Applications of antibody-drug conjugates

Abstract

The present invention provides antibody-drug conjugates, as well as pharmaceutically acceptable salts, hydrates, solvates, stereoisomers or isotope-labeled forms thereof, pharmaceutical compositions comprising the same, and uses thereof for the treatment of tumors and methods for treating tumors.

Description

[Technical Field]

[0001] This application belongs to the biopharmaceutical field and specifically relates to the applications of antibody-drug conjugates. [Background technology]

[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. Ligands confirmed to bind to EGFR include epidermal growth factor (EGF), transforming growth factor α (TGFα), amphiregulin, heparin-binding EGF, and epiregulin. In human tissue, EGF and TGFα are considered the two most important ligands for EGFR.

[0003] EGFR is a constitutively expressed component in many normal epithelial tissues, such as skin and hair follicles. Furthermore, EGFR is highly expressed in a wide variety of human malignant tumor tissues, including 40-80% in lung cancer, 14-91% in breast cancer, 33-74% in gastric cancer, 25-77% in colorectal cancer, 30-50% in pancreatic cancer, 40-80% in prostate cancer, 50-90% in kidney cancer, 35-70% in ovarian cancer, and 36-100% in head and neck cancer. EGFR is overexpressed and / or mutated in numerous tumors and is associated with poor prognosis. In vitro studies and animal model studies have shown that EGFR activation is involved in several cellular functions crucial for cell transformation and tumor cell progression: proliferation / differentiation, survival, induction of angiogenesis, formation of metastatic potential, and regulation of resistance to chemotherapy and radiotherapy (Normanno et al., 2003). Therefore, EGFR has become a noteworthy target in the development of antitumor drugs.

[0004] Numerous studies have already demonstrated that EGFR-targeted inhibitors possess antitumor activity, and several drugs have been approved and marketed in China and other countries. These include small molecule tyrosine kinase inhibitors (e.g., gefitinib, afatinib, osimertinib), monoclonal antibodies (e.g., cetuximab, nimotuzumab), bispecific antibodies (e.g., amuivantomab), and antibody-drug conjugates (e.g., cetuximab sarotalocan). Indications include EGFR-expressing and / or mutated non-small cell lung cancer, head and neck squamous cell carcinoma, colorectal cancer, and nasopharyngeal cancer. Cetuximab and other EGFR monoclonal antibodies have also been extensively explored in other tumors such as tripnegative breast cancer, gastric cancer, and pancreatic cancer, suggesting antitumor effects of EGFR monoclonal antibodies against these tumor types.

[0005] Antibody-drug conjugate (ADC) products targeting EGFR are attracting attention as they overcome resistance to epiderma 1 growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) and EGFR monoclonal antibodies. Currently, one EGFR-ADC product on the market is Akalux (cetuximab sarotalocan) from Rakuten Medical, which was approved in Japan on September 25, 2020, under accelerated approval for the treatment of unresectable locally advanced or recurrent head and neck cancer, becoming the world's first approved photoimmunotherapy drug. In China, development of Miracogen's MRG003 is relatively advanced and has already entered Phase II clinical trials.

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

[0007] [ka]

[0008] Here, Ab is an antibody targeting EGFR.

[0009] In vitro studies have shown that the ADC has excellent inhibitory effects on a variety of tumor cells. However, further research is awaited regarding the therapeutic effect of the ADC on malignant tumors, particularly advanced malignant tumors (such as advanced solid cancers).

Summary of the Invention

Problems to be Solved by the Invention

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

Means for Solving the Problems

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

[0012]

Chemical Formula

[0013] Here, Ab is selected from SWY2110, SWY2111, SWY2112, SWY2113;

[0014] 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;

[0015] 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;

[0016] 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;

[0017] 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;

[0018] n is selected from integers between 1 and 8 or decimals between 1 and 8.

[0019] Furthermore, n is selected from integers between 4 and 8 or decimals between 4 and 8.

[0020] Furthermore, if n is selected from integers between 1 and 8, it may be 1, 2, 3, 4, 5, 6, 7, or 8. If n is selected from integers between 4 and 8, it may be 4, 5, 6, 7, or 8.

[0021] Furthermore, the antibody-drug conjugate represented by formula I has the structures shown in formulas I-1 to I-4.

[0022] [ka]

[0023] The present invention provides applications for the manufacture of pharmaceuticals for the treatment of tumors, including antibody-drug conjugates represented by formula I, and pharmaceutically acceptable salts, hydrates, solvates, stereoisomers, or isotope-labeled forms thereof, wherein the tumors are 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 carcinoma of the lung, pancreatic cancer, cervical cancer, non-squamous non-small cell lung cancer, head and neck squamous cell carcinoma (including nasopharyngeal cancer), renal cancer, and the like.

[0024] Furthermore, the tumors are selected from triple-negative breast cancer with moderate / high EGFR expression, 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., as well as RAS / BRAF-mutated colorectal cancer, squamous cell carcinoma of the lung with moderate / high EGFR expression (Sq-NSCLC), EGFR-mutated non-squamous non-small cell lung cancer (Nsq-NSCLC), squamous cell carcinoma of the head and neck with moderate / high EGFR expression (including nasopharyngeal cancer), RAS / BRAF wild-type colorectal cancer with moderate / high EGFR expression, etc.

[0025] The present invention provides antibody-drug conjugates represented by formula I, as well as pharmaceutically acceptable salts, hydrates, solvates, stereoisomers, or isotope-labeled forms thereof, for use in the prevention and treatment of tumors, said tumors being 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, pulmonary squamous cell carcinoma, non-squamous non-small cell lung cancer, head and neck squamous cell carcinoma (including nasopharyngeal cancer), colorectal cancer, kidney cancer, and the like.

[0026] Furthermore, the tumors are selected from triple-negative breast cancer with moderate / high EGFR expression, 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., as well as RAS / BRAF-mutated colorectal cancer, squamous cell carcinoma of the lung with moderate / high EGFR expression (Sq-NSCLC), EGFR-mutated non-squamous non-small cell lung cancer (Nsq-NSCLC), squamous cell carcinoma of the head and neck with moderate / high EGFR expression (including nasopharyngeal cancer), RAS / BRAF wild-type colorectal cancer with moderate / high EGFR expression, etc.

[0027] The antibody-drug conjugate represented by formula I, as well as its pharmaceutically acceptable salts, hydrates, solvates, stereoisomers, or isotope-labeled forms, are administered to patients in need of treatment by clinically acceptable methods such as oral administration or injection (e.g., intravenous injection, intramuscular injection), preferably intravenous injection.

[0028] Antibody-drug conjugates represented by Formula I, as well as pharmaceutically acceptable salts, hydrates, solvates, stereoisomers, or isotope-labeled forms thereof, are prepared into clinically acceptable pharmaceutical formulations for oral and injectable preparations. The oral formulations include tablets and capsules, and the injectable formulations include injection solutions, lyophilized preparations for injection, etc. Optionally, the pharmaceutical formulations may include pharmaceutically acceptable adjuvants. The amount of the antibody-drug conjugate represented by formula I, and its pharmaceutically acceptable salt, hydrate, solvate, stereoisomer, or isotope-labeled form contained in each dose unit of the pharmaceutical preparation is 1 mg to 1000 mg, preferably 5 mg to 500 mg, or 10 mg to 300 mg, or 20 mg to 200 mg, or 50 mg to 150 mg, or 50 mg to 300 mg, or 50 mg to 400 mg, or 100 mg to 200 mg, or 100 mg to 300 mg, or 100 mg 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 aforementioned pharmaceutical preparation can be administered as a single dose or in divided doses.

[0029] The antibody-drug conjugate represented by Formula I, and its pharmaceutically acceptable salts, hydrates, solvates, stereoisomers, or isotope-labeled forms, are administered to subjects or patients in need of treatment in a therapeutically effective dose for tumor therapy. The therapeutically effective dose refers to a dose that mitigates, inhibits, or eliminates tumor growth, thereby benefiting the subject or patient, but without causing unbearable toxic side effects.

[0030] In some embodiments, the therapeutically effective dose per single dose (calculated as the antibody-drug conjugate represented by Formula I) is 1 mg / dose to 1000 mg / dose, preferably 5 mg / dose to 500 mg / dose, or 10 mg / dose to 300 mg / dose, or 20 mg / dose to 200 mg / dose, or 50 mg / dose to 150 mg / dose, or 50 mg / dose to 300 mg / dose, or 50 mg / dose to 400 mg / dose, or 100 mg / dose to 200 mg / dose, or 100 mg / dose to 300 mg / dose. The dosage is one dose, or 100 mg / dose to 500 mg / dose, more preferably 5 mg / dose, 6 mg / dose, 8 mg / dose, 10 mg / dose, 15 mg / dose, 20 mg / dose, 25 mg / dose, 30 mg / dose, 40 mg / dose, 50 mg / dose, 60 mg / dose, 70 mg / dose, 80 mg / dose, 90 mg / dose, 100 mg / dose, 150 mg / dose, 200 mg / dose, 250 mg / dose, 300 mg / dose, 500 mg / dose, 600 mg / dose, 700 mg / dose, 800 mg / dose, 900 mg / dose, etc.

[0031] In some embodiments, the therapeutically effective dose (calculated as an antibody-drug conjugate represented by formula I) is 1 mg / day to 1000 mg / day, preferably 5 mg / day to 500 mg / day, or 10 mg / day to 300 mg / day, or 20 mg / day to 200 mg / day, or 50 mg / day to 150 mg / day, or 50 mg / day to 300 mg / day, or 50 mg / day to 400 mg / day, or 100 mg / day to 200 mg / day, or 1 The dosages are 00mg / day to 300mg / day, or 100mg / day to 500mg / day, or more preferably 10mg / day, 20mg / day, 30mg / day, 40mg / day, 50mg / day, 60mg / day, 70mg / day, 80mg / day, 90mg / day, 100mg / day, 150mg / day, 200mg / day, 250mg / day, 300mg / day, 500mg / day, 600mg / day, 700mg / day, 800mg / day, 900mg / day, etc.

[0032] In some embodiments, the therapeutically effective dose (calculated based on the antibody-drug conjugate represented by formula I and patient weight, where mg is the dose of the antibody-drug conjugate represented by formula I and kg is patient weight) is 0.05 mg / kg to 50 mg / kg, preferably 0.1 mg / kg to 20 mg / kg, or 0.2 mg / kg to 20 mg / kg, or 0.5 mg / kg to 20 mg / kg, or 0.5 mg / kg to 15 mg / kg, or 0.5 mg / kg to 10 mg / kg, or 0.5 mg / kg to 8 mg / kg, or 0.5 mg / kg to 6 mg / kg, or 0.5 mg / kg to 4 mg / kg, or 0.5 mg / kg to 2 mg / kg, or 1 mg / kg to 20 mg / kg, or 1 mg / kg to 10 mg / kg, or 1 mg / kg to 8 mg / kg, or 2 mg / kg to 20 mg / kg, or 2 mg / kg to 10 mg / kg, or 2 mg / kg to 8 mg / kg, or 3 mg / kg to 20 mg / kg, or 3 mg / kg to 10 mg / kg, or 3 mg / kg to 8 mg / kg, or 5 mg / kg to 20 mg / kg, or 5 mg / kg to 10 mg / kg, or 0.3 mg / kg, or 0.4 mg / kg , or 0.6 mg / kg, or 0.8 mg / kg, or 1.5 mg / kg, or 1.8 mg / kg, or 2.0 mg / kg, or 2.2 mg / kg, or 2.4 mg / kg, or 2.5 mg / kg, or 2.8 mg / kg, or 3 mg / kg, or 3.2 mg / kg, or 3.5 mg / kg, or 3.6 mg / kg, or 3.8 mg / kg, or 4.0 mg / kg, or 4.2 mg / kg, or 4.4 mg / kg, or 4.5 mg / kg, or 4.6 mg / kg, or 4.8 mg / kg, or 5.0 mg / kg, or 5.2 mg / kg g, or 5.4 mg / kg, or 5.5 mg / kg, or 5.6 mg / kg, or 5.8 mg / kg, or 6.0 mg / kg, or 6.2 mg / kg, or 6.4 mg / kg, or 6.8 mg / kg, or 7.0 mg / kg, or 7.2 mg / kg, or 7.5 mg / kg, or 7.8 mg / kg, or 8.0 mg / kg, or 8.5 mg / kg, or 9.0 mg / kg, or 9.5 mg / kg, or 11 mg / kg, or 11.5 mg / kg, or 12.0 mg / kg, or 16.8 mg / kg, or 18.0 mg / kg.

[0033] The above effective therapeutic dose may be administered once daily (QD), or the above effective therapeutic dose may be divided into multiple doses per day as the total daily dose, for example, twice daily (BID) or three times daily (TID). The above effective therapeutic dose may be administered once weekly (QW), or the above effective therapeutic dose may be divided into multiple doses over several days of the week as the total weekly dose, for example, twice weekly (BIW) or three times weekly (TIW). The above effective therapeutic dose may be administered once every two weeks (Q2W), or the above effective therapeutic dose may be divided into multiple doses over two weeks as the total two-week dose, for example, twice every two weeks (BI2W) or three times every two weeks (TI2W). The above effective therapeutic dose may be administered once every three weeks (Q3W), or the total effective therapeutic dose may be divided and administered over multiple days within a three-week period, for example, twice every three weeks (BI3W, e.g., D1 and D8 for one three-week cycle) or three times every three weeks (TI3W). The above effective therapeutic dose may be administered once a month (QM), or the total effective therapeutic dose may be divided and administered over multiple days within a month, for example, twice a month (BI2M) or three times a month (TIM). The above effective therapeutic dose may be administered at intervals, for example, once every 2 to 7 days, or once every 3 days, 4 days, 5 days, or 6 days, or once every 2 to 4 weeks, for example, once every 2 weeks (once every 3 weeks, 3 weeks as one cycle), once every 1 week (once every 2 weeks, 2 weeks as one cycle or 4 weeks as one cycle), and once every 3 weeks (once every 4 weeks, 4 weeks as one cycle), or once every 1 to 3 months, for example, once every 1 month (once every 2 months), or it may be administered in a 4-week cycle consisting of 3 weeks of continuous administration and 1 week of rest, or it may be administered in a 3-week cycle consisting of 2 weeks of continuous administration and 1 week of rest, or it may be administered in a 4-week cycle consisting of 2 weeks of continuous administration and 2 weeks of rest, or it may be administered in a 4-week cycle consisting of 1 week intervals (once every 2 weeks). An example of a dosage regimen includes administering the drug once on the first day of each cycle, with each cycle lasting three weeks.

[0034] To provide a more concise description, the term “approximately” is not used with some of the quantitative figures in this specification. Each figure presented herein should be understood to include not only the actual given value (constant value) but also equivalents and approximations of such constant value that a person skilled in the art could reasonably estimate and that result from experimental and / or measurement conditions. The approximations are preferably in the range of ±20%, ±15%, ±10%, ±8%, ±6%, ±5%, ±4%, ±3%, ±2%, and ±1% based on the constant value.

[0035] Clinical studies have shown that antibody-drug conjugates represented by Formula I, as well as their pharmaceutically acceptable salts, hydrates, solvates, stereoisomers, or isotope-labeled forms, effectively treat advanced malignancies, particularly EGFR-positive advanced solid tumors, overcome resistance to EGFR-TKIs and EGFR monoclonal antibodies, and provide superior clinical benefit to patients with advanced solid tumors, including non-small cell lung cancer, enabling disease control or mitigation.

[0036] Description of antibody sequences 1. EGFR antibody SWY2110 Light chain (SEQ ID NO:30): EIVLTQSPDFQSVTPKEKVTITCRASQSIGTNIHWYQQKPDQSPKLLIKYASESISGIPSRFSGSGSGTDFTLTINSLEAEDAATYYCQQNNEWPTSFGQGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

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

[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 QVQLQESGPGLVKPSETLSLTCTVSGFSLSNYDVHWVRQAPGKGLEWLGVIWSGGNTDYNTPFTSRLTISVDTSKNQFSLKLSSVTAADTAVYYCARALDYYDYEFAYWGQGTLVTVSS

[0045] SWY2110 LV: SEQ ID NO:28 EIVLTQSPDFQSVTPKEKVTITCRASQSIGTNIHWYQQKPDQSPKLLIKYASESISGIPSRFSGSGSGTDFTLTINSLEAEDAATYYCQQNNEWPTSFGQGTKLEIK

[0046] 2. EGFR antibody SWY2111 Light chain (SEQ ID NO:30): EIVLTQSPDFQSVTPKEKVTITCRASQSIGTNIHWYQQKPDQSPKLLIKYASESISGIPSRFSGSGSGTDFTLTINSLEAEDAATYYCQQNNEWPTSFGQGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0047] Heavy chain (SEQ ID NO:34): QVQLQESGPGLVKPSETLSLTCTVSGFSLSDYDVHWVRQAPGKGLEWLGVIWSGGNTDYNTPFTSRLTISVDTSKNQFSLKLSSVTAADTAVYYCARALDDYDYEFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0048] SWY2111 HCDR1:DYDVH SEQ ID NO:31

[0049] SWY2111 HCDR2:VIWSGGNTDYNTPFTS SEQ ID NO:22

[0050] SWY2111 HCDR3:ALDDYDYEFAY SEQ ID NO:32

[0051] SWY2111 LCDR1:RASQSIGTNIH SEQ ID NO:24

[0052] SWY2111 LCDR2:YASESIS SEQ ID NO:25

[0053] SWY2111 LCDR3:QQNNEWPTS SEQ ID NO:26

[0054] SWY2111 HV:SEQ ID NO:33 QVQLQESGPGLVKPSETLSLTCTVSGFSLSDYDVHWVRQAPGKGLEWLGVIWSGGNTDYNTPFTSRLTISVDTSKNQFSLKLSSVTAADTAVYYCARALDDYDYEFAYWGQGTLVTVSS

[0055] SWY2111 LV:SEQ ID NO:28 EIVLTQSPDFQSVTPKEKVTITCRASQSIGTNIHWYQQKPDQSPKLLIKYASESISGIPSRFSGSGSGTDFTLTINSLEAEDAATYYCQQNNEWPTSFGQGTKLEIK

[0056] 3. EGFR antibody SWY2112 Light chain (SEQ ID NO:30): EIVLTQSPDFQSVTPKEKVTITCRASQSIGTNIHWYQQKPDQSPKLLIKYASESISGIPSRFSGSGSGTDFTLTINSLEAEDAATYYCQQNNEWPTSFGQGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0057] Heavy chain (SEQ ID NO:37): QVQLQESGPGLVKPSETLSLTCTVSGFSLSEYDVHWVRQAPGKGLEWLGVIWSGGNTDYNTPFTSRLTISVDTSKNQFSLKLSSVTAADTAVYYCARALDDYDYEFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0058] SWY2112 HCDR1:EYDVH SEQ ID NO:35

[0059] SWY2112 HCDR2:VIWSGGNTDYNTPFTS SEQ ID NO:22

[0060] SWY2112 HCDR3:ALDDYDYEFAY SEQ ID NO:32

[0061] SWY2112 LCDR1:RASQSIGTNIH SEQ ID NO:24

[0062] SWY2112 LCDR2:YASESIS SEQ ID NO:25

[0063] SWY2112 LCDR3:QQNNEWPTS SEQ ID NO:26

[0064] SWY2112 HV:SEQ ID NO:36 QVQLQESGPGLVKPSETLSLTCTVSGFSLSEYDVHWVRQAPGKGLEWLGVIWSGGNTDYNTPFTSRLTISVDTSKNQFSLKLSSVTAADTAVYYCARALDDYDYEFAYWGQGTLVTVSS

[0065] SWY2112 LV:SEQ ID NO:28 EIVLTQSPDFQSVTPKEKVTITCRASQSIGTNIHWYQQKPDQSPKLLIKYASESISGIPSRFSGSGSGTDFTLTINSLEAEDAATYYCQQNNEWPTSFGQGTKLEIK

[0066] 4. EGFR antibody SWY2113 Light chain (SEQ ID NO:30): EIVLTQSPDFQSVTPKEKVTITCRASQSIGTNIHWYQQKPDQSPKLLIKYASESISGIPSRFSGSGSGTDFTLTINSLEAEDAATYYCQQNNEWPTSFGQGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0067] Heavy chain (SEQ ID NO:40): QVQLQESGPGLVKPSETLSLTCTVSGFSLSHYDVHWVRQAPGKGLEWLGVIWSGGNTDYNTPFTSRLTISVDTSKNQFSLKLSSVTAADTAVYYCARALDDYDYEFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0068] SWY2113 HCDR1:HYDVH SEQ ID NO:38

[0069] SWY2113 HCDR2:VIWSGGNTDYNTPFTS SEQ ID NO:22

[0070] SWY2113 HCDR3:ALDDYDYEFAY SEQ ID NO:32

[0071] SWY2113 LCDR1:RASQSIGTNIH SEQ ID NO:24

[0072] SWY2113 LCDR2:YASESIS SEQ ID NO:25

[0073] SWY2113 LCDR3:QQNNEWPTS SEQ ID NO:26

[0074] SWY2113 HV:SEQ ID NO:39 QVQLQESGPGLVKPSETLSLTTCTVSGFSLSHYDVHWVRQAPGKGLEWLGVIWSGGNTDYNTPFTSRLTISVDTSKNQFSLKLSSVTAADTAVYYCARALDDYDYEFAYWGQGTLVTVSS

[0075] SWY2113 LV:SEQ ID NO:28 EIVLTQSPDFQSVTPKEKVTITCRASQSIGTNIHWYQQKPDQSPKLLIKYASESISGIPSRFSGSGSGTDFTLTINSLEAEDAATYYCQQNNEWPTSFGQGTKLEIK [Brief explanation of the drawing]

[0076] [Figure 1] Figure 1 shows the DAR value measurement results for SWY2110-JSSW-001. [Figure 2] Figure 2 shows the DAR value measurement results for SWY2111-JSSW-001. [Figure 3] Figure 3 shows the DAR value measurement results for SWY2112-JSSW-001. [Figure 4] Figure 4 shows the DAR value measurement results for SWY2113-JSSW-001. [Figure 5] Figure 5 shows the DAR value measurement results for SWY2110-Dxd. [Figure 6] Figure 6 shows the DAR value measurement results for SWY2110-Vc MMAE. [Figure 7] Figure 7 shows the effect of SWY2110-ADC on tumor volume in human colorectal cancer DiFi cell-grafted tumors in NU / NU mice. [Figure 8] Figure 8 shows the effect of SWY2110-ADC on tumor volume in human lung adenocarcinoma PC9-GR (gefitinib-resistant cell) cell transplanted tumors in NU / NU mice. [Figure 9] Figure 9 shows the results of measuring the proliferation inhibitory activity of SWY2110-ADC against DiFi cells. [Figure 10]Figure 10 shows the results of measuring the proliferation inhibitory activity of SWY2110-ADC against PC9-GR cells. [Figure 11] Figure 11 shows the effect of SWY2110-ADC on tumor volume in human lung adenocarcinoma PC9-AR (PC9-Del19 / T790M / C797S, osimertinib-resistant cells) cell-grafted tumors in NU / NU mice. [Figure 12] Figure 12 shows the effect of SWY2110-ADC, SWY2111-ADC, SWY2112-ADC, and SWY2113-ADC on the tumor volume of human lung adenocarcinoma NCI-H1975 cell-grafted tumors in NU / NU mice. [Figure 13] Figure 13 shows the effect of SWY2110-ADC, SWY2111-ADC, SWY2112-ADC, and SWY2113-ADC on the tumor volume of human breast cancer MDA-MB-468 cell-grafted tumors in NOD-SCID mice. [Figure 14] 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-grafted tumors in NU / NU mice. [Figure 15] Figure 15 shows the effect of SWY2110-ADC, SWY2111-ADC, SWY2112-ADC, and SWY2113-ADC on the tumor volume of human lung adenocarcinoma PC9-GR (gefitinib-resistant cell) cell transplanted tumors in NU / NU mice. [Modes for carrying out the invention]

[0077] Next, the present invention will be further described in conjunction with specific embodiments. It should be understood that these embodiments are for illustrative purposes only and do not limit the scope of the present invention. In the following embodiments, experimental methods for which specific conditions are not described will typically employ the conditions described in general conditions or the conditions recommended by the manufacturer. Unless otherwise defined herein, all technical terms used in these embodiments have the same meaning as those known to those skilled in the art. Furthermore, any methods and materials similar or equivalent to those described herein may be used in the present invention. More preferred methods and materials described herein are illustrative only.

[0078] The structures of the compounds of the present invention were identified by nuclear magnetic resonance (NMR) and / or liquid chromatography-mass spectrometry (LC-MS) and / or high-performance liquid chromatography (HPLC). A Bruker Avance III 400 MHz nuclear magnetic resonance spectrometer was used for NMR measurements, a SHIMADZU LC-20AD-PDA-LCMS-2020 was used for LC-MS measurements, and a SHIMADZU LC-20AD-PDA high-performance liquid chromatography system was used for HPLC measurements.

[0079] The starting materials used in the examples of the present invention are known compounds that are commercially available or can be synthesized using or in accordance with methods known in the art.

[0080] The antibodies in this invention can be produced using hybridoma technology, first described in Nature (Kohler et al., 1975), or by recombinant DNA methods (e.g., U.S. Patent No. 4,816,567).

[0081] [Table 1-1]

[0082] [Table 1-2]

[0083] Preparation Examples Linker - Preparation of drug compounds 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]indaridino[1,2-b]quinoline-1-yl)amino)-2-methyl-1,6,9,12,15-pentaoxo-3-oxa-5,8,11,14-tetraazahexadecane-16-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)hexaneamide (JSSW-001)

[0084] [ka]

[0085] Scheme 1:

[0086] [ka]

[0087] Step 1: Preparation of compound B2-1 In a 100 ml three-necked flask, 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. The solution turned orange and was heated under reflux. After 1 hour, the solution became colorless and a white solid was formed. After 3 hours of reaction, the reaction mixture was filtered through diatomaceous earth and the solid was washed with ethyl acetate. The filtrate was dried using a rotary evaporator. The resulting crude product was purified by column chromatography to obtain a solid (640 mg). LC-MS[M+Na] + : m / z 391.1.

[0088] Step 2: Preparation of compound A2-1 In a 100 ml three-necked flask, A1-1 (2.7 g, 30.0 mmol) and N,N-dimethylformamide (30 mL) were added, followed by potassium carbonate (4.17 g, 30.0 mmol) and benzyl bromide (2.4 mL, 20.0 mmol). After reacting at room temperature for 14 hours, the reaction mixture was added dropwise to water and extracted with ethyl acetate. The organic layers were combined, 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 2.61 g of the target compound as a colorless liquid.

[0089] 1 H NMR (400 MHz, CDCl3): δ 7.33-7.41 (m, 5H), 5.21 (s, 2H), 4.29-4.35 (m, 1H), 2.77 (d, J = 5.6 Hz, 1H), 1.44 (d, J = 6.8 Hz, 3H).

[0090] Step 3: Preparation of compound A3-1 In a 100 ml three-necked flask, A2-1 (720 mg, 4.0 mmol), B2-1 (368 mg, 1.0 mmol), and dichloromethane (5 mL) were added, and pyridinium p-toluenesulfonate (75 mg, 0.30 mmol) was added. The reaction mixture was heated under reflux and left overnight. After the reaction was complete, ethyl acetate (20 mL) was added to the reaction mixture, and after washing three times with water, the mixture was dried over anhydrous magnesium sulfate. After filtering off the anhydrous magnesium sulfate, the solution was concentrated, and the resulting crude product was purified by column chromatography (PE / EA = 2:1) to obtain a white solid A3-1 (290 mg).

[0091] LC-MS[M+Na] + : m / z 511.1. 1H NMR (400 MHz, CDCl3): δ 7.77 (d, J = 7.2 Hz, 2H), 7.58 (d, J = 7.6 Hz, 2H), 7.40 (t, J = 7.6 Hz, 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 = 6.8 Hz, 1H), 4.19-4.27 (m, 2H), 3.69-3.85 (m, 2H), 1.41 (d, J = 6.8 Hz, 3H).

[0092] Step 4: Preparation of compound A4-1 In a 100 ml three-necked flask, A3-1 (291 mg, 0.60 mmol) was dissolved in tetrahydrofuran:ethyl acetate (4 mL / 2 mL), and Pd / C (10% w / w, 60 mg) was added. After three hydrogen substitutions, a hydrogen balloon was attached and the mixture was stirred overnight. After the reaction was complete, the reaction mixture was filtered through diatomaceous earth and washed with methanol. The organic layer was concentrated to obtain product A4-1. LC-MS[M+Na] + : m / z 399.1.

[0093] Step 5: Preparation of compound A6-1 In a 25 ml single-neck flask, A4-1 (50 mg, 0.13 mmol, 1.3 eq), exatecan mesylate (A5-1) (50 mg, 0.049 mmol, 1.0 eq), 2-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (48 mg, 0.13 mmol, 1.3 eq), and N,N-diisopropylethylamine (36 mg, 0.28 mmol, 3.0 eq) were added and dissolved in N,N-dimethylformamide (2 ml). After stirring overnight at room temperature, the mixture was diluted with ethyl acetate and washed with semisaturated citrate. Subsequently, it was washed with saline solution, sodium bicarbonate solution, and saline solution. The organic layer was dried over anhydrous sodium sulfate, filtered to remove anhydrous sodium sulfate, and concentrated. The product was purified by prep-TLC (eluent system: DCM / MeOH = 15:1) to obtain product A6-1 (56 mg). LC-MS[M+H] + : m / z 816.3.

[0094] Step 6: Preparation of compound A7-1 In a 25 ml single-neck flask, A6-1 (56 mg, 0.069 mmol), N,N-dimethylformamide (2 ml), and piperidine (12 mg, 0.14 mmol) were added and stirred for 2 hours. The solution was concentrated to obtain a white solid. LC-MS[M+H] + : m / z 594.3.

[0095] Step 7: Preparation of compound JSSW-001 A solution of A7-1 (0.069 mmol), C7 (66 mg, 0.14 mmol), 2-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (53 mg, 0.14 mmol), and N,N-diisopropylethylamine (36 mg, 0.28 mmol) in N,N-dimethylformamide (2 ml) was added to a 25-ml single-neck flask and stirred at room temperature for 2 hours. It was diluted with ethyl acetate (30 ml) and washed with semi-saturated citric acid. Then, it was washed with brine, washed with sodium bicarbonate solution, and washed with brine again. The organic layer was dried over anhydrous sodium sulfate, filtered to remove anhydrous sodium sulfate, and concentrated. It was purified by prep-TLC (developing solvent system: DCM / MeOH = 15:1) to obtain the product JSSW-001 (21 mg).

[0096] LC-MS[M+H] + : m / z 1048. 1H NMR (400 MHz, DMSO-d6): δ 8.63 (t, J = 6.4 Hz, 1H), 8.53 (d, J = 8.8 Hz, 1H), 8.28 (t, J = 6.0 Hz, 1H), 8.03-8.10 (m, 2H), 7.98 (t, J = 6.0 Hz, 1H), 7.77 (d, J = 11.2 Hz, 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), 4.09-4.15 (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 = 7.2 Hz, 3H).

[0097] スキーム2:

[0098]

change

[0099] Project 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, and dimethylacetamide (DMAc) (40 mL, 5 v / w) was added and stirred to obtain a homogeneous solution. The temperature was controlled to 0-10°C, and N,N-diisopropylethylamine (DIEA) (34.45 g, 266.6 mmol) was added dropwise. After the addition was complete, the temperature was allowed to rise naturally to approximately 25°C and stirred at this temperature for 16 hours. After confirming that the reaction of the starting materials had proceeded completely by TLC, the reaction solution was poured into 120 mL of ice water, methyl tert-butyl ether (MTBE) (40 mL) was added and stirred, and after standing, the solution was separated. The aqueous layer was extracted four times with MTBE (40 mL x 4 times), and the organic layers were combined. The combined organic layers were washed once with 0.5 M hydrochloric acid solution (40 mL), once with water (40 mL), and twice with saturated saline solution (40 mL x 2 times). After drying over anhydrous sodium sulfate, the layers were filtered, concentrated, and purified by column chromatography using the wet sampling method. Elution was performed using hexane:ethyl acetate = 30:1 → 20:1 → 10:1 solvent systems to obtain a pale yellow liquid C3-1. LC-MS[M+Na] + : m / z 203.

[0100] Step 2: Under a nitrogen atmosphere, 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) and cooled to 10-15°C. p-toluenesulfonic acid (TsOH) (281 mg, 1.63 mmol, 0.1 eq) was added, and the reaction was allowed to proceed at 14-18°C for 4 hours. The progress of the reaction was monitored by TLC. After the reaction was complete, the reaction mixture was poured into ice water (60 mL) and extracted with ethyl acetate (EA) (60 mL x 3 times). The organic layers were combined and washed sequentially with saturated NaHCO3 aqueous solution (60 mL), water (60 mL x 2 times), and saturated saline solution (60 mL). After drying with anhydrous sodium sulfate, the solution was filtered, concentrated, and the solvent was removed by distillation. After mixing with silica, the solution was purified by column chromatography. Elution was performed using hexane:ethyl acetate in the following solvent systems: 10:1 → 5:1 → 3:1 → 2:1 → 1:1, yielding a colorless oily substance C5-1. LC-MS[M+Na] + : m / z 511.

[0101] Step 3: Under a nitrogen atmosphere, C5-1 (2.8 g, 5.74 mmol, 1.0 eq) was dissolved in DMAc (28 mL, 10 v / w) and the temperature was cooled to 14-18°C. 1,8-Diazabicyclo[5.4.0]undeca-7-ene (DBU) (436.6 mg, 2.87 mmol, 0.5 eq) was added dropwise, and the mixture was stirred at this temperature for 1.5 hours. After confirming that the reaction of the starting materials had proceeded completely by TLC, the temperature was cooled to 0-10°C. Pyridinium p-toluenesulfonate (PPTS) (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 the mixture was reacted at 0-10°C for 3-4 hours. After confirming that the reaction of C7-1 had proceeded completely by TLC, the reaction mixture was poured into ice water (100 mL) and extracted with 2-methyltetrahydrofuran (2-Me THF) (100 mL x 3 times). The organic layers were combined and sequentially washed with 0.5 M hydrochloric acid (150 mL x 2 times), saturated sodium bicarbonate aqueous solution (100 mL x 3 times), water (100 mL), and saturated brine (100 mL). After drying over anhydrous sodium sulfate, the mixture was filtered, concentrated, and the solvent was removed by distillation. After mixing with silica, the mixture was purified by column chromatography. Elution was performed in the solvent systems DCM → DCM:MeOH = 80:1 → 60:1 → 50:1 → 40:1 → 30:1 → 20:1 to obtain 3.1 g of white foamy solid C8-1 (yield 72%). LC-MS[M+Na] + : m / z 772.

[0102] Step 4: Under a nitrogen atmosphere, C8-1 (3.1 g, 4.14 mmol, 1.0 eq) was dissolved in dichloromethane (DCM) (46.5 mL, 15 v / w), and DBU (314.6 mg, 2.07 mmol, 0.5 eq) was added dropwise at room temperature (25-30°C). The mixture was stirred at this temperature for 16 hours. After confirming that the reaction of the starting materials had proceeded completely by TLC, the reaction mixture was subjected to direct wet column chromatography. Elution occurred in the solvent systems DCM → DCM:MeOH = 50:1 → 30:1 → 20:1 → 10:1 to obtain a white foamy solid C9-1. LC-MS[M+H] + : m / z 528.

[0103] Step 5: Under a nitrogen atmosphere, C9-1 (1.7 g, 3.2 mmol, 1.0 eq) was dissolved in water (25.5 mL, 15 v / w) and tert-butanol (8.5 mL, 5 v / w), and stirred to obtain a homogeneous solution. After one nitrogen substitution, Pd / C (0.34 g, 20% w / w) was added, and the mixture was substituted three times with hydrogen. The hydrogenation reaction was carried out at room temperature (25-30°C) for 12 hours, and the complete progress of the reaction of the starting materials was confirmed by LC-MS. The reaction solution was filtered, and the filtrate was washed with water (25 mL x 2 times). After filtering the filtrate again, it was concentrated to remove tert-butanol, and the aqueous layer was directly freeze-dried to obtain 1.5 g of white powdered solid C10-1 (100% yield). LC-MS[M+H] + : m / z 438.

[0104] Step 6: Under a nitrogen atmosphere, 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), and C11-1 (1.86 g, 6.097 mmol, 1.1 eq) was added and stirred to obtain a homogeneous solution. The temperature was cooled to 0-10°C, and DIEA (330.24 mg, 2.56 mmol) was added dropwise. After the addition was complete, the reaction was stirred at this temperature for 16 hours. The complete progress of the reaction of the starting materials was confirmed by LC-MS. Na2HPO4 (260 mg) and NaH2PO4 (5.6 g) were added to 20 mL of water and stirred to dissolve and prepare a buffer solution, and the temperature of this buffer solution was cooled to 0-5°C. The reaction mixture was poured into a pre-cooled buffer solution and extracted with a mixed solvent of DCM / isopropanol = 4 / 1 (50 mL x 4 times). After drying the combined organic layers, they were filtered, concentrated, and the solvent was removed by distillation. The solution was then purified by column chromatography. Elution was performed using the solvent systems DCM → DCM:MeOH = 50:1 → 30:1 → 20:1 → 15:1 to obtain the yellow solid C12-1.

[0105] Step 7: Under a nitrogen atmosphere, A5-1 (547.4 mg, 1.03 mmol, 1.0 eq) was dissolved in 5% anhydrous sodium sulfate aqueous solution (6.5 mL) and THF (7.8 mL), and stirred until completely dissolved. The temperature was cooled to 0-10°C, and N-methylmorpholine (NMM) (104 mg, 1.03 mmol, 1.0 eq) was added, and the mixture was reacted at this temperature for 1 hour. Subsequently, C12-1 (650 mg, 1.03 mmol, 1.1 eq), EDCI (296.2 mg, 1.545 mmol, 1.5 eq), and 2-oxime cyanoethyl acetate (73.9 mg, 0.52 mmol, 0.5 eq) were added sequentially, and the mixture was reacted at 0-10°C for 4-5 hours. After confirming that the reaction of the starting materials had proceeded completely by TLC, the reaction mixture was poured into 0.1 M hydrochloric acid (13 mL, 20 v / w) cooled in an ice bath and extracted with 2-methyltetrahydrofuran (2-Me THF) (13 mL x 3 times). The organic layers were combined and washed once with 0.05 M hydrochloric acid (13 mL, 20 v / w) and twice with water (13 mL x 2 times). After drying, filtering, and concentrating, the solvent was removed by distillation, and the mixture was purified by column chromatography. Elution was performed in the solvent systems DCM → DCM:MeOH = 50:1 → 30:1 → 20:1 → 15:1 → 12:1 to obtain the yellow foamy solid JSSW-001. LC-MS[M+H] + : m / z 1048.

[0106] Example 2: Antibody preparation Using techniques known to those skilled in the art, plasmids encoding the light chain and heavy chain genes were transiently transfected into HEK293 cells, expressed, and purified to obtain SWY2110.

[0107] Using computer simulation-assisted technology, modeling and docking analysis of SWY2110 and EGFR protein were performed to obtain antigen-antibody complexes. Amino acid residues within 3 angstroms of the complex were scanned with D, E, and H to obtain several pH-dependent antibodies that showed decreased affinity under pH 7.4 conditions and nearly maintained or showed minimal affinity decrease under pH 6.0 conditions. Several of the obtained antibody sequence genes were cloned into mammalian expression vectors, and antibody expression was performed by transfection into HEK-293 cells. After expression, the culture supernatant was collected, and the antibodies were purified using an AKTA system with a Protein A prepacked column to obtain the target antibodies SWY2111, SWY2112, and SWY2113.

[0108] Example 3 Preparation of Antibody-Drug Conjugates SWY2110-JSSW-001 ADC:

[0109] [ka]

[0110] Step 1: Antibody reduction: Using known techniques, plasmids encoding the light and heavy chain genes were transiently transfected into HEK293 cells, expressed, and purified. The resulting SWY2110 antibody medium was then replaced with PBS 6.0 / EDTA to adjust the antibody concentration to 10 mg / mL. This solution (1.0 mL) was placed in a 1.5 mL polypropylene tube, and 10 mM TCEP (Bailingwei Science and Technology Co., Ltd) aqueous solution (66.6 μL; 10 equivalents per antibody molecule) and 1 M dipotassium hydrogen phosphate aqueous solution (Tianjin Guangfu Science and Technology Development Co., Ltd.) were added. After confirming that the pH of the solution was 7.0 ± 0.1, the solution was incubated at 37°C for 3 hours to reduce the disulfide bonds within the antibody molecule.

[0111] Step 2: Coupling of antibody and linker-drug compound: At room temperature, a 10 mM dimethyl sulfoxide solution of compound JSSW-001 obtained in Example 1 (87 μL; 13 equivalents per antibody molecule) was added to the above solution and mixed. The mixture was then reacted at room temperature for 30 minutes to conjugate the drug linker to the antibody. Subsequently, a 100 mM N-NAC (Bailingwei Science and Technology Co., Ltd) aqueous solution (10.2 μL) was added, and the mixture was stirred at room temperature for another 20 minutes to stop the reaction.

[0112] Step 3: Purification of antibody-drug conjugates: The reaction mixture was purified by ultrafiltration using an ultrafiltration centrifuge tube (Merck, Ultracel® Regenerated Cellulose (30kDa MWCO), 15 mL sample volume). A buffer solution consisting of L-histidine 0.89 mg / mL, L-histidine hydrochloride 4.04 mg / mL, Tween80 0.03%, sucrose 90 mg / mL, pH=5.5 was added to the reaction mixture to remove unreacted drug linker 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.

[0113] Step 4: Measurement of the drug-antibody binding ratio (DAR) of the antibody-drug conjugate: A 2 mg / kg antibody-drug conjugate was treated with dithiothreitol to a final concentration of 20 mM, and the sample was treated in a 37°C water bath for 30 minutes to cleave the interchain disulfide bonds of the antibody-drug conjugate. The sample was then subjected to HPLC analysis. The HPLC system used was an Agilent Technologies 1260 Infinity HPLC, with a PLRP-S column (5 μm particle size; 2.1 mm × 50 mm; Agilent Technologies), at a column temperature of 80°C. Mobile phase A was a 0.1% trifluoroacetic acid (TFA) aqueous solution, and mobile phase B was a 0.1% TFA acetonitrile solution. The injection volume was 10 μL, and the gradient program was as follows: 0-3 min 27%-27%, 3-8 min 27%-35%, 8-25 min 35%-43%, 25-26 min 43%-95%, 26-31 min 95%-95%, 31-32 min 95%-27%, and 32-40 min 27%-27%. Unbound light chain (L0) and heavy chain (H0) were eluted in the order of L0, L1, H0, H1, H2, H3, while drug-bound light chains (L1, bound to one drug molecule) and heavy chains (H1, bound to one drug molecule; H2, bound to two drug molecules; H3, bound to three drug molecules) were eluted in the order of L0, L1, H0, H1, H2, H3, because hydrophobicity increases with the number of bound drug molecules. The DAR value was calculated based on the peak area at 280 nm. Unless otherwise specified in this specification and claims, the DAR value was calculated from the peak area at 280 nm by HPLC analysis as described above.

[0114]

number

[0115] By substituting the antibody SWY2110 with SWY2111, SWY2112, and SWY2113, respectively, using the same method as for preparing SWY2110-JSSW-001 ADC, we obtained SWY2111-JSSW-001 ADC (DAR: 7.48, DAR value results shown in Figure 2), SWY2112-JSSW-001 ADC (DAR: 7.51, DAR value results shown in Figure 3), and SWY2113-JSSW-001 ADC (DAR: 7.50, DAR value results shown in Figure 4).

[0116] [ka]

[0117] SWY2110-DXD ADC (DAR: 6.93, DAR value results are shown in Figure 5) was obtained by substituting compound JSSW-001 with GGFG-DXD using the same method as for preparing SWY2110-JSSW-001 ADC.

[0118] [ka]

[0119] Preparation of antibody-drug conjugate (SWY2110-VC MMAE ADC, DAR=4.53):

[0120] [ka]

[0121] Step 1: Antibody reduction: Using known techniques, plasmids encoding the light and heavy chain genes were transiently transfected into HEK293 cells, expressed, and purified. The resulting SWY2110 antibody medium was then replaced with PBS 6.0 / EDTA to adjust the antibody concentration to 10 mg / mL. This solution (1.0 mL) was placed in a 1.5 mL polypropylene tube, and 10 mM TCEP (Bailingwei Science and Technology Co., Ltd) aqueous solution (17 μL; 2.5 equivalents per antibody molecule) and 1 M dipotassium hydrogen phosphate aqueous solution (Tianjin Guangfu Science and Technology Development Co., Ltd.) were added. After confirming that the pH of the solution was 7.4 ± 0.1, it was incubated at 37°C for 1 hour to reduce the disulfide bonds of the antibody.

[0122] Step 2: Coupling of antibody and linker-drug compound: At room temperature, a 10 mM dimethyl sulfoxide solution (31.28 μL; 4.6 equivalents per antibody molecule) of the purchased mc-vc-PAB-MMAE (CAS No.: 646502-53-6, abbreviated as Vc MMAE) was added to the above solution and mixed. The mixture was then allowed to react at room temperature for 30 minutes to conjugate the linker-drug compound to the antibody. Subsequently, a 100 mM aqueous solution of NAC (Bailingwei Science and Technology Co., Ltd) (10.2 μL) was added, and the reaction was stopped by stirring for another 20 minutes at room temperature.

[0123] Step 3: Purification of antibody-drug conjugates: The reaction mixture was purified by ultrafiltration using an ultrafiltration centrifuge tube (Merck, Ultracel® Regenerated Cellulose (30kDa MWCO), 15 mL sample volume). A purified buffer consisting of 25 mM 2-(N-morpholino)ethanesulfonic acid (MES) and pH=6.5 was added to the reaction mixture, and the solution was concentrated to 1-2 mL. Then, the purified buffer was added again and the mixture was replaced more than 1000 times to remove unreacted linker-drug compounds and other low molecular weight reagents, yielding a purified antibody-drug conjugate (SWY2110-VC MMAE ADC, DAR=4.53).

[0124] Step 4: Measurement of the drug-antibody binding ratio (DAR) of the antibody-drug conjugate: A 10 μg sample of SWY2110-Vc MMAE ADC was attached to a column (2.5 μm particle size; 4.6 mm × 10 cm; TSKgel Butyl-NP). The elution mobile phase A was 20 mM phosphate buffer (PB), pH 7.0, 1.5 M (NH4)2SO4 aqueous solution, and the elution mobile phase B was 20 mM PB, pH 7.0, 25% isopropanol solution. Analysis was performed at a flow rate of 0.8 mL / min, column temperature 30°C, and detection wavelength 280 nm. The gradient program involved 0-100% mobile phase B from 0-20 minutes, 100% mobile phase B from 20-25 minutes, and 100% mobile phase A from 25-30 minutes. Unbound molecules were designated as DAR0, molecules bound to two small molecules as DAR2, molecules bound to four molecules as DAR4, molecules bound to six molecules as DAR6, and molecules bound to eight molecules as DAR8. Therefore, elution occurred in the order of DAR0, DAR2, DAR4, DAR6, and DAR8. The DAR values ​​were calculated based on the percentage of each peak area at 280 nm (A), and the results are shown in Figure 6.

[0125] DAR = 2 × A DAR2 +4×A DAR4 +6×A DAR6 +8 × A DAR8

[0126] Biological Examples Test 1: In vitro cell activity of antibody-drug conjugates.

[0127] 1. Test Objectives: The objective of this study was to detect the inhibitory activity of the antibody-drug conjugate of the present invention against in vitro 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)). Cells were treated in vitro with different concentrations of the compound, and after culturing, laserzlin was added and fluorescence values ​​at excitation wavelength 550 nm / fluorescence wavelength 610 nm were measured. The obtained data were fitted using a 4-parameter model and IC50 was calculated. 50 The values ​​were determined, and the biological activity of the compound was calculated based on them.

[0128] 2. Test materials and equipment:

[0129] [Table 1-3]

[0130] [Table 1-4]

[0131] 3. Test Procedure 1: 3.1 Medium: DMEM, 10% FBS, 1x bispecific antibody

[0132] 3.2 Cell Culture: Remove one vial of cryopreserved SK-BR-3 / MDA-MB-468 cells from liquid nitrogen and culture for 75 cm. 2 The cells were cultured in culture flasks and allowed to recover. They were cultured until cell confluence reached >75% and at least three passages had been performed.

[0133] 3.2.1 When cells were not subcultured, the culture medium was changed every 3-4 days.

[0134] 3.2.2 If cell amplification was required, the cells were subcultured into larger culture flasks to ensure that cell confluence reached >75% before use in the assay.

[0135] 3.3 Cell recovery: SK-BR-3 / MDA-MB-468 cells were recovered when the culture flask was nearly full.

[0136] 3.3.1 The culture medium was removed and washed with PBS to remove dead cells and residual medium.

[0137] 3.3.2 Add 2-3 mL of 0.25% Trypsin-EDTA, gently shake the culture flask, and then incubate at 37°C for 2-3 minutes to digest the cells.

[0138] 3.3.3 Immediately add 5 mL of culture medium to the culture flask and gently aspirate and discharge it using a pipette to disperse the cells.

[0139] 3.3.4 The cell suspension was transferred to a sterile centrifuge tube and centrifuged at 200 × g for 3 minutes.

[0140] 3.3.5 Remove the culture medium from the centrifuge tube and resuspend the cells in 5-10 mL of fresh culture medium.

[0141] 3.4 Cell density measurement: The number of cells was counted using a cell counting chamber under a microscope.

[0142] 3.5 Seeding onto assay plates 3.5.1 Cells in culture medium 1 × 10 5 The solution was diluted to cells / mL and 100 μL / well was seeded into each well of the assay plate, excluding columns A and H.

[0143] 3.5.2 120 μL of culture medium was added to each well in columns A and H as a blank control.

[0144] 3.5.3 The cells were incubated at 37°C and 5% CO2 for 4-6 hours to allow them to adhere.

[0145] 3.6 Preparation of diluents for compound samples The compound was diluted to a starting concentration of 18 μg / mL, and then the 3-fold dilution was repeated to prepare a total of 11 concentration gradient solutions, with the 12th column used as a blank control.

[0146] 3.7 Processing of drug addition 3.7.1 20 μL of each drug diluent was taken from rows 1-11 of the dilution plate and added to the corresponding rows 1-11 of the assay plate.

[0147] 3.7.2 20 μL of fresh culture medium was added to each well in columns 12, A, and H.

[0148] 3.7.4 After gently shaking the plate on a plate shaker for 10-15 seconds, the culture plate was incubated at 37°C for 3 days.

[0149] 3.8 Analysis 3.8.1 After incubation was complete, 20 μL of 0.03% Lezazlin (diluted with 1× PBS) was added to each well and gently shaken for 10-15 seconds.

[0150] 3.8.2 After incubation at 37°C for 3-4 hours, measurements were taken using a microplate reader. The parameter settings were as follows:

[0151] Excitation wavelength: 550 nm Fluorescence wavelength: 610 nm Integration time: 50 Shake: 15 seconds, swirling Reading method: Top read Number of reads / well: 1 Gain: Optimized setting (should be between 35 and 42)

[0152] When reading multiple plates, ensure that the same gain setting is used for all plates.

[0153] 3.8.3 Create graphs using Excel data and compare the ICs of reference standards and samples. 50 I fitted it.

[0154] 3.8.3.1 Data points were plotted using Model 201.

[0155] 3.8.3.2 For the Fit parameter, the following command was used:

[0156] a. A: Fit in advance b. B: Fit in advance c. C: Fitted in advance d. D: Fit in advance e. All constraints have no value.

[0157] 3.8.3.3 Output parameter C to IC 50 The unit was set to ng / mL.

[0158] Test procedure 2: This study investigated the inhibitory effects of SWY2110 monoclonal antibody (SWY2110 mAb), SWY2110-DXD ADC (DAR=6.93), and SWY2110-JSSW-001 ADC (DAR=7.12) on the proliferation of two cell lines: DiFi (human colorectal cancer cells) and PC9-GR (human lung cancer gefitinib-resistant cells). The specific concentration settings were as follows:

[0159] DiFi: SWY2110 mAb, SWY2110-DXD ADC (DAR=6.93), and SWY2110-JSSW-001 ADC (DAR=7.12) were used with a starting concentration of 3000 ng / mL and were diluted 3-fold to create a total of 10 concentration gradient solutions (3000, 1000, 333.33, 111.11, 37.04, 12.35, 4.12, 1.37, 0.46, 0.15 ng / mL), with a drug action duration of 144 hours.

[0160] PC9-GR: SWY2110 mAb, SWY2110-DXD ADC (DAR=6.93), and SWY2110-JSSW-001 ADC (DAR=7.12) were administered at a starting concentration of 1000 ng / mL, and then diluted 5-fold to create a total of 8 concentration gradient solutions (1000, 200, 40, 8, 1.6, 0.32, 0.064, 0.013 ng / mL), with a drug action duration of 144 hours.

[0161] A fixed number of cells in the logarithmic growth phase were seeded into a 96-well plate (100 μL / well). Adherent cells adhered to the plate, and 24 hours later, on the same day, 100 μL of medium containing SWY2110 mAb, SWY2110-DXD ADC (DAR=6.93), or SWY2110-JSSW-001 ADC (DAR=7.12) at different concentration gradients was added to each well. Three duplicate wells were set up for each drug concentration, with corresponding blank wells (medium only) and normal wells (drug concentration 0). After 144 hours of drug action, 20 μL / well of MTT working solution (5 mg / mL) was added. After 4 hours at 37°C, the plate was shaken to remove the supernatant, and 150 μL of DMSO (analytical purity) was added. After shaking and mixing with a microplate shaker and wiping the plate clean, the absorbance (OD) at 550 nm was measured using a microplate reader.

[0162] The cell proliferation inhibition rate was calculated using the following formula:

[0163] Suppression rate (%) = (OD value) 正常ウェル -OD value 薬物投与ウェル ) / (OD value 正常ウェル -OD value ブランクウェル ) × 100%

[0164] Based on the inhibition rate at each concentration, the 50% inhibitory concentration (IC) of the drug was determined using SPSS 19.0. 50 The following calculations were performed. The results are shown in Table 2 and Figures 9 and 10.

[0165] [Table 2]

[0166] From the data in Table 2, the antibody-drug conjugates SWY2110-JSSW-001 ADC (DAR=7.12) and SWY2110-DXD ADC (DAR=6.93) of the present invention showed similar IC50s against the EGFR-high-expressing cell line DiFi and the gefitinib-resistant cell line PC9-GR. 50 The values ​​were observed, and it was found that it showed superior cell suppression and proliferation effects compared to SWY2110 mAb.

[0167] Study 2: Efficacy study of SWY2110-ADC against human colorectal cancer DiFi cell transplanted tumors in NU / NU mice.

[0168] 1. Test animals NU / NU mice, 5-6 weeks old, 14 mice

[0169] 2. Test Objectives The in vivo antitumor effects of two SWY2110-ADC drugs were investigated using a transplanted tumor model in human colorectal cancer DiFi cells in NU / NU mice.

[0170] 3. Drug dosage and group classification

[0171] [Table 3]

[0172] 4. Test Method In this study, a nude mouse tumor transplantation model was constructed using human colorectal cancer DiFi cells. The tumor volume was approximately 110 mm². 3Upon reaching this point, the animals were divided into three groups (d0) by equalizing tumor volume according to Table 3. Five animals each were assigned to the solvent group and the SWY2110-JSSW-001 ADC (DAR=7.12) group, and four animals to the SWY2110-Vc MMAE ADC (DAR=4.53) group. A single intravenous dose was administered. The administration volume was 10 mL / kg, and the solvent control group (Vehicle) received 0.9% sodium chloride injection. After 27 days of observation, the inhibitory effects of the two SWY2110-ADCs listed in the table on human colorectal cancer DiFi nude mouse transplanted tumors were compared.

[0173] 5. Observation Indicators 5.1 Evaluation Metrics

[0174] (1) Tumor volume: V = 1 / 2 × A × B 2

[0175] (2) Relative tumor volume:

[0176]

number

[0177] (3) Relative tumor volume growth rate:

[0178]

number

[0179] (4) Tumor suppression rate:

[0180]

number

[0181] Note: V: Tumor volume A: Tumor diameter B: Short diameter of the tumor RTV: Relative Tumor Volume TV nd : Tumor volume on day n TV 0d Tumor volume on day 0 RTV xnd : Average relative tumor volume on day n TV Xn Mean tumor volume on day n of the treatment group TV X0 Mean tumor volume on day 0 of the treatment group TV Mn : Mean tumor volume of solvent group on day n TV M0 : Mean tumor volume on day 0 of the solvent group

[0182] 6. Results 6.1 Tumor Volume

[0183] At the end of the study, both SWY2110-Vc MMAE ADC (DAR=4.53) (P<0.05) and SWY2110-JSSW-001 ADC (DAR=7.12) (P<0.05) significantly suppressed the volume of transplanted tumors compared to the solvent group. The tumor suppression rate at 27 days in the SWY2110-JSSW-001 ADC (DAR=7.12) group reached 74.6%, followed by SWY2110-Vc MMAE ADC (DAR=4.53) at a high 69.7%. Since the toxicity of the JSSW-001 small molecule is lower than that of Vc MMAE, it was suggested that SWY2110-JSSW-001 ADC (DAR=7.12) can achieve a more efficient and less toxic effect. See Figure 7 and Table 4 for details.

[0184] [Table 4]

[0185] Study 3: Efficacy study of SWY2110-ADC in NU / NU mice with human lung adenocarcinoma PC9-GR (gefitinib-resistant cells) cell transplantation tumors.

[0186] 1. Test animals NU / NU mice, 5-6 weeks old, 30 mice

[0187] 2. Test Objectives The in vivo antitumor effects of SWY2110 monoclonal antibody and three types of SWY2110-ADC drugs were investigated using a human lung adenocarcinoma PC-9-GR (gefitinib-resistant cell) cell NU / NU mouse transplantation tumor model.

[0188] 3. Drug dosage and group classification

[0189] [Table 5]

[0190] 4. Test Method In this study, a nude mouse tumor transplantation model was constructed using human lung adenocarcinoma PC9-GR (gefitinib-resistant cells) cells. The tumor volume was approximately 110 mm². 3 Upon reaching the target tumor volume, the animals were divided into five groups (d0) by equalizing the tumor volume according to Table 5. Each group consisted of six animals, and intravenous administration was administered once a week for four consecutive weeks. The administration volume was 10 mL / kg, and the solvent control group (Vehicle) was administered 0.9% sodium chloride injection. The study was completed 28 days after administration, and the inhibitory effects of SWY2110 monoclonal antibody and three types of SWY2110-ADC drugs on human lung adenocarcinoma PC-9-GR (gefitinib-resistant cells) nude mouse transplanted tumors were compared.

[0191] 5. Observation Indicators 5.1 Evaluation Metrics I referred to section 5.1, Evaluation Criteria, of Exam 2.

[0192] 6. Results 6.1 Tumor Volume At the end of the study, 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) all suppressed the volume of transplanted tumors compared to the solvent group. The tumor suppression effect of the antibody SWY2110 was low, with a tumor suppression rate of only 20.3%. The SWY2110-JSSW-001 ADC (DAR=7.12) group showed the most significant suppression effect on PC9-GR transplanted tumor volume, reaching a tumor suppression rate of 77.7%. The tumor suppression rates for SWY2110-Vc MMAE ADC (DAR=4.53) and SWY2110-DXD ADC (DAR=6.93) were 40.4% and 50.2%, respectively. The tumor suppression effect of SWY2110-JSSW-001 ADC (DAR=7.12) against PC9-GR was 154% of that of SWY2110-DXD ADC (DAR=6.93) and 192% of that of SWY2110-Vc MMAE ADC (DAR=4.53), suggesting that SWY2110-JSSW-001 ADC (DAR=7.12) also has a significant inhibitory effect against gefitinib-resistant tumor cells. See Figure 8 and Table 6 for details.

[0193] [Table 6]

[0194] Study IV: Efficacy study of SWY2110-ADC in human lung adenocarcinoma PC9-AR (PC9-Del19 / T790M / C797S, osimertinib-resistant cells) cell transplant tumors. 1. Test animals Balb / c nude mice, 5-6 weeks old, 24 mice

[0195] 2. Test Objectives The in vivo antitumor effects of three SWY2110-ADC drugs were investigated using a NU / NU mouse transplanted tumor model of human lung adenocarcinoma PC9-Del19 / T790M / C797S (osimertinib-resistant cells, hereafter abbreviated as PC9-AR) cells.

[0196] 3. Drug dosage and group classification

[0197] [Table 7]

[0198] 4. Test Method In this study, a nude mouse tumor transplantation model was constructed using human lung adenocarcinoma PC9-Del19 / T790M / C797S (osimertinib-resistant cells) cells. The tumor volume was approximately 110 mm². 3 Upon reaching the target tumor volume, the animals were divided into four groups (d0) by equalizing the tumor volume according to Table 7. Each group consisted of 6 animals, and a single intravenous dose was administered. The dose volume was 10 mL / kg, and the solvent control group (Vehicle) was administered 0.9% sodium chloride injection. The study was completed 24 days after administration, and the inhibitory effects of the three SWY2110-ADC drugs on human lung adenocarcinoma PC9-Del19 / T790M / C797S (osimertinib-resistant cells) nude mouse transplanted tumors were compared.

[0199] 5. Observation Indicators 5.1 Evaluation Metrics I referred to section 5.1, Evaluation Criteria, of Exam 2.

[0200] 6. Results 6.1 Tumor Volume At the end of the test, compared with the solvent 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) all inhibited the volume of transplanted tumors. The SWY2110-JSSW-001 ADC (DAR = 7.12) group showed the most significant inhibitory effect on the volume of PC9-AR transplanted tumors, and the tumor inhibition rate reached 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 inhibitory effect of SWY2110-JSSW-001 ADC (DAR = 7.12) was 128% of that of SWY2110-DXD ADC (DAR = 6.93) and 109% of that of SWY2110-Vc MMAE ADC (DAR = 4.53), suggesting that SWY2110-JSSW-001 ADC (DAR = 7.12) also has a significant inhibitory effect on osimertinib-resistant tumor cells. For details, refer to Figure 11 and Table 8.

[0201]

Table 8

[0202] Test Five: Pharmacodynamic Test of SWY2110-ADC, SWY2111-ADC, SWY2112-ADC, and SWY2113-ADC against Human Lung Adenocarcinoma NCI-H1975 (Human Lung Adenocarcinoma Cells) Cell Transplant Tumors 1. Test Animals NU / NU mice, 5 - 6 weeks old, 30 mice

[0203] 2. Test Objectives Using a human lung adenocarcinoma NCI-H1975 (human lung adenocarcinoma cells) cell NU / NU mouse transplant tumor model, the in vivo antitumor effects of SWY2110-JSSW-001, SWY2111-JSSW-001, SWY2112-JSSW-001, and SWY2113-JSSW-001 drugs were examined.

[0204] 3. Drug Dosage and Grouping

[0205]

Table 9

[0206] 4. Test Method In this test, a nude mouse tumor transplantation model was constructed using human lung adenocarcinoma NCI-H1975 (human lung adenocarcinoma cells). When the tumor volume reached approximately 110 mm 3 , the animals were divided into 5 groups with balanced tumor volumes according to Table 9 (d0). Six animals were used in each group, and each dosage group was administered once at 1 mg / kg.

[0207] It was administered once intravenously, and the administration volume was 10 mL / kg. The vehicle control group (Vehicle) was administered 0.9% sodium chloride injection. The test was terminated 21 days after administration, and the inhibitory effects on human lung adenocarcinoma NCI-H1975 nude mouse transplanted tumors of SWY2110-JSSW-001, SWY2111-JSSW-001, SWY2112-JSSW-001, and SWY2113-JSSW-001 were compared.

[0208] 5. Observation Indicators 5.1 Evaluation Indicators Refer to Section 5.1 Evaluation Indicators of Test 2.

[0209] 6. Results 6.1 Tumor Volume At the end of the study, 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 superior antitumor effects compared to the solvent group, with tumor suppression rates reaching 84.1%, 83.0%, 80.9%, and 74.2%, respectively (P<0.001). All four types of ADC showed significant inhibitory effects against human lung adenocarcinoma NCI-H1975 tumor cells. See Figure 12 and Table 10 for details.

[0210] [Table 10]

[0211] Study VI: Efficacy study of SWY2110-ADC, SWY2111-ADC, SWY2112-ADC, and SWY2113-ADC on human breast cancer MDA-MB-468 (human breast cancer cell) cell transplant tumors. 1. Test animals NOD-SCID mice, 5-6 weeks old, 28 mice

[0212] 2. Test Objectives The in vivo antitumor effects of the drugs SWY2110-JSSW-001, SWY2111-JSSW-001, SWY2112-JSSW-001, and SWY2113-JSSW-001 were investigated using a NOD-SCID mouse transplanted tumor model of human breast cancer MDA-MB-468 (human breast cancer cells).

[0213] 3. Drug dosage and group classification

[0214] [Table 11]

[0215] 4. Test Method In this study, a mouse tumor transplantation model was established using human breast cancer MDA-MB-468 (human breast cancer cells) cells. When the tumor volume reached approximately 150 mm 3 3 , the animals were divided into 5 groups according to tumor volume as shown in Table 11 (d0). The control group consisted of 8 animals, and each experimental group consisted of 5 animals. They were administered once intravenously. The administration volume was 10 mL / kg. The vehicle control group (Vehicle) was administered 0.9% sodium chloride injection. The test was terminated 28 days after administration, and the in vivo antitumor effects of 4 types of ADC drugs were examined.

[0216] 5. Observation indicators 5.1 Evaluation indicators Refer to the 5.1 evaluation indicator section of Test 2.

[0217] 6. Results 6.1 Tumor volume At the end of the test, compared with the vehicle group, the tumor inhibition rates by the administration 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 65.8%, 65.1%, 58.3%, and 47.1% (P<0.001) respectively, all showing good tumor inhibition activity. It was suggested that all 4 types of ADCs had significant inhibitory effects on human breast cancer MDA-MB-468 tumor cells. For details, refer to Figure 13 and Table 12.

[0218]

Table 12

[0219] Test 7 Pharmacodynamic study of SWY2110-ADC, SWY2111-ADC, SWY2112-ADC, and SWY2113-ADC on transplanted tumors of human colorectal cancer DiFi (human colorectal cancer cells) 1. Test animals NU / NU mice, 5 - 6 weeks old, 35 animals

[0220] 2. Test Objectives The in vivo antitumor effects of the drugs SWY2110-JSSW-001, SWY2111-JSSW-001, SWY2112-JSSW-001, and SWY2113-JSSW-001 were investigated using a human colorectal cancer DiFi (human colorectal cancer cell) NU / NU mouse transplanted tumor model.

[0221] 3. Drug dosage and group classification

[0222] [Table 13]

[0223] 4. Test Method In this study, a mouse tumor transplantation model was constructed using human colorectal cancer DiFi (human colorectal cancer cells). The tumor volume was approximately 100 mm². 3 Upon reaching the target tumor volume, the animals were divided into five groups according to Table 13 (d0). Each group consisted of 7 animals, and intravenous administration was performed twice (once a week). The administration volume was 10 mL / kg. The solvent control group (Vehicle) was administered 0.9% sodium chloride injection. The study was completed 21 days after administration, and the in vivo antitumor effects of the four ADC drugs were examined.

[0224] 5. Observation Indicators 5.1 Evaluation Metrics I referred to section 5.1, Evaluation Criteria, of Exam 2.

[0225] 6. Results 6.1 Tumor Volume At the end of the study, compared to the solvent group, the tumor suppression rates for 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), all demonstrating good tumor suppression activity. All four ADCs were suggested to have a significant inhibitory effect on human colorectal cancer DiFi tumor cells. See Figure 14 and Table 14 for details.

[0226] [Table 14]

[0227] Study 8: Efficacy study of SWY2110-ADC, SWY2111-ADC, SWY2112-ADC, and SWY2113-ADC on human lung adenocarcinoma PC9-GR (human lung adenocarcinoma gefitinib acquired resistance strain) cell transplant tumors. 1. Test animals NU / NU mice, 5-6 weeks old, 30 mice

[0228] 2. Test Objectives The in vivo antitumor effects of the drugs SWY2110-JSSW-001, SWY2111-JSSW-001, SWY2112-JSSW-001, and SWY2113-JSSW-001 were investigated using a human lung adenocarcinoma cell PC9-GR NU / NU mouse transplanted tumor model.

[0229] 3. Drug dosage and group classification

[0230] [Table 15]

[0231] 4. Test Method In this study, a mouse tumor transplantation model was constructed using human lung adenocarcinoma cells (PC9-GR cells). The tumor volume was approximately 110 mm².3 Upon reaching the target tumor volume, the animals were divided into five groups according to Table 15 (d0). Each group consisted of six animals, and a single intravenous dose was administered. The dose volume was 10 mL / kg. The solvent control group (Vehicle) was administered 0.9% sodium chloride injection. The study was completed 28 days after administration to examine the in vivo antitumor effects of the four ADC drugs.

[0232] 5. Observation Indicators 5.1 Evaluation Metrics I referred to section 5.1, Evaluation Criteria, of Exam 2.

[0233] 6. Results 6.1 Tumor Volume At the end of the study, the tumor suppression rates for 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% (P<0.001), respectively, compared to the solvent group, all showing good tumor suppression effects. It was suggested that all four types of ADCs have a significant inhibitory effect on human lung adenocarcinoma cells (PC9-GR tumor cells). See Figure 15 and Table 16 for details.

[0234] [Table 16]

[0235] Test 9: Safety Evaluation Study In this study, six cynomolgus monkeys of appropriate age (3 males and 3 females) were intravenously administered SWY2110-JSSW-001, SWY2111-JSSW-001, and SWY2113-JSSW-001. The dosage was designed as shown in the table below, with intravenous injections administered once every 3 weeks for 3 consecutive doses. Continuous observation was performed for 7 days after administration, with general observations twice daily and detailed observations once daily. Weight changes were observed before and after administration, and hematological tests were performed. From the experimental results, ADCs prepared with pH-dependent antibodies (SWY2111-JSSW-001 and SWY2113-JSSW-001) showed reduced skin and gastrointestinal toxicity compared to SWY2110-JSSW-001, and no clear target-related toxicity (skin ulcers, scabbing, loose stools, etc.) was observed. As a result, pH-denatured ADCs showed reduced toxicity to normal tissues, but no significant differences were observed in hematological toxicity among the three types of low-molecular-weight ADCs.

[0236] [Table 17]

[0237] [Table 18]

[0238] Clinical examples Example 1 1. Experimental Design This study is an open-label, multicenter Phase I clinical trial in patients with advanced solid tumors, consisting of two stages: Stage I, which includes a dose-escalation study and a pharmacokinetic (PK) expansion study, and Stage II, which is a cohort expansion study. The purpose of this study was to evaluate the safety, tolerability, pharmacokinetic (PK) profile, and initial efficacy of the antibody-drug conjugate of the present invention in patients with advanced solid tumors.

[0239] 1.1 Stage I 1.1.1 Dose escalation study In patients with advanced solid tumors, dose escalation studies were conducted using both an accelerated dose escalation design and a conventional 3+3 design. The initial dose of the antibody-drug conjugate of the present invention was set at 0.6 mg / kg, and the maximum dose was set at 8 mg / kg. The dose-limiting toxicity (DLT) and maximum tolerated dose (MTD) were determined through the dose escalation studies. The specific dose designs for each dose escalation stage were based on a modified Fibonacci design and are shown in the table below.

[0240] [Table 19]

[0241] 1.1.2 PK Expansion Test Based on safety, tolerability, and pharmacokinetic (PK) data from each dose group in the dose escalation study, 2-3 dose groups were selected for PK expansion studies. Each dose group was expanded to include 8-12 subjects (including subjects from the same dose group in the dose escalation study).

[0242] 1.2 Stage II A cohort expansion study was conducted using the recommended doses initially determined in Phase I, and the subjects were divided into the following six cohorts according to tumor type:

[0243] Cohort A: EGFR moderate / high expression colorectal cancer Cohort B: Triple-negative breast cancer with moderate / high EGFR expression. Cohort C: Head and neck squamous cell carcinoma (including nasopharyngeal carcinoma) with moderate / high EGFR expression. Cohort D: EGFR-mutated non-squamous non-small cell lung cancer (Nsq-NSCLC) Cohort E: Squamous / High-Expression EGFR Lung Squamous Cell Carcinoma (Sq-NSCLC) Cohort F: Other advanced solid tumors with moderate / high EGFR expression (gastric cancer, esophageal cancer, pancreatic cancer, cervical cancer, biliary tract cancer, non-squamous non-small cell lung cancer, RAS / BRAF variant colorectal cancer, etc.)

[0244] All subjects underwent safety evaluations after their first dose of the investigational drug. PK and immunogenic blood samples were collected after single-dose administration and during the continuous administration period. Furthermore, tumor imaging evaluations were performed based on the criteria for evaluating the efficacy of solid tumors (RECIST v1.1).

[0245] 2. Subject Selection Participants had to meet all of the following selection criteria: 18 years of age or older (including 18 years of age), any sex; patients with pathologically diagnosed advanced solid tumors who had failed or were intolerant of standard treatment, had no standard treatment available, or refused standard treatment; subjects with EGFR activating mutations, moderate / high expression, or amplification; subjects with at least one measurable lesion confirmed by CT or MRI based on the Criteria for Evaluating the Response of Solid Tumors (RECIST) v1.1.

[0246] 3. Clinical trial interventions and combination therapies 3.1 Test drug (Dar value detected by hydrophobic interaction chromatography)

[0247] [ka]

[0248] Dosage form: Lyophilized preparation for injection Specifications: 100 mg / vial Storage conditions: 2~8℃, protected from light

[0249] 3.2 Implementation of the trial intervention The initial dose of the investigational drug was calculated based on the subjects' weight at screening / baseline. If a subject's weight change during the study treatment period was less than 10% compared to the screening / baseline weight, recalculation of the dose was not required. The drug was administered by intravenous infusion, once every three weeks (Q3W), with a 21-day treatment cycle. Infusion time was 60 minutes ± 10 minutes, and if no infusion-related reactions occurred after the initial dose, subsequent infusion times were shortened to 30 minutes ± 5 minutes. Administration was continued until the treatment discontinuation criteria specified in the clinical trial protocol were met.

[0250] If administration was delayed, the administration date for subsequent cycles was calculated from the actual administration date of the previous cycle.

[0251] 4. Evaluation Items 4.1 Pharmacokinetic (PK) evaluation

[0252] As planned in Table 20, PK blood samples were collected from each subject at the designated sampling time points and time windows in each study phase, and PK measurements of the test drug were performed. The measured items included toxin-bound antibodies, total antibodies, and free toxins.

[0253] For dose escalation studies and PK expansion studies, non-compartmental analysis was used to analyze the PK parameters (Cmax, Tmax, t1 / 2, MRT, Vd, CL, AUC) for each subject. 0-t AUC 0-∞ We calculated Cmax, ss, Cmin, ss, Tmax, ss, AUCss, Rac, etc. We created a descriptive statistical summary and list of PK parameters. We created box plots of dose-normalized major PK parameters (AUC and Cmax, etc.) and examined the relationship between major PK parameters and dose using an analysis of variance model and / or a power model.

[0254] 4.2 Immunogenicity Assessment As planned in Table 20, immunogenic blood samples were collected from each subject at the designated sampling time points and time windows in each study phase, and the immunogenicity of the test drug was measured. The immunogenic samples were used for setting the in-study cutoff value, anti-drug antibody (ADA) analysis, and targeted interference studies.

[0255] All ADA results were compiled into a table. The proportion of ADA-positive subjects, the timing of the first ADA positive test onset, and the duration of the test were analyzed. For ADA-confirmed positive samples, neutralizing antibody measurements were performed as needed, and the results were compiled into a table. The impact of immunogenicity on the drug's pharmacokinetics, safety, and efficacy was analyzed.

[0256] [Table 20]

[0257] 4.3 Biomarker Analysis EGFR status was detected using tumor tissue or peripheral blood samples from the subjects.

[0258] For EGFR-mutated Nsq-NSCLC, tumor tissue samples were provided simultaneously, and IHC measurements were performed. Furthermore, the correlation between EGFR expression levels in Nsq-NSCLC and the efficacy of the test drug was analyzed. During NSCLC screening, tumor tissue or peripheral blood samples were provided, and NGS measurements were performed to analyze factors influencing the efficacy of the test drug.

[0259] 4.4 Safety Evaluation The severity of adverse events was recorded using the National Cancer Institute Common Terminology Criteria for Adverse Events (NCI-CTCAE, v5.0). Adverse events were collected and evaluated, and the causal relationship between adverse events and drugs was examined.

[0260] All adverse events (AEs), including clinical symptoms, abnormal vital signs, and abnormal laboratory findings, were observed and recorded in all subjects, and their association with the test drug was determined.

[0261] In subjects participating in dose-escalation studies, DLT events were observed during the DLT observation period. Additionally, delayed DLTs were observed within 84 days of the initial dose.

[0262] Participants underwent safety evaluations (physical examination, ECOG score, clinical tests, electrocardiogram, etc.) at screening / baseline and after administration.

[0263] We investigated and evaluated skin toxicity, interstitial pneumonia / non-infectious pneumonia toxicity, hematological toxicity, gastrointestinal toxicity, ocular toxicity, hepatotoxicity, fluid-related reactions / allergic reactions, and other toxicities.

[0264] We observed whether any adverse skin events occurred in the subjects, classified the adverse skin events by severity based on CTCAE V5.0, and addressed any adverse events that occurred.

[0265] Participants were observed for changes in imaging findings consistent with interstitial lung disease / non-infectious pneumonia, acute onset, or the appearance of new / worsened lungs or related signs / symptoms (such as dyspnea, cough, and fever), and the presence or absence of interstitial lung disease / non-infectious pneumonia was evaluated. Pulmonary function tests (including hepatic parameters related to ventilatory and diffusion functions) were performed during screening.

[0266] The subjects' hematological tests were monitored regularly.

[0267] We observed whether the subjects experienced symptoms such as diarrhea, loose stools, vomiting, and nausea.

[0268] Participants were observed for the occurrence of ocular toxicity (acute or worsened eye inflammation, lacrimation, photosensitivity, decreased visual acuity, eye pain, and / or eye redness, etc.), and adverse events were classified according to CTCAE V5.0. Ophthalmic examinations (including visual acuity, slit-lamp examination, and fundus examination) were performed on participants during screening.

[0269] We observed whether the subjects developed abnormal liver function.

[0270] Subjects were observed for the occurrence of fluid-related / allergic reactions (fever, itching, rash, angioedema, chills, hypotension, dyspnea, chest discomfort, wheezing or tachycardia, anaphylaxis, etc.), and adverse events were classified according to CTCAE V5.0.

[0271] 4.5 Effectiveness Evaluation Efficacy evaluation was conducted according to RECIST 1.1 criteria, using CT / MRI imaging. Subjects underwent tumor imaging every 6 weeks until 24 weeks after the start of the first dose, and every 9 weeks thereafter. A tolerance time window of ±7 days was set for imaging examinations. Efficacy evaluations were conducted at the specified time points, regardless of or without delay in administration.

[0272] Efficacy endpoint: Objective response rate (ORR): Defined as the percentage of subjects whose best overall response, as assessed by the principal investigator based on RECIST v1.1, was a complete response (CR) or a partial response (PR) (i.e., CR+PR) during the period from the start of investigational drug use to the discontinuation of the study. Disease control rate (DCR): Defined as the percentage of patients whose response at the best response time, as assessed according to RECIST v1.1 criteria, was complete response (CR), partial response (PR), or stable disease (SD) (i.e., CR+PR+SD) during the period from the start of investigational drug use to the discontinuation of the study. Progression-free survival (PFS) was defined as the period from the start of treatment with the investigational drug to the date on which the first disease progression (PD) was recorded, or the date of death from any cause (whichever came first). Duration of response (DoR): Defined as the period from the first time the tumor was evaluated as CR or PR until the first time it was evaluated as PD, or the date of death from any cause (whichever comes first). Overall survival (OS) was defined as the period from the start of drug use to the date of death from any cause.

[0273] In Phase I, efficacy was described for each dose group. In Phase II, best overall response (BOR), objective response rate (ORR), and disease cycle response rate (DCR) were aggregated and analyzed. Progression-free survival (PFS), duration of treatment (DR), and overall survival (OS) were also analyzed. The outline of the analysis method is as follows:

[0274] Based on RECIST 1.1 criteria, BORs were compiled for each cohort, the number and proportion of cases for ORR and DCR were calculated, and the 95% confidence interval (CI) was calculated using the Clopper-Pearson method.

[0275] For PFS, DoR, and OS, the Kaplan-Meier method was used for analysis, the median and its 95% CI were estimated, and Kaplan-Meier curves were plotted.

[0276] 5. Test Results A total of 25 subjects were enrolled, and safety data were obtained from 23 subjects. Sixteen subjects completed at least one efficacy evaluation. Of these, 4 showed a partial response (PR), 11 showed stable disease (SD), and 1 showed progressive disease (PD).

[0277] 0.6 mg / kg dose group: 1 SD case (EGFR mutation, non-squamous NSCLC) 1.8 mg / kg dose group: 3 cases of SD (stable disease) (2 cases of EGFR-mutated non-squamous NSCLC, 1 case of EGFR-moderate / high-expression colorectal cancer) 3.6 mg / kg dose group: 2 SD cases (1 case of EGFR-moderate / high-expression squamous cell lung cancer, 1 case of EGFR-moderate / high-expression non-squamous NSCLC), 1 PD case (EGFR-moderate / high-expression colorectal cancer, RAS / BRAF wild-type). 4.8 mg / kg dose group: PR 3 cases (EGFR mutation / amplification non-squamous NSCLC 1 case; EGFR moderate / high expression / mutation lung squamous cell carcinoma 1 case; EGFR moderate / high expression nasopharyngeal carcinoma 1 case), SD 3 cases (EGFR mutation non-squamous NSCLC 2 cases, EGFR mutation / amplification non-squamous NSCLC 1 case) 6.4 mg / kg dose group: PR 1 case (EGFR mutation / amplification non-squamous NSCLC), SD 2 cases (EGFR moderate / high expression squamous cell carcinoma of the lung 1 case, EGFR moderate / high expression non-squamous NSCLC 1 case)

[0278] Among 13 cases of non-small cell lung cancer (NSCLC), there were 3 cases of partial response (PR) and 10 cases of stable disease (SD).

[0279] One case of nasopharyngeal cancer resulted in a partial response (PR).

[0280] Of the two cases of colorectal cancer, one was stable disease (SD) and the other was progressive disease (PD).

[0281] Typical case Case 1: A 69-year-old male was initially diagnosed with non-small cell lung cancer. After surgery (left upper lobectomy + mediastinal lymph node dissection), he received 4 cycles of vinorelbine + cisplatin therapy, but the disease progressed. Subsequently, oral osimertinib was initiated, followed by osimertinib therapy with paclitaxel + carboplatin, but the disease progressed. Radiotherapy was then performed for bone metastases. Afatinib was then administered, but the disease progressed. Paclitaxel + cisplatin therapy was then initiated, but the disease progressed. Finally, tislerizumab therapy with paclitaxel + cisplatin was performed, but the disease progressed.

[0282] Subsequently, informed consent was signed. At the time of screening, the patient was in stage IV of illness, with moderate / high EGFR expression and the T790M mutation. The baseline target lesion was 168 mm (maximum intrapulmonary lesion 84 mm, with new cavity formation). The investigational drug was administered as the first dose, and the response evaluation result was a partial response (PR).

[0283] Case 2: A 56-year-old male was diagnosed with non-small cell lung cancer. He received JNJ-61186372 + lazertinib combination therapy. After disease progression, he participated in another clinical trial, where his disease progressed further. Subsequently, he received pemetrexed + carboplatin therapy, but chemotherapy was discontinued due to myelosuppression.

[0284] Subsequently, informed consent was signed. At the time of screening, the clinical stage was stage IV, and the patient had an EGFR mutation (with amplification). The investigational drug was administered as the first dose, and the initial response evaluation result was a partial response (PR), which was confirmed at the second response evaluation.

[0285] Case 3: A 48-year-old woman was diagnosed with non-small cell lung cancer. She participated in other drug trials, but her disease progressed.

[0286] Subsequently, informed consent was signed. At the time of screening, the clinical stage was stage IV, and the patient had an EGFR mutation (with amplification). The investigational drug was administered as the first dose, and the initial response evaluation result was a partial response (PR).

[0287] Case 4: A 55-year-old male was initially diagnosed with nasopharyngeal cancer. He underwent gemcitabine + cisplatin therapy, followed by chemoradiotherapy, and then capecitabine maintenance therapy, but the disease progressed. Subsequently, he received Camrelizumab treatment, but the disease progressed. He then received docetaxel + nedaplatin therapy, but the disease progressed.

[0288] Subsequently, informed consent was signed. At the time of screening, the clinical stage was stage IV, and EGFR expression was moderate / high. The investigational drug was administered as the first dose, and the initial response evaluation result was a partial response (PR).

[0289] Clinical studies have shown that the antibody-drug conjugate represented by Formula I, as well as its pharmaceutically acceptable salts, hydrates, solvates, stereoisomers, or isotope-labeled forms, effectively treat advanced malignancies, particularly EGFR-positive advanced solid tumors, overcome resistance to EGFR-TKIs and EGFR monoclonal antibodies, and provide clinical benefits exceeding existing therapies, including control or mitigation of disease progression, in patients with advanced solid tumors such as non-small cell lung cancer.

Claims

1. Antibody-drug conjugates represented by formula I for tumor treatment, and pharmaceutically acceptable salts, hydrates, solvates, stereoisomers or isotope-labeled forms thereof, 【Chemistry 1】 However, Ab is selected from SWY2110, SWY2111, SWY2112, and SWY2113; SWY2110 is an antibody having two heavy chains having the amino acid sequence shown in SEQ ID NO:29 and two light chains having the amino acid sequence shown in SEQ ID NO:30; SWY2111 is an antibody having two heavy chains having the amino acid sequence shown in SEQ ID NO:34 and two light chains having 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 selected from integers between 1 and 8 or decimals between 1 and 8, preferably a number between 7 and 8 (including the endpoints). 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, lung squamous cell carcinoma, non-squamous non-small cell lung cancer, head and neck squamous cell carcinoma (including nasopharyngeal cancer), colorectal cancer, urothelial carcinoma, kidney cancer, etc., and preferably the tumor is colorectal cancer with moderate / high EGFR expression, triple-negative breast cancer with moderate / high EGFR expression, head and neck squamous cell carcinoma with moderate / high EGFR expression ( The following are selected from EGFR-mutated non-squamous non-small cell lung cancer (Nsq-NSCLC), including nasopharyngeal carcinoma, EGFR-mutated non-squamous non-small cell lung cancer (Nsq-NSCLC), EGFR-moderate / highly expressive squamous cell lung cancer (Sq-NSCLC), other EGFR-moderate / highly expressive advanced solid tumors such as gastric cancer, esophageal cancer, pancreatic cancer, cervical cancer, biliary tract cancer, non-squamous non-small cell lung cancer, and RAS / BRAF-mutated colorectal cancer, as well as drug-resistant lung adenocarcinoma (gefitinib-resistant and / or osimertinib-resistant lung adenocarcinoma).

2. Based on the antibody-drug conjugate represented by Formula I, the dose per dose is 1 mg / dose to 1000 mg / dose, preferably 5 mg / dose to 500 mg / dose, or 10 mg / dose to 300 mg / dose, or 20 mg / dose to 200 mg / dose, or 50 mg / dose to 150 mg / dose, or 50 mg / dose to 300 mg / dose, or 50 mg / dose to 400 mg / dose, or 100 mg / dose to 200 mg / dose, or 100 mg / dose to 300 mg / dose, or 100 mg / dose to 500 mg / dose, more preferably 5 mg / dose, 6 mg / dose, or 8 mg / dose. An antibody-drug conjugate represented by formula I as described in claim 1, characterized in that the dosage is 10 mg / dose, 15 mg / dose, 20 mg / dose, 25 mg / dose, 30 mg / dose, 40 mg / dose, 50 mg / dose, 60 mg / dose, 70 mg / dose, 80 mg / dose, 90 mg / dose, 100 mg / dose, 150 mg / dose, 200 mg / dose, 250 mg / dose, 300 mg / dose, 500 mg / dose, 600 mg / dose, 700 mg / dose, 800 mg / dose, 900 mg / dose, etc., and a pharmaceutically acceptable salt, hydrate, solvate, stereoisomer or isotope-labeled product thereof.

3. Based on the antibody-drug conjugate represented by Formula I and the patient's body weight, the single dose is 0.05 mg / kg to 50 mg / kg, preferably 0.1 mg / kg to 20 mg / kg, or 0.2 mg / kg to 20 mg / kg, or 0.5 mg / kg to 20 mg / kg, or 0.5 mg / kg to 15 mg / kg, or 0.5 mg / kg to 10 mg / kg, or 0.5 mg / kg to 8 mg / kg, or 0.5 mg / kg to 6 mg / kg, or 0.5 mg / kg to 4 mg / kg, or 0.5 mg / kg to 2 mg / kg, or 1 mg / kg to 20 mg / kg, or This includes 1 mg / kg to 10 mg / kg, or 1 mg / kg to 8 mg / kg, or 2 mg / kg to 20 mg / kg, or 2 mg / kg to 10 mg / kg, or 2 mg / kg to 8 mg / kg, or 3 mg / kg to 20 mg / kg, or 3 mg / kg to 10 mg / kg, or 3 mg / kg to 8 mg / kg, or 5 mg / kg to 20 mg / kg, or 5 mg / kg to 10 mg / kg, or 0.3 mg / kg, or 0.4 mg / kg, or 0.6 mg / kg, or 0.8 mg / kg, or 1.5 mg / kg, or 1.8 mg / kg, or 2.0 mg / kg, or 2.2 mg / kg, or 2.4 mg / kg, or 2.5 mg / kg, or 2.8 mg / kg, or 3 mg / kg, or 3.2 mg / kg, or 3.5 mg / kg, or 3.6 mg / kg, or 3.8 mg / kg, or 4.0 mg / kg, or 4.2 mg / kg, or 4.4 mg / kg, or 4.5 mg / kg, or 4.6 mg / kg, or 4.8 mg / kg, or 5.0 mg / kg, or 5.2 mg / kg, or 5.4 mg / kg, or 5.5 mg / kg, or 5.6 mg / kg, or 5.8 mg / kg, or 6.0 mg / kg, or 6.2 mg An antibody-drug conjugate represented by formula I as described in claim 1, characterized in that the concentration is 1 / kg, or 6.4 mg / kg, or 6.8 mg / kg, or 7.0 mg / kg, or 7.2 mg / kg, or 7.5 mg / kg, or 7.8 mg / kg, or 8.0 mg / kg, or 8.5 mg / kg, or 9.0 mg / kg, or 9.5 mg / kg, or 11 mg / kg, or 11.5 mg / kg, or 12.0 mg / kg, or 16.8 mg / kg, or 18.0 mg / kg, and a pharmaceutically acceptable salt, hydrate, solvate, stereoisomer, or isotope-labeled product thereof.

4. The antibody-drug conjugate represented by formula I, and its pharmaceutically acceptable salts, hydrates, solvates, stereoisomers, or isotope-labeled compounds may be administered once daily (QD), or in multiple divided doses per day, for example, twice daily (BID) or three times daily (TID), once weekly (QW), or the above therapeutically effective dose may be administered in multiple doses over several days of the week, for example, twice weekly (BIW) or three times weekly (TIW), or once every two weeks (Q2W). It may be administered as a single dose, or divided over multiple days within a two-week period, for example, twice every two weeks (BI2W) or three times every two weeks (TI2W), or once every three weeks (Q3W), or divided over multiple days within a three-week period, for example, twice every three weeks (BI3W, e.g., D1 and D8 for a three-week cycle) or three times every three weeks (TI3W), or once a month (QM), or divided over multiple days within a month, for example, twice a month (BI2M) or three times a month (TIM). It may also be administered at intervals, for example, once every 2 to 7 days, or once every 3, 4, 5, or 6 days, or at intervals of 2 to 4 weeks, for example, once every 2 weeks (once every 3 weeks, 3 weeks as one cycle), once every 1 week (once every 2 weeks, 2 weeks as one cycle or 4 weeks as one cycle), once every 3 weeks (once every 4 weeks, 4 weeks as one cycle), or at intervals of 1 to 3 months, for example, once every 1 month (every 2 months). Alternatively, the drug may be administered in a cycle of 4 weeks consisting of 3 weeks of continuous administration followed by 1 week of rest, or in a cycle of 3 weeks consisting of 2 weeks of continuous administration followed by 1 week of rest, or in a cycle of 4 weeks consisting of 2 weeks of continuous administration followed by 2 weeks of rest, or in a cycle of 4 weeks consisting of administration at 1-week intervals (once every 2 weeks), with 3 weeks forming one cycle and the drug being administered once on the first day of each cycle, characterized in that the antibody-drug conjugate represented by formula I as described in claim 1, and a pharmaceutically acceptable salt, hydrate, solvate, stereoisomer or isotope-labeled product thereof.

5. The antibody-drug conjugate represented by formula I, and a pharmaceutically acceptable salt, hydrate, solvate, stereoisomer, or isotope-labeled thereof, as described in claim 4, is characterized in that the antibody-drug conjugate represented by formula I, and a pharmaceutically acceptable salt, hydrate, solvate, stereoisomer, or isotope-labeled thereof, are administered in the doses described in claims 2 to 3 during each administration cycle.

6. Applications of antibody-drug conjugates represented by formula I, and pharmaceutically acceptable salts, hydrates, solvates, stereoisomers, or isotope-labeled forms thereof, in the manufacture of pharmaceuticals for the treatment of tumors, 【Chemistry 2】 However, Ab is selected from SWY2110, SWY2111, SWY2112, SWY2113. SWY2110 is an antibody having two heavy chains having the amino acid sequence shown in SEQ ID NO:29 and two light chains having the amino acid sequence shown in SEQ ID NO:30; SWY2111 is an antibody having two heavy chains having the amino acid sequence shown in SEQ ID NO:34 and two light chains having 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 selected from integers between 1 and 8 or decimals between 1 and 8, preferably a number between 7 and 8 (including the endpoints). 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, lung squamous cell carcinoma, non-squamous non-small cell lung cancer, head and neck squamous cell carcinoma (including nasopharyngeal cancer), colorectal cancer, urothelial carcinoma, kidney cancer, etc., and preferably the tumor is colorectal cancer with moderate / high EGFR expression, triple-negative breast cancer with moderate / high EGFR expression, head and neck squamous cell carcinoma with moderate / high EGFR expression ( The following are selected from EGFR-mutated non-squamous non-small cell lung cancer (Nsq-NSCLC), including nasopharyngeal carcinoma, EGFR-mutated non-squamous non-small cell lung cancer (Nsq-NSCLC), EGFR-moderate / highly expressive squamous cell lung cancer (Sq-NSCLC), other EGFR-moderate / highly expressive advanced solid tumors such as gastric cancer, esophageal cancer, pancreatic cancer, cervical cancer, biliary tract cancer, non-squamous non-small cell lung cancer, and RAS / BRAF-mutated colorectal cancer, as well as drug-resistant lung adenocarcinoma (gefitinib-resistant and / or osimertinib-resistant lung adenocarcinoma).

7. A drug comprising an antibody-drug conjugate represented by formula I, and a pharmaceutically acceptable salt, hydrate, solvate, stereoisomer, or isotope-labeled thereof, 【Transformation 3】 However, Ab is selected from SWY2110, SWY2111, SWY2112, SWY2113. SWY2110 is an antibody having two heavy chains having the amino acid sequence shown in SEQ ID NO:29 and two light chains having the amino acid sequence shown in SEQ ID NO:30; SWY2111 is an antibody having two heavy chains having the amino acid sequence shown in SEQ ID NO:34 and two light chains having 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 selected from integers between 1 and 8 or decimals between 1 and 8, preferably a number between 7 and 8 (including the endpoints). The aforementioned drug is an oral drug (e.g., tablets, capsules) or an injectable drug (e.g., an injectable solution, a lyophilized preparation for injection).

8. The drug according to claim 7, characterized in that, per unit of the preparation, the amount of the antibody-drug conjugate represented by formula I, and its pharmaceutically acceptable salt, hydrate, solvate, stereoisomer, or isotope-labeled product is calculated as 1 mg to 1000 mg, preferably 5 mg to 500 mg, or 10 mg to 300 mg, or 20 mg to 200 mg, or 50 mg to 150 mg, or 50 mg to 300 mg, or 50 mg to 400 mg, or 100 mg to 200 mg, or 100 mg to 300 mg, or 100 mg 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.

9. A method for treating a tumor by administering an antibody-drug conjugate represented by formula I, and a pharmaceutically acceptable salt, hydrate, solvate, stereoisomer, or isotope-labeled thereof, in a therapeutically effective dose to a subject or patient in need of treatment, 【Chemistry 4】 However, Ab is selected from SWY2110, SWY2111, SWY2112, SWY2113. SWY2110 is an antibody having two heavy chains having the amino acid sequence shown in SEQ ID NO:29 and two light chains having the amino acid sequence shown in SEQ ID NO:30; SWY2111 is an antibody having two heavy chains having the amino acid sequence shown in SEQ ID NO:34 and two light chains having 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 selected from integers between 1 and 8 or decimals between 1 and 8, preferably a number between 7 and 8 (including the endpoints). The therapeutically effective dose refers to a dose that alleviates, inhibits, or eliminates tumor growth, providing a benefit to the subject or patient without causing unbearable toxic side effects. 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 carcinoma of the lung, non-squamous non-small cell lung cancer, head and neck squamous cell carcinoma (including nasopharyngeal cancer), colorectal cancer, urothelial carcinoma, renal cancer, etc. Preferably, the tumor is colorectal cancer with EGFR medium expression / high expression, EGFR medium The following are selected from EGFR-expressing / high-expression triple-negative breast cancer, EGFR-moderate / high-expression head and neck squamous cell carcinoma (including nasopharyngeal carcinoma), EGFR-mutated non-squamous non-small cell lung cancer (Nsq-NSCLC), EGFR-moderate / high-expression lung squamous cell carcinoma (Sq-NSCLC), other EGFR-moderate / high-expression advanced solid tumors such as gastric cancer, esophageal cancer, pancreatic cancer, cervical cancer, biliary tract cancer, non-squamous non-small cell lung cancer, and RAS / BRAF-mutated colorectal cancer, as well as drug-resistant lung adenocarcinoma (gefitinib-resistant and / or osimertinib-resistant lung adenocarcinoma).

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

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

12. The antibody-drug conjugate represented by formula I, and its pharmaceutically acceptable salts, hydrates, solvates, stereoisomers, or isotope-labeled compounds may be administered once daily (QD), or in multiple divided doses per day, for example, twice daily (BID) or three times daily (TID), or once weekly (QW), or the above therapeutically effective dose may be administered in multiple doses over several days of the week, for example, twice weekly (BIW) or three times weekly (TIW), or It may be administered once every two weeks (Q2W), or divided into doses over several days within a two-week period, for example, twice every two weeks (BI2W) or three times every two weeks (TI2W), or once every three weeks (Q3W), or divided into doses over several days within a three-week period, for example, twice every three weeks (BI3W, e.g., D1 and D8 for a three-week cycle) or three times every three weeks (TI3W), or once a month (QM), or divided into doses over several days within a month, for example, twice a month It may be administered in two divided doses (BI2M) or three times a month (TIM), or with intervals between doses, for example, once every 2 to 7 days, or once every 3, 4, 5, or 6 days, or at intervals of 2 to 4 weeks, for example, once every 2 weeks (once every 3 weeks, 3 weeks as one cycle), once every 1 week (once every 2 weeks, 2 weeks as one cycle or 4 weeks as one cycle), once every 3 weeks (once every 4 weeks, 4 weeks as one cycle), or 1 to 3 months The method according to claim 9, characterized in that it may be administered at monthly intervals, for example, once every two months, or in a cycle of four weeks consisting of three weeks of continuous administration and one week of rest, or in a cycle of three weeks consisting of two weeks of continuous administration and one week of rest, or in a cycle of four weeks consisting of two weeks of continuous administration and two weeks of rest, or in a cycle of four weeks consisting of weekly administration (once every two weeks), with three weeks forming one cycle and one dose administered on the first day of each cycle.

13. The method according to claim 12, characterized in that an antibody-drug conjugate represented by formula I, and a pharmaceutically acceptable salt, hydrate, solvate, stereoisomer, or isotope-labeled thereof, are administered in the doses described in claims 10 to 11 during each administration cycle.

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