USE OF MEDICAMENT IN TREATMENT OF TUMOR DISEASE

Abstract

The present invention relates to the use of a medicament in treatment of a tumor disease. In particular, the present invention provides the use of a biologically active conjugate represented by formula (I) in treatment of a tumor disease. The tumor disease particularly and preferably refers to an unresectable locally advanced or metastatic solid tumor that is refractory according to existing treatment standards, including but not limited to breast cancer, gastric cancer, lung cancer, ovarian cancer, urinary tract epithelial cancer, esophageal cancer, liver cancer, colorectal cancer, cervical cancer, endometrial cancer, pancreatic cancer, and brain tumor.

Claims

1. Use of a biologically active conjugate represented by formula (I) in preparation of a medicament for treating a tumor disease; ##STR00020## wherein, L.sub.1 is ##STR00021## wherein R.sub.1 and R.sub.2 are each independently hydrogen (such as protium or deuterium), halogen, carboxylate, sulfonate, cyano, C.sub.1-6 alkyl, halogenated C.sub.1-6 alkyl, cyano-substituted C.sub.1-6 alkyl (such as CH.sub.2CN), C.sub.1-6 alkoxy, C.sub.2-10 alkenyl or C.sub.2-10 alkynyl; Z.sub.1 is an amino acid or a peptide consisting of 2-10 amino acids; x.sub.1 and x.sub.2 are each independently 0, 1, 2, 3, 4, 5 or 6; and the position 1 of L.sub.1 is attached to D, and the position 2 of L.sub.1 is attached to L.sub.2; L.sub.2 is ##STR00022## wherein ?.sub.1 is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; and the position 1 of L.sub.2 is attached to L.sub.1, and the position 2 of L.sub.2 is attached to L.sub.3; L.sub.3, is selected from 5-12-membered heteroaromatic rings; L.sub.4 is ##STR00023## wherein Z.sub.2 is selected from C.sub.1-6 alkylene, C.sub.2-10 alkenylene, C.sub.2-10 alkynylene and C.sub.3-8 cycloalkylene; R.sub.3 is selected from H and C.sub.1-6 alkyl; Z.sub.3 is absent or selected from C.sub.1-6 alkylene; or, R.sub.3 and Z.sub.3 together with the nitrogen atom to which they are attached form 4-8-membered heterocyclyl; ? is 0, 1, 2, 3, 4, 5 or 6, and the position 2 of L.sub.4 is attached to E, and the position 1 of L.sub.4 is attached to L.sub.3; E is ##STR00024## wherein each R.sub.4 is independently hydrogen (such as protium or deuterium) ? is 0, 1 or 2, and the position 2 of E is attached to A (such as with sulfhydryl on A), and the position 1 of E is attached to L.sub.4; m.sub.1, m.sub.2 and m.sub.3 are each independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; D is a bioactive molecular fragment; ? is selected from integers from 1 to 10; preferably, ? is selected from integers from 3 to 8 (such as 3, 4, 5, 6, 7 or 8); and A is a monoclonal antibody against Trop-2 or an antigen-binding fragment thereof.

2. The use according to claim 1, wherein the tumor disease is an unresectable locally advanced or metastatic solid tumor that has failed standard treatments, has no standard treatment regimen, or is not suitable for standard treatments at the present stage.

3. The use according to claim 1 or 2, wherein the tumor disease includes but is not limited to breast cancer (such as Luminal type A, Luminal type B, Her-2 positive and triple negative type breast cancers), gastric cancer, lung cancer, ovarian cancer, urinary tract epithelial cancer, esophageal cancer, liver cancer, colorectal cancer, cervical cancer, endometrial cancer, pancreatic cancer, bladder cancer, or brain tumor; preferably breast cancer (such as triple negative breast cancer or Her2 positive breast cancer), ovarian cancer (such as ovarian epithelial cancer), gastric cancer, lung cancer, pancreatic cancer, bladder cancer, or urinary tract epithelial cancer; more preferably, the tumor disease is triple negative breast cancer, Her2 positive breast cancer, ovarian cancer, gastric cancer, lung cancer or pancreatic cancer; further preferably, the tumor disease is triple negative breast cancer, Her2 positive breast cancer, ovarian cancer or gastric cancer: preferably, the ovarian cancer is selected from platinum-sensitive ovarian cancer and platinum-resistant ovarian cancer; preferably, the gastric cancer is selected from gastric adenocarcinoma, gastric adenosquamous carcinoma, gastric squamous cell carcinoma, gastric undifferentiated carcinoma and gastric neuroendocrine tumor; preferably, the pancreatic cancer is selected from pancreatic epithelial tumor, pancreatic exocrine tumor, pancreatic borderline tumor, pancreatic ductal adenocarcinoma pancreatic endocrine tumor, pancreatic mature teratoma, pancreatic mesenchymal tumor, pancreatic malignant lymphoma and pancreatic secondary tumor; preferably, the bladder cancer is selected from urothelial (transitional cell) carcinoma, bladder squamous cell cancer and bladder adenocarcinoma; preferably, the urinary tract epithelial cancer is selected from basal type, lumen type and wild type urinary tract epithelial cancers; and preferably, the lung cancer is selected from small cell lung cancer and non-small cell lung cancer.

4. The use according to any one of claims 1 to 3, wherein the conjugate has the following structure: L.sub.1 is selected from ##STR00025## and the position 1 of L.sub.1 is attached to D, and the position 2 of L.sub.1 is attached to L.sub.2; L.sub.2 is ##STR00026## wherein ?.sub.1 is 3, 4, 5, 6, 7, 8, 9 or 10; and the position 1 of L.sub.2 is attached to L.sub.1, and the position 2 of L.sub.2 is attached to L.sub.3; L.sub.3 is selected from 5-6-membered heteroaromatic rings, such as pyrazole or triazole; L.sub.4 is ##STR00027## wherein Z.sub.2 is selected from C.sub.1-3 alkylene; R.sub.3 is H; Z.sub.3 is selected from C.sub.1-3 alkylene; ? is 1, and the position 2 of L.sub.4 is attached to E, and the position 1 of L.sub.4 is attached to L.sub.3; E is ##STR00028## wherein each R.sub.4 is independently hydrogen (such as protium or deuterium), ? is 0, 1 or 2, and the position 2 of E is attached to A (such as with sulfhydryl on A), and the position 1 of E is attached to L.sub.4; m.sub.1, m.sub.2 and m.sub.3 are all 1; the bioactive molecule is selected from ##STR00029## preferably, the bioactive molecule is attached to the position 1 of L.sub.1 through its own hydroxyl; ? is selected from integers from 3 to 8 (for example, 3, 4, 5, 6, 7 or 8); and A is Sacituzumab or an antigen-binding fragment thereof.

5. The use according to any one of claims 1 to 4, wherein the structure of the conjugate is as follows: ##STR00030## wherein ? is an integer from 1 to 10; preferably, ? is selected from integers from 5 to 8; preferably, the DAR value of the conjugate is 1-12; more preferably, the DAR, value of the conjugate is 1-10; further preferably, the DAR value is 5-8; for example, the DAR value is 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9 or 8.0.

6. A method for treating a tumor disease, comprising a step of administering a therapeutically effective amount of the biologically active conjugate according to any one of claims 1, 4 or 5 and/or a pharmaceutical composition comprising the biologically active conjugate according to any one of claims 1, 4 or 5 to an individual in need thereof; wherein the tumor disease is selected from the tumor diseases as claimed in any one of claims 1 to 3.

7. The method according to claim 6, wherein the pharmaceutical composition comprises the biologically active conjugate and a pharmaceutically acceptable carrier and/or excipient.

8. The method according to claim 6 or 7, wherein the biologically active conjugate or the pharmaceutical composition is administered once every 7-35 days, preferably once every 7-28 days, such as once every 7 days, 14 days, 21 days, 28 days or 35 days.

9. The method according to any one of claims 6 to 8, wherein the administration route of the conjugate or pharmaceutical composition includes but is not limited to oral administration, percutaneous injection, rectal administration, mucosal administration, intramuscular injection, intramedullary injection, intravenous injection, intraperitoneal injection, and preferably, intravenous injection.

10. The method according to any one of claims 6 to 9, wherein the dose of the biologically active conjugate each time based on the body weight of a patient is 1 mg/kg to 30 mg/kg; preferably 1 mg/kg to 20 mg/kg; more preferably 2 mg/kg to 12 mg/kg; further preferably 2-5 mg/kg, 4-7 mg/kg, 6-9 mg/kg, 8-11 mg/kg, 10-13 mg/kg, or 12-15 mg/kg.

11. The method according to any one of claims 6 to 10, wherein the method is divided into one or more administration stages (for example, one, two, three or four stages), the administration cycle at each stage is as claimed in claim 8, and/or the administration dose at each stage is as claimed in claim 10.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0102] The drawing described herein is used to provide a further understanding of the present invention and constitute a part of the present application. The illustrative examples of the present invention and their descriptions are used to explain the present invention and do not constitute an undue limitation of the present invention. In the drawings:

[0103] FIG. 1 shows the in vitro plasma stability of the conjugate A and a commercially available drug Trodelvy?.

DETAILED DESCRIPTION

[0104] In the following, the technical solutions in the examples of the present invention will be clearly and completely described with reference to the drawings in the examples of the present invention. Obviously, the described examples are only a part of but not all the examples of the present invention. The following description of at least one exemplary example is merely illustrative in nature and is in no way intended to limit the present invention, its applications or uses. Based on the examples in the present invention, all other examples obtained by those of ordinary skill in the art without creative work fall within the scope of protection of the present invention.

Example 1. 4-((S)-2-(4-aminobutyl)-35-(4-((6-(2-(methylsulfonyl)pyrimidin-5-yl)hexan-5-amido)methyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonaoxa-3,9-diazapentatriacontanamino)benzil((S)-4-ethyl-11-(2-(N-isopropylmethylsulfonanmido)ethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3,4:6,7]indolizino[1,2-b]quinolin-4-yl)carbonate

[0105] ##STR00018##

Step 1: Synthesis of 6-(2-(methylsulfonyl)pyrimidin-5-yl)-N-(propan-2-alkyn-1-yl)hexa-5-alkynamide

[0106] At 25? C., prop-2-yn-1-amine (189 mg, 3.4 mmol) and compound 3-4 (800 mg, 2.83 mmol) were dissolved in dichloromethane (10 mL), N,N-diisopropylethylamine (738 mg, 5.67 mmol) and O-(7-azabenzotriazol-1-yl)-N,N,N,N-tetramethylurea hexafluorophosphate (1.63 g, 4.25 mmol) were added in sequence, and the mixture was stirred to react for 2 h. The reaction mixture was concentrated under reduced pressure, and the residue was purified by rapid silica gel column (ethyl acetate/petroleum ether=3/1) to obtain the title compound (700 mg). ESI-MS (m/z): 306.1[M+H]+.

Step 2: Synthesis of 4-((S)-35-azido-2-(4-((4-methoxyphenyl)diphenylmethyl)amino)butyl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonaoxa-3,9-diazapentatriacontanamino)benzyl((S)-4-ethyl-11-(2-(N-isopropylmethanesulfonamido)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-2H-pyrano[2,3-b]-1H-pyrano[3,4:6,7]indazino[1,2-b]quinolin-4-yl)carbonate

[0107] Under the protection of nitrogen at 25? C., T-030 (250 mg, 0.49 mmol) was dissolved in dichloromethane (10 mL), and cooled to 0? C., then 4-dimethylaminopyridine (478 mg, 3.91 mmol) in dichloromethane (3 mL) was added, and then triphosgene (72 mg, 0.24 mmol) in dichloromethane (10 mL) was slowly added dropwise. Thereafter, the mixture was stirred at 0? C. for 20 min and reaction mixture was blown with nitrogen for 20 min. (S)-2-(32-azido-5-oxo-3,9,12,15,18,21,24,27,30-nonaoxa-6-azatriacetamidyl)-N-(4-(hydroxymethyl)phenyl)-6(((4-methoxyphenyl)diphenylmethyl)amino)acetamide (518 mg, 0.49 mmol) in dichloromethane (7 mL) was added. Thereafter, the mixture was stirred to react for 1 h. The reaction mixture was concentrated under reduced pressure, and the residue was purified by preparative HPLC to obtain the title compound (500 mg). ESI-MS (m/z): 1597.5[M+H]+.

Step 3: Synthesis of (S)-4-ethyl-11-(2-(N-isopropylsulfonamido)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3,4:6,7]indazino[1,2-b]quinolin-4-yl(4-((S)-2-(4-(((4-methoxyphenyl)diphenylmethyl)amino)butyl)-35-(4-((6-(2-(methylsulfonyl)pyrimidin-5-yl)hexan-5-amido)methyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonoxy-3,9-diazapentatriacontanamino)benzyl)carbonate

[0108] At room temperature, compound 33-1 (14 mg, 0.05 mmol) was dissolved in dimethyl sulfoxide and water (2.0 mL: 0.5 mL), cuprous bromide (11 mg, 0.08 mmol) was added, and the mixture was stirred to react for 1 h. The system was purified by preparative HPLC to obtain the title compound (30 mg). ESI-MS (m/z): 815.9[(M-273)/2+H]+.

Step 4: Synthesis of 4-((S)-2-(4-aminobutyl)-35-(4-((6-(2-(methylsulfonyl)pyrimidin-5-yl)hexan-5-amido)methyl)-1?1=1,2,3-triazol-1-yl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonaoxa-3,9-diazapentatriacontanamino)benzyl((S)-4-ethyl-11-(2-(N-isopropylmethylsulfonanmido)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3,4:6,7]indolizino[1,2-b]quinolin-4-yl)carbonate (compound IM-1)

[0109] Compound 33-2 (30 mg, 0.02 mmol) was dissolved in dichloromethane (1.0 mL) and trifluoroacetic acid (0.2 mL) was added to the reaction to react at room temperature for 30 min. After purification by preparative high performance liquid chromatography (method C), the trifluoroacetate of the title compound was obtained (20.0 mg). Its structure is characterized as follows:

[0110] .sup.1H NMR (400 MHZ, DMSO-d6) ? 10.18 (s, 1H), 9.10 (s, 2H), 8.38 (t, J=5.56 Hz, 1H), 8.32 (d, J=8.40 Hz, 1H), 8.22-8.20 (m, 2H), 8.09 (t, J=5.68 Hz, 1H), 7.91-7.87 (m, 2H), 7.82-7.78 (m, 1H), 7.69 (brs, 3H), 7.61 (d, J=8.56 Hz, 2H). 7.32 (d, J=8.56 Hz, 2H). 7.06 (s, 1H), 5.56 (d, J=16.96 Hz, 1H), 5.51 (d, J=16.96 Hz, 1H), 5.47 (d, J=19.28 Hz, 1H), 5.42 (d, J=19.28 Hz, 1H), 5.14 (d, J=12.20 Hz, 1H), 5.07 (d, J=12.16 Hz, 1H), 4.48 (t, J=5.24 Hz, 2H), 4.46-4.43 (m, 1H), 4.29 (d, J=5.60 Hz, 211), 4.08-3.95 (m, 5H), 3.79 (t, J=5.28 Hz, 2H). 3.51-3.43 (m, 32H). 3.40 (s, 3H), 3.39-3.35 (m, 2H), 3.30-3.26 (m, 2H), 3.00 (s, 3H), 2.82-2.74 (m, 2H), 2.56 (t, J=7.08 Hz, 2H), 2.29 (t, J=7.36 Hz, 2H), 2.23-2.13 (m, 2H), 1.82 (p, J=7.24 Hz, 2H), 1.78-1.63 (m, 2H), 1.61-1.49 (m, 2H), 1.42-1.27 (m, 2H), 1.15 (d, J=6.80 Hz, 3H), 1.13 (d, J=6.76 Hz, 3H), 0.90 ((, J=7.32 Hz, 3H). ESI-MS (m/z):

816.0[M/2.sup.+H].sup.+[?].sub.D.sup.2D is ?19.55?(c=1.000 g/100 mL,CH.sub.3CN)

Example 2. Preparation of Conjugate A

[0111] 0.3 mL of Sacituzumab antibody (anti-Trop-2, 33.5 mg/mL) was taken, diluted with 0.25 mL of a solution (pH 7.6) containing 20 mM PB, 150 mM NaCl and 20 mM sodium edetate, then 0.45 mL of a solution (pH 7.6) containing 20 mM PB and 150 mM NaCl was added and the mixture was mixed well. pH of the reaction mixture was adjusted to 7.4 with | M Na.sub.2HPO.sub.4 solution, 10 mM TCEP (tris(2-carboxyethyl)phosphine) solution was added, the mixture was mixed well, and left to stand at room temperature for 30 min. 10 times in moles of IM-1 trifluoroacetate dissolved in dimethyl sulfoxide was added to the above solution system, the system was mixed well, and left to stand at room temperature for 2 h. Thereafter. 6.1 ?l of 100 mM cysteine was added to terminate the reaction. Finally. G-25 gel column was used to replace the buffer solution with PBS buffer solution at pH 6.5, and a product of IM-1 coupled with Sacituzumab antibody was obtained, which was named conjugate A.

##STR00019##

[0112] The molecular weight of the conjugate A was analyzed by LCMS, and the measured molecular weight of light chain and heavy chain of the conjugate A was correlated with the theoretical molecular weight of light chain and heavy chain coupled with different numbers of toxins. It was determined that each antibody molecule in the conjugate A was coupled with 1-10 toxins (i.e., ? was 1-10). Then the average coupling ratio (DAR) was calculated to be about 6.9 according to the respective percentages of conjugate molecules coupled with different numbers of toxins.

[0113] With reference to Example 2, conjugate A samples with DAR value ranging from 6 to 8 (such as 7.3 or 7.4) were prepared in batches, and the following non-clinical and clinical studies were carried out.

Experimental Example 1. Detection of the Inhibitory Effect of Conjugate on the Proliferation of Pancreatic Cancer Cell Line

[0114] The effect of the conjugate A on the proliferation of BxPC-3 cells (pancreatic cancer cell line, from ATCC, TROP2 positive cells) was detected by the CellTiter-Glo? chemiluminescence cell viability assay (i.e., CTG). On the first day of the experiment, BxPC-3 cells in exponential growth phase were collected, the concentration of the cell suspension was adjusted with the culture medium, and the suspension was added to a 96-well cell culture plate, the final cell concentration being 2000 cells/well. The cells were cultured overnight in a 5%) CO.sub.2 incubator at 37? C. On the second day of the experiment, the conjugate A (final concentration 0.152-1000 nM) was diluted to 10-fold dilute working solution with a gradient of 1:3, and 10 ?l of the corresponding 10-fold dilute working solution was added to each well, 3 replicate wells for each drug concentration. After adding the drug, the cells were cultured in a 5% CO.sub.2 incubator at 37? C. for 72 h. On the fifth day of the experiment, 50 ?l (? culture volume) of CTG solution melted in advance and equilibrated to room temperature was added to each well, and the mixture was mixed well with a microplate shaker for 2 min. After standing at room temperature for 20 min, the fluorescence signal value was measured using an Envision 2104 plate reader.

[0115] The results show that the conjugate A significantly inhibited the proliferation of TROP2 positive cell BxPC-3, with the IC.sub.50 of 14.3 nM, and the maximum inhibition rate of 95.3%. This indicates that the conjugate A can inhibit the proliferation of TROP2-positive pancreatic cancer cells, suggesting that it has therapeutic effects on pancreatic cancer.

Experimental Example 2. Detection of the Effects of Conjugate on Cardiovascular and Respiratory Functions

[0116] The cynomolgus monkeys were divided into 10 (half male and half female)/group. The conjugate A was injected intravenously at doses of 25 mg/kg, 50 mg/kg and 75 mg/kg respectively, once every two weeks, for a total of 4 times. The II-lead ECG and respiratory frequency were detected by a large animal noninvasive physiological signal telemetry system, and arterial blood pressure was measured by a noninvasive sphygmomanometer, so as to evaluate the effects of the conjugate A on cardiovascular and respiratory functions of cynomolgus monkeys.

[0117] The results show that prior to the first dosing, 2-3 h, 24-25 h, 72-73 h, 7 d after the first dosing, about 2-3 h after the last dosing and prior to the end of the recovery period, for the monkeys in each group receiving the conjugate A, there was no arrhythmia in II-lead ECG, and heart rate, R-R interval, P-wave time, P-R interval, QRS wave time, Q-T interval, corrected Q-T interval and other parameters of II-lead electrocardiogram, and systolic blood pressure, diastolic blood pressure, mean arterial pressure and respiratory rate were not significantly abnormal. These results indicate that the conjugate A has no effect on the cardiovascular system and respiratory system of cynomolgus monkeys, and has a good prospect of clinical drug safety.

Experimental Example 3. Detection of Toxic Metabolic Behavior of Conjugate in Animals

[0118] The cynomolgus monkeys were divided into four groups according to different doses: control group, 25 mg/kg dose group, 50 mg/kg dose group and 75 mg/kg dose group, with 5 males and 5 females in each group. At the dose levels of 25 mg/kg and 50 mg/kg, the cynomolgus monkeys were injected with the conjugate A intravenously once every two weeks for four consecutive times. For the monkeys in each group, blood samples were collected before and immediately after, and 4 h, 24 h, 48 h, 96 h and 168 h after the first and last dosing (0-1 min after the end of dosing), 1 h before and immediately after the second and third dosing (0-1 min after the end of dosing), and 336 h after the last dosing. For the monkeys in the 75 mg/kg dose group, additionally, blood samples were collected at 4 h, 24 h, 48 h, 96 h and 168 h after the third dosing, so as to detect the toxicokinetic behavior of the conjugate and toxin molecules released by it in vivo.

[0119] The following table records the peak plasma concentration (C.sub.max) of and exposure (AUC) to the conjugate A and the toxin molecules released by the conjugate A in male and female monkeys after administration in the above manner. The results show that the highest non-serious toxic dose (HNSTD) of the conjugate A was 50 mg/kg, and after the last dosing with this dose, the exposure to the toxin molecules in female and male monkeys was 3.85 h*?g/mL and 5.86 h*?g/mL, respectively, and the exposure to the conjugate A in female and male monkeys was 45.8 h*mg/mL and 64.2 h*mg/mL, respectively.

TABLE-US-00001 Dose (mg/kg, QW) 0 25 50 75 Sex of animal Male Female Male Female Male Female Male Female C.sub.max (?g/mL) of conjugate A Day 1 NA NA 599 641 1220 1170 1820 2000 Day 43 NA NA 601 518 1270 1370 1790 1820 C.sub.max (ng/mL) of toxins Day 1 NA NA 49.0 38.3 94.0 113 132 132 Day 43 NA NA 64.5 65.1 146 127 110 137 AUC.sub.last (hr*mg/ml) of conjugate A Day 1 NA NA 25.9 26.9 63.2 46.3 87.5 85.5 Day 43 NA NA 19.2 14.9 64.2 45.8 78.5 87.9 AUC.sub.last (hr*?g/mL) of toxins Day 1 NA NA 1.79 1.43 4.49 3.97 5.42 5.12 Day 43 NA NA 2.57 1.86 5.86 3.85 4.08 4.68

[0120] According to the experimental results, it can be seen that the exposure or C.sub.max data of ADC and toxins in all of the groups of animals were close after the first and last dosing, indicating that the conjugate A has no obvious toxicity accumulation after continuous intravenous administration, and has good tolerance in animals, so that it has a good prospect for clinical drug safety.

Experimental Example 4. Metabolic Stability of Conjugate In Vitro

[0121] In this experiment, firstly, the conjugate A and Trodelvy? were respectively formulated into a. 3.4 mg/mL working solution with physiological saline, and then the conjugate A and Trodelvy? working solutions were added into human blank plasma respectively to obtain 0.05 mg/ml human plasma samples. The plasma samples were incubated at 37? C, and the release of toxin molecules was determined at 1 h, 3 h, 24 h, 48 h, 72 h and 144 h. The conjugate A and the commercially available drug Trodelvy? were compared for the difference in plasma stability in vitro. The results are shown in FIG. 1.

[0122] As shown in FIG. 1 of the specification, with the increase of incubation time, the yield of free toxins in human plasma increased, and after incubation for 24 h, the release percentage of toxin molecules of the conjugate A and the commercially available ADC drug Trodelvy? in human plasma was 28.5% and 93.4%, respectively; after incubation for 48 h, the release percentage of toxin molecules of the conjugate A and the commercially available ADC drug Trodelvy? in human plasma was 44.6% and 109.1%, respectively; after incubation for 72 h, the release percentage of toxin molecules of the conjugate A and the commercially available ADC drug Trodelvy? in human plasma was 53.7% and 100.6%, respectively; and after incubation for 144 h, the release percentage of toxin molecules of the conjugate A and the commercially available ADC drug Trodelvy? in human plasma was 65.3% and 104.8%, respectively.

[0123] The results showed that the release rate of toxin molecules of conjugate A in human plasma was significantly lower than that of Trodelvy?, which indicates that the conjugate A is relatively stable in human plasma, and releases less toxins in plasma, effectively achieving the goal of releasing toxins after reaching the target tumor sites. Moreover, compared with the commercially available drug Trodelvy?, the conjugate A can reduce the risk of serious adverse reactions caused by rapid and excessive release of toxins in plasma.

Trial Example 1. Phase T Clinical Study

1. Protocol

[0124] Patients with malignant tumor of epithelial origin diagnosed by histology were treated with the conjugate A All the patients had unresectable locally advanced or metastatic solid tumors that had failed standard treatments, or had no standard treatment regimens or were not suitable for standard treatments at the present stage. Based on the patient's weight, five dose levels of 2, 4, 6, 9 and 12 mg/kg were designed, which were injected intravenously once every two weeks, every 28 days as a cycle, and were given every cycle until the disease progressed or an intolerable toxic reaction occurred. The toxicity of all planned dose groups and some intermediate dose groups was evaluated by BLRM. Based on the model analysis, the sponsor and the researcher jointly decided the decision of dose increase.

II. Safety Results

[0125] During the treatment, there was no Treatment Emergent Adverse Events (hereinafter referred to as TEAEs) of grade 3 or above in the 2 mg/kg dose group. TEAEs of grade 3 appeared in the 4 mg/kg dose group, including oral mucositis, anemia, etc.: TEAEs of grade 3 or above appeared in the 6 mg/kg dose group, including decreased neutrophil count, reduced white blood cell count, etc.; the abovementioned TEAEs of grade 3 or above can be recovered after symptomatic treatment and the drug can be administered continuously, which shows that the conjugate A has good clinical safety.

III. Efficacy Results

[0126] The treatment results for various indications are as follows:

1. Triple Negative Breast Cancer (TNBC)

[0127] In all the patients with triple negative breast cancer, some patients showed partial response.

[0128] A patient who had progressive disease after previous adriamycin/cyclophosphamide/paclitaxel neoadjuvant chemotherapy, left mastectomy and left chest radiotherapy was injected with the conjugate A intravenously at a dose of 4 mg/kg based on the patient's body weight according to the above-mentioned administration cycle. After 18 weeks, partial response was observed, which lasted for 6 weeks. In several times of efficacy evaluation, the total volume of the target lesions could be reduced by 40%. The patient did not have serious adverse events.

2. Ovarian Cancer

[0129] In all the patients with ovarian cancer, some patients showed partial response.

[0130] A patient with ovarian cancer who had failed previous hysterectomy, ovarian tumorectomy, carboplatin/paclitaxel, rucaparib, carboplatin/docetaxel/bevacizumab was injected with the conjugate A intravenously at a dose of 4 mg/kg based on the patient's body weight according to the above administration cycle. After 21 weeks, partial response was observed, which lasted for 15 weeks. In several times of efficacy evaluation, the total volume of the target lesions was reduced by 64.8%, and one of the target lesions completely disappeared. The patient did not have serious adverse events.

3. HER2 Positive Breast Cancer

[0131] A patient with HER2 positive breast cancer who had previously failed modified radical mastectomy, epirubicin/paclitaxel, HER2 monoclonal antibody/docetaxel and capecitabine was injected with the conjugate A intravenously at a dose of 6 mg/kg based on the patient's body weight according to the above administration cycle. After 8 weeks, partial response was observed. In several times of efficacy evaluation, the total volume of the target lesions could be reduced by 49.6%.

4. Gastric Cancer

[0132] A patient with gastric cancer who had previously failed total gastrectomy, paclitaxel/tegafur, anlotinib/tegafur, anlotinib/capecitabine, oxaliplatin/fluorouracil and oxaliplatin/raltitrexed was injected with the conjugate A intravenously at a dose of 4 mg/kg based on the patient's body weight according to the above administration cycle. After 15 weeks, partial response was observed, which had lasted for 10 weeks and was still benefiting. In several times of efficacy evaluation, the total volume of the target lesions could be reduced by 62.8%, and one of the target lesions disappeared. No serious adverse events occurred in the patient.

5. Pancreatic Cancer

[0133] A patient with pancreatic cancer who had previously failed multi-line therapy of distal resection of pancreatic cancer, radiotherapy, gemcitabine/capecitabine, gemcitabine/irinotecan/fluorouracil, gemcitabine/albumin paclitaxel/oxaliplatin/fluorouracil, and nivolumab/cabiralizumab was injected with the conjugate A intravenously at a dose of 4 mg/kg based on the patient's body weight according to the above-mentioned administration cycle, and stable disease was observed after 7 weeks, which lasted for 30.3 weeks. In several times of efficacy evaluation, the total volume of the target lesions could be reduced by 8.8%, and no serious adverse events occurred in the patient. To sum up, the phase I clinical trial showed that the conjugate A had a good efficacy at least in the above-mentioned unresectable metastatic TNBC, ovarian cancer, HER2 positive breast cancer, gastric cancer and pancreatic cancer that had failed standard treatments, without inducing serious toxicity that may hinder clinical use. These results prove that the conjugate A has good safety and efficacy in treatment of tumor diseases, and conjugates A with DAR ranging from 6 to 8 (such as DAR of 7.3 or DAR of 7.4) prepared in different batches have basically consistent efficacy and safety.

[0134] Various modifications of the present invention other than those described herein will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. All the reference documents cited in the present application (including all the patents, patent applications, journal articles, books and any other publications) are incorporated herein by reference in their entirety.