Oligosaccharide linker, linker-load and sugar chain remodeled antibody conjugate drug comprising oligosaccharide linker, methods of making and uses thereof

Abstract

The present invention relates to linker-load compounds comprising an oligosaccharide group, in particular a disaccharide group, wherein the oligosaccharide group is linked to the remainder of the compound by an amide linkage. The present invention also relates to an antibody-conjugated drug (ADC) comprising the linker-load compound, wherein the sugar chain in the antibody is remodeled with the oligosaccharide group in the linker-load compound. The invention also relates to a preparation method and application of the substance.

Description

Oligosaccharide linker, linker-loaded substance comprising oligosaccharide linker and sugar chain remodeled antibody-drug conjugate, preparation method and use thereof Technical Field

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

[0002] Cancer is one of the leading causes of death worldwide, accounting for approximately one in six deaths worldwide each year. In 2017, there were 24.5 million new cancer cases and 9.6 million cancer deaths worldwide. Cancer treatments primarily include surgery, radiotherapy, and drug therapy. The development and application of these therapies have significantly improved the survival of cancer patients. Drug therapy for cancer has evolved through three generations: chemotherapy, targeted therapy, and immunotherapy. While chemotherapy plays a crucial role in cancer treatment, it also carries significant non-therapeutic side effects, killing cancer cells while also injuring a significant number of normal cells. Targeted therapy has mitigated the severe side effects of traditional chemotherapy to some extent. These therapies primarily utilize small molecule targeted drugs (currently primarily tyrosine kinase inhibitors) or monoclonal antibodies to target specific genes or proteins (i.e., targets) involved in tumor cell growth and survival, thereby killing tumor cells and tissues. Antibody-drug conjugates (ADCs) are a new class of targeted drugs, consisting of highly active small molecule drugs chemically linked to monoclonal antibodies. 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, kidneys, nerves and heart to a certain extent, and overcome the limitations of antibody therapy in treating solid tumors. It has become one of the current hot directions in the research and development of anti-tumor drugs.

[0003] The development of ADC drugs involves a systematic engineering process in which four elements, including monoclonal antibodies, bioactive small molecules, linkers, and conjugation methods, are carefully regulated. The conjugation method largely affects drug-related properties such as efficacy stability, metabolic consistency, and quality control (Tsuchikama K, An Z. Antibody-drug conjugates: recent advances in conjugation and linker chemistries. Protein Cell 2018, 9, 33-46). ADC drugs currently on the market and in the clinical stage mainly use random conjugation based on lysine or cysteine ​​residues, which often results in highly heterogeneous mixtures with random conjugation sites and heterogeneous drug-antibody ratios (DAR). This is prone to problems in process stability, quality control, drug stability, metabolic consistency, safety, etc.

[0004] To overcome the aforementioned problems caused by random conjugation, academia and industry have conducted extensive research on the development and application of site-specific conjugation strategies since 2008, achieving some encouraging progress. These site-specific conjugation methods can be roughly divided into three categories: conjugation based on the introduction of specific amino acids through engineered mutations, enzymatic conjugation based on peptide insertion, and site-specific conjugation technologies based on enzymatic sugar chain remodeling. The exploration and application of these site-specific conjugation technologies in ADC drug development have effectively solved many of the problems caused by random conjugation. However, the first two categories of site-specific conjugation technologies typically require engineering or modification of antibodies, lacking the versatility to prepare ADCs for antibodies with different targets. Each new antibody requires a large amount of repetitive and tedious cell engineering, which greatly reduces the efficiency of new drug development. Site-specific conjugation based on antibody Fc sugar chain remodeling, on the other hand, eliminates the need for antibody modification and cell engineering, greatly reducing the difficulty and workload in development, and has the potential to become a universal platform technology for antibody site-specific conjugation.

[0005] There is a highly conserved glycosylation at asparagine 297 in the Fc region of antibodies (N-297 Glycan). By remodeling the sugar chain at this site, different molecules can be linked to the antibody at a specific site (Wang LX, Tong X, Li C. Glycoengineering of Antibcdies for Modulating Functions. Annu Rev Biochem 2019, 88, 433-459). There are two main types of site-specific coupling technologies based on antibody Fc sugar modification:

[0006] 1) Chemical method: sodium periodate is used to oxidize core fucose (Zuberbuhler K, Casi G, Bernardes GJ, et al. Fucose-specific conjugation of hydrazide derivatives to a vascular-targeting monoclonal antibody in IgG format. Chem Commun 2012, 48, 7100-7102) or the vicinal diol of the glycosyl-terminal sialic acid (Zhou Q, Stefano JE, Manning C, et al. Site-specific antibody-drug conjugation through glycoengineering. Bioconjugate Chem 2014, 25, 510-520) to generate the corresponding aldehyde. The aldehyde carbonyl group can be used to conjugate with small molecule toxin fragments to prepare ADCs. However, this method is limited in that the N-297 glycosyl group is structurally diverse and not all monoclonal antibody glycosyl groups contain reactive sites, resulting in significant substrate limitations.

[0007] 2) Enzymatic method, using the deglycosylation-transglycosylation relay catalysis of tool enzymes such as endoglycosidase and glycoside transferase to achieve sugar chain remodeling and introduce bioorthogonal reaction groups, and then through subsequent chemical reactions, achieve site-specific coupling preparation of ADC (Wang LX, Tong X, Li C. Glycoengineering of antibodies for modulating functions. Annu Rev Biochem 2019, 88, 433-459; and Zeng Y, Tang F, Shi W, et al. Recent advances in synthetic glycoengineering for biological applications. Current Opinion in Biotechnol. 2022, 74, 247-255).

[0008] In 2012, Wang Laixi et al. reported a site-specific coupling technology for antibody glycan remodeling catalyzed by the endoglycosidase Endo S and its mutants (Huang W, Giddens J, Fan SQ, et al. Chemoenzymatic glycoengineering of intact IgG antibodies for gain of functions. J. Am. Chem. Soc. 2012, 134, 12308). This technology utilizes the selective endo-deglycosylation of wild-type Endo S on the β-1,4-glycosidic bond between GlcNAc-GlcNAc in the N-297 oligosaccharide structure and the transglycosylation of the mutant Endo S D233Q to synthesize glycan-remodeled antibodies with a single glycoform structure and containing azide modifications. Based on this work, Wang Laixi, Huang Wei and others developed a large class of ADC site-directed conjugation technologies based on the three-step deglycosylation-transglycosylation-click chemistry reaction catalyzed by tool enzymes such as endoglycosidase Endo S or Endo S2 and their mutants (Zeng Y, Tang F, Shi W, et al. Recent advances in synthetic glycoengineering for biological applications. Current Opinion in Biotechnol. 2022, 74, 247-255.). In 2021, Wang Laixi et al. reported a one-pot Endo S2-catalyzed site-specific conjugation of antibody glycans, introducing a bioorthogonal azido functional group onto the antibody, followed by a one-step click chemistry reaction to obtain an ADC molecule (Zhang X, Ou C, Liu H, et al. General and robust chemoenzymatic method for glycan-mediated site-specific labeling and conjugation of antibodies: facile synthesis of homogeneous antibody-drug conjugates. ACS Chem. Biol. 2021, 16, 11, 2502-2514). Currently, the above technologies are all in the basic research stage, and there have been no reports of clinical development of ADC drugs based on the corresponding technologies.

[0009] Another commonly used tool enzyme in the site-specific coupling technology of sugar chain remodeling is β-1,4-galactosyltransferase (β-1,4-Gal-T1) and its mutant (β-1,4-Gal-T1 Y289L). This enzyme uses uridine diphosphate galactose (Gal-UDP) as a donor to transfer galactose Gal to the non-reducing end of acetylglucosamine (Glc-NAc) of glycoproteins. In 2009, Qasba et al. (Boeggeman E, Ramakrishnan B, Pasek M, et al. Site specific coniugation of fluoroprobes to the remodeled Fc N-glycans of monoclonal antibodies using mutant glycosyltransferases: application for cell surface antigen detection. Bioconjugate Chem. 2009, 20, 6, 1228-1236) first reported the antibody site-specific conjugation technology using β-1,4-Gal-T1 and β-1,4-Gal-T1 Y289L as tool enzymes and C2-keto-Gal-UDP or N-azidoacetylgalactosamine-UDP (GalNAz-UDP) as donors to prepare antibodies modified with ketocarbonyl or azido groups. On this basis, in 2014, they reported the first ADC molecule synthesis method based on the three-step deglycosylation-transglycosylation-bioorthogonal reaction catalyzed by β-1,4-Gal-T1 and its mutants. It is worth noting that the corresponding ADC molecule still retains a considerable degree of affinity with FcγRIIIa and FcγRI receptors, and its antibody-dependent cellular cytotoxicity (ADCC) effect is retained to a certain extent. The molecule showed potential lethality in the Her2-positive JIMT-1 breast cancer cell line (Zhu Z, Ramakrishnan B, Li J, et al. Site-specific antibody-drug conjugation through an engineered glycotransferase and a chemically reactive sugar. mAbs 2014, 6, 1190-1200).Based on the above work, Synaffix developed a three-step ADC synthesis method of deglycosylation-transglycosylation-bioorthogonal reaction using a combination of endoglycosidase Endo S and β-1,4-Gal-T1 Y289L tool enzyme (Van Geel R, Wijdeven MA, Heesbeen R, et al. Chemoenzymatic conjugation of toxic payloads to the globally conserved N-glycan of native mAbs provides homogeneous and highly efficacious antibody-drug conjugates. Bioconjugate Chem. 2015, 26, 2233-2242). In addition, a four-step synthesis method for ADCs, which involves deglycosylation, transglycosylation, and bioorthogonal reaction, has been reported in the literature (Li X, Fang T, Boons GJ. Preparation of well-defined antibody-drug conjugates through glycan remodeling and strain-promoted azide-alkyne cycloadditions. Angew. Chem. Int. Ed. 2014, 53, 7179-7182). This method uses three tool enzymes, namely β-1,4-Gal-T1, β-1,4-Gal-T1 Y289L, and sialyltransferase.

[0010] The above-mentioned sugar chain remodeling reaction avoids the problem of other site-specific coupling technologies requiring engineering modifications of antibodies, but it still has very large limitations. On the one hand, its coupling steps are lengthy and cumbersome, requiring at least two enzymatic reactions plus one chemical reaction, that is, at least three reactions and three complete purifications are required to obtain ADC. On the other hand, even trace amounts of residual tool enzymes in the product may cause deglycosylation of the ADC product and the shedding of toxin molecules, resulting in serious toxic reactions, posing major challenges to drug research and development and production, and also posing a major hidden danger to the safety of ADC drugs. More importantly, in the above-mentioned sugar chain remodeling reaction, the connection methods of sugar groups and toxin molecules are still limited, and additional reactions may be required to make the connection method more desirable, and these additional reactions may lead to many adverse byproducts. The present invention aims to solve these problems.

[0011] Summary of the Invention

[0012] The present invention provides a novel oligosaccharide (especially disaccharide) linker, and its preparation method and use. Also provided is a linker-load compound comprising an oligosaccharide group, wherein the oligosaccharide group is connected to the rest of the compound via an amide bond. Specifically, the present invention provides a linker-load compound having formula (I):

[0013] in

[0014] P is the load;

[0015] DC(O)-L- is a linker;

[0016] DC(O)- is an oligosaccharide structure, which is -the first hexose moiety or its derivative part-(the second hexose moiety or its derivative part) f -β-DN-acetylglucosamine moiety or -first hexose moiety or its derivative moiety-(second hexose moiety or its derivative moiety) f -β-D-glucose oxazoline moiety, wherein the 6-carbon of the first hexose group or its derivative is in the form of -C(O)-, which is -C(O)- in DC(O)-, and the β-DN-acetylglucosamine moiety is The β-D-glucose oxazoline part is and f is 0, 1, 2, 3, 4, 5 or 6;

[0017] L is a linker, for example, it can be disconnected from P chemically (for example, hydrolytically) or biologically (for example, enzymatically) to release P, and L is directly connected to the carbonyl group in DC(O)- via -NH- therein, wherein when L is an unbranched linker, it is connected to 1 P, and t is 1, and when L is a branched linker, each branch can be connected to 1 P, and t is an integer greater than 1 (for example, 2, 3, 4, 5, 6, 7, 8, 9 or 10).

[0018] The present invention also relates to an antibody-drug conjugate (ADC) comprising the linker-load compound, wherein the oligosaccharide groups in the linker-load compound are used to reshape the sugar chains in the antibody. Specifically, the present invention provides an antibody-drug conjugate having formula (II) based on site-specific attachment of the N-glycosylation site in the Fc region of the antibody:

[0019] in

[0020] P is the load;

[0021] R is hydrogen or α-L-fucosyl;

[0022] q is 1 or 2;

[0023] Ab is an antibody or antigen-binding fragment (for example, -NHC(O)CH2- in formula (II) is derived from asparagine at position 297 of the antibody Fc region);

[0024] wherein the carbon 6 of the first hexose moiety or its derivative is in the form of -C(O)-;

[0025] L is a linker (for example, it can be chemically (for example, hydrolytically) or biologically (for example, enzymatically) disconnected from P to release P), and L is directly connected to the carbonyl group in the first hexose group or its derivative portion via -NH- therein, wherein when L is an unbranched linker, it is connected to 1 P, and t is 1, and when L is a branched linker, each branch can be connected to 1 P, and t is an integer greater than 1 (for example, 2, 3, 4, 5, 6, 7, 8, 9 or 10).

[0026] f is 0, 1, 2, 3, 4, 5, or 6;

[0027] The present invention also relates to a preparation method and application of the substance.

[0028] The present invention expands the scope of ADCs obtained by sugar chain remodeling technology by adopting a novel oligosaccharide structure that forms an amide bond. In the expected case, compounds with NH2 (e.g., with L'-(P) t Compounds of the structure wherein P and t are as defined herein, and L' is the same as L defined herein, except that -NH- in L connected to DC(O)- is H2N- in L') can form a linker-loaded substance comprising an oligosaccharide linker through a simple amide reaction of the above oligosaccharide structure. Then form ADC with antibody, and The coupling with the antibody can be carried out by one-pot enzyme catalysis. In addition, the ADC provided by the present invention with a novel oligosaccharide structure forming an amide bond can be efficiently delivered to the target cell and efficiently release the cargo in the target cell. In the present invention, the unique oligosaccharide carboxylic acid substrate (e.g., ) can be well connected with amino compounds through a mild and compatible amide bonding method in one step, simplifying and accelerating the synthesis of linker-load compounds without the need for additional reactions that can lead to many undesirable byproducts. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1. SDS-PAGE analysis of ADC-1

[0030] Figure 2. HIC-HPLC analysis of ADC-1

[0031] Figure 3. SEC-HPLC analysis of ADC-1

[0032] Figure 4. Detection of affinity of ADC-1 to ErbB2 / HER2 on cell surface (EC 50 , nM)

[0033] Figure 5. Inhibitory effects of ADC-1 and other drugs on the proliferation of tumor cells BT474 (IC 50 , nM)

[0034] Figure 6. Inhibitory effects of ADC-1 and other drugs on the proliferation of tumor cells NCI-N87 (IC 50 , nM)

[0035] Figure 7. Inhibitory effects of ADC-1 and other drugs on the proliferation of HepG2 tumor cells (IC 50 , nM)

[0036] Figure 8. Inhibitory effect of ADC-1 on the NCI-N87 CDX mouse model

[0037] Figure 9. SDS-PAGE analysis of ADC-2

[0038] Figure 10. HIC spectrum of ADC-2

[0039] Figure 11. SEC spectrum of ADC-2

[0040] Figure 12. Inhibitory effects of ADC-2 and different drugs on the proliferation of tumor cells SK-BR-3 (IC 50 , nM)

[0041] Figure 13. Inhibitory effects of ADC-2 and different drugs on the proliferation of tumor cells HCC1954 (IC 50 , nM)

[0042] Figure 14. Inhibitory effects of ADC-2 and different drugs on the proliferation of tumor cells MDA-MB-468 (IC 50 , nM)

[0043] Figure 15. SEC-HPLC analysis of ADC-3

[0044] Figure 16. High-resolution mass spectrum deconvolution of ADC-3

[0045] Figure 17. Inhibitory effects of ADC-3 and different drugs on the proliferation of tumor cells BxPC-3 (IC 50 , nM)

[0046] Figure 18. Inhibitory effects of ADC-3 and different drugs on the proliferation of FaDu tumor cells (IC 50 , nM)

[0047] Figure 19. Inhibitory effects of ADC-3 and different drugs on the proliferation of HepG2 tumor cells (IC 50 , nM)

[0048] Figure 20. Tumor inhibition effect of ADC-3 on NCI-N87 CDX mice

[0049] Figure 21. Effect of ADC-3 on body weight of NCI-N87 CDX mice

[0050] Figure 22. HIC-HPLC analysis of ADC-4

[0051] Figure 23. SEC-HPLC analysis of ADC-4

[0052] Figure 24. HIC-HPLC analysis of ADC-5

[0053] Figure 25. SEC-HPLC analysis of ADC-5

[0054] Figure 26. In vitro inhibitory activity of ADC-4 and ADC-5 against BxPC-3

[0055] Figure 27. In vitro inhibitory activity of ADC-4 and ADC-5 against FaDu

[0056] Figure 28. In vitro inhibitory activity of ADC-4 and ADC-5 against HepG2

[0057] Figure 29. HIC-HPLC analysis of ADC-6

[0058] Figure 30. SEC-HPLC analysis of ADC-6

[0059] Figure 31. In vitro inhibitory activity of ADC-6 against SK-BR-3

[0060] Figure 32. In vitro inhibitory activity of ADC-6 against NCI-N87 DETAILED DESCRIPTION

[0061] General Definition

[0062] Unless otherwise defined below, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art. The technology used herein refers to the technology generally understood in the art, including variations and equivalent substitutions that are obvious to those skilled in the art. Although it is believed that the following terms are readily understood by those skilled in the art, the following definitions are set forth to better illustrate the present invention. When a trade name appears herein, it refers to the corresponding commercial product or its active ingredient. All patents, published patent applications and publications cited herein are incorporated herein by reference.

[0063] When a certain amount, concentration or other numerical value or parameter is set forth in the form of a range, a preferred range or a preferred upper limit or a preferred lower limit, it should be understood to be equivalent to specifically disclosing any range formed by combining any upper limit or preferred value with any lower limit or preferred value, regardless of whether the range is clearly stated. Unless otherwise stated, the numerical ranges listed herein are intended to include all integers and fractions (decimals) within the endpoints and ranges of the range. For example, the expression "i is an integer from 1 to 20" means that i is any integer from 1 to 20, for example, i can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20. Other similar expressions such as j, g, k, etc. should also be understood in a similar manner.

[0064] Unless the context clearly dictates otherwise, singular forms such as "a", "an", and "the" include plural forms. The expression "one or more" or "at least one" may mean 1, 2, 3, 4, 5, 6, 7, 8, 9 or more.

[0065] The terms "about" and "approximately" when used with a numerical variable generally mean that the value of the variable and all values ​​of the variable are within the range of experimental error (e.g., within a 95% confidence interval about the mean) or within ±10% or more of the stated value.

[0066] The term "optionally" means that the subsequently described event may or may not occur, and that the description includes instances where said event or circumstance occurs or does not occur.

[0067] The expressions "comprising," "including," "containing," and "having" are open ended and do not exclude additional unrecited elements, steps, or ingredients. The expression "consisting of excludes any elements, steps, or ingredients not specified. The expression "consisting essentially of means that the scope is limited to the specified elements, steps, or ingredients, as well as the optional presence of elements, steps, or ingredients that do not materially affect the basic and novel characteristics of the claimed subject matter. It should be understood that the expression "comprising" encompasses the expressions "consisting essentially of" and "consisting of."

[0068] The term "targeting molecule" refers to a molecule that has an affinity for a specific target (e.g., a receptor, a cell surface protein, a cytokine, a tumor-specific antigen, etc.). Targeting molecules can deliver a payload to a specific site in the body through targeted delivery. Targeting molecules can recognize one or more targets. A specific target is defined by the target it recognizes. For example, a receptor-targeting targeting molecule can deliver a cytotoxin to a site containing a large number of receptors. Examples of targeting molecules include, but are not limited to, antibodies, binding proteins for a given antigen, antibody mimics, scaffold proteins with affinity for a given target, ligands, etc. Targets recognized by targeted molecules include but are not limited to CD19, CD22, CD25, CD30 / TNFRSF8, CD33, CD37, CD44v6, CD56, CD70, CD71, CD74, CD79b, CD117 / KIT, CD123, CD138, CD142, CD174, CD227 / MUC1, CD352, CLDN18.2, DLL3, ErbB2 / HER2, CN33, GPNMB, ENPP3, Nectin-4, EGFRv III, SLC44A4 / AGS-5, CEACAM5, PSMA, TIM1, LY6E, LIV1, Nectin4, SLITRK6, HGFR / cMet, SLAMF7 / CS1, EGFR, BCMA, AXL, NaPi2B, GCC, STEAP1, MUC16, Mesothelin, ETBR, EphA2, 5T4, FOLR1, LAMP1, Cadherin 6, FGFR2, FGFR3, CA6, CanAg, Integrin αV, TDGF1, Ephrin A4, TROP2, PTK7, NOTCH3, C4.4A, FLT3, B7H3 / 4, TF (Tissue Factor), and ROR1 / 2.

[0069] HER2 refers to human epidermal growth factor receptor-2, which belongs to the epidermal growth factor (EGFR) receptor tyrosine kinase family. In this application, the terms ErbB2 and HER2 have the same meaning and can be used interchangeably.

[0070] TROP2 is a transmembrane glycoprotein encoded by the Tacstd2 gene. TROP2 is an intracellular calcium sensor and is overexpressed in various tumors.

[0071] CLDN18.2 (Claudin-18 isoform 2) is a member of the human claudin family. CLDN18.2 is a pan-cancer target expressed in primary and metastatic lesions of several human cancer types.

[0072] As used herein, the term "antibody" is used in a broad manner, and its definition covers conventional antibodies, recombinant antibodies / genetically engineered antibodies, particularly complete monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies) and antibody fragments, as long as they have the desired biological activity. The antibody can be of any subtype (e.g., IgG, IgE, IgM, IgD, and IgA) or subclass, and can be derived from any suitable species. In some embodiments, the antibody is human or mouse. The antibody can also be a fully human antibody, a humanized antibody, or a chimeric antibody prepared by a recombinant method.

[0073] As used herein, a monoclonal antibody refers to an antibody obtained from a substantially homogeneous antibody population, i.e., the individual antibodies comprising the population are identical except for a few possible naturally occurring mutations. Monoclonal antibodies are highly specific for a single antigenic site. The term "monoclonal" refers to the characteristic of an antibody as being derived from a substantially homogeneous antibody population and should not be construed as requiring some specific method for producing the antibody.

[0074] Intact antibodies or full-length antibodies essentially comprise an antigen-binding variable region and a light chain constant region (CL) and a heavy chain constant region (CH), which may include CH1, CH2, CH3, and CH4, depending on the subtype of the antibody. The antigen-binding variable region (also referred to as a fragment variable region, an Fv fragment) typically comprises a light chain variable region (VL) and a heavy chain variable region (VH). The constant region may be a constant region with a native sequence (e.g., a constant region with a human native sequence) or an amino acid sequence variant thereof. The variable region recognizes and interacts with the target antigen. The constant region can be recognized by the immune system and interacts with it.

[0075] Antibody fragments may comprise a portion of an intact antibody, preferably its antigen binding region or variable region. Examples of antibody fragments include Fab, Fab′, F(ab′)2, Fd fragments consisting of VH and CH1 domains, Fv fragments, single domain antibody (dAb) fragments, and isolated complementarity determining regions (CDRs). Fab fragments are antibody fragments obtained by digesting a full-length immunoglobulin with papain, or fragments having the same structure produced, for example, by recombinant expression. Fab fragments comprise a light chain (comprising VL and CL) and another chain, wherein the other chain comprises the variable region (VH) of the heavy chain and one constant region (CH1) of the heavy chain. F(ab′)2 fragments are antibody fragments obtained by digesting an immunoglobulin with pepsin at pH 4.0-4.5, or fragments having the same structure produced, for example, by recombinant expression. F(ab′)2 fragments essentially comprise two Fab fragments, wherein each heavy chain portion comprises several additional amino acids, including cysteine ​​that forms a disulfide bond linking the two fragments. Fab' fragment is a fragment comprising half of a F(ab')2 fragment (a heavy chain and a light chain). The antibody fragment may comprise multiple chains linked together, for example, by disulfide bonds and / or by peptide linkers. Examples of antibody fragments also include single-chain Fv (scFv), Fv, dsFv, diabody, Fd and Fd' fragments, as well as other fragments, including modified fragments. Antibody fragments typically comprise at least or about 50 amino acids, typically at least or about 200 amino acids. Antigen-binding fragments may include any antibody fragment that, when inserted into an antibody framework (e.g., by replacing the corresponding region), can obtain an antibody that immunospecifically binds to an antigen.

[0076] Specifically, the antibody-drug conjugates of the present application are site-specifically conjugated to any naturally N-glycosylated site within the Fc region of an antibody. Molecules containing carbohydrate chains within the Fc region of an antibody (including but not limited to antibodies, bispecific antibodies, Fc fusion proteins, single-chain antibodies, nanobodies, etc.) can be prepared in a one-step manner using the oligosaccharide-containing linker payload of the present application. Therefore, the antibodies of the present application are not particularly limited, as long as their Fc region contains carbohydrate chains, and can be native antibodies.

[0077] In addition, the antibodies of the present invention can also be prepared using techniques well known in the art, such as the following techniques or combinations thereof: recombinant technology / genetic engineering technology, phage display technology, synthetic technology, or other techniques known in the art. For example, genetically engineered recombinant antibodies can be expressed in a suitable culture system (e.g., E. coli or mammalian cells). The genetic engineering can refer to, for example, the introduction of a ligase-specific recognition sequence at its terminus.

[0078] As used herein, the term "targeting molecule-drug conjugate" is referred to as a "conjugate." Examples of conjugates include, but are not limited to, antibody-drug conjugates.

[0079] Small molecule compounds refer to molecules of a size comparable to organic molecules commonly used in pharmaceuticals. The term does not encompass biological macromolecules (e.g., proteins, nucleic acids, etc.), but does encompass low molecular weight peptides or derivatives thereof, such as dipeptides, tripeptides, tetrapeptides, pentapeptides, etc. Typically, small molecule compounds have a molecular weight of, for example, about 100 to about 2000 Da, about 200 to about 1000 Da, about 200 to about 900 Da, about 200 to about 800 Da, about 200 to about 700 Da, about 200 to about 600 Da, or about 200 to about 500 Da.

[0080] Cytotoxins refer to substances that inhibit or prevent the expression activity, cell function, and / or cause cell damage in cells. Cytotoxins commonly used in ADCs are more toxic than chemotherapy drugs. Examples of cytotoxins include, but are not limited to, drugs targeting the following targets: microtubule cytoskeleton, DNA, RNA, kinesin-mediated protein transport, and regulation of apoptosis. Drugs targeting the microtubule cytoskeleton can, for example, be microtubule stabilizers or tubulin polymerization inhibitors. Examples of microtubule stabilizers include, but are not limited to, taxanes. Examples of tubulin polymerization inhibitors include, but are not limited to, maytansinoids, auristatins, vinblastines, colchicines, and dolastatins. Drugs targeting DNA can, for example, be drugs that directly damage DNA structure or topoisomerase inhibitors. Examples of drugs that directly damage DNA structure include, but are not limited to, DNA double strand breakers, DNA alkylating agents, and DNA intercalators. DNA double strand breakers can, for example, be enediyne antibiotics, including, but not limited to, daneimycin, esperamicin, neocarzinostatin, and uncialamycin. DNA alkylating agents can be, for example, DNA bis-alkylators (i.e., DNA cross linkers) or DNA mono-alkylators. Examples of DNA alkylating agents include, but are not limited to, pyrrolo[2,1-c][1,4]benzodiazepines. (PBD) dimer, 1-(chloromethyl)-2,3-dihydro-1H-benzo[e]indole (CBI) dimer, CBI-PBD heterodimer, dihydroindole benzodiazepine (IGN) dimers, duocarmycin-like compounds, etc. Examples of topoisomerase inhibitors include, but are not limited to, exatecan and its derivatives (e.g., DX8951f, DXd-(1) and DXd-(2), the structures of which are described below), camptothecins, and anthracyclines. Drugs targeting RNA may, for example, be drugs that inhibit splicing, examples of which include, but are not limited to, pladienolide. Drugs targeting kinesin-mediated protein transport may, for example, be mitotic kinesin inhibitors, including, but not limited to, kinesin spindle (KSP) inhibitors.

[0081] "Spacer" refers to a structure located between different structural modules that can spatially separate the structural modules. The definition of a spacer does not limit whether it has a certain function, nor does it limit whether it can be cut or degraded in vivo. Examples of spacers include, but are not limited to, amino acid and non-amino acid structures, wherein the non-amino acid structure can be, but is not limited to, an amino acid derivative or analog. "Spacer sequence" refers to an amino acid sequence that serves as a spacer, examples of which include, but are not limited to, a single amino acid, a sequence containing multiple amino acids, for example, a sequence containing two amino acids, such as GA, or for example, GGGGS, GGGGSGGGGS, GGGGSGGGGSGGGGS, etc. A self-immolative spacer (e.g., self-immolative spacer Sp1) is a covalent component that causes two chemical bonds to be cleaved sequentially after activation of a protective portion in the precursor: the protective portion (e.g., the cleavable sequence) is removed after activation, triggering a cascade of decomposition reactions, resulting in the release of smaller molecules in a sequential order. Examples of self-immolative spacers include, but are not limited to, PABC (p-aminobenzyloxycarbonyl), acetals, heteroacetals, and combinations thereof.

[0082] As used herein, the term "amino acid" includes "natural amino acids" and "unnatural amino acids."

[0083] The term "natural amino acids" refers to amino acids, which are protein-building amino acids, including the common twenty amino acids (alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine), as well as the less common selenocysteine ​​and pyrrolysine.

[0084] As used herein, the term "unnatural amino acid" refers to an amino acid that is not a protein-forming amino acid. Specifically, the term refers to an amino acid that is not a natural amino acid as defined above.

[0085] The term "alkyl" refers to a straight or branched chain saturated aliphatic hydrocarbon group consisting of carbon atoms and hydrogen atoms, which is connected to the rest of the molecule by a single bond. Alkyl groups can have 1 to 20 carbon atoms and refer to "C1-C 20 "alkyl", such as C1-C4 alkyl, C1-C3 alkyl, C1-C2 alkyl, C3 alkyl, C4 alkyl, C3-C6 alkyl. Non-limiting examples of alkyl include, but are not limited to, methyl, ethyl, propyl, butyl, pentyl, hexyl, isopropyl, isobutyl, sec-butyl, tert-butyl, isopentyl, 2-methylbutyl, 1-methylbutyl, 1-ethylpropyl, 1,2-dimethylpropyl, neopentyl, 1,1-dimethylpropyl, 4-methylpentyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 2-ethylbutyl, 1-ethylbutyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1 -dimethylbutyl, 2,3-dimethylbutyl, 1,3-dimethylbutyl or 1,2-dimethylbutyl, or isomers thereof. A divalent free radical refers to a group obtained by removing a hydrogen atom from a carbon atom with free valence electrons of a corresponding monovalent free radical. A divalent free radical has two attachment sites connected to the rest of the molecule. For example, "alkylene" or "alkylene group" refers to a saturated straight or branched divalent hydrocarbon radical. Examples of "alkylene" include, but are not limited to, methylene (-CH2-), ethylene (-C2H4-), propylene (-C3H6-), butylene (-C4H8-), pentylene (-C5H8-), thiazolinyl ... 10 -), hexamethylene (-C6H 12 -), 1-methylethylene (-CH(CH3)CH2-), 2-methylethylene (-CH2CH(CH3)-), methylpropylene or ethylpropylene, etc.

[0086] The term "cycloalkyl" refers to a cyclic saturated aliphatic group consisting of carbon atoms and hydrogen atoms, which is connected to the rest of the molecule by a single bond. A cycloalkyl group can have 3 to 10 carbon atoms, i.e., "C3-C 10 "Cycloalkyl" refers to a divalent cycloalkyl radical, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl or cyclodecyl. "Cycloalkylene" refers to a divalent cycloalkyl radical.

[0087] The term "heterocyclyl" refers to a cycloalkyl group in which one or more carbon atoms are replaced by a heteroatom selected from nitrogen, oxygen, and sulfur, such as azepine, oxa-, or thiirane, azepine, oxa-, or thietanyl, pyrrolidinyl, pyrazolidinyl, imidazolidinyl, tetrahydrofuranyl, tetrahydrothiophenyl, piperidinyl, piperazinyl, tetrahydropyranyl, or tetrahydrothiopyranyl. "Heterocyclylene" refers to a divalent cycloalkyl group.

[0088] When "substituted" is mentioned herein, unless otherwise indicated, the relevant substituent is selected from alkyl, halogen, amino, monoalkylamino, dialkylamino, nitro, cyano, formyl, alkylcarbonyl, carboxyl, alkyloxycarbonyl, alkylcarbonyloxy, aminocarbonyl, monoalkylaminocarbonyl, dialkylaminocarbonyl, formylamino, alkylcarbonylamino, formyl(monoalkyl)amino or alkylcarbonyl(monoalkyl)amino.

[0089] As used herein, when a group is combined with another group, the connection between the groups can be linear or branched, provided that a chemically stable structure is formed. The structure formed by such a combination can be connected to other parts of the molecule through any suitable atom in the structure, preferably through a specified chemical bond. For example, when two or more groups selected from -CR 1 R 2 、C 1-10 Alkylene, C 4-10 Cycloalkylene, C 4-10 The heterocyclic group and the divalent group of -(CO)- are combined to form a combination, and two or more divalent groups can form a linear connection with each other, such as -CR 1 R 2 -C 1-10 Alkylene-(CO)-, -CR 1 R 2 -C 4-10 Cycloalkylene-(CO)-, -CR 1 R 2 -C 4-10 Cycloalkylene-C 1-10 Alkylene-(CO)-, -CR 1 R 2 -CR 1 R 2’ -(CO)-、-CR 1 R 2 -CR 1′ R 2′ -CR 1″ R 2″ -(CO)-, etc. The resulting bivalent structure can be further linked to other parts of the molecule.

[0090] When multiple identical letters representing chemical groups appear in the same chemical formula, they are independently selected and not necessarily the same. For example, multiple M in Formula I-2 are independently selected from LKa-L 2 -L 1 -BP; and multiple L 2 They are also independent of each other and not necessarily the same.

[0091] As used herein, the expressions "antibody-drug conjugate" and "antibody-drug conjugate" have the same meaning.

[0092] Linker-load compound

[0093] In a first aspect, the present invention provides a linker-load compound having formula (I):

[0094] in

[0095] P is the load;

[0096] DC(O)-L- is a linker;

[0097] DC(O)- is an oligosaccharide structure, which is -the first hexose moiety or its derivative part-(the second hexose moiety or its derivative part) f -β-DN-acetylglucosamine moiety or -first hexose moiety or its derivative moiety-(second hexose moiety or its derivative moiety) f -β-D-glucose oxazoline moiety, wherein the 6-carbon of the first hexose group or its derivative is in the form of -C(O)-, which is -C(O)- in DC(O)-, and the β-DN-acetylglucosamine moiety is The β-D-glucose oxazoline part is and f is 0, 1, 2, 3, 4, 5, or 6;

[0098] L is a linker (for example, it can be disconnected from P chemically (for example, hydrolytically) or biologically (for example, enzymatically) to release P), and L is directly connected to the carbonyl group in DC(O)- via -NH- therein, wherein when L is an unbranched linker, it is connected to 1 P, and t is 1, and when L is a branched linker, each branch can be connected to 1 P, and t is an integer greater than 1 (for example, 2, 3, 4, 5, 6, 7, 8, 9 or 10).

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

[0100] in

[0101] B is independently absent, or is 1) below, or is 2) below, or is a combination of 1) and 2) below: 1) a self-immolative spacer Sp1; 2) a divalent group, or a combination of two or more divalent groups, wherein the divalent group is selected from: -CR 1 R 2 -、C 1-10Alkylene, C 4-10 Cycloalkylene, C 4-10 heterocyclylene and -(CO)-;

[0102] L 1 Independently absent; or a non-cleavable sequence, for example, coupling the load to the antibody via a thioether bond; or a cleavable sequence comprising an amino acid sequence cleavable by an enzyme, the amino acid sequence cleavable by the enzyme comprising 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids;

[0103] L 2 Independently absent; or the following 1); or the following 2); or the following 1) and 2) combination:

[0104] 1)-NH-C 2-20 Alkylene, wherein one or more -CH2- structures in the alkylene are optionally replaced by: -CR 3 R 4 , -O-, -(CO)-, -S-, -S(=O)2-, -NR 5 -、 C 4-10 Cycloalkylene, C 4-10 Heterocyclylene, phenylene, wherein cycloalkylene, heterocyclylene and phenylene are each independently unsubstituted or selected from halogen, -C 1-10 Alkyl, -C 1-10 Haloalkyl, -C 1-10 Alkylene-NH-R 8 and -C 1-10 Alkylene-OR 9 is substituted with at least one substituent;

[0105] 2) Amino acid residue sequence, i.e. -*(AA) n **-, n is an integer from 1 to 100, AA is independently an amino acid residue at each occurrence, * represents the N-terminus of the corresponding amino acid, ** represents the C-terminus of the corresponding amino acid, and -(C2H4-O) is optionally present between the amino group and the α-carbon of an amino acid m -(CH2) p -, wherein m is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; p is 0, 1, 2 or 3, and the * end forms an amide bond with the carbonyl group in the oligosaccharide structure;

[0106] R 1 、R 2 、R 3 、R 4 、R 5 、R 6 、R 7 、R 8 、R9 are each independently selected from hydrogen, halogen, substituted or unsubstituted -C 1-10 Alkyl, C 4-10 Cycloalkylene; or R 1 and R 2 and together with the carbon atoms to which they are attached form a 3-6 membered cycloalkylene group, and / or R 3 and R 4 and together with the carbon atoms to which they are attached form a 3-6 membered cycloalkylene group;

[0107] P is with B part, or L 1 Part, or L 2 Partially connected load.

[0108] In another embodiment, --L--(P) t for That is, formula (I) is:

[0109] in,

[0110] Ld2 and each Ld1 are independently a bond; or selected from -NH-C 1-20 Alkylene-(CO)-, -NH-(PEG) i -(CO)-; or each independently unsubstituted or -CO-(PEG) on the side chain j -R 11 Substituted natural amino acids or oligomeric natural amino acids having a degree of polymerization of 2-10 (i.e., 2, 3, 4, 5, 6, 7, 8, 9 or 10);

[0111] -(PEG) i -and-(PEG) j - Each is a PEG fragment comprising a specified number of consecutive -(O-C2H4)- structural units or consecutive -(C2H4-O)- structural units, optionally with C appended at one end 1-10 alkylene;

[0112] M is hydrogen or LKa-L 2 -L 1 -BP;

[0113] Q is NH2 or L 2 -L 1 -BP;

[0114] Provided that the following is not included: M is hydrogen and at the same time Q is NH2;

[0115] Each LKa is independently selected

[0116] opSu is or a mixture thereof; wherein * represents and L 2 The connecting part;

[0117] Each L 2 Independently absent; or the following 1); or the following 2); or the following 1) and 2) combination:

[0118] 1)-NH-C 2-20 Alkylene, wherein one or more -CH2- structures in the alkylene are optionally replaced by: -CR 3 R 4 -, -O-, -(CO)-, -S-, -S(=O)2-, -NR 5 -、 C 4-10 Cycloalkylene, C 4-10 Heterocyclylene, phenylene, wherein cycloalkylene, heterocyclylene and phenylene are each independently unsubstituted or selected from halogen, -C 1-10 Alkyl, -C 1-10 Haloalkyl, -C 1-10 Alkylene-NH-R 8 and -C 1-10 Alkylene-OR 9 substituted with at least one substituent; preferably, -NH-(C2H4O)2-(C2H4)CO-;

[0119] 2) Amino acid residue sequence, i.e. -*(AA) n **-, n is an integer from 1 to 100, AA is independently an amino acid residue at each occurrence, * represents the N-terminus of the corresponding amino acid, ** represents the C-terminus of the corresponding amino acid, and -(C2H4-O) is optionally present between the amino group and the α-carbon of an amino acid m -(CH2) p -, wherein m is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; p is 0, 1, 2 or 3, and the * end forms an amide bond with the carbonyl group in the oligosaccharide structure; preferably, it is -Gly-Gly-Gly-;

[0120] L 1 Independently absent; or a non-cleavable sequence, for example, coupling the load to the antibody via a thioether bond; or a cleavable sequence comprising an amino acid sequence cleavable by an enzyme, the amino acid sequence cleavable by the enzyme comprising 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids;

[0121] Each B is independently absent, or is 1) below, or is 2) below, or is a combination of 1) and 2) below: 1) a self-immolative spacer Sp1; 2) a divalent group, or a combination of two or more divalent groups, wherein the divalent group is selected from: -CR1 R 2 -、C 1-10 Alkylene, C 4-10 Cycloalkylene, C 4-10 heterocyclylene and -(CO)-;

[0122] P is with B part, or L 1 Part, or L 2 partially connected loads;

[0123] R 1 、R 2 、R 3 、R 4 、R 5 、R 6 、R 7 、R 8 、R 9 are each independently selected from hydrogen, halogen, substituted or unsubstituted -C 1-10 Alkyl, C 4-10 Cycloalkylene; or R 1 and R 2 and together with the carbon atoms to which they are attached form a 3-6 membered cycloalkylene group, and / or R 3 and R 4 and together with the carbon atoms to which they are attached form a 3-6 membered cycloalkylene group;

[0124] R 11 It is C 1-10 alkyl;

[0125] d is 0, 1, 2, 3, 4, 5, or 6;

[0126] Each i is independently an integer from 1 to 100 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10), preferably 1 to 20; preferably, each i is independently an integer from 1 to 12; more preferably, 2 to 8; especially 4;

[0127] Each j is independently an integer from 1 to 100 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10), preferably 1 to 20; preferably, each j is independently an integer from 1 to 12; more preferably, from 8 to 12; especially, 8, 9, 12, or 13.

[0128] In one embodiment, B, L 1 and L 2 At least one of them is not "not present".

[0129] In one embodiment, L 2 Selected from: -NH-(CH2) a -(CH2)2(CO)-, a is an integer of 0, 1, 2, 3, 4 or 5; b is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In one embodiment, L 2 The carbonyl group and L in the structure 1 In one embodiment, a is 0, 1, 2 or 3, preferably 3.

[0130] In one embodiment, L 2 is the amino acid residue sequence, i.e. -*(AA) n **-, n is an integer from 1 to 100, AA is independently an amino acid residue at each occurrence, * represents the N-terminus of the corresponding amino acid, ** represents the C-terminus of the corresponding amino acid, and -(C2H4-O) is optionally present between the amino group and the α-carbon of an amino acid m -(CH2) p -, wherein m is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; p is 0, 1, 2 or 3, and the * terminus forms an amide bond with a carbonyl group in the oligosaccharide structure. In one embodiment, AA is independently any one of Phe, Lys, Gly, Ala, Leu, Asn, Val, Ile, Pro, Trp, Ser, Tyr, Cys, Met, Asp, Gln, Glu, Thr, Arg, His, or any combination thereof. 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.

[0131] In one embodiment, L 1 A cleavable sequence comprising an amino acid sequence cleavable by an enzyme, wherein the amino acid sequence cleavable by the enzyme comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids. In one embodiment, the amino acid sequence cleavable by the enzyme is selected from -Gly-Gly-Phe-Gly-, -Phe-Lys-, -Val-Cit-, -Val-Lys-, -Gly-Phe-Leu-Gly-, -Ala-Leu-Ala-Leu-, -Ala-Ala-Ala- and combinations thereof; a preferred amino acid sequence cleavable by the enzyme is -Gly-Gly-Phe-Gly-. In one embodiment, L 1is 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 It means -Val-Cit-.

[0132] In one embodiment, Sp1 is selected from PABC (p-aminobenzyloxycarbonyl), acetal, heteroacetal and a combination thereof; preferably, Sp1 is acetal, heteroacetal or PABC; further preferably, the heteroacetal is selected from N,O-heteroacetal; more preferably, Sp1 is -O-CH2-U- or -NH-CH2-U-, wherein -O- or -NH- is linked to an amino acid sequence that can be cleaved by the enzyme, U does not exist, or is CH2, O, S or NH, preferably O or S.

[0133] In one embodiment, B is absent, or is -NH-CH2-U-, or is -NH-CH2-U-(CH2) g -(CO)-, wherein g is 1, 2, 3, 4, 5 or 6, U is absent or is CH2, O, S or NH, preferably O or S. In one embodiment, B is absent. In one embodiment, B is 1), 2), or a combination of 1) and 2): 1) a self-immolative spacer Sp1; 2) a divalent group, or a combination of two or more divalent groups, wherein the divalent group is selected from: -CR 1 R 2 -、C 1-10 In one embodiment, B is -NH-CH2-U- or -NH-CH2-U-(CH2) g -(CO)-, U is absent or is CH2, O, S or NH, preferably O or S. In one embodiment, B is connected to the load via an amide bond, an ester bond or an ether bond. In one embodiment, B is selected from: (-PABC-), -NH-CH2-U-, or -NH-CH2-U-(CH2) g -(CO)-; wherein g is 1, 2, 3, 4, 5 or 6; U is absent or is CH2, O, S or NH, preferably O or S.

[0134] In one embodiment, -L 1 -B- represents -Val-Cit-PABC- or -Gly-Gly-Phe-Gly-.

[0135] 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-.

[0136] In one embodiment, Ld2 and each Ld1 are independently selected from a bond, or

[0137] Each i, j and k is independently selected from an integer from 1-100.

[0138] In one embodiment, each i, j, and k is independently selected from an integer from 1 to 20. In one embodiment, each i, j, and k is independently selected from an integer from 1 to 12.

[0139] In one embodiment, each i is independently selected from an integer from 2 to 8; particularly 4.

[0140] In one embodiment, each j is independently selected from an integer from 8 to 12; particularly 8 or 12.

[0141] In one embodiment, each k is independently selected from an integer of 1-7; in particular 1 or 3 or 5.

[0142] In one embodiment, Ld2 and each Ld1 are independently selected from a bond; or a C with an amino group and a carbonyl group at both ends. 1-20 Alkylene, or a PEG fragment of a certain length with an amino group and a carbonyl group at both ends (represented as -(PEG) i -), or one or more natural amino acids, each of which is independently unsubstituted or substituted with a PEG fragment of a certain length (represented as -CO-(PEG) j -)replace.

[0143] In one embodiment, -(PEG) i -Contains -(O-C2H4) i -or-(C2H4-O) i -, and optionally appended with C at one end 1-10 Alkylene; -(PEG) j -Contains -(O-C2H4) j -or-(C2H4-O) j -, and optionally appended with C at one end 1-10 In a very specific embodiment, -(PEG) i - contains -C2H4-(O-C2H4) i-or-(C2H4-O) i -C2H4-.

[0144] In one embodiment, the load can be selected from the group consisting of small molecule compounds (e.g., small molecule drugs with various mechanisms of action, including various traditional small molecule drugs, photoacoustic dynamic therapy drugs, photothermal therapy drugs, etc., such as chemotherapy drugs, small molecule targeted drugs, immune agonists, etc., such as traditional cytotoxic drugs, such as cisplatin, paclitaxel, 5-fluorouracil, cyclophosphamide and bendamustine, etc.; small molecule targeted drugs, such as imatinib mesylate, gefitinib and anlotinib, etc.; immune agonists such as STING agonists, TLR agonists, etc.), nucleic acids and nucleic acid analogs, tracer molecules (including fluorescent molecules, biotin, fluorophores, chromophores, spin resonance probes and radioactive labels, etc.), short peptides, polypeptides, peptidomimetics and proteins. In one embodiment, the load is selected from the group consisting of small molecule compounds and nucleic acid molecules. In a preferred embodiment, the load is selected from small molecule compounds. In a more preferred embodiment, the load is selected from the group consisting of cytotoxins and fragments thereof.

[0145] In one embodiment, the cargo is a cytotoxin or a fragment thereof, optionally derivatized to attach 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 part.

[0146] In one embodiment, the cytotoxin is selected from the group consisting of drugs that target the microtubule cytoskeleton. In a preferred embodiment, the cytotoxin is selected from the group consisting of taxanes, maytansinoids, auristatins, epothilones, combretastatin A-4 phosphate, combretastatin A-4 and its derivatives, indole-sulfonamides, vinca alkaloids such as vinblastine, vincristine, vindesine, vinorelbine, vinflunine, vinglycinate, anhy-droyinblastine, dolastatin 10 and its analogs, halichondrin B, indole-3-oxyacetamides, podophyllotoxins, 7-diethylamino-3-(2′-benzoxazolyl)-coumarin (DBC), discodermolide, and laulimalide. In another embodiment, the cytotoxin is selected from the group consisting of DNA topoisomerase inhibitors such as camptothecins and their derivatives, mitoxantrone, and mitoguanidine. In a preferred embodiment, the cytotoxin is selected from the group consisting of nitrogen mustards such as chlorambucil, naphthyl nitrogen mustard, cholophosphamide, estramustine, ifosfamide, nitrogen mustard, nitrogen oxide hydrochloride, melphalan, new nitrogen mustard, methionine, phenylephrine, prednimustine, trofosfamide, and uramustine. In another preferred embodiment, the cytotoxin is selected from the group consisting of nitrosoureas such as carmustine, flubenzuron, formoterol, lomustine, nimustine, and ranimustine. In one embodiment, the cytotoxin is selected from the group consisting of aziridines. In a preferred embodiment, the cytotoxin is selected from the group consisting of benzodopa, carboquinone, metodepa and uredepa. In one embodiment, the cytotoxin is selected from the group consisting of antitumor antibiotics. In a preferred embodiment, the cytotoxin is selected from the group consisting of enediyne antibiotics. In a more preferred embodiment, the cytotoxin is selected from the group consisting of dynemicin, esperamicin, neocarzinostatin, aclarubicin.In another preferred embodiment, the cytotoxin is selected from the group consisting of actinomycin, anthramycin, bleomycins, actinomycin C, carabinin, carminomycin, carminomycin, actinomycin D, daunorubicin, detorubicin, doxorubicin, epirubicin, esorubicin, idarubicin, mexicomycin, mitomycins, noramycin, olivomycin, peplomycin, porfibrinocin, puromycin, ferroxorubicin, rhodorubicin, streptozocin, streptozocin, zoloft, and daunorubicin. In yet another preferred embodiment, the cytotoxin is selected from the group consisting of trichothecenes. In a more preferred embodiment, the cytotoxin is selected from the group consisting of T-2 toxin, verracurin A, baculozolin A, and anguidine. In one embodiment, the cytotoxin is selected from the group consisting of antitumor amino acid derivatives. In a preferred embodiment, the cytotoxin is selected from the group consisting of ubenimex, azaserine, and 6-diazo-5-oxo-L-norleucine. In another embodiment, the cytotoxin is selected from the group consisting of folic acid analogs. In a preferred embodiment, the cytotoxin is selected from the group consisting of folate, methotrexate, pteropterin, trimetrexate, and edatrexate. In one embodiment, the cytotoxin is selected from the group consisting of purine analogs. In a preferred embodiment, the cytotoxin is selected from the group consisting of fludarabine, 6-mercaptopurine, thiabendine, and thioguanine. In yet another embodiment, the cytotoxin is selected from the group consisting of pyrimidine analogs. In a preferred embodiment, the cytotoxin is selected from the group consisting of ancitabine, gemcitabine, enocitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, and floxuridine. In one embodiment, the cytotoxin is selected from the group consisting of androgens. In a preferred embodiment, the cytotoxin is selected from the group consisting of captestosterone, drostanolone propionate, cyclothiocarbamate, melastosane, and testolactone. In another embodiment, the cytotoxin is selected from the group consisting of anti-adrenal drugs. In a preferred embodiment, the cytotoxin is selected from the group consisting of aminoglutethimide, mitotane, and trilostane. In one embodiment, the cytotoxin is selected from the group consisting of antiandrogens. In a preferred embodiment, the cytotoxin is selected from the group consisting of flutamide, nilutamide, bicalutamide, leuprolide acetate, and goserelin. In yet another embodiment, the cytotoxin is selected from the group consisting of protein kinase inhibitors and proteasome inhibitors.In another embodiment, the cytotoxin is selected from the group consisting of vinca alkaloids, colchicines, taxanes, auristatins, maytansinoids, calicheamicin, doxonubicin, duocarmucin, SN-38, cryptophycin analogs, deruxtecan, duocarmazine, calicheamicin, centanamycin, dolastansine, pyrrolobenzodiazepine (PBD), and exatecan. In one embodiment, the cytotoxin is selected from the group consisting of vinca alkaloids, colchicines, taxanes, auristatins, and maytansinoids.

[0147] In one embodiment, the cytotoxin is exatecan or a derivative thereof, such as DX8951f and the like.

[0148] In one embodiment, the cytotoxin is a maytansinoid, such as DM1. It should be noted that when a cytotoxin comprising a sulfhydryl moiety is used, the sulfhydryl moiety can react with a maleimide moiety to form a sulfosuccinimide, such as a maytansinoid, such as DM1, and the cytotoxin can be directly linked via the sulfosuccinimide. In this case, it will be understood that in some embodiments, the cargo and the sulfhydryl moiety together constitute the cytotoxin, and thus in this case, the cargo represents the remainder of the cytotoxin molecule excluding the sulfhydryl moiety.

[0149] In one embodiment, the cytotoxin is an auristatin, such as MMAE (monomethyl auristatin E), MMAF (monomethyl auristatin F), MMAD (monomethyl auristatin D), etc. The synthesis and structure of auristatin compounds are described in US20060229253, the entire disclosure of which is incorporated herein by reference.

[0150] The carrier contains an active group that can react with the active group in the compound of formula (I) and thus covalently conjugate the carrier to the compound of formula (I). Compounds without active groups require appropriate derivatization to obtain the carrier.

[0151] In one embodiment, the cytotoxin is a compound of formula (i)

[0152] Where g is any integer from 1 to 6;

[0153] In one embodiment, g is any integer from 1 to 3, preferably 1.

[0154] In one embodiment, the cytotoxin is selected from the following compounds 1-15; wherein the wavy bond represents the site of attachment to the compound of formula (I).

[0155] In some embodiments, the loading species is selected from DX8951f (Compound 9), DXd-(1) (Compound 10) and DXd-(2) (Compound 14), Compound 15, preferably DX8951f or DXd-(1), more preferably DXd-(1).

[0156] In one embodiment, the first hexosyl moiety or its derivative is selected from glucosyl, mannosyl, galactosyl, fructosyl, gulosyl, idosyl or derivatives thereof, and the carbon at position 6 thereof is in the form of -C(O)-.

[0157] In one embodiment, the second hexaglycosyl or derivative thereof moiety is independently selected at each occurrence from glucosyl, mannosyl, galactosyl, fructosyl or derivatives thereof.

[0158] In one embodiment, the monosaccharide moieties in the oligosaccharide structure are linked by β-(1→4) glycosidic bonds.

[0159] In one embodiment, the derivatives are independently selected from derivatives in which the hydroxyl group of uronic acid or monosaccharide is replaced by an acylamino group (eg, an alkanoylamino group, such as formylamino, acetylamino, propionylamino, etc., especially acetylamino).

[0160] In one embodiment, the first hexose moiety or its derivative is selected from Preferably, selected from

[0161] In one embodiment, f is 0.

[0162] In one embodiment, DC(O)- is a disaccharide structure

[0163] In one embodiment, DC(O)- is a disaccharide structure

[0164] In one embodiment, the linker-load compound having formula (I) is:

[0165] For example,

[0166] In another aspect, the present invention provides a linker-load compound obtained by a method comprising the steps of:

[0167] (i) oxidizing the 6-primary alcohol of the terminal first hexose unit in an oligosaccharide containing a terminal first hexose unit and a terminal N-acetylglucosamine (GlcNAc) unit to a carboxyl group to obtain an intermediate compound (a) having a carboxyl group, wherein 1, 2, 3, 4, 5 or 6 second hexose units or derivatives thereof are optionally present between the terminal first hexose unit and the terminal N-acetylglucosamine (GlcNAc) unit;

[0168] (ii) reacting the carboxyl group in the intermediate compound (a) obtained in step (i) with the reactive group in the linker-carrier compound (b) having a reactive group at the end to obtain the linker-carrier compound.

[0169] In one embodiment of this other aspect,

[0170] The first hexose unit is selected from glucosyl, mannosyl, galactosyl or fructosyl; and / or

[0171] The second six-carbon glycosyl unit is independently selected at each occurrence from glucosyl, mannosyl, galactosyl or fructosyl; and / or

[0172] The monosaccharide moieties in the oligosaccharide are linked by β-(1→4) glycosidic bonds; and / or

[0173] The derivatives are independently selected from derivatives in which the hydroxyl groups of the monosaccharide are replaced by acylamino groups (eg alkanoylamino groups, such as formylamino, acetylamino, propionylamino, etc., especially acetylamino groups).

[0174] In one embodiment of this other aspect,

[0175] The first hexose unit is mannosyl or glucosyl; and / or

[0176] The second hexose syl unit or its derivative part is not present.

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

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

[0179] In one embodiment of this other aspect, the reactive group of the linker-carrier compound (b) with a reactive group at the end in step (ii) is an amino group.

[0180] In one embodiment of this other aspect, the method may further include the step of converting the intermediate compound (a) having a carboxyl group into an acyl halide, and further reacting it with a linker-carrier compound (b) having a reactive group at the end to obtain the linker-carrier compound.

[0181] In one embodiment of this further aspect, the linker-load compound is a linker-load compound as defined in the first aspect or various embodiments thereof.

[0182] Preparation method of linker-load compound

[0183] In a second aspect, the present invention provides methods of preparing linker-load compounds having formula (I), wherein unless otherwise indicated, the various variables are as defined in the first aspect or various embodiments thereof.

[0184] In one embodiment, the method comprises reacting DC(O)-OH with L'-(P) t An amide formation reaction is performed to form The step of preparing the method of claim 1, wherein L' is the same as L defined in the first aspect or various embodiments thereof, except that the -NH- in L connected to DC(O)- is H2N- in L'.

[0185] In one embodiment, when DC(O)- in the prepared linker-load compound of formula (I) is When the method can further include Condensation In one embodiment, the condensation is carried out in the presence of water, a base and 2-chloro-1,3-dimethylimidazolinium chloride (DMC, cas: 37091-73-9). In one embodiment, the base is an inorganic base or an organic base. In one embodiment, the inorganic base is potassium carbonate, potassium phosphate, etc. In one embodiment, the organic base is an amine, such as a tertiary amine, such as triethylamine (Et3N).

[0186] In one embodiment, the amide formation reaction is carried out in the presence of an organic solvent, an organic base and a condensation reagent. In one embodiment, the organic solvent is selected from N,N-dimethylformamide (DMF) and N,N-dimethylacetamide (DMA). In one embodiment, the organic base is selected from diisopropylethylamine (DIPEA) and N-methylmorpholine (NMM). In one embodiment, the condensation reagent is selected from HATU, HBTU, TBTU and PyBOP.

[0187] In one embodiment, DC(O)-OH is

[0188] In one embodiment, Depend on Preparation. In one embodiment, the preparation is carried out under conditions where the substrate is debenzylated. In one embodiment, the deprotection is carried out in the presence of hydrogen and a palladium catalyst. In one embodiment, the palladium catalyst is selected from palladium carbon and palladium hydroxide on carbon.

[0189] In one embodiment, Depend on Preparation. In one embodiment, the preparation is carried out in the presence of an oxidant and an optional oxidation catalyst. In one embodiment, the oxidant is iodophenyl diacetic acid. In one embodiment, the oxidation catalyst is 2,2,6,6-tetramethyl-1-piperidinyl oxide.

[0190] In one embodiment, Depend on Preparation. In one embodiment, the preparation is carried out in the presence of an acid. In one embodiment, the acid is p-toluenesulfonic acid.

[0191] In one embodiment, Depend on Preparation. In one embodiment, the preparation is carried out in the presence of thioacetic acid (AcSH) and an organic solvent. In one embodiment, the organic solvent is selected from chloroform and pyridine or a mixture thereof.

[0192] In one embodiment, Prepared by forming a β-(1→4) glycosidic bond. In one embodiment, the preparation is carried out under glycosidic bond forming conditions. In one embodiment, the glycosidic bond forming conditions include using trifluoromethanesulfonic anhydride in an anhydrous environment.

[0193] The present invention also provides the following compounds:

[0194] Antibody-drug conjugates

[0195] In a third aspect, the present invention provides an antibody-drug conjugate having formula (II) based on site-directed attachment of the N-glycosylation site in the Fc region of an antibody:

[0196] in

[0197] P is the load;

[0198] R is hydrogen or α-L-fucosyl;

[0199] q is 1 or 2;

[0200] Ab is an antibody or antigen-binding fragment (for example, -NHC(O)CH2- in formula (II) is derived from asparagine at position 297 of the antibody Fc region);

[0201] wherein the carbon 6 of the first hexose moiety or its derivative is in the form of -C(O)-;

[0202] L is a linker (for example, it can be chemically (for example, hydrolytically) or biologically (for example, enzymatically) disconnected from P to release P), and L is directly connected to the carbonyl group in the first hexose group or its derivative portion via -NH- therein, wherein when L is an unbranched linker, it is connected to 1 P, and t is 1, and when L is a branched linker, each branch can be connected to 1 P, and t is an integer greater than 1 (for example, 2, 3, 4, 5, 6, 7, 8, 9 or 10).

[0203] f is 0, 1, 2, 3, 4, 5, or 6;

[0204] In one embodiment, the first hexosyl moiety or its derivative is selected from glucosyl, mannosyl, galactosyl, fructosyl, gulosyl, idosyl or derivatives thereof, and the carbon at position 6 thereof is in the form of -C(O)-.

[0205] In one embodiment, the second hexaglycosyl or derivative thereof moiety is independently selected at each occurrence from glucosyl, mannosyl, galactosyl, fructosyl or derivatives thereof.

[0206] In one embodiment, the individual monosaccharide moieties are linked by β-(1→4) glycosidic bonds.

[0207] In one embodiment, the derivatives are independently selected from derivatives in which the hydroxyl group of uronic acid or monosaccharide is replaced by an acylamino group (eg, an alkanoylamino group, such as formylamino, acetylamino, propionylamino, etc., especially acetylamino).

[0208] In one embodiment, the first hexose moiety or its derivative is selected from

[0209] In one embodiment, f is 0.

[0210] In one embodiment, -NHC(O)CH2- in formula (II) is derived from asparagine at position 297 of the Fc region of an antibody.

[0211] In one embodiment, the targets recognized by Ab 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, EGFRvIII, SLC44A4 / AGS-5, CEACAM5, PSMA, TIM1, LY6E, LIV1, Nectin4, SLITRK6, HGFR / cMet, SLAMF7 / CS1, EGFR, BCMA, AXL, NaPi2B, GCC, STEAP1, MUC16, Mesothelin, ETBR, EphA2, 5T4, FOLR1, LAMP1, Cadherin 6, FGFR2, FGFR3, CA6, CanAg, Integrin αV, TDGF1, Ephrin A4, Trop2, PTK7, NOTCH3, C4.4A, FLT3, B7H3 / 4, TF (Tissue Factor) and ROR1 / 2.

[0212] In some embodiments, Ab is a monoclonal antibody. In some embodiments, Ab is an anti-human HER2 antibody or its antigen-binding fragment. Examples of anti-human HER2 antibodies include but are not limited to Pertuzumab and Trastuzumab. In one embodiment, Ab is Trastuzumab.

[0213] In one embodiment, formula (II) is formula (II-1):

[0214] In one embodiment, formula (II) is formula (II-2):

[0215] In one embodiment, formula (II) is formula (II-3):

[0216] In one embodiment, formula (II) is formula (II-4):

[0217] In one embodiment, formula (II) is formula (II-5):

[0218] in

[0219] R is hydrogen or α-L-fucosyl;

[0220] q is 1 or 2;

[0221] Ab is an antibody or antigen-binding fragment (for example, -NHC(O)CH2- in formula (II-1) is derived from asparagine at position 297 of the antibody Fc region);

[0222] The remaining variables are as defined in the first aspect or various embodiments thereof.

[0223] In one embodiment, formula (II) is selected from formula (II-1), (II-2), (II-3), (II-4), (II-5):

[0224] in

[0225] R is hydrogen or α-L-fucosyl;

[0226] q is 1 or 2, for example, q is 2;

[0227] Ab is 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, and 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-Nectin4 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-Cadherin6 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-PT K7 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 anti-CD19 antibody, anti-ErbB2 / HER2 antibody, anti-CLDN18.2 antibody, anti-Nectin-4 antibody, anti-FGFR3 antibody, anti-Trop2 antibody; more preferably anti-ErbB2 / HER2 antibody, anti-Trop2 antibody; particularly preferably anti-ErbB2 / HER2 antibody (e.g., trastuzumab);

[0228] (P) t -L- is selected from:

[0229] In one embodiment, R in formula (II) is α-L-fucosyl;

[0230] q is 2;

[0231] Ab is trastuzumab;

[0232] (P) t -L- is

[0233] For example,

[0234] -NHC(O)CH2- in formula (II) is derived from asparagine at position 297 in the Fc region of an antibody.

[0235] Method for preparing antibody-drug conjugates

[0236] In a fourth aspect, the present invention provides a method for preparing an antibody-drug conjugate having formula (II), comprising conjugating a linker-load compound having formula (I) to an antibody Ab, wherein unless otherwise indicated, each variable is as defined in the third aspect or its various embodiments.

[0237] In one embodiment, the method comprises the steps of:

[0238] a) Under the catalysis of glycosidase or its mutant, the antibody Ab is cleaved from its N-glycan chain to obtain an antibody whose Fc region N-glycosylation site is modified with N-acetylglucosamine or fucosyl-α-1,6-N-acetylglucosamine;

[0239] b) coupling the modified antibody obtained in step a) with the linker-load compound described above under the catalysis of glycosidase or a mutant thereof;

[0240] The glycosidase or its mutant used in step a) and step b) may be the same or different.

[0241] In one embodiment, the glycosidase or its mutant used in steps a) and b) is a fucosylate, an N-acetylglucosamine endohydrolase, or a mutant thereof. In one embodiment, the N-acetylglucosamine endohydrolase comprises at least one selected from the group consisting of Endo-S (Streptococcus pyogenes endoglycosidase-S), Endo-F3 (Elizabethkingia miricola endoglycosidase-F3), Endo-S2 (Endoglycosidase-S2, Streptococcus pyogenes endoglycosidase-S2), Endo-Sd (Endoglycosidase-Sd, Streptococcus pyogenes endoglycosidase-Sd), and Endo-CC (Endoglycosidase-CC, Streptococcus pyogenes endoglycosidase-CC); preferably, the endoglycosidase is Endo H, Endo D, Endo F2, Endo F3, Endo M, Endo CC1, Endo CC2, Endo Om, Endo S, and Endo S2. In one embodiment, the enzyme is Endo S2.

[0242] In one embodiment, steps a) and b) are performed by one-pot enzymatic catalysis.

[0243] In one embodiment, steps a) and b) are performed by one-pot enzymatic catalysis, and the enzyme is Endo S2.

[0244] Pharmaceutical compositions and pharmaceutical preparations

[0245] Another object of the present invention is to provide a pharmaceutical composition comprising a preventively or therapeutically effective amount of the conjugate of the present invention and at least one pharmaceutically acceptable carrier.

[0246] The pharmaceutical composition of the present invention can be administered in any manner as long as it achieves the effect of preventing, alleviating, preventing or treating the symptoms of humans or animals. For example, various suitable dosage forms can be prepared according to the route of administration, particularly injections such as lyophilized powder injections, injections or sterile injection powders.

[0247] The term "pharmaceutically acceptable" means that it does not produce undue toxicity, irritation or allergic reaction when in contact with patient tissues within the scope of normal medical judgment, has a reasonable ratio of advantages to disadvantages, and is effective for the intended use.

[0248] The term pharmaceutically acceptable carrier refers to any carrier material that is pharmaceutically acceptable and does not interfere with the biological activity and properties of the conjugate. Examples of aqueous carriers include, but are not limited to, buffered saline. Pharmaceutically acceptable carriers also include substances that allow the composition to approach physiological conditions, such as pH adjusters and buffers, toxicity modifiers, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, and the like.

[0249] In one embodiment, the drug / antibody ratio (DAR) of the pharmaceutical composition of the invention is an integer or non-integer of 1-20, such as about 1 to about 10, about 1 to about 8, about 1 to about 6, about 1 to about 4, about 1 to about 3, about 1 to about 2.5, or about 1 to about 2. In a particular embodiment, the conjugate of the invention has a DAR of about 2, about 4, about 6, or about 8.

[0250] Treatment methods and uses

[0251] The antibody-drug conjugates of the present invention can be used to treat tumors and / or autoimmune diseases. Tumors sensitive to antibody-drug conjugate therapy include those characterized by specific tumor-associated antigens or cell surface receptors. These tumor cells can be recognized by the targeting molecules in the antibody-drug conjugate and, in turn, killed by the cargo / cytotoxin in the antibody-drug conjugate.

[0252] Therefore, in another aspect, the present invention also provides use of the antibody-drug conjugate of the present invention or the pharmaceutical composition of the present invention in the preparation of a medicament for treating a disease, disorder or condition selected from a tumor or an autoimmune disease.

[0253] In another aspect, the present invention provides an antibody-drug conjugate or a pharmaceutical composition of the present invention for use in treating tumors or autoimmune diseases.

[0254] In a further aspect, the present invention provides a method for treating tumors or autoimmune diseases, comprising administering an effective amount of the antibody-drug conjugate of the present invention or the pharmaceutical composition of the present invention to an individual in need thereof.

[0255] In one embodiment, the antibody-drug conjugate provided by the present invention, formed by linking an anti-human HER2 or Trop2 antibody to a small molecule cytotoxin, can specifically bind to HER2 or Trop2 on the surface of tumor cells, selectively killing tumor cells expressing HER2. In another embodiment, the present invention provides the use of the antibody-drug conjugate or pharmaceutical composition of the present invention in the preparation of a medicament for treating a disease, disorder, or condition selected from HER2- or Trop2-positive tumors. In a more preferred embodiment, the disease, disorder, or condition is selected from the group consisting of breast cancer, gastric cancer, lung cancer, ovarian cancer, and urothelial carcinoma.

[0256] The dosage of the antibody-drug conjugate administered to a subject can be adjusted to a considerable extent. The dosage can vary depending on the specific route of administration and the needs of the subject and can be subject to the judgment of a healthcare professional.

[0257] Example

[0258] 1. Preparation of disaccharide linkers

[0259] Example 1: Preparation of disaccharide substrate compound 1

[0260] The following steps were used to prepare compound 1, whose structure is as follows:

[0261] (1) Preparation of compound 1c

[0262] Under the condition of oil pump vacuum, bake 100mL Schlenk reaction bottle for 5 minutes with a heat gun, add activated molecular sieve to the system after cooling, and bake for another 5 minutes. Vacuum nitrogen replacement three times, add compound 1a (4.41g, 7.96mmol) to the system under nitrogen protection, stir for 3 minutes, add anhydrous dichloromethane (30mL) and stir for 0.5 hour. In addition, according to the same operation, add compound 1b (1.89g, 3.98mmol, dissolved in 20mL anhydrous dichloromethane) to another 50mL Schlenk reaction bottle to which activated molecular sieves have been added, stir for 1 hour and pre-dry to remove residual moisture in the system.

[0263] Under nitrogen, 1-(phenylsulfinyl)piperidine (BSP, 1.37 g, 6.56 mmol) and 2,4,6-tri-tert-butylpyrimidine (TTBP, 2.94 g, 11.94 mmol) were added to the dried solution of Compound 1a at room temperature and stirred for an additional 20 minutes. The reaction flask was placed in a dry ice / ethyl acetate bath and cooled to -65°C. Trifluoromethanesulfonic anhydride (1.2 mL, 7.16 mmol) was then added. Two minutes later, a pre-dried dichloromethane solution of Compound 1b was added to the mixture. The resulting reaction mixture was stirred at -65°C until the reaction was complete as determined by TLC (developing solvent: EtOAc / PE = 1 / 8) (approximately 3 hours). Saturated sodium bicarbonate solution was added to the system to quench the reaction, and the liquid was extracted with dichloromethane (150 mL × 3). The combined organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, wet loaded, 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). 1H 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 = 12.1Hz, 1H), 4.37 (d, J = 8.1Hz, 1H, H 1 ), 4.22-4.10 (m, 2H), 4.05 (t, J=9.3Hz, 1H), 3.80 (d, J=3.1Hz, 1H), 3.72 (dd, J=11.2, 2.2Hz, 1H), 3.67-3.52 (m, 3 H), 3.49 (dd, J=9.8, 3.1Hz, 1H), 3.41 (t, J=9.3Hz, 1H), 3.35 (dt, J=9.8, 2.9Hz, 1H), 3.16 (td, J=9.7, 4.8Hz, 1H). C 54 H 56 N3O ，0 + [M+H] + MS (ESI) m / z calculated: 906.4, found: 906.7.

[0264] (2) Preparation of compound 1d

[0265] At room temperature, compound 1c (228 mg, 0.252 mmol), chloroform (1.2 mL), pyridine (1 mL), and AcSH (1.2 mL) were sequentially added to a 25 mL Schlenk reaction flask and dissolved. The flask was sealed with a stopper and stirred at 60°C until the reaction was essentially complete as determined by HPLC, which took approximately 18 hours. After concentration under reduced pressure to remove most of the solvent, ethyl acetate (50 mL) was added to the system. The mixture was washed sequentially with saturated sodium bicarbonate solution (30 mL), 1 M hydrochloric acid (10 mL x 4), and saturated sodium bicarbonate solution (30 mL). The organic phase was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by column chromatography (eluent: EtOAc / PE = 1 / 10-1 / 1) to afford compound 1d (200 mg, 86% yield, as a white solid). 1H NMR (400 MHz, chloroform-d) δ 7.57-7.49 (m, 2H), 7.48-7.39 (m, 5H), 7.39-7.24 (m, 23H), 5.80 (d, J = 8.1 Hz, 1H), 5.59 (s, 1H), 5.00 (d, J = 6.7Hz, 1H), 4.97-4.87(m, 3H), 4.87-4.75(m, 2H), 4.69-4.59(m, 4H), 4.57(s, 1H), 4.45(d, J=12.0Hz, 1H), 4.19-4.05(m, 3H), 3.96(t, J=7.3Hz, 1H), 3.85-3.76 (m, 2H), 3.73-3.59 (m, 4H), 3.49 (dd, J=9.8, 3.1Hz, 1H), 3.18 (td, J=9.7, 4.8Hz, 1H), 1.77 (d, J=1.2Hz, 3H).C 56 H 60 NO 11 + [M+H] + MS (ESI) m / z calculated: 922.4, found: 922.4.

[0266] (3) Preparation of compound 1e

[0267] To a 100 mL single-necked flask at room temperature, compound 1d (168 mg, 0.168 mmol), p-toluenesulfonic acid monohydrate (35 mg, 0.168 mmol), methanol (5 mL), and tetrahydrofuran (5 mL) were added sequentially. The nitrogen atmosphere was replaced three times, and the reaction was stirred at room temperature overnight until complete as determined by HPLC. The reaction system was quenched with saturated sodium bicarbonate solution (20 mL), and the liquids were extracted with dichloromethane (20 mL × 3). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. After purification by column chromatography (eluent: MeOH / DCM = 1 / 20), compound 1e (136 mg, 89% yield) was obtained as a white solid. 1H NMR (400MHz, chloroform-d) δ7.43-7.24 (m, 29H), 5.76 (d, J=7.9Hz, 1H), 5.01 (d, J=6.9Hz, 1H), 4.9 4 (dd, J=11.8, 2.3Hz, 2H), 4.85 (d, J=11.7Hz, 1H), 4.77 (d, J=11.8Hz, 1H), 4.70-4.59 (m, 3H ), 4.58-4.47 (m, 3H), 4.36 (d, J = 11.7Hz, 1H), 4.20 (t, J = 7.9Hz, 1H), 3.94 (t, J = 7.5Hz, 1H) , 3.91-3.80(m, 3H), 3.80-3.71(m, 3H), 3.63-3.50(m, 2H), 3.22-3.14(m, 2H), 1.78(s, 3).C 49 H 56 NO 11 + [M+H] + MS (ESI) m / z, calculated value 834.4, found value 834.5.

[0268] (4) Preparation of Compound 1f

[0269] At room temperature, compound 1e (130 mg, 0.156 mmol), dichloromethane, tert-butyl alcohol, and water were added sequentially to a 100 mL single-necked flask. Iodophenyl diacetic acid (0.5-10 equivalents) and 2,2,6,6-tetramethyl-1-piperidinyl oxide (0.01-1 equivalents) were added to the system, and the reaction was stirred at room temperature overnight until complete as determined by TLC. The separated liquids were extracted with dichloromethane, and the combined organic phases were dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to afford compound 1f (100 mg, 75.1% yield) as a white solid. 1H NMR (400MHz, chloroform-d) δ7.42-7.31 (m, 18H), 7.30-7.27 (m, 2H), 7.25-7.20 (m, 3H), 7.15 (dd, J=6.7, 2.9Hz, 2H), 6.06 (d, J= 8.3Hz, 1H), 4.96 (d, J=6.6Hz, 1H), 4.93 (d, J=11.9Hz, 1H), 4.86 (d, J=11.1Hz, 1H), 4.81 (d, J=12.1Hz, 1H), 4.73 (d, J=11 .1Hz, 1H), 4.71-4.65 (m, 3H), 4.65-4.57 (m, 3H), 4.49 (s, 1H), 4.46 (d, J=12.0Hz, 1H), 4.23 (t, J=7.1Hz, 1H), 4.15 (t, J= 9.5Hz, 1H), 3.89 (t, J=6.5Hz, 1H), 3.86-3.65 (m, 6H), 3.54 (d, J=9.7Hz, 1H), 3.30 (dd, J=9.3, 2.8Hz, 1H), 1.65 (s, 3H).C 49 H 52 NO 12 - [MH + ] - MS (ESI) m / z calculated value 846.3, found value 846.3.

[0270] (5) Preparation of Compound 1

[0271] At room temperature, compound 1f (160 mg, 0.189 mmol), tetrahydrofuran, methanol, and palladium-carbon catalyst were added sequentially to a 50 mL single-necked flask. The reaction system was stirred under a hydrogen atmosphere until all the starting material disappeared as determined by TLC. The mixture was filtered, concentrated under reduced pressure, and pumped dry to obtain compound 1 (75 mg, 100% yield, as a white solid). 14 H 22 NO 12 - [MH + ] - MS (ESI) m / z calculated: 396.1, found: 396.1.

[0272] Example 2: Preparation of disaccharide substrate compound 2

[0273] The following steps were used to prepare compound 2, the structure of which is as follows:

[0274] (1) Synthesis of Compound 2-1

[0275] Step A: Synthesis of compound 2-1b

[0276] 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 to a single-necked flask and stirred at room temperature under a nitrogen atmosphere. The reaction progress was monitored by TLC. After the reaction was completed, 1 M hydrochloric acid was added to quench the reaction and adjust the reaction to neutrality. The solvent was removed under reduced pressure, and a small amount of toluene was added. The water was removed by azeotropic effect under reduced pressure rotary evaporation to obtain the crude product 2-1b as a light yellow viscous oily liquid, which was directly used in the next reaction without further purification.

[0277] Step B: Synthesis of compound 2-1c

[0278] To a single-necked flask, crude product 2-1b (1 equivalent), acetonitrile, camphorsulfonic acid (0.1 equivalent), and benzaldehyde dimethyl acetal (4 equivalents) were added sequentially, and the reaction system was stirred at room temperature overnight. TLC monitored the reaction progress. After completion (approximately 24 hours), saturated sodium bicarbonate solution was added to the system to quench the reaction. The liquid phases were extracted with ethyl acetate, and the combined organic phases were washed with water and saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by wet-loading silica gel column chromatography (eluent: PE / EtOAc = 5:1) to afford 2-1c (white solid, 83% yield). 1 H NMR (400MHz, chloroform-d) δ7.51-7.49 (m, 2H, Ar-H), 7.45 (d, J=8.0Hz, 2H, Ar-H), 7.42-7.37 (m, 5H, Ar-H), 7 .36-7.31 (m, 3H, Ar-H), 7.15 (d, J=8.0Hz, 2H, Ar-H), 5.56 (s, 1H, PhCH), 4.97 (d, J=11.6Hz, 1H, PhCH2 ), 4.81 (d, J=11.6Hz, 1H, PhCH2), 4.58 (d, J=9.6Hz, 1H), 4.40 (dd, J=10.2, 4.8Hz, 1H), 3.80 (dd, J=10 .0, 10.4Hz, 1H), 3.70 (dd, J=9.2, 9.2Hz, 1H), 3.65 (dd, J=9.2, 9.2Hz, 1H), 3.53-3.48 (m, 2H), 2.64 (br s, 1H, -0H), 2.36 (s, 3H).C 27 H 29 O5S + [M+H] +The MS (ESI) m / z was calculated to be 465.2 and found to be 465.3. The NMR data were consistent with those reported in the literature for compound 2b1 in Nature 2007, 446, 896.

[0279] Step C: Synthesis of compound 2-1d

[0280] To a single-necked flask were added 2-1c (1 equivalent), dichloromethane, and triethylamine (5 equivalents) in sequence. The system was cooled to 0°C, and acetic anhydride (2 equivalents) was added. After stirring for 10 min, the reaction system was warmed to room temperature and stirred. The reaction progress was monitored by TLC. After the reaction was completed (about 6 h), saturated sodium bicarbonate solution was added to the system to quench the reaction. The mixture was extracted with dichloromethane, and the combined organic phase was washed with water, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by wet-loading column chromatography (eluent: PE / EtOAc = 5:1) to give 2-1d (white solid, 89% yield). 1 H NMR (400MHz, chloroform-d) δ7.51-7.48 (m, 2H, Ar-H), 7.43-7.36 (m, 5H, Ar-H), 7.34-7.25 (m, 5H, Ar-H), 7.13 (d, J =8.0Hz, 2H, Ar-H), 5.58 (s, 1H, PhCH), 5.00 (dd, J = 8.0, 8.0Hz, 1H, H-2), 4.87 (d, J = 12.0Hz, 1H, PhCH2), 4. 67 (d, J=12.0Hz, 1H, PhCH2), 4.64 (d, J=10.4Hz, 1H, H-1), 4.39 (dd, J=10.8Hz, J=5.2Hz, 1H), 3.81 (t, J=10 .4Hz, 1H), 3.78-3.70 (m, 2H), 3.48 (ddd, J=9.6, 5.4, 10.0Hz, 1H), 2.35 (s, 3H, Ar-CH3), 2.05 (s, 3H, 0Ac). C 29 H 31 O6S + [M+H] + The MS (ESI) m / z was calculated to be 507.2, and the actual value was 507.4. The NMR data were consistent with those reported in the literature (see Nature 2007, 446, 896 for compound 3b1).

[0281] Step D: Synthesis of compound 2-1e

[0282] Under nitrogen atmosphere, compound 2-1d (1 equivalent), dichloromethane, and borane tetrahydrofuran (10 equivalents, 1M in THF) were added sequentially to a 100 mL two-necked flask. The system was cooled to 0°C, and dibutylborane trifluoromethanesulfonate (1.4 equivalents, 1.0M in DCM) was added dropwise. The temperature was maintained at 0°C until TLC showed completion of the reaction (about 5 hours). Subsequently, triethylamine solution was added to the system at 0°C to quench the dibutylborane trifluoromethanesulfonate in the system, and methanol was slowly added dropwise to quench the borane tetrahydrofuran. After the system no longer produced a large amount of bubbles, water was added to ensure sufficient quenching, and then the liquid was extracted with ethyl acetate. The combined organic phase was washed with water, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, wet-loaded, and flash column chromatography (eluent: eluent: PE / EtOAc = 4:1) to give the crude product 2-1e (white solid). LCMS confirmed the structure of the product, C 29 H 33 O6S + [M+H] + The MS (ESI) m / z was calculated to be 509.2 and the actual value was 509.3. The crude product was used directly in the next reaction without fine purification.

[0283] Step E / F: Synthesis of compound 2-1f

[0284] Step E: 2-1e (1 equivalent), iodobenzene acetate (3 equivalents), TEMPO (0.5 equivalents) and tert-butanol / dichloromethane / water (volume ratio 4:4:1) were added to a single-necked flask in sequence. The resulting mixture was stirred at room temperature and the reaction progress was monitored by TLC (developing solvent: PE / EtOAc = 2 / 1, containing 1 v / v% acetic acid). After the reaction was complete, saturated sodium thiosulfate was added to the system to quench the reaction. The mixture was extracted with dichloromethane and the combined organic phases were washed with water and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The resulting carboxylic acid intermediate was used directly in the next reaction without further purification.

[0285] Step F: The carboxylic acid intermediate obtained in the previous step was dissolved in DMF, and iodomethane (3 equivalents) and potassium carbonate (5 equivalents) were added. The mixture was stirred at room temperature and the reaction progress was monitored by TLC (developing solvent: PE / EtOAc = 5 / 1). After the reaction was complete (approximately 2 hours), water was added to the system and the mixture was extracted with ethyl acetate. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography (eluent: PE / EtOAc = 7:1) to obtain compound 2-1f (white solid, total yield of three steps 59%). 1H NMR (400MHz, chloroform-d) δ, 7.42-7.26 (m, 12H, Ar-H), 7.16 (d, J=7.6Hz, 2H, Ar-H), 5.02 (dd, J=8.4, 8 .0Hz, 1H), 4.83 (d, J=11.6Hz, 1H, PhCH2), 4.79 (d, J=11.2Hz, 1H, PhCH2), 4.70 (d, J=11.6Hz, 1H ), 4.64 (d, J = 11.2Hz, 1H), 4.62 (d, J = 10.0Hz, 1H), 3.97 (d, J = 10.0Hz, 1H), 3.90 (dd, J = 8.4, 8.0 Hz, 1H), 3.79 (s, 3H, 0Me), 3.72 (dd, J=8.8, 8.8Hz, 1H), 2.38 (s, 3H, Ar-CH3), 2.04 (s, 3H, 0Ac). C 30 H 33 O7S + [M+H] + MS (ESI) m / z calculated value: 537.2, found value: 537.4.

[0286] Step G: Synthesis of compound 2-1g

[0287] To a 50 mL single-necked flask, 2-1f (1.0 equivalent) and acetone were added sequentially. The system was cooled to 0°C, and N-bromosuccinimide (1.4 equivalents) was added. Stirring was continued at 0°C for approximately 1 hour. TLC (developing solvent: PE / EtOAc = 4:1) indicated the reaction was complete. Saturated sodium thiosulfate solution was added to the system to quench the reaction. The acetone was removed by rotary evaporation under reduced pressure, and water was added. Extraction was then performed with ethyl acetate. The organic phases were combined, washed with water and 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, 90% yield). 23 H 27 O8 + [M+H] + MS (ESI) m / z, calculated value 431.2, found value 431.5.

[0288] Step H: Synthesis of compound 2-1

[0289] Under a nitrogen atmosphere, 2-1 g (1 equivalent), 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU, 0.1 equivalent), and dichloromethane were added sequentially to a 50 mL single-necked flask. The system was cooled to 0°C and stirred evenly. Trichloroacetonitrile (4 equivalents) was then added. The ice-water bath was removed and the temperature was naturally raised to room temperature with stirring. The reaction was monitored by TLC (developing solvent: PE / EtOAc = 2 / 1). After the reaction was complete (approximately 2 hours), the solvent was removed by rotary evaporation under reduced pressure. The product was then purified by wet-loading column chromatography (eluent: PE / EtOAc = 4:1) to obtain compound 2-1 (a light yellow viscous oily liquid, yield 87%). 23 H 25 O7 + [M-Cl3CC(NH)O - ] + MS (ESI) m / z calculated: 413.2, found: 413.2.

[0290] (2) Synthesis of Compound 2-2

[0291] Step A: Synthesis of compound 2-2a

[0292] 2-2a (CAS: 1235137-45-7) is commercially available and was prepared by referring to the synthesis method of compound 2 in Carbohydr Res 2016, 426, 33.

[0293] At room temperature, compound 2-2a (1 equivalent), methanol and sodium methoxide (0.1 equivalent, 5M 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 the reaction was complete (about 1 hour), dilute hydrochloric acid (1M) was added to neutralize the solution to pH = 7. After the reaction solution was concentrated, toluene was added and the residual water in the system was removed by rotary evaporation under reduced pressure using the azeotropic effect to obtain a light brown oily crude product 2-2b, which was used directly in the next reaction without purification.

[0294] Step B: Synthesis of compound 2-2c

[0295] At room temperature, to the crude product 2-2b (1 equivalent) from the previous step, monohydrated p-toluenesulfonic acid (0.2 equivalent) and anhydrous acetonitrile were added sequentially and stirred thoroughly. The system was replaced with nitrogen atmosphere, and benzaldehyde dimethyl acetal (5 equivalents) was added. The resulting reaction liquid was stirred at room temperature overnight until TLC (developing solvent: PE / EtOAc = 5 / 1) showed the reaction was complete. Then, saturated sodium carbonate solution was added to the reaction system to quench the reaction. The liquid was extracted with dichloromethane, and the combined organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, wet-loaded, and purified by silica gel column chromatography (eluent: PE / EtOAc = 5 / 1) to give compound 2-2c (white solid, total yield of two steps 85%). 1 H NMR (400MHz, chloroform-d) δ7.63-7.54 (m, 2H), 7.52-7.39 (m, 5H), 7.25-7.14 (m, 2H), 5.60 (s, 1H), 5.53 (d, J=5.4Hz, 1H), 4.52-4.39 (m, 1H), 4. 28 (dd, J=10.4, 4.9Hz, 1H), 4.07 (t, J=9.5Hz, 1H), 3.92 (dd, J=10.0, 5.6Hz, 1H), 3.79 (t, J=10.3Hz, 1H), 3.60 (t, J=9.3Hz, 1H), 3.08 (br s, 1H), 2.40 (s, 3H).C 20 H 22 N3O4S + [M+H] + The MS (ESI) m / z of the product was calculated to be 400.1 and found to be 399.9. The NMR data were consistent with those reported in the literature, see compound 47 in Angew. Chem. Int. Ed. 2021, 60, 12413.

[0296] Step C: Synthesis of compound 2-2d

[0297] Under nitrogen atmosphere, compound 2-2c (1 equivalent) and anhydrous tetrahydrofuran (reaction concentration 0.2 M) were added sequentially to a dry two-necked flask, and the mixture 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, the ice bath was removed and the mixture was warmed to room temperature with stirring. After 1 hour, tetrabutylammonium iodide (0.1 equivalent) and benzyl bromide (1.5 equivalents) were added. The resulting reaction system was stirred at room temperature until TLC (developing solvent: PE / EtOAc = 8 / 1) showed that the reaction was complete (about 6 hours). After completion of the reaction, water was added dropwise to quench the reaction, and the liquid was extracted with ethyl acetate. The combined organic phases were washed 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 give compound 2-2d (white solid, yield 97%).1 H NMR (400MHz, chloroform-d) δ7.63-7.55 (m, 2H), 7.53-7.33 (m, 10H), 7.20 (d, J=8.4Hz, 2H), 5.67 (s, 1H), 5.56 (d, J=4.6Hz, 1H), 5.05 (d, J=10. 9Hz, 1H), 4.90 (d, J=10.9Hz, 1H), 4.59-4.47 (m, 1H), 4.31 (dd, J=10.4, 4.9Hz, 1H), 4.10-3.97 (m, 2H), 3.90-3.77 (m, 2H), 2.41 (s, 3H). C 27 H 28 N3O4S + [M+H] + The MS (ESI) m / z of the compound was calculated to be 490.2 and found to be 490.5. The NMR data were consistent with those reported in the literature, see compound 20 in reference Bioorg. Med. Chem. 2011, 19, 30.

[0298] Step D: Synthesis of compound 2-2e

[0299] Compound 2-2d (1 equivalent), tetrahydrofuran / methanol (v / v = 1:1, reaction concentration 0.5 M), and p-toluenesulfonic acid monohydrate (0.2 equivalent) were added to a single-necked flask and stirred overnight at room temperature. The reaction progress was monitored by TLC (developing solvent: PE / EtOAc = 8 / 1). After completion (approximately 12 hours), saturated sodium bicarbonate solution was added to the system to quench the reaction. The liquid phases were extracted with ethyl acetate, and the combined organic phases were washed with water and saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and wet-loaded. Purification by silica gel column chromatography (eluent: EtOAc / PE = 1 / 1) afforded 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). 20 H 27 N4O4S + [M+NH4] + MS (ESI) m / z calculated: 419.2, found: 419.2.

[0300] Step E: Synthesis of compound 2-2

[0301] Compound 2-2e (1 equivalent), dichloromethane (reaction concentration 0.5 M), imidazole (2 equivalents), and tert-butyldiphenylsilyl chloride (1.5 equivalents) were added sequentially to a single-necked flask and stirred at room temperature. The reaction progress was monitored by TLC (developing solvent: EtOAc / PE = 1 / 12). After the reaction was completed (about 6 hours), saturated ammonium chloride solution was added to the system to quench the reaction, and the mixture was extracted with dichloromethane. The combined organic phase was washed with water, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, wet-loaded, and purified by silica gel column chromatography (eluent: PE / EtOAc / DCM = 15 / 1 / 1) to give compound 2-2 (yellow viscous oily liquid, yield 66%). 1 H NMR (400MHz, chloroform-d) δ7.73-7.68 (m, 4H), 7.48-7.35 (m, 13H), 7.06 (d, J=7.6 Hz, 1H), 5.49 (d, J=5.2Hz, 1H), 4.94 (ABq, J=10.8Hz, 2H), 4.32-4.27 (m, 1H ), 3.96-3.90 (m, 2H), 3.87 (dd, J=4.8, 5.2Hz, 1H), 3.82 (dt, J=2.8, 8.8Hz, 1H), 3.74-3.69 (m, 1H), 2.70 (d, J=2.8Hz, 0H), 2.33 (s, 3H), 1.09 (s, 9H).C 36 H 42 N3O4SSi + [M+H] + MS (ESI) m / z, calculated value 640.3, found value 640.2.

[0302] (3) Synthesis of Compound 2

[0303] Step A: Synthesis of compound 2a

[0304] Add pre-activated molecular sieves to a dry two-necked flask, bake with a heat gun under oil pump vacuum conditions, cool naturally, replace nitrogen, repeat the above vacuum-exchange nitrogen operation three times, then add compound 2-1 (2 equivalents), compound 2-2 (1 equivalent) and dry toluene to the system under nitrogen protection, and stir the resulting system at room temperature for 0.5h to fully remove residual water in the system. The reaction system was then cooled to -40°C and stirred, and a certain amount of TMSOTf was added. Stirring was maintained at this temperature until TLC showed that the reaction was complete (about 3h). Triethylamine was added to the system to quench the reaction, filter, concentrate, wet-load, 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), 1H), 4.74 (d, J=10.9Hz, 1H), 4.66-4.60 (m, 3H), 4.20 (dd, J=9.2, 9.2Hz, 1H), 4.05-4.02 (m, 2H), 3.97 (dd, J=9.3, 9.2Hz, 1H ), 3.92-3.76 (m, 3H), 3.74-3.67 (m, 1H), 3.60 (s, 3H), 3.51 (dd, J=9.2, 9.2Hz, 1H), 2.32 (s, 3H), 1.82 (s, 3H), 1.07 (s, 9H).C 59 H 66 N3O 11 SSi + [M+H] + MS (ESI) m / z, calculated value 1052.4, found value 1052.7.

[0305] Step B: Synthesis of compound 2b

[0306] Compound 2a (1 equivalent), thioglycolic acid (CAS: 68-11-1) / pyridine / chloroform (v / v / v = 1:1:1) were added to a reaction flask. The mixture was heated to 60°C and stirred. The reaction progress was monitored by TLC. After completion of the reaction (approximately 12 h), the solvent was mostly removed by concentration under reduced pressure. An appropriate amount of ethyl acetate was added, and the mixture was washed with saturated sodium bicarbonate solution. After extraction and separation, the organic phase was washed with 1M hydrochloric acid solution and then washed with saturated sodium bicarbonate solution. The resulting organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain compound 2b (white solid, 71% yield). 1H 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 = 7.9Hz, 2H), 5.68 (d, J=4.8Hz, 1H), 5.36-5.30 (m, 1H), 5.09 (dd, J=9.5, 8.1Hz, 1H), 4.92 (d, J=12.4Hz, 1H), 4.85 (d, J=11.5Hz, 1H), 4.75-4.66 (m, 3H), 4.63-4.57 (m, 2H), 4.29 (ddd, J=9.3, 7.7, 4.8Hz, 1H), 4 .10 (t, J=7.8Hz, 1H), 4.01 (dd, J=11.5, 3.2Hz, 1H), 3.94-3.89 (m, 2H), 3.86-3.84 (m, 1H), 3.80 (dd, J= 11.5, 2.5Hz, 1H), 3.67 (s, 3H), 3.60-3.51 (m, 2H), 2.28 (s, 3H), 1.85 (s, 3H), 1.82 (s, 3H), 1.07 (s, 9H). C 61 H 70 NO 12 SSi + [M+H] + MS (ESI) m / z, calculated value 1068.4, found value 1068.4.

[0307] Step C: Synthesis of compound 2c

[0308] Compound 2b (1 equivalent) and tetrahydrofuran were added to the reaction flask, and then TBAF (2 equivalents, 1M in THF) was added to the system. The mixture was stirred at room temperature and the reaction progress was monitored by TLC. After the reaction was complete (about 10 h), saturated ammonium chloride solution was added to the system to quench the reaction. The mixture was then extracted with ethyl acetate. The combined organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and wet loaded. Compound 2c was initially purified by silica gel column chromatography to obtain compound 2c (white solid, 56% crude yield). 45 H 52 NO 12 S + [M+H] + MS (ESI) m / z calculated value 830.3, found value 830.6.

[0309] Step D: Synthesis of compound 2d

[0310] Compound 2c was dissolved in THF / MeOH (v / v = 3:1), and 1 M aqueous sodium hydroxide solution was slowly added to adjust the system to pH = 12. The resulting reaction solution was stirred at room temperature and the reaction progress was monitored by HPLC. After the reaction was sufficient (about 12 h), 1 M hydrochloric acid was added to the system to adjust the pH to 7-8. The solution was concentrated under reduced pressure to remove most of the organic solvent. The resulting crude product was purified by HPLC to obtain compound 2d (white solid, 82% yield). 1 H 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 (m, 4H). (d, J=12.2Hz, 1H), 4.36 (ddd, J=10.6, 7.9, 5.0Hz, 1H), 4.25-4.22 (m, 1H), 4.13-4.02 (m, 3H), 3.88 (t, J=8.6Hz, 1H), 3.81 (dd, J=12.7, 2.2Hz, 1H), 3.74 (dd, J=10.7, 8.5Hz, 1H), 3.68-3.59 (m, 2H), 3.40 (br s, 1H), 2.30 (s, 3H), 1.77 (s, 3H). 13 C 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 (three carbon signals in the aromatic region overlap and do not appear). 42 H 46 NO 11 S - [MH] - MS (ESI) m / z, calculated value 772.3, found value 772.2.

[0311] Step E: Synthesis of compound 2e

[0312] Weigh compound 2d and dissolve it in acetone. Stir in an ice-water bath for 5 minutes to cool. Weigh NBS and add it to the reaction mixture. Incubate in an ice-water bath for approximately 1 hour. Take a sample and send it to HPLC to confirm the completion of the reaction. After quenching with a saturated sodium thiosulfate solution, concentrate to remove the acetone, prepare by prep-HPLC, and lyophilize to obtain 2e (white solid, 45% yield). 35 H 40 NO 12 - [MH] - MS (ESI) m / z calculated: 666.3, found: 666.4.

[0313] Step F: Synthesis of compound 2

[0314] At room temperature, compound 2e, tetrahydrofuran, methanol, and palladium-carbon catalyst were added sequentially to a 50 mL single-necked flask. The reaction system was stirred under a hydrogen atmosphere until all the starting materials disappeared as determined by TLC. The mixture was filtered, concentrated under reduced pressure, and pumped dry to obtain compound 2 (white solid, 95% yield). 14 H 22 NO 12 - [MH + ] - MS (ESI) m / z calculated: 396.1, found: 396.1.

[0315] Example 3: Preparation of disaccharide substrate compound 3

[0316] The following steps were used to prepare compound 3, the structure of which is shown below:

[0317] The preparation process of compound 3 refers to the synthesis method of compound 2, and the specific route is as follows:

[0318] The final product, compound 3, was confirmed by mass spectrometry. 14 H 22 NO 12 - [MH + ] - MS (ESI) m / z calculated: 396.1, found: 396.0.

[0319] Example 4: Preparation of disaccharide substrate compound 4

[0320] The following steps were used to prepare compound 4, whose structure is as follows:

[0321] The preparation process of compound 4 is similar to the synthesis method of compound 2, and the specific route is as follows:

[0322] The final product, compound 4, was confirmed by mass spectrometry. 14 H 22 NO 12 - [MH + ] - MS (ESI) m / z calculated: 396.1, found: 396.1.

[0323] Example 5: Preparation of disaccharide substrate compound 5

[0324] The following steps were used to prepare compound 5, whose structure is as follows:

[0325] The preparation process of compound 5 refers to the synthesis method of compound 2, and the specific route is as follows:

[0326] The final product, compound 5, was confirmed by mass spectrometry. 14 H 22 NO 12 - [MH + ] - MS (ESI) m / z calculated: 396.1, found: 396.0.

[0327] 2. Preparation of linker-payload (LP)

[0328] Example 6: Preparation of LP-1

[0329] The structure of linker payload 1 (LP-1) is as follows:

[0330] (1) Preparation of compound LP-1b

[0331] At room temperature, compound 1 (0.5-5.0 equivalents), compound LP-1a (1.0 equivalent, GGG-VC-PAB-MMAE, CAS No. 2684216-48-4, commercially available), DMF, DIPEA (1-10 equivalents), and HATU (0.5-10 equivalents) were added sequentially to a 10 mL single-necked vial. The resulting reaction solution was stirred at room temperature until the reaction was complete as monitored by HPLC. The reaction solution was purified by semi-preparative HPLC to obtain compound LP-1b (white solid, yield 81.2%). 78 H 126 N 14 O 26 2+ [M+2H] 2+MS (ESI) m / z calculated value 837.4, found value 837.9.

[0332] (2) Preparation of compound LP-1

[0333] At room temperature, 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) were added sequentially to a 10 mL single-necked vial. The resulting reaction solution was stirred at room temperature and monitored by HPLC until the reaction was complete. The reaction solution was purified by semi-preparative HPLC to obtain compound LP-1 (15.3 mg, 71.3% yield, as a white solid). 78 H 124 N 14 O 25 2+ [M+2H] 2+ MS (ESI) m / z calculated value 828.4, found value 828.6.

[0334] Example 7: Preparation of LP-2

[0335] The structure of linker payload 2 (LP-2) is as follows:

[0336] The preparation process is as follows:

[0337] 20.1 Step A: Preparation of Compound LP-2a

[0338] At room temperature, compound 2 (0.5-5.0 equivalents), compound LP-1a (1.0 equivalent, GGG-VC-PAB-MMAE, CAS No. 2684216-48-4, commercially available), DMF, DIPEA (1-10 equivalents), and HATU (0.5-10 equivalents) were added sequentially to a 10 mL single-necked vial. The resulting reaction solution was stirred at room temperature until the reaction was complete as monitored by HPLC. The reaction solution was purified by semi-preparative HPLC to obtain compound LP-2a (white solid, 84% yield). 78 H 126 N 14 O 26 2+ [M+2H] 2+ MS (ESI) m / z, calculated value 837.4, found value 837.8.

[0339] 20.2 Step B: Preparation of Compound LP-2

[0340] At room temperature, 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) were added sequentially to a 10 mL single-necked vial. The resulting reaction solution was stirred at 0°C and monitored by HPLC until the reaction was complete. The reaction solution was purified by semi-preparative HPLC to obtain compound LP-2 (yield 87%, white solid). 78 H 124 N 14 O 25 2+ [M+2H] 2+ MS (ESI) m / z calculated value 828.4, found value 828.7.

[0341] Example 8: Preparation of LP-3, LP-4, and LP-5

[0342] Using similar steps to prepare LP-2, linker payloads LP-3, LP-4, and LP-5 with the following structures were prepared.

[0343] Example 9: Preparation of LP-6

[0344] (1) Synthesis of LP-6-1

[0345] Step A: Synthesis of intermediate LP-6-1b

[0346] Compound LP-6-1a (1.0 equivalent) and DMF (dissolved at 1 g / mL) were added to a reaction flask and stirred to dissolve under nitrogen. After cooling to 0-5°C, DIEA (3 equivalents) was added dropwise. The resulting system was then stirred at 5°C for 10 minutes, followed by benzyl bromide (1.3 equivalents). After the addition was complete, the reaction system was allowed to naturally warm to room temperature and stirred for 16 hours. The reaction solution was slowly poured into ice water, and methyl tert-butyl ether was added, stirred, and allowed to stand for separation. The aqueous phase was extracted four times with methyl tert-butyl ether. The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude yellow oil. The product was wet-loaded and purified by silica gel column chromatography (eluent: PE / EA = 6:1) to obtain product LP-6-1b (pale yellow oil, quantitative yield).

[0347] Step B: Synthesis of intermediate LP-6-1d

[0348] Under nitrogen, intermediate LP-6-1b (2.0 equivalents), compound LP-6-1c (1.0 equivalents), and THF (dissolved at 10 g / mL) were added to a reaction flask and stirred to dissolve. TsOH (0.1 equivalents) was added to the reaction, and the system was allowed to react at room temperature for 4 hours. The reaction solution was slowly poured into ice water and extracted three times with ethyl acetate. The combined organic phases were washed sequentially with saturated sodium bicarbonate aqueous solution, water, and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. The product was purified by silica gel column chromatography (eluent: PE / EA = 1:1) to obtain the product LP-6-1d (white solid, 40% yield).

[0349] Step C: Synthesis of intermediate LP-6-1e

[0350] Under nitrogen protection, compound LP-6-1d and N,N-dimethylacetamide (DMAc, dissolution concentration of 10 g / mL) were added to the reaction flask and stirred to dissolve. The system was cooled to 14-18°C, DBU (0.5 equivalent) was added dropwise, and the reaction was maintained at this temperature until TLC showed that the reaction was complete (about 1.5 h) to obtain the intermediate LP-6-1e, which was directly used in the next reaction without purification.

[0351] Step D: Synthesis of intermediate LP-6-1g

[0352] The reaction mixture from the previous step was cooled to 0-5°C, and 4-methylbenzenesulfonate pyridinium (PPTS, 0.5 equivalent), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCI, 1.0 equivalent), 1-hydroxybenzotriazole (HOBT, 1.0 equivalent), and LP-6-1f (0.85 equivalent) were added in sequence. The reaction system was reacted at 0-10°C until LCMS showed the reaction was complete (about 4 hours). The reaction mixture was added to ice water and extracted once with 2-methyltetrahydrofuran. The aqueous phase was extracted twice with 2-methyltetrahydrofuran. The organic phases were combined and washed in sequence with 0.5M hydrochloric acid, saturated aqueous NaHCO3, water, and saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by silica gel column chromatography (eluent: DCM / MeOH) to obtain the product LP-6-1g (white solid, yield 78%).

[0353] Step E: Synthesis of intermediate LP-6-1h

[0354] Under nitrogen, LP-6-1g and DMAc (dissolved at 10g / mL) were added to a reaction flask and stirred to dissolve. The temperature was cooled to 14-18°C, and DBU (0.5 equivalent) was added dropwise. The reaction was stirred at this temperature for 1.5 hours. The reaction progress was monitored by TLC. Upon completion, the intermediate LP-6-1h was obtained and used directly in the next reaction without purification.

[0355] Step F: Synthesis of intermediate LP-6-1j

[0356] The reaction solution of LP-6-1h in the previous step was cooled to 0-5°C, and PPTS (0.5 equivalent), EDCI (1 equivalent), HOBT (1 equivalent) and compound 3i (0.85 equivalent) were added. The mixture was reacted at 0-10°C for 3-4 hours. The reaction progress was monitored by LCMS. After the reaction was complete, the reaction solution was added to ice water and extracted once with 2-methyltetrahydrofuran. The aqueous phase was extracted twice with 2-methyltetrahydrofuran. The organic phases were combined and washed with 0.5M hydrochloric acid, saturated NaHCO3 aqueous solution, water, and saturated brine in sequence, dried over anhydrous sodium sulfate, filtered, concentrated, dry-mixed, and purified by column chromatography.

[0357] (Eluent: DCM / MeOH) to give LP-6-1j (white solid, yield 50%).

[0358] Step G: Synthesis of compound LP-6-1

[0359] Under nitrogen, intermediate LP-6-1j was dissolved in DCM (15 g / mL) and DBU (0.5 equivalent) was added dropwise at 20°C. The reaction was stirred and maintained at this temperature until HPLC indicated completion. DCM was then added to dilute the reaction solution, and the sample was directly wet-loaded and purified by column chromatography (eluent: DCM / MeOH) to obtain compound LP-6-1 (white solid, 82% yield). 34 H 47 N6O 10 + [M+H] + MS (ESI) m / z calculated: 699.4, found: 699.6.

[0360] (2) Synthesis of LP-6-2

[0361] LP-6-2 was synthesized using peptide solid phase synthesis method. The steps are as follows:

[0362] Step 1: Preparation of NH2-Asp(OtBu)-Rink amide resin

[0363] Weigh 400g of Rink amide resin into a reactor and soak it in 2400mL of DCM for 0.5h to fully swell the resin. Drain and drain. Add 2400mL of decapping reagent and wash. Drain and drain. Add 2400mL of decapping reagent and stir under nitrogen at 25±1°C for 0.5h. Drain and drain. Wash twice with 2400mL of DMF, 2400mL of anhydrous ethanol, 2400mL of DCM, and 2400mL of DMF. Drain and drain. Kaiser detector indicates blue.

[0364] Weigh 88.87g of Fmoc-Asp(OtBu)-OH and 29.19g of HOBT and dissolve them in 2000mL of DMF solution and 80ml of DIC. Place in a -10℃ ice bath for 0.5h, then slowly add them to the reactor for reaction. Stir and react under nitrogen bubbling at room temperature for 2h, then drain. Wash the mixture twice with 2400mL of DMF, 2400mL of anhydrous ethanol, 2400mL of DCM, and 2400mL of DMF, drain. Kaiser assay results in a light blue color. Add 2400mL of DCM and 60ml of capping reagent, stir and react under nitrogen bubbling at 25±1℃ for 1h, then drain. Wash the mixture twice with 2400mL of DMF, 2400mL of anhydrous ethanol, 2400mL of DCM, and 2400mL of DMF, then drain. Kaiser assay results in a colorless color.

[0365] Add 2400 mL of decapping reagent to wash and drain. Add 2400 mL of decapping reagent and stir under nitrogen at 25±1°C for 0.5 h, then drain. Wash twice with 2400 mL of DMF, 2400 mL of anhydrous ethanol, 2400 mL of DCM, and 2400 mL of DMF, drain. Kaiser assay results in blue.

[0366] Step 2: Preparation of NH2-PEG4-Asp(OtBu)-Rink amide resin

[0367] Weigh 131.64g of Fmoc-PEG4-OH and 48.64g of HOBT and dissolve them in 2000mL of DMF and 80.0ml of DIC. Place in a -10°C ice bath for 0.5h, then slowly add the mixture to the reactor. Stir under nitrogen purge at room temperature for 2-4h, then drain. Wash the mixture twice with 2400mL of DMF, 2400mL of anhydrous ethanol, 2400mL of DCM, and 2400mL of DMF, then drain. Kaiser assay indicates colorless.

[0368] Add 2400 mL of decapping reagent to wash and drain. Add another 2400 mL of decapping reagent and stir under nitrogen at 25±1°C for 0.5 h. Drain. Wash twice with 2400 mL of DMF, 2400 mL of anhydrous ethanol, 2400 mL of DCM, and 2400 mL of DMF, drain. Kaiser assay indicates blue.

[0369] Step 3: Preparation of NH2-Asp(OtBu)-PEG4-Asp(OtBu)-Rink amide resin

[0370] Weigh 222.18g of Fmoc-Asp(OtBu)-OH and 72.96g of HOBT and dissolve them in 2000mL of DMF and 80mL of DIC. Place in a -10°C ice bath for 0.5h, then slowly add the mixture to the reactor. Stir and react under nitrogen bubble for 2-4h at room temperature, then drain. Wash the mixture twice with 2400mL of DMF, 2400mL of anhydrous ethanol, 2400mL of DCM, and 2400mL of DMF, then drain. Kaiser assay indicates a colorless solution.

[0371] Add 2400 mL of decapping reagent, wash, and drain. Add another 2400 mL of decapping reagent, stir and react at 25±1°C with nitrogen for 0.5 h, and drain. Wash twice with 2400 mL of DMF, 2400 mL of anhydrous ethanol, 2400 mL of DCM, and 2400 mL of DMF, drain. Kaiser assay indicates blue.

[0372] Step 4: Preparation of Dde-Lys(NH2)-Asp(OtBu)-PEG4-Asp(OtBu)-Rink amide resin

[0373] Weigh 191.75g ​​of Dde-Lys(Fmoc)-OH and 48.64g of HOBT and dissolve them in 2000mL of DMF and 80.0ml of DIC. Place in a -10°C ice bath for 0.5h, then slowly add the mixture to the reactor. Stir and react under nitrogen bubbling at room temperature for 2-4h, then drain. Wash the mixture twice with 2400mL of DMF, 2400mL of anhydrous ethanol, 2400mL of DCM, and 2400mL of DMF, then drain. Kaiser assay indicates colorless.

[0374] Add 2400 ± 100 mL of decapping reagent to wash and drain. Add another 2400 mL of decapping reagent and stir under nitrogen at 25 ± 1°C for 0.5 h. Drain. Wash twice with 2400 mL of DMF, 2400 mL of anhydrous ethanol, 2400 mL of DCM, and 2400 mL of DMF, drain. Kaiser assay indicates blue.

[0375] Step 5: Preparation of Dde-Lys(mPEG12)-Asp(OtBu)-PEG4-Asp(OtBu)-Rink amide resin

[0376] Weigh 170.84g of m-PEG12-CH2CH2COOH and 48.64g of HOBT and dissolve them in 2000mL of DMF and 80.0ml of DIC. Place in a -10°C ice bath for 0.5h, then slowly add the mixture to the reactor. Stir under nitrogen at room temperature for 2-4h, then drain. Wash the mixture twice with 2400mL of DMF, 2400mL of anhydrous ethanol, 2400mL of DCM, and 2400mL of DMF, then drain. Kaiser assay indicates colorless.

[0377] Step 6: Preparation of NH2-Lys(PEG12)-Asp(OtBu)-PEG4-Asp(OtBu)-Rink amide resin

[0378] Add 2400 mL of de-DDE reagent and stir at 25±1°C for 10 min under nitrogen bubble. Drain and repeat this operation three times. Wash twice with 2400 mL of DMF, 2400 mL of anhydrous ethanol, 2400 mL of DCM and 2400 mL of DMF respectively. Drain and the Kaiser detector shows blue.

[0379] Step 7: Preparation of Fmoc-Gly-Gly-Gly-Lys(PEG12)-Asp(OtBu)-PEG4-Asp(OtBu)-Rink amide resin

[0380] Weigh 111.08g of Fmoc-Gly-Gly-Gly-OH and 48.64g of HOBT and dissolve them in 2000mL of DMF solution and 80.0ml of DIC. Place in a -10℃ ice bath for 0.5h, then slowly add them to the reactor for reaction. Stir and react under nitrogen bubbling at room temperature for 2-4h, then drain. Wash with 2400mL of DMF, 2400mL of anhydrous ethanol, 2400mL of DCM, and 2400mL of DMF twice each, drain, and Kaiser test. The resin peptide is colorless. Wash three times with 2400mL of anhydrous ethanol, drain, and wait for cleavage.

[0381] Step 8: Preparation of LP-6-2

[0382] Add 10,000 mL of cleavage reagent (TFA:TIS:H2O = 95:2.5:2.5) to a 10-L reactor and cool to -10 ± 2°C. Add the dried, weighed resin and heat to 20 ± 5°C, stirring under nitrogen for 2 h. Filter and wash the resin once with 100 mL of TFA. Combine the filtrate and wash.

[0383] Add 40 L of pre-cooled (below -10°C) cold ether, stir for 10 minutes and then centrifuge to precipitate. After centrifugation, discard the supernatant, mix the precipitate with cold ether and shake it evenly, then centrifuge again (repeat this step 3 times, each time using 10 L, 10 L, and 10 L. Set the centrifugal speed to 3600 rpm, the centrifugation time to 5 minutes, and the temperature of the centrifuge cavity to -5°C).

[0384] The precipitate was collected to obtain the crude product of LP-6-2, which was purified by Prep-HPLC and lyophilized to obtain LP-6-2. 74 H 121 N9O 31 2+ [M+2H] 2+ MS (ESI) m / z, calculated value 815.9, found value 816.3.

[0385] (3) Synthesis of LP-6-3

[0386] Step A: Synthesis of intermediate LP-6-3a

[0387] Compound LP-6-1 (2.2 equivalents) and compound LP-6-2 (1.0 equivalents) were added to the reaction flask and dissolved in DMF. DIPEA (5.0 equivalents) was then added and stirred evenly. HATU (2.5 equivalents) was added to the system and the reaction was allowed to proceed at room temperature. HPLC monitoring was performed until the reaction was complete (approximately 2 hours). The reaction system was directly prepared by prep-HPLC, and the prepared solution was lyophilized to obtain compound LP-6-3a (white solid, yield 52%). 143 H 212 O 48 N 21 3+ [M+3H] 3+ The MS (ESI) m / z was calculated to be 997.2 and the actual value was 875.9 (some functional groups would be detached after ionization).

[0388] Step B: Synthesis of intermediate LP-6-3b

[0389] Compound LP-6-3a was dissolved in purified water, and a certain amount of palladium hydroxide (10 wt% Pd(OH)2 on carbon) was added. The system was replaced with hydrogen three times and stirred at room temperature for 1.5 h. During this period, the reaction progress was monitored and the reaction was stopped immediately after the starting material disappeared to prevent the increase of de-Fmoc products. The reaction solution was filtered and prep-HPLC was prepared to obtain compound LP-6-3b (white solid, yield 76%). 128 H 200 O 48 N 213+ [M+3H] 3+ The MS (ESI) m / z of the product was calculated to be 937.1 and found to be 875.9 (some functional groups will be detached after ionization).

[0390] Step C: Synthesis of intermediate LP-6-3d

[0391] Compound LP-6-3b (1.0 equivalent) and compound LP-6-3c (2.2 equivalents) were dissolved in DMF, DIPEA (5.0 equivalents) was added and stirred uniformly, followed by the addition of HATU (2.5 equivalents). The reaction was allowed to proceed at room temperature and monitored by HPLC until the reaction was complete (approximately 16 hours). The reaction system was directly prepared by prep-HPLC, and the preparative solution was lyophilized to obtain compound LP-6-3d (yellow solid, yield 66%). 174 H 232 O 55 Cl2F2N 27 3+ [M+3H] 3+ MS (ESI) m / z, calculated value 1229.2, found value 1229.3.

[0392] Step D: Synthesis of compound LP-6-3

[0393] 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 the reaction was complete (about 0.5 h). After the reaction was completed, the pH was adjusted to neutral, and then prep-HPLC was used for preparation. After freeze-drying, compound LP-6-3 (yellow solid, yield 73%) was obtained. 159 H 222 O 53 Cl2F2N 27 3+ [M+3H] 3+ MS (ESI) m / z, calculated value 1155.2, found value 1155.3.

[0394] For the synthesis of compound LP-6-3c, please refer to patent CN202211428194.6.

[0395] (4) Synthesis of LP-6

[0396] Synthesis of LP-6a

[0397] Compound 1 (0.5-5.0 equivalents) and compound LP-6-3 (1.0 equivalents) were dissolved in DMF, DIPEA (1-10 equivalents) was added and stirred at 0°C. HATU (0.5-10 equivalents) was added and the reaction system was stirred at 0°C. The reaction progress was monitored by HPLC until the reaction was complete (approximately 2 hours). The reaction system was directly prepared by prep-HPLC, and the preparative solution was lyophilized to obtain compound LP-6a (yellow solid, 63% yield). 173 H 243 O 64 Cl2F2N 28 3+ [M+3H] 3+ MS (ESI) m / z, calculated value 1281.5, found value 1281.8.

[0398] Synthesis of LP-6

[0399] 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 reaction progress was monitored by HPLC. After the reaction was complete, it was purified by prep-HPLC. The prepared solution was lyophilized to obtain compound LP-6 (light yellow solid, yield 74%). 173 H 241 O 63 Cl2F2N 28 3+ [M+3H] 3+ MS (ESI) m / z, calculated value 1275.5, found value 1275.9.

[0400] Example 9: Preparation of LP-7

[0401] The structure of LP-7 is as follows:

[0402] The synthetic route is as follows:

[0403] Step A: Synthesis of compound LP-7b

[0404] Compound LP-7a was synthesized by a peptide solid phase synthesis method, referring to a similar method to LP-6-2.

[0405] Compound LP-7a (1.0 equivalent) and compound LP-6-1 (3.6 equivalents) were added to the reaction flask and dissolved in DMF. DIPEA (6.0 equivalents) was then added and stirred evenly. HATU (3.6 equivalents) was added to the system and the reaction was allowed to proceed at room temperature. The reaction was monitored by HPLC until the reaction was complete (about 2 h). The reaction system was directly prepared by prep-HPLC, and the preparative solution was lyophilized to obtain compound LP-7b (white solid, yield 56%). 191 H 280 O 66 N 29 3+ [M+3H] 3+ MS (ESI) m / z, calculated value 1345.3, found value 1345.8.

[0406] Step B: Synthesis of intermediate LP-7c

[0407] Compound LP-7b was dissolved in purified water, and a certain amount of palladium hydroxide (10 wt% Pd(OH)2 on activated carbon) was added. The system was replaced with hydrogen three times and stirred at room temperature for 1.5 h. During this period, the reaction progress was monitored. The reaction solution was filtered and directly prepared by prep-HPLC to obtain compound LP-7c (white solid, yield 80%). 170 H 262 O 66 N 29 3+ [M+3H] 3+ MS (ESI) m / z, calculated value 1255.3, found value 1255.9.

[0408] Step C: Synthesis of intermediate LP-7d

[0409] Compound LP-7c (1.0 equivalent) and compound LP-6-3 (1-10 equivalents) were dissolved in DMF, and DIPEA (1-20 equivalents) was added and stirred evenly. HATU (1-10 equivalents) was then added and reacted at room temperature. The reaction was monitored by HPLC until the reaction was complete (about 2 h). The reaction was directly used for the next step. 239 H 313 O 75 Cl3F3N 38 3+ [M+3H] 3+ MS (ESI) m / z, calculated value 1692.4, found value 1692.9.

[0410] Step D: Synthesis of compound LP-7e

[0411] Compound LP-7d was dissolved in DMF, diethylamine was added, and the reaction was carried out at room temperature. The reaction was monitored by HPLC until the reaction was complete (about 0.5 h). After the reaction was completed, the pH was adjusted to neutral, and then prep-HPLC was used for preparation. After freeze-drying, compound LP-7e (yellow solid, yield 73%) was obtained. 224 H 303 O 73 Cl3F3N 38 3+ [M+3H] 3+ MS (ESI) m / z, calculated value 1618.3, found value 1618.5.

[0412] Step E: Synthesis of compound LP-7f

[0413] Compound 1 (0.5-5.0 equivalents) and compound LP-7e (1.0 equivalent) were dissolved in DMF, DIPEA (1-10 equivalents) were added, and the mixture was stirred at 0°C. HATU (0.5-10 equivalents) was added, and the reaction system was stirred at 0°C. The reaction progress was monitored by HPLC until the reaction was complete (approximately 2 hours). The reaction system was directly prepared by prep-HPLC, and the preparative solution was lyophilized to obtain compound LP-7f (yellow solid, 65% yield). 238 H 325 O 84 Cl3F3N 39 4+ [M+4H] 4+ MS (ESI) m / z, calculated value 1308.8, found value 1309.0.

[0414] 15.6Step F: Synthesis of Compound LP-7

[0415] Compound LP-7f 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 reaction progress was monitored by HPLC. After the reaction was complete, it was purified by prep-HPLC. The prepared solution was lyophilized to obtain compound LP-7 (light yellow solid, 50% yield).

[0416] 3. Selection or Preparation of Antibodies

[0417] Antibody selection

[0418] The macromolecular portion of the antibody-drug conjugate prepared using the substrate and conjugation method of the present invention can be any molecule containing a sugar chain in the FC region of an antibody, including but not limited to antibodies / diabodies / FC fusion proteins / single-chain antibodies, etc.

[0419] In the following specific examples, trastuzumab can be selected, which is commercially available.

[0420] In addition, antibodies can also be selected that have been engineered. Examples of their preparation are as follows:

[0421] Example 10: Preparation of engineered anti-HER2 and Trop2 antibodies

[0422] The production, purification, and identification of the anti-human ErbB2 / HER2 antibody mAb-1 and the anti-human TROP2 antibody mAb-2 were described in Example 1 of patent CN 106856656 B, which is incorporated herein by reference in its entirety.

[0423] ADC Preparation, Characterization, and Activity Testing

[0424] Example 11: Preparation and Characterization of ADC-1

[0425] In the following, endoglycosidase is used to promote the remodeling of the sugar chain at the Fc end of the antibody mAb-1, and the linker payload is specifically coupled to the antibody to form the corresponding ADC drug.

[0426] 1) Treatment of mAb-1: The antibody was treated by ultrafiltration, dialysis, or desalting, and its storage buffer was replaced with 50 mM Tris-HCl (pH 5-8), 150 mM NaCl.

[0427] 2) Preparation of ADC-1: ADC-1 is prepared by coupling the mAb-1 antibody with LP-1 using the endoglycosidase Endo S2. In 1× endoglycosidase buffer, the mAb-1 antibody and LP-1 are thoroughly mixed at an appropriate molar ratio (1:1 to 1:100). Endoglycosidase Endo S2 is then added and mixed thoroughly. The coupling reaction is carried out at 4-40°C for 0.5-20 hours. After completion of the reaction, purification is performed. The purified ADC-1 is stored in 1× PBS, pH 7.4, at 4°C or -80°C.

[0428] The characterization data of the antibody-drug conjugate ADC-1 are described below.

[0429] SDS-PAGE analysis of ADC-1

[0430] After the conjugation reaction, the purity and conjugation efficiency of ADC-1 were determined by SDS-PAGE. The SDS-PAGE results of ADC-1 are shown in Figure 1. The conjugation reaction occurred specifically on the heavy chain of the antibody, and the heavy chain conjugated to the cytotoxin exhibited a significant molecular weight shift compared to the unconjugated heavy chain. The purity of the conjugated product met expectations.

[0431] HIC-HPLC analysis of ADC-1

[0432] HIC-HPLC: A Proteomix HIC Butyl-NP54.6*100mm 5um Non-Porous column was used; the column temperature was 30°C; 1.5M ammonium sulfate + 20mM phosphate buffer, pH 7.0, was used as mobile phase A; 20mM phosphate buffer: isopropanol = 7:3 (v / v) was used as mobile phase B; the flow rate was 0.8mL / min; a gradient method was used: phase B increased from 10% to 100% over 8 minutes; the detection wavelength was 280nm, and the DAR distribution of ADC-1 was detected.

[0433] The test results are shown in Figure 2. The amount of antibody not conjugated to LP-1 was less than 1%; the conjugated products were mainly DAR 2, and the average DAR value of the ADC drug was 1.93.

[0434] High-precision molecular weight mass spectrometry (ESI-MS) analysis of ADC-1

[0435] ADC-1 was analyzed and detected using high-precision molecular weight mass spectrometry. The results showed that its apparent molecular weight was 150539.27 and the theoretical molecular weight was 150538.78, which was in line with expectations and confirmed that each heavy chain Fc end was coupled to a cytotoxin molecule.

[0436] SEC-HPLC analysis of ADC-1

[0437] SEC column analysis was used to examine the high-molecular-weight aggregation of ADC-1. As shown in Figure 3, the high-molecular-weight aggregates in ADC-1 were less than 5% at 6.7 min, indicating that the ADC sample existed primarily as a monomer. Damage to the antibody caused by the coupling reaction was negligible.

[0438] Detection of ADC-1 affinity for ErbB2 / HER2 on cell surface

[0439] 1) Prepare HER2 ECD and coat with CBS coating solution (0.1M carbonate buffer, pH 9.6) at 1 μg / mL, 100 μL / well, and coat at 25°C for 60 min. 2) After coating, wash the plate three times with PBST and block with blocking solution (5% skim milk powder) (300 μL / well) at 25°C, 200 rpm, and incubate for 60 min. 3) After blocking, wash the plate three times with PBST, add mAb-1 and ADC-1 (100 μL / well), and incubate at 25°C, 200 rpm, for 60 min. 4) After incubation with the sample, wash the plate three times with PBST, add anti-human IgG Fc-HRP (100 μL / well), and incubate at 25°C, 200 rpm, and incubate for 60 min. 5) After 60 min incubation, TMB (100 μL / well) was used for color development for 5 min, and then the reaction was terminated with stop solution and the OD450 reading was read.

[0440] The ELISA test results showed that there was no significant difference in the antigen affinity of mAb-1 naked monoclonal antibody and ADC-1 for HER2 ECD, as shown in Figure 4.

[0441] Example 12: In vitro activity test of ADC-1 (BT-474, NCI-N87, HepG2)

[0442] The effect of ADC-1 on the proliferation of tumor cells with different ErbB2 / HER2 expression levels was tested as follows:

[0443] 1) 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 were seeded into 96-well cell plates at 100 μL per well (containing 1,000 to 10,000 cells) and cultured overnight in a cell culture incubator at 37°C, 5% CO2, 95% air, and 100% humidity.

[0444] 2) Different concentrations (10, 3.3, 1.1, 0.37, 0.12, 0.041, 0.014, 0.0046, 0.0015, 0.00051 nM) of ADC-1 or antibody mAb-1 or different concentrations (30, 10, 3.3, 1.1, 0.37, 0.12, 0.041, 0.014, 0.0046, 0.0015 nM) of MMAE (monomethyl auristatin E) were added to ErbB2 / HER2 positive cells cultured overnight; ErbB2 / HER2-negative cells were treated with various concentrations (100, 10, 3.3, 1.1, 0.37, 0.12, 0.041, 0.014, 0.0046, 0.0015 nM) of ADC-1 or mAb-1, or various concentrations (30, 10, 3.3, 1.1, 0.37, 0.12, 0.041, 0.014, 0.0046, 0.0015 nM) of MMAE overnight. Puromycin was added to a control group at a final concentration of 5 μM. Incubation was continued at 37°C for 72–120 h.

[0445] 3) Remove the cell plate from the 37°C cell culture incubator and equilibrate to room temperature for approximately 30 minutes. Add 100 μL of CellTiter Glo reagent to each well, shake on a shaker for 2 minutes, and then let it rest at room temperature in the dark for 10 minutes. Measure the luminescence value (RLU) using a Cytation 3 microplate reader.

[0446] 4) The results of the inhibitory effects of different drugs on tumor cell proliferation are shown in Tables 1 and 2 and Figures 5-7. ADC-1 and the MMAE small molecule toxin both showed significant inhibitory effects on ErbB2 / HER2-positive cells. mAb-1 showed a slight inhibitory effect on ErbB2 / HER2-positive cells, with ADC-1 being significantly superior to mAb-1 and MMAE. ADC-1 and mAb-1 had no inhibitory effects on ErbB2 / HER2-negative cells.

[0447] Table 1. Inhibitory effects of different drugs on tumor cell proliferation (IC 50 , nM)

[0448] Table 2. Inhibitory effects of different drugs on tumor cell proliferation (maximum killing percentage relative to the control group)

[0449] Example 13: In vivo activity testing of ADC-1 (NCI-N87 CDX mouse model)

[0450] The effect of ADC-1 on tumor growth in the ErbB2 / HER2 NCI-N87 CDX mouse model was tested as follows:

[0451] 1) Cell culture: NCI-N87 human gastric cancer cells (ATCC, Manassas, VA, cat # CRL-5822) were collected in the logarithmic growth phase and the cell density was adjusted with Matrigel buffer (PBS:Matrigel = 1:1). 7-9 week old SPF female BALB / c nude mice were subcutaneously injected with 0.2 mL of the prepared NCI-N87 cell suspension at the right scapula. The cell inoculation size was 10 × 10 6 / Only.

[0452] 2) Measure the tumor diameter with a vernier caliper and calculate the diameter using the formula V = 0.5axb 2 Tumor volume was calculated (where a is the longest diameter of the tumor and b is the shortest diameter of the tumor). Five days after cell inoculation, when the average tumor volume ranged from 100 to 300 mm 3 Animals were randomly divided into a vehicle control group and an ADC-1 3 mg / kg group, with 5 animals in each group. Animals were dosed via tail vein injection. The control group received an equal volume of vehicle (Vehicle). The day of group administration was defined as Day 0. Tumor volumes were measured twice weekly for 35 days after administration. Tumor volumes on Day 35 were compared between groups, and T / C and TGI values ​​were calculated using tumor volumes. The calculation formula is as follows: T / C% = T RTV / C RTV ×100%(T RTV :RTV in treatment group; C RTV : vehicle control group RTV). The relative tumor volume (RTV) was calculated based on the results of tumor measurement. The calculation formula was RTV = Vt / VO, where VO is the average tumor volume measured during grouping, Vt is the average tumor volume at a certain measurement, and T RTV with C RTV Data were collected on the same day. Calculation of TGI (%): TGI (%) = [1 - (average tumor volume of a treatment group at the end of treatment - average tumor volume of the treatment group at the start of drug administration) / (average tumor volume of the vehicle control group at the end of treatment - average tumor volume of the vehicle control group at the start of treatment)] × 100%.

[0453] 3) Independent sample t-test was used to analyze the data between groups. All analyses were performed using SPSS 17.0. P < 0.05 was considered statistically significant.

[0454] 4) Table 3 and Figure 8 show that ADC-1 can significantly inhibit tumor growth in the NCI-N87 CDX mouse model compared with the vehicle control group.

[0455] Table 3 Inhibitory effect of ADC-1 on the NCI-N87 CDX mouse model

[0456] Remark:

[0457] 1. Mean ± SEM;

[0458] 2. T / C% = T RTV / C RTV x100%(T RTV :RTV in treatment group; C RTV : RTV of vehicle control group). Relative tumor volume (RTV) was calculated based on the results of tumor measurement. The calculation formula is RTV=V 35 / V0, where V0 is the average tumor volume measured at the time of group administration (i.e., D0), V 35 T is the average tumor volume on day 35 after administration. RTV with C RTV Get data from the same day;

[0459] 3. TGI (%) = [1-(T 35 -T0) / (V 35 -V0)]×100)

[0460] 4. P values ​​were calculated based on tumor volume.

[0461] Example 14: Preparation and Characterization of ADC-2

[0462] The preparation and characterization were performed according 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 described below.

[0463] SDS-PAGE analysis of the antibody-drug conjugate ADC-2: After the conjugation reaction, the purity and conjugation efficiency of ADC-2 were determined by SDS-PAGE. The SDS-PAGE results of ADC-2 are shown in Figure 9. The conjugation reaction occurred specifically on the heavy chain of the antibody, and the ADC-2 heavy chain conjugated with the linker-payload exhibited a significant molecular weight shift compared to the heavy chain of the monoclonal antibody with the glycans removed, demonstrating successful site-specific conjugation of the linker-payload to the monoclonal antibody heavy chain. Unconjugated antibody was virtually absent from the conjugated product, resulting in a conjugation efficiency exceeding 95%, demonstrating the expected purity of the conjugated product.

[0464] HIC-HPLC analysis of ADC-2 was performed, and the test results are shown in Figure 10. As shown in the figure, the unconjugated cytotoxic antibody was less than 5%; the conjugated product was mainly DAR4, and the overall DAR value of ADC-2 drug was about 3.8.

[0465] The SEC-HPLC analysis of ADC-2 is shown in FIG11 : the high molecular weight aggregates in the ADC drug are less than 5%, and the ADC sample exists mainly in the monomer form at 8.1 min.

[0466] Plasma stability testing of ADC-2

[0467] Experimental Design:

[0468] Mixed healthy human plasma (equal volumes of 5 males and 5 females) was collected, ADC-2 was added to a specific final concentration, and the plasma was divided into 4 portions, each containing 450 μL. The samples were incubated in a 37°C incubator. The samples were collected at 0 h, 24 h, 48 h, and 96 h, and stored in a refrigerator at -60 to -90°C after collection to detect the free load shedding rate and DAR change rate.

[0469] LC-MS detection of small molecule toxins (the small molecule toxin of LP-6 involved in this embodiment is represented by payload)

[0470] For each sample (except Double Blank), 40 μL was added with a certain amount of internal standard precipitant (1 ng / mL DXd) and shaken for at least 10 min. For Double Blank, 40 μL of blank matrix was added with 120 μL of blank matrix without internal standard precipitant and shaken for at least 10 min. Centrifuge at 3600 g, 4°C, for 15 min, and 60 μL of supernatant was transferred to an injection vial. A certain amount of 0.1% FA ultrapure water was added and shaken for at least 3 min. Detection was performed by HPLC.

[0471] DAR detection by hybridization LC-MS

[0472] Weigh 1 g of CNBr-activated agarose (Sigma, Cat# C9142) into a 50 mL centrifuge tube. Add 50 mL of pre-chilled 1 mM HCl and incubate on a rotating mixer for at least 30 minutes at 4°C, 10 RPM. Remove the 1 mM HCl by centrifugation for a desired time. Wash the sol once with 5-10 volumes of deionized water and then three times with 0.1 M NaHCO₃. Take an appropriate amount of HER2 ECD and replace the buffer with 0.1 M NaHCO₃ using a 30 kD ultrafiltration tube. Mix the replaced HER2 ECD with the filler and incubate on a rotating mixer at 25°C, 10 RPM for 2 h ± 10 min for coupling. Add 5 mL of 0.1 M NaHCO₃ buffer and wash by centrifugation to remove uncoupled protein. Centrifuge for a desired time and wash three times. Add a desired amount of glycine (pH 8.0) to the filler and incubate at 2-8°C for 16 hours to block unreacted chemical groups on the filler. First, wash the filler with 5 mL of 0.1 M NaHCO₃ and centrifuge for a desired time. Then, wash the filler with a desired amount of acetate buffer and centrifuge for a desired time. Repeat this wash cycle several times. Transfer 0.1 mL of sample to a 1.5 mL EP tube and add 0.1 mL of HER2 ECD-conjugated filler. Incubate at 25°C, 10 RPM, for 2 hours. Wash three times with 1 mL of PBST and centrifuge for a desired time. After elution, centrifuge the supernatant, determine the concentration, and perform LC-MS analysis.

[0473] Result analysis:

[0474] ADC-2 incubated in healthy pooled human plasma for 0, 24, 48, and 96 hours showed DAR changes of 100.00%, 106.7%, 104.2%, and 106.5%, respectively. The shedding ratios of the small molecule toxin were only 0.006%, 0.179%, 0.373%, and 1.07%, respectively. These results demonstrate that after 96 hours of incubation at 37°C in healthy pooled human plasma, ADC-2 exhibited extremely low toxin shedding and a stable DAR. Low toxin shedding indicates a significant reduction in clinical side effects caused by free toxin. High DAR stability suggests that the antibody molecule can deliver more toxin molecules to the tumor, thereby improving drug efficacy. These two synergistic effects could significantly broaden the therapeutic window of ADC-2.

[0475] Example 15: In vitro activity test of ADC-2 (SK-BR-3, HCC1954, MDA-MB-468)

[0476] The effect of ADC-2 on the proliferation of tumor cells with different ErbB2 / HER2 expression levels was tested as follows:

[0477] Referring to the method described in Example 12, the antibody-drug conjugate ADC-2 was tested for its proliferative effects on cancer cells with varying ErbB2 / HER2 expression levels. For example, ErbB2 / HER2-positive human tumor cells, such as SK-BR-3 and HCC1954, and ErbB2 / HER2-negative human tumor cells, such as MDA-MB-468, were selected. The results of the inhibitory effects of different drugs on tumor cell proliferation are shown in Tables 4 and 5 and Figures 12-14. ADC-2 and the small molecule toxin both showed significant antiproliferative effects on ErbB2 / HER2-positive cells, while the antibody mAb-1 showed some antiproliferative effects on ErbB2 / HER2-positive cells, with ADC-2 significantly outperforming mAb-1. ADC-2 and mAb-1 had no inhibitory effects on ErbB2 / HER2-negative cells, demonstrating good targeting.

[0478] Table 4. Inhibitory effects of different drugs on tumor cell proliferation (IC50, nM)

[0479] Table 5 Inhibitory effects of different drugs on tumor cell proliferation (maximum killing percentage relative to the control group)

[0480] Example 16: Preparation and Characterization of ADC-3

[0481] The antibody-drug conjugate ADC-3 was prepared and characterized according to the method described in Example 11, except that the antibody used was the anti-TROP2 antibody, mAb-2, and the linker-payload used was LP-6. The characterization data of the antibody-drug conjugate ADC-3 are described below.

[0482] SEC-HPLC analysis of ADC-3, using a SEC column, examined the high-molecular-weight aggregation of ADC-1. The results, shown in Figure 15, showed <5% high-molecular-weight aggregates, indicating that the ADC sample existed primarily as a monomer, indicating negligible damage to the antibody from the coupling reaction.

[0483] High-resolution mass spectrometry (ESI-MS) DAR value analysis of antibody-drug conjugate ADC-3

[0484] The molecular weight of ADC-3 was analyzed by a high-resolution mass spectrometer, and the deconvoluted mass spectrum is shown in Figure 16. Based on the measured molecular weight information, by comparing it with the theoretical molecular weight, the major molecular weight variants can be assigned, and the mass spectrum abundance of each major molecular weight variant is used to perform DAR value analysis, and the average DAR value is calculated to be 3.93.

[0485] Example 17: In vitro activity test of ADC-3 (BxPC-3, FaDu, HepG2)

[0486] Similar operations were performed using the method described in Reference Example 12 to test the inhibitory effect of the antibody-drug conjugate ADC-3 on cancer cells with various TROP2 expression levels at different concentration gradients. For example, human tumor cells, such as BxPC-3, FaDu, and HepG2, were selected. The results of the inhibitory effects of different drugs on tumor cell proliferation are shown in Table 6 and Figures 17-19. ADC-3 and small molecule toxins both had significant inhibitory effects on TROP2-positive cells, while the TROP2 antibody, mAb-2, had no significant inhibitory effect on TROP2-positive cells. ADC-3 was significantly superior to both the monoclonal antibody and the small molecule toxin. Neither ADC-3 nor mAb-2 had an inhibitory effect on TROP2-negative cells, demonstrating good targeting.

[0487] Table 6. Inhibitory effects of different drugs on tumor cell proliferation (IC50, nM)

[0488] Example 18: In vivo activity testing of ADC-3 (NCI-N87 CDX mouse model)

[0489] Referring to a method similar to that described in Example 13, the effect of ADC-3 on tumor growth inhibition in the NCI-N87 CDX mouse model was evaluated. The tumor growth curves and body weight change curves after administration are shown in Figures 20-21. ADC-3 demonstrated good tumor growth inhibition and a good safety profile, and the experimental mice did not show any toxicity related to weight loss.

[0490] Example 19: Preparation and Characterization of ADC-4

[0491] ADC-4 was prepared and characterized by referring to the methods described for ADC-1, except that the antibody used was the anti-TROP2 antibody mAb-2. The characterization data of the antibody-drug conjugate ADC-4 are described below.

[0492] HIC-HPLC analysis of ADC-4, as shown in Figure 22, showed less than 5% unconjugated cytotoxic antibody; the conjugated product was primarily DAR2, with an overall DAR value of 1.89 for ADC-4. SEC-HPLC analysis of ADC-4, as shown in Figure 23, showed less than 5% high-molecular-weight aggregates in the ADC drug substance, indicating that the ADC sample was primarily present in monomeric form.

[0493] Example 20: Preparation and Characterization of ADC-5

[0494] ADC-5 was prepared and characterized using the methods described for ADC-1, except that the antibody used was the anti-TROP2 antibody mAb-2 and the linker-payload used was LP-2. The characterization data for the antibody-drug conjugate ADC-5 are described below.

[0495] HIC-HPLC analysis of ADC-5, as shown in Figure 24, shows that the unconjugated cytotoxic antibody is less than 5%; the conjugated product is primarily DAR2, with an overall DAR value of 1.87 for ADC-5. SEC-HPLC analysis of ADC-5, as shown in Figure 25, shows that high molecular weight aggregates are less than 5% in the ADC drug, indicating that the ADC sample exists primarily in monomeric form.

[0496] Example 21: Comparison of in vitro activity of ADC-4 and ADC-5 (BxPC-3, FaDu, HepG2)

[0497] With reference to the method described in Example 12, the antibody-drug conjugates ADC-4 and ADC-5 were tested for their effects on cancer cell proliferation at various levels of TROP2 expression. For example, TROP2-positive human tumor cells such as BxPC-3 (human pancreatic cancer cells) and FaDu (human laryngeal cancer cells), and TROP2-negative HepG2 (human liver cancer cells) were selected. The results of the inhibitory effects of different drugs on tumor cell proliferation are shown in Tables 7-8 and Figures 26-28. ADC-4, ADC-5, and the MMAE small molecule toxin all had significant inhibitory effects on positive cells, and there was no significant difference in activity between ADC-4 and ADC-5. The monoclonal antibody had no significant inhibitory effect on TROP2-positive cells. ADC-4, ADC-5, and the monoclonal antibody had no inhibitory effect on antigen-negative cells, reflecting good targeting.

[0498] Table 7 Inhibitory effects of different drugs on tumor cell proliferation (IC50, nM)

[0499] Table 8 Inhibitory effects of different drugs on tumor cell proliferation (maximum killing percentage relative to the control group)

[0500] Example 22: Preparation and Characterization of ADC-6

[0501] ADC-6 was prepared and characterized using the same methods as described for ADC-1, except that mAb-3 (trastuzumab) was used as the antibody and LP-6 as the linker-payload. Characterization data for the antibody-drug conjugate ADC-6 are described below. HIC-HPLC analysis of ADC-6, as shown in Figure 29, showed <5% unconjugated cytotoxic antibody; the conjugated product was primarily DAR4, with an overall DAR value of 3.93 for ADC-6. SEC-HPLC analysis of ADC-6, as shown in Figure 30, showed <5% high-molecular-weight aggregates in the ADC drug substance, indicating that the ADC sample existed primarily in monomeric form.

[0502] Example 23: In vitro activity test of ADC-6 (SK-BR-3, NCI-N87)

[0503] Referring to the method described in Example 12, the antibody-drug conjugate ADC-6 was tested for its proliferative effects on various cancer cells with varying HER2 expression levels. For example, two HER2 cell lines, SK-BR-3 and NCI-N87, were selected to compare the activity of ADC-2 (containing the Srt A tag) and ADC-6 (without the Srt A tag). The results showed that the target ADC had no difference in cell cytotoxicity in the presence or absence of the Srt A enzyme-specific recognition peptide sequence. See Figures 31-32.

[0504] The sequences involved in this application are as follows:

[0505] SEQ ID No. 1: mAb-1 light chain:

[0506] SEQ ID No. 2: mAb-1 heavy chain:

[0507] SEQ ID No. 3: mAb-2 light chain:

[0508] SEQ ID No. 4: mAb-2 heavy chain:

[0509] SEQ ID No. 5: mAb-3 light chain:

[0510] SEQ ID No. 6: mAb-3 heavy chain:

Claims

1. A linker-load compound having formula (I): in P is the load; DC(O)-L- is a linker; DC(O)- is an oligosaccharide structure, which is -the first hexose moiety or its derivative part-(the second hexose moiety or its derivative part) f -β-DN-acetylglucosamine moiety or -first hexose moiety or its derivative moiety-(second hexose moiety or its derivative moiety) f -β-D-glucose oxazoline moiety, wherein the 6-carbon of the first hexose group or its derivative is in the form of -C(O)-, which is -C(O)- in DC(O)-, and the β-DN-acetylglucosamine moiety is The β-D-glucose oxazoline part is and f is 0, 1, 2, 3, 4, 5, or 6; L is a linker, and L is directly connected to the carbonyl group in DC(O)- via -NH- therein, wherein when L is an unbranched linker, it is connected to 1 P, and t is 1, and when L is a branched linker, each branch can be connected to 1 P, and t is an integer greater than 1 (for example, 2, 3, 4, 5, 6, 7, 8, 9 or 10).

2. The linker-load compound of claim 1, wherein -L-(P) t -L 2 -L 1 -BP, that is, formula (I) is: in B is independently absent, or is 1) below, or is 2) below, or is a combination of 1) and 2) below: 1) a self-immolative spacer Sp1; 2) a divalent group, or a combination of two or more divalent groups, wherein the divalent group is selected from: -CR 1 R 2 、C 1-10 Alkylene, C 4-10 Cycloalkylene, C 4-10 heterocyclylene and -(CO)-; L 1 independently absent; or a non-cleavable sequence; or a cleavable sequence comprising an enzymatically cleavable amino acid sequence comprising 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids; L 2 Independently absent; or the following 1); or the following 2); or the following 1) and 2) combination: 1)-NH-C 2-20 Alkylene, wherein one or more -CH2- structures in the alkylene are optionally replaced by: -CR 3 R 4 -, -O-, -(CO)-, -S-, -S(=O)2-, -NR 5 -、 C 4-10 Cycloalkylene, C 4-10 Heterocyclylene, phenylene, wherein cycloalkylene, heterocyclylene and phenylene are each independently unsubstituted or selected from halogen, -C 1-10 Alkyl, -C 1-10 Haloalkyl, -C 1-10 Alkylene-NH-R 8 and --C 1-10 Alkylene-OR 9 is substituted with at least one substituent; 2) Amino acid residue sequence, i.e. -*(AA) n **-, n is an integer from 1 to 100, AA is independently an amino acid residue at each occurrence, * represents the N-terminus of the corresponding amino acid, ** represents the C-terminus of the corresponding amino acid, and -(C2H4-O) is optionally present between the amino group and the α-carbon of an amino acid m -(CH2) p -, 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; R 1 、R 2 、R 3 、R 4 、R 5 、R 6 、R 7 、R 8 、R 9 are each independently selected from hydrogen, halogen, substituted or unsubstituted -C 1-10 Alkyl, C 4-10 Cycloalkylene; or R 1 and R 2 and together with the carbon atoms to which they are attached form a 3-6 membered cycloalkylene group, and / or R 3 and R 4 and together with the carbon atoms to which they are attached form a 3-6 membered cycloalkylene group; P is with B part, or L 1 Part, or L 2 Partially connected load.

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

4. The linker-load compound of claim 3, wherein AA is independently any one of Phe, Lys, Gly, Ala, Leu, Asn, Val, Ile, Pro, Trp, Ser, Tyr, Cys, Met, Asp, Gln, Glu, Thr, Arg, His, or any combination thereof at each occurrence.

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

6. The linker-load compound according to any one of claims 2 to 5, wherein L 1 The cleavable sequence comprises an amino acid sequence that can be cleaved by an enzyme, wherein the amino acid sequence that can be cleaved by the enzyme comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids.

7. The linker-load compound according to any one of claims 2 to 6, wherein L 1 It is any one of Val, Cit, Phe, Lys, Gly, Ala, Leu, Asn or any combination thereof, preferably, it is -Val-Ala, -Gly-Gly-Phe-Gly-, -Phe-Lys-, -Val-Cit-, -Val-Lys-, -Gly-Phe-Leu-Gly-, -Ala-Leu-Ala-Leu-, -Ala-Ala-Ala- and combinations thereof.

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

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

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

11. The linker-load compound according to any one of claims 2 to 10, wherein -L 2 -L 1 -B- represents -Gly-Gly-Gly-Val-Cit-PABC- or -HN-(C2H4-O) m -(CH2) p -Gly-Gly-Phe-Gly-.

12. The linker-load compound according to any one of claims 1 to 11, wherein the load P is selected from the group consisting of small molecule compounds (e.g., small molecule drugs with various mechanisms of action, including various traditional small molecule drugs, photoacoustic dynamic therapy drugs, photothermal therapy drugs, etc., such as chemotherapy drugs, small molecule targeted drugs, immune agonists, etc., such as traditional cytotoxic drugs such as cisplatin, paclitaxel, 5-fluorouracil, cyclophosphamide and bendamustine; small molecule targeted drugs such as imatinib mesylate, gefitinib and anlotinib; immune agonists such as STING agonists, TLR agonists, etc.), nucleic acids and nucleic acid analogs, tracer molecules (including fluorescent molecules, biotin, fluorophores, chromophores, spin resonance probes and radioactive labels, etc.), short peptides, polypeptides, peptidomimetics and proteins.

13. The linker-cargo compound according to any one of claims 1 to 12, wherein the cargo P is a cytotoxin or a fragment thereof, optionally derivatized to attach to the L moiety in the compound of formula (I) according to claims 1 to 12; Preferably, the cytotoxin is selected from the group consisting of taxanes, maytansinoids, auristatins, epothilones, combretastatin A-4 phosphate, combretastatin A-4 and its derivatives, indole-sulfonamides, vinca alkaloids such as vinblastine, vincristine, vindesine, vinorelbine, vinflunine, vinglycinate, anhy-drovinblastine, dolastatin 10, 10) and its analogs, halichondrin B, indole-3-oxyacetamides, podophyllotoxins, 7-diethylamino-3-(2′-benzoxazolyl)-coumarin (DBC), discodermolide, laulimalide, camptothecins and their derivatives, mitoxantrone, mitoguanidine, nitrogen mustards, nitrosoureas, aziridines, benzodopa, carboquinone, metodepa, uredepa, dynemicin, esperamycin, icin), neocarzinostatin, aclarubicin, dactinomycin, anthramycin, bleomycin, actinomycin C, caramidocin, carminomycin, carminomycin, dactinomycin D, daunorubicin, detoxib, doxorubicin, epirubicin, esorubicin, idarubicin, mexicomycin, mitomycins, noramycin, olivomycin, peplomycin, porfiromycin, puromycin, ferroxorubicin, rhodorubicin, streptozocin, streptozocin, zorubicin, trichothecenes, T-2 toxin, verracurin A, baculosporin A, anguidine, ubenimex, azaserine, 6-diazo-5-oxo-L-norleucine, dimethylfolate, methotrexate, pteropterin, trimetrexate, edatrexate, fludarabine, 6-mercaptopurine, thiamidine, thioguanine, ancitabine, gemcitabine, enocitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, dooxyfluridine, floxuridine, captestosterone, drostanolone propionate, cyclothiosteroid, methotrexate, testolactone, aminoglutethimide, mitotane, trilostane, flutamide, nilutamide, bicalutamide, leuprolide acetate, protein kinase inhibitors and proteasome inhibitors; and / or selected from the group consisting of vinca alkaloids, colchicines, taxanes, auristatins, maytansinoids, calicheamicin, doxonubicin, duocarmucin, SN-38, cryptophycin analogs, deruxtecan, duocarmazine, calicheamicin, centanamycin, dolastansine, pyrrolobenzodiazepine, and exatecan, and derivatives thereof; and / or selected from auristatins, in particular MMAE, MMAF or MMAD; and / or Selected from exatecan and its derivatives, such as DX8951f; and / or Selected from DXd-(1) and DXd-(2); preferably DXd-(1).

14. The linker-carrier compound of any one of claims 2 to 13, wherein the carrier is selected from:

15. The linker-load compound of claim 2, which is selected from:

16. The linker-load compound of claim 1, wherein -L-(P) t for That is, formula (I) is: in, Ld2 and each Ld1 are independently a bond; or selected from -NH-C 1-20 Alkylene-(CO)-, -NH-(PEG) i -(CO)-; or each independently unsubstituted or -CO-(PEG) on the side chain j -R 11 Substituted natural amino acids or oligomeric natural amino acids having a degree of polymerization of 2-10 (i.e., 2, 3, 4, 5, 6, 7, 8, 9 or 10); -(PEG) i -and-(PEG) j - Each is a PEG fragment comprising a specified number of consecutive -(O-C2H4)- structural units or consecutive -(C2H4-O)- structural units, optionally with C appended at one end 1-10 alkylene; M is hydrogen or LKa-L 2 -L 1 -BP; Q is NH2 or L 2 -L 1 -BP; Provided that the following is not included: M is hydrogen and Q is NH2; Each LKa is independently selected opSu is or a mixture thereof; wherein * represents and L 2 The connecting part; Among them L 2 、L 1 , B is 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-(C2H4-O) m -(CH2) p -Gly-Gly-Phe-Gly-; R 11 It is C 1-10 alkyl; d is 0, 1, 2, 3, 4, 5, or 6; Each i is independently an integer from 1 to 100 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10); Each j is independently an integer from 1 to 100 (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10).

17. The linker-load compound of claim 16, wherein -L-(P) t Selected from 18. The linker-load compound of any one of claims 1 to 14 and 16, wherein The first hexosyl or its derivative is selected from glucosyl, mannosyl, galactosyl, fructosyl, gulosyl, idosyl or their derivatives, and the carbon at position 6 is in the form of -C(O)-; and / or The second hexasyl moiety or its derivative is independently selected at each occurrence from glucosyl, mannosyl, galactosyl, fructosyl or derivatives thereof; and / or The monosaccharide moieties in the oligosaccharide structure are linked by β-(1→4) glycosidic bonds; and / or The derivatives are independently selected from uronic acid or monosaccharide derivatives in which the hydroxyl group is replaced by an acylamino group.

19. The linker-load compound of any one of claims 1-14, 16, and 18, wherein The first hexose moiety or its derivative is selected from and / or, f is 0; and / or DC(O)- is a disaccharide structure and / or DC(O)- is a disaccharide structure 20. A method for preparing a linker-load compound according to any one of claims 1 to 19, comprising reacting DC(O)-OH with L'-(P) t An amide formation reaction is performed to form steps, where L' is the same as L defined in any one of claims 1 to 19, except that the -NH- attached to DC(O)- in L is H2N- in L'.

21. An antibody-drug conjugate based on site-directed attachment of an N-glycosylation site in the Fc region of an antibody, having a structure of formula (II): in R is hydrogen or α-L-fucosyl; q is 1 or 2; Ab is an antibody or antigen-binding fragment; -L-(P) t and f is as defined in any one of claims 1-19.

22. The antibody-drug conjugate of claim 21, wherein The first hexaglycosyl moiety or derivative thereof is as defined in claim 18 or 19; and / or The second hexaglycosyl moiety or derivative thereof is as defined in claim 18; and / or The derivative is as defined in claim 18; and / or The monosaccharide moieties are linked by β-(1→4) glycosidic bonds; and / or f As defined in claim 19.

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

24. The antibody-drug conjugate of claim 21, wherein formula (II) is selected from formula (II-1), (II-2), (II-3), (II-4), (II-5): in R is hydrogen or α-L-fucosyl; q is 2; Ab is 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-Nect in-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-Nectin4 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 anti-CD19 antibody, anti-ErbB2 / HER2 antibody, anti-CLDN18.2 antibody, anti-Nectin-4 antibody, anti-FGFR3 antibody, anti-Trop2 antibody; more preferably anti-ErbB2 / HER2 antibody, anti-Trop2 antibody; particularly preferably anti-ErbB2 / HER2 antibody (e.g., trastuzumab); (P) t -L- is selected from:

25. The antibody-drug conjugate according to any one of claims 20 to 24, wherein the preparation process comprises the following steps: a) Under the catalysis of glycosidase or its mutant, the antibody Ab is cleaved from its N-glycan chain to obtain an antibody whose Fc region N-glycosylation site is modified with N-acetylglucosamine or fucosyl-α-1,6-N-acetylglucosamine; b) coupling the modified antibody obtained in step a) with the linker-load compound according to any one of claims 1 to 19 under the catalysis of a glycosidase or a mutant thereof; in, The glycosidase or its mutant used in step a) and step b) may be the same or different.

26. A method for preparing the antibody-drug conjugate according to any one of claims 20 to 24, comprising the following steps: a) Under the catalysis of glycosidase or its mutant, the antibody Ab is cleaved from its N-glycan chain to obtain an antibody whose Fc region N-glycosylation site is modified with N-acetylglucosamine or fucosyl-α-1,6-N-acetylglucosamine; b) coupling the modified antibody obtained in step a) with the linker-load compound according to any one of claims 1 to 19 under the catalysis of a glycosidase or a mutant thereof; in, The glycosidase or its mutant used in step a) and step b) may be the same or different.

27. The method of claim 26, wherein: In steps a) and b), the glycosidase or its mutant is fucosylated enzyme, N-acetylglucosamine endohydrolase or its mutant.

28. The method of claim 27, wherein: 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 EndoS2.

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 in the preparation of a medicament for treating or preventing tumors or autoimmune diseases.

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

32. A compound having the structure 33. A method for preparing the compound as claimed in claim 32, comprising preparation steps.

34. The method of claim 33, further comprising: preparation steps.

35. The method of claim 34, further comprising: preparation steps.

36. The method of claim 35, further comprising: preparation steps.

37. The method of claim 36, further comprising: Prepared by forming a β-(1→4) glycosidic bond steps.

38. A linker-load compound obtained by a method comprising the steps of: (i) oxidizing the 6-primary alcohol of the terminal first hexose unit in an oligosaccharide containing a terminal first hexose unit and a terminal N-acetylglucosamine (GlcNAc) unit to a carboxyl group to obtain an intermediate compound (a) having a carboxyl group, wherein 1, 2, 3, 4, 5 or 6 second hexose units or derivatives thereof are optionally present between the terminal first hexose unit and the terminal N-acetylglucosamine (GlcNAc) unit; (ii) reacting the carboxyl group in the intermediate compound (a) obtained in step (i) with the reactive group in the linker-carrier compound (b) having a reactive group at the end to obtain the linker-carrier compound.

39. The linker-load compound of claim 38, wherein The first hexose unit is selected from glucosyl, mannosyl, galactosyl, fructosyl, gulosyl, and idosyl; and / or The second six-carbon glycosyl unit is independently selected at each occurrence from glucosyl, mannosyl, galactosyl or fructosyl; and / or The monosaccharide moieties in the oligosaccharide are linked by β-(1→4) glycosidic bonds; and / or The derivatives are independently selected from derivatives in which the hydroxyl groups of monosaccharides are replaced by acylamino groups.

40. The linker-load compound of claim 38 or 39, wherein The first hexose unit is selected from mannose, glucosyl; and / or The second hexose syl unit or its derivative part is not present.

41. The linker-load compound of claim 38, wherein The oligosaccharide in step (i) has the following structure:

42. The linker-load compound of any one of claims 38 to 41, wherein The reactive group of the linker-carrier compound (b) with a reactive group at the end in step (ii) is an amino group.

43. The linker-load compound according to any one of claims 38 to 42, wherein The method further comprises the step of converting the intermediate compound (a) having a carboxyl group into an acyl halide, and further reacting the intermediate compound (a) with a terminal reactive group to obtain the linker-load compound.

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