Application of antibody coupling medicine

Abstract

The invention provides an antibody-conjugated drug, a pharmaceutical composition of a pharmaceutical salt, a hydrate, a solvate, a stereoisomer or an isotope label of the antibody-conjugated drug, an application of the antibody-conjugated drug in tumor treatment and a method for treating the tumor.

Description

Use of an antibody-drug conjugate Technical Field

[0001] The present invention relates to the field of biomedicine and specifically relates to the use of an antibody-drug conjugate. Background Art

[0002] The epidermal growth factor receptor (EGFR) is a member of the HER family. EGFR is a 170-kDa transmembrane glycoprotein composed of 1186 amino acids. It consists of three parts: an extracellular receptor domain, a transmembrane domain, and an intracellular tyrosine kinase domain. Confirmed ligands that can bind to EGFR include epidermal growth factor (EGF), transforming growth factor-α (TGFa), bidirectional regulatory factors, heparin-binding EGF, and cytokine. In human tissues, EGF and TGFa are considered the two most important ligands for EGFR.

[0003] EGFR is constitutively expressed in many normal epithelial tissues, such as skin and hair follicles. Furthermore, EGFR is highly expressed in a variety of human malignant tumors, including 40%–80% of lung cancer, 14%–91% of breast cancer, 33%–74% of gastric cancer, 25%–77% of colon cancer, 30%–50% of pancreatic cancer, 40%–80% of prostate cancer, 50%–90% of renal cancer, 35%–70% of ovarian cancer, and 36%–100% of head and neck cancer. EGFR overexpression and / or mutation in most tumors is associated with poor prognosis. In vitro studies and experimental animal models have demonstrated that EGFR activation regulates several cellular functions important for cell transformation and tumor progression, including proliferation / differentiation, survival, angiogenesis induction, metastatic capacity, and resistance to chemotherapy and radiotherapy (Normanno et al., 2003). Consequently, EGFR has become a hot topic in the development of anti-tumor drugs.

[0004] Multiple studies have confirmed that EGFR-targeted inhibitors have anti-tumor activity, and a number of drugs have been approved for marketing in China and other countries. These include small molecule tyrosinase inhibitors (such as gefitinib, afatinib, osimertinib, etc.), monoclonal antibodies (cetuximab, nimotuzumab, etc.), bispecific antibodies (ervantuzumab), and antibody-drug conjugates (cetuximab sarotalocan). Indications include non-small cell lung cancer with positive and / or mutated EGFR expression, head and neck squamous cell carcinoma, colorectal cancer, nasopharyngeal carcinoma, etc. Cetuximab and other EGFR monoclonal antibodies have also been explored in other tumors, including triple-negative breast cancer, gastric cancer, pancreatic cancer, etc., suggesting the anti-tumor effect of EGFR monoclonal antibodies in these tumor types.

[0005] Antibody Drug Conjugate (ADC) products targeting EGFR targets have become a hot spot in drug research and development to overcome resistance to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKI) and EGFR monoclonal antibodies. Currently available EGFR-ADC products include Rakuten Medical's Akalux (cetuximab sarotalocan), which was accelerated approved in Japan on September 25, 2020 for the treatment of unresectable locally advanced or recurrent head and neck cancer. It is the world's first approved photoimmunotherapy drug. The fastest progress in China is Miyake's MRG003, which has entered Phase II clinical trials.

[0006] CN202211461614.0 discloses an antibody-drug conjugate targeting EGFR:

[0007] Among them, Ab is an antibody targeting EGFR.

[0008] In vitro studies have shown that this ADC has a good inhibitory effect on various tumor cells. However, the therapeutic effect of this ADC on malignant tumors, especially advanced malignant tumors such as advanced solid tumors, needs further study.

[0009] Summary of the Invention

[0010] The present invention provides an application of an antibody-drug conjugate.

[0011] In one aspect, the present invention provides an antibody-drug conjugate of Formula I, and pharmaceutically acceptable salts, hydrates, solvates, stereoisomers, or isotope-labeled products thereof:

[0012] wherein Ab is selected from SWY2110, SWY2111, SWY2112, and SWY2113;

[0013] SWY2110 is an antibody having two heavy chains with the amino acid sequence shown in SEQ ID NO: 29 and two light chains with the amino acid sequence shown in SEQ ID NO: 30;

[0014] SWY2111 is an antibody having two heavy chains with the amino acid sequence shown in SEQ ID NO: 34 and two light chains with the amino acid sequence shown in SEQ ID NO: 30;

[0015] SWY2112 is an antibody having two heavy chains with the amino acid sequence shown in SEQ ID NO: 37 and two light chains with the amino acid sequence shown in SEQ ID NO: 30;

[0016] SWY2113 is an antibody having two heavy chains with the amino acid sequence shown in SEQ ID NO:40 and two light chains with the amino acid sequence shown in SEQ ID NO:30;

[0017] n is an integer or a decimal selected from 1-8.

[0018] Furthermore, n is selected from an integer of 4-8 or a decimal of 4-8.

[0019] Further, when n is an integer selected from 1-8, it can be 1, 2, 3, 4, 5, 6, 7, 8; when n is an integer selected from 4-8, it can be 4, 5, 6, 7, 8.

[0020] Further, the antibody-drug conjugate of Formula I has the structures shown in Formula I-1 to Formula I-4:

[0021] The present invention provides an antibody-drug conjugate of Formula I, and use of a pharmaceutically acceptable salt, hydrate, solvate, stereoisomer, or isotope-labeled product thereof in the preparation of a medicament for treating a tumor, wherein the tumor is selected from colorectal cancer, triple-negative breast cancer, gastric cancer, esophageal cancer, biliary tract cancer, HR-positive breast cancer, liver cancer, ovarian cancer, endometrial cancer, prostate cancer, urothelial carcinoma, squamous cell lung cancer, pancreatic cancer, cervical cancer, non-squamous non-small cell lung cancer, head and neck squamous cell carcinoma (including nasopharyngeal carcinoma), renal cancer, and the like.

[0022] Furthermore, the tumor is selected from triple-negative breast cancer with medium to high expression of EGFR, gastric cancer, esophageal cancer, pancreatic cancer, cervical cancer, biliary tract cancer, non-squamous non-small cell lung cancer, HR-positive breast cancer, liver cancer, ovarian cancer, endometrial cancer, prostate cancer, urothelial carcinoma, renal cancer, etc. with medium to high expression of EGFR, as well as RAS / BRAF mutant colorectal cancer, EGFR medium to high expression of squamous cell lung cancer (Sq-NSCLC), EGFR mutant non-squamous non-small cell lung cancer (Nsq-NSCLC), EGFR medium to high expression of head and neck squamous cell carcinoma (including nasopharyngeal carcinoma), RAS / BRAF wild-type EGFR medium to high expression of colorectal cancer, etc.

[0023] The present invention provides an antibody-drug conjugate of Formula I, and uses of pharmaceutically acceptable salts, hydrates, solvates, stereoisomers, or isotope-labeled products thereof in the prevention and treatment of tumors, wherein the tumor is selected from triple-negative breast cancer, gastric cancer, esophageal cancer, pancreatic cancer, cervical cancer, biliary tract cancer, HR-positive breast cancer, liver cancer, ovarian cancer, endometrial cancer, prostate cancer, urothelial carcinoma, squamous cell lung cancer, non-squamous non-small cell lung cancer, head and neck squamous cell carcinoma (including nasopharyngeal carcinoma), colorectal cancer, renal cancer, and the like.

[0024] Furthermore, the tumor is selected from triple-negative breast cancer with moderate to high expression of EGFR, gastric cancer, esophageal cancer, pancreatic cancer, cervical cancer, biliary tract cancer, non-squamous non-small cell lung cancer, HR-positive breast cancer, liver cancer, ovarian cancer, endometrial cancer, prostate cancer, urothelial carcinoma, renal cancer with moderate to high expression of EGFR, squamous cell lung carcinoma (Sq-NSCLC) with moderate to high expression of EGFR, non-squamous non-small cell lung cancer (Nsq-NSCLC) with mutant EGFR, head and neck squamous cell carcinoma (including nasopharyngeal carcinoma) with moderate to high expression of EGFR, colorectal cancer with wild-type RAS / BRAF with moderate to high expression of EGFR, and colorectal cancer with mutant RAS / BRAF, etc.

[0025] The antibody-drug conjugate of Formula I, and its pharmaceutically acceptable salts, hydrates, solvates, stereoisomers or isotope-labeled products are administered to patients in need of treatment in a clinically accepted manner, such as orally or by injection (eg, intravenous injection, intramuscular injection), preferably by intravenous injection.

[0026] The antibody-drug conjugate of Formula I, and its pharmaceutically acceptable salts, hydrates, solvates, stereoisomers, or isotope-labeled products, can be formulated into clinically acceptable pharmaceutical preparations, such as oral preparations and injectable preparations. Oral preparations include tablets and capsules; injectable preparations include injection solutions and lyophilized powders for injection. Optionally, the pharmaceutical preparations contain pharmaceutically acceptable excipients. Each dosage unit of the pharmaceutical preparation contains the antibody-drug conjugate of Formula I, and its pharmaceutically acceptable salts, hydrates, solvates, stereoisomers or isotope-labeled substances in an amount of 1 mg to 1000 mg, preferably 5 mg to 500 mg, or 10 to 300 mg, or 20 to 200 mg, or 50 to 150 mg, or 50 to 300 mg, or 50 to 400 mg, or 100 to 200 mg, or 100 to 300 mg, or 100 to 500 mg, more preferably 1 mg, 5 mg, 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 500 mg, 1000 mg, etc. The pharmaceutical preparation can be administered in a single dose or in divided doses.

[0027] The antibody-drug conjugate of Formula I, and its pharmaceutically acceptable salts, hydrates, solvates, stereoisomers, or isotope-labeled forms, is administered to a subject or patient in need of treatment in a therapeutically effective amount to treat a tumor. The therapeutically effective amount refers to a dose that can alleviate, inhibit tumor growth, or eliminate the tumor, benefiting the subject or patient without causing intolerable toxic side effects.

[0028] In some embodiments, the therapeutically effective amount per administration (based on the antibody-drug conjugate of Formula I) is 1 mg / time-1000 mg / time, preferably 5 mg-500 mg / time, or 10-300 mg / time, or 20-200 mg / time, or 50-150 mg / time, or 50-300 mg / time, or 50-400 mg / time, or 100-200 mg / time, or 100-300 mg / time, or 100-500 mg / time, more preferably 5 mg / time , 6mg / time, 8mg / time, 10mg / time, 15mg / time, 20mg / time, 25mg / time, 30mg / time, 40mg / time, 50mg / time, 60mg / time, 70mg / time, 80mg / time, 90mg / time, 100mg / time, 150mg / time, 200mg / time, 250mg / time, 300mg / time, 500mg / time, 600mg / time, 700mg / time, 800mg / time, 900mg / time, etc.

[0029] In some embodiments, the therapeutically effective amount (based on the antibody-drug conjugate of Formula I) is 1 mg / day-1000 mg / day, preferably 5 mg-500 mg / day, or 10-300 mg / day, or 20-200 mg / day, or 50-150 mg / day, or 50-300 mg / day, or 50-400 mg / day, or 100-200 mg / day, or 100-300 mg / day, or 100-500 mg / day, more preferably 10 mg / day, 20 mg / day, 30 mg / day, 40 mg / day, 50 mg / day, 60 mg / day, 70 mg / day, 80 mg / day, 90 mg / day, 100 mg / day, 150 mg / day, 200 mg / day, 250 mg / day, 300 mg / day, 500 mg / day, 600 mg / day, 700 mg / day, 800 mg / day, 900 mg / day, etc.

[0030] In some embodiments, the therapeutically effective amount (based on the antibody-drug conjugate shown in Formula I and the patient's body weight, mg is the dosage of the antibody-drug conjugate shown in Formula I, and kg is the patient's body weight) is 0.05 mg / kg-50 mg / kg, preferably 0.1 mg / kg-20 mg / kg, or 0.2 mg / kg-20 mg / kg, or 0.5 mg / kg-20 mg / kg, or 0.5 mg / kg-15 mg / kg, or 0.5 mg / kg-10 mg / kg, or 0.5 mg / kg-8 mg / kg, or 0.5 mg / kg-6 mg / kg, or 0.5 mg / kg- 4mg / kg, or 0.5mg / kg-2mg / kg, or 1mg / kg-20mg / kg, or 1mg / kg-10mg / kg, or 1mg / kg-8mg / kg, or 2mg / kg-20mg / kg, or 2mg / kg-10mg / kg, or 2mg / kg-8mg / kg, or 3mg / kg-20mg / kg, or 3mg / kg-10mg / kg, or 3mg / kg-8mg / kg, or 5mg / kg-20mg / kg, or 5mg / kg-10mg / kg, or 0.3mg / kg, or 0.4mg / kg , or 0.6mg / kg, or 0.8mg / kg, or 1.5mg / kg, or 1.8mg / kg, or 2.0mg / kg, or 2.2mg / kg, or 2.4mg / kg, or 2.5mg / kg, or 2.8mg / kg, or 3mg / kg, or 3.2mg / kg, or 3.5mg / kg, or 3.6mg / kg, or 3.8mg / kg, or 4.0mg / kg, or 4.2mg / kg, or 4.4mg / kg, or 4.5mg / kg, or 4.6mg / kg, or 4.8mg / kg, or 5.0mg / kg, or 5.2mg / kg, or 5.4mg / kg, or 5.5mg / kg, or 5.6mg / kg, or 5.8mg / kg, or 6.0mg / kg, or 6.2mg / kg, or 6.4mg / kg, or 6.8mg / kg, or 7.0mg / kg, or 7.2mg / kg, or 7.5mg / kg, or 7.8mg / kg, or 8.0mg / kg, or 8.5mg / kg, or 9.0mg / kg, or 9.5mg / kg, or 11mg / kg, or 11.5mg / kg, or 12.0mg / kg, or 16.8mg / kg, or 18.0mg / kg.

[0031] The above-mentioned therapeutically effective amount can be applied once a day (QD), or the above-mentioned therapeutically effective amount can be divided into multiple applications in one day as a daily total dose, for example, divided into twice a day (BID) or three times a day (TID) for application. The above-mentioned therapeutically effective amount can be applied once a week (QW), or the above-mentioned therapeutically effective amount can be applied as a weekly total dose in multiple days within a week, for example, divided into twice a week (BIW) or three times a week (TIW) for application. The above-mentioned therapeutically effective amount can be applied once every two weeks (Q2W), or the above-mentioned therapeutically effective amount can be applied as a weekly total dose in multiple days within two weeks, for example, divided into twice two weeks (BI2W) or three times two weeks (TI2W) for application. The above-mentioned therapeutically effective amount can be applied once in three weeks (Q3W), or the above-mentioned therapeutically effective amount can be applied as a total dose in multiple days within three weeks, for example, divided into twice three weeks (BI3W, such as D1, D8 every three weeks cycle) or three times three weeks (TI3W) for application. The above-mentioned therapeutically effective amount can be administered once a month (QM), or the above-mentioned therapeutically effective amount can be administered as a total dose over multiple days within a month, for example, twice a month (BI2M) or three times a month (TIM). The above-mentioned therapeutically effective amount can also be administered at intervals, such as once every 2-7 days, or once every 3 days, or 4 days, or 5 days, or 6 days, or once every 2-4 weeks, such as once every 2 weeks (once every 3 weeks, a cycle every 3 weeks), once every 1 week (once every 2 weeks, a cycle every 2 weeks or every 4 weeks) and once every 3 weeks (once every 4 weeks, a cycle every 4 weeks), or once every 1-3 months, such as once every 1 month (every 2 months); or continuously administer for 3 weeks, stop for 1 week, and a cycle every 4 weeks, or continuously administer for 2 weeks, stop for 1 week, and a cycle every 3 weeks, or continuously administer for 2 weeks, stop for 2 weeks, and a cycle every 4 weeks, or administer at intervals of 1 week (once every 2 weeks), a cycle every 4 weeks. An exemplary administration method includes administering once on the first day of each 3-week cycle.

[0032] In order to provide a more concise description, some quantitative data in this article do not use the term "about". It should be understood that, regardless of whether the term "about" is explicitly used or not, each numerical value given here includes not only the actual given value (given value), but also means to include an approximate value of such given value reasonably inferred by a person of ordinary skill in the art, including equivalents and approximate values ​​of such given value resulting from experimental and / or measurement conditions. The approximate value is preferably ±20%, ±15%, ±10%, ±8%, ±6%, ±5%, ±4%, ±3%, 2%, ±1% based on the given value.

[0033] Clinical studies have shown that the antibody-drug conjugate represented by Formula I, and its pharmaceutically acceptable salts, hydrates, solvates, stereoisomers or isotope labels can effectively treat advanced malignant tumors, especially EGFR-positive advanced solid tumors, overcome resistance to EGFR-TKI and EGFR monoclonal antibodies, and can bring clinical benefits to patients with advanced solid tumors, including non-small cell lung cancer, that are superior to existing treatments, and the disease can be controlled or alleviated.

[0034] Antibody sequence description

[0035] 1.EGFR antibody SWY2110

[0036] Light chain (SEQ ID NO: 30):

[0037] Heavy chain (SEQ ID NO: 29):

[0038] SWY2110 HCDR1:NYDVH SEQ ID NO:21

[0039] SWY2110 HCDR2:VIWSGGNTDYNTPFTS SEQ ID NO:22

[0040] SWY2110 HCDR3:ALDYYDYEFAY SEQ ID NO:23

[0041] SWY2110 LCDR1:RASQSIGTNIH SEQ ID NO:24

[0042] SWY2110 LCDR2:YASESIS SEQ ID NO:25

[0043] SWY2110 LCDR3:QQNNEWPTS SEQ ID NO:26

[0044] SWY2110 HV: SEQ ID NO: 27

[0045] SWY2110 LV: SEQ ID NO: 28

[0046] 2.EGFR antibody SWY2111

[0047] Light chain (SEQ ID NO: 30):

[0048] Heavy chain (SEQ ID NO: 34):

[0049] SWY2111 Heavy Chain Complementary Determining Region 1: DYDVH SEQ ID NO:31

[0050] SWY2111 Heavy Chain Complementary Determining Region 2: VIWSGGNTDYNTPFTS SEQ ID NO:22

[0051] SWY2111 Heavy Chain Complementary Determining Region 3: ALDDYDYEFAY SEQ ID NO:32

[0052] SWY2111 Light Chain Complementary Determining Region 1: RASQSIGTNIH SEQ ID NO:24

[0053] SWY2111 Light Chain Complementary Determining Region 2: YASESIS SEQ ID NO:25

[0054] SWY2111 Light Chain Complementary Determining Region 3: QQNNEWPTS SEQ ID NO:26

[0055] SWY2111 Heavy Chain Variable Region: SEQ ID NO:33

[0056] SWY2111 Light Chain Variable Region: SEQ ID NO:28

[0057] 3. EGFR Antibody SWY2112

[0058] Light Chain (SEQ ID NO:30):

[0059] Heavy Chain (SEQ ID NO:37):

[0060] SWY2112 Heavy Chain Complementary Determining Region 1: EYDVH SEQ ID NO:35

[0061] SWY2112 Heavy Chain Complementary Determining Region 2: VIWSGGNTDYNTPFTS SEQ ID NO:22

[0062] SWY2112 Heavy Chain Complementary Determining Region 3: ALDDYDYEFAY SEQ ID NO:32

[0063] SWY2112 Light Chain Complementary Determining Region 1: RASQSIGTNIH SEQ ID NO:24

[0064] SWY2112 Light Chain Complementary Determining Region 2: YASESIS SEQ ID NO:25

[0065] SWY2112 LCDR3:QQNNEWPTS SEQ ID NO:26

[0066] SWY2112 HV: SEQ ID NO: 36

[0067] SWY2112 LV: SEQ ID NO: 28

[0068] 4.EGFR antibody SWY2113

[0069] Light chain (SEQ ID NO: 30):

[0070] Heavy chain (SEQ ID NO: 40):

[0071] SWY2113 HCDR1:HYDVH SEQ ID NO:38

[0072] SWY2113 HCDR2:VIWSGGNTDYNTPFTS SEQ ID NO:22

[0073] SWY2113 HCDR3:ALDDYDYEFAY SEQ ID NO:32

[0074] SWY2113 LCDR1:RASQSIGTNIH SEQ ID NO:24

[0075] SWY2113 LCDR2:YASESIS SEQ ID NO:25

[0076] SWY2113 LCDR3:QQNNEWPTS SEQ ID NO:26

[0077] SWY2113 HV: SEQ ID NO: 39

[0078] SWY2113 LV: SEQ ID NO: 28 BRIEF DESCRIPTION OF THE DRAWINGS

[0079] Figure 1 shows the DAR value test results of SWY2110-JSSW-001

[0080] Figure 2 shows the DAR value test results of SWY2111-JSSW-001

[0081] Figure 3 shows the DAR value test results of SWY2112-JSSW-001

[0082] Figure 4 shows the DAR value test results of SWY2113-JSSW-001

[0083] Figure 5 shows the DAR value test results of SWY2110-Dxd

[0084] Figure 6 shows the SWY2110-Vc MMAE DAR value test results

[0085] Figure 7 shows the effect of SWY2110-ADC on the tumor volume of human colorectal cancer DiFi cell transplanted tumors in NU / NU mice

[0086] Figure 8 shows the effect of SWY2110-ADC on the tumor volume of human lung adenocarcinoma PC9-GR (Gefitinib-resistant cells) transplanted tumors in NU / NU mice

[0087] Figure 9 shows the results of the inhibitory activity test of SWY2110-ADC on DiFi cells

[0088] Figure 10 shows the results of the inhibitory activity test of SWY2110-ADC on PC9-GR cells

[0089] Figure 11 shows the effect of SWY2110-ADC on the tumor volume of human lung adenocarcinoma PC9-AR (PC9-Del19 / T790M / C797S, Osimertinib-resistant cells) transplanted tumors in NU / NU mice

[0090] Figure 12 shows the effects of SWY2110-ADC, SWY2111-ADC, SWY2112-ADC, and SWY2113-ADC on the tumor volume of human lung adenocarcinoma NCI-H1975 cell transplanted tumors in NU / NU mice.

[0091] Figure 13 shows the effects of SWY2110-ADC, SWY2111-ADC, SWY2112-ADC, and SWY2113-ADC on the tumor volume of human breast cancer MDA-MB-468 cell transplanted tumors in NOD-SCID mice.

[0092] Figure 14 shows the effects of SWY2110-ADC, SWY2111-ADC, SWY2112-ADC, and SWY2113-ADC on the tumor volume of human colorectal cancer DiFi cell transplanted tumors in NU / NU mice

[0093] Figure 15 shows the effects of SWY2110-ADC, SWY2111-ADC, SWY2112-ADC, and SWY2113-ADC on the tumor volume of human lung adenocarcinoma PC9-GR (Gefitinib-resistant cells) transplanted tumors in NU / NU mice DETAILED DESCRIPTION

[0094] 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 present invention. Experimental methods in the following examples where specific conditions are not specified are generally performed under conventional conditions or according to the conditions recommended by the manufacturer. Unless otherwise defined, all professional and scientific terms used herein have the same meaning as those familiar to professionals in the field. In addition, any methods and materials similar or equivalent to those described herein can be applied to the present invention. The preferred embodiments and materials shown herein are for demonstration purposes only.

[0095] The structures of the compounds of the present invention are determined by nuclear magnetic resonance (NMR) and / or liquid chromatography-mass spectrometry (LC-MS) and / or liquid chromatography (HPLC). The NMR measurements were performed using a Bruker Avance III 400 MHz nuclear magnetic resonance spectrometer; the LC-MS measurements were performed using a SHIMADZU LC-20AD-PDA-LCMS-2020 instrument; and the HPLC measurements were performed using a SHIMADZU LC-20AD-PDA high performance liquid chromatograph.

[0096] The starting materials in the examples of the present invention are known and can be purchased commercially, or can be synthesized using or according to methods known in the art.

[0097] The antibodies of the present invention can be prepared using the hybridoma technique first described by Kohler et al., Nature (1975), or by recombinant DNA methods (US Pat. No. 4,816,567, etc.).

[0098] Table 1: Abbreviations

[0099] Preparation Example

[0100] Example 1: Preparation of Linker-Drug Compounds

[0101] N-((2R,10S)-10-Benzyl-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2-methyl-1,6,9,12,15-pentaoxo-3-oxa-5,8,11,14-tetraazahexadec-16-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide (JSSW-001)

[0102] Option 1:

[0103] Step 1: Preparation of compound B2-1: B1-1 (1.0 g, 2.28 mmol), anhydrous tetrahydrofuran (30 mL), toluene (10 mL), pyridine (0.5 mL), and lead tetraacetate (1.83 g, 4.12 mmol) were added to a 100 mL three-necked flask. The solution turned orange and was heated under reflux. After one hour, the solution became colorless and a white solid was formed. After three hours of reaction, the solution was filtered through diatomaceous earth, the solid was rinsed with ethyl acetate, and the filtrate was dried using a rotary evaporator. The crude product was purified by column chromatography to obtain a solid (640 mg). LC-MS [M+Na] + :m / z 391.1.

[0104] Step 2: Preparation of Compound A2-1: To a 100ml three-necked flask, add A1-1 (2.7g, 30.0mmol) and N,N-dimethylformamide (30mL), followed by potassium carbonate (4.17g, 30.0mmol) and benzyl bromide (2.4mL, 20.0mmol). After reacting at room temperature for 14 hours, the reaction solution was added dropwise to water and extracted with ethyl acetate. The organic layers were combined and washed with saturated brine, dried over anhydrous magnesium sulfate, filtered, and concentrated. The crude product was purified by column chromatography (PE / EA = 3:1) to obtain 2.61g of the target compound as a colorless liquid.

[0105] 1 H NMR (400MHz, CDCl3): δ7.33-7.41(m,5H),5.21(s,2H),4.29-4.35(m,1H),2.77(d,J=5.6Hz,1H),1.44(d,J=6.8Hz,3H).

[0106] Step 3: Preparation of compound A3-1: A2-1 (720 mg, 4.0 mmol), B2-1 (368 mg, 1.0 mmol) and dichloromethane (5 mL) were added to a 100 ml three-necked flask, and 4-methylbenzenesulfonic acid pyridinium (75 mg, 0.30 mmol) was added. The reaction solution was heated under reflux overnight. After the reaction was completed, ethyl acetate (20 mL) was added to the reaction solution, washed three times with water, and dried over anhydrous magnesium sulfate. The anhydrous magnesium sulfate was removed by filtration, and the solution was concentrated. The crude product was purified by column chromatography (PE / EA=2:1) ​​to obtain A3-1 (290 mg) as a white solid.

[0107] LC-MS[M+Na] + :m / z 511.1. 1 H NMR (400MHz, CDCl3): δ7.77(d,J=7.2Hz,2H),7.58(d,J=7.6Hz,2H),7.40(t,J=7.6Hz,2H),7.26-7.37(m,6H),6.70(brs,1H ),5.12-5.30(m,3H),4.76-4.89(m,2H),4.45(d,J=6.8Hz,1H),4.19-4.27(m,2H),3.69-3.85(m,2H),1.41(d,J=6.8Hz,3H).

[0108] Step 4: Preparation of Compound A4-1: In a 100ml three-necked flask, A3-1 (291mg, 0.60mmol) was dissolved in tetrahydrofuran:ethyl acetate (4mL / 2mL). Pd / C (10% w / w, 60mg) was added. The atmosphere was replaced with hydrogen three times, then covered with a hydrogen balloon and stirred overnight. After the reaction, the reaction mixture was passed through celite and rinsed with methanol. The organic layer was concentrated to obtain product A4-1. LC-MS [M+Na] + :m / z 399.1.

[0109] Step 5: Preparation of compound A6-1: A4-1 (50 mg, 0.13 mmol, 1.3 eq), isotecan mesylate (A5-1) (50 mg, 0.049 mmol, 1.0 eq), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (48 mg, 0.13 mmol, 1.3 eq), N,N-diisopropylethylamine (36 mg, 0.28 mmol, 3 0eq), dissolved in N,N-dimethylformamide (2 ml), stirred at room temperature overnight, diluted with ethyl acetate, washed with half-saturated citric acid, then washed with brine, washed with sodium bicarbonate solution, washed with brine, the organic layer was dried over anhydrous sodium sulfate and filtered to remove anhydrous sodium sulfate, concentrated, and purified using a prep-TLC developing solvent system (DCM / MeOH=15:1) to obtain product A6-1 (56 mg), LC-MS [M+H] + :m / z 816.3.

[0110] Step 6: Preparation of compound A7-1: Add A6-1 (56 mg, 0.069 mmol), N,N-dimethylformamide (2 ml), and piperidine (12 mg, 0.14 mmol) to a 25 ml single-necked bottle, stir for 2 hours, and concentrate the solution to obtain a white solid. LC-MS [M+H] + :m / z 594.3.

[0111] Step 7: Preparation of compound JSSW-001: A7-1 (0.069 mmol), C7 (66 mg, 0.14 mmol), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (53 mg, 0.14 mmol), and a solution of N,N-diisopropylethylamine (36 mg, 0.28 mmol) in N,N-dimethylformamide (2 ml) were added to a 25 ml single-necked bottle and stirred at room temperature for 2 hours. The mixture was diluted with ethyl acetate (30 ml) and washed with half-saturated citric acid, followed by washing with brine, sodium bicarbonate solution, and brine. The organic layer was dried over anhydrous sodium sulfate and filtered to remove anhydrous sodium sulfate. The mixture was concentrated and purified using a prep-TLC developing solvent system (DCM / MeOH = 15:1) to obtain the product JSSW-001 (21 mg).

[0112] LC-MS[M+H] + :m / z 1048. 1H NMR (400MHz, DMSO-d6): δ8.63(t,J=6.4Hz,1H),8.53(d,J=8.8Hz,1H),8.28(t,J=6.0Hz,1H),8.03-8.10(m,2H),7.98(t,J =6.0Hz,1H),7.77(d,J=11.2Hz,1H),7.30(s,1H),7.14-7.25(m,5H),6.99(s,2H),6.51(s,1H),5.56-5.62(m,1H),5.40(s, 2H),5.08-5.23(m,2H),4.66-4.71(m,1H),4.42-4.56(m,2H),4.09-4.1 5(m,1H),3.54-3.76(m,7H),3.09-3.27(m,1H),2.97-3.02(m,1H),2.67- 2.77(m,1H),2.38(s,3H),2.16-2.21(m,2H),2.05-2.10(m,2H),1.83-1 .89(m,2H),1.37-1.46(m,7H),1.14-1.33(m,4H),0.87(t,J=7.2Hz,3H).

[0113] Option 2:

[0114] Step 1: Compounds C1-1 (8 g, 88.86 mmol) and C2-1 (22.66 g, 133.33 mmol) were added to a 250 mL reaction flask. Dimethylacetamide (DMAc) (40 mL, 5 v / w) was added and stirred until dissolved. The temperature was maintained at 0-10°C, followed by the dropwise addition of N,N-diisopropylethylamine (DIEA) (34.45 g, 266.6 mmol). After the addition was complete, the mixture was allowed to warm naturally to approximately 25°C and stirred at this temperature for 16 hours. TLC was used to monitor the complete reaction of the starting materials. The reaction mixture was poured into 120 mL of ice water, and methyl tert-butyl ether (MTBE) (40 mL) was added and stirred. The mixture was allowed to stand for separation. The aqueous phase was extracted four times with MTBE (40 mL x 4). The organic phases were combined and washed sequentially with 0.5 M hydrochloric acid (40 mL), water (40 mL), and saturated brine (40 mL x 2). The mixture was dried over anhydrous sodium sulfate, filtered, concentrated, and wet loaded onto a column for purification using a mixture of petroleum ether:ethyl acetate = 30:1 → 20:1 → 10:1 to obtain a light yellow liquid C3-1. LC-MS [M+Na] + :m / z 203.

[0115] Step 2: Under nitrogen protection, C4-1 (6 g, 16.29 mmol, 1.0 eq) and C3-1 (3.23 g, 17.92 mmol, 1.1 eq) were dissolved in tetrahydrofuran (THF) (60 mL, 10 V / w), cooled to 10-15 ° C, and p-toluenesulfonic acid (TsOH) (281 mg, 1.63 mmol, 0.1 eq) was added. After the addition was complete, the reaction was carried out at 14-18 ° C for 4 h and monitored by TLC. After the reaction was completed. The reaction solution was added to ice water (60 mL), and then extracted with ethyl acetate (EA) (60 mL*3). The organic phases were combined, washed with saturated NaHCO3 aqueous solution (60 mL), washed with water (60 mL*2), and washed with saturated brine (60 mL). The product was dried over anhydrous sodium sulfate, filtered, concentrated and evaporated to dryness. The sample was mixed and purified by column chromatography. The system of n-hexane:ethyl acetate = 10:1 → 5:1 → 3:1 → 2:1 → 1:1 was used to obtain colorless oil C5-1. LC-MS [M+Na] + :m / z 511.

[0116] Step 3: Under nitrogen protection, C5-1 (2.8 g, 5.74 mmol, 1.0 eq) was dissolved in DMAc (28 mL, 10 V / w), cooled to 14-18 ° C, and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) (436.6 mg, 2.87 mmol, 0.5 eq) was added dropwise. The mixture was stirred at this temperature for 1.5 h. The reaction of the raw materials was completed by TLC monitoring. The temperature was cooled to 0-10 ° C, and 4-methylbenzenesulfonic acid pyridinium (PPTS) was added. )(721.23 mg, 2.87 mmol, 0.5 eq), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) (1.1 g, 5.74 mmol, 1.0 eq), 1-hydroxybenzotriazole (HOBT) (775 mg, 5.74 mmol, 1.0 eq) and C7-1 (2.45 g, 4.88 mmol, 0.85 eq) were added and reacted at 0-10 ° C for 3-4 h. After the reaction of C7-1 was completed, TLC was monitored. The reaction solution was added to ice water (100 mL), and then extracted with 2-methyltetrahydrofuran (2-MeTHF) (100 mL*3). The organic phases were combined, washed with 0.5 M hydrochloric acid (150 mL*2), saturated NaHCO3 aqueous solution (100 mL*3), water (100 mL), saturated brine (100 mL), dried over anhydrous sodium sulfate, filtered, concentrated and evaporated to dryness. The mixed sample was purified by column chromatography with a system of DCM→DCM:MeOH=80:1→60:1→50:1→40:1→30:1→20:1 to obtain C8-1 as a white foamy solid, 3.1 g, with a yield of 72%. LC-MS [M+Na] + :m / z 772.

[0117] Step 4: Under nitrogen, C8-1 (3.1 g, 4.14 mmol, 1.0 eq) was dissolved in dichloromethane (DCM) (46.5 mL, 15 V / w). DBU (314.6 mg, 2.07 mmol, 0.5 eq) was added dropwise at room temperature (25-30°C). The reaction was stirred at this temperature for 16 h. TLC monitored the complete reaction of the starting material. The reaction solution was directly wet-filtered through a column using a DCM→DCM:MeOH ratio of 50:1→30:1→20:1→10:1 ratio to obtain C9-1 as a white foamy solid. LC-MS [M+H] + :m / z 528.

[0118] Step 5: Under nitrogen, dissolve C9-1 (1.7 g, 3.2 mmol, 1.0 eq) in water (25.5 mL, 15 V / w) and tert-butanol (8.5 mL, 5 V / w). Stir and dissolve until clear. Replace the atmosphere with nitrogen once, then add Pd / C (0.34 g, 20% w / w). Replace the atmosphere with hydrogen three times. Hydrogenate at room temperature (25-30°C) for 12 h. LCMS monitors the reaction for complete reaction. The reaction mixture is filtered, and the filter cake is washed with water (25 mL x 2). The filtrate is filtered again and concentrated to remove the tert-butanol. The aqueous phase is then lyophilized to yield 1.5 g of C10-1 as a white powdery solid, with a yield of 100%. LC-MS [M+H] + :m / z 438.

[0119] Step 6: Under nitrogen, C10-1 (1.4 g, 3.2 mmol, 1.0 eq) was dissolved in acetonitrile (14 mL, 10 V / w) and water (28 mL, 20 V / w). C11-1 (1.86 g, 6.097 mmol, 1.1 eq) was added and stirred to dissolve. The mixture was cooled to 0-10°C and DIEA (330.24 mg, 2.56 mmol) was added dropwise. After addition, the mixture was stirred at the same temperature for 16 h. LC-MS monitored the complete reaction of the starting material. Na2HPO4 (260 mg) and NaH2PO4 (5.6 g) were added to 20 mL of water and stirred to dissolve to prepare a buffer solution. The buffer solution was cooled to 0-5°C. The reaction solution was poured into chilled buffer solution and extracted with DCM / isopropanol = 4 / 1 (50 mL x 4). The organic phases were combined, dried, filtered, concentrated, evaporated, and purified by column chromatography. The system of DCM→DCM:MeOH=50:1→30:1→20:1→15:1 was passed through the column to obtain yellow solid C12-1.

[0120] Step 7: Under nitrogen, A5-1 (547.4 mg, 1.03 mmol, 1.0 eq) was dissolved in 5% anhydrous sodium sulfate in water (6.5 mL) and THF (7.8 mL). Stirring was not effective until the solution was dissolved. The mixture was cooled to 0-10°C and N-methylmorpholine (NMM) (104 mg, 1.03 mmol, 1.0 eq) was added. The mixture was reacted at this temperature for 1 h. C12-1 (650 mg, 1.03 mmol, 1.1 eq), EDCI (296.2 mg, 1.545 mmol, 1.5 eq), and ethyl 2-oximecyanoacetate (73.9 mg, 0.52 mmol, 0.5 eq) were added in sequence. The mixture was reacted at 0-10°C for 4-5 h. The reaction was monitored by TLC to confirm the complete reaction of the starting material. The reaction mixture was added to 0.1M hydrochloric acid (13 mL, 20 V / w) under an ice bath and extracted with 2-methyltetrahydrofuran (2-MeTHF) (13 mL*3). The organic phases were combined and washed once with 0.05M hydrochloric acid (13 mL, 20 V / w), washed with water (13 mL*2), dried, filtered, concentrated and evaporated to dryness, and purified by column chromatography. The column chromatography was performed using a DCM→DCM:MeOH ratio of 50:1→30:1→20:1→15:1→12:1 to obtain JSSW-001 as a yellow foamy solid. LC-MS [M+H] + :m / z 1048.

[0121] Example 2 Preparation of Antibodies

[0122] SWY2110 was obtained by transiently transfecting HEK293 cells with plasmids encoding light and heavy chain genes using known techniques, and then expressing and purifying the cells.

[0123] Through computer simulation-assisted technology, SWY2110 and EGFR protein were modeled and docked to obtain the antigen-antibody complex. D, E, and H scans were performed on amino acids within 100 μg / mL, yielding several pH-dependent antibodies with reduced affinity at pH 7.4 and essentially unchanged or with a minimal decrease in affinity at pH 6.0. The resulting antibody sequences were cloned into mammalian expression vectors and expressed via transfection of HEK-293 cells. After expression, the supernatant was collected and purified using an AKTA system equipped with a prepacked Protein A column to obtain the target antibodies SWY2111, SWY2112, and SWY2113.

[0124] Example 3 Preparation of Antibody-Drug Conjugates

[0125] SWY2110-JSSW-001 ADC:

[0126] The first step is the reduction of antibodies:

[0127] The SWY2110 antibody medium obtained after transient transfection of HEK293 cells expressing and purifying the plasmid encoding the light and heavy chain genes was replaced with PBS6.0 / EDTA by known techniques to prepare an antibody concentration of 10 mg / mL. The solution (1.0 mL) was placed in a 1.5 mL polypropylene tube, and a 10 mM TCEP (B&K Technology Co., Ltd.) aqueous solution (66.6 uL; 10 equivalents relative to one molecule of antibody) and a 1 M potassium dihydrogen phosphate aqueous solution (Tianjin Guangfu Technology Development Co., Ltd.) were added thereto. After confirming that the pH of the solution was within 7.0 ± 0.1, it was incubated at 37 ° C for 3 hours to reduce the disulfide bonds in the antibody.

[0128] Step 2: Conjugation of Antibody to Linker-Drug Compound:

[0129] To the above solution, a 10 mM dimethyl sulfoxide solution (87 μL; 13 equivalents per antibody molecule) of the compound JSSW-001 obtained in Example 1 was added at room temperature. The mixture was mixed and allowed to react at room temperature for 30 minutes to attach the drug linker to the antibody. Next, a 100 mM aqueous solution of NAC (J&K Technology Co., Ltd.) (10.2 μL) was added and stirred at room temperature for another 20 minutes to terminate the reaction.

[0130] Step 3: Purification of Antibody-Drug Conjugate:

[0131] Ultrafiltration centrifuge tube (Merck, The reaction solution was purified by ultrafiltration through Regenerated Cellulose (30kDa MWCO), 15mL sample volume. A buffer solution consisting of 0.89mg / ml L-histidine, 4.04mg / ml L-histidine hydrochloride, 0.03% Tween 80, and 90mg / ml sucrose (pH 5.5) was added to the reaction solution to remove unconjugated drug linkers and other low-molecular-weight reagents, yielding a purified antibody-drug conjugate (SWY2110-JSSW-001 ADC, DAR = 7.12). The DAR value is shown in Figure 1.

[0132] Step 4: Determination of the Drug-Antibody Coupling Ratio (DAR) of the Antibody-Drug Conjugate:

[0133] Dithiothreitol was added to a final concentration of 20 mM to 2 mg / kg of the antibody-drug conjugate, and the sample, which had broken the interchain disulfide bonds of the antibody-drug conjugate, was incubated at 37°C in a water bath for 30 minutes. The sample was then used for HPLC analysis. The HPLC system used was an Agilent Technologies 1260 Infinity HPLC, and the column was a PLRP-S (5 μm particle size; 2.1 mm × 50 mm; Agilent Technologies). The column temperature was 80°C, and the mobile phase A consisted of 0.1% trifluoroacetic acid (TFA) in water, and the mobile phase B consisted of 0.1% trifluoroacetic acid (TFA) in acetonitrile. The sample volume was 10 μL, and the gradient program was 27%-27% from 0-3 minutes, 27%-35% from 3-8 minutes, 35%-43% from 8-25 minutes, 43%-95% from 25-26 minutes, 95%-95% from 26-31 minutes, 95%-27% from 31-32 minutes, and 27%-27% from 32-40 minutes. Compared to unlinked light chains (L0) and heavy chains (H0), the hydrophobicity of drug-linked light chains (light chains linked to one drug, L1) and heavy chains (heavy chains linked to one drug, H1; heavy chains linked to two drugs, H2; and heavy chains linked to three drugs, H3) increases with the number of linked drugs, resulting in elution in the order of L0, L1, H0, H1, H2, and H3. DAR values ​​were calculated based on peak areas at 280 nm. In the specification and claims of the present application, unless otherwise specified, the DAR value is calculated based on the peak area at 280 nm by HPLC analysis, as described above.

[0134] Using the same method as that used to prepare SWY2110-JSSW-001 ADC, the antibody SWY2110 was replaced with SWY2111, SWY2112, and SWY2113 to obtain SWY2111-JSSW-001 ADC (Dar: 7.48, DAR value results are shown in Figure 2), SWY2112-JSSW-001 ADC (Dar: 7.51, DAR value results are shown in Figure 3), and SWY2113-JSSW-001 ADC (Dar: 7.50, DAR value results are shown in Figure 4), respectively:

[0135] The same method as that used to prepare SWY2110-JSSW-001 ADC was used to replace the antibody JSSW-001 with GGFG-DXD to obtain SWY2110-DXD ADC (Dar: 6.93, the DAR value results are shown in Figure 5):

[0136] Preparation of antibody-drug conjugate (SWY2110-VC MMAE ADC, DAR = 4.53):

[0137] The first step is the reduction of antibodies:

[0138] The SWY2110 antibody medium obtained after transient transfection of HEK293 cells expressing and purifying the plasmid encoding the light and heavy chain genes was replaced with PBS6.0 / EDTA by known techniques to prepare an antibody concentration of 10 mg / mL. The solution (1.0 mL) was placed in a 1.5 mL polypropylene tube, and a 10 mM TCEP (B&K Technology Co., Ltd.) aqueous solution (17 uL; 2.5 equivalents relative to one molecule of antibody) and a 1 M potassium dihydrogen phosphate aqueous solution (Tianjin Guangfu Technology Development Co., Ltd.) were added thereto. After confirming that the pH of the solution was within 7.4 ± 0.1, it was incubated at 37 ° C for 1 hour to reduce the disulfide bonds of the antibody.

[0139] Step 2: Conjugation of Antibody to Linker-Drug Compound:

[0140] To the above solution, a 10 mM dimethyl sulfoxide solution (31.28 μL; 4.6 equivalents per antibody molecule) of commercially available mc-vc-PAB-MMAE (CAS No. 646502-53-6, abbreviated as Vc MMAE) was added at room temperature. Mix thoroughly and react at room temperature for 30 minutes to attach the linker-drug compound to the antibody. Next, a 100 mM aqueous solution of NAC (J&K Technology Co., Ltd.) (10.2 μL) was added and stirred at room temperature for 20 minutes to terminate the reaction.

[0141] Step 3: Purification of Antibody-Drug Conjugate:

[0142] Ultrafiltration centrifuge tubes (Merck, The reaction solution was purified by ultrafiltration through Regenerated Cellulose (30kDa MWCO), 15mL sample volume). 25mM 2-(N-morpholino)ethanesulfonic acid (MES), pH 6.5, was added as a purification buffer to the reaction solution. The solution was concentrated to 1-2mL and the purification buffer was added again, with a displacement factor greater than 1000. This removed unconjugated linker-drug compound and other low molecular weight reagents, yielding a purified antibody-drug conjugate (SWY2110-VC MMAE ADC, DAR = 4.53).

[0143] Step 4: Determination of the Drug-Antibody Coupling Ratio (DAR) of the Antibody-Drug Conjugate:

[0144] A 10 μg sample of SWY2110-Vc MMAE ADC was loaded onto a chromatographic column (2.5 μm particle size; 4.6 mm × 10 cm; TSKgel Butyl-NPR). Mobile phase A consisted of 20 mM PB, pH 7.0, in a 1.5 M (NH₄)₂SO₄ aqueous solution, and mobile phase B consisted of 20 mM PB, pH 7.0, in a 25% isopropanol solution. The flow rate was controlled at 0.8 mL / min, the column temperature was 30°C, and the detection wavelength was 280 nm. The gradient program was 0-20 min, with mobile phase B increasing from 0% to 100%, then 20-25 min, 100% mobile phase B, and then 25-30 min, 100% mobile phase A. The unattached small molecules are designated DAR0, the attached small molecules are designated DAR2, the attached small molecules are designated DAR4, the attached small molecules are designated DAR6, and the attached small molecules are designated DAR8. Therefore, the elution order is DAR0, DAR2, DAR4, DAR6, and DAR8. The DAR value is calculated based on the peak area at 280 nm, where A represents the percentage of each peak area. The DAR value results are shown in Figure 6.

[0145] DAR=2×A DAR2 +4×A DAR4 +6×A DAR6 +8×A DAR8

[0146] Biological Examples

[0147] Test 1: In vitro cell activity of antibody-drug conjugates

[0148] 1. Test objectives:

[0149] The purpose of this study is to detect the inhibitory activity of the antibody-drug conjugate of the present invention on the proliferation of SK-BR-3 (ATCC / HTB-30) tumor cells, human colorectal cancer cells DiFi, human lung cancer gefitinib-resistant cells PC9-GR, and human lung cancer mutant cells PC-9 (Del19-T790M-C797S) in vitro. The cells were treated with different concentrations of the compound in vitro, cultured, and then Resazurin was added to read the fluorescence values ​​at ex550nm / em610nm. The IC values ​​were obtained by four-parameter fitting of the data. 50 The biological activity of the compound was calculated.

[0150] 2. Test materials and equipment:

[0151] Material

[0152] equipment

[0153] 3. Test operation method 1:

[0154] 3.1 Culture medium: DMEM, 10% FBS, 1× double antibody

[0155] 3.2 Cell culture: Take out a frozen SK-BR-3 / MDA-MB-468 cell from liquid nitrogen and resuscitate to 75cm 2 Culture in culture flasks. Grow until the cells are >75% confluent and have been passaged at least 3 times.

[0156] 3.2.1 If cell passage is not performed, replace the culture medium every 3 to 4 days.

[0157] 3.2.2 If cell expansion is required, subculture the cells into larger culture flasks and allow the cells to reach a confluency of >75% before use in the assay.

[0158] 3.3 Cell collection: When the culture flask is nearly full, collect SK-BR-3 / MDA-MB-468 cells.

[0159] 3.3.1 Discard the culture medium and wash with PBS to remove dead cells and residual culture medium.

[0160] 3.3.2 Add 2-3 mL of 0.25% Trypsin-EDTA, gently shake the culture flask, and incubate at 37°C for 2-3 minutes to digest the cells.

[0161] 3.3.3 Immediately add 5 mL of culture medium to the culture flask and gently pipette up and down to disperse the cells.

[0162] 3.3.4 Transfer the cell suspension into a sterile centrifuge tube and centrifuge at 200×g for 3 minutes.

[0163] 3.3.5 Discard the culture medium in the tube and add 5-10 mL of fresh culture medium to resuspend the cells.

[0164] 3.4 Cell density determination: Count the cells using a cell counting plate under a microscope.

[0165] 3.5 Inoculation of assay plates

[0166] 3.5.1 Dilute the cells to 1×10 5 cells / mL, and 100 μL / well was seeded into the assay plate (except rows A and H).

[0167] 3.5.2 Add 120 μL of culture medium to each well in rows A and H as blank controls.

[0168] 3.5.3 Incubate at 37°C, 5% CO2 for 4-6 hours to allow cells to adhere.

[0169] 3.6 Prepare diluted compound samples

[0170] The compound was diluted to a starting concentration of 18 μg / ml and then diluted 3-fold to obtain a total of 11 gradient solutions, with the 12th column being a blank control.

[0171] 3.7 Dosing treatment

[0172] 3.7.1 Take 20 μL of each drug dilution in columns 1 to 11 of the dilution plate and add them to columns 1 to 11 of the assay plate.

[0173] 3.7.2 Add 20 μL of fresh culture medium to column 12 and rows A and H.

[0174] 3.7.4 Gently shake the plate on a plate shaker for 10-15 seconds, then incubate the plate at 37°C for 3 days.

[0175] 3.8 Analysis

[0176] 3.8.1 After incubation, add 20 μL of 0.03% resazurin (diluted with 1× PBS) to each well and gently shake for 10-15 seconds.

[0177] 3.8.2 After incubation at 37°C for 3-4 hours, read the plate reader. Set the parameters as follows:

[0178] Excitation light: 550nm

[0179] Emission light: 610nm

[0180] Points time: 50

[0181] Shake: 15 seconds, vortex

[0182] Reading: Top reading

[0183] Read times / well: 1

[0184] Gain: Set to optimize (should be between 35 and 42)

[0185] If you are reading multiple boards, make sure that the gain settings used are consistent across all boards.

[0186] 3.8.3 Use Excel data to draw a graph and fit the IC values ​​of the reference standard and sample 50 .

[0187] 3.8.3.1 Use Model 201 to plot data points.

[0188] 3.8.3.2 For the Fit parameter, use the following command:

[0189] aA: Provisioning

[0190] bB: Provisioning

[0191] cC: Provisioning

[0192] dD: Provisioning

[0193] e. No constraints for all

[0194] 3.8.3.3 Output parameter C is IC 50 , unit is ng / mL.

[0195] Test operation method 2:

[0196] This study validated the inhibition of cell proliferation of DiFi (human colorectal cancer cells) and PC9-GR (gefitinib-resistant human lung cancer cells) by SWY2110 monoclonal antibody (SWY2110 mAb), SWY2110-DXD ADC (DAR=6.93), and SWY2110-JSSW-001 ADC (DAR=7.12). The specific concentrations were set as follows:

[0197] DiFi: SWY2110 mAb, SWY2110-DXD ADC (DAR = 6.93), and SWY2110-JSSW-001 ADC (DAR = 7.12) were diluted to 3000 ng / ml. A total of 10 concentrations were set at 3× descending dilutions, namely 3000, 1000, 333.33, 111.11, 37.04, 12.35, 4.12, 1.37, 0.46, and 0.15 ng / ml, and the drug exposure lasted for 144 h.

[0198] PC9-GR: SWY2110 mAb, SWY2110-DXD ADC (DAR = 6.93), and SWY2110-JSSW-001 ADC (DAR = 7.12) were diluted to a starting concentration of 1000 ng / ml and 5× descending dilutions were used to set a total of 8 concentrations: 1000, 200, 40, 8, 1.6, 0.32, 0.64, and 0.013 ng / ml. The drug exposure lasted for 144 h.

[0199] Logarithmically growing cells were seeded in 96-well plates (100 μL / well) at a specific density. After 24 hours of adherent attachment, 100 μL of culture medium containing varying concentrations of SWY2110 mAb, SWY2110-DXD ADC (DAR = 6.93), or SWY2110-JSSW-001 ADC (DAR = 7.12) was added to each well. Three replicates were set for each drug concentration, along with corresponding blank wells (medium alone) and normal wells (drug concentration 0). After 144 hours of drug exposure, 20 μL of MTT working solution (5 mg / mL) was added to each well. The cells were incubated at 37°C for 4 hours, the supernatant was removed, and 150 μL of DMSO (analytical grade) was added. The cells were shaken and mixed on a microplate shaker. The plates were wiped clean, and the optical density (OD) was measured at 550 nm on a microplate reader.

[0200] The cell growth inhibition rate was calculated using the following formula:

[0201] Inhibition rate (%) = (OD value 正常孔 -OD value 给药孔 ) / (OD value 正常孔 -OD value 空白孔 )×100%

[0202] According to the inhibition rate of each concentration, the drug half-maximal inhibitory concentration IC was calculated using SPSS19.0 50 The results are shown in Table 2 and Figures 9 and 10.

[0203] Table 2: IC of SWY2110-ADC against two cell lines 50 Value (ng / ml)

[0204] “--” indicates that the results showed no anti-cell proliferation effect.

[0205] As can be seen from the data in Table 2, the antibody-drug conjugate SWY2110-JSSW-001 ADC (DAR=7.12) and SWY2110-DXD ADC (DAR=6.93) of the present invention have similar IC values ​​for the EGFR high-expressing cell line DiFi and the gefitinib-resistant cell line PC9-GR. 50 The results showed that SWY2110 mAb has a better anti-cell proliferation effect than SWY2110 mAb.

[0206] Study 2: Efficacy of SWY2110-ADC against human colorectal cancer DiFi cell transplants in NU / NU mice

[0207] 1. Experimental Animals

[0208] NU / NU mice, 5-6 weeks old, 14

[0209] 2. Purpose of the test

[0210] The in vivo antitumor effects of two SWY2110-ADC drugs were investigated using a human colorectal cancer DiFi cell NU / NU mouse xenograft tumor model.

[0211] 3. Drug dosage and grouping

[0212] Table 3 Animal grouping and dosage table Note: iv: intravenous injection.

[0213] 4. Test methods

[0214] In this study, human colorectal cancer DiFi cells were used to construct a nude mouse tumor transplant model. 3 At d0, the animals were divided into three groups balanced by tumor volume according to Table 3: a vehicle group and a SWY2110-JSSW-001 ADC (DAR = 7.12) group of 5 animals each, and a SWY2110-Vc MMAE ADC (DAR = 4.53) group of 4 animals. A single intravenous administration of 10 mL / kg was performed. The vehicle control group (Vehicle) was given 0.9% sodium chloride injection. The animals were observed for 27 days after administration. The inhibitory effects of the two SWY2110-ADCs listed in the table on human colorectal cancer xenografts in DiFi nude mice were compared.

[0215] 5. Observation indicators

[0216] 5.1 Evaluation Metrics

[0217] (1) Tumor volume: V = 1 / 2 × A × B 2

[0218] (2) Relative tumor volume:

[0219] (3) Relative tumor volume growth rate:

[0220] (4) Tumor inhibition rate:

[0221] Note: V: tumor volume

[0222] A: Tumor growth

[0223] B: Tumor width

[0224] RTV: relative tumor volume

[0225] TV nd : Tumor volume on day n

[0226] TV 0d : Tumor volume on day 0

[0227] RTVxnd: average relative tumor volume on day n

[0228] TV Xn : Average tumor volume on day n in the drug-treated group

[0229] TV X0 : Average tumor volume on day 0 of the drug-treated group

[0230] TV Mn : Average tumor volume on day n in the solvent group

[0231] TV M0 : Average tumor volume on day 0 in the solvent group

[0232] 6 Results

[0233] 6.1 Tumor volume

[0234] At the endpoint of the trial, compared with the vehicle group, both SWY2110-Vc MMAE ADC (DAR = 4.53) (P < 0.05) and SWY2110-JSSW-001 ADC (DAR = 7.12) (P < 0.05) significantly inhibited the volume of transplanted tumors. The 27-day tumor inhibition rate in the SWY2110-JSSW-001 ADC (DAR = 7.12) group was as high as 74.6%, while the SWY2110-Vc MMAE ADC (DAR = 4.53) had the second highest inhibition rate, at 69.7%. Since the toxicity of the JSSW-001 small molecule is lower than that of Vc MMAE, this suggests that the SWY2110-JSSW-001 ADC (DAR = 7.12) can achieve a more effective and less toxic effect. See Figure 7 and Table 4 for details.

[0235] Table 4 Tumor parameters of each group after 27 days of SWY2110-ADC administration *: P < 0.05 compared with the vehicle group

[0236] Experiment 3: Efficacy of SWY2110-ADC on human lung adenocarcinoma PC9-GR (Gefitinib-resistant cells) transplanted tumors in NU / NU mice

[0237] 1. Experimental Animals

[0238] NU / NU mice, 5-6 weeks old, 30

[0239] 2. Purpose of the test

[0240] The in vivo antitumor effects of SWY2110 monoclonal antibody and three SWY2110-ADC drugs were investigated using a NU / NU mouse xenograft tumor model of human lung adenocarcinoma PC-9-GR (Gefitinib-resistant cells).

[0241] 3. Drug dosage and grouping

[0242] Table 5 Animal grouping and dosage table Note: iv: intravenous injection, qw*4w: once a week for four weeks.

[0243] 4. Test methods

[0244] In this study, human lung adenocarcinoma PC9-GR (Gefitinib-resistant cells) cells were used to construct a nude mouse tumor transplantation model. 3 At d0, the animals were divided into 5 groups (d0) according to tumor volume according to Table 5, with 6 animals in each group. The drugs were administered intravenously once a week for 4 consecutive weeks at a dosage of 10 mL / kg. The vehicle control group (Vehicle) was given 0.9% sodium chloride injection. The experiment was terminated 28 days after administration to compare the inhibitory effects of SWY2110 monoclonal antibody and three SWY2110-ADC drugs on human lung adenocarcinoma PC-9-GR (Gefitinib-resistant cells) xenografts in nude mice.

[0245] 5. Observation indicators

[0246] 5.1 Evaluation Metrics

[0247] See Experiment 2, 5.1, Evaluation Indicators

[0248] 6 Results

[0249] 6.1 Tumor volume

[0250] At the end of the experiment, compared with the vehicle group, SWY2110-Vc MMAE ADC (DAR=4.53) (P<0.05), SWY2110-DXD ADC (DAR=6.93) (P<0.05) and SWY2110-JSSW-001 ADC (DAR=7.12) (P<0.001) could inhibit the volume of transplanted tumors. The antibody SWY2110 had a poor inhibitory effect on tumors, with a tumor inhibition rate of only 20.3%. The SWY2110-JSSW-001 ADC (DAR = 7.12) group had the most significant inhibitory effect on the volume of PC9-GR transplanted tumors, with a tumor inhibition rate of up to 77.7%. The tumor inhibition rates of SWY2110-Vc MMAE ADC (DAR = 4.53) and SWY2110-DXD ADC (DAR = 6.93) were 40.4% and 50.2%, respectively. The tumor inhibition effect of SWY2110-JSSW-001 ADC (DAR = 7.12) on PC9-GR was 154% of that of SWY2110-DXD ADC (DAR = 6.93). The tumor inhibition effect of SWY2110-JSSW-001 ADC (DAR = 7.12) on PC9-GR was 154% of that of SWY2110-DXD ADC (DAR = 6.93). The tumor inhibition effect of ADC (DAR=4.53) on PCR-GR was 192%, indicating that SWY2110-JSSW-001 ADC (DAR=7.12) still has a significant inhibitory effect on gefitinib-resistant tumor cells. See Figure 8 and Table 6 for details.

[0251] Table 6 Tumor parameters of each group after 28 days of SWY2110-ADC administration *: P < 0.05 compared with the vehicle group, ***: P < 0.001 compared with the vehicle group

[0252] Experiment 4: Efficacy of SWY2110-ADC on human lung adenocarcinoma PC9-AR (PC9-Del19 / T790M / C797S, Osimertinib-resistant cells) cell transplants

[0253] 1. Experimental Animals

[0254] Balb / c nude mice, 5-6 weeks old, 24

[0255] 2. Purpose of the test

[0256] The anti-tumor effects of three SWY2110-ADC drugs in vivo were investigated using a NU / NU mouse xenograft tumor model derived from human lung adenocarcinoma PC9-Del19 / T790M / C797S (osimertinib-resistant cells, hereinafter referred to as PC9-AR).

[0257] 3. Drug dosage and grouping

[0258] Table 7 Animal grouping and dosage table Note: iv: intravenous injection, iv / 1 time: administration once.

[0259] 4. Test methods

[0260] In this study, human lung adenocarcinoma PC9-Del19 / T790M / C797S (Osimertinib-resistant cells) were used to construct a nude mouse tumor transplantation model. 3 At d0, the animals were divided into four groups (6 animals per group) based on tumor volume according to Table 7. The animals were intravenously administered once at a volume of 10 mL / kg. The vehicle control group (Vehicle) was given 0.9% sodium chloride injection. The experiment was terminated 24 days after administration to compare the inhibitory effects of the three SWY2110-ADC drugs on nude mouse xenografts of human lung adenocarcinoma PC9-Del19 / T790M / C797S (osimertinib-resistant cells).

[0261] 5. Observation indicators

[0262] 5.1 Evaluation Metrics

[0263] See Experiment 2, 5.1, Evaluation Indicators

[0264] 6 Results

[0265] 6.1 Tumor volume

[0266] At the end of the experiment, compared with the vehicle group, SWY2110-Vc MMAE ADC (DAR=4.53) (P<0.001), SWY2110-Dxd ADC (DAR=6.93) (P<0.001) and SWY2110-JSSW-001 ADC (DAR=7.12) (P<0.001) could inhibit the volume of transplanted tumors. The SWY2110-JSSW-001 ADC (DAR = 7.12) group had the most significant inhibitory effect on PC9-AR transplanted tumor volume, with a tumor inhibition rate of up to 97.5%. The tumor inhibition rates of SWY2110-Vc MMAE ADC (DAR = 4.53) and SWY2110-DXD ADC (DAR = 6.93) were 89.7% and 76.2%, respectively. The tumor inhibition effect of SWY2110-JSSW-001 ADC (DAR = 7.12) was 128% of that of SWY2110-DXD ADC (DAR = 6.93), and the tumor inhibition effect of SWY2110-JSSW-001 ADC (DAR = 7.12) was 109% of that of SWY2110-Vc MMAE ADC (DAR = 4.53), indicating that SWY2110-JSSW-001 ADC (DAR = 7.12) still has a significant inhibitory effect on osimertinib-resistant tumor cells. See Figure 11 and Table 8 for details.

[0267] Table 8 Tumor parameters of each group after 24 days of SWY2110-ADC administration ***: Statistically significant p < 0.001 relative to vehicle group

[0268] Experiment 5: Efficacy of SWY2110-ADC, SWY2111-ADC, SWY2112-ADC, and SWY2113-ADC on human lung adenocarcinoma NCI-H1975 (human lung adenocarcinoma cell) transplanted tumors

[0269] 1. Experimental Animals

[0270] NU / NU mice, 5-6 weeks old, 30

[0271] 2. Purpose of the test

[0272] The human lung adenocarcinoma NCI-H1975 (human lung adenocarcinoma cell) NU / NU mouse transplant tumor model was used to investigate the in vivo anti-tumor effects of SWY2110-JSSW-001, SWY2111-JSSW-001, SWY2112-JSSW-001, and SWY2113-JSSW-001 drugs.

[0273] 3. Drug dosage and grouping

[0274] Table 9 Animal grouping and dosage table Note: iv: intravenous injection, iv / 1 time: one administration;

[0275] 4. Test methods

[0276] In this study, human lung adenocarcinoma NCI-H1975 cells (human lung adenocarcinoma cells) were used to construct a nude mouse tumor transplantation model. 3 At 37 ℃, the animals were divided into 5 groups (d0) according to the tumor volume according to Table 9, with 6 animals in each group. Each dose group received a single dose of 1 mg / kg.

[0277] The drug was administered intravenously once at a volume of 10 mL / kg. The vehicle control group (Vehicle) was given 0.9% sodium chloride injection. The experiment was terminated 21 days after administration to compare the inhibitory effects of SWY2110-JSSW-001, SWY2111-JSSW-001, SWY2112-JSSW-001, and SWY2113-JSSW-001 on human lung adenocarcinoma NCI-H1975 xenografts in nude mice.

[0278] 5. Observation indicators

[0279] 5.1 Evaluation Metrics

[0280] See Experiment 2, 5.1, Evaluation Indicators

[0281] 6 Results

[0282] 6.1 Tumor volume

[0283] At the end of the trial, compared with the vehicle group, SWY2110-JSSW-001 ADC (DAR = 7.12), SWY2112-JSSW-001 ADC (DAR = 7.51), SWY2111-JSSW-001 ADC (DAR = 7.48), and SWY2113-JSSW-001 ADC (DAR = 7.50) (P < 0.001) all showed excellent tumor inhibition effects, with tumor inhibition rates reaching 84.1%, 83.0%, 80.9%, and 74.2% (P < 0.001), respectively. This indicates that all four ADCs had significant inhibitory effects on human lung adenocarcinoma NCI-H1975 tumor cells. See Figure 12 and Table 10 for details.

[0284] Table 10 Tumor parameters of each group after 21 days of SWY2110-SWY2113-ADC administration ***: P < 0.001 compared with the vehicle group

[0285] Experiment 6: Efficacy of SWY2110-ADC, SWY2111-ADC, SWY2112-ADC, and SWY2113-ADC on human breast cancer MDA-MB-468 (human breast cancer cells) transplanted tumors

[0286] 1. Experimental Animals

[0287] NOD-SCID mice, 5-6 weeks old, 28

[0288] 2. Purpose of the test

[0289] The anti-tumor effects of SWY2110-JSSW-001, SWY2111-JSSW-001, SWY2112-JSSW-001, and SWY2113-JSSW-001 were investigated in vivo using a NOD-SCID mouse xenograft tumor model of human breast cancer MDA-MB-468 (human breast cancer cells).

[0290] 3. Drug dosage and grouping

[0291] Table 11 Animal grouping and dosage table Note: iv: intravenous injection, iv / 1 time: one administration;

[0292] 4. Test methods

[0293] In this study, human breast cancer MDA-MB-468 cells (human breast cancer cells) were used to construct a mouse tumor transplant model. 3 At d0, the animals were divided into 5 groups according to tumor volume according to Table 11, with 8 animals in the control group and 5 animals in the experimental group. The drugs were intravenously administered once with a dosage of 10 mL / kg. The vehicle control group (Vehicle) was given 0.9% sodium chloride injection. The experiment was terminated 28 days after administration to investigate the anti-tumor effects of the four ADC drugs in vivo.

[0294] 5. Observation indicators

[0295] 5.1 Evaluation Metrics

[0296] See Experiment 2, 5.1, Evaluation Indicators

[0297] 6 Results

[0298] 6.1 Tumor volume

[0299] At the endpoint, compared to the vehicle group, the tumor inhibition rates of SWY2110-JSSW-001 (DAR = 7.12), SWY2111-JSSW-001 ADC (DAR = 7.48), SWY2112-JSSW-001 ADC (DAR = 7.51), and SWY2113-JSSW-001 ADC (DAR = 7.50) were 65.8%, 65.1%, 58.3%, and 47.1%, respectively (P < 0.001), demonstrating excellent tumor inhibitory activity. This indicates that the four ADCs have significant inhibitory effects on human breast cancer MDA-MB-468 tumor cells. See Figure 13 and Table 12 for details.

[0300] Table 12 Tumor parameters of each group after 28 days of SWY2110-SWY2113-ADC administration ***: P < 0.001 compared with the vehicle group

[0301] Experiment 7: Efficacy of SWY2110-ADC, SWY2111-ADC, SWY2112-ADC, and SWY2113-ADC on human colorectal cancer DiFi (human colorectal cancer cell) transplanted tumors

[0302] 1. Experimental Animals

[0303] NU / NU mice, 5-6 weeks old, 35

[0304] 2. Purpose of the test

[0305] The human colorectal cancer DiFi (human colorectal cancer cells) NU / NU mouse transplant tumor model was used to investigate the in vivo anti-tumor effects of SWY2110-JSSW-001, SWY2111-JSSW-001, SWY2112-JSSW-001, and SWY2113-JSSW-001 drugs.

[0306] 3. Drug dosage and grouping

[0307] Table 13 Animal grouping and dosage table Note: iv: intravenous injection, iv / qw*2 times: administration twice a week;

[0308] 4. Test methods

[0309] In this study, human colorectal cancer DiFi (human colorectal cancer cell) cells were used to construct a mouse tumor transplant model. 3At d0, the animals were divided into five groups according to tumor volume according to Table 13, with seven animals in each group. The drugs were intravenously administered twice at a volume of 10 mL / kg. The vehicle control group (Vehicle) was given 0.9% sodium chloride injection. The experiment was terminated 21 days after administration to investigate the in vivo anti-tumor effects of the four ADC drugs.

[0310] 5. Observation indicators

[0311] 5.1 Evaluation Metrics

[0312] See Experiment 2, 5.1, Evaluation Indicators

[0313] 6 Results

[0314] 6.1 Tumor volume

[0315] At the endpoint, compared to the vehicle group, the tumor inhibition rates of SWY2110-JSSW-001 (DAR = 7.12), SWY2111-JSSW-001 ADC (DAR = 7.48), SWY2112-JSSW-001 ADC (DAR = 7.51), and SWY2113-JSSW-001 ADC (DAR = 7.50) reached 86.5%, 82.3%, 83.5%, and 76.8%, respectively (P < 0.001), demonstrating excellent tumor inhibitory activity. This suggests that the four ADCs have significant inhibitory effects on human colorectal cancer DiFi tumor cells. See Figure 14 and Table 14 for details.

[0316] Table 14 Tumor parameters of each group after 21 days of SWY2110-SWY2113-ADC administration ***: P < 0.001 compared with the vehicle group

[0317] Experiment 8: Efficacy of SWY2110-ADC, SWY2111-ADC, SWY2112-ADC, and SWY2113-ADC on human lung adenocarcinoma PC9-GR (gefitinib-acquired resistance strain) cell transplants

[0318] 1. Experimental Animals

[0319] NU / NU mice, 5-6 weeks old, 30

[0320] 2. Purpose of the test

[0321] The human lung adenocarcinoma cell line PC9-GR NU / NU mouse transplant tumor model was used to investigate the in vivo anti-tumor effects of SWY2110-JSSW-001, SWY2111-JSSW-001, SWY2112-JSSW-001, and SWY2113-JSSW-001.

[0322] 3. Drug dosage and grouping

[0323] Table 15 Animal grouping and dosage table Note: iv: intravenous injection, iv / 1 time: one administration;

[0324] 4. Test methods

[0325] In this study, human lung adenocarcinoma cell line PC9-GR was used to construct a mouse tumor transplant model. 3 At d0, the animals were divided into 5 groups according to tumor volume according to Table 15, with 6 animals in each group. The drugs were intravenously administered once at a volume of 10 mL / kg. The vehicle control group (Vehicle) was given 0.9% sodium chloride injection. The experiment was terminated 28 days after administration to investigate the anti-tumor effects of the four ADC drugs in vivo.

[0326] 5. Observation indicators

[0327] 5.1 Evaluation Metrics

[0328] See Experiment 2, 5.1, Evaluation Indicators

[0329] 6 Results

[0330] 6.1 Tumor volume

[0331] At the endpoint of the trial, compared with the vehicle group, the tumor inhibition rates of SWY2110-JSSW-001 (DAR = 7.12), SWY2111-JSSW-001 ADC (DAR = 7.48), SWY2112-JSSW-001 ADC (DAR = 7.51), and SWY2113-JSSW-001 ADC (DAR = 7.50) were 99.0%, 100.1%, 92.4%, and 89.3%, respectively (P < 0.001), all demonstrating a positive tumor inhibition effect. This indicates that the four ADCs have a significant inhibitory effect on human lung adenocarcinoma cell lines PC9-GR. See Figure 15 and Table 16 for details.

[0332] Table 16 Tumor parameters of each group after 28 days of SWY2110-SWY2113-ADC administration ***: P < 0.001 compared with the vehicle group

[0333] Test 9: Safety Assessment Study

[0334] This study enrolled six age-appropriate cynomolgus macaques, half male and half female, and administered SWY2110-JSSW-001, SWY2111-JSSW-001, and SWY2113-JSSW-001 intravenously. The dosage regimen is shown in the table below: Intravenous injections were administered once every three weeks for three consecutive doses. Patients were observed for seven consecutive days after administration, with general observations twice daily and detailed observations once daily. Body weight changes were observed before and after administration, and hematological tests were performed. The experimental results showed that ADCs prepared with pH-dependent antibodies (SWY2111-JSSW-001 and SWY2113-JSSW-001) exhibited reduced skin and gastrointestinal toxicity compared to SWY2110-JSSW-001, with no significant target-related toxicity (skin rupture, scabs, loose stools, etc.). This suggests that the pH-modified ADCs reduced toxicity to normal tissues, but there was no significant difference in the hematological toxicity caused by the three ADC small molecules.

[0335] Table 17 Pre-toxicity experiment design

[0336] Table 18 Pre-toxicity test results HGB: hemoglobin; WBC: white blood cell count; LYMP: absolute lymphocyte count.

[0337] Clinical Examples

[0338] Example 1

[0339] 1. Study Design

[0340] This study is an open-label, multicenter Phase I clinical study conducted in patients with advanced solid tumors. It is divided into two phases: Phase I (Stage I) includes a dose-escalation study and a PK expansion study, and Phase II (Stage II) is a cohort expansion study. The study aims to evaluate the safety, tolerability, pharmacokinetic characteristics, and preliminary efficacy of the antibody-drug conjugate of this invention in patients with advanced solid tumors.

[0341] 1.1 Stage Ⅰ

[0342] 1.1.1 Dose escalation study

[0343] A dose escalation study was conducted in patients with advanced solid tumors using an accelerated titration and a traditional 3+3 design. The starting dose of the antibody drug conjugate of the present invention was 0.6 mg / kg, and the highest escalating dose was set to 8 mg / kg. The dose-limiting toxicity (DLT) and maximum tolerated dose (MTD) were determined through the dose escalation study. The specific dose group settings for the dose escalation phase were based on a modified Fibonacci method, as shown in the table below:

[0344] Table 19 Dose escalation group settings

[0345] 1.1.2 PK extension study

[0346] Based on the safety, tolerability, and PK data from the different dose groups in the dose-escalation study, 2-3 dose groups will be selected for the PK expansion study. Each dose group will be expanded to 8-12 subjects (including subjects in the same dose group as in the dose-escalation study).

[0347] 1.2 Stage ⅠI

[0348] The recommended dose initially determined in the first phase was used for the cohort expansion study, and the patients were divided into 6 cohorts according to tumor types, namely:

[0349] Cohort A: colorectal cancer with moderate to high EGFR expression;

[0350] Cohort B: triple-negative breast cancer with moderate to high EGFR expression;

[0351] Cohort C: head and neck squamous cell carcinoma (including nasopharyngeal carcinoma) with moderate to high EGFR expression;

[0352] Cohort D: EGFR-mutant non-squamous non-small cell lung cancer (Nsq-NSCLC);

[0353] Cohort E: EGFR-high expression squamous cell lung cancer (Sq-NSCLC);

[0354] Cohort F: Other advanced solid tumors with high expression of EGFR, such as gastric cancer, esophageal cancer, pancreatic cancer, cervical cancer, biliary tract cancer, non-squamous non-small cell lung cancer, and RAS / BRAF mutant colorectal cancer.

[0355] All subjects underwent safety assessments after their first dose of the trial drug, and PK and immunogenicity blood samples were collected after a single dose and during continuous dosing. In addition, tumor imaging was assessed according to the Response Evaluation Criteria in Solid Tumors (RECIST v1.1).

[0356] 2. Subject Selection

[0357] Subjects must meet all of the following inclusion criteria to be included in this study: age 18 years or older, regardless of gender; patients with pathologically confirmed advanced solid tumors who have failed or are intolerant to standard treatment, have no standard treatment, or refuse standard treatment; have EGFR activating mutations, moderate to high expression, or amplification; and have at least one measurable lesion confirmed by CT or MRI according to the Response Evaluation Criteria in Solid Tumors (RECIST) v 1.1.

[0358] 3. Research interventions and combined treatments

[0359] 3.1 Test drug (Dar value is determined by hydrophobic chromatography)

[0360] Drug dosage form: lyophilized powder for injection;

[0361] Specification: 100mg / bottle;

[0362] Storage conditions: 2-8°C, protect from light.

[0363] 3.2 Implementation of the study intervention

[0364] The initial dose of the trial drug was calculated based on the subject's screening / baseline weight. If the subject's weight change during the study treatment was <10% compared to the screening / baseline weight, the dose did not need to be recalculated. The drug was administered by intravenous infusion every three weeks, with a 21-day treatment cycle. The infusion duration was 60 min ± 10 min. No infusion-related reactions occurred after the initial dose, and subsequent infusions were shortened to 30 min ± 5 min. Dosing continued until the protocol-specified discontinuation criteria were met.

[0365] If delayed administration occurs, the administration time of the subsequent cycle will be calculated based on the actual date of administration of the previous cycle.

[0366] 4 Evaluation indicators

[0367] 4.1 Pharmacokinetics

[0368] PK blood samples were collected from each subject according to the sampling time points and time windows of different study stages as planned in Table 20 for PK testing of the test drug. The test components included toxin-bound antibodies, total antibodies, and free toxins.

[0369] For dose escalation and PK expansion studies, non-compartmental models were used to calculate the pharmacokinetic parameters of each test component for each subject, including: Cmax, Tmax, t1 / 2, MRT, Vd, CL, AUC 0-t , AUC 0-∞ Descriptive statistics were summarized and tabulated for pharmacokinetic parameters, including Cmax,ss, Cmin,ss, Tmax,ss, AUCss, and Rac. Box plots were drawn for dose-normalized key PK parameters (e.g., AUC and Cmax), and analysis of variance and / or power models were used to explore the relationship between key PK parameters and dose.

[0370] 4.2 Immunogenicity Evaluation

[0371] Collect immunogenicity blood samples from each subject at the sampling time points and time windows planned for different study phases as shown in Table 20 for immunogenicity testing of the investigational drug. Immunogenicity samples are used for in-study cutpoint definition, anti-drug antibody (ADA) analysis, and target interference studies in clinical studies.

[0372] Tabulate all anti-drug antibody (ADA) results. Analyze the proportion of subjects with ADA-positive results, the time of first ADA-positive results, and the duration of ADA-positive results. If necessary, further neutralizing antibody testing will be performed on samples with confirmed ADA-positive results, and the results will be tabulated. Analyze the impact of immunogenicity on drug PK, safety, and efficacy.

[0373] Table 20 Pharmacokinetic and immunogenicity sample collection 1: If the interval between the end of treatment and the 30-day visit after the last dose is within 14 days, only one PK and immunogenicity sample will be collected.

[0374] 4.3 Biomarker Analysis

[0375] The EGFR status of the subjects' tumor tissue or peripheral blood samples was tested.

[0376] For EGFR mutant Nsq-NSCLC, tumor tissue samples are provided for IHC testing, and the correlation between Nsq-NSCLC EGFR expression levels and the efficacy of the test drug is analyzed. During the NSCLC screening period, tumor tissue or peripheral blood samples are provided for NGS testing to analyze the factors affecting the efficacy of the test drug.

[0377] 4.4 Safety evaluation

[0378] The severity of adverse events was recorded using the National Cancer Institute's Common Terminology Criteria for Adverse Events (NCI-CTCAE, version 5.0). Adverse events were collected and evaluated to examine the causal relationship between the adverse event and the drug.

[0379] Observe and record any AEs of all subjects, including abnormal clinical symptoms and vital signs, abnormalities in laboratory tests, and determine their relevance to the trial drug.

[0380] For subjects in the dose-escalation study, DLT events were observed during the DLT observation period; delayed DLTs were observed within 84 days after the first dose.

[0381] Subjects are required to undergo safety evaluation during the screening period / baseline and after administration, including physical examination, ECOG score, laboratory tests and electrocardiogram examination.

[0382] Skin toxicity, interstitial pneumonia / non-infectious pneumonia toxicity, blood toxicity, gastrointestinal toxicity, eye toxicity, liver toxicity, infusion-related reactions / allergic reactions and other toxicities were studied and evaluated.

[0383] Observe whether the subjects have adverse skin events, grade them according to CTCAE V5.0, and deal with any adverse events that occur.

[0384] Subjects will be observed for imaging changes consistent with interstitial lung disease / non-infectious pneumonia, or acute onset, or new or worsening lung or other related signs / symptoms, such as dyspnea, cough, or fever, and their development of interstitial lung disease / non-infectious pneumonia will be assessed. During the screening period, pulmonary function tests will be performed, including core parameters related to ventilation and gas exchange function.

[0385] The subjects' blood routine tests were monitored regularly.

[0386] Observe whether the subjects have symptoms such as diarrhea, loose stools, vomiting, and nausea.

[0387] Subjects were observed for ocular toxicity, such as acute or worsening ocular inflammation, tearing, light sensitivity, blurred vision, eye pain, and / or redness, and adverse events were classified according to CTCAE V5.0. During the screening period, subjects underwent an ophthalmological examination, including visual acuity, slit lamp examination, and fundus examination.

[0388] Observe whether the subjects have abnormal liver function.

[0389] Observe whether the subjects have infusion reactions / allergic reactions, such as fever, itching, rash, angioedema, chills, hypotension, dyspnea, chest discomfort, wheezing or tachycardia, allergic reactions, etc., and classify adverse events according to CTCAE V5.0.

[0390] 4.5 Efficacy evaluation

[0391] Efficacy evaluation was performed using RECIST 1.1 criteria, with CT / MRI imaging assessments performed every 6 weeks for the first 24 weeks after the first dose, and every 9 weeks thereafter. The imaging window allowed was ±7 days. Efficacy evaluations were performed at the prescribed time points and were not delayed by any delay in dosing.

[0392] Efficacy endpoints include:

[0393] Objective response rate (ORR) is defined as the proportion of subjects whose best overall response in the study was complete remission (CR) or partial remission (PR) (i.e., CR+PR) as assessed by the investigator according to RECIST v1.1 from the start of the trial drug to the end of the study.

[0394] Disease control rate (DCR): defined as the proportion of patients with CR, PR, or stable disease (SD) (i.e., CR+PR+SD) at the best time point, as evaluated according to RECIST v1.1 criteria, from the start of trial drug use to study exit.

[0395] Progression-free survival (PFS): defined as the time from the start of trial drug to the date of first documented disease progression (PD) or death from any cause, whichever occurs first.

[0396] Duration of response (DoR) was defined as the time from the first tumor assessment as CR or PR to the first assessment as PD or death from any cause, whichever occurred first.

[0397] Overall survival (OS) is defined as the time interval from the start of trial drug to the date of death due to any cause.

[0398] In the first phase, descriptive summaries of efficacy were performed by dose group. In the second phase, best overall response (BOR), ORR, and DCR were summarized and analyzed. PFS, DoR, and OS were also analyzed. The analysis methods are summarized as follows:

[0399] According to RECIST1.1 criteria, BOR was summarized by cohort, and the number and percentage of ORR and DCR were calculated, and the 95% CI was calculated using the Clopper-Pearson exact method.

[0400] The Kaplan-Meier method was used to analyze PFS, DoR, and OS, including estimating the median time and its 95% CI, and drawing the Kaplan-Meier curve.

[0401] 5. Research Results

[0402] A total of 25 subjects were enrolled, with safety data available for 23 subjects. A total of 16 subjects completed at least one efficacy assessment, with 4 subjects experiencing a PR, 11 experiencing SD, and 1 experiencing a PD.

[0403] In the 0.6 mg / kg dose group, one case developed SD (EGFR mutation, non-squamous NSCLC)

[0404] In the 1.8 mg / kg dose group, 3 cases had SD (2 cases with EGFR-mutated non-squamous NSCLC and 1 case with colorectal cancer with moderate to high EGFR expression).

[0405] In the 3.6 mg / kg dose group, there were 2 cases of SD (1 case of EGFR medium-to-high expression lung squamous cell carcinoma, 1 case of EGFR medium-to-high expression non-squamous NSCLC), and 1 case of PD (EGFR medium-to-high expression colorectal cancer, RAS / BRAF wild type);

[0406] In the 4.8 mg / kg dose group, 3 cases of PR (1 case of EGFR mutation / amplification, non-squamous NSCLC; 1 case of EGFR medium-high expression / mutation lung squamous cell carcinoma; 1 case of EGFR medium-high expression nasopharyngeal carcinoma) and 3 cases of SD (2 cases of EGFR mutation non-squamous NSCLC and 1 case of EGFR mutation / amplification non-squamous NSCLC) were observed.

[0407] In the 6.4 mg / kg dose group, there was 1 PR (EGFR mutation / amplification non-squamous NSCLC) and 2 SD (1 EGFR medium-to-high expression lung squamous cell carcinoma and 1 EGFR medium-to-high expression non-squamous NSCLC). In the 13 cases of non-small cell lung cancer, 3 had PR; 10 had SD; 1 had nasopharyngeal carcinoma, which was PR; 2 had colorectal cancer, 1 had SD, and 1 had PD.

[0408] Typical Cases

[0409] Case 1: A 69-year-old male patient was initially diagnosed with non-small cell lung cancer. He underwent surgery (left upper lobectomy with mediastinal lymph node dissection) and received four cycles of vinorelbine plus cisplatin, but his disease progressed. Subsequently, he began oral osimertinib, followed by paclitaxel, carboplatin, and osimertinib, but his disease progressed. Radiotherapy was then performed for bone metastases. He was subsequently treated with afatinib, but his disease progressed. He was then started on paclitaxel and cisplatin, but his disease progressed. He was then treated with paclitaxel, cisplatin, and tislelizumab, but his disease progressed.

[0410] After signing an informed consent form, the patient's clinical stage during the screening period was IV, with moderate to high EGFR expression and a T790M mutation. The baseline target lesion was 168 mm, with the largest lung lesion measuring 84 mm and containing a new cavity. The trial drug was administered for the first time, and the efficacy evaluation result was a PR.

[0411] Case 2: A 56-year-old male patient was diagnosed with non-small cell lung cancer. He was treated with JNJ-61186372 combined with lazertinib. After disease progression, he participated in another clinical trial and received pemetrexed combined with carboplatin. He discontinued chemotherapy due to myelosuppression.

[0412] After signing an informed consent form, the patient's clinical stage during the screening period was IV, with EGFR mutation and amplification. The first dose of the trial drug resulted in a PR in the first efficacy evaluation, which was confirmed at the second efficacy evaluation.

[0413] Case 3: A 48-year-old female patient was diagnosed with non-small cell lung cancer. She participated in other drug trials and her disease progressed.

[0414] After signing an informed consent form, the patient's clinical stage during the screening period was IV, with EGFR mutation and amplification. The trial drug was administered for the first time, and the first efficacy evaluation result was PR.

[0415] Case 4: A 55-year-old male patient was initially diagnosed with nasopharyngeal carcinoma. He received gemcitabine and cisplatin with concurrent chemoradiotherapy, followed by capecitabine maintenance therapy, but his disease progressed. He then received camrelizumab, but his disease progressed. He then received docetaxel and nedaplatin, but his disease progressed.

[0416] After signing the informed consent form, the patient's clinical stage during the screening period was IV, with moderate to high EGFR expression. The trial drug was administered for the first time, and the first efficacy evaluation result was PR.

[0417] Clinical studies have shown that the antibody-drug conjugate represented by Formula I, and its pharmaceutically acceptable salts, hydrates, solvates, stereoisomers or isotope-labeled products can effectively treat advanced malignant tumors, especially EGFR-positive advanced solid tumors, overcome resistance to EGFR-TKIs and EGFR monoclonal antibodies, and can bring clinical benefits superior to existing treatments to patients with advanced solid tumors, including non-small cell lung cancer, and can control or alleviate the disease.

Claims

1. The antibody-drug conjugate of formula I, and its pharmaceutically acceptable salts, hydrates, solvates, stereoisomers or isotope-labeled substances, for use in treating tumors: wherein Ab is selected from SWY2110, SWY2111, SWY2112, and SWY2113; SWY2110 is an antibody having two heavy chains with the amino acid sequence shown in SEQ ID NO:29 and two light chains with the amino acid sequence shown in SEQ ID NO:30; SWY2111 is an antibody having two heavy chains with the amino acid sequence shown in SEQ ID NO:34 and two light chains with the amino acid sequence shown in SEQ ID NO:30; SWY2112 is an antibody having two heavy chains with the amino acid sequence shown in SEQ ID NO:37 and two light chains with the amino acid sequence shown in SEQ ID NO:30; SWY2113 is an antibody having two heavy chains with the amino acid sequence shown in SEQ ID NO:40 and two light chains with the amino acid sequence shown in SEQ ID NO:30; n is an integer selected from 1-8 or a decimal selected from 1-8; preferably, n is selected from a number between 7 and 8, including the two endpoints of 7 and 8; The tumor is selected from triple-negative breast cancer, gastric cancer, esophageal cancer, pancreatic cancer, cervical cancer, biliary cancer, HR-positive breast cancer, liver cancer, ovarian cancer, endometrial cancer, prostate cancer, lung squamous cell carcinoma, non-squamous non-small cell lung cancer, head and neck squamous cell carcinoma (including nasopharyngeal carcinoma), colorectal cancer, urothelial carcinoma, renal cancer, etc. Preferably, the tumor is selected from colorectal cancer with medium to high expression of EGFR; triple-negative breast cancer with medium to high expression of EGFR; head and neck squamous cell carcinoma (including nasopharyngeal carcinoma) with medium to high expression of EGFR; EGFR mutant non-squamous non-small cell lung cancer (Nsq-NSCLC); EGFR medium to high expression of lung squamous cell carcinoma (Sq-NSCLC); other advanced solid tumors with medium to high expression of EGFR: such as gastric cancer, esophageal cancer, pancreatic cancer, cervical cancer, biliary cancer, non-squamous non-small cell lung cancer, and RAS / BRAF mutant colorectal cancer; resistant lung adenocarcinoma (such as lung adenocarcinoma resistant to Gefitinib and / or Osimertinib).

2. The antibody-drug conjugate of formula I for treating tumors according to claim 1, and its pharmaceutically acceptable salts, hydrates, solvates, stereoisomers or isotope-labeled substances, characterized in that: The dosage of each administration of the antibody-drug conjugate represented by Formula I is: 1 mg / time-1000 mg / time, preferably 5 mg-500 mg / time, or 10-300 mg / time, or 20-200 mg / time, or 50-150 mg / time, or 50-300 mg / time, or 50-400 mg / time, or 100-200 mg / time, or 100-300 mg / time, or 100-500 mg / time, more preferably 5 mg / time, 6 mg / time, 8mg / time, 10mg / time, 15mg / time, 20mg / time, 25mg / time, 30mg / time, 40mg / time, 50mg / time, 60mg / time, 70mg / time, 80mg / time, 90mg / time, 100mg / time, 150mg / time, 200mg / time, 250mg / time, 300mg / time, 500mg / time, 600mg / time, 700mg / time, 800mg / time, 900mg / time, etc.

3. The antibody-drug conjugate of formula I for treating tumors according to claim 1, and its pharmaceutically acceptable salts, hydrates, solvates, stereoisomers or isotope-labeled substances, characterized in that: The dosage of each administration based on the antibody-drug conjugate shown in Formula I and the patient's body weight is: 0.05 mg / kg-50 mg / kg, preferably 0.1 mg / kg-20 mg / kg, or 0.2 mg / kg-20 mg / kg, or 0.5 mg / kg-20 mg / kg, or 0.5 mg / kg-15 mg / kg, or 0.5 mg / kg-10 mg / kg, or 0.5 mg / kg-8 mg / kg, or 0.5 mg / kg-6 mg / kg, or 0.5 mg / kg-4 mg / kg, or 0.5 mg / kg-2 mg / kg, or 1 mg / kg g / kg-20mg / kg, or 1mg / kg-10mg / kg, or 1mg / kg-8mg / kg, or 2mg / kg-20mg / kg, or 2mg / kg-10mg / kg, or 2mg / kg-8mg / kg, or 3mg / kg-20mg / kg, or 3mg / kg-10mg / kg, or 3mg / kg-8mg / kg, or 5mg / kg-20mg / kg, or 5mg / kg-10mg / kg, or 0.3mg / kg, or 0.4mg / kg, or 0.6mg / kg, or 0.8mg / kg, or 1.5mg / kg, or 1.8mg / kg, or 2.0mg / kg, or 2.2mg / kg, or 2.4mg / kg, or 2.5mg / kg, or 2.8mg / kg, or 3mg / kg, or 3.2mg / kg, or 3.5mg / kg, or 3.6mg / kg, or 3.8mg / kg, or 4.0mg / kg, or 4.2mg / kg, or 4.4mg / kg, or 4.5mg / kg, or 4.6mg / kg, or 4.8mg / kg, or 5.0mg / kg, or 5.2mg / kg, or 5.4mg / kg, or 5.5mg / kg, or 5.6mg / kg, or 5.8mg / kg, or 6.0mg / kg, or 6.2mg / kg, or 6.4mg / kg, or 6.8mg / kg, or 7.0mg / kg, or 7.2mg / kg, or 7.5mg / kg, or 7.8mg / kg, or 8.0mg / kg, or 8.5mg / kg, or 9.0mg / kg, or 9.5mg / kg, or 11mg / kg, or 11.5mg / kg, or 12.0mg / kg, or 16.8mg / kg, or 18.0mg / kg.

4. The antibody-drug conjugate of formula I for treating tumors according to claim 1, and its pharmaceutically acceptable salts, hydrates, solvates, stereoisomers or isotope-labeled substances, characterized in that: The antibody-drug conjugate shown in Formula I, and its pharmaceutically acceptable salts, hydrates, solvates, stereoisomers or isotope labels can be administered once a day (QD), or divided into multiple administrations within a day, for example, divided into twice a day (BID) or three times a day (TID); can be administered once a week (QW), or the above-mentioned therapeutically effective amount can be administered as a total weekly dose on multiple days within a week, for example, divided into twice a week (BIW) or three times a week (TIW); can be administered once every two weeks (Q2W), or divided into multiple days within two weeks, for example, divided into twice two weeks (BI2W) or three times two weeks (TI2W); can be administered once three weeks (Q3W), or divided into multiple days within three weeks, for example, divided into twice three weeks (BI3W, such as D1, D8 every three weeks) or three times three weeks (TI3W); can be administered once a month (QM), or can be administered within a month Multiple-day divided administration, for example, divided into twice a month (BI2M) or three times a month (TIM); can be intermittent administration, such as once at an interval of 2-7 days, or once at an interval of 3 days, or 4 days, or 5 days, or 6 days, or once at an interval of 2-4 weeks, such as once at an interval of 2 weeks (once every 3 weeks, a cycle of 3 weeks), once at an interval of 1 week (once every 2 weeks, a cycle of 2 weeks or every 4 weeks) and once at an interval of 3 weeks (once every 4 weeks, a cycle of 4 weeks), or once at an interval of 1-3 months, such as once at an interval of 1 month (every 2 months); or continuous administration for 3 weeks, rest for 1 week, a cycle of 4 weeks, or continuous administration for 2 weeks, rest for 1 week, a cycle of 3 weeks, or continuous administration for 2 weeks, rest for 2 weeks, a cycle of 4 weeks, or interval administration for 1 week (once every 2 weeks), a cycle of 4 weeks; every 3 weeks is 1 cycle, and 1 administration is given on the first day of each cycle.

5. The antibody-drug conjugate of formula I for treating tumors according to claim 4, and its pharmaceutically acceptable salts, hydrates, solvates, stereoisomers or isotope-labeled substances, characterized in that: The antibody-drug conjugate of formula I, and its pharmaceutically acceptable salts, hydrates, solvates, stereoisomers or isotope-labeled substances are administered in the dosage of claims 2-3 in each administration cycle.

6. The antibody-drug conjugate of formula I, and its pharmaceutically acceptable salts, hydrates, solvates, stereoisomers or isotope-labeled substances: wherein Ab is selected from SWY2110, SWY2111, SWY2112, and SWY2113; SWY2110 is an antibody having two heavy chains with the amino acid sequence shown in SEQ ID NO:29 and two light chains with the amino acid sequence shown in SEQ ID NO:30; SWY2111 is an antibody having two heavy chains with the amino acid sequence shown in SEQ ID NO:34 and two light chains with the amino acid sequence shown in SEQ ID NO:30; SWY2112 is an antibody having two heavy chains with the amino acid sequence shown in SEQ ID NO:37 and two light chains with the amino acid sequence shown in SEQ ID NO:30; SWY2113 is an antibody having two heavy chains with the amino acid sequence shown in SEQ ID NO:40 and two light chains with the amino acid sequence shown in SEQ ID NO:30; n is an integer selected from 1-8 or a decimal selected from 1-8; preferably, n is selected from a number between 7 and 8, including the two endpoints of 7 and 8; Use in the preparation of a drug for treating tumors, wherein the tumor is selected from triple-negative breast cancer, gastric cancer, esophageal cancer, pancreatic cancer, cervical cancer, biliary cancer, HR-positive breast cancer, liver cancer, ovarian cancer, endometrial cancer, prostate cancer, lung squamous cell carcinoma, non-squamous non-small cell lung cancer, head and neck squamous cell carcinoma (including nasopharyngeal carcinoma), colorectal cancer, urothelial carcinoma, renal cancer, etc. Preferably, the tumor is selected from colorectal cancer with medium to high expression of EGFR; triple-negative breast cancer with medium to high expression of EGFR; head and neck squamous cell carcinoma (including nasopharyngeal carcinoma) with medium to high expression of EGFR; EGFR mutant non-squamous non-small cell lung cancer (Nsq-NSCLC); EGFR medium to high expression of lung squamous cell carcinoma (Sq-NSCLC); other advanced solid tumors with medium to high expression of EGFR: such as gastric cancer, esophageal cancer, pancreatic cancer, cervical cancer, biliary cancer, non-squamous non-small cell lung cancer, and RAS / BRAF mutant colorectal cancer; resistant lung adenocarcinoma (such as lung adenocarcinoma resistant to Gefitinib and / or Osimertinib).

7. A pharmaceutical preparation comprising an antibody-drug conjugate of formula I, and a pharmaceutically acceptable salt, hydrate, solvate, stereoisomer or isotope-labeled product thereof: wherein Ab is selected from SWY2110, SWY2111, SWY2112, and SWY2113; SWY2110 is an antibody having two heavy chains with the amino acid sequence shown in SEQ ID NO:29 and two light chains with the amino acid sequence shown in SEQ ID NO:30; SWY2111 is an antibody having two heavy chains with the amino acid sequence shown in SEQ ID NO:34 and two light chains with the amino acid sequence shown in SEQ ID NO:30; SWY2112 is an antibody having two heavy chains with the amino acid sequence shown in SEQ ID NO:37 and two light chains with the amino acid sequence shown in SEQ ID NO:30; SWY2113 is an antibody having two heavy chains with the amino acid sequence shown in SEQ ID NO:40 and two light chains with the amino acid sequence shown in SEQ ID NO:30; n is an integer selected from 1-8 or a decimal selected from 1-8; preferably, n is selected from a number between 7 and 8, including the two endpoints of 7 and 8; The pharmaceutical preparation is an oral preparation (such as tablets, capsules), or an injectable preparation (such as injection solution, lyophilized powder for injection).

8. The pharmaceutical preparation according to claim 7, characterized in that Each dosage unit of the pharmaceutical preparation contains the antibody-drug conjugate of Formula I, and its pharmaceutically acceptable salts, hydrates, solvates, stereoisomers or isotope labels in an amount of 1 mg-1000 mg, preferably 5 mg-500 mg, or 10-300 mg, or 20-200 mg, or 50-150 mg, or 50-300 mg, or 50-400 mg, or 100-200 mg, or 100-300 mg, or 100-500 mg, more preferably 1 mg, 5 mg, 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 500 mg, 1000 mg, etc.

9. A method for treating tumors, characterized in that The antibody-drug conjugate of Formula I, and its pharmaceutically acceptable salts, hydrates, solvates, stereoisomers or isotope-labeled substances: wherein Ab is selected from SWY2110, SWY2111, SWY2112, and SWY2113; SWY2110 is an antibody having two heavy chains with the amino acid sequence shown in SEQ ID NO:29 and two light chains with the amino acid sequence shown in SEQ ID NO:30; SWY2111 is an antibody having two heavy chains with the amino acid sequence shown in SEQ ID NO:34 and two light chains with the amino acid sequence shown in SEQ ID NO:30; SWY2112 is an antibody having two heavy chains with the amino acid sequence shown in SEQ ID NO:37 and two light chains with the amino acid sequence shown in SEQ ID NO:30; SWY2113 is an antibody having two heavy chains with the amino acid sequence shown in SEQ ID NO:40 and two light chains with the amino acid sequence shown in SEQ ID NO:30; n is an integer selected from 1-8 or a decimal selected from 1-8; preferably, n is selected from a number between 7 and 8, including the two endpoints of 7 and 8; The invention is administered to a subject or patient in need of treatment in a therapeutically effective amount to treat a tumor, wherein the therapeutically effective amount refers to a dose that can alleviate, inhibit tumor growth or eliminate the tumor, benefit the subject or patient, but does not cause intolerable toxic side effects, and the tumor is selected from triple-negative breast cancer, gastric cancer, esophageal cancer, pancreatic cancer, cervical cancer, biliary tract cancer, HR-positive breast cancer, liver cancer, ovarian cancer, endometrial cancer, prostate cancer, squamous cell lung cancer, non-squamous non-small cell lung cancer, head and neck squamous cell carcinoma (including nasopharyngeal carcinoma), colorectal cancer, urothelial carcinoma, renal cancer, etc. Preferably, the tumor is selected from EGFR Highly expressed colorectal cancer; EGFR-highly expressed triple-negative breast cancer; EGFR-highly expressed head and neck squamous cell carcinoma (including nasopharyngeal carcinoma); EGFR-mutant non-squamous non-small cell lung cancer (Nsq-NSCLC); EGFR-highly expressed lung squamous cell carcinoma (Sq-NSCLC); other advanced solid tumors with high expression of EGFR: such as gastric cancer, esophageal cancer, pancreatic cancer, cervical cancer, biliary tract cancer, non-squamous non-small cell lung cancer, and RAS / BRAF mutant colorectal cancer; resistant lung adenocarcinoma (such as lung adenocarcinoma resistant to gefitinib and / or osimertinib).

10. The method according to claim 9, characterized in that The dosage of each administration of the antibody-drug conjugate represented by Formula I is: 1 mg / time-1000 mg / time, preferably 5 mg-500 mg / time, or 10-300 mg / time, or 20-200 mg / time, or 50-150 mg / time, or 50-300 mg / time, or 50-400 mg / time, or 100-200 mg / time, or 100-300 mg / time, or 100-500 mg / time, more preferably 5 mg / time, 6 mg / time, 8mg / time, 10mg / time, 15mg / time, 20mg / time, 25mg / time, 30mg / time, 40mg / time, 50mg / time, 60mg / time, 70mg / time, 80mg / time, 90mg / time, 100mg / time, 150mg / time, 200mg / time, 250mg / time, 300mg / time, 500mg / time, 600mg / time, 700mg / time, 800mg / time, 900mg / time, etc.

11. The method according to claim 9, characterized in that The dosage of each administration based on the antibody-drug conjugate shown in Formula I and the patient's body weight is: 0.05 mg / kg-50 mg / kg, preferably 0.1 mg / kg-20 mg / kg, or 0.2 mg / kg-20 mg / kg, or 0.5 mg / kg-20 mg / kg, or 0.5 mg / kg-15 mg / kg, or 0.5 mg / kg-10 mg / kg, or 0.5 mg / kg-8 mg / kg, or 0.5 mg / kg-6 mg / kg, or 0.5 mg / kg-4 mg / kg, or 0.5 mg / kg-2 mg / kg, or 1 mg / kg g / kg-20mg / kg, or 1mg / kg-10mg / kg, or 1mg / kg-8mg / kg, or 2mg / kg-20mg / kg, or 2mg / kg-10mg / kg, or 2mg / kg-8mg / kg, or 3mg / kg-20mg / kg, or 3mg / kg-10mg / kg, or 3mg / kg-8mg / kg, or 5mg / kg-20mg / kg, or 5mg / kg-10mg / kg, or 0.3mg / kg, or 0.4mg / kg, or 0.6mg / kg, or 0.8mg / kg, or 1.5mg / kg, or 1.8mg / kg, or 2.0mg / kg, or 2.2mg / kg, or 2.4mg / kg, or 2.5mg / kg, or 2.8mg / kg, or 3mg / kg, or 3.2mg / kg, or 3.5mg / kg, or 3.6mg / kg, or 3.8mg / kg, or 4.0mg / kg, or 4.2mg / kg, or 4.4mg / kg, or 4.5mg / kg, or 4.6mg / kg, or 4.8mg / kg, or 5.0mg / kg, or 5.2mg / kg, or 5.4mg / kg, or 5.5mg / kg, or 5.6mg / kg, or 5.8mg / kg, or 6.0mg / kg, or 6.2mg / kg, or 6.4mg / kg, or 6.8mg / kg, or 7.0mg / kg, or 7.2mg / kg, or 7.5mg / kg, or 7.8mg / kg, or 8.0mg / kg, or 8.5mg / kg, or 9.0mg / kg, or 9.5mg / kg, or 11mg / kg, or 11.5mg / kg, or 12.0mg / kg, or 16.8mg / kg, or 18.0mg / kg.

12. The method of claim 9, characterized in that The antibody-drug conjugate shown in Formula I, and its pharmaceutically acceptable salts, hydrates, solvates, stereoisomers or isotope labels can be administered once a day (QD), or divided into multiple administrations within a day, for example, divided into twice a day (BID) or three times a day (TID); can be administered once a week (QW), or the above-mentioned therapeutically effective amount can be administered as a total weekly dose on multiple days within a week, for example, divided into twice a week (BIW) or three times a week (TIW); can be administered once every two weeks (Q2W), or divided into multiple days within two weeks, for example, divided into twice two weeks (BI2W) or three times two weeks (TI2W); can be administered once three weeks (Q3W), or divided into multiple days within three weeks, for example, divided into twice three weeks (BI3W, such as D1, D8 every three weeks) or three times three weeks (TI3W); can be administered once a month (QM), or can be administered within a month Multiple-day divided administration, for example, divided into twice a month (BI2M) or three times a month (TIM); can be intermittent administration, such as once at an interval of 2-7 days, or once at an interval of 3 days, or 4 days, or 5 days, or 6 days, or once at an interval of 2-4 weeks, such as once at an interval of 2 weeks (once every 3 weeks, a cycle of 3 weeks), once at an interval of 1 week (once every 2 weeks, a cycle of 2 weeks or every 4 weeks) and once at an interval of 3 weeks (once every 4 weeks, a cycle of 4 weeks), or once at an interval of 1-3 months, such as once at an interval of 1 month (every 2 months); or continuous administration for 3 weeks, rest for 1 week, a cycle of 4 weeks, or continuous administration for 2 weeks, rest for 1 week, a cycle of 3 weeks, or continuous administration for 2 weeks, rest for 2 weeks, a cycle of 4 weeks, or interval administration for 1 week (once every 2 weeks), a cycle of 4 weeks; every 3 weeks is 1 cycle, and 1 administration is given on the first day of each cycle.

13. The method of claim 12, characterized in that The antibody-drug conjugate of formula I, and its pharmaceutically acceptable salts, hydrates, solvates, stereoisomers or isotope-labeled substances are administered in the dosage of claims 10-11 in each administration cycle.

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