An antibody-drug conjugate, its preparation method and application

Abstract

The present invention provides an antibody-drug conjugate, a preparation method thereof, and an application thereof. The structural formula of the antibody-drug conjugate is as follows. Among them, mAb is the anti-HER2 antibody trastuzumab; the conjugate is obtained by Michael addition of the thiol group obtained by reducing the inter-chain disulfide bond of mAb with the linker-irinotecan conjugate LD038; n ranges from 1 to 10. The binding activity and internalization activity of the antibody-drug conjugate to various tumor target cells are not affected; compared with the same type of antibody-drug conjugate, the antibody-drug conjugate PRO1102 synthesized by the preparation method of the antibody-drug conjugate of the present invention shows certain advantages in terms of in vitro cytotoxicity to tumor cells, anti-tumor activity in rats in vivo, and in vivo pharmacokinetics.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceuticals, and particularly relates to an antibody-drug conjugate, a preparation method thereof, and an application thereof. Background Art

[0002] An antibody-drug conjugate (ADC) is a conjugate in which a monoclonal antibody or an antibody fragment is linked to a bioactive small molecule cytotoxic drug through a chemical linker. Compared with traditional chemotherapy, this novel antibody-based drug can effectively deliver cytotoxic drugs to tumor target cells, combining the high specificity of antibodies and the high toxicity of cytotoxic drugs to tumors. Therefore, antibody-drug conjugates can more precisely bind to tumor cells and reduce damage to normal cells.

[0003] An ADC comprises three components: an antibody, a linker, and a cytotoxic drug. By targeting specific antigens, ADCs can penetrate into tumor tissues and be phagocytosed by tumor cells and enter lysosomes, where the cytotoxic drugs are released. Although nearly 15 ADC drugs have been successively marketed at present, the structural design of ADCs still needs to be further optimized. For example, an ideal linker should remain stable in the circulatory system and effectively release the payload (e.g., cytotoxic drug) in tumors. However, existing linkers usually release the payload non-specifically and inevitably cause off-target toxicity. It can be seen that linker design plays a key role in regulating the stability of ADCs in systemic circulation and the payload release efficiency in tumors, greatly affecting the pharmacokinetics (PK), efficacy, and toxicity profiles of ADCs.

[0004] Human epidermal growth factor receptor 2 (HER2, also known as ERBB2) is a member of the epidermal growth factor receptor family (EGFR). The HER2 gene is located on chromosome 17q21, and its encoded product is a transmembrane glycoprotein with tyrosine kinase activity, with a molecular weight of approximately 185 kD, also known as p185. HER2 forms homodimers or heterodimers with other EGFR receptors HER1 (EGFR, ErbB-1), HER3 (ErbB-3), and HER4 (ErbB-4), thereby autophosphorylating intracellular tyrosine residues to activate itself, and then activating downstream signals to promote the invasion and metastasis of cancer cells.

[0005] Currently, the marketed anti-HER2 targeted drugs mainly include: 1. Monoclonal antibodies, such as trastuzumab and Pertuzumab; 2. ADCs, such as trastuzumab emtansine (T-DM1) and trastuzumab deruxtecan (T-DXd). However, there is still a need for an anti-HER2 targeted drug with better efficacy and more effective in inhibiting tumor growth, especially ADC drugs. SUMMARY OF THE INVENTION

[0006] According to one aspect of the present specification, there is provided an antibody-drug conjugate having a structure shown in Formula Ia:

[0007] Wherein, mAb is the anti-HER2 antibody trastuzumab; the conjugate is obtained by Michael addition of the thiol group obtained by reducing the interchain disulfide bond of the mAb with the linker-irinotecan conjugate LD038 ; n ranges from 1 to 10.

[0008] In some embodiments, n ranges from 5 to 10.

[0009] In some embodiments, n is 8.

[0010] In some embodiments, the linker is a cleavable hydrophilic linker.

[0011] According to another aspect of the present specification, there is provided a method for preparing the above antibody-drug conjugate, the method comprising:

[0012] a) Preparing the linker-irinotecan conjugate LD038

[0013]

[0014] b) After reducing trastuzumab, performing a coupling reaction with the LD038 to obtain the antibody-drug conjugate.

[0015] In some embodiments, after reducing trastuzumab and performing a coupling reaction with the LD038 to obtain the antibody-drug conjugate may include: Michael addition of the thiol group obtained by reducing the interchain disulfide bond of trastuzumab with the LD038.

[0016] In some embodiments, preparing the linker-irinotecan conjugate LD038 includes:

[0017] i) Synthesizing compound 38-6

[0018] ii) React the compound 38-6 with the compound 38-7

[0019]

[0020] to obtain the conjugate 38-9 of irinotecan and

[0021] iii) React the compound 38-9 with MC-OSu to obtain the LD038.

[0022] In some embodiments, the compound 38-6 is obtained by reacting the compound 38-1 N-hydroxysuccinimide (HOSu), the compound 38-3 and D-glucose.

[0023] In some embodiments, the compound 38-1 is mixed with the N-hydroxysuccinimide (HOSu), dichloromethane and EDCl are added, and the reaction is carried out at room temperature to obtain the compound 38-2

[0024] The compound 38-2, DIPEA and DMF are mixed, the compound 38-3 is added, and the reaction is carried out at room temperature to obtain the compound 38-4

[0025]

[0026] The Fmoc deprotection reaction is carried out on the compound 38-4 to obtain the compound 38-5

[0027]

[0028] The compound 38-5, the D-glucose, acetic acid and methanol are mixed and heated to 50 °C, after reacting for 30 minutes, NaCNBH3 is added, and the reaction is carried out at 50 °C for 16 h to obtain the compound 38-6.

[0029] According to another aspect of the present specification, there is provided a pharmaceutical composition comprising the above antibody-drug conjugate, and one or more pharmaceutically acceptable excipients, diluents or carriers.

[0030] According to still another aspect of the present specification, there is provided the use of the antibody-drug conjugate or the pharmaceutical composition in the preparation of a drug for treating HER2-overexpressing cancer.

[0031] In some embodiments, the cancer is selected from breast cancer, ovarian cancer and pancreatic cancer. Description of the Drawings

[0032] This specification will be further described by way of exemplary embodiments, which will be described in detail with reference to the accompanying drawings. These embodiments are not restrictive. In these embodiments, the same reference numerals represent the same structures, where:

[0033] Figure 1 is a chromatogram showing the determination of the content of PRO1102 by SEC-HPLC according to some embodiments of this specification;

[0034] Figure 2 is a chromatogram showing the determination of the content of trastuzumab-deruxtecan (8) conjugate by SEC-HPLC according to some embodiments of this specification;

[0035] Figure 3 is a chromatogram showing the determination of the content of trastuzumab-vedotin (4) conjugate by SEC-HPLC according to some embodiments of this specification;

[0036] Figure 4 is a graph showing the detection results of the binding activities of PRO1102, trastuzumab, and other trastuzumab conjugates to breast ductal carcinoma cells HCC1954 according to some embodiments of this specification;

[0037] Figure 5 is a graph showing the detection results of the binding activities of PRO1102, trastuzumab, and other trastuzumab conjugates to human ovarian cancer cells SKOV-3 according to some embodiments of this specification;

[0038] Figure 6 is a graph showing the detection results of the binding activities of PRO1102, trastuzumab, and other trastuzumab conjugates to breast cancer cells JIMT-1 according to some embodiments of this specification;

[0039] Figure 7 is a graph showing the detection results of the binding activities of PRO1102, trastuzumab, and other trastuzumab conjugates to human pancreatic cancer cells Capan-1 according to some embodiments of this specification;

[0040] Figure 8 is a graph showing the detection results of the internalization rates of trastuzumab in multiple tumor target cells according to some embodiments of this specification;

[0041] Figure 9 is a graph showing the detection results of the internalization rates of PRO1102 in multiple tumor target cells according to some embodiments of this specification;

[0042] Figure 10 It is a graph showing the cytotoxicity test results of PRO1102, trastuzumab, and other trastuzumab conjugates against breast ductal carcinoma cells HCC1954 as shown in some embodiments of this specification;

[0043] Figure 11 It is a graph showing the cytotoxicity test results of PRO1102, trastuzumab, and other trastuzumab conjugates against human ovarian cancer cells SK-OV-3 as shown in some embodiments of this specification;

[0044] Figure 12 It is a graph showing the cytotoxicity test results of PRO1102, trastuzumab, and other trastuzumab conjugates against breast cancer cells JIMT-1 as shown in some embodiments of this specification;

[0045] Figure 13 It is a graph showing the cytotoxicity test results of PRO1102, trastuzumab, and other trastuzumab conjugates against human pancreatic cancer cells Capan-1 as shown in some embodiments of this specification;

[0046] Figure 14 It is a tumor growth curve of PRO1102, trastuzumab, and other trastuzumab conjugates against breast ductal carcinoma cells HCC1954 after subcutaneous transplantation into nude mice as shown in some embodiments of this specification;

[0047] Figure 15 It is a tumor growth curve of PRO1102, trastuzumab, and other trastuzumab conjugates against human ovarian cancer cells SK-OV-3 after subcutaneous transplantation into nude mice as shown in some embodiments of this specification;

[0048] Figure 16 It is a tumor growth curve of PRO1102, trastuzumab, and other trastuzumab conjugates against breast cancer cells JIMT-1 after subcutaneous transplantation into nude mice as shown in some embodiments of this specification;

[0049] Figure 17 It is a tumor growth curve of PRO1102, trastuzumab, and other trastuzumab conjugates against human pancreatic cancer cells Capan-1 after subcutaneous transplantation into nude mice as shown in some embodiments of this specification;

[0050] Figure 18It is a graph showing the pharmacokinetics (PK) of PRO1102, trastuzumab, and other trastuzumab conjugates in rats as shown in some embodiments of this specification. Detailed implementation manners

[0051] To more clearly illustrate the technical solutions of the embodiments of this specification, the following will briefly introduce the accompanying drawings required for the description of the embodiments. Obviously, the accompanying drawings in the following description are only some examples or embodiments of this specification. For those of ordinary skill in the art, without creative efforts, this specification can also be applied to other similar scenarios based on these drawings.

[0052] As shown in this specification and the claims, unless the context clearly indicates otherwise, words such as "a", "an", "one", and / or "the" are not specifically singular and may also include plural. Generally speaking, the terms "comprising" and "including" only indicate the inclusion of the clearly identified steps, elements, and / or substances, and these steps, elements, and / or substances do not constitute an exclusive list and may also include other steps, elements, and / or substances.

[0053] Some commonly used abbreviations and English words in this application have the following meanings:

[0054] EDCl: 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride

[0055] HOSu: N-Hydroxysuccinimide

[0056] DCM: Dichloromethane

[0057] Fmoc: 9-Fluorenylmethoxycarbonyl

[0058] Boc: tert-Butyloxycarbonyl

[0059] DIPEA: N,N-Diisopropylethylamine

[0060] DMF: N,N-Dimethylformamide

[0061] DEA: Diethanolamine

[0062] D-glucose: D-Glucose

[0063] NaCNBH3: Sodium cyanoborohydride

[0064] MeOH: Methanol

[0065] THF: Tetrahydrofuran

[0066] HATU: 2-(7-Azabenzotriazol)-N,N,N',N'-tetramethyluronium hexafluorophosphate

[0067] TFA: Trifluoroacetic acid

[0068] DMSO: Dimethyl sulfoxide

[0069] EDTA: Ethylenediaminetetraacetic acid

[0070] TCEP: Tris(2-carboxyethyl)phosphine

[0071] LCMS: Liquid chromatography-mass spectrometry

[0072] SMCC-DM1: A small molecule formed by linking the linker 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid succinimidyl ester (SMCC) and the cytotoxic drug maytansine (DM1)

[0073] Mc-vcMMAE: A small molecule formed by linking the linker maleimidocaproyl (Mc), the cleavable dipeptide valine-citrulline (vc), and the cytotoxic drug monomethyl auristatin E (MMAE)

[0074] Definitions of some terms in this application:

[0075] The terms "compound / conjugate of the present specification", "antibody-drug conjugate of the present invention" refer to the compound shown in Formula Ia. This compound is named PRO1102 or trastuzumab-LD038. This term also includes various crystalline forms, pharmaceutically acceptable salts, hydrates or solvates of the compound of Formula Ia.

[0076] The term "pharmaceutically acceptable salt" refers to a salt formed by the compound shown in Formula Ia with an acid or a base, which is suitable for use as a drug. Pharmaceutically acceptable salts include inorganic salts and organic salts.

[0077] The term "drug" refers to cytotoxic drugs, specifically chemical molecules that can strongly disrupt the normal growth of tumor cells within tumor cells. In principle, cytotoxic drugs can kill tumor cells at a sufficiently high concentration, but due to the lack of specificity, while killing tumor cells, they will also cause apoptosis of normal cells, resulting in serious side effects. This term includes toxins, such as small molecule toxins or enzyme-active toxins of bacterial, fungal, plant or animal origin, radioactive isotopes, cytotoxic drugs, chemotherapeutic drugs, antibiotics and nucleases, preferably cytotoxic drugs. Cytotoxic drugs can be maytansine, auristatin, anthracyclines and camptothecin analogs, etc. Camptothecin analogs, as topoisomerase I (TOP1) inhibitors, inhibit DNA synthesis in vivo and play an anti-tumor role. Exemplary camptothecin analogs include topotecan, irinotecan (CPT-11), belotecan, exatecan, deruxtecan, etc. In some embodiments, the drug is exatecan, and its structural formula is

[0078]

[0079] The term "linker" 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, or can be connected to other linkers and then to the drug.

[0080] The term "antibody" generally refers to a Y-shaped tetrameric protein comprising two heavy chains (H) and two light chains (L) polypeptide chains held together by covalent disulfide bonds and non-covalent interactions. For example, the light chains of an antibody can be divided into κ and λ light chains. The heavy chains can be divided into μ, δ, γ, α, and ε, which define the isotypes of the antibody as IgM, IgD, IgG, IgA, and IgE, respectively. In both the light and heavy chains, the variable region is linked to the constant region by a "J" region of about 12 or more amino acids, and the heavy chain also contains a "D" region of about 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of 3 domains (CH1, CH2, and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The VH and VL regions can be further divided into hypervariable regions (called complementarity-determining regions (CDR)) separated by relatively conserved regions (called framework regions (FR)). Each VH and VL typically consists of 3 CDRs and 4 FRs in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from the N-terminus to the C-terminus. The variable regions (VH and VL) of each heavy chain / light chain pair form the antigen-binding site. Antibodies can have different antibody isotypes, such as IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM antibodies.

[0081] In the antibody-drug conjugates in this specification, the antibody refers to an anti-HER2 antibody. In some embodiments, the anti-HER2 antibody is trastuzumab.

[0082] The term "DAR value" refers to the average number of drugs attached to each antibody in the molecule of formula (Ia), and can also be expressed as the ratio of the amount of drug to the amount of antibody. In some embodiments of the present application, the DAR value is expressed as n. Exemplarily, n can be any integer or decimal value between 1 and 10, such as 1, 3, 5, 7, 8, 9, 10, etc. Conventional methods such as UV / visible light spectroscopy, mass spectrometry, ELISA assays, and HPLC can be used to characterize the DAR value of each ADC molecule after the conjugation reaction.

[0083] In the present invention, the term "pharmaceutically acceptable" ingredient refers to a substance that is suitable for humans and / or animals without excessive adverse side effects (such as toxicity, irritation, and allergic reactions), that is, a substance with a reasonable benefit / risk ratio.

[0084] The terms "about" and "around" can describe a certain range of values around a certain value, such as plus or minus 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of that value, etc. For example, the term "about 150 mm" 3 " can include 135 mm3 to 165 mm 3 。

[0085] "Between a - b" means any value between a and b, including decimals or integers. Between a - b includes the boundary values a and b. For example, "between 1 - 10" means any value between 1 and 10, such as 1, 5, 6.5, 8, 10, etc.

[0086] One aspect of the present specification provides an antibody - drug conjugate. Its structure is shown in Formula Ia

[0087]

[0088] wherein, mAb is the anti - HER2 antibody trastuzumab; the conjugate is obtained by Michael addition of the thiol group generated after reduction of the inter - chain disulfide bond of the mAb with the linker - irinotecan conjugate LD038 and n is between 1 - 10.

[0089] In some embodiments, the value of n is between 5 - 10. In some embodiments, the value of n is between 6 - 9. In some embodiments, the value of n is between 7 - 9. In some embodiments, the value of n is 8.

[0090] In some embodiments, the antibody - drug conjugate (ADC) shown in Formula Ia is composed of a linker, the DNA topoisomerase I inhibitor irinotecan, and the HER2 antibody trastuzumab.

[0091] In ADC drug design, a suitable linker can fully balance the interaction forces between the antibody and the small molecule, and the produced ADC has better stability to achieve the expected effect of the ADC drug. In some embodiments of the present specification, the linker connecting irinotecan and trastuzumab is a cleavable hydrophilic linker. Since this linker contains more hydroxyl groups, it can improve the hydrophilicity of the antibody - drug conjugate, thereby controlling the aggregation of the ADC, making the high - loaded ADC drug (DAR = 8) have good physicochemical properties. This linker contains a valine - citrulline (Val - Cit) dipeptide structure This dipeptide structure can prevent the premature release of small molecule drugs and increase the stability of the antibody - drug conjugate in the in - vivo circulatory system. This dipeptide structure shows broad sensitivity to a variety of cathepsins, including cathepsin B, cathepsin K, cathepsin L, etc. Cathepsin B is highly expressed specifically in tumor cells. Therefore, the dipeptide structure in the cleavable hydrophilic linker can release irinotecan inside the tumor.

[0092] In some embodiments, the cleavable hydrophilic linker further comprises a long-chain polyethylene glycol structure connected by an amide linking group. Such a linking group forms an amide bond after the reaction. The conjugate formed through the amide bond is relatively stable and is not easily cleaved in the systemic circulation, which can improve the stability of the ADC, thereby expanding the therapeutic window and enhancing the body's tolerance to the drug.

[0093] Irinotecan is an inhibitor of DNA topoisomerase I. Topoisomerase inhibitors act on topoisomerase and exhibit inhibitory activity against many tumors. DNA topoisomerase is a key enzyme for DNA replication and plays an important role in cell transcription, recombination, and repair. Compared with normal cells, the content and activity of topoisomerase in tumor cells are significantly increased. Therefore, topoisomerase is an excellent target for anti-tumor drugs. For example, camptothecin derivatives, podophyllotoxin derivatives, doxorubicin, etc. all target topoisomerase and interfere with DNA replication to exert anti-tumor effects. As a water-soluble camptothecin derivative, irinotecan has excellent anti-tumor functions.

[0094] Trastuzumab is a recombinant DNA-derived humanized IgG1 monoclonal antibody developed against the P185 glycoprotein regulated by the HER2 gene in the cell nucleus. The variable region of its light chain consists of a murine part and can recognize the P185 glycoprotein, while the constant region of the heavy chain and most of the light chain region are human parts. After entering the human body, trastuzumab can selectively bind to the P185 glycoprotein and inhibit the growth of HER2-positive tumor cells. Trastuzumab is a potential mediator of antibody-dependent cell-mediated cytotoxicity (ADCC), has anti-tumor effects itself, and can also enhance the sensitivity of tumor cells to chemotherapy, thereby improving the efficacy of chemotherapy.

[0095] The antibody-drug conjugate of the present specification uses a monoclonal antibody as a carrier to efficiently transport small molecule cytotoxic drugs to target tumor cells in a targeted manner, thereby exerting anti-tumor effects. The antibody-drug conjugate of the present specification can be used to treat HER2 overexpressing cancers, which may include breast cancer, ovarian cancer, lung cancer, pancreatic cancer, gastric cancer, colon cancer, endometrial cancer, colorectal cancer, etc. The cancers are preferably breast cancer, ovarian cancer, and pancreatic cancer.

[0096] Another aspect of the present specification provides a method for preparing the above-mentioned antibody-drug conjugate. The method may include the following steps:

[0097] a) Prepare the linker-irinotecan conjugate LD038

[0098]

[0099] b) After reducing trastuzumab, it is coupled with the LD038 to obtain the antibody-drug conjugate.

[0100] In some embodiments, after reducing trastuzumab, coupling it with the LD038 to obtain the antibody-drug conjugate may include: the thiol groups obtained by reducing the interchain disulfide bonds of trastuzumab undergo Michael addition with LD038. In some embodiments, one of the thiol groups in the disulfide bond after reduction undergoes a Michael addition reaction with LD038.

[0101] In some embodiments, the preparation of the linker-irinotecan conjugate LD038 includes the following steps:

[0102] i) Synthesize compound 38-6

[0103] ii) React the compound 38-6 with compound 38-7

[0104]

[0105] to obtain the irinotecan linker 38-9 and

[0106] iii) React the compound 38-9 with MC-OSu to obtain the LD038.

[0107] In some embodiments, the compound 38-6 is obtained by reacting compound 38-1 N-hydroxysuccinimide (HOSu), compound 38-3 and D-glucose.

[0108] In some embodiments, the compound 38-1 is mixed with the N-hydroxysuccinimide (HOSu), dichloromethane and EDCl are added, and the reaction is carried out at room temperature to obtain compound 38-2

[0109] The compound 38-2, DIPEA and DMF are mixed, the compound 38-3 is added, and the reaction is carried out at room temperature to obtain compound 38-4

[0110]

[0111] The Fmoc group of the compound 38-4 is removed by reaction to obtain compound 38-5

[0112]

[0113] Mix the compound 38-5, D-glucose, acetic acid and methanol and heat to 50 °C. After reacting for 30 minutes, add NaCNBH3 and react at 50 °C for 16 h to obtain the compound 38-6. In some embodiments, after adding NaCNBH3 and reacting at 50 °C for 4 h, NaCNBH3 and D-glucose can be supplemented and the reaction can continue for 12 h.

[0114] After trastuzumab and irinotecan are conjugated through a linker according to the above preparation method, the binding activity and internalization activity of the obtained ADC product to tumor target cells are not affected; compared with drug conjugates of the same type of trastuzumab, for example, trastuzumab-deruxtecan (8), the specific preparation method is shown in Example 2, trastuzumab-monomethyl auristatin E conjugate (trastuzumab-vedotin (4), the specific preparation method is shown in Example 4) and trastuzumab-maytansine conjugate (trastuzumab-emtansine (4), the specific preparation method is shown in Example 3), trastuzumab-LD038 (PRO1102) synthesized by the preparation method of the antibody-drug conjugate in this specification shows certain advantages in terms of in vitro cytotoxicity to tumor cells, anti-tumor activity in rats in vivo, and in vivo pharmacokinetics.

[0115] For more content about the preparation method, reference can be made to the content of Example 1. It should be noted that those skilled in the art can carry out large-scale or small-scale production according to the preparation method of the present invention. For example, if a large amount of antibody-drug conjugate needs to be produced, the amounts of each reagent can be increased and the reaction conditions can be modified accordingly. The replacement of various reagents in the method or the modification or replacement of their concentrations and ratios are all within the protection scope of the present invention.

[0116] According to another aspect of this specification, a pharmaceutical composition is provided, which includes the antibody-drug conjugate shown in the above formula Ia, and one or more pharmaceutically acceptable excipients, diluents or carriers.

[0117] The pharmaceutical composition includes a safe and effective amount of the antibody-drug conjugate. The effective amount may vary with the mode of administration and the severity of the disease to be treated, etc. The selection of the preferred effective amount can be determined according to various factors (for example, the weight of the patient, the immune status of the patient, the route of administration, pharmacokinetic parameters such as bioavailability, metabolism, half-life, etc.) (for example, through clinical trials).

[0118] In some embodiments, an excipient is an additive other than the active ingredient in a pharmaceutical composition, and can also be referred to as an adjuvant. Excipients can include, but are not limited to, binders, fillers, disintegrants, lubricants; the matrix portion in semi-solid preparations such as ointments and creams; preservatives, antioxidants, flavoring agents, fragrances, solubilizing agents, emulsifiers, solubilizers, osmotic pressure regulators, coloring agents, etc. in liquid preparations.

[0119] Diluents are used to increase the weight and volume of a pharmaceutical composition (e.g., in tablet form). Diluents can include, but are not limited to, starches, lactose, inorganic salts of calcium, microcrystalline cellulose, etc.

[0120] Pharmacologically acceptable carriers can include coating layers, capsules, microcapsules, nanocapsules, etc. or any combination thereof. In some embodiments, the carrier can protect the key components in the composition and reduce or avoid the inactivation or decomposition of the key components under some negative conditions (such as denaturation caused by oxidation, strong acids or strong bases, etc.). For example, enzymes in gastric juice or a relatively low pH value may cause the decomposition or inactivation of key components. The carrier can help maintain or improve the efficacy of the pharmaceutical composition by protecting the key components in the composition.

[0121] In some embodiments, the carrier can be used for the controlled release of key components (e.g., irinotecan). Controlled release can include, but is not limited to, slow release, sustained release, targeted release, etc. For example, the carrier can include hydrogel capsules, microcapsules or nanocapsules made of collagen, gelatin, chitosan, alginate, polyvinyl alcohol, polyethylene oxide, starch, cross-linked starch, etc. or any combination thereof.

[0122] In some embodiments, pharmaceutically acceptable carriers can include dispersion media (such as solvents), coatings, buffers, stabilizing agents, isotonic agents, absorption delaying agents, etc. Exemplary pharmacologically acceptable carriers can include phosphate buffered saline solutions, water, emulsions (such as oil / water emulsions), various types of wetting agents, sterile solutions, gels, bioadsorbable matrix materials, etc., or other suitable materials, or any combination thereof.

[0123] The pharmaceutical composition can be administered to a subject suffering from HER2-overexpressing cancer, e.g., a human or an animal. The pharmaceutical composition can be in the form of a sterile injectable aqueous solution. Acceptable solvents and vehicles for use include water, Ringer's solution, and isotonic sodium chloride solution. The sterile injectable preparation can be a sterile water-in-oil microemulsion in which the active ingredient is dissolved in the oil phase. For example, the active ingredient is dissolved in a mixture of soybean oil and lecithin. Then the oil solution is added to a mixture of water and glycerol and processed to form a microemulsion. The injection solution or microemulsion can be injected into the patient's blood by local bolus injection. In some embodiments, the composition can be stored at a suitable temperature, which can include room temperature (about 20 °C), 4 °C, -20 °C, -80 °C, etc. The composition can also be made into various forms that are conducive to storage and transportation, such as a powder. The powder can be a sterile powder, and a solvent can be added to the sterile powder and mixed evenly before use to prepare a solution for injection or topical administration.

[0124] According to another aspect of the present specification, there is provided the use of the antibody-drug conjugate or the pharmaceutical composition in the preparation of a drug for treating HER2-overexpressing cancer. In some embodiments, HER2-overexpressing cancer can include breast cancer, ovarian cancer, lung cancer, pancreatic cancer, gastric cancer, colon cancer, endometrial cancer, colorectal cancer, etc. In some embodiments, the cancer is preferably breast cancer, ovarian cancer, and pancreatic cancer.

[0125] Examples

[0126] Unless otherwise specified, the experimental methods in the following examples are all conventional methods. Unless otherwise specified, the test materials used in the following examples are all purchased from conventional biochemical reagent companies. In the following examples, all quantitative tests are set up with three repeated experiments, and the results are averaged.

[0127] Example 1 Preparation of the PRO1102 (trastuzumab-LD038) antibody-drug conjugate

[0128] (1) Synthesis of LD038

[0129]

[0130] Step 1: Add compound 38-1 (650 mg, 0.774 mmol) and N-hydroxysuccinimide (HOSu) (177.98 mg, 1.548 mmol) into a reaction flask, add dry dichloromethane (8 mL), stir at room temperature, add EDCl (296.69 mg, 1.548 mmol), stir at room temperature until the reaction is complete, add water for washing, extract twice repeatedly with DCM (10 mL * 2). Combine the extraction solutions, dry over anhydrous sodium sulfate, filter, and concentrate to dryness to obtain 38-2 (552 mg, 0.589 mmol, yield 76.12%), which is directly used for the next step reaction. LCMS: m / z = 959.4 (M+Na) + 。

[0131]

[0132] Step 2: Add compound 38-2 (300 mg, 0.357 mmol) and DIPEA (138.22 mg, 1.071 mmol) into a reaction flask, add dry DMF (2 mL), stir at room temperature, add compound 38-3 (87.97 mg, 0.357 mmol) into the reaction flask, stir at room temperature until the reaction is complete. The reaction solution is directly purified by reversed-phase preparative method (40 g C18 column, mobile phase is acetonitrile and 0.01% TFA aqueous solution). Combine the purified preparative solutions, concentrate and lyophilize to obtain compound 38-4 (260 mg, 0.243 mmol, yield 68.14%). LCMS ((M-100) / 2+H) + = 484.9。

[0133]

[0134] Step 3: Add compound 38-4 (260 mg, 0.243 mmol), diethylamine and acetonitrile into a reaction flask, stir at room temperature for 2 hours. The reaction solution is directly purified by reversed-phase preparative method (12 g C18 column, mobile phase is acetonitrile and 0.01% TFA aqueous solution). Combine the purified preparative solutions, concentrate and lyophilize to obtain compound 38-5 (170 mg, 0.201 mmol, yield 82.54%). LCMS, ESI m / z = 846.6 (M+H) + 。

[0135]

[0136] Step 4: Add compound 38-5 (170 mg, 0.201 mmol), D-glucose (217.08 mg, 1.206 mmol), acetic acid (1.21 mg, 0.020 mmol) and methanol (5 mL) into a reaction flask, heat to 50 °C, and react at this temperature for 30 minutes. Then add NaCNBH3 (75.98 mg, 1.206 mmol) and continue to react at this temperature for 4 hours. Add additional NaCNBH3 (75.98 mg, 1.206 mmol) and D-glucose (217.08 mg, 1.206 mmol), and react at 50 °C overnight. Remove methanol by concentration, prepare an aqueous solution, and purify by reversed-phase preparation to obtain compound 38-6 (106 mg, 0.090 mmol, yield 44.92%). LCMS, ESI m / z = 537.9 ((M - 100) / 2 + H) + 。

[0137]

[0138] Step 5: Add compound 38-6 (250 mg, 0.213 mmol), HATU (121.45 mg, 0.319 mmol), DIPEA (82.41 mg, 0.639 mmol) and DMF (2 mL) into a reaction flask, stir at room temperature for 5 minutes, then add compound 38-7 (178.88 mg, 0.213 mmol), and stir at room temperature for 2 hours until the reaction is complete. Purify the reaction solution directly by reversed-phase preparation (40 g C18 column, acetonitrile and 0.01% TFA aqueous solution as the mobile phase) to obtain compound 38-8 (270 mg, 0.135 mmol, yield 63.48%). LCMS, ESI m / z = 666.6 (M / 3 + H) + ,999.2 (M / 2 + H) + 。

[0139]

[0140] Step 6: Add compound 38-8 (120 mg, 0.060 mmol), dichloromethane and TFA (2 mL) into a reaction flask, react at room temperature for 1 hour until the raw materials disappear. Then add an aqueous sodium bicarbonate solution and continue to stir at room temperature for 1 hour to destroy all the trifluoroacetates. Concentrate the aqueous phase after removing dichloromethane and purify by reversed-phase preparation (C18 column, mobile phase is acetonitrile and 0.01% TFA aqueous solution) to obtain compound 38-9 (80 mg, 0.042 mmol, yield 70.19%). LCMS, ESI m / z = 633.2 (M / 3 + H) + ,949.2 (M / 2 + H).

[0141]

[0142] Step 7: Add compound 38-9 (20 mg, 0.011 mmol), DIPEA (4.08 mg, 0.032 mmol) and dry DMF (1 mL) into a reaction flask, stir the reaction at room temperature, slowly drip a DMF (1 mL) solution of compound 38-10 (4.88 mg, 0.016 mmol) into the reaction solution, react at room temperature overnight until the reaction is complete, and directly purify the reaction solution by reverse-phase preparation (C18 column, mobile phase: acetonitrile and 0.01% aqueous TFA solution) to obtain compound LD038 (PB038) (11 mg, 0.005 mmol, yield 49.91%), a white solid. LCMS, m / z = 697.7 (M / 3 + H) + . The NMR data of LD038 are as follows:

[0143] 1 1H NMR (400 MHz, DMSO-d6): δ 10.03 (s, 1H), 8.19 - 8.11 (m, 2H), 8.07 (d, J = 8.8 Hz, 1H), 7.96 (d, J = 7.6 Hz, 1H), 7.82 - 7.77 (m, 2H), 7.66 (d, J = 8.4 Hz, 1H), 7.60 (d, J = 8.4 Hz, 2H), 7.36 (d, J = 8.0 Hz, 2H), 7.32 (s, 1H), 7.00 (s, 2H), 6.53 (s, 1H), 5.99 (t, J = 5.6 Hz, 1H), 5.45 - 5.43 (m, 6H), 5.30 - 5.24 (m, 3H), 5.08 (s, 2H), 4.84 - 4.74 (m, 2H), 4.65 - 4.49 (m, 4H), 4.45 - 4.35 (m, 3H), 4.27 - 4.17 (m, 2H), 4.04 - 3.95 (m, 2H), 3.80 - 3.77 (m, 2H), 3.71 - 3.67 (m, 2H), 3.62 - 3.55 (m, 9H), 3.53 - 3.43 (m, 44H), 3.27 - 3.21 (m, 2H), 3.16 - 3.07 (m, 2H), 3.07 - 2.93 (m, 6H), 2.38 (s, 3H), 2.29 (t, J = 6.4 Hz, 2H), 2.23 - 2.13 (m, 2H), 2.13 - 2.08 (m, 2H), 2.00 - 1.82 (m, 4H), 1.73 - 1.54 (m, 4H), 1.54 - 1.40 (m, 7H), 1.40 - 1.30 (m, 4H), 1.30 - 1.14 (m, 5H), 0.90 - 0.81 (m, 9H) ppm.

[0144] (2) Preparation of PRO1102 (trastuzumab-LD038) conjugate

[0145] To the buffer solution of 20 mg of trastuzumab antibody, add 10 equivalents of aqueous TCEP-HCl solution. The buffer solution is 50 mM phosphate and 5 mM EDTA, pH = 6.9. React at 25 °C for 2 hours. The excess TCEP needs to be removed by ultrafiltration with a buffer solution of 50 mM phosphate and 5 mM EDTA, pH = 6.9. Add 9.5 equivalents of a 20 mg / mL aqueous solution of LD038 to the reduced antibody, and the conjugation reaction is carried out at 25 °C for 2 hours. The excess LD038 and its by-products are removed by ultrafiltration with a buffer solution of 50 mM phosphate and 5 mM EDTA, pH = 6.9. The ultrafiltered trastuzumab-LD038 conjugate is stored in a 20 mM histidine solution with 6% sucrose and 0.02% Tween 20 at pH = 5.6. Finally, the purity of the trastuzumab-LD038 conjugate measured by SEC-HPLC is 98%, and the DAR value measured by LCMS is 8.0. The obtained trastuzumab-LD038 conjugate is named PRO1102.

[0146] Example 2 Preparation of trastuzumab-deruxtecan (8) conjugate

[0147] To the buffer solution of 20 mg of trastuzumab antibody, add 10 equivalents of aqueous TCEP-HCl solution. The buffer solution is 50 mM phosphate and 5 mM EDTA, pH = 6.9. React at 25 °C for 2 hours. The excess TCEP needs to be removed by ultrafiltration with a buffer solution of 50 mM phosphate and 5 mM EDTA, pH = 6.9. Add 9.5 equivalents of a 20 mg / mL DMSO solution of deruxtecan to the reduced antibody, and the conjugation reaction is carried out at 25 °C for 8 hours. The excess deruxtecan and its by-products are removed by ultrafiltration with a buffer solution of 50 mM phosphate and 5 mM EDTA, pH = 6.9. The ultrafiltered trastuzumab-deruxtecan (8) conjugate is stored in a solution containing 6% sucrose and 0.02% Tween 20 at pH = 5.6 of 20 mM histidine. Finally, the purity of the trastuzumab-deruxtecan (8) conjugate measured by SEC-HPLC is 96%, and the DAR value measured by LCMS is 6.5.

[0148] Example 3 Preparation of trastuzumab-emtansine (4)

[0149] To a 20 mg trastuzumab antibody buffer, add a DMSO solution of 12 equivalents of SMCC-DM1. The buffer is 50 mM phosphate and 5 mM EDTA, pH = 7.5. React at 25 °C for 5 hours. Excess SMCC-DM1 and other by-products are removed by ultrafiltration using a buffer of 50 mM phosphate, 5 mM EDTA, and pH = 6.9. Store the ultrafiltered trastuzumab-emtansine (4) in a 20 mM histidine solution with 6% sucrose and 0.02% Tween 20 at pH = 5.6. Finally, the purity of trastuzumab-emtansine (4) measured by SEC-HPLC is 98%, and the DAR value measured by LCMS is 4.1.

[0150] Example 4 Preparation of trastuzumab-vedotin (4) conjugate

[0151] To a 20 mg trastuzumab antibody buffer, add an aqueous solution of 2.2 equivalents of TCEP-HCl. The buffer is 50 mM phosphate and 5 mM EDTA, pH = 6.9. React at 25 °C for 2 hours. Add a DMSO solution of 5.0 equivalents of 20 mg / mL Mc-vcMMAE to the reduced antibody, and the conjugation reaction is carried out at 25 °C for 2 hours. Excess Mc-vcMMAE and its by-products are removed by ultrafiltration using a buffer of 50 mM phosphate and 5 mM EDTA, pH = 6.9. Store the ultrafiltered trastuzumab-vedotin (4) conjugate in a 20 mM histidine solution with 6% sucrose and 0.02% Tween 20 at pH = 5.6. Finally, the purity of the trastuzumab-vedotin (4) conjugate measured by SEC-HPLC is 97%, and the DAR value measured by LCMS is 3.7.

[0152] Example 5 In vitro binding activity of antibody-drug conjugate to HER2-positive tumor cell lines

[0153] (1) Cell culture

[0154] HCC1954 (Kobio, catalog number CBP60374) is a human breast cancer cell line, SK-OV-3 (Kobio, catalog number CBP60291) is a human ovarian cancer cell line, and JIMT-1 (Kobio, catalog number CBP60378) is a human breast cancer cell line. These cell lines were purchased from Kobio. Raji (ATCC, catalog number CCL-86) is a lymphoma cell line purchased from ATCC, and Capan-1 is a human pancreatic cancer cell line, which was a gift from a friend. These cell lines were cultured in a CO2 incubator at 37°C and 5% CO2. HCC1954 and Raji were cultured in RPMI1640 (Gibco, catalog number 11875093) medium containing 10% FBS (Cell max, catalog number SA211.02), SK-OV-3 was cultured in McCoy's 5a Modified (Gibco, catalog number 16600082) medium containing 10% FBS (Cell max, catalog number SA211.02), JIMT-1 was cultured in DMEM (Gibco, catalog number C11995500BT) medium containing 10% FBS (Cell max, catalog number SA211.02), and Capan-1 was cultured in IMDM (Gibco, catalog number 12440053) medium containing 20% FBS (Cell max, catalog number SA211.02). The cells were passaged every 1 - 2 days. When passaging, 0.25% trypsin was used to digest the cells and ensure a reasonable passage density. When the cells were in the logarithmic growth phase and the number met the experimental requirements, the cells were collected and counted for subsequent cell experiments.

[0155] (2) Copy number of the tumor cell lines measured

[0156] The target copy number was detected by QIFKIT (DAKO, K0078). Cells were labeled with a mouse monoclonal antibody targeting the target antigen, and a fluorescein-conjugated anti-mouse antibody was used to label the cells, the set microspheres, and the calibration microspheres in the kit in parallel. The fluorescein is related to the number of mouse monoclonal antibody molecules bound to the cells and microspheres. The samples were analyzed on a flow cytometer, and the copy number was calculated according to the standard curve formula. The copy number of HER2 molecules on the surface of the detected tumor cell lines is shown in Table 1.

[0157] Table 1 Target copy number in tumor cell lines

[0158] Cell line Tumor type <![CDATA[HER2 copy number (×10 3 per cell)]]> HCC1954 Breast cancer 365 SK-OV-3 Ovarian cancer 467 JIMT-1 Breast cancer 70 Capan-1 Pancreatic cancer 33 Raji Lymphoma 0.18

[0159] As can be seen from Table 1, the copy number of SK-OV-3 cells is the largest, that of HCC1954 cells is the second, and the copy number of Raji is lower. Therefore, HCC1954, SKOV-3 HER2, JIMT-1, and Capan-1 are used as HER2-positive tumor cell lines (tumor target cells) for subsequent cell experiments.

[0160] (3) Evaluate the binding activity by flow cytometry

[0161] The binding activities of the antibody trastuzumab or the antibody-drug conjugates (ADCs) prepared in Examples 1-4 to the HER2-positive tumor cell lines HCC1954, SK-OV-3, JIMT-1, and Capan-1 were evaluated by flow cytometry (Beckman, Cytoflex). Cells were seeded at 3×10 5 cells / well on a V-bottom 96-well plate and then incubated with 100 μL of serially diluted trastuzumab or trastuzumab conjugate. After incubation at 4°C for 30 minutes, the cells were washed twice with PBS, stained with 100 μL of 1:200 diluted PE-anti-human Fc in FACS buffer (1×PBS containing 1% BSA), and then incubated at 4°C for 30 minutes. After incubation, the cells were washed twice with PBS and subjected to flow cytometry.

[0162] The results are shown in Figure 4 - Figure 7 and Table 2. The binding activities of PRO1102 and other trastuzumab conjugates to multiple tumor target cells are similar to that of trastuzumab, indicating that these trastuzumab conjugates after conjugation have not changed the binding activity of trastuzumab.

[0163] Table 2 EC of trastuzumab and its antibody-drug conjugates against multiple tumor cell lines 50

[0164]

[0165] Study on the internalization rate of the antibody-drug conjugate PRO1102 in HER2-positive tumor cell lines

[0166] The internalization ability of trastuzumab and its conjugate PRO1102 in HER2-positive tumor cell lines was detected using a flow cytometer. Cell culture was as described in (1) of Example 5 above. Using FACS buffer (1×PBS containing 0.1% BSA) containing 10 μg / mL trastuzumab or trastuzumab conjugate PRO1102, 3×10 5Cells were cultured at 4 °C for 30 minutes. Then the cells were washed at 4 °C to remove unbound substances and placed on ice or transferred to 37 °C as needed. The cells were stained with PE - anti - human Fc for 30 minutes at 4 °C at progressive time points (0 h, 0.5 h, 1 h, 2 h, 3 h, 4 h) and analyzed using a flow cytometer.

[0167] The internalization rate was calculated as follows: Subtract the mean fluorescence intensity (MFI) of cell - surface - bound antibody at each time point at 37 °C from the MFI of cell - surface - bound antibody at the 0 - hour time point at 4 °C, and then divide by the MFI of cell - surface - bound antibody at the 0 - hour time point at 4 °C.

[0168] The results are shown in Figure 8 - Figure 9 , and the internalization ability of PRO1102 in multiple tumor target cells is similar to that of trastuzumab, indicating that the internalization activity of trastuzumab is not changed after conjugation.

[0169] Example 7 Cytotoxicity Study of the Antibody - Drug Conjugate of the Present Invention against HER2 - Positive Tumor Cell Lines

[0170] Cell culture was as described in (1) of Example 5 above. One day before adding trastuzumab or the trastuzumab conjugates of Examples 1 - 4, the cells were harvested and seeded onto a 96 - well pure - white flat - bottom culture plate. The next day, the cells were exposed to trastuzumab or trastuzumab conjugates in samples with a range of 100 μg / mL to 0.05 μg / mL (3 - fold serial dilution). The culture plate was incubated at 37 °C for 96 - 120 hours. Subsequently, 40 μL of Cell - titer Glo (CTG) was added to each well of the culture plate, incubated for 5 minutes, and the luciferase was read using a microplate reader for analysis. All readings were normalized to the percentage of live cells in the untreated control wells, and the IC 50 value was calculated using Prism software.

[0171] The results are shown in Figure 10 - Figure 13 and Table 3. From the IC 50 data in Table 3, it can be seen that the cell activity data of PRO1102 with a TOP1 inhibitor as the pharmacodynamic molecule is comparable to that of trastuzumab - deruxtecan (8), and trastuzumab - emtansine (4) and trastuzumab - vedotin (4) with a tubulin inhibitor as the pharmacodynamic molecule both have strong activities, which is related to the activities of the pharmacodynamic molecules applied in ADC drugs.

[0172] Table 3 IC of trastuzumab and its antibody - drug conjugates against multiple tumor cell lines50

[0173]

[0174]

[0175] Study on the in vivo antitumor activity of an antibody-drug conjugate against HER2-positive tumor cell lines in a murine xenograft model

[0176] The in vivo antitumor activities of trastuzumab conjugates (trastuzumab-deruxtecan (8), trastuzumab-emtansine (4), trastuzumab-vedotin (4), and PRO1102) were evaluated in nude mice subcutaneously implanted with tumor cell lines HCC1954 (breast cancer), SK-OV-3 (ovarian cancer), JIMT-1 (breast cancer), and Capan-1 (pancreatic cancer).

[0177] Tumor models were established by injecting tumor cells suspended in 0.1 mL of medium (see Table 4). Tumor growth was observed daily after tumor inoculation, and mice with an average tumor size of approximately 150 mm 3 were selected and divided into groups according to tumor volume using stratified randomization. Administration of the drug was initiated on the day of randomization (randomization day defined as D0), and the mice received a single intravenous injection (on day 0) at the doses listed in Table 5.

[0178] Table 4 Experimental parameters of the tumor model

[0179]

[0180] This study was conducted according to a research protocol approved by the Institutional Animal Care and Use Committee (IACUC). During routine monitoring, all effects of tumor growth and drug administration on animal behavior were examined, such as activity, food intake, and water intake (by visual inspection), weight gain / loss (body weight measured 2 or 3 times per week), dull eyes / hair, and any other abnormal effects. Deaths and observed clinical signs were recorded. Animals that were observed to be in a continuously deteriorating condition or with a tumor size exceeding 3000 mm 3 were euthanized.

[0181] The primary endpoint was to observe whether tumor growth could be delayed or the mice could be cured. Tumor size was measured in two directions using calipers, and the volume (mm 3 ) was calculated using the following formula: V = 0.5 × a × b 2 , where a and b are the major and minor diameters of the tumor, respectively.

[0182] The tumor volume among groups was analyzed using one-way ANOVA. Tukey's post hoc test was used for comparison with the vehicle group.

[0183] All data were graphed and analyzed using the software GraphPad Prism 8.4.2. A P value < 0.05 was considered statistically significant.

[0184] Table 5 Administration regimens of trastuzumab conjugate in 4 mouse human tumor xenograft models

[0185]

[0186]

[0187] Figure 14 - Figure 17 They were the tumor growth curves of HCC1954, SK-OV-3, JIMT-1, and Capan-1 subcutaneously transplanted into nude mice, respectively. Compared with trastuzumab-deruxtecan (8), trastuzumab-emtansine (4), and trastuzumab-vedotin (4), PRO1102 showed stronger tumor inhibitory effects in multiple tumor models. For JIMT-1 and Capan-1, trastuzumab-emtansine (4) did not show significant tumor growth inhibitory effects, while PRO1102 showed significantly stronger inhibition of tumor growth than trastuzumab-deruxtecan (8). For Capan-1, PRO1102 showed significantly stronger inhibition of tumor growth than other conjugates and trastuzumab.

[0188] Through the method for preparing an antibody-drug conjugate provided in the examples of this specification, the linker-irinotecan conjugate LD038 was obtained, and LD038 was conjugated to the anti-HER2 antibody to obtain the antibody-drug conjugate PRO1102, which improved the killing ability of the antibody-drug conjugate against tumor target cells without changing the antibody affinity.

[0189] Example 9 Pharmacokinetic (PK) study of antibody-drug conjugate in rats

[0190] Male Sprague Dawley rats (n = 3 rats / group) received intravenous administration of 3 mg / kg trastuzumab and its conjugates (trastuzumab-deruxtecan (8), trastuzumab-vedotin (4), PRO1102). Orbital blood was cross-collected from each rat at 10 minutes, 4 hours, 1 day, 4 days, 7 days, 10 days, 14 days, and 21 days after dosing. The total anti-concentration in plasma was detected by an in-house developed assay and calculated using Winnonlin 8.2 software.

[0191] The results are shown in Figure 18 . PRO1102 exhibited excellent PK characteristics comparable to those of the un-conjugated parental trastuzumab.

[0192] The antibody-drug conjugate PRO1102 in the examples of this specification has good targeting function and good plasma stability, is not rapidly cleared by plasma, can effectively deliver small molecule drugs to tumor target cells. The cleavable hydrophilic linker used can make the prepared antibody-drug conjugate PRO1102 have PK behavior similar to that of naked anti-trastuzumab, with similar elimination half-life and clearance rate, and has good efficacy, and can be further used in later clinical studies.

[0193] Those skilled in the art should understand that the above examples are only for illustrating the present invention and do not constitute a limitation to the present invention. Any modifications, equivalent substitutions, and changes made within the spirit and principle of the present invention should be included within the protection scope of the present invention.

Claims

1. An antibody-drug conjugate, characterized in that, The conjugate has the structure shown in Formula Ia, wherein, mAb is the anti-HER2 antibody trastuzumab; The conjugate is obtained by Michael addition of the mercapto group obtained after reduction of the interchain disulfide bond of the mAb and the linker-irinotecan conjugate LD038 ; n ranges from 1 to 10.

2. The antibody-drug conjugate according to claim 1, wherein, n ranges from 5 to 10.

3. The antibody-drug conjugate according to claim 2, wherein n is 8.

4. The antibody-drug conjugate according to any one of claims 1-3, characterized in that, The linker is a cleavable hydrophilic linker.

5. A method for preparing the antibody-drug conjugate as claimed in claim 1, comprising the following steps: a) Preparing a linker-irinotecan conjugate LD038 b) After reducing trastuzumab, performing a conjugation reaction with the LD038 to obtain the antibody-drug conjugate.

6. The method for preparing the antibody-drug conjugate according to claim 5, wherein, The step of reducing trastuzumab and then performing a conjugation reaction with the LD038 to obtain the antibody-drug conjugate includes: The thiol groups obtained after reduction of the interchain disulfide bonds of trastuzumab undergo Michael addition with the LD038.

7. The method for preparing the antibody-drug conjugate according to claim 5, wherein, The step of preparing the linker-irinotecan conjugate LD038 includes: i) Synthesis of Compound 38-6 ii) Combine the said compound 38-6 with compound 38-7 React to obtain the conjugate 38-9 of irinotecan and iii) React the said compound 38-9 with MC-OSu to obtain the said LD038.

8. The method for preparing the antibody-drug conjugate according to claim 7, wherein, The compound 38-6 is obtained by reacting the compound 38-1 N-hydroxysuccinimide (HOSu), the compound 38-3 with D-glucose.

9. According to the method for preparing an antibody-drug conjugate as claimed in claim 8, characterized in that The compound 38-1 is mixed with the N-hydroxysuccinimide (HOSu), dichloromethane and EDCl are added, and the reaction is carried out at room temperature to obtain the compound 38-2 Mixing the compound 38-2, DIPEA and DMF, adding the compound 38-3, and reacting at room temperature to obtain the compound 38-4 Performing a de-Fmoc reaction on the compound 38-4 to obtain the compound 38-5 and Mixing the compound 38-5, the D-glucose, acetic acid and methanol, heating to 50 °C, reacting for 30 minutes, then adding NaCNBH3, and reacting at 50 °C for 16 h to obtain the compound 38-6.

10. A pharmaceutical composition comprising the antibody-drug conjugate as claimed in any one of claims 1-4, and one or more pharmaceutically acceptable excipients, diluents or carriers.

11. Use of the antibody-drug conjugate as claimed in any one of claims 1-4 or the pharmaceutical composition as claimed in claim 10 in the preparation of a drug for treating HER2-overexpressing cancers.

12. The application according to claim 11, wherein The cancers are selected from breast cancer, ovarian cancer and pancreatic cancer.

Images