]

[0146] (1) Preparation of Compound 1c [Chemistry] Under the condition of evacuating with an oil pump, a 100 mL Schlenk reaction flask was baked with a heat gun for 5 minutes, cooled, and then activated molecular sieves were added to the system and baked for another 5 minutes. Evacuation and nitrogen gas replacement were performed three times. Under nitrogen protection, compound 1a (4.41 g, 7.96 mmol) was added to the system and stirred for 3 minutes, then anhydrous dichloromethane (30 mL) was added and stirred for 0.5 hour. Separately, following the same procedure, compound 1b (1.89 g, 3.98 mmol, dissolved in 20 mL of anhydrous dichloromethane) was added to another 50 mL Schlenk reaction flask with activated molecular sieves added, and stirred for 1 hour for preliminary drying to remove the residual moisture in the system.

[0147] Under nitrogen protection, 1-(phenylsulfinyl)piperidine (BSP, 1.37 g, 6.56 mmol) and 2,4,6-tri-t-butylpyrimidine (TTBP, 2.94 g, 11.94 mmol) were added to the above-mentioned dried compound 1a solution at room temperature, and the mixture was further stirred for 20 minutes. The reaction flask was placed in a dry ice / ethyl acetate bath and cooled to -65 °C, and trifluoromethanesulfonic anhydride (1.2 mL, 7.16 mmol) was added to the system. After 2 minutes, a dichloromethane solution of pre-dried compound 1b was added to the system, and the resulting reaction mixture was stirred at -65 °C until the reaction was completed (about 3 h) as determined by TLC (developing solvent: EtOAc / PE = 1 / 8). Saturated sodium bicarbonate solution was added to the system to terminate the reaction, and the mixture was extracted with dichloromethane and separated (150 mL × 3). The combined organic phases were washed successively with water and saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, sample-fed by the wet method, and separated by column chromatography (eluent: EtOAc / PE = 1 / 12 - 1 / 10) to obtain compound 1c (2.72 g, yield 75.4%, colorless viscous oily liquid). 1 H NMR (400 MHz, chloroform-d) δ 7.55 (dd, J = 7.6, 2.1 Hz, 2H), 7.51 - 7.28 (m, 28H), 5.60 (s, 1H), 5.14 (d, J = 10.4 Hz, 1H), 5.00 (d, J = 12.0 Hz, 1H), 4.94 (d, J = 11.8 Hz, 1H), 4.90 - 4.80 (m, 2H), 4.76 (d, J = 12.0 Hz, 1H), 4.74 - 4.63 (m, 3H), 4.57 (s, 1H, H 1’ ), 4.48 (d, J = 12.1 Hz, 1H), 4.37 (d, J = 8.1 Hz, 1H,H 1), 4.22 - 4.10 (m, 2H), 4.05 (t, J = 9.3 Hz, 1H), 3.80 (d, J = 3.1 Hz, 1H), 3.72 (dd, J = 11.2, 2.2 Hz, 1H), 3.67 - 3.52 (m, 3H), 3.49 (dd, J = 9.8, 3.1 Hz, 1H), 3.41 (t, J = 9.3 Hz, 1H), 3.35 (dt, J = 9.8, 2.9 Hz, 1H), 3.16 (td, J = 9.7, 4.8 Hz, 1H). C 54 H 56 N3O 10 + [M+H] + The calculated value of the MS(ESI) m / z of [M+H] was 906.4, and the measured value was 906.7.

[0148] (2) Preparation of Compound 1d

Chemical Structure

[0149] (3) Preparation of Compound 1e

Chemical Structure

[0150] (4) Preparation of Compound 1f

Chemical Structure

[0151] (5) Preparation of Compound 1

Chemical Structure

[0152] Example 2 Production of Disaccharide Substrate Compound 2 Compound 2 was produced using the following procedure, and its structure is as follows:

Chemical formula

[0153] (1) Synthesis of Compound 2-1

Chemical formula

[0154] Step A: Synthesis of Compound 2-1b In a one-neck flask, compound 2-1a (1.0 equivalent, commercially available, CAS: 959153-39-0), MeOH, and sodium methoxide (0.1 equivalent, 5 mol / L in MeOH) were added. Under a nitrogen atmosphere, the mixture was stirred at room temperature, and the progress of the reaction was monitored by TLC. When the reaction was complete, 1 M hydrochloric acid was added to stop the reaction and adjust the reaction system to neutral. The solvent was removed under reduced pressure, a small amount of toluene was added, and water was removed by azeotropic effect under the condition of rotary evaporation under reduced pressure to obtain a pale yellow viscous oily liquid of crude compound 2-1b, which did not need to be further purified and was used directly in the next reaction.

[0155] Step B: Synthesis of Compound 2-1c ​To a one-necked flask, the crude product of compound 2-1b (1 eq), acetonitrile, camphorsulfonic acid (0.1 eq), and benzaldehyde dimethyl acetal (4 eq) were sequentially added, and the reaction system was stirred at room temperature overnight. The progress of the reaction was monitored by TLC. When the reaction was completed (about 24 h), saturated sodium bicarbonate solution was added to the system to terminate the reaction, and the mixture was extracted with ethyl acetate and separated. The combined organic phases were washed with water, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, supplied as a sample by the wet method, and purified by silica gel column chromatography (eluent: PE / EtOAc = 5:1) to obtain compound 2-1c (white solid, 83% yield). 1 H NMR (400 MHz, chloroform-d) δ 7.51-7.49 (m, 2H, Ar-H), 7.45 (d, J = 8.0 Hz, 2H, Ar-H), 7.42-7.37 (m, 5H, Ar-H), 7.36-7.31 (m, 3H, Ar-H), 7.15 (d, J = 8.0 Hz, 2H, Ar-H), 5.56 (s, 1H, PhCH), 4.97 (d, J = 11.6 Hz, 1H, PhCH2), 4.81 (d, J = 11.6 Hz, 1H, PhCH2), 4.58 (d, J = 9.6 Hz, 1H), 4.40 (dd, J = 10.2, 4.8 Hz, 1H), 3.80 (dd, J = 10.0, 10.4 Hz, 1H), 3.70 (dd, J = 9.2, 9.2 Hz, 1H), 3.65 (dd, J = 9.2, 9.2 Hz, 1H), 3.53-3.48 (m, 2H), 2.64(br s, 1H, -OH), 2.36 (s, 3H).C 27 H 29 O5S + [M+H] + In the MS (ESI) m / z of [M+H], the calculated value was 465.2, and the measured value was 465.3. The NMR data was consistent with the literature report (see compound 2b1 described in Nature 2007, 446, 896).

[0156] Step C: Synthesis of Compound 2-1d To a single-necked flask, sequentially add Compound 2-1c (1 equivalent), dichloromethane, and triethylamine (5 equivalents). Cool the system to 0 °C, add acetic anhydride (2 equivalents), stir for 10 min, then warm the reaction system to room temperature and stir. Monitor the progress of the reaction by TLC. When the reaction is complete (about 6 h), add saturated sodium bicarbonate solution to the system to terminate the reaction, extract with dichloromethane and separate the layers. Wash the combined organic phases with water, then with saturated brine, dry over anhydrous sodium sulfate, filter, concentrate, supply the sample by the wet method, and purify by column chromatography (eluent: PE / EtOAc = 5:1) to obtain Compound 2-1d (white solid, 89% yield). 1 H NMR (400 MHz, chloroform-d) δ 7.51-7.48 (m, 2H, Ar-H), 7.43-7.36 (m, 5H, Ar-H), 7.34-7.25 (m, 5H, Ar-H), 7.13 (d, J = 8.0 Hz, 2H, Ar-H), 5.58 (s, 1H, PhCH), 5.00 (dd, J = 8.0, 8.0 Hz, 1H, H-2), 4. 87 (d, J = 12.0 Hz, 1H, PhCH2), 4.67 (d, J = 12.0 Hz, 1H, PhCH2), 4.64 (d, J = 10.4 Hz, 1H, H-1), 4.39 (dd, J = 10.8 Hz, J = 5.2 Hz, 1H), 3.81 (t, J = 10.4 Hz, 1H), 3.78-3.70 (m, 2H), 3.48 (ddd, J = 9.6, 5.4, 10.0 Hz, 1H), 2.35 (s, 3H, Ar-CH3), 2.05 (s, 3H, OAc). C 29 H 31 O6S + [M+H] +In the MS(ESI) m / z, the calculated value was 507.2 and the measured value was 507.4. The NMR data was consistent with the literature report (see compound 3b1 described in Nature 2007, 446, 896).

[0157] Step D: Synthesis of compound 2-1e Under a nitrogen atmosphere, to a 100 mL two-necked flask were sequentially added compound 2-1d (1 equivalent), dichloromethane, borane tetrahydrofuran (10 equivalents, 1 M in THF), and the system was cooled to 0 °C. Dibutylboron trifluoromethanesulfonate (1.4 equivalents, 1.0 M in DCM) was added dropwise, and 0 °C was maintained until TLC indicated the completion of the reaction (about 5 hours). Then, a triethylamine solution was added to the system at 0 °C to terminate the reaction of dibutylboron trifluoromethanesulfonate in the system. Further, methanol was slowly added dropwise to terminate the reaction of borane tetrahydrofuran. After the system no longer produced a large amount of bubbles, water was added to ensure sufficient termination of the reaction. Then, extraction was performed with ethyl acetate and the layers were separated. The combined organic phases were washed with water, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, supplied as a sample by the wet method, and purified by flash column chromatography (eluent: PE / EtOAc = 4:1) to obtain the crude product of 2-1e (white solid). The structure of the product was confirmed by LCMS to be correct. C 29 H 33 O6S + [M+H] + In the MS(ESI) m / z, the calculated value was 509.2 and the measured value was 509.3. It was not necessary to precisely purify the crude product, which was directly used in the next reaction.

[0158] Step E / F: Synthesis of compound 2-1f Step E: To a one-necked flask, sequentially add compound 2-1e (1 equiv), iodoacetophenone (3 equiv), TEMPO (0.5 equiv), and t-butyl alcohol / dichloromethane / water (volume ratio 4:4:1). Stir the resulting mixture at room temperature and monitor the progress of the reaction by TLC (developing solvent: PE / EtOAc = 2 / 1, containing 1 v / v% acetic acid). When the reaction is complete, add saturated sodium thiosulfate to the system to terminate the reaction, extract with dichloromethane, separate the layers, wash the combined organic phase with water, then with saturated brine, dry over anhydrous sodium sulfate, filter, concentrate. The resulting carboxylic acid intermediate did not require further purification and was used directly in the next reaction as it was.

[0159] Step F: Dissolve the carboxylic acid intermediate obtained in the previous step in DMF, add methyl iodide (3 equiv) and potassium carbonate (5 equiv), stir at room temperature, and monitor the progress of the reaction by TLC (developing solvent: PE / EtOAc = 5 / 1). After the reaction is complete (about 2 hours), add water to the system, extract with ethyl acetate, separate the layers, wash the organic phase with water, then with saturated brine, dry over anhydrous sodium sulfate, filter, concentrate, and purify by column chromatography (eluent: PE / EtOAc = 7:1) to obtain compound 2-1f (white solid, total yield of three steps 59%). 1 H NMR (400 MHz, chloroform-d) δ, 7.42-7.26 (m, 12H, Ar-H), 7.16 (d, J = 7.6 Hz, 2H, Ar-H), 5.02 (dd, J = 8.4, 8.0 Hz, 1H), 4.83 (d, J = 11.6 Hz, 1H, PhCH2), 4.79 (d, J = 11.2 Hz, 1H, PhCH2), 4.70 (d, J = 11.6 Hz, 1H), 4.64 (d, J = 11.2 Hz, 1H), 4.62 (d, J = 10.0 Hz, 1H), 3.97 (d, J = 10.0 Hz, 1H), 3.90 (dd, J = 8.4, 8.0 Hz, 1H), 3.79 (s, 3H, OMe), 3.72 (dd, J = 8.8, 8.8 Hz, 1H), 2.38 (s, 3H, Ar-CH3), 2.04 (s, 3H, OAc). C 30 H 33 O7S + [M+H] + In the MS(ESI) m / z of , the calculated value was 537.2 and the measured value was 537.4.

[0160] Step G: Synthesis of Compound 2-1g To a 50 mL single-necked flask were successively added Compound 2-1f (1.0 equivalent) and acetone, and the system was cooled to 0 °C. N-Bromosuccinimide (1.4 equivalents) was added, and the mixture was stirred at 0 °C for about 1 hour until TLC (developing solvent: PE / EtOAc = 4 / 1) indicated the completion of the reaction. A saturated sodium thiosulfate solution was added to the system to terminate the reaction, and the mixture was rotary evaporated under reduced pressure to remove acetone. Water and ethyl acetate were added, and the mixture was separated and extracted. The organic phases were combined, washed with water, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography (eluent: PE / EtOAc = 6:1) to obtain Compound 2-1g (white solid, yield 90%). C 23 H 27 O8 + [M+H] + In the MS(ESI) m / z of , the calculated value was 431.2 and the measured value was 431.5.

[0161] Step H: Synthesis of Compound 2-1 Under a nitrogen atmosphere, 2-1 g (1 equivalent) of the compound, 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU, 0.1 equivalent), and dichloromethane were sequentially added to a 50 mL single-necked flask. After cooling the system to 0 °C and stirring uniformly, trichloroacetonitrile (4 equivalents) was added. The ice-water bath was removed, and the temperature was allowed to rise naturally to room temperature with stirring. The reaction was monitored by TLC (developing solvent: PE / EtOAc = 2 / 1). After completion of the reaction (about 2 hours), the solvent was removed by rotary evaporation under reduced pressure, and the sample was supplied by the wet method and purified by column chromatography (eluent: PE / EtOAc = 4:1) to obtain compound 2-1 (pale yellow viscous oily liquid, yield 87%). C 23 H 25 O7 + [M-Cl3CC(NH)O - + In the MS (ESI) m / z of [[M-Cl3CC(NH)O]], the calculated value was 413.2, and the measured value was 413.2.

[0162] (2) Synthesis of compound 2-2

Chemical formula

[0163] Step A: Synthesis of compound 2-2b Compound 2-2a (CAS: 1235137-45-7) is commercially available and was prepared with reference to the synthesis method of compound 2 described in the literature Carbohydr Res 2016, 426, 33.

[0164] At room temperature, compound 2-2a (1 equivalent), methanol, and sodium methoxide (0.1 equivalent, 5 M in MeOH) were added to a single-necked flask and stirred at room temperature. The reaction was monitored by TLC (developing solvent: PE / EtOAc = 2 / 1). After completion of the reaction (about 1 hour), dilute hydrochloric acid (1 M) was added to neutralize the solution to pH = 7. After concentrating the reaction solution, toluene was added, and the mixture was rotary evaporated under reduced pressure to remove the residual moisture in the system by the azeotropic effect, obtaining a light brown oily crude product 2-2b, which was used directly in the next reaction without purification.

[0165] ​Step B: Synthesis of Compound 2-2c At room temperature, to the crude reaction product 2-2b (1 equiv) from the previous step were sequentially added p-toluenesulfonic acid monohydrate (0.2 equiv) and anhydrous acetonitrile, and the mixture was stirred well until homogeneous. The system was purged with nitrogen atmosphere, and benzaldehyde dimethyl acetal (5 equiv) was added. The resulting reaction liquid was stirred at room temperature overnight until TLC (developing solvent: PE / EtOAc = 5 / 1) indicated the completion of the reaction. Then, saturated sodium carbonate solution was added to the reaction system to terminate the reaction, and the mixture was extracted with dichloromethane and separated. The combined organic phases were washed with water, then with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, fed into the sample by the wet method, and purified by silica gel column chromatography (eluent: PE / EtOAc = 5 / 1) to obtain Compound 2-2c (white solid, total yield of two steps 85%). 1 H NMR (400 MHz, chloroform-d) δ 7.63 - 7.54 (m, 2H), 7.52 - 7.39 (m, 5H), 7.25 - 7.14 (m, 2H), 5.60 (s, 1H), 5.53 (d, J = 5.4 Hz, 1H), 4.52 - 4.39 (m, 1H), 4.28 (dd, J = 10.4, 4.9 Hz, 1H), 4.07 (t, J = 9.5 Hz, 1H), 3.92 (dd, J = 10.0, 5.6 Hz, 1H), 3.79 (t, J = 10.3 Hz, 1H), 3.60 (t, J = 9.3 Hz, 1H), 3.08 (br s, 1H), 2.40 (s, 3H).C 20 H 22 N3O4S + [M+H] + For the MS (ESI) m / z of [[M+H]], the calculated value was 400.1 and the measured value was 399.9. The NMR data was consistent with the literature report (see Compound 47 described in the literature Angew.Chem.Int.Ed.2021,60,12413).

[0166] Step C: Synthesis of Compound 2-2d Under a nitrogen atmosphere, to two dry-necked flasks in sequence, compound 2-2c (1 equivalent), anhydrous tetrahydrofuran (reaction concentration 0.2 M) were added, and it was placed in an ice bath and cooled to 0 °C. Sodium hydride (1.2 equivalents, 60% content, dispersed in mineral oil) was added. After the addition was completed, the ice bath was removed, the temperature was raised to room temperature and stirred. After 1 hour, tetrabutylammonium iodide (0.1 equivalent) and benzyl bromide (1.5 equivalents) were added, and the resulting reaction system was stirred at room temperature until TLC (developing solvent: PE / EtOAc = 8 / 1) indicated the completion of the reaction (about 6 hours). After the reaction was completed, water was added dropwise to stop the reaction, and it was extracted with ethyl acetate and separated. The combined organic phases were washed successively with water and saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by silica gel column chromatography (eluent: PE / EtOAc = 5 / 1) to obtain compound 2-2d (white solid, yield 97%). 1 H NMR (400 MHz, chloroform-d) δ 7.63 - 7.55 (m, 2H), 7.53 - 7.33 (m, 10H), 7.20 (d, J = 8.4 Hz, 2H), 5.67 (s, 1H), 5.56 (d, J = 4.6 Hz, 1H), 5.05 (d, J = 10.9 Hz, 1H), 4.90 (d, J = 10.9 Hz, 1H), 4.59 - 4.47 (m, 1H), 4.31 (dd, J = 10.4, 4.9 Hz, 1H), 4.10 - 3.97 (m, 2H), 3.90 - 3.77 (m, 2H), 2.41 (s, 3H). C 27 H 28 N3O4S + [M+H] + For the MS (ESI) m / z of , the calculated value was 490.2 and the measured value was 490.5. The NMR data was consistent with the literature report (see compound 20 described in the reference Bioorg. Med. Chem. 2011, 19, 30).

[0167] Step D: Synthesis of compound 2-2e To a one-necked flask, compound 2-2d (1 equiv), tetrahydrofuran / methanol (v / v = 1:1, reaction concentration 0.5 M), and p-toluenesulfonic acid monohydrate (0.2 equiv) were added, and the mixture was stirred at room temperature overnight. The progress of the reaction was monitored by TLC (developing solvent: PE / EtOAc = 8 / 1). When the reaction was complete (about 12 h), saturated sodium bicarbonate solution was added to the system to quench the reaction, and the mixture was extracted with ethyl acetate and separated. The combined organic phases were washed with water, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, sample-fed by the wet method, and purified by silica gel column chromatography (eluent: EtOAc / PE = 1 / 1) to obtain compound 2-2e. 1 H NMR (400 MHz, chloroform-d) δ 7.41 - 7.34 (m, 7H), 7.14 (d, J = 8.0 Hz, 2H), 5.49 (d, J = 5.2 Hz, 1H), 5.01 (d, J = 11.2 Hz, 1H), 4.77 (d, J = 11.2 Hz, 1H), 4.24 - 4.20 (m, 1H), 3.89-3.85 (m, 1H), 3.79-3.77 (m, 2H), 3.69-3.65 (m, 2H), 2.42 (br s, 1H), 2.34 (s, 3H).C 20 H 27 N4O4S + [M+NH4] + For the MS (ESI) m / z of , the calculated value was 419.2 and the measured value was 419.2.

[0168] Step E: Synthesis of compound 2-2 To a one-necked flask, compound 2-2e (1 equiv), dichloromethane (reaction concentration 0.5 M), imidazole (2 equiv), and t-butyldiphenylchlorosilane (1.5 equiv) were sequentially added, and the mixture was stirred at room temperature. The progress of the reaction was monitored by TLC (developing solvent: EtOAc / PE = 1 / 12). When the reaction was completed (about 6 h), a saturated ammonium chloride solution was added to the system to terminate the reaction. Then, the mixture was extracted with dichloromethane and separated. The combined organic phases were washed with water, saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, supplied as a sample by the wet method, and purified by silica gel column chromatography (eluent: PE / EtOAc / DCM = 15 / 1 / 1) to obtain compound 2-2 (yellow viscous oily liquid, yield 66%). 1 H NMR (400 MHz, chloroform-d) δ 7.73 -7.68 (m, 4H), 7.48-7.35 (m, 13H), 7.06 (d, J = 7.6 Hz, 1H), 5.49 (d, J = 5.2 Hz, 1H), 4.94 (ABq, J = 10.8 Hz, 2H), 4.32-4.27 (m, 1H), 3.96-3.90 (m, 2H), 3.87 (dd, J = 4.8, 5.2 Hz, 1H), 3.82 (dt, J = 2.8, 8.8 Hz, 1H), 3.74-3.69 (m, 1H), 2.70 (d, J = 2.8 Hz, OH), 2.33 (s, 3H), 1.09 (s, 9H).C 36 H 42 N3O4SSi + [M+H] + For the MS (ESI) m / z of [M+H], the calculated value was 640.3, and the measured value was 640.2.

[0169] (3) Synthesis of compound 2

Chemical formula

[0170] Step A: Synthesis of compound 2a To a dried two-necked flask, add pre-activated molecular sieves, evacuate with an oil pump under conditions of baking with a heat gun, cooling, and then replacing with nitrogen gas. After repeating the above evacuation-nitrogen gas replacement procedure three times, add compound 2-1 (2 equivalents), compound 2-2 (1 equivalent), and dried toluene to the system under nitrogen protection, and stir the resulting system at room temperature for 0.5 h to sufficiently remove the remaining moisture in the system. Then, cool the reaction system to -40 °C, stir, add a certain amount of TMSOTf, and maintain this temperature and stir until TLC measurement indicates the completion of the reaction (about 3 h). Add triethylamine to the system to terminate the reaction, filter, concentrate, supply the sample by the wet method, and separate by silica gel column chromatography to obtain compound 2a (white solid, 60% yield). 1 H NMR (400 MHz, chloroform-d) δ 7.72 (d, J = 6.4 Hz, 2H), 7.64 (d, J = 6.4 Hz, 2H), 7.49 - 7.47 (m, 2H), 7.44 - 7.23 (m, 21H), 7.04 (d, J = 7.8 Hz, 2H), 5.57 (d, J = 7.2 Hz, 1H), 5.17 (d, J = 7.2 Hz, 1H), 5.10 (dd, J = 9.7, 8.1 Hz, 1H), 4.93 (d, J = 8.0 Hz, 1H), 4.82 (d, J = 11.4 Hz, 1H), 4.74 (d, J = 10.9 Hz, 1H), 4.66 - 4.60 (m, 3H), 4.20 (dd, J = 9.2, 9.2 Hz, 1H), 4.05 - 4.02 (m, 2H), 3.97 (dd, J = 9.3, 9.2 Hz, 1H), 3.92 - 3.76 (m, 3H), 3.74 - 3.67 (m, 1H), 3.60 (s, 3H), 3.51 (dd, J = 9.2, 9.2 Hz, 1H), 2.32 (s, 3H), 1.82 (s, 3H), 1.07 (s, 9H).C 59 H 66 N3O 11 SSi + [M+H] +In MS(ESI) m / z, the calculated value was 1052.4 and the measured value was 1052.7.

[0171] Step B: Synthesis of Compound 2b To the reaction flask, add Compound 2a (1 equivalent), thioglycolic acid (CAS: 68 - 11 - 1) / pyridine / trichloromethane (v / v / v = 1:1:1), heat the system to 60 °C and stir, monitor the progress of the reaction by TLC. When the reaction is complete (about 12 h), concentrate under reduced pressure to remove most of the solvent, add an appropriate amount of ethyl acetate, wash with saturated sodium bicarbonate solution, extract and separate the layers. Then wash the organic phase with 1 M hydrochloric acid solution, separate the layers and wash again with saturated sodium bicarbonate solution. Dry the obtained organic phase over anhydrous sodium sulfate, filter, concentrate, and purify by column chromatography to obtain Compound 2b (white solid, 71% yield). 1 H NMR (400 MHz, chloroform - d) δ 7.70 (d, J = 6.8 Hz, 2H), 7.66 (d, J = 7.2 Hz, 2H), 7.44 - 7.28 (m, 20H), 7.26 - 7.21 (m, 3H), 7.00 (d, J = 7.9 Hz, 2H), 5.68 (d, J = 4.8 Hz, 1H), 5.36 - 5.30 (m, 1H), 5.09 (dd, J = 9.5, 8.1 Hz, 1H), 4.92 (d, J = 12.4 Hz, 1H), 4.85 (d, J = 11.5 Hz, 1H), 4.75 - 4.66 (m, 3H), 4.63 - 4.57 (m, 2H), 4.29 (ddd, J = 9.3, 7.7, 4.8 Hz, 1H), 4.10 (t, J = 7.8 Hz, 1H), 4.01 (dd, J = 11.5, 3.2 Hz, 1H), 3.94 - 3.89 (m, 2H), 3.86 - 3.84 (m, 1H), 3.80 (dd, J = 11.5, 2.5 Hz, 1H), 3.67 (s, 3H), 3.60 - 3.51 (m, 2H), 2.28 (s, 3H), 1.85 (s, 3H), 1.82 (s, 3H), 1.07 (s, 9H). C61 H 70 NO 12 SSi + [M+H] + In the MS(ESI) m / z of + [M+H] , the calculated value was 1068.4 and the measured value was 1068.4.

[0172] Step C: Synthesis of Compound 2c After adding Compound 2b (1 equivalent) and tetrahydrofuran to the reaction flask, TBAF (2 equivalents, 1 M in THF) was added to the system, and the mixture was stirred at room temperature. The progress of the reaction was monitored by TLC. When the reaction was completed (about 10 h), a saturated ammonium chloride solution was added to the system to terminate the reaction. Then, the mixture was extracted with ethyl acetate and separated. The combined organic phases were washed with water, saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, supplied as a sample by the wet method, and preliminarily purified by silica gel column chromatography to obtain Compound 2c (white solid, 56% crude yield). 45 H 52 NO 12 S + [M+H] + In the MS(ESI) m / z of + [M+H] , the calculated value was 830.3 and the measured value was 830.6.

[0173] Step D: Synthesis of Compound 2d Compound 2c was dissolved in THF / MeOH (v / v = 3:1), and a 1 M aqueous sodium hydroxide solution was slowly added to adjust the pH of the system to 12. The resulting reaction solution was stirred at room temperature, and the progress of the reaction was monitored by HPLC. When the reaction was sufficient (about 12 h), 1 M hydrochloric acid was added to the system to adjust the pH to 7 - 8, and the mixture was concentrated under reduced pressure to remove most of the organic solvents. The obtained crude product was purified by HPLC to obtain Compound 2d (white solid, 82% yield). 11H NMR (400 MHz, chloroform-d) δ 7.43 - 7.27 (m, 17H), 7.07 (d, J = 7.9 Hz, 2H), 5.54 (d, J = 5.0 Hz, 1H), 5.33 (d, J = 8.0 Hz, 1H), 4.93 (d, J = 12.1 Hz, 1H), 4.86 (d, J = 11.3 Hz, 1H), 4.82 - 4.77 (m, 3H), 4.74 (d, J = 6.8 Hz, 1H), 4.69 (d, J = 12.2 Hz, 1H), 4.36 (ddd, J = 10.6, 7.9, 5.0 Hz, 1H), 4.25 - 4.22 (m, 1H), 4.13 - 4.02 (m, 3H), 3.88 (t, J = 8.6 Hz, 1H), 3.81 (dd, J = 12.7, 2.2 Hz, 1H), 3.74 (dd, J = 10.7, 8.5 Hz, 1H), 3.68 - 3.59 (m, 2H), 3.40 (br s, 1H), 2.30 (s, 3H), 1.77 (s, 3H). 13 13C NMR (100 MHz, chloroform-d) δ 170.73, 169.33, 138.20, 138.06, 137.81, 137.55, 132.37, 129.99, 129.44, 128.84, 128.76, 128.58, 128.42, 128.30, 127.90, 103.24, 88.40, 83.43, 78.89, 77.85, 77.35, 77.24, 77.03, 76.92, 76.72, 75.17, 74.93, 74.57, 74.02, 73.93, 72.95, 60.66, 52.95, 23.08, 21.06 (In the aromatic region, three carbon signals overlapped and no peak was observed). C 42 H 46 NO 11 S - [M-H] - In the MS (ESI) m / z of [M-H], the calculated value was 772.3 and the measured value was 772.2.

[0174] Step E: Synthesis of Compound 2e Compound 2d was weighed and dissolved in acetone, and the solution was stirred in an ice-water bath for 5 min to cool down. NBS was weighed and added to the reaction solution, and the reaction was carried out in an ice-water bath for about 1 h. Samples were taken for HPLC measurement to confirm the completion of the reaction. After the reaction was terminated with a saturated sodium thiosulfate solution, it was concentrated to remove acetone, prepared by prep-HPLC, and freeze-dried to obtain compound 2e (white solid, 45% yield). C 35 H 40 NO 12 - [M-H] - For the MS (ESI) m / z of , the calculated value was 666.3 and the measured value was 666.4.

[0175] Step F: Synthesis of Compound 2 At room temperature, compound 2e, tetrahydrofuran, methanol, and palladium-carbon catalyst were sequentially added to a 50 mL single-necked flask, and the reaction system was stirred under a hydrogen atmosphere until all the raw materials disappeared as determined by TLC measurement. It was filtered, concentrated under reduced pressure, and extracted and dried with an oil pump to obtain compound 2 (white solid, 95% yield). C 14 H 22 NO 12 - [M-H + - For the MS (ESI) m / z of , the calculated value was 396.1 and the measured value was 396.1.

[0176] Example 3 Production of Disaccharide Substrate Compound 3 Compound 3 was produced using the following procedure, and its structure is as follows:

Chemical formula

[0177] The production process of compound 3 referred to a similar method for the synthesis of compound 2, and the specific route is as follows:

Chemical formula

[0178] ​ The final product, compound 3, was verified by mass spectrometry and has the formula C 14 H 22 NO 12 - [M-H + - The MS(ESI) m / z of [M-H] was calculated to be 396.1 and the measured value was 396.0.

[0179] Example 4 Preparation of the disaccharide substrate compound 4 Compound 4 was prepared using the following procedure and has the structure as follows:

Chemical formula

[0180] The process for preparing compound 4 referred to a similar method for synthesizing compound 2, and the specific route is as follows:

Chemical formula

[0181] The final product, compound 4, was verified by mass spectrometry and has the formula C 14 H 22 NO 12 - [M-H + - The MS(ESI) m / z of [M-H] was calculated to be 396.1 and the measured value was 396.1.

[0182] Example 5 Preparation of the disaccharide substrate compound 5 Compound 5 was prepared using the following procedure and has the structure as follows:

Chemical formula

[0183] The process for preparing compound 5 referred to a similar method for synthesizing compound 2, and the specific route is as follows:

Chemical formula

[0184] The compound 5 of the final product was verified by mass spectrometry, C 14 H 22 NO 12 - [M-H + - In the MS(ESI) m / z of [[M-H]], the calculated value was 396.1 and the measured value was 396.0.

[0185] II. Production of linker-payload (hereinafter abbreviated as LP)

[0186] Example 6 Production of LP-1 The structure of linker-payload 1 (LP-1) is as follows:

Chemical formula

[0187] (1) Production of compound LP-1b

Chemical formula

[0188] (2) Production of compound LP-1 ​ [Chemical formula] At room temperature, to a 10 mL single-necked flask were sequentially added compound LP-1b (21.7 mg, 0.013 mmol, 1 equivalent), H2O, Et3N (1 - 100 equivalents), and DMC (2-chloro-1,3-dimethylimidazolinium chloride, CAS: 37091-73-9, 1 - 100 equivalents). The reaction was monitored by HPLC, and the resulting reaction solution was stirred at room temperature until the reaction was complete. The reaction solution was purified by semi-preparative HPLC to obtain compound LP-1 (15.3 mg, yield 71.3%, white solid). C 78 H 124 N 14 O 25 2+ [M + 2H] 2+ For the MS (ESI) m / z of [[M+2H]], the calculated value was 828.4 and the measured value was 828.6.

[0189] Example 7 Production of LP-2 The structure of linker-payload 2 (LP-2) is as follows: [Chemical formula]

[0190] The production process is as follows: [Chemical formula]

[0191] 20.1 Step A: Production of compound LP-2a At room temperature, to a 10 mL single-necked flask were sequentially added compound 2 (0.5 - 5.0 equivalents), compound LP-1a (1.0 equivalent, GGG-VC-PAB-MMAE, CAS: 2684216-48-4, commercially available), DMF, DIPEA (1 - 10 equivalents), and HATU (0.5 - 10 equivalents). The resulting reaction solution was stirred at room temperature under HPLC monitoring until the reaction was complete. The reaction solution was purified by semi-preparative HPLC to obtain compound LP-2a (white solid, yield 84%). C 78 H126 N 14 O 26 2+ [M + 2H] 2+ In the MS(ESI) m / z of [[M + 2H]], the calculated value was 837.4 and the measured value was 837.8.

[0192] 20.2 Step B: Preparation of Compound LP-2 At room temperature, to a 10 mL single-necked flask were sequentially added compound LP-2a (1 equivalent), H2O, Et3N (1 - 100 equivalents), and DMC (2-chloro-1,3-dimethylimidazolinium chloride, CAS: 37091-73-9, 1 - 100 equivalents). The resulting reaction solution was stirred at 0 °C, and the reaction was monitored by HPLC until completion. The reaction solution was purified by semi-preparative HPLC to obtain compound LP-2 (yield 87%, white solid). C 78 H 124 N 14 O 25 2+ [M + 2H] 2+ In the MS(ESI) m / z of [[M + 2H]], the calculated value was 828.4 and the measured value was 828.7.

[0193] Example 8 Preparation of LP-3, LP-4, and LP-5 Using a procedure similar to the preparation of LP-2, linkers-payloads LP-3, LP-4, and LP-5 with the following structures were prepared.

Chemical Structure

[0194] Example 9 Preparation of LP-6

Chemical Structure

[0195] (1) Synthesis of LP-6-1

Chemical Structure

[0196] Step A: Synthesis of Intermediate LP-6-1b Add compound LP-6-1a (1.0 equiv) and DMF (dissolution concentration 1 g / mL) to the reaction flask, stir and dissolve under a nitrogen atmosphere, cool to 0 - 5 °C, then add DIEA (3 equiv) dropwise. After that, stir the resulting system at 5 °C for 10 min, then add benzyl bromide (1.3 equiv) dropwise. After the addition is complete, allow the reaction system to warm naturally to room temperature and stir for 16 h. Slowly pour the reaction solution into ice water, add methyl-t-butyl ether and stir, then let it stand for liquid separation. Extract the aqueous phase 4 times with methyl-t-butyl ether, combine the organic phases, wash with saturated brine, dry over anhydrous sodium sulfate, filter, concentrate to obtain a crude product as a yellow oil, supply the sample by the wet method, and purify by silica gel column chromatography (eluent: PE / EA = 6:1) to obtain the product LP-6-1b (pale yellow oil, quantitative yield).

[0197] Step B: Synthesis of Intermediate LP-6-1d Under nitrogen protection, add intermediate LP-6-1b (2.0 equiv), compound LP-6-1c (1.0 equiv) and THF (dissolution concentration 10 g / mL) to the reaction flask, stir and dissolve, take TsOH (0.1 equiv) and add it to the reaction, and react the system at room temperature for 4 h. Slowly pour the reaction solution into ice water, extract 3 times with ethyl acetate, wash the combined organic phases successively with saturated aqueous sodium bicarbonate solution, water and saturated brine, dry over anhydrous sodium sulfate, filter and concentrate to obtain a crude product, and purify by silica gel column chromatography (eluent: PE / EA = 1:1) to obtain the product LP-6-1d (white solid, yield 40%).

[0198] Step C: Synthesis of Intermediate LP-6-1e Under nitrogen protection, add compound LP-6-1d and N,N-dimethylacetamide (DMAc, dissolution concentration 10 g / mL) to the reaction flask, stir and dissolve, cool the system to 14 - 18 °C, add DBU (0.5 equiv) dropwise, and keep the temperature at this level to react until TLC indicates the completion of the reaction (about 1.5 h) to obtain intermediate LP-6-1e, which does not need to be purified and is used directly in the next reaction.

[0199] Step D: Synthesis of Intermediate LP-6-1g Cool the reaction solution of the previous step to 0 - 5 °C, and sequentially add pyridinium 4-methylbenzenesulfonate (PPTS, 0.5 equivalent), 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide (EDCI, 1.0 equivalent), 1-hydroxybenzotriazole (HOBT, 1.0 equivalent) and LP-6-1f (0.85 equivalent). React the reaction system at 0 - 10 °C until LCMS indicates the completion of the reaction (about 4 h). Add the reaction solution to ice water, add 2-methyltetrahydrofuran for extraction once, extract the aqueous phase with 2-methyltetrahydrofuran twice more, combine the organic phases, wash successively with 0.5 M hydrochloric acid, saturated NaHCO3 aqueous solution, water, saturated brine, dry over anhydrous sodium sulfate, filter, concentrate, and purify by silica gel column chromatography (eluent: DCM / MeOH) to obtain the product LP-6-1g (white solid, yield 78%).

[0200] Step E: Synthesis of Intermediate LP-6-1h Under nitrogen protection, add LP-6-1g and DMAc (dissolution concentration 10 g / mL) to the reaction flask, stir to dissolve. Cool the temperature to 14 - 18 °C, dropwise add DBU (0.5 equivalent), stir and react at this temperature for 1.5 h, monitor the progress of the reaction by TLC. After the reaction is completed, obtain the intermediate LP-6-1h, which does not need to be purified and can be directly used in the next reaction.

[0201] Step F: Synthesis of Intermediate LP-6-1j Cool the reaction solution of LP-6-1h in the previous step to 0 - 5 °C, add PPTS (0.5 equiv), EDCI (1 equiv), HOBT (1 equiv) and compound 3i (0.85 equiv), react at 0 - 10 °C for 3 - 4 h, monitor the progress of the reaction by LCMS. After the reaction is complete, add the reaction solution to ice water, add 2-methyltetrahydrofuran for extraction once, extract the aqueous phase with 2-methyltetrahydrofuran twice more, combine the organic phases, wash successively with 0.5 M hydrochloric acid, saturated aqueous NaHCO3, water, saturated brine, dry over anhydrous sodium sulfate, filter, concentrate, stir by dry method, and purify by column chromatography (eluent: DCM / MeOH) to obtain LP-6-1j (white solid, yield 50%).

[0202] Step G: Synthesis of compound LP-6-1 Under nitrogen protection, dissolve intermediate LP-6-1j in DCM (concentration 15 g / mL), add DBU (0.5 equiv) dropwise at 20 °C, and stir and react at this temperature until HPLC indicates the completion of the reaction. Then, add DCM to the system to dilute the reaction solution, directly supply the sample by wet method, and purify by column chromatography (eluent: DCM / MeOH) to obtain compound LP-6-1 (white solid, yield 82%). C 34 H 47 N6O 10 + [M+H] + For the MS (ESI) m / z of, the calculated value was 699.4 and the measured value was 699.6.

[0203] (2) Synthesis of LP-6-2

Chemical Structure

[0204] Step 1: Preparation of NH2-Asp(OtBu)-Rink amide resin Weighed 400 g of Rink amide resin and put it into a reaction kettle, immersed it in 2400 mL of DCM for 0.5 h to fully swell the resin, and then extracted and dried it. Added 2400 mL of decapping reagent, washed it, and extracted and dried it. Added 2400 mL of decapping reagent, stirred and reacted it at 25 ± 1 °C for 0.5 h through nitrogen gas, and then extracted and dried it. Washed it twice with 2400 mL of DMF, 2400 mL of absolute ethanol, 2400 mL of DCM, and 2400 mL of DMF respectively, extracted and dried it, and it was blue in the Kaiser measurement.

[0205] Weighed 88.87 g of Fmoc-Asp(OtBu)-OH and 29.19 g of HOBT, dissolved them in 2000 mL of DMF solution and 80 mL of DIC, placed them in an ice bath at -10 °C for 0.5 h, then slowly added them to the reaction kettle to react, stirred and reacted them at room temperature through nitrogen gas for 2 h, and then extracted and dried it. Washed it twice with 2400 mL of DMF, 2400 mL of absolute ethanol, 2400 mL of DCM, and 2400 mL of DMF respectively, extracted and dried it, and it was light blue in the Kaiser measurement. Added 2400 mL of DCM, further added 60 mL of capping reagent, stirred and reacted it at 25 ± 1 °C for 1 h through nitrogen gas, and then extracted and dried it. Washed it twice with 2400 mL of DMF, 2400 mL of absolute ethanol, 2400 mL of DCM, and 2400 mL of DMF respectively, extracted and dried it. It was colorless in the Kaiser measurement.

[0206] Added 2400 mL of decapping reagent, washed it, and extracted and dried it. Added 2400 mL of decapping reagent, stirred and reacted it at 25 ± 1 °C for 0.5 h through nitrogen gas, and then extracted and dried it. Washed it twice with 2400 mL of DMF, 2400 mL of absolute ethanol, 2400 mL of DCM, and 2400 mL of DMF respectively, extracted and dried it, and it was blue in the Kaiser measurement.

[0207] Step 2: Preparation of NH2-PEG4-Asp(OtBu)-Rink amide resin Weighed 131.64 g of Fmoc-PEG4-OH and 48.64 g of HOBT, dissolved them in 2000 mL of DMF solution and 80.0 mL of DIC, placed them in an ice bath at -10 °C for 0.5 h, then slowly added them to the reaction kettle for reaction, and stirred and reacted at room temperature with nitrogen gas passing through for 2 - 4 h, followed by extraction and drying. Washed twice with 2400 mL of DMF, 2400 mL of absolute ethanol, 2400 mL of DCM, and 2400 mL of DMF respectively, and then extracted and dried. It was colorless in the Kaiser measurement.

[0208] Added 2400 mL of de-capping reagent, washed, extracted, and dried. Further added 2400 mL of de-capping reagent, stirred and reacted at 25 ± 1 °C with nitrogen gas passing through for 0.5 h, then extracted and dried. Washed twice with 2400 mL of DMF, 2400 mL of absolute ethanol, 2400 mL of DCM, and 2400 mL of DMF respectively, and then extracted and dried. It was blue in the Kaiser measurement.

[0209] Step 3: Preparation of NH2-Asp(OtBu)-PEG4-Asp(OtBu)-Rink amide resin Weighed 222.18 g of Fmoc-Asp(OtBu)-OH and 72.96 g of HOBT, dissolved them in 2000 mL of DMF solution and 80 mL of DIC, placed them in an ice bath at -10 °C for 0.5 h, then slowly added them to the reaction kettle for reaction, and stirred and reacted at room temperature with nitrogen gas passing through for 2 - 4 h, followed by extraction and drying. Washed twice with 2400 mL of DMF, 2400 mL of absolute ethanol, 2400 mL of DCM, and 2400 mL of DMF respectively, and then extracted and dried. It was colorless in the Kaiser measurement.

[0210] Added 2400 mL of de-capping reagent, washed, extracted, and dried. Further added 2400 mL of de-capping reagent, stirred and reacted at 25 ± 1 °C with nitrogen gas passing through for 0.5 h, then extracted and dried. Washed twice with 2400 mL of DMF, 2400 mL of absolute ethanol, 2400 mL of DCM, and 2400 mL of DMF respectively, and then extracted and dried. It was blue in the Kaiser measurement.

[0211] Step 4: Preparation of Dde-Lys(NH2)-Asp(OtBu)-PEG4-Asp(OtBu)-Rink amide resin Weighed 191.75 g of Dde-Lys(Fmoc)-OH and 48.64 g of HOBT, dissolved them in 2000 mL of DMF solution and 80.0 mL of DIC, placed them in an ice bath at -10 °C for 0.5 h, then slowly added them to the reaction kettle for reaction, and stirred and reacted at room temperature through nitrogen gas for 2 - 4 h, followed by extraction and drying. Washed twice with 2400 mL of DMF, 2400 mL of absolute ethanol, 2400 mL of DCM, and 2400 mL of DMF respectively, and then extracted and dried. It was colorless in the Kaiser measurement.

[0212] Added 2400 ± 100 mL of de-capping reagent, washed, extracted, and dried. Further added 2400 mL of de-capping reagent, stirred and reacted at 25 ± 1 °C through nitrogen gas for 0.5 h, then extracted and dried. Washed twice with 2400 mL of DMF, 2400 mL of absolute ethanol, 2400 mL of DCM, and 2400 mL of DMF respectively, and then extracted and dried. It was blue in the Kaiser measurement.

[0213] Step 5: Preparation of Dde-Lys(mPEG12)-Asp(OtBu)-PEG4-Asp(OtBu)-Rink amide resin Weighed 170.84 g of m-PEG12-CH2CH2COOH and 48.64 g of HOBT, dissolved them in 2000 mL of DMF solution and 80.0 mL of DIC, placed them in an ice bath at -10 °C for 0.5 h, then slowly added them to the reaction kettle for reaction, and stirred and reacted at room temperature through nitrogen gas for 2 - 4 h, followed by extraction and drying. Washed twice with 2400 mL of DMF, 2400 mL of absolute ethanol, 2400 mL of DCM, and 2400 mL of DMF respectively, and then extracted and dried. It was colorless in the Kaiser measurement.

[0214] Step 6: Preparation of NH2-Lys(PEG12)-Asp(OtBu)-PEG4-Asp(OtBu)-Rink amide resin Add 2400 mL of dde removal reagent, stir and react at 25 ± 1 °C for 10 min by passing nitrogen gas, extract and dry. After repeating this operation three times, wash successively with 2400 mL of DMF, 2400 mL of absolute ethanol, 2400 mL of DCM, and 2400 mL of DMF twice each, extract and dry. It was blue in the Kaiser measurement.

[0215] Step 7: Preparation of Fmoc-Gly-Gly-Gly-Lys(PEG12)-Asp(OtBu)-PEG4-Asp(OtBu)-Rink amide resin Weigh 111.08 g of Fmoc-Gly-Gly-Gly-OH and 48.64 g of HOBT, dissolve them in 2000 mL of DMF solution and 80.0 mL of DIC, place in an ice bath at -10 °C for 0.5 h, then slowly add to the reaction kettle to react, stir and react at room temperature by passing nitrogen gas for 2 - 4 h, extract and dry. Wash successively with 2400 mL of DMF, 2400 mL of absolute ethanol, 2400 mL of DCM, and 2400 mL of DMF twice each, extract and dry. It was colorless in the Kaiser measurement. Wash the resin peptide three times with 2400 mL of absolute ethanol, extract and dry, and prepare for cleavage.

[0216] Step 8: Preparation of LP-6-2 Add 10000 mL of cleavage reagent (TFA:TIS:H2O = 95:2.5:2.5) to a 10 L reaction kettle, cool to -10 ± 2 °C, add the resin weighed after drying, then raise the temperature, stir at 20 ± 5 °C by passing nitrogen gas for 2 h. Filter, wash the resin once with 100 mL of TFA, and combine the filtrate and the washing solution.

[0217] Add 40 L of pre-cooled (to -10 °C or below) cold ethyl ether, stir for 10 min, then centrifuge and precipitate. After centrifuging and precipitating, discard the supernatant, mix the precipitate with cold ethyl ether and shake evenly, then centrifuge and precipitate again (repeat this process three times, with the usage amount each time being 10 L, 10 L, 10 L. Set the centrifugation speed at 3600 rpm, the centrifugation time at 5 min, and the chamber temperature of the centrifuge at -5 °C each time).

[0218] The precipitate was collected, i.e., the crude product of LP-6-2, purified by Prep-HPLC, and freeze-dried to obtain LP-6-2. C 74 H 121 N9O 31 2+ [M+2H] 2+ In the MS(ESI) m / z of , the calculated value was 815.9 and the measured value was 816.3.

[0219] (3) Synthesis of LP-6-3

Chemical Structure

[0220] Step A: Synthesis of intermediate LP-6-3a Compound LP-6-1 (2.2 equivalents) and compound LP-6-2 (1.0 equivalent) were added to a reaction flask, dissolved in DMF, then DIPEA (5.0 equivalents) was added and stirred uniformly. After that, HATU (2.5 equivalents) was added to the system and reacted at room temperature. The reaction was monitored by HPLC until completion (about 2 h). The reaction system was directly prepared by prep-HPLC, and the prepared solution was freeze-dried to obtain compound LP-6-3a (white solid, yield 52%). C 143 H 212 O 48 N 21 3+ [M+3H] 3+ In the MS(ESI) m / z of , the calculated value was 997.2 and the measured value was 875.9 (some functional groups may fall off after ionization).

[0221] Step B: Synthesis of intermediate LP-6-3b Compound LP-6-3a was dissolved in purified water, a certain amount of palladium hydroxide (10 wt% Pd(OH)2 supported on carbon) was added, the system was replaced with hydrogen gas three times, stirred at room temperature for 1.5 h, and the progress of the reaction was monitored during the reaction. The reaction was stopped immediately after the raw materials were consumed to prevent the increase of the de-Fmoc product. The reaction solution was filtered and prepared by prep-HPLC to obtain compound LP-6-3b (white solid, yield 76%). C 128 H 200 O48 N 21 3+ [M + 3H] 3+ In the MS(ESI) m / z of [[M + 3H]], the calculated value was 937.1 and the measured value was 875.9 (some functional groups may fall off after ionization).

[0222] Step C: Synthesis of Intermediate LP - 6 - 3d Compound LP - 6 - 3b (1.0 equivalent) and compound LP - 6 - 3c (2.2 equivalents) were weighed and dissolved in DMF, DIPEA (5.0 equivalents) was added and stirred uniformly, then HATU (2.5 equivalents) was added and reacted at room temperature. The reaction was monitored by HPLC until completion (about 16 h), and the reaction system was directly prepared by prep - HPLC. After freeze - drying the preparation solution, compound LP - 6 - 3d (yellow solid, yield 66%) was obtained, C 174 H 232 O 55 Cl2F2N 27 3+ [M + 3H] 3+ In the MS(ESI) m / z of [[M + 3H]], the calculated value was 1229.2 and the measured value was 1229.3.

[0223] Step D: Synthesis of Compound LP - 6 - 3 Compound LP - 6 - 3d was dissolved in DMF, diethylamine was added, and the reaction was carried out at room temperature. The reaction was monitored by HPLC until completion (about 0.5 h). After the reaction was completed, the pH was adjusted to neutral, then prepared by prep - HPLC and freeze - dried to obtain compound LP - 6 - 3 (yellow solid, yield 73%), C 159 H 222 O 53 Cl2F2N 27 3+ [M + 3H] 3+ In the MS(ESI) m / z of [[M + 3H]], the calculated value was 1155.2 and the measured value was 1155.3.

[0224] Among them, for the synthesis of compound LP - 6 - 3c, please refer to Chinese Patent CN202211428194.6.

[0225] (4) Synthesis of LP - 6 [Chemical]

[0226] Synthesis of LP-6a Compound 1 (0.5 - 5.0 equivalents) and compound LP-6-3 (1.0 equivalent) were taken and dissolved in DMF, DIPEA (1 - 10 equivalents) was added, and the mixture was stirred uniformly at 0 °C. Then HATU (0.5 - 10 equivalents) was added, and the reaction system was stirred at 0 °C. The reaction was monitored by HPLC until completion (about 2 h). The reaction system was directly prepared by prep-HPLC. After the preparation solution was lyophilized, compound LP-6a (yellow solid, yield 63%) was obtained, C 173 H 243 O 64 Cl2F2N 28 3+ [M + 3H] 3+ In the MS (ESI) m / z of , the calculated value was 1281.5 and the measured value was 1281.8.

[0227] Synthesis of LP-6 Compound LP-6a was weighed and dissolved in purified water. After stirring and cooling in an ice-water bath, Et3N (1 - 100 equivalents) and DMC (2-chloro-1,3-dimethylimidazolinium chloride, CAS: 37091-73-9, 1 - 100 equivalents) were added. The progress of the reaction was monitored by HPLC. After the reaction was completed, it was purified by prep-HPLC. After the preparation solution was lyophilized, compound LP-6 (light yellow solid, yield 74%) was obtained, C 173 H 241 O 63 Cl2F2N 28 3+ [M + 3H] 3+ The MS (ESI) m / z of was calculated to be 1275.5 and the measured value was 1275.9.

[0228] Example 9 Production of LP-7 The structure of LP-7 is as follows: [Chemical]

[0229] The synthetic route is as follows:

Chemical Structure

[0230] Step A: Synthesis of Compound LP-7b Compound LP-7a was synthesized by the method of peptide solid-phase synthesis, referring to a similar method of LP-6-2.

[0231] Compound LP-7a (1.0 equivalent) and compound LP-6-1 (3.6 equivalents) were added to a reaction flask, dissolved in DMF, then DIPEA (6.0 equivalents) was added and stirred uniformly. After that, HATU (3.6 equivalents) was added to the system, and the reaction was carried out at room temperature. The reaction was monitored by HPLC until completion (about 2 h). The reaction system was directly prepared by prep-HPLC, and the prepared solution was freeze-dried to obtain compound LP-7b (white solid, yield 56%). For the MS (ESI) m / z of C 191 H 280 O 66 N 29 3+ [M + 3H] 3+ , the calculated value was 1345.3 and the measured value was 1345.8.

[0232] Step B: Synthesis of Intermediate LP-7c Compound LP-7b was dissolved in purified water, a certain amount of palladium hydroxide (10 wt% Pd(OH)₂ supported on activated carbon) was added, the system was replaced with hydrogen gas three times, stirred at room temperature for 1.5 h, the progress of the reaction was monitored during the reaction, the reaction solution was filtered, directly prepared by prep-HPLC, and compound LP-7c (white solid, yield 80%) was obtained. For the MS (ESI) m / z of C 170 H 262 O 66 N 29 3+ [M + 3H] 3+ , the calculated value was 1255.3 and the measured value was 1255.9.

[0233] Step C: Synthesis of Intermediate LP-7d Weigh compound LP-7c (1.0 equivalent) and compound LP-6-3 (1 - 10 equivalents), dissolve them in DMF, add DIPEA (1 - 20 equivalents), stir uniformly, then add HATU (1 - 10 equivalents), react at room temperature, monitor by HPLC until the reaction is complete (about 2 h), and use the reaction product directly in the next reaction, C 239 H 313 O 75 Cl3F3N 38 3+ [M + 3H] 3+ In the MS(ESI) m / z of , the calculated value was 1692.4 and the measured value was 1692.9.

[0234] Step D: Synthesis of compound LP-7e Dissolve compound LP-7d in DMF, add diethylamine, react at room temperature, monitor by HPLC until the reaction is complete (about 0.5 h). After the reaction is complete, adjust the pH to neutral, then prepare by prep-HPLC and lyophilize to obtain compound LP-7e (yellow solid, yield 73%), C 224 H 303 O 73 Cl3F3N 38 3+ [M + 3H] 3+ In the MS(ESI) m / z of , the calculated value was 1618.3 and the measured value was 1618.5.

[0235] Step E: Synthesis of compound LP-7f Take compound 1 (0.5 - 5.0 equivalents) and compound LP-7e (1.0 equivalent), dissolve them in DMF, add DIPEA (1 - 10 equivalents), stir uniformly at 0 °C, add HATU (0.5 - 10 equivalents), stir the reaction system at 0 °C, and monitor by HPLC until the reaction is complete (about 2 h). The reaction system was directly prepared by prep-HPLC, and the prepared solution was lyophilized to obtain compound LP-7f (yellow solid, yield 65%), C 238 H 325 O 84 Cl3F3N 39 4+ [M + 4H] 4+In the MS(ESI) m / z, the calculated value was 1308.8 and the measured value was 1309.0.

[0236] 15.6 Step F: Synthesis of Compound LP-7 Compound LP-7f was weighed and dissolved in purified water, and after cooling by stirring in an ice-water bath, Et3N (1 to 100 equivalents) and DMC (2-chloro-1,3-dimethylimidazolinium chloride, CAS: 37091-73-9, 1 to 100 equivalents) were added, and the progress of the reaction was monitored by HPLC. After completion of the reaction, it was purified by prep-HPLC, and the production solution was freeze-dried to obtain Compound LP-7 (pale yellow solid, yield 50%).

[0237] III. Selection or Production of Antibodies Selection of Antibodies In the antibody-drug conjugate produced by the substrate and conjugation method of the present invention, the polymer part may select a molecule containing any antibody FC region sugar-containing chain, including but not limited to antibodies / bispecific antibodies / FC fusion proteins / single-chain antibodies, etc.

[0238] In the following specific examples, commercially available trastuzumab may be selected.

[0239] Note that as the antibody, a genetically engineered antibody may be selected. The production examples are as follows:

[0240] Example 10 Production of Genetically Engineered Anti-HER2 and Trop2 Antibodies The production, purification and identification of anti-human ErbB2 / HER2 antibody mAb-1 and anti-human TROP2 antibody mAb-2 refer to Example 1 of Chinese Patent CN106856656B, and the entire content of CN106856656B is incorporated herein by reference.

[0241] IV. Production, Characterization and Activity Testing of ADCs

[0242] Example 11 Production and Characterization of ADC-1 Hereinafter, endoglycosidase was used to promote the remodeling of the sugar chain at the Fc terminus of antibody mAb-1, and the linker-payload was specifically bound to the antibody to form the corresponding ADC drug.

[0243] 1) Treatment of antibody mAb-1: The antibody was treated by ultrafiltration, or dialysis, or desalting column method, and its storage buffer was changed to 50 mM Tris-HCl (pH between 5 and 8), 150 mM NaCl.

[0244] 2) Production of ADC-1: The complex reaction of antibody mAb-1 and LP-1 was promoted using endoglycosidase Endo S2 as a catalyst to produce ADC-1. In 1× endonuclease buffer, antibody mAb-1 and LP-1 were sufficiently mixed at an appropriate molar ratio (1:1 to 1:100), and endoglycosidase Endo S2 was added and mixed uniformly. The complex reaction in the uniformly mixed state was carried out at 4 to 40 °C for 0.5 to 20 hours. After the reaction was completed, purification was performed. The purified ADC-1 was stored at 4 °C or -80 °C in 1× PBS with a pH of 7.4.

[0245] The characteristic evaluation data of the antibody-drug conjugate ADC-1 are as follows.

[0246] SDS-PAGE measurement analysis of ADC-1 After the complex reaction was completed, the purity and complex efficiency of ADC-1 were measured by SDS-PAGE method. As shown in Figure 1, the SDS-PAGE measurement results of ADC-1 showed that the complex reaction occurred at a predetermined position on the heavy chain of the antibody, and the heavy chain bound to the cytotoxin showed an obvious molecular weight shift compared with the heavy chain not bound to the cytotoxin. The purity of the complex product was as expected.

[0247] HIC-HPLC measurement analysis of ADC-1 HIC-HPLC: Using a Proteomix HIC Butyl-NP5 4.6×100mm 5μm non-porous chromatography column; setting the column temperature at 30 °C; using 1.5M ammonium sulfate + 20mM phosphate buffer with a pH of 7.0 as mobile phase A; using 20mM phosphate buffer: isopropanol = 7:3 (v / v) as mobile phase B; setting the flow rate at 0.8 mL / min; gradient method: increasing the B phase from 10% to 100% within 8 min; setting the measurement wavelength at 280 nm, the DAR distribution of ADC-1 was measured.

[0248] The measurement results are shown in Figure 2. The antibody not bound to LP-1 was <1%; the composite product was mainly DAR 2, and the average DAR value of the ADC drug was 1.93.

[0249] High-precision molecular weight mass spectrometry (ESI-MS) analysis of ADC-1 As a result of analyzing and measuring ADC-1 by high-precision molecular weight mass spectrometry, it was found that its apparent molecular weight was 150539.27, the theoretical molecular weight was 150538.78, which was as expected, and it was proved that one cytotoxin molecule was bound to the Fc end of each heavy chain.

[0250] SEC-HPLC measurement analysis of ADC-1 The high molecular weight aggregation degree of ADC-1 was analyzed and measured using an SEC chromatography column. The measurement results, as shown in Figure 3, were that in ADC-1, 6.7 min was the high molecular weight polymer and <5%, and the ADC sample mainly existed as a monomer, and the damage to the antibody in the complex reaction was almost negligible.

[0251] Measurement of the affinity of ADC-1 for cell surface ErbB2 / HER2 1) Take HER2 ECD, prepare it to 1 μg / mL with CBS coating solution (0.1 M carbonate buffer, pH 9.6), coat at 100 μL / well for 60 min at 25 °C. 2) After coating, wash the plate 3 times with PBST, block with blocking solution (5% non-fat dry milk) (300 μL / well), incubate at 25 °C, 200 rpm for 60 min. 3) After blocking, wash the plate 3 times with PBST, add antibody mAb-1 and ADC-1 (100 μL / well), place at 25 °C, incubate at 200 rpm for 60 min. 4) After incubation with the sample, wash the plate 3 times with PBST, add anti-human IgG Fc-HRP 100 μL / well, incubate at 25 °C, 200 rpm for 60 min. 5) After 60 min of incubation, further develop the color with TMB (100 μL / well) for 5 min, stop the reaction with the stop solution, and read at OD450.

[0252] Referring to Figure 4, it can be seen from the results of the Elisa measurement that there is no obvious difference in the antigen affinity of mAb-1 naked monoclonal antibody and ADC-1 for HER2 ECD.

[0253] Example 12 In vitro activity test of ADC-1 (BT-474, NCI-N87, HepG2) According to the following method, the effect of ADC-1 on the proliferation of tumor cells with different expression levels of ErbB2 / HER2 was measured.

[0254] 1) Inoculate ErbB2 / HER2-positive human breast cancer cells BT-474, ErbB2 / HER2-positive human gastric cancer cells NCI-N87, and ErbB2 / HER2-negative human liver cancer cells HepG2 into a 96-well cell plate at 100 μL (containing 1000 - 10000 cells) / well, and culture overnight in a cell incubator at 37 °C, 5% CO2, 95% air, 100% humidity.

[0255] 2) To each of the ErbB2 / HER2-positive cells cultured overnight, ADC-1 at different concentrations (10, 3.3, 1.1, 0.37, 0.12, 0.041, 0.014, 0.0046, 0.0015, 0.00051 nM), or antibody mAb-1, or MMAE (monomethyl auristatin E) at different concentrations (30, 10, 3.3, 1.1, 0.37, 0.12, 0.041, 0.014, 0.0046, 0.0015 nM) was added; to each of the ErbB2 / HER2-negative cells cultured overnight, ADC-1 at different concentrations (100, 10, 3.3, 1.1, 0.37, 0.12, 0.041, 0.014, 0.0046, 0.0015 nM), or antibody mAb-1, or MMAE at different concentrations (30, 10, 3.3, 1.1, 0.37, 0.12, 0.041, 0.014, 0.0046, 0.0015 nM) was added; to the control group, puromycin with a final concentration of 5 μM was added. Incubation was continued at 37 °C for 72 - 120 h.

[0256] 3) The cell plates were taken out from the 37 °C cell incubator and equilibrated to room temperature for about 30 minutes. 100 μL of CellTiter Glo reagent was added per well, shaken on a shaker for 2 min, then left standing in the dark at room temperature for 10 min, and the luminescence value (RLU) was measured with a Cytation 3 microplate reader.

[0257] 4) As shown in Table 1, Table 2, and Figures 5 - 7, the results of the inhibitory effects of different drugs on tumor cell proliferation showed that both ADC-1 and the MMAE low molecular toxin had obvious growth inhibitory effects on ErbB2 / HER2-positive cells, the antibody mAb-1 monoclonal antibody had a slight growth inhibitory effect on ErbB2 / HER2-positive cells, and ADC-1 was clearly superior to the mAb-1 monoclonal antibody and the MMAE low molecular toxin. On the other hand, ADC-1 and the mAb-1 monoclonal antibody had no inhibitory effect on ErbB2 / HER2-negative cells.

[0258]

Table 1

[0259]

Table 2

[0260] Example 13 In vivo activity test of ADC-1 (NCI-N87 CDX mouse model) According to the following method, the effect of ADC-1 on the tumor growth of the NCI-N87 CDX mouse model of ErbB2 / HER2 was measured.

[0261] 1) Cell culture: NCI-N87 human gastric cancer tumor cells (ATCC, Manassas, VA, cat # CRL-5822) in the logarithmic growth phase were collected, the cell density was adjusted with Matrigel buffer (PBS: Matrigel = 1:1), and 0.2 mL of the prepared NCI-N87 cell suspension was subcutaneously injected into the right scapular region of 7- to 9-week-old SPF female BALB / c nude mice, and the cell seeding amount was 10×10 6 / mouse.

[0262] 2) The tumor diameter was measured with calipers, and the tumor volume was calculated using the formula V = 0.5a×b 2 (where a is the longest diameter of the tumor and b is the shortest diameter of the tumor). Five days after cell seeding, when the average tumor volume ranged from 100 to 300 mm 3 animals were randomly divided into a solvent control group and an ADC-1 3 mg / kg group, with 5 animals in each group. The animals were administered by tail vein injection, the control group was given an equal volume of solvent (vehicle), and the day of grouped administration was defined as Day0. During the 35 days after administration, the tumor volume of the animals in each group was measured twice a week, the tumor volume of the animals on the 35th day was compared between the groups, and the T / C value and TGI value were calculated using the tumor volume. The calculation formulas are as follows: T / C% = T RTV / C RTV ×100% (T RTV : RTV of the treatment group, C RTV: Solvent control group (RTV). Relative tumor volume (RTV) was calculated from the results of tumor measurement. The calculation formula is RTV = Vt / VO, where VO is the average tumor volume measured at the time of grouping, Vt is the average tumor volume at a certain measurement time, and T RTV and C RTV used the data of the same day. Calculation of TGI(%): TGI(%) = [1 - (average tumor volume at the end of treatment in a certain treatment group - average tumor volume at the start of administration in the same treatment group) / (average tumor volume at the end of treatment in the solvent control group - average tumor volume at the start of treatment in the solvent control group)] × 100%.

[0263] 3) Independent sample t-tests were used to statistically analyze the data between each group, and all analyses were performed using SPSS 17.0. When P < 0.05, the difference is considered statistically significant.

[0264] 4) Table 3 and Figure 8 show that compared with the solvent control group, ADC-1 can significantly inhibit tumor growth in the NCI-N87 CDX mouse model.

[0265]

Table 3

[0266] Example 14 Manufacture and Characterization of ADC-2 Manufacture and characterization were carried out with reference to the method described in Example 11, except that the linker-payload used was LP-6. The characterization data of the antibody-drug conjugate ADC-2 are as follows.

[0267] SDS-PAGE measurement and analysis of antibody-drug conjugate ADC-2: After the conjugation reaction, the purity and conjugation efficiency of ADC-2 were measured by SDS-PAGE. As shown in Figure 9, the SDS-PAGE measurement results of ADC-2 indicated that the conjugation reaction occurred at a predetermined position on the heavy chain of the antibody, and the ADC-2 heavy chain bound to the linker-payload showed an obvious molecular weight shift compared with the monoclonal antibody heavy chain with the sugar chain removed, which proved that the linker-payload could be bound to the monoclonal antibody heavy chain molecule at a predetermined position; almost no antibody not bound to the linker-payload was found in the conjugation product, the conjugation efficiency was as high as over 95%, and the purity of the conjugation product was as expected.

[0268] In the HIC-HPLC measurement and analysis of ADC-2, the measurement results were as shown in Figure 10. As shown in the figure, the antibody not bound to the cytotoxin was <5%, the conjugation product was mainly DAR4, and the overall DAR value of the ADC-2 drug was about 3.8.

[0269] In the SEC-HPLC measurement and analysis of ADC-2, as shown in Figure 11, the high molecular weight polymer in the ADC drug was <5%, and the ADC sample mainly existed as a monomer at 8.1 min.

[0270] Stability measurement of ADC-2 in plasma Test design: Healthy human mixed plasma (equal volume mixture of 5 males and 5 females) was taken, ADC-2 was added to reach a specific final concentration, divided into 4 groups, 450 μL per group, incubated in a 37 °C incubator, the sampling times of the samples were 0 h, 24 h, 48 h, and 96 h, and after sampling, they were stored in a refrigerator at -60 to -90 °C for the measurement of the free load dropout rate and the DAR change rate.

[0271] LC-MS measurement of low molecular weight toxin (the low molecular weight toxin of LP-6 according to this example is represented by payload) Take 40 μL of each group of samples (except Double Blank), add a certain amount of internal standard precipitant (1 ng / mL of DXd), and shake for at least 10 min; for Double Blank, take 40 μL of blank matrix, add 120 μL of precipitant without internal standard, and shake for at least 10 min; centrifuge at 3600 g for 15 min at 4 °C, aspirate 60 μL of the supernatant into the injector, add a certain amount of 0.1% FA ultrapure water, shake for at least 3 min, and perform HPLC measurement.

[0272] Measurement of DAR by Hybrid LC-MS Method Weigh 1 g of CNBr-activated agarose (Sigma, Cat# C9142) into a 50 mL centrifuge tube, add 50 mL of pre-cooled 1 mM HCl, place it on a rotary mixer and incubate for at least 30 min. The incubation conditions are 4 °C and 10 RPM. Centrifuge for a certain time to remove 1 mM HCl by centrifugation. Wash the gel once with 5 - 10 volumes of deionized water, and then wash it three times with 0.1 M NaHCO3. Take an appropriate amount of HER2 ECD, use a 30 kD ultrafiltration tube to replace the buffer with 0.1 M NaHCO3, then mix the replaced HER2 ECD with the packing material, place it on a rotary mixer, and incubate at 25 °C and 10 RPM for 2 h ± 10 min to bind. Further add 5 mL of 0.1 M NaHCO3 buffer, centrifuge and wash to remove unbound proteins, centrifuge for a certain time, and wash three times. Take a certain amount of glycine (pH 8.0) and add it to the packing material, let it stand at 2 - 8 °C for 16 h to block, and block the unreacted chemical groups on the packing material. First, wash the packing material with 5 mL of 0.1 M NaHCO3, centrifuge for a certain time, then wash the packing material with a certain amount of acetate buffer, centrifuge for a certain time, and repeat the washing cycle several times. Take 0.1 mL of each sample into a 1.5 mL EP tube, add 0.1 mL of the packing material with HER2 ECD bound, incubate at 25 °C and 10 RPM for 2 h, wash three times with 1 mL of PBST, and centrifuge for a certain time. Elute and centrifuge to take the supernatant, measure the concentration, and perform LC-MS measurement.

[0273] Result analysis: When ADC-2 was incubated in healthy human pooled plasma for 0, 24, 48, and 96 h, the DAR change rates were 100.00%, 106.7%, 104.2%, and 106.5%, respectively; the low molecular weight toxin dropout rates were only 0.006%, 0.179%, 0.373%, and 1.07%, respectively. As can be seen from the above results, after ADC-2 was incubated at 37 °C for 96 h in healthy human pooled plasma, the amount of toxin dropout was extremely low, the DAR maintained stability, and the low toxin dropout suggested that the toxic side effects caused by free toxins were significantly reduced clinically. The high DAR stability indicated that the antibody molecule could target and deliver more toxin molecules to reach the tumor site, thereby improving the drug efficacy. The synergistic effect of both could significantly expand the therapeutic range of ADC-2.

[0274] Example 15 In vitro activity test of ADC-2 (SK-BR-3, HCC1954, MDA-MB-468) According to the following method, the effect of ADC-2 on the proliferation of tumor cells with different expression levels of ErbB2 / HER2 was measured.

[0275] With reference to the method described in Example 12, the inhibitory effect of the antibody-drug conjugate ADC-2 on the proliferation of cancer cells with various different ErbB2 / HER2 expression levels was measured. For example, ErbB2 / HER2-positive human tumor cells such as SK-BR-3 and HCC1954, and MDA-MB-468 ErbB2 / HER2-negative human tumor cells were selected. As shown in Tables 4, 5 and Figures 12 to 14, the results of the inhibitory effect of different drugs on tumor cell proliferation showed that both ADC-2 and the small molecule toxin had an obvious growth inhibitory effect on ErbB2 / HER2-positive cells. The antibody mAb-1 monoclonal antibody had a certain growth inhibitory effect on ErbB2 / HER2-positive cells, and ADC-2 was clearly superior to mAb-1. On the other hand, ADC-2 and the mAb-1 monoclonal antibody had no inhibitory effect on ErbB2 / HER2-negative cells, indicating good targeting properties.

[0276]

Table 4

[0277]

Table 5

[0278] Example 16 Manufacture and Characterization of ADC-3 The antibody-drug conjugate ADC-3 was manufactured and characterized with reference to the method described in Example 11, except that the antibody used was an anti-TROP2 antibody, i.e., mAb-2, and the linker-payload used was LP-6. The characterization data of the antibody-drug conjugate ADC-3 are as follows.

[0279] In the SEC-HPLC measurement analysis of ADC-3, the high molecular weight aggregation degree of ADC-3 was analyzed and measured using an SEC chromatography column. As shown in Figure 15, the measurement results showed that the high molecular weight polymer was <5%, and the ADC sample mainly existed as a monomer, indicating that the damage to the antibody during the conjugation reaction was almost negligible.

[0280] Analysis of the DAR value of antibody-drug conjugate ADC-3 by high-resolution mass spectrometry (ESI-MS) The molecular weight of ADC-3 was analyzed by a high-resolution mass spectrometer, and the mass spectrum after deconvolution is shown in Figure 16. Based on the measured molecular weight information, each main molecular weight variant can be assigned by comparing with the theoretical molecular weight. The DAR value analysis was performed using the abundance of each main molecular weight variant determined by mass spectrometry, and the calculated average DAR value was 3.93.

[0281] Example 17 In vitro activity test of ADC-3 (BxPC-3, FaDu, HepG2) With reference to a similar operation of the method described in Example 12, the inhibitory effects of different concentration gradients of the antibody-drug conjugate ADC-3 on the growth of cancer cells with various different TROP2 expression levels were measured. For example, human tumor cells such as BxPC-3, FaDu, and HepG2 were selected. The results of the inhibitory effects of different drugs on the growth of tumor cells are shown in Table 6 and Figures 17 to 19. Both ADC-3 and the low-molecular-weight toxin had obvious growth inhibitory effects on TROP2-positive cells, while the TROP2 antibody, that is, mAb-2, had no obvious growth inhibitory effect on TROP2-positive cells, and ADC-3 was clearly superior to the monoclonal antibody and the low-molecular-weight toxin. Neither ADC-3 nor the mAb-2 monoclonal antibody had an inhibitory effect on TROP2-negative cells, indicating good targeting properties.

[0282]

Table 6

[0283] Example 18 In vivo activity test of ADC-3 (NCI-N87 CDX mouse model) Referring to the similar method described in Example 13, the inhibitory effect on tumor growth in the NCI-N87 CDX mouse model of ADC-3 was evaluated. The tumor growth curve and body weight change curve after administration were as shown in Figures 20-21. ADC-3 showed a good inhibitory effect on tumor growth and good safety, and no toxicity related to weight loss was observed in the experimental mice.

[0284] Example 19 Manufacture and Characterization of ADC-4 In the manufacture and characterization of ADC-4, except that the antibody used was the anti-TROP2 antibody, i.e., mAb-2, the manufacture and characterization were carried out with reference to the method described for ADC-1. The characterization data of the antibody-drug conjugate ADC-4 are as follows.

[0285] In the HIC-HPLC measurement analysis of ADC-4, as shown in Figure 22, the antibody not bound to the cytotoxin was <5%; the composite product was mainly DAR2, and the overall DAR value of the ADC-4 drug was 1.89. In the SEC-HPLC measurement analysis of ADC-4, as shown in Figure 23, the high molecular weight polymer in the ADC drug was <5%, and the ADC sample mainly existed as a monomer.

[0286] Example 20 Manufacture and Characterization of ADC-5 In the manufacture and characterization of ADC-5, except that the antibody used was the anti-TROP2 antibody mAb-2 and LP-2 was used for the linker-payload, the manufacture and characterization were carried out with reference to the method described for ADC-1. The characterization data of the antibody-drug conjugate ADC-5 are as follows.

[0287] In the HPLC measurement and analysis of ADC-5, as shown in Figure 24, the antibody not bound to the cytotoxin was less than 5%; the complex product was mainly DAR2, and the DAR value of the overall ADC-5 drug was 1.87. In the SEC-HPLC measurement and analysis of ADC-5, as shown in Figure 25, the high molecular weight polymer in the ADC drug was less than 5%, and the ADC sample mainly existed as a monomer.

[0288] Example 21 In vitro activity comparison of ADC-4 and ADC-5 (BxPC-3, FaDu, HepG2) With reference to the operation method described in Example 12, the effects of the antibody-drug conjugates ADC-4 and ADC-5 on the growth of cancer cells with various different TROP2 expression levels were measured. For example, TROP2-positive human tumor cells such as BxPC-3 (human pancreatic cancer cells), FaDu (human pharyngeal cancer cells), and TROP2-negative tumor cells of HepG2 (human liver cancer cells) were selected. The results of the inhibitory effects of different drugs on tumor cell growth are shown in Tables 7-8 and Figures 26-28. Among them, ADC-4, ADC-5, and the MMAE low molecular toxin all had obvious growth inhibitory effects on positive cells, and there was no obvious difference in the activities of ADC-4 and ADC-5, and the monoclonal antibody had no obvious growth inhibitory effect on TROP2-positive cells. ADC-4, ADC-5, and the monoclonal antibody had no inhibitory effect on antigen-negative cells and showed good targeting.

[0289]

Table 7

[0290]

Table 8

[0291] Example 22 Manufacture and property evaluation of ADC-6 For the production and characterization of ADC-6, the method described for ADC-1 was referred to for production and characterization, except that the antibody used was mAb-3 (Trastuzumab) and the linker-payload used was LP-6. The characterization data of the antibody-drug conjugate ADC-6 are as follows. In the HIC-HPLC measurement analysis of ADC-6, as shown in Figure 29, the antibody not bound to the cytotoxin was <5%; the composite product was mainly DAR4, and the overall DAR value of the ADC-6 drug was 3.93. In the SEC-HPLC measurement analysis of ADC-6, as shown in Figure 30, the high molecular weight polymer in the ADC drug was <5%, and the ADC sample mainly existed as a monomer.

[0292] Example 23 In vitro Activity Test of ADC-6 (SK-BR-3, NCI-N87) With reference to the operation method described in Example 12, the effect of the antibody-drug conjugate ADC-6 on the growth of cancer cells with different expression levels of each HER2 was measured. For example, two groups of HER2 cell lines, SK-BR-3 and NCI-N87, were selected, and the difference in the activities of ADC-2 (containing Srt A tag) and ADC-6 (not containing Srt A tag) was compared. As a result, it was found that there was no difference in the cell killing activity of the target ADC in the above two types of cell lines regardless of the presence or absence of the specific recognition oligopeptide sequence of the Srt A enzyme (see Figures 31 - 32).

[0293] The sequences related to this application are as follows: SEQ ID No.1: mAb-1 light chain: DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGALPETGG SEQ ID No.2: mAb-1 heavy chain: EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID No.3: mAb-2 light chain: DIQLTQSPSSLSASVGDRVSITCKASQDVSIAVAWYQQKPGKAPKLLIYSASYRYTGVPDRFSGSGSGTDFTLTISSLQPEDFAVYYCQQHYITPLTFGAGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGALPETGG SEQ ID No.4: mAb-2 Heavy Chain QVQLQQSGSELKKPGASVKVSCKASGYTFTNYGMNWVKQAPGQGLKWMGWINTYTGEPTYTDDFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFCARGGFGSSYWYFDVWGQGSLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID No.5: mAb-3 Light Chain DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID No.6: mAb-3 Heavy Chain EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Claims

1. A linker-payload compound having formula (I). 【Chemical Formula 1】 In the formula, P is the payload; D-C(O)-L- is the linker; D-C(O)- is an oligosaccharide structure, - the first hexose group or its derivative moiety - (the second hexose group or its derivative moiety) ｆ -β-D-N-acetylglucosamine moiety, or, - the first hexose group or its derivative moiety - (the second hexose group or its derivative moiety) ｆ -β-D-glucose oxazoline moiety, the 6-position carbon of the first hexose group or its derivative moiety assumes the form of -C(O)-, which is the -C(O)- in D-C(O)-, and the β-D-N-acetylglucosamine moiety is 【Chemical Formula 2】 and the β-D-glucose oxazoline moiety is 【Chemical Formula 3】 and f is 0, 1, 2, 3, 4, 5, or 6, L is the linker terminus, and L is directly linked to the carbonyl group in D-C(O)- via -NH- therein. When L is an unbranched linker terminus, L is linked to one P and t is 1. When L is a branched linker terminus, each branch may be linked to one P, and t is an integer greater than 1 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10).

2. -L-(P) ｔ is -L ２ -L １ -B-P, that is, formula (I) is 【Chemical Formula 4】 and: In the formula, B either does not exist independently or is one of the following 1), or one of the following 2), or a combination of the following 1) and 2): 1) a self-destructive spacer Sp1; 2) one divalent group, or a combination of two or more divalent groups, the divalent group being selected from -CR １ R ２ , C １～１０ alkylene group, C ４～１０ cycloalkylene group, C ４～１０ heterocyclylene group, and -(CO)-; L １ either does not exist independently; or is an uncleavable sequence; or is a cleavable sequence containing an amino acid sequence cleavable by an enzyme, the amino acid sequence cleavable by the enzyme containing 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids; L ２ either does not exist independently; or is the following 1); or is the following 2); or is a combination of the following 1) and 2): 1) -NH-C ２～２０ alkylene group, one or more -CH ２ - structure in the alkylene group being -CR ３ R ４ -, -O-, -(CO)-, -S-, -S(=O) ２ -, -NR ５ -; [Chemical Formula 5] C ４～１０ cycloalkylene group, C ４～１０ heterocyclylene group, phenylene group may be substituted, and the cycloalkylene group, heterocyclylene group, and phenylene group are each independently unsubstituted or substituted with at least one substituent selected from halogen, -C １～１０ alkyl group, -C １～１０ haloalkyl group, -C １～１０ alkylene-NH-R ８ , and -C １～１０ alkylene-O-R ９ ; 2) an amino acid residue sequence, i.e., -*(AA) ｎ**–, where n is an integer from 1 to 100, AA is an amino acid residue that is independent each time it appears, * represents the N-terminus of the corresponding amino acid, ** represents the C-terminus of the corresponding amino acid, and there may be a –(C ２ H ４ –O) ｍ –(CH ２ ) ｐ – may exist. In the formula, m is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, p is 0, 1, 2, or 3, and the * end forms a carbonyl group and an amide bond in the oligosaccharide structure; 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, a substituted or unsubstituted –C １～１０ alkyl group, a C ４～１０ cycloalkylene group, or R １ and R ２ together with the carbon atom to which they are attached form a 3- to 6-membered cycloalkylene group, and / or R ３ and R ４ together with the carbon atom to which they are attached form a 3- to 6-membered cycloalkylene group; P is a B moiety, or an L １ moiety, or a payload that links to the L ２ moiety. The linker-payload compound according to claim 1.

3. L ２ is an amino acid residue sequence, i.e., –*(AA) ｎ **–, where n is an integer from 1 to 100, AA is an amino acid residue that is independent each time it appears, * represents the N-terminus of the corresponding amino acid, ** represents the C-terminus of the corresponding amino acid, and between the amino group of one amino acid and the α-carbon, there is a –(C ２ H ４ –O) ｍ –(CH ２ ) ｐ - may be present, where 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 oligosaccharide structure. The linker-payload compound according to claim 2.

4. AA is independently, each time it appears, 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 arbitrary combination thereof. The linker-payload compound according to claim 3.

5. 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. The linker-payload compound according to any one of claims 2 to 4.

6. L １ is a cleavable sequence containing an amino acid sequence cleavable by an enzyme, and the amino acid sequence cleavable by the enzyme contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids. The linker-payload compound according to any one of claims 2 to 5.

7. L １ is any one of Val, Cit, Phe, Lys, Gly, Ala, Leu, Asn or any arbitrary 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. The linker-payload compound according to any one of claims 2 to 6.

8. L １The linker-payload compound according to any one of claims 2 to 7, wherein it is -Val-Cit-, or -Gly-Gly-Phe-Gly-.

9. B is 【Chemical Formula 6】 (-PABC-), -NH-CH ２ -U-, or -NH-CH ２ -U-(CH ２ ) ｇ selected from -(CO)-: wherein g is 1, 2, 3, 4, 5, or 6, and U is absent or is CH ２ , O, S, or NH, preferably O or S, the linker-payload compound according to any one of claims 2 to 8.

10. -L １ -B- represents -Val-Cit-PABC- or -Gly-Gly-Phe-Gly-, the linker-payload compound according to any one of claims 2 to 9.

11. -L ２ -L １ -B- represents -Gly-Gly-Gly-Val-Cit-PABC- or -HN-(C ２ H ４ -O) ｍ -(CH ２ ) ｐ -Gly-Gly-Phe-Gly-, the linker-payload compound according to any one of claims 2 to 10.

12. The payload P is selected from the group consisting of small molecule compounds (for example, small molecule drugs of each mechanism of action including each conventional small molecule drug, photodynamic / sonodynamic therapy drug, photothermal therapy drug, etc., for example, chemotherapeutic drugs, small molecule target drugs, immune agonists, etc., for example, conventional cytotoxic drugs such as cisplatin, paclitaxel, 5-fluorouracil, cyclophosphamide, and bendamustine; small molecule target drugs such as imatinib mesylate, gefitinib, and anlotinib; immune agonists such as STING agonists and TLR agonists), nucleic acids and nucleic acid analogs, probe molecules (including fluorescent molecules, biotin, fluorophores, chromophores, spin resonance probes, and radiolabels), oligopeptides, polypeptides, peptidomimetics, and proteins, the linker-payload compound according to any one of claims 1 to 11.

13. The payload P is a cytotoxin or a fragment thereof and has any derivatization for linking to the L moiety in the compound of formula (I) according to claims 1 to 12, Preferably, the cytotoxin is a taxane-based, maytansinoid-based, auristatin-based, epothilone-based, combretastatin A-4 phosphate, combretastatin A-4 and its derivatives, indole-sulfonamide-based, vinblastine-based such as vinblastine, vincristine, vindesine, vinorelbine, vinflunine, vinglycinate, anhydrovinblastine, dolastatin 10 and its analogs, halicondrin B, indole-3-oxoacetamide-based, podophyllotoxin-based, 7-diethylamino-3-(2'-benzoxazolyl)-coumarin (DBC), discodermolide, laulimalide, camptothecin-based and its derivatives, mitoxantrone, mitoguazone, nitrogen mustard-based, nitrosourea-based, aziridine-based, benzodopa, carbocon, metsuredepa, uredepa, dynemicin, esperamicin, neocarzinostatin, aclacinomycin, actinomycin, anthramycin, bleomycin, actinomycin C, carabicin, calminomycin, sarcomycin, calminomycin, actinomycin D, daunorubicin, detorubicin, doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin-based, nogalamycin, olivomycin, peplomycin, porfiromycin, promycin, keramycin, rhodrubicin, streptozocin, dinostatin, zorubicin, trichothecene-based, T-2 toxin, verracurin AA), loridin A, anguidine, ubenimex, azaserine, 6-diazo-5-oxo-L-norleucine, denopterin, methotrexate, pteropterin, trimethoprim, edatrexate, fludarabine, 6-mercaptopurine, thiampurine, thioguanine, ancitabine, gemcitabine, enocitabine, azacitidine, 6-azauridine, carmofur, cytarabine, didoxyridine, doxifluridine, floxuridine, calusterone, drostanolone propionate, epithioestanol, mepitiostane, testolactone, aminoglutethimide, mitotane, trilostane, flutamide, nilutamide, bicalutamide, leuprorelin acetate, protein kinase inhibitor, and proteasome inhibitor, and / or, selected from the group consisting of vinblastine, colchicine, taxane, auristatin, maytansinoid, calicheamicin, doxorubicin, duocarmycin, SN-38, cryptophycin analog, deruxtecan, duocarmazine, calicheamicin, centamycin, dolastatin, pyrrolobenzodiazepine, and exatecan, and derivatives thereof, and / or, selected from auristatin, particularly MMAE, MMAF, or MMAD, and / or, selected from exatecan and its derivatives, for example, DX8951f, and / or, selected from DXd-(1) and DXd-(2), preferably DXd-(1), the linker-payload compound according to any one of claims 1 to 12.

14. The payload is 【Chemical Formula 7】 A linker-payload compound according to any one of claims 2 to 13, selected from

15. 【Chemical Formula 8】 A linker-payload compound according to claim 2, selected from

16. -L-(P) ｔ is 【Chemical Formula 9】 that is, namely, formula (I) is 【Chemical Formula 10】 that is, wherein Ld2 and each Ld1 are each independently a bond; or -NH-C １～２０ alkylene-(CO)-, -NH-(PEG) ｉ -(CO)-selected; or each independently unsubstituted or -CO-(PEG) ｊ -R １１ substituted natural amino acids, or oligomeric natural amino acids with a degree of polymerization of 2 to 10 (i.e., 2, 3, 4, 5, 6, 7, 8, 9, or 10); -(PEG) ｉ -and -(PEG) ｊ -are each a PEG fragment, containing a predetermined number of consecutive -(O-C ２ H ４ )-structural units, or consecutive -(C ２ H ４ -O)-structural units, and may have a C １～１０ alkylene group added to one end; M is hydrogen, or LKa-L ２ -L １ -B-P; Q is NH ２ or L ２ -L １ -B-P; provided that the case where M is hydrogen and at the same time Q is NH ２ is excluded; Each LKa is independently, [Chemical Formula 11] selected from, opSu is, [Chemical Formula 12] or a mixture thereof; wherein, * represents the linking moiety with L ２ ; L ２ L １ L, B are defined as in claim 2, or -L １ -B- represents -Val-Cit-PABC- or -Gly-Gly-Phe-Gly-, or -L ２ -L １ -B- represents -Gly-Gly-Gly-Val-Cit-PABC- or -HN-(C ２ H ４ -O) ｍ -(CH ２ ) ｐ -Gly-Gly-Phe-Gly-; R １１ is a C １～１０ alkyl group; d is 0, 1, 2, 3, 4, 5, or 6; each i is independently an integer from 1 to 100 (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10), each j is independently an integer from 1 to 100 (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10), the linker-payload compound according to claim 1.

17. -L-(P) ｔ is [Chemical Formula 13] selected from, the linker-payload compound according to claim 16.

18. The first hexosyl group or its derivative moiety is selected from a glucosyl group, a mannosyl group, a galactosyl group, a fructosyl group, a glucosyl group, an idosyl group, or derivatives thereof, and its 6-position carbon assumes the form of -C(O)-; and / or, The second hexosyl group or its derivative moiety is independently selected, each time it appears, from a glucosyl group, a mannosyl group, a galactosyl group, a fructosyl group, or a derivative thereof; and / or, each monosaccharide moiety in the oligosaccharide structure is linked via a β-(1→4) glycosidic bond; and / or, the derivative is independently selected from a derivative in which a uronic acid or a hydroxyl group of a monosaccharide is replaced with an acylamino group, A linker-payload compound according to any one of claims 1 to 14 and 16.

19. The first hexosyl group or its derivative moiety is 【Chemical Formula 14】 selected from; and / or, f is 0; and / or, D-C(O)- is a disaccharide structure 【Chemical Formula 15】 wherein; and / or, D-C(O)- is a disaccharide structure 【Chemical Formula 16】 wherein. A linker-payload compound according to any one of claims 1 to 14, 16 and 18.

20. Reacting D-C(O)-OH with L'-(P) ｔ by an amide formation reaction to 【Chemical Formula 17】 form a method for producing a linker-payload compound according to any one of claims 1 to 19. In the formula, the -NH- linked to D-C(O)- in L is H ２ N- in L', and L' is the same as L defined in any one of claims 1 to 19.

21. An antibody-drug conjugate by site-specific binding to the N-glycosylation site of the antibody Fc region having the structure of formula (II). 【Chemical 18】 Wherein, R is hydrogen or an α-L-fucosyl group; q is 1 or 2; Ab is an antibody or an antigen-binding fragment; -L-(P) ｔ and f is as defined in any one of claims 1 to 19.

22. The first hexasaccharide group or its derivative moiety is as defined in claim 18 or 19; and / or, The second hexasaccharide group or its derivative moiety is as defined in claim 18; and / or, The derivative is as defined in claim 18; and / or, Each monosaccharide moiety is linked via a β-(1→4) glycosidic bond; and / or, f is as defined in claim 19, The antibody-drug conjugate according to claim 21.

23. The antibody-drug conjugate according to claim 21, wherein formula (II) is selected from formula (II-1), formula (II-2), formula (II-3), formula (II-4), formula (II-5). 【Chemical 19】 Wherein, R is hydrogen or an α-L-fucosyl group; q is 1 or 2; Ab is an antibody or an antigen-binding fragment; -L-(P) ｔ is as defined in any one of claims 1 to 17.

24. The antibody-drug conjugate according to claim 21, wherein formula (II) is selected from formula (II-1), formula (II-2), formula (II-3), formula (II-4), formula (II-5). [Chemical Formula 20] In the formula, R is hydrogen or an α-L-fucosyl group; q is 2; Ab is an anti-CD19 antibody, anti-CD20 antibody, anti-CD22 antibody, anti-CD25 antibody, anti-CD30 / TNFRSF8 antibody, anti-CD33 antibody, anti-CD37 antibody, anti-CD44v6 antibody, anti-CD56 antibody, anti-CD70 antibody, anti-CD71 antibody, anti-CD74 antibody, anti-CD79b antibody, anti-CD117 / KITk antibody, anti-CD123 antibody, anti-CD138 antibody, anti-CD142 antibody, anti-CD174 antibody, anti-CD227 / MUC1 antibody, anti-CD352 antibody, anti-CLDN18.2 antibody, anti-DLL3 antibody, anti-ErbB2 / HER2 antibody, anti-CN33 antibody, anti-GPNMB antibody, anti-ENPP3 antibody, anti-nectin-4 antibody, anti-EGFRvIII antibody, anti-SLC44A4 / AGS-5 antibody, anti-CEACAM5 antibody, anti-PSMA antibody, anti-TIM1 antibody, anti-LY6E antibody, anti-LIV1 antibody, anti-nectin-4 antibody, anti-SLITRK6 antibody, anti-HGFR / cMet antibody, anti-SLAMF7 / CS1 antibody, anti-EGFR antibody, anti-BCMA antibody, anti-AXL antibody, anti-NaPi2B antibody, anti-GCC antibody, anti-STEAP1 antibody, anti-MUC16 antibody, anti-mesothelin antibody, anti-ETBR antibody, anti-EphA2 antibody, anti-5T4 antibody, anti-FOLR1 antibody, anti-LAMP1 antibody, anti-cadherin-6 antibody, anti-FGFR2 antibody, anti-FGFR3 antibody, anti-CA6 antibody, anti-CanAg antibody, anti-integrin αV antibody, anti-TDGF1 antibody, anti-ephrin A4 antibody, anti-TROP2 antibody, anti-PTK7 antibody, anti-NOTCH3 antibody, anti-C4.4A antibody, anti-FLT3 antibody, anti-B7H3 / 4 antibody, anti-TF (tissue factor) antibody, anti-ROR1 / 2 antibody; preferably, an anti-CD19 antibody, anti-ErbB2 / HER2 antibody, anti-CLDN18.2 antibody, anti-nectin-4 antibody, anti-FGFR3 antibody, anti-Trop2 antibody; more preferably, an anti-ErbB2 / HER2 antibody, anti-Trop2 antibody; particularly preferably, an anti-ErbB2 / HER2 antibody (e.g., trastuzumab); (P) ｔ -L- is selected from [Chemical Formula 21] the following.

25. The manufacturing process comprises a) under the catalysis of a glycosidase or a variant thereof, the antibody Ab excises the N-glycan moiety thereof, and the N-glycosylation site in its Fc region is modified with N-acetylglucosamine or fucosyl-α-1,6-N-acetylglucosamine to obtain an antibody; and b) under the catalysis of a glycosidase or a variant thereof, binding the modified antibody obtained in step a) to a linker-payload compound according to any one of claims 1 to 19; and the glycosidase or its variant used in step a) and step b) may be the same or different, an antibody-drug conjugate according to any one of claims 20 to 24.

26. A method for manufacturing an antibody-drug conjugate according to any one of claims 20 to 24, comprising a) under the catalysis of a glycosidase or a variant thereof, the antibody Ab excises the N-glycan moiety thereof, and the N-glycosylation site in its Fc region is modified with N-acetylglucosamine or fucosyl-α-1,6-N-acetylglucosamine to obtain an antibody; and b) under the catalysis of a glycosidase or a variant thereof, binding the modified antibody obtained in step a) to a linker-payload compound according to any one of claims 1 to 19; and the glycosidase or its variant used in step a) and step b) may be the same or different.

27. In steps a) and b), the glycosidase or its variant is a fucose hydrolase, an N-acetylglucosamine endohydrolase, or a variant thereof, the method according to claim 26.

28. The N-acetylglucosamine endohydrolase includes at least one selected from Endo-S (Streptococcus pyogenes endoglycosidase-S), Endo-F3 (Elizabethkingia miricola endoglycosidase-F3), Endo-S2 (Endoglycosidase-S2, Streptococcus pyogenes endoglycosidase-S2), Endo-Sd (Endoglycosidase-Sd, Streptococcus pyogenes endoglycosidase-Sd), and Endo-CC (Endoglycosidase-CC, Streptococcus pyogenes endoglycosidase-CC); 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, the method according to claim 27.

29. A pharmaceutical composition comprising the antibody-drug conjugate according to any one of claims 20 to 24.

30. Use of the antibody-drug conjugate according to any one of claims 20 to 24 for the manufacture of a medicament for the treatment or prevention of a tumor or an autoimmune disease.

31. A method for the treatment or prevention of a tumor or an autoimmune disease, comprising administering to a subject in need thereof a therapeutically effective dose of the antibody-drug conjugate according to any one of claims 20 to 24.

32. The structure is 【Chemical 22】 a compound.

33. A method for producing the compound according to claim 32, comprising 【Chemical 23】 from 【Chemical 24】 a step of producing.

34. 【Chemical 25】 from [Chemical Formula 26] The method according to claim 33, further comprising the step of producing

35. [Chemical Formula 27] from [Chemical Formula 28] The method according to claim 34, further comprising the step of producing

36. [Chemical Formula 29] from [Chemical Formula 30] The method according to claim 35, further comprising the step of producing

37. [Chemical Formula 31] from, by forming a β-(1→4) glycosidic bond, [Chemical Formula 32] The method according to claim 36, further comprising the step of producing

38. The following steps: (i) oxidizing the primary alcohol at the 6-position of the terminal first hexosyl unit in an oligosaccharide containing a terminal first hexosyl unit and a terminal N-acetylglucosamine (GlcNAc) unit to a carboxyl group to obtain an intermediate compound (a) having a carboxyl group, wherein there may be 1, 2, 3, 4, 5, or 6 second hexosyl units or derivative moieties thereof between the terminal first hexosyl unit and the terminal N-acetylglucosamine (GlcNAc) unit; (ii) reacting the carboxyl group in the intermediate compound (a) obtained in step (i) with a reactive group in a linker-terminal - payload compound (b) having a reactive group at the terminal to obtain the linker - payload compound; A linker - payload compound obtained by a method comprising

39. The first hexosyl unit is selected from a glucosyl group, a mannosyl group, a galactosyl group, a fructosyl group, a glucosyl group, and an idosyl group; and / or, The second hexosyl unit is independently selected from a glucosyl group, a mannosyl group, a galactosyl group, or a fructosyl group each time it appears; and / or, Each monosaccharide moiety in the oligosaccharide is linked via a β-(1→4) glycosidic bond; and / or, The derivatives are independently selected from derivatives in which the hydroxyl group of the monosaccharide is replaced with an acylamino group, The linker-payload compound according to claim 38.

40. The first hexosyl unit is selected from a mannosyl group and a glucosyl group; and / or, The second hexosyl unit or its derivative moiety is absent, The linker-payload compound according to claim 38 or 39.

41. The oligosaccharide in the step (i) has the following structure: 【Chemical formula 33】 The linker-payload compound according to claim 38, which has the above structure.

42. The reactive group of the linker-terminal-payload compound (b) having a reactive group at the terminal in the step (ii) is an amino group. The linker-payload compound according to any one of claims 38 to 41.

43. The method further includes a step of converting an intermediate compound (a) having a carboxyl group into an acid halide and further reacting it with a linker-terminal-payload compound (b) having a reactive group at the terminal to obtain the linker-payload compound. The linker-payload compound according to any one of claims 38 to 42.

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