Antibody drug conjugate as well as preparation method and application thereof

Abstract

The invention provides a novel antibody drug conjugate based on camptothecin derivatives. The antibody drug conjugate has a structure as shown in the specification. The antibody drug conjugate shows higher spectator killing activity and higher tumor growth inhibiting activity, meanwhile, the killing effect on positive cells is weaker, and higher effectiveness and safety are achieved. # imgabs0 #

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to Chinese patent application No. 2024103064013, filed on March 15, 2024, entitled “Antibody-drug conjugates, preparation methods and applications thereof”. The entire contents of the above patent application are incorporated herein by reference. Technical Field

[0003] The present disclosure belongs to the field of biopharmaceuticals, and specifically relates to an antibody-drug conjugate based on camptothecin derivatives, and a preparation method and application thereof. Background Art

[0004] ADCs (Antibody-Drug Conjugates) are a new type of targeted anticancer therapy that chemically links highly selective antibodies to highly toxic chemotherapy drugs. The design concept of ADCs is to leverage the targeting properties of antibodies to deliver chemotherapy drugs directly to tumor cells, thereby minimizing damage to normal cells. Compared to traditional chemotherapy drugs, ADCs offer advantages such as strong targeting, minimal side effects, and a wide therapeutic window.

[0005] Camptothecins hold great promise for application in ADCs. Trastuzumab deruxtecan (trade name: Enhertu), an antibody-drug conjugate containing DXd, was approved for marketing by the US FDA on December 20, 2019. As the first marketed camptothecin ADC, it demonstrated superior efficacy to Kadcyla (trastuzumab deruxtecan) in clinical trials in breast cancer patients.

[0006] A key component of Enhertu's anti-tumor mechanism is the bystander effect. Tumor cells are highly heterogeneous, with significant variations in antigen expression levels across cells, including a large number of antigen-negative tumor cells. When ADC molecules enter tumor cells through antigen-mediated internalization, they release toxins within the endosomes and lysosomes, killing the tumor cells. These toxins then spread to neighboring tumor cells, further killing cells with low antigen expression and antigen-negative activity. Therefore, the continuous discovery and engineering of novel toxins is crucial to enhance their bystander effect, improve their safety, and develop ADC technologies with a wider therapeutic window, thereby further benefiting cancer patients. Summary of the Invention

[0007] To address the shortcomings of the prior art, the present disclosure has discovered an antibody-drug conjugate based on a novel camptothecin-class topoisomerase I inhibitor. This antibody-drug conjugate exhibits enhanced bystander killing activity and higher tumor growth inhibition activity. Furthermore, because the antibody-drug conjugate disclosed herein has a weaker killing effect on HER2-positive cells, it is expected to exhibit a higher tolerable dose in humans. The antibody-drug conjugate disclosed herein has higher efficacy and safety.

[0008] One aspect of the present disclosure provides an antibody drug conjugate or a pharmaceutically acceptable salt, isotope-labeled compound, solvate, tautomer, mesomer, racemate, enantiomer, diastereomer or mixture thereof, wherein the antibody drug conjugate has a structure shown in formula (I-1),

[0009]

[0010] In formula (I-1),

[0011] R1 is selected from substituted or unsubstituted C1-C6 alkylene, C2-C6 alkenylene and C2-C6 alkynylene,

[0012] Ab is an antibody or its antigen-binding fragment,

[0013] n is an integer from 2 to 8,

[0014] L" is a divalent linker having a linker connected to Ab.

[0015] In some embodiments, the antibody drug conjugate has a structure shown in formula (I-2),

[0016]

[0017] In formula (I-2), Ab, n, R1 and L" are the same as defined in formula (I-1).

[0018] In some embodiments, L" has a structure shown in formula (I-3),

[0019]

[0020] In formula (I-3), R2 is selected from substituted or unsubstituted C6-C10 aryl groups, p is selected from integers of 1-10, and q, r, s, t, u, and v are each independently selected from integers of 1-5.

[0021] In some embodiments, the substituted C1-C6 alkylene, C2-C6 alkenylene, C2-C6 alkynylene or C6-C10 aryl is substituted with one or more substituents independently selected from deuterium, halogen, C1-C3 alkyl, C1-C3 haloalkyl or C1-C3 alkoxy. In some embodiments, the halogen is selected from F, Cl, Br or I.

[0022] In some embodiments, R1 is selected from substituted or unsubstituted C1-C3 alkylene, C2-C3 alkenylene, and C2-C3 alkynylene. Preferably, R1 is selected from substituted or unsubstituted C1-C3 alkylene. More preferably, R1 is selected from C1-C3 alkylene, such as methylene, ethylene, or propylene.

[0023] In some embodiments, R2 is selected from substituted or unsubstituted phenyl and naphthyl. Preferably, R2 is phenyl.

[0024] In some embodiments, p is selected from an integer of 3 to 7, for example, 3, 4, 5, 6 or 7. Preferably, p is selected from an integer of 4 to 6. More preferably, p is 5.

[0025] In some embodiments, q, r, s, t, u, and v are each independently selected from an integer of 1-3, for example, 1, 2, or 3.

[0026] In some embodiments, the antibody drug conjugate has a structure shown in formula (I-4),

[0027]

[0028] wherein Ab and n are the same as those defined in the above scheme.

[0029] In some embodiments, the Ab is selected from a murine antibody, a chimeric antibody, a humanized antibody, a fully human antibody, an antibody fragment, a bispecific antibody, a multispecific antibody, or an antigen-binding fragment thereof.

[0030] In some embodiments, the Ab is selected from an antibody or an antigen-binding fragment thereof that targets human HER2.

[0031] In some embodiments, the Ab is selected from trastuzumab.

[0032] In some embodiments, in the antibody drug conjugate, the linker is to L" through the sulfur atom in the disulfide bond of Ab.

[0033] In some embodiments, the linker is a maleimide group, and the sulfur atom of the cysteine ​​residue in Ab is linked to the maleimide group in L".

[0034] In some embodiments, n is selected from an integer of 4-8, more preferably 6, 7 or 8, and most preferably 8.

[0035] In some embodiments, antibody molecules (e.g., IgG) are partially reduced to break interchain disulfide bonds, exposing free sulfhydryl groups on cysteine ​​residues for Michael addition to maleimide groups in the linker to complete the conjugation. In some embodiments, if the amount of reducing agent added is sufficient to break all four disulfide bonds, followed by the addition of an excess of linker-toxin, a uniform antibody-drug conjugate with a DAR value of 8 can be obtained.

[0036] The antibody-drug conjugates disclosed herein can be prepared by coupling the toxin drug-linker (drug compound disclosed herein) to the antibody through conventional preparation methods in the art.

[0037] Another aspect of the present disclosure provides a method for preparing an antibody-drug conjugate, comprising the following steps:

[0038] (1) reacting a camptothecin derivative represented by formula (II-1) with a linker L to obtain a compound represented by formula (II-2), wherein the linker L has a linker connected to Ab; and (2) coupling the compound represented by formula (II-2) with Ab to obtain an antibody-drug conjugate represented by formula (I-1),

[0039]

[0040] In formula (II-1), R1 is the same as defined in formula (I-1) above, and R3 is selected from hydrogen, deuterium,

[0041]

[0042] In formula (II-2), R1 is the same as defined in formula (I-1) above, L' is a monovalent linker,

[0043]

[0044] In formula (I-1), L", Ab, R1, and n are the same as those defined in formula (I-1) above.

[0045] In some embodiments, the camptothecin derivative has a structure shown in formula (II-3),

[0046]

[0047] In formula (II-3), R1 and R3 are the same as defined in formula (II-1).

[0048] In some embodiments, the linker L has a structure shown in formula (II-4), and the compound shown in formula (II-2) has a structure shown in formula (II-5);

[0049]

[0050] In formula (II-4), R2, p, q, r, s, t, u, and v are the same as those defined in formula (I-3).

[0051]

[0052] In formula (II-5), R1, R2, p, q, r, s, t, u, and v have the same definitions as in formula (I-2) and formula (I-3) above.

[0053] In some embodiments, the camptothecin derivative has a structure shown in formula (II-6),

[0054]

[0055] In some embodiments, the linker has a structure shown in formula (II-7),

[0056]

[0057] In some embodiments, step (1) comprises reacting the camptothecin derivative represented by formula (II-1) with a linker L in the presence of a polypeptide condensing agent and an organic solvent to obtain a compound represented by formula (II-2).

[0058] In some embodiments, the molar ratio of the camptothecin derivative represented by formula (II-1) to the linker L is 1:(0.5-2), for example, 1:0.5, 1:1 or 1:2, preferably 1:1.

[0059] In some embodiments, the reaction time is 20-30°C, preferably 22-28°C.

[0060] In some embodiments, the reaction time is 5-20 min, preferably 8-15 min.

[0061] In some embodiments, the organic solvent includes N,N-diisopropylethylamine and / or N,N-dimethylformamide.

[0062] In some embodiments, the polypeptide condensing agent comprises 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate.

[0063] In some embodiments, step (2) comprises first subjecting Ab to a reduction reaction in the presence of a thiol reducing agent, and then coupling the reduced Ab with a compound represented by formula (II-2) to obtain an antibody-drug conjugate represented by formula (I-1).

[0064] In some embodiments, the reduction reaction time is 30-45°C, preferably 35-40°C.

[0065] In some embodiments, the reduction reaction time is 0.5-5 h, preferably 1-3 h.

[0066] In some embodiments, the coupling temperature is 15-30°C, preferably 20-25°C.

[0067] In some embodiments, the coupling time is 0.5-5 h, preferably 0.5-2 h.

[0068] In some embodiments, the thiol reducing agent includes tris(2-carboxyethyl)phosphine.

[0069] In some embodiments, the molar ratio of the Ab to the thiol reducing agent is 1:(2-20), preferably 1:(10-15).

[0070] In some embodiments, the molar ratio of the reduced Ab to the compound represented by formula (II-2) is 1:(2:30), preferably 1:(10-20).

[0071] Another aspect of the present disclosure provides a pharmaceutical composition comprising the antibody-drug conjugate described in the present disclosure or the antibody-drug conjugate obtained by the preparation method, or a pharmaceutically acceptable salt, isotope-labeled compound, solvate, tautomer, meso-racemate, racemate, enantiomer, diastereomer or mixture thereof, and a pharmaceutically acceptable carrier.

[0072] Another aspect of the present disclosure provides use of the antibody-drug conjugate described herein, or a pharmaceutically acceptable salt, isotope-labeled compound, solvate, tautomer, meso-racemate, racemate, enantiomer, diastereomer, or mixture thereof, or the pharmaceutical composition described herein, in the preparation of a medicament for treating or preventing solid tumors or hematological tumors.

[0073] In some embodiments, the solid tumor or hematological tumor is selected from breast cancer, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, kidney cancer, urethral cancer, bladder cancer, liver cancer, gastric cancer, endometrial cancer, salivary gland cancer, esophageal cancer, lung cancer, colon cancer, rectal cancer, colorectal cancer, bone cancer, skin cancer, thyroid cancer, pancreatic cancer, melanoma, glioma, neuroblastoma, glioblastoma multiforme, sarcoma, lymphoma and leukemia.

[0074] Another aspect of the present disclosure provides a method for treating or preventing solid tumors or hematological tumors, comprising: administering to a subject an effective amount of the antibody-drug conjugate or the antibody-drug conjugate obtained by the preparation method, or a pharmaceutically acceptable salt, isotope-labeled compound, solvate, tautomer, mesoform, racemate, enantiomer, diastereomer or mixture thereof, or the pharmaceutical composition. BRIEF DESCRIPTION OF THE DRAWINGS

[0075] Figure 1 The effect of freeze-thaw on the purity of the ADC molecule is shown.

[0076] Figure 2 The effect of 4°C incubation on the purity of the ADC molecule is shown.

[0077] Figure 3 The effect of incubation at 25°C on the purity of the ADC molecule is shown.

[0078] Figure 4 The effect of incubation at 40°C on the purity of the ADC molecules is shown.

[0079] Figure 5 The results show the killing effect of Herceptin-DXd, isotype control molecule Isotype control-DXd and free toxin DXd on NCI-N87 cells.

[0080] Figure 6 The results of the killing of MDA-MB-468 cells by Herceptin-DXd, isotype control molecule Isotype control-DXd and free toxin DXd are shown.

[0081] Figure 7 The results of the killing of NCI-N87 cells by Herceptin-DXd and Herceptin-Compound 1 are shown.

[0082] Figure 8 The results of the killing of MDA-MB-468 cells by Herceptin-DXd and Herceptin-Compound 1 are shown.

[0083] Figure 9Shown are the results of detecting the bystander killing effect of ADC by flow cytometry.

[0084] Figure 10 Shown are the results of detecting the bystander killing effect of ADC using luciferase assay.

[0085] Figure 11 Shown are the changes in total antibody and intact compound concentrations over time in blood ADC samples from a rat PK study.

[0086] Figure 12 Shows the tumor growth curve in the in vivo efficacy experiment of the ADC molecule.

[0087] Figure 13 Shows the changes in mouse body weight during in vivo efficacy experiments of ADC molecules. DETAILED DESCRIPTION

[0088] To make the objectives, technical solutions, and advantages of the present invention more clearly understood, the present invention is further described in detail below with reference to the accompanying drawings and examples. The specific embodiments described herein are intended only to illustrate the present invention and are not intended to limit the present invention in any way. In addition, descriptions of known structures and techniques are omitted in the following description to avoid unnecessary confusion about the concepts of the present disclosure. Such structures and techniques are also described in many publications.

[0089] definition

[0090] Unless otherwise defined, all technical and scientific terms used in this disclosure have the same meaning as commonly used in the field to which this disclosure belongs. For the purpose of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural form, and vice versa.

[0091] Unless the context clearly dictates otherwise, as used herein, the expressions "a" and "an" include plural references. For example, reference to "a cell" includes a plurality of such cells and equivalents thereof known to those skilled in the art, and so forth.

[0092] As used herein, the term "about" refers to a range of ±20% of the value that follows. In some embodiments, the term "about" refers to a range of ±10% of the value that follows. In some embodiments, the term "about" refers to a range of ±5% of the value that follows.

[0093] The term "antibody" refers to a macromolecular compound that can recognize and bind to an antigen or receptor associated with a target cell. The function of an antibody is to present a drug to the target cell population to which the antibody binds. In the embodiments of the present disclosure, the antibody is represented by Ab. The antibody can form a bond with a linker unit through a heteroatom on the antibody, preferably an antibody or an antigen-binding fragment thereof, and the antibody is selected from a chimeric antibody, a humanized antibody, a fully human antibody, or a murine antibody; preferably a monoclonal antibody. The antibody unit is a targeting agent that specifically binds to a target moiety. The antibody is capable of specifically binding to a cellular component or to a cellular component or to another target molecule of interest. The target moiety or target is typically on the cell surface. In some aspects, the function of the antibody unit is to deliver the drug unit to the specific target cell population with which the antibody unit interacts. Suitable antibody units include, for example, antibodies, such as full-length (intact) antibodies and antigen-binding fragments thereof. In embodiments where the antibody unit is a non-antibody targeting agent, it can be a peptide or polypeptide, or a non-protein molecule. Examples of such targeting agents include interferons, lymphokines, hormones, growth factors and colony-stimulating factors, vitamins, nutrient transport molecules, or any other cell-binding molecule or substance.

[0094] The term "linker" or "linker fragment" or "linker unit" refers to a chemical structure fragment or bond that is connected to an antibody at one end and to a drug at the other end, and can also be connected to a drug after being connected to other linkers. In some embodiments, the linker is covalently linked to a sulfur atom of an antibody. In some aspects, the sulfur atom is the sulfur atom of a cysteine ​​residue that forms an interchain disulfide bond of an antibody. In another aspect, the sulfur atom is the sulfur atom of a cysteine ​​residue that has been introduced into an antibody unit, which forms an interchain disulfide bond of an antibody. In another aspect, the sulfur atom is the sulfur atom of a cysteine ​​residue that has been introduced into an antibody unit (e.g., by site-directed mutagenesis or chemical reaction). In other aspects, the sulfur atom to which the linker is bound is selected from a cysteine ​​residue that forms an interchain disulfide bond of an antibody or an additional cysteine ​​residue that has been introduced into an antibody unit (e.g., by site-directed mutagenesis or chemical reaction).

[0095] As used herein, "antibody" or "antibody unit" includes any part of the antibody structure within the scope thereof. This unit can bind, reactively associate with, or complex with a receptor, antigen, or other receptor unit possessed by the target cell population. An antibody can be any protein or protein-like molecule that can bind, complex, or react with a portion of the cell population to be treated or biomodified. The antibodies that constitute the antibody drug conjugates in the present disclosure retain their original antigen binding ability in the wild-type state. Therefore, the antibodies in the present disclosure are able to specifically bind to antigens. The antigens involved include, for example, tumor-associated antigens (TAAs), cell surface receptor proteins and other cell surface molecules, cell survival regulatory factors, cell proliferation regulatory factors, molecules related to tissue growth and differentiation (such as those known or predicted to be functional), lymphokines, cytokines, molecules involved in cell cycle regulation, molecules involved in angiogenesis, and molecules related to angiogenesis (such as those known or predicted to be functional). Tumor-associated factors can be cluster differentiation factors (such as CD proteins). Antibodies used in antibody drug conjugates include, but are not limited to, antibodies against cell surface receptors and tumor-associated antigens.

[0096] The term "antibody drug conjugate" refers to an antibody linked to a biologically active drug via a stable linker. In the present disclosure, "antibody drug conjugate" is preferably an antibody-drug conjugate (ADC), which refers to a monoclonal antibody or antibody fragment linked to a biologically active toxic drug via a stable linker.

[0097] The term "antibody" refers to immunoglobulins, which are tetrapeptide chains composed of two identical heavy chains and two identical light chains connected by interchain disulfide bonds. The amino acid composition and order of the constant region of immunoglobulins' heavy chains vary, resulting in different antigenicity. Consequently, immunoglobulins can be divided into five classes, or isotypes, namely IgM, IgD, IgG, IgA, and IgE, corresponding to the μ, δ, γ, α, and ε heavy chains, respectively. Within the same class, Ig can be further divided into subclasses based on the amino acid composition of the hinge region and the number and location of heavy chain disulfide bonds. For example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4. Light chains are classified as either kappa or lambda chains based on differences in the constant region. Each of the five Ig classes can have either kappa or lambda chains.The antibodies disclosed herein are preferably specific antibodies against cell surface antigens on target cells, and non-limiting examples include the following antibodies: anti-EGFRvIII antibody, anti-DLL-3 antibody, anti-PSMA antibody, anti-CD70 antibody, anti-MUC16 antibody, anti-ENPP3 antibody, anti-TDGF1 antibody, anti-ETBR antibody, anti-MSLN antibody, anti-TIM-1 antibody, anti-LRRC15 antibody, anti-LIV-1 antibody, anti-CanAg / AFP antibody, anti-cladin antibody, anti- 18.2 antibody, anti-Mesothelin antibody, anti-HER2 (ErbB2) antibody, anti-EGFR antibody, anti-c-MET antibody, anti-SLITRK6 antibody, anti-KIT / CD117 antibody, anti-STEAP1 antibody, anti-SLAMF7 / CS1 antibody, anti-NaPi2B / SLC34A2 antibody, anti-GPNMB antibody, anti-HER3 (ErbB3) antibody, anti-MUC1 / CD227 antibody, anti-AXL antibody, anti-CD166 antibody, anti-B7-H3 (CD276) antibody, anti-PTK7 / CCK4 antibody, anti-PRLR antibody, anti-EFNA4 antibody, anti-5T4 antibody, anti-NOTCH3 antibody, anti-Nectin 4 antibodies, anti-TROP-2 antibody, anti-CD142 antibody, anti-CA6 antibody, anti-GPR20 antibody, anti-CD174 antibody, anti-CD71 antibody, anti-EphA2 antibody, anti-LYPD3 antibody, anti-FGFR2 antibody, anti-FGFR3 antibody, anti-FRα antibody, anti-CEACAMs antibody, anti-GCC antibody, anti-Integrin Av antibody, anti-CAIX antibody, anti-P-cadherin antibody, anti-GD3 antibody, anti-Cadherin 6 antibody, anti-LAMP1 antibody, anti-FLT3 antibody, anti-BCMA antibody, anti-CD79b antibody, anti-CD19 antibody, anti-CD33 antibody, anti-CD56 antibody, anti-CD74 antibody, anti-CD22 antibody, anti-CD30 antibody, anti-CD37 antibody, anti-CD138 antibody, anti-CD352 antibody, anti-CD25 antibody or anti-CD123 antibody or more; preferably trastuzumab (Trastuzumab, trade name Herceptin), pertuzumab (Pertuzumab, also known as 2C4, trade name Perjeta), nimotuzumab (Nimotuzumab, trade name Taixinsheng), enoblituzumab, emibetuzumab, inotuzumab, pinatuzumab, brentuximab, gemtuzumab, bivatuzumab, lorvotuzumab, cBR96 and glematumamab.

[0098] The term "drug loading" refers to the average amount of cytotoxic drug loaded per antibody, and can also be expressed as the ratio of the amount of drug to the amount of antibody. In the present disclosure, it is characterized by the DAR (drug-to-antibody ratio), which is defined as the drug / antibody ratio, i.e., the average amount of drug loaded per antibody. In the present disclosure, the DAR value can range from 0 to 8, preferably 2 to 8, cytotoxic drugs (D) attached per antibody (Ab). In embodiments of the present disclosure, the drug loading is expressed as n, which can be an average of 1, 2, 3, 4, 5, 6, 7, or 8, for example. The average amount of drug per ADC molecule after the conjugation reaction can be characterized by conventional methods such as UV / visible spectroscopy, mass spectrometry, ELISA assays, and HPLC.

[0099] The term "pharmaceutically acceptable salt" or "pharmaceutically acceptable salt" refers to a salt of the antibody-drug compound of the present disclosure, or a salt of the compound described in the present disclosure. Such salts are safe and effective when used in mammals and have the desired biological activity. The antibody-drug conjugate compound of the present disclosure contains at least one amino group and can therefore form salts with acids. Non-limiting examples of pharmaceutically acceptable salts include: hydrochloride, hydrobromide, hydroiodide, sulfate, bisulfate, citrate, acetate, succinate, ascorbate, oxalate, nitrate, sorbate, hydrogen phosphate, dihydrogen phosphate, salicylate, hydrogen citrate, tartrate, maleate, fumarate, formate, benzoate, methanesulfonate, ethanesulfonate, benzenesulfonate, and p-toluenesulfonate.

[0100] The term "isotope-labeled compound" refers to an antibody drug conjugate of the present disclosure in which certain atoms are replaced by isotope atoms. Based on the properties of the isotope atoms, the presence of the compound or its fragments can be tracked by physical, chemical or biological processes, usually using radioactive isotopes (e.g. 3 H. 14 C. 32 P. 35 S. 45 Ca, 51 Cr, 59 Fe, 125 I. 131 I. 198 Ag, etc.) or stable isotopes (e.g. 2 H. 13 C. 15 N. 18 O, etc.) as tracers.

[0101] The term "solvate" or "solvate compound" refers to a pharmaceutically acceptable solvate formed between the antibody drug conjugate of the present disclosure and one or more solvent molecules. Non-limiting examples of solvent molecules include water, ethanol, acetonitrile, isopropanol, DMSO, and ethyl acetate.

[0102] The term "alkyl" refers to a saturated aliphatic hydrocarbon group, which is an alkyl group containing 1 to 6 carbon atoms. Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, etc. Alkyl can be substituted or unsubstituted, and when substituted, the substituent can be substituted on any available point of attachment, and the substituent is preferably one or more of the following groups, which are independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkyloxy, heterocycloalkyloxy, cycloalkylthio, heterocycloalkylthio, and oxo.

[0103] The term "alkylene" refers to a saturated straight or branched aliphatic hydrocarbon group having two residues derived from the same carbon atom or two different carbon atoms of a parent alkane, which is an alkylene group containing 1 to 6 carbon atoms. Non-limiting examples of alkylene groups include, but are not limited to, methylene (-CH2-, 1,1-ethylene (-CH(CH3)-), 1,2-ethylene (-CH2CH2)-, 1,1-propylene (-CH(CH2CH3)-), 1,2-propylene (-CH2CH(CH3)-), 1,3-propylene (-CH2CH2CH2-), 1,4-butylene (-CH2CH2CH2CH2-), and 1,5-butylene (-CH 2CH2CH2CH2CH2-) etc. Alkylene may be substituted or unsubstituted. When substituted, the substituent may be substituted at any available attachment point. The substituent is preferably independently selected from one or more substituents selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkyloxy, heterocycloalkyloxy, cycloalkylthio, heterocycloalkylthio and oxo.

[0104] The term "alkoxy" refers to -O-(alkyl) and -O-(cycloalkyl), wherein the definition of alkyl or cycloalkyl is as described above. Non-limiting examples of alkoxy include: methoxy, ethoxy, propoxy, butoxy, cyclopropyloxy, cyclobutyloxy, cyclopentyloxy, cyclohexyloxy. Alkoxy can be optionally substituted or unsubstituted, and when substituted, substituents are preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, sulfhydryl, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkyloxy, heterocycloalkyloxy, cycloalkylthio, heterocycloalkylthio.

[0105] The term "cycloalkyl" refers to a saturated or partially unsaturated monocyclic or polycyclic hydrocarbon substituent, wherein the cycloalkyl ring contains 3 to 20 carbon atoms, preferably 3 to 12 carbon atoms, more preferably 3 to 10 carbon atoms, and most preferably 3 to 8 carbon atoms. Non-limiting examples of monocyclic cycloalkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptatrienyl, cyclooctyl, and the like; polycyclic cycloalkyls include spirocyclic, fused, and bridged cycloalkyls.

[0106] The term "heterocyclyl" refers to a saturated or partially unsaturated monocyclic or polycyclic hydrocarbon substituent containing 3 to 20 ring atoms, one or more of which is a heteroatom selected from nitrogen, oxygen or S. Preferably, it contains 3 to 12 ring atoms, 1 to 4 of which are heteroatoms; more preferably, the cycloalkyl ring contains 3 to 10 ring atoms. Non-limiting examples of monocyclic heterocyclyls include pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, homopiperazinyl, etc. Polycyclic heterocyclyls include spirocyclic, fused ring and bridged ring heterocyclyls.

[0107] Example

[0108] Example 1 Preparation of ADC molecules

[0109] 1. Preparation of Compound 1

[0110]

[0111]

[0112] Step 1: Under an ice bath and nitrogen atmosphere, dissolve a solution of boron trichloride in n-hexane (1 mol / L, 32.0 mL, 32.0 mmol) in 1,2-dichloroethane (160 mL). Add compound 1-1 (5.00 g, 39.95 mmol) portionwise. After stirring for 10 minutes, add chloroacetonitrile (3.62 g, 47.95 mmol) and aluminum trichloride (7.99 g, 59.93 mmol) sequentially. Remove the ice bath, stir for 10 minutes, then raise the temperature to 85°C and stir for 24 hours. Monitor the reaction by LCMS and TLC. Cool the reaction system to room temperature, slowly add ice water (200 mL) to quench, and then add aqueous hydrochloric acid (5%, 50 mL). After stirring for 30 minutes, extract with dichloromethane (70 mL x 3). The resulting organic phases are combined, washed with saturated brine (100 mL), dried, and concentrated to yield the crude product. The crude product was purified by silica gel chromatography: 5%-10% ethyl acetate / petroleum ether gradient to obtain a mixture of 1-1-P1 and 1-1-P2. MS m / z (ESI): = 202.6 [M+H] + .

[0113] Step 2: Under nitrogen, a mixture of 1-1-P1 and 1-1-P2 (2.00 g, 9.92 mmol), (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indolizine-3,6,10(4H)-trione (3.13 g, 11.9 mmol), and anhydrous p-toluenesulfonic acid (0.68 g, 3.97 mmol) was dissolved in toluene (80 mL) and heated to 120°C with stirring for 18 hours. The reaction was monitored by LCMS and TLC. The reaction system was cooled to room temperature, quenched by the addition of water (200 mL), and extracted with ethyl acetate (80 mL × 3). The resulting organic phases were combined, washed with saturated brine (50 mL), dried, and concentrated to obtain the crude product. The crude product was purified by silica gel chromatography: 5%-10% methanol / dichloromethane gradient to give compound 1-2-P1 (MS m / z (ESI): =429.2 [M+H] + , retention time 1.142 minutes) and obtained compound 1-2-P2 (MS m / z (ESI): =429.2 [M+H] + , retention time 1.123 minutes).

[0114] Step 3: Dissolve compound 1-2-P1 (100 mg, 0.23 mmol) and tert-butyl 3,8-diazabicyclo[3.2.1]octane-3-carboxylate (49.5 mg, 0.23 mmol) in tetrahydrofuran (5 mL), heat to 70°C, and stir for 24 hours. Monitor the reaction by LCMS. Concentrate to afford compound 1-3. MS m / z (ESI): 605.2 [M+H] + .

[0115] Step 4: Compound 1-3 (130 mg, 0.21 mmol) was dissolved in dichloromethane (3 mL) at room temperature, trifluoroacetic acid (0.6 mL) was added, and the mixture was stirred for 1 hour. The reaction was monitored by LCMS. Concentration afforded a crude product, which was then purified by reverse phase chromatography using a gradient of 18% to 95% acetonitrile / buffer (0.1 mol / L formic acid in water) to afford compound 1-4. MS m / z (ESI): 505.2 [M+H] + . 1H NMR (400MHz, DMSO-d6) δ8.53(d,J=8.4Hz,1H),7.94(d,J=10.9Hz,1H),7.39(s,1H),6.57(s,1H),5.50(d,J=2.2Hz,4H),4.12(s,2H),3.22- 3.14(m,4H),2.85(d,J=11.7Hz,2H),2.63(s,3H),2.20(dt,J=6.5,2.7Hz,2H),2.01-1.90(m,2H),1.92-1.83(m,2H),0.95(t,J=7.3Hz,3H).

[0116] Step 5: Compound 1-4 (15.0 mg, 0.030 mmol), compound L-1 (Biode Pharmaceuticals, 18.3 mg, 0.030 mmol), and N,N-diisopropylethylamine (10 μL, 0.06 mmol) were dissolved in N,N-dimethylformamide (1 mL). 2-(7-Azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (11.3 mg, 0.03 mmol) was added and stirred at 25°C for 10 minutes. The reaction was monitored by LCMS. The crude product was purified by reverse phase chromatography using a gradient of 5% to 95% acetonitrile / buffer (0.1% formic acid in water) to afford compound 1. MS m / z (ESI): =1103.5 [M+H] + . 1H NMR (400MHz, DMSO-d6) δ8.55(t,J=6.6Hz,1H),8.46(d,J=8.4Hz,1H),8.31(t, J=6.1Hz,1H),8.14(d,J=8.0Hz,1H),8.07(t,J=5.7Hz,1H),8.02(t,J=5.7Hz, 1H),7.88(d,J=10.8Hz,1H),7.32(s,1H),7.22(d,J=5.9Hz,4H),7.18-7.12(m ,1H),6.99(s,2H),6.54(s,1H),5.41(d,J=12.9Hz,4H),4.67-4.39(m,3H),4. 24-4.09(m,3H),4.00(d,J=14.0Hz,1H),3.91(d,J=12.4Hz,1H),3.78-3.53(m ,6H),3.26-3.22(m,2H),3.17(d,J=11.8Hz,2H),3.04(dd,J=13.7,4.5Hz,2H) ,2.82-2.65(m,3H),2.51(s,3H),2.09(t,J=7.4Hz,4H),1.93-1.79(m,2H),1. 76-1.66(m,1H),1.53-1.38(m,5H),1.22-1.11(m,2H),0.87(t,J=7.3Hz,3H).

[0117] 2. The control compound (deruxtecan, purchased from MedChemExpress, product number HY-13631E) has the following structural formula:

[0118]

[0119] 3. Preparation of ADC molecules

[0120] Compound 1 and the control compound prepared by the aforementioned method were used as the linker-toxin portion of the ADC molecule, respectively.

[0121] The purified antibody Ab trastuzumab (Herceptin, purchased from Baiying Bio, product number B7432) with a monomer rate greater than 95% was exchanged into phosphate buffer using an ultrafiltration centrifuge tube at a concentration of 5 mg / ml. Tris(2-carboxyethyl)phosphine (TCEP) 12 times the molar number of the antibody was added and reacted at 37°C for 2 hours to open the disulfide bonds between the antibody chains. Then, 20 times the molar number of the antibody linker-toxin was added and reacted at 22°C for 1 hour. After the reaction, the solution was exchanged into PBS using an ultrafiltration centrifuge tube with a molecular weight cutoff of 30KDa, and the uncoupled linker-toxin was removed. The product after the liquid exchange was filtered using a 0.22 micron sterilizing filter and set aside to obtain the ADC molecule.

[0122] 4. DAR value and purity testing of ADC molecules

[0123] (1) Hydrophobic interaction chromatography DAR value:

[0124] The average drug-antibody ratio (DAR) of the conjugated products was determined by hydrophobic interaction chromatography using an Agilent 1260 Infinity II chromatograph with a TSKgel Butyl-NRP column. Mobile phase A consisted of phosphate buffer containing 1.5 M ammonium sulfate, and mobile phase B consisted of phosphate buffer containing 25% isopropanol. The mobile phase gradient was from 100% phase A to 100% phase B, as shown in Table 1. Peaks with different retention times corresponded to antibody molecules with different numbers of toxins attached, from which the DAR value was calculated.

[0125] Table 1

[0126] Time (minutes) A phase ratio B phase ratio 0 100％ 0％ 12 0％ 100％ 17 0％ 100％ 18 100％ 0％ 20 100％ 0％

[0127] (2) Size exclusion chromatography to detect monomer purity:

[0128] The monomer purity of the coupled product was determined by size exclusion chromatography using an Agilent 1260 Infinity II chromatograph, a TSKgel G300SWXL column, and an isocratic elution using a phosphate buffer solution containing 15% isopropanol as the mobile phase.

[0129] The relevant information, DAR value and purity of the obtained ADC molecules are shown in Table 2 below.

[0130] Table 2

[0131] Linker-toxin Antibody ADC molecules DAR Monomer purity Control compound Herceptin Hercetin-DXd 8.0 98.89％ Compound 1 Herceptin Herceptin-Compound 1 8.0 98.74％

[0132] Example 2 Physicochemical stability test of ADC molecules

[0133] The ADC molecules were prepared at a final concentration of 5 mg / mL in PBS (purchased from Gibco, Catalog No. 70011-044, pH 7.4) and preformulated histidine buffer (10 mM histidine, pH 6.0). The molecules were frozen and thawed repeatedly up to 6 times and incubated at 4°C, 25°C, and 40°C for up to 14 days. The freeze-thaw stability and high temperature stability of the ADC molecules were investigated using the aforementioned methods used in the preparation of the ADC molecules: size exclusion chromatography to determine monomer purity and hydrophobic interaction chromatography to determine the DAR value.

[0134] (1) Effect of freeze-thaw on DAR value

[0135] The effects of freeze-thaw on the DAR value of ADC molecules are shown in Table 3 below, and the effects on the purity of ADC molecules are shown in Table 4 and Figure 1 shown.

[0136] Table 3

[0137]

[0138] Table 4

[0139]

[0140] (2) Effect of different incubation temperatures on DAR values

[0141] The effects of incubation at 4°C, 25°C and 40°C on the DAR value of ADC molecules are shown in Table 5 below, and the effects on the purity of ADC molecules are shown in Tables 6 and Figure 2-Figure 4 shown.

[0142] Table 5

[0143]

[0144] Table 6

[0145]

[0146] From the above data, it can be seen that the two ADC molecules, Herceptin-DXd and Herceptin-Compound 1, performed very well under repeated freeze-thaw and high-temperature incubation conditions. The linker did not break, and the ADC molecules did not undergo significant breakage and aggregation.

[0147] Example 3 ADC molecule anti-tumor cell activity test

[0148] In this disclosure, NCI-N87 (HER2 antigen-positive cells, purchased from Nanjing Kebai, CBP60491) and MDA-MB-468 (HER2 antigen-negative cells, purchased from Nanjing Kebai, CBP60387) were used as in vitro drug efficacy testing systems. Tumor cells were evenly seeded at 1000 cells per well in a 96-well plate and incubated in a CO2 incubator. After 24 hours, the cells were microscopically confirmed to be normal, and drug treatment was performed.

[0149] The drug (ADC, isotype control-DXd (ABINVIVO, Catalog No. B117901), or DXd toxin (Biodex Pharmaceuticals, Catalog No. 20220909-2)) was diluted in culture medium (RPMI1640 + 10% fetal bovine serum) at a starting concentration of 100 nM. The dilution factor was 5-fold, with a total of 8 concentration points (100 nM, 20 nM, 4 nM, 0.8 nM, 0.16 nM, 0.032 nM, 0.0064 nM, and 0.00128 nM, in duplicate wells). After mixing, the cells were added to the corresponding wells and incubated in a CO2 incubator at 37°C for 5 days. After 5 days, 50 μL of CellCounting-Life 2.0 (Vazyme, DD1101-02) was added to each well and incubated at room temperature for 15 minutes. Fluorescence values ​​were read using a microplate reader (TECAN-Spark).

[0150] Inhibition rate = (MAX-X) / MAX×100%

[0151] Where X is the average fluorescence intensity of the wells at each drug concentration, and MAX is the average fluorescence intensity of the control wells when the drug concentration is 0.

[0152] The half-maximal inhibitory concentration, IC50, was calculated using a standard four-parameter curve fitting method in GraphPad Prism 10 software.

[0153] The IC50 values ​​of Herceptin-DXd, isotype control molecule Isotype control-DXd and free toxin DXd are shown in Table 7 and Figure 5-Figure 6 As shown:

[0154] Table 7

[0155] cell lines Herceptin-DXd Isotype control-DXd DXD NCI-N87 0.32nM NA 6.70nM MDA-MB-468 25.21nM 19.83nM 0.54nM

[0156] Note: NA means not applicable.

[0157] The primary pathway for ADCs to enter cells is antigen-mediated endocytosis. As shown in Table 7 above, Isotype control-DXd has no cytotoxic activity against HER2 antigen-positive NCI-N87 cells, while Herceptin-DXd has significant cytotoxic activity, which is 20-fold higher than that of free toxin DXd. Against HER2 antigen-negative MDA-MB-468 cells, free toxin DXd exhibits potent cytotoxic activity, 40-50 times higher than that of ADCs, due to its inherent membrane permeability and independent of antigen-mediated endocytosis. Both Herceptin-DXd and Isotype control-DXd exhibit weak cytotoxic activity, likely due to the nonspecific endocytosis of ADCs by HER2 antigen-negative MDA-MB-468 cells (endocytosis, Fc receptor effects, etc.).

[0158] The cell killing activities of Herceptin-DXd and Herceptin-Compound 1 are shown in Table 8 and Figure 7-Figure 8 shown.

[0159] Table 8

[0160] cell lines Herceptin-DXd Herceptin-Compound 1 NCI-N87 0.38nM 1.18nM MDA-MB-468 21.19nM 18.68nM

[0161] The results showed that the cytotoxic activity of Herceptin-compound 1 on the HER2 antigen-positive cell line NCI-N87 was significantly weaker than that of Herceptin-DXd.

[0162] Example 4 ADC bystander killing effect test

[0163] (1) ADC bystander killing effect test based on flow cytometry

[0164] In this disclosure, NCI-N87 (HER2 antigen-positive) and MDA-MB-468 (HER2 antigen-negative) cells were used as the in vitro drug efficacy testing system. NCI-N87 and MDA-MB-468 cells were evenly seeded in a 3:1 cell ratio in 24-well plates and incubated in a CO2 incubator. After 24 hours, the cells were confirmed to be normal under a microscope and then treated with the drug.

[0165] The drug (ADC molecule final concentration of 3 nM) was diluted in culture medium (RPMI1640 + 10% fetal bovine serum), mixed thoroughly, and added to the corresponding wells. The last column served as the control group (i.e., cells + culture medium, no drug treatment). The cells were incubated in a CO2 incubator at 37°C for 5 days. After 5 days, the cells were harvested from each well, washed three times with PBS, and a mixture of LIVE / DEAD dye (Invitrogen, L34964) and goat anti-human IgG (H+L) dye (Life Technologies, A11013) was added at 100 μL / well. The cells were incubated at 4°C for 30 minutes. After washing three times with PBS, the cells were resuspended in FACS buffer (PBS + 2% FBS) and analyzed using a flow cytometer (BD FACS Celesta).

[0166] The bystander effect of ADC drugs was evaluated by measuring the number of viable MDA-MB-468 cells. The results are shown in Table 9 and Figure 9 shown.

[0167] Table 9

[0168]

[0169] The results showed that the ADC molecule had little killing effect on HER2 antigen-negative cells. However, when HER2 antigen-positive cells (NCI-N87) and antigen-negative cells (MDA-MB-468) were co-incubated, the toxin released by the killing of HER2 antigen-positive cells caused killing of HER2 antigen-negative cell fluid, which is called bystander killing effect. After the co-incubated cells were treated with Herceptin-DXd and Herceptin-Compound 1, the remaining HER2 antigen-negative living cells (MDA-MB-468) were 3% of those after isotype control-DXd treatment, indicating that both Herceptin-DXd and Herceptin-Compound 1 exhibited a strong bystander killing effect, and when the ADC molecule concentration was 3nM, the bystander killing effect of the two was comparable.

[0170] (2) ADC bystander killing effect test based on luciferase method

[0171] In this disclosure, NCI-N87 (HER2 antigen-positive cells) and MDA-MB-468-luc (HER2 antigen-negative cells, with the luciferase gene transferred into their DNA) were used as the research system for in vitro drug efficacy testing. The positive group NCI-N87, the negative group MDA-MB-468-luc, and the experimental group NCI-N87 / MDA-MB-468-luc (6:1 cell ratio) were evenly seeded in 96-well plates and incubated in a CO2 incubator. After 24 hours, the cells were confirmed to be normal under a microscope and then treated with the drug.

[0172] The drug was diluted with culture medium (RPMI1640 + 10% fetal bovine serum) (the starting concentration of the ADC molecule was 200nM, the dilution factor was 5 times, and there were 8 concentration points, namely 200nM, 40nM, 8nM, 1.6nM, 0.32nM, 0.064nM, 0.0128nM and 0.00256nM, in duplicate wells), mixed and added to the corresponding cell wells, and placed in a carbon dioxide incubator at 37°C for incubation for 5 days. After 5 days, 50uL CellCounting-Life 2.0 (Vazyme, DD1101-02) was added to each well of the positive group, and 50uL ONE-Step was added to each well of the negative group and the experimental group. TM Luciferase Assay System (BPS, 60690-2), incubate at room temperature for 15 minutes, and read the fluorescence value using a microplate reader (TECAN-Spark).

[0173] Inhibition rate = (MAX-X) / MAX×100%

[0174] Where X is the average fluorescence intensity of the wells at each drug concentration, and MAX is the average fluorescence intensity of the control wells when the drug concentration is 0.

[0175] The half-maximal inhibitory concentration, IC50, was calculated using a standard four-parameter curve fitting method in GraphPad Prism 10 software.

[0176] The bystander effect of the ADC molecule was evaluated by detecting the luciferase activity of MDA-MB-468-luc. The results are shown in Table 10 and Figure 10 shown.

[0177] Table 10

[0178] IC50(nM) Herceptin-DXd Herceptin-Compound 1 MDA-MB-468-luc 1.218 0.331

[0179] From the above results, it can be seen that the bystander killing activity of Herceptin-Compound 1 is about 3.7 times higher than that of Herceptin-DXd.

[0180] From the ADC molecule anti-tumor cell activity test experiment in Example 3, it can be seen that the killing activity of Herceptin-Compound 1 against positive cells is weaker than that of Herceptin-DXd. However, from the ADC bystander killing effect test experiment based on the luciferase method in Example 4, it can be seen that the bystander killing activity of Herceptin-Compound 1 is stronger than that of Herceptin-DXd. This may be related to the fact that Compound 1-4, as a toxin different from DXd, may have a stronger membrane permeation efficiency.

[0181] Example 5 ADC molecule rat PK experiment

[0182] To evaluate the pharmacokinetic properties of two ADC molecules, Herceptin-DXd and Herceptin-Compound 1, in rats, each ADC was intravenously injected at a dose of 3 mg / kg into three SD male rats (provided by Zhejiang Weitong Lihua Laboratory Animal Technology Co., Ltd., Certificate No.: 20230927Aazz0619000115). Before administration of the test article, approximately 80 μL of blank plasma was collected. Each animal then received a single dose of the test article for each group. Approximately 160 μL of blood was collected from the jugular vein of each rat at 15 minutes, 8 hours, 24 hours, 48 ​​hours, 96 hours, 168 hours, 240 hours, 336 hours, 504 hours, and 672 hours after test article administration. The blood was then transferred to a centrifuge tube containing EDTA-2K, stored on wet ice, and centrifuged within 1 hour (8000 rpm, 7 minutes, 4°C). The collected plasma was stored at -80°C for subsequent analysis.

[0183] The total antibody concentration was determined using human HER2 antigen protein (purchased from AcroBiosystems, Cat. No. HE2-H5225) and goat anti-human Fc secondary antibody-HRP (Sigma, Cat. #: A0170) as detection antibody (LLOQ: 15.6 ng / mL). The concentration of the complete compound was measured using a combination of anti-DXd antibody (Acro, Cat. #: DXD-S322) and goat anti-human Fc secondary antibody-HRP (LLOQ: 7.8 ng / mL).

[0184] Data were processed using Graphpad Prism 8.3.0 software to calculate sample concentrations, and drug metabolism parameters (non-compartmental analysis) were analyzed using Phoenix Win Nonlin 8.3 (Pharsight Inc.) software.

[0185] The concentration data of total antibody and intact compound of ADC samples in blood are shown in Table 11 and Figure 11 shown.

[0186] Table 11

[0187]

[0188]

[0189] Note: BQL stands for below detection limit and NA stands for not applicable.

[0190] A summary of the pharmacokinetic parameters of total antibody and intact compound following administration of Herceptin-Compound 1 and Herceptin-DXd to rats is provided in Table 12 below.

[0191] Table 12

[0192]

[0193] Comparison of the PK parameters of the total antibody and intact compound of Herceptin-DXd and Herceptin-Compound 1 revealed that both ADCs were very stable in rats, with no significant linker breakage or toxin shedding. Furthermore, comparison of various PK parameters between Herceptin-DXd and Herceptin-Compound 1 revealed that the PK performance of these two ADCs was very similar.

[0194] Example 6 In vivo efficacy experiment of ADC molecules

[0195] To evaluate the efficacy and preliminary safety of Herceptin-Compound 1 in mice, the pharmacological and pharmacodynamic effects of the test products, Herceptin-Dxd and Herceptin-Compound 1, were investigated in the NCI-N87 subcutaneous human gastric cancer xenograft model. Female BALB / c nude mice, 6-8 weeks old or weighing 18-22 g, were provided by Weitong Lihua Laboratory Animal Technology Co., Ltd.

[0196] All experimental animals were housed in a barrier-free animal room free of specific pathogens. Experiments were conducted after acclimation for 3 days. Human gastric cancer NCI-N87 tumor cells (from ATCC) were cultured in RPMI-1640 complete medium in a monolayer at 37°C in an atmosphere containing 5% CO2. Tumor cells were routinely subcultured 2-3 times per week. Cells in the exponential growth phase were collected and counted for use in tumor cell inoculation. 2.5x10 6 NCI-N87 tumor cells were resuspended in 0.05 ml PBS and mixed with 0.05 ml matrigel, and then inoculated subcutaneously on the right side of each mouse to form tumors. 3At about 14 days, appropriate tumor-bearing mice were selected according to the animal body weight and tumor volume, and then randomly divided into groups: blank control group, Herceptin-Dxd (0.5 mg / kg) low-dose group, Herceptin-Dxd (2 mg / kg) medium-dose group, Herceptin-Dxd (8 mg / kg) high-dose group, Herceptin-compound 1 (0.5 mg / kg) low-dose group, Herceptin-compound 1 (2 mg / kg) medium-dose group and Herceptin-compound 1 (8 mg / kg) high-dose group. The blank control group was intravenously administered with a solvent (DPBS), and the other groups were intravenously administered with the corresponding test article. All groups were administered with the test article once. During the experiment, the tumor volume and animal body weight of each group were monitored weekly, and the monitoring frequency was twice a week. The experimental results are shown in Tables 13 and 13 below. Figure 12-13 shown.

[0197] Table 13

[0198]

[0199] The in vivo efficacy studies in ADC mice demonstrated that Herceptin-Compound 1 exhibited clear antitumor activity in tumor-bearing mice, with average tumor volume significantly lower than that of the blank control. Compared with Herceptin-DXd, Herceptin-Compound 1 exhibited superior tumor growth inhibition at doses of 0.5 mg / kg and 2 mg / kg, likely due to the enhanced bystander cytotoxicity of Herceptin-Compound 1. Mouse body weight remained unchanged during dosing, and no mice died within the group, demonstrating the favorable safety profile of Herceptin-Compound 1 and its significant research and application value.

[0200] The technical solution of the present disclosure is not limited to the above-mentioned specific embodiments. Any technical variations made according to the technical solution of the present disclosure fall within the protection scope of the present disclosure.

Claims

1. An antibody-drug conjugate or a pharmaceutically acceptable salt, isotope-labeled compound, solvate, tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, wherein the antibody-drug conjugate has the structure represented by formula (I-1). In formula (I-1), R1 is selected from substituted or unsubstituted C1-C6 alkylene, C2-C6 alkenylene and C2-C6 alkynylene, Ab is an antibody or its antigen-binding fragment, n is an integer from 2 to 8, L" is a divalent linker having a linker connected to Ab.

2. The antibody-drug conjugate according to claim 1, or a pharmaceutically acceptable salt, isotope-labeled compound, solvate, tautomer, mesoform, racemate, enantiomer, diastereomer, or mixture thereof, wherein: The antibody-drug conjugate has a structure shown in formula (I-2), In formula (I-2), Ab, n, R1 and L" are the same as those defined in claim 1, Preferably, L" has the structure shown in formula (I-3), In formula (I-3), R2 is selected from substituted or unsubstituted C6-C10 aryl groups, p is selected from integers of 1-10, and q, r, s, t, u, and v are each independently selected from integers of 1-5.

3. The antibody-drug conjugate according to claim 1 or 2, or a pharmaceutically acceptable salt, isotope-labeled compound, solvate, tautomer, mesoform, racemate, enantiomer, diastereomer or mixture thereof, characterized in that: The substituted C1-C6 alkylene, C2-C6 alkenylene, C2-C6 alkynylene or C6-C10 aryl is substituted with one or more substituents independently selected from deuterium, halogen, C1-C3 alkyl, C1-C3 haloalkyl or C1-C3 alkoxy; and / or R1 is selected from substituted or unsubstituted C1-C3 alkylene, C2-C3 alkenylene and C2-C3 alkynylene, more preferably selected from substituted or unsubstituted C1-C3 alkylene, most preferably selected from C1-C3 alkylene; and / or R2 is selected from substituted or unsubstituted C6-C10 aryl, more preferably selected from substituted or unsubstituted phenyl, naphthyl, most preferably phenyl; and / or p is an integer selected from 3 to 7, more preferably an integer selected from 4 to 6, and most preferably 5; and / or q, r, s, t, u, and v are each independently selected from an integer of 1-3.

4. The antibody-drug conjugate according to any one of claims 1 to 3, or a pharmaceutically acceptable salt, isotope-labeled compound, solvate, tautomer, mesoform, racemate, enantiomer, diastereomer, or mixture thereof, characterized in that: The antibody-drug conjugate has a structure shown in formula (I-4), In formula (I-4), Ab and n are the same as defined in claim 1.

5. The antibody-drug conjugate according to any one of claims 1 to 4, or a pharmaceutically acceptable salt, isotope-labeled compound, solvate, tautomer, mesoform, racemate, enantiomer, diastereomer, or mixture thereof, characterized in that: The Ab is selected from a murine antibody, a chimeric antibody, a humanized antibody, a fully human antibody, an antibody fragment, a bispecific antibody, a multispecific antibody, or an antigen-binding fragment thereof, Preferably, the Ab is selected from an antibody or antigen-binding fragment thereof targeting human HER2, More preferably, the Ab is selected from trastuzumab.

6. The antibody-drug conjugate according to any one of claims 1 to 5, or a pharmaceutically acceptable salt, isotope-labeled compound, solvate, tautomer, mesoform, racemate, enantiomer, diastereomer, or mixture thereof, wherein: In the antibody-drug conjugate, the linker is connected to L" via the sulfur atom in the disulfide bond of Ab; Preferably, the linker is a maleimide group, and the sulfur atom of the cysteine ​​residue in Ab is connected to the maleimide group in L"; and / or Said n is selected from integers of 4-8, more preferably 6, 7 or 8, most preferably 8.

7. A method for preparing an antibody-drug conjugate, comprising the following steps: (1) The camptothecin derivative represented by formula (II-1) reacts with a linker L to obtain a compound represented by formula (II-2), wherein: The linker L has a linker connected to Ab; (2) coupling the compound represented by formula (II-2) with Ab to obtain the antibody-drug conjugate represented by formula (I-1); In formula (II-1), R1 is as defined in claims 1-3, and R3 is selected from hydrogen, deuterium, In formula (II-2), R1 is the same as defined in claims 1-3, L' is a monovalent linker, In formula (I-1), L", Ab, R1, and n are the same as those defined in claims 1-6.

8. The preparation method according to claim 7, characterized in that The camptothecin derivative has a structure shown in formula (II-3), In formula (II-3), R1 and R3 are the same as defined in claim 7; Preferably, the linker L has a structure represented by formula (II-4), and the compound represented by formula (II-2) has a structure represented by formula (II-5); In formula (II-4), R2, p, q, r, s, t, u, and v are the same as those defined in claims 2 to 6. In formula (II-5), R1, R2, p, q, r, s, t, u, and v have the same definitions as in claims 1 to 6.

9. The preparation method according to claim 7 or 8, characterized in that The camptothecin derivative has a structure shown in formula (II-6), Preferably, the linker has a structure shown in formula (II-7), 10. The preparation method according to any one of claims 7 to 9, characterized in that: Step (1) comprises reacting a camptothecin derivative represented by formula (II-1) with a linker L in the presence of a polypeptide condensing agent and an organic solvent to obtain a compound represented by formula (II-2); Preferably, the molar ratio of the camptothecin derivative represented by formula (II-1) to the linker L is 1:(0.5-2); Preferably, the reaction time is 20-30°C, more preferably 22-28°C; Preferably, the reaction time is 5-20 min, more preferably 8-15 min; Preferably, the organic solvent comprises N,N-diisopropylethylamine and / or N,N-dimethylformamide; Preferably, the polypeptide condensing agent comprises 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate; and / or Step (2) comprises first subjecting Ab to a reduction reaction in the presence of a thiol reducing agent, and then coupling the reduced Ab with the compound represented by formula (II-2) to obtain an antibody-drug conjugate represented by formula (I-1); Preferably, the reduction reaction time is 30-45°C, more preferably 35-40°C; Preferably, the reduction reaction time is 0.5-5h, more preferably 1-3h; Preferably, the coupling temperature is 15-30°C, more preferably 20-25°C; Preferably, the coupling time is 0.5-5h, more preferably 0.5-2h; Preferably, the thiol reducing agent comprises tris(2-carboxyethyl)phosphine; Preferably, the molar ratio of Ab to the thiol reducing agent is 1:(2-20), more preferably 1:(10-15); Preferably, the molar ratio of the reduced Ab to the compound represented by formula (II-2) is 1:(2-30), more preferably 1:(10-20).

11. A pharmaceutical composition comprising the antibody-drug conjugate according to any one of claims 1 to 6 or the antibody-drug conjugate obtained by the preparation method according to any one of claims 7 to 10, or a pharmaceutically acceptable salt, isotope-labeled compound, solvate, tautomer, mesoform, racemate, enantiomer, diastereomer or mixture thereof, and Pharmaceutically acceptable carrier.

12. Use of the antibody-drug conjugate according to any one of claims 1 to 6, or the antibody-drug conjugate obtained by the preparation method according to any one of claims 7 to 10, or a pharmaceutically acceptable salt, isotope-labeled compound, solvate, tautomer, mesoform, racemate, enantiomer, diastereomer, or mixture thereof, or the pharmaceutical composition according to claim 11, in the preparation of a medicament for treating or preventing solid tumors or hematological tumors; Preferably, the solid tumor or blood tumor is selected from breast cancer, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, kidney cancer, urethral cancer, bladder cancer, liver cancer, gastric cancer, endometrial cancer, salivary gland cancer, esophageal cancer, lung cancer, colon cancer, rectal cancer, colorectal cancer, bone cancer, skin cancer, thyroid cancer, pancreatic cancer, melanoma, glioma, neuroblastoma, glioblastoma multiforme, sarcoma, lymphoma and leukemia.

13. A method for treating or preventing solid tumors or hematological tumors, comprising: Administering to a subject an effective amount of the antibody-drug conjugate of any one of claims 1 to 6 or the antibody-drug conjugate obtained by the preparation method of any one of claims 7 to 10, or a pharmaceutically acceptable salt, isotope-labeled compound, solvate, tautomer, mesoform, racemate, enantiomer, diastereomer or mixture thereof, or the pharmaceutical composition of claim 11.

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