Tumor microenvironment-activated drug conjugate and antibody-drug conjugate

Abstract

The present invention relates to a tumor microenvironment-activated drug conjugate structure and an antibody-drug conjugate. Specifically, the present invention provides a drug conjugate represented by the following formula, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof. The present invention also provides a pharmaceutical composition of the drug conjugate and use thereof.

Description

Tumor microenvironment-activated drug conjugates and antibody-drug conjugates Technical Field

[0001] The present invention belongs to the field of medicinal chemistry, and specifically relates to a tumor microenvironment activated drug conjugate and an antibody drug conjugate. Background Art

[0002] To expand the therapeutic window of antibody or protein-drug conjugates, researchers have been working to improve their tumor specificity and release efficiency. In recent years, driven by advances in tumor molecular biology and other basic disciplines, the research and development of targeted tumor therapeutics has made significant progress in many areas.

[0003] The method of linking effector molecules to antibodies by utilizing the natural cysteine ​​residues in antibody sequences has been widely used, such as cytotoxins, immune agonists, and immune antagonists, further promoting the use of antibodies. Typically, antibody-drug conjugates must first enter tumor cells through endocytosis to produce their efficacy, where they are then degraded to release the cell-killing effector molecules, thereby achieving tumor inhibition. However, after years of development, only two types of linkers that can be activated by cathepsin B have been successfully used, and these must enter tumor cells through endocytosis, which has become a major factor limiting the drug development of various antibodies.

[0004] The tetrapeptide Gly-Gly-Phe-Gly (GGFG) is a cathepsin B-activatable linker, used in the ADC Enhertu. Daiichi Sankyo's Enhertu is a plasma-stable ADC with a DAR of 7.7. It undergoes proteolytic degradation in lysosomes, releasing the DX-8951f derivative, a potent topoisomerase I inhibitor derived from exatecan. Achieving such a high DAR is remarkable due to the unique properties of the GGFG linker, demonstrating that the structure-activity relationship between the linker and the toxin molecule is crucial for ADC development. However, the GGFG linker combined with the DX-8951f derivative has been associated with interstitial pneumonitis, a toxicity seen in clinical trials with Enhertu (T-Dxd), Datopotamab deruxtecan (Dato-DXd), Raludotatug deruxtecan (R-DXd), and Ifinatamab deruxtecan (I-DXd). Comprehensive analysis of 8 T-DXd monotherapy trials: 879 patients with different tumor types, 139 patients (15.8%) have been diagnosed with ILD. 108 people experienced grade 1 or 2 events, and 21 people experienced grade 5 events. This may be because cathepsin B is expressed in lung epithelial cells, and GGFG is the linker that activates cathepsin B, which will produce a certain release in the lungs. Comprehensive analysis of 23 PD-1 monotherapy trials: 3284 patients with different tumor types, 4% of patients developed ILD. In the future, if T-DXd is combined with PD-1 treatment, the incidence of ILD may be further increased.

[0005] We discovered that leucopenia is highly expressed in tumors and developed a new leucopenia-activated small molecule drug conjugate, leucopenia. Clinical trials have shown that hundreds of patients treated with novel chemotherapy have experienced no interstitial pneumonia, demonstrating that leucopenia activation is more tumor-specific than cathepsin B. DX-8951f derivatives and the immune agonist T785 are significantly more toxic than conventional chemotherapy drugs such as doxorubicin and paclitaxel, making their development into broad-spectrum small molecule drug conjugates more challenging. By leveraging the diverse linkages and enzymatic activation of leucopenia linkers and toxins, we identified promising AAN-toxin combinations. By screening linker and toxin combinations using R1 and R2, we identified small molecule drug conjugates with promising preclinical therapeutic indices. These small molecules can be developed as standalone drugs or conjugated to antibodies via EMC or other conjugation methods to form antibody-drug conjugates. Summary of the Invention

[0006] The present invention aims to provide drug conjugates and antibody drug conjugates with strong specificity and high stability, and their use in treating and / or preventing cancer and / or inflammation. The drug conjugates and antibody drug conjugates of the present invention are activated only in pathological microenvironments (e.g., tumor microenvironments or inflammatory sites), releasing active molecules, overcoming drug resistance and reducing toxicity.

[0007] Specifically, the present invention provides a drug conjugate represented by the following formula, its stereoisomers or pharmaceutically acceptable salts thereof:

[0008] The present invention also provides a pharmaceutical composition comprising: (i) the drug conjugate according to any embodiment herein, its stereoisomers or pharmaceutically acceptable salts; and (ii) a pharmaceutically acceptable carrier.

[0009] The present invention also provides use of the drug conjugate described in any embodiment herein, its stereoisomers, or pharmaceutically acceptable salts thereof in the preparation of a medicament for treating and / or preventing tumors and / or inflammation. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1: Tumor volume in the human fibrosarcoma HT-1080 nude mouse xenograft model after administration of S41.

[0011] Figure 2: Tumor volume of human fibrosarcoma HT-1080 nude mouse xenograft model after administration of S41.

[0012] Figure 3: Tumor volume in the CT-26 nude mouse colon cancer xenograft model after administration of S41.

[0013] Figure 4: Tumor volume in the CT-26 nude mouse colon cancer xenograft model after administration of S41 and / or anti-PD1 antibodies.

[0014] Figure 5: Tumor volume in the CT-26 nude mouse colon cancer xenograft model after administration of S41. DETAILED DESCRIPTION

[0015] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a preferred technical solution.

[0016] It must be noted that, as used herein and in the appended claims, the singular forms "a," "an," "and," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "an antibody" includes a plurality of antibodies, and reference to "the antibody" includes reference to one or more antibodies and equivalents thereof known to those skilled in the art, and so forth. It should also be noted that claims may be drafted to exclude any optional element. Thus, this statement is intended to serve as antecedent basis for use of the terminology "solely," "only," and the like in connection with the recitation of claim elements, or for use of a "negative" limitation.

[0017] The inventors previously developed the small molecule drugs legumain activated by the enzyme legumain, containing AANL-DOX, and legumataxel, containing AAN-PABC-taxel and paclitaxel. These drugs have been patented in China and are currently undergoing key Phase II / III and Phase I clinical trials, respectively. However, the toxicity of doxorubicin and paclitaxel cannot meet the more severe toxicity requirements used in ADCs, necessitating the innovation of conjugate structures with novel structure-activity relationships. Studies have found that legumain forms complexes with integrins, which can efficiently activate specific substrate structures on the surface of tumor cells. Therefore, the inventors used legumain as the activating enzyme and conducted extensive structural screening in combination with various toxin and immune agonist chemical structures, as well as auxiliary structures and linker structures. Through biological mechanisms of action and in vitro and in vivo efficacy, they obtained restrictive, specialized conjugate structures. These conjugate structures are highly stable in human plasma and can efficiently activate and release the compatible chemical molecules in the tumor microenvironment (outside of tumor cells). Therefore, compared with traditional ADCs, this new release method can better exert the bystander effect and activate adjacent immune cells when the combined payload drug is an immune agonist. It has a different drug-making mechanism and scalability from traditional ADCs.

[0018] Drug conjugates

[0019] Provided herein are compounds represented by the following formula, stereoisomers thereof, or pharmaceutically acceptable salts thereof:

[0020] Pharmaceutical composition

[0021] The pharmaceutical composition of the present invention comprises: (i) a drug conjugate or a stereoisomer thereof or a pharmaceutically acceptable salt thereof; and (ii) a pharmaceutically acceptable carrier. The carrier can be any pharmaceutically acceptable carrier or excipient, which can vary depending on the dosage form and mode of administration. Pharmaceutically acceptable carriers are generally safe and non-toxic and can include any known substance used in the pharmaceutical industry to formulate pharmaceutical compositions, including fillers, diluents, coagulants, binders, lubricants, glidants, stabilizers, colorants, wetting agents, and disintegrants. Suitable pharmaceutically acceptable carriers include sugars, such as lactose or sucrose, mannitol or sorbitol; cellulose preparations and / or calcium phosphates, such as tricalcium phosphate or dibasic calcium phosphate; starches, including corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth gum, methylcellulose, hydroxypropyl methylcellulose, sodium carboxymethylcellulose and / or polyvinylpyrrolidone; silicon dioxide, talc, stearic acid or a salt thereof, such as magnesium stearate or calcium stearate; and / or polyethylene glycol, etc. When selecting a pharmaceutically acceptable carrier, the primary consideration is the mode of administration of the pharmaceutical dosage form. This is well known in the art.

[0022] The pharmaceutical composition may comprise a therapeutically or prophylactically effective amount of a drug conjugate or a stereoisomer thereof or a pharmaceutically acceptable salt thereof. An "effective amount" means an amount of a component sufficient to produce the desired reaction. The specific effective amount depends on various factors, such as the specific disease to be treated, the patient's physical condition, such as weight, age and sex, the duration of treatment, co-administered treatments (if any), and the specific formulation used. Typically, an "effective amount" as described herein is a conventional amount of a biomolecule. However, in some embodiments, the therapeutically or prophylactically effective amount of the conjugate contained in the pharmaceutical composition of the present invention may be lower than the conventional amount of the biomolecule but may produce a better therapeutic or prophylactic effect because the biomolecule is protected by a protecting group before reaching the pathological microenvironment to bind to its ligand or receptor.

[0023] The pharmaceutical composition of the present invention can be formulated into various suitable dosage forms, including but not limited to tablets, capsules, injections, etc., and can be administered by any suitable route to achieve the intended purpose. For example, it can be administered parenterally, subcutaneously, intravenously, intramuscularly, intraperitoneally, transdermally, orally, intrathecally, intracranially, intranasally, or topically. The dosage of the drug can depend on the patient's age, health status and weight, concurrent treatments, and the frequency of treatment. The pharmaceutical composition of the present invention can be administered to any subject in need thereof, for example, a mammal, particularly a human.

[0024] use

[0025] The drug conjugates disclosed in the present invention, or their stereoisomers, or their pharmaceutically acceptable salts can be used to treat and / or prevent tumors or inflammation, or can be used as active ingredients for preparing drugs for treating tumors or inflammation.

[0026] The diseases that can be treated by the drug conjugate disclosed in the present invention, or its stereoisomers, or pharmaceutically acceptable salts thereof, are related to the active ingredients contained in the conjugate. The indications of these active ingredients are well known in the art.

[0027] In some embodiments, the tumors described herein may include hematological tumors and solid tumors, including but not limited to sarcomas (such as fibrosarcomas), bladder cancer, brain cancer, breast cancer, cervical cancer, colorectal cancer, esophageal cancer, kidney cancer, liver cancer, lung cancer, nasopharyngeal cancer, pancreatic cancer, prostate cancer, skin cancer, stomach cancer, uterine cancer, ovarian cancer, testicular cancer and blood cancer, etc.

[0028] The present invention also includes a method for treating or preventing tumors or inflammation, comprising administering to a subject in need thereof a therapeutically or prophylactically effective amount of a drug conjugate as described herein, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition as described herein. This method can be used in combination with any known radiotherapy or immunotherapy.

[0029] The present invention also provides the use of the drug conjugate described herein, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, in the preparation of an anti-tumor drug. Preferably, the tumor is selected from a blood tumor and a solid tumor. Preferably, the tumor includes, but is not limited to, sarcoma (such as fibrosarcoma), bladder cancer, brain cancer, breast cancer, cervical cancer, colorectal cancer, esophageal cancer, kidney cancer, liver cancer, lung cancer, nasopharyngeal cancer, pancreatic cancer, prostate cancer, skin cancer, stomach cancer, uterine cancer, ovarian cancer, testicular cancer, and blood cancer.

[0030] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the invention. The experimental methods in the following examples, for which specific conditions are not specified, are generally based on conventional conditions or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

[0031] It should be understood that the terms "comprising" and "including" or similar expressions used in the present invention also mean "consisting of..." etc. The sum of all weight percentages or volume percentages should equal 100%. Unless otherwise stated, the various reagents and products used in the examples are commercial products. Unless otherwise stated, the methods mentioned in the examples are carried out according to conventional techniques. The following examples are not intended to limit the scope of the present invention.

[0032] Example 1: Preparation of drug conjugate S41

[0033] A typical preparation method of the compound is as follows:

[0034] Synthesis route general formula (III)

[0035] 1) Synthesis of the compound Fmoc-Ala-Ala-Asn-AM-OAc

[0036] Dissolve Fmoc-Ala-Ala-Asn(Trt)-Gly-OH (1.0 eq) in dichloromethane / tetrahydrofuran (15 / 5 by volume) and cool to 0°C. Add AcOH (1.0 eq), lead acetate (1.2 eq), and copper acetate (0.2 eq) under nitrogen. Stir at 40-50°C under nitrogen for 2-3 hours. Monitor the reaction for completion by TLC. Dilute the reaction solution with dichloromethane, wash with water, separate the layers, extract the aqueous phase with dichloromethane, combine the organic phases, dry over anhydrous sodium sulfate, spin dry, and analyze by column chromatography to obtain Fmoc-Ala-Ala-Asn-AM-OAc.

[0037] 2) Synthesis of the compound Fmoc-Ala-Ala-Asn(Trt)-AM-Ac-OBn

[0038] To a three-necked flask, add Fmoc-Ala-Ala-Asn-AM-OAc (1.0 eq) and benzyl glycolate (2.0 eq). Under nitrogen and in an ice bath, add trifluoroacetic acid / dichloromethane (TFA / DCM, volume ratio = 1 / 5) and stir at room temperature for 2-3 hours. After completion of the reaction, the reaction mixture is concentrated and separated by high-pressure preparative chromatography to afford Fmoc-Ala-Ala-Asn-AM-Ac-OBn as a white solid.

[0039] 3) Synthesis of compound Fmoc-Ala-Ala-Asn-AM-Ac-OH

[0040] The compound Fmoc-Ala-Ala-Asn-AM-Ac-OBn (1.0 eq) was dissolved in an appropriate amount of MeOH, and Pd / C (10 wt%, 0.1 eq) was slowly added. The mixture was stirred under hydrogen for 2-3 hours. The compound Fmoc-Ala-Ala-Asn-AM-Ac-OH was obtained as a light yellow product under high pressure.

[0041] 4) Synthesis of compound Fmoc-Ala-Ala-Asn-AM-DXd

[0042] The compound Fmoc-Ala-Ala-Asn-AM-Ac-OH (1.0 eq) and isoproterenol methanesulfonate (1.0 eq) were dissolved in DMF and cooled to 0°C. Under nitrogen, the condensing agent DEPBT (1.2 eq) and the organic base DIPEA (2.0 eq) were added sequentially, and the mixture was stirred at room temperature for 2-3 hours. TLC confirmed the reaction was complete. The reaction mixture was concentrated in vacuo, redissolved in DMF, filtered, and purified by high-pressure liquid chromatography to obtain the compound Fmoc-Ala-Ala-Asn-AM-DXd as a pale yellow product.

[0043] 5) Synthesis of compound H-Ala-Ala-Asn-AM-DXd

[0044] The compound Fmoc-Ala-Ala-Asn-AM-DXd (1.0 eq) was dissolved in DMF, and piperidine (2.0 eq) was added. The mixture was stirred at room temperature under nitrogen for 2-3 hours. TLC confirmed the reaction was complete. The reaction solution was redissolved, filtered, and then purified by high-pressure liquid chromatography to obtain the compound H-Ala-Ala-Asn-AM-DXd as a pale yellow product.

[0045] 6) Synthesis of compound R1-R2-Ala-Ala-Asn-AM-DXd

[0046] Compound R1-R2-OH (1.0 eq) was dissolved in an appropriate amount of DMF, and H-Ala-Ala-Asn-AM-DXd (1.0 eq) was added. HBTU (1.2 eq) and DIPEA (2.0 eq) were then added under nitrogen, and the mixture was stirred at room temperature for 1-2 hours. TLC confirmed the reaction was complete. The crude reaction mixture was filtered, and then purified by high-pressure liquid chromatography to yield compound R1-R2-Ala-Ala-Asn-AM-DXd as a pale yellow product.

[0047] Using the corresponding raw materials, the S41 compound shown in Table 1 can be obtained according to the above method. The properties of S41 are shown in Table 2.

[0048] Table 1

[0049] Table 2

[0050] Example 2: Enzyme cleavage assay of S41 drug conjugate

[0051] The methods for testing the stability and enzymatic cleavage efficiency of the drug conjugate compounds are as follows, and the test results are shown in Table 3.

[0052] Accurately weigh each drug conjugate compound and add a sufficient amount of DMSO solution to a stock solution concentration of 10 pmol / mL. Then, a 10 μL aliquot was diluted to 1 μmol / mL by adding 80 μL of DMSO. This was then diluted to a sample solution concentration of 0.2 μmol / mL by adding purified water at a 1:4 ratio. After the sample clarified, it was placed in a 25°C / 37°C water bath. Samples were taken at 0 and 24 hours, and analyzed by HPLC (Agilent 1260, Eclipse Plus C18 column, 4.6 x 250 mm, 5 μm, mobile phase A: 0.1% TFA + H2O, mobile phase B: CH3CN, flow rate: 1.0 ml / min, column temperature: 30°C, detection wavelength: 214 nm). The drug conjugate content relative to the 0-hour concentration was used to obtain solution stability data for each compound.

[0053] An appropriate amount of sample was weighed and prepared into a 1 mM solution with DMSO. Human plasma was added at a volume ratio of 1:9 and the solution was incubated in a 37°C thermostat. Samples were taken after 0, 2, 4, 6, and 24 h. DMSO / MeOH (1:1) was added at a volume ratio of 1:3 to remove protein. The solution was centrifuged at 12,000 rpm for 5 min and the supernatant was collected. The HPLC method was as described above (Agilent 1260, chromatographic column: Eclipse Plus C18, 4.6*250 mm, 5 μm, mobile phase A: 0.1% TFA+H2O, mobile phase B: CH3CN, flow rate: 1.0 ml / min, column temperature: 30°C, detection wavelength: 214 nm).

[0054] Weigh a certain amount of drug conjugate, dissolve it, and dilute it tenfold to a concentration of 0.1mM / ml. Add the drug conjugate at a concentration of 1mg / ml to 100μg of acidified CT-26 tumor tissue homogenate (pH 5.0) at 37°C. The enzyme in the tumor tissue homogenate can be released and detected by HPLC to compare the activation efficiency of the linker by the tumor tissue.

[0055] Table 3

[0056] Control compound C

[0057] As can be seen in Table 3, compound S41 of the present invention exhibits high stability, with a 24-hour aqueous solution stability exceeding 85%, a 24-hour plasma stability exceeding 75%, and a 2-hour enzymatic cleavage efficiency exceeding 90%. The 24-hour aqueous solution stability, plasma stability, and 2-hour enzymatic cleavage efficiency of control compound C (compound S1 disclosed in CN104262455B) were all lower than those of compound S41 of the present invention. This is because the amide and urethane bonds between the linker and the payload in the present invention are significantly more stable than the carbonate bond in control compound C, while the ester bond is more susceptible to hydrolysis.

[0058] These results demonstrate that compound S41 provided by the present invention exhibits enhanced stability in aqueous solution and plasma, is not easily degraded or transformed into other compounds upon entry into the body, and exhibits a stable targeted therapeutic effect upon entry into tumor cells or tumor tissue. Furthermore, compound S41 provided by the present invention exhibits a higher tumor cell killing rate after entry into tumor cells and cleavage by a specific enzyme within 2 hours, releasing a toxic drug.

[0059] Example 3

[0060] 1. Animals: Nude mice, 6-8 weeks old, all female (Shanghai Lingchang Biotechnology Co., Ltd.).

[0061] 2. Cells: Cells were purchased from the American Type Culture Collection (ATCC) and authenticated according to ATCC instructions. Cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum at 37°C in 5% CO2. Cells were passaged every 3 days and used within 15 passages.

[0062] 3. Tumor generation, 1.5×10 6 HT1080 cells were subcutaneously injected into the back of nude mice. When the tumor grew to about 100 mm3, the mice were randomly divided into groups and treatment began. The day of treatment was regarded as the first day.

[0063] 4. Treatment Process

[0064] The dose was 5 μmol / Kg and administered once a week for 3 weeks.

[0065] The groups and outcome measures are shown in Table 4 .

[0066] Table 4: Effects of corresponding compounds and control group on tumor inhibition

[0067] 5. Results and Discussion: The compound S41 of the present invention has a greatly improved therapeutic effect compared to the exitecan derivatives and can almost achieve the effect of curing tumors.

[0068] Example 4

[0069] 1. Experimental purpose: To understand the acute toxicity of the drug of the present invention in vivo by determining the MTD experiment of intravenous administration in mice.

[0070] 2. Test drug: Exitecan derivatives, compound S41, were dissolved in DMSO and diluted with normal saline to the corresponding dose during the test.

[0071] 3. Animals: BALB / C mice (purchased from Shanghai Slake Laboratory Animal Co., Ltd.), weighing 19-21 g, all female.

[0072] 4. Methods and Results: Forty-eight female BALB / C mice, weighing 19-21 g, were randomly divided into seven groups of six mice each. A single intravenous injection of an exitecan derivative or compound S41 was performed at the doses shown in Table 20. A saline control group was also administered, with each mouse receiving a 0.2 ml dose. The animals were observed daily for 17 days for signs of erect piloerection, matted luster, lethargy, hunched back, or hyperreactivity. Body weight and mortality were recorded. Blood samples were collected on days 3, 5, and 14 for complete blood counts. On day 14, the animals were dissected and the heart, liver, kidneys, lungs, spleen, and pancreas were stained with hematoxylin and eosin.

[0073] Table 5: Comparison of mortality rates of mice receiving different doses of compound injections and saline

[0074] 5. Results and Discussion: Compared with the ixetine derivatives, when the animals were injected with S41 of the present invention, there was no evidence of piloerection, matted luster, lethargy, hunchback, overreaction or death, indicating that the toxicity of the small molecule conjugated drug was significantly lower than that of the unconjugated drug.

[0075] Example 5: Pharmacodynamics and safety evaluation of S41 (QHL-1618) on human fibrosarcoma cells HT-1080 in a BALB / c nude mouse subcutaneous tumor model

[0076] In this study, 32 female Balb / c nude mice were subcutaneously inoculated with human fibrosarcoma cells HT-1080 to construct a transplanted tumor model. 3The animals were randomly divided into four groups, each with eight mice, and given vehicle (Vehicle), 2.47 mg / kg or 5 μmol / kg CPT-780 (positive control, S41 payload Dxd), 7.12 mg / kg or 5 μmol / kg S41, and 14.25 mg / kg or 10 μmol / kg S41, respectively. The drugs were injected into the tail vein at a volume of 10 ml / kg once a week for three weeks.

[0077] The results are shown in Figure 1. S41 showed a significant inhibitory effect on the growth of HT-1080 tumor cells. On day 38 of the experiment, 10 μmol / kg of S41 was an effective dose, and no significant drug toxicity was observed in mice.

[0078] Example 6: In vivo pharmacodynamic study of the test compound on human fibrosarcoma HT-1080 in a BALB / c nude mouse subcutaneous transplant tumor model

[0079] This study used 30 female Balb / c mice and subcutaneously inoculated human fibrosarcoma HT-1080 cells to construct a transplanted tumor model. The average volume of the transplanted tumor reached 161 mm. 3 The animals were randomly divided into five groups, each with six mice, and given lysozyme (Vehicle), 1.63 mg / kg or 3.3 μmol / kg CPT-780 (positive control, S41 payload Dxd), 1.57 mg / kg or 1.1 μmol / kg S41, 4.70 mg / kg or 3.3 μmol / kg S41, and 14.25 mg / kg or 10 μmol / kg S41, respectively. The drugs were injected into the tail vein at a volume of 10 μl / g once a week for 17 days.

[0080] The results are shown in Figure 2. The results showed that S41 significantly inhibited the growth of HT-1080 xenograft tumor cells. On day 17 of the study, tumor regression was observed in all mice (N=6) treated with S41 at 4.70 mg / kg and 14.25 mg / kg, with 80% of the mice in both the S41 4.70 mg / kg and 14.25 mg / kg treatment groups experiencing complete tumor regression.

[0081] Example 7: Pharmacodynamics and safety evaluation of S41 against mouse colon cancer cell line CT-26 in a BALB / c mouse subcutaneous tumor model

[0082] This study used 30 female Balb / c mice and subcutaneously inoculated them with mouse colon cancer cells CT-26 to construct a transplanted tumor model. The average volume of the transplanted tumor reached 127 mm. 3The animals were randomly divided into five groups, with six mice in each group. They were given lysozyme (Vehicle), 3 mg / kg or 6 μmol / kg CPT-780 (positive control, S41 payload Dxd), 3 mg / kg or 6 μmol / kg S41, and 9 mg / kg or 18 μmol / kg S41 by tail vein injection, with a dosage of 10 ml / kg, once a week for four weeks.

[0083] The results are shown in Figure 3. The results show that S41 exhibited a significant inhibitory effect on the growth of CT-26 tumor cell transplants. On day 27 of the experiment, S41 (18 μmol / kg) was an effective dose. At the same time, no significant drug toxicity was observed in mice.

[0084] Example 8: Pharmacodynamics and safety evaluation of combined treatment with S41 and anti-mPD-1 on mouse colon cancer cell CT-26 in a BALB / c mouse subcutaneous tumor model

[0085] This study used 24 female Balb / c mice and subcutaneously inoculated them with mouse colon cancer cells CT-26 to construct a transplanted tumor model. The average volume of the transplanted tumor reached 127 mm. 3 The mice were randomly divided into four groups, each with six mice. Each group was given lysozyme (Vehicle), 9 mg / kg or 18 μmol / kg S41, 5 mg / kg anti-mPD-1 antibody, or 9 mg / kg S41 combined with 5 mg / kg anti-mPD-1 antibody, all via tail vein injection at a volume of 10 ml / kg. S41 was administered once a week, and anti-mPD-1 antibody was administered twice a week for four weeks.

[0086] The results are shown in Figure 4. The results showed that S41 and anti-mPD-1 exhibited a synergistic inhibitory effect on the growth of tumor cell CT-26 transplanted tumors.

[0087] Example 9: Pharmacodynamics and safety evaluation of S41 against mouse colon cancer cell line CT-26 in a BALB / c mouse subcutaneous tumor model

[0088] This study used 24 female Balb / c mice subcutaneously inoculated with mouse colon cancer cells CT-26 to construct a transplanted tumor model. The average volume of the transplanted tumor reached 150-200 mm. 3 The mice were randomly divided into 4 groups, with 6 mice in each group, and given lysozyme (Vehicle), 10 mg / kg T-DXD analog (positive control), 16 mg / kg S41, and 24 mg / kg S41 (HSNTD dose) respectively. The drugs were injected into the tail vein with a volume of 10 ml / kg, once a week for 2 weeks.

[0089] The results are shown in Figure 5. The results show that S41 significantly inhibited the growth of CT-26 xenograft tumor cells. Based on rat toxicity studies, 16 mg / kg S41 and 10 mg / kg T-DXD showed similar toxicity. At high doses, S41 was significantly more effective than T-DXD.

[0090] Example 10: Toxicity test of SD rats with intravenous injection of S41 for four weeks and repeated administration for four weeks during the recovery period

[0091] This study selected 160 rats and randomly divided them into 4 groups, each with 40 rats, half male and half female, including 30 main test groups and 10 TK groups. They were given normal saline, 3, 6, and 12 mg / kg of S41, respectively, once a week for 4 consecutive weeks. The drug was then stopped and recovered for 4 weeks. The following examinations were performed during the experiment, including general observation, body weight, food intake, clinical pathology (including hematology, blood biochemistry, coagulation), body temperature, electrocardiogram, ophthalmological examination, urine, gross anatomy, organ weight, bone marrow smear, histopathological examination, and toxicokinetics. The results showed that the HNSTD dose was 12 mg / kg.

[0092] Example 11: Toxicity study of Beagle dogs after intravenous administration of S41 for four weeks with a recovery period of four weeks

[0093] This study selected 40 beagle dogs and randomly divided them into 4 groups, with 10 dogs in each group, half male and half female. They were given normal saline, 1, 2.5, and 5 mg / kg of S41, respectively, once a week for 4 consecutive weeks. The drugs were then discontinued for 4 weeks. The following examinations were performed during the experiment: general observation, body weight, food intake, clinical pathology (including hematology, blood biochemistry, and coagulation), body temperature, electrocardiogram, ophthalmological examination, urine, gross anatomy, organ weight, bone marrow smear, histopathological examination, and toxicokinetics. The results showed that the HNSTD dose was 2.5 mg / kg.

[0094] In summary, the antibody-drug conjugate provided by the present invention can target and aggregate around tumor cells, and can only activate the drug on the surface of tumor cells, effectively killing tumors, and the toxicity of the drug is also reduced to a certain extent, which has very good application prospects.

Claims

1. A drug conjugate represented by the following formula, its stereoisomer or a pharmaceutically acceptable salt thereof:

2. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises: (i) the drug conjugate according to claim 1, its stereoisomer or pharmaceutically acceptable salt; and (ii) a pharmaceutically acceptable carrier.

3. Use of the drug conjugate according to claim 1, its stereoisomer or pharmaceutically acceptable salt in the preparation of a medicament for treating and / or preventing tumors and / or inflammation.

4. The use according to claim 3, characterized in that The tumor is a blood tumor or a solid tumor.

5. The use according to claim 3, characterized in that The tumor is selected from the group consisting of sarcoma (such as fibrosarcoma), bladder cancer, brain cancer, breast cancer, cervical cancer, colorectal cancer, esophageal cancer, kidney cancer, liver cancer, lung cancer, nasopharyngeal cancer, pancreatic cancer, prostate cancer, skin cancer, stomach cancer, uterine cancer, ovarian cancer, testicular cancer and blood cancer.

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